Exosomes in human semen restrict HIV-1 transmission by vaginal cells and block intravaginal replication of LP-BM5 murine AIDS virus complex

Abstract

Exosomes are membranous extracellular nanovesicles secreted by diverse cell types. Exosomes from healthy human semen have been shown to inhibit HIV-1 replication and to impair progeny virus infectivity. In this study, we examined the ability of healthy human semen exosomes to restrict HIV-1 and LP-BM5 murine AIDS virus transmission in three different model systems. We show that vaginal cells internalize exosomes with concomitant transfer of functional mRNA. Semen exosomes blocked the spread of HIV-1 from vaginal epithelial cells to target cells in our cell-to-cell infection model and suppressed transmission of HIV-1 across the vaginal epithelial barrier in our trans-well model. Our in vivo model shows that human semen exosomes restrict intravaginal transmission and propagation of murine AIDS virus. Our study highlights an antiretroviral role for semen exosomes that may be harnessed for the development of novel therapeutic strategies to combat HIV-1 transmission.

Introduction

Exosomes originate as a result of inward budding of endosomal membranes within the late endosomal compartment of many cell types (Gruenberg and Stenmark, 2004). Multivesicular bodies (MVB) are generated from this mechanism of inward budding with a large repertoire of exosomes and other membranous vesicles within them (Gruenberg and Stenmark, 2004). Exosomes are endogenous carriers of genetic and proteinaceous cargo, including proteins and RNAs (Madison et al., 2014; Schorey and Bhatnagar, 2008; Simpson et al., 2008; Vojtech et al., 2014b, a) and they can deliver their cargo (Alvarez-Erviti et al., 2011) through endocytosis or fusion with target cells (Li et al., 2013; Nanbo et al., 2013; Newton et al., 2006; Orth et al., 2002; Skog et al., 2008; Sun et al., 2010) resulting in the conditioning of target cells. Thus, exosomes have been implicated in intercellular communication (Danzer et al., 2012; Demory Beckler et al., 2012), modulation of immune response (Kelly et al., 1991; Li et al., 2013), and regulation of microbial pathogenesis (Delorme-Axford et al., 2013; Khatua et al., 2009; Li et al., 2013; Madison et al., 2014). For example, exosomes derived from H9 cells, a T lymphocyte cell line, contain the antiviral factor, Apobec3G, which endows H9 exosomes the ability to partially protect recipient cells from HIV-1 infection (Khatua et al., 2009). Similar to H9 exosomes, semen exosomes (SE) from healthy human semen contain antiviral mRNA and was shown to inhibit infection of target cells by retroviruses, such as murine AIDS virus complex LP-BM5 and HIV-1 (Madison et al., 2014).

Subsequent to crossing the host genital mucosa entry portal, sexually transmitted Retroviridae including SIV, MLV, and HIV-1 utilize various mechanisms to gain a foothold and establish persistent infection in the host. Worldwide, HIV/AIDS is a pandemic with three million new cases each year. HIV-1 is predominantly sexually transmitted between discordant sexual partners and semen is the principal vector (Byrn et al., 1997; Clumeck et al., 1989; Royce et al., 1997; Vernazza et al., 1999). The genital tract may serve as a haven for HIV-1 to undergo selective pressures (Byrn et al., 1997; Delwart et al., 1998; Kroodsma et al., 1994; Taylor et al., 2003). Such pressure may facilitate the evolution of drug resistant HIV-1 variants, which are subsequently sexually transmitted (Boden et al., 1999; Hecht et al., 1998; Little et al., 1999). It has been shown that HIV-1 patients whose viremia is persistently suppressed by antiretroviral therapy shed viral RNA into the genital tract (Cu-Uvin and Caliendo, 2011; Cu-Uvin et al., 2010), suggesting that shedding of HIV-1 in semen can occur in the absence of detectable viremia. However, sexual transmission of HIV-1 requires a high number of exposures (O'Brien et al., 1994; Varghese et al., 2002), an indication that not all viral particles in semen retain infectivity or that some anti-HIV-1 activity is operative in the mucosa or semen.

Despite decades of research, the component of semen that enhances or decreases efficacy of sexual transmission of HIV-1 is not fully understood. Previously, we showed that exosomes purified from the semen of healthy human donors inhibit replication of HIV-1 and LP-BM5 murine AIDS virus complex (mAIDS), but does not have effect on propagation of Herpes simplex virus types −1 and −2 (HSV-1 and HSV-2) (Madison et al., 2014), suggesting that the antiviral effect of SE may be retroviral specific.

In this study, we report the role of SE on retroviral transmission using in vitro and in vivo models. We show that SE are internalized by human vaginal epithelial cells ex vivo via endocytosis and by murine vaginal cells in vivo. In addition, SE is internalized by HIV susceptible T (SUPT1) and monocytic (U937) cell lines. Functionally, SE blocked trans infection and cell-to-cell spread of HIV-1 and potently protected mice from intravaginal infection and propagation of mAIDS. Furthermore, our data show that SE-mediated inhibition of retroviral propagation involves impairment of viral RNA reverse transcription process necessary for synthesis of nascent viral copy DNA required for establishing persistent infection. Thus, our data identify SE as a critical factor that may reduce efficacy of sexually transmitted retroviruses, suggesting new opportunities for the development of therapeutics against such viruses.

Results

Human vaginal epithelial cells internalize semen-derived exosomes

To examine whether cells of the female reproductive tract (FRT) internalize SE, E6/E7 transformed human vaginal epithelial cells (V428) (Peterson et al., 2005) were exposed to PKH67Green-labeled SE for 3 h followed by confocal microscopy (Figure 1A). V428 cells predominantly exhibited a punctate SE staining pattern, but some diffuse staining was also observed (Figure 1A). PKH67Green was not transferred to V428 cells co-incubated with vehicle control (Figure 1B). To examine whether differences exist in the ability of transformed and primary vaginal epithelial cells to internalize SE, we labeled primary human vaginal epithelial cells V428 with PKH26Red and co-incubated the cells with PKH67Green-labeled SE. At 3 h post-exposure, confocal microscopy indicated that PKH26Red-labeled V428 cells internalized PKH67Green-labeled SE (Supplemental Figure 1A) and exhibited more diffuse SE staining and less punctate SE staining pattern. Similar to transformed V428 cells, PKH67Green was not transferred to V428 cells co-incubated with vehicle control (Supplemental Figure 1B), signifying that exosome labeling and internalization was specific. These results show that both fusion between SE and the V428 plasma membrane as well as V428 cellular uptake of intact SE occur in primary and transformed vaginal epithelial cells with some minor differences. Since both primary and transformed V428 cells take up SE, transformed V428 cells were utilized for the remainder of the study due to ease of culturing/ availability.

Figure 1. Human vaginal epithelial cells internalize semen exosomes.

