Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol711 into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.

Acacia catechu (Mimosa family) stem bark extracts have been used traditionally as a dietary supplement as well as a folk medicine given its reported anti-inflammatory, immunomodulatory, hepatoprotective, antioxidant, anti-microbial and anti-tumor activities. The present study was undertaken to evaluate the anti-HIV-1 activity of the extracts from stem bark of A. catechu.

The delivery of plasmid DNA to the skin can target distinct subsets of dermal dendritic cells to confer a superior immune response. The needle-free immunization technology offers a reliable, safe and efficient means to administer intradermal (ID) injections. We report here that the ID injection of DNA vectors using an NF device (NF-ID) elicits a superior cell-mediated immune response, at much lesser DNA dosage, comparable in magnitude to the traditional intramuscular immunization. However, the humoral response is significantly impaired, possibly at the stage of B cell isotype switching. We found that the NF-ID administration deposits the DNA primarily on the epidermis resulting in a rapid loss of the DNA as well as the synthesized antigen due to the faster regeneration rate of the skin layers. Therefore, despite the immune-rich nature of the skin, the NF-ID immunization of DNA vectors may be limited by the impaired humoral response. Additional booster injections are required to augment the antibody response. As an alternative and a viable solution, we rescued the IgG response by coadministration of a Toll-like receptor 9 agonist, among other adjuvants examined. Our work has important implication for the optimization of the emerging needle-free technology for ID immunization.

Recent spread of the promoter variant (4-κB) Human immunodeficiency virus-1 clade C (HIV-1C) strain is attributed to duplication of the Nuclear Factor Kappa B (NF-κB) binding sites and potential increased heroin consumption in India. To study the underlying biology of 4-κB HIV-1C in rhesus macaques, we engineered a promoter-chimera variant (4NF-κB) Simian Human Immunodeficiency Virus (SHIV) by substituting the HIV-1C Long Terminal Repeat (LTR) region consisting of 4 NF-κB and 3 Sp-1 sites with the corresponding segment in the LTR of SHIV AD8EO. The wild-type (3NF-κB) promoter-chimera SHIV was generated by inactivating the 5' proximal NF-κB binding site in SHIV 4NF-κB. CD8-depleted rhesus macaque PBMCs (RM-PBMCs) were infected with the promoter-chimera and AD8EO SHIVs to determine the effects of opioid-exposure on inflammation, NF-κB activation, neurotoxicity in neuronal cells and viral replication. Morphine-exposure of RM-PBMCs infected with SHIVs 4NF-κB, 3NF-κB, and AD8EO altered cellular transcript levels of monocyte chemoattractant protein 1, interleukin 6, interleukin 1β, and Tumor Necrosis Factor α. Of note, divergent alteration of the cytokine transcript levels was observed with these promoter-chimera wild-type and variant SHIVs. NF-κB activation was observed during infection of all three SHIVs with morphine-exposure. Finally, we observed that SHIV AD8EO infection and exposure to both morphine and naloxone had the greatest impact on the neurotoxicity. The promoter-chimera SHIV 4NF-κB and SHIV 3NF-κB did not have a similar effect on neurotoxicity as compared to SHIV AD8EO. All SHIVs replicated efficiently at comparable levels in RM-PBMCs and morphine-exposure did not alter viral replication kinetics. Future in vivo studies in rhesus macaques will provide greater understanding of 4-κB HIV-1C viral immunopathogenesis and onset of disease in the central nervous system during morphine-exposure.

Human immunodeficiency virus type 1 (HIV-1) following integration hijacks host cell machineries where chromatinization of the viral genome regulates its latency, transcription, and replication. The cooperation among ATP-dependent chromatin remodeling factors, posttranslational modifying enzymes, and histone chaperones is well established during transcriptional activation in eukaryotes. However, the role of histone chaperones in transcription of the HIV promoter is poorly understood. Previous studies from our group have established the role of the human histone chaperone nucleophosmin (NPM1) in the acetylation-dependent chromatin transcription. NPM1 is known to interact with HIV-Tat. Here, we report that infection by HIV-1 induces the acetylation of histone chaperone NPM1. Acetylation of NPM1 was found to be critical for nuclear localization of Tat as well as Tat-mediated transcription alluding to the critical role for the host factor towards viral pathogenesis. Furthermore, knockdown experiments mediated by small interfering RNA identified the critical role played by the chaperone NPM1 in transcriptional activation of the integrated provirus. These results shed further insights into the possible role of histone chaperone NPM1 acetylation in viral gene transcription, which could be a potential therapeutic target.

