Antigenic Profile of African Horse Sickness Virus Serotype 4 VP5 and Identification of a Neutralizing Epitope Shared with Bluetongue Virus and Epizootic Hemorrhagic Disease Virus

Abstract

African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coli using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes.

Expression and Functional Characterization of Bluetongue Virus VP5 Protein: Role in Cellular Permeabilization

“…1A, compare lane 2 to lane 3) and was identified as VP5 by Western blotting (Fig. 1B) with the anti-VP5 MAb 10AE12 (46). GST-VP5 was purified from extracts by glutathione affinity chromatography (Fig.…”

A Modified Vaccinia Ankara Virus (MVA) Vaccine Expressing African Horse Sickness Virus (AHSV) VP2 Protects Against AHSV Challenge in an IFNAR −/− Mouse Model

“…We have previously demonstrated induction of VN antibodies in ponies vaccinated with the same recombinant virus [19] and others have succeeded in protecting horses against AHS using VP2 based vaccines, either in the form of baculovirus expressed protein [14], [16], [17] or pox-virus vectors [18], [29]. VP2 is the main target of neutralising antibodies on AHSV, although neutralising epitopes have also been identified on VP5 of AHS virus serotype 4 [30] and this protein seems to exert some conformational effect on VP2 epitopes. There are BTV and AHSV vaccination studies showing that co-expression of both outer capsid proteins induces a more effective protective immunity than either protein alone.…”

Adaptive strategies of African horse sickness virus to facilitate vector transmission

“…As a result, the genes encoding these proteins show substantial genetic variation, and the VP2 gene is the most variable segment of the orbivirus genome [49, 50]. Although VP2 is thought to contain most of the major serotype-determining antigens of AHSV, VP5 can also induce low levels of neutralising antibodies in the absence of VP2 [54]. …”