A Genetically Engineered Live Attenuated Vaccine of ... · Received 23 April 2009/Returned for modification 9 May 2009/Accepted 23 May 2009 Coccidioidomycosis (also known as San

INFECTION AND IMMUNITY, Aug. 2009, p. 3196–3208 Vol. 77, No. 80019-9567/09/$08.00�0 doi:10.1128/IAI.00459-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Genetically Engineered Live Attenuated Vaccine ofCoccidioides posadasii Protects BALB/c Mice

against Coccidioidomycosis�†Jianmin Xue,1‡ Xia Chen,1§ Dale Selby,2 Chiung-Yu Hung,1 Jieh-Juen Yu,1 and Garry T. Cole1*

Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas, San Antonio, Texas 78249,1

and Department of Pathology, Wilford Hall Medical Center, Lackland Air Force Base, Texas 782362

Received 23 April 2009/Returned for modification 9 May 2009/Accepted 23 May 2009

Coccidioidomycosis (also known as San Joaquin Valley fever) is an occupational disease. Workers exposedto outdoor dust which contains spores of the soil-inhabiting fungus have a significantly increased risk ofrespiratory infection. In addition, people with compromised T-cell immunity, the elderly, and certain racialgroups, particularly African-Americans and Filipinos, who live in regions of endemicity in the southwesternUnited States have an elevated incidence of symptomatic infection caused by inhalation of spores of Coccid-ioides posadasii or Coccidioides immitis. Recurring epidemics and escalation of medical costs have helped tomotivate 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 providedfull protection to coccidioidal disease-susceptible BALB/c mice. Here we describe the first genetically engi-neered, live, attenuated vaccine that protects both BALB/c and C57BL/6 mice against coccidioidomycosis. Twochitinase genes (CTS2 and CTS3) were disrupted to yield the attenuated strain, which was unable to endo-sporulate and was no longer infectious. Vaccinated survivors mounted an immune response characterized byproduction of both T-helper-1- and T-helper-2-type cytokines. Histology revealed well-formed granulomas andmarkedly diminished inflammation. Significantly fewer organisms were observed in the lungs of survivors thanin 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 thecausative agent of a human respiratory disease known as coc-cidioidomycosis or San Joaquin Valley fever. Results of mo-lecular epidemiological studies have suggested that C. posa-dasii (the “non-Californian” species) originated from a singleNorth American population in Texas and subsequently under-went rapid population growth in the southwestern UnitedStates (especially Texas and Arizona), Central Mexico, andregions of Venezuela, Brazil, and Argentina (15). On the otherhand, C. immitis appears to have remained geographically iso-lated to central and southern California. In spite of the geneticdiversity revealed by comparative genomic sequence analysesof these two species (43), laboratory animal studies have shownno significant difference in either the virulence or the growthand development of the organisms. Human infection typicallyoccurs by inhalation of the spores (arthroconidia) released intothe air from the saprobic phase of the soilborne fungus. In the

approximately 40% of human exposures that result in symp-tomatic infection, the initial clinical manifestation is typified byonset of an acute respiratory response that occurs about 1 to 3weeks after inhalation of the pathogen. These early symptomsare also characteristic of acute community-acquired pneumo-nia (CAP). Valdivia and coworkers (50) reported that 29% ofpatients diagnosed with CAP in Tucson, AZ, had contractedcoccidioidomycosis. Physicians are strongly advised to considerCoccidioides infection in their differential diagnosis of all pa-tients with CAP who reside in or have recently visited a regionto which coccidioidal disease is endemic (26). Coccidioidalinfections can progress to life-threatening, chronic progressivepneumonia, extrapulmonary nonmeningeal disease, or menin-gitis, the most feared complication of coccidioidomycosis (36).Although few patients develop these severe forms of the dis-ease (�1%), the number of reported cases of primary pulmo-nary infection in Arizona and California has significantly in-creased during the last decade (2, 47). Escalations in the costof long-term antifungal therapy, which is frequently requiredfor symptomatic coccidioidomycosis, argue for a way to bettercontrol this disease (17).

Several reported studies have presented evidence for thefeasibility of generating a vaccine against coccidioidal infec-tions (reviewed in references 5, 7, and 35). A compelling ar-gument for such a vaccine is the retrospective clinical obser-vation that natural human infection with Coccidioides results inlifelong immunity against the mycosis (46). Cell-mediated im-munity has been shown to be essential for an effective responseto infection by the fungal pathogen, and recent attempts todevelop a defined vaccine have focused on the identification of

* Corresponding author. Mailing address: Department of Biology,University of Texas at San Antonio, One UTSA Circle, San Antonio,TX 78249-0662. Phone: (210) 458-7017. Fax: (210) 458-7015. E-mail:[email protected].

