Targeting Trojan Horse leukocytes for HIV prevention

Introduction ….is sure design'd, by fraud or force: trust not their presents, nor admit the horse. Virgil, Aeneid Human immunodeficiency virus type 1 (HIV-1), the lymphotropic virus that causes AIDS, has infected more than 60 million people worldwide since its clinical appearance in 1981. Despite intensive prevention efforts, the HIV/AIDS epidemic continues to spread, particularly in developing countries in sub-Saharan Africa, southeast Asia and the Caribbean, as well as the developed world 1. Although HIV can be transmitted very efficiently parenterally, the advent of routine blood screening prior to transfusion and harm reduction programs for injection drug users, have made this mode of transmission much less common than mucosal transmission. Most new HIV infections are attributable to mucosal transmission: through genital and rectal mucosae in the case of sexual transmission and through oral or gastrointestinal mucosae in the case of mother-to-child transmission 2. Much has been learned about HIV pathogenesis and infection mechanisms at the molecular level, but the scientific community has yet to develop an effective vaccine or microbicide for HIV prevention. Many unanswered questions remain concerning HIV-1 sexual transmission. In 1983, barely 2 years into the AIDS epidemic, we hypothesized that the agent that was subsequently identified as HIV-1 may be sexually transmitted by infected 'Trojan Horse' leukocytes in semen 3. This hypothesis was based on our knowledge at the time that human semen contains substantial numbers of T lymphocytes and macrophages, which could host a T-cell tropic virus, and the following assumptions: intracellular virus would be better protected than free virus from adverse effects of antiviral factors in the genital environment such as antiviral antibodies likely to be present in genital secretions of the virus-infected transmitter, as well as antimicrobial peptides that play an important role in genital innate immune defense; and virus-infected allogeneic cells could also escape early detection by major histocompatibility complex (MHC)-restricted cytotoxic T cells in a new host. Over the intervening 25+ years, others have also championed this cause 4,5, and convincing evidence has emerged from clinical research as well as in-vitro and animal studies that infected leukocytes indeed play a role in HIV transmission. Yet, most recent research on sexual HIV transmission has focused on cell-free HIV in genital secretions because of the wide availability of HIV RNA quantification assays. Furthermore, the majority of HIV vaccines and microbicides have been designed to block transmission of cell-free virus and have been tested in animal and in-vitro models that use cell-free virus as the only infectious inoculum. As the molecular events underlying cell-associated HIV transmission differ from those underlying cell-free virus transmission, many of the current vaccine and microbicide candidates might not be expected to protect against cell-associated HIV transmission. The failure of several recent vaccine and microbicide clinical trials may be due in part to this oversight. It should be possible to design strategies that block cell-associated HIV transmission as well as cell-free HIV transmission. In this article, we present an overview of research that has been conducted on cell-associated HIV mucosal transmission and recommendations for future research. We focus on sexual HIV transmission, but this review also has relevance for mother-to-child HIV transmission, which may occur through mucosal transmission of cell-associated HIV from maternal genital or mammary gland secretions 6–8. We review published reports that describe and enumerate HIV-infected cells in genital secretions, and compelling evidence from clinical, animal and in-vitro studies demonstrating that such cells can transmit HIV across genital tract epithelial surfaces; potential molecular mechanisms underlying cell-associated HIV transmission that could be specifically targeted by future HIV prevention strategies; and in-vitro and animal cell-associated HIV transmission models currently used for studies on cell-associated HIV transmission mechanisms and for testing vaccine and microbicide candidates. Using this information as a foundation, we discuss the evidence and probability that various current microbicide and vaccine approaches prevent cell-associated HIV transmission, and suggest additional microbicide and vaccine concepts and experiments that will move this field forward. Putative cellular vectors of HIV mucosal transmission Seminal leukocytes Cell populations The principal cell types in human semen are spermatozoa, immature germ cells, and white blood cells (WBCs) (Fig. 1). WBCs enter semen from various sites along the reproductive tract, including the rete testis, epididymis, prostate, and urethra, where they play an immunosurveillance role 9. WBCs in semen have been enumerated and characterized by immunohistology and FACS analysis. Most of these studies indicate that semen from healthy non-HIV-infected men contains on the order of 105 WBCs/ml, the majority of which are polymorphonuclear leukocytes, although substantial numbers of macrophages