(A) Purified human semen-derived exosomes (SE, 100µg/ml) were labeled with PKH67Green, added to E6/E7 transformed V428 human vaginal epithelial cells and incubated at 37°C for 3 h. Unbound exosomes were removed by washing and cells were fixed in 2% paraformaldehyde. Uptake of green fluorescent exosomes into V428 cells was detected by confocal microscopy. DAPI was added to cells to detect the nucleus of the cells (blue). Exosomes fused with vaginal cells present with diffuse green fluorescence and intact exosomes endocytosed into cells present with punctate green fluorescence. Scale bar: 30µm. (B) Internalization of PKH67Green-labeled PBS vehicle by V428 human vaginal epithelial cells analyzed by confocal microscopy. DAPI labels cell nucleus (blue). (C) Uptake of PKH67Green-labeled PBS vehicle or 25, 50 or 100 µg/ml of PKH67Green-labeled SE into V428 cells at 24 h post exposure was examined by FACS analysis. (D) Uptake of PKH67Green-labeled PBS vehicle or SE into VK2 human vaginal epithelial cells at 1 h, 3 h and 6 h post exposure was examined by FACS analysis. The latest PKH67Green treated PBS control time point is indicated on the histograms in Figures 1D; there was no change in the MFI of these control samples for earlier time points (not shown). (E) Vesicle uptake efficiency in V428 cells incubated for 24 h with PKH67Green-labeled PBS vehicle or 25 µg/ml of PKH67Green-labeled blood exosomes (BE), liposomes (LIPO) or SE were examined by FACS analysis. (F) Uptake efficiency of PKH67Green-labeled PBS vehicle or PKH67Green-labeled vesicles including BE, LIPO and SE into VK2 human vaginal epithelial cells at 24 h post exposure was examined by FACS analysis. (G) Uptake of PKH67Green-labeled PBS vehicle or 25 µg/ml of PKH67Green-labeled SE into V428 cells pretreated with endocytosis inhibitor Dynasore or macropinocytosis inhibitor EIPA at 24 h post exposure was examined by FACS analysis.

To confirm that SE are incorporated and retained within cells rather than at the cellular surface, V428 cells exposed to increasing concentrations of PKH67Green-labeled SE for 24 h were trypsinized. FACS analysis was used to enumerate the level of SE uptake by trypsinized V428 cells post exposure to SE (Figure 1C). To confirm that vaginal epithelial cells take up SE, we utilized VK2 cells, another immortalized human vaginal epithelial cell line. FACS analysis show that, similar to V428 cells, VK2 cells incorporate PKH67Green-labeled SE but not PK67Green-labeled vehicle in a time (1 h, 3 h, and 6 h post exposure) dependent manner (Figure 1D).

To compare V428 cell uptake efficiency of SE with other types of vesicles, we incubated V428 cells with 25 µg/ml of PKH67Green-labeled blood exosomes (BE), liposomes (LIPO), or SE (Figure 1E). FACS analysis after 24 h incubation reveal that SE was most efficiently taken up (MFI 119), compared to LIPO (MFI 85.2) and BE (MFI 4.4) in that order (Figure 1E). A similar pattern of uptake efficiency was observed with VK2 vaginal epithelial cells (Figure 1F). Increases in MFI across all samples in VK2 cells (Figure 1F) compared to V428 cells (Figure 1E) may indicate that VK2 cells more efficiently incorporate vesicles than V428 cells.

To further demonstrate that SE are internalized by vaginal epithelial cells, we pre-treated V428 cells with inhibitors of endocytosis (Dynasore) or macropinocytosis (EIPA). Treatment with Dynasore but not EIPA inhibited internalization of PK67Green-labeled SE (Figure 1G), suggesting that the majority of SE uptake by V428 cells may occur via endocytosis. In all experiments, internalization of SE by vaginal epithelial cells was shown to be specific as PKH67Green-labeled vehicle was not taken up (Figures 1B to 1G and Supplemental Figure 1B).

Semen-derived exosomes inhibit cell-to-cell spread of HIV-1

During mucosal HIV-1 infection, it is likely that epithelial cells in the FRT are the first cells to come into contact with HIV-1. It is still debatable whether or not these cell types support active HIV-1 infection (Asin et al., 2004). Although epithelial cells of the FRT may or may not support active HIV-1 infection, they have been shown to transmit infectious virus to target cells (Stoddard et al., 2007).

To evaluate the role of SE on direct cell-to-cell spread of HIV-1 from vaginal V428 cells to target cells of infection, as a way to model infection during sexual transmission, we first sought to determine the properties of the vaginal V428 cells with respect to HIV-1 primary receptor CD4 and co-receptors CCR5 or CXCR4. RT-qPCR analysis show that in comparison to CD4+CCR5+ monocytic cells (U937) or CD4+CXCR4+ T lymphocytes (SupT1), V428 cells have little or no CD4 (Figure 2A), CCR5 (Figure 2B), and CXCR4 (Figure 2C) encoding mRNA.

Figure 2. Semen-derived exosomes inhibit cell-to-cell spread of HIV-1.

(A) HIV-1 primary receptor CD4 (B) co-receptor CCR5 and (C) co-receptor CXCR4 expression detected by RT-qPCR with RNA purified from U937, SupT1, and V428 cells. (D) Uptake of PKH67Green-labeled PBS vehicle or SE into U937 monocytes at 3 h and 24 h post exposure was examined by FACS analysis. (E) Uptake of PKH67Green-labeled PBS vehicle or SE into SupT1 T lymphocytes at 3 h and 24 h post exposure was examined by FACS analysis. The latest PKH67Green treated PBS control time point is indicated on the histograms in Figure 2D–E; there was no change in the MFI of the control sample for the 3 h time point (not shown). (F) U937 monocytes and SupT1 T lymphocytes or (G) V428 vaginal epithelial cells were exposed to 0 – 400 µg/ml of SE and cell viability was expressed as fold change over controls. For cell-to-cell spread, V428 cells were exposed to 50 ng p24 antigen of HIV-1_SF162 or HIV-1_NL4.3 pre-incubated with 100 µg/ml of SE or vehicle control for 24 h. V428 cells were then washed thrice in PBS. (H to K) V428 cells co-cultured for 24 h with HIV-1 susceptible, target U937 or SupT1 cells. Target cells were washed and re-plated without V428 cells 24 h post co-culture. Integrated proviral DNA was quantitated in target cells 72 h post co-culture by Alu-LTR nested PCR using chromosomal DNA from the target (H) U937 and (I) SUPT1 cells. Total cellular RNA purified from target (J) U937 and (K) SUPT1 cells 72 h post co-culture was used to synthesize cDNA and detect HIV Gag sequences by RT-qPCR. For panels H to K, equivalent concentrations of DNA (20 ng) or cDNA (500 ng) were used for ALU PCR or RT-qPCR. Data are normalized to GAPDH and presented as fold change. (L) Final PBS wash from HIV-1 exposed V428 cells was used to inoculate TZM-bl reporter cells as control to ensure the removal of free input virus from cells prior to cell-to-cell contact. Statistical significant differences were calculated using Student's t test (**, p <0.05).