‡ Present address: Division of Pulmonary Allergy and Critical CareMedicine, University of Pittsburgh, 3459 Fifth Ave., Pittsburgh, PA15213.

§ Present address: Department of Surgery, University of Wisconsin,WIMR 5118, 1111 Highland Ave., Madison, WI 53705.

† Supplemental material for this article may be found at http://iai.asm.org/.

� Published ahead of print on 1 June 2009.

3196

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

recombinant protein candidates which elicit protective T-cellresponses in immunized mice (45, 48). Earlier studies usedattenuated strains of Coccidioides of undefined genetic originas live vaccines and showed success in protecting BALB/c mice,the murine strain that is most susceptible to disseminated coc-cidioidomycosis following intranasal challenge (5). More re-cently a spontaneous, temperature-sensitive, auxotrophic mu-tant of C. immitis generated by exposure to UV radiation wasevaluated as a live, attenuated vaccine (14). In the absence ofa defined genetic mechanism of attenuation, however, the pos-sibility of reversion to the virulent phenotype restricts the prac-tical application of these live vaccines. Nevertheless, for nu-merous microbial diseases in which natural infection conferslifelong protection to the host, live vaccines have proved to bemost successful (13, 42). The major advantage is that the live,attenuated microorganism stimulates an immune responsesimilar to that elicited by the natural infection. The essentialfactor in a live vaccine is that its disease-causing capacity isvirtually eliminated by biological or technical manipulations.The parasitic cycle of Coccidioides is unique among the med-ically important fungi (5). Asexual reproduction of the patho-gen within the host occurs by a sequence of morphogeneticsteps that result in production of a multitude of endospores(approximately 100 to 300) which develop into new genera-tions of parasitic cells (spherules). We employed electron mi-croscopy combined with substrate labeling methods to examinesuccessive stages of spherule maturation (3). The results sug-gested that activation of chitinolytic enzymes is an essentialevent that occurs during early stages of endospore differenti-ation. We proposed that inhibition of chitinase activity at thispivotal stage of the parasitic cycle would render the pathogensterile. In this report, we describe a genetically engineeredmutant of C. posadasii which is unable to reproduce asexuallyduring its parasitic phase. The attenuated strain was evaluatedas a live vaccine in BALB/c and C57BL/6 mice and proved tobe superior to defined, recombinant protein vaccines so fartested against coccidioidomycosis.

MATERIALS AND METHODS

Fungal strains, media, and growth conditions. The fungal pathogen used inthis study is a clinical isolate (C735) of C. posadasii (16). The saprobic phases ofboth 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 generatea confluent layer of arthroconidia (spores) on the agar surface. Spores werecollected by washing plate cultures with sterile, endotoxin-free saline, and thesuspension was filtered to remove hyphal fragments. The spores were used toinoculate a defined glucose/salts medium (29) for growth of the parasitic phaseand 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 GenomicResearch, now the J. Craig Venter Institute (Rockville, MD) (www.jcvi.org). Thegenomic database for both C. posadasii and C. immitis (RS isolate), as well as theannotation for the latter, are available on the Broad Institute (Cambridge, MA)website (www.broad.mit.edu). Based on our hypothesis that chitinase productionduring the parasitic cycle plays a pivotal role in endospore differentiation (3), weconducted separate BLAST (basic local alignment search tool) searches (1) ofthe translated C. posadasii genome database using amino acid sequences of thetwo reported chitinases of this fungal pathogen as queries (Cts1 and Cts2) (37).A tBLASTn analysis was conducted using the Coccidioides databases (http://www.broad.mit.edu/annotation/genome/coccidioides_group/Blast.html) and resultedin retrieval of six homologs of the two putative chitinases of C. posadasii. Gene-specific oligonucleotide primers were synthesized and used for the rapid ampli-fication of cDNA ends procedure to obtain full-length cDNA sequences of eachof these homologs as described previously (23). Comparison of the genomic and

cDNA sequences confirmed the locations of the exons and introns and validatedeach of the respective C. posadasii genomic sequences in the database. Structuralcomparison of the eight deduced protein homologs was conducted by alignmentof amino acid sequences using the CLUSTALX algorithm (49). The PROSITEalgorithm was used to identify conserved motifs of the translated polypeptideswith homology to reported fungal chitinases in the nonredundant protein data-base available from the National Center for Biotechnology Information as pre-viously reported (9). Phylogenetic studies of both the C. posadasii and C. immitissubfamilies of chitinases were performed using maximum-likelihood methods asimplemented in the program (available at http://www.phylogeny.fr/) for compar-ison of the structure of the catalytic domains (11, 25). The PSORT and PSORTII algorithms were used for prediction of cellular localization sites of the putativechitinases (33).

