Studies on the Influenza A Virus Hemagglutinin and Immune Responses to Influenza Vaccine Vectors and RNA Viral Infections in Mice 公开
Langley III, William Arthur (2010)
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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