Since V428 cells internalize SE, we evaluated the ability of HIV-1 target monocytic (U937) and lymphocytic (SUPT1) cells to incorporate and retain SE. Thus, U937 and SupT1 cells were exposed to PKH67Green-labeled PBS or SE for 3 h or 24 h. Following trypsinization and PBS washing, FACS analysis confirms that SE are incorporated and retained within U937 (Figure 2D) and SUPT1 (Figure 2E) cells. Although both cell lines internalized SE, differences exist in level of SE incorporation, which was more efficient in U937 cells and increased with time from 3 h (MFI 7.79) to 24 h (MFI 10.8) post exposure (Figure 2D). In contrast, SUPT1 cells did not internalize SE efficiently and there were no differences in incorporation rate at 3 h (MFI 6.23) and 24 h (MFI 6.39) post exposure (Figure 2E). These data are consistent with a previous report showing that T lymphocytes are inefficient at incorporation of exosomes (Chivero et al., 2014). Given the differences in SE internalization between U937 and SUPT1 cells, we assessed whether such differences exist in internalization of other vesicles including BE and LIPO (used as positive control). FACS analysis reveal that U937 (Supplemental Figure 2C) and SUPT1 (Supplemental Figure 2D) cells incorporated BE and LIPO more efficiently than SE at 24 h post exposure. Similar to SE, SUPT1 cells incorporated lower BE (MFI 39.3) than U937 (MFI 68.7) cells. However, rate of LIPO incorporation was similar between U937 (MFI 58.3) and SUPT1 (MFI 56.5) cells.

V428, U937, and SUPT1 cells exposed to increasing concentrations of SE were used to assess the effect of SE on cell viability. FACS analysis showed that SE mildly stimulated U937 and SUPT1 cells (Figure 2F), as well V428 cells (Figure 2G) to proliferate. Modest cytotoxicity was observed in SE treated SupT1 at high concentrations (200 – 400 µg/ml) of SE. Based on these experiments, studies with SE on V428, U937, and SUPT1 cells were performed with noncytotoxic concentration of SE (100 µg/ml) as previously reported (Madison et al., 2014). This concentration of SE is significantly lower than physiological concentrations of 6 mg/ml of semen (Madison et al., 2014).

To determine whether vaginal epithelial cells exposed to R5 (HIV_SF162)- or X4 (HIV-1_NL4.3)- tropic HIV-1 strains transmit the virus in a cell-to-cell mode to target U937 monocytic and SupT1 T lymphocyte respectively, we performed an integration assay using Alu-LTR nested PCR. Alu-HIV Gag specific amplicons from the first PCR were subjected to nested PCR using HIV-1 LTR– specific primers (Asin et al., 2004). We observed integrated HIV-1 proviral DNA in cultures of U937 and SUPT1 cells exposed to either HIV-1 or HIV-1/SE. SE blocked HIV-1 infection of U937 by 33% (Figure 2H) and SUPT1 by 62% (Figure 2I) because the level of integrated proviral DNA was significantly reduced in the presence of SE compared to HIV-1 alone.

To further characterize the effect of SE on cell-to-cell transmission of HIV-1 from vaginal epithelial cells to target cells, we evaluated transcription of the HIV Gag gene by reverse transcription RT-qPCR. As shown in Figures 2J and 2K, transcription of HIV-1 Gag was significantly suppressed by SE in U937 cells by 99.99% (Figure 2J) and SUPT1 cells by 58% (Figure 2K). The presence of provirus and absence of viral RNA in U937 monocytes infected with HIV/SE may be indicative of abortive infection in U937 cells. More viral RNA than provirus was detected in SupT1 cells infected with HIV in the presence of SE, indicating that viral RNA was transcribed in the presence of SE, albeit 53% less than infection with HIV vehicle control.

To ensure that the vaginal cells mediated cell-to-cell HIV transmission without contribution from free-input virus, we inoculated TZM-bl reporter cells with the last wash from the vaginal V428 cells. A p24 normalized input virus was used as positive control. Absence of luminescence from the reporter TZM-bl cells inoculated with the last wash from the V428 cells confirms that free-input virus did not mediate direct infection (Figure 2L). Together, these results indicate that SE inhibit cell-to-cell transmission of both X4- and R5- tropic HIV-1 isolates.

Semen-derived exosomes inhibit trans HIV-1 infection

Previous studies have demonstrated the ability of female genital epithelial cells to transcytose HIV-1 particles. Aside from cell-to-cell HIV-1 transmission, trans infection may play a critical role in the propagation of sexual transmission of HIV-1 (Bobardt et al., 2007; Bomsel, 1997; Devito et al., 2000; Ganor et al., 2013; Gupta et al., 2013; Kinlock et al., 2014; Kohli et al., 2014; Saidi et al., 2007). To determine whether SE are capable of eliciting a block on HIV-1 trans infection, we utilized a trans well chamber system. As depicted in the methodological illustration (Figure 3A), a confluent, polarized monolayer of V428 cells was grown on a cell impermeant 0.45 µm pore size filter support forming a virus impermeable interface between the apical and basal chambers. In this regard, trans infection with HIV-1 will occur via transcytosis of virus through the V428 cell monolayer in the apical chamber to the target cells in the basal chamber.

Figure 3. Semen-derived exosomes inhibit trans HIV-1 infection.

(A) Schematic of trans HIV-1 infection model. Vaginal epithelial V428 cells were seeded on the apical chamber of a trans well cell culture semi-impermeable insert (0.45 µm pore size) in KSFM. HIV-1 target cells including U937 (B, C), SupT1 (D, E) and PBLs (G, H) from three different healthy human donors were seeded into the basal chamber. (F) PBLs from the same donors were exposed to 0 – 400 µg/ml of SE and cell viability was expressed as fold change over controls. HIV_SF162 (B, C), HIV_NL4.3 (D, E) or the clinical transmitted founder molecular infectious clone HIV_REJO (G, H) was pre-incubated for 1 h at 37°C in the presence or absence of SE and added to the apical chamber containing the primary V428 cell monolayer. All cultures were exposed to virus pre incubated with 100 µg/ml of SE (B - E) except PBLs exposed to 7.5 µg/ml of SE (G, H) to maintain cell viability. Forty-eight hours post challenge; total cellular DNA (B, D, G) or RNA (C, E, H) was purified and used to detect HIV-1 Gag gene expression by RT-qPCR analysis. Data is normalized to GAPDH and presented as fold change over control. Error bars are indicative of standard deviations between triplicate experiments. Statistical significant differences were calculated using Student's t test (**, p <0.05).

