Viral Systems Biology

**33**

**Chapter 3**

**Abstract**

gene therapy.

**1. Introduction**

Up Manufacture

*Juan C. Ramirez*

Lentiviral Vectors Come of Age?

Hurdles and Challenges in Scaling

The pharmaceutical industry has been attracted to the gene therapy field and is starting to support clinical trials, establishing collaborative strategies to develop commercial products which in many cases are based on lentiviral vectors. The predictable widespread use of lentiviral vectors in next-generation gene therapy scenarios aimed at dealing with not only rare diseases raises important challenges and hurdles regarding their manufacture. The author reflects on this in the chapter on the state of the art in the manufacture of lentiviral vectors, addressing some current manufacturing processes, their achievements, and the uncertainties in ensuring a validated process capable of releasing consistent vector quality that meets global health authorities' requirements. In summary, the proposal looks at the goals and challenges that must be addressed in manufacturing lentiviral vectors, in order to satisfy supply in the commercial stage, before we reach the next stage in

**Keywords:** lentiviral vector, large-scale manufacturing, gene therapy

The practice of medicine is undergoing a revolution, moving from a focus on the treatment of symptoms, toward targeting the genetic cause of the disease. The huge development of disciplines, including but not restricted to molecular and cellular biology and genetic sciences, provides the framework for the advancement of individualized precision medicine. This new conception of medicine is based on the novel paradigm: the genes represent medicines themselves. Gene therapy is the groundbreaking strategy, which uses genes as medicines. Gene therapy is no longer an experimental approach [1, 2], and as with any novel therapy, patients' benefits must be balanced against the nonzero risk of the therapeutical approach. Most products currently assayed in clinical trials of gene therapy are viral vectors [3], i.e., biological products that challenge both the manufacturing processes in order to guarantee the supply of adequate quantities of the active vector and the regulatory requirements from the medicine agencies of target countries. In summary, viral vector production on a large scale implies novel challenges for a multidisciplinary field, in order to accommodate such specific requirements within the industrial process. The first gene therapy experiments took advantage of the strategy that members of the *Retroviridae* family of viruses evolved to spread and remain stable in

#### **Chapter 3**

## Lentiviral Vectors Come of Age? Hurdles and Challenges in Scaling Up Manufacture

*Juan C. Ramirez* 

#### **Abstract**

The pharmaceutical industry has been attracted to the gene therapy field and is starting to support clinical trials, establishing collaborative strategies to develop commercial products which in many cases are based on lentiviral vectors. The predictable widespread use of lentiviral vectors in next-generation gene therapy scenarios aimed at dealing with not only rare diseases raises important challenges and hurdles regarding their manufacture. The author reflects on this in the chapter on the state of the art in the manufacture of lentiviral vectors, addressing some current manufacturing processes, their achievements, and the uncertainties in ensuring a validated process capable of releasing consistent vector quality that meets global health authorities' requirements. In summary, the proposal looks at the goals and challenges that must be addressed in manufacturing lentiviral vectors, in order to satisfy supply in the commercial stage, before we reach the next stage in gene therapy.

**Keywords:** lentiviral vector, large-scale manufacturing, gene therapy

#### **1. Introduction**

The practice of medicine is undergoing a revolution, moving from a focus on the treatment of symptoms, toward targeting the genetic cause of the disease. The huge development of disciplines, including but not restricted to molecular and cellular biology and genetic sciences, provides the framework for the advancement of individualized precision medicine. This new conception of medicine is based on the novel paradigm: the genes represent medicines themselves. Gene therapy is the groundbreaking strategy, which uses genes as medicines. Gene therapy is no longer an experimental approach [1, 2], and as with any novel therapy, patients' benefits must be balanced against the nonzero risk of the therapeutical approach. Most products currently assayed in clinical trials of gene therapy are viral vectors [3], i.e., biological products that challenge both the manufacturing processes in order to guarantee the supply of adequate quantities of the active vector and the regulatory requirements from the medicine agencies of target countries. In summary, viral vector production on a large scale implies novel challenges for a multidisciplinary field, in order to accommodate such specific requirements within the industrial process.

The first gene therapy experiments took advantage of the strategy that members of the *Retroviridae* family of viruses evolved to spread and remain stable in

 the host, with the integration of their genomes. Since those experiments we have assisted to a fast development of the viral vector field fuelled by promising data raised from early studies until the achievement of the current scenario [4, 5]. The gene therapy field is witnessing a sort of gold rush that is boosting personalized medicine by confronting many diseases as genetically treatable traits. Early vector developments focused on *Gammaretrovirus* as integrating entities for the delivery of a stable expression of the correctable gene, but since the last decade, they have been displaced by vectors deriving from the *Lentivirus* genus. As opposed to *Gammaretrovirus*, the *Lentivirus* displayed preferred integration sites in coding genomic regions rather than in transcription regulatory regions, and this has become a major safety feature to exploit. Nowadays, non-replicative self-inactivating lentiviral (SIN) vectors are used in the vast majority of novel gene therapy clinical trials using integrative vectors and are considered by now the optimal tools for ex vivo gene therapy and the safest and easiest-to-use vectors available for the delivery of genes into mammalian tissues [6].

 The use of retroviral vectors in gene therapy as an emerging technology is following the Gartner hype cycle. Hope and expectation were seen when gene therapy entered the clinic in the early 1990s, but due to a lack of profound success and the unexpected death in 2000 of two patients, caused by the treatment, the expectations slowed down. In the following decade (2000–2010), two approaches coexisted: first, follow-up and ongoing clinical trials that were using an integrative type of vector (gammaretroviruses, gRV) used in those clinical trials resulting in unexpected fatal deaths and, second, intensive academic research focused on the development of new viral vectors and methodical exploration of the clinical procedure. This resulted in the advent of a novel type of vector called SIN-LV, derived from the causative agent of AIDS, the *Lentivirus* HIV, properly modified and engineered to render them safer (see Vectorizing HIV). In the current decade (2010 to the present), we have been recording investigative clinical trials using both vectors in several hematological and neurodegenerative rare diseases with a conclusion: the feasibility of the second-generation gene therapy approaches [4, 5]. This is mostly due to the huge development of SIN-LV vectors, due to their safer profile, in comparison with gammaretroviruses [6].

Gene therapy products have entered the commercialization phase, and a dozen treatments have been approved since 2012 in EU and the USA [1]. Up to last year, all of them were treatments for rare or ultrarare conditions, but in August 2017, a new key milestone for gene therapy development can be added to the chart, reinforcing the concept of gene therapy use in frequent pathologies in which current treatments are failing: the Novartis receives the first ever FDA approval for a CAR-T cell therapy, Kymriah® (tisagenlecleucel), for children and young adults with B-cell acute lymphoid leukemia (ALL). The goals reached in the past two decades in the gene therapy field open novel expectations offering a cure to rare genetic diseases, cancer, infectious diseases, and vaccine development, and in the short-medium term, innovations in the field will make affordable genetic intervention covering an array of diseases with a gene-defined cause [7].

The aim of this review is to recapitulate specific problems related to the manufacture of lentiviral vectors in particular during production stages, known as the upstream process (USP), focusing on the limitations that exist when scaling up, due to the nature of the virus, the particularities of the lentivector life cycle, and the producer cell line commonly used for production. There are excellent reviews [13–16] that provide a detailed description of the methodologies that can be followed in order to produce lentivectors on a large scale. There is a growing interest in the lentiviral vector field, and there are many topics worthy of a description. For those interested in more detailed, specific topics, i.e., manufacturing of CAR-T *Lentiviral Vectors Come of Age? Hurdles and Challenges in Scaling Up Manufacture DOI: http://dx.doi.org/10.5772/intechopen.81105* 

approaches, purification strategies, or specific problems linked to the target cell, I recommend references [10, 11, 17].

#### **2. Delivering genes with** *Lentivirus*

The virus-derived vector as delivery system lies at the heart of most currently employed forms of gene therapy; without the viral vectors, there is no treatment. These viruses must be custom-made in specialized facilities for each treatment, but manufacturing them is costly and onerous: it requires great expertise and multidisciplinary teams and specialized facilities with stringent conditions both for safety/ containment and demanding production methods, under good manufacturing practice with regard to compliance (GMP) [8].

 Viral vectors are complex bioproducts with an ordered architecture and are very sensitive to handling and environmental conditions. For these reasons, there are stringent requirements aimed at preserving biological activity during all stages of the manufacturing and delivery process. Then, during the production, purification, storage, and transportation stages, it is necessary to maintain specific rigorous control aimed at minimizing the loss of biological activity, in addition to controls which are common to other bioproducts, such as sterility. This implies that the manufacture of large amounts of a viral vector cannot simply be produced by transferring the know-how and well-stablished procedures developed in the pharmaceutical industry for the production of monoclonal antibodies or recombinant proteins.

The large-scale manufacture of lentivectors for use in humans is becoming the bottleneck in the success of ongoing or planned gene therapy development to be launched in the near future [9]. Indeed, the manufacturing capabilities of the companies to satisfy the short-medium term markets are central to decision-making for backers and investors, who are becoming cautious with regard to biotech firms developing gene therapy products that do not have a secure virus source [9]. Several papers have recently reported the need to succeed in developing a global manufacturing process for lentiviral vectors, driven by a deep understanding of both the product and the process, in order to establish the viral vector product profile and critical quality attributes [10, 11]. In addition, lentiviral products require the creation of a worldwide accepted and adopted international standard, suitable for the standardization of trials, in particular quantitation trials related to the potency of the target product, allowing a comparison of cross-manufacturing results for any lentiviral platform [12].

#### **3. Vectorizing HIV**

 Human immunodeficiency virus (HIV) belongs to the family *Retroviridae*, subfamily *Orthoretrovirinae*, and genus *Lentivirus* of animal viruses. According to Baltimore's classification, it is an RNA reverse-transcribing virus (group VI). *Retroviridae* members are among the more exclusive entities in the virus taxon, and research in the field has provided outstanding insights into key concepts on biology, which were the basis demonstrating that dogmas do not stand up in science (the central dogma on molecular biology), providing one of the most useful tools in molecular biology (retrotranscriptase), supporting the concept of the existence of genes that provoke cancer (viral oncogenes), and finally, demonstrating that viral-genome integration also evolved in animal viruses as an efficient method of transmission. This breaking concept drove the original idea of gene therapy.

