Evaluation of expression and titration of recombinant nucleocapsid protein as an immunogenic candidate against SARS-CoV2

Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate. Materials and Methods: B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Results: The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high- frequency distribution was improved to 85%. Expression of recombinant protein in E. coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000. Conclusion: According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.

Preparation and identification of broad-spectrum monoclonal antibodies against N protein of avian infectious bronchitis virus

[Objective] To prepare broad-spectrum monoclonal antibody against N protein of avian infectious bronchitis virus (IBV), so as to lay a foundation for identifying conservative domain epitope of N protein and establish a universal IBV detection method. [Method] N protein of GX-YL5, a representative strain of IBV dominant serotype in Guangxi, was expressed in prokaryote. BALB/c mice were immunized with the purified protein. After the serum titer of the immunized mice reached 104 or more, the splenocytes were fused with SP2/0 myeloma cells. After screening by indirect ELISA, monoclonal antibody was prepared by ascites-induced method. Western blotting, IFA and indirect ELISA were used to identify the titer, subtype, reaction specificity and cross-reaction spectrum. And the prepared monoclonal antibody was used for immunohistochemical detection. And the prepared monoclonal antibody was used to detect the IBV in the trachea and kidney tissues of SPF chickens artificially infected with 4 representative IBV variants (GX-N130048, GX-N160421, GX-QZ171023 and GX-QZ170728). [Result] The prepared monoclonal antibody N2D5 had a titer greater than 217 and its subtype was IgG2b. The Western blotting and IFA results showed that the monoclonal antibody N2D5 only reacted with IBV, and were negative with Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV) and Marek's disease virus (MDV). Monoclonal antibody N2D5 reacted with many genotypes in China and all 7 serotypes of IBV currently prevalent in Guangxi, including commonly used standard strains, vaccine strains and field strains. Immunohistochemistry showed that the virus signals could be detected in the trachea and kidney tissues of SPF chickens at different time after artificial infection of 3 representative IBV strains from chicken and 1 isolated strain from duck, which further proved its broad spectrum. [Conclusion] The monoclonal antibody N2D5 of IBV prepared based on hybridoma technology belongs to the IgG2b subtype. It has the characteristics of high specificity, wide response spectrum and strong binding ability with IBV. It can be used as a specific diagnostic antibody for clinical diagnosis of IBV and the study of virus distribution.

Synthesis of killer T cell epitopes for peptide-based vaccine of SARS-CoV-2. (Special Issue: Chemistry and global challenges (Part B).)

COVID-19 is a current global pandemic, which has prompted many countries to develop ways to deal with it. Peptides have many medicinal and diagnostic benefits, so recently, many researchers have been developing peptide-based vaccines against COVID-19. In peptide-based vaccines, peptides act as specific antigens that will provide a faster immune response because they do not go through the process of cutting proteins in the Major Histocompatibility complex (MHC) antigen-presenting cells (APC) and can be directly presented outside the cells so that they can be recognized by the host killer T cells (CTL). Vaccine development can be accelerated with the help of immunoinformatic to predict specific epitopes to induce the CTL. We have predicted the CTL epitope through the immunoinformatic method. This study aims to synthesize candidate CTL epitopes as a candidate for the SARS-CoV-2 vaccine using the SPPS method with the Fmoc/t-Bu strategy. In this study, two CTL epitopes were synthesized through a conventional solid-phase peptide synthesis (SPPS) method, and another CTL epitope was synthesized using a semi-automated peptide synthesizer. The SPPS method is faster because the purification is only carried out at the final stage, while the Fmoc/t-Bu strategy was applied because it provides a mild reaction condition. Both synthetic approaches were compared. The semi-automated peptide synthesizer made the synthesis faster and more efficient due to the use of an inert gas (N2) during the synthesis. The synthetic peptides were characterized by TOF-ESI-MS. The three peptides showed ion peaks at m/z 1137.5509 (M+H)+, 1064.3468 (M+H)+, and 916.5859 (M+H)+, indicating correct molecular ion peaks for EILDITPCSF, IPIGAGICASY, and FIAGLIAIV, respectively.

