Pooled sera per group were 500-fold diluted and used in IPMA to immunostain BSR monolayers infected with each of the nine reference AHSV strains. As expected, guinea pig sera raised against single VP2 proteins immunostained monolayers infected with the homologous AHSV serotype (Table 2). Similar to cross-neutralization of genetically related AHSV serotypes, some monolayers infected AT13387 clinical trial with genetically related AHSV serotypes were also immunostained. In contrast to the cross-neutralization results (Table 1), AHSV-6 was not recognized by α-AHSV-9 VP2 serum (Table 2). In addition to immunostaining of genetically related
AHSV serotypes, some unrelated AHSV reference strains were also recognized in IPMA; e.g. AHSV-8 was recognized not only by α-VP2 sera of AHSV-5 and -8 but also by AHSV-4. AHSV-5 was also recognized by α-VP2 of AHSV-3. In general this immunostaining was weaker than for the respective homologous AHSV serotype (Table 2). VP2 protein of orbiviruses is the major Z-VAD-FMK order determinant of
eliciting nAbs and has been used as recombinant protein-based vaccine in previous studies , , ,  and . Particularly, VP2 of AHSV serotype 4 has been studied extensively by European research groups, as the last European AHS outbreak was caused by this serotype . In this report we studied the immunogenicity of VP2 proteins of all nine AHSV serotypes as a first step in the development of AHS subunit vaccines. This is the first report to show that VP2 of all nine AHSV serotypes
induce serotype specific nAbs with slight cross-neutralizing antibodies. The baculovirus expression system was used to produce recombinant VP2 protein of all nine serotypes for induction of nAbs. Further, some VP2 genes were optimized to increase protein expression. Still, quantities of soluble VP2 significantly varied between the different serotypes. Since it is generally known that recombinant VP2 protein of orbivirus is highly Sodium butyrate insoluble, it is likely that quantities of soluble VP2 proteins vary by differences in expression or solubility . VP2 proteins of each AHSV serotype were produced in insect cells and each induced detectable nAb titers in guinea pigs as an alternative animal model. Previously, purified AHSV VP2 seemed to be less immunogenic in rabbits , but as little as 5 μg of VP2 protein in insect cell lysate could protect horses from AHS by induction of nAbs . In this study, guinea pigs were immunized with insect cell lysate containing 50 μg of VP2 to elicit detectable antibodies. Each VP2 elicited serotype specific Abs, but nAb titers varied considerably among different AHSV serotypes, from 37 for AHSV-2 to 1365 for AHSV-6. Further, cross-neutralization antibodies between genetically related serotypes were detected, but most of those cross-neutralizing Abs titers were considerably lower than for the respective serotype. Moreover, some expected cross-reactive nAbs were not detected.