 The HIV pandemic in the 1980s alerted humanity to the silent spreading of a deadly disease. HIV, a *Lentivirus*, was revealed to be a highly sophisticated virus with fine-tuned regulation, and it was mostly deciphered a decade after its discovery [5]. Scientists took advantage of the impressive knowledge gained about the virus in a decade transforming a dangerous virus into a safe viral vector. It is worth mentioning that HIV was described as a new virus in 1987, and barely 10 years later, a safe version of HIV-derived vectors was demonstrated as efficient in animal models [23].

 Taking advantage of the previous studies with *Gammaretrovirus* vectors, the HIV-derived vectors were engineered to contain a mere 15–20% of the original virus, allowing plenty of room for transgenes/regulatory sequences. A method for production was established, and subsequent improvements ensure that the currently available format of the third generation of self-inactivating (SIN) vector [18] is generated as the safest and easiest-to-use vector available. It has multipurpose uses, from research and preclinical studies to clinical trials as there is a commercial product based on this type of vector. In all cases the procedure followed for production is almost the same, representing one of the main drawbacks, as manufacturing is not fully established, and a series of major concerns must be resolved in order to cover large mid- to long-term market requirements. Below is a brief summary of several relevant factors which must be addressed.

 Production of HIV-derived vectors is a poorly optimized process, and a major hurdle to large-scale manufacturing is due in part to a deficient production of fully biologically active virions recovered in the culture media [19]. This is a dynamic process involving both production and inactivation rates, which ultimately renders on average 3–10 virus per producer cell [20], whereas during natural lentiviral infection, the number is close to 103 /cell [21] and far removed from other vector systems such as AAV or *Adenovirus*, which render 104 –105 viral particles per producer cell. There are at least four major issues during production that can result in this poor yield: (a) transfection robustness, (b) protein interactions during morphogenesis, (c) the nature of the cell system used for production, and (d) extremely labile essential components within the viral particles that lose activity during the production testing [22, 23].

#### **3.1 DNA transfection**

 Production of HIV-derived lentivectors, and likewise other retroviral vectors derived from feline or equine *Lentivirus* or from gammaretroviruses, is based on DNA transfection of producer cells. The overall method was firstly demonstrated as feasible in pioneering research using poliovirus [24, 25], and it is based on the concept that the viral genome cloned in plasmids can recapitulate the genetic and morphogenetic instructions upon introduction in a eukaryotic cell in order to produce viral progeny. Early gene therapy studies developed a further step by splitting viral components in different plasmids, allowing the generation of non-replicative viral vectors as nonstructural/replication instructions which were no longer packaged in the progeny. These systems are currently also used to produce AAV-derived vectors. The basis is that packaging signals acting in cis are encoded in discrete regions of the viral genome. By including those sequences in the *transfer plasmid*  bearing the therapeutic/reporter gene, transgenes are encapsidated in the virions. All the accessory functions are expressed during production from the so-called helper plasmids but are not licensed for encapsidation, as they do not carry the packaging signals, nor are they encoded in the transfer plasmid. The current model of production on large or small scales is based on DNA transfection of three or four plasmids. For a full, detailed description of the plasmid used in the production of

HIV-derived vectors, see [8]. As a result, efficiency is compromised by the proportion of cells transfected with the proper combination of plasmids, and indeed earlier second-generation production systems that use just three plasmids are more efficient in production [6].

#### **3.2 Pseudotyping**

Lentiviral vectors can be designed to carry heterologous envelope proteins. This pseudotyping allows the selective targeting of specific cells, conferring broader uses on the vector. Thus, lentivectors bearing RD114 [26], CD105 [27], and more recently measles virus [28] envelope glycoproteins, among others, have been described as conferring specific targeting in B cells, T cells, and hematopoietic stem cells, respectively. However, most of the lentivectors that have entered into the clinic and are commonly being used in research are pseudotyped with *Vesicular stomatitis* virus g protein (VSVg). There are two reasons for this. First, a wide number of different cell types are targeted by such an envelope, and second, it confers robustness on the viral particle during the purification stages [29].

However, the presence of the VSV receptor in the producer cell line contributes to diminishing the viral burden in the harvest [30]. Envelope proteins are membrane proteins that pass through the secretory pathway involving the endoplasmic reticulum cisternae and the Golgi apparatus, before they reach the plasma membrane, a system also used for the synthesis and recycling of the membrane receptors. Prevention of a premature encounter between ligand and receptor is mandatory, in order to increase the env protein available for the morphogenetic program. Indeed, the impact of this phenomenon has evolved in the natural infection of HIV. Vpu, an HIV accessory protein (see below), plays a dual role in the viral cycle, firstly by promoting egress in a cell-type-dependent manner and secondly by controlling the recircularization of envelope proteins during the synthesis and preventing premature binding of the HIV gp160 env protein with the CD4 natural receptor during T-cell infection [31, 32]. To our knowledge no data has been published which accounts for the impact of such a process (autotransduction) during HIV-derived VSV-pseudotyped processes, but data obtained in our company indicate that this phenomenon is actually taking place in the producer cell. By specific quantitation of retrotranscribed RNA and integrated copies of cDNA in the producer cell, we have been able to quantitate that 30–50% loss of the actual viral particles produced are lost by reentering in the producer cell. Different lines are currently being developed at VIVEbiotech to minimize or fully prevent this phenomenon.

#### **3.3 Helper functions**

In the case of HIV, unlike to *Gammaretrovirus*, there is an array of six viral products collectively named accessory proteins. Their roles are different throughout the viral life cycle. Some are related to controlling the innate/cellular immune response (vif), others modulate the adaptive immune response (nef), while others are mandatory for a fully regulated genetic program (tat, rev), morphogenesis and the egress (vpu), or viral infectivity (vif, nef).

 HIV-derived vectors are produced in the absence of five of the six accessory proteins encoded by the wild-type virus: with the exception of rev, the other five are not expressed in helper plasmids. The rationale is to minimize the presence of viral sequences in the transfer plasmid, enabling safer vectors and minimizing the recombination between viral sequences in order to limit the risk of rescue of wildtype virus during production. Tat protein was unnecessary within the design of

 third-generation vectors, as no viral promoter was used in production. However, as has been demonstrated, the effect of those other accessory proteins is not negligible, and their function during vector production is controversial [31, 32].

#### **3.4 Manufacturing virus from DNA**

As described above DNA transfection is the current and unique manner to produce lentiviral vectors. Three major concerns must be considered about this approach:


### **4. Manufacturing** *Lentivirus***: the VIVEbiotech approach**

There are several excellent reviews on the specific steps during the manufacture of lentiviral vectors, the approaches to consider when scaling up and the critical points to consider for decision-making [13, 14, 34]. However, all of the processes must conciliate at least the next three considerations: (a) potency, meaning the capability of producing large quantities of vector; (b) robustness, i.e., highly reproducible; and (c) versatility, as demand changes according to project needs.

Manufacturing follows a process of production, purification, and concentration. At every step, specific features of the lentiviral vectors must be taken into account. Critical concerns to be considered include:

1.*Production*. It is worth remembering that lentiviral vectors are enveloped, and both cellular proteins and lipid content can vary depending on the culture conditions [22]. As previously mentioned, pseudotyping is of major relevance, as it can affect the fate of the produced virions and also contribute to the

*Lentiviral Vectors Come of Age? Hurdles and Challenges in Scaling Up Manufacture DOI: http://dx.doi.org/10.5772/intechopen.81105* 

physical properties of the particles [35] and interaction with the solid phase in bioreactors.


Herein is an overview of the manufacturing process developed at VIVEbiotech, which focuses on some of the critical steps. VIVEbiotech has obtained authorization from the European Medicines Agency (EMA) to provide cGMP batches of lentiviral vectors under the manufacturing process the company has fully developed (see **Figure 1**). VIVEbiotech is currently releasing batches of lentiviral vectors produced under these conditions to clients in EU and the USA. VIVEbiotech's manufacture of lentivectors is based on the fixed-bed bioreactor iCELLis™ supplied by Pall. High compaction in the solid phase allows for a large culture surface, ranging from 0.53 to 2.6 m2 (Nano™ configuration) in 1 liter disposable bioreactor, and is further scalable up to approximately 300 m<sup>2</sup> (500 + ™ configuration) based on chromatography principles. In the 1 liter small configuration, the carriers made of PET are fixed in a 40 mL chamber, and the process is monitored by probes controlling cell growth, and physical–chemical conditions are monitored by in-process BioXpert software. The harvest is collected by perfusion and purified by ion-exchange chromatography. It is then concentrated by tangential flow filtration, rendering a final concentration factor close to 300-fold and reaching a yield on a per-surface basis in accordance with market standards [39]. This process has been fully optimized in two remarkable steps:

**Figure 1.**  *Manufacturing process of lentiviral vectors optimized at VIVEbotech.* 


#### **5. Challenging by numbers**

As mentioned above, vector production is becoming a roadblock which is hitting gene therapy capabilities. Let us examine a few numbers in order to understand the size of the problem. So, what does lentivector-based gene therapy need to do in order to ensure it can be applied in the future? Lentiviral vectors for what? In their current design, lentivectors are capable of being used in the treatment of blood disorders, central nervous system disorders, immune therapy for certain cancers, and neurological conditions that can be treated with stem cells delivering a cargo of corrector genes. How can this be transformed into numbers? Certain statements require understanding, before setting out the main points which need to be addressed.

 Leaving aside the fact that for every condition treatable by gene therapy, the number of patients is highly variable; estimations can be made using a highly prevalent disease under phase III by bluebird bio (www.bluebird.com) such as beta-thalassemia/sickle-cell trait (SCT). According to NIH data, 1100 infants are born every year among the African American community, and more than 100,000 individuals are estimated to have SCD in the USA; in Africa 15 million Africans are estimated to have SCD, and there are 200–300,000 affected births per year worldwide (https://www.ncbi.nlm.nih.gov/books/NBK1377/). Current conditions for transduction efficiently into hematopoietic stem cells require around >1 × 1010 biologically active particles per vector dose per infant patient though these data can vary depending on specific features of each treatment. According to current standards of the manufacturing process to achieve such production, harvest produced from 2 square meters and equivalent to 4–6 liters of harvest per patient would need to be produced, representing a huge quantity of 10,000 liters per year to treat new infant cases in the USA for SCD. Current platforms of production and, significantly, purification procedures are not capable of addressing this situation.

