Research Team Takes Aim at Moving Target of Influenza Virus

Imagine you are a target shooter, with your target always moving. It’s more difficult, but not for a seasoned marksman. But what if your target also frequently changed shape and flight characteristics in mid-air? What if it altered camouflage schemes and direction without warning, or randomly increased and decreased speeds?

Suddenly your challenge would go from difficult to near impossible, yet Dr. Constantinos Kyriakis, assistant professor in the Auburn University College of Veterinary Medicine’s Department of Pathobiology, and his fellow Sugg Laboratory researchers face a similar challenge every day. Their target is indeed a shifting one, as well as one that has defied the best efforts of science for many years — the Influenza A virus (IAV).

IAV and Influenza B are the two flu virus types that routinely circulate among humans, and two subtypes of the A virus, H1N1 and H3N2, are those that cause seasonal epidemics. Of course, there are vaccines for IAV, but they provide a measure of protection from severe disease rather than prevent infection altogether. Moreover, because influenza viruses are shifting targets, and the large amounts of vaccines required for mass inoculations must be produced far in advance of the arrival of flu season, vaccines are developed based on previous seasonal virus strains. As a result, vaccine efficacy usually ranges between 19-60 percent.

“Current flu shots are based on older technologies,” said Kyriakis, whose team’s ongoing influenza research has Auburn preparing to become the newest member of the National Institutes of Health/National Institute of Allergy and Infectious Diseases NIH/NIAID Centers of Excellence for Influenza Research and Response (CEIRR). “Live virus is propagated in chicken eggs or cells, inactivated, and split before being given in measured doses to stimulate an immune response. More recently, subunit vaccines are also available that only contain the IAV hemagglutinin (HA) protein.”

“Production using these old methods is a time-consuming, but well-proven process,” he added. “But just like SARS-CoV-2, IAV is constantly evolving, escaping previous immunity. A vaccine that works one year may be ineffective the next. These changes in the virus over time are known as antigenic drift.”

To better understand antigenic drift and its potential outcomes, Kyriakis and his fellow Auburn researchers study IAVs in swine — a natural host of influenza and comparable to people in how they become infected and respond — in order to predict how new virus iterations will behave in humans. “Pigs are unique in their response to IAVs due to their commonalities with humans,” Kyriakis explained. “Transmission methods, immune responses and morbidity are all very alike. Human and swine flu viruses are also similar. Due to close interaction between people and pigs, the virus has often jumped between them.”

From the left: Drs. Huber, Kyriakis and Criado

Together with his colleagues Drs. Miriã Criado and Laura Huber, also from the Department of Pathobiology, Kyriakis is studying the mechanism of how influenza viruses evolve and evade preexisting immunity in swine. “We have groups of pigs that were previously infected or vaccinated with different viruses,” he said. “We then introduce a given virus and collect samples from the same animals over several days. We sequence each sample and record how the virus evolves over time and which specific mutations help it escape antibody responses. Combining our data with sequences from related human and swine viruses from the field, we aim to develop a model to predict virus evolution and aid in the design of more efficient vaccines.”

Monitoring the ecology and emergence of IAVs in animal populations, including swine and birds, is also of critical importance for pandemic preparedness. IAVs can jump from one animal species to another or exchange gene segments with other flu viruses in a phenomenon known as antigenic shift, or reassortment, resulting in the emergence of novel viruses capable of infecting and transmitting to new animal species.

In conjunction with the Alabama Department of Conservation and Natural Resources, Auburn researchers routinely sample state feral pig populations to monitor for signs of reassortment. “Pigs can be infected with swine, human and avian influenza viruses,” Kyriakis noted, “which makes them a critical species in the ecology of IAVs. For this reason, we study IAV reassortment in primary swine respiratory cells, trying to better understand the frequency and mechanism of the phenomenon.”

In addition to studying IAVs and how they evolve and reassort, Kyriakis and the Auburn team also study new ways to combat them. Influenza vaccines have traditionally been aimed at hemagglutinin (HA), the most abundant of two proteins found on the surface of the virus and the one which helps it bind to target cells. If that binding process can be reduced or eliminated, then infection can be inhibited.

Auburn researchers have shown that vaccines targeting the second protein, neuraminidase (NA), can show similar effectiveness. Neuraminidase aids in the final stage of viral infection by weakening the target cell surface and facilitating virus release from infected cells. The highly popular anti-viral drug Tamiflu is actually an NA-inhibitor, but NA-based vaccine development remains in the research stage.

While current flu vaccines are produced with what Kyriakis describes as first-generation technology, he is quick to note vaccine development is currently undergoing a renaissance. “The Covid pandemic acted as a catalyst for vaccine development worldwide,” he said, “stimulating a tremendous investment of money and new research.

Bottom photo: Graduate students J. Fletcher North and Philip Strate are mentored by Dr. Constantinos Kyriakis in their research activities.

“In the CVM, we are constantly studying both new and proven vaccine technologies,” he added, “but once a virus mutates, the effectiveness of any vaccine is reduced — even a third-generation product like mRNA vaccines. Even so, most are still effective at preventing severe disease.” And for now, Kyriakis sees that prevention of serious disease — or, even better, infection, as the end game for influenza vaccines. It is a target he and the other Auburn researchers continue to work toward, along with the broader influenza research community.

“The ideal goal has always been a one-size-fits-all vaccine that will protect against all strains,” he concluded, “both seasonal types and new viruses emerging from animal populations. Due to the way the virus constantly changes, however, that is probably not possible. But a more universally protective vaccine that provides protection against disease from drifting viruses is a more realistic target and a worthy goal.”