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.

Tentaclins-A Novel Family of Phage Receptor-Binding Proteins That Can Be Hypermutated by DGR Systems.

Diversity-generating retroelements (DGRs) are prokaryotic systems providing rapid modification and adaptation of target proteins. In phages, the main targets of DGRs are receptor-binding proteins that are usually parts of tail structures and the variability of such host-recognizing structures enables phage adaptation to changes on the bacterial host surface. Sometimes, more than one target gene containing a hypermutated variable repeat (VR) can be found in phage DGRs. The role of mutagenesis of two functionally different genes is unclear. In this study, several phage genomes that contain DGRs with two target genes were found in the gut virome of healthy volunteers. Bioinformatics analysis of these genes indicated that they encode proteins with different topology; however, both proteins contain the C-type lectin (C-lec) domain with a hypermutated beta-hairpin on its surface. One of the target proteins belongs to a new family of proteins with a specific topology: N-terminal C-lec domain followed by one or more immunoglobulin domains. Proteins from the new family were named tentaclins after TENTACLe + proteIN. The genes encoding such proteins were found in the genomes of prophages and phages from the gut metagenomes. We hypothesized that tentaclins are involved in binding either to bacterial receptors or intestinal/immune cells.

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.

The Smallest Isoform of Metridia longa Luciferase as a Fusion Partner for Hybrid Proteins.

Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepod Metridia longa. In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner. We demonstrate that, whereas fusion of a single-chain antibody to either N- or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (KD = 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (KD = 87.6 nM). The detection limit of 14D5a-MLuc7 hybrid protein was estimated to be 45 pg of the recombinant glycoprotein E. Although the smallest isoform of M. longa luciferase was tested as a fusion partner only with a single-chain antibody, it is reasonable to suppose that MLuc7 can also be successfully used as a partner for genetic fusion with other proteins.

Raoultella bacteriophage RP180, a new member of the genus Kagunavirus, subfamily Guernseyvirinae.

A novel lytic Raoultella phage, RP180, was isolated and characterized. The RP180 genome has 44,851 base pairs and contains 65 putative genes, 35 of them encoding proteins whose functions were predicted based on sequence similarity to known proteins. The RP180 genome possesses a gene synteny typical of members of the subfamily Guernseyvirinae. Phylogenetic analysis of the RP180 genome and similar phage genomes revealed that phage RP180 is the first member of the genus Kagunavirus, subfamily Guernseyvirinae, that is specific for Raoultella sp. The genome of RP180 encodes a putative protein with similarity to CRISPR-like Cas4 nucleases, which belong to the pfam12705/PDDEXK_1 family. Cas4-like proteins of this family have been shown to interfere with the bacterial host type II-C CRISPR-Cas system.

New human single chain anti-idiotypic antibody against benzo[a]pyrene.

The nal¨ve library from the lymphocytes of healthy humans was screened by murine single-stranded idiotypic antibodies against benzo[a]pyrene (pSh). The phage clone which contained of anti-idiotypic antibody against benzo[a]pyrene, designated as A4, was chosen for further work because of highly specific to pSh. The available protein databases were searched. The A4 amino acid sequence was unique and 76% identical to a sequence in antibody against interferon g. The A4 protein was expressed in bacteria and purified by two different methods: His-tagged A4 and CBD-fusion A4. Both the A4 bound to pSh and also to the human single chain idiotypic antibody against the benzo[a]pyrene (T72) by ELISA. The Kd values of A4 for pSh and T72 were very close: 4.44 × 10-7 M and 5.71 × 10-7M, respectively. A4 was a competitor with benzo[a]pyrene for binding sites of both idiotypic pSh and T72 in competitive ELISA. Thus, A4 was a high affinity anti-idiotypic against benzo[a]pyrene which recognised pSh and T72 active sites.

Biological evaluation of tetracationic compounds based on two 1,4-diazabicyclo[2.2.2]octane moieties connected by different linkers.

A series of 1,4-diazabicyclo[2.2.2]octane derivatives differing by linker moiety was evaluated for activity against several strains of both Gram-positive and Gram-negative bacteria including drug-resistant strains, one strain of fungus and influenza virus A/Puerto Rico/8/34 (H1N1). All compounds exhibited high antibacterial activity against all bacteria except Proteus vulgaris. The minimum inhibitory concentrations (MICs) of compound 1c with an o-phenylenebismethyl linker and compound 1e with a propylene aliphatic linker were found to be low and were comparable or better to the reference drug ciprofloxacin for Pseudomonas aeruginosa and Staphylococcus aureus. Additionally, a time-kill assay was performed to examine the bactericidal kinetics. Compounds 1c and 1e displayed rapid killing effects against St. aureus and Ps. aeruginosa after 2h. Furthermore, compounds 1a-c with aromatic linkers and compound 1e showed the highest antiviral activity.

