Development and Evaluation of Nanoparticle-Based Intranasal Inactivated

Development and Evaluation of Nanoparticle-based Intranasal Inactivated

Influenza Virus Vaccine Candidates in Pigs

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Santosh Dhakal, B.V.Sc.&A.H., MS

Graduate Program in Comparative and Veterinary Medicine

The Ohio State University

2018

Dissertation Committee:

Dr. Renukaradhya J. Gourapura, Advisor

Dr. Chang-Won Lee

Dr. Prosper N. Boyaka

Dr. Jordi B. Torrelles

Dr. Scott P. Kenney

Copyrighted by

Santosh Dhakal

2018

Abstract

Swine influenza A virus (SwIAV) causes severe economic loss to the swine industry globally. Pigs are also regarded as mixing vessel for influenza A viruses (IAVs) of human, avian and swine origin, generating viruses capable of human infections.

Vaccination is one of the effective means to prevent influenza in pigs. Currently available

SwIAV vaccines in pigs are predominantly monovalent or multivalent whole inactivated virus (WIV) vaccines administered by intramuscular (IM) route with potent adjuvants.

IM WIV vaccines provide homologous protection, but limited heterologous protection against continuously evolving field viruses, attributable to the induction of inadequate levels of mucosal IgA and cellular immune responses in the respiratory tract.

Additionally, IM WIV vaccines are not effective in the presence of maternally-derived antibodies (MDA) and often lead to vaccine-associated enhanced respiratory disease

(VAERD) when vaccine virus antigenically mismatches with challenge virus. Therefore, an alternative vaccine delivery approach is required to develop efficient SwIAV vaccine.

A novel vaccine delivery platform using biodegradable and biocompatible polymer-based nanoparticles (NPs) administered through intranasal (IN) route, has the potential to elicit strong mucosal and cellular immune responses in pigs and overcome the limitations of current IM WIV vaccines.

In this study, we developed poly(lactic-co-glycolic acid) (PLGA), polyanhydride, chitosan and Nano-11 NPs-based SwIAV vaccine candidates. Inactivated/killed SwIAV

H1N2 (δ-lineage) antigens (KAg) were either encapsulated within or adhered onto the surface of NPs. The vaccine candidates were administered twice IN as mist to nursery pigs. Vaccinates and controls were then challenged with a zoonotic and virulent heterologous SwIAV H1N1 (γ-lineage) via IN and intratracheal routes. None of the nanovaccines enhanced respiratory disease after virus infection. The IN PLGA-based

nanovaccine resulted in robust cross-reactive cell-mediated immunity, protected pigs from fever and cleared infectious virus from lungs but not from the nasal cavity, probably due to its inability to improve mucosal antibody response. Polyanhydride NPs-based influenza nanovaccine also enhanced cell-mediated immune response, protected pigs from fever and lowered infectious virus from nasal swabs 6 to 8 times. In another study, mucoadhesive chitosan NPs-based influenza nanovaccine exhibited an enhanced mucosal, humoral and cellular immune responses and lowered infectious challenge virus both from nasal cavity and lungs. We also showed the adjuvant potential of corn-derived

Nano-11 NPs in pigs after IN administration with influenza KAg which can be a safe, potent and cost-effective adjuvant for mucosal immunizations. In summary, diverse

immunogenic properties of these NPs can be used for improving the breadth of protective

efficacy of mucosal swine influenza vaccines. Future studies should explore the

combinatorial potential of these IN nanovaccine candidates, evaluate their ability to

override MDA effect and compare the vaccine efficacy with commercial vaccines against

SwIAV challenge with antigenic variant.

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Acknowledgments

I express my deep gratitude and appreciation to Dr. Renukaradhya J Gourapura

(Aradhya) for the continuous guidance, mentorship, encouragement and motivation during my five-year stay at The Ohio State University (OSU). I thank my committee

members Dr. Chang-Won Lee, Dr. Prosper N Boyaka, Dr. Jordi B Torrelles and Dr. Scott

P Kenney for their continuous and timely support, encouragement and constructive

criticisms.

I thank all the past and present members of Dr. Aradhya’s lab with whom I got

the opportunity to work together, including Rose Schleppi, Kathy Bondra, Jagadish

Hiremath, Basavaraj Binjawadagi, Kang Ouyang, DL (Bruce) Shyu, Sankar Renu,

Yashavanth SL, Shristi Ghimire, Christina Misch, Brad Hogshead and Jennifer Rank.

I appreciate Mrs. Hannah Gehman, Robin Weimer and Kathy Froilan for their

help in administrative issues. My sincere acknowledgements to Dr. Juliette Hanson,

Megan Strother, Sara Tallmadge, Ronna Wood and other staffs of animal care without

whom all these studies would have not been possible. My appreciation also goes to

summer students and students from Agricultural Technical Institute (ATI) including

Cecilia, Nino, Amanda, Kaitlyn, Payton, Taylor, Bridgette, Ally and Cody for their help

during my experiments. Acknowledgement also goes to lab members of Dr. Chang-Won

Lee, namely Drs. John Ngunjiri, Mohamed Elaish and Hyesun Jang for their help during

pig experiments.

I had a great opportunity to work with collaborators from different institutions:

Dr. Balaji Narasimhan and his group from Iowa State University; Dr. Harm HogenEsch and Fangjia Lu from Purdue University; Dr. Xingguo Cheng and his team from

SouthWest Research Institute, Texas; and Dr. Steven Krakowka from OSU. I owe my

deep respect and gratitude to all of them. I would also like to thank USDA-NIFA, The

Ohio State University, SouthWest Research Institute, I-Corps@Ohio program,

Nanovaccine Institute, Ames, Iowa and other agencies for their financial support for my

PhD projects. I thank Dr. Shauna Brummet for introducing us with I-Corps initiatives and

mentoring our projects.

My special thanks to the friends at Wooster who created homely environment

during my stay including Dr. Ajaya Shah, Sami Khanal, Bhupendra Acharya, Anita

Acharya, Mahesh KC, Dipak Kathayat, Ashish Shrestha, Isha Shrestha, Nawa Raj Baral,

Pratikshya Neupane, Vishal Srivastava and others.

I am always indebted to my parents, sisters, in-laws and other family members

who have always been my motivation and inspiration to do better even if we are far-away from each-other. At last but most importantly, immense respect and love to my dear wife

Shristi Ghimire, who is always there in my journey encouraging and supporting to reach my goal. Overall, it has been a wonderful expedition both personally and professionally,

filled with great learnings, and I am looking forward to make use of these for the

betterment of human and animal health in future.

Vita

2006 to 2011 ...... B.V.Sc.&A.H., Tribhuvan University, Nepal

2011 to May, 2013 ...... Veterinary Officer, National Zoonoses and

Food Hygiene Research Center, Nepal

June, 2013 to August, 2015 ...... M.S. Comparative and Veterinary Medicine

(CVM), The Ohio State University, USA

August, 2015 to present ...... PhD student, CVM, The Ohio State

University, USA

Publications

1. S. Dhakal, S. Renu, S. Ghimire, Y.S. Lakshmanappa, B.T. Hogshead, N.

Feliciano-Ruiz, F. Lu, H. HogenEsch, S. Krakowka, C.W. Lee, G.J.

Renukaradhya. 2018. Mucosal Immunity and Protective Efficacy of Intranasal

Inactivated Influenza Vaccine is Improved by Chitosan Nanoparticle Delivery in

Pigs. Frontiers in Immunology; 2018; 9: 934.

2. S. Renu, S. Dhakal, E. Kim, J. Goodman, Y.S. Lakshmanappa, M.J.

Wannemuehler, B. Narasimhan, P.N. Boyaka, G.J. Renukaradhya. 2018.

Intranasal Delivery of Influenza Antigen by Nanoparticles, but not NKT-cell

Adjuvant Differentially Induces the Expression of B-cell Activation Factors in

Mice and Swine. Cellular Immunology; 329:27-30.

3. S. Dhakal, J. Goodman, K. Bondra, Y.S. Lakshmanappa, J. Hiremath, D.L. Shyu,

K. Ouyang, K.I. Kang, S. Krakowka, M.J. Wannemuehler, C.W. Lee, B.

Narasimhan, G.J. Renukaradhya. 2017. Polyanhydride nanovaccine against swine

influenza virus in pigs. Vaccine; 35(8):1124-1131.

4. S. Dhakal, J. Hiremath, K. Bondra, Y.S. Lakshmanappa, D.L. Shyu, K. Ouyang,

K.I. Kang, B. Binjawadagi, J. Goodman, K. Tabynov, S. Krakowka, B.

Narasimhan, C.W. Lee, G.J. Renukaradhya. 2017. Biodegradable Nanoparticle

Delivery of Inactivated Swine Influenza Virus Vaccine Provides Heterologous

Cell-mediated Immune Response in Pigs. Journal of Controlled Release;

247:194-205.

5. M. Elaish, J.M. Ngunjiri, A. Ali, M. Xia, M. Ibrahim, H. Jang, J. Hiremath, S.

Dhakal, Y.A. Helmy, X. Jiang, G.J. Renukaradhya, C.W. Lee. 2017.

Supplementation of Inactivated Influenza Vaccine with Norovirus P particle-M2e

Chimeric Vaccine Enhances Protection against Heterologous Virus Challenge in

Chickens. PLoS ONE; 12(2): e0171174.

6. V. Dwivedi, C. Manickam, S. Dhakal, B. Binjawadagi, K. Ouyang, J. Hiremath,

M. Khatri, J.G. Hague, C.W. Lee, G.J. Renukaradhya. 2016. Adjuvant Effects of

Invariant NKT Cell Ligand Potentiates the Innate and Adaptive Immunity to an

Inactivated H1N1 Swine Influenza Virus Vaccine in Pigs. Veterinary

Microbiology; 186:157-163.

vii

7. B. Binjawadagi, Y.S. Lakshmanappa, Z. Longchao, S. Dhakal, J. Hiremath, K.

Ouyang, D.L. Shyu, J. Arcos, S. Pengcheng, A. gilbertie, F. Zuckermann, J.B.

Torrelles, D. Jackwood, Y. Fang, G.J. Renukaradhya. 2016. Development of a

Porcine Reproductive and Respiratory Syndrome Virus-like-particle-based

Vaccine and Evaluation of its Immunogenicity in Pigs. Archives of Virology;

161(6):1579-1589.

8. K. Ouyang, J. Hiremath, B. Binjawadagi, D.L. Shyu, S. Dhakal, J. Arcos, R.

Schleappi, L. Holman, M. Roof, J.B. Torrelles, G.J. Renukaradhya. 2016.

Comparative Analysis of Routes of Immunization of a Live Porcine Reproductive

and Respiratory Syndrome Virus (PRRSV) Vaccine in a Heterologous Virus

Challenge Study. Veterinary Research; 47:45.

9. K. Tabynov, A. Sansyzbay, Z. Tulemissova, K. Tabynov, S. Dhakal, A.

Samoltyrova, G.J. Renukaradhya, M. Mambetaliyev. 2016. Inactivated Porcine

Reproductive and Respiratory Syndrome Virus Vaccine Adjuvanted with

MontanideTM Gel 01 ST Elicits Virus-specific Cross-protective Inter-genotypic

Response in Piglets. Veterinary Microbiology; 192:81-89.

10. Y. Li, D.L. Shyu, P. Shang, J. Bai, K. Ouyang, S. Dhakal, J. Hiremath, B.

Binjawadagi, G.J. Renukaradhya, Y. Fang. 2016. Mutations in a Highly

Conserved Motif of nsp1β Protein Attenuate the Innate Immune Suppression

Function of Porcine Reproductive and Respiratory Syndrome Virus. Journal of

Virology; 90(7):3584-3599.

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11. J. Hiremath, K.I. Kang, M. Xia, M. Elaish, B. Binjawadagi, K. Ouyang, S.

Dhakal, J. Arcos, J.B. Torrelles, X. Jiang, C.W. Lee, G.J. Renukaradhya. 2016.

Entrapment of H1N1 Influenza Virus Derived Conserved Peptides in PLGA

Nanoparticles Enhances T cell Response and Vaccine Efficacy in Pigs. PLoS

ONE; 11(4): e0151922.

12. K. Ouyang, D.L. Shyu, S. Dhakal, J. Hiremath, B. Binjawadagi, Y.S.

Lakshmanappa, R. Guo, R. Ransburgh, K.M. Bondra, P. Gauger, J. Zhang, T.

Specht, A. Gilbertie, W. Minton, Y. Fang, G.J. Renukaradhya. 2015. Evaluation

of Humoral Immune Status in Porcine Epidemic Diarrhea Virus (PEDV) Infected

Sows under Field Conditions. Veterinary Research; 46:140.

13. S. Ghimire, S. Dhakal. 2015. Japanese Encephalitis: Challenges and Intervention

Opportunities in Nepal. Veterinary World; 8(1):61-65.

14. S. Dhakal, D.D. Joshi, A. Ale, M. Sharma, M. Dahal, Y. Shah, D.K. Pant, C.

Stephen. 2014. Regional Variation in Pig Farmers Awareness and Actions

Regarding Japanese Encephalitis in Nepal: Implications for Public Health

Education. PLoS ONE 9(1): e85399.

15. L. Ghimire, D.K. Singh, H.B. Basnet, R.K. Bhattarai, S. Dhakal, B. Sharma.

2014. Prevalence, Antibiogram and Risk Factors of Thermophilic Campylobacter

spp. in Dressed Porcine Carcass of Chitwan, Nepal. BMC Microbiology; 14:85.

16. S. Dhakal, C. Stephen, A. Ale, D.D. Joshi. 2012. Knowledge and Practices of Pig

Farmers Regarding Japanese Encephalitis in Kathmandu, Nepal. Zoonoses and

Public Health; 59(8):568-574.

Fields of Study

Major Field: Comparative and Veterinary Medicine

Immunology and Vaccine Development

Table of Contents Abstract ...... ii

Acknowledgments...... iv

Vita ...... vi

List of Tables ...... xv

List of Figures ...... xvi

Chapter 1: Influenza in pigs and potential use of biodegradable polymers in intranasal swine influenza vaccine development ...... 1

1.1 Introduction to influenza A virus (IAV) ...... 1

1.1.1 History and evolution of swine influenza A virus (SwIAV) ...... 3

1.1.2 Influenza disease in pigs ...... 6

1.1.3 Role of pigs in human influenza infection...... 9

1.1.4 Immune development after IAV infection in pigs ...... 11

1.1.5 Current swine influenza vaccines ...... 14

1.1.6 Alternative vaccine technologies against swine influenza ...... 18

1.2 Nanoparticles (NPs) in vaccine delivery ...... 20

1.2.1 Poly(lactic-co-glycolic acid) (PLGA) ...... 24

1.2.2 Polyanhydrides ...... 26

1.2.3 Chitosan ...... 27

1.2.4 Phytoglycogen ...... 30

1.3 Mucosal immunization through intranasal route ...... 31

Chapter 2: Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs ...... 39

2.1. Abstract ...... 39

2.2. Introduction ...... 40

2.3. Materials and methods...... 43

2.4 Results ...... 54

2.5 Discussions ...... 62

2.6 Conclusions ...... 68

2.7 Acknowledgements ...... 68

Chapter 3: Polyanhydride nanovaccine against swine influenza virus in pigs ...... 81

3.1 Abstract ...... 81

3.2 Introduction ...... 82

3.3 Materials and Methods ...... 84

3.4 Results ...... 88

3.5 Discussion ...... 92

xii

3.6 Acknowledgements ...... 95

Chapter 4: Mucosal immunity and protective efficacy of intranasal inactivated influenza

vaccine is improved by chitosan nanoparticle delivery in pigs ...... 102

4.1 Abstract ...... 102

4.2 Introduction ...... 103

4.3 Materials and methods...... 107

4.4 Results ...... 116

4.5 Discussion ...... 124

4.6 Ethics statement...... 130

4.7 Acknowledgements ...... 130

4.8 Funding ...... 131

4.9 Conflict of interest statement ...... 131

Chapter 5: Evaluation of corn-derived alpha-D-glucan nanoparticles as adjuvant for intramuscular and intranasal immunization in pigs ...... 143

5.1 Abstract ...... 143

5.2 Introduction ...... 144

5.3 Materials and methods...... 147

5.4 Results ...... 152

5.5 Discussions ...... 157

xiii

5.6 Conclusion ...... 162

Chapter 6: Summary and future directions ...... 175

Bibliography ...... 182

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List of Tables

Table 1.1 Protein(s) encoded by different genes of IAVs and their functions…………..38

Table 2.1 Experimental design showing assignments of pigs in each group………….....70

Table 2.2 Rectal temperature of pigs from DPC 0 to DPC 6………………………….....71

Table 2.3 Summary of pathological lung lesions scores and challenge virus titers……..72

Table 2.4 Humoral immune response in pigs pre and post-challenge…………………...73

Table 3.1: Experimental design showing assignment of pigs in each group………….....96

Table 4.1: Experimental design showing different vaccine groups…………………….132

Table 5.1 Adsorption efficiency and antigen release profile of Nano-11+KAg at different ratios…………………………………………………………………………………….164

Table 6.1 Summary of physical characteristics, immunogenicity and protective efficacies of different intranasal swine influenza nanovaccines in nursery pigs………………….181

List of Figures

Figure 1.1 Schematic diagram of influenza A virus showing surface glycoproteins and the gene segments……………………………………………………………………………37

Figure 2.1 In vitro physical characteristics of PLGA-KAg NPs and their role in maturation of APCs………………………………………………………………………74

Figure 2.2 Cellular and humoral immune responses in pigs pre-challenge……………...75

Figure 2.3 IN vaccine delivery device for pigs…………………………………………..76

Figure 2.4 Lung lesions in pigs at necropsy……………………………………………..77

Figure 2.5 Gating patterns of pig lymphocytes………………………………………...... 78

Figure 2.6 Lymphocytes recall response in vaccinated and challenged pigs..…………..79

Figure 2.7 IFNγ secretion and recall T cell response in PLGA-KAg vaccinated and virus

challenged pigs…………………………………………………………………………...80

Figure 3.1 Physical characterizations of polyanhydride nanoparticles…………………..97

Figure 3.2 Cellular and humoral immune responses in vaccinated pigs pre-challenge.....98

Figure 3.3 Clinical and pathological changes and SwIAV H1N1 titration in vaccinated

pigs post-challenge…………………………………………………………………….....99

Figure 3.4 Immunophenotyping of activated (IFN-γ+) lymphocytes in PBMCs post-

challenge………………………………………………………………………………..100

xvi

Figure 3.5 Humoral immune responses in vaccinated pigs post-challenge…………….101

Figure 4.1 In vitro characteristics of CNPs-KAg……………………………………….133

Figure 4.2 Cytokine responses in vitro………………………………………………....134

Figure 4.3 Antibody responses after prime-boost vaccination…………………………135

Figure 4.4 Expression of Th1 and Th2 transcription factors in PBMCs after prime-boost

vaccination……………………………………………………………………………...136

Figure 4.5 Mucosal IgA antibody responses in the respiratory tract at DPC 6....……...137

Figure 4.6 Serum IgG response and BAL fluid HI antibody titers in pigs at DPC 6…..138

Figure 4.7 Cell-mediated immune responses after prime-boost vaccination…………...139

Figure 4.8 Clinical and pathological changes in pigs post-challenge…………………..140

Figure 4.9 Cell-mediated immune responses in TBLN-MNCs at DPC 6……………....141

Figure 4.10 Infectious virus titers in the respiratory tract………………………………142

Figure 5.1 Physical characterizations of Nano-11 and Nano-11+KAg………………...165

Figure 5.2 Physical characterization of Nano-11 adsorbed with OVA at different ratio……………………………………………………………………………………..166

Figure 5.3 Size of Nano-11+KAg nanovaccine at Nano-11 and KAg ratios (A) 4:1 and

(B) 8:1 determined by DLS……………………………………………………………..167

Figure 5.4 Biological characterization of Nano-11 and Nano-11+KAg using porcine antigen presenting cells…………………………………………………………………168

Figure 5.5 Uptake of influenza antigen by porcine BAL cells (macrophages)………...169

Figure 5.6 Systemic IgG1 and IgG2 antibody responses in serum of pigs immunized twice IM with OVA or Nano-11+OVA………………………………………………...170

xvii

Figure 5.7 Expression of transcription factors involved in augmenting the adaptive immune response to Nano-11+KAg……………………………………………………171

Figure 5.8 Humoral immune responses in serum of pigs………………………………172

Figure 5.9 Virus specific cell-mediated immune responses in PBMCs at DPC 6……...173

Figure 5.10 Clinical and pathological changes and viral load in the respiratory tract …174

xviii

Chapter 1: Influenza in pigs and potential use of biodegradable polymers in

intranasal swine influenza vaccine development

1.1 Introduction to influenza A virus (IAV)

Influenza A virus (IAV) is a zoonotic pathogen that affects both humans and animals. Aquatic waterfowls and shorebirds are the major natural reservoirs of IAV, but it can infect a wide range of avian and mammalian species (1, 2). In birds, intestinal tract is the major site of replication for IAV while in mammals, it replicates mainly in the respiratory tract (3). IAVs is one of the major causes of human respiratory infections globally leading to deaths of over half a million people annually and substantial hospitalizations (3, 4). The symptoms of influenza virus infection in humans include fever, cough, sore throat, runny nose, muscle or body ache, headache, fatigue and occasional vomition and diarrhea (5).

IAV is 80-120nm sized, enveloped, negative-sense, single stranded RNA virus of

Orthomyxoviridae family (6). The genome of IAV has eight RNA segments that encode

10-11 proteins (Table 1.1) (6, 7). Hemagglutinin (HA) and neuraminidase (NA) are the two important envelope glycoproteins and the major antigenic determinants of IAV

(Figure 1.1). HA is the receptor binding and membrane fusion protein of IAV responsible for virus entry into the host cell (8). NA also promotes virus entry by mucous degradation

and mediates release of progeny virions by cleaving sialic acid residues from the

carbohydrate moieties on the cell surface (9-11). So far, 18 HA and 11 NA subtypes are

reported for IAV and the antigenic characteristics of these surface glycoproteins are used

to further subdivide members of IAV (12). Matrix protein 2 (M2) is the third envelope

glycoprotein of IAVs that acts as an ion channel for acidification of endosomes and leads

to uncoating and release of viral nucleoprotein into the host cell (13). The other proteins

of IAV include matrix protein 1 (M1), polymerase basic 1 (PB1) and 2 (PB2),

nucleoprotein (NP), polymerase acidic (PA), PB1-F2, nonstructural protein 1 (NS1) and nuclear export protein (NEP) which have their own distinct functions (Table 1.1).

IAV poses a constant threat of annual epidemics in humans, epizootics in animal

population and occasional human pandemics owing to the continued molecular evolution

and generation of new antigenic variants (14). It undergoes rapid evolutionary changes ranging from 1x10-3 to 8x10-3 substitutions per site per year and exists as quasispecies

attributed to the lower fidelity of RNA polymerase that cannot perform exonuclease

proofreading activity (15, 16). When mutations occur in the antigenic domains of HA and

NA glycoproteins, it is referred as ‘antigenic drift’ (17). Antigenic drift involves minor

changes and is the outcome of immune pressure exhibited by host to IAV (14). The

genetic diversity of IAV is also attributed by ‘antigenic shift’ which involves replacement

of entire HA and/or NA gene segment of the virus (14). Since IAV has segmented genome, reassortment of any gene segments is possible when different IAV infect same host cell and this event can confer cross-species transmission potential to IAV (17-19).

The high antigenic diversity of influenza virus is responsible for wide variation in

protective efficacy of influenza vaccines in humans where complete protection is assured

only when the vaccine virus strain matches with the virus strain that infected the host (20,

21). Thus, attempts are underway to develop influenza vaccines that account for the viral

antigenic diversity and provide protection against broad range of influenza viruses (22).

1.1.1 History and evolution of swine influenza A virus (SwIAV)

The first observation and documentation of influenza or ‘flu-like’ illness in pigs was made in 1918 from the swine herds of Iowa (23). The incidence of ‘flu-like’ respiratory illness in pigs coincided with the 1918 pandemic spread of human IAV that

killed 40 to 50 million people globally, suggesting that influenza virus circulated

simultaneously in humans and swine population (23, 24). In 1931, the causative agent of

‘swine flu’ or ‘hog flu’ was confirmed to be a ‘virus’ when a veterinarian, Robert Shope,

successfully reproduced the disease in healthy pigs after transferring the filtered secretions of sick pigs (25, 26). It was soon (in 1933) re-confirmed by Smith, Andrewes and Laidlaw when they reproduced influenza disease in ferret model after intranasal instillation of throat washings of human patients as well as swine influenza virus specimens provided by Shope (27). In fact, the diseases caused by human and swine influenza viruses in ferrets were indistinguishable and the viruses had close antigenic relatedness as shown by cross-immunity tests (27, 28). The virus, Shope isolated in

1930s, was the ancestor of what is now identified as ‘classical’ H1N1 (cH1N1) lineage of

SwIAV (29).

The cH1N1 influenza virus predominantly circulated and caused disease

outbreaks in North American swine population over 7 decades. Earlier, swine influenza

showed a predictable seasonal pattern with outbreaks recorded during late fall and early

winter months (14). During 1998 influenza season, several outbreaks of flu-like illness

were reported in swine population of North Carolina, Texas, Minnesota and Iowa. These

virus isolates were unique from the cH1N1 in pigs as they acquired components of

human H3N2 IAV. The isolate from North Carolina had HA, NA and PB1 gene segments

derived from human H3N2 IAV and other segments of swine cH1N1 IAV (double

reassortant virus) while isolates from other states additionally acquired PA and PB2 gene

segments from avian IAV (triple reassortant virus) (30). Within a year, triple reassortant

H3N2 SwIAV were widespread in the US swine population (31). In November 1999, shortly after the emergence and spread of triple reassortant H3N2 SwIAV, an H1N2 subtype of influenza virus was found to be associated with respiratory illness and abortion cases in swine farms of Indiana. This virus was the result of reassortment between cH1N1 and triple reassortant H3N2 SwIAV (32). The introduction of reassortant

H3N2 changed the complexity of SwIAV in the US swine population where cH1N1 was also replaced by reassortant H1N1 (rH1N1) virus that contained triple reassortant genes

(14, 33).

In general, SwIAV endemic in the US swine population from the beginning of

21st century belong to rH1N1, H1N2 and H3N2 subtypes that carry similar triple

reassortant internal genes (TRIG) cassettes. TRIG includes avian origin PA and PB2;

classical swine virus origin NS, NP and M; and human origin PB1 gene segments. The

TRIG bearing viruses are more liable to antigenic drift and shift compared to cH1N1

SwIAV making them able to escape the established herd immunity (14). The HA, NA or

both genes of these viruses are often derived from seasonal human IAV (34, 35). After

the introduction of pandemic H1N1, 2009 (H1N1pdm09) virus in the US swine

population via human to pig transmission, multiple reassortments occurred among

H1N1pdm09 and endemic SwIAV (36). Besides these striking reassortment events, antigenic diversity of SwIAV is also contributed by the mutations in virus surface proteins (antigenic drift) accumulated over time (37).

To address the expanding diversity of SwIAV in North American swine population, phylogenetic ‘cluster’ terminology was introduced and at least 10 phylogenetically separate HA clusters, 6 for H1 and 4 for H3, are identified cocirculating in North American swine population. The H1 clusters include H1α, H1β, H1γ,

H1δ1, H1δ2 and H1pdm09; while H3 clusters include H3I, H3II, H3III and H3IV. The

H3 IV cluster is further subdivided into 6 clades from A to F. The δ clusters of H1

SwIAV bear HA similar to human seasonal H1 IAV while α, β and γ clusters have HA derived from cH1N1 SwIAV (38). Members of SwIAV are continuously evolving as evidenced by the identification of two new genetic clades H1-δ1a and H1-δ1b in recent years. Such genetic and antigenic evolutions of contemporary SwIAV demand for updates in swine influenza vaccines and development of a vaccine that can provide better cross-protection against multiple viruses (39, 40).

1.1.2 Influenza disease in pigs

Influenza is an acute, contagious, respiratory infection of pigs characterized by

clinical signs such as sudden onset of fever, loss of appetite, jerky or thumpy respiration,

coughing, watery or gummy ocular discharge and nasal secretions (41-43). SwIAV is transmitted through direct contact of susceptible pigs with nasal secretions of infected pigs and through inhalation of aerosolized virus in droplets (41, 42). Historically, influenza infections in pigs showed seasonal pattern of occurrence which still persists as greater peaks of infections observed commonly in fall and smaller peaks in spring. The weather conditions of winter favors the survival and aerosol transmission of the virus

(42). However, with emergence of novel viruses, changes in farming pattern and availability of susceptible pigs, influenza now is considered to be a year-round disease

(41, 44, 45). The incubation period for influenza is very short and the infected pigs develop high fever (>1050F, 40.50C), anorexia and lethargy within 1 to 2 days of

infection (42). Fever persists for 3 to 4 days in infected pigs and by this time deep barking cough can be observable as a result of extensive bronchitis and bronchiolitis (42).

In the absence of secondary viral or bacterial infections, pigs can recover from influenza

within a week period (42). It can also lead to abortions in naïve pregnant sows or gilts

and the cases are more commonly associated with SwIAV H3N2 subtype (46). Influenza

is a herd disease as the morbidity rate is up to 100% but mortality rate is quite less (<5%)

(42, 43). In experimental challenge studies, fever and other clinical signs are more

6 apparent if pigs are challenged with higher dose of the virus (>10 TCID50) and using

intratracheal route compared to lower dose and intranasal inoculations (42, 43). Viruses

can be detected in nasal shedding of infected pigs from 1 to 7 days post-infection.

Bronchoalveolar lavage (BAL) fluids and lung homogenate/lysate are also used in experimental studies to compare the virus burden in lungs. IAV can also be detected in porcine oral fluid samples (47). The pig is an excellent model for IAV infection and vaccine studies because humans and pigs have similar receptor distribution patterns in respiratory tract epithelial cells, the same influenza subtypes are endemic in both populations, virus transmission between pigs and humans is frequently reported, and similarities exist in pathogenesis and disease pattern (48, 49).

SwIAV infects the epithelial lining of entire respiratory tracts starting from nasal passages to alveoli (42). Infection of porcine respiratory epithelium with influenza virus and its multiplication is rapid. Infection of bronchial epithelial cells of pigs is observed within 2h of infection while budding of pleomorphic virus particles from alveolar epithelium begins as early as 5h post-infection (50, 51). The peak infection of bronchi and bronchioles in experimental studies is observed after 2-3 days of infection, while it occurs in alveolar epithelium at 3-4 days of infection (51, 52). Influenza virus causes necrosis and sloughing of alveolar epithelium within 24 to 96h post-infection in pigs leading to alveolar edema and interstitial pneumonia (43). Macroscopically, influenza infection in pigs is characterized by cranioventral pneumonia where medium to large sized dark red consolidated lesions are observed in variable portions of the lungs (42, 53).

Airways are often filled with blood-tinged foams and lymph nodes associated to respiratory tract (e.g. trachea-bronchial lymph node) are often enlarged and hyperemic

(46). Microscopically different lesions can be observed such as – (i) atelectasis due to

7 collapse of pulmonary parenchyma; (ii) interstitial pneumonia due to swelling and thickening of alveolar capillary walls, extravascular fluid and protein accumulation and infiltration with vascular-origin leukocytes, and resultant alveolar airway collapse; (iii) peri-bronchial and peri-vascular accumulation of mononuclear inflammatory cells; (iv) bronchial exudates composed of dead sloughed epithelial cells and mononuclear inflammatory cells; and (v) visible changes to bronchial epithelium such as variable swelling, sloughing and necrosis together with formation of syncytium (42, 43, 46, 53-

55).

Influenza causes substantial economic burden to pig farms especially when the infection rate are high (42, 56). The economic losses are mainly associated with loss of body weight gain, increased time to market and medication and veterinary expenses.

Influenza infection also increases susceptibility of pigs to secondary bacterial infections such as Mycoplasma hyopneumoniae or viral infections such as porcine reproductive and respiratory syndrome virus (PRRSV). Such complicated cases of influenza further increase the economic loss in affected farms (57-59). Co-infection of SwIAV with

PRRSV, porcine circovirus 2 (PCV 2) and other bacteria constitute the porcine respiratory disease complex (PRDC), a serious health and economic challenge in the swine industry (58, 60, 61). Influenza can be managed in pig farms through vaccination of pigs and pig caretakers with seasonal influenza vaccines to prevent spillover of virus from humans to pigs, using strict biosecurity measures in farms, practicing good hygiene, and using proper ventilation systems (62). Influenza is basically introduced in the pig populations during and after movement of the animals and hence growing sows, nursery

pigs and grower-finisher pigs in a segregated rearing system through an all-in all-out

approach can minimize the risk of virus transmission from older immune pigs to younger

newly introduced naïve pigs (42).

1.1.3 Role of pigs in human influenza infection

Influenza viruses of swine origin can cause sporadic human infections. Antibodies

against SwIAV were first demonstrated in humans in 1958 when a female laboratory worker in Czechoslovakia was exposed to swine. Epidemiological evidences suggested

that, virus was subsequently transmitted to other family members who did not have swine

exposure (63, 64). The first isolation of SwIAV from infected humans, however, was

done only in 1974 when a 16-year-old farm boy died with extensive infiltration and consolidation of both lungs (65). Until 2006, 50 cases of apparent zoonotic SwIAV

infection were reported throughout the globe with case fatality rate of 14% (64). The first triple reassortant (tr) H1N1 SwIAV was isolated in the US from a 17-year-old boy in

2005 and more cases have since been reported to the Center for Disease Control (CDC)

(66, 67). Till mid-May 2018, 468 people in the United States have been infected with various types of IAV that are genetically similar to viruses circulating in swine, known as variant (v) viruses. Out of the reported 468 cases; 434, 21 and 13 cases are caused by vH3N2, vH1N1 and vH1N2 IAV, respectively, and all of them are ‘tr’ viruses from pigs

(62).

Moreover, pigs are regarded as the ‘mixing vessel’ for avian, human, and swine origin IAV. This concept was originated in the 1980s following genetic and biological

observations that pigs can be infected experimentally with different avian and human

origin IAV (18, 68-70). Sialyloligosaccharides (SA) serve as the host cell surface

receptors for IAV where avian and human viruses prefer binding respectively to N- acetylneuraminic acid-α2,3-galactose (SAα2,3Gal) and N-acetylneuraminic acid-α2,6-

galactose (SAα2,6Gal) linkage (71, 72). Molecular evidences suggest that the trachea of

pigs contain both SAα2,3Gal and SAα2,6Gal receptors making them susceptible to

infection with avian as well as human origin IAV (72). Further, the shift in receptor

specificity of avian origin viruses over time from SAα2,3Gal to SAα2,6Gal was also

observed in pigs which suggests the likely mechanisms of adaptation of avian viruses in

pigs before transmission to humans (73). Recent studies have shown that receptors for avian viruses (SAα2,3Gal) are predominantly present in bronchi and alveoli of pigs, while all other parts of the respiratory tract are dominated by SAα2,3Gal receptors that is preferred by human and swine IAVs (74). Since pigs are susceptible to infection with

IAV of human, swine and avian origins, they can act as the intermediate host for genetic reassortment (antigenic shift) of different IAV producing novel viruses of epidemic and pandemic potential (75, 76). The genetic reassortments between avian and human IAV in

European pigs were reported in the 1990s through phylogenetic analyses (75, 77). The resultant reassortant viruses were later isolated from humans in different parts of the world which indicated that pigs can produce virus with potential of infection for other mammalian species (64, 78). The 2009 influenza pandemic is the most striking example of multiple reassortments of IAV in pig population and generation of virus with cross-

species transmission potential (79, 80). This virus was generated by reassortments of

North American triple reassortant and avian-like Eurasian lineages of swine influenza viruses, and lead to over 200,000 deaths globally (19, 81). The economic burden of influenza in the pig industry and the associated risk of human infections highlight the need of increased influenza surveillance in pigs and development of efficient vaccine technology.

1.1.4 Immune development after IAV infection in pigs

Once IAV infects the respiratory epithelial cells, cellular pattern recognition receptors (PRRs) will sense the influenza viral RNA that serves as the pathogen associated molecular pattern (PAMP). Toll-like receptors (TLRs) 3 and 7, retinoic acid inducible gene –I (RIG-I), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are the major PRRs involved in innate sensing of IAVs (82, 83) . RIG-I recognizes viral RNA within the virus infected cells and NLRs are activated by M2 ion channel activity, while TLRs are activated when virus-infected cells are taken up and processed by macrophages or dendritic cells (83). Activation of PRRs leads to induction of expression of nuclear-factor-κB (NF-κB) and activation of caspase-1 resulting in synthesis of pro-inflammatory cytokines and type-I interferons (83, 84). These innate cytokines on one hand exert antiviral activity and limit virus replication and on the other hand stimulate dendritic cells (DCs) and contribute to adaptive immune response development (83, 84). In vitro stimulation of porcine alveolar macrophages with H3N2

SwIAV increased expression of TLRs 3 and 7 and RIG-I and in turn increased expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β mRNAs (85). Consistent to that,

11 induction of pro-inflammatory cytokines and interferons were observed in pigs experimentally infected with IAV (86). Interferon (IFN)-α, TNF-α, and IL-1 cytokines in

H1N1 IAV infected pigs were upregulated and positively correlated with disease progression and virus titers in pigs (87). Follow-up study indicated that cytokines IFN-α,

TNF-α and IL-6 but not IL-1 were positively correlated with virus titers in pigs (88).

Besides the cytokines, higher levels of acute phage proteins such as C-reactive proteins

(CRPs) and haptoglobin (Hp) were also observed after H1N1 virus infection in pigs (89,

90). Infection with H1N2 subtypes of IAVs also induced synthesis of innate cytokines in pigs where TNF-α was positively correlated with body temperature and lung lesions (91,

92).

Humoral immune response is efficiently induced after influenza infection in pigs and is shown to provide protective efficacy against re-infection with the same virus (93).

All three major immunoglobulins; IgM, IgG and IgA develop within 1 to 2 week post- infection both at the respiratory tract and in serum (94). Infection of pigs with H3N2

SwIAV through nebulization induced peak IgM antibody titer by 7 day post-infection

(dpi) while IgA and IgG antibodies peaked at 15 dpi. Virus specific activities of all three antibodies were much higher in respiratory mucosa than in serum. Once reached the peak, mucosal IgA remained static until the end of the experiment (44 dpi) while serum

IgA rapidly declined after 2 weeks (94). Similarly, when influenza seronegative pigs were infected with H1N1 SwIAV via intranasal route, virus specific IgG and IgA antibody titers peaked in 2-3 weeks post-infection. As in the experiment with H3N2

SwIAV, IgG was the predominant antibody isotype in serum while IgA was predominant

both in upper and lower respiratory tracts (95). IgM, IgG and IgA antibodies control

influenza virus infection and replication through (i) activation of complement fixation

pathway; (ii) enhancing the phagocytosis by polymorphonuclear cells; (iii) activation of antibody mediated cellular cytotoxicity; and (iv) neutralization of infectious virus

particles (84, 96-98). In pigs infected with IAVs, hemagglutination inhibition (HI)

antibodies were detected within the first week of infection and peaked after 2 to 3 weeks

(95, 99). HI titers are considered as the gold standard of immunogenicity and protective

efficacy of traditional influenza vaccines as these antibodies can successfully neutralize

IAVs (84). Antibodies induced after infection or vaccination and reactive with HA, NA

or M2 proteins have shown the ability to cross-react and protect against heterologous

influenza virus infections, indicating that they can be the target for developing more

efficient influenza vaccines (100-105). Several attempts are ongoing to develop universal

influenza vaccines using highly conserved epitopes of the major surface glycoproteins of

IAVs (106-109). The experimental studies showed that antibodies were locally produced

in the respiratory mucosa of pigs after IAVs infections and hence vaccines which activate

immune cells at respiratory mucosa and produce mucosal antibody response can provide

better protective efficacy in pigs against IAVs (94, 95).

Frequencies of T-helper (CD4+) cells, cytotoxic T (CD8+) lymphocytes (CTLs)

cells and regulatory T (Tregs) cells are expanded in lungs and tracheobronchial lymph

node (TBLN) of pigs within 6 days of infection with a triple reassortant and zoonotic

H1N1 SwIAV (48). Increased frequencies of CD4+ and CD8+ T cells after H1N1 subtype of IAV infection were also observed in pigs in other studies (93, 110). In a recent study,

H1N2 subtype of SwIAV elicited virus specific CD4+ and CD8+ T cells accumulation in

lungs from 4 dpi which secreted IFNγ and TNF-α. (111). Such an accumulation of

multifunctional and cross-reactive T cells in the lungs indicated their potential to combat

heterologous influenza virus infection in pigs (111). CD4+ T cells mediate antiviral immunity by stimulating the production of virus specific antibodies and supporting proliferation of CTLs (112-114). CTLs mediate virus clearance by eliminating virus- infected cells through release of perforins and granzymes which lyse infected cells.

Moreover, CTLs are known to provide broad range of protection against heterosubtypic

IAV (93, 115-118). Vaccines that efficiently activate cross-reactive T cell response can provide better protective efficacy in pigs and reduce pig to human transmission and genetic reassortments of different IAVs (111).

1.1.5 Current swine influenza vaccines

Vaccination is one of the important means to prevent SwIAV related losses in pigs and humans. Vaccines against SwIAV became available in the US since the 1990s and are being used at variable rates. Vaccination is commonly practiced by large producers in breeding herds (>70% vaccinated) compared to weaned piglets (~20% vaccinated) (14). Commercial vaccines in the US against swine influenza are

predominantly whole inactivated virus (WIV) vaccines (7, 119). However, since 2012,

alphavirus replicon-based influenza virus hemagglutinin vaccine against H3N2 SwIAV is

also licensed by the United States Department of Agriculture (USDA) (120, 121). USDA

also licensed a modified-live virus (MLV) vaccine in 2017 (122).

The conventional WIV vaccines are prepared by virus propagation in eggs or

mammalian cells followed by inactivation with chemicals such as formaldehyde or binary

ethylinemine (BEI) (7). Autogenous or custom-made vaccines are the most commonly

practiced (~100% of grower-finisher vaccines, 50% overall) vaccination strategy in the

US swine industry, wherein virus is isolated from the field cases, propagated, inactivated and used in the same herd (7, 119, 123). Regulatory approvals of an inactivated virus vaccine by USDA require successful demonstration of safety and efficacy against identical or heterologous challenge virus strain. The autologous vaccines should also be tested for proper virus inactivation, safety in laboratory animals, and purity but potency and efficacy tests are not mandatory (7). In order to address the growing genetic diversity

of contemporary SwIAV, since 2007, USDA allowed vaccine manufacturers with current

licensing to update the strains included in their vaccine formulations to match the field strains (119).

Commercial WIV vaccines in the US contain single or multiple strains of IAVs combined in their formulation. For example, ‘FLUSURE PANDEMIC’ vaccine marketed by Zoetis contains single strain of pandemic H1N1 while ‘FLUSURE XP’ of the same manufacturer contains δ1-H1N2, γ-H1N1, cluster IV-A H3N2 and cluster IV-B H3N2

SwIAVs in the vaccine formulation (7, 124). The current SwIAV WIV vaccines are administered with potent oil-in-water adjuvant through IM route. The primary aim of the adjuvanted WIV vaccines is to induce robust antibody response which can neutralize the infecting virus (7, 119). Some of the commercial WIV vaccines are also available in combination with antigens of other pathogens where the manufacturer needs to

demonstrate that the combination of antigens does not affect the safety and efficacy of

each other (7).

The WIV vaccines provide protective efficacy against identical or closely related

SwIAV, while protection against unrelated heterologous viruses is incomplete (125-128).

Even if multiple strains are included in vaccine formulations, the protective effect is not always guaranteed in the field scenario especially because the contemporary SwIAV are very diverse and continuously evolving antigenically (119, 129, 130). The long-existing approach of immunization in pigs against influenza is to vaccinate breeding sows so as to transfer passive immunity to newborn piglets via colostrum (7, 119). Though, maternal antibodies can provide certain level of protection during natural infections, they are also shown to mask or block efficient immune development after natural infection or vaccination in young piglets raising a concern over the effectiveness of this approach

(131-135). Both B and T cell development is affected by pre-existing maternal antibodies in young piglets (135, 136). Maternally-derived antibodies (MDAs) are even in some instances, shown to backfire by enhancing the respiratory disease development in pigs

(137, 138). Due to this fact, the commercial WIV vaccine manufacturers recommend farmers to use vaccines once the pre-existing MDAs decay in the piglets (124). This is one of the factors responsible for less number of finisher piglets (~20%) being immunized by large producers compared to vaccination of high number of breeding sows

(>70%) (119).

Vaccine associated enhanced respiratory disease (VAERD) is another important concern associated with adjuvanted WIV vaccines administered by IM route, wherein

pigs develop more pronounced disease compared to unvaccinated controls after a

heterologous virus challenge (127, 137, 139). VAERD is often encountered when the

vaccine virus and challenge virus are antigenically divergent but still belong to the same

subtype (139, 140). VAERD is most likely governed by multiple factors. The most

consistent factor is induction of excessive cross-reactive but non-neutralizing IgG antibodies observed in VAERD studies. These observation indicate that such antibodies might activate inflammatory immune mechanisms leading to enhanced tissue damage instead of virus clearance (127, 139, 141, 142).

Furthermore, commercial killed IM vaccines do not induce adequate mucosal antibody response and also fail to efficiently activate virus-specific CTLs response in pigs (130). Induction of a robust mucosal immune response is directly associated with increased breadth of protective response against IAVs (143, 144). Studies have shown that, IgA producing B cells and local production of IgA in the respiratory tract is possible during natural intranasal infection of pigs, but not through IM WIV vaccination (145,

146). Besides that, killed IM vaccines produce antibodies primarily in serum which needs to be transported to respiratory mucosa through transudation to neutralize virus during infection (130). Such antibody transudation is observed to be more efficient in lower respiratory surfaces (lungs) than in nasal mucosa (147). Thus, it is likely that antibodies induced by IM WIV vaccine after transudation into respiratory tract will prevent virus replication in lungs but not in upper respiratory mucosa, which is necessary for prevention of disease establishment and transmission of IAVs to susceptible hosts (130).

Regarding the cell-mediated immunity, the killed antigens are processed and presented

through endogenous major histocompatibility complex (MHC) II mediated pathway of

antigen processing. This pathway induces primarily the T-helper cell activity and poorly the CTL response (130).

