Setting the stage – HIV host invasion

Abstract

For more than two decades, HIV has infected millions of people worldwide each year through mucosal transmission. Our knowledge of how HIV secures a foothold at both the molecular and cellular levels has expanded by recent investigations that have applied new technologies and used improved techniques to isolate ex vivo human tissue and generate in vitro cellular models, as well as more relevant in vivo animal challenge systems. Here, we review the current concepts of the immediate events that follow viral exposure at genital mucosal sites where most documented transmissions occur. Furthermore, we discuss the gaps in our knowledge that are relevant to future studies, which will shape strategies for effective HIV prevention.

Introduction

HIV vaccines and microbicides hold promise of preventing the acquisition of HIV-1 and HIV-2, the two viruses that cause AIDS, but the success of designing such agents necessitates a clear understanding of where HIV first encounters its target cells — primarily T cells, macrophages and dendritic cells (DCs) — and how it gains entry at various sites to eventually establish infection. HIV infection has rapidly spread since the early 1980’s to become an epidemic that is maintained by sexual transmission through the lower genital and rectal mucosa, and it is these routes of infection that account for the vast majority of current and new infections (Figure 1, Table 1)¹. Here, we have endeavored to synthesize the current knowledge on the acquisition of HIV at these mucosal sites, confining our discussion to the lower genital mucosa. We 1 clearly recognize that other sites of entry, such as blood, placenta and gastrointestinal mucosa, are important considerations but these are beyond the scope of this Review.

Figure 1. HIV invasion sites.

HIV mucosal invasion sites of the lower genital tract, the rectum, and the upper intestinal tract. In women, viral invasion occurs mostly through the non-keratinized squamous epithelium of the vagina and ectocervix, as well as through the single-layer columnar epithelium of the endocervix. The endocervical canal is filled with mucus, providing a barrier against ascent of pathogens. However, ovulation is accompanied by hydration and alkalinization of the mucus plug, possibly decreasing its barrier function. Infection in women can also ensue when HIV-1 invades the single-layer columnar epithelium of the rectum following receptive anal intercourse. In men, viral invasion occurs most frequently through the inner foreskin and the penile urethra as a consequence of penile–vaginal or penile–anal intercourse. Thinly stratified columnar epithelial cells line most of the urethra except for the fossa navicularis near the external meatus (exit hole), which is covered by non-keratinized squamous epithelium. The glans penis and the outer foreskin are protected by keratinized squamous epithelium, which provides a strong mechanical barrier against HIV invasion. By contrast, a thin and poorly keratinized squamous epithelium covers the inner foreskin, rendering this site vulnerable to HIV invasion. Men are also infected by viral invasion through the rectum. In fact, receptive anal intercourse carries the highest per exposure probability of infection among all mucosal transmission sites. The upper gastrointestinal (tract, lined by non-keratinized squamous epithelium in the oropharynx and the esophagus, and by single layer columnar epithelium in the stomach and the small intestine, is another site of mucosal HIV invasion. In adults, transmission in the upper gastrointestinal tract occurs following contact with HIV-containing semen during fellatio, but the efficiency of this route is low. In infants, HIV invasion in the upper gastrointestinal tract occurs after exposure to or ingestion of infected maternal blood and genital secretions during birth as well as infected milk during breast feeding.

Table 1.

Contribution of HIV invasion sites to global HIV infections (adapted from¹,¹²³–¹³⁰)

HIV invasion site	Anatomical sub-location	Type of epithelium	Transmission medium	Transmission probability per exposure event	Estimated contribution to HIV cases worldwide
Female genital tract	Vagina	Squamous, non-keratinized
	Ectocervix	Squamous, non-keratinized
	Endocervix	Columnar, single layer	Semen	1 in 200 - 1 in 2,000	12.6 million
	Other	Various epithelia
Male genital tract	Inner foreskin	Squamous, poorly keratinized
	Penile urethra	Columnar, stratified	Cervicovaginal and rectal secretions and desquamations	1 in 700 - 1 in 3,000	10.2 million*
	Other	Various epithelia
	Rectum	Columnar, single layer	Semen	1 in 20 - 1 in 300	3.9 million**
Intestinal tract			Semen	1 in 2,500	1.5 million
	Upper GI tract	Various epithelia	Maternal blood, genital secretions¶	1 in 5 - 1 in 10	960,000***
			Breast milk¶¶	1 in 5 - 1 in 10	960,000***
Placenta	Chorionic villi	Two layer epithelium(cyto- and syncytiotrophoblast)	Maternal blood¶¶¶	1 in 10 - 1 in 20	480,000***
Blood stream			Blood products, sharps	95 in 100 - 1 in 150	2.6 million****

^*Includes men having sex with men (MSM), bisexual men and heterosexual men

^**Includes MSM, bisexual men and women infected via anal receptive intercourse

^***Mother-to-child transmission:

^¶intrapartum

^¶¶breastfeeding

^¶¶¶intrauterin

^****Mostly intravenous drug use (IDU), but includes infections by transfusions and health care related accidents

Many elegant studies have provided insights into selective aspects of how HIV and simian immunodeficiency virus (SIV) enter the mucosa through detailed examination of the relevant tissues and target cells following in vivo, ex vivo and in vitro virus exposure. We have organized the discussion in this Review based on the experimental system used from the same mucosal source to alleviate confusion and inconsistencies that often emerge when findings from one system are extrapolated to those of another. When appropriate, we have emphasized the benefits and limitations of the experimental approaches, important considerations in the interpretation of findings and their relevance for future studies.

