Understanding Factors That Modulate HIV Infection at the Female Genital Tract Mucosae for the Rationale Design of Microbicides

Abstract

Women are now becoming the pivot of the epidemiological spread of HIV infection worldwide, especially in developing countries. Therefore, research to develop an efficient microbicide is now a priority for the prevention of HIV-1 acquisition in exposed women. However, recent disappointing failures in microbicide clinical trials revealed major gaps in basic and applied knowledge that hinder the development of effective microbicide formulations. Indeed, the inhibitory power of microbicide molecules may be affected by several physiological and immunological factors present in male and female genital tracts. Furthermore, mucosal crossing of HIV-1 to increase the ability to reach the submucosal target cells (macrophages, lymphocytes, and dendritic cells) may be modulated by supraepithelial factors such as seminal complement components (opsonized HIV-1), by epithelial factors released in the submucosal microenvironment such as antimicrobial soluble factors, cytokines, and chemokines, and by potent intraepithelial and submucosal innate immunity. The design of vaginal microbicide formulations should take into account an understanding of the intimate mechanisms involved in the crossing of HIV through the female genital mucosae, in the context of a mixture of both male and female genital fluids.

Introduction

A ccording to the World Health Organization reports, the majority of HIV infections occur through heterosexual intercourse resulting in an estimated 34 million people living with HIV-1 worldwide, with almost 2.7 million new infections per year. A growing discrepancy exists in infection rates between men and women, most notably in sub-Saharan Africa where more than 60% of the people living with HIV-1 are women of child-bearing age.

The incidence of HIV is declining globally albeit slowly.¹ This decline in incidence is related to the reduction in risky behavior that has occurred in key sub-Saharan countries.² In these countries abstinence, monogamy, and a reduction in the number of sexual partners, and the use of barrier methods such as male and female condoms, are widely advocated. Condom use currently offers the best method to prevent HIV-1 transmission, but it requires partner consent. It seems that abstinence is not a realistic choice for many women, and consistent condom use remains low, especially in primary partnerships, as well as in couples expecting to have children. Likewise, research to develop biomedical approaches can then constitute an alternative strategy to prevent acquisition and transmission of HIV-1. Recently, postexposure and preexposure prophylaxis (PrEP) with antiretroviral drugs (ARVs),³ treatment of sexually transmitted infections,⁴ and male circumcision⁵ have been shown to significantly decrease the likelihood of HIV transmission from HIV-positive individuals to their HIV-negative partners. However, before the implementation of circumcision programs, behavioral and social research is needed to find out how people perceive the practice and what messages are needed to persuade them, and to assess the risk of an increase in unsafe sex after circumcision.

Among newly designed preventive methods, ARVs have been highly effective in preventing perinatal transmission. Results of the iPrEx trial demonstrated that the daily oral administration of emtricitabine/tenofovir reduced the risk of HIV acquisition among men who have sex with men by 44%.^6,7 The Partners PrEP Study in HIV-serodiscordant couples from Kenya and Uganda showed that the daily oral administration of tenofovir or an emtricitabine/tenofovir combination reduced HIV-1 infection by 67% and 75%, respectively.^7

–10 Among biomedical approaches, microbicides hold the greatest hope of curtailing AIDS worldwide. Recently, the CAPRISA 004 clinical trial showed, for the first time, that a 1% vaginal gel formulation of tenofovir, a nucleoside analog reverse transcriptase inhibitor (NRTI), protected women from HIV-1 acquisition with an overall efficacy of 39% and 54% observed in women who were high adherers¹¹ (i.e., reported to use the gel more than 80% of the time).

Effective prevention strategies involving blockade of HIV-1 mucosal transmission by using topical compounds such as microbicides will afford women the ability to protect themselves with a method that may also serve as a contraceptive. These agents were described initially as “virucides” because the first compounds evaluated such as detergents and acid buffering agents acted directly on the viral particles in a nonspecific manner by disrupting the viral envelope.¹² The name now commonly accepted for such products is “microbicides,” reflecting their ability to offer protection not only against viruses, such as HIV-1 and genital herpes, but also against bacteria that could modulate HIV-1 transmission, such as Gonorrhea or Chlamydia. ¹² Although the term microbicides has been entrenched in the field, it does encompass specific antiretroviral agents that are also described as “topical PrEP.” Effective microbicide formulations must be safe to use, chemically and physically stable, compatible with latex and other materials used in barrier devices, affordable, and acceptable to the end user. They will be formulated as rings, gels, foams, creams, impregnated sponges, suppositories, or films, for insertion prior to sexual intercourse.

The recent disappointing failure of large-scale vaccines¹³ and the need to improve microbicide efficiency^14

–17 revealed that major gaps in basic science remain to the development of effective microbicide formulations.¹⁸ Indeed, it seems to be difficult to translate in vitro and animal studies to clinical outcomes.¹⁹ We will here review current hypotheses concerning the crossing of HIV-1 through the genital mucosa with the aim of developing a design for microbicide formulations.

Transport of HIV-1 from the Female Genital Tract Toward the Lymph Nodes

Details concerning HIV-1 mucosal transmission have not been determined in humans but are essential for the design of effective vaginal microbicides. In the inferior female genital tract, cervicovaginal mucosal tissues include the columnar monolayer epithelium of the endocervix and the squamous multilayer epithelium of the vagina and ectocervix. The monolayer zone of transformation (between ectocervix and endocervix) and endocervix were described as potential mucosal sites of HIV entrance.^20,21 Recently, noninvasive imaging combining serial single-photon emission-computed tomography (SPECT/CT) and magnetic resonance (MR) analysis showed a radiolabeled cell-free and cell-associated HIV-1 surrogate distribution with the highest intensity in the vaginal pericervical area, without detectable signal in the uterus.²² However, these are not the exclusive site of entry, as SIV has been transmitted to hysterectomized animals and HIV-1 in the congenital absence of a cervix.²¹ Since women with HIV-1 detected in their cervical secretions were shown to have a thinner cervical epithelium and lower number of cellular layers,²³ we cannot exclude the possibility that exposure to HIV-1 impairs mucosal epithelial integrity.²⁴

The mechanism by which the virus crosses a tight layer of epithelial cells is still not known, and five potential mechanisms were thought to mediate HIV-1 mucosal transmission. These mechanisms included direct infection of epithelial cells, rapid transcellular transport through epithelial cells or “transcytosis,” epithelial transmigration of infected donor cells, uptake by intraepithelial dendritic cells, and circumvention of the epithelial barrier through physical breaches.²⁵ Since there is no evidence in vivo of transient infection of epithelial cells,^26,27 HIV-1 may cross a monolayer of epithelial cells by transcytosis, as suggested by experiments showing a link between protection of macaques against vaginal challenges and local production of antibodies that specifically inhibit HIV-1 transcytosis.²⁸ However, the extent to which these individual mechanisms contribute to mucosal HIV-1 infection is a matter of intensive investigation. A clear understanding of early events in sexual transmission, the immunological microenvironment, the target cells, and the type of transmitted viruses indeed constitutes the foundation for rationally designing products able to block specific viral entry pathways.

Role of genital inflammation in facilitating HIV-1 transmission

In macaques, SHIV_SF162P3 vaginal inoculation was followed by proinflammatory cytokine production in cervicovaginal lavages (CVL) and plasma²⁹ and recruitment of CD4⁺ T cells that were necessary for mucosal viral spread and dissemination to the systemic compartment.³⁰ Notably, elevated genital cytokine concentrations, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8, were associated with higher viral load set points, replication and shedding of HIV, and CD4 depletion, suggesting that strategies aimed at reducing genital inflammation may limit disease progression.³¹ The magnitude of cytokine induction in CVL seems to be higher in infected macaque when compared to uninfected macaques.²⁹ Concentrations of cytokines/chemokines in cervicovaginal fluids may also be regulated by pregnancy and the menstrual cycle (e.g., IL-1β, IL-10, IL-6, and IL-8) and/or bacterial vaginosis and sexually transmitted infections (STIs) [Nesseiria gonorrhoeae, Chamydia trachomatis, Trichomonas vaginalis, Candida albicans, Mycoplasma hominis, Peptostreptococcus ascharolyticus, or herpes simplex virus-2 (HSV-2)].^32,33 However, the number and distribution of immune cells in the cervicovaginal mucosa were found to remain constant throughout the menstrual cycle, indicating that sex steroid hormones modulate the metabolic activity of immune cells that are already present in the tissue.^34,35

Interestingly, a window of most frequent transmission of virus was estimated in the late luteal phase when progesterone levels are high and when local immunity may be low,³⁶ supporting findings concerning the higher susceptibility to HIV in women during progesterone-dominated periods including pregnancy and contraceptive use. Indeed, progesterone is known to inhibit cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, decrease and alter glycosylation of IgG and IgA antibodies, modulate cytokine production, and up-regulate HIV-1 receptors at the surface of CD4⁺ T cells.^37,38 In multiple studies in humans and macaques, high progesterone levels were associated with a higher viral burden, increased viral shedding,^39,40 and therefore accelerated HIV-1 disease progression.⁴¹

However, activation of the mucosal innate immune system is necessary to control early and local viral replication. Indeed, it was suggested that the lack of a protective effect of rDC-SIGN gel may be due to DC-SIGN binding to the virus, thereby reducing viral antigenic exposure to both innate and adaptive immunity, which lowered antigenic exposure and then the magnitude of early proinflammatory mucosal responses.²⁹ Several lines of evidence suggest an important role for specific polyfunctional T cell responses in immune control of HIV infection.⁴² Nevertheless, the induction of HIV-specific CD8⁺ effector cells was not clearly related to a better control of genital HIV-1 shedding.³¹ Several studies showed that by reducing the inoculum dose used in a mucosal challenge infection model, the time to the first positive peripheral plasma SIV RNA level was lengthened.⁴³ Furthermore, two recently published studies showed that the combined use of a microbicide and a T cell-based vaccine limited the early mucosal transmission of HIV-1,^44,45 suggesting that a vaccine established sufficient immunity for control of SHIV or SIVmac251 infection if assisted by a microbicide molecule that expands the potential window of vaccine effectiveness by slowing local virus expansion and replication.

