Superior Control of HIV-1 Replication by CD8+ T Cells Targeting Conserved Epitopes: Implications for HIV Vaccine Design

<div><p>A successful HIV vaccine will likely induce both humoral and cell-mediated immunity, however, the enormous diversity of HIV has hampered the development of a vaccine that effectively elicits both arms of the adaptive immune response. To tackle the problem of viral diversity, T cell-based vaccine approaches have focused on two main strategies (i) increasing the breadth of vaccine-induced responses or (ii) increasing vaccine-induced responses targeting only conserved regions of the virus. The relative extent to which set-point viremia is impacted by epitope-conservation of CD8⁺ T cell responses elicited during early HIV-infection is unknown but has important implications for vaccine design. To address this question, we comprehensively mapped HIV-1 CD8⁺ T cell epitope-specificities in 23 ART-naïve individuals during early infection and computed their conservation score (CS) by three different methods (prevalence, entropy and conseq) on clade-B and group-M sequence alignments. The majority of CD8⁺ T cell responses were directed against variable epitopes (p<0.01). Interestingly, increasing breadth of CD8⁺ T cell responses specifically recognizing conserved epitopes was associated with lower set-point viremia (r = - 0.65, p = 0.009). Moreover, subjects possessing CD8⁺ T cells recognizing at least one conserved epitope had 1.4 log₁₀ lower set-point viremia compared to those recognizing only variable epitopes (p = 0.021). The association between viral control and the breadth of conserved CD8⁺ T cell responses may be influenced by the method of CS definition and sequences used to determine conservation levels. Strikingly, targeting variable versus conserved epitopes was independent of HLA type (p = 0.215). The associations with viral control were independent of functional avidity of CD8⁺ T cell responses elicited during early infection. Taken together, these data suggest that the next-generation of T-cell based HIV-1 vaccines should focus on strategies that can elicit CD8⁺ T cell responses to multiple conserved epitopes of HIV-1.</p></div

Pooled-Peptide Epitope Mapping Strategies Are Efficient and Highly Sensitive: An Evaluation of Methods for Identifying Human T Cell Epitope Specificities in Large-Scale HIV Vaccine Efficacy Trials

<div><p>The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7–30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.</p></div

Correlation of responses to peptide pools and the peptides they contain.

<p>Rank-based correlations were assessed between the magnitudes of T-cell responses to positive peptide pools that contain a single positive peptide and the individual response to the positive peptide (A, one dot per pool, dashes indicate line of unity) and positive pools containing >1 positive peptide and the sum of the individual responses to the positive peptides they contain (B). Positive pools containing no positive 15-mer and >1 false negative 15-mer were plotted against the sum of the negative peptides they contained (C). 95% confidence intervals and p-value were computed using a participant-based bootstrap (n = 15 responders).</p

Evaluation of epitope mapping strategies.

<p>Epitope mapping strategies were assessed based on the number of positive 15-mers (A) and the number of epitopes (B) that were identified. By definition, all 15-mers were identified by the Test-all strategy. Epitopes detected using an optimal peptide were not necessarily identified by the Test-all strategy, but may have been detected by a pool strategy if all pools containing the epitope were positive. Potential benefits were explored of using computational HLA binding predictors to identify the epitope-containing 15-mer within a positive mini-pool (C, n = 20 pools) and to identify the optimal peptide within a positive 15-mer (D, n = 25 peptides). Ranks of the 15-mers in the mini-pool or the optimal peptides within the 15-mer were based on predicted HLA binding with each participant’s alleles. Observed average (line) and 95% confidence interval (dashed lines) are illustrated relative to a distribution of averages computed using random rankings.</p

Targeting conserved epitope (bCSp) was independent of possession of favorable alleles.