HIV invasion in the female genital tract

Anatomical sites

An estimated 30–40% of all new HIV-1 infections occur in women through vaginal intercourse, which carries a lower HIV transmission probability per exposure event than anal intercourse or parenteral inoculation (Table 1). Although HIV-1 can infect the vaginal, ectocervical and endocervical mucosa (Figure 1), the relative contribution of each site to the establishment of the initial infection is not known. The multilayered squamous epithelium that covers the vagina and ectocervix, when intact, provides better mechanical protection against HIV invasion than the single-layer columnar epithelium that lines the endocervix. However, the greater surface area of the vaginal wall and ectocervix, which often exceeds 15 times that of the endocervix, provides more potential access for HIV entry, particularly when breaches occur in the epithelial-cell layer. HIV or SIV can establish an initial infection solely in the vagina, as shown in women who lack a uterus at birth² and in female macaques after surgical removal of the uterus³. In fact, selective transmission of HIV through the vaginal mucosa rather than the cervix may commonly occur, as suggested by a recent large randomized, controlled prevention clinical trial in African women. In this study, no significant reduction in HIV-1 acquisition occurred in women using a diaphragm compared with the control group⁴. However, the observed potential benefit of blocking HIV-1 exposure to the cervix may have been undermined because the sexual partners reported lower condom usage in the diaphragm intervention group than the control group.

The region where the ectocervix transforms into the endocervix (Figure 1) can be enriched with CD4⁺ T cells and may be a particularly susceptible site of HIV entry. Whether HIV can cross the endocervical mucus plug, reach the uterine cavity and invade through the mucosa of the upper genital tract has not been well examined. In principle, uterine tissue is susceptible to infection if directly inoculated with HIV⁵. Uterine simian–HIV (SHIV) infection has been shown in one monkey following vaginal inoculation two days earlier, an interval that is probably too short for stromal or lymphatic spread from the lower genital tract⁶. This indicated that ascent of virus through the endocervical mucus plug may be possible, but confirmation in humans is lacking. Conceivably, the upper mucosal tract may become more vulnerable to HIV-1 penetration during ovulation, a period when rising estrogen levels alter the endocervical mucus to a less viscous and more alkaline consistency.

HIV entry through the female genital epithelium

Both free HIV and SIV virions, and HIV and SIV virions from seminal leukocytes (cell-associated virus) can establish mucosal infection (Figure 2)^7–11. This has been shown directly in vivo in female macaques¹² and in mice¹³, and indirectly in humans through genetic sequence comparisons of viral isolates from acutely infected women and from seminal cells and plasma from their infected source partners¹⁴. Ex vivo studies using human cervical explants have also confirmed transmission of cell-free and cell-associated HIV-1^15,16. Initially, cervical mucus can trap seminal cells or free virions^17,18. Conceivably, this could facilitate transmission by prolonging mucosal contact time. However, while immobilized, the virions may also become more susceptible to innate antiviral substances.

Figure 2. Pathways of HIV invasion in the mucosa of the vagina and uterine ectocervix, part A.

The human vagina and ectocervix are covered by non-keratinized squamous epithelium. Shearing during sexual intercourse can lead to physical abrasions of the epithelium, in particular in microanatomical regions where the stromal papillae, enriched with stromal DCs, reach close to the luminal surface of the mucosa. Depicted are two stromal papillae containing arterioles, venules and lymphatic vessels. The stromal papilla on the right signifies the afferent arm shuttling blood cells to the mucosa. Monocytic precursor cells differentiate upon arrival either into macrophages or dendritic cells (DCs), and DCs may differentiate further into subsets. Three stromal DC subsets have been identified in human skin, distinguished by BDCA-1, CD1 and CD14 expression patterns⁶⁵, but their presence and susceptibility to HIV have not been determined in the mucosa. An abrasion of the outer epithelium exposes the stromal papilla tip (left), as well as several epithelial cells located close to or within the basal layer. Infected donor cells and free virions may migrate along such an abrasion, as shown here, and directly contact various target cells in the mucosal epithelium and stroma. Resident mucosal leukocytes such as DCs and T cells tend to cluster in these regions (see Figure 3e), creating susceptible foci for infection. Possible pathways for HIV penetration are depicted on the left side of the illustration and are indicated by letters. Characteristic phenotypic cell receptors and receptors relevant for HIV binding and infection are shown on the top of the figure. a. Trapping of free HIV virions or HIV-infected donor cells in mucus covering the mucosa. b. Attachment of HIV-infected donor cells to the luminal surface of the mucosa and secretion of virions upon contact. c. Penetration of virions into gaps between epithelial cells. d. Capture of penetrating virions by Langerhans cells (LCs) residing within the epithelium, which extend processes toward the vaginal lumen. e. Internalization of virions into endocytic compartments of LCs. f. Fusion of HIV with the surface of intraepithelial CD4⁺ T cells, followed by productive infection. g. Transcytosis of virions through epithelial cells located close to or within the basal layer of the squamous epithelium (Figure 3a–c). h. Productive infection of basal epithelial cells. i. Internalization of virions into endocytic compartments of basal epithelial cells. j. Immigration of infected donor cells along physical abrasions of the epithelium into the mucosal stroma, where they are taken up by lymphatic or venous microvessels and transported to local lymph nodes or the blood circulation. k. Immigration of free virions along microabrasions into the stroma, where they can make direct contact with stromal DCs. l. Productive infection of stromal DCs by HIV. m. Internalization of virions into endocytic compartments of stromal DCs. n. Passage of virus from stromal DCs to CD4⁺ T cells across an infectious synapse (see also Figure 4). o. Massive productive infection of mucosal CD4⁺ T cells activated by contact with antigen-presenting DCs. p. Productive infection of resting mucosal CD4⁺ memory T cells. q. Binding of HIV and possibly productive infection of stromal macrophages. r. Emigration of productively infected CD4⁺ T cells and DCs into the submucosa and the draining lymphatic and venous microvessels. T cells may derive from the epithelium or the stroma. Likewise, emigrating DCs may originate from intraepithelial LCs or stromal DCs. DCs and T cells often form conjugates, and HIV may accumulate between the two cells along an infectious synapse. DCs carry virions in endocytic compartments and some are also productively infected, but it remains unclear at which differentiation stage this occurs.

