Anti-HIV potency of T-cell responses elicited by dendritic cell therapeutic vaccination

Abstract

Identification and characterization of CD8+ and CD4+ T-cell epitopes elicited by HIV therapeutic vaccination is key for elucidating the nature of protective cellular responses and mechanism of the immune evasion of HIV. Here, we report the characterization of HIV-specific T-cell responses in cART (combination antiretroviral therapy) treated HIV-1 infected patients after vaccination with ex vivo-generated IFNα Dendritic Cells (DCs) loaded with LIPO-5 (HIV-1 Nef 66-97, Nef 116-145, Gag 17-35, Gag 253-284 and Pol 325-355 lipopeptides). Vaccination induced and/or expanded HIV-specific CD8+ T cells producing IFNγ, perforin, granzyme A and granzyme B, and also CD4+ T cells secreting IFNγ, IL-2 and IL-13. These responses were directed against dominant and subdominant epitopes representing all vaccine regions; Gag, Pol and Nef. Interestingly, IL-2 and IL-13 produced by CD4+ T cells were negatively correlated with the peak of viral replication following analytic treatment interruption (ATI). Epitope mapping confirmed that vaccination elicited responses against predicted T-cell epitopes, but also allowed to identify a set of 8 new HIV-1 HLA-DR-restricted CD4+ T-cell epitopes. These results may help to better design future DC therapeutic vaccines and underscore the role of vaccine-elicited CD4+ T-cell responses to achieve control of HIV replication.

Fig 1. Immunogenicity of DALIA vaccine.

PBMC from patients (n = 16) collected before (W-4) and after vaccination (W16) were stimulated with individual 15-mer peptide (n = 36) spanning the vaccine sequence. Cytokine production was measured in culture supernatant using a Luminex assay at 48 hours. (A) Comparison of frequency of responders for each cytokine at W-4 and W16 using McNemar’s test. A patient was considered responder when he had at least one positive response. Positivity threshold was defined for each cytokine as fluorescence intensity (FI) > 90th percentile of all Non-Stimulated (NS) values and > 3-fold background (NS condition). (B) Comparison of the breadth of the responses for each cytokine at W-4 and W16 (Median, Wilcoxon matched-pairs signed rank test). Response breadth was defined as the number of individual peptides inducing a positive response. (C) Heatmap showing Spearman P values for correlations between maximum of plasma HIV RNA measured in patients after ATI and cytokine responses quantified by Luminex assay after vaccination at W16 from supernatants of PBMC stimulated with individual 15-mer HIV peptides. Magnitude was defined as the sum of FI for all peptide-stimulations.

Fig 2. Magnitude of IFNγ, IL-2 and IL-13 responses at W16.

PBMC from patients (n = 16) collected after vaccination (W16) were stimulated with individual 15-mer peptide (n = 36) spanning the vaccine sequence. Cytokine production was measured in culture supernatant using a Luminex assay at 48 hours. (A) Magnitude (FI) of IFNγ, IL-2 and IL-13 responses (median). Dotted line represents an arbitrary threshold of 500 FI for the definition of strong responses. (B) Magnitude (pg/ml) of IFNγ, IL-2 and IL-13 responses (median). Dotted line represents an arbitrary threshold of 15pg/ml for the definition of strong responses. (C) Heatmap showing Spearman P values for correlations between maximum of plasma HIV RNA measured in patients after ATI and IFNγ, IL-2 and IL-13 cytokine responses assessed by Luminex assay after stimulations of PBMC with individual 15-mer HIV peptides. *Peptides of interest showing at least one inverse correlation (in bold). ^#Peptide showing at least one inverse correlation but did not passed positivity criteria described in methods section (in bold). Immunodominant peptides are highlighted within red rectangles.

Fig 3. DALIA peptide vaccine coverage.

PBMC from patients (n = 14) collected after vaccination (W16) were stimulated at day 0 with individual 15-mer peptides (previously inducing a positive response in Luminex assay), cultured with rIL-2, and restimulated by the same peptides at day 7. Cytokine production was measured using an ICS assay. (A) Cytometry dot plots of PBMC from one representative individual (D7 patient) stimulated with G253-4, N116-6 or P325-5 15-mer peptides. (B) Mapping of CD4⁺ and CD8⁺ T-cell responses observed for each of the 14 patients using 15-mer peptides. Light orange: low CD8⁺ T-cell responses, dark orange: high CD8⁺ T-cell responses, light green: low CD4⁺ T-cell responses, dark green: high CD4⁺ T-cell responses, light purple: low CD4⁺ and CD8⁺ T-cell responses, dark purple: high CD4⁺ and CD8⁺ T-cell responses. An ICS response was considered positive for a given cytokine if > 3-fold the negative control (unstimulated cells) and >0.05%. Cut off for high and low T-cell responses was defined < or > 5%.

Fig 4. Identification and characterization of cytokine-producing cells by ICS assay.

PBMC from patients (n = 14) collected after vaccination (W16) were stimulated at day 0 with individual 15-mer peptides (previously inducing a positive response in Luminex assay), cultured with rIL-2, and restimulated by the same peptides at day 7. Cytokine production was measured using an ICS assay. (A) Mean % of HIV-specific IFNγ and/or IL-2 and/or IL-13 CD3⁺CD56^-CD4⁺ and CD3⁺CD56^-CD8⁺ T cells to sixteen individual 15-mer HIV-1 peptides. (B) Mean % of cytotoxic marker-expressing cells among cytokine-positive HIV-1-specific CD3⁺CD56^-CD4⁺ and CD3⁺CD56^-CD8⁺ T cells to sixteen individual 15-mer HIV-1 peptides. (C) Cytometry dot plots from one representative individual (D18 patient) showing cytotoxic profile of HIV-1-specific CD4⁺ and CD8⁺ T cells after PBMC stimulation with G253-4 peptide.