and CD4+ T cells are also present 10–14. Macrophages usually outnumber CD4+ T cells in semen. This is especially the case in HIV-infected men in whom seminal CD4+ lymphocytes are depleted; in one study of 98 antiretroviral therapy (ART)-naive HIV-positive men, the median ratio of macrophages to CD4+ lymphocytes in semen was 22: 1 15 (Table 1). These data indicate that macrophages are the most abundant HIV-susceptible host cell in semen and a likely principal mediator of cell-associated HIV transmission.Fig. 1: Leukocytes in human genital secretions, identified by immunohistochemistry. (a) CD4+ T cell in semen. (b) CD68+ macrophages in semen. (c) CD45+ leukocytes in semen from a man with leukocytospermia. (d) CD68+ macrophages in cervicovaginal secretions. Magnification ×400.Table 1: White blood cell concentrations in semen.Concentrations of WBCs in semen are highly variable. Leukocytospermia, an asymptomatic genital inflammatory condition characterized by more than 106 WBCs/ml semen 16,17 occurs in approximately 5–10% of healthy non-HIV-infected men 18–20 and as many as 24% of HIV-infected men 21. Leukocytospermic semen contains substantially elevated concentrations of macrophages and CD4+ T cells 22. In some HIV-positive leukocytospermic men, the seminal macrophage cell count has exceeded 107 cells/ml and the CD4+ T-cell count exceeded 2 × 106 cells/ml (these cases are described in more detail below). HIV-infected cells have also been detected in pre-ejaculatory fluid, a urethral secretion secreted from the glands of Littre and Cowper glands during sexual stimulation, and these may also facilitate the sexual transmission of HIV 23,24. Other important HIV-susceptible host cells such as dendritic and Langerhans cells have not been detected in semen, although it is possible that some viable HIV-infected Langerhans cells from penile skin, especially the inner foreskin 25–27, are shed in the vagina or rectum during intercourse. Prevalence and quantity of HIV-infected leukocytes in semen Most quantitative studies of HIV in semen have used commercially available HIV RNA assays to measure copy numbers of cell-free virions in seminal plasma; only a few have used HIV DNA PCR assays to assess the prevalence or number of HIV-infected cells in semen. In these studies, the prevalence of HIV proviral DNA in semen samples has ranged from 21 to 65% and the amount of HIV DNA has ranged from not detectable to 80 000 copies/ml (Table 2) 28–38. Interestingly, in two of the larger studies that assessed both HIV RNA and DNA copy numbers in semen, these two parameters were not correlated 34,35. Elevated proviral HIV DNA levels in semen have been associated with reduced peripheral CD4 cell counts 32, acute HIV infection 39, leukocytospermia and recent sexually transmitted infection (STI) 32,40, and vasectomy 34. After initiation of HAART, levels of both HIV RNA and DNA are reduced in semen, although HIV proviral DNA-bearing cells can persist in semen for several months 35,37 and have been shown to be infectious in vitro33.Table 2: Studies on HIV DNA in semen.The percentage of HIV-infected WBCs in semen has not been previously determined. To perform this calculation, we returned to a database that was used in a publication on factors associated with elevated HIV proviral DNA levels in semen 32. Semen from 38 HIV-positive men from this study had measurable levels of both HIV DNA and HIV-susceptible host cells (macrophages and CD4+ T cells, quantified by immunohistology); making assumptions that only a single HIV DNA copy was present in each infected cell and that only macrophages and CD4+ T cells were infected, the median infection rate of this seminal HIV-susceptible host cell population was 0.2% (range 0.002–16%). Infectiousness of semen cells Since the pioneering discovery in 1983 that HIV-1 could be cultured from seminal cells 41, a number of laboratories have cultured HIV from both seminal cells and cell-free seminal plasma (Table 3) 42–53. Overall, the recovery rate of infectious HIV from seminal cells has been much higher (median 20%, range 4–55%) than that from seminal plasma (median 5.9%, range 3–11%, P < 0.0001). The relatively low HIV recovery rate from seminal plasma contrasts with quantitative PCR data indicating that HIV prevalence rates and viral copy numbers are higher in seminal plasma than in the semen cell fraction 14,34–36. This discrepancy suggests that much of the cell-free HIV in semen is replication incompetent or inactivated. A number of factors have been identified in seminal plasma that may inactivate HIV, including anti-HIV antibodies 54,55, X4/R5 chemokines 20, SLPI, lactoferrin, and defensins 56. The low culture rate could also reflect the toxicity of seminal plasma to peripheral blood mononuclear cell (PBMC) target cells used for culturing HIV 57–61.Table 3: HIV culture rate from semen fractionsa.Factors that affect the abundance and infectiousness of HIV-infected leukocytes in semen Although WBCs can be detected in semen from virtually all men, several factors may affect the types, abundance, and infectiousness of WBCs in semen. Symptomatic bacterial genital tract infections and inflammation are often associated with increased urethral/seminal WBC numbers 62,63. However, chronic