Abstract

The goal of the present study was to develop a nonhuman primate model of intravaginal human immunodeficiency virus (HIV) transmission with cell-associated virus. Reproductively mature, cycling cynomolgus macaques with or without chemically induced, transient ulcers of the lower female reproductive tract repeatedly received challenge with a variable amount of in vitro simian immunodeficiency virus mac239–infected peripheral blood mononuclear cells. Persistent viremia was established with surprisingly few infectious lymphocytes containing physiologically relevant quantities of cell-associated virus. This model will be indispensable for the testing of vaccines and topical agents that are aimed toward the prevention of heterosexual transmission of HIV

Young women are the most vulnerable segment of the population affected by the AIDS pandemic [1]. Medical conditions that compromise the tight epithelial barrier of vaginal and cervical mucosa have been implicated in increased rates of HIV transmission [2]

The semen of HIV-infected men can contain both cell-free and cell-associated virus, the latter of which is associated with CD4+ T cells and macrophages [3]. In urogenital inflammations, even in asymptomatic persons, the concentration of lymphocytes can be up to 106 cells/mL of ejaculate, and the concentration of macrophages can be up to 107 macrophages/mL of ejaculate [4]

Models of in vivo transmission show that as few as 2 infected peripheral blood mononuclear cells (PBMCs) can transmit simian immunodeficiency virus (SIV) via the intravenous route [5]. In vitro assays involving human cervical cell lines, as well as tissue explants, have provided evidence that transmission of HIV with cell-associated virus is possible if there is a breach in the mucosal integrity [6, 7]. SCID mice reconstituted with human PBMCs also support the concept of male-to-female heterosexual transmission of HIV via cell-associated virus [8]. However, to date, nonhuman primate models have delivered contradictory results. In one study in which 4 chimpanzees were exposed to cell-associated HIV-1LAI (subtype IIIB), one animal developed persistent viremia, and another animal developed transient viremia [9]. In another study that involved rhesus macaques and cell-associated SIVmac251, all the animals remained virus free [5]. Because the menstrual cycle and female reproductive tract are similar in nonhuman primates and humans, any result derived from nonhuman primate models should be relevant to HIV transmission in humans

Understanding all the mechanisms employed by HIV during heterosexual transmission of the virus is critical for designing effective preventive strategies. In the present study, we demonstrate that intravaginal transmission of SIV is possible with cell-associated virus in nonhuman primates and that systemic viremia is established with an unexpectedly small number of infectious cells

Animals, materials, and methodsMature, female cynomolgus macaques (Macaca fascicularis) were housed at the Wisconsin National Primate Research Center at the University of Wisconsin–Madison, according to guidelines from the National Institutes of Health (NIH) regarding the care and use of laboratory animals [10]. All procedures were performed on anesthetized animals according to an experimental protocol approved by the Research Animal Resource Committee of the University of Wisconsin–Madison

The lower female reproductive tract was inspected for signs of preexisting inflammation, by use of a rigid fiberoptic scope. Such signs included heavy vaginal discharge, cervical erythema, or disruption of the mucosal epithelium. After inspection, the vaginal lumen was filled with 1 mL of surgical lubricant that contained 3% benzalkonium chloride (Sigma). The animals were kept in prone position for 15 min. One application of the detergent resulted in visible ulceration 24 h later, which was detected by fiberoptic scope. The location of the ulcers was limited to the entry of the vagina and to the cervix. A total of 200 μL of inoculum that contained cell-associated virus was deposited on the ulcer at the vaginal entry by use of a sterile plastic 1-mL pipette. To avoid possible trauma to the mucosal epithelium, control animals were challenged with virus without visualization of the reproductive tract by fiberoptic scope. Viral inocula were deposited in the vaginal entry. All animals were kept in a supine position for 15 min before anesthesia was reversed

