Studies on the Influenza A Virus Hemagglutinin and Immune Responses to Influenza Vaccine Vectors and RNA Viral Infections in Mice Open Access

Langley III, William Arthur (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/2j62s5070?locale=en
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Abstract

Influenza is a major health concern for humans and despite intensive efforts, strategies to prevent and/or cure it have not proven to be very successful. Viral mutants resistant to available drugs are common and antigenic changes in the glycoproteins make it necessary to frequently reformulate vaccine components. The work presented herein examines numerous aspects of influenza and immune responses to viruses in hopes of providing insights that may aid in the development of improved anti-viral strategies and vaccines. Drugs and vaccines that are able to effectively target conserved components of influenza are likely to prove effective at targeting multiple influenza subtypes. We have extended studies on one of these potential targets, the fusion peptide of the HA, and demonstrated that single residue deletions in this domain prevent it from mediating fusion. These studies demonstrate length constraints for the fusion peptide and may help lead to the design of drugs that can target this conserved region of the virus. Further work focuses on the ability of candidate influenza vaccines containing disrupted NS1 proteins to elicit CTL responses. We show that, although these viruses are severely attenuated, they are able to generate potent memory CTLs and these are able to mediate viral clearance. Several properties make influenza attractive as a vaccine vector. We demonstrate that recombinant influenza vectors containing inserts of Bacillus anthracis are able to elicit antibody responses against these inserted domains and that these responses can be boosted by heterologous vectors to levels that are able to neutralize the anthrax toxin. However, CTLs recognizing multiple strains of influenza inhibit previously infected mice from developing antibody or CTL responses after immunization with influenza vectors, although, these cross-reactive cells are able to protect mice from challenges with heterosubtypic viruses. Finally, we look at the longevity of humoral responses to influenza, LCMV, and VSV in mice. We show that depletion of naïve and memory B cells by rituximab results in a decrease of virus-specific plasma cells. These data indicate that plasma cells are intrinsically long-lived, but that some re-seeding by memory B cells may be necessary to maintain their numbers long-term.

Table of Contents


Table of Contents

Abstract
Acknowledgements
Table of Contents
List of Figures and Tables

Table of Contents

Chapter 1: Introduction
Influenza background.............................................................................................. 1
Influenza vaccines and antivirals............................................................................. 16
Influenza virus vaccine vectors............................................................................... 28
Maintenance of humoral immunity against viruses................................................. 35

Chapter 2: Single residue deletions along the length of the influenza HA fusion
peptide lead to inhibition of membrane fusion function
................................... 41
Abstract................................................................................................................... 43

Introduction............................................................................................................. 44

Results and Discussion............................................................................................ 48
Materials and Methods............................................................................................ 72
Acknowledgements................................................................................................. 75
References............................................................................................................... 75

Chapter 3: Immunization with Live Attenuated Influenza Viruses That Express
Altered NS1 Proteins Results in Potent and Protective Memory CD8+ T-Cell
Responses
............................................................................................................... 86
Abstract................................................................................................................... 88
Introduction............................................................................................................. 89
Materials and Methods............................................................................................ 92
Results..................................................................................................................... 96
Discussion................................................................................................................ 111
Acknowledgements.................................................................................................. 113
References................................................................................................................ 114

Chapter 4: Induction of neutralizing antibody responses to anthrax PA using
broadly-applicable influenza vectors: Implications for disparate immune system
priming pathways
................................................................................................... 122
Abstract.................................................................................................................... 124
Introduction.............................................................................................................. 125
Materials and Methods............................................................................................. 129
Results and Discussion............................................................................................. 131
Acknowledgements.................................................................................................. 146
References................................................................................................................ 146



Chapter 5: The effects of preexisting immunity to influenza on responses to
influenza vectors in mice

...................................................................................... 154
Abstract................................................................................................................... 156
Introduction............................................................................................................. 157
Materials and Methods............................................................................................ 161
Results..................................................................................................................... 164
Discussion................................................................................................................ 178
Acknowledgements.................................................................................................. 181
References................................................................................................................ 182

