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.

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.

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.

New Neutralizing Epitope Exposed on the Domain II of Tick-Borne Encephalitis Virus Envelope Glycoprotein E.

Orthoflavivirus encephalitidis, formerly tick-borne encephalitis virus (TBEV), belongs to the Orthoflavivirus genus. TBEV is transmitted by tick bites and infection with TBEV can lead to serious disorders of the central nervous system. In this study, a new protective monoclonal mouse antibody (mAb) FVN-32, with high binding activity to glycoprotein E of TBEV, was selected and examined in post exposure prophylaxis in a mouse model of TBEV infection. BALB/c mice were injected mAb FVN-32 at doses of 200 µg, 50 µg, and 12.5 µg per mouse one day after a TBEV challenge. mAb FVN-32 showed 37.5% protective efficacy when administered at doses of 200 µg and 50 µg per mouse. The epitope for protective mAb FVN-32 was localized in TBEV glycoprotein E domain I+II, using a set of truncated fragments of glycoprotein E. Additionally, the target site recognized by mAb FVN-32 was defined using combinatorial libraries of peptides. Three-dimensional modeling revealed that the site is dspatially close to the fusion loop, but does not come into contact with it, and is localized in a region between 247 and 254 amino acid residues on the envelope protein. This region is conserved among TBEV-like orthoflaviviruses.

A Novel Podophage StenR_269 Suggests a New Family in the Class Caudoviricetes.

Stenotrophomonas rhizophila was first discovered in soil; it is associated with the rhizosphere and capable of both protecting roots and stimulating plant growth. Therefore, it has a great potential to be used in biocontrol. The study of S. rhizophila phages is important for a further evaluation of their effect on the fitness and properties of host bacteria. A novel phage StenR_269 and its bacterial host S. rhizophila were isolated from a soil sample in the remediation area of a coal mine. Electron microscopy revealed a large capsid (~Ø80 nm) connected with a short tail, which corresponds to the podovirus morphotype. The length of the genomic sequence of the StenR_269 was 66,322 bp and it contained 103 putative genes; 40 of them encoded proteins with predicted functions, 3 corresponded to tRNAs, and the remaining 60 were identified as hypothetical ones. Comparative analysis indicated that the StenR_269 phage had a similar genome organization to that of the unclassified Xanthomonas phage DES1, despite their low protein similarity. In addition, the signature proteins of StenR_269 and DES1 had low similarity and these proteins clustered far from the corresponding proteins of classified phages. Thus, the StenR_269 genome is orphan and the analyzed data suggest a new family in the class Caudoviricetes.

StenM_174: A Novel Podophage That Infects a Wide Range of Stenotrophomonas spp. and Suggests a New Subfamily in the Family Autographiviridae.

Stenotrophomonas maltophilia was discovered as a soil bacterium associated with the rhizosphere. Later, S. maltophilia was found to be a multidrug-resistant hospital-associated pathogen. Lytic bacteriophages are prospective antimicrobials; therefore, there is a need for the isolation and characterization of new Stenotrophomonas phages. The phage StenM_174 was isolated from litter at a poultry farm using a clinical strain of S. maltophilia as the host. StenM_174 reproduced in a wide range of clinical and environmental strains of Stenotrophomonas, mainly S. maltophilia, and it had a podovirus morphotype. The length of the genomic sequence of StenM_174 was 42,956 bp, and it contained 52 putative genes. All genes were unidirectional, and 31 of them encoded proteins with predicted functions, while the remaining 21 were identified as hypothetical ones. Two tail spike proteins of StenM_174 were predicted using AlphaFold2 structural modeling. A comparative analysis of the genome shows that the Stenotrophomonas phage StenM_174, along with the phages Ponderosa, Pepon, Ptah, and TS-10, can be members of the new putative genus Ponderosavirus in the Autographiviridae family. In addition, the analyzed data suggest a new subfamily within this family.

