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A band of optimists considers the road towards a distant goal: eradication; Report from HIV Eradication Conference, 3rd Intl Workshop on HIV Persistence during Therapy, West Indies, St Maartin
 
 
  David Margolis MD, University of North Carolina at Chapel Hill
 
The Third International Workshop on HIV Persistence during Therapy convened from December 4th to 7th 2007 in the West Indies on Dutch side of the isle of St. Maarten. Organized by the tireless Alain Lafeuillade, the meeting, though small, featured a murderer's row of the HIV researchers involved in describing HIV persistence and latency since the earliest days of HIV virology, as well as young investigators who have fearlessly committed themselves to this difficult field. The meeting was marked by few breakthrough scientific advances, but instead by an exciting combination of prominent industry support for research and development in this area --- something cynics suspicious of the motivations of drug companies might not expect --- and a declaration from several prominent HIV research and advocacy foundations that they would commit new support to research towards therapies that might eradicate HIV infection. Given the huge obstacles and disappointments recently in the field of HIV prevention research (vaccines and microbicides), it would seem reasonable to invest a bit more in Plan C. Although these foundations cannot muster the financial power of the NIH or the Gates foundation, any support generates excitement in the current dreadful funding environment.
 
Although the answers as to where and how and why HIV infection can persist despite successful antiretroviral therapy (ART) were not uniformly agreed upon, as has been the case for more than a decade, there was general agreement on what the most important questions were, and hope that new therapeutics that are entering the clinic will allow studies to probe these important issues.
 
ANIMAL MODELS OF VIRAL PERSISTENCE (Session 1)
 
Due to the rarity of latent, persistent HIV infection in patients successfully treated with ART, model systems to study latency are a continuing need. Equally important is the contribution of patients willing to participate in research studies of latency, unlikely to offer them any clinical benefit in the foreseeable future, but critical if therapies to clear HIV infection are ever to be developed.
 
Short of human trials and studies of patients' cells, non-human primate models of HIV infection, ART, and latency will speed the study of the pathogenesis of persistence and treatments to overcome it. Ron Veazey (Tulane) reported on recent studies of tissues in chronically infected macaques that are controlling viral replication, compared to those that are not. As intestinal tract is the major site for viral replication in SIV infection, his studies aimed to assess whether the intestine is also a major reservoir in animals that control infection, at least as evidenced by low or undetectable plasma viremia.
 
To compare tissue reservoirs in progressors and nonprogressors, his work highlighted the use of rhesus macaques from China. Unlike monkeys of India origin, approximately 1/3rd of Chinese origin macaques inoculated with pathogenic strains of SIVmac239 become long-term nonprogressors (LTNP). Such animals offer the potential for studying viral reservoirs in tissues.
 
Veazey also compared the cell-associated levels of virus in the blood, lymph nodes, jejunum and colon of progressors and nonprogressors throughout infection. Specific regions of the intestine were major sites for viral persistence and replication, particularly in animals that have low or undetectable levels of plasma viremia. The level of viral replication in tissues was directly correlated with levels of cellular activation in these tissues. Veazey concluded that the intestine is a major reservoir for viral persistence and that anti-retroviral therapy and vaccine research must specifically targeting mucosal tissue sites
 
Jeff Lifson of the AIDS Vaccine Program at the National Cancer Institute, reported significant success in using a Merck Integrase Inhibitor to recreate truly potent and effective ART in the SIV-Infected Macaque model. Although SIV infection of macaques provides an excellent animal model of human HIV-1 infection, many potent anti-HIV-1 drugs have decreased or no activity against SIV. Maximal suppression of viral replication, as required for investigations of latently infected cell reservoirs, has not been previously achieved in this animal model.
 
Lifson's team used a four-drug regimen including Tenofovir and Emtricitabine (the components of Truvada) and two inhibitors of retroviral integrase enzymes ("812" and "564," integrase inhibitors developed and studied by Merck but not developed sufficiently for use in humans). 812 and 564 were shown previously to be active against the SIV integrase enzyme. Treatment was started 8-10 weeks after intravenous infection with the highly pathogenic SIVmac239 strain. A new and very sensitive plasma virion associated RNA viral load assay specifically designed for SIV was used.
 
