Inhibitors of Signaling Pathways That Block Reversal of HIV-1 Latency.

Signaling pathways play a key role in HIV-1 latency. In this study, we used the 24ST1NLESG cell line of HIV-1 latency to screen a library of structurally diverse, medicinally active, cell permeable kinase inhibitors, which target a wide range of signaling pathways, to identify inhibitors of HIV-1 latency reversal. The screen was carried out in the absence or presence of three mechanistically distinct latency-reversing agents (LRAs), namely, prostratin, panobinostat, and JQ-1. We identified inhibitors that only blocked the activity of a specific LRA, as well as inhibitors that blocked the activity of all LRAs. For example, we identified 12 inhibitors targeted toward protein kinase C or downstream kinases that blocked the activity of prostratin. We also identified 12 kinase inhibitors that blocked the reversal of HIV-1 latency irrespective of the LRA used in the screen. Of these, danusertib, an Aurora kinase inhibitor, and PF-3758309, a PAK4 inhibitor, were the most potent. The 50% inhibitory concentrations in the 24ST1NLESG cells ranged from 40 to 147 nM for danusertib (selectivity indices, >150) and from 0.1 to 1 nM for PF-3758309 (selectivity indices, >3,300). Both danusertib and PF-3758309 inhibited latency reversal in CD4+ T cells isolated from HIV-1-infected donors. Collectively, our study describes a chemical approach that can be applied to elucidate the role of signaling pathways involved in LRA activity or the maintenance of HIV-1 latency and also identifies inhibitors of latent HIV-1 reactivation that could be used with antiretroviral therapy to reduce residual viremia.

Identification of the transcripts associated with spontaneous HCV clearance in individuals co-infected with HIV and HCV.

BACKGROUND: Infection with human immunodeficiency virus (HIV) influences the outcome and natural disease progression of hepatitis C virus (HCV) infection. While the majority of HCV mono-infected and HCV/HIV co-infected subjects develop chronic HCV infection, 20-46% of mono- and co-infected subjects spontaneously clear HCV infection. The mechanism underlying viral clearance is not clearly understood. Analysis of differential cellular gene expression (mRNA) between HIV-infected patients with persistent HCV infection or spontaneous clearance could provide a unique opportunity to decipher the mechanism of HCV clearance. METHODS: Plasma RNA from HIV/HCV co-infected subjects who cleared HCV and those who remained chronically infected with HCV was sequenced using Ion Torrent technology. The sequencing results were analyzed to identify transcripts that are associated with HCV clearance by measuring differential gene expression in HIV/HCV co-infected subjects who cleared HCV and those who remained chronically infected with HCV. RESULTS: We have identified plasma mRNA, the levels of which are significantly elevated (at least 5 fold, False Discovery Rate (FDR) <0.05) before HCV infection in subjects who cleared HCV compared to those who remained chronically infected. Upon further analysis of these differentially expressed genes, before and after HCV infection, we found that before HCV infection 12 genes were uniquely upregulated in the clearance group compared to the chronically infected group. Importantly, a number of these 12 genes and their upstream regulators (such as CCL3, IL17D, LBP, SOCS3, NFKBIL1, IRF) are associated with innate immune response functions. CONCLUSIONS: These results suggest that subjects who spontaneously clear HCV may express these unique genes associated with innate immune functions.

Dendritic cells restore CD8+ T cell reactivity to autologous HIV-1.

UNLABELLED: Recall T cell responses to HIV-1 antigens are used as a surrogate for endogenous cellular immune responses generated during infection. Current methods of identifying antigen-specific T cell reactivity in HIV-1 infection use bulk peripheral blood mononuclear cells (PBMC) yet ignore professional antigen-presenting cells (APC) that could reveal otherwise hidden responses. In the present study, peptides representing autologous variants of major histocompatibility complex (MHC) class I-restricted epitopes from HIV-1 Gag and Env were used as antigens in gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and polyfunctional cytokine assays. Here we show that dendritic cells (DC) enhanced T cell reactivity at all stages of disease progression but specifically restored T cell reactivity after combination antiretroviral therapy (cART) to early infection levels. Type 1 cytokine secretion was also enhanced by DC and was most apparent late post-cART. We additionally show that DC reveal polyfunctional T cell responses after many years of treatment, when potential immunotherapies would be implemented. These data underscore the potential efficacy of DC immunotherapy that aims to awaken a dormant, autologous, HIV-1-specific CD8+ T cell response. IMPORTANCE: Assessment of endogenous HIV-1-specific T cell responses is critical for generating immunotherapies for subjects on cART. Current assays ignore the ability of dendritic cells to reveal these responses and may therefore underestimate the breadth and magnitude of T cell reactivity. As DC do not prime new responses in these assays, it can be assumed that the observed responses are not detected without appropriate stimulation. This is important because dogma states that HIV-1 mutates to evade host recognition and that CD8+ cytotoxic T lymphocyte (CTL) failure is due to the inability of T cells to recognize the autologous virus. The results presented here indicate that responses to autologous virus are generated during infection but may need additional stimulation to be effective. Detecting the breadth and magnitude of HIV-1-specific T cell reactivity generated in vivo is of the utmost importance for generating effective DC immunotherapies.

