Development and evaluation of a bivalent Lumpy skin disease vaccine and vaccine vector

Abstract

Lumpy skin disease (LSD), an economically significant disease of cattle and, to a lesser extent, water buffalo, is caused by the poxvirus, lumpy skin disease virus (LSDV). Vaccination with an attenuated strain of LSDV is the preferred method of disease control in endemic countries. Using serial passage of field strains, homologous and heterologous live-attenuated vaccines (LAVs) against LSD were developed, resulting in various mutations in several genes throughout the genome. Although effective vaccines are available, post-vaccinal reactions like fever and vaccination site reactions are occasionally observed, and a small percentage of animals develop mild, generalised disease. This study aimed to use a targeted approach to attenuate a field strain of LSDV (LSDV_WB) to produce an improved (safer) LSD vaccine, using knockout (KO) technology and homologous recombination to knockout/disrupt single putative LSDV immunomodulatory genes. One of the generated KO constructs would then be used as a vector to construct a bivalent vaccine by inserting the protective glycoproteins of the Rift Valley fever virus (RVFV) in another putative immunomodulatory gene of LSDV, thus inactivating a second LSDV gene. The resulting vaccine construct would then be evaluated for its ability to protect cattle against Rift Valley fever (RVF) and LSD. A small animal model is not yet available to study the pathogenesis of LSDV, although early reports indicated that rabbits are susceptible to LSDV. To investigate, New Zealand White rabbits were inoculated with a high dose of LSDV_WB or the LSD OBP vaccine via the intradermal (ID), intravenous (IV) or subcutaneous (SC) routes. Only ID-inoculated rabbits developed a reaction at the vaccination site. None of the SC- or IV-inoculated rabbits developed clinical reactions. LSDV-neutralising antibodies were not detected in any of the ID- IV- and SC-inoculated rabbits. Therefore, rabbits were considered unsuitable as a small animal model for studies of the pathogenesis of LSDV. The LSDV_WB005KO and LSDV_WB008KO constructs were generated by knocking out the IL-10-like and IFN-γR-like genes from LSDV_WB, respectively. Calves were inoculated subcutaneously with a high dose of either KO construct or the LSD OBP vaccine. Several calves in both KO groups developed severe vaccination site swellings, fever and viraemia. Humoral and cell-mediated immune (CMI) responses were observed in calves in both KO groups. After LSDV challenge, all calves in the KO groups were non-viraemic. In conclusion, both KO constructs were insufficiently attenuated, requiring further manipulation. To this end, a double-KO recombinant was generated, but with the addition of the protective RVF virus (RVFV) glycoproteins, as a dual vaccine against LSD and RVF. The IL-10-like gene was deleted from LSDV_WB, followed by inactivation of the putative serpin gene by inserting the RVFV GnGc glycoproteins. Calves were inoculated with a low-dose (LSD-O.RVF_Lo) or high-dose (LSD-O.RVF_Hi) of the vaccine construct. Most of the calves in both groups developed fever and vaccination site reactions, and generalised disease was observed in a low percentage of calves in either KO group. Viraemia was observed in a high percentage of calves in both KO groups, and most calves developed LSDV-neutralising antibodies after vaccination. However, four calves in the LSD-O.RVF_Hi group presented with severe vaccination-site swellings. Four LSD-O.RVF_Lo-vaccinated calves were protected against LSDV challenge. RVFV-neutralising antibodies were observed in a low percentage of calves in both groups after vaccination, including RVFV-specific CMI responses in several calves. All calves in the LSD-O.RVF_Hi group were protected against virulent RVFV challenge. In conclusion, a dual LSD and RVF vaccine was successfully generated; however, further manipulation is required to minimise post-vaccinal reactions and increase RVFV-neutralising antibody titres after vaccination.