HIV virions that are initially free or following their release from infected donor cells interact with epithelial cells and traverse the epithelium through several pathways, including transcytosis, endocytosis, or productive infection, or they merely penetrate through gaps between epithelial cells (Figure 2). Understanding these events has been hindered by inconsistent findings, largely because experimental systems have used epithelial cell types derived from different anatomical sites, and primary and immortalized cell lines. Several reports demonstrate that HIV-1 binds to and enters epithelial cells from the lower female genital tract^19–21. Transcytosis has been shown to occur in both cell lines and primary cells, but not definitively within intact tissue. Upon release, the virions readily infect susceptible leukocytes^22,23. Interestingly, cell-associated virions secreted from infected leukocytes appear markedly more efficient in transcytosis than cell-free virions^8,11,22. Productive infection may also occur within cervical epithelial cells themselves⁷, although this remains controversial^19,24. Conceivably, HIV-1 can also be transported through the cervicovaginal epithelium to the draining lymphatics by donor lymphocytes and macrophages, as has been suggested in mouse studies^7,25.

Our ex vivo experiments using sheets of isolated vaginal epithelium, devoid of mucosal stroma, confirmed that the sequestration of HIV-1 virions in endocytic compartments and in the cytosol of epithelial cells occurs (Figure 3a–c). However, although the experimental conditions permitted HIV-1 access to both the luminal and basal sides of the epithelium, the virions were detected exclusively in the basal and suprabasal epithelial cells (F. Hladik, P. Sakchalathorn, M. J. McElrath, unpublished observations). Therefore, rather than entering and traversing superficial epithelial cells in the vagina and ectocervix, HIV-1 probably disperses through the narrow gaps between them¹⁷, as depicted in Figure 2. This route might permit HIV-1 to directly contact and then infect intraepithelial Langerhans cells and CD4⁺ T cells²⁶ (see later), or it might allow HIV-1 to reach suprabasal or basal epithelial cells that are susceptible to viral sequestration and transcytosis. Importantly, factors in human semen, most notably amyloid fibrils forming from naturally occurring fragments of seminal prostatic acidic phosphatase, can capture virions and promote attachment to epithelial cells and leukocytes, thus increasing infectivity²⁷.

Figure 3. Pathways of HIV invasion in the mucosa of the vagina and uterine ectocervix, part B.

A–C. Likely HIV transcytosis in a vaginal epithelial cell in situ located one layer above the basal cell layer. Virions can be seen in the cytoplasm on both sides of the nucleus. Desmosomes and keratin fibers identify the cell as epithelial.

Several proteins expressed on the surface of epithelial cells may mediate attachment of HIV-1. Two cell surface glycosphingolipids, sulfated lactosylceramide expressed by vaginal epithelial cells²⁸ and galactosylceramide expressed by ectocervical epithelial cells^19,29, bind HIV-1 gp120 and foster transcytosis²². Interactions of HIV-1 gp120 with transmembrane heparan sulfate proteoglycans (syndecans) expressed by genital epithelial cells can also contribute to HIV-1 attachment and entry^20,23. Recently, glycoprotein 340, a splice variant of salivary agglutinin expressed by cervical and vaginal epithelial cells, was shown to specifically bind to the HIV envelope protein and to enhance the passage of HIV through the epithelium to susceptible leukocytes³⁰. One group found that the β₁ subunit of integrins expressed by explant cervical epithelial cells bound virions that were presumably coated with fibronectin, which is abundant in human semen, although this was not observed across all explants¹⁷. Detection of HIV-1 chemokine co-receptor expression has been inconsistent: one study did not detect the expression of either CC-chemokine receptor 5 (CCR5) or CXC-chemokine receptor 4 (CXCR4) by cervical epithelial cells¹⁹, another reported the expression of CXCR4 by these cells²¹, and another reported the exclusive expression of CCR5²⁹.

Regardless of the mode, the penetration of virus through the cervicovaginal epithelium in vivo occurs rapidly within 30–60 minutes of exposure, as shown in SIV infected macaques³¹. Once within the epithelium, HIV encounters CD4⁺ T cells as well as Langerhans cells (LCs). LCs have dendrites that extend and retract through the intercellular spaces³², and even reach up to the epithelium surface³³ where HIV can bind directly to these cells (T. Hope, personal communication). Based on observations of gut dendritic cells^34,35, this could be particularly true for DCs that are located just beneath the endocervical columnar epithelium. However, direct sampling of luminal pathogens by endocervical DCs or vaginal LCs, which could be exploited by HIV to bypass the epithelial-cell barrier, has not yet been formally demonstrated.

