New design could make seasonal influenza vaccinations more broadly effective

Stanford Medicine scientists have designed a way to make our seasonal influenza vaccinations more broadly effective and possibly to protect us from new flu variants with pandemic potential. In a study set to publish Dec. 20 in Science, they've shown in cultured human tonsil tissue that the method works.

Flu season is upon us, and flu is no joke. Every year, the influenza virus kills hundreds of thousands of people and sends millions to the hospital. The seasonal flu vaccine many of us get is intended to keep that from happening, by giving our immune system a heads-up that speeds its readiness for combat with the virus. A key component of that response is the development of antibodies: specialized proteins that can bind selectively to a targeted virus like a piece of a puzzle to its next-door neighbor and, when the fit is tight enough and in the right place, prevent that virus from getting into our cells and replicating inside of them.

Any classical vaccine displays, in a non-threatening way, one or more of a pathogen's immune-system-arousing biochemical features, or antigens, to various cells of the immune system whose job is to carefully note and memorize particular antigens belonging to the pathogen of interest -; the one the vaccine targets. When the real thing comes along, that memory will kick in and rouse those otherwise dormant immune cells to jump up, pump up and punch out the pest's lights -; preferably before it can invade any cells.

The influenza virus is studded with molecular hooks that it uses to latch on to vulnerable cells in our airways and lungs. This hook-like molecule, called hemagglutinin, is the principal antigen in the influenza vaccine.

The standard flu vaccine contains a mix of four versions of hemagglutinin -; one for each of four commonly circulating influenza subtypes. The goal is to protect us from whichever of those subtypes eventually slips through our nostrils and takes up residence in our airways.

The vaccine's efficacy isn't as high as it could be, though. In recent years its effectiveness has ranged between about 20% and 80%, said Mark Davis, PhD, professor of microbiology and immunology and the Burt and Marion Avery Family Professor of Immunology.

That's largely because many vaccinated people fail to develop enough antibodies to one or more of the subtypes represented in the vaccine, said Davis, the study's senior author. The lead author is Vamsee Mallajosyula, PhD, a basic science research associate in Davis' lab.

Strangely, most of us develop a robust antibody response to only one of them, Davis said. But he and his colleagues have figured out why that happens and have found a way to force our immune systems to mount a strong antibody response to all four subtypes. That could make a huge difference in the vaccine's ability to keep us from suffering even mild consequences from influenza infections, let alone more severe ones.

How it works

It's widely believed that individuals' immune responses are partially due to what immunologists refer to, tongue in cheek, as "original antigenic sin," Davis said. "The idea is that our first exposure to a flu infection predisposes us to mount a response to whatever subtype that infecting virus belonged to. Subsequent influenza exposures, regardless of which viral subtype is now assaulting us, will trigger a preferential or even exclusive response to that first subtype." It's been thought that we're marked for life, immunologically speaking, by that initial encounter regardless of which subtype is bugging us now.

But that's not true. An analysis conducted by Mallajosyula showed that it's mostly our genes, not our first exposure, that push our immune systems to mount an antibody response to one or another of a flu shot's four subtypes. Mallajosyula found this uneven immune response to different influenza subtypes (what immunologists call "subtype bias") in most people, including 77% of identical twins -; and 73% of newborns, who've had no previous exposure to the flu virus or the vaccine for it.

Davis' group has found a way to trick our immune systems into paying attention to all four subtypes represented in the vaccine. Here's how it works.

B cells -; the immune cells that serve as our body's antibody factories -; are ultrapicky about exactly which antibodies they make. An individual B cell will produce only a single species of antibody fitting a mere one or very few antigenic shapes. That B cell is just as picky about what antigen it will pay attention to: that is, precisely the antigen the B cell's antibodies will stick to. When this antigen comes along, the B cell recognizes it and gobbles it up.

That's step one.