Comparative real time-PCR examinations of CTS gene expression. To test ourhypothesis that chitinolytic activity is essential for completion of the endosporu-lation phase of the parasitic cycle, we first evaluated expression of each CTS geneduring successive stages of parasitic cell development in vitro. We expected toobserve elevated expression of one or more of these genes during early stages ofendospore formation. As shown previously, in vitro growth and differentiation ofspherules during the first generation of the parasitic cycle are nearly synchronous(23). This permitted comparison of expression profiles of the eight CTS genesduring selected stages of spherule development. We examined levels of geneexpression during spherule septation (72-h parasitic-phase cultures), digestion ofthe septal wall complex and initiation of endospore formation (96 h), and en-dospore release from ruptured spherules (120 to 132 h) (32) by quantitative realtime-PCR (QRT-PCR) as reported previously (22). The generation of cDNAfrom parasitic cell-derived total RNA preparations was conducted as reportedelsewhere (9). The nucleotide sequences of the CTS-specific sense and antisenseprimers used for QRT-PCR are provided in the supplemental material (seeTable S1).

Gene disruption strategies for generation of an attenuated strain of C. posa-dasii. To further test our hypothesis that chitinases play a key role in reproduc-tion 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 andQRT-PCR analyses. Both single and combined gene disruptions were per-formed. Plasmids were constructed to disrupt the CTS2 and CTS3 genes of C.posadasii by homologous recombination (10). To generate the plasmid constructfor disruption of CTS2 (37), a pair of synthesized oligonucleotide primers wasfirst used to amplify the gene from genomic DNA of C. posadasii by PCR. Thesequences of the sense and antisense primers were 5�-CCGTCGAGGGAGTCTGATAG-3� and 5�-GTATGTACAAGTACAGCACC-3�, respectively. The3,041-bp PCR product of CTS2 was cloned into the PCR 2.1 Topo vector(Invitrogen, San Diego, CA). The CTS2 gene disruption construct was obtainedby 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 vectorwith 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 termi-nator in C. posadasii (38) confers resistance to the growth inhibitor hygromycinB (HmB) as reported previously (40). The final 6.9-kb plasmid construct(pcts2�::HPH) used to disrupt the CTS2 gene was linearized by digestion of themodified Topo vector with ApaI and DraI and purified for subsequent transfor-mation of the C. posadasii parental strain as described previously (32). The6,891-bp ApaI/DraI plasmid construct was comprised of a 3,597-bp fragment ofpAN7.1, 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 1,161bp, respectively) were also present in this construct.

To generate the plasmid construct for disruption of CTS3, a pair of oligonu-cleotide primers were synthesized to amplify a 2,936-bp genomic DNA fragmentby 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 theORF. The sequences of the sense and antisense primers used to amplify the2.9-kb genomic fragment were 5�-GTGCGCTGTAAGACCATGATC-3� and 5�-CTTCGAGTAAAGCGCAGAAG-3�, respectively. As a result of ongoing an-notation of the C. immitis and closely related C. posadasii genomes, it was evidentthat what we originally interpreted as a 2.0-kb fragment of the 5�-UTR of CTS3actually 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 frag-ment was cloned into the Topo plasmid vector (Invitrogen). The disruptionconstruct 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-kbNheI/SspI fragment of the pAN7.1 plasmid, which includes the HPH gene. The

