Study Toward the Development Of

Study toward the Development of

Broad Spectrum Live Attenuated Influenza Vaccine

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the

Graduate School of The Ohio State University

Hyesun Jang

Graduate Program in Comparative and Veterinary Medicine

The Ohio State University

2017

Dissertation Committee

Dr. Chang-Won Lee, Advisor

Dr. Daral J. Jackwood

Dr. Renukaradhya Gourapura

Dr. Andrew S. Bowman

Copyrighted by

Hyesun Jang

2017

Abstract

Control of avian influenza (AI) has been continuously challenged by the extreme diversity and complicated ecology of the causative agent, avian lineage type A influenza virus. Poultry species serves unique roles in AI ecology as providing an environment for viral evolution and act as an intermediate host to spread novel viral strains to other animal species including humans. Thus, to minimize the socio-economic impact from the emergence of novel strains and zoonotic infection, development of a strong immunity in poultry is desirable as a preventive measure. But currently available influenza vaccines for poultry, inactivated vaccines (IV) or recombinant viral vector vaccines, provide limited protection range confined to a specific target antigen and they require periodic updates to keep pace with the frequent viral mutations. As an alternative, live attenuated influenza vaccine (LAIV) can supplement the current vaccines for providing a broader protective immunity against diverse strains.

The long-term aim of this study is to develop a broadly effective LAIV for the use in poultry. Among diverse strategies to produce LAIV candidates, we focused on influenza A virus mutants that encode C-terminally truncated nonstructural protein 1

(NS1-truncated mutants). Nonstructural protein 1 (NS1 protein) is a multifunctional protein which provides the virus a favorable environment, primarily by interfering with host type I interferon (IFN) induction and signaling system. In turn, the NS1-truncated

ii mutants can be a promising live vaccine candidate for their highly attenuated phenotype as well as an induction of a boosted immune response following the unimpeded type I

IFN response. Previously, four genetically stable plaque purified NS1 truncated mutants, pc1- to pc4-LAIV, were evaluated for their potential as LAIV candidates in chickens. All the four candidates showed limited replication in chickens, but interestingly, the protective efficacy was not equal among viruses; only the pc3-and pc4-LAIV induced a high level serum antibody response and provided better heterologous protection. In a later study, the in vitro type I IFN induction ability of pc3-, or pc4-LAIV was observed to be higher than pc1- or pc2-LAIV, suggesting a correlation between in vitro type I IFN induction ability and the in vivo protective efficacy among NS1 truncated mutants.

Extended from the previous findings, we hypothesized that the higher immunogenicity of pc4-LAIV than pc2- LAIV is due to enhanced induction of type I IFN responses. The first phase of this study was aimed to demonstrate a correlation between type I IFN response and efficacy of pc4-LAIV in vivo. We first demonstrated that pc4-

LAIV could induce higher levels of type I IFN stimulated genes (ISGs), 2’,5’-OAS and

Mx genes, in chicken trachea and spleen than pc2-LAIV. Also, we unexpectedly found that pc4-LAIV not only induce higher level of serum antibody response than pc2-LAIV, but also accelerated the serum antibody response which was noticeable as early as 7 days post vaccination. For further demonstration of the role of a type I IFN response in the efficacy of pc4-LAIV, we treated chickens with recombinant chicken type I IFN and assessed its effect on IFN/ ISGs transcription, serum antibody response, and heterologous protective efficacy. The type I IFN treatment induced an accelerated serum antibody iii response against IV and upregulation of ISGs as observed with pc4-LAIV. More interestingly, type I IFN treatment directly enhanced the protective efficacy of pc2-LAIV to a similar level as pc4-LAIV. The results showed that the higher immunogenicity of pc4-LAIV than pc2-LAIV is correlated with the type I IFN related responses. Also, in addition to the previously observed serum antibody response and protective efficacy, the pc4-LAIV was efficient in stimulating an innate type I IFN related signaling and accelerating the adaptive immune response.

Based on the findings from the first phase of our study, we expected that the pc4-

LAIV is less likely to be affected by the maturity of the chicken immune system due to its innate immune stimulatory effect and the ability to accelerate an adaptive immune response. In the second phase of this study, we aimed to compare the efficacy of pc4-

LAIV in immunologically immature 1-day-old chickens to IV, which is known to be less immunogenic in young birds. As a result, 1-day-old vaccination with pc4-LAIV induced higher innate signaling sensitization, mucosal IgA response and better clearance of a heterologous challenge virus than the IV. But a serum antibody response was observed to be higher in IV groups, and the lower antibody response by pc4-LAIV seems to be due to the lack of proper mucosal adjuvant that can be used with pc4-LAIV. These findings indicate that each vaccine provides a distinctive advantage; pc4-LAIV has a higher priming effect on 1-day-old chickens but a serum antibody response can be more effectively elicited by the IV.

Based on the findings from the second part of study, we hypothesized that the most ideal protective efficacy could be achieved by combining the advantageous traits of each vaccine. So in the third phase of study, we tested the prime-boost strategy of pc4-LAIV vaccination at one-day-old followed by a IV vaccination at 3-week-old. The priming effect of pc4-LAIV vaccination at 1-day of age synergistically enhanced the efficacy of the boost vaccination at 3-week of age in that the prime boost regimen showed the highest serum antibody production. Also as a combined effect, the prime boost regimen produced early serum antibody response, broadened the cross reactivity of the antibody and induced a mucosal IgA response.

In summary, our study demonstrated that pc4-LAIV is efficient in eliciting an in vivo innate signaling sensitization and accelerating the adaptive immune response which correlated with heightened protection in both immunologically mature and immature birds. Also, pc4-LAIV priming was successfully incorporated into the current IV application without hindrance of each other’s efficacy and the effect of two vaccines could be synergistic. Our findings serve as a basis for the development of a broad spectrum LAIV, and the pc4-LAIV may be synergistically used with other vaccines as an essential component of the prime-boost regimen.

Acknowledgments

I would like to appreciate my advisor, Dr. Chang-Won Lee, for the encouragement, support, patience and enthusiastic guidance throughout my doctoral training.

I sincerely thank my dissertation committee members, Drs. Daral J. Jackwood,

Renukaradhya Gourapura and Andrew S. Bowman for their invaluable advices and critical review of my thesis.

Special thanks to Dr. John M. Ngunjiri for generous support and positive influence. My gratitude also extended to my colleagues, Dr. Mohamed Elaish, Mahesh K C, Michael

Abundo, and Amir Ghorbani for sharing valuable moments.

Also, I want to acknowledge the help and supports from our animal care team, Megan

Strother, Rachelle Root, and Dr. Juliette Hanson.

Finally, I thank my parents, Changhwan Jang and Yoosun Seo, for their endless support and faith in me which made my dissertation possible.

Vita

January 12, 1987……………………………...Born-Seoul, Republic of Korea 2005-2011……………………………...……..D.V.M. College of Veterinary Medicine, Chungbuk National University, Cheong-ju, Republic of Korea 2011-2013………………………………….…M.S. College of Veterinary Medicine, Chungbuk National University, Cheong-ju, Republic of Korea 2013-present……………………………….…Graduate Research Associate, Food Animal Health Research Program, Comparative and Veterinary Medicine, The Ohio State University, OH, U.S.A.

Publications

1. Jang H, Jackson YK, Daniels JB, Ali A, Kang KI, Elaish M, Lee CW. Seroprevalence and risk factors of seasonal human flu (H1N1 and H3N2) and canine flu (H3N8) in dogs in Ohio. J Vet Sci. 2017; Suppl 1: 291–8.

2. Jang H, Ngunjiri JM, Lee CW. Correlation between interferon response and protective efficacy of NS1-truncated mutants as influenza vaccine candidates in chickens. PloS ONE. 2016;11(6):e0156603

vii

3. Han MS, Kim JN, Jeon EO, Lee HR, Koo BS, Min KC, Lee SB, Bae YJ, Mo JS, Cho SH, Jang H, Mo IP. The current epidemiological status of infectious coryza and efficacy of PoulShot Coryza in specific pathogen-free chickens. J Vet Sci. 2016;17(3):323-30

4. Jang H, Koo BS, Jeon EO, Lee HR, Lee SM, and Mo IP, Altered pro-inflammatory cytokine mRNA levels in chickens infected with infectious bronchitis virus. Poult Sci. 2013;92:2290–8

5. Elaish M, Ngunjiri JM, Ali A, Xia M, Ibrahim M, Jang H, Hiremath J, Dhakal S, Helmy YA, Jiang X, Renukaradhya GJ, Lee CW. Supplementation of inactivated influenza vaccine with norovirus P particle-M2e chimeric vaccine enhances protection against heterologous virus challenge in chickens. PloS ONE. 2017;12(2):e0171174

6. Koo BS, Lee HR, Jeon EO, Jang H, Han MS, and Mo IP, An unusual case of concomitant infection with chicken astrovirus and group A avian rotavirus in broilers with a history of severe clinical signs. J Vet Sci. 2013;14(2):231–3.

Fields of Study

Major Field: Veterinary Preventive Medicine

Virology and Immunology

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Table of Contents

Abstract ...... ii Acknowledgments...... vi Vita ...... vii Table of Contents ...... ix List of Tables ...... xi List of Figures ...... xii Introduction ...... 1 Biology of influenza ...... 5 Host barriers against viral invasion and the escaping strategy of virus ...... 7 Development of broadly effective influenza vaccine ...... 13 Chapter 1. Literature Review ...... 16 1.1. Current influenza control in poultry ...... 16 1.2. Live attenuated influenza vaccine (LAIV) ...... 20 1.2.1. Immunology of influenza virus infection and vaccination ...... 21 1.2.2. The LAIV application in human and the estimated epidemiologic effectiveness of vaccine (vaccine effectiveness) ...... 30 1.2.3. LAIV development strategies ...... 32 1.3. LAIV in poultry ...... 39 1.3.1. The use of live vaccine in poultry industry ...... 39 1.3.2. Application of LAIV in poultry ...... 43 1.4. Conclusion ...... 47 Chapter 2. Association between Interferon Response and Protective Efficacy of NS1- Truncated Mutants as Type A Influenza vaccine Candidates in Chickens ...... 48 2.1. Abstract ...... 48 2.2. Introduction ...... 49 2.3. Materials and Method...... 51 ix

2.4. Results ...... 56 2.5. Discussion ...... 60 Chapter 3. Immunogenicity and protective efficacy of live attenuated influenza vaccine and inactivated influenza vaccine in 1-day-old chickens ...... 71 3.1. Abstract ...... 71 3.2. Introduction ...... 72 3.3. Materials and Method...... 73 3.4. Results ...... 78 3.5. Discussion ...... 81 Chapter 4. Synergy of Live Attenuated and Inactivated Influenza Vaccines in a Prime- Boost Regimen ...... 90 4.1. Abstract ...... 90 4.2. Introduction ...... 91 4.3. Materials and Method...... 93 4.4. Results ...... 97 4.5. Discussion ...... 102 Bibliography ...... 113

List of Tables

Table 3. 1. Design of Study ...... 85

Table 4. 1. Design of Study ...... 106

Table 4. 2. The effect of antisera production method on the percent antigenic relatedness value between TK/OR/71 (H7N3) and CK/NJ/150383-7/02 (H7N2) antigens ...... 107

List of Figures

Figure 2. 1. Serum antibody response in chickens following vaccination with LAIV candidates or infection with rgWT virus ...... 65

Figure 2. 2 Comparison of virus replication in trachea ...... 66

Figure 2. 3. IFN and ISG responses after vaccination with LAIV candidates or infection with rgWT virus ...... 67

Figure 2. 4. Effect of per-oral rChIFN-α treatment to naïve chickens or chickens vaccinated with inactivated vaccine...... 68

Figure 2. 5. Pre-challenge antibody responses ...... 69

Figure 2. 6. Replication and shedding of heterologous challenge virus...... 70

Figure 3. 1. The ISG and IFN responses in 1-day-old chickens vaccinated with pc4-LAIV or IV...... 86

Figure 3. 2. Serum antibody response by 1-day-old vaccination...... 87

Figure 3. 3. Tear antibody responses...... 88

Figure 3. 4. Replication level of heterologous challenge virus...... 89

Figure 4. 1. Serum antibody response to homologous (A/turkey/Oregon/71 (H7N3)) and heterologous (A/chicken/NJ/150383-7/02 (H7N2))strains at 1 and 2 week post boost vaccination ...... 108

Figure 4. 2. Differences in titer between homologous and heterologous strain ...... 109 xii

Figure 4. 3. Mucosal antibody response ...... 110

Figure 4. 4. Reduction in heterologous challenge virus replication in vaccinated birds 111

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Introduction

Influenza is an infectious respiratory viral disease in human and animals (1, 2). The socio-economic losses range from the direct illness to indirect expenses spent on prevention endeavors (3, 4). Within the U.S., millions of people are hospitalized and die by influenza associated illness (5, 6). In the poultry business, the cost of influenza is estimated to be billions of U.S. dollars and the loss from highly pathogenic avian influenza (HPAI) can literally dampen the nation’s GDP growth level (7, 8). Moreover, the occasional zoonotic transmission and the periodic emergence of pandemic strains are formidable threats to public health (9, 10).

The causative agent influenza virus is divided into type A, B, C and D (11, 12).

Type A influenza is known for the widest host spectrum and for distinctive lineages adapted in human, poultry, swine, wild birds, horses, seals, canine and feline species (13,

14). Type B and C influenza are primarily identified as epidemics in human populations, except the occasional isolation from dogs, pigs (influenza C) or seals (influenza B) (15-

18). Influenza D virus has recently added as a new member after first being isolated in swine and subsequently found to be associated with respiratory disease in cattle (12).

Among the four types, type A gets the most attention due to the diverse subtypes/ pathogenicity, the broad geographical distribution and broad host spectrum (19). Type A

1 influenza strains are further categorized into subtypes determined by the surface glycoprotein, hemagglutinin (HA) and neuraminidase (NA) (20). So far, 18 HA and 11

NA subtypes were identified in diverse host species (20).

Influenza viruses can be also categorized by their circulating pattern in a natural host. For human, the strains circulating with predictable seasonal pattern are referred as “seasonal influenza” (22). Among unpredictable outbreaks of epidemic strains, a novel strain intruding in global human population is referred as “pandemic influenza” (22). Currently,

H1N1, H3N2 viruses and influenza B virus are circulating as seasonal strains (23).

Currently circulating seasonal H1N1 was originally derived from the novel H1N1 virus which was responsible for the pandemic in the year 2009 (H1N1 pam09 virus) (23).

Among diverse host species, wild aquatic birds are considered primary natural host considering that most subtypes (16 HA and 9 NA) are found without apparent clinical signs (14). The phylogenetic studies also showed that wild aquatic birds are the ancestral host for all known type A influenza viruses (14). In addition, wild aquatic birds play critical role in global distribution of novel viral strains, by perpetuating diversity in influenza gene pool and by spreading throughout the migration (14).

Another major spreader of type A influenza virus is the domestic poultry (24, 25).

Chickens, turkeys, ducks, quail, and guinea fowls, and many other species are reported to be susceptible to type A influenza virus (26). The direct or indirect contact with wild birds is thought to be the main transmission route (27). Once the virus adapted in domesticated species, the high titers of viruses are mainly excreted through feces and also from nasal, ocular discharge (28, 29). The virus particles in those secretions or vehicles

2 are transmitted birds to birds, or farm to farm (28, 29). The airborne transmission is also suggested as a possible mode of transmission when the birds are proximate each other and the air flow is optimal (30). While most influenza infections in wild bird species tend to show no apparent clinical signs, diverse clinical signs including respiratory illness, depression, lowered egg production, and even mortalities are observed in domesticated poultries (29). Based on the pathogenicity in chickens, AI virus is further categorized into low pathogenic avian influenza (LPAI) and high pathogenic avian influenza (HPAI) strains (29). The replication of LPAI strains is restricted in respiratory tract and the clinical outcomes usually are limited to respiratory signs and stress-induced egg production drop (29). HPAI strains can replicate in multiple organs and result in extreme clinical sequel, including multi-organ failure, systemic hemorrhages, neurologic signs and high mortality (>75%) within ten days (29).

The detailed mechanism on generation of HPAI strain is not well understood but the role of wild bird in HPAI outbreaks has been repeatedly reported (27, 31, 32). For example, the incidences of HPAI outbreak are often geographically correlated with migratory route of wild birds (33). Also, the low pathogenic version of same subtype is observed to circulate in wild bird population habituated in neighboring region in prior to the epidemics of HPAI in poultries (34). In recent years, the concerns on HPAI have been augmented due to the changes in outbreak patterns. The H7N9 is reported to be increased in its incidences and to be associated with multiple fatal human infection cases (10, 35,

36). Also, multiple H5 subtypes (H5Nx) are reported in new geographic ranges with increased frequencies and unprecedented virulence (32, 37-39). In several cases, those

3 outbreaks were not correlated with the exposure to wild birds and raised concern on unrevealed route of HPAI introduction. (33).

Avian species occupy unique roles not only in influenza ecology but also in public health for its zoonotic risk. Due to the restricted host tropism of virus and the species barrier, the interspecies transmission between human and avian species is not very frequent (40). However, during the HPAI epidemics, a significant number of poultry associated personnel are associated with fatal infection of avian lineage type A influenza

(18). Also, the four historic pandemics were associated with avian strains in a direct or indirect manner (18). Thus, the health authorities have been putting massive amount of effort on preventive measures on avian influenza in a global scale (41-43). The main control strategy depends on continuous monitoring of circulating strains in wild and domestic birds, securing biosecurity and vaccination of birds (41-43). Despite of all those laborious and costly efforts, those preventive measures has been restricted to partial success (44). The current control strategy needs to be bolstered with more effective preventive measures. Currently, the most proactive measure for influenza control is a vaccination (41-43). However, the narrow protection range and inefficient production/administration process of current inactivated vaccine raises questions whether it can promptly react to the outbreaks with novel strains (45-47). As an alternative, this study proposes the application of LAIV. In prior to detailed discussion about the prospectus of our LAIV, this introduction will cover the basic knowledge on influenza including epidemiology, biology, host-viral interaction and current state for novel influenza vaccine development.

Biology of influenza

Type A influenza virus is an enveloped negative-sense, single-stranded RNA virus belonging to family Orthomyxoviridae (48, 49). The 8 segments of viral RNA encode 8 structural proteins and at least 7 non-structural proteins (50).

During non-infectious phase, the virion exists as a three-layered structure; 1) the outer lipid bilayer envelope spiked with two major glycoproteins (hemagglutinin, HA; and neuraminidase, NA) and one transmembrane protein (matrix 2, M2); 2) the middle matrix 1 (M1) protein layer which stabilizes overall viral structure; 3) the core, viral ribonucleic protein (vRNP) complex, consisted with the each eight gene segments bound to monomers of nucleoprotein (NP) and trimeric polymerase complex (PB1 and 2

(polymerase basic 1 and 2), PA (polymerase acidic)) (50, 51).