Bioinformatics analysis of mutations in the spike protein of SARS-CoV-2 and their effects

In December 2019, a new type of pneumonia, coronavirus disease 2019 (COVID-19), caused by a novel coronavirus, SARS-CoV-2, was detected in hospitals in Wuhan, Hubei Province, China. The World Health Organization announced on 11 March 2020 that COVID-19 can be characterized as a pandemic, and since then COVID-19 has wrought havOc on public-health systems worldwide. The surface "spike" protein CS protein of SARS-CoV-2 mediates host-cell attachment and membrane fusion. The S protein is a key target for urgent development of vaccines, therapeutic antibodies, and diagnostics. To analyze the mutations and their effects on protein structure and function of the S protein, bioinformatics software has been used to analyze its nucleotide and amino-acid sequences, and Wuhan-Hu-1 (GenBank accession number: MN908947.3) was used as standard strain. As of 17 April 2020, there were 1, 002 SARS-CoV-2 strains in the GenBank database, of which 12 strains had mutations in the amino-acid sequence of the S protein. Some of these mutations could affect the physicochemical properties and secondary structures of the S protein. The R4081 mutation was located in the receptor-binding domain (RBD) and displayed on the surface, and could affect the RBD structure. The mutated amino acids 48, 74, 181, 221 and 655 were located in predicted linear epitopes of B cells, and 74, 181 and 655 mutations could greatly affect the structures and properties of linear epitopes of B cells.. The S protein of SARS-CoV-2 isolated from humans, dogs, cats and lions was highly conserved, whereas the D614G mutation was found in the isolated strain from tigers. Furthermore, the unique Flynn protease recognition site was presented in the S protein of SARS-CoV-2 compared with the coronavirus from bats. These results suggest that the S protein of SARS-CoV-2 is relatively conserved within and between species, whereas there are some mutations that can affect the physicochemical properties and structures of the S protein, which may also affect the linear epitopes of B cells. Taken together, these data provide a basis for the research and development of drugs, antibodies and vaccines against SARS-CoV-2.

Preparation of monoclonal antibody against porcine saperovirus VP1 protein and identification of its antigenic epitope

As a member of the family Picornaviridae, porcine sapelovirus (PSV) is often infected with porcine epidemic diarrhea virus, teschovirus and so on. In recent years, PSV has been isolated from porcine in many provinces of China. It suggests that it is necessary to strengthen the research on PSV. In this study, according to the sequence of PSV HuN2 strain, VP1 gene was inserted into the pGEX-6 P-1 vector, and expressed the recombinant protein. BALB/c mice aged 6-8 weeks were immunized according to the standard procedure. After the third immunization, the mouse orbital blood was collected to identify the antibody level. The highly positive mouse spleen cells were selected for cell fusion. The positive hybridoma cells and two subclones were screened by IFA method, and then a PSV VP1 monoclonal antibody was obtained, named as 33-2 A. The results of IFA showed that PSV could be recognized by 33-2 A MAb, and specific green fluorescence appeared in the cytoplasm;The results of WB and IP showed that PSV infected porcine cell could specifically bind to 33-2 A, and there was a specific band at 32 ku. We also identified the B-cell antigen epitope of 33-2 A, it was at amino acids 40-46 of PSV VP1 protein, and the polypeptide sequence was 40PALTAAE46. The results showed that the monoclonal antibody can react with PSV VP1 protein. The epitope was analyzed with the PSV sequences uploaded in NCBI, 33-2 A antibody can react with most PSV strains and has a certain universal to PSV. This study laid a foundation for the study of the etiology and pathogenesis of PSV.

Identification of T cell epitopes in BALB/c mice immunized with COVID-19 vaccine

Objective: To identify the T cell epitopes of the COVID-19 vaccine carrying SARS-CoV-2 S, N and M genes in BALB/c mice.

Temporal variations in country-specific mutational profiles of SARS-CoV-2: effect on vaccine efficacy

Aim: In order to curb the transmission of SARS-CoV-2, nation-wide travel restrictions at different levels were implemented in different countries. Country-specific mutational profile may exist and have an impact on vaccine efficacy. Materials & methods: We identified nonsynonymous mutations in approximately 215,000 SARS-CoV-2 sequences during the first year of the pandemic in 35 countries. Mutational profiles on a bimonthly basis were traced over time. We also examined the mutations that overlapped with the spike protein vaccine epitopes.

