Construção, caracterização, antigenicidade e imunogenicidade de proteína multiepítopo dos Alphaherpesvirus bovinos 1 e 5

Abstract

Bovine herpesviruses are significant microorganisms responsible for respiratory, reproductive, and encephalic disorders that continue to affect cattle farming worldwide, with potential impacts on buffaloes, sheep, and goats as well. Vaccination is the most effective method for controlling and reducing diseases caused by these microorganisms. In this context, various vaccine platforms have been developed over the years to achieve this goal. The objective of this study was the development of a multiepitope protein derived from the union of immunodominant epitopes of glycoproteins B, C, and D of BoAHVs 1 and 5, and its evaluation for use in diagnostic tests and vaccination of cattle under field conditions. Initially, the retrieval (NCBI) and curation (MUSCLE and BLASTp) of the glycoprotein sequences of the target organisms were performed, followed by the prediction of epitopes recognized by B lymphocytes (BepiPred 2.0), cytotoxic T cells (NetMHCpan EL 4.1), and helper T cells (NetMHCIIpan 4.0). Subsequently, after the antigenicity analysis (VaxiJen 2.0), the multiepitope protein, which would serve as the basis for the multiepitope vaccine, was constructed using GGGGS and EAAAK linkers between epitopes of the same glycoprotein or between epitopes of different glycoproteins, respectively. Following this, analyses of physicochemical parameters and allergenicity profiles were conducted using ProtParam and AlgPred servers, respectively, followed by another antigenicity analysis (VaxiJen 2.0). Protein modeling and molecular docking were performed using the I-TASSER and ClusPro servers, respectively, with the bovine TLR-7 receptor model obtained from the AlphaFoldDB database. For the expression of the multiepitope protein, the pET24a vector containing the gene of interest was used to transform the E. coli BL21 (DE3) Star strain via heat shock. Cultivation was carried out in 500 mL of liquid LB medium under optimal conditions for growth and expression of the multiepitope protein. The sample was then processed and subsequently purified using affinity chromatography on an ÄKTA Purifier system, followed by dialysis and quantification using the BCA method. Characterization was performed through Western blotting, and antigenicity was evaluated via indirect ELISA using an in-house protocol. Immunogenicity was assessed by immunizing five cattle with 400 µg of the multiepitope protein conjugated with Montanide™ ISA 50 V2 adjuvant, administered intramuscularly (multiepitope vaccine). The placebo group was inoculated with phosphate-buffered saline (PBS). To determine total IgG antibody levels, an indirect ELISA test using an in-house protocol was employed. The multiepitope protein consists of 321 amino acids, has a molecular weight of 33.24 kDa, a theoretical pI of 9.88, an estimated half-life in E. coli of >10 hours, a GRAVY score of -0.507, antigenicity of 1.1543, and is characterized as unstable and non-allergenic. The multiepitope protein was also capable of interacting with a high binding energy (-1264.7 kcal/mol) with 88 cluster members. Its soluble and insoluble fractions were recognized by anti-HIS6x antibodies in a Western blotting assay, confirming its molecular weight of approximately ~33 kDa. The multiepitope protein was strongly recognized by IgG anti-Alphaherpesvirus antibodies from bovine sera, demonstrating its antigenicity. The multiepitope vaccine successfully induced a humoral immune response at the total IgG level. The multiepitope protein can be employed in diagnostic tests and vaccines against bovine Alphaherpesviruses 1 and 5.