COVID-19 in Russia: Clinical and Immunological Features of the First-Wave Patients.

The coronavirus disease outbreak in 2019 (COVID-19) has now achieved the level of a global pandemic and affected more than 100 million people on all five continents and caused over 2 million deaths. Russia is, needless to say, among the countries affected by SARS-CoV-2, and its health authorities have mobilized significant efforts and resources to fight the disease. The paper presents the result of a functional analysis of 155 patients in the Moscow Region who were examined at the Central Clinical Hospital of the Russian Academy of Sciences during the first wave of the pandemic (February-July, 2020). The inclusion criteria were a positive PCR test and typical, computed tomographic findings of viral pneumonia in the form of ground-glass opacities. A clinical correlation analysis was performed in four groups of patients: (1) those who were not on mechanical ventilation, (2) those who were on mechanical ventilation, and (3) those who subsequently recovered or (4) died. The correlation analysis also considered confounding comorbidities (diabetes, metabolic syndrome, hypertension, etc.). The immunological status of the patients was examined (levels of immunoglobulins of the M, A, G classes and their subclasses, as well as the total immunoglobulin level) using an original SARS-CoV-2 antibody ELISA kit. The ELISA kit was developed using linear S-protein RBD-SD1 and NTD fragments, as well as the N-protein, as antigens. These antigens were produced in the prokaryotic E. coli system. Recombinant RBD produced in the eukaryotic CHO system (RBD CHO) was used as an antigen representing conformational RBD epitopes. The immunoglobulin A level was found to be the earliest serological criterion for the development of a SARS-CoV-2 infection and it yielded the best sensitivity and diagnostic significance of ELISA compared to that of class M immunoglobulin. We demonstrated that the seroconversion rate of "early" N-protein-specific IgM and IgA antibodies is comparable to that of antibodies specific to RBD conformational epitopes. At the same time, seroconversion of SARS-CoV-2 N-protein-specific class G immunoglobulins was significantly faster compared to that of other specific antibodies. Our findings suggest that the strong immunogenicity of the RBD fragment is for the most part associated with its conformational epitopes, while the linear RBD and NTD epitopes have the least immunogenicity. An analysis of the occurrence rate of SARS-CoV-2-specific immunoglobulins of different classes revealed that RBD- and N-specific antibodies should be evaluated in parallel to improve the sensitivity of ELISA. An analysis of the immunoglobulin subclass distribution in sera of seropositive patients revealed uniform induction of N-protein-specific IgG subclasses G1-G4 and IgA subclasses A1-A2 in groups of patients with varying severity of COVID-19. In the case of the S-protein, G1, G3, and A1 were the main subclasses of antibodies involved in the immune response.

Production of Recombinant Human Transferrin in Eukaryotic Pichia pastoris Expression System.

The development and manufacturing of serum-free culture media allowing reducing the costs of preparations and standardizing the biotechnological process are important trends in biotechnology. Substitution of protein compounds in the serum-free media with recombinant analogues reduces the risk of contamination with various infectious agents. Human transferrin is a protein component of serum-free media responsible for the transport of Fe3+ ions into cells. We generated a producing strain P. pastoris secreting human transferrin to the culture medium. The use of constitutive GAP promoter and maintenance of medium pH at 6.5 allows attaining maximum level of transferrin expression (20 mg/liter).

Loading Rate of Exogenous and Autoantigenic Determinants on Major Histocompatibility Complex Class II Mediates Resistance to Multiple Sclerosis.

Genetic analysis of thousands of patients with multiple sclerosis (MS) and healthy Russian donors showed that the carriage of groups of HLA-DRB1*15 and HLA-DRB1*03 alleles is associated with the risk of MS, whereas the carriage of groups of HLA-DRB1*01 and HLA-DRB1*11 alleles is protective. Recombinant HLA-DRB1*01:01 with a high affinity can recognize the fragments of myelin basic protein (MBP), one of the autoantigens in MS. However, the comparison of the kinetic parameters of the load of MBP and viral HA peptides on HLA-DRB1*01:01, which is catalyzed by HLA-DM, showed a significantly lower rate of exchange of CLIP for MBP peptides. We assume that the observed protective properties of the group of HLA-DRB1*01 alleles may be directly associated with the ability of HLA-DRB1*01:01 to kinetically distinguish peptides of exogenous and endogenous nature.

