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Infect Immun. 2009 August; 77(8): 3196–3208.
Published online 2009 June 1. doi:  10.1128/IAI.00459-09
PMCID: PMC2715678

A Genetically Engineered Live Attenuated Vaccine of Coccidioides posadasii Protects BALB/c Mice against Coccidioidomycosis[down-pointing small open triangle]


Coccidioidomycosis (also known as San Joaquin Valley fever) is an occupational disease. Workers exposed to outdoor dust which contains spores of the soil-inhabiting fungus have a significantly increased risk of respiratory infection. In addition, people with compromised T-cell immunity, the elderly, and certain racial groups, particularly African-Americans and Filipinos, who live in regions of endemicity in the southwestern United States have an elevated incidence of symptomatic infection caused by inhalation of spores of Coccidioides posadasii or Coccidioides immitis. Recurring epidemics and escalation of medical costs have helped to motivate production of a vaccine against valley fever. The major focus has been the development of a defined, T-cell-reactive, recombinant protein vaccine. However, none of the products described to date have provided full protection to coccidioidal disease-susceptible BALB/c mice. Here we describe the first genetically engineered, live, attenuated vaccine that protects both BALB/c and C57BL/6 mice against coccidioidomycosis. Two chitinase genes (CTS2 and CTS3) were disrupted to yield the attenuated strain, which was unable to endosporulate and was no longer infectious. Vaccinated survivors mounted an immune response characterized by production of both T-helper-1- and T-helper-2-type cytokines. Histology revealed well-formed granulomas and markedly diminished inflammation. Significantly fewer organisms were observed in the lungs of survivors than in those of nonvaccinated mice. Additional investigations are required to further define the nature of the live, attenuated vaccine-induced immunity against Coccidioides infection.

Coccidioides is a fungal pathogen which includes two species, Coccidioides posadasii and Coccidioides immitis (16), and is the causative agent of a human respiratory disease known as coccidioidomycosis or San Joaquin Valley fever. Results of molecular epidemiological studies have suggested that C. posadasii (the “non-Californian” species) originated from a single North American population in Texas and subsequently underwent rapid population growth in the southwestern United States (especially Texas and Arizona), Central Mexico, and regions of Venezuela, Brazil, and Argentina (15). On the other hand, C. immitis appears to have remained geographically isolated to central and southern California. In spite of the genetic diversity revealed by comparative genomic sequence analyses of these two species (43), laboratory animal studies have shown no significant difference in either the virulence or the growth and development of the organisms. Human infection typically occurs by inhalation of the spores (arthroconidia) released into the air from the saprobic phase of the soilborne fungus. In the approximately 40% of human exposures that result in symptomatic infection, the initial clinical manifestation is typified by onset of an acute respiratory response that occurs about 1 to 3 weeks after inhalation of the pathogen. These early symptoms are also characteristic of acute community-acquired pneumonia (CAP). Valdivia and coworkers (50) reported that 29% of patients diagnosed with CAP in Tucson, AZ, had contracted coccidioidomycosis. Physicians are strongly advised to consider Coccidioides infection in their differential diagnosis of all patients with CAP who reside in or have recently visited a region to which coccidioidal disease is endemic (26). Coccidioidal infections can progress to life-threatening, chronic progressive pneumonia, extrapulmonary nonmeningeal disease, or meningitis, the most feared complication of coccidioidomycosis (36). Although few patients develop these severe forms of the disease (<1%), the number of reported cases of primary pulmonary infection in Arizona and California has significantly increased during the last decade (2, 47). Escalations in the cost of long-term antifungal therapy, which is frequently required for symptomatic coccidioidomycosis, argue for a way to better control this disease (17).

Several reported studies have presented evidence for the feasibility of generating a vaccine against coccidioidal infections (reviewed in references 5, 7, and 35). A compelling argument for such a vaccine is the retrospective clinical observation that natural human infection with Coccidioides results in lifelong immunity against the mycosis (46). Cell-mediated immunity has been shown to be essential for an effective response to infection by the fungal pathogen, and recent attempts to develop a defined vaccine have focused on the identification of recombinant protein candidates which elicit protective T-cell responses in immunized mice (45, 48). Earlier studies used attenuated strains of Coccidioides of undefined genetic origin as live vaccines and showed success in protecting BALB/c mice, the murine strain that is most susceptible to disseminated coccidioidomycosis following intranasal challenge (5). More recently a spontaneous, temperature-sensitive, auxotrophic mutant of C. immitis generated by exposure to UV radiation was evaluated as a live, attenuated vaccine (14). In the absence of a defined genetic mechanism of attenuation, however, the possibility of reversion to the virulent phenotype restricts the practical application of these live vaccines. Nevertheless, for numerous microbial diseases in which natural infection confers lifelong protection to the host, live vaccines have proved to be most successful (13, 42). The major advantage is that the live, attenuated microorganism stimulates an immune response similar to that elicited by the natural infection. The essential factor in a live vaccine is that its disease-causing capacity is virtually eliminated by biological or technical manipulations. The parasitic cycle of Coccidioides is unique among the medically important fungi (5). Asexual reproduction of the pathogen within the host occurs by a sequence of morphogenetic steps that result in production of a multitude of endospores (approximately 100 to 300) which develop into new generations of parasitic cells (spherules). We employed electron microscopy combined with substrate labeling methods to examine successive stages of spherule maturation (3). The results suggested that activation of chitinolytic enzymes is an essential event that occurs during early stages of endospore differentiation. We proposed that inhibition of chitinase activity at this pivotal stage of the parasitic cycle would render the pathogen sterile. In this report, we describe a genetically engineered mutant of C. posadasii which is unable to reproduce asexually during its parasitic phase. The attenuated strain was evaluated as a live vaccine in BALB/c and C57BL/6 mice and proved to be superior to defined, recombinant protein vaccines so far tested against coccidioidomycosis.


Fungal strains, media, and growth conditions.

The fungal pathogen used in this study is a clinical isolate (C735) of C. posadasii (16). The saprobic phases of both the parental and mutant strains of this isolate were grown on GYE medium (1% glucose, 0.5% yeast extract, 1.5% agar) at 30°C for 3 to 4 weeks to generate a confluent layer of arthroconidia (spores) on the agar surface. Spores were collected by washing plate cultures with sterile, endotoxin-free saline, and the suspension was filtered to remove hyphal fragments. The spores were used to inoculate a defined glucose/salts medium (29) for growth of the parasitic phase and to challenge or vaccinate mice as described below.

