Early and Strong Antibody Responses to SARS-CoV-2 Predict Disease Severity in COVID-19 Patients

Background Antibody response to SARS-CoV-2 is a valuable biomarker for the assessment of the spread of the virus in a population and evaluation of the vaccine candidates. Recent data suggest that antibody levels also may have a prognostic significance in COVID-19. Most of the serological studies so far rely on testing antibodies against spike (S) or nucleocapsid (N) protein, however antibodies can be directed against other structural and nonstructural proteins of the virus, whereas their frequency, biological and clinical significance is unknown. Methods A novel antigen array comprising 30 SARS-CoV-2 antigens or their fragments was developed and used to examine IgG, IgA, IgE and IgM responses to SARS-CoV-2 in sera from 103 patients with COVID-19 including 34 patients for whom sequential samples were available, and 20 pre-pandemic healthy controls. Results Antibody responses to various antigens are highly correlated and the frequencies and peak levels of antibodies are higher in patients with severe/moderate disease than in those with mild disease. This finding supports the idea that antibodies against SARS-CoV-2 may exacerbate the severity of the disease via antibody-dependent enhancement. Moreover, early IgG and IgA responses to full length S protein may be used as an additional biomarker for the identification of patients who are at risk of developing severe disease. Importantly, this is the first study reporting that SARS-CoV-2 elicits IgE responses and their serum levels positively correlate with the severity of the disease thus suggesting a link between high levels of antibodies and mast cell activation. Conclusions This is the first study assessing the prevalence and dynamics IgG, IgA, IgE and IgM responses to multiple SARS-CoV-2 antigens simultaneously. Results provide important insights into the pathogenesis of COVID-19 and have implications in planning and interpreting antibody-based epidemiological studies.

RelCoVax®, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice

The COVID-19 pandemic has spurred an unprecedented movement to develop safe and effective vaccines against the SARS-CoV-2 virus to immunize the global population. The first set of vaccine candidates that received emergency use authorization targeted the spike (S) glycoprotein of the SARS-CoV-2 virus that enables virus entry into cells via the receptor binding domain (RBD). Recently, multiple variants of SARS-CoV-2 have emerged with mutations in S protein and the ability to evade neutralizing antibodies in vaccinated individuals. We have developed a dual RBD and nucleocapsid (N) subunit protein vaccine candidate named RelCoVax® through heterologous expression in mammalian cells (RBD) and E. coli (N). The RelCoVax® formulation containing a combination of aluminum hydroxide (alum) and a synthetic CpG oligonucleotide as adjuvants elicited high antibody titers against RBD and N proteins in mice after a prime and boost dose regimen administered 2 weeks apart. The vaccine also stimulated cellular immune responses with a potential Th1 bias as evidenced by increased IFN-γ release by splenocytes from immunized mice upon antigen exposure particularly N protein. Finally, the serum of mice immunized with RelCoVax® demonstrated the ability to neutralize two different SARS-CoV-2 viral strains in vitro including the Delta strain that has become dominant in many regions of the world and can evade vaccine induced neutralizing antibodies. These results warrant further evaluation of RelCoVax® through advanced studies and contribute towards enhancing our understanding of multicomponent subunit vaccine candidates against SARS-CoV-2.

Inhibition of glycogen synthase kinase-3-beta (GSK3β) blocks nucleocapsid phosphorylation and SARS-CoV-2 replication

GSK3β has been proposed to have an essential role in Coronaviridae infection. Screening of a targeted library of GSK3β inhibitors against SARS-CoV-2 and HCoV-229E resulted in identification of high proportion of active compounds with low toxicity to host cells. A select lead compound, T-1686568, showed dose-dependent activity against SARS-CoV-2 transcription, translation and viral particle release in multiple cell lines and primary organoids. A protein kinase substrate profiling assay combined with western blot analysis showed that SARS-CoV-2 nucleocapsid is phosphorylated by GSK3β on S180/S184, S190/S194 and T198 which have already been primed in the adjacent phospho-sites S188, T198 and S206 respectively. Inhibition by T-1686568 resulted in reduction of the S1 Spike protein levels, an accumulation of the Nucleocapsid (N) protein and maintenance of the non-structural (NSP2) level in infected Huh-7.5.1 cells, indicating that N phosphorylation might serve as a critical precursor for processing and release of mature viruses.

