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Clinical Trial
. 2014 Sep 16;111(37):13439-44.
doi: 10.1073/pnas.1400446111. Epub 2014 Sep 2.

Preferential infection of human Ad5-specific CD4 T cells by HIV in Ad5 naturally exposed and recombinant Ad5-HIV vaccinated individuals

Haitao Hu  1 , Michael A Eller  2 , Shah Zafar  2 , Yu Zhou  3 , Mengnan Gu  4 , Zhi Wei  4 , Jeffrey R Currier  5 , Mary A Marovich  6 , Hannah N Kibuuka  7 , Robert T Bailer  8 , Richard A Koup  8 , Merlin L Robb  2 , Nelson L Michael  9 , Jerome H Kim  9 , Silvia Ratto-Kim  2
Affiliations
Clinical Trial

Preferential infection of human Ad5-specific CD4 T cells by HIV in Ad5 naturally exposed and recombinant Ad5-HIV vaccinated individuals

Haitao Hu et al. Proc Natl Acad Sci U S A. .

Abstract

Efficacy trials of adenovirus 5-vectored candidate HIV vaccines [recombinant Ad5 (rAd5)-HIV] were halted for futility due to lack of vaccine efficacy and unexpected excess HIV infections in the vaccine recipients. The potential immunologic basis for these observations is unclear. We comparatively evaluated the HIV susceptibility and phenotypes of human CD4 T cells specific to Ad5 and CMV, two viruses that have been used as HIV vaccine vectors. We show that Ad5-specific CD4 T cells, either induced by natural Ad5 exposure or expanded by rAd5 vaccination, are highly susceptible to HIV in vitro and are preferentially lost in HIV-infected individuals compared with CMV-specific CD4 T cells. Further investigation demonstrated that Ad5-specific CD4 T cells selectively display a proinflammatory Th17-like phenotype and express macrophage inflammatory protein 3α and α4β7 integrin, suggestive of gut mucosa homing potential of these cells. Analysis of HIV p24 and cytokine coexpression using flow cytometry revealed preferential infection of IL-17- and IL-2-producing, Ad5-specific CD4 T cells by HIV in vitro. Our data suggest a potential mechanism explaining the excess HIV infections in vaccine recipients after rAd5-HIV vaccination and highlight the importance of testing the HIV susceptibility of vaccine-generated, vector and insert-specific CD4 T cells in future HIV vaccine studies.

Keywords: AIDS; antigen-specific CD4 T cells; viral vectors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Ad5-specific CD4 T cells are preferentially lost or greatly reduced in HIV-infected individuals. IFN-γ-ELISpot measurement of the magnitudes of HIV gag-, Ad5 hexon, EBV-LMP2, and CMV pp65-specific T-cell responses in PBMCs from HIV-uninfected (n = 24, red) and ART-naïve, HIV-infected (n = 24, blue) subjects. Results are shown as box and whisker plots showing the median and quartile spot-forming cells per 106 PBMCs. The Mann-Whitney test was used to analyze the statistical difference for the same antigen between groups, and the Wilcoxon test was used to examine the difference between antigens within the same group. Two-tailed P values were denoted.
Fig. 2.
Fig. 2.
Ad5-specific CD4 T cells from Ad5 naturally exposed healthy volunteers are highly susceptible to HIV in vitro compared with CMV-specific CD4 T cells. In vitro HIV infection of Ad5- and CMV-specific CD4 T cells in antigen-stimulated PBMCs of HIV- volunteers. (Left) Representative flow cytometry plots (CD3+CD8− T cells) show p24+ percentages in CFSE-low and CFSE-high CD4 T cells between Ad5 and CMV stimulation. (Right) Cumulative results (n = 7) for comparing p24+ rate in CFSE-low, CD4 T cells between Ad5 and CMV within the same individual. The Wilcoxon test was used to compare the difference. Two-tailed P values were denoted.
Fig. 3.
Fig. 3.
HIV infection of vaccine-expanded, rAd5 vector-specific CD4 T cells compared with CMV-specific CD4 T cells from DNA/rAd5-HIV vaccinated individuals. (A) Kinetics of peripheral rAd5- (Left) and CMV-specific (Right) CD4 T-cell responses in 18 Ugandan rAd5-HIV vaccine recipients measured by intracellular IFN-γ staining. Box and whisker plots showing the median and quartile IFN-γ expression at baseline, 28 d, and 336 d after vaccination with a trend line connecting the time points at the median response are presented. (B) In vitro HIV infection of Ad5- and CMV-specific CD4 T cells in antigen-stimulated PBMCs from rAd5 vaccine recipients (n = 7). Representative flow cytometry plots (Left) and cumulative results for comparing p24+ percentages in CFSE-low CD4 T cells (n = 7) (Right) are shown. Two-tailed P values were denoted.
Fig. 4.
Fig. 4.
Ad5-specific CD4 T cells selectively manifest a Th17-like phenotype with gut mucosal homing potential. (A) Expression of α4β7 integrin on Ad5- and CMV-specific CD4 T cells from rAd5-vaccinated individuals. Representative flow cytometry plots comparing α4β7+ percentages (Left) or MFI (Center), and box and whisker plot (Right) comparing the median and quartile of α4β7 MFI from seven donors between Ad5 and CMV are shown. (B) Cytokine profiles of Ad5- and CMV-specific CD4 T cells. PBMCs were restimulated with PMA and ionomycin on day 6 after initial Ag-specific stimulation. Expression of IL-17A, IFN-γ, and IL-2 are shown, with number in each plot representing percentage of cytokine+ in CFSE-low CD4 T cells. (C–E) Gene expression profiling of FACS-sorted, Ad5- and CMV-specific CD4 T cells from the same PBMCs by microarray. (C) Global view of fold changes and the associated P values for genes expressed at significantly higher (n = 205) and lower (n = 233) levels, respectively, in Ad5- relative to CMV-specific CD4 T cells (P value < 0.05). (D) Heat map comparison of gene expression changes between Ad5- and CMV-specific CD4 T cells from three subjects. Fold change for higher (red) and lower (blue) expression is shown. (E) List of selected genes expressed at significantly higher or lower levels in Ad5-specific CD4 T cells compared with CMV-specific CD4 T cells. Fold change (gray bar) and P value (black dot) for each gene are shown.
Fig. 5.
Fig. 5.
HIV preferentially infects IL-17- and IL-2-producing, Ad5-specific CD4 T cells in vitro. Antigen-stimulated, HIV-exposed PBMCs (CFSE labeled) were restimulated with PMA and ionomycin for de novo cytokine synthesis on day 6 after initial antigen stimulation. Coexpression of intracellular p24 (red) and cytokines within CFSE-low, Ad5-specific CD4 T cells (blue) was analyzed by flow cytometry. Representative plots (Left) and percentage of p24+ in each cytokine subsets (Right) are shown.
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