<p>(A) The median CS of epitopes by HLA group: favorable alleles (subjects possessing B*27 and B*57 alleles), unfavorable alleles (subjects possessing B*35Px alleles: B*35Px: B*35∶02, B*35∶03, B*35∶04, and B*53∶01) and neutral alleles (subjects not possessing B*27, B*57 or B*35Px alleles) (Kruskal-Wallis, p = 0.215). Horizontal lines indicate median value. (B) The median plasma VL set point in individuals (subjects not recognizing at least one conserved epitopes were excluded on this analysis) recognizing at least one conserved epitope by possession of favorable allele (Mann Whitney, p = 0.662). (C) The median plasma VL set point in individuals (not possessing favorable alleles, subjects possessing favorable alleles were excluded) who elicited CD8⁺ T cell responses against at least one conserved epitope (Mann Whitney, p = 0.067). (B–C) Subjects possessing B*35Px, B*27 and B*57 alleles are represented by red circles, green triangles and inverted green triangles respectively.</p

Method of determining CS influences significance of T cell association with and viral control.

<p>(A) The plasma VL set point was compared to breadth of conserved epitopes based on mCSp (Spearman Rank Correlation, r = −0.40, p = 0.139). (B, C) The plasma VL set point was compared to breadth of conserved epitopes based on bCSe (Spearman Rank Correlation, r = −0.52, p = 0.048) and mCSe (Spearman Rank Correlation, r = −0.52, p = 0.043) respectively. Subject possessing B*35Px, B*27 and B*57 allele are represented by red circles, green triangles and inverted green triangles respectively.</p

CD8⁺ T cell responses against conserved epitopes (bCSp) are associated with viral control.

<p>(A) The plasma VL set point was compared to breadth of conserved epitopes (Spearman Rank Correlation, r = −0.65, p = 0.009). The solid line represents a regression line. (B) The median plasma viral set point in individuals who mounted CD8⁺ T cell responses against at least one conserved epitope (Mann Whitney, p = 0.018). (A–B) Subjects possessing B*35Px, B*27 and B*57 alleles are represented by red circles, green triangles and inverted green triangles respectively.</p

T-cell responses to small pools of 15-mer peptides.

<p>ELISpot responses to peptide pools were categorized based first on their positivity (A, positive; B, negative; C, response magnitudes, one dot indicates the mean of a triplicate assay) and subsequently on whether or not they contained a positive 15-mer or a 15-mer containing the sequence of a positive optimal peptide.</p

Breadth of HIV-1-specific CD8⁺ T cell responses to conserved Gag epitopes correlate with lower viremia.

<p>VL set point was compared to breadth of CD8⁺ T cell responses. (A and B) Correlation between breadth of CD8⁺ T cell responses against conserved Gag or variable Gag epitopes (clade-B) with plasma VL set point (Spearman Rank Correlation, r = −0.65, p = 0.009 and r = −0.32, p = 0.250 respectively). (A–B) The solid line represents a regression line. (C) The median plasma VL set point in individuals who mounted CD8⁺ T cell responses against at least one conserved (bCSp) Gag epitope (Mann Whitney, p = 0.019). (A–C) Subject possessing B*35Px, B*27 and B*57 allele are represented by red circles, green triangles and inverted green triangles respectively.</p

CD8⁺ T cell functional avidity and control of viral replication.

<p>(A) Correlation between functional avidity and CS of CD8⁺ T cell epitopes. (B) The functional avidity of CD8⁺ T cells by CS (clade-B) grouping (Kruskal-Wallis, p = 0.681). Horizontal lines indicate median values. (C–E) Correlation between average (C), maximum (D) and immunodominant (E) functional avidity of CD8⁺ T cells in each subject with average plasma viral set point (Spearman Rank Correlation, r = −0.07, p = 0.800; r = −0.18, p = 0.516 and r = −0.44, p = 0.105 respectively). (C–E) The solid line represents a regression line. Subject possessing B*35Px, B*27 and B*57 allele are represented by red circles, green triangles and inverted green triangles respectively.</p