Finally, mechanical microabrasions of the mucosal surface induced by intercourse may allow HIV to directly access target cells, such as DCs, T cells and macrophages, at the basal epithelium and the underlying stroma³⁶. Areas above the stromal papillae, where the epithelium is relatively thin and where LCs on the epithelial-cell side (F. Hladik, L. Ballweber, M. J. McElrath, unpublished observations) and T cells and macrophages on the stromal side²⁹ congregate, appear particularly vulnerable to viral invasion. Consistent with this notion, in vivo SIV infection of the genital mucosa of macaques is initially established in a highly focal manner, and continuous seeding from this nidus of infection is crucial for establishing systemic infection¹⁸. Similarly, chemical microabrasions with the use of certain topical microbicides and microabrasions due to genital ulcers caused by sexually transmitted diseases (for example, syphilis, chancroid and those caused by infection by Herpes simplex virus) are also likely to expose vulnerable target cells in the basal epithelium and stroma³⁷.

Importance of cervicovaginal LCs in HIV invasion

LCs are a DC subtype residing within the outer squamous epithelium of the skin or mucosae. For many years, HIV-1 acquisition in the lower genital tract has been assumed to occur through internalization of HIV-1 by LCs. This view was supported by evidence that skin LCs are susceptible to infection by HIV-1^38–41 and that genital mucosal LCs harbour SIV virions within 24 hours of intravaginal inoculation of macaques³¹. However, soon after ex vivo organ culture, LCs migrate out of the epithelium^{15,24,42–44}; therefore, examination of LC infection specifically within the human vaginal epithelium has been technically difficult. For example, one landmark study demonstrated that after exposure of human complete cervical mucosa to HIV-1, emigrating DCs had efficiently captured HIV and were capable of transmitting the virus in trans⁴³. However, determining whether the cells originated from the epithelium as LCs or the underlying stroma as DCs was impossible. More recently, we resolved this issue by preparing sheets of vaginal epithelium separated from the underlying stroma, and observed that vaginal LCs efficiently internalized HIV-1 into their cytoplasmic compartments²⁶. As LCs exit the epithelium at the basal side, they transport intact virions, thereby enabling infection to spread beyond the site of viral entry (Figure 2).

The ability of LCs in the cervicovaginal epithelium to produce and release new HIV-1 virions is uncertain. HIV-specific receptors are expressed by these LCs, including CD4, CCR5 and the C-type lectin langerin (CD207), but not CXCR4 and dendritic-cell-specific ICAM3-grabbing non-integrin (DC-SIGN, CD209)^26,45–48. Antibodies that bind CD4 and CCR5 partially block the uptake of R5-tropic HIV-1 by LCs, but blocking the binding of C-type lectin to mannan on HIV-1 had little effect on uptake²⁶. Although low-level CD4- and CCR5-mediated productive infection of LCs in human skin explants has been shown^40,41,49, we were unable to confirm this finding in our imaging studies of vaginal LCs²⁶. Therefore, if de novo production of virions occurs, it appears relatively inefficient in contrast to the high capacity of vaginal LCs to endocytose HIV-1. Nevertheless, even low levels of productive HIV-1 infection of cutaneous DCs and LCs lead to profound viral replication in co-cultured T cells^41,49,50. Therefore, future investigations must conclusively determine if LCs in cervicovaginal epithelium can support productive HIV infection in vivo and if this property is required for the passage of the virus to T cells, as has been reported for other types of DCs^51–54.

The relative inefficiency of mannan, a mannose polymer, to block binding and endocytosis of HIV-1 by vaginal LCs was surprising²⁶, because C-type lectins, which recognize mannose containing carbohydrate structures, mediate viral entry in other types of DCs⁴⁷. However, HIV-1 can bind DC subsets independent of C-type lectins and CCR5^55–57. So, although HIV is efficiently captured by langerin expressed by epidermal LCs⁵⁸, HIV appears to largely bypass langerin expressed by vaginal LCs in favour of alternative endocytic routes. This distinction may be highly relevant for mucosal HIV transmission. Langerin expressed by epidermal LCs can direct HIV-1 to Birbeck granules for degradation⁵⁸. By contrast, by gaining entry to the vaginal LCs independently of langerin, HIV may survive by reaching endocytic compartments, such as early phagosomes, where antigens are preserved for cross-presentation⁵⁹. This is consistent with our observations that intact virions were still present in LCs that had migrated out of the vaginal epithelium at 60 hours after viral challenge²⁶. Thus, it appears that HIV-1 enters vaginal LCs through a different route than skin LCs, resulting in a distinct fate of the endocytosed virions. More detailed studies are now warranted to uncover which endocytic pathways HIV uses in vaginal LCs, and how this can be harnessed therapeutically.

Infection of DCs in the cervicovaginal stroma

Unlike genital LCs, stromal DCs express both DC-SIGN^47,48 and CCR5^60,61 and have been implicated in SIV and HIV infection, but their exact role in mucosal transmission is not clear. In situ studies in the human explant model have failed to identify DCs in the cervicovaginal stroma as foci for productive HIV infection^15,24,42,44. By contrast, SIV-infected DCs were present in the lamina propria of the cervicovaginal mucosa of macaques shortly after intravaginal SIV challenge^31,62, as well as in chronically infected animals⁶³. Likewise, HIV-infected DCs were identified in tissue biopsies of the vaginal stroma of asymptomatic HIV-1 infected women⁶⁴. The failure to reveal infection of stromal DCs in the human explant models may have been due to the relatively low sensitivity of the detection methods employed and the migration of stromal DCs from the tissue, which may drastically decrease the number of infected cells in situ over time. Indeed, when DCs were harvested from the culture supernatants of human cervical explants that were challenged with HIV-1, significant in trans infectivity was detected⁴³ and massive budding of virions was observed among emigrant DCs five days after virus exposure⁶¹ (Figure 4b–d). However, it could not be determined if the original source of DCs was from the epithelium proper or from the stromal tissue. In addition, inferring the initial susceptibility of DCs while confined to the mucosal stroma, based on findings from emigrated DCs that undergo phenotypic changes as they exit the mucosa, may be less reliable. Thus, stromal DCs exhibit different HIV-1 receptor expression patterns than LCs, potentially permitting different HIV-1 entry pathways than in the epithelial LCs. Much still remains to be learned about stromal DCs in the human genital mucosa in general, about whether different subsets exist similar to those found in skin dermis⁶⁵, and about the interaction of these DC subsets with HIV in particular. More sensitive in situ detection methods of HIV infection, as well as assays distinguishing de novo virus production from endocytically engulfed virions, as recently reported⁶⁶, will be helpful in sorting out the contribution of stromal DCs to HIV-1 propagation.

