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.

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: SARS-CoV-2 vaccine immunogenicity is evaluated in neutralization test with live virus. It is performed in a biosafety level 3 zone because requires live virus stage. Therefore, control laboratories should be certified for this class of work. The development of technology based on enzyme-linked immunosorbent assay as an analogue of the neutralization reaction makes it possible to create an immunobiological product in a shorter time and in conditions without special requirements for control laboratories. AIM: Development of an enzyme-linked immunosorbent assay for assessing SARS-CoV-2 vaccine immunogenicity by measuring neutralizing antibodies production in immunized animals. MATERIALS AND METHODS: Recombinant receptor-binding domain fused to a С-terminal hexahistidine sequence was produced in Escherichia coli cells and purified via metal-affinity chromatography on WorkBeads NiMAC (Bio-Works). Purified protein was used in enzyme-linked immunosorbent assay as an antigen for sorption. The sera of mice immunized with the vaccine preparation were tested for neutralizing activity against the SARS-CoV-2, as well as in the developed enzyme-linked immunosorbent assay. RESULTS: Sera with high neutralizing titers showed a high degree of binding to recombinant receptor-binding domain fused to a С-terminal hexahistidine sequence in enzyme-linked immunosorbent assay, while sera from non-immunized animals or sera with neutralization titers less than 1:8 were not reactive in enzyme-linked immunosorbent assay. The Spearman and Pearson correlation coefficients for neutralization test titers and optical density in enzyme-linked immunosorbent assay were 0.759 and 0.76, respectively. The developed assay can be used as a semi-quantitative method for assessing the immunogenicity of a vaccine against coronavirus infection. CONCLUSIONS: The developed platform makes it possible to reliably assess the immunogenicity of an inactivated coronavirus vaccine under conditions that do not require a high biosafety conditions.