Functional and transcriptional heterogeneity within the massively expanding HLADR+CD38+ CD8 T cell population in acute febrile dengue patients.

IMPORTANCE: CD8 T cells play a crucial role in protecting against intracellular pathogens such as viruses by eliminating infected cells and releasing anti-viral cytokines such as interferon gamma (IFNγ). Consequently, there is significant interest in comprehensively characterizing CD8 T cell responses in acute dengue febrile patients. Previous studies, including our own, have demonstrated that a discrete population of CD8 T cells with HLADR+ CD38+ phenotype undergoes massive expansion during the acute febrile phase of natural dengue virus infection. Although about a third of these massively expanding HLADR+ CD38+ CD8 T cells were also CD69high when examined ex vivo, only a small fraction of them produced IFNγ upon in vitro peptide stimulation. Therefore, to better understand such functional diversity of CD8 T cells responding to dengue virus infection, it is important to know the cytokines/chemokines expressed by these peptide-stimulated HLADR+CD38+ CD8 T cells and the transcriptional profiles that distinguish the CD69+IFNγ+, CD69+IFNγ-, and CD69-IFNγ- subsets.

Immunophenotyping and Transcriptional Profiling of Human Plasmablasts in Dengue.

Plasmablasts represent a specialized class of antibody-secreting effector B cells that transiently appear in blood circulation following infection or vaccination. The expansion of these cells generally tends to be massive in patients with systemic infections such as dengue or Ebola that cause hemorrhagic fever. To gain a detailed understanding of human plasmablast responses beyond antibody expression, here, we performed immunophenotyping and RNA sequencing (RNA-seq) analysis of the plasmablasts from dengue febrile children in India. We found that plasmablasts expressed several adhesion molecules and chemokines or chemokine receptors that are involved in endothelial interactions or homing to inflamed tissues, including skin, mucosa, and intestine, and upregulated the expression of several cytokine genes that are involved in leukocyte extravasation and angiogenesis. These plasmablasts also upregulated the expression of receptors for several B-cell prosurvival cytokines that are known to be induced robustly in systemic viral infections such as dengue, some of which generally tend to be relatively higher in patients manifesting hemorrhage and/or shock than in patients with mild febrile infection. These findings improve our understanding of human plasmablast responses during the acute febrile phase of systemic dengue infection. IMPORTANCE Dengue is globally spreading, with over 100 million clinical cases annually, with symptoms ranging from mild self-limiting febrile illness to more severe and sometimes life-threatening dengue hemorrhagic fever or shock, especially among children. The pathophysiology of dengue is complex and remains poorly understood despite many advances indicating a key role for antibody-dependent enhancement of infection. While serum antibodies have been extensively studied, the characteristics of the early cellular factories responsible for antibody production, i.e., plasmablasts, are only beginning to emerge. This study provides a comprehensive understanding of the transcriptional profiles of human plasmablasts from dengue patients.

Chikungunya-specific IgG and neutralizing antibody responses in natural infection of Chikungunya virus in children from India.

Chikungunya virus (CHIKV) infection is endemic in many different countries. CHIKV outbreaks are emerging in new areas and re-emerging in previously exposed geographical regions, thus making it a significant public health concern. CHIKV infections are often clinically inapparent, especially in children, which poses a challenge to testing and evaluating any vaccine. During CHIKV infection, CHIKV-specific antibodies are produced, and some of these antibodies can neutralize viruses released from infected cells before they can enter uninfected cells. In this study, we evaluated IgG binding and neutralizing antibody responses in paired serum samples from CHIKV-infected children and those with other febrile illness, using a recombinant truncated E2 protein and whole CHIKV particles as test antigens. Antibody detection using the truncated E2 protein showed a significant overlap between CHIKV-infected subjects and those with other febrile illnesses. This overlap was greater when binding antibody titers were determined using fixed CHIKV particles as the test antigen. Acute- and convalescent-phase sera collected from children after CHIKV infection showed significant differences in their neutralizing capacity. The neutralizing and binding antibody response showed a significant positive correlation. We detected IgG antibodies in most cases during the acute phase of infection. This was observed at two different geographical locations, one of which is not considered highly endemic. Conventional wisdom would suggest this to be a marker of re-infection (secondary infection). However, dissenting opinions have been voiced in other viral diseases (such as Ebola) where studies have detected IgG in acute illness. In the absence of any significant body of work documenting secondary CHIKV infections, we believe further work is needed to understand the early IgG response that we observed.

