A genetically edited form of a herpes simplex virus (HSV) has outperformed a leading vaccine candidate in a new preclinical study by researchers at the University of Cincinnati, Northwestern University, and the University of Nebraska-Lincoln. The vaccine, called R2, is a form of the herpes simplex virus type 1 (HSV-1) virus that causes cold sores around the lip, but can cross-protect against HSV type 2 (HSV-2), the sexually transmitted type of HSV that is usually responsible for genital herpes. To generate the vaccine, the virus was effectively engineered to keep it from taking refuge in the nervous system and eluding an immune response.

The newly reported study showed that vaccinating guinea pigs using the modified live virus vaccine significantly increased the production of virus-combating antibodies. And when challenged with a virulent strain of HSV-2, vaccinated animals displayed fewer genital lesions, reduced viral replication, and less of the viral shedding that spreads infection to others.

“The fact that the viral shedding was knocked down so much with the R2 vaccine is really important, because it’s the viral shedding—even if it doesn’t cause lesions—that can then pass on the virus,” said Gary Pickard, PhD, professor of veterinary medicine and biomedical sciences at Nebraska. “If you have genital herpes, you can pass that on to your significant other, not knowing that you’re doing it. It’s very problematic. So the fact that the shedding was knocked down so much is a really good sign.” Pickard and colleagues reported on their studies in npj Vaccines, in a paper titled, “The R2 non-neuroinvasive HSV-1 vaccine affords protection from genital HSV-2 infections in a guinea pig model.”

The World Health Organization estimates that more than 500 million people have HSV-2, an infection that persists for a lifetime and often flares up in response to stress. Both HSV-1 and HSV-2 can cause neonatal herpes, and may cause blindness, as well as other serious infections in immunocompromised people, the authors wrote. HSV infection can also increase the risk of HIV infection, and may contribute to Alzheimer’s disease or other forms of dementia. However, there is currently no vaccine against HSV-1 or HSV-2.

“The development of an effective vaccine for HSV is a priority because it is a common infection that causes physical and emotional stress as well as increasing the risk for HIV infection,” the team noted. “The recent failure of subunit HSV vaccines has highlighted the need for vaccines that present a diverse array of antigens, including the development of next-generation live-attenuated vaccines.”

Part of the difficulty in developing HSV vaccines relates to the way that alpha-herpesviruses, which include HSV, have evolved a sophisticated mechanism for evading immune responses that might destroy them. After infecting mucosal tissues of the mouth or genitourinary tract, HSV works its way to the tips of sensory nerves that transmit signals responsible for sensations such as pain and touch. With the help of a specialized molecular switch, the virus then breaks into the nerve cell, hitching a ride on molecules that transport it along a nerve fiber and into the nucleus of the sensory neuron. So while the mucosal infection is soon cleared by the immune response, the infected neurons become a sanctuary from the body’s immune system, with HSV leaving only when stirred by rises in steroids or other stress-elevated hormones in the host.

Nebraska’s Pickard and Patricia Sollars, PhD, alongside Northwestern’s Gregory Smith, PhD, and Tufts University’s Ekaterina Heldwein, PhD, have spent years studying how to prevent HSV from reaching the safety of the nervous system. Heldwein advanced those efforts when she characterized the architecture of a certain alpha-herpesvirus protein, pUL37, which the team suspected was integral to the virus moving along nerve fibers. Computer analyses based on that architecture suggested that three regions of the protein might prove important to the process.

Smith replaced five codons—the fundamental coding information in the DNA—from each region of the viral genome, which the researchers hoped might help impede the virus from invading the nervous system. “The R2 vaccine evaluated here is a live-attenuated HSV-1 strain encoding pUL37 tegument protein mutated in region 2, which is essential for neuroinvasion,” the authors wrote. Research had indicated that “… the R2 approach showed potential as a new live-attenuated HSV vaccine capable of amplified presentation of the entire cohort of HSV antigens in the absence of nervous system complications or the establishment of latent infections.”

A genetically edited form of a herpes simplex virus has outperformed a leading vaccine candidate in a recent study. Nebraska researchers Patricia Sollars, PhD, (left) and Gary Pickard, PhD, teamed with colleagues from the University of Cincinnati and Northwestern University to develop and test a form of HSV that generates high levels of neutralizing antibodies while limiting viral replication and shedding. [Craig Chandler, University of Nebraska-Lincoln]

When Pickard and Sollars injected mice with a virus modified in region 2, or R2, of the protein, they found that, rather than advancing deeper into the nervous system, the virus was stuck at the nerve terminal. But the team recognized that modifying HSV wasn’t enough, and could have unintended consequences. “You can keep the virus from getting into the nervous system,” said Pickard,  “that’s not that hard to do by making broadly debilitating mutations. But when you knock down the virus so much that it doesn’t replicate well, you are not rewarded with a robust immune response that can protect you from future exposures.”

Fortunately, the team’s studies in mice showed that the R2-mutated virus performed well as a vaccine in mice. “The R2 vaccine evaluated here is a live-attenuated HSV-1 strain encoding pUL37 tegument protein mutated in region 2, which is essential for neuroinvasion,” the authors wrote. “Consistent with the axonal transport deficit, the R2 attenuated viruses were specifically ablated of neuroinvasive potential and were avirulent in mice,” they noted. … the R2 approach showed potential as a new live-attenuated HSV vaccine capable of amplified presentation of the entire cohort of HSV antigens in the absence of nervous system complications or the establishment of latent infections.”

