Factors influencing the prevalence of resistance-associated substitutions in NS5A protein in treatment-naive patients with chronic hepatitis C

Direct-acting antivirals (DAAs) revolutionized treatment of hepatitis C virus (HCV) infection. Resistance-associated substitutions (RASs) present at the baseline impair response to DAA due to rapid selection of resistant HCV strains. NS5A is indispensable target of the current DAA treatment regimens. We evaluated prevalence of RASs in NS5A in DAA-naïve patients infected with HCV 1a (n = 19), 1b (n = 93), and 3a (n = 90) before systematic DAA application in the territory of the Russian Federation. Total proportion of strains carrying at least one RAS constituted 35.1% (71/202). In HCV 1a we detected only M28V (57.9%) attributed to a founder effect. Common RASs in HCV 1b were R30Q (7.5%), L31M (5.4%), P58S (4.4%), and Y93H (5.4%); in HCV 3a, A30S (31.0%), A30K (5.7%), S62L (8.9%), and Y93H (2.2%). Prevalence of RASs in NS5A of HCV 1b and 3a was similar to that worldwide, including countries practicing massive DAA application, i.e., it was not related to treatment. NS5A with and without RASs exhibited different co-variance networks, which could be attributed to the necessity to preserve viral fitness. Majority of RASs were localized in polymorphic regions subjected to immune pressure, with selected substitutions allowing immune escape. Altogether, this explains high prevalence of RAS in NS5A and low barrier for their appearance in DAA-inexperienced population.Fil: Kyuregyan, Karen K.. Russian Academy Of Sciences; Rusia. Russian Medical Academy Of Continuous Professional Education; Rusia. I. I. Mechnikov Research Institute For Vaccines And Sera; RusiaFil: Kichatova, Vera S.. Russian Medical Academy Of Continuous Professional Education; Rusia. Russian Academy Of Sciences; Rusia. I. I. Mechnikov Research Institute For Vaccines And Sera; RusiaFil: Karlsen, Anastasiya A.. I. I. Mechnikov Research Institute For Vaccines And Sera; Rusia. Russian Medical Academy Of Continuous Professional Education; Rusia. Russian Academy Of Sciences; RusiaFil: Isaeva, Olga V.. I. I. Mechnikov Research Institute For Vaccines And Sera; Rusia. Russian Medical Academy Of Continuous Professional Education; RusiaFil: Solonin, Sergei A.. N.V. Sklifosovsky Research Institute for Emergency Medicine; RusiaFil: Petkov, Stefan. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Isaguliants, Maria G.. Russian Academy Of Sciences; Rusia. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Mikhailov, Mikhail I.. Russian Medical Academy Of Continuous Professional Education; Rusia. I. I. Mechnikov Research Institute For Vaccines And Sera; Rusi

Inhibitors of the RBD-ACE-2 Found among a Wide Range of Dyes by the Immunoassay Method

Angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, plays a crucial role in the pathogenesis of COVID-19. ACE2 targeting holds the promise for preventing and inhibiting SARS-CoV-2 infection. In this work, we describe the development and use of a test system based on competitive ELISA for the primary screening of potential antiviral compounds. We studied the activity of the library of dyes of different groups. Several dyes (ortho-cresolphthalein, eosin (free acid), eosin (Na salt)) that inhibited the interaction of ACE2 with the spike proteins of SARS-CoV-2 have been identified among the candidates. A potential antiviral drug, methylene blue, did not show activity in our study. We believe that our results can help in the further search for inhibitors of interaction between the coronavirus spike protein and ACE2 receptor

Universal Single-Dose Vaccination against Hepatitis A in Children in a Region of High Endemicity

Since August 2012, universal single-dose vaccination in children aged at least three years has been implemented in the Republic of Tuva, which was previously the region most affected by hepatitis A in Russia. The objective of this cross-sectional study was the assessment of the immunological and epidemiological effectiveness of vaccination program five years following its implementation. In the pre-vaccination period, anti-HAV antibody detection rates in Tuva was 66.0% [95% CI: 56.3–74.6%] in children aged 10–14 years and reached a plateau (>95%) by age 20–29 years. Annual incidence rates in children under 18 years of age peaked at 450–860 per 100,000 in pre-vaccination years but dropped to 7.5 per 100,000 in this age group and to 3.2 per 100,000 in the total population one year after the start of vaccination. Since 2016, no cases of hepatitis A has been reported in Tuva. Serum anti-HAV antibodies were quantified in samples from healthy children following single-dose vaccination. Protective anti-HAV antibody concentrations (≥10 mIU/mL) were detected in 98.0% (95% CI: 96.2–99.0% (442/451)) of children tested one month after single-dose immunization, in 93.5% (95% CI: 91.0–95.4% (477/510)) and in 91.1% (95% CI: 88.2–93.4% (422/463)) of children one year and five years after single-dose immunization, respectively. Anti-HAV antibody geometric mean concentrations were similar in sera collected one month, one year, and five years following single-dose vaccination: 40.24 mIU/mL, 44.96 mIU/mL, and 57.73 mIU/mL, respectively (p > 0.05). These data confirm that single-dose vaccination is an effective method of bringing hepatitis A under control in a short period of time in a highly endemic region