Samples of mucosal secretion were collected and were stored at −80°C by use of premoistened Weck-Cel sponges (Medtronic). Secretion fluid was retrieved from the sponges by means of centrifugation performed at 4°C. The sponges were washed with 200 μL of PBS and underwent centrifugation again. Cytokine levels were determined using the BD Cytometric Bead Array Non-Human Primate Th1/Th2 Cytokine kit (BD), according to the manufacturer’s instructions, by use of the FACScalibur flow cytometer and BD CBA software

PBMCs obtained from an uninfected donor animal were stimulated overnight in vitro with 5 μg/mL phytohemagglutinin (Sigma) and then were incubated with SIVmac239 at an MOI of 5×10-5 for 4 h. The cells were washed twice, seeded in a 24-well plate at a concentration of 106 cells/well, and incubated for 7–9 days in the presence of 50 U of interleukin (IL)–2 (the source of which was the NIH AIDS Research and Reference Reagent Program). Before inoculation, dead cells were removed by ficoll-Histopaque (Sigma) density-gradient centrifugation. To remove cell-free virus, the cells were washed 2 times. A fraction of the inoculum was processed for intracellular SIVmac239 Gag p27 analysis, as described elsewhere [11]

Serial 2-fold dilutions (in duplicate) of the SIV-infected PBMCs were cocultured with CEM×174 host cells in 10% FBS-RPMI 1640 medium. The cocultures were maintained for 4 weeks and were monitored weekly for the production of p27 Gag antigen by use of the SIV p27 Core Antigen ELISA kit (Beckman Coulter). The end point results were expressed as the TCID per the number of PBMCs in the inocula

Genomic DNA was isolated using the DNeasy Tissue kit (Qiagen) according to the manufacturer’s recommendations. Proviral DNA was detected by real-time polymerase chain reaction amplification of the viral gag gene, by use of the primers 5′-GTCTGCGTCATCTGGTGCATTC-3′ and 5′-CACTAGCTGTCTCTGCACTATGTGTTTTG-3′ and probe 5′-6FAM-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-TAMRA-3′. The LightCycler TaqMan Master kit (Roche Diagnostics) was used for amplification on the LightCycler 2.0, with primers at a final concentration of 400 nM and with probes at a concentration of 100 nM. Cycling conditions were as follows: 95°C for 10 s; followed by 45 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s; and a final cycle at 40°C for 30 s. Viral DNA was quantified by comparison to a standard curve of log dilutions of DNA standards ranging from 15 to 15×106 copies. Samples were run in triplicate

To determine the mean number of cells that contained proviral DNA, we used a parallel amplification of ɛ-globin. Because genomic DNA from a cell contains 2 copies of ɛ-globin, we could determine the number of cellular equivalents in each reaction by amplifying this gene. Reaction conditions were identical to those detailed above. We used primers 5′-TGGCAAGGAGTTCACCCCT-3′ and 5′-AATGGCGACAGCAGACACC-3′ and probe 5′-6FAM-TGCAGGCTGCCTGGCAGAAGC-TAMRA-3′

Plasma and cellular elements of the EDTA anticoagulated blood were separated by centrifugation performed for 10 min at 3000 rpm. The plasma was stored at −80°C, until it underwent further processing for viral load quantification. The plasma virus concentration was determined as described elsewhere [11]

ResultsChallenges of animals with cell-free virus are usually performed using frozen aliquots of defined virus stock. However, cryopreservation of SIVmac239-infected PBMCs decreases the number of CD4+ T cells by ∼30% (our unpublished observation). Freezing may also exert unknown effects on the functional integrity of adhesion receptors and cell migratory functions. Therefore, we performed viral challenges using noncryopreserved, SIV-infected PBMCs 7–9 days after initiation of the tissue culture. Because the animals were inoculated on multiple occasions, we characterized each viral inoculum