VOL. 77, 2009 LIVE GENETICALLY DEFINED COCCIDIOIDOMYCOSIS VACCINE 3197

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

final 5,634-kb plasmid construct (pard1�cts3�::HPH) was linearized by digestionwith ApaI and KpnI. The plasmid was comprised of a 3,804-bp fragment ofpAN7.1, with a 965-bp 5� flanking component that included the partial 5�-UTRand an upstream portion of the ORF of ARD1, and a 741-bp 3�-flanking com-ponent that included the partial ORF and 3�-UTR of CTS3. In addition, theconstruct 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 asreported previously (40). Putative cts2�::HPH and ard1�/cts3�::HPH transfor-mants were selected on GYE agar plates supplemented with 75 �g/ml of HmBand subsequently maintained on a growth medium containing GYE plus 100�g/ml HmB. Southern hybridization was employed as reported previously (32) toconfirm the targeted disruption of the CTS2 and ARD1/CTS3 genes. In brief,genomic DNA isolated from candidate transformants was digested with endo-nucleases selected on the basis of the restriction maps of the genes (see Fig. 2).The digestion products were separated by agarose gel electrophoresis, trans-ferred to a nitrocellulose membrane, and hybridized with either a HPH, CTS2, orARD1 oligonucleotide probe (600, 628, or 601 bp, respectively). The probes weregenerated 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�) andan ARD1 primer pair (sense primer, 5�-CACCGGCTGACTAGTGATAC-3�;antisense primer, 5�-GCCGACGTGTGCCTTG-3�).

To generate a mutant in which both the CTS2 and CTS3 genes were disrupted,the cts2�::HPH transformant was used as the parental strain. Analysis of thegenomic database confirmed that CTS2 and the contiguous ARD1/CTS3 genesare located on separate chromosomes. The ARD1/CTS3 genomic fragment,which was cloned into the Topo plasmid as described above, was digested withNheI/SspI, and the 1.2-kb deletion product was replaced with a 3.1-kb NheI/SspIfragment of the pAN8.1 plasmid (GenBank accession no. Z32751). The lattercontains the BLE gene, which encodes a phleomycin binding protein (39) thatconfers resistance to the fungal growth inhibitor phleomycin (22, 41). The ARD1/CTS3 disruption plasmid (pard1�/cts3�::BLE) was linearized with ApaI andKpnI and used to transform the cts2�::HPH strain. Transformants(cts2�::HPH-ard1�/cts3�::BLE) were selected on GYE agar supplemented with3 �g/ml of phleomycin and subsequently maintained on GYE agar plus 5 �g/mlof phleomycin. Southern hybridization of the phleomycin-resistant transformantswas conducted using the CTS2 and ARD1 probes described above, as well as a346-bp BLE-specific probe. The sense and antisense primers employed to am-plify the BLE probe were 5�-AGTGCCGTTCCGGTGCTCACC-3� and 5�-CGGCCACGAAGTGCACGCAGT-3�, respectively.

The designation of the single, double, and triple gene knockout strains de-scribed above are henceforth referred to as the cts2�, ard1/cts3�, and cts2/ard1/cts3� mutants, respectively.

In vitro and in vivo growth of mutant strains. The parental strain and threemutant strains were cultured as both the saprobic and parasitic phases as de-scribed above and examined by light microscopy. In vivo growth of the parentalstrain and that of the cts2/ard1/cts3� mutant were compared by infecting BALB/cmice (females, 8 weeks old; supplied by the National Cancer Institute, Bethesda,MD) by the intranasal route with 500 viable spores suspended in saline asreported previously (32). Mice were sacrificed at 21 days postchallenge, andbiopsy specimens of lung abscesses were stained with blankophor, an opticalbrightener that binds to glucan and chitin in the fungal cell wall (53). Tissuesmears on glass slides were examined directly by fluorescence microscopy. Thinsections of abscesses obtained from the same infected lung tissue were alsoexamined by electron microscopy as described previously (32).

Virulence studies. To compare the virulence of the parental strain and threemutant strains, spores (50 viable cells) were obtained from GYE plate culturesof each, suspended in saline, and used to inoculate BALB/c mice (8-week-oldfemales; 12 mice per group) by the intranasal route as described above. Thesurvival plots of each group examined over a 40-day period were subjected toKaplan-Meier statistical analysis as described previously (32). Evaluation of thevirulence of the cts2/ard1/cts3� mutant in BALB/c mice was repeated but with a20-fold increase in the intranasal challenge dose (approximately 1,000 viablespores). Survivors were sacrificed at 60 days postchallenge to determine theresidual CFU of the mutant strain in homogenates of the murine lungs andspleen as reported previously (32).

The genetically engineered cts2/ard1/cts3� mutant is referred to below as theattenuated strain.

