Antiviral Therapy of COVID-19.

Since the beginning of the COVID-19 pandemic, the scientific community has focused on prophylactic vaccine development. In parallel, the experience of the pharmacotherapy of this disease has increased. Due to the declining protective capacity of vaccines against new strains, as well as increased knowledge about the structure and biology of the pathogen, control of the disease has shifted to the focus of antiviral drug development over the past year. Clinical data on safety and efficacy of antivirals acting at various stages of the virus life cycle has been published. In this review, we summarize mechanisms and clinical efficacy of antiviral therapy of COVID-19 with drugs based on plasma of convalescents, monoclonal antibodies, interferons, fusion inhibitors, nucleoside analogs, and protease inhibitors. The current status of the drugs described is also summarized in relation to the official clinical guidelines for the treatment of COVID-19. In addition, here we describe innovative drugs whose antiviral effect is provided by antisense oligonucleotides targeting the SARS-CoV-2 genome. Analysis of laboratory and clinical data suggests that current antivirals successfully combat broad spectra of emerging strains of SARS-CoV-2 providing reliable defense against COVID-19.

Immunogenicity and In Vivo Protective Effects of Recombinant Nucleocapsid-Based SARS-CoV-2 Vaccine Convacell®.

The vast majority of SARS-CoV-2 vaccines which are licensed or under development focus on the spike (S) protein and its receptor binding domain (RBD). However, the S protein shows considerable sequence variations among variants of concern. The aim of this study was to develop and characterize a SARS-CoV-2 vaccine targeting the highly conserved nucleocapsid (N) protein. Recombinant N protein was expressed in Escherichia coli, purified to homogeneity by chromatography and characterized by SDS-PAGE, immunoblotting, mass spectrometry, dynamic light scattering and differential scanning calorimetry. The vaccine, formulated as a squalane-based emulsion, was used to immunize Balb/c mice and NOD SCID gamma (NSG) mice engrafted with human PBMCs, rabbits and marmoset monkeys. Safety and immunogenicity of the vaccine was assessed via ELISA, cytokine titer assays and CFSE dilution assays. The protective effect of the vaccine was studied in SARS-CoV-2-infected Syrian hamsters. Immunization induced sustainable N-specific IgG responses and an N-specific mixed Th1/Th2 cytokine response. In marmoset monkeys, an N-specific CD4+/CD8+ T cell response was observed. Vaccinated Syrian hamsters showed reduced lung histopathology, lower virus proliferation, lower lung weight relative to the body, and faster body weight recovery. Convacell® thus is shown to be effective and may augment the existing armamentarium of vaccines against COVID-19.

Dissection of Antibody Responses of Gam-COVID-Vac-Vaccinated Subjects Suggests Involvement of Epitopes Outside RBD in SARS-CoV-2 Neutralization.

Millions of people have been vaccinated with Gam-COVID-Vac but fine specificities of induced antibodies have not been fully studied. Plasma from 12 naïve and 10 coronavirus disease 2019 (COVID-19) convalescent subjects was obtained before and after two immunizations with Gam-COVID-Vac. Antibody reactivity in the plasma samples (n = 44) was studied on a panel of micro-arrayed recombinant folded and unfolded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and 46 peptides spanning the spike protein (S) and by immunoglobulin G (IgG) subclass enzyme-linked immunosorbent assay (ELISA). The ability of Gam-COVID-Vac-induced antibodies to inhibit binding of the receptor-binding domain (RBD) to its receptor angiotensin converting enzyme 2 (ACE2) was investigated in a molecular interaction assay (MIA). The virus-neutralizing capacity of antibodies was studied by the pseudo-typed virus neutralization test (pVNT) for Wuhan-Hu-1 and Omicron. We found that Gam-COVID-Vac vaccination induced significant increases of IgG1 but not of other IgG subclasses against folded S, spike protein subunit 1 (S1), spike protein subunit 2 (S2), and RBD in a comparable manner in naïve and convalescent subjects. Virus neutralization was highly correlated with vaccination-induced antibodies specific for folded RBD and a novel peptide (i.e., peptide 12). Peptide 12 was located close to RBD in the N-terminal part of S1 and may potentially be involved in the transition of the pre- to post-fusion conformation of the spike protein. In summary, Gam-COVID-Vac vaccination induced S-specific IgG1 antibodies in naive and convalescent subjects in a comparable manner. Besides the antibodies specific for RBD, the antibodies induced against a peptide close to the N-terminus of RBD were also associated with virus-neutralization.