When the infectious cycle initiated, the first step is the binding of HA protein to sialic-acid (SA) on glycan structure on the cell surface (50). Once the virus succeeds to bind to cellular receptor and to be located close to cell surface membrane, the virus particle can be internalized via receptor-mediated endocytosis and engulfed into endosome (50, 52). The endosome contains diverse protease and at the late stage, the endosomal cavity is acidified to digest engulfed materials (50, 52). Type A virus uses this digestion process to open up its outer envelope/middle matrix layer and to release its vRNP complex into cytoplasm (50, 52). The host digestive enzyme is used to cleave disulfide bond which links the HA1 and HA2 subunits of HA protein. Once the HA protein is cleaved into two subunits, the amino (N) terminal of HA2 protein (as known as

5 fusion peptide) is exposed and inserted into target endosomal membrane, which destabilize the structure (50, 52). At the late stage of endosome, the endosomal cavity is acidified, and the M2e protein accordingly transports hydrogen ion (H+) into internal side of virus to destabilize the bind of vRNP to matrix layer (50, 52). Once the vRNP particle released from virus and is located in cell cytoplasm, vRNP complex interacts with host active transport system to enter the host cell nucleus (50, 52). This is a very unique feature of type A influenza virus different from other RNA viruses and it provides numerous advantages (52, 53). For example, by translocating the vRNP into cell nucleus, type A influenza virus minimizes chance to expose their RNA to cellular scavenger system scattered in cytoplasm (52, 53). Also, in cell nucleus, the capacity of viral RNA transcription/processing can be expanded by shutting down host mRNA transcription, processing, and export (52, 54). However, translocation of vRNP via nuclear pore requires highly complexed transport system, which involves tightly regulated action by nuclear transport receptors (Karyophrenins) which recognize the specific signal on protein (55). Detailed mechanism is not yet fully understood, but the nuclear locating signal (NLS) encoded in NP or nuclear export signal (NES) encoded on NP protein and nuclear export protein (NEP), previously known as NS2 protein, appear to play important role in the transportation (55).

Once the vRNP is located in the host nucleus, the RNA-dependent RNA polymerase starts to transcribe viral mRNA (52). For the transcription, vRNP complex snatches the

5’terminus cap part of host mRNAs and uses as the primer (cap-snatching) so viral mRNA with cap structure can use the host ribosome for translation of diverse proteins in

6 the cytoplasm (52). The newly synthesized viral RNA segments are exported into cytoplasm, processed to vRNP complex, assembled into matrix layer, and coated with host cell membrane where HA, NA and M2e proteins are expressed (52). At the final egress step, the action of NA protein is required to cut off the bound of HA (52).

Host barriers against viral invasion and the escaping strategy of virus

Multiple barriers blocking invasion of virus: To block the multiplication of foreign agents at the initial infection stage, host system built up diverse innate barriers (53, 56).

The multiple layered skin, anti-microbial peptides and lysozymes in mucosal secretions and muco-cilliary movements in airway are the compelling examples of the innate barriers (53, 56). In addition, diverse innate immune cells such as neutrophils, macrophage-monocytic system, and natural killer (NK) cells, localize the infection and trigger the following adaptive immune system (53, 56). Diverse cytokines secreted from those innate immune cells or virus infected epithelial cells are also important components of innate response (53, 56, 57). Type I IFN is the representative of innate cytokines and formidable threat to influenza viruses, which creates antiviral environment by activation of hundreds of interferon stimulated genes (ISGs), and influencing on immune cells

(dendritic cells, NK cells, monocytes, T- and B-cells) (57, 58). Type A influenza virus also evolved to counteract the type I IFN system via its nonstructural proteins (59, 60).

The detailed description on those proteins will be discussed in later part of this section.

In addition to the innate barriers, the pre-existing secretory immunoglobulin G or A in mucosal surface and serum block the repetitive infection (56). Compared to the action of innate response which localizes and minimize the viral infection, the neutralization activity of the antibody can block the binding of virus at the very beginning stage of infection (61). But unlike the innate response whose effective spectrum is broad over diverse foreign agents, the efficacy of humoral antibodies is specified on previously exposed agents or strains (56, 62).

Factors associated in receptor binding and un-coating steps: The HA protein of each influenza stain is known to have preference on the linkage pattern of SA on glycan structure and the different linkage pattern among species acts as species barrier to hinder the interspecies transmission of influenza virus (52, 63). For example, avian influenza viruses have preference on α 2,3-linkage SA(52, 63). But a surface of tracheal epithelial cells of human airway only expresses α 2,6-linkage SA and α 2,3-linkage SA is only limitedly expressed on ciliated cells or type II alveolar cells of a apical surface of lung

(52, 63). In turn, most avian influenza strains fail to grow in human respiratory system

(52, 63). Still, due to the frequent mutation of HA protein, avian influenza virus can obtain ability to bind to a human-type receptor (α 2,6-linkage SA) and cause serious respiratory disease in human (329). Also, the avian type receptor (2,3-linkage SA) can be found in lower respiratory tract of human and avian influenza viruses reached to the deep area of human lung can replicate and cause serious respiratory disease (330)

Even after the successful receptor binding and internalization, influenza virus needs to complete conformational change and uncoating process to proceed on further infection cycle (52). Also, the susceptibility to digestive enzyme can be the main determinant of infectivity and pathogenicity (64). For HPAI strains, the HA protein can be cleaved by the ubiquitous protease which can be found from all kinds of cell types, so the virus replicates in diverse host organs and causes multiple organ failure and extreme pro-inflammatory immune responses (64). Meanwhile, low pathogenic avian influenza

(LPAI) strains require the specific trypsin-like enzymes which exist only in respiratory system or kidney, so their replication occurs limitedly in specific organs so does the resultant impact (64, 65).

Fast-apt evolution strategy of virus during transcription/replication step: Influenza virus is located inside host cell nucleus and initiates its replication by the action of vRNP complex (52). The fidelity of the RNA polymerases is very low, so the transcription/replication cycle frequently produces the mutations on viral RNA and the resultant defective progenies (66). For example, Nobusawa and Sato (326) reported the mutation rates of 2.0 × 10−6 mutations per site per infectious cycle, during the growth of a six plaque clones from three human type A viruses in MDCK cells. The fatal mutation often results in the deletion of defective progenies from circulating influenza pool, but the frequent mutations often act as a driving force to develop diversity in circulating type A influenza virus pool (67). So, type A influenza virus can constitute the viral quasi- species, which refers the genetic pool of highly variable and diverse co-circulating strains

(68). The mutations in HA or NA proteins can be tolerated and tend to be accumulated.

Since the HA and NA protein located in outer surface and are the most abundant structure, they become the major target of humoral antibody responses; so the accumulated mutation on the HA and NA proteins results in change of antigenicity

(antigenic drift) and nullifies the efficacy of pre-existing immune response (21, 69). The frequent antigenic drift is main survival strategy of type A influenza virus and current vaccine program which provides specific immunity against HA and NA protein cannot efficiently prevent the drift strains (69, 70).

Generation of hybrid strains during assembly and egress step: At the final egress step, the action of NA protein is required to cut off the bound of HA. If the cell is coinfected with two or more influenza strains, the vRNP segments derived from different origin can be mixed at the assembly stage and egressed as one hybrid virus (genetic reassortant) (71,

72). Especially when the coinfected strains are derived from different host species, the resultant hybrid virus can obtain the capacity to infect originated host species (71-73).

The 2009 pandemic H1N1 (H1N1 pam09) virus is the most compelling example of the genetic reassortant (72, 74, 75). The H1N1 pam09 virus was derived by quadruple reassortment, originated from one human, two swine and one avian lineage influenza viruses, respectively (72, 74, 75). Since human population had never been exposed to this new variant but the hybrid virus was already well adapted to human through genetic reassortment, the virus could rapidly spread into naïve human population (72, 74, 75).

Actions of nonstructural proteins throughout the whole replication processes: The aforementioned antigenic drift and the genetic reassortant are the major driving force of the influenza evolution, whereas they are the major challenges for the control of influenza. In addition to fast-apt evolutionary strategy, influenza virus increases survivability through the action of non-structural proteins which counteract host innate and adaptive immune responses (76, 77).

At least 7 proteins has been identified to be synthesized in influenza A virus infected cells but do not constitute viral structure (78, 79). Due to their conditional presence during productive phase and restricted existence in certain influenza strains, most of the researches on those proteins were conducted in highly limited range (79). So far, the main function of those seven non-structural proteins is reported to antagonize host defense system, to shut off host transcription and to enhance viral transcription/replication (79).

The nonstructural 1 and 2 (NS1 and NS2) proteins are ubiquitously produced across type A influenza strains and relatively well studied than other nonstructural proteins. The NS1 and NS2 proteins are transcribed from the same NS gene, but diverged into two distinct proteins during post-transcription splicing process (alternative splicing)

(60, 80, 81). The NS1 protein is a multifunctional protein involved in diverse host-virus protein interactions to promote viral replication, such as inhibition of type I interferon

(IFN) system, enhancing viral translation and inhibition of mRNA processing mechanism

(81). Especially, the antagonizing type I IFN effect of NS1 protein can be key virulence factor, since influenza viruses are vulnerable to the strong induction of type I interferon 11

(IFN), followed by broad activation of the hundreds of antiviral genes (IFN stimulated genes; ISGs) and facilitated adaptive immune response (81).

Another nonstructural protein, PB1-F2 which is translated from the PB1 gene but separated from PB1 protein by leaky scanning, which refers the translation initiated from alternative initiation codon resulted from skip of the first weak initiation codon (78, 83,

84). With the NS1 protein, PB1-F2 was also suggested to possess the anti-type I IFN effect but it is not clear whether the function is universal across the strains (78, 83, 84).

The mutation studies indicated that the PB1-F2 is involved in delay of the IFN induction and interference of RIG-1/MAIVs proteins which trigger the induction of type I IFN response (78, 83, 84).

The other proteins, PB1-N40, PA-X, PA-N155/N182, and M42, are also known to be synthesized in influenza A virus infected cells and to interact with other proteins (79,

85). However, their presence was identified only in specific type A strains and the function was not fully determined (79, 85).

Since host-viral interaction has not yet well addressed until now, it is not surprising that current prevention strategy could not much evolve from the vaccination. So far, influenza control for poultry species in the U.S. is mainly operated by prevention of viral introduction into poultry farm and stamping-out/ emergency vaccination in case of HPAI outbreaks (41-43). This ‘prevention first’ strategy can be too laborious and costly to keep pace with increased incidences of HPAI outbreak. (44). Currently available vaccines, inactivated or viral vector vaccines, can only provide limited range of protection and they

12 require periodic update on their target strains (45-47). To effectively deal with a capricious nature of type A influenza virus, the best strategy should provide broader protection range, such as by development of broadly effective influenza vaccines. Next chapter will briefly introduce several vaccine development strategies, especially in aspect of providing broad protection spectrum.

Development of broadly effective influenza vaccine

Current strategies to utilize conserved region of type A influenza virus: One of the most encouraging approaches in broadly effective vaccine development is to use conserved portion of influenza A virus, expecting to induce immune response with broad cross- reactivity (86-90). The external portion of M2 protein (M2e) and HA stalk is often used as the target antigen since those proteins are highly conserved among type A virus (86-

90). However, the relatively lower immunogenicity of those proteins is the major drawback to be applied as universal vaccine (86-90). To enhance the immunogenicity, those peptide/proteins are genetically or chemically integrated to highly immunogenic carriers or require strong adjuvant to boost immune response to the peptide/proteins (87,

88). Several strategies were developed for this purpose using different vaccine platforms such as peptide, recombinant, vector, or DNA vaccines (91).

The peptide vaccine uses minimal epitope peptide encapsulated by liposome or virosome which act as both adjuvant and carrier (87, 91). Recent studies on peptide vaccines showed promising results, especially to elicit influenza specific T-cell response

13 and combined use with current IVs (92). However, the formulation of the peptide can be challenging and expensive, which needs further modification to be commercialized (91,

93).

The recombinant vaccines use the recombinant technology to produce virus like proteins (VLPs) that express HA stalk or M2e proteins covalently bound to carrier or adjuvant proteins (86, 94, 95). Currently, promising clinical trials are on process but most study results are focused on mice so its efficacy needed to be tested for more diverse and relevant host species (91).

The vector vaccine uses a live virus to serve as carrier of type A influenza virus genes, and the infection of small amount of vector vaccine results in comprehensive immune response especially in cross-reactive T cell responses (87, 91). The adenovirus, hepatitis virus B, or pox viruses are known to be suitable vector system for many pathogens (96). But in clinical trials, the pre-existing immunity to vector virus can reduce the efficacy of this delivery system (96, 97).

For DNA/RNA vaccines, the plasmid which encodes the protein of interest is inserted, expecting that host cell synthesizes protein and elicits the immune response against endogenously synthesized antigen (87, 91). This innovative concept proved its potential at phase I efficacy and safety study, but its usage in real world could be challenging since it requires special administration techniques and the insertion of exogenous genetic material can induce unwanted genetic changes, such as the tumor growth (91).

LAIV as practical alternative for influenza control: Some of the universal vaccine development strategies are very innovative and promising as future vaccines. However, there are still numerous steps to prove their efficacy and overcome the high production cost and parenteral administration appears not to be suitable for the commercialization, especially in farm animals. One of the practical alternatives to current inactivated vaccine can be the live attenuated influenza vaccine (LAIV) and current usage of LAIV in human proved its numerous advantages over IV. First, since the replication of LAIV mimics the natural infection of type A influenza virus with minimal or no pathogenicity, it efficiently elicits the innate, mucosal, cellular response as well as long lasting humoral antibody response (98-100). Owing to the stimulation of broad range of immune response, LAIV is known to show high cross-reactivity to broader range of influenza strains and not limited its efficacy in elderly or unprimed individuals (101-107).

This study aims to develop safe and efficacious LAIV candidate and their application strategies, especially in poultry. To begin with, next chapter will introduce the literatures about LAIVs focused on their mechanism of action, application in different host species and the prospectus for future use in poultry species.

Chapter 1. Literature Review

1.1. Current influenza control in poultry

Influenza is a formidable transmissible disease that affects both veterinary and public health (1, 2, 41). In the poultry industry, the economic impact from avian influenza is not limited to direct expenses, but also includes negative impacts on consumer motivation, export to the global market, and long-term costs of implementing the measures needed to prevent virus introduction in the farms (3, 7, 41).

According to the report from FAO (Food and Agriculture Organization), direct losses from HPAI outbreaks during 2003~2004 had significant impacts on the GDPs of six countries (7). For example, in Vietnam, the cost was estimated to be around 76 million~450 million U.S. dollars in the year 2004 (7). Even after the peak season, the prolonged cost for vaccination and implantation of biosecurity on small holder farms was

39 million and 500 million US dollars, respectively (7).

Novel strains of H5 HPAI virus have been emerging in the U.S. since 2014 (31,

32). The strains involved in recent outbreaks are reassortant viruses with hemagglutinin genes from the Asian lineage of H5N1 virus and neuraminidase genes from North

American wild bird lineages (38, 39, 108). The recent incidences of hybrid strains directly cost 1.6 billion U.S. dollars to euthanize turkeys and the economy-wide impact was estimated at 3.3 billion U.S. dollars (8). Considering the continuous evolution of

Asian-lineage HPAI viruses and the lack of clear knowledge about the mechanisms involved in virus reassortment and transmission route (31, 109), it appears that the severity of HPAI incidences will not be lessened in near future.

The losses incurred from LPAI viruses also cannot be underestimated, especially for the strains with potential to gain high pathogenicity or zoonotic risk. In many cases of the LPAI H5 and H7 epidemics, eradication measures are taken to prevent the LPAI virus from mutating to HPAI virus (110). The economic impact of LPAI H5 or H7 epidemics includes the direct loss in meat or egg production and costs associated with continuous maintenance of vaccination and monitoring programs (111, 112). More importantly, the consequential losses, such as cost from banning on restocking and losses of hatching eggs, chicks and poultry meat are devastating outcomes of LPAI epidemics (112, 113).

Sartore et al (112) reported that the overall loss from LPAI outbreaks during 2000~2007 in Italy was even higher than HPAI outbreaks during 1999~2000 in Italy, when consequential losses are included. The LPAI H9N2 virus is one of the endemic avian strains in poultry across Asian countries and North-American wild bird species (114). In

Asian countries, high rates of evolution of the H9N2 virus and reassortment with other circulating strains are frequently reported, and some of the novel strains have gained ability to replicate in human respiratory system (115-118).

The strategy for avian influenza control has been the “prevention first” strategy

(41, 119) despite the change in outbreak patterns and the severity of disease caused by recent strains (120, 121). The “prevention first” strategy primarily relies on maintenance of high level of biosecurity and depopulation of affected flocks or high-risk farms (41,

122). To minimize the affected area, all farms within a 6 mile radius from the affected farm are quarantined and tested for virus (122). Biosecurity and culling work most efficiently when the rearing density is low and the incidence is not too high (41).

Until mid-1990s, the incidences of influenza in domestic poultry were considered as novel introductions and the risk of endemic was not high (123). However, with increased incidences and diverted outbreak patterns, this control strategy became too laborious and costly to keep up with the rapidly spreading novel strains (41, 119). With the massive HPAI outbreaks that occurred during the first decade of this century serving as a turning point, a need has arisen for proactive modification of avian influenza control strategies (41, 44, 119, 124). Since 2003, the Asian lineage HPAI H5N1 has circulated in

Asia, Middle East, Europe and Africa. Billions of U.S. dollars were spent to control the spread of virus by culling of millions of poultry (125, 126). After 8 years of continuous outbreaks, the United Nations Food and Agriculture Organizations acknowledged that six countries (Bangladesh, China, Egypt, India, Indonesia, and Vietnam) are endemic for

HPAI H5N1 in poultry (127). Vaccination with strain-specific inactivated vaccines (IV) has been the main method of influenza control in the endemic areas (42, 119, 124). It appears that countries using vaccination have managed to reduce the economic burden of influenza outbreaks (43, 128). As detailed later in this Chapter, the current IV has many shortcomings and new vaccines are needed for influenza control in chickens (45-47, 129,

130).

Unlike the endemic countries, most developed countries experience relatively fewer avian influenza outbreaks and are able to maintain good biosecurity & culling

18 programs (119, 123). However, the utility of this control strategy has become questionable owing to the complexity of the ecology of type A virus. Recent reports warrant us to monitor the interaction among intercontinental strains and introduction of novel strains (38, 121, 126). As the most proactive preventive measure, novel vaccines need to be developed to prepare for the worst avian influenza pandemic scenario and to overcome the current limitations of IV.

As alternatives to the IV, recombinant vector vaccines were developed and commercialized in the poultry industry (128, 131). Recombinant vector vaccines use live viruses as vehicles to express the targeted proteins of influenza virus in the vaccinated animal. Currently, the vectored influenza vaccines used in the poultry industry are based on three viruses: fowlpox virus, herpesvirus of turkeys (HVT), and Newcastle Disease

(ND) viruses (128, 131). Vector vaccines are advantageous over IV in terms of the ease of administration and mass production (131). Also, the vector vaccines can provide immunity against both the wild type virus used to generate the vector and influenza virus through a single vaccination by eliciting a broad range of mucosal and cellular immune responses (98, 100, 132). Still, recombinant vector vaccines have a number of drawbacks.

First, vectored vaccines provide protection against a narrow range of influenza virus strains because only one or two foreign proteins can be inserted in the vector (131). In case of the fowl-pox recombinant vaccine, the efficacy was seriously reduced by pre- existing immunity against the vector virus and inability of the vaccine to induce high levels of antibodies (97). The HVT vaccine showed good efficacy and ability to overcome maternally derived antibody interference in 1 day old vaccination (131, 133,

134). However, the onset of peak immune response was delayed and still needed to be administered via the subcutaneous route (133-135). Also, the concerns and high regulation on genetically modified organisms can hamper active development and distribution of vector vaccines (136).

Recombinant vector vaccines are designed to mimic the natural infection and are administered on the mucosal surfaces of the respiratory system to induce local immune responses (137). Live attenuated vaccine (LAIV) also mimics natural infection and provides a broader range of immunity directed to all viral proteins (it is not limited in surface antigens) (138). The next section will review the current knowledge on LAIV.

