Objectives: Most antiviral vaccines are created by inactivating the virus using chemical methods. The inactivation and production of viral vaccine preparations after the irradiation of viruses with accelerated electrons has a number of significant advantages. Determining the integrity of the genome of the resulting viral particles is necessary to assess the quality and degree of inactivation after irradiation. Methods: This work was performed on the Sabin 2 model polio virus. To determine the most sensitive and most radiation-resistant part, the polio virus genome was divided into 20 segments. After irradiation at temperatures of 25 °C, 2–8 °C, −20 °C, or −70 °C, the amplification intensity of these segments was measured in real time. Results: The best correlation between the amplification cycle and the irradiation dose at all temperatures was observed for segment 3D, left. Consequently, this section of the poliovirus genome is the least resistant to the action of accelerated electrons and is the most representative for determining genome integrity. The worst dependence was observed for the VP1 right section, which, therefore, cannot be used to determine genome integrity during inactivation. The electrochemical approach was also employed for a comparative assessment of viral RNA integrity before and after irradiation. An increase in the irradiation dose was accompanied by an increase in signals indicating the electrooxidation of RNA heterocyclic bases. The increase in peak current intensity of viral RNA electrochemical signals confirmed the breaking of viral RNA strands during irradiation. The shorter the RNA fragments, the greater the peak current intensities. In turn, this made the heterocyclic bases more accessible to electrooxidation on the electrode. Conclusions: These results are necessary for characterizing the integrity of the viral genome for the purpose of creating of antiviral vaccines.

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.

Objectives: Most antiviral vaccines are created by inactivating the virus using chemical methods. Inactivation and production of viral vaccine preparations after irradiation of viruses with accelerated electrons has a number of significant ad-vantages. Determining the integrity of the genome of the resulting viral particles is necessary to assess their quality and degree of inactivation after irradiation. Methods: The work was performed on the Sabin 2 model polio virus. To deter-mine the most sensitive and most radiation-resistant part, the polio virus genome was divided into 20 segments. After irradiation at temperatures of 25°C, 2-8°C, -20°C, or -70°C, the amplification intensity of these segments was measured in re-al time. Results: The best correlation between the amplification cycle and the irra-diation dose at all temperatures was observed for segment 3D left. Consequently, this section of the poliovirus genome is the least resistant to the action of acceler-ated electrons and is the most representative for determining genome integrity. The worst dependence was observed for the VP1 right section, which, therefore, cannot be used to determine genome integrity during inactivation. The electro-chemical approach was also employed for a comparative assessment of viral RNA integrity before and after irradiation. An increase in the irradiation dose was accompanied by an increase in signals indicating the electrooxidation of RNA heterocyclic bases. The increase of peak current intensity of viral RNA electrochemical signals confirmed breaking of viral RNA strands during irradia-tion. The shorter the RNA fragments, the greater the peak current intensities. In turn, this made the heterocyclic bases more accessible to electrooxidation on the electrode. Conclusions: These results are necessary for characterizing the integrity of the viral genome for the purpose of creating of antiviral vaccines.

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.