Coccidioides genome database analysis and gene discovery.

The genome of C. posadasii (isolate C735) was originally sequenced at The Institute for Genomic Research, now the J. Craig Venter Institute (Rockville, MD) ( The genomic database for both C. posadasii and C. immitis (RS isolate), as well as the annotation for the latter, are available on the Broad Institute (Cambridge, MA) website ( Based on our hypothesis that chitinase production during the parasitic cycle plays a pivotal role in endospore differentiation (3), we conducted separate BLAST (basic local alignment search tool) searches (1) of the translated C. posadasii genome database using amino acid sequences of the two reported chitinases of this fungal pathogen as queries (Cts1 and Cts2) (37). A tBLASTn analysis was conducted using the Coccidioides databases ( and resulted in retrieval of six homologs of the two putative chitinases of C. posadasii. Gene-specific oligonucleotide primers were synthesized and used for the rapid amplification of cDNA ends procedure to obtain full-length cDNA sequences of each of these homologs as described previously (23). Comparison of the genomic and cDNA sequences confirmed the locations of the exons and introns and validated each of the respective C. posadasii genomic sequences in the database. Structural comparison of the eight deduced protein homologs was conducted by alignment of amino acid sequences using the CLUSTALX algorithm (49). The PROSITE algorithm was used to identify conserved motifs of the translated polypeptides with homology to reported fungal chitinases in the nonredundant protein database available from the National Center for Biotechnology Information as previously reported (9). Phylogenetic studies of both the C. posadasii and C. immitis subfamilies of chitinases were performed using maximum-likelihood methods as implemented in the program (available at for comparison of the structure of the catalytic domains (11, 25). The PSORT and PSORT II algorithms were used for prediction of cellular localization sites of the putative chitinases (33).

Comparative real time-PCR examinations of CTS gene expression.

To test our hypothesis that chitinolytic activity is essential for completion of the endosporulation phase of the parasitic cycle, we first evaluated expression of each CTS gene during successive stages of parasitic cell development in vitro. We expected to observe elevated expression of one or more of these genes during early stages of endospore formation. As shown previously, in vitro growth and differentiation of spherules during the first generation of the parasitic cycle are nearly synchronous (23). This permitted comparison of expression profiles of the eight CTS genes during selected stages of spherule development. We examined levels of gene expression during spherule septation (72-h parasitic-phase cultures), digestion of the septal wall complex and initiation of endospore formation (96 h), and endospore release from ruptured spherules (120 to 132 h) (32) by quantitative real time-PCR (QRT-PCR) as reported previously (22). The generation of cDNA from parasitic cell-derived total RNA preparations was conducted as reported elsewhere (9). The nucleotide sequences of the CTS-specific sense and antisense primers used for QRT-PCR are provided in the supplemental material (see Table S1).

Gene disruption strategies for generation of an attenuated strain of C. posadasii.

To further test our hypothesis that chitinases play a key role in reproduction of the parasitic cells, we selected two CTS genes to disrupt (CTS2 and CTS3) on the basis of their predicted protein structure and results of phylogenetic and QRT-PCR analyses. Both single and combined gene disruptions were performed. Plasmids were constructed to disrupt the CTS2 and CTS3 genes of C. posadasii by homologous recombination (10). To generate the plasmid construct for disruption of CTS2 (37), a pair of synthesized oligonucleotide primers was first used to amplify the gene from genomic DNA of C. posadasii by PCR. The sequences of the sense and antisense primers were 5′-CCGTCGAGGGAGTCTGATAG-3′ and 5′-GTATGTACAAGTACAGCACC-3′, respectively. The 3,041-bp PCR product of CTS2 was cloned into the PCR 2.1 Topo vector (Invitrogen, San Diego, CA). The CTS2 gene disruption construct was obtained by replacing a 966-bp StuI/SpeI restriction fragment of the CTS2 genomic insert (GenBank accession no. L41662; nucleotides no. 836 to 1,802) in the Topo vector with a 3.6-kb StuI/XbaI fragment of the pAN7.1 plasmid (GenBank accession no. Z32698). The 3.6-kb fragment includes a hygromycin B phosphotransferase gene (HPH) which when expressed with the appropriate fungal promoter and terminator in C. posadasii (38) confers resistance to the growth inhibitor hygromycin B (HmB) as reported previously (40). The final 6.9-kb plasmid construct (pcts2Δ::HPH) used to disrupt the CTS2 gene was linearized by digestion of the modified Topo vector with ApaI and DraI and purified for subsequent transformation of the C. posadasii parental strain as described previously (32). The 6,891-bp ApaI/DraI plasmid construct was comprised of a 3,597-bp fragment of pAN7.1, with flanking 5′ and 3′ regions of the CTS2 gene (828-bp and 1,242-bp, respectively). Terminal 5′ and 3′ sequences of the Topo vector (63 bp and 1,161 bp, respectively) were also present in this construct.

To generate the plasmid construct for disruption of CTS3, a pair of oligonucleotide primers were synthesized to amplify a 2,936-bp genomic DNA fragment by PCR, originally considered to represent the 5′-untranslated region (5′-UTR), open reading frame (ORF), and short 3′-UTR of the CTS3 gene of C. posadasii. The putative active site of the deduced CTS3 gene was identified within the ORF. The sequences of the sense and antisense primers used to amplify the 2.9-kb genomic fragment were 5′-GTGCGCTGTAAGACCATGATC-3′ and 5′-CTTCGAGTAAAGCGCAGAAG-3′, respectively. As a result of ongoing annotation of the C. immitis and closely related C. posadasii genomes, it was evident that what we originally interpreted as a 2.0-kb fragment of the 5′-UTR of CTS3 actually included an 874-bp ORF of a gene which encodes a homolog of d-arabinotol-2-dehydrogenase (ARD1) (19). The 2.9-kb ARD1/CTS3 genomic fragment was cloned into the Topo plasmid vector (Invitrogen). The disruption construct was obtained by replacing a 1.2-kb fragment of the contiguous ARD1/CTS3 genes excised from the Topo vector by NheI/SspI digestion with a 3.8-kb NheI/SspI fragment of the pAN7.1 plasmid, which includes the HPH gene. The final 5,634-kb plasmid construct (pard1Δcts3Δ::HPH) was linearized by digestion with ApaI and KpnI. The plasmid was comprised of a 3,804-bp fragment of pAN7.1, with a 965-bp 5′ flanking component that included the partial 5′-UTR and an upstream portion of the ORF of ARD1, and a 741-bp 3′-flanking component that included the partial ORF and 3′-UTR of CTS3. In addition, the construct consisted of 5′- and 3′-terminal sequences of the Topo plasmid vector (63 bp and 55 bp, respectively).