1.2. Live attenuated influenza vaccine (LAIV)

Because of the continuous spread and extremely versatile nature, type A influenza virus has established diverse host-specific lineages in diverse host species, including avian, swine, equine, human, canine and sea mammals (18, 27, 49). Also, influenza viruses periodically undergo reassortment and exchange the genetic material among different lineages resulting in drastic antigenic shifts (14). Influenza virus continues to evolve and broaden its host range (70, 114, 115, 120). The current IV has contributed to reduction in the severity and incidence of disease, but has often failed to protect due to frequent emergence of escape mutants in addition to the complex nature of viral ecology (42, 115,

124). The limitations of the IV are mainly attributed to the narrow breadth of its biased immunogenicity, which mainly induces systemic humoral immune response (139). On the contrary, live attenuated influenza vaccine (LAIV) mimics the natural infection and

20 provokes a broad range of immune responses including mucosal, cellular and antibody response without harmful effect by infection (139). This section will review the current knowledge on the basic host defense systems against the influenza infection and the immunological mechanisms of LAIV vaccination.

1.2.1. Immunology of influenza virus infection and vaccination

Immune responses to type A influenza virus infection: The most effective defense strategy against type A influenza virus is to establish local immune responses at the main portal of viral entry, the respiratory tract mucosa (140). The front line of defense in the mucosal system includes the non-specific anatomical and physiological barriers, such as the mucous membrane, complement system, and anti-microbial proteins (140, 141). At the same time, other arms of the immune system are activated by pathogen-specific signals to promote immediate pathogen clearance and protect from recurrent infection by the same pathogen (140, 142, 143). Pathogen-specific signals are recognized by cellular receptors such as pattern recognition receptors (PRRs). The PRRs comprise the innate sensing system which detects pathogen associated molecular patterns (PAMPs) commonly expressed by diverse pathogens (144). The toll like receptors 3, 7, and 8 (TLR

3, 7, and 8), retinoic acid-inducible gene I (RIG-I) and NOD-like receptor family are the currently identified PRRs involved in influenza infection (53). Recognition of PAMPs by

PRRs promptly alerts the immune system to trigger an extensive antiviral signaling involving factors such as type I interferon (type I IFNs/ IFN- α/β), pro-inflammatory cytokines, eicosanoids and chemokines (53). The main role of type I IFNs is to stimulate 21 a cascade of hundreds of genes involved in antiviral response (interferon stimulated genes

= ISGs), which is an important barrier for influenza infection (53, 144). Pro- inflammatory cytokines and eicosanoids trigger local inflammatory responses and induce subsequent adaptive immune responses (53). Chemokines recruit immune cells to the site of infection (53, 141, 144). Innate immune cells, such as macrophage, NK cells, dendritic cells (DCs), and granulocytes (neutrophil, basophil and eosinophil) are also an important component of the innate immune system (53, 141, 144). NK cells induce apoptosis of influenza infected cells to block the spread of virus, whereas the cells with phagocytic activity (neutrophils and macrophage) can engulf virus infected cells or the debris from the apoptotic cells (53, 141, 144). When the infection is not severe, the innate immune response can resolve the infection on its own; but when the intensity of infection overrides the capacity of innate response, a specialized adaptive immune response needs be promptly triggered before the infection is spread to uncontrollable scale (140, 144).

Unlike the innate responses which provide broad non-specific protection, adaptive immune responses are targeted to specific pathogens (56, 140, 144). To provoke pathogen-specific adaptive immune responses, the antigenic information of the invading organism needs to be delivered from the site of infection to secondary lymphoid organs where lymphocytes are gaining specialized effector functions (56, 144). Two types of cells play the role of presenting antigenic information to lymphocytes (56, 144). The first type is the professional antigen presenting cells (APCs) such as DCs, macrophages and

B-lymphocytes (145). The professional APCs first obtain viral antigen by phagocytosis of infected epithelial cells or directly by getting infected by the virus (146, 147). The intake 22 virus particle is processed into small peptides and presented in the context of class II major histocompatibility complexes (MHC-II) on the cell surface (144, 147). Later in secondary lymphoid organs, the peptide bound in MHC-II is recognized by T cell receptors (TCRs) on helper T cells (146, 147). The interaction between MHC-II and TCR triggers the proliferation and maturation into antigen specific effective T cells or B cells producing antigen specific antibodies (146, 147). Antigen presentation is also conducted by diverse nucleated cells, including influenza infected epithelial cells (145). As described above for the professional APCs, non-professional APCs process viral antigen into small peptides and present them on the cell surface in the context of MHC-I (145).

The difference is that the MHC-I-antigen complex is recognized by cytotoxic T lymphocytes which signal the infected cells to undergo apoptosis (147, 148). Also, non- professional APCs are known to be less effective than professional APCs, probably due to the lack of constitutive expression of MHC II and lack/lower level of costimulatory molecule expression (327).

In the mammalian immune system, the professional APCs deliver and present the antigen at secondary lymphoid organ, where naïve lymphocytes mature into effector cells

(144-146). The bone marrow (bursa for avian species) derived lymphocytes (B cells) and thymus-derived lymphocytes (T cells) are two distinctive lymphocytes whose mature forms serve the effector and memory functions in adaptive immune system (149).

Effector form of T-cells are further categorized by their surface clusters of differentiation

(CD): CD4+ helper T cells, which recognize antigens presented via MHC II expressed on

APCs and assist activation of other cells whereas CD8+ cytotoxic T cells recognize

23 antigen via MHC I to facilitate lysing of virus infected cells (146, 149, 150). Once the virus infections start to diminish, the immune system is reset by regulatory T cells and macrophages (151). Most of the activated lymphocytes are also removed at the end of immune response but some are retained as long-term memory T cells to respond quickly against recurrent infections (146, 152).

The B cells mature into plasma cells upon recognition of antigen presented directly by professional APCs or upon activation by CD4+ helper T cells (152). During maturation, the Fab part of B-cell receptor (BCR) transforms into a secretory antigen- specific receptor (152). Secreted BCRs are called antibodies (or immunoglobulins) and are transported in the blood stream to reach diverse locations in the body (152). In the initial phase of BCR maturation, primordial immunoglobulin (Ig) M (IgM) is produced, but as the infection progresses, the plasma cells produce immunoglobulins such as IgG

(IgY for chickens) and IgA which have higher affinity to the antigen, (152). Antibodies are an integral part of influenza virus clearance by the immune system. The antibody- mediated clearance involves antibodies that target the prominent viral glycoproteins (HA and NA) to block virus entry in susceptible cells (153). Influenza virus-specific antibodies are also known to trigger or amplify phagocytosis, antibody dependent cellular cytotoxicity (ADCC) responses, and activation of the compliment system (154, 155).

Secreted immunoglobulins can reside in the mucosal surface or blood stream for several months to years (156). In addition, subsets of B-cells are reserved as memory cells to promptly produce antibodies during re-infection (152, 156).

Immune responses associated with parenteral administration of IIV and mucosal administration of LAIV: The route of vaccine administration has a significant influence on the kind of immune response induced. Vaccine administration through the parenteral route involves penetration of multiple layers of skin to deliver the antigen.

Three parenteral routes are employed for IV delivery: subcutaneous (SC), intramuscular

(IM), and intradermal (ID) routes (157, 158). The SC administration delivers vaccine antigen right below the cutaneous layer. Since the subcutaneous area has loosely organized connective, adipose tissue and only a few vascular vessels, the antigen is slowly taken up by the lymphatic system (159). Due to the slow absorption rate and residual space in the subcutaneous layer, the SC administration allows a steady exposure of the immune system to the antigen for a long period of time (159). The IM injection delivers the antigen to the area with a good network of lymphatic and vascular systems allowing the antigen to be absorbed faster than SC route (159, 160). The ID route also delivers the antigen via lymphatic and vascular systems but it requires relatively less amount of dose since dermis and epidermal areas are rich in APCs (159). However, the

ID administration is not popularly used in the poultry industry since it requires special syringe for a delivery into the dermis (159, 161).

The main advantage of parenteral administration is that there are numerous choices for adjuvants (162). Adjuvants are chemical or biological substances which are administered along with vaccine antigen to enhance immune responses (162, 163). IVs do not elicit sufficient immunologic responses without co-administration with adjuvants

(163). Adjuvants for commercial vaccines (parenteral) include mineral salt (alum- or

25 calcium based), bacterial components, oil in water emulsion, liposomes, cytokines, carbohydrate, and microspheres (163). However, parenteral administration is associated with side effects such as inflammation at the injection site. In addition, parenteral administration is not an economical option for commercial animals because it is not only labor intensive but also associated with vaccine failure due to incomplete administration during mass vaccination (164).

To overcome the drawbacks of parenteral administration, mucosal delivery is actively investigated to overcome the drawbacks from parenteral vaccines (165, 166). The advantages of mucosal delivery is the easiness of application, non-invasiveness and economic for mass administration (163, 166). The gastrointestinal tract (GIT), respiratory tract, urogenital tract and ocular region are the target sites of mucosal delivery (163, 166).

Mass administration of mucosal vaccine in poultry can be conducted by mixing in feed or drinking water and aerosolization using a spray system (167).

The immunogenicity of mucosal vaccines is negatively affected by several factors

(165). First, it is difficult to achieve a steady antigen exposure due to inefficient uptake and rapid clearance on the mucosal surface (165). Replicating vaccines such as live attenuated vaccines or live vector vaccines are relatively free from this concern since they naturally have the ability to colonize and to replicate on the mucosal surface (165). The main challenge with replicating vaccines is attaining an optimal balance between attenuation and immunogenicity, the vaccine agent needs to replicate efficiently enough to elicit immune response but also be attenuated enough to prevent pathologic effects or transmission to other individuals (165, 166). Genetic modifications can be applied to

26 generate vaccines an optimal balance between safety and efficacy. For non-replicating vaccines, there are several strategies under investigation to increase the chance of antigen uptake on mucosal surface, such as mixing with muco-adhesive material (chitosan, carbopol, and sodium alginate) or particulating the antigen by specialized delivery system

(liposome, virosome, immune-stimulating complexes (ISCOMs) or lipopeptide) (165,

166). Another challenge of mucosal delivery of vaccine is that only a few options of adjuvants are available (165, 166). Some of the few adjuvants that have been studied for mucosal vaccination include: CpG oligodeoxynucleotides (CpG ODN), Fms-related tyrosine kinase 3 ligand (FLT3 ligand), monophosphoryl lipid A (MLA), and bacterial lipopolysacharides (LPS) are one of the few adjuvants that have been extensively investigated as potential mucosal adjuvants (165, 166).

Once the antigen has crossed the natural physical barriers, it needs to be transported to the lymphoid organs to elicit antigen specific effector B- or T-cell response

(47, 139, 168). The parenterally delivered IV is mainly carried by dendritic cells residing around the application site and transported to secondary lymphoid organs (159, 163).

After activation by parenterally-delivered antigen, B cells tend to mature into plasma cells which mainly secrete IgG antibodies (159, 169). The secreted IgG circulates all over the body, including mucosal surface, via the blood stream (46). However, parenteral IV seems to be less effective in provoking CD8+ cytotoxic T cell responses or local mucosal response (46, 170). On the other hand, the mucosal immunization activates the mucosa- associated lymphoid tissues (MALTs), which are specialized and compartmentalized for local effector function (166, 171). For example, the BALT (bronchus-associated

27 lymphoid tissue) and NALT (nasal-associated lymphoid tissue) are spread along the respiratory tract while GALT (gut-associated lymphoid tissue) can be found in the gastrointestinal tract (171). In MALTs, the major antibody produced by affinity maturation is secretory IgA (sIgA) (171). Due to the difficulty in sampling and lack of appropriate quantitative measurements, the characteristics of sIgA are not well understood but recent studies have revealed its unique role in mucosal immunity (172).

Due to its Fab region-dependent action, the sIgA is known to utilize its heavy chain and side chains as competitive inhibitors for pathogen binding and to interact with commensal microbes (173). Also, sIgA is involved in antigen uptake and transport on the luminal surfaces of intestinal and respiratory tracts and control of proinflammatory responses

(173, 174).

In addition to triggering local sIgA responses, mucosal administration of LAIV can also induce effector CD4+ and CD8+ T cell responses. He et al (104) reported that a virus-specific IFN-r+ CD4+ and CD8+ T cells were significantly increased only after

LAIV vaccination but not after IIV vaccination. Another study investigated T cell responses in children who received LAIV and/or trivalent inactivated influenza vaccine

(TIV) combinations and found that only regimens with LAIV could induce influenza specific CD4+, CD8+ and gamma delta T cell responses (105). Using a sensitive

ELISPOT, Forrest et al (175) reported an induction of CMI from seronegative (naïve) children after LAIV vaccination while such response was not observed in TIV-vaccinated children, unless the children were previously exposed to influenza virus or vaccination.

These findings may explain why protection by LAIV does not often correlate with serum

28 influenza virus-specific antibody responses. The higher and long term mucosal IgA response by LAIV than IV has been also reported in numerous animal and human clinical studies. The preferential induction of CMI by live vaccine is not limited to the mammalian species. Using IFN-γ as an indicator of CMI responses in chickens, Lambert et al (176) reported that live vaccine could stimulate the ChIFN-γ production in all chickens while only a few of the chickens vaccinated with inactivated vaccine showed an increase in ChIFN-γ level .

Taken together, the above studies show that IIV and LAIV provide different ranges of protection through distinctive immunologic mechanisms. While the role of mucosal immune response in vaccine efficacy is yet to be fully understood, it is apparent that the local mucosal immunity is critical to block infection at the virus entry step (143,

166, 174). Still, in field application, numerous unexpected factors can influence on the effect of vaccine and even overrides the immunologic scheme (163, 166). In humans, influenza vaccination has been conducted since 80 years ago and owing to the advances in meta-analysis, the epidemiologic observation studies on vaccine effectiveness (vaccine effectiveness study) has been most actively addressed than other host species (177, 178).

By reviewing the precedent cases in human, we may estimate the prospectus of LAIV application in the poultry industry. Human influenza vaccination is reviewed with focus on protective efficacy of live and IV.

1.2.2. The LAIV application in human and the estimated epidemiologic

effectiveness of vaccine (vaccine effectiveness)

The first influenza vaccine to be applied in public health was a LAIV, obtained by passaging A/PR8/34 H1N1 virus 30 times in eggs in 1930’s (177). However that LAIV had multiple problems such as the instability of attenuations, lower efficacy in adult/elderly population, interference from pre-existing immunity, and the risk of recombination with wild type strains circulating at that time (177). Since the virus could be adapted to grow to high titers in eggs, the scientists decided to switch to IV to overcome the problems of LAIV (177). In 1940, multiple subtypes and strains were found to be circulating and mismatches between the vaccine strain and circulating strains resulted in a significant drop in vaccine efficacy (177). From 1940’s onward, the monovalent H1N1 influenza vaccine was replaced with tri- or quadri-valent vaccines which include H3N2 strain and influenza B virus (177).

The production procedure of influenza vaccine has not undergone much innovation and the egg-based IV is still the most popular human influenza vaccine (90).

After the 2009 pandemic, concerns were raised on the egg-dependent process of vaccine production (122, 179, 180). The whole process of egg-based IIV production takes at least

4-6 month, and it may not keep up with the increased demands for vaccine in emergency or pandemic situations (181). The LAIV was suggested as alternative to IIV because the vaccine seed production process became revolutionarily shortened by the current technology, it is possible to produce influenza mutants with specific modifications in the target genes through mutagenesis and reverse genetics (107). In the U.S., the temperature 30 sensitive (ts)-, cold adapted (ca)- LAIV strain has been approved for human vaccination since 2003 (107). The vaccine was initially distributed in a frozen form that included

H1N1, H3N2 and one influenza B strain but it is currently distributed as a liquid nasal spray (107). Also, to tackle the increasing impact of influenza B virus, a quadri-valent vaccine with two influenza B lineages and two influenza A viruses is now available

(107).

It has been over a decade from the introduction of LAIV and there have been accumulated data about LAIV efficacy in diverse populations. For example, the Cochrane

Collaboration group evaluated the effect from IV or aerosol LAIV, in terms of the ability to reduce influenza like illness (ILI) (effectiveness) and to prevent influenza A/B infection (efficacy) in healthy individuals (16~65-year-old) (182). In that study, the parenteral IIV vaccination showed a 30 % overall effectiveness against the development of ILI for matching strain but no effectiveness was observed when the strain was either not matched or unknown (182). The efficacy of IV was 73 % to matching strains and 44

% to non-matching strains (182). Interestingly, the performance of LAIV was not significantly influenced by the strains and the overall effectiveness was 10 % and efficacy was 68% (182). In another study conducted by Tricco et al (183), the mismatched LAIV and IV could significantly reduce the risk of laboratory confirmed influenza cases by 60 % and 56 %, respectively.

Overall, the effectiveness and efficacy of LAIV against non-matched strains is similar or higher than IV. According to Carter et al (103), the efficacy of LAIV against matching strain was observed to be significantly better than IV in 6~59-months-old-children

31 whereas there was no significant difference in the adult population. A meta-analysis on 8 randomized controlled studies also showed that the efficacy of LAIV was higher in 2~17- year-old age group children compared to IV (101). Based on the consistent reports of superior effectiveness of LAIV in children, the CDC recommended use of LAIV in children 2 through 8 years of age during 2014-2015 flu seasons (184). However, data from recent studies have suggested that the efficacy of the currently licensed LAIV should be revisited. The Influenza Clinical Investigation for Children (ICICLE) study in the U.S. and studies in the United Kingdom and Finland have found LAIV to be less efficacious than IV (185, 186). Since the efficacy of H3N2 and influenza B components of LAIV was still high for LAIV, they suspected that new a/H1N1 pdm 09 LAIV strain was not genetically stable or has substantially drifted from circulating strains (187). In

2016, the CDC recommended against the use of LAIV except in cases when the patient cannot receive IIV or when there is a shortage of IV or recombinant vaccines (188). The accumulated data clearly demonstrates that LAIV is superior to IV in terms of effectiveness in young subjects and more effective and protection against antigenically mismatched strains (101, 103).

1.2.3. LAIV development strategies

The ideal LAIV candidate should be highly attenuated in its replication, free from virulence and risk of reversal to wild type virus. At the same time, LAIV immunogenicity should not be compromised by the attenuation process. This section will review the

32 different strategies employed for LAIV attenuation, from the classic serial passage method to genetic manipulation.

Serial passages : The classical approach for LAIV attenuation involves passaging the

“vaccine strains” in atypical hosts (e.g. eggs) or other conditions, such as temperatures that are lower than the host body temperature (177, 189). During adaptation in the new environment, the vaccine strains lose their fitness and become attenuated in replication ability in the original host (189). The very first LAIV was obtained by 30 blind passages in embryonated chicken eggs (177). Although the rescued strains showed reduced replication ability, they were not stably attenuated and often reverted back to the wild type strain (177). To produce more stably attenuated viruses, Chanock et al (190) used chemical mutagenesis to generate mutant strains which were manipulated to grow in lower temperature (32-34°C) and to shut off the replication at the core body temperature

(37-38°C). For induction of mutation, the virus was incubated at lower temperature (32-

34°C) in the presence of 5-fluorouracil to simultaneously facilitate gene mutation and adapt in low temperature. Among the diverse mutants generated by this method, a stable strain could be selected and used as temperature sensitive variant (190). Massab et al

(191, 192) developed cold-adapted influenza virus which was rescued by serial passage at gradually lowered temperatures. The rescued virus could actively replicate at 25°C but was attenuated at higher temperature (191, 192). It was later shown that those temperature-sensitive (ts-), and cold-adapted (ca-) phenotypes were correlated with specific mutations in PB1, PB2, PA, M, and NS genes (193, 194).

