IgA quantification as a good predictor of the neutralizing antibodies levels after vaccination against SARS-CoV-2.

Background: Vaccination against COVID-19 was implemented very quickly, but the emergence of new variants that can evade the previous acquired immunological protection highlights the importance of understanding the mechanisms involved in the immune response generated after SARS-CoV-2 infection or vaccination. Objectives: Since most of our knowledge on the humoral immunity generated against SARS-CoV-2 has been obtained from studies with infected patients before vaccination, our goal here was to evaluate seroconversion and its correlation with the titers of neutralizing antibodies (NAbs) in individuals who received the complete initial recommended vaccination schedule with three different vaccines. Study design: We analyzed serum IgG, IgA and total NAbs against the trimeric SARS-CoV-2 Spike (S) protein or its receptor binding domain (RBD) in blood samples collected from 118 healthy individuals without known previous infection, before and after receiving the first and the second dose of CoronaVac (n = 18), ChAdOx-1 (n = 68) or BNT162b2 (n = 32) vaccines. Results: We found that although IgG titers were high in all sera collected after the two doses of these vaccines, NAbs amounts varies among the groups. In contrast, serum NAbs concentrations were much more comparable to the IgA levels, indicating that these antibodies would have a major neutralizing capacity against SARS-CoV-2. Conclusions: Altogether our data suggest that quantification of serum anti-S or anti-RBD IgA, rather than IgG, may be a valuable tool to screen NAbs and may be considered for surveillance of vaccine coverage.

Comprehensive Fragment Screening of the SARS‐CoV‐2 Proteome Explores Novel Chemical Space for Drug Development

SARS‐CoV‐2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti‐virals. Within the international Covid19‐NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR‐detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure‐based drug design against the SCoV2 proteome.

On the caveats of a multiplex test for SARS-CoV-2 to detect seroconversion after infection or vaccination.

The Covid-19 pandemic, caused by SARS-CoV-2, has resulted in over 6 million reported deaths worldwide being one of the biggest challenges the world faces today. Here we present optimizations of all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay to a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals and 103 individuals without a confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals from our cohort before and after receiving ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It is also a valuable tool for elucidating the immunological consequences of new viral strains and monitoring vaccination coverage and duration of response to different immunization regimens.

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids.

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.

Dynamics of the SARS-CoV-2 nucleoprotein N-terminal domain triggers RNA duplex destabilization.

The nucleocapsid (N) protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. The N protein N-terminal domain (N-NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRSs), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of the severe acute respiratory syndrome coronavirus 2 N-NTD to nonspecific (NS) and TRS dsRNAs. We probed dsRNAs' Watson-Crick basepairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, triggering melting initiation. dsRNA destabilization driven by N-NTD was more efficient for dsTRSs than dsNS. N-NTD dynamics, especially a tweezer-like motion of ß2-ß3 and Δ2-ß5 loops, seems to play a key role in Watson-Crick basepairing destabilization. Based on experimental information available in the literature, we constructed kinetics models for N-NTD-mediated dsRNA melting. Our results support a 1:1 stoichiometry (N-NTD/dsRNA), matching MD simulations and raising different possibilities for N-NTD action: 1) two N-NTD arms of dimeric N would bind to two different RNA sites, either closely or spatially spaced in the viral genome, in a cooperative manner; and 2) monomeric N-NTD would be active, opening up the possibility of a regulatory dissociation event.

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

The 1H, 15N, and 13C resonance assignments of the N-terminal domain of the nucleocapsid protein from the Middle East respiratory syndrome coronavirus.

During the past 17 years, the coronaviruses have become a global public emergency, with the first appearance in 2012 in Saudi Arabia of the Middle East respiratory syndrome. Among the structural proteins encoded in the viral genome, the nucleocapsid protein is the most abundant in infected cells. It is a multifunctional phosphoprotein involved in the capsid formation, in the modulation and regulation of the viral life cycle. The N-terminal domain of N protein specifically interacts with transcriptional regulatory sequence (TRS) and is involved in the discontinuous transcription through the melting activity of double-stranded TRS (dsTRS).