Coreceptor Dependent Signaling in Individual Primary Resting CD4+ T-cells Mediated by Low Levels of HIV Binding

In order to enter into the target cell, HIV requires functional contact with CD4 and CCR5 or CXCR4. The last two are G-protein coupled receptors that when activated with chemokines, or HIV envelope can initiate a wide range of biological responses, including Ca2+ mobilization, cytoskeletal rearrangements and cell migration. To determine the specificity of X4-tropic gp120-mediated signaling through CXCR4, we have chosen a microscopybased approach to observe the response at the level of individual cells providing greater sensitivity. Target cells were able to activate a signaling cascade in response to both monomeric recombinant gp120 and virion-bound trimeric gp120. C2+ elevation was a direct measurement of CXCR4 engagement because it was dependent on the tropism of the envelope, engagement of CD4, and sensitive to the CXCR4 antagonist AMD-3100. Signaling required much lower levels of envelope when virion associated. Imaging analysis allowed the correlation of the pattern of virion-mediated C2+ fluxing with the exact number of viral particles bound to cells. This analysis revealed that an average of four virions, and as few as two virions associating with a primary resting T cell could mediate C2+ mobilization. The ability of several virions to stimulate signaling in primary resting T cells is physiologically relevant and has important implications for AIDS pathogenesis. Funded in part by DHHS NO1-CO-12400 and RO1AI052051. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005


Retrovirology 2005, 2(Suppl 1):S3
In order to enter into the target cell, HIV requires functional contact with CD4 and CCR5 or CXCR4. The last two are G-protein coupled receptors that when activated with chemokines, or HIV envelope can initiate a wide range of biological responses, including Ca 2+ mobilization, cytoskeletal rearrangements and cell migration. To determine the specificity of X4-tropic gp120-mediated signaling through CXCR4, we have chosen a microscopy-based approach to observe the response at the level of individual cells providing greater sensitivity. Target cells were able to activate a signaling cascade in response to both monomeric recombinant gp120 and virion-bound trimeric gp120. C 2+ elevation was a direct measurement of CXCR4 engagement because it was dependent on the tropism of the envelope, engagement of CD4, and sensitive to the CXCR4 antagonist AMD-3100. Signaling required much lower levels of envelope when virion associated. Imaging analysis allowed the correlation of the pattern of virion-mediated C 2+ fluxing with the exact number of viral particles bound to cells. This analysis revealed that an average of four virions, and as few as two virions associating with a primary resting T cell could mediate C 2+ mobilization. The ability of several virions to stimulate signaling in primary resting T cells is physiologically relevant and has important implications for AIDS pathogenesis. Funded in part by DHHS NO1-CO-12400 and RO1-AI052051.
have investigated the molecular basis of this resistance through genetic and phenotypic studies. We find that different genetic pathways leading to small molecule inhibitor resistance can be taken by the same starting virus. Additionally, these viruses can exhibit different levels of cross resistance to RANTES derivatives. These studies provide insight into potential mechanisms of CCR5 inhibitor resistance. chemokine beta-hairpin loops, suggests that the V3 region may direct coreceptor choice through structural mimicry of the chemokines that bind to either coreceptor. Some of our recent research based on available chemokine structure-activity data has produced evidence that challenges this model. HIV entry inhibition. The native ligands of HIV coreceptors prevent viral entry via a combination of steric blockade and removal of receptors from the cell surface (receptor sequestration). Our work on synthetic and semi-synthetic chemokine analogues has led to the discovery of potent HIV entry inhibitors such as PSC-RANTES, which owe their activity to a greatly enhanced capacity to sequester CCR5. PSC-RANTES has shown promise as a microbicide candidate, but as a totally synthetic protein it is likely to be too expensive for use in the developing world. We are now focused on discovering similarly potent molecules that are either partly or wholly accessible by biosynthesis and thus much cheaper to produce. Here, we report on the mechanisms of interactions of HIV-1 with several such microbes in the context of ex vivo infected human lymphoid tissue. Blocks of human tonsils maintained ex vivo were coinfected with R5 or X4 HIV-1 and with another microbe, measles virus (MV), vaccinia, herpesvirus 6 (HHV-6), herpesvirus 7 (HHV-7), cytomegalovirus (CMV), or a parasite, Toxoplasma gondii (TG). In ex vivo tissues, all the above-listed microbes, except CMV, inhibited replication of R5 HIV-1 whereas replication of X4 HIV-1 was affected mildly. In spite of similarity of the effects, the mechanisms of R5 inhibition by coinfecting microbes are strikingly diverse. HHV-6 and MV upregulate CC-chemokines, in particular RANTES to the levels sufficient to inhibit replication of R5 HIV-1; Toxoplasma gondii seems to secret its own soluble factor, cyclophyline, that also binds to CCR5 coreceptor, HHV-7 downregulates the expression of CD4 on T cells. In conclusion, soluble factors encoded by microbes, including their components, or secreted by infected and bystander cells in response to microbial invasion (in particular cytokines/ chemokines) constitute a universal network through which microbes interact with human body creating a microenvironment that is beneficial for them. However, what is beneficial for one microbe can be detrimental for another. Deciphering the molecular mechanisms by which pathogens alter tissue microenvironment so that it becomes detrimental to HIV, may significantly contribute to the development of efficient anti-HIV therapies.
Retrovirology 2005, 2(Suppl 1):S10 The fusion inhibitor T20 marks the beginning of a new era in the management of HIV-1 disease. By inhibiting viral entry, T20 suppress viral replication in patients carrying strains resistant to reverse transcriptase or protease inhibitors. However, its antiviral activity is compromised by mutations in gp41. Based on our previous work demonstrating that Rapamycin (RAPA) inhibits R5 HIV-1 by downregulating CCR5 surface expression, we now show that RAPA and T20 synergize in antiviral activity against R5 strains. Synergy studies using the Median Effect analysis revealed that the IC50 values of RAPA and T20 in the RAPA/T20 combination were reduced 9-and 3-fold, respectively. Three-Dimensional modeling confirmed the observed synergy (synergy volume of 253.85; 95% CL : 91-147). We also show that the RAPA/T20 combo, but not T20 alone, prevented the emergence of T20 resistance upon continuous passage of R5 HIV-1 ADA on PBMCs for 24 weeks under subinhibitory concentrations of T20. In addition, R5 ADA and YU-2 clones carrying T20 single mutations 36D, 38 M or 43 K (4-10 fold resistance) or the double mutation 36D/38 M (65-fold resistance), were all inhibited in the presence of RAPA. In conclusion, our results demonstrating that the RAPA/T20 combination has synergistic antiviral activity, prevents the emergence of T20 resistance, and inhibits T20 resistant strains, suggest a novel therapeutic approach to enhance the antiviral activity of T20 in patients carrying R5 strains of HIV-1. Maraviroc is a novel CCR5 antagonist and is the most advanced clinical candidate in Pfizer's CCR5 discovery and development programme. It is exquisitely selective for the CCR5 receptor and demonstrates potent activity in vitro against both labadapted and primary clinical HIV isolates spanning all of the clades, including viruses that are resistant to current classes of HIV agents. Maraviroc has been evaluated in >400 volunteers and in 66 HIV patients where it is well tolerated at doses in excess of those required to block the CCR5 receptor and those providing free drug levels above the in vitro concentrations for potent antiviral activity. Consistent with this, Maraviroc has demonstrated encouraging short-term (10 day), single agent efficacy as measured by reductions in viral loads in asymptomatic HIV patients; doses of 300 mg QD and 300 mg BID resulted in mean maximum HIV RNA reductions of 1.60log 10 and 1.84log 10 , respectively. Studies both with CYP 3A4 inhibitors and inducers have demonstrated that Maraviroc will have manageable drug interactions when used in the setting of HIV patients receiving HAART. In summary, Maraviroc has potency, pharmacokinetic and toleration profiles that merit its further evaluation as a new therapy for patients with HIV/AIDS. dosing. Variability in absorption is modest (20-40%). Exposure to vicriviroc, a substrate for CYP3A4, is ''boosted'' by CYP3A4 inhibition with ritonavir (RTV), without being affected by other metabolizing enzymes or pGp. Because of the high oral bioavailability and highly predictable exposure, particularly when boosted with as little as 100 mg RTV, potent HIV suppression of 1.5 log 10 is anticipated with as little as 10 mg vicriviroc daily with RTV. Dosage adjustments are not expected in combination regimens. Vicriviroc shows particular promise as an HIV therapeutic due to pharmaceutic, PK/PD properties that support its potent activity and convenient once daily dosing. S13 Update on Aplaviroc: An HIV Entry Inhibitor Targeting CCR5 James F Demarest 1,2 and the Aplaviroc Project Team 1 GlaxoSmithKline 2 Duke University AIDS Center, Durham, North Carolina, USA Retrovirology 2005, 2(Suppl 1):S13 Aplaviroc (873140) is a novel spirodiketopiperazine CCR5 antagonist that binds specifically to human CCR5 and allosterically inhibits HIV entry. Aplaviroc has exhibited potent in vivo antiviral activity (1.66 log decrease in viral load at nadir) following 10 days of monotherapy. In vitro studies of antiviral activity demonstrate that aplaviroc is active against HIV isolates from a variety of clades as well as those resistant to current HIV therapies targeting RT, PR, and gp41. In vitro studies suggest prolonged CCR5 receptor occupancy (RO) by aplaviroc with an offset half-life of >100 hours. In vivo studies following short term aplaviroc administration using CCR5specific mAb demonstrate substantial and prolonged CCR5 RO (>50%) by aplaviroc, when plasma drug levels were undetectable, observed for approximately 5 days. In the 10 day monotherapy study of aplaviroc in HIV+ subjects, one subject whose virus was R5-tropic at baseline and day 5 showed that R5X4-tropic (dual/mixed) virus was present at Day 10. Subsequent analysis revealed reversion to an R5-tropic only phenotype by day 24, with no decrease in sensitivity to aplaviroc (Fold IC50). The change in tropism at the population level observed on day 10 was the result of the emergence of pre-existing dual-tropic virus(-es) that were below the limits of detection on day 1. Viruses present in a subject's quasispecies that are below the limits of detection with currently available tropism assays may become detectable following monotherapy with a CCR5 antagonist; however, whether similar changes may occur on combination therapy with a CCR5 antagonist remains to be determined. Aplaviroc has demonstrated potent anti-HIV activity in vitro and in vivo. Furthermore, aplaviroc exhibits a unique allosteric interaction with CCR5 and demonstrates prolonged receptor occupancy. CCR5 antagonists show promise for inhibiting entry of CCR5-using viruses in the clinical setting.
GATA3 has been implicated as a key factor in commitment of CD4 T cells to the Th2 phenotype. Naïve CD4 T cells cultured without exogenous cytokines induce GATA3 and IL-4 transcription only when stimulated with low concentrations of cognate peptide in the presence of IL-2. Naïve CD4 T cells from mice with a conditional deletion of Gata3 fail to induce IL-4 in response to TCR engagement as do cells cultured in the presence of anti-IL-2. High concentration inhibition of GATA3 and IL-4 expression and of Stat5 signaling is rescued by MEK inhibitors implying that inhibition is mediated through erk action. Infection of cells cultured under Thnull conditions with retroviruses containing GATA3 and constitutively active Stat5a induce a Th2 phenotype and result in full accessibility of the Il4 gene. Further, ChIP analyses reveals that in Th2 cells, Stat5a is bound to DNase I hypersensitive sites in the second exon of the Il4 gene. Thus, both GATA3 and STAT5 are key factors in ''opening'' the Il4 gene and in inducing the Th2 phenotype. Furthermore, GATA3 proved to be important in Th2 growth. In vivo deletion of Gata3 using OX40-Cre eliminated Th2 responses and allowed the development of IFNg-producing cells in mice infected with Nippostrongylus brasiliensis. Thus, GATA3 serves three functions in Th2 biology; it induces the Th2 fate, represses the Th1 fate and it promotes selective outgrowth of Th2 cells.
HIV protease inhibitors and the newest class -HIV entry inhibitors. Current antiretroviral regimens are effective in suppressing viral replication, enhancing immune function, and preventing clinical progression of HIV disease. However, current antiretroviral drugs may be compromised by suboptimal antiretroviral activity; drug resistance and cross-resistance; complexity; acute, chronic and life-threatening toxicities; and drug-drug interactions. Newer antiretroviral agents, such as the HIV entry inhibitors, are needed to improve antiretroviral activity (particularly against drug-resistant strains), avoid the selection of drug resistance, improve convenience, improve tolerability and reduce toxicity, and minimize drug-drug interactions. With demonstrated safety and effectiveness against drug-resistant viruses, HIV entry inhibitors quickly may become part of treatment regimens for treatment-experienced patients. With additional safety, tolerability, convenience, and antiretroviral activity data, HIV entry inhibitors could become part of standard treatment regimens for treatment-naïve patients. EBV and KSHV both are associated with malignancies. In tumors the latent form of infection predominates and there is little or no expression of lytic genes. FIAU is a nucleoside analogue that is selectively phosphorylated by EBV and KSHV thymidine kinases. We have used a variety of pharmacologic agents to induce lytic infection in xenografts of Burkitt's lymphoma and primary effusion lymphoma cell lines in SCID mice. Using 125I labeled FIAU, we have been able to visualize lytic gene reactivation. gag/pol/nef with clades A, B, and C envelope was recently completed in 50 healthy adults in the U.S. CD4+ and CD8+ T cell responses were detected in the majority of vaccinees using IFN-(ELISpot) and flow cytometric detection of intracellular IL-2 or IFN-. HIV-specific antibody response was detected in about one third of vaccinees. Combination modality regimens using a DNA vaccine prime followed by a viral vector boost have shown promise in nonhuman primate models of HIV infection. Phase I clinical trials to test the safety and immunogenicity of an adenoviral vaccine expressing proteins similar to the DNA, and a DNA prime, adenoviral boost regimen are in progress. The initial adenoviral vector vaccine uses an Ad5 serotype vector. However, pre-existing immunity can mitigate the efficacy, so alternate serotypes and modifications to the adenoviral fiber regions are being explored. Today, nearly twenty-five years since the first AIDS cases were identified, the AIDS pandemic is recognized as a global public health priority. With 14,000 new HIV infections every day, the best hope for stemming the insidious spread of HIV and for ending the pandemic remains the development of a safe and effective AIDS vaccine. The search for an AIDS vaccine can be viewed from past, present, and future perspectives. Since the identification in 1983 of HIV as the etiologic cause of AIDS, the field has gained significant knowledge on the pathogenesis of HIV relevant for vaccine development, several vaccine approaches have been designed and tested in clinical trials, and infrastructure has been established both in developed and developing countries to assess HIV incidence, molecular epidemiology, host immune response to early infection, and to conduct Phase I, II and III trials. Yet despite current global investment of nearly $650 million per year, the HIV vaccine pipeline remains inadequate. Vaccine candidates designed by empiric approaches and tested thus far in human efficacy trials have failed to prevent HIV infection or suppress HIV viral load. Current candidates approaching human efficacy trials have shown some benefit in certain monkey models but not in others. There is considerable potential that these current candidates will achieve no more than limited success if any, since they have provided little or no protection from pathogenic SIV challenge in monkeys, are markedly impeded in their capacity to elicit cell mediated immune responses in humans due to anti-vector immunity, and have not been designed to elicit effective neutralizing antibodies. In order to significantly accelerate global efforts in AIDS vaccine development and shorten the timetable for a licensed and widely accessible AIDS vaccine, the following issues should be addressed. First of all, key scientific problems, known to the field for more than a decade, need focused and direct efforts to inform rational vaccine design. These problems include: the lack of understanding of how best to design vaccines to elicit broadly neutralizing antibodies to HIV; the lack of safe and suitable candidates for clinical development which mimic the protective efficacy thus far only achieved by live attenuated SIV vaccines; and the lack of candidates in the pipeline which adequately address the hypervariability of HIV. Secondly, an ''industrial model'' for applied research and product development needs to be incorporated into global AIDS vaccine R&D efforts, to facilitate an expedited transfer of leading vaccine candidates demonstrating feasibility/proof of concept to major vaccine-pharmaceutical companies for advanced development. Finally, a shift from business as usual risk-benefit paradigms common to vaccine R&D needs to be established to encourage innovative product development and accelerated clinical testing of AIDS vaccine candidates without compromising safety.

S29 Abstract withdrawn
(immunogens, vector, delivery, adjuvants) that induce immunologic responses relevant to preventing HIV infection and/or controlling HIV disease progression. The research agenda targets evaluation of candidates at all stages of product development from phase 1 first in human trials to proof of concept and efficacy trials. Innovative trial designs are driven by investigators and statisticians to efficiently advance products and information that seeks fundamental insight into correlates of protective immunity. HVTN laboratories quantitate adaptive and innate immune responses employing highly reproducible and standardized endpoint assays that meet GLP validation standards for core assessment as well as exploratory assays to understand correlate mechanisms. Currently 24 HIV vaccine candidates are slated for evaluation over the next 24 months including proof of concept and expanded phase 2 evaluation in preparation for larger efficacy trials in the 2007-2008 time period. Kaposi's sarcoma (KS) is characterized by an abnormal growth of blood vessels. KS was found mainly in older men of Mediterranean or African origin (classic KS) or in patients after organ transplantation (iatrogenic KS). However, in the early 1980s, an aggressive epidemic form, linked to AIDS, was noticed and was one of the first clues to the existence of HIV-1 pandemy. The link between KS occurrence and HIV has raised multiple hypotheses. The drastic reduction of KS after the introduction of HAART, suggests HIV as a powerful co-factor for KS progression. We and others have contributed to the elucidation of KS cell nature and the possible involvement of extracellular HIV Tat. Tat is proangiogenic and is a true promoter of KS lesions acting as a VEGFR2 ligand both on KS and endothelial cells, in addition Tat is able to bind and activate chemokine receptors on monocytes and granulocytes causing a proinflammatory status. Evaluation of the effects of extracellular Tat on KS cells by microarray analysis after 24 h of incubation shows an interesting clustering of gene products involved in signal transduction, especially GTP-ase, Kinase and cAMP activity, confirming that Tat acts extracellularly by ways that are probably unrelated to its nuclear activity. KS occurrence is reduced by HAART but still present and in Africa is one of the most frequent oncologic disease. To find suitable drugs with low toxic impact on KS patients, we have tested several drugs and gene therapy approaches in in vivo models. In addition to the B-cell derived Burkitt lymphoma and the immunoblastomas of immunodefective patients, most of the rare nasal NK lymphomas and about half of the Hodgkin's lymphomas (HL) carry EBV. In the 2 latter malignancies, the neoplastic cells are embedded in granulation tissue (with evidence for the contribution of the microenvironment to the maintenance of the disease). Their viral expression pattern is similar, EBNA-1 and LMP-1 pos and EBNA-2 neg (type II). In contrast to the EBV-driven immunoblastomas, EBV cannot be held directly responsible for the growth of these 2 malignancies that also differ among themselves.

Roles of EBV in Haemopoetic Malignancies
Hodgkin's lymphomas: The few existing HL derived cell lines are EBV negative. When from one line a forcibly converted EBV positive subline was established, it expressed EBNA-1 only (Type I).LMP-1 could be induced by exposure to CD40L and IL-4. Conceivably, the in vivo phenotype (Type II) is imposed by the cytokines that abound in the granulation tissue. According to one present view EBV is important in the early stage of HL development, in that it rescues B lymphocytes with non functional Ig-rearrangements from apoptosis. Nasal NK/T lymphomas: Normal NK/T cells do not carry EBV receptors The mechanism of infection of the NK/T cells is not known. EBV carrying cell lines exist and they express the Type II pattern, corresponding thus to the in vivo phenotype. They require IL-2 for in vitro proliferation. Conceivably IL-2 can be provided by activated T cells in vivo. Treatment with cytokines (IL-10, IFN g) upregulates LMP-1 expression that leads to more efficient growth response to IL-2. Kaposi's sarcoma represents a vascular proliferative process. Characterization of genes expressed in vascular endothelial cells in particuar arterial and venous endothelial cells when examined in KS indicate that KS displays profile that resembles arterial endothelial cells. These include certain Notch receptors and their ligands including Delta like 4 (dll4), neuropilin-1, ephrinB2, connexin 37, but not venous specific marker (ephB4). Furthermore HHV8 infection, and viral proteins vGPCR, leads to the induction of several proteins that preferentially induce genes expressed in artery endothelial cells (ephrinB2) but not venous specific (EphB4). KS gene expression profile also shows expression of lymphatic endothelial cell specific markers as well. Thus the phenotype of KS is distinct from the profile in resting or aniogenic response in physiological responses as in wound healing or tumor vasculature. Distinct phenotypic characteristics provide opportunities for novel targeting and therapeutics. For example inhibition of EphrinB2 expression with siRNA leads to detachment of the cells from matrix, and loss of cell viability represents a prototype. Growing understanding of KS biology is thus likely to offer novel targets for therapy. The HIV protease inhibitor ritonavir has been reported to have activities unrelated to inhibition of HIV protease, including anti-tumor activity in vivo and in vitro, induction of lipodystrophy in vivo, inhibition of the 20S proteasome, and inhibition of NFkB activation. Here we show that ritonavir also inhibits activation of NF-AT by PMA plus ionomycin and by the HHV-8 vGPCR. Inhibition of NF-AT activation occurs through the PI-3 kinase/ Akt/GSK-3 pathway, since ritonavir treatment leads to decreased Akt phosphorylation and a resultant decrease in GSK-3 phosphorylation. Treatment with ritonavir also inhibits the expression of NF-AT-dependent pro-inflammatory factors. Inhibition of multiple signaling pathways may help to explain the anti-tumor and other effects of ritonavir that are unrelated to its anti-retroviral activity. Angiogenesis plays a critical in the pathogenesis of Kaposi's sarcoma. Platelet derived growth factor (PDGF), vascular endothelial growth factor, fibroblast growth factor and matrix metalloproteinases (MMP) are among the angiogenic pathways that have been implicated in the development of Kaposi's sarcoma. The introduction of specific therapies such as imatinib and Col-3 that target the PDGF pathway and MMPs, respectively, provide a mechanism to test the importance of these pathways in Kaposi's sarcoma in vivo. In this session the rationale for targeted therapy in Kaposi's sarcoma will be discussed. The results from recent trials involving anti-angiogenic agents and signal transduction inhibitors will be reviewed. Additionally, trials of targeted therapies that are underway or in development will be outlined.

