New p35 (H3L) Epitope Involved in Vaccinia Virus Neutralization and Its Deimmunization

Vaccinia virus (VACV) is a promising oncolytic agent because it exhibits many characteristic features of an oncolytic virus. However, its effectiveness is limited by the strong antiviral immune response induced by this virus. One possible approach to overcome this limitation is to develop deimmunized recombinant VACV. It is known that VACV p35 is a major protein for B- and T-cell immune response. Despite the relevance of p35, its epitope structure remains insufficiently studied. To determine neutralizing epitopes, a panel of recombinant p35 variants was designed, expressed, and used for mice immunization. Plaque-reduction neutralization tests demonstrated that VACV was only neutralized by sera from mice that were immunized with variants containing both N- and C- terminal regions of p35. This result was confirmed by the depletion of anti-p35 mice sera with recombinant p35 variants. At least nine amino acid residues affecting the immunogenic profile of p35 were identified. Substitutions of seven residues led to disruption of B-cell epitopes, whereas substitutions of two residues resulted in the recognition of the mutant p35 solely by non-neutralizing antibodies

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

Cross-Reactive Antibodies to the NS1 Protein of Omsk Hemorrhagic Fever Virus Are Absent in the Sera of Patients with Tick-Borne Encephalitis

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis virus (TBEV) complex of the Flaviviridae family. Currently, there are no data on the cross-reactivity of antibodies to the NS1 proteins of OHFV and TBEV. Such data are of major interest for monitoring viral encephalitis of unknown etiology due to the increasing geographical distribution of OHFV. In this study, a recombinant OHFV NS1 protein was produced using the Escherichia coli expression system and purified. The recombinant OHFV NS1 protein was recognized by specific mice immune ascetic fluids to the native OHFV NS1 protein. A Western blot analysis and ELISA of the recombinant NS1 proteins of OHFV and TBEV were used to study the cross-reactivity of antibodies from immune ascites fluid obtained from OHFV-infected mice and mAbs against TBEV NS1. Anti-TBEV NS1 mouse monoclonal antibodies (mAbs) have been shown to not be cross-reactive to the OHFV NS1 protein. Sera from patients with confirmed tick-borne encephalitis (TBE) were examined by ELISA using recombinant OHFV NS1 and TBEV NS1 proteins as antigens. It was shown for the first time that cross-reactive antibodies to the OHFV NS1 protein were not detected in the sera of TBE patients, whereas the sera contained antibodies to the TBEV NS1 protein

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

International audienceβ-(1→3)-D-Glucan is an essential component of the fungal cell wall. Mouse monoclonal antibodies (mAbs) against synthetic nona-β-(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-β-(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 β-glucans with different lengths of oligo-β-(1→3)-D-glucoside chains, demonstrated that linear tri-, penta- and nonaglucoside, as well as a β-(1→6)-branched octasaccharide, were recognized by mAb 5H5. By contrast, only linear oligo-β-(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 β-glucan-specific mAbs could be useful in combinatorial antifungal therapy

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

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

Novel mouse monoclonal antibodies specifically recognize <i>Aspergillus fumigatus</i> galactomannan

<div><p>A panel of specific monoclonal antibodies (mAbs) against synthetic pentasaccharide β-D-Gal<i>f</i>-(1→5)-[β-D-Gal<i>f</i>-(1→5)]₃-α-D-Man<i>p</i>, structurally related to <i>Aspergillus fumigatus</i> 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 <i>A</i>. <i>fumigatus</i> cells in culture efficiently, but not the mutant strain lacking galactomannan. In addition, confocal microscopy demonstrated that <i>Candida albicans</i>, <i>Bifidobacterium longum</i>, <i>Lactobacillus plantarum</i>, 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 <i>A</i>. <i>fumigatus</i>, which is highly demanded for medical and environmental controls.</p></div

Binding of fungal and bacterial cultures with mAbs 7B8 and 8G4.

<p>(A) Sandwich enzyme-linked immunosorbent assay (ELISA) with 7B8 mAb: the wells of microtiter plates were coated with 7B8 mAb and incubated with serial dilutions of microbial supernatants; ELISA was performed with horseradish peroxidase-conjugated 7B8 mAb. (B) Sandwich ELISA with 8G4 mAb: the wells of microtiter plates were coated with 8G4 mAb and incubated with serial dilutions of microbial supernatants; ELISA was performed with horseradish peroxidase-conjugated 8G4 mAb.</p

Investigation of oligosaccharide specificity of mAbs 7B8 and 8G4 using ELISA.

<p>(A) Composition of thematic glycoarray built using oligosaccharide ligands representing key structural elements of <i>A</i>. <i>fumigatus</i> galactomannan chain, and (B) assay for carbohydrate specificity of 7B8 and 8G4 mAbs.</p

Structure of <i>Aspergillus fumigatus</i> galactomannan and its synthetic analogs.

<p>(A) Structural fragments of <i>A</i>. <i>fumigatus</i> galactomannan (summarized from refs. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193938#pone.0193938.ref006" target="_blank">6</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193938#pone.0193938.ref008" target="_blank">8</a>]). (B) Pentasaccharide <b>GM-1</b> and its BSA <b>(GM-1-BSA)</b> and biotinylated <b>(GM-1-Biot)</b> conjugates used in mice immunization and mAb screening. The carbohydrate sequences are represented according to symbol carbohydrate nomenclature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193938#pone.0193938.ref026" target="_blank">26</a>].</p