Figure 4. Significance of DC–T-cell interactions for HIV-1 transmission.

A. Pathways of HIV-1 passage between dendritic cells (DCs) and T cells. DCs can store HIV-1 in three forms for eventual infection of CD4⁺ T cells. (1) Endocytosed intact virions. Endocytic entry via C-type lectins such as DC-specific ICAM3-grabbing non-integrin (DC-SIGN, CD209) directs HIV to early and late endosomes. From there it enters multivesicular bodies and remains intact, or traffics to lysosomes where it is degraded. (2) Integrated provirus. Entry of HIV-1 by CD4- and co-receptor-mediated fusion leads to productive infection of DCs. (3) Surface-bound intact virions. Binding and trapping of intact virions on the cell surface can also occur by C-type lectins such as DC-SIGN.

Passage of HIV-1 from DCs to CD4⁺ T cell occurs most effectively across an infectious synapse, formed by concentration of HIV-1 on the DC side and of HIV receptors such as CD4 and CC-chemokine receptor 5 (CCR5) on the T cell side. HIV is released into the infectious synapse either by exocytosis of stored virions from multivesicular bodies (MVBs) or by budding of newly formed virions following active viral replication. Surface bound virions may also accumulate at the infectious synapse. Migration of HIV toward the T cell may be further enhanced by “surfing” of virions along the outer surface of filopodia or cytonemes that are extended from the T cell toward the DC. Coupling of virions with exosomes as they are being released from MVBs may also increase their infectivity. Exosome-associated virions are likely to be transmitted to CD4⁺ T cells through membrane binding and fusion, either within the infectious synapse or over longer distances. In parallel to transmission of virus from the DC to the CD4⁺ T cell, the DC also presents antigenic peptides through MHC class II molecules to the T-cell receptor CD3. During peptide recognition, additional receptor–ligand pairs that are important for T-cell stimulation accumulate in this region and form an immunological synapse. Signals delivered through the immunological synapse lead to T-cell activation, which ultimately causes transcription factors such as nuclear factor-κB (NF-κB) and nuclear factor of activated T cells (NFAT) to translocate into the nucleus of the T cell. There, they bind to the enhancer region of the viral long terminal repeat (LTR) and activate viral gene transcription, driving HIV-1 replication. B–D. Example of infectious synapse formation between DCs and T cells in the human vagina (taken from Hladik F. et al.⁶⁹). HIV-1 buds from the surface of the productively infected DC toward the contact zone between the DC and the two T cells. Viral budding is also seen along other areas of the DC surface. The DC contains the typical veiled nucleus as well as multiple large mitochondria, and one large cytoplasmic process is formed at the top right. C–D. The two contact zones with the lymphocytes are further magnified, displaying virus budding from the DC into the infectious synapse.

HIV infection of cervicovaginal CD4⁺ T cells

CD4⁺ T cells are dispersed throughout the lamina propria of the human vagina, ectocervix and endocervix, often clustering just beneath the basal membrane^67,68. They also reside at variable numbers within the vaginal and ectocervical squamous epithelium^67,68. The majority are memory T cells that express higher levels of CCR5 than T cells that circulate in peripheral blood^26,69–71. One day post HIV-1 inoculation of vaginal, ectocervical and endocervical tissue cultures, infected CD4⁺ T cells were shown to be confined to the mucosal stroma^15,17,24,43. This result was surprising, given the presence of CCR5⁺ CD4⁺ T cells within the squamous epithelium. However, by analyzing the fate of fluorescence-tagged virions as early as two hours after viral exposure, we observed that R5-tropic HIV-1 bound to intraepithelial vaginal CD4⁺ T cells very efficiently, followed by fusion and productive infection²⁶. Therefore, infected T cells must rapidly leave the epithelium, and those found in the stroma may be the same or early progeny of intraepithelial T cells.

Findings in the human explant studies show that HIV-1 very effectively targets CD4⁺ T cells in the genital mucosa for productive infection^15,26,43, and that the initial infection of intraepithelial CD4⁺ T cells is probably independent of LCs²⁶. The central role for genital CD4⁺ T cells in early infection and propagation is also evident from SIV challenge experiments in macaques^63,72,73. Interestingly, not only does SIV productively infect activated T cells, characterized by HLA-DR and Ki67 expression, but also T cells that are in the HLA-DR- and Ki67-negative resting state⁷². Consistent with this finding, we observed binding of HIV-1 to both HLA-DR⁺ and HLA-DR⁻ intraepithelial T cells in our human vaginal explant model (M. J. McElrath, P. Sakchalathorn, L. Ballweber, F. Hladik, unpublished observations). In addition, the contribution of resting CD4⁺ T cells to viral production is substantial during the very earliest stages of infection⁷⁴. The fact that vaginal CD4⁺ T cells are rapidly depleted following intravenous SIV inoculation of macaques^6,73, similar to that observed in CD4⁺ T cells of the gut during acute SIV infection⁷⁵, further illustrates their high susceptibility to infection in vivo.

