HIGH-RESOLUTION MAPPING OF T-CELL HYBRID IMMUNITY TO THE ENTIRE SARS-CoV-2 PROTEOME

Background: To understand T-cell responses to SARS-CoV-2, it is essential to define the contribution of infection versus immunization to virus-specific hybrid immunity. Here, we characterized the breadth and magnitude of T-cell responses to the entire SARS-CoV2 proteome over a 2-year follow-up period in infected and vaccinated (CoV2+Vac+) and vaccinated and infected (Vac+CoV2+) individuals. Method(s): We selected samples from 38 (19 CoV2+ and 19 CoV2-, time1, T1) ProHEpiC-19 cohort participants, a prospective, longitudinal study starting in March 2020 involving 7,776 healthcare workers in Spain. Longitudinal samples were available from 10 of them after a 3-dose mRNA vaccination, including 5 CoV2+Vac+ and 5 Vac+CoV2+, at 824.5 and 250.5 days from symptoms onset (DfSO, time 2, T2). We measured the breadth and magnitude of IFN-y T-cell responses by ELISpot assay in cryopreserved PBMCs, using a 15-mer overlapping peptide (OLP) library of 2,790 SARS-CoV-2 peptides in 100 pools. Result(s): We identified immunodominant T-cell responses in S1, S2, nsp3, Env, NC, and M proteins across the SARS-CoV2 proteome. We observed an increased breadth of T-cell responses (responding pools over the entire region) to S1 (44 - 30%) and S2 (31 - 40%) in CoV2+Vac+ and Vac+CoV2+, respectively. In addition, CoV2+Vac+ had an exclusive and sustained response to M. We found significantly stronger responses in CoV2+Vac+ (P=0.0313). Particularly the total magnitude was greater in CoV2+Vac+ vs. Vac+CoV2+ in S1 (4476.88 vs. 1498.53), Env (457.34 vs. 250.50), and M (455.13 vs. 0.00) but not in S2 and nsp3. The total number of peptides for deconvolution was higher in CoV2+Vac+ (32 peptides) than in Vac+CoV2+ (3 peptides) during the follow-up. Seventy-five percent of the responses targeted S, and 25% M, ORF1a, and Env. Conclusion(s): These results profile immunodominant T-cell responses in S1, S2, nsp3, Env, NC, and M proteins across the entire SARS-CoV2 proteome. The data delineate differences in the number of T-cell responses primed hybrid immunity by infection previous to vaccination (CoV2+Vac+), being broader and of higher magnitude and underlining an exclusive T-cell response to the M region. Overall, these findings identify differences in long-term T-cell hybrid immunity primed by infection or vaccination, which may have implications in protection from re-infection and vaccine design.

The Concept of Repurposing in COVID-19 Infection

The strategy of drug repurposing has been proved successful in response to the current corona-virus pandemic, with remdesivir becoming the first drug of choice, an antiviral drug approved for the treatment of COVID-19. In parallel to this, several drugs, such as antimalarial, corticosteroids, and antibi-otics, like azithromycin, are used to treat the severe condition of hospitalized COVID-19 patients, while clinical testing of additional therapeutic drugs, including vaccines, is going on. It is reasonably expected that this review article will deliver optimized and specific curative tools that will increase the attentive-ness of health systems to the probable outlook of epidemics in the future. This review focuses on the ap-plication of repurposed drugs by studying their structure, pharmacokinetic study, different mechanisms of action, and Covid-19 guidelines, which can potentially influence SARS-CoV-2. For most of the drugs, direct clinical evidence regarding their effectiveness in the treatment of COVID-19 is missing. Future clinical trial studies may conclude that one of these can be more potential to inhibit the progression of COVID-19.Copyright © 2022 Bentham Science Publishers.

