Patogenesis Del Vih

11th Conference on Retroviruses and Opportunistic Infections Pathogenesis of HIV Infection CME/CE

Contents of This CME/CE Activity

1. HIV Immunopathogenesis and Correlates of Protection Michael Lederman, MD

2. Virus and Host Factors in HIV Pathogenesis Mario Stevenson, PhD

3. Sexual Transmission of HIV Myron S. Cohen, MD

HIV Immunopathogenesis and Correlates of Protection Michael Lederman, MD

Introduction In the more than 20 years since the recognition of AIDS and HIV, substantial progress has been made both in understanding the mechanisms of HIV replication and in designing treatment strategies that have decreased HIV-related mortalities substantially. Nevertheless, the pathogenesis of immune deficiency in HIV infection is incompletely understood, and the determinants (as opposed to the correlates) of protection against and endogenous control of HIV replication are poorly understood. It is clear that an understanding of the determinants of protection will be critical to the design of prevention strategies, while an understanding of host determinants of virologic control may help in the design of strategies to suppress HIV replication.

New Data on HIV Immunopathogenesis Speelmon and colleagues[1] from the University of Washington in Seattle examined the susceptibility to HIV replication of CD4+ T cells obtained from exposed HIV-seronegative persons to support in vitro HIV replication. Although the group as a whole did not show differential susceptibility to HIV replication, a small number of subjects had cells that were reproducibly less capable of supporting HIV replication. The mechanisms that underlie these effects remain to be elucidated. Very recently, a subset of CD4+ T cells that provide regulatory (inhibitory) function has been described in both mice and humans. These T regulatory (Treg) CD4+ cells express the alpha chain of the interleukin (IL)-2 receptor (CD25), although they are distinguishable from the CD25+ CD4+ T cells that are expanded by treatment with high levels of IL-2. In healthy conditions, these cells are thought to play key roles in the prevention of autoreactivity; in a number of infectious diseases, these cells may attenuate the magnitude of immune responses. Audrey Kinter and colleagues[2] from the National Institutes of Health and Department of Health and Human Services in Bethesda, Maryland, reported increased numbers of these cells in HIVinfected persons; by depleting these cells, both the cytokine responses and the proliferation

responses of CD4+ and CD8+ T-cell responses to HIV antigens were enhanced. This enhancement appeared to be specific for HIV responses and not for responses to Candida. These findings confirm and extend findings of other groups who have studied this cell population in HIV disease.[3] Both the genesis of these cells and their role in HIV disease are not well understood. Do they contribute to the immune deficiency of HIV disease, or does their presence attenuate the heightened immune activation that is likely central to the pathogenesis of cell losses in HIV infection? Is the immunopathogenesis of HIV infection in men and women really different? Although this question is not easily resolved and is hotly debated, increasing evidence from earlier work and studies presented at this meeting suggest that differences are likely to exist. Earlier studies have indicated that women tend to present for HIV care with lower plasma HIV RNA levels, yet their rates of disease progression appear to be comparable to those experienced by men, and mortalities have seemed comparable. Garcia de la Hera and colleagues[4] of University Miguel Hernandez in Alicante, Spain, reported their findings concerning the risk for disease progression and death among Spanish male and female intravenous-drug users with comparable durations of known infection. Risk for both AIDS and death was apparently lower in women than in men. Correction for confounding differences in these groups must be done since most other groups have not been able to show an enhanced disease risk in men. Another report by Lisgaris and colleagues[5] of Case Western Reserve University in Cleveland, Ohio, found that, overall, HIVinfected women experienced opportunistic infections at lower CD4+ cell counts and at higher levels of HIV replication than did men. If these interesting observations are confirmed independently in other datasets, then we must ask if CD4+ cells are better helpers on a per-cell basis, if effector cells are greater in number or activity, or if other nonadaptive defenses are more active in women -- and why these differences exist. Two studies examined immune mechanisms in sooty mangabeys, monkeys that are susceptible to infection with SIV but that do not develop SIV-related immune deficiency. Wang and colleagues[6] of the New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts, demonstrated that these animals were capable of recognizing and generating cytokines in response to SIV peptides. This finding suggests that it is not the failure of immune recognition (as some had suggested) that protects these animals from the consequences of SIV infection. Ribeiro and colleagues[7] of University of Oxford, Oxford, United Kingdom, showed that the turnover of T cells as measured by incorporation of bromodeoxyuridine was not increased in SIV-infected mangabeys when compared with T-cell turnover in uninfected animals. This finding strongly supports current concepts in HIV disease pathogenesis that suggest that immune activation is a critical determinant of the immune deficiency that is the source of morbidity in HIV infection.

New Data on Anti-HIV Immune Responses: Mechanisms and Viral Control The determinants of successful survival with HIV infection are poorly understood. Studies of socalled long-term nonprogressors (LTNPs) may help to identify correlates or determinants of virologic control. Addo and colleagues[8] from Partners AIDS Research Center, Massachusetts General Hospital, Boston, Massachusetts, studied a group of 73 LTNPs and found that neither the breadth nor the magnitude of CD4+ or CD8+ T-cell responses to HIV distinguished these patients from patients with progressive disease. Of note, LTNPs with very low levels of HIV replication (viral load < 50 copies/mL) tended to have lesser HIV-specific CD4+ and CD8+ T-cell responses than did LTNPs with viral loads between 50 and 2000 copies/mL. In these patients and at this time in the course of disease, it appears that these readouts of immune responsiveness likely reflect more the consequence of viral replication than the determinants of its control. More details on the relationships among host genetics, adaptive immune responses, and virologic control were provided by Simon Mallal[9] from the Center for Clinical Immunology and Biomed Statistics at the Royal Perth Hospital, Perth, Australia. Dr. Mallal has been studying these issues in a large cohort of HIV-infected Australians. A balance between the advantage of viral evolution to escape peptide binding by class I MHC -- or possibly even intracellular processing of these sequences -- vs a replicative cost of this mutation have been modeled to

determine the ultimate effect of viral evolution on control of viral replication. At a time when some vaccine researchers are uncertain about the host determinants of virologic control in HIV infection, these studies support the importance of peptide recognition by CD8+ T cells in the control of HIV replication. This approach was proposed as a model to predict the design of HIV vaccines.

