Modeling nikkomycin Z dosing and pharmacology in murine pulmonary coccidioidomycosis preparatory to phase 2 clinical trials.

Nikkomycin Z (NikZ) is a chitin synthase inhibitor with activity against Coccidioides species that is being developed as a first-in-class orphan product for treatment of coccidioidomycosis. It has previously been shown to reduce lethal respiratory infections in mice to undetectable levels when treatment is begun 48 hours after infection. The studies described here focus on bracketing NikZ doses for phase 2 and 3 clinical trials, using an established mouse respiratory infection as a model and starting treatment 120 hours after infection. A dose of 80 mg/kg/day, divided into 2 doses, nearly eradicated infection, and larger doses did not improve fungal clearance. Increasing the duration of treatment from 1 week to 3 weeks resulted in a greater percentage of culture-negative mice. Comparative data show that plasma levels of NikZ that nearly eradicate Coccidioides in mice are achievable in patients and provide a plausibly effective dose range for initial phase 2 clinical studies.

Vaccine-induced cellular immune responses differ from innate responses in susceptible and resistant strains of mice infected with Coccidioides posadasii.

Susceptibility to Coccidioides spp. varies widely in humans and other mammals and also among individuals within a species. Among strains of mice with various susceptibilities, immunohistopathology revealed that C57BL/6 mice were highly susceptible to the disease following intranasal infection, DBA/2n mice were intermediate, and Swiss-Webster mice were innately resistant. Resistant Swiss-Webster mice developed prominent perivascular/peribronchiolar lymphocytic cuffing and well-formed granulomas with few fungal elements and debris in the necrotic center, surrounded by a mantle of macrophages, lymphocytes, and fibrocytes. Susceptible C57BL/6 mice became moribund between 14 and 18 days postinfection, with overwhelming numbers of neutrophils and spherules and very few T cells, the drastic reduction of which was associated with failure and death, while intermediate DBA/2n mice controlled the fungal burden but demonstrated progressive lung inflammation with prominent suppuration, and they deteriorated clinically. Vaccinated C57BL/6 mice had an early and robust lymphocyte response, which included significantly higher Mac2(+), CD3(+), and CD4(+) cell scores on day 18 than those of innately resistant SW mice and DBA/2n mice; they also had prominent perivascular/peribronchiolar lymphocytic infiltrates not present in their unvaccinated counterparts, and they appeared to be resolving lesions by day 56 compared to the other two strains, based on significantly lower disease scores and observably smaller and fewer lesions with few spherules and neutrophils.

Improved protection of mice against lethal respiratory infection with Coccidioides posadasii using two recombinant antigens expressed as a single protein.

Two recombinant antigens which individually protect mice from lethal intranasal infection were studied in combination, either as a mixture of two separately expressed proteins or as a single chimeric expression product. Mice vaccinated with either combination survived longer than mice given single antigens. Immunized mice also exhibited specific IgG immunoglobulins and yielded splenocytes which produced interferon-gamma in response to either antigen. The chimeric antigen has the practical advantage of offering enhanced protection from multiple components without increasing production costs.

Localization within a proline-rich antigen (Ag2/PRA) of protective antigenicity against infection with Coccidioides immitis in mice.

Subunits of a proline-rich coccidioidal antigen (Ag2/PRA) of Coccidioides immitis were analyzed by comparison as vaccines in mice. The optimal dose of plasmid vaccine encoding full-length Ag2/PRA was determined to be between 10 and 100 microg. Mice vaccinated with plasmids encoding amino acids (aa) 1 to 106 were as protective as full-length Ag2/PRA (aa 1 to 194). The subunit from aa 27 to 106 was significantly but less protective. Plasmids encoding aa 90 to 151 or aa 90 to 194 were not protective. Analogous results were obtained with recombinant vaccines of the same amino acid sequences. In addition, mixtures of aa 90 to 194 with either aa 1 to 106 or aa 27 to 106 did not enhance protection compared to the active single-recombinant subunits alone. Humoral response of total immunoglobulin G (IgG) and subclasses IgG1 and IgG2a were detectable in subunit vaccinations but at significantly (100-fold) lower concentrations than after vaccination with plasmids encoding full-length Ag2/PRA. Since virtually all protection by vaccination with full-length Ag2/PRA can be accounted for in the first half of the protein (aa 1 to 106), this subunit could make a multicomponent vaccine more feasible by reducing the quantity of protein per dose and the possibility of an untoward reactions to a foreign protein.

Protection of mice against Coccidioides immitis intranasal infection by vaccination with recombinant antigen 2/PRA.

Subcutaneous vaccination with recombinant antigen 2/PRA (rAg2/PRA) protected BALB/c mice against intranasal infection with Coccidioides immitis. Subcutaneously vaccinated C57BL/6 mice and intranasally vaccinated BALB/c mice were protected against larger numbers of infecting spores. Weight loss correlated with lethality, but histologic appearance did not. These studies support rAg2/PRA vaccination to prevent coccidioidomycosis.