Researchers at the Perelman School of Medicine at the University of Pennsylvania have developed an experimental multivalent mRNA-based vaccine against all 20 known subtypes of influenza virus. Their approach differs from previous attempts to craft a universal flu vaccine, by including antigens specific to each subtype, rather than just a smaller set of antigens shared among subtypes. This strategy harnesses the same mRNA technology as that employed in the Pfizer and Moderna SARS-CoV-2 vaccines. The mRNA technology that enabled those COVID-19 vaccines was pioneered at Penn.

Tests in animal models showed that the vaccine dramatically reduced signs of illness and protected from death, even when the animals were exposed to flu strains different from those used in making the vaccine.

The team suggests that their technology could lead to the development of a universal flu vaccine that protects against potential future pandemics. “The idea here is to have a vaccine that will give people a baseline level of immune memory to diverse flu strains, so that there will be far less disease and death when the next flu pandemic occurs,” said study senior author Scott Hensley, PhD, a professor of microbiology at in the Perelman School of Medicine. Hensley and colleagues reported on the development of their mRNA vaccine in Science, in a report titled, “A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes.” Hensley and his laboratory collaborated in the study with the laboratory of mRNA vaccine pioneer Drew Weissman, MD, PhD, the Roberts Family professor in vaccine research and director of vaccine research at Penn Medicine.

Influenza viruses periodically cause pandemics with enormous death tolls. The best known of these was the 191819 “Spanish flu” pandemic, which killed tens of millions of people worldwide. Flu viruses can circulate in birds, pigs, and other animals, and pandemics can start when one of these strains jumps to humans and acquires mutations that adapt it better for spreading among humans. The authors explained, “There are at least 18 different influenza A virus (IAV) subtypes that circulate in animal reservoirs, and these viruses occasionally enter the human population and cause a pandemic.”

Current flu vaccines are “seasonal” vaccines that protect against recently circulating strains, but would not be expected to protect against new, pandemic strains. And even with increased global surveillance, it is difficult to predict which flu strain will cause the next flu pandemic, making a universal vaccine important. As the investigators noted, “Although surveillance programs and modeling studies have increased our knowledge of pandemic risk, we cannot accurately predict which influenza subtype will cause the next pandemic.”

There are several universal influenza vaccines in development to provide protection against diverse influenza virus subtypes, the team continued. Most of these vaccine candidates include a limited number of antigens that have epitopes that are conserved across different influenza virus subtypes.

In contrast, the strategy employed by the Penn Medicine researchers is to vaccinate using immunogens—a type of antigen that stimulates immune responses—from all known influenza A and influenza B virus (IBV) subtypes in order to elicit broad protection. This vaccination strategy is not expected to provide “sterilizing” immunity that completely prevents viral infections. “Instead of focusing on immunogens to elicit antibodies against epitopes that are conserved among many different influenza virus strains, we designed a vaccine that encodes separate immunogens from all known IAV subtypes and IBV lineages,” the team explained.

Their newly reported study confirmed that the vaccine elicited a memory immune response that can be quickly recalled and adapted to new pandemic viral strains, significantly reducing severe illness and death from infections. “It would be comparable to first-generation SARS-CoV-2 mRNA vaccines, which were targeted to the original Wuhan strain of the coronavirus,” Hensley said. “Against later variants such as Omicron, these original vaccines did not fully block viral infections, but they continue to provide durable protection against severe disease and death.”

For their flu vaccine, the researchers prepared 20 different nanoparticle encapsulated mRNAs, each encoding a different hemagglutinin antigen. The experimental mRNA-lipid nanoparticle vaccine developed by Hensley and colleagues encoded hemagglutinin (HA) antigens from all 20 known influenza A and B virus subtypes.

When injected and taken up by the cells of recipients, the vaccine then resulted in production of copies of the key flu virus hemagglutinin protein, for all 20 influenza hemagglutinin subtypes—H1 through H18 for influenza A viruses, and two more for influenza B viruses. “For a conventional vaccine, immunizing against all these subtypes would be a major challenge, but with mRNA technology, it’s relatively easy,” Hensley said.

Tested in mice, the mRNA vaccine elicited high levels of antibodies, which stayed elevated for at least four months, and reacted strongly to all 20 flu subtypes. Multivalent protein vaccines produced using more traditional methods elicited fewer antibodies and were less protective compared to the multivalent mRNA vaccine in the animals.

Moreover, the new vaccine seemed relatively unaffected by prior influenza virus exposures, which can skew immune responses to conventional influenza vaccines. The researchers observed that the antibody response in the mice was strong and broad whether or not the animals had been exposed to flu virus before. The team also carried out tests in ferrets that were vaccinated using a prime-boost approach, and challenged with an avian H1N1 virus, to mimic a pandemic caused by an unknown viral strain. The results of these experiments confirmed that compared with unvaccinated animals challenged by the same virus, the vaccinated ferrets lost less weight, and all survived, whereas two of the four unvaccinated ferrets died. Unvaccinated animals also displayed more clinical signs of disease relative to vaccinated animals after infection.

“Further studies will be required to fully elucidate the mechanisms by which the 20-HA mRNA vaccine provides protection,” the authors acknowledged. Their reported findings suggested that protection against antigenically matched strains is mediated by neutralizing antibodies, whereas protection against mismatched viral strains may occur through non-neutralizing mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC).

Hensley and his colleagues currently are designing human clinical trials. The researchers envision that, if those trials are successful, the vaccine may be useful for eliciting long-term immune memory against all influenza subtypes in people of all age groups, including young children. “We think this vaccine could significantly reduce the chances of ever getting a severe flu infection,” Hensley said. As the team noted in their report, “It is likely that mRNA influenza vaccines that are imperfectly matched to novel pandemic influenza virus strains will not provide sterilizing immunity but will instead limit disease severity and protect against death through non-neutralizing mechanisms.”

In an accompanying perspective, Alyson A Kelvin, PhD, and Darryl Fallzarano, PhD, at the University of Saskatchewan, noted that the strengths of the mRNA platform for pandemic vaccine production include flexibility of antigen design, increased numbers of potential viral targets, speed of production, and inexpensive, scalable manufacturing. “These strengths are important when designing and producing vaccines for a highly diverse, unpredictable family of viruses that can easily spread globally in a matter of weeks,” they noted. However, they commented, “questions remain regarding the regulation and approval pathway of such a vaccine that targets viruses of pandemic potential but are not currently in human circulation.”

In principle, Hensley added, the same multivalent mRNA strategy could be used for other viruses with pandemic potential, including coronaviruses. “Multivalent mRNA-LNP vaccines may be applied against other variable pathogens, such as coronaviruses and rhinoviruses,” the scientists concluded. “For example, SARS-CoV-2mRNA vaccines are being updated to include multiple spike components to combat antigenically distinct strains. Additional studies will be required to determine the maximum number of antigens that can be simultaneously delivered through mRNA-LNP vaccines and the underlying immunological mechanisms that allow for the induction of responses against multiple antigens.”

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