**3. Development of live influenza vaccines against potentially pandemic avian influenza**

The LAIVs preparation against potentially pandemic avian influenza viruses is conducted in two directions: the preparation of vaccine strains using classical genetic reassortment in chick embryos or through reverse genetics (RG) technique. The first attenuated A/Ann Arbor/6/60 (H2N2)-based vaccine strains were obtained by reverse genetics shortly after H5 influenza outbreaks in Hong Kong in 1997 [41].

The vaccine candidates containing internal genes from the attenuation donor, and the surface antigens from viruses A/Hong Kong/156/97 (H5N1) or A/Hong Kong/483/97 (H5N1) with RG-modified HA demonstrated an attenuated phenotype for ferrets and chickens. Both reassortants caused seroconversions in chickens, which confirm the sensitivity of chickens to these vaccine strains despite the *att-*phenotype [42]. By RG methods, three reassortant strains based on the A/Ann Arbor/6/60 (H2N2) were prepared in the Vero cell line [43]. As a source of surface antigens, viruses H5N1 of 1997, 2003, and 2004 years of isolation with RG-modified HA were used. It was shown that a double immunization with LAIV from a strain isolated in 1997 completely protected mice from infection with later "wild" isolates, including the isolate obtained in 2005—A/Indonesia/05/2005 (H5N1). The use of HP viruses requires increased biosecurity level laboratories, certified cell lines, and RG techniques. The HP avian viruses found in nature cannot be used directly to prepare influenza vaccines because they would not grow in eggs and might be dangerous to people. The RG-modified viruses do not cause severe illness in birds and that also will grow well in chicken eggs (so that vaccine manufacturers can use it to produce vaccine). An alternative approach is to use low pathogenic surrogate viruses that show antigenic similarity to HP viruses. In this regard, the identification of non-pathogenic variants, which are antigenically close to potentially pandemic strains, may be very important.

Another vaccine candidate based on A/Ann Arbor/6/60, containing HA and NA from virus A/ duck/Hokkaido/69/2000 (H5N3), A/chicken/Hong Kong/G9/97 (H9N2), or A/Chicken/British Columbia/CN-6/04 (H7N3) was prepared by classical genetic reassortment methods in the chick embryos (CE) [44–46]. The vaccine strains exhibited *ts-*, *ca-*, and *att-*phenotype and provided protection against infection with the wild-type virus in mice and ferrets.

#### **3.1. Development of reassortant vaccine strains based on a/Leningrad/134/17/57 (H2N2) MDV**

To prepare vaccines based on A/Leningrad/134/17/57 (H2N2) MDV, several non-pathogenic avian viruses of different subtypes (A/duck/Potsdam/1402–6/1986 (H5N2), A/mallard /The Netherlands/12/2000 (H7N3), A/Hong Kong/1073/99 (H9N2), A/quail/Hong Kong/G1/1997 (H9N2), and А/herring gull/Sarma/51 s/2006 (H6N1)) were used. The HP avian influenza viruses of subtypes H5 and H7 contain a HA insertion from several positively charged amino acid residues (lysine and arginine) in the proteolytic cleavage site [47], which causes effective cleavage of HA by intracellular proteases expressed in most organs and tissues of birds and mammals. Unlike HP avian influenza viruses, non-pathogenic viruses contain a single arginine residue (R) in the cleavage site [44]. For non-pathogenic viruses proteolytic activation, the presence of trypsin-like enzymes is required, which is expressed by a limited range of cells and is found in the airways.