Other leukocyte targets for HIV in the female genital tract

Macrophages in the female genital mucosa are also susceptible targets for early HIV-1 infection, as demonstrated in studies using human explant models^24,42,44, and in two reports were the major cell type infected by R5-tropic HIV-1^24,44. Whether or not resident macrophages in the female genital tract constitutively express CCR5 in situ is not known, but most macrophages do so when harvested from supernatants of vaginal organ cultures⁶¹, suggesting that the expression of CCR5 by macrophages may occur during the period of activation and emigration from the mucosa⁷⁶. By contrast, SIV-infected macrophages in genital tissues were either rare⁷², or undetectable^31,62. Likewise, macrophages in the human intestinal mucosa were reported to lack CCR5 expression and to possess low permissibility for HIV-1 infection⁷⁷. These discrepancies illustrate a potential limitation of organ cultures. If indeed explantation activates stromal macrophages and as a consequence increases surface CCR5 expression, this would lead to an overestimation of their susceptibility to infection in vivo. Of note, in addition to chemokine receptor-mediated fusion, monocyte-derived macrophages can also trap intact virions through syndecans⁷⁸, or even without specific envelope-receptor interactions through a process known as macropinocytosis⁷⁹. Once captured, HIV-1 can be archived for several days and then transmitted to T cells in trans^80,81. If genital macrophages similarly archive infectious virions, their role in viral propagation once HIV-1 invades the stroma may be significant.

Other leukocyte subpopulations also interact with HIV. For example, productive infection of natural killer (NK) cells has been reported⁸², as well as transmission of virus through DC-SIGN expressed by B cells to T cells in trans⁸³. The significance of B cells for HIV invasion in the genital mucosa remains unknown. Lastly, monocyte precursor cells enter the mouse dermis in large numbers following intracutaneous infection with Leishmania major and differentiate into stromal DCs⁸⁴. This finding raises the possibility that the influx of inflammatory cells into the genital tract following HIV-1 exposure may create new potential target cells that normally do not reside in the mucosa, and further heighten the initial local infection.

The role of DCs in enhancement of infection

A growing body of literature suggests that HIV exploits DCs to enhance its infectivity of T cells⁸⁵. First reported in 1992, DCs, even when seemingly uninfected themselves, invoked vigorous cytopathic infection to CD4⁺ T cells⁸⁶. The potential relevance of these findings for the transmission of HIV at mucosal sites was subsequently highlighted by reports of increased HIV-1 replication in DC–T-cell conjugates derived from human skin⁸⁷ and cervicovaginal mucosa⁶¹. The enhancement of HIV transmission by DCs probably occurred by facilitating T-cell activation^72,88 as well as de novo T-cell infection.

Four mechanisms for how DCs can augment de novo infection of T cells have been proposed. In classic HIV trans infection⁴⁷, the DCs are not productively infected but trap and preserve the virus, which is subsequently transferred to T cells across an “infectious synapse”, which is a zone of DC–T-cell contact where HIV itself and the HIV receptors are concentrated^89,90. Alternatively, trans infection may occur by HIV association with DC-derived exosomes, which, intriguingly, appear to markedly increase the infectivity of virions that are coupled to them⁹¹. In a third pathway, productively infected DCs transmit new viral progeny across the infectious synapse to T cells^49,52–54. In this case, the contact zone has also been termed a “virological synapse”, to signify that the donor cell, in analogy to cell-associated HIV transmission between CD4⁺ T cells⁹², is productively infected⁹³. Furthermore, efficient retroviral transfer between cells has recently been described, in which retroviruses, including HIV-1, “surf” along the outer surface of filopodia or cytonemes that extend from an uninfected cell and interact through their tips with an infected cell⁹⁴. These narrow filopodial contact zones may be special cases of virological synapses⁹⁵, or may be analogous to nanotubules that are formed between immune cells⁹⁶. In T cells, migration of HIV-1 also occurs within nanotubules¹³⁰. Nanotubules have been shown to functionally connect DCs with other cells⁹⁶, but their significance for viral transfer from DCs remains to be determined.

Clear evidence for any of these described modes of viral transmission from DCs to CD4⁺ T cells in the genital mucosa is still lacking. We have shown that HIV concentrates along the cell–cell junction between emigrant LC–T-cell conjugates from human vaginal epithelium, supporting the formation of an infectious synapse²⁶. No consensus has been reached over whether in trans infection occurs primarily from surface bound virions^47,97, internalized virions⁹⁸ or both⁵⁷. At any rate, once HIV makes its way into the genital DC–T-cell conjugate, a profound productive infection ensues⁶¹. Of note, in these conjugates viral budding was not only observed from the surface of T cells but also of the DCs. Visualization of budding by electron microscopy signifies massive infection. So, in DC–T-cell conjugates not only T cells but also DCs, which by themselves are generally weak producers of virus⁸⁵, acquire the ability to produce large amounts of viral progeny. This is consistent with findings obtained in co-cultures of monocyte-derived DCs and T cells⁹⁹. Thus, DC–T-cell cross-talk in the genital tract appears to drive productive HIV infection in both cell types. Moreover, transmission from DCs to T cells may provide a means for HIV to avoid antibody-mediated neutralization^100–102. Taken together, unravelling the precise mechanisms by which the interaction between genital DCs and CD4⁺ T cells enhance HIV transmission will be important in developing effective strategies to counteract this process.