Attenuation by genetic modification: With the advancement of whole genome sequencing, cloning, and transfection techniques, researchers are now able to produce influenza virus mutants by targeting specific genes (177). The development of reverse genetics systems for de novo virus synthesis has not only enabled stable production of attenuated influenza virus but also eliminated the concern of unpredictable genetic changes that often emerge during passages (177). It also means that the exhaustive passaging process can be shortened and scientist can selectively produce targeted phenotype (195).

Temperature sensitive/ cold adapted/ attenuated (att) vaccine: The first approach to apply the reverse genetics in construction of attenuated vaccine strain was to reproduce the cold-adapted (ca-) and temperature sensitive (ts-) phenotype by manipulating the

PB1, PB2, and NP genes (196). Interestingly, the six genes carrying ca-/ts- phenotype could be used as the internal backbone to rescue reassortant viruses with foreign HA and

NA proteins (196). The Flumist was the first commercial LAIV constructed by genetic modification and reverse genetics (196). The ca-/ts-phenotype has been used together with diverse genetic modification to create attenuated vaccine viruses (196).

HA cleavage site modification: The modification of the cleavage site of HA protein can seriously impede virus replication efficiency and produce an attenuated phenotype (197, 198). Since the HA cleavage site is known to influence pathogenicity of influenza virus, the HA cleavage site modification is the preferable strategy to attenuate

34 highly pathogenic strains (197-199). Stech et al (200) modified the1 HA cleavage site of

A/WSN/33 H1N1 virus to use elastase for activation, instead of trypsin. In mouse lung where the elastase is not available, the modified virus was highly attenuated in replication and the virus was only detected during the first passage in mouse lung (200). The authors suggested that this strategy can be applied to produce rapid homologous vaccination in case of novel variant epidemics (200). Stech et al (201) also used a similar strategy to produce an influenza B vaccine candidate and proved the potential of this strategy with diverse strains. However, this approach may be restricted to the mouse model and not useful in poultry species where influenza viruses replicate more actively in the upper respiratory tract. For poultry vaccination, Cai et al (202) found that in ovo vaccination with LAIV candidate whose HA cleavage site was modified showed increased hatchability and protective efficacy.

NS1 protein truncated variants: NS1 protein is involved in regulation of transcription of diverse genes, especially type I IFN related genes (59). Due to the crucial role played by wild type NS1 in anti-IFN activity, mutants with truncation or defects in the NS1 are unable to block type I IFN responses and IFN signaling in the infected host

(45, 60). In turn, influenza viruses with NS1 mutation are attenuated in replication, and more interestingly, induce high levels of adaptive immune responses (45, 60). Solorzano et al (203) used reverse genetically constructed NS1 truncated variants from wild type swine influenza virus, A/Swine/Texas/4199-2/98. The NS1 truncated variants showed high type I IFN induction in tissue culture and a highly attenuated replication in pigs

(203). In a follow up study, the NS1 truncated variants were able to elicit high antibody 35 response for both serum antibodies and mucosal IgA and protect pigs from heterosubtypic challenge (204). Equine influenza virus was also modified as NS1 truncated LAIV and showed good efficacy in horses (205, 206). For mouse and chicken models, Steel et al (207) constructed NS1-trucated influenza variants of HPAI A/Viet

Nam/1203/04 (H5N1) with diverse length and further deleted the poly basic cleavage site of HA gene, and mutated PB2 genes. The resultant viruses were highly attenuated, immunogenic and fully protective against lethal homologous challenge in mice (207). In a trial with chickens, the vaccinated group showed complete homologous protection and partial heterologous protection (207). One interesting finding is that the NS1 truncated mutants usually shows highly attenuated phenotype but not all NS1 truncations could elicit proper immunologic responses. For the case of A/turkey/Oregon/71 (H7N3) virus,

NS1 truncation occurred during unknown number of egg passages (208). This virus is genetically stable in IFN-deficient culture systems such as Vero cells. When Wang et al

(45) further passaged the virus in an IFN-competent system, they rescued diverse mutants with different levels of NS1 truncation. In the same study, four genetically stable variants

(pc1~pc4) were selected, plaque purified, and tested as vaccine candidates in chickens

(45). Interestingly, all four variants showed highly attenuated replication but only two of the variants (pc3 and pc4) could induce high antibody responses and protect the birds from a heterologous challenge virus (45). A follow up study in mice and pigs showed that pc2, which was not immunogenic in the chicken model, induced a higher antibody response than pc4 (209). The in vitro type I IFN induction ability in each species

36 appeared to be correlated with their efficacy in corresponding animal model, even though the direct induction of type I IFN could not be observed in vivo (209, 210).

M2 protein truncated variant: The M2 proteins form ion channels in the envelope shell of influenza virus (211). The ion channels are proton pumps, which acidify the internal viral structure inside an endosome and help to uncoat the virion to release gene segments into the cytoplasm during the initial stages of the replication cycle

(211). Watanabe et al (212) truncated the cytoplasmic tail of M2 protein and deleted HA poly basic cleavage site of A/Vietnam/1203/2004 virus. The resultant virus was able to protect mice from lethal homologous and heterologous challenges viruses (212). The a/H1N1 pdm 09 virus was also modified as M2 cytoplasmic tail mutant and showed good efficacy to homologous challenge in mice (213). In a related study, a mutant A/Puerto

Rico/8/34 that lacked the transmembrane and cytoplasmic tail domains of M2 protein was shown to provide good protection against a lethal dose of homologous challenge virus in mice (214).

PB2 knock-out variants: PB2 protein constitutes the viral polymerase complex and plays a crucial role in the process of mRNA cap snatching (215). Ozawa et al (216) created PB2 knockout influenza virus which could replicate only in cell lines expressing

PB2 proteins (AX4/PB2). The PB2 knock-out (PB2-KO) virus was attenuated and immunogenic in mouse model (217). In a lethal challenge study, although mice that were immunized with PB2-KO had similar levels of immunogenicity and protective efficacy as

IV with regard to alleviation of clinical signs, PB2-KO vaccine was superior to IV in terms of protection against virus replication and shedding (217).

Development of single cycle infectious viruses: Single cycle infectious viruses

(sciIV) are designed to undergo only one replication cycle to produce non-infectious progeny virions to serve as antigen (218). The sciIV can be generated by modifying sequences in the packaging signal and the non-coding regions of gene segments, such as

HA, NA, PB2 and PB1 (218). There have been numerous in vivo studies in mice which addressed the safety and immunogenicity (serum antibodies, mucosal, and CD8 T cell responses) of sciIV (218). In swine, Masic et al (219) produced a chimeric SIV/TX98

H3N2 mutant with an ORF of HA ectodomain that is flanked by the NA-specific packaging signal from SIV/SK/02 H1N1 virus. The chimeric virus showed good potential as a LAIV in pigs, it was highly attenuated and induced robust immune responses (219).

The efficacy from single dose of sciIV has not been always successful. While a single dose of the HA-defective SciIV constructed by Guo et al (220) could elicit strong CD8 T cell and heterologous protection in mice, a construct generated by Baker et al (221) required two vaccinations in mice.

In theory, genetic modification can stably attenuate the viruses without exhaustive passages and even provide immune boosting effects. However, those strategies are still under investigation for further optimization and most of the strategies cannot provide universal efficacy to diverse influenza strains. For example, in the study of Watanabe et al (212), truncation of cytoplasmic tail of M2 protein was not enough to attenuate the

HPAI A/Vietnam/1203/2004 virus and the deletion of the polybasic cleavage site of HA protein was required. For NS1 truncated variants, not all NS1 truncations lead to enhanced protective efficacy and further experiments were needed to select efficacious variant (45). PB2 knockout variants or single cycle infectious viruses need special cell systems for propagation and may require very high doses to elicit immune response (217,

221). The HA cleavage site modification is limited to HPAI strains which has polybasic cleavage site. To be applied in the development of universal vaccine, each strategy still needs to overcome their limitations. Future directions for the genetically modified LAIV candidate should be toward the high adaptiveness across the diverse influenza strains.

1.3. LAIV in poultry

1.3.1. The use of live vaccine in poultry industry

The best policy for infectious disease control in farm animal should aim strict disease prevention based on good management practice, high level of hygiene and biosecurity system (41, 42). However, under the wide spread locality, global-scaled market, and highly compartmented production process of poultry industry, it is impossible to establish perfect biosecurity system against unpredictable challenges (41, 119, 167). As the most proactive prevention approach, vaccination has been contributed to lessen the severity and incidences of major poultry diseases (41, 119, 167). Unlike human application, economic burden is a major factor in decision making process of vaccination in poultry farm (167). Usually, the decision is affected by the type of production, density, the prevailing disease status, available resources and cost (167). Especially for the case of

39 influenza virus, the zoonotic risk should be seriously considered even though it can impede the active innovation of prevention measures to prevent possible adverse effect on integrity of public health (21, 222).

Vaccination has prevented and reduced the major infectious diseases in poultry

(167). Diverse choices are available for multiple agents and the decision making can vary depending on the production type (breeder, broiler, and layer), epidemiologic situation, and the scale of production (167).

In chicken production system with intensive rearing settings, live vaccine is often used for mass administration with relatively low cost (167, 223, 224). Currently, live vaccines are developed and applied for the control of Newcastle disease (ND), infectious bronchitis (IB), infectious bursal disease (IBD), and Marek’s disease (MD) (93, 225-

227).

Newcastle disease (ND) is an avian disease caused by infection of avian

Paramyxovirus type I (ND virus, NDV) and associated with respiratory, intestinal or neurologic illness (225, 228). After the first outbreak report from Indonesia and England, it has been one of the most formidable disease in poultry, and three panzootic cases of virulent strain occurred during three decades (1960s~80s) (225). Until now, the low pathogenic ND is endemic in worldwide among wild and domesticated birds (225). With the success of eradication program by maintenance of high standard of biosecurity and active vaccination program, the virulent strain has not been isolated in the U.S. (229).

Mostly, the vaccination of ND is conducted by delivering live vaccine strains via drinking water, spray or conjunctival administration on 1 day-old-chickens or 2-4 week

40 old chickens (225, 228). The single vaccination with lentogenic strain produces good seroconversion and the higher antibody titer is commonly obtained by boost vaccination with IV (228). Novel vaccines such as the recombinant or subunit vaccines have been developed and show promising efficacy for future use (228).

IB is a respiratory viral disease of chickens which results in diverse range of respiratory signs, reduction in egg quality and mortality from secondary bacterial infection or nephrotic disease (230, 231). The causative agent is one of gamma coronaviruses, IB virus (IBV), and known for its high antigenic diversity (230). Due to the high variation of virus, the vaccination for IB is required to be conducted multiple times with multiple vaccine strains (231, 232). Both live and IV are available for IBV, but the live vaccine is considered to provide better immunity in respiratory tract and wider protection range (231, 232). Still, since single vaccination cannot cover the extreme antigenic variability of IBV, the IV is applied as autogenous boost vaccine

(vaccine produced by inactivation of local isolate) for local endemic strains (231). Also,

IV is frequently used as booster vaccine for layer hens to prevent negative effect on egg production (231).

Infectious bursal disease (IBD) or Gumboro disease causes immunodepression in young birds less than 10 weeks of age (93, 233). The causative agent is Avibirnavirus which is known to primarily replicate at bursa of Fabricius and cause lymphoid depletion

(93, 233). The bird infected with IBD virus has severely reduced humoral antibody response and diarrhea, reluctance of move, anorexia and infection of secondary agents

(93, 233). In case of very virulent strain, the affected flock can show high mortality that

41 can reach 30-40% or more (233). Four kinds of vaccines are available for IBD: live attenuated, immune complex, live recombinant, and IV. Live attenuated vaccines is derived from attenuation of IBDv and depending on the attenuation level, they are divided into mild, intermediate, and intermediate plus vaccines (93, 233). While mild or intermediate vaccines are used as priming for the breeder chickens at 8 weeks of age, intermediate and intermediate plus strains are used for broiler chickens especially when there is a high risk of virulent strain (233). Even though those live strains are supposed to be highly attenuated in their virulence, there is a potential of mild immune suppression followed by secondary bacterial infection or interference with other vaccine efficacy

(233, 234). Live recombinant vaccine, which carries only VP2 antigen of IBDV in viral vector, can be free from safety issue related to attenuation of live vaccine (233). Immune complex vaccine is consisted of live vaccine mixed with IBDV-specific antibodies and mostly used for hatchery via in ovo or 1 day old vaccination (93, 233). This hybrid method has advantage to overcome the interference of maternally derived antibody (93,

233). IVhas relatively lower immunogenicity so it is combined with priming vaccination with live or another IV (233). The breeder chickens with high, consistent, and long lasting antibody response by this vaccine program successfully deliver the antibody to their offspring (233).

Marek’s disease (MD) virus (MDV) is also immune suppressive agent but mostly associated with cell-mediated immune response by causing lymphomatous and neurotropic disease (227, 235). The causative agent, MDV, belongs to alpha herpesviruses and transmitted via airborne virus particles (235). Due to its pathogenicity

42 associated with cell-mediated immune response, only live attenuated vaccine is available and administered via in ovo vaccination (227, 235).

As stated above, live vaccine has been proven its multiple advantages over IV in poultry industry, especially when the causative agent has highly variable characteristics.

Control of AI has been also struggled with its capricious nature and the narrow protection range of IV regimen (45-47). However, implementation of LAIV is not as simple as the case of other poultry diseases, since the avian species occupies a unique position in influenza ecology, which can result in devastating damage in both veterinary and public health (236). Next chapter will describe this concern and review the possible strategies to overcome the shortcoming.

1.3.2. Application of LAIV in poultry

Obstacles for application of LAIV in poultry: Application of live agent as vaccine strains has potential safety risk. The back mutation into wild type strain and regaining the pathogenicity will be the most common issue of live vaccine (237). In addition, for H5 or

H7 influenza strains, the potential change of low pathogenic vaccine strain to novel highly pathogenic strain is a serious concern (29, 238). Also, the zoonotic potential and its menacing consequence is another big concern of LAIV use in poultry (29). Even though the incidence of direct poultry to human transmission is low and the human to human transmission of avian origin virus is not efficient, influenza viruses can gain adaptability in human via the genetic reassortment (239). The genetic reassortment refers a phenomenon that the gene segments of different viruses are exchanged during co- 43 infection (240). Historically, there have been four pandemics which are thought to be caused by hybrid viruses generated from reassortment between mammalian and avian lineage of influenza viruses (239). Since the human population could not establish protective immunity against the avian lineage of virus, the novel reassortant virus can rapidly spread and result in devastating consequence (241). Thus, the discussion about the future directions of LAIV in poultry industry can be addressed when it is accompanied with the counteracting strategies against reassortment.

Strategies to block genetic reassortment: There have been several studies to develop strategy to prevent genetic reassortment. The first strategy is to avoid chance of the co- infection with wild type and vaccine strains, by delivering the vaccine virus before hatching (in-ovo delivery) or to generate a single cycle infectious virus. Another novel strategy is to construct vaccine strain which has genetic defect on reassortment

(reassortment-incompetent virus)

In ovo vaccination is the technique to deliver vaccine antigen into developing chicken embryo (202, 238). The vaccine antigen is administered via the hole in blunt edge of egg shell and deposited into amniotic sac or directly infected into embryonic tissue (242). Currently, broiler industry in the U.S. widely uses the in ovo delivery system for MD and IBD, and it has shown advantages in rapid and homogenous mass application, protection in earlier age, and reduced labor and contamination (243). Also, in ovo vaccination can avoid the risk of reassortment with wild type virus since the peak

44 replication of vaccine virus is finished before hatching and there is a minimal chance of co-infection with wild type viruses. Steel et al (244) demonstrated that in ovo vaccination on 18-day-old embryonated egg with a bivalent recombinant influenza vaccine provide the protection against lethal challenge of H5N1 and NDV. Toro et al (245) used defective adenoviral vector expressing HA from H5N9 for in ovo vaccination and proved the heterologous protection against H5N2 and H5N1 challenge. In a later study, Mesonero et al (99) showed that the in ovo vaccination with adenovirus vectored H5 vaccine can elicit both antibody independent protection and the antibody response lasted longer than 12 weeks. However, the in ovo vaccination can be intervened by the presence of maternal antibody titer. In the study of Mesonero (99), the chickens with high maternal antibody titer were not seroconverted by in ovo vaccination. Another problem of in ovo vaccination is that the vaccine virus can adversely effect on hatchability of chickens

(202). In a study where NS1 protein truncated variant was used as a vaccine strain, the hatchability was significantly reduced after the in ovo vaccination (238). For the application in poultry species, in ovo delivery of LAIV will be the most practical approach to solve the concern of genetic reassortmentif there is no adverse effect on the hatchability.

The single cycle infectious influenza virus (sciIV) can be a good example as a reassortment-incompetent virus, which was previously mentioned in LAIV development strategy section (218, 220). The sci virus can go through only one cycle of replication and no infectious progeny is produced; which prevent the risk of reassortment with wild type viruses (218, 220).

Also, modulation of packaging signal can be applied to prevent the reassortment by inducing mismatch with naturally encoded signals in wild type virus. For example, scientists applied the knowledge that reassortment between type A and type B influenza viruses is not naturally occurring, probably due to inter-typic incompatibility of protein functions and incorporation process (246). Baker et al (246) created chimeric influenza

B/A reassortant virus expressing chimeric HA protein between influenza B and A strain and showed the potential as LAIV candidate without concern of reassortment. But it appear not to be efficient on application for poultry since 1) the genetic incompatibility between influenza A and B could be a high barrier to create diverse chimeric viruses and

2) the backbone influenza B virus cannot be efficiently adapted to poultry species.

Another strategy is to use the specificity of packaging signal of each gene segment. To avoid redundant incorporation of each segment more than once into new progeny, segment specific packaging signals are encoded in both non-coding regions and open reading frame (247). Thus, switching packaging signal among gene segments originated from one influenza virus, or between influenza and other viruses can be applied to prevent reassortment with wild type influenza virus (248). Gao and Palese

(248) switched packaging signal between HA and NS genes (in turn, HA segment possesses packaging signal of NS gene, while NS gene has packaging signal from HA gene). In the study, the simple swapping of ORF of packaging signal could not inhibit reassortment, but the addition of silent mutation could prevent the reassortment with the segments with normal ORF (248). White et al (249) tried to swap heterologous packaging

46 into HA, NA and NS genes between human H1N1 and H3N2 viruses. Interestingly, only swapping of HA packaging signal could inhibit the reassortment.

As listed above, there are many innovative strategies that can relieve the concern of genetic reassortment. The problem is that most of strategies were intended for human influenza virus and it will need extensive research and optimization to be applied for poultry use. Still, in the long term perspective, invest on innovative approach will become a benefit considering the advantages from the application of LAIV in poultry species.

1.4. Conclusion

The live vaccine has been widely used to control diverse poultry infectious diseases. As we learned from the LAIV application in human, LAIV will be the most realistic alternate to current IV. Also, the induction of mucosal and cytotoxic T cell response by

LAIV will be effective strategy to develop broader protection range and to cope with continuous evolution of influenza virus. The use of live influenza virus as vaccine in poultry will require stronger safety assurance than other poultry agents, but the advances in reverse genetics shows the optimistic options to create LAIV candidates free from the reassortment risk. The researches on development of LAIV candidate and its application strategies for poultry species will not only lessen the burden created from influenza viruses but it will encourage developing innovative approaches on influenza control in other animal species including swine and equine.