Transformation of C. posadasii was conducted using the protoplast method as reported previously (40). Putative cts2Δ::HPH and ard1Δ/cts3Δ::HPH transformants were selected on GYE agar plates supplemented with 75 μg/ml of HmB and subsequently maintained on a growth medium containing GYE plus 100 μg/ml HmB. Southern hybridization was employed as reported previously (32) to confirm the targeted disruption of the CTS2 and ARD1/CTS3 genes. In brief, genomic DNA isolated from candidate transformants was digested with endonucleases selected on the basis of the restriction maps of the genes (see Fig. Fig.2).2). The digestion products were separated by agarose gel electrophoresis, transferred to a nitrocellulose membrane, and hybridized with either a HPH, CTS2, or ARD1 oligonucleotide probe (600, 628, or 601 bp, respectively). The probes were generated by PCR amplification using an HPH gene primer pair (sense primer, 5′-ACAGCGTCTCCGACCTGATG-3′; antisense primer, 5′-CCTCGCTCCAGTCAATGACC-3′), a CTS2 primer pair (sense primer, 5′-CCTTCCACCGAAAGTATCAC-3′; antisense primer, 5′-CCACCGGGTTGTTGTATTCC-3′) and an ARD1 primer pair (sense primer, 5′-CACCGGCTGACTAGTGATAC-3′; antisense primer, 5′-GCCGACGTGTGCCTTG-3′).

FIG. 2.
(A) Restriction maps of chromosomal loci of C. posadasii (isolate C735) that contain the CTS2 gene (left) and the contiguous ARD1 and CTS3 genes (right). CTS2 and ARD1 probes were used for Southern hybridization analyses of transformants. (B and C) Hypothetical ...

To generate a mutant in which both the CTS2 and CTS3 genes were disrupted, the cts2Δ::HPH transformant was used as the parental strain. Analysis of the genomic database confirmed that CTS2 and the contiguous ARD1/CTS3 genes are located on separate chromosomes. The ARD1/CTS3 genomic fragment, which was cloned into the Topo plasmid as described above, was digested with NheI/SspI, and the 1.2-kb deletion product was replaced with a 3.1-kb NheI/SspI fragment of the pAN8.1 plasmid (GenBank accession no. Z32751). The latter contains the BLE gene, which encodes a phleomycin binding protein (39) that confers resistance to the fungal growth inhibitor phleomycin (22, 41). The ARD1/CTS3 disruption plasmid (pard1Δ/cts3Δ::BLE) was linearized with ApaI and KpnI and used to transform the cts2Δ::HPH strain. Transformants (cts2Δ::HPH-ard1Δ/cts3Δ::BLE) were selected on GYE agar supplemented with 3 μg/ml of phleomycin and subsequently maintained on GYE agar plus 5 μg/ml of phleomycin. Southern hybridization of the phleomycin-resistant transformants was conducted using the CTS2 and ARD1 probes described above, as well as a 346-bp BLE-specific probe. The sense and antisense primers employed to amplify the BLE probe were 5′-AGTGCCGTTCCGGTGCTCACC-3′ and 5′-CGGCCACGAAGTGCACGCAGT-3′, respectively.

The designation of the single, double, and triple gene knockout strains described above are henceforth referred to as the cts2Δ, ard1/cts3Δ, and cts2/ard1/cts3Δ mutants, respectively.

In vitro and in vivo growth of mutant strains.

The parental strain and three mutant strains were cultured as both the saprobic and parasitic phases as described above and examined by light microscopy. In vivo growth of the parental strain and that of the cts2/ard1/cts3Δ mutant were compared by infecting BALB/c mice (females, 8 weeks old; supplied by the National Cancer Institute, Bethesda, MD) by the intranasal route with 500 viable spores suspended in saline as reported previously (32). Mice were sacrificed at 21 days postchallenge, and biopsy specimens of lung abscesses were stained with blankophor, an optical brightener that binds to glucan and chitin in the fungal cell wall (53). Tissue smears on glass slides were examined directly by fluorescence microscopy. Thin sections of abscesses obtained from the same infected lung tissue were also examined by electron microscopy as described previously (32).

Virulence studies.

To compare the virulence of the parental strain and three mutant strains, spores (50 viable cells) were obtained from GYE plate cultures of each, suspended in saline, and used to inoculate BALB/c mice (8-week-old females; 12 mice per group) by the intranasal route as described above. The survival plots of each group examined over a 40-day period were subjected to Kaplan-Meier statistical analysis as described previously (32). Evaluation of the virulence of the cts2/ard1/cts3Δ mutant in BALB/c mice was repeated but with a 20-fold increase in the intranasal challenge dose (approximately 1,000 viable spores). Survivors were sacrificed at 60 days postchallenge to determine the residual CFU of the mutant strain in homogenates of the murine lungs and spleen as reported previously (32).

The genetically engineered cts2/ard1/cts3Δ mutant is referred to below as the attenuated strain.

Vaccination and assessment of protection.

BALB/c mice or C57BL/6 mice (females, 8 weeks old; supplied by the National Cancer Institute) were immunized subcutaneously in the abdominal region with different numbers of freshly isolated or stored spores of the attenuated strain. The spores were stored in sterile saline at 4°C for up to 6 months. Separate groups of mice (12 per group) were vaccinated initially with 1.0 × 103, 5.0 × 103, or 5.0 × 104 viable spores suspended in 100 μl of saline, followed 14 days later with an immunization boost of 0.5 × 103, 2.5 × 103, or 2.5 × 104 live spores, respectively. Two separate groups of BALB/c mice were vaccinated with either a saline suspension of formalin-killed, mature spherules (72-h parasitic-phase culture) of the C. posadasii parental isolate (C735) (FKS vaccine; three immunizations of 106 cells (0.9 mg) each at 10-day intervals) as reported previously (30), or a saline suspension of mature spherules of the attenuated strain (same cell concentration and vaccination protocol). Control mice were immunized with saline alone using the same protocol as employed above for vaccination with live spores. Mice were challenged by the intranasal route with 50 to 70 viable spores of the parental strain 4 weeks after completion of the vaccination schedule as reported previously (32). Statistical analysis of survival of the vaccinated versus nonvaccinated groups of mice was conducted as described above. Control mice were sacrificed at 15 days postchallenge, while mice vaccinated with the attenuated strain were sacrificed at 75 days postchallenge. The CFU were expressed on a log scale, and the Mann-Whitney U test was used to compare differences in the median CFU values for statistical significance as described previously (32). Four separate experiments were conducted as described above with each mouse strain to evaluate survival and pathogen clearance.