Proinflammatory environment may be induced by interactions between epithelial cells and viral particles

Epithelial cells constitute the main cell population in the initial contact with HIV-1 during its crossing through mucosae. As the initial barrier against viral entry, epithelial cells may hamper the crossing of the virus by their intrinsic integrity and solidity. Indeed, the disruption of the reproductive tract epithelium, as observed with nonoxynol-9, may facilitate HIV infection and serve as a portal for human HIV transmission.⁴⁶ However, the intrinsic mucosal properties also seem to be important since the penetrability of the rhesus macaque mucosa to the virus was reported to be dependent on the origin of mucosal tissues.⁴⁷ Indeed, it was found that the rectal route requires the smallest quantities of virus to infect macaques, followed by the vaginal route and then the oral route, reflecting the risk order of sexual HIV-1 acquisition among humans.⁴⁷ Moreover, the tightness and the integrity of the epithelium change markedly during the menstrual cycle,³⁰ as reported by the enhancement of SIV vaginal transmission observed in progesterone pretreated macaques. Furthermore, the viral gp120 seems to alter the integrity of the mucosal epithelial barrier, which then allows virus translocation by disrupting tight junctions by a mechanism that is TNF-α dependent.²⁴ With the lowest tightness, single epithelial monolayers, such as the endocervix and rectal mucosae, may support rapid transcellular transport or “transcytosis” from the apical to basolateral pole of the cell, and thus provide viral access to the cells of the submucosa. We have previously reported that less than 1% of HIV-1 can cross through the epithelial monolayer by transcytosis.⁴⁸

Vaginal epithelial cells may also modulate HIV-1 transmission by secreting monokines or proinflammatory factors such as thymic stromal lymphopoietin (TSLP) that enhance the maturation, activation, or recruitment of HIV-sensitive submucosal cells.⁴⁹ We have shown that CCR5-tropic HIV-1 interacting closely with the apical pole of epithelial cells induced the basolateral release of macrophage-attracting chemokines and proinflammatory cytokines.⁵⁰ In agreement with our data, inflammatory responses (including IL-1β, TNF-α, IL-10, and IL-12) in genital secretions are found to be correlated with infection with multiple HIV-1 variants, suggesting that the virus may use epithelial cells to ensure its own spread.³¹ Notably, a proinflammatory mucosal environment may modulate the expression of HIV receptors at the surface of submucosal target cells.^51,52 More recently, it was shown that glycerol monolaurate, a widely used antimicrobial compound with inhibitory activity against the production of MIP-3α and other proinflammatory cytokines, can inhibit in vitro mucosal innate and inflammatory responses to HIV-1 and SIV, and in vivo it can protect rhesus macaques from acute infection even after repeated intravaginal exposure to high doses of SIV.⁵³ Overall, this study is consistent with studies showing that the establishment of a systemic infection requires a local expansion of a small founder population of infected cells in the cervicovaginal mucosa.^54,55 Altogether, these findings highlight the necessity of developing microbicide molecules that would prevent the early replication of the virus in mucosal tissues.

Submucosal replication of HIV-1

Early after HIV exposure, a small population retained the virus in the submucosa; this event was followed by local replication, which then resulted in expansion of virus replication mediated by the inflammatory environment.⁵³ Langerhans cells (LCs), defined by the cell surface expression of CD1a and langerin, are found within the epithelium. CD11c⁺ DCs, CD68⁺ macrophages, and CD4⁺ T cells are localized at the intraepithelial level and beneath the epithelial layer in the submucosa.^56

–59 Infected DCs, LCs, and macrophages have been observed shortly after sexual transmission,^{53,57,60

–63} strongly suggesting that these innate immune cells are initial targets for HIV-1. Thus, identification of cell receptors mediating virus attachment may be important to specifically prevent the initial infection of these HIV-1 target cells. Some lectins such as the mannose receptor (MR), the DC-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN; CD209), and the DC immunoreceptor (DCIR) were identified as attachment receptors for HIV-1 on specific DCs and macrophage subsets.^64
–66 In addition, attachment of HIV-1 on epithelial cells^50,67 and submucosal target cells (T cells,⁶⁸ macrophages,⁶⁹ and DCs) was found to be mediated by glycosaminoglycans (GAG) such as syndecan.⁷⁰ Several studies showed that syndecans together with other lectins or host proteins, by acting as attachment receptors, facilitate HIV-1 entry.

Some of these attachment receptors also mediate de novo virus production. Indeed, DC-SIGN, MR, and DCIR promoted infection of innate immune cells as well as transmission of HIV-1 to target T cells, while langerin prevented infection through viral degradation.^71,72 Therefore, the surface expression of specific lectins influences target cell infection as well as virus internalization, and may participate in the involvement of innate immune cells in HIV-1 transmission through mucosae. Gringhuis et al. demonstrated that virus production by DC required some defined innate signaling events.⁷³ The first signal resulted from the interaction between HIV-1 genomic RNA and Toll-like receptor 8 (TLR8) that then promoted transcription initiation from the integrated provirus. The second signal, mediated by the close interaction between HIV-1 glycoprotein gp120 and the C-type lectin DC-SIGN, was necessary to activate the kinase Raf-1 and to lead to transcription elongation and productive virus expression. Immature DCs (iDCs) in the mucosa are among the first cells that capture HIV-1 and play a critical role in the early dissemination of HIV-1 to CD4⁺ T cells following sexual transmission.^60,74 Blocking the interaction between HIV-1 and DC-SIGN may also prevent viral transmission.

We have previously demonstrated a synergistic activity between entry inhibitors and an RT inhibitor against CCR5-tropic HIV-1 infection,⁷⁵ suggesting that a combination of microbicide molecules would be a most promising strategy to block the viral cycle at different stages.⁷⁶ However, it remains very speculative to currently identify the best antiretroviral strategy to block the initial capture of HIV-1 by DCs, and, in addition, its transmission from DCs to CD4⁺ T cells and even its replication by DCs.⁷⁷

Role of innate immunity

During inflammatory responses, neutrophils are one of the first innate immune subsets to be recruited to the site of infection where they generate inflammatory responses.⁷⁸ Neutrophils were identified in the cervix from both HIV-infected and uninfected women.⁷⁹ A correlation was found between cervical neutrophil numbers and concentration of IL-1β, TNF-α, IL-10, and IL-6, suggesting that the HIV-induced increase of those cytokines in vaginal fluids is related to the activation of neutrophils.⁸⁰ Their role in mucosal HIV transmission needs to be further investigated.

Early stages of viral infections were also associated with local recruitment and activation of other effectors of innate immunity, i.e., NK cells and DCs. The maturation and homeostasis of DCs seem to be controlled by a crosstalk between DCs and NK cells. During contact between activated NK cells (aNK) and iDCs, HMGB1 was found to promote the maturation of iDCs and the induction of IL-12-dependent T-helper-1 responses.⁸¹ HMGB1 could act as an alarmin, a danger signal to alert the innate immune system to initiate the host defense, promoting the global immune activation reported in HIV-1 patients.⁸² Following infection of iDCs with HIV-1, DCs were no longer susceptible to NK-dependent IL-12 polarization, and thus were no longer able to induce a Th1 response, which constitutes a strategy used by the virus to escape to the immune system. In addition, NK-dependent DC maturation and survival were associated with increased production of the HIV-1 p24 antigen and expression of viral DNA by DCs.⁸¹ Interestingly, significantly high levels of HMGB1 were released in the cervicovaginal secretions of HIV-1-infected or HSV-2-infected women,⁸³ suggesting that coinfection with HSV-2 may increase HIV-1 transmission. In addition, a positive correlation between HMGB1 concentrations and disease progression was found,⁸⁴ suggesting that HMGB1 plays a central role in the global immune activation observed in HIV-1 infection.

