Adjuvanticity of the oil-in-water emulsion MF59 is independent of Nlrp3 inflammasome but requires the adaptor protein MyD88 - PubMed (original) (raw)

Comparative Study

. 2011 Jul 5;108(27):11169-74.

doi: 10.1073/pnas.1107941108. Epub 2011 Jun 20.

Samuele Calabro, Laura Santini, Barbara Galli, Alessia Genovese, Sara Valentini, Susanna Aprea, Annalisa Colaprico, Ugo D'Oro, Marzia M Giuliani, Michele Pallaoro, Mariagrazia Pizza, Derek T O'Hagan, Andreas Wack, Rino Rappuoli, Ennio De Gregorio

Affiliations

Comparative Study

Adjuvanticity of the oil-in-water emulsion MF59 is independent of Nlrp3 inflammasome but requires the adaptor protein MyD88

Anja Seubert et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Oil-in-water emulsions have been successfully used to increase the efficacy, immunogenicity, and cross-protection of human vaccines; however, their mechanism of action is still largely unknown. Nlrp3 inflammasome has been previously associated to the activity of alum, another adjuvant broadly used in human vaccines, and MyD88 adaptor protein is required for the adjuvanticity of most Toll-like receptor agonists. We compared the contribution of Nlrp3 and MyD88 to the adjuvanticity of alum, the oil-in-water emulsion MF59, and complete Freund's adjuvant in mice using a three-component vaccine against serogroup B Neisseria meningitidis (rMenB). Although the basal antibody responses to the nonadjuvanted rMenB vaccine were largely dependent on Nlrp3, the high-level antibody responses induced by alum, MF59, or complete Freund's adjuvant did not require Nlrp3. Surprisingly, we found that MF59 requires MyD88 to enhance bactericidal antibody responses to the rMenB vaccine. Because MF59 did not activate any of the Toll-like receptors in vitro, we propose that MF59 requires MyD88 for a Toll-like receptor-independent signaling pathway.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

IL-1β cleavage and release from BM-DC. BM-DC were primed overnight with serial dilutions of LPS (10 ng/mL, 1 ng/mL, 0.1 ng/mL, 0 ng/mL). MF59 or alum were added to the cells at the indicated concentrations and cells were incubated for further 24 h. Cleavage of pro–IL-1β was assessed by Western blotting (A) (M: protein weight marker) and cytokine release into the culture supernatant was measured by multiplex-bead ELISA (B). BM-DC from Nlrp3−/− or WT mice were primed with 10 ng/mL of LPS and incubated overnight with MF59 (1:100, vol:vol) or alum (400 μg/mL) (C).

Fig. 2.

Fig. 2.

Antibody titers in Nlrp3−/− mice and WT controls after vaccination with plain or adjuvanted MenB antigens. (A) Total IgG antibody titers to all antigens contained in the vaccine: NHBA-GNA1030, GNA2091-fHBP, and NadA. Values represent means of Log10 titers of eight mice per group plus SD. Unpaired, two-tailed Student's t test was performed comparing WT vs. Nlrp3−/− and adjuvant vs. PBS injection (in WT mice). *P < 0.05 are considered significant and **P < 0.001 highly significant. (B) SBA of mouse polyclonal antibodies in the serum of eight single mice (black symbols) and their median value (line). Statistical analysis of WT vs. KO mice was performed using unpaired, two-tailed Mann-Whitney test. *P < 0.05.

Fig. 3.

Fig. 3.

Antibody titers in MyD88−/− mice and WT controls after vaccination with plain or adjuvanted MenB antigens. (A) Total IgG antibody titers to all antigens contained in the vaccine: NHBA-GNA1030, GNA2091-fHBP, and NadA. (B) IgG subclasses IgG1, IgG2b, and IgG3 for one representative antigen (NHBA-GNA1030). Values represent means of Log10 titers of eight mice per group plus SD. Unpaired, two-tailed Student's t test. *P < 0.05; **P < 0.01. (C) SBA of mouse polyclonal antibodies in the serum of eight single mice (black symbols) and their median value (line). Unpaired, two-tailed Mann-Whitney test. *P < 0.05.

Fig. 4.

Fig. 4.

Innate immune responses after adjuvant stimulation in MyD88−/− mice or WT controls. (A) Cytokine content in intraperitoneal washfluid 4 h after intraperitoneal adjuvant injection. Black bars: WT mice; gray bars: MyD88−/− mice. Values represent mean of three mice plus SD. Unpaired, two-tailed Student's t test was performed comparing WT vs. MyD88−/− and adjuvant vs. PBS injection (in WT mice). *P < 0.05 **P < 0.001. (B) Cytokine content in serum at different timepoints postinjection of MF59. Black symbols: WT mice; gray symbols: MyD88−/− mice. Values represent mean of two to three mice ± SD. (C) Cell recruitment events in the peritoneal cavity 4 h after adjuvant injection. Values are given per 10E6 total peritoneal cells and represent means of three mice per group plus SD.

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