Treatment with the integrase inhibitor containing 4-drug regimen resulted in suppression of viral replication to <100 copies/ml over 45 weeks of therapy. However, the assay is sensitive to 50 copies/ml, and detected viremia in most animals above 50 copies. It is surprising that the 4 drug regimen was not able to achieve <50 copy suppression, but one speculation was that the monkeys went untreated for too long, and that earlier treatment might result in full suppression. SIV DNA was also measured and declined more than 1 log during treatment, suggesting a potent treatment effect. It is hoped that if full suppression can be achieved in this model, SIV infected rhesus macaques could be used to test strategies to decrease the size of latently infected cell reservoirs.
 
While not likely to win friends among the cat-loving crowd, Savarino of the Istituto Superiore di Sanit, Rome presented renewed work in the Feline Immunodeficiency Virus (FIV) model. Again, data was shown suggesting the combination therapy with nucleoside reverse transcriptase inhibitors (NRTIs) and integrase strand transfer inhibitors (INSTIs) may allow for fully suppressive ART in the feline model and studies of latency and eradication approaches. If viable, this small-animal model would be much less expensive than the monkey model. Although not presented at this meeting, several research groups are also pursuing this approach in immunodeficient mouse models in which the mice are reconstituted with human immune cells and infected with HIV-1.
 
CELLULAR BIOLOGY & RESERVOIRS (Session 2)
 
The next session focused on molecular mechanisms that allow the HIV virus to establish and maintain latency or quiescence. The major focus of this writer's laboratory research, it is thought that an understanding of these mechanisms will allow us to design rational therapies to disrupt latent infection. The session began with an elegant discussion by Warner Greene, director of the Gladstone Institute of Virology and Immunology at UC San Francisco, of the kinetic relationship between the induction of NF-_B and the activation of latent HIV-1 gene expression. NF-_B is a key factor that binds at the promoter of HIV, also called the long terminal repeat of LTR. The LTR is the virus's molecular on-off switch, and NF-_B docking at the LTR begins the process that allows virus expression. Sensitive molecular assays that measured the occupancy of the NF-_B protein RelA at the HIV promoter revealed an oscillating pattern of RelA recruitment to the HIV LTR after sustained activating stimulation. Transient stimulation led to a round of RelA binding but failed to induce robust expression of latent HIV-1. Conversely, sustained induction of NF-_B was required for strong HIV expression. These observations suggested that latency might result in cells that lacked sufficiently durable stimulation, and that strategies to break latency must be sufficient to induce sustained viral expression. The challenge will be to find how to give enough stimulation without giving so much that control of the virus by ART is lost, or that over-stimulation leads to immune damage.
 
Next, this writer discussed the research of his group and collaborators at UNC, Merck, Abbott, and the NCI Drug Resistance Program. We summarized our prior work showing that histone deacetylase (HDAC) contributes to the maintenance of viral quiescence in T cells, and that the HDAC inhibitor valproic acid (VPA) perturbs latent infection in resting CD4+ cells cultured from aviremic HIV-infected donors, allowing recovery of replication-competent HIV from these cells.
 
In a pilot clinical experiment in 2005, we found significant depletion of resting CD4+ T cell infection (RCI) in 3 of 4 volunteers given intensified ART and VPA. Over the last 2 years, we found that when VPA was added to standard (not intensified) ART, a decrease in RCI was observed in only 4 of the 9 patients. Low-level viremia (> 5 copies/ml) was not observed in the patients with RCI depletion, but was observed in all but one patient with stable RCI after VPA therapy. Our current studies have returned to examine the effect of VPA when combined with raltegravir-intensified ART.
 
We then presented pre-clinical data suggesting that Vorinostat, a more potent HDAC inhibitor, can induce latent proviral expression from patients' cells, but the clinical testing of this drug may be challenging given the possible toxicities of Vorinostat. We hope that pulsatile exposure, rather than continuous dosing, with Vorinostat will make it tolerable in patients that are doing well and suppressed on ART, and have no certainty of benefit from such an experiment.
 
