Elucidating Immune, Pathological, and Molecular Responses to Influenza A Virus Infection During Concurrent House Dust Mite Allergen Exposure Restricted; Files Only
Mills, Lisa (Spring 2020)
Abstract
Asthma is a risk factor for influenza virus infection associated morbidity and mortality. Studies on influenza virus pathogenesis using the house dust mite (HDM) mouse model of asthma have routinely revealed aberrant immune responses to influenza virus, however, allergic sensitization is discontinued prior to infection. In this dissertation, we produced a chronic asthma mouse model in which exposure to HDM was concurrent with exposure to either H1N1 or H3N2 influenza viruses to evaluate lung tissue pathology and repair, the underlying physiological changes affecting the host response to infection as well as local and systemic innate and adaptive responses to influenza virus. Infected HDM-treated mice demonstrated increased viral titers, extensive lung pathology and early signs of morbidity compared to PBS-treated controls, independent of influenza virus strain; however, the HDM cohort experienced decreased mortality. Persistent elevation of matrix metalloproteinases MMP2, MMP9, and MMP14 in the lungs was observed in H1N1 or H3N2 infected HDM-treated mice, although the PBS control group showed comparable levels of expression at 14 DPI, suggesting a wound healing rather than a pathogenic role. Measurement of lung COX-2, PGE2, and PGD2 secretion kinetics throughout infection revealed differences based on influenza virus strain rather than sensitization status. HDM-treated mice infected with either H1N1 or H3N2 demonstrated elevated lung IL-17A when compared to pre-infection status and to infected PBS-cohort, and a negative correlation with cytokines necessary for viral clearance such as IL-1α, IL-1β, IL-2, IFNγ, and TNFα. Concurrent exposure to HDM during influenza virus infection did not result in the impairment of influenza-specific T cell responses though decreased lung IFNg was observed in both H1N1 and H3N2-infected mice. While similar HAI titers were observed between HDM-treated and PBS-treated groups infected with either H1N1 or H3N2 influenza virus titer, exposure to H1N1 strain induced higher influenza-specific total IgG and IgG2a serum titers than H3N2 strain. Additionally, elevated serum influenza-specific IgG1 was demonstrated in HDM-treated H1N1-infected mice, exclusively, suggesting a role for IgG1 in viral clearance. To our knowledge this is the first study in which influenza A virus infection has been extensively assessed within an asthmatic phenotype and the first study to compare responses to different influenza virus strains within an asthmatic setting.
Table of Contents
Abstract 4
Table of Contents 6
CHAPTER I: Introduction 15
CHAPTER II: Immune responses to Influenza A Virus During Concomitant House Dust Mite Exposure 29
Abstract 30
Introduction 32
Materials and Methods 35
Results 41
Discussion 64
Acknowledgments 71
Figure 1. Pathological and immunological evaluation of asthmatic and control mice prior to infection. 72
Figure 2. Measurement of MMPs and COX2 73
Figure 3. Survival, weight loss and viral titers 74
Figure 4. H&E staining of lung sections at 1, 4, 7 and 14 DPI 75
Figure 5. Infected mice give HDM throughout infection display aberrant innate population cell numbers 76
Figure 6. CD8+ T Cell Responses in the Lung at 7 DPI. 77
Figure 7. CD4 T Cell Responses in the Lung at 7 DPI 78
Figure 8. B cell and humoral immune responses 79
Figure 9. Cytokine and chemokine analysis. 80
Figure 10. Measurement of MMPs, COX2, PGE2, and PGD2 81
Supplemental Figure 1. Table of pathology scores 82
Supplemental Figure 2. Time course of MHCII- populations throughout infection 83
Supplemental Figure 3. Table of means and standard errors for each innate cell population measured in each experimental group 84
Supplemental Figure 4. Kinetics of lung arginase activity throughout the duration of infection 85
Supplemental Figure 5. NP147 stimulated T cell populations at 14 DPI. 86
Supplemental Figure 6. Heat-inactivated stimulated CD4 T cell responses at 14DPI 87
Supplemental Figure 7. Western blot images of MMP9, MMP2, MMP14, COX2, PGE2, and PGD2. 88
CHAPTER III: Conclusions 89
CHAPTER IV: References 96
APPENDIX I: Stable incorporation of GM-CSF into dissolvable microneedle patch improves skin vaccination against influenza 128
Abstract 129
Introduction 130
Materials and Methods 133
Results 141
Discussion 151
Conclusions 154
Acknowledgments 155
References 156
Figure 1. Adjuvantation with 100 ng GM-CSF in intradermal H1N1 A/California/07/09 subunit vaccination improves antibody responses and enhances protection to lethal virus challenge 196
