| Summary: | Understanding of the viral and host factors that determine time for progression to acquired
immunodeficiency syndrome (AIDS) in individuals infected with human immunodeficiency
virus type 1 (HIV-1) could aid in the design of an effective HIV-1 vaccine. Human
leukocyte antigen (HLA) class I profile is strongly and consistently associated with
differential rates of HIV-1 disease progression, however the mechanisms explaining this are
not well understood. It has been hypothesised that “protective” HLA alleles select escape
mutations in functionally important epitopes in the conserved group specific antigen (Gag)
protein resulting in HIV-1 attenuation, which may result in slower disease progression.
Many of the studies investigating the fitness cost of Gag escape mutations have concentrated
on a few pre-selected mutations and have not assessed fitness consequences in the natural
sequence background. Furthermore, the majority of studies have focussed on HIV-1 subtype
B, while HIV-1 subtype C is the most prevalent subtype worldwide. Therefore, in the
present study, a large population-based approach and clinically-derived Gag-protease
sequences were used to comprehensively investigate the relationship between immunedriven
sequence variation in Gag, viral replication capacity and markers of disease
progression in HIV-1 subtype C chronic infection. The influence of Gag function on HIV-1
disease progression was further investigated in early HIV-1 subtype C infection. It was also
hypothesised that Gag may contribute significantly to overall HIV-1 fitness and towards
fitness differences between HIV-1 subtypes.
Materials and Methods
Recombinant viruses encoding Gag-protease, derived from antiretroviral naïve HIV-1
subtype C chronically (n=406) and recently (n=60) infected patients as well as a small subset
of HIV-1 subtype B chronically infected patients (n=25), were generated by electroporation of an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived
gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid.
The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from
peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C
(n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication
capacity was defined as the slope of increase in percentage infected cells from days 3-6
following infection, normalised to the growth of a wild-type NL4-3 control. Replication
capacities were related to patient HLA alleles and markers of disease progression (viral load,
CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral
set point and rate of CD4+ T cell decline in recently infected patients). Replication
capacities were compared between isolates and recombinant viruses encoding Gag-protease
from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses
matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease
amplicons was performed and mutations were identified that were significantly
associated with altered viral replication capacity. The fitness effect of some of these
mutations was directly tested by site-directed mutagenesis followed by assay of the mutant
viruses.
Results
In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81
(p<0.0001), were associated with lower replication capacities. HLA-associated mutations at
low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with
lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in
the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed
mutagenesis experiments (p<0.001), and the co-varying mutations tested did not
significantly compensate for this fitness cost. Replication capacity also correlated positively of an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived
gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid.
The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from
peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C
(n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication
capacity was defined as the slope of increase in percentage infected cells from days 3-6
following infection, normalised to the growth of a wild-type NL4-3 control. Replication
capacities were related to patient HLA alleles and markers of disease progression (viral load,
CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral
set point and rate of CD4+ T cell decline in recently infected patients). Replication
capacities were compared between isolates and recombinant viruses encoding Gag-protease
from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses
matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease
amplicons was performed and mutations were identified that were significantly
associated with altered viral replication capacity. The fitness effect of some of these
mutations was directly tested by site-directed mutagenesis followed by assay of the mutant
viruses.
Results
In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81
(p<0.0001), were associated with lower replication capacities. HLA-associated mutations at
low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with
lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in
the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed
mutagenesis experiments (p<0.001), and the co-varying mutations tested did not
significantly compensate for this fitness cost. Replication capacity also correlated positivelyof an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived
gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid.
The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from
peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C
(n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication
capacity was defined as the slope of increase in percentage infected cells from days 3-6
following infection, normalised to the growth of a wild-type NL4-3 control. Replication
capacities were related to patient HLA alleles and markers of disease progression (viral load,
CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral
set point and rate of CD4+ T cell decline in recently infected patients). Replication
capacities were compared between isolates and recombinant viruses encoding Gag-protease
from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses
matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease
amplicons was performed and mutations were identified that were significantly
associated with altered viral replication capacity. The fitness effect of some of these
mutations was directly tested by site-directed mutagenesis followed by assay of the mutant
viruses.
Results
In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81
(p<0.0001), were associated with lower replication capacities. HLA-associated mutations at
low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with
lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in
the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed
mutagenesis experiments (p<0.001), and the co-varying mutations tested did not
significantly compensate for this fitness cost. Replication capacity also correlated positively with baseline viral load (p<0.0001) and negatively with baseline CD4+ T cell count
(p=0.0004), but not with subsequent rate of CD4+ T cell decline (p=0.73).
In HIV-1 subtype C recent infection, replication capacities of the early viruses did not
correlate with subsequent viral set points (p=0.37) but were significantly lower in individuals
with below median viral set points (p=0.03), and there was a trend of correlation between
lower replication capacities and slower rates of CD4+ T cell decline (p=0.09). Overall, the
proportion of host HLA-specific Gag polymorphisms in or adjacent to epitopes was
negatively associated with replication capacities (p=0.04) but host HLA-B-specific
polymorphisms were associated with higher viral set points (p=0.01), suggesting a balance
between effective Gag CD8+ T cell responses and viral replication capacity in influencing
viral set point.
A moderate statistically significant correlation was found between the replication capacities
of whole isolates and their corresponding Gag-protease recombinant viruses (p=0.04) and
the replication capacities of the subtype C recombinant viruses were significantly lower than
that of the subtype B recombinant viruses (p<0.0001). The subtype-specific difference in the
consensus amino acids at Gag codons 483 and 484 was found in site-directed mutagenesis
experiments to largely contribute to the fitness difference between subtypes, possibly by
influencing budding efficiency.
Discussion
The data support that protective HLA alleles, in particular HLA-B*81, attenuate HIV-1
through HLA-restricted CD8+ T cell-mediated selection pressure on Gag. Results suggest
that viral replication capacity determined by sequence variability in Gag-protease has an
impact on HIV-1 disease progression, but also indicate that a balance between HLA-driven
fitness costs and maintenance of effective CD8+ T cell responses is important in determining clinical outcome. Gag-protease was observed to significantly contribute to overall HIV-1
replication capacity and variability in this region between HIV-1 subtypes B and C is
suggested to partly explain the difference in viral fitness between these subtypes. Specific
mutations in Gag-protease associated with viral attenuation were identified and it was also
observed that mutations in conserved Gag regions carried the greatest cost to HIV-1
replication capacity. Overall, the data support the concept of, and may assist in the rational
design of, an HIV-1 vaccine in which immune responses are directed towards several
conserved epitopes, particularly in Gag, with the aim to constrain immune escape (thereby
maintaining effective CD8+ T cell responses) and attenuate HIV-1 (in the event of partial
escape), resulting in slower disease course and reduced HIV-1 transmission at the population
level. === Thesis (Ph.D.)-University of KwaZulu-Natal, 2011.
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