Mario Stevenson (U Mass Med Ctr, Worcester) discussed infection of macrophages, another major site of HIV persistence. His group's work suggested that macrophages express uniques cellular factors that restrict the expression of HIV and that the Vpx proteins of HIV-2/SIVSM promote virus infection by antagonizing an antiviral restriction in macrophages.
 
Una O'Doherty (U Penn) presented a new assay to quantitated integrated HIV genomes. Although most of these genomes represent defective viruses rather than latent, persistent infection, it is assumed that a sensitive, accurate integrant assay would provide a simpler and clinically tractable tool to measure progress towards eradication of infection. O'Doherty improved her previously published assay methodology by incorporating the technique of repetitive sampling. Using real-time PCR to measure and re-measure integration up to 40 times in the same sample, and including numerous controls, O'Doherty suggested that she could detect one integrated genome in 10,000 cells. Should this assay be fully validated with an adequate set of clinical samples, it may provide an important tool for clinical studies that seek to purturb persistent infection.
 
The Kashanchi laboratory at GWU has recently suggested that RNAs made by HIV can be processed into negative regulators of gene expression called micro-RNAs. These small RNAs can drive remodeling of chromatin, and may create a negative feedback loop that inhibits HIV. Zach Klase presented findings suggesting that HIV miRNA derived for the TAR stem-loop structure, the first part of the HIV genome to express RNA, is detectable in cells. Expression of this miRNA can limit LTR driven gene expression through recruitment of HDAC-1 and silencing of the viral promoter. Additionally, this viral miRNA renders the cells resistant to cell death induced by cellular stress. If this negative regulatory mechanism gains the upper hand before enough of the viral activator Tat is produced, it could drive the virus into latency rather than productive infection.
 
Kara Lassen (Case) presented work that she is extending in the laboratory of Jonathan Karn, studying a selective block to the expression of HIV RNAs in resting CD4+ T cells. Although there are several obstacles to HIV exression in resting cells that have been described, Lassen's work suggests that post-transcriptional mechanisms also make a major contribution to latency. The polypyrimidine tract binding protein (PTB) has been identified as a positive factor capable of reactivating latent HIV-1 without inducing cellular stimulation.
 
PTB expression in resting CD4+ T cells is extremely low and cellular stimulation results in a several fold increase in PTB levels. Overexpression of PTB in latently infected resting CD4+ T cells caused a dramatic reversal of HIV-1 latency by allowing cytoplasmic accumulation of HIV-1 RNAs and subsequent production of replication-competent virus. Insufficient levels of PTB can reduce the accumulation of HIV-1 proteins in the cell and that this reduction can contribute to the establishment of HIV-1 latency. Perturbing the levels of PTB in vivo may be an additional approach to purge the latent reservoir and eradicating HIV-1 in infected individuals.
 
IMMUNOLOGY & RESERVOIRS (Session 3)
 
Satya Dandekar (UC Davis) extended the discussion begun by Veazey of SIV immunopathogenesis in the gut-associated lymphoid tissue (GALT) of the monkey. GALT is an early target for SIV and HIV infection and is a site for severe CD4+ T cell depletion. The impact of acute CD4+ T cell loss on the gut micro-environment and mucosal defenses against bacterial pathogens has not been characterized.
 
Dandekar and collaborators began to address this using a intestinal ligated loop model in SIV infected rhesus macaques. When contained portions of the intestine were challenged with a dose of pathogenic Salmonella typhimurium, early mucosal cytokine responses, especially that of the recruiting cytokine IL-17, were blunted. This led to increased Salmonella translocation and dissemination.
 
In a second part of her presentation, Dandekar compared recovery of the GALT after ART in the SIV monkey model. She found fuller and more rapid immune reconstitution in the GALT when ART was started within 1 week of experimental SIV infection. CD4 cells that returned in the GALT after early ART possessed a greater diversity of immune response capabilities, as measured by their ability to secrete different cytokines, than the GALT of monkey treated 4 weeks after SIV infection. Although a clinical benefit of early ART in acute HIV infection has not been clearly demonstrated, these findings provide some rationale for studies attempting to test early ART in acute HIV infection.
 