Figure 2. GM-CSF is an effective adjuvant at low doses in H3N2 vaccinations. 198
Figure 3. GM-CSF retains proliferative capacity in bone marrow cells following MN patch fabrication and dissolution. 200
Figure 4. GM-CSF retains bioactivity following fabrication with a range of MN excipients. 201
Figure 5. The inclusion of GM-CSF in MN patches in H1N1 influenza vaccination is dose-sparing and generates superior IgG expression against homologous and heterologous HAs compared to ID and IM vaccination 203
Figure 6. H1N1 subunit vaccination with GM-CSF improved protection of mice from lethal H1N1 influenza challenge 205
Figure 7. GM-CSF improved antibody responses and avidity when included in MN patch vaccination. 207
Figure 8. Inclusion of GM-CSF enhanced activation of B cells in spleen and bone marrow. 209
Fig 9. MN patch vaccination with GM-CSF increased CD8+ T cell responses in inguinal lymph nodes (ILN) and vaccine-specific IFN-γ responses in the spleen. 210
Fig. 10. GM-CSF adjuvanted MN patch immunization improved cross-reactive neutralization responses between homologous and heterologous viral strains. 212
Fig. 11. Th1 cytokine responses to viral infection were increased in GM-CSF-adjuvanted MN vaccinated mice. 214
Supplementary Figure 1. Gating strategies for flow cytometry. 215
Supplementary Figure 2. Activation of T cells in inguinal lymph nodes (ILN) and spleen. 216
Supplementary Figure 3. Cross-reactive antibody titers against H1N1 and H3N2 viruses 217
APPENDIX II: Microneedle vaccination against Zika virus confers superior cellular and humoral immunity while protecting against neuroinflammation 218
Abstract 219
Introduction 220
Results 222
Discussion 234
Materials and Methods 240
Acknowledgments 249
References 251
Figure 1. ZVIP-MNP results in higher antibody concentrations, greater binding avidity, and neutralize live flaviviruses better than IM counterparts.256
Figure 2. ZVIP-MNP specific antibodies resulted in reduced antibody-mediated infection by ZIKV or DENV viruses of U937 cells compared to IM or placebo groups. 258
Figure 3. ZVIP-MNP vaccinations resulted in greater B and T cell responses 2 weeks post-prime vaccination. 260
Figure 4. ZVIP-MNP vaccination confers protection of systemic and immune- privileged organs after infectious challenge. 262
Figure 5. A single ZVIP-MNP immunization conferred better protection against ocular and motor/neural symptoms than IM vaccination. 264
Figure 6. Vaccination with ZVIP-MNP protects against infection-induced decreases in MBP, myelin PLP, and Vimentin. 266
Figure 7. A single vaccination with ZVIP-MNP protects the ocular tract from ZIKV infection, tissue pathology, and apoptosis at both early and late time-points. 268
Figure 8. A prime/booster vaccination with ZVIP-MNP protects the ocular tract from ZIKV infection, infection-induced damage, and apoptosis at both early and late time-points. 270
Figure 9. ZVIP-MNP vaccination resulted in lower transcription of chronic inflammation markers in brain and eye tissues, as well as less systemic inflammation during the course of infection. 272
Figure 10. Anti-ganglioside antibodies were produced during ZIKV infection but are limited after vaccination with ZVIP-MNP. 274
Supplemental Figure 1. Vaccination via ZVIP-MNP generated greater vaccine-specific antibody titers following infection. 276
Supplemental Figure 2. Gating strategies utilized for flow cytometry experiments. 277
Supplemental Figure 3. MNP vaccination generated higher frequencies of germinal center B cells, TFH, and follicular CD8 cells compared to IM vaccination. 279
Supplemental Figure 4. ZVIP-MNP vaccination protected against infection-induced decreases in myelin and limited viral load in brains 100 DPI. 280
Supplemental Figure 5. ZVIP-MNP vaccination protected against infection-induced decreases in myelin PLP and limited viral load in hippocampal regions 100 DPI. 281
Supplemental Figure 6. ZVIP-MNP immunization protected against infection-induced decreases in vimentin and limited viral load in cortical and hippocampal regions of the brain 100 DPI. 282
Supplemental Figure 7. ZVIP-MNP immunization resulted in decreased viral load within corneal tissues of the optic tract compared to the IM immunization route at early and late infectious time-points. 283
Supplemental Figure 8. ZVIP-MNP immunization resulted in lower infection-induced apoptosis within corneal tissues compared to IM immunization at early and late infectious time-points. 284
Supplemental Table 1. All antibodies utilized in flow cytometry experiments are listed in panels according to the scope of each experiment, including information such as fluorophore, manufacturer, catalogue number, and clone number. 285
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