During more than one-quarter of a century of the HIV-1

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1 Vol 464j11 March 2010jdoi: /nature08757 Targeting early infection to prevent HIV-1 mucosal transmission Ashley T. Haase 1 Measures to prevent sexual mucosal transmission of human immunodeficiency virus (HIV)-1 are urgently needed to curb the growth of the acquired immunodeficiency syndrome (AIDS) pandemic and ultimately bring it to an end. Studies in animal models and acute HIV-1 infection reviewed here reveal potential viral vulnerabilities at the mucosal portal of entry in the earliest stages of infection that might be most effectively targeted by vaccines and microbicides, thereby preventing acquisition and averting systemic infection, CD4 T-cell depletion and pathologies that otherwise rapidly ensue. During more than one-quarter of a century of the HIV-1 pandemic, millions have died from AIDS, millions have been orphaned, and more than 30 million people worldwide are living with HIV-1 infection 1. Although advances in antiretroviral therapies and access to treatment are countering this great and continued toll in the developed world and sub-saharan Africa, the epicentre of the pandemic 2, prevention of sexual mucosal transmission, the principal route of acquisition 1, is clearly needed to contain the continuing growth of the pandemic and ultimately eradicate AIDS 3. The prevention record is mixed. Circumcision has proven effective in preventing 50% to 60% of female to male transmission 4 6, and there are the first hints recently of vaccine and microbicide candidates that could prevent sexual transmission to men and women, albeit at low efficacy of about 30% 7,8. However, vaccine and microbicide candidates have not proved efficacious or have even enhanced acquisition in previous trials 9,10. These disappointing failures and limited successes reinforce the view of leading investigators 11 that it is now especially timely to reinvigorate and broaden research on the virus, the immune response to it, and the pathogenesis of transmission and infection to identify the correlates of prevention that will enable the design of fully protective measures against acquisition of HIV-1. In this review, I provide a personal perspective and synthesis of studies on sexual mucosal transmission relevant to this goal. I largely focus on the results of in vivo analyses of relevant tissues, and on transmission to women, because of the increasing feminization of the pandemic in sub-saharan Africa 12, and because of insights into HIV-1 transmission to women derived from extensive studies of the simian immunodeficiency virus (SIV) rhesus macaque nonhuman primate model. I conclude from these studies that prevention strategies should target the earliest stage of infection because of: (1) viral vulnerabilities at this stage at the portal of entry in the small, infected founder populations; (2) local expansion necessary to establish systemic infection; and (3) the ill effects that otherwise rapidly ensue from systemic infection. Fast stage of slow lentivirus infections The retrovirus genus to which HIV-1 and SIV belong was long ago named Lentivirinae 13 based on the long incubation period and slow clinical course of disease that typify infection. However, it is now clear from the SIV rhesus macaque model that local events critical to establishing systemic infection take place very quickly in the early stages of lentivirus infections. CD4 T-cell depleting and pathological processes are also very quickly set in motion in the early stages of systemic infection that then play out over the months and years in the slow phase of infection. SIV rhesus macaque nonhuman primate model. These conclusions are drawn from studies in the SIV rhesus macaque nonhuman primate (NHP) model of transmission of HIV-1 to women 14, which provides a window through which mucosal transmission and the first 2 weeks of infection can be viewed in vivo in ways that, for practical and ethical reasons, are not possible in HIV-1 infection, which is only clinically manifest after this time 15. From NHP studies of the earliest stages of infection 16, we have the following picture (Fig. 1) of the critical events following mucosal exposure to high doses of SIV: virus can cross the mucosal epithelial barrier within hours 17 to establish what has turned out to be a small founder population of infected cells 16,18. This founder population then undergoes a necessary local expansion during the first week of infection to generate sufficient virus and infected cells to disseminate and establish a self-propagating systemic infection throughout the secondary lymphoid organs 16. Beginning in the second week of infection, replication explodes in the lymphatic tissues where virus has access to many more susceptible target cells in close spatial proximity, compared to the dispersed lowdensity populations of susceptible target cells at the portal of entry. Virus levels in blood and tissues peak near the end of this second week of infection, before declining towards relatively stable lower levels by 4 weeks after exposure. At this time, these infected lymphatic tissues comprise a reservoir where virus is produced and stored and where proviruses are harbored in latently infected cells in SIV-infected rhesus macaques 19, just as in HIV-1 infected humans 20. Furthermore, this reservoir is already the site of CD4 T-cell depletion and other pathological processes that will eventually lead to progression to disease (Fig. 2). This description of transmission, early infection and ill effects of systemic infection (Figs 1 and 2) is derived from studies in the SIV high-dose vaginal challenge NHP model in which rhesus macaques are atraumatically inoculated twice in the same day with two doses of 10 5 TCID 50 (50% tissue culture infectious dose) and billions of viral particles 16. The strengths of this model are: (1) the window (Fig. 1) it provides on critical events that precede the earliest time clinical signs and symptoms of HIV-1 infection disease are manifest 15 ; (2) infection of a high proportion of animals with a known time of exposure; (3) access to relevant tissues in a relevant time frame, which increases chances to observe directly virus host cell interactions and critical 1 Department of Microbiology, University of Minnesota, Minnesota 55455, USA. 217

