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African swine fever, a fatal disease of pigs, has been around for decades. Believed to have originated in sub-Saharan Africa, it’s made several visits to other continents, with outbreaks surfacing in Russia, Brazil, and various parts of Europe—where it still maintains a stronghold in wild boar populations.

But it only escalated to what Dirk Pfeiffer calls “the biggest animal disease outbreak ever” when it reached China last August, spreading like wildfire across the world’s largest pig congregations. “There’s so many pigs in China, it was just a matter of time,” says Pfeiffer, a veterinary epidemiologist at the City University of Hong Kong and the UK’s Royal Veterinary College.

The disease not only threatens the world’s largest pork industry, but also the global supply of the blood thinner heparin, most of which is produced by Chinese pigs.

Alarmed, Chinese officials have reportedly culled more than 1.2 million pigs to date in an attempt to prevent new infections, but the disease is spreading and has jumped to Vietnam and Cambodia in recent months. Pfeiffer reckons that anywhere between 10 percent and 40 percent of Chinese pigs could have been infected with the virus so far, although official statistics have not yet been released.

Desperate for a vaccine, China has put around $15 million towards research on the virus, according to Nature News, spurring researchers to find one quickly. There are several routes researchers are taking to do so, but this is proving challenging—in part because of the very nature of the virus.

Early failures of an ASFV vaccine

The sheer complexity of African swine fever virus (ASFV) is one reason why it’s so hard to tackle. Its double-stranded DNA genome can span an impressive 190 kilobases and codes for almost 170 proteins, dwarfing many other viruses, such as Ebola (some strains have only 7 proteins).

ASFV infects and replicates in macrophages, but also induces cell death in uninfected B and T lymphocytes. “It effectively wipes out the immune system so there’s not an effective response,” explains Linda Dixon, a virologist at the UK’s Pirbright Institute, part of the government’s Biotechnology and Biological Sciences Research Council. Ultimately, ASFV kills pigs by causing extreme hemorrhagic fever and massive destruction of lymphocytes in lymph tissues.

Both early studies in 1967 and more recent ones have shown that the classical and most obvious strategy of developing a vaccine doesn’t work for ASFV: killing or inactivating the virus and injecting it into healthy animals to prompt their immune system to generate antibodies that protect against future infections was attempted, but it failed. The protective antibodies produced just weren’t enough to ward off ASFV infection.

Scientists have instead learned that one of the most effective ways to produce immunity against ASFV is to expose animals to a less virulent strain of the virus. This can be produced through passaging the virus in culture until it loses its virulence, a strategy that has been successful in containing the spread of a different virus that causes similar symptoms in pigs, classical swine fever. Alternatively, attenuated viruses can be isolated from animals: in wild boar populations across Europe, for instance, many ASFV strains have naturally lost their potency to kill over time.

Some groups have shown that injecting a weaker ASFV strain isolated in 2017 from a wild boar in Latvia can protect domestic pigs against a virulent form of the virus, explains José Manuel Sánchez-Vizcaíno, a virologist at the World Organization for Animal Health’s reference laboratory for ASF in Madrid. He and his colleagues recently demonstrated that the same strain could also protect wild boars, an approach he thinks may be helpful in preventing spillovers of the disease to domestic pigs.

However, the main concern with live attenuated vaccine candidates is safety. Researchers realized this as early as the 1960s, when they tried to vaccinate large numbers of pigs in Portugal and Spain with a naturally attenuated form of ASFV. Although the animals didn’t die, many of them developed a debilitating, chronic form of the disease. “This is the biggest problem: it’s good protection, but not very safe,” explains Sánchez-Vizcaíno. He and his colleagues are currently evaluating the safety of their attenuated virus.

African swine fever virus The Pirbright Institute

Genetically modified viruses

As researchers have amassed more knowledge about ASFV’s biology and its genome, they have adopted a more targeted approach to attenuate ASFV: genetically modifying the virus by deleting genes that make it so virulent and then vaccinated animals with it. “It’s a case of trying to disarm the virus so that the host has a chance to respond and control replication and induce an adaptive immune response that will be a memory response,” explains Dixon.

In 2016, her team deleted several ASFV genes that are thought to inhibit its hosts’ type 1 interferon response, which induces factors that restrict virus replication in cells and stimulates other components of the immune system. Immunizing five pigs with this strain caused no symptoms—demonstrating that the virus had lost its virulence—and after being challenged with a lethal dose of the original virus, all animals survived. Dixon is now working to develop a live attenuated vaccine based on this approach.

Manuel Borca, a microbiologist based at Plum Island Animal Disease Center in New York—part of the USDA’s Agricultural Research Service’s Foreign Animal Disease Research Unit (FADRU)—has seen similar success with three gene-deleted attenuated candidate vaccines, with one or a combination of deletions. Some of the deleted genes are thought to be involved in ASFV’s regulation of hosts’ immune genes, although it’s not clear if this is why their deletion causes attenuation. Pigs that had received one of the vaccine candidates were by and large “all protected” against a lethal dose of a virulent form of the virus three weeks later, explains Borca’s colleague Luis Rodriguez, research leader at FADRU.