Abasic phosphorothioate oligomers inhibit HIV-1 reverse transcription and block virus transmission across polarized ectocervical organ cultures.

In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2' deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

The CD8 antiviral factor (CAF) can suppress HIV-1 transcription from the long terminal repeat (LTR) promoter in the absence of elements upstream of the CATATAA box.

BACKGROUND: The CD8 Antiviral Factor (CAF) suppresses viral transcription from the HIV-1 Long Terminal Repeat (LTR) promoter in a non-cytolytic manner. However, the region on the LTR upon which CAF acts is unknown. Our objective was to determine the region on the LTR upon which CAF acts to suppress HIV-1 transcription. METHODS: Serial deletions of the LTR from the 5' end and inactivating point mutations were made. RESULTS: Serial deletions of the LTR from the 5' end indicated the importance of a short ~120 bp segment, containing the 3 SpI sites, CATA box (used by HIV-1 instead of the TATA box) and TAR region, in the suppressive process. Introduction of deletions or inactivating point mutations in the SpI sites or deletion of the TAR region did not abolish CAF-mediated transcriptional suppression. Yet, CAF-mediated transcriptional suppression was still retained in the HIV-1 CATA-TAR segment. CONCLUSION: CAF is able to suppress transcription from the LTR lacking all the elements upstream of the CATA box. Our results suggest that the HIV-1 CATA box may be responsible for CAF-mediated suppression of transcription from the HIV-1 LTR.

Effect of early anti-retroviral therapy on the pathogenic changes in mucosal tissues of SIV infected rhesus macaques.

BACKGROUND: The gastrointestinal tissue plays an important role in the pathogenesis of HIV/SIV infection and serves as a viral reservoir in infected individuals under antiretroviral therapy (ART). However, the effect of ART administration in the very early stage of infection on HIV/SIV replication and pathogenesis in gastrointestinal tissue has not been fully studied. In this current study, rhesus monkeys infected with SIV were treated with ART starting at day 7 post-infection. The effect of early ART on SIV replication and infection-related pathogenic changes in mucosal tissues of the infected monkeys was examined. METHODS: Nuclear acids were extracted from snap frozen ileum and colon tissues and mesentery lymph nodes from SIV infected monkeys with or without ART. SIV RNA and DNA loads as well as levels of CD3, CD4 and cytokine mRNA were measured by PCR and RT PCR from the isolated nuclear acids. Tissue sections were stained by immuno-fluorescence labeled antibodies for CD3 and CD4. RESULTS: Without ART treatment, these monkeys underwent a mild SIV infection with low viral loads and slightly decreased CD4+ T cell counts in peripheral blood. In ART treated monkeys, SIV RNA loads were undetectable in blood with normal CD4+ T cell counts, however, SIV RNA and DNA were detected in the intestinal tissues and mesentery lymph nodes although the levels were lower than those in untreated monkeys. The levels of CD3 and CD4 positive cells in the tissues were similar between the infected untreated monkeys and infected ART treated monkeys based on RT-PCR and immune-fluorescence staining of the tissue sections. Furthermore, compatible levels of IL-6, TNF-a, IL-1b and MyD88 mRNAs were detected in most of intestinal tissues and mesentery lymph nodes of infected ART treated and infected untreated monkeys. CONCLUSIONS: These results suggest that early ART administration could not effectively inhibit SIV replication in intestinal tissues and mesentery lymph nodes and could not reduce the immune activation induced by SIV infection in the intestinal tissues.

