A Candidate Antigen of the Recombinant Membrane Protein Derived from the Porcine Deltacoronavirus Synthetic Gene to Detect Seropositive Pigs.

Porcine deltacoronavirus (PDCoV) is an emergent swine coronavirus which infects cells from the small intestine and induces watery diarrhea, vomiting and dehydration, causing mortality in piglets (>40%). The aim of this study was to evaluate the antigenicity and immunogenicity of the recombinant membrane protein (M) of PDCoV (rM-PDCoV), which was developed from a synthetic gene obtained after an in silico analysis with a group of 138 GenBank sequences. A 3D model and phylogenetic analysis confirmed the highly conserved M protein structure. Therefore, the synthetic gene was successfully cloned in a pETSUMO vector and transformed in E. coli BL21 (DE3). The rM-PDCoV was confirmed by SDS-PAGE and Western blot with ~37.7 kDa. The rM-PDCoV immunogenicity was evaluated in immunized (BLAB/c) mice and iELISA. The data showed increased antibodies from 7 days until 28 days (p < 0.001). The rM-PDCoV antigenicity was analyzed using pig sera samples from three states located in "El Bajío" Mexico and positive sera were determined. Our results show that PDCoV has continued circulating on pig farms in Mexico since the first report in 2019; therefore, the impact of PDCoV on the swine industry could be higher than reported in other studies.

Production of Sars-Cov-2 Membrane Protein as Better Target Protein for Diagnostic Application

Intro: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, an enveloped RNA of the coronavirus family. The advancement in molecular technology and biochemistry has accelerated the development of diagnostic reagents and assays. Much attention has been focused on the S protein, but the high mutation rate in this region could lead to false negative results. Thus, a better target protein for diagnostic application is needed for accurate detection. Method(s): Nucleotide sequences encoded for membrane (M) glycoprotein gene region of SARS-CoV-2 from Malaysian isolates were extracted from GISAID, aligned, and selected accordingly. The DNA plasmid was commercially synthesized with codon optimization for Escherichia coli (E. coli), and the presence of the M gene was confirmed by PCR. The plasmid was then transformed into E. coli. Later, the expression of M glycoprotein was induced, separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane, followed by immunostaining. Finding(s): The analysis of the M glycoprotein against the Omicron strains demonstrated that the amino acid is conserved (99.5%). The M glycoprotein was successfully expressed and detected with antibodies from SARS-CoV-2 infected patients at ~26 kDa. The protein is currently upscale for the generation of monoclonal Ab (Mab). Discussion(s): The M protein of SARS-CoV-2 is more conserved among the virus and also has been reported to confer antigenic properties. Selection of M protein perhaps a better option compared to current detection assays that use spike (S) protein, which could lead to false negative results, as this gene region particularly the ribosome-binding domain (RBD) rapidly undergoes mutations. The utilization of M protein potentially improves negative predictive value (NPV) of the diagnostic test. Conclusion(s): Further development of diagnostic reagents is needed to improve the assay's specificity. The newly developed M protein and the MAb can be used to generate a more accurate viral detection assay.Copyright © 2023

Development of a New Epitope Immunogenic Structure Based on the Coronavirus Membrane Glycoprotein M Using Immunoinformatics Tools

Backgrounds: Although conventional therapies have played an essential role in the treatment of many diseases, emerging diseases require new treatment methods with less complications. Therefore, it is important to develop an effective vaccine for infections caused by the coronavirus to prevent mortality and create immunity the community. Material(s) and Method(s): In this research bioinformatics tools were used to design a vaccine against the M membrane protein of SARS-CoV-2. A total of 27 epitopes confined to B cells and MHC I and II alleles were structurally constructed in M protein for immune stimulation and antibody recognition which were used in the construction of a chimeric peptide vaccine. Finding(s): The vaccine was predicted to be a stable, antigenic, and non-allergenic compound. TRL5/vaccine complex analysis and docking simulation indicated a sufficiently stable binding with appropriated receptor activation. The immune response simulation following hypothetical immunization indicated the potential of this vaccine to stimulate the production of active and memory B cells, CD8 + T and, CD4 + T cells, and effective immunological responses induced by Th2 and Th1. Conclusion(s): The analysis of in-silico processes showed that the vaccine structure induced high antigenicity and good cellular immunity in the host body and stimulates various immune receptors such as TLR5, MHC I, and MHC II. Vaccine function was also associated with an increase in IgM and IgG antibodies and a set of Th1 and Th2 cytokines. But the final confirmation of the effectiveness of the designed vaccine requires clinical processes.Copyright © 2022, TMU Press.