To determine the role of SE on HIV-1 transmission by trans infection, designated uninfected U937 and SupT1 cells were seeded in the basal chamber and bathed in RPMI. V428 cells were bathed in KSFM in the apical chamber and exposed for 3 h to cell-free virus (HIV_SF162, HIV-1_NL4.3, or HIV-1_REJO) that was pre incubated for 1 h in the presence of either SE (100 µg/ml) or PBS vehicle control. Trans infection of HIV-1 was examined 48 h post infection in target cells within the basal chamber by RT-qPCR detection of HIV-1 Gag sequences within purified cellular genomic DNA (Figures 3B and 3D) and HIV-1 Gag mRNA within purified total cellular RNA (Figures 3C and 3E). We discovered that transcytosis across the V428 cell monolayer was successful as manifested by detection of HIV-1 Gag sequences in cellular DNA and RNA in target cells within the basal chamber. Compared to vehicle control, trans infection with HIV_SF162 in the presence of SE was almost completely blocked by SE in U937 monocytic cells by 99.9% in gDNA or 82% in cellular RNA (Figures 3B and 3C, respectively). Trans infection with HIV-1_NL4.3 in the presence of SE was significantly reduced in SupT1 T lymphocyte cells by 84% in DNA and 64% in RNA (Figure 3D and 3E, respectively).

To evaluate the effect of SE on HIV-1 transmission by trans infection from V428 cells to peripheral blood leucocytes (PBL), we first assessed viability of healthy donor PBL exposed to increasing concentrations of SE as described in Figures 2F and 2G. Unlike V428, U937, and SUPT1 cells, PBLs are more sensitive to SE because mild cytotoxic effect was detected with 12.5 µg/ml of SE. Higher concentrations (25 – 400 µg/ml) of SE exhibited cytotoxic effects on human PBL (Figure 3F). Therefore, 7.5 µg/ml of SE was utilized for all transcytosis experiments involving PBL. Designated uninfected healthy donor PBLs were seeded in the basal chamber and bathed in RPMI. V428 cells were bathed in KSFM in the apical chamber and exposed for 3 h to cell-free virus (HIV-1_REJO) that was pre incubated for 1 h in the presence of either SE (7.5 µg/ml) or PBS vehicle control. Trans infection of HIV-1 was examined 48 h post infection in PBLs within the basal chamber by RT-qPCR detection of HIV-1 Gag sequences within purified cellular genomic DNA and RNA. Trans infection with the transmitted/founder isolate HIV-1_REJO was reduced in PBLs (Figures 3G and 3H) from three different healthy human donors in the presence of SE by 47%, 82% and 79% in purified DNA and by 19%, 51% and 33% in purified cellular RNA. The differences in PBL response to SE inhibition reflect donor variability and not SE efficacy. All experiments were performed with noncytotoxic concentrations of SE at 7.5 – 100 µg/ml.

Semen-derived exosomes donate genetic material to mouse cells in vivo

During host cell synthesis of exosomes, cytoplasmic proteins and RNA are incorporated into the exosome lumen. Secreted exosomes contain a diverse repertoire of RNA species, including small RNA, miRNA, tRNA, and mRNA (Keller et al., 2011; Vojtech et al., 2014a), and exosomal cargo can be transferred to target cells following fusion with such cells (Li et al., 2013; Skog et al., 2008; Sun et al., 2010). We previously reported that SE isolated from healthy human semen contains a range of mRNA, such as Apobec3 and that SE are taken up by recipient cells (Madison et al., 2014). To determine whether SE donate their cargo to cells of the FRT in vivo, we first examined the ability of C57Bl/6 murine vaginal epithelial cells to internalize human SE. As described in the methodological illustration (Fig. 4A), healthy age-matched female mice were intravaginally (IVag) inoculated with 100 µg/30 µl of PKH67Green-labeled SE or vehicle (Figure 4A) and sacrificed 3 h or 24 h post-inoculation. FACS analysis of single cells obtained from vaginal tissues showed that human SE were internalized by murine vaginal cells in vivo within 24 h of inoculation (Figure 4B).

Figure 4. Murine vaginal cells internalize human semen exosomes.

(A) Schematic of experimental procedure. Healthy female C57BL/6 mice received intravaginal (IVag) inoculation of 100 µg PKH67Green-labeled SE or PKH67Green-labeled PBS control. (B) At either 3 h or 24 h post inoculation mice were sacrificed. At necropsy, vaginal cells were collected, homogenized to obtain single cell suspension and screened for in vivo exosome uptake by FACS analysis. The latest PKH67Green treated PBS control time point is indicated on the histograms in Figures 4B; there was no change in the MFI of the control sample for the 3 h time point (not shown). (C) Human Apobec3G (A3G) and Apobec3F (A3F) mRNA were detected by RT-qPCR of cDNA synthesized from total RNA isolated from murine vaginal cells alone or murine vaginal cells exposed to human SE by IVag inoculation. Data ± SD are plotted as Fold change in expression of human Apobec3G sequences detected in murine vaginal cells exposed to SE (set at 1 for C). (D) Human Apobec3G and Apobec3F amplified from SE alone. Data ± SD are plotted as Fold change in expression of Apobec3G set at 1 for D and E. RT-qPCR was normalized to GAPDH (C – E). Error bars are indicative of standard deviations (SD) between triplicate experiments. Statistical significant differences were calculated using Student's t test (**, p <0.05).

Following internalization, exosomes can donate functional mRNA to recipient cells (Kogure et al., 2011; Li et al., 2013; Skog et al., 2008). We used Apobec3G and Apobec3F TaqMan RT-qPCR to examine whether human SE donated Apobec3G and Apobec3F mRNA to mice. We selected these antiviral Apobec3 genes because i) Apobec3G incorporated into H9 exosomes was shown to block HIV-1 replication (Khatua et al., 2009), ii) both Apobec3G and Apobec3F mRNA are enwrapped in human semen exosomes (Madison et al., 2014) and iii) mice do not express human Apobec3G/F. Human Apobec3G mRNA was amplified from murine vaginal tissues exposed to SE for 24 h, but not in control murine vaginal tissues exposed to PBS control (Figure 4C). Surprisingly, human Apobec3F was not detected in murine vaginal tissues exposed to SE (Figure 4C) despite that both human Apobec3F and human Apobec3G were detected in SE alone (Figure 4D). We did not observe the presence of Apobec3G or Apobec3F protein in our pooled donor SE prep (data not shown), suggesting that i) Apobec3 proteins may not be part of SE cargo and/or ii) the antibodies used were not sensitive enough to detect limited protein cargo. The absence of A3F in murine vaginal cells exposed to SE could not be explained. However, it is plausible that detection of transferred mRNA may partly depend on the level/amount of mRNA present in SE. Accordingly, more Apobec3G than Apobec3F mRNA is present in SE (Figure 4D). Whether or not SE-mediated donation of Apobec3G mRNA plays any antiviral role is yet to be determined. Taken together, our data reveal that murine vaginal cells internalize human SE and that SE are capable of shuttling functional mRNA between heterologous species in vivo.

