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ART influences HIV persistence in the female reproductive tract and cervicovaginal secretions
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"....when analyzed ex vivo, cells isolated from the FRT [female reproductive tract] and CVS [cervicovaginal secretions] of ART-suppressed BLT mice did not transmit HIV in a coculture assay. Thus, our results provide in vivo evidence supporting the hypothesis behind the success of HPTN 052 (12) for limiting sexual transmission from HIV-infected women......we treated infected mice with an ART regimen that consisted of TDF, emtricitabine (FTC), and raltegravir (RAL). This triple-drug combination has been shown to strongly suppress viral load in both humans and BLT mice with continuous dosing (51-53). However, ART discontinuation in patients results in a rapid rebound of plasma viremia.....Our results showed (i) that infectious cells were readily detected in PB, CVS, and the FRT of infected BLT mice; (ii) that ART suppresses the number of infectious cells in each of these 3 compartments; and (iii) that this reduction was statistically significant (P = 0.04)......Our results revealed that the number of HIV-RNA+ cells in all 3 compartments analyzed from ART-suppressed BLT mice is well below what is needed to establish vaginal HIV transmission in this model."
DISCUSSION
In this manuscript, we provide data demonstrating (i) that regardless of the route of infection (vaginal, rectal, or oral) and whether the inoculum is cell-free or cell-associated HIV, local viral replication occurs in the FRT and CVS during acute HIV infection, the time when secondary transmission is most likely to occur (Figure 3); (ii) that this is followed by a transient increase in CD4+ T cell levels in CVS that can serve to provide additional target cells to sustain or promote infection (Figure 5); and (iii) that this is itself followed by a somewhat delayed increase in CD8+ T cell levels in CVS (Figure 5). In addition, our study provides in vivo evidence supporting the hypothesis behind the success of HPTN 052 (12): that initiating ART can reduce the risk of secondary HIV transmission by efficiently suppressing HIV levels in the genital tract (Figures 6 and 7).
Interestingly, our results also highlighted a potentially important dichotomy between the levels of cell-free virus and cell-associated HIV-RNA in the FRT and CVS of ART-suppressed mice. Specifically, in animals with undetectable cell-free HIV-RNA in plasma and CVS, significant levels of HIV-infected cells producing viral RNA remain in the FRT and CVS of some ART-treated mice (Figure 7). The presence of cell-associated HIV-RNA has been demonstrated in other secretions of ART-suppressed women. Valea et al. demonstrated the presence of cell-associated HIV-RNA in breast milk obtained from ART-treated mothers with undetectable levels of cell-free HIV-RNA in plasma and breast milk (58). These results are in agreement with our observations in BLT mice demonstrating that cell-associated HIV-RNA can persist in mucosal secretions despite ART. Collectively, our results and those of Valea et al. may have important implications for the design of effective HIV prevention and curative approaches. In the future, it will be important to determine if cell-associated HIV-RNA persists in other mucosal secretions and tissues despite suppression of cell-free HIV-RNA.
Worldwide, the majority of new HIV infections occur after heterosexual exposure (59). In vaginally exposed women, the primary ports of HIV entry are the mucosal surfaces of the vagina, cervix, and uterus (60-62). The identity and the location of the initial cells involved in HIV-1 transmission are a subject of great debate (29). The DC-T cell milieu is a highly permissive site for virus growth, and DCs likely contribute to driving the productive infection in CD4+ T cells (63-67). Hence, both intraepithelial Langerhans cells and DCs have potential important roles in vaginal HIV transmission (65-69). However, the mucosa of the human FRT contains an abundance of CD4+ T cells (24, 25, 34, 68), and experiments in both NHPs (nonhuman primates) and human explant models suggest that the first productively infected cells are likely T cells (61, 68, 70-73). Regardless, each of the most relevant human HIV target cells (CD4+ T cells, macrophages, and DCs) are present throughout the entire FRT of BLT mice (Figure 1, Supplemental Figures 1 and 2, and ref. 21). Reconstitution of the FRT of BLT mice with the appropriate human hematopoietic cells renders BLT mice susceptible to vaginal HIV transmission (21). The susceptibility of BLT mice to vaginal HIV infection has allowed this model to be used to evaluate novel approaches of HIV prevention (21, 23, 74).