New Data on Immune-Based Therapies Kinloch and colleagues[10] from the Royal Free and University College Medical School, London, United Kingdom, presented the results of the QUEST study. This international collaborative trial included persons who had been recognized very shortly after acute HIV infection and who were started immediately on a combination antiretroviral regimen. After an average of approximately 2 years of virologic control, 79 patients were randomized to received immunization with ALVAC VcP1452, ALVAC plus Remune, or placebo. Median CD4+ cell counts in these groups exceeded 700 cells/mcL at the start of immunizations. After the 24-week immunization period, antivirals were stopped and patients were observed for evidence of virologic rebound. There was no difference between the 3 groups in the levels of plasma HIV RNA at 24 weeks after treatment discontinuation or in the timing of virologic rebound. Of note, approximately half of the patients in each group maintained plasma HIV RNA levels < 10,000 copies/mL and 20% had plasma HIV RNA levels < 1000 copies/mL. These findings suggest the possibility that early treatment of HIV infection permits the restoration or retention of the ability to better control HIV replication but that these immunization strategies were futile in this setting. Of course this study was not designed to ask the first question, and therefore there was no control group that did not receive antivirals early in the course of infection. Controlled studies are needed to determine whether administration of highly active antiretroviral therapy (HAART) early in the course of acute HIV infection affects subsequent virologic control. David Cooper and colleagues[11] from the National Centre in HIV Epidemiology and Clinical Research, Sydney, Australia, reported the results of another smaller vaccine trial that also studied persons who were treated with HAART shortly after acquisition of HIV infection. After an average of 4 years of antiviral therapies, 35 persons with controlled HIV replication were randomized to vaccination with a fowlpox vector containing no HIV sequences, a vector containing HIV gag/pol sequences, or a vector containing HIV gag/pol and a gene encoding human interferon gamma. Surprisingly, there was not much difference among the groups in terms of ELISPOT cell responses or cytolytic responses after vaccination and before treatment discontinuation, and 10 immunized subjects did not enter the treatment discontinuation phase of the study. Nonetheless, there was no difference in the degree of virologic control between the control group and the group immunized with the HIV gag/pol constructs. Still, the subjects immunized with the HIV gag/pol and human interferon gamma sequences seemed to experience better control of HIV replication, with average plasma HIV RNA levels about 0.8 log10 lower than levels in the other groups. The absence of detectable immune responses to the immunization is disappointing, and the responses in the interferon groups are surprising. Does this represent an effect of interferon alone or of host defenses? And if so, is the effector cell within the T-cell compartment or a component of the innate immune defense? Garcia and colleagues[12] of Barcelona University, Barcelona, Spain, presented a final dataset on a controlled study examining the use of monocyte-derived dendritic cells pulsed with plasmaderived autologous virus as a therapeutic vaccine strategy in persons with established HIV infection. Although there were no clinically significant effects on control of HIV replication, there were tantalizing hints of nominally significant activity, such as a delay in virologic rebound in the group and an apparent and marginally better control of HIV replication in the vaccinated subjects. The use of autologous sequences in established infection was an important piece of this well-designed study, yet this approach was limited by the availability of antigen with which to pulse the ex vivo matured dendritic cells. It will be very interesting to see whether higher doses of autologous antigen will provide greater immunogenicity and antiviral activity. IL-15 is a cytokine that is capable of expanding memory CD8+ T-cell populations in vitro and enhancing effector function without enhancing HIV replication. Petrovas and colleagues[13] from Drexel University College of Medicine, Philadelphia, Pennsylvania, reported that IL-15

administered twice weekly for 4 weeks to SIV-infected cynomolgus macaques increased the proliferation and expansion of CD8+ T cells without affecting levels of SIV replication. No increases in the frequency of gag-reactive interferon-gamma-producing cells were observed, but no note was made as to whether the absolute numbers of these cells were increased. Henry and colleagues[14] from the University of Minnesota, Minneapolis, reported the results of a pilot study (ACTG 5102) that asked whether intermittent treatment with high doses of IL-2 while on HAART could prolong the period off antiretroviral therapy after treatment interruption. Using defined criteria for the resumption of antiretroviral therapies, Dr. Henry's group found that IL-2treated patients maintained higher CD4+ cell counts and similar plasma HIV RNA levels after treatment interruption, and were less likely to "require" treatment reinitiation. Numbers are small to date, and the study is ongoing. While this strategy may permit longer treatment-free intervals after IL-2 administration, it should be recognized that irrespective of prolonging the treatmentfree interval in this setting, it remains uncertain as to whether IL-2 administration confers clinical benefit. Nonetheless, this team has taken an interesting approach to exploring potential utilities of this strategy and the concept of treatment interruption enhanced by IL-2-mediated CD4+ Tcell expansion as a means to spare exposure to antiretroviral therapies and their consequences.

Conclusion We are at an early stage in learning how to harness host mechanisms to treat HIV infection and its consequences. That said, host-targeted strategies (eg, targeting CD4 and CCR5) will undoubtedly prove to be clinically useful approaches to attenuating HIV replication. However, we have only the most limited understanding of the mechanisms that protect persons from acquisition of HIV infection and only modest understanding of the mechanisms and best measures of virologic control and immune deterioration. Taken together with our inability (at least to date) to generate broadly cross-reactive neutralizing antibodies against HIV after immunization, it is fair to conclude that we are, at this point, far from having an effective protective vaccine strategy. Likewise, the therapeutic vaccine successes to date have been modest. In the face of these findings, we must redouble our efforts to understand better the fundamental determinants of protection from infection, virologic control, and immune deterioration. This likely means a more detailed examination of innate defenses, intrinsic and virus-induced mechanisms of cellular activation and turnover, and a broader exploration of novel approaches to virus neutralization.

References

1. Speelmon E, Desbien A, Livingston-Rosanoff D, McElrath MJ. Diminished CD4+. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract M215.

2. Kinter A, Hennessey M, Bell A, et al. CD4+CD25+ Regulatory T-like cells suppress HIVspecific CD4+ and CD8+ T cell immune responses in vitro. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract M14.

3. Aandahl EM, Michaelsson J, Moretto WJ, Hecht FM, Nixon DF. Human CD4(+) CD25(+) regulatory T cells control T-cell responses to Human Immunodeficiency Virus and cytomegalovirus antigens. J Virol. 2004;78:2454-2459.

4. García de la Hera M, Ferreros I, del Amo J, et al. Gender differences in progression to AIDS and death from HIV seroconversion in a cohort of intravenous drug users from 1986 to 2001. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 152.

5. Lisgaris M, Rodriguez B, Yadavalli G, et al. AIDS-defining illnesses develop with lower

CD4 cell Counts and higher plasma HIV RNA levels in HIV-infected women than in men. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 951.

6. Wang ZC, McClure HM, Kaur A. Th1-type SIV-specific cellular immune responses targeting structural proteins are consistently detected in naturally SIV-infected Sooty Mangabeys. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 130.

7. Ribeiro RM, Di Mascio M, McClure HM, Johnson RP, Kaur A, Perelson AS. Modeling Tcell labeling with BrdU in SIV-infected sooty mangabeys. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 129.

8. Addo MM, Rathod A, Draenert R, et al. Immunological and genetic determinants in HIV1 controllers and long-term non-progressors. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 11.

9. Mallal S. HLA imprinting: implications for selection of vaccine immunogens. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 60.

10. Kinloch S, Perrin L, Hoen B, et al. Evaluation of 2 therapeutic HIV vaccination regimens in HAART-treated primary HIV infection subjects following analytical treatment interruption: QUEST PROB3005, a randomized, placebo-controlled study. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 168.

11. Cooper D, Workman C, Puls R, et al. Randomized, placebo-controlled, phase1/2a evaluation of the safety, biological activity and antiretroviral properties of an avipox virus vaccine expressing HIV gag-pol and interferon-gamma in HIV-1 infected subjects. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 169.

12. Garcia F, Lejeune M, Climent N, et al. Final results of a phase I study of a therapeutic vaccine using autologous dendritic cells primed with autologous virus in patients with chronic HIV infection and CD4 T cells above 400/mm3. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 518.

13. Petrovas C, Mueller YM, Bojczuk P, et al. IL-15 treatment of SIV-infected non-human primates. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 512.

14. Henry K, Tebas P, Cherng D, et al. Interleukin-2 prior to stopping effective antiretroviral therapy prolongs time off treatment: initial results of a pilot study utilizing CD4+ T-cell count <350 cells/mm3 as the threshold for restarting ART (ACTG A5102). Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 510.

