Corrigendum: Development of Foot-and-Mouth Disease Virus-Neutralizing Monoclonal Antibodies Derived From Plasmablasts of Infected Cattle and Their Germline Gene Usage.

[This corrects the article DOI: 10.3389/fimmu.2019.02870.].

Development of Foot-and-Mouth Disease Virus-Neutralizing Monoclonal Antibodies Derived From Plasmablasts of Infected Cattle and Their Germline Gene Usage.

Cattle are susceptible to foot-and-mouth disease virus (FMDV), and neutralizing antibodies are critical for protection against FMDV infection in this species. However, more information is needed on the host specific antigenic structure recognized by the FMDV-specific monoclonal antibodies (mAbs) and on the functional properties of the mAb that are produced in the natural host, cattle. Herein, we characterized 55 plasmablast-derived mAbs from three FMDV-infected cattle and obtained 28 FMDV-neutralizing antibodies by the single B cell antibody technique. The neutralizing mAbs (27/28) mainly recognized conformational epitopes that differ from the well-characterized immunodominant antigenic site 1 of FMDV as defined by murine mAbs. Of these FMDV-neutralizing mAbs, 13 mAbs showed intra-type broadly neutralizing activity against the three topotypes of FMDV serotype O (ME-SA, SEA, and Cathay topotypes). Moreover, all these intra-type broadly neutralizing antibodies competed with sera from FMDV infected or vaccinated cattle, which indicates their binding to native dominant epitopes, as revealed by a blocking ELISA. We further analyzed the germline V(D)J gene usage of the 55 FMDV-specific mAbs and found cattle IgG antibodies containing ultralong HCDR3 were exclusively restricted to usage of the germline gene segment VH 1-7*02. In addition, the restricted germline gene segments of VH 1-7*02 and VL1-47*01 or 1-52*01 pairing were observed in all IgG antibodies with ultralong HCDR3. Furthermore, antibodies with longer HCDR3 were more inclined to display FMDV-neutralizing activity. This study presents a novel method for screening FMDV-specific cattle mAbs which then provide the most useful tools for studying FMDV antigenic structure and variation.

A scale-down model of 4000-L cell culture process for inactivated foot-and-mouth disease vaccine production.

The anticipated increasing demand for inactivated foot-and-mouth (FMD) disease vaccine calls for its larger production capacity, while development of a large-scale process typically requires high running cost and has very limited experimental throughput at manufacturing scale. Thus, an economic scale-down model of representing a large-scale process becomes necessary and essential. In this study, we used a systematic approach to establish a scale-down model representing a 4000-L culture process for FMD vaccine production by suspension BHK-21 cells. In detail, we firstly compared hydrodynamic properties of three bioreactors (14-L, 800-L and 4000-L) under three different conditions (equivalent mixing time, equivalent shear stress and equivalent volumetric power). We figured out equivalent volumetric power (P/V) potentially as an appropriate scale-down strategy, since it resulted in comparable calculated hydrodynamic parameters among three bioreactors. Next, we used computational fluid dynamics (CFD) simulation to provide more details about hydrodynamic environments inside the bioreactors, which supports the reliability of this scale-down strategy. Finally, we compared cell growth, metabolites, vaccine productivity and product quality attributes during FMD vaccine production by BHK-21 cells and observed very close performances among three bioreactors, which once again demonstrates the robustness of this scale-down model. This scale-down strategy can be applied to study variations and critical quality attributes (CQAs) in the resultant production process based on quality by design (QbD) principles, aiming at further more efficient optimization of vaccine production.

Quality by Design-Driven Process Development of Cell Culture in Bioreactor for the Production of Foot-And-Mouth Veterinary Vaccine.

Quality by design (QbD) principle has been established as a guideline to emphasize the understanding of the relationship of product quality with process control. Vaccine product have characteristics of security and high efficiency, but it also has features such as complexity and rigorous regulatory for production. This case study describes an example of QbD-driven process development for manufacturing a veterinary vaccine produced with baby hamster kidney-21 cells. The study revealed that cell culture duration was the most significant factor affecting 50% tissue culture infectious doses (TCID50) and antigenic titer, and the factors of culture temperature and pH at infection phase exhibited less effect. Culture temperature at infection phase was the only significant factor for total protein. Through the Monte Carlo simulation, the design spaces of process parameters were determined. Meanwhile, the excellent and robust performance in manufacturing scale (4000-L) validated the effectiveness of this strategy. A reliable and robust multivariate process parameter range, that is, design space, was identified by this systematic approach. Our investigation presents a successful case of QbD principle, which encourages other researchers to combine the methodology into other biopharmaceutical manufacturing process.

[Screening and stability of Madin-Darby bovine kidney cell strain co-expressing the capsid precursor protein P1-2A gene and the protease 3C gene of foot-and-mouth disease virus].

OBJECTIVE: To screen the Madin-Darby bovine kidney [CO1] cell strains for stable expression of capsid protein of foot-and-mouth disease virus (FMDV [CO2]). METHODS: We obtained two genes coding for capsid precursor protein (P1-2A) and protease (3C) of FMDV by PCR from recombinant plasmids pMD-P1-2A and pMD-3C.The recombinant retroviral vector pBABE-puro/P1-2A-3C was constructed by inserting P1-2A gene and 3C gene into pBABE-puro. Both the recombinant plasmid pBABE-puro/P1-2A-3C and the plasmid pVSV-G were co-transfected into packaging cells GP2-293 by liposome-mediated transfection method. As a result, the recombinant pseudovirus was produced. The pseudovirus infected the interesting target cell MDBK. The infected MDBK cells were selected by puromycin (2.5 tg/mL) for 2 weeks. The monoclonal cells were selected using cloning rings. The expression of capsid protein was detected by indirect immunofluorescence and sandwich-ELISA. The empty capsids of FMDV were observed under electron microscope. RESULTS: The capsid protein of FMDV was expressed in MDBK cells. The expression levels in screened cell strains of various passages showed no significant difference. CONCLUSION: The MDBK cell strains for stable expression capsid protein of FMDV were successfully screened, which laid a foundation of development of FMDV subunit vaccine.