Vaccination and assessment of protection. BALB/c mice or C57BL/6 mice(females, 8 weeks old; supplied by the National Cancer Institute) were immu-nized subcutaneously in the abdominal region with different numbers of freshly

isolated or stored spores of the attenuated strain. The spores were stored insterile 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 sporessuspended in 100 �l of saline, followed 14 days later with an immunization boostof 0.5 � 103, 2.5 � 103, or 2.5 � 104 live spores, respectively. Two separategroups of BALB/c mice were vaccinated with either a saline suspension offormalin-killed, mature spherules (72-h parasitic-phase culture) of the C. posa-dasii parental isolate (C735) (FKS vaccine; three immunizations of 106 cells (0.9mg) each at 10-day intervals) as reported previously (30), or a saline suspensionof mature spherules of the attenuated strain (same cell concentration and vac-cination protocol). Control mice were immunized with saline alone using thesame protocol as employed above for vaccination with live spores. Mice werechallenged by the intranasal route with 50 to 70 viable spores of the parentalstrain 4 weeks after completion of the vaccination schedule as reported previ-ously (32). Statistical analysis of survival of the vaccinated versus nonvaccinatedgroups of mice was conducted as described above. Control mice were sacrificedat 15 days postchallenge, while mice vaccinated with the attenuated strain weresacrificed at 75 days postchallenge. The CFU were expressed on a log scale, andthe Mann-Whitney U test was used to compare differences in the median CFUvalues for statistical significance as described previously (32). Four separateexperiments were conducted as described above with each mouse strain toevaluate survival and pathogen clearance.

Gross examination, histology of vaccination sites, and persistence of theattenuated strain. Separate groups of BALB/c mice (three per group) werevaccinated with spores of the live, attenuated strain or the FKS preparation ofeither the parental or attenuated strain as described above and examined tocompare the intensity of reactogenicity (the capacity to produce adverse effects)at sites of subcutaneous immunization. A delipidation reagent (Nair, Church andDwight Co., Inc., Princeton, NJ) was applied to the abdominal region of the micefor removal of hair immediately prior to gross examination on day 5 aftercompletion of the respective vaccination protocols. Comparative histologicalexaminations of sections (5 �m thick) of paraffin-embedded skin biopsy speci-mens obtained from sites of immunization of these same three groups of micewere carried out to determine the intensities of immune responses to the re-spective vaccines. Tissue fixation and embedding procedures were performed asdescribed previously (32), and sections were stained with hematoxylin and eosin(H&E) by standard procedure. Two separate groups of BALB/c mice (12 miceper 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 thevaccine strain at sites of immunization in infected mice and whether the vaccinestrain disseminated from these sites to other body organs. The mice were sacri-ficed at either 15 or 75 days postchallenge, skin biopsy specimens at vaccinationsites were obtained, and lungs and spleens were excised, homogenized, andplated on GYE plus 50 �g/ml chloramphenicol (Sigma, St. Louis, MO) plus 100�g/ml HmB. The numbers of CFU of the attenuated strain in the tissue homog-enates were determined.

Cytokines in BALFs of vaccinated mice. Cytokine production in bronchoal-veolar lavage fluids (BALFs) was compared in three groups of BALB/c mice(four per group), which were either untreated (i.e., normal mice), not vaccinatedbut intranasally challenged with Coccidioides, or vaccinated and challenged. Themice were vaccinated subcutaneously with two doses of live spores of the atten-uated strain (total, 7.5 � 104) suspended in saline as described above. BALFsfrom untreated mice were used to determine the basal level of production ofeach cytokine examined in this study. Vaccinated, infected mice were sacrificedat 8, 25, or 75 days postchallenge. Nonvaccinated, infected mice were sacrificedat 8 days. OptEIA mouse cytokine kits (Pharmingen, San Diego, CA) were usedfor enzyme-linked immunosorbent assays (ELISAs) of interleukin 5 (IL-5), IL-6,IL-10, IL-12p70, and gamma interferon concentrations in lavage fluids as re-ported previously (53). ELISAs of samples from individual mice were carried outin triplicate. Standard curves were generated using purified, recombinant cyto-kines supplied by the manufacturer of the kits. Standard curves were used tocalculate the amounts of specific cytokines in the BALF samples.

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

3198 XUE ET AL. INFECT. IMMUN.

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Nucleotide sequence accession numbers. The C. posadasii nucleotide se-quences of the CTS3 to CTS8 genes described in this article have been depositedin GenBank under accession no. AF492472, AF510393, AY454340, AF519181,AY454339, and FJ801037, respectively. CTS1 and CTS2 sequences were previ-ously deposited under accession no. L41663 and L41662, respectively.