Virus and Host Factors in HIV Pathogenesis Mario Stevenson, PhD Pathogenic primate lentivirus infections are characterized by progressive loss of immune function, a heightened state of immune activation, and high-level viral replication. In contrast, in naturally infected African green monkeys, SIV infection is characterized by high viral loads, but there is no apparent loss of immune function nor any evidence of immune system hyperactivation. This has led to the proposal that what distinguishes pathogenic and nonpathogenic primate lentivirus infections is the degree of immune activation, and that it is this, rather than overt viral replication, that creates conditions for pathogenicity. However, the viral

and cellular effectors that contribute to immune activation in pathogenic HIV infection have not been clearly elucidated. Presentations at the 11th CROI may provide some clues as to how HIV infection induces a chronic state of immune activation. The process of T-cell activation is suppressed by a CD4+/CD25+ T-cell subset, also known as Treg cells. It is known that Treg cells express CD4. Therefore, one study examined whether Treg cells were potential targets for HIV infection.[1] The study demonstrated that Treg cells obtained from healthy seronegative individuals as well as T cells induced to a Treg phenotype in vitro were highly susceptible to HIV infection. This supports the notion that the infection and destruction of the Treg cell subset in HIV infection may remove the normal control mechanisms that regulate T-cell activation, and as such, contribute to the chronic activation state of T cells that is characteristic of pathogenic HIV infection. Continuing in this vein, another study indicated that the difference between pathogenic and nonpathogenic infections may relate to target-cell availability, which might affect the distribution of viral reservoirs.[2] In pathogenic SIV infections, the gastrointestinal tract is the major site of viral replication and lymphocyte depletion. In this reservoir, the major target cells are CD4+, CCR5+, and CD45RA-. Therefore, the phenotype of lymphocytes from the peripheral blood and intestinal and lymph node biopsies were examined in African green monkeys (which did not exhibit pathogenic manifestations of SIV infection) and rhesus macaques (which do show pathogenic manifestations of SIV infection). The study authors observed low levels of CD4+, CCR5+, and CD45RA- cells in all compartments of African green monkeys compared with rhesus macaques. The authors proposed that the limited availability of target cells in tissues such as the intestine may contribute to the diminished immune responses and lack of disease progression in this natural host. This generates a paradox because the African green monkey still supports levels of viral replication that parallel those in pathogenic infections of rhesus macaques, and raises questions regarding the major source of viral replication in these different models. In contrast to the well-characterized T-cell defects observed in HIV infection, B-cell dysfunction -- wherein B cells undergo activation-induced terminal differentiation into a plasma cell-like morphology -- remains a poorly explained feature of HIV pathogenesis. The same is true for the hypergammaglobulinemia that accompanies it. Essentially, the underlying basis for the B-cell defects in AIDS is not well understood. One study presented at the conference made a comparison of gene-expression profiles of B cells from HIV-viremic patients and B cells from aviremic and seronegative patients.[3] Of more than 40 genes that were found to be upregulated in the B cells of the former group, several were associated with B-cell terminal differentiation as well as members of the tumor necrosis factor (TNF) superfamily of receptors that regulate apoptosis. The study authors concluded that HIV replication may trigger increased expression of TNF superfamily receptors, making them susceptible to, for example, Fas-mediated cell death. The mechanism through which HIV infection actually causes the changes in B-cell phenotype awaits elucidation.

Viral Reservoirs, Replication, and Rebound In vivo, depending upon the activation state of the cell, CD4+ T lymphocytes exhibit profound qualitative and quantitative differences with respect to the sequelae of infection by HIV. In fully activated lymphocytes, HIV replication is rapid and efficient and invariably leads to the death of the infected cell. At the opposite end of the spectrum, quiescent T cells in the G0 stage of the cell cycle harbor HIV-1 in a latent or dormant state. When that cell subsequently becomes activated, the latent virus is activated along with it, and efficient viral replication resumes. The ability of the virus to reside in a latent state in quiescent T cells has been proposed to be the main obstacle to viral eradication by highly active antiretroviral therapy (HAART). A longstanding paradox regards the mechanism through which the latently infected cell is first established. Since quiescent lymphocytes are refractory to viral infection due to blocks early in the viral life cycle, it has been proposed that the precursor to the latently infected cell is a cycling cell that allows HIV entry and subsequently returns to a quiescent state after the virus is established.

One study suggested that HIV can, in fact, establish infection in a quiescent cell and directly enter a state of latency.[4] In their presentation, the investigators used a procedure, known as spinoculation, in which virus-cell contact is promoted by centrifugal force, thereby increasing the efficiency of infection. Under those conditions, the viral genome could be detected in approximately 10% of the quiescent T cells. This observation is at odds with a number of studies suggesting that quiescent lymphocytes do not allow the virus to establish an integrated state. One possibility is that the spinoculation acted as a subtle stimulus which induced the lymphocytes to enter an early stage of cell cycle; this would be consistent with the results of other studies that have shown that lymphocytes in the G1 phase of the cell cycle and beyond are permissive of HIV infection and integration. Nevertheless, should it be confirmed that the infection was established in a truly quiescent cell, this has important implications for understanding the source of latent viral reservoirs in HIV-infected individuals. Another study attempted to distinguish the origin of virus in the context of viral rebounds following treatment interruption.[5] Although HAART is very effective at suppressing viral replication to below the level of detection (based on plasma viral RNA measurements), viral replication rapidly rebounds when therapy is interrupted irrespective of the period of suppression. The prevailing view is that the rebound virus originates from the latent reservoir. However, other lines of evidence, such as continued viral evolution and the presence of replication intermediates in well-suppressed patients, raises the possibility that viral replication may persist in the face of apparently suppressive therapy. The study authors characterized transcription patterns in order to distinguish ongoing replication in patients with high-level replication, patients on HAART, and patients who underwent structured treatment interruptions. Genotypically, extracellular virus was highly comparable with viruses obtained from lymph nodes, implying that viruses are produced and sequestered locally. Furthermore, the identity of the rebound virus was consistent with the notion that viral rebound is due to random activation of latently infected cells, rather than ongoing, low-level replication.

HIV:Cell Interactions in the Viral Replication Cycle Arguably, the major research theme of the conference related to the cellular forces that favor or oppose viral replication. Primate lentiviruses such as HIV-1 have a limited armamentarium of genes and encoded proteins with which to carry out various aspects of their replication cycle. Therefore, it is not surprising that these viruses exploit cellular proteins in order to carry out their respective replicative functions. Perhaps the greatest progress in terms of understanding how cellular factors promote viral replication relates to cofactors for late steps in viral replication, specifically virus release. The final step in the viral replication cycle is the physical detachment of assembled virions from the surface of the infected cell, which is characteristic of type C lentiviruses. The closest parallel to this process is the formation of multivesicular bodies (MVB) in which membranes bud into cytoplasmic vesicles. It is now apparent that cellular proteins which govern MVB formation also control the process of virus detachment. Several of these proteins have now been identified and were discussed at the conference.[6-8] The proteins identified to date belong to a family of Class E Vps proteins that have well-orchestrated roles in formation of the MVB. These cellular proteins are recruited to sites of virus assembly through their interaction with the structural Gag protein in the virus. It is speculated that they assist in promoting curvature of the plasma membrane in order to wrap around structural virion proteins during the viral budding and detachment processes. Although, as is typical with type C lentiviruses, viruses assemble at and detach from the plasma membrane, there are instances in which viruses assemble within cytoplasmic vacuoles of infected macrophages. Recent studies have indicated that these cytoplasmic vesicles are, in fact, MVBs. This unusual pattern of virus assembly can thus be reconciled with research demonstrating that the machinery of MVB formation is exploited for HIV budding. The open question is why virus budding is intracellular in macrophages, but extracellular in T cells. One possibility is that the ability to bud into MVBs, which ultimately exocytose their contents after cell/cell contact, facilitates dissemination of the virus between cells. Support of this theory was seen in 2 presentations that indicated that viruses assemble within MVBs and MVBs are subsequently localized to the cell surface at the point of contact between the infected cell and the neighboring T cell.[9,10]