In general, the shortcomings associated with current IM WIV vaccines are as follows – (i) they provide narrow spectrum of protection; (ii) they do not induce adequate mucosal antibodies and CTL responses; (iii) the efficacy of current vaccines is compromised by MDAs; and (iv) these vaccines can even enhance the respiratory disease

(7, 119, 130, 148). Thus, novel approaches are needed to develop vaccines which can overcome these limitations to provide better protective efficacy against a multitude of

IAV likely to be generated through continuous antigenic evolution of SwIAV.

1.1.6 Alternative vaccine technologies against swine influenza

Different alternative techniques are being considered experimentally to develop better SwIAV vaccines including vectored vaccines, modified-live or live-attenuated vaccines and DNA vaccines (7). Alphavirus replicon particles (RPs) based vaccine carrying HA of cluster IV-H3N2 SwIAV is the first of its kind licensed by the USDA (7,

119). RPs is generated by deletion of structural genes of attenuated equine encephalitis virus (149). It confers adjuvant potential, induces robust HI antibodies in serum and protects against homologous virus challenge (120, 150-152). However, it was not able to overcome maternal antibody interference (150). A replication-defective human adenovirus 5 vector (Ad5) carrying the HA and NP genes of IAV is another vector based approach to develop swine influenza vaccine. IM priming of pigs with Ad5 based vaccine

was able to develop active HI antibody response and override the effects of MDA (153).

Likewise, IN immunization of pigs with Ad5-vectored vaccine provided homologous and

partial heterologous protection while overcoming the VAERD associated with IM WIV

vaccines (140).

Live-attenuated influenza virus (LAIV) or modified-live virus (MLV) vaccines in

swine have also demonstrated great promise of improved protective efficacies under

experimental conditions compared to inactivated vaccines (154-156). The three types of

LAIV/MLV used in swine influenza experiments include – (I) LAIV with elastase

dependent HA (eHA); (II) LAIV with NS1 truncation (ΔNS1); and (III) temperature-

sensitive PB2 and PB1 mutants with HA tagged to PB1 (tsHAtag) (119). LAIV vaccines

administered via the IN route can induce both mucosal and cell-mediated immune

responses in pigs (154, 156-158). These vaccines are shown to be effective against heterologous virus challenge and T cell-mediated immune response was likely to provide a protective response in pigs (154, 159). Improved antibody and cell-mediated immunity and better protection were also observed in another study where bivalent LAIV vaccine was administered via IN route in pigs (156). Importantly, IN LAIV vaccines have the potential to override the effects of maternal antibodies and also do not induce VAERD as found to occur with inactivated IM vaccines (137, 159). Overall, LAIV vaccines have the

promise of providing cross-protective efficacy by inducing mucosal and cellular

immunity, overcoming MDA interference and avoiding VAERD. However, the

regulatory concerns of safety and environmental impact should be taken under

19 considerations before successful use of these vaccines in the field as there will be possibility of reassortment of LAIV with wild type SwIAV (119).

DNA vaccines carrying single or multiple different genes of IAVs induced cross- reactive humoral and cell mediated immune response in pigs after needle-free intradermal immunization (160-162). Such influenza DNA vaccines provided protective efficacy in pigs against pandemic and classical H1N1 influenza virus infections (160, 162, 163).

However, despite the beneficial properties observed with influenza DNA vaccines, their commercial viability in swine industry is questionable because materials and labor costs associated with these vaccines are very high (7).

1.2 Nanoparticles (NPs) in vaccine delivery

Nanoparticles (NPs)-based or nano-scale vaccines (nanovaccines) have received increased attention in recent years in vaccine delivery studies as they provide several advantages over soluble antigens (164). Nanovaccines are prepared either by encapsulating the antigens within the NPs or decorating them on the surface (165). They protect encapsulated antigens from enzymatic and hydrolytic degradation. For example, when insulin loaded nanocapsules (<300nm) were orally administered in diabetic rats, they were protected from enzymatic degradation, transported efficiently through intestinal epithelium and successfully produced their effect as evidenced by marked reduction in glycemia from second day of administration to over two weeks period (166).

Encapsulation in NPs also increases the bioavailability of the encapsulated cargo which would otherwise be cleared from the system within a short period (167). The controlled,

slow and sustained release of the antigens by NPs also helps in induction of better

immune response compared to ‘fast released’ or soluble antigens (168).

The size of the particles play an important role in the antigen uptake by antigen presenting cells (APCs) and downstream events of immune activation (169). Particulate antigens are internalized by cells more efficiently in comparison to soluble antigens as shown in in vitro study of poly(lactic-co-glycolic acid) (PLGA) encapsulated PRRSV antigen uptake by porcine alveolar macrophage (PAM) cells (170). PLGA was also shown to induce maturation of human monocyte derived dendritic cells (MoDCs) (171).

In general nano-sized particles are preferentially internalized by dendritic cells (DCs) while microparticles (>1000nm) are taken up by macrophages (169, 172). In an experiment, PLGA NPs of size ranging from 300nm to 17µm were used to immunize mice with model antigens where higher internalization and better activation of DCs followed by highest antigen-specific T cell responses were observed with 300nm sized particles (173). Similarly, in another study, 360-470nm sized polyanhydride NPs were found to be appropriate for efficient pulmonary distribution in mice after IN administration (174).

NPs can also serve the function of adjuvant (175, 176). Adjuvants are commonly used in vaccine formulations to potentiate the immune responses. They exert these functions through activation of innate immune receptors, production of different cytokines and modulation of the antigen uptake, processing and presentation by APCs

(177-179). The innate immune activation by adjuvants determines the fate of adaptive immune response to be T helper 1 (Th1) or T helper 2 (Th2) biased (178). The commonly

used aluminum-based adjuvants in humans, despite being safe, inexpensive and

compatible with multiple antigens, occasionally induce adverse reactions at the site of

injection, induce weak cell-mediated immunity, and are susceptible to freezing (180,

181). Likewise, the oil-in-water emulsions or saponins used in animals as adjuvants also show adverse reactions at the site of injection (182, 183). Moreover, these adjuvants are

suitable for IM injections and not for alternative routes of antigen delivery (177, 184,

185). Hence, alternative, safe, and inexpensive adjuvants are necessary both for human

and animal vaccine use, and they should be compatible with different antigens and

suitable for different routes of immunization. NPs made of liposomes, chitosan, and

polyanhydrides can be the alternative adjuvants to use in humans and animals (186-188).

The importance of NPs as adjuvant is highlighted further with the current shift in use of

subunit or recombinant antigens and preference of mucosal immunization techniques

where such antigens require potent delivery vehicles to exert their immunogenic

properties (165, 184, 189).

Multiple antigens or combination of antigens and adjuvants can be encapsulated

or adhered together in nano or micro-particles. Such a combination approach delivers

both antigen and adjuvant at the same time to APCs allowing simultaneous occurrence of

antigen uptake and activation of APCs by adjuvant and induces potent antigen specific

immune response (190-193). Combination of two or three TLR ligands in biodegradable

polymer-based pathogen-like particles was shown to synergistically enhance both innate

and adaptive immune responses to ovalbumin (OVA) in vitro and in vivo in mice (194).

Similarly, co-delivery of cancer antigens and TLR4 ligand in PLGA NPs induced potent

CTLs response and anti-tumor immunity in mice (195). NPs can be surface modified to

develop vaccines for targeted delivery to immune cells or organs (196). PLGA NPs

encapsulating hepatitis B vaccine antigens were surface coated with lectin molecules to

target mucosal M cells (197). After oral delivery in mice, the lectinized NPs

predominantly associated with M cells and also produced better mucosal and cellular

immune responses compared to IM soluble antigen with alum adjuvant (197). Likewise,

DCs targeted chitosan NPs were designed using Severe Acute Respiratory Syndrome

Coronavirus (SARS-CoV) nucleocapsid antigens. IN delivery of DC-targeted vaccine

induced better mucosal IgA and systemic IgG responses against virus nucleoprotein

compared to unentrapped antigens (198). NPs are modified and explored in different

ways to target M cells, macrophages or DCs for oral, nasal or pulmonary deliveries (199-

201).

Cell-mediated immune response is critical in protection against intracellular and

pathogens and viruses such as influenza (202-204). The currently used aluminum-based

adjuvants are poor inducers of cell-mediated immunity, while different NPs are observed

to be potent inducers of cellular response (165, 180). PLGA NPs, encapsulating

inactivated/killed antigen of PRRSV, and conserved epitopes of IAV induced robust cell-

mediated immune response in pigs resulting in enhanced protective efficacy against

respective challenge infection (170, 205-207). Similar results were observed in mice when highly conserved CTL epitopes of IAV were encapsulated in PLGA and used in a vaccine-challenge study (208). Polyanhydride based nanovaccines are also shown to improve cell mediated immune responses in mice (187, 209). The inherent properties of

these NPs can be utilized to develop vaccine platforms that can induce better cell

mediated immune responses in higher mammals (164, 173, 196).

Since many pathogens enter the body through mucosal sites, immunization

through mucosal routes can be the ideal strategy to induce protective immunity at the site

of infection (177, 184). NPs can be used as efficient vaccine delivery vehicles to various

alternative routes of immunization including oral and intranasal (185, 210-212).

Nanovaccines are safe and non-toxic especially those prepared using biodegradable and biocompatible polymers. Biodegradable polymers undergo degradation, subsequent metabolism and elimination from the body after serving their intended purpose (213).

They are degraded either by chemical hydrolysis or enzymatic-catalyzed processes. They are considered ‘biocompatible’ if they do not induce toxicity or any adverse reactions inside the body. Biodegradable and biocompatible polymers can be of natural origin such as proteins (e.g. collagen) and polysaccharides (e.g. chitosan, phytoglycan) or synthetic materials like PLGA and polyanhydrides which carry their own inherent unique physicochemical properties (214).

1.2.1 Poly(lactic-co-glycolic acid) (PLGA)

PLGA is synthetic polyester composed of lactic acid and glycolic acid molecules.

It is one of the most widely studied polymers in drug and vaccine deliveries (215). The monomeric products formed after PLGA hydrolysis are easily metabolized in the body and hence PLGA is safe and non-toxic (216). It has been approved for different types of drug deliveries in humans by the US Food and Drug Administration (FDA) and European

Medicine Agency (EMA) (217). PLGA NPs are synthesized mostly by using ‘water in oil

(W/O)’ emulsification or ‘water in oil in water (W/O/W)’ double emulsification methods.

PLGA polymer and antigens are dissolved in organic solvent (e.g. dichloromethane) and emulsified with surfactant such as poloxamer-188 (218, 219). Nanoprecipitation technique can also be used for preparation of PLGA NPs where polymer and antigens are dissolved in organic solvent and added dropwise into water (217).

PLGA NPs activate antigen presenting cells (APCs) and induce their maturation.

Encapsulation of peptides in PLGA, enhanced murine and human DCs-stimulatory capacity (220). PLGA based particles also enhanced maturation of murine and human

MoDCs and porcine macrophages (171, 206, 221). PLGA NPs can maintain slow and sustained release of antigens which is essential for efficient expansion and memory differentiation of T cells (168, 222). At neutral pH, PLGA NPs are negatively charged

and are internalized into the cells partially through fluid phase pinocytosis and

endocytosis (223). The endocytosed NPs undergo reversal of charge (negative to

positive) at acidic pH of endosomes leading to endo-lysosomal escape of NPs into the cytosol. NPs are internalized into the cytosol within 10min of incubation with cells (224).

Cytosolic NPs are then processed and presented through the MHC-I pathway facilitating cross-presentation of antigens to CD8+ T cells (225, 226). In murine bone marrow-

derived dendritic cells (BMDCs), MHC-I presentation of PLGA-encapsulated OVA

stimulated IL-2 cytokine secretion at 1000 times lower concentration compared to soluble

antigen alone indicating the functional significance of endo-lysosomal escape and antigen

cross-presentation (226). PLGA NPs-based delivery in a murine model induced potent T

cell mediated immune responses against different antigens (195, 227, 228). Similarly, in

pigs, when inactivated PRRSV antigens were encapsulated and coadministered IN with a

potent adjuvant, cross-reactive cellular and humoral immune responses were upregulated

(170, 206, 229). Delivery of PLGA NPs encapsulated IAV subunit vaccine by the IN route was shown to induce peptide specific cellular but not humoral immune responses in pigs (205). Study in mice using PLGA delivery of conserved IAV peptides, also showed improved cell mediated immune response and protective efficacy after virus challenge

(208).

1.2.2 Polyanhydrides

Anhydride based biodegradable synthetic polymers have been intensely studied in recent past for their physicochemical properties and application in controlled release of the drugs and vaccine antigens (230). Sebacic acid (SA), 1,6-bis(p- carboxyphenoxy)hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaocatane

(CPTEG) are the monomers most widely used in formulation of polyanhydride NPs (231,

232). They are synthesized by polycondensation reactions or emulsification process (230,

233). Polyanhydride NPs are biocompatible and safe as they caused less inflammatory changes at the site of injection compared to Alum and incomplete Freund’s adjuvant

(234). Their safety was also evidenced through in vivo study in mice where carbohydrate functionalized CPH and CPTEG based polyanhydride NPs did not induce any damage in liver, kidney or lungs (235). Polyanhydride NPs activated DCs in a manner similar to lipopolysaccharide (LPS) simulation as observed through the expression of costimulatory

markers and cytokines (236). The pathogen mimicking property of these NPs can be

enhanced by surface decoration with carbohydrate moieties that ensures efficient

targeting of antigen-loaded cargo to C-type lectin receptors on DCs (237). These surface

eroding polymers can provide a better microenvironment to loaded antigens and retain the biological activity of varieties of antigens being encapsulated (232, 233, 238-240).

Polyanhydride NP-based vaccines are shown to induce both cellular and humoral

immune responses against viral, bacterial and parasitic pathogens (185). The

polyanhydride nanovaccine induced antigen-specific CTL response in mice immunized

with model antigen OVA (241). Immunization of mice with hemagglutinin-based

polyanhydride nanovaccine against H5N1 influenza elicited neutralizing antibodies and

cell-mediated immune response and elicited protective response in a virus challenge trial

(209). Sustained neutralizing antibodies up to 70 days post-immunization were observed

in mice after combinatorial vaccination with HA-based polyanhydride NPs and

pentablock copolymer-based hydrogels (242). Single dose intranasal immunization with

polyanhydride NP-based Yersinia pestis vaccine induced high titer and high avidity

antibodies compared to antigen alone or antigen with other adjuvant (243).

1.2.3 Chitosan

Chitosan is a polysaccharide made up of copolymers of glucosamine and N-

acetylglucosamine (244). It is a natural polymer manufactured by partial deacetylation of

chitin, which is the second-most abundant polysaccharide in nature and found in

exoskeleton of insects, fungal cell walls and crustacean shells (245). It is insoluble at

neutral and alkaline pH but can be dissolved easily in acidic solutions and is a

biodegradable and biocompatible polymer that degrades into non-toxic constituents by

lysozyme (246). Chitosan polymers can have wide range of molecular weights (50kDa to

2000kDa) and degree of deacetylation (40% to 98%), and their physicochemical and

biological properties depend on these factors (247, 248). Chitosan has amino and

carboxyl groups in the molecule and protonation of its free amino group at acidic

environment imparts net positive charge (249, 250). The positively charged chitosan

polymers can interact with negatively charged sialic acid residues on the glycoproteins of

epithelial cell wall which decreases the rate of mucociliary clearance and increases

antigen retention time at the epithelial surfaces. Further, chitosan molecules can

temporarily open the tight junction proteins facilitating paracellular and intracellular

transport of particulate antigens through epithelium where they come into contact with

APCs (251, 252). Chitosan greatly enhances the absorption of insulin across the nasal

epithelium of rat and sheep (253). Also, in a rabbit model, chitosan NPs (CNPs) of size

300-400nm enhanced the nasal absorption of insulin to a greater extent compared to an

aqueous solution of chitosan (254).

Chitosan has adjuvant and immunomodulatory properties (255, 256). CNPs

encapsulation enhances the internalization of antigens by APCs, increases the activation

marker expression on APCs and induces secretion of pro-inflammatory cytokines (257).

CNPs (~370nm) encapsulation of dengue virus antigens resulted in higher antigen uptake,

significantly higher maturation markers expression and induction of different cytokine

and chemokine synthesis by immature human DCs compared to treatment with soluble

antigens (258). Such activation of DCs by chitosan is plausible to be mediated through a

TLR-4 dependent mechanism (259). Compared to alum, chitosan is superior in enhancing cell-mediated immune responses (260). The enhancement in cell-mediated immune response by chitosan is mediated through the type I IFN dependent pathway (261).

Depending on the nature of the polymer and vaccine formulation, CNPs can act as both

Th1 and Th2 adjuvant (186). CNPs are often synthesized by an ionotropic gelation technique where polyanions such as tripolyphosphate (TPP) helps in proper encapsulation of antigens into the NPs (262, 263). This process is simple, mild and avoids use of high temperature and organic solvents, thus providing better environment to the antigens to be loaded (262). In rat, chitosan (CS) and TPP (CS/TPP) NPs loaded tetanus toxoid antigens were efficiently transported through the nasal epithelium, while in mice, induction of long-lasting systemic and mucosal immune responses were observed compared to soluble antigen immunization via IN route (264). CS/TPP NPs encapsulating Streptococcus equi antigens administered IN in mice induced both cell-mediated and antibody mediated immune responses (265). Similarly, IN delivery of CS/TPP-based influenza split virus vaccine also produced better antibody responses at systemic and mucosal sites in mice and also enhanced IFNγ secreting cell frequencies in spleen (266). Murine studies have shown that, IN administration of CNPs-based vaccines can induce strong local mucosal and systemic antibody responses against hepatitis B virus, Pneumococcus spp.,

Diphtheria spp., and Bordetella spp. (211, 267-270).

1.2.4 Phytoglycogen

Phytoglycogen (PG) is a plant-derived natural polymer that can be used to synthesize novel functionalized NPs with positive or negative surface charge (271).

Dendrimer-like-α-D-glucan (Nano-11) is one such PG-based NP synthesized from kernel of a genetic variant of sweet corn, sugary-1, through few simple chemical modifications

(272). Sugary-1 has large quantities of PG and lacks starch debranching enzymes which results in the formation of dense, highly branched, dendrimer-like NPs replacing starch granules (271, 273). The very first study conducted in mice using Nano-11 NPs showed its potential to be an effective vaccine adjuvant (272). Nano-11 NPs are spherical in shape with an irregular cauliflower-like appearance, range in size from 30 to 110 nm and have positive surface charge (+7 to +15mV). They effectively bind OVA and other negatively charged protein antigens on their surfaces through electrostatic interactions.

Nano-11 NPs enhance antigen uptake by DCs and induce their maturation in vitro. After

IM injection of OVA with Nano-11, significantly increased OVA-specific IgG antibodies were observed in mice. The inflammation at the site of injection with Nano-11 resulted in accumulation of monocytes as opposed to high counts of neutrophils accumulation in aluminum-based adjuvants (272). In a subsequent study, biodistribution and fate of Nano-

11 NPs were investigated after IM injection in mice (274). Nano-11 adsorbed recombinant protective antigen (rPA) of Bacillus anthracis, induced antibody responses similar to or higher than formulations with alum adjuvant, induced transient inflammatory responses and prolonged the retention of antigen at the injection site, effectively targeting antigens to DCs. This study indicated that, Nano-11 has the potential

to be an inexpensive, safe, and effective adjuvant for humans and animals (274).

However, further evaluation of Nano-11 compatibility with different varieties of antigens

and immunomodulatory properties in other animal models, using well designed vaccine-

challenge studies through alternative routes of immunization (e.g. IN), is necessary to

ascertain the broad adjuvant property of Nano-11.

1.3 Mucosal immunization through intranasal route

Mucosal surfaces cover enormous surface areas of the body including the

respiratory, gastrointestinal and urogenital tracts through which most pathogens enter

into the body (275). Therefore, mucosal immune responses are important for protection

against invading pathogens at the port of entry (276). Efficient mucosal immune

responses are developed when vaccines are administered to mucosal surfaces compared

to the vaccines administered systemically (277, 278). The mucosal immune system

consists of anatomically and functionally distinct immune inductive and effector sites

(275). The immune inductive sites in mucosa include mucosa-associated lymphoid tissue

(MALT) and associated draining lymph nodes. These are the sites where antigens are

sampled and stimulation of naïve T and B lymphocytes occurs (279). MALT consists of

gut-associated lymphoid tissue (GALT), nasopharynx-associated lymphoid tissue

(NALT) and other lymphoid tissues (280). These inductive sites are covered with follicle- associated epithelium (FAE) and contain specialized cells known as microfold or M cells

(275). M cells have thin mucus layer and poorly organized brush border with short irregular microvilli at the apical surface which provides greater access to antigens in

mucosal surfaces and hence they are involved in antigen uptake and transfer to APCs

located in pockets within M cell clusters via transcytosis (281-283). APCs, mainly DCs, then carry the antigen to the associated lymph nodes such as Payer’s patches to activate mucosal T and B cell responses including IgA class-switch recombination (284, 285).

Terminal differentiation of plasma cells occurs at mucosal effector sites such as intestinal lamina propria and upper respiratory tract to synthesize secretary immunoglobulin (sIgA) antibodies (286).

Effector molecules at the mucosal surfaces include sIgA antibodies, mucosal IgG antibodies and CTLs which are either produced locally or derived from systemic circulation (276). Locally produced sIgA antibodies in mucosal surfaces are dimeric or multimeric in nature, resistant to degradation in protease-rich environments and function as a first line of defense at the mucosal surfaces (287). sIgA mediated immunity is mainly through – (i) immune exclusion – sIgA binds to antigens and blocks attachment with epithelial cells; (ii) antigen excretion – sIgA containing immune complexes is cleared from the subepithelium; and (iii) intracellular neutralization within epithelial cell endosomes (288). IgG antibodies are either locally produced or transcytosed from serum and they can also neutralize pathogens which enter the mucosa and prevent systemic spread (276, 289). CTLs have important role in clearance or containment of mucosal pathogens when they establish infection in mucosa (276).

Mucosal administration of vaccine antigens can induce both cell mediated (Th1) and humoral (Th2) immune responses at local and distant mucosal surfaces as well as systemically (290, 291). Further, long-lived T and B-cell memory responses can also be

32 generated following mucosal immunization (292, 293). Examples of successful mucosal vaccines include vaccines against rotavirus, poliovirus, Salmonella and cholera delivered through an oral route, and live-attenuated influenza vaccine delivered into the nostrils

(294). Despite the importance of mucosal immunization and increased interest in this field, lesser success is achieved, in part, due to lack of suitable mucosal adjuvants and vaccine delivery vehicles (276). An antigen delivery platform that can gain easy access to the mucosal inductive sites, adhere to mucosal surfaces or M cells, and initiate early immune responses are required for successful mucosal immunization; and nanotechnology based particulate vaccines can best serve these purposes (276, 295).

Mucosal immunization can be done through different routes including oral, intranasal, rectal and vaginal (276). Among them, the interest in IN immunization is higher due to the several advantages it offers. IN route is straightforward, convenient, non-invasive, needle free, and provides large surface area for antigen deposition (296,

297). Since proteolytic enzymes are absent or minimal in nasal compared to oral administration, the former provides better antigen stability and availability to the mucosal surface and in turn reduces the dose of antigens required (298). Nasal mucosa is highly vascularized and contains large number of microvilli covering the nasal epithelium which facilitate rapid antigen uptake and processing (298, 299). As with other mucosal routes, both mucosal and systemic immune responses can be induced after IN delivery of vaccines (296, 298). Further, IN immunization can enhance antibody response both at the local mucosal sites (e.g. nasal cavity or respiratory tract) and distant mucosal sites (e.g. genital mucosa) (296, 298, 300).

NALT is the primary immune induction site after IN vaccination. In rodents, it is

situated on both sides of the nasopharyngeal duct dorsal to the cartilaginous soft palate

(300). NALT is comprised of well-organized immune tissue located underneath the mucosal surfaces including follicle-associated epithelium (FAE) containing M cells,

APCs and T and B-cell enriched areas (300-302). M cells at the nasal epithelium uptake the antigens and actively transport to the submucosa where APCs are present (303). Some small soluble molecules can even directly penetrate through nasal epithelium (298).

Subsequently, APCs will process and present antigens to the T helper cells and such antigen-specific T helper cells interact with B cells. B cells committed to produce IgA antibodies are then transported to effector sites such as nasal passage and respiratory mucosa where their terminal differentiation occurs into IgA producing plasma cells. sIgA produced in this way are transported by polymeric Ig receptor to effector sites where they perform their immune-protective function (304, 305).

The organogenesis, structure and function of NALT are not well defined in pigs, however there is evidence to suggest the existence of such a mucosal immune system in pigs as in rodents and humans (306). Examination of nasal cavity of Gottingen minipigs

from day 1 to 6 month of age indicated that NALT is present in minipigs as few scattered

lymphoid cell accumulations at the very first day of their lives. Gradually, it increased in size and within 3 days became activated with prominent germinal center development.

This tissue was observed until the pigs were sacrificed at 6 month of age (307). A recent study in Bama minipigs also showed the presence of lymphoid tissues at random locations in the nasal cavity (308). The roof of nasopharyngeal meatus had abundant

34 lymphoid tissues that continued with pharyngeal lymphoid tissues (308). The NALT structure observed in these studies are similar with diffused type of NALT structure described in humans (309). Moreover, vaccination or infection studies in pigs through IN routes have shown induction of local mucosal immune responses in respiratory tracts, systemic immune responses in serum, mucosal immunity in distal mucosal sites and cell- mediated immune response providing the experimental evidences that common mucosal immune system exists in pigs (306).

Despite the advantages, IN route of immunization suffers from limitations such as rapid mucociliary clearance of antigen from nasal cavity, inefficient uptake of soluble antigens by nasal epithelial cells and lack of compatible mucosal adjuvants to initiate the innate immune response (299, 310, 311). Therefore, an effective vaccine delivery system is necessary for IN immunization which can maintain antigen in stable form, ensures availability of antigens in nasopharynx for longer duration, facilitates antigen uptake at

NALT and stimulates the immune system (310, 312). Particulate vaccines prepared by using biodegradable and biocompatible NPs can be an attractive approach for IN vaccine delivery as they maintain stability of antigen, facilitate controlled antigen release, and activate the immune system (185, 313). Particulate antigens are readily taken up by M cells compared to soluble antigens (314). Chitosan NPs (CNPs), for example, are mucoadhesive in nature and hence can increase the local retention time of antigen in the nasal epithelium and decrease the rate of antigen clearance on mucosal surfaces. CNPs also open the tight junction of nasal epithelium to increase transport of particulate antigens to the area rich with APCs and, in addition, these particles exert adjuvant

properties (251, 252). A murine studies showed that IN delivery of CNPs-based vaccine induced robust mucosal and cellular immune responses (269). Polyanhydride NPs-based

IN delivered respiratory syncytial virus vaccine in neonatal calves induced both antibody

and cell-mediated immunity and provided better protection compared to unvaccinated

controls (315). Likewise, in pigs, PLGA NPs were able to successfully induce cellular

and humoral immune responses when whole inactivated PRRSV antigen and conserved

peptides of IAVs were administered through IN route and improved the protective

immunity against respective virus challenge infections (170, 205, 206, 229).

Figure 1.1 Schematic diagram of influenza A virus showing the surface glycoproteins and the gene segments. Figure modified from Vincent et. al.; 2008 (14).

Gene Encoded Function Reference Segment Protein 1 Polymerase basic 2 Involved in mRNA cap recognition. (316) (PB2) 2 Polymerase basic 1 Involved in RNA elongation process and (316, 317) (PB1) has endonuclease activity.

PB1-F2 Pro-apoptotic activity. Kills host (317) immune cells. 3 Polymerase acidic Has protease function. (318) (PA) 4 Hemagglutinin Surface glycoprotein and major (8) (HA) antigenic determinant of IAVs. Performs receptor binding and membrane fusion activity. 5 Nucleoprotein (NP) Binds RNA to form ribonucleoprotein (319) (RNP) structure. Regulates nuclear import. 6 Neuraminidase Surface glycoprotein, has sialidase (9, 320) (NA) activity. Involved in release of virus particles after replication. 7 Matrix protein 1 Interacts with viral RNP, involved in (321) (M1) nuclear export of RNA and virus release by budding. Matrix protein 2 Acts as an ion channel and facilitates (322) (M2) virus uncoating and assembly. 8 Non-structural Alters the normal cell RNA splicing, (323-325) protein 1 (NS1) involved in mRNA nucleo-cytoplasmic transport. Performs interferon antagonist function. Nuclear export Helps in nuclear export of RNA. (326) protein (NEP)/NS2

Table 1.1 Protein(s) encoded by different genes of IAVs and their functions.

Chapter 2: Biodegradable nanoparticle delivery of inactivated swine influenza virus

vaccine provides heterologous cell-mediated immune response in pigs

2.1. Abstract

Swine influenza virus (SwIAV) is one of the most important zoonotic pathogens.

Current flu vaccines have failed to provide cross-protection against evolving viruses in the field. Poly (lactic-co-glycolic acid) (PLGA) is a biodegradable FDA approved polymer and widely used in drug and vaccine delivery. In this study, inactivated SwIAV

H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA-KAg) were prepared,

which were spherical in shape with 200 to 300 nm diameter, and induced maturation of

antigen presenting cells in vitro. Pigs vaccinated twice with PLGA-KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA-KAg vaccinated and heterologous SwIAV H1N1

challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for

four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass

in the lung sections with clearance of infectious challenge virus in most of the PLGA-

KAg vaccinated pig lung airways was observed. Immunologically, PLGA-KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the

frequency of IFN-γ secreting total T cells, T-helper and CTLs against both H1N2 and

H1N1 SwIAV. In summary, inactivated influenza virus delivered through PLGA-NPs reduced the clinical disease and induced cross-protective cell-mediated immune response in a pig model. Our data confirmed the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans.

2.2. Introduction

Swine influenza is an acute respiratory infection of pigs caused by influenza A

virus (IAV) of Orthomyxoviridae family. At present H1N1, H1N2 and H3N2 subtypes of

IAV cause majority of infection in pigs. Owing to the presence of both avian (α2,3 Gal)

and human (α2,6 Gal) IAV receptors, pigs can potentially act as mixing vessel for

different IAV (73, 327). Acute clinical signs in influenza infected pigs include high fever, anorexia, respiratory distress, nasal discharge and coughing. Therefore, influenza in pigs causes significant economic loss to the porcine industry through morbidity, loss of body weight gain, increased time to market, susceptibility to secondary bacterial and viral infections like mycoplasma and porcine reproductive and respiratory syndrome (PRRS), medication and veterinary expenses (42, 59). Some of the swine influenza viruses

(SwIAV) can also be transmitted from pigs to humans (zoonotic) creating public health

risk. For example, the 2009 pandemic H1N1 swine influenza virus infected

approximately 20% of the global population and caused around 200,000 deaths (81, 328-

331), in addition to approximately 500,000 deaths due to seasonal annual influenza

infection (4, 20).

Vaccination is one of the most effective means of controlling influenza, and swine

influenza vaccines are commercially available to use in pigs. Due to high mutation rates

in circulating influenza viruses in animals the efficacy of commercial vaccines in the

field is always poor (332, 333). Commercial multivalent vaccines coadministered with an

adjuvant intramuscularly as prime-boost strategy provide homologous, but weak

heterologous protection. Intramuscular vaccination does not induce the required levels of

local mucosal antibody and cell-mediated immune responses; moreover, there are reports

of inactivated vaccine associated enhanced respiratory disease (126, 127, 139). Thus,

persistent economic burden of swine influenza in the pig industry and its potential risk of zoonotic transmission to humans warrant the development of broadly cross-protective vaccine platforms.

Biodegradable and biocompatible polymer, poly(lactic-co-glycolic acid) (PLGA), based nanoparticles (PLGA-NPs) are being widely used for controlled vaccine and drug delivery (215, 217, 334). PLGA has been approved by Food and Drug Administration

(FDA) and European Medicines Agency (EMA) for use in humans (including children) as a vehicle. PLGA-NPs encapsulated vaccine antigens are preserved inside the particles from degradation for a long period of time (4-8 weeks) under physiological conditions, which is critical when vaccine is delivered to mucosal sites; while the benefit of slow

release of the cargo in vivo when administered by parenteral route helps in prolonged

immune activation (335). Moreover, PLGA-NPs assist in internalization of the antigen by professional antigen presenting cells (APCs) such as dendritic cells (DCs), macrophages

(Mɸs) and B cells. PLGA-NPs also facilitate antigen processing and presentation by

APCs to naive lymphoctyes (336). PLGA-NPs of size up to 500 nm are readily uptaken by APCs and induce production of virus neutralizing antibodies as well as cell-mediated immune response in mice models (165, 337, 338). Antigens encapsulated in PLGA-NPs and delivered by intranasal route are protected from proteolytic degradation at mucosal surfaces and readily uptaken by immune cells at the mucosal sites of the respiratory tract, and thus has the potential of inducing strong mucosal immune response. Thus, particulate delivery of influenza vaccine can be a better alternative over existing parenteral vaccine delivery platforms to effectively control infectious diseases (295).

A previous vaccine trial carried out in our laboratory has shown that PLGA-NPs encapsulated porcine reproductive and respiratory syndrome virus (PRRSV) coadministered intranasally with a potent adjuvant significantly reduced challenge heterologous virus induced lung pathology, virus titers and protected pigs against the disease (170). Recently, we found that PLGA-NPs encapsulating a cocktail of five total highly conserved H1N1 IAV T and B cell peptides administered intranasally, without an adjuvant, elicited peptide specific T cell response, but not the antibody response. Despite the lack of antibody response, the PLGA-NPs still helped in the clearance of a heterologous challenge virus from the lungs of pigs (205). These findings suggested that the whole inactivated influenza viral antigens delivered in PLGA-NPs have the potential to further enhance the breadth of immunity and protection against influenza in pigs.

Therefore, in this study we prepared and evaluated the immunogenicity and efficacy of

PLGA-NPs encapsulated inactivated SwIAV (PLGA-KAg) vaccine in a heterologous virus challenge trial in pigs. Our results suggested that intranasal administration of

PLGA-KAg vaccine induced strong T cell response against both homologous and heterologous viruses detected at pre-challenge as well as post-challenge and substantially reduced the heterologous challenge virus induced clinical disease, lung pathology and virus load in the lungs.

2.3. Materials and methods

2.3.1 Cells and viruses

Stable mycoplasma-free Madin-Darby canine kidney epithelial cells (MDCK,

CRL-2285, ATCC, VA) were maintained in Dulbecco’s modified eagle medium

(DMEM) (Gibco) supplemented with 10% fetal bovine serum (Sigma) and antibiotic-

0 antimycotic (Gibco) at 37 C in 5% CO2 incubator. Field isolates of swine influenza virus

(SwIAV), SW/OH/FAH10-1/10 (H1N2-δ1 lineage) (339) and SW/OH/24366/2007

(H1N1-γ) (340) were used in inactivated virus vaccine preparation and challenge

infection of pigs, respectively. The H1N2 virus (SW/OH/FAH10-1/10) has NP and M

genes derived from the 2009 pandemic H1N1 (339), and the A/swine/Ohio/24366/07 was

a zoonotic virus isolated from swine and also was shown at the CDC to have 100%

identical genome sequence to the human virus associated in the Ohio county fair (340).

SwIAV stocks (passage 3) were obtained from the repository at FAHRP, Wooster, Ohio.

Both viruses were propagated on MDCK cells by infecting at MOI 0.005 and maintaining in serum free DMEM supplemented with 1 µg/ml TPCK-trypsin (Sigma, MO).

2.3.2 Vaccine preparation

SwIAV isolate SW/OH/FAH10-1/10 (H1N2-δ1) culture fluid was harvested and clarified to remove cell debris by centrifugation at 2000 xg for 30 min and subjected to

10-fold concentration using Pellicon-2 cassette filtration (Millipore, MA) followed by

ultra-centrifugation using OptimaTM L-100XP ultracentrifuge (Beckman Coulter) with

20% sucrose cushion at 107, 000 xg for 4 hrs without break. Virus pellet was suspended in PBS containing protease inhibitor (Sigma, MO), titrated and stored at -800C. Virus was

inactivated using binary ethyleneimine (BEI) (Sigma, MO) by treating with 10 mM BEI

for 6 hrs at 370C followed by treatment with 10 mM sodium thiosulphate (Sigma, MO) for additional 2 hrs at 370C to neutralize the unused BEI, and the virus inactivation was

confirmed in MDCK cells. Total protein concentration in the virus pellet was estimated

using micro BCA protein assay kit (Thermo Scientific, MA) as per the manufacturer’s

protocol.

Inactivated SwIAV antigen (KAg) was encapsulated in PLGA-NPs by

water/oil/water double emulsion solvent evaporation technique as described previously

(170, 205). Briefly, 5 mg of KAg in 500 µl PBS and 250 µl of 2% (w/v) polyvinyl

alcohol (PVA) with protein stabilizers, 50 µl of 20% sucrose (w/v) and 50 µl of 20%

Mg(OH)2 (w/v), were emulsified in 180 mg of PLGA polymer solution in 4.5 ml of

dichloromethane using high intensity ultrasonic processor (Sonics and Materials Inc., CT)

for 30 sec at duty cycle 30% and output control 3. The resulting water-in-oil (w/o)

primary emulsion was poured into a mixture of 23 ml 2% w/v PVA (Sigma) and 2 ml

12.5% (w/v) polaxmer 188 (Sigma, MO) to form an aqueous solution. The mixture was

divided equally into two tubes and emulsified again by sonication for 60 sec to obtain

secondary w/o/w emulsion, and it was emulsified by magnetic stirring overnight at 400

rpm in cold (4°C) to allow evaporation of the organic solvents. Resulting polymeric

particles were washed thrice using cold sterile Milli-Q water by centrifugation at 10,976

xg (Beckman Coulter, FX6100 rotor) for 30 min. Finally, PLGA-NP pellet was suspended in 5% sucrose in milli Q water, frozen at -800C for 30 min, freeze-dried

(Labconco, MO) for 18-20 hrs and aliquots were stored at -200C. The inactivated KAg

encapsulated in PLGA-NP is henceforth called as PLGA-KAg.

2.3.3 Characterization of PLGA-KAg

Particle size and morphology was examined by a FEI Quanta 250 scanning

electron microscope (SEM, Kyoto, Japan) after coating with 2 nm of iridium using a

Quorum Q150TS sputter coater (Lewes, UK). Nanoparticle size distribution was

characterized using ImageJ image software (National Institutes of Health, MD) with an

average of 200 nanoparticles per image. Quasi-elastic light scattering experiments

(QELS) were used to measure the ζ-potential of the nanoparticles using a Zetasizer Nano

(Malvern Instruments Ltd., Worchester, UK). NPs 100 µg were suspended in cold nanopure water and thoroughly dispersed using a probe sonicator (Ultra Sonic Processor

VC 130PB, Sonics Vibra Cell, CT) before analysis. Three independent measurements were taken in order to get an average ζ-potential value. Protein encapsulation efficiency and in vitro protein release profile at days 0,1, 3, 5, 7, 10, 15, 20, 25 and 30 were

estimated and expressed as the cumulative percentage release of SwIAV antignes at each

time point using the methods described previously (170, 205).

2.3.4 In vitro activation of APCs by PLGA-KAg

Monocyte derived dendritic cells (MoDCs) and alveolar macrophages were used

as APCs for in vitro activation study. To generate MoDCs – (i) peripheral blood

mononuclear cells were isolated from blood; (ii) CD172a+ myeloid cells were

magnetically sorted from PBMCs using MACS Large cell column (Miltenyi Biotec, CA);

and (iii) cells were treated with cytokines GM-CSF (50 ng/ml) and IL-4 (25 ng/ml)

(Kingfisher biotech, MN) for a week. As a source of alveolar macrophages, porcine

bronchoalveolar lavage (BAL) cells were collected by infusion of PBS through trachea of

heatlhy 6-8 week old pigs and collected the lavage fluid. Porcine BAL cells constitutes

over 90% of macrophages (341, 342), like in other species of animals (343). BAL cells

(0.5 million) and MoDCs (0.1 million) were cultured with: (i) RPMI enriched with 10%

FBS (E-RPMI) only; (ii) KAg (2 µg/ml) in E-RPMI; (iii) PLGA-KAg (KAg 2 µg/ml

equivalent of NPs); or (iv) PLGA-NPs (equivalent weight of NPs) in E-RPMI for 24 hrs

0 at 37 C in a 5% CO2 incubator. Stimulated cells were fixed and immunostained using

CD172a (Southernbiotech, AL) and CD152-muIg (Ancell, MN) antibodies, and 50,000

events were acquired by flow cytometry (BD FACS Aria II, BD Pharmingen CA) and

analyzed using the FlowJo software (Tree Star, OR). CD152-muIg is a human specific

antibody shown to cross-react with porcine APCs costimulatory molecules CD80/86

(344).

2.3.5 Experimental design, vaccination, viral challenge and collection of samples

Caesarian delivered colostrum deprived (CDCD) and bovine colostrum fed Large

White-Duroc crossbred 4-5 weeks old piglets (n=32) were raised in our BSL2 facility at

OARDC as described previously (205) and used in our study. Piglets were confirmed seronegative for hemagglutination (HI) antibodies against influenza virus subtypes H1N1 and H1N2 and were randomly divided into 4 experimental groups (n= 7-9 pigs/group)

(Table 2.1). For randomization, piglets derived from 4 sows on the same day were raised in a temperature regulated BSL2 isolation room in 4 elevated crates. At 4 weeks of age piglets were randomly assigned in to 1 of 4 groups (Table 2.1), wherein every experimental group had piglets from all the 4 sows (i.e., 1 to 3 pigs of each sow).

Animals were allowed an additional week of acclimation before used in the study. Pigs were maintained, inoculated and euthanized in accordance with the standards of the

Institutional Laboratory Animal Care and Use Committee at The Ohio State University.

Animals were vaccinated at 4-5 weeks, boosted after 3 weeks and challenged after 2

7 weeks of boost. Pigs were vaccinated with 10 TCID50 equivalent of H1N2 KAg or

PLGA-KAg suspended in 2 ml DMEM and delivered intranasally as a mist using a multi-

dose delivery device (Prima Tech USA, NC) (Fig. 3A), and challenged using the

6 heterologous H1N1 SwIAV (6x10 TCID50) in 2 ml, 1ml administered intranasally and

another 1 ml intratracheally (340).

Plasma samples were collected at days post-vaccination (DPV) 0, 21 and 35.

From the day of challenge to euthanasia, pigs were observed twice daily for clinical signs

and rectal temperature was recorded daily. Nasal swab samples were collected at days

post-challenge (DPC) 4 and 6 in 2 ml DMEM containing antibiotics. Pigs were

euthanized at DPC 6 and during necropsy the lungs were examined and scored for the

gross lesions (48). A board certified veterinary pathologist involved in scoring the lung

lesions of pigs was blinded by not providing any details of the experimental pig

groupings. Blood and BAL fluid samples were collected and aliquots of plasma and BAL fluid stored at -800C. BAL fluid was collected by infusing 20 ml PBS (containing 2%

EDTA) through the trachea and collected the fluid after gentle massaging of all the lung lobes. For preparation of lung lysate, 1gm of lung tissue from the right apical lobe was homogenized in 3ml DMEM (with protease inhibitor) and supernatant was collected after centrifugation and aliquots were stored at -800C as described previously (345). Lung

tissue samples were collected by one person uniformly from the identical regions of the

right apical, cardiac and diaphragmatic lobes of each pig irrespective of the presence or

absence of visible gross lung lesion/s in that area, and fixed in 10% neutral buffered

formalin for detailed histopathological and immunohistochemical evaluations of every

tissue section. Average scores of all the lung lobes sections were considered for final

grading as described previously (127, 346). PBMCs were isolated at DPC 0 and 6 and

used in cell proliferation assays upon stimulation with SwIAV as well as used in flow

cytometry analysis

2.3.6 Cell proliferation and flow cytometry assays

At DPC 0 antigen specific T cells proliferation was carried out in PBMCs using

cell titer 96 aqueous non-radioactive proliferation assay kit (Promega, WI) as per the

manufacturer's instructions. Briefly, 1x106 PBMCs/well were plated in a 96 well U-

bottom plate (Greiner bio-one, NC) in 100 µl of E-RPMI medium. Both the SwIAV

H1N2 and H1N1 used in the vaccine preparation and pig challenge, respectively, were

5 used at MOI of 0.1 in 100 µl (1x10 TCID50/well) for stimulation. Plates were incubated

0 at 37 C in a 5% CO2 incubator, and after 72h plates were centrifuged at 2000 rpm for 2

min and the supernatant was collected, and added 100 µl E-RPMI and 20 µl MTS + PMS

0 solution to the cell pellet and incubated for another 4h at 37 C in a 5% CO2 incubator.

The optical density (OD) at 490 nm was recorded using the ELISA plate reader

(Spectramax plus384, Molecular Devices, CA). Stimulation index (SI) was determined

by dividing OD of stimulated PBMCs from OD of cell control of the same pig, and

average SI values of 7 to 9 pigs of each group were compared among each other. At DPC

0 unstimulated PBMCs were also evaluated to determine the frequency of different T cell

subsets by flow cytometry analysis.

At DPC 6 PBMCs of pigs were restimulated with SwIAV H1N2 and H1N1 at

MOI 0.1 and subjected to cell proliferation as described above. The supernatant harvested

from 72 hr of restimulated PBMCs culture were analyzed for IFN-γ by ELISA, and cells were subjected to immunophenotyping and analyzed by flow cytometry to determine the frequency of activated T cell subsets as described previously (205). Briefly, PBMCs were blocked with 2% pig serum and surface-labeled with pig lymphocyte specific purified, fluorochrome or biotin conjugated mAbs followed by treatment with fluorochrome labeled anti-mouse isotype specific or streptavidin antibody. For intracellular IFNγ staining, GolgiPlugTM (BD Biosciences, CA) and Brefeldin A (Sigma, MO) were added

during the last 6 hr of incubation of PBMCs treated with or without indicated stimulants.

The surface immunostained cells were fixed with 1% paraformaldehyde and

permeabilized with cell-permeabilization buffer (85.9% deionized water, 11% PBS

+ + without Ca2 or Mg2 , 3% formaldehyde solution, and 0.1% saponin) overnight at 4°C.

