**Meet the editor**

Manal Mohammad Baddour is a Professor of Medical Microbiology and Immunology, Faculty of Medicine, Alexandria University, Egypt. She serves as a Clinical Microbiologist and Microbiology Consultant in the Alexandria University Hospital as well as in several private sector hospitals. She has been Principal Investigator or Coinvestigator of 18 research projects from various aca-

demic granting agencies. She has supervised 4 PhD and 13 master's degree theses. She has reviewed manuscripts for 18 international journals. She has served as Editor and Associate Editor on the board of several international journals. She has published 32 microbiology articles mostly in renowned journals and has authored 2 internationally published books with an h-index of 9. She has also edited an open-access book. She shares in many conferences and workshops related to microbiology and infection control.

## Contents

## **Preface XI**

**Section 1**  Chapter 1 Chapter 2 Chapter 3 Chapter 4 **Section 2**  Chapter 5 Chapter 6 **Diagnostic Approaches 1 Influenza Diagnosis with a Specific Emphasis on the M2e Antigen as a Diagnostic Tool 3**  Yasemin Budama‐Kilinc and Rabia Cakir‐Koc **Application of the New Generation of Sequencing Technologies for Evaluation of Genetic Consistency of Influenza A Vaccine Viruses 21**  Ewan Peter Plant, Tatiana Zagorodnyaya, Elvira Rodionova, Alin Voskanian‐Kordi, Vahan Simonyan, Zhiping Ye and Majid Laassri **Electrochemical Sensors for Detections of Influenza Viruses: Fundamentals and Applications 47**  Hanna Radecka and Jerzy Radecki **Biosensors for Rapid Detection of Avian Influenza 61**  Ronghui Wang and Yanbin Li **Vaccine Development 85 Steps toward a Universal Influenza Vaccine: Research Models and Comparison of Current Approaches 87**  Terianne Wong and Ted M. Ross **Influenza Inactive Virus Vaccine with the Fusion Peptide (rTα1- BP5) Enhances Protection Against Influenza Through Humoral and Cell-Mediated Immunity 119**

Chen Wang, Chengshui Liao, Wufan Zhang, Deyuan Li and Puyan Chen


Chapter 11 **Chronic Obstructive Pulmonary Disease (COPD): Clinical and Immunological Effects of Mono-Vaccination Against Influenza Using an Immunoadjuvant Vaccine of a New Class Versus Combined Administration S. pneumoniae, H. influenzae, and Influenza Vaccines 239** Andrey Dmitrievich Protasov, Mikhail Petrovich Kostinov, Alexander Victorovich Zhestkov, Mikhail L'vovich Shteiner, Svetlana

Vyacheslavovna Kazharova, Yuriy Vladimirovich Tezikov and Igor Stanislavovich Lipatov

## Preface

 Continuous sporadic transmission of avian influenza H5N1 and swine influenza H1N1 to large numbers of humans has prompted concerns that conditions are suitable for emergence of a pandemic influenza virus.

 There have been five influenza pandemics during the past 100 years, and each has been caused by the emergence of a novel virus. Adaptability of influenza virus to various host species and evasion of natural immunity make this ubiquitous pathogen particularly diffi‐ cult to eradicate. We are always prone to influenza pandemics and their consequences. In‐ fluenza epidemics and pandemics have an enormous toll on human health and economy. Estimates of potential global mortality related to pandemic avian influenza are as high as 62 million deaths.

 Rapid and accurate diagnosis is a key factor in order to timely instate therapeutic and con‐ trol measures. Current diagnostic techniques are not as rapid and as sensitive/specific as we would like them to be, and their application demands high-quality laboratories. Biosensors are attractive alternatives. They are self-contained integrated analytical instruments, which are capable of providing specific quantitative or semiquantitative analytical information ap‐ plying a biological recognition element, which is in direct spatial contact with a transducer element. Main parameters describing the quality of biosensors are selectivity, sensitivity, re‐ producibility, and time of response. Novel approaches using biosensors and electrochemical sensors are being studied and presented within the context of this book.

 Immunization against avian influenza remains an active area of research, despite a plethora of research articles and a wealth of information. The diversity of influenza virus strains and subtypes exacerbates the challenge for generating a universal vaccine against influenza. WHO makes recommendations for influenza vaccine composition for each flu season based on international surveillance systems and comparesthe ability of monovalentvaccineproto‐ types to elicit cross-reactive antibodies against prevalent circulating strains. Development of a universal vaccine to protect against the diverse and continuously evolving virus would be a shining beacon in the scientific virology community.

 Additionally, production of influenza vaccine has been hampered by manufacturing diffi‐ culties and modest immunogenicity in humans. High-priority research goals include im‐ proving production speed and increasing quantity of vaccine. Areas of research include the use of cell culture systems, dose-sparing approaches,theuse of adjuvantsand live-attenuat‐ ed viruses to induce more robust immune responses, as well as more contemporary delivery systems such as nanoparticles and recombinant vectors. Several such issues related to influ‐ enza vaccines are reviewed here.

 Vulnerable populations such as pregnant females, the elderly, and those suffering from lung diseases warrant special attention regarding vaccine efficacy and safety. Thus vaccination outcomes in these populations are among the topics discussed in this book.

## **Dr. Manal Mohammad Baddour**

Microbiology Dept., Faculty of Medicine Alexandria University Alexandria, Egypt **Diagnostic Approaches** 

## **Influenza Diagnosis with a Specific Emphasis on the M2e Antigen as a Diagnostic Tool**

Yasemin Budama‐Kilinc and Rabia Cakir‐Koc

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64786

#### **Abstract-**

 Thetherapy,observation,inclusiveness,andpreclusionofrelateddiseasesallinfluence- thediagnosisofinfluenza.-Particularly,thepandemicdurationanddiagnosistimefor- influenzaareextremelyimportant.-Aftertheappearanceofsymptoms,antiviral- medicationmustbeinitiatedwithin-48h.-Cellculture,real‐timepolymerasechain- reaction-(PCR),flowcytometry,directandindirectimmunofluorescencemethods,and- thequickdiagnosistestareallvaluableapproachesforthediagnosisofinfluenza.- Differentinstruments,differenttimedurationsfortheresults,anddifferentspecialists- characterizealltheseapproaches.-Antigenselectionisofcriticalimportancewithregard- tothespecificityandsensitivityofthesemethods,especiallytheserologicalandrapid- diagnosistests.-M2e,thehighlyconservedexternaldomainoftheinfluenza A M2- protein,isapotentialdifferentialdiagnosticmarkerforinfluenzavirusinfection.-This- chapterreviewsthestudiesthatuse-M2easadiagnosisagent,anditilluminatesthe- roleandimportanceof-M2einthediagnosisofinfluenza.-

**Keywords:** M2e, diagnosis, peptide, antigen, virus culture, rapid immunochromoto‐ graphic test, serological test, ELISA, PCR-

#### **1. Introduction-**

 Influenzavirusescancauserespiratoryinfections,anditisamajorcauseofmorbidityand- mortalityworldwide-[1].-Influenzaistypicallyamilddiseasethatlastsfor-1–2weeksinmost- people [2], although in some cases it can be fatal [3]. The likelihood of developing complications- ishigherincertainriskgroups,suchaspeoplewithchronichealthproblems,childrenunder-2- yearsold,andtheelderly-[4].-While-45–77%ofthosehospitalizedduetoinfluenzaareunder-65-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 yearsold,thepatientswhoareolderthan-65yearsarethemostlikely-(60–90%)todieasaresult- of influenza-[5–8].-

The variable nature of the influenza has already caused several pandemics. The accurate and- rapid diagnosis of influenzais therefore of great importance to the effectivemanagement of- epidemic and pandemic periods [4]. As there are several respiratory pathogens that may cause- clinical symptoms similar to those caused by influenza, afinaldiagnosis is difficultfor doctors- to establish [9]. As a result, sensitive and rapid diagnosis methods are needed to confirm the- clinical diagnosis of influenza,as well as to improve the quality of monitoring systems. A- variety of laboratory methods for the diagnosis of influenzaare available (**Table 1**). Each of- these methods has both advantages and disadvantages, and some or all of these factors may- affect the selection of an appropriate diagnosis method [2].-


**Table 1.** Comparison of the diagnostic methods available for the influenza A virus.-

In this chapter, the methods that have developed and used in the diagnosis of influenza will- be evaluated in terms of their principles, sensitivity, specificity,time, advantages and disad‐ vantages, and the antigens used for the diagnosis of influenza A.-

## **2. Diagnosis methods for influenza A-**

There are four basic methods available for the diagnosis of influenza [10], each of which is- detailed in **Table 1**.-

#### **2.1. Virus cultures-**

#### *2.1.1. Classic virus cultures-*

Symptoms such as the deformation of cell morphology, cell necrosis, pouring from the cell- location to the cell culture, and fusion in cells that are infected with the virus are, depending- on the virus proliferation, referred to as the cytopathic effects (CPE) [11].-

Virus cultures that use primary rhesus monkey kidney cells (PMK) or Madin‐Darby canine- kidney cells (MDCK) are commonly accepted to be one of the "gold standards" for laboratory- diagnosis. Whether or not the cells are infected with the virus is determined by the cytopathic- effectsin the cell cultures and hemadsorption using immunofluorescencemonoclonal anti‐ bodies against influenza [10].-

## *2.1.2. Rapid shell vial virus cultures-*

Coverglasses are used in the rapid shell vial cell culture method to passage the cell lines onto- them. The specimens are inoculated after the monolayer cell lines occur. The coverglasses are- then stained with cause‐specific-FITC‐marked monoclonal antibodies for about 24–48 h.- Cytopathic effects are not expected with this method, unlike in standard tube cultures [12].-

Influenzaisolation with the rapid shell vial culture method provides an advantage with regard- to its simplicity and speed when compared to traditional culture method [13]. Influenza shell- vial cultures show results within 1–3 days after virus inoculation [12] (**Figure 1**).-

**Figure 1.** Schematic presentation of shell vial method.-

The specificityof the rapid shell vial test is 100%. Although the time necessary to obtain the- results is much shorter in this method than in traditional cell cultures, in many cases, the time- is actually insufficient to begin optimal antiviral treatment [10].-

#### **2.2. Molecular methods-**

 Thegenomeoftheinfluenzavirusesconsistsofeightdifferentpartsofsingle‐stranded- negativesense-RNA.-Thisstructurechangescontinuouslyanditcontributestotheevaluation- ofthevirus-[14].-Majorchangesoccurinthegenesencodingthetwomajorsurface- glycoproteins,namelythehemagglutinin-(HA)andneuraminidase-(NA)antigens.-The-HA- antigensplayaroleintheconnectionofthevirustothecell,-NAplaysanessentialrolein- releaseandspreadofprogenyvirions,followingtheintracellularviralreplicationcycle-[15].- Thereare-18differenthemagglutininsubtypesand-11differentneuraminidasesubtypes,-H1–- H18and-N1–N11,respectively-[16].-Thehighmutationratecausesthedevelopmentof- subtypes-[17].-

The extreme genetic variability of the influenzaviruses leads to challenges in the design of- molecular‐based diagnosis tests. Most conserved sequences in the genome used to determine- the RT‐PCR primers can be used to identify individual strains of influenza virus [12]. A PCR- that has been modifiedby the addition of the reverse transcription step is known as a reverse- transcription‐PCR (RT‐PCR). In addition, the use of specificprimers for significanttypes of- HA and NA antigens can aid in the identification of subtypes of influenza viruses [17].-

A RT‐PCR can be performed in two differentways: (1) the one‐step approach (reverse tran‐ scription and PCR), which is performed in a single tube; and (2) the two‐step approach, which- involves the transformation of RNA to cDNA and the amplificationof the tested sequences.- The two‐step reaction process is more sensitive when compared with the one‐step reaction.- On the other hand, the one‐step RT‐PCR reaction is fast, and the minimal number of steps- reduces the risk of contamination and so improves the reproducibility of the obtained results- [12].-

Many RT‐PCR tests of the subtypes of influenzahave shown greater sensitivity than other- rapid diagnosis tests and conventional cell cultures. The RT‐PCR activity does not change- depending on the age of the patients. The rotation period of the RT‐PCR for influenzais- between 1 and 2 days. In addition, molecular tests require considerable skill and expertise, and- they should hence be integrated into the laboratory processes [10].-

#### **2.3. Rapid immunochromatographic tests-**

Rapid diagnosis kits are immunochromatographic methods that include the use of monoclonal- antibodies against preserved antigens or nucleoproteins that are localized with a membrane- or impregnated onto a strip of influenza A or A and B [18].-

Brieflyput, according to these methods, the respiratory specimen is primarily treated with an- extraction buffer and it is then applied to a filter paper or test strip depending on the test format.- If influenzaviral antigens are available, a visible color change is generated by the reaction of- the antigens and influenza‐specific monoclonal antibodies [19].-

The sensitivity of the rapid diagnostic kits is approximately 70%, depending on the particular- test kit used, the patient's age, and the sample collection time [20]. The specificityof the rapiddiagnostic kits ranges from 76 to 100% [21]. In addition, many kits can distinguish between- the influenza A and B strains, although they cannot subtype further [20].-

Influenza-A has a higher sensitivity in the rapid diagnosis test kits when compared with- influenza-B. Such rapid diagnosis kits are very convenient, and they have a high positive- predictive value because of the spread of influenzain the community [21]. The most important- advantage of these tests is the fact that they can provide results in approximately 10–30 min- [10].-

 However,duringperiodsoflowinfluenzaactivity,thepredictivevalueoftherapiddiagnostic- kitsisalsolow,andfalsepositiveresultsaremorelikely.-Therefore,theuseofthesetests- duringperiodsofhighinfluenzaactivityissuggested-[21].-Inaddition,morespecifictesting- methodsmustbeperformedonpatientswhopresentwithnegativeresultsaccordingtothe- rapidinfluenzadiagnostickitsduringhighinfluenzaperiods.-Themajordisadvantageof- thesetestsistheirlimitedshelflifeofapproximately-1–2years.-Theinadequatecollectionof- samplesandthemisinterpretationofteststripsbyinexperiencedpersonnelmayalsoleadto- errors-[10].-

#### **2.4. Serological tests-**

Serological methods are all based on determining the antibody response in the sera or specific- antigens or gene sequences of influenzavirus. Specific,sensitive, and validated serological- assays can be used for diagnosis of influenza, the identificationof the source of infection, the- epidemiology studies, and the identificationof asymptomatic cases. Serological methods have- also been utilized at prescreening of influenza disease [22].-

The immunology of tested population and sensibility and specificityof the tests have major- role on interpret results of serological tests [23]. Each type of serological methods have own- advantages, disadvantages, and unique characteristics [24]. The serological diagnosis of- influenza infection is based on agar gel immunodiffusion, radioimmunoassay, immunofluor‐ escenceantibody tests, hemagglutination inhibition, enzyme‐linked immunoassay (EIA),- complement fixation,and an increase of specificantibody titer between acute and healing- serum samples as measured by neutralization tests [10, 17, 25].-

#### *2.4.1. Direct immunofluorescence antibody test (DFA)-*

In this method, antibodies are labeled with fluorescentcompounds called fluorophores.- Fluorophores are generally organic molecules with cyclic structure. Fluorescein and rhoda‐ mine are examples of the most used molecules.-

In this method, the antigen in the suspected material is fixedon slides and specifically labeled- antibodies placed onto the antigen. Fluorescence labeled (FITC) "monoclonal antibodies" are- considered positive due to the luminescence in the presence of agent in patients' samples [12,- 25–27]. DFA test is developed for antigen detection in tissue or body fluids and used success‐ fully for diagnosis of influenza.-Among children who have high fever and spread lots of- influenza viruses, DFA sensitivity is commonly higher [26] (**Figure 2**).-

**Figure 2.** Schematic presentation of direct immunofluorescence antibody test.-

## *2.4.2. Indirect immunofluorescence antibody test (IFAT)-*

In an indirect immunofluorescenceassay, antibodies against influenzalabeled with fluores‐ cencedyes are used [10, 27, 28]. Influenza-A virus particles are fixedon a slide and then the- suspected serum is added. FITC conjugated anti‐human IgG antibodies are added and- examination is made under a microscope [29]. If there is a homologous antibody against the- virus on the slide in the influenzasuspected serum, a yellow‐green glow is seen under a- microscope (i.e., a positive reaction) [10, 12].-

In principle, the serum antibodies react with immunofluorescenceallow for the rapid diag‐ nosis. The sensitivity of the IFA test is 70–90%, while the specificityis above 90% for diagnosis- of influenza [20]. There are individual differencesin the reporting methods for the different- immunofluorescencetests, since this test faces the issue of subjectivity in the reading of slides.- To overcome this problem, the IFAT should be reported by experienced clinical laboratorians- [30, 31] (**Figure 3**).-

**Figure 3.** Schematic presentation of indirect immunofluorescence antibody test.-

#### *2.4.3. Radioimmunoassay (RIA)-*

Radioimmunoassay test is used to search for both an antibody against influenza and virus or- viral antigen, immunoglobulins are conjugated with radioactive material (radioisotopes 14C,- 125I, etc.). In positive reactions, the immune complex that occurs when the influenzaantigen- and specificantibody couple gains radioactivity. This radioactivity is determined by special- counters (such as a gamma counter detector). When the results are evaluated according to a- curve known as the standard curve, an overall result can be seen. The degree of determinedInfluenza Diagnosis with a Specific Emphasis on the M2e Antigen as a Diagnostic Tool 9 http://dx.doi.org/10.5772/64786

**Figure 4.** Principle of RIA. Labeled antigen competes with patient antigen for a limited number of binding sites on- solid‐phase antibody.-

radioactivity could be measured in this way. In negative reactions, radioactivity cannot be- detected [27, 32].-

RIA is a very sensitive and precise technique for detection low amounts of analytes. Otherwise,- the health hazard of radioactive substances is most important limitation of all RIA techniques- due to disposal problems, short half‐life, and the need for expensive equipment [31] (**Figure 4**).-

#### *2.4.4. Agar gel immunodiffusion (AGID)-*

The AGID detects antibodies against all influenza-A viruses, regardless of subtype. The- principle of the AGID test is the simultaneous migration of antigen and antibodies toward each- other through an agar gel matrix. Influenzaantigens and antibodies form a precipitate in to- gel matrix and precipitation line is visualized. Various parameters such as electrolyte concen‐ tration, pH, and temperature can affect precipitate formation [33]. Also, concentrations of the- antigen or antibodies cause shift the location of the line [34]. The immunodiffusionmethod- can be performed in three differentways, namely simple diffusion-(the Oudin method),- unidirectional diffusion-(the Mancini method), and bidirectional diffusion-(Ouchterlony) [35].- The advantages of this method are simplicity, low cost, and inessential specialized laboratory- equipment [36].-

## *2.4.5. Neutralization test-*

The neutralization assay determines that patients' antibody can neutralize the infectivity of a- given influenzavirus strain. Sera that contain specificneutralizing antibodies prevent the- cytopathic effectsof influenzavirus [27]. Influenzaviruses cause CPE via proliferation in cells- form plaques (i.e., the CPE focus) on nutrient agarose‐coated cells [19]. If the virus is cultured- on monolayer cells after being stirred with antiviral homologous antibodies formed against- itself and then left at room temperature for 30–40 min, plaques either do not form (i.e., plaque- neutralization) or else the number of plaques decreases in those cells coated with nutrient agar- (i.e., plaque reduction). A reduction in the number of plaques of 50% or more is considered a- positive reaction. If the virus encounters a homologous antibody carrying serum, then plaque- formation is seen because the neutralization does not occur [12].-

The influenzasubtypes can be definedby using a type‐specificantiviral immune serum. The- neutralization reaction is also utilized as a protection test in animals [27].-

The neutralization test has several advantages which it can identify functional, strain‐specific- antibodies in sera [37]. However, it cannot be used routinely since it is time‐consuming and- laborious.-

#### *2.4.6. Complement fixation (CF) test-*

The complement fixationtest (CFT) is one of the classical influenzadiagnostic assays, which- mainly detects IgG antibodies [38].-

In this test, the primarily influenzasuspected serum (titrated), then the known viral antigen- (titrated), and later the complement (fresh guinea pig serum titer) are added. They are- thoroughly mixed and then allowed to stand. At this stage, if a homologous antibody against- influenzais present in the serum, the antibody couples with the antigen and the complement- binds with this complex. If there is no antibody present, coupling with the antigen does not- occur and hence the complement cannot connect and so remains free. A mixture known as the- hemolytic system (hemolytic serum + sheep red blood cells) is added to the tubes to determine- whether or not the complement is attached.-If there is no hemolysis in the erythrocytes, the- complement is held in the firststage or, in other words, in the antibody‐antigen complex. This- indicates the presence of an antibody in the influenzasuspected patient's serum (i.e., a positive- reaction) [12, 39].-

CFT is useful test for diagnosis of acute virus infection, however, it is quite complex, less- sensitive, very labor intensive, and is not suitable for automation.-

## *2.4.7. Hemagglutination (HA) vs. hemagglutination inhibition (HI)-*

The hemagglutination inhibition test is an important diagnostic tool in certain infections- especially influenza.-The hemagglutinin protein on the surface of the virus may cause agglu‐ tination in the presence of erythrocytes [40]. Blocking of this aggregation by specificantibodies- in the patient's serum is named as hemagglutination inhibition test [37].-

The HA agglutination test is the conventional method for the determination of influenza- viruses, while the HI test is commonly used for typing [40]. If the sera from individuals- suspected of carrying the disease have virus‐specificantibodies against the virus, then these- antibodies prevent HA by neutralizing the HA property of the virus when the antibodies and- the virus come into contact with each other. In test tubes, erythrocyte aggregate occurs on the- bottom in the form of a round point [19].-

In the HI test, the numbers of antibodies within the serum are proportional to the amount of- HI titers. The HI titers in the serum may remain at the initial dilution if there is less antibody- present, although the titers in the serum will reach differentdilutions if there is too much- antibody present [39, 41].-

The hemagglutination inhibition assay is a reliable, relatively simple, and inexpensive- technique to antigenically characterize isolates of influenzaviruses [37]. Whereas the HI testis very useful in epidemiological surveys, it is not suitable for routine diagnosis of influenza- [42]. Nowadays, this assay is widely used and is replaced by more modern immunoassays [38].-

#### *2.4.8. Enzyme‐linked immunosorbent assay (ELISA)-*

In enzyme immunoassays, enzyme‐conjugated antibodies are generally used for detection of- viral antigens. These groups of tests, antigen or antibody are bound to solid phase as microtiter- plates, nitrocellulose membranes, and magnetic latex beads [31]. Patient serum is added to- antigen bounded solid phase and an enzyme‐labeled antiglobulin is added. If anti‐influenza- antibody is present in patient's serum, enzyme‐labeled secondary antibody reacts and- chromogenic substrate causes color change [27].-

The specificantigens as HA, NA, NP, and M protein of influenzaare widely used ELISA. The- sensitivity of EIA varies between 64 and 78% [43]. Because of the high specificity, sensitivity,- simplicity, and low cost, ELISA is one of the most common used immunoassays in the clinical- laboratory [31].-

#### **3. Antigens that are used in diagnosis-**

The methods used in the diagnosis of influenza-(except for viral cultures) are based on the- determination of the antigen of the virus, the determination of antibodies against the antigen- in the patient, or the determination of the gene region of the selected antigen as in molecular- methods [40]. In all these methods, the target antigenic structures are used as important- selection criteria that affectthe specificityand sensitivity of the method. Nucleoproteins and- neuraminidase are used in the viral commercial diagnostic kits (rapid tests) popular in- commercial studies and academic research. Due to the virus undergoing changes, the use of- these antigens in the diagnosis tests affectsthe obtained results. For this reason, it is also- important to use the conserved regions of influenza A in diagnosis [44].-

Therefore, in this part of the chapter, the M2 protein, the most conserved structure of influenza- A, will be evaluated in terms of its diagnostic importance by considering its biological- functions.-

#### **3.1. Definition of M2e protein-**

In influenza A viruses, one of the most important structural proteins is matrix protein 2 (M2)- [45]. The M2 protein, the internal membrane protein, is made up of an ectodomain part, which- is a single membrane spanning domain located at the N‐terminus, and a cytoplasmic tail, which- is found in the C‐terminal [46].-

While the N‐terminus domain is 24 amino acids long, part of it is located at the outer side of- the membrane surface (M2e), while the 54 amino acids long C‐terminus is situated in the- cytoplasmic side and the remaining 19 amino acids sequence the lipid bilayer [46] (**Figure 5**).-

From the firsthuman influenza-A strain that was isolated in 1933 to the present day, no amino- acid mutation has been found in the extracellular domain of the M2 protein [47]. In all subtypesof the influenza-A viruses, the extracellular domain of the M2 protein, M2e, is significantly- conserved with an unchanged SLLTEVET (residues 2e9) sequence at the N‐terminal [48]. The- M2e protein consists of a homotetramer structure with disulfidelinkages that are held together- by noncovalent interactions [49]. The M2e protein is conserved in all types of influenza A- viruses. Despite appearing in small amounts on the surface of mature virions, this protein is- majorly expressed on the surface of infected cells [46].-

The peptide epitope of the influenza-A virus M2e protein is: Met‐Ser‐Leu‐Leu‐Thr‐Glu‐Val‐ Glu‐Thr‐Pro‐Ile‐Arg‐Asn‐Glu‐Trp‐Gly‐Cys‐Arg‐Cys‐Asn‐Asp‐Ser‐Ser.-

**Figure 5.** Schematic presentation of influenza A virus structure.-

#### **3.2. Biological function of M2e-**

The main functions of the M2e protein are: (i) functioning as a proton channel; (ii) playing a- role in viral replication; and (ii) defending the maturation of HA and structural integrity [48].-

The M2e protein is not required for viral replication, but during viral disintegration, it does- act as an ion channel that allows the entry of protons into the virions [50]. M2 also plays an- effective role in virus morphogenesis and assembly.-

M2e stabilizes the newly synthesized HA molecules by adjusting the pH toward the secretion- pathway [51]. M2 transport protons reduce the interior pH of the virions via a receptor‐ mediated endocytosis procedure during the entry of the virus [52].-

#### **3.3. Immunological responses against M2e-**

Mucosal and systemic immunity play a role in the body's resistance to infection. Influenza‐ specificlymphocytes have been detected in the blood and lower respiratory secretions of- influenzapatients [53]. A short‐term primary cytotoxic T‐cell response can be detected after- 1–2 weeks (infection with influenzaviruses leads to virus‐specific-B cell as well as T‐cell- responses) [54]. Antibodies occur against type‐specificinternal proteins, NA, and M1, as well- as against viral surface glycoproteins such as HA and NA. The neutralizing antibodies against- HA are the firstimmune constituents to protect the host from infections caused by the influenza- viruses [55, 56].-

M2 is a significantlyexpressed structural protein on the surface of infected cells. M2e is a small- portion with a low immunogenicity in its native form [52]. The relatively small size of M2e- and its low abundance in virions when compared to other glycoproteins like HA and NA are- possible explanations for the low reactivity of M2e [57]. Anti‐M2e antibodies can develop in- some infected patients [52]. The host's adaptive immune system attractsthe virus. At the same- time, the infection of humans with influenza-A viruses stimulates a weak anti‐M2 antibody- response for a short duration of time [58]. The anti‐M2e antibodies' seroprevalence is increased- with age, which is an auxiliary factor in this pre‐existing immunity against M2 [59].-

M2e‐specificantibodies act through the antibody dependent cell cytotoxicity, and the innate- or complementary immune system promotes the killing of infected cells. Specific-M2e- antibodies prevent the release of viral particles toward the extracellular fluidor else stimulate- the uptake by phagocytic cells through the Fc receptors via connecting to the viral cell [44, 52].-

#### **3.4. Studies that have used M2e in the diagnosis of influenza-**

Ingrole et al. chose to use M2e in their study based on its preserved region. The use of an elastin‐ like particle led to the increase in size of the produced antibody. M2e can easily be recognized- by the M2e‐specificantibodies due to the conjugation of M2e and ELP (elastin‐like particles)- [60].-

Khurana et al. conducted an experiment based on HA2 (488–516), PB1‐F2 (2–75), and M2 (2–- 24) peptides, which have the most conserved regions of the H5N1 strain. In their study,- experiments were carried out with ELISA and rapid diagnostic kits, and the effectsof these- peptides were investigated. In the study that used rapid diagnostic kits, the attachmentto the- HA2 and M2 peptide bands was observed to be positive in the serum of infected patients. In- the ELISA study, there was no reactivity against H5N1 in patients who were vaccinated with- a vaccine that did not include the M2e peptide [61].-

Denis et al. investigated the use of the papaya mosaic virus as a carrier of the conserved and- small amount M2 peptide instead of HA and NA peptides in a vaccine study. Papaya mosaic- virus conjugated virus‐like particles have been observed to increase the production of the- antibody against the M2 peptide. Specificbindings were observed against M2e in both ELISA- and MCDK cell culture experiments [62].-

Wolf examined the effectof M2e‐based multiple antigenic peptides on the production of- antibodies via ELISA and ELISPOT techniques. The specificityto the M2e peptide was- measured in antibody secreting cells by ELISPOT assay and, as a result, a higher formation of- immunoglobulin levels was observed [63].-

Hadifar et al. used an ELISA method based on the distracted intravenous access (DIVA) test- against H5N1 in their study. The M2e antigenicity of the monomers that form when infected- in a natural way to be limited is developed with the DIVA test method. The use of the tetramer- structure of M2e instead of the M2e monomer increased the efficiencyof the ELISA and other- tests mentioned in this study [64].-

Tarigan et al. used the single M2e peptide (sp‐M2e) ELISA and multiple antigenic forms of the- M2e ELISA methods, and the sensitivity of these tests in the diagnosis of M2e was compared.- The degrees of M2e were measured by ELISA in order to use the M2e as a target in the DIVA- test during pandemics. Although both ELISA techniques have specificityin the diagnosis of- the M2e peptide, the MAP‐M2e ELISA technique has higher specificityand sensitivity in the- diagnosis of M2e [65].-

Black et al. determined the influenza-A (H2N2) M2 expression of recombinant baculovirus via- an indirect immunofluorescentantibody assay (IFA) in a study conducted in 1993. The- formation of an antibody against M2e was determined using the EIA test [54].-

## **4. Future aspects-**

In a study conducted by the US Food and Drug Administration (FDA), the analytical per‐ formance of the 11 most widely used influenzadiagnostic tests on the market was evaluated- in a comprehensive manner. In experiments conducted using 23 differentinfluenza subtypes- that have been in circulation recently, although most brands could detect the viral antigen in- samples that included a high concentration of influenza virus, it was found that detection based- on subtypes of the virus is limited in lower concentrations. It was further suggested that- physicians should be careful of negative results when using rapid diagnosis tests for the- diagnosis of influenza [66].-

Peptide‐based enzyme immunoassays are widely used in the sero‐diagnosis of bacterial and- viral infections. These tests can be easily applied due to their enhanced advantages, namely- simplicity, specificity, sensitivity, and relative inexpensiveness.-

Despite the genetic differentiationof the influenza-A virus, it has been shown that the peptide- region formed by the 24 amino acids of the M2e protein located on the surface of the virus is- the most conserved region in all strains, and it has therefore been used as a potential diagnosis- marker in various studies [56, 57, 67, 68]. Although M2e is normally found in small quantities- on virus particles, it is secreted abundantly on the cell surface infected by the virus and, in- particular, it will provide a significantadvantage in the detection of the disease during- pandemic periods [61]. Therefore, diagnosis tests that are developed based on M2e will be able- to detect different subtypes of the influenza viruses.-

#### **Acknowledgements-**

The authors would like to thank The Scientificand Technological Research Council of Turkey- (TUBİTAK) for their support the named "Development of Rapid Diagnostic Kit with Immu‐ nochromatographic Method of Depending on IgY Antibody Specificin (M2e) Peptide for- Diagnosis of Influenza A Infection" (Project No. 115S132).-

#### **Author details-**

Yasemin Budama‐Kilinc\* and Rabia Cakir‐Koc-

 \*Addressallcorrespondenceto:yaseminbudama@gmail.com-

 Departmentof-Bioengineering,-Chemicaland-Metallurgical-Engineering-Faculty,-Yildiz- Technical-University,-Esenler,-Istanbul,-Turkey-

## **References-**


and Reports: Morbidity and Mortality Weekly Report. Recommendations and Reports/- Centers for Disease Control, 2002. 51(RR‐3): p. 1–31.-


identifying infected among vaccinated chickens. Avian Pathology, 2015. 44(4): p. 259–-268.-


## **Application of the New Generation of Sequencing Technologies for Evaluation of Genetic Consistency of Influenza A Vaccine Viruses**

Ewan Peter Plant, Tatiana Zagorodnyaya, Elvira Rodionova, Alin Voskanian‐Kordi, Vahan Simonyan, Zhiping Ye and Majid Laassri

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64371

#### **Abstract-**

 Foralmosthalfacentury,-Sangersequencinghasbeentheconventionalmethodfor- sequencing-DNA.-However,itsutilityforsequencingheterogeneousviralpopulations- islimitedbecauseitcanonlydetectmutationsthatarepresentinasignificantportion- ofthe-DNAmolecules.-Severalmolecularmethodsthatquantifymutationspresentat- lowlevelsinviralpopulationswereproposedforevaluationofgeneticconsistencyof- viralvaccines;however,thesemethodsareonlysuitableforsinglesitepolymorphisms,- andcannotbeusedtoscreenforunknownmutations.-

 Next‐generation-(deep)sequencingmethodshaveenabledthedeterminationof- sequencesoftheentireviralpopulation,includingminoritycomponents.-Theyenable- notonlysequencing,butalsoaccuratequantificationofmutations.-Thistechniquehas- greatvalueformonitoringthegeneticconsistencyofviralvaccines.-Recently,anumber- ofnewdeepsequencingplatforms wereintroduced-(MiSeq,-Iron Torrent,etc.)thatmade- suchananalysisquiteaffordableforindividualresearchlabs.-Here,wereviewtheuse- ofcurrentdeepsequencingapproachesforinfluenzavirusstudies,focusingonthe- evaluationofthegeneticconsistencyofinfluenza-Avaccineviruses.-Wealsodescribea- newbioinformatictooltoanalyzedeepsequencingdataandidentifyartifactsfromthe- truemutants.-

**Keywords:** deep sequencing, DNA and RNA libraries, influenzaviruses, mutational- profiles, sequence heterogeneity-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1. Introductory comments on influenza viruses and vaccines-**

## **1.1. Influenza viruses-**

Influenza-A viruses are the causative agents of seasonal epidemics and periodic pandemics.- There are many serotypes that infect birds, especially waterfowl, and a few serotypes that infect- mammals, including humans. Although some influenza-A strains from birds and pigs have- jumped the species barrier to infect humans, the majority of human infections are caused by- the spread of endemic strains. The endemic strains are continually evolving being one of the- reasons that influenza-A infections remain a persistent problem. Strains of influenzavirus that- are used in vaccine production are prone to mutations during the manufacturing process,- which can cause breaches in the consistency of vaccine quality. Such mutations can lead to- changes in the antigenic structure of the virus and thus affect vaccine effectiveness.-

Influenza-A viruses belong to the Orthomyxoviridae family of viruses [1]. There are several- common features shared among the viruses in this family: they are all negative sense, single‐ stranded RNA viruses that replicate in the nucleus of the host cell. The influenza-A virus- genome is comprised of eight RNA segments that encode more than 11 proteins. Two of the- segments each encode a major antigenic protein. The fourth largest segment encodes a- hemagglutinin (HA) protein and the sixth largest segment encodes a neuraminidase (NA)- protein. The differentsubtypes and strains of influenza-A viruses are distinguished by the HA- and NA proteins that coat the surface of the virus. There are at least 18 different-HA types and- 11 NA types [2]. The segmented nature of the viral genome enables two viruses co‐infecting- the same cell to exchange their segments to produce reassortant progeny. Replication of- influenza-A viruses also results in mutated viral genomes because of the high error frequency- of the RNA polymerase and actions by host defensive elements [3, 4]. Mutations contribute to- the emergence of new endemic strains and reassortment may lead to the emergence of- epidemic or pandemic strains.-

 The presence of mutations in influenza-A populations has been examined in a variety of- contexts. Several groups have isolated clones and used Sanger sequencing to identify muta‐ tions. Isolation of a sufficientnumber of clones has resulted in estimates of the mutation- frequencies ranging from 6 × 10 −4to 2 × 10−6-[3, 5, 6]. Although the information about hetero‐ geneity is of great interest, caution must be exercised to ensure that it is accurately reflected in- sequence databases. The presence of errors in influenzadatabases has been noted [7, 8]. To- limit discrepancies, some groups have used next‐generation sequencing (NGS) to identify- sequence heterogeneities [9–12]. Additional technologies such as multisegment reverse‐ transcription PCR have also been employed [13]. These studies revealed several interesting- things. For example, it has been found that differencesin viral sequences may occur after a- single passage [14], that the same antigenic variants can be detected in differentindividuals- [15], and that oseltamivir resistant and sensitive viruses can be found together as part of- heterogeneous viral populations [16].-

Most human infections are caused by influenza-B viruses and the influenza-A serotypes H1N1- and H3N2. In addition to the endemic human influenzatransmission, there are cases reportedeach year of influenza-A infections originating from an animal. Influenza-A (H3N2) variant- viruses from swine are sometimes transmitted to humans, especially those in close contact with- pigs in agricultural settings [17]. Poultry workers are also frequently seropositive for a variety- of differentavian influenzastrains [18–20] suggesting infrequent but detectable transmission.- In most cases, there is no person‐to‐person transmission of the animal viruses. Nevertheless- candidate vaccine viruses (CVVs) are prepared each year against some of these viruses to- provide a prophylactic option in the event of an outbreak (http://www.who.int/influenza/en/).- The CVVs for vaccines against endemic strains and potentially pandemic strains are provided- to vaccine manufacturers for use as seed viruses in the manufacturing process.-

#### **1.2. Influenza vaccine production-**

There are several differentmethods that are used to produce influenzavaccines. Errors may- be introduced into the antigenic protein during the replication of the seed virus, no matter- which production method is used. The differentlicensed vaccines are produced from influenza- virus grown in eggs or cell culture, or from recombinant viruses expressing the influenza HA- from an alternative viral backbone grown in cell culture. Contemporary strains isolated from- patients during the current epidemic season normally do not always grow well in cell sub‐ strates used for vaccine production. To increase virus yields they are recombined with- reference high‐growth strains, such that the CVVs have HA and NA‐coding RNA segments- from the contemporary strain, and RNA segments coding for replicative proteins from the- high‐growth reference virus. HA is the primary protective antigen and is responsible for- binding the cellular receptor. Receptor properties in human and chicken cells differ,forcing- the virus HA to adapt to the new receptor, leading to changes in the antigenic specificity- potentially affecting vaccine potency.-

The most common manufacturing process used in FDA‐licensed vaccines is to grow influenza- virus in eggs and then inactivate the virus. The inactivated virus is purified and then diluted- to the desired potency for fillingvials or syringes. Live attenuatedinfluenza vaccines (LAIV)- are also grown in eggs but are administered as a nasal spray. Codon deoptimization has also- been proposed as a method for creating attenuatedviruses [21, 22]. Unlike the current licensed- products these may be grown in cell culture.-

Some inactivated influenzavaccines are grown in cells. Production of cell‐grown vaccines- currently uses the same egg isolated seed virus that is used for egg inactivated vaccine- production. The cell‐grown viruses are harvested, inactivated, and filled into vials or syringes- for distribution in a similar manner to the egg grown viruses. The production of recombinant- viruses to prepare HA does not require a live seed virus. The HA sequence is cloned into the- virus used for production. Although the frequency of errors during replication may differfrom- that of an influenzavirus, the concern still remains. Even if vaccine strains are produced using- cloned DNA sequences or synthetic sequences, there is still the possibility of errors arising- during amplificationof the seed virus. Errors may emerge because of inaccuracies inherent in- the replication system or as a response to the host cell defenses.-

#### **1.3. Influenza vaccine seed viruses-**

The seed viruses used to produce influenzavaccines are derived from differentsources.- Because the influenza virus spreads throughout the world and strains are continually evolving,- a network of academic, governmental, and commercial organizations work together to- produce new seed viruses. New virus isolates are collected by National Influenza-Centers and- sent to the World Health Organization Collaborating Centers (WHO CCs). The viruses are- typed according to strain and subtype using antigenic and genetic analyses. Viruses are usually- isolated in Madin‐Darby Canine Kidney Epithelial Cells (MDCK cells) and then amplified in- eggs. It is important that the seed virus is as close in sequence and antigenicity to the original- isolate as possible. To this end, the egg‐amplifiedvirus is used to immunize ferrets for the- production of antiserum. The antiserum is then used for antigenic typing of viruses. Such- analyses are used to determine how well the strains used in the influenzavaccine are matched- to the currently circulating strains.-

To produce sufficient quantities of vaccine, the CVVs must have good growth characteristics.- Ideally, the viruses should replicate efficiently, have a high antigen (primarily hemagglutinin)- to total protein ratio and not have increased pathogenicity. Many viruses do not produce high- yields in eggs without adaptation. Some viruses have been propagated for many years and- have well‐known growth characteristics. They include the influenza-A strains Puerto Rico/- 8/1934, cold‐adapted A/Ann Arbor/6/1960, A/Leningrad/134/17/1957 and variants of these- viruses. Where appropriate these strains have been used as a backbone for influenza-A CVVs.- Combining the high yield characteristics with the antigenic characteristics of contemporary- strains facilitates vaccine production. Reassortant viruses with the desired antigenicity and- growth characteristics are produced by two differentmethods; classical reassortment and- genetic reassortment. Dr. Kilbourne of the New York Medical College (NYMC) developed the- classical method to create reassortant viruses that expressed the HA and NA from a seasonal- strain in the background of a high‐growth virus [23]. This method involves co‐culture of a- contemporary virus and a high yield strain with antibodies to select against the HA and NA- of the high yielded strain. Although the resulting viruses have the desired HA and NA genomic- segments, the remaining segments may come from either the high‐growth strain or the- contemporary strain. Another approach based on genetic engineering allows the production- of viruses from plasmids expressing the eight influenzavirus segments. Genetic engineering- also allows the expression of HA and NA proteins in a vector‐based system such as a baculo‐ virus. The desired genetic sequence of HA engineered in this system may be incompatible with- baculovirus components, which can result in changes as more efficientmutants displace the- parental virus (the so‐called gene constellation effect) [24].-

All CVVs go through several rounds of replication at manufacturers' facilities as they producetheir own virus stocks, working seed, and the finalproduct. Some changes to the virus mayoccur during manufacture so tests to verify the identity, purity, potency and stability of vaccinelots are required. The potency of inactivated influenzavaccines and influenzavaccine pro‐ duced from recombinant viruses is determined using standardized reagents supplied bynational regulatory authorities. The potency of a live‐attenuatedvirus is calculated from theamount of viable attenuatedvirus. Genetic characterization of the vaccine viruses is currently- achieved by partial genome sequencing or restriction analysis.-

It has been suggested that most of the differencesbetween natural isolates and vaccine seed- viruses occur during the selection and clonal isolation of the candidate virus prior to manu‐ facture [25]. The fidelityof replication will vary among viruses and es and will depend on other- factors such as the host cell line and multiplicity of infection used. There are some limits on- the number of times that seed viruses may be passaged so that mutations are less likely to- occur. The European Pharmacopoeia monograph 0158 for inactivated influenzavaccines states- that the seed virus should not be passaged more than 15 times. However, because regulations- tend to lag behind scientificdevelopment, there is no universally accepted guideline for- influenzavaccine manufacture that covers egg‐derived, cell‐derived, and synthetic reassor‐ tant viruses.-

#### **2. Importance of the evaluation of genetic consistency of influenza A- vaccine viruses-**

Virus populations are comprised of genetically variable viruses and this can affecttheir- replication, evolution, attenuation,and pathogenesis [26, 27]. Having an understanding of the- mutations present, even at low levels, in a virus population is important for our understanding- of how the viruses grow and cause infections. It has recently been shown that an influenza- population containing two variants involved in cell exit grows betterthan populations- containing either variant alone [28]. Although good growth properties are a desirable feature- in vaccine seed viruses, it is critical that other parts of the genome, such as those causing- attenuationor encoding the major antigenic regions, remain stable. Consistency of manufac‐ ture is important and having suitable means to assess genetic consistency is valuable. New- assays capable of assessing entire viral genomes, and detecting mutations present at a low- level, are needed.-

The emergence of mutations in the course of vaccine manufacture was shown to contribute to- partial reversion to virulence in the oral polio vaccine (OPV). Mutant analysis by PCR and- restriction enzyme cleavage (MAPREC) is used to control batches of oral polio vaccine for the- presence of neurovirulent mutations [29, 30] and has been expanded to be used for other- viruses [31, 32]. Mutations emerging during virus growth may also change antigenic properties- and therefore affectprotective potency of live and inactivated vaccines. New approaches that- can be used not only for monitoring genetic stability of live vaccines, but also for controlling- consistency of inactivated vaccines are needed. Influenzavaccines are manufactured in- embryonated chicken eggs or cell culture. There is evidence that vaccine seed viruses adapt to- grow efficientlyin the differentsubstrates and this can lead to changes in the receptor‐ recognition site of viral hemagglutinin, which is the major protective antigen [33–36]. For this- reason, it is important to monitor the changes that may take place in major protective epitopes- of the virus. It is also important to know that mutations responsible for attenuatedphenotypes- are maintained. Knowing which mutations are emerging during virus growth in productionsubstrates could also be used to optimize genetic structure of vaccine strains. Consistently- accumulating mutations have higher fitness,and if they have no deleterious properties, their- incorporation into the genome of vaccine virus could increase its yield and improve vaccine- potency. Given these concerns it is imperative to screen genomes of viral vaccines for emerging- mutations.-

#### **3. Methods used for evaluation of genetic consistency of vaccine viruses-**

As mentioned above viral populations are highly heterogeneous, and even small quantities of- mutants in virus stocks may affecttheir biological properties. Although PCR and restriction- analysis of reassortant influenzaviruses can demonstrate which parental strain each genomic- segment was derived from, it cannot detect new mutations. Even traditional sequencing- methods are not sensitive enough to detect small amounts of mutants, and highly sensitive- PCR‐based methods can only analyze one or few known mutations at a time.-

Conventional sequencing approaches are suitable for discovery of mutations that are present- in substantial amounts, usually around 20–25% [37]. Determining the actual frequency using- conventional sequencing requires the labor‐ and time‐intensive analysis of a large number of- virus clones (plaques).-

