Anti-COVID-19 Activity of FDA Approved Drugs through RNA G-quadruplex Binding (preprint)

The COVID-19 pandemic caused by SARS-CoV-2 has caused millions of infections and deaths worldwide. Limited treatment options and the threat from emerging variants underline the need for novel and widely accessible therapeutics. G-quadruplexes (G4s) are nucleic acid secondary structures known to affect many cellular processes including viral replication and transcription. We identified heretofore not reported G4s with remarkably low mutation frequency across >4 million SARS-CoV-2 genomes. The G4 structure was targeted using FDA approved drugs that can bind G4s - Chlorpromazine (CPZ) and Prochlorperazine (PCZ). We found significant inhibition in lung pathology and lung viral load of SARS-CoV-2 challenged hamsters when treated with CPZ, PCZ that was comparable to the widely used antiviral drug Remdesivir. In support, in vitro G4 binding, inhibition of reverse transcription from RNA isolated from COVID-infected humans, and attenuated viral replication and infectivity in vero cell cultures were clear in case of both CPZ/PCZ. Apart from the wide accessibility of CPZ/PCZ, targeting relatively invariant nucleic acid structures poses an attractive strategy against fast mutating viruses like SARS-CoV-2.

Prophylactic treatment of Glycyrrhiza glabra mitigates COVID-19 pathology through inhibition of pro-inflammatory cytokines in the hamster model and NETosis. (preprint)

Severe coronavirus disease (COVID-19) is accompanied with acute respiratory distress syndrome & pulmonary pathology, and is presented mostly with inflammatory cytokine release, dysregulated immune response, skewed neutrophil/ lymphocyte ratio, and hypercoagulable state. Though vaccinations have proved effective in reducing the COVID-19 related mortality, the limitation of use of vaccine against immunocompromised, comorbidity, and emerging variants remains a concern. In the current study we investigate for the first-time the efficacy of Glycyrrhiza glabra (GG) extract, a potent immunomodulator, against SARS-CoV-2 infection in hamsters. Prophylactic treatment with GG showed protection against loss in body weight and 35-40% decrease in lung viral load along with reduced lung pathology in the hamster model. Remarkably, GG reduced the mRNA expression of pro-inflammatory cytokines and Plasminogen activator inhibito-1 (PAI-1). In-vitro, GG acted as potent immunomodulator by reducing Th2 and Th17 differentiation and IL-4 and IL-17A cytokine production. In addition, GG also showed robust potential to suppress ROS, mtROS and NETs generation in a concentration dependent manner in both human polymorphonuclear neutrophils (PMNs) and murine bone marrow derived neutrophils (BMDNs). Taken together, we provide evidence for the protective efficacy of GG against COVID-19 and its putative mechanistic insight, which might be developed as a future immunomodulatory approach against various pathologies with high cytokine production, aberrant neutrophil activation including coronavirus infection.

SARS-CoV-2 and its variants, but not Omicron, induces severe thymic atrophy and impaired T cell development (preprint)

Pathogenic infections cause thymic atrophy, perturb thymic-T cell development and alter immunological response. Previous studies reported dysregulated T cell function and lymphopenia in coronavirus disease-19 (COVID-19) patients. However, immune-pathological changes, in the thymus, post severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection have not been elucidated. Here, we report SARS-CoV-2 infects thymocytes, depletes CD4 + CD8+ (double positive; DP) T cell population associated with an increased apoptosis of thymocytes, which leads to severe thymic atrophy in K18-hACE2-Tg mice. CD44 + CD25- T cells were found to be enriched in infected thymus, indicating an early arrest in the T cell developmental pathway. Further, Interferon gamma (IFN-γ) was crucial for thymic atrophy, as anti-IFN-g antibody neutralization rescued the loss of thymic involution. Therapeutic use of remdesivir (prototype anti-viral drug) was also able to rescue thymic atrophy. While Omicron variant of SARS-CoV2 caused marginal thymic atrophy, delta variant of SARS-CoV-2 exhibited most profound thymic atrophy characterized by severely depleted DP T cells. Recently characterized broadly SARS-CoV-2 neutralizing monoclonal antibody P4A2 was able to rescue thymic atrophy and restore thymic developmental pathway of T cells. Together, we provide the first report of SARS-CoV-2 associated thymic atrophy resulting from impaired T cell developmental pathway and also explains dysregulated T cell function in COVID-19.

SARS-CoV-2 and its variants, but not Omicron, induces thymic atrophy and impaired T cell development (preprint)

Pathogenic infections cause thymic atrophy, perturb thymic-T cell development and alter immunological response. Previous studies reported dysregulated T cell function and lymphopenia in coronavirus disease-19 (COVID-19) patients. However, immune-pathological changes, in the thymus, post severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection have not been elucidated. Here, we report SARS-CoV-2 infects thymocytes, depletes CD4+CD8+ (double positive DP) T cell population associated with an increased apoptosis of thymocytes, which leads to severe thymic atrophy in K18-hACE2-Tg mice. CD44+CD25- T cells were found to be enriched in infected thymus, indicating an early arrest in the T cell developmental pathway. Further, Interferon gamma was crucial for thymic atrophy, as anti-IFN{gamma}; antibody neutralization rescued the loss of thymic involution. Therapeutic use of remdesivir (prototype anti-viral drug) was also able to rescue thymic atrophy. While Omicron variant of SARS-CoV2 caused marginal thymic atrophy, delta variant of SARS-CoV-2 exhibited most profound thymic atrophy characterized by severely depleted DP T cells. Recently characterized broadly SARS-CoV-2 neutralizing monoclonal antibody P4A2 was able to rescue thymic atrophy and restore thymic developmental pathway of T cells. Together, we provide the first report of SARS-CoV-2 associated thymic atrophy resulting from impaired T cell developmental pathway and also explains dysregulated T cell function in COVID-19.

SARS-CoV-2 variants’-Alpha, Delta, and Omicron D614G and P681R/H mutations impact virus entry, fusion, and infectivity (preprint)

SARS-CoV-2 variants acquire mutations to survive within the host and evade immunity. In addition to harboring D614G mutation in spike domain, P681R/H mutation at the junction of the S1/S2 furin cleavage site, is found to be the key mutation in variants of concerns (VoC); Alpha, Delta, and Omicron (B.1.1.519). The impact of these acquired mutations on entry, transmissibility, and infectivity of SARS-CoV2 VoC is not clearly identified. Here, using the spike-based pseudovirus, Delta and D614G+P681R synthetic mutants showed a significant increase in the pseudovirus entry, fusion, and infectivity. In contrast, Omicron spike-based pseudovirus and a synthetic P681H mutant showed preferential hACE2-mediated virus entry over TMPRSS2, less fusion, and highly susceptible to Cathepsin L inhibitor. Taken together, these results indicate while the Delta variant utilizes both ACE2 and TMPRSS2 mediated entry, thus causing systemic infection; Omicron has favored growth in ACE2 expressed cells thus mainly replicating in the upper respiratory tract.