Cells were washed and immunostained using fluorochrome-conjugated anti-pig IFNγ or its isotype control mAb (BD Biosciences, CA) in 0.1% saponin containing fluorescence-

activated cell-sorting (FACS) buffer. Immunostained cells were acquired using the flow

cytometer BD Aria II (BD Biosciences, CA) and analyzed using the FlowJo software

(Tree Star, OR). All specific cell population frequencies were presented as the percent of

total CD3+/CD3- lymphocytes. To define CTLs (CD8αβ+ T cells), cells were gated first

for CD8α marker followed by CD8β and T cells double positive were considered as

CTLs. Antibodies used in the flow cytometry were: anti-porcine CD3 (Southernbiotech,

AL), CD4α (Southernbiotech, AL), CD8α (Southernbiotech, AL), CD8β (BD

Biosciences, CA) and δ-chain (BD Pharmingen, CA).

2.3.7 Virus titration

Serial 10-fold dilutions of test samples in serum-free DMEM containing TPCK- trypsin (1 µg/ml) in quadruplicates were transferred to monolayer of MDCK cells cultured overnight in 96 well cell culture plates. Plates were incubated for 48 hr at 370C

in a 5% CO2 incubator and fixed using 80% acetone in water and immunostained using

IAV nucleoprotein specific primary antibody (#M058, CalBioreagents, CA) followed by

Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L) antibody (Life technologies,

OR). Immunofluorescence was recorded using fluorescent microscope (Olympus, NY)

and infectious virus titer was calculated using Reed and Muench method (347).

2.3.8 Antibody titration

Hemagglutination inhibition (HI) titers and specific antibody levels were

determined as described previously (205). Briefly, HA units of SwIAV H1N1 was first

determined and the virus stock was diluted to get 8 HA units in 50 µl volume and used in

a standard HI assay. Plasma and BAL fluid samples were incubated at 560C for 30 min to

inactivate innate complement activity. The starting dilution of plasma and BAL fluid for

HI assay was 1:2. SwIAV specific IgG and IgA antibodies in nasal swab, BAL fluid, lung

lysate and plasma samples were determined by ELISA. Briefly, flat bottom high binding

96 well plates (Greiner bio-one, NC) were coated with semipurified pretitrated SwIAV

H1N1 or H1N2 antigens (5 µg/ml) and incubated at 40C overnight. Plates were blocked

with 5% skim milk in PBST for 2 hr at RT and washed three times with PBST. Samples

diluted in 2.5% skim milk were added 50 µl/well in duplicate and incubated at RT for 2

hrs. After three washes goat anti-pig IgA conjugated with HRP (Bethyl Laboratories Inc.,

TX) or goat anti-pig IgG (γ) conjugated with HRP (KPL, MD) was added at 50 µl/well

(both the antibodies were diluted at 1:1000 in 2.5% skim milk in PBST) and incubated at

RT for 2 hrs. The Ag-Ab reaction was developed calorimetrically by adding 1:1 mixture of peroxidase substrate solution B and TMB peroxidase substrate (KPL, MD) 50 µl/well.

The reaction was stopped after 10-20 min by adding 1M phosphoric acid (50 µl/well).

Optical density (OD) was measured at 450 nm using the Spectramax microplate reader,

51 and corrected OD value was obtained after subtracting blank OD from mean OD of different treatment groups. Virus neutralization titer (VNT) in BAL fluid was determined using the procedures described previously (205).

2.3.9 Histopathology and Immunohistochemistry analyses

Five µm sections of apical, cardiac and diaphragmatic lung lobes of pigs were stained with hematoxylin and eosin and examined microscopically for histopathological changes as described previously (48). Peribronchial and perivascular accumulation of mononuclear inflammatory cells (MNCs) as well as bronchial exudates composed of dead sloughed epithelial cells and MNCs were scored as follows: 0, no change from normal; 0.5, changes present but too mild; 1, minimal changes from normal; 2, moderate changes from normal; and 3, marked changes from normal. Final lung pathology score for each pig was determined by taking the average of scores from the three lung lobes and the group averages were compared.

SwIAV specific antigens were detected in the lungs by IHC method as described previously with a few modifications (206, 346), and were henceforth called as the antigenic mass. Briefly, 5 µm tissue sections were deparaffinized and hydrated in

Dulbecco’s PBS (D-PBS) and incubated in 0.05% sodium borohydride solution for 10 min to break aldehyde bonds, washed twice and incubated in Protease VII (diluted 1:6.5 with D-PBS) for 30 min at RT for antigen retrieval. Slides were washed thrice and quenched in 3% H2O2 solution for 5 min, followed by three washes slides were blocked by incubating with 4% normal horse serum for 20 min at RT. Further, slides were

incubated with SwIAV nucleoprotein specific antibody (#MO58, CalBioreagents, CA)

for 60 min at RT, washed thrice, and incubated with biotinylated secondary antibody for

60 min at RT. To detect positive signals, slides were incubated in VECTASTAIN elite

ABC reagent (#PK-7100, Vector Lab., CA) followed by treatment with ImmPACT™

DAB Substrate (#SK-4105, Vector Lab., CA) as per manufacturer’s instructions. The slides were rinsed in tap water, counterstained with hematoxylin, rinsed well in tap water, dehydrated and mounted. Positive IHC signals on bronchial epithelium of apical, cardiac and diaphragmatic lung lobes were scored according to the following criteria: 0, no changes comparable to mock control - normal; 0.5, suggestive but not definite; 1, minimal changes from normal; 2, moderate changes from normal; and 3, marked changes from normal. IHC score of each pig was determined by taking the average scores from all three lung lobes, and treatment pig group average scores were compared. Microscopic and IHC slides were read by a board certificated veterinary pathologist who was blinded to the experimental design and SwIAV infection status.

2.3.10 Ethics Statement

This study was carried out in strict accordance with the recommendations by the

Public Health Service Policy, USDA Regulations, National Research Council’s Guide for the Care and Use of Laboratory Animals and the Federation of Animal Science Societies’

Guide for the Care and Use of Agricultural Animals in Agricultural Research and

Teaching. All the pigs were maintained, samples collected and euthanized, and all efforts were made to minimize the suffering of pigs as per the approved institutional, state and

federal regulations and policies regarding animal care and use at The Ohio State

University on the Ethics for Animal Experiments (Protocol Number: 2014A00000099).

2.3.11 Statistical analysis

Data were presented as mean ± standard error of mean (SEM) of 7-9 pigs. For HI and VN titers data were presented as geometric mean ±SEM of 7-9 pigs. For virus titer,

0 1 titers of 10 were used for less than 10 values; transformed to a log10 scale and analyzed

(346). In each assay, the differences of means among the groups were determined by one-

way analysis of variance (ANOVA) followed by Tukey’s post-hoc comparison test in

GraphPad Prism 5 (GraphPad Software, Inc., CA). A p-value less than 0.05 was

considered statistically significant.

2.4 Results

2.4.1 In vitro characterization of PLGA-KAg NPs

The encapsulation efficiency of KAg in PLGA-NPs was 57%. This result was

comparable to our previous results of PLGA encapsulation of peptides as well as

inactivated PRRSV with 50-60% encapsulation efficiency (205, 229). Morphology of

PLGA-KAg was determined using scanning electron microscope and size distribution

was calculated by analyzing 200 NPs using the ImageJ software. PLGA-KAg was

spherical in shape (Figure 2.1A) with the mean diameter of 313 nm and standard

deviation of 105 nm. Most of the NPs were in the size range of 200-300 nm diameters.

For efficient uptake of NPs by APCs and M cells at mucosal surface, the ideal particle

size is < 500 nm (165, 337), and approximately 95% PLGA-KAg particles were < 500

nm (Figure 2.1B). The charge of NPs was determined by a Quasi elastic light scattering experiment and found to be -18±0.56mV. The particle size and charge were comparable

to our previous PLGA NPs (170, 205, 229). During viral antigen encapsulation in NPs a

fraction of the antigen is always associated on the surface of particles, which gets

released immediately (≤ 30 min) after reconstitution in physiological buffers like PBS

and it is called burst release (348). We observed burst release of 22%, and after 24 hrs

27% of encapsulated cumulative quantity of KAg was released. Further, slow and

sustained release of antigen was observed over a period of 4 week and the total

cumulative release of KAg was approximately 50% after one month (Figure 2.1C). This

result was comparable to our earlier PLGA-NPs preparations encapsulated with IAV

peptides and inactivated PRRSV (170, 205).

2.4.2 PLGA-KAg NPs induced maturation of antigen presenting cells in vitro

Mɸs and DCs are the major APCs. Like in other species, porcine BAL cells

contain greater than 90% Mɸs, and hence BAL cells were not further purified and used as

a source of Mɸs along with MoDCs to investigate the adjuvant properties of PLGA-KAg

in vitro. Mɸs and MoDCs were treated with medium only, soluble KAg (2 µg/ml) or

PLGA-KAg (containing 2 µg/ml of KAg) and analyzed for the expression of APCs

maturation marker, costimulatory molecules CD80/86. MoDCs were also treated with

control empty PLGA-NPs at the same w/v concentration present in the PLGA-KAg to

determine the adjuvant role of PLGA-NPs alone. Our results showed that in PLGA-KAg

55 treated MoDCs expression of CD80/86 was significantly higher (40%) compared to medium control, KAg alone and empty PLGA-NPs treatment (< 25%) (Figure 2.1D).

Similarly, in Mɸs also expression of CD80/86 was significantly higher in PLGA-KAg treated compared to medium control. The percentage of CD80/86 expression in PLGA-

KAg treated Mɸs (14%) was higher than KAg only treatment (8.5%) (Figure 2.1E). This trend was similar when Mɸs and MoDCs were treated with higher concentration of the

KAg (20µg/ml) (data not shown). Furthermore, empty PLGA-NPs and KAg only also induced slightly increased CD80/86 expression in MoDCs (26%) compared to medium control (21%) (Figure 2.1D). PLGA-KAg particles compared to control empty PLGA-

NPs induced significantly higher adjuvant effects on treated MoDCs, suggesting the additive adjuvant effects of KAg when encapsulated in NPs.

2.4.3 PLGA-KAg NPs vaccine induced antigen specific cellular response in pigs

pre-challenge

Stimulation index (SI) of PLGA-KAg vaccinated pigs was significantly higher compared to mock and KAg vaccinated animals stimulated with either the SwIAV H1N2

(Figure 2.2A) or H1N1 (Figure 2.2B), indicating that PLGA-KAg vaccine recipient pigs had immune cells sensitized even against the heterologous SwIAV. Flow cytometry analysis of PBMCs at DPV 35 (without any SwIAV stimulation) demonstrated that

PLGA-KAg vaccination induced generation of significantly higher frequency of

CD3+CD4+CD8α+ T cells (Figure 2.2C), which are called as activated/memory T helper cells in pigs (349), and aslo CTLs (CD3+CD4+CD8αβ+) compared to KAg received pig

56 group (Figure 2.2D). We also observed significantly increased frequency of γδ T cells in

PLGA-KAg vaccinated pigs compared to KAg group (Figure 2.2E).

Significantly higher HI titer was observed both in KAg and PLGA-KAg vaccinated pig groups compared to mock group. However, we did not find any statistical difference in HI titer between KAg and PLGA-KAg treatment groups (Table 2.4).

Interestingly, plasma IgG antibody response was significantly higher in KAg vaccinated pigs compared to PLGA-KAg recipients (Table 2.4). In both KAg and PLGA-KAg vaccinated pig groups, HI titers against the vaccine virus (SwIAV H1N2) at DPV 21 were low (Table 2.4), and the virus specific IgG response was also low (data not shown).

Also we did not observe any difference in IgA antibody titers in nasal swab samples collected at DPV 35/DPC 0 among different vaccine groups (data not shown). Overall, our pre-challenge data demonstrated that PLGA encapsulation of inactivated SwIAV delivered intranasally in pigs induced a strong cell-mediated with moderate to weak humoral immune response.

2.4.4 PLGA-KAg NPs vaccine rescued pigs from clinical flu symptoms, lung

pathology and viral load in the lungs post-challenge

Pigs were vaccinated intranasally using the multidose aerosoal device which provides fine mist particles (Figure 2.3). In SwIAV H1N1 challenged, mock and KAg vaccinated pigs we observed fever with the mean rectal temperature until DPC 4 remained >1040F, and also most of those pigs were anorexic and lethargic during those four days post-challenge (Table 2.2). While PLGA-KAg vaccinated pigs had fever only

57 until DPC 1 (Table 2.2) with mild flu symptoms, and from DPC 2 onwards they were apparently normal and comparable to mock pigs.

On the day of necropsy (DPC 6) lungs were examined and scored for percentage consolidation due to influenza infection. The gross lung lesion scores in PLGA-KAg vaccinated pigs (mean value 12.1) was lower (not significant) than KAg group (mean value 20.8), but it was significantly lower than mock-infected animals (mean value 23.9)

(Table 2.3). The representative lung pictures showing gross lung lesions are shown

(Figure 2.4A). Though the lung lesion scores were not statistically significant between

KAg and PLGA-KAg group due to large variations among the pigs (an outbred species), but we still observed PLGA-KAg vaccinated pigs had 40% reduced average lung lesion scores compared to KAg group. Microscopic lung lesions scores indicated lower percentage of inflammatory cell infiltration around the bronchioles and bronchial epithelium of PLGA-KAg vaccinated pigs compared to both the control groups, suggesting that the PLGA-KAg vaccine relatively enhanced the protection levels in the lungs of pigs against a heterologous SwIAV challenge (Figure 2.4B; Table 2.3).

The antigenic mass in PLGA-KAg vaccinated and virus challenged pigs was significantly lower than mock as well as KAg vaccinated and virus challenged animals

(Table 2.3). The IHC scores and H&E results revealed that the lungs of PLGA-KAg vaccinated pigs were least affected by the virulent heterologous challenge virus, and they were comparable to mock uninfected pigs at DPC 6 in terms of influenza antigenic mass.

The representative IHC pictures from each of respective pig groups are shown (Figure

2.4C).

We also determined the infectious SwIAV H1N1 virus titer in the BAL fluid at

DPC 6, and all the mock vaccinated and virus challenged pigs were found positive for

virus (8/8), while 5 of 8 KAg and only 2 of 9 PLGA-KAg vaccinated pigs were positive for the SwIAV H1N1. Though the average challenge SwIAV titers of 8 to 9 pigs of both

2.4 0.8 KAg (10 TCID50/ml) and PLGA-KAg (10 TCID50/ml) vaccinated pigs were

5.3 significantly reduced in the BAL fluid compared to mock-infected (10 TCID50/ml)

animals. PLGA encapsulation of SwIAV KAg led to a substantial reduction (40-fold) in

the infectious lung virus titer compared to soluble KAg vaccination in pigs (Table 2. 3).

Further, we also tested virus shedding in nasal swabs at DPC 4 and 6, but surprisingly

unlike in the BAL fluid, nasal viral shedding at DPC 4 was comparable in all the

vaccinated and mock-infected pig groups; and by DPC 6 it was equally reduced across all

the groups. Overall, there was no difference in the nasal virus shedding between the KAg

and PLGA-KAg vaccine recipient pigs (Table 2.3). Our data suggested that intranasal

delivery of PLGA encapsulated SwIAV KAg provided clinical protection against a

heterologous virus challenge and reduced the lung pathology and replicating infectious

virus load in the lungs of pigs.

2.4.5 PLGA-KAg vaccination induced enhanced IFNγ secretion and activated

recall T cell response in virus challenged pigs

At pre-challenge DPC 0 augmented cellular immune response in PLGA-KAg

vaccinated pigs in PBMCs was detected (Figure 2.2), therefore we performed a similar

analysis post-challenge at DPC 6 in pig groups. To reveal the recall cellular response,

PBMCs were stimulated ex vivo with either the vaccine (H1N2) or challenge (H1N1)

SwIAV and analyzed for the activated (IFNγ+) T lymphocyte subsets. A representative

graph showing gating pattern followed for analysis of different T cell subsets in pigs by

flow cytometry is shown (Figure 2.5).

Total IFNγ producing T cells (CD3+) were significantly higher in PLGA-KAg

treatment pig group compared to KAg treated animals stimulated with both vaccine and

challenge viruses (Figure 2.6A). CD3-IFNγ+ cells were significantly higher in KAg than

PLGA-KAg vaccinated pig groups, irrespective of virus restimulation conditions (Figure

2.6B). CD3+CD4+CD8α+IFNγ+ (activated T-helper/memory) cells were significantly

higher in mock-challenge pig group in response to ex vivo stimulation with challenge virus, but not with vaccine virus, suggesting the activation of memory cells in pigs

(Figure 2.6C). CD3+CD4+CD8α-IFNγ+ (activated T-helper) cells were significantly

higher in PLGA-KAg vaccinated pig group compared to both mock-challenge and KAg

treatment groups cells stimulated with challenge virus, but significantly enhanced

compared to mock-challenge group on stimulation with vaccine virus (Figure 2.6D).

IFNγ producing CD3+CD4-CD8αβ+IFNγ+ (activated CTLs) cells were significantly

augmented in PLGA-KAg vaccinated pig group compared to both mock-challenge and

KAg vaccinated animals stimulated with both vaccine and challenge viruses (Figure

2.6E). Consistent with increased CD3-IFNγ+ cell subset response observed in KAg

vaccinated pig group, IFNγ producing NK cell subsets (CD3-CD4-CD8a+IFNγ+) were

also significantly higher compared to mock challenged animals (Figure 2.6F). Consistent

with the flow cytometry data, IFNγ secretion by restimulated PBMCs was also

significantly higher in PLGA-KAg vaccinated and virus challenged pigs compared to both mock-challenge and KAg vaccinated animals (Figure 2.7A). Further, IFNγ secretion into the cell culture supernatant was significantly higher in H1N2 stimulated PBMCs of

PLGA-KAg vaccinated pig group compared to unstimulated control cells (CC) (Figure

2.7B), indicating the presence of antigen specific recall Th1 response. Overall, our

lymphocyte response analysis data both at pre- and post-challenge in PLGA-KAg

vaccinated pigs suggested induction of a strong cellular immune response.

2.4.6 KAg compared to PLGA-KAg vaccination induced higher IgA and IgG

responses, but HI and virus neutralization titers were comparable

Intranasal delivery of vaccine is thought to induce strong local mucosal immunity.

Therefore, we determined IgA response in nasal swab, BAL fluid and lung lysate samples, and surprisingly, found significantly higher response against SwIAV H1N1 in

KAg compared to PLGA-KAg vaccinated and virus challenged pigs (Table 2.4). We also checked specific IgG antibody levels against SwIAV H1N1 and did not find any difference in the plasma among all the experimental pig groups (Table 2.4), but the levels were significantly higher in BAL fluid of KAg vaccinated pigs (Table 2.4). IgA and IgG antibody response against vaccine virus (SwIAV H1N2) also had a similar higher trend in

KAg rather than PLGA-KAg vaccinated pigs (data not shown). We observed absence of statistical difference in HI titer against SwIAV H1N1 in the BAL fluid and plasma between KAg and PLGA-KAg vaccinated pig groups (Table 2.4). Similarly, there was no difference in HI titer against SwIAV H1N2 in plasma, but HI titer in BAL fluid against

SwIAV H1N2 was significantly higher in PLGA-KAg compared to KAg vaccinated pig groups (data not shown). Surprisingly, irrespective of significantly higher IgG and IgA antibody response detected in KAg vaccinated pigs, the virus neutralization titers were comparable to that of PLGA-KAg vaccinated animals (Table 2.4). Overall, our cellular and humoral immune response data suggested that intranasal vaccination with both the

KAg and PLGA-KAg failed to induce strong HI and virus neutralizing antibody responses locally at the lungs as well as systemically in blood of pigs. However, PLGA encapsulation of SwIAV KAg elicited strong cross-protective cell-mediated immunity.

2.5 Discussions

PLGA polymer is extensively used in drug and vaccine delivery studies due to its

non-toxic, biodegradable and biocompatible properties (217). PLGA based candidate

viral vaccines delivered intranasally in rodent models in the absence of any adjuvant

have shown great promise against hepatitis B, equine encephalitis, influenza and

parainfluenza by inducing strong cellular and humoral immune responses (350-355). In

dairy calves immunized intranasally with PLGA encapsulated bovine parainfluenza virus,

enhanced virus specific antibody response was observed (356). Our earlier studies in pigs

indicated that intranasal delivery of PLGA-NPs based inactivated PRRSV induced

enhanced cross-protective both cellular and humoral immune responses only when

coadministered with a potent adjuvant (170, 206, 207). In a recent study in pigs,

vaccinated intranasally with PLGA-NPs encapsulated conserved IAV pooled T and B cell

peptides cocktail, peptide specific cellular (but not humoral) immune response was

62 upregulated, and the pigs were rescued from clinical flu and cleared the infectious replicating challenge virus from the lungs better than the control animals (205). In this study, our goal was to both improve virus specific humoral and cell-mediated immune responses and augment cross-protective efficacy of PLGA-NPs delivered inactivated

SwIAV. This approach should provide greater numbers of potential B and T cell epitopes to the pig immune system compared to a selected few peptides used earlier

(205). Our results in pigs indicated that PLGA-NPs delivered inactivated SwIAV failed to augment humoral response in spite of significantly boosting the cell-mediated response, suggesting that important cytokines and chemokines required for activation and interaction of B cells with T-helper cells were likely not secreted. This was different from

PLGA-NPs delivered inactivated PRRSV coadministered with a potent mucosal adjuvant which intranasally boosted both humoral and cell-mediated responses (170, 207). Thus, it is unlikely that B cell epitopes were damaged during the vaccine formulation process or they were masked by the NPs when phagocytosed by APCs.

Particulate delivery of antigens in NPs enhances its uptake by APCs (357, 358).

PLGA encapsulated PRRSV in comparison to soluble antigen was efficiently uptaken by

Mφs (170). Size of the NPs plays a critical role in rate of uptake and fate of antigen in

APCs, wherein < 500 nm sized NPs are readily internalized and processed by APCs (165,

337, 338, 359, 360). Consistent with that PLGA-KAg particles were around 300 nm and found efficiently internalized by pig APCs. Sustained slow release of vaccine antigens when delivered by parenteral route provides long-lasting immune response, avoid the risk of tolerance and need of additional boosts; and also essential for expansion of antigen

63 specific CD4 and CD8 T cells and differentiation of memory T cells (222, 361). But when particulate NPs entrapped vaccine is delivered intranasal the expected benefit is protection of entrapped antigens from proteolytic degradation at mucosal surfaces and facilitation of its uptake by immune cells present at the mucosal sites, which is critical for induction of strong mucosal immunity. PLGA-KAg had approximately 50% entrapped vaccine antigens left even after 4 weeks of its suspension in physiological saline conditions in vitro, suggesting that the prolonged availability of PLGA-KAg particles facilitated uptake by APCs present at the mucosal surfaces. This nature of PLGA-KAg was comparable to PLGA-NPs encapsulated PRRSV and influenza viral peptides (170,

205).

PLGA-NPs also possess inherent adjuvant properties by inducing maturation of

APCs (362, 363). PLGA-NPs encapsulated PRRSV was shown to induce increased expression of CD80/86 on treated Mφs (206). Similarly, PLGA based particles were shown to induce maturation of human MoDCs and murine bone marrow derived DCs

(171, 221). Consistent with that, PLGA-KAg also induced expression of CD80/86 on treated pig Mɸs and DCs. Interestingly, synergistic adjuvant effect was observed in

PLGA-KAg treated DCs compared to empty PLGA-NPs treatment, suggesting the innate adjuvant effects of inactivated KAg when presented in NPs. DCs pulsed with PLGA-NPs were shown to increase CD86 expression at modest level, which was synergistically increased when immunogen monophosphoryl lipid A was encapsulated in PLGA-NPs

(364). Overall, PLGA based inactivated SwIAV NPs vaccine had desirable properties of an ideal particulate vaccine.

Cell-mediated immune response is vital for clearing intracellular pathogens, and

antigen presentation by the MHC class I pathway mediates CTL response (365).

Inactivated soluble vaccine antigens are not processed through MHC class I pathway and

thus elicit poor cellular immune response, but the PLGA-NP delivery system overcomes

that lacunae by facilitating cross-presentation of encapsulated soluble antigens by APCs through an endolysosomal escape mechanism (225, 366, 367), mediated through rapid

reversal of NP surface charge from anionic to cationic inside the endolysosomal

compartment (224). In this study, though we did not test such a mechanism operating in

the pig system, the augmented T cell response in both pre- and post-challenged PLGA-

KAg vaccinated pigs suggested operation of such a mechanism. Unlike in mice and humans, in pigs some of the T cell subsets are defined slightly different (368-370),

CD3+CD4+CD8α+ T cells are regarded as activated/memory T-helper cells, and

CD3+CD4+CD8α- cells as naïve T-helper cells (371). We observed enhanced frequency

of activated/memory T helper cells in PLGA-KAg vaccinated pre-challenge pigs. PLGA-

KAg vaccinated pig lymphocytes were found to be primed against influenza virus antigens, indicated by increased antigen specific lymphocyte proliferation. Cells bearing

CD3+CD4-CD8αβ+ markers are CTLs which help in complete clearance of virus infected

cells in pigs (369). In PLGA-KAg vaccinated pigs at DPC 6, frequency of activated CTLs

and naïve T-helper cells were upregulated, but not memory T-helper cells. We do not

know why upregulated levels of memory T-helper cells observed in pigs pre-challenge

were not maintained at higher frequency post-challenge in PLGA-KAg vaccinated pigs.

However, our results are consistent with earlier studies wherein upregulation of CTLs

mediated through PLGA delivery system was associated with augmented immunity (195,

227, 372).

γδ T cells are innate immune cells which play a vital role in mediating antiviral

immune responses (373), and in pigs they are relatively abundant compared to other

species of animals (374). PLGA-KAg vaccinated pigs showed higher frequency of γδ T

cells which also would have contributed in induction of protective immune function

against influenza. PLGA-KAg elicited protective efficacy against a virulent heterologous

challenge virus, indicated by the absence of clinical flu symptoms, reduced gross and

microscopic lung pathology and challenge viral clearance from the lungs. Overall,

effector and memory recall T cell responses in PLGA-KAg vaccinated pigs were stronger and robust, particularly CTLs response compared to PLGA encapsulated conserved peptides cocktail vaccinated pigs (205); and this was comparable to inactivated PRRSV encapsulated PLGA vaccine coadministered with a potent adjuvant (170, 207).

For prevention of influenza virus transmission and induction of protection, wherein the virus primarily infects respiratory tract epithelial cells, induction of strong mucosal antibody response in the upper respiratory tract is critical, and vaccine delivery through nostrils has that potential (297, 375). In our study, specific HI titer in plasma was not increased in PLGA-KAg vaccinated pigs compared to KAg group. Surprisingly, though plasma IgG and BAL fluid IgA and IgG responses were significantly higher in

KAg compared to PLGA-KAg vaccinated pigs at both pre-and post-challenge, but the virus neutralization titer against the challenge virus in BAL fluid was comparable in KAg and PLGA-KAg vaccinated pigs. Clearance of replicating virus in 40% of pig lungs in

KAg vaccinated compared to mock-infected pigs appears to be contributed by antibodies

and increased innate NK cells, but the clinical disease and lung pathology remained

comparable to mock-challenged animals. Therefore, induction of strong cell-mediated

immune response (CTLs) in inactivated SwIAV vaccinated pigs is essential to limit the severity of influenza in pigs. Hence innovative vaccination strategies should explore T cell immunity to provide broad protective response (203, 376). Activated lymphocytes produce IFN-γ, which play a significant role in influenza virus clearance (205, 377).

Although PLGA-KAg vaccination induced less of IgA and IgG antibody response than

KAg at mucosal and systemic sites, the HI and VN titers were comparable. However, the

nasal viral shedding was not reduced and found comparable in all three virus-challenged pig groups, suggesting that mucosal IgA antibody response was weak in PLGA-KAg vaccinated animals.

The particulate vaccines trials to advance the potential of nanotechnology based

approach demand the evaluation of a candidate vaccines in multiple animal models,

including in pig before choosing the right model for translational studies to revolutionize

vaccine development against highly demanding respiratory infections like influenza in

humans (185). In mice using well-defined and highly conserved IAV-derived CTLs

peptides encapsulated in PLGA microspheres induction of strong CTLs response and

complete protection against a viral challenge was achieved (208). Overall our data in pigs

showed that induction of strong CTL response by using PLGA-KAg vaccine has helped

in reducing the clinical flu symptoms, substantially cleared the challenged heterologous

virus from the lungs, but failed to reduce the nasal viral shedding; suggesting the

necessity of concurrent secretion of enhanced levels of mucosal IgA antibody response

together with generation of specific CTLs through modifying the PLGA-KAg vaccine formulation.

2.6 Conclusions

In conclusion, we demonstrated that intranasal delivery of PLGA based inactivated SwIAV vaccine induced a strong CTL response resulting in protection from a virulent heterologous influenza virus induced clinical disease, reduced the lung pathology, and substantially cleared the virulent zoonotic heterologous challenge virus from the lungs of pigs. Since the strong CTL response is capable of providing heterosubtypic immunity in influenza infections in mice, PLGA based vaccination approaches form an ideal platform to use the pig model for translation of particulate candidate flu vaccines to effectively control flu pandemics in humans. Our future studies will focus on enhancing mucosal IgA antibody response to produce balanced Th1/Th2 immunity by incorporating Th2 based adjuvants in our candidate PLGA SwIAV vaccine

formulation to improve the vaccine efficacy.

2.7 Acknowledgements

We are thankful to Dr. Juliette Hanson and Megan Strother who provided help in

animal studies. This work was supported by Agriculture and Food Research Initiative

Competitive Grant no. 2013-67015-20476 from the USDA-NIFA and Nanovaccine

Research Initiative, Iowa state University. Dr. Artur Summerfield (Institute of Virology

68 and Immunology, Mittelhäusern, Switzerland) has shared his protocol for our pig dendritic cell study. Salaries and research support were provided by state and federal funds appropriated to OARDC, The Ohio State University.

Experimental groups Pig Vaccine formulations Nos First vaccination Second vaccination Day of Challenge (DPV 0 / DPC -35) (DPV 21 / DPC -14) (DPV 35 / DPC 0) Mock 7 Mock inoculum Mock inoculum Mock inoculum Mock + Ch. 8 Mock inoculum Mock inoculum SwIAV OH7 (H1N1) KAg + Ch. 8 Inactivated SwIAV OH10 (H1N2) Inactivated SwIAV OH10 (H1N2) SwIAV OH7 (H1N1) PLGA-KAg + Ch. 9 PLGA encapsulated PLGA encapsulated SwIAV OH7 inactivated SwIAV OH10 (H1N2) inactivated SwIAV OH10 (H1N2) (H1N1)

Table 2.1 Experimental design showing assignments of pigs in each group

DPC Mock Mock + Ch. KAg + Ch PLGA-KAg + Ch 0 102.2±0.1 A 102.7±0.1 B 102.6±0.1 AB 102.5±0.2 AB 1 102.9±0.1 A 105.1±0.2 B 104.5±0.3 BC 104.0±0.3 C 2 102.7±0.2 A 104.1±0.2 B 103.6±0.2 B 102.9±0.2 A 3 102.5±0.1 A 104.9±0.2 B 104.0±0.3 C 102.5±0.1 A 4 102.7±0.2 A 103.8±0.3 B 104.1±0.4 B 102.3±0.2 A 5 102.4±0.1 A 103.6±0.2 B 103.2±0.3 B 102.4±0.1 A 6 102.0±0.1 A 102.8±0.2 BC 102.6±0.2 BC 102.5±0.1 AC

Table 2.2 Rectal temperatures of pigs from DPC 0 to DPC 6. Each data in the table is the mean value of 7 or 9 pigs ± SEM. Letters A, B and C represent groups of means under each parameter significantly different from each other (P < 0.05). Means labeled with the same letter are not significantly different, while those with different letters are significantly different. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test.

Treatment group Gross lung H&E IHC Virus titerb Nasal Swab lesion score scorea scorea BAL fluid DPC 4 DPC 6 (DPC 6) Mock 0.0±0.0 C (0/7) 0.1±0.0 A (6/7) 0.1±0.1 A 0.0±0.0 A (0/7) 0.0±0.0 A (0/7) 0.0±0.0 A (0/7) (1/7) Mock + Ch. 23.9±3.1 B (8/8) 1.5±0.2 B (8/8) 2.2±0.4 B 5.3±0.3 B (8/8) 5.1±0.1 B (8/8) 2.3±0.6 B (6/8) (8/8) KAg + Ch. 20.8±3.7 AB 1.3±0.1 B (8/8) 1.3±0.3 B 2.4±0.7 C (5/8) 5.0±0.4 B (8/8) 1.8±0.5 AB (5/8) (8/8) (7/8) PLGA-KAg + 12.1±2.3 A (9/9) 1.0±0.1 B (9/9) 0.3±0.1 A 0.8±0.5 AC 4.7±0.1 B (9/9) 1.6±0.5 AB (5/9) Ch. (5/9) (2/9)

Table 2.3 Summary of pathological lung lesions scores and challenge virus titers. Lungs of vaccinated and virus challenged pigs were examined for gross lung lesions, microscopic lung lesions, immunohistochemistry (IHC) scores and viral titers in BAL fluid and nasal swab samples. Each data in the table is the mean value of 7 or 9 pigs ± SEM. The numbers in the parentheses are the number positive / total number of pigs. aRight apical, cardiac and diaphragmatic lobes were examined for microscopic lesions in H&E / virus reactivity in IHC of each pig and average score of 7 or 9 pigs under indicated pig group is b shown. Values have been transferred into log10 scale. Letters A, B and C represent groups of means under each parameter significantly different from each other (P < 0.05). Means labeled with the same letter are not significantly different, while those with different letters are significantly different. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test.

Pre-challenge Post-challenge Plasma DPC 0 / IgA antibody at DPC 6 IgG antibody at HI titer at DPC 6 VN titer at DPV 35 (DPV21a) against H1N1virus - DPC 6 against H1N1 against H1N1 DPC 6 in against H1N2 OD450 nm virus - OD450 nm virus BAL fluid virus against HI titer IgG Nasal BAL Lung Plasma BAL Plasma BAL H1N1 virus OD450 swab fluid lysate fluid fluid Mock 1.6±0.2 A 0.2±0.1A 0.06±0.03A 0.18±0.04 0.03±0. 0.40±0.13 0.09±0.01 3.6±1.0A 2.2±0.3A 1.3±0.4 A 01A A A A Mock + 2.4±0.3 A 0.2±0.1A 0.10±0.03A 0.37±0.06 0.09±0. 0.29±0.11 0.12±0.02 64±0.0 45.3±16 22.6±9.4 Ch A 02A A A B BC AC KAg + 16.0±2.4 B 1.1±0.2 0.41±0.07B 1.77±0.09 0.71±0. 0.41±0.05 0.80±0.18 76.1±13.4 64±15.9 53.8±14.8 a Ch 6.5±0.7 B B 17B A B B BC BC PLGA- 20.2±2.7 B 0.5±0.1 0.12±0.03A 0.71±0.11 0.17±0. 0.61±0.07 0.16±0.02 74.7±14.1 34.6±5.3 43.5±17.2 a KAg + Ch 6.2+0.7 A C 04A A A B AC BC

Table 2.4 Humoral immune response in pigs pre and post-challenge. Each data in the table is the mean value of 7 or 9 pigs ± SEM. aPlasma DPV 21 HI titer against H1N2 virus. Letters A, B and C represent groups of means under each parameter significantly different from each other (P < 0.05). Means labeled with the same letter are not significantly different, while those with different letters are significantly different. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test.

Figure 2.1 In vitro physical characterizations of PLGA-KAg NPs and their role in maturation of APCs. (A) Surface morphology of PLGA-KAg (10 Kx magnification). (B) Size distribution of PLGA-KAg. Percentages were calculated based on determining the size of 200 NPs. (C) In vitro protein release profile of PLGA-KAg over a period of 4 weeks. Effect of treatment of PLGA-KAg on the expression of costimulatory molecule CD80/86 on pig (D) MoDCs and (E) macrophages. CD172a is a porcine pan-myeloid marker and it is expressed on the surface of both dendritic cells and macrophages of pigs. Hence, CD80/86 expression is shown as the percentage of CD172a+ dendritic cells and macrophages in Fig. 1D and 1E, respectively. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Asterisk refers to statistical significant difference between the two indicated pig groups (* p<0.05; ** p<0.01; and *** p<0.001).

Figure 2.2 Cellular and humoral immune responses in pigs pre-challenge. Isolated PBMCs after prime-boost vaccination at DPV 35 / DPC 0 were restimulated and specific lymphocyte proliferation was determined against (A) homologous vaccine virus (SwIAV H1N2) and (B) heterologous challenge virus (SwIAV H1N1). Frequencies of (C) CD3+CD4+CD8α+cells; (D) CD3+CD4+CD8αβ+ cells; and (E) CD3+δ+ γδ T cells in PBMCs were determined at DPC 0 by flow cytometry analysis. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Asterisk refers to statistical difference between two indicated pig groups (* refers p<0.05; ** refers p<0.01; and *** refers p<0.001).

Figure 2.3 IN vaccine delivery device for pigs. Pigs were vaccinated with KAg or PLGA- KAg intranasally as a mist using a custom built multidose vaccine delivery device.

Figure 2.4 Lung lesions in pigs at DPC 6. A representative lung picture of every experimental pig groups is shown: (A) Gross lung lesions of consolidation are indicated by arrows; (B) microscopic lung sections stained by H&E; and (C) Immunohistochemistry analysis of lung sections for SwIAV antigens.

Figure 2.5 Gating patterns of pig lymphocytes. PBMCs isolated at DPC 6 from PLGA- KAg vaccinated and virus challenged pigs were restimulated with SwIAV H1N1 and treated with Golgiplug and golgiblock, and immunostained using pig specific lymphocyte surface markers followed by intracellular IFNγ, and estimated the frequency of activated (IFNγ+) lymphocyte subpopulations. Gating pattern of isotype and lymphocyte specific markers stained with CD3έ, CD4α, CD8α, CD8β and IFNγ to identify the frequency of CD3− IFNγ+, CD3− CD4− CD8α+ IFNγ+, CD3+ IFNγ+, CD3+ CD4+ CD8α− IFNγ+, CD3+ CD4+ CD8α+ IFNγ+ and CD3+ CD4− CD8αβ+ IFNγ+ cells are shown.

Figure 2.6 Lymphocytes recall response in vaccinated and challenged pigs. On the day of necropsy (DPC 6) isolated PBMCs were restimulated with vaccine or challenge virus and the frequency of activated (IFNγ+) lymphocytes were determined by flow cytometry. Average frequency of lymphocytes: (A) CD3+ IFNγ+; (B) CD3− IFNγ+; (C) CD3+ CD4+ CD8α+ IFNγ+; (D) CD3+ CD4+ CD8α− IFNγ+; (E) CD3+ CD4− CD8αβ+ IFNγ+; and (F) CD3− CD4− CD8α+ IFNγ+ from all the experimental pig groups were quantified. Each bar indicates the average frequency of indicated lymphocyte subset of 7 or 9 pigs ± SEM. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test. Asterisk refers to statistical significant difference between the two indicated pig groups (*p < 0.05; **p < 0.01; and ***p < 0.001).

Figure 2.7 IFNγ secretion and recall T cell response in PLGA-KAg vaccinated and virus challenged pigs. PBMCs isolated at DPC 6 were restimulated with vaccine or challenge virus for 3 days. (A) Cell culture supernatant was harvested and determined the levels of secreted IFNγ by ELISA. The recall cellular response in PBMCs of only PLGA-KAg vaccinated pigs restimulated (SwIAV H1N1 or H1N2) or unstimulated (cell control, CC) are shown in terms of (B) secreted IFNγ in cell culture supernatant. Each bar indicates the average frequency of indicated lymphocyte subset of 7 or 9 pigs ± SEM. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test. Asterisk refers to statistical significant difference between the two indicated pig groups (*p < 0.05; **p < 0.01).

Chapter 3: Polyanhydride nanovaccine against swine influenza virus in pigs

3.1 Abstract

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ~200 nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4+CD8αα+ T helper and CD8+ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8 fold

reduction in nasal shedding of SwIAV four days post-challenge compared to control

animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against

both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized

pigs compared to the animals immunized with KAg alone. However, in the KAg

nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody

responses, and virus neutralization titers were comparable to that in the animals

immunized with KAg alone. Overall, our data indicated that intranasal delivery of

polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity.

3.2 Introduction

Swine influenza A virus (SwIAV) causes considerable economic losses in the pig industry worldwide (378). Currently, multiple antigenically diverse strains of three major

SwIAV subtypes H1N1, H1N2 and H3N2 are circulating in pig populations. Since pigs serve as a mixing vessel for human and avian IAV, numerous distinct SwIAV strains are frequently generated, and some of these have zoonotic potential (379). An effective vaccination strategy can prevent economic losses in the pig industry and limit zoonotic transmission of SwIAVs to humans. Vaccination against SwIAV is frequently practiced on pig farms using either bivalent or multivalent whole virus inactivated (WIV) vaccines which protect against homologous virus but are ineffective against heterologous strains

(126, 127, 139, 380). Since SwIAV undergoes frequent mutation with antigenic drift and shift, there is an urgent need to develop broadly cross-protective vaccines. Moreover,

WIV vaccines do not elicit high levels of antigen-specific secretory IgA antibody

response in the respiratory tract where the disease is localized. It is also known that

strong mucosal immunity can correlate with cross-protective efficacy against influenza

(143, 144). Recently, WIV vaccine formulations were reported to enhance the severity of

lung lesions in pigs infected with heterologous IAV, raising concerns over judicious

selection and use of vaccines (126, 127, 139). To overcome these limitations, a novel

vaccine delivery platform is needed for prevention and control of influenza in pigs.

Biodegradable and biocompatible polyanhydrides have been widely used for

vaccine antigen delivery due to safety (234, 235, 381) and their adjuvant properties (382).

The most well-studied polyanhydride copolymers are based on sebacic acid (SA), 1,6-

bis(p-carboxyphenoxy)hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaocatane

(CPTEG) monomers. Polyanhydrides are surface eroding polymers, which minimize the exposure of encapsulated antigen to moisture providing a better microenvironment for the encapsulated vaccine antigen(s) (382, 383). Polyanhydride nanoparticles retain the structural and biological activity of released vaccine antigens (232, 233, 238-240, 384)

and also have pathogen mimicking properties to activate dendritic cells and enhance

innate immune response (233, 236, 237, 385). Recent studies have shown induction of

high virus neutralizing antibody titer and enhanced cell-mediated immune responses

against IAV in mice vaccinated with a hemagglutinin-based polyanhydride nanovaccine

(209, 386). In this study, we analyzed the immunogenicity and protective efficacy of

20:80 CPTEG:CPH nanoparticles encapsulating whole inactivated SwIAV vaccine against a heterologous and virulent zoonotic SwIAV H1N1 challenge in pigs. Our results

indicated that nanovaccine encapsulation of SwIAV augmented the virus specific cell-

mediated immune response and reduced the virus load and fever in pigs.

3.3 Materials and Methods

3.3.1 Vaccine preparation

The SwIAV isolates, SW/OH/FAH10-1/10 (H1N2) - a δ lineage virus bearing human like HA and NA genes, swine triple reassortant virus internal genes PB2, PB1, PA and NS and pandemic H1N1 lineage NP and M genes (339), and SW/OH/24366/2007

H1N1 a triple reassortant γ lineage virus having swine origin HA, NA, NP, M and NS genes, human origin PB1 and avian origin PB2 and PA genes (340) were used in vaccine

preparation and challenge infection, respectively.

For vaccine preparation, Madin-Darby canine kidney (MDCK) cell grown H1N2

virus culture fluid was concentrated by sucrose gradient ultra-centrifugation, and the

virus was inactivated by binary ethyleneimine (BEI). Inactivated/killed SwIAV (KAg)

was encapsulated in 20:80 CPTEG:CPH polyanhydride nanoparticles (KAg nanovaccine)

as described previously (387, 388). Particle size and morphology were examined by a

FEI Quanta 250 scanning electron microscope (SEM, Kyoto, Japan) and size distribution

was characterized by using ImageJ software with an average of 200 nanoparticles and

with quasi-elastic light scattering experiments (QELS) using a Zetasizer Nano (Malvern

Instruments Ltd., Worchester, UK). The SwIAV encapsulation efficiency in the

nanoparticles was determined as described previously (238).

3.3.2 Experimental design and sample collection

Caesarian-delivered colostrum-deprived (CDCD) and bovine colostrum-fed Large

White-Duroc crossbred piglets (n=30) were raised in the BSL2 facility at OARDC as

described previously (205). Piglets were confirmed seronegative for hemagglutination

inhibition (HI) antibodies against SwIAV H1N1 and H1N2, and were randomly divided

into 4 experimental groups (n=7 or 8 pigs/group) (Table 3.1). Maintenance of pigs and all

experimental procedures were conducted in accordance with the guidelines of the

Institutional Animal Care and Use Committee at The Ohio State University.

Animals were vaccinated at 4-5 weeks, boosted after 3 weeks, and challenged

after 2 weeks of boost i.e., day post-vaccination (DPV) 35. For each vaccination dose,

7 pigs intranasally received 10 TCID50 equivalent of inactivated H1N2 virions (KAg) or

KAg entrapped within the nanoparticles (KAg nanovaccine) suspended in 2 mL DMEM.

The challenge SwIAV inoculum consisted of a virulent, zoonotic, and heterologous

6 SwIAV H1N1 (6x10 TCID50) of which 1 mL was administered intranasally and 1 mL

intratracheally (340). Plasma samples were collected at the time of vaccination and

necropsy. After challenge the rectal temperature was recorded daily, and nasal swab

samples were collected in 2 mL of DMEM at four days post-challenge (DPC). Pigs were

euthanized at six DPC and lungs were examined and scored for gross lesions (48).

Broncho-alveolar lavage (BAL) fluid was collected for virus titration and lung lysate

(prepared using 1 g of tissue from the right apical lobe suspended in 3 mL of DMEM,

which was homogenized and the supernatant was collected) was analyzed for antibody

response (345). Lung tissues were formalin fixed for histopathological and

immunohistochemical evaluations. PBMCs were isolated by using density gradient

medium Lymphoprep in SepMate-50 tubes (Stemcell, BC, Canada) at DPC 0 and 6 for

lymphocyte proliferation and flow cytometry assays.

3.3.3 Cell proliferation and flow cytometry assays

SwIAV antigen-specific lymphocyte proliferation was assessed using PBMCs and

the cell titer 96 aqueous non-radioactive proliferation assay kit (Promega, WI) as per the

manufacturer's instructions. One million cells per well were seeded in triplicates in 96 well U-bottom plate (Greiner bio-one, NC) and stimulated with live SwIAV H1N2 at 0.1

0 MOI or with medium control. After 72 h of incubation at 37 C in a 5% CO2 incubator,

MTS+PMS solution was added and the OD490nm was measured after 4 h using an ELISA

plate reader (Spectramax Plus384, Molecular Devices, CA). Stimulation index (SI) was

determined by dividing OD of stimulated PBMCs by OD of cell control of the same pig.