#### **6. Conclusions**

Gene therapy is no longer an experimental approach to treat genetic diseases. Several medicine agencies worldwide have approved the commercialization of medicinal products based on viral vectors as intermediate medicinal products. This raises the need to manufacture large quantities of viral vectors under costly cGMP manufacturing environment. There are a limited number of pharmaceutical and biotechnology companies capable to manufacture and release lentiviral vectors of defined composition and quality control in quantities to attend the foreseeable market needs. Challenges for the development of more controlled and cost-effective manufacturing process have yet to be overcome. The complicated manufacturing

*Lentiviral Vectors Come of Age? Hurdles and Challenges in Scaling Up Manufacture DOI: http://dx.doi.org/10.5772/intechopen.81105* 

process needs to be simplified to promote standardization and yield products of increased defined composition. However, there are still open questions that arise from the system employed for production, principally related to the model of production based on DNA-transfected produced cells. Automatization of the manufacturing process is also required, in order to increase capabilities leading to an industrialization process. This will contribute to developing global manufacturing processes for lentiviral vectors and help to establish the target product profile and quality attributes. In summary, efforts in modifying the current manufacturing model of lentivectors are needed to facilitate the entry into commercialization stages.

#### **Acknowledgements**

 The dedication, hard work, and helping discussions of all the team working at VIVEbiotech are strongly acknowledged. The author wants to give special thanks to the cGMP producer team Maite Azcarate and Janire Arizeta for their excellent work and continuous effort to develop the manufacturing process of the company. The meticulous work of the QA/QP management by Estibaliz Perez and Rakel Lopez de Maturana is also acknowledged.

The projects developed at VIVEbiotech have been partially defrayed by project RTC-2015-3393-1 by the Spanish Ministry of Economy.

#### **Conflict of interest**

The author declares no conflict of interest.

#### **Author details**

Juan C. Ramirez VIVEbiotech, Donostia/San Sebastian, Spain

\*Address all correspondence to: jcramirez@vivebiotech.com

© 2018 The Author(s). Licensee IntechOpen. 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.

### **References**

[1] BioPharma Dealmakers. Gene therapy ready to deliver. Nature Reviews Drug Discovery. 2017;**16**:B24-B25

[2] Kumar SRP, Markusic DM, Biswas M, High KA, Herzog RW. Clinical development of gene therapy: Results and lessons from recent successes. Molecular Therapy. Methods & Clinical Development. 2016;**3**:16034:1-11. DOI: 10.1038/mtm.2016.34

[3] Kotterman MA, Chalberg TW, Schaffer DV. Viral vectors for gene therapy: Translational and clinical outlook. Viral Vectors for Gene Therapy. 2015;**17**:63-89. DOI: 10.1146/ annurev-bioeng-071813-104938

[4] Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K, Sadelain M. Gene therapy comes of age. Science. 2018;**359**:1-10. DOI: 10.1126/science. aan4672

[5] Naldini L, Trono D, Verma IM. Lentiviral vectors, two decades later. Science. 2016;**353**:1101-1102. DOI: 10.1126/science.aah6192

[6] White M, Whittaker R, Gandara C, Stoll EA. A guide to approaching regulatory considerations for lentiviralmediated gene therapies. Human Gene Therapy Methods. 2017;**28**:163-176. DOI: 10.1089/hgtb.2017.096

 [7] Mavilio F. Developing gene and cell therapies for rare diseases: An opportunity for synergy between academia and industry. Gene Therapy. 2017;**24**:590-592. DOI: 10.1038/gt.2017.36

 [8] Gandara C, Affleck V, Stoll EA. Manufacture of third-generation lentivirus for preclinical use, with process development considerations for translation to good manufacturing practice. Human Gene Therapy Methods. 2018;**29**:1-15. DOI: 10.1089/ hgtb.2017.098

 [9] Kolata G. Gene therapy's strange roadblock. The New York Times. 2017:D12

[10] Levine BL, Miskin J, Wonnacott K, Keir C. Global manufacturing of CAR T cell therapy. Molecular Therapy. Methods & Clinical Development. 2017;**4**:92-101. DOI: 10.1016/j. omtm.2016.12.006

[11] Wang X, Isabelle Rivière J. Clinical manufacturing of CAR T cells: Foundation of a promising therapy. Molecular Therapy—Oncolytics. 2016;**3**:16015. DOI: 10.1038/mto.2016.15

 [12] Zhao Y, Stepto H, Schneider CK. Development of the first World Health Organization lentiviral vector standard: Toward the production control and standardization of lentivirus-based gene therapy products. Human Gene Therapy Methods. 2017:205-214. DOI: 10.1089/hgtb.2017.078

[13] Merten OW, Scheweizer M, Chahal P, Karmen AA. Manufacturing of viral vectors: Part I. Upstream processing. Pharmaceutical Bioprocessing. 2014;**2**:183-202. DOI: 10.4155/PBP.1416

[14] Merten OW, Scheweizer M, Chahal P, Karmen AA. Manufacturing of viral vectors: Part II. Downstream processing and safety aspects. Pharmaceutical Bioprocessing. 2014;**2**:237-251. DOI: 10.4155/PBP.1415

[15] Schweizernd M, Merten OW. Largescale production means for the manufacturing of lentiviral vectors. Current Gene Therapy. 2010;**10**:474-486

[16] Merten OW, Charrier S, Laroudie N, Fauchille S, Dugue S, Jenny C, et al. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Human Gene Therapy. 2011;**22**:343-356. DOI: 10.1089/hum.2010.060

*Lentiviral Vectors Come of Age? Hurdles and Challenges in Scaling Up Manufacture DOI: http://dx.doi.org/10.5772/intechopen.81105* 

[17] Bandeira V, Peixoto C, Rodrigues AF, Cruz PE, Alves PM, Coroadinha AS, et al. Downstream processing of lentiviral vectors: Releasing bottlenecks. Human Gene Therapy Methods. 2011;**23**:255-263. DOI: 10.1089/ hgtb.2012.059

 [18] Zufferey R, Dull T, Mandel RJ, et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. Journal of Virology. 1998;**72**: 9873-9880

[19] Thomas JA, Ott DE, RJ G. Efficiency of HIV post-entry infection process: Evidence against disproportionate numbers of defective virus. Journal of Virology. 2007:4367-4370

[20] Andreadis S, Lavery T, Davis H, Le Doux JM, Yarmush ML, Morgan JR. Toward a more accurate quantitation of the activity of recombinant retroviruses: Alternatives to titer and multiplicity of infection. Journal of Virology. 2000;**74**:1258-1266

[21] Chen YH, Di Mascio M, Perelson AS, Ho DD, Zhang L. Determination of virus burst size in vivo using a singlecycle SIV in rhesus macaques. PNAS. 2007;**104**:19079-19084. DOI: 10.107/ pnas.0707449104

[22] Carmo M, Alves A, Rodrigues AF, Coroadinha AS, Carrondo MJT, Alves PM, et al. Stabilization of gammaretroviral and lentiviral vectors: From production to gene transfer. The Journal of Gene Medicine. 2009;**11**:670-678. DOI: 10.1002/ jgm.1353

[23] Coroadinha AS, Ribeiro J, Rolda A, Cruz PE, Alves PM, Merten OW, et al. Effect of medium sugar source on the production of retroviral vectors for gene therapy. Biotechnology and Bioengineering. 2006;**94**:24-36. DOI: 10.1002/bit.20778

[24] http://www.sciencemag.org/ news/2002/07/poliovirus-baked-scratch

 [25] Cello J, Paul AV. Wimmer E chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template. Science. 2002;**297**:1016-1018. DOI: 10.1126/ science.1072266

[26] Bell AJ Jr, Fegen D, Ward M, Bank A. RD114 envelope proteins provide an effective and versatile approach to pseudotype lentiviral vectors. Experimental Biology and Medicine. 2010;**235**:1269-1276. DOI: 10.1258/ ebm.2010.010053

 [27] Vanden Driessche T, Chuah MK. Targeting endothelial cells by gene therapy. Blood. 2013;**122**:1993-1994. DOI: 10.1182/blood-2013-08-518266

[28] Levy C, Amirache F, Girard-Gagnepain A, Frecha C, Roman-Rodrıguez FJ, Bernadin O, et al. Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent. Blood Advances. 2017;**1**:2088-2104. DOI: 10.1182/ bloodadvances.2017007773

 [29] Frecha C, Lévy C, Cosset F-L, Verhoeyen E. Advances in the field of lentivector-based transduction of T and B lymphocytes for gene therapy. Molecular Therapy. 2010;**18**:1748-1757. DOI: 10.1038/mt.2010.178

[30] Ohishi M, Shioda T, Sakuragi J-i. Retro-transduction by virus pseudotyped with glycoprotein of vesicular stomatitis virus. Virology. 2007;**362**:131-138. DOI: 10.1016/j.virol.2006.12.030

[31] Damme NV, Guatelli J. HIV-1 Vpu inhibits accumulation of the envelope glycoprotein within clathrin-coated, Gag-containing endosomes. Cellular Microbiology. 2008;**10**:1040-1057. DOI: 10.1111/j.1462-5822.2007.01101

[32] Neil SJD, Eastman SW, Jouvenet N, Bieniasz PD. HIV-1 Vpu promotes release and prevents endocytosis of

nascent retrovirus particles from the plasma membrane. PLoS Pathogens. 2006;**2**:354-367. DOI: 10.1371/journal. ppat.0020039.g011

 [33] Manceur AP, Kim H, Misic V, Andreev N, Dorion-Thibaudeau J, Lanthier S, et al. Scalable lentiviral vector production using stable HEK293SF producer cell lines. Human Gene Therapy Methods. 2017;**28**:330-339. DOI: 10.1089/ hgtb.2017.086

[34] Ausubel LJ, Hall C, Sharma A, Shakeley R, Lopez P, QuezadaV, et al. Production of cGMP-grade lentiviral vectors. Bioprocess International. 2012;**10**(2):32-43

[35] Richieri SP, Bartholomew R, Aloia RC, Savary J, Gore R, Halt J, et al. Characterization of highly purified, inactivated HIV-l particles isolated by anion exchange chromatography. Vaccine. 1998;**16**:119-129