In this manuscript, we utilized BLT mice to elucidate and study critical events occurring in the FRT and CVS during HIV infection. Specifically, by performing comprehensive analyses of the T cell subsets present in the FRT and CVS, we have gained insight into the human immune cell populations in this organ. Consistent with observations made in healthy women, the majority of the human lymphocytes present in both the FRT and CVS of BLT mice are memory T cells (Figure 2D and refs. 31, 32). Also, consistent with the preferential vaginal transmission of CCR5-tropic viruses, a high percentage of CD4+ T cells present in the FRT and CVS express CCR5 (Figure 2C and refs. 31, 32). Furthermore, consistent with humans, a significant number of the memory CD4+ T cells present in the FRT and CVS of BLT mice expressed α4β7 (Supplemental Figure 3). The similarities between the phenotypes of hematopoietic cells present in the FRT of humans and BLT mice emphasize the utility of BLT mice as an in vivo model for the study of events occurring at the site where HIV exposure occurs.
Our results demonstrating parallel reductions in the percentage of CD4+ T cells in the FRT and CVS of BLT mice indicate that the cell populations are closely linked throughout the course of infection (Figure 4). These striking similarities between the dynamics of T cells present in CVS and the FRT after HIV infection suggest that cells from CVS could be potentially used as a surrogate for monitoring some of the changes that occur in the FRT. Thus, these results may have significant implications that could facilitate and simplify future studies of transmission and prevention in both humans and in NHP models by minimizing the need to harvest or biopsy the FRT.
In order to study the dynamics of human CD4+ and CD8+ T cells and viral replication that occur in the FRT after vaginal HIV infection, we vaginally exposed BLT mice to HIV. Our results demonstrate an increase of CD4+ T cells in CVS during the first 2 weeks after infection (Figure 5A), providing additional HIV target cells to sustain and potentially spread the initial infection. These results are consistent with HIV-RNA being present in CVS within 1 week after exposure (Figure 3B), suggesting that local HIV replication occurs in the FRT and/or CVS followed by the establishment of systemic infection in all mice by 2 weeks after exposure (Figure 3B). Especially noteworthy is the timing of viral shedding into CVS after vaginal exposure, which is characterized by an early peak in viremia followed by a gradual decline (Figure 3B) that mimics the HIV genital shedding profile observed in CVS of women during acute HIV infection (39). Notably, we also observed an increase in CD8+ T cells in CVS after vaginal infection (Figure 5A). However, in contrast to the increase of CD4+ cells occurring within one to 2 weeks after exposure, the increase of CD8+ T cells was first detected 2 weeks after exposure and did not peak until week 5-7 (Figure 5A). These results are consistent with reports from vaginal-infection studies in NHPs demonstrating that initial SIV infection takes place in a few CD4+ T cells, resulting in local inflammation and recruitment of additional CD4+ T cells (61, 72, 75). Clusters of SIV-infected cells are present within inflammatory infiltrates, which increase in size during days 4-10 after exposure (61, 72, 75). In addition, genital CD8+ T cell influx was detected 2-3 weeks after vaginal SIV infection of NHPs, a time by which all animals had become systemically infected (72, 76). Thus, the timing of CD4+ and CD8+ T cell increase in CVS, as well as the timing of local viral replication and systemic infection that we have observed in BLT mice, is strikingly similar to NHPs. Together, these results support the hypothesis that the increase of CD8+ T cells in the CVS/FRT is delayed after exposure, potentially preventing effective suppression of HIV replication at early stages after exposure (75, 76).