"Shielding" of Cytokine Induction by the Periodontal Microbiome in Patients with Periodontitis Associated with Type 2 Diabetes Mellitus.

Periodontal diseases, especially those with polymicrobial etiology, are often associated with type 2 diabetes mellitus, proceeding more severely and affecting the course of diabetes mellitus. Recently, this feature has been associated with the ability of periodontopathogen microflora to cause not only a local infectious process in the oral cavity, but also to interact with the human immune system and induce various systemic effects. We investigated changes in the salivary cytokine profile of patients with chronic periodontitis, associated and not associated with type 2 diabetes mellitus. We observed a statistically significant decrease of MCP-1/CCL2, GM-CSF, IL-5, IL-6, and IFN-γ in the saliva of patients with chronic periodontitis associated with type 2 diabetes mellitus in comparison with patients with chronic periodontitis only. All of these cytokines are associated with macrophage activation. These data are an important contribution to the elucidation of the mechanism of periodontopathogens involvement in the manifestation of the systemic effects of type 2 diabetes.

Genetic Engineering of Native Chain Combinations of B-Cell Repertoires on the Surface of Methylotrophic Yeasts Pichia pastoris.

We designed genetic constructs for exposing Fab-fragment library of natively paired single cell B-cell receptors on the surface of Pichia pastoris yeast cells. We have previously obtained the A17 antibody in our laboratory [6]. In this study we showed that the newly designed genetic constructs provide a compatible level of A17 antibody Fab fragment on the surface of yeast cells as well as in the case of vectors containing DNA fragments corresponding to each chain of the antibody. The data suggest that the developed approach for constructing immunoglobulin gene libraries is adequate and fully convenient for studying properties of the real human B-lymphocyte repertoire.

Three-dimensional structures of noncovalent complexes of Citrobacter freundii methionine γ-lyase with substrates.

Crystal structures of Citrobacter freundii methionine γ-lyase complexes with the substrates of γ- (L-1-amino-3-methylthiopropylphosphinic acid) and ß- (S-ethyl-L-cysteine) elimination reactions and the competitive inhibitor L-norleucine have been determined at 1.45, 1.8, and 1.63 Å resolution, respectively. All three amino acids occupy the active site of the enzyme but do not form a covalent bond with pyridoxal 5'-phosphate. Hydrophobic interactions between the active site residues and the side groups of the substrates and the inhibitor are supposed to cause noncovalent binding. Arg374 and Ser339 are involved in the binding of carboxyl groups of the substrates and the inhibitor. The hydroxyl of Tyr113 is a potential acceptor of a proton from the amino groups of the amino acids.

Interaction of tyrosine phenol-lyase with phosphoroorganic analogues of substrate amino acids.

The phosphinic analogues of tyrosine and pyruvate were first demonstrated to be substrates in the reactions of elimination and synthesis catalyzed by tyrosine phenol-lyase. Kinetic parameters of the enzymatic process were determined, and the first enzymic synthesis of an aminophosphinic acid was carried out. Replacement of the planar HOOC-group by the tetrahedral (HO)(O)PH-group in the substrate slightly affected its affinity for the enzyme but substantially diminished the conversion rate. For phosphonic analogues, containing (HO)2(O)P group, the affinity to the enzyme was decreased considerably while the conversion was completely prevented. Thus, the structural parameters of the acid group are important not only for the affinity for the enzyme, but also for the formation of the catalytically competent conformation of the active site.

[Phosphinic analog of methionine inhibits growth of leucosis cell L1210 and transforms to phosphinic analog of S-adenosylmethionine]. / Fosfinovyi analog metionina tormozit rost leikoznykh kletok L1210 i prevrashchaetsia v fosfinovyi analog S-adenozilmetionina.

A phosphinic analogue of methionine bearing a phosphinic H(OH)(O)P fragment in place of the carboxyl group inhibited the growth of the L1210 cells and was intracellularly transformed to the phosphinic analogue of S-adenosylmethionine.