[36] Le Doux JM, Davis HE, Morgan JR, Yarmush ML. Kinetics of retrovirus production and decay. Biotechnology and Bioengineering. 1999;**63**:654-662

[37] Carmo M, Panet A, Carrondo MJT, Alves PM, Cruz PE. From retroviral vector production to gene transfer: Spontaneous inactivation is caused by loss of reverse transcription capacity. The Journal of Gene Medicine. 2008;**10**:383-391. DOI: 10.1002/jgm.1163

[38] Carmo M, Dias JD, Panet A, Coroadinha AS, Carrondo MJT, Alves PM, et al. Thermosensitivity of the reverse transcription process as an inactivation mechanism of lentiviral vectors. Human Gene Therapy. 2009;**20**:1168-1176. DOI: 10.1089=hum.2009.068

[39] Valkama AJ, Leinonen HM, Lipponen EM, Turkki V, Malinen J, Heikura T, et al. Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor. Gene Therapy. 2018;**25**:39-46. DOI: 10.1038/gt.2017.91

#### **Chapter 4**

## Orf Virus: A New Class of Immunotherapy Drugs

*Ruixue Wang and Shuhong Luo* 

#### **Abstract**

 Orf is one of the most widespread viral diseases worldwide, usually benign and self-limiting, and mainly affects not only sheep and goats, but also various other ruminants and mammals. The causative agent, orf virus (ORFV) is a member of the genus parapoxvirus, owing to its zoonotic importance and ability to crossinfect other species sporadically. ORFV encodes virulence and immunomodulatory factors that interfere with host inflammatory effect and antiviral immune mechanisms and induces a transient and complex cytokine response, initially represented by Th1-related cytokines followed by Th2-related cytokines. The ORFV has evolved several mechanisms to survive in the presence of the immune system, resulting in repeated infections. Currently, ORFV has been developed as vaccines in veterinary field. The unique host immune escape ability obtained by ORFV has made it one of the important candidates for prevention and treatment of various diseases (including chronic viral diseases, tumor, and liver fibrosis).

**Keywords:** immunoregulation, immunotherapy, orf virus, vaccine, oncolysis virus

#### **1. Introduction**

 Orf was first discovered in Europe in 1920. At present, the main geographical distribution of the pathogen is not clear, which is considered to be prevalent worldwide since orf exists in all areas where sheep exists. The United States of America, Germany, Korea, Japan, India, Argentina, Malaysia, Egypt, and China have reported the occurrence and prevalence of the disease, which has brought a certain degree of loss to the sheep industry. The causative agent, orf virus (ORFV), also known as contagious pustular stomatitis (contagious ecthyma) virus, belongs to the parapoxvirus genus of poxvirus family and causes nonsystemic cutaneous disease by mainly infecting sheep and goats. In recent years, the cases of human, camel, yak, red squirrel, cat, domestic reindeer, etc. infected by ORFV have been reported. This indicates that the host range of the virus is expanding.

After being infected by ORFV, the infected animals begin with the appearance of erythema on the lips, tongue, nose, and breast of sheep, then develop into papules, blisters, and pustules, and finally form crusts, characterized by proliferative inflammation (**Figure 1**). The course of the disease is mostly an acute infection, healed within 1-2 months, but there are also cases of chronic persistent infection records. The disease rarely causes animal death unless host immunosuppression or secondary infection occurs, but there are also reports of a high mortality rate of 93% in young goats.

**Figure 1.** 

*Typical clinical signs of ORFV infection in sheep (cited from reference [1]). Proliferative skin lesions of two infected sheep around the mouth, nostrils (arrows), and the eyelids (arrowheads).* 

#### **2. ORFV genome biology**

 ORFV belongs to the subfamily parapoxvirus of the poxvirus family. Other members of this genus include pseudovaccinia virus (PCPV), bovine papular stomatitis virus (BPSV), and parapoxvirus of red deer in New Zealand (PVNZ). The mature ORFV particles are 250–280 mm in length and 170–200 nm in width, and elliptical and coiled shaped, while the immature virus particles are conical, brick-shaped, and special coiled spherical particles (**Figure 2**). The surface of the virus particles showed a characteristic braided helical structure of cross-arranged around the long axis of the virus particles for eight-shaped winding. There are other ways of winding, and the virus particles encapsulated outside the capsule. ORFV replicates and matures in the cytoplasm, encoding polymerases associated with virus replication and transcription.

 ORFV is a linear double-stranded (ds) DNA virus with a genome size of 134– 139 kb. The average G + C content of the virus genome was approximately 64%. The content of G + C in the ORFs in the central coding region of the genome is not very different, but the ORFs in the two ends of the genome are very different, which are even less than 50% in some regions, such as ORFV127. Mercer believes that the terminal is based on the conservation of the genome sequence (OVSA00, OVIA82, and NZ2) and the transcriptional initiation of the gene in three strains of the virus. Even though the G + C content of seven ORFVs in ORFV102–104, 109–112 is quite different from the genome average G + C content, and the homology of the encoded proteins is low, but the G + C content between different strains is very close [2]. The content of G + C in BPSV genome terminal variant region is similar, too, which is a marker of poxvirus members. Like other members of the poxvirus family, it has a large central coding region in the middle of the genome and an inverted terminal repeat (ITR) at both ends, which had covalently closed terminal hairpin structures (**Figure 3**).

*Orf Virus: A New Class of Immunotherapy Drugs DOI: http://dx.doi.org/10.5772/intechopen.81535* 

#### **Figure 2.**

*The electron microscopy of the ORFV (cited from reference [1]). The predominantly immature virions (arrows and arrowheads) (A) and intracellular mature virions (B) under transmission electron microscopy. (C) The extracellular virions under atomic force microscopy. Scale bars = 500 nm.* 

#### **Figure 3.**

*The structure of the genome of ORFV strain NA1/11. The genome of ORFV is 138 kb, encoding 132 genes, and includes highly variable terminal regions, responsible for virus virulence and pathogenesis, and relatively conserved central regions with a high GC content for viral replication and virus morphogenesis. There are 16 novel genes unique to parapoxvirus, with putative virulence-host range functions.* 

There are many studies on the whole gene sequencing and analysis. The first genome of two strains (SA00 and IA82) was sequenced in 2003 by the Mei Dao Animal Disease Center of the US Department of Agriculture. OVSA00 was identified as the reference sequence of the virus. Mercer submitted the full genome sequence of the NZ2 strain in 2006, and McGuire sequenced strain (D1701) and submitted it to GenBank. Luo submitted the full genome sequence of the ORFV


#### **Table 1.**

*Summary of complete genomic sequence data of 10 ORFV strains.* 

strains of China. Up to now, NCBI has included 10 complete genome sequences of ORFV (**Table 1**).

 Delhon et al. conservatively estimated about 130 coding genes in the ORFV genome [5]. Through analysis of the genome sequence of NZ2 strain, 132 possible coding genes were found in the genome of this virus strain. Transcription initiation elements (TAAAT) existed before the coding regions of the two genes and were found in BPSV. Similar conserved sequences also exist, but these two genes only exist in parapoxvirus but not in orthopoxvirus. Mercer et al. checked the ORFs of 24 genes of ORFV, which showed high interspecific variability mainly in the two terminal variant regions [7]. Many genes located in the core region have been identified. The ORFV050 gene, similar to L4R of vaccinia virus (VACV), encodes the DNAbinding virion core protein VP8 [8]. ORFV057 encodes protein OH1, analogous to the VACV structural protein VH1, that can dephosphorylate phosphatidylinositol 3, 5-bisphosphate, and plays a role in virion maturation [9]. The ORFV011 (B2L) gene, a homolog of the F13 L gene of VACV, encodes a major envelope protein of 42 kDa, which is thought to be a lipase. Additionally, the viral A32L gene (ORFV108) encodes an ATPase involved in virion DNA packaging [10]. Virulence genes, coding genes related to host pathogenesis and immunoregulatory genes are located in the ITR regions of the ORFV genome, such as ORFVs 007, 020, 112, 117, 119, 125, and 132.

#### **3. The immunomodulatory ability of ORFV**

#### **3.1 ORFV and immunomodulatory ability**

ORFV is widely recognized as having a powerful host immunoregulatory function. ORFV can quickly mediate humoral and adaptive immune responses. After being infected with ORFV, many cells of the innate immune system are activated and induce the secretion of chemokines and cytokines. Neutrophils, natural killer (NK) cells, and dendritic cells (DC) are recruited at the site of infection. In the early stage of infection, ORFV mainly induces the Th1-type immune response. Peripheral immune cells secrete IFN-γ, TNF-a, IL-6, IL-8, IL-12, IL-18, and then Th2-type immune response appears, mainly inducing secreting of IL-4, IL-10, IL-1 receptor antagonists (IL-1RA). The conditioning of complement and antigen-presenting-cell (APC)-mediated antigen presentation are important steps to activate the immune response.

#### *Orf Virus: A New Class of Immunotherapy Drugs DOI: http://dx.doi.org/10.5772/intechopen.81535*

 ORFV and some of its encoded proteins have a good immunomodulatory function. Homologous alignment analysis of the host sequence and the viral gene sequence has identified that some ORFV genes have corresponding immunoregulatory functions, including coding for IL-10 homologous proteins, chemokine binding proteins, secretory inhibitors of GM-CSF and IL-2, vascular endothelial factor (VEGF), and interferon resistance protein. The main targets of IFN resistance genes are host cytokines, chemokines, NF-κB signaling pathway, and apoptosis pathway. The synergistic effect of these proteins has strong immunomodulatory effects on ORFV.

 ORFV is the only virus that contains the gene encoding lL-10 found in the poxvirus family. vIL-10, a 21.7 kDa protein with remarkable homology to IL-10, which is encoded by ORFV127 gene, plays an important role in immunosuppression through inhibition of cytokine syntheses, such as TNF-α and IL-8, IFN-γ [11], suppression of the maturation and functionality of DC [12, 13], blockage of Th1 cell activation indirectly through weakening the antigen processing, and presentation ability of APC. The direct role of the vIL-10 gene in virulence was demonstrated using an ORFV lacking the IL-10 gene, which showed attenuated properties in animal experiments [14], while vIL-10 can exert immunostimulatory effects by inducing moderate compensatory immune activation [15].

