Chimeric Antibody 14D5 Protects Mice against the Far-Eastern, Siberian, and European Tick-borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV), belonging to the Flaviviridae family, is the most significant pathogen transmitted by Ixodes ticks and causing one of the most severe human neuroinfections. In Russia, serum immunoglobulin produced from the donor blood is currently used for post-exposure prophylactic and therapy of tick-borne encephalitis virus. However, it is known that preparations obtained from donated blood have certain disadvantages, and therefore development of novel preparations for post exposure prophylaxis and therapy of tick-borne encephalitis is required. To develop an alternative preparation, which does not include donor blood, a chimeric antibody ch14D5 against glycoprotein E of TBEV was constructed.This study was aimed to investigate protective efficacy of the chimeric antibody ch14D5 against the Far-Eastern, Siberian, and European subtypes of TBEV in in vivo experiments.A peripheral mouse model of tick-borne encephalitis was used in this study: the chimeric antibody ch14D5 was administrated intravenously in mice one day after their intraperitoneal infection with TBEV strains Sofjin, Vasilchenko, and Absettarov. Anti-TBEV serum immunoglobulin was used as a control preparation, which was administered in the same way. Protective efficacy of the chimeric antibodies 14D5 was assessed using the log-rank test. In the study, the presence or absence of antibody-dependent enhancement of infection (ADE) was examined when mice, infected with different subtypes of the TBEV, got the antibody ch14d5.Obtained results demonstrated high efficacy of the ch14D5 antibody in post-exposure prophylaxis of the disease in mice infected with any of the used TBEV strains, as well as the absence of ADE.It was shown that protective efficacy of antibody ch14D5 is higher than that of the anti-TBEV serum immunoglobulin, and antibody ch14D5 could be used for development of a therapeutic preparation for post-exposure prophylaxis

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

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 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

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

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

Specific binding of mAbs 7B8 and 8G4 with <i>A</i>. <i>fumigatus</i>, <i>A</i>. <i>flavus</i>, and <i>C</i>. <i>albicans</i>.

<p>Cells were grown in Sabouraud broth, fixed, and incubated with mAbs 7B8 and 8G4. Binding of mAbs with fungal cells was detected with Alexa Fluor 488-conjugated anti-mouse IgG antibody staining in (A) confocal microscopy, and (B) DIC microscopy. Scale bar = 10 μm.</p