The Challenges of Vaccine Development against Betacoronaviruses: Antibody Dependent Enhancement and Sendai Virus as a Possible Vaccine Vector.

To design an effective and safe vaccine against betacoronaviruses, it is necessary to use their evolutionarily conservative antigenic determinants that will elicit the combination of strong humoral and cell-mediated immune responses. Targeting such determinants minimizes the risk of antibody-dependent enhancement of viral infection. This phenomenon was observed in animal trials of experimental vaccines against SARS-CoV-1 and MERS-CoV that were developed based on inactivated coronavirus or vector constructs expressing the spike protein (S) of the virion. The substitution and glycosylation of certain amino acids in the antigenic determinants of the S-protein, as well as its conformational changes, can lead to the same effect in a new experimental vaccine against SARS-CoV-2. Using more conservative structural and accessory viral proteins for the vaccine antigenic determinants will help to avoid this problem. This review outlines approaches for developing vaccines against the new SARS-CoV-2 coronavirus that are based on non-pathogenic viral vectors. For efficient prevention of infections caused by respiratory pathogens the ability of the vaccine to stimulate mucosal immunity in the respiratory tract is important. Such a vaccine can be developed using non-pathogenic Sendai virus vector, since it can be administered intranasally and induce a mucosal immune response that strengthens the antiviral barrier in the respiratory tract and provides reliable protection against infection.

[The Challenges of Vaccine Development Against Betacoronaviruses: Antibody Dependent Enhancement and Sendai Virus as a Possible Vaccine Vector].

To design an effective and safe vaccine against betacoronaviruses, it is necessary to elicit a combination of strong humoral and cell-mediated immune responses as well as to minimize the risk of antibody-dependent enhancement of viral infection. This phenomenon was observed in animal trials of experimental vaccines against SARS-CoV-1 and MERS-CoV that were developed based on inactivated coronavirus or vector constructs expressing the spike protein (S) of the virion. The substitution and glycosylation of certain amino acids in the antigenic determinants of the S-protein, as well as its conformational changes, can lead to the same effect in a new experimental vaccine against SARS-CoV-2. This review outlines approaches for developing vaccines against the new SARS-CoV-2 coronavirus that are based on non-pathogenic viral vectors. For efficient prevention of infections caused by respiratory pathogens the ability of the vaccine to stimulate mucosal immunity in the respiratory tract is important. Such a vaccine can be developed using non-pathogenic Sendai virus vector, since it can be administered intranasally and induce a mucosal immune response that strengthens the antiviral barrier in the respiratory tract and provides reliable protection against infection. The mucosal immunity and the production of IgA antibodies accompanying its development reduces the likelihood of developing an antibody-dependent infection enhancement, which is usually associated only with immunopathological IgG antibodies.

Change of Antigenic Determinants of SARS-CoV-2 Virus S-Protein as a Possible Cause of Antibody-Dependent Enhancement of Virus Infection and Cytokine Storm.

A hypothesis is proposed that the cytokine storm syndrome, which complicates COVID-19 in some patients, is a consequence of antibody-dependent enhancement of virus infection, which is in turn happens due to a change in dominant antigenic determinants of SARS-CoV-2 S-protein. The antibody-dependent enhancement of virus infection is a phenomenon in which virus-specific antibodies that are not neutralizing enhance the entry of infectious virus into immune cells causing their death. Antibody-dependent enhancement has been reported for different coronaviruses. This phenomenon happens due to a decrease in the binding strength of neutralizing antibodies to the virus, which converts these antibodies into suboptimal-not neutralizing ones. According to our hypothesis, such a decrease in affinity may be associated with a change in the conformation of the viral S-protein. We believe that this conformational change is the major factor in the switching of antibodies affinity, which triggers antibody-dependent enhancement. However, other factors that contribute to antigen drift and antigenic determinant changes may also play a role.