Following HIV capture, extensive in vitro and in vivo studies have shown that the transfer of the virus from DCs and/or LCs toward CD4⁺ T cells may have an important role in HIV transmission.^56,85 Indeed, Perreau et al. showed that the failure of a recent vaccine was due to the induction of the maturation of DCs by the vaccine, which was associated with better activation of CD4⁺ T cells by DCs, which in turn efficiently replicated HIV-1.⁸⁶ Transmission of the virus from DCs to CD4⁺ T cells seems to be mediated through a tight synapse by a DC-SIGN-dependent mechanism⁸⁷ or through exosome traffic.⁸⁸ In fact, exosomes are antigen-bearing vesicles released by DCs. They constitute a way for DCs that have captured antigen to transfer it to other DCs for presentation and to subsequently elicit efficient specific T cell responses.⁸⁹ Similarities in composition and size between exosomes and HIV particles as well as in their colocalization in DC compartments suggest that the development of a blocking reagent against this kind of transfer would be very difficult.

We and others found that the microenvironment may differentiate macrophages into distinct populations that would facilitate the dissemination of the virus and the emergence of reservoirs. An interferon (IFN)-γ-induced Th1 environment⁵² and IL-13-induced Th2 environment⁹⁰ resulted in the differentiation of macrophages into subsets that archived HIV-1 within a latent stage and resulted in the persistence of the virus by either favoring the recruitment of HIV-1-sensitive T cells or enhancing the viral production by these recruited T cells. However, differentiated macrophages in the presence of the Th2 cytokine IL-4 dramatically enhance the spread of HIV by capturing, integrating, producing, and transferring (toward CCR4⁺ IL-13⁺ T cells) higher levels of HIV-1.⁹⁰ High levels of lipopolysaccharides (LPS) were found in the serum of HIV-1-infected patients. LPS-treated macrophages released high doses of IL-10 that dramatically decrease the activation of CD4⁺ T cells by a PD-1/PDL-1-dependent mechanism.⁹¹ Taken together, these data suggest that the possible modulatory properties of microbicide formulations on mucosal innate immunity may, by itself, favor or decrease HIV replication. However, to our knowledge, no evaluation of the effect of microbicides on the antiviral function of cells on innate immunity has yet been proposed in the preclinical evaluation of microbicide formulations.

In spite of these recent advances in the understanding of the role of innate immunity in the control of HIV-1 replication, further work is needed to determine the role of other innate immune effectors such as NK T cells and γδ T cells. Indeed, γδ T cells may play an important role in mucosal protection against microorganisms.⁹² These cells are also able to release Th1 and Th2 cytokines that may modulate the activation of CD4⁺ T cell responses.⁹³ γδ⁺ T cells located in cervicovaginal tissues were found to release anti-HIV factors such as RANTES, MIP-1α, and MIP-1β that may prevent SIV infection.⁹⁴ Notably, their frequency was up-regulated by the treatment of macaques with GM-CSF and heat shock proteins, suggesting that mucosal immunization with heat shock proteins to activate γδ ⁺ T cells may also be a promising strategy.

Factors Modulating HIV Transmission

The nature of the transmitting virus

The selection of single or limited variants during transmission across the cervical/vaginal mucosa is still not clear. It was suggested that the mucosa exerts a selection pressure on the transmitted/founder (T/F) virus or that a competitive selection among transmitted variants occurs during the crossing from the mucosa to the systemic compartment.⁹⁵ There are several challenges to the characterization of T/F virus properties including identification of acutely infected individuals, development of an optimal robust and validated set of models to generate T/F molecular clones, and development of sensitive in vitro assays to detect phenotypic differences that could impact transmission fitness in vivo. ⁹⁶ Pseudoparticles expressing the viral envelope glycoprotein^97,98 found in viruses isolated weeks to month after infection⁹⁹ were used to study viral properties associated with mucosal transmission. At the mucosal level, an immune response against the virus may occur rapidly,¹⁰⁰ and induce mutations in the viral envelope leading to viral escape, suggesting that “early”-isolated viruses may differ from true T/F viruses.¹⁰¹ However, a limited viral evolution prior to the peak of viremia was observed,⁹⁷ suggesting a finite window of potential vulnerability of HIV-1 to specific engineered antibodies in microbicide formulation. A recent study involving the analysis of HIV-1 variants of transmitter–recipient pairs indicated selection for a transmitting virus variant with a set of essential biological properties, such as fewer glycosylation sites, more compact envelope domains, and a histidine residue at position 12 of the leader sequence of the envelope protein,¹⁰² and in some cases, enhanced sensitivity to neutralization by CD4 binding site monoclonal antibodies.¹⁰³

Several studies showed that T/F and chronic viruses used CD4 and CCR5 with equal efficiency,¹⁰⁴ whereas other studies showed that the overall replication efficiency of T/F viruses was significantly lower in monocyte-derived macrophages compared to prototypic "highly macrophage-tropic" virus strains.¹⁰⁵ Notably, these T/F viruses replicated more efficiently in CCR5⁺ CD4⁺ T cells than in macrophages.^106,107 Finally, a viral subpopulation is archived as proviral DNA in the female genital tract early in primary infection, suggesting that HIV-1 variants from the male donor are selected in the female mucosal site during male-to-female transmission of HIV-1.¹⁰⁸

Furthermore, R5 and X4 viruses were found to cross through a tight monolayer of epithelial cells with equal efficiency.⁹⁴ Contrary to SHIVHxBc2, which replicates only in macrophages, the T lymphocyte restricted SIVmac239-induced systemic infection in vaginally challenged macaques,¹⁰⁹ indicating that there is no correlation between the tropism of the virus and its capacity to replicate and to induce systemic infection in the macaque model. Since most of the HIV-1 sequences in semen are CCR5 tropic^110,111 and transmitting donors are mostly asymptomatic, it was expected that transmitting HIV-1 would be predominantly CCR5 tropic. Other studies have also shown that epithelial cells were able to specifically sequester X4 viruses and to likely promote the preferential transmission of R5 viruses.^48,112 Saba et al. found that productive infection of cervicovaginal T lymphocytes with R5 viruses is much more efficient than with X4 viruses, suggesting that HIV-1 gatekeeping mechanisms exist in the cervicovaginal mucosa, preventing X4-HIV-1 from replicating as efficiently as R5-HIV-1.¹¹³ Interestingly, the diversity of viruses transmitted intravaginally seems to be more restricted than those transmitted through the intravenous route, suggesting that host mucosal factors are mainly implicated in this selection.¹¹⁴

Altogether, these current data utilizing a large number of envelope constructs strongly suggest that the mucosal bottleneck in the vagina is not the result of selective transmission of viruses with highly efficient CD4 or CCR5 use or with increased efficiency of entry into particular CD4⁺ CCR5⁺ mucosal cell subsets.¹⁰⁴ Therefore, it is possible that the mucosal immune system acts on the selection of HIV-1 variants and that the selection of viral variants occurs at the regional draining lymph nodes after crossing mucosal tissues. Further studies with cell-associated SIV and semen in a macaque model would be useful in defining the pattern of cell-associated virus transmission compared to cell-free virus transmission, and the impact of semen on the selection of R5 variants in the mucosa and of its subsequent transmission.

In addition, it is still unclear whether infection occurs via a cell-associated or cell-free mechanism. Both cervicovaginal secretion and sperm contain HIV-1-infected T cells and macrophages that are able to produce viral particles. Notably, a study of four transmission pairs from a well-characterized cohort of recently HIV-infected men who have sex with men revealed that in all these cases, the virus establishing infection in the recipient originated from the seminal plasma, suggesting that cell-free rather than cell-associated viruses are the origin of sexually transmitted HIV-1.¹¹⁵ In addition, transmission via cell-associated SIV was found to be much less efficient than cell-free virus.^116,117 However, in vivo experiments showed that infected cells reached to cross through vaginal epithelial cells.¹¹⁸ In this study, cells from the spleen and not isolated from sperm were used to infect female macaques through the vaginal route. Cells from sperm and spleen may differentially express receptors such as those implicated in adhesion, viral attachment, and migration of cells, which may modulate their transmigration. Upon budding of the virus from host cells, viral particles incorporated cellular proteins such as ICAM-1, HLA-DR, CD40, CD40L, and CD86, and this enhanced their infectivity.¹¹⁹ Thus, the composition of the surface viral particle appears to influence the selection and the transmission of the most replicative variants.

Further analyses are required to characterize the cells that are able to transmigrate and to define the source of the seminal plasma virus. In addition, the viral load in the ejaculate needed for productive mucosal infection is not known yet.