Molecular epidemiology of noroviruses associated with sporadic gastroenteritis in children in Novosibirsk, Russia, 2003-2012.

Noroviruses (NoVs) are an important cause of acute gastroenteritis worldwide. To monitor the molecular epidemiology of NoVs genogroup II (GII) in Novosibirsk, Russia, a total of 10,198 stool samples from young children hospitalized with acute gastroenteritis and two asymptomatic comparison groups were collected from 2003 to 2012. All samples were screened for the presence of NoV GII, rotavirus, and astrovirus by RT-PCR. The prevalence of NoV in gastroenteritis cases was 13.1%, varying from 7.1% to 21.3% in different seasons. Rotavirus and/or astrovirus were detectable in 25% of the NoV-positive samples. NoV was detected throughout the year with a seasonal increase during winter months. Based on sequence analysis of regions D and/or C within the VP1 gene, 892 identified NoV strains were divided into nine genotypes­GII.3 (51%), GII.4 (44%), GII.6 (2%), as well as GII.1, GII.2, GII.5, GII.7, GII.16, and GII.21 (totally, 3%). The prevalence of NoV in the comparison groups was considerably lower (∼2.5%); only GII.4 (n = 6), GII.21 (n = 2) and GII.1 (n = 1) genotypes were revealed. Based on phylogenetic analysis of the ORF1/ORF2 junction region sequences, GII.P21/GII.3 recombinant and GII.P4/GII.4 were prevalent genotypes (totally, 93%) and their ratio changed every season. The median age of children with NoV infection was 6.6 months (range, <1-35 months), but it was different depending on NoV genotype. Children infected with the NoV GII.3 were younger (median 6.2 months) than GII.4-positive patients (median 9.1 months). This is the first long-term systematic study of NoV molecular epidemiology in Russia.

Generation and Characterization of Human Single-Chain Antibodies Against Polycyclic Aromatic Hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs and their epoxides are highly toxic, mutagenic and/or carcinogenic to microorganisms as well as to higher systems including humans. BP is one of the most toxicologically active PAHs and is often used as a prototype for this entire class of contaminants. In order to select anti-BP antibodies, the conjugate of BP with BSA (BP-BSA) was used to screen naïve combinatorial phage library of human scFvs. Seven unique scFvs against BP-BSA were selected after three rounds of selection. Analysis of the genes encoding the scFvs subdivided them to gene families and subfamilies. Homology with the closest germline ranged from 80.21% to 97.57% for heavy chains and 88.89% to 98.57% for the light chains. Four of the seven scFv amino acid residues sequences without stop codons in frame were selected for proteomic analysis with each other. Four scFvs encoded unique non-related proteins with low-sequence identity among them. All CDRs and the boundaries in the CDR3 formation were carried out. Two of the scFvs (T68 and T72) with the highest binding capabilities to PAHs were expressed in E. coli and purified using a nickel resin. The KDs of T68 to BP-BSA, chrysene, pyrene, and benzo[a]anthracene were almost similar, approximately 10(-7 )M. The KDs of T72 to benzo[a]anthracene and chrysene were 9.42 × 10(-8 )M and 2.63 × 10(-7 )M, respectively. The computational models of T68 and T72 active centers were different.

Bioluminescent detection probe for tick-borne encephalitis virus immunoassay.

To facilitate the detection of the tick-borne encephalitis virus (TBEV), the causative agent of one of the most severe human neuroinfections, we have developed an immunoassay based on bioluminescent hybrid protein 14D5a-Rm7 as a detection probe. The protein containing Renilla luciferase as a reporter and a single-chain variable fragment (scFv) of murine immunoglobulin to TBEV as a recognition element was constructed, produced by bacterial expression, purified, and tested. Both domains were shown to reveal their specific biological properties-affinity to the target antigen and bioluminescent activity. Hybrid protein was applied as a label for solid-phase immunoassay of the antigens, associated with the tick-borne encephalitis virus (native glycoprotein E or extracts of the infected strain of lab ticks). The assay demonstrates high sensitivity (0.056 ng of glycoprotein E; 10(4)-10(5) virus particles or 0.1 pg virions) and simplicity and is competitive with conventional methods for detection of TBEV.

Genetic diversity of Ixodes pavlovskyi and I. persulcatus (Acari: Ixodidae) from the sympatric zone in the south of Western Siberia and Kazakhstan.

