Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes’ presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300–400 μg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 μg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80–100 μg per gram of fresh leaf tissue; the yield after purification was up to 20 μg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen.

Introduction. COVID-19 is characterized by a varied clinical course. The aim of the work was to identify associations of SNPs of hemostatic system genes with COVID-19. Materials and methods. DNA was isolated from patients (n=117) and healthy participants (n=104). All infected patients were divided into 3 groups, depending on disease severity assessment, which was appreciated by NEWS2. Another group consisted of participants, who had asymptomatic infection in the past. Determination of SNPs of the genes FGB (-455 G/A), FII (20210 G/A), FV (1691 G/A), FVII (10976 G/A), FXIIIA1 (103 G/T), ITGA2 (807 C/T), ITGB3 (1565 T/C), SERPINE1 (-675 5G/4G) were performed by PCR using the “Genetics of Hemostasis” kit (“DNA-Technology”, Russia). Results. In analyzed SNPs, no significant differences were detected between the group of infected patients and healthy participants. But significant association was revealed in gene SERPINE1 (-675 5G/4G), when patient groups, differing in the disease severity, were analyzed relative to the group of participants with asymptomatic infection (p=0.0381; p=0 .0066; p=0.0009). It was found, that as COVID-19 severity scores increased, the proportion of 5G allele of gene SERPINE1 decreased, and the proportion of the 4G allele increased (p=0.005; p=0.009; p=0.0005). Similar processes were observed for genotypes 5G/5G and 4G/4G. Discussion. The gene SERPINE1 (-675 5G/4G) is associated with the severity of COVID-19. Conclusion. For the first time, it was discovered that 5G/5G genotype of gene SERPINE1 (-675 5G/4G) can be a marker of a milder course of COVID-19, and the 4G/4G genotype as a more severe one.

Introduction. Chronic viral hepatitis C (CHC) is a ubiquitous infectious disease, a significant limitation of which WHO attributes to the use of a new highly effective antiviral therapy. Previously, two B-cell epitopes were identified in NS4a antigen of the hepatitis C virus (HCV). It was shown that certain titers of antibodies (ABs) to the extended C-terminal epitope (16871718 a.a.) can predict a high probability of achieving a sustained virological response (SVR) to standard therapy with pegylated interferon- and ribavirin. The aim of the work was to determine immunoreactivity of two B-cell epitopes (middle and C-terminal) of NS4a antigen, and to estimate a possible association of ABs to them with the achievement of SVR after standard interferon therapy and treatment with direct antiviral drugs (DAAs) daclatasvir and sofosbuvir (velpanat). Materials and methods. Blood serum samples of patients with CHC (n = 113), of which 55 participants received standard interferon therapy, 50 received velpanate treatment, the remaining 8 received no therapy were examined. The middle B-cell epitope (positions 2434 a.a.) of NS4a was synthesized by the solid-phase method, while the C-terminal epitope (3454 a.a.) was obtained using genetically engineered techniques. Enzyme immunoassay (ELISA) testing of the sera collected before treatment was performed for the two selected epitopes according to the conventional methods. Results. The antibodies to the C-terminal epitope were detected significantly more frequently than those to the middle one (p = 0.01) when analyzing the blood sera of patients (n = 113). The presence of ABs to the C-terminal epitope in the serum samples of participants who completed standard interferon therapy was associated with the achievement of SVR (p = 0.0245). In the blood sera of participants who completed therapy with velpanate, an association of the presence of ABs to the C-terminal epitope with the achievement of SVR was also established (p 0.0001). The presence of ABs to the middle B epitope was not associated with the achievement of SVR, regardless of the therapy used. Discussion. The observed difference in the immunoreactivity of the two B-cell determinants may be associated with the localization of the nearest Th-epitopes, the sensitivity of NS4a antigen to proteolytic enzymes, and the peculiarities of epitope presentation by antigen-presenting cells. However, it should be noted that the immunoreactivity of the middle B-epitope is poorly studied. Although the association of ABs to the C-terminal epitope with the achievement of SVR has been shown by several scientific teams, the detailed molecular mechanism of their influence on the effectiveness of therapy is unclear. Conclusion. In CHC, ABs to the C-terminal epitope of NS4a are produced more frequently than those to the median epitope. The presence of ABs to the C-terminal epitope is a predictive marker of a high probability of achieving SVR, regardless of the type of therapy and antibody titer.