Semen-derived exosomes decrease intravaginal murine AIDS virus replication, viremia, and systemic spread of replicating virus

Since SE are incorporated into cells of the human and murine FRT, donate exosomal cargo, and suppress HIV-1 cell-to-cell and trans infection, we predict that SE may inhibit mucosal replication of LP-BM5 mAIDS retrovirus complex. Healthy age-matched female mice were intravaginally (IVag) inoculated with LP-BM5 pre-incubated with human SE or vehicle control as described in the methodological illustration (Figure 5A). Blood was collected biweekly from the tail vein to evaluate viremia. Animals were sacrificed at four weeks post inoculation to evaluate viral replication and spread using primers specific to LP-BM5 mAIDS virus. The level of LP-BM5 sequences in purified cellular gDNA (Figure 5B) and RNA (Figure 5C) in murine vaginal tissues was significantly reduced by SE (77% and 57% suppression, respectively). These data indicate that SE inhibit LP-BM5 replication at the portal of viral entry into the host.

Figure 5. Human semen-derived exosomes decrease murine susceptibility to LP-BM5 mAIDS virus infection.

(A) Schematic representation of experimental procedure. Healthy, wild-type, female C57BL/6 mice were intravaginally inoculated with LP-BM5 mAIDS virus pre incubated for 1 h at 37°C ±SE (100 µg) or an equivalent volume of PBS control. Blood was collected biweekly and at four weeks post challenge, the mice were sacrificed. At necropsy, whole blood was collected and PBMCs were isolated, organs including the vagina, draining lymph nodes (inguinal/subiliac), and spleen were harvested and homogenized to generate a single cell suspension. (B) Total DNA and (C) RNA were isolated from murine vaginal cells for analysis of LP-BM5 infection at the site of inoculation by detection of LP-BM5 sequences by RT-qPCR. (D) Equivalent volumes of cell-free plasma collected at two and four weeks post IVag inoculation was used to infect naïve C57BL/6 splenocytes ex vivo followed by isolation of DNA and RT-qPCR analysis of LP-BM5 DNA. GAPDH was used to normalize data plotted as % Secondary infectivity. Cells infected with plasma collected from control mice are set at 100%. (E to G) LP-BM5 DNA is detected by RT-qPCR in (E) PBMCs, (F) lymph nodes (G) spleen. Error bars are indicative of standard deviations (SD) between triplicate experiments. Data ± SD for B, C, E, F and G are expressed as fold change of vehicle values set at 1 for all panels. Statistical significant differences were calculated using Student's t test (**, p <0.05).

Following infection, retroviruses enter the blood stream resulting in viremia. To determine the effect of SE on the amount of infectious LP-BM5 mAIDS virus in circulation, we assessed viral load in the cell-free component of blood plasma using a cell-based infection assay. Infection of naïve murine splenocytes ex vivo with equal volumes of cell-free, blood plasma obtained from infected mice at two and four weeks post-challenge revealed that plasma from control mice is much more infectious than plasma from the SE group (Figure 5D). Secondary infectivity of cell-free, plasma was higher in the control plasma group compared to SE plasma group by 77% at 2 weeks and 65% at 4 weeks post infection (Figure 5D). These data suggest that SE decreased the concentration of infectious virus in circulation in infected mice.

Because SE impaired the amount of infectious virus in circulation, we assessed whether SE inhibit lymphatic dissemination of LP-BM5 mAIDS virus by evaluating the effect of SE on viral load in peripheral tissues that are targets of LP-BM5 (Jones et al., 2012b). DNA isolated from murine PBMCs (Figure 5E), inguinal/subiliac draining lymph nodes (Figure 5F), and splenocytes (Figure 5G) of mice sacrificed four weeks post infection was examined by RT-qPCR of LP-BM5 sequences for quantitation of viral load. SE diminished the expression of LP-BM5 sequences in purified gDNA by 84%, 46% and 46% respectively in PBMCs, lymph node, and splenocytes (Figures 5E to 5G) of mice intravaginally exposed to LP-BM5 that was pre-incubated with SE compared to vehicle control. These results reveal the ability of SE to limit the rate of lymphatic viral replication and spread of replicating virus when infection occurs via the vaginal mucosa in an intact organism.

Semen-derived exosomes impair reverse transcriptase activity of progeny LP-BM5 mAIDS virus

We previously showed that SE impair RT activity of progeny HIV-1 produced in various cell types (Madison et al., 2014). To determine the effect of SE on RT activity of LP-BM5 progeny produced in the presence and absence of SE, we performed RT activity assay on cell-free, blood plasma from LP-BM5 infected mice at two and four weeks after infection. As shown in Figure 6A, RT activity in cell-free blood plasma from LP-BM5 infected mice steadily increased as infection progressed from two to four weeks in both groups of animals. However, compared to the control mice, SE severely impaired RT activity of progeny LP-BM5 both at two and four weeks post infection by 91% and 82%, respectively (Figure 6A). These data support and extend our previous observation on the effect of SE on HIV-1 RT activity (Madison et al., 2014).

Figure 6. Human semen-derived exosomes impair LP-BM5 RT activity.

(A) Equivalent volumes of cell-free plasma collected at two and four weeks post IVag inoculation was used to examine RT activity in cell free progeny LP-BM5 within plasma. (B) RT activity of input virus was examined within LP-BM5 stock virus pre-incubated for 1 h at 37°C with PBS or 100 µg/ml SE. (C) RT activity of input virus was examined at 3 h post infection intracellularly and within the culture supernatant. (D) RT activity of progeny virus was examined at 24 h post infection within the supernatant and intracellularly within splenocytes infected ex vivo with LP-BM5 stock virus pre-incubated for 1 h at 37°C with PBS or SE. Error bars are indicative of standard deviations (SD) between triplicate experiments. Data ± SD for panels B, C and D are expressed as Fold change of vehicle values set at 1. Statistical significant differences were calculated using Student's t test (**, p <0.05). ns means non-significant.

Because SE impaired LP-BM5 RT activity, we sought to determine at what time point SE mediated suppression of LP-BM5 RT activity occurs. We first analyzed RT activity of LP-BM5 stock virus subsequent to 1 h co-incubation at 37°C with PBS or SE in a cell-free system. We did not observe differences in RT activity (Figure 6B), indicating that incubation of LP-BM5 with SE prior to cell infection does not impair RT activity.

Next, we determined whether equal amounts of LP-BM5 entered cells in the presence and absence of SE, and if there was a change in RT activity at an early time point after cell entry. Thus, we examined RT activity within cellular extract and supernatant of naïve splenocytes exposed to LP-BM5 incubated with SE or PBS at 3 h post infection. Results show that equal amount of LP-BM5 entered cells in the presence and absence of SE because we observed equal intracellular and extracellular RT activities in both treatment conditions (Figure 6C).

Although the level of RT activity in cells and in culture supernatant at 3 h after infection were not different between vehicle and SE treated LP-BM5 (Figure 6C), we found a significant reduction in RT activity of intracellular and progeny virus at 24 h post infection. SE suppressed intracellular RT activity in clarified cellular extracts by 52% and in cell-free culture supernatants by 45% (Figure 6D). This data indicates that SE mediate inhibition of LP-BM5 replication by blockade of reverse transcription within 24 h post infection. Our data further suggest that SE-mediated suppression of LP-BM5 retroviral infectivity culminates in the generation of LP-BM5 progeny with deleterious effects on RT activity and viral fitness.

