Efficacy of Liposomal M2e Vaccines in Combination With

EFFICACY OF LIPOSOMAL M2E VACCINES IN COMBINATION WITH

ADJUVANTS OR LIPOSOMAL ADJUVANT VACCINES WITHOUT M2E IN

MICE INFECTED WITH INFLUENZA H1N1 OR H3N2

A Thesis

Presented to the Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

Biological Sciences

Paulina G. Villanueva

2017

SIGNATURE PAGE

THESIS: EFFICACY OF LIPOSOMAL M2E VACCINES IN COMBINATION WITH ADJUVANTS OR LIPOSOMAL ADJUVANT VACCINES WITHOUT M2E IN MICE INFECTED WITH INFLUENZA H1N1 OR H3N2

AUTHOR: Paulina G. Villanueva

DATE SUBMITTED: Spring 2017

Biological Sciences Department

Dr. Jill Adler-Moore Thesis Committee Chair Biological Sciences

Dr. Nancy Buckley Biological Sciences

Dr. Jamie Snyder Biological Sciences

ii ACKNOWLEDGEMENTS

I want to thank all the members of Dr. Adler’s laboratory for always being supportive. Thank you Jenny, Hernan, Jon, Eden, Fady, Cole, and Sherleen for always helping me during my spleen collections. Jenny, thank you for becoming one of my great friends. I am so happy to know that we will pursue our dreams in Los Angeles. Don’t forget to come visit me. Thank you Fady for being the best influenza team partner I could have asked for. You are a kind, intelligent, and a thoughtful person and I am so happy to know that your dream of becoming a physician is coming true. You deserve all the good that is coming your way. Thank you Eden for coming into the lab during the most challenging time of my career at Cal Poly Pomona. I know that you are going to do amazing things for the influenza team. Most importantly, thank you for being my friend.

Jonathan, thank you for being my research mentor and friend. I will never forget that you always made time to help me during your most stressful times at Cal Poly Pomona. Dr.

Adler, thank you for accepting me as a graduate student. Without your mentorship and support, my dream of becoming a physician would not be a reality. Thank you for always pushing me to do more and for always making time to get to know me as a person. To my

committee members, Dr. Adler, Dr. Buckley, and Dr. Snyder, thank you from the bottom

of my heart for working so hard to help me complete my thesis. I feel blessed to have

been mentored by three incredibly accomplished women in science. I came to Cal Poly

Pomona to find the kind of mentorship that you have given me. I hope that one day, I can

shape someone’s future the way you help me shape mine. To my boyfriend, Nick

Shadwell, thank you for always supporting me and for believing in my dreams. It is

because of your motivation and support that I will soon be a medical student at UCLA.

iii Thank you for always being by my side. I am so happy for everything that is coming our way. Lastly, Mom and Dad, thank you for helping me become the person that I am today.

You brought me to the United States with big dreams for me. Because of your sacrifices, my dreams are coming true. You are my inspiration.

iv ABSTRACT

Introduction: Influenza A viruses are a major cause of acute respiratory diseases in

humans. Different influenza A strains have been responsible for seasonal infections

(epidemics) and worldwide pandemics. All of these pandemics and epidemics arise from

the mutations or genetic reassortment of viral RNA coding for hemagglutinin (HA) and

neuraminidase (NA) surface proteins of influenza A viruses, which inevitably leads to

antigenic shift (pandemics) and antigenic drift (epidemics). Because of this, we have

worked in collaboration with Molecular Express Inc. on the VesiVax® delivery system

which incorporates the viral protein M2e epitope, a 15 amino acid (aa) residue at the

amino end of the highly conserved region of the M2 protein, into a unilamellar liposome

less than 100 nm in size. The M2e protein can be conjugated to the surface of the liposomes via a maleimide linker (CALV) or fused to a hydrophobic protein (HD), which allows the M2e to be incorporated into the liposome bilayer. This technology was used to investigate various combinations of vaccines composed of M2e and different immunological adjuvants or adjuvants without the M2e protein for their efficacy in Swiss

Webster female mice against challenge with Influenza A/Puerto Rico/8/1934 (PR8) H1N1 or X-31 H3N2.

Methods: Study 1, the vaccine formulations were as follows: L-CMI-100 μg mycoviral

dsRNA-M2e, L-CMI-15 μg MPL-M2e, L-CMI-15μg MPL, L-CMI-100 μg mycoviral

dsRNA and PBS. The mycoviral dsRNA was obtained from the mycovirus present in

Saccharomyces cerevisiae. Mice were vaccinated subcutaneously on d0 and intranasally

(IN) boosted on d28 and d56. Sera (n=5/gp) were collected 3 days after the last boost and

tested for anti-whole virus IgG and anti-M2e IgG isotype concentrations. The remaining

v mice were IN challenged 2 weeks post-boost with 10X LD50 Influenza A/Puerto

Rico/8/1934 (PR8) H1N1 or 10X LD50 (X-31) H3N2. Lungs were collected 5 days after

infection (n=5/gp) and tested for viral burden via foci assay. The remaining mice

(n=10/gp) were monitored for morbidity to day 28 post-challenge. In Study 2, the vaccine

formulations were as follows: L-CMI-50 μg mycoviral dsRNA (UC)-M2e, L-CMI-25 μg mycoviral dsRNA (UC)-M2e, L-CMI-25 μg mycoviral dsRNA(C)-M2e, L-CMI-15 μg mycoviral dsRNA (C)-M2e, L-CMI-5 μg mycoviral dsRNA (C)-M2e, Non-CMI-50 μg mycoviral dsRNA (UC) and PBS. The experiment was performed as in Study 1, but only the H1N1 challenge was used and lungs were not collected for this study. In Study 3, the vaccine formulations were as follows: L-MPL-M2e (1-12), L-MPL-M2e (1-12)cD, L- mycoviral dsRNA-M2e (1-12), L-MPL-M2e (1-15), L-6 μg LT1-M2e (1-15), L-3 μg

LT1/7.5 μg MPL-M2e (1-15), L-3 μg LT1-M2e (1-15), L-Pam3CAG-M2e (1-15), L-MPL

(No CMI), and PBS. As in Study 3, mice were only challenged with H1N1. In Study 4, the vaccine formulations were as follows: L-MPL-M2e-HD, L-CMI-Pam3CAG, L-CMI-

MPL, L-CMI-mycoviral dsRNA, L-CMI-1V270, L-CMI-LT1 and L-MPL (No CMI).

Mice were primed and boosted as in Study 1, but viral challenge was done 1 week after the last boost with 10X LD50 H1N1. Mice (n=10/gp) were monitored for morbidity to day

28 post-challenge.

Results: L-CMI-100 μg mycoviral dsRNA-M2e provided significantly better protection

compared to MPL and PBS control groups against an H1N1 and H3N2 challenge as

measured by survival and decreased viral burden (p<0.014). L-CMI-100 μg mycoviral

dsRNA also provided significantly better protection compared to the MPL control group

(p<0.024) against H1N1 challenge and MPL and PBS control groups (p<0.01) and

vi against H3N2 challenge as measured by survival (p<0.024). L-CMI-50 μg mycoviral dsRNA (UC)-M2e provided significantly better protection compared to L-CMI-25 μg mycoviral dsRNA (C)-M2e as measured by survival against an H1N1 challenge

(p<0.024). Vaccine formulations L-Pam3CAG-M2e (1-15), L-LT1/MPL-M2e (1-15) and

L-mycoviral dsRNA-M2e (1-12) provided the most protection as measured by survival.

Lastly, L-MPL-M2e-HD, L-CMI-Pam3CAG, L-CMI-mycoviral dsRNA and L-CMI-MPL provided significantly better protection L-MPL (No CMI) as measured by survival, against an H1N1 challenge (p<0.048).

Conclusions: These studies demonstrated for the first time that a CMI liposomal M2e vaccine formulation containing 100 μg of mycoviral dsRNA could provide cross protection against two strains of influenza virus (H1N1 and H3N2) in Swiss Webster female mice. We also showed that the antigen M2e (1-15) was more effective than the shorter antigen M2e (1-12) when incorporated into a CMI liposomal vaccine.

Additionally, we observed that CMI liposomal vaccine formulations containing only an adjuvant without a protein antigen could provide protection against a lethal challenge of influenza virus.

vii TABLE OF CONTENTS

Signature Page…………………………………………………………………………….ii

Acknowledgments……………………………………………………………………..iii-iv

Abstract………………………………………………………………………………..v-vii

List of Tables ………………………………………………………………………xvi-xxi

List of Figures ……………………………………………………………………xxii-xxvi

Chapter 1: Introduction……………………………………………………………...…1-68

I. Influenza Infection: Transmission and Disease Symptoms…………………..1-5

II. Orthomyxoviridae Viruses …………………………………………………..5-9

A. Types of Influenza Viruses……………………………………………5-9

1. Influenza A…………………………………………………...6-7

2. Influenza B …………………………………………………..7-8

3. Influenza C …………………………………………………..8-9

III. Influenza A Outbreaks……………………………………………………..9-14

A. Overview…………………………………………………………….9-10

B. Influenza Pandemic of 1918………………………………………..10-11

C. Influenza Pandemic of 1957………………………………………..11-12

D. Influenza Pandemic of 1968………………………………………..12-13

E. Influenza Pandemic of 2009 …………………………………………..13

F. Continuous Threats: H5N1 and H7N9 Influenza Virus ……………13-14

IV. Antigen Drift and Antigen Shift in Influenza…………………………….14-16

V. Influenza Viral Proteins ………………………………………………….16-21

VI. Viral Nomenclature………………………………………………………21-22

viii VII. Influenza Replication Cycle……………………………………………..22-25

A. Viral Entry………………………………………………………….22-23

B. Intracellular Replication……………………………………………23-24

C. Viral Budding………………………………………………………24-25

VIII. Antivirals…………………………………………………………….…25-29

A. Amantadine and Rimantadine……………………………………...26-27

B. Oseltamivir and Zanamivir…………………………………………27-29

IX. Immunity to Influenza……………………………………………………29-46

A. Innate Immune System……………………………………………..30-37

1. Cellular Innate Immune Response…………………………30-34

2. Inflammasome……………………………………………..34-35

3. Complement………………………………………………..36-37

B. Adaptive Immune Response………………………………………..38-46

1. T Cell Maturation………………………………………….39-40

2. Th1 Response/Cell Mediated………………………………40-42

3. Th2 Response/Antibody Mediated………………………...42-44

4. Th17/Inflammatory Response……………………………..44-45

5. Treg/Immunosuppressive Response……………………………46

X. Influenza Vaccination…………………………………………………..…46-53

A. History of Influenza Vaccination………………………………….46-48

B. Types of Influenza Vaccines………………………………………48-53

1. Live Attenuated or Inactivated Vaccines…………………48-50

2. Vaccines Against The Conserved HA Stalk………………50-51

3. M2 Vaccines……………………………………………….51-53

XI. Previous Work on Liposomal Based M2e Vaccines Using The

Vesivax® System……………………………………………….………..54-59

A. Liposomes………………………………………………………….54-56

B. VesiVax ® Liposomal Vaccines…………………………………...56-57

C. M2e Vaccines Formulated Using L-M2eA-HD……………………57-59

XII. Adjuvants………………………………………………………………..59-61

XIII. Aims of the Present Study……………………………………………...61-65

A. Monophosphoryl Lipid A (MPL)……………………………………...62

B. Mycoviral dsRNA……………………………………………….…62-63

C. Tucaresol…………………………………………………………...63-64

D. Pam3CAG………………………………………………………….64-65

E. 1V270………………………………………………………………….65

XIV. Hypotheses of Present Studies…………………………………………66-68

A. Study I………………………………………………………………....68

B. Study II……………………………………………………………..68-67

C. Study III………………………………………………………………..67

D. Study IV……………………………………………………………67-68

Chapter 2: Materials and Methods………………………………………………..…69-113

I. Tissue and Virus Culture…………………………………………………..69-76

A. Cell Culture: Madin-Darby Canine Kidney-ATTC#34-CCL- (MDCK) Cell…………………………………………...…………69-70

B. Culturing MDCK Cells………………………………………………..70

C. Trypan Blue Exclusion………………………………………….…70-71

x D. Refreezing MDCK Cells………………………………………..….71-72

E. Termination of MDCK Cells…………………………………………..72

F. Viral Stocks………………………………………………………….…72

G. MDCK Cell Passaged H1N1 and H3N2 Stock…………………….73-74

H. Lung Passaged H1N1 Stock………………………………………..74-76

II. In-Vivo………………………………………………………………………...77

Study I. Investigation of a CMI liposomal M2e vaccine containing the adjuvant, mycoviral dsRNA or MPL, against H1N1 or H3N2 influenza A challenge…………………………………...... 77-79

Study II. Investigation of ultracentrifugation and column chromatography isolation methods to obtain mycoviral dsRNA for use in the CMI liposomal M2e vaccine……………………..…..79-80

Study III. Comparative efficacy of CMI liposomes containing M2e (1-12) vs M2e (1-15) and MPL, and testing other adjuvant molecules in Swiss Webster female mice…………..….....80-82

Study IV. Investigation of CMI liposomal vaccines containing adjuvants without the antigen protein M2e…………………………………………………...………82-85

III. Liposomal Vaccine Preparation………………………………………….85-86

A. M2e-HD Containing MPL…………………………………………….85

B. Conjugateable Adjuvant Lipid Vesicles (CALV) CMI Liposomes…...86

IV. Adjuvants in Vaccine Efficacy Studies…...... 86-88

1. MPL…………………………………………………………………....86

2. Mycoviral dsRNA………………………………………………….86-88

3. Pam3CAG……………………………………………………………...88

4. Lipidated-Tucaresol……………………………………………………88

5. 1V270………………………………………………………………..…88

xi V. Vaccination……………………………………………………………..…88-89

A. Subcutaneous (SC)…………………………………………………88-89

B. Intranasal (IN)………………………………………………………….89

VI. Intranasal Influenza A Challenge of Mice……………………………….89-90

VII. Cardiac Puncture…………………………………………………………....90

VIII. Splenocyte Collection………………………………………………..…90-91

IX. Lung Collection………………………………………………………..…91-92

X. Spleen Collection………………………………………………………….….92

XI. CO2 Asphyxiation………………………………………………………...92-93

XII. In-Vitro Techniques………………………………………………………....93

A. Cytolysis Assay…………………………………………………….93-95

B. Foci Assay……………………………………………………….…95-99

C. Enzyme Linked Immunosorbent Assay (ELISA)……………...…99-103

D. H1N1 or H3N2 Whole Virus Enzyme Linked Immunosorbent Assay (ELISA)…………………………………………………..103-107

E. Enzyme Linked Immunosorbent Spot Assay……………………107-111

F. Multiplex Cytokine Secretion Assay (Luminex)………………...111-113

XIII. Statistical Analysis………………………………………………………..113

Chapter 3: Results………………………………………………………………….114-194

Study I. Investigation of a CMI liposomal M2e vaccine containing the adjuvant, mycoviral dsRNA or MPL, against H1N1 influenza A challenge………………………………………..…..114-140

A. CMI liposomal M2e vaccine containing mycoviral dsRNA protects against H1N1 viral challenge………………………..…115-118

B. Mice vaccinated with L-CMI-100 μg mycoviral dsRNA-M2e or L-CMI-15 μg MPL-M2e had decreased lung

xii viral burden………………………………………………………118-120

C. CMI liposomal M2e vaccine containing mycoviral dsRNA had a predominantly Th1 response and CMI liposomal M2e vaccine containing MPL had a predominantly Th2 response based on anti-M2e IgG isotype characterization……….…….….120-122

D. L-CMI-15 μg MPL-M2e had an increased number of IFN-γ secreting splenocytes compared to the PBS control group……………………………………………….....…122-124

E. CMI liposomal M2e vaccine containing mycoviral dsRNA or MPL or mycoviral dsRNA without M2e protects against H3N2 viral challenge………………………………….……………...…124-127

F. Mice vaccinated with the liposomal M2e vaccine containing mycoviral dsRNA had less lung viral burden compared to all other vaccine groups……………………………………………..127-129

G. CMI liposomal M2e vaccine containing mycoviral dsRNA had a predominantly Th1 response and CMI liposomal M2e vaccine containing MPL had a predominantly Th2 response based on anti-M2e IgG isotype characterization…...….129-131

H. Mice vaccinated with the CMI liposomal M2e vaccine containing mycoviral dsRNA or MPL had an increased production of IgG antibodies that could bind to the M2e present on the whole virus…………………………………………………..131-133

I. Mice vaccinated with CMI liposomal vaccine containing mycoviral dsRNA and no M2e had higher levels of IFN-γ secreting splenocytes compared to mice vaccinated with the liposomal vaccine containing MPL with M2e……………...…....133-135

J. CMI liposomal M2e vaccine with mycoviral dsRNA had elevated levels of IFN-γ, IL-4, IL-6 and IL-1α and this correlated with its ability to produce the most protection following H1N1 or H3N2 challenge………………………………………….……135-140

Study II. Investigation of the effects of different purification methods to obtain mycoviral dsRNA for use in the CMI liposomal M2e vaccine……………………………………..…...140-156

A. Mice vaccinated with CMI liposomal M2e containing 50 μg mycoviral dsRNA obtained via ultracentrifugation had increased survival compared to mice vaccinated with CMI liposomal M2e

xiii containing 25 μg mycoviral dsRNA obtained via column chromatography……………………………………....…141-146

B. All CMI liposomal vaccines containing M2e and mycoviral dsRNA (UC or C) had more pronounced Th1 responses than Th2 responses based on the increased levels of anti-M2e IgG2a versus anti-M2e IgG1………………………………………………....…147-151

C. Only mice vaccinated with the CMI liposomal M2e vaccines and mycoviral dsRNA UC had elevated levels of anti-M2e antibodies that could bind with the whole virus compared to control groups……………………………………...151-153

D. Only mice vaccinated with a CMI liposomal M2e vaccine containing 50 μg or 25 μg of mycoviral dsRNA (UC) or 15 μg mycoviral dsRNA (C) had increased IL-4 secreting splenocytes compared to control groups……………………………………...153-156

Study III. Comparative efficacy of CMI liposomes containing M2e (1-12) vs M2e (1-15) and MPL, and testing other adjuvant molecules in Swiss Webster female mice…………………..…156-175

A. Mice vaccinated with L-M2 (1-12)-mycoviral dsRNA, L-M2e (1-15)-LT1/MPL or L-M2e (1-15)-Pam3CAG had increased survival compared to the other vaccines……………...157-162

B. L-CMI-M2e (1-12) vaccines had an enhanced Th1 cell mediated immune response while in general, L-CMI-M2e (1-15) vaccines had an enhanced Th2 antibody mediated immune response…………….……………...163-169

C. Mice vaccinated with the smaller more conserved M2e (1-12) had increased levels of IL-6, IL-1α and IL-10 compared to almost all other vaccines……………………..……169-175

Study IV. Investigation of CMI liposomal vaccine formulation containing adjuvants without the antigen protein M2e (1-15)………………………………………………………175-194

A. Mice vaccinated with L-MPL-M2e-HD, L-CMI-Pam3CAG (no M2e) or L-CMI-mycoviral dsRNA (no M2e) had the most survival………………………………………………...176-179

B. Vaccine formulations L-CMI-M2e-HD and L-Pam3CAG (no M2e) provided protection against H1N1 but only L-CMI-M2e-HD provided protection against H3N2……………178-187

xiv

C. Groups with higher survival had lower lung viral burdens……...187-189

D. Cytokine levels did not differ between L-MPL-M2e-HD vaccine and the liposomal adjuvant vaccines without M2e……………………………………………..…...….189-194

Chapter 4: Discussion……………………………………………………………...195-212

I. Swiss Webster female mice were better protected from a lethal challenge of H1N1 or H3N2 influenza virus when vaccinated with a CMI liposomal M2e vaccine containing mycoviral dsRNA compared to mice vaccinated with a CMI liposomal M2e vaccine with MPL or a CMI liposome with mycoviral dsRNA or MPL without M2e…..195-200

II. CMI liposomal M2e vaccines with mycoviral dsRNA (isolated by ultracentrifugation, UC) provided better protection in Swiss Webster female mice challenged with H1N1 influenza virus compared to mice vaccinated with CMI liposomal M2e vaccine with mycoviral dsRNA (isolated by column chromatography, C)…………………………………………………....200-203

III. CMI liposomes containing the larger M2e (amino acids 1-15) when used in combination with the adjuvant Pam3CAG or MPL + Tucaresol provided increased protection in Swiss Webster female mice challenged with H1N1 influenza virus compared to mice vaccinated with CMI liposomes containing a smaller M2e (amino acids 1-12) with the MPL adjuvant…………..203-206

IV. CMI liposomes containing Pam3CAG and no M2e protein provided protection in mice challenged with H1N1 influenza virus………………………………………………………...207-211

Future Studies……………………………………………………………....211-212

References…………………………………………………………………………213-221

List of Tables

Table 1. Comparison Between Influenza A and Influenza B Proteins…………………... 7

Table 2. Influenza Vaccines Available in the United States for the 2016-2017 Influenza Season………………………………………………….………….....51

Table 3. M2e-based Vaccines Under Clinical Investigation…………………………… 52

Table 4. Adjuvants Under Clinical Investigation For Prophylactic Use in Vaccine Formulations……………………………………………………………………61

Table 5. CMI Liposomal Formulations Containing M2e (1-15) Tested in Swiss Webster Female Mice Challenged with H1N1 Influenza Virus…………….….78

Table 6. CMI Liposomal Formulations Containing M2e (1-15) Tested in Swiss Webster Female Mice Challenged with H3N2 Influenza Virus…………….….79

Table 7. CMI Liposomal Formulations Containing M2e (1-15) and mycoviral dsRNA Purified Via Ultracentrifugation (U) or Column Chromatography (C) and Tested in Swiss Webster Female Mice Challenged with H1N1 Influenza Virus………………...... 80

Table 8. CMI Liposomal Formulations Containing M2e (1-12) or M2e (1-15) in Combination with Various Adjuvants Tested in Swiss Webster Female Mice Challenged with H1N1 Influenza Virus…………………………………….….82

Table 9. CMI Liposomal Formulations Containing Various Adjuvants and No M2e Protein Tested in Swiss Webster Female Mice Challenged with H1N1 Influenza Virus…………………………………………….……………………83

Table 10. CMI Liposomal Formulations Containing Pam3CAG or mycoviral dsRNA and No M2e Protein Tested in Swiss Webster Female Mice Challenged with H1N1 Influenza Virus……………………………………………..…………..84

Table 11. CMI Liposomal Formulations Containing Pam3CAG or mycoviral dsRNA and No M2e Protein Tested in Swiss Webster Female Mice Challenged with H3N2 Influenza Virus…………………………………………….……..85

Table 12. Typical 96-Well Plate Set Up for ELISA……………………………………102

Table 13. Typical 96-Well Plate Set Up for ELISA……………………………………106

Study I: Investigation of a CMI liposomal M2e vaccine containing the adjuvant, mycoviral dsRNA or MPL against H1N1 influenza challenge………………………………………………………..………..114-140

xvi

Table 14. Log-Rank (Mantel-Cox) Test of Survival for H1N1 Challenged Swiss Webster Female Mice………………………………………………………..116

Table 15. Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H1N1 Challenged Swiss Webster Female Mice…………………118

Table 16. Mann Whitney Non-Parametric Two-Tailed T-Test of Disease Scores on Day 28 for H1N1 Challenged Swiss Webster Female Mice……..………..…118

Table 17. Mann Whitney Non-Parametric Two-Tailed T-Test of Viral Burden in H1N1 Challenged Swiss Webster Female Mice……………………………..120

Table 18. Isotype Ratio of IgG1/IgG2a Used to Determine the Dominant Adaptive Immune Response Induced by the Liposomal M2e Vaccines with mycoviral dsRNA or MPL…………………………………………………...121

Table 19. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG1 in Swiss Webster Female Mice…………………………………………………122

Table 20. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG2a in Swiss Webster Female Mice…………………………………………..…….122

Table 21. Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ Secreting Splenocytes………………………………………………………………….123

Table 22. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-4 Secreting Splenocytes……………………………………………………………….…124

Table 23. Log-Rank (Mantel-Cox) Test of Survival for H3N2 Challenged Swiss Webster Female Mice……………………………………..…………………125

Table 24. Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H3N2 Challenged Swiss Webster Female Mice…………………...…127

Table 25. Mann Whitney Non-Parametric Two-Tailed T-Test of Disease Scores on Day 28 for H3N2 Challenged Swiss Webster Female Mice…………………127

Table 26. Mann Whitney Non-Parametric Two-Tailed T-Test of Viral Burden in H3N2 Challenged Swiss Webster Female Mice…………………….……….129

Table 27. Isotype Ratio of IgG1/IgG2a Used to Determine the Dominant Adaptive Immune Response Induced by the Liposomal M2e Vaccines with mycoviral dsRNA or MPL………………………………………………...…130

Table 28. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG1

xvii in Swiss Webster Female Mice……………………………………………...131

Table 29. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG2a in Swiss Webster Female Mice………………………………………….…...131

Table 30. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-Whole Virus M2e IgG in Swiss Webster Female Mice……………………………………133

Table 31. Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ Secreting Splenocytes………………………………………………………………….134

Table 32. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-4 Secreting Splenocytes………………………………………………………………….135

Table 33. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1β Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein…………137

Table 34. Mann Whitney Non-Parametric Two-Tailed T-Test of TNF-α Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….138

Table 35. Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein……….…138

Table 36. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-6 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….138

Table 37. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-4 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….139

Table 38. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1α Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….139

Table 39. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-10 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….139

Table 40. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-12 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….140

Study II: Investigation of the effects of different purification methods to obtain mycoviral dsRNA for use in the CMI liposomal M2e vaccine……...... 140-156

Table 41. Log-Rank (Mantel-Cox) Test of Survival for H1N1 Challenged Swiss Webster Female Mice……………………………………………………..…144

Table 42. Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day

xviii 28 for H1N1 Challenged Swiss Webster Female Mice…………………...…146

Table 43. Mann Whitney Non-Parametric Two-Tailed T-Test of Disease Scores on Day 28 for H1N1 Challenged Swiss Webster Female Mice………………....146

Table 44. Isotype Ratio of IgG1/IgG2a Used to Determine the Dominant Adaptive Immune Response Induced by CMI Liposomal M2e Vaccines with Mycoviral dsRNA Obtained Via UC or C………………………...…………148

Table 45. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG1 in Swiss Webster Female Mice………………………………………………....150

Table 46. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG2a in Swiss Webster Female Mice………………………………………………...150

Table 47. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG3 in Swiss Webster Female Mice………………………………………………....151

Table 48. Mann Whitney Non-Parametric One-Tailed T-Test of Anti-Whole Virus M2e IgG in Swiss Webster Female Mice…………………………………....153

Table 49. Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ Secreting Splenocytes…………………………………………………………………..155

Table 50. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-4 Secreting Splenocytes…………………………………………………………………..156

Study III: Comparative efficacy of CMI liposomes containing M2e (1-12) vs M2e (1-15) and MPL, and testing other adjuvant molecules in Swiss Webster female Mice…………………………………………..156-175

Table 51. Log-Rank (Mantel-Cox) Test of Survival for H1N1 Challenged Swiss Webster Female Mice………………………………………………………..160

Table 52. Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H1N1 Challenged Swiss Webster Female Mice……………………...162

Table 53. Mann Whitney Non-Parametric Two-Tailed T-Test of Disease Scores on Day 28 for H1N1 Challenged Swiss Webster Female Mice…………………162

Table 54. Anti-M2e IgG1: IgG2a Ratio of Liposomal Vaccine Formulations…………164

Table 55. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG1 in Swiss Webster Female Mice…………………………………………………166

Table 56. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG2a in

xix Swiss Webster Female Mice…………………………………………………167

Table 57. Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG3 in Swiss Webster Female Mice………………………………………………....168

Table 58. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1β Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein……….…171

Table 59. Mann Whitney Non-Parametric Two-Tailed T-Test of TNF-α Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….172

Table 60. Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….172

Table 61. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-6 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….173

Table 62. Mann Whitney Non-Parametric T-Test of IL-4 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………………………..173

Table 63. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1α Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….174

Table 64. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-10 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein……….…174

Table 65. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-12 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein………….175

Study IV: Investigation of CMI liposomal vaccine formulations containing adjuvants without the antigenic protein M2e (1-15)……………….175- 194

Table 66. Log-Rank (Mantel-Cox) Test of Survival for H1N1 Challenged Swiss Webster Female Mice……………………………………………………..…178

Table 67. Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H1N1 Challenged Swiss Webster Female Mice……………………...179

Table 68. Mann Whitney Non-Parametric Two-Tailed T-Test of Disease Scores on Day 28 for H1N1 Challenged Swiss Webster Female Mice………………....179

Table 69. Log-Rank (Mantel-Cox) Test of Survival for H1N1 Challenged Swiss Webster Female Mice………………………………………………………..183

Table 70. Long-Rank (Mantel-Cox) Test of Survival for H3N2 Challenged Swiss Webster Female Mice………………………………………………………..183

Table 71. Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H1N1 Challenged Swiss Webster Female Mice……………………...186

Table 72. Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H3N2 Challenged Swiss Webster Female Mice…………………...…186

Table 73. Mann Whitney Non-Parametric Two-Tailed T-Test for Disease Scores on Day 28 for H1N1 Challenged Swiss Webster Female Mice…………………186

Table 74. Mann Whitney Non-Parametric Two-Tailed T-Test for Disease Scores on Day 28 for H3N2 Challenged Swiss Webster Female Mice…………………187

Table 75. Mann Whitney Non-Parametric Two-Tailed T-Test of Viral Burden in H1N1 Challenged Swiss Webster Female Mice……………………………..188

Table 76. Mann Whitney Non-Parametric Two-Tailed T-Test of Viral Burden in H3N2 Challenged Swiss Webster Female Mice……………………………..189

Table 77. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1β in Spleen Homogenate Prior to H1N1 Challenge……………………………………....191

Table 78. Mann Whitney Non-Parametric Two-Tailed T-Test of TNF-α in Spleen Homogenate Prior to H1N1 Challenge……………………………….…...…192

Table 79. Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ in Spleen Homogenate Prior to H1N1 Challenge……………………………………....192

Table 80. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-6 in Spleen Homogenate Prior to H1N1 Challenge……………………………………....192

Table 81. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-4 in Spleen Homogenate Prior to H1N1 Challenge………………………………….…...193

Table 82. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-10 in Spleen Homogenate Prior to H1N1 Challenge……………………………………....193

Table 83. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1α in Spleen Homogenate Prior to H1N1 Challenge……………………………………….193

Table 84. Mann Whitney Non-Parametric Two-Tailed T-Test of IL-12 in Spleen Homogenate Prior to H1N1 Challenge…………………………………..…..194

xxi List of Figures

Figure 1. Known emergence events of influenza viruses in mammals…………………....2

Figure 2. Subtype of influenza transmission during peak influenza season for the Northern Hemisphere…………………………………………………………....4

Figure 3. Subtype of influenza transmission during peak influenza season for the Southern Hemisphere……………………………………………………………4

Figure 4. Comparison between influenza C and influenza A/B proteins………………....9

Figure 7. Hemagglutinin protein and conformational changes that lead to exposure of the fusion peptide and membrane fusion……………………………..……..17

Figure 8. Cartoon representation of the neuraminidase protein of the influenza virus…..18

Figure 9. Structure of M2 tetrameric protein (residues 22-62) at pH 7.5………………..19

Figure 10. Influenza virus entry into the host cell……………………………………….23

Figure 11. M2 mediated membrane scission leading to viral budding from host cell…...25

Figure 12. Structure of amantadine and rimantadine…………………………………….27

Figure 13. Oseltamivir and Relenza inhibit influenza by binding to NA and preventing the virus from budding from the host cell…………………...……28

Figure 14. Targets of antiviral drugs against influenza virus infection………………….29

Figure 15. PRRs of innate immune cells include TLRs, NOD and other types of receptors………………………………………………………………………32

Figure 16. Response of the innate immune system to influenza infection…………...….33

Figure 17. Components of the inflammasome…………………………………………...35

Figure 18. Functions of complement………………………………………………….....37

xxii Figure 19. T cell receptor (TCR) is composed of an αβ heterodimer which interacts with CD3 molecules………………………………………………………….38

Figure 20. B cell receptor (BCR) is a membrane bound immunoglobulin M which interacts with Igβ and Igα……………………………………………………39

Figure 21. Adaptive immune response to a viral infection……………………………....44

Figure 22. Action of cytokines produced by Th17 cells include granulocyte recruitment and may contribute to chronic inflammation……………….…....45

Figure 23. Unilamellar liposome representation of the different ways that molecules can be associated with the liposomes……………………………..55

Figure 24. M2e protein linked to a unilamellar liposome via a CMI linkage…………..57

Figure 25. Monophophory lipid A is devired from the cell wall of gram-nagative Bacteria……………………………………………………………….………62

Figure 26. Comparison of Tucaresol and lipidated Tucaresol……………………...……64

Figure 27. The lipopeptide containing the above mentioned side chains (R1, R2, R3, X) is called Pam3CAG……………………………………………………..……65

Figure 28. Structure of 1V270…………………………………………………………...65

Figure 29. MDCK cell morphology……………………………………………………..69

Figure 30. Cell counting under hemocytometer……………………………………..…..71

Figure 31. Timeline for Swiss Webster female mice challenged with H1N1 influenza virus………………………………………………………………..78

Figure 32. Timeline for Swiss Webster female mice challenged with H3N2 influenza virus…………………………………………………………....78-79

Figure 33. Timeline for Swiss Webster female mice challenged with H1N1 influenza virus………………………………………………………………..80

Figure 34. Timeline for Swiss Webster female mice challenged with H1N1 influenza virus………………………………………………………………..81

Figure 35. Timeline for Swiss Webster female mice challenged with H1N1 influenza virus……………………………………………….…………….…82

Figure 36. Timeline for Swiss Webster female mice challenged with H1N1

xxiii influenza virus…………………………………………………………....83-84

Figure 37. Timeline for Swiss Webster female mice challenged with H3N2 influenza virus………………………………………………………………..84

Figure 38. Example of a typical cytolysis plate……………………………………..…...94

Figure 39. Example of a Foci assay plate set up…………………………………………97

Figure 40. Example of a Foci assay as seen through an inverted microscope………...…98

Figure 41. Example of an ELISpot plate…………………………………………...…..111

Study I: Investigation of a CMI liposomal M2e vaccine containing the adjuvant, mycoviral dsRNA or MPL, against H1N1 influenza A challenge……………...... 114-140

Figure 42. Survival in H1N1 challenged Swiss Webster female mice…………………116

Figure 43. Weight change and disease signs in H1N1 challenged Swiss Webster female mice…………………………………………………………………117

Figure 44. Lung viral burden of Swiss Webster female mice challenged with H1N1…119

Figure 45. Anti-M2e IgG1 and IgG2a production in Swiss Webster female mice vaccinated with CMI liposomal vaccine formulations……………………...121

Figure 46. ELISpot assay to determine the number of IFN-γ and IL-4 secreting splenocytes………………………………………………………………..…123

Figure 47. Survival in H3N2 challenged Swiss Webster female mice…………………125

Figure 48. Weight change and disease signs in H3N2 challenged Swiss Webster female mice…………………………………………………………………126

Figure 49. Lung viral burden of Swiss Webster female mice challenged with H3N2....128

Figure 50. Anti-M2e IgG1 and anti-M2e IgG2a production in mice vaccinated with CMI liposomal vaccines…………………………………………………….130

Figure 51. Anti-whole virus M2e IgG production in Swiss Webster female mice vaccinated with CMI liposomal vaccines……………………………….….132

Figure 52. ELISpot assay to determine the number of IFN-γ and IL-4 secreting splenocytes…………………………………………………………………..134

xxiv Figure 53. Multiplex Luminex cytokine assay to determine concentrations of cytokines secreted by splenocytes stimulated with M2e……………...……137

Study II: Investigation of the effects of different purification methods to obtain mycoviral dsRNA for use in the CMI liposomal M2e vaccine……...140-156

Figure 54. Survival in H1N1 challenged Swiss Webster female mice………………....143

Figure 55. Weight and disease scores in H1N1 challenged Swiss Webster female Mice………………………………………………………………….……..145

Figure 56. Anti-M2e IgG1, anti-M2e IgG2a, and anti-M2e IgG3 production in mice vaccinated with CMI liposomal M2e vaccines and mycoviral dsRNA (UC or C)……………………………………………………………………149

Figure 57. Anti-whole virus M2e IgG production in Swiss Webster female mice vaccinated with CMI liposomal vaccines…………………………………..152

Figure 58. ELISpot assay to determine the number of IFN-γ and IL-4 secreting splenocytes……………………………………………………………..154-155

Study III: Comparative efficacy of CMI liposomes containing M2e (1-12) vs M2e (1-15) and MPL, and testing other adjuvant molecules in Swiss Webster female mice………………………………………….156- 175

Figure 59. Survival in H1N1 challenged Swiss Webster female mice………………....159

Figure 60. Weight change and disease scores in H1N1 challenged Swiss Webster female mice………………………………………………………………....161

Figure 61. Anti-M2e IgG1:IgG2a ratio of L-CMI-M2e (1-12) vs L-CMI-M2e (1-15) Vaccines………………………………………………...……….164-166

Figure 62. Anti-M2e IgG3 Production in Mice Vaccinated with CMI Liposomal Vaccines…………………………………………………………………….168

Figure 63. Cytokine production by splenocytes from vaccinated female mice prior to H1N1 challenge……………………………...……………..…169-171

Study IV: Investigation of CMI liposomal vaccine formulations containing adjuvants without the antigenic protein M2e (1-15)………………..175-194

Figure 64. Survival in H1N1 challenged Swiss Webster female mice…………………177

Figure 65. Survival in H1N1 challenged Swiss Webster female mice……………178-179

xxv Figure 66. Survival in H1N1 or H3N2 challenged Swiss Webster female mice….182-183

Figure 67. Weight change in H1N1 or H3N2 challenged Swiss Webster female mice…………………………………………………………………………184

Figure 68. Disease signs in H1N1 or H3N2 challenged Swiss Webster female mice…………………………………………………………………………185

Figure 69. Lung viral burden of Swiss Webster female mice challenged with H1N1 or H3N2………………………………………………………………………..188

Figure 70. Cytokines present in spleens of female mice vaccinated prior to H1N1 challenge……………...... 190-191

xxvi CHAPTER 1

Introduction

I. Influenza Infection: Transmission and Disease Symptoms

The influenza virus is a respiratory pathogen transmitted by respiratory droplets

and contact with contaminated surfaces that infects the nose, throat, and lungs (Deng,

Cho, Fiers, & Saelens, 2015; Center for Disease Control [CDC], 2013). Infection begins when flu particles gain access to the host’s epithelial cells in the upper or lower respiratory tracts (CDC, 2015; Medina & Garcia-Sastre, 2011). Symptoms of influenza infection will manifest 1-4 days following exposure and viral shedding can begin before the onset of symptoms and continue for 10 days (Fiore et al., 2011). Symptoms can include fever, myalgia, cough, headache, nasal congestion, rhinorrhea, sore throat and dyspnea (“Writing Committee”, 2010). For the majority of people who become sick, the infection will resolve itself in 3 to 7 days after the first symptoms appear (Fiore et al.,

2011). However, complications such as viral pneumonia and secondary bacterial

pneumonia can occur (Fiore et al., 2011). Generally, influenza poses a greater threat to the elderly (> 65 years old), children (<2 years old), pregnant women and those with

chronic health conditions such as asthma and cardiovascular disease (Fiore et al., 2011;

Grohskopg et al., 2016). This is likely due to their compromised immune responses.

