ABSTRACT
In this study, we report on longitudinal kinetics of cellular immune subsets following SARS-CoV-2 infection in a cohort of hospitalized individuals and evaluate the interplay of these profiles with infecting viral variants, humoral immunity including neutralizing responses, vaccination history and clinical outcomes. A cohort of 121 SARS-CoV-2 infected individuals exhibiting varying disease states were prospectively evaluated for lymphopenic profiles, anti-viral humoral responses and infecting viral variants for a period of up to 90 days spanning the period, February 2021-January 2022 (2nd and 3rd waves of infection). A total of 51 participants received at least one vaccine dose of indigenous vaccines (Covishield or Covaxin) prior to recruitment. When stratified in terms of mortality, B and NK cells, in contrast to the T cell compartment, did not recover from nadir levels in non-survivors who were largely unvaccinated. No discriminatory signature was identified for non-survivors in terms of anti-NC or anti-S1-RBD IgG CLIA profiles including GenScript S1-RBD assays. Evaluation of sVCAM and sMAdCAM revealed opposing dynamics that correlated with disease severity and convalescence respectively. Viral variant analysis revealed delta and omicron variants to comprise majority of the infections which reflected national transmission kinetics during the period of recruitment. Our results demonstrate the importance of monitoring circulating biomarkers for convalescence as well as mortality in COVID-19 progression. Delta variants of SARS-CoV-2 clearly demonstrated increased pathogenicity and warrants sustained viral surveillance for re-emergence of these strains. Our findings with respect to vaccination advocate for continued vaccine development and administration of COVID-19 vaccines.
ABSTRACT
Immune cell dysregulation and lymphopenia characterize COVID-19 pathology in moderate to severe disease. While underlying inflammatory factors have been extensively studied, homeostatic and mucosal migratory signatures remain largely unexplored as causative factors. In this study, we evaluated the association of circulating IL-6, soluble mucosal addressin cell adhesion molecule (sMAdCAM), and IL-15 with cellular dysfunction characterizing mild and hypoxemic stages of COVID-19. A cohort of SARS-CoV-2 infected individuals (n = 130) at various stages of disease progression together with healthy controls (n = 16) were recruited from COVID Care Centres (CCCs) across Mumbai, India. Multiparametric flow cytometry was used to perform in-depth immune subset characterization and to measure plasma IL-6 levels. sMAdCAM, IL-15 levels were quantified using ELISA. Distinct depletion profiles, with relative sparing of CD8 effector memory and CD4+ regulatory T cells, were observed in hypoxemic disease within the lymphocyte compartment. An apparent increase in the frequency of intermediate monocytes characterized both mild as well as hypoxemic disease. IL-6 levels inversely correlated with those of sMAdCAM and both markers showed converse associations with observed lympho-depletion suggesting opposing roles in pathogenesis. Interestingly, IL-15, a key cytokine involved in lymphocyte activation and homeostasis, was detected in symptomatic individuals but not in healthy controls or asymptomatic cases. Further, plasma IL-15 levels negatively correlated with T, B, and NK count suggesting a compensatory production of this cytokine in response to the profound lymphopenia. Finally, higher levels of plasma IL-15 and IL-6, but not sMAdCAM, were associated with a longer duration of hospitalization.
Subject(s)
COVID-19 , Interleukin-15/blood , Lymphopenia , CD8-Positive T-Lymphocytes , Cell Adhesion Molecules , Cytokines , Humans , Interleukin-6 , Lymphopenia/etiology , SARS-CoV-2ABSTRACT
The role of sMAdCAM, an important gut immune migratory marker, remains unexplored in COVID-19 pathogenesis considering recent studies positing the gut as a sanctuary site for SARS-CoV-2 persistence. Thus, assimilating profiles of systemic inflammatory mediators with sMAdCAM levels may provide insights into the progression of COVID-19 disease. Also, the role of these markers in governing virus specific immunity following infection remains largely unexplored. A cohort (n = 84) of SARS-C0V-2 infected individuals included a group of in-patients (n = 60) at various stages of disease progression together with convalescent individuals (n = 24) recruited between April and June 2020 from Mumbai, India. Follow-up of 35 in-patients at day 7 post diagnosis was carried out. Th1/Th2/Th17 cytokines along with soluble MAdCAM (sMAdCAM) levels in plasma were measured. Also, anti-viral humoral response as measured by rapid antibody test (IgG, IgM), Chemiluminescent Immunoassay (IgG), and antibodies binding to SARS-CoV-2 proteins were measured by Surface Plasmon Resonance (SPR) from plasma. IL-6 and sMAdCAM levels among in-patients inversely correlated with one another. When expressed as a novel integrated marker-sMIL index (sMAdCAM/IL-6 ratio)-these levels were incrementally and significantly higher in various disease states with convalescents exhibiting the highest values. Importantly, sMAdCAM levels as well as sMIL index (fold change) correlated with peak association response units of receptor binding domain and fold change in binding to spike respectively as measured by SPR. Our results highlight key systemic and gut homing parameters that need to be monitored and investigated further to optimally guide therapeutic and prophylactic interventions for COVID-19.