There are indirect approaches based on analysis of electrophoretic mobility in gels [38], which- are insufficientlysensitive and do not allow mutations to be located accurately. Mass spec‐ trometry (MALDI‐TOF) [39] and hybridization with microarrays of short oligonucleotides [40–- 42] are sensitive, but are laborious and may require follow‐up by direct sequencing. A highly- sensitive mutant analysis by PCR and MAPREC [29–31] can detect and quantify mutants at- levels as low as 0.1% of the viral population. Recently we developed a quantitative allele‐ specific-PCR (asqPCR) [43] for detection of a low level of mutants in viral vaccines. RT‐PCR- has been proposed as a method for checking the homogeneity of influenzavaccine seed- candidates [44]. However, these methods are only suitable for analysis of one known mutation- at a time.-

Several versions of high‐throughput sequencing technology, known also as deep or massively- parallel sequencing (MPS) have been used to assess influenza vaccine viruses. These technol‐ ogies enable rapid generation of large amounts of sequence information [45]. Three different- platforms for deep sequencing are used widely at present time: the Roche/454 FLX [46] (http://- www.454.com/)-(http://454.com/products/technology.asp),the Illumina/Solexa Genome- Analyzer [47]) (http://www.illumina.com/technology/next‐generation‐sequencing/sequenc‐ ing‐technology.html),and the Applied Biosystems SOLiD TM System (http://www.thermo‐ fisher.com/us/en/home/brands/applied‐biosystems.html).-Two new sequencing platforms that- are improved to sequence long reads have been developed recently: the Pacific-Biosciences- SMRT Sequencing (http://www.pacb.com/smrt‐science/smrt‐sequencing/) [48] and Oxford- Nanopore Technologies MinION (https://www.nanoporetech.com/technology/the‐minion‐ device‐a‐miniaturised‐sensing‐system/the‐minion‐device‐a‐miniaturised‐sensing‐system).-

These systems are also called "single molecule" sequencers and do not require any amplifica‐ tion of DNA fragments prior to sequencing.-

The deep sequencing technologies were shown to be suitable for analysis of heterogeneities in- viral populations [49]. It can produce huge sequencing information in one run. They are used- for *de novo*sequencing of large genomes, metagenomics studies (virome, microbiome, etc.),- screening for genomic markers, and many other applications [50–58]. Previously, it was- demonstrated that deep sequencing can be used to monitor the genetic stability of oral polio- vaccines, and could replace the WHO‐recommended MAPREC assay for lot release of OPV- [59]. Recently we showed that deep sequencing is suitable for evaluation of genetic consistency- of influenza vaccine viruses [36, 60]**.-**

#### **4. Description of the most used deep sequencing platforms-**

Deep or massively parallel sequencing refers to several high‐throughput methods for DNA- sequencing that are often referred to as NGS. They have dramatically improved the ability of- biotechnology, scientific,and healthcare researchers to analyze viruses by allowing users to- have massive sequencing information for the entire genomes. The high‐throughput sequenc‐ ing fieldhas witnessed the rise of many technologies capable of massive genomic analysis. In- the virology field,deep sequencing has made it simple to sequence full viral genomes.- Likewise, identificationand classificationof novel and known viruses, unbiased characteriza‐ tion of viral populations without the need for virus culturing (viromes), molecular epidemi‐ ology, viral diversity and evolution, transmission and pathogenesis, and medical virology have- greatly benefitedfrom the use of deep sequencing. The cost of deep sequencing has decreased- to an affordableprice due to the competition between vendors and the ability to analyze- multiple samples run in one lane of the sequencing flowcell. This has allowed virologists to- study a huge number of viral samples, including mixture of viral populations, and study low‐ level mutants in a wide range of viruses [36, 59–61].-

There are several platforms for deep sequencing. The most widely used sequencing platforms- are the Roche/454 FLX [46] (http://www.454.com/),the Illumina/Solexa Genome Analyzer [47]- (http://www.illumina.com/technology/next‐generation‐sequencing/solexa‐technology.html),- and the Applied Biosystems SOLiD System (http://www.appliedbiosystems.com/absite/us/en/- home/applications‐technologies/solid‐next‐generation‐sequencing/next‐generation‐systems/- solid‐4‐system.html?CID=FL‐091411\_solid4).-

The differencesbetween these platforms include DNA library preparation procedures and- chemistry, the sequencing reactions on the amplified strands, the length of reads, the amount- of data generated per run, the hardware, the software engineering and the technology used to- amplify single strands of a fragment from the library.-

In general the DNA libraries of fragment targets are generated, and adaptors containing- universal priming sites are ligated to the fragmented target ends, allowing complex genomes- to be amplifiedwith PCR primers. After ligation, the DNA is separated into single strands andattachedor immobilized to a solid surface or support. The immobilization of spatially- separated template sites allows thousands to billions of sequencing reactions to be performed- simultaneously.-

Immobilization and separation of the millions of molecules to differentsurfaces can be- achieved by a variety of methods including the Polonator and PicoTiter Plate [47, 62–64].- Attachmentof forward and reverse primers to a slide and use of solid‐phase amplificationalso- result in the enrichment and amplificationof separate template strands [47] (Illumina/Solexa).-

Two newer sequencing platforms with longer reads differfrom those described above. They- are sometimes referred to as "single molecule" sequencers because they sequence molecule by- molecule and do not require any amplificationof DNA fragments prior to sequencing. The- Pacific Biosciences system involves the attachment of a DNA polymerase to the DNA molecule.- During the sequencing phase the polymerase adds bases labeled with a fluorophore.-The- fluorescenceunique to each base is recorded and, as each new base is added, the fluorescent- label is removed [48]. It generates long sequencing reads (10–15 kb long) from single molecules- of DNA, very quickly.-

The Oxford Nanopore system runs the sample through very small (1 nm wide) pores. As the- DNA passes through these nanopores, the Oxford machine records the electrical charge that- is associated with each individual base pair of DNA, like a signature. It produces longer reads- (>100 kb long).-

A detailed description of the most used two deep sequencing platforms for analysis of- influenza viruses and their vaccines is given below.-

## **4.1. Roche/454 FLX pyrosequencer-**

The Roche/454 FLX sequencing [46] is based on the use of the pyrosequencing technology- (http://my454.com/products/technology.asp), in which the incorporation of each nucleotide by- DNA polymerase results in the release of pyrophosphate that initiates a cascade of enzymatic- reactions that converts the pyrophosphate to a light signal. This light is recorded by CCD- camera. This approach, as with most NGS procedures, starts with DNA library preparation;- the library DNAs with 454‐specificadaptors are denatured into single strands and mixed with- agarose beads whose surfaces carry oligonucleotides complementary to the 454‐specific- adapter sequences on the fragment library, so each bead is associated with a single fragment.- The DNA fragments captured by beads are amplifiedby emulsion PCR (ePCR) [65] to produce- approximately one million copies of each DNA fragment on the surface of each bead. These- amplifiedsingle molecules are then sequenced on a picotiter plate (a fused silica capillary- structure) that holds a single bead in each of several hundred thousand single wells, which- provides a fixed location at which each sequencing reaction can be monitored.-

Individual dNTPs are added to the template in the presence of a DNA polymerase. The- sequencing reaction releases pyrophosphate (PPi) after the incorporation of a complementary- nucleotide. The released PPi is used by an ATP sulfurylase to release ATP from adenosine 5'‐ phosphosulfate. The ATP is then used to generate light by converting luciferin into oxyluci‐ ferin [66]. Unincorporated dNTPs are degraded by an apyrase, and dATPαS (which is not asubstrate for luciferase) is used instead of dATP. This pyrosequencing reaction is repeated- during the sequence of the entire target DNA. This sequencing technology can now produce- sequencing reads with up to 1000 bp in length (http://454.com/products/gs‐flx‐system/).-

These raw reads are processed by the 454 analysis software and then filtered to remove poor‐ quality sequences, mixed sequences, and sequences without the initiating TCGA sequence.- Recently, the 454‐FLX system was upgraded to reach 99.9% of accuracy after filterand an- output of 14 Gb of data per run within 24 h.-

#### **4.2. Illumina genome sequencer-**

The Illumina sequencing [47] method begins with Illumina library preparation flankedwith- Illumina‐specificadapters. Sequencing templates are immobilized on a proprietary flowcell- surface that contains immobilized oligos with sequence complementary to those of the- adapters, and designed to present the DNA in a manner that facilitates access to enzymes while- ensuring high stability of surface‐bound template and low non‐specificbinding of fluores‐ cently labeled nucleotides. Solid‐phase amplificationof each single strand DNA from library- is performed by bridge amplification,which results in the generation of several million dense- clusters of single‐stranded DNA in each channel of the flow cell.-

The Illumina system sequences DNA in the presence of four reversible terminator‐bound- dNTPs [47]. At each sequencing step, a fluorescentlylabeled dNTP is added to the molecule.- The fluorescentsignal is recorded and then the fluorophoreis removed to allow sequencing- to continue. The base calls correlate with the signal intensity. Illumina sequencing technology- can now produce sequencing reads with up to 600 bp in length (http://www.illumina.com/- systems/sequencing.html).-The sequencing results are generated in filesin which each raw- read base has an assigned quality score so that the software can apply a weighting factor in- calling differencesand generating confidencescores. Illumina data collection software enables- users to align sequences to a reference in resequencing applications. This software suite- includes the full range of data collection, processing, and analysis modules to streamline- collection and analysis of data.-

#### **4.3. Deep sequencing data analysis-**

The massively parallel scale of sequencing implies a similarly massive scale of computational- analysis. The conventional pipeline for analysis of next‐generation sequencing data includes- the following stages: quality control and source data filtering;alignment (mapping); reference- profiling-(variant‐calling, pileup); followed by single‐nucleotide polymorphism (SNP) calling- (genotyping); and some form of clusterization or classificationanalysis of samples to discover- up or down expression of genes, detect overabundance of SNP positions and correlate those- with function and phenotype. Because of the sheer size of the data and amount of calculations- needed, such analyses place significantdemands on the information technology (IT) infra‐ structure. Lack of computational power, insufficiencyof actively accessible storage facilities in- laboratory information management systems (LIMS) and deficiencyin network capacity to- move data add significantlyto the overhead required for high‐throughput data production.- The hardware aspect of next‐generation sequencing is complicated by the imperfections of- current sequence analysis tools, which are suited to shorter sequence read data. There are- multiple implementations for all of the stages of the analysis, and some of those are considered- to be industry standard tools, running formidable amount of bio‐medical analytics. Large‐scale- analysis of thousands of samples using variety of available tools highlighted important issues- with data quality, pre‐analytic quality controls, software reproducibility and post‐analytic- quality controls. Existing data analysis pipelines and algorithms must be modified to accom‐ modate extra‐large amounts of short read sequences and combination of shorter and longer- read technologies.-

To analyze the deep sequencing data for genetic consistency evaluation of influenzavaccine- viruses, we have used the corresponding viral reference sequences from NCBI GenBank as a- template for alignment of individual sequencing reads. First, sequencing reads with low- quality (Phred) score are removed from the data set, and the remaining sequences aligned with- reference influenzavirus sequence using custom software: The High‐performance Integrated- Virtual Environment (HIVE, https://hive.biochemistry.gwu.edu/dna.cgi?cmd=main) comput‐ er cluster [67, 68].-

To create a quantifiablemeasure comparing the quality of the sequencing and mapping at- differentpositions on a genome, we developed a metrics for assessing positional variant‐call- quality. To do that, firsta histogram is built at every position of a genome where the number-

**Figure 1.** Explanation of entropy computation. For all bases aligned to a particular reference position, the coordinates- on a read‐frame are accumulated into a positional frequency histogram. For a non‐biased position, such distribution is- more or less uniform (A). For terminally biased base calls such frequency distribution is skewed towards ends (B) and- the Shannon's entropy values drop closer to zero.-

of times a base has occurred at a given read‐position is accumulated. Additionally, positions- of insertion and deletions are also collected in similar histogram. The underlying assumption- of the next‐generation sequencing method is that the DNA amplificationand digestion- procedure is random and the short sequences produced by DNA digestion are not strongly- biased and not sequence dependent. The default assumption is that a particular variant call- should be confirmedby differentpositions on many reads thus rendering the histogram- distribution to be uniform along the entire length of sequence read.-

Post‐alignment quality control includes identificationof mutations distributed non‐randomly- along individual sequencing reads, which may indicate artifacts in PCR amplificationor DNA- sequencing procedures (**Figure 1A**and **B**). Biased distribution of mutations along sequencing- reads was revealed by calculating Shannon entropy values [69]. Low entropy value suggests- that a mutation could be an artifact produced in sequencing procedures. This means that there- is an abnormal bias in distribution of this mutation. This entropy‐based post‐alignment quality- control value is calculated on the basis of the equation below. It is based on the normalized- firstorder momentum of logarithmic probability distribution for a particular base (*b*) at a- particular position (*r*) of reference genome:-

$$Entropy\left(b, r\right) = -\frac{\sum\_{L} p\_i\left(b\right) \times \log\left(p\_i\left(b\right)\right)}{\log\left(L^{-1}\right)}$$

where *L* is the length of the longest read, *pi* (b) is the frequency distribution of a base *b* in the- reference frame of the reads mapped at the location *r*. The index *i* runs over all of the available- positions from 1 to *L*. The denominator makes sure the Shannon's entropy is normalized to a- unit value of 1 as the maximum value for entirely uniform distribution. In contrast singular- value distribution would have a value for entropy equal to zero. This value is computed for- all of the reference positions for every base.-

Finally, aligned sequencing reads were used to compute SNP profilesfor the entire viral- genome.-

#### **5. The use of deep sequencing for evaluation of genetic consistency of- influenza A vaccine viruses-**

Influenza-A viruses are enveloped, single‐stranded RNA viruses belonging to the Orthomyx‐ oviridae family [70], which also contains four other viral species: influenza B virus, influenza- C virus, thogotovirus, and isavirus. The segmented genome of influenza-A virus is about 13.6- kb in size and encodes for at least 11 proteins. Its genome is highly variable due to the low- fidelityof RNA polymerase and reassortment between co‐infecting strains [71]. New virus- mutants emerge continuously allowing viruses to survive in presence of the host immunity- and cause repeated annual epidemics and occasionally pandemics. Because of this frequent- change of the antigens, influenzavaccines must be frequently reformulated to include antigensof the currently circulating strains. Both live and inactivated influenza vaccines are produced- mostly by reassortment with high‐growth strains for vaccine production [72, 73]. As stated- above, adaptation to growth in differentcells can lead to changes in viral receptor‐binding- region, and also in protective epitopes. Therefore, it is desirable to monitor genetic stability of- viruses used in vaccine manufacture to ensure that their antigenic structure remains un‐ changed.-

Deep sequencing technology has opened up the possibility for the characterization of viral- genomes directly from samples [74, 75]. The viral metagenome or "virome" refers to the- collection of viruses found in a particular sample from humans, animals, plants or from a- specificenvironmental sample. Virome studies can lead to the discovery of new viruses- and/or to their association with known or novel diseases. Numerous viruses have been- identified as part of the virome study, including influenza A viruses [2].-

The deep sequencing technologies are a great tool to investigate genetically complex popula‐ tions of influenzaviruses and to detect minority mutant variants with clinical or epidemio‐ logical relevance. Deep sequencing‐based methods have recently been applied for the- assessment of influenza-A viruses diversity and their dynamics of evolution [60, 76, 77]. Others- have focused on the evolution of avian influenzastrains with potential to become pandemic- in humans [78–82], as well as the detection of virulence signatures [83] and reassortment- patterns [84]. Other studies have investigated the transmission and adaptation of avian- influenzaviruses to humans, as part of preparedness for a potential influenzapandemic [12,- 53, 85–98].-

As it is crucial to study transmission and adaptation of avian influenzaviruses, and swine- strains for epidemics and pandemics in humans, many studies based on the use of deep- sequencing techniques have described avian and swine influenzavirus evolution [99–103].- Other studies have investigated the predominance and spread of differenthuman influenza- viruses in specific geographic areas [16, 104–106].-

Study of drug escape variants is an important aspect of epidemiological and clinical virology.- Sanger sequencing can only detect mutations present in around 20% of the viral population- [37, 107–109], which excludes it for quantitation of low‐level viral mutant variants. Using deep- sequencing to detect low portion of mutant drug resistant variants at levels as low as 0.1% of- the virus population has been demonstrated [110–116]. Other studies have focused on the use- of deep sequencing for surveillance of drug resistance‐associated mutations for both NA- inhibitors and adamantanes [117–127]. Deep sequencing has also been used for the detection- and subtyping of human influenza A viruses and reassortants [61, 84].-

Recently, deep sequencing‐based methods have been proposed for the assessment of influenza- A viruses antigenic stability [128] using complete influenza-A genomes and exploiting the- ability to detect and quantify mutations in heterogeneous viral populations. Deep sequencing- was used to study the evolution of influenza-A viruses in the vaccinated pigs. The genetic- diversity and evolution of the virus at an intra‐host level was analyzed directly from nasal- swabs collected during infection [129]. The obtained results demonstrated remarkable- diversity of influenza-A viruses, and rapid change of these viruses during infection of vacci‐

nated pigs. These types of complex studies can be done only by high throughput sequence- analysis.-

To evaluate the genetic stability in influenzavaccine viruses, we have used a deep sequencing- approach that was recently qualifiedfor quantitation of all mutants in the entire genome- including those that are present at low level in viral populations [59]. Recently, we explored- the utility of deep sequencing methods for monitoring the consistency of influenza-A vaccines- [36, 60]. Also in the same study, we proposed new protocols for simultaneous amplification of- all segments of influenza-A genomes and new bioinformatic tools to analyze the data and to- identify artifacts generated during PCR amplificationand deep sequencing procedures.- Amplificationof the entire genome of influenzaviruses presents a challenge because of the- difference in size and sequence composition of the eight genomic segments.-

**Figure 2.** General steps followed for RNA/DNA libraries sequencing of influenza-A/California/07/2009 (H1N1) vaccine- viruses.-

We described PCR conditions that allow to amplify all genomic segments of influenza-A virus- in one reaction [60] that was optimized subsequently during an analysis of the A/California/- 07/2009 (H1N1) vaccine viruses (derived from X‐179A, X‐181, 121XP viruses) [36]. We have- used both the total RNA (without specificamplificationof viral cDNA) and DNA amplicon,- for RNA and DNA libraries preparation, respectively, and both protocols were compared for- consistency in mutant variants quantitation.-

The protocols for deep sequencing of viral DNA libraries and whole‐RNA libraries used to- determine quantitative profilesof mutations along the entire genome of viruses of influenzaA/California/07/2009 (H1N1) vaccine viruses were described [36]. The steps followed to- perform the deep sequencing are presented in **Figure 2**and can be summarized as follows:- The PCR product was purifiedby QIAquick PCR Purification-Kit (Qiagen) and fragmented by- an ultrasonicator (Covaris) to generate the optimal fragment sizes needed for Illumina- sequencing, then the fragmented DNAs were used for library preparation with NEBNext®- DNA Sample Prep Reagent Set 1 (New England BioLabs).-

For preparation of Illumina sequencing libraries from total RNA, the NEBNext mRNA Sample-Prep Master Mix Set 1 (New England BioLabs) was used. Briefly,total RNA (extracted asmentioned above) was fragmented as described above to generate the optimal fragment sizes.-Double‐stranded cDNA was prepared and ligated to the Illumina paired end adaptors. Finally,the libraries were amplifiedusing 15 cycles of PCR with multiplex indexed primers andpurifiedwith magnetic beads using Agencourt Ampure Beads (Beckman Coulter). Deepsequencing was performed at Macrogen (Seoul, Korea) using HiSeq2000 (Illumina) or at ourlaboratory using MiSeq (Illumina).-

**Figure 3.** Sequencing analysis of the entire genome of the influenza-A/California/07/2009 (H1N1) vaccine virus reassor‐ tant tenth passage in eggs sequenced by HiSeq (Macrogen) using 101 bp paired‐end Illumina/Solexa sequencing tech‐ nology. Computations were made by in‐house custom software HIVE‐align. For each segment, the charts of the depth- of sequencing coverage distribution (the number of times every nucleotide was sequenced on ordinate plottedagainst- the position on genome—abscissa), SNP profile-(the ratio of mutants on ordinate plottedagainst the position on ge‐ nome—abscissa), and entropy were built.-

The sequencing data analysis was done using custom software, a highly integrated virtual- environment (HIVE) computer cluster (https://hive.biochemistry.gwu.edu/dna.cgi?- cmd=main)as described above. The RNA sequences of X‐179A, X‐181, 121XP, A/California/- 07/2009 (H1N1), and A/PR/08/34 viruses deposited in NCBI GenBank were used as referencesfor alignment of the viral sequence reads. We analyzed the depth of sequencing, the single‐ nucleotide polymorphism profile,and entropy (that allow us to distinct between bias and true- mutation) for each segment of influenzavirus (see **Figure 3**, for example), the data analysis- resulted also on generation of consensus sequences for each segment.-

The deep sequencing results revealed several heterogeneities in most genomic segments, and- several mutations led to amino acid changes. Deep sequencing of whole‐RNA libraries was- found to be more reproducible than sequencing of DNA libraries. This may be due to errors- introduced during PCR amplificationby DNA polymerase and non‐specificalignment of- primers [36].-

The deep sequencing of the X‐179A passaged viruses [36] identified several mutations in HA- and NA genes. In HA two non‐synonymous mutations; Pro314Gln (in 17% of the virus popu‐ lation) and Asn146Asp (in 78% of the virus population) were identified. The Asn146Asp mutation- is found in the antigenic site Sa; it was detected at 11% in the A/California/07/2009 (H1N1)- strain and as the dominant residue in the X‐181 virus [36]. One X‐179A stock contained the- Lys328Thr mutation at a low level (9%). Viruses derived from X‐179A were heterogeneous and- contained some complete nucleotide substitutions in comparison to their published sequences- in PB2, PB1, NP, and in NS segments [36]. The X‐181 virus was developed from the X‐179A- seed lot by another round of reassortment, and also is subjected to several passages in eggs.- Deep sequencing results [36] showed that the G756T (Glu252Asp, present at 47%) mutation- emerged in HA of the passaged X181 virus, and it is located in the conserved region of the- antigenic site Ca [36].-

**Figure 4.** Percent of mutations (≥5%) emerged in X‐179A‐M1, X‐181‐M4, and 121XP‐M4 viruses (derived from X‐179A,- X‐181, and 121XP viruses, respectively) passaged 10 times in embryonated chicken eggs.-

Unlike the X‐179A and X‐181 viruses, 121XP was developed by reverse genetics [130]. The deep- sequencing of 121XP virus passaged 10 times in eggs (121XP‐M4 virus) showed that this virus- is more heterogenic than X‐179A and X‐181 viruses passaged 10 times in eggs (X‐179A‐M1 and- X‐181‐M4 viruses respectively; **Figure 4**) [36]. In the passaged 121XP virus, the mutation- Lys226Glu was emerged at low level (18%) in Ca antigenic site of HA, which is very close to the- region that participates in the modulation of HA receptor specificityand that enables H3- influenzaviruses to switch specificityfrom avian to human [131–133]; another mutation- Lys136Asn was emerged at a high level (78%) close to the HA antigenic site Sa within the sialic- acid‐binding pocket [134]. Recently a similar deep sequencing approach was used to study the- genetic and potential antigenic diversity of influenzaviruses infecting humans, some of whom- became infected despite recent vaccination [15].-

We found that the deep sequencing approach based on RNA library preparation was effective- and reproducible for detection of low quantities of mutants in the entire genome of influenza- A vaccine viruses [36]. The deep sequencing approach revealed that the viruses derived from- three pandemic A/Ca/07/2009 (H1N1) vaccine viruses have varying levels of sequence- heterogeneities some of them in antigenic sites, which may affect their efficacy.-

## **6. Conclusions-**

In the last few years, the use of deep sequencing has expanded largely to tackle problems in- many fieldsof virology. The greatest benefitof deep sequencing is its ability to detect minor- mutant variants, as low as 0.1% of virus population [36, 59, 60, 110, 111, 113, 114]. The deep- sequencing approach based on RNA library preparation is effectiveand reproducible for- detection of low quantities of mutants in the entire genome of influenza-A vaccine viruses [36],- and eliminates the need for full‐length amplification.-The deep sequencing platforms are- improving continuously to combine low error rates with long reads and relatively low cost. It- played a key role in the discovery of many new viruses, the characterization of virus popula‐ tions in humans and the potential of their association with the pathogenesis of several diseases.- As described here, there is no doubt that the deep sequencing is facilitating and accelerating- the evaluation of the genetic consistency of vaccine viruses. It is an important tool for moni‐ toring vaccine consistency during manufacture and after vaccination. Deep sequencing‐based- assays are already being implemented for the genetic consistency evaluation of oral polio- vaccine and influenza-A vaccine viruses [36, 59, 60]. The ability to quantify potentially- undesirable mutations in vaccine batches makes this method suitable for quality control to- ensure manufacture of safe and effective vaccines.-

## **Acknowledgements-**

We thank Dr. Konstantin Chumakov and Dr. Christian Sauder for their critical review of this- chapter. The contents of this chapter represent solely the opinion of authors and do not- represent the official view of FDA.-

#### **Author details-**

Ewan Peter Plant, Tatiana Zagorodnyaya, Elvira Rodionova, Alin Voskanian‐Kordi,- Vahan Simonyan, Zhiping Ye and Majid Laassri\*-

 \*Addressallcorrespondenceto:majid.laassri@fda.hhs.gov-

 Centerfor-Biologics-Evaluationand-Research,-US-Foodand-Drug-Administration,-Silver- Spring,-MD,-USA-

## **References-**


## **Electrochemical Sensors for Detections of Influenza Viruses: Fundamentals and Applications**

Hanna Radecka and Jerzy Radecki

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64423

#### **Abstract-**

 Avianinfluenzaviruses-(AIVs)couldcauseseverediseasesand,astheconsequence,- seriouseconomic losses aswell asa riskfor potentialtransmission to humans. Therefore,- thedetectionofvirusesandtheirfragmentsorspecific-DNAsequencesbecomesan- importantapproachinmoleculardiagnosis.-Here,wepresentelectrochemical- genosensorsdevotedfordetectionofinfluenzavirus-H5N1genesequence.-Wefocus- ourattentiononion-channelmechanism,-E-DNAsensors,andgenosensorsbasedon- redox-activelayer.-Thenoveldual-DNAelectrochemicalsensorwith-"signal-off"and- "signal-on"architectureforsimultaneousdetectionoftwodifferentsequencesof-DNA- derivedfromavianinfluenzavirustype-H5N1bymeansofoneelectrodeispresented.- Immunosensorsarealsoadequateanalyticaldevicesfordetectionofpathogenssince- antibodiesarenaturalreceptorsresponsibleforbindingofantigens.-Thus,thebinding- selectivityandefficiencyarenaturallyhigh.-Theimmunosensorspresentedcouldbe- dividedintotwomaingroups:ion-channelmimeticandbasedonredox-active- monolayer.-

**Keywords:** Gold electrodes, Carbon electrodes, Electrochemical immunosensors, Genosensors, Influenza virus detection-

#### **1. Introduction-**

 Infectiousviraldisease,whichisspreadamongbirds,inparticularavianinfluenza-(AI),could- affectotheranimals,aswellashumans-[1].-

The contact with infected live or dead poultry is the main source of risk of people's AI infection- [2].-

Controlling the AI in animals is the firststep in decreasing risks to humans. Therefore, there- is a high need for the development of the analytical methods allowing the fast and reliable AI- virus detection.-

The real-time polymerase chain reaction (PCR) [3], enzyme-linked immunoassay (ELISA) [4],- and reverse transcription-polymerase chain reaction (RT-PCR) [5] are the most frequently used- methods. Their main drawbacks are being time-consuming and demanding high-quality- laboratories.-

The biosensors are very good alternative. They are self-contained integrated analytical- instruments which are capable of providing specific quantitative or semiquantitative analytical- information applying a biological recognition element, which is indirect spatial contact with- a transducer element. Main parameters describing the quality of biosensors are selectivity,- sensitivity, reproducibility, and time of response.-

Electrochemical biosensors belong to a subclass of biosensors, which contain an electrochemical transductor responsible for converting of energetic signal coming from an intermolecular- recognition process into electrical on. Their main advantages are as follows: (1) the direct- conversion of a biological event to an electronic signal, (2) excellent detection limits, (3) small- analyte volumes in μl range, (4) ability to be used in turbid biofluids,-(5) suitability for rapid- measurements of analytes from human and animal samples, and (6) easy miniaturization.-

The complete electrochemical biosensor should be cheap, small, portable, and capable of being- used by semiskilled operators. In order to achieve this finalgoal, we have been working on- several types of geno- and immunosensors.-

In the ion-channel mimetic immunosensors and genosensors, the presence of redox marker in- the sample solution is necessary. The antigen-antibody complex formation as well as hybridization process suppresses the accessibility of redox marker toward the electrode surface. This- phenomenon, which is the base of analytical signal generation by ion-channel mimetic mode,- was observed using the Osteryoung square-wave voltammetry (OSWV) or electrochemical- impedance spectroscopy (EIS) in the presence of [Fe(CN)6] 3−/4−as an electroactive marker [6–14].-

In the genosensors and immunosensors based on redox-active monolayer, the strategy for- immobilization of the specificrecognition elements involved their interactions with transition- metal centers complexed on the electrode surface [15–19]. So, the presence of electroactive- markers in the sample solution is not necessary. This is very important for analytical procedure- involving naturally occurring molecules in which properties might be influencedby redox- markers.-

## **2. Electrochemical genosensors-**

In general, electrochemical genosensors monitor the DNA duplex formation at the surface of- electrode through changes of current or potential values either using electrochemical labels or- label-free system [20–25].-

#### **2.1. Methods for immobilization of recognition of ssDNA probe-**

The immobilization of single-stranded DNA (ssDNA) probe at the surface of electrode plays- a crucial role for future genosensor analytical parameters. Various electrode materials such as- gold, glassy carbon, carbon nanotubes, and graphene-modified electrodes have been applied- for ssDNA immobilization. The physical adsorption, the simplest immobilization method,- relies on the electrostatic interactions between ssDNA and surface of electrode. But such- sensing layers are not stable. In addition, ssDNA strands are not well ordered, and because of- this, they are not sufficiently accessible for target molecules.-

The alternative method for immobilization of oligonucleotides on the electrode surface is their- entrapping into polymer filmdeposited on the surface. The layer prepared according to this- procedure is much more stable in comparison to the previous one. The weak point of this- approach is the difficultyto control the flexibilityof ssDNA probes and, as a consequence, their- availability for target DNA.-

The next method of DNA immobilization exploits the natural affinityof avidin to biotin. This- method allows to create stable sensing layers with controlled density of ssDNA probes.-

The most popular protocols of electrode modificationare based on the formation of covalent- bonds between the functional group introduced into ssDNA strand and functional group- located at the surface of electrode. Therefore, the regulation of ssDNA probe density, as well- as their stable (covalent) immobilization and proper orientation, is relatively easy to achieve.-

#### **2.2. Methods of hybridization process detection: selected examples of different types of- genosensors-**

Differentapproaches for detection of the probe-analyte hybridization processes have been- applied in various genosensors. One of them is based on changes of electrochemical activity- of nucleobases upon the hybridization events. This concept has been proposed by Palecek and- coworkers [26]. Oxidation of adenine (A) and guanine (G) can be readily observed using carbon- electrodes or hanging mercury drop electrodes (HMDEs), which are suitable for investigation- of reduction of nucleic acids. Their main drawback is background current at the relatively high- potentials required for direct oxidation of DNA. In the case of reduction, the serious limitation- is the necessity to use mercury electrode.-

Another approach for voltammetric signal generation was presented by Umezawa and- coworkers [6]. In their approach, the mechanism for generation of an analytical signal was- connected with the binding event between target compound and recognition element immobilized at the electrode surface. Because of the creation of steric hindrance, as well as changing- the surface charge, the accessibility of redox marker present in the sample solution toward the- electrode is changed. Thus, creation of analyte-receptor supramolecular complex affected the- electron transfer from marker to surface of electrode. The heterogeneous rate constant of- electron transfer from marker to electrode surface became large or smaller; therefore, the redox- current increased or decreased. The electrochemical sensors based on this mechanism are- called ion-channel mimetic sensors.-

Recently, in our laboratory, we have developed two genosensors working according to this- mechanism based on the modifiedgold electrode intended for the detection of specific DNA- sequence of avian influenzavirus (AIV) H5N1 using NH2-ssDNA or HS-ssDNA probes for the- modification of gold electrode [11, 12].-

NH2-ssDNA probes were immobilized on the surface of mixed thioacid-thioalcohol monolayer- via EDC/NHS coupling, whereas HS-ssDNA probes were immobilized directly to the gold- surface via S-Au bonds. As marker ions in both cases, [Fe(CN)6] 3−/4−was employed. When SH-NC3 probe was used, detection limit in the 10 pM range was achieved. The much lower- detection limit in fM range was recorded when NH2-NC3 probe was applied. These data- confirmedthat the application of DNA probe with longer spacer part increased the hybridization signal. The betteraccessibility of target ssDNA toward more flexible-DNA probe is the- main reason of this phenomenon. But at the same time, the lower genosensor selectivity was- observed. When SH-NC3 DNA probe with shorter spacer was applied, the sensor was able to- distinguish between the PCR products with differentpositions of complementary parts,- whereas the electrode modified with longer spacer molecules was not able to do this [11, 12].-

Plaxo and coworkers [25] introduced another type of hybridization detection technique that- exploits the differencein physical flexibilitybetween single-stranded oligonucleotides and- double-stranded ones. Upon hybridization, the physical changes in the probe structures,- including the change in distances of the labeled electroactive moieties to the electrode surface,- result in the switching "on/off" of the electrochemical signal.-

The main advantages of E-DNA sensors are the conformational changes caused by hybridization, potential activation of redox label in the "safe" range separated from potential activation of majority of electroactive biomolecules present in the clinical and environmental- samples, and detection limits in the picomoles of target DNA range.-

The genosensors working on described mechanism belong to very wide "signal-off"mode- family. It is worth to note that detection limit of E-DNA working according to "signal-off"- mechanism is in the range of 10 pM. In case of genosensors that generated analytical signal- according to "signal-on" mechanism, detection limit is around 200 pM [25].-

The "signal-off"sensors have numerous advantages such as sequence specificity, reusability,- and suitability for direct measurement in serum. But the suppression of signal generated after- target binding is their main disadvantage, because only 100 % suppression of the original- current could be detected. On the other hand, the "signal-on" sensors have a potential for great- improvement of sensitivity, because the stimulation of these types of sensors with the target- does not cause a limited increase of signal. A weak point of these types of E-DNA sensors is- the use of rather complicated, but not very stable architectures. This approach does not work- properly in the complex samples.-

Today, more and more often, biosensor is required to be not only miniaturized and costeffective but also capable of simultaneous detection of multiple analytes.-

Recently, a novel dual E-DNA sensor working on "signal-off"and "signal-on" mode has been- developed [20]. This sensor was suitable for simultaneous detection of two different oligonucleotide sequences present in avian influenzavirus (AIV) type H5N1 with using one electrode.- The ssDNA probe represented by hemagglutinin was functionalized with ferrocene (ssDNA-Fc). The functionalization with methylene blue (ssDNA-MB) was applied for sequence derived- from neuraminidase. Both of them were covalently immobilized on the gold electrode surface.- Hybridization process going at the electrode surface was controlled by the Osteryoung squarewave voltammetry. Detection limits determined by graphic method were 4.0 x 10−8and 2.0 x- 10−8-M, for simultaneous analysis of both sequences and for single one, respectively. These- values, in particular the detection limits for parallel determinations of two sequences, are very- promising from diagnosing point of view. The selectivity of duo-genosensor was similar for- both targets. The limits of detection were in the range of 18–21 nM. The duo-genosensor was- free from interferences. The presence of oligonucleotide sequences complementary to SHssDNA-Fc probe does not influence the function of the probe decorated with methylene blue- and vice versa. The main advantage of duo-sensor is diminishing false-positive determinations- which may appear in the case of non-perfect hybridization with component(s) present in noninfected host samples. The probability for existence in native samples of components efficiently- interacting with two independent DNA probes is limited.-

Recently, in our laboratory, we have developed the new group of electrochemical DNA sensors- in which the analytical signal generation is based on the changes of accessibility of redox center- attacheddirectly to the analytical active layer or at the "foot" of the oligonucleotide probe,- very close to the electrode surface [16, 17, 20–22]. Such localization exclude the changes of- distance of redox marker from the electrode surface after hybridization. As a redox center, we- have used the complexes of transition metals with dipyrromethene, porphyrin, and phenanthroline.-

A novel mechanism of electrochemical signal generation based on changes of the ion-barrier- energy "switch-off"system has been proposed. According to this mechanism, the proposed- sensors generate an analytical signal because of changes in the environment surrounding the- redox center occurring as a result of hybridization processes.-

For the sensor based on the Cu(II) complex, during the redox cycle, Cu(II) is reduced to Cu(I).- As a consequence, a single negative charge of the reduced form appears at the surface of the- electrode. The precondition of redox reaction run is the compensation of this charge by ions- from the supporting electrolyte, involving transport of cations. For the electrode modifiedwith- the Co(II) complex, an oxidation process is possible, which generates an extra positive charge- of the oxidized form. For its neutralization, anions from the supporting electrolyte will be- involved. The mechanism of analytical signal generation by this type of sensors relies on- changes in accessibility of ions present in the supporting solution to the redox centers in order- to neutralize the charge occurring as a result of oxidation/reduction processes. The good- illustration of such sensors is genosensor intended for detection of the sequence specificof- avian influenzavirus type H5N1. A 20 mer probe (NH2-NC3) was covalently attachedto the- gold electrode surface.-

The detection limit of 1.39 pM for fully complementary single-stranded DNA target was- achieved with genosensor based on (dipyrromethene)2Cu(II) complex. A linear dynamic range- was observed from 1 to 10 pM. The good discrimination between fully complementary,- partially complementary (with only six complementary bases), and totally non-complementary to the probe was also recorded [16].-

The Fe(III)-phenanthroline complex is the base of genosensor suitable for detection of target- DNA as well as RNA. An efficientclick reaction, carried out under mild conditions, between- the NH2-NC3 probe and the epoxy groups from Fe(III)-phenanthroline complex deposited on- the gold electrode surface, has been successfully applied for the genosensor construction. The- good detection limits of 73 and 0.87 pM for the 20 mer c-NC3 and the 283 mer RNA1, respectively, were achieved. The sensitivity of RNA sequence detection was about one hundred times- betterthan the DNA sequence detection. The 20 mer nc-NC3 non-complementary to the probe- generated a weak response. In the case of non-complementary to the probe 277 mer RNA3- fragment, even the opposite signal, an increase of the Fe(III)/Fe(II) peak currents was observed.- These data confirmedthe genosensor selectivity. The main advantage of genosensor based on- Fe(III)-phenanthroline complex is its suitability for determination of RNA and distinguishing- of the different positions of the complementary parts. Thus, it could be applied for the detection- of the H5N1 genetic material [17].-

## **3. Electrochemical immunosensors-**

## **3.1. Methods for immobilization of recognition element-**

The right immobilization of the proteins as the recognition elements on the transducer surface- (e.g., gold, platinum, indium tin oxide, carbon materials) is a crucial factor to fully maintain- their right conformation and activity.-

Physical protein immobilization is based mainly on electrostatic forces and hydrophobic- interactions. The entrapping of proteins into the polymer matrix, iridium oxide films [27], and- nanotextured zinc oxide [28] belongs to this strategy.-

The colloidal gold layers create friendly environment for proteins. Therefore, they are frequently used in immunosensor creation [7–10, 29–31].-

In this approach, proteins are mainly immobilized on colloidal gold layers based on the- electrostatic interactions, which could be enhanced by selecting the proper pH conditions. The- drawbacks of this method are random orientation and rather weak attachment.-In order to- overcome these weak points, covalent antibody immobilization has been applied. Two- polypeptide chains F(ab')2present in immunoglobulin molecule are responsible for antigen- binding. Fc domain, which is not involved in this interaction, could be removed by enzyme- digestions [32]. The presence of disulfideor thiol group allow the F(ab')2or F(ab') fragments- to covalent self-assembling on the gold nanoparticle surface [33, 34]. This approach has been- successfully applied for development of immunosensor destined for selective binding of- antigen rSPI2-His6present in the sample solution by F(ab') fragment of antibody immobilized- on a surface of the electrode using electrochemical impedance spectroscopy (EIS) and surface- plasmon resonance (SPR) [30] as well as for selective detection of hemagglutinin from avian- influenzavirus H5N1 [9, 10]. The proteins A and G possessing high affinitytoward the Fc partof immunoglobulins are frequently used for their covalent and oriented immobilization on the- electrode surface [35]. The immunosensor based on glassy carbon electrode incorporating the- protein A has been successfully applied to distinguish between sera of unvaccinated and- vaccinated chickens against the avian influenzavirus. Sensitivity of EIS immunosensor was- almost 104 times much better than ELISA [8].-

#### **3.2. Methods of detection of immunoreaction: selected examples of different types of- immunosensors-**

The majority of electrochemical immunosensors incorporate not only recognition element- (antibody or antigen) but also secondary enzyme-labeled antibody, which follows the addition- of proper enzymatic substrate. The antibody-antigen reaction is detected in no-direct manner- but by electroactive molecules produced by enzymatic reaction [36, 37]. Such approach- demands very precious labeled biological materials, which increase the cost of analysis. Also,- it involved numerous modificationsteps. The no-direct detection of immunoreaction is the- main drawback of sandwich type of immunoassays.-

In order to overcome the above weak points, the immunosensors allowing the direct electrochemical detection of immunoreactions have been developed in our laboratories.-

The whole antibodies or antibody-binding fragments (Fab') have been immobilized on the- surface of gold nanoparticle layers, which create friendly environment for proteins keeping- their physiological activity. The formation of immunoreactions has been detected by the- changes of accessibility of redox marker present in the sample solution to the electrode surface.- The deposition of antibody-antigen complex on the electrode surface increases substantially- the resistance of the analytical system, and as a consequence, faradaic current is very difficult- to measure by voltammetric techniques. Therefore, electrochemical impedance spectroscopy- (EIS) has been widely applied. EIS is an ac method that describes the response of an electrochemical cell to a small amplitude, sinusoidal voltage signal, and a function of frequency. The- most popular mode for analyzing electrochemical impedance data is the plot of the imaginary- impedance component versus real impedance component at each excitation frequency (the- Nyquist plot). Numerous impedimetric immunosensors have been already reported [7, 30, 31,- 38]. This approach has been successfully applied for detection of fragments of H5 hemagglutinin from avian influenzaviruses [9, 10]. The antibody-binding fragments have been covalently attachedto the gold nanoparticle layers. Application of 4,4'-thiobisbenzenethiol selfassembled monolayer [10] improved substantially immunosensor performance in the- comparison to one incorporated 1,6-hexanedithiol [9]. Taking into account the immunosensor- sensitivity with detection limit of 0.6 pg/mL and specificity (negative control H7 hemagglutinin- generate negligible response), they could be recommended for direct electrochemical detection- of H5 hemagglutinin from influenza virus in the field conditions.-

Direct impedimetric immunosensor has been successfully applied for detection of antibodies- generated against H5N1 virus [8]. The base of this immunosensor was glassy carbon electrode- incorporated fragment of H5 hemagglutinin. The interaction with specificantibodies was- detected electrochemically by changes of [Fe(CN)6] 3−/4−accessibility toward electrode surface.- The immunosensor was able to distinguish the sera from vaccinated and unvaccinated hen- with sensitivity 104 better than ELISA.-

In order to avoid the necessity of the redox marker present in the sample solution, the- immunosensor-incorporated redox-active layer has been developed [39].-

The Cu(II) complex with dipyrromethene deposited onto the gold electrode surface plays- double roles. It is a site for covalent immobilization of the His-tagged fragment of H5 hemagglutinin, as well as redox centers for sensing the antigen-antibody interaction.-

This type of immunosensor was also suitable for direct antibody detection in hen sera with 200- better sensitivity than ELISA.-

#### **4. Future perspective-**

The analytical chemists mainly search for improving sensitivity and selectivity of sensing- devices. The intensive development of nanotechnology gives wide possibility of using- nanomaterial labels for signal amplificationgenerated upon immunoreaction [18, 19, 40–43].- Nanomaterials could be used for modificationof electrochemical transducers in order to- improve their electrochemical properties by lowering background current and signal to noise- ratio, as well as increasing electron transfer rate.-

The most frequently used nanomaterials are colloidal gold and silver, semiconductor quantum- dots, carbon nanotubes, and graphene.-

They are very promising in the development of ultrasensitive immunosensors. Could they be- applied in point-of-care and clinical diagnoses? Taking into account so intensive effortdone- in this research area, the answer for this question is "yes."-

## **5. Conclusions-**

The main advantage of ion-channel mimetic sensors is the possibility of their application for- exploring the recognition processes occurring at the water/solid interface. It is very important- from biological as well as medical point of view. The electrochemical sensors based on redoxactive layer are new direction in sensing device development. Their main advantage is the lack- of the necessity of using the external redox marker. The redox centers can simultaneously act- as sites for host molecule immobilization and transducers.-

Taking into account the following parameters of electrochemical biosensors presented such as- very good sensitivity, very low sample consumption (in μl level), lack of matrix influence,- simple operation, and reasonable cost, it might be concluded that they are analytical tools- suitable for detection of viruses in the environmental sample.-

#### **Acknowledgements-**

This work was supported by a project from the National Centre for Research and Development- No. PBS2/A7/14/2014 and Institute of Animal Reproduction and Food Research of Polish- Academy of Sciences, Olsztyn, Poland.-

#### **Author details-**

Hanna Radecka\* and Jerzy Radecki-

 \*Addressallcorrespondenceto:h.radecka@pan.olsztyn.pl-

 Instituteof-Animal-Reproductionand-Food-Researchof-Polish-Academyof-Sciences,-Olsztyn,- Poland-

#### **References-**


the base of genosensor for the detection of specific DNA and RNA sequences of avian- influenza virus H5N1, Biosens Bioelectron. 2015;65:427–434-


## **Biosensors for Rapid Detection of Avian Influenza**

Ronghui Wang and Yanbin Li

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64353

### **Abstract-**

 Thescopeofthischapterwastoreviewtheadvancementsmadeintheareaofbiosensors- forrapiddetectionofavianinfluenzaviruses-(AIVs).-Itisintendedtoprovidegeneral- background about biosensor technology and to discuss important aspects for developing- biosensors,suchasselectionofthesuitablebiologicalrecognitionelements-(anti-AIV- bioreceptors)aswellastheirimmobilizationstrategies.-Amajorconcernofthischapter- isalsotocriticallyreviewthebiosensors'workingprinciplesandtheirapplicationsinAIV- detection.-Atablecontainingthetypesofbiosensor,bioreceptors,target-AIVs,methods,- etc.isgiveninthischapter.-Anumberofpapersforthedifferenttypesofbiosensorsgive- hintsonthecurrenttrendsinthefieldofbiosensorresearchforitsapplicationon-AIV- detection.-Bydiscussingrecentresearchandfuturetrendsbasedonmanyexcellent- publicationsandreviews,itishopedtogivethereadersacomprehensiveviewonthis- fast-growingfield.-

