However, VLPs are thought to be relatively unstable see more and have a limited shelf life. Other experimental subunit-vaccines for BTV include vectored-virus vaccines such as modified vaccinia Ankara (MVA), capripox virus, canarypox virus, bovine herpes virus, equine herpesvirus or myxomavirus [43], [44], [47], [48], [49], [50], [51], [52], [53] and [54]. However, simple bacterial expression

systems have not been fully explored, due to difficulties generating larger BTV proteins (such as VP2 ∼112 kDa) in a native and soluble form for use as subunit-vaccine antigens [55]. Previous findings suggested that VP2 of BTV (∼110 kDa), evolved through duplication and may therefore exist as two related domains, VP2D1 and VP2D2

[18]. Sera from Balb/c mice immunised with the soluble recombinant VP2D1 of BTV-4, neutralised Akt inhibitor the homologous virus, while significantly lower NAb titres were observed with sera of mice immunised with soluble VP2D2. This suggests that the majority of the dominant neutralising epitopes are located in the amino terminal half of VP2. However, when both domains were mixed together on an equimolar basis, higher titres of neutralising antibodies were elicited. There is published evidence that neutralisation epitopes are located in the first ∼350 amino acids (domain 1) of VP2 of BTV-10 [56]. IFNAR−/− mice immunised with VP2D1 + VP2D2 and challenged with live BTV-4 survived until the end of the experiment with a transient viraemia (∼0.3–9 pfu/ml detected by RT-PCR only) which was cleared subsequently. It was not possible to isolate virus in cell cultures from these blood samples, potentially reflecting presence of neutralising antibodies. many The CAPS-denatured (from insoluble fraction) VP2 domains did not raise any neutralising antibody response as compared to the soluble domains in bacteria. This strongly suggests that at least some neutralisation epitopes are conformational, which have been lost by dissolving the insoluble VP2 domains in a detergent such as CAPS. Several studies identified linear epitopes in VP2 which are serotype specific, some of which when used in the form

of peptides prevented virus neutralisation [57], [58] and [59]. Although BTV-VP2 is the primary determinant of serotype, the smaller outer capsid protein VP5, stimulates the neutralisation response, possibly through interactions with VP2 in the virus capsid [14] and [15]. Mice vaccinated with a combination of expressed VP5Δ1–100 and VP2 domains of BTV-4, generated higher neutralising antibody titres (P < 0.05) (against BTV-4, but not BTV-8) and delayed the transient viraemia (detected by RT-PCR, while no virus could be isolated by KC or BSR cell cultures) observed in some animals after homologous challenge than mice vaccinated with VP2 domains alone. However, addition of VP5 did not have significant differences in terms of protection.