HIV modulating factors found in the female genital tract

Epithelial cells from the genital tract produce several biological factors including a hydrophilic surface layer of glycoproteins and glycolipids, called the glycocalyx, and thick hydrophobic glycoprotein mucus¹²⁰ that may act as an initial barrier. Cervicovaginal mucus obtained from donors with lactobacilli dominant flora efficiently trapped HIV-1.¹²⁰ Hydrogen peroxide-producing (H₂O₂ ⁺) Lactobacillus, a key regulator of the vaginal ecosystem, was found to decrease HIV-1 replication mostly through direct effects, whereas lactic acid was found to suppress bacteria associated with bacterial vaginosis.¹²¹ However, cervicovaginal fluids and semen showed significant H₂O₂-blocking activity.¹²² Similar to inflammatory-associated diseases, bacterial vaginosis and diseases that alter epithelial integrity increased HIV-1 transmission. Microbicides and other topical applications prevented vaginal transmission of HIV, indicating that an efficient microbicide could preserve the vaginal flora.¹²³

Others antimicrobial compounds were produced such as secretory IgA or natural IgG antibodies and lactoferrin. Recently, it was found that following immunization and HIV reexposure, IgG and IgA antibody-secreting cells were significantly correlated with ADCC killing, ADCVI activity, and inhibition of transcytosis.¹²⁴ Upon reexposure to antigen, strong anamnestic antibody responses were detected with the rapid increase in antigen-specific circulating antibody levels, which was associated with weaker viral replication. However, some natural antibodies were found to enhance the capture of HIV-1 by dendritic cells via the Fc receptor, resulting in increased viral production, which constitutes a new way for the virus to use the immune system for own advantage.¹²⁵ The activity of these HIV-1-enhancing factors may be unbalanced by released mucosal antiretroviral factors, including RANTES, elafin, secretory leukocyte peptidase inhibitor (SLPI), histatins, statherin, lysozymes, and cystatins. Efficient microbicide formulations should not affect the abundance and/or effectiveness of these mucosal antiviral factors.

Several studies suggested that active immune regulatory mechanisms, rather than intrinsically attenuated innate immune responses, underlie the low levels of immune activation characteristic of resistance to HIV infection in humans¹²⁶ and in the macaque model.¹²⁷ Compared with HIV-unexposed individuals, it was reported that people who have been repeatedly exposed to HIV but are resistant to infection have reduced frequencies of activated T cells and elevated frequencies of regulatory CD4⁺ T cells (Treg).¹²⁶ A recently published paper suggests that a combination of a modest vaccine-induced CD8⁺ T cell response in the context of immunoregulatory suppression of T cell activation protected against vaginal HIV transmission.¹²⁸ This study highlighted the need to induce a protective adaptive immunity against the virus with a suppressive environment preventing virus replication in activated mucosal cells susceptible to HIV.¹²⁸ Moreover, the soluble factor PD-1 was able to negatively modulate immune activation by limiting the activation of both CD8⁺ T cells¹²⁹ and CD4⁺ T cells,⁹¹ indicating its potential use as a therapeutic/preventive target. In addition, the release of plasma proapoptotic factors [such as TNF-related apoptosis-inducing ligand (TRAIL), Fas ligand, TNF receptor type 2 (TNFR-2), and microparticles] early after infection was shown to have a suppressive effect on T cells, B cells, and macrophages.¹³⁰ The release of products of cell death from mucosal cells and the subsequent immunosuppression following HIV-1 transmission could potentially narrow the window of opportunity to block HIV amplification in mucosal tissues.¹³⁰ A comparison of the nonpathogenic and pathogenic models also revealed other factors that may contribute to regulating immune activation, including (1) IDO, an immunosuppressive molecule produced by macrophages and DCs regulating the proliferation of T cells and antagonizing IFN activity,¹³¹ (2) ADAR, an adenosine deaminase-suppressing IFN-stimulated gene (ISG) expression,^127,132 and (3) TIM3, a marker of T cell exhaustion, and its ligand galectin-9, a proapoptotic ISG.¹³³ Therefore, the activation of mucosal immunoregulatory mechanisms constitutes a new area of investigation to prevent early events in HIV-1 mucosal transmission.

HIV-1-inducing factors found in sperm

It has been reported that semen may enhance HIV infection.¹³⁴ HIV-1 concentrations in the semen are one of the predictive factors of HIV-1 transmission. Semen contains cell-free and cell-associated HIV-1 and then represents the main vector of HIV-1 dissemination. Within hours of coitus, macrophages, DCs, granulocytes, neutrophils, and memory CD8⁺ T cells are recruited into the endometrial stroma and lumen.¹³⁵ An accompanying increase in GM-CSF, IL-6, IL-8, and IL-1 was also induced in cervical tissues, which is consistent with seminal-plasma-induced cytokines acting to control leukocyte recruitment and activation in the female genital tract.^136,137 Indeed, seminal TGF-β was shown to stimulate GM-CSF production and inflammatory cell recruitment in the murine uterus.¹³⁸ In all semen samples from healthy men, IL-8, IL-7, SDF-1a, and MCP1 were present in high concentrations whereas IL-5, IL-6, IL-13, IL-17, IFN-α, RANTES, MIP-1β, IL-1α, IL-1α, G-CSF, and M-CSF were detected at low concentrations.¹³⁹ MIP-1α, IL-2, IL-10, IL-12, TNF-α, IFN-γ, and GM-CSF are detected in a minority of semen samples.¹³⁹

Some cytokines activated regulatory T cell responses, such as TGF-β, which is known to facilitate preparation of the female reproductive tissues for pregnancy.¹⁴⁰ This regulatory property may limit HIV replication. However, it has also been shown that TGF-β can potentiate the recruitment of immature CCR5⁺ Langerhans cells and can then facilitate the transport of viruses through epithelial cells toward the lymph nodes.¹⁴¹ These studies suggest that as a first step, proinflammatory factors found in semen and those induced in the female genital tract following HIV-1 exposure may facilitate recruitment of HIV-1 target cells and their subsequent infection. In addition, they indicate that as a second step, the local activation of immunosuppressive mechanisms may limit dissemination of the virus as well as the induction of a protective immune response, facilitating the escape of several viral strains and their subsequent dissemination. Cytokines may also activate the local replication of the virus in mucosal target cells (dendritic cells, macrophages, and T cells) soon after its crossing through the vagina. To block the replication of HIV-1 in the first cells infected in mucosal tissues, RTIs have demonstrated efficacy as microbicides.¹⁴² Compared to NRTIs, non-NRTIs (NNRTIs) seem to be more potent, in part because of their long half-life due to their capacity to bind irreversibly to RT, disrupting the binding of substrates to the active site of the enzyme. Recent examples of NNRTIs in clinical evaluations as microbicide candidates include dapivirine and UC781 (http://www.ipmglobal.org/publications/first-efficacy-trial-microbicide-ring-prevent-hiv-underway).¹⁴³

In addition to free viruses and infected leukocytes, spermatozoa-associated virions are also one of the major sources of infectious virus in semen. Heparin sulfate moieties, expressed in spermatozoa, were found to play an important role in the capture of HIV-1 and to mediate efficiently the transmission of HIV-1 to DCs, macrophages, and T cells.¹⁴⁴ Notably, at low values of extracellular pH, similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced, suggesting that spermatozoa may act in the early transmission of HIV-1 infection. In addition, opsonization of free HIV-1 particles by complement components found in semen enhanced in vitro infection of macrophages and DCs and also the viral transfer to CD4⁺ T cells.⁵⁰ We have recently shown that HIV-1 opsonization may modulate in vitro the efficiency of candidate microbicides to inhibit HIV-1 infection of mucosal target cells, as well as its crossing through the mucosa.¹⁴⁵ Sperm contains other HIV-1-inducing proteins such as fibril-forming prostatic acidic phosphatase (PAP),¹³⁴ basic polyamines (spermin and spermidin), and free nucleotides (dNTP) that may protect HIV-1 virions from the threat of acid inactivation in the vaginal tract and enhance the transcription of viruses or TGF-β1.

However, sperm also contains antimicrobial compounds such as SLPI,¹⁴⁶ β-defensins, and natural antibodies against DC-SIGN or CCR5.^51,87 Further work is needed to identify all these semen-activating factors, which will then provide an alternate strategy to limit HIV-1 transmission by specifically blocking all these HIV-1-inducing factors.

Conclusions

Well-defined mechanisms of HIV-1 mucosal transmission are an essential component of rational microbicide development. Despite years of basic science, methods by which HIV-1 crosses through epithelial cells, identification of HIV-1 target cells that replicate the virus locally in the female mucosal tissue, and characterization of cells that disseminate the virus are still not well defined (Fig. 1).

FIG. 1.

Microbicides would delay mucosal viral replication or decrease the infectious dose of the virus, which helps the induction of efficient and specific anti-HIV mucosal responses. Five potential mechanisms are thought to mediate HIV-1 mucosal transmission: direct infection of epithelial cells, transcytosis, epithelial transmigration, uptake by intraepithelial dendritic cells, and circumvention of the epithelial barrier through physical breaches. Mucosal crossing of HIV and further reaching of submucosal HIV-sensitive cells may be modulated by supraepithelial factors, such as seminal complement component (opsonized HIV), and by epithelial factors released in the submucosal environment, such as antimicrobial factors, cytokines, and chemokines, and by the submucosal innate immunity. Once HIV-1 has reached the submucosal environment, numerous HIV target cells may support the local replication of the virus [macrophages (Mϕ), DCs, and CD4⁺ T cells]. This HIV replication may be dramatically increased by several cells of the innate immune system (neutrophils and NK cells). More specifically, NK cells may promote viral dissemination by enhancing HIV replication by DCs and their migration to the lymph nodes where they may transfer the virus to T cells. To prevent HIV mucosal transmission, a microbicide would act on the HIV crossing through mucosae (1) by limiting its attachment of the virus to epithelial cells (1a) and/or their migration (1b and 1c), but also on the local replication (3) by inhibiting infection of submucosal cells (2). If the microbicide is not 100% efficient, the delay of viral replication would be efficient to the activation of the adaptive immune system. Cytokines and chemokines released by HIV-activated innate cells may elicit the induction of anti-HIV CD4⁺ and CD8⁺ T cells, and the release of specific antibodies that may neutralize directly the virus or induce antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI). The time of induction of these anti-HIV-specific responses may be shortened by a prevaccination of individuals that activates efficient memory-specific responses. However, there is no HIV vaccine currently available and poor understanding of the subtle orchestration of the numerous viruses, cell factors, and HIV target cells involved in HIV mucosal transmission renders the design of an effective microbicide formulation difficult. DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin; MBP, mannose-binding protein; NK, natural killer; NKT, natural killer T cells.

