Vertical T cell immunodominance and epitope entropy determine HIV-1 escape.

HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8+ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell–mediated in vivo control of HIV-1. Primary HIV-1–specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or “vertical” immunodominance as the primary determinant of in vivo CD8+ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8+ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance

Early Low-Titer Neutralizing Antibodies Impede HIV-1 Replication and Select for Virus Escape

Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC50) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1–V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one

Impact of immune escape mutations on HIV-1 fitness in the context of the cognate transmitted/founder genome

<p>Abstract</p> <p>Background</p> <p>A modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method.</p> <p>Results</p> <p>The T/F and mutant viruses were competed in CD4⁺ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations which restored viral replication fitness were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but V247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region.</p> <p>Conclusions</p> <p>These findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.</p

Adsorption of plasma Nabs by autologous T/F Env monomers and trimers.

<p>Plasma from CH40 (A), CH77 (B), and CH58 (C) was incubated with magnetic-bead bound gp120 or tethered gp140 protein corresponding to the T/F sequence from each subject. Beads were removed and neutralization assessed by TZM-bl assay (BSA, bovine serum albumen; b12 broadly neutralizing mAb positive control). Results are the mean +/− SD of three independently performed experiments each performed in duplicate.</p

Comparison with Poisson model of random sequence evolution.

a<p>Number of reads in Poisson distance distribution.</p>b<p>APOBEC sites were included or excluded from the analysis.</p>c<p>Number of nucleotides used to compute pairwise sequence distances.</p>d<p>Mean Hamming distance in pairwise sequence distance distributions.</p>e<p>Maximum Hamming distance in pairwise sequence distance distributions.</p>f<p>P-value for goodness-of-fit to the Poisson distribution by chi-squared test; where P>0.01, the sample fits the Poisson null model.</p>g<p>Nab epitope includes the 12 amino acids encompassing phenotypically-proven Nab escape mutations; see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002721#ppat-1002721-g007" target="_blank">Figure 7</a>.</p>h<p>Non-epitope includes the 12 remaining amino acids in V1 not included in Nab epitope region; see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002721#ppat-1002721-g007" target="_blank">Figure 7</a>.</p

Sequence entropy and viral dynamics at the CH40 V1 Nab epitope.

<p>(A) 454 sequence entropies inside (grey) and outside (white) of a 12-amino acid Nab epitope region in V1. Bar length indicates Shannon entropy computed bidirectionally (a and b) (one-sided Wilcoxon analysis). (B and C) Plasma viral RNA (black asterisks) consist of epitope variants in various abundances as determined by SGA or 454 sequencing. Error bars depict 95% confidence intervals from the binomial distribution.</p

Sequences and autologous neutralization sensitivities of consensus infectious molecular clones.

<p>A. 6 mo and 6 mo-Nab IMC sequences are aligned to the T/F sequence with red and blue tics indicating non-synonymous changes implicated in CTL and Nab escape, respectively. Green tics denote synonymous changes and aqua tics changes in non-coding regions. B. Neutralization of IMCs by autologous 6 month plasma is reported as mean (+/− SD) reciprocal plasma dilutions (IC₅₀). Experiments were conducted in triplicate and repeated three times.</p

Env models.

<p>Models of gp120 molecules from (A) CH40, (B) CH77, and (C) CH58 are shown as white surface projections, with the V3 and V4 loop regions colored in blue and cyan. Models of the V1/V2 regions of CH40 and CH77 are shown as yellow ribbons with parts of the variable V1 and V2 loops shown as dotted lines. Potential N-linked glycans are modeled as grey spheres. Nab escape mutations are shown in red (HXB2 numbering), with mutations removing or adding a potential N-linked glycosylation site marked with an asterisk. (D) Schematic of putative V1/V2 (yellow dot), V3 (blue dot), and V4 (cyan dot) locations on the Env trimer.</p

<i>Highlighter</i> analysis and <i>env</i> sequence alignments of putative Nab epitopes in subject CH40.

<p>A. <i>Highlighter</i> plot traces acquired mutations in gp160 <i>env</i> against the T/F sequence at top. Nucleotide differences from the T/F sequence are indicated (red: non-synonymous, green: synonymous) according to days post-seroconversion. CTL epitopes previously confirmed in T cell assays, are indicated by blue triangles. Mutations responsible for Nab escape are highlighted in yellow. B. Amino acid alignments of the V1 and V3 regions (HXB2 numbering). The two amino acid span interrogated by PASS is underlined. SGA sequences were from 6 independent experiments.</p