HIV-1 low-level viraemia predicts virological failure in first-line and second-line ART-experienced individuals in India: A retrospective longitudinal study.

OBJECTIVE: To study the prevalence of low-level viraemia (LLV) and its association with virological failure (VF). METHODS: We conducted a retrospective analysis of 3498 participants at YRG CARE, Chennai, India (2013-2018) on antiretroviral therapy (ART) for ≥6 months with two or more plasma viral load (pVL) measurements. Results were stratified for those with pVL <1000 copies/mL: fully suppressed (FS) (pVL <40), low-LLV (pVL 40-199), mid-LLV (pVL 200-399), and high-LLV (pVL 400-999). The study assessed the association with VF (pVL >1000 copies/mL) using Cox proportional hazard model. RESULTS: Among 3498 participants, 2965 (84.8%) were FS and 533 (15.2%) were LLV. During the follow-up, 348 (10%) experienced VF, with 222 (6.3%) experienced after LLV (42% of LLV) and 126 (3.6%) experienced after FS (4.3% of FS). When compared with FS, those with LLV had a greater risk of VF [adjusted hazard ratio (aHR) = 12.7; 95% confidence interval (CI): 10.2-15.9]. First-line participants had a higher VF incidence (aHR = 15.8, 95% CI: 11.4-21.9) than second-line participants (aHR = 5.6, 95% CI: 4.1-7.7). Those with high-LLV had the highest VF risk (aHR = 22.856, 95% CI: 15.204-34.359 vs. aHR = 8.186, 95% CI: 5.564-12.043, for first-line vs. second-line participants, respectively), followed by those with mid-LLV (aHR = 13.375, 95% CI: 8.327-21.483 vs. aHR = 6.261, 95% CI: 4.044-9.695) and low-LLV (aHR = 12.976, 95% CI: 7.974-21.118 vs. aHR = 4.158, 95% CI: 2.826-6.119). CONCLUSIONS: The prevalence of LLV was intermediate in our study population. There was a higher risk of VF among individuals with LLV, and this risk increased with the increasing levels of LLV. Close monitoring of individuals experiencing LLV could help in the early identification of VF.

Role of Circulating T Follicular Helper Cells and Stem-Like Memory CD4+ T Cells in the Pathogenesis of HIV-2 Infection and Disease Progression.

CD4+ T cells are critical players in the host adaptive immune response. Emerging evidence suggests that certain CD4+ T cell subsets contribute significantly to the production of neutralizing antibodies and help in the control of virus replication. Circulating T follicular helper cells (Tfh) constitute a key T cell subset that triggers the adaptive immune response and stimulates the production of neutralizing antibodies (NAbs). T cells having stem cell-like property, called stem-like memory T cells (Tscm), constitute another important subset of T cells that play a critical role in slowing the rate of disease progression through the differentiation and expansion of different types of memory cell subsets. However, the role of these immune cell subsets in T cell homeostasis, CD4+ T cell proliferation, and progression of disease, particularly in HIV-2 infection, has not yet been elucidated. The present study involved a detailed evaluation of the different CD4+ T cell subsets in HIV-2 infected persons with a view to understanding the role of these immune cell subsets in the better control of virus replication and delayed disease progression that is characteristic of HIV-2 infection. We observed elevated levels of CD4+ Tfh and CD4+ Tscm cells along with memory and effector T cell abundance in HIV-2 infected individuals. We also found increased frequencies of CXCR5+ CD8+ T cells and CD8+ Tscm cells, as well as memory B cells that are responsible for NAb development in HIV-2 infected persons. Interestingly, we found that the frequency of memory CD4+ T cells as well as memory B cells correlated significantly with neutralizing antibody titers in HIV-2 infected persons. These observations point to a more robust CD4+ T cell response that supports B cell differentiation, antibody production, and CD8+ T cell development in HIV-2 infected persons and contributes to better control of the virus and slower rate of disease progression in these individuals.