In women, technical challenges associated with obtaining genital tissue limit our understanding of the important role of the cervicovaginal environment in the induction of innate and adaptive immunity to prevent HIV-1 infection. Invasive biopsy approaches to isolate immune cells from mucosal tissues and approaches for sampling mucosal tissues from the genital tract, such as cervical lavages and cervical cytobrushes, are not sufficient to explore details of HIV-1 mucosal transmission. Recently, Louissaint and colleagues demonstrated the feasibility of using radiolabeled HIV-free or cell-associated particles to determine the distribution of HIV-1 and semen surrogates after simulated intercourse without disrupting the vaginal contents and inducing local inflammation.²²

Several studies showed clearly that cervicovaginal and seminal factors may modulate transmission of the virus. It seems that the presence of high levels of proinflammatory cytokines/chemokines in mucosal tissues may favor replication of the virus and is correlated with disease progression. However, induction of anti-HIV-specific responses needs the activation of innate effectors that release proinflammatory monokines. It will then be a challenge to differentiate the proinflammatory response that facilitates the initial viral seeding/spread and the proinflammatory response resulting from the activation of the innate immune system that elicits specific antiviral responses. In the macaque model, the reduction of mucosal T cell activation by immunosuppressive mechanisms combined with a modest vaccine-induced CD8⁺ T cell response would allow the antiviral CD8⁺ T cells to control mucosal SIV replication before the infection could disseminate.¹²⁸ These latter findings suggest that an HIV-induced proinflammatory environment constitutes the first step in the local amplification of the T/F viruses, which would help its escape from the mucosal immune system induced more recently in a second step (emergence of new virus variants, infection of the cellular reservoir, and migration of infected cells from mucosal tissues to lymph nodes).

A recent proof of concept study also demonstrates that the combined use of a microbicide to lower the initial viral spread and a vaccine to elicit a strong mucosal immune response holds promise as a preventive strategy to limit HIV-1 mucosal transmission.⁴⁴ Other studies raised the possibility of combining microbicide molecules, which would (1) decrease the toxicity of each compound and their adverse effects, (2) have potential synergistic activity against distinct and complementary pathways used by HIV-1 to disseminate, (3) decrease the risk of selection of antiretroviral drug-resistant HIV-1 strains, and (4) be more advantageous not only against HIV-1 but also against other pathogens (such as genital herpes).^{75,147
–149} Since gels containing nonspecific compound (such as polyanion-based microbicides and buffering agents) showed modest potency in phase III efficacy trials,^16,17 product development has recently turned to microbicides containing highly potent HIV-specific antiretroviral compounds, including entry inhibitors and RT inhibitors. Thus, several entry inhibitors under development as microbicide candidates work to block the CCR5 chemokine receptor, such as the CCR5 antagonists maraviroc and L-167,^150,151 and the specific interaction between gp120 and CD4, such as the Cyanovirin-N lectin, the L-644 peptide, and the BMS-378806 compound.^152,153

CCR5 antagonists may be more efficient in blocking the transmucosal crossing of CCR5-tropic HIV-1 strains, which are considered to be the primary viruses to be transmitted, due to their mucosal selection and their concentration within the genital secretions.²⁵ Based on this, CCR5 antagonists could constitute another very efficient mucosal barrier against the virus, acting synergistically with the other physiological existing barriers. However, in addition to CCR5-blocking agents, the RT inhibitors blocking the conversion of viral RNA to proviral DNA, and then preventing the infection of target cells and the subsequent establishment of reservoirs, may also be used as possible microbicides,¹⁴² acting after cell entry in a more classical antiviral process. Both CCR5 antagonists and antiretroviral molecules targeting the HIV replication cycle are likely to be complementary and could be associated. Further studies are required to compare the effectiveness of several combinations of topical microbicides with partially effective preventive strategies (male circumcision, treatment of curable STIs, and use of antiretroviral drugs as prevention).

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

UNAIDS: World AIDS Day Report. www.unaids.org/en/media/unaids/contentassets/documents/unaidspublication/2011/JC2216_WorldAIDSday_report_2011_en.pdf 2011.

Halperin

, Mugurungi

, Hallett

et al.

A surprising prevention success: Why did the HIV epidemic decline in Zimbabwe?

PLoS Med, 2011; 8:e1000414.

Bunnell

, Ekwaru

, Solberg

et al. Changes in sexual behavior and risk of HIV transmission after antiretroviral therapy and prevention interventions in rural Uganda. AIDS, 2006; 20:85–92.

Serwadda

, Gray

, Sewankambo

et al. Human immunodeficiency virus acquisition associated with genital ulcer disease and herpes simplex virus type 2 infection: A nested case-control study in Rakai, Uganda. J Infect Dis, 2003; 188:1492–1497.

Katz

, Wright

. Circumcision—a surgical strategy for HIV prevention in Africa. N Engl J Med, 2008; 359:2412–2415.

Grant

, Lama

, Anderson

et al. Preexposure chemoprophylaxis for HIV prevention in men who have sex with men. N Engl J Med, 2010; 363:2587–2599.

Celum

. HIV preexposure prophylaxis: New data and potential use. Top Antivir Med, 2011; 19:181–185.

Baeten

, Donnell

, Ndase

et al. Antiretroviral prophylaxis for HIV prevention in heterosexual men and women. N Engl J Med, 2012; 367:399–410.

Baeten

, Donnell

, Ndase

et al. ARV PrEP for HIV-1 Prevention among Heterosexual Men Women. The Conference on Retroviruses and Opportunistic Infections (CROI), Seattle, WA, 2012.

10.

Mujugira

, Baeten

, Donnell

et al. Characteristics of HIV-1 serodiscordant couples enrolled in a clinical trial of antiretroviral pre-exposure prophylaxis for HIV-1 prevention. PLoS One, 2011; 6:e25828.

11.

Abdool Karim

, Abdool Karim

, Frohlich

et al. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science, 2010; 329:1168–1174.

12.

Stone

. Microbicides: A new approach to preventing HIV and other sexually transmitted infections. Nat Rev Drug Discov, 2002; 1:977–985.

13.

Watkins

, Burton

, Kallas

, Moore

, Koff

. Nonhuman primate models and the failure of the Merck HIV-1 vaccine in humans. Nat Med, 2008; 14:617–621.

14.

Feldblum

, Adeiga

, Bakare

et al. SAVVY vaginal gel (C31G) for prevention of HIV infection: A randomized controlled trial in Nigeria. PLoS One, 2008; 3:e1474.

15.

Ramjee

, Govinden

, Morar

, Mbewu

. South Africa's experience of the closure of the cellulose sulphate microbicide trial. PLoS Med, 2007; 4:e235.

16.

Van Damme

, Govinden

, Mirembe

et al. Lack of effectiveness of cellulose sulfate gel for the prevention of vaginal HIV transmission. N Engl J Med, 2008; 359:463–472.

17.

Skoler-Karpoff

, Ramjee

, Ahmed

et al. Efficacy of Carraguard for prevention of HIV infection in women in South Africa: A randomised, double-blind, placebo-controlled trial. Lancet, 2008; 372:1977–1987.

18.

Saidi

. Microbicides: An emerging science of HIV-1 prevention in women—15th Conference on Retroviruses and Opportunistic Infections, Boston, MA, 3–6 February 2008. Rev Med Virol, 2009; 19:69–76.

19.

Turville

, Aravantinou

, Miller

et al. Efficacy of Carraguard-based microbicides in vivo despite variable in vitro activity. PLoS One, 2008; 3:e3162.

20.

Kell

, Barton

, Edmonds

, Boag

. HIV infection in a patient with Meyer-Rokitansky-Kuster-Hauser syndrome. J R Soc Med, 1992; 85:706–707.

21.

Miller

, Alexander

, Vogel

, Anderson

, Marx

. Mechanism of genital transmission of SIV: A hypothesis based on transmission studies and the location of SIV in the genital tract of chronically infected female rhesus macaques. J Med Primatol, 1992; 21:64–68.

22.

Louissaint

, Fuchs

, Bakshi

et al. Distribution of cell-free and cell-associated HIV surrogates in the female genital tract after simulated vaginal intercourse. J Infect Dis, 2012; 205:725–732.

23.

Cavellani

, Rocha

, da Silva Rosa

et al. The influence of AIDS on the morphometric and immune status of the uterine cervix of autopsied patients. Curr HIV Res, 2011; 9:606–612.

24.

