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Since the beginning of the AIDS pandemic, and following the discovery of the human immunodeficiency virus (HIV) as the etiological agent of the disease, it was clear that the virus gains access to the human host predominantly through the mucosal tissue after sexual exposure. As a consequence, the female genital tract (vaginal and cervical), as well as the rectal, penile, and oral mucosae have been extensively studied over the last thirty years towards a better understanding of - and to develop strategies to prevent - sexual HIV transmission. This review seeks to describe the biology of the events leading to HIV infection through the human mucosa and introduce some of the approaches attempted to prevent the sexual transmission of HIV.
HIV mucosal infection plays a critical role not only in virus transmission but also in AIDS pathogenesis, affecting mucosal surfaces of the gastrointestinal tract early on by depleting it of CD4+ T helper cells independently of the virus transmission route . Although current antiretroviral therapy helps controlling HIV infection in most patients, it cannot eradicate the virus from the human host . In addition, an effective HIV vaccine seems to still be difficult to achieve, at least in the near future . Therefore, the development and use of HIV microbicides (i.e., topical pre-exposure prophylaxis) has become the most promising approach to prevent HIV transmission.
Most HIV infections occur as consequence of unprotect sexual contact , with the risk of transmission being higher during anal intercourse (0.65% to 1.7%)[5, 6] than via heterosexual intercourse (0.03% to 0.5%)[4, 6]. Interestingly, HIV transmission rate through the oral mucosa varies from 0.1% to 2% during oral sex [7, 8] and it could be as high as 5% to 20% during breastfeeding [9, 10]. Here we review the multiple host and viral factors associated with HIV mucosal infection and transmission as a preamble to the numerous approaches described in this issue as current and potential HIV microbicides.
The AIDS pandemic has arisen largely through heterosexual transmission of HIV, yet the probability of infection for each encounter with the virus is quite small, i.e., less than 0.0014 based in studies of discordant couples [11-14]. This poor efficiency in viral transmission may be the consequence of low amounts of virus inoculum, restricted access to target cells, selective transmission, and/or outgrowth of a subset of viral variants within the population. There is clear evidence from cross-sectional studies of individuals with acute and early infections that transmission is associated with a population bottleneck [15-19]. Early studies of mother to infant HIV transmission first demonstrated that a restricted subset of viruses is present soon after infection [20, 21]. In these limited lineages harmful mutations accumulate over time causing the average fitness of the virus population to decline, a process commonly associated with Muller's ratchet [22-26]. A detailed molecular understanding of the transmission and the early evolution of HIV, including a precise description of the transmitted or early founder virus, is critical in the development of not only microbicides but a potential HIV vaccine .
HIV gains entry into the body mainly during sexual intercourse by crossing epithelial barriers covering the mucosal surfaces of the female and male (penis) genital tracks as well as the anal/rectal epithelia. In the female genital tract virus transmission involves early capture by epidermal Langerhans cells (LCs) in the vagina and endocervix stratified epithelia, a process facilitated by their close proximity to the mucosal surface. Langerhans cells bind the envelope gp120 of HIV through their C-type unique langerin, a process that at low viral loads might lead to the degradation of HIV [27, 28]. On the contrary, if the protective effect of langerin is inhibited due to higher viral concentrations, the internalized virus will be transferred to T-cells leading to a productive infection of these cells within the mucosae or in draining lymph nodes following migration of LCs to secondary lymphoid organs [29-31].
As described above, within hours of infection, HIV crosses the mucosal epithelial barrier to establish a founder population of infected cells [32, 33]. This population expands locally during the first week of infection generating a viral pool that establishes a self-propagating systemic infection in the secondary lymphoid organs . As virus access to more susceptible target cells increase during the second week of infection, a significant increase in replication in the lymphatic tissues occurs. The peak of virus replication in blood and tissue happens during the second week after infection before declining to stable levels around fours weeks post exposure . The infected lymphatic tissues act as reservoirs for virus production and storage as well as harboring of proviruses in latently infected cells . Interestingly, although it is estimated that a single virion is effectively transmitted, HIV diversity increases over the course of infection due to the continuous replication of the virus and the error prone nature of the reverse transcriptase . Such diversity results in natural selection caused partly by host immune response, which eventually facilitates viral persistence [15, 18].