We next investigated the effect of ART on HIV levels in CVS of BLT mice. Consistent with results obtained in humans, ART treatment of infected BLT mice resulted in a significant decrease in the levels of HIV in both PB and CVS (15, 16). However, our finding showing the absence of cell-free HIV in CVS during ART, concurrent with the continued presence of infected cells producing HIV-RNA, could have important implications for HIV prevention and eradication strategies. Consistent with the lack of transmission observed in heterosexual couples where the infected partner is undergoing ART (12), our analysis showed that the residual levels of HIV-RNA+ cells present in mice receiving ART were too low to transmit HIV in vitro. This lack of HIV transmission could have been due to too few infected cells for cell-to-cell transmission and/or too little cell-free replication-competent virus produced from the residual HIV-RNA+ cells for in vitro infection of target cells. The residual levels of HIV-RNA+ cells detected in the CVS and FRT of ART-suppressed mice were well below the number of HIV-infected cells required for HIV transmission in BLT mice. The availability of a small animal model that so accurately recapitulates key aspects of the human condition represents a unique tool for the in vivo study of the intricate cellular dynamics occurring during HIV infection in the FRT and to address critical questions in the field, such as whether cell-to-cell transmission contributes sexual transmission of HIV or whether all transmission is via cell-free, replication-competent virions. In addition, this model could prove helpful in the evaluation of novel approaches to prevent cell-free and cell-associated HIV transmission.
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ART influences HIV persistence in the female reproductive tract and cervicovaginal secretions
https://www.jci.org/articles/view/64212
J Clin Invest. Feb 8 2016
Rikke Olesen1,2, Michael D. Swanson1, Martina Kovarova1, Tomonori Nochi1, Morgan Chateau1, Jenna B. Honeycutt1, Julie M. Long1, Paul W. Denton1,Michael G. Hudgens3, Amy Richardson3, Martin Tolstrup2, Lars stergaard2, Angela Wahl1, and J. Victor Garcia1
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.
Introduction
Most clinical trials of HIV prevention have aimed at preventing HIV acquisition by topical or systemic administration of preventative antiretroviral drugs to uninfected individuals (1-10). Results from these clinical trials have shown either partial or no protection. The basis for these discordant results are not yet clear and have been postulated to be due to a combination of a lack of adherence and inadequate drug levels at the site of exposure (5, 7, 11). In contrast, the HIV prevention trials network study 052 (HPTN 052) demonstrated 93% protection against secondary heterosexual transmission when infected individuals received early antiretroviral therapy (ART) (12). Importantly, no linked partner infections were observed when the HIV-infected participant was stably suppressed by ART. The prevailing hypothesis for the success of HPTN 052 is that ART reduces genital cell-free and/or genital cell-associated HIV to levels that are too low to support HIV transmission (12). This hypothesis is supported by observational studies suggesting a strong correlation between plasma/genital HIV-RNA levels and risk of heterosexual transmission (13, 14); it is also supported by the ability of ART to decrease the genital levels of HIV in both men and women (15-17). There is very limited data in the literature to determine whether transmission occurs from cell-free virus only or if productively infected cells themselves can transmit HIV in the absence of cell-free virions (18).
In order to better understand the ability of ART to prevent secondary transmission of HIV, we used a small animal model of HIV infection to further characterize key virological and immunological events that occur in the female reproductive tract (FRT) during ART. We designed the following experiments using BM/liver/thymus humanized mice (BLT mice). First, we performed a detailed and comprehensive phenotypic characterization of the human lymphocyte subsets present in the FRT and cervicovaginal secretions (CVS). Next, we analyzed HIV levels and cellular dynamics in CVS during HIV infection. Finally, we evaluated virological suppression and cellular dynamics in the FRT and CVS during ART. We provide data demonstrating that HIV replication occurs in CVS soon after exposure and continues during the course of infection. This is followed by an increase of CD4+ T cells in CVS, providing additional target cells for infection. This CD4+ T cell increase is followed by a delayed increase of CD8+ T cells in CVS. Surprisingly, despite the strong suppressive effect of ART on the viral load in CVS, HIV-RNA+ cells were still present in both the FRT and CVS. However, when analyzed ex vivo, cells isolated from the FRT and CVS of ART-suppressed BLT mice did not transmit HIV in a coculture assay. Thus, our results provide in vivo evidence supporting the hypothesis behind the success of HPTN 052 (12) for limiting sexual transmission from HIV-infected women.
ART efficiently suppresses HIV in CVS and restores CD4+ and CD8+ T cell numbers.