In the case of dendritic cells, it was demonstrated that extracellular viral particles could be internalized into the MVB and then shunted to contact points between the dendritic cell and T cell.[9] This is likely to provide a highly efficient mechanism for the transfer of virus particles onto new substrate T cells. It is important to note that the virus-containing vesicles did not contain markers characteristic of early and late endosomes, and as such are unlikely to undergo acidification. This further suggests that these compartments may retain the virus in a stable and infectious form. In macrophages, evidence was presented to suggest that the MVB is the exclusive pathway for the assembly of virus and release into extracellular fluids.[10] During virus budding and detachment, the viral membrane exhibits the same composition as the cellular membrane from which it detached. Therefore, the viral membrane will also contain membrane proteins common to the cellular membrane from which it originated. When the composition of membrane proteins in viruses released from infected macrophages was analyzed, the membrane proteins were found to be compositionally very similar to the membranes of the MVB. Therefore, in cells such as macrophages, there is likely to be a dynamic and continuous process of virus budding into the MVB and movement of the MVB to the plasma membrane where MVB contents (such as virus particles) are subsequently released. While these studies are provocative, it is too early to gauge their roles in viral persistence and dissemination. However, if one is to speculate, it is possible that virus particles contained within MVBs of macrophages or dendritic cells are specifically channeled to sites of contact with neighboring T cells. This may be the favored route of viral dissemination, as opposed to the random release of virions into the extracellular space and chance encounters with new substrate T cells. Therefore, the immunologic synapse that occurs between an antigenpresenting cell and a T cell may present a highly favorable environment for viral dissemination. It is important to determine whether MVBs actually represent a stable reservoir for viral persistence. If, as was suggested at the conference, virus-containing MVBs do not undergo acidification, then this may represent a stable compartment for virus sequestration and as such may provide a sanctuary site for infectious virus particles. Although conflicting results were represented, 2 studies described another potential positiveacting factor for HIV replication that acts at an early stage in viral replication cycle, namely at the point of viral DNA integration.[11,12] When lentiviruses infect cells, they must synthesize their cDNA and translocate this cDNA to the vicinity of cellular DNA where it integrates to form a provirus. However, very little is known about the mechanisms governing the nuclear translocation of viral cDNA or the viral and cellular factors that control it. The 2 presentations focused on a cellular factor called lens epithelium-derived growth factor (LEDGF), which was found to form a specific interaction with the viral integrase protein, an enzyme that catalyzes the integration of viral cDNA with the host cell DNA. LEDGF was shown to govern the ability of the integrase protein to localize to the nucleus. The level of expression of LEDGF in the cell was reduced using RNA interference, and the susceptibility of those cells to infection was monitored. In one study, the ability of the virus to integrate within cellular DNA was blocked when LEDGF was absent, while in the other study, there was no effect. While additional work is needed to confirm the exact importance of LEDGF in viral replication, these findings nonetheless point to a potentially new cofactor that regulates a preintegration step in viral replication. These studies illustrate the fact that with the advent of RNA interference, it is now possible to validate the importance of cellular cofactors in virus biology. This will be an invaluable tool to further dissect virus-host cell interactions that could represent future targets for drug discovery.

Cellular Antiviral Factors and Effects While it is clear that a number of cellular proteins are positive factors for viral replication, it is now apparent that there are cellular proteins that oppose viral infection. Research into host defense mechanisms that counteract the replication of viruses such as HIV has focused on the role of the humoral and cellular arms of the immune response. However, the discovery approximately a year and a half ago, of a cellular protein called APOBEC 3G that counteracts viral infection, has highlighted the existence of far more potent and specific defense mechanisms that are mounted by the cell to protect itself from virus infection. APOBEC 3G is the cellular target of the viral protein Vif, which is essential for viral replication in primary cells such as lymphocytes and macrophages. Many presentations shed light on the mechanism by

which APOBEC 3G blocks viral replication and strategies used by the virus to counteract this block.[13-18] APOBEC 3G inhibits viral replications by inducing extensive deamination of cDNA as it is being synthesized by reverse transcription. This deamination of deoxycytidine to deoxyuridine likely promotes the degradation of the viral cDNA in the cell. It should be emphasized that this is a very potent mechanism of viral inactivation and in fact, the antiviral activity of APOBEC 3G does not appear to be restricted to viruses such as HIV. It also works with diverse viruses such as hepatitis virus. Obviously, in order to establish infection in humans, HIV has come up with a way to counteract APOBEC 3G. This is where the viral Vif protein comes in, and evidence presented at the conference indicated that the virus uses its Vif protein to drag APOBEC 3G to the proteosome in order to affect its degradation. This, of course, relies on the ability of Vif to interact directly or indirectly with APOBEC 3G. It now appears that the interaction of Vif with APOBEC 3G is species-specific. While some SIV Vif proteins interact with human APOBEC 3G, others do not. This illustrates the fact that HIV is a zoonosis, and that the SIV ancestor to HIV during its introduction into the human population had to evolve its Vif protein in order to interact with the human APOBEC 3G. Therefore, the goal for the field is to find ways to block the interaction of Vif with APOBEC 3G, using, for example, small-molecule inhibitors, thereby rendering the host nonpermissive to viral replication. It is also apparent that there are many new cellular resistance factors awaiting discovery. One such factor, termed Lv1 (the identity of the actual factor is as yet unknown), protects cells such as those from owl monkeys from HIV infection. This factor appears to act very early in the replication cycle by targeting the Gag protein and preventing efficient reverse transcription of viral cDNA. Studies are beginning to shed light on how these additional Gag-targeted restriction factors operate and how viruses such as HIV may counteract them.[19-21] Cyclophilin A is a cellular protein that was found to interact with HIV-1 Gag and promote viral infectivity. It now appears that cyclophilin A may regulate the susceptibility of the virus to restriction factors such as Lv1. While owl monkey cells are resistant to HIV infection because of Lv1 restriction, treating the cells with drugs such as cyclosporin A, which blocks the interaction of cyclophilin with the viral capsid, markedly augments HIV infection. In contrast, when interaction of cyclophilin A with capsid is blocked in human cells, HIV infectivity is reduced. Therefore, strategies to manipulate the virus' ability to interact with cyclophilin may restore the antiviral activities of these endogenous restriction factors. There is likely to be a great deal of research activity in this area in the next several years.

References 1. Oswald-Richter K, Grill SM, Shariat N, et al. HIV infection of naturally occurring and genetically reprogrammed human regulatory T cells. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 124LB. 2. Pandrea I, Apetrei C, Dufour J, et al. Lack of SIV target cells in African non-human primate: host or virus adaptation? Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 417. 3. Moir S, Malaspina A, Donoghue E, et al. Decreased survival of B cells of HIV-viremic patients mediated by altered expression of receptors of the TNF superfamily. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 126.