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.

ANALYSIS OF EPITOPE-SPECIFIC T CELLS IN SARS-CoV-2 INFECTION AND VACCINATION

Background: T cells play a critical role in the adaptive immune response to SARS-CoV-2 in both infection and vaccination. Identifying T cell epitopes and understanding how T cells recognize these epitopes can help inform future vaccine design and provide insight into T cell recognition of newly emerging variants. Here, we identified SARS-CoV-2 specific T cell epitopes, analyzed epitope-specific T cell repertoires, and characterized the potency and cross-reactivity of T cell clones across different common human coronaviruses (HCoVs). Method(s): SARS-CoV-2-specific T cell epitopes were determined by IFNgamma ELISpot using PBMC from convalescent individuals with mild/moderate disease (n=25 for Spike (S), Nucleocapsid (N) and Membrane (M)), and in vaccinated individuals (n=27 for S). Epitope-specific T cells were isolated based on activation markers following a 6-hour peptide stimulation, and scRNAseq was performed for TCR repertoire analysis. T cell lines were generated by expressing recombinant TCRs in Jurkat cells and activation was measured by CD69 upregulation. Result(s): We identified multiple immunodominant T cell epitopes across S, N and M proteins in convalescent individuals. In vaccinated individuals, we detected many of the same dominant S-specific epitopes at similar frequencies as compared to convalescent individuals. T cell responses to peptide S205 (amino acids 817-831) were observed in 56% and 59% of individuals following infection and vaccination, respectively, while 20% and 19% of individuals responded to S302 (a.a. 1205-1219) following infection and vaccination, respectively. For S205, a CD4+ T cell response, we confirmed 8 unique TCRs and determined the minimal epitope to be a 9mer (IEDLLFNKV). While TCR genes TRAV8-6*01 and TRBV30*01 were commonly utilized across the TCRs, we did identify TCRs with unique immunogenetic properties with different potencies of cross-reactivity to other HCoVs. For S302, a CD8+ T cell response, we identified two unique TCRs with different immunogenetic properties that recognized the same 9mer (YIKWPWYIW) and cross-reacted with different HCoV peptides (Figure 1). Conclusion(s): These data identify immunodominant T cell epitopes following SARS-CoV-2 infection and vaccination and provide a detailed analysis of epitope-specific TCR repertoires. The prospect of developing a vaccine that broadly protects against multiple human coronaviruses is bolstered by the identification of conserved immunodominant SARS-CoV-2 T cell epitopes that cross react with multiple other HCoVs.

Evaluation of the immunogenicity of an inactivated vaccine prototype against SARS-COV- 2 virus in vitro