**Keywords:** biosensor, avian influenzavirus, rapid detection, bioreceptor, nanobiosensor-

## **1. Introduction-**

 Influenzaviruses,whichbelongtothe-*Orthomyxoviridae* family,areclassified as A, B, and C- based on antigenic differences in their nucleoprotein (NP) and matrix (M1) protein [1]. All avian- influenza viruses (AIV) are classifiedas type A. Type A viruses are further subtyped on the basis- ofantigenicdifferencesofthesurfaceglycoproteins,thehemagglutinin-(HA),andtheneuraminidase-(NA)proteins-[2].-Sofar,seventeen-HA-(H1through-H17)andten-NA-(N1through- N10)subtypeshavebeenidentified-[3].-**Figure-1**presentsaschematicdiagramofinfluenza A- virus,and-**Figure-2**showsatransmissionelectronmicroscopyphotograph.-Influenza-Avirus- isanenveloped-RNAvirusapproximately-80–120nmindiameter-[1,-2].-Therearetwomajor-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 surfaceglycoproteins-(HAand-NA)andasmallnumberof-M2protein,whichhasionchannel- activity.-Theratioof-HAand-NAisapproximately-4:1.-Thegenomeoftype-Avirusconsistsof- eightsegmentsofnegative-sensesingle-stranded-RNA,whichareassociatedwithmany-NP- andthetranscriptasecomplex-(RNApolymerasecomponents-PB1,-PB2and-PA)toform- ribonucleoproteins(RNPs).ThematrixproteinM1hasaninteractionwiththeRNPsandisunder- thelipidbilayer.NS1 isfoundonlyininfectedcellsandisnotthoughttobeastructuralcomponent- ofthevirus,butsmallamountof-NS2arepresentinpurifiedviruses.-

**Figure 1.** A schematic diagram of the structure of the influenza A virus particle.-

**Figure 2.** A transmission electron micrograph of influenza A virus particles.-

AIVs have a large impact on the poultry each year and also represent a threat to human health.- The highly pathogenic H5N1 avian influenza-(HPAI H5N1), which originally emerged in- Southeaster Asia in late 1990s, cost the poultry industry an estimated \$10 billion between 1997- and 2008 [4]. HPAI H5N1 has also caused global concerns for public health and continues to- spread throughout the world. Since 2003, a total of 62 countries (regions) have been affected- by HPAI H5N1 and new human and animal cases are continuously being confirmedand- reported [5]. The link between human and AI has raised concern among public health- authorities and the scientificcommunity about the prevalence and pandemic potential of AI- virus. The H5N1 virus has caused 449 deaths of 846 people infected since 2003, according to- the World Health Organization [5]. Therefore, advanced technologies for more rapid and- sensitive AIV detection are needed for better surveillance and control of the outbreaks.-

Current virus detection techniques are available, such as virus isolation, immunofluorescence- antibody (FA) test, immunohistochemistry (IHC), and ELISA as well as rapid procedures based- on molecular tools, such as polymerase chain reaction (PCR)-based assays, gene sequencing,- and microarrays. Among these test methods, virus isolation has been the "gold standard" as- its highest sensitivity and reliability, but it is a very time-consuming procedure (4–7 days).- ELISA is a simple and rapid test in testing mass serum samples; thus, it is commonly used for- serum antibody detection, but not used for virus detection in practice or diagnostics. Other- immunoassays of FA or IHC can be used as a direct virus detection test from tissue specimens,- but they either require labels or have limitations of low sensitivity and cross-reactions in certain- circumstances. The molecular assays of RT-PCR and real-time RT-PCR methods have the- advantages of high sensitivity and good specificity,but possess disadvantages of requiring- expensive PCR equipment and reagents, and well-trained skillful technical personnel.- Therefore, in order to minimize the social and economic costs, the development of rapid- detection methods is essential. These rapid detection methods should meet the following- requirements: (1) high throughput, (2) possibility of multiple target detection, (3) high- sensitivity, (4) good specificity,-(5) high speed, (6) simple operation, (7) suitability for on-site- and/or in-fielduse, and (8) less cost. Biosensors have shown a great potential to meet such- criteria.-

The biosensor research began in the early 60 s of the twentieth century, when a glucose- biosensor was proposed by Clark and Lyons [6] at Children's Hospital in Cincinnati. From then- on, biosensor applications have expanded throughout the medical diagnosis fieldand also into- the fieldsof environmental monitoring, agricultural production, food safety, pharmaceutical- screening, and biodefense. A biosensor can be definedas a "compact analytical device or unit- incorporating a biological or biologically derived sensitive recognition element integrated or- associated with a physiochemical transducer" [7]. This device is composed of a biological- recognition element (often called bioreceptor) and directly interfaced signal transducer- (**Figure 3**). The selective and reversible process of interaction between the analyte and- bioreceptor is transduced into a measurable signal, for example, electrical signal, which is- proportional to the concentration or activity of an analyte in any type of samples. Biosensors- can be classifiedbased on the transduction element or the biological element. According to the- basic principles of signal transduction, there are three main biosensor types, which have beenstudied for use in AIVs detection: piezoelectric, optical, and electrochemical biosensors. If we- follow the types of biological element or bioreceptors, AIV biosensors can be classifiedinto- immunosensors, aptasensors, DNA or RNA-probe sensors, and more. In this chapter, we focus- on these types of biosensors studied and developed recently for rapid detection of AIVs. It- offersa survey of the principles, fabrication, operation, and the most popular types of biosensing devices in research or application today for AIVs detection. By discussing recent- research and future trends based on many excellent publications and reviews, it is hoped to- give the readers a comprehensive view on this fast-growing field.-

**Figure 3.** A schematic diagram of biorecognition and signal transduction in a typical biosensor.-

## **2. Biosensors for detection of AIVs-**

As shown in **Figure 4**, the number of papers published in the fieldof biosensor research for- detection of AI has kept increasing from year 2001 to 2015 based on PubMed. Biosensors offer- the advantages of simple operation, rapid response, low cost, portability, automation, and easy- to integrate with nanomaterials/nanostructure, Micro-Electro-Mechanical System(MEMS)/- Nano-Electro-Mechanical System(NEMS), biotechnology, Global Position System(GPS)/- wireless, and image technologies. One of the major requirements in developing a biosensor- for AIVs is the need for a sensitive analytical device that can easily go down to very low- detection levels without significantchanges in selectivity. AIVs will spread rapidly through a- community before any symptoms appear for the identification.-A biosensor that can rapidly,- sensitively and selectively detect target virus will be invaluable. In addition, a simple, robust,- rapid, cost-effective,and potable biosensor, suitable for use in the field,is urgently needed.- While a variety of biosensors have been developed for use in differentapplications, three types- of biosensors have been mainly studied for AIV detection: piezoelectric, optical, and electrochemical biosensors.-

**Figure 4.** Numbers of papers published in the fieldof biosensor research for detection of AIVs between year 2001 and- 2015.-

## **2.1. Piezoelectric biosensors-**

 Piezoelectric biosensors utilize crystals capable of generating a piezoelectric fieldto detect- mass changes in the sensing environment [8]. The crystal is sandwiched between two excitation- electrodes, which apply an electrical fieldthat causes the crystal to undergo dimensional- changes, or oscillations, at the crystals natural resonant frequency. An increase in the mass on- the surface of the crystal, such as antibody immobilization or capture of antigen, decreases the- resonant frequency. Piezoelectric biosensors are useful because they are low cost, label free,- sensitive, and have extremely low detection levels [8]. The most intensively studied piezoelectric biosensor is the quartzcrystal microbalance (QCM), which uses a thin wafer of quartz- as the transducing crystal. Quartzcrystals have the advantages of being widely available,- relatively inexpensive, durable, direct detection, and real-time output. Other techniques are- often coupled with QCM to increase the performance and capability of the biosensor. QCM- biosensors have attractedinterest in applications for AIVs detection. **Table 1**summarizes the- main analytical features of QCM biosensors for detection of AIVs. Differentstudies used- differentconcentration units for AIV. It would be helpful to know the comparable relationship- between them. The common concentration units for AIV contain EID50-(50% egg infectious- does), ELD50-(50% egg lethal does), PFU (plague-forming units), and HAU (hemagglutination- unit). 1 × 103-EID50 ml−1equals to ~0.0128 HAU/50 μl [9], 1 × 106.2-ELD50 ml−1equals to 128 HAU/- 50 μl [10], and 1 × 105 PFU equals to 1 HAU [11].-


**Table 1.** QCM biosensors for detection of AIVs.-

Antibodies are the most common bioreceptor of choice for sensing, and they are generated by- immunizing an animal system with antigen. The interaction between antibody and antigen- can be transduced into measurable signal changes. Early studies focused on the development- of QCM immunosensor based on antibody-antigen interaction for influenzavirus detection- [12–14]. For example, the characterization of a QCM biosensor for the direct detection of- influenza-A viruses was reported by Owen et al. [12]. Self-assembled monolayers (SAMs) of- mercaptoundecanoic acid (MUA) were formed on QCM gold electrodes for the immobilization- of anti-influenza-A antibodies, and the limit of detection was estimated to be 4 virus particles/- ml. QCM immunosensor has been used also to detect avian influenzalabel free in nasal- washings with a lower detection limit of 104pfu/ml, although, with the addition of a gold- nanoparticle conjugate, the detection limit was reduced to 103pfu/ml, which is comparable tothe sensitivity and specificityof viral isolation techniques [13]. Li et al. [14] reported a nanobeads amplified-QCM immunosensor with polyclonal antibody as the recognition ligand for- detection of AIV H5N1.-

 In recent years, aptamers have been investigated as an alternative of sensing elements, which- have the potential to replace the antibodies. This is possible due to the unique features of- aptamers (sensitivity, specificity,reusability, stability, nonimmunogenicity), which can be- easily exploited in biosensor technology. Aptamers are single-stranded RNA or DNA oligonucleotides which rely on hydrogen bonding, electrostatic and hydrophobic interactions- rather than Watson-Crick base pairing for recognition to their target. Aptamers can fold into- distinct secondary and tertiary structures, bind to their targets with high affinity (dissociation- constants on the order of nano- to picomolar), and recognize their targets with a specificity- that challenges antibodies and other biological ligands. They are selected in vitro through- systematic evolution of ligands by exponential enrichment (SELEX). The selection procedure- involves the iterative isolation of ligands out of the random sequence pool with affinityfor a- definedtarget molecule and PCR-based amplificationof the selected RNA or DNA oligonucleotides after each round of isolation. As biorecognition ligands, aptamers possess numerous- advantages, including small size, rapid and reproducible synthesis, simple and controllable- modificationto fulfilldifferentdiagnostic and therapeutic purposes, slow degradation- kinetics, nontoxicity, and a lack of immunogenicity.-

The majority of aptamers developed for AIVs have focused on inhibition of the hemagglutinin- protein preventing viral infection. For example, a DNA aptamer was developed by Jeon and- co-workers [15]. RNA aptamers that inhibit membrane fusion of AIV H3N2 and influenza B- virus hemagglutinin also were developed [16, 17]. DNA and RNA aptamers that target HA1- proteins of influenzavirus hemagglutinin subtype H5 were investigated in two different- studies [18, 19]. An aptamer that binds efficientlyto the HA of highly pathogenic AIV H5N1- and H7N7 was studied by Suenaga and Kumar [20], which inhibits HA-glycan interactions.- These aptamers were developed to inhibit function of the hemagglutinin protein and prevent- or treat influenzainfection. Based on the function and likely binding sites of these aptamers,- it seems unlikely these aptamers would be optimal sequences to use in a biodetection assay.- A DNA aptamer for avian influenza H5N1 was developed by Wang et al. [21] using a combination of protein and whole virus targets. It is unique in its binding affinity in that it specifically- targets the H5N1 subtype, having no binding activity with other H5 or N1 subtype viruses.- This suggests that the binding site be at an intersection of the H5 and N1 proteins.-

Aptamer-based QCM biosensors, also called QCM aptasensors, have been developed for the- detection of AIVs. Brockman et al. [22] studied a QCM aptasensor for detection of AIV H5N1- using magnetic nanobeads labels as mass amplifiers.-The biosensor was found to have a lower- detection limit of 1 HAU, although the detection time was reduced by half compared to a- similar QCM biosensor using antibodies [14]. A hydrogel-based QCM aptasensor (**Figure 5**)- was able to detect AIV H5N1 at 0.0128 HAU in 30 min [9]. The QCM aptasensor was based on- swelling of the hydrogel after AIV H5N1 capture due to dissolution of the cross-linked- aptamers and ssDNA in the hydrogel polymer. The developed aptasensor was both rapid and- sensitive.-

**Figure 5.** (a) Schematic of the QCM aptasensor; (b) a photograph of the QCM aptasensor setup. The aptamer hydrogelis immobilized on the gold electrode surface. The quartzin the electrode acts as a transducer, converting the mass andviscosity change to a frequency signal.-

 Influenzavirus specificityfor the host is mediated by the viral surface glycoprotein hemagglutinin (HA), which binds to receptors containing glycans with terminal sialic acids. This- molecular recognition process leads to the host cell-virus adhesion stage [23]. The sialic acid- receptor of various influenzavirus strains differsin affinityto sialic acids terminally linked- either in α(2,3) or α(2,6) position to the galactose (Gal) residues. Human influenza-A viruses- preferentially recognize the α(2,6)-linkage, while avian viruses have preference for the α(2,3) linkage [24, 25]. Each monovalent sialic acid-binding site of HA is weak, with dissociation- constants in the millimolar range, while the high virus-cell affinity is due to multivalency.-

Takahashi and co-workers [26] demonstrated kinetics of HA binding to sulfatide and ganglioside GD1a by QCM analysis. QCM analysis showed that the HA bound with the *Kd* of- 1.44 × 10−8-M to sulfatide immobilized on a sensor chip, which indicated that sulfatide directly- binds to the ectodomain of HA with high affinity.-N-Acetylglucosamine (GlcNAc) is a natural- ligand and is part of the oligosaccharide ligand responsible for the influenzavirus binding- firststep. In a report by Wangchareansak et al. [27], GlcNAc was employed as bioreceptor- immobilized on QCM gold sensor surface for detection of influenza-A virus (H5N3, H5N1,- H1N3), which displayed high binding affinitywith *Ka* values of 2.03 × 1010, 4.35 × 1010, and- 2.56 × 1012 M, respectively.-

QCM biosensor has advantages, such as simplicity, cost-effectiveness,real-time output, and- direct detection. But there also exists some disadvantages, such as the lack of stability, difficulty- with sensor surface regeneration, loss of the immobilized ligands after multiple washing and- regeneration, nonspecificbinding of other nontarget biomaterials, and relatively long incubation time. Further improvements are required to address these limitations before the QCM- biosensor technology can be routinely employed for AIVs detection.-

#### **2.2. Optical biosensors-**

Optical biosensors rely on visual phenomena to detect the interaction between the biological- element and the target analyte. Examples of optical biosensors include surface plasmon- resonance (SPR), absorption, luminescence, and fluorescentsensors. Detection by optical- biosensors can occur in two ways: by the analyte directly affectingthe optical properties of the- sensing environment, such as in SPR or absorption methods, or by the analyte being tagged- with a label that produces an optical phenomenon, such as in fluorescencemethods. Optical- biosensors, which are sometimes referred to as "optodes," have received considerable interest- in the detection of AIVs. The main analytical features of optical biosensors for detection of- AIVs are summarized in **Table 2-**[3, 28–38]. The target "virus" in **Table 2**is the entire virus- particle and target "HA" is the HA protein or recombinant HA protein of virus.-


**Table 2.** A list of optical biosensors reported for the detection of AIVs.-

Various modes of optical measurement exist (i.e., absorption, reflection,fluorescence, chemiluminescence, and phosphorescence); however, biosensors based on surface plasmon resonance (SPR) and fluorescenceprinciples are the most common and promising methods for- AIVs detection.-

The Kretschmann configuration [39] is the most popular optical setup for SPR applications.- SPR biosensors measure the change in refractive index due to binding of biomolecules near- the sensor surface. Refractive index is definedas the ratio of the speed of light in vacuum and- the phase velocity of light in the medium. SPR is one of very few techniques that are able to- provide noninvasive, real-time kinetic data on association, and dissociation rates, along with- equilibrium binding constants for receptor or ligand systems. Estmer-Nilsson et al. [40] were- able to utilize SPR to quantify influenzavirus for vaccine production via an antibody inhibition- assay using HA proteins immobilized on the sensor surface. Some research showed SPR- biosensors for avian influenza-DNA hybridization [41], adamantane binding sites in the- influenza-A M2 ion channel [42], influenzavirus hemagglutinin monitoring [43], and binding- kinetics study [44]. Studies by Gopinath et al. [28] revealed that SPR-based biosensor is a useful- tool for detection of human and avian influenza viruses.-

SPR-based Biacore technology has been designed to investigate biomolecular interactions,- which was initiated in 1984. The commercial available systems include Biacore 1000, Biocore- 3000, BIAlite, and Biacore T100. Biacore is the dominant SPR technique used for AIVs detection.- The main advantage of this technology is its capacity to monitor weak macromolecular- interactions that cannot be detected by other sensors and its subject ability to automation. SPR- was used to evaluate the binding of influenza-A virus H1N1 directly to a neomembrane of- bovine brain lipid or an egg yolk lecithin fraction [29] and to monitor the interactions between- influenza-HA and glycan [30] or apatmers [28]. However, Biacore instruments are expensive- and need proper maintenance. Bai and co-workers [31] have shown that a portable hand-held- SPR-based biosensor (Spreeta™, Texas Instruments, Dallas, TX, USA) (**Figure 6**) can be- employed for the detection of AIV H5N1. The fabrication of the SPR biosensor was based on- the streptavidin-biotin binding. The streptavidin was directly adsorbed on the gold surface,- and then, biotinylated ssDNA aptamers were immobilized. Target AIVs were captured by the- immobilized aptamers and resulted in an increase in the refraction index. It was able to detect- AIV H5N1 in poultry swab samples with a lower detection limit of 0.128 HAU in 1.5 h.-

The working principle of a waveguide mode sensor is similar to that of a SPR sensor. The only- differenceis that the measurement is conducted using a waveguide mode rather than a surface- mode [32]. Based on the properties of light-guiding and dimensions, there are two general- classes, fiberoptical waveguides and planar waveguides, which can be further divided into- single mode and multimode [45]. Waveguide mode biosensors were developed using antibody- as bioreceptor for the detection of HA from H3N2 [32] and H3N2 virus [3].-

Of the optical methods requiring labels for detection, fluorescenceis the most widely studied- [34–36, 46, 47]. Fluorescence measurements are of particular interest in biosensor systems due- to their high sensitivity. Commonly used labels in fluorescentbiosensors are dyes, quantum- dots (QDs), and fluorescentproteins, with the lattertwo becoming more popular as they are- further researched [46]. A fluorescentaptasensor was developed by Pang and co-workers [34]- for detection of AIV H5N1. The Ag@SiO2core-shell nanoparticles were coated with the anti-HA aptamers. The binding of aptamer-HA protein formed a G-quadruplex complex, which- captured thiazole orange (TO) and reported the fluorescentsignal of TO. Moreover, it caused- a surface plasmon resonance enhancement and performed as a metal-enhanced fluorescence- sensing. The detection of HA protein of the AIV H5N1 could be operated both in aqueous- bufferand human serum with the detection limit of 2 and 3.5 ng/mL, respectively. The total- detection time was only 30 min. Using antibodies as bioreceptors, Li et al. [35] developed a- highly sensitive fluorescentimmunosensor for detection of H1N1, which was based on Ag- autocatalysis. It had a detection range of 1.0 × 10−12to 1.0 × 10−8g/ml with a detection limit of- 10−13 g/ml.-

**Figure 6.** Configuration for measuring AIV H5N1using the portable SPR aptasensor (with permission from MDPI).-

Fluorescence resonance energy transfer (FRET) is used in the design of a biosensor, which is- based on the energy transfer between two light-sensitive molecules, a donor and an acceptor- chromophore. In the nucleic acid-based FRET method, a reporter and a quencher are conjugated at the terminals of a nucleic acid probe. Without the target, the duplex is formed, bringing- these two molecules in close proximity, which results in fluorescence quenching. The present- of a target can cause the conformational change of the probe, which changes the position of- the reporter and quencher and emits fluorescence.-A QD-induced FRET system was developed- by Chou and Huang [47] using two oligonucleotides. These two oligonucleotides were- designed to specificallyrecognize two regions of the AIV H5 sequences and were employed- as the capturing and reporter probes, respectively. They were conjugated to QD655 (donor)- and Alexa Fluor 660 dye (acceptor), respectively. At target concentrations ranging from 0.5 nM- to 1 μM, the QD emission decreased at 653 nm and dye emission increased at 690 nm. Another- luminescence resonance energy transfer (LRET)-based biosensor was developed by Ye et al.- [37] for rapid and ultrasensitive detection of AIV H7 subtype. In this work, BaGdF5:Yb/Er- upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs) were used as the pair- of donor and acceptor. The oligonucleotides with H7 HA gene sequence were conjugated with- thiol and then were assembled on the AuNPs surfaces. The complementary sequence probes- were modifiedwith amino group and then were covalently bond to poly(ethylenimine)- modified-BaGdF5:Yb/Er UCNPs. The hybridization process brought these two molecules in- close proximity, resulting in fluorescence quenching.-

QDs are a type of semiconductor with a diameter typically between 2 and 10 nm whose excitons- are confinedin three spatial dimensions, giving them properties of both unconfined semiconductors and discrete molecules. In comparison with organic dyes and fluorescentproteins,- QDs have unique optical and electronic properties including size-tunable light emission,- improved signal brightness, high resistance against photobleaching, and simultaneous- excitation of multiple fluorescencecolors. QD-based bioconjugates used for optical sensing- could dramatically increase the sensitivity that would permit simultaneous measurement of- several targets. Lee and co-workers [38] developed a plasmon-assisted fluoro-immunosensor- by the conjugation of antibody onto the surface of cadmium telluride QDs and the Au- nanoparticle-decorated carbon nanotubes (AuCNTs) for the detection of H1N1 and H3N2. A- detection limit of 0.1 pg/ml and 50 pfu/ml was obtained for H1N1 and H3N2, respectively.-

Fluorescent biosensors are simple and show improved sensitivity and reduced detection time.- But they have a drawback of requiring relatively expensive reagents. In order to apply the- fluorescentbiosensor for detection of real samples with complex background, such as tracheal- and cloacal swabs from birds, further work would be required to combine an effective sample- pretreatment method with the fluorescent biosensor for the detection of AIVs in the field.-

## **2.3. Electrochemical biosensors-**

 More than half of the biosensors used for the detection of AIVs are based on electrochemical- transducers. Electrochemical biosensors use changes in the electrical properties caused by- biochemical reactions to detect an analyte [48]. Electrochemical transducers offerseveral- advantages: low cost, high sensitivity, ease of miniaturization, independence from solution- turbidity, low power requirements, and simplicity of use [49]. These characteristics make- electrochemical detection methods highly attractivefor fieldmonitoring of infectious diseases- and biological warfare agents. Electrochemical biosensors can be divided by the electrical- parameter that they measure: amperometric, potentiometric, conductimetric, and impedimetric. Amperometric biosensors work by applying a constant potential across an electrode and- measuring the current associated with either the reduction or oxidation of an electroactive- species created by the interaction of the biological element and the analyte. Amperometric- biosensors are often used with an enzyme capable of catalyzing the production of an ionic- product and increasing the selectivity and the sensitivity [50]. Potentiometric biosensors gather- data by converting a biological reaction into a potential signal with the use of ion-selective- electrodes [51]. Conductimetric biosensors simply measure the conductivity change in a- medium caused by the analytes. Impedimetric biosensors measure a combination of theresistive and capacitive or inductive properties of a material in response to a small amplitude- sinusoidal excitation signal [52].-

A detailed review of the electrochemical biosensors for detection of AIVs before year 2014 can- be found in the paper by Grabowska et al. [53], which covered the electrochemical genosensors- and electrochemical immunosensors for detection of AIVs. A total of 69 references were cited- in their review paper. Within the context of this chapter, the electrochemical biosensors- reported prior to 2014 are referred to the review paper by Grabowska et al. and the electrochemical biosensors reported in the years 2014 and 2015 plus some impedance biosensors not- mentioned by Grabowska et al. are reviewed in detail.-

Impedance biosensors are a class of electrical biosensors that measure the electrical impedance- of an interface in AC steady state with constant DC bias conditions. This is accomplished by- imposing a sinusoidal voltage at a given frequency and measuring the current. This measurement can be done over a range of frequencies or at a given frequency [54]. Many impedance- biosensors utilize a capture probe on the detecting surface to hold the target molecule, thereby- stabilizing the point of detection. Due to the ease of miniaturization, low energy usage and- relatively low cost, impedance biosensors show promise for AIVs applications. Impedance- biosensors can use a variety of biological sensing elements but the most commonly used are- antibodies. When using antibodies as the biological element the biosensor is often referred to- as an impedance immunosensor. Impedance immunosensors rely on the interaction of the- antibody and the antigen to generate a detectable signal for the transducing element. This- allows the immunosensor to detect either indirect or direct impedance measurements. Direct- impedance measurement, or label-free detection, is dependent on monitoring the changes in- the electrical properties of the sensing environment caused by the antibody-antigen interaction. A label-free detection method has several advantages over an indirect detection method,- including reduced detection time, lower cost, and simpler detection protocol.-

There are two general classes of impedance biosensors, Faradic and non-Faradic impedance- biosensor, which have been used for the detection of AIV H5N1 or subtype H5. A Faradic- impedance biosensor was developed by Wang and co-workers [55] using an interdigitated- array microelectrode. Polyclonal antibody against H5 was oriented immobilized on the gold- surface through protein A to capture target virus, and red blood cells (RBCs) were used as- biolabels for signal amplification.-The biosensor had a lower detection limit of 103 EID50 ml−1.- Wang et al. [10] also reported a non-Faradic impedance biosensor for H5N2 avian influenza- detection, which was based on the combination of an immunomagnetic nanobeads separation- and a microfluidicchip with an interdigitated array microelectrode. A lower detection limit- of 103 EID50 ml−1was achieved with detection time less than 1 h. Then, Lum et al. [56] further- improved the non-Faradic impedance biosensor for H5N1 detection by a combination of- immunomagnetic nanoparticles, a microfluidicchip, an interdigitated array microelectrode,- and RBCs for signal amplification.-The specificityof the biosensor was improved due to the- use of anti-H5 as a capture antibody and anti-N1 as a detection antibody.-

A miniaturized biosensor was developed by Diouani et al. [57] for the detection of H7N1.- Polyclonal antibodies against AIV H7N1 were attachedto a gold electrode using a selfassembled monolayer. The impedance measurement was carried out in the presence of a redoxprobe and inside a Faraday cage. The lower detection limit of this biosensor was determined- to be 5 μg/ml of specificantigen. Hassen et al. [58] developed an impedance biosensor capable- of quantifying influenza A virus in a media comprised of numerous other proteins and viruses.- The biosensor had a lower detection limit of 8 ng/ml, even in the presence of nonspecific viruses- and proteins.-

In **Table 3**, the electrochemical biosensors developed for the detection of AIVs in year 2014 and- 2015 are summarized [59–79].-



**Table 3.** A list of electrochemical biosensors reported in 2014 and 2015 for the detection of AIVs.-

For the past two years, the immunosensors have been the dominant electrochemical biosensors- for detection of AIVs [59–68]. For example, universal anti-M1 antibodies, which allow detection- of all serotypes of influenza-A virus, were used for the development of an universal immunosensor for detection of the influenza-A virus. It showed similar sensitivity (80–100 virus- particles/μl) to molecular methods [59]. Miodek and co-workers [60, 61] developed an- electrochemical immunosensor, which was based on conductive polypyrrole modifiedwith- ferrocenyl groups as a redox marker for the detection of PB1-F2, a nonstructural accessory- protein of influenza-A virus. Lin et al. [62] demonstrated that an impedance immunosensor,- which was based on low-cost microelectrodes and specificmonoclonal antibodies for rapid- detection of AIV H5N1 in chicken swabs. Studies by Jarocka and co-workers [63, 64] revealed- that the immobilization of the recombinant His-tagged HA was able to detect antibodies- against AIV H5N1 in hen serum.-

Although electrochemical biosensors for avian influenzadetection have mostly been investigated for whole virus or virus protein detection, some groups have developed biosensors for- the detection of avian influenzanucleic acid sequences [69–73]. These biosensors are based on- the use of nucleic acid probes, which bind to specificsequence in the influenzagenome. Several- genosensors have been studied for the detection of the full length H5 gene of AIV H5N1. In- the study of genosensors by Grabowska et al. [69, 70], redox active compounds (such as cobalt- porphyrins or 3-iron bis(dicarbollide) were conjugated with the DNA probes, which were very- close to the electrode surface. These developed genosensors showed a sensitivity in the fM- range. Malecka and co-workers [71] reported an AET/Phen-Epoxy/Fe(III)/(Phen-Epoxy-NH2- NC3)2 genosensor, which has a working principle similar to this already reported by Grabowska et al. [69]. The hybridization process caused the formation of the duplex structure on- the electrode surface, which resulted in the thickness changes of the double layer at the- interface between the electrode and solution.-

A new trend in the development of biosensors is the use of nanomaterials which exhibit unique- and attractivechemical, physical, and electronic properties. Various nanomaterials have been- used as an electrode platform in highly sophisticated electroanalytical biosensing devices. Theworking electrodes (actual physical transducers) upon modificationwith these materials gain- large effectivesurface area, high catalytic capabilities, and high conductivity. Thus, these- transducers could act as effectivemediators and facilitate electron transfer between an active- site on the receptor and the electrode surface. Nanoparticles [74, 75], carbon nanotubes [65],- magnetic nanobeads [68, 74], or quantum dots [76] enhance sensitivity and selectivity of the- electrochemical detection. Among the variety of metal nanoparticles, gold nanoparticles- (GNPs) have been extensively utilized in recent years, mainly because of their nanoscopic size,- good conductivity, and biocompatibility. One of the most commonly used is single-walled (SW)- or multiwalled (MW) carbon nanotubes (CNTs) as well as graphene and graphene nanosheets.- Thanks to their fast electron transfer ability, mechanical strength, chemical stability, catalysis- effect,and thermal and electrical conductivity, they are attractinggreater interest than other- applied technologies.-

A magnetic nanobeads-based impedance aptasensor was designed and developed by Fu and- co-workers [74] (**Figure 7**). Briefly,-H5N1 aptamers were coated on the magnetic nanobeads- (MBs) for specificallycapturing target AIV H5N1. Then, bionanocomposites (BNCs) were- added using Au nanoparticles (AuNPs) as carriers, which were conjugated with concanavalin- A (ConA) and glucose oxidase (GOx). The BNCs attachedon the captured target virus through- ConA-glycan interaction formed a sandwich complex. Finally, the sandwich complex was- transferred to a glucose solution, resulting in an efficient enzymatic reaction and the impedance- decreased correspondingly on a screen-printed interdigitated array electrode. This method- took advantages of the high efficiencyof enzymatic catalysis and the high susceptibility of- electrochemical impedance on the ion strength and endowed the aptasensor with high- sensitivity.-

**Figure 7.** Illustration of the aptasensing mechanism and construction of the aptasensor (with permission from ACS).-

Wang and co-workers [77] developed an aptamer-based bifunctional bionanogate, which could- selectively respond to target molecules and control enzymatic reaction for electrochemical- measurements (**Figure 8**). A nanoporous gold filmwith a pore size of ~20 nm was prepared- by a metallic corrosion method and then was functionalized with two types of thiol-modified- single-stranded oligos (SH-ssDNAs) by means of Au–thiolate bonding. The bases of the two- immobilized SH-ssDNAs were selected to be partly complementary to that of the two ends of- an aptamer, respectively. Then, aptamers were added and hybridized with the two immobilized SH-ssDNAs, resulting in the nanopore covered with aptamers, a "closed" bionanogate.- Finally, the aptamer covered nanopore filmwas placed onto an enzyme precoated glassycarbon electrode to form an aptasensor. The working principle of the bionanogate-based- aptasensor for detection of AIV H5N1 is illustrated in **Figure 8**. Initially, without the target- AIV, the bionanogate was kept "closed," which isolated coenzymes and substrates in the- testing solution from the enzymes immobilized on the electrode so that the enzymatic reaction- was restricted (**Figure 8a**). Upon the target virus binding, the aptamers dissociated into- solution from their ssDNAs and formed aptamer–virus complexes, which triggered the- bionanogate "open" (**Figure 8b**). This "open" state allowed coenzymes and substrates to- diffusefreely through the opened nanopore and to create contact with the immobilized- enzymes, resulting in an efficient enzymatic reaction (**Figure 8c**).-

**Figure 8.** Principle of the bionanogate-based aptasensor for detection of AIV H5N1 (with permission from Elsevier).-

Despite the remarkable sensitivity, rapid response, miniaturization capability, and low cost of- the electrochemical biosensors for AIV detection, there are still problems with long-term- stability and selectivity in real samples with complex background. Obviously, further work- would be required to demonstrate that the electrochemical biosensors do not sufferfrom- stability and selectivity problems when handling real samples in the field.-

## **3. Conclusion-**

Biosensors have attractedtremendous interest in AIVs detection. They are characterized by- good selectivity and sensitivity with a wide dynamic range from subfemtomolar to nanomolar,- easy and rapid experimental protocol, reasonable cost, and usage of the sample volume in a- μl range. Rapid development of nanotechnology has opened a new way for design and- construction of biosensors with even betterfeatures. However, many contributions to the field- of biosensors for AIVs detection still are at the "proof-of-concept" stage. Thus, authors hope- that this chapter will promote lively and valuable discussions in order to generate new ideasand make new approaches toward the development of innovative biosensors for applications- in AIVs detection.-

## **Author details-**

Ronghui Wang1,2 and Yanbin Li1,2,3\*-

 \*Addressallcorrespondenceto:yanbinli@uark.edu-

 1-Collegeof-Biosystems-Engineeringand-Food-Science,-Zhejiang-University,-Hangzhou,-China-

2-Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR, USA-

3 Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR, USA-

## **References-**


surface plasmon resonance. Anal Chim Acta 2008;623(1):66–75. doi:10.1016/j.aca.-2008.06.005-


nomagnetic separation and enzyme-induced metallization. Biosens Bioelectron- 2015;68:586–592. doi:10.1016/j.bios.2015.01.051-


## **Vaccine Development**

## **Steps toward a Universal Influenza Vaccine: Research Models and Comparison of Current Approaches**

Terianne Wong and Ted M. Ross

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64369

#### **Abstract-**

 Theabilityofinfluenzavirustoadapttovariousspeciesandevadenaturalimmunity- makestheubiquitouspathogenparticularlydifficulttoeradicate.-Annualreformulation- ofinfluenzavaccinesiscostlyandtime-consumingandhasvaryingefficacyagainst- influenzavirusstrains.-Therefore,worldwideeffortsaimtodevelopauniversal- influenzavaccinetopreventpotentialhealthcareemergenciessuchaspandemic- influenzathreats,suchasthe-1918-Spanish-Fluandpandemic-Swine-Fluof-2009.-Efficacy- ofauniversalinfluenzavaccinemustovercomecurrentchallengeswithsubtype- diversity,antigenicdrift,andadequatelyprotectagainstemergingreassortantsfrom- bothenvironmentalandagriculturalsources.-Furthermore,themanufacturingand- productionofvaccineslargelyinfluencetheeffectivenessofavaccineandtechnological- advancementsmaysoonrivalcurrentvaccinestrategies.-Thisreviewdiscussesthe- evolutionanddiversityofinfluenzaviruses,howviralglycoproteinhemagglutinin- playsadominantroleininfluenzasurveillanceandassessmentofprotectionand- comparesthemethodologiesofcurrentandupcomingvaccineoptions.-Whilethe- obstaclesremaindaunting,growingknowledgeofinfluenzaevolutionandimmunity- mayleadtomoreviablecandidatesthatprotectagainstbroadervarietiesofinfluenza- virusesandhelppreventfutureinternationalhealthcrises.-

**Keywords:** influenza, vaccine development, universal flu,flu strategies, immunology-

## **1. Introduction-**

 The-World-Health-Organization-(WHO)estimates-3–5millioncasesofsevereinfluenza- worldwidewillresultbetween250,000and500,000deathsannually[1].WithintheUnitedStates,- 24.7millioncasesofseasonalinfluenzaofvariousseveritiesarepredictedandanestimated-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 3,300–49,000deathsoccurredperyearbetween-1979and-2001-[2].-From-2013to-2014,the- hospitalizationrateinthe-United-Stateswas-24.6per-100,000persons-[3],althoughratesashigh- as-309per-100,000havebeenreportedfrom-1993to-2008,amongelderly->65yearsofage-[4].- Includingdirectmedicalcostsandlossofproductivityandearningsassociatedwithwork- absentness,seasonalinfluenzacontributesan-\$87billionburdenonthe-USeconomy-[5].- Furthermore,thespreadofpandemicswineinfluenzavirusfrom-2009to-2010attributedto-42–- 89millioncasesofpandemic-H1N1-(pdm-H1N1)andanestimated-8870–18,300deaths.- Consequently,thereisstillaneedtoenhanceourunderstandingofinfluenzavirusevolution- andmanagethespreadofpandemicinfluenzaviruses.-

Global influenzavirus monitoring and surveillance systems track significanttrends or realtime changes to influenzaepidemiology and identify predominant viral subtypes during a- particular season. Clinical symptoms include acute onset of cough, fever, and myalgia [6–9],- and viral presence is confirmedby WHO-mandated laboratory standard operating procedures, such as immunostaining, cell culture growth, real-time polymerase chain reaction (RT-PCR), and detection of hemagglutination-inhibiting or viral neutralizing antibodies [10]. While- viral culture in mammalian cells remains vital for characterization, RT-PCR remains the most- rapid and sensitive diagnostic test available, with detection rates enhanced twofold over cell- culture [8, 11, 12]. Alternatively, increases in hemagglutination-inhibiting antibody titers more- than fourfold are counted as positive seroconversion and following viral infection or vaccination, which assists with monitoring immunogenicity. Collectively, data are shared through the- WHO's Global Influenza-Surveillance and Response System (GISRS), which includes 143- institutions in 113 WHO Member States [13], to help alert the emergence of antigenic variants- or the beginning of a pandemic.-

 In April 2009, the Centers of Disease Control and Prevention (CDC) cited the firstincidence of- human-to-human transmission of pdmH1N1, also referred to as swine influenza-A, which was- antigenically distinct from other circulating human H1N1 [14]. As the firstinfluenzapandemic- of the twenty-firstcentury, pandemic (pdm) H1N1 was not included in the annual trivalent- vaccine regimen, leaving a large majority of the population unprotected from the newly- emerging pathogen. In response to the increasing number of illnesses associated with pdm- H1N1, satellite surveillance sites throughout the world rapidly collected clinical samples,- surveyed seroconversion rates, and monitored the spread of the pandemic. Under the recommendation of WHO and CDC, the standard trivalent vaccine regimen of 2009–2010 [15] was- reformulated to accommodate for the pdm H1N1 influenza strain (A/California/07/2009-like)- [16, 17], and a median seroconversion rate of 59% was observed among healthy individuals- ranging from 18 to 65 years of age [18]. During the 2010–2011 season, predicted efficacyin- Sweden among individuals between 6 months and 64 years was between 63% and 80%, when- 60% of the Swedish population received a monovalent AS03-adjuvanted vaccine against pdm- H1N1 [19]; importantly, laboratory diagnosis of pdm09 H1N1 was utilized in this study. As of- 2016, A/California/07/2009-like pdm H1N1 remains the predominant H1N1 strain circulating- in the human population and remains a component in the annual trivalent or quadrivalent- vaccine schedule. The remaining components of the annual vaccine, including prototype- strains of Group B influenza strains and subtype H3N2, are frequently updated due to frequentantigenic changes to the hemagglutinins (HA). Ultimately, the WHO and CDC provide- recommendations for vaccine formulation and identify what particular health concerns need- to be address by the public, healthcare workers, and pharmaceutical/biomedical research- industry.-

#### **2. Influenza diversity, tropism, and evolution-**

Currently, there are three types of influenza-(A, B, and C), however, only types A and B infect- humans. Influenzabelongs to the family of Orthomyxoviridae and contains a negative sense,- single segmented RNA genome. The influenzavirion is pleomorphic or filamentousin shape,- budding to approximately 80–120 nm in diameter, and attaches to sialic acid receptors found- on numerous tissues in a diverse variety of hosts, including birds and mammals, such as swine,- equine, canine, ferrets, and primates [20–23]. Upon budding from host cells, a lipid bilayer- envelopes the viral ribonucleoprotein complex and presents surface viral glycoproteins, which- are various combinations of hemagglutinins (HA, H1-H18) and neuraminidases (NA, N1- N11); the arrangement of HA and NA help distinguish the large diversity of influenza- subtypes [24].-

Avian influenza strains are ubiquitous in the wild; circulating in predominantly the wild ducks- Anatidae bird populations [25–27] through an indirect fecal-oral transmission route and often- results in littleto no pathology [25, 28]. Replication of avian influenzavirus (AIV) occurs in- the intestinal tract of waterfowl and is shed in feces, such that contaminated waters infect other- hosts; certain strains of AIV can persist in aquatic environments for 9 to 200 days, depending- on salinity, pH, and temperature [29–31]. Consequently, poor water quality control and- cohabitation of wild species increase the likelihood of AIV transmission into domestic birds- markets [27, 32, 33] and livestock. Surveillance of live poultry markets in Asia indicate high- prevalence of low pathogenic avian influenzastrains, such as H3N2, H5N2, and H9N2 [32, 34–- 37]; isolates collected from poultry populations in Korea were used to experimentally infect- mice and demonstrate zoonotic adaptation for mammalian hosts [38].-

The human influenza virus threats are derived from avian [21, 39–42] or swine [14, 43] influenza- predecessors, but these viruses have adapted to transmit and to replicate in the human host.- Highly pathogenic avian influenza-H5N1 (HPAIV H5N1) outbreaks during the end of the- twentieth century led to lethal disease in domestic poultry and waterfowl, and eventually- human intervention was needed to quarantine and cull millions of contaminated flocks- worldwide [41, 44, 45]. Although most avian influenzastrains typically do not cause disease- in humans, HPAIV H5N1 was spread to humans in Hong Kong in 1997. From 2003 to January- 2014, the WHO reported 386 human deaths out of the 650 confirmed-H5N1 cases [46]. In- February 2013, a novel avian influenzavirus subtype, H7N9, was also detected in humans [47]- and was thought to have arisen from nonpathogenic H7 strains.-

In humans, influenzaspreads through aerosolized respiratory droplet transmission and the- virus attachesto primarily columnar epithelial cells lining the respiratory tract. Infection of- influenzaspreads to airway epithelial cells [48–50] and alveolar macrophages [51], althoughdendritic cells [52], natural killer cells [53], and mast cells are semi-permissive for influenzainfection in vitro and in vivo. Typical incubation period for influenza virus is 1–4 days, in whichan afflictedindividual begins shedding virus and continues to shed up to 10 or more days afterthe onset of symptoms [9, 54]. Unlike human seasonal influenza, most AIV transmits directlyfrom animal-to-human (zoonotic), but infrequently transmits between human hosts; however,since 1918, four influenzapandemics occurred after antigenic shift enabled human adaptionand no prior immunity existed in the population. Since influenzahemagglutinin binds to sialicacid receptors that are on respiratory epithelial as well as erythrocytes, a rapid assay for sialicacid receptor binding is the red blood cell agglutination and hemagglutination inhibition (HAI)assays. Developed in the 1940s and based on the ability of influenzahemagglutinin to bindreceptors of target cells and agglutinate red blood cells (RBCs), the HAI assay has become thestandard serological assay for screening influenzahemagglutinin-reactive antibodies in sera- [10]. Serial dilutions of heat-inactivated, receptor-destroying enzyme (RDE)-treated sera areincubated with a fixed amount of virus titrated in hemagglutination units and then RBCs are- added to the virus-sera mixture to permit agglutination. The presence of influenza-specific,- receptor-binding antibodies prevents RBC agglutination and potentially reduces infectivity.-By standard, an antibody titer of >32, or most commonly 40, is correlated with up to 50%protection from influenzadisease [14], and seroconversion is assumed when greater thanfourfold increase is observed when comparing of pre- and post-vaccination HAI titers [13].-However, as important as the standard agglutination and HAI assays are for vaccine immunogenicity surveillance, new challenges arise when viruses have reduced species-specific-RBCagglutination [55, 56] or when HAI titers do not correlate with protection [15]. Consequently,additional verificationapproaches are needed and applied to assess efficacybeyond immunogenicity of a vaccine candidate; the greatest obstacle, however, is overcoming the antigenicdiversity in influenzastrains and eliciting vaccine-induced protection prior to antigenic driftsor shifts.-

 Influenzaglycoprotein hemagglutinin (HA) amino acid sequence variation fluctuates rapidlyamidst cross-species infections, leading to shifts within HA subtypes. For example, theinfluenzaviruses that spread internationally in 2009 were antigenically unique to seasonal-H1N1 strains, but resembled the pandemic H1N1 strains isolated in 1918. The emergence of- H1N1 into the human population in 1918 caused the firstinfluenzapandemic of the twentiethcentury and caused ~50 million deaths worldwide [57]. Cross-reactive antibodies to the pdm-H1N1 strains of 2009 were identifiedin the sera of older people [58] and therefore this impliedthat people infected with the 1918 H1N1 viruses elicited long-lasting antibodies that crossreacted with the pdm H1N1 infecting people 90 year later. H3N2 influenzastrains emerged in-1968 as a human pathogen after reassortment of HA & PB1 genes with avian source (1918 H1N1- (avian/human)→1957 H2N2 (avian)→1968 H3N2 (avian/human)) [43] leading to the loss of- immune recognition among human hosts. The timeline in **Figure 1**illustrates the reassortment- events that transitioned between avian influenzaviruses into human pathogens and theemergence of pdm H1N1 or swine fluremained circulating in the swine population since earlytwentieth century and was reintroduced in the early 2000s.-

Steps toward a Universal Influenza Vaccine: Research Models and Comparison of Current Approaches 91 http://dx.doi.org/10.5772/64369