Treatment of COVID-19 patients with a SARS-CoV-2-specific siRNA-peptide dendrimer formulation.

BACKGROUND: Severe acute respiratory syndrome corona virus (SARS-CoV-2) infection frequently causes severe and prolonged disease but only few specific treatments are available. We aimed to investigate safety and efficacy of a SARS-CoV-2-specific siRNA-peptide dendrimer formulation MIR 19® (siR-7-EM/KK-46) targeting a conserved sequence in known SARS-CoV-2 variants for treatment of COVID-19. METHODS: We conducted an open-label, randomized, controlled multicenter phase II trial (NCT05184127) evaluating safety and efficacy of inhaled siR-7-EM/KK-46 (3.7 mg and 11.1 mg/day: low and high dose, respectively) in comparison with standard etiotropic drug treatment (control group) in patients hospitalized with moderate COVID-19 (N = 52 for each group). The primary endpoint was the time to clinical improvement according to predefined criteria within 14 days of randomization. RESULTS: Patients from the low-dose group achieved the primary endpoint defined by simultaneous achievement of relief of fever, normalization of respiratory rate, reduction of coughing, and oxygen saturation of >95% for 48 h significantly earlier (median 6 days; 95% confidence interval [CI]: 5-7, HR 1.75, p = .0005) than patients from the control group (8 days; 95% CI: 7-10). No significant clinical efficacy was observed for the high-dose group. Adverse events were reported in 26 (50.00%), 25 (48.08%), and 28 (53.85%) patients from the low-, high-dose and control group, respectively. None of them were associated with siR-7-EM/KK-46. CONCLUSIONS: siR-7-EM/KK-46, a SARS-CoV-2-specific siRNA-peptide dendrimer formulation is safe, well tolerated and significantly reduces time to clinical improvement in patients hospitalized with moderate COVID-19 compared to standard therapy in a randomized controlled trial.

Vaccine based on folded receptor binding domain-PreS fusion protein with potential to induce sterilizing immunity to SARS-CoV-2 variants.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global COVID-19 pandemic. One possibility to control the pandemic is to induce sterilizing immunity through the induction and maintenance of neutralizing antibodies preventing SARS-CoV-2 from entering human cells to replicate in. METHODS: We report the construction and in vitro and in vivo characterization of a SARS-CoV-2 subunit vaccine (PreS-RBD) based on a structurally folded recombinant fusion protein consisting of two SARS-CoV-2 Spike protein receptor-binding domains (RBD) fused to the N- and C-terminus of hepatitis B virus (HBV) surface antigen PreS to enable the two unrelated proteins serving as immunologic carriers for each other. RESULTS: PreS-RBD, but not RBD alone, induced a robust and uniform RBD-specific IgG response in rabbits. Currently available genetic SARS-CoV-2 vaccines induce mainly transient IgG1 responses in vaccinated subjects whereas the PreS-RBD vaccine induced RBD-specific IgG antibodies consisting of an early IgG1 and sustained IgG4 antibody response in a SARS-CoV-2 naive subject. PreS-RBD-specific IgG antibodies were detected in serum and mucosal secretions, reacted with SARS-CoV-2 variants, including the omicron variant of concern and the HBV receptor-binding sites on PreS of currently known HBV genotypes. PreS-RBD-specific antibodies of the immunized subject more potently inhibited the interaction of RBD with its human receptor ACE2 and their virus-neutralizing titers (VNTs) were higher than median VNTs in a random sample of healthy subjects fully immunized with registered SARS-CoV-2 vaccines or in COVID-19 convalescent subjects. CONCLUSION: The PreS-RBD vaccine has the potential to serve as a combination vaccine for inducing sterilizing immunity against SARS-CoV-2 and HBV by stopping viral replication through the inhibition of cellular virus entry.