The proviral DNA content of such cultures was between 104 and 105 copies/million cells (figure 1A). The inocula mainly consisted of T cells (>90% CD3+ T cells), and 35%–45% of the cells expressed CD4 coreceptor (figure 1B and 1C). A total of 4%–10% of the cells in the lymphocyte gate were positive for the SIVmac239 Gag p27 antigen. The majority of these cells did not express detectable levels of CD3 and CD4 T cells (figure 1C and 1D), likely because of nef-mediated down-regulation of these proteins. The number of infectious cells ranged from as few as 7 cells to as many as 8192 cells, depending on the actual number of PBMCs in the inoculum (table 1)

Figure 1

Proviral DNA copy content, cell composition, and relative frequency of simian immunodeficiency virus mac239 Gag p27-positive cells of the cell-associated virus inoculum. A Proviral DNA copy content of in vitro–infected donor cynomolgus macaque peripheral blood mononuclear cells at days 7–9 after initiation of the culture. Results are the mean values (±SD) of 3 independent experiments. CD3 antigen expression (B) relative frequency of Gag p27-positive cells on the second day of viral challenge in macaques at day 8 after in vitro infection (C) and relative frequency of p27-positive cells among uninfected control cells in the same experiment (D) as assessed by fluorescence-activated cell sorter analysis. Data were acquired using the FACSCalibur flow cytometer (Becton Dickinson). Between 30,000 and 200,000 events were collected and were displayed in the lymphocyte gate, as determined by the forward and side scatter parameters. Representative plots are for 1 of 11 independent stainings. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin

Figure 1

Proviral DNA copy content, cell composition, and relative frequency of simian immunodeficiency virus mac239 Gag p27-positive cells of the cell-associated virus inoculum. A Proviral DNA copy content of in vitro–infected donor cynomolgus macaque peripheral blood mononuclear cells at days 7–9 after initiation of the culture. Results are the mean values (±SD) of 3 independent experiments. CD3 antigen expression (B) relative frequency of Gag p27-positive cells on the second day of viral challenge in macaques at day 8 after in vitro infection (C) and relative frequency of p27-positive cells among uninfected control cells in the same experiment (D) as assessed by fluorescence-activated cell sorter analysis. Data were acquired using the FACSCalibur flow cytometer (Becton Dickinson). Between 30,000 and 200,000 events were collected and were displayed in the lymphocyte gate, as determined by the forward and side scatter parameters. Representative plots are for 1 of 11 independent stainings. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin

Table 1

Parameters regarding the infectious cell content of the individual simian immunodeficiency virus mac239 cell-associated virus inocula and the presence of female reproductive tract inflammation in recipient macaques at the time of viral challenges

Table 1

Parameters regarding the infectious cell content of the individual simian immunodeficiency virus mac239 cell-associated virus inocula and the presence of female reproductive tract inflammation in recipient macaques at the time of viral challenges

Previous attempts to infect macaques with 1 intravaginal inoculation of 10,000 infectious cells yielded negative results [5]. Therefore, our viral challenge experiments included 3 animal cohorts (table 1). The first cohort was exposed to 500,000 in vitro–infected PBMCs on 3 consecutive days. The number of infectious cells in these inocula was 384–8192 cells. The second cohort was inoculated with 5000 PBMCs on 3 consecutive days (7–82 infectious cells/inoculum). The third cohort was challenged only once with an inoculum of 5000 PBMCs (4–8 infectious cells/inoculum). Each cohort involved 1 or 2 control animals and animals that had chemically induced ulcers. One of the control animals (macaque Cy0083) had heavy vaginal discharge, which is one type of evidence of preexisting inflammation of the female reproductive tract. Two of the other control animals (macaques Cy0094 and Cy0084) developed minor inflammation by the second or third day of receiving inoculations; this inflammation was only detected on the basis of slightly increased IL-6 levels in cervicovaginal secretions (table 1). Because macaque Cy0084 started menstruating on the third day of receiving inoculations, her elevated IL-6 level in cervicovaginal secretions can be attributed to this physiological event