Gross examination, histology of vaccination sites, and persistence of the attenuated strain.

Separate groups of BALB/c mice (three per group) were vaccinated with spores of the live, attenuated strain or the FKS preparation of either the parental or attenuated strain as described above and examined to compare the intensity of reactogenicity (the capacity to produce adverse effects) at sites of subcutaneous immunization. A delipidation reagent (Nair, Church and Dwight Co., Inc., Princeton, NJ) was applied to the abdominal region of the mice for removal of hair immediately prior to gross examination on day 5 after completion of the respective vaccination protocols. Comparative histological examinations of sections (5 μm thick) of paraffin-embedded skin biopsy specimens obtained from sites of immunization of these same three groups of mice were carried out to determine the intensities of immune responses to the respective vaccines. Tissue fixation and embedding procedures were performed as described previously (32), and sections were stained with hematoxylin and eosin (H&E) by standard procedure. Two separate groups of BALB/c mice (12 mice per group) were vaccinated with the attenuated strain (a total of 7.5 × 104 spores) and challenged intranasally with the parental isolate as described above. The purpose of these studies was to determine the degree of persistence of the vaccine strain at sites of immunization in infected mice and whether the vaccine strain disseminated from these sites to other body organs. The mice were sacrificed at either 15 or 75 days postchallenge, skin biopsy specimens at vaccination sites were obtained, and lungs and spleens were excised, homogenized, and plated on GYE plus 50 μg/ml chloramphenicol (Sigma, St. Louis, MO) plus 100 μg/ml HmB. The numbers of CFU of the attenuated strain in the tissue homogenates were determined.

Cytokines in BALFs of vaccinated mice.

Cytokine production in bronchoalveolar lavage fluids (BALFs) was compared in three groups of BALB/c mice (four per group), which were either untreated (i.e., normal mice), not vaccinated but intranasally challenged with Coccidioides, or vaccinated and challenged. The mice were vaccinated subcutaneously with two doses of live spores of the attenuated strain (total, 7.5 × 104) suspended in saline as described above. BALFs from untreated mice were used to determine the basal level of production of each cytokine examined in this study. Vaccinated, infected mice were sacrificed at 8, 25, or 75 days postchallenge. Nonvaccinated, infected mice were sacrificed at 8 days. OptEIA mouse cytokine kits (Pharmingen, San Diego, CA) were used for enzyme-linked immunosorbent assays (ELISAs) of interleukin 5 (IL-5), IL-6, IL-10, IL-12p70, and gamma interferon concentrations in lavage fluids as reported previously (53). ELISAs of samples from individual mice were carried out in triplicate. Standard curves were generated using purified, recombinant cytokines supplied by the manufacturer of the kits. Standard curves were used to calculate the amounts of specific cytokines in the BALF samples.


Comparative histopathology was conducted with lung tissue of nonvaccinated and vaccinated BALB/c mice (five mice per group) which were challenged intranasally with a potentially lethal inoculum of spores (50 viable cells) derived from the parental isolate (C735) of C. posadasii. The nonvaccinated control mice were injected subcutaneously with saline, challenged intranasally, and sacrificed 18 days later. Mice vaccinated with spores of the live, attenuated mutant strain (total, 7.5 × 104 viable spores) were sacrificed at 75 days postchallenge. Paraffin sections of infected lung tissue were stained either with H&E as above or with periodic acid-Schiff reagent.

Nucleotide sequence accession numbers.

The C. posadasii nucleotide sequences of the CTS3 to CTS8 genes described in this article have been deposited in GenBank under accession no. AF492472, AF510393, AY454340, AF519181, AY454339, and FJ801037, respectively. CTS1 and CTS2 sequences were previously deposited under accession no. L41663 and L41662, respectively.


Discovery of eight members of a subfamily of C. posadasii genes encoding chitinolytic enzymes.

As a result of tBLASTn analysis of the C. posadasii (C735) genome database and alignment of matching amino acid sequences, we retrieved six protein homologs of the previously reported chitinases (Cts1 and Cts2) (37). Each of the eight amino acid sequences contained the fully conserved consensus domains of family 18 chitinolytic enzymes (EC (Table (Table1)1) (21). A BLASTp search was conducted using each of the deduced amino acid sequences to query against the nonredundant NCBI database as described previously (9). The results revealed highest sequence similarities and identities with fungal chitinases of other ascomycetous fungi (not shown). CLUSTALX analysis of the subfamily of chitinases in C. posadasii and C. immitis predicted the existence of a ninth chitinase of C. immitis, which is absent from the genome of C. posadasii (not shown). Phylogenetic analyses were conducted using the eight deduced chitinase sequences of C. posadasii and orthologs of the annotated genome of C. immitis (RS isolate). The combined phylogenetic tree suggested that Cts2, -3, and -4 are more closely related to each other than to the other members of this subfamily (see Fig. S1 in the supplemental material). On the basis of structural analysis of the deducted proteins, Cts2 and Cts4 were predicted to have signal sequences while Cts3 apparently lacks a signal peptide and may be localized to the cytoplasm of spherules. The structural domains of Cts2 are similar to those of a Saccharomyces cerevisiae chitinase (28). Both proteins contain a conserved hydrolytic region upstream of a Ser/Thr-rich domain. The yeast chitinase also contains a C-terminal chitin binding signature sequence, which is required for localization of the enzyme in the yeast cell wall but is absent from Cts2 of C. posadasii. However, Coccidioides Cts2 is predicted to have a glycosylphosphatidylinositol anchor, which could be an alternative structural component that targets the protein to the fungal cell wall.

Conserved domains of deduced chitinases (Cts1 to CTS8) of C. posadasii (isolate C735)a

Comparison of levels of CTS gene expression during the parasitic cycle suggests functional differences.