**Figure 1.** Timeline of influenza viruses and circulation among avian, swine and human populations.-

The emergence of the 1918 Spanish Flu into the human population resulted from divergent- reassortment of avian-like influenzaand caused substantial morbidity and mortality worldwide. Further reassortment of the HA subsequently led to H2N2 and H3N2, which currently- circulates abundantly in the human population. Introduction of H3N2 into the swine population during the 1970s and circulation of H1N2 permittedadditional reassortment in the form- of novel Swine Flu or pdm H1N1. The recent incidence of avian strains H5N1 and H7N1 is- further evidence that zoonotic exchange of influenzaviruses increases the likelihood of- introduction to human populations.-

Influenza-HA mediates the binding to host cell surface receptors, dominantly sialic acid, which- tends to be specificto species and tissue. HA proteins expressed by avian influenzastrains- preferentially bind to the sialic acids bound by an alpha 2-3 glycosidic bond, whereas an alpha- 2-6 linkage is preferred by human adapted strains. Importantly, the upper human respiratory- tract is lined with epithelial cells expressing sialic acids with alpha 2-6, whereas the lower- respiratory tract contains sialic acids containing the alpha 2-3[41] potentially increasing- susceptibility to lower respiratory tract infections by avian influenzaviruses [42]; however,- direct droplet transmission between humans is inefficient and remains limited.-

In addition to receptor-binding specificity, the influenza-HA sequence contains a critical- cleavage site that is necessary for initiating pH-dependent fusion into the host cell. Influenzavirus HA is synthesized as a precursor HA0, which is composed of a globular surface "head"- HA1 and the stalk-like HA2. The HA2 portion contains a transmembrane domain spanning- the viral membrane [23, 59, 60]. Within the HA1 subunit is the receptor-binding site (RBS) and- a vestigial esterase domain that alters pH stability and ultimately, confers high or moderate- pathogenicity [61, 62]. The exposure of the fusion peptide sequence is essential for conformational changes in the coiled-coil trimeric HA structure during a prefusion event, leading to- extension of the HA, fold-back, hemifusion and eventual fusion of viral membrane to host- membrane [63]. **Figure 2**is reprinted from a study [63] that analyzed the fusion event between- influenzahemagglutinin and cell membrane of the infected cell, which utilizes a similar coiledcoil hemifusion state as other viral pathogens, such as HIV, respiratory syncytial virus, and- Ebola virus [64].-

**Figure 2.** Fusion event of influenza hemagglutinin on host cell. Adapted from Ivanovic et al. [63].-

Utilizing the coiled-coil motif to extend into a host cell, the hemagglutinin fusion peptide serves- as an anchoring "hook" to attachto the cell membrane; through pH-mediated hairpin folding,- the hemifusion event occurs, followed by complete fusion of the two membranes and release- of viral components into the host cytoplasm.-

The majority of low pathogenic influenzastrains have HA0 containing a monobasic arginine- residue that is cleaved by extracellular enzymes; within the human respiratory tract, the- transmembrane protease, serine 2 (TMPRSS2) and human airway trypsin-like protease (HAT)- are capable of mediating mono-basic cleavage to yield disulfide-bonded-HA1 and HA2- subunits [65]. Several highly pathogenic avian influenza strains (H1N1v, H5 and H7) contain- multi-arginine motifs that are cleaved by subtilisin-like cellular proteases and are often present- in the intestinal and respiratory tracts of birds and mammals; cleavage by host proteinases,- such as furin and PC6, occurs intracelluarly upon exit of the endoplasmic reticulum, and- proteolysis is blocked by inhibitors of serine proteases, such as aprotinin [50, 66]. Interestingly,- within a human large intestine carcinoma cell line Caco-1, aprotinin treatment and disruption- of the late Golgi transport mechanisms by brefeldin A inhibit cleavage of HA0, regardless- whether the influenzastrain contains mono- or multi-basic proteolytic sites [66]. **Figure 3-** illustrates the domains HA1 and HA2 in a homotrimer of HA from A/Fujian/411/2002-H3N2- influenzapredicted using SWISS-MODEL service [67–70], with antigenic sites highlighted.- Sites in H3 HA have been best characterized, with antigenic epitopes definedas A, B, C, D,- and E [71–73]. Alternatively, H1 contains fiveantigenic sites, Cb, Sa, Sb, Ca1, and Ca2, that- were originally modeled from H3 structure [74].-

**Figure 3.** Structure of influenza hemagglutinin with antigenic sites and domains highlighted.-

Influenzahemagglutinin is a homotrimer of a globular head and stalk domains. The HA of A/- Fujian/411/2002-H3N2 influenzawas modeled to PDB 2yp2.1.a using structure homology- software SWISS-MODEL and further manipulated with PyMol. One monomer is shown with- surface, whereas the remaining two monomers illustrated in ribbon-cartoon form. Highlighted- antigenic sites A (red), B (yellow), C (green), D (blue), and E (purple) are present in H3 HA- (shown), whereas Cb, Sa, Sb, Ca1, and Ca2 are the known antigenic epitopes in H1 influenza- subtypes. Sites used were derived from 73.-

Incomplete cleavage of HA0 yields lower viral titers [50, 65, 75] and virions incapable of HA0- cleavage are less infectious upon subsequent replication cycles, with more than 4 log-units- lower than cleaved HA [50, 76]. Current cell-based approaches to influenzapropagation using- Madin-Darby canine kidney (MDCK) cells require artificialaddition of trypsin or serine- protease treatment to yield appreciable viral titers, but native enzymes in egg-based culture- sufficientlycleave HA; therefore, no addition treatment is required [77, 78]; consequently, posttranslational HA processing is substantially differentbetween the two standard methods [79].- Recent human isolates, particularly H3N2 subtype, are difficult to propagate in conventional- egg or MDCK cell lines; advancements in stable-overexpression of α2–6 sialic acids on the- MDCK cell line (MDCK-SIAT1) have improved virus isolation rates[56] and typically retains- HA sequence identity betterthan the aforementioned egg or MDCK approaches. These- characteristic of HA and sialic acid binding affinitydetermine species- and tissue-specificity,- and further complicate the likelihood of zoonotic diseases and transmission among humans.-

Fears of an avian HA and NA reassortment with a human seasonal influenzavirus are well- justifiedgiven the emergence of avian-to-human and human-to-human transmissible HPAI- H5N1 [45, 80] and subsequent fatality rates averaging 56% [81]. Human infection with H7N9- [47, 82–85] and H10N8 [86, 87] is high risks to individuals working or residing near live poultry- markets, and mammalian receptor adaptation remains a significant concern for health officials.- While H10N8 does not immediately appear to pose an imminent threat to global health, there- are at least 450 confirmedcases of H7N9 by June 2014 since its initial emergence in China in- March 2013. Historically associated with a highly pathogenic avian influenzastrain, the H7- subtype strains, such as H7N2, H7N3, and H7N7 [47, 88, 89], have the preferential binding to- α2–6-linked sialic acids, which increase their affinityto human upper respiratory epithelial- cells [88, 90]; however, incidence of H7 avian and rare human infections remained isolated [91].- Experimental aerosol transmission studies in 2012 [92, 93] and 2013 [94, 95] using viruses in- subtypes H5 and H7 showed that, similar HPAI H5N1, the H7N9 viruses potentially poses- greater threat to humans since it has a limited capacity to transmit through aerosol droplets- (REFS) and therefore further investigations are needed to elucidate H7 pathogenesis and HA- evolution.-

## **3. Challenges of generating a universal influenza vaccine-**

The diversity of influenzavirus strains and subtypes exacerbates the challenge for generating- a universal vaccine against influenza. The WHO makes recommendations for influenza vaccine- composition for each fluseason based on international surveillance systems and compares the- ability of monovalent vaccine prototypes to elicit cross-reactive antibodies against prevalent- circulating strains [16, 96, 97]. Unfortunately, delays in surveillance and the generation and- evaluation of serology data hinder the selection of the optimal candidates and seasonal efficacy- depends on prompt production by vaccine manufacturers and subsequent distribution.- Moreover, the emergence of antigenic drift variants after selection of vaccine candidates may- result in poor efficacy and wasted production time and resources [98]. Consequently, there is- a demand for a universal, or more broadly reactive, vaccine against influenza virus. Utilizing- various manufacturing platforms, adjuvants, and targets, researchers worldwide are currently- seeking ways to improve current influenzavaccination strategies [99]. The following traits are- desired in an ideal universal vaccine candidate: [1] recognition of antigenic drift variants; [2]- elicitation of long-lasting memory responses; and [3] minimal manufacturing lag time between- vaccine formulations. Additional desirable features may include cost-efficientproduction- methods, predictive measures to protect against future antigenic variants, and efficacyamong- immunocompromised target populations, infants and elderly over the age of 65. Despite nearly- a century of research, human influenzavaccine production is largely based on the traditional- technologies. The rapid emergence of antigenically unique subtypes continues to challenge- the current pace of diagnosis, prototype selection, and egg-based vaccine distribution.- Furthermore, the importance of pre-existing immunity determines the level of protection from- novel strains and depends on the presence of circulating cross-reactive antibodies. For- example, individuals born prior to 1957 reportedly had fewer complications associated withpdm H1N1 than the younger, naïve population potentially because the elderly population- encountered similar historical H1N1 strains during their lifetime and therefore had preexisting neutralizing antibodies to antigenically similar strains [100].-


**Table 1.** A list of FDA-approved influenza vaccines in US market as of March 2016.-

 Incontrast,recentevidencesuggeststhatsequentialexposuretoseasonalstrainsconfers- greaterprotectionagainstnovelantigenicdriftvariants;consequently,thequestionremains- whethertheexistenceofprotectiveneutralizingantibodiesintheelderlyisduetoimmunologicalrecall,orrather,enhancedbroadlyreactiveresponseduetoimmunologicalboostsof- heterologousstrainsoveralifetime.-Thishassincebeenmodeledintheferretanimalmodels- and,withoutpriorexposuretothepdm-H1N1,ferretssequentiallychallengedwithseasonal- H1N1-[101,-102]orimmunizedwithinactivatedseasonalvaccine-[103]strainsmount- protectiveresponsestopdm-H1N1.-Insummary,seroprotectionagainstinfluenzamayalso- dependontheindividual'spre-existingimmunity,aswellastheplatformandstrategy- implemented.-

Current 2016–2017 influenzavaccines recommended by the WHO are available as an inactivated trivalent or quadrivalent formulation delivered intramuscularly or as live attenuated- mixture of influenza strains (LAIV) administered through intranasal route [104]. The various- doses and formulations may or may not contain ovalbumin or mercury, and eligibility for some- formulations is age restricted. Vaccine manufacturers have proprietary methods for virus- propagation and inactivation, which often alters the antigenic properties of the vaccine- candidate. Vaccine antigens may be prepared as whole virus, detergent or solvent disrupted- split virions [105, 106], or subunit vaccines [107–109]. **Table 1**displays the 2016–2017 FDAapproved influenzavaccines used in the United States and exhibits formulation differences as- described in the package inserts of each product [110–118]. Essentially, all current formulations- aim to elicit protective antibody responses, predominantly against HA, and therefore protect- against antigenically similar viral strains to the vaccine strain. Since the vaccine is often- generated as a reassortant (6:2) vaccine strain from a high yield genetic backbone A/Puerto- Rico/8/34 (H1N1), the HA and NA selected as a seasonal vaccine recommendation must be- reformulated each season and validated for cross-reactivity to antigenically similar strains- using HAI assay [98].-

Adverse reactogenicity remains a safety concern for the young, immunocompromised, and- elderly, therefore split or subunit vaccines are preferred over live or whole inactivated virus,- despite enhanced humoral responses elicited by whole virus preparations [119]. Effortsto- reduce the risks associated with LAIV are particularly important since humoral and crossreactive T-cell responses are superior to that of inactivated vaccines in experimental swine and- ferret studies [120–122]; among children, LAIV, but not inactivated vaccine, led to enhanced- CD4+, CD8+, and γδ T-cell responses [123]. In contrast to inactivated vaccines that require- reformulation each year, there is evidence that LAIV induces cross-reactivity with heterologous strains. This cross-reactivity phenomenon makes LAIV a promising candidate for the- development of a universal vaccine platform. The route of administration, absence of active- viral replication, and process of inactivation are different between influenza vaccine formulations. Further studies are needed to understand the mechanisms that promote influenzaspecific T-cell responses upon LAIV immunization.-

The WHO recommends propagating influenzaviruses in embryonic chicken eggs, and this- methodology is predominant source of high yield vaccine strains [124]. A list of FDA-approvedinfluenza vaccines, majority of which are derived from embryonated chicken eggs, is provided in **Table 1**.-

**Table 1**shows differences in inactivation, detergents, and platforms used in the current vaccine- market.-

Despite the frequent use of the relatively inexpensive but highly productive virus production- egg-based platform, some challenges are arising. Spontaneous mutations due to egg adaptation were recognized as early as 1951 [125] and, as a recent vaccine efficacy-(VE) study showed,- may present unanticipated challenges to vaccine development. In the 2012–2013 influenza- season, MDCK-grown prototypes were selected for vaccine production and classical reassortant methodology was performed; in brief, co-culture of high-yield A/Puerto Rico/8/34 (H1N1)- reassortant (6:2) with the HA and NA gene products from A/Victoria/361/2011 (H3N2) in the- presence of antiserum to A/Puerto Rico/8/34 yielded reassortant A/Victoria/361/2011(H3N2)- IVR-165 [126]. During vaccine production, the high growth strain acquired several substitutions within the antigenic site B, which reduced reactogenicity against the original prototype- A/Victoria/361/2011 and antigenically similar circulating fieldstrains, resulting in a disappointing 39% (95% CI, 29%–47%) rate in humans vaccinated with the 2012–2013 egg-based- preparations [127]. Consequently, the low VE for the 2012–2013 H3N2 season is attributed to- changes in the HA due to vaccine manufacturing, not antigenic drift. In contrast, growth in- mammalian cell lines results in fewer mutations than in eggs, with up to two mutations in the- hemagglutinin of H3N2 strains after three passages in MDCK [128–130] or MDCK-SIAT1 [56]- but no genotypic changes reported after 10 passages of avian influenzausing human colorectal- adenocarcinoma cell line, Caco-2 [131]. These examples suggest that alternative methods for- influenzapropagation are expanding and optimization could ensure more consistency and- efficacy.-

In 2013, the FDA approved the first-MDCK cell-culture influenzavaccine (CCIV) called- Flucelvax®, originally developed by Novartis Vaccines and Diagnostics, Inc., Cambridge, MA,- USA [115] but acquired by CSL Limited, Melbourne, Australia, as of December 2015 [132],- which is tolerable and safe, and demonstrates comparable, if not greater, protection to that of- traditional egg-based technologies [133–137]. Furthermore, trials involving trivalent preparation of influenzaviruses through growth in green monkey kidney cell line, Vero, is another- new alternative to egg-based propagation [138]. Although not yet FDA approved in the United- States, the Vero cell-based technology licensed as Vepacel® from Baxter AG, Vienna, Austria,- is a pre-pandemic whole, inactivated virus H5N1 vaccine designed to elicit strong immunological responses in healthy, as well as immunocompromised populations [139]. Also in 2013,- the FDA approved a recombinant HA (rHA) only influenzavaccine (Flubok®) from Protein- Sciences Corporation, Meriden, CT, USA [118]. Flubok is generated from insect cells using a- baculovirus expression system to produce high yield rHA vaccine against each seasonal- influenzasubtype. Further studies are still needed to assess cost-effectivenessin transitioning- the current platforms of egg-derived influenzavirus to mammalian cell culture. Modifications- to standard method of virus propagation have economical and practical challenges, but federal- initiatives suggest divergence from traditional egg-based approaches.-

#### **4. Alternatives to current influenza vaccine strategies-**

 Adisadvantageofmammaliancellcultureistheneedforregulatedgrowthconditionsand- reagents,whichhinderimmediateproductionandincreasecost;however,advancementsin- high-throughputbioreactorsareongoing-[140].-However,thesametypesoftechnologies- involvedinmammaliancellcultureviruspropagationmayfurtheradvancenovelalternativestoeggs.-Virus-likeparticles-(VLPs)arenon-replicating,self-assemblingnanostructures- thatmimicvirussurfaceproteinpresentationandaresynthesizedthroughco-expressionof- recombinant-DNAininsectormammaliancellculture-[141–147].-Multipleadvantagesare- associatedwith-VLPoverstandardmethods-[141],including-[1]theyarenon-infectious- materialandthereforenotabiohazardriskandinactivationisnotrequired,-[2]-VLPsselfassembleintoproperconformationandmultivalency,and-[3]influenza-VLPshaveenhanced- stabilityand-HApotencyupto-12monthswithnodegradation-[148].-Severalinvestigators- haveutilizedthisplatformforevaluatingimmunogenicityofinfluenza-HA-[149–151],-NA- [144],-M2-[152],andcombinationsofviralproteins.-Amammalian-VLPderivedin-Verocells,- H5N1-VLP-(RG-14)-[153],wascomposedoffourinfluenzavirusstructuralproteinsand- protectedagainstalethal-H5N1challengeinmice,yielding-H5-specific-IgG1antibodieswith- adoseaslowas-2.5μginaprime-boostregimen.-Similarly,baculovirus-derived-VLPs- expressing-H3N2protectedagainstalethalchallengeofmouse-adaptedinfluenza-H3N2-A/- Hong-Kong/68-[149],withintranasalinstillationprovidingthegreatest-HA-specificantibody- titersand protectionascomparedto intramuscularadministration ortwodosesof sublethal- intranasal challenges.-Thebaculovirus/insect cellexpression systemsgeneratehighyieldsof- VLPs,andtrivalentvaccinepreclinicalstudiesinmiceandferretsdemonstratethebaculovirus-derived-VLPseffectivelyelicitserum-HAIantibodiesagainst-H1N1,-H3N2,and- influenza-B,althoughcross-reactivitytoheterologousstrainswaspoor-[154].-Importantly,- side-by-sidecomparisonofbaculovirus-derived-VLPsversuswholevirionvaccineor- recombinant-HAsuggests-VLPselicitcomparable,ifnothigher,-IgGtiters,butalsoyielded- antibodiesthatcross-reacttoabroaderpanelofnon-homologousinfluenzaviruses-[146].- Thebaculovirus-derived-VLPshavebeenfullycharacterizedby-Novavax-,-Inc.from- Rockville,-MD,-USA,forthe-HAcontent,-NAactivity,stability,and-VLPpurity-[155],and- safetyandreactogenicitywereevaluatedintwoclinicaltrialswith-H5N1and-H1N1- baculovirus-derived-VLPs-[156,-157].-

 Proprietary application of recombinant RNA bacteriophage Qbeta by Cytos Biotechnology- Ltd., Switzerland,offersan alternative strategy for conformational antigen presentation on the- surface of Qbeta-derived virus-like particles through covalent chemical linkage [158]. Since- presentation on Qbeta-derived VLPs does not involve post-translational modificationsfrom- the VLP-producing cell, potential cell type-specificalterations to the viral surface glycoprotein- are avoided [159]; moreover, chemical linkage of antigens to Qbeta-derived VLPs overcomes- the steric hindrance associated with masked immunodominant epitopes and efficiently elicits- B cell responses in the absence of an adjuvant [160]. Importantly, specificsites or domains can- be exclusively presented on the Qbeta-VLPs, such as the globular head of influenzahemagglutinin to induce hemagglutination inhibiting antibodies [161]. Consequently, this approach- advanced to Phase I clinical trial in Singapore through sponsorship of the manufacturer CytosBiotechnology; the findingssuggest that the Qbeta-VLP is tolerable and elicits HAI-specific- antibodies comparable to the standard influenzavaccines [162]. However, as with any new- approach, insect-, mammalian-, or Qbeta-VLP manufacturing processes still need to undergo- more thorough safety evaluation and quality control; therefore, marketing and production are- still a few years away.-

 Modernrecombinanttechnologyoffersalternativestoliveviruspreparation.-Syntheticor- subunitviralpeptidevaccinesstimulatetheimmuneresponseinsimilarmechanismassplit- inactivatedvaccinesandmaybeformulatedwithpatternrecognitionreceptoragonistsor- emulsionstoenhanceadjuvantactivity.-Plasmid-DNAencodinganimmunogenicviral- proteincanbeelectroporated-[163],administeredintranasallyinconjugationwithnanoparticles-[164],ordeliveredwithabiolisticsystem.-Particle-mediatedepidermaldeliveryof-DNA- encodinginfluenza-HAwasrecentlyoptimizedinanexperimentalferretmodeland,when- administeredtotheabdomenortongue,yielded-HAItitersgreaterthan-1:40-[165];sublingual- routeofvaccineadministrationhaspotentialtoelicitmucosalimmunityandmount- protectivesecretoryimmunoglobulin-A-(sIgA)-[166,-167].-Previouslyinpreclinicalstudies,- deliveryofrecombinant-IgAprotectsagainstinfectionintheexperimentalanimalinfluenza- model-[168]whileelicitationof-IgGcontrolsdiseaseseverity,thuselicitationof-IgAbyany- universalvaccinecandidatewouldbehighlybeneficial.-Intheefforttoreduceinfant- morbidityassociatedwithinfluenzainfections,ongoingstudieswilltestthesafety,efficacy,- andtolerabilityofvaccinationofpregnantmothersandtheirnewborns-[169–171];subsequent- protectionthroughmaternal-IgGandsIgAlastingupto-6monthscouldreduceinfection- withininfluenzavirus-[172].-

In addition to the HA, DNA vaccination of the neuraminidase N1 from H1N1, A/New- Caledonia/20/99, also partially protects against heterologous challenge with H5N1, A/- Vietnam/1203/04, in the mouse model; similarly, passive immunization of heterologous anti-N1 sera in naïve mice provided modest protection against H5N1 challenge [173]. NA-DNA- vaccination also conferred crossprotection against heterologous H3N2 strains in a lethal H3N2- influenzachallenge [174], further suggesting neuraminidase may be an additional target for- eliciting protection against drift variants within a influenzasubtype. In addition to the- neuraminidase, viral targets such as the nucleoprotein (NP), matrix 1 and 2 (M1 and M2), and- polymerase (PB1) are differentimmunogens for vaccine candidates, resulting in varied- immunological responses. Internal NP is the predominant target antigen for cytotoxic T- lymphocyte (CTL) activity and is well conserved among influenza-A viruses; peptide-based- and DNA vaccination strategies have demonstrated promising protection against homologous- and heterologous challenge with influenza-A viruses. However, as with epitope-specific-T cell- responses, binding and affinitybetween epitope and human leukocyte antigen (HLA) could- be restricted to highly polymorphic HLA alleles and could reduce efficacyamong particular- populations [175]. Consequently, promiscuous T cell epitopes that bind sufficiently to a wide- range of HLA alleles remain to be identified.-Recent studies also suggest cooperative T helper- cell assistance to mount B cell immunological responses to promiscuous epitopes on the- ectodomain of M2 protein, M2e [176]. Modified-M2e, termed M2e multiple antigenic peptides- (M2e-MAPs), appears to protect against influenzachallenges among mice of various geneticbackground, providing proof of concept that M2e-MAPs are not necessarily MHC allelerestricted. Multiple effortsremain ongoing to test the feasibility of M2e-MAP-based vaccinestrategies against influenza.-


**Table 2.** Clinical trials ongoing the US testing novel influenza vaccine or antiviral approaches.-

Since monovalent HA reactivity is thought to limit broad use of an influenzavaccine, alternative strategies aimed at less conventional tactics aside from full-length, wild-type HA. Directed- reactogencity to either the globular head or stalk have been examined in mice, ferrets, and- human clinical studies [150, 151, 156]. Broad protection against heterologous influenza virus- strains is thought to rely on elicitation of well-conserved, HA2, stalk- or stem-specificantibodies [177]. Cross-protection against heterologous strains and crossreactivity to various- subtypes appears the most rational direction toward a universal influenzavaccine. Immunization with recombinant HA2 construct that maintains a neutral pH conformation conferred- protection against homologous H3-type virus strains, such as A/Philadelphia/2/82 and A/HK/- 68, but not heterologous H1-type viruses [178]. Importantly, prime-boost immunization of- chimeric hemagglutinin expressing mismatched globular HA heads on homologous HA stalk- (cH6/1, cH9/1, and cH5/1) elicited stalk-reactive, neutralizing antibodies and conferred- protection against lethal H1N1challenge [179]. Protection, however, remained limited within- a particular influenzagroup; therefore, further investigation remains to overcome such large- diversity of influenzaviruses. In an attemptto address the vast diversity of globular HA,- computationally optimized broadly reactive antigens (COBRA) have been utilized in mammalian VLP platforms against H5N1 infection in mice, ferrets, and nonhuman primates [180–- 182]. Through multiple layers of consensus sequences of influenzahemagglutinin, COBRA- methodology avoids database input bias associated with influenzasurveillance systems;- constructs generated have been validated to stimulate greater immunological breadth than- monovalent vaccine strategies while successfully eliciting seroconversion, yielding hemagglutination-inhibiting antibody titers more than fourfold. **Table 2**is a summary of several- clinical trials that investigated novel influenzavaccine strategies with various targets and- approaches for eliciting protective immunity. In conclusion, the efficacyof the immunogen,- route of administration, and types of immune responses elicited will further determine- whether novel approaches could replace current vaccination methods.-

The ClinicalTrial.gov database provides latest information regarding past and current clinical- trials and the status toward future applications. Unlike the conventional influenzavaccines- that dominant to HA-mediated immune responses, recent clinical trials test antiviral therapies- targeting influenzamatrix (M and M2e, matrix ectodomain) or highly conserved nucleoprotein- (NP). These strategies come from various technologies, including virus-like particles (VLPs)- and platforms, which do not rely on the egg-based vaccine manufacturing processes. The- routes of delivery also vary, including intramuscular (IM), intradermal (ID), and subcutaneous- (SC).-

## **5. Concluding remarks-**

The pursuit of a universal vaccine against influenzais a constant battle between influenzavirus- evolution and current technologies. Since influenzainfections remain ubiquitous among- humans, as well as wildlife and domestic livestock, reassortment of genomic segments can still- yield combinations that the human species has not previously encountered; alternatively,- gradual antigenic variation of the hemagglutinin also results in poor cross-reactivity andprotection against heterologous strains. As such, protective measures such as effective- vaccination and more thorough understanding of HA evolution are needed to prevent- forthcoming pandemics. Current practices rely on relatively traditional technologies for- vaccine selection and manufacturing, and as witnessed during the pandemic H1N1 outbreak- of 2009, it took months to identify and manufacture a new vaccine [183, 184]. In addition,- inadequate quality control led to sterility breaches of millions of doses Fluvirin®, which attime was manufactured by Chiron, acquired firstby Novartis Vaccines and Diagnostics- Limited, Speke, Liverpool, UK [185, 186] but now recently under Seqirus™, a CSL Limited- company. During such shortages, it was evident that influenzavaccine resources were- insufficientgiven the substantial health risks. To minimize future damages, both in morbidity- and economical losses, the international community must disseminate new findingsrapidly- and cooperatively strive for more effective and readily available preventative actions.-

In addition to the current vaccine approaches, innovative strategies are being tested for- feasibility, safety, and the elicitation of broad immunogenicity to influenzastrains and- subtypes. Overcoming the antigenic diversity remains the greatest challenge to the efforts of- designing a universal influenzavaccine. Moreover, changes in manufacturing and rapid- implementation are critical for vaccine efficacyand may require divergence from the currently- accepted methodologies. The recombinant technology and nonconventional vaccine platforms- aforementioned demonstrate potential and may rival the current standard of care. As our- understanding of immune responses to influenzaand antigenic variability furthers, improvements to influenzavaccine strategies are expected to prevent significantmorbidity and- unnecessary disease through preventative measures.-

## **Author details-**

Terianne Wong and Ted M. Ross\*-

 \*Addressallcorrespondenceto:tedross@uga.edu-

 Departmentof-Infectious-Diseases,-Collegeof-Veterinary-Medicine,-Universityof-Georgia,- Athens,-GA,-United-States-

#### **References-**


charides and gangliosides and by a higher conservation of the HA receptor-binding- site. Virology. 1997;233(1):224 - 234.-


induce diverse T-cell responses in young children. The Journal of Infectious Diseases.- 2011;204(6):845-853.-


**Influenza Inactive Virus Vaccine with the Fusion Peptide (rTα1-BP5) Enhances Protection Against Influenza Through Humoral and Cell-Mediated Immunity**

Chen Wang, Chengshui Liao, Wufan Zhang, Deyuan Li and Puyan Chen

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64403

### **Abstract-**

 Thymosinα1-(Tα1)and-Bursopentin-(BP5)arebothimmunopotentiators.-Toexplore- whetherthethymosinα1-Bursopentin-(rTα1-BP5)isanadjuvantornot,weclonedthe- geneof-Tα1-BP5andprovidedevidencethatthegeneof-Tα1-BP5inarecombinant- prokaryoticexpressionplasmidwassuccessfullyexpressedin-*Escherichiacoli-*BL21.-To- evaluatetheimmuneadjuvantpropertiesofrTα1-BP5,chickenswereimmunizedwith- rTα1-BP5combinedwith-H9N2avianinfluenzawhole-inactivatedvirus-(WIV).-The- titersof-HIantibody,antigen-specificantibodies,-Avianinfluenzavirus-(AIV)-neutralizingantibodies,levelsof-Th1-typecytokines-(gammainterferon-(IFN-γ))and-Th2-type- cytokines-(interleukin-4-(IL-4)),andlymphocyteproliferationresponsesweredetermined.-WefoundthatrTα1-BP5enhanced-HIantibodyandantigen-specificimmunoglobulin-G-(IgG)antibodiestiters,increasedthelevelof-AIV-neutralizingantibodies,- inducedthesecretionof-Th1- and-Th2-typecytokines,andpromotedtheproliferation- of-Tand-Blymphocyte.-Furthermore,viruschallengeexperimentsconfirmedthat- rTα1-BP5contributedtotheinhibitionreplicationofthevirus-(H9N2-AIV-(A/chicken/- Jiangsu/NJ07/05)fromchickenlungs.-Altogether,thesefindingssuggestthatrTα1-BP5- isanoveladjuvantsuitablefor-H9N2avianinfluenzavaccine.-

**Keywords:** thymosin α1 (Tα1), Bursopentin (BP5), fusion peptide, avian influenza- vaccine, adjuvant-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction-**

 Avianinfluenzavirus-(AIV)isanenvelopedvirusthatbelongstothe-*Orthomyxoviridae* family- andhasaneight-segmented,single-stranded,negative-sense-RNAgenome.-Amongthe- proteinsencodedbythegenome,therearetwosurfaceglycoproteins,hemagglutinin-(HA)- andneuraminidase-(NA)-[1].-AIVsareclassifiedintosubtypesaccordingtothecombination- of-16-HAandnine-NAmolecules.-Amongthemanysubtypesof-AIV,-H9N2isthoughttohave- originatedfromshorebirdsandgulls,andhasrapidlyspreadtobecomeoneofthemost- prevalentdiseasesindomesticpoultryworldwide.-Italsocausesseriouseconomiclossinthe- poultryindustry-[2]-(see-**Table-1**).-



**Table 1.** Abbreviations for full name.-

In domestic avian species in North America, H9N2 influenzaviruses occur primarily in- turkeys, occasionally in quail, and rarely if ever in chickens. The H9N2 virus subtype was first- isolated from turkeys in 1966 [3], when the virus was associated with mild respiratory disease.- In Asia, long-term surveillance in live poultry markets in Hong Kong from 1975 to 1985- detected H9N2 influenzaviruses in apparently healthy ducks but not in chickens [4]. Since the- early 1990s, H9N2 influenzaviruses have become widespread in domestic chickens in Asia [5].- Among the avian influenza-A virus subtypes, H9N2 viruses have the potential to cause an- influenzapandemic because they are widely prevalent in avian species in Asia and have- demonstrated the ability to infect humans [6]. In April 1999, two World Health Organization- reference laboratories independently confirmedthe isolation of avian influenza-A (H9N2)- viruses for the first time in humans [7].-

The best protection against influenzavirus infection remains effective vaccination [8]. Inactivated vaccines have been undergoing clinical trials as pandemic vaccine candidates, and it has- been shown that inactivated vaccines elicit strong humoral responses; however, it is commonly- accepted that no adequate mucosal or cellular immunity is achieved [9]. Adjuvants are able to- improve the quantity and quality of innate immune responses by enhancing their speed and- duration, and by inducing adequate adaptive immunity [10]. To improve methods for- influenzavaccine production, the current strategy of many investigators is to increase the- efficacyof pandemic influenzavaccines by the addition of adjuvants to boost immune- responses, such as aluminum salts, MF59, IC31®, and chitosan [11–14].-

A definedpeptide sequence able to stimulate specificimmune cell subsets has the potential to- act as an adjuvant for a variety of immunogens. The thymus is an important central immune- organ for T-lymphocyte differentiationand maturation [15]. It is capable of secreting many- peptides with the functions of regulating the development of differentphenotypic markersand lymphocyte [16]. Thymosin alpha 1 (Tα1), an immunomodulatory peptide consisting of- 28 amino acid residues, was isolated originally from calf thymus [17]. As a biological response- modifier (BRM), Tα1 has multiple biological activities in the immune system. It can promote- specificlymphocyte functions, stimulate the production of lymphokines such as gamma- interferon (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin 2 (IL-2), macrophage migration inhibitory factor (MIF), and precursor stem cell into the CD4+/CD8+ T cells, increase Tcell proliferation, differentiationand maturation, and so on [18, 19]. Furthermore, it has the- activities of antitumor and protection against oxidative damage [20]. Consequently, Tα1 is- widely used in clinic treating various diseases including immunodeficiencydiseases, severe- sepsis, and systemic infectious disorder [21].-

The bursa of Fabricius (BF) is a primary humoral immune organ unique to birds and is the site- of B-lymphocyte development and differentiation.-The tripeptidebursin (LysHisGlyNH2) has- been described as an endogenous B-cell stimulant or differentiationfactor [22]. BS and bursinlike peptide T-X-N-L-K-H-G significantlyenhance the JEV subtype vaccine-induced immune- response in immunized mice [23]. Bursin-like epitope peptide (BLP) is one of bursin-like- peptides and enhances immune responses in mice immunized with inactivated H9N2 avian- influenzavaccine [24]. Our previous study has been reported that Bursopentin (BP5) is a small- peptide separated from BF, which amino acid sequence is CKDVY. We found that BP5 not only- promotes T-cell and B-cell proliferation, enhances humoral immunity and cellular immunity- but also balances Th1 and Th2 immune responses [25, 26].-

Although both Tα1 and BP5 have the potent adjuvant effects,this study designed and- synthesized Tα1-BP5 fusion gene according to the preferential codons of *Escherichia coli*, fused- with prokaryotic expression vector pET-32a, and then transferred into *E. coli-*BL21 to induce- its expression. Then, we tested whether rTα1-BP5 could enhance immune responses in chicken- upon vaccination with H9N2 avian influenza whole-inactivated virus (WIV).-

#### **2. Materials and methods-**

## **2.1. Plasmid, viruses, and reagents-**

pET-32a (+), *E. coli-*DH5α, *E. coli-*BL21 (DE3), pET32a (+)-BP5, and avian influenzavirus A/- Chicken/Jiangsu/JS-1/2002(H9N2) were maintained in our laboratory. Avian influenzavirus- A/Chicken/Jiangsu/NJ08/05(H9N2) was kindly provided by Dr. Qi-Sheng Zheng. Virus titers- were determined in MDCK cells. H9N2 avian influenzawhole-inactivated virus (WIV) was- prepared by diluting the virus 1:4000 (v/v) in formalin [27, 28]. All restriction enzymes and *Taq-* polymerase were purchased from TakaRa Biotechnology (Dalian, China). RPMI 1640 medium- and fetal bovine serum (FBS) were purchased from Gibco (New York, NY, USA). Isopropyl-βd-thiogalactoside (IPTG), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide- (MTT), TEMED, dNTP, Concanavalin A (ConA), Phorbol-12-myristate-13-acetate (PMA), and- tetramethylbenzidine (TMB) were purchased from Jiu Shi Corporation (Zhengzhou, China).- Horseradish peroxidase (HRP)-conjugated goat anti-mice IgG was obtained from Boshide- Corporation (Wuhan, China). Control Standard Tα1 and BP5 peptides were synthesized byShanghai Science Peptide Biological Technology Co., Ltd. (Shanghai, China), and the purity- was over 95%.-

#### **2.2. Chicken embryos, animals, and vaccines-**

Specificpathogen-free (SPF) Roman chicken and chicken embryos were obtained from the- Henan Experimental Animal Research Center. Avian influenzavirus A/Chicken/Jiangsu/- NJ08/05(H9N2) (107-TCID50/0.1 mL) was inoculated into the allantoic cavities of 10-day-old SPF- chicken embryos; the embryos that died within 24 h were discarded, and the allantoic fluids- were harvested from the infected embryos at 48 h postinfection and inactivated by treatment- with 0.2% formalin. The inactivated virus was emulsifiedwith mineral oil to make an oilformulated inactivated H9N2 AIV vaccine. One dose of the vaccine contained 107-TCID50/0.1- mL, which was equal to it before inactivation. Procedure and test of inactivated vaccine were- described according to OIE Terrestrial Manual 2012 [29].-

#### **2.3. Gene cloning and expression of the recombinant fusion peptide Tα1-BP5-**

Gene of the recombinant fusion peptide thymosin α1-Bursopentin (Tα1-BP5) was designed- according to the preferential codons of *E. coli*and amplifiedby splicing overlap extension- polymerase chain reaction (SOE-PCR) method [30]. Sequences of the primers used for the- synthetic Tα1-BP5 are as follows: F1: 5′-CCG GAA TTC AGC GAC GCT GCT GTT GAC ACT- AGC AGC GAA ATC ACT ACTA AAG ACT TG-3′; F2: 5′-GTT CGG GGT GCTG CCG CCG- CCG CCG TTT TCA GCT TCT TCA ACA ACT TCT TTT TTT TCT TTC AAG TCT TTA GTA- GT-3′; and F3: 5′-GGC GGC GGC GGC AGC TGC AAA AAT GTG TAT TAA GTC GAC- TCG-3′, with *Eco*R I and *SaI-*I site (underlined). The genes of Tα1 and BP5 were connected with- the gene of the GGGGS linker to form the Tα1-BP5 fusion gene. And then, the amplified-DNA- fragment was digested by *Eco*R I and *SaI-*I, and then ligated into the expression vector pET32a.- The ligation mix was transformed into competent DH5α cells and the single bacterial colony- was selected by overnight growth on Luria broth (LB) agar plates containing 100-μg/mL- ampicillin. The obtained recombinant plasmid pET32a-Tα1-BP5 was confirmed by restriction- endonuclease digestion and DNA sequencing. The pET32a-Tα1-BP5 plasmid was transformed- into *E. coli-*BL21 (DE3) for inducing expression. The expression products were examined by- sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). And the recombinant proteins were purifiedon a Ni-affinitychromatography column (Amersham Bioscience- HiTrap chelating HP 5 mL × one column) following the manufacturer's instructions.-

#### **2.4. Activity testing of fusion peptide Tα1-BP5 (rTα1-BP5) in vitro-**

Thymus and spleens from 4 to 6-week-BALB/c mice with (20 ± 2) g were collected aseptically,- put them at 200-mesh stainless screen mesh cells, and gently minced into single cell suspension- with a syringe followed by adding Hank's solution. The red blood cells were removed by- centrifugation at 500 rpm for 5 min. The supernatant was centrifuged at 500 rpm for 5 min.- The obtained pellet was washed with Hank's solution twice. The density of lymphocytes was- adjusted to around 5 × 106cells/mL using RPMI-1640 medium containing 10% FBS. ConA and- PMA were added into thymic lymphocytes and splenic lymphocytes to make the concentrations reach 5 μg/mL and 300 ng/mL, respectively. The two kinds of solutions were subpackaged- into a 96-well plate with 100 μL/well, respectively, and three parallel samples were set for each- well. The plates were incubated in CO2incubator at 37°C for 6 h, followed by adding 100-μL/- well rTα1-BP5 (affinitychromatography purifiedthrough Ni column) with different concentrations (1.25, 2.5, 5.0, 10.0, and 20.0 μg/mL) and continued culturing for 72 h. Control groups- (phosphate-bufferedsaline (PBS), 10.0 μg/mL thioredoxin, 10.0 μg/mL Tα1, and 10.0 μg/mL- BP5) were used following the same procedures. MTT method was used to test the effectof- rTα1-BP5 effecton thymic and splenic lymphocytes proliferation. Relative ratio of cell- proliferation (%) = (experimental group OD570/control group OD570) × 100% [31, 32].-

#### **2.5. Immunization of chickens-**

All animal experiments were approved by the Henan University of Science and Technology- Animal Care and Use Committee.-Twenty-one-day-old SPF Roman chickens were randomly- divided into six experimental groups of 25 chickens each and intramuscularly immunized two- times on days 0 and 14 with (i) 100 μL PBS as a negative control, (ii) 100 μL H9N2 WIV (A/- Chicken/Jiangsu/NJ08/05, 107-TCID50/0.1 mL), (iii) a mixture of 100 μL H9N2 WIV and Tα1 (50- μg), (iv) a mixture of 100 μL H9N2 WIV and BP5 (50 μg), (v) a mixture of 100 μL H9N2 WIV- and rTα1-BP5 (50 μg), and (vi) 100 μL oil-formulated inactivated H9N2 AIV vaccine (A/- Chicken/Jiangsu/NJ08/05, 107 TCID50/0.1 mL) as a positive control (**Table 2**).-


a H9N2 WIV, inactivated H9N2 avian influenza whole-inactivated virus; H9N2 AIV vaccine, H9N2 avian influenzavirus vaccine prepared with oil/water as an adjuvant.-

**Table 2.** Animal groups and the experimental design.-

The details of the animal experiment time points are shown in **Figure 1**.-

## **2.6. Detection of antibodies in serum-**

Chicken (*n* = 5 per group) sera were collected on 7 and 21 days after the firstimmunization.- Serum antibody (HI and antigen-specificantibodies) titers were determined using standard- HI microliter and enzyme-linked immunosorbent assay (ELISA) as described [25, 33]. Briefly,- to detect the HI titers in chicken serum, sera were inactivated by incubation for 30 min at 56°C- and serially diluted twofold in PBS, then transferred in duplicate to 96-well round-bottomedInfluenza Inactive Virus Vaccine with the Fusion Peptide (rTα1-BP5) Enhances Protection Against Influenza... 125 http://dx.doi.org/10.5772/64403

**Figure 1.** Experimental scheme of immunization, sample collection, and challenge.-

plates. Standard avian influenzavirus (A/Chicken/Shandong/6/96(H9N2)) antigen with four- units was then added to each diluted serum samples in a volume of 50 μL, and followed by- an equal volume of 0.5% chicken erythrocyte suspensions. The mixture was incubated for 1 h- at room temperature before the results were read. The HI titers were definedas the highest- serum dilution capable of preventing hemagglutination.-

To evaluate the antigen-specificantibodies titers, ELISA plates were coated with 10-μg/mL- recombinant influenza-HA protein (expressed in *E. coli-*BL21) and blocked with 1% bovine- serum albumin (BSA) for 2 h at 37°C. Aliquots of diluted chicken sera were added to the plates,- which were then incubated overnight, washed, and incubated with HRP-conjugated goat antichicken IgG. Finally, TMB was added and the reaction was stopped by the addition of 2N- H2SO4 and the absorbance was read at OD450. Each serum sample was repeated in quintuplicate.- The results were plottedas OD versus dilution (log scale). Titers at half maximal OD were- determined by linear interpolation [34].-

#### **2.7. Determination of AIV-neutralizing antibodies-**

Inactivated sera were incubated with 100 plaque-forming unit (pfu) of avian influenzavirus- (A/Chicken/Jiangsu/JS-1/2002(H9N2), and the titers of AIV-neutralizing antibodies determined as described [35].-

#### **2.8. Cytokine assays-**

On 7 and 21 days after the firstimmunization, the serum levels of Th1-type cytokine (IFN-γ)- in chickens were determined using commercial Chicken cytokines gamma interferon ELISA- kits (Cusabio Biotech, MD, USA), whereas Th2-type cytokine (IL-4) was determined with- another commercial Chicken cytokines interleukin 4 ELISA kits (Cusabio Biotech, USA). The- procedure followed the manufacturer's instructions.-

## **2.9. Lymphocyte proliferation response-**

To detect changes in cellular immunity, lymphocyte proliferation response was performed.- Thymus and bursa of Fabricius were collected from immunized chickens at 7 and 21 days after- the firstimmunization. The thymus and BF lymphocytes were isolated and maintained in 1640- medium supplemented with 10% FBS at 37°C with 5% CO2. The thymus lymphocytes (5 × 106- cells/mL) were seeded in a 96-well plate and incubated with 50 μL of ConA (40 μg/mL) at 40°C/- 5% CO2for 48 h, whereas the BF lymphocytes (5 × 106cells/mL) were treated with 50 μL of- PMA (1 μg/mL) in a 96-well plate at 40°C/5% CO2for 24 h. Then, the lymphocyte proliferation- assay was performed using a standard MTT method as described previously [36, 37]. Then,- the plate was incubated with 10 μL of 5 mg/mL MTT for 3 h. Finally, 100 μL of 10% (w/v) SDS- in 0.01 M HCl was added into the plate and allowed to incubate for 2 h. A spectrophotometric- measurement was taken at A570.-

#### **2.10. Virus challenge experiment-**

 Two weeks after the second vaccination, chickens (*n-*= 15 per group) were intranasally- challenged with 2.5 × 106-TCID50avian influenza virus A/chicken/Jiangsu/JS-1/2002(H9N2) in- 100 μL PBS. Five chickens per group were humanely sacrificedat 3, 5, and 7 days after virus- challenge and the viral titers in their lungs assessed by plaque formation assays using MDCK- cells as described [35].-

#### **2.11. Statistical analysis-**

Statistical analyses were performed using unpaired *t*-tests or one-way analysis of variance- (ANOVA) *F*-statistics followed by GraphPad Prism 6 software. Data are presented as the mean- ± standard deviation (SD). Turkey multiple comparison tests were used to assess differences- among the five experimental groups, with differences being considered significant at *P-*< 0.05- or *P* < 0.01.-

#### **3. Results-**

#### **3.1. Expression of the recombinant fusion peptide Tα1-BP5-**

The gene of Tα1-BP5 was amplifiedby SOE-PCR with the primers F1, F2, and F3. The PCR- products were identifiedby electrophoresis, and then about 114bp strip was observed. The- recombinant plasmid was extracted and identified with *Hind-*III enzyme. The results showed- that the recombinant plasmid was not digested by *Hind-*III, indicating that the recombinant- plasmid had deleted the *Hind-*III restriction site (**Figure 2A**). Sequencing result showed that- the gene of the recombinant fusion peptide Tα1-BP5 was inserted into pET32a vector, and it- was consistent to the expected size (**Table 3**), which was suggested that the recombinant- expression vector was constructed successfully, and it was named pET32a-Tα1-BP5. Then,- pET32a-Tα1-BP5 was transferred into *E. coli-*BL21 (DE3) for its expression, and the expressed- products were detected by using SDS-PAGE. The result showed that Tα1-BP5 was expressedand purifiedwith the molecular weight of 31.4 kDa (**Figure 2B**), which is consistent with its- predicted molecular weight.-


