This randomized study assessed eight different formulations of influenza vaccine, adjuvanted with AS03 (squalene and tocopherol oil-in-water emulsion) with or without MPL, and a contour plot model was used to identify an optimal formulation for vaccination of older adults based on immunogenicity. Age-related declines in innate, and adaptive humoral and cell-mediated immunity, is thought to impair the ability to resist influenza infection and response to vaccination [26, 27], therefore, the rationale for formulating seasonal influenza vaccine with the adjuvant was to enhance immunogenicity and potentially improve protection of existing vaccines.
HI antibody responses with non-adjuvanted vaccine in the younger (18–40 years) control group were consistent with responses reported by randomized immunogenicity trials with this vaccine (Fluarix™), and responses in older (≥65 years) people to non-adjuvanted control vaccine were relatively low; seroprotection rates (proportion with titer ≥1:40) were 92.4–100% in the 18–40 year old group compared with 70.2–97.3% in the older group. In older participants, HI antibody responses to all adjuvanted formulations appeared slightly higher than responses to non-adjuvanted vaccine; however, because 200 participants per group did not provide sufficient power to compare the groups, a statistical model based on HI GMTs was used to objectively rank the different formulations. In the model, the lower limit of the 90% CI for the GMT ratio for each adjuvanted formulation versus non-adjuvanted was used to assign a ‘desirability index’ for each formulation, which involved transforming the CI to a value between 0 and 1, where 0 was undesirable and 1 was most desirable. Contour plots were used to characterize the desirability of each formulation based on the immunogenicity against each vaccine strain, and all three strains.
The contour plots showed that the effect of adjuvantation on the humoral response to vaccination was strain-dependent. For HI antibody responses against the A/Solomon Islands H1N1 and A/Wisconsin H3N2 strains, all AS03 formulations had the highest desirability, but for the B/Malaysia strain, an increase in desirability was observed when the AS03 dose approached a maximum level. The model also indicated that whereas inclusion of MPL appeared to increase desirability based on HI responses against the B/Malaysia strain, no clear effect was shown for the A/Solomon Islands H1N1 and the A/Wisconsin H3N2 strains.
The unexpected observations for the influenza A strains lead to a lack of discrimination within the initial plots, so we performed post hoc analyses applying more stringent criteria to further evaluate potential differences between the formulations. In the post hoc model, a higher AS03 dose, but not MPL, increased the desirability based on HI responses against both of the influenza A strains. Overall for the three vaccine strains, the dosage of MPL had a limited effect in discriminating the formulations in terms of HI response despite the fact that the AS03A-MPL25 formulation had the highest score. The contour plots showed that AS03 dosage had a clear effect with relatively high AS03 content formulations needed to achieve an optimal HI antibody response. Increased immune responses with increasing AS03 dosage was further confirmed by the HI immunogenicity results, although the sample size in each group did not allow definite statistical conclusions.
In addition to the provision of CD4 T cell help for B cell differentiation, both CD4 effector and memory T cells appear to have multifaceted roles in the protective responses to influenza infection [23, 28]. CD4+ T-helper cells are polarized into T helper 1 or T helper 2 cells, and T helper 1 cells predominantly secrete IFN-γ, which activates macrophages and facilitates clearance of intracellular pathogens [29–32]. Impairment of T cell responses due to aging is therefore considered to be an important factor that limits the responses to vaccination, and it has been proposed that correlates of vaccine-mediated protection against influenza in older adults should include measures of cell-mediated responses [5, 33]. In our study, adjuvanted vaccine also enhanced CD4 T cell-mediated responses to all three vaccine strains compared with non-adjuvanted vaccine in elderly people, with the most marked impact on responses to the pooled strains was observed for the AS03A, AS03B and AS03A-MPL25 formulations. This indicates that the presence of MPL does not appear, in this setting, to be essential to achieve maximal enhancement of CD4 T cell responses to influenza antigens.