One of the characteristics of ORFV infection is the proliferation of capillaries and the increasing of permeability in the dermis, which is caused by viral VEGF. The deletion of VEGF gene leads to vascular permeability reduction, inhibition of epidermal cells and inactivation of VEGF receptors (VEGF-2) [16, 17]. Viral VEGF, sharing 16–27% of amino acid identity with its homolog VEGFs (VEGF-A, VEGF-B, VEGF-C, VEGF-D), has the same function as VEGFs: promoting the proliferation of epidermal cells, inducing the proliferation of host vascular endothelial cells and increasing the permeability of capillary vessel wall. Interestingly, VEGF variants are observed in different strains of ORFV. Despite such variation existing, the functional domains of the protein exhibit conserved structure. Studies reported that a recombinant virus strains lacking VEGF gene reduced vascular changes characteristic of natural infections, with less proliferation of blood vessels and dermal edema, pustule, and scab formation in ORFV pathogenesis [18].

In the early stage of ORFV infection, the orf virus interferon resistance protein (OVIFNR) encoded by ORFV20 binds to the viral replication intermediates and prevents the termination of IFN-induced virus-carrying protein translation. OVIFNR shares 31% sequence similarity with the E3L protein of VACV, and the C-terminal region with the binding activity of dsRNA (or viral replication intermediates) was the necessary region to prevent the antiviral activity of IFN and associated with pathogenicity and host tropism. OVIFNR eliminates the antiviral effect of IFN through the synthesis of the dsRNA-dependent protein, like protein kinase (PKR) [19]. The dsRNA-activated PKR is one of the main antiviral proteins induced by IFN. Activated PKR phosphorylates the translation initiation factor eIF2-α and impairs protein synthesis to inhibit viral replication [20]. OVIFNR not only compete with PKR to bind to viral replication intermediates but also inhibit the activity of PKR, thus preventing host cell interference from terminating the translation of viral proteins.

The ORFV007-encoded dUTPase clusters with mammalian counterparts and is more similar to mammalian dUTPases than to dUTPases from other poxviruses [21]. The virulence of ORFV with the 007-gene deletion is significantly lower than that of natural ORFV.

Chemokine-binding protein (CBP), the coding product of ORFV112, has similarities in structure and function with CBP II of orthopoxvirus and rabbit poxvirus. It can bind and inhibit chemokine and prevent chemokine-receptor

interaction. When chemokines bind to their receptor, the G protein-coupled receptor, the white blood cells were recruited and activated in viral infection. In addition, chemokines interact with glycosaminoglycan (GAG) and establish a gradient liquid phase that guides leukocytes through the endothelium into tissues. ORFV-CBP and CBP-II have high affinity to some CC chemokines, such as monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1 (MIP1), and regulated on activation, normal T cell expressed and secreted (RANTES), which can produce chemotactic effects of both nucleus/macrophage and T lymphocyte toward inflammation. Although ORFV encodes a number of secreted anti-inflammatory factors, the deletion of the CBP gene severely attenuated viral virulence and pathogenesis [22].

GIF is encoded by ORFV117, expressed in the late stage of infection, and has the dual activity of inhibiting host GM-CSF and IL-2, thus inhibiting host immune activity [23]. The gene is conserved in different ORFV strains and also exists in other parapoxvirus strains, but the amino acid sequence similarity between ORFV and BPSN is only 40%. However, the function of GIF in virulence and pathogenesis is not yet known.

#### **3.2 ORFV and immune evasion**

After infection, sheep produces antibodies to four or five immunodominant antigens [24–26]. Murine monoclonal antibodies recognizing 42 kDa envelope proteins, the 10 kDa putative fusion protein, and 65 kDa antigens have been described that can discriminate between the different parapoxvirus species [27, 28]. In spite of an apparently normal immune response to infection, sheep can be repeatedly infected, suggesting that, in common with other large DNA viruses, ORFV has evolved an immune evasion strategy [29, 30].

ORFV infection stimulated hyperplasia of epidermal cells and capillaries growth with increased vascular permeability, which allows increased virus replication and formation of scabs on wound healing. Scabs are rich in virus particles and provide temporary refuge for viruses to escape from immunization. The antiviral effect of IFN is the first line of defense against viral infection; ORFV evades immune clearance by inhibiting IFN-stimulated genes expression mediated by the JAK/STAT signaling pathway [31]. In addition, ORFV also can induce apoptosis mediated by CD95 pathway [15] or inhibit the pro-inflammatory NF-κB signaling, a crucial regulator of host innate immune responses. For pathogens, interfering with the activation of NF-κB is a particular strategy against host defense mechanisms. The regulation of NF-κB includes the regulation of IκB in the cytoplasm, and posttranslational phosphorylation, acetylation, and methylation in the nucleus. The ORFV 002, 024, 073, 119, and 121 genes have been reported that play roles in NF-κB pathway regulation [32–38].

ORFV002 is an early and late stage virus gene, mainly located in the nucleus. ORFV002-encoded protein can inhibit the activation of NF-κB pathway induced by TNF-α and ORFV virus infection, which may through interfering the interaction between NF-κB-p65 and P300 in the nucleus block the acetylation of NF-κB-p65 Lys310 when phosphorylation occurs at ORFV002 Ser 276 [32, 33]. The 52 amino acids of ORFV002 N terminal may interact with protein S100A4 [34].

The ORFV024-encoded protein combines with LAGE3 to inhibit the phosphorylation of IKKs complex and then affects the phosphorylation of NF-κB-p65, inhibits the host immune cells to secrete some important cytokines, and regulates the host's immune response [35].

ORFV119 blocks the NF-κB signaling largely in a pRb-dependent manner, by inhibiting IKK complex activation early in infection [36]. ORFV119 interacted

#### *Orf Virus: A New Class of Immunotherapy Drugs DOI: http://dx.doi.org/10.5772/intechopen.81535*

with TNF receptor-associated factor 2 (TRAF2), an adaptor protein recruited to signaling complexes upstream of IKK in infected cells, in a LxCxE motif-dependent manner, which leads to inhibition of NF-κB signaling.

The ORFV073 protein, 188 amino acids with a molecular weight of 21.8 kDa, whose protein at 149, 160, and 166 locations contained three predicted and partially overlapping nuclear localization signals, is located in nucleus during viral replication and be related to gene expression regulation. When the ORFV073 gene was deleted in the ORFV genome, the expression of chemokines and other pro-inflammatory genes was significantly increased, and most of the gene expression changes were regulated by the NF-κB transcription factor family [37].

After infected with an ORFV121 gene deletion mutant, NF-κB-mediated gene transcription was increased, while the expression of ORFV121 in cell cultures significantly decreased NF-κB-regulated reporter gene expression, suggesting that NF-κB inhibitor binds to and inhibits the phosphorylation and nuclear translocation of NF-κB-p65 in the cell cytoplasm, thus providing a mechanism for the inhibition of NF-κB-p65 phosphorylation and nuclear translocation [38].

#### **4. Diagnosis and treatment**

At present, there is no international standard for the diagnosis of amniotic aphthous ulcer, mainly based on the typical clinical symptoms and laboratory tests to diagnose. As shown in **Figure 4**, current laboratory diagnoses include PCR, ELISA, electron microscopy, histopathology, Western blotting.

The orf is mainly observed by the morphology of virus particles. A human case of orf was identified by transmission electron microscopy in a 20-year-old woman with two painful pruritic lesions on her left index finger [39]. Under the transmission electron microscopy, multiple typical ORFV particles existed with brickshaped morphology, consisting of a central DNA-containing core surrounded by a bilayered capsid.

**Figure 4.**  *The diagnosis methods and treatment of orf.* 

#### *Systems Biology*

Histopathological features of the orf lesion include vacuole and swelling of keratinocytes, interstitial degeneration, marked epidermal hyperplasia, microswelling in the epidermis, aggregation of neutrophils, DC, T cells and B cells in the subcutaneous tissue, and formation of the crust. Eosinophilic inclusion bodies were also evident in the infected cytoplasm, but not at all stages of infection. There are mononuclear cells infiltrating into the dermis, such as phagocytes, lymphocytes, and eosinophils. In secondary infection, a dermal infiltration of neutrophils appeared [40].

 A variety of primary cells and cell lines can be used to isolate ORFV. Primary lamb testicular cells and primary lamb kidney-trophoblast cells, which were originally used by Plowright et al., are the most common in the prozonal cells [41]. The primary fetal lamb muscle cells [42] and turbinate cells [5], fetal bovine lung cells [43], Madin-Darby bovine kidney cells [44], and vero cells [45] were also used. ORFV can induce cytopathy in these cells after inoculation of the first generation or blindly transmitted for one to two generations. The common cytopathic effects are aggregation, fusion, shrinkage, and cell detachment. Eosinophils in the cytoplasm can be observed by staining of infected cell lines. Parapoxvirus culturing, in general, is considered to be difficult, with a need for many passages before observing cytopathic effects, such as ballooning, wounding, degeneration of cells, etc.

Serum neutralization tests, commonly used for the detection of antibodies, are not considered to be the method of choice for primary diagnosis, as immunity to ORFV is mainly cell-mediated, and neutralizing antibodies are usually at small concentrations. Suspected serum with a titer of 8 and 20 or above is considered as positive for orf infection, in serum neutralization test and complement-fixation test, respectively [46].

Indirect ELISA with a purified virus as coating antigen, peroxidase complex protein A, G and chimeric A/G as secondary antibodies can be used to detect antibody levels in different animals [47]. The method has been successfully applied to detect the virus in camels [48], lambs [49], and humans [50] suspected to be infected with the disease.

A 40 kDa immunogenic protein has been found in the positive sera of infected animals by Western blotting. Similarly, two proteins of approximately 22 and 20 kDa have been found by this method.

 Different PCR methods can be used for rapid diagnosis of ORFV. A conventional PCR assay based on amplification of the ORFV B2L gene, a homolog of the F13 L gene of VACV, encodes a major envelope protein of 42 kDa, which is supposed to be a lipase, and was used to detect parapoxvirus species. A duplex PCR assay using A29 gene (413 bp) and H3L gene (708 bp) has the potential to differentiate capripoxviruses from ORFV [51]. A single-step PCR method was applied for the rapid differential diagnosis of ORFV infections [52]. Primers targeting the A32L gene, besides, the complete sequences of another two viral genes were also investigated: the B2L, and E3L genes, which encodes a dsRNAbinding protein. A conventional PCR assay combined with DNA sequencing can be used to distinguish among the different parapoxvirus species [53]. A sensitive and specific SYBR Green I real-time PCR assay was performed to quantitatively detect ORFV [54].

