The seasonal influenza vaccine contains strains of viruses from distinct virus subtypes, but most people who are vaccinated will mount a strong immune response to one strain, leaving them vulnerable to infection by the others. Researchers have long wondered whether host genetics or prior exposure to virus strains impacts more on these variable responses.
A study by Stanford Medicine scientists now indicates that host genetics is a stronger driver of individual differences in influenza vaccine response. The team’s reported study also presents a novel vaccine platform that improved protection against diverse influenza subtypes when tested in animal models and in human tonsil-derived organoids. The research suggests that the new approach might also be used to help protect against new flu variants with pandemic potential.
Research lead Mark Davis, PhD, professor of microbiology and immunology and the Burt and Marion Avery Family Professor of Immunology, and colleagues, reported on their findings in Science, in a paper titled “Coupling antigens from multiple subtypes of influenza can broaden antibody and T cell responses.”
The influenza virus kills hundreds of thousands of people every year and sends millions to hospitals, the authors explained. 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 that can bind selectively to prevent a particular virus from infecting and replicating inside our cells.
There are many different known influenza subtypes, but human infections are primarily restricted to influenza A (H1N1 and H3N2 subtypes) and B viruses (Victoria and Yamagata lineages), the authors continued. “Each influenza subtype includes several viral strains. A seasonal vaccine is formulated each year with the strains of each type that are predicted to be most prevalent in circulation.”
Any classical vaccine displays one or more of a pathogen’s antigens to various cells of the immune system whose job is to carefully note and memorize specific antigens belonging to that target pathogen. In the event of future infection by the pathogen, the immune system memory will kick in and rouse dormant immune cells to mount an attack.
The influenza virus is studded with the hemagglutinin (HA) molecule that acts like a molecular hook to latch on to vulnerable cells in our airways and lungs. 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 we may be exposed to.
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 senior study author Davis. “This has led to calls for the development of a more effective vaccine,” the team continued.
Interestingly, the authors continued, a large fraction of vaccinated individuals exhibit a higher response to one strain in the vaccine formulation, and are vulnerable to infection by the others. “The development of an effective influenza vaccine remains challenging. A major issue with the current vaccines is that most individuals respond better to the strain that they are biased for and thus may have little protection against infection by other strains.”
It’s widely believed that individuals’ immune responses are partially due to what immunologists refer to 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 we are subsequently exposed to. “OAS refers to the preferential induction of antibodies with higher affinity to the priming immunogen resulting from prior exposures as opposed to the boosting immunogens present in the seasonal vaccine formulation,” the authors further explained. “Thus, memory from past exposures can divert and limit the response to influenza strains in circulation.”
Genetic variations in the human leukocyte antigen (HLA) system also shape how individuals process and present vaccine antigens, influencing immune outcomes. But, as the team noted, “The relative contributions of previous heterologous exposures and host genetics are poorly understood.”
To address this question, first author Vamsee Mallajosyula, PhD, a basic science research associate in Davis’ lab, and colleagues analyzed antibody responses in monozygotic twins, vaccinated infants, and mouse models, to find that this uneven immune response to different influenza subtypes—what immunologists call “subtype bias”—is primarily driven by the individual’s genetics, particularly major histocompatibility complex (MHC) class-II polymorphisms, and that prior exposure plays a secondary role.
The investigators found this 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. “By studying a monozygotic twin cohort, we found that although prior exposure is a factor, host genetics are a stronger driver of subtype bias to influenza viral strains,” the scientists wrote in their paper.
B cells are the immune cells that serve as our body’s antibody factories, and an individual B cell will produce only a single species of antibody that fits only one or very few antigenic shapes. The B cell will also recognize the antigen that the B cell’s antibodies will stick to. When the B cell finds and recognizes this antigen it breaks it up into peptides that are then displayed on the B cell surface and recognized by helper T cells, which are critical for turning the antigen-displaying B cells into antibody-producing B cells.
In the standard flu vaccine formulation, the four antigens corresponding to the four common subtypes are delivered as separate particles in a mix. “The seasonal influenza vaccine contains strains of viruses from distinct subtypes that are grown independently and then combined,” the authors explained. They hypothesized that B cells expressing cell-surface B cell receptors (BCRs) that are subtype-specific would thus bind to and primarily internalize only their target HA in the current vaccines. “Consequently, these B cells would present peptides from only one antigen, which could limit CD4+ T cell responses owing to a bias in MHC-II peptide presentation,” they wrote. They reasoned that “ … covalently coupling heterologous HA antigens from distinct influenza subtypes might allow B cells to internalize the entire complex and recruit help from a much broader array of T cells to induce an antibody response to multiple strains after vaccination.”
Due to this, Davis, Mallajosyula, and their colleagues designed a vaccine in which the four hemagglutinin varieties are chemically conjoined on a molecular matrix scaffolding. That way, any B cell that recognized and started ingesting one of the four HA antigens in the vaccine, would internalize the entire matrix and then display bits of all four antigens on its surface, persuading the immune system to react to all.
Forcing B cells to internalize all four hemagglutinin subtypes instead of just one then multiplies the number of B cells displaying hemagglutinin-derived peptides from every subtype on their surfaces. This, in turn, makes helper T cells much more likely to encounter their target antigen, ultimately activating antibody production and B cell proliferation, culminating in bulk production of antibodies that are likely to stop the influenza virus—whatever its subtype.
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 acting just like a lymph node—the ideal environment for antibody manufacturing.
Tests showed that B cells in these organoids that recognized any of the four conjoined hemagglutinin molecules swallowed the complete 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. “We found that covalent coupling of heterologous hemagglutinin (HA) from different viral strains could largely eliminate subtype bias in an animal model and in a human tonsil organoid system,” the scientists wrote.
There is considerable concern about the potential for an avian flu strain detected in parts of the United States to represent a future pandemic. While this type of flu is not yet easily transmitted between human beings, it could mutate to gain this ability and thus is considered a major risk-in-waiting. “Highly pathogenic avian influenza, with its very high mortality rate, represents a major pandemic threat if it becomes transmissible through air,” the researchers wrote. “Thus, we tested whether we could augment the response to an avian influenza HA by providing additional T cell help.”
Through their tests, the scientists confirmed 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. This boosted response contrasted with the tepid response when vaccinating with just the bird-flu hemagglutinin or combining it with the four seasonal antigens on different constructs. “Whereas tonsil organoids stimulated with H5N1 HA alone induced very weak antibody responses, a coupled heterologous antigen with an optimal ratio of H5N1 HA and seasonal HA that maximized cross-subtype T cell help induced higher antibody responses.” Davis added, “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.”
In their paper, the team pointed out, “For many decades, the OAS hypothesis has influenced the explanation of subtype bias.” However, they further noted, that it doesn’t suggest any practical way that influenza vaccines could be altered to overcome this problem. “By contrast, our study shows that coupling heterologous antigens may broaden T cell help and improve vaccine efficacy. This strategy to augment T cell help is readily applicable to vaccines for other pathogens for which multistrain coverage is needed.”
The authors also pointed out that immune organoids such as the tonsil-cell cultures used in their study could offer a fast way to test vaccine constructs and hypotheses. 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.