We recognize that transcription is subject to multiple regulatory mechanism and attempts to correlate temporal gene expression with a proposed function can be misleading. Nevertheless, we used QRT-PCR to predict which CTS genes of C. posadasii play a role in the digestion of the septal wall during the early stages of endospore differentiation. We focused on differences in the amounts of CTS transcripts between the 72-h culture stage, when spherule septal wall formation is under way, and the 96-h stage, when the septal wall complex undergoes digestion and endospore differentiation is initiated. Of the eight deduced C. posadasii chitinase genes, only CTS2 and CTS3 revealed a significant increase in expression during this developmental transition. CTS2 showed a 3.0-fold increase while CTS3 revealed a 6.5-fold increase in the amount of respective transcript during this period of spherule development (Fig. (Fig.1).1). The level of expression of CTS2 sharply decreased upon spherule maturation and endospore formation, while the expression level of CTS3 remained elevated.

FIG. 1.
QRT-PCR of CTS1 to CTS8 expression in vitro during selected stages of the parasitic cycle. Expression of each CTS gene was compared to that of the constitutive GAPDH gene. [box with upper left to lower right fill], spherule septation stage; [filled square], septal digestion and early endospore ...

Single and combined disruption of CTS2 and CTS3 genes.

On the basis of the structural and phylogenetic analyses of the subfamily of chitinases of C. posadasii together with the results shown in Fig. Fig.1,1, we decided to test whether expression of CTS2 and/or CTS3 is essential for digestion of the septal wall and subsequent formation of endospores within the maternal spherules. The two chitinase genes are located on separate chromosomes, and their restriction maps are shown in Fig. Fig.2A.2A. Two plasmid constructs were employed to separately disrupt the CTS2 and CTS3 genes. A plasmid construct containing the HPH gene replaced the putative substrate binding and active site domains of CTS2 (Fig. (Fig.2B).2B). Results of PCR screening of these candidate transformants suggested that four had undergone homologous recombination. One of these transformants, referred to as the cts2Δ strain, was selected for subsequent analysis by Southern hybridization. Disruption of the CTS3 gene was complicated by the presence of a contiguous, upstream gene (ARD1) separated by a 697-bp UTR (Fig. (Fig.2A).2A). The plasmid construct, which also contained the HPH gene, replaced a 342-bp ORF fragment of ARD1 and a 189-bp ORF component of CTS3. As a result, this transformation plasmid (pard1Δ/cts3Δ::HPH) disrupted both the ARD1 and CTS3 genes. PCR screening of the putative transformants identified four that had apparently undergone homologous recombination based on results of PCR analysis. One of the transformants, referred to as the ard1/cts3Δ strain, was selected for further analysis by Southern hybridization.

Examination of in vitro growth of the cts2Δ and ard1/cts3Δ mutant strains, as described below, indicated that disruption of either the CTS2 gene alone or the combined ARD1/CTS3 genes had no significant effect on endosporulation. On this basis, we decided to generate a mutant strain in which both the CTS2 and CTS3 genes were disrupted. The cts2Δ strain was used as the parent for disruption of CTS3, and the same plasmid construct was employed as described above, except that the pAN7.1 fragment was replaced with a 3.1-kb NheI/SspI-restricted fragment of pAN8.1 containing the BLE gene, which confers resistance to phleomycin (Fig. (Fig.2C).2C). One of the transformants, designated the cts2/ard1/cts3Δ strain, was selected for further study. The cts2Δ, ard1/cts3Δ, and cts2/ard1/cts3Δ mutant strains were subjected to Southern hybridization for confirmation of disruption of the respective target genes (Fig. (Fig.2D).2D). Restriction maps of CTS2 and ARD1/CTS3 were constructed by reference to the C. posadasii genomic database. Restriction sites were mapped in a 12.0-kb locus of chromosome 1, which contained the CTS2 gene, and a 6.0-kb locus of chromosome 3, which included the contiguous ARD1/CTS3 genes (Fig. (Fig.2A).2A). Maps were also constructed for the hypothetical chromosomal loci which contained the integrated pcts2Δ::HPH, pard1Δ/cts3Δ::HPH (not shown), or pard1Δ/cts3Δ::BLE plasmid DNA (Fig. 2B and C). Southern hybridization was conducted using gene-specific probes (CTS2, ARD1, and BLE) to test by reference to the above restriction maps whether single-site, gene-targeted integration of the plasmid constructs had occurred (Fig. (Fig.2D).2D). The data derived from Southern hybridization revealed that homologous recombination occurred at a single chromosomal locus in each of the transformants examined and confirmed that the mutant strains are homokaryotic.

Targeted CTS gene disruption generated a sterile mutant.

The morphogenesis of the saprobic and parasitic phases of the three mutant strains of C. posadasii described above were compared to that of the parental (C735) isolate. Mycelial growth rate, spore production, and development of endosporulating spherules of the cts2Δ and ard1/cts3Δ mutants were comparable to these developmental features of the parental isolate (Fig. (Fig.3A).3A). However, the time required for endospore differentiation and release from maternal spherules of these two mutants showed a significant delay compared to the parental-phase cultures. Light-microscopic examinations of spherule development in parasitic-phase cultures of the cts2/ard1/cts3Δ strain, on the other hand, revealed the production of sterile spherules that were unable to form endospores even after 3 weeks of incubation in liquid growth medium (Fig. (Fig.3B).3B). BALB/c mice challenged intranasally with a potentially lethal inoculum of 500 viable spores of the cts2/ard1/cts3Δ strain all survived, and sterile spherules (approximately 60 to 80 μm in diameter) were observed in their lungs at 21 days postchallenge (Fig. (Fig.3C).3C). Thin sections of lung tissue from mice challenged with this mutant strain and examined by transmission electron microscopy showed spherules with atypically thickened septal walls and an absence of endospores (Fig. (Fig.3D3D).

FIG. 3.
(A) In vitro-grown spherules of the C. posadasii parental isolate (C735) which had ruptured and released their endospores. This same development stage was observed in cultures of the cts2Δ and ard1/cts3Δ mutants. (B to D) Sterile spherules ...

The sterile mutant was confirmed to be avirulent in mice.

In spite of the delay in endosporulation of the cts2Δ and ard1/cts3Δ mutants in vitro, BALB/c mice challenged intranasally with 50 viable spores from saprobic cultures of each of these mutant strains showed survival plots that were nearly identical to that of mice challenged with the parental isolate (Fig. (Fig.4A).4A). On the other hand, all BALB/c mice challenged intranasally with the same number of viable spores obtained from the cts2/ard1/cts3Δ mutant survived over a 40-day period after challenge. To further evaluate whether the sterile cts2/ard1/cts3Δ mutant was avirulent, BALB/c mice were challenged intranasally with 1,000 spores and both the percent survival and fungal burden in the lungs and spleen were determined. All mice survived and were sacrificed at 60 days postchallenge. No fungal elements were detected in cultured homogenates of the lungs or spleens of these animals (Fig. (Fig.4B).4B). None of the BALB/c mice challenged intranasally with a suspension of 50 viable spores obtained from the parental (C735) strain survived beyond 28 days.