Oil-in-water adjuvants have been shown to enhance the response to vaccination by triggering chemokine production resulting in recruitment of immunocompetent cells to the injection site, cell maturation into a dendritic phenotype, the stimulation of antigen uptake into these cells, and facilitation of subsequent migration to the lymph nodes [22, 34–36]. The adjuvant activity of AS03 is highly dependent on the presence of the immunostimulant α-tocopherol (an isoform of vitamin E) , which distinguishes AS03 from other oil-in-water emulsion-based adjuvants. The oil-in-water adjuvant MF59™ contains polysorbate 80, sorbitan trioleate, trisodium citrate dehydrate, citric acid monohydrate, and has been shown to enhance immune responses to seasonal influenza vaccine compared with non-adjuvanted vaccine [10, 38]. An MF59™-adjuvanted influenza vaccine (Fluad™) is licensed and widely used, and has been shown to significantly reduce hospitalization for influenza and pneumonia compared with non-adjuvanted vaccine in elderly people . However, there have been no prospective, randomized clinical trials of vaccine efficacy of this formulation in people aged ≥65 years .
Seasonal influenza vaccines are licensed based on demonstrating the induction of HI antibody titers above a defined threshold; a HI antibody titer of 1:40 is generally accepted as corresponding to a 50% reduction in the risk of influenza, which is based on a challenge study conducted forty years ago . However, although humoral immune responses measured by HI may be a good correlate of protection in younger adults, it has been suggested that HI titers may not be an adequate surrogate for protection against influenza in older adults. Indeed, vaccine efficacy data from field trials are considered to provide a more meaningful measure of the benefits of influenza vaccination than serological data in elderly populations. Based on the results of our Phase II study, AS03B (5.93 mg tocopherol) without MPL was selected for further evaluation as it provided the best balance between the immune response and reactogenicity; AS03-adjuvanted versus non-adjuvanted trivalent influenza vaccine was evaluated in a Phase III study including 43,000 people in 15 countries, representing the largest field study to date to assess influenza vaccine efficacy in people aged ≥65 years (NCT00753272). In addition to vaccine efficacy, the study evaluated clinical outcomes, immunogenicity, reactogenicity and safety .
In addition to enhancing immunogenicity, the incorporation of adjuvant components into a vaccine can also have an impact on the safety/reactogenicity profile. We observed that in the 7 days following vaccination, overall reactogenicity was higher for the eight adjuvanted influenza formulations than for non-adjuvanted influenza vaccine in ≥65 year old participants. In general, the incidence of most reactions in the adjuvanted vaccine groups remained within the same range as those induced by non-adjuvanted control vaccine in the young adult group. A dose-range effect was observed, with the highest incidence of reactions associated with the AS03A formulations. The presence of MPL also tended to increase reactogenicity of the influenza vaccine. Nevertheless, the majority of adverse events reported following administration of all eight adjuvanted formulations were mild to moderate in nature, and no clinically observable safety concerns were raised. Ultimately, assuming similar safety profiles, the public acceptability of the reactogenicity profiles for the different formulations in the days immediately following vaccination will depend upon the extent of the clinical benefit. The selection of final candidate formulations must therefore be based on a balance between improvement in immunogenicity and increase in reactogenicity.
Limitations of the current study included that the randomization procedure did not take previous vaccination history into account, or that there may have been a tendency for more healthy participants to be recruited, which cannot be adjusted for in the analyses. Another possible limitation of the study is that a relatively low sample size was used in the testing of cell-mediated immunity. Although it is not a limitation, it should be noted that the GMT ratios used in the primary endpoint was calculated using 90% CIs instead of the more usual 95% CIs. The desirability model was based only on the GMT ratios, and the reactogenicity results were descriptive. In addition, descriptive immunogenicity (SCR, SCF and SPR) was based on HI antibody assays, as this measure is the basis of the established correlate of protection in seasonal influenza; other measures of immunogenicity such as microneutralization assay and single radial haemolysis were not used.