Background: Belarus started developing a vaccine against SARS-CoV- 2 in 2021. The aim of the first stage of investigation was to evaluate the immunogenicity of the vaccine prototypes (VP) in vitro. Method(s): SARS-CoV- 2 strains (n = 7) were isolated using Vero E6 cells, inactivated with beta-propiolactone and purified. Antigens (Ag) were adsorbed on adjuvants: Al(OH)3 (Ag+AH group) or Al3PO4 (Ag+AP group). The single dose of VP (500 ul) was composed of 10 mug of Ag adsorbed on adjuvants (200 mug of Al3+). Blood samples from SARS-CoV- 2 recovered donors (n = 18) and healthy controls having no history of COVID-19 infection (n = 5) were used. Whole blood and Tag-it Violet labeled PBMCs were cultivated with VP, pure Ag, adjuvants (0.25-1 mug of Ag, 20 mug of Al3+ for probe) or pool of peptides, covering sequence of SARS-CoV- 2 N, S, M-proteins (PP), for 6 h and 7 days respectively. INF-gamma production and proliferation of CD3+ T-cells were assayed by FACS. Result(s): Counts of CD3+IFN-gamma+ T cells were 3.14(2.72-5.13)/ 1x105 CD3+ T cells in negative control (NC), and 12.73(10.09-33.95)/ 1x105 CD3+ T cells in specific positive control (PP) (n = 18, p < 0.0001), proving presence of antigen-specific T cells (ASCs) in donor blood. Samples were considered positive for VP and Ag immunogenicity when numbers of CD3+IFN-gamma+ T cells were 1.5 times greater compared with NC. Both VP types (Ag+AP, Ag+AH) and pure SARS-CoV- 2 isolates stimulated the production of INF-gamma by ASCs, responses ranged from 1 to 4 isolates of 7 studied per donor. Immunogenicity of Ag+AP, Ag+AH was confirmed by proliferation assay. Proliferation level was 1.07(0.97-2.38)% in Ag group with no differences from NC (n = 7, p > 0.5). Proliferation was significantly greater in VP groups compared with Ag: 2.47(1.65-2.68)% in Ag+AH, 4.03(2.56-4.61)% in Ag+AP (p = 0.009 and 0.002, respectively), stimulation of T cell was stronger by Ag+AP compared with Ag+AH (p = 0.009, M-U test). Pure adjuvants did not induce T cells response. There was no T-cell stimulation by Ag and VP in samples obtained from COVID-19 negative donors. Conclusion(s): The VP against SARS-CoV- 2 infection composed of inactivated virus adsorbed on Al(OH)3 and Al3PO4 adjuvants has immunogenic properties proven in two different immunological assays. VP stimulated activation and proliferation of ASCs in vitro suggesting this VP can be used for further preclinical in vivo evaluation.

Assessment of specific T-cell immunity in patients who have been ill and vaccinated against COVID-19.

Introduction. In the context of a pandemic of a new coronavirus infection (COVID-19), research on the peculiarities of the formation of an immune response to SARS-CoV-2 in patients who have been ill and vaccinated is of particular relevance. However, most studies are currently devoted to evaluating only the humoral link of immunity, and its cellular component remains insufficiently studied. The aim of the study was to evaluate the features of the formation and changes of the T-cell link of immunity in patients with a new coronavirus infection and vaccinated against this disease. Materials and methods. The study was performed on the basis of the clinical and diagnostic laboratory of the European Medical Center "UMMC-Health "LLC. Specific T-cell immunity was evaluated using ELISPOT technology. In the course of the study, 72 blood samples of employees of medical organizations were analyzed, including 26 from those who had a new coronavirus infection, 23 from persons who were intact according to COVID-19 before vaccination and 23 from the same employees after vaccination (<<Gam-Covid-Vac>>). In addition, each of the study participants was examined to determine specific class G antibodies (IgG) by solid-phase enzyme immunoassay using SARS-CoV-2-IgG-ELISA-BEST test systems (manufactured by VECTOR-BEST JSC). Results and discussion. In the group of patients (26 people), T-lymphocytes capable of specifically reacting to SARSCoV-2 antigens were detected in 100% of cases, even in individuals with IgG elimination. It should be noted that the response was more pronounced when meeting with M-and N-pepdids, compared with S-protein. 22 out of 23 COVID-19 intact individuals had no T-cell immunity to coronavirus infection before vaccination, but one employee had a response to 3 proteins-M, N, S, which indicates that he had previously encountered the SARS-CoV-2 virus. After vaccination with the drug "Gam-Covid-Vac", 22 (95.6%) employees revealed a T-cell response, while 21-only to S-protein, and an employee with a previously detected immune response-after vaccination, the response to M -, N-proteins remained almost at the same level, and the cellular response to S-peptide doubled. Conclusion. Thus, based on the results of the study, important materials were obtained on the peculiarities of the formation of a specific T-cell immune response to a new coronavirus infection. The obtained data provide a broader understanding of the immune response in new coronavirus infection in patients who have been ill and vaccinated and can be used in the future when planning preventive and anti-epidemic measures.Copyright © 2022 Interregional public organization Association of infectious disease specialists of Saint-Petersburg and Leningrad region (IPO AIDSSPbR). All rights reserved.