S46
Virus (KSHV/HHV8) Infection and Genomic Aberrations in Developing AIDS Kaposi's Sarcoma (KS) P Biberfeld 1 , P Pyakurel 1 , F Pak 1 , C Massambu 1,2 , AR Mwakigonja 1,2 , T Heiden 1 , E Castanos-Velez 1 and EE Kaaya 1,2 1 Immunopathology Lab, Karolinska Institute, Stockholmn 2 Dept. of Pathology, Muhimbili University College, Dar-es-Salaam, Tanzania E-mail: peter.biberfeld@onkpat.ki.se Background: AKS is the most frequent AIDS tumor and like endemic (EKS) always associated with HHV8/KSHV but it is still controversial whether KS represents a monoclonal cell proliferation with distinct recurrent genomic changes or a polyclonal, hyperplastic, reactive process. Material and Methods: Biopsies of AKS and EKS were compared by triple immunoflourscence for possible stage related phenotypic differences in HHV8 (LANA) infected tumor spindle cells (CD34+SC) and proliferating (Ki67+) cells. Histological tumor areas were alsolaser microdissected and the DNA analyzed by CGH and interphase FISH for cytogenetic changes in early and late stages of tumor development.
Results: LANA and CD34 tumor spindle cells (SC) varied concordantly with stage of AKS and EKS. However among CD34+SC approximately 30-40% were LANA negative, but onlya minor population of LANA cells were CD34-(3-4%) in all KS forms and stages Cell proliferation (Ki67+) was relative constant (4. [5][6][7][8][9][10][11].5%) at all KS stages but usually more frequent in early AKS and EKS. Most Ki67+ cells were LANA+/CD34+ but a few were LANA+/CD34-. CGH analysis of KS tumors (n = 27) showed mostly apparent random but no recurrent chromosomal aberrations. Fewer chromosomal aberrations were observed in AKS compared to EKS. Conclusion: Apparently there is either a heterogeneity among SC for HHV8 infection or a continuous recruitment of noninfected precursor SC to the lesions. The LANA+ SC appeared to have a proliferative advantage compared to LANA-SC. Comparison of random chromosomal aberrations in AKS and EKS appears to indicate that genomic instability could be a more important factor for the development of EKS than for AKS. Most likely AKS development is also promoted by various cytokines and growth factors produced during HIV infection and by the compromised state of host immune response in HIV infection. Thailand has experienced a major epidemic of HIV/AIDS since 1988. Currently over 650,000 persons are HIV-infected and 400,000 have died of AIDS in Thailand. However, Kaposi's Sarcoma (KS) is very rare. Among the 101,945 adults AIDS cases reported between 1994 and 1998 only 0.2% had KS. We have ruled out the possibility that HHV-8 infections are rare; in a study of 992 persons at risk or positive for HIV we found an HHV-8 antibody prevalence of 24.2%. Another hypothesis to explain the rarity of KS is that endothelial cells in Thai's are relatively resistant to HHV-8 infection. We obtained umbilical cord endothelial cell cultures from 10 Thai women who were HIV negative and analyzed their DNA for novel single nucleotide polymorphisms (SNPs) in the coding region for the promoter and 3' UTR region of the DC-SIGN gene. These results were compared to other Asian (11), Caucasian (n = 120), and African (n = 206) samples. No novel SNPs were found in the Thai samples, however some haplotypes that differed from the Caucasian samples were found. Three Thai samples were homozygous for a complete absence of SNPs in the UTR and reduced diversity in the promoter. This was not seen in the Caucasian samples. Additional analysis of linkage disequilibrium and HHV-8 infectivity analysis of these cell cultures are in progress. It is possible that genetic differences in the endothelial cell receptors for HHV-8 or resistance of cells to HHV-8 among Thai's could partially explain the rarity of AIDS-related KS among Asians. Infections with high-risk human papillomaviruses (HPVs) are associated with the vast majority of cervical carcinoma. A fraction of other anogenital tract malignancies such as penile cancer in males and vulvovaginal cancers in females as well as anal carcinomas in immunosuppressed patients and some oropharyngeal carcinomas are also associated with high-risk HPV infections. These cancers generally arise as a consequence of a molecular accident whereby a small genomic high-risk HPV fragment is irreversibly integrated into a host cell chromosome resulting in dysregulated expression of the HPV E6 and E7 oncogenes. Expression of HPV E6/E7 in epithelial cells recapitulates key steps of cervical neoplasia and cancer, which allowed for the creation of tissue culture and animal models of cervical cancer. High-risk HPV E6 and E7 proteins target important cellular growth regulatory circuits among them the p53 and retinoblastoma tumor suppressors, respectively. In addition, E6/E7 expression is a driving force for malignant progression through induction of genomic instability. Hence, high-risk HPVs are the first-ever identified, necessary and molecularly defined causative agents of a major human cancer. The EBV latent membrane protein 1 (LMP1) is expressed in most of the EBV-associated malignancies including nasopharyngeal carcinoma (NPC), Hodgkin's Lymphoma, and immunosuppression-associated lymphoma. have been identified by distinguishing amino acids. We have identified seven sequence variants of LMP1 that can be distinguished using a heteroduplex tracking assay and have determined that most healthy individuals are infected with multiple strains of EBV. Striking differences were found between NPC and matching blood samples with one specific variant, China 1, prevalent in NPC samples and multiple other variants of LMP1 prevalent in the blood samples. The possible selection against some strains appearing in the tumor was highly significant with a p < 0.0001. Many of the LMP1 variants had changes in predicted HLA epitopes of various restrictions. The potential negative selection of the immune system on strains detected in the blood would be reflected in the striking predominance of the China 1 strain in the tumors. In lymphoma samples, changes were also frequently detected in known HLA-restricted epitopes of EBNA3A, 3B, or 3C, in addition to LMP1. Variation in potential immune recognition could contribute to the development of EBV-associated diseases in distinct populations and individuals. Epstein-Barr virus persists latently within resting memory B lymphocytes. To gain access to this compartment the virus first infects and activates a naïve B cell and then drives it to differentiate into a resting memory B cell. To achieve this the virus uses four different viral latent gene transcription programs which are also expressed in EBV associated lymphomas e.g. the growth program in immunoblastic lymphoma, the default program in Hodgkin's disease and the EBNA1 only program in Burkitt's lymphoma. This suggests: 1. that the EBV associated lymphomas arise when the normal progress of a latently infected, activated, naïve B cell to a resting, memory B cells is blocked. 2. the viral transcription program used by the tumor reflects the normal cellular counterpart that it is derived from. The mechanism of EBV persistence will be described and the origins of the EBV lymphomas, predicted from this model, will be discussed. A significant manifestation of tumor-endothelium interactions is the release of the proinflammatory cytokines IL-1 or TNF from tumor cells. These cytokines induce the expression of Eselectin molecules on endothelial cells (EC). The expression of E selectin on EC facilitates contact with selectin ligand-expressing cancer cells thus promoting their transendothelial migration. We reported previously that factors released from cultured head and neck squamous carcinoma cells into the culture medium, induced the release of monocyte chemoattractants from EC. In view of the potential significance of this finding to tumor progression, we asked whether colorectal cancer (CRC) cells also secrete factors capable of inducing up regulating the expression of chemokines in, and their release from EC. A cDNA-microarray analysis of EC treated with culture supernatants of CRC cells revealed that the expression of several CXC chemokines, including CXCL-1 and CXCL-8, was up regulated in EC exposed to the tumor-derived factors. These results were confirmed by RT-PCR. The treated EC secreted higher amounts of CXCL-1 and CXCL-8 than untreated, control EC. These chemokines are involved in tumor progression. Several lines of evidence suggest that E-selectin is involved in the delivery of the CRC-derived chemokine-secretion-enhancing signals to the EC. Experiments to characterize the CRC-derived molecules that mediate these signals are under way. Background: Human xenograft tumor models are widely used for evaluation of potential cancer targets, by assessing the antitumor effects of specific agents, such as siRNA. siRNA is usually stably introduced into tumor cells prior to transplantation. However, oncogene silencing results in reduced cell growth/ survival in vitro and/or failure to establish tumors in vivo, thus hindering tumor response-based efficacy evaluation that is more clinically relevant. We therefore explored a new tumor response model based on regulated RNAi. Methods: A unique RNAi vector was generated to express shRNA only after induction with doxycylcine. Using this vector, we created a novel xenograft tumor model, in which tumors are established under non-induced conditions, followed by induced target inactivation upon oral dosing of the inducer. Three genes were evaluated, a known oncogene (mTOR), and two novel cancer targets (HE7 and HE26), by assessing the tumor response to their silencing. Results: We demonstrate a significant response of staged tumor regression to silencing of all three target genes. For early staged tumors, inactivation of each of the three targets caused dramatic tumor regression (100% regressed and 50% became tumor-free for both mTOR and HE7, and 100% for HE26). Advanced staged tumors also demonstrated significant responses (100% regression for mTOR, and 75% for HE7, 85% HE26). Conclusion: Our results demonstrate the utility of this unique and powerful model for efficacy evaluation of cancer targets; our data also provide robust in vivo efficacy validations of HE7 and HE26 as novel cancer therapeutic targets.

S51
HIV-1 integration resulted in induction of STAT3 and possibly promoted lymphoma formation. This suggests that HIV-1 insertional mutagenesis may be associated with some cases of AIDS lymphoma. Many HIV associated diseases such as KS have resolved or dramatically decreased since the institution of HAART. Two diseases, HIV associated dementia (HAD) and AIDS related lymphoma (ARL) continue to occur and represent two diseases where HIV infected macrophages have been implicated in disease pathogenesis. In order to test whether persistent macrophage reservoirs of HIV might in part be responsible for subsets of these diseases, a survey of tissues obtained from the AIDS and Cancer Specimen Resource (ACSR) was performed. HIV copy and HIV genetic diversity studies were carried out on DNA extracted from HAD brain, ARL and KS specimens. In this pilot study, all HAD involved sections of brain, 10/14 ARL's and 1/11 KS tissues contained > 1 copy of HIV/2000 genomic equivalents. Phylodynamic analysis of the HIV in the HAD and ARL cases demonstrated the presence of dominant/monophyletic forms of compartmentalized HIV is diseased tissues. By comparison only diverse forms of HIV were observed within uninvolved tissues from the same (HAD or ARL) patient, or within KS tissues. Further genetic analysis of HIV from one patient with both HAD and primary CNS ARL, revealed distinctly different LTR's associated with the ARL as compared to the HAD. The ARL LTR was missing an NFk-B site whereas the HAD LTR carried both sites consistent with B-clade forms of HIV. These data suggest that a persistent macrophage based reservoir of HIV may contribute to ARL.  that the murine mammary adenocarcinoma cell line TS/A, a highly  malignant MHC-II-negative tumor, is rejected in vivo if genetically  engineered to express MHC-II molecules by transfer of the MHC-II  transactivator CIITA. TS/A-CIITA cells are fully rejected by 93% of the syngeneic recipients and have a significantly lower growth rate in the remaining 7% of animals. Rejection requires CD4+ and CD8+ cells. CD4+ T cells are fundamental in the priming phase, whereas CTLs are the major anti-tumor effectors. All tumor rejecting animals are protected against rechallenge with the parental TS/A tumor. Immunohistochemical data at day 5 post-inoculation showed an higher infiltrate of CD4+ T cells in mice bearing TS/A-CIITA, than in mice bearing the TS/A tumor. Subsequently, from day 7 trough day 10, TS/A-CIITA tumors showed higher number of both CD4+ and CD8+ cells, dendritic cells, together with massive necrosis. The frequency of IFN--secreting splenocytes early after inoculations was also assessed by an ex vivo ELISPOT assay. Only the rejecting TS/A-CIITA animals showed an high frequency of IFN--secreting cells (between 80 and 120/10 6 splenocytes). Importantly, CD4 and CD8 depletion experiments revealed that at the time of tumor resolution the major cell population recognizing the TS/A-CIITA cells was of CD4 origin. This is the first example of successful tumor vaccination by genetic transfer of CIITA. These results open the way to a possible use of CIITA for increasing both the inducing and the effector phase of the anti-tumor response. To detect asymptomatic subjects with acute HIV infection (before seroconversion) we developed a cross sectional detection strategy. We tested 109,000 samples over 9 months at public testing sites in North Carolina. We found 606 people with undiagnosed HIV infection, including 23 with acute HIV infection (HIV antibody negative, serum RNA positive); the majority of subjects were screened in STD clinics. The median blood HIV concentration was 209,000 copies/ml, more than 10 times higher than in subjects with established HIV infection. We conducted two similar studies in our STD Clinic in Lilongwe, Malawi. Between 1 and 2% of subjects had acute HIV infection and the median blood viral load was greater than 106 copies/ml. We also found very high concentrations of HIV in the genital tract secretions of these subjects, and in patients with untreated STDs. Viral diversification was observed in serial samples from subjects with acute HIV infection after 11 weeks. The viral load in genital tract decreased more rapidly and to a greater extent than in blood. These results suggest that successful HIV prevention will require increased focus on the most infectious subjects, and Vaccines based on plasmid DNA (pDNA) or recombinant vesicular stomatitis virus (rVSV) vectors elicit HIV-1-specific cellular and humoral immune responses in small animal models. However, hurdles remain with each of these vaccine modalities before they can be considered for widespread clinical use. For pDNA vaccines, early clinical studies indicate that additional measures are needed to improve immunogenicity. For rVSVbased vaccines, issues related to the potential neurovirulence of the prototype vector represent a safety concern. Adjuvant development and pDNA vector optimization are two important elements of current DNA vaccine research. Cytokine expression plasmids may function as potent adjuvants, and in fact, we have observed that plasmid-encoded IL-12 can substantially enhance immune responses in non-human primates. In addition, it is hypothesized that broad immune responses against multiple HIV antigens will be required to protect against infection and/or disease progression. Results from studies will be presented in which several pDNA vaccine designs were tested for their ability to elicit broadly reactive immune responses to multiple HIV antigens. To address safety concerns related to use of rVSV vectors in humans, a range of further attenuated vector candidates has been developed and screened for neurovirulence in small animal models and nonhuman primates. Attenuated vectors have been identified that cause minimal CNS lesions after intracranial inoculation similar to those seen in control animals. Importantly, a number of these further attenuated vectors retained levels of immunogenicity in mice equivalent to prototype rVSV vectors, and have been advanced into nonhuman primate immunogenicity studies. To overcome the problem of pre-existing anti-Ad5 immunity, rAd vectors are being developed from rare Ad serotypes such as Ad35. However, studies have suggested that rAd35 vectors are less immunogenic than rAd5 vectors. We therefore constructed novel chimeric rAd35 vector incorporating the Ad5 fiber knob (rAd35k5). Both rAd35 and rAd35k5 vectors proved immunogenic in mice with and without anti-Ad5 immunity. In rhesus monkeys, rAd5 vectors elicited potent Gag/Env-specific immune responses, but a homologous boost immunization failed to enhance these responses as a result of high Ad5-specific NAbs. The rAd35 vectors elicited lower antigen-specific immune responses as compared with rAd5 vectors, but these responses increased substantially following a homologous boost immunization, consistent with lower vector-specific NAbs induced in these animals. Interestingly, rAd35k5 vectors elicited antigen-specific immune responses and vector-specific NAb titers that were between those induced by rAd5 and rAd35 vectors following the initial immunization. After the boost immunization, rAd35k5 vectors elicited potent cellular immune responses that were 2-3-fold higher than those elicited by both rAd5 and rAd35 vectors. These data demonstrate the immunogenicity of rare serotype and chimeric rAd vectors in rhesus monkeys. Moreover, these studies suggest that chimeric rAd vectors can be constructed to combine the beneficial immunologic and serologic properties of different Ad serotypes. Background: Gene-based vaccine delivery is an important strategy for induction of T cell responses that may be critical for a successful AIDS vaccine. Despite promising results in animal models, evidence of immune responses to DNA and rAd vaccines in humans has been limited. Materials and methods: Three Phase I studies have evaluated a series of DNA and rAd vaccine candidates expressing constructs encoding clades A, B, and C Envelope and clade B Gag and Pol with or without Nef, as fusion proteins or individually. Results: T cell and antibody responses are detected by IFN-ELISpot and FACS detection of intracellular IL-2 or IFN-in the large majority of vaccinees. Env peptide pools elicit the strongest response, but the 6-plasmid and rAd product also induced robust responses to Gag, Pol, and Nef. Both T cell and humoral responses were dose dependent. The T cell responses induced by DNA are detectable for at least 52 weeks, and the pattern of cytokine expression evolves over time with fewer IFN-and more IL-2 producing T cells at one year. Conclusion: DNA and rAd5 vaccine candidates are well tolerated and induce broad, durable immune responses. The combination will be tested in Phase II trials beginning 4Q2005. treatment naïve HIV-1 infected individuals with > 400 CD4+ T cells/ml for at least 5 years including 9 patients with low viral load (VL, < 5000 copies/ml) and 8 with high VL (> 5000 copies/ ml). HIV-1 specific IFN--production and cytolytic activity were higher in subjects with low VL. The differences between the two groups were statistically significant for CD4+ T-cell responses to Gag and Nef peptides, tested by a long-term (48 h) ICS assay and of border-line significance for the Gag-specific cytolytic responses measured by a flow-cytometry assay and a chromium release assay. We also found a significant inverse correlation between VL and IFN--production by CD8+ T-cells in response to Gag as measured by ICS. The ELISpot IFN-response was not significantly different in patients with high and low VL. During a median follow-up period of 2.4 years, 6 of 8 subjects with high VL and 1 of 9 with low VL showed decreasing CD4+ T-cell counts, and ARV treatment was more frequently initiated in the former patient group (5 of 8 versus 1 of 9). The CD4 and CD8 T cell immune responses found to be associated with low VL and stable CD4 counts may be of importance for protection. In the giant struggle with the long chronic HIV infection the immune system has generated unique antibodies matured to neutralize a virus which has evolved to escape them. I will describe unique features of a CD4bs (m18), and two CD4i (m12, X5) antibodies selected from immune phage libraries developed from long-term nonprogressors with high levels of broadly neutralizing antibodies. The m18 H3 shows striking similarity to the Ig CDR2-like C'C'' region of the CD4 domain 1 which dominates the binding to gp120. The X5 H3 is exceptionally flexible -IgG X5 inhibits efficiently infections of cells with low surface concentrations of CCR5. M12 is the only HIV-specific antibody identified which does not express its light chain but still binds gp120 -it was engineered to a single domain antibody that neutralized isolates from different clades. Mei-Yun Zhang, Vidita Choudhry, and Ponraj Prabakaran from my group, and our collaborators X. Ji, P. Kwong, C. Broder, G. Quinnan, R. Blumenthal, D. Montefiori, and their groups contributed to these results.  The potent, persistent immunity needed to prevent HIV infection might be best achieved by priming cellular immunity with replicating vectors and boosting antibodies with optimally designed envelopes. Replicating Ad vaccines infect epithelial cells on mucosal surfaces and thus also elicit mucosal immunity. In chimpanzees, compared to non-replicating Ad vaccines, at the same or lower dose replicating Ad vaccines were better at eliciting cellular immunity and priming antibody responses. Mismatched envelope boosts induced broad neutralizing activity to diverse R5 viruses and cross-clade ADCC activity. Multigenic Ad-SIV vaccines and SIV envelope subunit boosts elicited strong protection in 39% of rhesus macaques challenged mucosally with SIV mac251 . Durability of protection against a second challenge was established in 73% of previously protected animals, associated with persistent cellular immunity. Induction of memory cells and broad, strong functional antibodies illustrates the promise of this prime-boost vaccine strategy.

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Retrovirology 2005, 2(Suppl 1):S67 The Tat protein has been recently explored as a prospective vaccine candidate against HIV-1 with broad, subtype non-specific action. A truncated version of Tat()Tat) with the basic loop, involved in immunosuppression, removed has been previously demonstrated as efficacious as the full-size Tat protein. We produced both full-size Tat and truncated _Tat in plants, including one edible species -spinach, thus simultaneously addressing problems of an inexpensive Tat production and a direct delivery through the mucosal route. We tested this oral delivery route in a mouse model. Tat and )Tat genes were assembled from a set of synthetic overlapping oligonucleotides, and subsequently cloned into a plant virus-based expression vector. Codon optimization allows production of up to 300-500 mg of Tat or )Tat antigen per 1 g of leaf tissue in spinach. Spinach plants inoculated with the Tat-producing constructs were  collected and fed to mice 7-14 days post inoculation with or  without mucosal adjuvants. Mice were fed with the Tatproducing or control vector-inoculated spinach. After 3  voluntary feedings, 1 week apart, 1 g per mice, no differences were detected in the growth rate or behavior of the animals fed with these two types of spinach. None of the animals developed measurable Tat antibodies. Challenge DNA vaccination with a homologous Tat-expressing construct was performed using a gene gun. Following DNA vaccination, however, mice previously receiving oral Tat with cholera toxin as an adjuvant, developed higher antibody titers to Tat than did the controls, with the titers peaking at four weeks post-vaccination. Thus, our data suggested that oral Tat primed for the development of Tat antibodies when mice were challenge-vaccinated with plasmid DNA for expression of Tat. Most of the HIV-1 vaccines under development contain multiple viral genes or proteins. As a result, many vaccine-recipients react positive in licensed HIV-1 detection assays. This will have negative impact on future efficacy trials of prophylactic HIV vaccines that require early detection of intercurrent HIV infections. It will also exclude all vaccinees from blood/plasma donations, and may contribute to other social harms. Therefore, it is important to design new strategies for vaccine trial participants that will clearly discriminate between vaccine-induced antibodies and true HIV-1 infection. We identified new HIV-1 epitopes that: 1) Do not contain important neutralizing or CTL epitopes, and can be omitted from future HIV vaccine candidates, 2) Recognized by antibodies from early HIV infected individuals, 3) Highly conserved among HIV-1 clades and subtypes. Using Phage Display libraries constructed from whole HIV-1 genome, combined with panning over antibodies from early seroconvertors, we identified new immunodominant epitopes, in the gp41 intracytoplasmic tail and in p6, which conform to the above criteria. These peptides were used for the development of new HIV-1 EIA. To date the assay specificity and sensitivity are at >99%. Based on reactivity of several well-characterized panels of seroconvertors it was demonstrated that these peptides could detect antibodies within 4 weeks of HIV-1 infection. Testing of diverse serum samples (>2100) from around the world supports the utilization of our assay in detecting antibodies from infected individuals with clades A, B, C, D, E, F, and multiple recombinants. Importantly, testing of sera from HIV-1 vaccine trials gave mostly negative reactivity in our assay while scoring positive in one or more of the currently FDA-licensed HIV-1/2 EIA kits. Furthermore, our assay detected intercurrent HIV infections among vaccine recipients in 4 different vaccine trials. This assay could be added to the HIV detection kits used in prophylactic vaccine trials and blood/plasma collection centers. There is growing interest in using antigens that replicate the envelope transition state structures that occur during HIV entry as vaccine immunogens. Three such immunogens have been developed -complexes between gp120 and sCD4 (CD4/gp120), gp120 and a human monoclonal antibody A32 (A32/gp120), and gp120 and a CD4 mimic molecule CD4M9, SCBaL/M9. Antigenic comparisons of these immunogens revealed key differences between these complexes. Coreceptor binding is 3-fold higher with the gp120/sCD4 over gp120/A32 and gp120/CD4M9 complexes. However, the CD4 induced epitopes (CD4i) recognized by 17b and FabX5 are expressed equally between all three complexes. 19e, which recognizes an epitope that is completely dependent upon CD4 binding (CD4d), binds to gp120/sCD4 but not to A32/gp120 or SCBaL/M9 complexes. Another CD4d epitope recognized by ED47 is similarly prominent in CD4/gp120 complexes but significant less so in A32/gp120 and SCBaL/M9. These data indicate that the antigenic features of the A32/gp120 and SCBaL/M9 are more consistent with a transition structure between unligated gp120 and the CD4/gp120. Chemical crosslinking can obscure these CD4i and CD4d epitopes. These antigenic differences may also explain the differences in the neutralizing antibody profiles generated by CD4/gp120 and A32/gp120 complexes in animal experiments.