Epidemiology and molecular characterization of chikungunya virus from human cases in North India, 2016.

Chikungunya virus (CHIKV), an arthropod-borne Alphavirus is responsible for chikungunya disease. Arthralgia and arthritis are the major symptom. Some patients recover early while others for a very long time. This study provides, epidemiology and molecular characterization of three whole-genome sequences of CHIKV and assessed phylogenetic analysis, physiological properties, antigenicity, and B-cell epitope prediction by in silico. We report the clinical epidemiology of 325 suspected patients. Of these, 118 (36.30%) were confirmed CHIKV positive by either PCR or ELISA. Clinical analysis showed joint pain, joint swelling and headache were frequent and significant features. Phylogenie analysis showed the currently circulating strain is in close clustring to Africa, Uganda, and Singapore CHIKV strains. Molecular characterization by WGS was done. Thirty eight amino acid changes in the nonstructural proteins were found with respect to the S27 (ECSA) strain. Of these five located in nsP2. Similarly, 34 amino acid changes in structural proteins were observed. The major change was notice; in E3 protein hydropathicity -0.281 to -0.362, in E2 isoelectric point (pI) 8.24 to 8.37, instability index 66.08 to 71.062, aliphatic index varied from 74.69 to 68.59 and E3 75.79 to 70.05. In nsP1 protein pI varies from 6.62 to 8.04, while no other change was observed in structural and nonstructural protein. The linear B-cell epitopes, position, and number varied with the mutation. The molecular characterizations of WGS demonstrate the observation of protein, antigenicity with respect to the mutation.

Plant-expressed Fc-fusion protein tetravalent dengue vaccine with inherent adjuvant properties.

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly-immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor-targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell-mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen-specific CD4+ and CD8+ T-cell proliferation, IFN-γ and antibody production. The purified polymeric fraction of dengue PIGS (D-PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low-affinity Fcγ receptors on antigen-presenting cells. These results show that the plant-expressed D-PIGS have the potential for translation towards a safe and easily scalable single antigen-based tetravalent dengue vaccine.

Clinical, Serological, and Virological Analysis of 572 Chikungunya Patients From 2010 to 2013 in India.

Background: Chikungunya fever (CHIK) is a major public health concern in India. Characterized by acute fever with joint pain and swelling, most patients recover from this self-limiting illness in 7-10 days, with cessation of joint pain post-acute episode. However, in some patients, joint pain persists, lasting for months or even years. The precise correlates to the chronic phase of this debilitating illness and/or this remarkable heterogeneity in disease manifestation are poorly understood. Methods: We evaluated 572 chikungunya patients from India who were recruited on the basis of positive real-time polymerase chain reaction and/or CHIK virus immunoglobulin (IgM) after receiving consent. Arthralgic conditions were monitored using visual analog score (VAS) 12 weeks after onset of fever in 130 patients. Initial viral load, IgG, and initial neutralization response were assayed and correlated with clinical and VAS information in 40 patients. Results: Our extensive screening revealed that patients with higher initial viral loads during the acute phase of illness had poor prognosis at the post-acute phase with more restricted joint movement and higher VAS. Additionally, patients who showed early seroconversion to neutralizing IgG responses had better prognosis, as many of these patients did not manifest restricted joint movements at the post-acute phase. Conclusions: Our study sheds light on chikungunya disease with respect to disease progression and assesses clinical, virological, and serological parameters of chikungunya disease severity. Importantly, it reveals that initial high viral load and neutralizing IgG response may function in a seemingly contrasting manner to negatively or positively dictate disease outcome.

Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India.

Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR- CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue. IMPORTANCE: Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects.

Mycobacterial antigen driven activation of CD14++CD16- monocytes is a predictor of tuberculosis-associated immune reconstitution inflammatory syndrome.