Computational and Rational Design of Single-Chain Antibody against Tick-Borne Encephalitis Virus for Modifying Its Specificity.

Tick-borne encephalitis virus (TBEV) causes 5-7 thousand cases of human meningitis and encephalitis annually. The neutralizing and protective antibody ch14D5 is a potential therapeutic agent. This antibody exhibits a high affinity for binding with the D3 domain of the glycoprotein E of the Far Eastern subtype of the virus, but a lower affinity for the D3 domains of the Siberian and European subtypes. In this study, a 2.2-fold increase in the affinity of single-chain antibody sc14D5 to D3 proteins of the Siberian and European subtypes of the virus was achieved using rational design and computational modeling. This improvement can be further enhanced in the case of the bivalent binding of the full-length chimeric antibody containing the identified mutation.

Influence of Caudovirales Phages on Humoral Immunity in Mice.

Bacteriophages are promising antibacterial agents. Although they have been recognized as bacterial viruses and are considered to be non-interacting with eukaryotic cells, there is growing evidence that phages may have a significant impact on the immune system via interactions with macrophages, neutrophils, and T-cell polarization. In this study, the influence of phages of podovirus, siphovirus, and myovirus morphotypes on humoral immunity of CD-1 mice was investigated. In addition, tissue distribution of the phages was tested in these mice. No common patterns were found either in the distribution of phages in mice or in changes in the levels of cytokines in the sera of mice once injected with phages. Importantly, pre-existing IgM-class antibodies directed against capsid proteins of phages with myovirus and siphovirus morphotypes were identified in mice before immunization. After triple immunization of CD1-mice with phages without any adjuvant, levels of anti-phage serum polyclonal IgG antibodies increased. Immunogenic phage proteins recognized by IgM and/or IgG antibodies were identified using Western blot analysis and mass spectrometry. In addition, mice serum collected after immunization demonstrated neutralizing properties, leading to a substantial decrease in infectivity of investigated phages with myovirus and siphovirus morphotypes. Moreover, serum samples collected before administration of these phages exhibited some ability to reduce the phage infectivity. Furthermore, Proteus phage PM16 with podovirus morphotype did not elicit IgM or IgG antibodies in immunized mice, and no neutralizing activities against PM16 were revealed in mouse serum samples before and after immunization.

Characterization of neutralizing monoclonal antibody against tick-borne encephalitis virus in vivo.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted pathogen in the family Flaviviridae and causes one of the most severe human neuroinfections. In this study, a neutralizing mouse mAb 14D5, which was previously shown to have cross-reactive binding to several flaviviruses belonging to the TBEV group, was examined for its prophylactic and therapeutic effects in BALB/c mice infected with TBEV. Before and after infection, mice were administrated mAb 14D5 at doses 100 µg and 10 µg per mouse. mAb 14D5 showed clear protective efficacy when injected at the high dose one day after infection, with survival rates that were TBEV dose-dependent. Prophylactic administration of mAb 14D5 was more effective than post-exposure administration and complete protection was documented when the mAb was administered one day before infection. The protective efficacy of mAb 14D5 was significantly higher than that of the anti-TBE serum immunoglobulin. However, no protection was observed in mice received the low dose of mAb 14D5 independent of the timing of mAb injection and TBEV dose. The ability of species-matched mAb 14D5 to mediate TBEV infection in mice was also investigated, and the results indicated that mAb 14D5 did not augment TBEV infection independent of the time of mAb administration. The neutralizing epitope for mAb 14D5 was localized in domain III of glycoprotein E of TBEV in a region between residues 301-339, which is conserved among flaviviruses from the TBEV group.

The Ca2+ -Regulated Photoprotein Obelin as a Tool for SELEX Monitoring and DNA Aptamer Affinity Evaluation.

