Novel B-Cell Epitopes of Non-Neutralizing Antibodies in the Receptor-Binding Domain of the SARS-CoV-2 S-Protein with Different Effects on the Severity of COVID-19.

Antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein (RBD S-protein) contribute significantly to the humoral immune response during coronavirus infection (COVID-19) and after vaccination. The main focus of the studies of the RBD epitope composition is usually concentrated on the epitopes recognized by the virus-neutralizing antibodies. The role of antibodies that bind to RBD but do not neutralize SARS-CoV-2 remains unclear. In this study, immunochemical properties of the two mouse monoclonal antibodies (mAbs), RS17 and S11, against the RBD were examined. Both mAbs exhibited high affinity to RBD, but they did not neutralize the virus. The epitopes of these mAbs were mapped using phage display: the epitope recognized by the mAb RS17 is located at the N-terminal site of RBD (348-SVYAVNRKRIS-358); the mAb S11 epitope is inside the receptor-binding motif of RBD (452-YRLFRKSN-459). Three groups of sera were tested for presence of antibodies competing with the non-neutralizing mAbs S11 and RS17: (i) sera from the vaccinated healthy volunteers without history of COVID-19; (ii) sera from the persons who had a mild form of COVID-19; (iii) sera from the persons who had severe COVID-19. Antibodies competing with the mAb S11 were found in each group of sera with equal frequency, whereas presence of the antibodies competing with the mAb RS17 in the sera was significantly more frequent in the group of sera obtained from the patients recovered from severe COVID-19 indicating that such antibodies are associated with the severity of COVID-19. In conclusion, despite the clear significance of anti-RBD antibodies in the effective immune response against SARS-CoV-2, it is important to analyze their virus-neutralizing activity and to confirm absence of the antibody-mediated enhancement of infection by the anti-RBD antibodies.

Antibodies Capable of Enhancing SARS-CoV-2 Infection Can Circulate in Patients with Severe COVID-19.

Antibody-dependent enhancement (ADE) has been shown previously for SARS-CoV-1, MERS-CoV, and SARS-CoV-2 infection in vitro. In this study, the first monoclonal antibody (mAb) that causes ADE in a SARS-CoV-2 in vivo model was identified. mAb RS2 against the SARS-CoV-2 S-protein was developed using hybridoma technology. mAb RS2 demonstrated sub-nanomolar affinity and ability to neutralize SARS-CoV-2 infection in vitro with IC50 360 ng/mL. In an animal model of SARS-CoV-2 infection, the dose-dependent protective efficacy of mAb RS2 was revealed. However, in post-exposure prophylaxis, the administration of mAb RS2 led to an increase in the viral load in the respiratory tract of animals. Three groups of blood plasma were examined for antibodies competing with mAb RS2: (1) plasmas from vaccinated donors without COVID-19; (2) plasmas from volunteers with mild symptoms of COVID-19; (3) plasmas from patients with severe COVID-19. It was demonstrated that antibodies competing with mAb RS2 were significantly more often recorded in sera from volunteers with severe COVID-19. The results demonstrated for the first time that in animals, SARS-CoV-2 can induce antibody/antibodies that can elicit ADE. Moreover, in the sera of patients with severe COVID-19, there are antibodies competing for the binding of an epitope that is recognized by the ADE-eliciting mAb.

SARS-CoV-2 Seroprevalence Structure of the Russian Population during the COVID-19 Pandemic.

The SARS-CoV-2 pandemic, which came to Russia in March 2020, is accompanied by morbidity level changes and can be tracked using serological monitoring of a representative population sample from Federal Districts (FDs) and individual regions. In a longitudinal cohort study conducted in 26 model regions of Russia, distributed across all FDs, we investigated the distribution and cumulative proportions of individuals with antibodies (Abs) to the SARS-CoV-2 nucleocapsid antigen (Ag), in the period from June to December 2020, using a three-phase monitoring process. In addition, during the formation of the cohort of volunteers, the number of seropositive convalescents, persons who had contact with patients or COVID-19 convalescents, and the prevalence of asymptomatic forms of infection among seropositive volunteers were determined. According to a uniform methodology, 3 mL of blood was taken from the examined individuals, and plasma was separated, from which the presence of Abs to nucleocapsid Ag was determined on a Thermo Scientific Multiascan FC device using the "ELISA anti-SARS-CoV-2 IgG" reagent set (prod. Scientific Center for Applied Microbiology and Biotechnology), in accordance with the developer's instructions. Volunteers (74,158) were surveyed and divided into seven age groups (1-17, 18-29, 30-39, 40-49, 59-59, 60-69, and 70+ years old), among whom 14,275 were identified as having antibodies to SARS-CoV-2. The average percent seropositive in Russia was 17.8% (IQR: 8.8-23.2). The largest proportion was found among children under 17 years old (21.6% (IQR: 13.1-31.7). In the remaining groups, seroprevalence ranged from 15.6% (IQR: 8-21.1) to 18.0% (IQR: 13.4-22.6). During monitoring, three (immune) response groups were found: (A) groups with a continuous increase in the proportion of seropositive; (B) those with a slow rate of increase in seroprevalence; and (C) those with a two-phase curve, wherein the initial increase was replaced by a decrease in the percentage of seropositive individuals. A significant correlation was revealed between the number of COVID-19 convalescents and contact persons, and between the number of contacts and healthy seropositive volunteers. Among the seropositive volunteers, more than 93.6% (IQR: 87.1-94.9) were asymptomatic. The results show that the COVID-19 pandemic is accompanied by an increase in seroprevalence, which may be important for the formation of herd immunity.