During sexual intercourse, HIV transmission from male to female is more efficient than from female to male, thereby making women a disproportionately more susceptible population to HIV infection . As consequence, 76% of young people (aged 15–24 years) living worldwide with HIV are female with vaginal intercourse remaining the most prevalent route of infection . Vaginal and cervical epithelia are most likely the primary sites where HIV from male semen and cells from the female genital tract first encounter. Interestingly, although most studies on mucosal HIV transmission have focused on cell-free virions , all genital fluids that transmit HIV such as seminal plasma and cervicovaginal secretions also carry HIV-infected cells . Seminal plasma may contain up to 105 white blood cells/mL including substantial numbers of HIV susceptible macrophages and CD4+ T-cells . This is particularly important due to recent studies suggesting that HIV transcytosis across simple epithelia is more efficient if the viruses bud via viral synapses rather than infecting via cell-free fluids [41, 42]. In addition, several factors present in seminal plasma can either enhance or inhibit HIV infection of T-cells. For example, human plasma abrogates the capture and transmission of HIV to CD4+ cells mediated by DC-SIGN  and semen-derived amyloid fibrils drastically enhance HIV infection .
The mucosal epithelium provides a robust physical and immunological barrier that acts as the first line of defense against viral entry in the endocervix and the upper female reproductive tract. It does mediate innate defenses against microbes under hormonal control and functions by the recognition and secretion via Toll-like receptors of host-derived defensins and other antimicrobial peptides and enzymes that respond to pathogen-associated molecular patterns . Chemokines and cytokines also recruit plasmacytoid dendritic cells (pDC) that mediate innate defenses and inflammation . These defenses provide a series of inhibitory activities inhibiting HIV entry and replication. For example, SDF-1 blocks viral entry mediated by CXCR4 while MIP-1α/β and RANTES blocks CCR5-based entry . Following vaginal inoculation, MIP-3α recruits interferon (IFN)-α/β producing pDCs to the endocervix and lead to an increase in IFN-γ expression in the vaginal tissues . In non-human primates (e.g., macaques), such innate immune defenses could cumulatively lead to a limited number of infecting founder viruses despite a large dose of initial virus inoculum. Interestingly, a recent study used proteomics to identify serpin and cystatin antiproteases as host factors that could be playing a role in providing protection against HIV in the female genital tract of exposed, but uninfected, female sex workers . This would explain the low probability, although still effective and probable, of HIV transmission in humans, i.e., approximately 1 in 100 or 1,000 per coital act [12, 13].
Most HIV microbicides tested during the last 20 years have failed to demonstrate meaningful protection against HIV infection . There is no doubt that topical pre-exposure prophylaxis aimed to prevent vaginal/cervical HIV transmission would be a major step towards controlling the HIV/AIDS epidemic by allowing women self protection against HIV infection. Sub-Saharan Africa accounts for approximately 70% of the global burden of HIV and women in this region are disproportionately affected by AIDS , mostly because of their inability to successfully negotiate mutual monogamy or condom use. Although there is still a lot of work to do in order to develop effective microbicides to reduce HIV acquisition among women, promising data from the CAPRISA 004 trial showed that a 1% vaginal gel formulation of tenofovir reduced HIV acquisition by an estimated 39% overall and by 54% in women with high gel adherence . Thus, it is possible that a combination of condom use and microbicides based on one or more antiretroviral drugs could have a considerable impact in preventing HIV transmission via the female genital tract.