Antibodies to the Spike Protein Receptor-Binding Domain of SARS-CoV-2 at 4–13 Months after COVID-19

Identification of factors behind the level and duration of persistence of the SARS-CoV-2 antibodies in the blood is assumed to set the direction for studying humoral immunity mechanisms against COVID-19, optimizing the strategy for vaccine use, antibody-based drugs, and epidemiological control of COVID-19. Objective: This study aimed to study the relationship between clinical and demographic characteristics and the level of IgG antibodies to the RBD of SARS-CoV-2 spike protein after COVID-19 in the long term. Residents of the Altai Region of Western Siberia of Russia, Caucasians, aged from 27 to 93 years (median 53.0 years), who recovered from COVID-19 between May 2020 and February 2021 (n = 44) took part in this prospective observational study. The titer of IgG antibodies to the RBD of SARS-CoV-2 spike protein was measured repeatedly in the blood at 4–13 months from the beginning of the clinical manifestation of COVID-19 via the method of enzyme-linked immunosorbent assay. The antibody titer positively correlated with age (p = 0.013) and COVID-19 pneumonia (p = 0.002) at 20–40 and 20–24 weeks from the onset of COVID-19 symptoms, respectively. Age was positively associated with antibody titer regardless of history of COVID-19 pneumonia (beta regression coefficient p = 0.009). The antibody titer decreased in 15 (34.1%) patients, increased in 10 (22.7%) patients, and did not change in 19 (43.2%) patients from the baseline to 48–49 weeks from the onset of COVID-19 symptoms, with seropositivity persisting in all patients. Age and COVID-19 pneumonia are possibly associated with higher IgG antibodies to the spike protein RBD of SARS-CoV-2 following COVID-19 in the long term. Divergent trends of anti-RBD IgG levels in adults illustrate inter-individual differences at 4–13 months from the onset of COVID-19 symptoms

Comparative Immunogenicity of the Recombinant Receptor-Binding Domain of Protein S SARS-CoV-2 Obtained in Prokaryotic and Mammalian Expression Systems

The receptor-binding domain (RBD) of the protein S SARS-CoV-2 is considered to be one of the appealing targets for developing a vaccine against COVID-19. The choice of an expression system is essential when developing subunit vaccines, as it ensures the effective synthesis of the correctly folded target protein, and maintains its antigenic and immunogenic properties. Here, we describe the production of a recombinant RBD protein using prokaryotic (pRBD) and mammalian (mRBD) expression systems, and compare the immunogenicity of prokaryotic and mammalian-expressed RBD using a BALB/c mice model. An analysis of the sera from mice immunized with both variants of the protein revealed that the mRBD expressed in CHO cells provides a significantly stronger humoral immune response compared with the RBD expressed in E.coli cells. A specific antibody titer of sera from mice immunized with mRBD was ten-fold higher than the sera from the mice that received pRBD in ELISA, and about 100-fold higher in a neutralization test. The data obtained suggests that mRBD is capable of inducing neutralizing antibodies against SARS-CoV-2

Self-Assembled Particles Combining SARS-CoV-2 RBD Protein and RBD DNA Vaccine Induce Synergistic Enhancement of the Humoral Response in Mice

Despite the fact that a range of vaccines against COVID-19 have already been created and are used for mass vaccination, the development of effective, safe, technological, and affordable vaccines continues. We have designed a vaccine that combines the recombinant protein and DNA vaccine approaches in a self-assembled particle. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 was conjugated to polyglucin:spermidine and mixed with DNA vaccine (pVAXrbd), which led to the formation of particles of combined coronavirus vaccine (CCV-RBD) that contain the DNA vaccine inside and RBD protein on the surface. CCV-RBD particles were characterized with gel filtration, electron microscopy, and biolayer interferometry. To investigate the immunogenicity of the combined vaccine and its components, mice were immunized with the DNA vaccine pVAXrbd or RBD protein as well as CCV-RBD particles. The highest antigen-specific IgG and neutralizing activity were induced by CCV-RBD, and the level of antibodies induced by DNA or RBD alone was significantly lower. The cellular immune response was detected only in the case of DNA or CCV-RBD vaccination. These results demonstrate that a combination of DNA vaccine and RBD protein in one construct synergistically increases the humoral response to RBD protein in mice

Are Hamsters a Suitable Model for Evaluating the Immunogenicity of RBD-Based Anti-COVID-19 Subunit Vaccines?

Currently, SARS-CoV-2 spike receptor-binding-domain (RBD)-based vaccines are considered one of the most effective weapons against COVID-19. During the first step of assessing vaccine immunogenicity, a mouse model is often used. In this paper, we tested the use of five experimental animals (mice, hamsters, rabbits, ferrets, and chickens) for RBD immunogenicity assessments. The humoral immune response was evaluated by ELISA and virus-neutralization assays. The data obtained show hamsters to be the least suitable candidates for RBD immunogenicity testing and, hence, assessing the protective efficacy of RBD-based vaccines

Structure- and Interaction-Based Design of Anti-SARS-CoV-2 Aptamers

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity using small-angle X-ray scattering, cytometry, and fluorescence polarization. Using a new iterative design procedure, Interaction Based Drug Design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.peerReviewe