Epitope length variants balance protective immune responses and viral escape in HIV-1 infection

Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4–14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition

Sequential Broadening of CTL Responses in Early HIV-1 Infection Is Associated with Viral Escape

BACKGROUND: Antigen-specific CTL responses are thought to play a central role in containment of HIV-1 infection, but no consistent correlation has been found between the magnitude and/or breadth of response and viral load changes during disease progression. METHODS AND FINDINGS: We undertook a detailed investigation of longitudinal CTL responses and HIV-1 evolution beginning with primary infection in 11 untreated HLA-A2 positive individuals. A subset of patients developed broad responses, which selected for consensus B epitope variants in Gag, Pol, and Nef, suggesting CTL-induced adaptation of HIV-1 at the population level. The patients who developed viral escape mutations and broad autologous CTL responses over time had a significantly higher increase in viral load during the first year of infection compared to those who did not develop viral escape mutations. CONCLUSIONS: A continuous dynamic development of CTL responses was associated with viral escape from temporarily effective immune responses. Our results suggest that broad CTL responses often represent footprints left by viral CTL escape rather than effective immune control, and help explain earlier findings that fail to show an association between breadth of CTL responses and viral load. Our results also demonstrate that CTL pressures help to maintain certain elements of consensus viral sequence, which likely represent viral escape from common HLA-restricted CTL responses. The ability of HIV to evolve to escape CTL responses restricted by a common HLA type highlights the challenges posed to development of an effective CTL-based vaccine

HIV-1 Adaptation to Antigen Processing Results in Population-Level Immune Evasion and Affects Subtype Diversification

Summary: The recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8+ T cell responses to two HIV-Gag regions are uniquely associated with delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ∼30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ∼60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins. : CD8+ T cell responses against HIV-1 effectively delay disease progression in a minority of patients with relatively rare HLA variants but are ineffective in most. Here, Tenzer et al. identify fundamental HIV-1 adaptation to the conserved human antigen-processing machinery that feeds epitopes to HLA. This adaptation occurs at subtype-specific motifs, facilitates subtype diversification, is predictable, and results in CD8 epitope abundances that correlate inversely with the HLA allele frequencies in affected populations. Thus, HIV vaccine immunogenicity might be increased by unnatural substitutions at subtype-specific motifs

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

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

<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

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

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