Enhanced Bovine Herpesvirus Type 1 Neutralization by Multimerized Single-Chain Variable Antibody Fragments Regardless of Differential Glycosylation

ABSTRACTSingle-chain variable antibody fragments (scFvs) with a 2-amino-acid linker capable of multimerization as di-, tri-, or tetrabodies that neutralize bovine herpesvirus type 1 (BoHV-1)in vitrowere constructed and expressed inPichia pastoris. In contrast to the monomeric form, multimeric scFvs had a higher virus neutralization potency, as evidenced by a 2-fold increase in their ability to neutralize BoHV-1 due to avidity effects. Mass spectrum (quadrupole time of flight [Q-TOF]) analyses of multimeric scFv demonstrated extensive heterogeneity due to differential cleavage, variable glycosylation (1 to 9 mannose residues), and the incorporation of minor unidentified adducts. Regardless of the differential glycosylation patterns, the scFvs recognized non-gB or -gE target viral epitopes in the BoHV-1 envelope fraction in a Western blot and also neutralized BoHV-1 in infected Madin-Darby kidney (MDBK) cellsin vitro. Indirect evidence for the noncovalent multimerization of scFv was the presence of a major peak of multimerized scFv without a His tag (due to differential cleavage) in the Q-TOF profile, unlike monomeric scFv, which copurified with normally His-tagged scFv and recognized the target antigen. Overall, differentially glycosylated recombinant scFvs against BoHV-1 with a short linker (2 amino acids) are capable of assembly into functional multimers that confer high avidity, resulting in increased virus neutralizationin vitrocompared to that of monovalent scFv with a long (18-amino-acid) flexible linker. Overall, recombinant multimerized scFv5-2L potentially provides a high-potency therapeutic and immunodiagnostic reagent against BoHV-1, which is suitable for passive immunization and topical application.

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Characterization of Monoclonal Antibodies against Bovine herpesvirus type 1 selected by Phage Display Technology

Semina Ciências Agrárias ◽

10.5433/1679-0359.2017v38n6p3915 ◽

2017 ◽

Vol 38 (6) ◽

pp. 3915

Author(s):

Greice Japolla ◽

Ana Flávia Batista Penido ◽

Greyciele Rodrigues Almeida ◽

Luiz Artur Mendes Bataus ◽

Jair Pereira Cunha Junior ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Phage Display ◽

Bovine Herpesvirus ◽

Single Chain ◽

Phage Display Technology ◽

Herpesvirus Type ◽

Wide Range ◽

Display Technology ◽

Bovine Herpesvirus Type 1

The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.

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2,3,7,8-tetrachlorodibenzo-p-dioxin increases bovine herpesvirus type-1 (BHV-1) replication in madin-darby bovine kidney (MDBK) cells in vitro

Journal of Cellular Biochemistry ◽

10.1002/jcb.21398 ◽

2007 ◽

Vol 103 (1) ◽

pp. 221-233 ◽

Cited By ~ 14

Author(s):

F. Fiorito ◽

U. Pagnini ◽

L. De Martino ◽

S. Montagnaro ◽

R. Ciarcia ◽

...

Keyword(s):

Bovine Herpesvirus ◽

Bovine Kidney ◽

Herpesvirus Type ◽

Mdbk Cells ◽

Bovine Herpesvirus Type 1 ◽

Tetrachlorodibenzo P Dioxin

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297 ASSESSMENT OF THE TRANSPORTATION OF BOVINE OOCYTES EXPERIMENTALLY EXPOSED TO BOVINE HERPESVIRUS TYPE 1 (BoHV-1) DURING THE PERIOD OF MATURATION IN IN VITRO OVIDUCTS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab297 ◽

2010 ◽

Vol 22 (1) ◽

pp. 305

Author(s):

M. M. Piccolomini ◽

M. L. Batista ◽

A. C. Góes ◽

D. L. Pavão ◽

M. F. Alves ◽

...

Keyword(s):

Los Angeles ◽

Bovine Herpesvirus ◽

Control Group ◽

Potential Hazard ◽

Herpesvirus Type ◽

Oocyte Recovery ◽

Significant Difference ◽

Bovine Herpesvirus Type 1

The necessity of greater elucidation about oocyte pathogen interactions made us examine the interaction of bovine herpesvirus type 1 (BoHV-1), Los Angeles strain, with oocytes matured in vitro to assess the potential hazard of transmission of such infectious agent through IVF. The process of capacitation and transportation of oocytes through the oviduct depends on the quality of the female gamete. Keeping in mind that the BoHV-1 causes morphophysiological modification in oocytes, the aim of this study was to evaluate, in an experimental model of in vitro oviduct, whether the pathogen interferes in the transportation of oocytes. This was determined using the percentage of oocyte recovery and assessment of the alteration in the morphology of the oviduct lumen. Oviducts and oocytes were collected from bovine ovaries derived from slaughterhouse cows. The oviducts were dissected, washed, and placed in Petri dishes (100 × 20 mm). On the previous portions of the infundibulum, in vitro-matured control oocytes or oocytes exposed to the virus (105.5 TCID50 mL-1) were introduced (20 oocytes per group) and then immersed in 100 mL of TCM-199 medium. Each dish was incubated at 38°C, 5% CO2, and 95% humidity for 24 h. Then, we introduced in the infundibulum, through intramammary infusion probe, 5 mL from a physiologic solution containing 1% of bovine fetal serum. Each oviduct effluent was collected separately and evaluated through a stereoscope for the recovery and counting of oocytes. Immediately after the recovery, both oviduct groups were sectioned longitudinally and observed through an optical microscope (100 ×) for a morphological evaluation of the luminar area. For the control group, the percentage of recovery was 14.9% (65/437), whereas in the infected group, the oocyte recovery was 23.4% (100/428). The statistical analysis was made according to Student’s t-test (P < 0.05), and presented a significant difference in the final results. The previous results show that the group of oocytes that was exposed to the virus presented a higher percentage of recovery compared with the control group. The oviducts that received the exposed oocytes presented areas where it seemed there has been cytopathic effect represented by dark and lumpy sections, in which there were round and degenerating cells mainly located in the oviducts’ peripheral area. The morphophysiological modifications in contaminated oocytes and oviducts probably complicate their transportation and also interferes in the process of their liberation. It is relevant to elucidate the importance of the studies above because of their participation in the recovery of these gametic cells in bovine oviducts, as well as the transmission during IVF processes. Vitrocel/Embriolife.

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