HIV invasion in the male genital tract

Of the nearly 15 million infected men, an estimated 70–75% acquired HIV-1 through vaginal intercourse (Table 1), making the male genital tract the second leading site of HIV invasion following the cervicovaginal mucosa. HIV-1 target cells are abundant in the foreskin, and include CD1a⁺ LCs and CD4⁺ T cells in the squamous lining, as well as T cells, macrophages and DCs in the underlying stroma^46,103–105. Variable fractions of these cells express CD4, CCR5 and CXCR4^46,103,104. As in the vagina, LCs do not express DC-SIGN, whereas at least some of the stromal DCs do¹⁰³. Therefore, similar mechanisms that have been described for viral invasion into the female genital tract above are likely to occur in the male genital tract as well.

The highly protective effect of circumcision^106,107, which has been reviewed comprehensively elsewhere¹⁰⁸, suggests that the penile foreskin is particularly vulnerable to HIV infection. The foreskin is lined by stratified squamous epithelium, and the external surface is more heavily keratinized than the internal surface^103,104. Consequently, the inner foreskin may be more susceptible to infection, a view that was supported by investigations of penile autopsy tissues¹⁰³, and corroborated in an in vitro foreskin explant model that showed infectious foci, predominantly containing LCs and CD4⁺ T cells, at the base of the epithelial-cell layer, exclusively in the inner foreskin¹⁰⁴. Alternatively, circumcision may reduce the risk for genital tears, abrasions and ulcer disease, resulting in decreased HIV infection risk^108,109.

Although circumcision appears to provide a protective effect against HIV infection, entry sites other than the foreskin must exist for circumcised males. The glans penis has a heavily keratinized squamous epithelium similar to the outer foreskin, and effective viral penetration seems relatively improbable here. By contrast, the penile urethra is a more likely candidate as it is lined by a narrowly stratified, non-keratinized columnar epithelium and contains high numbers of CD4⁺ and CD8⁺ T cells, and macrophages, within the epithelium and the lamina propria¹¹⁰. Interestingly, DCs are not observed in the urethral mucosa^46,110. The presence of CCR5 and CXCR4 mRNA in urethral swabs indicates that these HIV-1 co-receptors may be expressed by urethral cells¹¹¹, particularly the predominant CD4⁺CD45RO⁺ memory T cells¹¹⁰. Of note, intraurethral infusion of SIV resulted in infection of all six inoculated male macaques¹¹². In HIV-1 infected men, antibiotic treatment of urethral Neisseria gonorrhoeae infection reduces HIV-1 shedding in semen but not the viral load in blood¹¹³. This finding, as well as HIV-1 shedding in the ejaculate of vasectomized HIV-positive men¹¹⁴ and in the pre-ejaculatory fluid^115,116 suggest a distal source for virus, pointing to the urethra as a site harboring significant numbers of cells susceptible to HIV-1 infection. Taken together, these data suggest that CD4⁺ T cells and macrophages in the male urethra provide a suitable portal of entry for HIV, although the specific events of urethral invasion are still unknown.

Conclusions

Although studies that focus on mucosal HIV infection continue to be painstaking, many investigators have overcome some of the technical difficulties that have previously precluded active research in this area. Recent investigations summarized here provide invaluable insights into the distinct cellular and molecular interactions of mucosal HIV infection. Clearly, the challenge emerging from these findings is to counteract the rapid acceleration of infection in local reservoirs of the lower genital and gastrointestinal tracts. Two key questions to address in future studies lie in determining whether DC–T-cell interactions that markedly amplify HIV-1 production, which are consistently observed in vitro, are relevant in the mucosal epithelium in vivo, and whether common mechanisms of HIV-1 entry apply to both the lower genital and gastrointestinal tract. This information can guide the development of innovative strategies to protect susceptible target cells from HIV-1 infection in both women and men, such as with barrier protection methods, topical microbicides and mucosal immunization.

Although there are an increasing number of large-scale HIV prevention clinical trials that have recently reported a lack of efficacy, one favourable approach has been male circumcision. The opportunity to gain a more thorough understanding of how HIV-1 invades its target cells in the human foreskin should be exploited. The recent emphasis of CD4⁺ T-cell depletion by HIV-1 in the gut and vagina during acute infection has sparked a renewed interest in abating infection at the sites where massive replication occurs¹¹⁷. Although important in counteracting HIV disease, CD4⁺ T-cell depletion appears to be a secondary event that commonly occurs through various routes of transmission. Therefore, the best opportunity to prevent HIV disease clearly lies at the sites of mucosal entry, and investigations to directly counterattack HIV infection must continue to focus on these portals.

Box 1. HIV invasion in the lower gastrointestinal tract

HIV-1 and SIV infection commonly targets the lower gastrointestinal tract as an initial site following receptive anal intercourse in humans and direct inoculation in macaques, and as a secondary infection site following rapid dissemination from mucosal foci¹⁸ or acute systemic infection^118–120. The rectal mucosa contains simple columnar epithelial cells, and the lamina propria is a rich source of lymphoid cells and lymphoid nodules. Numerous reports have documented the pathogenic effect of the virus in the gastrointestinal tract, and have shown a severe depletion of CD4⁺ T cells that express CCR5 in the gut, regardless of the route of infection¹¹⁷. The relevant target cells for infection in the lower gastrointestinal tract are thus likely to be primarily CD4⁺ memory T cells¹²¹. At present, detailed features of HIV-1 entry into the lower gastrointestinal tract that may be distinct from the genital tract have not been elucidated, but this remains an important question to address for prevention strategies.