A Method to Monitor Activity of SARS-CoV-2 Nsp3 from Cells

SARS-CoV-2 protease Nsp3 is a therapeutic target for developing anti-SARS-CoV-2 drugs. Nsp3 is a large multi-spanning membrane protein, and its characterization in vitro has been challenging. Here we describe an in vitro assay to characterize the biochemical activity of full-length Nsp3 isolated from cells. The assay can be used to evaluate Nsp3 inhibitors. © 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Exploration of Luteolin as Potential Anti-COVID-19 Agent: Molecular Docking, Molecular Dynamic Simulation, ADMET and DFT Analysis

Background: Coronavirus disease-2019 (COVID-19) has recently emerged as a pandemic respiratory disease with mild to severe pneumonia symptoms. No clinical antiviral agent is available so far. However, several repurposing drugs and vaccines are being given to individuals or in clinical trials against SARS-CoV-2 Objective: The aim of this study is to uncover the potential effects of Luteolin (Lut) as an inhibitor of SARS-CoV2 encoded proteins via utilizing computational tools. Methods: Molecular modelling to unfold the anti-SARS-CoV2 potential of Lut along with reference drugs namely remdesivir and nafamostat was performed by the use of molecular docking, molecular dynamic (MD) simulation, absorption, distribution, metabolism, excretion, toxicity (ADMET) and density functional theory (DFT) methods against the five different SARS-CoV-2 encoded key proteins and one human receptor protein. The chemical reactivity of Luteolin is done through prediction of HOMO-LUMO gap energy and other chemical descriptors analysis. Results: In the present study, Lut binds effectively in the binding pockets of spike glycoprotein (6VSB), ADP phosphatase of NSP3 (6W02), and RNA dependent RNA polymerase (7AAP) protein receptors with significant values of docking scores-7.00,-7.25, and-6.46 respectively as compared to reference drugs remdesivir and nafamostat. Conclusion: Thus, Lut can act as a therapeutic agent and is orally safe for human consumption as predicted by molecular modelling against SARS-CoV-2 in the treatment of COVID-19.

My Journeys with Three Microbes in the Reproductive Tract

Problem: The placenta performs various functions of the lung/GI/GU tract for the developing fetus, while also moderating host defenses of the fetus against infections in utero, and likely educates the developing fetal immune system. It thus has long-term impacts on the health of both the woman and the child. Knowledge is limited about the underlying mechanisms that enable the placenta to serve as a protective barrier for the fetus against infection. The long-term goals of my research program are to, 1) elucidate the normal barriers to infection in the placenta and show how dysfunction in barrier function can lead to adverse maternal-fetal outcomes, 2) define how viral infections impact placental biology, and 3) characterize possible functional roles for the newly described microbiota at the maternal-fetal interface. Method of Study: To address the above questions, our research includes the use human placentas, primary human trophoblasts and immune cells derived from term placentas, cultured placental cells, trophoblast organoids, and mousemodels. Results: We found that placentas from women who gave birth prematurely exhibit reduced autophagy activity. Prematurity and reduced autophagy levels were also strongly associated with maternal infection. In a mouse model of pregnancy, we showed that placentas from mice deficient for Atg16L1 were significantly less able to withstand infection, and the deficient mice gave birth prematurely upon an inflammatory stimulus. We have also shown that the autophagy pathway plays a key role in ZIKV vertical transmission from mother to fetus. We demonstrated that hydroxychloroquine (HCQ), an autophagy inhibitor approved for use in pregnant women, can attenuate placental and fetal ZIKV infection and ameliorate adverse placental and fetal outcomes. More recently, we have identified a small molecule inhibitor that targets the NS2B-NS3 protease of ZIKV and inhibits viral replication. It has recently become evident that SARS-CoV-2 infection is also associated with adverse outcomes for pregnant women, including preterm birth, preeclampsia, and fetal growth restriction. We localized SARS-CoV-2 to the placenta and showed that infection alters the Renin Angiotensin System (RAS) that regulates blood pressure, thereby increasing risk for preeclampsia. In new work, we are showing that SARS-CoV-2 non-structural proteins affect autophagy in different ways than in Zika virus. Finally, we have discovered that the maternal fetal interface of the placenta harbors intracellular resident microbes, and functionally demonstrated that they do not induce any inflammatory response or cell death but may promote immune tolerance and support normal pregnancy outcomes. Conclusions: For the past 10 years of my career, I have been working on host microbial interactions at the maternal fetal interface. Our work has led to new insights into viral infections, showing how they co-opt host defenses, and that tolerance may have microbial drivers. We have shown how cellular pathways in the placenta such as autophagy and RAS mechanistically regulate host defenses against pathogens, including ZIKV and SARS-CoV-2. Additionally, our studies provide a foundation for understanding possible 'commensal' microbial- placental interactions and hint at the functional importance of microbes at the fetal maternal interface in maintaining placental health and supporting fetal development.

