Stanislav A. Cherepushkin

INTRODUCTION. Rotavirus vaccines based on virus-like particles (VLPs), non-infectious recombinant proteins of human rotavirus A that mimic the structure of the native virus, show promise for preventive vaccination. Presumably, the optimal method to determine the potency of VLP-based rotavirus vaccines is the enzyme-linked immunosorbent assay (ELISA) method used to measure the titre of specific IgG antibodies to rotavirus A proteins in serum samples from vaccinated animals.AIM. This study aimed at assessing the potency of a VLP-based rotavirus vaccine by developing and validating an analytical procedure using ELISA to determine the levels of antibodies to the VP2 and VP6 proteins of rotavirus A in serum samples from vaccinated guinea pigs.MATERIALS AND METHODS. The potency of the VLP-based rotavirus vaccine was determined in vivo in three types of experimental animals, including BALB/c mice, agouti guinea pigs, and newborn minipigs. The animals received three intramuscular injections of the vaccine at a dose of 30 µg. This study used the indirect ELISA method to quantify VP2- and VP6-specific immunoglobulin G (IgG) antibodies and the virus-neutralisation test to measure neutralising antibodies (nAbs) to rotavirus A in animal serum samples. The study involved calculating the geometric mean titres (GMTs) of antibodies. The authors validated the analytical procedure for potency assessment on two batches of the vaccine using standard statistical analysis methods.RESULTS. The study compared VP2- and VP6-specific IgG and nAb levels 14 days after the first, second, and third vaccinations. The authors observed a significant increase in antibody titres and statistically significant (pCONCLUSIONS. The authors developed and validated an ELISA-based analytical procedure for assessing the potency of VLP-based preventive rotavirus vaccines. According to the study results, ELISA is applicable to the control of the potency of VLP-based preventive rotavirus vaccines.

Introduction. The COVID-19 pandemic caused by SARS-CoV-2 has created serious health problems worldwide. The most effective way to prevent the occurrence of new epidemic outbreaks is vaccination. One of the modern and effective approaches to vaccine development is the use of virus-like particles (VLPs). The aim of the study is to develop a technology for production of VLP based on recombinant SARS-CoV-2 proteins (E, M, N and S) in insect cells. Materials and methods. Synthetic genes encoding coronavirus proteins E, M, N and S were used. VLP with various surface proteins of strains similar to the Wuhan virus, Delta, Alpha and Omicron were developed and cloned into the pFastBac plasmid. The proteins were synthesized in the baculovirus expression system and assembled into VLP in the portable Trichoplusia ni cell. The presence of insertion in the baculovirus genome was determined by PCR. ELISA and immunoblotting were used to study the antigenic activity of VLP. VLP purification was performed by ultracentrifugation using 20% sucrose. Morphology was assessed using electron microscopy and dynamic light scattering. Results. VLPs consisting of recombinant SARS-CoV-2 proteins (S, M, E and N) were obtained and characterized. The specific binding of antigenic determinants in synthesized VLPs with antibodies to SARS-CoV-2 proteins has been demonstrated. The immunogenic properties of VLPs have been studied. Conclusion. The production and purification of recombinant VLPs consisting of full-length SARS-CoV-2 proteins with a universal set of surface antigens have been developed and optimized. Self-assembling particles that mimic the coronavirus virion induce a specific immune response against SARS-CoV-2.

