Peripheral lymphocyte subset alteration in patients with COVID-19 having differential clinical manifestations.

Background & objectives: The COVID-19 disease profile in Indian patients has been found to be different from the Western world. Changes in lymphocyte compartment have been correlated with disease course, illness severity and clinical outcome. This study was aimed to assess the peripheral lymphocyte phenotype and subset distribution in patients with COVID-19 disease from India with differential clinical manifestations. Methods: Percentages of peripheral lymphocyte subsets were measured by flow cytometry in hospitalized asymptomatic (n=53), mild symptomatic (n=36), moderate and severe (n=30) patients with SARS-CoV-2 infection, recovered individuals (n=40) and uninfected controls (n=56) from Pune, Maharashtra, India. Results: Percentages of CD4+Th cells were significantly high in asymptomatic, mild symptomatic, moderate and severe patients and recovered individuals compared to controls. Percentages of Th memory (CD3+CD4+CD45RO+), Tc memory (CD3+CD8+CD45RO+) and B memory (CD19+CD27+) cells were significantly higher in the recovered group compared to both asymptomatic, mild symptomatic patient and uninfected control groups. NK cell (CD56+CD3-) percentages were comparable among moderate +severe patient and uninfected control groups. Interpretation & conclusions: The observed lower CD4+Th cells in moderate+severe group requiring oxygen support compared to asymptomatic+mild symptomatic group not requiring oxygen support could be indicative of poor prognosis. Higher Th memory, Tc memory and B memory cells in the recovered group compared to mild symptomatic patient groups might be markers of recovery from mild infection; however, it remains to be established if the persistence of any of these cells could be considered as a correlate of protection.

Mycobacterium tuberculosis Lineages Associated with Mutations and Drug Resistance in Isolates from India.

Current knowledge on resistance-conferring determinants in Mycobacterium tuberculosis is biased toward globally dominant lineages 2 and 4. In contrast, lineages 1 and 3 are predominant in India. In this study, we performed whole-genome sequencing of 498 MDR M. tuberculosis isolates from India to determine the prevalence of drug resistance mutations and to understand the genomic diversity. A retrospective collection of 498 M. tuberculosis isolates submitted to the National Institute for Research in Tuberculosis for phenotypic susceptibility testing between 2014 to 2016 were sequenced. Genotypic resistance prediction was performed using known resistance-conferring determinants. Genotypic and phenotypic results for 12 antituberculosis drugs were compared, and sequence data were explored to characterize lineages and their association with drug resistance. Four lineages were identified although lineage 1 predominated (43%). The sensitivity of prediction for isoniazid and rifampicin was 92% and 98%, respectively. We observed lineage-specific variations in the proportion of isolates with resistance-conferring mutations, with drug resistance more common in lineages 2 and 3. Disputed mutations (codons 430, 435, 445, and 452) in the rpoB gene were more common in isolates other than lineage 2. Phylogenetic analysis and pairwise SNP difference revealed high genetic relatedness of lineage 2 isolates. WGS based resistance prediction has huge potential, but knowledge of regional and national diversity is essential to achieve high accuracy for resistance prediction. IMPORTANCE Current knowledge on resistance-conferring determinants in Mycobacterium tuberculosis is biased toward globally dominant lineages 2 and 4. In contrast, lineages 1 and 3 are predominant in India. We performed whole-genome sequencing of 498 MDR M. tuberculosis isolates from India to determine the prevalence of drug resistance mutations and to understand genomic diversity. Four lineages were identified although lineage 1 predominated (43%). The sensitivity of prediction for isoniazid and rifampicin was 92% and 98%, respectively. We observed lineage-specific variations in the proportion of isolates with resistance-conferring mutations, with drug resistance more common in lineages 2 and 3. Disputed mutations (codons 430, 435, 445, and 452) in the rpoB gene were more common in isolates other than lineage 2. Phylogenetic analysis and pairwise SNP difference revealed high genetic relatedness of lineage 2 isolates. WGS based resistance prediction has huge potential, but knowledge of regional and national diversity is essential to achieve high accuracy for resistance prediction.

An influence of dew point temperature on the occurrence of Mycobacterium tuberculosis disease in Chennai, India.