RESULTS

Discovery of eight members of a subfamily of C. posadasiigenes encoding chitinolytic enzymes. As a result of tBLASTnanalysis of the C. posadasii (C735) genome database and align-ment of matching amino acid sequences, we retrieved six pro-tein homologs of the previously reported chitinases (Cts1 andCts2) (37). Each of the eight amino acid sequences containedthe fully conserved consensus domains of family 18 chitinolyticenzymes (EC 3.2.1.14) (Table 1) (21). A BLASTp search wasconducted using each of the deduced amino acid sequences toquery against the nonredundant NCBI database as describedpreviously (9). The results revealed highest sequence similar-ities and identities with fungal chitinases of other ascomyce-tous fungi (not shown). CLUSTALX analysis of the subfamilyof chitinases in C. posadasii and C. immitis predicted the exis-tence of a ninth chitinase of C. immitis, which is absent fromthe genome of C. posadasii (not shown). Phylogenetic analyseswere conducted using the eight deduced chitinase sequences ofC. posadasii and orthologs of the annotated genome of C.immitis (RS isolate). The combined phylogenetic tree sug-gested that Cts2, -3, and -4 are more closely related to eachother than to the other members of this subfamily (see Fig. S1in the supplemental material). On the basis of structural anal-ysis of the deducted proteins, Cts2 and Cts4 were predicted tohave signal sequences while Cts3 apparently lacks a signalpeptide and may be localized to the cytoplasm of spherules.The structural domains of Cts2 are similar to those of a Sac-charomyces cerevisiae chitinase (28). Both proteins contain aconserved hydrolytic region upstream of a Ser/Thr-rich do-main. The yeast chitinase also contains a C-terminal chitinbinding signature sequence, which is required for localizationof the enzyme in the yeast cell wall but is absent from Cts2 ofC. posadasii. However, Coccidioides Cts2 is predicted to have aglycosylphosphatidylinositol anchor, which could be an alter-

native structural component that targets the protein to thefungal cell wall.

Comparison of levels of CTS gene expression during theparasitic cycle suggests functional differences. We recognizethat transcription is subject to multiple regulatory mechanismand attempts to correlate temporal gene expression with aproposed function can be misleading. Nevertheless, we usedQRT-PCR to predict which CTS genes of C. posadasii play arole in the digestion of the septal wall during the early stagesof endospore differentiation. We focused on differences in theamounts of CTS transcripts between the 72-h culture stage,when spherule septal wall formation is under way, and the 96-hstage, when the septal wall complex undergoes digestion andendospore differentiation is initiated. Of the eight deduced C.posadasii chitinase genes, only CTS2 and CTS3 revealed asignificant increase in expression during this developmentaltransition. CTS2 showed a 3.0-fold increase while CTS3 re-vealed a 6.5-fold increase in the amount of respective tran-script during this period of spherule development (Fig. 1). Thelevel of expression of CTS2 sharply decreased upon spherulematuration and endospore formation, while the expressionlevel of CTS3 remained elevated.

Single and combined disruption of CTS2 and CTS3 genes.On the basis of the structural and phylogenetic analyses of thesubfamily of chitinases of C. posadasii together with the resultsshown in Fig. 1, we decided to test whether expression of CTS2and/or CTS3 is essential for digestion of the septal wall andsubsequent formation of endospores within the maternalspherules. The two chitinase genes are located on separatechromosomes, and their restriction maps are shown in Fig. 2A.Two plasmid constructs were employed to separately disruptthe CTS2 and CTS3 genes. A plasmid construct containing theHPH gene replaced the putative substrate binding and activesite domains of CTS2 (Fig. 2B). Results of PCR screening ofthese candidate transformants suggested that four had under-gone homologous recombination. One of these transformants,referred to as the cts2� strain, was selected for subsequentanalysis by Southern hybridization. Disruption of the CTS3

FIG. 1. QRT-PCR of CTS1 to CTS8 expression in vitro duringselected stages of the parasitic cycle. Expression of each CTS gene wascompared to that of the constitutive GAPDH gene. p, spherule sep-tation stage; f, septal digestion and early endospore formation stage;

, spherule rupture and endospore release stage.

TABLE 1. Conserved domains of deduced chitinases (Cts1 toCTS8) of C. posadasii (isolate C735)a

ChitinaseSequence

Box1 Box2

Cts1 ktllSiGG lmkdlgfDGiDiDwEypCts2 tillSlGG pfgeasvDGfDfDiEkgCts3 kvmgMvGG mirtrnlNGlDlDvEedCts4 tiilSiGG pfgdatvDGfDlDfEatCts5 ktviAlGG fmrqyafDGvDfDwEypCts6 killSiGG lladcgfDGiDiDwEypCts7 kvllSiGG lmldmgmDGlDvDwEypCts8 kvilSiGG mvdrfglDGlDiDwEhp

a The consensus sequences in Box1 and Box2 correspond to the �3 and �4barrel folds of the family 18 chitinases, respectively. The glycines (G) in Box1 areproposed to be components of the chitin binding domain. The adjacent aspar-tate/glycine residues (DG) in Box2 are fully conserved in all family 18 sequences.Glutamic acid (E) in Box2 is essential for catalytic activity of family 18 chitinases(51). Conserved residues are bold and uppercase.