Intraocular Cytokines and Their Correlations with Clinical Parameters in Patients with Myopic Choroidal Neovascularization.

PURPOSE: To study cytokine levels in aqueous humor of patients with myopic choroidal neovascularization (mCNV), their correlations with each other and ocular parameters. METHODS: Ophthalmological examination and immunological study of aqueous humor with cytokine levels measurement (Bio-Plex™ Human Cytokine 27-Plex panel; Bio-Rad Laboratories, USA) were performed in 19 patients (19 eyes) with ranibizumab-treated mCNV and compared to 15 patients (15 eyes) with myopia without CNV. RESULTS: The levels of 10 cytokines were significantly different in patients with mCNV compared to the controls: the vascular endothelial growth factor (VEGF) level was 2 times lower (191.15 ± 142.30 and 320.06 ± 170.05 pg/mL, respectively), and the levels of PDGF, IL-2, IL-5, IL-13, IL-15, IL-17А, TNF-α, IL-8, and RANTES were elevated. Strong correlations between morphological and functional parameters and cytokines, including VEGF, were found. The VEGF level inversely correlated with the myopia degree and the cytokines IL-13, INF-γ, and RANTES. CONCLUSION: The decrease in VEGF levels accompanied by imbalance of other cytokines may suggest additional mCNV development pathways.

Dextran loading protects macrophages from lipid peroxidation and induces a Keap1/Nrf2/ARE-dependent antioxidant response.

AIMS: Linear dextrans are often proposed as drug delivery systems with milder adverse effects and lower effective drug concentrations. Linear dextrans are polysaccharides that can potentially be used to load macrophages with drugs to transport them to a site of inflammation. Recently, it was reported that dextrans may exert a protective effect vis-à-vis drug cytotoxicity and during wound healing. The aim of the current work was to evaluate molecular mechanisms of action of dextrans that may be relevant to the cytoprotective effects. MAIN METHODS: We determined the effect of treatment with 40- or 70-kDa dextran on production of reactive oxygen species, lipid peroxidation, and lysosomal pH in the J774 macrophage cell line. In addition, induction of Keap1/Nrf2/ARE and autophagic activity were evaluated. KEY FINDINGS: Dextrans of both molecular weights protected the cells from oxidative stress induced by cumene hydroperoxide and from lysosomal stress induced by ammonium chloride. The effect was associated with induction of the Keap1/Nrf2/ARE signaling pathway. Furthermore, dextran stimulated autophagy in a dose-dependent manner but inhibited the autophagosome-lysosome fusion in a time-dependent manner. SIGNIFICANCE: This study shows possible cytoprotective effects of dextran under oxidative stress, and these findings may be used for the development of novel (dextran-based) drug delivery approaches.

Maturation of the translation inhibitor microcin C.

Microcin C (McC), an inhibitor of the growth of enteric bacteria, consists of a heptapeptide with a modified AMP residue attached to the backbone of the C-terminal aspartate through an N-acyl phosphamidate bond. Here we identify maturation intermediates produced by cells lacking individual mcc McC biosynthesis genes. We show that the products of the mccD and mccE genes are required for attachment of a 3-aminopropyl group to the phosphate of McC and that this group increases the potency of inhibition of the McC target, aspartyl-tRNA synthetase.

Ammonium salicylate: a synchrotron study.

The structure of the title salt, NH(4) (+)·C(7)H(5)O(3) (-), is stabilized by substantial hydrogen bonding between ammonium cations and salicylate anions that links the components into a two-dimensional array.

Anomalous photoluminescence in CdSe quantum-dot solids at high pressure due to nonuniform stress.