However, during the 1918 H1N1 pandemic and the 2009 H1N1 pandemic, the majority

of deaths occurred in those under 65 years of age (“Writing Committee”, 2010; Potter,

2001). Between 32% to 45% of those hospitalized in 2009 were under the age of 18

(“Writing Committee”, 2010). In these cases, it is generally thought that the more severe

1 illness in the younger population was a result of these individuals having an excessive

immune response to the influenza infection.

Influenza virus is a zoonotic virus originating from bats and aquatic birds that

infects humans, swine, dogs, horses, seals and domestic poultry as well as other

mammals (Webster & Govorkova, 2014; Parrish, Murcia, & Holmes, 2015). Because

influenza viral polymerase lacks a proofreading mechanism, they accumulate mutations

that allow them to adapt and replicate in different species resulting in a wide spectrum of

hosts (Figure 1).

Figure 1. Known emergence events of influenza viruses in mammals. Influenza viruses are assumed to have emerged from avian reservoirs and then infected other species including humans, swine, horses, and dogs. Red lines indicate transmission that lead to epidemics in the respective host. Reprinted from “Influenza Virus Reservoirs and Intermediate Hosts: Dogs, Horses, and New Possibilities for Influenza Virus Exposure of

2 Humans,” by C. R. Parrish, P. R. Murcia, and E. C. Holmes, 2015, Journal of Virology, 89, 2991. Copyright by “American Society of Microbiology.”

These mutations can also allow the virus to escape the immune response of their host (antigenic drift) leading to epidemics and occasionally pandemics (Webster &

Govorkova, 2014). Pandemics are usually caused by avian and human-adapted influenza viruses infecting the same cells of an intermediate host, like the pig, resulting in reassortment of viral genes to produce a new virus strain (antigenic shift).

According to the World Health Organization (WHO), influenza epidemics begin as early as September and run through early February in the Northern Hemisphere of

Africa, America, Asia, Europe and Oceania (Figure 2). The influenza activity peaks during late November to early December in most of these regions (WHO, 2016). In the

Southern Hemisphere, the influenza epidemics begin in March and extend through

September with the peak periods varying by continent (Figure 3) (WHO, 2016).

Worldwide, seasonal influenza is estimated to cause 3 to 5 million cases of severe illness and between 250,000 to 500,000 deaths (WHO, 2014). In the United States, between the years of 1976 to 2007, estimated deaths ranged from 3,349 to 48,614 cases (Molinari et al., 2007). Additionally, a study in the United States, which considered just the influenza infections in 2003, determined that seasonal influenza resulted in a total economic cost of

$87.1 billion (Molinari et al., 2007).

Figure 2. Subtype of influenza transmission during peak influenza season for the Northern Hemisphere. Reprinted from “Recommended composition of influenza virus vaccines for use in the 2016-2017 northern hemisphere influenza season,” by WHO, 2016. Copyright 2016 by “WHO.”

Figure 3. Subtype of influenza transmission during peak influenza season for the Southern Hemisphere. Reprinted from “Recommended composition of influenza virus

II. Orthomyxoviridae Viruses

The orthomyxoviridae family of viruses is composed of influenza A virus, influenza B virus, influenza C virus, isavirus, quaranjavirus and thogotovirus

(International Committee on Taxonomy of Viruses [ICTV], 2015). The genomes of

Orthomyxoviridae range from six to eight segments of negative-sense, single-stranded

RNA (Presti et al., 2009; Briese et al., 2014). These viruses replicate in the nucleus of the host by utilizing the viral polymerase proteins to synthesize viral RNA, positive-sense complementary RNA and mRNA (Briese et al., 2014). Due to the nature of their segmented genomes, these viruses are capable of undergoing reassortment creating new viral strains that can lead to epidemics or pandemics (Presti et al., 2009). Within the family of Orthomyxoviridae, influenza viruses are responsible for thousands to millions of human deaths annually (Presti et al., 2009).

A. Types of Influenza Viruses

Influenza A and B viruses are responsible for the annual influenza epidemics

(Hay, Gregory, Douglas, & Yin, 2001). It is estimated that 75% of confirmed influenza cases are caused by influenza A virus, while 25% of influenza cases are caused by influenza B virus (Nyirenda, Omori, Tessmer, Arimura, & Ito, 2016). Due to its greater genetic diversity, influenza A virus has a greater impact in terms of annual epidemics and pandemics (Hay et al., 2001). Influenza C virus causes limited numbers of infections, but it has been found that the majority of people will develop antibodies against this virus early in life (Wang & Veit, 2016).

5 1. Influenza A

Influenza A virus has eight negative-sense RNA genome segments that encode for

17 proteins. It also has a wide range of hosts which include avian species, humans and other mammals (Webster & Govorkova, 2014; Parish et al., 2015). Transcription of the viral genome segments is carried out by the RNA-dependent RNA polymerase complex:

PB1, PB2, PA (Schnitzler & Schnitzler, 2009; Medina & Garcia-Sastre, 2011). Segment

1 encodes basic polymerase 2 (PB2) and Segment 2 encodes basic polymerase 1 (PB1),

PB1- F2 and N40 protein (Ebrahimi & Tebianian, 2011; Wise et al., 2009). Segment 3 encodes acidic protein (PA), PA-X, PA-N155 and PA-N182 (Ebrahimi & Tebianian,

2011; van de Sandt, Bodewes, Rimmelzwaan, & de Vries, 2015). Segment 4 encodes hemagglutinin (HA) divided into two domains, HA1 and HA2 (Ebrahimi & Tebianian,

2011). Segments 5 and 6 encode nucleoprotein (NP) and neuroaminidase (NA) proteins, respectively (Medina & Garcia-Sastre, 2011; Ebrahimi & Tebianian, 2011). Segment 7 encodes the viral matrix proteins 1 (M1), ion channel matrix protein 2 (M2) and M42

(Medina & Garcia-Sastre, 2011; van de Sandt, 2016). Segment 8 encodes the viral nuclear export protein (NEP/NS2), as well as the host antiviral response non-structural protein (NS1) and NS3 (Medina & Garcia-Sastre, 2011; van de Sandt; 2015).

Each viral RNA segment forms a ribonucleoprotein complex composed of viral

RNA associated with the viral nucleoprotein and the viral polymerase protein (Medina &

Garcia-Sastre, 2011; Ebrahimi & Tebianian, 2011). Influenza A virus can be further classified based on its surface glycoproteins, hemagglutinin and neuraminidase (Deng et al., 2015). Currently, 18 HA subtypes and 11 NA subtypes have been identified (Deng et

6 al., 2015). Although many influenza A virus subtypes exist, only the H1N1, H3N2 and

H2N2 have caused widespread epidemics in humans (Cox & Subbarao, 2000).

Table 1.

Comparison Between Influenza A and Influenza B Proteins

Note. Reprinted from “Influenza B viruses: not to be discontinued,” by C. E. van de Sandt, R. Bodewes, G. F. Rimmelzwaan, and R. D. de Vries, 2015, Future Microbiology, 10, 1448. Copyright 2015 by “Future Medicine Ltd.”

2. Influenza B

Influenza virus B has eight negative-sense RNA genome segments that encode for

11 proteins. Segments 1, 2 and 3 encode proteins PB2, PB1 and PA, respectively, which

collectively function as the RNA polymerase complex (Lindstrom et al., 1991; van de

Sandt et al., 2015). Segments 4 and 5 encode the HA and NP proteins, respectively (van

7 de Sandt et al., 2015). Segment 6 encodes the NA protein as well as the NB protein

(Lindstrom et al., 1991; van de Sandt, 2015). Segment 7 codes for the matrix protein M1 and BM2 which is an ion channel protein critical for viral uncoating upon entry into the host cell (Lindstrom et al., 1991; van de Sandt, 2015). Segment 8 encodes the proteins

NS1 and NS2/NEP (van de Sandt, 2015). Based on the genetic differences of the HA protein, influenza virus B can be classified into the Yamata lineage and the Victoria lineage which co-circulate with seasonal influenza A H3N2 and H1N1 (Nyirenda et al.,

2016; Lindstrom et al., 1991). Influenza virus B has been isolated from dogs, harbor seals and grey seals (van de Sandt et al., 2015). This indicates that influenza virus B can infect different species but unlike influenza A, there is no evidence of an animal reservoir that could cause human infections (van de Sandt et al., 2015).

3. Influenza C

Influenza virus C has seven negative-sense, single-stranded RNA genome segments (Wang & Veit, 2016). Genomic segments 1, 2 and 3 encode for PB2, PB1 and

P3 which function as the RNA polymerase complex (Wang & Veit, 2016). Segment 4 encodes the membrane glycoprotein Hemagglutinin-Esterase-Fusion (HEF) which functions in a similar manner as the HA and NA of influenza A virus (Wang & Veit,

2016). Segment 5 encodes the NP protein and Segment 6 encodes the matrix protein

(M1) and CM2 (Wang & Veit, 2016). Segment 7 encodes non-structural proteins NS1 and NS2 (Wang & Veit, 2016). Influenza virus C mainly infects humans, however, pigs and dogs can also be infected (Wang & Veit, 2016). A new virus isolated from cattle showed significant amino acid sequence differences when compared to human Influenza virus C (Wang & Veit, 2016). Additionally, this new virus did not reassort or cross-react

8 with antisera from human influenza virus C (Wang & Veit, 2016). Because of these differences, it has been suggested that this virus should be classified as a new genus and given the name influenza virus D (Wang & Veit, 2016).

Figure 4. Comparison between influenza C and influenza A/B proteins. Reprinted from “Hemagglutinin-esterase-fusion (HEF) protein of influenza C virus,” by M. Wang and M. Veit, 2016, Protein Cell, 7, 30. Copyright 2015 by “M. Wang and M. Veit.”

III. Influenza A Outbreaks

A. Overview

The baseline level of a disease that is expected to affect a community is referred to as the endemic level of disease (CDC “Introduction to Epidemiology”, 2012). When there is an increase in the prevalence of a disease above the endemic level for a given geographic area, this is referred to as an epidemic (CDC “Introduction to Epidemiology”,

2012). Epidemics arise for several reasons including increase in the amount of a contagious pathogen, introduction of a pathogen into a naïve population, enhanced transmission of the disease to new hosts and an increase in susceptibility of new hosts

(CDC “Introduction to Epidemiology”, 2012).

In general, pandemics occur when there is widespread infection across several countries. In the case of influenza, pandemics are believed to arise when influenza strains

9 from different hosts (e.g. pigs, humans or birds) infect the same host cell, allowing the

HA and NA proteins from the different hosts to reassort and form a new more virulent strain of influenza. This virulent strain has the ability to readily infect human hosts which then get a severe infection (Schnitzler & Schnitzler, 2009). The Pandemic Severity Index

(PSI) was introduced as a way to predict the severity of future influenza pandemics

(Holloway, Rasmussen, Zaza, Cox, & Jernigan, 2014). The PSI is based on categories

(categories 1-5) of increasing severity for influenza and it is intended to guide prepandemic planning efforts in case of an emerging pandemic (Holloway, 2014).

B. Influenza Pandemic of 1918

The H1N1 Influenza Pandemic of 1918-1920 also known as the Spanish Flu was one of the most devastating human infections (Potter, 2001). Although the origin of this avian-like influenza is unknown, evidence suggests that the infection could have started in the United States and spread to the rest of the world as military personnel traveled to

Europe (Schnitzler & Schnitzler, 2009; Potter, 2001). After its arrival in France, the infection spread to Italy, Spain, Britain, Germany and eventually reached Russia (Potter,

2001). From Russia, the infection spread to North Africa, China, New Zealand and the

Philippines (Potter, 2001). The infection reached Australia in January 1919 (Potter,

2001). It was estimated that 33% to 50% of the world’s population of 1.5 billion people were infected, with a total mortality between 40-50 million people (Potter, 2001; WHO

“2017 Southern Hemisphere influenza season,” 2016). Additionally, several countries experienced second and third waves of infection with a more virulent form of the virus

(Potter, 2001). One of the most unique aspects of this infection was that the majority of deaths occurred in individuals between 20-40 years of age (Potter, 2001). This was

10 unexpected since previously most of the deaths due to influenza infection occurred in very young or older individuals. It has been hypothesized with some evidence that those over >45 yeas of age could have been protected by a circulating virus that was genetically similar to the 1918 influenza virus (Morens & Fauci, 2007). The pronounced immune response to the influenza infection of people between the ages of 20-40 was excessive and contributed to the deaths in this population (Morens & Fauci, 2007).

Figure 5. Mortality reported in Breslau, Silesia (now Wroclaw, Poland) from June 1918 through December 1922 which demonstrates the increased mortality for those 20-40 years of age. Reprinted from “The 1918 Influenza Pandemic: Insights for the 21 st century,” by D. M. Morens and A. S. Fauci, 2007, Journal of Infectious Diseases, 195, 1021.

C. Influenza Pandemic of 1957

This H2N2 influenza pandemic originated in the Yuman Province of China

(Potter, 2001). From China, it reached Hong Kong and then spread to Singapore, Taiwan,

11 Japan, United States and United Kingdom (Schnitzler & Schnitzler, 2009; Potter, 2001).

It is believed that most of the infections were transmitted through sea routes across the world (Potter, 2001). Within 6 months from its outbreak, it is believed that the pandemic had reached the rest of the world (Potter, 2001). In 1958, a second wave of infection was observed in Europe, North America, Russia and Japan (Potter, 2001). This pandemic was caused by reassortment of avian H2 HA and N2 NA with the human influenza virus that then infected the human population (Schnitzler & Schnitzler, 2009). The total mortality was estimated to have exceeded 1 million people with the majority of deaths occurring in the very young and very old (Potter, 2001). H2N2 virus continues to circulate throughout the world in aquatic birds. Because much of the world’s population is immunologically naïve to this virus, H2N2 could potentially cause another pandemic (Webster &

Govorkova, 2014).

D. Influenza Pandemic of 1968

In 1968, the H3N2 virus was isolated in Hong Kong (Webster & Govorkova,

2014; Schnitzler & Schnitzler, 2009). Within a few weeks, the virus had spread to

Vietnam and Singapore (Tognotti, 2009). Eventually, the virus reached the Philippines,

India, Australia, the United Kingdom, Europe and the United States (Tognotti, 2009). By

1969, the infection had reached South Africa and South America (Tognotti, 2009).

Molecular analysis of the virus causing this pandemic revealed that reassortment of avian

HA and PB1 replaced the circulating H2N2 with a new virus which was referred to as the pandemic H3N2 influenza virus (Taubenberger & Morens, 2010). The other gene segments originating from the 1957 H2N2 subtype were retained by the H3N2 virus

12 (Taubenberger & Morens, 2010). The 1968 pandemic had a low mortality rate, in some areas, it was lower than during non-pandemic years (Taubenberger & Morens, 2010).

E. Influenza Pandemic of 2009

In 2009, a virus that was genetically and antigenically different from seasonal

H1N1 influenza was detected in California and was later discovered to be the cause of major respiratory disease outbreaks in Mexico (Schnitzler & Schnitzler, 2009). Further research demonstrated that this new strain of H1N1 was the result of triple reassortment between genome segments of North American H3N2 and H1N1 swine viruses and

Eurasian avian-like swine viruses which then crossed the species barrier from pigs to humans (Webster & Govorkova, 2014; Schnitzler & Schnitzler, 2009; Clark & Lynch,

2011). Unlike seasonal influenza, during the 2009 pandemic, less than 10% of reported infections were in those older than 60 years of age (Clark & Lynch, 2011), and 90% of deaths occurred in those younger than 65 years of age (Clark & Lynch, 2011). This infection was severe in individuals not previously exposed to this strain, pregnant women and obese people, causing 100,000 deaths worldwide (Webster & Govorkova, 2014).

However, most illnesses caused by the virus was mild with an overall mortality rate of

>0.5% (Clark & Lynch, 2011).

F. Continuous Threat: H5N1 and H7N9 Influenza Viruses

In 1997, 18 people in Hong Kong were infected with H5N1 avian influenza and this resulted in six fatalities or 1/3 of the infected people (Schnitzler & Schnitzler, 2009).

Although the outbreaks were controlled, there was a new appearance of this same virus in

2002 (Schnitzler & Schnitzler, 2009). As of 2014, there have been 650 reported infections resulting in 386 deaths or almost 60% of the infected people (Webster &

13 Govorkova, 2014). Currently, this virus has spread to Europe and Africa (Schnitzler &

Schnitzler, 2009). So far, this infection has been caused by direct contact with infected avian species (Medina & Garcia-Sastre, 2011).

H5N1, which is referred to as a Highly Pathogenic Avian Influenza virus (HPAI), has poor human-to-human transmissibility and thus far, has not been able to cause a pandemic (Schnitzler & Schnitzler, 2009). It is believed that this highly pathogenic avian influenza arose from mutations in the HA cleavage sites (Medina & Garcia-Sastre, 2011).

The danger posed by this virus is that it could gain the ability for effective human-to- human transmission by the respiratory route which could lead to a high mortality pandemic (Schnitzler & Schnitzler, 2009).

In the past two decades, there has been an emergence of the H7 and H9 virus subtypes that have caused infection in Europe, Asia and the Americas (Medina & Garcia-

Sastre, 2011). In 2013, an H7N9 virus emerged with the ability to sporadically infect humans (Webster & Govorkova, 2014). As of 2014, there have been 259 reported infections which resulted in 71 deaths or 25% of infected people (Webster & Govorkova,

2014).

IV. Antigenic Drift and Antigenic Shift in Influenza

Hemagglutinin (HA) which is responsible for entry of the virus into the host cells and Neuraminidase (NA) which is responsible for the spread of the virus from cell to cell are critical surface viral glycoproteins (Treanor, 2004). The accumulation of point mutations in the antigenic region of these surface glycoproteins or reassortment of these proteins between human and avian influenza viruses can provide the virus with an

14 advantage that might allow it to escape preexisting immunity (Taubenberger & Morens,

2010).

Figure 6. Location of five antibody-binding sites against hemagglutinin and point mutations in this antibody-binding sites that can allow the influenza virus to escape host immunity. Reprinted from “Influenza Vaccine—Outmaneuvering Antigenic Shift and Drift,” by J. Treanor, 2004, The New England Journal of Medicine, 350, 219. Copyright 2005 by “Massachusetts Medical Society.”

Antigenic drift can occur in influenza A and influenza B viruses (Treanor, 2004).

Antigenic drift is caused by amino acid substitutions, usually in five antigenic sites on the globular head of HA, that allow the new virus to escape host immunity and replace the circulating strain, potentially leading to an epidemic (Figure 6) (Webster & Govorkova,

2014). Usually, when a host has been infected with influenza, antibodies to HA or NA will be produced to neutralize the virus (Treanor, 2004). However, the accumulation of

15 mutations on the HA and/or NA antibody-binding sites will prevent antibodies from neutralizing the virus (Treanor, 2004).

Antigenic shift is only seen in influenza A viruses because influenza B viruses do not have an animal reservoir (Treanor, 2004). Antigenic shift is caused by reassortment of avian influenza virus with circulating human influenza virus in a given infected cell.

This will lead to the introduction of novel gene segments, potentially creating a new virus strain (Webster & Govorkova, 2014). Usually the source of the influenza hemagglutinin or neuraminidase is waterfowl (Treanor, 2004).

Because of the constant mutations and possibility of reassortment of the HA and

NA proteins between human and avian influenza viruses, developing a vaccine that focuses on a more conserved influenza protein could provide a universal vaccine that would be unaffected by the constant evolution of the surface glycoproteins. Several researchers have suggested that Matrix Protein 2 Ectodomain (M2e) is an ideal candidate for a universal vaccine because it is highly conserved across many influenza strains.

V. Influenza Viral Proteins

Influenza A and B viruses both contain the surface glycoproteins hemagglutinin

(HA) and neuraminidase (NA) (Air & Laver, 1989). Hemagglutinin is a homotrimeric protein that is synthesized from a precursor, HA0 (Kang, Kim, & Compans, 2012; Skehel

& Wiley, 2000). HA0 is cleaved and its C terminus region becomes the HA1 subunit and the N terminus region becomes the HA2 subunit (Skehel & Wiley, 2000). The cleavage of HA0 that produces H1, H2 or H3 subtypes is believed to be mediated by a serine protease synthesized by Clara cells of the bronchiolar epithelium (Skehel & Wiley,

2000). Cleavage of the HA0 is required for membrane fusion and virus infectivity of the

16 host cell (Skehel & Wiley, 2000). The HA1 subunit contains the globular head domain

which is a major target of neutralizing antibodies (Kang et al., 2012). The HA2 subunit

contains the fusion peptide, long alpha-helix domain and the transmembrane cytoplasmic

tail (Kang et al., 2012).

HA1 and HA2 are anchored in the viral envelope by a series of hydrophobic amino acids near their C-terminus (Air & Laver, 1989). HA is responsible for viral entry into the host cell by binding to cell receptors that contain the alpha-2-6-linked or alpha-2-

3-linked sialic acid receptors (Medina & Garcia-Sastre, 2011). Additionally, once the virus is inside endosomes, which have a pH between 5 and 6, the conformation of HA is changed which leads to exposure of a fusion peptide that initiates fusion of the viral envelope and the host endosomal membrane (Skehel & Wiley, 2000).

Figure 7. Hemagglutinin protein and conformational changes that lead to exposure of the fusion peptide and membrane fusion. Reprinted from “Structural insights into the coupling of virion assembly and rotavirus replication,” by S. D. Trask, S. M. McDonald and J. T. Patton, 2012, Nature Reviews Microbiology, 10, 21. Copyright 2012 by “Rights Managed by Nature Publishing Group.”

Neuraminidase accounts for 5 to 10% of the proteins that are on the surface of the

virus (Air & Laver, 1989). NA is composed of a single polypeptide chain that creates a

tetramer of four spherical subunits and a hydrophobic stalk by the N-terminus which

embeds the proteins into the viral envelope (Air & Laver, 1989). The role of NA is

17 cleavage of the alpha-ketosidic linkage between terminal host sialic acid and adjacent

sugar residues destroying the hemagglutinin receptor on host cells (Air & Laver, 1989).

This function is essential for proper viral budding from the host cell and to prevent self-

aggregation of the virus (Medina & Garcia-Sastre, 2011; Air & Laver, 1989).

Figure 8. Cartoon representation of the neuraminidase protein of the influenza virus. The catalytic activity will cleave the sialic acid receptors to release the virus from the host cells. Protease cleavage site will release the NA heads which retain their antigenic activity. Reprinted from “The Neuraminidase of Influenza Virus,” by G. M. Air and W. G. Laver, 1989, Proteins: Structure, Function, and Genetics, 6, 343. Copyright 1989 by “ALAN R. LISS, INC.”

The third glycoprotein present on the surface of the influenza A virus is the matrix

protein 2, M2. M2 protein is composed of 96 amino acids which form the amino-terminal

extracellular domain, a transmembrane domain, and a cytoplasmic domain (Ebrahimi &

Tebianian, 2011). It is estimated that only 23 to 60 M2 molecules are incorporated into

the viral envelope (Ebrahimi & Tebianian, 2011). In an extracellular domain study, it was demonstrated that 17 out of 24 amino acids are conserved at a rate of over 94% in 716 influenza viruses from isolated human, avian, equine and other hosts (Liu et al., 2005).

18 Additionally, the N-terminal epitope (residues 2-9) was conserved at a rate of 100% from influenza A virus subtypes isolated from humans (Ebrahimi & Tebianian, 2011).

M2 functions as an ion channel that allows virus uncoating and virus maturation

(Ebrahimi & Tebianian, 2011). After receptor-mediated endocytosis, M2 reduces the pH

of the virion interior by transporting protons from the host endosome across the viral

envelope (Ebrahimi & Tebianian, 2011; Schotsaert et al., 2009). The reduction in pH

allows for the dissociation between matrix protein (M1) and the ribonucleoprotein

(Ebrahimi & Tebianian, 2011; Schotsaert et al., 2009). In some influenza subtypes (H5

and H7), M2 increases the pH of the trans-Golgi network of the host cell preventing the

pre-mature activation of newly synthesized HA (Schotsaert et al., 2009).

Figure 9. Structure of M2 tetrameric protein (residues 22-62) at pH 7.5. Reprinted from “Viroporins, Examples of the Two-Stage Membrane Protein Folding Model,” by L. Martinez-Gil and I. Mingarro, 2015, Viruses, 7, 3474.

19 Influenza C virus contains only one glycoprotein. Hemagglutinin-Esterase-Fusion

(HEF) recognizes surface receptors to allow viral entry, catalyzes the fusion of the viral

envelope with the host cell membranes, and is the receptor-destroying enzyme (Figure 4)

(van de Sandt et al., 2015). However, unlike influenza A and B viruses A, HEF binds to

and cleaves N-acetyl-9-O-acetylneuraminic acid which is another derivative of the neuraminic acid (van de Sandt et al, 2015).

Influenza A virus nucleoprotein (NP) binds to the vRNA to create viral

ribonucleoproteins (RNPs) and helps transit the virus from the cytoplasm to the nucleus

of the host cell (Marklund, Ye, Dongm Tao & Krung, 2012; Cianci, Gerritz, Deminie, &

Krystal, 2013). NP is composed of a head domain and a body domain (Marklund et al.,

2012). The two domains are separated by a groove of basic amino acids which bind

single-stranded RNA (Marklund et al., 2012). There is approximately one NP for 24

bases of RNA (Cianci et al., 2013). Extending from the head domain is a tail loop that

allows the NP to form oligomers by looping into nearby NP molecules (Marklund et al.,

2012). H1N1 and H5N1 NPs crystallize as trimers (Cianci et al., 2013). However,

cryogenic electron microscopic data shows that NP associates as monomers in RNPs

(Cianci et al., 2013).

Matrix 1 Protein (M1) provides structural support for the viral envelope and it is

one of the most highly conserved proteins of influenza A virus (Rossman & Lamb, 2011;

Mosier, Chiang, Lin, & Gao, 2016). It is composed of 252 amino acids that create three

domains of alpha-helices separated by linker sequences (Rossman & Lamb, 2011). M1 forms a three-dimensional helical layer underneath the viral envelope, with regularly spaced holes that function to connect the envelope proteins HA and NA with the viral

20 ribonucleoprotein core (Rossman & Lamb, 2011; Mosier et al., 2016). Under neutral pH,

M1 monomers will self-associate to maintain viral capsid integrity (Mosier et al., 2016).

However, in the acidic pH caused by proton entry into the viral core by M2, M1 will

dissociate to allow release of the viral ribonucleoprotein (Mosier et al., 2016). M1 is an

essential protein with diverse roles that include viral assembly and budding, virion

uncoating and nuclear export of the viral ribonucleoprotein into the cytoplasm (Mosier et

al., 2016).

Nuclear Export Protein (NEP) is composed of 121 amino acids and is responsible

for mediating the transport of the viral ribonucleoprotein from the nucleus to the

cytoplasm of the host cell (Lara-Sampablo et al., 2014). Recently, it has been recognized that NEP participates in viral transcription and translation via interactions with the viral polymerase complex as well as regulating the accumulation of viral mRNA, cRNA

(complementary), and vRNA which has been implicated in the adaptation of some avian

H5N1 viruses to replicate in mammalian cells (Lara-Sanpablo et al., 2014; Peterson &

Fodor, 2012).

VI. Viral Nomenclature

Eighteen hemagglutanin (HA) and eleven neuraminidase (NA) surface

glycoproteins have been identified (Webster & Govorkova, 2014). Sixteen HA and nine

NA circulate in aquatic birds and the other two HA and NA proteins are found in bats

(Webster & Govorkova, 2014). HA subtypes are divided into two groups. Group 1

includes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18 (Webster &

Govorkova, 2014). Group 2 includes H3, H4, H7, H10, H14, and H15 (Webster &

Govorkova, 2014). Influenza virus strains nomenclature is exemplified as follows: Virus

21 type (A), geographic origin (Fujian), strain number (411), year of isolation (2002) virus subtype (H3N2) = A/Fujian/411/2002 (H3N2) (“A Revision of the system,” WHO,

1980).

VII. Influenza Replication Cycle

A. Viral Entry

Viral entry into the host cell begins when the HA, on the surface of the virus, binds to the host cell sialic acid receptors that are bound to galactose in an α(2, 3) or α(2,

6) conformation (Samji, 2009). This conformation determines host specificity of the HA.

HA molecules that bind to human cells recognize primarily the α(2, 6) conformation while those that bind to avian cells recognize the α(2, 3) conformation (Samji, 2009).

After HA binds to the host cell, receptor-mediated endocytosis allows the virus to enter the cell (Samji, 2009). The low pH inside the endosome triggers a conformational change in the HA protein that causes the initiation of the fusion of the viral envelope with the endosomal membranes (Samji, 2009). Additionally, the acidic environment of the endosomes causes a conformational change which opens the proton-selective ion channel of the M2 protein (Samji, 2009). This allows the acidification of the viral core which releases the vRNP from M1 allowing the vRNP to enter the cytoplasm of the host cell

(Schotsaert et al., 2009).

Figure 10. Influenza virus entry into the host cell. The HA protein will bind the receptors containing an α-2,3-SA or α-2,6-SA. Eventually the viral genomic segments will enter the nucleus of the host cell where viral replication and transcription will take place. Virus will bud from the host cell by utilizing the NA protein to cleave the sialic acid receptors. Reprinted from “Influenza A viruses: new research developments,” by R. A. Medina and A. Garcia-Sastre, 2011, Nature Reviews Microbiology, 9, 591. Copyright 2011 by “Macmillan Publishers Limited.”

B. Intracellular Replication

Next, nuclear localization sequences on vRNP, NP, PA, PB1 and PB2 allow these proteins to bind to the host’s cellular nuclear import mechanism and enter the nucleus

(Samji, 2009). Once in the nucleus, the negative-sense single stranded RNA genome must be converted into positive-sense RNA to serve as the template for the production of viral RNA (Samji, 2009). This process is performed without the need for a primer via the viral RNA dependent RNA polymerase (Samji, 2009). To begin transcription of viral mRNA, the viral RNA dependent RNA polymerase cleaves the 5’ methylated caps of cellular mRNA and uses these nucleotides as primers for viral transcription (Samji,

2009). Proteins such as M2 and NEP which are the product of splicing events are created by using the host’s splicing machinery (Samji, 2009). To mediate transport of vRNP from

23 the nucleus, the cellular nuclear export protein Crm1 interacts with the N-terminal domain of NEP (Peterson & Fodor, 2012). The C-terminus of the NEP binds the nuclear localization signal on the M1 protein which in turn binds with the nucleoprotein of the vRNP (Peterson & Fodor, 2012) (Figure 10).

C. Viral Budding

To form a viral particle, HA, NA, and M2 are transported to the host cell plasma membrane (Samji, 2009). Some hypothesize that viral genomic segments are randomly packaged into the virion, while others believe that there are signal sequences guiding the genomic segments (Samji, 2009). It is hypothesized that HA initiates virus budding by assembling into lipid rafts (Deng, 2015). M2 is then recruited to the boundaries of the lipid raft to start membrane scission and release of virus (Deng, 2015). Lastly, NA cleaves sialic acid receptors allowing the newly formed viruses to infect other cells

(Samji, 2009).

Figure 11. M2 mediated membrane scission leading to viral budding from host cell. After HA and NA concentrate at the budding membrane, M1 will bring viral ribonucleoproteins and M2 into the budding virus. M2 accumulates at the boundary between the budding virion and the host plasma membrane promoting budding of the virus from the cell membrane. Reprinted from “Influenza Exits the Cell Without ESCRT,” by Heinrich G. Göttlinger, 2010, Cell, 142, 840. Copyright 2010 by “Elsevier Inc.”

VIII. Antivirals

In the United States, amantadine, rimantadine, oseltamivir and zanamivir are

antiviral drugs approved for the treatment or prophylaxis of influenza infection (Clark &

Lynch, 2011). These medications provide optimal benefits when they are delivered

within 48 hours after symptoms occur and they can decrease illness and reduce the

chances of developing severe complications (Clark & Lynch, 2011). Treatment is

recommended for patients with confirmed or suspected cases of influenza who are at risk

25 of complications based on age or other medical conditions (Clark & Lynch, 2011).

However, resistance to these drugs has occurred and has prompted the surveillance of

influenza virus strains to identify their drug resistance (Clarn & Lynch, 2011).

A. Amantadine and Rimantadine

Amantadine and rimantadine are antiviral drugs that inhibit influenza A virus

infection by blocking the M2 proton-selective ion channel (Clark & Lynch, 2011) (Figure

12). These two drugs do not prevent infection by influenza B virus (Clark & Lynch,

2011). These drugs block the M2 pore, by binding to an amantadine/ramantadine-binding

site inside the pore around Ser31 or possibly by binding to the lipid-facing pocket around

Asp44 inhibiting M2 function allosterically (Deng et al., 2015) (Figure 14). These drugs are administered at a dose of 100 to 200 mg per day and have been shown to reduce the duration of illness (Clark & Lynch, 2011). However, amandatine is known to cause severe adverse side effects to the central nervous system such as anxiety and seizures

(Clark & Lynch, 2011). Additionally, amantadine and rimantadine cause nausea, vomiting, and dyspepsia in some patients (Clark & Lynch, 2011). During the 2005 influenza season, the CDC determined that 92% of H3N2 influenza viruses isolated from patients were resistant to amantadine and rimantadine and this resistance has continued to increase (Fiore et al., 2011).

Figure 12. Structure of amantadine and rimantadine. These drugs inhibit influenza by blocking the M2 ion channel thus preventing the acidification of the viral core inhibiting the release of the viral ribonucleoprotein into cytoplasm of the host cell. Reprinted from “An update on swine-origin influenza A virus /H1N1 a review,” by S. U. Schintzler and P. Schnitzler, 2009, Virus Genes, 39, 288. Copyright 2009 by “Springer Science+Business Media, LLC.”

B. Oseltamivir and Zanamivir

Oseltamivir and Zanamivir inhibit the enzymatic activity of NA by mimicking the

sialic acid receptor located on the surface of host cells (Clark & Lynch, 2011) (Figure

10). By inhibiting NA, these drugs prevent virus from detaching from the host cell and infecting new cells (Clark & Lynch, 2011) (Figure 13). Additionally, oseltamivir and zanamivir are active against influenza A and B viruses (Clark & Lynch, 2011). Both drugs are approved for treatment and prophylaxis of influenza infection (Clark & Lynch,

2011). For therapeutic treatment of influenza, oseltamivir is delivered orally at a dose of

75 mg twice per day for those over 40kg in weight. The dose can be doubled for severe infection (Clark & Lynch, 2011). Zanamivir is delivered therapeutically via inhalation at a dose of 10 mg twice per day for children and adults for 5 days (Clark & Lynch). As a prophylactic treatment, oseltamivir can be delivered at 75 mg orally once per day for 10 days and zanamivir at 10 mg once per day via inhalation for 10 days (Clark & Lynch,

2011). Resistance to oseltamivir and zanamivir is much more limited compared to

27 amantadine and rimantadine discussed above. Sporatic oseltamivir-resistant 2009 H1N1

virus was identified but had limited transmission (Fiore et al., 2011). Several other

studies report the appearance of oseltamivir-resistant seasonal influenza A and neuroaminidase-inhibitor resistant influenza B viruses (Fiore et al., 2011).

Figure 13. Tamiflu (oseltamivir) and Relenza (zanamivir) inhibit influenza by binding to NA and preventing the virus from budding from the host cell. Reprinted from “An update on swine-origin influenza A virus /H1N1 a review,” by S. U. Schintzler and P. Schnitzler, 2009, Virus Genes, 39, 288. Copyright 2009 by “Springer Science+Business Media, LLC.”

Figure 14. Targets of antiviral drugs against influenza virus infection. Image obtained from [91]. Reprinted from “The war against influenza: discovery and development of sialidase inhibitors,” by M. von Itzstein, 2007, Nature Reviews, 6, 968. Copyright 2007 by “Nature Publishing Group.”

IX. Immunity to Influenza

Although antivirals are approved for the treatment and prophylaxis of influenza and have been shown to lessen symptoms and decrease the chances of complications due to severe illness, the high mutation rate of influenza increases the opportunity for influenza to gain resistance to drugs that target M2 or NA. Current vaccines target the surface glycoproteins HA or NA and these target glycoproteins are constantly mutating.

This means that the protection provided by a vaccine will not be guaranteed for the next influenza season, as the influenza virus could have gained enough mutations to escape the adaptive host immunity created by the vaccine. This issue has brought vaccinologists and

29 immunologists to investigate influenza vaccines that target a more conversed surface protein. Several researchers have focused on the M2e protein because it is highly conserved among different influenza strains and it has been shown to induce the production of anti-M2e antibodies and antibodies that limit viral replication (Lee, Kim,

Lee, Lim, & Kang, 2015; Ernest et al., 2006).

A. Innate Immune System

A review of the immune system is needed to understand the advantages and disadvantages of the immunological responses evoked by different vaccines. The first part of the immune system that is stimulated by a vaccine is the innate immune response.

The innate immune system has anatomical (e.g. skin and mucus membranes), physiological (e.g. temperature and low pH), and cellular responses other than T cells and

B cells, which are effective against inhibiting pathogens that enter the body (Warrington,

Watchon, Kim, & Antonetti, 2011). Most microbes like the influenza virus that enter through the respiratory tract are trapped by the nasal mucosa which is the first line of defense against particles in the air (Fokkens & Scheeren, 2000). Particles trapped in the mucus layer coating the nasal epithelium and respiratory tract are transported by ciliary motion to the pharynx where they are swallowed by the host (McCormack & Whitsett,

2002).. If this first line of defense fails, the immune system will then summon immune cells along with complement activation (Fokkens & Scheeren, 2000).

1. Cellular Innate Immune Response

The cellular component of the innate immune response is composed of dendritic cells, macrophages, and neutrophils (Iwasaki & Medzhitov, 2010). These cells are capable of identifying pathogens by recognizing the Pathogen-Associated Molecular

30 Patterns (PAMPs) on the surface of pathogens by using Pattern Recognition Receptors

(PRRs) (Iwasaki & Medzhitov, 2010). PRRs can be secreted by immune cells, incorporated into their plasma membrane or endosomal membrane or located in their cytosol (Iwasaki & Medzhitov, 2010).

