Adjuvant Selection for Influenza and RSV Prefusion Subunit Vaccines

Abstract

Subunit vaccines exhibit favorable safety and immunogenicity profiles and can be designed to mimic native antigen structures. However, pairing with an appropriate adjuvant is imperative in order to elicit effective humoral and cellular immune responses. In this study, we aimed to determine an optimal adjuvant pairing with the prefusion form of influenza haemagglutinin (HA) or respiratory syncytial virus (RSV) fusion (F) subunit vaccines in BALB/c mice in order to inform future subunit vaccine adjuvant selection. We tested a panel of adjuvants, including aluminum hydroxide (alhydrogel), QS21, Addavax, Addavax with QS21 (AdQS21), and Army Liposome Formulation 55 with monophosphoryl lipid A and QS21 (ALF55). We found that all adjuvants elicited robust humoral responses in comparison to placebo, with the induction of potent neutralizing antibodies observed in all adjuvanted groups against influenza and in AdQS21, alhydrogel, and ALF55 against RSV. Upon HA vaccination, we observed that none of the adjuvants were able to significantly increase the frequency of CD4⁺ and CD8⁺ IFN-γ⁺ cells when compared to unadjuvanted antigen. The varying responses to antigens with each adjuvant highlights that those adjuvants most suited for pairing purposes can vary depending on the antigen used and/or the desired immune response. We therefore suggest that an adjuvant trial for different subunit vaccines in development would likely be necessary in preclinical studies.

Keywords: RSV; adjuvant; influenza; microbiology; vaccine; virology.

Figure 1

Serum analysis of terminal bleed from mice immunized with clamped antigen with different adjuvants. (A) Schematic of immunization regime. (B) ELISA EC₅₀ of serum against each vaccine antigen on day 43. Dotted line shows limit of detection. (C) Serum samples from final cardiac bleed (day 43) tested against homologous H1N1pdm (A/Auckland/1/2009, left) virus or respiratory syncytial virus (RSV) A2 (right) in a plaque reduction neutralization test (PRNT). Data represents geometric mean (n = 4) with geometric standard deviation (SD). Dotted line shows limit of detection. p-values were calculated using a one-way Tukey’s multiple comparison ANOVA, where * indicates p < 0.05, ** p < 0.005, *** p < 0.0005, and **** p < 0.0001; β = p < 0.0001 compared to all groups except PBS, and α = p < 0.0001 compared to all groups unless otherwise specified. (D) Linear regressions of PRNT IC₅₀ vs. total IgG EC₅₀ from groups vaccinated with RSV fusion (F) clamp (left) and haemagglutinin (HA) clamp (right).

Figure 2

Antibody isotyping of mouse sera post antigen immunization with various adjuvants. Data shown is from the final terminal bleed. Sera from individual mice (n = 4) were isotyped by ELISA against the vaccine antigen with secondary antibodies specific for mouse IgG1 (A), IgG2a (B), IgG2b (C), and IgG3 (D) with PBS background subtracted. Data represents geometric mean with geometric SD. Dotted line shows limit of detection. A heat map is shown for each isotype, with values representing means of each group. Statistics calculated using a one-way Tukey’s multiple comparison ANOVA, where * indicates a p < 0.05, ** p < 0.005, *** p < 0.0005, and **** p < 0.0001; β = p < 0.0005 compared to all groups except PBS, and α = p < 0.0001 compared to all groups unless otherwise specified.

Figure 3

CD4⁺ and CD8⁺ T-cell cytokine profiling of mice immunized with HA clamp alongside various adjuvants. Data shown is from day 43 spleen harvest. Data shown is after subtraction of PBS-stimulated cells. Data represents mean (n = 4) with SD. Statistics calculated using a one-way Tukey’s multiple comparison ANOVA, where * indicates a p < 0.05, ** p < 0.005, *** p < 0.0005, and **** p < 0.0001.