Development of a liposome microbicide formulation for vaginal delivery of octylglycerol for HIV prevention.

The feasibility of using a liposome drug delivery system to formulate octylglycerol (OG) as a vaginal microbicide product was explored. A liposome formulation was developed containing 1% OG and phosphatidyl choline in a ratio that demonstrated in vitro activity against Neisseria gonorrhoeae, HSV-1, HSV-2 and HIV-1 while sparing the innate vaginal flora, Lactobacillus. Two conventional gel formulations were prepared for comparison. The OG liposome formulation with the appropriate OG/lipid ratio and dosing level had greater efficacy than either conventional gel formulation and maintained this efficacy for at least 2 months. No toxicity was observed for the liposome formulation in ex vivo testing in a human ectocervical tissue model or in vivo testing in the macaque safety model. Furthermore, minimal toxicity was observed to lactobacilli in vitro or in vivo safety testing. The OG liposome formulation offers a promising microbicide product with efficacy against HSV, HIV and N. gonorrhoeae.

Preformulation and stability in biological fluids of the retrocyclin RC-101, a potential anti-HIV topical microbicide.

BACKGROUND: RC-101, a cationic peptide retrocyclin analog, has in vitro activity against HIV-1. Peptide drugs are commonly prone to conformational changes, oxidation and hydrolysis when exposed to excipients in a formulation or biological fluids in the body, this can affect product efficacy. We aimed to investigate RC-101 stability under several conditions including the presence of human vaginal fluids (HVF), enabling the efficient design of a safe and effective microbicide product. Stability studies (temperature, pH, and oxidation) were performed by HPLC, Circular Dichroism, and Mass Spectrometry (LC-MS/MS). Additionally, the effect of HVF on formulated RC-101 was evaluated with fluids collected from healthy volunteers, or from subjects with bacterial vaginosis (BV). RC-101 was monitored by LC-MS/MS for up to 72 h. RESULTS: RC-101 was stable at pH 3, 4, and 7, at 25 and 37°C. High concentrations of hydrogen peroxide resulted in less than 10% RC-101 reduction over 24 h. RC-101 was detected 48 h after incubation with normal HVF; however, not following incubation with HVF from BV subjects. CONCLUSIONS: Our results emphasize the importance of preformulation evaluations and highlight the impact of HVF on microbicide product stability and efficacy. RC-101 was stable in normal HVF for at least 48 h, indicating that it is a promising candidate for microbicide product development. However, RC-101 stability appears compromised in individuals with BV, requiring more advanced formulation strategies for stabilization in this environment.

Noncytotoxic suppression of human immunodeficiency virus type 1 transcription by exosomes secreted from CD8+ T cells.

CD8(+) T cells display a noncytotoxic activity that suppresses transcription of human immunodeficiency virus type 1 (HIV-1) in an antigen-independent and major histocompatibility complex-unrestricted manner. To date, the precise cellular and molecular factors mediating this CD8(+) T-cell effector function remain unsolved. Despite evidence indicating the dependence of the activity on cell-cell contact, the possibility of a membrane-mediated activity that represses transcription from the viral promoter remains unexplored. We therefore investigated whether this inhibition of HIV-1 transcription might be elicited by a membrane-bound determinant. Using a CD8(+) T-cell line displaying potent noncytotoxic HIV-1 suppression activity, we have identified a membrane-localized HIV-1-suppressing activity that is concomitantly secreted as 30- to 100-nm endosome-derived tetraspanin-rich vesicles known as exosomes. Purified exosomes from CD8(+) T-cell culture supernatant noncytotoxically suppressed CCR5-tropic (R5) and CXCR4-tropic (X4) replication of HIV-1 in vitro through a protein moiety. Similar antiviral activity was also found in exosomes isolated from two HIV-1-infected subjects. The antiviral exosomes specifically inhibited HIV-1 transcription in both acute and chronic models of infection. Our results, for the first time, indicate the existence of an antiviral membrane-bound factor consistent with the hallmarks defining noncytotoxic CD8(+) T-cell suppression of HIV-1.

Multisite comparison of anti-human immunodeficiency virus microbicide activity in explant assays using a novel endpoint analysis.

Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development.

Detection of HIV-1 RNA/DNA and CD4 mRNA in feces and urine from chronic HIV-1 infected subjects with and without anti-retroviral therapy.