2 NATUREjVol 464j11 March 2010 Viral vulnerabilities Opportunities for prevention Window Time frame Hours Crossing the barrier Local propagation Small R 0 <1 Vagina Infected founder population Cervix Weeks 2-4 Establishment Lymphatic tissue reservoir GALT Thoracic duct Spleen Brain Lung Liver Lymph node Activated CD4 + T-cell target Immune activation Suppress Early T Reg cells Days Local expansion Infected cells Virus Massive depletion of gut lamina propria CD4 + T cells Prevent Too little too late Virus-specific CD8 + T cells Weeks Sufficient production Dissemination Week 1 Establishment Lymphatic tissue reservoir Selfpropagating systemic infection GALT Thoracic duct Draining lymph node Lymph node Battlefield map Week 2 Large numbers of SIV-specific CD8 + T cells + Large numbers of SIV-infected cells E:T ratio <2 Spleen Brain Lung Liver Figure 1 Time frame, sites and major events in vaginal transmission and the fast phase of lentivirus infection. The SIV rhesus macaque animal model provides a window through which to view early infection. Within hours, virus in the inoculum may gain access through breaks in the mucosal epithelial barrier to susceptible target cells. The small focal infected founder population is initially composed mainly of infected resting CD4 T cells lacking conventional markers of activation. The founder population expands locally in these resting and in activated CD4 T cells. Local expansion is necessary to disseminate infection to the draining lymph node, and subsequently through the bloodstream to establish a self-propagating infection in secondary lymphoid organs. Crossing the barrier, small founder populations (with the associated risk that the basic reproductive rate, R 0, will fall below one), and local expansion are vulnerabilities for the virus in week 1 of infection. These vulnerabilities create opportunities for prevention targeting this stage. The male (blue) and female (pink) figures at the lower left are positioned in the time frame marked weeks, after systemic infection has been established. It is at this time that HIV-1 infection is first clinically manifest, and, hence, the need for the animal model to view sexual mucosal transmission and the earlier stages of infection. The larger female figure symbolizes the disproportionate acquisition of HIV-1 infection and focus of the review on transmission to women. events; and (4) similarities in anatomy, physiology and immunology of the female reproductive tract of rhesus macaques to humans, and the general similarities of pathogenic SIV infection to HIV-1 infection in CD4 T-cell depletion, pathology and AIDS 14,21. This animal model also has weaknesses. First, the SIV challenge dose exceeds the highest dose of HIV-1 to which humans are exposed in semen, even in the acute stage of HIV-1 infection where virus concentrations and transmission rates are the highest 22. We thus have to assume that virus host interactions documented in tissue analyses in the high dose challenge also occur with lower dose challenges at a local level, but at such low frequencies that they would escape detection in tissue samples. In further defence of the high dose challenge, single genome analysis (see below) in multiple low-dose intra-rectal challenges that more closely approximate typical doses of HIV-1 in semen provides evidence consistent with one of the key findings to emerge from the high dose model: that infected founder populations are small as a consequence frequently of infection with a single virus genotype 23. In addition, this cell-free virus model of course cannot provide insight into cell-associated virus transmission. An animal model of the latter potential mode of HIV-1 transmission would certainly be valuable, but would have to be developed, as infected cell inocula 218 SIV-specific effector CD8 + T cell SIV-infected target cell Effector target conjugate Figure 2 Ill effects of systemic infection and too little too late immune response. After dissemination and establishment of systemic infection in weeks 2 4 (upper left panel), peak viral replication in gut-associated and other lymphatic tissues results in massive depletion of CD4 T cells in gut lamina propria. Immune activation (upper right panel) has drawbacks in supplying activated CD4 T-cell targets for viral replication and inducing a T reg response that suppresses the immune response. Immune activation does elicit a virus-specific CD8 T-cell response that is too little and too late to prevent gut CD4 T-cell depletion or clear infection. Although there are large numbers of SIV-specific CD8 T cells in, for example, lymph nodes (lower panel), there are also large numbers of infected cell targets so that at the effector to target (E:T) ratio achieved (,2), infection is only partially controlled. have so far proved an inefficient way to infect animals reliably 24, except when facilitated by genital ulceration 25. I later discuss the implications and relevance of rapid dissemination from the portal of entry under these conditions. Opportunities and lost opportunities. Studies in this high-dose vaginal challenge model support the conclusion that prevention strategies should target the first week of infection both to take advantage of viral vulnerabilities and two opportunities for prevention (Fig. 1) and to avert the ill effects of systemic infection in the second week and beyond (Fig. 2). The first viral vulnerability and opportunity for prevention is related to the establishment of the small founder population, which must be sustained at a basic reproductive rate, R 0, $ 1, otherwise infection will be aborted. Thus, early intervention measures that reduce the growth rate to,1 will have that desired outcome. The second opportunity is preventing local expansion so that insufficient virus and infected cells are produced to disseminate and establish systemic infection. This second opportunity for prevention may not hold for rectal and oral transmission, given the evidence that dissemination beyond the portal of entry occurs within a few days of rectal or oral exposure, without apparent local expansion The ill effects depicted in Fig. 2 of losing those opportunities to prevent systemic infection are similar in SIV and acute HIV infections, and include: massive depletion of memory CD4 T cells in the

3 NATUREjVol 464j11 March 2010 lamina propria of the gut mediated by direct effects of infection and apoptosis of bystander cells ; acute enteropathy induced by proinflammatory cytokines and virotoxic effects of the virus that induce intestinal epithelial apoptosis ; and immune activation, the downside of which includes a new supply of activated CD4 T cells to support virus replication, and a T regulatory (T reg ) response that has been called premature, because the immunosuppressive effects that measurably dampen the SIV-specific cellular immune response precede clearance or control of viral replication 39. Although immune activation does elicit a virus-specific CD8 T-cell response, it is too little and too late to prevent gut CD4 T-cell depletion or do more than partially control infection 40. These serious ill effects on the host s immune system in early infection are only the beginning of immunopathological processes set in motion at this stage that will have their greatest impact in the later chronic stages of SIV and HIV-1 infection: microbial translocation associated with continued damage to the gut lining that contributes to chronic immune activation and the continued loss of CD4 T cells 41 ; and TGF-b 1 T reg cells 42 that initiate a fibrotic process, which disrupts the lymphatic tissue architecture that supports the migration, growth and survival of T cells, thereby contributing both to CD4 T-cell depletion and limiting immune reconstitution with antiretroviral therapies 43. Small founder population and local expansion. The