Role of Transmitted Gag CTL Polymorphisms in Defining Replicative Capacity and Early HIV-1 Pathogenesis

<div><p>Initial studies of 88 transmission pairs in the Zambia Emory HIV Research Project cohort demonstrated that the number of transmitted HLA-B associated polymorphisms in Gag, but not Nef, was negatively correlated to set point viral load (VL) in the newly infected partners. These results suggested that accumulation of CTL escape mutations in Gag might attenuate viral replication and provide a clinical benefit during early stages of infection. Using a novel approach, we have cloned <em>gag</em> sequences isolated from the earliest seroconversion plasma sample from the acutely infected recipient of 149 epidemiologically linked Zambian transmission pairs into a primary isolate, subtype C proviral vector, MJ4. We determined the replicative capacity (RC) of these Gag-MJ4 chimeras by infecting the GXR25 cell line and quantifying virion production in supernatants via a radiolabeled reverse transcriptase assay. We observed a statistically significant positive correlation between RC conferred by the transmitted Gag sequence and set point VL in newly infected individuals (p = 0.02). Furthermore, the RC of Gag-MJ4 chimeras also correlated with the VL of chronically infected donors near the estimated date of infection (p = 0.01), demonstrating that virus replication contributes to VL in both acute and chronic infection. These studies also allowed for the elucidation of novel sites in Gag associated with changes in RC, where rare mutations had the greatest effect on fitness. Although we observed both advantageous and deleterious rare mutations, the latter could point to vulnerable targets in the HIV-1 genome. Importantly, RC correlated significantly (p = 0.029) with the rate of CD4+ T cell decline over the first 3 years of infection in a manner that is partially independent of VL, suggesting that the replication capacity of HIV-1 during the earliest stages of infection is a determinant of pathogenesis beyond what might be expected based on set point VL alone.</p> </div

Cohort statistics generated from the 149 transmission pairs selected from the ZEHRP cohort.

a<p>Median (Inter-Quartile Range).</p>b<p>Measured at or within one month of seroconversion of LR.</p>c<p>LR for whom we have longitudinal CD4+ T cell counts (n = 63).</p

Replicative capacity is correlated to viral load in recipients and donors.

<p>(A) The RC of Gag-MJ4 chimeras generated from <i>gag</i> sequences isolated from epidemiologically linked recipients at acute time points correlates to early set point VL in the same acutely infected recipients (Spearman correlation, r = 0.17, p = 0.02). Replicative capacity scores were generated by normalizing the log₁₀-transformed slopes of replication curves from days 2 through 6 for each Gag-MJ4 chimeric virus to the log₁₀-transformed slope of wild-type MJ4. (B) The RC of Gag-MJ4 chimeric viruses also correlates to the VL near the estimated date of infection in chronically infected donors (Spearman correlation, r = 0.18, p = 0.01). Trend lines were generated using linear regression analysis, and are shown in order to facilitate visualization of correlations.</p

Cox proportional hazard models demonstrate the independent effects of RC and VL on CD4 decline.

a<p>as compared to RC<1.</p>b<p>Model in which log₁₀ set point viral load in the linked recipient is taken into account.</p>c<p>The ratio of hazard rates between the variables RC>2 and RC<1.</p>d<p>The upper and lower bounds of confidence.</p

The balance of fitness increasing and decreasing HLA-associated polymorphisms strongly correlates with RC.

<p>(A) The total number of HLA-associated polymorphisms positively correlates with RC. HLA-associated polymorphisms were defined as non-consensus residues at any amino acid position known to have polymorphisms statistically linked to HLA-I alleles. (B) RC of Gag-MJ4 chimeras is highly correlated to the summed polymorphism score in Gag. Each amino acid polymorphism was given a score of 1 (fitness increasing) or −1 (fitness decreasing). Summed polymorphism scores were generated by summing these scores for each Gag protein. Trend lines were generated using linear regression analysis, and are shown in order to facilitate visualization of correlations.</p

Insertion of the <i>gag</i> gene from newly infected individuals dramatically alters the replicative capacity of MJ4.

