Characterization and expression of domains of Alphaherpesvirus bovine 1/5 envelope glycoproteins B in Komagataella phaffi

Abstract

Background Bovine herpes virus (BoHV 1 and BoHV-5) are the causative agents of infectious bovine rhinotracheitis (IBR). IBR is responsible for important economic losses in the cattle industry. The envelope glycoprotein B (gB) is essential for BoHV infection of cattle's upper respiratory and genital tract. gB is one of the main candidate antigens for a potential recombinant vaccine since it induces a strong and persistent immune response. Results In this study, gB of BoHV-1 and BoHV-5 was characterized in terms of function, structure, and antigenicity through bioinformatics tools. gB showed conserved sequence and structure, so, both domains named PH Like 1 and 2 domains of each virus were selected for the design of a bivalent vaccine candidate. The immunoinformatic study showed that these two domains have epitopes recognizable by B and T lymphocytes, followed by this, the cDNA domains from BoHV-1/5 gB (Domains-gB) were transformed into the yeast Komagataella phaffii GS115 (previously known as Pichia pastoris). A recombinant protein with molecular weight of about 110 kDa was obtained from the culture media. The vaccine candidate protein (Domains-gB) was recognized by a monoclonal antibody from a commercial ELISA kit used for IBR diagnostic, which may suggest that the epitopes are conserved of the entire infectious virus. Conclusion Overall, it was shown that the recombinant domains of BoHV-1/5 gB have antigenic and immunogenic properties similar to the native gB. This vaccine candidate is promising to be used in future studies to assess its immunogenicity in an animal model.

Humoral Immune Response of Mice against a Vaccine Candidate Composed of a Chimera of gB of Bovine Alphaherpesviruses 1 and 5

“…Considering previous studies and the different problems with other prokaryotic organisms [ 9 , 10 , 11 ] the yeast K. phaffii was selected as a host for the recombinant production of the vaccine candidate “gB Domains”. At shaker scale, the expression of the protein of interest was confirmed, which was efficiently purified through affinity chromatography with a final concentration of 873 µg/mL of the gB domains at the shaker with four cultures of 500 mL [ 21 ]. Other studies demonstrated lower purification yields for BoHV glycoproteins using K. phaffii expression of 190 µg/mL or less [ 9 ].…”

“…The higher antibody titers against BoHV-5 compared to BoHV-1 might be explained by the higher scores of possible epitopes recognized by B and T lymphocytes for the PH-domain-like 2 of BoHV-5, as observed when we applied immune-informatics approaches, where scores ranged from 8 to 10, with 10 being the maximum possible score for recognizable B lymphocyte conformational and lineal epitopes [ 21 ].…”

“…The chimera gB vaccine candidate, composed of PH-like domain 1 from BoHV-1 and PH-like domain 2 from BoHV-5 (hereafter DgB), was expressed in Komagataella phaffii GS115 and produced as previously described [ 21 , 22 , 23 ]. After induction phase, the recombinant protein was concentrated from the culture supernatant by using Centriprep 50YM (Millipore, Burlington, MA, USA) and purified by affinity chromatography using both HisTrap™ HP 1 mL columns pre-packed with pre-charged Ni Sepharose™ and the ÄKTAprime™ Automated Liquid Chromatography system (GE Healthcare, Chicago, IL, USA).…”

“…Looking for a product capable of increasing protection time with as little administration as possible, we performed the immune-informatic characterization of a chimeric protein composed of glycoprotein B domains from BoHV-1 and BoHV-5, obtaining high scores in the different searches for epitopes recognized by B and T lymphocytes. This vaccine candidate was produced by recombinant techniques in the yeast Komagataella phaffii , which was recognized by a monoclonal antibody from a commercial ELISA kit used for IBR diagnostics, suggesting that the epitopes are conserved among the entire infectious virus [ 21 ]. Due to its ease and lower cost, yeasts are the main candidates for the production of glycoprotein subunits as vaccine candidates; in addition to this, being a eukaryotic organism, post-translational rearrangements such as disulfide bridges, glycosylations, foldings, etc., are important for the antigenicity of the candidate [ 9 , 11 , 21 ].…”

“…This vaccine candidate was produced by recombinant techniques in the yeast Komagataella phaffii , which was recognized by a monoclonal antibody from a commercial ELISA kit used for IBR diagnostics, suggesting that the epitopes are conserved among the entire infectious virus [ 21 ]. Due to its ease and lower cost, yeasts are the main candidates for the production of glycoprotein subunits as vaccine candidates; in addition to this, being a eukaryotic organism, post-translational rearrangements such as disulfide bridges, glycosylations, foldings, etc., are important for the antigenicity of the candidate [ 9 , 11 , 21 ].…”