ABSTRACT
The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is related with the COVID-19 pandemic. Recent spike protein variations have had an effect on the transmission of the virus. In addition to ACE-2, spike proteins can employ DC-SIGN and its analogous receptor, DC-SIGNR, for host evasion. Spike variations in the DC-SIGN interaction region and role of DC-SIGN in immune evasion have not been well defined. To understand the spike protein variations and their binding mode, phylogenetic analysis of the complete GISAID (Global Initiative for Sharing Avian Influenza Data) data of the SARS-CoV-2 spike protein was considered. In addition, an in silico knockout network evaluation of the SARS-CoV-2 single-cell transcriptome was conducted to determine the key role of DC-SIGN/R in immunological dysregulation. Within the DC-SIGN-interacting region of the SARS-CoV spike protein, the spike protein of SARS-CoV-2 displayed remarkable similarity to the SARS-CoV spike protein. Surprisingly, the phylogenetic analysis revealed that the SARS-CoV-2's spike exhibited significantly diverse variants in the DC-SIGN interaction domain, which altered the frequency of these variants. The variation within the DC-SIGN-interacting domain of spike proteins affected the binding of a limited number of variants with DC-SIGN and DC-SIGNR and affected their evolution. MMGBSA binding free energies evaluation differed for variants from those of the wild type, suggesting the influence of substitution mutations on the interaction pattern. In silico knockout network analysis of the single-cell transcriptome of Bronchoalveolar Lavage and peripheral blood mononuclear cells revealed that SARS-CoV-2 altered DC-SIGN/R signaling. Early surveillance of diverse SARS-CoV-2 strains could preclude a worsening of the pandemic and facilitate the development of an optimum vaccine against variations. The spike Receptor Binding Domain genetic variants are thought to boost SARS CoV-2 immune evasion, resulting in its higher longevity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00820-3.
ABSTRACT
Introduction: The emergence of SARS-CoV-2 virus, which causes COVID-19, is a major global health hazard. Therefore, a comprehensive characterization of the humoral and cellular immune responses to this virus is essential to combat the COVID-19 pandemic. Our goal was to develop reliable methods and tools for the analysis of humoral and cellular B- and T- cell responses, which will facilitate scientific research for prediction of disease progression, long-term immunity and will support vaccine development. Methods: Plasma samples and PBMCs of COVID-19 convalescent and healthy donors were obtained. For the detection of SARS-CoV-2 specific antibodies and identification of antigen-specific B cells, we manufactured recombinant mono-biotinylated protein variants of the Spike (S), Receptor Binding Domain (RBD) and Nucleoprotein (N). To identify antigen-reactive T cells, SARS-CoV-2 peptide pools were synthetized for the S, N and Membrane (M) antigens and used for stimulation. The peptide pools consist of mainly 15-mer peptides having an 11-mer amino acid overlap and thereby overspan a whole protein sequence. Results: To determine the presence of SARS-CoV-2 reactive antibodies a flow-based bead assay using recombinant, mono-biotinylated SARS-CoV-2 antigens loaded onto Streptavidin (SAV)-coated-PMMA beads was set up. The beads were incubated with plasma samples and fluorochrome conjugated anti-human isotype specific antibodies for flow cytometric analysis. All the antigens tested were shown to be suitable for the detection of antibodies to SARS-CoV-2 in COVID-19 convalescent plasma. To assess the feasibility of recombinant antigens for the detection and isolation of antigen-specific B cells, the mono-biotinylated Spike and RBD antigens were tetramerized on fluorochrome-conjugated SAV. These tetramers were used for staining, magnetic enrichment and flow cytometric sorting of B cells specific to SARS-CoV-2 antigens. We were able to demonstrate that our recombinant antigens can be used to assess the presence and enable the phenotyping and isolation of rare antigen-specific B cells. For further characterization of the SARS-CoV-2 reactive T cell immunity PBMCs were short term stimulated with the S, M and N peptide pools. After intracellular staining of IFNg, TNFa, IL-2 and CD154, reactive T cells were detected using flow cytometry. We could demonstrate T cell reactivity towards each peptide pool. However, strengths of T cell responses towards the S, M and N peptide pools were heterogeneous between different COVID-19 convalescent individuals. Conclusion: To support and improve current research activities for the identification and characterization of SARS-CoV-2 reactive humoral and cellular B- and T- cell responses, potent tools and assays were developed. Described here research solutions offer the opportunity to successfully address and contribute to the investigation on healthy and dysfunctional immune reactions towards SARS-CoV-2.