The reassortant vaccine strains were prepared in the Virology Department, Institute of Experimental Medicine, using classical genetic reassortment in CE as previously described [48]. The H5N2 reassortant virus inherited only the HA gene from the H5N2 parent virus, and the remaining seven genes from the Len/17 MDV (7,1 genome composition) [49]. The reassortants of subtypes H7N3, H9N2, and H6N1 inherited the HA and NA from parental avian influenza viruses (6,2 genome composition). All the reassortant strains were studied for temperaturesensitive (*ts-*) and cold-adapted (*ca-*) phenotype [49–52]. For those purposes, the reassortant viruses were propagated in CE for 2 days at 25, 34, and 40°C. The yield of "wild-type" avian influenza viruses at 40°C was the same or greater than at 34°C. Only when the temperature was increased to 41°C, the reproduction of these strains was partially limited. Thus, the high degree of temperature resistance of all the above viruses was demonstrated. In contrast to parental avian viruses, all vaccine candidates poorly reproduced at 40°C in titers not exceeded 1.5–1.8 log10 EID50/ml. At the same time, these reassortant strains grew well at low temperatures. Thus, all obtained reassortants acquired the genes of internal and nonstructural proteins from the A/Leningrad/134/17/57 (H2N2) MDV inherited the *ts-* and *ca-*phenotype. The pronounced difference in optimal reproductive conditions between the temperature-resistant viruses of avian influenza and the cold-adapted attenuation donor is due to the properties of viral polymerases [53]. This difference in the temperature optimum of the parental viruses may facilitate the isolation of the reassortant viruses possessing the desired gene composition after selective passages at a lower temperature.

#### **3.2. Immunogenicity and cross-protection in mice**

number of zoonotic and potentially pandemic influenza viruses to schedule candidates for

The LAIVs preparation against potentially pandemic avian influenza viruses is conducted in two directions: the preparation of vaccine strains using classical genetic reassortment in chick embryos or through reverse genetics (RG) technique. The first attenuated A/Ann Arbor/6/60 (H2N2)-based vaccine strains were obtained by reverse genetics shortly after H5 influenza

The vaccine candidates containing internal genes from the attenuation donor, and the surface antigens from viruses A/Hong Kong/156/97 (H5N1) or A/Hong Kong/483/97 (H5N1) with RG-modified HA demonstrated an attenuated phenotype for ferrets and chickens. Both reassortants caused seroconversions in chickens, which confirm the sensitivity of chickens to these vaccine strains despite the *att-*phenotype [42]. By RG methods, three reassortant strains based on the A/Ann Arbor/6/60 (H2N2) were prepared in the Vero cell line [43]. As a source of surface antigens, viruses H5N1 of 1997, 2003, and 2004 years of isolation with RG-modified HA were used. It was shown that a double immunization with LAIV from a strain isolated in 1997 completely protected mice from infection with later "wild" isolates, including the isolate obtained in 2005—A/Indonesia/05/2005 (H5N1). The use of HP viruses requires increased biosecurity level laboratories, certified cell lines, and RG techniques. The HP avian viruses found in nature cannot be used directly to prepare influenza vaccines because they would not grow in eggs and might be dangerous to people. The RG-modified viruses do not cause severe illness in birds and that also will grow well in chicken eggs (so that vaccine manufacturers can use it to produce vaccine). An alternative approach is to use low pathogenic surrogate viruses that show antigenic similarity to HP viruses. In this regard, the identification of non-pathogenic variants, which are antigenically close to potentially pandemic strains, may be very important. Another vaccine candidate based on A/Ann Arbor/6/60, containing HA and NA from virus A/ duck/Hokkaido/69/2000 (H5N3), A/chicken/Hong Kong/G9/97 (H9N2), or A/Chicken/British Columbia/CN-6/04 (H7N3) was prepared by classical genetic reassortment methods in the chick embryos (CE) [44–46]. The vaccine strains exhibited *ts-*, *ca-*, and *att-*phenotype and pro-

**3. Development of live influenza vaccines against potentially** 

vided protection against infection with the wild-type virus in mice and ferrets.

**3.1. Development of reassortant vaccine strains based on a/Leningrad/134/17/57** 

To prepare vaccines based on A/Leningrad/134/17/57 (H2N2) MDV, several non-pathogenic avian viruses of different subtypes (A/duck/Potsdam/1402–6/1986 (H5N2), A/mallard /The Netherlands/12/2000 (H7N3), A/Hong Kong/1073/99 (H9N2), A/quail/Hong Kong/G1/1997 (H9N2), and А/herring gull/Sarma/51 s/2006 (H6N1)) were used. The HP avian influenza viruses of subtypes H5 and H7 contain a HA insertion from several positively charged amino

the development of appropriate vaccines [40].