**Table 3.** Amino acid sequence of recombinant fusion peptide Tα1-BP5.-

**Figure 2.** Identificationof the recombinant plasmid pET32a-Tα1-BP5 and the expression of fusion peptide Tα1-BP5 in- *E. coli*. (A) 1: DL2000 marker; 2: the recombinant plasmid pET32a-Tα1-BP5 after *Hind-*III enzyme digestion; and 3: the- recombinant plasmid pET32a-Tα1-BP5. (B) 1: low molecular weight protein marker; 2: not induced *E. coli-*BL21 (DE3);- 3: Not induced recombinant *E. coli-*BL21 (DE3)/pET32a-Tα1-BP5; 4: induced recombinant *E. coli-*BL21 (DE3)/pET32a-Tα1-BP5.-

#### **3.2. Activity of rTα1-BP5 in vitro-**

The expressed product of TBP5 recombinant bacteria was affinitychromatography purified- through protein Ni column and quantified through spectrophotometer. MTT method was used- to test the effectof rTα1-BP5 on the proliferation of mouse thymic and splenic lymphocytes.- The results showed that all rTα1-BP5 with differentconcentrations (1.25, 2.5, 5.0, 10.0, and 20.0- μg/mL) could promote the proliferation of thymic and splenic lymphocytes compared to PBS- group. rTα1-BP5 could stimulate thymic and splenic lymphocytes proliferation stronger than- TP5 and BP5. The differences were significant (*P-*< 0.05) on the concentrations of 5.0 and 20.0- μg/mL, and the differenceswere more significant (*P-*< 0.01) on the concentrations of 10.0- μg/mL (**Figure 3A**and **B**). All these data demonstrated that rTα1-BP5 could promote the- proliferation of mouse thymic T lymphocytes and splenic B lymphocytes.-

**Figure 3.** The effectsof rTα1-BP5 on the proliferation of thymic lymphocytes (A) or splenic lymphocytes (B) from immunized mice. The data presented are of fivereplicates. \*, *P* < 0.05, compared with mice immunized with PBS, and \*\*,- *P* < 0.01, compared with mice immunized with BP5 or Tα1.-

**Figure 4.** Effectof rTα1-BP5 to H9N2 AIV vaccination on antigen-specific-HI titers and anti-HA IgG antibodies. Chickens were immunized two times, and chicken sera were collected on days 7 and 21 after the firstimmunization, and theserum HI titers (A) and IgG titers (B) were analyzed by HI assay and ELISA, respectively. The data presented aremeans ± SD of results from fivereplicates. \*, *P* < 0.05, and \*\*, *P* < 0.01, compared with chickens immunized with H9N2-WIV alone.-

### **3.3. rTα1-BP5 stimulates significant antigen-specific immune responses-**

To determine antigen-specificimmune responses to immunization, chickens were immunized- two times, then sera were taken on days 7 and 21 after the firstimmunization and detected for- HI and anti-HA antibody titers. HI antibody titers of chickens immunized with inactivated- vaccine, Tα1 combined with H9N2 WIV and BP5 combined with H9N2 WIV increased- significantlycompared with chickens immunized with the H9N2 WIV alone at days 7 and 21- (*P* < 0.05). However, HI antibody titers in chickens immunized with rTα1-BP5 combined with-H9N2 WIV were significantlyhigher than in chicken immunized with BP5 combined withH9N2 WIV at days 7 and 21 (*P* < 0.05) (**Figure 4A**). Anti-HA IgG antibody was observed in- immunized chickens on days 7 and 21 after the firstimmunization. rTα1-BP5 enhanced the- secretion of IgG antibody on day 7 after the firstimmunization, and the effectwas greater than- that induced by H9N2 AIV vaccine, Tα1 combined with H9N2 WIV and BP5 combined with- H9N2 WIV. On day 21 after the first immunization, BP5 significantly enhanced IgG antibody- secretion levels compared with that induced by H9N2 vaccine, Tα1 combined with H9N2 WIV- (*P* < 0.05), while the effectof rTα1-BP5 was the greatest than the other groups (*P* < 0.01) (**Figure- 4B**). These results suggested that rTα1-BP5 stimulates significantantigen-specificimmune- responses.-

#### **3.4. rTα1-BP5 promoted the production of AIV-neutralizing antibody-**

To assess whether rTα1-BP5 can effectively enhance virus-neutralizing antibodies, chicken sera- were collected on days 7 and 21 after the firstimmunization and the titers of AIV-neutralizing- antibody were assessed. The result showed that the titers of neutralizing antibody of chickens- immunized with Tα1 plus H9N2 WIV, BP5 plus H9N2 WIV, and H9N2 AIV vaccine were higher- than that in chickens immunized with H9N2 WIV alone on day 7, while it was higher in- chickens immunized with Tα1-BP5 plus H9N2 WIV than that of other groups. Consistent with- this, AIV-neutralizing antibody titers of chicken injected with rTα1-BP5 plus H9N2 WIV were- the highest on day 21 (**Table 4**). These results indicated that rTα1-BP5 significantly stimulates- the production of AIV-neutralizing antibodies.-


a Chickens were vaccinated on days 0 (first boost) and 14 (second boost). Chicken sera (*n* = 5) were collected on days 7- and 21, and plaque-reducing neutralizing antibody titers were determined. The 50% plaque-reducing neutralizing titer- (PRNT50) was reported as the geometrical reciprocal of the serum dilution resulting in a 50% reduction in plaques. The- data presented are means ± SD of results from five replicates.-

\*, *P* < 0.05, and-

\*\*, *P* < 0.01, compared with chickens immunized with H9N2 WIV alone.-

**Table 4.** Titers of plaque-reducing neutralizing antibody in groups of chicken.-

#### **3.5. rTα1-BP5 increases the production of both Th1- and Th2-type cytokines-**

We then examined the levels of Th1 (IFN-γ) and Th2 (IL-4) cytokines from immunized- chickens. Compared with stimulation with H9N2 WIV alone, both IFN-γ and IL-4 secretion- were remarkably increased after immunization with inactivated H9N2 AIV vaccine, Tα1 plusH9N2 WIV and BP5 plus H9N2 WIV at days 7 and 21, and the highest level of IFN-γ secretionwas observed in the vaccination group with rTα1-BP5 plus H9N2 WIV (*P* < 0.01) (**Figure 5A-** and **B**). Taken together, the results suggested that rTα1-BP5 promoted the secretion of both-Th1 and Th2 cytokines.-

**Figure 5.** Effectof rTα1-BP5 to H9N2 AIV vaccination on cytokine production in chicken sera. Chickens were immunized two times, and chicken sera were collected on days 7 and 21 after the firstimmunization. Cytokine release was- measured by using commercial chicken cytokines gamma interferon (IFN-γ) and interleukin 4 (IL-4) ELISA kits. The- data presented are means ± SD of results from fivereplicates. \*, *P* < 0.05, and \*\*, *P* < 0.01, compared with chickens immunized with H9N2 WIV alone.-

**Figure 6.** rTα1-BP5 significantlystimulates chicken T- and B-lymphocyte proliferation. Chickens were immunized twotimes, and chicken thymus and bursa of Fabricius were collected on days 7 and 21 after the firstimmunization. T- (A)and B (B)-lymphocyte proliferation assays were evaluated by MTT method. The data presented are means ± SD of results from five replicates. \*, *P* < 0.05, and \*\*, *P* < 0.01, compared with chickens immunized with H9N2 WIV alone.-

#### **3.6. rTα1-BP5 significantly enhances T- and B-lymphocyte proliferation-**

To investigate the effectsof rTα1-BP5 on T- and B-lymphocyte proliferation, thymus and BF- were collected from chickens immunized with rTα1-BP5 plus H9N2 WIV. T-lymphocyte- proliferation responses of chickens immunized with Tα1 plus H9N2 WIV, BP5 plus H9N2 WIV,- and H9N2 AIV vaccine were enhanced at 7 days compared with chickens immunized with- H9N2 WIV alone (*P* < 0.05), whereas it was higher for that immunized with rTα1-BP5 plus- H9N2 WIV (*P* < 0.01). On day 21, T-lymphocyte proliferation responses of chickens immunized- with Tα1 plus H9N2 WIV and H9N2 AIV vaccine were higher than chickens immunized with- H9N2 WIV alone (*P* < 0.05), while it was highest for that immunized with rTα1-BP5 plus H9N2- WIV (*P* < 0.01) (**Figure 6A**). Similarly, B-lymphocyte proliferation responses of chickens- immunized with BP5 plus H9N2 WIV and H9N2 AIV vaccine were enhanced at 7 and 21 days- compared with chickens immunized with H9N2 WIV alone (*P* < 0.05), whereas, it was highest- for that immunized with rTα1-BP5 plus H9N2 WIV (*P* < 0.01) (**Figure 6B**). The data indicated- that rTα1-BP5 promoted T- and B-lymphocyte proliferative responses.-

#### **3.7. rTα1-BP5 significantly promotes immune protection against H9N2 AIV challenge-**

To evaluate whether rTα1-BP5 promotes immune protection against H9N2 AIV infection, viral- titers in chicken lungs were evaluated at 3, 5, and 7 days after viral challenge by plaque- formation assays. Chickens immunized with Tα1 plus H9N2 WIV, BP5 plus H9N2 WIV, and- H9N2 AIV vaccine showed significantvirus removal from the lungs at 3, 5, and 7 days after- challenge compared with H9N2 WIV groups (*P* < 0.05), while the viral titers of lungs from- chicken immunized with rTα1-BP5 plus H9N2 WIV were significantlylower than that in the- H9N2 AIV vaccine group (*P* < 0.01). Moreover, chickens immunized with rTα1-BP5 plus H9N2- WIV had almost no detectable virus particles in the lungs 7 days after challenge (**Figure 7A–- C**). The data indicated that rTα1-BP5 significantlypromoted immune protection against H9N2- AIV challenge.-

**Figure 7.** AIV-viral titers of lung in chickens. Lung samples from individual chicken in each group (*n* = 5) were collected on days 3, 5, and 7 post challenge with 2.5 × 106 TCID50avian influenzavirus (A/chicken/Jiangsu/JS-1/2002(H9N2)).- Each lung sample was diluted to 1 mL with 1640 media. The titers are presented as pfu per mL. The data presented are- means ± SD of results from fivereplicates. \*, *P* < 0.05, and \*\*, *P* < 0.01, compared with chickens immunized with H9N2- WIV alone.-

## **4. Discussion-**

In the event of an influenzapandemic, vaccination is one of the most effective ways of- intervention in terms of reducing cost, disease, and even death. Appropriate adjuvant can- enhance the immunogenicity of the vaccine and improve the immune responses [38, 39].- However, most of the adjuvants used in conjugation with antigen have unacceptable levels of- side effects,only a few of them are used clinically [40]. Thus, we need to findnew and optimal- adjuvant candidates for vaccine. In recent years, some small peptide immunostimulants were- reported in use for vaccine adjuvants [41–43]. Both Tα1 and BP5 are associated with immune- regulation. Previous studies showed that both Tα1 and BP5 had high potential as an adjuvant- for vaccines [26, 44].-

In this study, the fusion peptide of rTα1-BP5 was designed and synthesized, and to investigate- it as an adjuvant for inducing immune responses in chickens upon vaccination with inactivated- H9N2 avian influenzavirus (WIV). An effective adjuvant should be able to enhance the levels- of both humoral and cell-mediated immunity. To investigate the effectof rTα1-BP5 on humoral- responses, chickens were immunized with H9N2 WIV combined with Tα1-BP5, and then titers- of HI antibody, antigen-specificantibodies, and AIV-neutralizing antibodies were assessed.- Then, we found that rTα1-BP5 significantlyenhanced HI antibody and antigen-specific-IgG- antibodies titers, promoted the secretion of AIV-neutralizing antibodies, which suggested that- rTα1-BP5 enhanced the levels of humoral immune responses in chickens when it was coimmunized with H9N2 WIV.-

 In addition to humoral immune responses, cellular immunity also plays an important role in- fighting influenza virus infections [45]. The levels of Th1- and Th2-type cytokines are important- references to measure cellular immunity. And lymphocyte homeostasis is required for the- maintenance of normal immune function [46]. Th1-type cytokines mainly include IL-2, TNFα, and IFN-γ, whereas Th2-type cytokines include IL-4, IL-5, and IL-10 [47]. Our study though- analyzed the production of Th1 (IFN-γ)- and Th2 (IL-4)-type cytokines, and T- and B-lymphocytes proliferation in vaccinated chickens post immunization to evaluate the cell-mediated- immunity. The results suggested that rTα1-BP5 promoted the secretion of both Th1 and Th2- cytokines and T- and B-lymphocyte proliferative responses. Overall, this study found that- rTα1-BP5 not only enhanced the humoral immune responses but also promoted the cellmediated immune responses, and it had the potential to use as an adjuvant.-

To further evaluate the influenceof rTα1-BP5 as an adjuvant on the immunity protection- provided by H9N2 AIV vaccine against AIV infection, chickens were intramuscularly challenged with H9N2 AIV (A/chicken/Jiangsu/JS-1/2002) on day 28 post immunization. After 3- days post challenge, the PBS group chickens that received the challenge virus were mildly- depressed. No other clinical signs were observed in that group or any of the other groups,- which is typical of low-pathogenicity AIV in chickens [48, 49]. At 7 days post challenge, only- the PBS-challenged group had mild, grossly detectable lesions in both the respiratory and- gastrointestinal tract. And we found that the viral titers of lungs from chicken immunized with- rTα1-BP5 plus H9N2 WIV were significantly lower than all the other groups at 3 days. Chickens- immunized with rTα1-BP5 plus H9N2 WIV had almost no detectable virus particles in thelungs at 7 days after challenge. Our data indicated that rTα1-BP5 could effectively inhibit the- replication of H9N2 AIV in chickens and promote virus clearance in the lungs of chickens.- Thus, rTα1-BP5 had the potential to be used in vaccine formulations to provide improved- protection against H9N2 AIV infection in poultry.-

In summary, this study demonstrated that inactivated H9N2 AIV vaccine with Tα1-BP5 as an- adjuvant enhanced strong immune responses at both humoral and cellular levels against AIV- infection in chickens. These data may provide a novel insight to findnew adjuvant in vaccines.-

## **Acknowledgements-**

This work was supported by Grant no. 31101792 from the National Natural Science Foundation- of China and no. 2012GGJS-077 from the Foundation for University Key Teacher by Higher- Education of Henan Province.-

#### **Author details-**

Chen Wang1\*, Chengshui Liao1 , Wufan Zhang1 , Deyuan Li2 and Puyan Chen2-

 \*Addressallcorrespondenceto:wangchen2001@126.com-

 1-Key-Laboratoryof-Veterinary-Oncological-Immunology,-Henan-Universityof-Scienceand- Technology,-Luoyang,-China-

2 Key Laboratory of Animal Bacteriology of China's Department of Agriculture, Nanjing- Agricultural University, Nanjing, China-

#C.WANG and C.S.LIAO equally contributed to this work.-

#### **References-**


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evidence that mitotic Bcl-2 phosphorylation is JNK-independent. The Journal of- Biological Chemistry. 2004; 279: 11957-11966. DOI: 10.1074/jbc.M304935200-


pathogenic potential of isolate MS96. Avian diseases. 2000; 44: 527–535. DOI:- 10.2307/1593091-

[49]-Soda K, Asakura S, Okamatsu M, Sakoda Y, Kida H. H9N2 influenzavirus acquires- intravenous pathogenicity on the introduction of a pair of di-basic amino acid residues- at the cleavage site of the hemagglutinin and consecutive passages in chickens. Virology- Journal. 2011; 8: 64. DOI: 10.1186/1743-422X-8-64-

## **Cholesteryl Pullulan Nanoparticles-Encapsulated TNF-α: An Effective Mucosal Vaccine Adjuvant Against Influenza**

## Tsunetaka Ohta

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64366

#### **Abstract-**

 Weencapsulatedtumornecrosisfactor-α-(TNF-α),amajorproinflammatorycytokine,- intocholesterylpullulan-(CHP)toprepare-TNF/CHPnanoparticles.-Inthischapter,the- immuneresponse-enhancingcapabilityofthenanoparticlestoactasavaccineadjuvant- againstinfluenzaisdescribed.-TNF/CHPnanoparticlesshowedexcellentstorage- stability,andtheyenhancedhostimmuneresponsestoexternalimmunogens.-We- appliedthenanoparticlesinamousemodelofinfluenzavirusinfectiontoinvestigate- theiradjuvantability.-Nasaladministrationof-TNF/CHPnanoparticlescombinedwith- aconventionalsplit vaccine was effective atinducing systemic-IgG1 as wellasmucosal- IgA,anditprotectedmiceagainstalethalchallengeof-A/PR/8/34-(H1N1)influenza- virus.-Mechanisticstudiesshowedthatthenanoparticlesenhancedantigenuptakeby- dendriticcells-(DCs)andmoderatelyinducedtheexpressionofinflammation-related- genesinnasal-associatedlymphoidtissue-(NALT),leadingtotheactivationofboth-B- and-Tcells.-Apreliminarysafetystudyrevealednoseveretoxicityto-TNF/CHP- nanoparticles.-Slight-to-moderateinfluencesinnasalmucosawereobservedonlyafter- repeatedadministrationandtheywerereversible.-Ourdatashowthat-TNF/CHP- nanoparticleseffectivelyenhancebothhumoralandcellularimmunityvianasal- administrationandcouldbeapotentialadjuvantforvaccinesagainstinfectiousdiseases- likeinfluenza.-

**Keywords:** adjuvant, mucosal, nanoparticle, CHP, TNF-α-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction-**

 Vaccinesarethemosteffectiveinterventionsagainstinfectiousdiseasessuchasinfluenza.-Manyvaccines,however,areonlyeffectiveatpreventingonsetandaggravationofsymptoms,- andlesseffectiveatpreventinginfection,particularlywithrespiratoryinfections.-Onereason- forthisisthatthemajoradministrationroutesofconventionalvaccines,includingsubcutaneous-(*s.c.*)andintramuscular-(*i.m.*),induceneutralizing-IgGantibodyinbloodbutnot- mucosal-IgAantibody,whichismoreeffectiveatpreventinginfection.-Theefficacyof-IgG- antibodyagainstvariantormutatedvirusesisverylimitedbecauseithashighlyrestricted- cross-protectivecapabilities.-Conversely,-IgAantibodyonmucosashowswidecross-protectionandcanblockinfection-[1,-2].-Whenimmunizationsaredeliveredatthemucosa,-IgA- antibodyisinducedonmucosalsurfacesthroughoutthebodyand-IgGantibodyisproduced- intheblood.-Sincemucosalvaccinationinducesimmunityinboththesystemicandmucosal- compartments-[3,-4],enhancedantigen-specificmucosalimmunityisacleargoalfornextgenerationvaccines.-Mucosal,especiallynasal,vaccinesareidealbecauseoftheireffectiveness- inpreventinginfectionviatherespiratorytract.-Nasalvaccineshavetheadditionalbenefit of- improvedpatientcomplianceandgreaterclinicalconvenienceaswell.-

One significantdrawback of mucosal vaccines is that they generally do not induce strongenough immune responses. The recent component-split vaccines, while avoiding manynegative patient reactions, tend to be less immunogenic by themselves even in the case ofintravenous (*i.v.*) or *i.m.* administration. Generally, children and the elderly tend to respondless to vaccinations, which may lower the preventive power of the population [5]. Therefore,adjuvants must be administered simultaneously with the vaccine in order to enhance vaccinespecificimmune responses. Alum salts are the most commonly used adjuvant, but they areneither suitable for all vaccines nor always capable of eliciting the desired immune responses.-Other types of adjuvants are being tested, such as liposomes, emulsions, and their combinations [6, 7]. The development of safe, effective, and suitable adjuvants is an important component in the future of mucosal vaccines.-

Several groups have examined the use of cytokines (proteinaceous bioactive substances) as anew type of vaccine adjuvant due to their potent effectson the immune system [8–10].-Although some encouraging results have been reported, cytokines are not yet in the practicaluse as adjuvants. One of the important points to consider is the type of drug delivery system-(DDS). Recently, our group tried to generate a new type of vaccine adjuvant by combiningcytokines and biocompatible saccharide materials. This chapter describes the creation ofhuman tumor necrosis factor-α (TNF-α) encapsulated by cholesteryl pullulan (CHP) resultingin TNF/CHP nanoparticles. We investigated the potential of the nanoparticles as a nasal vaccineadjuvant by examining its ability to protect against lethal influenzainfection in a mouse modeland conducting further mechanistic analyses on innate and acquired immunity.-

#### **2. Tumor necrosis factor-α (TNF-α)-**

TNF-α is a major proinflammatorycytokine primarily produced by T cells and macrophages,- and it is bioactive in a homotrimeric form [11]. It was firstdescribed as a potent anti-tumor- factor, but it is now known to play an integral role in host defense. It activates innate and- adaptive immunity by stimulating dendritic cell (DC) maturation and subsequent T cell- activation as well as contributing to inflammatory responses [12, 13] (**Figure 1**).-

Interestingly, it was recently shown that TNF-α exerted adjuvant activities against pathogenic- infections [14, 15]. Although there were some attemptsto develop TNF-α (as well as other- cytokines) as a vaccine adjuvant, successful practical results have not been reported. This is- probably because TNF-α causes unfavorable biological reactions when administered systemically, and it is rapidly degraded when delivered at the mucosal surface. To overcome these- obstacles, some investigators have attemptedto generate protease-resistant mutant TNF-α- molecules and have reported some potential as a vaccine adjuvant at the experimental level- [16, 17].-

#### **3. Pullulan and cholesteryl pullulan (CHP)-**

One method of establishing a safer and more effective way to administer bioactive substances- that is gaining popularity is a nanoparticle DDS. For nanoparticle materials, polysaccharides- have been shown to possess several favorable characteristics in comparison with synthetic- polymers currently used. Unlike synthetic polymers that could accumulate in the body to levels-

**Figure 1.** Various actions of TNF-α.-

beyond the renal clearance, saccharides are biocompatible, meaning they are degraded by- intrinsic enzymes inside the body [18, 19]. In this study, we used pullulan to create DDS- nanoparticles.-

Pullulan is a natural and chemically neutral homopolysaccharide consisting of α-1, 6-linked- maltotriose units (maltotriose is three glucose molecules linked with α-1, 4 glycosidic bonds).- It is produced primarily by fermentation of starch by strains of the fungus *Aureobasidium pullulans* [20]. By introducing hydrophobic moieties onto the hydrophilic pullulan molecule,- amphiphilic coploymers can be generated. A representative is cholesteryl pullulan (CHP) [21,- 22] (**Figure 2**).-

**Figure 2.** Chemical structure of CHP. m, n; integer values. In PUREBRIGHT CP-100T (NOF Co., Tokyo, Japan), 1–3% ofglucose units are modified with cholesterol residues.-

CHP self-assembles into nanoparticles in aqueous solution and entraps various molecules in- its internal space through hydrophobic interactions. The hydrophilic shell serves as a stabilizing interface between the hydrophobic core and the external aqueous environment. It also- protects the entrapped molecule from mechanical, chemical, or enzymatic attacksfrom outside- the particle, and it acts as a superior carrier for delivery. It also allows slow release of the- encapsulated materials [23–25]. Furthermore, the CHP nanoparticles showed prolonged- circulation and thermodynamic stability in animal models [26].-

Cholesteryl Pullulan Nanoparticles-Encapsulated TNF-α: An Effective Mucosal Vaccine Adjuvant Against Influenza 143 http://dx.doi.org/10.5772/64366

**Figure 3.** Schematic model of self-assembling of TNF/CHP nanoparticles. TNF-α is shown in the trimeric, bioactive- form. The diameter of the particle is approximately 20–30 nm.-

Pullulan-based nanoparticles have been used for the delivery of proteins, anticancer drugs,- imaging agents, and nucleotides. CHP nanoparticles are efficientlytransferred to antigenpresenting cells such as macrophages and/or DCs, and they elicit strong immune responses- [27, 28]. CHP is under vigorous investigation for establishing novel vaccine therapies against- several types of cancers [29–31]. We created CHP nanoparticles containing TNF-α, which are- described in the following sections (**Figure 3**).-

#### **4. TNF/CHP nanoparticles-**

## **4.1. Preparation of TNF/CHP nanoparticles-**

In this study, we used TNF-αderived from a human lymphoblastoid cell line, BALL-1 [32],- and CHP (PUREBRIGHT CP-100T) from NOF Corporation. CHP encapsulated active trimeric- TNF-α to form stable nanoparticles as schematically shown in **Figure 3**. The encapsulating- process was time- and temperature-dependent; at 37°C, more than 95% of the TNF-αwas- encapsulated into CHP complexes after 5 days of incubation; at 4°C, very few, if any, TNF/CHP- nanoparticles were formed.-

The resulting nanoparticles were relatively uniform. The mode and average sizes of the- particles were 27.2 and 42.4 nm based on dynamic light scattering-(DLS) results. This was not- very differentfrom the size of the blank CHP particles with a mode of 27.6 nm and an average- of 42.8 nm (**Figure 4**). Stoichiometric analyses showed that a TNF/CHP nanoparticle consisted- of a TNF-α active trimer (ca. 50 kDa) in a CHP tetrameric complex (ca. 400 kDa).-

**Figure 4.** Size distribution of TNF/CHP nanoparticles. Two hundred fiftyμg/mL TNF-α and 12 mg/mL CHP were- mixed, sterilized by filtration,and incubated at 37°C for 5 days. CHP self-assembled with TNF-α molecules to form- nanoparticles. The particle size was determined by DLS with a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern,- UK). (a) TNF/CHP nanoparticles, (b) blank CHP particles. Reproduced with permission from Nagatomo D. et al., [33].-

### **4.2. Storage stability of TNF/CHP nanoparticles-**

Stability of the nanoparticles was evaluated after various treatments by measuring the level of- TNF-α. To estimate the amount of encapsulated TNF-α, methyl-β-cyclodextrin (Me-β-CD) was- used to disrupt the CHP complex and release the TNF-αas previously described [26]. The- results showed that the nanoparticle retained its integrity and kept TNF-α molecules active- inside the complex in aqueous solution at room temperature for at least 21 days (**Figure 5a**).- Furthermore, even after fivecycles of freezing and thawing, 80% of the particles remained- intact (**Figure 5b**). These results show that the TNF/CHP nanoparticles have excellent storage- stability. However, upon contact with high concentrations of dissolved proteins, such as serum- albumin, the nanoparticles rapidly released the encapsulated TNF-α (data not shown),- probably replaced by proteins from the external environment as reported [34]. This calls an- attention to the usage of the nanoparticles, such as *i.v.* injection.-

Me-β-CD is known to interact with cholesteryl groups and disrupt CHP complexes to release- the substance inside the particles [26]. The amount of Me-β-CD required to disrupt the- TNF/CHP nanoparticles was approximately 100 mg/mL, much higher than the 0.3 mg/mL- reported for Interleukin-12 (IL-12)/CHP nanoparticles [35], suggesting that the affinitybetween TNF-α and CHP was much stronger than that of IL-12 and CHP. The molecular- interaction of TNF-αand CHP that creates this strength is an interesting area for further study.-

Many medical formulations, especially biologicals, require storage at low temperatures or- freezing. On the contrary, the TNF/CHP nanoparticles could be stored in solution and without- refrigeration. Our formulation offers improved convenience of handling and transportation.-

**Figure 5.** Stability of TNF/CHP nanoparticles *in vitro*. TNF/CHP nanoparticles were incubated in Dulbecco's phosphate- bufferat 25°C or repeatedly freeze-thawed (−80°C/25°C). An aliquot was examined for the amount of active TNF-α.- Samples were treated with 100 mg/mL Me-β-CD at 37°C for 2 h to release TNF-αfrom the particles. The amount of- TNF-α was determined by an enzyme-linked immunosorbent assay (ELISA) system. (a) storage stability at 25°C; (b)- stability through freeze-thaw cycles. Open circle, treated with Me-β-CD; closed circle, without Me-β-CD. (mean ± SD, *n-* = 3).-

#### **5. Immune responses induced by TNF/CHP nanoparticles administered- nasally-**

 Although TNF-α is known to have immune-enhancing activity [14], severe and unfavorable- effectshave hampered its practical use. Based on the stability described in the previous section,- we hypothesized that delayed release of TNF-α from TNF/CHP nanoparticles would promote- the beneficialeffectsof TNF-α while avoiding harmful events. We examined the adjuvant- activity of the TNF/CHP nanoparticles, for example, enhanced induction of antigen-specific- antibodies in mice, particularly in the case of nasal administration. We used a commercial- influenzavirus hemagglutinin vaccine (IVV), which is a component-split and trivalent vaccine- for seasonal influenza.-It consists of the inactivated hemagglutinin (HA) antigens from A/- Brisbane/59/2007 (H1N1), A/Uruguay/716/2007 (H3N2), and B/Brisbane/60/2008. The nasally- administered TNF/CHP nanoparticles combined with the IVV induced significantlevels ofIgA in the nasal wash, as well as IgG1in blood plasma (**Figure 6a, b**). These are comparable tothose of the positive control cholera toxin B subunit (CTB), the most powerful adjuvant inexperimental settings [35]. Furthermore, IVV with CHP alone (without TNF-α) or with free-TNF-α failed to induce significantlevels of antibodies when compared to IVV with noadjuvant. The TNF/CHP nanoparticles alone did not induce a measurable antibody responseagainst IVV. To further examine antigen specificity, we performed hemagglutinin (HA)-specifichemagglutination inhibition (HI) assay for the differenttypes of influenzavirus included inthe vaccine. The TNF/CHP nanoparticles with IVV induced significant-HI activity against alltypes of HA used (A/H1N1, A/H3N2, and B) (**Figure 6c**).-

**Figure 6.** Adjuvant effectsof TNF/CHP nanoparticles administered nasally. BALB/c mice were nasally given IVV-(SEIKEN, Denka Seiken Co., Ltd., Japan) (0.3 μg/mouse) and TNF/CHP nanoparticles (5 μg/mouse of TNF-α) or CTB-(0.8 μg/mouse) once a week for 4 weeks. The nasal wash and blood plasma were prepared from the mice, and the levels of IVV-specific-IgA and IgG1were determined by ELISA. (a) IgA levels in nasal wash, (b) IgG1levels in blood plasma, (c) HI titer in blood plasma against different HA types of influenza virus expressed in GMT (geometric mean titer).-Blue column, type A/H1N1; red column, type A/H3N2; green column, type B. (mean ± SEM, n = 8). \*, *P* < 0.05 vs. saline/IVV; #, *P* < 0.05 vs. free TNF/IVV. Adapted with permission from Nagatomo D. et al., [33].-

These data indicate that TNF/CHP nanoparticles administered nasally can induce not only- mucosal but also systemic immunity significantly and efficiently, comparable to the effects of- CTB. In addition, the nasal vaccination covers a broad range of antigenicity as previous reports- suggested [1, 2]. Also, just for reference, the induction of specificantibodies was seen for other- antigens, such as Hepatitis virus type A vaccine, diphtheria toxoid, and cedar pollen allergen- (data not shown). Those suggest that TNF/CHP nanoparticles have the potential as a vaccine- adjuvant with a broad range of applications, as well as influenza.-

**Figure 7.** Proliferation and cytokine production by splenocytes from mice nasally administered TNF/CHP nanoparticles and IVV. BALB/c mice were given IVV (0.3 μg/mouse) and TNF/CHP nanoparticles (5 μg/mouse as TNF-α) or CTB- (0.8 μg/mouse) by the nasal route as previously described. Splenocytes were prepared from the mice. Then, IVV-specificproliferation and IL-4/IFN-γ-producing cells were examined with alamarBlue and ELISpot, respectively. The results- represent the difference-(Δ) between data from experiments with and without IVV antigen stimulation. (a) proliferation response. (b) cytokine production. Blue column, IL-4 production; red column, IFN-γproduction (mean ± SEM, n =- 8). \*, *P* < 0.05 vs. saline/IVV. Adapted with permission from Nagatomo D. et al. [33].-

The antigen-specific-T cell responses of the vaccinated animals were examined by challenging- their isolated splenocytes with IVV. IVV-specificproliferation in the TNF/CHP nanoparticlegroup was comparable to that of CTB (**Figure 7a**). We also measured cytokine production tounderstand what kind of T cell response occurred. IVV alone increased IFN-γ production,while the addition of either adjuvant (TNF/CHP nanoparticle or CTB) suppressed IVV-induced-Interferon-γ(IFN-γ) production. The TNF/CHP nanoparticles combined with IVV alsoproduced IL-4 cytokine to a higher level than IVV with CTB (**Figure 7b**). These experimentssuggest that the nasally administered adjuvant shifted the Th1/Th2 balance to a Th2-dominantstate, which confirms previous results obtained with a mutant TNF-α [17].-

#### **6. Protective effect of TNF/CHP nanoparticles in lethal challenge of- influenza virus on mice-**

To directly address the stimulatory effectof the TNF/CHP nanoparticle adjuvant on protective- immunity, we challenged immunized mice with a lethal dose of influenzavirus. Mice were- nasally immunized with IVV with or without TNF/CHP nanoparticles once a week for 3 weeks.- Then, they were challenged with the antigenically distinct influenzavirus A/Puerto Rico/8/34- strain at a lethal dose (**Figure 8**). The mice that received only IVV died by 8 days post challenge,- which was comparable to mice without IVV immunization. The TNF/CHP nanoparticles- without the IVV slightly delayed the time to death, but all of the animals eventually died. On- the contrary, combined administration of IVV and TNF/CHP nanoparticles showed a highly- protective effectwith 90% of the mice surviving lethal challenge. The effectwas comparable- to that of the CTB adjuvant. Free TNF also showed a somewhat protective effect.-Interestingly,-

**Figure 8.** Protective effectof TNF/CHP nanoparticles adjuvant against lethal influenzavirus challenge in mice. BALB/cmice were nasally administered with IVV with or without an adjuvant once a week for 3 weeks. Seven days after thefinalimmunization, mice were challenged nasally with influenzavirus (Puerto Rico/8/34, 10 LD50) and then monitoreddaily. Mice that were moribund or that had lost more than 20% of their body weight were considered to have reachedan experimental endpoint and were humanely euthanized by anesthetization. Blue diamond, saline only; red square,-TNF/CHP nanoparticles only (5 μg/mouse of TNF-α); green triangle, IVV only (0.3 μg/mouse); purple cross, IVV withblank CHP nanoparticles (240 μg/mouse); blue square, IVV with TNF/CHP nanoparticles (5 μg/mouse of TNF-α); orange circle, IVV with free TNF (5 μg/mouse); purple dot, IVV with CTB (0.8 μg/mouse) (n = 10). Reproduced with permission from Nagatomo D. et al. [33].-

CHP only (without TNF-α) provided a certain level of protection as an adjuvant, as we- observed 50% survival. Importantly, the nasally administered TNF/CHP nanoparticles- induced significant protective immunity in spite of the distinct antigenicities [36], suggesting- that they have a potential for inducing broad cross-protection.-

Antibodies became detectable after the second or third vaccination and reached plateau levels- thereafter in mice vaccinated with TNF/CHP nanoparticles. The surviving animals immunized- with IVV and TNF/CHP nanoparticles had immunological memory, including IgG1in plasma,- and IgA in nasal/vaginal wash and feces. This memory was maintained at high levels for more- than 90 days, and these mice responded to a boosting challenge of the IVV to further elevate- the antibody levels (**Table 1**). These data indicate that the nanoparticles induced systemic- immunity and long-lived memory, a critical feature for successful vaccine adjuvants. Overall,- our data demonstrate that TNF/CHP nanoparticles are effectiveas a vaccine adjuvant for- nasally delivered IVV.-

TNF/CHP nanoparticles enhanced an IgA response not only at the site of application (e.g., in- the nasal wash) but also at distant mucosal sites, such as the intestine (feces), vaginal, and- salivary glands (data not shown). IgA antibody elicited at the mucosa is of vital importance as- the natural route of infection for influenzais via the respiratory mucosa. Hence, local mucosal- protection against pharyngeal carriage is likely to be decisive for preventing disease [37].- Conventional parenteral vaccines are not able to stimulate mucosal immune responses, thus- restricting their efficacyin infections of mucosal surfaces such as the respiratory tract [1]. Our- nasal vaccine/adjuvant formulation consisting of the IVV and TNF/CHP nanoparticles- effectively induced both systemic and mucosal protective immunity.-


BALB/c mice were nasally administered with IVV (0.3 μg/mouse), with or without TNF/CHP nanoparticles (5 μg/- mouse of TNF-α) or CTB (0.8 μg/mouse) once a week for 4 weeks. Ninety-one days after the immunization, the mice- were boosted with IVV (0.9 μg/mouse). Twenty-one days after the boosting challenge, the blood plasma was prepared- and the feces were extracted with 10 volumes of water. The IgG1 in blood plasma and IgA in feces extract were- examined (mean ± SEM, *n* = 8).- n.d., not detected.-

**Table 1.** Induction of immunological memory by TNF/CHP nanoparticles.-

Muraoka et al. proposed that CHP-based nanoparticles preferentially deliver antigen to- antigen-presenting cells in the lymph nodes, which potentiates effective immune responses- [38]. This might be the reason why the TNF/CHP nanoparticles induced excellent protective- immunity. They, however, reported that CHP itself did not show an adjuvant effectin the- context of a tumor vaccine [31]. Interestingly, CHP only (without TNF-α) showed a certain- level of protective efficacyin our study. The reason for the discrepancy between their results- and ours is not clear. It is unlikely, given the short time frame, that TNF-α was replaced *in vivo-* by IVV antigens to form IVV/CHP nanoparticles that were then delivered to the lymph node.- Another factor that is critical for adjuvant activity is particle size [39]; however, size is not an- issue in our case since the DLS analyses showed no differencein particle size between the TNF/- CHP nanoparticles and empty CHP particles (**Figure 4**). Most probably, the differencehas to- do with varying mechanisms to elicit protection against external pathogens, such as influenza- virus, and internal antigens, such as tumor antigen.-

#### **7. Mechanistic analyses of effects of TNF/CHP nanoparticles-**

## **7.1. Activation of immune cells in NALT-**

Being focused on the nasal route of vaccination, we examined immune cells in nasal tissues- after immunization. The mucosal surfaces contain abundant B cells, T cells, and plasma (or- DC) cells. After repeated immunization of animals for 3 weeks, cells from the nasal-associated- lymphoid tissues (NALT) were prepared from mice, and expression of a surface marker for- DCs (CD11c) and activation markers for B cells (CD80 and CD86) were examined by flow- cytometry. The ratios of CD80+ /CD11c+cells; CD86+ /CD11c+cells were 0.20; 0.33, 0.30; 0.44, and- 0.33; 0.50% for saline, IVV, and IVV with TNF/CHP nanoparticles, respectively (data not- shown). Even though the degree was small, IVV vaccination with or without TNF/CHP- nanoparticles activated DCs and B cells. Also, TNF/CHP nanoparticles did not have a prominent effect on DC or B cell activation.-

## **7.2. Antigen uptake and activation of NALT and nasal passage cells-**

We next focused on the early immune response of nasal mucosal tissues. Uptake of antigen by- the mucosal tissues is essential for the induction of immune responses [40]. Therefore, we- examined antigen uptake by NALT-resident and nasal passage DCs, the inductive sites of the- common mucosal immune system [41]. In these experiments, we used ovalbumin (OVA) as a- model antigen and assessed antigen uptake by DCs in the NALT and nasal passage cells by- flowcytometry at 6 h after immunization. Immunization of mice with OVA combined with- TNF/CHP nanoparticle activated antigen uptake by both NALT and nasal passage DCs. TNF/- CHP nanoparticles stimulated DCs most in the nasal passage mucosal immune tissue- (**Figure 9**). We also found that TNF/CHP nanoparticles enhanced expression of DC and B cell- activation markers (CD40, 80, and 86) in a bone marrow-derived immature DC preparation *in- vitro* (data not shown).-

Cholesteryl Pullulan Nanoparticles-Encapsulated TNF-α: An Effective Mucosal Vaccine Adjuvant Against Influenza 151 http://dx.doi.org/10.5772/64366

**Figure 9.** Antigen uptake of NALT and nasal passage DCs after TNF/CHP nanoparticles administration. BALB/c micewere nasally immunized with 10 μg of Alexa 647-labeled OVA antigen with or without TNF/CHP nanoparticles as anadjuvant. Six hours after the immunization the NALT and the nasal passage cells were prepared [42] and subjected toflowcytometric analysis. Antigen uptake was determined by detecting the Alexa 647 florescenceintensity and the DCmarker (CD11c+cells) in parallel, for example, ratio of Alexa 647+ /CD11c+ . (a) NALT DCs, (b) nasal passage DCs. MFI,mean fluorescence intensity (mean ± SD, n = 4). Adapted with permission from Nagatomo D. et al. [33].-

## **7.3. Expression of inflammatory signals in NALT-**

Vaccine adjuvants trigger the innate immune system allowing enhanced humoral and cellularresponses against the co-administered vaccine antigens. To understand innate immune systemactivation caused by the TNF/CHP nanoparticles after immunization, we conducted geneexpression profilingin NALT cells 2, 6, and 26 h after nasal administration of IVV antigen with- or without the nanoparticles. By scatteringanalyses, the gene expression of inflammation- andimmunity-related molecules was found to be significantlyupregulated (data not shown).-These included the triggering receptor expressed on myeloid cells 1, fibronectin-1, CD14, Tolllike receptor (TLR) 2, TLR3, IL-1β, IL-1 family 9, and IL-6. We confirmedthe level of inflammation-relatedmolecules in activated NALT cells by quantitative polymerase chain reaction-(PCR) analysis. Expression of the inflammatorymarkers was enhanced when an adjuvant wasincluded (TNF/CHP nanoparticles, free TNF, or CTB), while CHP itself did not show significantenhancing activity. Among the molecules tested, significantincreases in expression occurred- for IFN-γ, IL-1α, IL-1β, IL-6, CXCL2, IL-12β, CD14, and lipopolysaccharide (LPS)-binding- protein (**Figure 10**). The degree of enhancement varied from gene to gene, but the greatest- increase of expression was observed for IL-6 and IL-12β.-

Overall, free exogenous TNF-α elicited the strongest increase in expression of inflammatory- markers, and the enhancement tended to be highest at 2 h post-immunization. TNF/CHP- nanoparticles elicited a moderate increase in gene expression by comparison, but the pattern- over time was similar to that of free TNF-α. One reason for the discrepancy in gene expression- between free TNF and the nanoparticles could be that the nanoparticles cause a slow release- of TNF-α, prolonging the immune-stimulatory effect.-The patternof expression over time was- very differentfor most genes when stimulated with CTB adjuvant. For example, although the- expression of IL-12βwas prominent at 6 h post-immunization when CTB was used as an- adjuvant, the IL-12β response was much lower with TNF/CHP nanoparticles at the same time- point.-

 Taken together, TNF/CHP nanoparticles delayed activation of innate immunity. By prolonging- and dampening the stimulatory effectof TNF-α-CHP seemed to minimize the unfavorable- effectsof TNF-αwhile promoting its beneficialactivities. One important safety issue related- to the development of nasal vaccines is the potential dissemination of vaccine antigens to the- central nervous system (CNS). Past reports suggested that nasal administration of CTB allowed- it to reach the CNS and accumulate in olfactory tissues. It caused Bell's Palsy in clinical studies,- probably due to IL-12β production, and the use of CTB in humans was prohibited [43, 44]. In- this context, the lower expression level of IL-12βseen in our experiments with TNF/CHP- nanoparticles could be a beneficial safety feature.-

Regarding CHP itself, a shell of the nanoparticles did not show immune-enhancing activity,- such as increasing IgG1and IgA or the expression of inflammation-related genes (**Figure 10**).- However, CHP did confer a certain level of protection in a lethal influenzavirus challenge- (**Figure 8**). We cannot easily account for these conflictingobservations; there are other pathways and mechanisms likely involved and waiting to be clarified.-

#### **8. Safety study of TNF/CHP nanoparticles-**

General safety was preliminarily examined according to the OECD guidelines for testing- chemicals [45]. Mice nasally administered with a combination of TNF/CHP nanoparticles and- IVV either once or four times were subjected to an acute or a repeated toxicity study, respectively.-

#### **8.1. General symptoms, ophthalmic examination, body weight, and body temperature-**

No general symptoms or behavioral anomalies in either male or female animals were correlated with the IVV with TNF/CHP nanoparticles treatment during the study period. In- ophthalmic examinations, there were no test material-related ocular findings observed. Body-

**Figure 10.** Gene expression in NALT after TNF/CHP nanoparticles administration. mRNA was prepared from NALT- cells of BALB/c mice 2, 6, and 26 h after administration of TNF/CHP nanoparticles and IVV. Gene expression related to- innate and adaptive immune responses was analyzed by quantitative PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Data are shown as relative level vs. control (means of quadruple experiments). Red column, 2 h; orange column, 6 h; yellow column, 26 h post treatment. \*, *P* < 0.05; \*\*, *P* < 0.01; \*\*\*, *P* < 0.001- vs. saline control. Adapted with permission from Nagatomo D. et al. [33].-

weight and body temperature were not statistically different among the treatment groups (data- not shown).-

## **8.2. Hematology, blood biochemistry, and urinalysis-**

Hematology evaluations were performed during and at the end of study. There were no- differencesin any of the tested parameters (white blood cell, red blood cell, hematocrit,- lymphocyte, neutrophil, eosinophil, basophil, and monocyte) between controls and TNF/CHP- nanoparticles with the IVV treatment. For blood biochemistry, we examined total protein,- albumin, urea nitrogen, creatinine, Na+ , K+ , Cl− , Ca2+, inorganic phosphate, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), amylase- (AMY), γ-glutamyl transpeptidase (γ-GT), total cholesterol, triglyceride, high density lipoprotein (HDL)-cholesterol, total bilirubin, and glucose. Urobilinogen, bilirubin, ketone body,- glucose, protein, pH, specificgravity, nitrite salt, and leucocytes were examined during the- study period. All differencesfound during the study fell within historical control value ranges- and were not considered test material-related (data not shown).-

## **8.3. Pathology and major organ weights-**

Gross pathology of all the animals was examined at the end of the study. The major organs- (brain, heart, lung, kidney, liver, ovary, testis, spleen, adrenal, and thymus) were weighed at- the end of the study in all of the animals. No change was noted as test material-related (data- not shown).-