Bioluminescent solid-phase analysis was proposed to monitor the selection process and to determine binding characteristics of the aptamer-target complexes during design and development of the specific aptamers. The assay involves Ca2+ -regulated photoprotein obelin as a simple, sensitive and fast reporter. Applicability and the prospects of the approach were exemplified by identification of DNA aptamers to cardiac troponin I, a highly specific early biomarker for acute myocardial infarction. Two structurally different aptamers specific to various epitopes of troponin I were obtained and then tested in a model bioluminescent assay.

Isolation and Characterization of a Novel Klebsiella pneumoniae N4-like Bacteriophage KP8.

Klebsiella pneumoniae is a common pathogen, associated with a wide spectrum of infections, and clinical isolates of K. pneumoniae often possess multiple antibiotic resistances. Here, we describe a novel lytic N4-like bacteriophage KP8, specific to K. pneumoniae, including its genome, partial structural proteome, biological properties, and proposed taxonomy. Electron microscopy revealed that KP8 belongs to the Podoviridae family. The size of the KP8 genome was 73,679 bp, and it comprised 97 putative open reading frames. Comparative genome analysis revealed that the KP8 genome possessed the highest similarity to the genomes of Enquatrovirus and Gamaleyavirus phages, which are N4-like podoviruses. In addition, the KP8 genome showed gene synteny typical of the N4-like podoviruses and contained the gene encoding a large virion-encapsulated RNA polymerase. Phylogenetic analysis of the KP8 genome revealed that the KP8 genome formed a distinct branch within the clade, which included the members of Enquatrovirus and Gamaleyavirus genera besides KP8. The average evolutionary divergences KP8/Enquatrovirus and KP8/Gamaleyavirus were 0.466 and 0.447 substitutions per site (substitutes/site), respectively, similar to that between Enquatrovirus and Gamaleyavirus genera (0.468 substitutes/site). The obtained data suggested that Klebsiella phage KP8 differs from other similar phages and may represent a new genus within the N4-like phages.

Post-exposure administration of chimeric antibody protects mice against European, Siberian, and Far-Eastern subtypes of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted pathogen. It belongs to the Flaviviridae family and causes severe human neuroinfections. In this study, protective efficacy of the chimeric antibody chFVN145 was examined in mice infected with strains belonging to the Far-Eastern, European, and Siberian subtypes of TBEV, and the antibody showed clear therapeutic efficacy when it was administered once one, two, or three days after infection. The efficacy was independent of the TBEV strain used to infect the mice; however, the survival rate of the mice was dependent on the dose of TBEV and of the antibody. No enhancement of TBEV infection was observed when the mice were treated with non-protective doses of chFVN145. Using a panel of recombinant fragments of the TBEV glycoprotein E, the neutralizing epitope for chFVN145 was localized in domain III of the TBEV glycoprotein E, in a region between amino acid residues 301 and 359. In addition, three potential sites responsible for binding with chFVN145 were determined using peptide phage display libraries, and 3D modeling demonstrated that the sites do not contact the fusion loop and, hence, their binding with chFVN145 does not result in increased attachment of TBEV to target cells.

Novel mouse monoclonal antibodies specifically recognizing ß-(1→3)-D-glucan antigen.

ß-(1→3)-D-Glucan is an essential component of the fungal cell wall. Mouse monoclonal antibodies (mAbs) against synthetic nona-ß-(1→3)-D-glucoside conjugated with bovine serum albumin (BSA) were generated using hybridoma technology. The affinity constants of two selected mAbs, 3G11 and 5H5, measured by a surface plasmon resonance biosensor assay using biotinylated nona-ß-(1→3)-D-glucan as the ligand, were approximately 11 nM and 1.9 nM, respectively. The glycoarray, which included a series of synthetic oligosaccharide derivatives representing ß-glucans with different lengths of oligo-ß-(1→3)-D-glucoside chains, demonstrated that linear tri-, penta- and nonaglucoside, as well as a ß-(1→6)-branched octasaccharide, were recognized by mAb 5H5. By contrast, only linear oligo-ß-(1→3)-D-glucoside chains that were not shorter than pentaglucosides (but not the branched octaglucoside) were ligands for mAb 3G11. Immunolabelling indicated that 3G11 and 5H5 interact with both yeasts and filamentous fungi, including species from Aspergillus, Candida, Penicillium genera and Saccharomyces cerevisiae, but not bacteria. Both mAbs could inhibit the germination of Aspergillus fumigatus conidia during the initial hours and demonstrated synergy with the antifungal fluconazole in killing C. albicans in vitro. In addition, mAbs 3G11 and 5H5 demonstrated protective activity in in vivo experiments, suggesting that these ß-glucan-specific mAbs could be useful in combinatorial antifungal therapy.