Olivier Schwartz (Institut Pasteur, Paris) described studies of the virological synapse, the term used to describe cell-cell junctions between T cells and/or dendritic cells through which HIV can be physically transmitted. He suggested that HIV uses cellular filiopodia, specialized extensions of the cell membrane, to "reach out and touch" neighboring cells and transmit virus. This type of infection likely takes place in the lymphoid tissues, and may be resistant to certain classes of antiretroviral therapy, such as entry inhibitors or protease inhibitors.
 
Sharon Lewin (Monash University, Mellbourne) suggested that a subclass of memory CD4+ T cells called central memory cells may be more likely to establish a latent infection. The cells express the CCR7 receptor, signaled via the cytokines CCL19 and CCL21. She showed that signaling of these cells via CCL19 or CCL 21 might allow HIV infection without activating the cell. In this setting it is thought that latency is much more likely to result following infection, rather than viral production. Data presented by Chomont (University of Montreal) agreed that the central memory CD4+ cell compartment contained more replication-competent HIV than transitional memory or effector memory cells.
 
CK Hwang of Vanderbilt University used an Alu-PCR assay to quantitate integrated provirus in resting CD4+ cells. This assay detects integrated HIV genomes by amplifying from an Alu sequence, found repetitively in the human genome, to the nearest HIV integrant site. He hypothesized that cells from long-term HIV controllers would contain fewer integrated proviruses when compared with cells from ART-suppressed patients. This would be consistent with either establishment of a larger reservoir in ART-suppressed patients, or with residual HIV replication replenishing the latent reservoir in ART-suppressed patients more than in long-term controllers. Provirus copy numbers were 1 .4 log10 lower in PBMCs from long-term controllers than in cells from ART-suppressed patients and untreated viremic patients. The ART-suppressed patients and non-controlling untreated patients had similar levels of integrated provirus.
 
RESISTANCE & RESERVOIRS (Session 4)
 
Mark Wainberg (McGill) discussed his group's work with the PRMT6 arginine methylase. They have found that this host enzyme can post-transcriptionally modify several important HIV proteins: the transactivator Tat, the RNA export protein Rev, and the virion core nucleocapsid protein. Arginine methylation of Tat, Rev, or NC inhibits the function of these viral factors, potentially contributing to the establishment of latent infection.
 
Steve Deeks (UCSF) discussed studies in "Elite Controllers," a recently coined term for HIV-infected patients that innately control viremia to <50 copies/ml without the use of ART. Deeks proposed that insights regarding the nature of the virus/host relationship in these individuals might be informative regarding the feasibility of HIV eradication. The majority of these controllers have detectable plasma HIV RNA, albeit at very low levels, which remains stable over time. Vigorous HIV-specific immune responses are observed in many controllers, and when present is associated with modest but abnormal T cell activation. Deeks raised the concern that these levels of activation might lead to morbidity, and reported that carotid artery medial intimal thickness, a surrogate measure of general atherosclerosis, was increased in these patients when compared to HIV-seronegative controls.
 
Finally, John Coffin (NCI Drug Resistance Program) discussed the important implication of a series of studies performed by his group on "low-level" viremia. Using an advanced PCR assay able to quantitatively measure plasma HIV RNA down to 1 copy of RNA/ml, Coffin summarized the group's conclusions on the decay phases of plasma viremia after ART initiation. They added a fourth decay phase to the three phases previously described, although the slope of the 4rth phase was really no slope at all. The 4rth phase has likely been obscure previously as assays have not been sensitive enough to detect the 3rd phase of decay:
Phase I) week 0 to week 2; half-life of decay 1.5 days
Phase II) week 2 to week 60; half-life of decay 28 days
Phase III) week 60 to week 180; half-life of decay 39 weeks
Phase IV) after week 180; half-life of decay infinite
 