**pandemic avian influenza**

64 Influenza - Therapeutics and Challenges

outbreaks in Hong Kong in 1997 [41].

**(H2N2) MDV**

The ability of LAIV to induce antibodies not only to the homologous variant subtype but also to cross-reacting antibodies to antigenically different variants including HP variants was shown in several mouse studies [50–52, 54–56].

Among all vaccine candidates based on non-pathogenic avian influenza viruses, the H6N1 LAIV was characterized by the highest HI titers in mice after a single administration (GMT = 17.4). The LAIV of H7N3 subtype raised serum antibodies not only against the homologous virus but also against H7N9, which possessed the difference of 3% in the HA amino acid sequence. In the sera from mice double-vaccinated with H7N3 LAIV, serum HI titers against H7N9 were 20–40 times higher than against H7N3 (P < 0.05) [56]. At the same time, local IgA levels were higher against homologous H7N3 compared with H7N9 after vaccination with LAIV. The H5N2 LAIV induced detectable HI and neutralizing antibody titers only against the homologous H5N2 virus, perhaps due to the genetic differences between H5N2 vaccine strain and infectious viruses H5N1 isolated in 1997, 2003, and 2005 (10–12% differences of the HA1 amino acid sequence).

Nevertheless, immunization using virus H5N2 of 1986 resulted in a significant level of protection in experimental infection of mice (**Figure 1**).

Data on the protective efficacy of reassortant vaccine strains against intranasal challenge with avian influenza viruses are summarized in **Table 1**. When the mice were challenged with HP H5N1 viruses following immunization with H5N2 LAIV, the infectious viruses were not isolated from nasal passages or from the brain [54, 55]. Limited reproduction of HP viruses in the respiratory tract of mice and preventing a systemic infection, including neuro-infections, are important advantages of LAIV, especially in respect with data on the neurogenic pathway of generalization of infection caused by HP H5N1 viruses [58]. The absence of nasal infection correlated with high titers of secretory virus-specific IgA viruses in nasal swabs. The local immune response of the mucous membranes of the body serves as the first and most significant barrier for many viral infections, including influenza [59]. Due to their polymeric structure, IgAs have several times higher anti-hemagglutinating and neutralizing activity compared to IgG [60] and are also more stable and more cross-reactive. In addition, IgA can interact with the surface proteins of the influenza virus intracellularly, during trans-cytosis [61]. With respect to LAIV, it is still unclear how antibody-mediated immune response is related to protective efficacy. Mechanisms of cross-immunity in influenza are mediated by several factors, among which the cellular immune response is very important. Cellular immunity is involved in virus clearance and in activating the humoral immune response. In this regard, the production of Th1 and Th2 marker cytokines *in vitro* by splenocytes from mice immunized with H5N2 LAIV and whole-virion H5N2 IIV was compared [55].

Both LAIV and IIV caused the cytokines production by splenocytes of immunized mice in response to stimulation with both whole H5N1 virus and recombinant H5 HA. While immunization with LAIV caused higher levels of IFN-γ production by splenocytes of mice stimulated with H5N1 viruses, immunization with IIV induced IL-4 and IL-10 production. Interestingly,

after immunization with the H2N2 MDV, the IFN-γ production by splenocyte of mice occurred only in response to stimulation with whole virus H5N1, but not purified HA. This may indicate the directivity of hetero-subtypic immunity to conserved epitopes of viral proteins [55].