Discussion

Exosomes are small extracellular membrane bound nanovesicles secreted from numerous types of cells. When taken up by recipient cells, exosomes can donate their cargo to the recipient cells and subsequently modify cellular behavior. We previously characterized and showed that semen exosomes (SE) derived from healthy donors possess potent anti-HIV-1 activity (Madison et al., 2014). Here, we show that SE blocked intravaginal infection of mice with murine AIDS virus complex LP-BM5 and lymphatic spread of the virus. Importantly, SE inhibited HIV-1 transmission to target monocytic and lymphocytic cell lines from vaginal epithelial cells. Upon exposure to vaginal epithelial cells, SE were internalized by dynamin-dependent endocytic pathway and by direct fusion to the plasma membrane. Exosome internalization by endocytosis is a known mechanism for exosomal uptake and have previously been reported to occur in hippocampal neurons (Newton et al., 2006) and CNE1 nasopharyngeal carcinoma cells (Nanbo et al., 2013). However, exosomal uptake mechanism in vivo is unknown. The evidence that uptake of human SE in mice resulted in transfer of functional mRNA encoding human Apobec3G supports a mechanism of genetic exchange of mRNA between unrelated species. Since mice do not express human Apobec3G, amplification of human Apobec3G mRNA in murine vaginal cells suggests that SE are capable of shuttling RNA between heterologous species. This finding is consistent with previous reports that showed that exosomes could transfer anti- viral factors to target cells with resultant modulation of host cell response to mouse hepatitis virus (Li et al., 2013) and HIV-1 (Khatua et al., 2009).

During sexual transmission of HIV-1, the female genital mucosa is exposed to HIV-1 and exosomes contained in semen. HIV-1 may be propagated through i) direct cell-to-cell transmission, ii) trans infection by cell-free virions through transcytosis (Sato et al., 1992) and iii) direct infection by cell-free virions on permissive target cells within the mucosa. There is no consensus on the identity of the initial targets of HIV-1 infection in the mucosa. However, upon overcoming mucosal barrier, partly via transcytosis (Kinlock et al., 2014), HIV-1 comes in contact with susceptible target cells in the submucosa, including dendritic cells, Langerhans cells, macrophages, and CD4+ T cells. Of all the different cell types, CD4⁺ T cells represent the main population of leukocytes in the cervicovaginal tissues. These cells are also present in the lamina propria, subepithelial stroma, and the epithelium (Edwards and Morris, 1985; Hickey et al., 2011; Johansson et al., 1999). Findings using human vaginal explants show that CD4⁺ T cells are relevant to HIV-1 transmission because HIV-1 productively infects both activated and resting CD4⁺ T cells in the genital mucosa (Gupta et al., 2002; Hladik et al., 2007; Zhang et al., 1999). Whether DCs, Langerhans cells, and monocytes present in the cervicovaginal epithelium can support productive HIV-1 infection in vivo is controversial. It is known that these myeloid cells can sample antigens in the mucosa, capture the virus and transfer virus to susceptible CD4⁺ T cells (Ballweber et al., 2011; Hu et al., 2004; Wu and KewalRamani, 2006). Furthermore, macrophages in the mucosa express the HIV-1 receptor CD4 and co-receptor CCR5, but their role in the initial events of HIV-1 infection is yet to be defined (Shen et al., 2009; Wira et al., 2005). Therefore, our finding that SE blocked the transfer of HIV-1 particles from vaginal epithelial cells to target monocytes (U937) and leukocytes (SupT1 and PBLs) in both cell to cell and trans infection models is remarkable in the sense that the antiviral factor of SE can be exploited for therapeutic application.

Moreover, during the eclipse phase of intravaginal exposure to HIV-1 infected cells the virus replicates locally in the mucosa (Li et al., 2009; Miller et al., 2005). Following the eclipse phase, the virus disseminates. Virus and virus-infected cells leave infected mucosal tissues through afferent lymphatics to enter the draining lymph nodes. In the draining lymph node, HIV-1 and HIV-infected cells come in contact with large numbers of target cells, resulting in significant virus replication, virus dissemination, and establishment of systemic infection throughout the secondary lymphoid organs and the bloodstream (Li et al., 2009; Miller et al., 2005).

Interestingly, SE protected mice from intravaginal infection with LP-BM5, blocked the spread of replicating virus in vivo, and reduced plasma viral load. This observation is important because high viral load is the greatest predisposing factor for transmission of HIV-1 and viral load predicts transmission risk. Contextually, studies have shown that transmission of HIV-1 rarely occurs in people with less than 1500 copies/ml of viral RNA (Quinn et al., 2000; Tovanabutra et al., 2002). The impairment of progeny virus RT activity by SE could partly explain the reduction in viral load. It is also plausible that the reported immunosuppressive property of SE may contribute to inhibition of viral infection and reduction in viral load, since retroviruses, such as HIV-1 thrives under hyper-immune activated state.

Collectively, our results address the physiological role of SE in HIV-1 transmission and present a paradigm for the study of the effect of SE on HIV-1-induced immune activation. Future studies should focus on evaluating whether SE alters local and systemic HIV-1-induced hyperactivation.

Experimental Procedures

Ethics Statement

This study involves the use of existing, de-identified human specimens (semen, serum and PBLs) and therefore is not human subjects’ research. Semen samples were obtained either from the Reproductive Specialty Laboratory of Middle Tennessee, Nashville or University of Iowa In Vitro Fertilization and Reproductive Testing Laboratory. Healthy HIV-1-negative subjects were invited to participate in the study. Following written informed consent, blood was obtained for PBL isolations. This study was approved by the University of Iowa Institutional Review Board.

The University of Iowa Animal Care and Use Committee (IACUC) approved experiments involving mice and the mice were housed according to the policies of the Institutional Animal Care and Use Committee of the University of Iowa. Experiments involving the use of mice were performed in accordance to NIH guidelines, the Animal Welfare Act, and US federal law.

Exosome Purification

Semen or blood serum exosomes were purified using our previously described protocol (Madison et al., 2014). Briefly, healthy human donor semen samples were procured by dry manual stimulation and ejaculation into sterile polypropylene tubes followed by 30 min incubation at room temperature for promotion of liquefaction. Spermatozoa were pelleted by centrifugation for 10 min at 1000 × g at 4°C. Seminal plasma or blood serum samples were stored at −80°C until purification of exosomes using ExoQuick (SBI) exosome precipitation reagent as previously described (de Hoog et al., 2013; Madison et al., 2014; Quackenbush et al., 2014; Rekker et al., 2013) or sucrose gradient differential ultracentrifugation (Madison et al., 2014). Supernatant (exosome depleted seminal plasma or blood serum) was discarded, the SE or BE exosome pellet was washed in PBS three times for 30 minutes at 4°C and 100,000 × g, resuspended in PBS vehicle, aliquoted, quantified by the Bradford method and stored at −80°C until use. Liposomes were used as a positive control for vesicle uptake and were generated according to manufacturer protocol using Lipofectamine 2000 (Life Technologies).