asymptomatic genital viral infections do not generally produce elevated seminal WBC counts 64,65, and as mentioned above, HIV infection appears to deplete CD4+ and CD8+ lymphocytes in semen 15,66, an effect partially reversed by antiretroviral therapy 15. Epidemiologic studies indicate that STIs substantially enhance HIV transmission 67,68. Urethritis caused by Neisseria gonorrhoeae was associated with a 10-fold increase in HIV RNA copy numbers in semen, which declined following successful antibiotic treatment 69. Other studies have demonstrated increased HIV RNA shedding from genital ulcers caused by various STI pathogens 70–72. Most of these studies have only measured cell-free HIV RNA, but because symptomatic infections and inflammation are associated with elevated WBC levels in semen, it is probable that the number of HIV-infected cells in semen is also increased. One study to date has shown that both HIV RNA and proviral DNA levels were elevated in semen from men with a recent STI 40. Elevated polymorphonuclear leukocyte (PMN) counts and leukocytospermia have also been associated with increased levels of both cell-free and cell-associated HIV in semen 32,45,73, as well as increased levels of IL-1β, TNF-α, IL-6, and other proinflammatory cytokines that could activate HIV replication in infected cells 20,65,74. Some men may be particularly contagious due to abnormally elevated seminal leukocyte counts. In one study from our laboratory, semen samples from two HIV-positive persons without STI symptoms contained 15–25 million macrophages and 2–6 million CD4+ T cells per ml (the average human ejaculate comprises 2.5 million per ml). In addition, their semen was highly infectious when cultured with PBMC target cells 15. Both of these men had advanced HIV disease in the pre-HAART era and had high peripheral blood viral loads. Cases such as these may play an important role in the HIV epidemic. Men with acute HIV infection also have high levels of HIV RNA in semen, and epidemiologic studies indicate that they are highly infectious 75,76. Only one study thus far has measured HIV proviral DNA levels in semen of acutely infected men; 10 out of 13 samples from three HIV-infected men within 80 days of initial infection tested positive for HIV DNA 39. More research is needed to determine whether HIV-infected WBCs in semen contribute to the highly contagious profile of this group. HIV transmission by spermatozoa The question of whether spermatozoa transmit HIV infection has been controversial for several years 77–79. HIV and simian immunodeficiency viruses (SIV) apparently infect testicular germ cells 80–82, and early electron microscopy and in-situ hybridization studies provided evidence that human spermatozoa may contain HIV viral particles or RNA 83–85. However, these findings have not been confirmed 78,86, and most recent studies using PCR techniques have not detected HIV infection of viable spermatozoa 79,87. Viable, motile spermatozoa from HIV-infected men, separated from other cell types in semen by density gradient centrifugation and/or swim-up techniques, rarely contain detectable amounts of HIV DNA or RNA 31,36,79,86,88–96. Occasional positive results may be due to contamination of the sperm pellet with infected leukocytes or false-positive PCR reactions, or could indicate that HIV infection of sperm occurs but is exceedingly rare. We measured HIV DNA in isolated cell populations from semen of HIV-infected men and detected HIV DNA in immunobead-purified macrophage and CD4+ T-cell populations, but not in motile sperm 31. In the same study, we also compared the relative infectiousness of cell populations from semen of HIV-positive men and found that isolated CD4+ T cells and macrophages were highly infectious when cultured with PBMC target cells in vitro, whereas motile sperm from the same participants were not infectious 31. Reports from Assisted Reproduction Clinics that have used isolated motile sperm from HIV-infected men to inseminate HIV-uninfected partners provide further evidence that motile sperm are not infectious. Over 4500 inseminations have been performed with processed sperm from HIV-infected men without infection of the seronegative partners 96–105. However, even in light of substantial data to the contrary, one cannot conclude that sperm never transmit HIV following natural intercourse. As mentioned above, occasional detection of HIV DNA in purified sperm preparations could indicate rare HIV infection of sperm. Furthermore, several groups have reported that HIV virions can bind to sperm through mannose or glycolipid receptors 85,106–111. This interaction may be missed with processed sperm, as loosely-attached HIV may be stripped-off by gradient separation protocols, but this association could be relevant following normal intercourse as sperm could transport HIV to host cells in the lower as well as upper urogenital tract. In a recent study 112, abnormal/immotile ejaculated sperm from HIV-infected men were found to contain HIV DNA, suggesting that HIV-infected testicular germ cells produce immotile/nonviable sperm. These defective sperm could potentially introduce HIV to phagocytic macrophages or other cells in the female genital tract after intercourse 105,113. Leukocytes in female