In addition to the vaginal/cervical epithelia, the rectal epithelium represents the predominant route of HIV transmission during sexual intercourse. Worldwide, men having sex with men (MSM) is the population group with the highest risk of acquiring HIV, including developing countries . In 2009, men having sex with men (MSM) represented close to 60% of all new HIV infections in the United States and 40% in Canada [52, 53]. However, little information is known on the anal sexual practices of heterosexual individuals and its role in the spread of HIV in sub-Saharan Africa [54-56].
The relative ease by which HIV is transmitted rectally [54-56] makes this a particularly important, although relatively neglected, route to target with prevention strategies [57-59]. The gastrointestinal mucosa is a secondary lymphoid organ that contains the majority of the body's CD4+ lymphocyte population [60, 61] and, therefore, likely represents the largest reservoir of HIV and site of viral replication compared to other body compartments. Genital and rectal sub-epithelial stromal tissues are densely populated with dendritic cells, macrophages and T cells that express CD4, CCR5 and, to a lesser extent, CXCR4, all susceptible to HIV infection [14, 62]. Unlike the vagina, the rectal canal has only a single layer of columnar epithelium overlying tissue rich in activated lymphoid cells . Thus, any extensive breakdown in epithelial integrity allows HIV direct access to a rich source of target cells, allowing the establishment of infection in mucosal sites . A greater percentage of mucosal mononuclear cells (MMCs) than peripheral blood mononuclear cells (PBMCs) are infected by both CCR5- and CXCR4-tropic viruses [14, 63]. Passage of virus from the lumen to the cellular targets may be facilitated by binding of virus to dendritic cell projections that extend into the epithelial compartment with subsequent presentation to sub-epithelial target cells [64-66]. After initial infection, local viral replication is followed by dissemination of virus to the regional lymph nodes, at which point systemic infection is established. Studies using animal models suggest that initial infection can occur within one hour of exposure and dissemination can occur within 24 hours .
As described above, the recent success of the CAPRISA 004 trial  indicates the potential impact of this approach on the epidemic worldwide . Although these clinical findings are a breakthrough for vaginal microbicides, the differences between the microenvironments of the rectal and cervico-vaginal mucosal tissue may require that different formulations be used for the two routes [62, 66]. Several studies have demonstrated the efficacy of topical microbicides against rectal simian/human immunodeficiency virus (SHIV) transmission in macaque models [57, 58, 67]. More important, although resistance to tenofovir was not observed in people who became infected with HIV in the CAPRISA 004 trial, tenofovir is commonly used to treat HIV-infected individuals, posing a risk for the emergence and transmission of drug resistant viruses. There are currently over thirty clinically licensed antiretroviral drugs and many more in the pipeline (http://aidsinfo.nih.gov/DrugsNew/Default.aspx?MenuItem_Drugs). Ongoing  and future studies based on these novel anti-HIV drugs may lead to the clinical testing and usage of HIV microbicides specifically formulated for the vaginal/cervical and rectal epithelia.
As with the female genital tract and the rectal mucosae, the uncircumcised penis outer and inner foreskin is a portal of entry for HIV during sexual transmission [68, 69]. Langerhans and dendritic cells, as well as CD4+ T-cells are found in the penile foreskin [68, 70]. Uncircumcised men have a higher density and more superficial presence of Langerhans cells with reduced keratinization of the inner layer of the foreskin [68, 71]. HIV survival in the subpreputial environment increases viral entry and transmission following exposure to infected genital fluids in uncircumcised males. Moreover, retraction of the foreskin during intercourse exposes the lightly keratinized inner mucosa to vaginal/rectal secretions and to potential microtears . Following male circumcision, these vulnerable tissues are removed leaving the urethral meatus as the only unkeratinized mucosa, which offers a smaller surface area for infection . Therefore, circumcision has been shown to block female-to-male HIV transmission by 50% to 60% [74-76] and, consequently, is highly recommended by the WHO/UNAIDS as an HIV prevention strategy. In addition, it is possible that daily application of microbicide gels on the penis might provide protection against HIV transmission. Clinical trials will be needed to further explore the feasibility and success of this strategy compared to vaginal/cervical and rectal topical pre-exposure prophylaxis.