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an aberrant inflammatory response occurring in a subset of TB-HIV co-infected patients initiating anti-retroviral therapy (ART). Here, we examined monocyte activation by prospectively quantitating pro-inflammatory plasma markers and monocyte subsets in TB-HIV co-infected patients from a South Indian cohort at baseline and following ART initiation at the time of IRIS, or at equivalent time points in non-IRIS controls. Pro-inflammatory biomarkers of innate and myeloid cell activation were increased in plasma of IRIS patients pre-ART and at the time of IRIS; this association was confirmed in a second cohort in South Africa. Increased expression of these markers correlated with elevated antigen load as measured by higher sputum culture grade and shorter duration of anti-TB therapy. Phenotypic analysis revealed the frequency of CD14(++)CD16(-) monocytes was an independent predictor of TB-IRIS, and was closely associated with plasma levels of CRP, TNF, IL-6 and tissue factor during IRIS. In addition, production of inflammatory cytokines by monocytes was higher in IRIS patients compared to controls pre-ART. These data point to a major role of mycobacterial antigen load and myeloid cell hyperactivation in the pathogenesis of TB-IRIS, and implicate monocytes and monocyte-derived cytokines as potential targets for TB-IRIS prevention or treatment.

Severe disease during both primary and secondary dengue virus infections in pediatric populations.

Dengue is a global epidemic causing over 100 million cases annually. The clinical symptoms range from mild fever to severe hemorrhage and shock, including some fatalities. The current paradigm is that these severe dengue cases occur mostly during secondary infections due to antibody-dependent enhancement after infection with a different dengue virus serotype. India has the highest dengue burden worldwide, but little is known about disease severity and its association with primary and secondary dengue infections. To address this issue, we examined 619 children with febrile dengue-confirmed infection from three hospitals in different regions of India. We classified primary and secondary infections based on IgM:IgG ratios using a dengue-specific enzyme-linked immunosorbent assay according to the World Health Organization guidelines. We found that primary dengue infections accounted for more than half of total clinical cases (344 of 619), severe dengue cases (112 of 202) and fatalities (5 of 7). Consistent with the classification based on binding antibody data, dengue neutralizing antibody titers were also significantly lower in primary infections compared to secondary infections (P ≤ 0.0001). Our findings question the currently widely held belief that severe dengue is associated predominantly with secondary infections and emphasizes the importance of developing vaccines or treatments to protect dengue-naive populations.

Short communication: Virological and B cell profiles of chikungunya and Dengue virus co-infections in Delhi during 2017-2019.

With explosive epidemics of chikungunya in India since 2004, chikungunya virus (CHIKV) now co-circulates in geographical areas where Dengue virus (DENV) is already endemic and thus provides opportunity for the same mosquito to be infected with both viruses. Although there are excellent studies that have addressed the clinical of mono and co-infection, we have little to no knowledge on the current viral sequences that pre-dominate co-infections, and the B cell response elicited. In this study, we analyzed febrile patients that were confirmed to have DENV-CHIKV co-infections and asked the following questions: 1) what is the frequency of co-infections found in a single cycle of transmission; 2) what are the viral sequences associated with them; 3) what does the antibody secreting cell / plasmablast response look like in patients that are co-infected with both viruses. We report those co-infections occur at a frequency of 6.7% in the transmission cycle, and while DENV-3 is now frequently detected, we do not see a serotype bias in the patients that are co-infected with ESCA strain of CHIKV. Moreover, the effector B cell response (plasmablasts) observed are specific to both infecting viruses indicating no overt bias. Further studies to associate whether any of these properties have a bearing on clinical disease manifestation will be both timely and important.

Frequency of CXCR3+ CD8+ T-Lymphocyte Subsets in Peripheral Blood Is Associated With the Risk of Paradoxical Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome Development in Advanced HIV Disease.

Background: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is a clinical aggravation of TB symptoms observed among a fraction of HIV coinfected patients shortly after the start of antiretroviral therapy (ART). Of note, TB-IRIS is characterized by exacerbated inflammation and tissue damage that occurs in response to the elevated production of CD4+ T cell-derived IFN-γ. Nevertheless, the possible participation of CD8+ T cells in TB-IRIS development remains unclear. Methods: We performed a comprehensive assessment of the composition of CD8+ T cell memory subsets and their association with circulating inflammation-related molecules in TB-HIV coinfected patients initiating ART. Results: We found that TB-IRIS individuals display higher frequencies of Antigen-experienced CD8+ T cells during the onset of IRIS and that the levels of these cells positively correlate with baseline mycobacterial smear grade. TB-IRIS individuals exhibited higher frequencies of effector memory and lower percentages of naïve CD8+ T cells than their Non-IRIS counterparts. In both TB-IRIS and Non-IRIS patients, ART commencement was associated with fewer significant correlations among memory CD8+ T cells and cells from other immune compartments. Networks analysis revealed distinct patterns of correlation between each memory subset with inflammatory cytokines suggesting different dynamics of CD8+ T cell memory subsets reconstitution. TB-IRIS patients displayed lower levels of memory cells positive for CXCR3 (a chemokine receptor that plays a role in trafficking activated CD8+ T cells to the tissues) than Non-IRIS individuals before and after ART. Furthermore, we found that CXCR3+ naïve CD8+ T cells were inversely associated with the risk of TB-IRIS development. On the other hand, we noticed that the frequencies of CXCR3+ effector CD8+ T cells were positively associated with the probability of TB-IRIS development. Conclusion: Our data suggest that TB-IRIS individuals display a distinct profile of memory CD8+ T cell subsets reconstitution after ART initiation. Moreover, our data point to a differential association between the frequencies of CXCR3+ CD8+ T cells and the risk of TB-IRIS development. Collectively, our findings lend insights into the potential role of memory CD8+ T cells in TB-IRIS pathophysiology.