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Toll-like receptors signaling. The VLP-loaded MDDCs are able to induce a primary and secondary response in autologous T cells, using an in vitro immunization assay. Moreover, the genomic transcriptional profile of VLP-activated DCs show, by gene microarray analysis, the upregulation of several genes involved in the immune response. Conclusion: Our results show that baculovirus VLPs activate MDDCs and may ''cross'' over to the endogenous pathway to gain access to MHC class I, inducing CD8+ cytotoxic T cells activation. The intra-cellular Toll-like receptors appear to be involved in this process; additional signaling pathways induced by VLPs in the MDDCs are currently under evaluation. These data give an insight into the mechanisms of the cellular immunity induced in vivo by VLPs, which may be extremely useful to optimize and modulate the immune response. Neutralising antibodies recognising membrane proximal epitopes of gp41 have been isolated from HIV-infected patients. Since these epitope domains are highly conserved, the corresponding antibodies 2F5 and 4E10 neutralise a broad range of HIV strains. Numerous attempts by several laboratories to generate 2F5/4E10-like antibodies by vaccination have failed, obviously because the conformation of the domain is difficult to reproduce. Recently we reported induction of neutralising antibodies against the porcine endogenous retrovirus (PERV) and the feline leukaemia virus (FeLV) by immunisation with their transmembrane envelope (TM) proteins p15E. In both retroviral TM proteins two epitope regions were identified, one located in the N-terminal part (designated E1) and the other located in the C-terminal part of p15E (E2). E2 is related to the 2F5/4E10-epitope, and is located opposite E1 when the TM envelope protein has folded. An E1 domain was identified in the C-terminal part of gp41 and two strategies were developed to induce neutralising antibodies against HIV. First, immunisation was performed with a hybrid protein based on p15E of PERV, containing the E1 domain and the E2 (2F5/4E10 epitope) domain of gp41 of HIV-1. Second, immunisation was performed with a hybrid containing the N-terminal backbone of p15E of FeLV and only the E2 (2F5/4E10 epitope) domain of gp41. With both strategies antibodies neutralising laboratory and primary strains of HIV-1 were induced. These strategies may be used to generate a vaccine inducing broadly neutralising antibodies to prevent or curtail HIV infection.  For years now, AIDS researchers have suspected that most investigations have been focusing on the wrong site, i.e. blood. In fact, it has been well known for over a decade that lymphoid tissue is the key viral replication site from the start of infection, and also a major reservoir and source of virus at all stages of the disease. Lymphoid tissue studies in humans have been impeded by difficulties in obtaining material and, first and foremost, in reiterating sampling. Recent findings in the SIV-infected macaque model pinpoint the intestinal mucosal immune system as a key site of viral replication/persistence and CD4 depletion, even in subjects with undetectable blood virus during therapy. These studies demonstrate that acute SIV and HIV infections are coupled with a dramatic and selective loss of memory CD4 T cells in lymphoid tissue, due to direct virus-induced cytolysis or immune-mediated mechanisms (Mattapallil JJ et al, Nature 2005; 434: 1093-7). Treatment strategies based primarily on blood viral load and circulating CD4 cell counts are hence misguided. These findings plead for HAART initiation as early as possible, and will also have implications for vaccine development. Although failure to recover infectious virus from these patients certainly does not reflect the elimination of latently infected cells, these data are encouraging in the search of new strategies combining HAART and immune interventions. Despite its major effect on plasma viremia, HAART initiated at the chronic stage of the disease is known to have several major drawbacks, i.e. its inability to achieve HIV eradication due to poor targeting of the reservoir of latent but replicationcompetent virus, plus the inadequate diffusion of antiretroviral drugs in the various anatomic reservoirs. For example, it has been clearly demonstrated that some molecules in the combination are unable to reach the central nervous system or genital tract well enough to block viral replication, hence potentially creating sanctuaries of resistant virus. More recently, parallels have been made between the notion of cellular resistance to antiretroviral drugs and the resistance of some cancer cells to drugs. This resistance can involve efflux molecules like P-glycoprotein (MDR-1) and Multi-drug Resistance Protein (MRP). We investigated the expression of MDR-1, MPR-1 and MRP-4 mRNA levels in PBMC and lymph node cells (LNMC) in 15 HIV-infected patients on long-term effective PI-based HAART regimens (plasma viremia <20 copies/ml) versus controls (HIV-uninfected, HIV-infected HAART-naïve, HIV-infected on non-PI-based HAART). MDR-1, MPR-1 and MRP-4 mRNA levels were measured by PCR related to GAPDH expression and results given in arbitrary units. We found MDR-1 mRNA expression in PBMC to be much higher in HIV-infected but naïve patients than HIV-negative controls (46.64 vs 0.24), and higher in treated versus untreated patients, with no differences according to therapy type (no PI: 210.54, PI: 348.96). Surprisingly, MDR-1 expression in LNMC from PI-treated patients was significantly lower (0.97) than in paired PBMC. MRP-1 and MRP-4 expression showed significantly higher expression in HIV-treated versus HIV-negative patients, with no differences according to therapy type, and similar expression in HIV-infected untreated patients versus HIV-negative controls. No differences in MRP-1 and MRP-4 expression were found in LNMC and PBMC taken from the same patients on HAART. These advances should help us design new therapeutic approaches for HIV, capable of overcoming the reservoir problem.  CDNA synthesized from viral RNA was PCR amplified and sequenced with an ABI 3100 sequencer. Results: Over 60 strains were analyzed and all but one was subtype B. Phylogenetically there were no island-specific or Caribbean-specific genetic clusters, though a majority of new TT samples retained the signature threonine deletion in the V3 loop. One sample from DR was a unique BC recombinant having gag and env from subtype C and pol from subtype B. Glycosylation patterns in the Caribbean gp160's were different from those of other subtype B strains from the epidemic. Conclusion: The genetic isolation of the TT strains from the mid-1990's is no longer visible in the most recent samples, though the majority still retains the earlier signature in V3. Glycosylation differences between the Caribbean envs and those in North America may relate to natural selection of the Caribbean virus for heterosexual transmission. Early events leading to HIV infection across the mucosa likely involve HIV capture by a wide variety of molecules on the surface of epithelial cells and leukocytes followed by infection of permissive target cells within the tissues. In studying this biology, we are focusing on the contribution of dendritic cells (DCs) and T cells to these events and exploring effective ways to block these complex events in vitro and in vivo. Earlier work confirmed that there are at least two phases of DC-driven transmission of virus to T cells -one involves virus captured by (but not infecting) DCs that is handed directly over to the T cells and the other involves DC infection and the transmission of newly synthesized viruses. Virus captured by DCs is transmitted to CD4 T cells moving rapidly across the synapse naturally created between DCs and T cells. Inclusion of the fusion inhibitor T1249 reduces the amount of virus movement to the T cells, while increasing the amount of virus accumulating in the DCs. The immunologic and virologic consequences of this are under investigation. These data highlight how only blocking certain pathways of virus-DC interactions are suboptimal in preventing DC-driven HIV spread. As a result, additional studies are being performed to test the ability of more broad-acting carrageenan-based formulations for their ability to impede the complex virus-cell interplay needed to facilitate transmission. Carrageenan-based microbicides are promising due to their wide rage of activity against HIV/SIV and other sexually transmitted pathogens. Carrageenans impair virus capture by DCs in vitro and block infection of permissive DC-T cell mixtures. Recent in vivo data revealed that macaques were protected against vaginal SHIV challenge by carrageenan-based microbicides. These data are encouraging for future application of carrageenan-based formulations in preventing HIV spread. Heterosexual transmission through mucosal surfaces is one of the most common routes of HIV-1 spread. Topical microbicides are self-applied prophylactic agents, used to prevent vaginal and other mucosal transmission of HIV-1, which have the advantage of empowering vulnerable receptive partners to take effective measures for their own protection. In a search for candidate topical microbicides we discovered that retrocyclin, a unique -defensin synthetically constructed based on sequence data from its pseudogene, can potently prevent infection of CD4+ cells by both X4 and R5 HIV-1. While many studies have utilized simulants of mucosal fluid to test compounds, we are studying how whole human vaginal fluid and its functional components affect the activity and stability of peptide-based microbicides. We explored a novel, physiologically relevant approach to assess the ability of a candidate retrocyclin microbicide, RC-101, to inhibit HIV-1 infection of immunocompetent human cervicovaginal tissue. We revealed that 1) using a novel proteomic approach, human vaginal fluid contained at least 20 different cationic (poly)peptides with purported roles in innate host defense, 2) the cationic polypeptide fraction of vaginal fluid was required for innate anti-HIV-1 activity, 3) RC-101 retained full anti-HIV-1 activity in the presence of whole human vaginal fluid, 4) when applied apically to organotypical cervicovaginal epithelium, RC-101 was retained in the tissue, and 5) RC-101 prevented HIV-1 infection of immunocompetent organotypic cervicovaginal epithelium. Collectively, we have characterized the innate antiviral host defense factors within vaginal fluid, and developed a highly relevant ex vivo vaginal model suitable for peptide-based microbicide evaluation.

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CV-N dramatically decreases infectivity of CCR5-tropic HIV BaL and CXCR4-tropic HIV IIIB in vitro. We further demonstrate that this strain is genetically stable and can transiently colonize animal vaginal mucosa, while retaining important characteristics of the native bacterial phenotype. Conclusion: This live microbicide represents a novel approach in the development of an inexpensive and stable protein-based microbicide to curtail the HIV/AIDS pandemic worldwide. Starpharma focuses on the use of dendrimers as drugs in their own right -in contrast to dendrimers as drug delivery vehicles or diagnostics. Dendrimers offer a unique platform for exploring chemical diversity on the nanoscale and the production of dendrimer libraries covering a diverse array of macromolecular structures can be used in drug discovery and development. One pharmaceutical application of dendrimers that Starpharma is pursuing is the development of microbicides for the prevention of HIV and sexually transmitted infections (STIs). This presentation will describe the dendrimer drug discovery and lead candidate selection process from which SPL7013 emerged as a microbicide development candidate. Pivotal preclinical data will be presented that lead to Starpharma submitting an  Heterosexual RAI is common, with rates from 5-10% in the general population of women with up to 30-50% among women with other risk for HIV infection. RAI is considered to be the most common route of HIV transmission in men who have sex with men (MSM). Because of this broad and continuing HIV risk, topical HIV rectal microbicides are being developed to prevent rectal sexual transmission of HIV. The environment of the rectosigmoid region of the colon is very different to the vagina, serving different physiological roles and, as a consequence, have unique and distinct features that complicate microbicide development. In contrast to the robust, stratified squamous, epithelium lining the vagina, the intestinal mucosa is lined by a single layer of columnar epithelium that is very vulnerable to trauma or potential microbicide toxicity. Moreover, the gut associated lymphoid tissue (GALT), underlying the mucosal surface, has a physiological level of inflammation perfectly suited to HIV replication. The limited anatomical space of the vaginal cavity contrasts with the relative enormity of the intestinal colon and raises profound questions about drug delivery and distribution. Despite these significant challenges, recent animal studies have shown the feasibility of preventing rectal SIV transmission using a locally applied microbicide. Collaborative efforts are underway to begin to address these divergent needs in developing a safe rectal microbicide (RM). These include a recently concluded trial defining safety parameters (HPTN 056) and a focused NIH U19, now ending Year 1, with a targeted 5-year goal of initiating Phase 1 RM study. Failure to acknowledge the role that RAI plays in the global AIDS pandemic is likely to limit the success of intervention strategies and, more specifically, compromise attempts to develop safe and effective vaginal microbicides. Background: Co-infection of HIV with HVB and C is well described in medical literature from all over the world. The coexistence of these infections can worsen the evolution of anyone of them, therefore a fast diagnosis and treatment can help to achieve a better prognosis. Malvinas Argentinas County is a low resources seting located in NW of Buenos Aires Province. Objective: Determining the existence of co-infection with HIV and HVB and/or HVC in patients (p) included in the Municipal Program Against HIV and establishing the characteristics of the infected population. Methods: Prospective study of HIV+ p regarding the presence of positive serology for HVC (ELISA), HVB (determination of HBsAg) or both. Data obtained: Sex and risk factor for HIV infection (heterosexual, homo/bisexual, intravenous drug use (IDU). Results: Of 162 p, 15 were HVC+ (9.3%), 13 HbsAg+ (8%) y 2 HVC/HbsAg+ (1,2%). 13 HVC+ were male (87%) (p = 0.007), and among them, risk factor for HIV acquisition was IDU in 9 cases (60%) and heterosexual risk behaviour in 6 (RR 12 -IC: 4,83<RR<29.79; p0.0005). 11 HbsAg+ were male (84.5%) (p = 0.005) and among them risk factors for HIV acquisition were heterosexual risk behaviour in 5 cases, homosexual risk behaviour in 6 cases and IDU in 2 cases, none of this were significant as relative risks. Conclusion: Male sex was a significant relative for coinfection on HIV with HBV and HVC, maybe related to IDU in this population. IBU as a risk factor for acquisition of HIV was signicant for coinfection with HCV. Warning about HCV coinfection should be adopted in this improve the prognosis of both infections. Topical microbicides are considered an affordable choice for the prevention of sexually transmitted diseases in women. We have developed a comprehensive testing program for preclinical microbicide development. Over the years, we have tested several thousand compounds for use as topical microbicides in a series of cell-based assays addressing HIV-1 efficacy and toxicity. Recently, we compared historical data of the spermicide nonoxynol-9 (N-9) in a multi-center study and found that the HIV-1 efficacy paralleled its toxicity. Intra-assay, inter-assay, and inter-laboratory variability for toxicity were remarkably consistent. In a recent clinical trial, N-9 was found to enhance HIV-1 infection, thus confirming the preclinical toxicity data. In addition to N-9, lemon and lime juices have been proposed and used as contraceptives and were recently shown to exhibit in vitro activity against HIV-1. Therefore, we tested freshly prepared lemon juice, lime juice, and household vinegar (concentration = 100%) for HIV-1 efficacy and toxicity and for effect on beneficial Lactobacillus species. In all assays, the therapeutic index was <10, due to toxicity of the juices and vinegar to cells (mean TC 50 of lemon juice = 5.6%, mean TC 50 of lime juice = 4.9%, and mean TC 50 of vinegar 0.1%). Ten percent lemon or lime juice were not toxic to beneficial Lactobacillus species, in contrast to 10% vinegar which was highly toxic. Our preclinical data indicate that candidate topical microbicides should be moved forward into clinical trials with caution.  Results: The optimized CV-N expression cassette was stably integrated in single copy into the lactobacillus bacterial chromosome, and resolved from extraneous plasmid DNA and antibiotic resistance determinants. The L. jensenii-expressed CV-N dramatically decreased CCR5-trophic HIV BaL infectivity in vitro, with an IC 50 of 0.3 nM. Histological examination of CD1 mice, which were intra-vaginally inoculated with L. jensenii expressing CV-N, revealed that L. jensenii was associated with keratinized epithelium present during estrus or free in the vaginal lumen and secreted full-length CV-N in vivo. Conclusion: This live microbicide represents a major step towards developing inexpensive, durable protein-based microbicides to address the urgent need for female-controlled approaches to block the heterosexual transmission of HIV.

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Retrovirology 2005, 2(Suppl 1):S94 Background: We have previously shown that the tripeptide glycyl-prolyl-glycine-amide (GPG-amide) inhibits HIV-1 replication in vitro by affecting proper capsid assembly of HIV-1. However, GPG-amide failed in a phase II clinical trial on HIV-infected individuals. In the search for what went wrong we have now found that the tripeptide in itself does not exert the antiviral activity but is metabolised in two steps into the active compound by serum enzymes. The first step is cleavage of GPG-amide into GP and glycine-amide (G-amide) by the soluble di-peptidyl peptidase CD26. In the present study we show that G-amide is further metabolised to the actve antiviral substance by an enzyme present in foetel bovine serum but not in human serum. Material and Methods: Numerous methods have been employed including molecular biology, magnetic resonance (NMR). Results: The second step is an enzyme mediated oxidation of G-amide into the active anti-viral compound. By NMR the molecule was found to be alpha-hydroxy glycine amide (alphaHGA). The latter is a small molecule with a molecular mass of 90. The conversion of G-amide into alphaHGA does not take place in human or rodent serum but in the serum from most species including fetal calf and pig serum. Hence, GPG-amide does not affect HIV-1 replication if the infected cells are cultured in the presence of human serum only. We have now synthesized alphaHGA and been able show that the synthesized substance inhibits HIV-1 replication in the presence of human serum only or with heat inactivated fetal calf Factors relevant to potential changes in microbicide efficacy include the presence of mucins within the cervical mucus. We hypothesize that polycationic PEHMB molecules will interact with the anionic mucin molecules to form a lattice-like network that serves as a physical barrier to the movement of infectious virus and HIV-1-infected cells to the cervical and vaginal epithelia. In vitro experiments demonstrated that the anti-HIV-1 activity of PEHMB was increased almost two logs in the presence of mucin. In contrast, the activity of anionic dextran sulfate was unaffected. These results suggest that electrostatic interactions between PEHMB and mucin molecules may augment the inherent anti-HIV-1 activity of PEHMB by facilitating the formation of a physical barrier between HIV-1 and susceptible cells. This property would be expected to increase the in vivo efficacy of PEHMB. The remarkable successes achieved using combination therapy to treat systemic human immunodeficiency virus type 1 (HIV-1) infection suggest that combination microbicides, which include two or more active ingredients, may also provide a particularly effective means to prevent HIV-1 transmission. We have recently identified the compound PSMA, an alternating copolymer of polystyrene (PS) and maleic anhydride (MA), as a potential partner for our candidate microbicide polyethylene hexamethylene biguanide (PEHMB), a member of the polybiguanide family of compounds. In vitro studies of PSMA demonstrated that this compound is minimally cytotoxic and highly effective against both macrophage-and T cell-tropic strains of HIV-1. We hypothesize that the dissimilar mechanisms of action of PSMA and PEHMB may provide additive or synergistic activity against HIV-1. Experiments are now underway to identify optimal combinations of PSMA and PEHMB to be used in experiments to assess toxicity, anti-HIV-1 activity, and formulation strategies. These investigations will be used to confidently advance the preclinical development of PSMA and a combination microbicide containing both compounds toward human trials. Background and Objectives: Mycoplasma pneumoniae has been implicated with community-acquired pneumonia and mild to severe respiratory infections in the normal population. The prevalence of this mollicute in HIV infected patients has never been reported from India. Mycoplasmas have also been reported to act as cofactors in AIDS progression. Aims and Objectives: a) To divulge the incidence of Mycoplasma pneumoniae and other respiratory pathogens in the respiratory specimens of HIV infected patients. b) To compare the sensitivities of induced sputum and throat swab specimens for detecting Mycoplasma pneumoniae. c) To correlate the various haematological and immunological findings with infection due to M. pneumoniae in the HIV infected patients tested.

Materials and methods:
The present study has been carried out on 60 HIV infected patients presenting with underlying pulmonary complaints and whose clinical presentation was consistent with disease caused by Mycoplasma pneumoniae, after obtaining informed consent subsequent to approval by the Institutional Review Board (IRB) on human ethics in Chennai where the recovery rates of Mycoplasma pneumoniae from induced sputum and throat swab specimens of HIV infected patients were compared and the haematological and immunological findings were analysed. Patients screened were from the age groups ranging from 15 to 60 years, whose respiratory specimens were cultured on PPLO glucose agar and broth, the later with 1% methylene blue. Presumptive identification of Mycoplasma pneumoniae was carried out using guidelines The use of vaginal microbicides has gained support as a strategy for the protection of women against HIV-1 and other sexually transmitted disease (STD) pathogens. The preclinical Swiss Webster murine model has been developed to specifically measure cervicovaginal tissue integrity and inflammation following application of candidate vaginal microbicides, when potential exposure to an STD pathogen may occur. This model demonstrates both mechanistic and temporal differences in inflammatory responses following microbicide exposure. Currently, specific markers of inflammation, including proinflammatory cytokines, are being evaluated in the cervicovaginal mucosa. Safety profiles of polybiguanides (PBGs), which demonstrated significant in vitro efficacy against HIV-1, are being assessed in vivo. Intravaginal application of PEHMB (1%) resulted in little or no cervicovaginal toxicity after short-or long-term exposure. Collectively, these studies support the Swiss Webster model as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides. Furthermore, these results strongly support further development of polybiguanide derivatives as vaginal microbicidal agents. circulation. Also, the role of CTL avidity in mucosal AIDS viral transmission is unknown. To address these questions, we used delay in acute-phase peak viremia after intrarectal challenge as an indicator of systemic dissemination. We find that a peptide-prime/poxviral boost vaccine inducing high levels of high avidity mucosal CTL can impact dissemination of intrarectally administered pathogenic SHIV-ku2 in macaques, and that such protection correlates better with mucosal than with systemic CTL and particularly with levels of high avidity mucosal CTL.