Elevated Numbers of HIV-Specific Poly-Functional CD8+ T Cells With Stem Cell-Like and Follicular Homing Phenotypes in HIV-Exposed Seronegative Individuals.

HIV-specific CD8+ T cells are known to play a key role in viral control during acute and chronic HIV infection. Although many studies have demonstrated the importance of HIV-specific CD8+ T cells in viral control, its correlation with protection against HIV infection remains incompletely understood. To better understand the nature of the immune response that contributes to the early control of HIV infection, we analyzed the phenotype, distribution and function of anti-viral CD8+ T cells in a cohort of HIV-exposed seronegative (HESN) women, and compared them with healthy controls and HIV-infected individuals. Further, we evaluated the in vitro viral inhibition activity of CD8+ T cells against diverse HIV-1 strains. We found that the HESN group had significantly higher levels of CD8+ T cells that express T-stem cell-like (TSCM) and follicular homing (CXCR5+) phenotype with more effector like characteristics as compared to healthy controls. Further, we observed that the HESN population had a higher frequency of HIV-specific poly-functional CD8+ T cells with robust in vitro virus inhibiting capacity against different clades of HIV. Overall, our results demonstrate that the HESN population has elevated levels of HIV-specific poly-functional CD8+ T cells with robust virus inhibiting ability and express elevated levels of markers pertaining to TSCM and follicular homing phenotype. These results demonstrate that future vaccine and therapeutic strategies should focus on eliciting these critical CD8+ T cell subsets.

Deciphering the Role of Mucosal Immune Responses and the Cervicovaginal Microbiome in Resistance to HIV Infection in HIV-Exposed Seronegative (HESN) Women.

The female genital tract (FGT) is an important site of human immunodeficiency virus (HIV) infection. Discerning the nature of HIV-specific local immune responses is crucial for identifying correlates of protection in HIV-exposed seronegative (HESN) individuals. The present study involved a comprehensive analysis of soluble immune mediators, secretory immunoglobulins (sIg), natural killer (NK) cells, CXCR5+ CD8+ T cells, T follicular helper (Tfh) cells, and T regulatory cells (Tregs) in the vaginal mucosa as well as the nature and composition of the cervicovaginal microbiome in HESN women. We found significantly elevated antiviral cytokines, soluble immunoglobulins, and increased frequencies of activated NK cells, CXCR5+ CD8+ T cells, and Tfh cells in HESN females compared to HIV-unexposed healthy (UH) women. Analysis of the genital microbiome of HESN women revealed a greater bacterial diversity and increased abundance of Gardnerella spp. in the mucosa. The findings suggest that the female genital tract of HESN females represents a microenvironment equipped with innate immune factors, antiviral mediators, and critical T cell subsets that protect against HIV infection. IMPORTANCE The vast majority of human immunodeficiency virus (HIV) infections across the world occur via the sexual route. The genital tract mucosa is thus the primary site of HIV replication, and discerning the nature of HIV-specific immune responses in this compartment is crucial. The role of the innate immune system at the mucosal level in exposed seronegative individuals and other HIV controllers remains largely unexplored. This understanding can provide valuable insights to improve vaccine design. We investigated mucosal T follicular helper (Tfh) cells, CXCR5+ CD8+ T cells, natural killer (NK) cells subsets, soluble immune markers, and microbiome diversity in HIV-exposed seronegative (HESN) women. We found a significantly higher level of mucosal CXCR5+ CD8+ T cells, CD4+ Tfh cells, activated NK cell subsets, and antiviral immune cell mediators in HESN women. We also found a higher abundance of Gardnerella spp., microbiome dysbiosis, and decreased levels of inflammatory markers to be associated with reduced susceptibility to HIV infection. Our findings indicate that increased distribution of mucosal NK cells, CXCR5+ CD8+ T cells, Tfh cells, and soluble markers in HIV controllers with a highly diverse cervicovaginal microbiome could contribute effectively to protection against HIV infection. Overall, our findings imply that future vaccine design should emphasize inducing these highly functional cell types at the mucosal sites.

