Mechanisms of Viral Escape from Neutralizing Antibodies during Subtype C HIV- 1 Infection Open Access
Lynch, Rebecca Marie (2010)
Abstract
Human immunodeficiency virus type 1 (HIV-1) group M is
responsible for the
current AIDS pandemic and exhibits exceedingly high levels of viral
genetic
diversity around the world. This diversity reflects the remarkable
ability of the
virus to adapt to selective pressures, the bulk of which is applied
by the host
immune response, and represents an obstacle for developing a
vaccine capable of
broad coverage. Studying how the virus escapes this immune pressure
at both a
population as well as an individual level will ultimately aid in
vaccine design. The
V3 region of the HIV-1 envelope (Env) glycoprotein gp120 is a key
functional
domain yet it exhibits distinct mutational patterns across
subtypes. Here an
invariant residue in V3 (Ile 309) is replaced with Leu in subtype C
patient-
derived Envs. The results demonstrate that conservation of Ile 309
preserves a
V3-mediated masking function that occludes the CD4 binding site,
revealing a
novel immune evasion strategy that subtype C HIV-1 uses to protect
this immune
target. Within individual subjects, however, the virus can elicit
diverse
neutralizing antibody (Nab) responses, leading to different escape
pathways. In
order to elucidate this process in more detail, the early Nab
response in a
Zambian seroconverter is characterized for the first time at the
autologous
monoclonal antibody (Mab) level. Here five Mabs are described, and
autologous
neutralization by Mabs representative of three distinct B-cell
clones are mapped
to two residues (134 in V1 and 189 in V2). Mutational analysis
reveals cooperative
effects between glycans and residues at these two positions,
arguing that they
contribute to a single novel epitope. Further data demonstrates
that although
independent B cells in this subject repeatedly target a single
structure in V1V2,
this pressure is escaped by a single residue change with no
demonstrable effect
on replication. Together this thesis suggests that subtype C HIV-1
can evade the
potent but limited humoral immune response using both sequence
variation and
conservation, and thus I would propose that a successful vaccine
might need to
expand the narrow response of natural infection by targeting
multiple domains of
gp120 in order to achieve ultimate effectiveness.
Table of Contents
Table of Contents
Chapter One: Thesis Introduction 1
Origins of HIV 1
HIV Diversity 2
Function and structure of HIV Envelope 3
Subtype-specific Nab responses during HIV-1 infection 6
Targets of Nabs and subsequent viral escape 8
Consequences of viral escape 11
Summary 13
Chapter Two: Subtype-specific Conservation of Isoleucine 309 in the
Envelope V3 Domain is Linked to Immune Evasion in Subtype C
HIV-1 Infection 15
I. Abstract 15
II. Introduction 16
III. Materials and Methods 18
Env clones 18
PCR-based site mutagenesis 18
Virus neutralization and inhibition assays 20
Replication in CD4 and monocyte-derived macrophages (MDM) using an
NL4.3 proviral cassette 20
Receptor-dependent pseudovirus entry assay 22
Statistical analysis 22
IV. Results 23
I309L was created in a representative panel of diverse subtype C Envs 23
The I309L mutation does not decrease replication in primary CD4 T cells 26
The I309L mutation leads to a moderate enhancement of replication in
monocyte-derived macrophages 29
The I309L mutation confers increased entry into a cell line expressing low
CD4 32
The I309L mutation confers increased sensitivity to sCD4 35
The I309L mutation moderately increased sensitivity to monoclonal antibodies
directed against V3 and a CD4-induced epitope 38
V. Discussion 43
Replication is not dependent on conservation of I309 43
Conservation of I309 prevents exposure of neutralization targets 44
V3-mediated masking of the CD4 binding site 45
VI. Acknowledgements 50
VII. Contributors 51
Chapter Three: The B cell response is redundant and highly focused
on V1V2 during early subtype C infection in a Zambian
seroconverter 52
I. Abstract 52
II. Introduction 53
III. Materials and Methods 56
Env clones 56
PCR-based site mutagenesis and virus preparation 56
Generation of human monoclonal antibodies 58
Screening ELISA for Mabs 58
Neutralization Screening Assay for Mabs 59
Pseudovirus inhibition assays 59
Homology modeling of residues 134 and 189 in the V1V2 domain 60
Replication in CD4+ cells using an NL4.3 proviral cassette 60
IV. Results 62
Characterization of monoclonal antibodies isolated from a subtype C infected patient
and selected for neutralization activity against the founder Env 62
The monoclonal antibodies likely arose before eight months
post-seroconversion 66
Glycosylation in V1 can confer sensitivity to monoclonal antibodies 68
Amino acid sequence rather than glycosylation status in V2 defines neutralization
sensitivity to Mabs 71
Residues 134 and 189 may contribute to a single epitope near the V1V2 stem 74
Amino acid changes at residues 134 and 189 do not overtly affect replication fitness in
vitro 76
V. Discussion 79
By 8-months post-seroconversion, multiple B-cell clones produced somatic variations
of antibodies that neutralize the virus 79
Mutational analysis reveals that residues 134 and 189 contribute to a novel epitope
near the V1V2 stem 80
Glycosylation plays varying roles in neutralization during early infection 81
Replication kinetics of neutralization resistant Envs is similar to neutralization
sensitive Envs 82
VI. Acknowledgements 86
VII. Contributors 87
Chapter Four: Thesis Discussion 88
Conclusion 96
Bibliography 98
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