Contrasting behavior between the three human monocyte subsets in dengue pathophysiology.

Monocytes are known to play a critical role in dengue pathophysiology. However, which monocyte subset expresses what inflammatory mediator(s) and what transcriptional features distinguish each of the monocyte subset in vivo remain poorly understood. In this study we provide a detailed transcriptional analysis of the three human monocyte subsets in healthy children and in children with dengue febrile illness. Notably, we found that the CD14+ CD16high intermediate monocyte subset from dengue patients highly upregulated key genes involved in mediating inflammation, endothelial dysfunction, vascular permeability, tissue extravasation, and clot prevention compared to healthy children. The CD14+CD16low classical monocytes shared some of these features. These two subsets increased massively in patients with severe dengue. By contrast, the CD14-CD16high nonclassical monocyte subset upregulated key genes involved in vasoconstriction, endothelial barrier stability, and are involved in endothelial patrolling while showing a significant decline from circulation. These findings improve our understanding of monocyte responses in dengue.

Structural insights for neutralization of Omicron variants BA.1, BA.2, BA.4, and BA.5 by a broadly neutralizing SARS-CoV-2 antibody.

In this study, by characterizing several human monoclonal antibodies (mAbs) isolated from single B cells of the COVID-19-recovered individuals in India who experienced ancestral Wuhan strain (WA.1) of SARS-CoV-2 during early stages of the pandemic, we found a receptor binding domain (RBD)-specific mAb 002-S21F2 that has rare gene usage and potently neutralized live viral isolates of SARS-CoV-2 variants including Alpha, Beta, Gamma, Delta, and Omicron sublineages (BA.1, BA.2, BA.2.12.1, BA.4, and BA.5) with IC50 ranging from 0.02 to 0.13 µg/ml. Structural studies of 002-S21F2 in complex with spike trimers of Omicron and WA.1 showed that it targets a conformationally conserved epitope on the outer face of RBD (class 3 surface) outside the ACE2-binding motif, thereby providing a mechanistic insights for its broad neutralization activity. The discovery of 002-S21F2 and the broadly neutralizing epitope it targets have timely implications for developing a broad range of therapeutic and vaccine interventions against SARS-CoV-2 variants including Omicron sublineages.

Optimization of Flow-Cytometry Based Assay for Measuring Neutralizing Antibody Responses against Each of the Four Dengue Virus Serotypes.

Dengue is an important public health problem worldwide, with India contributing nearly a third of global dengue disease burden. The measurement of neutralizing antibody responses is critical for understanding dengue pathophysiology, vaccine development and evaluation. Historically, dengue virus neutralization titers were measured using plaque reduction neutralization tests (PRNTs), which were later adapted to focus reduction neutralization tests (FRNTs). Given the slow and laborious nature of both these assays, there has been interest in adapting a high-throughput flow cytometry based neutralization assay. However, flow cytometry based assays typically underestimate neutralization titers, and in situations where the titers are low they can even fail to detect neutralization activity. In this study, by evaluating graded numbers of input Vero cell numbers and viral inoculum, we optimized the flow cytometry based neutralization assay in such a way that it is sensitive and scores titers that are in concordance with focus reduction neutralization tests for each of the four dengue virus serotypes (p < 0.0001). Given that dengue is a global public health concern, and several research groups are making efforts to understand its pathophysiology and accelerate vaccine development and evaluation both in India and worldwide, our findings have timely significance for facilitating these efforts.

Dynamics of T-Lymphocyte Activation Related to Paradoxical Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome in Persons With Advanced HIV.