VOL. 77, 2009 LIVE GENETICALLY DEFINED COCCIDIOIDOMYCOSIS VACCINE 3199

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

gene was complicated by the presence of a contiguous, up-stream gene (ARD1) separated by a 697-bp UTR (Fig. 2A).The plasmid construct, which also contained the HPH gene,replaced a 342-bp ORF fragment of ARD1 and a 189-bp ORFcomponent of CTS3. As a result, this transformation plasmid(pard1�/cts3�::HPH) disrupted both the ARD1 and CTS3genes. PCR screening of the putative transformants identifiedfour that had apparently undergone homologous recombina-tion based on results of PCR analysis. One of the transfor-mants, referred to as the ard1/cts3� strain, was selected forfurther analysis by Southern hybridization.

Examination of in vitro growth of the cts2� and ard1/cts3�mutant strains, as described below, indicated that disruption ofeither the CTS2 gene alone or the combined ARD1/CTS3genes had no significant effect on endosporulation. On thisbasis, we decided to generate a mutant strain in which both theCTS2 and CTS3 genes were disrupted. The cts2� strain wasused as the parent for disruption of CTS3, and the same plas-mid construct was employed as described above, except thatthe pAN7.1 fragment was replaced with a 3.1-kb NheI/SspI-restricted fragment of pAN8.1 containing the BLE gene, whichconfers resistance to phleomycin (Fig. 2C). One of the trans-formants, designated the cts2/ard1/cts3� strain, was selectedfor further study. The cts2�, ard1/cts3�, and cts2/ard1/cts3�mutant strains were subjected to Southern hybridization forconfirmation of disruption of the respective target genes (Fig.

2D). Restriction maps of CTS2 and ARD1/CTS3 were con-structed by reference to the C. posadasii genomic database.Restriction sites were mapped in a 12.0-kb locus of chromo-some 1, which contained the CTS2 gene, and a 6.0-kb locus ofchromosome 3, which included the contiguous ARD1/CTS3genes (Fig. 2A). Maps were also constructed for the hypo-thetical chromosomal loci which contained the integratedpcts2�::HPH, pard1�/cts3�::HPH (not shown), or pard1�/cts3�::BLE plasmid DNA (Fig. 2B and C). Southern hybrid-ization was conducted using gene-specific probes (CTS2,ARD1, and BLE) to test by reference to the above restrictionmaps whether single-site, gene-targeted integration of the plas-mid constructs had occurred (Fig. 2D). The data derived fromSouthern hybridization revealed that homologous recombina-tion occurred at a single chromosomal locus in each of thetransformants examined and confirmed that the mutant strainsare homokaryotic.

Targeted CTS gene disruption generated a sterile mutant.The morphogenesis of the saprobic and parasitic phases of thethree mutant strains of C. posadasii described above were com-pared to that of the parental (C735) isolate. Mycelial growthrate, spore production, and development of endosporulatingspherules of the cts2� and ard1/cts3� mutants were compara-ble to these developmental features of the parental isolate(Fig. 3A). However, the time required for endospore differen-tiation and release from maternal spherules of these two mu-

FIG. 2. (A) Restriction maps of chromosomal loci of C. posadasii (isolate C735) that contain the CTS2 gene (left) and the contiguous ARD1and CTS3 genes (right). CTS2 and ARD1 probes were used for Southern hybridization analyses of transformants. (B and C) Hypotheticalrestriction maps of the same chromosomal loci shown in panel A after replacement of fragments of the CTS2 gene (B) and contiguous ARD1 andCTS3 genes (C) with the pAN7.1 plasmid construct and pAN8.1 plasmid construct, respectively. Replacement of the ARD1/CTS3 gene fragmentsin panel C was conducted using the cts2� transformant as the parental strain. The BLE probe was used to confirm by Southern hybridization thatinsertion of the pAN8.1 plasmid construct into the targeted chromosome had occurred. (D) Results of Southern hybridization using three separateprobes described above with restricted genomic DNA isolated from the original C. posadasii (C735) parental isolate (P) or the transformantcontaining the CTS2 gene disruption construct (cts2�), the contiguous ARD1/CTS3 gene disruption construct (ard/3�), or the combined CTS2 andARD1/CTS3 gene disruption constructs (2/a/3�).