Nazli

, Chan

, Dobson-Belaire

et al. Exposure to HIV-1 directly impairs mucosal epithelial barrier integrity allowing microbial translocation. PLoS Pathog, 2010; 6:e1000852.

25.

Margolis

, Shattock

. Selective transmission of CCR5-utilizing HIV-1: The ‘gatekeeper' problem resolved? Nat Rev Microbiol, 2006; 4:312–317.

26.

Dezzutti

, Guenthner

, Cummins

et al. Cervical and prostate primary epithelial cells are not productively infected but sequester human immunodeficiency virus type 1. J Infect Dis, 2001; 183:1204–1213.

27.

Furuta

, Eriksson

, Svennerholm

et al. Infection of vaginal and colonic epithelial cells by the human immunodeficiency virus type 1 is neutralized by antibodies raised against conserved epitopes in the envelope glycoprotein gp120. Proc Natl Acad Sci USA, 1994; 91:12559–12563.

28.

Keller

, Herold

. Understanding basic mechanisms and optimizing assays to evaluate the efficacy of vaginal microbicides. Sex Transm Dis, 2009; 36:S92–S95.

29.

Promadej-Lanier

, Srinivasan

, Curtis

et al. Systemic and mucosal immunological responses during repeated mucosal SHIV(162P3) challenges prior to and following infection in pigtailed macaques. Virology, 2008; 375:492–503.

30.

Poonia

, Walter

, Dufour

, Harrison

, Marx

, Veazey

. Cyclic changes in the vaginal epithelium of normal rhesus macaques. J Endocrinol, 2006; 190:829–835.

31.

Gumbi

, Nkwanyana

, Bere

et al. Impact of mucosal inflammation on cervical human immunodeficiency virus (HIV-1)-specific CD8 T-cell responses in the female genital tract during chronic HIV infection. J Virol, 2008; 82:8529–8536.

32.

Zara

, Nappi

, Brerra

, Migliavacca

, Maserati

, Spinillo

. Markers of local immunity in cervico-vaginal secretions of HIV infected women: Implications for HIV shedding. Sex Transm Infect, 2004; 80:108–112.

33.

Ryckman

, Williams

, Krohn

, Simhan

. Racial differences in cervical cytokine concentrations between pregnant women with and without bacterial vaginosis. J Reprod Immunol, 2008; 78:166–171.

34.

Fidel

Jr. , Luo

, Steele

, Chabain

, Baker

, Wormley

Jr . Analysis of vaginal cell populations during experimental vaginal candidiasis. Infect Immun, 1999; 67:3135–3140.

35.

, Lu

, Torten

, Miller

. The number and distribution of immune cells in the cervicovaginal mucosa remain constant throughout the menstrual cycle of rhesus macaques. Clin Immunol, 2001; 100:240–249.

36.

Vishwanathan

, Guenthner

, Lin

et al. High susceptibility to repeated, low-dose, vaginal SHIV exposure late in the luteal phase of the menstrual cycle of pigtail macaques. J Acquir Immune Defic Syndr, 2011; 57:261–264.

37.

Baeten

, Lavreys

, Overbaugh

. The influence of hormonal contraceptive use on HIV-1 transmission and disease progression. Clin Infect Dis, 2007; 45:360–369.

38.

Hel

, Stringer

, Mestecky

. Sex steroid hormones, hormonal contraception, and the immunobiology of human immunodeficiency virus-1 infection. Endocr Rev, 2010; 31:79–97.

39.

Marx

, Spira

, Gettie

et al. Progesterone implants enhance SIV vaginal transmission and early virus load. Nat Med, 1996; 2:1084–1089.

40.

Stringer

, Antonsen

. Hormonal contraception and HIV disease progression. Clin Infect Dis, 2008; 47:945–951.

41.

Stringer

, Kaseba

, Levy

et al. A randomized trial of the intrauterine contraceptive device vs hormonal contraception in women who are infected with the human immunodeficiency virus. Am J Obstet Gynecol, 2007; 197:144e141–e148.

42.

Ferre

, Hunt

, McConnell

et al. HIV controllers with HLA-DRB1*13 and HLA-DQB1*06 alleles have strong, polyfunctional mucosal CD4+ T-cell responses. J Virol, 2010; 84:11020–11029.

43.

Liu

, Keele

, Li

et al. Low-dose mucosal simian immunodeficiency virus infection restricts early replication kinetics and transmitted virus variants in rhesus monkeys. J Virol, 2010; 84:10406–10412.

44.

Cheng-Mayer

, Huang

, Gettie

et al. Delay of simian human immunodeficiency virus infection and control of viral replication in vaccinated macaques challenged in the presence of a topical microbicide. AIDS, 2011; 25:1833–1841.

45.

Barouch

, Klasse

, Dufour

, Veazey

, Moore

. Macaque studies of vaccine and microbicide combinations for preventing HIV-1 sexual transmission. Proc Natl Acad Sci USA, 2012; 109:8694–8698.

46.

Dayal

, Wheeler

, Williams

, Barnhart

. Disruption of the upper female reproductive tract epithelium by nonoxynol-9. Contraception, 2003; 68:273–279.

47.

Chenine

, Siddappa

, Kramer

et al. Relative transmissibility of an R5 clade C simian-human immunodeficiency virus across different mucosae in macaques parallels the relative risks of sexual HIV-1 transmission in humans via different routes. J Infect Dis, 2010; 201:1155–1163.

48.

Saidi

, Magri

, Nasreddine

, Requena

, Belec

. R5- and X4-HIV-1 use differentially the endometrial epithelial cells HEC-1A to ensure their own spread: Implication for mechanisms of sexual transmission. Virology, 2007; 358:55–68.

49.

Fontenot

, He

, Hanabuchi

et al. TSLP production by epithelial cells exposed to immunodeficiency virus triggers DC-mediated mucosal infection of CD4+ T cells. Proc Natl Acad Sci USA, 2009; 106:16776–16781.

50.

Bouhlal

, Chomont

, Requena

et al. Opsonization of HIV with complement enhances infection of dendritic cells and viral transfer to CD4 T cells in a CR3 and DC-SIGN-dependent manner. J Immunol, 2007; 178:1086–1095.

51.

Eslahpazir

, Jenabian

, Bouhlal

et al. Infection of macrophages and dendritic cells with primary R5-tropic human immunodeficiency virus type 1 inhibited by natural polyreactive anti-CCR5 antibodies purified from cervicovaginal secretions. Clin Vaccine Immunol, 2008; 15:872–884.

52.

Saidi

, Magri

, Carbonneil

, Nasreddine

, Requena

, Belec

. IFN-gamma-activated monocytes weakly produce HIV-1 but induce the recruitment of HIV-sensitive T cells and enhance the viral production by these recruited T cells. J Leukoc Biol, 2007; 81:642–653.

53.

, Estes

, Schlievert

et al. Glycerol monolaurate prevents mucosal SIV transmission. Nature, 2009; 458:1034–1038.

54.

Haase

. Targeting early infection to prevent HIV-1 mucosal transmission. Nature, 2010; 464:217–223.

55.

Zhang

, Schuler

, Zupancic

et al. Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science, 1999; 286:1353–1357.

56.

Geijtenbeek

, Kwon

, Torensma

et al. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell, 2000; 100:587–597.

57.

Hladik

, Sakchalathorn

, Ballweber

et al. Initial events in establishing vaginal entry and infection by human immunodeficiency virus type-1. Immunity, 2007; 26:257–270.

58.

Hirbod

, Kaldensjo

, Broliden

. In situ distribution of HIV-binding CCR5 and C-type lectin receptors in the human endocervical mucosa. PLoS One, 2011; 6:e25551.

59.

Hirbod

, Kaldensjo

, Lopalco

et al. Abundant and superficial expression of C-type lectin receptors in ectocervix of women at risk of HIV infection. J Acquir Immune Defic Syndr, 2009; 51:239–247.

60.

Spira

, Marx

, Patterson

et al. Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. J Exp Med, 1996; 183:215–225.

61.

Collins

, Patterson

, Naus

, Landers

, Gupta

. Development of an in vitro organ culture model to study transmission of HIV-1 in the female genital tract. Nat Med, 2000; 6:475–479.

62.

, Gardner

, Miller

. Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells. J Virol, 2000; 74:6087–6095.

63.

Patterson

, Landay

, Siegel

et al. Susceptibility to human immunodeficiency virus-1 infection of human foreskin and cervical tissue grown in explant culture. Am J Pathol, 2002; 161:867–873.

64.

Chehimi

, Luo

, Azzoni

et al. HIV-1 transmission and cytokine-induced expression of DC-SIGN in human monocyte-derived macrophages. J Leukoc Biol, 2003; 74:757–763.

65.

Nguyen

, Hildreth

. Involvement of macrophage mannose receptor in the binding and transmission of HIV by macrophages. Eur J Immunol, 2003; 33:483–493.

66.

Lambert

, Gilbert

, Richard

, Beaulieu

, Tremblay

. The C-type lectin surface receptor DCIR acts as a new attachment factor for HIV-1 in dendritic cells and contributes to trans- and cis-infection pathways. Blood, 2008; 112:1299–1307.

67.

Bobardt

, Chatterji

, Selvarajah

et al. Cell-free human immunodeficiency virus type 1 transcytosis through primary genital epithelial cells. J Virol, 2007; 81:395–405.