4. O'Doherty U, Baytop C, Yu J, Swiggard W. De Novo latent infection of quiescent CD4+ T cells in the absence of exogenous stimuli. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 123LB. 5. Fischer M, Joos B, Wong JK, et al. The presence of extracellular virion-associated HIVRNA in lymphoid tissue reflects local productive infection: a detailed analysis of lymphatic HIV transcription patterns in vivo. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 422.

6. Sundquist WI. Cellular factors and HIV budding. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 6. 7. Strack B, Calistri A, Popova E, Craig S, Gottlinger H. AIP1 and ESCRT-III are components of the HIV-1 budding machinery. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 64. 8. Ono A, Freed EO. Cell-type-specific targeting of HIV-1 gag: evidence of a role for PIP2. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 65. 9. McDonald D, Hope TJ. Enhancement of HIV infection by activated dendritic cells occurs via trafficking through a CD81 enriched compartment. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 44. 10. Marsh M, Pelchen-Matthews A, Kramer B, et al. HIV assembly in, and release from, primary macrophages. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 46. 11. Debyser Z, Emiliani S, Van Maele B, et al. Characterization of the role of LEDGF during HIV replication. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 68. 12. Marnett A, Nomura A, Shimba N, et al. Communication between the spatially separate active site and dimer interface of KSHV protease revealed by small molecule inhibition. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 337. 13. Neuberger M. DNA editing and host resistance factors. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 61. 14. Mehle A, Strack B, Ancuta P, Gabuzda D. HIV-1 vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome pathway. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 62. 15. Yu Q, Konig R, Pillai S, et al. Characterization of mutations generated by APOBEC3G on HIV-1 DNA. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 63. 16. Landau NR. HIV vif: deactivation of a deadly deaminase. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 103. 17. Kobayashi M, Takaori-Kondo A, Shindo K, et al. Species-specific target specificity of APOBEC3G. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 352. 18. Doehle B, Bogerd H, Wiegand H, Cullen B. A single amino acid change controls the ability of HIV-1 vif to discriminate between human and African green monkey APOBEC3G. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 353. 19. Towers GJ. Host factors controlling species-specific replication of lentiviruses. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 104. 20. Hatziioannou T, Cowan S, Bieniasz PD. Mapping the restriction determinants in HIV-1 capsid and defining the role of cyclophilin A in restriction. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 356. 21. Bobardt M, Saphire A, Gallay P. Natural resistance of HIV-1 primary isolates to ref1. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 357.

Sexual Transmission of HIV Myron S. Cohen, MD

Introduction Sexual transmission of HIV is dependent upon a multitude of interrelating viral, source, and host factors that affect the efficiency of transmission. The infectiousness of the source partner's virus is the clearest determinant of whether HIV is transmitted. Infectiousness of HIV appears to depend primarily on the concentration of HIV in relevant body fluids (eg, semen, cervicovaginal secretions).[1,2] Further, multiple lines of evidence support a positive correlation between plasma viral load and HIV transmission.[3] In this regard, it is important to note that several lines of evidence suggest that antiretroviral therapy can reduce sexual transmission of HIV by reducing excretion of HIV in semen and female genital secretions -- an effect which is to some extent related to the ability of individual antiviral agents to achieve suppressive concentrations in genital secretions.[4-10] Susceptibility of the exposed partner is the other major determinant of HIV transmission. Susceptibility to HIV infection depends on the availability of appropriate receptive cells; local replication of the virus; and hereditary, innate, and acquired resistance to infection. Cells concomitantly expressing CCR5, CD4 receptors, and DC-SIGN (a dendritic cell [DC]-specific HIV-1-binding protein that enhances transinfection of T cells) are most likely to be infected.[11] However, not all people are equally susceptible to HIV infection. For example, approximately 1 of 100 white persons has a deletion in a portion of the CCR5 receptor, which has been found to exert a considerable genetic resistance to HIV infection.[12] Susceptibility to HIV infection also depends on a variety of microenvironmental factors. For example, bacterial vaginosis has been associated with increased risk of HIV acquisition.[13] The biology of transmission of HIV has been extensively and recently reviewed on Medscape HIV/AIDS [http://www.medscape.com/viewprogram/2671]. The purpose of this review is to provide an update on new information presented in the symposia, oral sessions, and posters of the 11th Conference on Retroviruses and Opportunistic Infections.

Shedding of HIV in the Genital Tract Sexual transmission of HIV is closely associated with the concentration of the virus in the genital tract of the infecting partner. S. Pillai[14] of University of California, San Diego, presented analyses of envelope sequences from blood and semen isolates derived from 12 patients. Relative to blood HIV variants, less viral diversity was observed in semen variants, with a slower rate of molecular evolution, and reduced potential for CXCR4 usage. The degree of HIV glycosylation in semen and blood HIV is similar, but glycosylation sites were different. Another study was designed to examine populations of virus in semen that persist during therapy with a new nonnucleoside reverse transcriptase inhibitor (DM266I) and indinavir.[15] HIV mRNA was harvested from mononuclear cells in blood and semen. Resistance mutations were not observed, but reduced divergence after therapy in the envelope sequences suggested considerable restriction during replication. Different mutations were observed in semen and blood cells. In a rather remarkable piece of work by R Coombs and colleagues[16] of the University of Washington, Seattle, the investigators recruited 15 men who provided samples from -- in order -- a urethral swab, first-void urine, prostate massage secretions, postmassage urine, and 6 prostate biopsies. Materials were studied for HIV-1 RNA concentration and (in the case of biopsies) HIV-1 DNA. Five men were receiving antiretroviral therapy. The results suggest that HIV in the urethral secretions and (likely) in semen are derived from the prostrate as well as the urethral glands distal to the prostate.