Climate factors such as dew point temperature, relative humidity and atmospheric temperature may be crucial for the spread of tuberculosis. This study was conducted for the first time to investigate the relationship of climatic factors with TB occurrence in an Indian setting. Daily tuberculosis notification data during 2008-2015 were generated from the National Treatment Elimination Program, and analogous daily climatic data were obtained from the Regional Meteorological Centre at Chennai city, Tamil Nadu, India. The decomposition method was adopted to split the series into deterministic and non-deterministic components, such as seasonal, non-seasonal, trend and cyclical, and non-deterministic climate factors. A generalized linear model was used to assess the relation independently. TB disease progression from latent stage infection to active was supported by higher dew point temperature and moderate temperature. It had a significant association with TB progression in the summer and monsoon seasons. The relative humidity may be favored in the winter and post-monsoon. The water tiny dew droplets may support the TB bacterium to recuperate in the environment.

Whole genome sequencing based differentiation between re-infection and relapse in Indian patients with tuberculosis recurrence, with and without HIV co-infection.

INTRODUCTION: Differentiation between relapse and reinfection in cases with tuberculosis (TB) recurrence has important implications for public health, especially in patients with human immunodeficiency virus (HIV) co-infection. We compared Mycobacterial Interspersed Repeat Unit (MIRU) typing and spoligotyping with whole genome sequencing (WGS) to differentiate between relapse and reinfection in patients (HIV-positive and HIV-negative) with TB recurrence. We also assessed the value of WGS to track acquired drug resistance in those with relapse after successful treatment. METHOD: Forty-one paired M. tuberculosis isolates collected from 20 HIV-positive and 21 HIV-negative patients were subjected to WGS in addition to spoligotyping and MIRU typing. Phylogenetic and Single Nucleotide Substitution (SNP) clustering analyses were performed to determine whether recurrences were due to relapse or re-infection. RESULTS: Comparison of M. tuberculosis genomes indicated that 95% of TB recurrences in the HIV-negative cohort were due to relapse, while the majority of TB recurrences (75%) in the HIV-positive cohort was due to reinfection (P = 0.0001). New drug resistance mutations were acquired in 5/24 cases (20.8%) that experienced relapse. CONCLUSIONS: WGS provided increased resolution, but differentiation between relapse and reinfection was broadly consistent with MIRU and spoligotyping. The high contribution of reinfection among HIV infected patients experiencing TB recurrence warrants further study to explore risk factors for TB exposure.

Discovery of a highly potent novel rifampicin analog by preparing a hybrid of the precursors of the antibiotic drugs rifampicin and clofazimine.

Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb). The present work reports the design and synthesis of a hybrid of the precursors of rifampicin and clofazimine, which led to the discovery of a novel Rifaphenazine (RPZ) molecule with potent anti-TB activity. In addition, the efficacy of RPZ was evaluated in-vitro using the reference strain Mtb H37Rv. Herein, 2,3 diamino phenazine, a precursor of an anti-TB drug clofazimine, was tethered to the rifampicin core. This 2,3 diamino phenazine did not have an inherent anti-TB activity even at a concentration of up to 2 µg/mL, while rifampicin did not exhibit any activity against Mtb at a concentration of 0.1 µg/mL. However, the synthesized novel Rifaphenzine (RPZ) inhibited 78% of the Mtb colonies at a drug concentration of 0.1 µg/mL, while 93% of the bacterial colonies were killed at 0.5 µg/mL of the drug. Furthermore, the Minimum Inhibitory Concentration (MIC) value for RPZ was 1 µg/mL. Time-kill studies revealed that all bacterial colonies were killed within a period of 24 h. The synthesized novel molecule was characterized using high-resolution mass spectroscopy and NMR spectroscopy. Cytotoxicity studies (IC50) were performed on human monocytic cell line THP-1, and the determined IC50 value was 96 µg/mL, which is non-cytotoxic.

The recent trend in mycobacterial strain diversity among extra pulmonary lymph node tuberculosis and their association with drug resistance and the host immunological response in South India.