Introduction. In Russia, almost half of the cases of acute intestinal infections of established etiology in 2022 are due to rotavirus infection (RVI). There is no specific treatment for rotavirus gastroenteritis. There is a need to develop modern, effective and safe vaccines to combat rotavirus infection that are not capable of multiplying (replicating) in the body of the vaccinated person. A promising approach is to create vaccines based on virus-like particles (VLPs). Objective. Study of the safety and immunogenicity of a vaccine against rotavirus infection based on virus-like particles of human rotavirus A in newborn minipigs with multiple intramuscular administration. Materials and methods. Newborn minipigs were used as an animal model in this study. The safety of the tested vaccine was assessed based on thermometry data, clinical examination, body weight gain, clinical and biochemical blood parameters, as well as necropsy and histological examination. When studying the immunogenic properties of the Gam-VLP-rota vaccine in doses of 30 and 120 µg, the cellular, humoral and secretory immune response was studied. Results. The results of assessing the general condition of animals during the immunization period, data from clinical, laboratory and pathomorphological studies indicate the safety of the vaccine against human rotavirus infection based on VLP (Gam-VLP-rota) when administered three times intramuscularly. Good local tolerance of the tested vaccine was demonstrated. The results of the assessment of humoral immunity indicate the formation of a stable immune response after three-time immunization with Gam-VLP-rota, stimulation of the production of antigen-specific IgG antibodies and their functional activity to neutralize human rotavirus A. It was shown that following the triple immunization with the minimum tested concentration of 30 µg/dose, animals developed a cell-mediated immune response. The results of the IgA titer in blood serum and intestinal lavages indicate the formation of both a systemic immunological response and the formation of specific secretory immunity to human rotavirus A. Conclusion. Thus, three-time intramuscular immunization of minipigs with the Gam-VLP-rota vaccine forms stable protective humoral and cellular immunity in experimental animals. Evaluated vaccine is safe and has good local tolerability.

Introduction. Rotavirus infection is one of the main concerns in infectious pathology in humans, mammals and birds. Newborn piglets or rodents are usually being used as a laboratory model for the evaluation of immunogenicity and efficacy for all types of vaccines against rotavirus A (RVA), and the use of ELISA for the detection of virus-specific antibodies of specific isotype is an essential step of this evaluation. Objective. Development of indirect solid-phase ELISA with VP2/VP6 rotavirus VLP as an antigen to detect and assess the distribution of RVA-specific IgG, IgM and IgA in the immune response to rotavirus A. Materials and methods. VP2/VP6 rotavirus VLP production and purification, electron microscopy, PAGE, immunoblotting, ELISA, virus neutralization assay. Results. The study presents the results of development of a recombinant baculovirus with RVA genes VP2-eGFP/VP6, assessment of its infectious activity and using it for VLP production. The morphology of the VP2/VP6 rotavirus VLPs was assessed, the structural composition was determined, and the high antigenic activity of the VLP was established. VLP-based ELISA assay was developed and here we report results for RVA-specific antibody detection in sera of different animals. Conclusion. The developed ELISA based on VP2/VP6 rotavirus VLP as a universal antigen makes it possible to detect separately IgG, IgM and IgA antibodies to rotavirus A, outlining its scientific and practical importance for the evaluation of immunogenicity and efficacy of traditional vaccines against rotavirus A and those under development.

It has recently been shown that the titer of the SARS-CoV-2 virus decreases in a cell culture when the cell suspension is irradiated with electromagnetic waves at a frequency of 95 GHz. We assumed that a frequency range in the gigahertz and sub-terahertz ranges was one of the key aspects in the “tuning” of flickering dipoles in the dispersion interaction process of the surfaces of supramolecular structures. To verify this assumption, the intrinsic thermal radio emission in the gigahertz range of the following nanoparticles was studied: virus-like particles (VLP) of SARS-CoV-2 and rotavirus A, monoclonal antibodies to various RBD epitopes of SARS-CoV-2, interferon-α, antibodies to interferon-γ, humic–fulvic acids, and silver proteinate. At 37 °C or when activated by light with λ = 412 nm, these particles all demonstrated an increased (by two orders of magnitude compared to the background) level of electromagnetic radiation in the microwave range. The thermal radio emission flux density specifically depended on the type of nanoparticles, their concentration, and the method of their activation. The thermal radio emission flux density was capable of reaching 20 μW/(m² sr). The thermal radio emission significantly exceeded the background only for nanoparticles with a complex surface shape (nonconvex polyhedra), while the thermal radio emission from spherical nanoparticles (latex spheres, serum albumin, and micelles) did not differ from the background. The spectral range of the emission apparently exceeded the frequencies of the Ka band (above 30 GHz). It was assumed that the complex shape of the nanoparticles contributed to the formation of temporary dipoles which, at a distance of up to 100 nm and due to the formation of an ultrahigh strength field, led to the formation of plasma-like surface regions that acted as emitters in the millimeter range. Such a mechanism makes it possible to explain many phenomena of the biological activity of nanoparticles, including the antibacterial properties of surfaces.