Secreted PRRs include collectins, ficolins and pentraxins which bind to the surfaces of microbes and activate complement pathways and mark pathogens for phagocytosis by other immune cells (Iwasaki & Medzhitov, 2010). Transmembrane

PRRs include the toll-like receptors (TLR), C-type lectins and dectin 1 and dectin 2 as well as other types of receptors (Iwasaki & Medzhitov, 2010). TLRs can recognize

PAMPs that are at the surface of microbes, such as the lipopolysaccharides of gram- negative bacteria, lipoteichoic acid of gram-positive bacteria, fungi and flagellin (Iwasaki

& Medzhitov, 2010; Dunkelberger & Song, 2010). Endosomal TLRs detect microbial nucleic acids such as dsRNA or dsDNA (Iwasaki & Medzhitov, 2010). Dectin-1 and dectin-2 are transmembrane receptors that belong to the C-type lectin family that detect

PAMPs such as beta-glucan on fungal cell walls (Iwasaki & Medzhitov, 2010). Cytosolic

PRRs are expressed in many different nucleated cell types and include retinoic acid inducible gene I (RLR) and the nucleotide-binding domain and leucine-rich repeating receptors (NLRs) (Iwasaki & Medzhitov, 2010). NLRs are composed of a C-terminal leucin-rich repeat, an intermediate nucleotide-binding oligomerization domain and an N- terminal effector binding region (Wu, Metcalf, and Wu, 2011). NLRs help to assemble inflammasomes which lead to the autoactivation of caspase-1 (Pulendran & Maddur,

2015; Wu et al., 2011).

Figure 15. PRRs of innate immune cells include TLRs, NOD and other types of receptors. Reprinted from “ Adjuvants: Classification, Modus Operandi, and Licensing,” by de J. de Souza Apostolico, V. A. Alves Santos Lunardelli, F. C. Coirada, S. B. Boscardin, and D. Santoro Rosa, 2016, Journal of Immunology Research, 2016, 7. Copyright 2016 by “Julia de Souza Apostolico et al.”

In the lungs, influenza virus will first infect respiratory epithelial cells which will produce large amounts of virus that will then infect alveolar macrophages and dendritic cells (DC) (Ichino, Kye Lee, Ogura, Flavell, & Iwasaki, 2009). Epithelial cells will also produce IL-6, TNF-α, IL-8/CXCL, CXCL10, CCL2 and CCL5 in response to influenza infection (Pulendran & Maddur, 2015). After fusion and replication of the influenza virus inside the infected cell, the virus is recognized by the PRR known as RIG-1 (Ichinohe et al., 2009). Activated RIG-1 binds to the mitochondrial antiviral signaling protein which activates genes that lead to the activation of the NF-κβ (Wu et al., 2011). NF-κβ is important for the production of inflammatory cytokines, and activation of IRF3/7 which is important for the antiviral interferon response (Wu et al., 2011). This RIG-1 receptor is

32 used by most cell types including non-immune cells to respond to viral infection (Iwasaki

& Medzhitov, 2010; Ichinohe et al., 2009).

Figure 16. Response of the innate immune system to influenza infection. Influenza is detected by PRRs such as RIG-1 and TLRs. This will lead to the production of pro- inflammatory cytokines and chemokines that activate the antiviral response. Reprinted from “Innate immune response to influenza virus,” by S. Wu, J. P. Metcalf, and W. Wu, 2011, Current Opinion in Infectious Diseases, 24, 237. Copyright 2011 by “Wolters Kluwer Health.”

Once inside the endosome, influenza will release its single-stranded viral RNA and the viral RNA will be recognized by the PRR in plasmacytoid dendritic cells (pDCs),

33 known as TLR7 (Ichinohe, 2009) (Figure 16). Since no viral dsRNA is generated during

influenza virus replication, the role of PRR TLR3 expressed by myeloid dendritic cells

(mDCs), macrophages, and monocyte-derived dendritic cells (moDCs), remains unclear in stimulating an innate immune response (Pulendran & Maddur, 2015). Additionally, the

PRR TLR4 expressed by neutrophils, monocytes, mDCs, moDCs, and macrophages, can recognize the damage-associated molecular patterns (DAMPs) released from virus infected cells and can induce a pro-inflammatory response and cell-death (Pulendran &

Maddur, 2015).

Signaling through these receptors will cause the production of type I interferon

which will limit viral replication (Ichinohe, 2009). The increased production of IL-18 and

IL-1β by macrophages results in the recruitment of circulating monocytes and neutrophils

into the lungs to control the influenza viral replication (Wu et al., 2011). Natural killer

(NK) cells contain natural cytotoxicity receptors (NCR1 in mice and NKp30, Nkp44,

NKp46 in humans) which are involved in the recognition of viral associated molecules

and the activation of NK cells (Pulendran & Maddur, 2015).

2. Inflammasome

Another important aspect of the innate immunity against influenza infection is

the activation of an inflammasome. Inflammasomes are signaling platforms composed of

aggregates of inflammasome sensor molecules that connect caspase-1 to an adapter

protein called ASC (Latz, Xiao, & Stutz, 2013). Upon ASC interaction with the

inflammasome sensor molecules, ASC will bring monomers of pro-caspase 1 in close

proximity to each other, activating caspase-1 self-cleavage which results in activated caspase-1 (Latz et al, 2013). Infection with influenza leads to the activation of caspase-1

34 which is an important regulator of a pro-inflammatory response since it is capable of regulating the activation of pro-IL-1β, pro-IL-18 and pro-IL-33 by cleaving inactive cytokine precursors into their biologically active form (Ichinohe et al., 2009; Wu et al.,

2011). Data indicates that inflammasomes are activated in an NLRP3-dependent and independent fashion and that other factors such as viral RNA are required to elicit inflammasome activation (Ichinohe et al., 2009). Mice lacking NLRP3, ASC or caspase-1 have decreased inflammation in the lungs and this has been associated with increased mortality and decreased viral clearance (Wu et al., 2011).

Figure 17. Components of the inflammasome. ASC contains a pyridine domain and a caspase activation and recruitment domain (CARD) (Latz et al., 2013). Reprinted from “NLRP3 has a sweet tooth,” by D. K. Davis and J. P. Y. Ting, 2010, Nature Immunology, 11. Copyright 1969 by “Rights Managed by Nature Publishing Group.”

35 3. Complement

The complement system is an essential part of the innate immune response.

Complement is made up of serum proteins which can activate inflammatory and cytolytic immune responses to infection (Dunkelberger & Song, 2010). Complement can be activated via the classical, lectin or alternative pathways (Dunkelberger & Song, 2010).

Although these three pathways are activated via different mechanisms, they lead to generation of anaphylatoxins, the membrane attack complex (MAC) and opsonins

(Dunkelberger & Song, 2010).

The anaphylatoxins C4a, C3a, C5a are pro-inflammatory molecules which alert the immune system by binding to receptors found on basophils, eosinophils, neutrophils, monocytes, macrophages, mast cells and some dendritic cells (Dunkelberger & Song,

2010). MAC assembly begins when C5b associates with C6 and C7 at the targeted cell membrane (Dunkelberger & Song, 2010). Next, C8 will associate with the complex and is partially inserted into the membrane and this will allow C9 to insert into the lipid bilayer (Dunkelberger & Song, 2010). Twelve to fifteen, C9 molecules will assemble at the lipid bilayer to create a pore that will cause the targeted lysis of the cell

(Dunkelberger & Song, 2010).

Lastly, the generation of the C3b fragment and other molecules such as C1q and mannose-binding protein initiates opsonization which function as a tag to remove foreign bodies (Dunkelberger & Song, 2010). This process is mediated by the binding of the CR1 receptor on neutrophils and monocytes with the opsonization molecule and this then leads to phagocytosis of the foreign body by the neutrophils and monocytes (Dunkelberger &

Song, 2010). In addition, the interaction between CR1 and the opsonization molecules

36 promotes the secretion of pro-inflammatory molecules such as interleukin-1α, interleukin-1β and prostaglandins (Dunkelberger & Song, 2010).

Figure 18. Functions of complement. A) Demonstrates the assembly of the membrane attack complex (MAC). B) Complement mediated anaphylatoxins and inflammatory responses. C) Complemented mediated opsonization and phagocytosis of microbes. Reprinted from “Complement and its role in innate and adaptive immunity,” by J. R. Dunkelberger and W. C. Song, 2010, Cell Research, 20, 39. Copyright 2010 by “IBCB, SIBS, CAS.”

37 B. Adaptive Immune Response

The adaptive immune system is comprised of T and B lymphocytes which are

cells that utilize their antigen receptors to generate highly specific and long lasting

responses to a particular antigen (Iwasaki & Medzhitov, 2010; Pulendran & Maddur,

2015). The T cell receptors are referred to as TCR, and the B cell receptors are made up

of monomeric IgM (Travers et al., 2001).

Figure 19. T cell receptor (TCR) is composed of an αβ heterodimer which interacts with CD3 molecules. Reprinted from Immunology: The Immune System in Health and Disease, by C. A. Janeway Jr., P. Travers, M. Walport, and M. J. Shlomchik, 2001, New York: Garland Sciences. Copyright 2001 by “Garland Sciences.”

Figure 20. B cell receptor (BCR) is a membrane bound immunoglobulin M which interacts with Igβ and Igα. Reprinted from Immunology: The Immune System in Health and Disease, by C. A. Janeway Jr., P. Travers, M. Walport, and M. J. Shlomchik, 2001, New York: Garland Sciences. Copyright 2001 by “Garland Sciences.”

1. T Cell Maturation

T cells are derived from hematopoietic stem cells in the bone marrow that will migrate to the thymus (Warrington et al., 2011). To be activated, T cells require the presentation of an antigen by an antigen presenting cell (APC) such as dendritic cells, macrophages, B cells, fibroblasts or epithelial cells (Warrington et al., 2011). The activation of T cells begins when T cells encounter an APC that is presenting an antigen bound to its MHC molecule (Warrington et al., 2011). The major histocompatibility complex molecule (MHC) is a surface protein expressed on all nucleated cells (MHC I) or expressed on immune cells such as macrophages, DC, and B cells (MHC II)

39 (Warrington et al., 2011). The function of the MHC is to express antigenic portions of a

pathogen when a host cell has been infected with a pathogen or has taken up an antigen

(Warrington et al., 2011).

MHC I molecules present antigenic peptides that have been generated

intracellularly while MHC II molecules present antigenic peptides that have been taken

up from the extracellular environment (Warrington et al., 2011). The T cell receptor

(TCR) will bind with the MHC-antigen complex activating the T cell to secrete cytokines which can further activate other parts of the adaptive immune system (Warrington et al.,

2011). Stimulation by the APC will cause the differentiation of T cells into cytotoxic T cells (CD8+ Tc) or helper T cells (CD4+ Th) (Warrington et al., 2011).

Activation of helper T cells (Th) begins when the TCR interacts with MHC II of

APCs (Warrington et al., 2011). Th can further differentiate into Th1 or Th2 cells. It is hypothesized that naïve CD4+ T cells initially produce IL-2 and that differentiation into a

Th1, Th2, or Th17 cell depends on which cytokines the cell is exposed to during primary activation (London, Abbas, and Kelso, 1991). IL-12 produced by macrophages and

dendritic cells will lead to the differentiation into a Th1 cell while increased levels of IL-

4 leads to differentiation into a Th2 cell (London et al., 1991; Choi & Reiser, 1998;

Romagnani, 2000).

2. Th1 Response / Cell Mediated

The Th1 response is characterized by the production of INF-γ, IL-2, IL-12, TNF-β

and IgG2a and is traditionally thought to function as a cell-mediated response because of

the activation of macrophages, natural killer cells (NK), neutrophils and antigen specific

40 Tc that cause the apoptosis of infected cells (Warrington et al., 2011; London et al., 1991;

Kanai, Min, Ichim, Wang, & Zhong, 2007).

Upon binding of the IL-12 to the IL-12 receptor on the T cell, IL-12 will cause the activation of transcription factors Stat1, Stat2 and Stat4 leading to the production of INF-

γ (London et al., 1991). INF- γ will ensure Th1 differentiation by enhancing IL-12 secretion by macrophages and by maintaining expression of IL-12 receptors on T cells

(London et al., 1991). Additionally, IL-18 produced by macrophages and monocytes is involved in the activation of JNK, AP-1, NF-kβ and IRAK, which may independently activate the IFN- γ gene promotor (Romagnani, 2000; Dinarello, Novick, Kim, &

Kaplanski, 2013).

Tc activation begins when their TCR interacts with the MHC I of APCs or host cell antigens (Warrington et al., 2011). To complete activation, the Tc must be stimulated by cytokines, such as IFN- γ, produced by Th1 cells (London et al., 1991). Activated Tc will destroy infected cells by releasing perforin which induces lysis of target host cells and granzyme which induces apoptosis of target host cells (Warrington et al., 2011).

Although at the resolution of an infection most of the Tc will die, some will be retained as memory cells that will be available to defend the host against a future attack by the same antigen (Warrington et al., 2011).

During influenza virus infection, CD8+ T cells located in the lymph nodes are activated by presentation of viral epitopes via the MHC class I molecule (Bahadoran et al., 2016). Activation leads to the differentiation of CD8+ T cells into Tc cells which will subsequently migrate to the infected tissue (Bahadoran et al., 2016). Tc cells eliminate virus infected cells by releasing perforin and granzymes (Bahadoran et al., 2016).

41 Perforin will penetrate the cell membrane of infected cells allowing the entrance of

granzymes which causes the apoptosis of the infected cells (Behadoran et al., 2016).

Additionally, granzyme A has been shown to prevent viral spread by cleaving viral

proteins (Bahadoran et al., 2016). As a response to an influenza virus infection, memory

Tc cells are generated and are capable of rapidly responding to a secondary influenza

virus infection (Bahadoran et al., 2016).

3. Th2 Response/ Antibody Mediated

B cells, which will eventually mature into plasma cells, develop from hematopoietic stem cells in the bone marrow (Warrington et al., 2011). The activation of

B cells to produce antibodies begins when B cells recognize an antigen (Warrington et al., 2011). Next, B cells are further stimulated by cytokines such as IL-4 produced by Th2 cells (Warrington et al., 2011). B cells produce immunoglobulin alpha, delta, epsilon, gamma, and mu. These antibodies have different biological functions that lead to targeting pathogens and toxins for neutralization, classical complement activation, opsonin promotion of phagocytosis and pathogen elimination (Warrington et al., 2011).

Activated uncommitted CD4+ T cells expressing the IL-4 receptor will bind IL-4

which will cause the activation of the transcription factor Stat6 (London et al., 1991).

This will lead to the production of more IL-4 which can further stimulate the T cells,

committing them to become Th2 cells (London et al., 1991). Additionally, lack of IL-12

will cause the differentiation of T cells into Th2 cells (London et al., 1991). Production of

a Th2 cell and the subsequent activation of plasma cells to produce antibodies is

traditionally viewed as an antibody mediated response.

42 Th2 cells are characterized by the production of cytokines such as IL-4, IL-5, IL-

10 and IL-13 which stimulate antibody producing B cells (Warrington et al., 2011;

London et al., 1991). The cytokines produced during a Th2 response antagonize the actions of a Th1 response. IL-4 and IL-13 prevent the macrophage activating properties of INF- γ and IL-10 suppresses macrophage functions (London et al., 1991).

Since the influenza virus enters the body by crossing the mucosal barriers, IgA

antibodies located in the mucosal tissue are generated against the HA and NA viral

proteins during an influenza virus infection (Bahadoran et al., 2016). IgM and IgG

antibodies will also be generated (Bahadoran et al., 2016). It has been demonstrated that a

high IgM response is associated with higher viral clearance and stronger IgG production

(Bahadoran et al., 2016). Most antibodies against HA target the trimeric head of the viral

proteins but some antibodies also target the conserved stem region (Bahadoran et al.,

2016). IgG antibodies against HA and NA may be recognized through their Fc receptors

by natural killer cells (NK) initiating antibody-dependent cellular cytotoxicity killing of

virus infected cells (Bahadoran et al., 2016; Jegaskanda et al., 2013). Antibodies against

the NA viral protein are also generated and function to prevent viral activity (Bahadoran

et al., 2016). Lastly, antibodies against the M2 protein are generated but at lower titers

(Bahadoran et al., 2016). The M2e antibodies are capable of activating complement and

mediating cell lysis but the mechanisms remain unclear (Bahadoran et al., 2016).

Figure 21. Adaptive immune response to a viral infection. Th1 cells will activate Tc (CTL) to initiate a cell mediated immune response. Th2 cells will stimulate B cells to differentiate into plasma cells and produce antibodies against the viral antigen. Reprinted from “Evasion of Influenza A Viruses from Innate and Adaptive Immune Responses,” by C. E. van de Sandt, J. H. C. M. Kreijtz and G. F. Rimmelzwaan, 2012, Viruses, 4, 1446. Copyright 2012 by “C. E. van de Sandt, J. H. C. M. Kreijtz and G. F. Rimmelzwaan.”

4. Th17/ Inflammatory Response

Th17 cells are CD4+ Th cells characterized by their production of IL-17

(Romagnani, 2008). The differentiation of uncommitted T cells into Th17 cells is initiated by TGF-β with IL-6 being a critical co-factor produced by dendritic cells

(Romagnani, 2008). IL-23, IL-1, and TNF-α are involved in amplifying the Th17

44 response (Romagnani, 2008). In mice, the major roles of the cytokines IL-17 and IL-17F produced by Th17 cells are to recruit neutrophils and macrophages, produce pro- inflammatory cytokines such as IL-6, nitric oxide synthase, and establishment of chronic inflammation (Romagnani, 2008). The differentiation of T cells into Th17 cells is inhibited by a Th1 or Th2 response (Romagnani, 2008). Influenza A virus infection causes the production of type I IFN which inhibits IL-23 production (Kudva et al., 2014).

IL-23 is required to activate Th17 cells necessary for preventing bacterial infections in the lungs (Kudva et al., 2014). Thus, it has been speculated that down regulation of Th17 cells during influenza A virus infection could lead to development of secondary bacterial pneumonia (Kudva et al., 2014).

Figure 22. Action of cytokines produced by Th17 cells include granulocyte recruitment and may contribute to chronic inflammation. Reprinted from “Interleukin-17 in atherosclerosis and cardiovascular disease: the good, the bad, and the unknown,” by G. Liuzzo, F. Trotta, and D. Pedicino, 2013, European Heart Journal, 34, 557. Copyright 2012 by “G. Liuzzo, F. Trotta, and D. Pedicino.”

45 5. T reg/ Immunosuppressive Response

Treg cells are responsible for suppressing the immune response (Figure 22)

(Warrington et al., 2011). Treg cells develop from naïve T helper cells when TGF-β is

present in the absence of IL-6 (Romagnani, 2008). Data suggest that Treg cells can delay

IAV-induced mortality and that in combination with neutralizing antibodies, Treg cells

can prevent clinical disease signs after H1N1 viral infection (Zou et al., 2014). Another

study showed that H5N1 virus-primed CD8+ Treg cells inhibited CD8+ T cells in an IL-

10 dependent manner leading to higher mortality and viral load in the lungs of mice (Zou

et al., 2014). Although the mechanisms by which Treg cells regulate the immune response

remain unclear, they play an important role that will require further studying.

Based on the above immunology discussion, an effective vaccine against influenza probably needs to stimulate both the innate and adaptive T cell and B cell responses.

X. Influenza Vaccination

A. History of Influenza Vaccination

In general, the protection provided by most vaccines is through activation of B cells which produce antibodies against the pathogen of interest (Krieg, 2007). For a vaccine to elicit the production of antibodies B cells must be stimulated by antigen binding and a co-stimulatory signal provided to them by T cells which are activated by

APCs (Krieg, 2007). Early vaccines were live attenuated vaccines that would cause a mild infection to which the body would develop immunity (Krieg, 2007). However, people who were immunocompromised could become sick by these live attenuated vaccines and as a result, subunit vaccines were developed (Krieg, 2007). Subunit

46 vaccines use highly purified antigens derived from the pathogen that can be targeted by the immune system (Krieg, 2007). Although the exact mechanism of action of most vaccines remains unclear, it is known that the efficacy of a vaccine depends upon the activation of the adaptive and innate immune responses. The latter response is a result of the stimulation of the TLR and other receptors (Krieg, 2007).

The first clinical trials for an influenza vaccine occurred in the mid 1930s

(Barberis, Myles, Ault, Bragazzi, & Martini, 2016). The clinical trials used a live attenuated influenza monovalent vaccine which was protective against Influenza H1N1 type A (Barberis, Martini, Lavarone, & Orsi, 2016; Barberis et al., 2016). In 1940,

Influenza type B was isolated and this was subsequently combined with the influenza type A to create a bivalent influenza vaccine (Barberis & Martini et al., 2016). In 1957, the World Health Organization created the first surveillance system to track the mismatches between the current circulating influenza strains and the vaccines against influenza (Barberis et al., 2016). As a result of the 1968 pandemic, a trivalent inactivated influenza vaccine which included influenza A/H1N1, influenza A/H3N2 and influenza B was developed (Barberis et al., 2016). In the 1970s, genetic reassortment was introduced for the production of influenza vaccines which allowed researchers to produce viruses that had the genes for rapid replication in embryonated eggs and the genes for the target

HA and NA proteins that were present in the circulating influenza strains (Barberis et al.,

2016). This technique allowed viral strains to grow much more rapidly in embryonated eggs (Barberis et al., 2016). In 2003, the FDA approved FluMist which is a live attenuated vaccine intranasally administered which presently is not recommended for use

(see details below) (Barberis et al., 2016). After the 2009 influenza pandemic, the FDA

47 recommended the use of Fluzone which is a vaccine that contains 4X more HA antigen

compared to the trivalent seasonal influenza vaccine for those at high risk of

complications after an influenza infection (Barberis at al., 2016). In 2011, the FDA

approved the intradermal administration of Fluzone because it was shown to provide

better innate and adaptive immune responses (Barberis et al., 2016). In 2012, the FDA

approved Fluarix which is a quatrivalent influenza vaccine protective against two

influenza A strains and two influenza B strains (Barberis et al., 2016). In 2013, the FDA

approved FluBlock, which contained viral antigens made by baculovirus that had

recombinant DNA in it which coded for the viral proteins and this baculovirus was used

to infect and insect cells which produced the viral antigens (Barberis et al., 2016).

B. Types of Influenza Vaccines

There are several different types of influenza vaccines that are commercially available

today. They include the various types in Table 1. The following paragraphs will discuss

each of these vaccines.

1. Live Attenuated or Inactivated Vaccines

Influenza vaccination with a live attenuated influenza vaccine or a trivalent

influenza inactivated vaccine causes the production of memory B cells and antibody-

secreting cells (Dormitzer et al., 2011). CD4+ T lymphocytes activate CD8+ T cells and

the early clonal expansion of B lymphocytes (Dormitzer et al., 2011). Preclinical studies suggest that influenza-specific CD4+ T cells accelerate recovery from infection by

producing INF-γ, and by activating the innate response in infected tissues (Dormitzer et

al., 2011). As mentioned previously, influenza virus infection is recognized by the innate immune system via TLR7 and RIG-I (Pulendran & Ahmed, 2011). However, it remains

48 unknown to what extent a live attenuated influenza vaccine activates the innate immune

system compared to a natural influenza infection (Pulendran & Ahmed, 2011). It is

known that the efficacy of inactivated whole virus vaccines require TLR7-mediated production of type I interferon by plasmocytoid Dendritic Cells (Pulendran & Ahmed,

2011).

These influenza vaccines usually target two influenza A subtypes (H1N1 and

H3N2) and one or two influenza B virus strains (Reperant, Rimmerlzwaan, and

Osterhause, 2014). These viruses are selected by the WHO/Global Surveillance and

Response System for influenza which includes participation by many laboratories worldwide that predict the influenza viruses that are expected to circulate in the upcoming flu season (Ebrahimi & Tebianian, 2011). Current commercial vaccines are formulated to contain inactivated virus which are then prepared as split viruses, viral subunits, or viral proteins.

These vaccines provide protection by generating neutralizing antibodies against the constantly mutating HA and NA viral surface proteins (Reperant et al., 2014). HA on the virus surface is present in higher concentrations than the other surface proteins and is the prime vaccine target capable of providing protection because it prevents binding of the virus with the host cell. NA-specific antibodies will inhibit the release of new viral progenies from the host cell (Ebrahimi & Tebianian, 2011).

Currently, vaccine production requires that influenza virus strains used to formulate the vaccines match the circulating strain with respect to their HA and NA proteins (Ebrahimi & Tebianian, 2011). Additionally, most vaccines are produced by first growing the virus in embryonated chicken eggs (Ebrahimi & Tebianian, 2011; Reperant

49 et al., 2014). This limits the production capability of vaccine manufacturers as they rely on the availability of embryonated eggs (Reperant et al., 2014). This is also problematic for individuals who are allergic to egg products as they might develop an allergic reaction to the vaccine (Ebrahimi & Tebianian, 2011).

2. Vaccines Against the Conserved HA Stalk

Since the head region of the HA protein constantly mutates, researchers are investigating vaccines that would target the stem region of the HA protein which is more conserved across different influenza viruses (Cimons, 2016). Researchers at Janssen

Pharmaceutical Companies of Johnson & Johnson and Scripps Research Institute have removed the head region of the HA protein and introduced mutations to the stem region to increase the stem region stability in a vaccine (Cimons, 2016). Other scientists have created a new head region for the HA protein of the influenza virus to direct the immune system to the conserved stem region and the neuraminidase protein rather than the head region (Cimons, 2016). These vaccines have yet to be tested in humans (Cimons, 2016).

Developing a more universal vaccine targeted to a conserved protein, that could be made by using the faster recombinant protein approach, and one that is not limited to predicting the circulating influenza strain, is the ideal approach to prevent influenza virus infections. Since the current production of most influenza vaccines requires a long production cycle which includes growing the circulating virus in embryonated eggs, there is a limited capacity to respond to a pandemic outbreak or a mismatch in vaccine strains between the circulating virus and the virus selected for the vaccine that year.

50 Table 2.

Influenza Vaccines Available in the United States for the 2016-2017 Influenza Season

Note. Adapted from “US Department of Health and Human Services/Centers for Disease Control and Prevention,” 2016, Morbidity and Mortality Weekly Reports, 65, 19.

3. M2 Vaccines

Given that developing a vaccine to target the conserved portion of the HA stem protein is still in development, another approach to developing a universal vaccine would be to target the conserved portion of the extracellular influenza envelope M2 protein, referred to as M2e (aa 1-23). Many scientists have investigated M2e as a possible vaccine target because of its conserved nature across different influenza strains (Ebrahimi &

Tebianian, 2011).. M2 is not expressed in high concentrations on the virus, but it is expressed in high concentrations on influenza infected cells (Deng et al., 2015). M2e could be manufactured recombinantly in tissue culture cells or bacteria which would completely avoid the need to use embryonated eggs as a method to obtain the viral antigen.

51 Vaccines using M2e as a vaccine antigen have not been approved for commercial

use. Some M2e vaccines have been investigated in phase 1 clinical trails (Table 3) (Deng et al., 2015). A vaccine using a recombinant flagellin protein and four tandem copies of

M2e administered at 0.3 and 1.0 μg was shown to be safe and induce the production of 1

μg/mL of anti-M2e IgG antibodies (Deng et al., 2015). Another vaccine formulation tested was a recombinant protein fusing M2e and NP linked to a proprietary TLR 9 receptor ligand (Deng et al., 2015). This vaccine induced the production of antibodies against M2e and NP (Deng et al., 2015).

Table 3

M2e-based Vaccines Under Clinical Investigation

Note. Adapted from “M2e-based universal influenza A vaccines,” by Deng et al., 2015, Vaccines, 3.

M2e is shielded by the larger proteins HA and NA in the viral envelope (Ebrahimi

& Tebianian, 2011). This severely limits the production of M2e antibodies during a

52 natural infection (Ebrahimi & Tebianian, 2011). There is no evidence to suggest that those who do develop antibodies against M2e, as part of a natural influenza infection, have protection against infection with different influenza strains (Ebrahimi & Tebianian,

2011). Nonetheless, if a vaccine can be used to stimulate high levels of anti-M2e antibodies, evidence suggests that M2e-specific antibodies can provide protection through antibody dependent cellular cytotoxicity given the high expression of the M2 on infected cells enabling the anti-M2e antibodies to bind with the M2e on the surface of viral infected cells and prevent viral budding (Ebrahimi & Tebianian, 2011). There is also evidence that anti-M2e antibodies generated by vaccination with a liposomal

VesiVax® M2e vaccine can bind directly to the whole influenza A virus and this binding can be detected by an ELISA assay (Tringali, MS Thesis). And finally, several investigators using different types of M2e vaccines have reported that passive transfer of serum from M2e vaccinated mice to naïve mice has protected the naïve mice from influenza challenge (Deng et al., 2015; Ernest et al., 2006; Adler-Moore et al., 2011).

Although using M2e as a vaccine antigen is promising, M2e is not conserved in every strain of influenza. Thus, targeting the conserved M2e is not enough to create a completely universal vaccine which stimulates a potent adaptive immune response against all strains of influenza. This suggests that in order to maximize protection, enhanced stimulation of the innate immune response by the adjuvant added to the vaccine could be very helpful. In fact, it may be possible to enhance the innate immune response enough by the use of the appropriate adjuvant, to completely circumvent the need for an influenza protein in the vaccine.

53 XI. Previous Work on Liposomal Based M2e Vaccines Using the VesiVax® System

A. Liposomes

To introduce this topic, a background in liposomes is needed. Liposomes are microscopic amphipathic spherical vesicles composed of various types of phospholipids which could also include cholesterol (Akbarzadeh et al., 2013). Liposomes can contain one (unilamellar) or more (multilamellar) lipid bilayers which surround an aqueous center if it is a unilamellar liposome and aqueous layers located between the bilayers if it is a multilamellar liposome (Akbarzadeh et al., 2013). Liposome formulations can vary with respect to lipid composition, size, charge and location of antigen and adjuvants relative to the liposome phospholipid bilayer (Schwendener, 2013). For example, water- soluble antigens such as proteins, peptides, nucleic acids, carbohydrates and haptens will localize in the aqueous region of the liposome following passive entrapment

(Schwendener, 2013). On the other hand, lipophilic antigens and adjuvants can be entrapped within the lipid bilayers. Soluble antigens can be attached to the outer surface of the liposome bilayer via a lipid component that has a linker molecule and the antigen is then attached at the liposome surface via a covalent bond with the linker molecule

(Schwendener, 2013).

Figure 23. Unilamellar liposome representation of the different ways that molecules can be associated with the liposomes. Hydrophilic drugs localize to the aqueous center of liposomes. Hydrophobic molecules can be incorporated within the lipid bilayer. Some liposomes can include surface PEG (polyethylene glycol) molecules to prevent its rapid uptake by the reticuloendothelial cells, such as macrophages. Other liposomes can include lipids in the bilayer which have reactive groups that can be conjugated with different drugs or be conjugated with different targeting ligands. Reprinted from Application of Nanotechnology in Drug Delivery (2), by M. Cagdas, A. D. Sezer, and S. Bucak, 2014. Copyright 2014 by “M. Cagdas, A. D. Sezer, and S. Bucak.”

Liposomes can be manufactured with a high degree of specificity and as stated above, they are capable of trapping hydrophobic and hydrophilic molecules. Depending upon their lipid composition, they may also have adjuvant activity. Given these characteristics, certain types of liposomes have been used as vaccine delivery systems

(Akbarzadeh et al., 2013; Schwendener, 2014). Liposomal vaccine formulations have been investigated for infections caused by parasites, viruses, fungi, and bacteria

(Schwendener, 2014). A liposomal vaccine containing the adjuvant MPL and a 78 kDa

55 antigen of the parasite Leishmania donovani significantly decreased the parasite burden after challenge (Schwendener, 2014). Liposomes mixed with a DNA plasmid containing the M1 gene of influenza A virus generated a strong immune response and protection against influenza challenge in mice (Schwendener, 2014). A vaccine composed of liposomes mixed with a DNA plasmid encoding heat shock protein 65 was used against the pulmonary infection, paracoccidiomycosis, and resulted in decreased fungal burden

(Schwendener, 2014).

B. VesiVax® Liposomal Vaccines

Our partners at Molecular Express, Inc, developed two types of VesiVax® vaccine liposomes. One type allows for the liposomal delivery of an antigen fused to a proprietary hydrophobic domain (HD) that allows the antigen with the HD to be anchored into the phospholipid bilayer of the liposome. The other type of VesiVax® liposomes are called Conjugatable Adjuvant Lipid Vesicles (CALVs). In this case, a reactive maleimide group (CMI) is bound to some of the lipids and displayed at the surface of the liposomes.

The CMI will react with the sulfhydryl group on a cysteine at the terminal end of antigenic proteins or peptides. This results in the formation of a covalent bond between the maleimide and the antigen (Lockner et al., 2013). The antigen is thus presented at the surface of the liposome.

Figure 24. M2e protein linked to a unilamillar liposome via a CMI linkage.

Delivering the M2e protein linked to a liposome provides several advantages

compared to using other drug delivery methods. Using the reactive maleimide group, the

M2e can be attached to the outer portion of the liposome perhaps allowing a better

presentation of the M2e protein to APCs (Figure 24). Additionally, liposomes have been

shown to be non-toxic, biodegradable and can reduce the toxicity associated with a

specific drug such as in the case of amphotericin B (Akbarzadeh et al., 2013). These

reasons combined with the specificity to which liposomes can be manufactured make

liposomes an advantageous delivery method for the M2e protein vaccine.

C. M2e Vaccine Formulations Using L-M2eA-HD

Previously, my laboratory in collaboration with Molecular Express, Inc, used the liposomes with an M2e-HD fused protein (L-M2e-HD) to stimulate a protective immune response against an H1N1 and H6N2 influenza challenge (Ernst et al., 2006). This vaccine formulation was created by first recombinantly preparing three optimized M2e sequences, representative of three different strains of influenza A (M2eA1, M2eA2,

M2eA3), in Escherichia coli (Ernst et al., 2006).

Each of the M2eA gene sequences was ligated into a plasmid containing the HD gene (Ernst et al., 2006). The plasmid containing the M2eA-HD genetic sequence with a

57 histidine tag was then transformed into BL21 pLysS E. coli for expression (Ernst et al.,

2006). Bacterial cells were grown, lysed and centrifuged to remove cellular debris and the M2eA-HD protein antigen was subsequently selected using nickel affinity to bind with the histidine on the protein antigen (Ernst et al., 2006). Several subsequent steps were needed to purify the hydrophobic M2eA-HD.

To prepare the liposomes, phospholipids, cholesterol and the adjuvant monophosphoryl Lipid A (MPL), with or without the M2eA-HD protein, were dissolved in an organic solvent mixture of chloroform/methanol (Ernst et al., 2006). The solvent was then evaporated to create a thin film which was later hydrated with buffer, and then subjected to probe sonication (Ernst et al., 2006). The M2eA liposomal vaccine formulations used to immunize the mice provided an immune response resulting in increased survival in mice challenged with H1N1 (100% survival with M2eA1) or H9N2

(80% survival with M2eA3) (Ernst et al., 2006). Additionally, this vaccine formulation elicited high IgG antibody titers against M2eA1 and the serum from these mice provided passive protection to naïve mice which were subsequently challenged with a lethal dose of H6N2 influenza virus (Ernst et al., 2006). These results provide some support for targeting M2e as a universal vaccine candidate since the vaccine could protect against influenza caused by different influenza strains.

In a subsequent study, using a novel in vitro assay, it was demonstrated that M2e specific antibodies stimulated by L-M2e-HD/MPL liposomes inhibited viral cell lysis by multiple strains of influenza (H2N2, H3N2, H6N2, and H5N9) compared to the serum from L-control/MPL (Adler-Moore et al., 2011). In addition, splenocytes from mice vaccinated with L-M2e-HD/MPL and incubated with M2e had significantly higher IFN-γ

58 and IL-4 secreting splenocytes compared to splenocytes from the mice given L- control/MPL (Adler-Moore et al., 2011).

Given that soluble M2e protein antigens can be much more easily purified from the E. coli compared to purification of the hydrophobic M2e-HD, the M2e was mixed with the CALV liposomes and conjugated with the maleimide to be displayed on the surface of the liposome. Our laboratory then compared the efficacy of the M2e-HD/MPL liposomes with the CMI M2e/MPL liposomes in female Swiss Webster mice against a

10X LD50 challenge of influenza strain A/PR/8/34 (H1N1) (Joseph Henriquez, MS

Thesis). This study demonstrated that the M2e-HD/MPL liposomes and the CMI-

M2e/MPL liposomes were comparable with significantly better protection compared to the control group of MPL liposomes with no CMI or M2e. This protection was measured by increased survival, increased anti-M2e antibodies and decreased lung viral burden in most vaccine groups as measured by a viral foci assay. Interestingly, CMI/MPL liposomes without M2e also provided significant protection without stimulating the production of anti-M2e antibodies (Joseph Henriquez, MS Thesis). This indicated that the protection observed in this latter vaccine group was mediated through activation of the innate immune system.

XII. Adjuvants

Adjuvants are molecules that enhance the efficacy of vaccines by stimulating the innate immune response (Schwendener, 2014; de Souza Apostolico et al., 2016). Their mechanisms of action vary from adjuvant to adjuvant, but in general, adjuvants work by increasing antigen uptake by APCs, activating APCs, causing the production of

59 cytokines, activating inflammasomes, and inducing inflammation and cell recruitment (de

Souza Apostolico et al., 2016).

One of the first adjuvants to be used in vaccine formulations was mineral salts

(alum) (de Souza Apostolico et al., 2016). It was originally believed that this adjuvant

functioned by slowly releasing the antigen (de Souza Apostolico et al., 2016). However,

research has shown that it interacts with NOD-like receptors to activate the inflammasome complex (de Souza Apostolico et al., 2016). This adjuvant is licensed for vaccines against Hepatitis A, Hepatitis B, DTP, HVP, and others (de Souza Apostolico et al., 2016). Recently, the adjuvant MF59 (water-in-oil squalene based emulsion) was licensed to be used as part of Fluad ™ flu vaccine (de Souza Apostolico et al., 2016).

Although its mechanism of action is not understood, it is known that MF59 stimulates the production of cytokines from macrophages, monocytes and dendritic cells (de Souza

Apostolico et al., 2016). Several other adjuvants are currently under investigation in several vaccine formulations for various diseases.

60 Table 4

Adjuvants Under Clinical Investigation For Prophylactic Use in Vaccine Formulations

Note. Adapted from “Adjuvants: Classification, Modus Operandi, and Licensing,” by de Souza Apostolico et al., 2016, Journal of Immunology Research, 2016. Copyright 2016 by “Julia de Souza Apostolico et al.”

XIII. Aims of the Present Study

Based on the results from the previous work in our laboratory, in collaboration with Molecular Express, Inc, the current research focused on investigating the efficacy of liposomal M2e vaccines using different adjuvants to stimulate both the adaptive and innate immune response, and the use of liposomes with only adjuvants and no M2e or any other influenza protein to stimulate only the innate immune response. To target the innate immune response, we used the following adjuvants: MPL, mycoviral dsRNA, lipidated-Tucaresol, Pam3CAG, and 1V270.

61 A. Monophosphoryl Lipid A (MPL)

MPL is a non-toxic lipopolysaccharide derivative obtained from Gram-negative

bacteria (Figure 25) (de Souza Apostolico et al., 2016). It is an agonist for the TLR4

receptor located on cell membranes of dendritic cells (de Souza Apostolico et al., 2016).

MPL activates the TRIF pathway and leads to the production of IFN-β and increased IL-

10 (de Souza Apostolico et al., 2016; Castellas & Mitchell, 2008). Much of the literature reports that MPL can stimulate a Th1 and Th2 response depending upon the antigen with which it is associated (Castellas & Mitchell, 2008). MPL is currently licensed for use as an adjuvant system in vaccines against hepatitis B and HPV (Castellas & Mitchell, 2008).

