SIV escape mutants in rhesus macaques vaccinated with NEF-derived lipopeptides and challenged with pathogenic SIVmac251

BACKGROUND: Emergence of viral variants that escape CTL control is a major hurdle in HIV vaccination unless such variants affect gene regions that are essential for virus replication. Vaccine-induced multispecific CTL could also be able to control viral variants replication. To explore these possibilities, we extensively characterized CTL responses following vaccination with an epitope-based lipopeptide vaccine and challenge with pathogenic SIVmac251. The viral sequences corresponding to the epitopes present in the vaccine as well as the viral loads were then determined in every macaque following SIV inoculation. RESULTS: In most cases, the emergence of several viral variants or mutants within vaccine CTL epitopes after SIV challenge resulted in increased viral loads except for a single macaque, which showed a single escape viral variant within its 6 vaccine-induced CTL epitopes. CONCLUSION: These findings provide a better understanding of the evolution of CD8+ epitope variations after vaccination-induced CTL expansion and might provide new insight for the development of an effective HIV vaccine

Anti-HPV16 E2 Protein T-Cell Responses and Viral Control in Women with Usual Vulvar Intraepithelial Neoplasia and Their Healthy Partners

T-cell responses (proliferation, intracellular cytokine synthesis and IFNγ ELISPOT) against human papillomavirus 16 (HPV16) E2 peptides were tested during 18 months in a longitudinal study in eight women presenting with HPV16-related usual vulvar intraepithelial neoplasia (VIN) and their healthy male partners. In six women, anti-E2 proliferative responses and cytokine production (single IFNγ and/or dual IFNγ/IL2 and/or single IL2) by CD4+ T lymphocytes became detectable after treating and healing of the usual VIN. In the women presenting with persistent lesions despite therapy, no proliferation was observed. Anti-E2 proliferative responses were also observed with dual IFNγ/IL2 production by CD4+ T-cells in six male partners who did not exhibit any genital HPV-related diseases. Ex vivo IFNγ ELISPOT showed numerous effector T-cells producing IFNγ after stimulation by a dominant E2 peptide in all men and women. Since the E2 protein is absent from the viral particles but is required for viral DNA replication, these results suggest a recent infection with replicative HPV16 in male partners. The presence of polyfunctional anti-E2 T-cell responses in the blood of asymptomatic men unambiguously establishes HPV infection even without detectable lesions. These results, despite the small size of the studied group, provide an argument in favor of prophylactic HPV vaccination of young men in order to prevent HPV16 infection and viral transmission from men to women

Intracellular cytokine synthesis to HPV16 E2 peptides in proliferative T-cell female responders.

<p>The percentage of CD4+ T-cells synthesizing single IFNγ, dual IFNγ/IL2 or single IL2 was measured in response to E2 peptide pools or E2 peptide and compared to negative (no or irrelevant peptide) and positive controls (pool of non-HPV viral peptides). Single IFNγ responses are shown in blue, dual IFNγ/IL2 in red and single IL2 in green. Percentages of CD4+ responding cells are in the same color as the cytokine(s) synthesized. The white color represents the percentage of non responding cells among CD4+ T lymphocytes. Responses were considered as positive when the percentage of CD4+ T-cells synthesizing cytokines increased by at least 0.2% in the presence of the peptide as compared to negative control.</p

<i>Ex vivo</i> anti-HPV16-E2 peptide IFNγ ELISPOT responses in women presenting with usual VIN.

<p>IFNγ ELISPOT response to E2 peptide pools was studied using PBMCs from A: women presenting with usual VIN either during the whole study (F#1 and F#7) or at study entry and up to the healing after treatment (F#3, F#6, F#8 before M6, F#5 before M12) and B: asymptomatic women having cleared their usual VIN after treatment (F#2, F#4 before their entry in the study, F#3, F#6, F#8 from M6, F#5 from M12). Bars represent numbers of SFC/10⁶ PBMCs against a pool of E2 peptides, negative (0) and positive (+) controls obtained at M0 (blue), M6 (red), M12 (yellow) and M18 (turquoise blue). Standard deviation of triplicates appears for each bar. Responses were considered significant when the mean number of SFC per 10⁶ cells in the 3 experimental wells was >3-fold the mean number of SFC in the negative control wells (PBMC alone) and >30 SFC/10⁶ cells. Positive responses are identified by a black star above the corresponding bar.</p

Ex vivo IFNγ ELISPOT responses in women and men against HPV16, HPV2, HPV27 and HPV55 E2 309–323 peptide.

<p>A: number of SFC in negative control was substracted.</p><p>B: number of SFC was <3-fold the mean number of SFC in the negative control.</p

<i>Ex vivo</i> anti-HPV16-E2 peptide IFNγ ELISPOT responses in male partners of women presenting with usual VIN.

<p>IFNγ ELISPOT responses to E2 peptide pools were studied with PBMCs from male partners of women presenting with usual VIN. Bars represent numbers of SFC/10⁶ PBMCs against a pool of E2 peptides, negative (0) and positive (+) controls obtained at M0 (blue), M6 (red), M12 (yellow) and M18 (turquoise blue). Standard deviation of triplicates appears for each bar. Responses were considered significant when the mean number of SFC per 10⁶ cells in the 3 experimental wells was >3-fold the mean number of SFC in the negative control wells (PBMC alone) and >30 SFC/10⁶ cells. Positive responses are identified by a black star above the corresponding bar.</p

Clinical and biological characteristics of the eight women having usual VIN and their male partners.

<p>NA: not applicable.</p

Anti-HPV16 E2 peptide proliferative responses in women.

<p>T-cell proliferation induced by E2 peptide pools were studied with PBMCs from A: women presenting with usual VIN either during the whole study (F#1 and F#7) or at study entry and up to the healing after treatment (F#3, F#6, F#8 before M6, F#5 before M12) and B: asymptomatic women having cleared their usual VIN after treatment (F#2, F#4 before their entry in the study, F#3, F#6, F#8 from M6, F#5 from M12). SI is represented by the cpm in peptide-stimulated cells/cpm in negative control wells (without peptide). Each bar represents SI against a pool of E2 peptide or positive control (+) obtained from PBMCs sampled at M0 (blue), M6 (red), M12 (yellow) and M18 (turquoise blue). Proliferative responses with SI >3 were scored as positive, provided that cpm in the negative control was above 500. The values of positive SI for E2 pools are indicated in black near the corresponding bar. Responses to 15 mer peptides comprised in the various peptide pools are shown, superposed to the response to the pool, in another color. The SI values for these peptides are indicated using the same colors near the corresponding bars.</p

Anti-HPV16 E2 peptide proliferative responses in male partners of women presenting with usual VIN.

<p>Proliferative assays were performed using PBMCs in presence of pools of E2 peptides. SI is represented by the cpm in peptide-stimulated cells/cpm in negative control wells (without peptide). Each bar represents SI against a pool of E2 peptide or positive control (+) obtained from PBMCs sampled at M0 (blue), M6 (red), M12 (yellow) and M18 (turquoise blue). Proliferative responses with SI >3 were scored as positive, provided that cpm in the negative control was above 500. The values of positive SI for E2 pools are indicated in black near the corresponding bar. Responses to 15 mer peptides comprised in the various peptide pools are shown, superposed to the response to the pool, in another color. The SI values for these peptides are indicated in the same colors near the corresponding bars.</p

Peptides and pools spanning the whole E2 protein.

<p>Peptides and pools spanning the whole E2 protein.</p