Restriction fragment length polymorphism (RFLP) of the genome is a powerful tool for analyzing the molecular characteristics of poxvirus, which can potentially distinguish different strains of amniotic stomatitis virus. Restriction enzyme fragments are obtained by enzyme (EcoRI, BamHI, and HindIII) digestion. Commercialized kits with random amplified polymorphic DNA have been used to distinguish virus strains from large numbers of species. Loop-mediated isothermal

#### *Orf Virus: A New Class of Immunotherapy Drugs DOI: http://dx.doi.org/10.5772/intechopen.81535*

amplification targeting B2L, DNA polymerase, and F1L genes have been developed and proven to be effective diagnostic tools [55, 56].

The ORFV has a strong ability to adapt to the external environment and has a strong resistance, even after a year in the sheep pen around, the virus still has a strong infectivity. But the virus is sensitive to temperature changes, suitable for the humid environment, and can be killed at 60°C in 30 min. Besides, the use of antiviral drugs has been applied in human and animal orf infections with satisfying results, such as Cidofovir [57, 58].

 The virus is virulent for about a year when it is added to 50% glycerol saline and stored at 4°C. In the actual feeding process, generally choosing to use 20% hot grass and wood ash solution, 10% lime milk, 2% sodium hydroxide solution, and 1% acetic acid for disinfection can kill the virus, while using 2% sodium hydroxide solution to kill the virus in 5 min. The treatment for human orf is often focused on secondary infection. Previous reports have described speeding up the healing process with topical imiquimod [59] and the antiviral cidofovir cream [60]. Cryotherapy has also been used successfully to treat orf cases, especially in immunocompromised patients [61].

#### **5. Clinical applications**

 ORFV is widely recognized as a virus with powerful host immunoregulatory function, but neutralizing antibody in ORFV infection is rare [27], and passive transfer of antibody-rich colostrum or serum does not protect lambs from infection [62, 63].

In veterinary medicine, ORFV is widely used as a new type of preventive and immunomodulatory preparation. Activated or inactivated ORFV preparations have a dose-dependent immunomodulatory function. Therefore, based on ORFV, drugs for a variety of infectious animal diseases have been developed (Baypamun; Zylexis). For example, Baypamun was used to suppress stress-related infections in horses, and clinical data showed a significant 40% reduction in the incidence of stress-related infections in the medication group [64]. Its therapeutic effect has also been verified in other animals, such as the treatment of bovine herpesvirus type 1, chronic stomatitis or infectious peritonitis in cats, and breast tumors in dogs. Inactivated ORFV can induce spontaneous regulation of cytokine responses in mice, such as up-regulation of Th1 cytokines (IL-12, IL-18, and IFN-γ), activation of CD14 and TLR-mediated monocyte activation, and release of anti-inflammatory Th2-related cytokines.

#### **5.1 Antiviral preparations**

Activated or inactivated ORFV makes many kinds of animals to fight different viral diseases. The ORFV has significant antifibrous activity in CCL4 mediated liver fibrosis [65]. The inactivated ORFV agents with a low dose (only 500,000 virus particles) are more effective in transgenic mice than the standard 3TC for HBV infection [66]; thus, it can be used as the candidate antiviral agent for the treatment of human HBV. Inactivated ORFV has anti-HCV activity in vitro and transgenic mice model [67]. In addition, ORFV can prevent the recurrence of fatal herpes simplex virus (HSV) and recurrence of genital herpes in Guinea [68].

ORFV can be used as a carrier to produce new animal recombinant vaccines. Recombinant ORFV vector induces an antiviral response in various animals. Recombinant pseudorabies virus glycoprotein gC or gD can be used to prevent

 infection in mice and pigs [69, 70]. ORFV recombinant with protein P40 can induce the immune protection of rats from infection, and effectively eliminated the Borna disease virus in the brain [71]. ORFV recombinant classical swine fever virus protein E2 can also make pigs immune to classical swine fever virus (CSFV) [72]. Recombinant ORFV expressing hemagglutinin (HA) or nucleoprotein (NP) of highly pathogenic avian influenza virus H5N1 protects mice against H5N1 and H1N1 influenza viruses [73]. A recombinant virus strain D1701-VP1 of rabbit blast virus VP60 gene expression induced the infected cells releasing goblet-like particles to protect the rabbit from ORFV attack [74].

#### **5.2 Oncolytic virus**

 Live or inactivated ORFV induces antitumor immune responses in multiple tumor models. Fiebig et al. reported for the first time that inactivated ORFV has antitumor effects in a variety of tumor metastasis models, such as mousetransplanted malignant melanoma B16F10 and human breast cancer MDA-MB-231 models [75], and found that NK cells play an important role in the antitumor of ORFV. After neutralizing IFN-γ, the antitumor effect disappeared, while the anti-NK-1.1 antibody partially weakened the antitumor activity of ORFV by inhibiting the activity of NK and NKT cells. Inactivated ORFV inhibited tumor growth in a mouse MDA-MB-231 tumor model without NK and lacking functional T and B lymphocytes. Whether inactivated or active ORFV is used to treat mouse tumor models, NK cells play an important role in antitumor. A study by Rintoul further confirmed that ORFV inhibits tumor growth of melanoma and colorectal cancer, and proved that ORFV could play an antitumor role by activating NK cells and stimulating their secretion of cytokines IFN-γ and granzyme B [76]. Tai et al. found that surgery mediated the dysfunction of NK cells [77]. Intraoperative injection of ORFV improves the function of NK cells, thereby reducing intraoperative metastasis and prolong survival. Recently, a study of the virus strain CF189, which is the high similarity with the ORFV virus strain NZ2 obtained by homologous recombination, showed that CF189 effectively kills three negative breast cancer cells with time and dose dependence [78].

#### **6. Conclusion**

 ORFV causes orf, a nonsystemic, highly contagious, ubiquitous disease of sheep and goats [79], which is characterized by maculopapular and proliferative lesions affecting the skin around the mouths, nostrils, and teats. Virus virulence and immunomodulation genes of ORFV contribute to combat local inflammatory response, innate immunity (including apoptosis, NK cell activation, and antiviral response), and immune adaptation. Therefore, ORFV has been used in veterinary medicine as preventive and therapeutic immunomodulatory agents. Moreover, live or inactivated ORFV preparations exhibit immunomodulatory effects, with therapeutic efficacy demonstrated for various diseases, including infectious diseases and tumors.

#### **Acknowledgements**

 This work was partially supported by grants from the National Natural Science Foundation of China (NSFC) (nos. 81773271 and 31672536), the Foshan University Senior Talent Start Fund (20161110004, y2016-ky69, and Gg07043), the *Orf Virus: A New Class of Immunotherapy Drugs DOI: http://dx.doi.org/10.5772/intechopen.81535* 

Foshan University High-level University Fund (y2016-td148, 20170131020), and Guangdong Provincial Education Department of Education of Guangdong Province (2017KZDXM088). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

### **Conflict of interest**

The authors declare that no conflict of interest exists.

### **Author details**

Ruixue Wang1,2 and Shuhong Luo1 \*

1 Department of Laboratory Medicine, School of Stomatology and Medicine, Foshan University, Foshan, Guangdong Province, P.R. China

2 Department of Basic Medical Sciences, School of Stomatology and Medicine, Foshan University, Foshan, Guangdong Province, P.R. China

\*Address all correspondence to: sluo815@gmail.com

© 2018 The Author(s). Licensee IntechOpen. 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.

#### **References**

[1] Li W, Ning Z, Hao W, Song D, Gao F, Zhao K, et al. Isolation and phylogenetic analysis of orf virus from the sheep herd outbreak in northeast China. BMC Veterinary Research. 2012;**8**:229. DOI: 10.1186/1746-6148-8-229

[2] Chen H, Li W, Kuang Z, Chen D, Liao X, Li M, et al. The whole genomic analysis of orf virus strain HN3/12 isolated from Henan province, central China. BMC Veterinary Research. 2017;**13**(1):260. DOI: 10.1186/ s12917-017-1178-1

[3] McGuire MJ, Johnston SA, Sykes KF. Novel immune-modulator identified by a rapid, functional screen of the parapoxvirus ovis (Orf virus) genome. Proteome Science. 2012;**10**(1):4. DOI: 10.1186/1477-5956-10-4

[4] Chi X, Zeng X, Li W, Hao W, Li M, Huang X, et al. Genome analysis of orf virus isolates from goats in the Fujian province of southern China. Frontiers in Microbiology. 2015;**6**:1135. DOI: 10.3389/fmicb.2015.01135

 [5] Delhon G, Tulman ER, Afonso CL, Lu Z, de la Concha-Bermejillo A, Lehmkuhl HD, et al. Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus. Journal of Virology. 2004;**78**(1):168-177. DOI: 10.1128/ JVI.78.1.168-177.2004

[6] Li W, Hao W, Peng Y, Duan C, Tong C, Song D, et al. Comparative genomic sequence analysis of Chinese orf virus strain NA1/11 with other parapoxviruses. Archives of Virology. 2015;**160**(1):253-266. DOI: 10.1007/ s00705-014-2274-1

 [7] Mercer AA, Ueda N, Friederichs SM, Hofmann K, Fraser KM, Bateman T, et al. Comparative analysis of genome sequences of three isolates of Orf virus reveals unexpected sequence variation.