Subject(s)
COVID-19/immunology , Cell Adhesion Molecules/blood , Interleukin-6/blood , Mucoproteins/blood , Adolescent , Adult , Aged , Biomarkers/blood , COVID-19/physiopathology , Cohort Studies , Cytokines/blood , Disease Progression , Female , Humans , Intestines/immunology , Male , Middle Aged , Surface Plasmon Resonance , Young Adult , COVID-19 Drug TreatmentABSTRACT
Integrin α4ß7 expressing CD4+ T cells are preferred targets for HIV infection and are thought to be predictors of disease progression. Concurrent analysis of integrin α4ß7 expressing innate and adaptive immune cells was carried out in antiretroviral (ART) therapy naïve HIV infected women in order to determine its contribution to HIV induced immune dysfunction. Our results demonstrate a HIV infection associated decrease in the frequency of integrin α4ß7 expressing endocervical T cells along with an increase in the frequency of integrin α4ß7 expressing peripheral monocytes and central memory CD4+ T cells, which are considered to be viral reservoirs. We report for the first time an increase in levels of soluble MAdCAM-1 (sMAdCAM-1) in HIV infected individuals as well as an increased frequency and count of integrin ß7Hi CD8+ memory T cells. Correlation analysis indicates that the frequency of effector memory CD8+ T cells expressing integrin α4ß7 is associated with levels of both sMAdCAM-1 and TGF-ß1. The results of this study also suggest HIV induced alterations in T cell homeostasis to be on account of disparate actions of sMAdCAM-1 and TGF-ß1 on integrin α4ß7 expressing T cells. The immune correlates identified in this study warrant further investigation to determine their utility in monitoring disease progression.
Subject(s)
Cell Adhesion Molecules/blood , HIV Infections/immunology , HIV-1/immunology , Mucoproteins/blood , T-Lymphocytes, Cytotoxic/immunology , Transforming Growth Factor beta1/blood , Adolescent , Adult , CD4-Positive T-Lymphocytes/immunology , Cell Adhesion Molecules/immunology , Disease Progression , Female , HIV Infections/blood , HIV Infections/virology , Humans , Integrins/metabolism , Lymphocyte Count , Middle Aged , Mucoproteins/immunology , T-Lymphocytes, Cytotoxic/metabolism , Transforming Growth Factor beta1/immunology , Young AdultABSTRACT
BACKGROUND: India bears the second largest burden of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A multitude of reverse transcription polymerase chain reaction (RT-PCR) detection assays with disparate gene targets, including automated high-throughput platforms, are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays, leading to suboptimal disease management. This article reports the development of a novel ORF1a-based SARS-CoV-2 RT-PCR assay - Viroselect - that shows high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India. METHODS: A unique target region within SARS-CoV-2 ORF1a - the non-structural protein 3 (nsp3) region - was used to design and develop the assay. This hypervariable region (1923-3956) between SARS-CoV-2, SARS-CoV-1 and Middle East respiratory syndrome coronavirus was utilized to design the primers and probes for the RT-PCR assay. The concordance of this assay with commonly used emergency use authorization (US Food and Drug Administration) manual kits and an automated high-throughput testing platform was evaluated. Further, a retrospective analysis was carried out using Viroselect on samples reported as 'inconclusive' between April and October 2020. RESULTS: In total, 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect with both manual (87.6%) and automated (84.7%) assays. Also, in the retrospective analysis of 224 additional samples reported as 'inconclusive', Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) of samples which had inconclusive results on manual and automated high-throughput platforms, respectively. CONCLUSION: Viroselect had high concordance with conventional assays, both manual and automated, and has potential to resolve inconclusive samples.