## **8.4. Histopathology-**

The histopathology of tissues from animals in each group was examined. As discussed in- Section 7.3, there were some concerns of possible harmful effectson the CNS. However, no- abnormalities in the brain, especially olfactory bulb, were noted in any animals after histological examination. There were some effectsobserved at the administration site (nasal mucosal- tissue). While single administration showed no effect,repeated administration with IVV alone- showed a slight infusion and TNF/CHP nanoparticles combined with IVV induced slight-tomoderate infusion and infiltrationof inflammatorycells (lymphocytes, neutrophils, eosinophils, and mast cells). However, the response was reversed with time since the infusion- diminished to trace proportions after the 2-week cessation period. No excessive inflammatory- symptoms, such as formation of edema or fibrosis, were noted (data not shown).-

Overall, no obvious immunotoxicity was detected. Although further evaluation is required,- our results demonstrate that the toxicity of TNF/CHP nanoparticles is relatively low and safe- as a nasal vaccine adjuvant against influenza.-

Very recently, Onishi et al. reported that hydroxypropyl-β-cyclodextrin (HP-β-CD), another- type of saccharide-based material that can form nanoparticles, exhibited adjuvant activity and- elicited a strong protective effectagainst influenzavirus in mice and cynomolgus macaques- [46]. They suggested the involvement of follicular helper T cells via myeloid differentiation- primary gene 88 (MyD88)- and TANK-binding kinase (TBK)-dependent pathways. Their- findingsmay shed some light on additional mechanisms at play with nanoparticles as vaccine- adjuvants. However, they mentioned the cytotoxicity of HP-β-CD at more than 0.5% *in vitro*,- probably because of β-CD's ability to extract cholesterol out of cell membranes [47]. TNF/CHP- nanoparticles might represent a preferable alternative.-

A probable precaution for the practical use of TNF/CHP nanoparticles is avoiding contact with- high concentrations of dissolved proteins as mentioned in Section 4.2. Unless, encapsulated- TNF-α would be released from the nanoparticles and the adjuvant activity would be diminished. This means that the nanoparticles is not suitable for *i.v*. administration and premixed- formulation with vaccine antigen. For the best performance, we recommend to administer the- TNF/CHP nanoparticles on mucosa, such as nasal surface, after mixing with the vaccine- antigen just before use.-

## **9. Conclusions-**

The results of this study demonstrate that TNF/CHP nanoparticles are effective as a vaccine- adjuvant against influenzawhen administered via the nasal mucosal route. Moreover, the- ability of TNF/CHP nanoparticles to stimulate comparatively balanced systemic and mucosal- immune responses makes them a potentially promising vaccine adjuvant for inducing- immunity against infectious pathogens. In the short term, TNF/CHP nanoparticles may aid- the development of new nasal influenzavaccines. Looking further ahead, we propose that- combining TNF/CHP nanoparticles with next-generation vaccine platforms that do not rely- on the cold chain will offer valuable alternatives for vaccination in a variety of settings.-

#### **Acknowledgements-**

The author is very grateful to Dr. Ayato Takada of Research Center for Zoonosis Control,- Hokkaido University for his expertise with influenzachallenge of mice. The author is also- thankful to the staffof R&D Center, Hayashibara Co., Ltd. for their excellent technical- assistance, especially Mr. Daiki Nagatomo and Ms. Madoka Taniai for the mechanistic analyses- of action, and Dr. Shigeyuki Arai for the histopathological examination.-

#### **Author details-**

Tsunetaka Ohta-

 Addressallcorrespondenceto:tsunetaka.ota@hb.nagase.co.jp-

 R&D-Center,-Hayashibara-Co.-Ltd.,-Okayama,-Japan-

## **References**


cytidine-phosphate-guanosine DNA drives T cell activation *in vitro*and therapeutic- anti-tumor immune responses *in vivo*. J. Immunol. 2000;165:6278–6286.-


poly(I:C) combined with mucosal vaccine protects against influenza virus infection. J.- Virol. 2005;79:2910–2919.-


## **Development of Vaccines for Poultry Against H5 Avian Influenza Based on Turkey Herpesvirus Vector**

Atsushi Yasuda, Motoyuki Esaki,

Kristi Moore Dorsey, Zoltan Penzes, Vilmos Palya,

Darrell R. Kapczynski and Yannick Gardin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64348

## **Abstract-**

 Avianinfluenza-(AI)remainsamajorthreattopublichealthaswellastothepoultry- industry.-AIvaccinesareconsideredasuitabletooltosupport-AIcontrolprogramsin- combinationwithothercontrolmeasuressuchasgoodbiosecurityandmonitoring- programs.-Weconstructedrecombinantturkeyherpesvirus-(HVT)vectorvaccines- expressingthehemagglutiningeneof-AIvirus-H5subtype-(rHVT‐H5)andevaluated- theircharacteristicsandefficacyagainst-AI.-Wefoundthatthecytomegalovirus-(CMV)- promoteristhemostsuitableforexpressionofthehemagglutiningeneamongthree- promotersweevaluated.-TherHVT‐H5vaccinedidnotcauseanyadversereactionsand- didnotreverttovirulenceafterpassagesinchicken.-Finally,efficacyoftherHVT‐H5- vaccinewasevaluated.-Wedemonstratedthatitprovidedprotectionagainstdiverse-AI- H5virusesbelongingtodifferentcladesandreducedvirussheddingfromthe- challengedchicken.-WealsoprovedthatefficacyprovidedbytherHVT‐H5vaccinewas- notsignificantlyaffectedbypresenceofmaternallyderivedantibodies-(MDA)against- AIvirus.-Furthermore,therHVT‐H5vaccinecouldbeapplicabletothedifferentiating- infectedfromvaccinatedanimals-(DIVA)strategy.-Insummary,wesuccessfully- developeda-HVTvector-AIvaccinethatpossessesfeaturesthatcouldbebeneficialto- AIcontrol.-

**Keywords:** avian influenza, turkey herpesvirus, vector vaccines, DIVA, hemagglutinin- gene-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction-**

 Avianinfluenza-(AI)isanimportantzoonoticdiseaseanditremainsamajorthreattopublic- healthandtothepoultryindustry.-Thehighlypathogenic-(HP)-H5N1outbreakswerefirst- reportedin-Chinain-1996andthenspreadtootherpartsoftheworld.-These-HPavian- influenzaviruses-(AIV)havebecomeendemicinseveralcountriesincluding-China,-Indone‐ sia,-Vietnam,and-Egypt-[1].-Between-2003and-2015,-846confirmedhumancasesof-AI-(H5N1)- havebeenreportedand-449ofthosehumanpatientshavedied-(WHO,-2016).-Mostrecently,- HP-H5N2and-H5N8virusescausedoutbreaksinthe-United-Statesfrom-December-2014to- June-2015,whichresultedindepopulationofmorethan-48millionchickensandturkeys.- Theseoutbreakscostfarmers,thegovernment,andconsumersinthe-United-Statesbillions- ofdollars.-Francehasalsobeenhitby-HP-H5virusessince-November-2015andtheoutbreaks- havecausedsignificantdamagestoitspoultryindustry.-Theserecent-HP-H5-AIVcontinued- toevolveintovariouscladesasdefinedbythe-World-Health-Organization-(WHO)/World- Organisationfor-Animal-Health-(OIE)/Foodand-Agriculture-Organization-(FAO)-H5N1- Evolution-Working-Groupaccordingtophylogenetictopologybasedon*hemagglutinin* (HA)- genesequences-[2].-Anothersignificanteventof-AIistheseriesofhumaninfectionsin-China- causedby-H7N9-AIVsince-March-2013.-Althoughthesevirusesarelowpathogenic-(LP)for- poultry,-277humandeathshavebeenreportedoutof-693confirmedcases-(WHO,-2016).-These- casesemphasizeimportanceofcontrolling-AIVinpoultryfortheindustryaswellasforpublic- health.-

Control of HP AI in poultry has been achieved traditionally through (1) education, (2)biosecurity, (3) diagnostics and surveillance, and (4) elimination of infected poultry (stamping‐ out) [3]. Viruses were successfully eradicated through a combination of those measures inmany countries affected by HP AIV. However, in endemically infected countries, viruses hadspread widely before executing effective measures, and therefore, it was impossible to identifyand eliminate all of infected birds. In such endemic situations, vaccines are considered suitableand powerful tools to support AI eradication or control programs in combination with othercontrol measures such as good biosecurity and monitoring programs [4–6]. When usedproperly, vaccines for AI have been demonstrated to protect poultry against clinical signs andmortality, increase resistance to infection, and reduce virus shedding markedly, thus decreas‐ ing the possibility of virus spreading among birds [7]. Most frequently used AI vaccines havebeen oil‐adjuvanted, inactivated whole‐virus vaccines and fowlpox virus or Newcastle diseasevirus (NDV)‐vectored vaccines are also available. However, efficacyof these vaccines is limitedespecially against antigenically distant viruses [8–10]. Also, efficacyof these vaccines is knownto be severely impaired by maternally derived antibodies (MDA) [11, 12]. Furthermore, oil‐ adjuvanted, inactivated whole‐virus vaccines impede virus surveillance programs based onserology because serological responses elicited by the inactivated whole‐virus vaccines areindistinguishable to those elicited by live fieldviruses. Therefore, development of novelvaccines which are efficaciousin the face of MDA and are compatible with so‐called differen‐ tiate infected from vaccinated animals (DIVA) strategy is necessary.-

Turkey herpesvirus (HVT), or the meleagrid herpesvirus 1, belongs to the family of *Herpesvir‐ idae*, the subfamily of *Alphaherpesvirinae*, and the genus *Mardivirus*. It is classifiedas part of the- Marek's disease virus (MDV) group and designated as serotype 3 MDV. HVT is non‐oncogenic- and antigenically related to oncogenic serotype 1 MDV. The virus has been utilized extensively- as a vaccine against Marek's disease for over 30 years and considered extremely safe [13, 14].- It is administered at hatcheries either to day‐of‐age chicks by subcutaneous (SQ) route or in- ovo to chicken embryos at 18–19 days of incubation and is known to elicit strong cell‐mediated- immunity as well as humoral immunity [15–17]. Furthermore, since HVT becomes latent and- persists in inoculated chickens [18], a longer period of protection is expected. Indeed, HVT‐ vectored Newcastle disease vaccine has been shown to be efficaciousfor 72 weeks after- inoculation into day‐of‐age chicks [19]. For aforementioned reasons and also because of its- large genome that can be potentially used for insertion of heterologous genes [20], HVT has- been evaluated as vectors expressing protective antigen gene(s) of various poultry pathogens- including NDV, infectious bursal disease virus (IBDV), and infectious laryngotracheitis virus- (ILTV) [21–26]. These HVT vector vaccines have shown to be very safe and induce effective- humoral and cellular immunity that is long lasting. Also, the efficacy of HVT vector vaccines- does not appear to be excessively affectedby the presence of MDA, probably because HVT- replicates in a cell‐associated manner [22]. Here, we intended to apply the HVT vector- technology to develop an effective poultry vaccine against AI. Several HVT vector vaccines- expressing *HA* gene of H5 AIV (rHVT‐H5) were constructed and evaluated for their charac‐ teristics and efficacy against AI.-

#### **2. Construction of rHVT‐H5 vaccines-**

#### **2.1. Construction of recombinant viruses-**

A number of HVT vector vaccines have been constructed and evaluated for their characteristics- and efficacyagainst avian pathogens [21–26]. There are three important elements in HVT- vector vaccines that can impact efficacyof these vaccines in chicken: insertion sites, antigen- genes, and promoters. Several insertion sites including US2, US10, UL39 [21], and an intergenic- region between UL45 and UL46 (UL45/46) [24–26] have been evaluated. Out of these potential- insertion sites, we demonstrated that insertion of extraneous genes at the UL45/46 site did not- alter their capacity to replicate [26]. Furthermore, using the UL45/46 as the insertion site, we- were successful in constructing HVT vector vaccines having antigen genes of NDV, IBDV, or- ILTV that are highly efficaciousagainst these pathogens [24–26]. Therefore, we decided to use- the UL45/46 site for insertion of antigen genes of AIV.-

Eight segments of genomic RNA of influenza-A virus encode three membrane‐associated- proteins, HA, neuraminidase (NA), and Matrix (M) 2, fiveinternal proteins, nucleoprotein- (NP), PB1, PB2, PA, and M1, and two nonstructural (NS) proteins, NS1 and NS2. Out of these- viral proteins, the HA surface glycoprotein is known to be the major antigen and elicits- neutralizing antibodies that provide protection against the disease [27]. We used the *HA* gene- cloned from LP A/turkey/Wisconsin/68 (H5N9) strain or HP A/Swan/Hungary/4999/2006(H5N1) clade 2.2 strain. The cleavage site of the *HA* gene from A/Swan/Hungary/4999/2006- (H5N1) strain was altered to a typical cleavage site sequence of LP AIV strains.-

Selection of promoters that control the expression of antigen genes is also an important factor.- Tsukamoto et al. compared the cytomegalovirus (CMV) promoter and CMV/chicken β‐actin- chimera (Pec) promoter for expression of IBDV *VP2* gene in HVT vectors and found that the- Pec promoter expressed more VP2 protein and provided superior protection against challenge- with IBDV [24]. In MDV serotype 1 (MDV1) vectors expressing NDV *fusion* gene, less potent- MDV glycoprotein B promoter gave betterprotection than the simian virus 40 (SV40) late- promoter when tested in chickens with MDA [28]. Another group compared fivedifferent- promoters for expression of the *HA* gene of AIV H9N2 subtype in MDV vectors and found- that two MDV endogenous promoters (pp38 and gB) with relatively low expression activities- provided betterprotection than the other three promoters (CMV, SV40, and p1.8 kb) [29]. In- this experiment, to findthe most appropriate promoter to express the *HA* gene of AIV H5- subtype in HVT vectors, we compared three differentpromoters: the CMV promoter, the- chicken β‐actin (Bac) promoter, and the Pec promoter.-

To prepare for construction of recombinant HVT, HVT insertion site sequences (*UL45* and *UL46* genes) were isolated by polymerase chain reaction (PCR) and cloned into pUC18. Homology- plasmids were then constructed by inserting the *HA* gene with the promoter into the insertion- site (between *UL45* gene and *UL46* gene) of the HVT sequences. The homology plasmid along- with HVT infectious genomic DNA was transfected into chicken embryo fibroblasts-(CEF),- where homologous recombination took place. Recombinant HVT with the inserted gene was- identifiedby an in situ immunostaining assay called black plaque assay (BPA) using anti‐HA- (H5) antibodies. Recombinant HVT was purifiedfrom HVT parent through several rounds of- screening process by the BPA.-

**Figure 1.** Genomic structure of rHVT‐H5 vaccines.-

## **2.2. In vitro characterization of rHVT‐H5-**

**Figure 1**shows genomic structures of the constructed rHVT‐H5 vaccines. The genomic- structures of the rHVT‐H5 vaccines were confirmedby PCR assays with one primer binding- to the insert sequence and the other primer binding to the HVT insertion site sequences (data- not shown). The genomic structures were further confirmedby Southern blot analysis usingDevelopment of Vaccines for Poultry Against H5 Avian Influenza Based on Turkey Herpesvirus Vector 165 http://dx.doi.org/10.5772/64348

**Figure 2.** (A) Gene structure of rHVT/H5 and expected annealing site of the HA probe. (B) Result of Southern blot us‐ ing the HA probe. Lane 1 = DNA molecular weight marker II, DIG‐labeled (Roche); Lane 2 = uninoculated CEF control;-Lane 3-= HVT parent; Lane 4 = rHVT/H5; Lane 5 = rHVT/H5 passage 5; Lane 6 = homology plasmid. (C) Gene structureof rHVT/H5 with CMV promoter and HVT parent and expected annealing site of the 45/46 probe. (D) Result of South‐ ern blot using the 45/46 probe. Lane 1 = DNA molecular weight marker II, DIG‐labeled (Roche); Lane 2 = uninoculated- CEF control; Lane 3 = HVT parent; Lane 4 = rHVT/H5 with CMV promoter; Lane 5 = rHVT/H5 with CMV promoter- passage 5; Lane 6 = homology plasmid.-

**Figure 3.** Black plaque assay on a rHVT/H5 plaque detecting expression of the HA protein of AIV H5 subtype. CEFmonolayer infected with rHVT/H5 was incubated for 5 days and fixedwith methanol:acetone. The monolayer withrHVT/H5 plaques was reacted with chicken anti‐AIV HA serum, then with biotinylated anti‐chicken IgG antibody andfinallywith streptavidin‐alkaline phosphatase conjugates. Plaques expressing HA protein were stained by addition of-BCIP/NBT solution.-

 digoxigenin‐labeled probes specificto the *HA* gene or HVT insertion site sequences (**Fig‐ ure 2**). Expression of the HA protein by the rHVT‐H5 vaccines was confirmedby BPA usingthe chicken anti‐HA serum (**Figure 3**). Western blot analysis using the chicken anti‐HA serumdetected a 75‐kDa band with CEF infected with the rHVT‐H5 (**Figure 4**). This band correspondsto non‐cleaved HA protein that is produced by the rHVT‐H5. Since the cleavage sites of theexpressed HA proteins are those of LP AIV strains, the HA protein was not cleaved to HA1and HA2 subunits in CEF.-

**Figure 4.** Western blot assay detecting expression of the HA protein by rHVT/H5. Lane 1 = Precision Plus Protein All- Blue Standards (Bio‐Rad Laboratories); Lane 2-= CEF control; Lane 3 = HVT FC126 parent strain; Lane 4 = rHVT/H5. An- arrow indicates the HA protein with a molecular weight of 75 kDa.-

In order to assess genetic and phenotypic stability of the rHVT‐H5 vaccines, the viruses were- passed in CEF 20 times. Viruses after the passages were characterized by PCR, Southern blot,- BPA, and western blot. Results obtained with the passed viruses were identical to those- obtained with the viruses before the passage (data not shown), and therefore, it was concluded- that these viruses were genetically and phenotypically stable.-

## **2.3. Evaluation of promoters for expression of HA gene-**

In order to identify the most suitable promoter among the CMV promoter, the Bac promoter,- and the Pec promoter for expression of the *HA* gene, we compared three rHVT‐H5 vaccines- harboring the *HA* gene from A/turkey/Wisconsin/68 (H5N9) strain with one of these promot‐ ers. The rHVT‐H5 vaccines with the CMV promoter, the Bac promoter, and the Pec promoter- were designated as HVT‐CMV‐H5Wis68, HVT‐Bac‐H5Wis68, and HVT‐Pec‐H5Wis68, respec‐ tively.-

One‐day‐old specificpathogen free (SPF) White Leghorn chicks were vaccinated subcutane‐ ously with one of the rHVT‐H5 vaccines. A group of chickens was held as a non‐inoculated- negative control group and another group of chickens was vaccinated subcutaneously with- inactivated A/turkey/Wisconsin/68 (H5N9) vaccine at 3 weeks of age as an inactivated vaccine- control group. Chickens in each group were bled each week between 3 and 7 weeks of age (6- and 7 weeks of age for the inactivated vaccine control group) and obtained sera were evaluated- by the AIV hemagglutination inhibition (HI) test and AIV enzyme‐linked immunosorbent- assay (ELISA). The AIV HI tests were conducted using four hemagglutination units of an- inactivated AIV homologous antigen of the A/turkey/Wisconsin/68 (H5N9) strain according- to the standard procedure [30].-

**Figure 5.** HI titers in chickens vaccinated with rHVT‐H5 vaccines with different promoters.-

**Figure 6.** ELISA titers in chickens vaccinated with three rHVT‐H5 vaccines using (A) FlockChek™ AIV Ab kit (Idexx-Laboratories) and (B) ProFLOK® AIV Ab test kit (Zoetis).-

As shown in **Figure 5**, the rHVT‐H5 vaccines induced increased HI titers as early as 3 weeks- of age and the increased titers were maintained through 7 weeks of age. Chickens vaccinated- with HVT‐CMV‐H5Wis68 had higher mean HI titers than HVT‐Pec‐H5Wis68 and HVT‐Bac‐ H5Wis68 vaccinated groups had between 4 and 7 weeks of age, with the differencesstatistically- significant at 5 and 6 weeks of age. As expected, the inactivated A/turkey/Wisconsin/68 (H5N9)- vaccine induced high HI titers at 6 and 7 weeks of age (3 and 4 weeks post‐vaccination),- confirmingvalidity of the assay. When tested with the commercial AIV ELISA kits, Flock‐ Chek™-AIV Ab kit (Idexx Laboratories) and ProFLOK®-AIV Ab test kit (Zoetis), sera collected- from the rHVT‐H5 vaccinated chickens were negative between 3 and 7 weeks of age, whereas- sera collected from the inactivated vaccine control chickens showed highly positive ELISA- titers with both kits (**Figure 6**).-

This result demonstrated that the CMV promoter was the most suitable for expression of the- *HA* gene in HVT vector vaccines. Furthermore, sera from the rHVT‐H5 vaccinated chickens- were found to be negative by the commercially available ELISA kits, although they were highly- positive by the AIV HI test. This is most likely because these ELISA kits are designed to detect- antibodies to more conserved internal protein, NP, of AIV, in order to detect antibodies to many- differentsubtypes of AIV. This feature of the rHVT‐H5 vaccines will be useful in differentiating- chicken infected with field AIV viruses from vaccinated chicken (DIVA).-

## **3. Safety of rHVT‐H5 vaccine-**

 Based on the results described above, the CMV promoter was selected for the expression of- the *HA* gene. The rHVT‐H5 vaccine with the CMV promoter attachedto the *HA* gene from A/- Swan/Hungary/4999/2006 (H5N1) strain (rHVT‐H5h) was evaluated further for its safety and- efficacy.-

Safety of the rHVT‐H5h vaccine was evaluated by an overdose study and a backpassage study.- For the overdose study, SPF embryos at 18 days of incubation or day‐of‐age SPF chicks received- rHVT‐H5h at 10 times the typical field dose. In ovo application of rHVT‐H5h at overdose did- not affecthatchability and the vaccinated chickens remained free from any clinical signs or- adverse reactions until 18 weeks of age. Chickens were necropsied at 18 weeks of age and no- gross lesions were observed in any of the vaccinated chickens. Similarly, rHVT‐H5h did not- cause any clinical signs, adverse reactions, or gross lesions in other avian species such as- turkeys, quail, pheasants, and pigeons.-

For the backpassage study, to confirmthat the rHVT‐H5h vaccine will not revert to virulence,- rHVT‐H5h was passed fivetimes in SPF chickens by using heparinized blood from chickens- in the previous passage to inoculate a new set of SPF chicks. Chickens inoculated with the virus- at the fifthpassage, that is, inoculated with the heparinized blood from the fourth passage,- were observed closely for any clinical signs for 45 days. Chickens were then necropsied and- observed for grossly observable lesions. No chickens had any clinical signs, adverse reactions,- or gross lesions. Therefore, it was concluded that rHVT‐H5h did not revert to virulence after- backpassages in chickens.-

We conducted another study to evaluate the ability of the rHVT‐H5h vaccine to transmit from- vaccinated chickens to non‐vaccinated chickens in contact. Day‐of‐age SPF chickens vaccinated- in ovo with rHVT‐H5h or parental HVT were commingled in isolators with non‐vaccinated- contact chickens. Virus isolation was attemptedfrom the peripheral mononuclear blood- lymphocytes collected from chickens at 10, 14, and 21 days of age. No virus was isolated from- any of the contact chickens at any time points while the viruses were isolated from the- vaccinated chickens, indicating that neither rHVT‐H5h nor parental HVT spread from- vaccinated chickens to non‐vaccinated contact chickens. It appears that transmissibility of HVT- parent and the rHVT‐H5h vaccine between chickens is negligible.-

In summary, similar to parent HVT vaccines and other HVT vector vaccines, the rHVT‐H5h- vaccine is extremely safe causing no adverse effectsand does not revert to virulence after- passages in chickens. The lack of transmission of the rHVT‐H5h vaccine further strengthens- the safety profileof this vaccine in terms of containment of the genetically modifiedorganism.-

## **4. Efficacy of rHVT‐H5 vaccine-**

## **4.1. Efficacy against homologous challenge-**

Finally, efficacyof the rHVT‐H5h vaccine was evaluated. After vaccination, humoral immune- response was evaluated by the AIV HI tests. After challenge with HP AIV, vaccine efficacy was- evaluated for (1) protection against mortality and clinical signs and (2) reduction of challenge- virus shedding from challenged birds. Both the criteria are important for AI vaccines because- these features will ensure that the vaccine will be beneficialas a measure to contain spread of- field-AI viruses as well as to reduce economical burdens in both endemic and emergent- situations [4, 31].-

 **Figure 7.** Individual HI titers (log2) and standard error for bird groups at 5 weeks post‐vaccination (prechallenge). Stat‐ istical significancebetween mean titers was determined with ANOVA using the Tukey's multiple comparison test (*p* <  0.05). NS = no significantdifference.-Modifiedfrom "Vaccine protection of chickens against antigenically diverse H5- highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene- derived from a clade 2.2 avian influenza virus" by D.R. Kapczynski et al., 2015, Vaccine, 33, p1200.-

Initial evaluation of the rHVT‐H5h vaccine efficacywas conducted using homologous AIV asa challenge virus [32]. Day‐of‐age SPF chicks were vaccinated subcutaneously with either afrozen, cell‐associated (ca) form or a lyophilized, cell‐free (cf) form of the rHVT‐H5h vaccine.- At 5 weeks of age before challenge, increased HI titers were observed in all the rHVT‐H5hvaccinated chicken with mean titers of 26.3 for the ca group and 25.1 for the cf group (**Figure 7**)when using homologous antigen.-

Chickens were challenged with 106mean embryo infectious dose (EID50) HP H5N1 AIV A/- Whooper Swan/Mongolia/3/2005 clade 2.2 strain at 6 weeks of age. The *HA* gene from the A/- Whooper Swan/Mongolia/3/2005 strain has 100% gene homology with the A/Swan/Hungary/- 4999/2006 (H5N1) *HA* gene sequence. All chickens in the diluent‐vaccinated, challenge control- group died within 3 days. However, none of the rHVT‐H5h vaccinated chickens succumbed- to challenge and all the chickens were free from clinical signs of AI.-

 **Figure 8.** Viral titers from oral (A) and cloacal swabs (B) on day 2 post‐challenge. Birds were vaccinated with a singledose rHVT‐H5h in cell‐associated (ca) or cell‐free (cf) form at 1 day‐of‐age and challenge at 6 weeks with homologous-HPAI H5N1. Viral titers are expressed as log10EID50per milliliter. The lower limit of detection is 0.9 log10EID50per milli‐ liter. Statistical significancebetween mean titers was determined with ANOVA using Tukey's multiple comparison test-(*p* < 0.05). NS = no significantdifference.-Modifiedfrom "Vaccine protection of chickens against antigenically diverse-H5 highly pathogenic avian influenzaisolates with a live HVT vector vaccine expressing the influenzahemagglutiningene derived from a clade 2.2 avian influenza virus" by D.R. Kapczynski et al., 2015, Vaccine, 33, p1200.-

We observed 3–6 log10reduction of challenge virus shedding in the rHVT‐H5h vaccinated- chickens. From oropharyngeal swabs at 2 days post‐challenge (dpc), only 4/30 (13%) chickens- in the ca rHVT‐H5h group and 3/20 (15%) chickens in the cf rHVT‐H5h group shed virus with- minimal virus titers (101 –103-EID50/ml), while all the challenge controls shed significant- amounts (105 –107 EID50/ml) of virus (**Figure 8A**). From cloacal swabs, no virus was isolated from- any of the rHVT‐H5h vaccinated chickens at 2 dpc, while all the challenge controls shed virus- at 102 –105 EID50/ml (**Figure 8B**).-

These results demonstrated high potential of the rHVT‐H5h vaccine as an excellent tool to- support control of AI. Therefore, we proceeded to further evaluation of the rHVT‐H5h,- especially for protection against heterologous virus challenge and effectsof MDA. Since the- ca form appeared to provide slightly betterimmunogenicity than the cf form, the following- evaluation was conducted using the ca form of the vaccine.-

#### **4.2. Efficacy against heterologous challenge-**

Since HP H5 viruses have become diverse and have evolved into various differentclades, it is- highly important that AI vaccines exert broad "cross‐clade" efficacyagainst diverse AIV- strains. Indeed, one of the limitations of conventional oil‐adjuvanted, inactivated vaccines is- that they are not as effective against heterologous viruses as homologous viruses [8–10].- Therefore, we tested efficacy of the rHVT‐H5h vaccine against various AIV HP H5 strains.-

Day‐of‐age SPF broiler chicks were vaccinated with the rHVT‐H5h vaccine. A commercially- available inactivated vaccine based on H5N2 Mexican strain (iH5N2) was used as a control- and injected into birds at 10 days of age. Challenge was conducted at 4 weeks of age using the- Indonesian A/chicken/WestJava Sbg/29/2007 H5N1 strain (CW07). The CW07 virus belongs to- clade 2.1.3 and sequence similarity of the HA gene between the rHVT‐H5h insert and the CW07- virus was 93%. After challenge, all chickens in the sham‐vaccinated, challenge control group- died within 2 days. In the group vaccinated with rHVT‐H5h, 80% (16/20) of the chickens- survived the challenge, while only one bird (5%) vaccinated with iH5N2 survived the chal‐ lenge. When rHVT‐H5h vaccinated chickens received boost with iH5N2 vaccine at 10 days of- age, protection increased to 90% (18/20). Reduction in virus shedding up to 3 log10 was observed- in rHVT‐H5h vaccinated chickens compared to the challenge control. When the homologous- antigen was used, rHVT‐H5h, either with or without boost with iH5N2 vaccine, it induced- average HI titers of 25 –26prior to challenge. HI titers were much lower with the heterologous- CW07 antigen with average between 21and 22 . These results indicate that cell‐mediated- immunity as well as mucosal immunity provided by HVT vector vaccines might be involved- in protective efficacy against heterologous CW07 strain.-

In the next trial, another AIV HP H5 strain, A/Viet Nam/1203/04 (H5N1) (VN04), was used for- challenge. The VN04 virus belongs to clade 1 and shares 96.5% HA gene similarity with the- rHVT‐H5h insert. Vaccination was conducted at the day of age to SPF layer chicks and- challenge was conducted at 4 weeks of age. Protection was 85% (17/20) while all challenge- control chickens died and reduction of virus shedding of 3 log10was observed in the rHVT‐ H5h group.-

Since these two trials using heterologous HP H5 strains demonstrated that the rHVT‐H5h- provided "cross‐clade" efficacy,we went further and conducted another efficacytrial using- Mexican H5N2 HP strain which shares only 82% HA gene similarity with the rHVT‐H5h insert.- Day‐of‐age chicks vaccinated with rHVT‐H5h were challenged with A/chicken/Queretaro/- 14588‐19/95 (H5N2) strain at 4 weeks of age. Nineteen out of 20 (95%) chickens survived the- challenge, while all the challenge control died. This result demonstrated very broad cross‐ protective efficacy provided by rHVT‐H5h.-

We further evaluated efficacyof the rHVT‐H5h vaccine against various HP AIV H5 isolates- including several Egyptian isolates and a recent 2014 H5N8 isolate from Germany, as sum‐ marized in **Figure 9**. Protection in rHVT‐H5h vaccinated chickens ranged between 60 and 100%- with significantreduction in virus shedding, further strengthening evidence of very broad- "cross‐clade" efficacy provided by the rHVT‐H5h vaccine. In one study where layer chickens- were vaccinated and raised under fieldconditions in Egypt, a single rHVT‐H5h vaccination at- day of age conferred a high level of protection (60–73%) for a relatively extended period (up- to 19 weeks of age) against an Egyptian isolate [33].-


**Figure 9.** Summary of challenge experiments conducted with the rHVT‐H5 vaccine. Modifiedfrom "Experimental and- field results regarding immunity induced by a recombinant HVT‐H5 vector vaccine against H5N1 and other H5 Highly- Pathogenic Avian Influenza Virus challenge." by Y. Gardin et al., 2016, Avian Dis, in press.-

To see if cellular immunity indeed is involved in this broad protective efficacy, we conducted- in vitro cytotoxicity assay. Splenic lymphocytes collected from chickens vaccinated with the- rHVT‐H5h vaccine were incubated with chicken lung cells infected with either H5N9, H6N2,- H7N2, or H9N2 low pathogenic AIV. The highest level of lysis by splenic cytotoxic T cells was- observed with H5‐infected target cells. Lysis was also observed with other heterologous AIV- (H6, H7, or H9), although to a lower degree. Negligible lysis was observed with naïve unin‐ fected lung cells. These results indicated that the rHVT‐H5h vaccine induced AIV‐specific- cytotoxic T‐cell activity and it may contribute in part to broad protective immunity induced- by the rHVT‐H5h.-

#### **4.3. Efficacy of rHVT‐H5 in chicken with maternally derived antibodies-**

In endemic countries, most breeders are vaccinated with AI vaccines and/or exposed to field- AIV challenge and therefore, their progeny possess MDA against AIV. Efficacyof oil‐adju‐ vanted, inactivated AIV vaccines and fowlpox‐vectored AI vaccines has been shown to be- significantlyimpaired in the presence of MDA. On the other hands, HVT vector vaccines have- been shown not to be excessively affectedby the presence of MDA against inserted antigens.- Indeed, with the rHVT‐H5h vaccine, several studies demonstrated lack of significant interfer‐ ence on its protective efficacyby the presence of MDA when administered to day‐of‐age chicks- [10, 34].-

## **4.4. DIVA-**

AI surveillance is conducted through serological assays including ELISA and HI. Except in- endemic countries, positive serological response in those assays will lead to immediate and- extreme actions including "stamping‐out." Also, there are trade implications because many- countries ban importation of poultry from AI‐positive countries. Therefore, when AI vaccina‐ tion is introduced, it is critical that AI vaccines do not interfere with the AI surveillance. It is- highly favorable that serological responses elicited by AI vaccines may be distinguished from- those elicited by infection of field AIV. Since conventional inactivated vaccines elicit humoral- immune responses that lead to positive titers in both ELISA and HI tests, these vaccines do- interfere with the surveillance [7].-

The rHVT‐H5h vaccine elicits antibodies against the HA protein and the antibody responses- can be detected by the HI tests. However, those sera from vaccinated chickens were negative- in commercial ELISA kits because the ELISA kits are designed to detect antibodies against- more conserved internal protein (NP) in order to offercoverage over differentsubtypes of AIV.- When we examined serological responses in chickens that were vaccinated with the rHVT‐H5h- and then challenged with HP AIV, we found positive ELISA titers in chickens that excreted- challenge viruses. These results demonstrated that the rHVT‐H5h vaccines may be applied to- the DIVA strategy and do not interfere with AI surveillance.-

## **5. Conclusions-**

Our studies demonstrated that the rHVT‐H5h vaccine possesses characteristics that could be- beneficialto control of AI in endemic countries and in emergency situations. Those character‐ istics are (1) broad "cross‐clade" protective efficacyagainst diverse AIV H5 isolates, (2) lack of- interference by MDA, (3) applicability to hatchery vaccination, and (4) applicability to DIVA.- The rHVT‐H5h vaccine has been approved by authorities in Egypt, Mexico, and Bangladesh- and is in use in the field.-An independent survey conducted by FAO, General Organization for- Veterinary Services in Egypt, and Centre International de Recherche en Agriculture pour le- Développement in France concluded that day‐of‐age vaccination utilizing the rHVT‐H5h- vaccine at hatcheries is more efficientthan the program using the inactivated vaccines at farms- and it would have a positive impact for disease control in Egypt [35].-

It is clear that vaccines alone cannot solve all the problems associated with AI. However, we- believe that in conjunction with active and efficientsurveillance and strict biosecurity meas‐ ures, the rHVT‐H5h vaccine can contribute to disease control by increasing resistance against- infection and decreasing the amount of virus shed to the environment. It would also remove- economical burdens from farmers and consumers and improve animal welfare by protecting- chickens from mortality and clinical signs. In conclusion, we successfully developed a HVT- vector AI vaccine that possesses many features that could be beneficialto AI control. It remains- to be seen whether this vaccine is truly useful in the field.-

## **Author details-**

Atsushi Yasuda1\*, Motoyuki Esaki1 , Kristi Moore Dorsey2 , Zoltan Penzes3 , Vilmos Palya3 ,- Darrell R. Kapczynski4 and Yannick Gardin5-


2 Ceva Animal Health, Lenexa, Kansas, USA-

3 Ceva Animal Health, Budapest, Hungary-

4 Exotic and Emerging Avian Viral Disease Research Unit, Southeast Poultry Research Labo‐ ratory, U.S. National Poultry Research Center, Athens, Georgia, USA-

5 Ceva Animal Health, Libourne, France-

## **References-**


**Response to Vaccines** 

## **Maternal Influenza: Infection, Vaccination, and Compelling Questions**

## Jill M. Manske

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64368

## **Abstract-**

 Influenzapresentsasignificantriskforincreasedmorbidityandmortalitytopregnant- womenandinfantsbasedonevidencefrompreviousinfluenzapandemics,seasonal- epidemics,andtherecent-H1N1pandemic.-Since-2004,influenzavaccinehasbeen- recommendedforpregnantwomenduringanytrimesterofpregnancytoreducethis- risk.-Thischapterpresentsanoverviewofinfluenzarisksassociatedwithpregnancy- aswellasareviewofourcurrentunderstandingofvaccineeffectivenessinpregnant- women,neonates,andyounginfants.-Inaddition,someofthecurrentcompelling- questionsrelatedtoinfluenzariskandpreventionacrossalltrimestersofpregnancy- areexplored.-

**Keywords:** influenza vaccine, pregnancy, effectiveness, maternal immunization-

#### **1. Introduction-**

 Influenzaisahighlycontagious,acuterespiratoryinfection-(ARI).-Eachyear,itisestimated- that-5–10%ofadultsand-20–30%ofchildrenworldwidewillbecomeinfectedwithinfluenza- [1].-Pregnantwomenandinfantsareatparticularriskforinfluenza.-Influenzainfection- duringpregnancyorduringthefirst-6monthsoflifeisasubstantialcauseofmorbidity.- Preventionofinfluenzainthesepopulationsisofglobalhealthimportance.-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Maternal influenza-**

Pregnant women are considered a high-risk group for serious illness and complications from- influenza.-While annual influenzaincidence rates in pregnant women are similar to those of- nonpregnant women [2–4], influenzainfection is associated with increased morbidity and- mortality in this subpopulation, with pregnant women having an increased risk of influenzaattributed hospitalizations compared to nonpregnant women [4, 5]. Most likely this is associated with the interaction of the infection with the physiologic and immunologic changes that- occur during pregnancy.-

Pregnancy-associated changes such as decreased lung capacity, reduced tidal volume, and- increased cardiac output likely reduce the capacity of the respiratory and cardiac system to- respond to the stress of influenzainfection [2, 6]. In addition, during pregnancy there is a- progressive suppression of cellular (T-cell mediated) immunity. While this immune suppression serves to protect the developing fetus from maternal cytotoxic T-cell immunity, it can- impair the maternal response to viral infections such as influenza [7–11].-

The combination of these factors contributes to an increased risk of negative outcomes from- influenzainfection. If respiratory disease develops, especially during the later stages of- pregnancy, it can lead to high morbidity among the pregnant women [6, 12–14]. In a study of- hospital admissions records of women admittedbetween 1994 and 2000 with respiratory- conditions during pregnancy, the hospitalization rate was 150/100,000, an admission rate- considerably higher than that of nonpregnant women (17/100,000) and corresponding to the- rate for people aged 65–69 [5]. Healthy pregnant women ages 20–34 were estimated to be 18- times more likely to be hospitalized for influenza than their nonpregnant peers [5].-

While hospitalization rates are increased in all trimesters of pregnancy [4, 15, 16], they are the- highest during the third trimester [5, 14, 16–18]. Likewise, there is a strong association of- maternal morbidity with this trimester of pregnancy. In a 2003 study, Hartert et al. showed that- cardiovascular hospitalization during influenzaseason increased with each trimester, nearly- threefold higher by the third trimester compared to the first trimester [18]. In a study of 8323- healthy pregnant and postpartum women, Lindsay et al. found that the strength of association- between influenzaexposure and influenza-likeillness (ILI) increased as the stage of pregnancy- progressed, reporting an odds ratio (OR) of 1.12 (CI, 0.79–1.59) during the first trimester, 1.30- (CI, 0.97–1.73) during the second trimester, and 1.84 (CI, 1.31–2.59) for the third trimester [14].- Other studies have reported that by the third trimester, healthy pregnant women with no- comorbidity have the same risk for ILI-associated hospitalization as nonpregnant women with- chronic or comorbid conditions [4, 5].-

Pregnant women with comorbid conditions such as asthma, diabetes, heart disease, or chronic- obstructive pulmonary disease are even more likely to be hospitalized than are pregnant- women without chronic conditions [5, 14, 16–18]. Neuzil et al. reported event rates for influenza- for low-risk women of 3, 6, and 10 per 10,000 women months in the first,second, and third- trimesters, respectively. The event rate in nonpregnant women was 2 per 10,000 women- months. Among women with chronic comorbid conditions, rates of 31, 16, and 21 per 10,000- women months were observed during these trimesters, respectively [4].-

While considerable data demonstrate increased morbidity and hospitalization from seasonal- influenzain pregnant women, mortality appears to be rare in healthy pregnant women during- non-pandemic seasons [7, 19, 20]. A study of seasonal influenzaamong pregnant women over- an 8-year period reported an average of fivedeaths per year and a mean mortality ratio of 2.9- per million live births [21]. However, during pandemics, influenzainfection presents a- significant increased risk of both morbidity and mortality in pregnant women.-

For example, during the pandemic of 1918, pneumonia was reported in 50% of previously- healthy pregnant women, leading to case-fatality rates of over 50% [22–26]. In the 1957- pandemic, 50% of the women of childbearing age who died of influenzawere pregnant.- Furthermore, 10% of all influenzadeaths during this pandemic occurred in pregnant women,- with the majority occurring during the third trimester [23, 24].-

Increased rates of morbidity and mortality in pregnant women also were observed more- recently during the 2009 influenza-A(H1N1)pdm09 pandemic. In fact, the firstreported death- of an adult in the United States during this pandemic was a pregnant woman [27]. Of the 45- deaths reported early in the pandemic, 6 (13.3%) were pregnant women, all of whom developed- viral pneumonia and respiratory distress syndrome [27–29].-

A study of pregnant US women with confirmed or probable influenza during the first month- of the outbreak reported 11/34 cases (32.4%) resulted in hospitalization—admissions rates four- times higher than those in the general population [30]. Deaths were reported in all three- trimesters and were independent of preexisting risk factors. In a California study, pregnant- women who were hospitalized with or died from pH1N1 were less likely than nonpregnant- women to have a predisposing or comorbid medical condition [31].-

A review of published studies following the 2009 pandemic documented that pregnant women- were disproportionately represented among hospitalizations, ICU admissions, and deaths.- The 120 papers that were included in the review reported 3110 pregnant women from 29- countries with A(H1N1)pdm09 influenzainfection, including 1625 (52.3%) who were hospitalized with 2009 H1N1, of whom 378 (23.3%) were admittedto an ICU and 130 (8%) died [32].- Pooling the data from all of the studies included in the review, the authors reported that- pregnant women, who represent approximately 1% of the population of United States and- Australia, accounted for 6.3% of hospitalizations, 5.9% of ICU admissions, and 5.7% of deaths- [32].-

 Using data from the Centers for Disease Control (CDC) and the Pregnancy Mortality Surveillance System, Callaghan et al. estimated the total burden of pregnancy-related mortality- resulting from the 2009 to 2010 pandemic. Confirmedand possible deaths resulting from- A(H1N1)pdm09 infection represented the leading cause of pregnancy-related mortality in the- United States between the months of April 2009 and June 2010. Of 915 total pregnancy-related- deaths during this period, 12% of pregnancy-related deaths were attributedto influenza,-75- (8.2%) classifiedas confirmedinfluenza-A(H1N1)pdm09 deaths, and 34 (3.7%) classifiedas- possible influenza infection deaths [33]. The authors calculated the pregnancy-related morality- ratio for confirmedand possible influenzadeaths at 2.2 per 100,000 live births. This represents- a significant burden of mortality. The number of deaths (109) during the 2009–2010 influenzaseason was 20 times greater than the mean number (5) of annual possible influenzadeaths- reported in a 1998–2005 cohort of pregnancy-related deaths in non-pandemic years [33].-

Infants born to influenza-infectedwomen during the pandemic also experienced increased- risks of poor clinical outcomes, mostly due to preterm birth. Reports of preterm birth rates- ranged from 15 to 30% among infected women [32, 34–36]. Infection with A(H1N1)pdm09 was- associated with increased risk of cesarean delivery [32]. In most cases, cesarean delivery was- an attemptto improve worsening maternal status rather than out of concern for the infant [32].- Siston et al. reported a cesarean delivery rate of 58% in pregnant women with 2009 H1N1- compared to a baseline cesarean rate of 30.5% [32, 34, 37]. While cesarean deliveries were- commonly described, it is likely that this rate was over-reported, since many of the studies- reported only severely affectedwomen. Many deliveries were emergencies and performed- outside of controlled operating room settings,indicating the urgent nature of these deliveries- and the critical status of the women [32].-

#### **3. Influenza infection and fetal development-**

While there is no clear consensus on transplacental transmission of influenzavirus or direct- viral effectson the fetus [2], the spread of virus beyond the respiratory tract during acute- infection is unusual, and vertical transmission, although documented [38–40], appears to be- rare [41, 42]. However, even in the absence of vertical transmission, adverse fetal effectscan- occur, most likely due to the systemic maternal immune response to the infection [43, 44].- Studies have documented inflammatoryresponses in fetal tissues in response to influenza- infection. Such responses could impact the maternal-fetal interface, the placenta, or the fetus- directly, leading to pregnancy loss [43]. In addition to direct damage from inflammatory- responses, it is hypothesized that maternal hyperthermia can result in adverse fetal outcomes- [28, 45]. Maternal hyperthermia during the firsttrimester of pregnancy, regardless of the cause,- has been associated with an increased risk for neural tube defects [46], while fever during labor- has been associated with adverse outcomes including neonatal seizures, cerebral palsy,- encephalopathy, and death [2, 47–50].-

In a large population-based study of influenzainfection in over 100,000 women, Hansen et al.- observed an increased risk for fetal abnormalities (e.g., central nervous system malfunctions- in the fetus, chromosomal abnormalities, suspected damage to the fetus from viral disease)- was present in both seasonal (OR 1.53, CI, 1.19–1.95) and pandemic infections (OR 1.48, CI,- 1.27–1.73) [51].-