Chapter 2. Association between Interferon Response and Protective Efficacy of

NS1-Truncated Mutants as Type A Influenza vaccine Candidates in Chickens

2.1. Abstract

Type A influenza virus mutants that encode C-terminally truncated NS1 proteins (NS1- truncated mutants) are attractive candidates for avian live attenuated influenza vaccine

(LAIV) development because they are both attenuated and immunogenic in chickens. We previously showed that a high protective efficacy of NS1-truncated LAIV in chickens corresponds with induction of high levels of type I interferon (IFN) responses in chicken embryonic fibroblast cells. In this study, we investigated the relationship between induction of IFN and IFN-stimulated gene responses in vivo and the immunogenicity and protective efficacy of NS1-truncated LAIV. Our data demonstrates that accelerated antibody induction and protective efficacy of NS1-truncated LAIV correlates well with upregulation of IFN-stimulated genes. Further, through oral administration of recombinant chicken IFN alpha in drinking water, we provide direct evidence that type I

IFN can promote rapid induction of adaptive immune responses and protective efficacy of influenza vaccine in chickens.

2.2. Introduction

Avian lineage of type A influenza viruses (AIVs) pose a constant threat to the poultry industry with avian influenza (AI) outbreaks resulting in significant economic losses

(123, 250-253). During the 2015 highly pathogenic avian influenza (HPAI) outbreak in the Midwest, more than 40 million birds were killed and 10% of the United States egg supply was affected (254). In addition to their devastating impact on the poultry industry, occasional direct transmission of AIVs from poultry to humans has resulted in serious outbreaks in the past that produced fatal outcomes (8, 255). The recent avian H5N1,

H7N7, and H7N9 human outbreaks in China and Europe have come with severe respiratory illness resulting in severe respiratory signs and death in some cases (35, 36,

255).

AI can be prevented, managed, or eradicated through programs that focus on education, diagnostics, surveillance, biosecurity, elimination of infected poultry, and reduction of host susceptibility to AIVs (256). While pre-emptive culling of affected flocks is the most preferred method of controlling the spread of HPAI virus during an outbreak, it inevitably results in huge monetary losses. Such losses can be prevented by decreasing host susceptibility through vaccination or, in the event of an outbreak, by selective culling followed by vaccination.

Whole inactivated virus influenza vaccines are the most commonly used vaccines in poultry (42). Although these vaccines provide excellent protection from homologous strains, they are less effective or completely unprotective against heterologous and heterosubtypic strains (42). In addition, the inactivated vaccines (IVs) do not elicit strong

49 cross-reactive T-cell and mucosal immune responses. Clearly, broadly protective AI vaccines need to be developed (42).

Novel influenza vaccine designs seek to increase the breadth of heterologous and heterosubtypic cross-protection. One approach is to develop inactivated vaccines that selectively induce broadly neutralizing antibodies that target the conserved regions of viral proteins, such as HA stalk or the ectodomain of M2 protein (M2e) (257, 258).

Another approach is to use live attenuated influenza vaccines (LAIV) with capacities to elicit long lasting immunity by stimulating mucosal, cellular, and systemic (IgG) responses that are cross protective against heterologous and heterosubtypic viral infections (42, 256-258).

The nonstructural protein 1 (NS1) of influenza virus has been an attractive target for attenuation in LAIV development strategies (60, 80). The NS1 protein is known to enhance virus replication by antagonizing antiviral host cell functions, especially by blocking type I interferon (IFN) responses (87). In this context, influenza viruses with truncation in the NS1 (NS1-truncated) provoke high type I IFN responses and replicate poorly in IFN competent hosts (59). However, we have observed that not all NS1- truncated variants are effective as LAIV candidates (60). Four NS1-truncated mutants were previously tested for their capacity to induce protective immunity in chickens (45).

Two of the mutants (pc3-LAIV and pc4-LAIV) were more efficacious than the other two

(pc1-LAIV and pc2-LAIV) in protecting chickens against heterologous challenge virus

(45).

A series of in vitro experiments were subsequently carried out to establish why these LAIV candidates differ in their protective efficacy (210, 259). The in vivo efficacy of vaccine candidates (45) was observed to correlate strongly with induction of high yields of type I IFN in vitro (210, 259). For example, infection of chicken embryonic fibroblasts with pc4-LAIV, the more efficacious LAIV in chickens, resulted in production of high levels of type I IFN compared to pc2-LAIV (the less effective vaccine) (210, 259). This finding is suggestive but does not prove that type I IFN is required to boost the efficacy of NS1-truncated LAIV in chickens.

In the current study, we sought to establish the relationship between the induction of

IFN and IFN-stimulated gene (ISG) responses in vivo and the immunogenicity and protective efficacy of NS1-truncated LAIV. Our data demonstrates that the level of antibody induction and protective efficacy of NS1-truncated LAIV correlates well with upregulation of ISG expression. Further, through oral administration of recombinant chicken IFN alpha (rChIFN-α) in drinking water, we provide direct evidence that type I

IFN is a potent adjuvant for influenza vaccine in chickens.

2.3. Materials and Method

Animals and ethics statement: All animals were maintained, vaccinated, challenged and euthanized in accordance with protocol #2009AG0002-R2 approved by The Ohio State

University Institutional Animal Care and Use Committee (IACUC). This protocol complies with the U.S Animal Welfare Act, Guide for Care and Use of Laboratory

Animals and Public Health Service Policy on Humane Care and Use of Laboratory

Animals. The Ohio State University is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). White leghorn chickens were obtained from our institutional (Food Animal Health Research Program,

Wooster, OH) specific pathogen free (SPF) flock. The chickens were housed in a BSL2 facility. The facility has forced air ventilation and adequate air exchanges to prevent ammonia build up. Air entering or leaving the facility is HEPA filtered. The birds were kept in large cages (2592 sq. inch) before infection and transferred to Model 934-1 isolators (900 sq. inch) (Federal Designs Inc., Comer, GA). The number of birds in each cage was calculated based on age and the space available after subtracting the space occupied by the feeder and the watering system. Room and isolator temperatures were maintained at 25±3 °C. Birds had ad libitum access to feed and water. The wellbeing and health status of the animals was monitored twice daily throughout the experiments.

Animals were humanely euthanized when they displayed clinical signs such as ruffled feathers and reluctance to move, not moving when prodded, respiratory distress, or injuries that were not related to experimental treatment. Euthanasia was actualized by exposure to carbon dioxide (CO2). Based on the age and body size, 1-10 animals were placed in the euthanasia chamber connected to a CO2 source. The CO2 flow was set at

10-30% displacement of chamber volume/minute. Birds were observed for respiratory arrest and the CO2 flow was maintained for at least one minute after the arrest was observed. The animals were checked for an absence of breathing and lack of heartbeat. If any respiration or heartbeat was detected, the animal was placed back into the chamber and additional CO2 was administered as described above. After death has been

52 confirmed, an additional secondary physical euthanasia (cervical dislocation or removal of a vital organ) was performed before collection of tissues and carcass disposal.

Vaccination with live-attenuated influenza vaccine (LAIV) candidates: Groups of four- week-old SPF chickens (n=23 per group) were intranasally mock-vaccinated with phosphate-buffered saline (PBS) or intranasally inoculated with NS1-truncated LAIV candidates (pc2-LAIV and pc4-LAIV, both derived from wildtype virus

A/turkey/Oregon/71 (TK/OR/71) (H7N3) or reverse genetically created wildtype

6 A/Turkey/Oregon/71 (H7N3) (rgWT) virus (45) at a dose of 10 EID50 per bird. Five birds per group were euthanized at 1, 2, and 3 days post-inoculation (dpi) to harvest trachea and spleen tissues for analysis of gene expression. The remaining 8 birds per group were bled at 8 and 14 dpi for detection and titration of hemagglutination-inhibition

(HI) antibodies (260).

Oral recombinant chicken IFN-α (rChIFN-α) treatment and vaccination with inactivated influenza vaccine: Cloning and expression of rChIFN-α in mammalian cells was described previously (261). Groups of four-week-old SPF chickens (n=20 per group) were mock-vaccinated or subcutaneously vaccinated with PBS or whole-inactivated rgWT virus vaccine and provided with plain drinking water or drinking water with rChIFN-α (105 Units/bird/day). The inactivated vaccine was prepared by treating the rgWT virus with betapropiolactone as previously described (262). Five birds per group were euthanized at 1, 3, and 8 days post treatment (dpt) of rChIFN-α treatment) for

53 transcription analysis. All the remaining birds (10 birds/group at 8 dpt and 5 birds/group at 14 dpt) were bled for detection and titration of HI antibodies.

Oral rChIFN-α or Poly I:C treatment and vaccination with NS1 variants: Four-week- old SPF chickens (n=35) were divided into 5 groups: 1. unvaccinated control; 2. pc2-

LAIV vaccinated; 3. pc2-LAIV vaccinated + per-oral rChIFN treated; 4. pc2-LAIV vaccinated + per-oral treated with high molecular weight (1.5-8 kb) VacciGrade polyinosinic-polycytidylic acid (poly(I:C)) (InvivoGen); and 5. pc4-LAIV vaccinated.

Vaccination with live virus and oral treatment with rChIFN-α were conducted as described above. At 14 dpv, all chickens were challenged with a heterologous strain

CK/NJ/150383-7/02 (H7N2), and the replication of challenge virus was evaluated from tracheal swab samples collected at 2 and 4 days post challenge (dpc).

Transcriptional analysis: Total RNA was extracted from trachea and spleen tissues using

Trizol and subjected to quantitative reverse transcription PCR (qRT-PCR) as previously described (263). The primer sets used in this study were published previously (264). The fold-change in gene expression was calculated using the ∆∆Ct method using GAPDH gene as the internal control (265). All groups were included in the statistical analyses where the unvaccinated (uninfected) and untreated control groups were used as references for the analysis of data shown in figures 3 and 4, respectively. To plot the figures, the expression fold change value was normalized by dividing with that of the

54 corresponding gene in the control group. Therefore, the normalized fold change of each gene in the control group is 1.

Virus replication in chickens: Tracheal swabs were collected at the indicated time points and eluted in 1 ml of PBS supplemented with gentamicin (10 µg/ml) for virus detection.

RNA was extracted from 100 µl of the sample using QIAamp Viral RNA Mini Kit

(Qiagen). The remaining sample was used for virus isolation. To allow interpolation of median egg infective dose (EID50) titers of swab samples by the qRT-PCR method (266,

267), a standard curve was created by plotting cycle threshold (Ct) values generated with

RNA extracted from serial 10-fold dilutions of the same virus stock (with known EID50 titer) used to inoculate the chickens as a function of virus dilution. The curve was used to convert Ct values of tracheal swab viral RNA to EID50 titers. EID50 titers derived by qRT-PCR are equivalent to EID50 titers measured in eggs (267). MDCK cells were used for virus isolation and median tissue culture infective dose (TCID50) calculation. The cells were propagated in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 10 µg/ml gentamicin. Serial 10-fold dilutions of tracheal swab eluate were prepared in serum-free DMEM containing 0.75 µg/ml TPCK trypsin. Confluent cell monolayers in 96-well tissue culture plates were washed two times with PBS, inoculated with 100 µl of the diluted sample (5 replicate wells per dilution), and incubated for 5 days at 37°C. Hemagglutination assay was used to detect virus in the supernatant medium. TCID50 was then calculated by the Reed-Muench method (268).

Statistical analysis: Differences in virus titers among groups were determined by the

One-way ANOVA since we are comparing the difference in multiple groups based on one factor (GraphPad Software, San Diego, CA,USA) (327). After confirmation of significant difference among groups, post-hoc Tukey test was used for the pair-wise comparison since we wanted to compare every mean with every other mean (GraphPad

Software, San Diego, CA,USA) (327). The Mann-Whitney U test (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY, USA) was used to determine differences between transcriptional fold-change values since we could not assume the normality of the fold-change values and based on the previous study on relative quantification of mRNA (328, 329).

2.4. Results

Serum antibody response in chickens vaccinated with experimental NS1-truncated

LAIV: We first looked at the development of adaptive immune responses in 4-week-old chickens (n = 8 per group) after intranasal vaccination with pc2-LAIV and pc4-LAIV.

The reverse genetically created wildtype (rgWT) virus was included for comparison.

Among birds vaccinated with pc4-LAIV, five had detectable levels of HI antibodies as early as 8 days post-vaccination/infection (dpv/dpi) (Fig. 2.1). This was in clear contrast with pc2-LAIV vaccination where none of the birds were HI positive or rgWT infection where only 2 birds had detectable antibodies at this point. At 14 dpv/dpi, only two birds in the pc2-LAIV-vaccination group were HI positive compared to 7 in the pc4-LAIV- vaccination group and 8 in the rgWT-infected group.

To rule out the possibility that the rapid and high ratio of seroconversion in the pc4-LAIV-vaccination group was due to high replication efficiency (or high antigen load) of the vaccine virus, viral titers were determined from tracheal swabs. Birds in all three groups had similar viral titers at 2 dpv/dpi (Fig 2.2). At 3 dpv/dpi, the rgWT viral titers were significantly higher than pc2-LAIV or pc4-LAIV by several folds and there was no difference between pc2-LAIV and pc4-LAIV even though not all the birds in the pc2-

LAIV group were PCR positive (Fig 2.2).

Gene expression in chickens vaccinated with experimental NS1-truncated LAIV: In a previous in vitro study, pc4-LAIV induced a higher level of type I IFN response in chicken embryonic fibroblasts than pc2-LAIV and rgWT virus (210). Thus, the rapid seroconversion of pc4-LAIV-vaccinated chickens (Fig. 2.1) could be due to high IFN response in vivo. We tested this possibility by analyzing the expression levels of IFN and

IFN-related genes in trachea and spleen tissues following vaccination with LAIV candidates or infection with rgWT virus. Two IFN-stimulated genes (ISGs), 2′,5′-OAS and Mx, were targeted due to their high sensitivity to the type I IFN stimulus. Figure 3 shows that at 1 dpv/dpi, the level of 2′,5′-OAS gene transcription was significantly increased in trachea and spleen tissues of chickens vaccinated with pc4-LAIV or infected with rgWT but not in the trachea of pc2-LAIV-vaccinated birds. Expression of the Mx gene was significantly upregulated only in 1 dpv tracheal tissues from pc4-LAIV- vaccinated chickens and downregulated in 3 dpv/dpi spleen samples from pc2-LAIV and rgWT vaccinated/infected birds. We could not detect any increase in type I IFN (IFN-

α/β) gene transcription although there was upregulation of the IFN-γ gene in spleen samples collected from the pc4-LAIV-vaccincation group at 3 dpv.

Oral IFN treatment induces a rapid antibody response to influenza vaccination in chickens: The observed correlation between antibody response and transcription of IFN- related genes in pc4-LAIV-vaccinated birds (Figs. 2.1. and 2.3.) is suggestive but does not prove that IFN is involved in rapid seroconversion. To assess the direct role of IFN in stimulating rapid seroconversion further, we subcutaneously inoculated chickens with inactivated influenza vaccine and provided them with rChIFN-α in drinking water at an average dose of 105 Units/bird/day for 14 days. This dose of type I IFN was previously shown to have biological effects in 33-day old chickens (269). Figure 2.4.A shows that 9 of 10 rChIFN-α-treated birds seroconverted by 8 days dpt compared to only 4 out of 10 birds provided with plain drinking water. All remaining birds (n=5) in the rChIFN-α- treatment group seroconverted by 14 dpt while one of the 5 birds provided with plain drinking water was HI negative. Further, a wider range of HI titers was observed in the untreated group (Log2 HI titer <1-7) relative to the treated birds (Log2 HI titer 2-6) at 14 dpt (Fig. 2.4.A). The rapid seroconversion of rChIFN-α-treated chickens is similar to that observed in the pc4-LAIV vaccination group (Fig. 2.1.).

Gene expression in chickens treated with rChIFN-α in drinking water: Biological activity of orally-administered rChIFN-α was previously reported to correlate with increase in the level of Mx1 and 2′,5′-OAS gene expression in trachea tissues (269). In

58 this study, we focused on the effect of oral rChIFN-α treatment on the expression of these two ISGs along with type I IFN (IFN-α/β) and IFN-γ genes. As shown in Figure 2.4.B, treatment with rChIFN-α resulted in significant upregulation of 2′,5′-OAS and IFN-γ genes in spleen tissues harvested from unvaccinated chickens at 3 dpt. The rChIFN-α treatment did not cause upregulation of 2′,5′-OAS, Mx1, and IFN-γ gene expression in spleen at 1 dpt. While the transcription levels of 2′,5′-OAS and IFN-γ genes were upregulated in birds administered with inactivated vaccine, there was no statistical difference between rChIFN-α-treated and untreated birds (Fig. 2.4.C).

Effect of type I IFN treatment on immunogenicity and heterologous protection efficacy of pc2-LAIV: Above data suggests that immunogenicity of NS1-truncated LAIV candidates is partially dependent on the levels of type I IFN induced in vivo. Thus, we hypothesized that the poor immunogenicity and inefficacy of pc2-LAIV is mainly due to the lower type I IFN induction capacity in chickens and tested whether oral treatment with type I IFN can boost the protective efficacy of pc2-LAIV. Thirty-five chickens were divided into five groups (n=7 per group): unvaccinated control; pc2-LAIV vaccination;

IFN treatment + pc2-LAIV vaccination (exogenous rChIFN-α treatment); poly I:C treatment + pc2-LAIV LAIV vaccination (endogenous IFN induction); and pc4-LAIV vaccination groups. Poly I:C was previously shown to enhance adaptive immune responses to influenza vaccine in chickens (270). Vaccine immunogenicity was first assessed by testing serum HI antibody titer. As described above (Fig. 2.1.), pc4-LAIV vaccination provoked an early antibody response at 8 dpv (5 out of 8 birds (65.5%)), but

59 no antibody response was detected in pc2-LAIV-vaccinated animals even after treatment with exogenous rChIFN-α or poly I:C (Fig. 2.5.). At 15 dpv, we challenged the birds with a heterologous (H7N2) virus and compared the protective efficacy among the vaccination groups. One bird in the unvaccinated control group was euthanized at 2 days post challenge (dpc) due to severe clinical signs (ruffled feathers and periorbital swelling).

Consistent with the previous report (45), pc4-LAIV vaccination consistently showed the highest degree of protection against the heterologous challenge virus as indicated by

EID50 equivalent titers detected by qRT-PCR (Fig. 2.6., top (p<0.001)) and confirmed by virus isolation in MDCK cells at 2 and 4 dpc (Fig. 2.6., bottom (2 dpc, p<0.001; 4 dpc, p<0.05)). In contrast, significant reduction of virus shedding by pc2-LAIV vaccination was only detected in 4 dpc samples using virus isolation method (Fig. 2.6., bottom right

(p<0.05)). Of note, treatment of birds with rChIFN-α prior to pc2-LAIV vaccination led to a significant reduction in the titer of re-isolated challenge virus at both time points

(Fig. 2.6., bottom (2 dpc, p<0.001; 4 dpc, p<0.05)) and virus detected by qPCR at 4 dpc

(Fig. 2.6., top right (p<0.001)). Poly I:C treatment did not enhance the efficacy of pc2-

LAIV, rather it appears to have increased the level of virus replication (Fig. 2.6.).

2.5. Discussion

We have compared two NS1-truncated mutants in terms of their immunogenicity, ability to induce IFN and ISG responses, and protective efficacy in four-week-old chickens.