BACKGROUND: Tuberculosis (TB) though primarily affects the lungs it may also affect the other parts of the body and referred as extra pulmonary (EPTB). This study is focused on understanding the genetic diversity and molecular epidemiology of Mycobacterium tuberculosis (M.tb) among tuberculous lymphadenitis (TBL), a form of EPTB patients identified in Chennai, Tamil Nadu. METHODS: The genetic diversity was identified by performing spoligotyping on the M.tb clinical isolates that were recovered from lymph node samples. A total of 71 M.tb isolates were recovered from extra pulmonary lymph node samples and subjected to Drug susceptibility testing and spoligotyping was carried out. In addition, immunological characterization from blood of same individuals from whom M.tb was isolated was carried out between the two major lineages groups East African Indian 3 (EAI3) and non-EAI3 strains by ELISA. The results of spoligotyping patterns were compared with the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4). RESULTS: We found 41 spoligotype patterns and their associated lineages. Out of 41 spoligotype pattern, only 22 patterns are available in the spoldB4 database with Spoligotype international Type (SIT) number and remaining patterns were orphan strains without SIT number. The most predominant spoligotype lineage that was found in lymph node sample in this region of India was EAI (36), followed by central Asian strain (CAS) (6), T1 (5), Beijing (3), Latin American & Mediterranean (LAM) (2), U (1), X2 (1) and orphan (22). In addition to EAI, CAS and Beijing, our study identified the presence of orphan and unique spoligotyping patterns in Chennai region. We observed six drug resistant isolates. Out of six drug resistant isolates, four were resistant to isoniazid drug and associated with EAI family. Moreover, we observed increased levels of type 2 and type 17 cytokine profiles between EAI3 and non-EAI family, infected individuals. CONCLUSIONS: The study confirms that EAI lineage to be the most predominant lineages in EPTB patients with lymphadenitis and were found to have increased type 1 and type 17 proinflammatory cytokine profiles.

Deep Profiling Identifies Selection of Nonsynonymous Amino Acid Substitutions in HIV-1 Envelope During Early Infection.

Understanding the evolutionary dynamics of the viruses within an individual at or near the moment of transmission can provide critical inputs for the design of an effective vaccine for HIV infection. In this study, high-throughput sequencing technology was employed to analyze the evolutionary rate in viruses obtained at a single time point from drug-naive recently infected infants and adults in the chronic stage of disease. Gene-wise nonsynonymous (pN) and synonymous (pS) mutation rates were estimated and compared between the two groups. Significant differences were observed in the evolutionary rates between viruses in the early and late stages of infection. Higher rates of adaptive mutations in the HIV-1 envelope gene (env) were found in the chronic viruses as compared with those in the early stages of HIV infection. Conversely, percentage of nonsynonymous substitutions in env was found to be higher in recently transmitted viruses. In addition, a positive correlation was found between mutation and the evolutionary rate, and infectivity titer in recent infection. Despite the small sample size, the study identified useful information about viral evolution on transmission-associated bottlenecks. The effect of intraindividual HIV-1 evolution at the population level was highly contemporary, and the higher percentage of nonsynonymous substitutions seen in env during recent HIV-1 infection has suggested a pattern of convergent evolution leading to a positive selection for survival fitness and disease progression.

Publisher Correction: In vitro replicative fitness of early Transmitted founder HIV-1 variants and sensitivity to Interferon alpha.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characteristics of paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome and its influence on tuberculosis treatment outcomes in persons living with HIV.

OBJECTIVE: The influence of tuberculosis (TB)-immune reconstitution inflammatory syndrome (IRIS) on TB treatment outcomes and its risk factors were investigated among people with human immunodeficiency virus (HIV) and co-infected with TB. METHODS: Newly diagnosed, culture-confirmed, pulmonary TB patients with HIV and enrolled in a clinical trial (NCT00933790) were retrospectively analysed for IRIS occurrence. Risk factors and TB outcomes (up to 18 months after initiation of anti-TB treatment [ATT]) were compared between people who experienced IRIS (IRIS group) and those who did not (non-IRIS group). RESULTS: TB-IRIS occurred in 82 of 292 (28%) participants. Significant baseline risk factors predisposing to TB-IRIS occurrence in univariate analysis were: lower CD4+ T-cell count, CD4/CD8 ratio, haemoglobin levels, presence of extra-pulmonary TB focus, and higher HIV viral load; the last two retained significance in the multivariate analysis. After 2 months of ATT commencement, sputum smear conversion was documented in 45 of 80 (56.2%) vs. 124 of 194 (63.9%) (p=0.23), culture conversion was in 75 of 80 (93.7%) vs. 178 of 194 (91.7%) (p=0.57) and the median decline in viral load (log10copies/mm3) was 2.7 in the IRIS vs. 1.1 in the non-IRIS groups (p<0.0001), respectively. An unfavourable response to TB therapy was detected in 17 of 82 (20.7%) and 28 of 210 (13.3%) in the IRIS and non-IRIS groups, respectively (p=0.14). CONCLUSIONS: TB-IRIS frequently occurred in people with advanced HIV infection and in those who presented with extra-pulmonary TB lesions, without influencing subsequent TB treatment outcomes.

