ART influences HIV persistence in the female reproductive tract and cervicovaginal secretions

Abstract

The recently completed HIV prevention trials network study 052 is a landmark collaboration demonstrating that HIV transmission in discordant couples can be dramatically reduced by treating the infected individual with antiretroviral therapy (ART). However, the cellular and virological events that occur in the female reproductive tract (FRT) during ART that result in such a drastic decrease in transmission were not studied and remain unknown. Here, we implemented an in vivo model of ART in BM/liver/thymus (BLT) humanized mice in order to better understand the ability of ART to prevent secondary HIV transmission. We demonstrated that the entire FRT of BLT mice is reconstituted with human CD4+ cells that are shed into cervicovaginal secretions (CVS). A high percentage of the CD4+ T cells in the FRT and CVS expressed CCR5 and therefore are potential HIV target cells. Infection with HIV increased the numbers of CD4+ and CD8+ T cells in CVS of BLT mice. Furthermore, HIV was present in CVS during infection. Finally, we evaluated the effect of ART on HIV levels in the FRT and CVS and demonstrated that ART can efficiently suppress cell-free HIV-RNA in CVS, despite residual levels of HIV-RNA+ cells in both the FRT and CVS.

Figure 1. Human CD4⁺ T cells are present throughout the FRT of BLT mice.

Immunohistochemical analysis of the entire FRT of a HIV^– BLT mouse demonstrates the presence of CD4⁺ cells in the vagina, cervix, and uterus. CD4⁺ cells are stained brown. Scale bars: 100 μm.

Figure 2. Human memory T cells expressing CCR5 are the main human hematopoietic cell population in CVS and the FRT of BLT mice.

(A) Flow cytometric analyses of cells from the PB, FRT, and CVS of a representative HIV^– BLT mouse demonstrate reconstitution with human CD4⁺ and CD8⁺ T cells in each compartment. (B and C) Further characterization of T cells in PB (dots), the FRT (diagonal stripes), and CVS (solid color). Box plot showing the percentages of CD4⁺ (blue) and CD8⁺ T cells (red) in PB (n = 60), the FRT (n = 6), and CVS (n = 57) of HIV^– BLT mice. The middle line of the box plot is the median; box extends from the 25th to the 75th percentiles, and error bars extend down to the lowest value and up to the highest value (B). CD4⁺ and CD8⁺ T cells in PB (n = 9), the FRT (n = 4), and CVS (n = 46, CD4⁺ T cells; n = 37, CD8⁺ T cells) were analyzed for CCR5 expression with flow cytometry (C). (D and E) Bars represent mean values (± SEM). Further flow cytometric analyses characterizing the CD4⁺ (D) and CD8⁺ (E) T cell subsets. Naive T cells (CD45RA⁺CD27⁺), green; CM T cells (CD45RA^–CD27⁺), pink; and EM T cells (CD45RA^–CD27^–), purple. Bars represent mean values for PB (n = 26), the FRT (n = 4), and CVS (n = 60, CD4⁺ T cells; n = 41, CD8⁺ T cells). Data represented as mean ± SEM. A Mann-Whitney U test with a Holm-Bonferroni step-down correction was used to compare the frequencies of immune cell populations within and between the PB, FRT, and CVS of BLT mice (*P < 0.05, **P < 0.01, ****P < 0.0001) (C–E).

Figure 3. HIV is present in the FRT and CVS of infected BLT mice, and the kinetics of HIV-RNA in CVS is similar to the kinetics of HIV-RNA in PB plasma.

(A) Immunohistochemical analysis of the FRT (vagina, cervix, and uterus) from one vaginally HIV-infected BLT mouse (3 weeks after exposure). HIV p24 gag–positive cells are stained brown. Scale bars: 100 μm. (B–D) HIV-RNA was detected in the plasma (black solid line, filled circles), and CVS supernatant (black dashed line, open circles) following vaginal (B) (n = 21), rectal (C) (n = 16), and oral (D) (n = 14) exposure. (E) Comparison of the viral load in plasma and CVS supernatant of all routes combined (mice infected vaginally, rectally, and orally combined; n = 51). The assay limit of detection is indicated with a dashed gray line. (B–E) Data represented as mean ± SEM. Associations between plasma and CVS supernatant viral loads were estimated with Spearman’s correlation coefficient.

Figure 4. After vaginal, rectal, or oral HIV infection, there is a decrease in the percentage of CD4⁺ T cells in CVS and the FRT.

