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The formaldehyde-killed, whole-spherule vaccine, which is protective against lethal challenge of laboratory animals withCoccidioides immitis, was fractionated. It yielded a soluble, multicomponent, subcellular fraction termed the 27K vaccine. This vaccine, when it was accompanied by adjuvant, protected mice against lethal intranasal and intravenous challenge withC. immitis.

A chitinase isolated from Coccidioides immitis was subjected to amino-terminal protein sequence analysis. The resulting 18-amino-acid sequence was compared with the previously reported amino acid sequence of coccidioidal immunodiffusion-complement fixation (IDCF) antigen. From the homology of the two sequences, the results support the identification of the IDCF antigen with a chitinase.

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A chitinase had been isolated from the culture filtrates of Coccidioides immitis endosporulating spherules and from hyphae and shown to be the coccidioidal complement fixation (CF) and immunodiffusion-CF antigen. In the present study, we made use of our previously determined amino-terminal (N-terminal) sequence of the CF-chitinase to design degenerate oligonucleotide primers and to amplify and sequence a PCR product that coded for the N-terminal portion of the CF-chitinase. The PCR product was used as a hybridization probe to screen a developing spherule-ZAP cDNA library, and three hybridizing clones were selected. These clones were converted into their pBluescript expression plasmid form in Escherichia coli and induced to express their recombinant proteins. Lysate from only one clone, pCTS 4-2A, yielded an enzymatically functional CF-chitinase and a line of identity with control immunodiffusion-CF-positive antigen. The pCTS 4-2A insert was sequenced and found to contain a deduced open reading frame coding for a 427-amino-acid polypeptide with an approximate molecular weight of 47 kDa. When purified by a chitin adsorption-desorption method, the recombinant protein exhibited virtually identical characteristics to those of the original C. immitis CF-chitinase. Nondenaturing gels of the pCTS 4-2A E. coli lysates and the purified C. immitis and recombinant CF-chitinase revealed proteins that had chitinase activity and similar relative electrophoretic mobilities. The appearance and relative levels of hybridizing RNA from the developing spherules-endospores (SEs) and hyphae correlated with the appearance or presence and level of CF-chitinase enzyme activity found in SEs culture filtrate and in cellular extracts of developing SE and hyphae. Thus, a functional recombinant CF-chitinase antigen was produced in E. coli and was used in serological diagnostic applications. These results also suggest a functional role for this chitinase in SE development and maturation.

The marked increase in the number of cases of coccidioidomycosis in California in 1992 led to a study of isolates from various patients and environmental sources by restriction fragment length polymorphism (RFLP) analysis. Of 15 different isolates, most of the isolates (13 of 15) from California and 1 from Venezuela yielded one main RFLP pattern with evidence of two subgroups. The other two isolates (both from patients in the San Joaquin Valley of California) yielded a different RFLP pattern. Coccidioides immitis is a biphasic, terricolous fungus found in the southwestern United States, Mexico, and Central and South America (14). In the soil, the fungus grows in a hyphalarthroconidial form. The arthroconidia produce infection in a susceptible human or other animal host, usually via the respiratory route. During the years 1991 through 1993, there was a marked increase in the incidence of coccidioidomycosis in California. This has occurred both in the usual high-risk areas and in areas where the disease is less prevalent (15). In the past, it has been possible to distinguish isolates of C. immitis from various patients and geographic locations by phenotypic characteristics (4, 5, 7-9, 16). However, little is known of the extent of genetic variation within C. immitis at the DNA sequence level (13). The ability to distinguish genotypes of isolates would be useful in investigating the source(s) of outbreaks of infection and in defining the relatedness of isolates recovered from different patients. As a first step, we used restriction fragment length polymorphisms (RFLPs) to compare different C. immitis isolates.

The activity of the chitin synthase inhibitor lufenuron was evaluated in vitro using the spherule-endospore (SE) phase of Coccidioides immitis. The lufenuron was also used to treat mice infected with C. immitis by the respiratory route. In vitro, lufenuron had no effect upon fungal cell growth. Two formulations of lufenuron were evaluated in vivo. Neither the oral nor the injectable lufenuron extended the survival of mice infected with C. immitis when compared with placebo-treated mice.