During the first two weeks post vaccination, the development of adaptive immune responses was monitored by measuring HI antibody titers in serum samples. In line with

60 our previous observation (45), pc4-LAIV was superior to pc2-LAIV in terms of inducing seroconversion and HI antibodies in chickens. In addition, the current study has provided new insight into induction of adaptive immune responses by NS1-truncated LAIV candidates. We have demonstrated that pc4-LAIV consistently induced a rapid antibody response within 8 days following intranasal vaccination (Figs 2.1. and 2.5.). This can shorten the risk period between vaccination and the development of protective immunity especially in young birds that do not respond well to inactivated vaccines (271). The inability of pc2-LAIV to induce seroconversion may be attributed to over-attenuation

(suboptimal replication) (Fig. 2.1.) and poor immunogenicity of the vaccine.

In general, NS1-truncated mutants are attenuated in avian and mammalian species partly due to induction of high type I IFN responses (60, 209). The type I IFN is also known to enhance the mucosal and systemic adaptive immune responses (272-274).

In chickens, rChIFN-α treatment was shown to induce more rapid seroconversion to natural infection by low-pathogenicity influenza virus (275). In mice, IFN-α/β treatment promoted fast and polyclonal antibody responses (276) and a recombinant rabies virus expressing IFN-α1 was shown to stimulate an antibody response that was more rapid compared to the isogenic wildtype virus (277). Thus, induction of rapid immune responses by the pc4-LAIV may be due to its capacity to trigger higher levels of type I

IFN compared to pc2-LAIV and rgWT virus (210).

Contrary to the high levels of type I IFN induced in primary chicken fibroblast cells (210), expression of IFN-α/β genes was generally not upregulated except for a small but statistically significant increase in IFN-α transcription in 3 dpv spleens of the pc2-

LAIV group (Fig. 2.3). The discrepancy between in vitro and in vivo IFN inducing capacities of our vaccine candidates is a subject for future study. Penski et al (278) reported a similar discrepancy where a set of NS1-truncated mutants were able to induce high levels of IFN in chicken cell cultures but were poor inducers in chickens, in vivo, in a manner that correlated with virus replication. The fact that our vaccine candidates and

3 the isogenic rgWT virus are very attenuated in chickens (<10 EID50/ml of swab eluate)

(Fig. 2.1.) could explain why we did not see upregulation of IFN genes in trachea and spleen tissues collected 1 and 3 dpv/dpi. However, it does not explain why there was significant upregulation of the 2',5' OAS gene in trachea and spleen tissues of chickens vaccinated with pc4-LAIV or infected with rgWT and Mx gene in tracheal tissues from pc4-LAIV-vaccinated chickens (Fig. 2.3.). It is possible that both pc4-LAIV and rgWT were able to induce some IFN that triggered ISG upregulation (279). We could have missed a critical time point for IFN-α/β gene detection or a cell population that produces large amounts of the cytokine in chickens (278). Alternatively, ISG transcription may have been triggered directly by the virus infection independently of IFN signaling (280,

281). For example, the 2′,5′-OAS gene can be activated by dsRNA independently of IFN signaling [43]. The level of ISG transcription can also be affected by the ability of truncated or full-size NS1 proteins to suppress epigenetic control of gene regulation (282,

283). Although our study focused on the 2′,5′-OAS and Mx genes, there are more than

300 ISGs (284). An in-depth study is required to identify ISGs that are critical for vaccine efficacy and to delineate the mechanism of ISG upregulation by the NS1-truncated LAIV candidates.

We reasoned that if the rapid seroconversion triggered by pc4-LAIV was due to ISG upregulation, a similar response could be produced through rChIFN-α treatment.

As observed in the study published by Meng et al (269), rChIFN-α treatment resulted in elevated levels of 2',5'-OAS gene expression in spleen at 3 dpt (Fig. 2.4.B). Thus, our orally-administered rChIFN-α was biologically active. Clearly, the rapid antibody development in birds treated with rChIFN-α and vaccinated with whole inactivated rgWT virus vaccine (Fig. 2.4.A) was similar to that observed in pc4-vaccinated birds (Figs 2.1. and 2.5.). It is worth noting that both the rgWT virus and pc4-LAIV have the same backbone genes and proteins except for the NS1 gene/protein (45) and humoral immune response to whole virus inactivated vaccine is mainly directed to HA and NA proteins

(not the NS1 protein) (49). Therefore, a whole inactivated pc4 vaccine is also expected to induce rapid seroconversion in rChIFN-α treated chickens. Future work should determine how ISGs, IFN, and pc4-LAIV interdependently or independently trigger the acceleration of adaptive immune responses.

The poor efficacy of pc2-LAIV may result from an inability to induce high levels of type I IFN (19) or ISGs (Fig. 2.3.). This prompted us to test whether direct

(exogenous) IFN treatment or endogenous IFN induction by poly I:C can enhance pc2-

LAIV efficacy. Heterologous protection by pc2-LAIV was significantly enhanced by rChIFN-α treatment (Fig. 2.6.). Although the in vivo half-life of rChIFN-α has yet to be determined, non-PEGylated interferons have short in vivo half-lives due to low stability.

For example, in humans, orally administered non-PEGylated IFN has a half-life of up to

8.5 hours (285). Since the rChIFN-α was administered for 4 days and withdrawn on the

63 day of vaccination and the chickens were challenged at 2 weeks post vaccination, it is less likely that reduction of virus shedding was due to the innate effects of IFN.

The failure of pc2-LAIV to induce an antibody response when administered together with rChIFN-α is intriguing since rChIFN-α was previously shown to facilitate seroconversion of chickens after natural infection by low pathogenicity avian influenza virus (275). The ability of IFN to facilitate seroconversion in the context of live virus may depend on the virus strain. The enhanced protective efficacy of pc2-LAIV in rChIFN-α treated chickens may be due to stimulation of cross-protective cell-mediated immunity (175, 286, 287).

We will address this possibility in a separate study. Another unexpected result was the observation that poly I:C treatment not only failed to enhance pc2-LAIV efficacy but also appeared to cause a slight increase in virus shedding at 4 dpc (Fig. 2.6.). We speculate that the dose of poly I:C (100 µg/bird) used in this study was not optimal for pc2-LAIV even though it was previously shown to enhance adaptive immune responses to inactivated avian H5N1 influenza vaccine in chickens (270).

In this study, we have demonstrated that the level of antibody induction and protective efficacy of NS1-truncated LAIV in chickens correlates well with upregulation of ISG expression. An in-depth analysis such as systems biology [52] is required to determine which ISGs need to be upregulated to enhance NS1-truncated LAIV efficacy.

8 1414 dpv dpi/dpi

8 DPV )

2 6 g

o 8 dpv8 dpi/dpi L ( 8 DPV

r 4

(HI (HI titer)

I Log H 2

Figure 2. 1. Serum antibody response in chickens following vaccination with LAIV candidates or infection with rgWT virus

Chickens were infected or vaccinated by rgWT virus or NS1-truncated variants (pc2-

LAIV and pc4-LAIV) via intranasal route. Serum was collected at 8 and 14 days post- vaccination/infection (dpv/dpi) and tested for the presence of influenza virus

A/Turkey/Oregon/71 specific hemagglutination inhibition (HI) antibodies. HI titers are presented as circles (rgWT), squares (pc2-LAIV), and triangles (pc4-LAIV). The thick horizontal lines represent median titers of the groups.

* *

Figure 2. 2 Comparison of virus replication in trachea

The EID50 equivalent titers were interpolated from qRT-PCR Ct values of tracheal swab viral RNA as described in Materials and Methods. Bar and Lines represent the average and standard deviation of EID50 equivalent titers in each group, respectively. Statistical significance, *p<0.05. EID50, median egg infectious doses.

Trachea – 1 dpv/dpi Spleen – 1 dpv/dpi 100 100

** ** * 10 10 ** ** *

1 1 Fold change over controlchangeover Fold

0.1 0.1 2',5'-OAS Mx IFN-α IFN-β IFN-γ 2',5'-OAS Mx IFN-α IFN-β IFN-γ

Trachea – 3 dpv/dpi Spleen – 3 dpv/dpi 100 100

10 10 *

* *

1 1 Fold Fold change over control

**** 0.1 0.1 2',5'-OAS Mx IFN-α IFN-β IFN-γ 2',5'-OAS Mx IFN-α IFN-β IFN-γ rg WT pc2-LAIV pc4-LAIV

Figure 2. 3. IFN and ISG responses after vaccination with LAIV candidates or infection with rgWT virus

See Materials and Methods for details on fold change calculation, statistical analysis and data normalization. Bar and Lines represent the average and standard deviation of EID50 equivalent titers in each group, respectively. dpv/dpi, days post vaccination/infection.

Error bars, mean ± S.D. Statistical significance, *p<0.05, **p<0.001.

A) 14 dpi

8 dpi

(HI (HI titer)

2 Log

B) C) Spleen - 1 dpt Spleen - 3 dpt Spleen -3dpt 6 6 ** 4 4 ** ** ** * 2 * 2

0 0 (Fold (Fold change over control)

-2 2 -2

Log Log2 (Fold Log2 (Fold change over control) -4 -4 IFN IFN Vaccine Vac+IFN 2',5'-OAS Mx IFN-α IFN-β IFN-ƴ 2',5'-OAS Mx IFN-α IFN-β IFN-γ

Figure 2. 4. Effect of per-oral rChIFN-α treatment to naïve chickens or chickens vaccinated with inactivated vaccineA) ISGs and IFN response to rChIFN-α at 1 and 3 days post treatment (dpt) in spleens of naïve chickens; B) Comparison of ISGs and IFN response in spleen of inactivated vaccinated chickens with or without rChIFN-α treatment at 3dpt; C)antibody response to inactivated vaccination with our without rChIFN-α treatment at 8 and 14 days post inoculation (DPI). dpi: days post-inoculation. Statistical significance, *p<0.05, **p<0.001

8 dpv/dpi 13 dpv/dpi

(HItiter)

2 Log

Figure 2. 5. Pre-challenge antibody responses

The development of antibody response was monitored at 8 and 13 days post-vaccination

(dpv). Horizontal bars represent mean antibody titer for the group (n=8).

2 DPC 4 DPC

8 8

PCR 6

- qRT 4

Equivalent/ml) 4

50 Virus Titer Titer Virus

EID 2

10 2

Measured by Measured (Log

10 10

8 8

/ml)

isolated

50 -

Equivalent/ml 6 6

50 TCID

Virus Titer Virus 4 4

TCID 10

In MDCK cells MDCK In 2 (Log

Measured in MDCK cells MDCK in Measured 2

Log Virus Titer Titer Virus

Figure 2. 6. Replication and shedding of heterologous challenge virus.

Top: Viral titers expressed as median (50%) egg-infectious dose equivalent by qRT-PCR

(see Materials and Methods) [27]. Bottom: viral titers re-isolated in MDCK cells.

Horizontal bars represent mean antibody titer for the group (n=8). DPC, days post challenge. TCID50, median (50%) tissue culture infectious dose. *p<0.05. **p<0.001.

Chapter 3. Immunogenicity and protective efficacy of live attenuated influenza

vaccine and inactivated influenza vaccine in 1-day-old chickens

3.1. Abstract

Until few weeks after hatching, the chicken immune system is not fully mature enough to protect the birds from opportunistic infections. While IV have limited efficacy in young birds, live vaccines against diverse pathogens have been effectively used at 1-day of age or in ovo. Along with the previous findings from vaccinations against other agents, we were interested to know whether our live influenza vaccine candidate, the pc4-LAIV, can elicit a better immune response than an IV in 1-day old chickens. In this study, 1 day old chickens were vaccinated with pc4-LAIV or IV, and the innate signaling sensitization, mucosal antibody response and heterologous protective efficacy were compared between the two vaccines. We observed that the pc4-LAIV elicits a higher innate signaling sensitization, mucosal IgA response and better clearance of heterologous challenge virus at 5 days post challenge. However, the level of serum antibody and mucosal IgG was higher in the IV vaccinated group. In this study, our pc4-LAIV was confirmed to provide a higher immune priming effect in immunologically immature chickens that can confer early protection and be used as a priming vaccine.

3.2. Introduction

In chickens, the first few weeks after hatching is the most vulnerable phase of opportunistic infection due to the immature immune system (288, 289). Also, the low responsiveness of immature immune system, inactivated vaccination is not efficacious at the early time point (271, 290). Still, to protect the young birds from diverse infectious disease, such as Newcastle disease, infectious bronchitis, Marek’s disease, and infectious laryngotrachitis virus, live vaccine has been conducted on day old or as an in ovo vaccination; and it has proved good immune response and protective efficacy in the early age (93, 225-227). But use of live influenza vaccine in poultry requires extra caution than any other disease, since the poultry can serve as host for a rapid evolution and interspecies transmission of influenza virus (29, 238, 239). Thus, the successful LAIV candidate to be applied in young bird should meet the high level of safety requirements as well as the good protective efficacy.

In our previous studies, we showed that our non-structural protein truncated variant, pc4-LAIV is a promising LAIV candidate in chickens (45, 291). First, the pc4-

LAIV is free from the concern of reversion to wild type strain since the truncation of the

NS1 protein is irreversible (45, 291). Also, in two independent pc4-LAIV vaccination studies, the pc4-LAIV was confirmed to be highly attenuated with poor replication and no transmission of virus to contact cage mates (45, 291). Interestingly, even though the virus could not replicate well, the pc4-LAIV induced good serum antibody response and heterologous protection (45, 291). Influenza NS1 protein is known to antagonize host type I IFN response to create virus favorable environment and thus, truncation in NS1

72 protein can result in increased level of type I IFN along with the impeded replication efficacy (206, 207). Following studies showed that infection with pc4-LAIV could induce high level of type I IFN production in vitro (210, 291). Also, the correlation of enhanced type I IFN related gene responses (interferon stimulated genes, ISGs) with accelerated serum antibody response and high level of heterologous protection was observed in 3- week-old chicken vaccination study (291).

Given that pc4-LAIV elicits good level of innate type I IFN related responses and has an ability to accelerate adaptive immune response, we hypothesized that pc4-LAIV will have advantage to induce early immune response from immunologically immature young chickens. This study was aimed to demonstrate the efficacy of pc4-LAIV in 1- day-old chickens in terms of induction of innate and mucosal immune response and the protection from heterologous challenge.

3.3. Materials and Method

Animals and ethics statement: All animals were maintained, vaccinated, challenged and euthanized in accordance with protocol #2009AG0002 approved by The Ohio State

University Institutional Animal Care and Use Committee (IACUC). This protocol complies with the U.S Animal Welfare Act, Guide for Care and Use of Laboratory

Animals and Public Health Service Policy on Humane Care and Use of Laboratory

Wooster, OH) specific pathogen free (SPF) flock. The chickens were housed in a BSL2 facility with forced air ventilation and adequate air exchanges to prevent ammonia build up. Air entering or leaving the facility is HEPA filtered. The birds were kept in large cages (2592 sq. inch) before infection and transferred to Model 934–1 isolators (900 sq. inch) (Federal Designs Inc., Comer, GA). The number of birds in each cage was calculated based on age and the space available after subtracting the space occupied by the feeder and the watering system. Room and isolator temperatures were maintained at

Based on the age and body size, 1–10 animals were placed in the euthanasia chamber connected to a CO2 source. The CO2 flow was set at 10–30% displacement of chamber volume/minute. Birds were observed for respiratory arrest and the CO2 flow was maintained for at least one minute after the arrest was observed. The animals were checked for an absence of breathing and lack of heartbeat. If any respiration or heartbeat was detected, the animal was placed back into the chamber and additional CO2 was administered as described above. After death has been confirmed, an additional secondary physical euthanasia (cervical dislocation or removal of a vital organ) was performed before collection of tissues and carcass disposal.

Experimental design: The design of study is summarized in Table 3.1. Briefly, fifty-four

1-day-old birds were split into three groups (pc4-LAIV, IV, and unvaccinated) (n = 18 birds per group). Each bird in the pc4-LAIV group was intranasally vaccinated with

6 1×10 EID50 doses of the vaccine virus in a 200 µL PBS. The IV group received a mixture of IV prepared from A/Turkey/Oregon/71 (H7N3) (the parental virus of pc4-

LAIV) and Montanide ISA 70 adjuvant (Seppic, France) (3:7, V/V ratio) via subcutaneous route. The unvaccinated control group did not receive any treatment. All birds were monitored for clinical signs and behavioral changes throughout the study. At 1 and 3 days post vaccination (dpv), five birds from each group were euthanized for collection of trachea and spleen samples to be used for transcriptional analysis as described below. At 14 dpv, 10 birds were randomly selected from each group and serum and tear samples were collected to measure systemic and mucosal antibody responses, respectively. After serum and tear collection, all birds were intranasally challenged with

6 1×10 EID50 doses of a heterologous virus, A/CK/NJ/150383-7/02 (H7N2), in a 200 µL volume. At 3 and 5 days post-challenge (dpc), six or seven birds from each group were euthanized to harvest tracheal tissue for titration of the challenge virus titers through real time RT-PCR as previously described (266, 267).

Transcriptional analysis: Trachea and spleen samples were homogenized in Trizol reagent and total RNA was extracted as previously described (263, 291). Messenger RNA

(mRNA) was selectively converted into cDNA using RT-PCR with oligo dT primer and subjected to quantitative PCR using SYBR GREEN system (Quanta, Gaithersburg, MD,

USA). The primer sequences used for amplification of 2’,5’-OAS, Mx, IFN- α, IFN-β, and IFN-γ genes were previously published (264). The ΔΔCt method was used to determine differential gene regulation using GAPDH gene as the internal control. Gene expression levels were calculated as fold changes over the unvaccinated control group as previously described (263, 291).

Serologic assays: For analysis of serum antibodies, chickens were bled via wing veins and the blood was incubated overnight at room temperature for serum separation to occur. The separated sera were heat inactivated and stored at -20°C until used for hemagglutination inhibition (HI) or enzyme-linked immunosorbent assay (ELISA) tests.

The HI test was conducted in accordance with the recommendation of World

Organization for Animal Health (OIE) (292). Briefly, 50 µl of heat inactivated samples was serially diluted and mixed with an equal volume of antigen preparation containing 8 hemagglutinating units of virus. The serum-antigen mixture was incubated for 30 min at room temperature to form antigen-antibody complexes. Then, 50 µl of 1 % turkey erythrocyte suspension was added in each well. The HI titer was reciprocally determined as the end-point dilution which showed complete inhibition of hemagglutination.

Mucosal antibody analysis: Mucosal antibody responses were measured using tear samples collected according to a previously described method with slight modification

(293). Briefly, chickens were comfortably held with their eyelids open and fine sodium chloride crystals (less than 0.01g) were sprinkled onto each eye. Once lachrymation was

76 induced, tears were carefully harvested using a micropipette attached with a sterile tip and immediately placed in tubes. For consistency, and to prevent antibody dilution, only the first 50 µL of induced-tears was collected. The tear samples were stored at -20 °C until used for ELISA tests.

Three different ELISA kits were used to detect antibodies in tears. Influenza A nucleoprotein (NP)-specific antibodies were measured using a competitive ELISA kit

(Influenza A NP antibody inhibitor ELISA; Virusys Corporation, Sykesville, MD, USA) according to instructions provided by the manufacturer. The NP Reduction Index (NPRI) value was calculated based on the formula: NPRI = (1- [Absorbance value (Abs) of samples – mean Abs value of diluent control]/[mean Abs of negative serum control- mean Abs value of diluent control]). Influenza virus-specific IgG response was measured by indirect ELISA kit (IDEXX AI Ab test; IDEXX Laboratories, Westbrook, ME, USA) in accordance with manufacturer’s instructions. Influenza virus-specific IgA response was measured using the same kit described for IgG except that the secondary antibody was replaced with 10000 fold diluted HRP-labeled Anti-Chicken IgA (α chain specific)

(Gallus Immunotech Inc., Fergus, Canada).The level of IgG was represented as sample to positive control ratio (S/P ratio) using the positive control samples provided by the manufacturer while raw OD values were used for the analysis of IgA levels.

