Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms

Abstract

Adjuvanted protein vaccines offer high efficacy, yet most potent adjuvants remain proprietary. Several adjuvant compounds are being developed by the Vaccine Formulation Institute in Switzerland for global open access clinical use. In the context of the R21 malaria vaccine, in a mouse challenge model, we characterize the efficacy and mechanism of action of four Vaccine Formulation Institute adjuvants: two liposomal (LQ and LMQ) and two squalene emulsion-based adjuvants (SQ and SMQ), containing QS-21 saponin (Q) and optionally a synthetic TLR4 agonist (M). Two R21 vaccine formulations, R21/LMQ and R21/SQ, offer the highest protection (81%-100%), yet they trigger different innate sensing mechanisms in macrophages with LMQ, but not SQ, activating the NLRP3 inflammasome. The resulting in vivo adaptive responses have a different T_H1/T_H2 balance and engage divergent innate pathways while retaining high protective efficacy. We describe how modular changes in vaccine formulation allow for the dissection of the underlying immune pathways, enabling future mechanistically informed vaccine design.

Keywords: NLRP3; QS-21; TLR4; adjuvants; emulsions; inflammasome; liposomes; malaria; saponin; vaccines.

Graphical abstract

Figure 1

LMQ and SQ promote strong protection against malaria through distinct humoral responses (A) Schematic of R21 antigen and adjuvants LQ, LMQ, SQ, and SMQ. (B) Summary of the experimental protocol. Vaccine dose: 1 μg of R21; 25 μL of each adjuvant (containing 5 μg of QS-21 saponin with or without 2 μg of TLR4 agonist 3D6AP). (C) Survival post-malaria challenge: BALB/c mice were vaccinated and challenged as in (B). Parasitemia was assessed by daily blood smears with 1% parasitemia taken as irreversible malaria infection. ∗p < 0.05, ∗∗p < 0.01, log rank (Mantel-Cox) test. (C), (D), (F) and (G) show pooled data from three independent experiments; n = 16 R21, LQ, SQ, SMQ; n = 24 naive, LMQ. ∗p < 0.05, ∗∗p < 0.01. 01. Each symbol represents an individual mouse. (D) Serum anti-NANP IgG, IgM, and IgA isotype titers were assessed by ELISA. (E) Serum anti-NANP total IgG titers of mice protected (+, n = 50) vs. unprotected (−, n = 22) from malaria challenge across all groups. Data pooled from three independent experiments; median + replicates; ∗∗p < 0.01, Mann-Whitney test. Correlation analysis for (E), (J), and (M) was performed by point-biserial correlation; r_pb and p values are shown in the panels. (F) Anti-NANP IgG subclasses by proportional ELISA (Mean + SEM). (G) T_H1/T_H2 index of adjuvant-induced IgG subclasses calculated as ([IgG2a + IgG3]/2)/IgG1. Increased T_H1/T_H2 index indicates T_H1-skewed immune response (median + replicates; ∗p < 0.05, ∗∗p < 0.01). (H) Total anti-NANP IgG avidity, measured by NaSCN displacement assay (n = 8). (I) Complement activation with serum anti-NANP IgG measured by C1q deposition assay (data of one experiment; median + replicates; n = 7, LQ; n = 8, LMQ, SQ; ∗p < 0.05). (J) Complement activation of mice protected (+) vs. unprotected (−) from malaria challenge. Graph includes mice from adjuvanted groups depicted in (I) (data of one experiment; median + replicates; n = 16, protected [+]; n = 7, unprotected [−]; ∗p < 0.05, Mann-Whitney test). (K) Summary of experimental protocol for inhibition of sporozoite invasion assay (ISI). (L) ISI assay of indicated groups. Graph shows reduction of sporozoite entry into hepatocytes compared to control in percent (data of one experiment; median + replicates; n = 8; ∗∗p < 0.01). Analysis for (F), (G), (I), and (L) was performed by Kruskal-Wallis ANOVA with Dunn’s multiple comparisons. (M) ISI assay of mice protected (+) vs. unprotected (−) from malaria challenge. Graph includes mice from adjuvanted groups depicted in (L) (data of one experiment; median + replicates; n = 16, protected [+]; n = 8, unprotected [−]; ∗∗p < 0.01, Mann-Whitney test).