evidence for a small, focal infected founder population of cells depicted in Fig. 1, with local expansion as an antecedent and prerequisite for dissemination and systemic infection, is based on in vivo tissue analyses of vaginal transmission. These analyses revealed only small foci of productively infected cells, defined as cells with detectable SIV RNA, at 3 to 4 days after inoculation 16,18. Infection then expands locally before detection of infection in the draining lymph nodes and systemically throughout the secondary lymphoid organs, consistent with the local expansion and staged dissemination model shown in Fig. 1. There is evidence that dendritic cells with detectable viral antigens reach the draining lymph nodes much earlier 18 to 24 h after exposure 17 but apparently the threshold for a self-propagating infection is not crossed until virus and infected cells are produced locally in sufficient quantities to establish infection distal to the portal of entry, because the draining lymph nodes are not the site where productive infection is first detected 18. Thus, dilution, trapping in cervical mucus, the physical mucosal epithelial barrier and other mechanisms transform exposure to a large quantity of virus in the inoculum to a small founder population of infected cells that must then expand locally for a few days before systemic infection is established. Genetic bottleneck in HIV-1 transmission. The earliest stages of mucosal transmission of HIV-1 have not been directly observed, but we can infer parallels to SIV s small, infected founder populations from the genetic bottleneck at HIV-1 transmission. It has been known for some time that in mother-to-child and heterosexual transmission, virus isolates from the newly infected individual are genetically much less diverse than viruses from the transmitter 44,45. More recently, sequencing viruses in heterosexual transmission pairs and in acute HIV-1 infection provided evidence that a single virus (or infected cell) initiated productive infection in close to 80% of the individuals tested, and two to five viruses in the other 20% 46,47. The implied small, infected founder populations in HIV-1 mucosal transmission again indicate that the greatest opportunities for prevention are strategies that target these initially small and genetically homogeneous foci of infection in the first week of infection. However, the small size of the infected founder population does not imply that the transmitted virus itself is inherently any easier to contain. To the contrary, phenotypic analysis of transmitted HIV-1 revealed masking of CCR5 co-receptor binding regions, and equivalent or enhanced resistance to fusion inhibitors and neutralizing antibodies compared to virus isolated from chronically infected individuals 47,48. Mucosal front line Crossing the barrier. I now return to a fuller description of the earliest steps in transmission and local infection based on direct observations on cervical vaginal tissues. Virus depicted in Fig. 1 enters at anatomical sites where the mucosal barrier is most easily breached, by mechanisms such as the microtrauma associated with sexual intercourse 49 that provide immediate access to target cells in the submucosa (Fig. 1). Only a single layer of columnar epithelium guards the endocervix, and the transformation zone between ectoand endocervix is also a single layer of epithelium and a site of high cell turnover that could facilitate entry 50,51. These anatomical differences and recent mapping studies of the sites where infected founder populations were identified in vaginal transmission of SIV are consistent with the transformation zone and endocervix as preferential sites of entry 52. This hypothesis could also explain the increased acquisition of HIV-1 associated with cervical ectopy extension of the simple columnar epithelium to cover a portion of the ectocervix observed in some studies 53. The transformation zone and endocervix, however, are not exclusive sites of entry, as SIV has been transmitted to hysterectomized animals 54, and HIV-1 in the congenital absence of a cervix 55, establishing that there are target cells and independent entry mechanisms for vaginal transmission. These observations, however, do not address preferred sites of entry and the role of inflammation and trauma in the individual described in ref. 55; nor does the failure of diaphragms to decrease HIV-1 transmission exclude cervix as a preferred site of entry, because of the confounding effects of poor adherence in that study 56. Initial target cells. In the SIV rhesus macaque model, the earliest focal collections of infected cells are CD4 T cells with a surprising phenotype. The initial expectation from growing HIV isolates and SIV in tissue culture was that they would be macrophage-tropic and/ or replicate in activated CD4 T cells, using the CCR5 co-receptor (reviewed in ref. 57). However, in vivo, it turned out that the initially infected cells were CD4 T cells with none of the expected markers of activation 18. They were therefore called 31 resting, with the implication that they probably had some residue of co-receptor expression and prior activated state to enable them to support productive infection, in contrast to truly resting CD4 T cells. In the gut, a4b7 may facilitate entry and preferential infection of T-helper 17 CD4 T cells with this minimally activated phenotype 58,59. Infected resting CD4 T cells comprise about 90% of the detectably productively infected cells in the initial foci and subsequent local expansion at the portal of entry, and the population of infected cells that produces peak levels of virus in SIV infections 18,31,60 in the lymphatic tissues in the second week of infection. Over 60% of CD4 T cells in gut with detectable SIV DNA in early infection have also been shown to be memory CD4 T cells with relatively low levels of CCR5 30 ; in early HIV-1 infection close to 90% of infected cells were typed as CD4 T cells and the majority had a resting phenotype 18,61. Moreover, replication-competent clones derived from single genome amplification of isolates in acute HIV-1 infection primarily infected CD4 T cells rather than macrophages in culture 48. Thus, CD4 T cells are probably the principal cell type infected at the portal of entry and throughout the lymphatic tissue reservoir from the earliest stages of infection. Target cell availability. One reason that this may be the case is target cell availability. CD4 T-cell susceptible target cell populations in the vagina, ecto- and endocervix in rhesus macaques 60,62 and humans 50,63 are largely spatially dispersed populations lying just beneath the epithelium, and, to a lesser extent, deeper submucosa, with some small focal aggregates and intraepithelial lymphocytes. There are also macrophages and dendritic cells in the submucosa, and dendritic cells within the epithelium in both species, but resting CD4 T cells outnumber macrophages and dendritic cells by 4 to 5:1 and two to three or more infected resting CD4 T cells are often found clustered in the small focal clustered infected founder populations