S101
Abstract withdrawn Background: Previously, we employed site-directed mutagenesis to introduce HIV-1 4E10 and 2F5 epitopes into the corresponding region of a functional HIV-2 envelope glycoproteins (AIDS Vaccine 2005, abstract #A210). The resulting ''chimeric'' viruses were used to screen HIV-1 infected human plasma for 4E10 or 2F5-like neutralizing antibodies (Nabs). Among 177 subjects infected with HIV-1 representing ten different subtypes or circulating recombinant forms, none had significant Nab titers directed toward these epitopes. Materials and methods: Here, we tested the same HIV-1 positive plasma specimens for neutralizing activity against chimeric HIV-2 viruses in which we substituted the complete 25 amino acid HIV-1 MPER (designated clone C1) or non-overlapping amino-terminal or carboxy-terminal portions of it (designated C3 and C4, respectively) using a single-cycle infectivity assay (Nature 422:307, 2003). Results: HIV-2 viruses containing the C1, C3 or C4 MPER, and the parental virus HIV-2/7312A, were infectious and equally susceptible to neutralization by T1249, sCD4, the anti-HIV-2 Env mAb 1.7A, and polyclonal anti-HIV-2 antibodies. This result demonstrates that none of the chimeric HIV-2 viruses was ''globally sensitive'' to neutralization. Surprisingly, 60 out of 165 (36%) of HIV-1 plasmas tested contained MPER specific Nabs. IC50 titers ranged from 0.0005-0.02 (mean 0.006; median 0.004; standard deviation 0.004). Anti-MPER Nab reactivities were mapped to C3 (6 subjects) or C4 (14 subjects) regions of the HIV-1 MPER or to epitopes spanning them (13 subjects). None of the 60 subjects with Nabs to C1, C3, or C4 had antibodies that neutralized HIV-2 viruses containing 2F5 or 4E10 epitopes only.

Conclusion:
These results indicate that the MPER of HIV-1 elicits Nab responses in a substantial proportion of infected patients and that the epitopes recognized by these Nabs are distinct from those recognized by 4E10 or 2F5.

S103 Abstract withdrawn
Retrovirology 2005, 2(Suppl 1):S103 HIV-1-specific CD8+ T cells in primary infection are associated with the dramatic decline of peak viremia to the viral set point, while their antiviral activity in chronic infection is less apparent. Here, we comparatively analyzed functional properties of HIV-1-specific CD8+ T cells in primary and chronic infection, and demonstrate that the functional avidity and TCR affinity of HIV-1-specific CD8+ T cells was consistently higher in primary infection than in chronic infection. The change of TCR affinities between primary and chronic infection was linked to an almost complete switch in the clonotypic composition of epitope-specific CD8+ T cells, resulting from the preferential loss of high-avidity CD8+ T cell clones. These data suggest that the initial recruitment of high-avidity HIV-1-specific CD8+ T cell may contribute to the control of HIV-1 viremia during primary infection, while their selective elimination during the subsequent disease process contributes to the loss of immune control during chronic infection.

S105 Abstract withdrawn
Retrovirology 2005, 2(Suppl 1):S105 S106 Cytotoxic T lymphocytes (CTL) induce target cell apoptosis when they release perforin (PFN) and granzymes (Gzm) into the immune synapse. PFN delivers Gzms into the target cell, but how PFN does this has been unclear. Because PFN forms pores in the plasma membrane, Gzms were originally thought to enter target cells via these pores. However, these pores were too small to allow even small dyes to disseminate into the target. Here we find that PFN dramatically perturbs the target cell membrane, creating pores that transiently allow Ca ++ and small dyes into the cell. However, the membrane is rapidly resealed and the dyes are circumscribed within membrane proximal blebs. The Ca ++ flux triggers a wounded membrane repair response in which internal vesicles, including lysosomes and endosomes, donate their membranes to reseal the damaged membrane. The target cell actively participates in determining its own fate during cell-mediated death. The target cell membrane repair response is necessary for target cells subjected to CTL attack to avoid necrosis and undergo the slower process of programmed cell death. However, Gzms do not pass into the cell via PFN plasma membrane pores. Instead PFN triggers the Background: Although human T cells enter the peripheral lymphoid tissues early during fetal development 1 , the adaptive immune system in the fetus has largely been regarded as functionally immature and unresponsive to stimulation. In adults, CD4CD25high regulatory T cells (TReg) are critical for maintenance of peripheral T cell tolerance, but their role in the developing fetus is unknown. Here, we demonstrate that a large population of human fetal FOXP3CD4CD25high TReg cells, present from the earliest stages of T cell colonization of the periphery, efficiently suppresses fetal T cell responses.
Results: Depletion of CD4 + CD25 high T Reg cells from fetal lymph node cells, but not adult lymph nodes, resulted in the proliferation and acquisition of effector functions in the absence of exogenous stimulation by a large subpopulation of T cells identifiable by the expression of CD69 in utero. A large population of fetal CD4 + CD25 high T Reg cells also expressed CD69+ and displayed a memory/effector phenotype, as indicated by low expression of CD45RA and CCR7. However, the CD69+ and CD69-CD4 + CD25 high T Reg cells did not differ in their suppression of T cell responses in the absence of exogenous stimulation, indicating that the activation status of these cells do not correlate with their suppressive function.
Conclusion: These studies demonstrate that the fetal T cells are, in the absence CD4 + CD25 high T Reg cells, highly responsive to stimulation, indicating that human fetal T cells are active and functionally mature. Strong evidence has also been obtained for an important role for CD4 + CD25 high T Reg cells in controlling T cell responses in utero. The implications of these findings for pediatric HIV infection will be discussed. Like HIV-infected humans, simian immunodeficiency virus rapidly and selectively infects, replicates in, and destroys memory CD4+ T cells co-expressing CCR5 (viral ''target'' cells) resulting in loss of the majority of the bodies CD4+ T cell pool within 21 days of infection. The vast majority of these cells reside in the intestinal tract and other mucosal tissues but selective loss of these target cells is detectable throughout the lymphoid system. Restoration of memory CD4+CCR5+ T cells directly correlates with improved clinical course and lower viremia, but these cells are never restored in macaques that progress to AIDS. Continuous and effective antiviral treatment initiated within days of SIV infection can rescue mucosal CD4+ T cells, but delaying therapy for a couple of weeks does not restore these vital helper memory cells, despite effective control of viremia. Similarly, monkeys that ''appear'' protected in vaccine challenge studies may in fact harbor smouldering infection in the intestine with continuous CD4+ T cell loss, despite undetectable plasma viremia. The rapidity and severity of the loss of memory CD4+ T cell function is likely the major reason no cure or vaccine is in sight. In fact, converging evidence suggests that other primate species changed fundamental properties of their immune system, such as eliminating the need for CD4+CCR5+ T cells (yet maintaining CD8+CCR5+ T cells), rather than cope with this subversive infection. This and other data suggest that conventional immune responses simply may not be adequate to control or prevent HIV infection. presence and level of viremia. In viremic individuals we also have characterized a population of aberrantly activated CD56-NK cells that manifest lower-than-normal expression of Natural Cytotoxicity Receptors and a normal or increased expression of inhibitory NK receptors. This dichotomy is reflected in decreased cytotoxic function and decreased secretion of TNF-alpha and interferongamma. In addition, NK cells from viremic individuals express Fas on their surfaces at significantly increased levels, and are more susceptible to apoptosis upon exposure to sFASL. Furthermore, viremia impacts the NK cell-dendritic cell interactions that are critical to normal immune responsiveness. Taken together, our data indicate that HIV pathogenesis involves both direct and indirect effects of HIV and a relentless cycle of aberrant immune activation that drives the disease process in HIV-infected individuals.

S110
Abstract withdrawn MHC class I-specific inhibitory receptors are expressed by a subset of memory-phenotype CD8+T cells. Similar to NK cells, MHC class I-specific inhibitory receptors might subserve on CD8 T cells an important negative control that participates to the prevention of autologous damage but may also contribute to viral escape. We found that the expression of CD94 and KIRs is increased on CD8 T cells from HIV patients, and the accumulation of CD94 +CD8+ T cells is driven by HIV replication.The expression of iNKR was found associated with a poor cytokine response (IFNg and TNFa) upon TCR triggering, not restored by IL-15. The expression of CD94 and KIRs on HIV-and CMV-specific CD8 T cells was investigated with specific tetramer staining, and these NKR were barely detectable at the surface of virusspecific T cells, in contrast to CD85j. Characterization of the maturation stage of CD85jCD8 T cells, and of the impact of CD85j on CD8 T cell response upon TCR triggering with HIVspecific peptides is currently under investigation. HIV infection results in an early and profound loss of Vg2Vd2 T cells, and this is the only T cell receptor-specific depletion that is common to all individuals with HIV/AIDS. A similar pattern of Vg2Vd2 T cell depletion occurs in mycobacterium infection and during malaria. We hypothesize that the loss of Vg2Vd2 T cells, irrespective of the primary cause for this loss, results in disease acceleration during HIV+ tuberculosis or HIV+ malaria coinfections and also leads to increased incidence of cancer in the context of HIV/AIDS. Using a macaque model for mycobacterium infection, we demonstrated the dynamics of Vg2Vd2 responses to infection with attenuated M. bovis (BCG). In this study, we confirmed that activation-induced cell death is the mechanism for Vg2Vd2 T cell depletion in vivo, and confirmed this with in vitro studies using human T cells. In vitro studies with human PBMC allowed us to understand the specificity of T cell receptor recognition of human lymphomas. Using AIDS-related and non-AIDS related B NHL, we defined the T cell receptor structures required for tumor recognition and showed they are indeed, missing in HIVinfected individuals. Lastly, we evaluated longitudinal specimens from HIV-infected individuals receiving HAART and showed that recovery of the Vg2 repertoire was occurring by the use of previously rare sequences that survived initial HIV-mediated depletion and were expanded during the treatment interval. Importantly, repertoire recovery occurred in the absence of new cell synthesis, consistent with observations on CD4 and CD8 T cell repertoire and changes during HIV infection and treatment. The Vg2Vd2 T cell subset is an example of indirect or bystander cell killing during HIV infection, and its impact on immunity to seemingly unrelated pathogens. Destruction of Vg2Vd2 T cells and the critical elimination of the Vg2-Jg1.2 expressing subset, likely accounts for the mutual acceleration of HIV, malaria and tuberculosis diseases, and may explain the specific of enhanced risk for AIDS-related neoplasia. We continue efforts to comprehend this unusual T cell subset both as a model for the Retrovirology 2005, 2(Suppl 1) http://www.retrovirology.com/supplements/2/S1 impact of HIV in host immunity, and to define new targets for

S112
Retrovirology 2005, 2(Suppl 1):S117 Background: HIV-1 envelope glycoprotein (Env) is the primary target for inducing neutralizing antibodies against the virus Env yet only a small fraction of antibodies elicited are directed against conserved epitopes. Thus, the antibodies produced during infection ad vaccination (to date) have been limited in their ability to neutralize heterologous primary isolates. Since interactions between the virus and its receptor and co-receptor are critical for virus entry into the cell, targeting conserved functional epitopes located in or near the receptor and co-receptor binding sites may be the key for developing an effective vaccine. We as well as others have shown that Env-CD4 complexes are capable of inducing broadly neutralizing antibodies, however use of sCD4 as part of the vaccine has the potential for inducing an autoimmune response. Materials and methods: Therefore, we are evaluating several approaches, including such as CD4 peptide mimetics (CD4M33), small molecules and novel scaffolds such as invasin and tat (onto which the CD4 binding domain is grafted). This may facilitate targeting of conserved functional epitopes on liganded forms of Env and also reduce immune responses directed towards CD4 Results: We have developed and characterized stable Env-CD4M33 complexes and evaluated them in rabbits for inducing neutralizing antibody responses. In a parallel approach, we have used BMS-853 as a filter to identify 100 structurally similar small molecules, and screened them for their ability to compete with CD4 and b12 for binding to Env as well as their ability to induce conformational change as reflected by enhanced binding to 17b binding. We have so far identified three classes of small molecules that: i) compete for CD4 binding only, ii) induce conformational change in Env without competing for CD4, and iii) compete for CD4 binding and induce conformational changes.
Conclusion: We plan to use small molecules for stabilizing Env in liganded or un-liganded forms for further evaluation of immunogenicity in rabbits. These studies should yield important structural information about the apo and liganded structure of Env and the resulting exposure of conserved epitopes for vaccine applications.

S118
Characterization of gp120 and Its Single-chain Derivatives, gp120-CD4 and gp120-M9: Implications for Targeting  Single-chain derivatives of gp120 linked to the first two domains of CD4 (gp120-CD4 D12 ) or to the CD4 analogue CD4M9 were assessed for their abilities to elicit CD4-induced neutralizing antibodies. Both complexes showed binding to a CD4i epitope as defined by the mAb 17b. Addition of exogenous CD4 did not increase 17b binding to gp120-CD4 D12 but augmented binding to gp120-M9 perhaps reflecting the lower binding affinity of M9 to gp120 compared to CD4 D12 or suggesting that M9 does not completely fill the CD4 binding site of gp120. Vaccination of guinea pigs and rhesus monkeys with recombinant protein or DNA prime followed by protein boosting generated broadly neutralizing antibodies only for sera generated against gp120-CD4 D12 . Passage of these sera over a CD4 D12 affinity column removed neutralizing activities. Rhesus monkeys were also immunized with gp120-human CD4 or gp120-rhesus CD4 complex. Virus-neutralizing antisera were observed for each of these groups, but titers were much greater for the gp120-human CD4 complex. Neutralizing antibody titers showed a significant correlation to CD4 antibody titer for both vaccine groups. These data suggest that most neutralizing antibodies generated by gp120-CD4 complexes are directed against CD4.

S119
C3d Enhancement of Anti-Env Immunity Using Background: DNA vaccines expressing the HIV-1 envelope (Env) have been relatively ineffective at generating high-titer, long-lasting, immunity. Conjugating the molecular adjuvant, C3d, to HIV-1 Env enhances both humoral and cellular immunity. Methods: BALB/c mice were vaccinated with DNA plasmids (weeks 0, 4, and 8) expressing wild-type or modified envelope proteins. Each Env immunogen was tested alone or conjugated to multiple copies of the molecular adjuvant, C3d. Both humoral and cellular immunity were analyzed. Results: DNA vaccines expressing a fusion protein of the soluble human CD4 (sCD4) and the Env gp120 enhanced the immunogenicity of the expressed fusion protein only when conjugated to mC3d3. Monoclonal antibodies that recognize CD4-induced epitopes on Envgp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d 3 . In addition, both molecules bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice vaccinated with DNA plasmids expressing either gp120-mC3d 3 or sCD4-gp Retrovirology 2005, 2(Suppl 1) http://www.retrovirology.com/supplements/2/S1 120-mC3d 3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d 3 -DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d 3 had antibodies that elicited cross-protective neutralizing antibodies. In a separate set of experiments, the unique sequence found in the crown of the V3 loop of the envelope from the CD4-independent isolate, HIV-1 R2 , was used to elicit crossclade neutralizing antibodies. The codons encoding for the V3 loop amino acids, Pro-Met, were introduced into the sequences encoding the gp120 ADA (R5) or gp120 89.6 (R5X4). Mice vaccinated with gp120 ADA -mC3d 3 -DNA with the Pro-Met mutation had antibodies that neutralized HIV-1 infection, but not the gp120 89.6 -mC3d 3 -DNA.
Conclusion: Therefore, the use of sequences that expose cryptic epitopes by CD4 or found in CD4-independent viral isolates expose neutralizing epitopes that can elicit broad, crossclade immunity.

S120 Abstract withdrawn
Retrovirology 2005, 2(Suppl 1):S120 S121 Background: The narrow spectrum of HIV-specific neutralizing antibodies points to the need for new immunogens based on highly conserved epitopes. HIV-1 infects host cells by membrane fusion: during this process conserved epitopes are exposed on the viral glycoprotein gp120/41 that may be used as targets for the induction of antibodies against HIV-1. Neutralizing antibodies against different heterologous HIV-1 isolates may be obtained by immunizing mice with fusion complexes on which conserved epitopes have been stabilized by fixation, or with gp120/41s with a CD4-independent phenotype on which these conserved epitopes may be already exposed.
Methods: Fusion complexes were prepared using cells expressing gp120/41 cocultivated with cells expressing the receptors CD4-CCR5 at different temperatures, corresponding to intermediate stages of the membrane fusion process, and stabilized using different fixatives. Mice were immunized to reveal the induction of HIV neutralizing antibodies and their spleen cells used to generate hybridoma clones to be tested for the production of neutralizing monoclonal antibodies. In addition, syngeneic balb/c mouse cells expressing gp120/41 with a CD4-independent phenotype have been prepared by transfection and using viral vectors and are being used to assess their capability to induce broad spectrum neutralizing antibodies.
Results: Results obtained indicate that: 1) fusion complexes were immunogenic and induced neutralizing antibodies against R5 and X4 HIV-1 heterologous isolates; 2) extensive purification of antibodies allowed the removal of any aspecific cytotoxic effect; 3) complexes prepared at higher temperatures were more immunogenic and induced higher titers of neutralizing antibodies; 4) titer of neutralizing antibodies was not affected by the fixative used; 5) neutralizing activity was retained after CD4-CCR5 antibody removal; 6) CD4-independent gp120s were expressed in syngeneic mice cells and recognized by HIV-1 positive human sera; mice immunizations are currently ongoing.
Conclusion: Results show that fusion complexes are immunogenic and induce neutralizing antibodies against heterologous HIV-1 isolates. Removal of non-specific inhibitors that confused early promising results is necessary to obtain a specific antibody response. The production and selection of neutralizing monoclonal antibodies will be useful to identify specific immunogenic structures and epitopes to be used to induce neutralizing antibodies when administered in a suitable delivery system. Antibodies obtained by immunizing with CD4-independent gp120s and antibody fragments isolated through phage display libraries panning will be evaluated for their broad-spectrum neutralizing activity against different HIV-1 isolates with and without the addition of sCD4 or CD4-like antibodies. Although many mutations may also revert to wild-type, a danger is that they will have become permanently archived in a patient's long-lived memory Tcells, and that this may preclude future therapeutic options. At the same time, many of the mutations associated with HIV drug resistance may cause diminished ''replicative capacity'' and, indeed, some clinical studies have shown that at least some individuals infected with multi-drug resistant strains may have lower viral loads over periods of several years than do individuals infected with wild-type strains. It is also interesting that some of the mutations associated with HIV drug resistance Retrovirology 2005, 2(Suppl 1) http://www.retrovirology.com/supplements/2/S1 may be less easily transmitted than others or are present at diminished frequency in newly infected hosts. In general, it appears as though thymidine analogue mutations (TAMs), associated with resistance to zidovudine and stavudine, as well as mutations associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs), may be transmitted fairly efficiently while mutations associated with resistance to other nucleosides, e.g. 3TC, TDF (M184V and K65R), may be transmitted much less frequently. This subject has relevance in view of the widespread use of co-formulated nevirapine/3TC/stavudine (TRImune) as a first line regimen favored by the World Health Organisation (WHO) for use in many developing countries. There is a strong possibility that resistance might develop over time against several of the drugs in this regimen that have low genetic barriers for resistance, meaning that only a single point mutation in the reverse transcriptase gene may yield significantly diminished levels of antiviral activity (3TC and NVP). Nonetheless, we should support this WHO initiative (3 Â 5) because this regimen is likely to have the greatest impact and save millions of lives during the next several years and, as well, will impact on rates of HIV transmission.

S123 yesThe Complexities of ART Which Prevent Durable Viral Suppression
Anthony Amoroso Institute of Human Virology E-mail: amoroso@umbi.umd.edu Retrovirology 2005, 2(Suppl 1):S123 The complexities of ART which prevent durable viral suppression should not be under estimated, and include the need for absolute adherence, the rapid development of resistance, and the impact of cross resistance, unequal potency of ARV's, pharmacokinetic limitations, and drug toxicities. Providers of HIV care in the U.S. are faced with the increased complexities of care as they encounter growing numbers of patients with drug resistance. Reliance on frequent use of viral load monitoring and genotypic analysis has become the recommended norms.
As the world prepares for antiretroviral scale up programs in resource limited nations, the need to achieve durable antiretroviral therapy (ART) success across multiple different patient populations and to decrease the reliance on frequent viral loads and genotypes to gauge treatment outcomes has become more apparent. Treatment programs in clinics which serve patients in resource limited settings are trying to address these complexities to improve long term treatment success. The availability of the Antiretroviral (ARV) generic formulations since 2000 has increased dramatically the demand to access care and ARV drugs in the developping countries. Most of the subsaharian African and Asian countries which reported an epidemic have started to benefit from the various international supports.

S124
Although there is a global consensus among the developped countries to make the ARV access easier for the developping countries, the frequent lack of sustained structures and well trained medical teams in addition to the lack of facilities are at high risk of emergence of resistant viral strains and compromission of the expected benefits.
In Burma the ARV implementation and scaling up have considered the possible mechanisms to decline the risk of emergence of a rapid resistance to the ARVs. Burma one of the poorest country of the world has 50 millions of inhabitants. The UNAIDS estimates that the prevalence of the HIV infection is between 2 and 3% of the global population. It is the third country of the South East Asian region (SEAR) in term of prevalence after Cambodia and Thailand.
Retrovirology 2005, 2(Suppl 1) http://www.retrovirology.com/supplements/2/S1 The mix of high level of poverty, low awareness, poor health care, low political involvement, and total lack of governmental funds dedicated to this infection led the few skills to collaborate closely to set up a comprehensive programme aimed at fighting HIV/AIDS in this country prevented from accessing to most of international supports because of the political sanctions. The french government signed an agreement in 1996 with the Burma's medical school to traine MDs in France for HIV/AIDS. 6 out of them have already been trained. The french oil company Total joined this programme in 2004 to provide medical scholarships. The 7th MD that the Total company has supported is the first Burma's peadiatrician trained especially for HIV/AIDS in children in Paris. After their return, these medical leaders have been posted in the 2 referral HIV/AIDS departments implemented in the 2 main cities (yangon and Mandalay) with the tecbnical support of WHO. The Yadana company operated by Total in Burma has provided the financial support for the purchase of the ARVs which are ordered by WHO to avoid any custom fees, to ensure a regular supply and avoid shortages. The recipient for the Total funds is the Union: an Int'NGO well implemented in numerous developping countries in charge of Tuberculosis (TB). TB is the most frequent infection encountered in HIVpatients and occurs at any time of the disease progression. This choice is a major contribution, since it has provided for the first time the opportunity to detect and treat HIV patients at an earlier disease stage. Up to now the government allowed to perform the HIV testing only in patients with evident clinical diagnosis of AIDS. The benefit of the ARV treatment started at a late disease stage is very limited compared to the one when started at an earlier stage. This earlier HIV testing possibility is the first step toward the voluntary testing in asymptomatic people who just whish to know their own status. The HIV testing center has been implemented for the first time outside the walls of the AIDS center in the TB center. The test kits are provided by UNAIDS. It is the first time that the test is done anonymously in in a public place. The setting for the programme is the General Hospital in Mandlay, the second city of the country. All the criteria required to start the programme are met at this place. The patients are monitored by the MDs trained in France. The organization of the management of the patients has been set up by the Union which delivers the drugs to the hospital, supports the cost of the laboratories facilities as well as the cost of the management of the opportunistic infections. 2MDs from this IntNGO specialized in TB and in HIV in the developping countries are responsible for this program. This is the first free access to the ARVs and to the care for HIV infection in a public hospital in this country. The anonymous testing (approved by the government), the monitoring of the patients by the well trained MDs, the management of the programme performed by the MDs of the Union, the funds provided by Yadana company, and the support of WHO is an exemple of a global commitment.
One major objective of the programme is to limit the resistant viral strains which should start in Burma in patients followed up in the private sector where the drugs prescribed : monotherapy, bitherapy or tripletherapy are related to the shortages and supplies, to the variable medical knowledge and to the patient's financial resources. The success of this programme should drive the few thousands patients followed up in the private sector to the public sector providing since recently confidentiality and free access to the ARV treatment and care. This programme is in the frame of the WHO 3 Â 5 initiative and contributes to the sustained development. This rare example of a synergistic and close collaboration between different structures should help to find additional funds to join the programme and expand it to other hospitals in Burma.