The application of static high pressure provides a means to precisely control and investigate many fundamental and unique properties of nanoparticles. CdSe is a model quantum-dot system, the behavior of which under high pressure has been extensively studied; however, the effect of nonuniform stresses on this system has not been fully appreciated. Photoluminescence data obtained from CdSe quantum-dot solids in different stress environments varying from purely uniform to highly nonuniform are presented. Small deviations from a uniform stress distribution profoundly affect the electronic properties of this system. In nonuniform stress environments, a pronounced flattening of the photoluminescence enegy is observed above 3 GPa. The observations are validated with theoretical calculations obtained using an all-atom semiempirical pseudopotential technique. This effect must be considered when investigating other potentially pressure-mediated phenomena.

A novel approach to local similarity of protein binding sites substantially improves computational drug design results.

We present a novel notion of binding site local similarity based on the analysis of complete protein environments of ligand fragments. Comparison of a query protein binding site (target) against the 3D structure of another protein (analog) in complex with a ligand enables ligand fragments from the analog complex to be transferred to positions in the target site, so that the complete protein environments of the fragment and its image are similar. The revealed environments are similarity regions and the fragments transferred to the target site are considered as binding patterns. The set of such binding patterns derived from a database of analog complexes forms a cloud-like structure (fragment cloud), which is a powerful tool for computational drug design. It has been shown on independent test sets that the combined use of a traditional energy-based score together with the cloud-based score responsible for the quality of embedding of a ligand into the fragment cloud improves the self-docking and screening results dramatically. The usage of a fragment cloud as a source of positioned molecular fragments fitting the binding protein environment has been validated by reproduction of experimental ligand optimization results.

CYP51 from Trypanosoma cruzi: a phyla-specific residue in the B' helix defines substrate preferences of sterol 14alpha-demethylase.

A potential drug target for treatment of Chagas disease, sterol 14alpha-demethylase from Trypanosoma cruzi (TCCYP51), was found to be catalytically closely related to animal/fungi-like CYP51. Contrary to the ortholog from Trypanosoma brucei (TB), which like plant CYP51 requires C4-monomethylated sterol substrates, TCCYP51 prefers C4-dimethylsterols. Sixty-six CYP51 sequences are known from bacteria to human, their sequence homology ranging from approximately 25% between phyla to approximately 80% within a phylum. TC versus TB is the first example of two organisms from the same phylum, in which CYP51s (83% amino acid identity) have such profound differences in substrate specificity. Substitution of animal/fungi-like Ile105 in the B' helix to Phe, the residue found in this position in all plant and the other six CYP51 sequences from Trypanosomatidae, dramatically alters substrate preferences of TCCYP51, converting it into a more plant-like enzyme. The rates of 14alpha-demethylation of obtusifoliol and its 24-demethyl analog 4alpha-,4alpha-dimethylcholesta-8,24-dien-3beta-ol(norlanosterol) increase 60- and 150-fold, respectively. Turnover of the three 4,4-dimethylated sterol substrates is reduced approximately 3.5-fold. These catalytic properties correlate with the sterol binding parameters, suggesting that Phe in this position provides necessary interactions with C4-monomethylated substrates, which Ile cannot. The CYP51 substrate preferences imply differences in the post-squalene portion of sterol biosynthesis in TC and TB. The phyla-specific residue can be used to predict preferred substrates of new CYP51 sequences and subsequently for the development of new artificial substrate analogs, which might serve as highly specific inhibitors able to kill human parasites.

A very large diversity space of synthetically accessible compounds for use with drug design programs.

We have constructed a very large virtual diversity space containing more than 10(13) chemical compounds. The diversity space is built from about 400 combinatorial libraries, which have been expanded by choosing sizeable collections of suitable R-groups that can be attached to each link point of their scaffolds. These R-group collections have been created by selecting reagents that have drug-like properties from catalogs of available chemicals. As members of known combinatorial libraries, the compounds in the diversity space are in general synthetically accessible and useful as potential drug leads. Hence, the diversity space can be used as a vast source of compounds by a de novo drug design program. For example, we have used such a program to generate inhibitors of HIV integrase enzyme that exhibited activity in the micromolar range.

Optical properties of CdSe superlattices.

Three-dimensional, faceted assemblies of CdSe nanocrystals were grown to microscopic sizes sufficient for identification and direct characterization. Analyses made by optical, fluorescence, and transmission electron microscopy showed that individual, faceted superlattices are composed of nearly single-size nanocrystals assembled into fcc lattices. Photoluminescence was measured in individual superlattices, and the results were compared to the same measurements made in amorphous solid layers and solutions of nanocrystals. Differences in the shape and peak positions of photoluminescence spectra are explained by energy transfer processes determined by nanocrystal size distribution, structure of solid layers, and presence of aggregates in solutions.