Effect of vaginal immunization with HIVgp140 and HSP70 on HIV-1 replication and innate and T cell adaptive immunity in women

Abstract

The international effort to prevent HIV-1 infection by vaccination has failed to develop an effective vaccine. The aim of this vaccine trial in women was to administer by the vaginal mucosal route a vaccine consisting of HIV-1 gp140 linked to the chaperone 70-kDa heat shock protein (HSP70). The primary objective was to determine the safety of the vaccine. The secondary objective was to examine HIV-1 infectivity ex vivo and innate and adaptive immunity to HIV-1. Protocol-defined female volunteers were recruited. HIV-1 CN54gp140 linked to HSP70 was administered by the vaginal route. Significant adverse reactions were not detected. HIV-1 was significantly inhibited ex vivo in postimmunization CD4(+) T cells compared with preimmunization CD4(+) T cells. The innate antiviral restrictive factor APOBEC3G was significantly upregulated, as were CC chemokines which induce downregulation of CCR5 in CD4(+) T cells. Indeed, a significant inverse correlation between the proportion of CCR5(+) T cells and the concentration of CCL-3 or CCL-5 was found. Importantly, the upregulation of APOBEC3G showed a significant inverse correlation, whereas CCR5 exhibited a trend to correlate with inhibition of HIV-1 infection (r = 0.51). Furthermore, specific CD4(+) and CD8(+) T cell proliferative responses were significantly increased and CD4(+) T cells showed a trend to have an inverse correlation with the viral load (r = -0.60). However, HIVgp140-specific IgG or IgA antibodies were not detected. The results provide proof of concept that an innate mechanism consisting of CC chemokines, APOBEC3G, and adaptive immunity by CD4 and CD8 T cells might be involved in controlling HIV-1 infectivity following vaginal mucosal immunization in women. (This study has been registered at ClinicalTrials.gov under registration no. NCT01285141.) Importance: Vaginal immunization of women with a vaccine consisting of HIVgp140 linked to the 70-kDa heat shock protein (HSP70) elicited ex vivo significant inhibition of HIV-1 replication in postimmunization CD4(+) T cells compared with that in preimmunization peripheral blood mononuclear cells. There were no significant adverse events. The vaccine induced the significant upregulation of CC chemokines and the downmodulation of CCR5 expression in CD4(+) T cells, as well as an inverse correlation between them. Furthermore, the level of CCR5 expression was directly correlated with the viral load, consistent with the protective mechanism in which a decrease in CCR5 molecules on CD4(+) T cells decreases HIV-1 envelope binding. Expression of the antiviral restriction factor APOBEC3G was inversely correlated with the viral load, suggesting that it may inhibit intracellular HIV-1 replication. Both CD4(+) and CD8(+) T cells showed HIVgp140- and HSP70-specific proliferation. A strong inverse correlation between the proportion of CC chemokine-modulated CCR5-expressing CD4(+) T cells and the stimulation of CD4(+) or CD8(+) T cell proliferation by HIVgp140 was found, demonstrating a significant interaction between innate and adaptive immunity. This is the first clinical trial of vaginal immunization in women using only HIVgp140 and HSP70 administered by the mucosal route (3 times) in which a dual innate protective mechanism was induced and enhanced by significant adaptive CD4(+) and CD8(+) T cell proliferative responses.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

Vaginal immunization at weeks 0, 4, and 12 induces increases in RANTES concentrations (ng/ml) in plasma (A) and MIP-1α concentrations (pg/ml) (B) and MIP-1β (pg/ml) concentrations (C) in the culture supernatants (SN) of PBMCs alone (□) or PBMCs stimulated with HIVgp140 (■) or HSP70 () (n = 8). The correlations between CCR5 and RANTES (D) and between CCR5 and MIP-1α after in vitro stimulation with HIVgp140 (E) and HSP70 (F) were assayed preimmunization and at 4, 8, 16, and 20 weeks postimmunization (all readings before and after each immunization), but only samples from 7 subjects were available because of the limited number of cells available at 4, 8, 16, and 20 weeks postimmunization. Statistical tests used the Wilcoxon rank sum test for panels A to C and Spearman correlation analyses for panels D to F. *, P < 0.05; ***, P < 0.005.

FIG 2

(A) Downregulation of CCR5+ CD4+ T cells after each immunization at 0, 4, and 12 weeks; (B) regulation of the corresponding CD4+ T cells showing no change; (C) APOBEC3G upregulation in CD4+ T cells following each immunization; (D) viral load (expressed as the area under the curve [AUC]) pre- and postimmunization in the PBMCs of the 8 female volunteers; (E) multiplicity of infection of HIV-1 (BaL) (expressed as the mean ± SEM) preimmunization (○) and postimmunization (●) with the HIVgp140-HSP70 vaccine by the mucosal route (3 times). (F to H) Correlation between the viral load (VL) and CCR5 expression (F), APOBEC3G expression (G), and CD4+ T cell proliferation (H) after the 3rd immunization (week 16). Statistical analyses were performed as described in the legend to Fig. 1. *, P < 0.05; **, P < 0.01, ***, P < 0.001.

FIG 3

Proliferative responses of PBMCs and CD4+ and CD8+ T cells of 8 women immunized at 0, 4, and 12 weeks. Blood samples were taken before and 4 weeks after each vaginal immunization, and the last reading was 8 weeks after the final (3rd) immunization. (A) Proliferative response of PBMCs and CD4+ and CD8+ T cells before and after each immunization with no stimulation in vitro. (B and C) Proliferative responses with in vitro stimulation with HIVgp140 (B) and HSP70 (C). (D) Proliferative responses before immunization □ and after the 3rd immunization () with stimulation in vitro with TT compared with that with HIVgp140. Pre- versus postimmunization statisticsl analyses were carried out by the Wilcoxon rank sum test. (E) The net difference between the pre- and postimmunization values for cytokines IL-12 and IL-6 (see Table S7 in the supplemental material) was plotted against the viral load (VL) as the area under the curve. *, P < 0.05; **, P ≤ 0.01; ***, P < 0.001; ****, P < 0.005. Im, immunization.

FIG 4

(A to C) Proliferative responses of CD4+ T cells plotted against the proportion of CCR5+ CD4+ T cells not stimulated (use of medium [Med] alone) (A), stimulated in vitro with HIVgp140 (B), or stimulated in vitro with HSP70 (C). (D to F) Proliferative responses of CD8+ T cells plotted against the proportion of CCR5+ CD4+ T cells not stimulated (D), stimulated with HIVgp140 (E), and stimulated with HSP70 (F). The statistical analyses used Spearman correlation.