FIG. 4.
(A) Survival plots of BALB/c mice challenged intranasally with 50 viable spores of the C. posadasii parental isolate (C735) versus those of the three mutant strains. Only mice challenged with the cts2/ard1/cts3Δ strain survived to the time of ...

Vaccination with the attenuated strain protects mice against coccidioidomycosis.

Since the cts2/ard1/cts3Δ mutant (attenuated strain) was able to remain viable and convert from spores to sterile spherules in vivo, we decided to test whether this live strain could be used as a vaccine to protect mice against a potentially lethal pulmonary challenge. Three different saline suspensions of viable spores were tested by subcutaneous immunization of BALB/c mice. The vaccination protocol employed was the same as that used in our previous evaluations of recombinant protein vaccines (48); a prime immunization was followed by a boost 2 weeks later. Either freshly isolated or stored viable spores of the attenuated strain were used to immunize mice. Four weeks after the boost, the mice were challenged intranasally with 50 to 70 spores obtained from the parental (C735) strain. Control mice were immunized with saline alone. All BALB/c mice immunized subcutaneously with a total of 7.5 × 103 or 7.5 × 104 spores of the attenuated strain survived to at least 75 days after a potentially lethal intranasal challenge (Fig. (Fig.5A).5A). The same results were obtained after vaccination with either freshly isolated spores of the attenuated strain or spores obtained from this same strain which had been stored in saline at 4°C for 6 months. Mice vaccinated with a total of 1.5 × 103 spores showed a slight decrease in percent survival, but this difference was not statistically significant compared to results for the other two groups of vaccinated mice. Mice immunized with saline alone died over a period of 12 to 28 days postchallenge. At 15 days after challenge, lung homogenates of these control mice revealed a mean fungal burden of 107.7 CFU, while the vaccinated BALB/c mice all showed a significant reduction of fungal burden in their lungs (102.5 to 103.6; P = 0.04) (Fig. (Fig.5B).5B). None of the vaccinated BALB/c mice had detectable CFU in their spleen, while the control mice typically had counts of 103 to 104 CFU (not shown). Although not statistically significant, the results suggested that mice vaccinated with a total of 7.5 × 104 spores showed the highest degree of pathogen clearance from infected lungs. This same number of spores of the attenuated strain was used to vaccinate C57BL/6 mice for evaluation of survival and clearance of the pathogen after intranasal challenge (Fig. 5C and D). All vaccinated mice survived to at least 75 days postchallenge, and the mean number of CFU in their lungs at this time was significantly lower than the number of CFU in the lungs of control mice sacrificed at 15 days postchallenge (P < 0.001).

FIG. 5.
(A and B) Representative evaluations of the protective efficacy of subcutaneous vaccination of BALB/c or C57BL/6 mice, respectively, with saline suspensions of different total numbers of viable spores isolated from the attenuated strain versus immunization ...

The live attenuated vaccine elicits moderate reactogenicity at sites of immunization.

A formalin-fixed, mature spherule (FKS) vaccine was previously evaluated in mice (30) and humans (34) for its ability to protect against coccidioidomycosis. The human clinical trial was terminated, in part because of severe reactogenicity at sites of immunization. The unacceptably high level of inflammatory response to this same FKS vaccine, consisting of mature spherules of the parental isolate of C. posadasii (isolate C735), is shown at sites of subcutaneous immunization of BALB/c mice at 5 days after completion of the vaccination protocol (Fig. 6A and B). A comparable degree of reactogenicity was observed after vaccination with an equal number of formalin-killed spherules of the attenuated strain (not shown). In contrast, vaccination with live spores of the attenuated strain elicited a moderate inflammatory response, as revealed by slight swelling at sites of immunization (arrow in Fig. Fig.6C)6C) and the lower concentration of neutrophils observed in paraffin sections of skin biopsy specimens compared to results for the FKS-vaccinated mice (Fig. (Fig.6D).6D). We examined cultured homogenates of skin biopsy specimens, lungs, and spleens obtained from BALB/c mice which were immunized with a total of 7.5 × 104 spores of the attenuated strain and then challenged with a potentially lethal inoculum of the parental strain by the intranasal route. The results indicated a prolonged viability of the vaccine strain at sites of immunization in the skin but an absence of the hygromycin-resistant, attenuated strain in the lungs and spleen. We detected a range of CFU (101.0 to 103.8) in skin biopsy specimens of 40% of the vaccinated mice at 15 days postchallenge and persistence of the attenuated strain in 20% of mice at 75 days after challenge (CFU range, 101.2 to 102.2).

FIG. 6.
Comparison of reactogenicity of the subcutaneously administered FKS vaccine (A and B) versus that of the live, attenuated vaccine (C and D) in the abdominal region of BALB/c mice. Both the gross anatomy and histology of H&E-stained paraffin sections ...

The live attenuated vaccine stimulates both Th1 and Th2 pathways of immune response.

Both clinical and murine studies of coccidioidomycosis have shown that T-cell immunity is pivotal for protection and that antigens which stimulate a T-helper-1 (Th1) pathway of host immune response are essential components of a vaccine (5). A dominant Th2-type response, on the other hand, is not protective against Coccidioides infection (31). The type and amount of cytokines detected in BALFs of vaccinated mice at increasing durations postchallenge have proved to be useful surrogates of the nature of the host response to an intranasal challenge with the fungal pathogen (53). In Fig. Fig.7,7, we report the results of ELISAs of selected cytokines detected in BALFs of BALB/c mice vaccinated with the live, attenuated strain and sacrificed at 8, 25, and 75 days postchallenge. Infected control mice were immunized with saline alone, and all of these animals died within 12 to 28 days postchallenge. Normal mice (N) were included for determination of baseline amounts of the cytokines in BALFs. The amounts of both Th1-type cytokines (IL-5 and IL-10) and Th2-type cytokines (IL-12 and gamma interferon) were significantly higher in vaccinated mice than in nonvaccinated mice at 8 days postchallenge (P < 0.001) and showed a near-linear increase in concentration over the 75-day period after intranasal challenge. However, the relative amounts of Th1-type cytokines were at least 2 to 15-fold higher than selected Th2-type cytokines at corresponding assay time points. Nonvaccinated, infected mice showed a high concentration of IL-6 in their BALFs at 8 days postchallenge, in contrast to the vaccinated, infected mice, which revealed approximately threefold less IL-6 after 8 days. The IL-6 concentration in the vaccinated mice was sustained at 25 days and increased slightly at 75 days postchallenge.