4 NATUREjVol 464j11 March 2010 These census analyses are consistent with the hypothesis that numbers and spatial proximity contribute to preferential propagation initially in this cell type 60. However, target cell availability is unlikely to be the sole determinant of the cell tropisms in early infection, because activated CD4 T cells are the other cell type most frequently infected at this stage, even though there are 70 times as many resting CD4 T cells and relatively more abundant CD4 1 CCR5 1 macrophages and dendritic cells in the vicinity. Mucosal epithelium: immunity s front line. Mucosal epithelial defences against viral entry are conventionally portrayed as a passive physical barrier protecting against invasion, but, in actuality, the epithelium lining endocervix and upper female reproductive tract is the active front line of the host immune system. This mucosal epithelial front line mediates innate defences against microbes under the hormonal control of oestradiol and progesterone 64 66, and functions as a sentinel and signalling system with Toll-like receptors that recognize and respond to pathogen-associated molecular patterns by secreting: (1) microbicidal defensins and other antimicrobial peptides; (2) secretory leukocyte protease inhibitor; (3) the microbicidal enzymes lactoferrin and lysozyme; (4) surfactant protein A; and (5) complement. Mucosal epithelial signalling through chemokines and cytokines also recruits plasmacytoid dendritic cells (pdcs) 67,68 and other cells that mediate innate defences and inflammation, and initiate and link innate and adaptive immunity. The balancing act in innate defences. The physical barrier and active mucosal front-line defences described above would be expected to inhibit SIV and HIV entry and to provide an array of inhibitory activities against viral replication: (1) SDF-1, MIP-1a/b and RANTES to block viral entry mediated by the co-receptors CXCR4 (blocked by SDF-1) and CCR5 (blocked by MIP-1a/b and RANTES) 57 ; (2) MIP-3a expression in response to vaginal inoculation of SIV to immediately recruit interferon (IFN)-a/b producing pdcs to the endocervix 52 ; and (3) large increases in IFN-c expression in cervical vaginal tissues by 6 days after exposure 69. These increases in inhibitory chemokines immediately after exposure and the first few days of infection and unknown contributions of innate defences cited in the preceding section presumably provide a cumulative level of protection that accounts in part for the small infected founder populations in SIV infection despite exposure to a large dose of virus. This innate immune response might also explain restricted cellular tropisms in vivo, for example protection of IFN-producing pdcs from infection. In HIV-1 transmission, similar defence mechanisms probably also contribute to the generally low probability of vaginal transmission of HIV-1 of,1 in 100 to 1,000, depending on the viral load of the transmitting partner 70,71. Paradoxically, these innate antiviral and inflammatory defence mechanisms in SIV and HIV-1 infections at the same time may facilitate transmission, by increasing target cell availability, and by creating conditions for highly efficient cell-to-cell spread of infection where HIV-1 replication has been shown to become less sensitive to inhibition by interferon 72. Mapping local expansion 52 revealed growth by accretion of newly infected cells around foci of infected founder cells, and spread of infection along tracts of infiltrating inflammatory cells. These findings led to the model shown in Fig. 3: SIV overcomes the problem of replication in the low density and dispersed populations of CD4 T cells at the portal of entry through exploiting the innate immune and inflammatory response that brings in large numbers of target cells to create a generally favourable environment to fuel expansion wherever the initial focus is established, and to generate conditions where SIV has been shown to propagate most efficiently by direct cell-to-cell spread or at close range 73. The mechanism inducing the influx of target cells fits nicely with the concept of mucosal epithelium functioning as a sentinel and signalling system 65. In this case, exposure to virus or other components of the inoculum stimulates increased expression of MIP-3a in the endocervical epithelium, attracting pdcs beneath the epithelium, which in turn recruit CD4 T cells through MIP-1b and other chemokines 52. The 220 Vagina Target cells Thinned/disrupted mucosal barrier Local expansion Pre-existing inflammation Cervix New target cells Fuel infection MIP-3α MIP-1β Interferons Anti-viral chemokines Restrict infection Figure 3 Inflammation, innate immunity, mucosal epithelial signalling and target cell availability at mucosal front lines. Exposure of endocervical epithelium to virus, a component of virus or inoculum initiates signalling (jagged arrow) that increases expression of MIP-3a in the epithelium that recruits pdcs. They in turn recruit, through chemokines such as MIP-1b, the CD4 T cells that fuel local expansion. Interferons from the pdcs and chemokines also suppress viral replication but the balance is tipped in favour of the virus by the cells that fuel the local expansion necessary for dissemination and establishment of systemic infection. Pre-existing vaginal inflammation also facilitates infection by thinning and disrupting the multilayered lining, and providing a pool of target cells for local expansion. scales in Fig. 3 are meant to convey the counterbalancing effects of the innate and inflammatory response in restricting viral infection, and facilitating it, through increasing target cell availability, with the greater target cell availability tipping the scales in favour of the virus. The influx of new target cells to fuel local expansion is also a vulnerability that can potentially be exploited in preventing transmission. Glycerol monolaurate inhibits female reproductive tract epithelial signalling and may have prevented local expansion to thereby protect animals exposed to high doses of SIV 52. Inflammation and mucosal transmission. Innate defences and associated inflammation generally facilitate transmission of SIV and HIV-1 by compromising the integrity of the mucosal barrier and increasing target cell availability. In the model illustrated in Fig. 3, pre-existing inflammation is shown in association with thinned and disrupted vaginal epithelium. This provides immediate access to large numbers of target cells in the underlying submucosa, and thus the association of initial vaginal SIV infections at such a site 52. The seemingly paradoxical enhancing effects of imiquimod and CpG ODN 74 applied vaginally on SIV transmission may also be explained by the effects on mucosal integrity and target cell availability of inflammation induced by these Toll-like receptor agonists. Pre-existing genital infections and ulcers may facilitate systemic infection not only by effects on the mucosal barrier and target cell availability, but also by providing a haematogeneous route for immediate systemic spread. For example, SIV-infected and other cells inoculated vaginally were quickly disseminated throughout the lymphatic tissues in the setting of experimentally induced genital ulceration 25. Co-existing genital infections also increase HIV-1 acquisition through macro- or micro-ulcerations compromising the integrity of the mucosal barrier and the increased availability of target cells associated with these infections 75,76. These conditions relieve the genetic bottleneck described above, such that multiple genetic variants rather than one or two are transmitted to individuals with underlying genital infections 77. Target cell availability may also explain the failure of acyclovir treatment of herpes simplex virus 2 (HSV-2) infection to reduce the associated acquisition of HIV-1, despite decreased shedding and healing of HSV-2 ulcers 78,79. This result, in line with the generally disappointing results in reducing HIV-1 acquisition by treating sexually transmitted pdcs