HIV-1 infects gut associated lymphoid tissues (GALT) very early after transmission by multiple routes. The infected GALT consequently serves as the major reservoir for HIV-1 infection and could constantly shed HIV-1 and CD4+ T cells into the intestinal lumen. To examine this hypothesis, we monitored HIV-1 RNA/DNA and CD4 mRNA in fecal samples of chronically infected subjects with and without antiretroviral therapy (ART). We compared this to levels of HIV-1 RNA/DNA in urine and blood from the same subjects. Our results show that HIV-1 DNA, RNA and CD4 mRNA were detected in 8%, 19% and 31% respectively, of feces samples from infected subjects with detectable plasma viral load, and were not detected in any of subjects on ART with undetectable plasma viral load. In urine samples, HIV-1 DNA was detected in 24% of infected subjects with detectable plasma viral load and 23% of subjects on ART with undetectable plasma viral load. Phylogenetic analysis of the envelope sequences of HIV-1 revealed distinct virus populations in concurrently collected serum, feces and urine samples from one subject. In addition, our study demonstrated for the first time the presence of CD4 mRNA in fecal specimens of HIV-1 infected subjects, which could be used to assess GALT pathogenesis in HIV-1 infection.

TZA, a Sensitive Reporter Cell-based Assay to Accurately and Rapidly Quantify Inducible, Replication-competent Latent HIV-1 from Resting CD4+ T Cells.

The latent HIV-1 viral reservoir in resting CD4+ (rCD4+) T cells represents a major barrier to an HIV-1 cure. There is an ongoing effort to identify therapeutic approaches that will eliminate or reduce the size of this reservoir. However, clinical investigators lack an assay to determine whether or not a decrease in the latent reservoir has been achieved. Therefore, it is critical to develop assays that can reproducibly quantify the reservoir size and changes therein, in participant's blood during a therapeutic trial. Quantification of the latent HIV viral reservoir requires a highly sensitive, cost-effective assay capable of measuring the low frequency of rCD4+ T cells carrying functional provirus. Preferably, such an assay should be such that it can be adopted for high throughput and could be adopted under conditions for use in large-scale clinical trials. While PCR-based assays are commonly used to quantify pro-viral DNA or intracellular RNA transcript, they cannot distinguish between replication-competent and defective proviruses. We have recently published a study where a reporter cell-based assay (termed TZA or TZM-bl based quantitative assay) was used to quantify inducible replication-competent latent HIV-1 in blood. This assay is more sensitive, cost-efficient, and faster than available technology, including the quantitative viral outgrowth assay or the Q-VOA. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in virally suppressed participants on ART is approximately 70-fold larger than previous estimates. We describe here in detail an optimized method to quantitate latently infected cells using the TZA.

Neisseria gonorrhoeae uses cellular proteins CXCL10 and IL8 to enhance HIV-1 transmission across cervical mucosa.

PROBLEM: Neisseria gonorrhoeae (NG) infection has been shown to increase sexual transmission of HIV-1. However, the mechanism of NG-induced enhanced HIV-1 transmission is unknown. METHODS: (a) The cervical tissues were exposed to NG, and cytokine induction was monitored by measuring cytokine proteins in culture supernatants and cytokine mRNAs in tissues. (b) Transcription and replication of HIV-1 in TZM-bl, U1, and ACH2 cells were measured by Beta-Gal activity and p24 proteins in the supernatant, respectively. (c) HIV-1 transmission was assayed in an organ culture system by measuring transmitted HIV-1 in supernatant and HIV-1 gag mRNA in the tissues. (d) Transcriptome analysis was done using second generation sequencing. RESULTS: (a) NG induced membrane ruffling of epithelial layer, caused migration of CD3+ cells to the intraepithelial region, and induced high levels of inflammatory cytokines IL-1ß and TNF-α. (b) NG-induced supernatants (NGIS) increased HIV-1 transcription, induced HIV-1 from latently infected cells, and increased transmission of HIV-1 across cervical mucosa. (c) Transcriptome analysis of the epithelial layer of the tissues exposed to NG, and HIV-1 showed significant upregulation of CXCL10 and IL8. IL-1ß increased the induction of CXCL10 and IL-8 expression in cervical mucosa with a concomitant increase in HIV-1 transmission. CONCLUSION: We present a model in which IL-1ß produced from cervical epithelium during NG exposure increases CXCL10 and IL8 in epithelia. This in turn causes upon HIV-1 infection, the migration of HIV-1 target cells toward the subepithelium, resulting in increased HIV-1 transcription in the sub-mucosa and subsequent enhancement of transmission across cervical mucosa.