Figure 2

Protective adjuvants LMQ and SQ trigger different innate pathways in vitro (A) Summary of the experimental protocol. (B) IL-1β and TNF-α secretion in supernatants after stimulation of WT BMDMs with indicated amounts of adjuvants was measured by ELISA. LDH release was measured using a colorimetric assay and is depicted as a percentage of lysed positive control (representative data from three independent experiments; cells are stimulated in triplicates; mean ± SEM are shown). (C) BMDMs derived from C57BL/6 WT and Nlrp3^−/− mice were stimulated with adjuvants (1:20 dilution). Cytokines and LDH were measured as described in (B). LPS/nigericin (100 ng/mL LPS for 6 h with 5 μM nigericin for the last 1 h) stimulation was used as positive control (pooled data from three independent experiments; cytokines were normalized to LPS/nigericin control set to 1; cell death/LDH was normalized to maximal cell death control, set to 100%; cells were stimulated in triplicates; mean ± SEM are shown). (D) Representative western blots for pro-caspase-1 (p46), cleaved caspase-1 (p20), NLRP3, and GAPDH in cell lysates and supernatants from cultures of WT and Nlrp3^−/− BMDMs stimulated with adjuvants (1:20 dilution) or LPS/nigericin (representative data from two independent experiments). (E) Summary of the experimental protocol. (F) HMDMs were stimulated for 6 h with adjuvants (1:20 dil.) in the presence or absence of R21 (given at 1/5 of mouse dose to preserve the ratio of Ag:adjuvant given in vivo), in the presence or absence of NLRP3 inhibitor MCC950 (10 μM) added 0.5 h before the adjuvants. IL-1β and TNF-α secretion in supernatants was measured by ELISA. LDH release was measured using a colorimetric assay. LPS/nigericin served as a positive control (100 ng/mL LPS for 6 h with 10 μM nigericin for the last 2 h) (pooled data from three independent experiments/donors for IL-1β and LDH and two independent experiments/donors for TNF-α; cells are stimulated in triplicates; cytokines were normalized to LPS/nigericin control set to 1; cell death/LDH was normalized to maximal cell death lysis control, set to 100%; mean ± SEM are shown). (G) Representative western blots for pro-caspase-1 (p46), cleaved caspase-1 (p20), NLRP3, and GAPDH in cell lysates and supernatants from HMDMs, stimulated as in (F) (representative data from two independent experiments/donors are shown). All statistical analyses were done using two-way ANOVA with Bonferroni’s correction for multiple comparisons; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. In (B), significant adjuvant effect is reported. In (C) and (F), significant genotype effect is reported.

Figure 3

Protective adjuvants LMQ and SQ trigger disparate innate responses in vivo (A) Summary of experimental protocol. Site of injection (SOI). (B) Indicated cytokines were measured by LEGENDplex assay (dashed line = mean of naive group; mean ± SEM; n = 5; ∗p < 0.05, ∗∗p < 0.01, Mann-Whitney test per time point between SQ/LMQ). (C) Heatmap of indicated cytokines, measured as described in (B). (D) Summary of experimental protocol. (E) Indicated cytokines were measured by LEGENDplex assay (mean ± SEM; n = 6; ∗p < 0.05, ∗∗p < 0.01, Mann-Whitney test per adjuvant between ± MCC950).

Figure 4

R21/LMQ and R21/SQ induce disparate CD4⁺ T cell responses (A) Summary of the experimental protocol. C57BL/6 mice require three vaccine doses to develop full response against R21. (B) Representative FACS plots of R21 peptide pool stimulated splenocytes from mice with indicated genotype and vaccine adjuvant used. Graphs show IFN-γ and TNF-α double-positive CD4⁺ T cells. Cells were pre-gated on live single cells, CD19⁻, CD3⁺, and CD8⁻. (B and C) Representative data from two experiments; mean ± SEM; n = 3 for WT/R21, WT/LMQ, and WT/SQ; n = 4 for Nlrp3^−/−/R21; n = 5 for Nlrp3^−/−/LMQ and Nlrp3^−/−/SQ. (C) Quantification and summary of data represented in (B). (D) IFN-γ and IL-13 in supernatants of R21 peptide pool stimulated splenocytes from mice with indicated genotype and vaccine adjuvant used, as measured by ELISA. Representative data from two experiments. (E and F) Pooled data from three experiments; median + replicates; n = 9, WT/R21; n = 13, Nlrp3^−/−/R21; n = 12, WT/LMQ; n = 22, Nlrp3^−/−/LMQ; n = 13, WT/SQ; n = 20, Nlrp3^−/−/SQ. (E) Anti-NANP serum antibody titers of indicated isotypes. C57BL/6 WT or Nlrp3^−/− mice were vaccinated and sampled as indicated in (A). Titers were assessed by ELISA. (F) Detection of anti-NANP IgG subclasses by proportional ELISA. Each sample was diluted and normalized to 80 ng/mL of total IgG. Graphs show optical density against indicated IgG subclasses. Pooled data from three independent experiments; mean ± SEM; n = 12, WT/LMQ; n = 22, Nlrp3^−/−/LMQ; n = 13, WT/SQ; n = 20, Nlrp3^−/−/SQ. (G) T_H1/T_H2 index of adjuvant-induced subclass patterns. Index was calculated as ([IgG2c + IgG3]/2)/IgG1. Increased T_H1/T_H2 index indicates T_H1-skewed immune response. Pooled data from three independent experiments; median + replicates; n = 12, WT/LMQ; n = 21, Nlrp3^−/−/LMQ; n = 13, WT/SQ; n = 19, Nlrp3^−/−/SQ; all statistical analyses were done using two-way ANOVA of adjuvanted groups with Bonferroni’s multiple comparisons; ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.