In order to examine the effect of ART on HIV levels and CD4+ and CD8+ T cell numbers in CVS, we treated infected mice with an ART regimen that consisted of TDF, emtricitabine (FTC), and raltegravir (RAL). This triple-drug combination has been shown to strongly suppress viral load in both humans and BLT mice with continuous dosing (51-53). However, ART discontinuation in patients results in a rapid rebound of plasma viremia as well as a loss of PB CD4+ T cells (54, 55). These phenomena are also fully recapitulated in BLT mice (53, 56). In BLT mice infected vaginally with cell-associated virus (Supplemental Table 1), ART administration resulted in a dramatic and sustained decrease in viral load in both plasma and CVS as early as 2 weeks after ART initiation (Figure 6A). Analysis of CD4+ T cells in PB and CVS from these mice prior to ART demonstrated the characteristic steep decrease in the percentage of CD4+ T cells in CVS described above and the less pronounced decrease observed in PB. During ART, there was a dramatic increase in the percentage of CD4+ T cells in CVS and an increase in the percentage of CD4+ T cells in PB (Figure 6B). These findings are in agreement with the fact that HIV+ women on ART have a significantly higher percentage of cervical CD4+ T cells than infected women not receiving treatment (57). In addition — as indicated above — prior to treatment, there was a dramatic increase in total CD8+ T cells in CVS (Figure 6C). However, in response to ART, there was a rapid and substantial decrease in the numbers of CD8+ T cells in this compartment. In contrast, while the numbers of CD8+ cells decreased, the numbers of CD4+ T cells increased, resulting in the eventual return to near-normal levels (Figure 6C). These results reveal that the observed increase in the percentage of CD4+ T cell in CVS during ART (Figure 6B) was caused by a concurrent decrease in the numbers of CD8+ T cells and an increase in the numbers of CD4+ T cells.
We next determined if ART suppression of cell-free HIV-RNA levels observed in plasma and CVS (Figure 7A) parallels a similar decrease in the levels of cell-associated HIV in PB, CVS, and the FRT. For this purpose, we isolated cells from PB, FRT, and CVS and analyzed their levels of cell-associated HIV-RNA (Figure 7B). Our results show that ART significantly decreased the amount of cell-associated HIV-RNA in all 3 compartments (PB ART vs. No ART, P = 0.0009; FRT ART vs. No ART, P = 0.0002; and CVS ART vs. No ART, P = 0.015) (Figure 7B). However, cell-associated HIV-RNA remained readily detectable in the FRT and/or CVS of the majority of mice undergoing ART. Therefore, despite the strong reduction in the levels of cell-free HIV-RNA in mice receiving ART, our analysis reveals an important dichotomy between the suppression of cell-free HIV-RNA levels observed in CVS and the continued presence of residual levels of cell-associated HIV-RNA in the FRT/CVS. To determine whether these residual HIV-RNA+ cells constitute an important source of infectious virus, we established a sensitive coculture assay to measure the number of infectious cells in PB, CVS, and the FRT of infected mice receiving ART (Figure 7, C and D). Our results showed (i) that infectious cells were readily detected in PB, CVS, and the FRT of infected BLT mice; (ii) that ART suppresses the number of infectious cells in each of these 3 compartments; and (iii) that this reduction was statistically significant (P = 0.04) (Figure 7D). Thus, our results demonstrate that ART efficiently suppresses levels of cell-free HIV and infectious cells in PB, CVS, and the FRT. Most importantly, we then determined if ART suppresses the number of HIV-RNA+ cells in the PB, CVS, and FRT of BLT mice to levels below those needed to prevent secondary mucosal HIV transmission. We assessed the ability of HIV-infected cells to establish infection in vivo by vaginally exposing BLT mice to 2 different doses of HIV-infected PB mononuclear cells (PBMCs) and monitoring their plasma level of HIV-RNA for 8 weeks. As shown in Figure 7E, all BLT mice exposed to 5,000 HIV-infected PBMCs remained negative for HIV-RNA. However, 50% of mice exposed to 10,000 HIV-infected cells became positive for plasma HIV-RNA. Our results revealed that the number of HIV-RNA+ cells in all 3 compartments analyzed from ART-suppressed BLT mice is well below what is needed to establish vaginal HIV transmission in this model.
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