The most epidemiologically significant tick species in Siberia involved in transmission of a large number of pathogens causing human infectious diseases is Ixodes persulcatus. Ixodes pavlovskyi, being more active, also poses epidemiological threats. These tick species share morphology, activity seasons and geographic distribution range. In this paper, we characterize the geographic and genetic structures of I. persulcatus and I. pavlovskyi populations inhabiting the southern part of Western Siberia (Russia and Kazakhstan)--the western part of I. pavlovskyi distribution range. The data are based on six distinct Ixodes tick populations. Analysis of the concatenated mitochondrial marker sequences (16S rRNA and COI) and the nuclear sequence (ITS2) showed genetic polymorphisms in both I. persulcatus and I. pavlovskyi ticks inhabiting the sympatric zone. We could not determine the phylogeographic structure of I. pavlovskyi populations whereas for I. persulcatus significant within-region variance was shown. Notably, the abundance of I. persulcatus ticks negatively correlates with nucleotide and haplotype diversity in the concatenated sequence of mitochondrial gene (16S rRNA and COI) fragments. This is the first description of the genetic polymorphism of I. persulcatus and I. pavlovskyi ticks coexisting in a sympatric zone based on analysis of mitochondrial and nuclear markers.

Successful Use of Phage and Antibiotics Therapy for the Eradication of Two Bacterial Pathogens from the Respiratory Tract of an Infant.

Phage therapy can be a useful approach in a number of clinical cases associated with multidrug-resistant (MDR) bacterial infections. In this study, we describe a successful consecutive phage and antibiotic application to cure a 3-month-old girl suffering from severe bronchitis after tracheostomy. Bronchitis was associated with two bacterial agents, MDR Pseudomonas aeruginosa and a rare opportunistic pathogen Dolosigranulum pigrum. The phage cocktail "Pyobacteriophage" containing at least two different phages against isolated MDR P. aeruginosa strain was used via inhalation and nasal drops. Topical application of the phage cocktail removed most of P. aeruginosa cells and contributed to a change in the antimicrobial resistance profile of surviving P. aeruginosa cells. As a result, it became possible to choose and administer an appropriate antibiotic that was effective against both infectious agents. Complete recovery of the infant was recorded.

Bacteriophage Treatment of Infected Diabetic Foot Ulcers.

Diabetic foot ulcers occur as a common complication of diabetes. The concomitant infection significantly delays the healing of the ulcers. Antibiotic treatment of infected ulcers is complicated by the formation of microbial biofilms, which are often heterogeneous and resistant to antibiotics. Bacteriophage therapy is considered an additional approach to the treatment of infected wounds. Here, we describe the basic method of application of bacteriophages for the treatment of infected diabetic foot ulcers, including very large ones.

Robust and Reproducible Protocol for Phage Genome "Rebooting" Using Transformation-Associated Recombination (TAR) Cloning into Yeast Centromeric Plasmid.

Production of infectious bacteriophage based on its genome is one of the necessary steps in the pipeline of editing phage genomes and creating synthetic bacteriophages. This process is called "rebooting" of the phage genome. In this chapter, we describe key steps required for successful genome "rebooting" using a native host or intermediate host. A detailed protocol is given for the "rebooting" of the genome of T7 bacteriophage specific to Escherichia coli and bacteriophage KP32_192 that infects Klebsiella pneumoniae.

Genome Analysis of Epsilon CrAss-like Phages.

CrAss-like phages play an important role in maintaining ecological balance in the human intestinal microbiome. However, their genetic diversity and lifestyle are still insufficiently studied. In this study, a novel CrAssE-Sib phage genome belonging to the epsilon crAss-like phage genomes was found. Comparative analysis indicated that epsilon crAss-like phages are divided into two putative genera, which were proposed to be named Epsilonunovirus and Epsilonduovirus; CrAssE-Sib belongs to the former. The crAssE-Sib genome contains a diversity-generating retroelement (DGR) cassette with all essential elements, including the reverse transcriptase (RT) and receptor binding protein (RBP) genes. However, this RT contains the GxxxSP motif in its fourth domain instead of the usual GxxxSQ motif found in all known phage and bacterial DGRs. RBP encoded by CrAssE-Sib and other Epsilonunoviruses has an unusual structure, and no similar phage proteins were found. In addition, crAssE-Sib and other Epsilonunoviruses encode conserved prophage repressor and anti-repressors that could be involved in lysogenic-to-lytic cycle switches. Notably, DNA primase sequences of epsilon crAss-like phages are not included in the monophyletic group formed by the DNA primases of all other crAss-like phages. Therefore, epsilon crAss-like phage substantially differ from other crAss-like phages, indicating the need to classify these phages into a separate family.

Characterization of a Thermostable Endolysin of the Aeribacillus Phage AeriP45 as a Potential Staphylococcus Biofilm-Removing Agent.