Macro domain F156 residue: a hallmark of SARS-CoV-2 de-MARylation specificity

The Coronavirus disease 2019 (Covid-2019) pandemic currently provokes a global health and economic crisis due to the generalized spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a large, enveloped and positive sense single stranded RNA virus. The SARS-CoV-2 genome encodes 16 non-structural proteins (nsp 1-16), forming a large membrane bound replication complex. The largest protein of this complex is nsp3, a multi-domain protein that contain a well conserved Macro domain (also called X domain or ADP-ribose phosphatase domain). The Macro domain can bind to mono-ADP-ribose (MAR) and poly-ADP-ribose (PAR) in their free form or conjugated to a protein or RNA substrates. Macro domains also carry hydrolase activities including de-MARylation and de-PARylation implicated in the inflammation process and the regulation of innate immunity. Herein, we report a mutagenesis study focusing on SARS-CoV-2 F156 and SARSCoV N157 residues, stipulated important for ADP-ribose orientation within the binding clef. Our data suggest that the exchange of these residues or their substitution to alanine slightly influence ADP-ribose binding, but drastically impact Macro domain de-MARylation activity.

Genetic and structure of novel coronavirus COVID-19 and molecular mechanisms in the pathogenicity of coronaviruses

The recently identified 2019 novel coronaviruses (2019-nCoV) has caused extra-human infections. 2019-nCoV identified a global threat that is causing an outbreak of unusual viral pneumonia in patients with severe acute respiratory syndrome (SARS)-coronaviruses 2 (SARS-CoV-2). Considering the relatively high identity of the receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS-CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. The zinc metallopeptidase angiotensin-converting enzyme 2 (ACE2) is the only known human homolog of the key regulator of blood pressure ACE. ACE2 also serves as the cellular entry point for the SARS virus, therefore, a prime target for pharmacological intervention. SARS-CoV-2 uses the SARS-CoV receptor for entry and the serine protease transmembrane protease serine 2 for spike (S) protein priming. That it is still necessary to develop novel mAbs that could bind specifically to 2019-nCoV RBD. Cell entry of coronaviruses depends on the binding of the viral S proteins to cellular receptors and S protein priming by host cell proteases. A transmembrane protease serine 2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention. This review will help understand the biology and potential risk of CoVs that exist in richness in wildlife such as bats. We provide a brief introduction to the pathogenesis of SARS-CoV and Middle East respiratory syndrome-CoV and interaction between the RBD of coronavirus spike protein and ACE2.

Analysis of a crucial interaction between the coronavirus nucleocapsid protein and the major membrane-bound subunit of the viral replicase-transcriptase complex.

The coronavirus nucleocapsid (N) protein comprises two RNA-binding domains connected by a central spacer, which contains a serine- and arginine-rich (SR) region. The SR region engages the largest subunit of the viral replicase-transcriptase, nonstructural protein 3 (nsp3), in an interaction that is essential for efficient initiation of infection by genomic RNA. We carried out an extensive genetic analysis of the SR region of the N protein of mouse hepatitis virus in order to more precisely define its role in RNA synthesis. We further examined the N-nsp3 interaction through construction of nsp3 mutants and by creation of an interspecies N protein chimera. Our results indicate a role for the central spacer as an interaction hub of the N molecule that is partially regulated by phosphorylation. These findings are discussed in relation to the recent discovery that nsp3 forms a molecular pore in the double-membrane vesicles that sequester the coronavirus replicase-transcriptase.

Current concepts in the development of therapeutics against human and animal coronavirus diseases by targeting NP.

The coronavirus (CoV) infects a broad range of hosts including humans as well as a variety of animals. It has gained overwhelming concerns since the emergence of deadly human coronaviruses (HCoVs), severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, followed by Middle East respiratory syndrome coronavirus (MERS-CoV) in 2015. Very recently, special attention has been paid to the novel coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 due to its high mobility and mortality. As the COVID-19 pandemic continues, despite vast research efforts, the effective pharmaceutical interventions are still not available for clinical uses. Both expanded knowledge on structure insights and the essential function of viral nucleocapsid (N) protein are key basis for the development of novel, and potentially, a broad-spectrum inhibitor against coronavirus diseases. This review aimed to delineate the current research from the perspective of biochemical and structural study in cell-based assays as well as virtual screen approaches to identify N protein antagonists targeting not only HCoVs but also animal CoVs.