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To explain a stable clinical outcome observed in a previous pilot clinical trial using a polyherbal formulation (PHF) for HIV–AIDS, we, in the present study, evaluated the T cell functions from fresh and stored blood samples. In three clinical groups–the anti-retroviral therapy, PHF and control arms–we compared the circulating levels of lipopolysaccharide, LPS-binding protein, soluble CD14, aspartate transaminase (AST) and alanine transaminase (ALT). Additionally, we evaluated the expression of T cell markers and gag-specific immune responses. The PHF treatment significantly reduced the levels of sCD14, AST and ALT. In a cross-sectional analysis at 30 months post-treatment, in comparison to the control group, the PHF arm showed significantly low per-cell expression of PD1, CD95 and HLA-DR. The PHF treatment appears to have attenuated general immune activation and hepatic inflammation in the study participants. Targeting the mediators of immune activation must be pursued as a useful strategy for HIV–AIDS management.

We demonstrate that at least three different promoter variant strains of HIV-1 subtype C have been gradually expanding and replacing the standard subtype C viruses in India, and possibly in South Africa and other global regions, over the past decade. The new viral strains contain an additional NF-κB, NF-κB-like, or RBEIII site in the viral promoter. Although the acquisition of an additional RBEIII site is a property shared by all the HIV-1 subtypes, acquiring an additional NF-κB site remains an exclusive property of subtype C. The acquired κB site is genetically distinct, binds the p50-p65 heterodimer, and strengthens the viral promoter at the levels of transcription initiation and elongation. The 4-κB viruses dominate the 3-κB "isogenic" viral strains in pairwise competition assays in T-cell lines, primary cells, and the ecotropic human immunodeficiency virus mouse model. The dominance of the 4-κB viral strains is also evident in the natural context when the subjects are coinfected with κB-variant viral strains. The mean plasma viral loads, but not CD4 counts, are significantly different in 4-κB infection suggesting that these newly emerging strains are probably more infectious. It is possible that higher plasma viral loads underlie selective transmission of the 4-κB viral strains. Several publications previously reported duplication or deletion of diverse transcription factor-binding sites in the viral promoter. Unlike previous reports, our study provides experimental evidence that the new viral strains gained a potential selective advantage as a consequence of the acquired transcription factor-binding sites and importantly that these strains have been expanding at the population level.

After screening a large number of clinical samples of HIV-1 subtype C in India, a subset of viral strains containing sequence insertions upstream of the viral enhancer has been identified. The sequence insertions contained binding sites for at least two different transcription factors NF-κB and RBEIII, importantly, in a mutually exclusive fashion. Furthermore, while some of the viral strains contained insertions of κB-like sites, a few others contained dual insertions of the RBEIII and κB sites together but only one of the two was intact. NF-κB acquisition appears to be the most common phenotype unique for subtype C with nearly half of the variant strains containing such insertions. Given that subtype C already contains three functional NF-κB sites in the viral enhancer, acquisition of a fourth NF-κB motif in some variant viral strains is intriguing. Further investigation is warranted to examine the significance of the sequence insertions for the replicative fitness of the variant viral strains.

Tat, an important regulatory protein of HIV-1, has been implicated in HIV-related pathogenesis. Immune responses to Tat, although underrepresented, confer protection against disease progression, in natural infection and experimental immunization, making Tat an attractive vaccine candidate. Information on immune responses to Tat from India which has the second largest HIV incidence has been lacking. Here we report a cross-sectional study evaluating the humoral response to Tat from a large number of samples from two southern states of India. 14% of the seropositive (63/447) and 4.6% of seronegative samples (7/150) harbored Tat-reactive antibodies. A significant number of the seropositive samples contained high levels of anti-Tat antibodies (31/447) which demonstrated class-switch to IgG1 and bound to Tat with high avidity. Cross-reactivity analysis showed that these antibodies interacted with Tat from different clades with variable degree with the highest interaction with subtype-AE and the least with subtype-B Tat. Importantly, a B-cell epitope in the cysteine-rich domain was found to be the most immunodominant one and antibodies interacting with this epitope blocked extracellular Tat efficiently. To the best of our knowledge this is the first report on immune responses to Tat from Indian populations and the data presented here could significantly contribute to HIV Tat vaccine design.