5 NATUREjVol 464j11 March 2010 infections 75,76, can now be attributed to persistence of enriched target cell populations after ulcer healing 80. Seminal contributions to transmission. Even without pre-existing inflammation, exposure of the mucosal epithelium to semen initiates signalling to orchestrate the necessary changes in the female reproductive tract for tolerance of allogeneic sperm, implantation and development of the fetus 81,82 that may also facilitate transmission. Exposure of cervical vaginal epithelium to semen elicits increases in chemokines such as MIP-3a 83 (similar to the effect of exposure to the viral inoculum in experimental SIV infection 52 ) and pro-inflammatory cytokines (GM-CSF, IL-1, IL-6 and IL-8) that recruit neutrophils, dendritic cells, macrophages and lymphocytes, which accumulate beneath the cervical and uterine epithelium. Thus, a microenvironment conducive to transmission is created by the recruited cells, the immunosuppressive effects of TGF-b and prostaglandin E in semen, other transmission-enhancing factors in semen such as the amyloid fibres derived from prostatic acid phosphatase 84, alterations in mucosal integrity effected by neutrophils migrating through the epithelium, and increased target cell availability mediated by mucosal epithelial signalling. Mucosal cellular immune response Too little and too late. I now turn from early infection and virus interactions with innate defences to the adaptive immune response. Even though this response is not detectable until the end of the second week of infection 40, its timing, location and magnitude are still instructive for designing effective preventive measures. The cellular immune response that has so far been characterized follows antigen expansion and peak replication, and therefore has been described 40 as too late and too little, because it is too late to clear virus locally or prevent systemic spread, and insufficient in magnitude to prevent the massive CD4 T-cell depletion in gut and other pathological consequences of early infection. Nonetheless, the robustness of the immune response in specific tissue compartments is a determinant of the extent of control of viral replication and thus rate of disease progression. Tissue-compartment-specific partial control. The changing relationships over time in numbers and locations of immune effectors virus-specific CD8 T cells and their infected targets in infected tissues can be visualized and quantified by combining in situ tetramer staining of the effectors with in situ hybridization to detect viral RNA 1 cells 85. This analysis, illustrated in Fig. 2, provides images of the spatial proximity and conjugates of effectors and targets, and a direct way to determine the in vivo effector/target ratio. It is immediately apparent in this infected lymph node that there are not only large numbers of effectors but also large numbers of targets, so the effector/target ratio is,2. Such a low effector/target ratio correlates with poor control of viral replication measured as reduction from peak viral load. By contrast, at high effector/target ratios $50 100:1 achieved at 3 weeks after vaginal exposure in cervical vaginal tissues, there was a commensurate,100-fold reduction in viral load. This analysis tells us that the extent of control by SIV-specific CD8 T cells is tissue-compartment-specific and related to the relative numbers of both effectors and targets. The opportunities for prevention in the earliest stages of infection tell us that both timing and location may be critical determinants of outcome. When infected founder populations are small and focal, and have not as yet expanded sufficiently to have disseminated and established systemic infection, a relatively small number of effectors at the mucosal site of entry might be at the right place at the right time to be enough and soon enough to clear infection 85. Targeting early infection. The soon enough part of this concept implies that a very rapid local recall response and local expansion of virus-specific cytotoxic T lymphocytes (CTLs) might eradicate founder populations before local expansion spreads infection. Whether the few days available in vaginal transmission would be sufficient or not is unclear from experience with conventional vaccines and ideas aboutimmunologicalmemory.itmaybethatavaccinewouldhave to induce and maintain a population of effectors at the portal of entry to be up to the challenge. This is more likely to be the case for rectal and oral transmission where infection is also even more quickly disseminated Consistent with the concept of a protective mucosal population of immune effectors, vaccine-induced high-avidity CTLs have been shown to delay simian-human immunodeficiency virus (SHIV) dissemination from mucosa following rectal challenge 86, and prior infection of rhesus macaques with an attenuated SHIV has been shown to provide protection against vaginal challenge associated with SIV-specific CTLs in cervical vaginal tissues 87. Furthermore, a persistent prior infection with rhesus cytomegalovirus expressing SIV antigens that elicited and maintained virus-specific effector memory CD4 and CD8 T cells at extra-lymphoid sites has recently been shown to provide partial protection against repeated intrarectal challenge 88. More generally, measures that target the establishment and expansion of small founder populations in the earliest stages of infection are most likely to be effective in prevention (Fig. 1). Thus, pharmacological interventions with antiretroviral therapies within the window in which the infected founder population has just been established can prevent or increase control and moderate the pathological consequences of infection 89. Microbicides that block binding and coreceptor-mediated entry and reverse transcription have been shown to protect against SHIV and SIV vaginal and rectal challenges in the rhesus macaque model Similarly, moderating the growth in target cells available to fuel the local expansion on which the virus depends can also prevent vaginal transmission 52. Challenges and gaps in knowledge Studies described herein reveal both challenges in