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BASIC SCIENCE: CONCISE COMMUNICATION

Human vaginal fluid contains exosomes that have an inhibitory effect on an early step of the HIV-1 life cycle

Smith, Johanna A.; Daniel, Rene

Author Information
doi: 10.1097/QAD.0000000000001236

Abstract

Introduction

Worldwide, around 16 million women are infected with HIV-1, with the majority of infections occurring through heterosexual contact, and with vaginal contact accounting for about half of these transmissions (http://www.who.int/gho/hiv/epidemic_status/cases_adults_women_children/en/, [1]). Consequently, a reduction in vaginal transmission will have a major impact on the reduction of new HIV-1 infections around the world. Although 24 potential vaginal microbicides are currently under development, there is no efficient method to eliminate vaginal transmission [2]. At the same time, data have shown that the pooled risk estimate for anal intercourse is 1.7%, which is at least five to six times higher than the risk estimate for vaginal sex [3]. These data suggest that there exist natural barriers to HIV-1 infection in the vaginal environment. Some of these barriers have been studied, but our understanding is yet in its infancy [4–9].

Very recently, HIV-1 infection efficiency was tied to the presence of exosomes in biological fluids [10–12]. Exosomes are nanovesicles that contain proteins, mRNA, and microRNAs [13]. Recent studies demonstrated that exosomes are secreted by multiple cell types, and a recent study also demonstrated exosomes in mouse oviduct during estrus [13–18]. It has been shown that semen and breast milk contain exosomes that may inhibit HIV-1 transmission [10–12].

We aimed to determine whether vaginal fluid contains exosomes, and whether these may play a role in HIV-1 infection. Our data support the hypothesis that biological fluid exosomes, in this case those discovered by us, in vaginal fluid, present one of the first lines of defense against HIV-1 infection in women.

Materials and methods

Vaginal fluid samples

Raw, unprocessed frozen vaginal fluid, and seminal fluid – nonreactive for HIV-1/HCV/HBV by nucleic acid testing, HBV surface antigen, HCV antibody, HIV-1&2 antibody and rapid plasma reagin by currently approved Food and Drug Administration methods – were purchased from Lee Biosolutions, Maryland Heights, Missouri, USA (vaginal fluid cat 991-10-C).

Exosome isolation and quantitation

To isolate vaginal fluid exosomes (VFE), we used ExoQuick Exosome Precipitation Solution (EXOQ5A-1) from System Biosciences, LLC (Palo Alto, California, USA) and followed the manufacturer's instructions. The volume of vaginal fluid was approximately 700 μl/sample following the initial centrifugation step to remove cells and cell debris. The final exosome-containing pellets were resuspended in a PBS volume of 100 μl (1/7 the original volume of fluid).

To confirm our results using an alternative method, exosomes were also isolated using qEV size exclusion column (cat SP1; iZon Science, Cambridge, Massachusetts, USA) following the manufacturer's instructions. Briefly, several fractions of the column output were collected, concentrated, and tested for its ability to inhibit the vector-mediated transduction of the green fluorescent protein (GFP) marker, as described in Fig. 2d. After the sample was added to the column, the first 1.5 ml through the column was discarded. The second 1.5 ml was collected as a control (as this is predicted to be the second half of the void volume according to the manufacturer's instructions), and we call this fraction, fraction 1. The fraction 2 (the next 1.75 ml through the column) is where the exosomes are concentrated as per the qEV column protocol, and their presence was confirmed using the NanoSight NS300 utilizing Nanoparticle Tracking Analysis software (Malvern Instruments Ltd, Malvern, UK). The presence of exosomes then decreases in the fraction 3 (the following 1.5 ml). Fractions were concentrated using Amicon Ultra-4 10 kDa nominal molecular weight centrifugal filter units (cat UFC501024; MilliporeSigma, Merck KGaA, Darmstadt, Germany).

A standard Bradford protein assay was conducted to quantify total protein concentration present in purified exosome samples.

Exosome characterization

VFE (isolated as described above) were analyzed for exosomal surface markers by fluorescence-activated cell sorting (FACS) analysis utilizing an Exo-Flow kit from SBI (cat EXOFLOW150A-1). Following the manufacturer's instructions, biotinylated capture antibodies (CD9 or CD63, provided with the kit, the biotinylated CD81 antibody was not available from the manufacturer) were conjugated to Exo-Flow FACS magnetic streptavidin 9.1 μm beads to allow for the efficient capture of exosomes expressing these surface markers. Then 70-μg exosomes were captured, stained with fluorescein isothiocyanate, and subjected to FACS analysis. As a control, beads were treated with all reagents in the kit, but instead of exosomes, plain PBS was added.

We also performed western blotting to detect the presence of exosomal surface markers. Semen exosome samples were used as a control. Exosome samples were lysed and approximately 20 μg of each sample was run on SDS–PAGE, transferred and blotted with exosome marker antibodies CD63 (SBI, Palo Alto, California, USA, cat EXOAB-CD63A-1) and CD81 (SBI, cat EXOAB-CD81A-1). As a negative and a purity control, filters were blotted with the ER-derived marker calnexin (Santa Cruz Biotechnology, Santa Cruz, California, USA, cat sc-11397).