Covid 19 Pandemic: An Overview

In the 1930's the corona virus was first identified as a highly contagious chicken respiratory virus. Two human coronaviruses were later identified, the human coronavirus 229E causing the flu and secondly the human coronavirus OC43. Others are also important as SARS-CoV. In late 2019 the outbreak of Pneumonia occurred in the Chinese city of Wuhan which was investigated as a result of the corona virus, renamed as 2019-nCoV by the World Health Organization (WHO) and. now called as SARS-CoV-2. The WHO has identified the global health problem as an epidemic. Respiratory droplets produced during coughing and sneezing are the main means of transmission of COVID-19. Infection with COVID-19 in an infected person may remain undetected. Common symptoms of fever and dry cough are less common in the production of sputum, fatigue and in some cases may be dyspnoea or shortness of breath. The COVID-19 virus is a type of RNA virus, the outer envelope containing a lipid bilayer in which various proteins are synthesized such as membrane (M), envelope (E) and spike (S). Hand washing, coughing, social isolation, wearing a face mask in public, disinfection areas, and isolation are various ways to prevent the disease. The diagnosis of COVID-19 can be made on the basis of symptoms and confirmed using reverse transcription polymerase chain reaction (RT-PCR) tests. There are currently no antiretroviral drugs approved for COVID-19, only symptomatic and supportive treatment is used to treat people with this viral infection. Drugs that have been approved for the purpose of treating other viral infections are under investigation. Vaccination is an ultimate prevention and protection;few vaccines are given emergency approval and some are in progressive development phase in various countries to prevent this deadly pandemic.

An adaptable in vitro cytokine release assay (CRA): Susceptibility to cytokine storm in COVID-19 as a model.

Translational in vitro models such as cytokine release assay (CRA) are essential to assess the susceptibility to cytokine storm or CRS in a non-interventional manner in a human in vitro laboratory setting. Such models are also helpful to unravel disease mechanisms, to study the effects of new therapeutics and vaccines thereon and to diagnose or monitor diseases. Such assay will be important in predicting, planning and preparing for hospital intensive care units that are needed during the course of a pandemic. We present a CRA that can be adapted for assessing acute cytokine release risk against viral antigens, and potentially be used for cytokine storm simulation in viral infection outbreaks. We have used SARS-CoV-2 antigens and COVID-19 as a model. The assay can be challenged by changed or mutated forms of a virus in follow on waves of the epidemic and it can easily be modified for other future pandemics. We show that the membrane protein of SARS-CoV-2 is playing a major role in cytokine release (CR), mainly that of IL-6, IFNγ, TNFα and IL-8, that may be associated with COVID-19. These results are in agreement with recent clinical findings and new vaccine designs.

Effect of cannabidiol on apoptosis and cellular interferon and interferon-stimulated gene responses to the SARS-CoV-2 genes ORF8, ORF10 and M protein.

AIMS: To study effects on cellular innate immune responses to ORF8, ORF10, and Membrane protein (M protein) from the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, in combination with cannabidiol (CBD). MAIN METHODS: HEK293 cells transfected with plasmids expressing control vector, ORF8, ORF10, or M protein were assayed for cell number and markers of apoptosis at 24 h, and interferon and interferon-stimulated gene expression at 14 h, with or without CBD. Cells transfected with polyinosinic:polycytidylic acid (Poly (I:C)) were also studied as a general model of RNA-type viral infection. KEY FINDINGS: Reduced cell number and increased early and late apoptosis were found when expression of viral genes was combined with 1-2 µM CBD treatment, but not in control-transfected cells treated with CBD, or in cells expressing viral genes but treated only with vehicle. In cells expressing viral genes, CBD augmented expression of IFNγ, IFNλ1 and IFNλ2/3, as well as the 2'-5'-oligoadenylate synthetase (OAS) family members OAS1, OAS2, OAS3, and OASL. CBD also augmented expression of these genes in control cells not expressing viral genes, but without enhancing apoptosis. CBD similarly enhanced the cellular anti-viral response to Poly (I:C). SIGNIFICANCE: Our results demonstrate a poor ability of HEK293 cells to respond to SARS-CoV-2 genes alone, but an augmented innate anti-viral response to these genes in the presence of CBD. Thus, CBD may prime components of the innate immune system, increasing readiness to respond to RNA-type viral infection without activating apoptosis, and could be studied for potential in prophylaxis.

Characterization of the Interaction Between SARS-CoV-2 Membrane Protein (M) and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target.

SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M-PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and PLA (Proximity Ligation Assay). In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase in PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

SARS-CoV-2 Amino Acid Mutations Detection in Greek Patients Infected in the First Wave of the Pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel virus that belongs to the Coronoviridae family, emerged in December 2019, causing the COVID-19 pandemic in March 2020. Unlike previous SARS and Middle East respiratory syndrome (MERS) outbreaks, this virus has a higher transmissibility rate, albeit a lower case fatality rate, which results in accumulation of a significant number of mutations and a faster evolution rate. Genomic studies on the mutation rate of the virus, as well as the identification of mutations that prevail and their impact on disease severity, are of great importance for pandemic surveillance and vaccine and drug development. Here, we aim to identify mutations on the SARS-CoV-2 viral genome and their effect on the proteins they are located in, in Greek patients infected in the first wave of the pandemic. To this end, we perform SARS-CoV-2 amplicon-based NGS sequencing on nasopharyngeal swab samples from Greek patients and bioinformatic analysis of the results. Although SARS-CoV-2 is considered genetically stable, we discover a variety of mutations on the viral genome. In detail, 18 mutations are detected in total on 10 SARS-CoV-2 isolates. The mutations are located on ORF1ab, S protein, M protein, ORF3a and ORF7a. Sixteen are also detected in patients from other regions around the world, and two are identified for the first time in the present study. Most of them result in amino acid substitutions. These substitutions are analyzed using computational tools, and the results indicate minor or major impact on the proteins' structural stability, which could probably affect viral transmissibility and pathogenesis. The correlation of these variations with the viral load levels is examined, and their implication for disease severity and the biology of the virus are discussed.

SARS-CoV-2 membrane protein is incompetent at inducing antibody-dependent enhancement though Fc receptor pathway

Objective To investigate the immune characteristics of SARS-CoV-2 membrane (M) protein, especially the possibility of inducing antibody-dependent enhancement effect (ADE) .Methods Full-length SARS-CoV-2 M protein was prepared by prokaryotic expression system and purified.BALB/ c mice were immunized subcutaneously three times (on day 1, day 14 and day 21) by purified M protein.Serum samples were collected before immunization and after each immunization.The specificity of immune sera against M protein was identified by Western blot, and the antibody titers were detected by ELISA and neutralization test.In the presence of anti-M protein serum, the proliferation of SARS-CoV-2 in dendritic cells, nature killer cells, T and B cells was detected in vitro.Results The immune sera from BALB/ c mice immunized with purified full-length M protein of SARS-CoV-2 specifically recognized viral M protein.The titer of anti-whole virus antibody in immune sera was about 1 ∶ 400, but the antibody could not neutralize live virus.Moreover, the antibody could not help the virus to infect and proliferate in the various types of immune cells with Fc receptor (FcR).Conclusions Non-neutralizing antibody induced by M protein could not cause ADE through FcR pathway.

TCR SEQUENCING of EPITOPE-SPECIFIC T CELLS from SARS-CoV-2 CONVALESCENT INDIVIDUALS

Background: T cells have been shown to play a role in the immune response to SARS-CoV-2. Identification of T cell epitopes and a better understanding of the T cell repertoire will provide important insights into how T cells impact antiviral immunity. Here, we identified T cell epitopes within the Spike (S), Nucleocapsid (N) and Membrane (M) proteins from SARS-CoV-2 convalescent individuals and performed TCR sequencing on epitope-specific T cells. Methods: Epitope mapping was performed by IFNγ ELISpot on PBMC from SARS-CoV-2 convalescent patients with mild/moderate disease (n = 19 for S;n=15 for N and M), and minimum epitopes were determined using truncated peptides and ICS. TCR sequence analysis was performed on a subset of individuals (n=9 donors;2-3 epitopes/donor), with longitudinal samples for 7 donors (2-3 time points/donor;33 to 236 days post-symptom onset). T cells were stimulated with individual peptides for 6 hours and sorted based on the expression of activation markers (CD4+: CD69, CD40L;CD8+: CD69, CD107a, surface TNF). scRNAseq was performed on sorted cells for TCR repertoire and transcriptome analysis. Results: We identified several peptides recognized by multiple individuals, including S42 (amino acids 165-179;7/19 donors), S302 (a.a. 1205-1219;6/19 donors), N27 (a.a. 106-120;6/14 donors) and M45 (a.a. 177-191;10/14 donors). S42 elicited both CD4+ (n=5) and CD8+ (n=1) T cell responses, with one individual having both a CD4+ and CD8+ response. The minimum epitope for S42 was determined to be a 9mer (FEYVSQPFL) for both CD4+ and CD8+ cells. TCR sequencing of S42-specific T cells identified a dominant gene pairing for TCRα across multiple donors (TRAV35;TRAJ42) and for both CD4+ and CD8+ T cells (Figure 1). In general, epitope-specific CD4+ responses (S42, M45) were more clonally diverse than CD8+ responses (S42, S302, N27). For both CD4+ and CD8+ T cells, conserved TCR gene usage and gene pairings could be identified within multiple donors responding to the same epitope. Conclusion: These data suggest that in SARS-CoV-2 convalescent people, epitope-specific CD4+ and CD8+ T cells can differ in their clonal diversity and that related TCRs can be identified across multiple donors. S42-specific T cell studies are ongoing to determine their transcriptional profile and pMHC presentation. Ongoing longitudinal analysis will provide a better understanding of different epitope-specific TCR repertoires and T cell transcriptional profiles, and how they evolve after infection.