Glossary terms

Simian–HIV (SHIV)

SHIVs are chimeric viruses that are created by inserting the envelope protein (Env), the transcriptional transactivator (Tat) and the regulator of virion gene expression (Rev) of HIV into the SIV_MAC239 clone. Depending on the particular HIV Env protein, these SHIVs have different in vivo characteristics. The SHIV chimeric viruses are best used for testing antibodies specific for HIV in non-human primate models.

Transcytosis

A process by which various macromolecules, including HIV-1 virions, are transported across the interior of a cell.

Syndecans

Single transmembrane domain proteins that carry three to five heparan sulfate and chondroitin sulfate chains which allow for interaction with a large variety of ligands including residues on the HIV-1 gp120 protein.

Langerhans cell

A type of dendritic cell that is localized in the squamous epithelial layer of the skin and certain mucosae.

Stromal papillae

Superficial areas of the mucosal stroma that interdigitate with the epithelium.

C-type lectin receptors

A large family of receptors that bind glycosylated ligands and have multiple roles, such as in cell adhesion, endocytosis, natural-killer-cell target recognition and dendritic-cell activation.

R5-tropic HIV-1

An HIV strain that uses CC-chemokine receptor 5 (CCR5) as the co-receptor to gain entry to target cells.

Birbeck granules

Membrane-bound rod- or tennis racket-shaped structures with a central linear density, found in the cytoplasm of Langerhans cells. The formation of Birbeck granules is induced by langerin, an endocytic C-type lectin specific to Langerhans cells.

Phagosomes

Vacuolar compartments that confine bacteria after enforced endocytosis or after phagocytosis. Unless counteracted by a bacterial survival strategy, the phagosome matures into a hostile environment that is designed to kill and digest microorganisms.

Cross-presentation

The initiation of a CD8⁺ T-cell response to an antigen that is not present within antigen-presenting cells (APCs). This exogenous antigen must be taken up by APCs and then re-routed to the MHC-class-I pathway of antigen presentation.

Lamina propria

Connective tissue that underlies the epithelium of the mucosa and contains various myeloid and lymphoid cells, including macrophages, dendritic cells, T cells and B cells.

Exosomes

Small lipid-bilayer vesicles that are released from activated cells. They comprise either plasma membrane or membrane derived from intracellular vesicles.

Filopodia

Slender cytoplasmic projections, which extend from the leading edge of migrating cells.

Cytonemes

Actin-based filopodial cell extensions.

Nanotubules

Cytonemes that connect blood cells over a distance of several cell diameters and transport membrane proteins, lipids and ions from one of the connected cells to another, thus executing long range intercellular communications.

Macropinocytosis

A mechanism of endocytosis in which large droplets of fluid are trapped underneath extensions (ruffles) of the cell surface. Can be exploited by some pathogens as a route for entry into cells.

Glans penis

Sensitive tip of the penis. When the penis is flaccid it is wholly or partially covered by the foreskin, except in men who have been circumcised.

Biographies

Florian Hladik

Florian Hladik obtained his M.D., Ph.D. and dermatology training at the University of Vienna in Austria. He carried out postdoctoral research at the Johannes Gutenberg University in Mainz, Germany, and the University of Washington, USA. He is currently Research Assistant Professor in the Departments of Gynecology and Medicine, University of Washington, and Affiliate Investigator at the Fred Hutchinson Cancer Research Center in Seattle. His research focuses on mucosal HIV transmission pathogenesis, microbicide development and infectious causes of fetal prematurity.

M. Juliana McElrath

After receiving her M.D., Ph.D. and internal medicine residency training at the Medical University of South Carolina, Dr. McElrath undertook a clinical fellowship in infectious diseases at Columbia University and a faculty position at the Rockefeller University. For nearly two decades she has focused her research efforts on HIV pathogenesis, mucosal immunity and vaccine development in Seattle. She currently is a Member and Co-Director of the Vaccine and Infectious Disease Institute at Fred Hutchinson Cancer Research Center and a Professor of Medicine at the University of Washington.

Online Summary

• HIV invasion through the mucosae of the female lower genital tract contributes the largest number of new HIV-1 infections worldwide. The second leading site of viral invasion is the lower male genital tract, followed by invasion via the rectal mucosa in both women and men.

• Explant models of human genital tissues have provided new insights into the mechanisms of sexual HIV transmission.

• Initial attachment of HIV-1 to the mucosa may be aided by cervical mucus and a variety of gp120-binding surface receptors on epithelial cells. HIV-1 penetration into the genital mucosa occurs rapidly after exposure and is possibly enhanced by microabrasions or genital ulcer disease.

• In the human vagina, intraepithelial CD4⁺ T cells and CD1a⁺ Langerhans cells are the first cells infected by HIV-1.

• Vaginal Langerhans cells exhibit a high capacity to endocytose HIV-1 virions. C-type lectins such as DC-SIGN (CD209) or langerin (CD207) appear to play little to no role in mediating this infection pathway.

• Genital CD4⁺ T cells express high levels of CCR5, are rapidly infected by HIV-1 and produce large quantities of viral progeny.

• Dendritic cells utilize several pathways to enhance viral propagation to CD4⁺ T cells for productive infection. Presumably these occur as well in the genital mucosa, but direct evidence is lacking.

• The highly protective effect of circumcision indicates that viral invasion in men occurs predominantly through the inner foreskin, where both CD1a⁺ Langerhans cells and CD4⁺ cells are abundant. The second leading site of viral invasion in the male genital tract is likely the penile urethra.