All the macaques with ulcers became persistently infected after receiving repeated inoculations. The infection was characterized with plasma viral load kinetics typical of mucosal challenge with SIVmac239: peak plasma viral loads of 106–107 copies/mL 2 weeks after viral challenge and set point plasma viral loads of 104–106 copies/mL

Surprisingly, 2 of the control animals that were inoculated 2 times (macaque Cy0084) or 3 times (macaque Cy0094) also became infected with similar viral load kinetics. None of the animals that were exposed to inoculum only once showed signs of persistent viremia

DiscussionThe purpose of the present studies was to introduce a nonhuman primate model of intravaginal transmission of HIV with cell-associated virus. Previous reports have implicated cell-associated virus in the sexual transmission of HIV [6–8], but experiments involving nonhuman primate model systems have not provided definitive results [5, 9]. An intact genital mucosa may be a formidable obstacle to HIV, because several in vitro experiments have demonstrated the importance of the integrity of the mucosal epithelium. Indeed, previous nonhuman primate models have focused on maintaining the integrity of the mucosal epithelium during viral inoculation, which may explain their difficulty in achieving systemic infection with both cell-free and, in particular, cell-associated virus inocula. We designed a model that involved the presence of ulcers of the female reproductive tract. Unexpectedly, some of the animals that had no clinical evidence of inflammation became infected. These results suggest that although major inflammation may increase the probability of infection, it is not an absolute requisite for this route of transmission. The presence of minor epithelial trauma in the control animals cannot be entirely excluded. However, the precedent merits consideration, because it may resemble certain conditions in humans when the probability of HIV transmission might be elevated. For example, in the female reproductive tract in healthy women, minor trauma of the mucosa that was instigated or exacerbated by a heterosexual encounter is well documented in several publications [12, 13]

Our data, similar to findings from a recent rhesus macaque model using intravaginal challenges with cell-free SIVmac251, show that repeated exposures to low-dose inocula result in systemic viremia within a short time [14]. In both of these low-dose models, a high transmission rate was achieved in the absence of hormonal thinning of the vaginal epithelium, demonstrating that the thick epithelial barrier can be overcome with frequent exposures to virus [15]

Semen from HIV-infected men with urethritis can contain 10–104 copies of proviral DNA/mL of ejaculate [3, 4]. One infected cell can harbor a mean number of 3–4 copies/cell [16], suggesting that 1 mL of ejaculate could comprise 3–3000 HIV-infected cells. Our inocula of 5000 PBMCs contained up to 1.4×103 copies of proviral DNA. Because ∼4%–10% of the lymphocytes were found to be positive for the SIVmac239 Gag p27 antigen, at least 200–500 cells harbored virus in these preparations. The cell-associated viral burden of our inocula was therefore similar to that of the semen of HIV-infected men

There are several mechanisms that could promote mucosal cell-associated HIV/SIV transmission. Infected cells can migrate through epithelial abrasion, or they may adhere to the mucosal epithelium, serving as a continued source of budding virions. Uninfected, virus-trapping cells can also prolong the presence of infectious virus in the vaginal cavity by adhering to the mucosa. Undoubtedly, major inflammation of the reproductive tract creates an environment that is immunologically different than that associated with minor mucosal abrasions

In the present study, we have provided evidence for in vivo vaginal SIV transmission in a nonhuman primate species with a clinically relevant amount of cell-associated virus. We have also found that reproductive tract inflammation manifested by overt clinical symptoms is not essential for this mode of transmission. Because cell-associated HIV is particularly difficult to neutralize, our viral challenge model will be critical for the testing of preventive measures against heterosexual transmission of HIV infection

Acknowledgments

We thank the animal care and veterinary staff of the Wisconsin National Primate Research Center at the University of Wisconsin–Madison. We are indebted to Dr. David Watkins for guidance and helpful discussions

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Financial support: Wisconsin National Primate Research Center (base grant P51 RR00167)
Potential conflicts of interest: none reported