Type 1-programmed dendritic cells drive antigen-specific latency reversal and immune elimination of persistent HIV-1.

BACKGROUND: Despite the success of antiretroviral therapy (ART), latent HIV-1 continues to persist in a long-lived population of resting memory CD4+ T cells within those who are infected. Finding a safe and effective means to induce latency reversal (LR) during ART to specifically expose this latent HIV-1 cellular reservoir for immune elimination has been a major barrier to a functional cure. METHODS: In this study, we test the use of antigen-presenting type 1-polarized, monocyte-derived dendritic cells (MDC1) generated from chronic HIV-1-infected individuals on ART as a means to induce HIV-1 latency reversal in autologous CD4+ T cells harboring replication-competent provirus. We use the same MDC1 for ex-vivo generation of autologous HIV-1 antigen-specific CD8+ cytotoxic T cells (CTL) and test their effector responses against the MDC1-exposed HIV-1- infected CD4+ T cell targets. FINDINGS: MDC1 presentation of either HIV-1 or cytomegalovirus (CMV) antigens to CD4+ T cells facilitated HIV-1 LR. This antigen-driven MDC1-mediated LR was sharply diminished with blockade of the CD40L/CD40 'helper' signaling pathway. Importantly, these antigen-presenting MDC1 also activated the expansion of CTL capable of killing the exposed HIV-1-infected targets. INTERPRETATION: Inclusion of virus-associated MHC class II 'helper' antigens in MDC1-based HIV-1 immunotherapies could serve both as a targeted means to safely unmask antigen-specific CD4+ T cells harboring HIV-1, and to support CTL responses that can effectively target the MDC1-exposed HIV-1 cellular reservoir as a functional cure strategy. FUND: This study was supported by the NIH-NAID grants R21-AI131763, U01-AI35041, UM1-AI126603, and T32-AI065380.

DC-SIGN on B lymphocytes is required for transmission of HIV-1 to T lymphocytes.

Infection of T cells by HIV-1 can occur through binding of virus to dendritic cell (DC)-specific ICAM-3 grabbing nonintegrin (DC-SIGN) on dendritic cells and transfer of virus to CD4+ T cells. Here we show that a subset of B cells in the blood and tonsils of normal donors expressed DC-SIGN, and that this increased after stimulation in vitro with interleukin 4 and CD40 ligand, with enhanced expression of activation and co-stimulatory molecules CD23, CD58, CD80, and CD86, and CD22. The activated B cells captured and internalized X4 and R5 tropic strains of HIV-1, and mediated trans infection of T cells. Pretreatment of the B cells with anti-DC-SIGN monoclonal antibody blocked trans infection of T cells by both strains of HIV-1. These results indicate that DC-SIGN serves as a portal on B cells for HIV-1 infection of T cells in trans. Transmission of HIV-1 from B cells to T cells through this DC-SIGN pathway could be important in the pathogenesis of HIV-1 infection.

Chromosomal engineering of Clostridium perfringens using group II introns.

Clostridium perfringens is a major natural pathogen of human and domestic animals owing to the production of multiple toxins. Defined clostridial mutants are essential for studying the role of toxins in disease pathogenesis. However, it has been very difficult to introduce mutations into C. perfringens. We recently developed a clostridia-modified targetron that can specifically and efficiently inactivate C. perfringens genes. The usefulness of this system has now been demonstrated by specifically inactivating four different C. perfringens toxin genes.

Human immunodeficiency virus infection induces lymphoid fibrosis in the BM-liver-thymus-spleen humanized mouse model.