Peculiarities of the T Cell Immune Response in COVID-19.

Understanding the T cell response to SARS-CoV-2 is critical to vaccine development, epidemiological surveillance, and control strategies for this disease. This review provides data from studies of the immune response in coronavirus infections. It describes general mechanisms of immunity, its T cell components, and presents a detailed scheme of the T cell response in SARS-CoV-2 infection, including from the standpoint of determining the most promising targets for assessing its level. In addition, we reviewed studies investigating post-vaccination immunity in the development of vaccines against COVID-19. This review also includes the peculiarities of immunity in different age and gender groups, and in the presence of a number of factors, for example, comorbidity or disease severity. This study summarizes the most informative methods for assessing the immune response to SARS-CoV-2 infection.

Differentiation of COVID-19 signs and symptoms from allergic rhinitis and common cold: An ARIA-EAACI-GA2 LEN consensus.

BACKGROUND: Although there are many asymptomatic patients, one of the problems of COVID-19 is early recognition of the disease. COVID-19 symptoms are polymorphic and may include upper respiratory symptoms. However, COVID-19 symptoms may be mistaken with the common cold or allergic rhinitis. An ARIA-EAACI study group attempted to differentiate upper respiratory symptoms between the three diseases. METHODS: A modified Delphi process was used. The ARIA members who were seeing COVID-19 patients were asked to fill in a questionnaire on the upper airway symptoms of COVID-19, common cold and allergic rhinitis. RESULTS: Among the 192 ARIA members who were invited to respond to the questionnaire, 89 responded and 87 questionnaires were analysed. The consensus was then reported. A two-way ANOVA revealed significant differences in the symptom intensity between the three diseases (p < .001). CONCLUSIONS: This modified Delphi approach enabled the differentiation of upper respiratory symptoms between COVID-19, the common cold and allergic rhinitis. An electronic algorithm will be devised using the questionnaire.

Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation.

BACKGROUND: First vaccines for prevention of Coronavirus disease 2019 (COVID-19) are becoming available but there is a huge and unmet need for specific forms of treatment. In this study we aimed to evaluate the anti-SARS-CoV-2 effect of siRNA both in vitro and in vivo. METHODS: To identify the most effective molecule out of a panel of 15 in silico designed siRNAs, an in vitro screening system based on vectors expressing SARS-CoV-2 genes fused with the firefly luciferase reporter gene and SARS-CoV-2-infected cells was used. The most potent siRNA, siR-7, was modified by Locked nucleic acids (LNAs) to obtain siR-7-EM with increased stability and was formulated with the peptide dendrimer KK-46 for enhancing cellular uptake to allow topical application by inhalation of the final formulation - siR-7-EM/KK-46. Using the Syrian Hamster model for SARS-CoV-2 infection the antiviral capacity of siR-7-EM/KK-46 complex was evaluated. RESULTS: We identified the siRNA, siR-7, targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) as the most efficient siRNA inhibiting viral replication in vitro. Moreover, we showed that LNA-modification and complexation with the designed peptide dendrimer enhanced the antiviral capacity of siR-7 in vitro. We demonstrated significant reduction of virus titer and lung inflammation in animals exposed to inhalation of siR-7-EM/KK-46 in vivo. CONCLUSIONS: Thus, we developed a therapeutic strategy for COVID-19 based on inhalation of a modified siRNA-peptide dendrimer formulation. The developed medication is intended for inhalation treatment of COVID-19 patients.