Later in the meeting Sarah Palmer continued the reporting on single-copy assays that had been begun by Coffin. Upon measurements of of viremia in patients up to seven years after the initiation of suppressive therapy using a real time RT-PCR assay with single HIV RNA copy sensitivity, she found that 77% of the patient samples had detectable low-level viremia ranging from 1-99 HIV RNA copies/ml and all patients had at least one sample with detectable viremia (≥1 copy/ml). The 3rd and 4rth phases of decay were significantly correlated with total baseline (pretherapy) viremia for each patient. She concluded that persistent viremia on treatment may result from virus production by cells that are infected prior to initiation of therapy. This low-level persistent viremia appears to arise from at least two cell compartments, one in which viral production decays over time and a second in which viral production remains stable for at least seven years.
 
Palmer also reported on low-level viremia in "elite controllers." 46 elite controllers who had three measured viral RNA levels of <50 copies/ml over at least a one year period without treatment, and 163 patients suppressed on therapy to <50 copies/ml for up to 333 weeks were compared. Viremia was detected in 75% of the samples from both elite controllers (ranging from 1-614 copies/ml) and patients on suppressive therapy (ranging from 1-43 copies/ml). The median HIV-1 RNA value for the elite controllers and treated patients (irrespective of their treatment regimens) was significantly different 5 .5 and 3 .0 copies/ml, respectively (p=0 .023, one-way ANOVA), and the overall distribution of HIV RNA levels was significantly different (p=0.013, Kolmogorov-Smirnov test). Overall, the levels of viremia below 50 copies/ml was higher for elite controllers compared to those in persons on suppressive therapy.
 
HAART POTENCY, PHARMACOLOGY, COMPARTMENTS & RESERVOIRS (Session 5)
 
Scott Letendre (UCSD) described the findings of CNS ART pharmacology studies carried out within the CNS HIV Antiretroviral Effects Research (CHARTER) study, some of which was presented at CROI 2007. Using an index of CNS ART penetration to estimate the total efficacy of a regimen within the CNS, Letendre showed that regimens with higher scores resulted in lower CSF viral loads. Although the data was convincing if the score was > 0, the linearity of the relationship was unconvincing with scores above 1. As the data matures, and the study expands, the relationships may become clearer.
 
Yilmaz (Sahlgrenska Academy at Goteborg) suggested that neopterin, a well-established marker of macrophage activation and pass marker of HIV infection used in the early days of the epidemic, are elevated in most HIV-1-infected individuals, and decrease significantly after initiation of antiretroviral therapy (ART). 157 chronically infected patients with stable ART and no neurological symptoms were compared to 193 untreated patients. HIV-1 RNA and CSF neopterin levels were markedly lower in patients on ART. No significant difference in CSF neopterin concentrations was found between those treated with protease- or non-nucleoside RT-inhibitor regimens in combination with 2 nucleoside analogues. Subjects with CSF HIV-1 RNA loads < 2. .5 copies/ml had the lowest CSF neopterin levels. . Plasma viral load had no impact on intrathecal immune activation in cases with CSF viral loads < 50 copies/ml, suggesting compartmentalized patients in some, but not all, patients.
 
PRIMARY HIV INFECTION & ERADICATION ISSUES (Session 6)
 
The HIV virion maturation inhibitor under development by Panacos, Bevirimat (dimethylsuccinyl betulinic acid), was discussed by C-H Chen (Duke). As the toxicology profile of the drug remains to be determined, Chen presented studies that attempted to identify the potential cellular targets of Bevirimat. Betulinic acid is a potent proteasome activator that preferentially activatess the chymotrypsin-like activity of the proteasome. Chemical modification of betulinic acid that resulted in Bevirimat transformed the drug into an inhibitor of the human 20S proteasome. Whether clinical concentrations of the drug are sufficient to inhibit proteosome function, which would seem likely to eventually lead to toxicity given the important role of the proteosome machinery in host cell protein metabolism, has yet to be determined.
 