Of the diverse subtypes of Human Immunodeficiency Virus Type-1 (HIV-1), subtype-C strains cause a large majority of infections worldwide. The reasons for the global dominance of HIV-1 subtype-C infections are not completely understood. Tat, being critical for viral infectivity and pathogenesis, may differentially modulate pathogenic properties of the viral subtypes. Biochemical studies on Tat are hampered by the limitations of the current purification protocols. Tat purified using standard protocols often is competent for transactivation activity but defective for a variety of other biological functions. Keeping this limitation in view, we developed an efficient protein purification strategy for Tat.

Tat/Rev Induced Limiting Dilution Assay (TILDA) is instrumental in estimating the size of latent reservoirs of HIV-1. Here, we report an optimized TILDA containing a broader detection range compared to the reported methods and high sensitivity. Giving priority to sequence conservation, we positioned the two forward primers and the probe in exon-1 of HIV-1. The reverse primers are positioned in highly conserved regions of exon-7. The optimized TILDA detected eight molecular clones belonging to five major genetic subtypes of HIV-1 with a comparable detection sensitivity. Using the optimized assay, we show that only a minor proportion of CD4+ T cells of primary clinical samples can spontaneously generate multiply spliced viral transcripts. A significantly larger proportion of the cells produced viral transcripts following activation. The optimized TILDA is suitable to characterize HIV-1 latent reservoirs and the therapeutic strategies intended to target the reservoir size.

The ability of HIV to establish long-lived latent infection is mainly due to transcriptional silencing of viral genome in resting memory T lymphocytes. Here, we show that new semi-synthetic ingenol esters reactivate latent HIV reservoirs. Amongst the tested compounds, 3-caproyl-ingenol (ING B) was more potent in reactivating latent HIV than known activators such as SAHA, ingenol 3,20-dibenzoate, TNF-α, PMA and HMBA. ING B activated PKC isoforms followed by NF-κB nuclear translocation. As virus reactivation is dependent on intact NF-κB binding sites in the LTR promoter region ING B, we have shown that. ING B was able to reactivate virus transcription in primary HIV-infected resting cells up to 12 fold and up to 25 fold in combination with SAHA. Additionally, ING B promoted up-regulation of P-TEFb subunits CDK9/Cyclin T1. The role of ING B on promoting both transcription initiation and elongation makes this compound a strong candidate for an anti-HIV latency drug combined with suppressive HAART.

The pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood. This situation is more complex in subtype C infection where coreceptor switch is either absent or extremely rare. To gain insights into the mechanisms that underlie coreceptor requirement of subtype C, we screened several primary viral isolates and identified a clinical sample that demonstrated a potential to grow on standard T-cell lines with no detectable CCR5 expression. The subject was diagnosed with HIV-1 associated dementia in the absence of opportunistic infections of the brain. To isolate molecular clones from this virus, we devised a novel strategy based on anchor primers that target a sequence in the reverse transcriptase, highly conserved among diverse subtypes of HIV-1.

HIV-1 subtype C demonstrates several biological properties distinct from other viral subtypes. One such variation is the duplication of PTAP motif in p6 Gag. PTAP motif is a key player in viral budding. Here, we studied the prevalence of PTAP motif duplication in subtype C viral strains in a longitudinal study.