Association of circulatory Tfh-like cells with neutralizing antibody responses among chronic HIV-1 subtype C infected long-term nonprogressors and progressors.

HIV-1 vaccine functioning relies on successful induction of broadly neutralizing antibodies (bNAbs). CXCR3- circulatory T-follicular helper (cTfh) cells are necessary for inducing B-cells for generating bNAbs. Recent studies have suggested that CXCR3+ Tfh cells might also influence bNAb production. Plasma samples from 34 ART-Naïve HIV-1 infected individuals [long-term nonprogressors (LTNP)-19; Progressors-13] were tested against a heterologous virus panel (n = 11) from subtypes A, B, C, G, AC, BC and AE. Frequencies of CXCR3+ and CXCR3- cTfh-like cells in peripheral circulation were studied using flow cytometry. LTNP showed significantly lower CXCR3+ and higher CXCR3- cTfh-like cell frequencies, while neutralization breadth was observed to be broader in progressors. A positive correlation was observed between bNAb breadth and potency with CXCR3+PD-1+ cTfh-like cells in LTNP. Based on neutralization breadth, 9 HIV-1 infected individuals were classified as 'top neutralizers' and 23 as 'low neutralizers' and they did not show any correlations with CXCR3+ and CXCR3- cTfh-like cells. These preliminary data suggest that CXCR3+ similar to CXCR3- might possess significant functional properties for driving B-cells to produce bNAbs. Hence, an HIV vaccine which is capable of optimal induction of CXCR3+ cTfh cells at germinal centers might confer superior protection against HIV.

Seroprevalence of hepatitis delta virus infection among subjects with underlying hepatic diseases in Chennai, southern India.

Four hundred million people are carriers of hepatitis B virus (HBV) worldwide and approximately 5% of these are reportedly positive for hepatitis delta virus (HDV). Several reports indicate a declining trend in the occurrence of HDV infection in the north of tropical India. To our knowledge, no study has been conducted to evaluate whether a similar epidemiological change is occurring in southern India. Therefore we evaluated the seroprevalence of HDV among 153 individuals with HBV-related liver diseases in Chennai, and assessed any change in epidemiological pattern by comparing the results with seroprevalence figures reported previously. Of the 153 patients screened, nine (5.9%) were reactive to anti-delta antibodies, six (3.9%) presented an evidence of past infection (IgG anti-delta positive) and three (2.0%) showed anti-HDV IgM, suggestive of recent HDV infection. Alanine transaminase elevation was not significant in HDV-associated infection compared with HBV alone-infected acute viral hepatitis (AVH) (P=0.82) and chronic liver disease (P=0.77) patients. The anti-HDV positivity in AVH was considerably low (6.6%), compared with previous Indian reports varying from 10.7% to >30%. HDV infection was relatively low and seems to play a minor determining factor of liver diseases in the tropical south Indian population.

Transmission of "a" determinant variants of hepatitis B virus in immunized babies born to HBsAg carrier mothers.

Hepatitis B virus (HBV) surface antigen mutations may lead to immune escape and eventually cause failure of immunization. In this report, we identified immune escape variants in immunized babies born to hepatitis B surface antigen (HBsAg) carrier mothers. A total of 68 babies were followed up for 2 years after the full course of vaccination; 2.9% (2/68) of babies were found to be infected with the variant HBV in spite of preexisting antibody to surface antigen (anti-HBs) at 24 months post immunization. Both infants were positive for HBV-DNA; sequencing results of the "a" determinant region of the surface gene revealed that both babies had point mutations at a different nucleotide position resulting in various amino acid substitutions. In addition, an intriguing variant having an addition-deletion mutation was observed in one of the babies. This is the first report to show the addition-deletion variant of HBV in India. However, the immunological significance of the above HBV variants needs to be further elucidated.