Prediction of the Effects of Nonsynonymous Variants on SARS-CoV-2 Proteins

Background: SARS-CoV-2 virus is a highly transmissible pathogen that causes COVID-19. The outbreak originated in Wuhan, China in December 2019. A number of nonsynonymous mutations located at different SARS-CoV-2 proteins have been reported by multiple studies. However, there are limited computational studies on the biological impacts of these mutations on the structure and function of the proteins. Methods: In our study nonsynonymous mutations of the SARS-CoV-2 genome and their frequencies were identified from 30,229 sequences. Subsequently, the effects of the top 10 nonsynonymous mutations of different SARS-CoV-2 proteins were analyzed using bioinformatics tools including co-mutation analysis, prediction of the protein structure stability and flexibility analysis, and prediction of the protein functions. Results: A total of 231 nonsynonymous mutations were identified from 30,229 SARS-CoV-2 genome sequences. The top 10 nonsynonymous mutations affecting nine amino acid residues were ORF1a nsp5 P108S, ORF1b nsp12 P323L and A423V, S protein N501Y and D614G, ORF3a Q57H, N protein P151L, R203K and G204R. Many nonsynonymous mutations showed a high concurrence ratio, suggesting these mutations may evolve together and interact functionally. Our result showed that ORF1a nsp5 P108S, ORF3a Q57H and N protein P151L mutations may be deleterious to the function of SARS-CoV-2 proteins. In addition, ORF1a nsp5 P108S and S protein D614G may destabilize the protein structures while S protein D614G may have a more open conformation compared to the wild type. Conclusion: The biological consequences of these nonsynonymous mutations of SARS-CoV-2 proteins should be further validated by in vivo and in vitro experimental studies in the future.

Characterization of Hantavirus N Protein Intracellular Dynamics and Localization

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation and several other crucial processes during hantavirus infection. In this study we have generated fluorescently tagged N protein constructs derived from Puumalavirus, the dominant hantavirus species in Central, Northern and Eastern Europe. We have comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We found a significant spatial correlation of N with vimentin, actin and P-bodies, but not with microtubules. N constructs also co-localized with Gn and Gc, albeit not as strong as the glycoproteins associated with each other. Moreover, we as-sessed oligomerization of N constructs, observing efficient and concentration-dependent multi-merization, with complexes comprising more than 10 individual proteins.

Temporal Patterns in the Evolutionary Genetic Distance of SARS-CoV-2 during the COVID-19 Pandemic

During coronavirus disease 2019 (COVID-19) pandemic, the genetic mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred frequently. Some mutations in the spike protein are considered to promote transmissibility of the virus, while the mutation patterns in other proteins are less studied and may also be important in understanding the characteristics of SARS-CoV-2. We used the sequencing data of SARS-CoV-2 strains in California to investigate the time-varying patterns of the evolutionary genetic distance. The accumulative genetic distances were quantified across different time periods and in different viral proteins. The increasing trends of genetic distance were observed in spike protein (S protein), the RNA-dependent RNA polymerase (RdRp) region and nonstructural protein 3 (nsp3) of open reading frame 1 (ORF1), and nucleocapsid protein (N protein). The genetic distances in ORF3a, ORF8, and nsp2 of ORF1 started to diverge from their original variants after September 2020. By contrast, mutations in other proteins appeared transiently, and no evident increasing trend was observed in the genetic distance to the original variants. This study presents distinct patterns of the SARS-CoV-2 mutations across multiple proteins from the aspect of genetic distance. Future investigation shall be conducted to study the effects of accumulative mutations on epidemics characteristics.

Alterations in B Cell and Follicular T-Helper Cell Subsets in Patients with Acute COVID-19 and COVID-19 Convalescents

Background. Humoral immunity requires interaction between B cell and T follicular helper cells (Tfh) to produce effective immune response, but the data regarding a role of B cells and Tfh in SARS-CoV-2 defense are still sparse. Methods. Blood samples from patients with acute COVID-19 (n = 64), convalescents patients who had specific IgG to SARS-CoV-2 N-protein (n = 55), and healthy donors with no detectable antibodies to any SARS-CoV-2 proteins (HC, n = 44) were analyses by multicolor flow cytometry. Results. Patients with acute COVID-19 showed decreased levels of memory B cells subsets and increased proportion plasma cell precursors compared to HC and COVID-19 convalescent patients, whereas for the latter the elevated numbers of virgin naïve, Bm2′ and “Bm3+Bm4” was found if compared with HC. During acute COVID-19 CXCR3+CCR6− Tfh1-like cells were decreased and the levels of CXCR3–CCR6+ Tfh17-like were increased then in HC and convalescent patients. Finally, COVID-19 convalescent patients had increased levels of Tfh2-, Tfh17- and DP Tfh-like cells while comparing their amount with HC. Conclusions. Our data indicate that COVID-19 can impact the humoral immunity in the long-term.