preventing mucosal HIV-1 transmission and opportunities to do so, particularly with strategies targeting early infection. Designing and assessing the effectiveness of countermeasures would be informed and enabled by addressing the following six issues. First, discovering ways to generate a resident or rapidly responding mucosal population to eliminate the small founder population at the portal of entry that avoids the immunosuppressive and target cell availability problems inherent in the immune activation necessary for that response. Second, identifying correlates of prevention in the recent Thai trial 7 of a two-component vaccine in which neither component alone had previously shown efficacy. Third, monitoring occult infections in trials of vaccines and microbicides. Occult infections are defined as HIV-1-exposed individuals who remain seronegative but who have detectable cellular immune responses to HIV-1 and DNA in CD4 T cells 93 ; or rhesus macaques repeatedly challenged vaginally or rectally with low or high doses of SIV that develop transient viraemia with low to undetectable antibodies or cellular immune response, and have detectable provirus in vaginal and lymphoid tissues 94,96. They may later be manifest as typical pathogenic systemic infections, have been documented after apparent protection with a microbicide 52, and thus represent a potential public health issue and point to the need for long-term follow up in testing vaccine and microbicide candidates. Fourth, defining mechanism(s) by which circumcision does or does not protect 4 6, and the lack of circumcision facilitates male transmission. Fifth, developing and investigating the following in NHP models: (1) cell-free virus transmission under conditions that more closely approximate human exposures; (2) cell-associated viral transmission; and (3) each route of mucosal transmission vaginal, rectal, oral and penile which will probably differ, based on their distinct anatomy and functions, both in local propagation and routes and speed of systemic spread. 221

6 NATUREjVol 464j11 March 2010 Sixth, advancing fundamental knowledge of mucosal immunology: (1) understanding why the interferon system and other innate immune mechanisms are not more effective against SIV and HIV-1 despite their massive upregulation in early infection 52,69,97 ; (2) the role of natural killer cells and antibody-dependent cellular cytotoxicity in host defences at mucosal sites; (3) CD8 T-cell mucosal immune responses and the trafficking, turnover and fate of these cells at mucosal sites; (4) the role of CD4 T cells in host defences at mucosal sites; and (5) discovering new mechanisms of host defences at mucosal sites by genomic analyses and systems biology. Looking to the future, a better understanding of mucosal-sitespecific infection and immunity, answering the fundamental immunological questions posed in ref. 98, and engaging a broader scientific community in the effort constitute the most promising path to an effective vaccine and other interventions to prevent sexual mucosal transmission of HIV UNAIDS Report on the global AIDS epidemic Æ KnowledgeCentre/HIVData/GlobalReport/2008/2008_Global_report.aspæ (2008). 2. Fauci, A. S. 25 years of HIV. Nature 453, (2008). 3. Cohen, J. Treatment and prevention exchange vows at international conference. Science 321, (2008). 4. Bailey, R. C. et al. Male circumcision for HIV prevention in young men in Kisumu, Kenya: a randomised controlled trial. Lancet 369, (2007). 5. Auvert, B. et al. Randomized, controlled intervention trial of male circumcision for reduction of HIV infection risk: the ANRS 1265 Trial. PLoS Med. 2, e298 (2005). 6. Gray, R. H. et al. Male circumcision for HIV prevention in men in Rakai, Uganda: a randomised trial. Lancet 369, (2007). 7. Rerks-Ngarm, S. et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N. Engl. J. Med. 361, (2009). 8. Karim, S. A. et al. Safety and effectiveness of vaginal microbicides BufferGel and 0.5% PRO 2000/5 gel for the prevention of HIV infection in women: Results of the HPTN 035 trial. Abstract 48LB (16th Conference on Retroviruses and Opportunistic Infections, 2009). 9. Buchbinder, S. P. et al. Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomized, placebo-controlled, testof-concept trial. Lancet 372, (2008). 10. Check, E. Scientists rethink approach to HIV gels. Nature 446, 12 (2007). 11. Fauci, A. et al. HIV vaccine research: the way forward. Science 321, (2008). 12. Quinn, T. C. & Overbaugh, J. HIV/AIDS in women: an expanding epidemic. Science 308, (2005). 13. Haase, A. The slow infection caused by visna virus. Curr. Top. Microbiol. Immunol. 72, (1975). 14. Miller, C. J. et al. Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus. J. Virol. 63, (1989). 15. Fiebig, E. W. et al. Dynamics of HIV viremia and seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. AIDS 17, (2003). 16. Miller, C. J. et al. Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus. J. Virol. 79, (2005). Comprehensive tissue analysis of the pathogenesis of transmission and early infection in the nonhuman primate model of vaginal transmission of HIV Hu, J., Gardner, M. B. & Miller, C. J. Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells. J. Virol. 74, (2000). 18. Zhang, Z.-Q. et al. Sexual transmission and propagation of simian and human immunodeficiency viruses in two distinguishable populations of CD4 1 T cells. Science 286, (1999). First description of the CD4 T cell as the principal target in acute SIV and HIV-1 infections, and the surprising ostensibly resting phenotype of a majority of the CD4 T cells initially infected. 19. Reinhart, T. et al. Simian immunodeficiency virus burden in tissues and cellular compartments during clinical latency and AIDS. J. Infect. Dis. 176, (1997). 20. Haase, A. T. Population biology of HIV-1 infection: viral and CD4 1 T cell demographics and dynamics in lymphatic tissues. Annu. Rev. Immunol. 17, (1999). 21. Miller, C. M. & Shattock, R. J. Target cells in vaginal HIV transmission. Microbes Infect. 5, (2003). 22. Christopher, D. P. et al. Brief but efficient: acute HIV infection and the sexual transmission of HIV. J. Infect. Dis. 189, (2004). 23. Keele, B. F. et al. Low-dose rectal inoculation of rhesus macaques by SIV sme660 or SIVmac251 recapitulates human mucosal infection by HIV-1. J. Exp. Med. 206, (2009). 24. Sodora, D. L., Gettie, A., Miller, C. J. & Marx, P. A. Vaginal transmission of SIV: assessing infectivity and hormonal influences in macaques inoculated with 222 cell-free and cell-associated viral stocks. AIDS Res. Hum. Retroviruses 14 (Suppl. 1), S119 S123 (1998). 25. Weiler, A. M. et al. Genital ulcers facilitate rapid viral entry and dissemination following intravaginal inoculation with cell-associated simian immunodeficiency virus SIVmac239. J. Virol. 82, (2008). 26. Miyake, A. et al. Rapid dissemination of a pathogenic simian/human immunodeficiency virus to systemic organs and active replication in lymphoid tissues following intrarectal infection. J. Gen. Virol. 87, (2006). 27. Stahl-Hennig, C. et al. Rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus. Science 285, (1999). 