HIV-specific T-cell Responses and Generalized Activation in HIV-1 Infected Long-term Non-progressors and Progressors from South India.

BACKGROUND: Anti-viral cytokine expressions by cytotoxic T-cells and lower activation rates have been reported to correlate with suppressed HIV replication in long-term non-progressors (LTNP). Immune mechanisms underlying disease non-progression in LTNP might vary with HIV-1 subtype and geographical locations. OBJECTIVE: This study evaluates cytokine expression and T-cells activation in relation to disease non-progression in LTNP. METHODS: HIV-1 Subtype C infected LTNP (n=20) and progressors (n=15) were enrolled and flowcytometry assays were performed to study HIV-specific CD8 T-cells expressing IL-2, IFN-γ, TNF-α and MIP-1ß against gag and env peptides. CD4+ T-cell activation was evaluated by surface expression of HLADR and CD38. RESULTS: Proportions of cytokines studied did not differ significantly between LTNP and progressors, while contrasting correlations with disease progression markers were observed in LTNP. CD4+ T-cell activation rates were significantly lower in LTNP compared to progressors which indicate the potential role of T-cell activation rates in disease non-progression in LTNP. CONCLUSION: LTNP and progressors showed similar CD8+ T-cell responses, but final conclusions can be drawn only by comparing multiple immune factors in larger LTNP cohort with HIV-1 infected individuals at various levels of disease progression. A possible role of HIV-1 subtype variation and ethnic differences in addition to host-genetic and viral factors cannot be ruled out.

Coinfection of hepatitis B and hepatitis C virus in HIV-infected patients in south India.

AIM: To screen for the co-infection of hepatitis B (HBV) and hepatitis C virus (HCV) in human immunodeficiency virus (HIV) infected patients in southern India. METHODS: Five hundred consecutive HIV infected patients were screened for Hepatitis B Virus (HBsAg and HBV-DNA) and Hepatitis C virus (anti-HCV and HCV-RNA) using commercially available ELISA kits; HBsAg, HBeAg/anti-HBe (Biorad laboratories, USA) and anti-HCV (Murex Diagnostics, UK). The HBV-DNA PCR was performed to detect the surface antigen region (pre S-S). HCV-RNA was detected by RT-PCR for the detection of the constant 5' putative non-coding region of HCV. RESULTS: HBV co-infection was detected in 45/500 (9%) patients and HCV co-infection in 11/500 (2.2%) subjects. Among the 45 co-infected patients only 40 patients could be studied, where the detection rates of HBe was 55% (22/40), antiHBe was 45% (18/40) and HBV-DNA was 56% (23/40). Among 11 HCV co-infected subjects, 6 (54.5%) were anti-HCV and HCV RNA positive, while 3 (27.2%) were positive for anti-HCV alone and 2 (18%) were positive for HCV RNA alone. CONCLUSION: Since the principal routes for HIV transmission are similar to that followed by the hepatotropic viruses, as a consequence, infections with HBV and HCV are expected in HIV infected patients. Therefore, it would be advisable to screen for these viruses in all the HIV infected individuals and their sexual partners at the earliest.

Epidemiological studies on pulmonary pathogens in HIV-positive and -negative subjects with or without community-acquired pneumonia with special emphasis on Mycoplasma pneumoniae.