68.

Bobardt

, Saphire

, Hung

et al. Syndecan captures, protects, and transmits HIV to T lymphocytes. Immunity, 2003; 18:27–39.

69.

Saphire

, Bobardt

, Zhang

, David

, Gallay

. Syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages. J Virol, 2001; 75:9187–9200.

70.

de Parseval

, Bobardt

, Chatterji

et al. A highly conserved arginine in gp120 governs HIV-1 binding to both syndecans and CCR5 via sulfated motifs. J Biol Chem, 2005; 280:39493–39504.

71.

de Witte

, Nabatov

, Geijtenbeek

. Distinct roles for DC-SIGN+-dendritic cells and Langerhans cells in HIV-1 transmission. Trends Mol Med, 2008; 14:12–19.

72.

van der Vlist

, van der Aar

, Gringhuis

, Geijtenbeek

. Innate signaling in HIV-1 infection of dendritic cells. Curr Opin HIV AIDS, 2011; 6:348–352.

73.

Gringhuis

, van der Vlist

, van den Berg

, den Dunnen

, Litjens

, Geijtenbeek

. HIV-1 exploits innate signaling by TLR8 and DC-SIGN for productive infection of dendritic cells. Nat Immunol, 2010; 11:419–426.

74.

Miller

, Vogel

, Alexander

, Sutjipto

, Hendrickx

, Marx

. Localization of SIV in the genital tract of chronically infected female rhesus macaques. Am J Pathol, 1992; 141:655–660.

75.

Jenabian

, Saidi

, Charpentier

et al.

In vitro synergistic activity against CCR5-tropic HIV-1 with combinations of potential candidate microbicide molecules HHA, KRV2110 and enfuvirtide (T20)

J Antimicrob Chemother, 2009; 64:1192–1195.

76.

Vermeire

, Brouwers

, Van Herrewege

et al. CADA, a potential anti-HIV microbicide that specifically targets the cellular CD4 receptor. Curr HIV Res, 2008; 6:246–256.

77.

Saïdi

, Jenabian

, Bélec

. Early events in vaginal HIV transmission: Implications in microbicide development. Future Virol, 2009; 4:259–269.

78.

Nathan

. Neutrophils and immunity: Challenges and opportunities. Nat Rev Immunol, 2006; 6:173–182.

79.

Nkwanyana

, Gumbi

, Roberts

et al. Impact of human immunodeficiency virus 1 infection and inflammation on the composition and yield of cervical mononuclear cells in the female genital tract. Immunology, 2009; 128:e746–e757.

80.

Belec

, Gherardi

, Payan

et al. Proinflammatory cytokine expression in cervicovaginal secretions of normal and HIV-infected women. Cytokine, 1995; 7:568–574.

81.

Saidi

, Melki

, Gougeon

. HMGB1-dependent triggering of HIV-1 replication and persistence in dendritic cells as a consequence of NK-DC cross-talk. PLoS One, 2008; 3:e3601.

82.

Gougeon

, Melki

, Saidi

. HMGB1, an alarmin promoting HIV dissemination and latency in dendritic cells. Cell Death Differ, 2012; 19:96–106.

83.

Borde

, Barnay-Verdier

, Gaillard

, Hocini

, Marechal

, Gozlan

. Stepwise release of biologically active HMGB1 during HSV-2 infection. PLoS One, 2011; 6:e16145.

84.

Nowak

, Barqasho

, Sonnerborg

. Elevated plasma levels of high mobility group box protein 1 in patients with HIV-1 infection. AIDS, 2007; 21:869–871.

85.

Ploquin

, Diop

, Sol-Foulon

et al. DC-SIGN from African green monkeys is expressed in lymph nodes and mediates infection in trans of simian immunodeficiency virus SIVagm. J Virol, 2004; 78:798–810.

86.

Perreau

, Pantaleo

, Kremer

. Activation of a dendritic cell-T cell axis by Ad5 immune complexes creates an improved environment for replication of HIV in T cells. J Exp Med, 2008; 205:2717–2725.

87.

Requena

, Bouhlal

, Nasreddine

et al. Inhibition of HIV-1 transmission in trans from dendritic cells to CD4+ T lymphocytes by natural antibodies to the CRD domain of DC-SIGN purified from breast milk and intravenous immunoglobulins. Immunology, 2008; 123:508–518.

88.

Izquierdo-Useros

, Naranjo-Gomez

, Erkizia

et al.

HIV and mature dendritic cells: Trojan exosomes riding the Trojan horse?

PLoS Pathog, 2010; 6:e1000740.

89.

, Reuter

, McDonald

. HIV traffics through a specialized, surface-accessible intracellular compartment during trans-infection of T cells by mature dendritic cells. PLoS Pathog, 2008; 4:e1000134.

90.

Saidi

, Carbonneil

, Magri

, Eslahpazir

, Sekaly

, Belec

. Differential modulation of CCR5-tropic human immunodeficiency virus-1 transfer from macrophages towards T cells under interleukin-4/interleukin-13 microenvironment. Hum Immunol, 2010; 71:1–13.

91.

Said

, Dupuy

, Trautmann

et al. Programmed death-1-induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection. Nat Med, 2010; 16:452–459.

92.

Modlin

, Pirmez

, Hofman

et al. Lymphocytes bearing antigen-specific gamma delta T-cell receptors accumulate in human infectious disease lesions. Nature, 1989; 339:544–548.

93.

McMenamin

, Pimm

, McKersey

, Holt

. Regulation of IgE responses to inhaled antigen in mice by antigen-specific gamma delta T cells. Science, 1994; 265:1869–1871.

94.

Lehner

, Mitchell

, Bergmeier

et al. The role of gammadelta T cells in generating antiviral factors and beta-chemokines in protection against mucosal simian immunodeficiency virus infection. Eur J Immunol, 2000; 30:2245–2256.

95.

Keele

, Estes

. Barriers to mucosal transmission of immunodeficiency viruses. Blood, 2011; 118:839–846.

96.

Salazar-Gonzalez

, Bailes

, Pham

et al. Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single-genome amplification and sequencing. J Virol, 2008; 82:3952–3970.

97.

Keele

, Giorgi

, Salazar-Gonzalez

et al. Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci USA, 2008; 105:7552–7557.

98.

Fisher

, Ensoli

, Looney

et al. Biologically diverse molecular variants within a single HIV-1 isolate. Nature, 1988; 334:444–447.

99.

Etemad

, Fellows

, Kwambana

et al. Human immunodeficiency virus type 1 V1-to- V5 envelope variants from the chronic phase of infection use CCR5 and fuse more efficiently than those from early after infection. J Virol, 2009; 83:9694–9708.

100.

Saag

, Hahn

, Gibbons

et al. Extensive variation of human immunodeficiency virus type-1 in vivo. Nature, 1988; 334:440–444.

101.

Goonetilleke

, Liu

, Salazar-Gonzalez

et al. The first T cell response to transmitted/founder virus contributes to the control of acute viremia in HIV-1 infection. J Exp Med, 2009; 206:1253–1272.

102.

Gnanakaran

, Bhattacharya

, Daniels

et al. Recurrent signature patterns in HIV-1 B clade envelope glycoproteins associated with either early or chronic infections. PLoS Pathog, 2011; 7:e1002209.

103.

Wilen

, Parrish

, Pfaff

et al. Phenotypic and immunologic comparison of clade B transmitted/founder and chronic HIV-1 envelope glycoproteins. J Virol, 2011; 85:8514–8527.

104.

Parrish

, Wilen

, Banks

et al. Transmitted/founder and chronic subtype C HIV-1 use CD4 and CCR5 receptors with equal efficiency and are not inhibited by blocking the integrin alpha4beta7. PLoS Pathog, 2012; 8:e1002686.

105.

Ochsenbauer

, Edmonds

, Ding

et al. Generation of transmitted/founder HIV-1 infectious molecular clones and characterization of their replication capacity in CD4 T lymphocytes and monocyte-derived macrophages. J Virol, 2012; 86:2715–2728.

106.

, Bar

, Wang

et al. High multiplicity infection by HIV-1 in men who have sex with men. PLoS Pathog, 2010; 6:e1000890.

107.

Salazar-Gonzalez

, Salazar

, Keele

et al. Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection. J Exp Med, 2009; 206:1273–1289.

108.

Chomont

, Hocini

, Gresenguet

et al. Early archives of genetically-restricted proviral DNA in the female genital tract after heterosexual transmission of HIV-1. AIDS, 2007; 21:153–162.

109.

Miller

, Hu

. T cell-tropic simian immunodeficiency virus (SIV) and simian-human immunodeficiency viruses are readily transmitted by vaginal inoculation of rhesus macaques, and Langerhans' cells of the female genital tract are infected with SIV. J Infect Dis, 1999; 179,Suppl 3:S413–S417.

110.

Zhang

, Dornadula

, Beumont

et al. Human immunodeficiency virus type 1 in the semen of men receiving highly active antiretroviral therapy. N Engl J Med, 1998; 339:1803–1809.

111.