genital secretions Cell populations Several studies have documented HIV-susceptible host cells in vaginal and cervical tissue (described below), but few have quantified or characterized these cell populations in human vaginal and cervical secretions. Macrophages and CD4+ T cells are often detectable but not numerous in cervicovaginal secretions from healthy uninfected 114,115 or HIV-infected women 116 (Table 4) 117. The viability of lymphocytes in vaginal secretions from healthy women is usually poor, probably due to the toxic effects of low pH conditions commonly found in the human vagina 118.Table 4: WBC concentrations in cervicovaginal secretions.Leukocyte counts are elevated in cervicovaginal secretions of women with certain STIs. Neisseria gonorrhoeae and Chlamydia trachomatis infections can induce massive inflammatory infiltrates 119. In contrast, bacterial vaginosis appears to have little or no effect on vaginal leukocyte counts 119–121, but these cells could have improved viability and higher infectiousness due to near neutral pH associated with this condition. Prevalence and quantity of HIV-infected leukocytes in female genital secretions Several studies on HIV in vaginal secretions have used qualitative HIV DNA assessment as an endpoint. An increased prevalence of HIV DNA in vaginal secretions has been associated with cervicitis, candidiasis, and STIs 122–133, hormonal contraception 129,134, and vitamin A or selenium deficiency 129,135–137. The prevalence of HIV-infected cells in vaginal secretions is reduced in women on antiretroviral therapy 138,139. Only a few studies have quantified HIV DNA in cells from cervicovaginal secretions 7,131,140–145 (Table 5). In these studies, maximum HIV proviral copies were on the order of 104 per lavage (103 copies/ml lavage fluid). Our laboratory quantified HIV RNA and DNA in cervicovaginal secretions from women in the WITS cohort during the third trimester of pregnancy; levels of HIV DNA, but not RNA, and proviral heterogeneity were positively associated with perinatal HIV transmission 7,140.Table 5: Studies on HIV DNA in cervicovaginal secretions.Infectiousness of cervicovaginal leukocytes Early studies on HIV isolation from cervical swabs did not separate cells from cell-free fractions; the culture rate averaged 43% 140,147–150. Due to heavy contamination of vaginal lavages with endogenous bacteria and fungus, HIV culture is now usually conducted with filtered cell-free fractions, yielding culture rates ranging from 11 to 22% 147,151,152. Only one study to date has compared the HIV culture rate from cell-free vs. cell-associated fractions of cervicovaginal lavage samples: HIV was cultured from 12 of 55 (22%) cell-free supernatants and five of 22 (23%) cell lysates 147. Although correlates of HIV culture from cervicovaginal cell pellets have not been studied, it is possible that HIV-infected leukocytes from reproductive aged women with normal vaginal flora are inactivated by lactic acid produced by lactobacilli and are, therefore, less infectious 118. We predict that HIV-infected genital leukocytes from women with neutral vaginal pH due to conditions such as bacterial vaginosis and low estrogen states 153 are more infectious than those from reproductive aged women with vaginal pH in the 3.5–5.0 range and are more capable of cell-associated HIV transmission. Recently a sensitive short-term MAGI culture assay was used to improve the detection rate of infectious HIV in filtered female genital secretions. Although the overall culture rate was 51%, there was only a weak correlation between MAGI plaque (infectious virus) numbers and HIV RNA viral load. In addition, 10 out of 32 women with more than 10 000 HIV RNA copies/lavage had undetectable levels of infectious HIV in the MAGI plaque assay. The investigators speculated that the discrepancy may indicate inactivation of cell-free virus in genital secretions, possibly by neutralizing antibodies, low pH or innate immune mediators 152. These data support the potential importance of cell-associated HIV transmission. HIV target cells in genital mucosae Following vaginal intercourse, HIV from an infectious partner enters an environment that contains a multitude of factors contributed from both male and female genital secretions. (The rectal environment is not as well studied but would be expected to contain many of the same components.) As discussed above, several factors in semen and cervicovaginal secretions (antimicrobial peptides, X4/R5 chemokines, anti-HIV antibodies) can inactivate cell-free HIV, but may not affect HIV-infected cells. Factors in this environment that have been determined to potentially affect cell-associated HIV transmission are mucins, large hydrophilic molecules that lubricate and protect genital mucosal epithelia, and endogenous vaginal lactobacilli that produce lactic acid to maintain a low pH 56. In an in-vitro model system, lymphocytes and activated seminal leukocytes were able to traverse midcycle cervical mucus, although they failed to penetrate thicker substrates representing the viscosity of mucus present during the luteal phase of the menstrual cycle and pregnancy 154. Macrophages and T cells were immobilized and eventually killed by low pH conditions commonly found in