The prevalence of Mycoplasma pneumoniae among HIV-positive patients with community-acquired pneumonia (CAP) remains unclear. We investigated 300 HIV-positive adults (200 with CAP and 100 with no respiratory illness) and 75 HIV-negative adults with CAP for the prevalence of respiratory pathogens using culture and serology. A growth inhibition test was employed to confirm the isolates of M. pneumoniae using species-specific typing sera. The prevalence of M. pneumoniae in HIV-positive subjects was 17% by induced sputum and 11.3% by throat swab culture. The seroprevalence of anti-M. pneumoniae IgM was 11.7% by ELISA and 14.3% by the gelatin microparticle agglutination test. The prevalence of M. pneumoniae among HIV-negative cases was relatively low. Streptococcus pneumoniae was predominant (28%) among subjects with lower respiratory disease, whereas Staphylococcus aureus (15%) was common among upper respiratory symptomatic cases. Rales (P=0.001), pharyngeal erythema (P=0.02), cervical adenopathy (P=0.004), skin rash (P=0.001), and crepitations (P=0.001) were each significantly related to M. pneumoniae positivity. Statistical significance was observed in relation to total lymphocyte count (P=0.02) and erythrocyte sedimentation rate (P=0.04), as well as M. pneumoniae positivity. This study shows that the prevalence of M. pneumoniae in HIV-positive subjects is comparatively higher than in HIV-negative subjects with pulmonary symptoms, and concords with previous pilot studies carried out in Chennai, South India.

Obtaining valid laboratory data in clinical trials conducted in resource diverse settings: lessons learned from a microbicide phase III clinical trial.

BACKGROUND: Over the last decade several phase III microbicides trials have been conducted in developing countries. However, laboratories in resource constrained settings do not always have the experience, infrastructure, and the capacity to deliver laboratory data meeting the high standards of clinical trials. This paper describes the design and outcomes of a laboratory quality assurance program which was implemented during a phase III clinical trial evaluating the efficacy of the candidate microbicide Cellulose Sulfate 6% (CS) [1]. METHODOLOGY: In order to assess the effectiveness of CS for HIV and STI prevention, a phase III clinical trial was conducted in 5 sites: 3 in Africa and 2 in India. The trial sponsor identified an International Central Reference Laboratory (ICRL), responsible for the design and management of a quality assurance program, which would guarantee the reliability of laboratory data. The ICRL provided advice on the tests, assessed local laboratories, organized trainings, conducted supervision visits, performed re-tests, and prepared control panels. Local laboratories were provided with control panels for HIV rapid tests and Chlamydia trachomatis/Neisseria gonorrhoeae (CT/NG) amplification technique. Aliquots from respective control panels were tested by local laboratories and were compared with results obtained at the ICRL. RESULTS: Overall, good results were observed. However, discordances between the ICRL and site laboratories were identified for HIV and CT/NG results. One particular site experienced difficulties with HIV rapid testing shortly after study initiation. At all sites, DNA contamination was identified as a cause of invalid CT/NG results. Both problems were timely detected and solved. Through immediate feedback, guidance and repeated training of laboratory staff, additional inaccuracies were prevented. CONCLUSIONS: Quality control guidelines when applied in field laboratories ensured the reliability and validity of final study data. It is essential that sponsors provide adequate resources for implementation of such comprehensive technical assessment and monitoring systems. TRIAL REGISTRATION: ClinicalTrials.gov NCT00153777 and Current Controlled Trials ISRCTN95638385.

Common protozoans as an uncommon cause of respiratory ailments in HIV-associated immunodeficiency.

Opportunistic infections (OIs) are the leading cause of mortality and morbidity among HIV-positive subjects. The breadth of reports of the rare occurrence of OIs in HIV/AIDS has been increasing over the years and more recent studies have outlined the changing trends in the emergence of newer pathogens. Recent reports of the association of certain protozoans that normally do not infect sites other than their normal sites of localization have generated huge interest among scientists. The complete depression of the immune system, followed by the onset of OIs, especially due to protozoans, i.e. toxoplasmosis, isosporiasis, leishmaniasis, cyclosporosis, microsporidiosis and cryptosporidiosis, is not uncommon in AIDS. The immunologic and pathologic basis behind the susceptibility of immunodepressed individuals to these 'non-site-specific parasites' is likely to have a huge impact on HIV disease progression. Certain possible shortcomings in the immunologic armory of immunodeficient subjects, their failure to contain the establishment of these 'uncommon' agents in the human host and their significance in HIV disease progression are discussed.