Analysis of Interaction Network Between Host Protein and M Protein of Swine Acute Diarrhea Syndrome Coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an enterovirus that can cause acute diarrhea and death in piglets and cause serious economic losses to the pig industry. SADS-CoV membrane (M) protein mainly plays a key role in biological processes, such as virus assembly, budding, and host innate immune regulation. Understanding the interaction between M protein and host proteins is very important to define the molecular mechanism of cells at the protein level and to understand specific cellular physiological pathways. In this study, 289 host proteins interacting with M protein were identified by glutathione-S-transferase (GST) pull-down combined with liquid chromatography-mass spectrometry (LC-MS/MS), and the protein-protein interaction (PPI) network was established by Gene Ontology (GO) terms and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways analysis. Results showed that SADS-CoV M protein was mainly associated with the host metabolism, signal transduction, and innate immunity. The Co-Immunoprecipitation (CO-IP) validation results of six randomly selected proteins, namely, Rab11b, voltage-dependent anion-selective channel 1 (VDAC1), Ribosomal Protein L18 (RPL18), RALY, Ras Homolog Family Member A (RHOA), and Annexin A2 (ANXA2), were consistent with LC-MS results. In addition, overexpression of RPL18 and PHOA significantly promoted SADS-CoV replication, while overexpression of RALY antagonized viral replication. This work will help to clarify the function of SADS-CoV M protein in the life cycle of SADS-CoV.

Cytotoxicity, Antioxidant Activity, Molecular Docking, and Dynamics Simulation Analysis Against SARS-CoV-2 M and N Protein Models of Phytoconstituents of Micromelum Minutum

Phytochemical investigations of the methanolic extract of the whole plant of Micromelum minutum provided two coumarins, namely micromelin and murrangatin, and one sterol stigmast-4-en-3-one, the latter being reported for the first time from M. minutum. To evaluate bioactivities, different fractions of the crude methanol extracts of the plant obtained by partitioning were screened for antioxidant and cytotoxic activity by DPPH radical scavenging method and the brine shrimp lethality bioassay, respectively. Among the different fractions of M. minutum tested, pet ether and chloroform soluble fractions showed prominent antioxidant activities with IC50 values of 49.46 and 67.53 μg/mL compared to the standard butylated hydroxytoluene (IC50 31.02 μg/ml). The pet ether and chloroform fractions of M. minutum showed good brine shrimp larvicidal activity with LC50 values of 1.15 and 1.50 μg/ml, respectively, compared to vincristine sulfate (LC50 0.27 μg/ml). The results obtained from molecular docking, Stigmast-4-en-3-one exerts the highest negative binding affinity (-9.1 kcal/mol) for interaction with SARS-CoV-2 M protein and develops a strong network with eleven hydrophobic bonds established by ADMET profile studies and YASARA Dynamics program. © 2022 by the authors.

Isolation of Functional SARS-CoV-2 Antigen-Specific T-Cells with Specific Viral Cytotoxic Activity for Adoptive Therapy of COVID-19.