FIG. 7.
ELISAs of cytokines in BALFs of BALB/c mice which were either vaccinated with the live attenuated strain or not vaccinated. BALFs were obtained from vaccinated mice at 8, 25, and 75 days postchallenge. BALFs from nonvaccinated mice were obtained at 8 ...

Lung histopathology of vaccinated/infected mice provides evidence of a protective host response.

Nonvaccinated BALB/c mice challenged intranasally with 50 viable arthroconidia of the virulent isolate of C. posadasii (C735) typically showed an intense suppurative response to lung infection within 1 to 3 weeks postchallenge. The H&E-stained paraffin section of lung tissue from a nonvaccinated mouse at 18 days after challenge in Fig. Fig.8A8A shows multiple spherules, some of which have ruptured and released their endospores (arrows). High concentrations of inflammatory cells are visible in association with these spherules and appear to be directed to the site of rupture (arrow in Fig. Fig.8B).8B). In contrast, BALB/c mice which had been vaccinated with the live, attenuated mutant showed an organized response to infection (Fig. 8C and D). No lesions were visible upon necropsy at 75 days postchallenge. Sections of lung tissue revealed well-differentiated granulomas, with extensive fibrosis at their perimeter and minimal neutrophil involvement. No parasitic cells were observed outside the lumen of the granulomas. The majority of spherules detected within the granulomas appeared to be in preseptation or septation stages of development (Fig. (Fig.8E)8E) rather than undergoing endosporulation.

FIG. 8.
Comparative histopathology of coccidioidal infection in the lungs of nonvaccinated BALB/c mice (A and B) sacrificed at 18 days postchallenge versus results for BALB/c mice which had been vaccinated with the live, attenuated strain of C. posadasii and ...


In this investigation, we have reported the first multiple gene disruption in Coccidioides, produced a genetically engineered mutant of C. posadasii which retained viability but lost its capacity to asexually reproduce within the host, and demonstrated that vaccination with this live, attenuated strain can protect coccidioidal disease-vulnerable BALB/c mice against disseminated coccidioidomycosis. The disease-causing capacity of the vaccine strain was apparently eliminated as a result of disruption of two chitinase genes located on separate chromosomes of Coccidioides. The experimental design used to generate the sterile mutant was based on our understanding of the morphogenetic events of the parasitic cycle of C. posadasii. Results of light- and electron-microscopic studies of the parasitic cycle in vitro and in vivo suggested that digestion of the chitin-rich septal wall complex of spherules is a pivotal event which coincides with the initiation of endospore formation (3, 4). It was reasonable to assume that chitinase activity played a key role in this morphogenetic process, but our discovery of eight members of family 18 chitinases (21) in the C. posadasii genome presented a challenge to validate this hypothesis. However, several features of the translated gene products supported our choice of candidates worthy of further evaluation. The structural domains of Cts2 of C. posadasii and the previously described Cts1 of S. cerevisiae (28) revealed striking similarities (37). The yeast chitinase was shown to be associated with the septal wall, and disruption of the gene which encodes this enzyme resulted in a defect in separation of the maternal and daughter yeast cells. Results of phylogenetic analyses of the subfamily of Coccidioides chitinases suggested that Cts2, -3, and -4 were most closely related. Two classes of fungal chitinases have been proposed based upon their structural similarity to family 18 chitinases of plants or bacteria (24). The plant-like fungal chitinolytic enzymes of Coccidioides include Cts2, -3, and -4 (25), which are suggested to function as endochitinases that cleave the chitin chain randomly (44). Both Cts2 and Cts4 were predicted to be secreted proteins based on the presence of signal peptides at their N termini. Cts3 lacked a signal peptide consensus sequence, suggesting that it is localized to the spherule cytoplasm. Electron-microscopic studies described above indicated that the final stages of digestion of the septal wall complex appear to involve chitinolytic activity in the matrix (residual spherule cytoplasm) surrounding the endospores. Both Cts2 and -3 showed the highest amino acid sequence similarity to chitinases of Aspergillus, many of which have been reported to be cell wall associated and suggested to perform morphogenetic roles (20). Finally, only CTS2 and CTS3 showed significant increases in expression during the transition period between spherule septation and initiation of endosporulation, when elevated chitinase activity within the spherules was expected to occur. On the basis of these observations, we decided to determine whether expression of either CTS2 or CTS3 was necessary for successful completion of the asexual reproductive process in C. posadasii.

Disruption of the CTS2 gene progressed without difficulty using a method which is now well established in our laboratory (22, 40). On the other hand, disruption of the CTS3 gene was problematic because of an error in construction of the plasmid employed in the knockout procedure. As a result of incomplete annotation of the C. posadasii (C735) genome, the plasmid was mistakenly designed to disrupt CTS3 as well as a contiguous, upstream gene, which was subsequently designated ARD1. Results of Southern hybridization confirmed that disruption of the CTS2 and ARD1/CTS3 genes had occurred by homologous recombination and that the two selected transformants were homokaryotic. The two mutant strains, the cts2Δ and ard1/cts3Δ strains, were still able to endosporulate in vitro, although both showed a significant delay in endospore formation compared to the parental strain. Nevertheless, BALB/c mice challenged intranasally with 50 viable spores of either mutant strain revealed survival plots which were nearly identical to that of mice challenged with the same number of spores of the parental isolate. We suggest that the transformants in which either the CTS2 or CTS3 gene was disrupted were reciprocally compensated by expression of the respective intact chitinase gene, and a loss of ARD1 expression did not influence endosporulation of the ard1/cts3Δ mutant. To test whether a loss of both CTS2 and CTS3 expression was necessary to inhibit endosporulation, we used the cts2Δ mutant as the parental strain and repeated the ARD1/CTS3 disruption. The phenotype of the parasitic phase of this cts2/ard1/cts3Δ mutant was sterile spherules when the mutant was grown either in vitro or in vivo. Confirming that disruption of the two chitinase genes is responsible for the observed phenotype necessitates generation of a revertant strain by complementation of the cts2/ard1/cts3Δ mutant with the wild-type CTS2 and CTS3 genes. Of relevance to this study, however, is the observation that the cts2/ard1/cts3Δ strain was avirulent in BALB/c mice challenged intranasally with either 50 or 1,000 viable spores. Particularly intriguing was that BALB/c mice totally cleared the cts2/ard1/cts3Δ mutant from their lungs and spleen after a potentially lethal challenge with 1,000 viable spores of this attenuated strain. On the basis of these results, we considered using the genetically engineered avirulent strain of C. posadasii as a live vaccine against coccidioidal infection.