In vitro replicative fitness of early Transmitted founder HIV-1 variants and sensitivity to Interferon alpha.

Type I interferons, particularly interferon-alpha (IFN-α), play a vital role in the host's anti-viral defenses by interfering with viral replication. However, the virus rapidly evolves to exploit the IFN-α response for its replication, spread, and pathogenic function. In this study, we attempted to determine IFN-α susceptibility and productivity of infectious transmitted/founder (TF) (n = 8) and non-transmitted (NT) viruses (n = 8) derived from HIV-1 infected infants. Independent experiments were carried out to determine IFN-α resistance, replication fitness, and viral productivity in CD4+ T cells over a short period. In vitro studies showed that TF viruses were resistant to IFN-α during the very near moment of transmission, but in the subsequent time points, they became susceptible to IFN-α. We did not observe much difference in replicative fitness of the TF viruses in cultures treated with and without IFN-α, but the difference was significant in the case of NT viruses obtained from the same individual. Despite increased susceptibility to IFN-α, NT viruses produced more viral particles than TF viruses. Similar results were also obtained in cultures treated with maraviroc (MVC). The study identified unique characteristics of TF viruses thus prompting further investigation into virus-host interaction occurring during the early stages of HIV infection.

Role of neutrophils in tuberculosis: A bird's eye view.

Neutrophils are innate immune cells implicated in the process of killing Mycobacterium tuberculosis early during infection. Once the mycobacteria enter the human system, neutrophils sense and engulf them. By secreting bactericidal enzymes and α-defensins like human neutrophil peptides loaded in their granule armory, neutrophils kill the pathogen. Peripheral blood neutrophils secrete a wide range of cytokines like IL-8, IL-1-ß and IFN-γ in response to mycobacterial infection. Thus they signal and activate distant immune cells thereby informing them of prevailing infection. The activated monocytes, dendritic cells and T cells further continue the immune response. As a final call, neutrophils release neutrophil extracellular traps in circulation which can trap mycobacteria in patients with active pulmonary tuberculosis. Extensive neutrophilic response is associated with inflammation, pulmonary destruction, and pathology. For example, inappropriate phagocytosis of mycobacteria-infected neutrophils can damage host cells due to necrosis of neutrophils, leading to chronic inflammation and tissue damage. This dual nature of neutrophils makes them double-edged swords during tuberculosis, and hence data available on neutrophil functions against mycobacterium are controversial and non-uniform. This article reviews the role of neutrophils in tuberculosis infection and highlights research gaps that need to be addressed. We focus on our understanding of new research ideologies targeting neutrophils (a) in the early stages of infection for boosting specific immune functions or (b) in the later stages of infection to prevent inflammatory conditions mediated by activated neutrophils. This would plausibly lead to the development of better tuberculosis vaccines and therapeutics in the future.

Plasma Metabolic Signature and Abnormalities in HIV-Infected Individuals on Long-Term Successful Antiretroviral Therapy.