Although HIV transmission through the oral mucosa is a relatively rare event , it can occur through genital-oral or breastfeeding routes. Numerous studies have described the transmission of the virus from mothers to children during lactation  demonstrating that, although uncommon compared with vaginal and rectal sexual transmission, HIV infection via the oral cavity is clearly possible. The presence of virions in saliva, salivary glands, and buccal epithelial cells is well documented ; however, it is still controversial whether saliva is a real route of HIV transmission. On the other hand, how the innate immune and related protective properties of oral mucosal epithelium might control HIV infection during clinical exposure to infectious virus is becoming an area of intense interest. This stems from multiple studies and epidemiological reports suggesting that the oral mucosa is not as permissive for efficient HIV replication as other mucosal epithelia (e.g., vagina/cervix and anal/rectal) and, therefore, may differ in susceptibility when compared to these mucosal sites. Discovering factors that explain the differential susceptibility and resistance to HIV infection in mucosal sites will allow for the identification and development of novel protective strategies.
The relative infrequency of oral HIV infection can be posited to involve: (i) a thick multilayered mucosal surface as the first line of defense against microbial invasion, (ii) low salivary HIV titers, and (iii) endogenous antiviral factors present in oral secretions. Many endogenous inhibitors of HIV in saliva have been proposed (e.g., amylase, lactoferrin, proline-rich peptides, salivary mucins, thrombospondin and secretory leukocyte protease inhibitor or SLPI)[80-82]. Remarkably, these agents are also found in seminal fluid and vaginal secretions, routinely harvested from sites that are more susceptible to HIV infection [83, 84]. Furthermore, fresh human saliva cannot inactivate HIV rapidly enough to prevent entry of infectious virus into oral keratinocytes . It is clear, therefore, that more information is needed to understand whether salivary innate immune factors contribute efficiently to the resistance against acquisition of HIV.
Mucosal epithelial cells also express antimicrobial peptides with antiretroviral activity. In addition to SLPI, oral epithelial cell-derived human β defensins (hBDs) inhibit HIV infection of immunocompetent cells in vitro [87, 88]. When compared with adult oral mucosae, the lack of hBD and SLPI expression in fetal oral mucosae appears to render underlying immunocompetent cells more susceptible to HIV infection . While transcytosis of the virions is not appreciably different in adult and fetal mucosal cells, the ease with which HIV infects the underlying lymphocytes in the fetal mucosal model, and the loss of protection by use of specific antibodies to hBDs and SLPI in the adult oral mucosae, strongly suggest that these peptides protect against HIV infection . Interestingly, a notable difference between oral epithelia and most other epithelia is the constitutive expression of hBDs. These defensins are expressed only in the presence of infection or inflammation in most tissues, including skin, trachea and gut epithelium [90, 91]. However, both hBD-2 and -3 are expressed in normal uninflamed gingival tissue , perhaps due to chronic exposure to specific oral commensal bacteria that promote hBD expression . What is less clear is whether the target cells for antiviral activity are the hBD-producing oral epithelial cells or the proximal immunocompetent cells.
Despite the myriad of studies on HIV transmission during the last thirty years, definitive information about the fate of the HIV virion in the mucosal epithelium is absent. Greater understanding of vulnerable versus less susceptible mucosal sites may help identify strategies to prevent HIV transmission. For example, it would be interesting to compare anatomic structure of different mucosal epithelia (i.e., vaginal, cervical, colorectal, intestinal, and oral) with function during HIV exposure, resting and, active HIV infection in naïve patients or treated with antiretroviral drugs. Deciphering the mechanisms underlying HIV infection through the human mucosal sites will hopefully aid in the development of novel and less invasive prophylactic strategies to prevent HIV transmission.
M.E.Q-M. was supported by research grant NIH-AI-71747. A.W. is supported by research grants NIH/NIDCR-R01-DE018276 and P01-DE019759.