Pillai

, Good

, Pond

et al. Semen-specific genetic characteristics of human immunodeficiency virus type 1 env. J Virol, 2005; 79:1734–1742.

112.

Berlier

, Bourlet

, Lawrence

et al. Selective sequestration of X4 isolates by human genital epithelial cells: Implication for virus tropism selection process during sexual transmission of HIV. J Med Virol, 2005; 77:465–474.

113.

Saba

, Grivel

, Vanpouille

et al. HIV-1 sexual transmission: Early events of HIV-1 infection of human cervico-vaginal tissue in an optimized ex vivo model. Mucosal Immunol, 2010; 3:280–290.

114.

Greenier

, Miller

, Lu

et al. Route of simian immunodeficiency virus inoculation determines the complexity but not the identity of viral variant populations that infect rhesus macaques. J Virol, 2001; 75:3753–3765.

115.

Butler

, Delport

, Kosakovsky Pond

et al. The origins of sexually transmitted HIV among men who have sex with men. Sci Transl Med, 2011; 2:18re11.

116.

Miller

, Marthas

, Torten

et al. Intravaginal inoculation of rhesus macaques with cell-free simian immunodeficiency virus results in persistent or transient viremia. J Virol, 1994; 68:6391–6400.

117.

Miller

, McChesney

, Moore

. Langerhans cells, macrophages and lymphocyte subsets in the cervix and vagina of rhesus macaques. Lab Invest, 1992; 67:628–634.

118.

Salle

, Brochard

, Bourry

et al. Infection of macaques after vaginal exposure to cell-associated simian immunodeficiency virus. J Infect Dis, 2010; 202:337–344.

119.

Denton

, Krisko

, Powell

et al. Systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous HIV-1 transmission in humanized BLT mice. PLoS One, 2010; 5:e8829.

120.

Lai

, Hida

, Shukair

et al. Human immunodeficiency virus type 1 is trapped by acidic but not by neutralized human cervicovaginal mucus. J Virol, 2009; 83:11196–11200.

121.

O'Hanlon

, Moench

, Cone

. In vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide. BMC Infect Dis, 2011; 11:200.

122.

O'Hanlon

, Lanier

, Moench

, Cone

. Cervicovaginal fluid and semen block the microbicidal activity of hydrogen peroxide produced by vaginal lactobacilli. BMC Infect Dis, 2010; 10:120.

123.

Lederman

, Offord

, Hartley

. Microbicides and other topical strategies to prevent vaginal transmission of HIV. Nat Rev Immunol, 2006; 6:371–382.

124.

Brocca-Cofano

, McKinnon

, Demberg

et al. Vaccine-elicited SIV and HIV envelope-specific IgA and IgG memory B cells in rhesus macaque peripheral blood correlate with functional antibody responses and reduced viremia. Vaccine, 2011; 29:3310–3319.

125.

Saidi

, Eslahpazir

, Carbonneil

et al. Differential modulation of human lactoferrin activity against both R5 and X4-HIV-1 adsorption on epithelial cells and dendritic cells by natural antibodies. J Immunol, 2006; 177:5540–5549.

126.

Card

, McLaren

, Wachihi

, Kimani

, Plummer

, Fowke

. Decreased immune activation in resistance to HIV-1 infection is associated with an elevated frequency of CD4(+)CD25(+)FOXP3(+) regulatory T cells. J Infect Dis, 2009; 199:1318–1322.

127.

Bosinger

, Li

, Gordon

et al. Global genomic analysis reveals rapid control of a robust innate response in SIV-infected sooty mangabeys. J Clin Invest, 2009; 119:3556–3572.

128.

Genesca

, Ma

, Wang

et al. Live-attenuated lentivirus immunization modulates innate immunity and inflammation while protecting RM from vaginal SIV challenge. J Virol, 2012; 77:3099–3118.

129.

Estes

, Gordon

, Zeng

et al. Early resolution of acute immune activation and induction of PD-1 in SIV-infected sooty mangabeys distinguishes nonpathogenic from pathogenic infection in rhesus macaques. J Immunol, 2008; 180:6798–6807.

130.

Gasper-Smith

, Crossman

, Whitesides

et al. Induction of plasma (TRAIL), TNFR-2, Fas ligand, and plasma microparticles after human immunodeficiency virus type 1 (HIV-1) transmission: Implications for HIV-1 vaccine design. J Virol, 2008; 82:7700–7710.

131.

Boasso

, Shearer

. How does indoleamine 2,3-dioxygenase contribute to HIV-mediated immune dysregulation. Curr Drug Metab, 2007; 8:217–223.

132.

Hartner

, Walkley

, Lu

, Orkin

. ADAR1 is essential for the maintenance of hematopoiesis and suppression of interferon signaling. Nat Immunol, 2009; 10:109–115.

133.

Chawla-Sarkar

, Lindner

, Liu

et al. Apoptosis and interferons: Role of interferon-stimulated genes as mediators of apoptosis. Apoptosis, 2003; 8:237–249.

134.

Munch

, Rucker

, Standker

et al. Semen-derived amyloid fibrils drastically enhance HIV infection. Cell, 2007; 131:1059–1071.

135.

Sharkey

, Tremellen

, Jasper

, Gemzell-Danielsson

, Robertson

. Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus. J Immunol, 2012; 188,5:2445–2454.

136.

Hubert

, van den Brule

, Giannini

, Franzen-Detrooz

, Boniver

, Delvenne

. Colonization of in vitro-formed cervical human papillomavirus-associated (pre)neoplastic lesions with dendritic cells: Role of granulocyte/macrophage colony-stimulating factor. Am J Pathol, 1999; 154:775–784.

137.

Shen

, Fahey

, Hussey

, Asin

, Wira

, Fanger

. Synergy between IL-8 and GM-CSF in reproductive tract epithelial cell secretions promotes enhanced neutrophil chemotaxis. Cell Immunol, 2004; 230:23–32.

138.

Tremellen

, Seamark

, Robertson

. Seminal transforming growth factor beta 1 stimulates granulocyte-macrophage colony-stimulating factor production and inflammatory cell recruitment in the murine uterus. Biol Reprod, 1998; 58:1217–1225.

139.

Politch

, Tucker

, Bowman

, Anderson

. Concentrations and significance of cytokines and other immunologic factors in semen of healthy fertile men. Hum Reprod, 2007; 22:2928–2935.

140.

Kafka

, Sheth

, Nazli

et al. Endometrial epithelial cell response to semen from HIV-infected men during different stages of infection is distinct and can drive HIV-1-long terminal repeat. AIDS, 2012; 26:27–36.

141.

Berlier

, Cremel

, Hamzeh

et al. Seminal plasma promotes the attraction of Langerhans cells via the secretion of CCL20 by vaginal epithelial cells: Involvement in the sexual transmission of HIV. Hum Reprod, 2006; 21:1135–1142.

142.

Hammer

, Eron

Jr , Reiss

et al. Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. JAMA, 2008; 300:555–570.

143.

Bunge

, Macio

, Meyn

et al. The safety, persistence, and acceptability of an antiretroviral microbicide candidate UC781. J Acquir Immune Defic Syndr, 2012; 60:337–343.

144.

Ceballos

, Remes Lenicov

, Sabatte

et al. Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells. J Exp Med, 2009; 206:2717–2733.

145.

Jenabian

, Saidi

, Charpentier

et al. Differential activity of candidate microbicides against early steps of HIV-1 infection upon complement virus opsonization. AIDS Res Ther, 2010; 7:16.

146.

Kazmi

, Naglik

, Sweet

et al. Comparison of human immunodeficiency virus type 1-specific inhibitory activities in saliva and other human mucosal fluids. Clin Vaccine Immunol, 2006; 13:1111–1118.

147.

Herrera

, Cranage

, McGowan

, Anton

, Shattock

. Colorectal microbicide design: Triple combinations of reverse transcriptase inhibitors are optimal against HIV-1 in tissue explants. AIDS, 2011; 25:1971–1979.

148.

Keller

, Tuyama

, Carlucci

, Herold

. Topical microbicides for the prevention of genital herpes infection. J Antimicrob Chemother, 2005; 55:420–423.

149.

Belec

, Jenabian

, Charpentier

, Saidi

. Combinatorial prevention of HIV transmission in women: The case for a vaginal microbicide. Future Microbiol, 2011; 6:731–737.

150.

Lieberman-Blum

, Fung

, Bandres

. Maraviroc: A CCR5-receptor antagonist for the treatment of HIV-1 infection. Clin Ther, 2008; 30:1228–1250.

151.

Veazey

, Klasse

, Schader

et al. Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion. Nature, 2005; 438:99–102.

152.

Mori

, Boyd

. Cyanovirin-N, a potent human immunodeficiency virus-inactivating protein, blocks both CD4-dependent and CD4-independent binding of soluble gp120 (sgp120) to target cells, inhibits sCD4-induced binding of sgp120 to cell-associated CXCR4, and dissociates bound sgp120 from target cells. Antimicrob Agents Chemother, 2001; 45:664–672.

153.

Singh

, Chauthe

. Small molecule HIV entry inhibitors: Part I. Chemokine receptor antagonists: 2004–2010. Expert Opin Ther Pat, 2011; 21:227–269.