28. Milush, J. M. et al. Rapid dissemination of SIV following oral inoculation. AIDS 18, 1 10 (2004). 29. Veazey, R. S. et al. Gastrointestinal tract as a major site of CD4 1 T cell depletion and viral replication in SIV infection. Science 280, (1998). 30. Mattapallil, J. et al. Massive infection and loss of memory CD4 1 T cells in multiple tissues during acute SIV infection. Nature 434, (2005). 31. Li, Q. et al. Peak SIV replication in resting memory CD4 1 T cells depletes gut lamina propria CD4 1 T cells. Nature 434, (2005). Tissue analysis that revealed the rapid kinetics of acute SIV infection after vaginal exposure, predominance of productive infection in resting memory CD4 T cells, and the loss of gut lamina propria CD4 T cells mediated mainly by apoptosis. 32. Clayton, F., Snow, G., Reka, S. & Kotler, D. P. Selective depletion of rectal lamina propria rather than lymphoid aggregate CD4 lymphocytes in HIV infection. Clin. Exp. Immunol. 107, (1997). 33. Mehandru, S. et al. Primary HIV-1 infection is associated with preferential depletion of CD4 1 T lymphocytes from effector sites in the gastrointestinal tract. J. Exp. Med. 200, (2004). 34. Brenchley, J. M. et al. CD4 1 T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J. Exp. Med. 200, (2004). 35. Guadalupe, M. et al. Severe CD4 1 T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J. Virol. 77, (2003). 36. Kotler, D. P., Gaetz, H. P., Lange, M., Klein, E. B. & Holt, P. R. Enteropathy associated with the acquired immunodeficiency syndrome. Ann. Intern. Med. 101, (1984). 37. Heise, C., Miller, C. J., Lackner, A. & Dandekar, S. Primary acute simian immunodeficiency virus infection of intestinal lymphoid tissue is associated with gastrointestinal dysfunction. J. Infect. Dis. 169, (1994). 38. Li, Q. et al. Simian immunodeficiency virus-induced intestinal cell apoptosis is the underlying mechanism of the regenerative enteropathy of early infection. J. Infect. Dis. 197, (2008). 39. Estes, J. D. et al. Premature induction of an immunosuppressive T regulatory response in acute SIV infection. J. Infect. Dis. 193, (2006). 40. Reynolds, M. R. et al. The CD8 1 T-lymphocyte response to major immunodominant epitopes after vaginal exposure to SIV: too late and too little. J. Virol. 79, (2005). Comprehensive tissue analysis after vaginal exposure that revealed the too late and too little CD8 T-cell response to prevent massive gut CD4 T-cell depletion in acute SIV infection but a robust response in cervical vaginal tissues that could potentially prevent acquisition if elicited earlier by a vaccine. 41. Brenchley, J. M. et al. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nature Med. 12, (2006). 42. Estes, J. D. et al. Simian immunodeficiency virus-induced lymphatic tissue fibrosis is mediated by transforming growth factor b1-positive regulatory T cells and begins in early infection. J. Infect. Dis. 195, (2007). 43. Estes, J. D., Haase, A. T. & Schacker, T. W. The role of collagen deposition in depleting CD4 1 T cells and limiting reconstitution of HIV-1 and SIV infections through damage to the secondary lymphoid organ niche. Semin. Immunol. 20, (2008). 44. Wolinsky, S. et al. Selective transmission of human deficiency virus type 1 variants from mothers to infants. Science 255, (1992). 45. Zhu, T. et al. Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission. J. Virol. 70, (1996). 46. Derdeyn, C. A. et al. Envelope-constrained neutralization-sensitive HIV-1 after heterosexual transmission. Science 303, (2004). First description of the monophyletic nature of viruses transmitted heterosexually. 47. Keele, B. F. et al. Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc. Natl Acad. Sci. USA 105, (2008). Comprehensive sequence analysis showing that a single virus genotype initiates the vast majority of HIV-1 infections. 48. Salazar-Gonzalez, J. F. et al. Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection. J. Exp. Med. 206, (2009). 49. Norvell, M. K., Benrubi, G. I. & Thompson, R. J. Investigation of microtrauma after sexual intercourse. J. Reprod. Med. 269, (1984). 50. Pudney, J., Quayle, A. J. & Anderson, D. J. Immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone. Biol. Reprod. 73, (2005).

7 NATUREjVol 464j11 March O Connor, D. M. A tissue basis for colposcopic findings. Obstet. Gynecol. Clin. North Am. 35, (2008). 52. Li, Q. et al. Glycerol monolaurate prevents mucosal SIV transmission. Nature 458, (2009). Tissue analysis of vaginal transmission revealing local expansion of small, infected founder populations, in vivo evidence of mucosal epithelial signalling that elicited an inflammatory response to fuel local expansion, and the ability of glycerol monolaurate to prevent transmission potentially by blocking signalling at mucosal front lines. 53. Myer, L., Wright, T. C. Jr, Denny, L. & Kuhn, L. Nested case-control study of cervical mucosal lesions ectopy, and incident HIV infection among women in Cape Town, South Africa. Sex. Transm. Dis. 33, (2006). 54. Miller, C. J., Alexander, N. J., Vogel, P., Anderson, J. & Marx, P. A. Mechanism of genital transmission of SIV: a hypothesis based on transmission studies and location of SIV in the genital tract of chronically infected female rhesus macaques. J. Med. Primatol. 21, (1992). 55. Kell, P. D., Barton, S. E., Edmonds, D. K. & Boag, F. C. HIV infection in a patient with Meyer-Rokitansky-Kuster-Hauser syndrome. J. R. Soc. Med. 85, (1992). 56. Padian, N. S. et al. Diaphragm and lubricant gel for prevention of HIV acquisition in southernafricanwomen: a randomisedcontrolledtrial. Lancet 370, (2007). 57. Berger, E. A., Murphy, P. M. & Farber, J. M. Chemokine receptors as HIV-1 coreceptors: Roles in viral entry, tropism, and disease. Annu. Rev. Immunol. 17, (1999). 58. Arthos, J. et al. HIV-1 envelope binds to and signals through integrin a4b7, the gut mucosalhoming receptor for peripheral T cells. Nature Immunol. 9, (2008). 59. Kader, M. et al. a4b7 hi CD4 1 memory T cells harbor most T H -17 cells and are preferentially infected during acute SIV infection. Mucosal Immunol. 2, (2009). 60. Zhang, Z.-Q. et al. Roles of substrate availability and infection of resting and activated CD4 1 T cells in transmission and acute simian immunodeficiency virus infection. Proc. Natl Acad. Sci. USA 101, (2004). 61. Schacker, T. S. et al. Productive infection of T cells in lymphoid tissues during primary and early human immunodeficiency virus infection. J. Infect. Dis. 183, (2001). 62. Ma, Z., Lü, F. X., Torten, M. & Miller, C. J. The number and distribution of immune cells in the cervicovaginal mucosa remain constant throughout the menstrual cycle of rhesus macaques. Clin. Immunol. 100, (2001). 63. Edwards, J. N. T. & Morris, H. B. Langerhans cells and lymphocyte subsets in the female genital tract. Br. J. Obstet. Gynaecol. 92, (1985). 64. Wira, C. R., Fahey, J. V., Sentman, C. L., Pioli, P. A. & Shen, L. Innate and adaptive immunity in female genital tract: cellular responses and interactions. Immunol. Rev. 206, (2005). 