A major pathogenic feature associated with HIV infection is lymphoid fibrosis, which persists during antiretroviral therapy (ART). Lymphoid tissues play critical roles in the generation of antigen-specific immune response, and fibrosis disrupts the stromal network of lymphoid tissues, resulting in impaired immune cell trafficking and function, as well as immunodeficiency. Developing an animal model for investigating the impact of HIV infection-induced lymphoid tissue fibrosis on immunodeficiency and immune cell impairment is critical for therapeutics development and clinical translation. Said model will enable in vivo mechanistic studies, thus complementing the well-established surrogate model of SIV infection-induced lymphoid tissue fibrosis in macaques. We developed a potentially novel human immune system-humanized mouse model by coengrafting autologous fetal thymus, spleen, and liver organoids under the kidney capsule, along with i.v. injection of autologous fetal liver-derived hematopoietic stem cells, thus termed the BM-liver-thymus-spleen (BLTS) humanized mouse model. BLTS humanized mouse model supports development of human immune cells and human lymphoid organoids (human thymus and spleen organoids). HIV infection in BLTS humanized mice results in progressive fibrosis in human lymphoid tissues, which was associated with immunodeficiency in the lymphoid tissues, and lymphoid tissue fibrosis persists during ART, thus recapitulating clinical outcomes.

Lack of differences in HIV-1 nef functional domains in infected chinese blood donors at different stages of disease.

HIV-1 nef regions were amplified by polymerase chain reaction and sequenced from DNA samples of five asymptomatic subjects and five AIDS patients from a cohort of HIV-1-infected Chinese plasma and blood donors. Sequence analysis revealed that regardless of the stage of disease, each patient's HIV-1 nef sequences belonged to the clade B' subtype. Although there are some differences between the sequences from different patients, no significant differences have been detected in nef nucleotide sequences or functional motifs in the deduced amino acid sequences from patients at different stages of the disease. Furthermore, the predicted binding motifs of HLA-A2 and HLA-A11 were highly conserved among patient nef sequences. These results will contribute to a better understanding of the pathogenesis of circulating HIV-1 in infected Chinese former blood donors and may have important implications in developing an epitope-based vaccine suitable for Chinese blood donors.

Genetic and functional characterization of the LTR of HIV-1 subtypes A and C circulating in India.

Genetic analysis of HIV-1 sequences circulating in different parts of India have shown that the predominant proportion of HIV-1 subtypes circulating in India is type C and a small fraction are subtypes A, B, E, and CRFs. We sequenced the HIV-1 LTR promoter region of seven subtype C and five subtype A isolates obtained from two major cities in India. Sequence analysis of the complete promoter and TAR regions revealed conserved subtype-specific variability in several major binding sites. Three NF-kappaB sites were present in all subtype C isolates and two isolates contained an insertion in the MFNLP. The transcriptional activity of one of these isolates may have been hindered due to this insertion. Despite the apparent variability between the LTRs we did not observe any significant difference in the transcriptional activity between subtype C and subtype A. To our knowledge, this is the first study characterizing the genetic structure and functional attributes of subtype A LTRs from India.

Preformulation and Vaginal Film Formulation Development of Microbicide Drug Candidate CSIC for HIV prevention.

PURPOSE: 5-chloro-3-[phenylsulfonyl] indole-2-carboxamide (CSIC) is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 which has been shown to have a more desirable resistance profile than other NNRTIs in development as HIV prevention strategies. This work involves generation of preformulation data for CSIC and systematic development of a cosolvent system to effectively solubilize this hydrophobic drug candidate. This system was then applied to produce a polymeric thin film solid dosage form for vaginal administration of CSIC for use in prevention of sexual acquisition of HIV. METHODS: Extensive preformulation, formulation development, and film characterization studies were conducted. An HPLC method was developed for CSIC quantification. Preformulation tests included solubility, crystal properties, stability, and drug-excipient compatibility. Cytotoxicity was evaluated using both human epithelial and mouse macrophage cell lines. Ternary phase diagram methodology was used to identify a cosolvent system for CSIC solubility enhancement. Following preformulation evaluation, a CSIC film formulation was developed and manufactured using solvent casting technique. The developed film product was assessed for physicochemical properties, anti-HIV bioactivity, and Lactobacillus biocompatibility during 12-month stability testing period. RESULTS: Preformulation studies showed CSIC to be very stable. Due to its hydrophobicity, a cosolvent system consisting of polyethylene glycol 400, propylene glycol, and glycerin (5:2:1, w/w/w) was developed, which provided a uniform dispersion of CSIC in the film formulation. The final film product met target specifications established for vaginal microbicide application. CONCLUSIONS: The hydrophobic drug candidate CSIC was successfully formulated with high loading capacity in a vaginal film by means of a cosolvent system. The developed cosolvent strategy is applicable for incorporation of other hydrophobic drug candidates in the film platform.