Effectiveness of a vaccine composed of heat-killed Candida albicans and a novel mucosal adjuvant, LT(R192G), against systemic candidiasis

Abstract

The incidence of fungal infections caused by the opportunistic yeast Candida albicans has increased significantly in recent years. The ability to vaccinate selected patients against the organism would be advantageous. In this paper we describe a potential anti-C. albicans vaccine consisting of heat-killed C. albicans (HK-CA) in combination with the novel mucosal adjuvant LT(R192G), a genetically detoxified form of the heat-labile toxin of enterotoxigenic Escherichia coli. Groups of male CBA/J mice were immunized intranasally on three occasions at weekly intervals with 2 x 10(7) HK-CA per dose, alone or in conjunction with 10 micrograms of LT(R192G) per dose. Two weeks following the last application of antigen, some animals were challenged intravenously (i.v.) with 10(4), 10(5), or 10(6) viable C. albicans to assess protection as measured by survival and/or culture. Some groups of animals were footpad tested with C. albicans mannan to assess delayed-type hypersensitivity (DTH), and all the animals were bled for antibody assays. In two independent studies, all the animals immunized with HK-CA plus LT(R192G) were able to eradicate 10(4) C. albicans completely, as determined by kidney culture 4 weeks after challenge. Animals immunized with HK-CA only had reduced levels of C. albicans compared to the adjuvant or saline-only control. Greatly enhanced survival was observed when mice immunized with HK-CA plus LT(R192G) were challenged with 10(5) live C. albicans as well. Animals immunized with HK-CA plus LT(R192G) developed a significant DH response, while those given HK-CA alone developed only marginal DH responses. High immunoglobulin G (IgG) levels to cytoplasmic antigens developed in mice immunized with HK-CA plus LT(R192G), but they were found only after i.v. challenge. Addition of adjuvant shifted the antibody isotype production in i.v.-challenged animals to a response dominated by IgG2a. Clearly, intranasal immunization with killed C. albicans in conjunction with LT(R192G) afforded significant levels of protection. This novel approach offers new possibilities for the development of an effective vaccine against candidiasis for use in humans.

FIG. 1

Demonstration of protective immunity by culture of kidneys 4 weeks following i.v. challenge with 10⁴ viable C. albicans. Animals were immunized i.n. with HK-CA (2 × 10⁷ organisms) with or without 10 μg of the adjuvant LT(R192G), adjuvant alone, or NPS on the same schedule. Other control groups included animals immunized by i.d. exposure to 2 × 10⁶ viable C. albicans, animals vaccinated i.g. with 2 × 10⁷ HK-CA, and animals given an i.g. immunization followed by an i.d. inoculation with live C. albicans as described in Materials and Methods. An i.v. challenge was performed 2 weeks following the last exposure to antigen. There were seven or eight animals per group.

FIG. 2

Survival of mice immunized with HK-CA plus LT(R192G), LT(R192G) alone, or NPS and challenged i.v. with 10⁵ or 10⁶ viable C. albicans 2 weeks following the last exposure to antigen. There were 7 to 12 animals per group.

FIG. 3

DTH to C. albicans mannan in mice immunized i.n. with HK-CA plus LT(R192G), HK-CA alone, NPS, LT(R192G) alone, or 10⁶ viable C. albicans administered i.d. Footpad testing was performed on animals 2 weeks following the last exposure to HK-CA plus LT(R192G), HK-CA, LT(R192G), or NPS or 1 week following the last i.d. exposure to viable C. albicans. There were five or six animals per group. SEM, standard error of the mean.

FIG. 4

ELISA analyses for total IgG (A) or IgG subclasses (B) specific for cytoplasmic antigens (SCS) of C. albicans in sera from animals immunized i.n. with HK-CA alone, HK-CA plus LT(R192G), LT(R192G) alone, or NPS or with viable C. albicans administered i.g.-i.d. Sera were obtained at the time of sacrifice for culture, i.e., 4 weeks following i.v. challenge of immunized animals with 10⁴ viable C. albicans (solid circles) or 100 days following i.v. challenge of immunized animals with 10⁵ viable C. albicans (open circles). There were 7 to 12 animals per group.

FIG. 5

Western blot analyses of sera from animals immunized i.n. with HK-CA plus LT(R192G) or HK-CA alone. Lanes: 1, molecular size markers; 2 to 9, sera from mice immunized with HK-CA plus LT(R192G) and challenged with 10⁵ viable C. albicans (sera were obtained 100 days postchallenge); 10 to 15, sera from mice immunized with HK-CA alone.