Targeted metabolomics studies reported metabolic abnormalities in both treated and untreated people living with human immunodeficiency virus (HIV) (PLHIV). The present study aimed to understand the plasma metabolomic changes and predicted the risk of accelerated aging in PLHIV on long-term suppressive antiretroviral therapy (ART) in a case-control study setting and its association with the plasma proteomics biomarkers of inflammation and neurological defects. Plasma samples were obtained from PLHIV on successful long-term ART for more than five years (n = 22) and matched HIV-negative healthy individuals (n = 22, HC herein). Untargeted metabolite profiling was carried out using ultra-high-performance liquid chromatography/mass spectrometry/mass spectrometry (UHPLC/MS/MS). Plasma proteomics profiling was performed using proximity extension assay targeting 184 plasma proteins. A total of 250 metabolites differed significantly (p < 0.05, q < 0.1) between PLHIV and HC. Plasma levels of several essential amino acids except for histidine, branched-chain amino acids, and aromatic amino acids (phenylalanine, tyrosine, tryptophan) were significantly lower in PLHIV compared to HC. Machine-learning prediction of metabolite changes indicated a higher risk of inflammatory and neurological diseases in PLHIV. Metabolic abnormalities were observed in amino-acid levels, energetics, and phospholipids and complex lipids, which may reflect known differences in lipoprotein levels in PLHIV that can resemble metabolic syndrome (MetS).

Systemic Inflammation and the Increased Risk of Inflamm-Aging and Age-Associated Diseases in People Living With HIV on Long Term Suppressive Antiretroviral Therapy.

The ART program in low- and middle-income countries (LMIC) like India, follows a public health approach with a standardized regimen for all people living with HIV (PLHIV). Based on the evidence from high-income countries (HIC), the risk of an enhanced, and accentuated onset of premature-aging or age-related diseases has been observed in PLHIV. However, very limited data is available on residual inflammation and immune activation in the populations who are on first-generation anti-HIV drugs like zidovudine and lamivudine that have more toxic side effects. Therefore, the aim of the present study was to evaluate the levels of systemic inflammation and understand the risk of age-associated diseases in PLHIV on long-term suppressive ART using a large number of biomarkers of inflammation and immune activation. Blood samples were obtained from therapy naïve PLHIV (Pre-ART, n = 43), PLHIV on ART for >5 years (ART, n = 53), and HIV-negative healthy controls (HIVNC, n = 41). Samples were analyzed for 92 markers of inflammation, sCD14, sCD163, and telomere length. Several statistical tests were performed to compare the groups under study. Multivariate linear regression was used to investigate the associations. Despite a median duration of 8 years of successful ART, sCD14 (p < 0.001) and sCD163 (p = 0.04) levels continued to be significantly elevated in ART group as compared to HIVNC. Eleven inflammatory markers, including 4E-BP1, ADA, CCL23, CD5, CD8A, CST5, MMP1, NT3, SLAMF1, TRAIL, and TRANCE, were found to be significantly different (p < 0.05) between the groups. Many of these markers are associated with age-related co-morbidities including cardiovascular disease, neurocognitive decline and some of these markers are being reported for the first time in the context of HIV-induced inflammation. Linear regression analysis showed a significant negative association between HIV-1-positivity and telomere length (p < 0.0001). In ART-group CXCL1 (p = 0.048) and TGF-α (p = 0.026) showed a significant association with the increased telomere length and IL-10RA was significantly associated with decreased telomere length (p = 0.042). This observation warrants further mechanistic studies to generate evidence to highlight the need for enhanced treatment monitoring and special interventions in HIV-infected individuals.

Current trends in tuberculosis vaccine.

Despite the global efforts made to control tuberculosis (TB) and the large number of available new anti-TB drugs, TB still affects one-third of the world population. The conventional vaccine bacille Calmette-Guérin (BCG) shows varying efficacy in different populations, and there are safety issues in immunocompromised patients. Hence, there is an urgent requirement for a new and better TB vaccine candidate than BCG. There are several alternate vaccines available for TB such as DNA, subunit, adjuvant, and live-attenuated vaccines. Use of auxotrophic vaccine is an emerging technology. Newer vaccine technologies include vaccine delivery methods such as adenovirus- and cytomegalovirus (CMV)-based vector delivery, chimeric monoclonal antibody, single-chain fragment variable, RNA-lipoplexes, and nanoparticle-based technology. Based on its application, TB vaccines are classified as conventional, prophylactic, booster, therapeutic, and reinfection preventive vaccines. Currently, there are 12 vaccine candidates in clinical trials. In this review, we have briefly discussed about each of these vaccines in different phases of clinical trials. These vaccines should be analyzed further for developing a safe and more efficacious vaccine for TB.