Introduction. Predisposition to different courses of the infectious process is largely associated with the polymorphisms in human genome, especially in genes encoding proteins of the immune system. In the early stages of influenza infection such components of innate immunity as interferons I (α/β) and III (λ) type play a significant role in limiting virus replication. The aim of the work was to investigate associations of single nucleotide polymorphism in IFNL3 (rs8099917 T/G) and IFNL4 (rs12979860 C/T) genes with different course of influenza, and identify genetic markers of influenza complicated by community-acquired pneumonia. The genes noted above affect the production of interferon-λ3, which is involved in restriction of the viral replication. Materials and methods. Samples from 456 patients with mild (n = 150), moderate (n = 173), and severe (n = 133) influenza were studied. The viral RNA was detected by reverse transcription and polymerase chain reaction (RT-PCR). Polymorphisms in IFNL3 (rs8099917 T/G) and IFNL4 (rs12979860 C/T) genes was detected by PCR. Statistical analysis was performed using SNPStats software. Results. Patients with the C/T or T/T genotype of IFNL4 gene (rs12979860 C/T) were more likely to have pneumonia than those with the C/C genotype (OR 2.47 (1.31–4.63); p = 0.0044; q = 0.0059). The presence of one T allele increased the risk of developing pneumonia (OR 2.02 (1.05–4.02); p = 0.006; q = 0.008). In the presence of the T/T genotype, the risk increased more than twofold: OR 2.14 (1.31–3.48). Analysis of the SNP of IFNL3 gene (rs8099917 T/G) revealed a weak association of the G allele with pneumonia (OR 1.86 (1.04–3.31); p = 0.03; q = 0.045). Conclusion. Genetic markers of increased risk of community-acquired pneumonia in influenza include the presence of the T allele in IFNL4 gene (rs12979860 C/T) and, to a lesser extent, the G allele in IFNL3 gene (rs8099917 T/G). Patients carrying these alleles have an increased risk of developing pneumonia, especially in old age.

Objective: The aim of this study was to identify antibodies to peptides representing conserved Bcell epitopes of hepatitis C virus (HCV) envelope proteins E1 and E2, and to evaluate their significance for predicting viral elimination in patients undergoing directacting antiviral (DAA) therapy. Methods: Nine peptides derived from HCV envelope proteins E1 and E2 were synthesized using the solidphase method. Immunoreactivity of the peptides was assessed via a solidphase enzymelinked immunosorbent assay (ELISA) using serum samples from 9 patients with acute hepatitis C and 67 patients with chronic hepatitis C who received DAA therapy. Statistical significance was evaluated using pvalues. Results and Discussion: The presence of antibodies to the E1E2 complex before treatment, as demonstrated by other researchers, is associated with the achievement of a sustained virological response (SVR) in 70% to therapy with DAAs, and thus may serve as a predictive marker (p = 0.012). However, the possible predictive role of individual B-epitopes of HCV envelope proteins has not yet been clarified. The study revealed that the presence of antibodies to two peptides representing conserved immunodominant Bepitopes of the E2 protein was associated with achieving SVR in 73.0 and 89.2% of patients (p = 0.00001 and p = 0.0086, respectively). These findings suggest that antibodies targeting specific Bepitopes of HCV envelope proteins may serve as positive prognostic markers for treatment outcome. The results highlight the potential of using antibody profiles to improve prediction of therapy success, enable personalized treatment approaches, and inform vaccine development. Conclusions: The presence of antibodies to conserved immunodominant Bepitopes of the HCV E2 protein is a promising positive prognostic marker for achieving SVR in patients treated with DAAs. The obtained data can be used to improve methods for predicting therapy outcomes, develop personalized approaches to treatment, and develop vaccines against viral hepatitis C.

Objective: Hepatitis C virus (HCV) is characterized by high genetic variability, which complicates the development of vaccines that protect against all viral genotypes and subtypes. Therefore, studying the immunogenicity of conserved B-cell epitopes is an important task. The aim of this study was to synthesize and analyze seven peptides reproducing conserved B-cell epitopes of HCV structural and nonstructural proteins: E1(313–327), E2(412–422), E2(434–446), E2(524–535), Core(1–17), NS4A(1689–1711), and NS4B(1722–1739). Methods: The selected peptides were obtained by solid-phase peptide synthesis using the Fmoc-strategy with high purity (>95%). To enhance peptide immunogenicity, liposomal formulations based on soybean phosphatidylcholine were constructed. To evaluate immunogenicity, BALB/c mice were immunized with the following preparations: peptides in saline, peptides with incomplete Freund's adjuvant (IFA), and liposomal formulations of the peptides. Antibodies against the peptides were detected by solid-phase enzyme-linked immunosorbent assay (ELISA). Results and Discussion: Liposomal nanoparticles containing the peptides were homogeneous, with an average size of 170–205 nm, high peptide entrapment efficiency (61–86%), and long-term stability. Analysis of mouse sera after immunization with peptides in saline revealed no humoral immune response. The use of IFA led to antibody production against only two peptides (E2(524–535) and NS4A(1689–1711)), with low titers (up to 1:56). In contrast, immunization with liposome-encapsulated peptides induced antibody production against five peptides (E2(434–446), E2(524–535), Core(1–17), NS4A(1689–1711), and NS4B(1722–1739)), with titers ranging from 1:24 to 1:448. Conclusions: These results demonstrate that liposomal peptide formulations are an effective strategy for enhancing the immunogenicity of HCV B-cell epitopes. This study provides a promising foundation for the development of a multi-epitope hepatitis C vaccine.