Next, the B cell chops the antigen up into tiny strips called peptides, which it displays on its surface for inspection by roving immune cells called helper T cells, whose follow-on stimulatory services are critical for turning antigen-displaying B cells into antibody-spewing B cells.

Helper T cells are just as finicky as B cells. A helper T cell will sprinkle its stardust only on B cells displaying antigen-derived peptides that particular T cell is designed to respond to -; and even then, only when that peptide is gripped by one of the matching molecular jewel cases that B cells produce in myriad varieties.

But different peptides require different jewel cases. And depending on their luck in the genetic draw, people's repertoires of those specialized jewel cases vary from one person to the next, leaving many of us with plenty of the jewel cases that match peptides from one influenza-subtype hemagglutinin but far fewer of those that match another flu subtype's peptides.

In the standard flu-vaccine formulation, the four antigens corresponding to the four common subtypes are delivered as separate particles in a mix. To overcome subtype bias, Davis, Mallajosyula and their colleagues stitched all four antigens together. They designed a vaccine in which the four hemagglutinin varieties are chemically conjoined on a molecular matrix scaffolding. That way, any B cell that recognizes and begins ingesting one or another of the vaccine's four hemagglutinin types ends up wolfing down the entire matrix and displaying bits of all four antigens on its surface, persuading the immune system to react to all of them despite its predisposition not to.

Forcing B cells to "eat their broccoli" -; internalize all four hemagglutinin subtypes instead of just the one that tastes best -; effectively multiplies the number of B cells displaying hemagglutinin-derived peptides from every subtype on their surfaces, albeit still in a ratio skewed by the B cells' uneven inventories of jewel-case molecules.

This, in turn, makes helper T cells much more likely to stumble on a sample from the antigen they love to hate. They fire up, start multiplying feverishly, branch out in pursuit of any B cells displaying that antigen and spur antibody production in them. These selected B cells also proliferate, culminating in bulk production of antibodies that are likely to stop the influenza virus -; whatever its subtype -; in its tracks.

Human tonsil organoids

Davis, Mallajosyula and their colleagues tested their four-antigen vaccine construct by putting it into cultures containing human tonsil organoids -; living lymph tissue originating from tonsils extracted from tonsillitis patients and then disaggregated. In a laboratory dish, the tissue spontaneously reconstitutes itself into small tonsil spheres, each a "mini-me" that acts just like a lymph node -; the ideal environment for antibody manufacturing.

Sure enough, B cells in these organoids that recognized any of the four conjoined hemagglutinin molecules swallowed the whole matrix and, potentially, displayed bits of all four subtypes, thus recruiting far more helper T cells to kick-start their activation. The result was solid antibody responses to all four influenza strains.

There is considerable concern about a viral strain that could cause the next devastating pandemic: namely avian or "bird flu," which recently has been detected in wastewater and milk in California, Texas and other parts of the United States. While this type of flu is not yet able to be transmitted easily between human beings, it could mutate to gain this ability and thus is considered a major risk-in-waiting.

The scientists further showed that they could substantially boost the antibody response to bird flu by vaccinating tonsil organoids with a five-antigen construct connecting the four seasonal antigens along with the bird-flu hemagglutinin, as opposed to getting a tepid response when vaccinating with just the bird-flu hemagglutinin or combining it with the four seasonal antigens on different constructs.

Overcoming subtype bias this way can lead to a much more effective influenza vaccine, extending even to strains responsible for bird flu. The bird flu could very likely generate our next viral pandemic."

Mark Davis, PhD, professor of microbiology and immunology and the Burt and Marion Avery Family Professor of Immunology

Davis and Mallajosyula are co-inventors on a patent Stanford's Office of Technology Licensing has filed for intellectual property related to their coupled-antigen methodology.

Researchers from the University of Cincinnati College of Medicine contributed to the work.

The study was funded by National Institutes of Health (grants 5U19AI090019, 5U19AI057229, 5U01AI144673, 75N93019C00051 and U01AI144616) and the Howard Hughes Medical Institute.