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African swine fever is a devastating disease of domestic swine and wild boar caused by a large double-stranded DNA virus that encodes for more than 150 open reading frames. There is no licensed vaccine for the disease and the most promising current candidates are modified live viruses that have been attenuated by deletion of virulence factors. Like many viruses African swine fever virus significantly alters the host cell machinery to benefit its replication and viral genes that modify host pathways represent promising targets for development of gene deleted vaccines. Autophagy is an important cellular pathway that is involved in cellular homeostasis, innate and adaptive immunity and therefore is manipulated by a number of different viruses. Autophagy is regulated by a complex protein cascade and here we show that African swine fever virus can block formation of autophagosomes, a critical functional step of the autophagy pathway through at least two different mechanisms. Interestingly this does not require the A179L gene that has been shown to interact with Beclin-1, an important autophagy regulator.

© 2020 by the authors., Classical approaches to African swine fever virus (ASFV) vaccine development have not been successful; inactivated virus does not provide protection and use of live attenuated viruses generated by passage in tissue culture had a poor safety profile. Current African swine fever (ASF) vaccine research focuses on the development of modified live viruses by targeted gene deletion or subunit vaccines. The latter approach would be differentiation of vaccinated from infected animals (DIVA)-compliant, but information on which viral proteins to include in a subunit vaccine is lacking. Our previous work used DNA-prime/vaccinia-virus boost to screen 40 ASFV genes for immunogenicity, however this immunization regime did not protect animals after challenge. Here we describe the induction of both antigen and ASFV-specific antibody and cellular immune responses by different viral-vectored pools of antigens selected based on their immunogenicity in pigs. Immunization with one of these pools, comprising eight viral-vectored ASFV genes, protected 100% of pigs from fatal disease after challenge with a normally lethal dose of virulent ASFV. This data provide the basis for the further development of a subunit vaccine against this devastating disease., C.L.N., L.C.G. and L.K.D. were supported by Department for Food, Environment and Rural Affairs (DEFRA) grants SE1514, SE1515 and SE1516 as well Biotechnology and Biological Sciences Research Council (BBSRC) grants, BBS/E/I/00007030, BBS/E/I/00007031, BBS/E/I/00007034, BBS/E/I/00007035, BBS/E/I/00007036, BBS/E/I/00007037, BBS/E/I/00007038 and BBS/E/I/00007039. G.T. was also supported by SE1514 and SE1515 and M.M. was supported by SE1515. A.L.R and R.P. were supported by DEFRA grants SE1515 and SE1516 respectively. H.G. was supported by DEFRA grant SE1516. G.L.S. was supported by BBSRC grant BBS/E/I/00002120.

Subversion of programmed cell death-based host defence systems is a prominent feature of infections by large DNA viruses. African swine fever virus (ASFV) is a large DNA virus and sole member of the Asfarviridae family that harbours the B-cell lymphoma 2 or Bcl-2 homolog A179L. A179L has been shown to bind to a range of cell death-inducing host proteins, including pro-apoptotic Bcl-2 proteins as well as the autophagy regulator Beclin. Here we report the crystal structure of A179L bound to the Beclin BH3 motif. A179L engages Beclin using the same canonical ligand-binding groove that is utilized to bind to pro-apoptotic Bcl-2 proteins. The mode of binding of Beclin to A179L mirrors that of Beclin binding to human Bcl-2 and Bcl-xL as well as murine &gamma, herpesvirus 68. The introduction of bulky hydrophobic residues into the A179L ligand-binding groove via site-directed mutagenesis ablates binding of Beclin to A179L, leading to a loss of the ability of A179L to modulate autophagosome formation in Vero cells during starvation. Our findings provide a mechanistic understanding for the potent autophagy inhibitory activity of A179L and serve as a platform for more detailed investigations into the role of autophagy during ASFV infection.

African swine fever (ASF) is a lethal haemorrhagic disease of domestic pigs caused by infection with African swine fever virus (ASFV). An outbreak in Russia in 2007 has since advanced into Europe and the Far East. With the spread of ASF expected to continue, the requirement for effective vaccines has become critical. The prospects for vaccine development are promising as infection with attenuated strains of ASFV can offer protection against closely related virulent strains. Autophagy is an essential cell process that sequesters cytoplasmic cargo into double-membrane vesicles called autophagosomes for degradation via lysosomes. Autophagy regulates multiple pathways that are vital to mounting an effective immune response and viruses specifically target this pathway for modulation. Experiments have shown that inhibiting the ability of viruses to regulate autophagy can lead to enhanced immune responses. The work in this thesis shows that autophagy is actively inhibited by ASFV and is not required for virus replication. Further investigation revealed that ASFV activates Akt and mTORC1 which causes a block in autophagosome assembly. Pharmacological studies show that autophagy can be induced during the very early stages of viral replication but at later times, additional measures of modulation are implemented, most likely via virally encoded genes. ASFV encodes A179L which binds to the key autophagy protein Beclin 1 leading to the inhibition of autophagosome formation. However, this work has demonstrated that additional protein modulators are also encoded. A gene library screen was conducted to identify these and unexpectedly, the results showed that some ASFV genes are able to stimulate elements of the autophagy pathway which was investigated further. This research will expand our understanding of the interaction between ASFV and the autophagy pathway with the potential that a low virulent ASFV strain with an altered ability to modulate autophagy will provide enhanced immunity against virulent isolates.

Foot-and-mouth disease (FMD) affects economically important livestock and is one of the most contagious viral diseases. The most commonly used FMD diagnostic assay is a sandwich ELISA. However, the main disadvantage of this ELISA is that it requires anti-FMD virus (FMDV) serotype-specific antibodies raised in small animals. This problem can be, in part, overcome by using anti-FMDV monoclonal antibodies (MAbs) as detecting reagents. However, the long-term use of MAbs may be problematic and they may need to be replaced. Here we have constructed chimeric antibodies (mouse/rabbit D9) and Fabs (fragment antigen-binding) (mouse/cattle D9) using the Fv (fragment variable) regions of a mouse MAb, D9 (MAb D9), which recognises type O FMDV. The mouse/rabbit D9 chimeric antibody retained the FMDV serotype-specificity of MAb D9 and performed well in a FMDV detection ELISA as well as in routine laboratory assays. Cryo-electron microscopy analysis confirmed engagement with antigenic site 1 and peptide competition studies identified the aspartic acid at residue VP1 147 as a novel component of the D9 epitope. This chimeric expression approach is a simple but effective way to preserve valuable FMDV antibodies, and has the potential for unlimited generation of antibodies and antibody fragments in recombinant systems with the concomitant positive impacts on the 3Rs (Replacement, Reduction and Refinement) principles.