Statistical analysis: Differences in virus titers among groups were determined by the

One-way ANOVA since we are comparing the difference in multiple groups based on one factor (GraphPad Software, San Diego, CA, USA) (327). After confirmation of

77 significant difference among groups, post-hoc Tukey test was used for the pair-wise comparison since we wanted to compare every mean with every other mean (GraphPad

Software) (327). The Mann-Whitney U test (IBM SPSS Statistics for Windows, Version

22.0. Armonk, NY, USA) was used to determine differences between transcriptional fold-change values since we could not assume the normality of the fold-change values and also based on the previous study on relative quantification of mRNA (328, 329).

3.4. Results

We previously demonstrated that upregulation of IFN-related genes by influenza vaccine correlates well with rapid induction of adaptive immune responses and enhancement of protective efficacy in 4-week-old chickens (291). To determine and compare the efficacy of live and inactivated vaccines in younger birds, 1-day-old birds were vaccinated intranasally with pc4-LAIV or subcutaneously with IV as shown in Table 3.1. One bird in the IV group died at 1 day after vaccination due to unknown reasons. The remaining birds, in all three groups, did not show abnormal behavior, clinical signs, or mortality for the entire duration of the experiment.

Interferon (IFN) and IFN stimulated gene (ISG) responses: The ability of pc4-LAIV and IV to stimulate innate immune responses in 1-day-old chickens was assessed by quantification of mRNA transcription level of type I/II IFNs and ISGs. Fig. 3.1 shows upregulation or downregulation of these genes as fold changes over the unvaccinated control group. The most consistent and apparent changes were observed in ISGs, 2’,5’-

OAS and Mx genes. Both vaccines were able to induce a significant upregulation of

2’,5’-OAS in 1 dpv trachea and spleen tissues (Fig. 3.1). However, there was no significant difference between the two vaccine groups despite having lower magnitude of upregulation in the IV group compared to the pc4-LAIV group. Transcription of Mx gene was also significantly increased by pc4-LAIV in trachea at both time points and in spleen at 1 dpv while induction of the Mx gene by IV vaccination was observed only in 1 dpv tracheal samples (Fig. 3.1). Birds that received pc4-LAIV had a significant decrease in IFN-α mRNA levels at both time points whereas those vaccinated with IV had downregulated levels of IFN-β and IFN-γ transcription in trachea (at 1 dpv and 3 dpv, respectively) and IFN-α transcription in spleen at 3 dpv. Even though the level of 2’,5’-

OAS gene transcription was consistently elevated in 1 dpv trachea and spleen samples by both kinds vaccinations, the result from 3 dpv spleen samples showed that a significant downregulation of 2’,5’-OAS gene transcription was induced by IV vaccination. Overall,

ISG transcription was upregulated in both types of tissues at 1 dpv and reduced or downregulated at 3 dpv. There were subtle changes in IFN gene transcription but the general trend was downregulation in trachea at 1 dpv and in spleen 3 dpv.

Pre-challenge antibody levels in serum and tears at 14 dpv: Both vaccines were poor in induction of serum antibody responses in 1-day-old chickens. Seroconversion to homologous virus (A/Turkey/Oregon/71 H7N3) was observed in eight out of ten birds in the IV group and only four out of ten birds in the pc4-LAIV group. Heterologous

(A/CK/NJ/150383-7/02 H7N2) HI antibodies were not detected in any of the groups (Fig.

3.2). Levels of antibodies in tear samples were measured using three kinds of ELISA kits

79 which detect anti-influenza nucleoprotein (NP) binding activity or the presence of influenza specific chicken immunoglobulins (IgG and IgA). The anti-NP ELISA kit is based on an inhibitory ELISA format, which detects any kind of binding activity to the

NP protein and presents the scale of binding activity as NP Reduction Index (NPRI). As shown in Fig. 3.3A, vaccinated groups had significantly higher NPRI values than the unvaccinated group. To further delineate the mucosal antibody responses induced by the two different forms of vaccine, the levels of influenza specific IgG and IgA responses were measured. Both pc4-LAIV and IV vaccinations significantly increased IgG response and the level of increase was higher in the IV vaccinated group than the pc4-LAIV group

(Fig. 3.3B). A significant increase of IgA response was observed in the pc4-LAIV group while only a slight increase in IgA response was observed in the IV group (Fig. 3.3C).

Heterologous protection efficacy: The above data (Figs. 3.1-3.3) suggested that, overall, pc4-LAIV and IV stimulated the immune system of 1-day-old chickens via distinctive mechanisms. Interestingly, neither pc4-LAIV nor IV could induce the high levels of serum antibodies that are usually associated with protective efficacy in older birds. To determine whether the observed immune responses can provide sufficient protection against heterologous virus challenge, the chickens were challenged at 2 weeks post vaccination (2 weeks of age) and the levels of challenge virus replication in trachea were compared among groups. At 3 dpc, the median challenge virus titer of the unvaccinated

4.69 control group was 10 EID50 Equivalent/ml. A significant reduction in challenge virus replication was observed in the IV group where virus was detected only from one out of

80 six birds (Fig. 4). The median challenge virus titer of the pc4-LAIV group was about 1

3.71 log lower than the unvaccinated group (10 EID50 Equivalent/ml) but the difference was not statistically significant. As expected based on our previous study (291), the unvaccinated control group showed the highest level of challenge virus replication (106.89

EID50 Equivalent/ml) at 5 dpc. Although virus replication was significantly reduced in vaccinated groups compared to the unvaccinated group, no virus was detected from more than half of the birds in pc4-LAIV group (4/7) while more than half of birds in IV group

3.96 (5/7) were virus positive with a median titer of 10 EID50 Equivalent/ml (Fig. 3.4).

3.5. Discussion

Many vaccine effectiveness studies have confirmed that LAIV has advantages over

IV in terms of stimulating immature immune systems (105, 175, 294). Live vaccines can efficiently trigger the innate immune system, via diverse pathogen-associated molecular patterns (PAMPs), and lead to regulation of several host genes including type I IFNs (53).

The type I IFN is a crucial component of the innate antiviral immune system which regulates several hundreds of ISGs and shapes the development of adaptive immune responses (57, 58, 295, 296). Two genes, 2’,5’-OAS and Mx, are common among the

ISGs induced by type I IFNs in chickens (269, 291). Upregulation of ISGs by pc4-LAIV correlated well with rapid induction of adaptive immune responses and enhancement of protective efficacy in 4-week-old chickens (291). We were interested whether pc4-LAIV can also be efficacious in younger, immunologically immature chickens. The evidence provided in the current study indicates that pc4-LAIV can elicit higher levels of innate responses, mucosal IgA antibodies, and heterologous protection in 1-day-old chickens

81 compared to IV. This regimen could be a big advantage in young birds considering their limited number and functionality of antigen presenting cells (143, 297). In addition to the general advantages of live vaccine, our pc4-LAIV is highly immunogenic via the action of truncated NS1 protein as proved in our previous studies (45, 291)

A single administration of pc4-LAIV in 1-day old chickens was sufficient to induce a significant increase in influenza virus-specific IgA antibodies in tears but failed to stimulate high serum antibody titers. Previous studies have repeatedly described serum

IgG and mucosal IgA responses as key features that distinguish live vaccines from parenterally administered IVs (102, 106, 170). The protection afforded by IV vaccination primarily depends on the action of neutralizing serum antibodies and the serum HI antibody titer is a strong indicator of IV efficacy (157). Serum antibody response does not always correlate with protective efficacy of live vaccines. The underlying mechanism of serum antibody-independent viral clearance by live vaccines is postulated to be driven by mucosal IgA or CD8+ and CD4+ T cell responses (157, 298-300). This serum antibody-independent viral clearance can be utilized to enhance vaccine efficacy in young chickens considering their poor antibody production ability (301, 302). According to a study of live infectious bronchitis (IB) virus vaccination in chickens of different ages, birds vaccinated immediately after hatching tended to produce lower levels of serum IgG antibodies which had lower avidity indices compared to the birds vaccinated at 4-weeks of age (303). However, there was no difference in mucosal IgA antibody avidity between the two age groups (303). Another study of live IB vaccination in 1-day- old chickens also showed an inefficient antibody response and the protective efficacy of

82 the vaccine mostly correlated with induction of high levels of CD4+, CD8+ and IgA bearing B cells (304). Also, the vaccination of 1 day old chickens with adenovirus vectored H5 and H7 influenza vaccine could induce IgA response in lachrymal fluid and increased interleukin-6 expression without inducing detectable levels of serum antibodies

(224). Therefore, our current findings are consistent with previous reports in that pc4-

LAIV enhances mucosal IgA response and provides viral clearance that is less dependent to serum antibody response in young chickens.

There were clear differences between the protective efficacy of pc4-LAIV and IV in 1-day-old chickens. It was surprising to see that IV could provide partial protection

(Fig. 3.4) without detectable levels of pre-challenge heterologous HI antibodies (Fig.

3.2B) considering that IV provides protection mainly via neutralizing serum antibodies

(305). It remains to be investigated why IV vaccination transiently provided an almost complete block of virus replication at 3 dpc (Fig. 3.4). Reduction of challenge virus by pc4-LAIV vaccination was apparent at 3 dpc and statistically significant at 5 dpc when most of the birds were completely protected (Fig. 3.4). Lau et al (306) also observed a similar protection trend with the cold-adapted live vaccine in mice challenged with a heterologous virus at 28 dpv. A significant reduction in challenge virus titer in lung was observed at 4 dpc, but not at 2 dpc (306). The differences between the protective efficacy of pc4-LAIV and IV in 1-day-old chickens may reflect differences in immunologic mechanisms. For the protection of young, immunologically immature, birds with a limited capacity to produce sufficient levels of serum antibodies, we believe that the pc4-

LAIV could be the better option. Additional optimization, such as using mucosal

83 adjuvant or modification of vaccination regimen and dosage, may further improve protective efficacy of pc4-LAIV in 1-day-old chickens.

One of the perceived risks of vaccinating commercial poultry against influenza is that the vaccine cannot fully prevent virus shedding and transmission to other birds. We have demonstrated that a single dose pc4-LAIV is able to induce stronger innate and mucosal IgA response, and protect young, immunologically immature, chickens than a single dose of conventional IV. The findings from this study demonstrated the distinctive features of each vaccine can provide supplemental, or even synergistic protective efficacy as part of a prime-boost vaccination.

Table 3. 1. Design of Study

Groups Age of vaccination Vaccination

1d pc4 1 day old pc4-LAIV Turkey/Oregon/71 H7N3

Inactivated Turkey/Oregon/71 H7N3 1d IV 1 day old + adjuvant

Unvaccinated - No treatment negative control The live vaccine, pc4-LAIV derived from Turkey/Oregon/71 (H7N3), was administered via intranasal route. Inactivated vaccine was prepared with the inactivated

Turkey/Oregon/71 virus mixed with adjuvant and the vaccination was done via subcutaneous route.

1 day post vaccination 3 days post vaccination 8 1 DPV Trachea

8 3 DPV Trachea

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Figure 3. 1. The ISG and IFN responses in 1-day-old chickens vaccinated with pc4-

LAIV or IV.

The bar represents the mean transcription level of ISGs and IFN genes. Significant difference compared with unvaccinated group are indicated with asterisks (*p<0.05,

**p<0.01, and *** p<0.001)

Homologous Heterologous 6 *** 6

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Figure 3. 2. Serum antibody response by 1-day-old vaccination.

HI antibody response to homologous (Turkey/Oregon/71, H7N3) and heterologous

(CK/NJ/150383-7/02, H7N2) antigens was measured with sera collected from 1-day-old vaccinated chickens at 14 days post vaccination. The individual and median HI titers are illustrated as symbols and horizontal lines, respectively. Significant difference compared with unvaccinated group were indicated with asterisks (**p<0.01, and *** p<0.001)

(A) Total antibody response (B) IgG response (C) IgA response

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Figure 3. 3. Tear antibody responses.

Tear samples were collected at 14 days post vaccination and total antibody against NP antigen, or influenza specific IgG and IgA were measured. Significant difference compared with unvaccinated group were indicated with asterisks (*p<0.05, **p<0.01, and *** p<0.001)

Figure 3. 4. Replication level of heterologous challenge virus.

One-day-old vaccinated chickens were challenged with heterologous virus

(CK/NJ/150383-7/02 H7N2) at 2 weeks post vaccination. At 3 and 5 days post challenge, tracheas were collected and the replication level of challenge virus was determined by matrix gene specific qRT-PCR. Significant difference compared with unvaccinated group were indicated with asterisks (*p<0.05, **p<0.01)

Chapter 4. Synergy of Live Attenuated and Inactivated Influenza Vaccines in a

Prime-Boost Regimen

4.1. Abstract

Since 2014, outbreaks of novel highly pathogenic avian influenza (HPAI) have been continuously reported in wild birds and poultry in the United States. This indicates that the current avian influenza (AI) control strategy based on biosecurity and inactivated vaccines (IV) is not adequate to combat the challenge of AI virus and more proactive measures are needed. Live attenuated influenza vaccine (LAIV) can be one of the alternative measures and provides numerous advantages, such as eliciting a broader range of immune response which covers humoral, cellular and mucosal immunity, ease of administration and broad reactivity to diverse strains. In previous studies, we showed that the pc4-LAIV, the non-structural protein 1 truncated variant, has potential to be a safe

LAIV candidate providing good heterologous protection. This study is an extension from the previous studies, focusing on optimization of the application strategy to maximize the potential use of the pc4-LAIV. The priming effect of pc4-LAIV vaccination at 1 day of age could synergistically enhance the efficacy of an IV vaccination at 3 weeks of age.

The chickens that received pc4-LAIV priming and IV boost showed the highest level of serum antibody production, broadened cross reactivity of the antibody, good mucosal antibody response and 100 % protection against heterologous challenge. Our study 90 demonstrated the potential application strategy of LAIV in a prime-boost regimen that can maximize the production of a broadly reactive protective immune response.

4.2. Introduction

Avian influenza (AI) is a major zoonotic viral disease that causes significant adverse impacts on poultry production, global trade market, and public health (4, 27, 70, 251).

Despite decades of research and control efforts, the incidences and severity of AI outbreaks have not been alleviated but rather increased (307, 308). The strategies currently being employed to control AI are focused on prevention of virus introduction by maintaining strict biosecurity procedures (41, 124, 257, 309). However, the current biosecurity systems have repeatedly failed to protect poultry farms from introduction of novel strains that continue to cause major outbreaks (310, 311). To overcome this challenge, some countries have incorporated vaccination with IV in their control strategies (41, 42). The United States has been using ‘stamping out’ as the primary control strategy but the occurrence of recent highly pathogenic avian influenza (HPAI) outbreaks (42, 312) further proves the limitations of biosecurity control and the country should consider adding vaccines to its AI control arsenal. The IV is currently in use in countries where HPAI viruses are endemic (124). Unfortunately, although IV can provide good protection from homologous field strains (42), it is weak against heterologous strains that arise from random mutations and not protective against heterosubtypic strains (131, 257, 313). In addition to having a narrow range of protection,

91 there are fear/concerns that long-term use of IV without eradication of heterologous

(mismatched) strains may result in the selection of the antigenically divergent strains.

Live vaccines have numerous advantages over IV that can be exploited further to develop new broadly protective vaccines and vaccination regimens. Since a live agent can mimic the natural infection, it can elicit a broad range of immune responses including humoral, cell-mediated and mucosal immunity (45, 60, 238). Importantly, live vaccine can be directly administrated on the mucosal surface by spray or through drinking water, which not only elicits local mucosal immunity but also significantly reduces the cost for mass administration (45, 314).

Currently, the only live influenza vaccines available for use in the poultry industry are live viral-vectored vaccines (41, 131). Live viral vaccine can induce a broader range of protection compared to IV, but they have several shortcomings including reduced efficacy due to preexisting immunity to the viral vector and the difficulty of expressing two or more influenza virus proteins in the same vector (97, 131,

315). Live-attenuated influenza vaccine (LAIV) is an excellent alternative to the vectored vaccine or IV since it contains all proteins that are naturally found in influenza virus particles (45, 157). In humans, LAIV has been used for more than a decade and it has been reported to protect young individuals better than IV (104, 105, 294). Importantly, recent studies showed that LAIV can pre-sensitize the population and, subsequently, synergistically boost the efficacy of IV (316, 317). It should be noted that the use of

LAIV in poultry requires strict safety standards due to concerns about the possibility of replication of wild type strains can replicate in domestic poultry, without apparent

92 clinical signs, and may undergo genetic reassortment with the vaccine virus and produce novel virulent strains (318, 319). Ideal poultry LAIV should not be able to revert to wild type virus or undergo reassortment with field strains.

In a previous chapter (chapter 3), we showed the promising aspect of pc4-LAIV to be used in poultry species in chickens, especially to induce better immune response from immunologically immature 1 day old chickens. However, single vaccination with pc4-

LAIV was not efficacious to provoke serum antibody response. IV vaccination has advantage over pc4-LAIV in eliciting serum antibody response, probably due to presence of proper adjuvant system. Considering the different characteristics of live and inactivated vaccines, we hypothesized that combined use of live and inactivated vaccines can supplement the limitations of each vaccine. Our previous findings addressed that pc4-

LAIV is good in eliciting innate type I IFN related responses while IV vaccination was more efficacious in provoking serum antibody response.

4.3. Materials and Method

Animals and ethics statement: All animals were maintained, vaccinated, challenged and euthanized in accordance with protocol #2009AG0002 approved by The Ohio State

University Institutional Animal Care and Use Committee (IACUC). This protocol complies with the U.S Animal Welfare Act, Guide for Care and Use of Laboratory

Animals and Public Health Service Policy on Humane Care and Use of Laboratory

25±3°C. Birds had ad libitum access to feed and water. The wellbeing and health status of the animals was monitored twice daily throughout the experiments. Animals were humanely euthanized when they displayed clinical signs such as ruffled feathers and reluctance to move, not moving when prodded, respiratory distress, or injuries that were not related to experimental treatment. Euthanasia was actualized by exposure to carbon dioxide (CO2). Based on the age and body size, 1–10 animals were placed in the euthanasia chamber connected to a CO2 source. The CO2 flow was set at 10–30% displacement of chamber volume/minute. Birds were observed for respiratory arrest and the CO2 flow was maintained for at least one minute after the arrest was observed. The animals were checked for an absence of breathing and lack of heartbeat. If any respiration or heartbeat was detected, the animal was placed back into the chamber and additional

CO2 was administered as described above. After death has been confirmed, an additional secondary physical euthanasia (cervical dislocation or removal of a vital organ) was performed before collection of tissues and carcass disposal.

Experimental design: Eight different vaccine regimens were tested as summarized in

Table 2. Three categories of vaccination regimens were investigated: unvaccinated, single dose (groups 1d pc4, 1d IV, 3w pc4, and 3w IV), and prime-boost (groups pc4-pc4, pc4-

IV, and IV-IV). The unvaccinated control group did not receive any treatment. The single vaccination groups were vaccinated only once either at 1 day (1d pc4 and 1d IV) or 3 weeks (3w pc4 and 3w IV) of age. The prime-boost groups (pc4-pc4, pc4-IV, and IV-IV) received the priming and boost vaccinations at 1 day and 3 weeks of age, respectively.