Figure 25. Monophosphoryl lipid A is derived from the cell wall of gram-negative bacteria. Reprinted from “Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant,” by R. C. Casella and T. C. Mitchell, 2008, Cellular and Molecular Life Sciences, 65, 14.

B. Mycoviral dsRNA

dsRNA binds to the endosomal TLR3 receptor leading to an inflammatory

response (Zhou et al., 2013). A recent study found that the surface integrin receptor

62 CD11b/CD18 (Mac-1) interacts with dsRNA to internalize dsRNA and induce a TLR3- dependent and a TLR3-independent but Mac-1 dependent, inflammatory oxidative signal

(Zhou et al., 2013). Using synthetic dsRNA, it has been demonstrated that activation of the TLR3 receptor leads to activation of dendritic cells which will produce IL-12, Type I interferon, unregulated MHC II expression and a Th1 response (de Souza Apostolico et al., 2016; Poteet et al., 2016). In the present study, we used a natural source of dsRNA derived from the dsRNA mycovirus that infects Saccharomyces cerevisiae which in previous studies has been shown to stimulate B cell and T cell responses better than the synthetic dsRNA Poly (I:C) (Wright & Adler-Moore, 1985).

C. Tucaresol

Tucaresol is a substituted benzaldehyde synthetic adjuvant (Collins et al., 2014;

Rhodes, 2002). It is believed that the aldehyde groups of tucaresol mimic the carbonyl groups of the lysines present on APC which then interact with T cells, providing the T cells with co-stimulatory signals (Collins et al., 2014). This leads to enhanced TCR- dependent IL-2 and IFN- γ production which increases CD4+ T cell and CD8+ T cell responses to antigens (Rhodes, 2002). Tucaresol can be lipidated to facilitate its integration into a liposome bilayer (Collins et al., 2002). Tucaresol is known to induce both Th1 and Th2 immune responses (Collins et al., 2014)

Tucaresol Lipid-Tucaresol

Figure 26. Comparison of Tucaresol and Lipidated Tucaresol. Reprinted from “Lipid Tucaresol as an adjuvant for methamphetamine vaccine development,” by Collins et al., 2014, Chemical Communications, 50, 6. Copyright 2014 by “The Royal Society of Chemistry.”

D. Pam3CAG

Pam3CysAlaGly (Pam3CAG) is an amphiphilic lipopeptide synthetic analogue of

the N-terminus of E. coli lipopeptide that can be incorporated into liposomes (Fernandes,

Frisch, Muller, & Schuber, 1997; Espuelas et al., 2005). Lipopeptides such as Pam3CAG

are known to bind to the TLR2 receptor expressed on the surface of dendritic cells

(Espuelas et al., 2005). Transmembrane TLR2 activation has a biased Th1 response

(Mukherjee, Karmakar, & Baby, 2016). Additionally, Pam3CAG has been utilized to enhance effector and memory CD8+ T cells, supporting the role of Pam3CAG as a Th1

biased adjuvant (Chua et al., 2014).

64 R1: C15H31CO R2: C15H31 R3: C15H31 X: OH

Figure 27. The lipopeptide containing the above mentioned side chains (R1, R2, R3, X) is called Pam3CAG. Reprinted from “Effects of synthetic lipopeptides formulated in liposomes on the maturation of human dendritic cells,” by Espuelas et al., 2005, Molecular Immunology, 42, 722. Copyright 2004 by “Elsevier Ltd.”

E. 1V270

1V270 is a small synthetic phospholipid-conjugated TLR7 agonist (Goff et al.,

2015). It is believed that 1V270 leads to a Th1 immune response (Goff et al., 2015).

Figure 28. Structure of 1V270. Reprinted from “Synthetic Toll-like Receptor 4 (TLR4) and TLR7 Ligands as Influenza Virus Vaccine Adjuvants Induce Rapid, Sustained, and Broadly Protective Responses,” by Goff et al., 2015, Journal of Virology, 89, 3222. Copyright 2015 by “American Society of Microbiology.”

65 XIV. Hypotheses of Present Study

A. Study I

In the first Study, we investigated various combinations of CMI liposomal vaccines composed of M2e (amino acids 1-15) and mycoviral dsRNA or MPL for their efficacy in Swiss Webster female mice against challenge with 10X LD50 Influenza A/

Puerto Rico/8/1934 (PR8) H1N1 or 10X LD50 (X-31) H3N2. This study allowed us to compare the protective efficacy between vaccines containing mycoviral dsRNA (100 μg) or MPL (15 μg) with M2e versus vaccines with MPL or mycoviral dsRNA alone which served as controls. The pathogens, H1N1 and H3N2, which were serially mouse passaged, were selected for these experiments since they are a common cause of seasonal influenza. We hypothesized that Swiss Webster female mice could be equally protected from a lethal challenge of H1N1 or H3N2 influenza virus when vaccinated with a liposomal M2e vaccine containing either mycoviral dsRNA or MPL.

B. Study II

To continue my investigation of mycoviral dsRNA as an adjuvant for the liposomal M2e (amino acids 1-15) vaccine, we tested whether different purification methods to obtain mycoviral dsRNA would generate comparable protection. We compared the efficacy of mycoviral dsRNA obtained through column chromatography delivered in combination with liposomes conjugated to M2e via CMI (L-CMI-M2e) at 25

μg, 15 μg or 5 μg per dose versus mycoviral dsRNA obtained through ultracentrifugation delivered in combination with L-CMI-M2e at 50 μg or 25 μg per dose. We hypothesized that CMI liposomal vaccines with M2e and mycoviral dsRNA purified using a column

66 chromatography method would provide comparable protection compared to our standard

method of using mycoviral dsRNA isolated by ultracentrifugation.

C. Study III

We tested a shortened, more conserved, M2e epitope composed of only twelve

amino acids, with MPL, mycoviral dsRNA, Pam3CAG or lipidated-Tucaresol delivered

at 3 or 6 μg per dose or the combination of lipidated-Tucaresol plus MPL. We

hypothesized that the CMI liposomal vaccines with the smaller M2e (amino acids 1-12)

would provide comparable protection to the larger M2e (amino acids 1-15) when used in

combination with these adjuvants.

D. Study IV

Based on observations from the proceeding studies, we screened for adjuvants

(MPL, mycoviral dsRNA, Pam3CAG, 1V270 and lipidated-Tucaresol) that would provide protection in our CMI liposomal vaccine which did not include the M2e. Our hypothesis for this screening study was that different CMI liposomal vaccines containing different adjuvants and no antigenic protein would vary in the degree of protection they provided to Swiss Webster female mice challenged against a 10X LD50 A/ Puerto

Rico/8/1934 (PR8) H1N1.

From these results, we then selected mycoviral dsRNA (50 μg per dose) and

Pam3CAG (50 μg per dose) adjuvants to test their efficacy against an H1N1 and H3N2

influenza infection in Swiss Webster female mice since they showed good protection

against H1N1. We hypothesized that Pam3CAG or mycoviral dsRNA in the CMI

liposomes without M2e, would be capable of protecting Swiss Webster female mice

67 against a 10X LD50 of PR8 H1N1 or X-31 H3N2 by using only the innate immune response since no antigen protein was added to the CMI liposomal vaccines.

68 CHAPTER 2

Materials and Methods

I. Tissue and Virus Culture

A. Cell Culture: Madin-Darby Canine Kidney-ATCC#34-CCL (MDCK) Cells

MDCK cells were used for all assays and were taken from our laboratory stock

maintained at -80oC, at cell passage number around 50 (P50), and were originally

purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were

stored in Gibco’s (Gibco-Invitrogen, Carlsbad, CA) freezing recovery media. Freezing

media contained 0.5% Dimethylsulfoxfide (DMSO) in Eagles Minimal Essential Media.

Cells were stored in 1 ml aliquots in Nalgene® cryo-freezing cryo-vials (Nalgene Nunc

International, NY).

Figure 29. MDCK cell morphology. Madin-Darby canine kidney (MDCK) cells have a distinctive epithelial morphology and display several functional and anatomical properties of normal kidney tubule cells. Reprinted from “MDCK (NBL-2) (ATCC® CCL-34)™”, n.d.

B. Culturing MDCK Cells

To culture MDCK cells, the frozen stock of MDCK cells were thawed rapidly by placing the vial directly into a warm (37oC) water bath with gentle agitation for about a

minute. To avoid any toxic effects of the DMSO, cells were centrifuged with Complete

Dulbecco’s Modified Essential Media (C-DMEM), the supernatant DMSO media was

poured off, and the cells were suspended in C-DMEM for seeding. The C-DMEM was

made complete by adding 89% DMEM, 10% Fetal Bovine Serum (FBS) (Atlanta

Biologicals, Lawrenceville, GA) and 1% Dulbecco penicillin/streptomycin (Mediatech,

Inc., Herndon, VA).

o MDCK cells were grown at 37 C and 5% CO2 in filter topped tissue culture flasks

from Sigma-Aldrich (St. Louis, MO) and passaged every 2-3 days or until cells became

90-98% confluent. MDCK cells were then passaged using 0.25% Trypsin/EDTA (Atlanta

Biologicals, Lawrenceville, GA) and the cell concentration was adjusted using Trypan

Blue Exclusion assay (described below).

C. Trypan Blue Exclusion

Trypan Blue Exclusion can be used for any cell type (MDCK, Splenocytes,

Macrophages, etc.) and is performed in the same manner for each cell type. First, 0.4% trypan blue dye diluted in sterile distilled water (Gibco-Invitrogen, Carlsbad, CA) is added to make a 1:5 dilution, in this case 10 μl of samples (cells) with 40 μl of 0.4% trypan blue is placed in a sterile microcentrifuge tube using a sterile tip on a p20

Eppendorf micropipetor. The mixture is then pipetted up and down and a 10 μl sample of the mixture is then added to a hemocytometer. The viable cells that are not stained blue

70 are counted using a hand counter and a microscope. The cell concentration is determined

using the following equation:

Figure 30. Cell counting under hemocytometer. Four corner squares of the hemocytometer as highlighted in the image are used to count viable cells that are not stained blue. Reprinted from “hemocytometer.org.” Copyright 2017 by “Hemocytometer blog.”

D. Refreezing MDCK Cells

Cell lines in continuous culture are prone to genetic drift and all cell cultures are

susceptible to microbial contamination. Because an established cell line is a valuable

resource and its replacement is expensive and time consuming, it is vitally important that

they are frozen down and preserved for long-term storage. MDCK cells were refrozen by

first adding 2-3 ml of trypsin/EDTA to a 2-3 day old confluent cell monolayer in a T-75

tissue culture flask (Sigma-Aldrich, St. Louis, MO). The flask with the MDCK cell

71 o monolayer was then placed in an incubator set at 37 C and 5% CO2 until the monolayer

broke up. The cells and the media was then sterilely removed and placed in a 50 ml

sterile centrifuge tube and spun in the centrifuge at 1000 rpm for 5 minutes. After

centrifugation, the supernatant was discarded and the pellet of MDCK cells was

resuspended in Cell Freezing Recovery media containing DMSO. DMSO is used in cell

freezing media to protect cells from ice crystal induced mechanical injury. The concentration of cells was set to 5 x 106 viable cells/ml using the trypan blue viability

assay described above. Cells were adjusted to the desired concentration in Cell Freezing

Recovery media.

One ml aliquots of the cell suspension were then added to labeled, round bottom

sterile cryotubes and placed into a cryo freezing chamber (Mr. Frosty, Thermo-Fisher

Scientific, Waltham, MA) which was then placed into a -80oC Revco freezer (Revco

Freezer Refrigeration System, DuPont Suva Refrigerants, Asheville, NC). This is done

because it is best to freeze the cells as slow as possible (1oC/minute) in order to minimize

osmotic shock, large crystal formation and avoid damage upon thawing.

E. Termination of MDCK cells

After 80 Passages (P80) the MDCK cells will detach from cell culture flasks

which can lead to unreliable results. Because of this, MDCK cells were never used past

P75 for the assays. To terminate the cell culture, MDCK cells at Passage 75 were killed

by adding 50% bleach to their tissue culture flasks and discarded.

F. Virus Stocks:

The viruses used were A/Puerto Rico/8/34 PR8 H1N1 and X-31 H3N2

72 G. MDCK Cell Passaged H1N1 and H3N2 Stocks

MDCK cells (P72) were grown in a T-75 tissue culture flask (Sigma Aldrich, St.

Louis, MO) to 90-95% confluency and rinsed with 4-6 ml warm (37oC), sterile Phosphate

Buffered Saline (PBS). Warm 1 ml sterile C-DMEM was then added to the flask. A 1 ml aliquot of our previously frozen H1N1 or H3N2 MDCK cell passaged viral stock was quickly thawed by gentle agitation while in the 37oC water bath, and then added to the

flask containing the MDCK cells. The flask was rocked to ensure even distribution of the

o virus over the MDCK monolayer and incubated at 37 C and 5% CO2 for 1 hour.

Following the incubation, 8 ml of warm, sterile C-DMEM was added to the flask and the

o flask was returned to the 37 C, 5% CO2 incubator for 40-47 hours. From hours 40 to 47,

cells were examined in the inverted microscope and evaluated for the level of infection.

The level of infection was assessed by changes in cellular morphology characterized by

elongated cells becoming ovoid in shape and approximately 10% of the cells becoming

detached (dead).

After visual microscopic confirmation that the cells were infected with virus, all

the media containing released virus and detached cells from the flask were removed and

saved in a sterile 15 ml conical tube, which was then placed on ice. Two ml of cold fresh

media was then added to the flask and the remaining cells scraped off using a cell scraper

(Thermo Scientific, Rochester, NY). The 2 ml of media containing the mechanically

detached cells were placed into a new sterile 15 ml conical tube and centrifuged for 2

minutes at 1000 rpm (Heraeus™ Labofuge ™ 400 R, Thermo-Fisher Scientific,

Waltham, MA).

The supernatant was removed and added to the first conical tube containing the

73 original 10 ml of media. The cell pellet was then sonicated twice with 1-minute bursts in ice water in the Branson 1510 water bath sonicator (Branson Ultrasonic Corporation,

Danbury, CT). The sonicated pellet was then added to the 15 ml conical tube containing

the media, mixed well and then aliquoted into 250 μl aliquots in sterile microcentrifuge

tubes making sure that the material was vortexed really well before each aliquot was

removed to ensure that all tubes contained similar amounts of the virus in the media. The

tubes with the aliquots were then frozen in an -80oC freezer (Revco Freezer Refrigeration

System, DuPont Suva Refrigerants, Asheville, NC). Lastly, to ensure that the aliquots had

viable virus, cytolysis assays were done to determine viral virulence.

H. Lung Passaged H1N1 Stock

Lung passaged H1N1 stock was used for the in vivo studies. Six-week old

BALB/c female mice (Harlan Laboratories, Indianapolis, IN) were utilized for generating

the lung-passaged stock of influenza A/Puerto Rico/8/1934 H1N1 virus. Mice were

infected with 10X LD50 (1:4000 dilution in cold sterile PBS) of the fourth lung passage of influenza stock (4LP). The mice were first sedated with 80 mg/kg Ketamine (Putney Inc.,

Portland, ME) and 16 mg/kg Xylazine (SoloPak Laboratories Inc., Elk Grove Village, IL) administered via intraperitoneal injection with a 30 gauge needle. Once sedated, 40 μl of

1:4000 4LP viral dilution was then added drop wise to the nares and mice were allowed to recover from sedation.

The day the mice displayed severe symptoms (around day 5 or 6 of infection)

mice were euthanized via CO2 asphyxiation, placed in a Biosafety Level 2 hood, sprayed

with 70% isopropanol, and their lungs were harvested. To harvest the lungs, the skin of

the mice was removed with sterile scissors exposing the rib cage. The ribs were removed

74 with sterile scissors to expose the lungs. Lungs from each mouse were then taken using

sterile forceps and scissors and placed into individual 5 ml round bottom tubes, which

were then stored at -80oC.

The next day, a handheld Dremel 300 homogenizer (Biospec Products

Incorporated, Bartlesville, OK) was sterilized by submersion of the probe in 70%

isopropyl alcohol for 30 minutes in a Biosafety Level 2 hood. The homogenizer was pulsed in the alcohol for 1 minute at medium RPM and then given several short pulses.

The probe was then removed from the alcohol and allowed to air dry inside of the BSL hood. During this time, the lungs were thawed and 1 ml of Complete Eagles Minimal

Essential Media (CMEM, 10% FBS, 1% Essential Amino Acids, 1% penicillin- streptomycin mixture) was added to each tube. Lungs were kept on ice for the remainder

of the procedure.

The lungs were homogenized using the homogenizer with three, 5-second bursts

at medium-high power with 1-minute intervals of no homogenization between each burst

to prevent viral inactivation. The probe of the homogenizer was rinsed between each

sample by dipping into four sequential test tubes containing cold sterile PBS. The probe

was pulsed with three to four bursts in each of the rinse tubes to remove any bits of lung

tissue that may have been lodged during the previous homogenization. Once all the lungs

were homogenized, the homogenates were pooled. The pooled homogenates were shaken

to prevent aggregation of lung tissue and aliquoted into 250 μl samples with through

vortexing between removing each aliquot to ensure that the same account of virus was

placed in each tube. The aliquots were placed into sterile, labeled microcentrifuge tubes.

The samples were then stored in the -80oC freezer. Because this was the fifth lung

75 passage of this virus stock, the new viral stock was designated as “5LP”.

To test the virulence of the new 5LP Influenza A/Puerto Rico/8/1934 H1N1 viral stock and the 4LP X31 H3N2 viral stock and determine the LD50 challenge dilution, a

series of viral dose comparisons were tested in 6-7 week old Swiss Webster female mice and BALB/c female mice since both mouse strains are used as model systems for influenza animal research performed in Dr. Adler’s laboratory. Groups of 6-7 week old

Swiss Webster and BALB/c mice (N=10 mice/group) were ordered from Harlan

Laboratories (Indianapolis, IN) to be used for dose comparison studies. Each group of

Swiss Webster and BALB/c mice were then challenged with different 5LP dilutions. The dilutions were made in cold, sterile PBS.

To challenge these mice with the new 5LP influenza stock, mice were first sedated with 80 mg/kg Ketamine (Putney Inc., Portland, ME) and 16 mg/kg Xylazine

(SoloPak Laboratories Inc., Elk Grove Village, IL) administered via intraperitoneal injection with a 30-gauge needle. Once the sedation of the mice was confirmed, mice were administered 40 μL of the appropriate viral stock dilution drop-wise to the nares and the mice were allowed to recover. The mice were then monitored for weight loss, disease progression and morbidity for the following 14 days. 40 μl of various dilutions starting at

1:1000 for H1N1 and 1:8 for H3N2 were tested to determine the 10X LD50 for Swiss

Webster and BALB/c female mice. The following viral dilutions were determined to be

10X LD50 challenge dose: 1:3500 5LP PR8 in BALB/c, 1:1500 5LP PR8 in Swiss

Webster, 1:100 4LP X31 in BALB/c and 1:64 4LP X31 in Swiss Webster.

76 II. In-Vivo

Swiss Webster female mice (6-7 weeks old) were ordered from Harlan

Laboratories (Indianapolis, IN). All the mice were housed in a pathogen-free

environment in an ALAAC (Assessment and Accreditation of Laboratory Animal Care)

approved facility in accordance with the California State Polytechnic University, Pomona

Animal Care and Use Committee (ACUC) approved protocols. Animals were housed in

microisolator cages and were fed a standard rodent diet (Teklad Laboratory Rodent diet

#2918 (18% protein), Harlan/Teklad, Madison, WI). Water was available ad libitum to

the mice.

Study I. Investigation of a CMI liposomal M2e vaccine containing the adjuvant, mycoviral dsRNA or MPL against H1N1 or H3N2 influenza A challenge.

For this experiment, ten groups (N=22 mice/groups) of Swiss Webster female mice

were vaccinated with CMI liposomes subcutaneously at day 0, and boosted intranasally at day 28 and 30 and day 56 and 58 following sedation. On day 60, blood and spleens from

7 mice/group were collected and the remaining mice (N =15 mice/groups) were challenged on day 70 with 10 XLD50 H1N1 (PR8) or H3N2 (X-31) Influenza A. On day

60, the blood was collected by cardiac puncture of sedated mice using a 30 gauge needle,

the mice were then euthanized by CO2 asphyxiation and the spleens collected and each

placed in a separate sterile tube. The blood was placed at 4oC for several hours, the cells then removed by centrifugation and the serum collected and frozen in the -80oC freezer in

labeled, sterile microcentrifuge tubes (1 tube/mouse). On day 75, 5 mice/group were

euthanized by CO2 asphyxiation and lungs were collected from the infected mice. The

remaining mice (N=10 mice/group) were monitored for morbidity to day 98 after which

time they were euthanized by CO2 asphyxiation. Spleens were used to obtain splenocytes

77 to test for cytokine production in response to M2e by the ELISpot assay and multiplex

Luminex assay. The serum obtained from the blood was tested for anti-whole virus M2e

IgG by ELISA and anti-M2e IgG1 and anti-M2e IgG2a isotyping by ELISA. The lungs

were used to test for viral burden using a foci assay.

Timeline for H1N1 Challenge

Figure. 31. Timeline for Swiss Webster female mice challenged with H1N1 influenza virus. 110 mice were subcutaneously primed on day 0 with 80 μl of CMI liposomes and intranasally boosted on days 28 and 30 and 56 and 58 with 40 μl/day with CMI liposomes. On days 60 and 61, 35 mice (n=7/group) were euthanized to obtain blood and spleens. Serum obtained from blood was used for anti-whole virus M2e IgG, anti-M2e IgG1 and anti-M2e IgG2a ELISA assay. Splenocytes were obtained from spleens to perform cytokine analysis using ELISpot and multiplex Luminex assays. The remaining 75 mice were IN challenged with 40 μl of 10XLD50 H1N1 (PR8). On day 75, 25 mice (n=5/group) were euthanized to obtain lungs which were used to determine lung viral burden using a foci assay. The remaining 50 mice (n=10 mice/ group) were monitored 2X per day for 28 days for weight change, disease signs, and morbidity to day 98.

Table 5

CMI Liposomal Formulations Containing M2e (1-15) Tested in Swiss Webster Female Mice Challenged with H1N1 Influenza Virus

Group Vaccine Adjuvant M2e (μg/dose) (μg/dose) 1 L-CMI-100 μg mycoviral dsRNA- 100 50 M2e 2 L-CMI-15 μg MPL-M2e 15 50 3 L-CMI-100 μg mycoviral dsRNA 100 0 4 L-CMI-15 μg MPL 15 0 5 PBS 0 0

Timeline for H3N2 Challenge

78 Figure 32. Timeline for Swiss Webster female mice challenged with H3N2 influenza virus. 110 mice were subcutaneously primed on day 0 with 80 μl of CMI liposomes and intranasally boosted on days 28 and 30 and 56 and 58 with 40 μl/day with CMI liposomes. On days 60 and 61, 35 mice (n=7/group) were euthanized to obtain blood and spleens. Serum obtained from blood was used for anti-whole virus M2e IgG, anti-M2e IgG1 and anti-M2e IgG2a ELISA assay. Splenocytes were obtained from spleens to perform cytokine analysis using ELISpot and multiplex Luminex assays. The remaining 75 mice were IN challenged with 40 μl of 10XLD50 H3N2 (X-31). On day 75, 25 mice (n=5/group) were euthanized to obtain lungs which were used to determine lung viral burden by foci assay. The remaining 50 mice (n=10 mice/ group) were monitored 2X per day for 28 days for weight change, disease signs, and morbidity to day 98.

Table 6

CMI Liposomal Formulations Containing M2e (1-15) Tested in Swiss Webster Female Mice Challenged with H3N2 Influenza Virus

Group Vaccine Adjuvant M2e (μg/dose) (μg/dose) 1 L-CMI-100 μg mycoviral dsRNA- 100 50 M2e 2 L-CMI-15 μg MPL-M2e 15 50 3 L-CMI-100 μg mycoviral dsRNA 100 0 4 L-CMI-15 μg MPL 15 0 5 PBS 0 0

Study II. Investigation of ultracentrifugation (UC) and column chromatography (C) isolation methods to obtain mycoviral dsRNA for use in the CMI liposomal M2e vaccine.

For this experiment, seven groups (N=12 mice/ group) of Swiss Webster female

mice were vaccinated with CMI liposomes subcutaneously at day 0, and boosted

intranasally at days 28 and 30 and day 56 and 58. On day 60, blood and spleens (N=5/

group) were collected and the remaining mice (N=7 mice/group) were challenged on day

70 with 10XLD50 H1N1 (PR8). The mice (N=7 mice/ group) were monitored for

morbidity to day 98. Spleens were used to obtain splenocytes to test for cytokine

production in response to M2e by the ELISpot assay and multiplex Luminex assay. The

79 serum obtained from the blood was tested for anti-whole virus M2e IgG, anti-M2e IgG1,

anti-M2e IgG2a and anti-M2e IgG3 isotyping by ELISA.

Timeline for H1N1 Challenge

Figure 33. Timeline for Swiss Webster female mice challenged with H1N1 influenza virus. 84 mice were subcutaneously primed on day 0 with 80 μl of CMI liposomes and intranasally boosted on days 28 and 30 and 56 and 58 with 40 μl/day with CMI liposomes. On days 60 and 61, 35 mice (n=5/group) were euthanized to obtain blood and spleens. Serum obtained from blood was used for anti-whole virus M2e IgG, anti-M2e IgG1, anti-M2e IgG2a and anti-M2e IgG3 by ELISA. Splenocytes were obtained from spleens to perform cytokine analysis using ELISpot and multiplex Luminex assays. The remaining 49 mice were IN challenged with 40 μl of 10XLD50 H1N1 (PR8). The mice were monitored 2X per day for 28 days for weight change, disease signs, and morbidity until day 98.

Table 7

CMI Liposomal Formulations Containing M2e (1-15) and mycoviral dsRNA Purified Via Ultracentrifugation (UC) or Column Chromatography (C) and Tested in Swiss Webster Female Mice Challenged with H1N1 Influenza Virus

Group Vaccine Mycoviral Mycoviral M2e dsRNA UC dsRNA C (μg/dose) (μg/dose) (μg/dose) 1 L-CMI-50 μg mycoviral dsRNA (UC)- 50 0 50 M2e 2 L-CMI-25 μg mycoviral dsRNA (UC)- 25 0 50 M2e 3 L-CMI-25 μg mycoviral dsRNA(C)- M2e 0 25 50 4 L-CMI-15 μg mycoviral dsRNA(C)-M2e 0 15 50 5 L-CMI-5 μg mycoviral dsRNA(C)-M2e 0 5 50 6 L-50 μg mycoviral dsRNA (UC) (No 50 0 50 CMI) 7 PBS 0 0 0

Study III. Comparative efficacy of CMI liposomes containing M2e (1-12) vs M2e (1- 15) and MPL, and testing other adjuvant molecules in Swiss Webster female mice.

For this experiment, ten groups (N=20 mice/group) of Swiss Webster female mice were vaccinated with CMI liposomes subcutaneously at day 0, and boosted intranasally

80 on days 28 and 30 and day 56 and 58. On day 60, serum and spleens (N= 7 mice/ group)

were collected and the remaining mice (N =13 mice/ group) were challenged IN on day

70 with 10XLD50 H1N1 (PR8). Lungs were taken out from infected mice (N=5

mice/group) on day 75. The remaining mice (N= 8 mice/group) were monitored for

morbidity to day 98. Spleens were used to obtain splenocytes to test for cytokine

production in response to M2e by the ELISpot assay and the multiplex Luminex assay.

The serum obtained from the blood was tested for anti-whole virus M2e IgG, anti-M2e

IgG1, anti-M2e IgG2a, and anti-M2e IgG3 isotyping by ELISA. The lungs were used to

test for viral burden using a foci assay.

Timeline for H1N1 Challenge

Figure 34. Timeline for Swiss Webster female mice challenged with H1N1 influenza virus. 200 mice were subcutaneously primed on day 0 with 80 μl of CMI liposomes and intranasally boosted on days 28 and 30 and 56 and 58 with 40 μl/day with CMI liposomes. On days 60 and 61, 70 mice (n=7/group) were euthanized to obtain blood and spleens. Serum obtained from blood was used for anti-whole virus M2e IgG, anti-M2e IgG1, anti-M2e IgG2a, and anti-M2e IgG3 by ELISA. Splenocytes were obtained from spleens to perform cytokine analysis in response to M2e using ELISpot and multiplex Luminex assays. The remaining 130 mice were IN challenged with 40 μl of 10XLD50 H1N1 (PR8). On day 75, 50 mice (n=5/group) were euthanized to obtain lungs which were used to determine lung viral burden using a foci assay. The remaining 80 mice (n=8 mice/ group) were monitored 2X per day for 28 days for weight change, disease signs, and morbidity to day 98.

81 Table 8

CMI Liposomal Formulations Containing M2e (1-12) or M2e (1-15) in Combination with Various Adjuvants Tested in Swiss Webster Female Mice Challenged with H1N1 Influenza Virus

Group Vaccine Adjuvant M2e M2e (μg/dose) (1-12) (1-15) (μg/dose) (μg/dose) 1 L-CMI-M2e (1-12)-MPL 15 50 0 2 L-CMI-M2e (1-12)cD-MPL* 15 50 0 3 L-CMI-M2e (1-12)-mycoviral dsRNA 25 50 0 4 L-CMI-M2e (1-15)-MPL 15 0 50 5 L-CMI-M2e (1-15)-LT1** 6 0 50 6 L-CMI-M2e (1-15)-LT1/MPL** 3/7.5 0 50 7 L-CMI-M2e (1-15)-LT1** 3 0 50 8 L-CMI-M2e (1-15)-Pam3CAG 50 0 50 9 L-MPL (No CMI) 15 0 0 10 PBS 0 0 0

Note. *cD: Cathepsin D added to M2e, **LT1: Lipidated Tucaresol.

Study IV: Investigation of CMI liposomal vaccine formulations containing adjuvants without the antigen protein M2e.

For this experiment, groups (N=10 mice/ group) of Swiss Webster female mice were

vaccinated with CMI liposomes subcutaneously at day 0, and boosted intranasally at days

28 and 30 and days 56 and 58. On day 63, mice were challenged IN with 10X LD50 H1N1

(PR8). The mice were monitored 2X per day for 28 days for weight change, disease

signs, and morbidity until day 91.

Timeline for H1N1 Challenge

Figure 35. Timeline for Swiss Webster female mice challenged with H1N1 influenza virus. 70 mice were subcutaneously primed on day 0 with 80 μl of CMI liposomes and intranasally boosted on days 28 and 30 and 56 and 58 with 40 μl/day with CMI liposomes. On day 63, mice were IN challenged with 40 μl of 10XLD50 H1N1 (PR8). Mice were monitored 2X per day for 28 days for weight change, disease signs, and morbidity until day 91.

Table 9

CMI Liposomal Formulations Containing Various Adjuvants and No M2e Protein Tested in Swiss Webster Female Mice Challenged with H1N1 Influenza Virus

Group Vaccine Adjuvant M2e-HD (μg/dose) (μg/dose) 1 L-MPL-M2e-HD 15 100 2 L-MPL 15 0 3 L-Pam3CAG 50 0 4 L-mycoviral dsRNA 50 0 5 L-1V270 25 0 6 L-lipidated-Tucaresol 6 0 7 L-MPL (no CMI) 15 0

Note. L-MPL-M2e-HD served as a positive control.

Based on increased survival, CMI liposomes L-Pam3CAG and L-mycoviral

dsRNA were selected to be tested for cross protection against H1N1 and H3N2. For this

experiment, four groups (N= 17 mice/group) of Swiss Webster female mice were

vaccinated with CMI liposomes on day 0, and boosted intranasally on days 28 and 30 and

day 56 and 58. On day 60, spleens (N=5/group) were collected for cytokine analysis

using the multiplex Luminex assay. On day 63, the remaining mice were IN challenged

with 10X LD50 H1N1 (PR8). On day 69, lungs were collected (N=5 mice/groug) to

analyze lung viral burden using a foci assay. The remaining mice were monitored 2X per

day for 28 days for weight change, disease signs, and morbidity until day 91. This same

procedure was repeated against 10X LD50 H3N2 (X-31) challenge.

Timeline for H1N1 Challenge

Figure 36. Timeline for Swiss Webster female mice challenged with H1N1 influenza virus. 68 mice were subcutaneously primed on day 0 with 80 μl of CMI liposomes and

83 intranasally boosted on days 28 and 30 and 56 and 58 with 40 μl/day with CMI liposomes. On day 60, mice (N=5 mice/ group) were euthanized to collect spleens. Spleens were homogenized and used to perform cytokine analysis using the multiplex Luminex assay. On day 63, mice were IN challenged with 40 μl of 10XLD50 H1N1 (PR8). On day 69, mice (N= 5 mice/ group) were euthanized to obtain lungs. Lungs were used to determine lung viral burden using a foci assay. The remaining mice were monitored 2X per day for 28 days for weight change, disease signs, and morbidity until day 91.

Table 10

CMI Liposomal Formulations Containing Pam3CAG or mycoviral dsRNA and No M2e Protein Tested in Swiss Webster Female Mice Challenged with H1N1 Influenza Virus

Group Vaccine Adjuvant M2e-HD (μg/dose) (μg/dose) 1 L-MPL-M2e-HD 15 100 2 L-Pam3CAG 50 0 3 L-mycoviral dsRNA 50 0 4 PBS 0 0

Note. L-MPL-M2e-HD served as a positive control.

Timeline for H3N2 Challenge

Figure 37. Timeline for Swiss Webster female mice challenged with H3N2 influenza virus. 68 mice were subcutaneously primed on day 0 with 80 μl of CMI liposomes and intranasally boosted on days 28 and 30 and 56 and 58 with 40 μl/day with CMI liposomes. On day 60, mice (N=5 mice/ group) were euthanized to collect spleens. Spleens were homogenized and used to perform cytokine analysis using the multiplex Luminex assay. On day 63, mice were IN challenged with 40 μl of 10XLD50 H3N2 (X- 31). On day 69, mice (N= 5 mice/ group) were euthanized to obtain lungs. Lungs were used to determine lung viral burden using a foci assay. The remaining mice were monitored 2X per day for 28 days for weight change, disease signs, and morbidity until day 91.

84 Table 11

CMI Liposomal Formulations Containing Pam3CAG or mycoviral dsRNA and No M2e Protein Tested in Swiss Webster Female Mice Challenged with H3N2 Influenza Virus

Group Vaccine Adjuvant M2e-HD (μg/dose) (μg/dose) 1 L-MPL-M2e-HD 15 100 2 L-Pam3CAG 50 0 3 L-mycoviral dsRNA 50 0 4 PBS 0 0

Note. L-MPL-M2e-HD served as a positive control.

III. Liposomal Vaccine Preparation

A. M2e-HD Containing MPL

Preparation of the vaccine formulations using liposomal HD technology were made exclusively by Molecular Express Inc. (Rancho Dominguez, CA). Liposomes were composed of phospholipids, cholesterol, MPL (Sigma-Aldrich, St. Louis, MO) and recombinant M2e fused to a proprietary hydrophobic domain (M2e-HD) dissolved in chloroform, methanol and water (10:10:1). Lipid films were produced by pipetting the organic solution into round bottom glass tubes and evaporating off the solvent at 65oC under nitrogen gas. Residual solvent was removed by placing the tubes under vacuum for

24 hours. The lipid films were hydrated with 9% sucrose/10mM Phosphate Buffer (pH

7.2) and incubated for 10 minutes at 65oC. The multilamellar lipid vesicles (MLV) were then either probe sonicated or extruded via microfluidization to form small unilamellar vesicles (SUVS) or liposomes (<150 nm in diameter). The liposomes were filter sterilized by passing them through a 0.22 μm filter, sized by dynamic light scattering (Microtrac,

Largo, FL) and the preparations stored at 4oC throughout the experimental period.

85 B. Conjugateable Adjuvant Lipid Vesicles (CALV) CMI Liposomes

The Conjugateable Adjuvant Lipid Vesicle (CALV) liposomal vaccine formulations

(utilized in the Conjugatable Lipid Vesicle Kit Study) were prepared by Molecular

Express Inc. (Rancho Dominguez, CA) using a proprietary method. For these studies,

M2e (1-15) was chemically linked using the CMI on some of the lipids of the liposome bilayer. Briefly, lipophilic adjuvants such as Pam3CAG and MPL are mixed with phospholipids, cholesterol, and CMI linker in chloroform/methanol (1:1 v/v). Organic solvents are removed under a stream of nitrogen gas at 65oC. Unilamellar liposomes are formed by hydrating the lipid films with 100 mM sodium phosphate, pH 7.0, and subjected to probe sonication. Water lipophilic adjuvants such as mycoviral dsRNA are added to the liposome as part of the hydration buffer or after the liposomes are prepared.

The liposomes were filter sterilized by passing them through a 0.22 μm filter and sized by dynamic light scattering (Microtrac, Largo, FL).

IV. Adjuvants in Vaccine Efficacy Studies

1. MPL: MPL, the TLR4 agonist, was purchased from Sigma-Aldrich, St. Louis,

MO.

2. Mycoviral dsRNA: The mycoviral dsRNA was prepared from mycoviruses

that infect Saccharomyces cerevisiae. Mycoviral dsRNA (UC) and mycoviral

dsRNA (C) was prepared by Molecular Express, Inc. (Rancho Dominguez,

CA). Below is a modified protocol used to obtain mycoviral dsRNA (UC) or

(C).

a. Mycoviral dsRNA obtained by ultracentrifugation (UC): Yeast cells

naturally infected with the mycoviruses were plated for growth and

86 incubated at 30oC for 24 hours. Yeast cells were inoculated with

mycovirus, incubated at 30oC for 24 hours. The grown yeast cells in

medica were centrifuged 15,200 x g at 4oC for 30 minutes. Pellets

were frozen at -80oC until needed. Pellets were mixed with 1/3 volume

of buffer and ½ volume of glass beads was added to the mixture. The

yeast cell solution was vortexed in 30-second intervals for 9-10

minutes with the beads. The broke up yeast cells were centrifuged for

15 minutes at 3500 x g. The supernatant was centrifuged at 27,200 x g

for 30 minutes at 4oC, twice. An equal volume of Trizol and

chloroform and adjusted to 2% with β-mercaptoethanol were stirred at

20,000 x g for 15 minutes at 4oC. The supernatant was collected and

adjusted to 16% ethanol (v/v) with Absolute Ethanol. The supernatant

in 16% ethanol was centrifuged at 20, 000 x g for 15 minutes at 4oC.

DNA was cleaved with DNAse with 2mM CaCl for 15 minutes at

37oC. b. Mycoviral dsRNA Column Chromatography: mycoviral dsRNA

isolated as described above was further purified by a cellulose column.

A 2-gram cellulose column was washed with 20 resin volumes of STE,

16 % ethanol, 2% β-mercaptoethanol. The column was loaded with the

prepared supernatant and washed with 20 resin volumes with STE, 16

% ethanol, 2% β-mercaptoethanol. The column was washed again with

20 resin volumes with STE and 16% ethanol but no β-

mercaptoethanol. Mycoviral dsRNA was eluted with STE buffer and

87 collected in 2 ml fractions. Isopropanol was added at a 1:1 ratio to the

aqueous supernatant, incubated for 1-2 hours and centrifuged at 10K-

15K x g for 10 minutes at 4oC. The supernatant was discarded. Pellets

were washed with 70% ethanol in RNase-free water and centrifuged at

12,000 x g for 5 minutes at 4oC. The supernatant was discarded.