In order to demonstrate the feasibility of preparing clinical-grade SARS-CoV-2-specific T-cells from convalescent donors and the ability of these cells to neutralize the virus in vitro, we used blood collected from two COVID-19 convalescent donors (before and after vaccination) that was stimulated with specific SARS-CoV-2 peptides followed by automated T-cell isolation using the CliniMacs Prodigy medical device. To determine cytotoxic activity, HEK 293T cells were transfected to express the SARS-CoV-2 M protein, mimicking SARS-CoV-2 infection. We were able to quickly and efficiently isolate SARS-CoV-2-specific T lymphocytes from both donors before and after they received the Pfizer-BioNTech vaccine. Althoughbefore vaccination, the final product contained up to 7.42% and 30.19% of IFN-γ+ CD3+ T-cells from donor 1 and donor 2, respectively, we observed an enrichment of the IFN-γ+ CD3+ T-cells after vaccination, reaching 70.47% and 42.59%, respectively. At pre-vaccination, the isolated SARS-CoV-2-specific T-cells exhibited cytotoxic activity that was significantly higher than that of unstimulated controls (donor 2: 15.41%, p-value 3.27 × 10-3). The cytotoxic activity of the isolated SARS-CoV-2-specific T-cells also significantly increased after vaccination (donor 1: 32.71%, p-value 1.44 × 10-5; donor 2: 33.38%, p-value 3.13 × 10-6). In conclusion, we demonstrated that SARS-CoV-2-specific T-cells can quickly and efficiently be stimulated from the blood of convalescent donors using SARS-CoV-2-specific peptides followed by automated isolation. Vaccinated convalescent donors have a higher percentage of SARS-CoV-2-specific T-cells and may be more suitable as donors. Although further studies are needed to assess the clinical utility of the functional isolated SARS-CoV-2-specific T-cells in patients, previous studies using the same stimulation and isolation methods applied to other pathologies support this idea.

SARS CoV-2;a closer look at the agent of the pandemic of modern times

Due to the COVID-19 pandemic, as of September 2021, a total of 222,309,456 people were infected in the world and a total of 4,592,685 patients were lost. The pandemic, which has a fatality rate of around 2%, has made and continues to make us live thhrough all experiences of epidemics that we have only read about in Annals of Medicine and Microbiology and that deeply affected the World at their times. The virus causing the pandemic has a positive polarity RNA genome of 30,000 bases and produces a total of 29 proteins. Of these proteins, 4 are structural, 16 are nonstructural, and 9 are accessory proteins. SARS-CoV-2 is an enveloped RNA virus with a diameter of 150-200 nm, has an S (spike-spike-tassel) glycoprotein on its surface, which, like other coronaviruses, creates the crown appearance unique to these viruses. After the S protein is synthesized as a polyprotein, it is cleaved into S1 and S2 subunits. The S1 subunit binds to the target cell, and the S2 subunit performs fusion with the cell membrane to be infected. Since these functions are critical features of a successful viral infection, the S protein is the main target of all interventions to prevent virus infection. In this context, the main target of neutralizing antibodies and drugs to stop virus infection before it starts is the S protein. The S protein has a trimer structure similar to hemagglutinin in influenza virus and contains the fusion peptide that becomes exposed during transition from the prefusion configuration to the fusion configuration and facilitates the fusion function with the cellular/endosomal membranes. Apart from the S protein, SARS-CoV-2 has structural proteins known as E (envelope), M (membrane), and N (nucleocapsid) proteins;The N protein binds to the RNA genome and together with the S, E and M proteins and the RNA genome form the virion. While SARS-CoV-2 S protein attaches to cells using Cellular Angiotensin Converting Enzyme 2 (HCoV- NL63, SARS-CoV and SARS-CoV-2), other coronaviruses use different receptors (Aminopeptidase N-HCoV-229E;dipeptidyl peptidase 4- MERS-CoV). Unlike viruses in this group, the SARS CoV-2 S1 protein with receptor binding domain (RBD) has a cleavage site made up of polybasic amino acids at the S1-S2 border and used by the cellular furin protease, which is believed to provide advantages to the virus in proteolytic cleavage, cell tropism, virulence and pathogenicity. ACE-2 is important in the renin-angiotensin-aldosterone system and although it is rarely found in the circulation, it is widely expressed in organs and is an enzyme involved in the regulation of blood pressure and fluid balance. Following intracellular entry and fusion of membranes, the SARS-CoV-2 genome is released into the cytoplasm and gene expression proceeds as a temporally and spatially well-regulated process. Non-structural proteins, which are produced from direct translation of ORF1a and ORF1b regions of positive sense genomic RNA, form the replication and transcription complex. These complexes establish the infrastructure for the next steps. The common features of coronaviruses such as cytoplasmic replication, viral gene expression through sub-genomic nested set messages, exocytosis of mature virions within vesicles occur in SARS-CoV-2 as well. One of the most important problems in the COVID-19 pandemic has been the emergence of variant viruses. These viruses adversely affecting the transmission rate, virulence, clinical course, and the effectiveness of the diagnostic or therapeutic methods carry mutations that lead to amino acid changes, especially in the RBD region. The World Health Organization and other authorities refer to these viruses as variants of concern or variants of interest. As of September 2021, WHO lists Alpha (UK, September 2020), Beta (South Africa, May 2020), Gamma (Brazil, November 2020), and Delta (India, October 2020) viruses as variants of concern. Also, Eta (December 2020), Iota (USA, November 2020), Kappa (India, October 2020), Lambda (Peru December, 2020) and Mu (Colombia, January 2021) mutant viruses are on he list variants of interest. In conclusion, less than 2 years of time has passed since the emergence of the COVID-19 agent SARS CoV-2 virus. However, this virus has been the most extensively studied viral agent in the history of medicine and the most detailed information has been gathered about the infection. Despite all these, it is difficult to indicate that the fight against this pathogen has been successful nor are we any closer to declare that the enormous danger the virus poses to humanity is reduced.