Cells and HIV-1 vector

293T/17 cells were purchased from the American Type Culture Collection. Vesicular stomatitis virus (VSV) G-pseudotyped HIV-based vector was described previously [19,20]. Vector DNA was obtained from Addgene (cat 8455, 8454, and 19319).

Infections and quantitative analysis of viral entry

The current analysis was done following established protocols [12]. Briefly, 293T/17 cells were infected in the presence of either VFE or controls. To prepare for infection, 50 or 90 μl of vaginal fluid exosomal fraction (corresponding to approximately 170 μg of protein, which was isolated from an equivalent of 630 μl of vaginal fluid following removal of cellular debris) or controls (PBS, or the ExoQuick reagent diluted in PBS) were mixed with the GFP-carrying HIV-1-based vector (see above) in a total volume of 0.6 ml with 6-μg DEAE-dextran. After exosome/vector incubation at 37°C for 1 h, mixtures were applied to cells (plated the day before at a density of 105 cells per well in 12 well plates) for a 6-h infection. For the quantitative experiment in Supplementary Fig. 1, https://links.lww.com/QAD/A974, the experiment was scaled down for infections in 48-well plates. To quantify the intracellular p24 levels, 6-h postinfection cells were harvested and lysed, and equivalent amounts of lysate of each sample were then subjected to western blotting analysis with the p24 antibody (cat ab63913; Abcam, Cambridge, Massachusetts, USA). Radiograph images (p24 bands) were then quantified using the ImageJ software (https://imagej.nih.gov/ij).

Real-time PCR analysis of vector DNA copies in infected cells and Alu-PCR

These experiments were performed as described previously [21,22].

Transmission efficiency

Cells were assayed 3–5 days post infection by FACS using a BD LSRII (BD Biosciences, San Jose, California, USA) to quantify GFP reporter gene expression.

Results

We have isolated the exosomal fraction of human vaginal fluid samples as described in the Materials and methods. The exosomal fraction was then examined using both flow cytometry and western blotting analysis for the presence of the tetraspanin proteins CD9, CD63, and CD81, which are established exosome markers [23]. As shown in Fig. 1a and b, the exosomal fraction contains particles containing the human exosomal markers CD9 and CD63 proteins on their surface. These results were confirmed by western blotting, which also showed the presence of CD81 in the exosomal fraction (Fig. 1c). In contrast, the samples are devoid of the presence of the endoplasmic reticulum-derived marker calnexin, which would otherwise indicate contamination with cells and cell debris (Fig. 1c).

F1-6
Fig. 1:
Analysis of the exosomal fraction of human vaginal fluid.(a) and (b) Detection of exosomal markers on the surface of particles isolated from vaginal fluid by flow cytometry analysis. Exosomes were purified from vaginal fluid as described in the Materials and methods, and surface markers were detected using an Exo-Flow Kit from SBI. Biotinylated capture antibodies CD9 (a) or CD63 (b) were conjugated to streptavidin beads to allow for the efficient capture of exosomes expressing these surface markers. As a control, beads were treated with all reagents in the kit, but instead of exosomes, plain PBS was added. A shift in GFP intensity was observed when vaginal fluid exosomes were captured with the CD9 antibody [(a), green histogram] and with the CD63 antibody [(b), green histogram] when compared with PBS controls (white histograms). (c) Detection of exosomal markers in the exosomal fraction of vaginal fluid using western blotting analysis. Exosomes were purified from vaginal fluid as described in the Materials and methods. As a control, semen samples, which are known to contain exosomes, were also purchased from Lee Biosolutions and exosomes isolated the same way. Samples were lysed and approximately 20 μg of each sample was run on the SDS–PAGE, transferred and blotted with exosome marker antibodies CD63 and CD81. As a negative and a purity control, filters were blotted with the ER-derived marker calnexin, which should be present only in cell lysates, and not the exosomal fraction. CL, cell lysate from 293T cells; SE, semen exosomal fraction; VF1, vaginal fluid exosomal fraction (sample 1); VF2, vaginal fluid exosomal fraction (sample 2, independent purification); CD63, CD63 protein; CD81, CD 81 protein; CXN, calnexin.