PBMCs isolated at DPC 0 were also analyzed to determine the frequency of different T

cell subsets by flow cytometry. At DPC 6, isolated PBMCs were stimulated with live

SwIAV H1N2 or H1N1 at MOI 0.1 for 72 h, and cells were immunostained and analyzed

by flow cytometry to determine the frequency of activated (IFN-γ+) T cell subsets as

described previously (205). Antibodies used in the assay were anti-porcine CD3έ, -CD4α and -CD8α (Southernbiotech, AL), -CD8β, -δ-chain, and anti-pig IFNγ (BD biosciences,

CA) along with their respective isotype controls.

3.3.4 Virus titration

Serial tenfold dilutions of BAL fluid and nasal swabs were prepared in DMEM

supplemented with TPCK-trypsin (1µg/mL) and transferred to MDCK cells grown on 96

0 well cell culture plates. After 48 h of incubation at 37 C in a 5% CO2 incubator, cells

were immunostained using IAV nucleoprotein specific primary antibody (#M058,

CalBioreagents, CA) followed by AlexaFluor 488 conjugated goat anti-mouse IgG (H+L)

antibody (Life technologies, OR). Immunofluorescence was recorded using fluorescent

microscope (Olympus, NY) and infectious virus titer was calculated using the Reed and

Muench method (205, 347).

3.3.5 Antibody titration

Hemagglutination inhibition (HI) titer and SwIAV-specific antibody responses

were determined as described previously (205). Undiluted nasal swab samples and 1:200

dilutions of BAL fluid, plasma and lung lysates were used in IgG and IgA antibody

response comparison among the pig groups. Virus neutralization titer (VNT) in BAL

fluid was also determined using procedures described previously (205).

3.3.6 Histopathology and Immunohistochemistry (IHC) analyses

Five micron sections of apical, cardiac and diaphragmatic lung lobes of pigs were stained with hematoxylin and eosin (H&E) and examined microscopically for histopathological changes as described previously (48). Briefly, lesion severity was scored by the distribution or by the extent of lesions within the sections and PMN

infiltration and graded 0 to 3. SwIAV-specific antigen in the lungs was detected by IHC

as described previously (206, 346) with a few modifications. SwIAV nucleoprotein

specific antibody (#MO58, CalBioreagents, CA) was used for immunostaining the viral

antigens followed by treatment with VECTASTAIN elite ABC reagent (#PK-7100,

Vector Labs, CA) to develop positive signal as per manufacturer’s instructions. The reactivity of viral antigenic mass in the airway epithelial cells was evaluated in IHC analysis.

3.3.7 Statistical analysis

Data are presented and compared as median and range of 7 or 8 pigs in different

groups. HI and VN titers are presented as geometric mean ± 95% CI. Virus titers were

log transformed and analyzed (346). Non-parametric Kruskal-Wallis test followed by

Dunn’s post-hoc test was used to compare the data in GraphPad Prism 5 (GraphPad

Software, Inc., CA) considering a P < 0.05 as statistically significant.

3.4 Results

3.4.1 Physical characteristics of SwIAV nanovaccine

The morphology of the synthesized polyanhydride nanoparticles was spherical and the size of the majority of particles was between 100 and 200 nm as determined by scanning electron photomicrographs (Figure 3.1A). The mean diameter of the antigen loaded nanoparticles as determined by ImageJ software (and confirmed with light

scattering) was 181 ± 56 nm (Figure 3.1B) (387, 388). The encapsulation efficiency of

SwIAV H1N2 antigens within the nanoparticles was determined to be 60%.

3.4.2 Pre-challenge cellular and humoral immune responses in pigs

PBMCs isolated from vaccinated pigs at DPV 35/DPC 0 were stimulated with the

vaccine virus (SwIAV H1N2) and lymphocyte proliferation was assessed. Our data

suggested that lymphocyte stimulation index in KAg nanovaccine vaccinated pigs was

significantly higher compared to that in the animals vaccinated with the KAg alone

(Figure 3.2A). KAg nanovaccine-immunized pigs but not the KAg alone vaccinated pigs

had significantly higher frequency of T helper/memory cells (CD3+CD4+CD8αα+)

(Figure 3.2B) and cytotoxic T lymphocytes (CTLs) (CD3+CD4-CD8αβ+) compared to the

mock group (Figure 3.2C). The HI titer in plasma at DPC 0 was significantly higher

against the vaccine virus in both KAg nanovaccine- and KAg-immunized pigs compared to mock controls, but no significant differences were observed between two vaccine groups (Figure 3.2D). The IgG antibody response to the homologous vaccine virus in plasma at DPC 0 was also similar between KAg- and KAg nanovaccine-immunized animals (Figure 3.2E) and the antibody response against the challenge heterologous virus was weak (data not shown).

3.4.3 Clinical and pathological changes and virus load post-challenge

The comparative protective efficacy of KAg nanovaccine vs. KAg alone was assessed in heterologous virus-challenged pigs. A body temperature of 104oF is accepted

as fever in pigs (389). Mock-challenged and KAg-vaccinated pigs had fever at DPC 1 to

4, while KAg nanovaccine-immunized pigs had fever only at DPC 1 (Figure 3.3A).

Though not statistically significant, the gross lesions involved a smaller area of the lungs

in KAg nanovaccine-immunized animals compared to the animals vaccinated with KAg

(Figure 3.3B). The H&E scores at DPC 6 were comparable among all three groups of

virus challenged pigs (Figure 3.3C). The mean IHC scores for SwIAV antigen reactivity

in the lung sections was significantly reduced in KAg nanovaccine-immunized animals

compared to the mock vaccinated and virus challenged pigs, but not in KAg alone

vaccinated group (Figure 3.3D); representative lung pictures of bronchial epithelial cells

with virus antigens is shown (Figure 3.3E).

Consistent with the lung IHC scores, the nasal virus shedding at DPC 4 in KAg

nanovaccine group was six- and eight-fold lower than KAg and mock vaccinated and

SwIAV H1N1 challenged groups, respectively (Figure 3.3F). However, at DPC 6 the

challenge virus titer in BAL fluid was reduced in both KAg (40 fold) and KAg

nanovaccine-immunized (37 fold) pig groups compared to mock vaccinated and virus

challenged pigs (Figure 3.3G), suggesting less replication/shedding in the airway

epithelium of the vaccinated pigs.

3.4.4 Activated recall IFNγ secreting lymphocyte response in the blood post-

challenge

To assess the recall cellular immune response post-challenge, isolated PBMCs

were stimulated with vaccine (H1N2) and challenge (H1N1) SwIAV virions to estimate

the frequencies of activated (i.e., IFNγ+) lymphocyte subsets. The frequency of activated

T cells (CD3+IFNγ+) (Figure 3.4A) and T helper cells (CD3+CD4+IFNγ+) (Figure 3.4D) were significantly higher in animals vaccinated with KAg nanovaccine stimulated with both the H1N1 and H1N2 viruses compared to animals vaccinated with KAg alone. The frequency of activated CTLs (CD3+CD4- CD8αβ+IFNγ+) were significantly higher in both

KAg nanovaccine and KAg vaccinated pig groups stimulated with both the H1N1 and

H1N2 viruses compared to mock control pig group (Figure 3.4C). The innate CD3-IFNγ+

non-T cells (Figure 3.4B) as well as CD3-CD4-CD8α+IFNγ+ NK cells (Figure 3.4E) were

found to be significantly higher in KAg- compared to KAg nanovaccine-immunized

animals.

3.4.5 Humoral immune response post-challenge

In post-challenged pigs, the specific antibody response against the challenge virus

was analyzed both locally and systemically (Figure 3.5). The virus-specific IgA antibody

response in nasal swabs (Figure 3.5A), BAL fluid (Figure 3.5B), and lung lysates (Figure

3.5C) were significantly higher in KAg (but not KAg nanovaccine) vaccinated compared

to both mock control and mock challenge pig groups; while the IgA levels between the

KAg and KAg nanovaccine-immunized pig groups was not statistically different

Specific IgG response in plasma (Figure 3.5D) and BAL fluid (Figure 3.5E), HI titers in

BAL fluid (Figure 3.5F) and plasma (Figure 3.5G), and VN titers in BAL fluid (Figure

3.5H) were comparable in the animals immunized with KAg and KAg nanovaccine

3.5 Discussion

Induction of protective immunity against influenza is possible under field conditions only by developing a vaccine that elicits robust immune responses against conserved viral antigens, but current SwIAV vaccines have failed to do that. To achieve that goal, we evaluated the immunogenicity and cross-protective efficacy of a polyanhydride-based nanoparticle encapsulated killed SwIAV vaccine administered intranasally in influenza antibody-free pigs. The KAg nanovaccine rescued pigs from heterologous virulent SwIAV induced clinical symptoms and fever, associated with reduced gross lung pathology, slightly reduced nasal virus shedding and antigenic load in the lungs. This is likely mediated by the induction of robust antigen-specific cell- mediated immune responses against the challenge virus, in spite of the lack of induction of an enhanced antibody response. The lack of an enhanced antibody response in the

KAg nanovaccine-immunized animals may be attributed to little to no disease being observed in those animals with only modest viral replication in the lungs. These observations would point to an absence of an anamnestic antibody response, which merits further investigation.

Polyanhydride micro/nanoparticles based on CPTEG, CPH and SA have been widely studied for vaccine and drug delivery against viral, bacterial and parasitic pathogens in rodent models (185). In this work, we used the 20:80 CPTEG:CPH-based nanoparticle formulation because it has been shown to be a potent adjuvant based on its ability to enhance both humoral and cellular immune responses to vaccine antigens (382,

387, 388, 390). Amphiphilic polyanhydrides based on CPTEG and CPH facilitate slow

release of antigen, conserve protein structure and stability, and maintain immunogenicity

of the antigenic epitopes (232). Polyanhydride nanovaccines have also induced antigen-

specific memory T cells and profound recall responses in mice (241, 391). A previous

study involving 20:80 CPTEG:CPH-based nanoparticle encapsulated recombinant H5

+ trimer (H53) vaccine against H5N1 influenza challenge in mice enhanced the CD4 T cell recall response and showed protective efficacy (209). Consistent with these findings, the

current study in pigs with intranasal immunization with KAg nanovaccine induced a

strong cell-mediated immune response by enhancing the antigen specific lymphocyte

proliferation and increasing the frequency of T helper/memory cells and CTLs.

Furthermore, in post-challenged pigs, the IFN-γ producing T cell sub-population was

increased in ex vivo cultures of PBMCs stimulated with antigens from both vaccine and

challenge viruses.

In addition, it is important to use low doses of antigens in vaccine formulations in

order to prevent unwanted side effects. Polyanhydride nanovaccines have been reported

to induce strong antibody response along with dose-sparing capabilities. For example, a

suboptimal dose of ovalbumin (25 µg) in polyanhydride particles delivered

subcutaneously in mice induced antibody response similar to that induced by delivering

400-1600 µg of the soluble antigen (392). A 20:80 CPTEG:CPH particle-based

formulation containing 20 µg hemagglutinin protein of H5N1 virus was also shown to

induce robust VN titer in a homologous challenge trial in mice (209). In the current study

in pigs the 20:80 CPTEG:CPH polyanhydride nanoparticle-encapsulated SwIAV KAg induced similar systemic and local antigen-specific VN and HI antibody titers compared

to pigs immunized with the KAg alone. Intranasal vaccination of mice has been shown to

induce better mucosal IgA responses compared to parenteral immunization. In mice,

intranasal delivery of 50:50 CPTEG:CPH polyanhydride based vaccine showed

protective efficacy against Yersinia pestis challenge mediated mainly by a high titer

antibody response (243). In contrast to mice, it appears that intranasal administration of

the 20:80 CPTEG:CPH polyanhydride nanovaccine formulation induces more of a

cellular than a humoral immune response in pigs.

The size, charge, and other characteristics of nanoparticles play a major role on favorable outcome of vaccination. Particle size of less than or equal to 500 nm is considered suitable to be taken up readily by APCs (337, 338). In mice, 360-470 nm

particles were found to be suitable for adequate pulmonary distribution (174). Thus, our

100-300 nm polyanhydride nanoparticles were of appropriate size for intranasal vaccine

delivery in pigs. We detected low and comparable titers of the challenge virus in the BAL

fluid of pigs that received KAg nanovaccine and soluble vaccine antigen at DPC 6. This

indicates that, despite the benefit of improved cellular immunity, there is a need to

improve the efficacy of polyanhydride nanoparticle-based vaccine delivery platform in pigs for induction of better cross-protection.

The controlled release of antigen, pathogen mimicking properties and in vivo immune modulation capabilities of polyanhydride nanoparticles are dependent on polymer chemistry (232, 388, 390, 393). Hence, the polymer formulation and route of delivery best suited in one animal model may or may not be suitable in other animal model or species. Our preliminary findings suggest that there was no adverse effect of

94 intranasal administration of 20:80 CPTEG:CPH polyanhydride nanoparticles in pigs.

Furthermore, this vaccine delivery platform provided clinical protection against a virulent heterologous virus challenge by inducing robust antigen-specific cell-mediated immune response, in spite of not inducing enhanced antibody response, indicating the immunological benefits of the nanovaccine in pigs. Our future studies will be focused on optimizing the nanoparticle chemistry and vaccine formulation so as to exploit the inherent adjuvant properties of polyanhydride nanoparticles aimed to further enhance the cross-protective efficacy of SwIAV vaccines in pigs.

3.6 Acknowledgements

We are thankful to Dr. Juliette Hanson and Megan Strother who provided help in animal studies. This work was supported by Nanovaccine Research Initiative, Iowa State

University and Agriculture and Food Research Initiative Competitive Grant no. 2013-

67015-20476 from the USDA-NIFA. Salaries and research support were provided by state and federal funds appropriated to OARDC, The Ohio State University.

Experimental groups N Vaccine formulations First vaccination Second vaccination Day of Challenge (DPV 0 / DPC -35) (DPV 21 / DPC -14) (DPV 35 / DPC 0) Mock 7 Mock inoculum Mock inoculum Mock inoculum Mock + Ch. 8 Mock inoculum Mock inoculum SwIAV OH7 (H1N1) KAg + Ch. 8 Inactivated SwIAV (H1N2) Inactivated SwIAV (H1N2) SwIAV OH7 (H1N1) KAg Nanovaccine + Ch. 7 SwIAV Nanovaccine (H1N2) SwIAV Nanovaccine (H1N2) SwIAV OH7 (H1N1) DPV - Day post-vaccination; DPC - Day post-challenge; Ch - SwIAV H1N1 Challenge

Table 3.1: Experimental design showing assignment of pigs in each group (n = number of pigs)

Figure 3.1 Physical characterizations of polyanhydride nanoparticles. (A) Surface morphology of KAg nanovaccine nanoparticles depicted by scanning electron photomicrograph (25Kx magnification). (B) Size distribution of KAg nanovaccine nanoparticles presented as percentage of total particles.

Figure 3.2 Cellular and humoral immune responses in vaccinated pigs pre-challenge. (A) PBMCs isolated at DPV35/DPC0 were stimulated with vaccine virus (SwIAV H1N2) and lymphocyte proliferation was determined. PBMCs isolated at DPV 35/DPC0 were also immunostained for phenotyping: (B) CD3+CD4+CD8αα+ (T helper/memory) and (C) CD3+CD4-CD8αβ+ (CTLs). For humoral immune response analyses: (D) HI titer and (E) total IgG antibody responses were determined as described in Methods. Each bar is the median and range value of 7 or 8 pigs. Panel D shows geometric mean ± 95% CI. Data were analyzed by non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc test. Asterisk refers to statistical difference between two indicated pig groups (*p<0.05; **p<0.01 and ***P<0.001).

Figure 3.3 Clinical and pathological changes and SwIAV H1N1 titration in vaccinated pigs post-challenge. (A) Graphs showing the average rectal body temperature recorded daily post-challenge; (B) Gross lung lesions in pigs recorded during necropsy at DPC 6; (C) Microscopic lung lesions scores of H&E stained lung sections; (D) Immunohistochemistry analysis of lung sections for SwIAV antigens; and (E) Representative pictures of lung sections of pigs analyzed for SwIAV antigens by Immunohistochemistry. Virus titers in (F) nasal swabs at DPC 4 and in (G) BAL fluid at DPC 6 are shown. The dashed line at temperature 1040C indicates fever in pigs. Each bar is the median and range value of 7 or 8 pigs. Data were analyzed by non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc test. Asterisk refers to statistical difference between two indicated pig groups (*p<0.05; **p<0.01 and ***P<0.001).

Figure 3.4 Immunophenotyping of activated (IFN-γ+) lymphocytes in PBMCs post- challenge. PBMCs were restimulated with the vaccine (H1N2) or challenge (H1N1) SwIAV ex vivo. Frequencies of (A) CD3+IFNγ+ (T cells); (B) CD3-IFNγ+ cells (non-T cells); (C) CD3+CD4-CD8αβ+IFNγ+ (CTLs); (D) CD3+CD4+IFNγ+ (T-helper cells); and (E) CD3-CD4-CD8α+IFNγ+ (NK cells) are shown. Each bar is the median and range value of 7 or 8 pigs. Data were analyzed by non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc test. Asterisk refers to statistical difference between two indicated pig groups (*p<0.05; **p<0.01 and ***P<0.001).

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Figure 3.5 Humoral immune responses in vaccinated pigs post-challenge. Antibody analysis was performed against challenge virus at DPC 6 by ELISA to determine the levels of humoral response: IgA in (A) nasal swab; (B) BAL fluid; (C) lung lysate; and IgG response in (D) plasma and (E) BAL fluid. SwIAV H1N2 specific titers of HI antibodies in (F) BAL fluid and (G) plasma; and (H) VN titers in BAL fluid were determined. Each bar is the median and range value of 7 or 8 pigs. Panels F, G, H show geometric mean ± 95% CI. Data were analyzed by non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc test. Asterisk refers to statistical difference between two indicated pig groups (*p<0.05; **p<0.01 and ***P<0.001).

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Chapter 4: Mucosal immunity and protective efficacy of intranasal inactivated

influenza vaccine is improved by chitosan nanoparticle delivery in pigs

4.1 Abstract

Annually, swine influenza A virus (SwIAV) causes severe economic loss to swine industry. Currently used inactivated SwIAV vaccines administered by intramuscular injection provide homologous protection, but limited heterologous protection against constantly evolving field viruses, attributable to induction of inadequate levels of mucosal IgA and cellular immune responses in the respiratory tract. A novel vaccine delivery platform using mucoadhesive chitosan nanoparticles administered through intranasal route has the potential to elicit strong mucosal and systemic immune responses in pigs. In this study, we evaluated the immune responses and cross-protective efficacy of

intranasally-delivered chitosan encapsulated inactivated SwIAV vaccine in pigs. Killed

SwIAV H1N2 (δ-lineage) antigens (KAg) were encapsulated in chitosan polymer-based

nanoparticles (CNPs-KAg). The candidate vaccine was administered twice intranasally as

mist to nursery pigs. Vaccinates and controls were then challenged with a zoonotic and

virulent heterologous SwIAV H1N1 (γ-lineage). Pigs vaccinated with CNPs-KAg

exhibited enhanced IgG serum antibody and mucosal secretory IgA antibody responses in

nasal swabs, bronchoalveolar lavage (BAL) fluids and lung lysates that were reactive

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against homologous (H1N2), heterologous (H1N1) and heterosubtypic (H3N2) IAV

strains. Prior to challenge, increased frequency of cytotoxic T lymphocytes, antigen-

specific lymphocyte proliferation and recall IFN-γ secretion by restimulated peripheral

blood mononuclear cells in CNPs-KAg compared to control KAg-vaccinates were

observed. In CNPs-KAg vaccinated pigs challenged with heterologous virus reduced

severity of macroscopic and microscopic influenza-associated pulmonary lesions were

observed. Importantly, the infectious SwIAV titers in nasal swabs (days post-challenge

4) and BAL fluid (days post-challenge 6) were significantly (p<0.05) reduced in CNPs-

KAg vaccinates but not in KAg vaccinates when compared to the unvaccinated challenge controls. As well, increased frequency of T-helper memory cells and increased levels of recall IFNγ secretion by tracheobronchial lymph nodes cells was observed. In summary, chitosan SwIAV nanovaccine delivered by intranasal route elicited strong cross-reactive mucosal IgA and cellular immune responses in the respiratory tract that resulted in reduced nasal viral shedding and lung virus titers in pigs. Thus, chitosan-based influenza nanovaccine may be an ideal candidate vaccine for use in pigs, and pig is a useful animal model for preclinical testing of particulate intranasal human influenza vaccines.

4.2 Introduction

Influenza is caused by influenza A virus (IAV) of Orthomyxoviridae family. It is an economically important disease in the global pig industry (86, 378). Virulent swine

IAV (SwIAV) infection leads to acute febrile respiratory disease which is often complicated with secondary bacterial infections (42). SwIAV increases its genetic

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diversity through frequent antigenic drift and antigenic shift. So far H1N1, H1N2 and

H3N2 subtypes are the major SwIAV circulating in pig populations (14). Since epithelial

cells lining the porcine respiratory tract bear receptors for both avian and human IAVs,

pigs can be infected with IAV from different hosts and this event favors genetic

assortment and adaptation of novel influenza strains of zoonotic and even pandemic

potential (73). The pandemic H1N1 virus of 2009 and more recent ‘H3N2 variant’ virus in the US are recent examples of swine-origin IAVs which cause infection and resultant pulmonary disease in humans (379, 394). Controlling influenza in pigs through vaccination serves dual benefits by protecting economic loss in swine industry and preventing possible public health risk that these reassorted SwIAV pose for humans.

Swine influenza vaccines are commercially available. These are multivalent whole inactivated virus (WIV) vaccines that are administered intramuscularly (IM) (119).

The WIV vaccines provide protection against homologous virus infections but do not induce adequate heterologous immunity against constantly evolving IAVs that develop by point mutation(s) (119, 395). Moreover, the IM route used for WIV vaccines does not elicit adequate mucosal immune responses which are essential for providing cross- protective immunity against multitude of variant IAVs (143, 144). Intranasal (IN) vaccine that targets mucosal immune system of the respiratory tract can be a useful alternative to the current IM influenza vaccines used in pigs. Nasal mucosal vaccination not only induces strong protective immune responses at mucosal sites in the respiratory tract but also enhances immunity at distal mucosal and systemic sites (396, 397).

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Biodegradable and biocompatible polymer-based nanoparticle (NP) formulation(s) provide an innovative strategy of vaccine antigen delivery to mucosal sites

(398). Particulate vaccines facilitate antigen uptake by professional antigen presenting cells (APCs), maintain slow and sustained antigen release, prevent the antigen(s) from undesirable enzymatic degradation and potentiate the levels of protective immunity (334,

398). Different types of nanoparticles are investigated for IN delivery of influenza vaccine antigens. For example, IN immunization in mice using liposome based DNA and subunit influenza nanovaccines are shown to elicit mucosal, cellular and humoral immune responses (399, 400). PLGA nanoparticles entrapping highly conserved H1N1 influenza virus peptides administered IN enhances the epitope specific T cell response and protective efficacy in pigs (205). Ferritin based IN influenza nanovaccine is shown to enhance mucosal secretary IgA and T cell responses and confers homo- and heterosubtypic protection in mice (401). In our previous study, killed SwIAV antigen (KAg) encapsulated in PLGA polymer-based NP and delivered IN induced a robust cross- reactive cell-mediated immune response associated with significant clearance of a heterologous challenge virus from the lungs of pigs (402). In another study, encapsulation of KAg in polyanhydride polymer-based NP also enhanced the cross-reactive cell- mediated immune response against SwIAV (403). However, both PLGA and polyanhydride polymer-based NP SwIAV vaccines used IN in these studies failed to elicit mucosal IgA and systemic IgG antibody responses, most likely due to their biased ability to induce strong T helper 1 (Th1) but not Th2 responses. This Th1-biased response failed to reduce the nasal virus shedding in pigs (402, 403).

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In the present study, we used chitosan, a natural mucoadhesive polymer for

encapsulation of SwIAV KAg (CNPs-KAg) and performed a heterologous vaccine-

challenge trial in nursery pigs. Due to its cationic nature, chitosan binds readily to

mucosal surfaces. Chitosan also possesses adjuvant properties, a feature which promotes

immune activation (404). Previous studies have shown that chitosan nanoparticles form

an attractive platform for mucosal vaccine delivery. For example, live Newcastle disease

virus (NDV) encapsulated in chitosan nanoparticles and delivered through oral and

intranasal routes in chickens induced higher secretary IgA antibody responses in

intestinal mucosa and enhanced protective efficacy against highly virulent NDV strain

challenge infection (405). Similarly, influenza subunit/split virus vaccine delivered in

chitosan nanoparticles by the IN route improves systemic and mucosal antibody and cell-

mediated immune responses in mice (354, 406, 407). Hence, we hypothesized that IN

delivery of chitosan-based nanovaccine would enhance both mucosal antibody and

cellular immune responses and provide better protective immunity against SwIAV in pigs

compared to soluble IN inactivated vaccine. Our results demonstrated that CNPs-KAg IN vaccination improved mucosal IgA response in the entire respiratory tract and also elicited cell-mediated immune response against different subtypes of SwIAV resulting in reduced nasal viral shedding and infectious virus burden in the pulmonary parenchyma.

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4.3 Materials and methods

4.3.1 SwIAV propagation and inactivation

Field isolates of IAVs A/Swine/OH/FAH10-1/10 (H1N2) (339),

A/Swine/OH/24366/2007 (H1N1) (340) and A/Turkey/OH/313053/2004 (H3N2) (408) were propagated in Madin-Darby canine kidney (MDCK) cells. The H1N2

A/Swine/OH/FAH10-1/10 (H1N2-OH10) was used for CNPs-KAg vaccine preparation and H1N1 A/Swine/OH/24366/2007 (H1N1-OH7) was used for the virulent virus challenge infection. The H3N2 A/Turkey/OH/313053/2004 (H3N2-OH4) was used together with H1N2-OH10 and H1N1-OH7 for ex vivo cross-reactive immune analysis.

The H1N2-OH10 vaccine virus and H1N1-OH7 challenge virus are heterologous to each other with 77% HA gene identity, whereas H3N2-OH4 virus, originally isolated from turkeys, is heterosubtypic to other two SwIAV with HA gene identity 63% (339, 340,

408). For vaccine preparation, cell culture fluid of H1N2-OH10 virus grown in MDCK cells was harvested and subjected to sucrose gradient ultracentrifugation. The virus pellet was suspended in phosphate-buffered saline (PBS), titrated for infectious virus titer and inactivated by using binary ethyleneimine (BEI) (Sigma, MO) as described previously

(402).

4.3.2 Preparation of chitosan based nanovaccine and in vitro characterization

Chitosan NP loaded killed SwIAV antigen (KAg) (CNPs-KAg) formulation was prepared by the ionic gelation method as described previously (405, 409-411) with some modifications. Briefly, 1.0% (w/v) low molecular weight chitosan polymeric (Sigma,

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MO) solution was prepared in an aqueous solution of 4.0% acetic acid under magnetic

stirring until the solution became clear. The chitosan solution was sonicated; pH was

adjusted to 4.3 and filtered via 0.44 µm syringe filter. Five mL of 1.0% chitosan solution

was added to 5.0 mL deionized water and incubated with 3.0 mg SwIAV KAg dissolved

in 1.0 mL 3-(N-morpholino) propanesulfonic acid (MOPS) buffer pH 7.4. Consequently,

2.5 mL of 1.0% (w/v) tripolyphosphate (TPP) (Sigma, MO) dissolved in 2.5 mL

deionized water was added into the chitosan polymer solution with continuous magnetic

stirring at room temperature (RT, 220C). The formulated SwIAV nanovaccine was centrifuged at 10,000 rpm for 10 min, dispersed in MOPS buffer pH 7.4, lyophilized with a cryoprotectant and stored at -800C.

Particle size and zeta potential of empty and vaccine antigen loaded nanoparticles

were measured after dispersion in physiological buffer saline (PBS, pH 7.4) and stored at

40C for at least 30h by dynamic light scattering (DLS) method using a zeta-sizer coupled with an MPT-2 titrator (Malvern) as described previously (412). During each vaccination,

CNPs-KAg nanoparticles were freshly prepared and used. The morphology of

nanoparticles was obtained by using the cold field emission Hitachi S-4700 scanning

electron microscope (SEM) (402). Briefly, the powder form of nanoparticles was loaded

on to aluminum stubs and coated with platinum prior to examination under the

microscope. Protein loading efficiency in CNPs-KAg was estimated indirectly by

determining the difference between initial amount of protein used for loading CNPs and

the protein left in the supernatant (405). In vitro protein release profile in CNPs-KAg

suspended in PBS for up to 15 days was estimated and expressed as the cumulative

108 percentage release of SwIAV antigen at each time point as described previously (402). In brief, CNPs-KAg suspended in 500µL PBS (pH 7.4) in triplicate in Eppendorf tubes was incubated at 370C in a revolving roller apparatus. At indicated time point tubes were centrifuged, supernatant collected and pellet was resuspended in fresh 500µL PBS.

Protein released in to the supernatant was estimated by micro BCA protein assay kit

(Thermo Scientific, MA) and expressed as the percentage of cumulative protein released over the initial amount at time zero in particles.

4.3.3 In vitro uptake of CNPs-KAg by antigen presenting cells (APCs)

Peripheral blood mononuclear cells (PBMCs) isolated from 9 to 10 week-old pigs were used for the in vitro antigen uptake study. Cells were suspended in enriched-

Roswell Park Memorial Institute (E-RPMI) medium and seeded 1 million cells/well in 96

0 well cell culture plates. After overnight incubation at 37 C in 5% CO2, unattached cells were removed. The attached monocyte/macrophage cells were treated with SwIAV KAg or CNPs-KAg containing the antigen at 10µg/mL concentration for 10 min, 30 min and

150 min. After the indicated period of incubation, the cells were fixed with 80% acetone, stained with IAV nucleoprotein-specific antibody (CalBioreagents, CA) followed by

Alexa Fluor 488 conjugated goat anti-mouse IgG antibody (Life technologies, OR). Cells were evaluated under fluorescent microscope (Olympus IX70) and photomicrographs were taken (20X). For evaluation of SwIAV antigen uptake from CNPs-KAg treated porcine monocyte/macrophages prepared from three pigs PBMCs separately were incubated in 48-well plates seeded with 2x106 cells per well overnight as described

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above. Cells were treated with KAg or CNPs-KAg at SwIAV antigen concentration

10µg/mL for 10 min, 30 min and 150 min. Positive control was MDCK cells infected with SwIAV H1N2-OH10 at MOI 1 for 12h. After indicated period of treatment cells

were fixed using 1% paraformaldehyde (PFA), permeabilized and stained with IAV

nucleoprotein-specific antibody (CalBioreagents, CA) followed by treatment with goat

anti-mouse IgG Alexa Flour488 conjugated secondary antibody. We acquired 50000

events in BD Aria II flow cytometer (BD Biosciences, CA) and analyzed the data by

using the FlowJo software (Tree Star, OR).

4.3.4 In vitro generation and stimulation of porcine dendritic cells

Porcine monocyte derived dendritic cells (MoDCs) were prepared from PBMCs isolated from seven pigs as described previously (413) with few modifications. Briefly,

25 million PBMCs per mL were seeded in each well of 6-well culture plates. After

0 overnight incubation at 37 C in a 5% C02 incubator, non-adherent cells were discarded and adhered cells were treated with GM-CSF (25ng/mL) and IL-4 (10ng/mL) cytokines.

Half of the culture media was replaced on every third day. On day 7 the plate was centrifuged at 2000 RPM at 40C for 5 min and the supernatant was harvested gently, and

generated immature MoDCs were stimulated in the same plate without seeding into fresh

plates with medium only, LPS control (10µg/mL), KAg (10µg/mL) and CNPs-KAg

containing 10µg/mL of KAg for 48h. The culture supernatant was harvested and

estimated the levels of innate, proinflammatory and Th1 cytokines, IFN-α, TNF-α. IL-1β,

IL-12, IL-6 and IL-10 by ELISA as described previously (414).

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4.3.5 Experimental design

Caesarian-delivered colostrum-deprived (CDCD) and bovine colostrum-fed

influenza antibody-free Large White-Duroc crossbred piglets were raised in our BSL2

animal facility at OARDC. Piglets at 4 weeks of age (male and female) were randomly

assigned into one of the three experimental groups and kept in separate isolation rooms

(Table 4.1). The first IN vaccination was performed at 5 weeks of age and the second IN

booster vaccination at 8 weeks of age. All piglets receiving virulent SwIAV were

challenged at 10 weeks of age. Separate groups of pigs were vaccinated IN with DMEM

7 (Gibco) or with 1x10 TCID50 equivalent of -KAg or CNPs-KAg suspended in 2 mL

DMEM by intranasal mist as described previously (402). The challenge infection was

6 done using heterologous H1N1-OH7 SwIAV (6×10 TCID50) in 2 mL, divided into 1 mL

administered IN and 1 mL intratracheally as described previously (205, 402, 403).

Serum samples were collected at days post-vaccination (DPV) 21 and 35. The rectal temperatures were recorded daily from day post-challenge (DPC) 0 onward and nasal swab samples were collected at DPC 0, DPC 4 and DPC 6. Pigs were euthanized at

DPC 6 and serum and bronchoalveolar lavage (BAL) fluid were collected. During necropsy, lungs were examined for macroscopic pneumonic lesions and scored as described previously (402). Lung lysates were prepared by homogenization of 1.0 g of lung tissue collectedly from the right apical lobe (402). Nasal swabs, sera, BAL fluid and lung lysate samples were stored at -800C until processed for antibody and virus titration.

The PBMCs were isolated from blood at DPV 35/DPC 0 and DPC 6 (402). Mononuclear

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cells were harvested from tracheobronchial lymph nodes (TBLN-MNCs) at DPC 6 as

described previously (414).

4.3.6 Antibody titration

Hemagglutination inhibition (HI) antibody titers against IAVs H1N1-OH7,

H1N2-OH10 and H3N2-OH4 in sera and BAL fluid samples were determined as

described previously (402). The SwIAV-specific IgG and IgA antibodies in nasal swabs,

sera, BAL fluids and lung lysates were determined by ELISA (402). Briefly, 96 well

plates (Greiner bio-one, NC) were coated overnight with respective pre-titrated IAV antigen (5µg/mL) and blocked with 5% skim milk powder containing 0.05% Tween-20

for 2hr at RT. After wash five-fold dilutions of nasal swab, serum , BAL fluid and lung

lysate samples in in PBS containing 2.5% skim milk powder and 0.05% Tween-20 were

added to marked duplicate wells, incubated for 2h at RT, washed and horse radish

peroxidase (HRP)-conjugated goat anti-pig IgA (Bethyl Laboratories Inc., TX) or goat

anti-pig IgG (KPL, MD) was added. Finally, the antigen and antibody interaction was

detected by using 1:1 mixture of peroxidase substrate solution B and TMB peroxidase

substrate (KPL, MD). The reaction was stopped using 1.0 M phosphoric acid and optical

density (OD) was measured at 450nm using Spectramax microplate reader (Molecular

devices, CA).

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4.3.7 Antigen specific cell proliferation assay

The PBMCs isolated at DPV 35/DPC 0 were cultured together with H1N1-OH7,

H1N2-OH10 or H3N2-OH4 SwIAV at 0.1 multiplicity of infection (MOI) and incubated

0 at 37 C in 5% CO2 incubator for 72h. Antigen-specific lymphocyte proliferation was determined by using the cell titer 96 aqueous non-radioactive proliferation assay kit

(Promega, WI). The cell proliferative response was compared among groups using lymphocyte stimulation index values as described previously (402).

4.3.8 Cytokine ELISA

PBMCs isolated at DPC 0 and TBLN-MNCs at DPC 6 were cultured with H1N2-

OH10, H1N1-OH7 or H3N2-OH4 SwIAV at 0.1 MOI. After 72h of stimulation the supernatant was collected and interferon gamma (IFNγ) secretion was determined by

ELISA as described previously (402). Similarly, production of interleukin-6 (IL-6) in

BAL fluid collected at DPC 6 was determined by ELISA (414).

4.3.9 Virus titration

Viral titers contained in nasal swabs and BAL fluids were determined in ten-fold dilution of the samples in DMEM containing TPCK-trypsin (1µg/mL). The samples were transferred to quadruplicate 96 well cell culture plates wells containing overnight

0 cultured monolayers of MDCK cells and incubated for 72h, 37 C, 5.0% CO2. Cells were fixed with acetone and immunostained with IAV nucleoprotein-specific antibody

(#M058, CalBioreagents, CA) followed by Alexa Fluor 488 conjugated goat anti-mouse

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IgG (H+L) antibody (Life technologies, OR). Virus replication in cells was determined

by using immunofluorescence technique as described previously (402).

4.3.10 Histopathology of lungs

For histopathological analysis of pulmonary tissues, 10% formalin-inflated apical,

cardiac and diaphragmatic lobes were collected and further emulsion-fixed in 10%

neutral buffered formalin. Five μm sections of formalin-fixed, paraffin embedded apical, cardiac, and diaphragmatic lung lobes were stained with hematoxylin and eosin (HE) as previously described (402). The H&E-stained tissues sections were examined for microscopic changes of interstitial pneumonia, peribronchial, and perivascular

accumulation of mononuclear cells, bronchial exudates and epithelial changes related to

influenza infection. All these parameters were scored by a board certified veterinary

pathologist (SK) who was not provided with any vaccination history of pig groups in a

scale of 0 (no change compared from normal) to 3 (marked changes from normal) as

described previously (402).

4.3.11 Flow cytometry

The PBMCs isolated at DPC 0 and TBLN-MNCs at DPC 6 were immunostained

for T lymphocyte subset phenotyping as described previously (402). Antibodies used in

the flow cytometry were: anti-porcine CD3 (Southernbiotech, AL), CD4α

(Southernbiotech, AL), CD8α (Southernbiotech, AL) and CD8β (BD Biosciences, CA).

Briefly, the cells were blocked with 2% pig serum in fluorescence activated cell sorting

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(FACS) buffer and surface labelled with pig lymphocyte-specific purified, biotin or fluorochrome-conjugated antibodies or their respective antibody isotypes. Cells were fixed using 1% paraformaldehyde, washed, suspended in FACS buffer and acquired using BD Aria II flow cytometer (BD Biosciences, CA). Data analysis was done using

FlowJo software (Tree Star, OR).

4.3.12 Quantitative reverse transcription PCR (RT-qPCR)

Total RNA was extracted from PBMCs at DPC 0 and TBLN at DPC 6 using

TRIzol reagent (Invitrogen, CA) as per the manufacturer's instructions. NanoDropTM

2000c Spectrophotometer (Thermo Fisher Scientific, MA) was used to determine the

concentration and purity of RNA. cDNA was prepared from 1µg of total RNA using the

QuantiTect Reverse Transcription Kit (QIAGEN). Primers of housekeeping gene (β

actin) and target genes (T-bet and GATA-3) used in this experiment were described

previously (415). The mRNA expression was analyzed by 7500 Real-Time qPCR system

(Applied Biosystems, CA) using the qScriptTM One-Step SYBR Green qRT-PCR kit,

Low ROXTM (Quantabio, MA). The target gene expression level was normalized with

housekeeping gene levels and the fold change was determined by comparative 2−ΔΔCT

method (416).

4.3.13 Statistical analysis

Statistical analysis was performed by using non-parametric Kruskal-Wallis test

followed by Dunn’s post-hoc test using the software GraphPad Prism 5 (GraphPad

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Software, Inc., CA). Pig rectal temperature data was analyzed by repeated measure

ANOVA using Friedman test followed by Dunn’s pairwise comparison. Cytokine data

(Fig. 2) between two groups were analyzed by Mann-Whitney test. A p-value of less than

0.05 was considered statistically significant. The infectious virus titer was determined

using Reed and Muench method. Data were presented as the mean ± SEM of 3-5 pigs

except for the HI titers which were expressed as geometric mean with 95% confidence

interval.

4.4 Results

4.4.1 Characterization of CNPs-KAg vaccine candidate

The encapsulation efficiency of SwIAV KAg in chitosan nanovaccine formulation

was 67%. This result was comparable to encapsulation efficiency of chitosan NPs

entrapped with Salmonella outer membrane protein antigens (70%) (Unpublished data).

As determined by DLS, the average size of the empty (Figure 4.1A) and antigen loaded

(Figure 4.1B) NPs was 414.2nm and 571.7nm, respectively. Empty NPs showed two

peaks at 36nm (~10%) and 323nm (~90%) with polydispersity index (PDI) 0.39.

Likewise, antigen loaded NPs also had two peaks at 70nm (~15%) and 468nm (~85%)

with PDI 0.60. Data shows that the CNPs-KAg nanoparticles were polydispersed in

nature. SEM analysis showed the morphology of the empty NPs were spherical with

smooth surface (Figure 4.1C), while antigen loaded NPs had relatively rough and

irregular surface (Figure 4.1D). The surface charge of empty and antigen loaded chitosan

NPs was +1.88mV and +1.69mV, respectively. We observed 6% burst release i.e.,

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surface associated antigen release during the first 1h, and on an average 9% of antigen

was released after 24h of incubation. Further, slow and sustained release of antigen was

observed with cumulative release of approximately 46% after 15 days (Figure 4.1E).

To determine whether chitosan encapsulation of KAg enhances the uptake of antigen by APCs, we prepared monocyte/macrophages from PBMCs and allowed for interaction with KAg or CNPs-KAg and stopped the reaction at three different time points. Internalization of CNPs-KAg vaccine by monocytes/macrophages was observed within 10 min of treatment indicated by higher number of influenza-specific fluorescent signals compared to KAg treatment (Figure 4.1F). Further the uptake of CNPs-KAg was substantially increased after 30 min and 150 min post-treatment compared to control

KAg-treated cells. We also performed flow cytometry analysis of monocyte/macrophages treated with KAg or CNPs-KAg to determine the frequency of specific uptake of influenza antigens in APCs. MDCK cells infected with SwIAV H1N2-OH10 were used as positive control (Figure 4.1G) and a representative picture of flow cytometry analysis of KAg or CNPs-KAg uptake by monocyte/macrophages is also shown (Figure 4.1H). In soluble KAg treated cells an average 2.7%, 7.1% and 10.1% cells positive for influenza antigen, and in CNPs-KAg treated cells 7.2%, 11.7% and 16% cells with uptaken influenza antigen after 10 min, 30 min and 150 min of incubation were noticed, respectively (Figure 4.1I). This data clearly demonstrated that CNPs-KAg was efficiently internalized by pig APCs better than soluble antigens.

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4.4.2 CNPs-KAg formulation induced the secretion of cytokines by porcine

MoDCs in vitro

In order to elucidate the adjuvant property of chitosan NPs in porcine APCs, we

treated porcine MoDCs with medium and LPS as control to compare the effect of soluble

KAg and CNPs-KAg treatment in inducing secretion of different cytokines. As expected,

the medium control cells had very little secretion of all the detected cytokines, while LPS

treatment induced the production of all the analyzed cytokines except IFN-α (Figure

4.2A-F). Cells treated with KAg secreted significantly higher levels of proinflammatory cytokines TNF-α (Figure 4.2B) and IL-6 (Figure 4.2E) and Th1 cytokine IL-12 (Figure

4.2D) compared to medium control. In DCs treated with CNPs-KAg production of innate

IFN-α (Figure 4.2A), TNF-α (Figure 4.2B), IL-1β (Figure 4.2C) and IL-12 (Figure 4.2D) were significantly higher in CNPs-KAg treated compared to soluble KAg treated cells. In

CNPs-KAg treated cells production of IL-6 (Figure 4.2E) and Th2 cytokine IL-10 (Figure

4.2F) were higher than medium control cells, but not significantly higher compared to

KAg treated cells. Our in vitro DCs treatment data suggest that chitosan nanovaccine formulation has potent adjuvant effect on porcine DCs.

4.4.3 CNPs-KAg vaccine augmented the IAV specific mucosal antibody response

in the respiratory tract of pigs

Secretary IgA antibody levels in nasal swab samples collected after IN prime- boost vaccination at DPV 35/DPC 0 was significantly higher (p<0.05) in CNPs-KAg- vaccinated pigs compared to pigs receiving soluble KAg when tested against the

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homologous H1N2-OH10 (Figure 4.3A), heterologous H1N1-OH7 (Figure 4.3B) and

heterosubtypic H3N2-OH4 IAVs (Figure 4.3C). A significant difference in antibody

response was observed between CNPs-KAg and KAg-vaccinates in serial two-fold

diluted nasal swab samples (Figure 4.3A-C). This data suggest that the CNPs-KAg IN delivery induced enhanced cross-reactive mucosal secretary IgA antibody response in pigs. Specific IgG antibody response in sera after prime-boost vaccination in KAg vaccinated pigs against the vaccine virus was comparable to the CNPs-KAg vaccine group (Figure 4.3D). While significantly higher (p<0.05) cross-reactive IgG response was observed in CNPs-KAg vaccinates against heterologous H1N1-OH7 (Figure 4.3E) and heterosubtypic H3N2-OH10 (Figure 4.3F) IAVs compared to KAg vaccinated animals. In

CNPs-KAg vaccinated pig sera, IAV- specific HI antibody titers against H1N2-OH10

(Figure 4.3G), H1N1-OH7 (Figure 4.3H) and H3N2-OH4 (Figure 4.3I) were significantly

higher (p<0.05) compared to mock pig group. The HI titers in CNPs-KAg-vaccinates

were around 2- fold higher compared to KAg-vaccinates against heterologous (Figure

4.3H) and heterosubtypic (Figure 3I) IAVs, but the data was not statistically significant

(p>0.05). The expression of T helper 2 (Th2) specific transcription factor GATA-3

mRNA in PBMCs of pigs at DPV 35/DPC 0 was 4-fold and 1.5-fold higher in CNPs-

KAg vaccinated pigs compared to unvaccinated control (p<0.05) and KAg-vaccinated

pigs (p>0.05) (Figure 4.4A). The expression of Th1 specific transcription factor T-bet in

PBMCs was not significantly different among the pig groups (Figure 4.4B).