<p>In order to generate replication curves, 5×10⁵ GXR25 cells were infected with Gag-MJ4 chimeras at an MOI of 0.05. Viral supernatants (100 µL) were collected on days 2, 4, and 6, and the amount of virus in supernatants was quantified using a radiolabeled reverse transcriptase assay. Replicative capacity scores were generated by dividing the log₁₀-transformed slope of replication of each Gag-MJ4 chimera by the log₁₀-transformed slope of MJ4. It is clear that the insertion of the <i>gag</i> gene alone can dramatically alter the <i>in vitro</i> RC, generating viruses with up to 1000-fold different replication curves. DLU, digital light units.</p

Identification of polymorphisms in Gag that significantly affect RC, several of which can be linked to HLA-class I alleles.

<p>(A) In a pair-wise analysis of all amino acids represented in the 149 <i>gag</i> sequences tested, we observed 49 amino acids at 31 unique positions that were statistically associated (p<0.05) with changes in RC (open circles RC decreasing, filled circles RC increasing), 3 of which were significant at (q<0.2) when adjusted for multiple comparisons (red for RC reducing and green for RC increasing). The x-axis shows the polymorphism position in the primary Gag sequence (HXB2 numbering), and the y-axis depicts the impact of the polymorphism on RC relative to the median RC of all viruses (∼1.5). (B) In a separate study analyzing 1899 subtype C gag sequences from Zambia and South Africa, 199 residues were linked to HLA-I alleles (q<0.2, Carlson, Schaefer <i>et al.</i>, manuscript in preparation). From this, a total of 7 polymorphisms associated with changes in RC (p<0.05) were found to be adapted to specific HLA-I alleles, adapted (amino acid is present when HLA-I allele is also present.) (* denotes q<0.2). (C) Six polymorphisms associated with changes in RC (p<0.05) were found to be non-adapted (amino acid is present only when HLA-I allele is absent) to specific HLA-I alleles (* denotes q<0.2).</p

The balance of HLA-associated fitness increasing and decreasing mutations strongly correlates with set point viral load in newly infected individuals.

<p>(A) The number of HLA-associated amino acid polymorphisms within well-defined epitopes for each Gag protein negatively correlates with set point VL. (B) The total number of HLA-associated polymorphisms (including those outside well-defined CTL epitopes) in Gag does not correlate to set point VL in newly infected individuals. (C) When the quality of HLA-associated polymorphisms is considered, a strong correlation between the summed polymorphism score (as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003041#ppat-1003041-g005" target="_blank">Figure 5</a>) and set point VL is observed. Trend lines were generated using linear regression analysis, and are shown in order to facilitate visualization of correlations.</p

Transmission pair information.

a<p>M: male; F: female.</p>b<p>D: donor, LR: virologically linked recipient.</p>c<p>Fiebig stage: Fiebig stage of the recipients at the time of enrollment into the IAVI protocol C early infection study, when plasma was first collected for viral sequence analysis.</p>d<p>pVL: Plasma viral loads for the recipients are shown at the time of screening prior to enrollment into the IAVI protocol C early infection study, when R880F was at Fiebig stage III and R463F at Fiebig stage IV. The plasma viral load in R463F at Fiebig stage V was 3,980,000 copies/ml. Plasma viral loads in the donors are shown at the timepoint when the recipients were at Fiebig stage III/IV.</p><p>Transmission pair information.</p

T cell response kinetics during acute and early HIV-1 infection.

<p>For both R880F (A) and R463F(B), individual peptide response magnitudes in an IFNγ ELISpot assay are shown as a percentage of the overall response. The insert shows the number of peptide responses detected and the total response magnitude (SFC/10⁶ PBMCs) at each of the time points tested. The sequences of the peptides are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004565#ppat-1004565-t002" target="_blank">Table 2</a>.</p