S130
HIV/AIDS Treatment in Developing Countries: Guyana's Approach Leslie Ramsammy

Minister of Health
Retrovirology 2005, 2(Suppl 1):S130 Diagnostic and treatment tools for HIV are well known and are widely available today. Equitable accessibility to these tools is a major area of concern. The WHO estimates that about 7 M persons are in need of ARV therapy in low and middle-income countries around the world. Viral load, CD4 cell count, weight, haematological and chemistry parameters were noted at baseline. These parameters were again determined at 3 and 6 months after combined therapy of ARV and antihelminthic drugs.
Results: A general correlation exists between HIV plasma viral load and the number of excreted worm eggs in stool of studied candidates. In group 'A' individuals with complete eradication from helminths, there were significant reduction in HIV/AIDS related symptoms notably, pallor, abdominal pains, diarrhoea and mean increase in CD4 count. Also, a mean reduction of 0.32log 10 copies/ml of viral load, weight-gain, increase haematological and decrease chemistry parameters were generally observed at 3 and 6 months follow-up from baseline. A median increase of 0.14 log 10 copies/ml of viral load was observed among group 'B' candidates who were either persistently helminth-negative or helminth-positive at 3 and 6 months follow-up from baseline. Two of the patients under study were lost to full-blown AIDS among group 'B' candidates.
Conclusion: Results of this study showed that, the combine use of ARV and antihelminthic drugs in the treatment of HIV/ AIDS patients is safe, well tolerated and offers effective viral load suppression with attendant increase in immunological values. This therapeutic approach is of great relief for patients in developing countries when cost and accessibility to ARV drugs are considered.  The interaction of HIV-1 envelope glycoproteins with CD4 and coreceptors triggers a barrage of conformational changes in HIV-1 gp120 and gp41, which lead to the gp41 six-helix bundle formation that drives the membrane merger and eventual fusion. Although significant progress has been made in understanding HIV fusion, little is known about the cell biological processes that impact on HIV entry. Manipulating lipids has yielded important insights into the role that membrane of the host cell plays in regulating the HIV-1 entry process. In this talk I will describe how lipid manipulation affects HIV-1 entry by 1) altering the disposition and lateral mobility of HIV-1 receptors, 2) endosomal re-routing, and 3) cytoskeletal remodeling. Manipulation of lipid metabolism may therefore constitute a promising avenue for the development of antiretrovirals.   We explored therapeutic immunization of ART-treated SIV-mac251 infected rhesus macaques using a new generation of optimized DNA-based vaccine vectors that produce either secreted or intracellularly degraded SIV antigens. Macaques infected for 15-70 wks were treated with a combination of 3 drugs (ART) for 13-23 wks. During this time, the animals were immunized via the IV route with the SIV DNAs and then released from ART. Macaques receiving DNA showed a significant decrease in viral load for long periods after therapy termination compared to controls (p < 0.001). DNA vaccination, but not ART alone, led to substantial decreases in viremia.

S138
Half of the animals (6/12) continue to control viremia levels for 2 years. Cellular immune responses were immediately boosted strongly by DNA vaccination and persisted despite lower virus loads. Thus, the combination of novel forms of DNA vaccines administered during ART treatment induced an immune response able to persistently decrease viremia after removal of ART. These DNA vectors used as therapeutic vaccine may be beneficial either alone or in combination with other vaccine modalities as an addition to antiretroviral treatment. Nef is an accessory protein of primate lentiviruses that is required for high levels of viremia and progression to AIDS in infected individuals. Our studies on Nef have centered around its effects on budding and release of optimally infectious virions from cells. To these ends, we have characterized interactions between Nef and the transframe portion of the GagPol polyprotein, AIP1 and cholesterol. Specific sequences in Nef were identified that mediate these interactions. For example, the flexible loop in Nef binds p6* that connects Gag and PR. It is via this interaction that Nef helps to aggregate viral structural proteins in lipid rafts and is itself incorporated into progeny virions. A sequence in the core of the protein binds AIP1 that helps HIV-1 form multivesicular bodies and be released from cells. Indeed, fusing Nef with a mutant Gag that lacks the late domain allows for the release of VLPs into the supernatant. Finally, at its very C-terminus, Nef binds newly synthesized cholesterol, which is incorporated into viral particles that are more infectious. To detemine which one of these interactions was more important for high levels of viremia and progression to AIDS in the rhesus macaque, multiple mutations were engineered into the nef gene in SIVmac239. Of interest, before high levels of viral replication could be observed in monkeys infected with the mutant SIVmac239, both the binding to GagPol and AIP1 had to be restored in the mutant Nef protein. From these studies, it appears that Nef also plays a critical role in the later phases of the viral replicative cycle and ensures that optimally infectious virions are released from infected cells. In order to identify the cellular gene target for Tat, we have performed gene expression profile analysis and found that Tat

S142
Retrovirology 2005, 2(Suppl 1) http://www.retrovirology.com/supplements/2/S1 upregulates the expression of 8-oxoguanine DNA glycosylase (OGG1) gene, encoding an enzyme responsible for repairing the oxidatively damaged guanine, 7,8-dihydro-8-oxoguanine (8-oxo-dG). We observed that Tat induces OGG1 gene expression by enhancing its promoter activity without changing the mRNA stability. We found that the upstream AP-4 site within the OGG1 promoter is responsible and that Tat interacts with AP-4 and removes AP-4 from OGG1 promoter by in vivo chromatin immunoprecipitation assay. Thus, Tat appears to activate OGG1 expression by sequestrating AP-4. Interestingly, although Tat induces oxidative stress known to generate 8-oxo-dG that causes the G:C to T:A transversion, we observed that the amount of 8-oxo-dG was reduced by Tat. When OGG1 was knocked-down by small interfering RNA (siRNA), Tat increased the amount of 8-oxo-dG, thus confirming the role of OGG1 in preventing the formation of 8-oxo-dG. These findings collectively indicate a possibility that Tat may play a role in the maintenance of genetic integrity of the proviral and host cellular genomes by upregulating OGG1 as a feed-forward mechanism.

S143
Abstract withdrawn Comparison of different cohorts of SIV-infected rhesus macaques that control viremia may help to identify the mechanisms that prevent progression towards AIDS. We study 3 groups of macaques able to control viremia to various extents. Group 1 was infected with live-attenuated Rev-independent SIV is able to persistently control viremia over more than 7 years. Group 2 are live-attenuated SIV-infected macaques additionally challenged with pathogenic SIVmac251 and controls the challenge to various levels (<10 5 copies/ml) for more than 4 years. Group 3 are SIVmac251-infected animals therapeutically immunized using DNA vectors during ART. These animals control viremia after release from ART for more than 18 months (<10 4 copies/ml). Animals in groups 1 and 2 developed long-lasting humoral and cellular immune responses. Animals in group 3 have persistent increases in cellular and humoral responses leading to virus containment and slower onset of disease. The understanding of the underlying mechanism leading to protective immune responses of these 3 cohorts of 'controllers' will be useful for rational vaccine design.

S146
Abstract withdrawn  Six morphine-dependent and 3 control male Indian rhesus macaques were intravenously inoculated with mixture of SHIV KU , SHIV 89.6 P and SIV/17E-Fr. These animals were followed for a period of 56 weeks for virus replication, disease progression and immune responses. Both morphine-dependent and control macaques showed precipitous loss of CD4+ T cells but CD4 recovery was found to better in more control animals than that in the morphine-dependent animals. The plasma and CSF viral load was significantly higher in morphine-dependent group than those in the control group. Four morphinedependent succumbed to SIV/SHIV-induced AIDS at week 18, 19, 20 and 51, post-infection with neurological disorders in 3 of those 4 animals. Other 2 morphine-dependent and 3 controls were still alive at the end of 56 week observation period. All 3 Retrovirology 2005, 2(Suppl 1) http://www.retrovirology.com/supplements/2/S1 viruses replicated in the blood of both morphine-dependent and control macaques, but cerebral compartment showed a selection phenomenon and only SIV/17E-Fr and SHIVKU crossed the blood brain barrier (BBB). The morphine-dependent macaques further favored the viral migration through blood brain barrier (BBB Acute SIV infection of macaques is a model for human AIDS typified by high levels of plasma viremia that decline with the onset of specific viral immunity. SIV infection limits the development of viral immunity and persists to allow for the establishment of chronic progressive disease. The destruction of CD4+ and CD4-lymphocytes argues that both direct and indirect killing mechanisms contribute to the loss of cells. Although early intervention with antiretroviral drugs reduces viremia and disease, it is still unclear whether protection is due to diminished viral cytopathicity or due to blocking a host mechanism for cell killing that is triggered by the high level of viral replication. We tested the role of FasL killing of bystander lymphocytes by injecting monkeys with a humanized monoclonal anti-FasL. Treatment with anti-FasL during acute infection reduced the level of apoptosis of circulating T and B cells, peak vRNA levels were unaffected but higher antibody and CTL responses to viral proteins lead to lower set-point viremia among treated macaques. Reduced bystander killing and increased viral immunity were associated with attenuated SIV disease and a significant increase in the life span of infected macaques after transient treatment with a monoclonal antibody against FasL.  Retrovirology 2005, 2(Suppl 1) http://www.retrovirology.com/supplements/2/S1

S154
Conclusion: Gag-specific CD8 + T-cells are detectable in some patients who have been successfully treated with HAART for more than 5 years. The frequency and absolute counts of Gagspecific CD8 + T-cells in patients with more than 5 years of successful HAART are different compared with untreated patients. These findings are relevant for the analysis of immune reconstitution following long-term successful HAART.

P2
The Optimal vaccination strategies against HIV-1 require the fulfilment of two conditions: a)-the choice of an immunogen including pathogen's antigenic epitopes against which an immune response with neutralizing characteristics can be generated; b) the optimisation of triggering and maintenance of the immune response against the vaccine. Our interest is focussed on the second aspect. The expression of the MHC class-II transactivator (CIITA) whose locus AIR-1 and corresponding function were discovered in our laboratory, is needed for continuous expression of MHC class-II molecules and consequent antigen presenting function by APC. Unexpectedly, and of great relevance for antiviral functions, we found that CIITA potently inhibits HIV-1 viral replication by a competitive action on the viral transactivator Tat for Cyclin T1, the cellular cofactor used by Tat to elongate viral transcripts. This effect is found in APC and, of greater importance, also in HIV-1-infected T cells. Molecular analysis has revealed that the inhibitory activity of CIITA for HIV-1 Tat maps to the N-terminal region, and particularly to the segment 200-285 included within the P/S/T region of the CIITA activation domain which is then the molecularly defined Cyclin-T1-interacting region. Thus CIITA has a dual role on HIV infection: 1)-it increases APC function for HIV-1 viral antigens; 2)-it decreases viral replication and thus viral spreading in infected individuals. Within this frame CIITA represents the necessary and ideal molecule to control both innate and adaptive immunity against the virus. Considering the functional importance of a sustained and persistent expression of CIITA in APC, the search for potential synthetic and natural mediators, drugs and biomolecules, that can act on CIITA expression at level of transcription as well as biosynthesis, will be of great importance in tailoring better vaccines against HIV-1, and in controlling and combating HIV-1 infection and spreading.

Retrovirology 2005, 2(Suppl 1):P5
The transcriptional activator CIITA is the master regulator of the expression of MHC class II genes. In addition to this major role, we have found that CIITA exerts an important inhibitory effect on the HTLV-2 replication, similar to our previously decribed effect on the HIV-1 replication. This inhibition is mediated by the N-terminal 1-321 region where we identified a minimal fragment of 80 aminoacids that specifically blocks the activity of the viral transactivator Tax2. This fragment does not inhibit the function of Tat, the transcriptional activator of HIV-1.
To unveil the biochemical basis of the CIITA-mediated inhibition of Tax2 we first focussed on the identification of the cellular cofactors used by Tax2 to transactivate the viral promoter. DNA-binding factors (CREB, NFYB) and several co-activators involved in chromatin remodeling (CBP, p300, PCAF, BRG1) are known to interact with or stimulate the transcriptional activity of both CIITA and Tax1, the HTLV-1 homologous of Tax2. Preliminary data indicate that the transactivation activity of Tax1 and Tax2 is differently influenced by the hystone acetyltransferases CBP, p300 and PCAF providing new informations on the biology of HTLV-2. Furthermore, none of these factors was able to reverse the inhibitory action of CIITA on Tax2 function. Interestingly, we found that the B and, to a lesser extent, the A subunits of the NFY complex inhibit Tax2 activity when iper-expressed in cells. On the basis of our results and of the reported physical interactions between NFY and both CIITA and Tax1, we propose a molecular model for the CIITA-mediated inhibition of Tax2 activity via the binding of the CIITA-NFY complex to Tax2. When expressed in cells CIITA interacts with the NFY complex; this interaction changes the conformation of NFY increasing its binding affinity for Tax2. Following this model the inhibition of Tax2 by CIITA it is not due to the squelching of a transcriptional positive co-activator, but instead to the recruitment of a cellular factor, NFY, with a negative regulatory action on Tax2. This as well as possible alternative models are presently under scrutiny. On the whole these results confirm that CIITA may represent a physiologic tool for novel therapeutic strategies aimed at counteracting HLTV-2 replication and spreading.

P7
HTLV-1-associated myelopathy/tropical spastic paraparesis is characterized by highly stimulated immune response that includes elevated levels of inflammatory cytokines/chemokines, and oligoclonal expansion of Tax-specific CD8 + cytotoxic T lymphocytes in the cerebrospinal fluid. Studies have shown that the HTLV-1 transactivator protein Tax is available for immune recognition by antigen presenting cells such as dendritic cells. In this study, we have shown that the treatment of monocytederived dendritic cells (MDDCs) with extracellular Tax induces the secretion of Th1 cytokines (IL-12, and TNF-) and -chemokines (MIP-I, MIP-I, and RANTES). A significant dose-dependent increase was observed with IL-12 (5-, 7-, and 24-fold) and TNF-(5-, 6-, and 9.6-fold) with Tax treatment for 24 hr at concentrations of 0.1, 1, and 10 mg/ml, respectively. All three chemokines exhibited both dose-and time-dependent increase in the presence of Tax. More specifically, after 24 hr treatment with Tax (0.1, 1 and 10 g/ml), MIP-1a was induced by 3.7-, 5.3-, and 6-fold, MIP-1b by 2-, 3.7-, and 3.7-fold, and RANTES by 7-, 8-, and 20-fold, respectively. The mRNA expression of these cytokines/chemokines was confirmed by real time PCR and was in direct correlation with observations regarding their protein expression. The surface marker expression after this treatment also correlated with a more differentiated phenotype. To begin exploring the potential of these cells to support productive HIV-1 replication, a series of stably transfected cell lines were developed. To this end, macrophage-, T cell-and dual-tropic long terminal repeats (LTRs) were coupled to the gene encoding green fluorescent protein. These cell lines were utilized to explore the functional properties of specific cis-acting regulatory elements in LTR function within the bone marrow precursor cell population. the basic knowledge required for safer sexual behavior. As this group requires having proper guidance regarding their health, which can make them beneficial for community and nation and so, they can play an innovative role for welfare and advancement of the country. We want to have a clear idea of young adult's knowledge about reproductive health, safe sex, and STDs. Youth's attitude toward risky behaviour is an interesting topic that has attracted our attention. The main reason for this is that it focuses on youth in particular rather than the public. Youth are faced with numerous problems, which are sometimes beyond their comprehension. They may have heard of the phrase ''Risky behaviours'', but not everyone understands the meaning of these words. For the purpose of this research project, we have decided to limit the definition of risky behaviours to those practises that may lead to unsafe sex. This is a quantitative study which will be done by the survey method. The major source of data in this study is a selfadministerd questionnaire which will be used to collect data. There is a little difference between women and men questionnaires. Due to sensitive nature of some questions and to make the respondents feel at ease, female interviewers will be responsible for interviewing female respondents while the male interviewers will ask questions of male respondents. The assurance will be given to the committee representatives and respondents specially, the identification, names will not be made public or published, code numbers are given to their names. The questionnaire will be pretested to provide information on the clarity of the questions and respondents' comprehension. mechanism(s) involved, our laboratory has been investigating the critical elements responsible for regulating this gene. We have shown that the CD3 gene is transcribed from an independent but weak, lymphoid-specific TATA-less promoter and demonstrated that a cluster of transcription initiation sites is present in the vicinity of the principal core promoter with the major start site situated in a classical initiator sequence. A GT box upstream of the initiator binds Sp family proteins and the general transcription machinery, with the activity of these contiguous elements enhanced by a second Sp binding GC box ten nucleotides further upstream. We found that two previously identified NFAT motifs positively (NFAT 1) or negatively (NFAT 1 and NFAT 2) regulate expression of the CD3 gene by their differential binding of NFATc1 plus NF-B p50 or NFATc2 containing complexes, respectively. Analysis of various mutant and deletion CD3 promoter constructs in a transient reporter assay revealed that a 53 bp region downstream from the major transcription start site is critical for positive gene expression. Deletion of ten nucleotides in this region results in a 50% decrease in promoter activity, while deletion of 39 nucleotides completely eliminates promoter activity. EMSA experiments using DNA or RNA probes covering the 53 bp region demonstrate that this element functions through an RNA rather than a DNA intermediate. At least three specific nuclear protein complexes bind to the RNA probe. Deletion of the U at position +9 and the U at +37 completely abrogate binding and promoter activity. Experiments are currently underway to determine whether the composition of the transcription factor(s) complexes bound to the CD3 element contain components of P-TEFb, which binds to HIV TAR.

P14
Abstract withdrawn  normal healthy subJects not belonging to any risk group for HIV infection completed the study. Saliva was collected at awakening before brushing teeth and the concentration of hemoglobin was determined. Hemoglobin concentration in saliva in basal conditions is higher in anti-HIV positive IVDA with respect to anti-HIV negative IVDA (p less than 0.05) and controls (p less than 0.01). In anti-HIV positive IVDA hemoglobin concentration in saliva is higher in subjects with CD4+ cells less than 200/ 10(6) l with respect to subjects with CD4+ greater than 200/10 (6) l (p less than 0.05) and in subjects with ARC/AIDS with respect to subjects with PGL or who are asymptomatic (p less than 0.01). SubJects with ARC/AIDS have a mean concentration of hemoglobin of 19 micrograms/0.1 ml saliva (range 0-153) which corresponds to 1.3 microliters of blood/ml saliva. If 10 ml of saliva are exchanged during kissing an average of 13 microliters of blood are transferred (110 microliters of whole blood at extreme range). Blood of symptomatic patients has an HIV titer of 7 TCID/microliters which for 10 ml saliva containing an average of 1.3 microliters blood/ml saliva corresponds to an average of 90 TCID (770 TCID at the extreme range). To extend our studies, we have expressed recombinant protein fragments that mimic the core-coiled-coil region and sixhelix bundle of fusion-active HTLV-1 envelope. Using these recombinant proteins as immunogens we have generated monoclonal antibodies (mAbs) against the fusion-active and post-fusion conformations of HTLV-1 envelope. Most importantly, we have now used these conformation-specific mAbs to probe the events that culminate in membrane fusion. We demonstrate that these monoclonal antibodies can be used to detect viral envelope on infected cells and to monitor the process of cell-to-cell viral transfer.
Our recent results will be presented, and the implications of our results for HTLV-1 pathogenesis will be discussed.

P20
Abstract withdrawn Naturally occurring human antibodies containing only heavy chain are very rare. All antibodies specific for HIV identified until now contain both light and heavy chains. By screening an immune HIV phage library we have identified an antibody, m12, that expresses only a heavy chain. The Fd of this heavy chain behaves as a CD4i antibody and binds gp120 complexed with CD4 better than gp120 alone. This antibody was further engineered to a single domain antibody, which is the smallest possible antibody fragment that still exhibits binding to the antigen. The domain m12 neutralized HIV isolates from different clades but had low solubility and was difficult to express. To further improve its solubility and potency we generated a mutant library. This library is being screened against gp120 and gp120-CD4.  Levels of CCR7 were intermediate (mean 29.8%) in the IR+VRgroup, and were almost absent in two patients with VL>100,000 copies who had a markedly reduced IFN-production in pDC. CD83, CD80, and TNF-a were expressed in all patients and were more pronounced in mDC than in pDC. Conclusion: The most striking finding was a reduced expression of CCR7, and is indicative of a defect in homing of the plasmacytoid DC to lymph nodes in HIV infected children who have ongoing active viral replication and poor immunologic control in spite of HAART. Background: The aim of this study is to identify conserved epitopes of HIV-1 neutralizing antibodies in polyclonal plasma from LTNP to finally derive vaccine candidates.