Most persons living with HIV (PLWH) experience a significant restoration of their immunity associated with successful inhibition of viral replication after antiretroviral therapy (ART) initiation. Nevertheless, with the robust quantitative and qualitative restoration of CD4+ T-lymphocytes, a fraction of patients co-infected with tuberculosis develop immune reconstitution inflammatory syndrome (TB-IRIS), a dysregulated inflammatory response that can be associated with significant tissue damage. Several studies underscored the role of adaptive immune cells in IRIS pathogenesis, but to what degree T lymphocyte activation contributes to TB-IRIS development remains largely elusive. Here, we sought to dissect the phenotypic landscape of T lymphocyte activation in PLWH coinfected with TB inititating ART, focusing on characterization of the profiles linked to development of TB-IRIS. We confirmed previous observations demonstrating that TB-IRIS individuals display pronounced CD4+ lymphopenia prior to ART initiation. Additionally, we found an ART-induced increase in T lymphocyte activation, proliferation and cytotoxicity among TB-IRIS patients. Importantly, we demonstrate that TB-IRIS subjects display higher frequencies of cytotoxic CD8+ T lymphocytes which is not affected by ART. Moreover, These patients exhibit higher levels of activated (HLA-DR+) and profilerative (Ki-67+) CD4+ T cells after ART commencenment than their Non-IRIS counterparts. Our network analysis reveal significant negative correlations between Total CD4+ T cells counts and the frequencies of Cytotoxic CD8+ T cells in our study population which could suggest the existance of compensatory mechanisms for Mtb-infected cells elimination in the face of severe CD4+ T cell lymphopenia. We also investigated the correlation between T lymphocyte activation profiles and the abundance of several inflammatory molecules in plasma. We applied unsupervised machine learning techniques to predict and diagnose TB-IRIS before and during ART. Our analyses suggest that CD4+ T cell activation markers are good TB-IRIS predictors, whereas the combination of CD4+ and CD8+ T cells markers are better at diagnosing TB-IRIS patients during IRIS events Overall, our findings contribute to a more refined understanding of immunological mechanisms in TB-IRIS pathogenesis that may assist in new diagnostic tools and more targeted patient management.

Characterization of neutralizing versus binding antibodies and memory B cells in COVID-19 recovered individuals from India.

India is one of the most affected countries by COVID-19 pandemic; but little is understood regarding immune responses to SARS-CoV-2 in this region. Herein we examined SARS-CoV-2 neutralizing antibodies, IgG, IgM, IgA and memory B cells in COVID-19 recovered individual from India. While a vast majority of COVID-19 recovered individuals showed SARS-CoV-2 RBD-specific IgG, IgA and IgM antibodies (38/42, 90.47%; 21/42, 50%; 33/42, 78.57% respectively), only half of them had appreciable neutralizing antibody titers. RBD-specific IgG, but not IgA or IgM titers, correlated with neutralizing antibody titers and RBD-specific memory B cell frequencies. These findings have timely significance for identifying potential donors for plasma therapy using RBD-specific IgG assays as surrogate measurement for neutralizing antibodies in India. Further, this study provides useful information needed for designing large-scale studies towards understanding of inter-individual variation in immune memory to SARS CoV-2 natural infection for future vaccine evaluation and implementation efforts.

Antibody response patterns in chikungunya febrile phase predict protection versus progression to chronic arthritis.

Chikungunya virus (CHIKV) infection causes acute febrile illness in humans, and some of these individuals develop a debilitating chronic arthritis that can persist for months to years for reasons that remain poorly understood. In this study from India, we characterized antibody response patterns in febrile chikungunya patients and further assessed the association of these initial febrile-phase antibody response patterns with protection versus progression to developing chronic arthritis. We found 5 distinct patterns of the antibody responses in the febrile phase: no CHIKV binding or neutralizing (NT) antibodies but PCR positive, IgM alone with no NT activity, IgM alone with NT activity, IgM and IgG without NT activity, and IgM and IgG with NT activity. A 20-month follow-up showed that appearance of NT activity regardless of antibody isotype or appearance of IgG regardless of NT activity during the initial febrile phase was associated with a robust protection against developing chronic arthritis in the future. These findings, while providing potentially novel insights on correlates of protective immunity against chikungunya-induced chronic arthritis, suggest that qualitative differences in the antibody response patterns that have evolved during the febrile phase can serve as biomarkers that allow prediction of protection or progression to chronic arthritis in the future.