The mucosal IgA response in pigs post-challenge at DPC 6 was determined and

the data indicate that specific IgA in CNPs-KAg vaccinated pig group was significantly

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higher (p<0.05) compared to unvaccinated challenged animals and remarkably higher

compared to KAg vaccinated and challenged animals against all three IAV subtypes in

nasal swabs (Figure 4.55A-C), BAL fluids (Figure 4.5D-F) and lung lysates (Figure

4.5G-I). These data indicated the secretion of robust mucosal IgA antibody in the upper

respiratory tract (nasal swabs), lower respiratory tract (BAL fluids) and lung parenchyma

(lung lysates) of pigs. Similarly, systemic IgG antibody response in serum at DPC 6 was

also enhanced in the CNPs-KAg-vaccinates compared to unvaccinated (p<0.05) and KAg

vaccinated and virus challenged animals (Figure 4.6A-C). However, HI antibody titers in

BAL fluid at DPC 6 were comparable between KAg and CNPs-KAg vaccinates (Figure

4.6D-F).

4.4.4 CNPs-KAg vaccine enhanced systemic specific cell-mediated immune

response against IAVs

To understand the role of chitosan delivered IAV nanovaccine in induction of specific cell-mediated immune response after IN vaccinations, isolated PBMCs at DPV

35 were restimulated with H1N2-OH10, H1N1-OH7 and H3N2-OH4 viruses. The harvested cell culture supernatants were analyzed for IFNγ secretion and significantly higher (p<0.05) levels of IFNγ in homologous H1N2-OH10 virus restimulated CNPs-

KAg compared to soluble KAg vaccinated pigs was observed (Figure 4.7A). Though not statistically significant, the IFNγ recall response in heterologous and heterosubtypic viruses restimulated cells were noticeably higher levels in CNPs-KAg vaccinates than in

KAg-vaccinated pigs (Figure 4.7B- C). The average IFNγ amounts in CNPs-KAg vaccine

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group against H1N1-OH7 and H3N2-OH4 viruses was 463pg/mL and 332pg/mL

compared to 91pg/mL and 16pg/mL in KAg vaccinates, respectively (Figure 4.7B-C).

We also performed phenotyping of PBMCs isolated at DPC 0 by flow cytometry.

The frequency of cytotoxic T cells (CTLs) in CNPs-KAg vaccinated pigs (average:

18.1%) was higher compared to KAg-vaccinated (average: 15.7%, p>0.05) and unvaccinated pigs (average: 13.4%, p<0.05) (Figure 4.7D). This finding is consistent with enhanced IFNγ response in CNPs-KAg vaccinated pigs, as activated CTLs are one of the major T cell subsets which secrete high levels of antiviral cytokine IFNγ. In addition, in PBMCs at DPC 0 virus specific cell proliferation was detected in an increased trend upon restimulation with homologous (Figure 4.7E) and heterologous

(Figure 4.7F), but not with heterosubtypic (Figure 4.7G) viruses in CNPs-KAg vaccinated pigs. Overall, these data suggested the presence of superior cross-reactive effector memory lymphocyte response in pigs induced by chitosan encapsulation of inactivated SwIAV antigen.

4.4.5 CNPs-KAg vaccine reduced the inflammatory changes in the lungs of

virulent and heterologous virus challenged pigs

Rectal temperature of pigs was recorded daily post-challenge until euthanized.

Pigs in all groups had fever (≥1040F) for first two days after challenge. However, there was no statistical difference in temperature profile among the pig groups (Figure 4.8A).

Macroscopic pulmonary lesions were scored for percent consolidation induced by influenza infection and observed lower pulmonary consolidation in CNPs-KAg

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vaccinates (mean score 15) compared to KAg (mean score 17) and unvaccinated animals

(mean score 19) (Figure 4.8B), but the data was not statistically significant (p>0.05).

Microscopic pulmonary lesions were subjectively scored on Hematoxylin and Eosin

(H&E) stained lung sections where a score of 0 = no change from normal, 1 = minimal

change from normal, 2 = moderate change from normal and 3 = severe change from

normal (Figure 4.8C). The mean scores of interstitial pneumonia (2, 1.6 and 0.8),

peribronchial inflammation (2, 1.8 and 1.8), perivascular inflammation (1.6, 0.3 and 0.5),

bronchial exudates (0.7, 0.1 and 0.2) and epithelial changes (0.3, 0.3 and 0.1) were

observed in virus challenged unvaccinated, KAg and CNPs-KAg-vaccinates, respectively. All the microscopic evaluation of pulmonary tissues was conducted by a board certified veterinary pathologist. A moderate reduction in inflammatory changes was observed in both the vaccinated pig groups when compared to the lesion scores in the unvaccinated and challenged group. In particular, the interstitial pneumonia and epithelial changes were much reduced in CNPs-KAg group compared to soluble KAg vaccinated pigs.

We also evaluated the levels of proinflammatory cytokine IL-6 secretion in the

BAL fluid and observed relatively lower levels in CNPs-KAg vaccinated pigs, consistent with the lower macroscopic and microscopic lung lesions (Figure 4.8D).

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4.4.6 CNPs-KAg vaccine enhanced the mucosal cellular immune response in the

tracheobronchial lymph nodes of virulent and heterologous virus-challenged

pigs

In the CNPs-KAg vaccinated pigs, the frequency of CTLs, IFNγ and specific lymphocyte proliferation index values were augmented in PBMCs (Figure 4.7). The cell-

mediated immune response in the lung draining TBLN was also examined in these

vaccinated pigs. Our data demonstrated significantly higher (p<0.05) secretion of IFNγ

by TBLN-MNCs restimulated with vaccine (H1N2-OH10) and challenge (H1N1-OH7)

viruses in CNPs-KAg, but not in KAg vaccinated compared to mock group (Figure 4.9A-

B). Cells similarly stimulated with heterosubtypic (H3N2-OH4) IAV, showed an increase

in IFNγ secretion in CNPs-KAg vaccinated pig group but this increase was not

statistically significant (Figure 4.9C). We performed flow cytometry analysis of TBLN-

MNCs isolated at DPC 6 and observed a significantly higher (p<0.05) frequency of T

helper/memory cells (CD3+CD4+CD8α+), one of the principle contributor of IFNγ

production in pigs (205), in CNPs-KAg vaccinated pig group compared to unvaccinated

and challenged animals (Figure 4.9D). The expression of Th1 and Th2 transcription

factors mRNA level in TBLN collected at DPC 6 were analyzed. Consistent with

augmented cellular response in TBLN-MNCs of CNPs-KAg vaccinated pigs, in frozen

TBLN tissues mRNA expression of the Th1 specific transcription factor T-bet was

significantly higher (p<0.05) in CNPs-KAg compared to KAg vaccinates (Figure 4.9E).

The expression of Th2 transcription factor GATA-3 mRNA was not increased in TBLN.

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4.4.7 CNPs-KAg vaccine reduced virus shedding in the nasal cavity and also

pulmonary viral titers in SwIAV challenged pigs

We observed significantly reduced (p<0.05) challenge virus shedding at DPC 4

from the nasal passage of CNPs-KAg vaccinates compared to unvaccinated and

challenged animals (Figure 4.10A). By DPC 6, infectious virus was detected in the nasal

passage of only 1 of 5 pigs (20%) vaccinated with CNPs-KAg vaccine, while all pigs in

KAg-vaccinated and unvaccinated groups were shedding virus ranging from 102.5 to 103.3

TCID50/mL (Figure 4.10B). The average virus titers in nasal swab at DPC 6 in

unvaccinated, KAg- and CNPs-KAg vaccinated and challenged pigs were 102.8, 102.5 and

0.5 10 TCID50/mL, respectively (Figure 4.10B). Similarly, virus titer in BAL fluid on DPC

6 was significantly reduced (p<0.05) in CNPs-KAg but not in KAg group compared to

unvaccinated virus challenge pigs (Figure 4.10C). The average virus titers in BAL fluid

at DPC 6 in unvaccinated, KAg- and CNPs-KAg vaccinated and IAV challenged pig

6.3 5 3 groups were 10 , 10 and 10 TCID50/mL, respectively.

4.5 Discussion

Chitosan is a natural polymer synthesized by deacetylation of chitin, one of the

most abundant polysaccharides in nature (417). Chitosan forms an attractive excipient for

drug and vaccine delivery as it bears biocompatible, biodegradable, mucoadhesive,

polycationic and immunomodulatory properties (417, 418). Chitosan is often coupled with tripolyphosphate (TPP), a polyanion that helps in encapsulation of the biochemical agents through inotropic gelation. The chitosan and TPP (CS/TPP) NPs formulation in

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mice was shown to induce both cell-mediated (Th1) and humoral (Th2) immune responses when immunized through IN route against Streptococcus equi (419). Similarly,

tetanus toxoid loaded in CS/TPP NPs IN delivered in rat were efficiently transported

through the nasal epithelium, and in mice it induced long-lasting systemic and mucosal antibody response compared to soluble antigen (420). Mice immunized through IN route using CS/TPP based influenza split virus vaccine was shown to induce higher systemic and mucosal antibody response than soluble antigens, and also enhanced the cell- mediated immune response indicated by increased IFNγ secreting cells frequency in spleen (407). Unlike the preparation of poly(lactic-co-glycolic) acid (PLGA) and polyanhydride nanoparticles, the process of preparing chitosan nanoparticles does not need any organic solvents and thus involves a simple and mild procedure protecting sensitive biochemical agents including proteins and provides scope for easy modification of particles (421-424).

In this study, we prepared chitosan-based influenza nanovaccine using TPP by ionotropic gelation technique. The resulting NPs were around 500nm in diameter which is adequate for efficient uptake by APCs (205, 337, 402, 403). The size of NPs was slightly increased after antigen loading like reported earlier (425). But the surface charge

of our NPs did not change much with or without antigen loading, and the charge

(+2.84mV) was comparable to NPs entrapped with Newcastle disease virus (NDV),

which was also loaded in CS:TPP at 2:1 ratio formulation like our CNPs-KAg (405). We evaluated the stability of nanoparticles in physiological buffer until 30h at 40C storage

condition. For vaccination of pigs, particles were freshly prepared and maintained on ice

125 until IN delivery (1-2h) which ensures stability of chitosan in nanoparticle form in this particular study. However, for better stability and long term storage of nanoparticles, the surface charge should be highly negative or positive (417, 426). Our CNPs-KAg nanoparticles were polydispersed in nature and had weak positive surface charge which suggest that we need to do screening for best ratios of CS:TPP:KAg in our future experiments to ensure better physicochemical properties of CNPs-KAg. The optimal

CS:TPP:KAg combination should have higher positive surface charge, monodispersed nature, relatively smaller size and better stability at different storage conditions. The encapsulation efficiency of KAg in chitosan NPs formulation was 67%, higher than the encapsulation efficiency of H1N2-OH10 KAg (~50-55%) in PLGA and polyanhydride

NPs (402, 403). The higher encapsulation efficiency of vaccine antigens is desirable to reduce the cost of vaccine production. The protein release from CNPs-KAg was slower than previously reported similar CS/TPP NPs formulation, wherein close to 50% of NDV antigens were released from CNPs within first three days (405). Chitosan nanoparticle encapsulation enhances the antigen uptake by APCs, increases expression of activation markers and secretion of proinflammatory cytokines by APCs (427). As expected,

SwIAV antigens delivered in chitosan NPs were efficiently internalized by porcine APCs compared to soluble KAg; and importantly, induced the production of innate, proinflammatory and Th1 cytokines compared to soluble KAg.

Induction of strong mucosal immunity is associated with increased breadth of protective efficacy against influenza, and inactivated IM vaccines do not elicit high levels of antigen-specific mucosal IgA antibody response in the respiratory tract (143, 144).

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Moreover, IM influenza vaccines in pigs have a limitation of not being effective in presence of maternal derived antibody (MDA) (153). However, successful IN vaccination has a potential to overcome MDA interference because of induction of robust local mucosal immunity in the respiratory tract with minimal MDA interference (428).

Chitosan is an attractive polymer for intranasal immunization (404). It enhances the absorption of vaccine particles across the nasal epithelium (253). Further, when compared to aqueous chitosan solution, insulin-loaded chitosan nanoparticles (300-

400nm diameter) increased the nasal absorption of insulin (254). Due to the positive charge of chitosan, it can interact with anionic components such as sialic acid of glycoproteins on epithelial cell surfaces thereby prolonging local retention time and decreasing antigen clearance on mucosal surfaces. In addition to its bioadhesive properties, chitosan enhances paracellular and intracellular transport of particulate antigens into the subepidermal space for optimal contact with APCs and other cells associated with immune responses (251, 429). In mice, intranasal delivery of chitosan nanoparticle-based hepatitis B vaccine enhances the mucosal IgA antibody response

(430, 431). Other murine studies have shown that intranasal immunizations with chitosan-based nanovaccine formulations induce robust mucosal and systemic antibody responses against Pneumococcus spp., Diphtheria spp., and Bordetella spp. (211, 432,

433).

An influenza subunit vaccine coadministered intranasally with chitosan delivery system enhanced both mucosal and systemic antibody response in mice (434). Intranasal delivery of chitosan delivered DNA vaccine against Coxsackievirus in mice enhanced the

127 secretion of both serum IgG and mucosal IgA as wells as CTLs activity in spleen (435).

Consistent with the previous studies in mice (211, 432, 433, 436), the prime-boost vaccination of CNPs-KAg in pigs improved the IgA antibody secretion in the nasal passage and lungs. Importantly, robust secreted antibodies were cross-reactive against heterologous and heterosubtypic IAV and helped in significant reduction in nasal virus shedding and lung load of a heterologous challenge virus. In a previous experiment,

PLGA- SwIAV KAg nanovaccine failed to reduce nasal virus shedding in spite of inducing robust specific cell-mediated immune response and reducing virus load in the lungs of most of the pigs. This anomaly was likely due to the inability of PLGA- encapsulated vaccine to induce mucosal IgA response (402). Similarly, polyanhydride-

SwIAV KAg nanovaccine also enhanced specific cell-mediated immunity but did not enhance mucosal antibody responses and hence did not significantly reduce the nasal virus shedding (403). Like earlier murine studies (437, 438), intranasal vaccination with

CNPs-KAg also induced influenza-specific systemic IgG antibody and HI titers.

Cell-mediated immunity is of prime importance for providing complete protection against intracellular pathogens. The Th1 cytokine IFN-γ is a critical cytokine involved in antiviral responses (439, 440). Chitosan is superior to alum adjuvant in enhancing the cell-mediated immune responses (441). It also induces type I IFN secretion from immature dendritic cells (DC) which helps in DC maturation and generation of Th1 mediated cellular immune responses (442). In this study, enhanced IFNγ secretion by activated lymphocytes in a recall response with genetically variant IAVs was observed in both PBMCs and TBLN-MNCs of CNPs-KAg-vaccinated pigs. The observed spike in

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IFNγ recall response was associated with enhanced virus-specific cellular response both

at mucosal sites and systemically. Activated T cell subsets such as T helper and CTLs

and innate NK cells are the sources of IFNγ (443). The prime-boost vaccination schedule

employed in this study with CNPs-KAg increased the CTLs in PBMC cultures, the major

source of IFNγ, the cytokine that clears virus from infected cells (440).

Another important T cell subset in pigs is T helper/memory cells

(CD3+CD4+CD8α+) (349) which possesses cytolytic function and also secretes IFNγ. The protective response against pseudorabies virus infection has been attributed to the increased frequency of T helper/memory cells (349, 444). The frequency of T helper/memory cells in TBLN-MNCs was significantly enhanced in CNPs-KAg vaccinated pigs. Thus, both T helper/memory and CTLs appear to contribute substantially in improving the cross-protective cellular immune response in pigs vaccinated with chitosan-based influenza nanovaccine.

In conclusion, the mucoadhesive chitosan based IAV nanovaccine formulation delivered as intranasal mist augmented cross-reactive T and B lymphocytes response in pigs at both mucosal (upper and lower respiratory tract and regional lymph nodes-TBLN) and systemic (blood) sites by augmenting secretary IgA, systemic IgG and T cell responses against highly variant IAVs. This augmented virus-specific cross-reactive immune response resulted in reduced nasal virus shedding, reduced viral titers in the pulmonary parenchyma, and relatively reduced inflammatory changes in the lungs. Thus, our study indicates that chitosan IAV nanovaccine might be an ideal vaccine candidate against constantly evolving influenza infections in swine herds. Future studies will focus

129 on optimization of CS:TPP:KAg combination to ensure monodispersed nature, higher positive charge and better stability of CNPs-KAg vaccine. The efficacy of IN CNPs-KAg vaccine will also be compared in future vaccine challenge studies with commercial IM killed and IN modified live IAV vaccines, and in MDA positive piglets against variant field IAV isolates.

4.6 Ethics statement

This animal study was carried out in strict accordance with the recommendations by Public Health Service Policy, United States Department of Agriculture Regulations, the National Research Council’s Guide for the Care and Use of Laboratory Animals and the Federation of Animal Science Societies’ Guide for the Care and Use of Agricultural

Animals in Agricultural Research and Teaching. We followed all relevant institutional, state and federal regulations and policies regarding animal care and use at The Ohio State

University. All the pigs were maintained, samples collected and euthanized, and all efforts were made to minimize the suffering of pigs. This study was carried out in accordance with the approved protocol of the Institutional Animal Care and Use

Committee at The Ohio State University (Protocol number 2014A00000099).

4.7 Acknowledgements

We are thankful to Dr. Juliette Hanson, Megan Strother, Sara Tallmadge, Hyesun

Jang, John M Ngunjiri and Mohamed Elaish for their help in animal studies. We also

130 thank Dr. Tea Meulia, Horst Leona and the team at MCIC, OARDC for their help in

SEM analysis.

4.8 Funding

This work was supported by OARDC OSU industry match grant and Agriculture and Food Research Initiative Competitive Grant no. 2013-67015-20476 from the USDA

National Institute of Food and Agriculture.

4.9 Conflict of interest statement

Authors declare absence of any conflict of interest.

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Experimental Pig First vaccination Second vaccination Day of challenge groups no. (DPV 0) (DPV 21) (DPV 35/DPC 0)

Unvaccinated 3 DMEM DMEM H1N1-OH7

KAg 4 Inactivated H1N2-OH10 Inactivated H1N2-OH10 H1N1-OH7

CNPs-KAg 5 Inactivated H1N2-OH10 Inactivated H1N2-OH10 encapsulated H1N1-OH7

encapsulated in chitosan nanoparticle in chitosan nanoparticle

Table 4.1 Experimental design showing different vaccine groups

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Figure 4.1 In vitro characteristics of CNPs-KAg. Diameter of (A) Empty chitosan nanoparticles (CNPs) and (B) SwIAV killed antigen (KAg) loaded CNPs (CNPs-KAg) determined by DLS. Scanning electron microscope (SEM) images of (C) Empty CNPs and (D) CNPs-KAg. (E) Release of KAg from CNPs-KAg suspended in PBS over a period of 15 days. (F) Uptake of soluble SwIAV KAg or CNPs-KAg formulation by monocytes/macrophages at indicated time points determined by fluorescent microscopy (Olympus, IX70, 20 X magnifications). Frequency of monocytes/macrophages uptaken SwIAV KAg treated with soluble antigen or CNPs-KAg determined by flow cytometry: (G) SwIAV infected MDCK cells as positive control; (H) a representative picture of SwIAV KAg or CNPs-KAg uptake by porcine monocytes/macrophages after 150 min treatment; and (I) percentage of cells with internalized SwIAV antigen at 10 min, 30 min and 150 min treatment.

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Figure 4.2 Cytokine responses in vitro. Production of innate, proinflammatory and Th1 cytokines by porcine MoDCs treated for 48h with medium, KAg, CNPs-KAg or LPS control were determined by ELISA. Levels of cytokines (A) IFN-α; (B) TNF-α; (C) IL- 1β; (D) IL-12; (E) IL-6; and (F) IL-10 were estimated in stimulated cell culture supernatant by ELISA. Data represents mean value of 7 pig derived DCs ± SEM. Statistical analysis between two groups was carried out using Mann-Whitney test. Asterisk refers to statistical significant difference between the indicated two pig groups (* p<0.05, ** p<0.01 and ***p<0.001).

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Figure 4.3 Antibody responses after prime-boost vaccination. Mucosal secretory IgA antibody response in nasal swab, systemic IgG antibody and hemagglutination inhibition (HI) titers in serum samples were determined at day post-vaccination 35/ day post- challenge 0 (DPV35/DPC0) against: (A,D,G) H1N2-OH10; (B,E,H) H1N1-OH7; and (C,F,I) H3N2-OH10 IAVs. Data represents mean value of 3 to 5 pigs ± SEM. Statistical analysis was carried out using Kruskal-Wallis test followed by Dunn’s post-hoc test. Asterisk refers to statistical significant difference between the indicated two pig groups (* p<0.05). In antibody dilution curves (A-F); A, B and C refers to significant difference between unvaccinated vs KAg-vaccinates; unvaccinated vs CNPs-KAg-vaccinates; and KAg vs CNPs-KAg-vaccinates, respectively, at the indicated dilution.

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Figure 4.4 Expression of Th1 and Th2 transcription factors in PBMCs after prime-boost vaccination. The expression of (A) Th2 transcription factor GATA-3 and (B) Th1 transcription factor T-bet in PBMCs of pigs at DPV 35/DPC 0 were determined by qRT- PCR. Data represents mean value of 3 to 4 pigs ± SEM. Statistical analysis was carried out using Kruskal-Wallis test followed by Dunn’s post-hoc test. Asterisk refers to statistical significant difference between the indicated two pig groups (* p<0.05).

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Figure 4.5 Mucosal IgA antibody responses in the respiratory tract at DPC 6. Specific IgA antibody response in nasal swab, BAL fluid and lung lysate samples against H1N2- OH10 (A,D,G), H1N1-OH7 (B,E,H) and H3N2-OH4 (C,F,I) IAVs. Data represents mean value of 3 to 5 pigs ± SEM at all indicated dilutions. Statistical analysis was carried out using Kruskal-Wallis test followed by Dunn’s post-hoc test where A, B and C refers to significant difference (p<0.05) between unvaccinated vs KAg-vaccinates; unvaccinated vs CNPs-KAg-vaccinates and KAg vs CNPs-KAg-vaccinates, respectively, at the indicated dilution.

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Figure 4.6 Serum IgG response and BAL fluid HI antibody titers in pigs at DPC 6. Specific IgG antibody response in serum and BAL fluid HI titers against H1N2-OH10 (A,D), H1N1-OH7 (B,E) and H3N2-OH10 (C,E) IAVs. Data represents mean value of 3 to 5 pigs ± SEM. Statistical analysis was carried out using Kruskal-Wallis test followed by Dunn’s post-hoc test. Asterisk refers to statistical significant difference between the indicated two pig groups (* p<0.05). In antibody dilution curves (A-C); A, B and C refers to significant difference between unvaccinated vs KAg-vaccinates; unvaccinated vs CNPs-KAg-vaccinates and KAg vs CNPs-KAg-vaccinates, respectively, at the indicated dilution.

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Figure 4.7 Cell-mediated immune responses after prime-boost vaccination. PBMCs isolated from blood were stimulated with different variant IAVs. IFNγ secretion in the culture supernatant and antigen specific lymphocyte proliferation was determined after 72h of stimulation with (A,E) H1N2-OH10, (B,F) H1N1-OH7 and (C,G) H3N2-OH4 IAVs. (D) Flow cytometry analysis of PBMCs showed enhanced frequency of CTLs (CD3+CD4-CD8αβ+) in CNPs-KAg vaccinated pigs. Data represents mean value of 3 to 5 pigs ± SEM. Statistical analysis was carried out using Kruskal-Wallis test followed by Dunn’s post-hoc test. Asterisk refers to statistical significant difference between the indicated two pig groups (* p<0.05).

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Figure 4.8 Clinical and pathological changes in pigs post-challenge. (A) Rectal temperature was recorded daily after challenge until the day of necropsy. (B) Gross pneumonic lesions in lungs determined at DPC 6. (C) Representative H&E-stained lung pictures showing bronchial exudates (dotted black circle), perivascular inflammation (black arrow), peribronchial inflammation (dashed black arrow) and interstitial pneumonia (small black triangle). (D) Secretion of cytokine IL-6 in BAL fluid. Data represents mean value of 3 to 5 pigs ± SEM.

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Figure 4.9 Cell-mediated immune responses in TBLN-MNCs at day post-challenge 6. TBLN-MNCs isolated on the day of necropsy were stimulated with different variant SwIAVs, and secreted IFNγ into the culture supernatant was measured by cytokine ELISA against: (A) H1N2-OH10; (B) H1N1-OH7; and (C) H3N2-OH4 IAVs. (D) The frequency of T helper/memory cells (CD3+CD4-CD8α+) in TBLN-MNCs of CNPs-KAg vaccinated pigs were analyzed by flow cytometery. Expression of Th1 (E) and Th2 (F) transcription factors were also determined in TBLN at DPC 6. Data represents mean value of 3 to 5 pigs ± SEM. Statistical analysis was carried out using Kruskal-Wallis test followed by Dunn’s post-hoc test. Asterisk refers to statistical significant difference between the indicated two pig groups (* p<0.05).

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Figure 4.10 Infectious virus titers in the respiratory tract. Titers of challenge SwIAV shedding through nostrils at (A) DPC 4 and (B) DPC 6; and in BAL fluid at DPC 6 (C) determined by using cell culture technique. Data represents mean value of 3 to 5 pigs ± SEM. Statistical analysis was carried out using Kruskal-Wallis test followed by Dunn’s post-hoc test. Asterisk refers to statistical significant difference between the indicated two pig groups (* p<0.05).

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Chapter 5: Evaluation of corn-derived alpha-D-glucan nanoparticles as adjuvant

for intramuscular and intranasal immunization in pigs

5.1 Abstract

We recently demonstrated the adjuvant potential of corn-derived dendrimer-like alpha-D-glucan nanoparticles (Nano-11). Intramuscular (IM) injection of Nano-11 induced transient and local inflammatory response, retained antigens at the injection site and effectively targeted them to dendritic cells in mice. In this study, adjuvant potential of Nano-11 was evaluated to electrostatically adsorbed killed influenza virus antigen

(KAg) (Nano-11+KAg) in pig system. Our in vitro data indicated that Nano-11 facilitated uptake of KAg by porcine antigen presenting cells and activated them to produce innate and proinflammatory cytokines. In prime-boost Nano-11+KAg vaccinated pigs by intranasal mist delivery, induction of the expression of T-helper 1 and T-helper 2 transcription factors associated with secretion of cross-reactive influenza specific mucosal IgA antibody response in the nasal cavity were detected. In vaccinated and heterologous virus challenged pigs, higher mucosal IgA and increased frequency of IFN-

γ secreting T-helper and cytotoxic T-cells responses were observed. Clinically, in vaccinated pigs reduced fever and decreased macroscopic pneumonic lesions, associated with a marginal reduction in the virus load by 2-3 times in the respiratory tract compared

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to soluble KAg-vaccinates were evident. In another independent experiment, IM

immunization of pigs with Nano-11 adsorbed ovalbumin (OVA) induced significantly higher levels of serum IgG1 and IgG2 antibodies compared to OVA alone. Overall, our results indicate that Nano-11 exert adjuvant effects to intranasally delivered influenza

antigens and IM delivered OVA in pigs, which can be considered as an alternative

adjuvant and delivery system for developing vaccines against other pathogens to improve

human and animal health.

5.2 Introduction

An adjuvant is an agent that potentiates the immune response to coadministered

vaccine antigen(s). The mechanisms underlying the immune potentiation by adjuvants include activation of innate immune receptors, production of proinflammatory cytokines and interferon responses, and increased uptake and presentation of antigens by professional antigen presenting cells (APCs) (177). The activation of innate immune responses guides the development of functionally appropriate T-helper 1 (Th1) and T- helper 2 (Th2) types of adaptive immune responses. Adjuvants also reduce the dose of antigen(s) or the number of immunizations required to elicit long-lasting protective immunity (178). The most commonly used adjuvants in human vaccines are aluminum- based products, while animal vaccines often contain oil emulsions (water-in-oil/oil-in-

water) and saponins as adjuvants (179, 183). Aluminum-based adjuvants are potent inducers of antibody responses, possess an excellent long-term safety profile (used over

80 years), show adjuvanticity to variety of antigens, and are inexpensive. However, in

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some individuals they can induce adverse reactions at the site of injection, augment IgE

antibody secretion, induce a weak cell-mediated immune response, and their activity is compromised by freezing (180, 445). Emulsions and saponins used in animal vaccines often cause local reactions at injection site, such as inflammation leading to granulomas or sterile abscess formation (182). Further, adjuvants used in vaccines administered by intramuscular (IM) injection may not be suitable for mucosal immunization. Many pathogens infect mucosal sites, thus delivery of vaccines to mucosal surfaces can be an ideal strategy to induce protective immune response at the site of infection to effectively prevent diseases. However, the progress in mucosal immunization is hampered, in part, by the lack of effective mucosal adjuvants (177, 184). In addition, there has been a shift in vaccine formulation from traditional whole microbe-based approaches to modern highly characterized subunit vaccine(s) which usually require adjuvants to be immunogenic (189). This underscores the need for novel adjuvants which are safe and inexpensive, trigger cellular and humoral immune responses, and are compatible with

various antigens, different animal species and routes of immunization.

In the 21st century, nanoparticles (NPs) have gained increased attention as vaccine

delivery systems, wherein vaccines are prepared either by encapsulating antigen(s) or

adsorbing them on the surface of NPs (165). NPs can incorporate multiple vaccine

antigen(s) or adjuvants in a single vaccine formulation; protect antigen from enzymatic

and proteolytic degradation; increase the duration of availability of antigen and slow

release (depot formation); stimulate APCs to produce proinflammatory cytokines;

augment internalization and processing of antigen by APCs; promote cross-presentation

145 of antigen; modulate the nature of adaptive immune response; and offer flexibility for modifications in its physicochemical properties to develop receptor or organ targeted vaccine products (165, 196). NP-based vaccines can also be used for non-traditional routes of delivery such as mucosal and intradermal immunizations (165). We recently developed dendrimer-like α-D-glucan NPs, named Nano-11, through a few simple chemical modifications of phytoglycogen derived from sweet corn (412). Nano-11 enhanced antigen uptake by dendritic cells (DCs), and promoted antibody responses against antigens indicating its adjuvant nature (412). In a subsequent study, biodistribution and fate of Nano-11 were investigated after IM injection in mice. Nano-11 had comparable adjuvanticity to aluminum hydroxide, induced transient inflammation, prolonged retention of antigen at the injection site, and effectively targeted antigens to

DCs in mice (446). This work indicated that Nano-11 is a safe and effective alternate vaccine adjuvant for IM immunization in a rodent model, but its adjuvant effect in larger mammals had not been tested. In this study, we investigated the adjuvant effect of Nano-

11 to electrostatically adsorbed OVA delivered IM and similarly adsorbed inactivated/killed influenza antigen (KAg) delivered as intranasal (IN) mist in a pig model. We also assessed the vaccine efficacy upon challenge using infectious zoonotic swine influenza A virus (SwIAV). Our results suggest that, as in murine model, Nano-11 confers potent adjuvant effects in pigs likewise.

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5.3 Materials and methods

5.3.1 Influenza A virus (IAV) propagation and vaccine antigen preparation

Swine IAV, A/SW/OH/FAH10-1/10 (H1N2-OH10) was used in vaccine

preparation and a zoonotic A/SW/OH/24366/2007 (H1N1-OH7) virus which is

heterologous to vaccine virus was used for challenge infection (402, 403). A turkey

isolate of IAV A/Turkey/OH/313053/2004 (H3N2-2004) which is also infectious in pigs

was used in immunological in vitro assays as a heterosubtypic virus. All the three IAVs were propagated in Madin-Darby Canine Kidney (MDCK) cells (402). Inactivated/killed

(KAg) H1N2-OH10 was prepared by inactivation of ultracentrifuged virus pellet using

binary-ethylinemine (BEI) as described previously (402). Endotoxin free OVA were purchased from Invivogen (San Diego, CA).)

5.3.2 Adsorption of KAg to Nano-11 NPs and determination of size and zeta

potential

Nano-11 NPs were prepared from phytoglycogen (PG) derived from sweet corn

kernels and SwIAV KAg or OVA was adsorbed electrostatically to Nano-11 as described previously (412). Particle size and zeta potential of Nano-11 preparations were determined by dynamic light scattering method using a Zeta-sizer coupled with an MPT-

2 titrator (Malvern). NPs images were obtained using Philips CM-100 transmission

electron microscope (TEM) (412, 446). Protein adsorption efficiency was determined by

subtracting the unbound protein in the supernatant from the initial amount used for

adsorption. For in vitro protein release study, supernatants collected at different time

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points were subjected to protein estimation and the data were shown as cumulative

protein release as described previously (402).

5.3.3 Antigen uptake and DCs stimulation study

Plate adhered monocyte/macrophages obtained from porcine peripheral blood mononuclear cells (PBMCs) and porcine bronchoalveolar lavage (BAL) fluid cells which contain approximately 80% macrophages (447) were used for evaluation of antigen uptake by APCs. Cells were untreated (medium control) or treated with 10µg/mL of soluble KAg or the same amount of KAg adsorbed on Nano-11 (Nan-11+KAg) for

10min, 30min and 150min and stained with IAV nucleoprotein specific antibody

(CalBioreagents, CA, USA) followed by Alexa Fluor conjugated goat anti-mouse IgG

(Life Technologies, OR, USA) (448). Porcine monocyte derived dendritic cells (MoDCs) were generated by treatment of plate adhered PBMCs (n=6 pigs) with cytokines (GM-

CSF and IL-4) as described previously (413). MoDCs were either untreated or treated with LPS (positive control) (10µg/mL), KAg (10µg/mL) or Nano-11+KAg (10µg/mL).

The culture supernatant was harvested after 48h stimulation and analyzed for cytokines by ELISA as described previously (414).

5.3.4 Influenza vaccine-challenge study in pigs

Caesarian-delivered and colostrum-deprived (CDCD) and bovine colostrum fed piglets (n=12) were raised in our BSL-2 facility at OARDC (402). Serum samples collected from pigs at 4 weeks of age were confirmed negative for influenza antibodies

148 against H1N2-OH10, H1N1-OH7 and H3N2-OH4 viruses used in this experiment, and randomly (male and female) divided into 3 groups: (i) unvaccinated group received

7 medium; (ii) KAg group received 1x10 TCID50 of inactivated H1N2-OH10 virus; and

7 (iii) Nano-11+KAg group received 1x10 TCID50 equivalent of KAg adsorbed on Nano-

11. Pigs were vaccinated IN with a spray mist. The first vaccination was done at 5 weeks of age and the booster using a similar dose when the pigs were 8 weeks old. The challenge infection was done by IN and intratracheal routes using SwIAV H1N1-OH7

6 (6x10 TCID50) at 10 week of age, with 50% virus dose by each route. Piglets were monitored daily for clinical flu signs and euthanized at day post-challenge (DPC) 6 (402).

5.3.5 Intramuscular vaccine study in pigs

In an independent study, 12 pigs at five-week age were recruited at the Animal

Sciences Research and Education Center at Purdue University and randomly assigned into two treatment groups (n=6 per group, 3 males and 3 females). Pigs were injected IM in the neck region with 25μg ovalbumin (OVA) or 25μg OVA combined with 1mg Nano-

11 in 0.5mL 20 mM Tris-buffered saline (0.02M). OVA was completely adsorbed at this dose (data not shown). Second immunization was similarly performed after 3 weeks.

Serum samples were collected 10 days after boost to determine OVA-specific immunoglobulins by ELISA (449). Briefly, plates were coated with OVA overnight at

4°C, incubated with serial dilutions of the serum samples, followed by treatment with mouse anti-pig IgG1 (clone K139 3C8) or mouse anti-pig IgG2 (clone K68 1g2, both from BIO-RAD) and HRP labeled goat anti-mouse IgG1 secondary antibody

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(ThermoFisher). Finally, wells were treated with 3,3',5,5'-tetramethylbenzidine (TMB)

substrate (Sigma) and the reaction was stopped using 2 M sulfuric acid. The optical

density (OD) values were recorded at 450nm wavelength in a microplate reader

(BioTEK, Winooski, VT). End-point was determined as the dilution where OD450 nm

reached 0.2.

5.3.6 Antibody assays and virus titration

Virus-specific hemagglutination inhibition (HI) titers and IgG and IgA antibody

responses were determined against homologous/vaccine virus (H1N2-OH10);

heterologous/challenge virus (H1N1-OH7); and heterosubtypic virus (H3N2-OH4) as described previously (450). Infectious virus titer in nasal swab and BAL fluid samples were determined by infecting MDCK cells with serial 10-fold diluted test samples followed by treatment with immunofluorescent staining with IAV nucleoprotein specific antibody, and the virus titers were determined using the Reed and Muench method (402).

5.3.7 Quantitative reverse transcription PCR (RT-qPCR)

Total RNA was extracted from PBMCs at day post-vaccination (DPV) 35 using

Trizol (Invitrogen) reagent, converted to cDNA to measure the mRNA expression of Th1 transcription factor (T-bet) and Th2 transcription factor (GATA-3) by using β-actin as the house keeping internal control gene as described previously (415, 451).

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5.3.8 Flow cytometry analysis of restimulated PBMCs and IFNγ ELISA on culture

supernatant

PBMCs isolated at DPC 6 were stimulated with IAVs H1N2-OH10, H1N1-OH7 or H3N2-OH4 at 0.1MOI. Cells were treated with Golgiplug (BD bioscience, CA) and

Brefeldin A (Sigma, MO) during the last 6h of the 72h stimulation period. Cells were labeled for activated T helper (CD3+CD4+IFNγ+) and cytotoxic T cells (CD3+CD4-

CD8+IFNγ+) using the procedure and specific antibodies as described previously (402).

Supernatant collected from stimulated PBMCs culture at 72h were subjected to IFNγ cytokine analysis by ELISA (402).

5.3.9 Ethics statement

Animals were maintained, cared, fed, inoculated, samples collected and euthanized in strict accordance with the approved protocols of the Institutional Animal

Care and Use Committee at The Ohio State University (Protocol number 201400000099) and Purdue University (1704001570).

5.3.10 Statistical analysis

Statistical analysis was performed by using non-parametric tests Mann-Whitney and Kruskal-Wallis followed by Dunn’s post-hoc test to compare two and three pig groups, respectively, using the GraphPad Prism 5 (GraphPad Software Inc., CA, USA).

Body temperature of pigs recorded after virus challenge was compared by Friedman test followed by Dunn’s pairwise comparison. A value of p < 0.05 was considered

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statistically significant and a value between 0.05 and 0.1 was considered to express the

statistical trend in the data.

5.4 Results

5.4.1 Physical characterization of Nano-11+KAg nanovaccine

Adsorption of SwIAV KAg on Nano 11 (Nano-11+KAg) was analyzed at 2:1,

1:1, 1:2 and 1:4 ratios of Nano-11 to KAg which yielded 84.2%, 79.3%, 73.6% and

61.6% adsorption efficiency, respectively. Protein release in first 24h ranged from 3 to

10% depending on the Nano-11 to KAg ratios, and cumulative Ag release after 15 days was 16 to 26% (Table 5.1). For the SwIAV IN vaccine-challenge study in pigs, we used

Nano-11+KAg at a 2:1 ratio (in mg/mL) of Nano-11 to SwIAV KAg, as it showed the highest adsorption. The empty Nano-11 NPs had average particle size 76nm (Figure

5.1A), while adsorption of SwIAV KAg at the 2:1 ratio (Nano-11+KAg) increased the average particle size to 487nm (range 200 to 800nm) (Figure 5.1B). Morphology of

Nano-11 was spherical with irregular surfaces, and the size of Nano-11+KAg was

increased due to formation of nano-sized aggregates as observed by TEM (Figure 5.1C-

D). The zeta potential for empty Nano-11 NPs and Nano-11+KAg was +21.8mV and

+19.2mV, respectively. The vaccine (Nano-11+KAg) protein release profile was

determined in triplicate samples and observed a slow and steady release over a period of

30 days (Figure 5.1E).

To understand the effect of Nano-11 to antigen ratios on the size of resulting

vaccine particles, we adsorbed Nano-11 with the model antigen OVA at 2:1; 1:1; 1:2 and

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1:4 ratios. Our results showed that with an increase in OVA concentration the size of NPs

also increased. At 1:2 and 1:4 ratios the diameter of the particles increased to >1000nm.

Zeta potential was gradually reduced from +17.8mV at 2:1 ratio to +8.81mV at 1:4 ratios

(Figure 5.2A-F). To determine whether increasing the amount of Nano-11 would reduce

the size of Nano-11+KAg NPs we mixed Nano-11 and KAg NPs at 4:1 and 8:1 ratio. The

average size was 238nm at both ratios with zeta potentials of +20.3mV and +20.4mV,

respectively (Figure 5.3A-B).

5.4.2 Biological characterization of Nano-11+KAg using porcine antigen

presenting cells

Plate adherent porcine monocytes/macrophages derived from healthy pig PBMCs

were treated with KAg or Nano-11+KAg for different time points, and then cells were fluorescently labeled for influenza antigen. Influenza-specific fluorescence was greater in

Nano-11+KAg treated cells compared to KAg treatment after 10min, 30min and 150 min

(Figure 5.4A). A similar result was observed in porcine BAL cells (Figure 5.5). BAL

cells comprised of approximately 80% macrophages (447).

Treatment of porcine MoDCs with LPS for 48 h induced secretion of cytokines

except IFNα (Figure 5.4B-G). Nano-11 (100µg/mL) induced the secretion of IFN-α,

TNF-α, IL-1β, IL-6, IL-10 and IL-12 from MoDCs at significantly higher (p<0.05) levels

compared with medium control (Figure 5.4B-G). MoDCs treated with Nano-11+KAg

induced the production of IFN-α at 3-fold higher levels than KAg treatment, but this

increase was not statistically significant (p>0.05) (Figure 5.4B). Nano-11+KAg induced

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significantly greater secretion of TNF-α (Figure 5.4C), IL-1β (Figure 5.4D) and IL-10

(Figure 5.4F) compared to KAg treatment.

5.4.3 Adjuvant effect of Nano-11 upon IM injection

The in vitro data with porcine APCs suggest that Nano-11 can activate these cells

similar to mouse DCs. To determine if Nano-11 is an effective adjuvant in pigs, we

injected nursery pigs IM with OVA alone or Nano-11+OVA. Serum samples collected 10

days after the second injection showed that pigs immunized with Nano-11+OVA had a

significantly greater anti-OVA IgG1 and IgG2 titers (p < 0.05) compared with animals

that had received OVA only (Fig. 5.6). In pigs, IgG1 and IgG2 correlate with Th2 and

Th1 biased response, respectively (452).

5.4.4 Expression of transcription factors involved in augmenting the adaptive

immune response to Nano-11+KAg and IAV specific antibody response in

pigs

PBMCs were isolated from blood after prime-boost IN vaccination at DPV

35/DPC 0 and the levels of Th1 (T-bet) and Th2 (GATA-3) transcription factors were

quantified. The expression of GATA-3 was significantly higher (p<0.05) in Nano-

11+KAg but not in KAg vaccinates compared to unvaccinated pigs (Figure 5.7A). There was a modest 1.6 fold increased expression of T-bet in Nano-11+KAg than KAg vaccinates with a p value close to significance (p=0.0571) (Figure 5.7B).

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We also evaluated the mucosal IgA antibody response in nasal swab after prime- boost immunization (Figure 5.7C-E). The IgA antibody response in nasal swabs of pigs immunized with Nano-11+KAg vaccine were significantly higher (p<0.05) than the KAg only group, and this increase was detected against the homologous vaccine virus (H1N2-

OH10), heterologous virus (H1N1-OH7) and heterosubtypic (H3N2-OH4) IAV (Figure

5.7C-E). Secretion of IgA antibody in the nasal passage was also evaluated against all those three IAVs at DPC 6. Like at DPC 0 (Figure 5.7C-E), secretion of specific IgA in nasal cavity was greater in Nano-11+KAg vaccinates compared to control unvaccinated and KAg vaccinates at DPC 6 (Figure 5.7F-H). Though IgA response in nasal cavity was better in Nano-11+KAg vaccinates, the HI titer in serum and systemic IgG antibody responses at DPV 35/DPC 0 and DPC 6 were not significantly different between the KAg and Nano-11+KAg groups (Figure 5.8A-I).

5.4.5 Virus-specific cell-mediated immune response in virus challenged pigs

PBMCs isolated at DPC 6 were stimulated using three different IAVs and determined the virus specific IFNγ recall response by Flow-cytometry. The frequency of

IFNγ+ T-helper cells (CD3+CD4+IFNγ+) (Figure 5.9A) and cytotoxic T cells (CD3+CD4-

CD8αβ+IFNγ+) (Figure 5.9D) in Nano-11+KAg vaccinates derived PBMCs stimulated with vaccine virus (H1N2-OH10) were significantly higher (p<0.05) compared to KAg- vaccinates. Relatively higher but non-significant increase of IFNγ+ T-helper cells was

detected in PBMCs after stimulation with heterologous (H1N1-OH7) challenge (Figure

5.9B). Frequency of T helper cells (Figure 5.9C) and cytotoxic T cells (Figure 5.9E-F)

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were not increased in PBMCs after stimulation with challenge virus (H1N1-OH7) and

heterosubtypic virus (H3N2-OH4). Supernatants of stimulated PBMCs had higher but

non-significant levels of IFNγ in the Nano-11+KAg group compared with unvaccinated and KAg only vaccinates (Figure 5.9G-I).

5.4.6 Clinical outcome, lung pathology and virus clearance in the respiratory tract

of pigs

The rectal temperature above 1040F in pigs is considered as fever. Pigs from

unvaccinated and KAg vaccinates had fever for 2 days, while Nano-11+KAg vaccine

group had fever for 1 day following the challenge with virulent heterologous virus

(Figure 5.10A). Specific brown areas of lung consolidation representing influenza

induced pneumonia in pigs were scored in unvaccinated, KAg and Nano-11+KAg

vaccinates and observed the scores 19, 17 and 11, respectively (Figure 5.10B). Overall,

lung consolidation in Nano-11+KAg group was 47% and 35% lower than unvaccinated

and KAg vaccinated pig groups, respectively.

The replicating virus titers in nasal swab samples of virus challenged pigs at DPC

4 were determined by using MDCK cells. Our results determined that Nano-11+KAg

vaccine group had nasal virus titers 5 times lower than unvaccinated group and 2 times

lower than KAg-vaccinates (Figure 5.10C). Likewise, virus titers in BAL fluid at DPC 6

in Nano-11+KAg vaccinates was 63 times lower than unvaccinated group and 3 times

lower than KAg-vaccinates (Figure 5.10D). Thus, our data indicated that Nano-11+KAg

156 vaccination resulted in slight reduction in influenza induced fever, pneumonic lesions and virus titers in the respiratory tract of pigs.