Moreover, it circumvented certain stubborn issues that have cropped up with other vaccine approaches. Some approaches have involved challenging the immune system with only a subset of HSV components, or antigens, priming the body to recognize them but potentially miss others. Some have modified the virus so that it can replicate just once, preventing long-term persistence in the nervous system but also reducing spread in mucosal tissues and, by extension, a stout immune response.

“So it’s the same story over and over again: Either your subunit vaccine doesn’t present enough antigens, or you make the live virus essentially so sick that it doesn’t work really well to generate an immune response,” Pickard said. “That’s why we’re so optimistic about our R2 platform, because it avoids all those problems.” Encouragingly, the new R2 vaccine was generating very promising results. “The R2 vaccine evaluated here is a live-attenuated HSV-1 strain encoding pUL37 tegument protein mutated in region 2, which is essential for neuroinvasion,” the authors wrote. “Consistent with the axonal transport deficit, the R2 attenuated viruses were specifically ablated of neuroinvasive potential and were avirulent in mice … the R2 approach showed potential as a new live-attenuated HSV vaccine capable of amplified presentation of the entire cohort of HSV antigens in the absence of nervous system complications or the establishment of latent infections.”

David Bernstein, a researcher at Cincinnati Children’s Hospital Medical Center who evaluates herpes virus vaccine candidates through a program supported by the National Institutes of Health, took note of the team’s success and reached out to Northwestern’s Smith in 2018. Bernstein decided to test the effectiveness of an R2-modified form of HSV-1 against HSV-2 infection in guinea pigs. As promising as their prior results had been, Pickard wasn’t sure an HSV-1 vaccine would be up to the task of generating immunity against HSV-2.

Encouragingly, the results showed that only one of the dozen R2-inoculated guinea pigs developed acute lesions after being injected with HSV-2, compared with five of 12 animals receiving another vaccine candidate that had recently failed a human clinical trial. And whereas the previously human-tested vaccine candidate had no discernible effect on the number of days that guinea pigs shed the virus, the team’s R2 vaccine cut the shedding period from 29 days to about 13. “Perhaps of greatest significance, recurrent virus shedding was reduced by 33–64% in each of the R2 vaccinated groups,” the investigators commented. “This is of particular importance because recurrent virus shedding is the main means of HSV transmission.” Unlike the guinea pigs receiving no vaccine or the failed candidate, those receiving the R2 vaccine showed no sign of HSV-2 in the cluster of brain cells that normally house it. Neutralizing antibodies, meanwhile, registered about three times higher in the R2-inoculated guinea pigs than in those inoculated with the other vaccine candidate. “The use of live-attenuated HSV vaccines that robustly replicate in mucosal tissues but are ablated for neuroinvasion offers a promising approach for HSV vaccines,” the scientists commented.

With an HSV-1 version of the R2 vaccine showing such promising cross-protection against its sexually transmitted counterpart, the researchers aim to develop and test an HSV-2 vaccine against the HSV-2 virus. “We consider these results highly encouraging, especially considering R2 is an HSV-1 virus and was evaluated for cross protection against HSV-2 challenge,” the investigators pointed out. Pickard added, “if you’re making antibodies against the proteins of that particular virus, it stands to reason (that) it would work better than if you’re making an antibody against something that’s slightly different. So that’s our expectation.”

Around the time that Bernstein and his NIH program were expressing interest in the R2 vaccine design, Pickard and Smith were launching a startup, Thyreos, to further develop and commercialize the R2 vaccine design. The team is working on vaccines for livestock—specifically cattle and hogs—that contend with alpha-herpesviruses of their own. In cattle, the bovine herpesvirus can cause respiratory disease, curb appetite, and even contribute to aborted calves, all of which add up to billions of dollars in lost revenue annually. Though a modified live-virus vaccine for cattle does exist, it also gets into the bovine nervous system. And that, Pickard said, can spell trouble in cattle just as easily as in people.

“What happens, then, is that when those cows are loaded on a truck and shipped to a feedlot, it’s a stressful environment,” he said. “The virus hiding in the immune system reactivates. They start shedding the virus from excretions in their nose, and they can then pass that virus on to other animals in that feedlot, and the cattle can get respiratory disease. “So the fact that our R2-modified viruses don’t enter the nervous system is not just an academic thing. Actually, it has a real, practical application for the cattle industry.”

Hoping that future studies will show the R2 design’s superiority to the current industry-wide vaccine, Pickard and Smith are embarking on an initial round of seed funding for the enterprise. In fact, the team initially developed the R2 design in the pseudorabies virus alphaherpesvirus that infects pigs, and Pickard has also expressed confidence in the design’s promise for protecting hogs. Although during the 1990s and early 2000s, a U.S. campaign successfully eradicated pseudorabies from the country—largely through vaccination—as with cattle, the vaccine can enter the nervous system of hogs and has proven less successful in countries that are less vigilant about outbreaks.

“Again, we are pretty confident that our pseudorabies virus R2 vaccine is going to be more effective than what’s been out there,” Pickard said. “In terms of protecting pigs, it’s going to have a big impact at some point. These pathogens can survive trans-Pacific transport in feed ingredients or feed products. When you talk to people who are concerned about biosecurity, they say that whatever is going on elsewhere in the world in terms of these viruses, eventually, they may show up here. It’s just a matter of time.”

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