**groups**

LAIV

LAIV

LAIV

LAIV

LAIV

LAIV

7 lg EID50 H7N3

**Protection Refs.**

1.9 ≤0.8 ≤0.8 0% [54]

1.8 Nd\* Nd Na\*\* [54]

1.6 0.8 0.8 0% [55]

Nd Nd Nd 13% [57]

≤1.5 ≤1.5 ≤1.5 Na [50]

3.4 1.1 Nd Nd [51]

**Lethality**

67

http://dx.doi.org/10.5772/intechopen.76980

**Virus titers (log10EID50/**

**Lung Noses Brain**

PBS 5.9 4.0 4.3 100%

PBS 5.3 Nd Nd Na

PBS 6.1 4.7 4.5 100%

PBS Nd Nd Nd 100%

PBS 5.7 4.2 ≤1.5 Na [50]

PBS 6.9 2.0 Nd Nd [51]

**ml)**

Preparing Live Influenza Vaccines against Potential Pandemic Influenza Using Nonpathogenic…

Several experiments with vaccine candidates H5N2 and H7N3 were performed at Southeast Poultry Research Laboratory, GA, USA. Those studies demonstrated that the *ca-* reassortants of avian viruses adapted to a lower temperature of reproduction were unable to either infect a bird or be released into the environment. This was confirmed by the absence of virus isolation from the gastrointestinal tract of birds, as well as the impossibility in the determination of

A high degree of attenuation of H5N2 and H7N3 reassortants in chickens (up to a total inability) to reproduce confirms the safety for poultry farms during the production and use of such strains.

If the genetically homogeneous population using linear mice is the most appropriate model for assessing the molecular mechanisms of pathogenicity, the use of genetically heterogeneous

**3.3. Pathogenicity for chickens**

A/mallard/The Netherlands/12/2000

The virus was not lethal for mice.

(H7N3)

\*

Nd, not done. \*\*Na, not applicable.

**Challenge virus Dosage Vaccine** 

A/Hong Kong/483/97 (H5N1) 50 LD50 H5N2

A/Hong Kong/213/2003 (H5N1) 100 MID50 H5N2

A/Vietnam/1203/2004 (H5N1) 200 LD50 H5N2

A/chicken/Kurgan/02/2005 (H5N1) 27 LD50 H5N2

A/chicken/Hong Kong/G9/97 (H9N2) 7 lg EID50 H9N2

**Table 1.** Protection against infection with avian influenza viruses.

specific antibodies (**Table 2**).

**3.4. Study in primates**

**Figure 1.** Influenza virus-specific serum antibodies and local IgA in mice after intranasal (i.n.) immunization with LAIV [50**–**52, 54**–**56]. \* Nd, not done.

Preparing Live Influenza Vaccines against Potential Pandemic Influenza Using Nonpathogenic… http://dx.doi.org/10.5772/intechopen.76980 67


\*\*Na, not applicable.

Nevertheless, immunization using virus H5N2 of 1986 resulted in a significant level of protec-

Data on the protective efficacy of reassortant vaccine strains against intranasal challenge with avian influenza viruses are summarized in **Table 1**. When the mice were challenged with HP H5N1 viruses following immunization with H5N2 LAIV, the infectious viruses were not isolated from nasal passages or from the brain [54, 55]. Limited reproduction of HP viruses in the respiratory tract of mice and preventing a systemic infection, including neuro-infections, are important advantages of LAIV, especially in respect with data on the neurogenic pathway of generalization of infection caused by HP H5N1 viruses [58]. The absence of nasal infection correlated with high titers of secretory virus-specific IgA viruses in nasal swabs. The local immune response of the mucous membranes of the body serves as the first and most significant barrier for many viral infections, including influenza [59]. Due to their polymeric structure, IgAs have several times higher anti-hemagglutinating and neutralizing activity compared to IgG [60] and are also more stable and more cross-reactive. In addition, IgA can interact with the surface proteins of the influenza virus intracellularly, during trans-cytosis [61]. With respect to LAIV, it is still unclear how antibody-mediated immune response is related to protective efficacy. Mechanisms of cross-immunity in influenza are mediated by several factors, among which the cellular immune response is very important. Cellular immunity is involved in virus clearance and in activating the humoral immune response. In this regard, the production of Th1 and Th2 marker cytokines *in vitro* by splenocytes from

mice immunized with H5N2 LAIV and whole-virion H5N2 IIV was compared [55].