5.5 Discussions

Our results demonstrated that the novel Nano-11 particle adjuvant has immune stimulatory properties in the porcine system by in vitro and in vivo analyses, consistent with the previous studies in mice. In vivo imaging experiments in mice indicate that

Nano-11 remains localized to the injection site and draining lymph nodes following IM injection, and induces a transient inflammatory response, suggesting that it is safe (446).

This combined with its biodegradability and relatively low production cost, support the potential utility of Nano-11 as an adjuvant in vaccines for animal health.

The adsorption efficiency of SwIAV KAg on Nano-11 NPs was approximately

80%, which was greater than encapsulation of the same antigen in polyanhydride, poly lactic-co-glycolic acid (PLGA) and chitosan NPs (50-70%) (402, 403, 448). Thus the higher antigen binding efficiency of Nano-11 minimizes antigen loss during vaccine preparation and reduces the vaccine cost. The high positive charge of Nano-11 (>

+20mV) is likely responsible for the strong adsorption of negatively charged vaccine antigens by electrostatic interactions, similar to adsorption of OVA and other negatively charged proteins (412). The irregular cauliflower like surface of Nano-11 also provides higher adsorption surface area for antigen binding (412). In general, NPs with either high positive or negative charge are stable, while NPs with near neutral charge tend to coagulate or flocculate (426). The high positive charge of Nano-11+KAg (> +19mV)

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conferred better stability and uniform dispersion of vaccine particles in diluent, and hence

we could successfully deliver Nano-11+KAg as mist using our IN vaccine delivery device in pigs (402). Nano-11 mediated slow and sustained release of influenza antigens which is required for induction of an optimal immune response compared with ‘fast released’ antigens (168).

Activation of innate immune system is an important early step essential after immunization to elicit an effective specific adaptive immune response, which begins from recognition and uptake of antigens by APCs such as DCs, macrophages and B cells

(453). An ideal and safe adjuvant initiates transient inflammatory response at the site of administration and attracts APCs for antigen uptake. In mice, Nano-11 was shown to induce such transient inflammatory response at the site of injection characterized by accumulation of predominantly monocytes and macrophages as opposed to mostly neutrophils with aluminum based adjuvant (446). Positively charged particles are efficiently internalized by APCs compared to particles with negative charge or neutral

(337, 454, 455). Nano-11 enhances the uptake of electrostatically adsorbed proteins by murine APCs (412, 446). Likewise, in this study influenza antigens adsorbed on Nano-11 were taken up efficiently by porcine macrophages compared to soluble antigen.

Adjuvants also enhance expression of costimulatory molecules in APCs and activate them to produce innate and proinflammatory cytokines, which respectively represent the second and third important signals for T cell activation and adaptive immune response development (456). Nano-11 induced secretion of proinflammatory cytokine IL-1β by murine DCs in caspase-1 dependent fashion indicating its intrinsic property to induce an

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inflammatory response (412). Similarly, Nano-11 enhanced synthesis of different innate and proinflammatory cytokines such as IFN-α, TNF-α and IL-1β from treated porcine

DCs. Thus, Nano-11 facilitates not only the antigen uptake by porcine APCs but also activates them to produce cytokines necessary for immune activation.

In Nano-11+KAg vaccinated pigs the expression of Th2 transcription factor

GATA3 was highly upregulated, which was associated with increase in the secretion of cross-reactive specific IgA antibody response in nasal cavity against different IAVs.

Systemic IgG antibody response was not significantly increased in IN immunized pigs, but it was enhanced after IM vaccination with OVA. This data is interesting and might indicate differential immune activation mechanisms when Nano-11 based vaccine was delivered by IN and IM routes in pigs or function of Nano-11 to Ag ratios used in IM

(40:1) versus IN (2:1) route.

There is a need of developing innovative influenza vaccine formulations capable of inducing robust protection against heterologous and heterosubtypic strains of viruses, because IAV is known for undergoing frequent genetic changes with emergence of variant pathogenic strains causing disease in animals and humans. Cell-mediated immunity is of prime importance in protection against viral pathogens including influenza, and aluminum-based adjuvants fail to induce such a response (180, 445).

Cytotoxic T cells kill the virus infected cells through different mechanisms including synthesis of granzymes and perforins (440). IFNγ is an important Th1 cytokine mediates antiviral response secreted by activated T helper and cytotoxic T cells (203, 457).

Adsorption of influenza KAg on Nano-11 also improved the cell-mediated immunity

159 evidenced through presence of higher frequencies of activated T helper and cytotoxic T cells and secretion of IFNγ. Together these data indicate the potential Th1/Th2 adjuvant effect of Nano-11 to influenza antigen in pigs vaccinated by IN route.

Influenza causes acute respiratory infection in pigs associated with typical clinical signs of flu such as high fever, respiratory distress, sneezing, coughing, ocular or nasal discharge and anorexia (42). Influenza infected pigs are prone to secondary bacterial and viral infections responsible for increasing the management and veterinary cost to pig farmers (59). Pigs can also act as mixing vessels as they permit reassortment of influenza viruses from different hosts leading to generation of novel viruses capable of causing epidemics and pandemics (73). Vaccination can be an effective means to control influenza in pigs and hence its transmission to humans. At present, multivalent whole inactivated virus (WIV) vaccines are used in pigs delivered by IM route (119). They provide protective response against homologous virus strains, but only partial or suboptimal protection against heterologous strains (127, 145, 380). Further, WIV vaccines are not effective in the presence of maternal antibodies, and also reported to enhance respiratory disease if mismatch occurs between vaccine virus and heterologous field virus infection (136, 458). Hence, development of a novel inactivated virus vaccine platform is necessary to mitigate swine influenza. In this study, we adsorbed inactivated influenza virus protein on Nano-11 and delivered IN, and analyzed the efficacy against a heterologous challenge virus infection. Nano-11+KAg vaccinates had low fever than control groups. Influenza damages pulmonary tissues which are macroscopically visible as variable sized medium to dark red consolidations (42). Flu specific pneumonic lesions

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were comparatively lower in Nano-11 based vaccine recipients, associated with reduced virus titers in the respiratory tract and lungs compared to controls. Overall, our vaccine- challenge trial indicated the potent adjuvant effects of Nano-11 in pigs, but the level of protection with the current formulation was not sufficient for induction of desired level of cross-protection, indicating the need of improving the Nano-11 vaccine formulation and inclusion of secondary adjuvant/s.

Improvement in Nano-11 based influenza vaccine formulation can be achieved, in part, by determining the optimal ratio of Nano-11 to influenza antigen for IN delivery, because significantly higher IgG antibody response was observed after IM immunization

(40:1 ratio of Nano-11 to OVA) versus IN vaccination (2:1 ratio of Nano-11 to KAg) in pigs. Importantly, higher ratio of Nano-11 to KAg likely reduce the size of particles from

~500nm to <300nm. Particle size is a critical factor in nanovaccines meant for IN immunization, as it greatly determines the type of cells that are targeted and the quality and magnitude of triggered immune responses (169). NPs size lower than 200nm are readily uptaken by DCs while larger particles are internalized by macrophages (169).

Smaller sized particles are shown to induce better immune response compared to larger particles (459, 460). In mice, particles of 360-470nm was found optimal for wide pulmonary distribution after IN delivery (174), but such an information is unavailable for large animals, thus further investigations are required to understand the fate and biodistribution of Nano-11 in pigs after IM and IN administration.

In summary, IAVs undergo high antigenic variation through antigenic drift or shift and hence require broadly protective immune response against different influenza

161 subtypes (461). Intranasal administration of potent vaccines may induce more broadly protective immunity than IM immunization demonstrated in mouse and ferret models

(462, 463). The mechanisms underlying heterosubtypic immunity following mucosal vaccination have not been completely elucidated, but it probably involves multiple factors including broadly reactive antibodies and memory T cell responses (464, 465).

Cross-presentation of antigen by DCs is critical to increase the breadth of immunity, and

PLGA-NPs vaccine delivery system has been shown to mediate cross-presentation of antigen in mice (336, 466, 467) and in pig vaccine studies (170, 205-207, 402). Our future IN vaccine studies will focus on development and evaluation of different ratios of

Nano-11 and influenza whole virus, subunit or split virus antigens, and inclusion of secondary adjuvant/s in the vaccine formulation to induce broad protective immune response against IAVs by augmenting the induction of cross-presentation of antigen in pigs.

5.6 Conclusion

Our data showed efficient uptake of Nano-11 NPs adsorbed influenza antigen by porcine APCs and activation of DCs to produce innate and proinflammatory cytokines.

Nano-11 upregulated the expression of Th2 transcription factor associated with increased secretion of mucosal IgA antibody response in the respiratory tract. The adjuvant role of

Nano-11 was comparable in pigs and mice indicating its applicability in different animal models. The observed adjuvant potential of Nano-11 in pigs delivered IN showed its compatibility to use in alternative routes of immunization as well. Thus, Nano-11 can be

162 used as a safe and inexpensive alternative adjuvant for vaccines against different pathogens of human and animal health importance.

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Nano-11 to Adsorption Cumulative release (%) of antigen at different time KAg ratio (%) 24h 3d 7d 15d 2:1 84.2 8.9 14.2 17.8 23.9 1:1 79.3 6.5 11.3 15.1 26.2 1:2 73.6 4.7 10.1 12.8 16.1 1:4 61.6 2.7 4.3 10.8 25.7

Table 5.1 Adsorption efficiency and antigen release profile of Nano-11+KAg at different ratios

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Figure 5.1 Physical characterizations of Nano-11 and Nano-11+KAg. Size of (A) Nano- 11 and (B) Nano-11+KAg NPs was determined by DLS method. TEM images of (C) Nano-11 and (D) Nano-11+KAg. (E) Cumulative release of KAg from Nano-11+KAg over a period of 30 days.

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Figure 5.2 Physical characterizations of Nano-11 adsorbed with OVA at different ratios. DLS images of Nano-11 and OVA adsorbed at (A) 2:1; (B) 1:1; (C) 1:2 and (D) 1:4 ratios. Likewise, representative TEM images of Nano-11 and OVA adsorbed at ratios (E) 2:1 and (F) 1:2.

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Figure 5.3 Size of Nano-11+ KAg nanovaccines at different ratios of Nano-11 and KAg. Size at (A) 4:1 and (B) 8:1 ratios determined by DLS.

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Figure 5.4 Biological characterization of Nano-11 and Nano-11+KAg using porcine antigen presenting cells. (A) Uptake of SwIAV KAg by porcine blood monocytes/macrophages after 10min, 30min and 150 min treatment with KAg or Nano- 11+KAg. Secretion of cytokines (B) IFN-α, (C) TNF-α, (D) IL-1β, (E) IL-6, (F) IL-10 and (G) IL-12 after 48h stimulation of porcine MoDCs with medium control, LPS control, Nano-11, KAg and Nano-11+KAg. Each bar represents mean ± SEM of 6 pig values. Data were analyzed by Mann-Whitney test and asterisks refer to statistical significance between two indicated treatment groups (*p<0.05 and ** p<0.01).

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Figure 5.5 Uptake of influenza antigen by porcine BAL cells (macrophages). Cells were treated for 10min, 30min and 150 min after treatment with KAg or Nano-11+KAg.

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Figure 5.6 Systemic IgG1 and IgG2 antibody responses in serum of pigs immunized twice IM with OVA or Nano-11+OVA. Each bar represents mean ± SEM of 6 pig values. Data were analyzed by Mann-Whitney test and asterisks refer to statistical significance between two indicated treatment groups (**p<0.01, ***p<0.001).

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Figure 5.7 Expression of transcription factors involved in augmenting the adaptive immune response to Nano-11+KAg. (A) Th2 transcription factor GATA-3 and (B) Th1 transcription factor T-bet. Mucosal IgA antibody response was measured in nasal swab (1:2 dilution) samples of pigs collected at DPV 35/DPC 0 (C-E) and DPC 6 (F-H) against homologous vaccine virus H1N2-OH10 (C,F); heterologous challenge virus H1N1-OH7 (D,G); and heterosubtypic H3N2-OH4 IAV (E,H). Each bar represents mean ± SEM of 3 to 5 pig values. Data were analyzed by Kruskal-Wallis test followed by Dunn’s post-hoc test and asterisks refer to statistical significance between two indicated treatment groups (*p<0.05 and ** p<0.01).

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Figure 5.8 Humoral immune responses in serum of pigs. (A-C) HI titer in serum at DPV 35/DPC 0; specific IgG antibody response in serum at (D-F) DPV 35/DPC 0 and (G-I) DPC 6 against vaccine/homologous virus H1N2-OH10 (A, D, G); heterologous/challenge virus H1N1-OH7 (B, E, H); and heterosubtypic virus H3N2-OH4 (C, F, I). Each bar represents mean ± SEM of 3 to 5 pig values. Data were analyzed by Kruskal-Wallis test followed by Dunn’s post-hoc test and asterisks refer to statistical significance between two indicated treatment groups (*p<0.05).

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Figure 5.9 Virus specific cell-mediated immune responses in PBMCs of pigs post- challenge. The frequency of IFNγ+ T helper cells (A-C) and cytotoxic T cells (D-F) in PBMCs stimulated with vaccine/homologous virus H1N2-OH10 (A, D); heterologous/challenge virus H1N1-OH7 (B, E); and heterosubtypic virus H3N2-OH4 (C, F). Secretion of the cytokine IFNγ in the supernatant of PBMCs restimulated with (G) H1N2-OH10; (H) H1N1-OH7 and (I) H3N2-OH4 IAVs were measured by ELISA. Each bar represents mean ± SEM of 3 to 5 pig values. Data were analyzed by Kruskal-Wallis test followed by Dunn’s post-hoc test and asterisks refer to statistical significance between two indicated treatment groups (*p<0.05).

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Figure 5.10 Clinical and pathological changes and virus titers in the respiratory tract of pigs. (A) Rectal temperature of pigs recorded from DPC 0 to DPC 6. (B) Macroscopic percentage consolidation of pig lungs scored during necropsy at DPC 6. Infectious replicating virus titers in (C) nasal swab samples at DPC 4; and in (D) BAL fluid at DPC 6. Each bar represents mean ± SEM of 3 to 5 pig values. Data were analyzed by Kruskal- Wallis test followed by Dunn’s post-hoc test.

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Chapter 6: Summary and future directions

Influenza A virus infection is consistently one of the top three health challenges in nursery, grower-finisher and breeding pig farms, and it causes significant economic loss to the pig industry through loss of body weight gain, increased time to market, and management and veterinary expenses. IAV of pig origin sporadically cause human infections. Due to the possibility of concurrent infection with avian and human origin

IAV in pigs, they can be the potential host for generation of novel reassortant influenza strains of pandemic potential.

Vaccination is the efficient means of preventing influenza in pigs and virus transmission to humans. The most widely used SwIAV vaccines in the US is whole inactivated virus (WIV) vaccines containing one or more strains of SwIAV administered through IM route. Over 50% of total vaccines used in the US are autogenous WIV vaccines. The IM-delivered WIV vaccines primarily induce systemic antibody response and provide homologous protection to highly similar virus strains contained in the vaccine formulation. However, protection against unrelated heterologous IAV is limited.

The cell-mediated and mucosal immune responses are weak with current WIV vaccines.

Further, several experimental and field reports have documented enhanced respiratory disease in WIV vaccinated pigs when exposed to heterologous virus infection. also 175

Maternal antibody interference does exist with IM-delivered WIV vaccinates. The

limitations of current vaccines warrant the development of novel vaccine delivery

technologies that induce robust mucosal and cellular immune responses, protect pigs

against broad-spectrum of SwIAV, avoid maternal antibody interference and do not

enhance respiratory disease.

Intranasal immunization presents influenza antigens to immune cells in a manner

akin to natural infection. IN mist delivery of vaccine provides larger surface area for

antigen deposition, has lesser enzymatic and chemical activity and is highly vascularized

with large number of M cells. It can elicit better mucosal antibody and cellular immune

responses and also avoids the vaccine associated respiratory disease and maternal

antibody interference than WIV IM vaccine. However, IN route has limitations such as rapid mucociliary clearance, inefficient uptake of soluble antigens and lack of proven and compatible mucosal adjuvants. Therefore, an efficient vaccine delivery platform is required for IN route that ensures antigen availability at nasal cavity for longer duration, facilitates antigen uptake by APCs and stimulates innate immune system. Particulate vaccine designed by using biodegradable and biocompatible NPs forms an attractive platform for IN vaccine delivery as it ensures stability and controlled release of antigens and also activates the immune system. Hence, in the series of experiments discussed in this dissertation, we developed different biodegradable NPs-based inactivated influenza virus vaccines and evaluated their immunogenicity and protective efficacy in heterologous vaccine-challenge studies in pigs following the immunization through IN route.

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In chapter 2, we developed PLGA-based inactivated influenza nanovaccine

(PLGA-KAg) which had 57% antigen encapsulation efficiency, 313±105 nm of average

NPs size and negative surface charge. PLGA-KAg NPs induced maturation of antigen presenting cells in vitro. After heterologous virus challenge in prime-boost IN vaccinated pigs, PLGA-KAg rescued the pigs from influenza induced fever and also reduced pneumonic lung lesions compared to KAg-vaccinates. PLGA-KAg-vaccinates had 40 times lower infectious virus titers in the lungs compared to KAg group, while the virus titer in nasal cavity was not reduced. Immunologically, PLGA-KAg nanovaccine resulted in robust cell-mediated immune response but antibody response was not improved compared to KAg-vaccinates. In chapter 3, we developed polyanhydride-based inactivated influenza vaccine (KAg-nanovaccine) which had 60% antigen encapsulation efficiency, 181±56 nm average NPs size and negative surface charge. KAg nanovaccine protected pigs from fever and slightly lowered pneumonic lesions in pigs compared to soluble KAg-vaccinates. The virus titer was 6 times lower in nasal cavity but comparable in lungs to that of KAg group. Immunologically, KAg-nanovaccine also showed improved cell-mediated but not the antibody response. In summary, both PLGA and polyanhydride were both negatively charged polymers and induced more of cell-mediated immunity in pigs vaccinated IN, which is likely due to their ability to induce robust Th1 biased immune response. The Th1 biased immune response in PLGA, in particular, is likely due to its inherent property of antigen cross-presentation through MHC class I pathway after endo-lysosomal escape of antigens. Both nanoparticles failed to reduce nasal virus shedding to a greater extent because they could not induce mucosal IgA

177

responses, which warrants for incorporation of additional adjuvants in such nanovaccine

formulations to achieve balanced Th1/Th2 immunity.

With the aim of inducing better mucosal antibody (IgA) response and to reduce

nasal virus shedding, chitosan-based inactivated influenza nanovaccine (CNPs-KAg) was

developed and evaluated in pigs in chapter 4. Chitosan is mucoadhesive in nature and

modulates tight junction protein in reversible manner to enhance paracellular antigen

uptake. The chitosan nanovaccine had 67% antigen encapsulation efficiency, 571.7nm

average particle size and weakly positive surface charge. CNPs-KAg enhance antigen

uptake in vitro and activated APCs to produce innate cytokines such as IFN-α, IL-1β and

IL-12. In heterologous SwIAV challenged pigs, CNPs-KAg-vaccinates lowered infectious virus titers both in nasal passage and lungs by 100 times compared to KAg- vaccinates. A moderate reduction in pneumonia was observed but temperature profile was similar between CNPs-KAg and KAg-vaccinates. Immunologically, CNPs-KAg induced robust mucosal IgA secretion (that helped in reduction of nasal virus shedding) and also improved mucosal and systemic cell-mediated immunity.

In chapter 5, we evaluated the adjuvant potential of novel corn-based NPs (Nano-

11) for its adjuvant potential after systemic (IM) and mucosal (IN) antigen delivery in pigs. The positively charged Nano-11 polymers adhere efficiently with negatively charged antigens including OVA and inactivated influenza virus. Nano-11 NPs had 80% antigen adsorption efficiency for inactivated influenza antigen (KAg) at 2:1 ratio of NPs and KAg, and were positively charged with average size of 487nm. Nano-11 NPs enhanced antigen uptake in vitro and induced secretion of innate cytokines by porcine

178

APCs. Prime-boost IN immunization of pigs with Nano-11+KAg upregulated both Th1 and Th2 transcription factors and improved both humoral and cellular immune responses.

Despite the immune-modulation, current Nano-11+KAg formulation did not provide efficient heterologous protection against influenza virus challenge infection compared to

KAg-vaccinates suggesting the need of optimization of this nanovaccine and conducting dose-response studies including additional adjuvants in the formulation The adjuvant potential of Nano-11 was evident even in IM injected Nano-11 adsorbed with OVA with production of significantly higher serum IgG response compared to OVA alone.

The physical chracteristics, immunogenicity and protective efficacy of different nanovaccine formulations delivered IN in pigs are compared in table 6.1. Overall, we did not observe enhanced respiratory disease in any of our IN vaccine-challenge studies.

PLGA and polyanhydride NPs primarily induced cell-mediated immunity while chitosan and Nano-11 induced both cellular and mucosal antibody (IgA) responses. The extent of cellular and humoral immune responses greatly differed among different NPs which was also evident in differences in protection parameters after challenge infection with heterologous virus. Our study highlighted that biodegradable and biocompatible polymer- based NPs can be used to improve the immune response and protective efficacy of intranasal inactivated influenza virus vaccine in pigs. The immunogenic properties of such NPs can be used to improve the breadth of protective immunity of current inactivated swine influenza vaccines and they can also be used to design NPs-based subunit swine influenza vaccines.

179

Further studies are warranted to understand the underlying mechanisms of immune-modulation by these NPs in pig system as such information will guide us in rational vaccine design. Follow up studies with each of the NPs vaccine formulations should focus on how to make them more stable at different storage conditions and methods to improve the specific arm of immune responses that they failed to elicit. For example, antibody response in PLGA and polyanhydride nanovaccines may be improved by incorporation of potent known mucosal adjuvants such as α-Galcer (activates iNKT cells) and mycobacterium whole cell lysate in pigs which induced robust mucosal IgA antibody response. Stronger and balanced Th1 and Th2 immune responses can also be generated after consecutive prime and boost vaccination with different NPs-based vaccines which showed robust cell-mediated immune response, such as PLGA and the one that showed strong mucosal IgA antibody response like chitosan NPs. Dose response study is also necessary to determine the optimal amount or dose of particular NPs (e.g.

Nano-11) to induce its inherent adjuvant potential in vivo in pigs. All of our studies were conducted in pigs that were caesarian-delivered and influenza antibody free. Evaluation of the immunogenicity and protective efficacy of these nanovaccines in commercial maternal antibody positive pigs will provide immediate commercial value as maternal antibody interference is one of the major factor hindering grower-finisher piglet immunization in the US. Likewise, vaccine-challenge studies with different virulent field strains of SwIAV and simultaneous comparison of nanovaccines with commercial influenza vaccines will further help in highlighting the benefits of IN immunization with biodegradable and biocompatible NPs-based swine influenza vaccines in pigs.

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Physical characteristics Antibody responses Cell-mediated Protective responses immune responses

Average Surface KAg HI titer Respiratory Systemic Mucosal Fever in Pneumonic Virus titer reduction compared size charge loading (serum and IgA response response pigs was lesions reduction to KAg group (fold) NPs used (nm) (mV) (%) BAL fluid) (PBMCs) (TBLN- absent after compared to KAg MNCs) DPC 1 group (%) Nasal swab BAL fluid PLGA 313 -18 57 NE Yes 40 2 40

Polyanhydride 181 -11 60 NE Yes 15 6 (20:80 CPTEG:CPH) Chitosan 572 +1.69 67 No 12 100 100 (2:1 CS/TPP) Nano-11 487 +19.2 80 NE Yes 35 2 3

Table 6.1: Summary of physical characteristics, immunogenicity and protective efficacies of different intranasal swine influenza nanovaccines in nursery pigs. Immune responses and protective efficacies are expressed in comparison to IN KAg-vaccinates used as control in each experiment. Symbols: = Not improved; = Improved; = Strongly improved; NE = Not evaluated.

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Bibliography

1. Webster, R. G., W. J. Bean, O. T. Gorman, T. M. Chambers, and Y. Kawaoka. 1992. Evolution and ecology of influenza A viruses. Microbiol Rev 56: 152-179. 2. Taubenberger, J. K., and D. M. Morens. 2009. Pandemic influenza--including a risk assessment of H5N1. Rev Sci Tech 28: 187-202. 3. Webster, R. G. 2002. The importance of animal influenza for human disease. Vaccine 20 Suppl 2: S16-20. 4. WHO. 2014. Influenza (seasonal) fact sheet. 5. Punpanich, W., and T. Chotpitayasunondh. 2012. A review on the clinical spectrum and natural history of human influenza. International Journal of Infectious Diseases 16: e714-e723. 6. Shaw, M. L., and P. Palese. 2013. Orthomyxoviridae. In Field's Virology. D. M. Knipe, and P. Howley, eds. Wolters Kluwer. 7. Sandbulte, M. R., A. R. Spickler, P. K. Zaabel, and J. A. Roth. 2015. Optimal Use of Vaccines for Control of Influenza A Virus in Swine. Vaccines (Basel) 3: 22-73. 8. Skehel, J. J., and D. C. Wiley. 2000. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69: 531-569. 9. Matrosovich, M. N., T. Y. Matrosovich, T. Gray, N. A. Roberts, and H. D. Klenk. 2004. Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. Journal of virology 78: 12665-12667. 10. Palese, P., J. L. Schulman, G. Bodo, and P. Meindl. 1974. Inhibition of influenza and parainfluenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N- trifluoroacetylneuraminic acid (FANA). Virology 59: 490-498. 11. Gottschalk, A. 1957. Neuraminidase: the specific enzyme of influenza virus and Vibrio cholerae. Biochim Biophys Acta 23: 645-646. 12. Tong, S., X. Zhu, Y. Li, M. Shi, J. Zhang, M. Bourgeois, H. Yang, X. Chen, S. Recuenco, J. Gomez, L. M. Chen, A. Johnson, Y. Tao, C. Dreyfus, W. Yu, R. McBride, P. J. Carney, A. T. Gilbert, J. Chang, Z. Guo, C. T. Davis, J. C. Paulson, J. Stevens, C. E. Rupprecht, E. C. Holmes, I. A. Wilson, and R. O. Donis. 2013. New world bats harbor diverse influenza A viruses. PLoS Pathog 9: e1003657. 13. Wang, C., K. Takeuchi, L. H. Pinto, and R. A. Lamb. 1993. Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block. Journal of virology 67: 5585-5594. 14. Vincent, A. L., W. Ma, K. M. Lager, B. H. Janke, and J. A. Richt. 2008. Swine influenza viruses a North American perspective. Adv Virus Res 72: 127-154. 15. Chen, R., and E. C. Holmes. 2006. Avian influenza virus exhibits rapid evolutionary dynamics. Mol Biol Evol 23: 2336-2341. 182

16. Domingo, E., E. Baranowski, C. M. Ruiz-Jarabo, A. M. Martin-Hernandez, J. C. Saiz, and C. Escarmis. 1998. Quasispecies structure and persistence of RNA viruses. Emerg Infect Dis 4: 521-527. 17. Taubenberger, J. K., and J. C. Kash. 2010. Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe 7: 440-451. 18. Scholtissek, C., W. Rohde, V. Von Hoyningen, and R. Rott. 1978. On the origin of the human influenza virus subtypes H2N2 and H3N2. Virology 87: 13-20. 19. Garten, R. J., C. T. Davis, C. A. Russell, B. Shu, S. Lindstrom, A. Balish, W. M. Sessions, X. Xu, E. Skepner, V. Deyde, M. Okomo-Adhiambo, L. Gubareva, J. Barnes, C. B. Smith, S. L. Emery, M. J. Hillman, P. Rivailler, J. Smagala, M. de Graaf, D. F. Burke, R. A. Fouchier, C. Pappas, C. M. Alpuche-Aranda, H. Lopez- Gatell, H. Olivera, I. Lopez, C. A. Myers, D. Faix, P. J. Blair, C. Yu, K. M. Keene, P. D. Dotson, Jr., D. Boxrud, A. R. Sambol, S. H. Abid, K. St George, T. Bannerman, A. L. Moore, D. J. Stringer, P. Blevins, G. J. Demmler-Harrison, M. Ginsberg, P. Kriner, S. Waterman, S. Smole, H. F. Guevara, E. A. Belongia, P. A. Clark, S. T. Beatrice, R. Donis, J. Katz, L. Finelli, C. B. Bridges, M. Shaw, D. B. Jernigan, T. M. Uyeki, D. J. Smith, A. I. Klimov, and N. J. Cox. 2009. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325: 197-201. 20. Lambert, L. C., and A. S. Fauci. 2010. Influenza vaccines for the future. N Engl J Med 363: 2036-2044. 21. Dos Santos, G., E. Neumeier, and R. Bekkat-Berkani. 2016. Influenza: Can we cope better with the unpredictable? Hum Vaccin Immunother 12: 699-708. 22. Erbelding, E. J., D. Post, E. Stemmy, P. C. Roberts, A. D. Augustine, S. Ferguson, C. I. Paules, B. S. Graham, and A. S. Fauci. 2018. A Universal Influenza Vaccine: The Strategic Plan for the National Institute of Allergy and Infectious Diseases. J Infect Dis. 23. Easterday, B. C. 2003. Swine influenza: historical perspectives. In 4th International Symposium on Emerging and Re-emerging Pig Diseases, Rome, Italy. 241-244. 24. Zimmer, S. M., and D. S. Burke. 2009. Historical Perspective — Emergence of Influenza A (H1N1) Viruses. New England Journal of Medicine 361: 279-285. 25. Shope, R. E. 1931. The Etiology of Swine Influenza. Science 73: 214-215. 26. Shope, R. E. 1931. Swine Influenza : Iii. Filtration Experiments and Etiology. J Exp Med 54: 373-385. 27. Smith, W., C. H. Andrewes, and P. P. Laidlaw. 1933. A VIRUS OBTAINED FROM INFLUENZA PATIENTS. The Lancet 222: 66-68. 28. Shope, R. E. 1936. The Incidence of Neutralizing Antibodies for Swine Influenza Virus in the Sera of Human Beings of Different Ages. J Exp Med 63: 669-684. 29. Olsen, C. W. 2002. The emergence of novel swine influenza viruses in North America. Virus Res 85: 199-210. 30. Zhou, N. N., D. A. Senne, J. S. Landgraf, S. L. Swenson, G. Erickson, K. Rossow, L. Liu, K. Yoon, S. Krauss, and R. G. Webster. 1999. Genetic reassortment of

183

avian, swine, and human influenza A viruses in American pigs. Journal of virology 73: 8851-8856. 31. Webby, R. J., S. L. Swenson, S. L. Krauss, P. J. Gerrish, S. M. Goyal, and R. G. Webster. 2000. Evolution of swine H3N2 influenza viruses in the United States. Journal of virology 74: 8243-8251. 32. Karasin, A. I., C. W. Olsen, and G. A. Anderson. 2000. Genetic characterization of an H1N2 influenza virus isolated from a pig in Indiana. J Clin Microbiol 38: 2453-2456. 33. Webby, R. J., K. Rossow, G. Erickson, Y. Sims, and R. Webster. 2004. Multiple lineages of antigenically and genetically diverse influenza A virus co-circulate in the United States swine population. Virus Res 103: 67-73. 34. Vincent, A. L., W. Ma, K. M. Lager, M. R. Gramer, J. A. Richt, and B. H. Janke. 2009. Characterization of a newly emerged genetic cluster of H1N1 and H1N2 swine influenza virus in the United States. Virus Genes 39: 176-185. 35. Anderson, T. K., M. I. Nelson, P. Kitikoon, S. L. Swenson, J. A. Korslund, and A. L. Vincent. 2013. Population dynamics of cocirculating swine influenza A viruses in the United States from 2009 to 2012. Influenza Other Respir Viruses 7 Suppl 4: 42-51. 36. Ducatez, M. F., B. Hause, E. Stigger-Rosser, D. Darnell, C. Corzo, K. Juleen, R. Simonson, C. Brockwell-Staats, A. Rubrum, D. Wang, A. Webb, J. C. Crumpton, J. Lowe, M. Gramer, and R. J. Webby. 2011. Multiple reassortment between pandemic (H1N1) 2009 and endemic influenza viruses in pigs, United States. Emerg Infect Dis 17: 1624-1629. 37. de Jong, J. C., A. P. van Nieuwstadt, T. G. Kimman, W. L. Loeffen, T. M. Bestebroer, K. Bijlsma, C. Verweij, A. D. Osterhaus, and E. C. Class. 1999. Antigenic drift in swine influenza H3 haemagglutinins with implications for vaccination policy. Vaccine 17: 1321-1328. 38. Rajao, D. S., T. K. Anderson, P. C. Gauger, and A. L. Vincent. 2014. Pathogenesis and vaccination of influenza A virus in swine. Curr Top Microbiol Immunol 385: 307-326. 39. Rajao, D. S., T. K. Anderson, P. Kitikoon, J. Stratton, N. S. Lewis, and A. L. Vincent. 2018. Antigenic and genetic evolution of contemporary swine H1 influenza viruses in the United States. Virology 518: 45-54. 40. Walia, R. R., T. K. Anderson, and A. L. Vincent. 2018. Regional patterns of genetic diversity in swine influenza A viruses in the United States from 2010 to 2016. Influenza Other Respir Viruses. 41. Easterday, B. C. 1980. Animals in the influenza world. Philos Trans R Soc Lond B Biol Sci 288: 433-437. 42. Janke, B. H. 2013. Clinicopathological features of Swine influenza. Curr Top Microbiol Immunol 370: 69-83. 43. Winkler, G. C., and N. F. Cheville. 1986. Ultrastructural morphometric investigation of early lesions in the pulmonary alveolar region of pigs during experimental swine influenza infection. Am J Pathol 122: 541-552.

184

44. Hinshaw, V. S., W. J. Bean, Jr., R. G. Webster, and B. C. Easterday. 1978. The prevalence of influenza viruses in swine and the antigenic and genetic relatedness of influenza viruses from man and swine. Virology 84: 51-62. 45. Corzo, C. A., M. Culhane, K. Juleen, E. Stigger-Rosser, M. F. Ducatez, R. J. Webby, and J. F. Lowe. 2013. Active surveillance for influenza A virus among swine, midwestern United States, 2009-2011. Emerg Infect Dis 19: 954-960. 46. Brockmeier, S. L., P. G. Halbur, and E. L. Thacker. 2002. Porcine Respiratory Disease Complex. . In Polymicrobial Diseases. K. A. Brogden, and J. M. Guthmiller, eds. ASM Press, Washingtion (DC). 47. Detmer, S. E., D. P. Patnayak, Y. Jiang, M. R. Gramer, and S. M. Goyal. 2011. Detection of Influenza A virus in porcine oral fluid samples. J Vet Diagn Invest 23: 241-247. 48. Khatri, M., V. Dwivedi, S. Krakowka, C. Manickam, A. Ali, L. Wang, Z. Qin, G. J. Renukaradhya, and C. W. Lee. 2010. Swine influenza H1N1 virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human H1N1 influenza virus. Journal of virology 84: 11210-11218. 49. Rajao, D. S., and A. L. Vincent. 2015. Swine as a model for influenza A virus infection and immunity. ILAR J 56: 44-52. 50. Nayak, D. P., M. J. Twiehaus, G. W. Kelley, and N. R. Underdahl. 1965. Immunocytologic and histopathologic development of experimental swine influenza infection in pigs. Am J Vet Res 26: 1271-1283. 51. Van Reeth, K., and M. B. Pensaert. 1994. Porcine respiratory coronavirus- mediated interference against influenza virus replication in the respiratory tract of feeder pigs. Am J Vet Res 55: 1275-1281. 52. Jung, K., Y. Ha, and C. Chae. 2005. Pathogenesis of swine influenza virus subtype H1N2 infection in pigs. J Comp Pathol 132: 179-184. 53. Janke, B. H. 2014. Influenza A virus infections in swine: pathogenesis and diagnosis. Vet Pathol 51: 410-426. 54. Dea, S., R. Bilodeau, R. Sauvageau, C. Montpetit, and G. P. Martineau. 1992. Antigenic variant of swine influenza virus causing proliferative and necrotizing pneumonia in pigs. J Vet Diagn Invest 4: 380-392. 55. Kothalawala, H., M. J. Toussaint, and E. Gruys. 2006. An overview of swine influenza. Vet Q 28: 46-53. 56. Schultz-Cherry, S., C. W. Olsen, and B. C. Easterday. 2013. History of Swine Influenza. In Swine Influenza. J. A. Richt, and R. J. Webby, eds. Springer Berlin Heidelberg, Berlin, Heidelberg. 21-27. 57. Richard, B. 2003. The ‘Direct Costs’of Livestock Disease: The Development of a System of Models for the Analysis of 30 Endemic Livestock Diseases in Great Britain. Journal of Agricultural Economics 54: 55-71. 58. Thacker, E. L., B. J. Thacker, and B. H. Janke. 2001. Interaction between Mycoplasma hyopneumoniae and swine influenza virus. J Clin Microbiol 39: 2525-2530. 59. Haden, C., T. Painter, T. Fangman, and H. D. 2012. Assessing production parameters and economic impact of swine influenza, PRRS and Mycoplasma 185

hyopneimoniae on finishing pigs in a large production system In Proceedings of AASV Annual Meeting. 75-76. 60. Ellis, J., E. Clark, D. Haines, K. West, S. Krakowka, S. Kennedy, and G. M. Allan. 2004. Porcine circovirus-2 and concurrent infections in the field. Vet Microbiol 98: 159-163. 61. Fablet, C., C. Marois-Crehan, G. Simon, B. Grasland, A. Jestin, M. Kobisch, F. Madec, and N. Rose. 2012. Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: a cross-sectional study. Vet Microbiol 157: 152- 163. 62. CDC. 2018. Information on Swine Influenza/Variant Influenza Virus. . 5-19-2017 ed. 63. Kluska, V., M. Macku, and J. Mensik. 1961. [Demonstration of antibodies against swine influenza viruses in man]. Cesk Pediatr 16: 408-414. 64. Myers, K. P., C. W. Olsen, and G. C. Gray. 2007. Cases of swine influenza in humans: a review of the literature. Clin Infect Dis 44: 1084-1088. 65. Smith, T. F., E. O. Burgert, Jr., W. R. Dowdle, G. R. Noble, R. J. Campbell, and R. E. Van Scoy. 1976. Isolation of swine influenza virus from autopsy lung tissue of man. N Engl J Med 294: 708-710. 66. Newman, A. P., E. Reisdorf, J. Beinemann, T. M. Uyeki, A. Balish, B. Shu, S. Lindstrom, J. Achenbach, C. Smith, and J. P. Davis. 2008. Human case of swine influenza A (H1N1) triple reassortant virus infection, Wisconsin. Emerg Infect Dis 14: 1470-1472. 67. Shinde, V., C. B. Bridges, T. M. Uyeki, B. Shu, A. Balish, X. Xu, S. Lindstrom, L. V. Gubareva, V. Deyde, R. J. Garten, M. Harris, S. Gerber, S. Vagasky, F. Smith, N. Pascoe, K. Martin, D. Dufficy, K. Ritger, C. Conover, P. Quinlisk, A. Klimov, J. S. Bresee, and L. Finelli. 2009. Triple-reassortant swine influenza A (H1) in humans in the United States, 2005-2009. N Engl J Med 360: 2616-2625. 68. Scholtissek, C., H. Burger, O. Kistner, and K. F. Shortridge. 1985. The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses. Virology 147: 287-294. 69. Hinshaw, V. S., R. G. Webster, B. C. Easterday, and W. J. Bean, Jr. 1981. Replication of avian influenza A viruses in mammals. Infect Immun 34: 354-361. 70. Kida, H., T. Ito, J. Yasuda, Y. Shimizu, C. Itakura, K. F. Shortridge, Y. Kawaoka, and R. G. Webster. 1994. Potential for transmission of avian influenza viruses to pigs. J Gen Virol 75 ( Pt 9): 2183-2188. 71. Rogers, G. N., and J. C. Paulson. 1983. Receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the H3 hemagglutinin based on species of origin. Virology 127: 361-373. 72. Rogers, G. N., T. J. Pritchett, J. L. Lane, and J. C. Paulson. 1983. Differential sensitivity of human, avian, and equine influenza A viruses to a glycoprotein inhibitor of infection: selection of receptor specific variants. Virology 131: 394- 408. 73. Ito, T., J. N. Couceiro, S. Kelm, L. G. Baum, S. Krauss, M. R. Castrucci, I. Donatelli, H. Kida, J. C. Paulson, R. G. Webster, and Y. Kawaoka. 1998. 186

Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. Journal of virology 72: 7367-7373. 74. Trebbien, R., L. E. Larsen, and B. M. Viuff. 2011. Distribution of sialic acid receptors and influenza A virus of avian and swine origin in experimentally infected pigs. Virol J 8: 434. 75. Castrucci, M. R., I. Donatelli, L. Sidoli, G. Barigazzi, Y. Kawaoka, and R. G. Webster. 1993. Genetic reassortment between avian and human influenza A viruses in Italian pigs. Virology 193: 503-506. 76. Hass, J., S. Matuszewski, D. Cieslik, and M. Haase. 2011. The role of swine as "mixing vessel" for interspecies transmission of the influenza A subtype H1N1: a simultaneous Bayesian inference of phylogeny and ancestral hosts. Infect Genet Evol 11: 437-441. 77. Brown, I. H., P. A. Harris, J. W. McCauley, and D. J. Alexander. 1998. Multiple genetic reassortment of avian and human influenza A viruses in European pigs, resulting in the emergence of an H1N2 virus of novel genotype. J Gen Virol 79 ( Pt 12): 2947-2955. 78. Gregory, V., W. Lim, K. Cameron, M. Bennett, S. Marozin, A. Klimov, H. Hall, N. Cox, A. Hay, and Y. P. Lin. 2001. Infection of a child in Hong Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs. J Gen Virol 82: 1397-1406. 79. Mena, I., M. I. Nelson, F. Quezada-Monroy, J. Dutta, R. Cortes-Fernandez, J. H. Lara-Puente, F. Castro-Peralta, L. F. Cunha, N. S. Trovao, B. Lozano-Dubernard, A. Rambaut, H. van Bakel, and A. Garcia-Sastre. 2016. Origins of the 2009 H1N1 influenza pandemic in swine in Mexico. Elife 5. 80. Smith, G. J., D. Vijaykrishna, J. Bahl, S. J. Lycett, M. Worobey, O. G. Pybus, S. K. Ma, C. L. Cheung, J. Raghwani, S. Bhatt, J. S. Peiris, Y. Guan, and A. Rambaut. 2009. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459: 1122-1125. 81. Dawood, F. S., A. D. Iuliano, C. Reed, M. I. Meltzer, D. K. Shay, P. Y. Cheng, D. Bandaranayake, R. F. Breiman, W. A. Brooks, P. Buchy, D. R. Feikin, K. B. Fowler, A. Gordon, N. T. Hien, P. Horby, Q. S. Huang, M. A. Katz, A. Krishnan, R. Lal, J. M. Montgomery, K. Molbak, R. Pebody, A. M. Presanis, H. Razuri, A. Steens, Y. O. Tinoco, J. Wallinga, H. Yu, S. Vong, J. Bresee, and M. A. Widdowson. 2012. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. The Lancet. Infectious diseases 12: 687-695. 82. Pang, I. K., and A. Iwasaki. 2011. Inflammasomes as mediators of immunity against influenza virus. Trends in immunology 32: 34-41. 83. Iwasaki, A., and P. S. Pillai. 2014. Innate immunity to influenza virus infection. Nat Rev Immunol 14: 315-328. 84. Kreijtz, J. H., R. A. Fouchier, and G. F. Rimmelzwaan. 2011. Immune responses to influenza virus infection. Virus Res 162: 19-30.

187

85. Zhang, J., J. Miao, J. Hou, and C. Lu. 2015. The effects of H3N2 swine influenza virus infection on TLRs and RLRs signaling pathways in porcine alveolar macrophages. Virol J 12: 61. 86. Crisci, E., T. Mussa, L. Fraile, and M. Montoya. 2013. Review: influenza virus in pigs. Mol Immunol 55: 200-211. 87. Van Reeth, K., H. Nauwynck, and M. Pensaert. 1998. Bronchoalveolar interferon- alpha, tumor necrosis factor-alpha, interleukin-1, and inflammation during acute influenza in pigs: a possible model for humans? J Infect Dis 177: 1076-1079. 88. Van Reeth, K., S. Van Gucht, and M. Pensaert. 2002. Correlations between lung proinflammatory cytokine levels, virus replication, and disease after swine influenza virus challenge of vaccination-immune pigs. Viral Immunol 15: 583- 594. 89. Brookes, S. M., A. Nunez, B. Choudhury, M. Matrosovich, S. C. Essen, D. Clifford, M. J. Slomka, G. Kuntz-Simon, F. Garcon, B. Nash, A. Hanna, P. M. Heegaard, S. Queguiner, C. Chiapponi, M. Bublot, J. M. Garcia, R. Gardner, E. Foni, W. Loeffen, L. Larsen, K. Van Reeth, J. Banks, R. M. Irvine, and I. H. Brown. 2010. Replication, pathogenesis and transmission of pandemic (H1N1) 2009 virus in non-immune pigs. PLoS One 5: e9068. 90. Barbe, F., K. Atanasova, and K. Van Reeth. 2011. Cytokines and acute phase proteins associated with acute swine influenza infection in pigs. Vet J 187: 48-53. 91. Kim, B., K. K. Ahn, Y. Ha, Y. H. Lee, D. Kim, J. H. Lim, S. H. Kim, M. Y. Kim, K. D. Cho, B. H. Lee, and C. Chae. 2009. Association of tumor necrosis factor- alpha with fever and pulmonary lesion score in pigs experimentally infected with swine influenza virus subtype H1N2. J Vet Med Sci 71: 611-616. 92. Jo, S. K., H. S. Kim, S. W. Cho, and S. H. Seo. 2007. Pathogenesis and inflammatory responses of swine H1N2 influenza viruses in pigs. Virus Res 129: 64-70. 93. Heinen, P. P., E. A. de Boer-Luijtze, and A. T. Bianchi. 2001. Respiratory and systemic humoral and cellular immune responses of pigs to a heterosubtypic influenza A virus infection. J Gen Virol 82: 2697-2707. 94. Heinen, P. P., A. P. van Nieuwstadt, J. M. Pol, E. A. de Boer-Luijtze, J. T. van Oirschot, and A. T. Bianchi. 2000. Systemic and mucosal isotype-specific antibody responses in pigs to experimental influenza virus infection. Viral Immunol 13: 237-247. 95. Larsen, D. L., A. Karasin, F. Zuckermann, and C. W. Olsen. 2000. Systemic and mucosal immune responses to H1N1 influenza virus infection in pigs. Vet Microbiol 74: 117-131. 96. Jayasekera, J. P., E. A. Moseman, and M. C. Carroll. 2007. Natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity. Journal of virology 81: 3487-3494. 97. Fernandez Gonzalez, S., J. P. Jayasekera, and M. C. Carroll. 2008. Complement and natural antibody are required in the long-term memory response to influenza virus. Vaccine 26 Suppl 8: I86-93.

