Identification of African swine fever virus proteins that activate T-cell immune responses

Abstract

African swine fever (ASF) is a highly contagious haemorrhagic disease of domestic pigs, which affects wild boar as well and for which there is currently no commercial vaccine. African swine fever virus (ASFV), the causal agent of ASF infects warthogs (Phacochoerus africanus), bush pigs (Potamochoerus porcus), giant forest hogs and members of the soft tick genus (Ornithodoros moubata complex) without causing disease. Virulent isolates of this virus causes mortality in domestic pigs within seven to ten days post infection, leading to negative economic consequences. Vaccination attempts using different strategies only protect against homologous strains with inadequate protection against heterologous strains of ASFV. The immunological characterisation of ASFV proteins could assist in the development of an effective vaccine against ASF. The aim of this study was therefore to immunologically characterise a selected range of ASFV proteins (p22, CD2V, pp220 fragments (F)1-F4, pS273R, pA104R, pE165R, pF334L, pK205R and pL11L) by identifing the cytokines they induce in peripheral blood mononuclear cells (PBMC) from a pig that was infected with ASFV via tick feeding (pig 1) and an incontact ASFV infected pig (pig 2). The benefits of identifying immunilogical characteristics of the cytokines can lead to possible vaccine development and decrease of animal losses due to ASF. Several proteins (p22, pp220 F1-4, pS273R, pA104R, pE165R and pK205R) from the genotype II ASFV isolate MAL/11/02 were successfully expressed using a pET102/D-Topo isomerase enzyme (TOPO)® bacterial expression system. Plasmids with gene inserts were amplified using the Invitrogen E. coli TOP10 cloning cells and proteins were expressed in BL21 DE3 cells using isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The protein product of genes EP402R (CD2V), pF334L and pL11L did not express and were excluded from futher experiments. The expressed proteins were purified by affinity chromatography using Nickel columns with affinity to the His-tag on the recombinant proteins. The immunological assays, interferon gamma (IFN)-γ enzyme-linked immunospot (ELISpot) and cytokine [Interleukin (IL)-2, IL-4, IL-8, IL 10, IL-12, IFN-γ and IFN-alpha (α)] reverse transcription (RT) quantitative real time polymerase chain reaction (RT-qPCR), were conducted using PBMCs. PBMC was collected from pig 1 after ten days post infection (pi) with the genotype XIX ASFV isolate RSA/12/15 strain of ASFV and pig 2 ten days after co-habitation with pig 1. Only four of the nine recombinant ASFV proteins produced significant cytokine levels. The recombinant p22 protein up-regulated cytokines IL-2, IL-4, IL-8, IL-10 and IFN-γ in pig 1 and IFN-α in pig 2. Fragment four (F4) of polyprotein (pp) 220 up-regulated IL-4, IL-8, IL-12 and IFN-α in pig 1 and IFN-α in pig 2. Recombinant proteins pA104R up-regulated IFN-α in pig 1 and pS273R significantly down-regulated IFN-α in pig 1. In conclusion, the study revealed that ASFV recombinant proteins p22 and pp220-F4 induced all the study cytokines which are important in antiviral immunity. This data can be applied in future ASFV vaccinology studies.