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping.

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies.

Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan.

A panel of specific monoclonal antibodies (mAbs) against synthetic pentasaccharide ß-D-Galf-(1→5)-[ß-D-Galf-(1→5)]3-α-D-Manp, structurally related to Aspergillus fumigatus galactomannan, was generated using mice immunized with synthetic pentasaccharide-BSA conjugate and by hybridoma technology. Two selected mAbs, 7B8 and 8G4, could bind with the initial pentasaccharide with affinity constants of approximately 5.3 nM and 6.4 nM, respectively, based on surface plasmon resonance-based biosensor assay. The glycoarray, built from a series of synthetic oligosaccharide derivatives representing different galactomannan fragments, demonstrated that mAb 8G4 could effectively recognize the parental pentasaccharide while mAb 7B8 recognizes its constituting trisaccharide parts. Immunofluorescence studies showed that both 7B8 and 8G4 could stain A. fumigatus cells in culture efficiently, but not the mutant strain lacking galactomannan. In addition, confocal microscopy demonstrated that Candida albicans, Bifidobacterium longum, Lactobacillus plantarum, and numerous gram-positive and gram-negative bacteria were not labeled by mAbs 7B8 and 8G4. The generated mAbs can be considered promising for the development of a new specific enzyme-linked assay for detection of A. fumigatus, which is highly demanded for medical and environmental controls.

A protective chimeric antibody to tick-borne encephalitis virus.

The efficiency of several mouse monoclonal antibodies (mAbs) specific to the tick-borne encephalitis virus (TBEV) glycoprotein E in post-exposure prophylaxis was assessed, and mAb14D5 was shown to be the most active of all those studied. It was proven that the hybridoma cell line 14D5 produced one immunoglobulin H chain and two L chains. They were used to construct chimeric antibodies ch14D5a and ch14D5b, the affinity constants of which were 2.6 × 10(10)M(-1) and 1.0 × 10(7)M(-1), respectively, according to the SPR-based ProteOn biosensor assay. The neutralization index (IC50) of ch14D5a was 0.04 µg/ml in the focus reduction neutralization test. In in vivo experiments, ch14D5a at a dose of 10 µg/mouse resulted in a 100% survival of the mice infected with 240 LD50 of TBEV. This chimeric antibody is promising for further development of prevention and therapeutic drugs against TBEV.

Chimeric antibodies against tick-borne encephalitis virus.

Two chimeric antibodies (ch) 13D6 and 10C2 against the glycoprotein E of tick-borne encephalitis virus (TBEV) were constructed by fusing variable regions of murine monoclonal antibodies (Mabs) 13D6 and 10C2 to human constant regions. Monovalent analogues of these antibodies in format of single-chain antibodies (scFv or sc) were developed, as well. The ch13D6, ch10C2, sc13D6 and sc10C2 exhibited binding characteristics similar to parental Mabs. Only the ch13D6 and sc13D6 were able to neutralize TBEV infectivity in vitro. The in vitro neutralization provided by ch13D6 suggests that this antibody can be further developed into a potent prophylaxis and therapy for tick-borne encephalitis (TBE) infection.