188

98. Renegar, K. B., P. A. Small, Jr., L. G. Boykins, and P. F. Wright. 2004. Role of IgA versus IgG in the control of influenza viral infection in the murine respiratory tract. J Immunol 173: 1978-1986. 99. Pomorska-Mol, M., I. Markowska-Daniel, and K. Kwit. 2012. Immune and acute phase response in pigs experimentally infected with H1N2 swine influenza virus. FEMS Immunol Med Microbiol 66: 334-342. 100. Sandbulte, M. R., G. S. Jimenez, A. C. Boon, L. R. Smith, J. J. Treanor, and R. J. Webby. 2007. Cross-reactive neuraminidase antibodies afford partial protection against H5N1 in mice and are present in unexposed humans. PLoS Med 4: e59. 101. Fan, J., X. Liang, M. S. Horton, H. C. Perry, M. P. Citron, G. J. Heidecker, T. M. Fu, J. Joyce, C. T. Przysiecki, P. M. Keller, V. M. Garsky, R. Ionescu, Y. Rippeon, L. Shi, M. A. Chastain, J. H. Condra, M. E. Davies, J. Liao, E. A. Emini, and J. W. Shiver. 2004. Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. Vaccine 22: 2993-3003. 102. Ding, H., C. Tsai, F. Zhou, P. Buchy, V. Deubel, and P. Zhou. 2011. Heterosubtypic antibody response elicited with seasonal influenza vaccine correlates partial protection against highly pathogenic H5N1 virus. PLoS One 6: e17821. 103. Neirynck, S., T. Deroo, X. Saelens, P. Vanlandschoot, W. M. Jou, and W. Fiers. 1999. A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nature medicine 5: 1157-1163. 104. Tompkins, S. M., Z. S. Zhao, C. Y. Lo, J. A. Misplon, T. Liu, Z. Ye, R. J. Hogan, Z. Wu, K. A. Benton, T. M. Tumpey, and S. L. Epstein. 2007. Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1. Emerg Infect Dis 13: 426-435. 105. Hancock, K., V. Veguilla, X. Lu, W. Zhong, E. N. Butler, H. Sun, F. Liu, L. Dong, J. R. DeVos, P. M. Gargiullo, T. L. Brammer, N. J. Cox, T. M. Tumpey, and J. M. Katz. 2009. Cross-reactive antibody responses to the 2009 pandemic H1N1 influenza virus. N Engl J Med 361: 1945-1952. 106. Fiers, W., M. De Filette, K. El Bakkouri, B. Schepens, K. Roose, M. Schotsaert, A. Birkett, and X. Saelens. 2009. M2e-based universal influenza A vaccine. Vaccine 27: 6280-6283. 107. Ebrahimi, S. M., and M. Tebianian. 2011. Influenza A viruses: why focusing on M2e-based universal vaccines. Virus Genes 42: 1-8. 108. Sautto, G. A., G. A. Kirchenbaum, and T. M. Ross. 2018. Towards a universal influenza vaccine: different approaches for one goal. Virol J 15: 17. 109. Hashem, A. M. 2015. Prospects of HA-based universal influenza vaccine. Biomed Res Int 2015: 414637. 110. Lange, E., D. Kalthoff, U. Blohm, J. P. Teifke, A. Breithaupt, C. Maresch, E. Starick, S. Fereidouni, B. Hoffmann, T. C. Mettenleiter, M. Beer, and T. W. Vahlenkamp. 2009. Pathogenesis and transmission of the novel swine-origin influenza virus A/H1N1 after experimental infection of pigs. J Gen Virol 90: 2119-2123.

189

111. Talker, S. C., M. Stadler, H. C. Koinig, K. H. Mair, I. M. Rodriguez-Gomez, R. Graage, R. Zell, R. Durrwald, E. Starick, T. Harder, H. Weissenbock, B. Lamp, S. E. Hammer, A. Ladinig, A. Saalmuller, and W. Gerner. 2016. Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung. Journal of virology 90: 9364-9382. 112. Mozdzanowska, K., M. Furchner, K. Maiese, and W. Gerhard. 1997. CD4+ T cells are ineffective in clearing a pulmonary infection with influenza type A virus in the absence of B cells. Virology 239: 217-225. 113. Riberdy, J. M., K. J. Flynn, J. Stech, R. G. Webster, J. D. Altman, and P. C. Doherty. 1999. Protection against a lethal avian influenza A virus in a mammalian system. Journal of virology 73: 1453-1459. 114. Topham, D. J., and P. C. Doherty. 1998. Clearance of an influenza A virus by CD4+ T cells is inefficient in the absence of B cells. Journal of virology 72: 882- 885. 115. Furuya, Y., J. Chan, M. Regner, M. Lobigs, A. Koskinen, T. Kok, J. Manavis, P. Li, A. Mullbacher, and M. Alsharifi. 2010. Cytotoxic T cells are the predominant players providing cross-protective immunity induced by {gamma}-irradiated influenza A viruses. Journal of virology 84: 4212-4221. 116. Nguyen, H. H., Z. Moldoveanu, M. J. Novak, F. W. van Ginkel, E. Ban, H. Kiyono, J. R. McGhee, and J. Mestecky. 1999. Heterosubtypic immunity to lethal influenza A virus infection is associated with virus-specific CD8(+) cytotoxic T lymphocyte responses induced in mucosa-associated tissues. Virology 254: 50-60. 117. Sambhara, S., A. Kurichh, R. Miranda, T. Tumpey, T. Rowe, M. Renshaw, R. Arpino, A. Tamane, A. Kandil, O. James, B. Underdown, M. Klein, J. Katz, and D. Burt. 2001. Heterosubtypic immunity against human influenza A viruses, including recently emerged avian H5 and H9 viruses, induced by FLU-ISCOM vaccine in mice requires both cytotoxic T-lymphocyte and macrophage function. Cell Immunol 211: 143-153. 118. Kreijtz, J. H., R. Bodewes, G. van Amerongen, T. Kuiken, R. A. Fouchier, A. D. Osterhaus, and G. F. Rimmelzwaan. 2007. Primary influenza A virus infection induces cross-protective immunity against a lethal infection with a heterosubtypic virus strain in mice. Vaccine 25: 612-620. 119. Vincent, A. L., D. R. Perez, D. Rajao, T. K. Anderson, E. J. Abente, R. R. Walia, and N. S. Lewis. 2017. Influenza A virus vaccines for swine. Vet Microbiol 206: 35-44. 120. Vander Veen, R. L., A. T. Loynachan, M. A. Mogler, B. J. Russell, D. L. Harris, and K. I. Kamrud. 2012. Safety, immunogenicity, and efficacy of an alphavirus replicon-based swine influenza virus hemagglutinin vaccine. Vaccine 30: 1944- 1950. 121. Vander Veen, R. L., M. A. Mogler, B. J. Russell, A. T. Loynachan, D. L. Harris, and K. I. Kamrud. 2013. Haemagglutinin and nucleoprotein replicon particle vaccination of swine protects against the pandemic H1N1 2009 virus. Vet Rec 173: 344. 122. USDA. 2018. Current Veterinary Biologics Product Catalogue. 190

123. USDA. 2016. Swine 2012. Part II: Reference of Swine Health and Health Management in the United States, 2012. . 124. Zoetis. Swine Influenza Virus (SIV). 125. Lee, J. H., M. R. Gramer, and H. S. Joo. 2007. Efficacy of swine influenza A virus vaccines against an H3N2 virus variant. Can J Vet Res 71: 207-212. 126. Kitikoon, P., A. L. Vincent, B. H. Janke, B. Erickson, E. L. Strait, S. Yu, M. R. Gramer, and E. L. Thacker. 2009. Swine influenza matrix 2 (M2) protein contributes to protection against infection with different H1 swine influenza virus (SIV) isolates. Vaccine 28: 523-531. 127. Vincent, A. L., K. M. Lager, B. H. Janke, M. R. Gramer, and J. A. Richt. 2008. Failure of protection and enhanced pneumonia with a US H1N2 swine influenza virus in pigs vaccinated with an inactivated classical swine H1N1 vaccine. Vet Microbiol 126: 310-323. 128. Vincent, A. L., K. M. Lager, K. S. Faaberg, M. Harland, E. L. Zanella, J. R. Ciacci-Zanella, M. E. Kehrli, Jr., B. H. Janke, and A. Klimov. 2010. Experimental inoculation of pigs with pandemic H1N1 2009 virus and HI cross-reactivity with contemporary swine influenza virus antisera. Influenza Other Respir Viruses 4: 53-60. 129. Bikour, M. H., E. Cornaglia, and Y. Elazhary. 1996. Evaluation of a protective immunity induced by an inactivated influenza H3N2 vaccine after an intratracheal challenge of pigs. Can J Vet Res 60: 312-314. 130. Van Reeth, K., and W. Ma. 2013. Swine Influenza Virus Vaccines: To Change or Not to Change—That’s the Question. In Swine Influenza. J. A. Richt, and R. J. Webby, eds. Springer Berlin Heidelberg, Berlin, Heidelberg. 173-200. 131. Allerson, M., J. Deen, S. E. Detmer, M. R. Gramer, H. S. Joo, A. Romagosa, and M. Torremorell. 2013. The impact of maternally derived immunity on influenza A virus transmission in neonatal pig populations. Vaccine 31: 500-505. 132. Renshaw, H. W. 1975. Influence of antibody-mediated immune suppression on clinical, viral, and immune responses to swine influenza infection. Am J Vet Res 36: 5-13. 133. Blaskovic, D., O. Jamrichova, V. Rathova, M. M. Kaplan, and D. Kociskova. 1970. Experimental infection of weanling pigs with A-swine influenza virus. 2. The shedding of virus by infected animals. Bull World Health Organ 42: 767-770. 134. Choi, Y. K., S. M. Goyal, and H. S. Joo. 2004. Evaluation of transmission of swine influenza type A subtype H1N2 virus in seropositive pigs. Am J Vet Res 65: 303-306. 135. Loeffen, W. L., P. P. Heinen, A. T. Bianchi, W. A. Hunneman, and J. H. Verheijden. 2003. Effect of maternally derived antibodies on the clinical signs and immune response in pigs after primary and secondary infection with an influenza H1N1 virus. Vet Immunol Immunopathol 92: 23-35. 136. Kitikoon, P., D. Nilubol, B. J. Erickson, B. H. Janke, T. C. Hoover, S. A. Sornsen, and E. L. Thacker. 2006. The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination. Vet Immunol Immunopathol 112: 117-128. 191

137. Vincent, A. L., W. Ma, K. M. Lager, J. A. Richt, B. H. Janke, M. R. Sandbulte, P. C. Gauger, C. L. Loving, R. J. Webby, and A. Garcia-Sastre. 2012. Live attenuated influenza vaccine provides superior protection from heterologous infection in pigs with maternal antibodies without inducing vaccine-associated enhanced respiratory disease. Journal of virology 86: 10597-10605. 138. Rajao, D. S., M. R. Sandbulte, P. C. Gauger, P. Kitikoon, R. Platt, J. A. Roth, D. R. Perez, C. L. Loving, and A. L. Vincent. 2016. Heterologous challenge in the presence of maternally-derived antibodies results in vaccine-associated enhanced respiratory disease in weaned piglets. Virology 491: 79-88. 139. Gauger, P. C., A. L. Vincent, C. L. Loving, K. M. Lager, B. H. Janke, M. E. Kehrli, Jr., and J. A. Roth. 2011. Enhanced pneumonia and disease in pigs vaccinated with an inactivated human-like (delta-cluster) H1N2 vaccine and challenged with pandemic 2009 H1N1 influenza virus. Vaccine 29: 2712-2719. 140. Braucher, D. R., J. N. Henningson, C. L. Loving, A. L. Vincent, E. Kim, J. Steitz, A. A. Gambotto, and M. E. Kehrli, Jr. 2012. Intranasal vaccination with replication-defective adenovirus type 5 encoding influenza virus hemagglutinin elicits protective immunity to homologous challenge and partial protection to heterologous challenge in pigs. Clin Vaccine Immunol 19: 1722-1729. 141. Gauger, P. C., A. L. Vincent, C. L. Loving, J. N. Henningson, K. M. Lager, B. H. Janke, M. E. Kehrli, Jr., and J. A. Roth. 2012. Kinetics of lung lesion development and pro-inflammatory cytokine response in pigs with vaccine- associated enhanced respiratory disease induced by challenge with pandemic (2009) A/H1N1 influenza virus. Vet Pathol 49: 900-912. 142. Khurana, S., C. L. Loving, J. Manischewitz, L. R. King, P. C. Gauger, J. Henningson, A. L. Vincent, and H. Golding. 2013. Vaccine-induced anti-HA2 antibodies promote virus fusion and enhance influenza virus respiratory disease. Sci Transl Med 5: 200ra114. 143. Tamura, S., and T. Kurata. 2004. Defense mechanisms against influenza virus infection in the respiratory tract mucosa. Jpn J Infect Dis 57: 236-247. 144. van Riet, E., A. Ainai, T. Suzuki, and H. Hasegawa. 2012. Mucosal IgA responses in influenza virus infections; thoughts for vaccine design. Vaccine 30: 5893-5900. 145. Heinen, P. P., A. P. van Nieuwstadt, E. A. de Boer-Luijtze, and A. T. Bianchi. 2001. Analysis of the quality of protection induced by a porcine influenza A vaccine to challenge with an H3N2 virus. Vet Immunol Immunopathol 82: 39-56. 146. Larsen, D. L., A. Karasin, and C. W. Olsen. 2001. Immunization of pigs against influenza virus infection by DNA vaccine priming followed by killed-virus vaccine boosting. Vaccine 19: 2842-2853. 147. Martin, J. E., and B. S. Graham. 2009. Immunization against Viral Diseases. . In Clinical Virology, 3rd ed. D. D. Richman, R. J. Whitley, and F. G. Hayden, eds. ASM Press, Washington, DC. 148. Chen, Q., D. Madson, C. L. Miller, and D. L. Harris. 2012. Vaccine development for protecting swine against influenza virus. Anim Health Res Rev 13: 181-195. 149. Vander Veen, R. L., D. L. Harris, and K. I. Kamrud. 2012. Alphavirus replicon vaccines. Anim Health Res Rev 13: 1-9. 192

150. Bosworth, B., M. M. Erdman, D. L. Stine, I. Harris, C. Irwin, M. Jens, A. Loynachan, K. Kamrud, and D. L. Harris. 2010. Replicon particle vaccine protects swine against influenza. Comp Immunol Microbiol Infect Dis 33: e99- e103. 151. Erdman, M. M., K. I. Kamrud, D. L. Harris, and J. Smith. 2010. Alphavirus replicon particle vaccines developed for use in humans induce high levels of antibodies to influenza virus hemagglutinin in swine: proof of concept. Vaccine 28: 594-596. 152. Vander Veen, R., K. Kamrud, M. Mogler, A. T. Loynachan, J. McVicker, P. Berglund, G. Owens, S. Timberlake, W. Lewis, J. Smith, and D. L. Harris. 2009. Rapid development of an efficacious swine vaccine for novel H1N1. PLoS Curr 1: RRN1123. 153. Wesley, R. D., and K. M. Lager. 2006. Overcoming maternal antibody interference by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of swine influenza virus. Vet Microbiol 118: 67-75. 154. Kappes, M. A., M. R. Sandbulte, R. Platt, C. Wang, K. M. Lager, J. N. Henningson, A. Lorusso, A. L. Vincent, C. L. Loving, J. A. Roth, and M. E. Kehrli, Jr. 2012. Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs. Vaccine 30: 280-288. 155. Loving, C. L., K. M. Lager, A. L. Vincent, S. L. Brockmeier, P. C. Gauger, T. K. Anderson, P. Kitikoon, D. R. Perez, and M. E. Kehrli, Jr. 2013. Efficacy in pigs of inactivated and live attenuated influenza virus vaccines against infection and transmission of an emerging H3N2 similar to the 2011-2012 H3N2v. Journal of virology 87: 9895-9903. 156. Pyo, H. M., and Y. Zhou. 2014. Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection. Vaccine 32: 3835-3842. 157. Masic, A., J. S. Booth, G. K. Mutwiri, L. A. Babiuk, and Y. Zhou. 2009. Elastase- dependent live attenuated swine influenza A viruses are immunogenic and confer protection against swine influenza A virus infection in pigs. Journal of virology 83: 10198-10210. 158. Vincent, A. L., W. Ma, K. M. Lager, B. H. Janke, R. J. Webby, A. Garcia-Sastre, and J. A. Richt. 2007. Efficacy of intranasal administration of a truncated NS1 modified live influenza virus vaccine in swine. Vaccine 25: 7999-8009. 159. Sandbulte, M. R., R. Platt, J. A. Roth, J. N. Henningson, K. A. Gibson, D. S. Rajao, C. L. Loving, and A. L. Vincent. 2014. Divergent immune responses and disease outcomes in piglets immunized with inactivated and attenuated H3N2 swine influenza vaccines in the presence of maternally-derived antibodies. Virology 464-465: 45-54. 160. Gorres, J. P., K. M. Lager, W. P. Kong, M. Royals, J. P. Todd, A. L. Vincent, C. J. Wei, C. L. Loving, E. L. Zanella, B. Janke, M. E. Kehrli, Jr., G. J. Nabel, and S. S. Rao. 2011. DNA vaccination elicits protective immune responses against 193

pandemic and classic swine influenza viruses in pigs. Clin Vaccine Immunol 18: 1987-1995. 161. Borggren, M., J. Nielsen, K. Bragstad, I. Karlsson, J. S. Krog, J. A. Williams, and A. Fomsgaard. 2015. Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans. Hum Vaccin Immunother 11: 1983-1990. 162. Borggren, M., J. Nielsen, I. Karlsson, T. S. Dalgaard, R. Trebbien, J. A. Williams, and A. Fomsgaard. 2016. A polyvalent influenza DNA vaccine applied by needle- free intradermal delivery induces cross-reactive humoral and cellular immune responses in pigs. Vaccine 34: 3634-3640. 163. Bragstad, K., L. Vinner, M. S. Hansen, J. Nielsen, and A. Fomsgaard. 2013. A polyvalent influenza A DNA vaccine induces heterologous immunity and protects pigs against pandemic A(H1N1)pdm09 virus infection. Vaccine 31: 2281-2288. 164. Mamo, T., and G. A. Poland. 2012. Nanovaccinology: the next generation of vaccines meets 21st century materials science and engineering. Vaccine 30: 6609- 6611. 165. Gregory, A. E., R. Titball, and D. Williamson. 2013. Vaccine delivery using nanoparticles. Front Cell Infect Microbiol 3: 13. 166. Damgé, C., C. Michel, M. Aprahamian, P. Couvreur, and J. P. Devissaguet. 1990. Nanocapsules as carriers for oral peptide delivery. Journal of Controlled Release 13: 233-239. 167. Pandey, R., Z. Ahmad, S. Sharma, and G. K. Khuller. 2005. Nano-encapsulation of azole antifungals: potential applications to improve oral drug delivery. Int J Pharm 301: 268-276. 168. Demento, S. L., W. Cui, J. M. Criscione, E. Stern, J. Tulipan, S. M. Kaech, and T. M. Fahmy. 2012. Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype. Biomaterials 33: 4957-4964. 169. Xiang, S. D., A. Scholzen, G. Minigo, C. David, V. Apostolopoulos, P. L. Mottram, and M. Plebanski. 2006. Pathogen recognition and development of particulate vaccines: does size matter? Methods 40: 1-9. 170. Binjawadagi, B., V. Dwivedi, C. Manickam, K. Ouyang, Y. Wu, L. J. Lee, J. B. Torrelles, and G. J. Renukaradhya. 2014. Adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs. Int J Nanomedicine 9: 679-694. 171. Yoshida, M., and J. E. Babensee. 2004. Poly(lactic-co-glycolic acid) enhances maturation of human monocyte-derived dendritic cells. J Biomed Mater Res A 71: 45-54. 172. Slutter, B., and W. Jiskoot. 2016. Sizing the optimal dimensions of a vaccine delivery system: a particulate matter. Expert Opin Drug Deliv 13: 167-170. 173. Joshi, V. B., S. M. Geary, and A. K. Salem. 2013. Biodegradable particles as vaccine delivery systems: size matters. AAPS J 15: 85-94. 174. Brenza, T. M., L. K. Petersen, Y. Zhang, L. M. Huntimer, A. E. Ramer-Tait, J. M. Hostetter, M. J. Wannemuehler, and B. Narasimhan. 2015. Pulmonary 194

biodistribution and cellular uptake of intranasally administered monodisperse particles. Pharm Res 32: 1368-1382. 175. Kreuter, J. 1995. Nanoparticles as adjuvants for vaccines. Pharm Biotechnol 6: 463-472. 176. Zhao, L., A. Seth, N. Wibowo, C. X. Zhao, N. Mitter, C. Yu, and A. P. Middelberg. 2014. Nanoparticle vaccines. Vaccine 32: 327-337. 177. Aoshi, T. 2017. Modes of Action for Mucosal Vaccine Adjuvants. Viral Immunol 30: 463-470. 178. Coffman, R. L., A. Sher, and R. A. Seder. 2010. Vaccine adjuvants: putting innate immunity to work. Immunity 33: 492-503. 179. Garcon, N., and A. Di Pasquale. 2017. From discovery to licensure, the Adjuvant System story. Hum Vaccin Immunother 13: 19-33. 180. Kool, M., K. Fierens, and B. N. Lambrecht. 2012. Alum adjuvant: some of the tricks of the oldest adjuvant. J Med Microbiol 61: 927-934. 181. Hogenesch, H. 2012. Mechanism of immunopotentiation and safety of aluminum adjuvants. Front Immunol 3: 406. 182. Spickler, A. R., and J. A. Roth. 2003. Adjuvants in veterinary vaccines: modes of action and adverse effects. J Vet Intern Med 17: 273-281. 183. Gerdts, V. 2015. Adjuvants for veterinary vaccines--types and modes of action. Berl Munch Tierarztl Wochenschr 128: 456-463. 184. Lycke, N. 2012. Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol 12: 592-605. 185. Renukaradhya, G. J., B. Narasimhan, and S. K. Mallapragada. 2015. Respiratory nanoparticle-based vaccines and challenges associated with animal models and translation. Journal of controlled release : official journal of the Controlled Release Society 219: 622-631. 186. Wen, Z. S., Y. L. Xu, X. T. Zou, and Z. R. Xu. 2011. Chitosan nanoparticles act as an adjuvant to promote both Th1 and Th2 immune responses induced by ovalbumin in mice. Mar Drugs 9: 1038-1055. 187. Tamayo, I., J. M. Irache, C. Mansilla, J. Ochoa-Reparaz, J. J. Lasarte, and C. Gamazo. 2010. Poly(anhydride) nanoparticles act as active Th1 adjuvants through Toll-like receptor exploitation. Clin Vaccine Immunol 17: 1356-1362. 188. Tandrup Schmidt, S., C. Foged, K. Smith Korsholm, T. Rades, and D. Christensen. 2016. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators. Pharmaceutics 8: 7. 189. Moyle, P. M., and I. Toth. 2013. Modern subunit vaccines: development, components, and research opportunities. ChemMedChem 8: 360-376. 190. Hu, C. M., S. Aryal, and L. Zhang. 2010. Nanoparticle-assisted combination therapies for effective cancer treatment. Ther Deliv 1: 323-334. 191. Zhang, X. Q., C. E. Dahle, N. K. Baman, N. Rich, G. J. Weiner, and A. K. Salem. 2007. Potent antigen-specific immune responses stimulated by codelivery of CpG ODN and antigens in degradable microparticles. J Immunother 30: 469-478.

195

192. Tan, S., T. Sasada, A. Bershteyn, K. Yang, T. Ioji, and Z. Zhang. 2014. Combinational delivery of lipid-enveloped polymeric nanoparticles carrying different peptides for anti-tumor immunotherapy. Nanomedicine (Lond) 9: 635- 647. 193. Diwan, M., M. Tafaghodi, and J. Samuel. 2002. Enhancement of immune responses by co-delivery of a CpG oligodeoxynucleotide and tetanus toxoid in biodegradable nanospheres. Journal of controlled release : official journal of the Controlled Release Society 85: 247-262. 194. Madan-Lala, R., P. Pradhan, and K. Roy. 2017. Combinatorial Delivery of Dual and Triple TLR Agonists via Polymeric Pathogen-like Particles Synergistically Enhances Innate and Adaptive Immune Responses. Scientific Reports 7: 2530. 195. Hamdy, S., O. Molavi, Z. Ma, A. Haddadi, A. Alshamsan, Z. Gobti, S. Elhasi, J. Samuel, and A. Lavasanifar. 2008. Co-delivery of cancer-associated antigen and Toll-like receptor 4 ligand in PLGA nanoparticles induces potent CD8+ T cell- mediated anti-tumor immunity. Vaccine 26: 5046-5057. 196. Salem, A. K. 2015. Nanoparticles in vaccine delivery. AAPS J 17: 289-291. 197. Gupta, P. N., K. Khatri, A. K. Goyal, N. Mishra, and S. P. Vyas. 2007. M-cell targeted biodegradable PLGA nanoparticles for oral immunization against hepatitis B. J Drug Target 15: 701-713. 198. Raghuwanshi, D., V. Mishra, D. Das, K. Kaur, and M. R. Suresh. 2012. Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein. Mol Pharm 9: 946-956. 199. Yun, Y., Y. W. Cho, and K. Park. 2013. Nanoparticles for oral delivery: Targeted nanoparticles with peptidic ligands for oral protein delivery. Advanced drug delivery reviews 65: 822-832. 200. Costa, A., B. Sarmento, and V. Seabra. 2015. Targeted Drug Delivery Systems for Lung Macrophages. Curr Drug Targets 16: 1565-1581. 201. Kunda, N. K., S. Somavarapu, S. B. Gordon, G. A. Hutcheon, and I. Y. Saleem. 2013. Nanocarriers targeting dendritic cells for pulmonary vaccine delivery. Pharm Res 30: 325-341. 202. Koszinowski, U. H., M. J. Reddehase, and S. Jonjic. 1991. The role of CD4 and CD8 T cells in viral infections. Curr Opin Immunol 3: 471-475. 203. La Gruta, N. L., and S. J. Turner. 2014. T cell mediated immunity to influenza: mechanisms of viral control. Trends in immunology 35: 396-402. 204. Thomas, P. G., R. Keating, D. J. Hulse-Post, and P. C. Doherty. 2006. Cell- mediated protection in influenza infection. Emerg Infect Dis 12: 48-54. 205. Hiremath, J., K. I. Kang, M. Xia, M. Elaish, B. Binjawadagi, K. Ouyang, S. Dhakal, J. Arcos, J. B. Torrelles, X. Jiang, C. W. Lee, and G. J. Renukaradhya. 2016. Entrapment of H1N1 Influenza Virus Derived Conserved Peptides in PLGA Nanoparticles Enhances T Cell Response and Vaccine Efficacy in Pigs. PLoS One 11: e0151922. 206. Dwivedi, V., C. Manickam, B. Binjawadagi, D. Joyappa, and G. J. Renukaradhya. 2012. Biodegradable nanoparticle-entrapped vaccine induces cross-protective

196

immune response against a virulent heterologous respiratory viral infection in pigs. PLoS One 7: e51794. 207. Binjawadagi, B., V. Dwivedi, C. Manickam, K. Ouyang, J. B. Torrelles, and G. J. Renukaradhya. 2014. An innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination. Int J Nanomedicine 9: 1519-1535. 208. Herrmann, V. L., C. Hartmayer, O. Planz, and M. Groettrup. 2015. Cytotoxic T cell vaccination with PLGA microspheres interferes with influenza A virus replication in the lung and suppresses the infectious disease. Journal of controlled release : official journal of the Controlled Release Society 216: 121-131. 209. Ross, K. A., H. Loyd, W. Wu, L. Huntimer, S. Ahmed, A. Sambol, S. Broderick, Z. Flickinger, K. Rajan, T. Bronich, S. Mallapragada, M. J. Wannemuehler, S. Carpenter, and B. Narasimhan. 2015. Hemagglutinin-based polyanhydride nanovaccines against H5N1 influenza elicit protective virus neutralizing titers and cell-mediated immunity. Int J Nanomedicine 10: 229-243. 210. Sahdev, P., L. J. Ochyl, and J. J. Moon. 2014. Biomaterials for nanoparticle vaccine delivery systems. Pharm Res 31: 2563-2582. 211. Xu, J., W. Dai, Z. Wang, B. Chen, Z. Li, and X. Fan. 2011. Intranasal vaccination with chitosan-DNA nanoparticles expressing pneumococcal surface antigen a protects mice against nasopharyngeal colonization by Streptococcus pneumoniae. Clin Vaccine Immunol 18: 75-81. 212. Primard, C., J. Poecheim, S. Heuking, E. Sublet, F. Esmaeili, and G. Borchard. 2013. Multifunctional PLGA-based nanoparticles encapsulating simultaneously hydrophilic antigen and hydrophobic immunomodulator for mucosal immunization. Mol Pharm 10: 2996-3004. 213. Piskin, E. 2002. Biodegradable Polymers in Medicine. . In Degradable Polymers. G. Scott, ed. Springer, Dordrecht. 214. El-Sherbiny, I. M., N. M. El-Baz, and A. H. Mohamed. 2015. Biodegradable and Biocompatible Polymers‐Based D rug D elivery Systems for Cancer Therapy. I n Handbook of Polymers for Pharmaceutical Technologies. V. K. Thakur, and M. K. Thakur, eds. 215. Makadia, H. K., and S. J. Siegel. 2011. Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers (Basel) 3: 1377-1397. 216. !!! INVALID CITATION !!! {}. 217. Danhier, F., E. Ansorena, J. M. Silva, R. Coco, A. Le Breton, and V. Preat. 2012. PLGA-based nanoparticles: an overview of biomedical applications. Journal of controlled release : official journal of the Controlled Release Society 161: 505- 522. 218. Scholes, P. D., A. G. A. Coombes, L. Illum, S. S. Daviz, M. Vert, and M. C. Davies. 1993. The preparation of sub-200 nm poly(lactide-co-glycolide) microspheres for site-specific drug delivery. Journal of Controlled Release 25: 145-153.

197

219. Zambaux, M. F., F. Bonneaux, R. Gref, P. Maincent, E. Dellacherie, M. J. Alonso, P. Labrude, and C. Vigneron. 1998. Influence of experimental parameters on the characteristics of poly(lactic acid) nanoparticles prepared by a double emulsion method. Journal of Controlled Release 50: 31-40. 220. Clawson, C., C. T. Huang, D. Futalan, D. M. Seible, R. Saenz, M. Larsson, W. Ma, B. Minev, F. Zhang, M. Ozkan, C. Ozkan, S. Esener, and D. Messmer. 2010. Delivery of a peptide via poly(D,L-lactic-co-glycolic) acid nanoparticles enhances its dendritic cell-stimulatory capacity. Nanomedicine 6: 651-661. 221. Yoshida, M., J. Mata, and J. E. Babensee. 2007. Effect of poly(lactic-co-glycolic acid) contact on maturation of murine bone marrow-derived dendritic cells. J Biomed Mater Res A 80: 7-12. 222. Blair, D. A., D. L. Turner, T. O. Bose, Q. M. Pham, K. R. Bouchard, K. J. Williams, J. P. McAleer, L. S. Cauley, A. T. Vella, and L. Lefrancois. 2011. Duration of antigen availability influences the expansion and memory differentiation of T cells. J Immunol 187: 2310-2321. 223. Panyam, J., and V. Labhasetwar. 2003. Dynamics of endocytosis and exocytosis of poly(D,L-lactide-co-glycolide) nanoparticles in vascular smooth muscle cells. Pharm Res 20: 212-220. 224. Panyam, J., W. Z. Zhou, S. Prabha, S. K. Sahoo, and V. Labhasetwar. 2002. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. FASEB J 16: 1217-1226. 225. Gerelchuluun, T., Y.-H. Lee, Y.-R. Lee, S.-A. Im, S. Song, J. S. Park, K. Han, K. Kim, and C.-K. Lee. 2007. Dendritic cells process antigens encapsulated in a biodegradable polymer, poly(D,L-lactide-co-glycolide), via an alternate class I MHC processing pathway. Archives of Pharmacal Research 30: 1440-1446. 226. Hong, S., A. A. L., C. Virginia, G. Alessandra, H. E. R., C. Peter, E. R. L., S. W. Mark, and H. D. J. 2006. Enhanced and prolonged cross‐presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles. Immunology 117: 78-88. 227. Zhang, Z., S. Tongchusak, Y. Mizukami, Y. J. Kang, T. Ioji, M. Touma, B. Reinhold, D. B. Keskin, E. L. Reinherz, and T. Sasada. 2011. Induction of anti- tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery. Biomaterials 32: 3666-3678. 228. Hanlon, D. J., P. B. Aldo, L. Devine, A. B. Alvero, A. K. Engberg, R. Edelson, and G. Mor. 2011. Enhanced stimulation of anti-ovarian cancer CD8(+) T cells by dendritic cells loaded with nanoparticle encapsulated tumor antigen. Am J Reprod Immunol 65: 597-609. 229. Dwivedi, V., C. Manickam, B. Binjawadagi, and G. J. Renukaradhya. 2013. PLGA nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs. Vet Microbiol 166: 47-58. 230. Basu, A., and A. J. Domb. 2018. Recent Advances in Polyanhydride Based Biomaterials. Adv Mater: e1706815.

198

231. Schmeltzer, R. C., and K. E. Uhrich. 2006. Synthesis and Characterization of Salicylic Acid-Based Poly(Anhydride-Ester) Copolymers. J Bioact Compat Polym 21: 123-133. 232. Lopac, S. K., M. P. Torres, J. H. Wilson-Welder, M. J. Wannemuehler, and B. Narasimhan. 2009. Effect of polymer chemistry and fabrication method on protein release and stability from polyanhydride microspheres. J Biomed Mater Res B Appl Biomater 91: 938-947. 233. Torres, M. P., A. S. Determan, G. L. Anderson, S. K. Mallapragada, and B. Narasimhan. 2007. Amphiphilic polyanhydrides for protein stabilization and release. Biomaterials 28: 108-116. 234. Huntimer, L., A. E. Ramer-Tait, L. K. Petersen, K. A. Ross, K. A. Walz, C. Wang, J. Hostetter, B. Narasimhan, and M. J. Wannemuehler. 2013. Evaluation of biocompatibility and administration site reactogenicity of polyanhydride-particle- based platform for vaccine delivery. Adv Healthc Mater 2: 369-378. 235. Vela-Ramirez, J. E., J. T. Goodman, P. M. Boggiatto, R. Roychoudhury, N. L. Pohl, J. M. Hostetter, M. J. Wannemuehler, and B. Narasimhan. 2015. Safety and biocompatibility of carbohydrate-functionalized polyanhydride nanoparticles. AAPS J 17: 256-267. 236. Petersen, L. K., A. E. Ramer-Tait, S. R. Broderick, C. S. Kong, B. D. Ulery, K. Rajan, M. J. Wannemuehler, and B. Narasimhan. 2011. Activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants. Biomaterials 32: 6815-6822. 237. Carrillo-Conde, B., E. H. Song, A. Chavez-Santoscoy, Y. Phanse, A. E. Ramer- Tait, N. L. Pohl, M. J. Wannemuehler, B. H. Bellaire, and B. Narasimhan. 2011. Mannose-functionalized "pathogen-like" polyanhydride nanoparticles target C- type lectin receptors on dendritic cells. Mol Pharm 8: 1877-1886. 238. Ross, K. A., H. Loyd, W. Wu, L. Huntimer, M. J. Wannemuehler, S. Carpenter, and B. Narasimhan. 2014. Structural and antigenic stability of H5N1 hemagglutinin trimer upon release from polyanhydride nanoparticles. J Biomed Mater Res A 102: 4161-4168. 239. Vela Ramirez, J. E., R. Roychoudhury, H. H. Habte, M. W. Cho, N. L. Pohl, and B. Narasimhan. 2014. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells. J Biomater Sci Polym Ed 25: 1387-1406. 240. Haughney, S. L., L. K. Petersen, A. D. Schoofs, A. E. Ramer-Tait, J. D. King, D. E. Briles, M. J. Wannemuehler, and B. Narasimhan. 2013. Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles. Acta Biomater 9: 8262-8271. 241. Huntimer, L. M., K. A. Ross, R. J. Darling, N. E. Winterwood, P. Boggiatto, B. Narasimhan, A. E. Ramer-Tait, and M. J. Wannemuehler. 2014. Polyanhydride nanovaccine platform enhances antigen-specific cytotoxic T cell responses. TECHNOLOGY 02: 171-175. 242. Ross, K., J. Adams, H. Loyd, S. Ahmed, A. Sambol, S. Broderick, K. Rajan, M. Kohut, T. Bronich, M. J. Wannemuehler, S. Carpenter, S. Mallapragada, and B. 199

Narasimhan. 2016. Combination Nanovaccine Demonstrates Synergistic Enhancement in Efficacy against Influenza. ACS Biomaterials Science & Engineering 2: 368-374. 243. Ulery, B. D., D. Kumar, A. E. Ramer-Tait, D. W. Metzger, M. J. Wannemuehler, and B. Narasimhan. 2011. Design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases. PLoS One 6: e17642. 244. Roberts, G. A. F. 1992. Structure of Chitin and Chitosan. In Chitin Chemistry. Macmillan Education UK, London. 1-53. 245. Hejazi, R., and M. Amiji. 2003. Chitosan-based gastrointestinal delivery systems. Journal of Controlled Release 89: 151-165. 246. Xing, L., Y.-T. Fan, T.-J. Zhou, J.-H. Gong, L.-H. Cui, K.-H. Cho, Y.-J. Choi, H.- L. Jiang, and C.-S. Cho. 2018. Chemical Modification of Chitosan for Efficient Vaccine Delivery. Molecules 23: 229. 247. Mun, S., E. A. Decker, and D. J. McClements. 2006. Effect of molecular weight and degree of deacetylation of chitosan on the formation of oil-in-water emulsions stabilized by surfactant–chitosan membranes. Journal of Colloid and Interface Science 296: 581-590. 248. Illum, L. 1998. Chitosan and its use as a pharmaceutical excipient. Pharm Res 15: 1326-1331. 249. Lee, D. W., C. Lim, J. N. Israelachvili, and D. S. Hwang. 2013. Strong adhesion and cohesion of chitosan in aqueous solutions. Langmuir : the ACS journal of surfaces and colloids 29: 14222-14229. 250. Lim, C., D. W. Lee, J. N. Israelachvili, Y. Jho, and D. S. Hwang. 2015. Contact time- and pH-dependent adhesion and cohesion of low molecular weight chitosan coated surfaces. Carbohydrate Polymers 117: 887-894. 251. Artursson, P., T. Lindmark, S. S. Davis, and L. Illum. 1994. Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2). Pharm Res 11: 1358-1361. 252. Dodane, V., M. Amin Khan, and J. R. Merwin. 1999. Effect of chitosan on epithelial permeability and structure. International Journal of Pharmaceutics 182: 21-32. 253. Illum, L., N. F. Farraj, and S. S. Davis. 1994. Chitosan as a novel nasal delivery system for peptide drugs. Pharm Res 11: 1186-1189. 254. Fernandez-Urrusuno, R., P. Calvo, C. Remunan-Lopez, J. L. Vila-Jato, and M. J. Alonso. 1999. Enhancement of nasal absorption of insulin using chitosan nanoparticles. Pharm Res 16: 1576-1581. 255. Illum, L., I. Jabbal-Gill, M. Hinchcliffe, A. N. Fisher, and S. S. Davis. 2001. Chitosan as a novel nasal delivery system for vaccines. Advanced Drug Delivery Reviews 51: 81-96. 256. Wang, J. J., Z. W. Zeng, R. Z. Xiao, T. Xie, G. L. Zhou, X. R. Zhan, and S. L. Wang. 2011. Recent advances of chitosan nanoparticles as drug carriers. International Journal of Nanomedicine 6: 765-774.

200

257. Koppolu, B. p., and D. A. Zaharoff. 2013. The effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells. Biomaterials 34: 2359-2369. 258. Hunsawong, T., P. Sunintaboon, S. Warit, B. Thaisomboonsuk, R. G. Jarman, I. K. Yoon, S. Ubol, and S. Fernandez. 2015. Immunogenic Properties of a BCG Adjuvanted Chitosan Nanoparticle-Based Dengue Vaccine in Human Dendritic Cells. PLoS Negl Trop Dis 9: e0003958. 259. Villiers, C., M. Chevallet, H. Diemer, R. Couderc, H. Freitas, A. Van Dorsselaer, P. N. Marche, and T. Rabilloud. 2009. From Secretome Analysis to Immunology: Chitosan Induces Major Alterations in the Activation of Dendritic Cells via a TLR4-dependent Mechanism. Molecular & Cellular Proteomics : MCP 8: 1252- 1264. 260. Andres, M., O. Ewa, S. F. A., C. Michelle, O. Yuki, S. Manmohan, O. H. D. T., T. Lidia, C. O. I., M. E. A., and L. E. C. 2012. The vaccine adjuvant alum inhibits IL‐12 by pr omoting PI3 kinase signaling while chitosan does not inhibit IL‐12 and enhances Th1 and Th17 responses. European Journal of Immunology 42: 2709-2719. 261. Carroll, E. C., L. Jin, A. Mori, N. Muñoz-Wolf, E. Oleszycka, H. B. T. Moran, S. Mansouri, C. P. McEntee, E. Lambe, E. M. Agger, P. Andersen, C. Cunningham, P. Hertzog, K. A. Fitzgerald, A. G. Bowie, and E. C. Lavelle. 2016. The Vaccine Adjuvant Chitosan Promotes Cellular Immunity via DNA Sensor cGAS-STING- Dependent Induction of Type I Interferons. Immunity 44: 597-608. 262. Rampino, A., M. Borgogna, P. Blasi, B. Bellich, and A. Cesàro. 2013. Chitosan nanoparticles: Preparation, size evolution and stability. International Journal of Pharmaceutics 455: 219-228. 263. Zhao, K., G. Chen, X.-m. Shi, T.-t. Gao, W. Li, Y. Zhao, F.-q. Zhang, J. Wu, X. Cui, and Y.-F. Wang. 2012. Preparation and Efficacy of a Live Newcastle Disease Virus Vaccine Encapsulated in Chitosan Nanoparticles. PLoS ONE 7: e53314. 264. Vila, A., A. Sánchez, K. Janes, I. Behrens, T. Kissel, J. L. V. Jato, and M. a. J. Alonso. 2004. Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice. European Journal of Pharmaceutics and Biopharmaceutics 57: 123-131. 265. Figueiredo, L., A. Cadete, L. M. D. Gonçalves, M. L. Corvo, and A. J. Almeida. 2012. Intranasal immunisation of mice against Streptococcus equi using positively charged nanoparticulate carrier systems. Vaccine 30: 6551-6558. 266. Sawaengsak, C., Y. Mori, K. Yamanishi, A. Mitrevej, and N. Sinchaipanid. 2014. Chitosan Nanoparticle Encapsulated Hemagglutinin-Split Influenza Virus Mucosal Vaccine. AAPS PharmSciTech 15: 317-325. 267. Borges, O., A. Cordeiro-da-Silva, J. Tavares, N. Santarém, A. de Sousa, G. Borchard, and H. E. Junginger. 2008. Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles. European Journal of Pharmaceutics and Biopharmaceutics 69: 405-416.

201

268. Lebre, F., G. Borchard, H. Faneca, M. C. Pedroso de Lima, and O. Borges. 2016. Intranasal Administration of Novel Chitosan Nanoparticle/DNA Complexes Induces Antibody Response to Hepatitis B Surface Antigen in Mice. Molecular Pharmaceutics 13: 472-482. 269. Jabbal-Gill, I., A. N. Fisher, R. Rappuoli, S. S. Davis, and L. Illum. 1998. Stimulation of mucosal and systemic antibody responses against Bordetella pertussis filamentous haemagglutinin and recombinant pertussis toxin after nasal administration with chitosan in mice. Vaccine 16: 2039-2046. 270. McNeela, E. A., D. O'Connor, I. Jabbal-Gill, L. Illum, S. S. Davis, M. Pizza, S. Peppoloni, R. Rappuoli, and K. H. G. Mills. 2000. A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM197) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 responses following nasal delivery. Vaccine 19: 1188-1198. 271. Huang, L., and Y. Yao. 2011. Particulate structure of phytoglycogen nanoparticles probed using amyloglucosidase. Carbohydrate Polymers 83: 1665-1671. 272. Lu, F., A. Mencia, L. Bi, A. Taylor, Y. Yao, and H. HogenEsch. 2015. Dendrimer-like alpha-d-glucan nanoparticles activate dendritic cells and are effective vaccine adjuvants. Journal of Controlled Release 204: 51-59. 273. Scheffler, S. L., L. Huang, L. Bi, and Y. Yao. 2010. In Vitro Digestibility and Emulsification Properties of Phytoglycogen Octenyl Succinate. Journal of Agricultural and Food Chemistry 58: 5140-5146. 274. Lu, F., Y.-Y. C. Mosley, R. J. Rodriguez Rosales, B. E. Carmichael, S. Elesela, Y. Yao, and H. HogenEsch. 2017. Alpha-D-glucan nanoparticulate adjuvant induces a transient inflammatory response at the injection site and targets antigen to migratory dendritic cells. npj Vaccines 2: 4. 275. McGhee, J. R., and K. Fujihashi. 2012. Inside the mucosal immune system. PLoS Biol 10: e1001397. 276. Neutra, M. R., and P. A. Kozlowski. 2006. Mucosal vaccines: the promise and the challenge. Nature Reviews Immunology 6: 148. 277. Levine, M. M. 2000. Immunization against bacterial diseases of the intestine. J Pediatr Gastroenterol Nutr 31: 336-355. 278. Lamm, M. E. 1997. INTERACTION OF ANTIGENS AND ANTIBODIES AT MUCOSAL SURFACES. Annual Review of Microbiology 51: 311-340. 279. Brandtzaeg, P., H. Kiyono, R. Pabst, and M. W. Russell. 2007. Terminology: nomenclature of mucosa-associated lymphoid tissue. Mucosal Immunology 1: 31. 280. Brandtzaeg, P., and R. Pabst. 2004. Let's go mucosal: communication on slippery ground. Trends in immunology 25: 570-577. 281. C., C. S., G. C. C.G.M., and H. Colin. 2008. M‐cells: origin, morphology and role in mucosal immunity and microbial pathogenesis. FEMS Immunology & Medical Microbiology 52: 2-12. 282. Ohno, H. 2016. Intestinal M cells. Journal of Biochemistry 159: 151-160. 283. Schulz, O., and O. Pabst. 2013. Antigen sampling in the small intestine. Trends in immunology 34: 155-161.

