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.

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.

Distribution of HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 allele frequencies in patients with COVID-19 bilateral pneumonia in Russians, living in the Chelyabinsk region (Russia).

In this population-based case-control study conducted in the Chelyabinsk region of Russia, we examined the distribution of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1, in a group of 100 patients with confirmed COVID-19 bilateral pneumonia. Typing was performed by NGS and statistical calculations were carried out with the Arlequin program. HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles were compared between patients with COVID-19 and 99 healthy controls. We identified that COVID-19 susceptibility is associated with alleles and genotypes rs9277534A (disequilibrium with HLA-DPB1*02:01, -02:02, -04:01, -04:02, -17:01 alleles) with low expression of protein products HLA-DPB1 (pc < 0.028) and homozygosity at HLA-C*04 (p = 0.024, pc = 0.312). Allele HLA-A*01:01 was decreased in a group of patients with severe forms of bilateral pneumonia, and therefore it may be considered as a protective factor for the development of severe symptoms of COVID-19 (p = 0.009, pc = 0.225). Our studies provide further evidence for the functional association between HLA genes and COVID-19.

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.

Naïve Regulatory T Cell Subset Is Altered in X-Linked Agammaglobulinemia.

The interplay between T- and B-cell compartments during naïve, effector and memory T cell maturation is critical for a balanced immune response. Primary B-cell immunodeficiency arising from X-linked agammaglobulinemia (XLA) offers a model to explore B cell impact on T cell subsets, starting from the thymic selection. Here we investigated characteristics of naïve and effector T cell subsets in XLA patients, revealing prominent alterations in the corresponding T-cell receptor (TCR) repertoires. We observed immunosenescence in terms of decreased diversity of naïve CD4+ and CD8+ TCR repertoires in XLA donors. The most substantial alterations were found within naïve CD4+ subsets, and we have investigated these in greater detail. In particular, increased clonality and convergence, along with shorter CDR3 regions, suggested narrower focused antigen-specific maturation of thymus-derived naïve Treg (CD4+CD45RA+CD27+CD25+) in the absence of B cells - normally presenting diverse self and commensal antigens. The naïve Treg proportion among naïve CD4 T cells was decreased in XLA patients, supporting the concept of impaired thymic naïve Treg selection. Furthermore, the naïve Treg subset showed prominent differences at the transcriptome level, including increased expression of genes specific for antigen-presenting and myeloid cells. Altogether, our findings suggest active B cell involvement in CD4 T cell subsets maturation, including B cell-dependent expansion of the naïve Treg TCR repertoire that enables better control of self-reactive T cells.

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.

The Frequency of CCR5del32 Mutation in Populations of Russians, Tatars and Bashkirs of Chelyabinsk Region, Russia.

The distribution of genetic variants associated with natural resistance to viral infections can vary among human ethnic groups due to evolutionary factors, defining the different epidemiologic background of world populations. The polymorphisms, defining the natural resistance to HIV-infection and the rate of progression up to AIDS, are very important since epidemic is still on rise. We have studied the distribution of allele and genotype frequencies of CCR5delta32 mutation in major populations inhabiting Chelyabinsk region of the Russian Federation. Genetic survey included the population of 509 potential blood marrow donors: Russians (N = 300), Bashkirs (N = 118) and Tatars (N = 91). The genotyping assay was performed using real-time polymerase chain reaction (real-time PCR). The genotypes were defined by melting curve analysis. The CCR5delta32 allele and CCR5delta32/delta32 genotype are presented in population of Russians in Chelyabinsk region with the frequencies of F x  = 10.83% and P x  = 1.67, for the CCR5delta32 allele and its homozygosity, respectively. In populations of Bashkirs and Tatars CCR5delta32 allele and CCR5delta32/delta32 genotype are presented at lower frequencies of F x  = 6.36%/P x  = 0.85 and F x  = 7.14%/P x  = 1.10, respectively. These data are consistent with the theory of northern origin of the CCR5delta32 mutation.