Pellets were dried under the biohazard hood. Pellets were resuspended

in RNase free water, incubated at 55-60oC for 10-15 minutes, and

centrifuged at 12,000 x g for 1 minute. Mycoviral dsRNA

concentration was assayed to determine the mycoviral dsRNA

concentration by UV spectrophotometry. Samples were run on a 1%

agarose gel to verify viral mycoviral dsRNA, purity and gross

concentration.

3. Pam3CAG: Palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-Ala-Gly-OH

from Bachem, Torrance CA.

4. Lipidated-Tucaresol: Synthesized by the laboratory of Professor Dennis

Carson of the University of California San Diego, San Diego CA.

5. 1V270: Obtained from Professor Kim Janda, The Scripps Institute, La Jolla,

CA.

V. Vaccination

The timing, volume, and other details are varied according to the needs of the study but the procedures remain unchanged.

A. Subcutaneous (SC)

The subcutaneous vaccine priming was administered on day 0. This was done by

88 placing a sterile 30-gauge needle onto a 1 ml syringe, and inserting the needle just

beneath the skin in the inguinal area of the mouse. Mice were injected with 40 μl in the

left side of the body and 40 μl in the right side of the body to prevent leakage of the

vaccine from the inguinal area. Mice were allowed to rest and were monitored for weight

changes every day for 7 days following subcutaneous vaccination. Subsequently, mice

were monitored once per week until the intranasal boosters were given.

B. Intranasal (IN)

Prior to intranasal boosting, mice were first sedated using 80 mg/kg Ketamine

(Putney Inc., Portland, ME) and 16 mg/kg Xylazine (SoloPak Laboratories Inc., Elk

Grove Village, IL) administered via intraperitoneal (IP) injection with a 30-gauge needle.

Once the mice were sedated, 40 μl of the vaccine was administered drop-wise using a p200 micropipette to the nares of the mouse. The head of the mouse was tilted slightly backward to ensure that the vaccine entered the nares. Mice were monitored as they recovered from sedation. Their weights were monitored for changes once a day every day for 7 days following the vaccine boost and then once per week until the mice were challenged.

VI. Intranasal Influenza A Challenge of Mice

The day of the challenge varied by study (see study timelines above) but was always performed as described below. The mouse lung passaged viral stock of Influenza 5LP

A/Puerto Rico/8/1934 H1N1 or 4LP X31 H3N2 was first thawed on ice and brought to the proper dilution (1:1500 viral dilution for H1N1 and 1:64 viral dilution for H3N2) to facilitate a 10X LD50 challenge using ice cold, sterile PBS. Mice were moved to the biohazard room of the Animal Facility (Building 92, California State Polytechnic

89 University, Pomona, CA) and sedated with Ketamine and Xylazine as explained above.

Once the mice were sedated, 40 μl of the viral dilution was added via p200 micropipette

drop-wise to the nares. The mice were monitored for recovery from sedation and then

monitored twice a day for 28 days for disease progression, weight loss, and survival. At

28 days post challenge, surviving mice were euthanized via CO2 asphyxiation.

VII. Cardiac Puncture

On the appropriate day indicated by the study timeline, mice were utilized for

splenocyte and blood collection. Mice were first sedated with Ketamine and Xylazine as

described above. To collect the blood, a 25-gauge needle was attached to a 1 ml syringe

as sterilely as possible. The needle was then inserted in the abdomen just posterior to the

xiphoid process of the animal and then moved into the cardiac cavity by keeping the

needle dorsal to the sternum. The needle will then penetrate the heart and blood was

collected until there was about 1 ml total. Once blood was collected, the mice were

usually dead and cervical dislocation was performed to ensure the death of the animal.

Once the mice were confirmed dead, the mice were prepared for the spleen collection

procedure. The blood was then stored at 4oC overnight and spun at 10,000 rpm for 5 minutes the next day. The serum was collected and stored at – 80oC until it was used for

ELISA antibody isotyping and ELISA anti-whole virus M2e IgG.

VIII. Splenocyte Collection

On the same day as blood collection (cardiac puncture), spleens were also collected for use in the ELISpot and multiplex Luminex assays (See below). In brief, the spleens from the mice utilized for blood collection were sterilely collected using sterile forceps and scissors and placed in a 70 μm cell strainer (Thermo-Fisher Scientific, Waltham,

90 MA) which was set on top of a 50 ml conical tube containing 50 ml of CMEM media.

The splenocytes were then removed from the spleen using a rubber plunger from a 1 ml

syringe by tapping motion, pushing the splenocytes through the strainer. The splenocytes

in the tube were pelleted by centrifugation at 1500 rpm for 10 minutes and re-suspended

in 1 ml of lysis buffer to lyse the erythrocytes for 1 minute. 10 ml of CMEM media was

then added to neutralize the lysis buffer and the cell suspension was centrifuged at 1500

rpm for 10 minutes. This process of lysing erythrocytes was done twice. On the last

centrifugation cycle, the splenocytes were re-suspended in 1 ml of CMEM media, and

counted with a hemocytometer for viable cells. This suspension was adjusted to a

concentration of 2x106 cells/ml using trypan blue. This suspension was then used in the

ELISpot and Luminex cytokine secretion assays as described below.

IX. Lung Collection

Lungs were collected from virally challenged mice when mice were seen to be extremely sick. Depending on whether H1N1 or H3N2 was used, this process typically took 4-6 days. Mice were euthanized via CO2 asphyxiation. The lungs were sterilely

removed using sterile forceps and scissors and placed in previously weighed 5 ml culture

tubes. The 5 ml culture tubes containing the lungs were weighed. This allowed us to

determine the weight of the lungs in grams. Before homogenizing the lungs, 1 ml of C-

DMEM was added to the 5 ml culture tube containing the weighted lung. The lungs were

placed on ice and then homogenized using a Dremel 300 hand homogenizer (Racine, WI)

with isopropanol sterilized 7 mm probe using 1 to 3 small burst lasting 3 to 5 seconds.

After homogenizing the lungs, the lung homogenates were stored in 200 μl aliquots and

frozen back at 80oC. The lung homogenates were used for the cytolysis and foci assays

91 which are described below.

X. Spleen Collection

For Study IV, spleens were collected 3 days after the last vaccine boost to perform a multiplex Luminex assay to analyze various cytokines. Mice were euthanized via CO2 asphyxiation. The spleens were sterilely removed using sterile forceps and scissors and placed in 5 ml culture tubes containing 0.5 ml of EMEM tissue culture media. The spleens were then homogenized using a Dremel 300 hand homogenizer

(Racine, WI) as described above with isopropanol sterilized 7 mm probe. After homogenizing, the spleens were stored in 200 μl aliquots and frozen back at -80oC. The spleen homogenates were used for the multiplex Luminex assay which is described below. For Study IV, the spleen homogenate was used rather than the splenocyte supernatant because the mice from this study did not receive an antigen protein that when delivered to the splenocytes could stimulate the production of cytokines.

XI. CO2 Asphyxiation

Mice were euthanized using compressed medical grade CO2 in gas cylinders since this is the only acceptable source of CO2 for euthanasia. Moribund and/or sick mice were euthanized separately from healthy mice. First, moribund and/or sick mice were separated from healthy mice and placed into a euthanasia chamber meeting the space guidelines as specified by “Guide for the Care and Use of Laboratory Animals 8th Ed.”

The CO2 cylinder was turned on (small mouse cages: 1 liter/minute; large mouse cages:

4.5 liters/minute) to slowly expose the mice to increasing levels of CO2. This is important because sudden exposure to high concentrations of CO2 may cause distress to mice. Flow was maintained for at least 1 minute after apparent clinical death. The CO2 valve was

92 turned off allowing excess CO2 remaining in the line to bleed out. Death was verified by cervical dislocation. Euthanasia chambers were cleaned between animals or groups of animals to minimize odors that might distress animals prior to being euthanized. Animals were not euthanized in animal housing rooms except during quarantine due to influenza infection. This procedure is in compliance with the 2013 AVMA Guidelines for

Euthanasia.

XII. In-Vitro Techniques

A. Cytolysis Assay

Purpose. The cytolysis assay gives a qualitative assessment of the level of virulence of a particular influenza virus stock based on the level of lysis of MDCK cells. The higher the viral dilution that causes cell lysis, the more cytolytic the virus stock.

Reagents. All reagents were prepared using ultrapure water and filter sterilized.

• CMEM

o 89% MEM, 10% FBS, 1% penicillin-strepomycin

• PBS

o 1.095 g Na2HPO4, 0.315 g NaH2PO4 and 8.5 g NaCl in ultrapure H20 (pH 7.2)

• Trypsin with ethylene-diamine-tetraacetic acid (EDTA)

• 100% Methanol

• 0.1% crystal violet in 20% ethanol

• 0.4% Trypan Blue Solution in sterile distilled water

Overview. All of the following procedures were done in the BSL2 Biosafety cabinet and performed using sterile technique. The viral stock was first thawed on ice to maintain viral integrity. Once thawed, two-fold dilutions of the virus were made in cold C-DMEM

93 in a sterile 24 well tissue culture plate (Thermo-Fisher Scientific, Waltham, MA), with a

final volume of 200 μl of each viral dilution per well. The plate was kept on ice while the

MDCK cells were prepared.

MDCK cells (P72-75) were grown in a T75 tissue culture flask (Sigma Aldrich, St.

Louis, MO) to 90-95% confluency. Cells were trypsinized as previously described. The

viable cell concentration was determined using Trypan Blue Exclusion (described above)

and adjusted using C-DMEM to a concentration of 1 x 106 cells/ml. After the cells were

prepared, 200 μl of the cell suspension was added to each well of the 24 well tissue

culture plate containing the aforementioned viral dilutions. The plate was then incubated

o at 37 C and 5% CO2 for 48 hours.

Figure 38. Example of a typical cytolysis plate. The 24 well flat bottom tissue culture plate illustrated here indicates a typical set up for a cytolysis assay. The numbers inside of the wells indicate the dilution of the virus used for the assay. The cell and virus controls, “Cell ctl” and “Virus Ctl”, respectively, are also indicated.

94 Following the incubation, each well was rinsed once with 300 μl of PBS per well for

1 minute. The PBS was removed via aspiration and 300 μl of 100% methanol was added

to the wells to fix the cells and then the cells were incubated for 20 minutes at room temperature. After incubation, methanol was removed via rapid inversion. The cells were then stained with 300 μl of 0.1% crystal violet in 20% ethanol and incubated at room temperature for 10 minutes. The cells were gently rinsed using deionized water by flooding the plate repeatedly until the wash water ran clear. The plates were then tapped dry, inverted, and allowed to air-dry overnight.

The titer for the cytolysis assay was the last well showing any cell lysis as compared to the MDCK cell control, which contained no virus and had no lysis. The cytolysis assay titer was represented as the reciprocal of the highest dilution of virus showing any cell lysis (e.g. 1:256 cytolysis titer would be represented as 256).

B. Foci Assay

Purpose. The Foci assay is used to determine the number of infectious virions found in lung tissue or in a viral stock. In this way the viral load of the tissue or viral stock can be quantitatively determined.

Reagents. All reagents were prepared using ultrapure water and filter sterilized.

• PBS

• Trypsin-EDTA

• Monoclonal mouse anti-NP influenza Antibodies

o 1:100 dilution in cold PBS

• 3% methylcellulose (diluted in DMEM)

• Dilution media

95 o PBS with 10 % FBS

• Developing substrate

o 25.7 ml Na2HP04 (0.1M) mixed with 24.3 ml citric acid (0.05M) and 20 mg of

diaminobenzidine and 20 µl H202

• Horse Radish Peroxidase-Labeled Goat anti mouse IgG

o 1:1000 dilution diluted with dilution media

• Complete Dulbecco’s Modified Eagles Medium

• 0.4% Trypan Blue Solution

• 4% Formaldehyde

• 0.5% Triton X (diluted in PBS)

Overview. All of the following procedures were done in the BSL2 Biosafety cabinet and performed using sterile technique. Frozen lung homogenates collected from mice 4-6

days post viral challenge were thawed on ice to maintain viral integrity. After the lung

homogenates were thawed, they were diluted to a 1:10-1:80 starting dilution, followed by

a series of 2 fold dilutions in 24 well plates using cold, sterile C-DMEM in a final volume

of 200 μl/well of lung homogenate. The 24 well plates were then placed on ice until the

MDCK cells were prepared.

MDCK cells (P72-75) were grown in a T75 tissue culture flask (Sigma Aldrich,

St. Louis, MO) to 90-95% confluency. Cells were trypsinized as previously described.

The viable cell concentration was determined using Trypan Blue Exclusion (described

above) and adjusted using C-DMEM to a concentration of 7x106 cells/ml. After the cells

were prepared, 200 μl of the cell suspension was added to each well of the 24 well tissue

96 culture plates containing the above-mentioned lung tissue homogenate dilutions (Figure

o 37). The plates were then incubated at 37 C and 5% CO2 for 6 hours.

During the 6-hour incubation time, a 3% w/v methylcellulose solution was prepared

in C-DMEM by heating the media and methylcellulose (Sigma-Aldrich, St. Louis, MO)

in a boiling water bath until solubilized and then placed in a 37oC water bath until it

cooled to 37oC (approximately 1 hour). Upon reaching a temperature of 37oC, 300 μl of

the 3% w/v methylcellulose solution in C-DMEM was added slowly to each well of the tissue culture plates, which had been incubating with the lung homogenate dilutions for

o the previous 6 hours. The plates were incubated at 37 C with 5% CO2 for 42 hours.

Figure 39. Example of a Foci assay plate set up. The lung homogenate from each mouse is diluted in one row. The numbers in each of the wells indicate the dilution of the lung homogenate. Here there is a 2 fold dilution series.

After the incubation period, the well contents from each plate were removed via rapid

inversion and cells were washed with 300 μl of PBS 3 times to remove the remainder of

the methylcellulose. The cells were then fixed using 400 μl per well of 4% formaldehyde

(Ted Pella Inc., Redding, CA) diluted in PBS, and incubated at room temperature for 30

97 minutes. Cells were washed again with PBS 3 times and then permeabilized with 300 μl of 0.5% Triton-X 100 (Sigma-Aldrich, St. Louis, MO) per well for 5 minutes. To make this solution, water should first be heated in a 37oC water bath as this allows the Triton-X to dissolve easily. Cells were washed 5 times with 300 μl per well of PBS. Cell monolayers were then blocked using 300 μl of a 10 % FBS solution in PBS at room temperature for 90 minutes. Following the blocking procedure, the cells were washed three times again with PBS and 150 μl of 1:100 diluted mouse anti-Nucleo Protein 1

(NP1) IgG (Millipore, Temecula, CA) in PBS was added to each well.

Figure 40. Example of a Foci assay as seen through an inverted microscope. The regions of dark brown indicate a high concentration of Mouse Anti-NP1 antibody bound to the Influenza NP-1 protein being produced by infected MDCK cells. Anti-NP is used because it is a conserved structural protein found in the viral nucleus. Each Foci is considered to be 1 infectious Influenza A unit (virion) within the lung homogenate. The arrows indicate what is considered a single foci. In this example there are 10 foci.

98 The plate was incubated for another 90 minutes at room temperature. Following the incubation, the cells were washed again (300 μl PBS/well 3 times) and 150 μl of a 1:1000 dilution of goat anti-mouse IgG conjugated Horse Radish Peroxidase (HRP) (Millipore,

Temecula, CA), diluted in 5% FBS in PBS solution, was added to each well and allowed to incubate for another 90 minutes at room temperature. Finally, the cells were washed with 300 μl of PBS 5 times and 200 μl of the developing solution (0.365 g 0.1 M

Na2HPO4 in 25.7 ml of PBS, 0.314 g 0.05 M citric acid in 24.3 ml of PBS, 20 mg diaminobenzidine, and 20 μl H2O2) was added to each well and the plates were allowed to develop in the dark for 10-30 minutes. Brown spots appeared in the wells when the plates were finished developing (Figure 39). The plates were flooded with deionized water to stop the reaction, inverted over a paper towel and allowed to air dry overnight.

Foci were counted using a hand counter and an inverted microscope by tedious examination. The counts from at least 3 wells were adjusted for the dilution factor and averaged to determine viral load of that mouse. The viral load was expressed as the number of Foci (foci of infection) per gram of lung homogenate.

C. Enzyme Linked Immunosorbent Assay (ELISA)

Purpose. Though the ELISA has many applications, in the present study, it was used to determine the anti-M2e IgG1, anti-M2e IgG2a, and anti-M2e IgG3 isotypes that were present in the serum of vaccinated mice.

Reagents. All reagents were prepared using ultrapure water and filter sterilized.

• Blocking buffer

o 5% Heat inactivated Fetal Bovine Serum (FBS) (Gibco Rochester, NY) in sterile PBS

99 • Coating Buffer

o 4.2 g NaHCO3 and 3.56 g Na2CO2 in ultrapure H20 (pH 9.5)

• Phosphate Buffered Saline (PBS)

o 1.095 g Na2HPO4, 0.315 g NaH2PO4 and 8.5 g NaCl in ultrapure H20 (pH 7.2)

• Assay Diluent (Blocking Buffer)

o 5% Heat inactivated Fetal Bovine Serum (FBS) (Gibco, Rochester, NY) in sterile PBS.

• Wash Buffer

o 0.05% Tween-20 (Sigma Aldrich, St. Louis, MO) diluted in PBS.

• Capture Antigen

o Lyophilized M2e (1-15 aa) prepared by Molecular Express Inc. (Rancho Dominguez, CA) was diluted in sterile ultrapure water to a specified

concentration (e.g. 2 mg/ml) and stored at -80o C. The aliquots were thawed

before use and added to Coating Buffer to a final concentration of 25 μg/ml of

M2e immediately before use.

• Detection Antibody

o The rat anti mouse IgG1, IgG2A, and IgG3 antibodies from BD Biosciences (San Diego, CA) were diluted from the stock solution to 2 μg/ml with assay

diluent just before use.

• Strepavidin Horse Radish Peroxidase (HRP)

o The streptavidin HRP from BD Biosciences (San Diego, CA) was diluted 1:1000 with assay diluent just before use.

• Substrate Reagent

100 o The TMB substrate reagent used for the ELISA was purchased from BD Biosciences (San Diego, CA) and stored at 4oC until its expiration date. It was

prepared by mixing equal parts of reagent A and B (proprietary formulation)

just before it was needed in the assay and kept in the dark at all times.

• Standards for IgG1, IgG2a, and IgG3 antibodies of known concentrations were

purchased from BD Biosciences (San Diego, CA) and were serially diluted to a

starting concentration of 960 pg/mL.

Overview. Each well of the 96-well, Immulon coated, flat bottom plates (Dynex

Technologies, Chantilly, VA) was coated with 100 μl of the capture antigen (M2e 1-15 aa

peptide, 2.5 μg/well) in coating buffer. The plate was sealed with parafilm and stored at

4oC for at least 8 hours or overnight. Also at this time, the Standards for the mouse IgG1

and IgG2a (BD Biosciences, San Diego, CA) were prepared by diluting the stock

solutions in coating buffer to a concentration of 960 pg/ml followed by 11 two-fold

dilutions. The Standard dilutions for IgG1 and IgG2a were used to coat 3 wells/dilution,

with 100 μl/well. The standard for IgG3 was prepared by diluting the stock solution in

coating buffer to a concentration of 500 ng/ml followed by 11 two-fold dilutions. It was

used to coat 3 wells/dilution, with 100 μl/well. The Standard plates were sealed with

parafilm and stored overnight at 4oC. Following incubation of the plates (test and

standards) at 4oC, the contents of the wells were removed by inversion and the plates were washed 3 times with wash buffer making sure to remove the well contents by gently tapping after the final wash. Each well was blocked using 300 μl of blocking buffer and incubated for 2 hours at room temperature.

101 Table 12

Typical 96-Well Plate Set Up for ELISA

Note. The numbers in each of the wells indicates the dilution factor of the serum in that well. As can be seen, a 2-fold serial dilution was used. Only 8 dilutions were used to minimize the amount of M2e protein needed to coat the plates. The blank wells were also used to obtain an average of the background.

During the incubation with the blocking buffer, sera from each of the test mice were

diluted in 96-well round bottom, microtiter plates in a 2-fold dilution series from 1:200 to

1:25600. The dilutions were done in assay diluent and the final volume of each well was

150 μl. Although prepared close to the time they were needed, they were stored on ice

until they were actively being used.

After the 2 hour incubation, the blocking buffer from the test plates was removed by

rapid inversion and the plates washed five times with washing buffer, being sure to tap

out the contents after the final wash. From each of the plates containing serum dilutions,

100 μl of each dilution was transferred to the test plate well that had been coated

with M2e. The test plates were then incubated at room temperature for 2 more hours

while the standard plates were blocked with blocking buffer.

Following this incubation, the contents of the wells were removed by rapid inversion

and the plates were washed 5 times with wash buffer. Biotinylated Rat anti-mouse IgG1,

IgG2a or IgG3 (BD Biosciences, San Diego, CA) detection antibodies were diluted in dilution buffer (assay buffer) to 2 μg/ml final concentration. Once diluted, 100 μl of the

102 detection antibody was then added into the appropriate wells and the plates were allowed

to incubate for another 2 hours at room temperature.

After the incubation with the detection antibody to IgG1, IgG2a, or IgG3 the plates

were inverted to remove the well contents and washed 7 times with washing buffer being

sure to blot after the final wash. The Strep-HRP 1:1000 dilution was added to each well

(100 μl/well) and the plates were allowed to incubate for 30 minutes at room temperature.

During this incubation the TMB substrate solution was prepared and kept in the dark.

Following the incubation with the Strep-HRP, the plates were washed 7 times with wash buffer, being sure to blot dry at the end, and then 100 μl of the TMB substrate was added to each well. The plates were allowed to develop between 5-30 minutes. The developing reaction was stopped with 100 μl of 1M Phosphoric Acid/ well. The plates were then read in a SpectraMax 250 Microplate Spectrophotometer (Molecular Devices Inc, Sunnyvale,

CA) at 450 nm. The serum concentrations for each antibody isotype were calculated using the standard curve for each of the standards and adjusting for serum dilution. The average concentration of at least 3 wells was used to make an accurate measurement. The results were represented as pg/ml or ng/ml of serum.

D. H1N1 or H3N2 Whole Virus Enzyme Linked Immunosorbent Assay (ELISA)

Purpose. The whole virus ELISA was used to determine the concentration of anti-

M2e IgG, from serum in vaccinated mice that binds to H1N1 or H3N2.

Materials. All reagents were prepared using ultrapure water and filter sterilized.

• Blocking buffer

o Made using filter sterilized PBS

• Coating Buffer

103 o 4.2 g NaHCO3 and 3.56 g Na2CO2 in ultrapure H20 (pH 9.5)

• Phosphate Buffered Saline (PBS)

o 1.095 g Na2HPO4, 0.315 g NaH2PO4 and 8.5 g NaCl in ultrapure H20 (pH 7.2)

• Assay Diluent (Blocking Buffer)

o 5% Heat inactivated Fetal Bovine Serum (FBS) (Gibco, Rochester, NY) in sterile PBS.

• Wash Buffer

o Tween-20 (Sigma Aldrich, St. Louis, MO) diluted to 0.05% with PBS.

• Capture Antigen Preparation

o o MDCK derived H1N1 or H3N2 virus was removed from the -80 C freezer and thawed on ice. The virus was then centrifuged for 10 minutes at 1000 rmp and

the supernatant which contained the virus was collected. The virus was then

diluted 1:8 H1N1 or 1:32 H3N2 in coating buffer. The diluted viral stocks

were then placed in a sterile, small polystyrene petri dish (50 x 15 mm) and

placed 14 cm from a UV lamp source located inside a level II Biohazard hood

for 45 minutes and then stored at 4oC. Right before coating the plate, the virus

was then further diluted in coating buffer to 1:32 H1N1 and 1:64 H3N2.

• Detection Antibody

o The rat anti mouse IgG antibodies from BD Biosciences (San Diego, CA) were diluted from the stock solution to 2 μg/ml with assay diluent just before

use. The stock solution was stored at 4oC until the expiration date.

• Strepavidin Horse Radish Peroxidase (HRP)

104 o The streptavidin HRP from BD Biosciences (San Diego, CA) was diluted 1:1000 with assay diluent just before use. The stock solution was stored at 4o

C until its expiration date.

• Substrate Reagent

o The TMB substrate reagent used for the ELISA was purchased from BD Biosciences (San Diego, CA) and stored at 4oC until its expiration date. It was

prepared by mixing equal parts of reagent A and B just before it was needed

in the assay and kept in the dark at all times.

Overview. Each well of the 96-well, Immulon coated, flat bottom plates (Dynex

Technologies, Chantilly, VA) was coated with 100 μl of the H1N1 1:32 or H3N2 1:64 virus in coating buffer. The plate was sealed with parafilm and stored at 4oC for at least 8

hours or overnight. Also at this time, the Standard for the mouse IgG (BD Biosciences,

San Diego, CA) was prepared by diluting the stock solution in coating buffer to a

concentration of 960 pg/ml followed by 11 two-fold dilutions. The standard dilution for

IgG was used to coat 3 wells/dilution, with 100 μl/well. The standard plates were sealed

with parafilm and stored overnight at 4oC. Following incubation of the plates (test and

standard) at 4oC, the contents of the wells were removed by inversion and the plates were

washed 3 times with wash buffer being sure to remove the well contents by gently

tapping after the final wash. Each well was blocked using 275 μl of blocking buffer and

incubated for 2 hours at room temperature.

105 Table 13

Typical 96-Well Plate Set Up for ELISA

Note. The numbers in each of the wells indicates the dilution factor of the serum in that well. As can be seen, a 2-fold series dilution was used. The last well in each row was left blank to get an average for the background.

During the incubation with the blocking buffer, sera from each of the test mice were diluted in 96-well round bottom, microtiter plates in a two-fold dilution series from 1:200 to 1:25600. The dilutions were done in assay diluent and the final volume of each well was 150 μl. Although prepared close to the time they were needed, they were stored on ice until they were actively being used.

After the 2 hour incubation, the blocking buffer from the test plates was removed by rapid inversion and the plates washed five times with washing buffer, being sure to tap out the contents after the final wash. From each of the plates containing serum dilutions,

100 μl of each dilution was transferred to the test plate well that had been coated with

H1N1 or H3N2 whole virus. The test plates were then incubated at room temperature for

2 more hours while the standard plates were blocked with blocking buffer.

Following this incubation, the contents of the wells were removed by rapid inversion and the plates were washed 5 times with wash buffer. Rat anti-mouse IgG-HRP (BD

Biosciences, San Diego, CA) detection antibodies were diluted in dilution buffer (assay buffer) to 2 μg/ml final concentration. Once diluted, 100 μl of the detection antibody was then added into the appropriate wells and the plates were allowed to incubate for another

106 2 hours at room temperature. During this incubation the TMB substrate solution was prepared and kept in the dark. After the incubation with the detection antibody to IgG, the plates were inverted to remove the well contents and washed 7 times with washing buffer being sure to blot after the final wash. Following the incubation, 100 μl of the TMB substrate was added to each well. The plates were allowed to develop between 5-30 minutes. The developing reaction was stopped with 100 μl of 1M Phosphoric Acid/ well.

The plates were then read in a SpectraMax 250 Microplate Spectrophotometer

(Molecular Devices Inc, Sunnyvale, CA) at 450 nm. The serum concentrations for IgG were calculated using the standard curve. The average concentration of at least 3 wells was used to make an accurate measurement. The results were represented as pg/ml of serum.

E. Enzyme Linked Immunosorbent Spot Assay (ELISpot)

Purpose. The ELISpot Assay was used to quantify the number of cells that secreted a specific cytokine. For these experiments, the ELISpot was used to quantify the number of

IFN-γ or IL-4 secreting splenocytes. In this way, the type of immune response being stimulated by a vaccine could be characterized (i.e. IFN-γ indicating a Th1 response and

IL-4 indicating a Th2 response). Each spot that develops on an ELISpot plate represents one cell actively secreting the cytokine of interest.

Reagents. All buffers were sterilized and kept sterile using sterile technique. Only the wash buffers were allowed to be non-sterile as they were required at the end of the procedure.

• Coating Buffer/ELISpot Phosphate Buffered Saline (ES-PBS)

107 o 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, in 1 L of DI H2O (pH 7.2)

• Blocking Buffer

o Eagles Minimal Essential Media (EMEM) (Media Tech Inc, Manassas, VA) made complete with 10% FBS, 1% penicillin and streptomycin, (Media Tech

Inc, Manassas, VA), and 1% Essential Amino Acids (Media Tech Inc,

Manassas, VA)

• Wash Buffer I

o PBS with 0.5% Tween-20 (Sigma Aldrich, St. Louis, MO)

• Wash Buffer II

o PBS

• Dilution Buffer

o PBS with 10% FBS

• Treatment Solutions

o Positive Control for Cell Stimulation: C-MEM with 5 μg/ml concanavalin A (con A) (Sigma-Aldrich, St. Louis, MO), M2e Test Media: C-MEM with 25

μg/ml M2e (1-15 aa), Negative Control for Cell Stimulation: C-MEM only

• Capture Antibodies

o Rat anti-mouse IFN-γ or IL-4 diluted 1:200 in dilution buffer

• Detection Antibodies

o Biotinylated rat anti-mouse INF- γ or IL-4 diluted 1:200 in dilution buffer

• Enzyme Conjugate

o 1:100 horseradish peroxidase conjugated to strepavidin diluted in dilution

108 buffer

o Substrate Solution

o AEC Substrate Reagent Set from BD BioSciences (San Diego, CA) was prepared according to the manufacturer’s instructions (1 drop of substrate

concentrate to 1 ml of substrate diluent) and kept in the dark

• Stopping solution

o Deionized water Overview. Each well of the 96 well, flat bottom, ELISpot plates (BD Biosciences,

San Diego, CA) were coated with either rat anti-mouse IFN-γ or IL-4 purified antibodies

(BD Biosciences, San Diego, CA) diluted to 1:200 in dilution buffer (100 μl/well). The plates were incubated at 4oC for a minimum of 8 hours or overnight. Following the incubation, the wells were blocked using 200 μl/well of blocking buffer for a minimum of

2 hours at room temperature. During the blocking period, spleens were collected from euthanized mice (see description above) and passed through 70 μm cell strainers

(Thermo-Fisher Scientific, Waltham, MA). Erythrocytes were lysed using AKC lysing

buffer (Gibco, Grand Island, NY). Following this step, 100 μl/well of splenocytes at a

concentration of 2 x 106 cells/ml were added to all wells of the ELISpot plates coated

with the capture antibody and containing 100 μl/well of con A (0.5 μg/well), M2e (2.5

o μg/well), or just C-MEM. The plates were incubated at 37 C with 5% CO2. The length of

time the plates were incubated depended upon the cytokine in question, i.e. 24 hours for

IFNγ and 48 hours for IL-4.

Following incubation, the well contents were removed via aspiration and the plates

were washed with deionized water 5 times and 3 times with wash buffer 1, respectively.

109 To each well 100 μl of a 1:250 dilution of biotinylated rat anti-mouse IFNγ or IL-4 (BD

Sciences, San Diego, CA) diluted in dilution buffer was added and allowed to incubate for 2 hours at room temperature. After the incubation, the plates were washed 3 times with wash buffer 1 and then 100 μl of 1:100 horseradish peroxidase with streptavidin conjugate (BD Biosciences, San Diego, CA) diluted in dilution buffer was added to each well and the plate was incubated in the dark for 1 hour at room temperature. Finally, following the incubation, the plates were washed 5 times with wash buffer II and then

100 μl of the AEC substrate added to each well in the plate. The plates were then allowed to develop in the dark for 5-60 minutes. The reaction was stopped by adding deionized water when the wells had developed completely but not overdeveloped. Development was indicated by the appearance of red spots, with a lot of spots in the con A wells. Wells were rinsed once with deionized water and then tapped to remove the deionized water.

Plates were then allowed to dry overnight by first taking off the removable backs of the plates, inverting them over a paper towel (to catch run-off) and air drying overnight in a dark location.

110

Figure 41. Example of an ELISpot plate. Pictured here is an example of what a typical ELISpot plate looks like after developing. The three treatments of Concanavalin A, M2e and C-EMEM are indicated along the top. Each spot indicates a cell that has secreted the cytokine of interest, which in this case was IFN-γ.

Plates were read by Cellular Technology Limited, Shaker Heights, OH. Results were recorded as the number of cytokine secreting splenocytes, either IFN-γ or IL-4, per 2x106 cells.

F. Multiplex Cytokine Secretion Assay (Luminex)

Purpose. The Luminex assay was used to determine how much of a given cytokine was present in a solution. For these studies, the Luminex assay was used to determine the amount of cytokines secreted by the splenocytes harvested from mice and incubated with concanavalin A, M2e or C-MEM. It was also used to determine the amount of different cytokines present in the spleen tissue of infected mice. Unlike the ELISpot assay, the

Luminex assay is able to detect multiple cytokines simultaneously by using antibodies

111 specific for different cytokines attached to fluorescent beads, which fluoresce at unique

wavelengths depending upon the particular anti-cytokine antibody. When the test solution

is added to this mixture of antibody-tagged fluorescent beads, a given cytokine will bind

to the beads, which have the antibody specific for that cytokine. Binding of this cytokine

to the beads is then detected by using a detection antibody. Known amounts of a given

cytokine are mixed with the beads and used to create a standard curve of fluorescence

versus cytokine concentration.

Reagents. The constituents of the reagents for the BioRad Multiplex Cytokine

Secretion Assay are standardized and supplied by BioRad (Hercules, CA) as part of a kit.

The reagents included in the supplied kit are: standard diluent, assay diluent, detection antibody diluent, and wash buffer.

Overview. The splenocyte supernatants were obtained from the same splenocyte suspensions used for the ELISpot assay but dispensed into round bottom 96-well plates

(100 μl/well at a concentration of 2 x 106 cells/ml). These splenocytes were incubated

with 0.5 μg/well of con A, 2.5 μg/well of M2e or 100 μl of C-EMEM for 48 hours. The supernatants were collected, labeled and stored at -80oC until analyzed. For Study IV,

instead of using the splenocyte supernatants, the spleens were homogenized and the

spleen homogenates used for the Luminex assay. To do this, harvested spleens were

added to a 5 ml culture tube containing 0.5 ml of EMEM and homogenized using a

Dremel 300 hand homogenizer (Racine, WI) with an isopropanol sterilized 7 mm probe.

After homogenizing, the spleen homogenates were divided into 200 μl aliquots and

frozen at -80o C.

The supernatants/spleen homogenates were thawed while the cytokine standards were

112 diluted using the standard diluent. Cytokine specific beads from BioRad (Hercules, CA)

were diluted in assay buffer and added to 96 well filter plates (50 μl/well) (Millipore,

Billerica, MA). 50 μl of each supernatant/spleen homogenate or standard was added into

each well. The plates were sealed with sealer tape and placed on a shaker for 30 seconds

at 1000 rpm and 30 minutes at 300 rpm. The tape was then removed and the well contents

removed via suction. The plates were washed 3 times with wash buffer and then

biotinlyated detection antibodies (anti-cytokine antibodies) were added (BioRad). The

plates were sealed again with sealing tape and the previous incubation steps repeated. The tapes were removed again and Strepavidin Phycoerythrin (PE) (BioRad) was diluted in

assay buffer and added to the plates. The plates were incubated on the shaker again for 10

minutes at 300 rpm. Finally, the plates were washed 3 times with wash buffer and then

assay buffer (100 μl/well) was added and used to resuspend the beads in the wells. The

plates were read on a Luminex 100 platform. Data from the Luminex were analyzed

using Masterplex 2.0 (Hitachi Solutions America, South San Francisco, CA).

XIII. Statistical Analysis

All data, either generated from in vivo studies or in vitro assays, were analyzed for

statistical significance using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla,

CA). The chosen statistical test for each of the data sets was selected based upon the

appropriate conditions for the test. Each of the tests used are indicated on the statistical

analysis tables found in the Results section (Chapter 3). p < 0.05 was considered

significant.

113 CHAPTER 3

Results

Study I: Investigation of a CMI liposomal M2e vaccine containing the adjuvant, mycoviral dsRNA or MPL, against H1N1 influenza A challenge.

Previous studies in our laboratory have shown that liposomes with the adjuvant

MPL and the M2e protein fused to a proprietary hydrophobic protein (M2e-HD) stimulated significant protection against (PR8) H1N1 infection. With the advent of the technology by Molecular Express Inc. to conjugate the M2e to the liposomal surface via comaleimide (CMI) linker, we needed to test the efficacy of this L-CMI-M2e in the

H1N1 infection model since purification of the hydrophilic M2e from the E. coli was markedly easier to perform than purification of the hydrophobic M2e-HD. In addition, based on the previous research done by Dr. Adler-Moore, it was reported that mycoviral dsRNA was more immunostimulating than synthetic Poly (I:C) dsRNA (Wright & Adler-

Moore, 1985), and recent literature reported that Poly (I:C) was a TLR3 agonist (Zhou et al., 2014). Therefore, we decided to compare vaccine formulations containing M2e and mycoviral dsRNA or MPL, and hypothesized that Swiss Webster female mice could be equally protected from a lethal challenge of H1N1 influenza virus when vaccinated with a CMI liposomal M2e vaccine containing either mycoviral dsRNA or MPL.

The experimental groups and timeline of the experiment were as follows:

1. L-CMI-100 μg mycoviral dsRNA-50 μg M2e

2. L-CMI-15 μg MPL-50 μg M2e

3. L-CMI-100 μg mycoviral dsRNA

4. L-CMI-15 μg MPL

5. PBS

114

A. CMI Liposomal M2e vaccine containing mycoviral dsRNA protects against H1N1 viral challenge.

Groups of mice were vaccinated with L-CMI-100 μg mycoviral dsRNA-M2e, L-

CMI-15 μg MPL-M2e, L-CMI-100 μg mycoviral dsRNA, L-CMI-15 μg MPL or PBS.

Mice given L-CMI-100 μg mycoviral dsRNA-M2e were the most protected as measured by significantly better survival (60%) than the L-CMI-15 μg MPL (10%) or PBS (10%)

groups (p< 0.012) (Figure 42, Table 14). Mice vaccinated with the other control, L-CMI-

100 μg mycoviral dsRNA with no M2e, had increased survival (40%) compared to L-

CMI-15 μg MPL control (10%) (p=0.010). There was no statistically significant

difference between the survival of mice vaccinated with L-CMI-15 μg MPL-M2e, L-

CMI-100 μg mycoviral dsRNA-M2e, or L-CMI-100 μg mycoviral dsRNA with no M2e

(Figure 42), underscoring the observation that even without the M2e, the mycoviral dsRNA adjuvant with the CMI-liposomes provided significant protection.