India has the third largest HIV-1 epidemic with 2.4 million infected individuals. Molecular epidemiological analysis has identified the predominance of HIV-1 subtype C (HIV-1C). However, the previous reports have been limited by sample size, and uneven geographical distribution. The introduction of HIV-1C in India remains uncertain due to this lack of structured studies. To fill the gap, we characterised the distribution pattern of HIV-1 subtypes in India based on data collection from nationwide clinical cohorts between 2007 and 2011. We also reconstructed the time to the most recent common ancestor (tMRCA) of the predominant HIV-1C strains.

The natural cysteine to serine variation at position 31 of Tat in HIV-1C disrupts the dicysteine motif attenuating the chemokine function of Tat. We ask if there exists a trade-off in terms of a gain of function for HIV-1C Tat due to this natural variation. We constructed two Tat-expression vectors encoding Tat proteins discordant for the serine 31 residue (CS-Tat vs. CC-Tat), expressed the proteins in Jurkat cells under doxycycline control, and performed the whole transcriptome analysis to compare the early events of Tat-induced host gene expression. Our analysis delineated a significant enrichment of pathways and gene ontologies associated with the angiogenic signaling events in CS-Tat stable cells. Subsequently, we validated and compared angiogenic signaling events induced by CS- vs. CC-Tat using human umbilical vein endothelial cells (HUVEC) and the human cerebral microvascular endothelial cell line (hCMEC/D3). CS-Tat significantly enhanced the production of CCL2 from HUVEC and induced an activated phenotype in endothelial cells conferring on them enhanced migration, invasion, and in vitro morphogenesis potential. The ability of CS-Tat to induce the activated phenotype in endothelial cells could be of significance, especially in the context of HIV-associated cardiovascular and neuronal disorders. The findings from the present study are likely to help appreciate the functional significance of the SAR (signature amino acid residues) influencing the unique biological properties.

We observe the emergence of several promoter-variant viral strains in India during recent years. The variant viral promoters contain additional copies of transcription factor binding sites present in the viral modulatory region or enhancer, including RBEIII, LEF-1, Ap-1 and/or NF-κB. These sites are crucial for governing viral gene expression and latency. Here, we infer that one variant viral promoter R2N3-LTR containing two copies of RBF-2 binding sites (an RBEIII site duplication) and three copies of NF-κB motifs may demonstrate low levels of gene expression noise as compared to the canonical RN3-LTR or a different variant R2N4-LTR (a duplication of an RBEIII site and an NF-κB motif). To demonstrate this, we constructed a panel of sub-genomic viral vectors of promoter-variant LTRs co-expressing two reporter proteins (mScarlet and Gaussia luciferase) under the dual-control of Tat and Rev. We established stable pools of CEM.NKR-CCR5 cells (CEM-CCR5RL reporter cells) and evaluated reporter gene expression under different conditions of cell activation.

HIV-1 Clade C (Subtype C; HIV-1C) is responsible for greater than 50% of infections worldwide. Unlike clade B HIV-1 (Subtype B; HIV-1B), which is known to cause HIV associated dementia (HAD) in approximately 15% to 30% of the infected individuals, HIV-1C has been linked with lower prevalence of HAD (0 to 6%) in India and Ethiopia. However, recent studies report a higher prevalence of HAD in South Africa, Zambia and Botswana, where HIV-1C infections predominate. Therefore, we examined whether Southern African HIV-1C is genetically distinct and investigated its neurovirulence. HIV-1 Tat protein is a viral determinant of neurocognitive dysfunction. Therefore, we focused our study on the variations seen in tat gene and its contribution to HIV associated neuropathogenesis.

The complementary and alternative medicines (CAM) have not been systematically evaluated for the management of HIV/AIDS patients. In a prospective, single-site, open-label, non-randomized, controlled, pilot trial, we evaluated a polyherbal formulation (PHF) for its safety and efficacy in treating subjects with HIV-AIDS.

Understanding co-receptor tropism of HIV-1 strains circulating in India will provide key analytical leverage for assessing the potential usefulness of newer antiretroviral drugs such as chemokine co-receptor antagonists among Indian HIV-infected populations. The objective of this study was to determine using in silico methods, HIV-1 tropism among a large number of Indian isolates both from primary clinical isolates as well as from database-derived sequences.