Flagellin is an effective adjuvant for immunization against lethal respiratory challenge with Yersinia pestis

Abstract

Gram-negative flagellin, a Toll-like receptor 5 (TLR5) agonist, is a potent inducer of innate immune effectors such as cytokines and nitric oxide. In the lung, flagellin induces a localized and transient innate immune response characterized by neutrophil infiltration and the production of cytokines and chemokines. In view of the extraordinary potency of flagellin as an inducer of innate immunity and the contribution of innate responses to the development of adaptive immunity, we evaluated the efficacy of recombinant Salmonella flagellin as an adjuvant in an acellular plague vaccine. Mice immunized intranasally or intratracheally with the F1 antigen of Yersinia pestis and flagellin exhibited dramatic increases in anti-F1 plasma immunoglobulin G (IgG) titers that remained stable over time. In contrast, control mice had low or undetectable antibody responses. The IgG1/IgG2a ratio of antibody titers against F1 in immunized mice is consistent with a Th2 bias. However, no significant antigen-specific IgE production was detected. Interferons, tumor necrosis factor alpha, and interleukin-6 were not essential for the adjuvant effects of flagellin. Preexisting antiflagellin antibodies had no significant effect on the adjuvant activity of flagellin. Importantly, intranasal immunization with flagellin and the F1 antigen was protective against intranasal challenge with virulent Y. pestis CO92, with 93 to 100% survival of immunized mice. Lastly, vaccination of cynomolgus monkeys with flagellin and a fusion of the F1 and V antigens of Y. pestis induced a robust antigen-specific IgG antibody response.

FIG. 1.

Immunization with flagellin and the F1 antigen of Yersinia pestis results in substantial anti-F1 antibody production. (a) Female BALB/c mice were immunized i.t. or i.n. with 10 μg F1 plus 1 μg flagellin (FliC). Control animals were immunized i.t. with 10 μg F1 alone or with 1 μg of the 229 mutant flagellin. Mice were boosted in an identical manner at 4 weeks, and plasma was collected 2 weeks later for analysis by ELISA. Numbers within the bars indicate ratio of IgG1/IgG2a isotypes. A single asterisk indicates statistical significance over controls, and a double asterisk indicates that i.n. titers are statistically greater than i.t. (P < 0.007). (b) Anti-F1 antibody titers from mice immunized i.n. with 10 μg F1 plus 1 μg flagellin. Each line represents one mouse, and arrows indicate booster immunizations. (c) Female BALB/c mice were immunized i.t. with 10 μg F1 and increasing amounts of FliC or 5 μg of 229 and boosted at 4 weeks. Plasma anti-F1 IgG titers were determined 2 weeks postboost. (d) A group of female BALB/c mice was immunized i.n. with 5 μg of flagellin alone and boosted in an identical manner at 4 weeks. Anti-FliC antibody titers were determined 2 weeks later (mean anti-FliC titer = 8.5 × 10⁵), and flagellin-immune mice were then immunized and boosted with 10 μg F1 plus 1 μg FliC i.n. Two weeks postboost, anti-F1 titers were determined and compared to titers of flagellin-naive animals immunized with 10 μg F1 plus 1 μg FliC or 229. Bars represent mean antibody titers ± standard errors of the means. Seven female BALB/c mice were used per immunization group.

FIG. 2.

Flagellin stimulates antigen-specific responses and requires T cells. (a) Groups of seven female BALB/c mice were immunized i.n. with 10 μg F1 antigen plus 1 μg flagellin (FliC) and boosted at 4 weeks with PBS, 1 μg FliC alone, 10 μg F1 alone, or 10 μg F1 plus 1 μg FliC. Plasma was collected 3 weeks after boosting for analysis by ELISA. A single asterisk indicates statistical significance over animals boosted with PBS or FliC alone, and a double asterisk indicates that boosting with F1 plus FliC results in antibody titers statistically greater than F1 antigen alone (P < 0.01). Bars represent mean antibody titers ± standard errors of the means. (b) A group of seven athymic nude mice (BALB/cAnNCr-nu/nu) was immunized and boosted i.n. with 10 μg F1 plus 1 μg FliC. Plasma was collected 2 weeks postboost for analysis of anti-F1 IgG titers by ELISA. A single asterisk indicates statistical significance compared to normal BALB/c mice immunized in an identical manner (P < 0.001).

FIG. 3.

Requirements for the adjuvant effects of flagellin. (a and b) TNFR^−/− or IL-6^−/− and wild-type B6;129 control mice were immunized i.t. with 10 μg F1 antigen plus 1 μg flagellin (FliC) or mutant flagellin (229). A single asterisk indicates that TNFR^−/− titers are statistically less than those of B6;129 control (P < 0.001). (c) C3H/HeJ (Tlr4 P712H mutant) and wild-type C3H/HeN mice were immunized with 10 μg F1 plus 1 μg FliC or 229. (d and e) IFN-α/βR^−/− and IFN-γ^−/− mice and corresponding wild-type controls were immunized i.n. with 10 μg F1 plus 1 μg FliC or 229. Seven female mice were used in each immunization group. Mice were boosted in the same manner at 4 weeks, and plasma was collected 2 weeks later for analysis of anti-F1 IgG titers by ELISA. Bars represent mean antibody titers ± standard errors of the means.

FIG. 4.

Flagellin promotes a protective response for intranasal infection with Yersinia pestis CO92. (a) Groups of 15 female BALB/c mice were immunized i.n. with 10 μg F1 antigen plus 1 μg flagellin (FliC) or PBS alone and boosted in an identical manner at 4 weeks. Plasma was collected 2 weeks postboost for analysis of antibody titers by ELISA (mean anti-F1 titer = 9.4 × 10⁵). One week later mice were challenged i.n. with a dose of Y. pestis CO92 equivalent to 100× the LD₅₀. Mice were monitored for 30 days postchallenge. (b) Groups of 10 antibody-deficient IgH^−/− mice were immunized i.n. with 10 μg F1 plus 1 μg FliC or PBS alone and boosted in an identical manner at 4 weeks. Mice were challenged 2 weeks later i.n. with a dose of Y. pestis equivalent to 155× the LD₅₀. (c and d) Groups of 10 female IFN-γ^−/− mice and wild-type C57BL/6 mice were immunized and boosted i.n. with 10 μg F1 plus 1 μg FliC. Plasma was collected 2 weeks postboost for analysis of antibody titers by ELISA (anti-F1 titer, ≥1 × 10⁶). One week later mice were challenged i.n. with a dose of Y. pestis equivalent to 150× the LD₅₀ and monitored for 16 days postchallenge.

FIG. 5.

Flagellin is an effective adjuvant in nonhuman primates. Female cynomolgus monkeys (Macaca fascicularis) were immunized intranasally (n = 6) or intramuscularly (n = 6) with 150 μg F1/V fusion protein plus 50 μg flagellin. Control animals (n = 3) were immunized i.n. and i.m. with PBS alone. No significant change in body temperature occurred over 12 h, and TNF-α was not detected in plasma collected at 4 h, 12 h, and 24 h postimmunization. Animals were boosted in an identical manner at 4 weeks, and plasma was collected 2 weeks later for analysis by ELISA. Bars indicate mean anti-F1/V antibody titers ± standard errors of the means, and a single asterisk indicates statistical significance over intranasal immunization (P < 0.006).