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BACKGROUND: Bacille Calmette-Guérin (BCG) vaccine is protective against Tuberculosis (TB) in children, but its efficacy wanes with age. Consequently, determining if BCG revaccination augments anti-TB immunity in young adults in TB endemic regions is vital. METHODS: Two hundred healthy adults, BCG vaccinated at birth, were tested for their IFN-γ release assay (IGRA) status. Of these, 28 IGRA(+) and 30 IGRA(–) were BCG revaccinated, and 24 IGRA(+) and 23 IGRA(–) subjects served as unvaccinated controls. T and innate cell responses to mycobacterial antigens were analyzed by 14-color flow cytometry over 34 weeks. RESULTS: IFN-γ and/or IL-2 Ag85A- and BCG-specific CD4(+) and CD8(+) T cell responses were boosted by revacciantion at 4 and 34 weeks, respectively, and were > 2-fold higher in IGRA(+) compared with IGRA(–) vaccinees. Polyfunctional Ag85A, BCG, and mycobacterium tuberculosis (Mtb) latency Ag–specific (LTAg-specific) CD4(+) T cells expressing up to 8 cytokines were also significantly enhanced in both IGRA(+) and IGRA(–) vaccinees relative to unvaccinated controls, most markedly in IGRA(+) vaccinees. A focused analysis of Th17 responses revealed expansion of Ag85A-, BCG-, and LTAg-specific total IL-17A(+),IL-17F(+),IL-22(+), and IL-10(+) CD4(+) T cell effectors in both IGRA(+) and IGRA(–) subjects. Also, innate IFN-γ(+) NK/γδ/NKT cell responses were higher in both IGRA(+) and IGRA(–) vaccinees compared with controls. This is the first evidence to our knowledge that BCG revaccination significantly boosts antimycobacterial Th1/Th17 responses in IGRA(+) and IGRA(–) subjects. CONCLUSION: These data show that BCG revaccination is immunogenic in IGRA(–) and IGRA(+) subjects, implying that Mtb preinfection in IGRA(+) subjects does not impact immunogenicity. This has implications for public health and vaccine development strategies. FUNDING: This work was funded principally by DBT-NIH (BT/MB/Indo-US/HIPC/2013).

Vaccines that confer protection through induction of adaptive T-cell immunity rely on understanding T-cell epitope (TCE) evolution induced by immune escape. This is poorly understood in tuberculosis (TB), an ancient, chronic disease, where CD4 T-cell immunity is of recognized importance. We probed 905 functionally validated, curated human CD4 T cell epitopes in 79 Mycobacterium tuberculosis (Mtb) whole genomes from India. This screen resulted in identifying 64 mutated epitopes in these strains initially using a computational pipeline and subsequently verified by single nucleotide polymorphism (SNP) analysis. SNP based phylogeny revealed the 79 Mtb strains to cluster to East African Indian (EAI), Central Asian Strain (CAS), and Beijing (BEI) lineages. Eighty-nine percent of the mutated T-cell epitopes (mTCEs) identified in the 79 Mtb strains from India has not previously been reported. These mTCEs were encoded by genes with high nucleotide diversity scores including seven mTCEs encoded by six antigens in the top 10% of rapidly divergent Mtb genes encoded by these strains. Using a T cell functional assay readout, we demonstrate 62% of mTCEs tested to significantly alter CD4 T-cell IFN gamma and/or IL2 secretion with associated changes in predicted HLA-DR binding affinity: the gain of function mutations displayed higher predicted HLA-DR binding affinity and conversely mutations resulting in loss of function displayed lower predicted HLA-DR binding affinity. Most mutated antigens belonged to the cell wall/cell processes, and, intermediary metabolism and respiration families though all known Mtb proteins encoded mutations. Analysis of the mTCEs in an SNP database of 5,310 global Mtb strains identified 82% mTCEs to be significantly more prevalent in Mtb strains isolated from India, including 36 mTCEs identified exclusively in strains from India. These epitopes had a significantly higher predicted binding affinity to HLA-DR alleles that were highly prevalent in India compared to HLA-DR alleles rare in India, highlighting HLA-DR maybe an important driver of these mutations. This first evidence of region-specific TCE mutations potentially employed by Mtb to escape host immunity has important implications for TB vaccine design.

Significant subtype-specific differences were observed in the protease (PR) region of the HIV-1 pol gene. Most of the previous studies were restricted to subtype B, although subtype C accounts for more than 50% of HIV infections worldwide. In this study we characterized PR sequences from primary clinical isolates from protease inhibitor (PI)-naive patients in South India (n=39) as well as database-derived HIV-1 subtype C sequences from India (n=542) and globally (n=2970). All the study sequences were identified as subtype C, which is predominant in India. Drug resistance genotyping analysis identified 2.6% (1/39) prevalence of major PI resistance (I54T) and 7.7% (3/39) of minor PI resistance (L10I, T74S, and A71T). Selection of T12S, I15V, L19I, M36I, R41K, H69K, L89M, and I93L was observed both in global and Indian subtype C while the L63P mutation was selected in Indian PR sequences. Three different codon-based maximum likelihood methods agreed on four sites (12, 19, 36, and 82) under positive selection in Indian sequences.