S.T. Sadiq of the Royal Free and University College Medical School, London, United Kingdom, and colleagues[17] measured HIV concentrations in the semen of 20 men before and after antibiotic therapy for urethritis. Men with gonococcal and chlamydial urethritis, but not nonspecific urethritis, had increased concentrations of HIV-1 RNA in semen relative to 35 control subjects; decreased excretion of HIV was observed after appropriate antibiotic therapy in men with gonorrhea and chlamydia. Blood plasma HIV-1 RNA levels were not affected. The absolute concentrations of HIV-1 RNA in semen were lower than those previously reported from Africa. Far less is known about shedding of HIV in the female genital tract, in part because of the difficulty in collecting specimens in a reliable and reproducible way, without disrupting the mucosa. M. Nowicki and coworkers[18] from the University of Southern California, Los Angeles, measured HIV concentrations in oral and cervicovaginal lavage (CVL) secretions of 102 women. HIV-1 RNA was detectable in 30% of oral secretions and 56% of CVL samples. Perhaps not surprising, HIV-1 RNA concentrations were higher in the pellets derived from CVL than in the whole (diluted) secretions. S. Cu-Uvin[19] of Brown University in Providence, Rhode Island, and coinvestigators looked at HIV-1 RNA levels in blood and the female genital tract over 36 months. Ninety-seven women provided 530 paired plasma and CVL samples. At each visit, CVL HIV levels were lower than blood plasma, but levels in the 2 compartments were associated; for example, among women on antiretroviral therapy, HIV-1 RNA was only detected in CVL samples during blood plasma breakthrough. B. Sha of Rush University Medical Center in Chicago, Illinois, and colleagues[20] examined the relationship between bacterial vaginosis (BV) and genital tract viral shedding. Of 406 samples from 362 women, 203 had evidence of BV using a variety of techniques, including PCR. Median HIV-1 RNA levels in blood and genital tract samples from women with BV were 4.58 and 2.88 log10 copies/mL, respectively, compared with 4.57 and 2.52 log10 copies/mL in women without BV. CD4+ cell counts were comparable in these women. The investigators concluded that BV flora increased HIV shedding, after correction for the plasma viral load. In addition, genital tract infection with Candida (n = 89) and Trichomonas (n = 41) was also associated with increased HIV shedding. The results of a study designed to compare resistance mutations in blood and plasma HIV isolates were presented by J. Mullen[21] of Guy 's, King 's, St. Thomas' School of Medicine in London. Twenty-six pregnant and 15 nonpregnant women were included. As seen in other studies, 94% of women had more HIV in blood plasma than in cervicovaginal secretions. Paired samples sufficient for genetic analysis were recovered from 13 women. HIV-1 subtypes were the same in both compartments. Phylogenetic analysis indicated genetic diversity between compartments, however. Antiretroviral drug resistance reflecting past history of therapy was observed in 3 women, and genotypes from blood and genital tract-derived virus were discordant in 1 of 3 women. The results of these numerous studies compliment a considerable body of literature regarding HIV in semen, and more limited information about HIV in the female genital tract. HIV in semen represents a heterogeneous population of HIV derived from replication by seminal cells and of HIV from the blood, prostate, and urethral glands. The source of HIV in the female genital tract is less well understood. Differences in concentration and genetic composition of HIV in blood and genital secretions can be expected. Local inflammation produced by some (but not all) sexually transmitted diseases can be expected to (reversibly) increase the concentration of HIV in genital secretions. The selective pressures that lead to viral diversity in the genital tract require continued study because it is the genital tract -- and not blood -- variants that will ultimately be transmitted to the susceptible host.

Dendritic Cells, HIV Transmission, and Selection of CCR5 Variants HIV in the genital tract must overcome all innate and acquired mucosal resistances and find a susceptible host cell to infect. It remains unclear as to whether HIV is transmitted by cell-free

virus (as is used routinely in the macaque model) or by infected genital tract cells. The first cell infected could be a macrophage, a dendritic cell (DC), a lymphocyte, or even a membranous epithelial cell (M cell), although the most compelling evidence focuses on the phagocytes. Increasingly, DCs are believed to play a critical role in the transfer of HIV to susceptible lymphocytes, leading to HIV expansion, dissemination, and established infection. The tight junction between cells involved in transfer may represent an "immunologic/infectious synapse," a popular neologism. C-type lectin receptors (eg, DC-SIGN) facilitate HIV binding to and endosomal uptake by DCs. Within the endosome, acid proteolytic degradation destroys some viruses, whereas others, including CCR5-tropic viruses, remain viable, replicate, and are ultimately transferred to susceptible T cells. A.L. Cunningham and colleagues[22] of the Centre for Virus Research in Westmead, Australia, presented data which suggest that HIV is transferred in 2 distinct phases: In phase 1 (ie, in the first 24 hours) HIV escapes/avoids the endolysosomal degradation and is transferred to the DC-T-lymphocyte synapse, whereas in phase 2, HIV produced by low-level replication within the DC is transmitted. The investigators argue that de novo CCR5-tropic viruses may be selected in this second phase. D. McDonald and T.J. Hope[23] of the University of Illinois, Chicago, were less sanguine about HIV replication actually occurring within DCs, and focused on the transfer itself. They found that lipopolysaccharide-activated DCs were best at capturing HIV and recruiting susceptible T cells. Based on their experiments, they proposed that HIV is sequestered in multivesicular bodies within DCs, sites normally reserved for the sequestration of intact antigens. They observed concentrations of HIV colocalized with surface markers (CD63, CD9, DC-SIGN, HLA-DR, CD86, and especially CD81), perhaps marking the site of transfer of HIV from DCs to lymphocytes. The selection of CCR5-tropic HIV variants in primary infection has not been explained. In a study presented by N. Gonzalez[24] of the Institut de Salud Carlos III in Madrid, Spain, replication of CCR5 viruses in lymphocytes was increased 20-fold when DCs were included in the system. This enhancement depended on the presence of DC-SIGN on DCs, as this receptor facilitates transfer of HIV to lymphocytes. Conversely, addition of DCs decreased replication of CRCX4tropic HIV variants. Addition of antibodies targeting the chemokine SDF-1 (stromal cell-derived factor-1), a natural ligand of the CRCX4 receptor, improved replication of CRCX4-tropic viruses in this model, suggesting that SDF-1 expression by DCs represents an inhibitory antiviral mechanism that blocks replication of CRCX4-tropic HIV variants, but not CCR5-tropic viruses. In a study presented by M. Cavrois[25] of the Gladstone Institute of Virology and Immunology, San Francisco, California, peripheral blood monocytes were put into culture, differentiated into DCs (with Iinterleukin-4 and granulocyte macrophage colony-stimulating factor), and forced to maturation with tumor necrosis factor-alpha and polyinosinic-polycytidylic acid (poly IC). Immature DCs were found to preferentially bind to CCR5. Another group from Gladstone compared the replication of CCR5- and CRCX4-tropic viruses in CD4+ cells.[26] After 7 days in culture, CD4+ cells growing CRCX4 variants underwent cell death, whereas CCR5-tropic virus replicated persistently at lower levels. In experiments conducted by S. Aquaro of the University of Rome, Tor Vergato, Italy, and colleagues,[27] peripheral blood monocytes were incubated with either CRCX4 or CCR5-tropic HIV. Infection of theses cells by CRCX4 viruses was abortive, leading to increased macrophage apoptosis, whereas replication of CCR5 viruses was sustained. Microarray analysis of macrophage gene expression demonstrated that CRCX4 variants induced proapoptotic gene expression, whereas CCR5 viruses did not. These findings and other current evidence add to a compelling picture of initial HIV infection, in which macrophages and DCs become infected through a combination of receptor (CCR5 and CD4) and C-type lectin usage (DC-SIGN). Some HIV is endocytosed and destroyed in endolysosomes while other viruses are permitted to replicate. CRCX4-tropic HIV appears to be too toxic to monocytes and lymphocytes to survive, thus allowing for selection of CCR5-tropic variants. Thus, CCR5-tropic HIV can cause persistent infection of CD4+ lymphocytes after they come into close contact with infected DCs. Using this information to design novel prevention strategies is the next great challenge.

