The Achille’s heel of influenza

Influenza virus evolves so rapidly that last year’s antibodies usually cannot protect us from this year’s viral strain. This is due to specific characteristics of the virus that make it highly prone to mutate. First, its RNA polymerase complex has no proofreading activity, so small mutations tend to accumulate randomly. Second, the segmented nature of the influenza genome (in which coding sequences are located on individual RNA segments) increases the risk of reassortment.

If this was not enough, it has become evident that the immune status of the host population can shape influenza virus evolution: in other words, flu viruses evolve in response to the antibodies our bodies produce each year. This is supported by the observation that the influenza virus undergoes faster antigenic changes in long-lived humans that accumulate immune memory than in short-lived swines that are mostly naïve.

Most of the antibodies that our body produces to neutralize the flu virus target epitopes on the head of influenza’s hemagglutinen (HA) protein. This region is highly susceptible of mutational change and, by natural selection, mutations that allows the viral particle to escape the immunodominant antibodies generated by our immune response will be favoured and will manage to replicate and spread. This process leads to the evolution of new viral strains and, therefore, to the requirement of annual changes in vaccine composition.

Different traits evolve at different rates

While those traits that experience strong selection evolve quickly, key traits that, if altered, would compromise the functioning of the virus tend to evolve slowly. Several studies have shown that the head domain of HA is more tolerant to mutations than the stalk domain; in fact, the stem of HA, which is involved in the process of viral fusion, is a relatively conserved region across a wide range of flu strains. Most of the influenza antibodies are directed against the variable regions of HA’s globular head, with only few available antibodies that target the stem region. These rare stem-targeting antibodies are able to broadly recognize and neutralize many viral strains.

This raises important questions, including: Is it possible to select antigenic variants using these broad stem-targeting antibodies or are these epitopes entirely refractory to change? In vitro, even viruses that we effectively vaccinate against can generate selectable mutants (e.g. measles virus and polio virus); therefore, the finding that it is possible to select influenza mutants resistant to broad antibodies, does not necessarily mean that, in vivo, influenza virus will just escape broad antibodies as easily as it escapes narrow strain-specific ones.

One obstacle to answering the above questions has been the absence of studies where the ease of viral escape has been quantified in a way that can be compared across antibodies. To address this, Doud and colleagues have mapped all viral escape mutations to H1 hemagglutinin that increase resistance to broad and narrow antibodies.

Quantifying the antigenic effects of mutations to study viral antibody escape

These authors found that the magnitudes of the antigenic effects vary greatly across antibodies. While single mutations can make the virus completely resistant to narrow strain-specific antibodies, no single mutation does more than modestly increase the virus’s resistance to broad antibodies against the HA stalk. Therefore, compared to strain-specific antibodies, broad anti-stalk antibodies are quantifiably more resistant to viral escape via single amino-acid mutations.

To determine the fraction of mutant virions that survive antibody neutralization for all mutations to HA, Doud et al. applied the following approach:

If a mutation escapes neutralization, then all virions with this mutation survive antibody treatment at a concentration where other virions are mostly neutralized. This resistance is manifested by a large shift (by orders of magnitude) in the neutralization curve for the mutant. Antibody selection on all viral mutations can be assayed in a single experiment by using mutational antigenic profiling. This involves generating viral libraries containing all mutations to the protein of interest, then selecting among these viruses using antibodies treatments, and finally using an accurate deep-sequencing method to determine the relative frequencies of each mutation.

These experiments were performed using anti-HA antibodies with a different range of target-breadths and epitopes: Doud et al. chose two broad antibodies that target the stalk of HA and three narrow strain-specific antibodies that bind the antigenic regions on HA’s globular head.

The narrow, strain-specific antibodies select mutations with large antigenic effects: for these antibodies, multiple sites in HA show mutations that enabled over a third of virions to infect at concentrations where virtually all wildtype virions were neutralized. In contrast, the stalk-targeting antibodies select no strong escape mutants: there are only a few sites where mutations slightly increase infection. Therefore, influenza viruses are far less capable of escaping these anti-stalk antibodies by single mutations than they are of escaping the narrow, strain-specific antibodies.

It is also interesting to notice that the antigenic changes induced by strain-specific antibodies occur at residues in or near the physical binding footprint of the antibody. Some of these mutations introduce glycosylation motifs near the epitope, and some others escape with other escamotages, for example, by altering HA expression levels, leading to lower HA density on virions and, therefore, antibody neutralization.

But why the anti-stalk antibodies do not select for effective escape mutants?

One possibility is that all HA sites in the antibody-binding footprint do not tolerate mutations, meaning that viruses with mutations at these sites cannot replicate and so are not present in the tested mutant virus libraries. Another possibility is that broadly neutralizing anti-stalk antibodies distribute their binding energetics in such a way that changes in mutationally-tolerant HA sites do not affect neutralization.

Broadly neutralizing antibodies hold particular promise in the search for antibodies that are resistance to viral evolutionary escape. Multiple studies now suggest that the use of these antibodies as a flu vaccine or a vaccine inducing antibodies against the stalk region may alleviate the need to reformulate and receive a flu shot every year. However, how long this broadly neutralizing effect would last and how long it would take before the emergence of escaping mutants (maybe through mutliple mutations) is currently not clear.