Succinyl phosphonate inhibits alpha-ketoglutarate oxidative decarboxylation, catalyzed by alpha-ketoglutarate dehydrogenase complexes from E. coli and pigeon breast muscle.

Effects of a set of alpha-ketoglutarate phosphoanalogues on the activity of alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) complexes from E. coli and pigeon breast muscle, as well as on alpha-ketoglutarate dehydrogenase isolated from the pigeon breast muscle, have been studied. alpha-Ketoglutarate phosphoanalogues (succinyl phosphonate and its monomethyl ester) were found to be effective inhibitors of alpha-ketoglutarate oxidative decarboxylation, catalyzed by both muscle and bacterial alpha-ketoglutarate dehydrogenase complexes, as well as muscle alpha-ketoglutarate dehydrogenase. The ability of glutamate phosphoanalogues to inhibit alpha-ketoglutarate oxidative decarboxylation has been shown in E. coli extract and a model system.

[Enzyme reamination of phosphororganic analogs of aspartate and glutamate]. / Fermentativnoe pereaminirovanie fosfororganicheskikh analogov aspartata i glutamata.

Phosphonous and phosphonic analogues of aspartate and glutamate are substrates of semireaction of enzymatic transamination catalysed by aspartate aminotransferase.

[Reaction of apo-aspartate aminotransferase with 5-thiophosphate analogues of the coenzyme. Split of the thiophosphate bond]. / Reaktsiia apo-aspartat-transaminazy s 5'-tiofosfatnymi analogami kofermentob. Rasshcheplenie tiofosfatnoi sviazi.

By the interaction of pyridoxamine- and pyridoxale-5'-thiophosphate with aspartate-aminotransferase complexes similar in their properties to corresponding forms of the native enzyme were obtained. Reversible convertions of the obtained complexes were performed by short time incubation with substrates. It was found that the thioester bond can be splitted as a result of incubation of the pyridoxamine-5'-thiophosphate form of the enzyme with the substrate mixture for several hours at pH 5.0. The same split took place during incubations of the complex of apo-enzyme with L-Nalpha-(pyridoxyl-5'-thiophosphate)-glutamic acid at pH 3.5 within several minutes. The split of the thioester bond was accomplished by formation of phosphate-enzyme bonds, the latter was found to be stable towards gel-filtration and denaturation, but unstable to proteolysis. The labilisation of the thiophosphate bond was explained in terms of changes of structure and specificity of the anchoring site of 5'-phosphoryl group according to the reaction coordinate.

Labilization of the phosphoester linkage in enzyme-inhibitor complexes of aspartate aminotransferase.

Individual enzyme-inhibitor complexes with characteristic absorption spectra have been obtained as a result of the reaction of the apoenzyme of aspartate aminotransferase with Nalpha-(5'-phosphopyridoxyl)-L-glutamic acid, Nalpha-(5'-phosphopyridoxyl)-D-glutamic acid, and Nalpha-(5'-phosphopyridoxyl)-L-pyroglutamic acid. The stability of the enzyme-inhibitor complexes has been investigated under various conditions, viz., reactivation by the coenzyme, denaturation by urea, variations in the pH. It has been shown that the complexes formed by the last two inhibitors are reactivated by pyridoxal-5'-phosphate and that the inhibitor can be released under mild conditions. The enzyme-inhibitor complex formed by Nalpha-(5'-phosphopyridoxyl)-L-glutamic acid, on the other hand, was not reactivated by the coenzyme. Pyridoxylglutamic acid has been isolate in attempts to release the inhibitor. The dephosphorylation of the inhibitor has been associated both with the hydrolysis of a phosphate bond involving the enzyme and with the phosphorylation of aspartate aminotransferase. A 32P peptide containing 13 amino acids has been isolated from the tryptic hydrolysate of the enzyme-inhibitor complex (formed by a 32P inhibitor). The data obtained have been interpreted on the basis of an assumption that the phosphate group of the coenzyme has an active role in the enzymatic transamination reaction.