Primary Infection: Diversity and Resistance The study of viruses recovered from people with acute infection (ie, antibody-negative, RNApositive) and early infection (ie, symptomatic, antibody-positive) provides crucial information about selective pressure and fitness. Selection of CCR5-tropic viruses has already been reviewed, but other parameters have been examined. For example, drug resistance in HIV recovered from untreated people indicates that drug-resistant virus has been transmitted. Increasing resistance in drug-naive patients has attracted great attention because it emphasizes the fragility of the antiretroviral drug arsenal currently available for therapy. In addition, examinations of viral genetic diversity can provide insights about the degree or specifics of viral fitness required for transmission. J-P Routy and colleagues[28] at McGill University in Montreal, Quebec, Canada, compared genetic analyses of HIV harvested from 585 chronically infected people with isolates from 182 recently infected patients. HIV recovered from patients with established infection had more mutations per patient, and more patients had resistant variants. The prevalence of mutations V1181, M184V, and K103N increased from 3% to 26%, 56% to 68%, and 14% to 33%, respectively. Protease inhibitor mutations were found to remain stable over time. However, a decrease has been observed in primary HIV drug resistance: from 13.4% (1996-2000) to 3.6% (2001-2003). The prevalence of M184V and D30N is 5 times greater in patients with established infection compared with those with recent infection. The study authors ascribed decreased de novo resistance to reduced concentrations of HIV in blood of patients who harbor mutants (0.70.8 log10 copies/mL) compared with wild-type. Similarly, in a separate study, newly infected patients (n = 100) were identified in Amsterdam, The Netherlands, between 1994 and 2002.[29] During the first 5 years of the study, de novo resistance was observed in 21% of subjects compared with 6% in the past 5 years. Ten subjects with recent HIV infection (mean, 50 days) transmitted by a total of 8 sexual partners (donor partners) were studied.[30] All subjects had clade B infection. Three donor subjects with drug resistance detected in blood plasma transmitted HIV to 4 partners. Genetic divergence between donor and partners was less than 2%. Fewer glycosylation sites were observed in the recipients than in the source partners, while HIV env sequences were either the same length or shorter in the recipient. Neutralizing antibody responses were weak, and virus from donors and recipients was resistant to polyclonal and monoclonal antibodies. Diversity in the V1/V2 and V3 variable regions of HIV env was studied in 109 subjects with early infection by K. Ritola of University of North Carolina, Chapel Hill, and colleagues.[31] Among women who acquired HIV, 4 of 7 had multiple V1/V2 variants compared with 6 of 7 men who had a single variant. However, 53% of men who acquired HIV through anal intercourse had diverse V1/V2 variants. Eighty-three percent of subjects had a single V3 variant. Ninety-seven percent of subjects were infected with CCR5 variants. The changes observed in the prevalence of drug resistance among newly infected patients must reflect the effects of antiretroviral therapy within the population. First, therapy that completely suppresses HIV likely eliminates transmission, so mutations related to the most potent drugs will only rarely be observed. Second, antiretroviral therapy that incompletely suppresses viral load in the genital tract might reduce the efficiency of transmission, but allow detection of mutations associated with such therapy. Third, some HIV mutants might not be fit for transfer. This latter explanation might help to account for differences in HIV env in patients with primary infection, an area that will receive far more attention in the future. Understanding viral characteristics required to establish infection is required for the development of effective prevention strategies.

Microbicides for the Prevention of Sexual Transmission of HIV Microbicides have become a "cause célèbre" in HIV prevention. This topic was emphasized in a passionate lecture in the Opening Ceremony by Stephen H. Lewis,[32] the United Nations Special Envoy for HIV/AIDS in Africa, in the opening plenary talk Dr. Robin Shattock[33] of St. George's Hospital Medical School in London, and in both a poster session (#121) and a microbicide

symposium (Session 30). The microbicide symposium included 3 lectures, which are reviewed here. In order to identify the first-line coreceptors used by HIV to attach to and infect cells of the cervix, H. Qu, from St. George's Hospital Medical School, and colleagues[34] used a variety of methods to block the entry and "spread" of HIV-1 strains into an ex vivo cervical explant model. Release of p24 antigen into the supernatant, or detection of HIV DNA in the cervical tissue, was used to determine the occurrence of primary infection. Infection, they found, could be blocked through inhibition of CD4 alone, or CCR5 and CXCR4 together, suggesting that primary infection occurs in macrophages and/or lymphocytes. Spread of HIV in tissue, represented by detection of HIV in DCs and lymphocytes in the culture supernatant, could be blocked by inhibition of CD4 and mannose C-type lectin receptors (eg, DC-SIGN). Neutralizing antibodies (mAB b12) and sCD4 fusion protein CD4-IgG2 blocked both localized and disseminated infection. These results were used to relate the explant model to infection in vivo, with the assumption that this model can be used to test microbicides. Using this model, their results suggest initial infection of macrophages with subsequent infection of DCs, which in turn infect lymphocytes. Ron Veazey[35] from Tulane University in New Orleans, Louisiana, reported on the use of the CCR5 receptor antagonist PSC-RANTES in a macaque challenge model. PSC-RANTES is a potent amino-terminus-modified analog of the chemokine RANTES, which inhibits HIV replication and downregulates CCR5 receptor expression. Macaques were given medroxyprogesterone (Depo-Provera) to increase susceptibility to HIV infection through changes in the endocervix. Animals were then treated with 1 of 3 doses of PSC-RANTES and challenged intravaginally with 300 TCID50s (approximately 1 million copies) of SHIV162P3 15 minutes after treatment. All 5 macaques who received the highest dose (1 mM) of PSCRANTES remained uninfected, compared with 4 of 5 animals treated with 330-mcM PSCRANTES and 3 of 5 treated with 100-mcM PSC-RANTES. In a control group, 4 of 5 untreated macaques became infected after SHIV challenge. The investigators concluded that PSCRANTES in high concentration is sufficient to block vaginal SHIV infection. In addition, clinical and tissue examination of the cervix failed to reveal evidence of inflammation after RANTES exposure. Ron Otten[36] of the US Centers for Disease Control and Prevention in Atlanta, Georgia, reported on the use of vaginal cellulose acetate phthalate (CAP) to prevent SHIV in a pig-tailed macaque model. Animals (n = 12) were repeatedly exposed over 16 weeks to 3 concentrations (1.2 x 105, 1.2 x 106, or 5.8 x 106 copies/mL) of SHIV162P3. Macaques exposed to these concentrations became infected after 3, 4-8, and 12 exposures, respectively. The higher concentration of SHIV was then used to test the efficacy of a 2-mL application of a 13% preparation of CAP provided 15 minutes prior to SHIV exposure. Three of 4 macaques treated with CAP remained virus-free after 8 exposures, but 1 animal became infected after 3 exposures. The peak plasma viral RNA level in this macaque was approximately 2 logs lower than in the control animals. CAP caused no clinical inflammation at the concentration used. The ideal microbicide will reliably prevent sexually transmitted diseases and HIV concomitantly, without inhibiting conception. It will be cheap to manufacture, prove simple to store and apply, and prove palatable to women and their sexual partners. A wide range of products, including detergents, charged compounds, acidic compounds, antibodies, antivirals, and protobiotics, are in development. However, none of these compounds fulfills all of the criteria desired. Further, Dr. Shattock predicted that using an optimistic timeline, no product would be clinically available before 2010. In general, the funding stream for microbicide development has improved and enthusiasm is high, but development of a successful product will require overcoming a variety of major biological restrictions and conducting very large, expensive clinical trials. The models described above, however, should facilitate the development of effective microbicides. Challenge of animals with HIV (or SHIV) in concentrations found in human semen should help to provide a good idea of the capacity of candidate microbicide compounds.