The exosomal fraction (now termed VFE) was then evaluated for its ability to affect HIV-1 infection using HIV-1 replication-defective vectors. We have first examined the VFE effect on the entry of the vector particles into cells. As shown in Fig. 2a, the efficiency of HIV-1 vector entry into target cells was not significantly different in the presence or absence of VFE, as evaluated by the presence of intracellular p24. We then examined the levels of total viral DNA and integrated viral DNA using our established quantitative real-time PCR assays. We observed that both integrated viral DNA and total viral DNA were decreased by 47 and 58.4%, respectively, when compared with controls (Fig. 2b and c). These latter results correlated with an overall drop in transduction efficiency (Fig. 2d). Similar results were obtained with four independently isolated samples in corresponding independent experiments, and also with semen exosomes (Fig. 2e and not shown). We note that, in Fig. 2e, we have used increasing amounts of exosomes (increasing by factor 2). We note that, at the highest concentration of VFE (column 2, which contains twice as much VFE as used in experiments described in Fig. 2a–d), we observed that VFE have a cytotoxic effect. We have not observed this effect at lower concentrations of VFE. To determine if our findings were cell type-specific, we also examined VFE effects on HIV-1 transmission in Jurkat Clone E6-1 T cells (ATCC, Manassas, Virginia, USA). We observed that the presence of VFE resulted in a similar reduction of HIV-1 transmission in these cells (data not shown). The latter results are consistent with those obtained on 293T cells (Fig. 2d and e). One possible issue with VFE is the purity of the exosomes. To purify VFE, we have used the ExoQuick method, which provides high exosome yields, but these also contain significant amounts of impurities [24]. To address this issue, we have employed an alternative purification method, which employs size exclusion columns [24]. We note that this method was demonstrated to yield exosomes of purity higher than the ExoQuick or even density gradient methods [24]. As shown in the Supplementary Fig. 1A, https://links.lww.com/QAD/A974, we have observed that VFE purified by the size exclusion column-based method suppress the vector transmission by 40% compared with negative controls, which is consistent with the ExoQuick results (Fig. 2d). In addition, we have observed comparable results when we used this method to purify semen exosomes (Supplementary Fig. 1B, https://links.lww.com/QAD/A974).

F2-6
Fig. 2:
Analysis of the vaginal fluid exosomes effect on the steps of the life-cycle of the HIV-1 vector.(a) Analysis of the vaginal fluid exosomes effect on the entry step. This analysis was essentially done as described [11]. Briefly, 293T/17 cells were infected at an m.o.i. of 0.1 with vaginal fluid exosomes or controls. Cells were then washed and trypsinized to remove extracellular proteins, as described [11]. The intracellular p24 levels were then quantified using western blotting analysis and ImageJ analysis. M, mock-infected cells; P, cells infected with vector and PBS control; E, cells infected with vector and diluted ExoQuick reagent control; VF, cells infected with the vector and vaginal fluid exosomes. Relative density in %. (b) Real-time PCR analysis of total virus DNA copies in infected cells. Cells were infected as described in the Materials and methods. One-day postinfection DNA was extracted from infected cells and vector DNA copies quantified using real-time PCR. Real-time PCR was performed as described in the Materials and methods. Samples labeled as in (a), V – cells infected with vector only. (c) Analysis of the integrated virus DNA in vaginal fluid exosomes treated cells. Infections were performed as in (b). Three days postinfection, genomic DNA was extracted. A nested Alu-PCR technique was conducted as described in the Materials and methods and by us previously [21,22]. Samples labeled as in (b). (d) Analysis of the HIV-1 mediated transduction of the GFP marker. Infections were performed as described in the Materials and methods. Fluorescence-activated cell sorting was performed 3 days postinfection to quantify GFP expression. Samples labeled as in (b). Error bars represent SDs. (e) Repeated analysis of the HIV-1 mediated transduction of the GFP marker in the presence of increasing amounts of vaginal fluid exosomes. The highest amount is equivalent to double that used in (d). Samples labeled as in (d), except the following: one – vaginal fluid exosomes used at the concentration that was used in (a)–(d), one/two – vaginal fluid exosomes diluted twofold, two – concentration of vaginal fluid exosomes increased twofold in comparison with (a)–(d).

Discussion

HIV-1 infection is a complex process that involves extensive intercellular communications. Very recently, new data appeared to suggest that this intercellular communication network might involve a new component, the exosome [10–12]. Very recent results by other laboratories have shown that human semen and breast milk contain exosomes that have inhibitory effect on HIV-1 replication. The results of our experiments suggest that HIV-1-inhibiting exosomes are also present in the human vagina.

Our results, obtained with replication-defective virus, suggest that VFE blocked a postentry step, most likely reverse transcription. The observed decrease in the integration efficiency is likely because of the decrease in reverse transcription. We did not observe any effect of exosomes on the entry step of the vector. However, we note that our HIV-1 vector is pseudotyped with the VSV G envelope, and we thus cannot exclude the possibility that VFE also block the entry of wild type HIV-1 virus. We also note that semen exosomes appear to inhibit HIV-1 infection at the reverse transcription step [10,11]. Extensive analysis will be needed to determine whether VFE represent a species of exosomes different from semen exosomes or breast milk exosomes or whether they represent a common defense against HIV-1 infection.

Finally, vaginal transmission plays a crucial role in the spread of HIV-1. Consequently, a number of vaginal microbicides are under development. The presence of vaginal natural defenses, such as VFE, suggests that these new drugs may need to be tested for their ability to synergize with VFE.

Acknowledgements

This study was supported by the NIH R21AI102684 grant to R.D.

Author's contributions: All experiments were performed by J.A.S., within the exception of the western blotting analysis, which was performed by R.D. The study design: R.D. manuscript was written by both J.A.S. and R.D.

Conflicts of interest

There are no conflicts of interest.

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Keywords:

exosome; HIV; innate defense; vaginal fluid; women

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