Virus Research. 2006;**116**(1-2):146-158. DOI: 10.1016/j.virusres.2005.09.011

 [8] Wang H, Jiang J, Ding R, Wang X, Liao M, Shao J, et al. Identification and characterization of Orf virus 050 protein proteolysis. Virus Genes. 2017;**53**(3):400-409. DOI: 10.1007/ s11262-017-1430-6

[9] Segovia D, Haouz A, Porley D, Olivero N, Martinez M, Mariadassou M, et al. OH1 from Orf virus: A new tyrosine phosphatase that displays distinct structural features and triple substrate specificity. Journal of Molecular Biology. 2017;**429**(18):2816- 2824. DOI: 10.1016/j.jmb.2017.07.017

 [10] Yogisharadhya R, Bhanuprakash V, Venkatesan G, Balamurugan V, Pandey AB, Shivachandra SB. Comparative sequence analysis of poxvirus A32 gene encoded ATPase protein and carboxyl terminal heterogeneity of Indian orf viruses. Veterinary Microbiology. 2012;**156**(1-2):72-80. DOI: 10.1016/j. vetmic.2011.10.021

[11] Imlach W, McCaughan CA, Mercer AA, Haig D, Fleming SB. Orf virusencoded interleukin-10 stimulates the proliferation of murine mast cells and inhibits cytokine synthesis in murine peritoneal macrophages. The Journal of General Virology. 2002;**83**(Pt 5):1049-1058. DOI: 10.1099/0022-1317-83-5-1049

[12] Chan A, Baird M, Mercer AA, Fleming SB. Maturation and function of human dendritic cells are inhibited by orf virus-encoded interleukin-10. The Journal of General Virology. 2006;**87**(Pt 11):3177-3181. DOI: 10.1099/ vir.0.82238-0

[13] Lateef Z, Fleming S, Halliday G, Faulkner L, Mercer A, Baird M. Orf virus-encoded interleukin-10 inhibits maturation, antigen presentation

*Orf Virus: A New Class of Immunotherapy Drugs DOI: http://dx.doi.org/10.5772/intechopen.81535* 

and migration of murine dendritic cells. The Journal of General Virology. 2003;**84**(Pt 5):1101-1109. DOI: 10.1099/ vir.0.18978-0

[14] Fleming SB, Anderson IE, Thomson J, Deane DL, McInnes CJ, McCaughan CA, et al. Infection with recombinant orf viruses demonstrates that the viral interleukin-10 is a virulence factor. The Journal of General Virology. 2007;**88**(Pt 7):1922-1927. DOI: 10.1099/ vir.0.82833-0

[15] Kruse N, Weber O. Selective induction of apoptosis in antigenpresenting cells in mice by Parapoxvirus ovis. Journal of Virology. 2001;**75**(10):4699-4704. DOI: 10.1128/ JVI.75.10.4699-4704.2001

 [16] Savory LJ, Stacker SA, Fleming SB, Niven BE, Mercer AA. Viral vascular endothelial growth factor plays a critical role in orf virus infection. Journal of Virology. 2000;**74**(22):10699-10706. DOI: 10.1128/JVI.74.22.10699-10706.2000

 [17] Ueda N, Wise LM, Stacker SA, Fleming SB, Mercer AA. Pseudocowpox virus encodes a homolog of vascular endothelial growth factor. Virology. 2003;**305**(2):298-309. DOI: 10.1006/ viro.2002.1750

 [18] Wise LM, Savory LJ, Dryden NH, Whelan EM, Fleming SB, Mercer AA. Major amino acid sequence variants of viral vascular endothelial growth factor are functionally equivalent during Orf virus infection of sheep skin. Virus Research. 2007;**128**(1-2):115-125. DOI: 10.1016/j.virusres.2007.04.018

[19] Tossici-Bolt L, Fleming JS, Conway JH, Martonen TB. An analytical technique to recover the third dimension in planar imaging of inhaled aerosols--2 estimation of the deposition per airway generation. Journal of Aerosol Medicine. 2007;**20**(2):127-140. DOI: 10.1089/jam.2007.0577

 [20] Li H, Zhu X, Zheng Y, Wang S, Liu Z, Dou Y, et al. Phylogenetic analysis of two Chinese orf virus isolates based on sequences of B2L and VIR genes. Archives of Virology. 2013;**158**(7):1477-1485

 [21] Cottone R, Buttner M, McInnes CJ, Wood AR, Rziha HJ. Orf virus encodes a functional dUTPase gene. The Journal of General Virology. 2002;**83**(Pt 5):1043-1048. DOI: 10.1007/ s00705-013-1641-7

[22] Fleming SB, McCaughan C, Lateef Z, Dunn A, Wise LM, Real NC, et al. Deletion of the chemokine binding protein gene from the parapoxvirus Orf virus reduces virulence and pathogenesis in sheep. Frontiers in Microbiology. 2017;**8**:46. DOI: 10.3389/ fmicb.2017.00046

[23] Deane D, McInnes CJ, Percival A, Wood A, Thomson J, Lear A, et al. Orf virus encodes a novel secreted protein inhibitor of granulocytemacrophage colony-stimulating factor and interleukin-2. Journal of Virology. 2000;**74**(3):1313-1320. DOI: 10.1128/ JVI.74.3.1313-1320.2000

[24] McKeever DJ, Jenkinson DM, Hutchison G, Reid HW. Studies of the pathogenesis of orf virus infection in sheep. Journal of Comparative Pathology. 1988;**99**(3):317-328. DOI: 10.1016/0021-9975(88)90052-7

 [25] Yirrell DL, Reid HW, Norval M, Howie SE. Immune response of lambs to experimental infection with Orf virus. Veterinary Immunology and Immunopathology. 1989;**22**(4):321-332. DOI: 10.1016/0165-2427(89)90168-2

[26] Chand P, Kitching RP, Black DN. Western blot analysis of virusspecific antibody responses for capripox and contagious pustular dermatitis viral infections in sheep. Epidemiology and Infection. 1994;**113**(2):377-385. DOI: 10.1017/S0950268800051803

[27] Czerny CP, Waldmann R, Scheubeck T. Identification of three distinct antigenic sites in parapoxviruses. Archives of Virology. 1997;**142**(4):807- 821. DOI: 10.1007/s007050050120

 [28] Housawi FM, Roberts GM, Gilray JA, Pow I, Reid HW, Nettleton PF, et al. The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein. Archives of Virology. 1998;**143**(12):2289-2303. DOI: 10.1007/ s007050050461

[29] Alcami A, Koszinowski UH. Viral mechanisms of immune evasion. Immunology Today. 2000;**21**(9):447-455. DOI: 10.1016/ S0167-5699(00)01699-6

[30] Haig DM. Subversion and piracy: DNA viruses and immune evasion. Research in Veterinary Science. 2001;**70**(3):205-219. DOI: 10.1053/ rvsc.2001.0462

 [31] Harvey R, McCaughan C, Wise LM, Mercer AA, Fleming SB. Orf virus inhibits interferon stimulated gene expression and modulates the JAK/STAT signalling pathway. Virus Research. 2015;**208**:180-178. DOI: 10.1016/j. virusres.2015.06.014

[32] Ning Z, Zheng Z, Hao W, Duan C, Li W, Wang Y, et al. The N terminus of orf virus-encoded protein 002 inhibits acetylation of NF-kappaB p65 by preventing Ser(276) phosphorylation. PLoS One. 2013;**8**(3):e58854. DOI: 10.1371/journal.pone.0058854

[33] Diel DG, Luo S, Delhon G, Peng Y, Flores EF, Rock DL. A nuclear inhibitor of NF-kappaB encoded by a poxvirus. Journal of Virology. 2011;**85**(1):264-275. DOI: 10.1128/JVI.01149-10

[34] Chen D, Zheng Z, Xiao B, Li W, Long M, Chen H, et al. Corrigendum: Orf virus 002 protein targets

ovine protein S100A4 and inhibits NF-kappaB signaling. Frontiers in Microbiology. 2017;**8**:160. DOI: 10.3389/ fmicb.2017.00160

[35] Diel DG, Delhon G, Luo S, Flores EF, Rock DL. A novel inhibitor of the NF-{kappa}B signaling pathway encoded by the parapoxvirus orf virus. Journal of Virology. 2010;**84**(8):3962- 3973. DOI: 10.1128/JVI.02291-09

[36] Nagendraprabhu P, Khatiwada S, Chaulagain S, Delhon G, Rock DL. A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-kappaB signaling. PLoS Pathogens. 2017;**13**(12):e1006779. DOI: 10.1371/ journal.ppat.1006779

[37] Khatiwada S, Delhon G, Nagendraprabhu P, Chaulagain S, Luo S, Diel DG, et al. A parapoxviral virion protein inhibits NF-kappaB signaling early in infection. PLoS Pathogens. 2017;**13**(8):e1006561

 [38] Diel DG, Luo S, Delhon G, Peng Y, Flores EF, Rock DL. Orf virus ORFV121 encodes a novel inhibitor of NF-kappaB that contributes to virus virulence. Journal of Virology. 2011;**85**(5):2037-2049. DOI: 10.1371/ journal.ppat.1006561

[39] Peng F, Chen Z, Zheng SY, Li HM, Du J, Zhang JZ. A case of Orf identified by transmission electron microscopy. Chinese Medical Journal. 2016;**129**(1):108-109. DOI: 10.4103/0366-6999.172606

[40] Vikoren T, Lillehaug A, Akerstedt J, Bretten T, Haugum M, Tryland M. A severe outbreak of contagious ecthyma (orf) in a free-ranging musk ox (Ovibos moschatus) population in Norway. Veterinary Microbiology. 2008;**127**(1-2):10-20. DOI: 10.1016/j. vetmic.2007.07.029

[41] Plowright W, Ferris RD. Studies with rinderpest virus in tissue culture. *Orf Virus: A New Class of Immunotherapy Drugs DOI: http://dx.doi.org/10.5772/intechopen.81535* 

II. Pathogenicity for cattle of culturepassaged virus. Journal of Comparative Pathology. 1959;**69**(2):173-184

 [42] McInnes CJ, Wood AR, Nettleton PE, Gilray JA. Genomic comparison of an avirulent strain of Orf virus with that of a virulent wild type isolate reveals that the Orf virus G2L gene is nonessential for replication. Virus Genes. 2001;**22**(2):141-150

[43] Inoshima Y, Murakami K, Wu D, Sentsui H. Characterization of parapoxviruses circulating among wild Japanese serows (Capricornis crispus). Microbiology and Immunology. 2002;**46**(8):583-587. DOI: 10.1111/ j.1348-0421.2002.tb02738.x

[44] Guo J, Zhang Z, Edwards JF, Ermel RW, Taylor CJ, de la Concha-Bermejillo A. Characterization of a North American orf virus isolated from a goat with persistent, proliferative dermatitis. Virus Research. 2003;**93**(2):169-179. DOI: 10.1016/ S0168-1702(03)00095-9

[45] Tedla M, Berhan N, Molla W, Temesgen W, Alemu S. Molecular identification and investigations of contagious ecthyma (Orf virus) in small ruminants, North west Ethiopia. BMC Veterinary Research. 2018;**14**(1):13. DOI: 10.1186/s12917-018-1339-x