References

1. Royce RA, Sena A, Cates W Jr, Cohen MS. Sexual transmission of HIV. N Engl J Med. 1997;336:1072-1078. 2. Quinn TC, Wawer MJ, Sewankambo N, et al. Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med. 2000;342:921-929. 3. Chakraborty H, Sen PK, Helms RW, et al. Viral burden in genital secretions determines male-to-female sexual transmission of HIV-1: a probabilistic empiric model. AIDS. 2001; 15:621-627. 4. Vernazza PL, Troiani L, Flepp MJ, et al. Potent antiretroviral treatment of HIV-infection results in suppression of the seminal shedding of HIV. The Swiss HIV Cohort Study. AIDS. 2000;14:117-121. 5. Coombs RW, Reichelderfer PS, Landay AL. Recent observations on HIV type-1 infection in the genital tract of men and women. AIDS. 2003;17:455-480. 6. Hart CE, Lennox JL, Pratt-Palmore M, et al. Correlation of human immunodeficiency virus type 1 RNA levels in blood and the female genital tract. J Infect Dis. 1999;179:871882. 7. Kashuba AD, Dyer JR, Kramer LM, Raasch RH, Eron JJ, Cohen MS. Antiretroviral-drug concentrations in semen: implications for sexual transmission of human immunodeficiency virus type 1. Antimicrob Agents Chemother. 1999;43:1817-1826. 8. Pereira AS, Kashuba AD, Fiscus SA, et al., Nucleoside analogues achieve high concentrations in seminal plasma: relationship between drug concentration and virus burden. J Infect Dis. 1999;180:2039-2043. 9. Reddy YS, Gotzkowsky SK, Eron JJ, et al. Pharmacokinetic and pharmacodynamic investigation of efavirenz in the semen and blood of human immunodeficiency virus type 1-infected men. J Infect Dis. 2002;186:1339-1343. 10. Cohen MS. Preventing sexual transmission of HIV--new ideas from sub-Saharan Africa. N Engl J Med. 2000;342:970-972. 11. Greene WC, Peterlin BM. Charting HIV's remarkable voyage through the cell: Basic science as a passport to future therapy. Nat Med. 2002;8:673-680. 12. Samson M, Libert F, Doranz BJ, et al. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature. 1996;382:722-755. 13. Taha TE, Hoover DR, Dallabetta GA, et al. Bacterial vaginosis and disturbances of vaginal flora: association with increased acquisition of HIV. AIDS. 1998;12:1699-1706.

14. Pillai S, Good B, Wong J, Strain M, Richman D, Smith D. Semen-specific genetic

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characteristics of HIV-1 env. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 302. Craigo K, Patterson B, Paranjpe S, et al. Persistent viral infection and sequence evolution in semen and blood compartments in HIV-infected patients following long-term potent antiretroviral therapy. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 647. Coombs R, Deutsch L, Ross S, et al. The distal lower genitourinary tract as a source of seminal HIV. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 420. Sadiq ST, Taylor S, Copas AJ, et al. The effects of gonococcal, chlamydial and nonspecific urethritis on seminal plasma HIV-1 RNA loads in the United Kingdom. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 458. Nowicki M, Kovacs A, DeGiacomo M, Chen Z-C, Navazesh M. HIV-1 distribution in oral and vaginal mucosa specimens. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 953. Cu-Uvin S, Snyder B, Hogan J, et al. Paired plasma and cervicovaginal HIV-1 RNA expression over 36 months. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 954.

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quantitative PCR for Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus is a sensitive indicator of bacterial vaginosis and correlates with genital tract HIV viral load. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 955. Mullen J, O'Shea S, Cormack I, et al. Antiretroviral drug resistance and genetic diversity of HIV-1 in the blood and female genital tract. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 956. Cunningham AL, Turville SG, Wilkonson J, et al. HIV capture and transmission by dendritic cells. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 43. McDonald D, Hope TJ. Enhancement of HIV infection by activated dendritic cells occurs via trafficking through a CD81-enriched compartment. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 44. Gonzalez N, Bermejo M, Pablos JL, Baleux F, Arenzana F, Alcami J. SDF-1/CXCL12 production by dendritic cells decreases infection of lymphocytes by X4 strains in the immune synapsis. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 255. Cavrois M, Neidleman JA, Callebaut C, Kreisberg JF, Fenard D, Greene WC. Fusion differences between R5 and X4 HIV-1 in dendritic cells: implications for selective transmission of R5 strains. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 316.

26. Schweighardt B, Meiklejohn DA, Grace EJ III, Nixon DF. R5 HIV-1 strains replicate more efficiently in primary CD4+ T cell cultures than X4 HIV-1 strains. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 317. 27. Aquaro S, Ranazzi A, Bellocchi M, et al. HIV-1-mediated Apoptosis Occurs in Macrophages Infected by X4, but Not by R5 Viruses. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 414. 28. Routy J-P, Machouf N, Edwardes MD, et al. Factors associated with a decrease in the prevalence of drug resistance in newly HIV-1-infected individuals in Montreal. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 678. 29. Bezemer D, Jurriaans S, Prins M, et al. Limited transmission of drug resistant HIV-1 and non-B subtypes in Amsterdam. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 679.

30. Little SJ, Liu Y, Wrin T, et al. env sequences and neutralization of HIV from transmission partners of primary HIV infection. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 384.

31. Ritola K, Pilcher C, Little S, et al. HIV-1 V1/V2 and V3 env diversity during primary infection suggests a role for multiply infected cells in transmission. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 386. 32. Lewis SH, Sundquist W, Ho D. Opening Session. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. 33. Shatttock R. How close are we to an effective microbicide? Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Plenary Session 6. 34. Hu Q, Watts P, Frank I, et al. Blockade of attachment and fusion receptors inhibits HIV-1 infection of human cervical tissue. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 157.

35. Veazey R, Offord R, Hartley O, et al. Intravaginal PSC-RANTES protects against vaginal transmission of SHIV162P to macaques; implications for HIV microbicide strategies and pathogenesis. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 158. 36. Otten R, Adams DR, Kim CN. Cellulose acetate phthalate protects macaques from multiple, low-dose vaginal exposures with an SHIV virus: New strategy to study HIV pre-clinical interventions in non-human primates. Program and abstracts of the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, California. Abstract 159.

Authors and Disclosures Author Myron S. Cohen, MD Professor, Division Chief, Director, Center for Infectious Disease, University of North Carolina at Chapel Hill; Chief, Division of Infectious Disease; Director, Center for Infectious Disease, UNC HealthCare Systems, Chapel Hill, North Carolina Disclosure: Myron S. Cohen, MD, has no significant financial interests to disclose. In this activity he has reported that he discusses the use of investigational microbicidal products. Michael Lederman, MD Scott R. Inkley Professor of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio; Director, Center for AIDS Research, Principal Investigator, AIDS Clinical Trials Unit, Case Western Reserve University, University Hospital of Cleveland, Cleveland, Ohio Disclosure: Michael Lederman, MD, has disclosed that he has received research grants from AnorMED, Berlex, Bristol-Myers Squibb, Chiron Corporation, Coley, Merck, Triangle Pharmaceuticals, and Roche. In this activity, he reported that he does not discuss any investigational or unlabeled uses of any commercial products. Mario Stevenson, PhD Professor, University of Massachusetts Medical School, Worchester, Massachusetts; Director, Center for AIDS Research, University of Massachusetts Medical School, Worchester, Massachusetts Disclosure: Mario Stevenson, PhD, has no significant financial interests to disclose. In this activity, he reported that he does not discuss any investigational or unlabeled uses of any commercial products.

Editor Craig Sterritt

Site Editor/Program Director, Medscape HIV/AIDS, Medscape Infectious Diseases Disclosure: Craig Sterritt has no significant financial interests or relationships to disclose.

Registration for CME credit, the post test and the evaluation must be completed online. To access the activity Post Test and Evaluation link, please go to: http://www.medscape.com/viewprogram/2985_index