Materials and methods:
The presence of neutralizing antibodies in 9 LTNP sera was proved by in vitro neutralization assays. Phage displayed peptide libraries were screened with LTNP IgG. HIV-specific mimotopes were analyzed for homology to the gp120 structure by a software (3DEX) especially developed for this purpose. Mice were immunized with interesting phages and their sera were analyzed for neutralizing activities against HIV-1.
Results: After biopannings, between 19% and 75% HIV-specific phage clones were identified by ELISA. Mimotope sequences were identified and could be aligned by 3DEX to linear or conformational epitopes on gp120. A peptide specific immune response was detected in sera of immunized mice. The first mice sera analyzed showed neutralizing activities against HIV-1.
Conclusion: Mimotopes could be selected from LTNP sera that represent conformational epitopes on gp120. Those ones inducing neutralizing antibodies upon immunization potentially are suited to derive vaccine candidates. Tax protein has been shown to play an integral role in HTLV-1induced diseases including adult T cell leukemia (ATL) and HTLV-1associated myelopathy/tropical spastic paraparesis (HAM/TSP). Extracellular Tax has been detected in the cerebrospinal fluid of HAM/TSP patients, suggesting that cell-free Tax may be physiologically involved in the progression of neurologic disease. We have previously demonstrated the secretion of full-length Tax and its co-localization with the cytoplasmic organelles relevant to secretion. The present study elucidates the mechanism of Tax secretion. To identify Tax interacting proteins within cell, we have used an antibody array spotted with antibodies directed against cellular secretory pathway proteins. Upon reaction with protein extracts from Tax-treated cells, antibody array analyses have suggested the interaction of Tax with SCAMP1, SCAMP2, SNAP23, and COPII; proteins that facilitate transport between nucleus and cytoplasm, and between endoplasmic reticulum, Golgi complex, and plasma membrane. Subsequently, these specific protein-protein interactions have been confirmed by co-immunoprecipitation and GST pulldown assays. Collectively, these studies have demonstrated the interaction of Tax with multiple proteins in the secretory pathway and that Tax may be secreted and act as an extracellular effector molecule. IFN-, a cytokine produced mainly by T lymphocytes, interferes with viral replication by acting as a powerful immunomodulator, and has a negative effect on HIV-1 Tatmediated viral transactivation in vitro. The production of this cytokine, which is upregulated during acute infection with HIV-1, seems to decrease during progressive HIV-1 infection, perhaps in part by de novo methylation of its promoter, yet the full mechanism for this decrease is still unclear. The HIV-1 Tat protein has been shown to induce the production of several cytokines and interferon-inducible proteins in high quantities, triggering a toxic cascade of events on surrounding cells, yet the role of Tat in the stimulation of these gene products is not fully known. We have assessed the effect of HIV-1 Tat on the production of IFN-, since it may provide an explanation for the modulation of these genes and perhaps the subsequent downregulation of IFN-itself. HIV envelope glycoprotein (Env) mediates infection by fusing virus with cellular membranes. Fusion inhibitors, a new class of antiretroviral drugs, inhibit HIV infection by binding to gp41 to form a peptide-gp41 6HB that is fusion-incompetent. To understand resistance mechanisms to peptide fusion inhibitors that will aid development of new drugs, we generated an escape-mutant virus against an N-peptide inhibitor. We found that two mutations in gp41, one each in the N-and C-heptad repeats, confer early resistance to the N peptide. These same mutations also confer resistance to a C peptide inhibitor. This is the first report of cross-resistance among peptide fusion inhibitors. Curiously, the N mutation alone or in combination with C mutation also conferred increased sensitivity to soluble CD4 and was associated with faster growth kinetics and larger syncytia. These results suggest global changes in Env involving receptor activation and fusion kinetics.

P32 A Model for Advocacy and Community Participation in Research
Using thermal denaturation studies, involving N and C peptides containing wild type (Nw or Cw) or resistance residues (Nm or Cm), which self-assemble into a 6HB, we showed that the N mutation improved the energetics of the viral 6HB, however, the energetics of the 6HB formed with the inhibitor and the N and C peptides is not afffected. Thus, our results demonstrate a resistance pathway that appears to involve both kinetic and thermodynamic factors that regulate virus entry and work indirectly to reduce the ability of fusion inhibitors to bind Env. We describe a novel chimera RNA expressing vif short-hairpin RNA (shRNA) and decoy trans-activation response region (TAR) RNA from a human U6 Pol II promoter, which enhanced the inhibition of human immunodeficiency virus (HIV) vif small-interfering RNA (siRNA) and arrested virus breakthrough by siRNA-generated escape variants in long-term culture assays. Our strategy was based on a second-generation anti-HIV-1 shRNA vector system, in which HIV-1 vif shRNA was fused to a decoy TAR RNA by a linker UU cleavage site to generate vif shRNA-decoy TAR RNA. Upon expression, the RNA molecule was cleaved and separated into vif siRNA and decoy TAR RNA. The synergistic effect of these molecules enhanced the inhibition of HIV-1 replication in a longterm culture assay and prevented virus breakthrough associated with siRNA-mediated escape variants. Combining shRNA with decoy TAR RNA as second-generation anti-HIV shRNA may provide practical basis for applying siRNA-based gene therapy to the treatment of HIV/AIDS. were the only variants examined that were found in low frequencies in PB-derived LTRs derived from patients at early stages of HIV-1 disease, and at increasing frequencies in patients representing later stages of disease. Sequence variation at these sites was also examined in LTRs derived from autopsied brain tissue of patients both with and without HIVD. The 3T C/EBP site I was identified in 25% of brain-derived LTRs from patients with HIVD, but was absent in patients without HIVD. This suggests that 3T C/EBP site I, and possibly 5T Sp site III may prove valuable in assessing the likelihood of an HIV-1-infected individual developing HIVD.

P35
HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/ TSP) is characterized by the generation of an intense cytotoxic T cell (CTL) response directed against oncoprotein Tax. Previous studies have suggested that Tax may be available for immune recognition by dendritic cells (DCs). In this study, we have shown that purified Tax protein efficiently bound and localized to the cell membrane of monocyte-derived dendritic cells (MDDCs) and was internalized within a few hours. After uptake, Tax-induced expression of DC activation markers MHC class I and II, and costimulatory molecules as well as the DC maturation marker, CD83. Tax has also promoted the production of major immune-directing cytokines IL-12, TNF-, and proinflammatory chemokines MIP-1, MIP-1, and RANTES. The inhibitors of NF-B have abrogated Tax-induced secretion of cytokines/chemokines indicating a role for NF-B signaling in Taxmediated immune response. Finally, Tax enhanced the allogenic and antigen-specific T cell proliferation capability of MDDCs. These results have indicated that extracellular Tax may selectively target MDDCs, be taken up by these cells and promote their maturation and antigen-presenting functions, driving a Th1-type immune response.
Numerous studies have demonstrated that patients diagnosed with HAM/TSP exhibit rapid activation and maturation of dendritic cells (DCs) while ATL is associated with their maturation defect. In addition to T cells, HTLV-1 is known to infect DCs. HTLV-1 infection of DCs could alter general DC function or the specific processing and/or presentation of HTLV-1-specific peptides, potentially playing a major role in the course of HTLV-1-associated disease. In this regard, we have demonstrated that an important antigen receptor on DCs, DC-SIGN serves as a receptor for HTLV-1 binding using a quantum dot-based fluorescent binding assay. We have also demonstrated that gene silencing of DC-SIGN inhibits the infection of DC in a DC/T cell co-infection system. Furthermore, expression of DC-SIGN in B cells enhances viral binding, integration, and infection. These investigations, which consider the involvement of DC surface molecules in HTLV-1 pathogenesis, are the first explorations of the intricate mechanisms that underlie the interactions between DCs and HTLV-1. Background: Sensitive/less-sensitive (S/LS) serologic assays that differentiate recent from established HIV infection may not be suitable for use in resource-limited and financially challenged countries. A more simple and affordable method is needed to address these limitations. Methods: The Serodia HIV-1/HIV-2 particle agglutination assay (PA) was modified to act as a S/LS assay. Antigen-coated gelatin particles were diluted 1:68, and sera were diluted at intervals from 1:10 to 1:80,000; HIV antibody status was confirmed at the 1:10 dilution. 37 clade B seroconversion panels from Trinidad and BBI (n = 309) were tested at each sample dilution to calibrate the PA assay; the last positive reaction (>1+) was considered the endpoint dilution (ED). The greatest sensitivity for correctly classifying recent and established infection samples was determined by ROC analyses. A subset of these panels (n = 181) was also tested by the Vironostika S/LS (DV) as a reference for comparison. Results: At a dilution of 1:40,000 and a days post SC cutoff of 190 days the PA test gave 97% sensitivity for classifying both recent and established infection samples, as compared with 82% and 53% on a subset tested by the DV; this resulted in a poor concordance of 60% and 73%. Conclusion: A low cost, simple to perform PA test was modified as a S/LS test and exhibited excellence in distinguishing recent and established HIV infection. The PA S/LS performed more accurately than the reference DV S/LS when testing samples with known times of seroconversion.

P40 Abstract withdrawn
Toxicity from methanol (MeOH), a potentially significant problem due to occupational, accidental, intentional, as well as daily ingestion of small amounts of the agent, only receives considerable attention after severe signs of intoxication have set in or death is imminent. While accidental and intentional exposures usually involve very high doses, the occupational and ingestion forms more often reflect small daily intakes. Still, even at the low levels, little is known about the potential immunotoxic implications (and less so in regard to mechanisms) from these ongoing exposures. This study has been focused on the effect of methanol on cell mediated and humoral immune function in non-immunized and immunized rats. The level of methanol used in this study was one fourth of the LD 50 values (2.37 gm/kg b.wt). The cell mediated and humoral immune function tests were carried out in seven different groups of albino rats, namely control, 1 day, 15 days, 30 days and corresponding immunized groups were used. Sheep red blood cells (SRBC 5 Â 10 9 cells/ ml) were used for immunizing the animals that belongs to the immunized groups. Food intake, urine output, animal and organs weight ratio, cellularity of lymphoid organs and foot pad thickness were significantly decreased when compared with respective controls. However, water intake, and leucocytes migration inhibition were significantly increased when compared with respective control groups. Antibody titre was significantly increased in non-immunized groups and significantly decreased in immunized groups. Therefore, the present study encompasses that repeated exposure of methanol profoundly suppressed the cell mediated and humoral immunity. Quantum dots (Qdots) are fluorescent semiconductor nanocrystals comprised of CdSe core with a semiconductor shell of zinc sulfide coated with a polymer shell allowing particles to be conjugated to biological molecules while retaining the optical properties of the particle. We have used this unique property of Qdots to develop a high throughput binding assay to study the attachment of HTLV-1 to host cells. To this end, we have biotinylated cell-free HTLV-1 (biot-HTLV-1) to facilitate viral detection using streptavidin-coated Qdots. B cells (BTHP-1 and Ramos) were exposed to biot-HTLV-1 with increasing concentrations of DEAE-dextran, a reagent known to enhance binding of other retroviruses. Unbound virus was removed by washing and cells were added with strep-Qdots and fluorescence readings were obtained at 605 nm. HTLV-1 bound efficiently to BTHP-1 and Ramos cells and this binding was significantly increased (3-fold) by DEAE-dextran. To confirm the specificity of viral binding, a competitive inhibition assay was performed wherein increasing amounts of non-biotinylated HTLV-1 was added to the binding assay along with a fixed amount of biot-HTLV-1. A dose-dependent inhibition in biot-HTLV-1 binding was observed in the presence of native virus. These results suggest that the Qdot-based assay may be useful in studying virus attachement to host cells, and the screening of inhibitors for viral binding and entry.

P47
Abstract withdrawn Since 1987, more than 25,000 individuals have been immunized with 65 HIV preventive vaccines. Current candidate HIV-1 vaccines are complex products containing multiple HIV genes or proteins. As a result, high proportion of vaccinees score positive in licensed HIV diagnostic kits. This will have negative impact on vaccine trials that require early detection of breakthrough infections. For vaccinees it may contribute to range of social harms (jobs, insurance, blood donors). Therefore, it is important to design new tests that will discriminate between vaccine induced reactivity and true HIV infection. Our goals were to identify new HIV epitopes that: 1) Do not contain important neutralizing or CTL epitopes, 2) Recognized by antibodies early after HIV infection. 3) Highly conserved among HIV clades. Using Phage Display libraries constructed from whole HIV-1 genomes, combined with affinity selection with antibodies from early seroconvertors, we identified new immunodominant epitopes, in gp41 cytoplasmic tail and in p6 that fit the above criteria. These peptides were used for development of new differential HIV-1 ELISA. To date, 100% specificity for gp41 and 99.4% with p6 peptide was observed with 1300 HIV seronegative samples. Africa, continent devastated by poverty and the civil wars, became one of the continents more touched by the HIV/AIDS. According to UNAIDS, Africa is home to 70% of adults and 80% of children living with HIV in the world. Not so long ago, testing positive for HIV meant an automatic death sentence. Now things have changed for the better. A combination of drugs introduced about seven years ago has turned AIDS from a death sentence to a treatable disease. These drugs are very expensive and many African governments do not have the funds to import these drugs. In Africa, fewer than 100,000 people living with HIV have access to antiretroviral treatment and everybody know that AIDS won't wait, this means that a majority of the 25.3 million Africans infected with AIDS won't get the best available treatment and many African HIV/AIDS patients have died and other may follow in the next five years if nothing is made to stop the progress of the AIDS.
To save the African continent, there are two possibilities, the first possibility is to allow African countries, especially those most affected by AIDS to declare a state of emergency and produce affordable generic versions of HIV/AIDS drugs to provide the much needed help to their citizens. The second is to try to slow new cases through preventive education and encouraging condom use, maybe reduce transmission from mothers to babies. Hardly enough to save African Retrovirology 2005, 2(Suppl 1) http://www.retrovirology.com/supplements/2/S1 continent, so figuring out how to save the millions who are infected remains an agonizing challenge.

P50
Abstract withdrawn The statistical probability of seroconversion is proportional to the number of needlesticks incurred and the likelihood that the needlesticks will be with HIV infected blood. Careful adherence to recommended operating room practices, combined with meticulous attention to handling needles and sharps, should result in few, if any, cases of occupational HIV seroconversion among OR personnel. HIV testing is not feasible in the management of emergency patients; these are often the individuals at highest risk for HIV infection and over whom the surgical team has the least control. Non-operative treatment of HIV-infected patients is not an option; many procedures are performed either to enable the individual to lead a more comfortable, productive life or for diagnostic purposes.

P52
Abstract withdrawn The purpose of this grounded theory study was to describe the experience of HIV/AIDS family caregiving in the palliative phase. Seven in-depth interviews were conducted and analyzed using the constant comparative method. The analysis resulted in a conceptualization of HIV/AIDS family caregiving. This paper describes the ''personal work'' of caregivers, including reconciling that a loved one would die, making life-and-death decisions, and letting go. The nature of support received to attend to this work is highlighted, with attention to its influences on HIV/AIDS caregiver bereavement. The findings of this study provide some insights into the HIV/AIDS family caregiver experience and reveal a significant need for interventions designed to support caregivers in establishing the mechanisms required for bereavement resolution. The need for the creation of supportive networks for HIV/AIDS caregivers cannot be overstated. Further research is required to help clarify and expand on how social support might have an effect on HIV/AIDS family caregiver bereavement. With this knowledge, health-care providers will be better prepared to anticipate difficulties faced by caregivers, plan appropriate interventions to address these difficulties, prevent future problems, and plan care based on theory and research. Conformational change in HIV-1 gp120 is a dynamic process essential for HIV-1 entry. It is not clear whether the dynamic nature of gp120 could be exploited to abort the viral entry machinery. Here we show that a small molecule entry inhibitor, IC9564, induces a conformational change in gp120 and locks the envelope into fusion incompetent conformation. Binding of IC9564 to HIV-1 envelope results in the exposure of CD4i epitopes that are ally concealed in gp120. As a result of the conformational effect, IC9564 significantly enhances the neutralizing activity of 17b that binds to an epitope overlapping chemokine receptor binding site. Unlike CD4, IC9564induced conformational change in gp120 does not trigger a conformational change in gp41. In fact, IC9564 inhibits CD4 induced conformational changes in gp41. The binding site of IC9564 remains elusive due to the fact that mutations in both gp120 and gp41 could change IC9564 sensitivity. Nevertheless, a common effect of these mutations is that conformation of gp120 is changed to conceal conserved epitopes such as CD4i. In summary, IC9564 exploits the dynamic nature of HIV-1 gp120 by inducing a nonproductive conformational change in gp120 and prevents HIV-1 from entering the cells.
ionomycin resulted in a small increase in the amount of Tax secreted suggesting that a fraction of cytoplasmic Tax was present in the regulated secretory pathway. These studies provide a link between Tax accumulation in the cytoplasm, the detection of Tax in the extracellular environment. Human immunodeficiency virus type 1 (HIV-1) has been transmitted worldwide and regional viral clades have been designated as subtype A through K. Subtype C, which is concentrated in southeast Asia and sub-Saharan Africa, is the most prevalent subtype worldwide. To date, no studies have examined the role of CCAAT/enhancer binding proteins (C/EBP) in LTR-directed viral gene expression in the clade C LTR. Within clade B viruses, two functional C/EBP sites upstream of the TATA box have been shown to be required for efficient viral replication in cells of monocyte/macrophage lineage. In order to assess the role of the C/EBP sites within the subtype C viral LTR, 211 HIV-1 subtype C LTR sequences were collected and aligned via the Clustal V method. From these analyses, three potential C/EBP sites were identified: two upstream binding sites and one downstream binding site. Interestingly, the putative downstream site was highly conserved between clades B and C, suggesting the presence of a functionally important cis-acting element that has yet to be characterized. Electrophoretic mobility shift analyses demonstrated that two of the three sites within the HIV-1 subytpe C were able to bind C/EBP factors. Additional studies focused on examining relative binding affinities of naturally occurring variants of these two sites. Future studies will examine the roles of these sites in the regulation of LTR activity.

P57
Abstract withdrawn Background: In South Africa, HIV-1 subtype C is the prevalent subtype and has been found to use CCR5 throughout the viral infection in most cases1. In contrast, HIV-1 subtype B often switches its coreceptor usage to CXCR4 due to mutations primarily within the V3 loop of the gp120 protein 2 . In order to assess whether subtype C is still able to infect T cells in late stages of the infection, FISH was used to localize HIV sequences within different blood cell types of AIDS patients. Methods and Materials: Fresh blood and bone marrow samples obtained upon informed consent from late stage, antiretroviral therapy-naïve, non-opportunistic infectious HIV patients were separated into CD4 + and CD4fractions using Dynabeads. The cells were washed, placed onto slides, and hybridized with HIV-gag-RNA-specific probes, labelled by nick translation with spectrum green. Following overnight incubation at 37˚C, the slides were washed, stained with DAPI and analyzed under fluorescent microscopy. DNA was also extracted from each sample, the HIV envelope gene was amplified using nested primers, subjected to sequencing to determine the V3 loop sequence and the resulting overall positive charges.
Results: In order to assess the site of HIV subtype C active infection in late stage of the disease FISH was performed on peripheral blood or bone marrow slides of 20 HIV + patients produced positive signals in and around the nucleus of CD4 + T cell as well as monocytes/macrophages, whereas control slides made from HIV-individuals and CD4fractions of HIV patients possessed no signals. Both CD4 + cell population exhibited high number of signals, but the T cells had a greater variation, ranging from none to over thirty. The V3 loop sequencing and analysis of the protein sequence from 11 of the 20 patients revealed the structure's overall positive charge and amino acid placements were compatible with HIV-1 subtype C CCR5-using viruses in these patients. Conclusion: Despite the use of CCR5 by HIV-1 subtype C, CD4 + T cells were found to exhibit positive signals in vivo in late stage HIV + patients. This may suggest that CCR5 expression is upregulated on CD4v T cells to allow the virus access, or that the virus is exploiting some other mechanism of invasion like the viral synapses that has been found to exist between infected macrophages and uninfected T cells, rather than a direct approach of infection where the use of chemokine receptors are necessary.
HAART. We studied HPV related parameters to identify markers of progression and HIV/HPV infections interaction. Methods: HIV pos women (N = 410) were followed since 1995 in a longitudinal study. Each one undergo periodic (6-12 months) colposcopy, PAP smear, biopsy if needed, and cervical sampling for HPV testing. HIV related parameters (CD4, HIV-RNA, ART) are recorded and related to cervical disease. HPV typing is performed by reverse hybridisation assays, viral load by in-house real time PCR and viral expression by E6/E7 mRNA detection. The Mann-Whitney rank sum test for non-parametric data and the association between discrete variables by Chisquare test of Fisher exact test were applied. In this study, the dynamics of CD4 cell depletion during tenofovir/ didanosine co-administration were analysed. Ninety-five HIVpositive patients were followed for 562 days, and 37 lost at least 50 CD4 cells, with a median delay of 274 days. Cox analysis showed that the CD4 cell decrease was associated with a duration of treatment by didanosine of more than 853 days and a didanosine dose of more than 5.50 mg/kg. Saliva specimens were tested for HIV antibody (anti-HIV) by an immunoglobulin G (IgG) antibody capture radioimmunoassay (GACRIA) and three sensitive commercial assays. In tests on 460 seronegative subjects and 196 seropositive subjects GACRIA was 99.8% specific and 100% sensitive. The Wellcome HIV monoclonal and Abbott recombinant DNA enzyme-linked immunosorbent assays (ELISAs) were also highly specific (99.8%, 100%) but they were less sensitive (90.9%, 82.0%). The Fujirebio particle agglutination assay was sensitive (97.8%) but its specificity was poor (84.1%). In testing saliva specimens from populations with an anti-HIV prevalence greater than 0.5%, sampling by GACRIA alone could provide a good estimate of the true prevalence. For true prevalences less than 0.5% good estimates could only be obtained if positive GACRIA reactions were confirmed by another independent salivary assay. Salivary testing for anti HIV is a convenient and potentially an accurate epidemiological tool. HTLV-1 oncoprotein Tax protein is known to be released from HTLV-1-transformed cells by a mechanism other than cell death; however, the mechanism of Tax secretion remains to be established. This study elucidates domains within Tax that contribute to its subcellular localization and secretion. Analysis of the amino acid sequence of Tax has revealed the presence of four putative secretory signals within the carboxy-terminal domain namely YTNI, LL, DHE and terminal V. Mutation of two putative signals (YTNI and DHE) resulted in aberrant subcellular localization of Tax, with cytoplasmic Tax accumulating in structures corresponding to the ER and Golgi. We have also studied the effect of these mutations on the secretion of Tax in baby hamster kidney cell (BHK-21) line. The results have demonstrated that mutating YTNI to ATNI resulted in approximately a 2-fold reduction in secretion. Mutation of LL to AA abrogated the intake of Tax, therefore, no secretion was observed. Mutating DHE and terminalV did not show any effect on Tax secretion, however, a combination of DHE mutation with YTNI mutation or AA mutation resulted in an altered intake and secretion of Tax. These studies substantiate a link between cytoplasmic Tax and its subsequent secretion and also indicate potential amino acid signals that might direct secretion of Tax.