Markowitz (Aaron Diamond AIDS Research Center) reviewed the studies of his group on immune cell losses and viral replication in acute and early HIV-1 infection in the GALT. Up to 60% of CD4+T cells in the lamina propria of the lower gastrointestinal tract (GI) are lost as early as 2 to 4 weeks after infection. 54 acute or early HIV-1 infected (AEI) subjects and 18 uninfected controls underwent colonic biopsy. 18 of the 54 AEI were studied with sequential biopsies longitudinally over 3 years after HAART and 22 were studied cross-sectionally after 1 year to 7 years of suppressive, uninterrupted therapy,
 
70% of the patients studied maintained a 50-60% depletion of lamina propria lymphocytes despite 1 to 7 years of ART. Recovery was seen in most after 1-3 years of therapy, with fuller recovery after 3-7 years of therapy, but never returned to levels prior to infection. Lymphocytes expressing CCR5 and both CCR5 and CXCR4 were persistently and preferentially depleted. Levels of immune activation in the memory cell population, CD45RO+ HLA-DR+, returned to levels seen in the uninfected controls in the peripheral blood but were elevated in the GI tract of patients with persistent CD4+ T cell depletion despite therapy. Rare HIV-1 RNA expressing cells were seen by in situ hybridization. Preliminarily, it appeared that lower levels of CD4 cell activation in the GALT were associated with greater GALT CD4 cell recovery. As ever, it is unclear if this observation is the chicken, or its egg. Studies of viral evolution in the GALT might demonstrate whether there is escape from ART drug pressure in the GALT, but have thus far been technically impossible.
 
Dean Hamer (Laboratory of Biochemistry, NCI) presented the history of the development of inducers of HIV gene expression that might be used to reduce the reservoir of latent HIV in resting CD4 T cells. Analogues of the lipid second messenger diacylglycerol can reactivate HIV transcription through the protein kinase pathway. Two DAG lactones with high potency but minimal side effects were identified. Valproic acid can alter chromatin configuration by inhibiting histone deacetylase. In model systems, the most efficient and complete induction of HIV was achieved using a combination of both types of agent, which work synergistically in reactivating HIV transcription. The combination of drugs also renders the cells maximally sensitive to killing by an anti-HIV envelope-targeted immunotoxin. Moreover, he found that 1.5 mM of valproic acid was specifically able to induce HIV-specific apoptosis in latently infected cell lines. Hamer suggested that this dual approach be tested clinically.
 
NEW THERAPEUTIC APPROACHES (Session 7)
 
Daria Hazuda, fresh from a victorious 10-year battle bringing an HIV integrase inhibitor from the test tube into the clinic, outlined the latest Sisyphusean taken on by her drug development team in a talk entitled "HIV-1 Latency: A Drug Discovery and Development Perspective." Although the point may be debated by some, she stated that while new agents in development may enhance the potency and durability of anti-retroviral therapy and perhaps even impact persistent HIV-1 replication, they will not address the potential latent reservoir of infection.
 
Hazuda explained that in vitro it has been well established that histone deacetylase (HDAC) inhibitors or other compounds that activate the HIV LTR can induce HIV-1 replication in latently infected cell lines. These observations suggest latent HIV-1 infection is maintained by cellular mechanisms that control chromatin structure and demonstrate de-repression of HIV-1 transcription can be achieved without activating T cells. Therefore, with the development of appropriate in vitro assays and a combination of small molecule screening and genomics technologies it should be possible to identify and exploit specific molecular targets that control latent infection.
 
She then described a high-throughput cell model system that was used to identify dozens of HDAC inhibitor compounds in the Merck chemical library, and the screening of selected compounds in various cell models of latency. Specific HDACs were validated as targets by specific targeted inhibition with siRNA. These studies suggested that HDACs 1, 2, and 3 were important in the maintenance of latency, and that inhibition of HDAC 3 was, in part, required for reactivation of quiescent HIV.
 
Later her colleague Herbert Dang illustrated the effects of selected HDAC inhibitors on HIV-1 expression in an in vitro system using isolated primary resting CD4+ T-cells infected with HIV ex vivo and induced to enter a non-productive state (Swiggard et al. J Virol. 2005). Application of either or both HDAC inhibitors or hexamethylbisacetamide (HMBA) induced p24 expression in this model system.
 