the human vagina 118. However, after intercourse, the pH of cervicovaginal secretions is neutralized for several by the of seminal plasma seminal and cervicovaginal leukocytes a of to the target genital Furthermore, bacterial vaginosis and conditions underlying and states are also associated with elevated vaginal pH levels it appears probable that infected leukocytes in genital secretions can remain viable at for several after intercourse in healthy reproductive aged women and in women with bacterial vaginosis and other conditions associated with elevated vaginal and are capable of HIV through genital secretions to the epithelial such as those and foreskin are of a epithelial whereas such as those penile urethra, and mucosae of a of epithelial cells. In the epithelial is infectious such as HIV traverse or target cells within the epithelial electron microscopy studies have demonstrated that HIV-infected T cells and bind to mucosal epithelial cells, and that their of HIV the epithelial where virions can within and enter within epithelial cells (Fig. 2) virions may be by epithelial cells to an to infect an target cell which could be to the through of chemokines or other proinflammatory by the cell and/or epithelial cell or virions may be across epithelial cells to infect cells in the We and others have also shown that macrophages and T cells can and epithelial (described in more detail infected with HIV, infect cells within or the 2: HIV-infected leukocyte interaction with epithelial and viral (a) electron HIV-infected lymphocytes to the of an epithelial cell (b) electron of HIV-infected macrophage from semen virus after with a genital tract epithelial cell provided by with from the (a) from is as well as and in the density of the leukocyte cell populations that may as HIV target cells in genital mucosae are usually few CD4+ T cells within the epithelial although they can be abundant inflammatory However, macrophages and Langerhans cells are abundant within and can potentially be infected by HIV and/or transport virus to target cells in The that the epithelial and that into the contain numerous HIV-susceptible host cells macrophages, and dendritic the from to cervical at the of the penile contain an especially population of HIV target cells HIV may also infect target cells in the and of HIV cells in the genital are substantially increased during In addition, use of such as the can the genital and rectal in inflammation and of macrophages, and other cells into the epithelial and secretions After intercourse, numbers of HIV-susceptible host cells are increased in cervicovaginal tissue and secretions, and potentially in rectal and secretions, due to chemokines and other in semen and effects of semen on mucosal epithelial cells These conditions would be expected to enhance cell-associated HIV transmission. for cell-associated HIV transmission studies The sexual transmission of HIV is a rare for the probability of HIV transmission per range from 1 in for transmission, 1 in 000 for transmission, and 1 in for transmission Studies on the of HIV from blood of infected with HIV-1 indicate that in the majority of of the transmission a single virus from an infected partner is for clinical infections This suggests that HIV is usually transmitted a single HIV or infected A transmission has been in studies of and STI where can an infection in the probably due to of the mucosal and/or increased numbers of HIV target cells at the infection HIV in semen often differ from those in peripheral blood and at two studies provide evidence that of cell-free HIV differ from those of cell-associated HIV in semen therefore, should be possible to determine whether the initial transmission is by a cell-free or an HIV-infected out to between these in acute and found that the of the virus that of HIV in semen cells of the in three out of five cases and two male More studies of this are needed to determine the prevalence and factors of cell-associated HIV transmission. Other evidence that seminal leukocytes can the vaginal in is provided by a study that that intercourse an allogeneic in women that is for their sexual human leukocyte This was not in that used and is likely by to seminal leukocytes because sperm do not As the human vagina is a for immune and the allogeneic was detected in peripheral it is probable that the leukocytes the mucosal to an immune in A weak but was also in the male partners and could be to to vaginal In this study, from women with allogeneic HIV-1 infection of activated T cells from their evidence that allogeneic could protect against cell-associated HIV transmission. studies immunodeficiency virus model The animal model of cell-associated transmission across vaginal and rectal mucosal was the immunodeficiency virus infection a with to HIV, T cells and causes an immunodeficiency disease in was one of the animal models used to mechanisms of HIV transmission and pathogenesis can be transmitted of infected T cells or cell-free virus vaginal or rectal and this model was used to the of early microbicide candidates against vaginal and rectal transmission of cell-associated The model has also been used for vaccine the numerous of developing a vaccine to protect against a T-cell tropic CD4+ T-cell of viral in