Tuberculosis preventive treatment: the next chapter of tuberculosis elimination in India.

The End TB Strategy envisions a world free of tuberculosis-zero deaths, disease and suffering due to tuberculosis by 2035. This requires reducing the global tuberculosis incidence from >1250 cases per million people to <100 cases per million people within the next two decades. Expanding testing and treatment of tuberculosis infection is critical to achieving this goal. In high-burden countries, like India, the implementation of tuberculosis preventive treatment (TPT) remains a low priority. In this analysis article, we explore potential challenges and solutions of implementing TPT in India. The next chapter in tuberculosis elimination in India will require cost-effective and sustainable interventions aimed at tuberculosis infection. This will require constant innovation, locally driven solutions to address the diverse and dynamic tuberculosis epidemiology and persistent programme monitoring and evaluation. As new tools, regimens and approaches emerge, midcourse adjustments to policy and practice must be adopted. The development and implementation of new tools and strategies will call for close collaboration between local, national and international partners-both public and private-national health authorities, non-governmental organisations, research community and the diagnostic and pharmaceutical industry. Leading by example, India can contribute to global knowledge through operational research and programmatic implementation for combating tuberculosis infection.

Unique Phenotypic Characteristics of Recently Transmitted HIV-1 Subtype C Envelope Glycoprotein gp120: Use of CXCR6 Coreceptor by Transmitted Founder Viruses.

Adequate information on the precise molecular and biological composition of the viral strains that establish HIV infection in the human host will provide effective means of immunization against HIV infection. In an attempt to identify the transmitted founder (TF) virus and differentiate the biological properties and infectious potential of the TF virus from those of the population of the early transmitted viruses, 250 patient-derived gp120 envelope glycoproteins were cloned in pMN-K7-Luc-IRESs-NefΔgp120 to obtain chimeric viruses. Samples were obtained from eight infants who had recently become infected with HIV through mother-to-child transmission (MTCT) and two adults who acquired infection through the heterosexual route and were in the chronic stage of infection. Among the 250 clones tested, 65 chimeric viruses were infectious, and all belonged to HIV-1 subtype C. The 65 clones were analyzed for molecular features of the envelope, per-infectious-particle infectivity, coreceptor tropism, drug sensitivity, and sensitivity to broadly neutralizing antibodies. Based on genotypic and phenotypic analysis of the viral clones, we identified 10 TF viruses from the eight infants. The TF viruses were characterized by shorter V1V2 regions, a reduced number of potential N-linked glycosylation sites, and a higher infectivity titer compared to the virus variants from the adults in the chronic stage of infection. CXCR6 coreceptor usage, in addition to that of the CCR5 coreceptor, which was used by all 65 chimeric viruses, was identified in 13 viruses. The sensitivity of the TF variants to maraviroc and a standard panel of neutralizing monoclonal antibodies (VRC01, PG09, PG16, and PGT121) was found to be much lower than that of the virus variants from the adults in the chronic stage of infection.IMPORTANCE Tremendous progress has been made during the last three and half decades of HIV research, but some significant gaps continue to exist. One of the frontier areas of HIV research which has not seen a breakthrough yet is vaccine research, which is because of the enormous genetic diversity of HIV-1 and the unique infectious fitness of the virus. Among the repertoire of viral variants, the virus that establishes successful infection (transmitted founder [TF] virus) has not been well characterized yet. An insight into the salient features of the TF virus would go a long way toward helping with the design of an effective vaccine against HIV. Here we studied the biological properties of recently transmitted viruses isolated from infants who acquired infection from the mother and have come up with unique characterizations for the TF virus that establishes infection in the human host.

Cross Type Neutralizing Antibodies Detected in a Unique HIV-2 Infected Individual From India.