[46] Zarnke RL, Dieterich RA, Neiland KA, Ranglack G. Serologic and experimental investigations of contagious ecthyma in Alaska. Journal of Wildlife Diseases. 1983;**19**(3):170-174. DOI: 10.7589/0090-3558-19.3.170

[47] Inoshima Y, Shimizu S, Minamoto N, Hirai K, Sentsui H. Use of protein AG in an enzyme-linked immunosorbent assay for screening for antibodies against parapoxvirus in wild animals in Japan. Clinical and Diagnostic Laboratory Immunology. 1999;**6**(3):388-391

[48] Azwai SM, Carter SD, Woldehiwet Z. Immune responses of the camel (*Camelus dromedarius*) to contagious ecthyma (Orf) virus infection. Veterinary Microbiology. 1995;**47**(1-2):119-131. DOI: 10.1016/0378-1135(95)00055-F

[49] McKeever DJ, Reid HW, Inglis NF, Herring AJ. A qualitative and quantitative assessment of the humoral antibody response of the sheep to orf virus infection. Veterinary Microbiology. 1987;**15**(3):229-241

[50] Yirrell DL, Vestey JP, Norval M. Immune responses of patients to orf virus infection. The British Journal of Dermatology. 1994;**130**(4):438-443. DOI: 10.1016/0378-1135(87)90077-0

[51] Zheng M, Liu Q, Jin N, Guo J, Huang X, Li H, et al. A duplex PCR assay for simultaneous detection and differentiation of Capripoxvirus and Orf virus. Molecular and Cellular Probes. 2007;**21**(4):276-281. DOI: 10.1016/j.mcp.2007.01.005

 [52] Chan KW, Hsu WL, Wang CY, Yang CH, Lin FY, Chulakasian S, et al. Differential diagnosis of orf viruses by a single-step PCR. Journal of Virological Methods. 2009;**160**(1-2):85-89. DOI: 10.1016/j.jviromet.2009.04.025

[53] Inoshima Y, Morooka A, Sentsui H. Detection and diagnosis of parapoxvirus by the polymerase chain reaction. Journal of Virological Methods. 2000;**84**(2):201-208. DOI: 10.1016/S0166-0934(99)00144-5

[54] Wang Y, Yang K, Bai C, Yin D, Li G, Qi K, et al. Development of a SYBR Green I real-time PCR for the detection of the orf virus. AMB Express. 2017;**7**(1):21. DOI: 10.1186/ s13568-016-0322-9

[55] Tsai SM, Chan KW, Hsu WL, Chang TJ, Wong ML, Wang CY. Development of a loop-mediated isothermal

amplification for rapid detection of orf virus. Journal of Virological Methods. 2009;**157**(2):200-204. DOI: 10.1016/j. jviromet.2009.01.003

 [56] Li J, Song D, He W, Bao Y, Lu R, Su G, et al. Rapid detection of orf virus by loop-mediated isothermal amplification based on the DNA polymerase gene. Archives of Virology. 2013;**158**(4):793- 798. DOI: 10.1007/s00705-012-1526-1

[57] De Clercq E. Cidofovir in the therapy and short-term prophylaxis of poxvirus infections. Trends in Pharmacological Sciences. 2002;**23**(10):456-458. DOI: 10.1016/ S0165-6147(02)02091-6

[58] Dal Pozzo F, Andrei G, Lebeau I, Beadle JR, Hostetler KY, De Clercq E, et al. In vitro evaluation of the anti-orf virus activity of alkoxyalkyl esters of CDV, cCDV and (S)-HPMPA. Antiviral Research. 2007;**75**(1):52-57. DOI: 10.1016/j.antiviral.2006.11.010

[59] Erbagci Z, Erbagci I, Almila Tuncel A. Rapid improvement of human orf (ecthyma contagiosum) with topicalimiquimod cream: Report of four complicated cases. The Journal of Dermatological Treatment. 2005;**16**:353-356

[60] De Clercq E. Clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir in treatment of DNA virus and retrovirus infections. Clinical Microbiology Reviews. 2003;**16**:569-596. DOI: 10.1128/ CMR.16.4.569-596.2003

[61] Degraeve C, De Coninck A, Senneseael J, Roseeuw D. Recurrent contagious ecthyma (Orf) in an immunocompromised host successfully treated with cryotherapy. Dermatology. 1999;**198**:162-163. DOI: 10.1159/000018095

[62] Buddle BM, Pulford HD. Effect of passively-acquired antibodies and vaccination on the immune response to contagious ecthyma virus. Veterinary Microbiology. 1984;**9**(6):515-522. DOI: 10.1016/0378-1135(84)90013-0

 [63] Mercer AA, Yirrell DL, Reid HW, Robinson AJ. Lack of crossprotection between vaccinia virus and orf virus in hysterectomy-procured, barrier-maintained lambs. Veterinary Microbiology. 1994;**41**(4):373-382. DOI: 10.1016/0378-1135(94)90033-7

 [64] Horohov DW, Breathnach CC, Sturgill TL, Rashid C, Stiltner JL, Strong D, et al. In vitro and in vivo modulation of the equine immune response by parapoxvirus ovis. Equine Veterinary Journal. 2008;**40**(5):468-472. DOI: 10.2746/042516408X322111

[65] Nowatzky J, Knorr A, Hirth-Dietrich C, Siegling A, Volk HD, Limmer A, et al. Inactivated Orf virus (Parapoxvirus ovis) elicits antifibrotic activity in models of liver fibrosis. Hepatology Research. 2013;**43**(5):535-546. DOI: 10.1111/j.1872-034X.2012.01086.x

[66] Weber O, Siegling A, Friebe A, Limmer A, Schlapp T, Knolle P, et al. Inactivated parapoxvirus ovis (Orf virus) has antiviral activity against hepatitis B virus and herpes simplex virus. The Journal of General Virology. 2003;**84**(Pt 7):1843-1852. DOI: 10.1099/ vir.0.19138-0

[67] Paulsen D, Urban A, Knorr A, Hirth-Dietrich C, Siegling A, Volk HD, et al. Inactivated ORF virus shows antifibrotic activity and inhibits human hepatitis B virus (HBV) and hepatitis C virus (HCV) replication in preclinical models. PLoS One. 2013;**8**(9):e74605. DOI: 10.1371/journal.pone.0074605

[68] Ons E, Van Brussel L, Lane S, King V, Cullinane A, Kenna R, et al. Efficacy of a Parapoxvirus ovisbased immunomodulator against equine herpesvirus type 1 and

*Orf Virus: A New Class of Immunotherapy Drugs DOI: http://dx.doi.org/10.5772/intechopen.81535* 

Streptococcus equi equi infections in horses. Veterinary Microbiology. 2014;**173**(3-4):232-240. DOI: 10.1016/j. vetmic.2014.07.015

[69] van Rooij EM, Rijsewijk FA, Moonen-Leusen HW, Bianchi AT, Rziha HJ. Comparison of different prime-boost regimes with DNA and recombinant Orf virus based vaccines expressing glycoprotein D of pseudorabies virus in pigs. Vaccine. 2010;**28**(7):1808-1813. DOI: 10.1016/j. vaccine.2009.12.004

[70] Fischer T, Planz O, Stitz L, Rziha HJ. Novel recombinant parapoxvirus vectors induce protective humoral and cellular immunity against lethal herpesvirus challenge infection in mice. Journal of Virology. 2003;**77**(17):9312-9323. DOI: 10.1128/ JVI.77.17.9312-9323.2003

 [71] Henkel M, Planz O, Fischer T, Stitz L, Rziha HJ. Prevention of virus persistence and protection against immunopathology after Borna disease virus infection of the brain by a novel Orf virus recombinant. Journal of Virology. 2005;**79**(1):314-325. DOI: 10.1128/JVI.79.1.314-325.2005

 [72] Voigt H, Merant C, Wienhold D, Braun A, Hutet E, Le Potier MF, et al. Efficient priming against classical swine fever with a safe glycoprotein E2 expressing Orf virus recombinant (ORFV VrV-E2). Vaccine. 2007;**25**(31):5915-5926. DOI: 10.1016/j. vaccine.2007.05.035

[73] Rohde J, Amann R, Rziha HJ. New Orf virus (Parapoxvirus) recombinant expressing H5 hemagglutinin protects mice against H5N1 and H1N1 influenza A virus. PLoS One. 2013;**8**(12):e83802. DOI: 10.1371/journal.pone.0083802

 [74] Rohde J, Schirrmeier H, Granzow H, Rziha HJ. A new recombinant Orf virus (ORFV, Parapoxvirus) protects rabbits against lethal infection with rabbit

hemorrhagic disease virus (RHDV). Vaccine. 2011;**29**(49):9256-9264. DOI: 10.1016/j.vaccine.2011.09.121

 [75] Fiebig HH, Siegling A, Volk HD, et al. Inactivated orf virus (Parapoxvirus ovis) induces antitumoral activity in transplantable tumor models. Anticancer Research. 2011;**31**(12):4185-4190

[76] Rintoul JL, Lemay CG, Tai LH, Stanford MM, Falls TJ, de Souza CT, et al. ORFV: A novel oncolytic and immune stimulating parapoxvirus therapeutic. Molecular Therapy. 2012;**20**(6):1148-1157. DOI: 10.1038/ mt.2011.301

[77] Tai LH, de Souza CT, Belanger S, Ly L, Alkayyal AA, Zhang J, et al. Preventing postoperative metastatic disease by inhibiting surgery-induced dysfunction in natural killer cells. Cancer Research. 2013;**73**(1):97-107. DOI: 10.1158/0008-5472.CAN-12- 1993 80

[78] Choi AH, O'Leary MP, Chaurasiya S, Lu J, Kim SI, Fong Y, et al. Novel chimeric parapoxvirus CF189 as an oncolytic immunotherapy in triplenegative breast cancer. Surgery. 2018;**163**(2):336-342. DOI: 10.1016/j. surg.2017.09.030

 [79] Spyrou V, Valiakos G. Orf virus infection in sheep or goats. Veterinary Microbiology. 2015;**181**(1-2):178-182. DOI: 10.1016/j.vetmic.2015.08.010

**63**

Section 4

Applications of Systems

Biology

### Section 4