Viruses

The plasmid encoding HIV-1_pNL4.3 was obtained from the NIH AIDS Reagent Program, plasmids expressing the clade B transmitted/founder HIV-1 infectious molecular clone REJO and R5 tropic HIV-1_pSF-162 were generously provided by Dr. Wendy Maury of University of Iowa (Ochsenbauer et al., 2012). Viral stocks were generated by transfecting HEK 293 cells using PEI or Lipofectamine according to the manufacturer’s instructions (Invitrogen). Virus was 0.45 µm filtered, treated with DNAse, then concentrated by ultracentrifugation at 100,000 × g for 2 h through a 20 % sucrose cushion as previously described (Liao et al., 2003). Viral stocks were resuspended in RPMI with 10% exosome free FCS, aliquoted, and stored at −80°C. The Lentivirus-Associated p24 ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA) and TZM-bl reporter cells were utilized to determine viral titer and infectivity, respectively. The G6 clone of chronically infected SC-1 cells (Cook et al., 2003; Jones et al., 2012b) obtained from NIH AIDS Research and Reference Reagent was cultured for 7 days and LP-BM5 murine AIDS virus complex was harvested from the supernatant.

Cells

TZM-bl and Sup-T1 cell lines were obtained through the NIH AIDS Reagent Program. Dr. Aloysius Klingelhutz from the University of Iowa generously provided primary and E6/E7 transformed human vaginal epithelial cells (V428) previously described (Peterson et al., 2005). VK2 human vaginal epithelial (HPV-16 E6/E7 transformed) cells and U937 human monocytic cells were obtained from the American Tissue Culture Collection (ATCC). Complete RPMI (cRPMI) 1640 (Gibco-BRL/Life Technologies) supplemented with 10% exosome depleted FCS (HyClone), 100 U/ml penicillin, 100 µg/ml streptomycin, sodium pyruvate and 0.3 mg/ml l-glutamine (Invitrogen, Molecular Probes) was utilized to maintain Sup-T1 and U937 cells whereas TZM-bl and HEK 293 cells were maintained in complete DMEM (cDMEM, Gibco-BRL/Life Technologies). Keratinocyte serum free media (KSFM, Life Technologies) containing l-glutamine and supplemented with prequalified human recombinant Epidermal Growth Factor 1–53, Bovine Pituitary Extract (BPE), 100 U/ml penicillin and 100 µg/ml streptomycin was used to maintain V428 and VK2 cells. PBLs from three healthy anonymous blood donors were isolated from whole human blood by Ficoll-Hypaque density centrifugation and depleted of macrophages by gelatin adherence as previously described (Maury, 1994). PBLs were then propagated in cRPMI 1640, stimulated for 48 h with 5 µg/ml phytohemagglutinin (PHA, Roche Applied Science), washed with cRPMI and resuspended in fresh cRPMI with IL-2 (10 U/ml). The G6 clone of chronically infected SC-1 cells was maintained in cDMEM. All cells were grown at 37 °C with 5% CO₂. Where indicated, cellular viability was examined using CellTiter-Glo luminescent cell viability assay (Promega) according to manufacturer instructions.

Exosome uptake by V428, VK2, U937 and SupT1 cells

Exosomes (SE, BE) or liposomes (LIPO) were labeled using the PKH67Green fluorescent kit (Sigma-Aldrich) according to the manufacturer’s instructions and as previously described (Madison et al., 2014). PBS vehicle control was labeled using the PKH67Green fluorescent kit and did not retain label. V428 cells were seeded in a tissue culture plate at 8 × 10³ cells per well on collagen coated coverslips overnight in KSFM. Where indicated, cells were labeled in the plate using the PKH26Red fluorescence kit (Sigma-Aldrich) according to the manufacturer’s instructions. PKH67 Green labeled SE (25 µg/ml) were added to PKH26Red-labeled or unlabeled V428 cells and incubated at 37°C for 3 h. The cells were washed three times on the coverslips with PBS, fixed in 2% paraformaldehyde for 15 min on ice, washed three times with PBS, mounted onto microscope slides with Vectashield antifade reagent (Invitrogen, Molecular Probes), and observed using a laser scanning confocal microscope (Nikon TE2000). Fluorescence was also analysed by use of a FACSCalibur flow cytometer (BD) to detect the PKH67Green transferred from SE to V428 or VK2 human vaginal epithelial cells during fusion and uptake at 1, 3, 6 and 24 h post 25 µg/mL SE exposure or to U937 and SupT1 cells 3h and 24h post exposure to 25 µg/mL SE. V428, VK2 , U937 and SupT1 cellular uptake efficiency of 25 µg/ml PKH67Green labeled BE or liposome positive control (LIPO) was compared to 25 µg/ml SE uptake in these cells by FACS analysis as described above. Cells were incubated with PKH67 Green treated PBS alone (unlabeled cells) for each time points indicated above and were used to determine positive staining and the flow cytometer determined background fluorescence. The latest PKH67 Green treated PBS control time point is indicated on the histograms in Figures 1D, 2F–G and 4B. There was no change in the MFI of these control samples for earlier time points (not shown). All cells examined for SE uptake by FACS analysis were trypsinised with 0.25% trypsin post exposure at the indicated time point to differentiate between cell surface bound vesicles and internalized vesicles. Cellular frequency and fluorescence intensity were determined by Flowjo analysis software (TreeStar).

To examine the role of endocytosis in SE uptake, V428 cells were pretreated with dimethyl sulfoxide (DMSO) vehicle control, 150 µM dynasore (Sigma-Aldrich), or 75 µM 5-(N-ethyl-N-isopropyl amiloride (EIPA) (Sigma-Aldrich) for 30 min at 37°C and then incubated with PKH67 Green-labeled SE (25 µg/ml) for 3 h as described above in the presence of inhibitors. V428 cell surface-bound exosomes were removed by treatment with 0.25% trypsin. The V428 cells were washed three times with PBS, fixed in 2% paraformaldehyde for 15 min on ice and internalized green fluorescence was examined by FACS analysis as described above.

Exosome uptake by murine vaginal cells in vivo

Isoflurane-anesthetized age and weight matched female C57BL/6 mice were inoculated intravaginally with 100 µg/30µl PKH67Green-labeled PBS or SE. Mice were sacrificed 3 h and 24 h post inoculation. Vaginal tissues were harvested, single cell suspensions were generated by homogenization and collagen digestion. Cells were fixed for 15 min on ice with 2% paraformaldehyde and immunofluorescence was analysed by use of a FACSCalibur flow cytometer (BD) to detect PKH67Green transferred from SE to murine vaginal cells during fusion and uptake. Cellular frequency and fluorescence intensity were determined by Flowjo analysis software (TreeStar). Donation of human genetic material from SE to murine vaginal cells was examined by RT-qPCR as described below.