Multidrug-resistant Gram-positive bacteria, including bacteria from the genus Staphylococcus, are currently a challenge for medicine. Therefore, the development of new antimicrobials is required. Promising candidates for new antistaphylococcal drugs are phage endolysins, including endolysins from thermophilic phages against other Gram-positive bacteria. In this study, the recombinant endolysin LysAP45 from the thermophilic Aeribacillus phage AP45 was obtained and characterized. The recombinant endolysin LysAP45 was produced in Escherichia coli M15 cells. It was shown that LysAP45 is able to hydrolyze staphylococcal peptidoglycans from five species and eleven strains. Thermostability tests showed that LysAP45 retained its hydrolytic activity after incubation at 80 °C for at least 30 min. The enzymatically active domain of the recombinant endolysin LysAP45 completely disrupted biofilms formed by multidrug-resistant S. aureus, S. haemolyticus, and S. epidermidis. The results suggested that LysAP45 is a novel thermostable antimicrobial agent capable of destroying biofilms formed by various species of multidrug-resistant Staphylococcus. An unusual putative cell-binding domain was found at the C-terminus of LysAP45. No domains with similar sequences were found among the described endolysins.

Binding of Natural Antibodies Generated after COVID-19 and Vaccination with Individual Peptides Corresponding to the SARS-CoV-2 S-Protein.

The rapid development of vaccines is a crucial objective in modern biotechnology and molecular pharmacology. In this context, conducting research to expedite the selection of a potent immunogen is imperative. The candidate vaccine should induce the production of antibodies that can recognize the immunogenic epitopes of the target protein, resembling the ones found in recovered patients. One major challenge in vaccine development is the absence of straightforward and reliable techniques to determine the extent to which the spectrum of antibodies produced after vaccination corresponds to antibodies found after recovery. This paper describes a newly developed method to detect antibodies specific to immunogenic epitopes of the target protein in blood plasma and to compare them with antibody spectra generated post vaccination. Comparing the antibody pool generated in the human body after recovering from an infectious disease with the pool formed through vaccination can become a universal method for screening candidate vaccines. This method will enable the identification of candidate vaccines that can induce the production of antibodies similar to those generated in response to a natural infection. Implementing this approach will facilitate the rapid development of new vaccines, even when faced with a pandemic.

Bacteriophage vB_SepP_134 and Endolysin LysSte_134_1 as Potential Staphylococcus-Biofilm-Removing Biological Agents.

Bacteria of the genus Staphylococcus are significant challenge for medicine, as many species are resistant to multiple antibiotics and some are even to all of the antibiotics we use. One of the approaches to developing new therapeutics to treat staphylococcal infections is the use of bacteriophages specific to these bacteria or the lytic enzymes of such bacteriophages, which are capable of hydrolyzing the cell walls of these bacteria. In this study, a new bacteriophage vB_SepP_134 (St 134) specific to Staphylococcus epidermidis was described. This podophage, with a genome of 18,275 bp, belongs to the Andhravirus genus. St 134 was able to infect various strains of 12 of the 21 tested coagulase-negative Staphylococcus species and one clinical strain from the Staphylococcus aureus complex. The genes encoding endolysin (LysSte134_1) and tail tip lysin (LysSte134_2) were identified in the St 134 genome. Both enzymes were cloned and produced in Escherichia coli cells. The endolysin LysSte134_1 demonstrated catalytic activity against peptidoglycans isolated from S. aureus, S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus warneri. LysSte134_1 was active against S. aureus and S. epidermidis planktonic cells and destroyed the biofilms formed by clinical strains of S. aureus and S. epidermidis.

Natural Antibodies Produced in Vaccinated Patients and COVID-19 Convalescents Hydrolyze Recombinant RBD and Nucleocapsid (N) Proteins.

Antibodies are protein molecules whose primary function is to recognize antigens. However, recent studies have demonstrated their ability to hydrolyze specific substrates, such as proteins, oligopeptides, and nucleic acids. In 2023, two separate teams of researchers demonstrated the proteolytic activity of natural plasma antibodies from COVID-19 convalescents. These antibodies were found to hydrolyze the S-protein and corresponding oligopeptides. Our study shows that for antibodies with affinity to recombinant structural proteins of the SARS-CoV-2: S-protein, its fragment RBD and N-protein can only hydrolyze the corresponding protein substrates and are not cross-reactive. By using strict criteria, we have confirmed that this proteolytic activity is an intrinsic property of antibodies and is not caused by impurities co-eluting with them. This discovery suggests that natural proteolytic antibodies that hydrolyze proteins of the SARS-CoV-2 virus may have a positive impact on disease pathogenesis. It is also possible for these antibodies to work in combination with other antibodies that bind specific epitopes to enhance the process of virus neutralization.

Special Issue "Bacteriophage Genomics": Editorial.

Virus genomics as a separate branch of biology has emerged relatively recently [...].