ASU study investigates new weapons against African swine fever

African swine fever is a highly contagious, viral infection affecting pigs. It has devastated domestic herds in many regions and brought economic hardship. The rapid spread of this disease has become a serious concern to wildlife biologists and veterinarians.

No adequate vaccine exists to protect against African Swine Fever Virus (ASFV), the cause of this ailment, and efforts to arrest the spread of ASFV have been largely ineffective.

In a new study, researchers from Arizona State University’s Biodesign Institute and the Pirbright Institute, UK have explored new options for creating a potent vaccine against ASFV. Their work involves identifying and ranking the most effective disease antigens in ASFV, extracting them from the viral genome and reintroducing them into pigs to stimulate an effective immune response.

A total of 47 genes or gene fragments coding for ASFV disease antigens were evaluated for their vaccine potential. These were packaged for delivery into the pigs in circular segments of DNA known as plasmids, delivered using a device known as a gene gun.

A subsequent “boost” dosage was delivered by incorporating ASFV antigens into another virus known as vaccinia. The recombinant vaccinia virus was then introduced, to provide additional immune stimulation to help guard against ASFV infection.

The results demonstrated improved immune protection in pigs following the one-two punch of ASFV antigens, delivered as plasmid DNA and recombinant vaccinia.

The research findings recently appeared in the Journal of Virology.

“ASF is like the ebola of pigs,” said Debra Hansen, a co-author of the study and an associate research professor at Biodesign. “It’s a highly lethal hemorrhagic disease, it’s extremely infectious, there are no treatments, and the virus is also in the wild populations. Outbreaks are inevitable and require constant monitoring and immediate quarantine and culling.”  

Bertram Jacobs, professor and director of the School of Life Sciences, and Kathryn Sykes, former ASU assistant professor and current Bidodesign Institute external collaborator, also contributed to the study.

Describing the experimental method used, Hansen said, “because the virus has an unusually large number of genes, we took a DNA immunization approach to evaluate effective immune responses to large subsets of the molecular components of the virus. This approach required collaboration between several groups with expertise in molecular biology, vaccine development, and ASFV pathogenesis.”  

Virus on the move

African swine fever, though harmless to humans, is highly lethal to both domestic pigs and wild boar, killing up to 90 percent of those animals exposed to the virus. The disease causes internal bleeding and fluid-filled lungs in infected animals.

ASFV, a complex virus encoding 150–167 proteins, is spread through secretions from sick animals. The virus is evidently capable of surviving for long periods and may be inadvertently carried on workers’ clothing and shoes or in hay, helping the pathogen to leapfrog from farm to farm. The disease is also transmitted to and carried by wild boar.

Once a pig is infected with ASFV, the disease is untreatable. Thwarting the spread of ASFV depends on ensuring that neither infected live pigs nor pig meat products manage to enter areas free of the disease. This combination of factors makes control of ASFV, in the absence of an adequate vaccine, highly challenging. Successful eradication programs rely on rapid diagnosis, slaughter, and disposal of all animals on infected premises.

The disease was first reported in domestic pigs in eastern Africa in 1921, but has recently spread to eastern Europe, with outbreaks occurring in the Russian Federation, Belarus, Ukraine, Lithuania, Estonia and Moldova. (In 2015, the crisis prompted the slaughter of 22,000 pigs in Estonia, causing pork prices to collapse and more than a third of pig farms to go out of business.) Neighboring European counties are now at acute risk of the rapidly spreading disease.

Vaccine challenge

The best solution to the dilemma would be a vaccine, but efforts to create one using inactivated virus particles have been unsuccessful. While attenuated live virus vaccine candidates are being explored, they may be decades away from reaching the market, and carry safety concerns for healthy animals.

The new study takes a different approach, by attempting to identify the most effective antigens in the ASFV genome for conferring protective immunity, and introducing these into pigs in a two-stage process. Exposure of pigs to the ASFV virus following vaccination with pools of candidate antigens allows the researchers to narrow the field to the most effective antigens, which could eventually be incorporated into a potent vaccine.

To accomplish this, the study constructed three ASFV libraries: a gene library used for DNA vaccination, a recombinant vaccinia virus library and a third library used for the expression and capture of recombinant proteins.

Pigs were first exposed to individual disease antigens to test immune response and then with pools of antigens. Next, the pigs were immunized with ASFV antigens, delivered via DNA gene gun, and recombinant vaccinia boost and exposed to whole ASFV virus. 

(As the authors note, recombinant vaccinia viruses are believed to have many of the properties of live attenuated viruses, though they can stimulate both humoral and cell-mediated immunity in a more natural and effective way. Further, vaccinia-based vaccines can be heat stabilized, produced at low cost and are easy to administer, making them an attractive alternative to conventional vaccines.)

Pathway to protection

Pools of up to 22 antigens at a time were delivered via the two-step process. Protection in the pigs was then evaluated by analyzing the response of lymphocyte cells — specialized sentinels, which are called into action when the immune system detects foreign infection. Responses to individual recombinant proteins as well as whole ASF virus were examined.

The 47 gene and gene fragments selected in the study coded for protein antigens that differed in terms of function and timing of expression within their disease host, in order to attempt to replicate the diversity of antigens expressed during an ASFV infection. The antigens examined in the study represent roughly 30 percent of the full ASFV genome.

The sequential method of testing, known as expression library immunization, is a powerful tool as it can identify vaccine candidates encoded in complex pathogens like ASFV, without prior knowledge of which antigens are protective. The trial and error technique can also identify important antigens that are only expressed in very low levels during infection, making them difficult to detect through conventional tests.

Results showed that pigs immunized with the target antigens had lower levels of ASFV-related symptoms, including lower levels of virus in blood, tonsil, spleen and submandibular lymph node compared to pigs immunized with control antigens following challenge with a lethal dose of ASFV. Indeed, viral load was reduced 10 to 100-fold in the submandibular lymph nodes, tonsils and blood of pigs receiving antigen vaccination, compared with those not receiving it.

The study has thus identified novel ASFV proteins and demonstrated their potential as candidates in future vaccine studies. Further testing will help refine the efficacy of these antigens and pinpoint potential side effects.