Real-time Quantitative PCR

Exosome and cellular total RNA and/or cellular genomic DNA were isolated using a ZR-Duet™ DNA/RNA Mini Prep Kit (Zymo Research). Isolated RNA was exposed to DNAse and subjected to cDNA synthesis (ABI) as previously described (Jones et al., 2013a; Jones et al., 2013c; Jones et al., 2012a; Jones and Okeoma, 2013; Mehta et al., 2012). Nucleic acid concentration and purity were measured spectrophotometrically at 260/280nm. A predetermined concentration of template genomic DNA or cDNA was amplified with primers specific to LP-BM5 BM5 (Jones et al., 2012b) or HIV-1 Gag sequences (Li et al., 2007) to quantify LP-BM5 mAIDS or HIV-1 viral gene expression respectively, by real-time quantitative PCR (RT-qPCR) using ABI 7500 FAST thermal cycler (Applied Biosciences) as previously described (Jones et al., 2013b, a; Jones et al., 2012a; Jones and Okeoma, 2013; Mehta et al., 2012; Okeoma et al., 2007; Okeoma et al., 2009). Integration of HIV-1 proviral DNA was examined by nested PCR using genomic DNA isolated as described above. The HIV-1 Gag antisense and Alu sense primers were used for the first PCR and 5 µl of this PCR product was then used as template for nested PCR using primers designed to detect HIV-1 LTR as previously described (Asin et al., 2004). For RT-qPCR analysis of SE-mediated donation of human genes to murine vaginal cells, total RNA was isolated from SE alone, murine vaginal cells alone or murine vaginal cells exposed to SE using the RNeasy Mini-Prep kit (Qiagen) followed by cDNA synthesis (ABI) and a predetermined concentration of cDNA was used to probe for the indicated human or changes in murine gene expression using TaqMan assays or QuantiFast Sybr green assays as previously described (Jones et al., 2013c; Jones and Okeoma, 2013; Mehta et al., 2012). GAPDH was used to normalize gene expression across all samples.

HIV-1 Trans Infection and Cell-to-Cell Transmission

For analysis of the effect of SE on trans HIV-1 infection, V428 cells were seeded at a density of 2 × 10⁶ on the apical side of 0.45 µm pore sized polyethylene terephthalate trans well cell culture inserts (BD Biosciences) and cultured overnight within a volume of 0.5 ml KSFM in 6-well trans well dishes. The indicated target cells (2 × 10⁵ cells/ml) were added in 1 ml complete RPMI 1640 to the basal chamber of the trans well cell culture dish. Semen is the vector for sexual transmission of HIV-1, and in an infected male, HIV-1 is expected to encounter SE in the male genital tract. Therefore, SE (7.5 µg/ml or 100 µg/ml) and HIV-1 (50 ng p24 antigen) were co-incubated for 1 h at 37°C prior to addition to cell cultures for trans infection to mimic physiologic conditions in the male genital tract. Thereafter, HIV-1 that had been pre incubated with vehicle (PBS) or 7.5 – 100 µg/ml of pooled SE (n = 20) for 1 h in a volume of 0.5 ml KSFM was added to the V428 cells in the apical chamber. Trans epithelial electrical resistance was measured using an EVOM Epithelial Voltohmmeter (World Precision Instruments, Inc.), which indicated equal viability and confluence of the V428 cell monolayer in the trans wells across different treatments. V428 mediated trans HIV-1 infection was analyzed 48 h post challenge by RT-qPCR analysis of HIV-1 Gag amplicons using DNA and cDNA from the target cells as described above.

For analysis of the effect of SE on cell-to-cell spread of HIV-1 we utilized a co-cultivation model as previously described (Asin et al., 2004). In brief, V428 cells were seeded in KSFM at a density of 0.5 × 10⁶ overnight. V428 cells were then exposed to HIV-1 _SF162 or HIV-1 _NL4.₃ (50 ng p24 antigen) pre-incubated with SE (100 µg/mL) or vehicle control. Twenty-four hours later, V428 cells were washed thrice in PBS and the final wash was used to inoculate TZM-bl reporter cells to ensure that residual input virus was washed away. V428 cells were then co-cultured for 24 h with 1 × 10⁵ U937 or SupT1 target cells in cRPMI. Target cells were washed thrice in PBS and re-plated without V428 cells 24 h post co-cultivation. Integrated proviral DNA and viral mRNA was quantitated in target cells 72 h post co-cultivation by ALU PCR using chromosomal DNA or by RT-qPCR using total cellular RNA to synthesize cDNA as described above.

LP-BM5 infection

Intravaginal infection of mice was performed as follows; Age and weight matched female C57BL/6 mice (n =11) were anesthetized with isoflurane and infected by intravaginal inoculation with 30 µl (300 RT units) of LP-BM5 mAIDS virus pre-incubated for 1 h at 37°C in the presence or absence of 100 µg SE or an equivalent volume of PBS vehicle. Blood was collected weekly to isolate plasma and examine viremia. At the indicated time depending on experiment, mice were sacrificed and relevant tissues obtained for downstream analysis of viral replication, viremia and spread by RT-qPCR as described above. Infection of naïve murine splenocytes with plasma from mice challenged with LP-BM5 murine leukemia virus in the presence or absence of SE was done in triplicates in 48-well plate format to examine viremia. Briefly, splenocytes were plated at 1 × 10⁵ cells per well and incubated with 20 µl of virus-containing plasma. Input virus was washed away and DNA was extracted from cells for examination of LP-BM5 DNA and RNA 72 h post viral challenge by RT-qPCR.

Statistical analysis

Analysis of statistically significant differences was examined by paired, two-tailed Student's t test. All p-values were calculated from triplicate experiments. A p-value of <0.05 was regarded as statistically significant. Standard deviations (SD) are represented by error bars between triplicate experiments. All experiments were repeated thrice in triplicate with similar results.

Highlights.

• Human and murine vaginal cells internalize human semen exosomes

• Human semen exosomes transfer functional mRNA to mice

• Semen exosomes inhibit trans infection and cell to cell transmission of HIV-1

• Human semen-exosomes restrict intravaginal transmission and propagation of LP-BM5 murine AIDS virus complex

• Semen exosomes diminish progeny virus fitness

List of abbreviations

HIV-1

human immunodeficiency virus type 1

AIDS

acquired immunodeficiency syndrome

mAIDS

murine acquired immunodeficiency syndrome

SIV

simian immunodeficiency virus

SE

semen-derived exosomes

PBMC

peripheral blood mononuclear cells

IVag

intra vaginal

FRT

female reproductive tract

RT

reverse transcriptase

A3

APOBEC3, Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3

PBL

peripheral blood leukocytes

BE

blood exosomes

LIPO

liposomes