Both LAIV and IIV caused the cytokines production by splenocytes of immunized mice in response to stimulation with both whole H5N1 virus and recombinant H5 HA. While immunization with LAIV caused higher levels of IFN-γ production by splenocytes of mice stimulated with H5N1 viruses, immunization with IIV induced IL-4 and IL-10 production. Interestingly,

**Figure 1.** Influenza virus-specific serum antibodies and local IgA in mice after intranasal (i.n.) immunization with LAIV

[50**–**52, 54**–**56]. \*

Nd, not done.

tion in experimental infection of mice (**Figure 1**).

66 Influenza - Therapeutics and Challenges

The virus was not lethal for mice.

**Table 1.** Protection against infection with avian influenza viruses.

after immunization with the H2N2 MDV, the IFN-γ production by splenocyte of mice occurred only in response to stimulation with whole virus H5N1, but not purified HA. This may indicate the directivity of hetero-subtypic immunity to conserved epitopes of viral proteins [55].

#### **3.3. Pathogenicity for chickens**

Several experiments with vaccine candidates H5N2 and H7N3 were performed at Southeast Poultry Research Laboratory, GA, USA. Those studies demonstrated that the *ca-* reassortants of avian viruses adapted to a lower temperature of reproduction were unable to either infect a bird or be released into the environment. This was confirmed by the absence of virus isolation from the gastrointestinal tract of birds, as well as the impossibility in the determination of specific antibodies (**Table 2**).

A high degree of attenuation of H5N2 and H7N3 reassortants in chickens (up to a total inability) to reproduce confirms the safety for poultry farms during the production and use of such strains.

#### **3.4. Study in primates**

If the genetically homogeneous population using linear mice is the most appropriate model for assessing the molecular mechanisms of pathogenicity, the use of genetically heterogeneous


animals were infected in a combined method using intratracheal and intranasal administration of 7.5 lg EID50/ml primate-adapted influenza virus A/Chicken/Kurgan/2/05 (H5N1). According to the summary data on clinical reactions and virus isolation from the respiratory tract, the vac-

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69

The randomized, double-blinded, placebo-controlled phase I trials were conducted in healthy adults at the St. Petersburg Institute of Influenza [65, 66]. Both H5N2 and H7N3 LAIV were safe for volunteers. In the genome of the isolated vaccine virus, all the mutations known for the MDV were conserved [65, 66]. Data on the LAIV when used in humans confirm the concept of attenuation of influenza viruses by reassortment with MDV and association of the ca- phenotype with an attenuation for people. For the vaccine virus isolation from the nasal washes, two to three passages were required on MDCK cell culture, indicating a very low content of the virus. These data confirmed LAIV implementation safety for contact persons. The post-vaccination antibody response was assessed using the HI test, which is still posing as the "gold standard" for the evaluation of influenza vaccine immunogenicity, the microneutralization (MN) test which is supposed to be more sensitive compared to the HI in the detection of serum antibodies after immunization against potential pandemic subtypes. Local

According to the results of three tests, more than 80% of the vaccinated subjects responded to immunization with a significant increase in serum or local antibodies [65, 66]. Moreover, after double vaccination with H5N2 LAIV, 30.8% of vaccinated volunteers responded to the HA antigen of the A/Indonesia/05/2005xPR8 IBCDC-RG (H5N1). When serum samples of volunteers vaccinated with H7N3 LAIV were tested for the anti-H7N9 HI antibodies, the

cine protected at least 50% of immunized animals against the H5N1 infection.

**3.5. Study of the H5N2 and H7N3 reassortants in phase I clinical trials**

IgA response in nasal washes was estimated using ELISA (**Figure 2**).

**Figure 2.** Immunogenicity of H5N2 and H7N3 LAIV in volunteers after boost immunization [65**–**67].

\* Groups of eight 5-week-old specific pathogen-free (SPF) chickens were infected intravenously (i.v.) with and observed daily for 10 days for clinical signs and death.

\*\*Groups of five chickens were infected intranasally (i.n.) with 6 log10 EID50/0.1 ml. The oropharyngeal and cloacal swabs were collected 3 days post infection (p.i.) and titrated in eggs for assessing viral replication. The chickens were observed for clinical signs of disease and death for 14 days. To determine infectivity, sera were collected 21 days p.i. and tested for the presence of antibodies by agar gel immunodiffusion (AGID) test.

