<b>Early viral escape driven by an HLA-A2-restricted response against HIV-Nef.</b> The interactions between viral evolution (within all defined targeted epitopes), anti-HIV-specific CTL responses, and viral load, was followed in subject OP177.

A<p>Estimated week from infection. ^B The HXB2 sequence is given as a reference with the HLA-A2 restricted epitopes AL9 and VL10 indicated with an unbroken line in bold face letters, the HLA-B40 restricted epitope KL9 with a dotted line, and the HLA-B15 restricted WF9 epitope with double lines. <b>^C</b> The frequency of each clone is given as a number of the total number of clones sequenced with the major autologous sequence at each time-point given in bold face letters. ^DThe autologous CTL responses are given for each peptide tested. The viral variant that generates the strongest CTL response at each time-point are indicated in bold face letters.</p

Increase of HIV-Gag responses over time.

<p>The cellular immune responses against the control peptide pools CEF (A) and HIV-Gag (B) were followed in longitudinally drawn PBMC samples. No statistically significant differences in CTL responses to non-HIV antigens included in the CEF peptide pool were seen over time (Paired T-test, p = 0.269). However, the increase in CTL response to the HIV-Gag (p55) peptide pool was borderline significant (Paired T-test, p = 0.064). The responses in subjects OP177, OP428 and OP599, who developed multiple HLA-A2-restricted CTL responses and showed evidence of viral escape within targeted epitopes, are indicated with dashed lines.</p

Phylogenetic tree illustrating viral evolution of the HIV-p17 <i>gag</i> region (HXB2 coordinates 790–1431) in subject OP428 under selective pressure.

<p>Positively selected amino acid sites within the HLA-A2 ((SL9, ⁷⁷SLYNTIATL⁸⁵ (patient consensus)), HLA-A1 (GY9, ⁷¹GSEELRSLY⁷⁹), and HLA-B8 (EL9, ⁹³EVKDTKEAL¹⁰¹) restricted epitopes. Amino acids corresponding to the consensus B sequence are shown in blue, while the Y79F mutation within SL9 and GY9 is given in red, and the E93D and V94I mutations within EL9 are shown in green on the tree. The variation found at the positively selected amino acid position 62, where a potential compensatory mutation (E62A) seems to occur prior to, and associated with, the Y79F substitution are as follows: variation at position 62: E, blue, G, orange, V purple and A red. Amino acid numbering corresponds to HXB2 Gag. Scale bars signify substitutions/site. w: corresponds to estimated week from infection (e.g. a clone named 428_w15 was obtained from a plasma sample drawn at week 15).</p

Viral escape from an intricate HIV-Gag specific CTL response restricted by three HLA-alleles followed in patient OP428.

A<p>Estimated weeks from infection. ^BThe Consensus B sequence is given as a reference for amino acid position 71 to 101 of the HIV-1 Gag p17 region. The HLA-A2 restricted epitope SL9 is underlined with a single unbroken line, the HLA-A1 restricted epitope GY9 with a dotted line, and the HLA-B8 restricted epitope EL9 with double lines. <b>^C</b>The frequency of each clone is given as a number of the total number of clones sequenced. The major autologous sequence at each time-point is given in bold face letters. ^DThe autologous CTL responses are given for each peptide tested. The at week 63 minor viral sequence, which by week 139 becomes the major viral sequence, are indicated by a black box. Likewise, the corresponding CTL responses are surrounded by black boxes for each of the HLA-A2-, HLA-A1-, and HLA-B8-restriced responses, respectively. The viral variant that generates the strongest CD8+ T cell response for every HLA-restriction at each time-point is given in bold.</p

Continuous development of HLA-A2-restricted CTL responses.