We report the cloning and sequence analysis of the long terminal repeat (LTR) of several primary HIV-1 subtype C strains of India. Phylogenetically, all the LTRs and the paired env sequences clustered with subtype C reference strains. The LTRs demonstrated extensive polymorphism in the transcription factor binding sites (TFBS) within the enhancer and the modulator regions. We generated reporter vectors under the control of a select subset of the subtype C LTRs. The reporter vectors are distinguished by the simultaneous expression of two independent reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescence protein (EGFP), in response to Tat. Expression of EGFP was facilitated by engineering an internal ribosome entry site (IRES) into the expression cassette. Although subtype C strains cause a large majority of the global infections, and important differences in the transcription factor binding sites have been identified in the subtype C promoter, few reporter vectors containing subtype C-LTR have been described. We analyzed gene expression from the C-LTR reporter vectors in different cell lines under diverse experimental conditions and compared it to the B-LTR reporter vector. The reporter vectors were responsive to Tat derived from diverse viral subtypes. Furthermore, a positive correlation was observed between the expression of the reporter genes and the viral structural protein p24 when the cells were infected with viral molecular clones. The LTR reporters we developed could be of significant use in the study of viral transactivation, in the evaluation of biological properties of viral subtypes, and in the screening for antiviral inhibitors.

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses are associated with nearly half of worldwide HIV-1 infections and are most predominant in India and the southern and eastern parts of Africa. Earlier reports from India identified the preponderance of subtype C and a small proportion of subtype A viruses. Subsequent reports identifying multiple subtypes suggest new introductions and/or their detection due to extended screening. The southern parts of India constitute emerging areas of the epidemic, but it is not known whether HIV-1 infection in these areas is associated with subtype C viruses or is due to the potential new introduction of non-subtype C viruses. Here, we describe the development of a specific and sensitive PCR-based strategy to identify subtype C-viruses (C-PCR). The strategy is based on amplifying a region encompassing a long terminal repeat and gag in the first round, followed by two sets of nested primers; one amplifies multiple subtypes, while the other is specific to subtype C. The common HIV and subtype C-specific fragments are distinguishable by length differences in agarose gels and by the difference in the numbers of NF-κB sites encoded in the subtype C-specific fragment. We implemented this method to screen 256 HIV-1-infected individuals from 35 towns and cities in four states in the south and a city in the east. With the exception of single samples of subtypes A and B and a B/C recombinant, we found all to be infected with subtype C viruses, and the subtype assignments were confirmed in a subset by using heteroduplex mobility assays and phylogenetic analysis of sequences. We propose the use of C-PCR to facilitate rapid molecular epidemiologic characterization to aid vaccine and therapeutic strategies.

Introduction Human Immunodeficiency Virus (HIV) has an estimated prevalence of 0.9% in India (5.2 million). Anti-retroviral drugs (ARV) are the treatments of choice and non-adherence is an important factor in treatment failure and development of resistance, as well as being a powerful predictor of survival. This study assesses adherence to ARV in HIV positive patients in Bangalore, India, a country where only 10% of those who need therapy are receiving it. Methods A cross-sectional anonymous questionnaire survey of 60 HIV antibody positive patients was carried out with patients attending HIV outpatient services at two centres: The Chest and Maternity Centre, Rajajinagar, and Wockhardt Hospital and Heart Institute, Bangalore. Consent was obtained. Translation was done by a translator and doctors where required. Data was analysed using SPSS statistical analysis. Results A response rate of 88% (53/60) was achieved. The mean patient age was 39.98 years, with 50% aged 30–40, and 73.6% of participants being male. Mean family size was 4.8 (1–13). 21% lived less than 50 kms and 21% greater than 400 kms from clinic. 60% reported they were fully adherent. Adherence was statistically significantly linked to regular follow-up attendance (70.5%, p = 0.002). No other results were statistically significant but trends were found. "100% adherence" trends were seen in older patients, male gender, those from larger families, those who had a previous AIDS defining illness, those taking fewer tablets, and without food restrictions. Commonest side-effects causing non-adherence were metabolic reasons (66%) and GI symptoms (50%). No trends were seen for education level, family income, distance travelled to clinic, time since diagnosis, or time on ART. Conclusion Regular attendance for follow up was statistically significant for 100% lifetime adherence. Positive trends were seen in those in larger families, older, those who had AIDS defining illness, simple regimes, and without side-effects. Education, income, distance travelled and length of time diagnosed or treated had no effect on adherence.