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

Langley III, William Arthur (2010)
Dissertation (301 pages)
Committee Chair / Thesis Advisers: Steinhauer, David; Ahmed, Rafi
Committee Members: Compans, Richard W ; Jacob, Joshy ; Katz, Jacqueline M ; Speck, Sam
Research Fields: Health Sciences, Immunology; Biology, Virology
Keywords: influenza; vaccines; viral vectors; plasma cells
Program: Laney Graduate School, Biological and Biomedical Sciences
Permanent url: http://pid.emory.edu/ark:/25593/7qjwx

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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