Experts in the field say that such gene-deletion approaches are currently the most advanced vaccine candidates. But it will likely be a matter of several years before these can be deployed, according to Borca and Dixon. First, they’ll have to go through a series of tests to ensure they’re effective and safe, and must be registered with relevant regulatory bodies.

They’re under so much pressure to do something that they might start using a vaccine before it’s been tested enough to make sure that it’s safe. —Dirk Pfeiffer, City University of Hong Kong and the UK’s Royal Veterinary College

Researchers working on the vaccine at China’s Harbin Veterinary Research Institute didn’t respond for comment, but Pfeiffer says they appear to be pursuing a gene-deleted attenuated vaccine based on research he saw them presented at a symposium in April.

Borca and Dixon say these approaches have an edge over naturally attenuated forms of the virus because they allow researchers to fine-tune genetic tweaks to make a harmless virus that is still able to replicate—which is important in triggering immunity as well as amplifying the virus in culture. Deleting parts of the genome also makes it more difficult for the virus to revert back to a virulent form.

However, a major difficulty with attenuated vaccines generally—whether they’re genetically modified or naturally occurring—is growing them in cell culture, which is essential for making vaccines in bulk. Macrophages don’t last very well in culture, Borca explains, so he needs to extract the lymphocytes directly from animals to make virus stocks. To get around this, his team is currently trying to find a stable cell line they can grow the viruses in.

A key concern with such live attenuated viruses is that, as they’re capable of replication, vaccinated animals can shed the virus and infect other animals. Sánchez-Vizcaíno says that in some cases that could help immunize other pigs—something he demonstrated in his study with wild boars. However, others are worried about the risk of side effects.

“There are not [much] experimental data about the development of chronic symptoms weeks/months after vaccination,” says Covadonga Alonso, a virologist at Spain’s National Institute for Agricultural and Food Research and Technology in Madrid, in an email. She says she’d also like to see more experiments investigating whether the attenuated viruses can mutate their way back to virulent forms after generations of replication in vaccinated animals. Dixon says she thinks this is unlikely. “The virus is genetically very stable as it replicates by an accurate DNA polymerase,” she writes to The Scientist in an email.

Protein cocktails

Alonso considers a subunit vaccine—based on the vaccination of viral proteins, such as antigens or proteins that bind to viral receptors—to be “the best alternative for a future vaccine.” As these don’t require cell lines to produce, they may be easier to make in bulk than attenuated vaccines.

Earlier this year, Waithaka Mwangi, a virologist at Kansas State University, and his team developed two different cocktails of viral antigens, which they delivered to pigs by incapsulating them in an inactivated human adenovirus. This way, the virus will infect any antigen-presenting blood cells and “the protein synthesis machinery kicks in, so you have de novo synthesis of the protein in the infected cell,” Mwangi explains. The proteins are then presented on blood cell surfaces and can be recognized by the immune system in a way that triggers a T-cell response as well as an antibody response. For him, the approach effectively mimics the way attenuated viruses induce immunity, “but in a much safer way.”

In his study, however, neither of the antigen cocktails showed much success: When challenged with a virulent form of ASFV, many vaccinated animals succumbed to the disease regardless. A study by Dixon’s group using a similar approach—encoding antigens in DNA plasmids and shuttling them into pigs using a vaccinia viral vector—showed that while this could reduce viral genome loads a little, it didn’t prevent a virulent form of the virus from replicating and causing disease. To Mwangi, these results indicate that researchers have yet to find and target the right viral proteins—or the right combination—that will induce a protective immune response.

Alonso’s group is currently working to find antiviral proteins that work against ASFV—based on natural or synthesized compounds or existing drugs—which she thinks will help reveal the right viral proteins to target. In addition, these compounds could be included in animal feed as supplements to help boost immunity against ASFV, she adds.

Beyond vaccines to biosecurity

For the US and Europe, developing a vaccine is not as high a priority as it is in mainland China, where scientists are working fast to bring one to market. Several researchers are worried that safety concerns won’t be sufficiently addressed before a vaccine candidate is deployed there. “They’re under so much pressure to do something that they might start using a vaccine before it’s been tested enough to make sure that it’s safe,” says Pfeiffer.

China’s pigs are, for the most part, scattered across small-scale farms, containing less than a hundred pigs each. These often have limited biosecurity—there’s little or no control mechanisms to ensure that the virus isn’t transmitted via trucks, on the clothes of people entering the farms, or in animal feed, in which the virus persists for long periods of time, recent research has confirmed. “ASFV is one of the hardy or robust viruses in pH extremes as well as temperature extremes, so it can survive for sustained periods and maintain its infectivity in various environmental conditions,” says Megan Niederwerder of Kansas State University, who led the research.

For that reason, a vaccine may only be a partial solution to China’s ASF epidemic, says Pfeiffer. “It is unrealistic to achieve the required vaccination coverage for the infection to fade out.” Vaccine efforts have to go alongside with increased biosecurity around farms to be effective, he says, particularly if ASFV has become endemic there and has infected wild boars.