\*\*\*Mean virus titers (EID50/0.1 ml).

**Table 2.** Pathogenicity and infectivity data for chickens.

animals (ferrets, primates) better allows one to assess the effect of natural host defense factors in mammalian infection by avian influenza viruses. The use of primates is one of the most promising areas in the study of human infectious pathology. The evolutionary relationship and biological similarity between humans and monkeys make them unique objects in the modeling of infectious diseases. However, the lower primates, while remaining closest to humans than other mammals, differ significantly in physiological characteristics from them. In experiments on the hybridization of nuclear DNA, it has been established that the similarity of man to chimpanzee reaches 90–98%, with lower monkeys—50–75% whereas in rodents, this index is not more than 20% (unpublished data).

The use of lower primates as models makes it possible to establish the duration and sequence of biochemical, metabolic, and physiological responses in the course of the development of the disease, which are then used to evaluate various preventive and therapeutic measures [62]. The use of primates for the modeling of the pathogenesis of influenza H5N1 in people of preclinical evaluation of vaccine preparations by a group of scientists from The Netherlands is described [63].

Before the clinical trials, the safety, immunogenicity, and protective properties of the LAIV based on strain A/17/duck/Potsdam/86/92 (H5N2) were studied by intranasal immunization of Java macaques [64]. None of the four monkeys immunized with H5N2 LAIV at a dose of 6.9 log10 EID50/ml showed no adverse reactions with either temperature or behavioral changes or weight loss. The vaccine virus multiplied in the upper respiratory tract and was isolated in two of four monkeys, on days 3–5 after the first vaccination with the maximum titer of 4.2 lg EID50/ml. The absence of viremia and a temperature reaction in the same period indicates the local immunization process. In three of four monkeys, double immunization caused neutralizing antibodies to H5 viruses in titers 1:40–1:160. Twenty-one days after the end of the immunization cycle, the animals were infected in a combined method using intratracheal and intranasal administration of 7.5 lg EID50/ml primate-adapted influenza virus A/Chicken/Kurgan/2/05 (H5N1). According to the summary data on clinical reactions and virus isolation from the respiratory tract, the vaccine protected at least 50% of immunized animals against the H5N1 infection.

#### **3.5. Study of the H5N2 and H7N3 reassortants in phase I clinical trials**

animals (ferrets, primates) better allows one to assess the effect of natural host defense factors in mammalian infection by avian influenza viruses. The use of primates is one of the most promising areas in the study of human infectious pathology. The evolutionary relationship and biological similarity between humans and monkeys make them unique objects in the modeling of infectious diseases. However, the lower primates, while remaining closest to humans than other mammals, differ significantly in physiological characteristics from them. In experiments on the hybridization of nuclear DNA, it has been established that the similarity of man to chimpanzee reaches 90–98%, with lower monkeys—50–75%

**Virus I.v. pathogenicity test\* I.n. pathogenicity and infectivity data\*\* Refs.**

Len17/Н7N3 0/8 0/8 0/5 0/5 0/5 0/5 0/5 [49]

\*\*\* 1/5(100.91)

Groups of eight 5-week-old specific pathogen-free (SPF) chickens were infected intravenously (i.v.) with and observed

\*\*Groups of five chickens were infected intranasally (i.n.) with 6 log10 EID50/0.1 ml. The oropharyngeal and cloacal swabs were collected 3 days post infection (p.i.) and titrated in eggs for assessing viral replication. The chickens were observed for clinical signs of disease and death for 14 days. To determine infectivity, sera were collected 21 days p.i. and tested for

Len17/H5N2 0/8 0/8 0/5 0/5 0/5 0/5 0/5 H5N2-wt 0/8 0/8 0/5 0/5 3/5 0/5 0/5

**Morbidity Mortality Oropharyngeal** 

the presence of antibodies by agar gel immunodiffusion (AGID) test.

**Table 2.** Pathogenicity and infectivity data for chickens.

H7N3-wt 5/8 5/8 2/5(101.1)

daily for 10 days for clinical signs and death.