Monitoring of HIV-infected individuals on antiretroviral treatment (ART) ideally requires periodic viral load measurements to ascertain adequate response to treatment. While plasma viral load monitoring is widely available in high-income settings, it is rarely used in resource-limited regions because of high cost and need for sophisticated sample transport. Dried blood spot (DBS) as source specimens for viral load measurement has shown promise as an alternative to plasma specimens and is likely to be a useful tool for Indian settings. The present study was undertaken to investigate the performance of DBS in HIV-1 RNA quantification against the standard plasma viral load assay.Between April-June 2011, 130 samples were collected from HIV-1-infected (n=125) and non-infected (n=5) individuals in two district clinics in southern India. HIV-1 RNA quantification was performed from DBS and plasma using Abbott m2000rt system after manual RNA extraction. Statistical analysis included correlation, regression and Bland-Altman analysis.The sensitivity of DBS viral load was 97 per cent with viral loads3.0 log 10 copies/ml. Measurable viral load (3.0 log 10 copies/ml) results obtained for the 74 paired plasma-DBS samples showed positive correlation between both the assays (r=0.96). For clinically acceptable viral load threshold values of5,000 copies/ml, Bland-Altman plots showed acceptable limits of agreement (-0.21 to +0.8 log 10 copies/ml). The mean difference was 0.29 log 10 copies/ml. The cost of DBS was $2.67 lower compared to conventional plasma viral load measurement in the setting.The significant positive correlation with standard plasma-based assay and lower cost of DBS viral load monitoring suggest that DBS sampling can be a feasible and economical means of viral load monitoring in HIV-infected individual in India and in other resource-limited settings globally.

Introduction: Human APOBEC3G/F (hA3G/F) restricts retroviral replication through G-to-A hypermutations, which can generate drug-resistant progenies in vitro . The clinical relevance is still inconclusive. To bridge this gap, we aim to study the role of these hypermutations in evolution of drug resistance; we characterised hA3G/F-mediated hypermutations in the RT region of the pol gene of patients with or without antiretroviral therapy (ART). Methods: In 88 HIV-1-positive individuals, drug resistance genotyping was carried out in plasma virus and provirus by population sequencing. Hypermutations were determined by three different approaches using Hypermut 2.0 software, cluster analysis and APOBEC3G-mediated defectives indices. Clinical and demographic characteristics of these individuals were studied in relation to these hypermutations. Results: hA3G/F-mediated hypermutated sequences in proviral DNA, but not in plasma virus, were identified in 11.4% (10/88) subjects. Proviral hypermutations were observed more frequently in patients with ART failure than in ART-naive individuals ( p = 0.03). In therapy failure patients, proviral hypermutation were associated with greater intra-compartmental genetic diversity ( p

Analysis of treatment response among HIV-infected children in India on first-line antiretroviral treatment for >2 years revealed that 85% were virologically suppressed. Of those with virologic failure, only 17% manifested immunologic failure, whereas majority had resistance-associated mutations. The presence of resistance highlights the need for virologic monitoring of children receiving antiretroviral treatment to optimize treatment success and preserve future treatment options.

BACKGROUND Coreceptor switch from CCR5 to CXCR4 is considered to be less common in HIV-1 subtype C even in advanced stages of infection. In this study, we have examined viral genotypic coreceptor tropism and its clinical, virological, and host genetic determinants among perinatally infected children in India. METHODS Genotypic coreceptor tropism analysis was conducted on env V3 sequences using Geno2pheno with a threshold of 10% false-positive rate. A total of 473 sequences were obtained from 72 isolates amplified from children aged 2-17 years. Factors associated with viral tropism in subtype C infections were studied using logistic regression. RESULTS Among the samples, 98.6% (71 of 72) were HIV-1 subtype C. Coreceptor tropism analysis determined 81.7% (58 of 71) as R5 tropic, 9.9% (7 of 71) as X4 tropic, and 8.5% (6 of 71) as R5/X4 tropic or dual-tropic HIV-1 strains. Children with X4 or R5/X4 strains were more likely to be older than those with R5-tropic strains (P < 0.05), have lower CD4 counts (P < 0.05), and have viral populations with greater intrapopulation viral divergence (P < 0.01). Older age was a significant independent predictor for X4 or R5/X4 tropism in these children (P < 0.05). None were identified as being heterozygous or homozygous for the CCR5[INCREMENT]32 deletion. CONCLUSIONS The high prevalence of X4 and R5/X4 tropic strains among older perinatally infected children with HIV-1 subtype C in India indicate that this phenomenon is not uncommon as previously thought and suggest that coreceptor transition can occur with longer duration of infection and greater disease progression in this population of perinatally infected children living with HIV-1 subtype C.

Background High plasma viremia in HIV-1 infection is associated with rapid CD4 cell decline and faster disease progression. Children with HIV infection have high viral loads, particularly in early childhood. In this study we sought to understand the relationship between duration of HIV-1 infection and viral dynamics among perinatally-infected children and adolescents in India along with transmitted drug resistance in this population. Methods During 2007–2011, cross-sectional samples were collected from ART-naïve children (n = 105) with perinatally-acquired HIV infection, aged 2–16 years from Bangalore, India. CD4 counts, viral load and in-house genotyping were performed and transmitted drug resistance mutations were identified using the World Health Organization recommendations for Surveillance of Drug Resistance Mutations (SDRM_2009) list. Results Among 105 children studied, 73.3% (77/105) were asymptomatic, but had a median viral load of 5.24 log copies/mL (IQR 4.62-5.66). In the adolescent age group, 54% (21/39) had high levels of viremia (median 5.14 log copies/mL) but were asymptomatic. HIV-1 subtyping identified 98% strains (103/105) as subtype C; one A1 and one unique recombinant form (URF). Transmitted NRTI resistance was 1.9% (2/105); NNRTI resistance was 4.8% (5/105) and overall prevalence of transmitted drug resistance was 5.7% (6/105). Conclusions The high burden of plasma viremia found among untreated asymptomatic adolescents needs to be addressed both from an individual angle to halt disease progression, and from a public health perspective to arrest horizontal transmission. The low level of transmitted drug resistance among perinatally-infected children is reassuring; however with improving ART access globally, regular genotyping surveillance is indicated.