Background: Infection with HIV-2, a retrovirus that is closely related to HIV-1, is characterized by slower disease progression and transmission, longer latency period and low or undetectable viremia. Host immunity, including production of potent neutralizing antibodies, may be one of the possible contributors to the distinction between the two infections. In an attempt to understand whether HIV-2 infection results in production of neutralizing antibodies and to characterize the nature of the neutralization response we screened plasma of 37 HIV-2 infected individuals for the presence of broadly neutralizing antibodies. Materials and Methods: Thirty seven asymptomatic, ART-naïve, HIV-2 infected individuals were recruited for the study. Plasma obtained from these individuals were screened for the presence of broadly cross reactive neutralizing antibodies (BCNabs) using the standard neutralization screening protocol with a panel of HIV-1 and HIV-2 pseudoviruses. Plasma exhibiting broad neutralization activity were assessed for their potency employing a titration assay. Further, an attempt was made to characterize the neutralization specificity of the plasma exhibiting broad and potent neutralization activity. Result: While majority of the samples tested were capable of neutralizing HIV-2 pseudoviruses with high to moderate potency, one unique sample demonstrated broad cross clade and cross type neutralization with ability to strongly neutralize the vast majority of both HIV-1 and HIV-2 viruses tested (19/20). Preliminary analyses indicate the possible presence of antibodies with multiple glycan epitope binding specificities. Conclusion: The study identified a unique HIV-2 sample with exceptional ability to neutralize HIV-2 viruses and cross-neutralize HIV-1 viruses with great breadth and potency. This sample holds promise for isolation of novel monoclonal antibodies that may exploited as potential therapeutic tools for HIV infection.

Molecular Characteristics of the Envelope of Vertically Transmitted HIV-1 Strains from Infants with HIV Infection.

Mother-to-child transmission (MTCT) of HIV offers a good opportunity to study the dynamics of early viral evolution in the host environment to which the virus has partially adapted. Such studies would throw light on the unique features of the infecting viruses, which will subsequently help to design preventive or therapeutic measures against the newly infecting and evolving strains of HIV. Therefore, we undertook a study to determine the genetic divergence of proviral envelope sequences from the HIV-infected infants (<2 years). Detailed analysis revealed unique features of potential N-linked glycosylation sites (PNGS) and their frequency of occurrence that built on the difference in length of the V1V2 region of the envelope sequences. Surprisingly, frequency of PNGS in the V5 region was found to revert rapidly, in about 75% of the sequences, which could surmise a fitness disadvantage in the variant forms. Further, a stable net charge was observed in the V2 and V3 regions prompting us to speculate on the established interaction of the transmitted variant with the integrin α4ß7 receptor and R5 co-receptor, respectively. In brief, our observations suggest that differences in the length of the variable regions and variation in the frequency of PNGS in the envelope of the viruses obtained from very recently infected individuals in our population could be important characteristics of the unique quasispecies that is responsible for the spread of HIV in the early stages of infection in MTCT.

Outcome of patients on second line antiretroviral therapy under programmatic condition in India.

BACKGROUND: The National AIDS Control Organization of India has been providing free second line antiretroviral therapy (ART) since 2008. This observational study reports the survival and virologic suppression of patients on second-line ART under programmatic condition and type of mutations acquired by those failing therapy. METHODS: 170 patients initiated on second-line therapy between 2008 and 2012 were followed up till 2013. Viral Load (VL) was repeated at 6 months for all patients and at 12 months for those with VL >400 copies/ml at 6 months. Adequate virological response was defined as plasma HIV-1 VL <400 copies/ml and virological failure was defined as VL >1000 copies/ml. Genotyping was done in 16 patients with virological failure. RESULTS: Out of 170 patients, 110 (64.7 %) were alive and on therapy and 35 (20.5 %) expired. In the first year the occurrence of death was 13.7 /100 person years while between 1 and 5 year it was 3.88 /100 person years. In the first year, duration of immunological failure >12 months, weight <45 kg, WHO clinical stage 3 and 4 and WHO criteria CD4 count less than pretherapy baseline [hazard ratio HR 4.2. 15.8, 11.9 & 4.1 respectively] and beyond first year poor first and second line adherence and first line CD4 count < 200/µL [HR 5.2,15.8, 3.3 respectively] had high risk of death. 119/152 (78.2 %) had adequate virological response and 27/152 (17.7 %) had virological failure. High viral load at baseline and poor second line adherence (Odds Ratio 3.4 & 2.8 respectively) had increased risk of virological failure. Among those genotyped, 50 % had major Protease Inhibitor mutation (M46I commonest) however 87.5 % were still susceptible to darunavir. CONCLUSIONS: Second line therapy has shown high early mortality but good virological suppression under programmatic conditions.