SARS-CoV-2 RBD Conjugated to Polyglucin, Spermidine, and dsRNA Elicits a Strong Immune Response in Mice.

Despite the rapid development and approval of several COVID vaccines based on the full-length spike protein, there is a need for safe, potent, and high-volume vaccines. Considering the predominance of the production of neutralizing antibodies targeting the receptor-binding domain (RBD) of S-protein after natural infection or vaccination, it makes sense to choose RBD as a vaccine immunogen. However, due to its small size, RBD exhibits relatively poor immunogenicity. Searching for novel adjuvants for RBD-based vaccine formulations is considered a good strategy for enhancing its immunogenicity. Herein, we assess the immunogenicity of severe acute respiratory syndrome coronavirus 2 RBD conjugated to a polyglucin:spermidine complex (PGS) and dsRNA (RBD-PGS + dsRNA) in a mouse model. BALB/c mice were immunized intramuscularly twice, with a 2-week interval, with 50 µg of RBD, RBD with Al(OH)3, or conjugated RBD. A comparative analysis of serum RBD-specific IgG and neutralizing antibody titers showed that PGS, PGS + dsRNA, and Al(OH)3 enhanced the specific humoral response in animals. There was no significant difference between the groups immunized with RBD-PGS + dsRNA and RBD with Al(OH)3. Additionally, the study of the T-cell response in animals showed that, unlike adjuvants, the RBD-PGS + dsRNA conjugate stimulates the production of specific CD4+ and CD8+ T cells in animals.

Natural IgG against S-Protein and RBD of SARS-CoV-2 Do Not Bind and Hydrolyze DNA and Are Not Autoimmune.

Since the onset of the COVID-19 pandemic, numerous publications have appeared describing autoimmune pathologies developing after a coronavirus infection, with several papers reporting autoantibody production during the acute period of the disease. Several viral diseases are known to trigger autoimmune processes, and the appearance of catalytic antibodies with DNase activity is one of the earliest markers of several autoimmune pathologies. Therefore, we analyzed whether IgG antibodies from blood plasma of SARS-CoV-2 patients after recovery could bind and hydrolyze DNA. We analyzed how vaccination of patients with adenovirus Sputnik V vaccine influences the production of abzymes with DNase activity. Four groups were selected for the analysis, each containing 25 patients according to their relative titers of antibodies to S-protein: with high and median titers, vaccinated with Sputnik V with high titers, and a control group of donors with negative titers. The relative titers of antibodies against DNA and the relative DNase activity of IgGs depended very much on the individual patient and the donor, and no significant correlation was found between the relative values of antibodies titers and their DNase activity. Our results indicate that COVID-19 disease and vaccination with adenoviral Sputnik V vaccine do not result in the development or enhancement of strong autoimmune reactions as in the typical autoimmune diseases associated with the production of anti-DNA and DNA hydrolyzing antibodies.

Role of the Hospital Pharmacist in the Evaluation of the Anti- COVID-19 Vaccine Safety Profile in the Post-Marketing Phase

Introduction: The European Medicines Agency, following positive evaluation regarding the safety, quality, and efficacy of anti-COVID- 19 vaccines, granted conditional marketing authorization (CMA) for these drugs on the condition that the developers would continuously provide additional data on their safety and efficacy even after marketing authorization in order to confirm the risk-benefit ratio. Following the start of the vaccination campaign in December 2020, special attention was paid to the occurrence of possible adverse reactions (ADRs). The pharmacist staff of Pugliese-Ciaccio Hospital, in order to monitor the safety of the new vaccine and to promptly report suspected vaccine ADRs, prepared a short questionnaire to be administered to the employees of the hospital at the time of administration of the second dose of vaccine. Objective(s): Monitoring vaccine safety & promptly reporting side effects. Method(s): The survey was conducted between January and April 2021. All employees were administered mRNA vaccine and, at the time of the administration of the second dose, were asked to answer the above questionnaire specifying the following information: biographical data, gender, date of administration of the two doses of vaccine, occurrence of any ADRs resulting from the first dose of vaccine, type of resolution, presence of concomitant diseases and related medication intake in the days before/following the vaccination. Result(s): The questionnaire was administered to 1,656 health care workers and all of them answered the questions comprehensively. Among them, 51.6 percent experienced adverse reactions after administration of the first dose of vaccine, and the predominantly noted symptoms included systemic diseases and conditions related to the site of administration, musculoskeletal and connective tissue disorders, nervous system disorders and gastrointestinal disorders. The frequency of reporting was higher among the young than the elderly population (58% vs. 38.67%). ADRs occurred approximately 1,7 times more frequently among women than men. Conclusion(s): The intense pharmacovigilance activity carried out by the Hospital Pharmacist was a pivotal moment during the pandemic emergency, as it allowed the safety profile of anti-COVID-19 vaccines to be readily confirmed with real-life data. Infact, it was found that the main symptoms detected were in line with what was reported in the safety data from the pre-registration studies [1], from which it was also found that the frequency of ADRs was higher among the young than the elderly, a finding that was also confirmed by our study. So, the questionnaire survey was able to substantiate the safety of vaccines, confirming that the benefits of vaccination outweigh the risks.