65. Wira, C. R., Grant-Tschudy, K. S. & Crane-Godreau, M. A. Epithelial cells in the female reproductive tract: a central role as sentinels of immune protection. Am. J. Reprod. Immunol. 53, (2005). 66. Fahey, J. V. et al. Estradiol selectively regulates innate immune function by polarized human uterine epithelial cells in culture. Mucosal Immunol. 1, (2008). 67. Dieu-nosjean, M. et al. Macrophage inflammatory protein 3a is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors. J. Exp. Med. 192, (2000). 68. Cremel, M. et al. Characterization of CCL20 secretion by human epithelial vaginal cells: involvement in Langerhans cell precursor attraction. J. Leukoc. Biol. 78, (2005). 69. Abel, K., Rocke, D. M., Chohan, B., Fritts, L. & Miller, C. J. Temporal and anatomic relationship between virus replication and cytokine gene expression after vaginal simian immunodeficiency virus infection. J. Virol. 79, (2005). 70. Gray, R. H. et al. Probability of HIV-1 transmission per coital act in monogamous, heterosexual, HIV-1-discordant couples in Rakai, Uganda. Lancet 357, (2001). 71. Wawer, M. J. et al. Rates of HIV-1 transmission per coital act, by stage of HIV-1 infection, in Rakai, Uganda. J. Infect. Dis. 191, (2005). 72. Vendrame, D., Sourisseau, M., Perrin, V., Schwartz, O. & Mamano, F. Partial inhibition of human immunodeficiency virus replication by type I interferons: Impact of cell-to-cell viral transfer. J. Virol. 83, (2009). 73. Rudnicka, D. et al. Simultaneous cell-to-cell transmission of human immunodeficiency virus to multiple targets through polysynapses. J. Virol. 83, (2009). 74. Wang, Y. et al. The toll-like receptor 7 (TLR7) agonist, imiquimod and the TLR9 agonist, CpG ODN, induce antiviral cytokines and chemokines but do not prevent vaginal transmission of simian immunodeficiency virus when applied intravaginally to rhesus macaques. J. Virol. 79, (2005). 75. Glavin, S. R. & Cohen, M. S. The role of sexually transmitted diseases in HIV transmission. Nature Rev. Microbiol. 2, (2004). 76. Kaul, R. et al. The genital tract immune milieu: an important determinant of HIV susceptibility and secondary transmission. J. Reprod. Immunol. 77, (2008). 77. Haaland, R. E. et al. Inflammatory genital infections mitigate a severe genetic bottleneck in heterosexual transmission of subtype A and C HIV-1. PLoS Pathog. 5, 1 13 (2009). 78. Celum, C. et al. Effect of acyclovir on HIV-1 acquisition in herpes simplex virus 2 seropositive women and men who have sex with men: a randomized, doubleblind, placebo-controlled trial. Lancet 371, (2008). 79. Watson-Jones, D. et al. Effect of herpes simplex suppression on incidence of HIV among women in Tanzania. N. Engl. J. Med. 358, (2008). 80. Zhu, J. et al. Persistence of HIV-1 receptor-positive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition. Nature Med. 15, (2009). Report of persistent target cells in healed HSV-2 ulcers after acyclovir treatment as the potential explanation for the failure of treatment to reduce HIV-1 acquisition. 81. Robertson, S. A. Seminal plasma and male factor signalling in the female reproductive tract. Cell Tissue Res. 322, (2005). 82. Robertson, S. A., Ingman, W. V., O Leary, S., Sharkey, D. J. & Tremellen, K. P. Transforming growth factor b a mediator of immune deviation in seminal plasma. J. Reprod. Immunol. 57, (2002). 83. Berlier, W. 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. 21, (2006). 84. Münch, J. et al. Semen-derived amyloid fibrils drastically enhance HIV infection. Cell 131, (2007). 85. Li, Q. et al. Visualizing antigen-specific and infected cells in situ predicts outcomes in early viral infection. Science 323, (2009). Report of the tissue-compartment-specific nature of the cellular immune response that reveals the importance of the relative numbers of both immune effectors and infected targets in determining whether viral infection is cleared, and the extent of control if infection is not cleared. 86. Belyakov, I. M. et al. Impact of vaccine-induced mucosal high-avidity CD8 1 CTLs in delay of AIDS viral dissemination from mucosa. Blood 107, (2006). 87. Genescà, M., Skinner, P. J., Bost, K. M., Lu, D. & Wang, Y. Protective attenuated lentivirus immunization induces SIV-specific T cells in the genital tract of rhesus monkeys. Mucosal Immunol. 1, (2008). 88. Hansen, S. G. et al. Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge. Nature Med. 15, (2009). Report of impressive protection against rectal transmission associated with effector memory T-cell responses elicited by a rhesus CMV vaccine. 89. Lifson, J. D. et al. Containment of simian immunodeficiency virus infection: Cellular immune responses and protection from rechallenge following transient postinoculation antiretroviral treatment. J. Virol. 74, (2000). 90. Veazey, R. S. et al. Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion. Nature 438, (2005). 91. Lederman, M. M. et al. Prevention of vaginal SHIV transmission in rhesus macaques through inhibition of CCR5. Science 306, (2004). 92. Cranage, M. et al. Prevention of SIV rectal transmission and priming of T cell responses in macaques after local pre-exposure application of tenofovir gel. PLoS Med. 5, (2008). 93. Zhu, T. et al. Persistence of extraordinarily low levels of genetically homogeneous human immunodeficiency virus type 1 in exposed seronegative individuals. J. Virol. 77, (2003). 94. McChesney, M. B. et al. Occult systemic infection and persistent simian immunodeficiency virus (SIV)-specific CD4 1 -T-cell proliferative responses in rhesus macaques that were transiently viremic after intravaginal inoculation of SIV. J. Virol. 72, (1998). 95. Ma, Z.-M., Abel, K., Rourke, T., Wang, Y. & Miller, C. J. A period of transient viremia and occult infection precedes persistent viremia and antiviral immune responses during multiple low-dose intravaginal simian immunodeficiency virus inoculations. J. Virol. 78, (2004). 96. Trivedi, P. et al. Intrarectal transmission of simian immunodeficiency virus in rhesus macaques: selective amplification and host responses to transient or persistent viremia. J. Virol. 70, (1996). 97. Li, Q. et al. Microarray analysis of lymphatic tissue reveals stage-specific, gene expression signatures in HIV-1 infection. J. Immunol. 183, (2009). 98. Virgin, H. W. & Walker, B. D. Immunology and the elusive AIDS vaccine. Nature. (in the press). Acknowledgements I thank J. V. Carlis, R. P. Johnson, Q. Li, J. D. Lifson, D. Masopust, S. Pambuccian, P. J. Southern, J. Estes, D. Douek, H. W. Virgin and B. D. Walker for discussions. Errors of commission are mine as are errors of omission, with apologies to the authors of work that I could not cite because of space limitations and exclusive focus on tissue analyses. I thank C. O Neill and T. Leonard for help with the manuscript and figures. Work from my laboratory cited in the review was supported by grants from the National Institutes of Health (AI 38565, AI 48484, AI 71976) and the International AIDS Vaccine Initiative. Author Information Reprints and permissions information is available at The author declares no competing financial interests. Correspondence should be addressed to the author (haase001@umn.edu). 223