202

284. Cerutti, A., K. Chen, and A. Chorny. 2011. Immunoglobulin Responses at the Mucosal Interface. Annual review of immunology 29: 273-293. 285. Mats, B., B. Preben, and L. N. Y. 2012. Induction of gut IgA production through T cell‐dependent and T cell‐ independent pathw ays. Annals of the New York Academy of Sciences 1247: 97-116. 286. Pabst, O. 2012. New concepts in the generation and functions of IgA. Nat Rev Immunol 12: 821-832. 287. Mestecky, J., I. Moro, M. A. Kerr, and J. M. Woof. 2005. Chapter 9 - Mucosal Immunoglobulins. In Mucosal Immunology (Third Edition). Academic Press, Burlington. 153-181. 288. Strugnell, R. A., and O. L. C. Wijburg. 2010. The role of secretory antibodies in infection immunity. Nature Reviews Microbiology 8: 656. 289. Parr, E. L., and M. B. Parr. 1997. Immunoglobulin G is the main protective antibody in mouse vaginal secretions after vaginal immunization with attenuated herpes simplex virus type 2. Journal of virology 71: 8109-8115. 290. Holmgren, J., and C. Czerkinsky. 2005. Mucosal immunity and vaccines. Nature medicine 11: S45. 291. Brandtzaeg, P. 2010. Function of Mucosa-Associated Lymphoid Tissue in Antibody Formation. Immunological Investigations 39: 303-355. 292. Brandtzaeg, P. 2007. Induction of secretory immunity and memory at mucosal surfaces. Vaccine 25: 5467-5484. 293. Sheridan, B. S., and L. Lefrançois. 2011. Regional and mucosal memory T cells. Nature immunology 12: 485-491. 294. Kim, S.-H., and Y.-S. Jang. 2017. The development of mucosal vaccines for both mucosal and systemic immune induction and the roles played by adjuvants. Clinical and Experimental Vaccine Research 6: 15-21. 295. Chadwick, S., C. Kriegel, and M. Amiji. 2010. Nanotechnology solutions for mucosal immunization. Adv Drug Deliv Rev 62: 394-407. 296. Watts, P. J., and A. Smith. 2011. Re-formulating drugs and vaccines for intranasal delivery: maximum benefits for minimum risks? Drug Discovery Today 16: 4-7. 297. Zaman, M., M. F. Good, and I. Toth. 2013. Nanovaccines and their mode of action. Methods 60: 226-231. 298. Davis, S. S. 2001. Nasal vaccines. Advanced Drug Delivery Reviews 51: 21-42. 299. Partidos, C. D. 2000. Intranasal vaccines: forthcoming challenges. Pharmaceutical Science & Technology Today 3: 273-281. 300. Kiyono, H., and S. Fukuyama. 2004. NALT- versus Peyer's-patch-mediated mucosal immunity. Nat Rev Immunol 4: 699-710. 301. Hameleers, D. M., M. van der Ende, J. Biewenga, and T. Sminia. 1989. An immunohistochemical study on the postnatal development of rat nasal-associated lymphoid tissue (NALT). Cell Tissue Res 256: 431-438. 302. Porgador, A., H. F. Staats, Y. Itoh, and B. L. Kelsall. 1998. Intranasal immunization with cytotoxic T-lymphocyte epitope peptide and mucosal adjuvant cholera toxin: selective augmentation of peptide-presenting dendritic cells in nasal mucosa-associated lymphoid tissue. Infect Immun 66: 5876-5881. 203

303. Fujimura, Y. 2000. Evidence of M cells as portals of entry for antigens in the nasopharyngeal lymphoid tissue of humans. Virchows Arch 436: 560-566. 304. Ogasawara, N., T. Kojima, M. Go, K. Takano, R. Kamekura, T. Ohkuni, J. Koizumi, T. Masaki, J. Fuchimoto, K. Obata, M. Kurose, T. Shintani, N. Sawada, and T. Himi. 2011. Epithelial barrier and antigen uptake in lymphoepithelium of human adenoids. Acta Otolaryngol 131: 116-123. 305. Johansen, F. E., and C. S. Kaetzel. 2011. Regulation of the polymeric immunoglobulin receptor and IgA transport: new advances in environmental factors that stimulate pIgR expression and its role in mucosal immunity. Mucosal Immunol 4: 598-602. 306. Wilson, H. L., and M. R. Obradovic. 2015. Evidence for a common mucosal immune system in the pig. Mol Immunol 66: 22-34. 307. Kuper, C. F., H. Ernst, L. C. van Oostrum, S. Rittinghausen, A. H. Penninks, N. C. Ganderup, and A. P. Wolterbeek. 2012. Nasal passages of Gottingen minipigs from the neonatal period to young adult. Toxicol Pathol 40: 656-666. 308. Yang, J., L. Dai, Q. Yu, and Q. Yang. 2017. Histological and anatomical structure of the nasal cavity of Bama minipigs. PLoS One 12: e0173902. 309. Debertin, A. S., T. Tschernig, H. Tonjes, W. J. Kleemann, H. D. Troger, and R. Pabst. 2003. Nasal-associated lymphoid tissue (NALT): frequency and localization in young children. Clin Exp Immunol 134: 503-507. 310. Zaman, M., S. Chandrudu, and I. Toth. 2013. Strategies for intranasal delivery of vaccines. Drug Deliv Transl Res 3: 100-109. 311. Renukaradhya, G. J., B. Narasimhan, and S. K. Mallapragada. 2015. Respiratory nanoparticle-based vaccines and challenges associated with animal models and translation. Journal of controlled release : official journal of the Controlled Release Society 219: 622-631. 312. Jabbal-Gill, I. 2010. Nasal vaccine innovation. J Drug Target 18: 771-786. 313. Marasini, N., M. Skwarczynski, and I. Toth. 2017. Intranasal delivery of nanoparticle-based vaccines. Ther Deliv 8: 151-167. 314. Illum, L. 2007. Nanoparticulate Systems for Nasal Delivery of Drugs: A Real Improvement over Simple Systems? Journal of Pharmaceutical Sciences 96: 473- 483. 315. McGill, J. L., S. M. Kelly, P. Kumar, S. Speckhart, S. L. Haughney, J. Henningson, B. Narasimhan, and R. E. Sacco. 2018. Efficacy of mucosal polyanhydride nanovaccine against respiratory syncytial virus infection in the neonatal calf. Scientific Reports 8: 3021. 316. Braam, J., I. Ulmanen, and R. M. Krug. 1983. Molecular model of a eucaryotic transcription complex: functions and movements of influenza P proteins during capped RNA-primed transcription. Cell 34: 609-618. 317. Chen, W., P. A. Calvo, D. Malide, J. Gibbs, U. Schubert, I. Bacik, S. Basta, R. O'Neill, J. Schickli, P. Palese, P. Henklein, J. R. Bennink, and J. W. Yewdell. 2001. A novel influenza A virus mitochondrial protein that induces cell death. Nature medicine 7: 1306.

204

318. Kawakami, K., and A. Ishihama. 1983. RNA polymerase of influenza virus. III. Isolation of RNA polymerase-RNA complexes from influenza virus PR8. J Biochem 93: 989-996. 319. O'Neill, R. E., R. Jaskunas, G. Blobel, P. Palese, and J. Moroianu. 1995. Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import. J Biol Chem 270: 22701-22704. 320. Palese, P., K. Tobita, M. Ueda, and R. W. Compans. 1974. Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61: 397-410. 321. Martin, K., and A. Helenius. 1991. Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell 67: 117-130. 322. Sugrue, R. J., and A. J. Hay. 1991. Structural characteristics of the M2 protein of influenza a viruses: Evidence that it forms a tetrameric channe. Virology 180: 617-624. 323. Fortes, P., A. Beloso, and J. Ortin. 1994. Influenza virus NS1 protein inhibits pre- mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J 13: 704-712. 324. Shimizu, K., A. Iguchi, R. Gomyou, and Y. Ono. 1999. Influenza virus inhibits cleavage of the HSP70 pre-mRNAs at the polyadenylation site. Virology 254: 213-219. 325. Dauber, B., G. Heins, and T. Wolff. 2004. The influenza B virus nonstructural NS1 protein is essential for efficient viral growth and antagonizes beta interferon induction. Journal of virology 78: 1865-1872. 326. Lamb, R. A., P. W. Choppin, R. M. Chanock, and C. J. Lai. 1980. Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome. Proc Natl Acad Sci U S A 77: 1857-1861. 327. Vincent, A., L. Awada, I. Brown, H. Chen, F. Claes, G. Dauphin, R. Donis, M. Culhane, K. Hamilton, N. Lewis, E. Mumford, T. Nguyen, S. Parchariyanon, J. Pasick, G. Pavade, A. Pereda, M. Peiris, T. Saito, S. Swenson, K. Van Reeth, R. Webby, F. Wong, and J. Ciacci-Zanella. 2014. Review of Influenza A Virus in Swine Worldwide: A Call for Increased Surveillance and Research. Zoonoses and Public Health 61: 4-17. 328. Khaled, Y. S., B. J. Ammori, and E. Elkord. 2013. Myeloid-derived suppressor cells in cancer: recent progress and prospects. Immunol Cell Biol 91: 493-502. 329. Lesokhin, A. M., T. M. Hohl, S. Kitano, C. Cortez, D. Hirschhorn-Cymerman, F. Avogadri, G. A. Rizzuto, J. J. Lazarus, E. G. Pamer, A. N. Houghton, T. Merghoub, and J. D. Wolchok. 2012. Monocytic CCR2(+) myeloid-derived suppressor cells promote immune escape by limiting activated CD8 T-cell infiltration into the tumor microenvironment. Cancer Res 72: 876-886. 330. Iclozan, C., S. Antonia, A. Chiappori, D. T. Chen, and D. Gabrilovich. 2013. Therapeutic regulation of myeloid-derived suppressor cells and immune response to cancer vaccine in patients with extensive stage small cell lung cancer. Cancer Immunol Immunother 62: 909-918. 205

331. Rodrigues, J. C., G. C. Gonzalez, L. Zhang, G. Ibrahim, J. J. Kelly, M. P. Gustafson, Y. Lin, A. B. Dietz, P. A. Forsyth, V. W. Yong, and I. F. Parney. 2010. Normal human monocytes exposed to glioma cells acquire myeloid-derived suppressor cell-like properties. Neuro Oncol 12: 351-365. 332. Ellebedy, A. H., and R. J. Webby. 2009. Influenza vaccines. Vaccine 27 Suppl 4: D65-68. 333. Savic, M., J. L. Dembinski, Y. Kim, G. Tunheim, R. J. Cox, F. Oftung, B. Peters, and S. Mjaaland. 2016. Epitope specific T-cell responses against influenza A in a healthy population. Immunology 147: 165-177. 334. Mahapatro, A., and D. K. Singh. 2011. Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines. Journal of Nanobiotechnology 9: 1-11. 335. Getts, D. R., L. D. Shea, S. D. Miller, and N. J. King. 2015. Harnessing nanoparticles for immune modulation. Trends in immunology 36: 419-427. 336. Woodrow, K. A., K. M. Bennett, and D. D. Lo. 2012. Mucosal vaccine design and delivery. Annu Rev Biomed Eng 14: 17-46. 337. Foged, C., B. Brodin, S. Frokjaer, and A. Sundblad. 2005. Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model. Int J Pharm 298: 315-322. 338. Chithrani, B. D., A. A. Ghazani, and W. C. Chan. 2006. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett 6: 662-668. 339. Ali, A., M. Khatri, L. Wang, Y. M. Saif, and C. W. Lee. 2012. Identification of swine H1N2/pandemic H1N1 reassortant influenza virus in pigs, United States. Vet Microbiol 158: 60-68. 340. Yassine, H. M., M. Khatri, Y. J. Zhang, C. W. Lee, B. A. Byrum, J. O'Quin, K. A. Smith, and Y. M. Saif. 2009. Characterization of triple reassortant H1N1 influenza A viruses from swine in Ohio. Vet Microbiol 139: 132-139. 341. van Leengoed, L. A., and E. M. Kamp. 1989. A method for bronchoalveolar lavage in live pigs. Vet Q 11: 65-72. 342. Ganter, M., and A. Hensel. 1997. Cellular variables in bronchoalveolar lavage fluids (BALF) in selected healthy pigs. Res Vet Sci 63: 215-217. 343. Collins, A. M., J. Rylance, D. G. Wootton, A. D. Wright, A. K. Wright, D. G. Fullerton, and S. B. Gordon. 2014. Bronchoalveolar lavage (BAL) for research; obtaining adequate sample yield. J Vis Exp. 344. Piriou-Guzylack, L., and H. Salmon. 2008. Membrane markers of the immune cells in swine: an update. Vet Res 39: 54. 345. Renukaradhya, G. J., K. Alekseev, K. Jung, Y. Fang, and L. J. Saif. 2010. Porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs. Viral Immunol 23: 457-466. 346. Richt, J. A., P. Lekcharoensuk, K. M. Lager, A. L. Vincent, C. M. Loiacono, B. H. Janke, W. H. Wu, K. J. Yoon, R. J. Webby, A. Solorzano, and A. Garcia-

206

Sastre. 2006. Vaccination of pigs against swine influenza viruses by using an NS1-truncated modified live-virus vaccine. Journal of virology 80: 11009-11018. 347. Reed, L. J., and L. Muench. 1938. A Simple Method of Estimating Fifty Per Cent Endpoints. The American Journal of Hygiene 27(3): 493-497. 348. Rawat, A., Q. H. Majumder, and F. Ahsan. 2008. Inhalable large porous microspheres of low molecular weight heparin: in vitro and in vivo evaluation. Journal of controlled release : official journal of the Controlled Release Society 128: 224-232. 349. Zuckermann, F. A. 1999. Extrathymic CD4/CD8 double positive T cells. Vet Immunol Immunopathol 72: 55-66. 350. Thomas, C., A. Rawat, L. Hope-Weeks, and F. Ahsan. 2011. Aerosolized PLA and PLGA Nanoparticles Enhance Humoral, Mucosal and Cytokine Responses to Hepatitis B Vaccine. Mol Pharm 8: 405-415. 351. Greenway, T. E., J. H. Eldridge, G. Ludwig, J. K. Staas, J. F. Smith, R. M. Gilley, and S. M. Michalek. 1998. Induction of protective immune responses against Venezuelan equine encephalitis (VEE) virus aerosol challenge with microencapsulated VEE virus vaccine. Vaccine 16: 1314-1323. 352. Shephard, M. J., D. Todd, B. M. Adair, A. L. Po, D. P. Mackie, and E. M. Scott. 2003. Immunogenicity of bovine parainfluenza type 3 virus proteins encapsulated in nanoparticle vaccines, following intranasal administration to mice. Res Vet Sci 74: 187-190. 353. Yassine, H. M., J. C. Boyington, P. M. McTamney, C. J. Wei, M. Kanekiyo, W. P. Kong, J. R. Gallagher, L. Wang, Y. Zhang, M. G. Joyce, D. Lingwood, S. M. Moin, H. Andersen, Y. Okuno, S. S. Rao, A. K. Harris, P. D. Kwong, J. R. Mascola, G. J. Nabel, and B. S. Graham. 2015. Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection. Nature medicine 21: 1065-1070. 354. Liu, Q., X. Zheng, C. Zhang, X. Shao, X. Zhang, Q. Zhang, and X. Jiang. 2015. Conjugating influenza a (H1N1) antigen to n-trimethylaminoethylmethacrylate chitosan nanoparticles improves the immunogenicity of the antigen after nasal administration. Journal of medical virology 87: 1807-1815. 355. Singh, M., M. Briones, and D. T. O'Hagan. 2001. A novel bioadhesive intranasal delivery system for inactivated influenza vaccines. Journal of controlled release : official journal of the Controlled Release Society 70: 267-276. 356. Mansoor, F., B. Earley, J. P. Cassidy, B. Markey, S. Doherty, and M. D. Welsh. 2015. Comparing the immune response to a novel intranasal nanoparticle PLGA vaccine and a commercial BPI3V vaccine in dairy calves. BMC Veterinary Research 11: 1-11. 357. Beaudette, T. T., E. M. Bachelder, J. A. Cohen, A. C. Obermeyer, K. E. Broaders, J. M. Frechet, E. S. Kang, I. Mende, W. W. Tseng, M. G. Davidson, and E. G. Engleman. 2009. In vivo studies on the effect of co-encapsulation of CpG DNA and antigen in acid-degradable microparticle vaccines. Mol Pharm 6: 1160-1169. 358. Jong, S., G. Chikh, L. Sekirov, S. Raney, S. Semple, S. Klimuk, N. Yuan, M. Hope, P. Cullis, and Y. Tam. 2007. Encapsulation in liposomal nanoparticles enhances the immunostimulatory, adjuvant and anti-tumor activity of 207

subcutaneously administered CpG ODN. Cancer Immunology, Immunotherapy 56: 1251-1264. 359. Manolova, V., A. Flace, M. Bauer, K. Schwarz, P. Saudan, and M. F. Bachmann. 2008. Nanoparticles target distinct dendritic cell populations according to their size. European Journal of Immunology 38: 1404-1413. 360. Blank, F., P. A. Stumbles, E. Seydoux, P. G. Holt, A. Fink, B. Rothen- Rutishauser, D. H. Strickland, and C. von Garnier. 2013. Size-dependent uptake of particles by pulmonary antigen-presenting cell populations and trafficking to regional lymph nodes. American journal of respiratory cell and molecular biology 49: 67-77. 361. Jiang, W., R. K. Gupta, M. C. Deshpande, and S. P. Schwendeman. 2005. Biodegradable poly(lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens. Adv Drug Deliv Rev 57: 391-410. 362. Babensee, J. E., and A. Paranjpe. 2005. Differential levels of dendritic cell maturation on different biomaterials used in combination products. Journal of Biomedical Materials Research Part A 74A: 503-510. 363. Yoshida, M., and J. E. Babensee. 2006. Differential effects of agarose and poly(lactic-co-glycolic acid) on dendritic cell maturation. J Biomed Mater Res A 79: 393-408. 364. Elamanchili, P., M. Diwan, M. Cao, and J. Samuel. 2004. Characterization of poly(d,l-lactic-co-glycolic acid) based nanoparticulate system for enhanced delivery of antigens to dendritic cells. Vaccine 22: 2406-2412. 365. Arens, R., and S. P. Schoenberger. 2010. Plasticity in programming of effector and memory CD8 T-cell formation. Immunol Rev 235: 190-205. 366. Shen, H., A. L. Ackerman, V. Cody, A. Giodini, E. R. Hinson, P. Cresswell, R. L. Edelson, W. M. Saltzman, and D. J. Hanlon. 2006. Enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles. Immunology 117: 78-88. 367. Yang, Y.-W., and P. Y.-J. Hsu. 2008. The effect of poly(d,l-lactide-co-glycolide) microparticles with polyelectrolyte self-assembled multilayer surfaces on the cross-presentation of exogenous antigens. Biomaterials 29: 2516-2526. 368. Thome, M., W. Hirt, E. Pfaff, M. J. Reddehase, and A. Saalmuller. 1994. Porcine T-cell receptors: molecular and biochemical characterization. Vet Immunol Immunopathol 43: 13-18. 369. Gerner, W., T. Kaser, and A. Saalmuller. 2009. Porcine T lymphocytes and NK cells - An update. Dev Comp Immunol 33: 310-320. 370. Talker, S. C., T. Kaser, K. Reutner, C. Sedlak, K. H. Mair, H. Koinig, R. Graage, M. Viehmann, E. Klingler, A. Ladinig, M. Ritzmann, A. Saalmuller, and W. Gerner. 2013. Phenotypic maturation of porcine NK- and T-cell subsets. Dev Comp Immunol 40: 51-68. 371. Saalmuller, A., T. Werner, and V. Fachinger. 2002. T-helper cells from naive to committed. Vet Immunol Immunopathol 87: 137-145. 372. Hanlon, D. J., P. B. Aldo, L. Devine, A. B. Alvero, A. K. Engberg, R. Edelson, and G. Mor. 2011. Enhanced Stimulation of Anti-Ovarian Cancer CD8+ T Cells 208

by Dendritic Cells Loaded with Nanoparticle Encapsulated Tumor Antigen. American Journal of Reproductive Immunology 65: 597-609. 373. Welsh, R. M., M.-Y. Lin, B. L. Lohman, S. M. Varga, C. C. Zarozinski, and L. K. Selin. 1997. αβ and γδ T-cell networks and their roles in natural resistance to viral infections. Immunological Reviews 159: 79-93. 374. Olin, M. R., L. Batista, Z. Xiao, S. A. Dee, M. P. Murtaugh, C. C. Pijoan, and T. W. Molitor. 2005. Gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus. Viral Immunol 18: 490-499. 375. Almeida, A. J., and H. O. Alpar. 1996. Nasal delivery of vaccines. J Drug Target 3: 455-467. 376. Moss, P. 2003. Cellular immune responses to influenza. Developments in biologicals 115: 31-37. 377. Bot, A., S. Bot, and C. A. Bona. 1998. Protective Role of Gamma Interferon during the Recall Response to Influenza Virus. Journal of virology 72: 6637- 6645. 378. Dykhuis, H. C., T. Painter, T. Fangman, and H. D. 2012. Assessing production parameters and economic impact of swine influenza, PRRS and Mycoplasma hyopneimoniae on finishing pigs in a large production system In Proceedings of AASV Annual Meeting. 75-76. 379. Vincent, A., L. Awada, I. Brown, H. Chen, F. Claes, G. Dauphin, R. Donis, M. Culhane, K. Hamilton, N. Lewis, E. Mumford, T. Nguyen, S. Parchariyanon, J. Pasick, G. Pavade, A. Pereda, M. Peiris, T. Saito, S. Swenson, K. Van Reeth, R. Webby, F. Wong, and J. Ciacci-Zanella. 2014. Review of influenza A virus in swine worldwide: a call for increased surveillance and research. Zoonoses Public Health 61: 4-17. 380. Vincent, A. L., J. R. Ciacci-Zanella, A. Lorusso, P. C. Gauger, E. L. Zanella, M. E. Kehrli, Jr., B. H. Janke, and K. M. Lager. 2010. Efficacy of inactivated swine influenza virus vaccines against the 2009 A/H1N1 influenza virus in pigs. Vaccine 28: 2782-2787. 381. Andrew, F. A., K. P. Latrisha, H. W. Jennifer, P. T. Maria, B. T. Jon, W. G. Stuart, K. M. Surya, J. W. Michael, and N. Balaji. 2009. High Throughput Cell- Based Screening of Biodegradable Polyanhydride Libraries. Combinatorial Chemistry & High Throughput Screening 12: 634-645. 382. Mallapragada, S. K., and B. Narasimhan. 2008. Immunomodulatory biomaterials. Int J Pharm 364: 265-271. 383. Determan, A. S., B. G. Trewyn, V. S. Lin, M. Nilsen-Hamilton, and B. Narasimhan. 2004. Encapsulation, stabilization, and release of BSA-FITC from polyanhydride microspheres. J Control Release 100: 97-109. 384. Carrillo-Conde, B., E. Schiltz, J. Yu, F. Chris Minion, G. J. Phillips, M. J. Wannemuehler, and B. Narasimhan. 2010. Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia pestis antigens. Acta Biomater 6: 3110-3119. 385. Phanse, Y., B. R. Carrillo-Conde, A. E. Ramer-Tait, R. Roychoudhury, N. L. Pohl, B. Narasimhan, M. J. Wannemuehler, and B. H. Bellaire. 2013. 209

Functionalization of polyanhydride microparticles with di-mannose influences uptake by and intracellular fate within dendritic cells. Acta Biomater 9: 8902- 8909. 386. Ross, K. A., J. R. Adams, H. Loyd, S. Ahmed, A. Sambol, S. R. Broderick, K. Rajan, M. Kohut, T. Bronich, M. J. Wannemuehler, S. Carpenter, S. Mallapragada, and B. Narasimhan. 2016. Combination nanovaccine demonstrates synergistic enhancement in efficacy against influenza. ACS Biomater. Sci. Eng. 2: 368-374. 387. Binnebose, A. M., S. L. Haughney, R. Martin, P. M. Imerman, B. Narasimhan, and B. H. Bellaire. 2015. Polyanhydride Nanoparticle Delivery Platform Dramatically Enhances Killing of Filarial Worms. PLoS Negl Trop Dis 9: e0004173. 388. Goodman, J. T., J. E. Vela Ramirez, P. M. Boggiatto, R. Roychoudhury, N. L. B. Pohl, M. J. Wannemuehler, and B. Narasimhan. 2014. Nanoparticle Chemistry and Functionalization Differentially Regulates Dendritic Cell–Nanoparticle Interactions and Triggers Dendritic Cell Maturation. Particle & Particle Systems Characterization 31: 1269-1280. 389. Risatti, G. R., J. D. Callahan, W. M. Nelson, and M. V. Borca. 2003. Rapid Detection of Classical Swine Fever Virus by a Portable Real-Time Reverse Transcriptase PCR Assay. Journal of Clinical Microbiology 41: 500-505. 390. Torres, M. P., J. H. Wilson-Welder, S. K. Lopac, Y. Phanse, B. Carrillo-Conde, A. E. Ramer-Tait, B. H. Bellaire, M. J. Wannemuehler, and B. Narasimhan. 2011. Polyanhydride microparticles enhance dendritic cell antigen presentation and activation. Acta Biomater 7: 2857-2864. 391. Joshi, V. B., S. M. Geary, B. R. Carrillo-Conde, B. Narasimhan, and A. K. Salem. 2013. Characterizing the antitumor response in mice treated with antigen-loaded polyanhydride microparticles. Acta Biomater 9: 5583-5589. 392. Huntimer, L., J. H. Wilson Welder, K. Ross, B. Carrillo-Conde, L. Pruisner, C. Wang, B. Narasimhan, M. J. Wannemuehler, and A. E. Ramer-Tait. 2013. Single immunization with a suboptimal antigen dose encapsulated into polyanhydride microparticles promotes high titer and avid antibody responses. J Biomed Mater Res B Appl Biomater 101: 91-98. 393. Petersen, L. K., L. Huntimer, K. Walz, A. Ramer-Tait, M. J. Wannemuehler, and B. Narasimhan. 2013. Combinatorial evaluation of in vivo distribution of polyanhydride particle-based platforms for vaccine delivery. Int J Nanomedicine 8: 2213-2225. 394. Schicker, R. S., J. Rossow, S. Eckel, N. Fisher, S. Bidol, L. Tatham, J. Matthews- Greer, K. Sohner, A. S. Bowman, J. Avrill, T. Forshey, L. Blanton, C. T. Davis, J. Schiltz, S. Skorupski, L. Berman, Y. Jang, J. S. Bresee, S. Lindstrom, S. C. Trock, D. Wentworth, A. M. Fry, S. de Fijter, K. Signs, M. DiOrio, S. J. Olsen, and M. Biggerstaff. 2016. Outbreak of Influenza A(H3N2) Variant Virus Infections Among Persons Attending Agricultural Fairs Housing Infected Swine - Michigan and Ohio, July-August 2016. MMWR Morb Mortal Wkly Rep 65: 1157-1160.

210

395. Van Reeth, K., and W. Ma. 2013. Swine influenza virus vaccines: to change or not to change-that's the question. Curr Top Microbiol Immunol 370: 173-200. 396. Neutra, M. R., and P. A. Kozlowski. 2006. Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 6: 148-158. 397. Kim, S. H., and Y. S. Jang. 2017. The development of mucosal vaccines for both mucosal and systemic immune induction and the roles played by adjuvants. Clinical and experimental vaccine research 6: 15-21. 398. Mishra, N., A. K. Goyal, S. Tiwari, R. Paliwal, S. R. Paliwal, B. Vaidya, S. Mangal, M. Gupta, D. Dube, A. Mehta, and S. P. Vyas. 2010. Recent advances in mucosal delivery of vaccines: role of mucoadhesive/biodegradable polymeric carriers. Expert Opin Ther Pat 20: 661-679. 399. Wang, D., M. E. Christopher, L. P. Nagata, M. A. Zabielski, H. Li, J. P. Wong, and J. Samuel. 2004. Intranasal immunization with liposome-encapsulated plasmid DNA encoding influenza virus hemagglutinin elicits mucosal, cellular and humoral immune responses. J Clin Virol 31 Suppl 1: S99-106. 400. Babai, I., S. Samira, Y. Barenholz, Z. Zakay-Rones, and E. Kedar. 1999. A novel influenza subunit vaccine composed of liposome-encapsulated haemagglutinin/neuraminidase and IL-2 or GM-CSF. II. Induction of TH1 and TH2 responses in mice. Vaccine 17: 1239-1250. 401. Qi, M., X. E. Zhang, X. Sun, X. Zhang, Y. Yao, S. Liu, Z. Chen, W. Li, Z. Zhang, J. Chen, and Z. Cui. 2018. Intranasal Nanovaccine Confers Homo- and Hetero- Subtypic Influenza Protection. Small. 402. Dhakal, S., J. Hiremath, K. Bondra, Y. S. Lakshmanappa, D. L. Shyu, K. Ouyang, K. I. Kang, B. Binjawadagi, J. Goodman, K. Tabynov, S. Krakowka, B. Narasimhan, C. W. Lee, and G. J. Renukaradhya. 2017. Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs. J Control Release 247: 194- 205. 403. Dhakal, S., J. Goodman, K. Bondra, Y. S. Lakshmanappa, J. Hiremath, D. L. Shyu, K. Ouyang, K. I. Kang, S. Krakowka, M. J. Wannemuehler, C. Won Lee, B. Narasimhan, and G. J. Renukaradhya. 2017. Polyanhydride nanovaccine against swine influenza virus in pigs. Vaccine 35: 1124-1131. 404. van der Lubben, I. M., J. C. Verhoef, G. Borchard, and H. E. Junginger. 2001. Chitosan for mucosal vaccination. Adv Drug Deliv Rev 52: 139-144. 405. Zhao, K., G. Chen, X. M. Shi, T. T. Gao, W. Li, Y. Zhao, F. Q. Zhang, J. Wu, X. Cui, and Y. F. Wang. 2012. Preparation and efficacy of a live newcastle disease virus vaccine encapsulated in chitosan nanoparticles. PLoS One 7: e53314. 406. Amidi, M., S. G. Romeijn, J. C. Verhoef, H. E. Junginger, L. Bungener, A. Huckriede, D. J. Crommelin, and W. Jiskoot. 2007. N-trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model. Vaccine 25: 144- 153.

211

407. Sawaengsak, C., Y. Mori, K. Yamanishi, A. Mitrevej, and N. Sinchaipanid. 2014. Chitosan nanoparticle encapsulated hemagglutinin-split influenza virus mucosal vaccine. AAPS PharmSciTech 15: 317-325. 408. Yassine, H. M., M. Q. Al-Natour, C. W. Lee, and Y. M. Saif. 2007. Interspecies and intraspecies transmission of triple reassortant H3N2 influenza A viruses. Virol J 4: 129. 409. Calvo, P., C. Remunan-Lopez, J. L. Vila-Jato, and M. J. Alonso. 1997. Chitosan and chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines. Pharm Res 14: 1431-1436. 410. Debnath, S. K., S. Saisivam, M. Debanth, and A. Omri. 2018. Development and evaluation of Chitosan nanoparticles based dry powder inhalation formulations of Prothionamide. PLoS One 13: e0190976. 411. Mirzaei, F., N. Mohammadpour Dounighi, M. R. Avadi, and M. Rezayat. 2017. A New Approach to Antivenom Preparation Using Chitosan Nanoparticles Containing EchisCarinatus Venom as A Novel Antigen Delivery System. Iran J Pharm Res 16: 858-867. 412. Lu, F., A. Mencia, L. Bi, A. Taylor, Y. Yao, and H. HogenEsch. 2015. Dendrimer-like alpha-d-glucan nanoparticles activate dendritic cells and are effective vaccine adjuvants. J Control Release 204: 51-59. 413. Nedumpun, T., P. Ritprajak, and S. Suradhat. 2016. Generation of potent porcine monocyte-derived dendritic cells (MoDCs) by modified culture protocol. Vet Immunol Immunopathol 182: 63-68. 414. Dwivedi, V., C. Manickam, S. Dhakal, B. Binjawadagi, K. Ouyang, J. Hiremath, M. Khatri, J. G. Hague, C. W. Lee, and G. J. Renukaradhya. 2016. Adjuvant effects of invariant NKT cell ligand potentiates the innate and adaptive immunity to an inactivated H1N1 swine influenza virus vaccine in pigs. Vet Microbiol 186: 157-163. 415. Meurens, F., M. Berri, G. Auray, S. Melo, B. Levast, I. Virlogeux-Payant, C. Chevaleyre, V. Gerdts, and H. Salmon. 2009. Early immune response following Salmonella enterica subspecies enterica serovar Typhimurium infection in porcine jejunal gut loops. Vet Res 40: 5. 416. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408. 417. Illum, L., I. Jabbal-Gill, M. Hinchcliffe, A. N. Fisher, and S. S. Davis. 2001. Chitosan as a novel nasal delivery system for vaccines. Adv Drug Deliv Rev 51: 81-96. 418. Wang, J. J., Z. W. Zeng, R. Z. Xiao, T. Xie, G. L. Zhou, X. R. Zhan, and S. L. Wang. 2011. Recent advances of chitosan nanoparticles as drug carriers. Int J Nanomedicine 6: 765-774. 419. Figueiredo, L., A. Cadete, L. M. Goncalves, M. L. Corvo, and A. J. Almeida. 2012. Intranasal immunisation of mice against Streptococcus equi using positively charged nanoparticulate carrier systems. Vaccine 30: 6551-6558.

212

420. Vila, A., A. Sanchez, K. Janes, I. Behrens, T. Kissel, J. L. Vila Jato, and M. J. Alonso. 2004. Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice. Eur J Pharm Biopharm 57: 123-131. 421. Rampino, A., M. Borgogna, P. Blasi, B. Bellich, and A. Cesaro. 2013. Chitosan nanoparticles: preparation, size evolution and stability. Int J Pharm 455: 219-228. 422. Peniche, H., and C. Peniche. 2011. Chitosan nanoparticles: a contribution to nanomedicine. Polymer International 60: 883-889. 423. Astete, C. E., and C. M. Sabliov. 2006. Synthesis and characterization of PLGA nanoparticles. J Biomater Sci Polym Ed 17: 247-289. 424. Torres, M. P., B. M. Vogel, B. Narasimhan, and S. K. Mallapragada. 2006. Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J Biomed Med Res A 76: 102-110. 425. Satzer, P., F. Svec, G. Sekot, and A. Jungbauer. 2016. Protein adsorption onto nanoparticles induces conformational changes: Particle size dependency, kinetics, and mechanisms. Engineering in life sciences 16: 238-246. 426. Yu, W., and H. Xie. 2012. A Review on Nanofluids: Preparation, Stability Mechanisms, and Applications. 427. Koppolu, B., and D. A. Zaharoff. 2013. The effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells. Biomaterials 34: 2359-2369. 428. Zhang, F., B. Peng, H. Chang, R. Zhang, F. Lu, F. Wang, F. Fang, and Z. Chen. 2016. Intranasal Immunization of Mice to Avoid Interference of Maternal Antibody against H5N1 Infection. PLoS One 11: e0157041. 429. Dodane, V., M. Amin Khan, and J. R. Merwin. 1999. Effect of chitosan on epithelial permeability and structure. Int J Pharm 182: 21-32. 430. Borges, O., A. Cordeiro-da-Silva, J. Tavares, N. Santarem, A. de Sousa, G. Borchard, and H. E. Junginger. 2008. Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles. Eur J Pharm Biopharm 69: 405-416. 431. Lebre, F., G. Borchard, H. Faneca, M. C. Pedroso de Lima, and O. Borges. 2016. Intranasal Administration of Novel Chitosan Nanoparticle/DNA Complexes Induces Antibody Response to Hepatitis B Surface Antigen in Mice. Mol Pharm 13: 472-482. 432. Jabbal-Gill, I., A. N. Fisher, R. Rappuoli, S. S. Davis, and L. Illum. 1998. Stimulation of mucosal and systemic antibody responses against Bordetella pertussis filamentous haemagglutinin and recombinant pertussis toxin after nasal administration with chitosan in mice. Vaccine 16: 2039-2046. 433. McNeela, E. A., D. O'Connor, I. Jabbal-Gill, L. Illum, S. S. Davis, M. Pizza, S. Peppoloni, R. Rappuoli, and K. H. Mills. 2000. A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM(197)) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 responses following nasal delivery. Vaccine 19: 1188-1198.

213

434. Bacon, A., J. Makin, P. J. Sizer, I. Jabbal-Gill, M. Hinchcliffe, L. Illum, S. Chatfield, and M. Roberts. 2000. Carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens. Infect Immun 68: 5764-5770. 435. Xu, W., Y. Shen, Z. Jiang, Y. Wang, Y. Chu, and S. Xiong. 2004. Intranasal delivery of chitosan-DNA vaccine generates mucosal SIgA and anti-CVB3 protection. Vaccine 22: 3603-3612. 436. Renegar, K. B., and P. A. Small, Jr. 1991. Immunoglobulin A mediation of murine nasal anti-influenza virus immunity. J Virol 65: 2146-2148. 437. Etchart, N., F. Wild, and D. Kaiserlian. 1996. Mucosal and systemic immune responses to measles virus haemagglutinin in mice immunized with a recombinant vaccinia virus. J Gen Virol 77 ( Pt 10): 2471-2478. 438. Bergquist, C., E. L. Johansson, T. Lagergard, J. Holmgren, and A. Rudin. 1997. Intranasal vaccination of humans with recombinant cholera toxin B subunit induces systemic and local antibody responses in the upper respiratory tract and the vagina. Infect Immun 65: 2676-2684. 439. Samuel, C. E. 2001. Antiviral actions of interferons. Clin Microbiol Rev 14: 778- 809, table of contents. 440. La Gruta, N. L., and S. J. Turner. 2014. T cell mediated immunity to influenza: mechanisms of viral control. Trends in immunology 35: 396-402. 441. Mori, A., E. Oleszycka, F. A. Sharp, M. Coleman, Y. Ozasa, M. Singh, D. T. O'Hagan, L. Tajber, O. I. Corrigan, E. A. McNeela, and E. C. Lavelle. 2012. The vaccine adjuvant alum inhibits IL-12 by promoting PI3 kinase signaling while chitosan does not inhibit IL-12 and enhances Th1 and Th17 responses. Eur J Immunol 42: 2709-2719. 442. Carroll, E. C., L. Jin, A. Mori, N. Munoz-Wolf, E. Oleszycka, H. B. T. Moran, S. Mansouri, C. P. McEntee, E. Lambe, E. M. Agger, P. Andersen, C. Cunningham, P. Hertzog, K. A. Fitzgerald, A. G. Bowie, and E. C. Lavelle. 2016. The Vaccine Adjuvant Chitosan Promotes Cellular Immunity via DNA Sensor cGAS-STING- Dependent Induction of Type I Interferons. Immunity 44: 597-608. 443. Farrar, M. A., and R. D. Schreiber. 1993. The molecular cell biology of interferon-gamma and its receptor. Annu Rev Immunol 11: 571-611. 444. De Bruin, T. G., E. M. Van Rooij, Y. E. De Visser, and A. T. Bianchi. 2000. Cytolytic function for pseudorabies virus-stimulated porcine CD4+ CD8dull+ lymphocytes. Viral Immunol 13: 511-520. 445. Hogenesch, H. 2013. Mechanism of immunopotentiation and safety of aluminum adjuvants. Front Immunol 3: 406. 446. Lu, F., Y. C. Mosley, R. J. Rodriguez Rosales, B. E. Carmichael, S. Elesela, Y. Yao, and H. HogenEsch. 2017. Alpha-D-glucan nanoparticulate adjuvant induces a transient inflammatory response at the injection site and targets antigen to migratory dendritic cells. Npj Vaccines 2: 4. 447. Gehrke, I., and R. Pabst. 1990. Cell composition and lymphocyte subsets in the bronchoalveolar lavage of normal pigs of different ages in comparison with germfree and pneumonic pigs. Lung 168: 79-92.

214

448. Dhakal, S., S. Renu, S. Ghimire, Y. Shaan Lakshmanappa, B. T. Hogshead, N. Feliciano-Ruiz, F. Lu, H. HogenEsch, S. Krakowka, C. W. Lee, and G. J. Renukaradhya. 2018. Mucosal Immunity and Protective Efficacy of Intranasal Inactivated Influenza Vaccine Is Improved by Chitosan Nanoparticle Delivery in Pigs. Frontiers in Immunology 9. 449. Lu, F., and H. Hogenesch. 2013. Kinetics of the inflammatory response following intramuscular injection of aluminum adjuvant. Vaccine 31: 3979-3986. 450. Beck, J. R., D. E. Swayne, S. Davison, S. Casavant, and C. Gutierrez. 2003. Validation of egg yolk antibody testing as a method to determine influenza status in white leghorn hens. Avian Dis 47: 1196-1199. 451. Renu, S., S. Dhakal, E. Kim, J. Goodman, Y. S. Lakshmanappa, M. J. Wannemuehler, B. Narasimhan, P. N. Boyaka, and G. J. Renukaradhya. 2018. Intranasal delivery of influenza antigen by nanoparticles, but not NKT-cell adjuvant differentially induces the expression of B-cell activation factors in mice and swine. Cell Immunol. 452. Guo, Y. J., S. H. Sun, Y. Zhang, Z. H. Chen, K. Y. Wang, L. Huang, S. Zhang, H. Y. Zhang, Q. M. Wang, D. Wu, and W. J. Zhu. 2004. Protection of pigs against Taenia solium cysticercosis using recombinant antigen or in combination with DNA vaccine. Vaccine 22: 3841-3847. 453. De Gregorio, E., E. Caproni, and J. B. Ulmer. 2013. Vaccine adjuvants: mode of action. Front Immunol 4: 214. 454. Mannhalter, J. W., H. O. Neychev, G. J. Zlabinger, R. Ahmad, and M. M. Eibl. 1985. Modulation of the human immune response by the non-toxic and non- pyrogenic adjuvant aluminium hydroxide: effect on antigen uptake and antigen presentation. Clin Exp Immunol 61: 143-151. 455. Morefield, G. L., A. Sokolovska, D. Jiang, H. HogenEsch, J. P. Robinson, and S. L. Hem. 2005. Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro. Vaccine 23: 1588-1595. 456. Kalinski, P., C. M. Hilkens, E. A. Wierenga, and M. L. Kapsenberg. 1999. T-cell priming by type-1 and type-2 polarized dendritic cells: the concept of a third signal. Immunol Today 20: 561-567. 457. Stambas, J., C. Guillonneau, K. Kedzierska, J. D. Mintern, P. C. Doherty, and N. L. La Gruta. 2008. Killer T cells in influenza. Pharmacol Ther 120: 186-196. 458. Rajao, D. S., H. Chen, D. R. Perez, M. R. Sandbulte, P. C. Gauger, C. L. Loving, G. D. Shanks, and A. Vincent. 2016. Vaccine-associated enhanced respiratory disease is influenced by haemagglutinin and neuraminidase in whole inactivated influenza virus vaccines. J Gen Virol 97: 1489-1499. 459. Li, X., B. R. Sloat, N. Yanasarn, and Z. Cui. 2011. Relationship between the size of nanoparticles and their adjuvant activity: data from a study with an improved experimental design. Eur J Pharm Biopharm 78: 107-116. 460. Fifis, T., A. Gamvrellis, B. Crimeen-Irwin, G. A. Pietersz, J. Li, P. L. Mottram, I. F. McKenzie, and M. Plebanski. 2004. Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. J Immunol 173: 3148-3154. 215

461. Berlanda Scorza, F., V. Tsvetnitsky, and J. J. Donnelly. 2016. Universal influenza vaccines: Shifting to better vaccines. Vaccine 34: 2926-2933. 462. Tumpey, T. M., M. Renshaw, J. D. Clements, and J. M. Katz. 2001. Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection. J Virol 75: 5141-5150. 463. Perrone, L. A., A. Ahmad, V. Veguilla, X. Lu, G. Smith, J. M. Katz, P. Pushko, and T. M. Tumpey. 2009. Intranasal vaccination with 1918 influenza virus-like particles protects mice and ferrets from lethal 1918 and H5N1 influenza virus challenge. J Virol 83: 5726-5734. 464. Benton, K. A., J. A. Misplon, C. Y. Lo, R. R. Brutkiewicz, S. A. Prasad, and S. L. Epstein. 2001. Heterosubtypic immunity to influenza A virus in mice lacking IgA, all Ig, NKT cells, or gamma delta T cells. J Immunol 166: 7437-7445. 465. Epstein, S. L., C. Y. Lo, J. A. Misplon, C. M. Lawson, B. A. Hendrickson, E. E. Max, and K. Subbarao. 1997. Mechanisms of heterosubtypic immunity to lethal influenza A virus infection in fully immunocompetent, T cell-depleted, beta2- microglobulin-deficient, and J chain-deficient mice. J Immunol 158: 1222-1230. 466. Heit, A., F. Schmitz, T. Haas, D. H. Busch, and H. Wagner. 2007. Antigen co- encapsulated with adjuvants efficiently drive protective T cell immunity. Eur J Immunol 37: 2063-2074. 467. Schliehe, C., C. Redaelli, S. Engelhardt, M. Fehlings, M. Mueller, N. van Rooijen, M. Thiry, K. Hildner, H. Weller, and M. Groettrup. 2011. CD8- dendritic cells and macrophages cross-present poly(D,L-lactate-co-glycolate) acid microsphere-encapsulated antigen in vivo. J Immunol 187: 2112-2121.

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