Dominant antigenic sites inducing serotype specific neutralizing SB431542 molecular weight antibodies (nAbs) are mainly located on VP2, however, other structural and non-structural proteins – VP3, VP5, VP7, NS1 and NS2 – also induce humoral and cellular immune responses [4], [5], [6], [7], [8] and [9]. Since there is no successful treatment for AHS, vaccination is the most important approach to protect horses against AHS. Live-attenuated vaccines (LAVs) obtained by serial passages of AHSV in cell culture are available commercially for most serotypes in South Africa [1]. Although LAVs have been extensively used in South Africa and

other African countries, there are still concerns as LAVs cause viremia and could be transmitted by midges. However, the biggest concern of using these vaccines is reassortment between LAVs or

with wild type AHSV, which could result in more pathogenic virus variants. Moreover, the recent outbreak of AHSV serotype 9 in Gambia is suspected to be derived from vaccine strains [10]. Currently, LAVs are not licensed in Europe. To overcome safety issues, alternative AHS vaccines are under Akt phosphorylation development including inactivated virus, recombinant VP2, DNA vaccine and vaccinia virus vectors expressing VP2 protein [11], [12], [13], [14], [15], [16], [17], [18] and [19]. Outer capsid protein VP2 of orbiviruses determines the serotype and is the main target of nAbs [20], [21], [22] and [23]. Vaccination with recombinant VP2 of AHSV serotype 4, 5 or 9 has been reported to induce nAbs and protect horses against homologous AHSV challenge infection [13], [14], [16], [18], [19], [22] and [24]. To date, there are no reports regarding the immunogenicity of VP2 proteins of other serotypes of AHSV. In this report, VP2 of all nine AHSV serotypes were produced individually using the baculovirus expression system and their immunogenic for activities were investigated by immunization of guinea pigs, singly or in cocktail mixtures. The results demonstrated that

recombinant VP2 proteins of all nine AHSV serotypes have the potential to be used as safe subunit vaccines for AHS either individually or in a multi-serotype cocktail. AHSV reference strains (obtained from ANSES, France) were passaged and amplified in BSR cells, a derivative of the BHK-21 cell line, in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (Invitrogen). Virus titers were determined by a plaque-forming assay in BSR cells and defined as plaque forming units per ml (pfu/ml) as described [25]. Insect cell lines of Spodoptera frugiperda, Sf9 and Sf21, were cultured at 28 °C in Insect-Xpress (Lonza, Basel, Switzerland) and TC100 medium (Biochrom AG, Berlin, Germany), respectively. TC100 medium was supplemented with 10% fetal bovine serum.