Background/Objectives: The World Health Organization (WHO) recommends the use of attenuated Sabin strains for the production of inactivated poliovirus vaccine (IPV), offering improved biosafety while retaining immunogenicity. To better characterize the antigenic composition of Sabin strain-based IPV (sIPV), including both the protective D-antigen and the non-protective H-antigen forms, we developed a method for purifying D- and H-antigens forms. Methods: D- and H-antigens of poliovirus Sabin strains types 1, 2, and 3 were purified using gradient ultracentrifugation and used to generate antigen-specific polyclonal antibodies. Results: The generated polyclonal antibodies demonstrated high specificity with neutralizing titers of antibodies against Sabin type 1 poliovirus—1:2048, against Sabin type 2 poliovirus—more than 1:2048, against Sabin type 3 poloivirus—1:2048. Conclusions: This antigen-specific antibody approach provides a valuable tool for routine quality control in sIPV manufacturing, enabling accurate quantification of immunogenic components and detection of potentially immunogenic degradation products during vaccine storage and distribution. Antibodies to the D-antigen allow assessment of immunogenic, neutralizing epitopes, while antibodies to the H-antigen provide a tool for detecting non-neutralizing components. This antigen-specific antibody approach offers a valuable tool for studying the antigenic structure of sIPV and for improving the accuracy of ELISA-based antigen quantification.

Various types of COVID-19 vaccines, including adenovirus, mRNA, and inactivated ones, have been developed and approved for clinical use worldwide. Inactivated vaccines are produced using a proven technology that is widely used for the production of vaccines for the prevention and control of infectious diseases, including influenza and poliomyelitis. The development of inactivated whole-virion vaccines commonly includes several stages: the production of cellular and viral biomass in cell culture; inactivation of the virus; filtration and ultrafiltration; chromatographic purification of the viral antigen; and formulation with stabilizers and adjuvants. In this study, the suitability of four resins for Size-Exclusion Chromatography was investigated for the purification of a viral antigen for the human COVID-19 vaccine.

Vaccination remains the primary strategy for poliomyelitis prevention. The D-antigen of poliovirus is a critical component of inactivated polio vaccines, as it induces the production of neutralizing antibodies and provides protective immunity. Therefore, the development of quantitative immunoassays for monitoring D-antigen content during vaccine production is an important task. In this study, we generated recombinant camelid single-domain antibodies (VHHs) specific for the D-antigen of Sabin poliovirus type 1 and evaluated their antigen specificity. The obtained VHH antibodies demonstrated strong binding to the native D-antigen of Sabin type 1 poliovirus. Furthermore, the use of clone S1-C7 as a capture antibody in combination with Sabin type 1-specific polyclonal antibodies for detection revealed differential recognition of the vaccine-derived Sabin type 1 and homologous wild-type Mahoney strains. Notably, such discrimination was not observed when polyclonal antibodies were used alone, indicating that incorporation of VHH S1-C7 into the assay enhances strain-specific antigen recognition. In a neutralization assay, clone S1-C7 exhibited in vitro neutralizing activity against Sabin type 1 poliovirus. One clone, S1-D9, demonstrated cross-reactivity with all three poliovirus serotypes, suggesting recognition of a conserved epitope in the capsid and potential utility as a universal detection antibody. The generated VHH antibodies represent promising analytical tools for poliovirus antigen characterization. Together with S1-C7, they enable the discrimination of both D- and C-antigen forms as well as vaccine-derived and wild-type strains.

A panel of monoclonal nanobodies (VHH) specific to poliovirus Sabin strains types 2 and 3 has been generated and characterized. For poliovirus Sabin type 2, a panel of four serotype-specific single-domain antibodies was obtained. Notably, two of these antibodies, S2-C5 and S2-D5, were able to discriminate between D- and H-antigens. Three nanobodies specifically neutralized poliovirus Sabin type 2 and exhibited no neutralizing activity against the homologous wild-type MEF-1 strain or other poliovirus serotypes. Among the four nanobodies selected against Sabin type 3, S3-A2 and S3-G1 demonstrated high serotype specificity in ELISAs. When used as a capture antibody in the ELISA, S3-G1 showed preferential binding to D-antigen over H-antigen. However, the type 3 nanobodies did not display virus-neutralizing activity. The obtained antibodies represent promising tools for improving existing immunoassays based on polyclonal antibodies by increasing D-antigen specificity and may facilitate the development of next-generation ELISA systems for the quality control of inactivated polio vaccines.

BACKGROUND: The emergence of new variants of the SARS-CoV-2 virus at the end of 2020, which became a source of increased risk for global public health, prompted the study of their molecular biological characteristics and pathogenic effects. It is known that one of the reasons for the viruses pathogenic action is their interaction with the defense mechanisms of the cell. Ire1 (inositol-requiring enzyme 1)-mediated splicing of Xbp1 mRNA (X-box binding protein 1) is a protective mechanism that is activated in response to the accumulation of misfolded proteins in the cell, a situation that occurs due to uncontrolled synthesis of viral proteins during infection. Studying the interaction of different variants of the SARS-CoV-2 virus with this protective mechanism will help to shed light on various aspects of the pathogenesis of a new coronavirus infection. AIM: Study of Ire1-Xbp1 defense mechanism activation and modulation in SARS-CoV-2 infected Vero cells. MATERIALS AND METHODS: We studied the activation of the Ire1 enzyme in Vero cells infected with various variants of the SARS-CoV-2 virus using immunoblotting and antibodies to various forms of this protein in the cell. In addition, we studied the activation of Xbp1 mRNA splicing under conditions of infection with various variants of the SARS-CoV-2 virus in a PCR reaction with specific primers. RESULTS: Reproduction of B.1.1.529 (Omicron) strain in Vero cells is slower than B.1.1.7 (Alpha) and B.1.617.2 (Delta) strains of SARS-CoV-2. The whole reproduction cycle is 48 hours. Ire1-dependent defense mechanism is activated after 12 hours of SARS-CoV-2 infection with either of three variants. However, despite the activation of the Ire1 endonuclease domain, there is no Xbp1 mRNA splicing in SARS-CoV-2 infected cells. Inhibition of Xbp1 mRNA splicing occurs slower in Vero cells infected with the B.1.1.529 Omicron variant. CONCLUSIONS: The paper describes the reproduction of various variants of the SARS-CoV-2 virus in Vero cell culture and the activation of the Ire1-Xbp1 defense mechanism during infection. The Ire1 endonuclease is phosphorylated, however, mRNA splicing of the Xbp1 transcription factor is impaired in SARS-CoV-2 infected cells. A decrease in the rate of inhibition of this protective mechanism in Vero cells infected with the Omicron (B.1.1.529) variant of the SARS-CoV-2 virus may be the reason for its lower pathogenicity described in various studies.