With respect to disease signs, the differences between the groups paralleled the

results from the survival data (Figure 43D, Table 16). In comparison, the weight loss data showed that all groups by day 28 had similar weights. This is probably the result of the severity of the infection leading to severe weight loss even in the groups that did survive, and these mice were slowly gaining weight by the end of the study on day 28.

The data indicates that L-CMI-100 μg mycoviral dsRNA-M2e generated a protective adaptive immune response while the L-CMI-100 μg mycoviral dsRNA without

the protein was stimulating a protective innate immune response. In contrast, the MPL

adjuvant in the CMI liposomes was only protective if the M2e protein was included.

115

Figure 42. Survival in H1N1 challenged Swiss Webster female mice. Six to seven week old Swiss Webster female mice (n=10/group) were subcutaneously primed on d0, and intranasally boosted on d28 and d56 with L-CMI-(100 μg/dose) mycoviral dsRNA-(50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose)M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. Two weeks after the last vaccination, mice were intranasally challenged with 10X LD50 H1N1 (A/ Puerto Rico/8/1934 PR8). Survival was monitored for 28 days.

Table 14

Log-Rank (Mantel-Cox) Test of Survival for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

116

Figure 43 A, B, C, and D. Weight change and disease signs in H1N1 challenged Swiss Webster female mice. Six to seven week old Swiss Webster female mice (n=10/group) were subcutaneously primed on d0, and intranasally boosted on d28 and d56 with L- CMI-(100 μg/dose) mycoviral dsRNA-(50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose)M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. Two weeks after the last vaccination, mice were intranasally challenged with 10X LD50 H1N1 (A/ Puerto Rico/8/1934 PR8). (A) Weight change over 28 days, (B) weight (g) of mice on day 28, (C) mean disease score over 28 days, (D) disease scores of mice on day 28. Bar indicates the mean ± SEM.

117 Table 15

Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 16

Mann Whitney Non-Parametric Two-Tailed T-Test of Disease Scores on Day 28 for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

B. Mice vaccinated with L-CMI-100 μg mycoviral dsRNA-M2e or L-CMI-15 μg MPL- M2e had decreased lung viral burden.

To analyze the lung viral burden of the different vaccine groups after H1N1

challenge, a viral foci assay was performed. Mice vaccinated with L-CMI-100 μg

mycoviral dsRNA-M2e or L-CMI-15 μg MPL-M2e had significantly less lung viral

burden compared to L-CMI-15 μg MPL and PBS groups (p <0.029) (Figure 44, Table

17). Additionally, mice vaccinated with L-CMI-100 μg mycoviral dsRNA-M2e had

significantly less viral burden when compared to mice vaccinated with L-CMI-100 μg

mycoviral dsRNA without the M2e protein (p < 0.029) (Figure 44, Table 17). These

results indicated that vaccine formulations containing the antigen M2e in combination

with mycoviral dsRNA or MPL were more effective in reducing the lung viral burden

118 after H1N1 challenge than the liposomes without M2e. These results also emphasize the

importance of evaluating treatments based not just on survival, but also by the ability of

the treatment to clear the viral infection from the tissues.

Figure 44. Lung viral burden of Swiss Webster female mice challenged with H1N1. Mice (n=4 or 5/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(100 μg/dose) mycoviral dsRNA-(50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose) M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. Two weeks after the last vaccination, mice were intranasally challenged with 10X LD50 H1N1 (A/ Puerto Rico/8/1934 PR8). Day 5 or 6 post- challenge, mice (n= 4 or 5/group) were euthanized and lungs were collected. The lungs were homogenized and incubated with MDCK cells for 48 hours. Virus was detected by using an anti-NP influenza antibody. An anti-NP IgG detection antibody was added followed by the addition of HRP-labeled goat anti-mouse IgG and the developing substrate. Foci of infection were positively identified by the appearance of an area of brown infected cells. Bar indicates the mean ± SEM.

119

Table 17

Mann Whitney Non-Parametric Two-Tailed T-Test of Viral Burden in H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

C. CMI Liposomal M2e vaccine containing mycoviral dsRNA had a predominantly Th1 response and liposomal M2e vaccine containing MPL had a predominantly Th2 response based on anti-M2e IgG isotype characterization.

The serum levels of anti-M2e IgG2a for the L-CMI-100 μg mycoviral dsRNA-

M2e were significantly higher than serum levels of anti-M2e IgG1, indicating that the response with the mycoviral dsRNA adjuvant favored a Th1 response (Table 18, 19, and

20, Figures 45 A and B). Investigators have reported that a IgG1:IgG2a ratio of <1 indicates a Th1 response (Kanai et al., 2007) and in this experiment, the ratio of IgG1:

IgG2a was 0.15. In comparison, L-CMI-15 μg MPL-M2e gave a IgG1:IgG2a ratio of

1.16 and this would correlate with a Th2 response, underscoring the difference in the way that the adjuvants stimulate the adaptive immune response. The other vaccine formulations without M2e, as one would expect, had no anti-M2e IgG of either isotype.

120

Table 18

Isotype Ratio of IgG1/IgG2a Used to Determine the Dominant Adaptive Immune Response Induced by the Liposomal M2e Vaccines With mycoviral dsRNA or MPL

Note. Values indicate the anti-M2e IgG1 or anti-M2e IgG2a concentration in pg/ml.

Figure 45 A and B. Anti-M2e IgG1 and IgG2a production in Swiss Webster female mice vaccinated with CMI liposomal vaccine formulations. Mice (n=6 or 7/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(100 μg/dose) mycoviral dsRNA-(50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose) M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. Blood was collected by cardiac puncture on all uninfected mice three days after the last boost, and the serum was separated by centrifugation. ELISA plates were coated with M2e protein and incubated with different dilutions of the serum from each mouse. Mouse Anti-IgG1 or Anti-IgG2a detection antibody was added to each well in the plate to determine the concentration of anti-M2e IgG1 or anti-M2e IgG2a. (A) anti-M2e IgG1 production, (B) anti-M2e IgG2a production. Bar indicates the mean ± SEM.

121

Table 19

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG1 in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 20

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG2a in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

D. L-CMI-15 μg MPL-M2e had an increased number of IFN-γ secreting splenocytes compared to the PBS control group.

To determine if the liposomal M2e vaccines containing mycoviral dsRNA or

MPL would cause a difference in the number of IFN-γ or IL-4 secreting splenocytes, an

ELISpot assay was performed. Mice vaccinated with L-CMI-15 μg MPL-M2e had

significantly higher numbers of IFN-γ secreting splenocytes compared to mice vaccinated

with PBS (p < 0.033) (Figure 46A, Table 21) but the amount of IFN-γ secreting

splenocytes was not different amongst the other vaccine groups. For the IL-4 secreting splenocytes, there was also no difference amongst all groups. Unlike the IgG isotyping data, the ELISpot data did not show a dominant adaptive Th1 or Th2 response.

122

Figure 46 A and B. ELISpot assay to determine the number of IFN-γ and IL-4 secreting splenocytes. Mice (n=6 or 7/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(100 μg/dose) mycoviral dsRNA-(50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose) M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. Splenocytes were collected from the spleen of all uninfected mice three days after the last boost. Plates were coated with an anti-IFN-γ or anti-IL-4 capture antibody. Splenocytes were added and stimulated with M2e protein. Anti-IFN-γ or anti-IL-4 detection antibody and developing substrate were added. Number of IFN-γ or IL-4 secreting splenocytes were identified by the appearance of a red spot on the plate which was quantified by Cellular Technology Limited, Shaker Heights, OH. (A) IFN-γ secreting splenocytes, (B) IL-4 secreting splenocytes. Bar indicates the mean ± SEM.

Table 21

Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ Secreting Splenocytes

Note. Value of p < 0.05 was considered statistically significant.

123 Table 21

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-4 Secreting Splenocytes

Note. Value of p < 0.05 was considered statistically significant.

E. CMI Liposomal M2e vaccine containing mycoviral dsRNA or MPL or mycoviral dsRNA without M2e protects against H3N2 viral challenge.

In the second part of this experiment, the effectiveness of vaccines containing

M2e and mycoviral dsRNA or MPL adjuvants was tested in mice given an H3N2 influenza A challenge. This allowed us to determine if these vaccine formulations could provide cross protection against two different strains of influenza A.

The experimental groups and timeline were as follows:

1. L-CMI-100 μg mycoviral dsRNA-50 μg M2e

2. L-CMI-15 μg MPL-50 μg M2e

3. L-CMI-100 μg mycoviral dsRNA

4. L-CMI-15 μg MPL

5. PBS

Mice vaccinated with L-CMI-100 μg dsRNA-M2e (90% survival), L-CMI-15 μg

MPL-M2e (60% survival) or L-CMI-100 μg mycoviral dsRNA (70% survival) had

significantly higher survival against an H3N2 influenza challenge compared to control

groups, L-CMI-15 μg MPL and PBS (p < 0.017) (Figure 47, Table 23). Disease scores on

124 day 28 paralleled survival results (Figure 48D, Table 25). This data indicates that

liposomal vaccine formulations containing the adjuvants mycoviral dsRNA or MPL and

the antigen M2e or the adjuvant mycoviral dsRNA without M2e provide protection

against an H3N2 influenza challenge.

Figure 47. Survival in H3N2 challenged Swiss Webster female mice. Six to seven week old Swiss Webster female mice (n=10/group) were subcutaneously primed on d0, and intranasally boosted on d28 and d56 with L-CMI-(100 μg/dose) mycoviral dsRNA-(50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose)M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. Two weeks after the last vaccination, mice were intranasally challenged with 10X LD50 H3N2 X-31. Survival was monitored for 28 days.

Table 23

Log-Rank (Mantel-Cox) Test of Survival for H3N2 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

125

Figure 48 A, B, C, and D. Weight change and disease signs in H3N2 challenged Swiss Webster female mice. Six to seven week old Swiss Webster female mice (n=10/group) were subcutaneously primed on d0, and intranasally boosted on d28 and d56 with L- CMI-(100 μg/dose) mycoviral dsRNA-(50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose)M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. Two weeks after the last vaccination, mice were intranasally challenged with 10X LD50 H3N2 X-31. (A) Weight change over 28 days, (B) weight (g) of mice on day 28, (C) mean disease score over 28 days, (D) disease scores of mice on day 28. Bar indicates the mean ± SEM.

126 Table 24

Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H3N2 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 25

Mann Whitney Non-Parametric Two-Tailed T-Test of Disease Scores on Day 28 for H3N2 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

F. Mice vaccinated with the liposomal M2e vaccine containing mycoviral dsRNA had less lung viral burden compared to all other vaccine groups

To measure the lung viral burden post-challenge, a viral foci assay was

performed. Mice vaccinated with L-CMI-100 μg mycoviral dsRNA-M2e had

significantly less lung viral burden when compared to all other vaccine groups and the

control, including the other M2e containing vaccine, L-CMI-15 μg MPL-M2e (p < 0.016)

(Figure 49, Table 26). This same group had the most survival following H3N2 infection

(Figure 47). Mice vaccinated with L-CMI-15 μg MPL-M2e or L-CMI-100 μg mycoviral dsRNA or L-CMI-15 μg MPL had significantly less viral burden when compared to the

PBS control group (p < 0.016). The L-CMI-15 μg MPL-M2e or L-CMI-100 μg

mycoviral dsRNA vaccines also had enhanced survival while in comparison, the L-CMI-

15 μg MPL vaccine had very poor survival indicating that the mycoviral dsRNA adjuvant

127 alone or with M2e was a better immunostimulator in this infection than the MPL adjuvant alone. These results again underscore the importance of examining both survival and viral burden when assessing the efficacy of a given treatment.

Figure 49. Lung viral burden of Swiss Webster female mice challenged with H3N2. Six to seven week old female Swiss Webster mice (n=10/group) were vaccinated d0 subcutaneously, d28 and d56 intranasally with L-CMI-(100 μg/dose) mycoviral dsRNA- (50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose)M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. On day 6, mice (n= 4 or 5/group) were euthanized and lungs were collected. The lungs were homogenized and incubated with MDCK cells for 48 hours. Virus was detected by using an anti-NP influenza antibody. An anti-NP IgG detection antibody was added followed by the addition of HRP-labeled goat anti-mouse IgG and the developing substrate. Foci of infection were positively identified by the appearance of an area of brown infected cells. Bar indicates the mean ± SEM.

128 Table 26

Mann Whitney Non-Parametric Two-Tailed T-Test of Viral Burden in H3N2 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

G. CMI Liposomal M2e vaccine containing mycoviral dsRNA had a predominantly Th1 response and CMI liposomal M2e vaccine containing MPL had a predominantly Th2 response based on anti-M2e IgG isotype characterization

The serum levels of anti-M2e IgG2a for L-CMI-100 μg mycoviral dsRNA-M2e

were significantly higher than the serum levels of anti-M2e IgG1, indicating that the

response with the mycoviral dsRNA adjuvant favored a Th1 response (Table 27, 28, and

29, Figures 50A). As mentioned above, investigators have reported that a IgG1:IgG2a

ratio of <1 indicates a Th1 response (Kanai et al., 2007) and in this experiment, the ratio

of IgG1: IgG2a was 0.36. In comparison, the L-CMI-15 μg MPL-M2e gave a

IgG1:IgG2a ratio of 3.88 and this would correlate with a Th2 response, underscoring the difference in the way that the adjuvants stimulate the immune response. The other vaccine formulations without M2e, as one would expect, had no anti-M2e IgG of either isotype. However, the L-CMI-100 mycoviral μg dsRNA without M2e stimulated an innate immune response which was as protective in the influenza model as the liposomal

M2e vaccine with MPL or mycoviral dsRNA that stimulated an adaptive immune response to the M2e. These results support the findings of the previous experiment.

129 Table 27

Isotype Ratio of IgG1/IgG2a Used to Determine the Dominant Adaptive Immune Response Induced by the Liposomal M2e Vaccines with mycoviral dsRNA or MPL

Note. Values indicate the anti-M2e IgG1 or anti-M2e IgG2a concentration in pg/ml.

Figure 50 A and B. Anti-M2e IgG1 and anti-M2e IgG2a production in mice vaccinated with CMI liposomal vaccines. Mice (n=6 or 7/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(100 μg/dose) mycoviral dsRNA- (50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose)M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. Blood was collected by cardiac puncture on all uninfected mice three days after the last boost, and the serum was separated by centrifugation. ELISA plates were coated with M2e protein and incubated with different dilutions of the serum from each mouse. Mouse anti-IgG1 or anti-IgG2a detection antibody was then added to each well in the plate to determine the concentration of anti-M2e IgG1 or anti-M2e IgG2a based on a standard curve of known concentrations of IgG1 and IgG2a. (A) Anti-M2e IgG1 production, (B) anti-M2e IgG2a production. Bar indicates the mean ± SEM.

130

Table 28

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG1 in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 29

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG2a in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

H. Mice vaccinated with the CMI liposomal M2e vaccine containing mycoviral dsRNA or MPL had an increased production of IgG antibodies that could bind to the M2e present on the whole virus.

Although the serum from the non-viral challenged mice vaccinated with the CMI

liposomal M2e formulations had high levels of anti-M2e IgG1 and anti-M2e IgG2a, it was important to evaluate if the M2e present on the whole virus was actually accessible to the anti-M2e antibodies that were detected in the isotyping assay. An ELISA assay was performed using a UV inactivated H3N2 virus as the capture antigen, rather than just the

M2e. In this experiment, mice vaccinated with L-CMI-100 μg mycoviral dsRNA-M2e or

L-CMI-15 μg MPL-M2e had elevated levels of IgG in their serum that bound with the

M2e on the whole virus (Figure 51, Table 30). Serum from the mice given non-M2e

vaccines or PBS exhibited background binding of IgG to the whole virus. This data

131 supports the conclusion that the anti-M2e IgG would be able to bind to the whole virus in

a viral challenged animal, and probably contributes to clearance of the virus.

Figure 51. Anti-whole virus M2e IgG production in Swiss Webster female mice vaccinated with CMI liposomal vaccines. Mice (n=5 or 7/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(100 μg/dose) mycoviral dsRNA-(50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose)M2e, L- CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. Blood was collected by cardiac puncture on all uninfected mice three days after the last boost, and the serum was separated by centrifugation. ELISA plates were coated with UV inactivated H3N2 virus and incubated with different dilutions of the serum from each mouse. Mouse anti-IgG detection antibody was added to each well in the plate to determine the concentration of serum IgG that could bind to the M2e protein present on the UV inactivated H3N2 virus based on a standard curve of known concentrations of IgG. Bar indicates the mean ± SEM.

132 Table 30

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-Whole Virus M2e IgG in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

To investigate if the CMI liposomal vaccines enhanced the number of IFN-γ (Th1

response) or IL-4 (Th2 response) secreting splenocytes, an ELISpot assay was

performed. There was no increase in the number of IL-4 secreting splenocytes from the

mice in any of the groups. Only the splenocytes from the mice given the L-CMI-100 μg

mycoviral dsRNA without M2e showed elevated IFN-γ levels that were significantly

higher than the splenocytes from the mice vaccinated with L-CMI-15 μg MPL-M2e or

PBS (Figure 52A, Table 31). Unlike the IgG isotyping data, the ELISpot data did not show a dominant Th1 or Th2 response and the results could not be correlated with the survival and morbidity data. It is possible that we just did not obtain enough splenocytes per mouse to stimulate an adequate response to the M2e.

133

Figure 52 A and B. ELISpot assay to determine the number of IFN-γ and IL-4 secreting splenocytes. Mice (n=5, 6 or 7/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(100 μg/dose) mycoviral dsRNA-(50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose)M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L- CMI-(15 μg/dose) MPL or PBS. Splenocytes were collected from the spleen of all uninfected mice three days after the last boost. Plates were coated with a IFN-γ or IL-4 capture antibody. Splenocytes were stimulated with M2e protein. IFN-γ or IL-4 detection antibody and developing substrate were added. Number of IFN-γ or IL-4 secreting splenocytes were identified by the appearance of a red spot on the plate which was quantified by Cellular Technology Limited, Shaker Heights, OH. A) IFN-γ secreting splenocytes, (B) IL-4 secreting splenocytes. Bar indicates the mean ± SEM.

Table 31

Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ Secreting Splenocytes

Note. Value of p < 0.05 was considered statistically significant.

134 Table 32

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-4 Secreting Splenocytes

Note. Value of p < 0.05 was considered statistically significant.

J. CMI Liposomal M2e vaccine with mycoviral dsRNA had elevated levels of IFN-γ, IL-4, IL-6 and IL-1α and this correlated with its ability to produce the most protection following H1N1 or H3N2 challenge

To measure the cytokines levels released by splenocytes collected 3 days after the

last boost, following stimulation with M2e, a multiplex Luminex assay was performed.

Overall, the data indicates that L-CMI-100 μg mycoviral dsRNA-M2e increased the

levels of IFN-γ, IL-4, IL-6 and IL-1α compared to mice vaccinated with L-CMI-15 μg

MPL-M2e or L-CMI-15 μg MPL (Figure 53C, D, E, and F, Table 35, 36, 37, and 38).

Interestingly, when the vaccine formulation lacked the M2e but had mycoviral dsRNA

(L-CMI-100 μg mycoviral dsRNA) the IL-6 levels were also significantly elevated in

these mice compared to mice vaccinated with L-CMI-15 μg MPL (p <0.048) (Figure

53D, Table 36). And finally, only the mice vaccinated with L-CMI-100 μg mycoviral

dsRNA-M2e had higher levels of IFN-γ compared to vaccine groups L-CMI-15 μg MPL-

M2e, L-CMI-100 μg mycoviral dsRNA or L-CMI-15 μg MPL (p <0.009) (Figure 53D,

Table 36). Given the elevated levels of both Th1 (IFN-γ) and Th2 (IL-4) cytokines, as well as the increased levels of pro-inflammatory cytokines IL-6 and IL-1α by the L-CMI-

100 μg mycoviral dsRNA-M2e, we hypothesize that these cytokines contributed to the better survival seen in the mice given this formulation.

135

136

Figure 53 A, B, C, D, E, F, G and H. Multiplex Luminex cytokine assay to determine concentrations of cytokines secreted by splenocytes stimulated with M2e. Mice (n=5 or 7/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(100 μg/dose) mycoviral dsRNA-(50 μg/dose) M2e, L-CMI-(15 μg/dose) MPL-(50 μg/dose)M2e, L-CMI-(100 μg/dose) mycoviral dsRNA, L-CMI-(15 μg/dose) MPL or PBS. Splenocytes were collected from the spleens of all uninfected mice three days after the last boost. Splenocytes were incubated with M2e protein for 48 hours. The M2e stimulated splenocytes were centrifuged to obtain the supernatant with the secreted cytokines. The supernatant was assayed for cytokine production using a multiplex Luminex assay. (A) IL-1β, (B) TNF-α, (C) IFN-γ, (D) IL-6, (E) IL-4, (F) IL-1α, (G) IL- 10, (H) IL-12. Bar indicates the mean ± SEM.

Table 33

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1β Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

137 Table 34

Mann Whitney Non-Parametric Two-Tailed T-Test of TNF-α Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

Table 35

Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

Table 36

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-6 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

138 Table 37

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-4 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

Table 38

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1α Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

Table 39

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-10 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

139 Table 40

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-12 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

In this study, it was shown that optimal protection was achieved by combining the

antigen M2e with the adjuvant mycoviral dsRNA. This protection was demonstrated by

increased survival, increased levels of anti-M2e IgG1 and IgG2a, increased levels of anti-

whole virus M2e IgG, decreased lung viral burden, and increased levels of IFN-γ, IL-4,

IL-6 and IL-1α. In addition, L-CMI-100 μg mycoviral dsRNA-M2e also provided cross protection in mice challenged with H1N1 or H3N2 influenza virus, underscoring the potential of this vaccine to function as a universal vaccine against influenza.

Interestingly, we also showed that L-100 μg mycoviral dsRNA was capable of providing protection against H1N1 and H3N2. Although this protection was less compared to L-

CMI-100 μg mycoviral dsRNA-M2e, the protection was significantly better compared to control groups L-15 μg MPL or PBS. This highlights the possibility that by activating the innate immune system, mice can be protected from a severe influenza infection.

Study II: Investigation of the effects of different purification methods to obtain mycoviral dsRNA for use in the CMI liposomal M2e vaccine.

Since mycoviral dsRNA was an effective adjuvant as seen in the previous experiment, different purification methods for extracting mycoviral dsRNA from the

Saccharomyces cerevisieae were investigated to determine if extracting more pure mycoviral dsRNA would require that less mycoviral dsRNA to be used in the CMI

140 liposomal vaccines. Additionally, it was important to determine if increasing the purity of

the mycoviral dsRNA would also enhance its efficacy as an adjuvant. We hypothesized

that liposomes with M2e and mycoviral dsRNA purified using a column chromatography

(C) method would provide at least comparable protection compared to the standard method of isolating mycoviral dsRNA by ultracentrifugation (UC). These two isolation techniques are described above in the Materials and Methods section.

The experimental groups and timeline for the experiment were as follows:

1. L-CMI-50 μg (UC) mycoviral dsRNA-50 μg M2e

2. L-CMI-25 μg (UC) mycoviral dsRNA-50 μg M2e

3. L-CMI-25 μg (C) mycoviral dsRNA-50 μg M2e

4. L-CMI-15 μg (C) mycoviral dsRNA-50 μg M2e

5. L-CMI-5 μg (C) mycoviral dsRNA-50 μg M2e

6. L-50 μg (UC) mycoviral dsRNA (No CMI)

7. PBS

A. Mice vaccinated with CMI liposomal M2e containing 50 μg mycoviral dsRNA obtained via ultracentrifugation had increased survival compared to mice vaccinated with CMI liposomal M2e containing 25 μg mycoviral dsRNA obtained via column chromatography.

To determine if the purification method used to obtain the mycoviral dsRNA from

mycoviruses in Saccharomyces cereviseae affects its efficacy as an adjuvant, liposomal

vaccine formulations containing mycoviral dsRNA obtained via UC versus mycoviral

dsRNA obtained via C were compared. Mice vaccinated with L-CMI-50 μg mycoviral

141 dsRNA (UC) M2e had higher survival (100%) when compared to mice vaccinated with

L-CMI-25 μg mycoviral dsRNA (C) M2e (43%) (p < 0.0243) (Figure 54, Table 41).

There was no other statistically significant difference in survival, disease signs or weight loss amongst the vaccine groups although it is clear that the UC groups with M2e did produce better survival that the C groups with M2e. In fact, when 25 μg mycoviral dsRNA (UC) was compared with 25 μg mycoviral dsRNA (C), the survival (71.4% vs

43%), disease signs and weight loss were improved with the mycoviral dsRNA (UC).

The only significant difference we did observe was that mice vaccinated with L-CMI-50

μg mycoviral dsRNA (UC) M2e had lower disease scores compared to mice vaccinated with L-CMI-5 μg mycoviral dsRNA (C) M2e (p <0.0408). The reason that there is also no significant difference in survival between the mice given L-CMI-50 μg mycoviral dsRNA (UC) M2e versus PBS is because the PBS group accidentally received a less lethal dose of virus due to an improper viral challenge. We concluded from this study that the mycoviral dsRNA obtained by UC was stimulating a better protective immune response than the mycoviral dsRNA obtained by C.

142

Figure 54. Survival in H1N1 challenged Swiss Webster female mice. Six to seven week old Swiss Webster female mice (n=10/group) were subcutaneously primed on d0, and intranasally boosted on d28 and d56 with L-CMI-(50 μg/dose) mycoviral dsRNA (UC)- (50 μg) M2e, L-CMI-(25 μg/dose) mycoviral dsRNA (UC)- (50 μg/dose) M2e, L-CMI- (25 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, L-CMI-(15 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, L-CMI-(5 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, Non-CALV-(50 μg/dose) mycoviral dsRNA (UC), or PBS. Two weeks after the last vaccination, mice were challenged intranasally with 10X LD50 PR8 H1N1.

143 Table 41

Log-Rank (Mantel-Cox) Test of Survival for H1N1 Challenged Swiss Webster Female Mice

Note. Value p < 0.05 was considered statistically significant.

144

Figure 55. Weight and disease scores in H1N1 challenged Swiss Webster female mice. Six to seven week old Swiss Webster female mice (n=10/group) were subcutaneously primed on d0, and intranasally boosted on d28 and d56 with L-CMI-(50 μg/dose) mycoviral dsRNA (UC)- (50 μg) M2e, L-CMI-(25 μg/dose) mycoviral dsRNA (UC)- (50 μg/dose) M2e, L-CMI- (25 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, L-CMI- (15 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, L-CMI-(5 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, Non-CALV-(50 μg/dose) mycoviral dsRNA (UC), or PBS. Two weeks after the last vaccination, mice were challenged intranasally with 10X LD50 PR8 H1N1. (A) Weight change over 28 days, (B) weight (g) of mice on day 28, (C) mean disease score over 28 days, (D) mean disease scores of mice on day 28. Bar indicates the mean ± SEM.

145 Table 42

Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 43

Mann Whitney Non-Parametric Two-Tailed T-Test of Disease Scores on Day 28 for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

146 B. All CMI liposomal vaccines containing M2e and mycoviral dsRNA (UC or C) had more pronounced Th1 responses than Th2 responses based on the increased levels of anti-M2e IgG2a versus anti-M2e IgG1

Overall, 50 μg of mycoviral dsRNA (UC) in the liposomes with M2e lead to significantly higher anti-M2e IgG1, but not anti-M2e IgG2a or IgG3, compared to mycoviral dsRNA (C) in the liposomes with M2e, except for IgG1 with 15 μg dsRNA

(C) (Table 44, Figure 56 A and B). However, the anti-M2e IgG2a levels (108) for all mycoviral dsRNA formulations with M2e were much higher than the anti-M2e IgG1 levels (107) and the isotype ratio of anti-M2e IgG1/anti-M2e IgG2a showed that all vaccine formulations with mycoviral dsRNA (UC) or mycoviral dsRNA (C) had a dominant Th1/cell mediated response (Table 44). This agrees with our previous experiments (Study 1) showing that CMI liposomal vaccine formulations with M2e and mycoviral dsRNA elicited a primarily Th1 immune response.

It has been reported that IgG3 is involved in viral infections, appearing first during the infection (Vidarsson, Dekkers, & Rispens, 2014). For this reason, we analyzed the levels of anti-M2e IgG3 production in mice vaccinated with the CMI liposomal M2e vaccines with mycoviral dsRNA (UC) or mycoviral dsRNA (C). The anti-M2e IgG3 levels were comparable for the M2e vaccines with mycoviral dsRNA (UC) or mycoviral dsRNA (C), although the anti-M2e IgG3 levels (105) were much lower than what we observed for anti-M2e IgG1 and anti-M2e IgG2a (Figure 56C, Table 47).

147 Table 44

Isotype Ratio of IgG1/IgG2a Used to Determine the Dominant Adaptive Immune Response Induced by CMI Liposomal M2e Vaccines with mycoviral dsRNA Obtained Via UC or C

Note. Values indicate the anti-M2e IgG1 or anti-M2e IgG2a concentration in pg/ml.

148

Figure 56 A, B, and C. Anti-M2e IgG1, anti-M2e IgG2a, and anti-M2e IgG3 production in mice vaccinated with CMI liposomal M2e vaccines and mycoviral dsRNA (UC or C). Mice (n=4 or 5/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(50 μg/dose) mycoviral dsRNA (UC)- (50 μg) M2e, L-CMI-(25 μg/dose) mycoviral dsRNA (UC)- (50 μg/dose) M2e, L-CMI- (25 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, L-CMI-(15 μg/dose) dsRNA (C)- (50 μg/dose) M2e, L- CMI-(5 μg/dose) dsRNA (C)- (50 μg/dose) M2e, Non-CALV-(50 μg/dose) mycoviral dsRNA (UC), or PBS. Blood was collected by cardiac puncture from all uninfected mice three days after the last boost, and the serum was separated by centrifugation. ELISA plates were coated with M2e protein and incubated with different dilutions of the serum from each mouse. Mouse anti-M2e IgG1 or anti-M2e IgG2a or anti-M2e IgG3 detection antibody was added to each well in the plate to determine the concentration of anti-M2e IgG1, anti-M2e IgG2a or anti-M2e IgG3.(A) anti-M2e IgG1 production, (B) anti-M2e IgG2a production, (C) anti-M2e IgG3 production. Bar indicates the mean ± SEM.

149 Table 45

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG1 in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 46

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG2a in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

150 Table 47

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG3 in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

C. Only mice vaccinated with the CMI liposomal M2e vaccines and mycoviral dsRNA UC had elevated levels of anti-M2e antibodies that could bind with the whole virus compared to control groups.

To investigate if the liposomal M2e vaccines containing various doses of

mycoviral dsRNA UC or C caused the production of IgG that would bind to the M2e

present on the whole virus, we performed an ELISA assay using UV inactivated H1N1 as

the capture antigen.

Mice vaccinated with the liposomal M2e vaccine containing 50 μg of mycoviral

dsRNA UC or 25 μg of mycoviral dsRNA UC produced significantly more anti-whole

virus M2e IgG compared to mice vaccinated with the controls containing no M2e, Non-

CALV-50 μg mycoviral dsRNA (UC) and PBS (p < 0.0337) (Figure 57, Table 48). This

was not the case for the mycoviral dsRNA (C) vaccine which had antibody levels

comparable to the controls. In addition, lower levels of anti-whole M2e virus antibodies

151 were produced by the mice given 5 μg mycoviral dsRNA (C) compared to the mice given

25 μg mycoviral dsRNA (UC). This data suggests that mycoviral dsRNA (UC) could produce anti-M2e antibodies that could bind to the M2e present on the whole virus, but the mycoviral dsRNA (C) vaccines with M2e did not do this. This data parallels the survival results since the two vaccine groups given M2e and mycoviral dsRNA (UC) had increased survival relative to all other groups. It also suggests that testing for the binding of the anti-M2e antibodies to the whole virus is a reasonable correlate of protection, unlike the binding of the anti-M2e antibodies to the M2e alone in the isotyping assay.

Figure 57. Anti-whole virus M2e IgG production in Swiss Webster female mice vaccinated with CMI liposomal vaccines. Mice (n=4 or 5/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(50 μg/dose) mycoviral dsRNA (UC)- (50 μg) M2e, L-CMI-(25 μg/dose) mycoviral dsRNA (UC)- (50 μg/dose) M2e, L-CMI- (25 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, L-CMI- (15 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, L-CMI-(5 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, Non-CALV-(50 μg/dose) mycoviral dsRNA (UC), or PBS. Blood was collected by cardiac puncture on all uninfected mice three days after the

152 last boost, and the serum was separated by centrifugation. ELISA plates were coated with UV inactivated H1N1 virus and incubated with different dilutions of the serum from each mouse. Anti-mouse IgG detection antibody was added to each well in the plate to determine the concentration of serum IgG that could bind to the M2e protein present on the UV inactivated H1N1 virus. Bar indicates the mean ± SEM.

Table 48

Mann Whitney Non-Parametric One-Tailed T-Test of Anti-Whole Virus M2e IgG in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

To analyze if there was a difference in the number of IFN-γ (Th1 response) or IL-

4 (Th2 response) secreting splenocytes between the different vaccine groups, an ELISpot

assay was performed. There was no difference amongst any of the groups with respect to

the numbers of IFN-γ secreting splenocytes. In comparison, mice vaccinated with the L-

CMI-50 μg mycoviral dsRNA (UC)-M2e or L-CMI-25 μg mycoviral dsRNA (UC)-M2e had increased levels of IL-4 secreting splenocytes compared to controls (p < 0.046)

(Figure 58B, Table 50). In contrast, only the mice vaccinated with the L-CMI-15 μg

153 mycoviral dsRNA (C)-M2e vaccine had increased levels of IL-4 secreting splenocytes compared to controls (p= 0.0079). This data indicates that mycoviral dsRNA (UC) in combination with the liposomal M2e delivered at 50 μg or 25 μg is better at enhancing the number of IL-4 secreting splenocytes compared to most doses of mycoviral dsRNA

Th2 response with the mycoviral dsRNA (UC), and that is different than what we observed when analyzing the anti-M2e IgG isotyping ratios which indicated that the mycoviral dsRNA (UC) and mycoviral dsRNA (C) stimulated more of a Th1 response. It needs to be emphasized that these two assays measure different aspects of the immune response, with the IgG isotyping focused on the B cell response and the ELISpot assay focused on the T cell response. One might conclude that both the Th1 and Th2 responses are involved in the protection generated by the mycoviral dsRNA adjuvant when it is given with the M2e.

Figure 58 A and B. ELISpot assay to determine the number of IFN-γ and IL-4 secreting splenocytes. Mice (n=4 or 5/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(50 μg/dose) mycoviral dsRNA (UC)- (50 μg) M2e,

154 L-CMI-(25 μg/dose) mycoviral dsRNA (UC)- (50 μg/dose) M2e, L-CMI- (25 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, L-CMI-(15 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, L-CMI-(5 μg/dose) mycoviral dsRNA (C)- (50 μg/dose) M2e, Non- CALV-(50 μg/dose) mycoviral dsRNA (UC), or PBS. Splenocytes were collected from the spleens of all uninfected mice three days after the last boost. Plates were coated with a IFN-γ or IL-4 capture antibody. Splenocytes were added and stimulated with M2e protein. IFN-γ or IL-4 detection antibody and developing substrate were added. Number of IFN-γ or IL-4 secreting splenocytes were identified by the appearance of a red spot on the plate which was quantified by Cellular Technology Limited, Shaker Heights, OH. (A) IFN-γ secreting splenocytes, (B) IL-4 secreting splenocytes. Bar indicates the mean ± SEM.

Table 49

Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ Secreting Splenocytes

Note. Value of p < 0.05 was considered statistically significant.

155 Table 50

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-4 Secreting Splenocytes

Note. Value of p < 0.05 was considered statistically significant.

Study III: Comparative efficacy of CMI liposomes containing M2e (1-12) vs M2e (1- 15) and MPL, and testing other adjuvant molecules in Swiss Webster female mice.

In the previous experiments, it was demonstrated that the M2e epitope composed of the first 15 amino acids of the M2e epitope used in a CMI liposomal vaccine formulation in combination with mycoviral dsRNA or MPL was protective. This M2e epitope was selected because it is highly conserved across various strains of influenza A virus (Schotsaert, De Filette, Fiers, & Saelens, 2009). In this experiment, we explored if using a more conserved M2e epitope composed of the first 12 amino acids would also provide protection when used in combination with MPL or mycoviral dsRNA. We hypothesized that the liposomes with the smaller M2e epitope (amino acids 1-12) would provide comparable protection to the larger M2e epitope (amino acids 1-15) when used in combination with various adjuvants.

156

The experimental groups and timeline for the experiment were as follows:

1. L-CMI- 50 μg M2e(1-15)-15 μg MPL

2. L-CMI- 50 μg M2e(1-15)-50 μg Pam3CAG

3. L-CMI- 50 μg M2e(1-12)-15 μg MPL

4. L-CMI- 50 μg M2e(1-12)-cD-15 μg MPL

5. L-CMI- 50 μg M2e(1-12)-25 μg mycoviral dsRNA

6. L-CMI- 50 μg M2e(1-15)-6 μg LT1

7. L-CMI- 50 μg M2e(1-15)-3 μg LT1/7.5 μg MPL

8. L-CMI- 50 μg M2e(1-15)-3 μg LT1

9. L- 15 μg MPL (no CMI)

10. PBS

A. Mice vaccinated with L-M2e (1-12)-mycoviral dsRNA, L-M2e (1-15)-LT1/MPL or L- M2e (1-15)-Pam3CAG had increased survival compared to mice given the other vaccines.

Mice vaccinated with L-CMI-M2e (1-15)-Pam3CAG (62.5% survival), L-CMI-

M2e (1-12)- mycoviral dsRNA (62.5% survival) or L-CMI-M2e (1-15)-LT1/MPL

(62.5% survival) had the highest survival but only L-CMI-M2e (1-15)-Pam3CAG was statistically significantly higher when compared to mice vaccinated with L-CMI-M2e (1-

12)-MPL (0% survival) or L-CMI-M2e (1-15)- (3 μg/dose)-LT1 (0% survival) (p <

0.0179) (Figure 59, Table 51). Mice vaccinated with L-CMI-M2e (1-15)-LT1/MPL or L-

CMI-M2e (1-15)-Pam3CAG had higher weights on day 28 compared to mice vaccinated

157 with L-CMI-M2e cD (1-12)-MPL (p<0.0463) (Figure 60B, Table 52). Mice vaccinated

with L-CMI-M2e (1-15)-Pam3CAG also had lower disease scores on day 28 than mice

vaccinated with L-CMI-M2e cD (1-12)-MPL, L-CMI-M2e(1-15)- (3 μg/dose)-LT1, L-

MPL (no CMI), or PBS (p<0.0466) (Figure 60D, Table 53).

This data indicates that vaccine formulations containing the antigen protein M2e

(1-15 amino acids) in combination with adjuvants Pam3CAG or LT1/MPL provided the

highest protection based on survival and increased weight by the end of the study on day

28. This data also showed that L-CMI-M2e (1-12)-mycoviral dsRNA was protective, as

indicated by survival that was comparable to that of L-CMI-M2e (1-15)-Pam3CAG. This

protection was probably mediated by the action of the adjuvant mycoviral dsRNA based

on previous experiments (Studies I and II) which have shown that 50 or 100 μg/dose of

mycoviral dsRNA delivered with a CMI liposomal M2e vaccine provided protection

against H1N1 or H3N2 influenza challenge. Notably, the lack of protection by the other

vaccines containing M2e (1-12) indicates that the absence of the amino acids 13, 14 and

15 are critical for stimulating a protective immune response even when they are incorporated into the liposome with the adjuvant MPL.