<p>(A) At study entry only two of eleven patients (OP177 and OP599) showed detectable responses to three of the 20 HLA-A2-restricted epitopes tested. * Both epitopes were recognized by OP177. (B) At early chronic infection (week 48–144 of follow-up) most patients had developed a response against the Gag 77-85 (SL9) epitope. (C) Three of the eleven subjects (OP177, OP428, and OP599) developed broad HLA-A2-restricted CTL responses targeting four to five epitopes. The magnitude of the CD8+ T cell responses is given as the percentage of cells producing IFN-γ and TNF-α after withdrawing the experimental background.</p

Broad HLA-A2-restricted CTL responses are associated with an early increase of viral load

<p>(A) The number of targeted epitopes in primary and early chronic infection increased significantly during the study-period (Paired T-test, p = 0.03). While only one patient recognized 2 epitopes in primary infection, three targeted at least 4 epitopes, situated in HIV-Gag, Env, Nef and Pol, in early chronic infection. (B) All three patients who targeted multiple epitopes (open circles) had a viral load increase >0.5 log₁₀ during the first year of infection, compared to just one of seven patients with narrow or absent HLA-A2-restricted responses (closed circles) (Fisher's exact test, p = 0.03). Patient OP478, identified as being superinfected during the first year of infection, was excluded from this analysis. (C) While the viral load increased more in the group with broad HLA-A2-restricted responses during the first year of infection (open circles), the absolute viral load increase was not significant in the group during year one (Paired T-test, p = 0.15).</p

Signature pattern and viral evolution within the HLA-A2-restricted AL9 epitope.

A<p>The follow up sequence was available from a mean of 96 weeks from base-line (range: 72–144 weeks). ^BThe Consensus B sequence of the AL9 epitope is given as a reference for amino acid position 83-91. <b>^C</b>Sequence obtained at estimated week 39 from infection. ^DViral evolution due to a confirmed case of HIV superinfection within the first year of infection (F.M.H., unpublished data). Dashes represent identity with the reference sequence.</p

Characterization of the HLA-A2-restricted SL9-specific (82I and 79F82I autologous variants) and HLA-A1-restricted GY9 specific CTL responses in OP428.

<p>Peptide titration of the autologous epitope variants was conducted to evaluate the functional avidity of the responses over time, at weeks 39, 88, and 139 from estimated infection. The CFC assay was used to measure the production of gamma interferon by antigen specific CD8+ T cells, with the indicated peptides in 10-fold dilutions.</p

Phylogenetic tree illustrating viral evolution of the HIV-<i>nef</i> region (HXB2 coordinates 8774–9540) in subject OP177.

<p>Positive selection occurs from the HLA-A2-restricted CTL response (AL9, ⁸³AAVDLSHFL⁹¹) at positions 83, 85, 87 and 91 and the variation at these sites are shown in color on the tree and branches (amino acid positions identical to the consensus B sequence are shown in blue and variations in red) as well as sporadic variation elsewhere in the epitope (shown in green). The patient's virus evolves from the consensus 83A, 85 V, 87L/I, 91L (all blue except 87I which is red) to 83G (red), 85L (red), 87L (blue), 91L (blue) over approximately three years during which various epitope variants with single, or linked mutations occur in the population (83K, 87I, 91I, shown in red). Of note is the temporary predominance of the 87I mutation, which is lost shortly after the 85L mutation develop. An X at the end of the clone name refers to changes in other epitopes; green X = KL9 epitope, E98D, green XX = KL9 epitope, G99E, red X = VL9 epitope and overlapping WF9 epitope, R185K, blue X = sporadic changes in WF9 epitope (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000225#pone-0000225-t003" target="_blank">Table 3</a>). Amino acid numbering corresponds to HXB2 Gag. Scale bars signify substitutions/site. w: corresponds to estimated week from infection (e.g. a clone named 177_w13 are obtained from a plasma sample drawn at week 13).</p

HLA-A2 restricted epitopes in HIV-Gag, -Env, -Nef and –Pol.

<p>HLA-A2 restricted epitopes in HIV-Gag, -Env, -Nef and –Pol.</p