Promising compounds from natural sources against COVID-19

The epidemic associated with the new Sars-CoV-2 coronavirus has affected almost all countries of the world and no reliable treatment for this infection exists yet. Many laboratories in the world are currently conducting intensive experimental and theoretical/in silico studies to find effective drugs specific for this disease (COVID-19), but unfortunately, it may take a long time before new drugs appear in the clinical practice. One of the currently widely accepted approaches for finding active compounds is based on the possibility of using existing drugs approved by government medical organizations (as the FDA). Their choice is based on screening, based on the use of computer models that evaluate the specific binding (energy minimization) of such drugs to target molecules that are important for the life cycle. Thus, a few well-known antiviral drugs against HIV, hepatitis C and others selected on this basis exerted an antiviral effect in vitro, but their real effectiveness was far from expected. It should be emphasized that the severe clinical manifestation of the disease is an acute respiratory distress syndrome, mediated by oxidative stress and an aggressive immune attack on its own cells. In this regard, the use of compounds with high antioxidant activity could have advantages both prophylactically and medically. There is a huge range of natural compounds, including official and traditional medicine, which represent valuable unlimited potential for COVID-19 therapy, the advantage of such compounds in their low toxicity. In this review, we tried to focus on the clinical and pharmacological properties of natural substances, mainly flavonoids, which can become promising drugs for the treatment and prevention of COVID-19. The review includes information on possible virus targets and antiviral drugs. Much attention is paid to the question of inhibition of viral activity. Based on published data, including structural features of various compounds, a prediction is made about the prospects of using these compounds as inhibitors of viral activity, as well as anti-inflammatory drugs for the treatment of COVID-19. An important step in the analysis of compounds was the study of the possibility of their interaction with cellular targets of the virus, as well as the ability to bind to the proteins of the Sars-CoV-2 virus itself. Эпидемия, связанная с новым коронавирусом Sars-CoV-2, поразила практически все страны земного шара, и надежных лечебных средств от этой инфекции пока не существует. Многие лаборатории в мире в настоящее время ведут интенсивные экспериментальные и теоретические исследования с целью поиска эффективных препаратов, специфичных для этого заболевания (COVID-19), но, к сожалению, может потребоваться много времени, прежде чем новые лекарства появятся в клинической практике. Один из самых популярных подходов основан на возможности использования для лечения существующих препаратов, одобренных правительственными медицинскими организациями. Их выбор основан на скрининге, в основе которого лежит использование компьютерных моделей, оценивающих специфическое связывание (минимизация энергии связывания) таких препаратов с молекулами-мишенями, важных для жизненного цикла. Так, ряд известных антивирусных препаратов против ВИЧ, гепатита С, выбранных подобным образом, оказывали противовирусный эффект in vitro, но их клиническая эффективность была невысокой. Следует подчеркнуть, что тяжелая форма клинического проявления заболевания представляет собой острый респираторный дистресс-синдром, опосредованный окислительным стрессом и агрессивной иммунной атакой на собственные клетки. В этой связи применение соединений с высокой антиоксидантной активностью может иметь преимущества как в профилактическом, так и в лечебном плане. Существует огромный спектр природных соединений, включая препараты официальной и традиционной медицины, которые представляют неограниченный потенциал, в том числе для терапии вирусных заболеваний. Основным преимуществом подобных соединений является их низкая токсичность. В данном обзоре мы постарались сделать акцент на клинические и фармакологические свойства природных веществ, преимущественно флавоноидов, которые могут стать перспективными препаратами для лечения и профилактики COVID-19. В обзор включена информация о возможных мишенях вируса и противовирусных препаратах. Большое внимание уделено вопросу ингибирования вирусной активности. На основе литературных данных, в том числе о структурных особенностях различных соединений, сделан прогноз о перспективности использования данных соединений в качестве ингибиторов вирусной активности, а также в качестве противовоспалительных средств для терапии COVID-19. Важным этапом при анализе соединений было изучение возможности их взаимодействия с клеточными мишенями вируса, а также способности связывания с белками самого вируса Sars-CoV-2.