158

Figure 59. Survival in H1N1 challenged Swiss Webster female mice. Six to seven week old female Swiss Webster mice (n=7 or 8/group) were vaccinated d0 subcutaneously, and d28 and d56 intranasally boosted with L-CMI-(50 μg/dose) M2e(1-12)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-12)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1- 12)-(25 μg/dose) mycoviral dsRNA, L-CMI-(50 μg/dose) M2e(1-15)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-15)-(6 μg/dose) LT1, L-CMI-(50 μg/dose) M2e(1-15)-(3 μg/dose) LT1/ (7.5 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-15)-(3 μg/dose) LT1, L- CMI-(50 μg/dose) M2e(1-15)-(50 μg/dose) Pam3CAG, L-CMI-(15 μg/dose) MPL(no CMI) or PBS. Two weeks after the last vaccination, mice were challenged intranasally with 10X LD50 H1N1 (PR8).

159 Table 51

Log-Rank (Mantel-Cox) Test of Survival for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

160

Figure 60 A, B, C, and D. Weight change and disease scores in H1N1 challenged Swiss Webster female mice. Six to seven week old female Swiss Webster mice (n=7 or 8/group) were vaccinated d0 subcutaneously, and d28 and d56 intranasally boosted with L-CMI-(50 μg/dose) M2e(1-12)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-12)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-12)-(25 μg/dose) mycoviral dsRNA, L-CMI- (50 μg/dose) M2e(1-15)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-15)-(6 μg/dose) LT1, L-CMI-(50 μg/dose) M2e(1-15)-(3 μg/dose) LT1/ (7.5 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-15)-(3 μg/dose) LT1, L-CMI-(50 μg/dose) M2e(1-15)-(50 μg/dose) Pam3CAG, L-CMI-(15 μg/dose) MPL(no CMI) or PBS. Two weeks after the last vaccination, mice were challenged intranasally with 10X LD50 H1N1 (PR8). Mice were monitored for 28 days. (A) Weight change over 28 days, (B) mean weight (g) on day 28, (C) disease scores over 28 days, (D) mean disease scores on day 28. Bar indicates the mean ± SEM.

161 Table 52

Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 53

Mann Whitney Non-Parametric Two-Tailed T-Test of Disease Scores on Day 28 for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

162 B. L-CMI-M2e (1-12) vaccines had an enhanced Th1 cell mediated immune response while in general, L-CMI-M2e (1-15) vaccines had an enhanced Th2 antibody mediated immune response.

The production of anti-M2e IgG1, anti-M2e IgG2a, and anti-M2e IgG3 was

determined for mice vaccinated with various vaccine formulations containing different

adjuvants and the antigen protein M2e (1-12) or M2e (1-15). The isotype ratios of

IgG1/IgG2a were analyzed to determine the dominant adaptive immune response generated by the different vaccines (Table 54). All vaccine formulations containing the smaller more conserved M2e (1-12) had similar patterns with a dominant Th1 cell- mediated immune response regardless of the adjuvant with which it was delivered

(mycoviral dsRNA or MPL) (<1.0). However, the scatter plots (Figures 61) show that skewing to a Th1 response seems to be the result of only a few samples that had elevated

IgG2a concentrations. More likely, the IgG1: IgG2a ratio without these few mice, would indicate that the response is both Th1 and Th2. In contrast, L-M2e(1-15)-LT1/MPL, gave a Th2 response with an ratio of IgG1: IgG2a of 113, while L-M2e(1-15)LT1(6 μg/dose) produced a Th1 response with a isotype ratio of 0.36. The remaining vaccines with the

M2e (1-15) and the adjuvants MPL, LT1 (3 μg/dose) or Pam3CAG seemed to favor a more balanced Th1 and Th2 response. This data indicates that independently of the adjuvant used, M2e (1-12) favored a Th1 and Th2 response. In contrast, the immune response of M2e (1-15) was more dependent on the adjuvant with which it was delivered.

163 Table 54

Anti-M2e IgG1:IgG2a (pg/ml) Ratio of Liposomal Vaccine Formulations

Note. Values indicate the anti-M2e IgG1 or anti-M2e IgG2a concentration in pg/ml.

164

165 Figure 61. Anti-M2e IgG1:IgG2a ratio of L-CMI-M2e (1-12) vs L-CMI-M2e (1-15) vaccines. Mice (n= 5-7/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(50 μg/dose) M2e(1-12)-(15 μg/dose) MPL, L- CMI-(50 μg/dose) M2e(1-12)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-12)-(25 μg/dose) mycoviral dsRNA, L-CMI-(50 μg/dose) M2e(1-15)-(15 μg/dose) MPL, L-CMI- (50 μg/dose) M2e(1-15)-(6 μg/dose) LT1, L-CMI-(50 μg/dose) M2e(1-15)-(3 μg/dose) LT1/ (7.5 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-15)-(3 μg/dose) LT1, L-CMI-(50 μg/dose) M2e(1-15)-(50 μg/dose) Pam3CAG, L-CMI-(15 μg/dose) MPL(no CMI) or PBS. Blood was collected by cardiac puncture on all uninfected mice three days after the last boost, and the serum was separated by centrifugation. ELISA plates were coated with M2e protein and incubated with different dilutions of the serum from each mouse. Mouse anti-IgG1 or anti-IgG2a detection antibody was then added to each well in the plate to determine the concentration of anti-M2e IgG1 or anti-M2e IgG2a. Bar indicates the mean ± SEM.

Table 55

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG1 in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

166 Table 56

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG2a in Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

The anti-M2e IgG3 data showed that mice vaccinated with L-M2e (1-15) LT1 (6

μg/dose) or L-M2e (1-15) LT1 (3 μg/dose) had significantly lower levels of anti-M2e

IgG3 compared to the other M2e (1-15) groups that had the adjuvants MPL, Pam3CAG, or LT1 with MPL (p=0.0006) (Figure 62, Table 57). This again emphasizes the role of the adjuvant in directing the antibody responses.

167

Figure 62. Anti-M2e IgG3 Production in Mice Vaccinated with CMI Liposomal Vaccines. Mice (n= 5-7/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(50 μg/dose) M2e(1-12)-(15 μg/dose) MPL, L- CMI-(50 μg/dose) M2e(1-12)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-12)-(25 μg/dose) mycoviral dsRNA, L-CMI-(50 μg/dose) M2e(1-15)-(15 μg/dose) MPL, L-CMI- (50 μg/dose) M2e(1-15)-(6 μg/dose) LT1, L-CMI-(50 μg/dose) M2e(1-15)-(3 μg/dose) LT1/ (7.5 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-15)-(3 μg/dose) LT1, L-CMI-(50 μg/dose) M2e(1-15)-(50 μg/dose) Pam3CAG, L-CMI-(15 μg/dose) MPL(no CMI) or PBS. Blood was collected by cardiac puncture on all uninfected mice three days after the last boost, and the serum was separated by centrifugation. ELISA plates were coated with M2e protein and incubated with different dilutions of the serum from each mouse. Mouse anti-IgG3 detection antibody was added to each well in the plate to determine the concentration of anti-M2e IgG3. Bar indicates the mean ± SEM.

Table 57

Mann Whitney Non-Parametric Two-Tailed T-Test of Anti-M2e IgG3 in Swiss Webster Female Mice

168

Note. Value of p < 0.05 was considered statistically significant.

C. Mice vaccinated with the smaller more conserved M2e (1-12) had increased levels of IL-6, IL-1α and IL-10 compared to almost all the other vaccines.

We performed a multiplex Luminex assay to analyze if the various vaccines enhanced or decreased the levels of pro-inflammatory and anti-inflammatory cytokines.

There were very few significant differences amongst the vaccine groups for the cytokines

IL-β, TNF-α, IFN-γ, IL-4, and IL-12. We did observe, however, that the vaccine formulations containing M2e (1-12) had a pattern of having significantly higher levels of

IL-1α (pro-inflammatory), IL-6 (pro-inflammatory) and IL-10 which can function as either an anti-or pro-inflammatory cytokine (p<0.039) (Figure 63 A, B, C, E, G, Table

58, 59, 60, 62 and 64). This data again underscores the difference in the immune response

to the M2e (1-12) and the M2e (1-15) and the importance of the molecular structure of

the antigenic epitopes.

169

Figure 63 A, B, C, D, E, F, G and H. Cytokine production by splenocytes from vaccinated female mice prior to H1N1 challenge. Mice (n=4- 5/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L-CMI-(50

170 μg/dose) M2e(1-12)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-12)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-12)-(25 μg/dose) mycoviral dsRNA, L-CMI-(50 μg/dose) M2e(1-15)-(15 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-15)-(6 μg/dose) LT1, L-CMI-(50 μg/dose) M2e(1-15)-(3 μg/dose) LT1/ (7.5 μg/dose) MPL, L-CMI-(50 μg/dose) M2e(1-15)-(3 μg/dose) LT1, L-CMI-(50 μg/dose) M2e(1-15)-(50 μg/dose) Pam3CAG, L-CMI-(15 μg/dose) MPL(no CMI) or PBS. Splenocytes were collected from the spleens of all uninfected mice three days after the last boost. Splenocytes were incubated with M2e protein for 48 hours. Then, the splenocytes and M2e plates were centrifuged to obtain the supernatant. The supernatant was assayed for cytokine production using a Luminex assay. (A) IL-β, (B) TNF-α, (C) IFN-γ, (D) IL-6, (E) IL-4, (F) IL-1 α, (G) IL-10, (H) IL-12. Bar indicates the mean ± SEM.

Table 58

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1β Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

171

Table 59

Mann Whitney Non-Parametric Two-Tailed T-Test of TNF-α Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

Table 60

Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

172

Table 61

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-6 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

Table 62

Mann Whitney Non-Parametric T-Test of IL-4 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

173

Table 63

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1α Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

Table 64

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-10 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

174

Table 65

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-12 Secreted by Splenocytes After a 48 Hour Incubation Period with M2e Protein

Note. Value of p < 0.05 was considered statistically significant.

In summary, the data from this set of studies demonstrated that the molecular structure of the antigen, the type of adjuvant, and the dose of adjuvant play a critical role in determining the degree of immune protection generated by the vaccine. We observed that the M2e (1-15) was a more protective antigenic epitope than the M2e (1-12) except for the M2e (1-12) with the mycoviral dsRNA adjuvant. When the M2e (1-15) was delivered with the LT1 (3 μg/dose)/MPL (7.5 μg/dose), LT1 (6 μg/dose) or Pam3CAG, the protection was significantly better than the groups with no survival. Notably, the immunological data with each of the vaccines varied considerably and could not be used to predict survival following H1N1 viral challenge.

175 Study IV: Investigation of CMI liposomal vaccine formulations containing adjuvants without the antigenic protein M2e (1-15).

When we investigated the efficacy of a liposomal M2e (1-15) vaccine containing mycoviral dsRNA, it was discovered that a liposomal vaccine containing mycoviral dsRNA and no M2e (1-15), used as a control in that experiment, provided significant protection of mice challenged with H1N1 and H3N2 (Study I). Based on these results, we wanted to investigate if liposomal vaccine formulations containing different adjuvants and no M2e protein would be capable of providing protection to mice challenged with

H1N1 or H3N2. To accomplish this, we first performed a screening study of various adjuvants to determine which adjuvants, if any, would provide protection to Swiss

Webster female mice challenged with H1N1 influenza A virus. We hypothesized that different adjuvants when used in a CMI liposomal vaccine without M2e would provide different levels of protection against H1N1 influenza challenge.

For the screening study, the experimental groups and timeline were as follows:

1. L-15 μg MPL-100 μg M2e-HD

2. L-CMI-50 μg Pam3CAG

3. L-CMI-50 μg mycoviral dsRNA

4. L-CMI-15 μg MPL

5. L-CMI-25 μg 1V270

6. L-CMI- 6 μg lipidated-Tucaresol

7. L-15 μg MPL (no CMI)

176 A. Mice vaccinated with L-MPL-M2e-HD, L-CMI-Pam3CAG (no M2e) or L-CMI- mycoviral dsRNA (no M2e) had the most survival.

Mice vaccinated with L-MPL-M2e-HD (75% survival), L-CMI-Pam3CAG (70% survival), L-CMI-mycoviral dsRNA (62.5% survival), L-CMI-MPL (50% survival) had the highest survival which was statistically significantly better than the L-MPL (no CMI)

(10% survival) control group (p<0.0474) (Figure 64, Table 66). Only mice vaccinated

with L-CMI-Pam3CAG or L-CMI-mycoviral dsRNA had increased weight on day 28 compared to the L-MPL (no CMI) group (p<0.0123) (Figure 65B, Table 67). Only mice

vaccinated with L-MPL-M2e-HD or L-CMI-Pam3CAG had significantly decreased

disease signs compared to the L-MPL (no CMI) group (p<0.017) (Figure 65D, Table 68),

but overall, there was a trend to having decreased disease scores on day 28 for all

vaccines that had increased survival.

Figure 64. Survival in H1N1 challenged Swiss Webster female mice. Six to seven week old female Swiss Webster mice (n=8 or 10/group) were vaccinated d0 subcutaneously, and d28 and d56 intranasally boosted with L-(15 μg/dose)MPL-(100 μg/dose)M2e-HD, L-CMI-(50 μg/dose)Pam3CAG, L-CMI-(50 μg/dose ) mycoviral dsRNA, L-CMI-(25 μg/dose )1V270, L-CMI-(25 μg/dose )lipid-Tucaresol, L-CMI-( 15 μg/dose )MPL, or L- (15 μg/dose)MPL(no CMI). One week after the last vaccination, mice were challenged intranasally with 10X LD50 H1N1 (PR8). Survival was monitored for 28 days.

177 Table 66

Log-Rank (Mantel-Cox) Test of Survival for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Figure 65 A, B, C, and D. Survival in H1N1 challenged Swiss Webster female mice. Six to seven week old female Swiss Webster mice (n=8 or 10/group) were vaccinated d0 subcutaneously, and d28 and d56 intranasally boosted with L-(15 μg/dose)MPL-(100 μg/dose)M2e-HD, L-CMI-(50 μg/dose)Pam3CAG, L-CMI-(50 μg/dose ) mycoviral dsRNA, L-CMI-(25 μg/dose )1V270, L-CMI-(25 μg/dose )lipid-Tucaresol, L-CMI-( 15 μg/dose )MPL, or L-(15 μg/dose)MPL(no CMI). One week after the last vaccination, mice were challenged intranasally with 10X LD50 H1N1 (PR8).(A)Weight change over

178 28 days, (B) mean weight (g) on day 28, (C) mean of disease score over 28 days, (D) mean disease scores on day 28. Bar indicates the mean ± SEM.

Table 67

Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 68

Mann Whitney Non-Parametric Two-Tailed T-Test of Disease Scores on Day 28 for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

B. Vaccine formulations L-MPL-M2e-HD and L-CMI-Pam3CAG (no M2e) provided protection against H1N1 but only L-MPL-M2e-HD provided protection against H3N2.

From the screening study, the CMI vaccines with the adjuvants Pam3CAG (TLR2 receptor) or mycoviral dsRNA (TLR3 receptor) with no M2e were selected to be investigated for possible cross protection in Swiss Webster female mice challenged with

H1N1 or H3N2 influenza A viruses. We hypothesized that Pam3CAG or mycoviral dsRNA in the CMI liposomes without M2e, would be capable of protecting Swiss

Webster female mice against a 10X LD50 of PR8 H1N1 or X-31 H3N2 challenge by

179 stimulating only the innate immune response since no antigen protein was added to the

liposomal vaccine formulations.

For the cross protection study, the experimental groups and timeline were as

follows:

1. L-15 μg MPL-100 μg M2e-HD

2. L-CMI-50 μg Pam3CAG

3. L-CMI-50 μg mycoviral dsRNA

4. PBS

With the H1N1 influenza challenge, vaccine formulations L-MPL-M2e-HD

(~43% survival) and L-CMI-Pam3CAG (~57% survival) provided significant protection

compared to the PBS control group (0% survival) (p<0.0161) (Figure 66A, Table 69),

although the results here for H1N1 are different than what we observed in the screening

study with H1N1. In that study, survival rates were higher for the L-MPL-M2e-HD

(75%) as well as the two vaccine formulations that did not contain M2e but had the adjuvant Pam3CAG (70%) or mycoviral dsRNA (62.5%). The reason for this was probably related to the amount of virus that was used for the challenge. The virus is obtained from a frozen stock of infected lung homogenate which is not a homogeneous suspension. Although we vortex the homogenate well between taking each aliquot, the amount of the suspension is not exactly the same in each vial aliquot. It is likely that the vial we used in this experiment had slightly more virus in it. Nevertheless, the Pam3CAG

180 without the M2e does show promise as an effective vaccine against H1N1 challenge which is probably due to its stimulation of the innate immune response.

In comparison, with H3N2 influenza challenge, only mice vaccinated with L-

MPL-M2e-HD had increased survival (100% survival) compared to the PBS control group (~14% survival) (p=0.0015) (Figure 66B, Table 70). It appears that protection against H3N2 by a liposomal vaccine which only contains an adjuvant and no M2e will require higher doses of the adjuvant or more doses of the adjuvant.

181

Figure 66 A and B. Survival in H1N1 or H3N2 challenged Swiss Webster female mice. Six to seven week old female Swiss Webster mice (n=7/group) were vaccinated d0 subcutaneously, and d28 and d56 intranasally boosted with L- (15 μg/dose) MPL-(100 μg/dose ) M2e-HD, L-CMI-(50 μg/dose )Pam3CAG, L-CMI-(50 μg/dose ) mycoviral

182 dsRNA or PBS. One week after the last vaccination, mice were challenged intranasally with 10X LD50 H1N1 (PR8) or H3N2 (X-31). (A) Survival of H1N1 challenged mice, (B) survival of H3N2 challenged mice.

Table 69

Log-Rank (Mantel-Cox) Test of Survival for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 70

Long-Rank (Mantel-Cox) Test of Survival for H3N2 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

183

Figure 67 A, B, C and D. Weight change in H1N1 or H3N2 challenged Swiss Webster female mice. Six to seven week old Swiss Webster female mice (n=7/group) were vaccinated d0 subcutaneously, and d28 and d56 intranasally boosted with L- (15 μg/dose) MPL-(100 μg/dose ) M2e-HD, L-CMI-(50 μg/dose )Pam3CAG, L-CMI-(50 μg/dose ) mycoviral dsRNA or PBS. One week after the last vaccination, mice were challenged intranasally with 10X LD50 H1N1 (PR8) or H3N2 (X-31). (A) Weight change over 28 days for H1N1 challenged mice, (B) mean weight (g) on day 28 for H1N1 challenged mice, (C) weight change over 28 days for H3N2 challenged mice, (D) mean weight (g) on day 28 for H3N2 challenged mice. Bar indicates the mean ± SEM.

184

Figure 68 A, B, C, and D. Disease signs in H1N1 or H3N2 challenged Swiss Webster female mice. Six to seven week old Swiss Webster female mice (n=7/group) were vaccinated d0 subcutaneously, and d28 and d56 intranasally boosted with L- (15 μg/dose) MPL-(100 μg/dose ) M2e-HD, L-CMI-(50 μg/dose )Pam3CAG, L-CMI-(50 μg/dose ) mycoviral dsRNA or PBS. One week after the last vaccination, mice were challenged intranasally with 10X LD50 H1N1 (PR8) or H3N2 (X-31). (A) Disease scores over 28 days for H1N1 challenged mice, (B) mean disease scores on day 28 for H1N1 challenged mice, (C) disease scores over 28 days for H3N2 challenged mice, (D) mean disease scores on day 28 for H3N2 challenged mice. Bar indicates the mean ± SEM.

185 Table 71

Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 72

Mann Whitney Non-Parametric Two-Tailed T-Test of Weight (g) on Day 28 for H3N2 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

Table 73

Mann Whitney Non-Parametric Two-Tailed T-Test for Disease Scores on Day 28 for H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

186 Table 74

Mann Whitney Non-Parametric Two-Tailed T-Test for Disease Scores on Day 28 for H3N2 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

C. Groups with higher survival had lower lung viral burdens

Mice vaccinated with L-MPL-M2e-HD or L-CMI-Pam3CAG and challenged with

H1N1 had decreased viral burden compared to mice vaccinated with PBS (p=0.0079)

(Figure 69A, Table 75). Similarly mice vaccinated with L-MPL-M2e-HD and challenged

with H3N2 had decreased viral burden compared to all other groups (p<0.0159) (Figure

69B, Table 76). For this study, viral burden was a predictor of survival since those groups

with the lowest viral burden had increased survival. Additionally, this data further

supports the observation that a liposomal Pam3CAG vaccine without any M2e stimulates

a protective innate immune response as evidenced by lowering of the lung viral burden.

187

Figure 69 A and B. Lung viral burden of Swiss Webster female mice challenged with H1N1 or H3N2. Six to seven week old female Swiss Webster mice (n=7/group) were vaccinated d0 subcutaneously, and d28 and d56 intranasally boosted with L- (15 μg/dose) MPL-(100 μg/dose ) M2e-HD, L-CMI-(50 μg/dose )Pam3CAG, L-CMI-(50 μg/dose ) mycoviral dsRNA or PBS. One week after the last vaccination, mice were challenged with H1N1 or H3N2. On day 6, mice (n= 4 or 5/group) were euthanized and lungs were collected. The lungs were homogenized and incubated with MDCK cells for 48 hours. Virus was detected by using an anti-NP influenza antibody. An anti-NP IgG detection antibody was added, followed by incubation with HRP-labeled goat anti-mouse IgG. The developing substrate was used to visualize the infected cells which appeared as a brown spot referred to as a foci. (A) Viral burden of H1N1 challenged mice, (B) viral burden of H3N2 challenged mice. Bar indicates the mean ± SEM.

Table 75

Mann Whitney Non-Parametric Two-Tailed T-Test of Viral Burden in H1N1 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

188 Table 76

Mann Whitney Non-Parametric Two-Tailed T-Test of Viral Burden in H3N2 Challenged Swiss Webster Female Mice

Note. Value of p < 0.05 was considered statistically significant.

D. Cytokine levels did not differ between L-MPL-M2e-HD vaccine and the liposomal adjuvant vaccines without M2e.

We did this assay in an attempt to see if the cytokines produced by the whole spleen tissue without any further incubation, would be different when we compared the splenic response from the L-MPL-M2e-HD vaccinated mice with the splenic response from the mice that were vaccinated with the liposomes containing adjuvant but without any M2e. The L-MPL-M2e-HD would have stimulated both an adaptive and innate immune response while the other vaccines without M2e would only be stimulating an innate immune response. We observed no differences in the cytokine production by the spleens of any of the mice. We did this same assay with the spleens from the mice that were later challenged with H3N2, and the results were the same. We have concluded that any differences between the adaptive immune response and the innate immune response should be examined by testing chemokines produced by macrophages or neutrophils, determining the ratio of M1 to M2 macrophages, and possibly NK cell activity.

189

190

Figure 70 A, B, C, D, E, F, G and H. Cytokine production for Swiss Webster female mice prior to H1N1 challenge. Mice (n=4-5/group) were subcutaneously primed on d0 and intranasally boosted on d28 and d56 with L- (15 μg/dose) MPL-(100 μg/dose ) M2e-HD, L-CMI-(50 μg/dose )Pam3CAG, L-CMI-(50 μg/dose ) mycoviral dsRNA or PBS. Spleens were collected from all uninfected mice three days after the last boost, homogenized and the spleen homogenate without any incubation assayed for cytokine production using a Luminex assay. (A) IL-β, (B) TNF-α, (C) IFN-γ, (D) IL-6, (E) IL-4, (F) IL-10, (G) IL-1α, (H) IL-12. Bar indicates the mean ± SEM.

Table 77

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1β in Spleen Homogenate Prior to H1N1 Challenge

Note. Value of p < 0.05 was considered statistically significant.

191 Table 78

Mann Whitney Non-Parametric Two-Tailed T-Test of TNF-α in Spleen Homogenate Prior to H1N1 Challenge

Note. Value of p < 0.05 was considered statistically significant.

Table 79

Mann Whitney Non-Parametric Two-Tailed T-Test of IFN-γ in Spleen Homogenate Prior to H1N1 Challenge

Note. Value of p < 0.05 was considered statistically significant.

Table 80

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-6 in Spleen Homogenate Prior to H1N1 Challenge

Note. Value of p < 0.05 was considered statistically significant.

192 Table 81

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-4 in Spleen Homogenate Prior to H1N1 Challenge

Note. Value of p < 0.05 was considered statistically significant.

Table 82

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-10 in Spleen Homogenate Prior to H1N1 Challenge

Note. Value of p < 0.05 was considered statistically significant.

Table 83

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-1α in Spleen Homogenate Prior to H1N1 Challenge

Note. Value of p < 0.05 was considered statistically significant.

193 Table 84

Mann Whitney Non-Parametric Two-Tailed T-Test of IL-12 in Spleen Homogenate Prior to H1N1 Challenge

Note. Value of p < 0.05 was considered statistically significant.

In summary, Pam3CAG in CMI liposomes without M2e has consistently shown protection against an H1N1 infection although the other adjuvants, including mycoviral dsRNA and MPL, in CMI liposomes without M2e also showed varying degrees of protection against H1N1 infection. Given that there is no M2e in these liposomes, we have to conclude that the protection has been generated by an innate immune response.

We did observe protection against H3N2 challenge when we used 100 μg mycoviral dsRNA, but not 50 μg mycoviral dsRNA, in CMI liposomes without M2e. This demonstrates the importance of the dose of mycoviral dsRNA needed to properly stimulate the innate immune response. Overall, however, the CMI liposomes with M2e and MPL performed as well as or better than the CMI liposomes with only adjuvant, underscoring the value of stimulating both the adaptive and innate immune response to influenza challenge.

194 CHAPTER 4

Discussion

The overall objective of these studies was to investigate the efficacy of CMI

liposomal vaccine formulations containing M2e (1-15) or M2e (1-12) in combination

with various adjuvants or CMI liposomal vaccines with adjuvants and no M2e protein in

Swiss Webster female mice challenged with influenza H1N1 or H3N2.

Previous studies in our laboratory had demonstrated that the liposomal M2e vaccine containing MPL provided protection in mice against an H1N1 and H6N2 influenza A challenge (Ernst et al., 2006). In addition, Dr. Adler-Moore demonstrated that mycoviral dsRNA could function as an adjuvant which provided better immune stimulation compared to synthetic dsRNA, Poly I:C (Wright & Adler-Moore, 1985).

These previous findings, lead us to investigate if CMI liposomal vaccines containing M2e and mycoviral dsRNA or MPL would provide comparable protection in Swiss Webster female mice against an H1N1 or H3N2 influenza A challenge. For this study and subsequent studies, Swiss Webster female mice were selected because they are an outbred strain of mice which provide a more heterogeneous population better representative of the response to influenza A infection by the heterogeneous human population. As our controls, we also investigated the effects of CMI liposomal vaccines containing MPL or mycoviral dsRNA without the antigen M2e protein. Using these various liposomal vaccine formulations would allow us to test if adjuvants enhanced

195 protection when delivered in combination with the antigen compared to delivering the adjuvant alone.

Adjuvants provide protection by enhancing the response of the innate immune system. It has been reported that combining adjuvants with a highly purified antigen such as M2e is necessary to generate protection because small proteins like M2e given alone are less immunogenic (de Sauza Apostolico et al., 2016). In this part of our studies we used the adjuvant mycoviral dsRNA or MPL with M2e, and demonstrated that the liposomal M2e vaccine containing mycoviral dsRNA provided the best protection in

Swiss Webster female mice against H1N1 or H3N2 influenza A challenge. This protection was better than that provided by the CMI liposomal M2e vaccine with MPL although the difference was not significant.

These two adjuvants target different toll-like receptors (TLRs). Monophosphoryl lipid A (MPL) binds to TLR4 which is the only TLR that results in activation of both the

MyD88 and TRIF signaling pathways (Casellas & Mitchell, 2008; Hernandez et al.,

2016). The TLR4 is an extracellular receptor and consequently responds to extracellular pathogens. Activation of the MyD88 and TRIF signaling pathways by MPL leads to neutrophil expansion and mobilization which is regulated by the chemokines CXCL1,

CXCL2, and the hemopoietic factor G-CSF. These pathways also increase MHCII and costimulatory molecule expression along with enhanced chemokine and cytokine secretion (Hernandez et al., 2016).

Mycoviral dsRNA binds with to TLR3 which is an intracellular receptor and

TLR3 is reported to play a role in detecting influenza virus infected cells (Iwasaki &

Pillai, 2014). Thus, utilizing an adjuvant such as mycoviral dsRNA that binds to this

196 intracellular receptor perhaps provides better activation of the innate immune response to

intracellular virus infection than MPL because it mimics the natural infection. This

allows cells to be optimally prepared for an influenza challenge. It is widely accepted that

negative-sense RNA viruses such as Influenza A virus do not produce detectable levels of dsRNA and yet they activate the TLR3 receptor (Weber, Wagner, Rasmussen, Hartmann,

& Paludan, 2006). This has lead to speculation that certain unknown dsRNA structures are produced during replication of the viral RNA by infected cells and these structures are detected by TLR3 (Weber et al., 2006). dsRNA binding to TLR3 activates the TRIF pathway which leads to the production of type I interferon and dendritic cell maturation

(Matsumoto & Seya, 2008).

It is generally accepted that the dsRNA generated during a viral infection and subsequently detected by TLR3 is generated intracellularly and perhaps only exposed to the extracellular space upon cell lysis (Zhou et al., 2013). Thus, it is important to investigate how mycoviral dsRNA enters innate immune cells such as macrophages and dendritic cells to begin the activation of the innate immune system. Our CMI liposomal vaccines containing M2e plus mycoviral dsRNA or only mycoviral dsRNA and no M2e raise the question of how the mycoviral dsRNA enters the immune cell. The CMI liposomal vaccines with M2e or without M2e are prepared by simply adding mycoviral dsRNA to the already made CMI liposomes before injecting the mice. Although it remains unknown exactly how this mycoviral dsRNA enters the cells, the MIC-1 receptor present on several immune cells has been demonstrated to play an important role in mediating TLR-3 dependent and TLR-3 independent innate immune responses to dsRNA

(Collins et al., 2014).

197 In the experiment being discussed, the lung viral burden of mice challenged with

H1N1 or H3N2 was decreased in mice vaccinated with a CMI liposomal M2e vaccine with mycoviral dsRNA or MPL. However, the vaccine formulations with just the mycoviral dsRNA or MPL and no M2e that provided protection did not decrease the viral burden as much as the vaccines which contained M2e. This indicates that survival does not necessarily reflect the ability to clear the viral infection. Thus, when we define a vaccine as being protective, we must look at survival, the ability to promote viral clearance, and decreased morbidity.

As expected, the anti-M2e IgG1 and anti-M2e IgG2a levels for mice vaccinated with the liposomal M2e vaccines containing mycoviral dsRNA or MPL were elevated with only background levels of IgG1 and IgG2a for the serum from the liposomal vaccines without M2e. These two M2e vaccine formulations, one with mycoviral dsRNA, a TLR3 intracellular agonist, and one with MPL, a TLR4 extracellular agonist, provided comparable survival and lung viral clearance, yet the dominant adaptive immune response was different based on the IgG isotype ratio. Mice vaccinated with the CMI liposomal M2e vaccine containing mycoviral dsRNA responded with a more dominant

Th1 cell mediated immune response. Mice vaccinated with the CMI liposomal M2e vaccine containing MPL responded with a more dominant Th2 antibody mediated immune response. The traditional way of developing a vaccine that would be protective has relied on demonstrating that the vaccine would stimulate a strong antibody response.

However, our results indicate that either a Th1 or Th2 response would be protective against influenza infection. Simply knowing that a vaccine stimulates a Th1 or Th2

198 response is not enough to predict if a vaccine will be protective in vivo. It is important to

carry out in vivo studies to test the vaccine in animals.

ELISpot data, which analyzes the number of IFN-γ or IL-4 secreting splenocytes, did not show a dominant adaptive Th1 or Th2 immune response. This was probably due to the low number of splenocytes used to perform this experiment limiting the amount of cytokines that were produced. In our experiments we aimed to achieve a concentration of

2x106 splenocytes per ml/mouse. However, sometimes we are unable to get this

concentration from each mouse. To get more splenocytes, we would need to pool

splenocytes from two animals and this would increase the number of animals needed to

perform the study and the cost of the study.

Like the ELISpot data, the Luminex assay did not show a dominant Th1 or Th2

adaptive immune response based on the level of IFN-γ or IL-4. However, the Luminex

data did show that mice vaccinated with the CMI liposomal M2e vaccine containing

mycoviral dsRNA had increased levels of IFN-γ, IL-6, and IL-1α compared to mice vaccinated with the CMI liposomal M2e vaccine containing MPL. This demonstrated that the adjuvant mycoviral dsRNA generated a pronounced pro-inflammatory immune response compared to the MPL adjuvant.

Overall, from this study we learned that CMI liposomal M2e vaccines containing

100 μg mycoviral dsRNA generated the most protection in mice challenged with H1N1 or H3N2 influenza virus compared to all other CMI liposomal vaccines. Additionally, this vaccine also provided cross protection against two influenza strains (H1N1 or

H3N2). We have shown that optimal protection against an influenza infection (H1N1 or

H3N2) is achieved by combining the antigen M2e and mycoviral dsRNA since this

199 vaccine provided both an adaptive immune response to the M2e and an innate immune response to the mycoviral dsRNA. It is also possible that the M2e is stimulating the innate immune response by binding with other cytosol receptors within the dendritic cells, such as RIG1, NLRP3 and NLR (Teijaro et al., 2014)

We also observed that CMI liposomes with mycoviral dsRNA and no M2e provided increased protection against H1N1 and H3N2 when compared to the other control groups. Although this protection was less than the one provided by the CMI liposome with M2e and mycoviral dsRNA, CMI liposomes with mycoviral dsRNA activated the innate immune system which does not require priming by a specific antigen to produce a protective response to the influenza infection. In addition, CMI liposomes with mycoviral dsRNA are less labor intensive to prepare since the mycoviral dsRNA is simply mixed with the CMI liposomes before administration and do not require the addition of an immunogenic protein. The CMI liposomal vaccine with mycoviral dsRNA and no M2e could be a powerful tool to fight future unexpected epidemics and pandemics since it would not require identifying the HA and NA proteins on the circulating influenza virus strain. In addition, this vaccine could potentially be combined with the current influenza HA vaccines to enhance protection in case of a mismatch of the HA and/or NA to the current circulating influenza strain.

II. CMI liposomal M2e vaccines with mycoviral dsRNA (isolated by ultracentrifugation, UC) provided better protection in Swiss Webster female mice challenged with H1N1 influenza virus compared to mice vaccinated with CMI liposomal M2e vaccines with mycoviral dsRNA (isolated by column chromatography, C).

Given the effectiveness of mycoviral dsRNA as an adjuvant in the CMI liposomal

M2e vaccine formulation when delivered at a dose of 100 μg mycoviral dsRNA, we

200 wanted to investigate if using a column chromatography (C) method to further purify the

mycoviral dsRNA would provide comparable protection to the mycoviral dsRNA

purified by ultracentrifugation (UC). Perhaps by using a more pure mycoviral dsRNA,

the dose of mycoviral dsRNA could be lowered and this would be beneficial because

obtaining mycoviral dsRNA from mycoviruses can be time consuming.

Survival data demonstrated that the CMI liposomal M2e vaccine with 50 μg

mycoviral dsRNA (UC) had significantly better survival compared to mice vaccinated

with the CMI liposomal M2e vaccine with 25 μg mycoviral dsRNA (C). We believe that

this difference was not due to the 50 μg mycoviral dsRNA (UC) dose compared to the 25

μg mycoviral dsRNA (C) dose since CMI liposomal M2e vaccines with 25 μg mycoviral

dsRNA (UC) also had increased survival compared to mice given the CMI liposomal

M2e vaccine with 25 μg mycoviral dsRNA (C), although this difference was not statistically significant.

We hypothesize that we can attribute the enhanced survival observed in the mice

given the 50 μg mycoviral dsRNA (UC) to be due to the purification method used to

isolate the mycoviral dsRNA. It is possible that by using ultracentrifugation to isolate

mycoviral dsRNA, some ssRNA from the yeast cell host remained. However, it is

unlikely that DNA or proteins remained because during the isolation process, DNase and

proteases were used to destroy any DNA or protein present in the sample. ssRNA is

recognized by the TLR7 receptor which leads to the production of type I interferon and

pro-inflammatory cytokines such as TNF-α and IL-6 via the MyD88 signaling pathway

(Brencicova & Diebold, 2013; Guiducci, Coffman, & Barrat, 2008). This would mean

that by using the liposomal M2e with mycoviral dsRNA (UC), we are activating two

201 TLRs and two signaling pathways that lead to increased protection for the mice. On the

other hand, by further purifying the mycoviral dsRNA using column chromatography,

this ssRNA could have been eliminated, reducing the potency of the mycoviral dsRNA

To further analyze the differences between UC and C mycoviral dsRNA, a gel

was performed (data not shown). The data showed that although both mycoviral dsRNA isolation types contained 4.5kb mycoviral dsRNA, the mycoviral dsRNA isolated via UC also had bands that indicated smaller pieces of mycoviral dsRNA. Tests were performed to identify the type of RNA (single-stranded or double-stranded) in these smaller pieces, but the results were inconclusive.

Interestingly, analysis of the anti-M2e IgG isotype ratios (IgG1: IgG2a) showed that liposomal M2e vaccines with mycoviral dsRNA (UC) or mycoviral dsRNA (C) both had a dominant Th1 cell mediated immune response. Although the survival for these two types of vaccines was different, the adaptive immune response that mediated this protection was not different. This underscores the importance of the innate immune system in providing protection against infection. We can hypothesize that although both types of vaccines responded with a Th1 immune response, the vaccines containing M2e with mycoviral dsRNA (UC) provided better activation of the innate immune response via TLR3 and potentially TLR7 receptors. It is also important to note that only liposomal

M2e vaccines with mycoviral dsRNA (UC) had increased anti-M2e that could bind to the whole virus compared to the PBS group. This would mean that liposomal M2e vaccines with mycoviral dsRNA (C) did not produce anti-M2e antibodies that could bind to whole virus since their antibody levels were not statistically different from background.

202 ELISpot data indicated that although the Th1 response was dominant as

demonstrated by the anti-M2e IgG isotype ratio, CMI liposomal M2e vaccines containing

mycoviral dsRNA (UC) had increased levels of IL-4 (Th2 response) relative to the PBS control. CMI liposomal M2e vaccines with mycoviral dsRNA (C) only had an increased number of IL-4 secreting splenocytes compared to the PBS control when delivered at 15

μg. These results demonstrate that both adaptive immune responses (Th1 and Th2) are likely activated by the CMI liposomal M2e vaccines containing mycoviral dsRNA (UC).