P62 HIV Surveillance By Testing Saliva
mother to child transmission of HIV among pregnant mothers attending antenatal care clinic in Southwestern Ethiopia. Patients and Methods: A cross-sectional survey was conducted among antenatal care attendants of Jimma university hospital from Jan. 25, 2004 to Feb 25, 2004 using a structured questionnaire. At the same time unlinked anonymous blood sample was taken during routine ANC investigations. The sera were tested for HIV antibody using rapid test algorithm. In addition, full VCT service was given for volunteers. The data was analyzed by using SPSS 12th edition and chi-square and binary logistic regression were used to see associations and p-value less than 0.05 was taken as cut of point for significance. Results: On the whole, 258 women were included in the study of which four were excluded due to incompleteness of the data. The over all HIV sero prevalence was 9.8%; being 10.4 % in urban & 7.7% in rural women. Logistic regression demonstrated low income groups, illiterates, low-level workers and those who have misconception about HIV/AIDS had higher chance of HIV infection accompanied by low likelihood of accepting HIV testing. Women who have history of STD & surgery were also found to be more likely to be HIV sero positive as compared to those with out history. Though, 92.1% of mothers were indebted about the importance of HIV screening test, only 31.9 % of them received VCT service. Similarly, only 17.3% of mothers who believed breastfeeding as a potential HIV mode of transmission to the baby if they were found to be infected could afford to buy formula milk for at least six months. Conclusion: The study found out higher HIV prevalence among pregnant mothers as compared to the national figure with highest infection rate in the age group 15 -24 yrs old, reflecting recent high rate of HIV infection. In addition, provision of VCT service only would not be enough for effective prevention of mother to child transmission of HIV, as it was made known from our study, unless it is supplemented with some form of activity which would promote the uptake of VCT service by the mothers. Provision of free formula milk shall also be considered as a part of intervention program in addition to antiretroviral drug administration for effective prevention of vertical transmission of HIV as most mothers couldn't afford to buy as reflected in this study.

P66
Abstract withdrawn Infants of infected mothers have at least a 60% probability of acquiring HIV in utero. The normal latent period after infection is between 2 and 5 years, and it is estimated that for every case of AIDS, 50-100 people may be infected. Extrapolation of these estimates suggest 1,000,000 may already be infected and the established risk group for AIDS may not reflect the pattern of present infection. In Central and East Africa there now appears to be an epidemic of enormous proportions. Oocytes and spermatozoa are not attacked by the HIV virus but associated lymphocytes or monocytes may be infected. Screening for HIV for semen donation is mandatory and precautions for infection with HIV should follow procedures adopted for hepatitis B virus. Human immunodeficiency virus (HIV) research will continue to face significant barriers unless solution to enrollment and follow up are found. 30 high-risk HIV negative individuals were recruited and followed up every three months for 9 months. Multiple approaches were used in recruitment. Community education seminars were conducted in nightclubs and the public gatherings. Posters and fliers were used to invite people to the seminars. Reading materials were provided to enhancing understanding. In every visit volunteers were counseled and information on their high-risk behaviour was collected. The study shows that the follow up was 90% for the 30 volunteers. Two of the volunteers lost follow up in their third visit. The success of the follow up was due to counseling and mobilization 2 of the community. Challenges encountered in recruitment and follow up include low literacy levels, poverty, gender inequality, stigma, fears and mistrust about the vaccine. The volunteers could be easily recruited and retained for future vaccine studies. We have previously demonstrated that the C/EBP site II consensus B (conB) variant was highly conserved in brain-derived HIV-1 LTRs and that LTRs containing C/EBP site II 4C and 6G variants were only found in brain tissue of patients with HIV-1-associated dementia (HIVD). Therefore, the regional distribution of LTRs containing the conB, 4C, or 6G variant of patients with and without HIVD was examined. A statistically significant difference was found in the regional distribution of LTRs containing the C/EBP site II conB, 4C, or 6G variant in brain regions derived from patients with and without HIVD. LTRs containing a low affinity C/EBP site II 4C were shown to accumulate in the cerebellum, a site of little viral gene expression, and in conjunction with a conB site I exhibited the lowest basal LTR activity of any of the LTRs examined. LTRs containing a high affinity C/EBP site II 6G variant accumulated in the mid-frontal gyrus, a site of highly productive replication which correlates with the C/EBP site II 6G variant with a conB site I exhibiting the highest basal LTR activity. In conclusion, distinct LTR populations with specific C/EBP site II configurations were found in different regions of the brain.  Background: Genetic variations in chemokine genes have been associated with protection against infection by HIV-1 or slow progression to AIDS in infected individuals. We carried out this study to determine the prevalence of polymorphisms in CCR-5 D32 and SDF-1 3' genes an in exposed but uninfected partners of HIV infected individuals in North India. Materials and methods: 25 exposed but uninfected (EU) individuals and 25 normal healthy controls (HC) with low risk of infection were studied. Genomic DNA was extracted from whole blood and PCR was performed for genotyping. CCR-5 D32 and SDF-1 3'A were studied by PCR-RFLP using EcoR I and Msp I enzyme, respectively. Results: No CCR-5 D32 mutations were detected in any individual. Both EU and HC showed a wild type CCR-5 gene. SDF-1 3'A gene polymorphism was frequently detected. Variant allele frequencies were 36% in EU and 20% in HC (P > 0.5). Out of 25 EU's, 16 (64%) wild type, 8 (32%) heterozygous and 1 (4%) were homozygous were detected while in HC, 20 (80%) wild type and 5 (20%) heterozygous were detected. No homozygous mutation was detected in the control group. Conclusion: CCR-5 D32 polymorphism, which was common among Caucasians, was not present in our population. Increased frequency of SDF-1 3'A polymorphism was detected in both EU's and controls. Additional factors must be operating in protecting exposed uninfected individuals in our population.

P78
Abstract withdrawn Recently, CCAAT/enhancer-binding protein (C/EBP) transcription factors have been shown to form heterodimers with cAMP-responsive element binding protein 2 (CREB-2), another bZIP transcriptional regulator known to play a critical role in driving basal and Tax-mediated transactivation of the HTLV-1 long terminal repeat (LTR). Herein, we describe that overexpression of C/EBPa and C/EBPb, including the endogenous isoforms liverenriched activation protein (LAP) and liver-enriched inhibitory protein (LIP) inhibits Tax-mediated transactivation of the HTLV-1 LTR. C/EBP-mediated inhibition was not the result of competition with the viral oncoprotein Tax for recruitment of co-activators CREB-binding protein (CBP)/p300 to the viral promoter. Electrophoretic mobility shift analyses demonstrated that C/EBP proteins derived from U-937 monocytic nuclear extracts directly interacted with the Tax-responsive element 1 repeat III as well as Tax-responsive element 2. However, deletion of these sequences within the context of the full-length LTR did not prevent C/EBP-mediated inhibition. Disruption of C/EBPb/CREB-2 heterodimerization by deletion of the C/EBPb leucine zipper prevented C/EBP inhibition of Tax-mediated transactivation of the LTR. These results suggest that C/EBP binding to Tax was not a requirement for C/EBP-mediated inhibition.

P80
Redirecting the Specificity of Naturally Occurring Antibodies Using gal-alpha1, 3 Introduction: Due to the great variability and high glycosylation of gp120, possible targets for a fusion inhibitor include the CD4 binding region of gp120. Here we describe a method by which peptides corresponding to residues 25 to 64 of the CD4 receptor have been coupled to a major antigen, the gal-alpha 1,3-gal disaccharide, towards which humans have natural antibodies. Use of these fusion-molecules should redirect the specificity of the antibodies towards gp120 and possibly help reducing the viral loads of infected individuals. Materials and methods: Binding of human anti gal-alpha 1,3gal antibody glycopeptide complexes to gp120 and the virus was analysed by ELISA and a neutralization assay. The latter was based on reduction of syncytia in U87 cells in the presence of heat inactivated human serum from healthy individuals. Noninactivated serum was also used to analyse the contribution of the complement dependent cytotoxicity to the system. Results: Binding of the molecules was confirmed both by ELISA and by neutralization using the HIV 1 IIIB virus at several concentrations of the peptides with a 1:10 or 1:20 dilution of the human serum. Furthermore, complement proteins contributed to the neutralization capacity of the human anti gal-alpha 1,3-gal antibody-glycopeptide complexes. Conclusion: Taking advantage of the innate response by redirecting the specificity of natural antibodies could be a complement to existing anti retroviral therapy of HIV infected individuals. Interleukin-7 (IL-7) is a survival factor for naïve and memory T lymphocytes and it also increases T cell proliferation during lymphopenic conditions. Elevated levels of IL-7 have been found in the blood of HIV+ patients, which was considered as a homeostatic response to peripheral T cell depletion. We showed that HIV infection is associated with an increased proportion of IL-7R low/negative peripheral T lymphocytes. Down-regulation of IL-7R on T cells was correlated with the depletion of CD4+ T cells and also with the increased concentration of serum IL-7. The decreased IL-7R expression resulted in the reduced survival capacity of T cells in presence of IL-7 and was associated with low Bcl-2 expression. Mostly the memory T cells down-regulated the IL-7R and we found a strong association between CD28 and IL-7R down-regulation. Accordingly, only CD28+ T cells responded to IL-7 with strong Bcl-2 upregulation. The positive effects of IL-7 on survival and homeostatic proliferation of T cells might be severely impaired in HIVinfected individuals due to the decreased IL-7R expression. Chronic T cell activation may lead to an overall decrease of IL-7 mediated survival signals in HIV-infected individuals.

P85
Increased IFN-γ Production by NK and NKT Cells From HIV-1-exposed But Uninfected Background: Innate immunity is very active at mucosal surfaces, that are the main port of viral entry; it is known that several components of the innate immune response have a direct anti-HIV-1 activity such IFNs and chemokines (1). Interestingly, a report (2) has indicated a high frequency of plasmocytoid dendritic cells and high production of IFN-in response to Herpex simplex virus infection in long-term non-progressors and long-term survivors, suggesting an important role of these innate mechanisms in the control of HIV-1 infection.
Objective: To establish a relationship between some components of the innate immune system and the phenomenon of natural resistance exhibited by individuals who are continuously exposed sexually to HIV-1 but remain seronegatives (ESNs). Results: Among the evaluated parameters of the innate response, only the expression of IFN-by NK and NKT cells was significantly higher in exposed-seronegative individuals when compared with controls. As previously reported, HIV-1-infected individuals exhibited a significant decrease in the frequency of myeloid and plasmacytoid dendritic cells, NK cells and invariant NKT cells.
Conclusion: Since it is well known that IFN-is effective against HIV-1 in vitro, and also activates dendritic cells, NK cells Taking in view the socio cultural context of the country, considerable effort and planning is required to ensure equitable number of women participate in trials and stay through the entire duration of trial. Gender specific issues are integral to and an essential pillar of a larger programme focus on ethics. This includes building a gender sensitive perspective in members of the ethics review committee and members of the team that will be conducting the trials. Background: There is increasing concern that synonymous genetic polymorphisms in HIV can influence drug resistance and molecular evolution. This study investigated the distribution of synonymous codons in HIV-1 and SIV reverse transcriptase enzymes. Materials and methods: HIV-1 and SIV pol gene sequences were translated and the resulting protein sequences used as a scaffold to align the corresponding nucleic acid sequences. Synonymous codon usage was assessed in order to find out whether the use of unfavored codons at a given site is conserved.   Several small molecule antagonists of the HIV co-receptors CCR5 and CXCR4 are being developed as HIV entry inhibitors, but side effects have been observed in clinical trials that are likely due to agonist activity and/or cross-reactivity with closely related receptors. In order to develop high resolution maps of the binding sites for these antagonists that can be exploited to improve the activity and specificity, we are synthesizing derivatives of CCR5 and CXCR4 antagonists which contain photo-crosslinking groups at a variety positions in the molecules and that retain high affinity and activity against the receptors. Derivatives of two CCR5 antagonists have been crosslinked to affinity-purified CCR5 or CCR5 expressed on cells, and the interaction sites are being mapped by mass spectrometry. Techniques for purification, crosslinking, CNBr and/or trypsin digestion, and LC/MS/MS and MALDI-TOF mass spectrometry of highly hydrophobic peptides initially developed using the rhodopsin GPCR system have been applied with some success to CCR5. The peptide photo-crosslinked to a derivative of the antagonist TAK-779 has been identified, and modeling of the interaction with CCR5 suggests that it binds within the transmembrane region of the receptor and is oriented parallel to the transmembrane helices -in striking contrast to the perpendicular orientation expected for GPCR agonists.
cell-associated forms of HIV-1. Flow cytometric analyses have shown that PEHMB exposure results in epitope-specific alterations in the detection of viral co-receptors CXCR4 and CCR5, suggesting that PEHMB acts by interfering with events critical to viral binding and entry. Changes in co-receptor detection have also been observed up to 24 hr after removal of PEHMB. Consistent with the latter result was the demonstration of persistent protection from infection in experiments in which cells were challenged with HIV-1 after removal of PEHMB. Cumulatively, these results suggest that HIV-1 co-receptors may play roles in PEHMB-mediated protection from HIV-1 infection, and that products containing PEHMB may provide both shortand long-term protection against HIV-1 transmission. Immunization with more than one immunogen (co-immunization) is an efficient regimen to induce immunity to multiple antigens. However, immune interference has been reported using multi-plasmid DNA immunizations (e.g. co-immunization using HIV-1 Gag-Pol p160 and Vpr). HIV-1 envelope (Env) and Gag gene products are the most predominant immunogens used in current AIDS vaccines, although, few studies have evaluated possible immune interference when these two antigens are co-administered. Therefore, in this study, immune interference during co-inoculation was examined using DNA vaccines expressing Env gp120 and Gag p55 from gene sequences optimized for efficient expression in mammalian cells (codon-optimized). BALB/c mice vaccinated with each plasmid individually elicited high titer immune responses, however, when these same plasmids were co-inoculated, there was a reduction in the immunity elicited to Gag p55 . In contrast, anti-Env gp120 immunity was not affected. To determine if the anti-Gag immune interference was specific to Env, mice were co-immunized with plasmids expressing a soluble form of hemagglutinin (sHA) from influenza virus (A/PR/8/34) and Gag p55 -DNA. Similar titers of anti-Gag p55 immunity were observed in mice co-immunized with sHA-DNA and Gag p55 -DNA, as mice vaccinated with Gag p55 -DNA only. Gag p55 -DNA co-immunization did not affect anti-Env gp120 or anti-sHA immune responses. The Env gp120 / Gag p55 immune interference elicited during co-immunizations was not dependent on the amount of protein expressed. In addition, this induced immune interference was observed in mice even when the immunizations were performed in separate locations. Therefore, Env gp120 specifically interferes with the elicitation of anti-Gag p55 immune responses following co-immunization. Since Env and Gag are the most commonly used HIV-1 antigens in AIDS vaccine designs, future vaccine development should consider the effect of each immunogen during evaluation of the effectiveness of AIDS vaccines.

P95
transcription factors, such as NFkB and NFAT for its transcription and expression. It has previously been shown that HIV-1 preferentially replicates in Th2 cells, but the mechanism of action for this finding remains unknown. c-maf is a Th2-restricted transcription factor that is critically important for differentiation along a Th2, but not Th1 lineage, and for transcription of the prototypic Th2 cytokine, IL-4. c-maf directly binds the proximal IL-4 promoter and acts synergistically with a neighboring NFAT site. We have demonstrated at the individual cell level that IL-4 positive cells (Th2) preferentially support HIV-1 replication compared to IFNg positive (Th1) cells. In studying the HIV-1 long terminal repeat (LTR)/promoter sequence, we identified a MARE (maf-recognition element) located just proximal (5' Similarly, over-expression of c-maf alone, or with NFAT1 or 2, increases HIV-1 replication, as measured by intracellular p24/gag expression, in CD4 T cells co-expressing GFP but not in GFP negative/ c-maf-negative controls within the same transfected population. Lastly, depletion of c-maf expression by siRNA in primed and HIV-1 infected CD4 T cells decreases p24/gag expression. In summary, the Th2-specific transcription factor, c-maf, binds the HIV-1 promoter in cooperation with NFAT transcription factors, primarily NFAT2, to augment HIV-1 transcription and replication in primary human CD4 T cells. These are the first data to mechanistically explain preferential HIV-1 transcription in IL-4 producing (Th2) cells.

P104
HIV- Results: In spite of limited fundings, 500 women have been trained on STI/HIV/AIDS, stigma discrimination and human rights. About 20 PLHA have received capacity on communication skills, self esteem and knowledge of the media environment, and three load chapters have been created. RWAN has matured into a respected independent and sustainable instutions, participates in various national and international conferences, and organizes national prevention campaign through radio advertisment and street banners. Conclusion: Educating, engaging and structuring rural women is an invaluable asset in combating stigma and discrimination, promoting human rights; engaging women to prooduce quantities of quality articles and raising self-esteem among PLHA's from victims to a profile of courage.
(LNACP) that the rate of infection on our growing population in term of those infected with the virus are increasing immensely. Many people do not really believe that AIDS is real. People are dying ignorantly from the virus. Other people just believe that something just have to kill someone. In the year 2004 the rate of infection was estimated to be 11-12 % of the approximately 3 million people in Liberia. By this time we are optimistic that there is an increase in the infection rate which is basically caused by the high rate of illiteracy, the influx of more foreigners or aliens on peace mission and business purposes, the high rate of poverty (80-90%) also cause by the high rate of unemployment leaving many people vulnerable to the virus.
Methods: A team of awareness on AIDS education took the city of Monrovia and its surroundings, congregating students from various high schools between the ages of 12-20 explicating the danger and prevention of AIDS from poster prints, tracts, and handouts for better understanding. Audio visual aids were actually needed to authenticate the information provided, but none was available. Questionaires were distributed to evaluate their comprehensions and for statistical purpose.
Results: Of the total number of students 55% of them now believe that AIDS is real whilst 35% believe AIDS does not exist; on the other hand 10% believe that abstinance is the best method of prevention. Conclusion: Peer education-one on one and one on group methods of awareness, engaging and structuring the press, promoting human rights, engaging journalists to produce quantities of quality articles, raising awareness among policy makers, high level government officials and the public in general are invaluable asset in combating HIV/AIDS. Capacity building workshops and conferences are most needed among the various tribes in worship centers. If these are considered and fully supported by international AIDS groups working in collaboration with local based groups Liberia will be a success story.

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Abstract withdrawn standing of RANTES structure-function relations and obtain more potent antiviral peptido-mimetics, we have extensively mutagenized the prototypic peptide, R11-29. This presents two clusters of hydrophobic residues at its termini, (corresponding to RANTES Nloop and b1-strand) connected by a positively-charged linker. Single or multiple alanine substitutions within the N-or C-terminal hydrophobic clusters resulted in a dramatic loss of antiviral activity, whereas deletion of selected residues within the hydrophilic linker had no major functional consequences. Based on RANTES 3D structure, we designed a series of modified peptides, resulting in a progressive increase in specific antiviral activity. These peptides also displayed anti-inflammatory properties blocking RANTES-elicited lymphocyte chemotaxis. Background: Vicriviroc (SCH 417690) inhibits HIV-1 infection by blocking the viral CCR5 co-receptor. Early HIV replication is associated with rapid depletion of CCR5+ CD4 T lymphocytes that predominate in gut-associated lymphoid tissue (GALT), an important site of early establishment of HIV infection. Given the rapid absorption of oral Vicriviroc, appreciable drug exposure to GALT is predicted, potentially protecting this important component of the immune system. Materials and methods: An oral 5 mg/125 Ci/kg dose of 14 C-labeled Vicriviroc was administered to rats and drug concentrations determined by autoradiographic techniques at various timepoints up to 168 hr. Results: Vicriviroc rapidly permeated the gut wall resulting in appreciable drug exposure to GALT. The rank-order in cumulative Vicriviroc exposure was GALTl > ymph node ! lungs > blood, although exposure to GALT<spleen. GALT drug concentrations up to 48 hr were 10-to 10 3 -fold higher than the targeted IC90 concentration (IC90 = 6 nM). Differences in cumulative drug exposure between GALT and other tissues was the result of differences in both the observed peak drug concentration and in clearance rates from the individual tissues. Conclusion: Results suggest that high and sustained Vicriviroc concentrations in GALT can be achieved by as little as a single oral dose, which may prevent viral replication in these tissues and reduce the depletion of CCR5+ CD4 T lymphocytes.  http://www.retrovirology.com/supplements/2/S1 In clinical trials, vicriviroc co-administered with !100 mg QD ritonavir (RTV), a potent CYP 3A4 inhibitor, resulted in a Cmax 2 to 3 times higher and an AUC(0-12 hr) 4 to 5 times higher than vicriviroc alone. In vitro pre-or co-incubation inhibition studies with HLM demonstrated that vicriviroc does not significantly inhibit the activities of CYPs 1A2, 2A6, 2D6, 2C9, 3A4, or 2C19 at concentrations up to 26.7 g/mL (100 X the expected human plasma Cmax following once daily oral doses of 10 mg + RTV), which suggests that vicriviroc is not an inhibitor of major CYP enzymes. The results suggest that formation of the major vicriviroc metabolites in human liver microsomes is primarily mediated via CYP3A4, and that vicriviroc, at clinically relevant doses, is unlikely to inhibit other co-administered drugs metabolized by the major CYP 450 enzymes.