Subject(s)
COVID-19 Testing/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Viral Proteins/genetics , Humans , Limit of Detection , Polyproteins/genetics , Retrospective Studies , SARS-CoV-2/isolation & purificationABSTRACT
Integrin α4ß7, a CD4 independent receptor of human immunodeficiency virus-1 (HIV-1) gp120, defines a subset of CD4+T cells preferentially targeted by HIV. It is also considered as a promising therapeutic target for HIV-1 infection. Despite its role in HIV acquisition and disease progression, HIV-1-mediated integrin α4ß7 signaling has not been elucidated so far. In view of this, we determined phosphoproteomic signatures of HIV-1 gp120 signaling as well as signaling mediated by the integrin α4ß7 ligand, mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1), in primary CD4+ T cells. This is the first comprehensive report on MAdCAM-1 signaling, which is believed to enhance HIV-1 replication. Importantly, we identified proteins associated with both classical and nonclassical integrin functions. We observed that HIV-1 gp120 signaling is associated with proteins that have previously not been associated with HIV-1 pathogenesis and thus, need to be explored further. There was a significant overlap in proteins identified by both MAdCAM-1 and HIV-1 gp120 signaling, which most likely represents cellular processes triggered upon interaction of HIV-1 gp120 with integrin α4ß7. Pathway analysis revealed enrichment of processes that could facilitate viral replication as well as viral entry through endocytosis. Although these results warrant independent replication and further validation, they suggest the presence of additional potential therapeutic targets. These results also suggest that combinatorial approaches for targeting both HIV-1 gp120 and MAdCAM-1 signaling may be necessary for efficient control of HIV-1 infection as well as novel innovation strategies in HIV therapeutics.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , HIV Infections/metabolism , HIV Infections/virology , HIV-1/physiology , Host-Pathogen Interactions , Integrins/metabolism , Signal Transduction , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , Cell Adhesion Molecules/metabolism , HIV Envelope Protein gp120/metabolism , HIV Infections/genetics , HIV Infections/immunology , Humans , Integrins/chemistry , Models, Biological , Mucoproteins/metabolism , Phosphoproteins/metabolism , Protein Binding , Protein Interaction Mapping , Protein Interaction Maps , Proteome , Proteomics/methods , Virus InternalizationABSTRACT
Bacterial vaginosis (BV) is a common polymicrobial infection affecting women in the reproductive age and is associated with adverse obstetric and gynaecological outcomes. Gardnerella vaginalis is the most virulent anaerobic bacterial species predominantly associated with BV. However, a clear understanding of the mechanisms by which it contributes to the pathogenesis and persistence of BV is lacking. In this report, we demonstrate for the first time, the isolation of membrane vesicles (MVs) from G. vaginalis ATCC 14019. These MVs are approximately 120-260â¯nm in diameter. Proteomic characterization of the MVs by LC-MS/MS led to the identification of 417 proteins, including proteins involved in cellular metabolism as well as molecular chaperones and certain virulence factors. Immunoblot analysis of the MVs confirmed the presence of vaginolysin, the most well-characterized virulence factor of G. vaginalis. The exposure of the vaginal epithelial cells, VK2/E6E7 to the G. vaginalis MVs resulted in the internalization of the MVs. The MVs induced cytotoxicity and an increase in the levels of the pro-inflammatory cytokine, IL-8 in VK2 cells as well lysis of erythrocytes. The results of the study indicate that G. vaginalis MVs may be involved in the delivery of cytotoxic proteins and other virulence factors to the host cells and could thereby contribute towards enhancing the cellular damage associated with pathogenesis of BV.
Subject(s)
Cytoplasmic Vesicles/metabolism , Epithelial Cells/microbiology , Gardnerella vaginalis/physiology , Vaginosis, Bacterial/microbiology , Bacterial Proteins , Cell Survival , Computational Biology/methods , Cytokines/metabolism , Cytoplasmic Vesicles/ultrastructure , Female , Gardnerella vaginalis/ultrastructure , Hemolysis , Humans , Mass Spectrometry , Proteome , Proteomics/methods , Vaginosis, Bacterial/pathologyABSTRACT
Seventy-five glorious years have passed since estradiol was discovered by Edward Doisy. From discovery in the ovaries to delineation of diverse physiological effects, research on estrogens has covered a lot of ground. Estrogen receptors that mediate estrogenic effects, have been detected not only in reproductive organs, but also in other body organs. Estrogen receptors function either as conventional transcription factors or as rapid signal transducers. These different modes of action are opted by estrogens to elicit an array of reproductive and non-reproductive functions. It is well established that estrogens promote cell proliferation in various tissues and hence are also linked to carcinogenesis. Anti-estrogens are being used as adjunct therapies for cancers since several years. On the other hand, estrogen-based strategies are used to alleviate adverse effects of menopause. Apart from estrogens synthesized in various organs, exposure to environmental estrogens can also impact physiology. Thus, too much or too less of estrogens can tip the balance and lead to unfavorable consequences. Multiple estrogen receptors with their tissue- or cell type-specific expression eliciting dose-dependent effects make it perplexing to 'unify' estrogenic actions in diverse tissues/organs. This warrants more research on estrogen-mediated effects and their regulation in somatic and reproductive tissues. This review presents physiological and pathological aspects of estrogens thus highlighting the good, bad, and ugly facets of estrogens.