\*\*\*Mean virus titers (EID50/0.1 ml).

68 Influenza - Therapeutics and Challenges

\*

**swabs**

**Virus isolation on day 3 p.i. Seroconversions** 

**Cloacal swabs** Len/17 0/8 0/8 0/5 0/5 0/5 0/5 0/5 [53]

**(AGID).**

\*\*\* 5/5 0/5 0/5

**Morbidity Mortality**

The use of lower primates as models makes it possible to establish the duration and sequence of biochemical, metabolic, and physiological responses in the course of the development of the disease, which are then used to evaluate various preventive and therapeutic measures [62]. The use of primates for the modeling of the pathogenesis of influenza H5N1 in people of preclinical evaluation of vaccine preparations by a group of scientists from The Netherlands

Before the clinical trials, the safety, immunogenicity, and protective properties of the LAIV based on strain A/17/duck/Potsdam/86/92 (H5N2) were studied by intranasal immunization of Java macaques [64]. None of the four monkeys immunized with H5N2 LAIV at a dose of 6.9 log10 EID50/ml showed no adverse reactions with either temperature or behavioral changes or weight loss. The vaccine virus multiplied in the upper respiratory tract and was isolated in two of four monkeys, on days 3–5 after the first vaccination with the maximum titer of 4.2 lg EID50/ml. The absence of viremia and a temperature reaction in the same period indicates the local immunization process. In three of four monkeys, double immunization caused neutralizing antibodies to H5 viruses in titers 1:40–1:160. Twenty-one days after the end of the immunization cycle, the

whereas in rodents, this index is not more than 20% (unpublished data).

is described [63].

The randomized, double-blinded, placebo-controlled phase I trials were conducted in healthy adults at the St. Petersburg Institute of Influenza [65, 66]. Both H5N2 and H7N3 LAIV were safe for volunteers. In the genome of the isolated vaccine virus, all the mutations known for the MDV were conserved [65, 66]. Data on the LAIV when used in humans confirm the concept of attenuation of influenza viruses by reassortment with MDV and association of the ca- phenotype with an attenuation for people. For the vaccine virus isolation from the nasal washes, two to three passages were required on MDCK cell culture, indicating a very low content of the virus. These data confirmed LAIV implementation safety for contact persons.

The post-vaccination antibody response was assessed using the HI test, which is still posing as the "gold standard" for the evaluation of influenza vaccine immunogenicity, the microneutralization (MN) test which is supposed to be more sensitive compared to the HI in the detection of serum antibodies after immunization against potential pandemic subtypes. Local IgA response in nasal washes was estimated using ELISA (**Figure 2**).

According to the results of three tests, more than 80% of the vaccinated subjects responded to immunization with a significant increase in serum or local antibodies [65, 66]. Moreover, after double vaccination with H5N2 LAIV, 30.8% of vaccinated volunteers responded to the HA antigen of the A/Indonesia/05/2005xPR8 IBCDC-RG (H5N1). When serum samples of volunteers vaccinated with H7N3 LAIV were tested for the anti-H7N9 HI antibodies, the

**Figure 2.** Immunogenicity of H5N2 and H7N3 LAIV in volunteers after boost immunization [65**–**67].


polybasic amino acid insertion in the cleavage site and therefore do not require modifica-

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71

• A high degree of attenuation of the reassortants of subtypes H5N2 and H7N3 in chickens, up to a total inability to reproduce, confirms the safety for poultry farms during the production and use of such strains. The high yield of the obtained reassortants in the CE makes it possible to produce a large amount of viral material, which allows their use for the

• In preclinical and clinical studies, LAIV based on non-pathogenic avian influenza viruses causes the formation of systemic and secretory antibodies including those against antigenically distant viruses. In animal models, LAIV based on non-pathogenic avian influenza viruses provided protection against HP variants that appeared much later. Protection from lethal infection with HP viruses was observed even in the absence of HI antibodies. This suggests that the use of LAIV may be effective against HP influenza viruses even in the case of incomplete antigenic correspondence between the vaccine strain and