Vaccine administration was conducted as described in Chapter 3. The birds were bled at

4 and 5 weeks of age to measure post-vaccination serum antibody titers. Tears were collected at 5 weeks of age to measure post-vaccination mucosal antibody responses. All birds were challenged with A/chicken/NJ/150383-7/02 (H7N2) virus as described above.

The level of challenge virus replication was monitored through real time RT-PCR titration of tracheal swab samples collected at 2 and 4 dpc.

The HI test was conducted in accordance with the recommendation of World

95 room temperature to form antigen-antibody complexes. Then, 50 µl of 1 % turkey erythrocyte suspension was added in each well. The HI titer was reciprocally determined as the end-point dilution which showed complete inhibition of hemagglutination.

To determine cross-reactivity of serum with homologous and heterologous antigens, cross HI test was conducted on live or beta-propiolactone-inactivated virus

A/turkey/Oregon/71 (TK/OR/71) (H7N3) and A/chicken/NJ/150383-7/02 (H7N2) with cross-matching antisera produced by vaccination with inactivated antigen or live virus infection. Based on the cross HI titer, the percent antigenic relatedness was calculated as previously described by Archetti and Horsfall (320).

Mucosal antibody analysis: Mucosal antibody responses were measured using tear samples collected according to a previously described method with slight modification

(293). Briefly, chickens were comfortably held with their eyelids open and fine sodium chloride crystals (less than 0.01g) were sprinkled onto each eye. Once lachrymation was induced, tears were carefully harvested using a micropipette attached with a sterile tip and immediately placed in tubes. For consistency, and to prevent antibody dilution, only the first 50 µL of induced-tears was collected. The tear samples were stored at -20 °C until used for ELISA tests.

Three different ELISA kits were used to detect antibodies in tears. Influenza A nucleoprotein (NP)-specific antibodies were measured using a competitive ELISA kit

(Influenza A NP antibody inhibitor ELISA; Virusys Corporation, Sykesville, MD, USA) according to instructions provided by the manufacturer. The NP Reduction Index (NPRI)

96 value was calculated based on the formula: NPRI = (1- [Absorbance value (Abs) of samples – mean Abs value of diluent control]/[mean Abs of negative serum control- mean Abs value of diluent control]). Influenza virus-specific IgG response was measured by indirect ELISA kit (IDEXX AI Ab test; IDEXX Laboratories, Westbrook, ME, USA) in accordance with manufacturer’s instructions. Influenza virus-specific IgA response was measured using the same kit described for IgG except that the secondary antibody was replaced with 10000 fold diluted HRP-labeled Anti-Chicken IgA (α chain specific)

(Gallus Immunotech Inc., Fergus, Canada). The level of IgG was represented as sample to positive control ratio (S/P ratio) using the positive control samples provided by the manufacturer while raw OD values were used for the analysis of IgA levels.

Statistical analysis: Differences in virus titers among groups were determined by the

One-way ANOVA since we are comparing the difference in multiple groups based on one factor (GraphPad Software, San Diego, CA, USA) (327). After confirmation of significant difference among groups, post-hoc Tukey test used for the pair-wise comparison since we wanted to compare every mean with every other mean (GraphPad

Software) (327).

4.4. Results

In chapter 3, although the types and levels of adaptive immune responses induced in young birds appeared to depend on the type of vaccine used, neither of the two vaccines could provide full protection at 5 dpc. This study was designed to determine whether the

97 beneficial effects from each vaccine can be exploited to produce a more efficacious vaccination regimen. Evidence obtained from our studies suggests that age is a critical determinant of the level of protection provided by heterologous influenza vaccines in chickens. For examples, pc4-LAIV is more efficacious than IV in 1-day-old birds

(Chapter 3) whereas IV is more efficacious in older birds (291). In this study, our focus was on vaccination schedule consisting of priming with pc4-LAIV at 1 day of age and boosting with IV at 3 weeks of age (Table 4.1., pc4-IV). This regimen was compared with other prime-boost regimens (IV-IV, pc4-pc4) or single (non-prime-boost) vaccinations (1d pc4, 1d IV, 3w pc4, and 3w IV) in terms of antibody response and heterologous protection efficacy (Table 4.1.).

Serum antibody responses induced by different vaccination schedules: Figure 4.1 shows antibody responses at 4 and 5 weeks of age. Seroconversion of birds vaccinated at

1 day of age (1d IV and 1d pc4) was age-dependent and reached 100% for homologous viral antigen in 5-weeks-old chickens. However, the concentration of heterologous serum antibodies remained either low level or completely undetectable (Figs 4.1. A-D). In birds vaccinated at 3 weeks of age, there was a clear difference between single vaccination regimens of live and inactivated vaccines (3w pc4 versus 3w IV). All but one bird in the

3w pc4 group seroconverted to homologous viral antigen by 1-week post vaccination

(wpv) and the median titer reached 24 HI units (Fig 4.1. A). At the same time point, three out of eight birds in the 3w IV group failed to show homologous seroconversion and the median titer was 23 HI units (Fig 4.1. A). Homologous seroconversion was observed in

98 all birds in both groups at 2 wpv but the trend of median HI titer was reversed: the 3w IV group (29 HI units) had a higher titer than the 3w pc4 group (26 HI units) (Fig 4.1. B).

This result indicates that pc4-LAIV had the advantage of accelerating seroconversion whereas the IV vaccination induced a higher level of antibody responses in chickens vaccinated at 3 weeks of age. As described above, although single vaccinations could induce homologous seroconversion in all birds at 5 weeks of age (5 wpv for 1d IV and 1d pc4; 2 wpv for 3w pc4 and 3w IV), they did not induce 100% heterologous seroconversion. For the prime-boost regimen, the primary and boost vaccinations were administered at 1-day and 3 weeks of age, respectively (Table 4.1). At 1-week post-boost vaccination (wpb), both pc4-pc4 and pc4-IV groups showed higher seroconversion rates

(100%) for both homologous and heterologous strains compared to the IV-IV group (Fig.

4.1. A and C). The performance of the pc4-IV regimen was outstanding in terms of inducing the most rapid seroconversion and the highest serum antibody titers compared to the other groups (Fig. 4.1).

Cross-reactivity of serum antibodies with homologous and heterologous viral antigens:

We routinely use the A/TK/OR/71 (H7N3) virus to generate vaccine strains and

CK/NJ/150383-7/02 (H7N2) virus for the heterologous challenge strain (45, 291). Both viruses belong to North American H7 lineage, but they show low cross-reactivity to each other in cross HI test. On average, HI titers against heterologous antigen were about 20- fold lower than homologous titers when hyper-immune sera prepared by IV vaccination are used (Fig 4.1. B and D). Interestingly, we observed in this study that the difference of

HI titer between the vaccine and heterologous challenge strains was lower in sera from the groups primed with pc4-LAIV (pc4-pc4 and pc4-IV) (Fig. 4.2). For example, the HI titer difference between the two strains at 2 wpb was 22.2 HI units in pc4-pc4 group and

23.1 HI units in pc4- IV group whereas HI titer differences in other groups were 24 HI unit or higher, except 1d pc4 (22.3HI unites) (Fig. 4.2). Since the HI titer difference for the IV-

IV group was not significantly different compared with IV single vaccinations (1d IV, 3w

IV), we reasoned that pc4-LAIV priming was responsible for the broadened reactivity of the sera and thus results in smaller HI titer differences in pc4-IV and pc4-pc4 groups. To further demonstrate the difference in reactivity of the sera among different vaccine groups, we conducted a cross-HI test and calculated the percent antigenic relatedness (R

%) as previously described (320). Table 4.2 summarizes the antigenic relatedness in a pairwise representation of the sera. We found consistently higher R values in antisera produced by live A/chicken/NJ/150383-7/02 (H7N2) virus infection than vaccination with inactivated vaccine (Table 4.2). R values were highest in the pc4-IV and pc4-pc4 groups and low in IV-IV and single vaccination groups (Table 4.2).

Induction of mucosal influenza virus-specific antibody responses by different vaccine regimens

To compare the levels of mucosal antibody responses, we tested tear samples collected at

2 wpb vaccination with the three different ELISAs, as described above. Figure 4.3. A and

4.3. B show that all vaccinated groups had higher levels of total anti-NP and IgG antibody responses compared with the unvaccinated control group. However, the increase

100 in IgG antibodies in the 3w pc4 group was not statistically significant (Fig 4.3. B).

Figure 4.3. C shows that vaccination with pc4-LAIV, as a priming or booster vaccine, resulted in induction of significantly higher levels of IgA responses compared with the groups vaccinated with IV alone or the unvaccinated control group.

Overall, birds in the pc4-pc4 group showed consistently high mucosal antibody responses across all three ELISA tests (Fig. 4.3). The pc4-IV group also showed a good level of antibody response that was somewhat biased toward IgG response despite that the IgA response observed in this group was not significantly different from the LAIV vaccinated groups (pc4-pc4 and 3w pc4). The single vaccination regimens administered at 3 weeks of age (3w IV and 3w pc4) showed similar trend with the 1-day-old vaccination result, no differences in anti-NP antibodies (compare Fig 4.3. A with Fig 3.3.

A), the 3w pc4 group showed a higher IgA response, and the 3w IV group showed a higher IgG response (Compare Figs 4.3. B and C with Figs 3.3. B and C).

Heterologous protection efficacy of prime-boost regimen: From the data presented above, it is clear that pc4-LAIV can increase the breadth of serum antibody reactivity and induce higher mucosal IgA responses than IV. The prime-boost regimen using live and inactivated vaccines (pc4-IV) resulted in a synergistic effect that provided the highest serum antibody titer, an enhanced cross-reactivity of serum antibodies, and high levels of tear antibody responses. We further tested how those immune responses correlate with heterologous protection efficacy of each vaccine regimen. At 2 dpc, replication of the challenge virus was not high enough to enable comparison of the protective efficacies

101 among vaccine regimens. At 4 dpc, the pc4-IV, IV-IV and 3w IV regimens provided complete protection against heterologous challenge virus while the pc4-pc4 regimen was partially, but significantly, protective (Fig. 4.4). The reduction in challenge virus replication was not significant in the 1d pc4 and the 1d IV groups, but still, two out of eight birds in the 1d pc4 group were able to prevent the replication of challenge virus

(Fig. 4.4). Interestingly, the 3w pc4 regimen was significantly protective (Fig. 4.4) despite its inability to induce heterologous HI antibodies (Fig. 4.1D) and having low R values (Table 4.2).

4.5. Discussion

In Chapter 3, neither of the two vaccines could protect all birds at 5 dpc (Fig. 3.4), and each vaccine provided protection by different mechanisms. We reasoned that these vaccines could supplement or even synergize each other if used in a prime-boost regimen.

We have provided data to prove the advantage of this approach. When 1-day-old chickens were intranasally primed with pc4-LAIV and subcutaneously boosted with IV three weeks later (pc4-IV vaccination), they showed a rapid, robust, and highly cross- reactive serum antibody response (Figs. 4.1 and 4.2, Table 4.2) and a high level of mucosal IgA response (Fig. 4.2). The pc4-IV regimen was remarkably synergistic. For example, the mean HI titers induced by pc4-IV were at least 7 times higher than the combined mean HI titers of 1d pc4 and 3w pc4 groups combined (Fig. 4.1). This kind of synergy between live and inactivated vaccines was previously described in humans.

Talaat et al (316) found that the frequency and level of antibody response to inactivated

102

H5N1 vaccination was significantly higher in subjects who were previously primed with homologous H5N1 LAIV. After prime vaccination, there was no detectable serum antibody response but the immune system was apparently sensitized to rapidly respond to

IV vaccination and produce high titers of broadly cross-reactive serum HI antibodies

(316). More recently, Pitisuttithum et al (317) have confirmed induction of high serum

HI antibody titers by IV in LAIV-experienced individuals and went further to demonstrate that the antibody boosting effect correlated strongly with an increase in circulating follicular T-helper cells and plasma B cells. It appears that the long-lasting priming effect of LAIV has no species barrier.

Using the concept of antigenic relatedness based on cross-HI test (320, 321), we have demonstrated that priming with pc4-LAIV leads to induction of antibodies with enhanced cross-reactivity to heterologous virus independently of whether the boosting vaccine is live or inactivated (Table 4.2, compare pc4-pc4 and pc4-IV with the other groups). In addition, the antigenic relatedness was higher with serum produced by live

A/chicken/NJ/150383-7/02 (H7N2) virus infection relative to the hyper-immune serum produced by a matched IV (Table 4.2, compare the columns). Therefore, the antibodies produced by live viral infection seem to have stronger cross-reactivity to heterologous antigen. However, pc4-LAIV was unable to enhance serum antibody cross-reactivity boosting with a live or inactivated vaccine and we suspect that the reason is relatively weaker antibody response by single pc4-LAIV vaccination than live CK/NJ/150383-7/02

H7N2 viral infection or IV. Enhancement of serum antibody cross- reactivity by LAIV vaccination or live virus infection has been reported in previous studies. Jang et al (322)

103 reported that the pandemic 2009 H1N1 influenza virus vaccine could elicit cross-reactive antibody response to seasonal H1N1 and H5 strains. Hancock et al (323) found that persons 60 years or older possess cross-reactive serum antibodies to pandemic 2009

H1N1 virus, which was not observed in younger adults or children. The authors speculated that the cross-reactive antibodies in elderly individuals were a result of priming by natural infection with H1N1 virus followed by vaccination with swine-origin

A/NJ/76 H1N1 vaccine (323). While the mechanism involved in induction of cross- reactive antibodies remain to be investigated, our findings in this study and previous studies mentioned above suggest the potential of LAIV priming as a strategy that can be exploited to develop broadly effective influenza vaccination.

The mucosal-derived immune response is known to provide broad range of immune response (171). Secretory IgA (sIgA) is a major neutralizing antibody in local mucosal immune response, preventing pathogen entry and inhibiting intracellular replication of virus (324, 325). This study showed that priming pc4-LAIV vaccination could supplement the limited mucosal IgA response of IV vaccine. It directs the further verification of increased cross protection by pc4-IV regimen via enhanced mucosal immune response.

All three groups that received IV at 3 weeks of age (pc4-IV, IV-IV, and 3w IV) developed high levels of serum HI antibodies (Fig. 4.1) and fully blocked heterologous challenge virus replication at 4 dpc (Fig. 4.4). Thus, the effects of the immunological advantages of the pc4-IV regimen (Figs .4.1 - 4.3, Table 4.2) could not be discerned at the level of challenge virus replication because full protection was observed in all 3

104 groups (Fig. 3.3). However, the pc4-IV regimen may be the best option under field settings where circulating strains may be much more distantly related than the challenge virus used in this study.

Priming with pc4-LAIV led to a synergistic serum antibody induction by IV and enhancement of antibody cross-reactivity, thereby increasing the chance of protection from distantly related strains. Also, priming vaccination with pc4-LAIV could supplement limited mucosal IgA response by IV vaccination. The identification of underlying mechanism involved in synergy of our regimen will provide fundamental information for the development of broadly effective influenza vaccine.

105

Table 4. 1. Design of Study

1-day-old priming Groups 3-weeks-old boost vaccination vaccination 1d pc4 pc4-LAIV -

1d IV Inactivated vaccine -

3w pc4 - pc4-LAIV

3w IV - Inactivated vaccine

pc4-pc4 pc4-LAIV pc4-LAIV

pc4-IV pc4-LAIV Inactivated vaccine

IV-IV Inactivated vaccine Inactivated vaccine

Unvaccinated - - negative control The live vaccine, pc4-LAIV, derived from TK/OR/71 (H7N3) virus was administrated through intranasal route, while inactivated vaccine, a mixture of inactivated TK/OR/71 virus and adjuvant was administrated into subcutaneous area.

106

Table 4. 2. The effect of antisera production method on the percent antigenic relatedness value between TK/OR/71 (H7N3) and CK/NJ/150383-7/02 (H7N2) antigens

H7N2 Antisera produced by

live virus infection inactivated vaccination

Group 3w IV 54.64 37.80

Group 3w pc4 49.42 34.18

Group IV + IV 56.65 39.19

Group pc4+pc4 63.09 43.64 ntiseraproduced by Group pc4+IV 67.49 46.69

H7N3 A H7N3 Group 1d IV 55.86 38.36

Group 1d pc4 52.86 36.57 The cross HI test between vaccine strain (TK/OR/71, H7N3) and challenge strain

(CK/NJ/150383-7/02, H7N2) was conducted separately among antisera produced by live virus infection or inactivated vaccination. The cross HI test result was converted into percentage antigenic relatedness as previously described method (Percentage antigenic relatedness (R %) = 100 X √r1 X r2; r1 = cross HI titer of H7N2 antisera to H7N3 antigen / HI titer of H7N3 antisera to H7N3 antigen; r2 = cross HI titer of H7N3 antisera to H7N2 antigen / HI titer of H7N2 antisera to H7N2 antigen (320).

107

Figure 4. 1. Serum antibody response to homologous (A/turkey/Oregon/71 (H7N3)) and heterologous (A/chicken/NJ/150383-7/02 (H7N2))strains at 1 and 2 week post boost vaccination

The antibody response of each vaccine regimen was monitored at 1 and 2 weeks post boost vaccination. The individual and median HI titers are illustrated as symbols and horizontal lines, respectively. Different letters (A~E) indicate statistical significance among groups (p<0.05).

108

Homologous Heterologous

16 3.1

14 4.6 12 4.4 4.4 10 4.4 2.2 8

(HI TITER) (HI 2.3 2

6 LOG 4

0 1d pc4 3w pc4 pc4-pc4 pc4-IV IV-IV 3w IV 1d IV

Figure 4. 2. Differences in titer between homologous and heterologous strain

The bars and lines represent the average and standard deviation of

Log2(Hemagglutination inhibition titer at 2 weeks post boost). Numbers indicated above the bars and lines show average differences between homologous and heterologous HI titers in each vaccine regimen.

109

(A) Total antibody response (B) IgG response (C) IgA response

1 0 0 C

) 2 .0 d C BC 0 .3

B d

e BC

(

B a

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m ABC

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a 0 .1

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2 5 O 0 .5 A

n A

a a

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A A e

N M 0 0 .0 M 0 .0

4 4 4 d V V V V c 4 4 d V V V V 4 4 4 d V V V V c c 4 e I I -I I c c e I I I I c c c e I I -I I c t - p p p t - - p t - p p a d w V 4 - a d w V 4 - p p n d w 4 - p n 1 3 I c 4 n 1 c 4 1 V c 4 w i p c w d i 3 I c w d ia 3 I c d c 3 1 c p 3 1 c p 3 1 c p c p c p a a a v v v n n n u u u

Figure 4. 3. Mucosal antibody response

Tear samples were collected at 3 weeks post boost vaccination and measured for the influenza NP protein binding activity, or influenza specific IgG and IgA response. Significant difference compared among all groups (p<0.05) is indicated with different letters

(A~C).

110

4 d a y s p o st c h a lle n g e 2 d a y s p o st c h a lle n g e

) 8

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V V V 4 V d I I I c 4 4 I e - - p c c t V V V 4 4 4 V d - p p d I I I c c c I e 4 V w a + p t c I 3 4 d 1 n + p p d a p c w 1 i 4 V w + 1 p 3 c c I 3 4 w d in c p c 3 1 c a p c v a n v U n U

Figure 4. 4. Reduction in heterologous challenge virus replication in vaccinated birds

111

At 2 weeks post boost vaccination (equivalent to 5 weeks after 1-day priming vaccination), chickens were challenged with heterologous virus. The tracheal swab samples were collected to determine the replication level of challenge virus at 2 and 4 days post challenge. Significant difference compared among all groups (p<0.05) is indicated with different letters (A and B).

112

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