Tae-Wok Chun of the NIAID presented recent updates from his cohort of patients treated and studied at the NIH. He pointed to both the persistence of latently infected, resting CD4+ T cells, and low levels of on-going viral replication despite modern ART as reasons for persistent infection. However, he holds that the activated CD4+ T cell compartment harbors the majority of persisting HIV in infected individuals who have had no detectable viremia for extended periods of time as a result of effective antiretroviral therapy. He suggested that the frequency of infected CD4+ T cells in gut-associated lymphoid tissue was significantly higher than that of cells in blood despite years of effective antiretroviral therapy. He found up to 10,000 molecules of HIV DNA/million GALT CD4+ cells in the ileum and sigmoid colon, but only up to 2000 copies in resting CD4+ cells of PBMCs. He also reported no significant differences between the GALT and PBMC sequences, arguing against effective replication of HIV in the GALT despite therapy.
 
Johansson (Karolinska Institutet, Stockholm) reported on the construction of a murine monoclonal antibody against the envelope antigen of HIV (P4/D10) conjugated with the conventional anti-cancer drug, doxorubicin. Doxorubicin-conjugated P4/D10 neutralized HIV-1IIIB and eliminated HIV spread and replication in cell lines in culture. infected Jurkat cells in vitro . The conjugate also protected mice from challenge with HIV-1IIIB/MuLV at an eight-fold lower concentration than needed for free antibody, while no effects were observed for comparable doses of free drug or irrelevant conjugate controls. Her colleague A. Vahlne then followed with a report of redirecting natural antibodies that recognize the epitope gal-_1,3-gal, part of a pentasaccharide present on cells of all mammals except primates. Such natural antibodies can induce antibody-dependent cellular cytotoxicity (ADCC). ADCC was directed to HIV-infected cells using CD4 peptides coupled to gal-_1,3-gal. These antibodies can trigger the activation of NK cells via CD16 recognition of the Fc fraction of the antibody. The use of these glycopeptides in the presence of serum from non-infected donors redirected the specificity of the natural anti-gal-_1,3-gal antibodies towards gp120 in ELISA and neutralized HIV-1 in all three assays down to picomolar concentrations.
 
Finally, novel antiviral compounds were discussed. Mahy (H-PHAR sa, Belgium) presented data on a new chemical entity, HPH116, is a modified synthetic form of azodicarbonamide. HPH116 is a zinc finger ejector molecule that targets the NCP7 protein within the viral capsid . The capsid depends on zinc ions to maintain its structure, and the NCP7 protein is required by HIV. HPH116 in combination with other ARVs induces a dramatic increase in viral replication in cell line model systems. Such molecules could represent an entirely new class of antiviral.
 
Alcami (Unidad de Inmunopatologia del SIDA, Majadahonda, Spain) described here the effect of SJ23B, a new natural compound isolated from Euphorbia Hyberna, on HIV reactivation and viral receptor expression in lymphocytes. This plant is related to the Samoan plant from which another similar inducer, prostratin, is derived. SJ23B induced the internalization of the HIV-1 receptors CD4, CXCR4 and CCR5 thus preventing R5 and X4 viral infection in human primary T cells at the nanomolar range. SJ23B was a potent antagonist of HIV-1 latency, activating HIV-1 gene expression in Jurkat-LAT-GFP cells with at least 10-fold greater potency than prostratin.
 
Overall, the attempts of scientists and clinicians to deal with the challenges of persistent HIV infection have met with only incremental success thus far. Better assays and drugs are available, and a new appreciation of the challenges posed by viral replication in the GALT and CNS has been gained. Many seem of the opinion that HDAC inhibitors might play some role is allowing depletion of persistent infection, but just as many feel that other approaches will be required. It is hoped that the Fourth International Workshop on HIV Persistence during Therapy in 2009 will feature reports of definite clinical advances.
 
 
 
 
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