IL-15 and sMAdCAM: Novel roles in COVID-19 pathogenesis (preprint)

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=125) 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 was 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, negative association of plasma IL-15 with depleted T, B and NK subsets suggested 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 longer duration of hospitalization.

Viroselect: A novel SARS-CoV-2 detection assay to resolve inconclusive samples (preprint)

ABSTRACT Background India bears the second largest burden of SARS-CoV-2 infection. A multitude of 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. Here, we report the development of a novel ORF-1a based SARS-CoV-2 RT-PCR assay, Viroselect, showing high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India. Methods We identified a unique target region within SARS-CoV-2 ORF1a, non-structural protein (nsp3), that was used to design and develop our assay. This hypervariable region (1933-3956) between SARS-CoV-2, SARS-CoV, and MERS-COV was utilized to design our primers and probe for RT-PCR assay. We further evaluated concordance of our assay with commonly used EUA (USFDA) manual kits as well as an automated high throughput testing platform. Further, a retrospective analysis using Viroselect on samples reported as ‘inconclusive’ during April-October 2020 was carried out. Results A total of 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect assay with both manual (87.6%; 95% CI) as well as automated (84.7%; 95% CI) testing 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) samples which were termed inconclusive by manual and automated high throughput platform respectively. Conclusion We show that Viroselect had high concordance with conventional assays, both manual and automated, as well as highlight its potential in resolving inconclusive samples.

sMAdCAM:IL-6 (sMIL Index): A novel signature associated with COVID-19 disease progression and development of anti-SARS-CoV-2 antibodies (preprint)

IMPORTANCE: Recent studies positing the gut as a sanctuary site for viral persistence in SARS-CoV-2 infection highlight the importance of assimilating profiles of systemic as well as gut inflammatory mediators to understand the pathology of COVID-19. Also, the role of these markers in governing virus specific immunity following infection remains largely unexplored. OBJECTIVE: To evaluate the role of systemic and gut inflammatory markers in disease progression and development of anti-viral humoral immunity following SARS-CoV-2 infection. DESIGN, SETTING AND PARTICIPANTS: This cohort study (n=58) of SARS-CoV-2 infected individuals included a group of in-patients (n=36) at various stages of disease progression together with convalescent individuals (n=22) recruited between April and June 2020 (peak of the epidemic) from a tertiary care hospital in Mumbai, India. Follow-up of 11 in-patients at day 7 post diagnosis was carried out, resulting in a total of 47 in-patient samples. EXPOSURES: Diagnosis of SARS-CoV-2 infections was confirmed by reverse transcriptase-polymerase chain reaction-based testing of nasopharyngeal/oropharyngeal samples. MAIN OUTCOMES AND MEASURES: Primary outcomes were the measurement of inflammatory markers including Th1/Th2/Th17 cytokines and levels of soluble mucosal addressin cell adhesion molecule (sMAdCAM) in plasma. Anti-viral humoral response was measured by rapid antibody test (IgG, IgM) and chemiluminescent immunoassay (CLIA) (IgG). Also antibodies binding to SARS-CoV-2 proteins were measured by surface plasmon resonance (SPR). Secondary outcomes were correlation of the inflammatory signature with clinical information, including age, sex, disease duration and co-morbidities. RESULTS: Twenty eight of 36 (78%) in-patients and 19 of 22 (86%) convalescents were males. Out of 47 in-patient samples, 22 (46%), 11 (23%) and 14 (30%) were IgG-/IgM-, IgG+/IgM+ and IgG+/IgM- respectively. Of 22 convalescent samples, 3 (14%), 1 (4%) and 17 (77%) were respectively IgG-/IgM-, IgG+/IgM+ and IgG+/IgM-. Two out of 22 (9%) convalescents showed high IL-6 levels (>100pg/ml) and 4 (18%) had high TNF levels (>30pg/ml). However, the convalescents (n =22) had significantly lower levels of IL-6 [Median=27.48 (IQR=23.54-39.92)] compared to followed up in-patients (n = 11) at day 0 [Median=111(IQR=68-129.7), p =0.0002] and higher levels of sMAdCAM [Median=1940 (1711-2174) pg/ml] compared to these individuals at day 0 [Median=1701 (IQR=1532-1836) pg/ml; p=0.032] and day 7 [Median=1534 (IQR=1236-1654) pg/ml; p=0.0007]. Further, IL-6 and sMAdCAM levels among in-patients inversely correlated with one another (r =-0.374, p = 0.009, CI = 95%). When expressed as a novel integrated marker, sMIL (sMAdCAM/IL-6 ratio) index, these levels were incrementally and significantly higher across various disease states with convalescents exhibiting the highest values [Median= 64.74 (IQR=47.33-85.58)]. Also, the sMIL index was significantly higher in convalescents (with class-switched responses) compared to IgG+/IgM+ individuals at early stages of infection [Median=28.65 (IQR=13.63-96.26), p = 0.034]. Real-time measurement by SPR of plasma antibody binding to viral nucleocapsid (NC), receptor binding domain (RBD) and spike (S) revealed waxing and waning of plasma antibody responses to all 3 targets. Importantly, sMAdCAM levels as well as sMIL index (fold change) correlated with peak association rates of RBD-binding (r = 0.462, p = 0.03, CI = 95%) and fold change in binding to S (r = 0.68, p = 0.050, CI = 95%) respectively. CONCLUSION AND RELEVANCE: Our results highlight key systemic and gut-associated immune parameters that need to be monitored and investigated further to optimally guide therapeutic and prophylactic interventions for COVID-19.

SARS-CoV-2 S protein ACE2 interaction reveals novel allosteric targets (preprint)

The Spike (S) protein is the main handle for SARS-CoV-2 to enter host cells through surface ACE2 receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, we have mapped the S:ACE2 interface and uncovered long-range allosteric propagation of ACE2 binding to sites critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 [A] away while dampening dynamics of the stalk hinge (central helix and heptad repeat) regions ~ 130 [A] away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the pre-fusion state. Our findings provide a mechanistic basis for S:ACE2 complex formation, critical for proteolytic processing and viral-host membrane fusion and highlight protease docking sites flanking the S1/S2 cleavage site, fusion peptide and heptad repeat 1 (HR1) as allosterically exposed cryptic hotspots for potential therapeutic development.

Persistence of SARS-CoV-2 in the first trimester placenta leading to vertical transmission and fetal demise from an asymptomatic mother (preprint)

Coronaviruses infect the respiratory tract and are known to survive in these tissues during the clinical course of infection. However, how long can SARS-CoV-2 survive in the tissues is hitherto unknown. Herein, we report a case where the virus is detected in the first trimester placental cytotrophoblast and syncytiotrophoblasts five weeks after the asymptomatic mother cleared the virus from the respiratory tract. This first trimester placental infection was vertically transmitted as the virus was detected in the amniotic fluid and fetal membranes. This congenitally acquired SARS-CoV-2 infection was associated with hydrops and fetal demise. This is the first study providing concrete evidences towards persistent tissue infection of SARS-CoV-2, its congenital transmission in early pregnancy leading to intrauterine fetal death.