Role of the Hospital Pharmacist in the Evaluation of the Anti- COVID-19 Vaccine Safety Profile in the Post-Marketing Phase: An International Journal of Medical Toxicology and Drug Experience

Introduction: The European Medicines Agency, following positive evaluation regarding the safety, quality, and efficacy of anti-COVID19 vaccines, granted conditional marketing authorization (CMA) for these drugs on the condition that the developers would continuously provide additional data on their safety and efficacy even after marketing authorization in order to confirm the risk-benefit ratio. Following the start of the vaccination campaign in December 2020, special attention was paid to the occurrence of possible adverse reactions (ADRs). The pharmacist staff of Pugliese-Ciaccio Hospital, in order to monitor the safety of the new vaccine and to promptly report suspected vaccine ADRs, prepared a short questionnaire to be administered to the employees of the hospital at the time of administration of the second dose of vaccine. Objective: Monitoring vaccine safety & promptly reporting side effects. Methods: The survey was conducted between January and April 2021. All employees were administered mRNA vaccine and, at the time of the administration of the second dose, were asked to answer the above questionnaire specifying the following information: biographical data, gender, date of administration of the two doses of vaccine, occurrence of any ADRs resulting from the first dose of vaccine, type of resolution, presence of concomitant diseases and related medication intake in the days before/following the vaccination. Results: The questionnaire was administered to 1,656 health care workers and all of them answered the questions comprehensively. Among them, 51.6 percent experienced adverse reactions after administration of the first dose of vaccine, and the predominantly noted symptoms included systemic diseases and conditions related to the site of administration, musculoskeletal and connective tissue disorders, nervous system disorders and gastrointestinal disorders. The frequency of reporting was higher among the young than the elderly population (58% vs. 38.67%). ADRs occurred approximately 1,7 times more frequently among women than men. Conclusion: The intense pharmacovigilance activity carried out by the Hospital Pharmacist was a pivotal moment during the pandemic emergency, as it allowed the safety profile of anti-COVID-19 vaccines to be readily confirmed with real-life data. Infact, it was found that the main symptoms detected were in line with what was reported in the safety data from the pre-registration studies [1], from which it was also found that the frequency of ADRs was higher among the young than the elderly, a finding that was also confirmed by our study. So, the questionnaire survey was able to substantiate the safety of vaccines, confirming that the benefits of vaccination outweigh the risks.

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.

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.

Cover Feature: Structure‐ and Interaction‐Based Design of Anti‐SARS‐CoV‐2 Aptamers (Chem. Eur. J. 12/2022)

Digital drug design reveals DNA aptamers binding SARS‐CoV‐2: A hybrid in silico et vitro approach, structure and interaction‐based drug design, has been developed to create highly specific DNA aptamers for the receptor‐binding domain of the SARS‐CoV‐2 spike protein. The structure and binding affinity of the aptamers were validated by small‐angle X‐ray scattering, flow cytometry, and fluorescence polarization. This approach offers a blueprint for the straightforward design of targeting molecules for new pathogens and emerging variants. More information can be found in the Research Article by Y. Alexeev, M. V. Berezovski, A. S. Kichkailo, et al. (DOI: 10.1002/chem.202104481).

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.

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 by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and 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.

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.

ORAL TELEMEDICINE: AN EXPERIENCE IN ORAL MEDICINE DURING COVID-19 PANDEMIA

The current coronavirus pandemic disease 2019 (COVID-19), has caused changes in patients' healthcare methods. In response to COVID-19, medical and dental centers have adopted digital tools and technologies such as telemedicine and virtual care, refer to the provision of digital or remote health care services through the use of information and communication technologies, for the evaluation, diagnosis and treatment of patients. Telemedicine is expected to provide timely care while minimizing exposure to protect physicians and patients. With this in mind, oral medicine specialists may consider incorporating telemedicine into their routine clinical practices to benefit patients who have to travel long distances or who have to rely on family members or transportation to attend clinical visits in person. Consequently, a literature review was conducted to provide theoretical and practical evidence on the importance of the use of telemedicine and virtual care for the remote treatment of patients;In turn, three cases of oral medicine carried out successfully via telemedicine are described. The aim of this study is to consolidate virtual care solutions in the current time, to integrate digital technologies in the care of the oral physician to his/her patients.

Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil.

Cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Manaus, Brazil, resurged in late 2020 despite previously high levels of infection. Genome sequencing of viruses sampled in Manaus between November 2020 and January 2021 revealed the emergence and circulation of a novel SARS-CoV-2 variant of concern. Lineage P.1 acquired 17 mutations, including a trio in the spike protein (K417T, E484K, and N501Y) associated with increased binding to the human ACE2 (angiotensin-converting enzyme 2) receptor. Molecular clock analysis shows that P.1 emergence occurred around mid-November 2020 and was preceded by a period of faster molecular evolution. Using a two-category dynamical model that integrates genomic and mortality data, we estimate that P.1 may be 1.7- to 2.4-fold more transmissible and that previous (non-P.1) infection provides 54 to 79% of the protection against infection with P.1 that it provides against non-P.1 lineages. Enhanced global genomic surveillance of variants of concern, which may exhibit increased transmissibility and/or immune evasion, is critical to accelerate pandemic responsiveness.