Incomplete immunological recovery following anti-tuberculosis treatment in HIV-infected individuals with active tuberculosis.

BACKGROUND & OBJECTIVE: Mycobacterium tuberculosis infection has been shown to result in increased HIV replication and disease progression in HIV-infected individuals through increased immune activation. The objective of this study was to correlate plasma levels of immune activation markers with the presence of tuberculosis (TB) in HIV-infected and uninfected individuals, and to study the changes following anti-tuberculosis treatment. METHODS: Plasma markers of immune activation - neopterin, beta-2-microglobulin (beta2M) and soluble tumour necrosis factor alpha receptor type I (sTNFalpha-RI) were measured by ELISA in 42 HIV positive TB patients (HIV+TB+) undergoing a six-month course of TB chemotherapy. Thirty seven HIV+ persons without active TB, 38 TB patients without HIV infection, and 62 healthy volunteers served as controls. RESULTS: Plasma levels of all three markers were elevated in HIV+ individuals, more so in those with active TB. When HIV+ individuals were further categorized based on CD4+ T cell counts, HIV+TB+ patients with CD4+ T cells counts

Emergence of new genotypes and lineages of dengue viruses during the 2012-15 epidemics in southern India.

OBJECTIVES: To genotypically characterize dengue virus (DENV) isolates among dengue-infected children from 2012-13/2014-15 outbreaks in southern India. METHODS: Children hospitalized with suspected dengue were tested for dengue RT-PCR targeting Capsid-preMembrane (C-prM) and Envelope (Env) regions. Following virologic confirmation (n=612), a representative selection of DENV isolates (n=99) were sequenced for C-prM, aligned using ClustalW and subjected to phylogenetic analysis by maximum-likelihood method in MEGA6. RESULTS: In 2012-13 (n=113), DENV-3 (44, 38.9%) and DENV-2 (43, 38.1%) predominated; DENV-1 (22, 19.5%) and DENV-4 (1, 0.9%) were less common. The pattern changed in 2014-15 (n=499), when DENV-1 (329, 65.7%) predominated, followed by DENV-2 (97, 21.2%), DENV-3 (36, 6.7%) and DENV-4 (10, 2.0%). Multiple-serotype co-infections occurred in 2.7% and 5.4% in 2012-13 and 2014-15, respectively. Genotype III (GIII) of DENV-1 predominated (85.7%) in 2012-13, ceding to GI predominance (80.8%) in 2014-15. Among DENV-2, 71.9% (23/32) showed distinct clustering suggesting a new lineage, 'GIVc'. All tested DENV-4 were GIC, whose clustering pattern showed the emergence of two distinct clades. CONCLUSIONS: New genotypic/lineage variations in DENV-1 and DENV-2 may have influenced the magnitude and severity of dengue epidemics in southern India during this period. These findings emphasize the role of active surveillance of DENV serotypes/genotypes in aiding outbreak control and vaccine studies.

Differential expression of CXCR3 and CCR6 on CD4+ T-lymphocytes with distinct memory phenotypes characterizes tuberculosis-associated immune reconstitution inflammatory syndrome.

Immune reconstitution inflammatory syndrome (IRIS) occurs in up to 40% of individuals co-infected with pulmonary tuberculosis (PTB) and HIV, primarily upon antiretroviral therapy (ART) initiation. Phenotypic changes in T-cells during TB-IRIS and their relationship with systemic inflammation are not fully understood. In this prospective cohort study, we followed 48 HIV-positive patients with PTB from South India before and after ART initiation, examining T-lymphocyte subsets and inflammatory biomarkers in peripheral blood. Quantification of naïve (CD27+CD45RO-) as well as effector memory CD4+ T cells (CD27-CD45RO+) at weeks 2-6 after ART initiation could distinguish TB-IRIS from non-IRIS individuals. Additional analyses revealed that ART reconstituted different quantities of CD4+ T lymphocyte subsets with preferential expansion of CXCR3+ CCR6- cells in TB-IRIS patients. Moreover, there was an expansion and functional restoration of central memory (CD27+CD45RO+) CXCR3+CCR6- CD4+ lymphocytes and corresponding cytokines, with reduction in CXCR3-CCR6+ cells after ART initiation only in those who developed TB-IRIS. Together, these observations trace a detailed picture of CD4+ T cell subsets tightly associated with IRIS, which may serve as targets for prophylactic and/or therapeutic interventions in the future.