A Case of ABO Discrepancy in a Multiple Myeloma Patient Misinterpreted as a Complication of COVID-19 Vaccination

Introduction: The effect of COVID-19 infection and its vaccines onthe immune system is still a subject of much research and debate.Unusual findings due to COVID-19 infection or COVID-19 vaccination are increasingly being reported. However, caution is warrantedto rule out known entities before considering these possibilities.Aims &Objectives: We report a case of incidentally detected ABOdiscrepancy attributed to complications of COVID-19 vaccination butdiagnosed ultimately as multiple myeloma.Materials &Methods: Case Report: A 52-year-old male presentedwith weakness, easy fatigability and chest pain following the seconddose of the COVID-19 vaccine. His hemoglobin fell progressivelyand he was advised blood transfusion.Result: Blood grouping revealed ABO discrepancy in forward andreverse grouping. Routine peripheral blood smear examinationshowed rouleaux formation and background blue-tinging. The patientalso had raised ESR. The patient was suspected to have a plasma celldyscrasia, which was confirmed on serum electrophoresis and thisexplained the ABO discrepancy.Conclusions: M protein in plasma cell myeloma is known to causegroup III ABO discrepancy due to rouleaux formation which can beinterpreted as pseudo-agglutination and can be a presenting feature.Though vaccine-induced blood group discrepancies have also beenreported in the literature, a detailed workup with a good interpretationof peripheral smear findings is necessary so as not to miss the primaryunderlying disease.

Probing the formation, structure and free energy relationships of M protein dimers of SARS-CoV-2.

The M protein of the novel coronavirus 2019 (SARS-CoV-2) is the major structural component of the viral envelope and is also the minimum requirement for virus particle budding. M proteins generally exist as dimers. In virus assembly, they are the main driving force for envelope formation through lateral interactions and interactions with other viral structural proteins that play a central role. We built 100 candidate models and finally analyzed the six most convincing structural features of the SARS-CoV-2 M protein dimer based on long-timescale molecular dynamics (MD) simulations, multiple free energy analyses (potential mean force (PMF) and molecular mechanics Poisson-Boltzmann surface area (MMPBSA)) and principal component analysis (PCA) to obtain the most reasonable structure. The dimer stability was found to depend on the Leu-Ile zipper motif and aromatic amino acids in the transmembrane domain (TMD). Furthermore, the C-terminal domain (CTD) effects were relatively small. These results highlight a model in which there is sufficient binding affinity between the TMDs of M proteins to form dimers through the residues at the interface of the three transmembrane helices (TMHs). This study aims to help find more effective inhibitors of SARS-CoV-2 M dimers and to develop vaccines based on structural information.