Identification of cytokines induced by two strains of African swine fever virus recombinant proteins p30 p54 and p72

Abstract

African swine fever virus (ASFV) is a virus that infects pigs causing haemorrhagic fever called African swine fever (ASF) with 100% mortality in domestic pigs. No effective vaccine is available that protects across genotypes. Cellular immune responses were demonstrated in pigs that were protected against a virulent strain challenge subsequent to infection with a moderately virulent ASFV strain. However, pigs did not survive challenge with a non-related virulent strain resulting in the development of ASF symptoms and death. The cellular immune response toward ASFV is not fully understood and needs further investigation in order to produce a vaccine that can cross-protect different ASFV genotypes. Structural proteins P30, P54 and P72 are the most immunogenic proteins to which neutralization antibodies have been observed. These were expressed previously in baculovirus for diagnostic purposes targeting ASFV antibodies in sera. The major structural protein P72, encoded by the B646L gene, contains conformational neutralizing epitopes and elicits robust immune responses. The proteins have been found to stimulate a humoral immune response that was confirmed when neutralizing antibodies against proteins were detected. However, this humoral response is still not effective in protecting swine against ASF. On the other hand, a cellular immune response can be stimulated with whole virus and has proven that its stimulation can protect against infection from a closely related strain. It is unknown whether these proteins also induce a cellular immune response even though they have been shown to induce humoral immunity due to the presence of neutralizing antibodies against these structural proteins. Therefore, it is hypothesised that if the cellular immune profiles of structural proteins P30, P54 and P72 are determined it could help in identifying potential vaccine candidate epitopes or give a platform by which other ASFV proteins can be screened for vaccine development.Protection against ASFV has been proven between virus strains from the same genotype. As an example, pigs challenged with a non-virulent strain (OURT88/3) were protected against challenge with the related virulent (OURT88/1) strain. However, when the pigs were challenged with virulent strains from different genotypes the pigs developed disease symptoms and succumbed to the virus. There is no effective vaccine against ASFV that confers cross protection between heterologous strains. This is one of the major obstacles in development of an effective vaccine against ASF. Therefore, the aim was to identify the cytokines induced by ASFV recombinant proteins P30, P54 and P72 from two divergent strains, one from Malawi (MAL), MAL/11/02 genotype II and one from the South Africa (RSA), RSA/12/15 from genotype XIX. Proteins from these strains were expressed using a pET102/D-TOPO® bacterial expression system, cloning was performed using the Invitrogen TOP10 cloning cells and expressed in BL21 (DE3) cells using isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The proteins were purified by affinity chromatography using Nickel columns with affinity to the His tag incorporated.