Chapter 6: The role of memory B cells in maintaining long-lived humoral immunity
.................. 189
Abstract.................................................................................................................... 191
Introduction.............................................................................................................. 192
Results...................................................................................................................... 195
Discussion................................................................................................................ 212
Materials and Methods............................................................................................. 215
References................................................................................................................ 220

Chapter 7: Discussion and Future Directions...................................................... 227

References
................................................................................................................ 238

























List of Figures and Tables


Chapter 2: Single residue deletions along the length of the influenza HA fusion
peptide lead to inhibition of membrane fusion function

Figure 2-1:

HA Fusion peptide sequences from representatives of each of the 16 HA
subtypes and nomenclature and fusion peptide sequences of the HA deletion mutants
addressed in this study..........................................................................................
51

Figure 2-2: Ribbon diagrams of the three conformations of the HA trimer........
52

Table 2-1: Antibody reactivity of cell-surface HAs by ELISA............................
54

Figure 2-3: Cell surface expression of HAs as assayed by trypsin cleavage of HA0 into
HA1 and HA2........................................................................................................
56

Figure 2-4: Graphs of ELISA experiments to demonstrate HA conformational changes
resulting from incubation at reduced pH...............................................................
61

Figure 2-5: Graphs of ELISA experiments showing reactivity with the low pH-specific
monoclonal antibody IIF4 as a function of pH...................................................... 62

Figure 2-6: Western blot analysis for the determination of the pH of conformational
change by trypsin susceptibility............................................................................. 63

Figure 2-7: Polykaryon formation by HA-expressing BHK cells following incubation at
pH 4.8..................................................................................................................... 66

Figure 2-8: Dye transfer assay for hemifusion and full fusion activity of HA mutants
............. 67

Chapter 3: Immunization with Live Attenuated Influenza Viruses That Express
Altered NS1 Proteins Results in Potent and Protective Memory CD8+ T-Cell
Responses

Figure 3-1:
Constructs used in this study............................................................... 99

Figure 3-2: T-cell responses in the spleen and lungs of mice 8 days after infection with
the NS1 mutant viruses............................................................................................ 102

Figure 3-3: Long-lived memory CD8 T-cell populations are primed by infection with the
NS1-mutant viruses.................................................................................................. 104

Figure 3-4: Recall of memory P14 cells primed by NS1 mutant viruses................ 106


Figure 3-5:

Accelerated prime-boost responses soon after infection with NS1 mutant
virus.......................................................................................................................... 110


Chapter 4: Induction of neutralizing antibody responses to anthrax PA using
broadly-applicable influenza vectors: Implications for disparate immune system
priming pathways

Figure 4-1:
Bacillus anthracis protective antigen and vector constructs................ 133

Figure 4-2: Antibody responses following immunization with viral vectors......... 138

Figure 4-3: Antibody responses following heterologous boosting.......................... 139

Figure 4-4: Antibody responses following intramuscular boosting......................... 141

Table 4-1: Anthrax toxin neutralizing titers............................................................. 145


Chapter 5: The effects of preexisting immunity to influenza on responses to
influenza vectors in mice

Figure 5-1:
The effect of previous exposure to influenza on antibody responses to
influenza vectors....................................................................................................... 166

Figure 5-2: The effect of previous exposure to influenza on CD8+ T cell responses to
influenza vectors....................................................................................................... 169

Figure 5-3: NP366-374 CD8+ T cell responses in previously infected mice prior to and
following administration of the inf-LEF vector........................................................ 172

Figure 5-4: The effect of the absence of preexisting influenza-specific CD8+ T cells on
the antibody response to the inf-LEF vector............................................................. 174

Figure 5-5: The effect of previous infection with homologous or heterologous influenza
subtypes on mice receiving a lethal challenge of H1N1............................................ 177


Chapter 6: The role of memory B cells in maintaining long-lived humoral immunity

Figure 6-1:
B cell depletion in PB of rituximab treated mice................................... 196

Figure 6-2: Memory B cell depletion in rituximab treated mice.............................. 198

Figure 6-3: Anti-viral IgG titers in rituximab treated mice...................................... 200

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