Group A rotavirus (RVA) infection is the leading cause of hospitalization of children under 5 years old, presenting with symptoms of acute gastroenteritis. The aim of our study was to explore the genetic diversity of RVA among patients admitted to Moscow Infectious Disease Clinical Hospital No. 1 with symptoms of acute gastroenteritis. A total of 653 samples were collected from May 2019 through March 2020. Out of them, 135 (20.67%) fecal samples were found to be positive for rotavirus antigen by ELISA. RT-PCR detected rotavirus RNA in 80 samples. Seven G-genotypes (G1, G2, G3, G4, G8, G9, and G12) and three P-genotypes (P[8], P[4], and P[6]) formed 9 different combinations. The most common combination was G9P[8]. However, for the first time in Moscow, the combination G3P[8] took second place. Moreover, all detected viruses of this combination belonged to Equine-like G3P[8] viruses that had never been detected in Russia before. The genotype G8P[8] and G9P[4] rotaviruses were also detected in Moscow for the first time. Among the studied rotaviruses, there were equal proportions of Wa and DS-1-like strains; previous studies showed that Wa-like strains accounted for the largest proportion of rotaviruses in Russia.

Introduction. Rotavirus infection is the leading cause of acute gastroenteritis among infants. The development of new vaccines against rotavirus A is urgent because the virus has many genotypes, some of which have regional prevalence. Virus-like particles (VLP) is a promising way to create effective and safe vaccine preparations.The purpose of the study is to develop the technology for the production of VLP, containing VP2, VP4, VP6 and VP7 of viral genotypes prevalent on the territory of the Russian Federation, and to give its molecular genetic and virological characteristics.Material and methods. The virulent strain Wa G1P[8] of human RV A adapted to MARC-145 cell culture has been used. It was cultured and purified according to the method described by the authors earlier. Standard molecular genetic and cytological methods were used: gene synthesis; cloning into transfer plasmids; recombinant baculoviruses production in Bac-to-Bac expression system; VLP production in the insect cells; centrifugation in sucrose solution; enzyme-linked immunosorbent assay (ELISA); electron microscopy (EM); polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis.Results. VP4 and VP7 of the six most represented in Russia genotypes: G1, G2, G4, G9, P4, P8, as well as VP2 and VP6 were selected for VLP production. Recombinant baculoviruses were obtained with codon frequencies optimized for insect cells. Cabbage loopper (Trichoplusia ni) cell culture was coinfected with different combinations of baculoviruses, and VLP consisting of 2-4 proteins were produced. VLP were purified by centrifugation. The size and morphology of the particles matched the rotavirus A virion (by EM). The presence of rotavirus A proteins in VLP was confirmed by the ELISA, SDS-PAGE and western blot analysis.Conclusion. The technology for the synthesis of three-layer VLP consisting of VP2, VP4, VP6 and VP7 has been developed and optimized. The resulting VLP composition represents 6 serotypes of VP4 and VP7, which are most represented on the territory of Russia, and can be used for vaccine development.

The use and development of biotherapeutics increases and the need for accurate, sensitive and robust bioanalytical methods is also increasing. ELISA and other ligand-binding assays are the most widely used methods for the quantification of macromolecules in complex biological samples. One of the alternatives to ELISA is AlphaLISA — a versatile chemiluminescent ligand binding assay using a homogeneous no-wash protocol. AlphaLISA assays are suited for automation and exhibit high sensitivity, high throughput and wide analytical range. Since the early 2000s, this method has been used in science, medicine, and drug development for wide variety of applications, including the quantification of analytes, immunogenicity, protein-protein interactions, enzyme activity, post-translational modifications and epigenetics. In this review, we describe the principles of the AlphaLISA assay and its application in bioanalytical studies (pharmacokinetics and immunogenicity) and high-throughput screening in drug development, medical diagnostics and pathogens detection.

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