• In general, studies in mice represent an adequate preclinical model for studying the properties of reassortants of non-pathogenic avian influenza viruses, since data on the safety, immunogenicity, and cross-reactivity of post-vaccinal antibodies obtained in mice were

• In the clinical trials of LAIV of potentially pandemic subtypes, the detection of only strainspecific HI antibodies is not sufficient to fully characterize the positive effect of immunization on the stimulation of antiviral immunity, which in this case is mediated by a variety of

In the face of a pandemic threat, only live vaccines can eliminate the risk of losses from increased morbidity and mortality, as it was demonstrated in the cases with smallpox eradication and polio control. The conducted studies clearly showed that the classical genetic reassortment method allows obtaining high-yield, harmless and immunogenic LAIVs on the basis of an attenuated donor virus. In the post-pandemic period, when the direct threat of infection recedes, the main task is the search for optimal regimens for the use of new pandemic vaccines, including (1) the possibility of including such vaccine strains in the composition of polyvalent live vaccines; (2) prime-boost schemes using both LAIV and IIV; (3) the development of recommendations for vaccination of people with an increased risk of influenza infection complications; (4) a comprehensive study of the immune mechanisms of vaccination with influenza vaccines against emerging variant viruses; (5) the development of the most reliable and standardized assays to measure

Currently, the FluMist LAIV, which was withdrawn from use in the USA and Europe in 2015 due to reduced LAIV effectiveness against A/H1N1pdm09, was returned to the practice by

tion by reverse genetics methods prior to reassortment.

production of both LAIV and IIV.

the infectious virus.

confirmed in clinical trials.

**5. Future perspectives**

post-vaccination immune response.

other factors, both humoral and cellular.

**Table 3.** Serum NA-inhibiting antibodies against H5N2 LAIV 21 days after second vaccination.

seroconversions were found among 44.8% of vaccinated persons [67]. These data indicate the substantial level of cross-reactive antibodies induced by LAIV against distant avian influenza viruses. The two doses of LAIV raised both CD4 and CD8 T-memory-cell responses in peripheral blood of healthy volunteers on day 21 after boost immunization [67].

Previously, when studying the immunogenicity of inactivated vaccines based on potentially pandemic avian influenza viruses, both the experiment and the clinical trials showed a low immunogenicity of such preparations, according to generally accepted criteria for seroconversion of HI antibodies. The European Committee for the Control of Medicines has established the following criteria for the immunogenicity of vaccine preparations based on both epidemic and potentially pandemic influenza viruses: the multiplicity of antibody growths of at least 2.5 for individuals 18–60 years old and the development of reliable seroconversion in 40% of the vaccinated [68]. Obviously, the detection of only strain-specific HI antibodies is not sufficient to fully characterize the immunogenicity of the LAIV [69]. Moreover, it remains unclear what antibody titer can be considered protective against potentially pandemic viruses—1:20 or 1:40. Recently, it was shown that neuraminidase-inhibiting (NI) antibody titers better correlate with protection and can be an independent predictor of reduction of influenza disease severity [70]. Therefore, neuraminidase immunity should be considered when studying susceptibility after vaccination as a critical target in future influenza vaccine platforms. In this connection, the NI antibodies in the sera of volunteers after H5N2 immunization were estimated (**Table 3**). The two doses of the monovalent LAIV H5N2 raised a statistically significant increase in the NI antibodies against vaccine strain. More than twofold increase in antibodies was obtained among 19.5**–**33.3% of those vaccinated. The MN test and NI assay titers in the same sera of the vaccinated volunteers were 73.2% corresponded and suggested a statistically significant correlation between the values in antibody titers revealed in both tests (p = 0.04).

#### **4. Conclusions**

• The use of non-pathogenic avian viruses as a source of surface antigens combined with the use of cold-adapted "donors" of attenuation can be a significant advantage in the development of vaccine strains for LAIV against potentially pandemic influenza using classical genetic reassortment in CE. Low pathogenic avian influenza viruses do not contain a polybasic amino acid insertion in the cleavage site and therefore do not require modification by reverse genetics methods prior to reassortment.