Vaccination of BALB/c and C57BL/6 mice was conducted by subcutaneous immunization with viable spores of the attenuated strain suspended in saline. Three different vaccination doses were tested, and although results of survival and pathogen clearance were not significantly different, a trend was suggested in which an initial immunization with 5 × 104 spores followed 2 weeks later with a boost of 2.5 × 104 spores yielded the best results. Both vaccinated strains of mice appeared healthy at 75 days postchallenge. This degree of protection of the two inbred strains of mice using the live vaccine was superior to that with all recombinant protein vaccines against coccidioidomycosis reported to date (5, 7, 45). Most striking was the ability of the live vaccine to protect BALB/c mice, the strain which is most susceptible to disseminated coccidioidomycosis following an intranasal challenge (27).

Pappagianis (34) has pointed out that in most human cases of coccidioidomycosis, recovery from infection is followed by immunity to exogenous reinfection. Recognition of this induced, protective immunity led to studies of the abilities of live, attenuated, and killed vaccines to protect mice, cynomolgous monkeys, and humans against coccidioidal infection (30, 34). Prominent in this list of early experimental vaccines was a formulation consisting of formaldehyde-killed spherules (FKS), which was shown to protect BALB/c mice and was tested in a human double blind “phase 3” study conducted between 1980 and 1985 (34). Protective studies of mice had shown that it was necessary for the FKS vaccine to consist of mature spherules for optimal results. Intramuscular injection of a total of 3.2 mg of the killed parasitic cells was claimed to be tolerated using the murine model. On the other hand, we observed an unacceptable level of reactogenicity in BALB/c mice following subcutaneous injections of a total of only 2.7 mg of the FKS vaccine, which required the sacrifice of these animals prior to completion of the protection experiment. In humans, unfavorable local reactions necessitated adoption of 1.75 mg/dose for the clinical trial (34), which may have been insufficient to mount a protective immune response to infection. There was no statistically significant difference in the incidence of coccidioidomycosis between the vaccinated and placebo groups of volunteers. An ideal vaccine should elicit protection against infection with minimal reactogenicity and only one or two doses of the immunogen (13). Our live, attenuated vaccine showed transient reactogenicity at sites of immunization of BALB/c mice over a period of 2 to 6 weeks after completion of the vaccination protocol. The vaccine strain appeared to remain localized to sites of subcutaneous immunization. We suggest that immunization with the live spores followed by their differentiation into full-size spherules resulted in associated dendritic cell maturation and activation as reported by others (12). We also propose that as a result of the persistence of the live vaccine strain, progressive, acquired immunity against Coccidioides was effectively induced, and the immune response was much better tolerated by the mammalian host than vaccination with FKS. We have not tested whether a single dose of 7.5 × 104 spores of the attenuated strain affords the same level of protection to BALB/c mice as the prime and boost vaccination protocol described in this study. However, given how long the vaccine strain persists, it is possible that a single immunization with the live, attenuated strain would suffice.

Analysis of cytokine production by vaccinated BALB/c mice at different times postchallenge suggested that stimulation of both Th1 and Th2 pathways of T-cell immunity had occurred. The Th1/Th2 dichotomy is based on evidence that Th1 cells secrete cytokines that initiate and participate in cell-mediated immune responses while the Th2 subset of T lymphocytes secretes cytokines that stimulate B cells to produce antibodies, activate mast cells and eosinophils, and can downregulate cellular immune responses (5). Clinical studies of patients with coccidioidomycosis have indicated that high pathogen-specific antibody titers are of little benefit since they typically correlate with a poor prognosis (5-7). On the other hand, there is a growing consensus that antibodies collaborate with phagocytic cells and T cells to reduce inflammation and polarize the Th1 response against systemic mycoses (8, 18). Disseminated coccidioidomycosis in our BALB/c model typically correlated with early and persistently high levels of production of the proinflammatory cytokine IL-6. Mice vaccinated with the live, attenuated strain, on the other hand, showed a more than threefold reduction in the IL-6 concentration in BALFs at 1 to 10 weeks postchallenge. The histopathology of the nonvaccinated versus vaccinated mice underscored this difference in host response. Vaccination with the attenuated strain significantly dampened host tissue damage, which in the nonvaccinated BALB/c mice is associated with an intense, persistent inflammatory response that most likely exacerbates the course of disease. Paraffin sections of infected lungs of control mice typically revealed an apparent migration of a large number of inflammatory cells to mature spherules which had ruptured and released their contents. Thin sections of infected murine lung tissue have shown this same host-pathogen association (22). Vaccinated mice at 75 days postchallenge showed no evidence of such an intense inflammatory response but instead had produced well-differentiated granulomas which restricted the metastasis of the pathogen.

Wüthrich and coworkers (52) reported a genetically engineered, live, attenuated strain of Blastomyces dermatitidis that conferred sterilizing immunity against lethal pulmonary infection. Practical concerns raised in their study were whether the mammalian host deficient in CD4+ T cells, as can be the case for a patient with AIDS, would respond poorly to this vaccine or be at risk for adverse effects. Results of their investigations showed that protective immunity required the presence of αβ T lymphocytes but vaccine-induced immunity could be achieved in the absence of CD4+ T cells, suggesting a role for CD8+ T cells in a vaccinated, immunocompromised host. Both CD4+ and CD8+ T cells have also been shown to mediate live-vaccine-induced immunity against Coccidioides infection in mice (14). Additional studies are required to evaluate whether our genetically engineered, live, attenuated strain of C. posadasii can provide protection against coccidioidal infection in a mammalian host which is depleted of CD4+ T cells.

Supplementary Material

[Supplemental material]


Support for this study was provided by Public Health Service grant AI071118 from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health. Additional support for this project was provided by the California HealthCare Foundation and the Margaret Batts Tobin Foundation, San Antonio, TX.

We are grateful to Kalpathi R. Seshan and Veronica M. Hearn for their technical assistance and to Jo Ann Michaelson for help with the preparation of the manuscript.


Editor: A. Casadevall


[down-pointing small open triangle]Published ahead of print on 1 June 2009.

Supplemental material for this article may be found at


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