A review and meta-analysis of 33 studies published from 1953 to 2013 of first-trimester- influenzaexposure found that influenzaexposure during the firsttrimester of pregnancy was- associated with an increase in congenital abnormalities [adjusted odds ratio (AOR) 2.00, CI,- 1.62–4.28]. Anomalies included neural tube defects [odds ratio (OR) 3.33, CI, 2.05–5.40],- hydrocephaly (OR 5.74, 1.10–30.00), congenital heart defects (OR 1.56, 1.13–2.14), cleft lip (OR- 3.12, CI, 2.20–4.42), digestive system anomalies (OR 1.72, CI, 1.09–2.68), and limb reduction- defects (OR 2.03, CI, 1.27–3.27) [52]. A major limitation of this study concerns the fact that it-

 definedinfluenzaexposure as any reported influenza,influenza-likeillness, or fever with or- without clinical confirmation.-The inclusion of wide clinical symptoms without laboratory- confirmationof influenzalikely resulted in overestimation of the number of infants exposed- to influenzaduring gestation. It therefore cannot be determined whether congenital abnormalities were associated with general, all cause hyperthermia or whether influenza infection- poses a unique and specificrisk for these outcomes. Nonetheless, these observations suggest- that prevention of influenzaduring the firsttrimester of pregnancy may reduce risk for- congenital abnormalities.-

Fetal demise is associated with influenza infection as well. Women with influenza, especially- those with pneumonia, had high rates of spontaneous abortion and preterm birth, with 52%- of pregnancies ending in spontaneous abortion or preterm delivery during the 1918 pandemic- [22, 23, 25].-

Using a large nationwide registry, Haberg et al. examined the risk of fetal death after maternal- exposure to pandemic influenzainfection. The researchers found that pregnant women with- a clinical diagnosis of influenzahad a nearly twofold increase in the risk of fetal death (adjusted- hazard ratio, 1.91; 95% CI, 1.07–3.41) as compared to women who were not exposed to influenza- [53].-

Pierce et al. assessed perinatal outcomes of maternal A(H1N1)pdm09 infection. The authors- found that perinatal mortality was higher in infants born to infected women than in infants of- uninfected women (39 per 1000 live births versus 7 per 1000 total births, respectively, *p* = 0.001).- This was principally explained by an increase in the rate of stillbirth (27 per 1000 total births- versus 6 per 1000 total births, *p* = 0.001) [54].-

## **4. Influenza infection in neonates and young infants-**

Infants younger than 6 months of age are at a heightened risk for serious illness from influenza,- exhibiting the highest rates of severe influenzacompared to other pediatric populations [41].- This age group has higher rates of hospitalization, more prolonged ICU stays, and higher- fatality rates (0.33 per 100,000 children) than almost any other age group [55–59]. In the United- States, estimates of hospitalization rates for young infants (less than 6 months of age) range- between 1.8 and 7.2 per 1000 infants, higher than reported rates of hospitalization for children- up to 4 years of age (0.14 per 1000) [60] and people 65–80 years of age (0.56–2.13 per 1000) [60–- 63]. Childhood deaths associated with influenzaare most frequent in infants during the first- months of life, with mortality rates in infants 0–6 months old more than four times higher than- those in older children [59].-

Libster et al. [64] documented particularly high mortality rates for infants during the 2009- H1N1 pandemic in Argentina. Of 251 infants and children hospitalized with confirmed- A(H1N1)pdm09 infection, 13 (5%) died, for an overall death rate of 1.1 per 100,000 children.- Infants were at particularly high risk for fatality, representing the highest death rate at 7.6 per- 100,000 children. By comparison, this death rate was 10 times the reported US infant death rate- during the relatively serious seasonal influenza season of 2003–2004 [59, 64].-

Taken together, these observations highlight the impact of influenzaon infants, especially the- youngest, and underscore the need for prevention in this vulnerable population.-

## **5. Maternal influenza vaccination-**

A large body of evidence collected over several decades demonstrates that pregnant women- and young infants are at increased risk for complications from influenza,making control of- influenzainfection in these populations an important public health challenge. Influenza- vaccines have been used since 1945 and currently are the primary strategy for preventing- influenza infection [65].-

Due to the frequent changes of the influenza viral antigens, a vaccine is formatted during the- end of the previous season to include the specificantigens of the influenzastrains expected to- circulate in the following season, with a goal of conferring protection against the upcoming- season's strains. Each year, the trivalent inactivated influenzavaccines (IIV3) are formulated- to contain three viral components: two influenza A subtypes and one influenza B virus. In 2012,- the US Food and Drug Administration (FDA) approved the use of a quadrivalent influenza- vaccine (IIV4). The quadrivalent vaccine contains two influenza-A subtypes and two influenza- B subtypes. In 2009, a monovalent H1N1 vaccine was prepared because the newly recognized- strain was identifiedtoo late in the season to be included in the trivalent seasonal vaccines [66].-

The current inactivated influenzavaccines (IIVs) are composed of inactivated (killed) virus- that has been chemically disrupted and purifiedto form a split-inactivated virus preparation.- Such split-virus vaccines contain purified-HA and NA antigens and have fewer side effects- and reactions than inactivated whole virus vaccines. The IIVs currently available in the United- States are split virus or subunit virus similar to the split virus [66].-

 The other vaccine available in the United States is a live, attenuatedinfluenzavaccine (LAIV).- This vaccine is composed of live, attenuated,cold-adapted, temperature-sensitive virus- administered directly into the nasal passage. The type A and B strains of influenzain this- vaccine can replicate in the nasal passages, stimulating an immune response, but cannot- replicate in the lower respiratory tract [67]. While LAIV has been licensed in the United States- since 2003, it is not recommended for use in pregnant women [68].-

A strategy to increase the effectivenessof IIVs is the addition of adjuvants. Adjuvants are- compounds that stimulate the immune system to mount a more robust and protective response- to the vaccine. The most commonly used adjuvants in influenzavaccines are the oil-based- compounds AS03 and MF59. Adjuvants can allow for the use of lower doses of antigen,- resulting in more available doses of vaccine, which may be useful in times of high demand- such as during pandemics. While adjuvanted vaccines are not approved for use in pregnant- women in the United States, in Canada, and in many European countries, the pandemic vaccine- was formulated with adjuvant and administered to pregnant women [69].-

Vaccination of pregnant women with IIV has taken place since the 1960s. Universal recommendation for vaccinating woman at all stages of pregnancy has been recommended by theCDC's Advisory Committeeon Immunization Practices (ACIP) since 2004 [65, 70, 71] and by- the World Health Organization (WHO) since 2005 [1]. The Advisory Committeeon Immunization Practices does not preferentially recommend a specificformulation—trivalent or- quadrivalent—of the influenzavaccine [72]. In response to the 2009 H1N1 pandemic, the WHO- placed pregnant women, along with caregivers of infants younger than 6 months old, healthcare and emergency services personnel, individuals between 6 months and 24 years, and those- aged 25 years or older with chronic medical conditions, in the highest priority group to receive- vaccines [73].-

The American College of Obstetrics and Gynecology (ACOG) considers prevention of- influenzato be an "essential element of prenatal care" [74]. In full support of the ACIP- recommendations, ACOG issued new guidelines in September of 2010 stating that all unvaccinated pregnant women at any gestational age be vaccinated against influenza [74].-

It is well documented that the antibody (Ab) response to influenzavaccine in pregnant women- is similar to that of age-matched, nonpregnant women [75–79]. These observations support a- conclusion that influenzavaccination will lead to an effective immune response in pregnant- women and thereby provide an important tool in prevention of influenza in this population.-

## **5.1. Effectiveness of maternal influenza vaccines-**

Pregnant women and neonates are high-risk groups for complications from influenzainfection. Control of influenzain this population is an important public health concern. However,- due in part to ethical concerns related to enrolling pregnant women in clinical studies,- experimental data on influenzavaccination during pregnancy has been scarce, and the quality- of the evidence is not consistently high overall [80].-

Prior to 2010, there were few well-designed studies specificallyaddressing vaccine effectivenessin pregnant women. From 1964 to 2008, four studies specificallyaddressed vaccine- effectivenessin pregnant women (**Table 1**). These studies covered eight differentinfluenza- seasons and included 51,547 pregnant women.-

During a 1962–1963 outbreak of Asian influenza,-Hulka measured vaccine effectiveness in- pregnant women by asking immunized and nonimmunized patients if they had experienced- influenzasymptoms during the influenzaseason. While fewer immunized than unimmunized- patients reported respiratory illness with fever (11% versus 20%, respectively), there was no- significant difference in reports of respiratory illness between these patients [76].-

Black et al. assessed vaccine effectivenessin almost 50,000 pregnant women across five- influenzaseasons. Vaccine effectivenesswas determined by the number of outpatient visits for- ILI or hospitalization for influenzaor pneumonia. Using these outcomes, the risk of medical- visit for respiratory symptoms was no differentbetween vaccinated and unvaccinated women,- and hospitalization was rare in both groups [81]. Using the adjusted hazard ratios- (AHR = 1.151; CI, 0.979–1.352) from this study, Skowronski and De Serres calculated a vaccine- effectiveness of −15% (CI, −35 to 2%) for this study population [7].-




**Table 1.** Clinical effectiveness of IIV and A(H1N1)pdm09 influenza vaccine during pregnancy.-

The authors of this study speculate that while the receipt of an influenzavaccine may not- prevent infection, it is likely to reduce the severity of the disease. However, they provided no- data on clinical severity or evidence of such an association with vaccination status. Since there- was no confirmation of influenza infection in most patients with ARI, it is impossible to confirmwhether clinical symptoms were associated with influenzainfection or with other respiratory viruses.-

In 2008, Zaman et al. published the firstrandomized, double-blind, controlled clinical trial- (RCT) of influenzavaccine in pregnant women. In this study, 340 pregnant women were- randomized to receive either trivalent influenzavaccine (IIV3) or pneumococcal polysaccharide vaccine during the third trimester of pregnancy. This study was part of The Mother's Gift- project, a randomized trial with the primary goal of assessing the safety and immunogenicity- of pneumococcal vaccines. Therefore, the control arm consisted of mothers who received the- pneumococcal vaccine, providing a control for an active, non-influenzavaccine. The authors- measured nonspecificrespiratory illness with fever and found that vaccinated women were- significantlyless likely to have respiratory illness, reporting a clinical effectiveness of 35.8%- (CI, 3.7–57.2%) for respiratory illness with any fever and 43.1% for fever over 38 °C (CI, −9.0- to 70.3%) [79].-

 All of these studies share the common weakness that they use clinical symptoms, and not- laboratory-confirmedinfluenza,as the primary outcome. Influenzavaccines are specifically- targeted to influenza viruses. Many other respiratory pathogens can cause symptoms similar- to influenza,but influenzavaccines are not designed to prevent other causes of influenza-like- illness. Hence, clinical symptoms without laboratory confirmation are nonspecific outcomes.- Interpretation and quantificationof true vaccine effectiveness using only clinical outcomes are- problematic, potentially leading to inaccurate estimates of effectiveness.-For this reason,- laboratory confirmation,either by reverse transcription polymerase chain reaction (RT-PCR)- or viral culture, remains the best diagnostic tool for confirminginfluenzaand evaluating- vaccine efficacy (VE) and effectiveness.-

In 2014, two studies began to address this deficiencyin the literature. The research teams- estimated influenzavaccine effectivenessin preventing illness among pregnant women using- laboratory-confirmed (RT-PCR) influenza as the primary outcome.-

Madhi et al. examined maternal and fetal outcomes in 2116 HIV-negative South African women- during the 2011–2012 influenzaseason. 1062 women who received IIV3 were compared to- women who received a placebo. Overall vaccine effectiveness at preventing laboratoryconfirmed (RT-PCR) influenza in this population was 50.4% (CI, 14.5–71.2) [83].-

 Using a large health plan database, the Pregnancy and Influenza-Project evaluated seasonal- vaccine efficacyduring the 2010–2011 and 2011–2012 influenzaseasons. The authors compared- the proportion of vaccinated women among 100 RT-PCR-confirmedinfluenzacases with the- proportion vaccinated among 192 women with ARI who tested negative for influenza,and 200- controls matched by season, site, and trimester [84]. The adjusted vaccine efficacyagainst- influenzawas of 44% (CI, 5–67%) using the influenza-negative controls and 53% (CI, 24–72%)- for the ARI-negative controls [84].-

The 2009 A(H1N1) pandemic provided an opportunity to improve understanding of influenza- vaccination during pregnancy. The pandemic allowed an evaluation of the effectiveness of- maternal vaccination during an influenzaseason in which there was a high rate of viral- circulation, as well as a close match between the vaccine strain and the circulating viral strain.- Pregnant women were prioritized to receive the vaccine and were strongly advised to be- vaccinated [85], resulting in higher than usual vaccination rates [86].-

Since the 2009 pandemic, a number of studies have examined monovalent pandemic A(H1N1)- vaccination of pregnant women. Synthesizing evidence from this new and expanding database- should increase our understanding of maternal influenzavaccination. While most of these- studies measured either seroconversion and hemagglutinin inhibition (HAI) titers [87–92] or- safety and/or birth outcomes [93–100], two studies specificallyexamined clinical efficacyof- pandemic A(H1N1) vaccine during pregnancy.-

In a retrospective cohort study, Richards et al. evaluated influenzainfection in 1125 vaccinated- and 1581 non-vaccinated women. In this study, influenzainfection was definedas testing- positive for influenza by RT-PCR or having a medical visit during pregnancy with influenzarelated *ICD-9* diagnosis code during the period of 2009 influenza-A(H1N1) virus circulation- [101]. The researchers reported a vaccine efficacy-(VE) of 61.5% (CI, 15.5–82.5%) for 2009 H1N1- influenza vaccine against diagnosed 2009 influenza A(H1N1) infection during the study period- among all mothers in the study cohort [101].-

A separate population-based study of 117,347 pregnant women in Norway estimated that- vaccination during the second or third trimester of pregnancy resulted in a 70% reduction in- influenzadiagnosis following vaccination with adjuvanted A(H1N1) vaccine (adjusted hazard- ratio, 0.30; CI, 0.25–0.34) [53].-

 While there is significantheterogeneity among these eight clinical studies, presenting wideranging estimates of vaccine effectiveness in pregnant women (from −15 to 70%), the evidence- of effectivenessdata based on laboratory-confirmedinfluenzais mounting and compelling.- The cumulative evidence to date provides three studies showing significant clinical effectivenessduring seasonal influenzayears, 35.8% against respiratory disease in a RCT [79], 50.4%- protection against laboratory-confirmedinfluenza in a RCT [83], and one large data-base study- documenting VE of 44–53% [84]. Two studies of vaccine effectiveness during the 2009 A(H1N1)- pandemic estimated VE of 61.5% (unadjuvanted) and 70% (adjuvanted), suggesting that- vaccination during a pandemic season may offermore benefitthan during non-pandemic- years.-

These more recent estimates using laboratory-confirmedendpoints are well within the- reported vaccine effectivenessfor healthy, nonpregnant adults. When combined with the welldocumented observation that the antibody response to influenza vaccine in pregnant women- is similar to that of nonpregnant women, all current evidence suggests that efficacy of influenza- vaccines in pregnant women is similar to the nonpregnant population.-

#### **5.2. Maternal vaccination to protect young infants-**

An important secondary benefitof maternal vaccination appears to be protection of infants- from influenzainfection during the firstmonths of life, providing an important two-for-one- benefitsince no influenzavaccine is licensed or recommended for infants younger than 6- months of age.-

Maternal vaccination has potential to protect newborns due to the transfer of maternal- immunity. Transplacental transfer of antibodies occurs throughout pregnancy, with highest- levels during the last 4–6 weeks of gestation [102, 103]. Antibodies (specifically IgG) cross the- placenta from mother to fetus during the finalweeks of pregnancy, while infants acquire- additional immune protection during breastfeeding, when the main class of immunoglobulin- transferred in breast milk is IgA [104].-

 Several studies have demonstrated transplacentally acquired antibodies after natural influenza- infection in the mother. One study examined the cord sera of 26 infants who had cultureconfirmedinfluenzawhen younger than 4 months of age. The authors found a direct correlation between the gestational age at the time of infection and the level of antibody in the cord- serum (*p* = 0.002), suggesting a protective effect of transplacentally acquired antibody [105].-

During the 1979 influenzaepidemic, infants of mothers with serum antibody to influenza- (seropositive) were compared to infants of mothers who were seronegative [106]. The infants- of seropositive mothers had higher specificserum antibody (IgG) titers against the HA- influenzaprotein than did infants of seronegative mothers. Good correlation was found- between maternal antibody titers and titers in infants (correlation, 0.81). No such correlation- was found for non-immune mothers and their infants (correlation, 0.24) [106]. However, this- study showed no differencein incidence of influenzainfection, although infants of immune- mothers showed delayed onset of symptoms and shorter duration of illness, suggesting that- passive maternal immunization may delay onset and severity.-

#### *5.2.1. Serological studies-*

When evaluating vaccination response to influenza,a serum antibody titer of ≥1:40 against the- HA protein is considered to be clinically relevant, resulting in a 50% decrease in symptomatic- infection and, therefore, serving as a correlate of protective immunity [107]. Recent analysis of- published data indicates an even higher level of clinical protection, estimating that 70% of- participants are protected at a titer of 1:40, with protection increasing gradually with higher- titers [108].-

In the absence of a widely accepted immune correlate of protection for influenza,the analysis- of hemagglutinin inhibition (HAI) seroprotection rates is generally considered a useful- indicator of protection in vaccinated persons. There are challenges associated with using this- antibody titer as a correlate of protection in infants, however, particularly since this correlate- was determined in healthy adults [41, 107]. In any event, the US Food and Drug Administration- (FDA) uses this HAI antibody titer in evaluation of influenzavaccines, making the HAI- antibody titer the standard used by most investigators [40], including those examining vaccine- protection in neonates.-

In a study examining transplacental transfer of antibody in response to vaccination, Englund- et al. studied women during the last trimester of pregnancy who were vaccinated with IIV3- (A/Sichuan/H3N2, A/Taiwan/H1N1, B/Victoria). Maternal immunization resulted in the- transfer of influenza-specific-IgG to the infants. When measured at the time of delivery, the- levels of antibody transferred were high, with between 87 and 99% of antibody detectable inthe mother also detectable in the infant [78]. The antibody titers to at least two of the three- influenza antigens (influenza-H3N2 and influenza-B) remained significantlyelevated up to 2- months of age [78].-

Sumaya and Gibbs vaccinated pregnant women and reported a similar correlation, finding- that cord blood HAI antibody in infants correlated with titers of vaccine-stimulated HAI- antibody in mothers, with HAI antibody detected in 54% of newborn serum and 73% of- maternal serum [75]. This study did not examine infant influenza infection as an outcome, so- the study was unable to correlate serum antibody levels with clinical outcome.-

While these previous studies demonstrated that maternal antibody appears to be transferred- to infants following both natural infection and vaccination, neither study discussed whether- the level maternal antibody was high enough to confer protective immunity to the infant [41,- 107]. Using HAI titer as a correlate of immunity, Steinhoffet al. did conduct an RCT of maternal- influenzavaccine. Serum samples were obtained from 311 mothers before vaccination and at- delivery and from 292 infants at birth at 10 weeks and 20–26 weeks later [109]. The proportion- of mothers with a protective antibody titer (HAI ≥ 1:40) at the time of delivery was 88% for A/- New Caledonia (H1N1), 98% for A/Fujian (H3N2), and 45% for B/Hong Kong. Similar- proportions of infants had protective antibody titers at birth [109].-

Yamaguchi et al. also examined the immune responses to influenzavaccination during- pregnancy by measuring the effectof influenzavaccination (IIV3) in pregnancy, including- maintenance of the specificantibody response, and the efficiencyof transplacental transfer of- the antibody to the fetus. The study included 125 pregnant women, 71 in their second trimester- and 54 during the third trimester of pregnancy. The authors reported that vaccination at any- time during pregnancy yielded protective levels of antibody in both maternal and fetal blood- [110].-

In a prospective study examining the immunogenicity and transplacental transmission of- antibodies in pregnant women in Asia, Lin et al. enrolled 46 pregnant women who received a- single dose of IIV3. Twenty-eight days after vaccination, the seroprotection rate in vaccinated- women against H1N1, H3N2, and influenza B was 91.3, 84.8, and 56.5%, respectively [111].-

Several additional studies have demonstrated transplacental antibody transfer following- maternal vaccination with pandemic H1N1 vaccine. Three studies evaluated adjuvanted- A(H1N1) vaccines, and two studied nonadjuvanted A(H1N1)vaccine. All fiveof the studies- documented protective levels of antibody in significant percent of infants (79–95%).-

Zuccottiet al. followed 69 mother-infant pairs. The women were vaccinated during their third- trimester of pregnancy with MF59-adjuvanted influenza-A(H1N1) vaccine. All of the mothers- had HAI antibody titers at or above 1:40 at the time of delivery and throughout the 5 months- of follow-up. Ninety-five percent of the infants had HAI antibody titers at or about 1:40 at both- birth and 2 months of age. By 5 months of age, the proportion of infants with titers of 1:40- dropped to 81.2% [89], suggesting that passively acquired antibody at levels thought to be- protective is transferred and persists for at least 5 months.-

In a separate study performed at three sites in the UK, researchers followed infants born to- mothers who were vaccinated with AS03-adjuvanted H1N1 vaccine during the second or thirdtrimester of pregnancy. This study found that 79% of the infants of vaccinated mothers had- serum antibody titers at or higher than 1:40 compared to a background immunity in 19% of- infants of unvaccinated women (*p* = 0.001) [112].-

Helmig et al. evaluated the serological response of a cohort of women immunized with- adjuvanted A(H1N1) vaccine or by natural infection with A(H1N1)pdm09. The authors- detected protective antibody levels (>1:40) in a significant number 17/19 (89.5%) of newborns- born to vaccinated mothers (*p-*= 0.001) [113]. Notably, while natural influenzainfection- conferred protective antibody levels in mothers, it did not contribute to protective levels in- their infants; only 15.8% of infants born to women who had natural influenzainfection during- pregnancy developed protective levels of antibody at birth.-

In a study of nonadjuvanted A(H1N1) vaccine, Tsatsaris et al. measured Ab titers in cord blood- of 88 infants born to women vaccinated with a single dose of monovalent A(H1N1) vaccine.- The researchers reported 95% (CI, 89–99%) of infants have protective levels of antibody (>1:40)- at birth [114], and Fisher et al. also reported protective levels of Ab in cord blood of infants- following maternal vaccination with 2009 monovalent A(H1N1) [115].-

Taken together, these studies provide an important proof of concept for transplacentally- acquired antibody, from either natural infection or vaccination of their mother, to potentially- protect young infants from influenzainfection and present maternal immunization as a- promising strategy for reducing influenza infection in infants.-

#### *5.2.2. Clinical studies-*

Over the past 11 years, nine studies have been published evaluating the effectof maternal- immunization on influenzain 97,656 infants across 15 influenzaseasons. Six of these studies- showed clinical protection; three studies did not (**Table 2**).-




**Table 2.** Clinical effectiveness of maternal vaccination for protection of infants.-

## *5.2.2.1. Studies finding no protective effect-*

In a retrospective cohort study of 48,639 infants, Black et al. found that infants born to- vaccinated women had the same risk of hospitalization for influenzaor pneumonia as infants- of unvaccinated women (CI, 0.889–1.029). They also reported that maternal vaccination was- not a significant determinant of risk for ILI or otitis media [81].-

France et al. followed 3160 infants of immunized mothers and 37,969 infants of nonimmunized- mothers from 1995–2001 and during four specificseasonal periods: peak influenza,respiratory- syncytial virus predominant, periseasonal, and summer weeks [116]. They found no difference- in medically attended-ARI (incident rate ratio for peak influenzaseason 0.96, CI, 0.86–1.07),- concluding "maternal influenzavaccination did not reduce visit rates during any of the four- time periods and did not delay the onset of first respiratory illness" [116].-

 In a study that included an examination of data over fiveinfluenzaseasons, Munoz et al.- reported similar results, findingno differencein hospitalizations for respiratory illness during- the peak of influenzaseason between infants of vaccinated mothers versus infants of nonvaccinated mothers [117].-

## *5.2.2.2. Studies finding clinical protection-*

While the three earlier studies above found no benefitto infants from maternal vaccination, a- more recent matched case-control study of infants less than 12 months old admitted to hospitals- for seasonal influenzabetween 2000 and 2009 reported maternal vaccination to be 91.5%- effective (CI, 61.7–98.1%, *p-*= 0.001) in preventing hospitalization of infants younger than 6- months of age [118]. Maternal vaccination was not effective in preventing hospitalization for- infants older than 6 months (−41.4% (CI, 0.153–0.918)). In this study, influenzacases from 2007- to 2009 were confirmedby direct fluorescentantibody (DFA) test, which was reported to be- 96.2% sensitive and 99% specificfor influenza [119]. A limitation to this study is that results- from nine influenzaseasons were combined, with no information regarding the strain match- of vaccine to circulating strain for each influenzaseason. The researchers did not type the strain- of influenzain infected infants, stating that they "did not have adequate power to assess thevaccine's effectivenessby influenzaseason…and did not type strains to determine whether- influenza infections were caused by strains included in the vaccine" [118].-

 Eick et al. conducted a study in the White Mountain and Navajo reservations over three- influenzaseasons, November 2002 to September of 2005. In this prospective cohort study, the- effectof influenzavaccine during pregnancy on influenzainfection in infants was compared- between infants of vaccinated women and those born to non-vaccinated women. The authors- analyzed 83 confirmed influenza cases. Of these cases, seven (86%) were confirmed by serology,- ten (12%) by viral culture, and two (2%) by rapid influenzatesting. Using these three measures- of influenzainfection, the authors reported a 41% reduction in the risk of laboratory-confirmed- influenzafor infants born to vaccinated women (RR 0.59, CI, 0.37–0.93) and a 39% reduction- in the risk of ILI hospitalization (RR 0.61, CI, 0.45–0.84) [120].-

The authors of this study pooled the results from three differentassay methods, each with- differentsensitivities, to document influenzainfection. Pooling assays with differingdiagnostics represents a limitation of this study. Likewise, the use of serology to document influenza- in most (86%) of cases represents a weakness of this study. The authors describe serological- documentation of influenza infection as a fourfold or greater increase in HAI antibody in serum- collected at 2–3 or 6 months compared with previous serum specimen [120]. Significant- limitations have been reported regarding the use of serology in diagnosing influenza in adult- patients that have been vaccinated with inactivated vaccines [121–123]. Using serology to- document influenza infection in young infants presents additional complications.-

Infants under 6 months of age have immature, immunologically inexperienced immune- systems. Since their ability to produce antibody in response to infection is often delayed or- completely absent, many of their serum antibodies are maternally derived. More than 50 years- ago, the use of serology for diagnosis of influenzain infancy was questioned. During an- influenzaoutbreak in Glasgow in 1953, fourfold increases in antibody were found in only 20%- of infants admittedto pediatric pneumonia wards (as compared to 30% of adult cases) [124].- In two different outbreaks, antibody production was absent or considerably delayed in infected- children under 18 months of age, even when virus could be isolated [125]. These observations- illustrate the difficulties inherent in using serology to document influenza infection in young- infants.-

 In the only randomized, blinded clinical study to assess infant protection, Zaman et al. reported- a vaccine effectivenessof 63% (CI, 5–85%) against laboratory-confirmedinfluenzain infants- up to 6 months of age [79]. Influenzawas confirmedby rapid test (Z Stat Flu), which was- reported to have a specificityof 80–90% and a sensitivity of 70–72% for type A and type B- influenza.-When respiratory illness and fever were used as measure of disease, the reported- effectiveness was 29% (CI, 7–46%) [79].-

 A recent study sought to determine whether maternal vaccination during pregnancy was- associated with a reduced risk or laboratory-confirmedinfluenzahospitalizations in 1510- infants over seven consecutive influenzaseasons (2002–2003 through 2008–2009) and across- three diverse geographic regions of the United States [126]. Of the 1510 infants hospitalized- with fever or respiratory symptoms, 151 (10%) had laboratory-confirmed-(by viral culture orRT-PCR) influenza.-The proportion of infants who were influenzapositive varied significantly- across influenzaseasons, from 3% in 2006–2007 to 15% in 2003–2004. The percentage of women- who were vaccinated also varied by season, from 10% in 2003–2004 to 38% in 2008–2009.-

 Among influenza-positive infants during all study years, 12% of their mothers reported being- vaccinated during pregnancy, while 20% of mothers of influenza-negative infants reported- receiving a vaccination [126], yielding an adjusted odds ratio of 0.52 (CI, 0.30–0.91) and- suggesting that infants whose mothers received influenzavaccines during pregnancy were- 48% less likely to have laboratory-confirmedinfluenzathan infants of unvaccinated women- [126].-

In a randomized, placebo-controlled trial of influenzavaccination in South Africa, Madhi et- al. followed 1026 infants born to women who received IIV3 and 1023 infants born to placebo- recipients. The attackrate of influenzawas lower among infants of vaccinated mothers (1.9%)- than among those whose mothers received placebo vaccine (3.6%), yielding a vaccine effectiveness of 57.5% (CI, 7.6–70.4) in this population [83].-

 In a prospective study, Sugimura et al. assessed the incidence of fever and laboratory-confirmedinfluenzain newborns whose mothers were vaccinated during pregnancy with IIV3.- Two hundred infants were followed from birth to 6 months of age. Fever was noted in 36 (34%)- of the infants in the vaccinated group and 47 (52.2%) of infants born to unvaccinated mothers- (*p* < 0.007). The incidence of influenzaas diagnosed by rapid test was 0 (0%) and 5 (5.6%),- respectively, (*p* = 0.019) [127].-

The reviewed studies assessed hospitalization, clinic visits, ARI, or laboratory-confirmed- influenzaas primary outcomes. Rationale for influenza vaccination often includes additional- secondary outcomes such as reduction of absenteeism for household contacts, secondary- infections, acute otitis media, or community transmission of influenza. While these outcomes- are important in measuring the broad impact of influenza,they do not provide accurate- assessment of vaccine efficacyor effectiveness.-Such an assessment is essential for evidencebased, reasoned development of public health policy and decision-making about influenza- prevention.-

 In summary, the evidence for newborn protection through maternal vaccination is encouraging, but several studies exhibit methodological limitations. The results of studies measuring- rates of ARI, clinic visit, or hospitalization range from no vaccine effectup to 42% effectiveness- [81, 116, 117]. The six studies that used some form of laboratory-confirmedinfluenza-(rapid- tests, viral culture, or PCR) as the primary outcome are more encouraging, reporting vaccination effectiveness ranging from 41 to 91.5% [79, 83, 118, 120, 126, 127].-

While outcomes and study designs differ,in general the data to date suggest that maternal- vaccination has the potential to decrease influenzaillness in newborns. As such, maternal- immunization during pregnancy should continue to be recommended and encouraged for all- women.-

## **6. Current challenges-**

Influenzais a complex disease. For centuries it has eluded complete understanding or control.- Constantly evolving and catching us by surprise, influenzais a perpetually emerging disease.- The main tool for prevention of influenzais vaccination, the cornerstone for prevention of- influenza disease. However, unlike many other vaccines, the effectiveness of influenza vaccines- remains moderate to marginal across all populations [128], and in addition, the vaccines need- to be administered every year. A universal influenzavaccine providing broad and long-lasting- protection continues to be elusive.-

These realities—difficultenough in the general population—are even more challenging to- understand during pregnancy. Particularly complex is determining how, or if, the changing- immune responses over pregnancy vis-a-vis influenzaseason and vaccination schedules- impact vaccine effectiveness and disease outcomes in pregnant women.-

A recent critique by Savitzet al. calls into question our most basic understanding of influenza- infection during pregnancy, with a particular critique on the quality of the evidence for a benefit- of maternal vaccination in prevention of harm from seasonal influenza [129]. Savitzet al.- discuss the complications inherent in many current research practices, which serve to limit our- understanding of the true picture of maternal influenza.-Central to this critique is the notion- that both pregnancy and influenzahave temporal components; influenzatypically is seasonal,- with a 2–3-month period of circulation each year. But pregnancy has trimesters, each with- distinct fetal developmental stages along with physiological and immunological changes in- the expectant mother. To complicate this further, the risk of adverse outcomes from influenza- infection is not equal across all trimesters of pregnancy. Therefore, optimizing risk and benefit- of vaccination during pregnancy is complicated and likely requires reexamination of influenza- risk and benefit across trimesters in association with months of influenza circulation [129].-

A recent WHO review of influenzathroughout pregnancy found insufficientdata from- comparable studies to discern which specificweeks, months, or trimesters influenzaposes- increased risk to pregnant women [130]. Savitzet al. assert that "As with all time dependent- states, pregnancies must be followed longitudinally. There needs to be a week-by-week- consideration of the pregnancy with regard to vaccination status and circulating influenza- viruses" [129].-

Aside from complications associated with potential differencesin virus exposure across the- trimesters of a pregnancy, risks posed to pregnant women also may differaccording to the- specificstrain circulating during a given season, and, likewise, the immune response to- vaccination also could differ based on strain [131].-

Even when taking yearly strain differencesinto account, variation in immune responses across- trimesters adds yet another complicating variable. It is widely accepted that the immune- response to influenzavaccine in pregnant women is indistinguishable from that of nonpregnant women and that gestational age appears to have no effecton antibody response [75, 76,- 132, 133].-

Findings from more recent studies suggest that we may need to entertain a more nuanced- approach to our understanding of the maternal immune response to influenza vaccination—- precisely because immunogenicity and seroconversion may not tell the entire story.-

 Ina-2011study,-Ohfujietal.reportedalowerseroprotectiveantibodyresponsetopandemic-A(H1N1)vaccineinpregnantwomenwhohadreceivedpriorseasonalinfluenzavaccine- andsuggestedthatthepotentialinterferencebetweenpH1N1andseasonalvaccination- neededadditionalinvestigation-[15].-Schlaudeckeretal.directlycomparedimmunogenicityofinactivatedinfluenzavaccineinpregnantversusnonpregnantwomenandfoundthat- pregnancymodifiedantibodyresponsestothevaccine-[134].-Theydemonstratedasignificantlydecreasedpostimmunization-HAIgeometricmeantiterandanonsignificantlydecreasedgeometricmeanratio-(foldincrease)toinfluenza-Aantigensafterinfluenza- vaccine,eventhoughoverallseroconversionandseroprotectionrateswerecomparablebetweenthetwogroupsofwomen.-Ina-2013blindedrandomizedcontrolstudy,-Bischoffet- al.foundthattheimmuneresponsetoanadjuvantedpandemic-A(H1N1)vaccineinpregnantwomenwasdecreasedcomparedwithnonpregnantwomen-[133].-Sperlingetal.examined-HAItitersoverpregnancyandfoundthattimingofvaccinationdidnotalter- response,althoughtherewasatrendtowardlowerresponsesduringthefirsttrimesterand- sixweekspostpartum-[135].-

A small study of 36 women during the 2012–2014 fluseasons suggests that T-follicular helper- (Tfh)cell response to vaccination was highest during the firsttrimester of pregnancy. Tfh cells- are required for the generation of high-quality antibody-producing B cells. Their expansion- has been shown to be a predictor of response to influenzavaccination outside of pregnancy- suggesting that immunologic changes during pregnancy may impact vaccine response.- Notably, there was no significantexpansion of Tfhafter vaccination during either the second- or third trimester [136].-

 Inastudyaimedtodeterminetheoptimaltimingforvaccinationwithinthesecondor- thirdtrimestersofpregnancy,-Yamaguchietal.reportedthatserumantibodylevelsinvaccinatedwomendependednotongestationalstagebutontheamountoftimeelapsed,since- vaccinationantibodytitersdecreasedwithtime-[110].-Thisobservationissupportedbyotherstudiesdocumentingthatinfluenza-specificantibodiesaftervaccinationaretypically- shortlived-[115].-

Two separate studies of vaccine effectivenessin nonpregnant adults during the 2011–2012- influenzaseason also suggest that vaccine effectivenesswanes with time since vaccination- [137, 138]. In both of these studies, vaccination effectiveness waned in people who were- vaccinated 93 days—around 3 months—or more before presentation of symptoms. A similar- observation was made by Fisher et al. who demonstrated a significantlinear decline over time- in HAI titers after pH1N1 infection or vaccination (*p* = 0.04 for infected and *p* = 0.009 for- vaccinated) [115].-

 Takentogether,theseobservationssupportaconsensusthatpregnantwomenarecapable- ofmountingarobustimmuneresponsetoinfluenzavaccinationduringthesecondand- thirdtrimesterofpregnancy.-Experimentalevidenceduringthefirsttrimesterislessclear,-

 dueinparttoalimitednumberofstudiesoninfluenzavaccinationduringthefirsttrimester.-

There is increasing evidence suggesting that—regardless of initial vaccine response—the level- of vaccine-mediated anti-influenza-specificantibodies decreases with time. This waning of- antibody implies that women vaccinated during the firsttrimester of pregnancy might be *less* well protected as their pregnancy progresses. It is well documented that serious influenzarelated morbidity in healthy pregnant women most often occurs during the second and third- trimester of pregnancy. So a reasonable goal appears to be ensuring the highest possible level- of protection during the second and third trimesters. Studies on waning immunity suggest- that this may be precisely opposite of what occurs in women who are vaccinated early in- pregnancy; protection wanes as risk increases.-

#### **6.1. Timing maternal vaccination for protection of neonates-**

Cross-placental transfer of immunity occurs when antibodies cross the placenta via active- transport, particularly in the finalweeks of pregnancy. It is logical to assume that vaccineinduced maternal antibody transmitted to the fetus will provide protection to the infant during- the firstmonths of life, and several studies document increased antibody titers in infants of- vaccinated mothers [120].-

Comparable to studies of vaccinated mothers, researchers have demonstrated that the titers of- influenza-specificantibodies in newborns, while not affectedby trimester of maternal vaccination (within second or third trimester), do wane with time and are short lived [75]. Tsatsaris- et al. also noted a trend toward lower cord blood antibody titer associated with longer intervals- between maternal vaccination and delivery. A similar observation was made by Yamaguchi et- al. who reported that transfer rate from the maternal blood to the fetal blood at time of delivery- tended to be inversely correlated with duration of gestation postvaccination [110]. Some- additional studies have hinted at lower cord blood titers among infants of women vaccinated- in the first trimesters, but it is unclear if this is clinically significant [131].-

Taken together, these studies serve to highlight the complicated nature of immunity particularly during pregnancy in response to vaccination and invite further virological and epidemiological studies to confirmand fully understand these observations and to correlate changes- in antibody titer with clinical outcomes in both mothers and infants. As our understanding- evolves, we may need to develop a more nuanced approach—one that takes into consideration- the trimester of exposure to both circulating virus and vaccines—to optimize vaccine effectiveness during the period of highest risk of influenza disease.-

The recommendation to vaccinate all pregnant women regardless of gestational age is- motivated in part by operational concerns. Timing of the influenza vaccine campaigns occurs- in the fall in temperate regions. Pregnancy, on the other hand, occurs throughout the year. Since- pregnancy presents a period of regularly scheduled and repeat visits, a more tailored approach- to influenzavaccine coverage may be appropriate in order to stack the odds in favor of the- highest level of protection for both mother and infant.-

## **7. Conclusions-**

The impact of influenzainfection on pregnant women and newborns is well documented. The- increased risk for morbidity and mortality has resulted in the universal recommendation that- pregnant women be vaccinated for influenzaat any stage of pregnancy [65]. Evidence to date- suggests that influenzavaccination during pregnancy is similarly effective as in the nonpregnant population. Pregnant women mount robust immune response that correlates with clinical- efficacy.-This immunity then is transferred to the fetus during gestation, providing clinical- benefitto infants less than 6 months of age. And while not a component of this review, there- is substantial evidence that influenzavaccines are safe during all stages of pregnancy with no- evidence of risk of adverse pregnancy outcome linked to influenzavaccines [3, 65, 131, 139,- 140].-

 Gapsremaininourunderstandingofinfluenzariskoverallseasonsandtrimesters,ofmaternalimmunity,andofoptimaltimingofvaccination.-Tothisend,in-Januaryof-2015,the- Billand-Melinda-Gates-Foundationheldameetingofglobalstakeholdersinthematernal- influenzafield.-Participantsidentifiedaneedforstrongerevidenceregarding,amongotherissues,theburdenofdiseaseandmaternalimmunizationefficacy-[141].-Expandingthis- evidencebasewillallowforadeeperunderstandingofinfluenzainthispopulationand- thedevelopmentofmorerobustandefficaciouspublichealthapproachestowardthisdisease.-

## **Author details-**

## Jill M. Manske-

 Addressallcorrespondenceto:jmmanske@stthomas.edu-

 Universityof-St.-Thomas,-Saint-Paul,-MN,-USA-

## **References-**


## **Antibody Responses after Influenza Vaccination in Elderly People: Useful Information from a 27-Year Study (from 1988–1989 to 2014–2015)**

Barbara Camilloni, Emilia Nunzi, Michela Basileo and Anna Maria Iorio

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/104559

### **Abstract-**

 Elderlypeoplearemorelikelythanyoungerpeopletogetflucomplicationsand- respondsuboptimallytoinfluenzavaccinationbecauseofthepresenceofcomorbidities- andimmunosenescence.-Inordertocollectinformationaboutthisissue,weevaluated- dataobtainedin-27wintersofstudy,from-1988–1989to-2014–2015,infrailelderly- institutionalizedpeople (≥60years)vaccinatedwithcommerciallyavailableseasonal- trivalentinactivatedinfluenzavaccines.-Theantibodyresponsewasexamined- comparinghemagglutinationinhibitionantibodytitersinseracollectedfrom-4461- volunteersbeforeand-30daysaftervaccination.-Examiningtheresultsascrudemean- responses,weevidencedtheabilityofinfluenzavaccinestoinducesignificantincreases- inantibodytitersagainstallthethreevaccineantigenssatisfyingatleastoneofthe- threecriteriaofthe-Committeefor-Medical-Productsfor-Human-Use-(CHMP).-Higher- responseswerefoundagainst-A/H3N2vaccinecomponentsand,examiningdifferent- subgroups,involunteersreceiving-45μgvaccineascomparedwith-30μgandinfemale- ascomparedwithmalesubjects.-Veryelderlypeople-(>75years)gavebetterresponses- thanyoungerelderly (≤75years)atleastagainst-A/H1N1strainandthelastlicensed- potentiatedvaccines-(MF59-adjuvantedandintradermal)weremoreimmunogenic- thantraditionalvaccines-(whole,subunit,andsplit).-

**Keywords:** influenzavaccination, vaccine immunogenicity, HI antibody titers, CHMP- criteria, elderly institutionalized people-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction-**

 Influenzavirusinfectionscanaffectallagegroups,andolderindividualsareparticularlyat- riskforinfluenzasince,despitehavingnohigherattackratethanyoungeradults,most- influenza-relateddeathsandseverecomplicationsoccurinthisagegroup.-Althoughinfluenza- vaccinationremainsthemainstayinprevention,nonetheless,uncertaintiesregardingthe- effectivenessoftheinfluenzavaccinesinelderlyadultsarepersistent-[1,-2].-

The higher rate of flu complications and the reduced vaccine efficacy are generally attributed- to both concomitant comorbidities and immunosenescence, i.e., the age-related weakening of- the immune system [3, 4].-

As reported by Lambert et al. [5], the measurement of vaccine efficacyagainst influenzaillness- is a difficulttask especially in older adults. Although influenza vaccine effectiveness depends- not only on vaccine-induced immune response but also on annual variations in influenza- incidence, circulating strain virulence, and the quality of the vaccine-to-circulating strain- match [6], previous studies have established that a high serum antibody level can prevent- infection at least in children and young adults [7–9], and serological studies based on the- evaluation of influenza-specificantibody titers have been widely accepted and used as a- surrogate marker for protection against influenza and vaccine efficacy.-

Chronic underlying diseases, particularly cardiac and respiratory diseases, were shown to- negatively influence the immune response after influenza vaccination in old people [10].-

Three previous reviews on serological responses to inactivated seasonal vaccines in elderly- people did not consider the possible role of chronic underlying illnesses, because there was- not the possibility of controlling for the presence of serious illnesses [11] or because the elderly- population was carefully selected to exclude any chronic diseases so that the results would- reflect the effect of ageing on comparison with young people [12, 13].-

In comparison with community-dwelling elderly people, residents of nursing homes are- considered to be at a higher risk of serious influenza-relatedcomplications, because they are- generally older, more debilitated, and more exposed to influenzainfection once the virus is- introduced because of the close environment in which they live [14]. However, evaluating- vaccine immunogenicity, results reported in the review of Goodwin et al. [12] and results- previously obtained in our laboratory [15] suggested that institutionalized elderly responded- better when compared with community-dwelling elderly.-

The aim of this chapter of the book is to examine the phenomenon of the decreased immunogenicity and efficacyof influenzavaccines in older persons from available data. We examined- the data obtained by our research group in 27 winter seasons, from 1988–1989 to 2014–2015,- of vaccine immunogenicity in a considerable number (4461) of elderly people (≥60 years of- age), most of them with underlying medical conditions, vaccinated with commercially- available seasonal trivalent inactivated influenzavaccines. Although some of the results- obtained in the differentwinters were previously published, in the present report the results- we obtained are cumulatively examined for the first time.-

#### **2. Materials and methods-**

#### **2.1. Study design and vaccination-**

The volunteers initially enrolled in the prospective study of antibody response to influenza- vaccination, conducted over a period of 27 consecutive winters, were 4461 elderly people,- aged ≥60 years (mean age 80.5 year, range 60–106 years). Eighty-six percent of them were- living in nursing homes in Central Italy.-

After providing informed consent, all the subjects received one dose of trivalent inactivated- influenzavaccine intramuscularly, in the deltoid, or intradermally. The vaccines used were- commercially available inactivated trivalent vaccines for the winters from 1988–1989 to 2014–- 2015 produced by propagation of the virus in embryonated hens' eggs. Each dose of vaccine- consisted of 10 μg (from 1988–1989 to 1991–1992) or 15 μg of hemagglutinin (HA) in a 0.5 ml- dose (for vaccines administered intramuscularly) or in a 0.1 ml dose (for vaccines administered- intradermally) for each of the three influenzastrain antigens (A/H3N2, A/H1N1, and B- influenzaviruses). At the time of recruitment of this study, demographic data, health status,- and history of influenzavaccination over the preceding year were obtained from each subject.- Serum samples were obtained from the same subject before and 1 month after vaccination.- Subjects were included in this study if they did not have a history of immediate hypersensitivity to eggs components. Subjects sufferingfrom specificillnesses or chronic condition were- not excluded. The study was conducted according to the Declaration of Helsinki and Good- Clinical Practices. Since vaccines were assigned by local health authorities within the annual- influenzacampaign and sera were leftover sera from samples collected for clinical routine- controls, the study did not need to be registered as a formal trial.-