(A–C) The percentage of CD4⁺ T cells in PB (filled diamond, solid line) and CVS (open triangle, dashed line) was measured in HIV^– (n = 28) and vaginally (A) (n = 18), rectally (B) (n = 11), or orally (C) (n = 12) infected BLT mice. Data is shown as mean ± SEM. A Mann-Whitney U test was used to compare CD4⁺ T cell levels between the PB and CVS (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (D) Additional analysis of the percentage of CD4⁺ T cells in the FRT was performed in HIV^– BLT mice (n = 7); vaginally infected BLT mice (filled circle) at weeks 1 (n = 1), 3 (n = 1), 5 (n = 1), and 7 (n = 1); rectally infected mice (asterisk) at weeks 6 (n = 2) and 10 (n = 1); and an orally infected mouse (filled square) at week 8 (n = 1) after exposure.

Figure 5. After vaginal, rectal, or oral HIV infection, there is an increase of CD8⁺ and CD4⁺ T cells in CVS.

(A–C) The CD8⁺ (filled upward triangle, dashed red line) and CD4⁺ T cell numbers (filled downward triangle, blue dashed line) in CVS were measured in HIV^– (n = 28) and vaginally (A) (n = 18), rectally (B) (n = 11), or orally (C) (n = 4) infected BLT mice. Data presented as mean ± SEM. Statistical analysis represents comparisons of the weeks indicated vs. the numbers of CD4⁺ or CD8⁺ T cells in naive mice. A Mann-Whitney U test was used to compare the numbers of CD4⁺ and CD8⁺ T cells in the CVS of naive and HIV-infected BLT mice (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 6. ART suppresses HIV-RNA in the plasma and CVS, leading to a reduction in the number of CD8⁺ T cells and an increase of CD4⁺ T cells in CVS.

Six HIV-infected BLT mice were treated with daily ART consisting of FTC, TDF, and RAL to evaluate the effect of ART on HIV-RNA levels in the plasma and CVS supernatant, as well as T cell levels in PB and CVS. ART was initiated in BLT mice 6–8 weeks after HIV exposure. Time point 0 corresponds to the day of ART initiation, and the time points corresponding to ART are shaded gray. (A) Viral load analyses of the plasma (black solid line, filled circles) and CVS supernatant (black dashed line, open circles). The assay limit of detection is indicated with a dashed gray line. (B) The percentage of CD4⁺ cells in PB (filled diamond, blue solid line) and CVS (open downward triangle, blue dashed line). (C) The number of CD4⁺ (filled downward triangle, blue dashed line) and CD8⁺ (filled upward triangle, dashed red line) T cells in CVS. Data represented as mean ± SEM.

Figure 7. ART suppresses cell-free HIV-RNA in CVS and PB but does not consistently suppress cell-associated HIV-RNA in the FRT and CVS.

(A) Viral load analyses of the plasma (black solid line, filled circles) and CVS supernatant (black dashed line, open circles) demonstrated sustained viral load in the plasma and CVS supernatant of 8 HIV-infected ART-naive mice (left panel) and a dramatic decrease in viral load to undetectable levels in both plasma and CVS supernatant in 5 representative ART-treated mice (right panel). Time points corresponding to ART are shaded gray. (B) Cell-associated HIV-RNA in the PB, FRT, and CVS of mice receiving ART for 5–8 weeks (n = 8) and of ART-naive mice (No ART) (n = 8). RNA was isolated from mononuclear cells, and the RNA determination for each sample was performed in triplicate. (C) The level of infectious cells in PB and CVS from BLT mice was determined in 8 ART-treated mice and 5 ART-naive mice. Samples from all mice in each group (ART vs. No ART) were pooled at each time point: week –1 (ART n = 8, No ART n = 5), week 2 (ART n = 4, No ART n = 5), and week 3 (ART n = 3, No ART n = 5). (D) The level of infectious cells in the FRT of mice treated with ART for 5 weeks and in ART-naive mice (ART n = 4, No ART n = 5). (B–D) Bars represent mean values. Data are represented as ± SEM. (C and D) Limit of detection was 2 infectious units per 1 × 10⁶ cells. (E) BLT mice were exposed vaginally to 2 different doses of HIV-infected PBMC (open symbols: 5,000 PBMC, n = 4; closed symbols: 10,000 PBMC, n = 4). Plasma levels of HIV-RNA were monitored for 8 weeks. A Mann-Whitney U test was used to compare levels of cell-associated HIV-RNA and infectious cells between ART-naive and ART-treated mice (*P < 0.05, ***P < 0.001) (B and D). The assay limit of detection is indicated with a dashed gray line.