Induction of antibody responses to African horse sickness virus (AHSV) in ponies after vaccination with recombinant modified vaccinia Ankara (MVA)

Abstract

Background: African horse sickness virus (AHSV) causes a non-contagious, infectious disease in equids, with mortality rates that can exceed 90% in susceptible horse populations. AHSV vaccines play a crucial role in the control of the disease; however, there are concerns over the use of polyvalent live attenuated vaccines particularly in areas where AHSV is not endemic. Therefore, it is important to consider alternative approaches for AHSV vaccine development. We have carried out a pilot study to investigate the ability of recombinant modified vaccinia Ankara (MVA) vaccines expressing VP2, VP7 or NS3 genes of AHSV to stimulate immune responses against AHSV antigens in the horse.

Methodology/principal findings: VP2, VP7 and NS3 genes from AHSV-4/Madrid87 were cloned into the vaccinia transfer vector pSC11 and recombinant MVA viruses generated. Antigen expression or transcription of the AHSV genes from cells infected with the recombinant viruses was confirmed. Pairs of ponies were vaccinated with MVAVP2, MVAVP7 or MVANS3 and both MVA vector and AHSV antigen-specific antibody responses were analysed. Vaccination with MVAVP2 induced a strong AHSV neutralising antibody response (VN titre up to a value of 2). MVAVP7 also induced AHSV antigen-specific responses, detected by western blotting. NS3 specific antibody responses were not detected.

Conclusions: This pilot study demonstrates the immunogenicity of recombinant MVA vectored AHSV vaccines, in particular MVAVP2, and indicates that further work to investigate whether these vaccines would confer protection from lethal AHSV challenge in the horse is justifiable.

Figure 1. Detection of MVAVP2 or MVAVP7-expressed VP2 and VP7 protein, respectively, within QT35 and ESF cells.

Cell lysates of uninfected cells (lanes 1,3,5 and 7) and cells infected at high MOI with MVA-VP2 (lanes 2 and 4) or MVA-VP7 (lanes 6 and 8), and harvested at 24 hours post-infection, were separated by SDS-PAGE on 10% gels. Immunoblotting was conducted with either anti-VP2 mAb (lanes 1–4) or anti-VP7 mAb (lanes 5–8).

Figure 2. Detection of NS3 RNA transcripts from MVANS3.

1% agarose gels showing RT-PCR and PCR products using RNA templates extracted from MVA infected QT35 and ESF cells 4 hours post infection. Tracks 1–3 QT35 RNA extract RT-PCR products. Tracks 4–6 QT35 RNA extract PCR products. Tracks 7–9 ESF RNA extract RT-PCR products. Tracks 10–12 ESF RNA extract PCR products. Expected product size 670 bp. Tracks 1, 4, 7, & 10 MVANS3; 2, 5, 8, & 11 MVA wild type; 3, 6, 9, & 12 pSC11NS3 plasmid.

Figure 3. Development of neutralising antibodies against MVA following vaccination.

MVA plaque reduction neutralisation titre of sera taken from ponies following initial vaccination with recombinant MVA and two subsequent boosts. Arrows denote days of vaccination.

Figure 4. Detection of immunoprecipitated, recombinant-baculovirus-expressed VP2V5 with MVAVP2-vaccinated pony serum.

Lysates of uninfected Sf9 cells (lanes 1,3,5,7 and 9) and Sf9 cells infected with recombinant baculovirus FBVP2-V5 were immunoprecipitated with anti-V5tag mAb and Protein G agarose. The immunoprecipitates were separated by SDS-PAGE on 10% gels, and immunoblotted with MVAVP2 vaccinated pony sera (4256, 5483) (lanes 1–8) or anti-V5tag mAb (lanes 9 & 10). The pony sera tested were derived from a pre-vaccination control bleed (lanes 1 & 2) and three post-vaccination bleeds (lanes 3–8) from days 21, 42, and 84, respectively. A non-specific band was present to some extent in each lane at ∼50 KDa.

Figure 5. Detection of recombinant baculovirus-expressed VP7 with MVA-VP7-vaccinated pony serum.

A semi-purified preparation of recombinant baculovirus FBVP7-expressed VP7 was separated by by SDS-PAGE on 10% gels, and immunoblotted with MVA-VP7-vaccinated pony sera (lanes 1–4) or anti-VP7 mAb (lane 5). The pony sera tested were derived from a pre-vaccination control bleed (lane 1) and three post-vaccination bleeds (lanes 2–4) from days 21, 42, and 84, respectively.