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Abstract withdrawn To improve their prognoses, these problems must be addressed.
hepatitis C virus (HCV). However the prevalence of HCV infection varies throughtout the world. HCV is transmitted primarily through blood or blood products or contact with infected tissue (blood transfusion intravenous immunoglobulins, intavenous drug abuse and tissue transplant). Since hepatitis C is a prevntable disease, accurate description of the prevalence and risk factors for the disease is a pre-requisite for prevention. This study is of public health importance as the National Transfusion Service has no policy to screen donated units of blood for hepatitis C. This is so due to the non availability of supporting epidemiological data and prohibitive costs of the HCV testing kits. Materials and methods: This was a cross-sectional study of 1600 regular health blood donors. These donors underwent a routine blood donor selection process. Blood sample of 5-8 mls was collected into a labelled vacutainer. Blood was allowed to clot, after centrifuging 2 mls of serum was stored at -20 degrees celcius for further analysis for confirmation of antibodies to HCV. Serum samples were also analysed for hepatitis B (HBV), Syphilis and HIV 1/2. All antibodies to HCV were determined using ELISA Abbott murex (version 4.0) which has 99% sensitivity and 99.9% specificity. Statistical analysis was performed using EPI Info version 6. Prevalence of HIV-HCV at 95% confidence interval (CI) were calculated. Chi-squared analysis was done to test for 5% level of significance. A p-value of less than 0.05 was considered significant. Fisher's test-2 tail probability was used to determine for the association between factors when the expected frequency was less than 5. Seropositve samples were not confirmed using molecular methods due to limited resources Results: The median age (Q1, Q3) of the blood donors was 31 (22,46) and 56% of them were male. ELISA assay was positive for 28 samples in the studied population yielding an overall prevalence of 1% (95% CI 0.7-1.5%). There was no dual infection for HBV and HCV. 1.2 % Anti-HCV individuals were also positive for HIV1/2. 1.7 % individuals tested positive for syphilis as well as positive to anti-HCV. No association was detected between age and seropositive status (p = 1.000). There was 18% risk of acquring infection from blood transfusion (RR = 1.18, 0.78<RR<1.79). Conclusion: Considering the nature of the population studied the prevalence of HIV-HCV co-infection of 1.2% is high. Screening all donated blood for HCV must be mandatory. There is also need to carry out a study of HCV co-infetion in patients living with HIV/AIDS since anaemia of chronic illness is a common trigger for blood transfusion in this group.

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Abstract withdrawn Infection of human cells by human T cell leukaemia virus (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein makes first contact with the target cell through binding to the cell surface receptor Glut-1, thereby allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. We have now used a soluble recombinant form of gp46 fused to the Fc-region of human IgG (sRgp46-Fc), and a panel of antibodies and inhibitory peptides to probe envelope function during cell-to-cell viral transfer. We have been able to recapitulate the transfer of HTLV-1 between cells through sites of tight cell-to-cell contact that have been termed the virological synapse. We now demonstrate that upon contact with HTLV-1 infected T-cells, the HTLV-1 receptor glucose transporter-1, Glut-1, is redistributed within the membrane of target cells. On the non-infected target cell Glut-1 is re-localized to, and enriched within, the point of synaptic T-cell contact. Importantly, this re-localization of Glut-1 on target cells occurs rapidly, and is specific for Glut-1, as irrelevant cell surface markers do not show polarized redistribution. Significantly, we find that redistribution of Glut-1 reflects the pattern of envelope accumulation on the HTLV-1 infected cell. Moreover, we find that sRgp46-Fc is also able to effect re-localization of Glut-1 on T cells, suggesting that envelope-mediated recruitment of Glut-1 to the site of synaptic transfer may be a crucial event in the cell-to-cell transfer of HTLV-1. Our recent results will be presented, and the implications of our findings for HTLV-1 pathogenesis will be discussed.
Progressors (LTNP), 70 Progressors, 135 HIV+ HAART treated, and 207 seronegative donors. We found anti-CCR5 Abs in a fraction of LTNP (22.9%), but not in the other populations studied (p < 0.0001). These Abs efficiently prevent infection of HIV-R5 strains representing subtypes B, C and A by inducing a stable and long last down regulation of CCR5 on surface of T lymphocytes. Follow-up studies showed that the loss of anti-CCR5 Abs, occurred in some subjects, was significantly associated with a progression toward disease. Thus, the anti-CCR5 Abs could be relevant to vaccine design and therapeutics. Plasmid DNA encoding human Interleukin-12 (IL-12) was produced under GMP conditions and injected into lesions of nine patients with malignant melanoma (stage IV) previously treated with both, standard and non-standard therapies. The treatment was based on efficacy in preclinical studies with melanoma in mice and gray horses. The DNA was applied in cycles, three injections per cycle, up to seven cycles. Three therapy arms comprised low (2 mg), medium (4 mg) and high (10 to 20 mg) amounts of total DNA. The therapy was well tolerated. Three out of nine patients experienced a clinical response, 2 SD and 1 CR. One patient receiving a low dose of DNA experienced a long-lasting stabilization of the disease for more than three years, while the other two responders received high doses of DNA. All patients but one (P9) experienced a transient response at the intratumoral injection site. Immunohistochemical staining of responders showed local reduction of angiogenesis and lymphocyte infiltrations. All patients, in particular the responders (P3, P7, and P8) exhibited an antigen-specific immune response against MAGE-1 and MART-1. Biopsies of responders showed some increase of IL-12, IP-10 and IFN-. (Hu. Gene Ther. 16, 35 (2005)). Combinations of IL-12 DNA with other therapies show significant increase of efficacy in preclinical studies and will be discussed.

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Abstract withdrawn Background: In West Africa, the high level of Stigma and discrimination attached to the victims of HIV/AIDS is due to the lack of many factors including the lack of free and cheaper antiretroviral drugs. This is due to poverty on the part of victims and the lack of good policies by the government to subsidise ARVs. Methods: Discussing sexuality issues publicly in West Africa is like a taboo, therefore several techniques have to be adopted inorder to collect adequate informationfor analysis. The information collected was drawn from four countries, namely; Liberia, Ivory Coast, Ghana and Benin. In the process, schools refugee camps internally displaced campsnight clubs bars and other public places including market grounds and youth centers. Interviews were conducted and questionaires were given at times and also group discussionswere held inorder to get the individual and general view of the masses.
Results: The assessment passed out well with just few minor obstacles relating to some discussions in some areas forbidden by social customs and traditions. In total 300 persons were surveyed, ages 12 to 25. Ninety percent of illiterate the people know that AIDS is a sickness but do not know how it is transmitted, whereas 50% of the students know that AIDS exist and it is real but do not know the actual root causes or what even the acronym stands for. About 20% of them do not know the difference between high risk behavior and low risk ones. About 98% of the students agree that stigma and discrimination affects the rate of growth of infected persons whilst 40% of sexually active youthsaid that they do not like the use of condoms. About 85% of them said that they can never befriend someone with AIDS i.e they cannot eat, talk, shakehandsor share clothes. Surprisingly 23% said that if the doctor tells them that they are HIV positive they will either commit suicide or get extraordinarily promiscuous to spread the disease and not to die alone. Conclusion: It is possible to decrease stigma and discrimination by providing information on HIV/AIDS. This should focus on what the virus is how it can be transmitted and prevented. There should be intensive education on how victims of the pandemic should be treated and cared for. Governments shoul subsidise the importation of cheaper and safer antiretroviral drugs and and do everything possible to reduce stigma and discrimination as relate to HIV/AIDS. Stigma and discrimination will be reduced because some hope, at least is provided for a longer and healthier life. If drugs are provided to buttress the counseling received, people will feel comfortable when affected.

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The Immunomodulatory Agent Rapamycin Potentiates the Antiviral Activity of the Fusion Inhibitor T20 Against R5 Strains of HIV-1 Alonso Heredia, Anthony Amoroso, Charles Davis, Nhut Le, Douty Bamba, Robert Gallo and Robert Redfield Institute of Human Virology, University of Maryland, Baltimore, MD Retrovirology 2005, 2(Suppl 1):P134 The fusion inhibitor T20 marks the beginning of a new era in the management of HIV-1 disease. By inhibiting viral entry, T20 suppress viral replication in patients carrying strains resistant to reverse transcriptase or protease inhibitors. However, its antiviral activity is compromised by mutations in gp41. Based on our previous work demonstrating that Rapamycin (RAPA) inhibits R5 HIV-1 by downregulating CCR5 surface expression, we now show that RAPA and Retrovirology 2005, 2(Suppl 1) http://www.retrovirology.com/supplements/2/S1 T20 synergize in antiviral activity against R5 strains. Synergy studies using the Median Effect analysis revealed that the IC50 values of RAPA and T20 in the RAPA/T20 combination were reduced 9-and 3-fold, respectively. Three-Dimensional modeling confirmed the observed synergy (synergy volume of 253.85; 95 % CL : 91-147). We also show that the RAPA/T20 combo, but not T20 alone, prevented the emergence of T20 resistance upon continuous passage of R5 HIV-1 ADA on PBMCs for 24 weeks under subinhibitory concentrations of T20. In addition, R5 ADA and YU-2 clones carrying T20 single mutations 36D, 38M or 43K (4-10 fold resistance) or the double mutation 36D/38M (65-fold resistance), were all inhibited in the presence of RAPA. In conclusion, our results demonstrating that the RAPA/T20 combination has synergistic antiviral activity, prevents the emergence of T20 resistance, and inhibits T20 resistant strains, suggest a novel therapeutic approach to enhance the antiviral activity of T20 in patients carrying R5 strains of HIV-1.

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Abstract withdrawn Background: It is extremely difficult to have a clear picture of HIV/AIDS in Liberia. Liberia has been ravaged by arm conflict for the past fourteen years. Due to this it has been very difficult to collect and analyze data in the length and breadth of this tiny West African republic. The bad roads and the constant back and forth movement of refugees and internally displaced persons from one place to another is a contributing factor. Methods: There were wide ranging places that our assessment covered. We were in the market places, Nightclubs and bars as well as schools and the most important of all we were from door to door. This was a long a hectic process that took more than six months. Questions were being asked in the form of conversation and at some time for the literates questionnaire were issued to be filled in. Gifts were given to people at times for encouragement. Children at various schools were allowed to have time to discuss AIDS and some causes among women and young girls.
Results: There are numerous problems that must be addressed in order to smoothly run programs related to AIDS. Firstly, a vast majority of the people is inaccessible; lack of roads has become a barrier separating advocates from victims and vulnerable people. These people lived in isolated areas and remote areas that can only be reached by foot. Secondly, there is a high level of illiteracy and this is due to poverty. There are very few trained personnel that are available to relate to people in the rural areas and the most vulnerable. Conclusion: One of the simplest methods in resolving this issue is by providing means by which local trainers can be trained because they know the terrain, language, culture and tradition of the local people. Poverty alleviation programs should be intensified through literacy programs. HIV/AIDS and other STD's awareness programs should be integrated into schools curricula as a subject and teachers as well as parents should abandoned that age-old legacy of not discussing issues relating to sexuality with adolescents and the public in general.

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The The objective of inducing broadly cross-reactive neutralizing antibodies against HIV-1 is promatic because of the high sequence variability of the viral envelope protins and the general resistance of primary isolates to neutralizating. The gP120 glycoprotein elicits both virus-neutralizing and non-neutralizing antibodies during natural infection. Non-neutralizing antibodies are often directed against the gP120 regions that are occlude on the assembled trimer which are exposed only upon shedding. Neutralizing antibodies must access the functional envelope glycoprotein complex and typically recognize conserved or variable epitopes near the receptor-binding regions.
HIV envelope glycoproteins that are conserved among diverseviral strains are poorly expose to the humoral immune system. The conserved gP120 surfaces involved in binding to its three minimally polymorphic ligands, gP41, CD4 and chemokine receptors, each exhibit particular problems with respect to the elicitation of or sensitive to neutralizing antibodies. The moieties involved in gP120-gP41 association are buried in the interior or of the functional envelope glycoprotein spike. The CD4 binding site is recessed, flanked by variable regions exhibiting considerable glycosylation. The chemokine receptor-binding site is masked by varial loops-V3 and V2. The relatively conserved HIV-1 gp120 core has been structurally analyzed, the outer domain exhibits a variable, heavily glycosylated surface. This concentrated glycosylation may reduce the potential of a large portion of the gP120 surface to serve as an immunogenic target.
costs, increase success rates, and identify failures during discovery and preclinical development. Moreover, the growth in cell-based screening has led to the development of novel cell lines and technologies to increase the throughput and data output of cell-based assays. Therefore, we have also developed a new reporter system that allows monitoring of HIV-1 protease activity and susceptibility to protease inhibitors in living cells.
Our aim was to construct a cell-based assay for assessing the activity of intracellular HIV protease, use it to screen protease inhibitors and test HIV susceptibility to these drugs. Functional bioassay for resistance was constructed without the need to culture infectious HIV. These assays are based on processing of recombinant reporter proteins in mammalian cells using wild-type PR or a pool of patient-derived PR sequences. Assays not involving infectious HIV should be simpler, faster, safer, and more economic and allow implementation in clinical routine labs, which are generally not equipped for virus culture. Moreover, its application for searching inhibitors of this important enzyme will provide faster, high-throughput and reliable results. The working hypothesis was to test if a transactivator protein conjugated to the cytoplasmic portion of a cellular receptor, via the respective cleavage peptide, could be used as an indicator of HIV protease susceptibility. This assay was constructed in a cell line that expresses an indicator protein under the inducible action of the transactivator. The cleavage of the transactivator protein by the action of the protease resulted in the expression of luciferase orgalactosidase. In preliminary experiments, the signal obtained correlates with the intracellular activity of the protease. The optimized cleavage assay is currently being used for testing current protease inhibitors to compare the IC50 values to reference virologic assays. We analyzed the association between evolution of the 5' exon of tat and disease progression in a SIV/SHIV macaque model of opiate-dependence and AIDS. We cloned tat sequences using RT-PCR of plasma virus from eight animals at three time points following infection. Six of these monkeys were part of a morphine-dependent cohort, while two served as non-drug using controls. We found a significant inverse correlation between disease progression and tat diversity in plasma by 20 weeks. The morphine cohort segregated into two classifications based on progression: a rapidly progressing group (Group A) and a second set (Group B) that progressed at a rate similar to the two non-morphine controls (Group C). The three animals in Group A exhibited À40% (p = 0.01) and À50% (p = 0.028) less diversity than Group B and C animals, respectively. Group A animals showed prominent re-emergence of the wild-type inoculum tat sequence as illness progressed. This suggests that the virus from the original infection represented the most pathogenic form in these cohorts throughout the first 20 weeks of infection. Our results indicate that in vivo morphine dependence can contribute to the pathogenesis of SIV/SHIV infection and that it may do so in conjunction with the evolution of viral proteins, such as Tat. It is unclear if this is a direct effect of morphine on the virus replication/evolution or if it is mediated indirectly through modulation of the immune response, or through the enhanced vulnerability of a protected compartment such as the CNS.
ubiquitinated 62-64 kDa CXCR4, and in the co-precipitation of the 62 kDa CXCR4 with CD4. Striping of CD4 from the cell surface prior to cell lysis only partially reduced co-precipitation of CD4 with the 62 kDa CXCR4, revealing a pool of intracellular CD4-CXCR4 complexes. Brefeldin A and monensin reduced co-precipitation of CXCR4 with CD4, suggesting that late endosomes play a role in intracellular association of CXCR4 with CD4. Our data demonstrated a correlation between the enhanced susceptibility of activated T cells to HIV-1 fusion and an increase in CXCR4-CD4 complexes that may shuttle between late endosomes and the cell surface. The HIV-1 Tat gene is required for virus replication and disease. Tat was reported to be released from infected cells. Recombinant soluble Tat may be taken up by many cell types and transported to the nucleus as an active transcription factor leading to upregulation of viral replication in bystander cells. However, numerous attempts to use Tat protein or Tat expressing constructs for protective immunization in non human primate model produced controversial results, leading us to ask whether the effects of Tat might be indirect and result from increased expression of secondary mediators like cytokines or growth factors. Immunization with Tat protein produces antibodies to a limited number of linear epitopes in animals and human beings, mainly located in the N-terminus of the molecule. We generated a unique prototypic monoclonal antibody TR1 that recognizes an epitope in the N-terminus of HIV-1 Tat and inhibits Tat uptake by reporter cells. This antibody strongly neutralizes recombinant Tat protein in a cellular assay for the induction of a latent Tat deficient HIV-1 provirus. Expression of Tat protein in different cell types leads to the accumulation of a ''transactivation activity'' in culture medium. However, 1) this activity cannot be inhibited by Tat-neutralizing antibody TR1; 2) Tat protein is detected in the supernatants only in low nanomolar concentrations; 3) Similar amounts of exogenous recombinant Tat protein are not sufficient to induce detectable transactivation of in our assay system. This activity is also distinct from proinflammatory cytokines like TNF-a, IL-1b or IL-6. We hypothesize that positive effect of Tat on viral replication in bystander cells in vitro and possibly in vivo may be indirect and mediated by an unknown secondary cytokine(s) or growth factor(s) released from Tat expressing cells. A likely process of chronic positive selection produces the highly biased peripheral blood T-cell repertoire of adult human beings. The major blood subset expresses the V2V2 T-cell receptor and responds to phosphoantigen stimulation in the absence of MHC restriction. Chronic expansion of T-cell pool is expected to produce a population of cells with the effector/memory phenotype. A CC chemokine RANTES is produced late after TCR stimulation of Tcells, accumulates into the cytoplasm and represents a marker for nonnaïve T-cells. We demonstrate here that the vastmajority of peripheral human T-cells contain RANTES in the cytoplasmic granules. In vitro expansion after non-peptidic phosphoantigen stimulation mimics the normal T cell response to pathogens, and produces polyclonal V2/ V2 cell population uniformly positive for cytoplasmic RANTES. These cells readily release RANTES from cytoplasmic depots into the culture medium after TCR stimulation. The presence of stored RANTES suggests a memory phenotype and may mediate effector functions of circulating V2/V2 cells. Phosphoantigen-responsive V2/V2 T cells represent 1 in 40 of circulating CD3+ lymphocytes; this is the dominant central memory population in primate peripheral blood that can evolve directly into an effector memory pool.

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and Ivory Coast couple with political instability in Nigeria, Guinea Bissau and recently Togo have put Women and children at risk to diseases and poverty. These women struggled with their in order to meet the basic necessity of life. We take this as a serious problem because according to Church World service refugees and internally displaced persons are six times more likely to get infected with the virus than their counterparts in normal condition.
Methods: There were wide ranging places that our assessment covered. We were in the market places, Night clubs and bars as well as schools and the most important of all we were from door to door. This was a long a hectic process that took more than six months. Questions were being asked in the form of conversation and at some time for the literates questionnaire were issued to be filled in. Gifts were given to people at times for encouragement. Children at various schools were allowed to have time to discuss AIDS and some causes among women and young girls. Results: After visiting the Liberian Refugee Camps in Ghana and the Ivory Coast and also displaced camps in Liberia Guinea and Sierra Leone several data were collected which are clear representation of the vulnerability of women and young girls. About 80% of the women told us that they get involved in risky sexual behaviour inorder to keep up their children. Also about 72% of young girls get involved in sexual behaviour because of the pressure from men and also rape. Ten percent told us that whenever they drink alcohol the feel like indulging in sexual activity. The location of wells and market places as well as the closeness of the houses are factors that increase their vulnerability. Conclusion: After this assessment it can be concluded that the main causes of the growing rate of infection in the sub region is poverty and instability. The only way to solve this is to first have political stability in the region where people will go through the normal education process and become soundly education. If they are sound they will not be too greedy for power to the detriment of the masses. There are enough resources that can be exploited in order to feed the people of the sub region. The international community should think on stability before fighting the virus. Elevated IL-6, IL-10, IL-8, gro-alpha and TNF-alpha and decreased IL-2 values have been regularly observed in HIV infected individuals. To study the influence of the transmembrane envelope protein gp41 of HIV-1 on the cytokine production by human blood donor PBMCs, additional cytokine arrays (RayBiotech) that measured the release of about 100 cytokines, were used. In parallel a synthetic peptide corresponding to a domain highly conserved amongst all retroviruses, the so-called immunosuppressive (isu-) domain, was studied. The isu-peptide was used as a homopolymer, since unconjugated peptides were inactive. The expression of cytokines such as IL-6, IL-8, IL-10, RANTES, MCP-1, MCP-2, gro-alpha, TNF-alpha, MIP-1alpha, MIP-1beta, MIP-3 increased upon exposure to the transmembrane envelope protein gp41 and the isu-peptide of HIV. In contrast, the expression of IL-2 decreased and the expression of the other cytokines remained unchanged. The extent of changes in the cytokine expression varied from donor to donor. These data confirm and extend previous data obtained with purified HIV-1 and porcine endogenous retrovirus (PERV) particles, the transmembrane envelope proteins gp41 of HIV-1 and p15E of PERV and their isu-peptides. These data indicate that retroviral transmembrane envelope proteins modulate the cytokine production of normal PBMCs and therefore may play an important role in retrovirus-induced immunopathogenesis.
generally measured by spectratyping and is defined as Vg2 chains encoded by sequences between 990 and 996 nucleotides in length. In a previous cross-sectional study (Bordon, et al. JID 189:1482, 2004 we demonstrated a relationship between the duration of therapy and recovery of Vg2 sequences between 990-996 nucleotides. Now using longitudinal specimens and extensive analysis of Vg2 chains by DNA sequencing, we show that prolonged HAART does indeed reconstitute the repertoire of 990-996 length Vg2 chains, but they are no longer restricted to using the Jg1.2 segment. Instead, we observe Jg1.1 and Jg2.3 sequences with unusual N regions sequences that are rare in healthy individuals. In this unique example, we show that HIV infection exhausts the population of T cell clones capable of expressing the Vg2-Jg1.2 chain, but that strong selection for Vg2 chains in the range of 990-996 nucleotides, forces the expansion of a previously minor population. Within the Vg2Vd2 T cell population, prolonged therapy enabled a functional reconstitution of the Vg2 repertoire. Even though repertoire selection for Vg2 is largely independent of thymic function, there was no evidence that T cell clones could be replaced from hematopoietic stem cells, despite the continued strong selection for Vg2 chains of the appropriate length. Caco-2 cells (passage 60 to 61) were grown for 3 weeks to confluency and the integrity of the monolayer was confirmed by TEER measurements in the presence of vicriviroc (50 to 400 mM). For bi-directional permeability studies, vicriviroc was placed on either the apical (A) or basolateral (B) compartment at a concentration of 40 mM and permeability (n = 3) was determined over 2 hrs with an LC/MS/MS assay. Total recovery exceeded 85% in all studies. The passive permeability (A to B) performance of the caco-2 cell monolayers were confirmed with atenolol (Pc = 3 AE 1.7 nm/s) and pindolol (Pc = 200 AE 9 nm/s). Functional expression of pGp was confirmed with the standard pGp substrate digoxin (bi-directional efflux ratios: 4-to 10-fold). Vicriviroc showed high A to B permeability (Pc = 400 AE 4 nm/s) consistent with its high in vivo oral absorption. The bi-directional efflux ratio of vicriviroc was only 0.6 indicating that it is not a pGp substrate in vitro. These data suggested that pGp is unlikely to affect the oral absorption of vicriviroc and that co-administration of vicriviroc with a pGp inhibitor is unlikely to cause significant drug-drug interactions.