Characteristics of the liver parenchyma according to the native CT examinations data at the stages of COVID-19 treatment (Full text in english)

Purpose. To assess the liver density according to the data of native CT studies in patients with COVID-19, depending on the severity of the pulmonary parenchyma damage and the prescribed treatment, to compare the data with biochemical indicators, and also to demonstrate changes in density indicators over time. Material and methods. Lung CT data from 200 patients with COVID-19 were retrospectively analyzed. The density of the liver, spleen, and subcutaneous fat tissue was measured in all patients on the images of the upper abdominal cavity that entered the scan area. The ratio of the density of the liver to the spleen and to the density of the fat tissue was assesed. These indicators were compared with each other in two groups of lung tissue damage: CT 1-2 and CT 3-4. The CT 3-4 group was assessed in detail: the density indicators of the liver were studied in dynamics, and their relationship with biochemical indicators - during the initial study. A comparison was also made between two subgroups: patients taking tocilizumab and those without tocilizumab. Results. A decrease in liver density and the ratio of liver density to spleen density was observed in 35.5% and 47.5% of patients respectively. Liver density and the ratio of liver density to spleen density were lower in the CT 3-4 group than in the CT 1-2 group, and amounted to 43.9 HU versus 49.3 HU (p < 0.008) and 0.9 versus 1.0 respectively (p < 0.014). In the initial study, there were a moderate (r = -0.30;p < 0.05) and weak (r = -0.26;p < 0.05) negative correlation of liver density and the ratio of liver density to spleen density with serum albumin. When assessing the dynamics in patients in the CT 3-4 group, with each subsequent study, an increase in the density of the liver parenchyma and the ratio of liver density to spleen density was noted. The difference between the mean values of liver density at the first and at the fourth CT examinations was 11.85 HU. Liver density values were independent of treatment with tocilizumab. Conclusion. Liver density values were lower in patients with COVID-19 with the degree of lung parenchyma lesion CT 3-4, increased during treatment and did not depend on the prescription of tocilizumab. Evaluation and monitoring of the dynamics of liver density could become a useful parameter in determining the severity of the disease course. No strong relationships were found between the density parameters during primary CT and any of the biochemical parameters. A more detailed analysis of these changes in dynamics is required, which may suggest the prevailing mechanism of liver damage in COVID-19. Цель. Оценить плотность печени при нативной КТ у больных COVID-19 в зависимости от степени поражения легочной паренхимы и назначенного лечения, сравнить данные с биохимическими показателями, а также продемонстрировать изменения плотности в динамике. Материал и методы. Ретроспективно анализировали данные КТ легких 200 пациентов с COVID-19. У всех пациентов измерили плотность печени, селезенки, подкожной жировой клетчатки (ПЖК) на вошедших в зону сканирования изображениях верхних отделов брюшной полости. Изучили отношение плотности печени к селезенке и к плотности ПЖК. Показатели сравнили между собой в двух группах поражения легочной ткани: КТ 1-2 и КТ 3-4. Детально рассмотрели группу КТ 3-4: плотность печени изучена в динамике, а ее связь с биохимическими показателями - при первичном исследовании. Также выполнено сравнение двух подгрупп: пациентов, принимавших тоцилизумаб, и без назначения тоцилизумаба. Результаты. Уменьшение плотности печени и коэффициента соотношения плотности печени к плотности селезенки отмечено у 35,5 и 47,5% пациентов соответственно. Плотность печени и коэффициент соотношения плотности печени к плотности селезенки были меньше в группе КТ 3-4, чем в группе КТ 1-2, и составляли 43,9 HU по сравнению с 49,3 HU (p < 0,008) и 0,9 по сравнению с КТ 1 соответственно (p < 0,014). При первичном исследовании была получена умеренная (r = -0,30;p < 0,05) и слабая (r = -0,26;p < 0,05) отрицательная корреляция плотности печени и коэффициента соотношения плотности печени к плотности селезенки с уровнем сывороточного альбумина. При оценке в динамике у пациентов в группе КТ 3-4 с каждым последующим исследованием отметили увеличение плотности паренхимы печени и коэффициента соотношения плотности печени к плотности селезенки. Разница между средними показателями плотности печени при певой и при четвертой КТ составила 11,85 HU. Плотностные показатели печени не зависели от лечения тоцилизумабом. Заключение. Значения плотности печени были меньше у больных COVID-19 при поражении легочной паренхимы КТ 3-4, увеличивались во время лечения и не зависели от назначения тоцилизумаба. Оценка плотности печени и изучение в динамике может стать полезным параметром в определении тяжести течения заболевания. Сильной взаимосвязи между плотностью при первичной КТ и биохимическими показателями не выявлено. Необходим более детальный анализ этих изменений в динамике, который, возможно, позволит предположить превалирующий механизм поражения печени при СOVID-19.