Neuraminidase Virus-like Particles as Candidate Influenza A Virus Vaccines Öffentlichkeit
Menne, Zachary (Fall 2021)
Published
Abstract
Influenza A viruses (IAV) are a seasonal cause of acute respiratory disease and a significant contributor to human morbidity and mortality worldwide. Current strategies to mitigate IAV risk focus on prophylactic seasonal vaccines manufactured primarily in embryonated chicken eggs, requiring long lead times and that vaccines be updated and re-administered on a yearly basis due to antigenic changes in the hemagglutinin (HA) protein. A clear need exists for influenza vaccines that can be rapidly manufactured and result in broader immunity and protection against disease caused by influenza virus infection. In this dissertation, I describe the production and characterization of novel IAV virus-like particle (VLP) vaccines containing neuraminidase (NA) from recent seasonal IAV strains produced using a recombinant baculovirus expression system in Trichoplusia ni insect cells. I demonstrate that NA virus-like particles produced using the NA sequence derived from the H1N1 virus A/California/04/2009 (NA1) and the H3N2 virus A/Perth/16/2009 (NA2) are immunogenic in a murine model. Additionally, I demonstrate that the bivalent administration of NA1 and NA2 VLPs is protective in a murine model against lethal challenge with both heterologous H3N2 mouse adapted A/Hong Kong/1/1968 virus and homologous H1N1 mouse adapted A/California/04/2009. Furthermore, I show that bivalent administration of NA1 and NA2 VLPs reduced viral lung replication after A/Hong Kong/1/1968 challenge compared to monovalent administration of NA2 VLPs.
I also investigate NA2 VLP vaccination in a swine infection model. I compared the immunogenicity and virological responses against a commercially available licensed swine influenza vaccine. Pigs vaccinated with NA2 VLPs demonstrated a superior anti-NA immune response compared to vaccination with the commercial vaccine or mock vaccination. Furthermore, pigs vaccinated with NA2 VLPs and the commercial vaccine had a similar and significant reduction in virus replication in lung tissue and pulmonary histopathology scores compared to mock vaccinated controls post challenge with the recently circulating H3N2 swine virus, A/swine/NC/KH15/2016.
This work has demonstrated that anti-NA immunity is capable of conferring protection against IAV challenge and could augment existing influenza virus vaccines. NA VLPs have the potential to supplement current influenza virus vaccines and should be considered in future vaccine development.
Table of Contents
Chapter I: Introduction 1
Influenza Disease 1
Influenza A Virus 2
Influenza Epidemiology 4
Influenza Treatment 7
Prophylaxis and Current Vaccine Strategies 9
Neuraminidase Function, Structure, and Immunogenicity 15
Virus-like Particles as a Vaccine Platform 26
Model Systems for Influenza Vaccine Research 29
Introduction to Dissertation Research 32
Chapter II: Bivalent vaccination with NA1 and NA2 neuraminidase virus-like particles is protective against challenge with H1N1 and H3N2 influenza A viruses in a murine model. 33
Abstract 34
Research Highlights 35
Introduction 36
Materials and Methods 39
Cells and virus stocks 39
Animals 40
VLP Production and Purification 40
VLP Characterization 41
Neuraminidase Activity Assay 42
Immunization and infection 42
Measurement of NA-specific antibody using ELISA 43
Plaque Assay for virus titration 44
Statistics 44
Results 45
Characterization of NA VLPs 45
Intramuscular vaccination with NA2 VLPs is immunogenic and protective in a heterologous challenge model 46
Vaccination with NA2 VLPs has a dose dependent effect on serum IgG levels and morbidity after challenge 47
Vaccination with NA2 VLPs was not protective in a heterosubtypic H1N1 lethal challenge model 50
Multivalent vaccination with NA1 and NA2 VLPs efficiently induce both anti-N1 and anti-N2 antibodies 50
Multivalent vaccination with NA1 and NA2 VLPs is protective against lethal challenge with IAVs containing group 1 or group 2 neuraminidases 52
Discussion 55
Acknowledgements 60
Funding Support 60
References 61
Figures 72
Figure 1: NA2 VLP purification and characterization. 72
Figure 2: The route of administration affects immune responses to NA2 VLPs 74
Figure 3: Dose response to intramuscular (IM) vaccination with differing amounts of NA2 VLPs. 76
Figure 4: Intramuscular vaccination with differing amounts of NA2 VLPs in a heterosubtypic H1N1 challenge model. 78
Figure 5: Serological responses to the combined administration of NA1 + NA2 VLPs. 80
Figure 6: Lethal challenge of mice vaccinated with combined administration of NA1 + NA2 VLPs using H3N2 and H1N1 viruses. 82
Chapter III: Neuraminidase Virus-Like Particle vaccine: protective efficacy and immunogenicity in the swine model 84
Introduction 86
Materials & Methods 91
Cells and virus stocks 91
Animals 92
VLP Production and Purification 92
Vaccine preparation 93
Swine Vaccination, Virus Challenge, and Sample Collection 93
Measurement of NA-specific IgG antibody titers 95
Neuraminidase Activity and Inhibition Assay 96
Hemagglutination Inhibition (HAI) assay 97
Virus Titration (TCID50) Assay 97
Cytopathological Evaluation of Bronchoalveolar Lavage Fluid 98
Macroscopic and microscopic evaluation of respiratory tissues 99
Statistical analysis 100
Results 101
NA2 VLPs are immunogenic in swine model 101
NA2 VLPs induce functional antibody responses directed at NA 102
Vaccination with QWIV vaccine induces antibodies directed at HA capable of inhibiting hemagglutination of erythrocytes 104
Challenge with A/Swine/NC/KH1552516/2016 did not induce significant clinical disease 105
QWIV vaccination reduced viral shedding post challenge 106
Vaccination with NA2 VLPs and QWIV reduces viral replication in swine lung tissue 106
Vaccination with NA2 VLPs and QWIV reduces pulmonary neutrophilic infiltration 107
Vaccination with NA2 VLPs and QWIV reduces pulmonary histopathology scores 108
Discussion 109
References 112
Figures 123
Figure 1: Vaccination with NA2 VLPS induces Anti-NA serum IgG titers in swine 123
Figure 2: Vaccination with NA2 VLPS induces functional Anti-NA antibodies capable of inhibiting A/Perth/16/2009 NA activity in vitro 125
Figure 3: Vaccination with NA2 VLPS induces functional Anti-NA antibodies capable of inhibiting A/swine/NC/HK1552516/2016 NA activity in vitro 127
Figure 4: Prime boost vaccination with commercial QWIV induces serum antibody responses capable of inhibiting hemagglutination of A/Swine/NC/HK1552516/2016 129
Figure 5: Swine clinical disease scores after challenge with A/swine/NC/HK1552516/2016 130
Figure 6: Nasal virus shedding after challenge with A/swine/NC/HK1552516/2016 131
Figure 7: Vaccination with NA2 VLPs and the commercially available QWIV swine IAV vaccine reduced viral replication in pig lungs post challenge 132
Figure 8: Vaccination with NA2 VLPs and the commercially available QWIV swine IAV vaccine reduced neutrophilic infiltration in bronchoalveolar lavage fluid collected from pig lungs post challenge 133
Figure 9: Figure 8: Vaccination with NA2 VLPs and the commercially available QWIV swine IAV vaccine reduced total histopathology scores in pig lungs post challenge 134
Chapter IV: Conclusions 135
References 144
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