African Swine Fever virus (ASFV) is the causal agent of a serious disease which affects domestic pigs and causes important economic losses to the affected countries. ASFV is the only DNA arbovirus known, and its perpetuation and transmission in Africa, where the virus remains endemic, involves a sylvatic cycle (i.e. occurring in the wild) between ticks and wild pigs that are resistant to the disease, becoming a continuous source of virus, therefore complicating its eradication.
Why are ASF vaccines not available? To our understanding, there are two main reasons explaining the lack of available vaccines against ASF. First of all, the high complexity of the virus has complicated this issue. ASFV is a large double stranded DNA virus that encodes more than 150 different proteins, with the ASFV particle containing at least 50 proteins arranged in several layers. In comparison, Porcine Circovirus type 2 particles are composed by one only polypeptide, the cap protein.
Additionally, ASFV encodes multiple virulence factors allowing its replication in porcine macrophages and the concomitant evasion from the host immune response, therefore complicating the development of efficient antiviral strategies.
Second, little effort has been made up to now to obtain a safe and efficient vaccine against ASF, leading to the probable false perception that this was an impossible task to pursue.
Thus, compared with the deep knowledge that we have today about different aspects of the complex biology of ASFV, efforts made to obtain an efficient vaccine have usually been scarce and most of them were performed more than ten years ago. Three different strategies have been followed in the past:
1. Inactivated vaccines
Inactivated vaccines of ASFV were capable of inducing antibody responses that, however, did not translate to efficient protection.
2. Attenuated strains
In clear contrast, immunization of pigs with classically attenuated strains of ASFV (natural isolates or tissue culture adapted viruses) induced very solid protection against the homologous viral challenge. Safety issues made the application of these live-attenuated viruses as vaccines impossible, they have however provided us with the most useful data existing today about immune parameters involved in protection. Thus, both antibodies and cytotoxic specific CD8+ T-cells were demonstrated to play important roles in the protection afforded by live-attenuated vaccines.
2a. Antibodies and T-cells
Neither antibodies nor T-cells seemed to be able, by themselves, to confer complete and sterilizing protection, indicating that an ideal vaccine against ASFV should be able to confer both kinds of immune responses. While specific antibodies could more efficiently neutralize and/ or inhibit the virus particles found in suspension (blood and other corporal fluids); CD8+ T-cells (cytotoxic T-cells) would be able to recognize and destroy ASFV infected cells.
2b. Deletion of genes
Deletion of specific virulence genes by homologous recombination allowed the construction of live attenuated ASFV viruses, albeit several genes should be simultaneously deleted in order to comply with the minimum security issues requested for any commercial vaccine. This alternative will require further research on ASFV pathogenesis, allowing a more rational selection of the virulence factors to be eliminated from the virus in order to obtain the safest and most efficient recombinant vaccine.
2c. Replication deficient strains
An attractive alternative to the classical deletion of genes is the use of inducible viruses as vaccines, a strategy that is currently being explored by the group of Dr Salas, at the CBMSO in Madrid, Spain. The idea behind this technology is to develop replication deficient ASFV vaccine strains. So far, in vitro experiments have demonstrated that these cells produce, under restrictive conditions, ‘empty’ viral particles that lack the inner contents.
Such virus-like particles possess all the external domains (inner envelope and capsid) and can exit efficiently from the infected cell, but they are not infectious.
This strategy should comply with all the requisites for an ideal vaccine against ASFV. Again, further research is needed.
3. Subunit vaccines
In terms of safety, subunit vaccines should be the preferred choice. However, the complexity of ASFV influences the task of selecting the optimal antigens to be included in a vaccine. Several reports exist describing antigenic viral proteins, but little has been reported about their protective efficacy. Immunization with peptide ‘cocktails’ showed a slight delay in the mortality found after experimental infection, while vaccination with entire viral proteins yielded contradictory results. Work done in the mid-1990’s described the protective potential of three ASFV structural proteins: p54, p30 and hemagglutinin (HA), when expressed in a baculovirus system and administered without further purification.
4. Partial protection
Partial protection against ASFV lethal challenge was demonstrated after DNA immunization of pigs with a plasmid encoding only three ASFV antigens: p54, p30 and the extracellular domain of the hemagglutinin (sHA), fused to ubiquitin. This plasmid was called pCMV-UbsHAPQ. As expected, the protection was afforded in the absence of antibodies, correlating with the expansion of CD8 T-cells that specifically recognized two peptides of nine aminoacids from the sHA, one of the three antigens encoded by the vaccine. Preliminary immunization experiments with these two synthetic peptides confirmed their protective capabilities. The identification for the first time of specific protective CD8 T-cell epitopes not only confirmed the relevance of this kind of T-cell response in protection against ASFV but also opened the possibility of generating peptide-based vaccines using more potent expression vectors (see below).
These results have been confirmed more recently by using an alternative vaccine delivery technology, named BacMam, a baculovirus vector that expresses the antigens of interest (in this case the sHA, p54, p30 from ASFV), under the control of a mammalian promoter. Immunization of pigs with the recombinant BacMam-sHAPQ was able to protect pigs against sub-lethal challenge in the absence of specific antibodies. Protection again, correlated with the presence of a high number of ASFV-specific T-cells in their blood. These results definitively demonstrated the key role that T-cells play in protection against ASFV.
Aiming to improve the efficacy of the vaccines, the CReSA scientists are currently extending their studies in two complementary directions. First, they are identifying additional protective determinants from within the ASFV-genome and second, they are also exploring alternative vaccination protocols aiming to optimize the immune responses induced, including the use of more powerful expression vectors, prime-boost regimes and the use of different adjuvants. In summary, results obtained at CReSA show the feasibility of obtaining safe and efficient vaccines against ASFV. Obtaining the optimal vaccine formulation is just a matter of time, investment and willingness.
Meanwhile, we need to control ASF with the tools that are currently available. Even if none products are a guaranteed protection against the disease, immunostimulants or medium chain fatty acids like mono-laurin in the feed can help to limit mortality and slowdown the spreading of the disease. One area to investigate as well is the acidification of the drinking water that seems to have a positive effect on the expression of the pathology. But more studies will be required before validating the protocol and confirm results.