The oil-in-water adjuvant MF59 was shown (Khurana et al., 2011) to broaden the hemagglutinin (HA) epitope repertoire and raise the binding affinity of polyclonal anti-HA antibodies after pandemic H1N1 and H5N1 vaccination, with affinity tightly correlating with in vitro virus-neutralizing titers; the paper explicitly left open why this qualitative enhancement occurs, whether it extends to broadly cross-reactive stem-directed neutralizing antibodies, and whether it improves protection in vivo. Later work established parts of the mechanism in mice (MF59 promotes T follicular helper and germinal-center responses and intranodal antigen-presenting-cell differentiation) and showed that MF59-adjuvanted vaccines elicit stalk-directed IgG and uniquely raise HA-specific IgG avidity among seasonal vaccine platforms in older adults. However, the causal mechanism of the affinity gain in humans has not been identified, MF59's effect on the affinity of broadly neutralizing stem antibodies has not been determined, and no prospective study has demonstrated that affinity gains per se (independent of titer) translate into superior protection or reduced immunopathology. The problem is to resolve these three linked questions: the mechanistic causation of affinity enhancement, its applicability to stem-directed broadly neutralizing antibodies, and its translational value for protection and safety.

Background

Influenza vaccines based on the surface glycoprotein hemagglutinin (HA) are poorly immunogenic in immunologically naïve populations and in the elderly, which motivated the addition of adjuvants — components formulated with the antigen that enhance the magnitude and quality of the immune response. MF59 is a squalene-based oil-in-water nanoemulsion adjuvant, the first non-aluminum adjuvant licensed in human vaccines; it is used in adjuvanted seasonal influenza vaccines for older adults and was used at pandemic scale in 2009–2010 H1N1 and stockpiled H5N1 vaccines. HA consists of an antigenically variable globular head domain (HA1) and a conserved stem/stalk domain (largely HA2); most preexisting antibodies in adults target the HA2-containing stem, while neutralizing responses to vaccination are dominated by HA1 head antibodies. A rare class of broadly cross-reactive, stem-directed antibodies can neutralize across influenza A subtypes and protect in animal models, making them a target for universal influenza vaccines.

The quality of antibody responses is shaped in germinal centers (GCs) — transient structures in lymphoid tissue where B cells proliferate and diversify their immunoglobulin genes by somatic hypermutation (SHM, the introduction of point mutations into antibody variable-region genes), with mutants competing for antigen and for help from CD4+ T follicular helper (Tfh) cells; the outcome is affinity maturation, the progressive increase in antibody binding strength. Khurana et al. (2010) had shown with genome-fragment phage display libraries (GFPDL, a technique mapping the epitopes targeted by polyclonal serum antibodies) that an MF59-adjuvanted H5N1 vaccine expanded the antibody repertoire toward protective HA1 sites. Khurana et al. (2011) extended this: using GFPDL, surface plasmon resonance (SPR, which infers polyclonal antibody affinity from the dissociation off-rate of the antigen-antibody complex), and 7 M urea-wash ELISA (an avidity index), they found that MF59 selectively enhanced HA1-head antibody titers and epitope diversity in adults and children, increased antibody affinity (≥10-fold slower off-rates) in toddlers and children receiving the 2009 pandemic H1N1 vaccine, and improved affinity maturation across a three-dose H5N1 series in H5N1-naïve adults; for both vaccines, affinity correlated closely with virus-neutralizing (VN) titers. The authors concluded that MF59 quantitatively and qualitatively enhances functional antibody responses, but posed questions they could not answer: the paper asks literally "How is an oil-in-water adjuvant such as MF59 able to increase antibody affinity?", hypothesizing either a preferential effect on naïve B cells (possibly increasing SHM rates) or a difference in the requirements for activating naïve HA1-specific as opposed to recall HA2-specific B cells, and citing prior evidence that MF59-adjuvanted H5N1 vaccine primes antigen-specific CD4+ T cell help; it flags the effect of MF59 on the recently described broadly cross-reactive stem-directed antibodies as an open topic "of great interest", particularly whether MF59 could increase their affinity; and it describes improved in vivo protection from the affinity gains as a prediction requiring demonstration, motivated partly by evidence that low-affinity, complement-fixing antibodies can contribute to enhanced respiratory disease after vaccination or infection.

Problem Statement

The common objective is to establish the mechanistic and translational basis of the qualitative (affinity and epitope-breadth) enhancement of anti-HA antibody responses that MF59 produces: not only that it happens, but why, whether it extends to the antibodies most valued for broad protection, and whether it matters clinically. Specifically, following the demonstration by Khurana et al. 2011 that MF59 broadens the HA1 epitope repertoire and increases polyclonal anti-HA antibody affinity (with off-rate constants improving by an order of magnitude or more in young children and after serial H5N1 dosing, and affinity correlating with in vitro virus-neutralizing titers):

  1. Mechanism. Determine the molecular and cellular mechanism(s) by which MF59 raises the affinity of polyclonal anti-HA antibodies in humans, the population in which the affinity gain was demonstrated, and establish which of the processes hypothesized in that work or subsequently implicated in animal and human studies actually account for it: state which candidate processes are supported and which are excluded, and name the discriminating experiment(s) that would settle those left open.

  2. Stem-directed broadly neutralizing antibodies. Determine whether MF59 can preferentially elicit, or increase the affinity of, broadly cross-reactive stem-directed neutralizing antibodies (the heterosubtypic anti-stem specificities), and whether such effects would enhance the breadth and potency of protection in vivo.

  3. Translational linkage. Determine, from prospective animal or human challenge or observational evidence (existing or newly proposed), whether MF59-driven increases in antibody affinity and HA1 targeting translate into superior protection and reduced pathological responses relative to the same vaccine given without MF59, judged by the disease-severity endpoints of the challenge model or surveillance setting used (for example, avoidance of the low-affinity, complement-fixing antibodies implicated in enhanced respiratory disease), distinguishing affinity-mediated from titer-mediated protection.

These three questions are scientifically linked: (1) explains the cellular basis of the phenomenon, (2) asks whether it can be steered toward the antibody class most relevant to broad protection, and (3) tests whether the affinity gain carries clinical value; together they decide how MF59 and similar oil-in-water adjuvants should be used, and paired with which immunogens, in next-generation influenza vaccines.

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