#### **2.2. Determination of hemagglutination-inhibiting (HI) antibody titers and measurement- results-**

HI antibody titers were determined using a standard microtiter method [16] with 0.5%- chicken (from 1988–1989 to 1996–1997) or turkey erythrocytes (after 1996–1997). Antigens- were prepared from the allantoic fluidsof embryonated hens' eggs inoculated 3 days earlier- with influenzavirus. All sera were heat-inactivated at 56°C for 30 min and treated with potassium periodate and trypsin (from 1988 to 1994) or with receptor-destroying enzyme- (RDE) of *Vibrio cholerae-*(after 1994) to remove nonspecific inhibitors. The first dilution for antibody titration was 1:10. Pre- and postvaccination sera from each of the vaccines were frozen at −30°C until used and tested simultaneously for HI antibody titers using the same- antigens as those in the vaccine. To eliminate any subjective bias, HI titers determinations- were carried on in a blind fashion, i.e., with the tester unaware of which treatment the donor- had received.-

#### **2.3. Criteria used for evaluating vaccines immunogenicity-**

HI antibody titers obtained by following the procedure indicated in the previous section- were reported as protection rate (percentage of volunteers showing HI titers ≥40, considered- to be associated with protection from influenzainfection) [9], geometric mean titers (GMT;- any HI antibody titer <10 was considered equal to 5 for GMT calculation), ratio of postvaccination to prevaccination GMT values (GMTR), and seroconversion rate (percentage of subjects with a fourfold or greater increase in titer and with a postvaccination titer at least equal- to 40 in seronegative volunteers). The antibody titers measured 1 month after vaccination- were also evaluated according to the criteria of the Committeefor Medicinal Products for- Human Use (CHMP) for approval of influenzavaccines, which require that for individuals- aged ≥60 years at least one of the following values must be met: seroprotection rate ≥60%,- GMTR ≥2, or seroconversion rate ≥30% [17].-

## **2.4. Statistical analyses-**

 Statistical analyses and subanalyses considered in this work were applied to populations- with a relatively large number of people, as a consequence both GMT and rate statistics- were well approximated by a log normal and normal distributions, respectively. Moreover,- since rates values were not close to 0 or 100%, thus significantdifferences between mean values of the groups were analyzed by Student's *t*-test. Both estimated mean values with their- corresponding 95% confidenceintervals (CI) the p-value of the *t*-statistic have been reported- in the paper. In particular, *p*-values <0.01 were considered highly statistically significant,- whereas *p*-values <0.05 were regarded as marginally statistically significant.-Values of postvaccination GMT observed against differentantigens and in differentyears were examined- as such and also corrected for prevaccination status according to Beyer et al. [18] in order to- verify that significantdifferencesin the postvaccination status were independent on the prevaccination HI titers. Vaccine response was evaluated also according to the dosage of vaccine antigens (30 and 45 μg), gender, and age (≤75 and >75). For each antigen, significant- differencesbetween subpopulations means were evaluated and the corresponding statistical- significance was indicated.-

A multiple comparison test between groups of vaccine type and between antigens was- executed by using one-way analysis of variance (ANOVA). Paired comparison values were- presented only when one-way ANOVA comparison identifiedpotentially significantdifferences.-All statistical analyses were carried out using MATLAB® of MathWorks Inc. release- 2014b.-

## **3. Results-**

#### **3.1. Study population and demographic characteristics-**

**Table 1**reports the baseline characteristics of the 4461 elderly volunteers, aged ≥60 years- (range 60–106) vaccinated with commercially available seasonal trivalent inactivated influenza vaccines for each year of the 27 consecutive winters (from 1988–1989 to 2014–2015)- studied. The number of volunteers examined each year varied from 64 to 372. The mean age- was lower in the firstyears studied (from 1988–1989 to 1998–1999) when a mixed population- of community-dwelling and institutionalized elderly was examined (60–80 years) than in the- other seasons when volunteers were totally recruited from nursing homes (82–86 years). Themajority of elderly subjects has been previously vaccinated (61–100%). Although not reported in **Table 1**, percentage of volunteers, ≥80%, presented underlying diseases or risk factors- for influenzaand as a consequence used chronic drugs. The most frequent chronic diseases- were cardiovascular, respiratory diseases, and diabetes. The most frequent drugs used were- antihypertensive/inotropic drugs and benzodiazepines.-



a Whole: whole-virus vaccine; Sub-u: sub-unit vaccine; Split: split-virus vaccine; MF59: subunit MF59-adjuvanted- vaccine; ID: Intradermal subunit vaccine.-

b I: Institutionalized elderly; M: mixed, both institutionalized and community living elderly.-

c Percent of elderly having received influenza vaccination in the previous year.-

na: not available-

**Table 1.** Characteristics of studied population and type of influenza vaccines in the 27 winter seasons studied (from- 1988/1989 to 2014/2015).-

## **3.2. Vaccines-**

As reported in **Table 1**, different formulations such as whole, split (composed by viruses disrupted, by a detergent, and containing the internal and external component of the virus),- and subunit (composed of just the purified surface glycoproteins of the virus, i.e., hemagglutinin (HA) and neuraminidase) of trivalent inactivated vaccines were used in the different- years or in the same year. In the firstfour studied years (from 1988–1989 to 1991–1992), the- HA concentration for each strain was lower (10 μg for each antigen) as compared with the- concentration (15 μg for each antigen) of the vaccines used in all the years after the winter- season 1991–1992. Whole and subunit formulations were administered respectively to 863- and 1094 volunteers in the first-13 years of the study (from 1988–1989 to 2001–2002). Nine- hundred ninety-six elderly people were vaccinated with split vaccine in many years studied- and, starting from the 1999–2000 season, 1343 volunteers received a subunit vaccine potentiated with MF59 adjuvant. In the last period of the study, a limited number of elderly people- was vaccinated with vaccine administered intradermally (165 volunteers from 2011–2012 to- 2014–2015). The percentages of previously influenza-vaccinatedpeople were high and- ranged from 88 to 100%, not considering 3 years (1990–1991, 69%; 1991–1992, 61%, and 1993–- 1994, data not available).-

The antigenic composition of the vaccines used is reported in **Figure 1**and each year was- formulated according to the recommendations of both "Ministero della Salute (Italy)" andWHO (Northern Hemisphere) for the corresponding studied winter. During the 27-year period- covered by our study (1988–2014), the WHO recommended 15 A/H3N2, 7 A/H1N1, and 12 B- new influenza strains for inclusion in seasonal vaccines.-

## **3.3. Overall response to influenza vaccination-**

The ability of licensed influenzavaccines to elicit an antibody response against vaccine antigens was examined comparing HI antibody titers in blood samples collected from the 4461- volunteers before and 1 month after vaccination with commercially available seasonal trivalent inactivated influenza vaccines in 27 consecutive winters (from 1988–1989 to 2014–2015).-

**Figure 1.** Recommended viruses for influenza vaccines by World Health Organization between 1988 and 2014.-


\*\*: *p*-value < 0.01 comparing pre- and postvaccination values.-

A: *p*-value <0.01 comparing A/H3N2 and A/H1N1 antigens.-

B: *p*-value <0.01 comparing A/H3N2 and B antigens.-

**Table 2.** Mean values of the HI antibody responses observed in the 27-years study of the total population to the three- influenza vaccine antigens and reachment CHMP criteria.-

The HI antibody response after one dose of influenza vaccine was evaluated for each antigen- (A/H1N1, A/H3N2, and B) and data obtained were processed in order to calculate, for each- population considered in the paper, pre- and postvaccination seroprotection rate, seroconversion rate, pre- and postvaccination GMT, and GMTR together to their corresponding 95%- confidenceintervals. For each antigen, the values of these parameters referred to the overall- population are reported in **Table 2**. One month after vaccination, statistically significant- increases were found in the percentage of seroprotected volunteers and in the values of their- corresponding GMT against all the three differentvaccine antigens. The three CHMP requirements were satisfied-1 month after vaccination against the A/H3N2 vaccine component,- whereas only the requested value of GMTR was reached against the A/H1N1 and B antigens.-

**Table 3**reports the results obtained examining the reachment of the CHMP criteria for each- studied year against the three vaccine antigens. The seroprotection rate (HI titer ≥40) was- higher than the requested 60% in 20 years against A/H3N2 (74%), 16 years against A/H1N1- (59%), and 14 years against B antigen (52%) of the 27 years studied. Values of GMTR satisfying- the requested value ≥2 were found in 22 (81%), 25 (93%), and 21 (78%) years against A/H3N2,- A/H1N1, and B vaccine components, respectively. The lower positive results were found for- seroconversion requested to be ≥30%. This value was reached in 13 years against A/H3N2- (48%), 10 years against A/H1N1 (37%), and 8 years against B virus (30%). In some years none- of the three CHMP criteria was satisfied,i.e., in 3 years against A/H3N2 (11%), 2 years against- A/H1N1 (7%), and in 7 years against the B antigen (26%). Years with responses satisfying all- the three CHMP criteria ranged between 22% (B antigen) and 48% (A/H3N2 antigen). Because- the use of a vaccine featuring a novel antigen might affectthe antibody response, considering- data reported about vaccine antigenic composition in **Figure 1**, we identifiedthe presence or- absence of a novel vaccine component in each year studied, but we could not evidence any- obvious association between vaccine HI antibody response and the presence of a new vaccine- component.-


**Table 3.** Reachment of the CHMP criteria in the total population in the 27 years examined.-

The data reported in **Table 2**evidenced differencesin the values of the HI antibody titers- against the three differentvaccine antigens. HI antibody values against A/H3N2 antigen were- in most instances significantlyhigher before and after vaccination as compared with those- found both against A/H1N1 and B vaccine components.-

Since the baseline serological status is considered to be important in evaluating immunogenicity of influenzavaccines and is regarded as capable of affectingthe serological outcomes,- in order to reduce the heterogeneity among the responses found against the three vaccine- antigens, we examined the GMT values of the overall population correcting the postvaccination titers for the prevaccination status according to Beyer (**Figure 2**) [18].-

**Figure 2.** Postvaccination GMT values of: (a) the overall population corrected for the average prevaccination status according to Beyer; and (b) subjects unprotected before vaccination. Comparison of antigens is also shown when differences are significant. The bars indicate the ranges of the 95% confidence limits.-

For comparison purposes, also the postvaccination GMT values of the prevaccination unprotected volunteers (HI < 40) are shown in **Figure 2**as indicated by the corresponding labels. The- data reported confirmedthat the responses against the A/H3N2 antigen were higher as- compared with those against A/H1N1 and B antigens.-

#### **3.4. Factors associated with vaccine response-**

Since differentfactors may have an impact on vaccine response, we controlled for a number- of variables for which we could obtain data. We did not consider the health status of the- study participants, previous vaccination histories, and living situation, since a high percentage of the subjects had chronic underlying disease, was previously vaccinated, and was living in a nursing home.-

#### *3.4.1. Subanalysis according to different influenza vaccine dosages-*

In Italy, as in most European countries, seasonal trivalent influenzavaccines containing- 10 μg HA for each antigen (30 μg) has been used until 1991. From 1992 onwards European- influenza vaccines contain 15 μg HA per strain (45 μg), according to the European Harmonization of Requirements for Influenza-Vaccines [17]. As a consequence, in the first-4 years of- the 27-year period examined in our study, we used 30 μg and, after the winter 1991–1992, 45- μg vaccines.-

Since previous observations suggested that increase in influenzavaccine dosage might be- associated with an increase in antibody titers, at least against some of the vaccine strains [19,- 20], we compared HI immune response following vaccination with 30 or 45 μg vaccines. As- reported in **Table 1**, 860 (19%) and 3601 (81%) of the 4461 elderly subjects received respectively- a 30 or a 45 μg trivalent influenzavaccine. **Table 4**reports the results obtained studying the- induced HI antibody response. Significant increases were observed against all the three vaccine- antigens comparing pre- and postvaccination data against all the three differentvaccine- antigens examining the percentages of seroprotected people and GMT values both after 30 and- 45 μg vaccine administration.-


\*\*: *p*-value <0.01 comparing pre- and postvaccination values.-

A: *p*-value <0.01 comparing response between vaccine dosages (30 and 45 μg).-

**Table 4.** HI antibody response in volunteers divided according to the vaccine dosage (30 or 45 μg).-

At least one of the three CHMP requirements, i.e., the value of GMTR (≥2), was always reached- using vaccine containing 30 μg of antigen but following 45 μg vaccine administration all the- three parameters were satisfiedagainst A/H3N2 antigen and two of them against the B antigen.-

Postvaccination results observed after 45 μg vaccine administration were always significantly- higher as compared with those after 30 μg vaccine.-

However, comparing values found in the two groups of people before vaccination, we observed- that the two groups were poorly comparable since there were differencesin the prevaccination-

Antibody Responses after Influenza Vaccination in Elderly People: Useful Information from a 27-Year Study... 225 http://dx.doi.org/10.5772/104559

**Figure 3.** Postvaccination GMT values of populations divided according to the vaccine dosage (30 or 45 μg)), as indicated by legend labels. Postvaccination GMT values calculated on the overall population have been corrected for the average prevaccination status according to Beyer. For comparison purposes, post-GMT values of subjects unprotectedbefore vaccination are also shown. The bars indicate the ranges of the 95% confidence limits.-

status. Volunteers vaccinated with 45 μg vaccine showed prevaccination HI titers in most- instances significantlyhigher as compared with the 30 μg volunteers. In order to have more- homogeneous and comparable data, we examined vaccine immunogenicity both correcting- the titers for prevaccination status of overall population [17], and considering only prevaccination unprotected volunteers (HI titers < 40). As shown in **Figure 3**, GMT corrected for- prevaccination status confirmedthat the increasing of the antigen dosage increments the- response to the vaccine antigens. Postvaccination values found considering only people- nonseroprotected before vaccination again evidenced a statistically significant higher response- induced by 45 μg vaccine as compared with 30 μg.-

#### *3.4.2. Subanalysis of immunogenicity within the elderly groups, i.e., younger elderly (≤75 years) and- very elderly (>75 years)-*

In a recent meta-analysis about the effectof age on the influenzavaccine–induced immune- response based on studies from the past 20 years, Goodwin at al. [12] concluded that aged- individual (>65 years) had a significantlyreduced antibody response to vaccination. The- studied elderly were categorized into two age groups, above or below 75 years. Antibody- responses among the very elderly (≥75 years of age) were especially impaired with seroconversion levels at 32%, 46%, and 29% to A/H1N1, A/H3N2, and influenza-B, respectively,- compared with 42%, 51%, and 35% observed in people aged <75 to >65 years of age [12].-

 Inordertohaveadditiveinformationweconsideredtheimmuneresponsesfoundinvolunteersofourstudyaged-≤75or->75years.-Theexactagewasavailableforonly-2712people- (61%)ofthe-4461participantsand-658-(24%)wereaged-≤75yearsand-2054-(76%)were- >75years.-Theresultsobtainedarereportedin-**Table-5**andshowthatinbothgroupsthe- vaccineadministrationinducedsignificantincreasesin-HItitersevaluatedaspercentageof- seroprotectedpeople-(HI-≥-40)andas-GMTvalues.-CHMPcriteriawerealwayssatisfiedfor- GMTRparameter (≥2)againstallthethreevaccineantigens.-Allthethreerequestedvalues- werereachedinbothgroupsagainst-A/H3N2antigenandonlyin->75yeargroupagainst-A/- H1N1antigen.-Againstthe-Bantigen,therequestedvalueforseroconversion (≥30%)wasnot- reachedinbothgroupsandthevalueforseroprotection (≥60%)wassatisfiedonlyin->75 yeargroup.-


\*\*: *p*-value <0.01 comparing pre- and post-vaccination values.-

A: *p*-value <0.01 comparing response between age groups.-

a: *p*-value <0.05 comparing response between age groups.-

**Table 5.** HI antibody response of populations divided according to the age (younger elderly, ≤75 years, and very- elderly, >75 years).-

Comparing results obtained in the two groups, the responses observed in the oldest group- (>75) were in most instances higher than those observed in the younger elderly (≤75). However,- since the prevaccination status of these two groups were not fully comparable, we evaluated- the values of GMT corrected for prevaccination status and GMT in people unprotected (HI < 40)- before vaccination. Again the values were higher in the very elderly as compared with theyounger against A/H1N1 for GMT corrected and against A/H1N1 and B for the GMT unprotected people (**Figure 4**).-

**Figure 4.** Postvaccination GMT values of populations divided according to the age class (younger elderly, ≤75 years,- and very elderly, >75 years), as indicated in legend labels. Postvaccination GMT values calculated on the overall population have been corrected for the average prevaccination status according to Beyer. For comparison purposes, post-GMT values of subjects unprotected before vaccination are also shown. The bars indicate the ranges of the 95%- confidence limits.-

## *3.4.3. Subanalysis according to responses found in females and males-*

Previous data indicated that receipt of trivalent inactivated influenzavaccines results in- significantlyhigher HI antibody titers among females than males, both in adults and elderly- people [21].-

In our study, sex data were available for about all the people studied (4457/4461) and the- volunteers were prevalently females (70%). We examined the vaccine immunogenicity in- females and males and the results are reported in **Table 6**. Postvaccination increases found- against all the three vaccine antigens were statistically significantin both groups. All the three- CHMP criteria were satisfiedagainst A/H3N2 antigen in female subjects, whereas only the- GMTR requirement was satisfiedin males against A/H3N2 and both in males and females- against A/H1N1 and B antigens. Comparison of postvaccination values evidenced statistically- higher values in the female compared with male group. However, since differences were found- also in the prevaccination values we compared the GMT corrected for the prevaccination status- and examined the GMT found considering only volunteers not seroprotected before vaccination. The female responses were again higher than those of male against all the three vaccineantigens (**Figure 5**).-


\*\*: *p*-value <0.01 comparing pre- and post-vaccination values.-

A: *p*-value <0.01 comparing response between M and F.-

**Table 6.** HI antibody response of populations divided according to gender (male: M; female: F).-

**Figure 5.** Postvaccination GMT values of populations divided according to gender (male: M and female: F) as indicated- in legend labels. Postvaccination GMT values calculated on the overall population have been corrected for the average- prevaccination status according to Beyer. For comparison purposes, post-GMT values of subjects unprotected before- vaccination are also shown. The bars indicate the ranges of the 95% confidence limits.-

## *3.4.4. Evaluation of vaccine immunogenicity in "strong responder"-*

Examining the antibody response after influenzavaccination, McElhaney et al. [22] considered as a vaccination efficiency-relatedparameter the HI antibody titer ratio between day 30- and day 0 and identifiedas weak/nonresponder people with a ratio 1– < 4 and as strong responders those with a ratio ≥4, i.e., people who seroconverted after vaccination. Using the- same parameter we decided to evaluate in the groups identifiedas strong responders the induction of HI antibody response evaluated as GMT values against the three vaccine antigens.-

The data obtained comparing results found in people who seroconverted after vaccination are- reported in **Table 7**, and in most instances confirmed the results obtained examining the overall- population of subgroups vaccinated with vaccine containing different dosages of antigens or- subdivided in male and female. The responses induced by a 45 μg vaccine or in female were- in most instances statistically higher than those induced by a 30 μg vaccine or in male volunteers, respectively. Moreover, the immune responses evaluated in volunteers with an age ≤ or- >75 years were similar against A/H1N1 and B antigens and higher against the A/H3N2 antigen- in people aged >75 years as compared with response in those ≤75 years.-


\*\*: *p*-value <0.01 comparing pre- and postvaccination values.-

A: *p*-value <0.01; a: *p*-value <0.05 comparing response between different groups.-

**Table 7.** HI antibody response of strong responder population divided according to the vaccine dosage (30 or 45 μg),- age class (younger elderly, μ75 years, and very elderly, >75 years), and gender (male: M; female: F).-

### *3.4.5. Subanalysis according to the different types of vaccine used-*

Finally, since differentvaccine formulations (whole, subunit, split, MF59-adjuvanted, and intradermally administered) were used in the 27 years studied, we compared the results obtained after administration of the differenttypes of vaccine. Chi-square and one-wayanalysis of variance (ANOVA) were used for evaluating multiple comparisons amonggroups vaccinated with the differentvaccine types. Estimates and comparison intervals areshown in **Figure 6**. Paired comparison *p*-values resulting from the multicomparison test arereported in **Tables 8**only when one-way ANOVA comparison identifiedpotentially significant differences.-

**Figure 6.** Values of CHMP parameters against the three vaccine antigens following vaccination with whole (*N-*= 863),split (*N-*= 996), subunit (*N-*= 1094), MF-59 adjuvanted (*N-*= 1343), and intradermally administered (*N-*= 165) influenzavaccines. The black-dashed bold line in each figurerepresents the CHMP threshold value for the corresponding parameter. The bars indicate the ranges of the 95% confidence limits.-

All vaccines used induced HI antibody responses satisfying at least one (prevalently GMTR- value ≥2) of the three CHMP criteria. The antibody response induced by whole vaccine was in- most instances lower as compared with responses induced by the others vaccines (**Table 8**).- However, as reported in **Table 1**, many of the volunteers vaccinated with whole vaccine in the- firstyears of the study received a vaccine with a low dose of antigen (30 μg). The responses- induced by split and subunit vaccines against A/H3N2 and B antigens were similar; on the- contrary against A/H1N1 antigen, the response induced by split vaccine was significantly- lower as compared with subunit.-

The two enhanced vaccines, MF59-adjuvanted and intradermal, induced similar and higherresponses compared with conventional vaccines against A/H3N2 antigen.-

Against A/H1N1, the response induced by MF59-adjuvanted vaccine was in most instanceshigher than conventional and intradermal vaccines.-

Against B antigen, intradermal vaccine induced higher HI response than that induced by- conventional and MF59-adjuvanted vaccines. In some cases the differenceswere statistically- significant.-


Whole: whole-virus vaccine; Sub-u: sub-unit vaccine; Split: split-virus vaccine; MF59: subunit MF59-adjuvanted- vaccine; ID: Intradermal subunit vaccine.-

**Table 8.** Paired comparison of results obtained in volunteers divided in groups according to the type of vaccine used- for immunization. *p*-values resulting from the multicomparison test are reported only when one-way ANOVA- comparison identified potentially significant differences.-

## **4. Discussion-**

This study describes the humoral antibody response of 4461 elderly frail institutionalized- volunteers prevalently vaccinated in the previous year after vaccination with influenza- inactivated trivalent vaccines commercially available for the differentyears studied during a- 27-year period (from winter season 1988–1989 to 2014–2015).-

The firstdata were obtained by examining the results found in the 27-year period studied as- crude mean responses and evidenced the ability of influenzavaccine administration to elicit- antibody response in elderly volunteers (**Table 2**). One month after vaccination, significant- increases were found against all the three vaccine antigens; however, vaccination induced- significantlyhigher HI antibody titers against A/H3N2 antigen as compared with A/H1N1 and- B strains. The higher responses against A/H3N2 strain were substantially confirmedconsidering the number of years in the 27-year period examined in which the CHMP criteria werefulfilled (**Table 3**) or comparing GMT values after correction for baseline titers or considering- responses in prevaccination unprotected people (**Figure 2**).-

In accordance with our results, higher titers after vaccination against A/H3N2 strain were- previously found by Sasaki et al. [23] and Ohmit et al. [24], but it was not possible to discriminate between the possibility that A/H3N2 antigen is more immunogenic than A/H1N1 and B- antigens or the possibility that the higher GMT and protection rate values might depend from- earlier contact with the A/H3N2 virus due to vaccination or natural infection. Since all the- volunteers were previously vaccinated, the possibility of the influenceof a differentcirculation- of A/H3N2 strains is more acceptable. The A/H3N2 viruses have the highest rate of evolution- among the three influenzasubtypes currently circulating, with antigenically distinct strains- emerging on average 2–5 years and capable of a betterdiffusion among the population [25].-

Further considerations about the results obtained derive from *post hoc*analyses conducted to- determine whether vaccine dose, age, sex, and type of vaccine might influencethe vaccineinduced humoral immune response.-

Although the issue of increase in the antibody titers following increase in influenzavaccine- dosage is not completely clarified [19, 20, 26], our data found using vaccines with 30 or 45 μg- of antigens for vaccine dose, suggested that the increase in influenzavaccine dosage is- generally associated with an increase in the induction of antibody titers. Significant antibody- titers increases were observed both administering vaccines with 30 or 45 μg of antigens for- vaccine dose against all the three vaccine antigens. However, postvaccination values following- vaccination with 45 μg vaccine were in most instances statistically higher as compared with- 30 μg both considering mean values for the overall population (**Table 4**) or GMT corrected for- prevaccination status or calculated in prevaccination unprotected volunteers (**Figure 3**). In- accordance with these observations, recently (December 2009) in the United States, a high-dose- (60 μg HA per strain) trivalent inactivated influenza vaccine was licensed for people 65 years- of age or older. The high dose vaccine was found to improve in people aged ≥65 years both- antibody response and protection against laboratory-confirmed influenza illness [27, 28].-

Considering vaccine immunogenicity in younger elderly (≤75 years) or in very elderly- (>75 years), vaccine administration induced statistically significantincreases in both groups.- Comparing the two groups, the values were in many instances slightly higher in the very- elderly as compared with younger elderly, and in some instances the differenceswere- statistically significant.-However, the differencespersisted against A/H1N1 antigen both after- correction for prevaccination status or calculation in unprotected volunteers before vaccination, and against B antigen only considering responses in unprotected people (**Figure 4**). The- highest response of very elderly people as compared with younger elderly volunteers might- be due to the fact that they probably represent a more selected group of elderly people capable- of longer surviving and with a possible lower degree of age-associated alteration of the- immune system [29].-

However, since the differenceswere particularly evident against the A/H1N1 strain and are in- accordance with previous data found in our laboratory showing in two different winter seasons- a higher ability to give HI antibody response against A/H1N1 strains of people born between1903 and 1919 as compared with volunteers born between 1920 and 1957, we cannot exclude- the possibility that the differencesmight be due to cross-reactivity generated from exposure- to the 1918 A/H1N1 virus or related A/H1N1 strains [30].-

As far as sex could influencethe immune response against influenzavaccines, our results- confirmedprevious data indicating that receipt of trivalent inactivated influenzavaccines- results in significantlyhigher HI titers among females than males, both in adults and elderly- people [21]. Significantrises in antibody titers were found after vaccination both in males and- females, but the values observed in females were significantlyhigher as compared with males- (**Table 6**) and the differencespersisted also considering only GMT of volunteers unprotected- before vaccination or GMT corrected for prevaccination status (**Figure 5**).-

Sex hormones have been considered to be the most important mediators of sex differencesand- males with high level of testosterone have been found to have low antibody responses after- influenza vaccination [31, 32].-

However, since our data were obtained in elderly people, i.e., after the reproductive senescence, they support the hypothesis that the sex hormones are not the only mediator of sex- differencesin humoral response to influenzavaccination and there is the possibility that- genetic differencesalso might underlie sex-based differencesin adaptive immune response to- viral vaccines [21, 33].-

These results (vaccine dose, age, and sex) were, at least in part, confirmedalso considering- responses evaluated in strong responder, i.e., in volunteers showing a positive response after- vaccination (**Table 7**).-

Comparison of the differenttype of vaccines used in the 27-year period evidenced higher- immunogenicity of the new "enhanced vaccines" specially licensed for elderly individuals,- i.e., adjuvanted and intradermally administered vaccines, as compared with traditional whole,- subunit, and split vaccines (**Table 7**, **Figure 6**) supporting previously published data [34, 35].-

Our study had several limitations. The most important are that our observations may apply- only to frail seniors living in care facilities and that the subanalysis groups were not fully- comparable. However, since institutionalized people represent a significanttarget group for- influenzavaccination, it is important to analyze their response to influenzavaccines. An- additional limitation is the lack of data demonstrating clinical efficacyagainst influenza- infection and illness. Although there is substantial evidence that HI antibody titers represent- a good correlate of protection from severe illness in young adults, the predictive value of these- measurements in older adults might be variable. Although the number of volunteers and of- winter seasons we examined was considerable and comparable to the data reported in a review,- differentlyfrom a review on influenzavaccine immunogenicity, the results obtained in each- year were considered cumulatively not taking into account of the differentcharacteristics of- the vaccines used through the 27-year period. Indeed, the antigenic composition of influenza- vaccines differ,even considerably, from one year to another, since it is updated each year to- match the strains circulating in the community and inactivated influenza vaccines are available- in differentformulations (whole, split, and subunit with or without adjuvants), which are- administered intramuscularly or intradermally. Moreover, a further aspect that should becarefully considered as compared with those of a review on HI antibody titers after influenza- vaccine administration is the HI assay itself. HI test is not standardized across laboratory and- was found to be highly variable and sensitive to factors such as reagents, erythrocyte source,- and virus passage history. The results reported in the present report were all obtained in the- same laboratory, although in differentyears and although, some changes were introduced in- the HI test used during the 27-year period as reported in Section 2.-

In conclusion, our data evidenced that the use of influenzavaccination appears to be an- appropriate strategy to address the challenge of influenzainfections of the elderly. However,- they underline the need of studies for new improved influenza vaccines, since, as previously- found, the vaccine-induced HI antibody responses against the three vaccine antigens were- different and resulted not satisfactory against A/H1N1 and B antigen, since the postvaccination- values of seroprotected volunteers were lower than the requested 60% (**Table 3**).-

Moreover, they underline the necessity to expand researches and approaches to understand- immunosenescence and its relationship to vaccine-induced immunity in order to have more- valid vaccines. The vaccine-induced stimulation of HI antibody response following vaccination- was found not only to be higher against one vaccine component as compared with the other- two, but also to be influencedby differentfactors as vaccine dose, age, sex, and type of vaccine.- It is therefore important, as suggested by Lambert et al. [5], both to understand the mechanisms- that result in these differencesand to use such information to devise more immunogenic- influenza vaccine candidates.-

## **Author details-**

Barbara Camilloni\* , Emilia Nunzi, Michela Basileo and Anna Maria Iorio-

 \*Addressallcorrespondenceto:barbara.camilloni@unipg.it-

 Departmentof-Experimental-Medicine,-Universityof-Perugia,-Perugia,-Italy-

## **References-**


**Chronic Obstructive Pulmonary Disease (COPD): Clinical and Immunological Effects of Mono-Vaccination Against Influenza Using an Immunoadjuvant Vaccine of a New Class Versus Combined Administration S. pneumoniae, H. influenzae, and Influenza Vaccines** 

Andrey Dmitrievich Protasov , Mikhail Petrovich Kostinov , Alexander Victorovich Zhestkov , Mikhail L'vovich Shteiner , Svetlana Vyacheslavovna Kazharova , Yuriy Vladimirovich Tezikov and Igor Stanislavovich Lipatov

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64292

## **Abstract**

 In-Russian-Federation,-27,300,000–41,200,000acuteupperandlowerrespiratory- infectionsarereportedannually.-Patientswithchronicobstructivepulmonarydisease- (COPD)areathigherriskofseverecourse,complications,andlethaloutcomesof- influenza.-About-30%of-COPDexacerbationsareduetoviralinfections,andinfluenza- Aand-Bvirusesareamongthemostcommoncauses.-Theaimofourstudywastoassess- exacerbationrate,numberofcoursesofantibioticchemotherapy,pulmonaryfunction,- andimmunologicaleffectsofmono-vaccinationwithanewimmunoadjuvantinfluenza- vaccinevs.combinedvaccinationagainstpneumococcalinfection,-Hemophilustypeb- infection,andinfluenzain-COPDpatients.-Bothcomplexvaccinationagainst-Streptococcuspneumoniae,-Haemophilusinfluenzaetypeb,andinfluenzaandmonovaccinationwithanewimmunoadjuvantinfluenzavaccineledtostatisticallysignificant- reductioninthenumberof-COPDexacerbationsandofantibioticchemotherapy- courses.-Basedontheobtainedresults,widespreadimplementationofmono-vaccina-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 tionagainstinfluenzawithanewimmunoadjuvantinfluenzavaccine,aswellas- complexvaccinationagainstbacterialrespiratoryinfectionsandinfluenzacanbe- recommendedfor-COPDpatients,asvaccinationisbeneficialfortheirfunctionalstatus,- thatis,improvesforcedexpiratoryvolumein-1s-(FEV1)and-6-minutewalktestresults.- Inourstudy,weevaluatedimmunogenicityofthenewinfluenzaimmunoadjuvant- vaccineadministeredasmono-vaccineto-COPDpatientsinaccordancewith-Committee- for-Proprietary-Medicinal-Products-(CPMP)requirements.-

**Keywords:** chronic obstructive pulmonary disease (COPD), vaccination, influenza,- clinical effects, immunological effects-

## **1. Introduction-**

 In-Russia,-27,300,000–41,200,000acuteupperandlowerrespiratoryinfectionsarereported- annually.-Patientswithchronicobstructivepulmonarydisease-(COPD)areathigherriskof- severe course, complications, and lethal outcomes of influenza. Therefore, influenza prevention- insuchpatientsisoneofthemosturgenttasks.-

About 30% of COPD exacerbations are due to viral infections, and influenza-A and B viruses- are among the most common causes.-

The best approach to prophylaxis is provided by vaccination because it combines the advantages of specificity, efficacy,safety, and cost-effectiveness.-The GOLD guidelines (Global- Initiative for Chronic Obstructive Lung Disease) recommend that influenzavaccination- preferentially with split or subunit vaccines must be integrated into treatment strategies for- all COPD patients regardless of the disease stage. However, further studies are needed to- evaluate the efficacy and immunogenicity of modern adjuvant influenza vaccines [1].-

It is not rarely that the risk of COPD exacerbation is associated not directly with the influenza- virus as such, but rather with the development of bacterial superinfection, mainly caused by- Streptococcus pneumoniae or Haemophilus influenzae type b [2–4].-

In the Russian Federation, both inactivated and live vaccines against influenzahave been- licensed [5]. Antigenic composition of these vaccines is modifiedannually to adopt current- epidemic situation and WHO guidelines. Recently, an innovative new class of immunoadjuvant influenzavaccines has evolved. The use of these vaccines in COPD patients will be- outlined below.-

According to CPMP criteria (Committeefor Proprietary Medicinal Products), a vaccine is- considered immunogenic, if at least one of the assessments meets the indicated requirements:-


**3.-** Mean titre increase after vaccination: ≥2.5 in individuals aging 18–60 years and ≥2 in- individuals above 60 years.-

Vaccination against influenzafor COPD patients is included into the National Immunization- Calendar of the Russian Federation.-

S. pneumoniae, H. influenzae,-M. catarrhalis, and influenzavirus are the most common causes- of COPD infective exacerbations [6]. The standard of care for COPD patients includes vaccination against influenzaand pneumococcal infection. Several serotypes of H. influenzaeare- known, and COPD exacerbation may be caused by each of them, including H. influenzaetype- b. Vaccine against H. influenzaetype b is now available; therefore, it is of interest to evaluate- a combined vaccination against S. pneumoniae, H. influenzae,and influenzain COPD patients- and the effects of these vaccines on exacerbation rate and pulmonary function tests.-

In adult COPD patients, the pioneer studies evaluating the therapeutic effectof PPV23 were- performed in 2004. Elimination of S. pneumoniae from the sputum was observed in 52.9%- cases, that is, at a lower rate compared to children. Other findings include increased levels of- IgG against S. pneumoniae serotypes 3, 6B, 9N, 23F; decreased total IgE, and increased Wright's- phagocytic index [7].-

Immunization of COPD patients with PPV23 contributed to a 2.2-fold decrease in exacerbation- rate by 18 months; at 12 months post vaccination, the duration of acute episodes decreased 1.8 fold compared to the control group [8].-

Though disputable, the preliminary results regarding therapeutic effects of vaccination against- respiratory infections were thus obtained. The common controversy is whether it is possible- to improve the respiratory function tests in COPD patients through vaccination.-

Study aim—to assess exacerbation rate, number of courses of antibiotic chemotherapy,- pulmonary function, and immunological effectsof mono-vaccination with a new immunoadjuvant influenzavaccine vs. combined vaccination against pneumococcal infection, Haemophilus type b infection, and influenza in COPD patients.-

#### **2. Material and methods-**

The study enrolled 170 patients with grade 1, 2, 3, 4 COPD (age 30–80 years) who had signed- informed consent according to the study protocol approved by the ethics committeeof the- Samara State Medical University (Russian Federation) and Research Institute of Pulmonology- (Moscow, Russian Federation). The diagnosis was determined according to GOLD guidelines- (2012) [9].-

Patients were divided into four groups. Group I enrolled 50 patients with COPD who continued to receive basic therapy for the main disease and were vaccinated with commercially- available vaccines against pneumococcal infection (Pneumo 23, France), H. influenzae type b- infection (Hiberix, Belgium), and influenza-(new immunoadjuvant vaccine Grippol® plus,- Russian Federation). Vaccines were administered once intramuscularly into various parts of- the body. Two patients from Group I did not complete the study per protocol (one patient died- after a trafficaccident, and one patient died from sudden massive pulmonary embolism).- Therefore, the data from these patients were not included into finalanalysis, and it was based- on 48 patients of Group I.-

Group II (control for patients who received complex vaccination against pneumococcal- infection, Haemophilus type b infection, and influenza)consisted of 80 patients with COPD- of similar grade, who were not vaccinated and received only basic therapy.-

Group III consisted of 20 COPD patients vaccinated against influenzawith a new immunoadjuvant vaccine. Group IV (control for patients who received mono-vaccination against- influenzawith a new immunoadjuvant vaccine) enrolled 20 unvaccinated COPD patients.- Groups II and IV were composed of patients who categorically rejected any vaccination,- despite the information provided. Nevertheless, these patients gave their consent to participate- in the study. All study patients were followed up for 1 year and subjected to function and- immunological tests at baseline and at 12 months.-

The use of two control groups was associated with enrollment of patients in two various study- centers. Each study center enrolled subjects either to Group I (group of complex vaccination- and control group) or to Group III (group of mono-vaccination against influenza and control- group). Another reason for the use of two control groups was the probability that baseline- characteristics of the patients in Group 1 and Group 3 will not be well balanced, and their- comparison with the total control group will be incorrect. Our study is not a direct comparison- between the groups of complex vaccination and mono-vaccination. We just compare each of- these groups with its own control.-

All study patients underwent a history taking (identificationof risk factors for COPD,- complaints for cough, sputum discharge, dyspnea of any grade worsened by physical- exercise). To verify the diagnosis of COPD, all patients were subjected to pulmonary function- tests and broncholytic test with 400 μg of salbutamol according to the standard techniques [8].- The study enrolled patients with Tiffeneauindex forced expiratory volume in 1 s/forced vital- capacity (FEV1/FVC) below 70%.-

#### **Inclusion criteria:-**


## **Exclusion criteria:-**


Patients were followed up by general practice physicians, pulmonologists, or allergologistsimmunologists in the outpatient context or in hospitals, if hospitalization was required. In- cases of COPD exacerbation, if necessary, patients were hospitalized to departments of- pulmonology.-

Patients meeting inclusion/exclusion criteria were divided into four groups. Groups II and IV- enrolled patients who categorically rejected any vaccination. Other patients were firstrecruited- to Group I to undergo complex vaccination and then to Group III to be vaccinated against- influenzausing a new immunoadjuvant vaccine. Sample size was determined by the number- of vaccines.-

All patients received basic bronchodilatory and anti-inflammatorytherapies in accordance- with the disease severity and GOLD guidelines (2012). At baseline, groups were well balanced- for age, gender, disease severity, and scope of basic therapy, which remained unchanged- throughout the study period. Vaccination was performed at remission in the outpatient- context, follow-up period lasted for 12 months after vaccination.-

PPV23 vaccine is a polyvalent pneumococcal vaccine manufactured by Sanofiaventis (France).- It contains purifiedcapsule S. pneumoniae polysaccharides of 23 serotypes. One dose of- vaccine is 0.5 ml.-

 Hiberixisaconjugatedvaccinetopreventinfectionscausedby-H.influenzaetypeb- (GlaxoSmithKline-Biologicalss.a.,-Belgium).-Onedosecontains-10μgofpurifiedcapsule- polysaccharideisolatedfromstr.-H.influenzaetypebconjugatedwith-30μgoftetanus- toxoid.-

Grippol plus vaccine (NPO Petrovax Pharm, Russia) is a trivalent polymeric subunit immunoadjuvant vaccine (Petrovax, Russia) containing protective antigens isolated from purified- influenzatype A and B viruses grown in chicken embryos. One immunization dose (0.5 ml)- contains at least 5 μg of hemagglutinin of epidemiologically relevant influenzasubtype A- (H1N1 and H3N2) and type B viruses, and polyoxidonium immunoadjuvant 500 μg in sodium- phosphate buffer. Vaccine is free of preservatives.-

Clinical efficacyof vaccination was assessed by the number of COPD exacerbations during the- year before vaccination and after vaccination. COPD exacerbation was definedas increased- dyspnea, cough, and sputum volume requiring medical advice and modificationof current- therapy documented by primary medical records.-

Ventilation function was investigated using a Spiro S-100 spirometer (Russian Federation). The- following parameters were measured: forced vital capacity (FVC), forced expiratory volume- in 1 s (FEV1), and a calculated ratio of these two parameters (FEV1%/FVC), that is, modified- Tiffeneautest. Exercise tolerance was assessed using the 6-minute walk test (6MWT) according- to the standard protocol.-

The levels of IgG antibodies against influenza virus strains were measured using standard HAI- assay (hemagglutination inhibition assay) in accordance with the manufacturer's instructions- to the kit.-

The results were studied statistically using StatPlus 2009 Professional 5.8.4. Measures of central- tendency and dispersion were chosen depending on the data distribution type. Continuous- variables corresponding to normal distribution are presented as means (SD); variables- differingfrom normal distribution as medians (interquartile distance). Categorical variables- are presented as proportions (%) and absolute values.-

The choice of statistical test depends on the data distribution type and evaluation of dispersion- equality. The hypothesis of data distribution normality was tested (Shapiro-Wilks test). In cases- of normal distribution of data in each sample, dispersion is to be compared between two- distributions (Levene's test). If both criteria are met, Student's t test is selected, if no, its nonparametric alternative is used (Mann-Whitney test). The same is applicable to paired tests for- comparisons of changing variables (pairwise Student's t test or Wilcoxon test for paired- comparisons). Comparative analysis of categorical variables was performed using exact twosided Fisher's test. Differences were statistically significant at p < 0.05 [10].-

Prospective study design allows calculating the following statistics:-


About 95% CI of the differenceof absolute risks (AR): the results are statistically significant, if- 95% CI will not contain 0.-

About 95% CI of the relative risk (RR): the results are statistically significant,if 95% CI will not- contain 1.-

About 95% CI for the odds ratio (OR): the results are statistically significant,if 95% CI will not- contain 1.-

## **3. Results and discussion-**

#### **3.1. Clinical effects of vaccination in COPD patients-**

The clinical effectsof vaccination (exacerbation rate) in COPD patients are characterized by- groups in **Table 1**.-


**Table 1.** Number of exacerbations (calculated statistics) in COPD patients per year before and after vaccination.-

Both complex and mono-vaccination resulted in a statistically significantreduction of the- number of COPD exacerbations. The risk of COPD exacerbation was reduced by 54% after- complex vaccination vs. 30% after mono-vaccination (p > 0.05 for all comparisons).-


**Table 2.** Number of courses of antibiotic chemotherapy (calculated statistics) in COPD patients per year before and- after vaccination in the groups considered.-

As compared to unvaccinated patients, complex vaccination reduced the risk of COPD- exacerbation by 59 vs. 38% reduction after mono-vaccination vs. increase by 6% in unvaccinated patients.-

In the group of complex vaccination against pneumococcal, Haemophilus type b infection, and- influenza,the chance of COPD exacerbation was 5% of that in unvaccinated patients (p < 0.05).- In the group of mono-vaccination, the chance of COPD exacerbation was 25% of that without- vaccination (p > 0.05).-

**Table 2**presents characteristics of the clinical effectof vaccination (i.e., number of courses of- antibiotic chemotherapy) in different groups of COPD patients.-

Both complex and mono-vaccination led to statistically significantreduction in the number of- antibacterial chemotherapy courses.-

The risk of antibiotic chemotherapy was reduced by 54% in the group of complex vaccination- against pneumococcal, Hemophilus type b infection, and influenzavs. 30% in the group of- mono-vaccination (p > 0.05 for all comparisons).-

As compared to no vaccination, complex vaccination reduced the risk of antibiotic chemotherapy in patients with COPD by 64%, whereas mono-vaccination reduced it by 35%.-

In the group of complex vaccination against pneumococcal, Hemophilus type b infection, and- influenza,the chance of antibiotic chemotherapy was 8% of that in unvaccinated patients (p <- 0.05). In the group of mono-vaccination, the chance of antibiotic chemotherapy was 21% of that- in unvaccinated (p < 0.05).-

#### **3.2. The effect of vaccination on the pulmonary function tests in COPD patients-**

In COPD patients from Groups I and II, FVC changes did not undergo any statistically- significantchanges over 12 months (**Table 3**). In Group I (patients vaccinated against pneumococcal, Hemophilus type b infection, and influenza),forced expiratory volume in 1 s (FEV1)- increased to 57.4% (2.0%) at 12 months vs. 53.9% (2.7%) at baseline (p < 0.05). In unvaccinated- patients (Group II), these parameters remained unchanged: 54.1% (1.9%) at baseline vs. 50.4%- (2.8%) at 12 months (p > 0.05), indicating that the rate of FEV1 decrease was lower in vaccinated- COPD patients (Group I). Detailed characteristics of FEV1 changes in Groups I and II depending on the disease severity are given in **Table 4**, and **Table 5**outlines the same for Tiffeneau- index.-


Data are presented as means (standard deviation).-

Group I patients in the numerator, Group II patients in the denominator.-

& p < 0.05—differences between Groups I and II (Student's test).-

**Table 3.** FVC changes in COPD patients from Groups I and II over 12 months.-


Data are presented as means (standard deviation).-

Group I patients in the numerator, Group II patients in the denominator.-

& p < 0.05; && p < 0.01—differences between Groups I and II (Student's test).-

**Table 4.** FEV1 changes in COPD patients from Groups I and II over 12 months.-


Data are presented as means (standard deviation).-

Group I patients in the numerator, Group II patients in the denominator.-

& p < 0.05—differences between Groups I and II (Student's test).-

**Table 5.** Tiffeneau index changes in COPD patients from Groups I and II over 12 months.-

One year post vaccination, vaccinated COPD patients (Group I) showed 6MWT results that- were 75.2% (2.8%) of the desired values vs. 60.4% (2.3%) in Group II patients (p < 0.001). Detailcharacteristics of the dynamics of the 6-minute walk test depending on the disease severity are- given in **Table 6**.-


Data are presented as means (standard deviation).-

Group I patients in the numerator, Group II patients in the denominator.-

\* p ˂ 0.05 vs. baseline values in Group I (Student's test).-
