Inflammasome-independent role of the apoptosis-associated speck-like protein containing CARD (ASC) in the adjuvant effect of MF59

Abstract

Clinical studies have indicated that subvirion inactivated vaccines against avian influenza viruses, particularly H5N1, are poorly immunogenic in humans. As a consequence, the use of adjuvants has been championed for the efficient vaccination of a naïve population against avian influenza. Aluminum salts (alum) and the oil-in-water emulsion MF59 are safe and effective adjuvants that are being used with influenza vaccines, but the mechanism underlying their stimulation of the immune system remains poorly understood. It was shown recently that activation of a cytosolic innate immune-sensing complex known as "NLR-Pyrin domain containing 3" (NLRP3) inflammasome, also known as "cryopyrin," "cold-induced autoinflammatory syndrome 1" (CIAS1), or nacht domain-, leucine-rich repeat-, and PYD-containing protein 3 (Nalp3), is essential for the adjuvant effect of alum. Here we show that the inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), an adapter protein within the NLRP3 inflammasome, is a crucial element in the adjuvant effect of MF59 when combined with H5N1 subunit vaccines. In the absence of ASC, H5-specific IgG antibody responses are significantly reduced, whereas the responses are intact in NLRP3(-/-) and caspase-1(-/-) mice. This defect is caused mainly by the failure of antigen-specific B cells to switch from IgM to IgG production. We conclude that ASC plays an inflammasome-independent role in the induction of antigen-specific humoral immunity after vaccination with MF59-adjuvanted influenza vaccines. These findings have important implications for the rational design of next-generation adjuvants.

Fig. 1.

Superior priming with MF59-adjuvanted H5N1 vaccines in comparison with the alum-adjuvant or the unadjuvanted formulations. Four groups of C57BL/6 WT mice (n = 5) were primed i.m. with 1 μg of H5N1 subvirion vaccine (MF59- or alum-adjuvanted or unadjuvanted) or with sublethal A/vietnam/1203/2004 (H5N1) influenza virus infection. All groups were boosted with 3 μg of H5N1 subvirion vaccine in PBS, and sera and other organs were harvested 5 d after the boost. (A) Mean circulating levels of H5-specific IgG antibodies. (B and C) Mean number of H5-specific IgG-forming cells in the bone marrow (B) and spleen (C). The results are representative of at least two separate experiments. Statistically significant differences between WT and mutant groups are indicated by asterisks; *P < 0.05. ns, not significant. nd, not detected. Error bars show SE.

Fig. 2.

Defective humoral immune response to MF59-adjuvanted H5N1 vaccines in ASC-KO mice. C57BL/6 WT (n = 5), Nlrp3^−/− (n = 4), ASC^−/− (n = 5), and caspase-1^−/− (n = 5) mice were immunized twice, 3 wk apart, with 1 μg of H5N1 subvirion vaccine adjuvanted with MF59. Sera were collected 3 wk after the boosting immunization. (A) Mean serum HI titers. (B–F) Mean circulating levels of H5-specific IgM (B), IgG (C), IgG1 (D) IgG2b (E), and (F) IgG2c antibodies. Results are representative of at least two separate experiments. Statistically significant differences between WT and mutant groups are indicated by asterisks; *P < 0.05. Error bars show SE.

Fig. 3.

Analysis of GC B-cell development after immunization with MF59-adjuvanted H5N1 subvirion vaccine. C57BL/6 WT (n = 5), Nlrp3^−/− (n = 4), ASC^−/− (n = 5), and caspase-1^−/− (n = 3) mice were immunized with 5 μg of H5N1 subvirion vaccine adjuvanted with MF59. Sera and draining (inguinal) lymph nodes were collected 7 d after immunization. (A and B) Mean circulating levels of H5-specific IgM (A) and IgG (B) antibodies were determined as in Fig. 1. (C) Mean number of live cells in the ipsilateral draining lymph node. (D) Representative FACS plots for lymphocytes from the draining lymph nodes stained with monoclonal antibodies to B220 and CD95 (Fas) on day 7 after immunization with 5 μg of H5N1 subvirion vaccine adjuvanted with MF59. The gated population (circled in red) from each plot represents GC B cells, which were defined as cells that were negative for surface CD4, CD8, and CD11b, with low expression of surface IgD and high expression of surface B220 and FAS (CD195). (E) Total number of GC B cells as determined by FACS analysis of the cells with the phenotype defined in D. (F) Total number of CD4⁺ lymphocytes. Statistically significant differences between WT and mutant groups are indicated by asterisks; *P < 0.05. Error bars show SE.

Fig. 4.

Poor H5-specific antibody recall responses in immunized ASC^−/− but not in WT, NLRP3^−/−, or caspase-1^−/− mice after antigen reexposure. C57BL/6 WT (n = 5), Nlrp3^−/− (n = 4), ASC^−/− (n = 3), and caspase-1^−/− (n = 3) mice were primed with 1 μg of H5N1 subvirion vaccine adjuvanted with MF59 and were boosted 3 wk later with 5 μg of H5N1 subvirion vaccine in PBS. Sera were collected before and 5 d after the boost. Ipsilateral draining lymph nodes and bone marrow were collected 5 d after the boost. (A) Mean number of live cells in the draining ipsilateral draining lymph nodes. (B) Mean number of plasma cells in the draining lymph nodes as determined by FACS analysis of cells that were negative for surface CD4, CD8, and CD11b, with low expression of surface IgD and high surface expression of CD138. (C) Mean number of GC B cells determined as in Fig. 2E. (D) Mean frequencies of H5-specific AFCs in the bone marrow of WT, Nlrp3^−/−, ASC^−/−, and caspase-1^−/− mice 5 d after antigen reexposure. (E) Fold increase in H5-specific IgG titers 5 d after antigen reexposure. Statistically significant differences between WT and mutant groups are indicated by asterisks; *P < 0.05. Error bars show SE.

Fig. 5.

Defective inflammatory response to MF59in ASC^−/− BMDCs. BMDCs were stimulated for 24 h with MF59 in complete medium (1:100 vol/vol), and the quantities of (A) MIP1β and (B) MIP2 were determined in cell-culture supernatants. Tests were assayed in triplicate or quadruplicate. Statistically significant differences between WT and mutant groups are indicated by asterisks; *P < 0.05. Error bars show SE.