In conclusion, we have demonstrated that mycoviral dsRNA isolated by

ultracentrifugation and delivered at 50 μg or 25 μg in a CMI liposomal M2e vaccine,

provides better protection than mycoviral dsRNA, isolated by column chromatography

delivered at 25 μg, 15 μg, or 5 μg doses, in the CMI liposomal M2e vaccine. These

results underscore the importance of both the adaptive and innate immune responses in

mediating protection against an influenza infection.

III. CMI Liposomes containing the larger M2e (amino acids 1-15) when used in combination with the adjuvant Pam3CAG or MPL + Tucaresol provided increased protection in Swiss Webster female mice challenged with H1N1 influenza virus compared to mice vaccinated with CMI Liposomes containing a smaller M2e (amino acids 1-12) with the MPL adjuvant.

To continue our investigation of the CMI liposomal M2e vaccine, we tested

whether a smaller M2e (amino acids 1-12) that is more conserved than the 1-15 M2e

amongst the different influenza strains, would provide comparable or better protection

than the larger M2e (amino acids 1-15). We also used four different adjuvants to test this,

which included Pam3CAG that binds with the surface TLR2 receptor on antigen

presenting cells, MPL that binds with the TLR4 surface receptor on antigen presenting

cells, and mycoviral dsRNA which binds with the intracellular TLR3 receptor within the

203 antigen presenting cells (Chua et al., 2014; Iwasaki and Pillai, 2014; Casellas & Mitchell,

2008; Hernandez et al., 2016). We also used lipidated Tucaresol which mimics the

carbonyl group on the surface of the antigen presenting cells that normally form Schiff

bases with lysine residue on the surface of T cells (Collins et al., 2014). Thus, the

Tucaresol interacts directly with the T cells.

Our results demonstrated that CMI liposomal M2e (1-15) vaccines containing

Pam3CAG or MPL + Tucaresol or CMI liposomal M2e (1-12) with mycoviral dsRNA provided the most protection as measured by increased survival, less weight change over

28 days, and decreased disease scores over 28 days. We concluded that overall, it appears that the M2e (1-15) is a better antigen since most vaccines containing M2e (1-15) had

increased survival relative to those vaccines containing M2e (1-12). The only exception

to this was the CMI liposomal M2e (1-12) with mycoviral dsRNA which also showed

increased survival. We hypothesize that the mycoviral dsRNA adjuvant and not the M2e

(1-12) antigen in this liposomal vaccine was responsible for this increased survival since

other vaccines containing M2e (1-12) with other adjuvants had 10% or less survival.

With respect to the adjuvant Tucaresol, we did not see protection when the Tucaresol

was used at a dose of 3 μg, but this same dose of Tucaresol when combined with MPL at

7.5 μg and not the usual 15 μg/dose of MPL, was protective. We could speculate that

most of the protection observed when both adjuvants were used was due to the MPL

alone since doses as low as 5 μg/dose MPL with the M2e (1-15) have been observed to be

protective (data not shown). However, one cannot eliminate the possibility that the

Tucaresol was also contributing to this protection.

204 In a comparative study analyzing 716 different strains of influenza that infect

human, avian, swine, equine, and unknown species, it was discovered that amino acids 10

to 20 of M2e contain the highest variability across different strains of influenza (Liu et al., 2005). Interestingly, it was also discovered that all 716 strains of influenza had a conserved tryptophan at position 15 (Liu et al., 2005). This means that our smaller M2e

(1-12) was missing the highly conserved Trp15. It is possible that Trp15 and the other two amino acids missing from the M2e (1-12) epitope changed the stability and

conformation of the M2e epitope, reducing its ability to act as an antigen. This could also

mean that the smallest M2e that could be immunogenic in the CMI liposomes is one

which contains the first 15 amino acids.

IgG isotype ratios showed that a dominant Th1 or Th2 immune response could

not be used to predict survival. Mice vaccinated with CMI liposomal M2e (1-15)-

Pam3CAG had a combined Th1 and Th2 immune response, mice vaccinated with CMI

liposomal M2e (1-15)-MPL + lipidated-Tucaresol had a predominant Th2 response, while mice vaccinated with CMI liposomal M2e (1-12)-mycoviral dsRNA had more of a

Th1 immune response. These three vaccines produced increased survival by utilizing

different adaptive immune responses. Additionally, vaccine formulations with low

survival also stimulated a Th1 or Th2 immune response.

When we analyzed IgG3, it was discovered that CMI liposomal M2e (1-15)

vaccines containing a high dose of lipidated-Tucaresol (6 μg/dose) or a low dose of

lipidated-Tucaresol (3 μg/dose) had decreased IgG3 levels compared to other CMI

liposomal M2e (1-15) vaccines. Based on the decreased levels of IgG3, one would expect

that both of these vaccines would have decreased survival. However, CMI liposomal M2e

205 (1-15)-lipidated-Tucaresol (6 μg/dose) had higher survival than the CMI liposomal M2e

(1-15)-lipidated-Tucaresol (3μg/dose). Taken together, these results support our previous

findings that immunological data alone cannot be used to predict the efficacy of a

vaccine. As mentioned above, vaccine formulations need to be tested in animal models to

study their efficacy.

Luminex data indicated that CMI liposomal M2e (1-12) vaccines had increased

levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-10 compared to the CMI liposomal M2e (1-15) vaccines. It should be noted that IL-10 can be either anti- or pro-

inflammatory depending upon the condition (Lauw et al., 2000). Here again, we observed

no distinctive set of cytokines that was exclusively elevated in those groups with high or

low survival, since the CMI liposomal M2e (1-12) with mycoviral dsRNA had increased

survival whereas the other CMI liposomal M2e (1-12) vaccines with the other adjuvants

did not have increased survival.

Overall, this study illustrated the importance of the structure of the M2e protein

for maintaining its immunogenicity. By removing three amino acids, the M2e (1-12) was

less protective. We attribute the high survival of mice vaccinated with the CMI liposomal

M2e (1-12) with 25 μg mycoviral dsRNA to be due to the innate and adaptive immune

response generated by the mycoviral dsRNA and the M2e (1-12). We also observed that

immunological data alone is not sufficient to predict the efficacy of a vaccine and that it

is important to perform survival studies to analyze survival and morbidity in combination

with immunological data.

206 IV. CMI Liposomes containing Pam3CAG and no M2e protein provided protection in mice challenged with H1N1 influenza virus.

We demonstrated that the CMI liposomal vaccine containing 100 μg of mycoviral dsRNA without M2e was able to provide significant protection to mice challenged with

H1N1 or H3N2 influenza virus. Thus, we wanted to investigate if the CMI liposomes containing other adjuvants without the M2e protein could also provide significant protection to mice. To begin this investigation, we performed a screening study in which we tested CMI liposomes containing Pam3CAG, mycoviral dsRNA, MPL, lipidated-

Tucaresol, or 1V270. As our positive control, we used the L-MPL-M2e-HD vaccine since it has been demonstrated that this vaccine is protective. As our negative control, we used a liposomal MPL vaccine without the CMI linker and with no M2e. From the screening study, L-Pam3CAG, L-mycoviral dsRNA, and L-MPL-M2e-HD provided the most protection (70%, 62.5%, and 75%, respectively) as measured by increased survival, increased weight on day 28, and decreased disease scores on day 28. These three vaccines were then tested for cross protection against H1N1 and H3N2 influenza virus.

The adjuvants Pam3CAG, mycoviral dsRNA, MPL, and 1V270 activated the innate immune system by binding to different toll-like receptors on the surface of innate immune cells. Lipopeptides such as Pam3CAG are known to bind to the TLR2 receptor on the surface of neutrophils, monocytes, and dendritic cells (Chua et al., 2014)

(Mukherjee et al., 2016). Activation of the TLR2 receptor leads to the production of the pro-inflammatory cytokines IL-12, TNF-α, and IFN-γ via the MyD88 pathway

(Mukherjee et al., 2016). Research has shown that when Pam3CAG is delivered with an antigen, it is capable of enhancing the priming abilities of dendritic cells leading to improved T-cell proliferation into effector cytotoxic T cells (Chua et al., 2014). It has

207 also been shown that liposomal Pam3CAG, similar to the one used in this study, was able to trigger the maturation of human derived dendritic cells (Espuelas et al., 2005). Our results from this study showed that L-Pam3CAG provided significant protection in mice against a lethal challenge with H1N1 influenza virus. Perhaps this protection was mediated by a superior activation of the innate immune system via the TLR2 receptor, which led to enhanced dendritic cell maturation, and ultimately a protective immune response.

L-mycoviral dsRNA also provided significant protection to mice challenged with

H1N1 influenza virus. As discussed above, mycoviral dsRNA is recognized by the intracellular TLR3 receptor and might provide protection by better mimicing a natural influenza infection since the TLR3 receptor recognizes an influenza virus infection

(Iwasaki and Pillai, 2014). This finding supports our previous finding from Study I in which we observed that L-mycoviral dsRNA provided significant protection in mice challenged with H1N1 or H3N2 influenza virus.

The lack of protection by L-1V270 and lipidated-Tucarasol underscores the importance of appropriately activating the innate immune system. L-1V270 binds to the intracellular TLR7 receptor which leads to the activation of the MyD88 signaling pathway (Goff et al., 2015). As previously mentioned, TLR7 is an important receptor involved in recognition of the influenza viral infection (Goff et al., 2015). One would expect that activating a signaling pathway involved in influenza infection recognition would yield increased survival. It has been found that when 1V270 is delivered in combination with the synthetic TLR4 agonist, 1Z105, and the antigen protein HA, this combination was able to provide protection to mice challenged with H1N1 influenza

208 virus (Goff et al., 2015). In this same study, the researchers found that when they

delivered the 1V270 with 1Z105 without the antigen HA, the morbidity and mortality of

the mice was similar to the group that received the control (Goff et al., 2015). This latter

result parallels the findings in our study.

Tucaresol is an adjuvant that will interact with the lysines on the surface of T cells

providing the same co-stimulatory signal that occurs when these lysines interact with the

carbonyl groups on the surface of antigen presenting cells (Collins et al., 2014).

However, our findings from Study III showed that Tucaresol in combination with the

M2e (1-15) protein was not able to provide protection to mice challenged with H1N1

influenza virus. Additionally, studies have shown Tucaresol’s ability to function as an

adjuvant is dose dependent. At higher concentrations, Tucaresol appears to be an

immunosuppressant and at lower concentrations it acts as an immunopotentiator (Collins

et al., 2015). In this study, Tucaresol was delivered at 6 μg per dose and it is possible that

this dose was high and immunosuppressed the mice challenged with H1N1. However,

other studies in our laboratory have shown that Tucaresol in combination with an HSV-2

epitope (gD3PEP) is very protective and these results emphasize how important it is to

develop unique vaccines for each type of infection.

In the cross protection study, only L-MPL-M2e-HD was able to provide

significant protection in mice challenged with H1N1 or H3N2 influenza virus. L-

Pam3CAG was protective only against H1N1 challenge and not H3N2 challenge. During

the screening study which was only against H1N1, L-Pam3CAG, L-mycoviral dsRNA, and L-MPL-M2e-HD provided 70%, 62.5%, and 75% survival, respectively against

H1N1 challenge. For the cross protection study L-Pam3CAG, L-mycoviral dsRNA, and

209 L-MPL-M2e-HD had 57%, 29%, and 43% survival, respectively, against H1N1. A possible explanation for this disparity in protection against H1N1 by the different adjuvants between the screening study and the cross protection study may involve the amount of virus with which the mice were challenged. Mice were infected with homogenized infected lung tissue. The lungs are initially collected from infected mice, homogenized, and then vortexed well before each aliquot of the homogenate is dispensed into small vials at 200 μl. The lung homogenate is not a clear solution and contains some small lung tissue debris which is why it has to be vortexed well as each aliquot is taken.

The vials are stored until needed at -80 C. Prior to challenge, a given vial is removed from the freezer, and vortexed well before about 100 μl is removed from the vial to make up the virus dilution needed to produce 10X LD50 dose. This challenge dose has been established by doing a previous LD50 study. Due to this procedure, it is possible that each vial of virus does not contain exactly the same concentration of virus, and some vials may contain more virus than others. Thus, in making up the viral dilution, this variation from vial to vial can affect the total amount of virus given to the mice. During the cross protection study, the mice could have received a greater H1N1 challenge than intended and this could have resulted in a decrease in the protection observed for the same liposomal vaccines tested in the screening study. This may also explain why across different studies, there is a difference in the survival rate in groups of mice receiving the same vaccine.

These results also underscore the importance of the dose of the adjuvant in appropriately activating the innate immune response. Research has shown that delivering mycoviral dsRNA at lower doses (15 μg/dose) causes immunosuppression (Hewitt and

210 Adler, 1982). During Study I, it was demonstrated that delivering mycoviral dsRNA at

100 μg per dose acted as an adjuvant that provided protection against H1N1 and H3N2.

For Studies II and III and the adjuvant screening study, mycoviral dsRNA was delivered

at 50 or 25 μg per dose, respectively. Protection was observed in all of these studies when

mycoviral dsRNA was used alone or in combination with M2e in the CMI liposomes.

This underscores the importance of determining the optimal dose of mycoviral dsRNA

that could provide optimal protection in the CMI liposomal vaccines. During the cross

protection study, however, the protection provided by 50 μg per dose of mycoviral

dsRNA decreased. This could indicate that the ideal dose of mycoviral dsRNA needed to

optimally activate the innate immune response is between 50 μg and 100 μg per dose.

Ultimately this study showed that by combining the antigen M2e-HD and the adjuvant MPL in liposomes, mice were cross protected against an H1N1 and H3N2 influenza challenge. This demonstrates that by utilizing both the adaptive and innate immune responses, we can achieve protection from different influenza viral strains.

However, an influenza vaccine that uses M2e as the target antigen will not provide protection against Influenza B virus since this virus does not have an M2 protein

(Lindstrom et al., 1991). It is therefore important to continue our investigation of the innate immune response since its nonspecific stimulation has the potential to protect against influenza B viruses and perhaps many other microbial pathogens which are inhibited by the innate immune response.

Future Studies

Future studies will focus on optimizing the vaccination schedule and doses for the

CMI liposomal vaccines containing adjuvants and no M2e protein. The vaccination

211 schedule used for the above mentioned studies was determined based on vaccines that would target the adaptive immune response. Since the CMI liposomes with adjuvants do not contain the antigen M2e, and instead target the innate immune response, the vaccination schedule needs to be adjusted to better target the innate immune response.

In addition, future studies will characterize the type of innate immune response generated by the CMI liposomes containing adjuvants and no M2e. This study will look at macrophage polarization into subsets of M1 and M2 macrophages. M1 macrophages are associated with Th1 immune responses while M2 macrophages are associated with

Th2 immune responses (Martinez and Gordon, 2014). To identify M1 macrophages, macrophages will be stained and analyzed using FACS markers including iNOS, CCR2,

CD11b, and CC11B. To identify M2 macrophages, the novel markers Egr2 and c-Myc could also be investigated (Jablonski et al., 2015).

Lastly, the CMI liposomal M2e vaccines containing adjuvants and the CMI liposomes with adjuvants and no M2e should be investigated in male mice. Thus far, our studies have only used female mice. This is because female mice are less aggressive which allows for easier housing conditions. However, studies have revealed that males and females have different immune responses to vaccines (Weiman, 2016). Therefore, it is important to investigate if the CMI liposomal vaccines will provide comparable protection in male mice.

212 References

1. A revision of the system of nomenclature for influenza viruses: WHO memorandum. (1980). Bulletin of the World Health Organization, 58(4), 585-591.

2. Adler-Moore, J., Munoz, M. Kim, H., Romero, J., Tumpeu, T. Zeng, H., Petro, C., Ernst, W., Kosina, S., Kimenez, G., & Fugii, G. (2011). Characterization of the murine Th2 response to immunization with liposomal M2e influenza vaccine. Vaccine, 29, 4460-4468.

3. Air, G. M. & Laver, W. G. (1989). The Neuraminidase of Influenza Virus. Proteins: Structure, Function, and Genetics, 6, 341-356.

4. Akbarzahed, A., Rezaei-Sadabady, R., Davaran, S., Woo Joo, S., Zarghami, N., Hanifehpour, Y., Samiel, M., Kouhi, M., & Nejati-Koshki, K. (2013). Liposome: classification, preparation, and applications. Nanoscale Research Letters, 8, 1-9.

5. Afluria Quadrivalent 2016 (Afluria Quadrivalent 2016): Side Effects, Interactions, Warning, Dosage & Uses. (n.d.). Retrieved March 13, 2017, from http://www.rxlist.com/afluria-drug.htm.

6. Ansaldi, F., de Florentiis, D., Durando, P., & Icardi, G. (2012). Fluzone® intradermal vaccine: a promising new chance to increase the acceptability of influenza vaccine in adults. Expert Reviews Vaccines, 11(1), 17-25.

7. Bahadoran, A., Lee, S. H., Wang, S. M., Manikam, R., Rajarajeswaran, J., Raju, C. S., Sekaran, S. D. (2016). Immune Response to Influenza Virus and Its correlation to Age and Inherited Factors. Frontiers in Microbiology, 7, 1-11.

8. Barberis, I., Martini, M., Iavarone. F., & Orsi, A. (2016). Available influenza vaccines: immunization strategies, history and new tools for fighting the disease. Journal of Preventative Medicine and Hygiene, 57, 41-46.

9. Barberis, P., Myles, S. K., ault, N. L., & Bragazzi, M. M. (2016). History and evolution of influenza control through vaccination: from the first monovalent vaccine to universal vaccines. Journal of Preventative Medicine and Hygiene, 57, 115-120.

10. Belkaird, Y. (2007). Regulatory T cells and infection: a dangerous necessity. Nature Reviews Immunology, 7, 875-888.

11. Brencicova, E., & Diebold, S. S. (2013). Nucleic acids and endosomal pattern recognition: how to tell friend from foe?. Frontiers in Cellular and Infection Microbiology, 3(37).

12. Briese, T., Chowdhary, R., Travassos da Rosa, A., Hutchison, S. T., Popov, V., Street, C., Tesh, R. B., & Lipkin, W. I. (2014). Upolu virus and Aransas Bay virus,

213 two presumptive Bunyaviruses, are novel members of the family Orthomyxoviridae. Journal of Virology, 88, 5298-5309.

13. Cagdas, M., Sezer, A. D., & Bucak, S. (2014). Liposomes as Potential Drug Carriers Systems for Drug Delivery (A. D. Sezer, Ed.). In Application of Nanotechnology in Drug Delivery. doi:10-5772/58459

14. Casellas, R. C., & Mitchell, T. C. (2008). Putting endotoxins to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant. Cell and Molecular Life Sciences, 65, 3231-3240.

15. Center for Disease Control and Prevention. (2016). Morbidity and Mortality Weekly Reports, 65(5).

16. Choi, P., & Reiser, H. (1998). IL-4: Role in Disease and Regulation of Production. Clinical and Experimental Immunology, 113(3), 317-319.

17. Cianci, C., Gerritz, S. W., Deminie, C., & Krystal, M. (2013). Influenza nucleoprotein: promising target for antiviral chemotherapy. Antiviral Chemistry & Chematherapy, 23, 77-91.

18. Cimons, M. (2016). Universal Influenza Vaccine: Quest in Sight?. Microbe, 11, 433- 437.

19. Clark, N. M., & Lynch, J. P. (2011). Influenza: Epidemiology, Clinical Features, Therapy, and Prevention. Seminars in Respiratory and Critical Care Medicine, 32, 373-392.

20. Collins, K. C., Schlosburg, J. E., Lockner, J. W., Bremer, P. T., Ellis, B. A., & Janda, K. D. (2014). Lipid Tucaresol as an adjuvant for methamphetamine vaccine development. Chemical Communications, 50, 4079-4081.

21. Cox, N. J., & Subbarao, K. (2000). Global epidemiology of Inluenza: Past and Present. Annual Review of Medicine, 51, 407-421.

22. Davis, B. K., & Ting, J. P. Y. (2010). NLRP3 has a sweet tooth. Nature Immunology, 11, 105-106.

23. De Souza Apostolico, J., Alves Santos Lunardelli, V. A., Caroline Coirada, F., Beatriz Boscardin, S., & Santoro Rosa, D. (2016). Adjuvants: Classification, Modus Operandi, and Licensing. Journal of immunology Research, 2016, 1-16.

24. Deng, L., Cho, K., Fiers, W., & Saelens, X. (2015). M2e-Based Universal Influenza A Vaccines. Vaccines, 3(1), 105-136. doi:10.3390/vaccines3010105

214 25. Dinarello, C. A. Novick, D., Kim, S., & Kaplanski, G. (2013). Interlukein-18 and IL- 18 Binding Protein. Frontiers in Immunology, 4.

26. Dormitzer, P. R., Galli, G., Castellino, F., Golding, H., Khurana, S., Del Giudice, G., & Rappuoli, R. (2011). Influenza vaccine Immunology. Immunological Reviews, 239, 167-177.

27. Dunkelberger, J. R., & Song, W. C. (2010). Complement and its role in innate and adaptive immunity. Cell Research, 20, 34-50.

28. E van de Sandt, C., Bodewes, R., Rimmelzwaan, G. F., & D de Vries, R. (2015). Influenza B viruses: not to be discontinued. Future Microbiology, 10, 1447-1465.

29. Ebrahimi, S. M., & Tebianian, M. (2011). Influenza A viruses: why focusing on M2e- based universal vaccines. Virus Genes, 42, 1-8.

30. Ernest, W. A., Kim, H. J., Tumpey, T. M., Jansen, A. D. A., Tai, W., Cramer, D. V., Adler-Moore, J. P., & Fugii, G. (2006). Protection against H1, H5, H6, and H9 influenza A infection with liposomal matrix 2 epitope vaccines. Vaccines, 24(24), 5158-5168.

31. Espuelas, S., Roth, A., Thumann, C., Frisch, B., & Schuber, F. (2005). Effects of synthetic lipopeptides formulated in liposomes on the maturation of human dendritic cells. Molecular Immunology, 42, 721-729.

32. Fernandes, I., Frisch, B., Muller, S., & Schuber, F. (1997). Synthetic lipopetides incorporated in liposomes: in vitro stimulation of the proliferation of murine splenocytes and in vivo induction of an immune response against a peptide antigen. Molecular Immunology, 34, 568-576.

33. Fiore, E. A., Fry, A., Shay, D., Gubareva, L., Bresee, J. S., & Uyeki, T. M. (2011). Morbidity and Mortality Weekly Report, 60 (1), 1-25.

34. Flublok (Influenza Vaccine for Intramuscular Injection): Side Effects, Interactions, Warning, Dosage & Uses. (n.d.). Retrieved March 13, 2017, from http://www.rxlist.com/flublok-drug.htm.

35. Flucelvax (Influenza Virus Vaccine): Side Effects, Interactions, Warning, Dosage & Uses. (n.d.). Retrieved March 13, 2017, from http://www.rxlist.com/flucelvax- drug.htm.

36. Fluzone (Influenza Virus Vaccine for Injection): Side Effects, Interactions, Warning, Dosage & Uses. (n.d.). Retrieved March 13, 2017, from http://www.rxlist.com/fluzone-drug.htm.

215 37. Flulaval (Influenza Virus Vaccine): Side Effects, Interactions, Warning, Dosage & Uses. (n. d.). Retrieved March 13, 2017, from http://www.rxlist.com/flulaval-drug- htm.

38. Fokkens, W. J., Scheeren, R. A. (2000). Upper airway defense mechanisms. Pediatric Respiratory Reviews, 1, 336-341.

39. Goff, P. H., Hayashi, T., Martinez-Gil, L., Corr, M., Crain, B., Yao, S., Cottam, H. B., Chan, M., Ramos, I., Eggink, D., Heshmati, M., Krammer, F., Messer, K., Pu, M., Fernandez-Sesman, A., Palese, P., & Carson, D. A. (2015). Synthetic Toll-Like Receptor 4 (TLR4) and TLR7 Ligands as Influenza Virus Vaccine Adjuvants Induce Rapid, Sustained, and Broadly Protective Responses. Journal of Virology, 89, 3221- 3235.

40. Grohskopg, L. A., Sololow, L. Z., Olsen, S. J., Karron, R. A., Jernigan, D. B., & Bresee, J. S. (2016). Prevention and Control of Season Influenza with Vaccines Recommendations of the Advisory Committee on Immunization Practices—United States, 2016-2017 Influenza Season. Morbidity and Mortality Weekly Reports, 65(5), 1-54.

41. Hay, A. J., Gregory, V., Douglas, A. R., & Lin, Y. P. (2001). The evolution of human influenza viruses. Philosophical Transactions of the Royal Society, 356, 1861-1870.

42. Hernandez, A., Bohannon, J. K., Luan, L., Fensterheim, B. A., Gua, Y., Pastil, N., K., McAdams, C., Wang, J., & Sherwood, E. (2016). The role of MyD88-andTRIF- dependent signaling in monophorphoryl lipid A-induced expansion and recruitment of innate immunocytes. Journal of Leukocyte Biology, 100(6), 1311-1322.

43. Heweitt, W. C., & Adler, J. P. (1982). Murine immunosuppression with mycoviral dsRNA. Immunopharmacology, 5, 103-109.

44. Holloway, R., Rasmussen S. A., Zaza, S., Cox, N. J., & Jernigan, D. B. (2014). Updated Preparedness and Response Framework for Influenza Pandemics. Recommendations and Reports, 63(6), 1-9.

45. How Flu Spreads. (2013, September 12). Retrieved May 21, 2017, from https://www.cdc.gov/flu/about/disease/spread.htm

46. Ichinoche, T., Kye, L. H., Ogura, Y., Flavell, R., & Iwasaki, A. (2009). Inflammasome recognition of influenza virus is essential for adaptive immune responses. Journal of Experimental Medicine, 206, 79-87.

47. Influenza (Seasonal). (n.d.). Retrieved May 21, 2017, from http://www.who.int/mediacentre/factsheets/fs211/en/

216 48. Interim prepandemic planning guidance: community strategy for pandemic influenza mitigation in the United States—early, targeted, layered use of nonpharmaceutical interventions. Atlanta, GA: CDC; 2007. Retrieved from https://flu.gov/planning- preparedness/community/community_mitigation.pdf.

49. International Committee on Taxonomy of Viruses. (2015, July). Taxonomy. Retrieved May 21, 2017, from https://talk.ictvonline.org/taxonomy/. Email Ratification 2016 (MSL #30).

50. Iwasaki, A., & Medzhitov, R. (2010). Regulation of adaptive immunity by the innate immune system. Science, 327, 291-295.

51. Iwasaki, A., & Pillai, P. S. (2014). Innate immunity to influenza virus infection. Nature Reviews Immunology, 4, 315-328.

52. Jegaskanda, S. Job, E. R., Kramski, M. Laurie, K., Isitman, G., de Rose, R., Winnall, W. R., Strotov, I., Brooks, A. G., Reading, P. C., & Kent, S. J. (2013). Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity Antibodies in the Absence of Neutralizing Antibodies. The Journal of Immunology, 190, 1837-1848.

53. Kanai, N., Min, W. P., Ichim, T. E., Wang, H., & Zhong, R. (2007). Th1/Th2 Xenogenic Antibody Responses are Associated with Recipient Dendritic Cells. Microsurgery, 27, 234-239.

54. Kang, S. M., Kim, M. C., & Compans, R. W. (2012). Virus-like particles as universal influenza vaccines. Expert Review of Vaccines, 11, 995-1007.

55. Krieg, M. A. (2007). Strong immune responses can be activated in the absence of a major immune pathway. Nature Biotechnology, 25, 303-305.

56. Kudva, A., Scheller, E., Robinson, K. M., Crowe, C. R., Choi, S. M., Slight, S. R., Khader, S. A., Dubin, P. J., Enelow, R. I., Kolls, J. K., Alcorn, J. F. (2014). Influenza A inhibits Th17-Mediated Host Defense Against Bacterial Pneumonia in Mice. Journal of Immunology, 186, 1666-1674.

57. Lats, E., Xiao, T. S., & Stutz, A. (2013). Activation and regulation of the inflammasome. Nature Reviews Immunology, 13(6).

58. Lara-Sampable, A., Flores-Alonso, J. C., Jesus-Ortega, N., Santos-Lopez, G. Vallejo- Ruiz, V., Rosas-Murrieta, N., Reyes-Carmona, S., Herrera-Camacho, I., & Reyes- Leyva, J. (2014). Transfection of influenza A virus nuclear export protein induces the expression of tumor necrosis factor alpha. Virus Research, 185, 1-9.

59. Lee, Y. N., Kim, M. C., Lee, Y. T., Kim, Y. J., & Kang, S. M. (2015). Mechanisms of Cross Protection by Influenza Virus M2e-based Vaccines. Immune Network, 15(5), 213-221.

217 60. Lindstrom, S. E., Hiromoto, Y., Nishimura, H., Saito, T., Nerome, R., & Nerome, K. (1991). Comparative Analysis of Evolutionary Mechanisms of the Hemagglutinin and Three Internal Proteins Genes of Influenza B Virus: Multiple Cocirculating Lineages and Frequent Reassortment of the NP, M, and NS Genes. Journal of Virology, 73, 4413-4426.

61. Lockner, J. W., Ho, S. O., McCague, K. C., Chiang, S. M., Do, T. Q., Fugii, G., & Janda, K. D. (2013). Enhancing nicotine vaccine immunogenicity with liposomes. Bioorganic & Medical Chemistry Letters, 23, 975-978.

62. London, C. A., Abbas, A. K., & Kelso, A. (1991). Helper T cell subsets: Heterogeneity, functions and development. Veterinary Immunology and Immunopathology, 63, 37-44.

63. Mark von Itzstein. (2007). The war against influenza: discovery and development of sialidase inhibitiors. Nature Reviews Drug Discovery, 6, 967-974.

64. Marklund, K. J., Ye, Q., Dong, J., Tao, Y. J., & Krung, R. M. (2012). Sequence in the Influenza A Virus Nucleoprotein Required for Viral Polymerase Binding and RNA Synthesis. Journal of Virology, 86, 7292-7297.

65. Martinez-Gil, L., & Mingarro, I. (2015). Viroporins, Examples of the Two-Stage Membrane Protein Folding Model. Miltidisciplinarity Digital Publishing Institute.

66. Matsumoto, M., & Seya, T. (2008). TLR3: Interferon induction by double-stranded RNA including poly(I:C). Advanced Drug Delivery Reviews, 60, 805-812.

67. McCormack, F. X., & Whitsett, J. A. (2002). The pulmonary collectins, SP-A and SP- D, orchestrate innate immunity in the lung. The Journal of Clinical Investigation, 109, 707-712.

68. McKeage, K. (2013). Inactivated quadrivalent split-virus seasonal influenza vaccine (Fluarix ® quadrivalent): a review of its use in the prevention of disease caused by influenza A and B. Drugs, 73(14), 1587-1594.

69. Medina, R. A., & García-Sastre, A. (2011). Influenza A viruses: new research developments. Nature Reviews Microbiology, 9(8), 590-603. doi:10.1038/nrmicro2613

70. Molinari, N. A. M., Ortega-Sanchez, I. R., Messonnier, M. L., Thompson, W. W., Wortley, P. M., Weintraub, E., & Bridges, C. B. (2007). The annual impact of seasonal influenza in the US: measuring disease burden and costs. Vaccine, 25, 5086- 5096.

71. Morens, D. M., & Fauci, A. S. (2007). The 1918 Influenza Pandemic: Insights for the 21st Century. Journal of Infectious Diseases, 195(7), 1018-1028.

218 72. Mosier, P. D., Chiang, M. J., Lin, Z., Gao, Y., Althufairi, B., Zhou, Q., Musayev, F., Safo, M. K., Xie, H., & Desai, U. R. (2016). Broad Spectrum Anti-Influenza Agents by Inhibiting Self-Association of Matrix Protein 1. Scientific Reports, 6, 1-10.

73. Mukherjee, S., Karmakar, S., & Babu, S. P. S. (2016). TLR2 and TLR4 mediated host immune responses in major infectious disease: a review. The Brazilian Journal of Infectious Disease, 20, 193-204.

74. Nyirenda, M., Omori, R., Tessmer, H. L., Arimura, H., & Ito, K. (2016). Estimating the Lineage Dynamics of Human Influenza B Viruses. PLoS ONE, 11. DOI: 10.1371/journal.pone.0166107.

75. Parrish, C. R., Murcia, P. R., & Holmes, E. C. (2015). Influenza Virus Reservoirs and Intermediate Hosts: Dogs, Horses, and New Possibilities for Influenza Virus Exposure of Humans. Journal of Virology, 89, 2990-2994.

76. Peterson, D., & Fodor, E. (2012). Emerging Roles for the Influenza A Virus Nuclear Export Protein (NEP). PLOS Pathogens, 8, 1-8.

77. Potter, C. W. (2001). A history of influenza. Journal of Applied Microbiology, 91, 572-579.

78. Presti, R. M., Zhao, G., Beatty, W. L., Mihindukulasuriya, K. A., Travossos da Rosa, A. P. A., Popov, V. L., Tesh, R. B., Virgin, H. W., & Wang, D. (2009). Quaranfil, Johnson Atoll, and Lake Chad Viruses are Novel Members of the Family Orthomyxoviridae. Journal of Virology, 83, 11599-11606.

79. Pulendran, B., & Asmed, R. (2011). Immunological mechanism of vaccination. Nature Immunology, 12, 509-517.

80. Pulendran, B., Maddur, M. S. (2015). Innate Immune Sensing and Response to Influenza. Current Topics in Microbiology and Immunology, 386, 23-71.

81. Recommended composition of influenza virus vaccines for use in the 2016-2017 northern hemisphere influenza season. (2016). Weekly Epidemiological Record, 91, 121-132. Retrieved from http://www.who.int/influenza/vaccines/virus/recommendations/2016_17_north/en/.

82. Recommended composition of influenza virus vaccines for use in the 2017 southern hemisphere influenza season. (n.d.). Retrieved May 21, 2017, from http://www.who.int/influenza/vaccines/virus/recommendations/2017_south/en/

83. Reperant, L. A., Rimmerlzwaan, G. F., & Osterhause, A. D. M. E. (2014). Advances in influenza vaccination. F1000Prime Reports, 6, 1-8.

219 84. Rhodes, J. (2002). Discovery of immunopotentiatory drugs: current and future strategies. Clinical and Experimental Immunology, 130, 363-369.

85. Romagnani, S. (2008). Human Th17 cells. Arthritis Research & Therapy, 10, 1-8.

86. Romagnani, S. (2000). T-cell subsets (Th1 versus Th2). Annals of Allergy, Asthma & Immunology, 85, 9-21.

87. Rossman, J. S., & Lamb, R. A. (2011). Influenza Virus Assembly and Budding. Virology, 411, 229-236.

88. Samji, T. (2009). Influenza A: Understanding the Viral Life Cycle. Yale Journal of Biology and Medicine, 82, 153-159.

89. Schnitzler, S. U., & Schnitzler, P. (2009). An update on swine-origin influenza virus A/H1N1 a review. Virus Genes, 39, 279-292.

90. Schwendener, R. A. (2014). Liposomes as vaccine delivery systems: a review of the recent advances. Therapeutic Advances in Vaccines, 2, 159-182.

91. Skehel, J. J., & Wiley, D. C. Receptor Binding and Membrane Fusion in virus Entry: The Influenza Hemagglutanin. Annual Review of Biochemistry, 69, 531-569.

92. Taubenberger, J. K., & Morens, D. M. (2010). Influenza: The Once and Future Pandemic. Public Health Reports, 125, 16-26.

93. Thompson, M. G., Shay, D. K., Zhou, H., Bridges, C. B., Cheng, P. Y., Burns, E., Bresee, J. S., & Cox, N. J. (2010). Estimates of Deaths Associated with Seasonal Influenza—United States, 1976-2007. Morbidity and Mortality Weekly Report, 59(33), 1057-1062.

94. Third Edition An Introduction to Applied Epidemiolgy and Biostatistics. Lesson 1: Introduction to Epidemiology. (2012, May 18). Retrieved May 21, 2017, from https://www2a.cdc.gov/TCEOnline/registration/detailpage.asp?res_id=2965. CDC.

95. Tognotti, E. (2009). Influenza pandemics: a historical retrospect. The Journal of Infection in Developing Countries, 3. 331-334.

96. Trask, S. D., McDonald, S. M., & Patton, J. T. (2012). Structural insights into the coupling of virion assembly and rotavirus replication. Nature Reviews Microbiology, 10, 165-177.

97. Treanor, J. (2004). Influenza Vaccine—Outmaneuvering Antigenic Shift and Drift. The New England Journal of Medicine, 350, 218-220.

220 98. Tsai, T. F. (2013). Fluad®-MF59®-Adjuvanted Influenza Vaccine in Older Adults. Infection & Chemotherapy, 45(2), 159-174.

99. Wang, M., & Veit, M. (2016). Hemagglutinin-esterase-fusion (HEF) protein of influenza C virus. Protein Cell, 7, 28-45.

100. Warrington,R. Watchon, W., Kim, H. L., & Antonetti, F. R. (2011). An introduction to immunology and immunopathology. Allergy, Asthma & Clinical Immunology, 7, 1-8.

101. Weber, F., Wagner, V., Rasmussen, S. B., Hartmann, R., & Paludan, S. R. (2006). Double-Stranded RNA is Produced by Positive-Stranded RNA Viruses and DNA Viruses but Not in Detectable Amounts by Negative-Stranded RNA Viruses. Journal of Virology, 80(10), 5059-5064.

102. Webster, R. G. & Govorkova, E. A. (2014). Continuing challenges in influenza. Annals of the New York Academy of Science, 1323, 115-139.

103. Weiman, S. (2016). Sex Hormones Can Trigger Differential Responses Between Females and Males to Infectious Agents and Vaccines. Microbe, 11(9), 383-387.

104. Wise, H. M., Foeglein, A., Sun, J., & Digard, P. (2009). A Complicated Message: Identification of a Novel PB-1 Related Protein Translated from Influenza A Virus Segment 2 nRNA. Journal of Virology, 83, 8021-8031.

105. Wright, C. L., & Adler-Moore, J. P. (1985). The adjuvant effects of mycoviral dsRNA and polyinosinic:polycytidylic acid on the murine immune response. Biochemical and Biophysical Research Communications, 131, 949-955.

106. Writing Committee of the WHO Consultation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza. (2010). Clinical Aspects of Pandemic Influenza A (H1N1) Virus Infection. The New England Journal of Medicine, 362, 1708-1719.

107. Wu, S., Metcalf, J. P., & Wu, W. (2011). Innate immune response to influenza virus. Current Opinion in Infectious Disease, 24, 235-240.

108. Zhou, H., Liao, J., Aloor, J., Nie, H., Wilson, B. C., Fessler, M. B., Gao, H. M., & Hong, J. S. (2013). CD11b/CD18(Mac-1) Is a Novel Surface Receptor for Extracellular Double-Stranded RNA to Mediate Cellular Inflammatory Response. Journal of Immunology, 190, 115-125.

109. Zou, Q., Wu, B., Xue, J., Fan, X., Feng, C., Geng, S., Wang, M., & Wang, B. (2014). CD8+ T reg cells suppress CD8+ T cell-responses by IL-10-depedent mechanism during H5N1 influenza virus infection. European Journal of Immunology, 44, 103-114.

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