3200 XUE ET AL. INFECT. IMMUN.

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

tants showed a significant delay compared to the parental-phase cultures. Light-microscopic examinations of spheruledevelopment in parasitic-phase cultures of the cts2/ard1/cts3�strain, on the other hand, revealed the production of sterilespherules that were unable to form endospores even after 3weeks of incubation in liquid growth medium (Fig. 3B).BALB/c mice challenged intranasally with a potentially lethalinoculum of 500 viable spores of the cts2/ard1/cts3� strain allsurvived, and sterile spherules (approximately 60 to 80 �min diameter) were observed in their lungs at 21 days post-challenge (Fig. 3C). Thin sections of lung tissue from micechallenged with this mutant strain and examined by trans-mission electron microscopy showed spherules with atypi-cally thickened septal walls and an absence of endospores(Fig. 3D).

The sterile mutant was confirmed to be avirulent in mice. Inspite of the delay in endosporulation of the cts2� and ard1/

cts3� mutants in vitro, BALB/c mice challenged intranasallywith 50 viable spores from saprobic cultures of each of thesemutant strains showed survival plots that were nearly identicalto that of mice challenged with the parental isolate (Fig. 4A).On the other hand, all BALB/c mice challenged intranasallywith the same number of viable spores obtained from thects2/ard1/cts3� mutant survived over a 40-day period afterchallenge. To further evaluate whether the sterile cts2/ard1/cts3� mutant was avirulent, BALB/c mice were challengedintranasally with 1,000 spores and both the percent survivaland 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 ofthe lungs or spleens of these animals (Fig. 4B). None of theBALB/c mice challenged intranasally with a suspension of 50viable spores obtained from the parental (C735) strain sur-vived beyond 28 days.

FIG. 3. (A) In vitro-grown spherules of the C. posadasii parental isolate (C735) which had ruptured and released their endospores. This samedevelopment stage was observed in cultures of the cts2� and ard1/cts3� mutants. (B to D) Sterile spherules of the cts2/ard1/cts3� mutant grownin vitro (B) or in vivo (C and D). A blankophor-stained, whole mount of lung tissue from a BALB/c mouse challenged intranasally with thects2/ard1/cts3� mutant revealed sterile spherules (C), and a thin section of this same tissue showed the thickened septal walls and absence ofendospores in a spherule of the same triple gene knockout strain (D). Bars in panels A to D represent 60 �m, 60 �m, 60 �m, and 15 �m,respectively.

VOL. 77, 2009 LIVE GENETICALLY DEFINED COCCIDIOIDOMYCOSIS VACCINE 3201

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Vaccination with the attenuated strain protects mice againstcoccidioidomycosis. Since the cts2/ard1/cts3� mutant (atten-uated strain) was able to remain viable and convert fromspores to sterile spherules in vivo, we decided to testwhether this live strain could be used as a vaccine to protectmice against a potentially lethal pulmonary challenge. Threedifferent saline suspensions of viable spores were tested bysubcutaneous immunization of BALB/c mice. The vaccina-tion protocol employed was the same as that used in ourprevious evaluations of recombinant protein vaccines (48); aprime immunization was followed by a boost 2 weeks later.Either freshly isolated or stored viable spores of the atten-uated strain were used to immunize mice. Four weeks afterthe boost, the mice were challenged intranasally with 50 to70 spores obtained from the parental (C735) strain. Controlmice were immunized with saline alone. All BALB/c miceimmunized subcutaneously with a total of 7.5 � 103 or 7.5 �104 spores of the attenuated strain survived to at least 75days after a potentially lethal intranasal challenge (Fig. 5A).The same results were obtained after vaccination with eitherfreshly isolated spores of the attenuated strain or sporesobtained from this same strain which had been stored insaline at 4°C for 6 months. Mice vaccinated with a total of1.5 � 103 spores showed a slight decrease in percent sur-vival, but this difference was not statistically significant com-pared to results for the other two groups of vaccinated mice.Mice immunized with saline alone died over a period of 12to 28 days postchallenge. At 15 days after challenge, lunghomogenates of these control mice revealed a mean fungalburden of 107.7 CFU, while the vaccinated BALB/c mice allshowed a significant reduction of fungal burden in theirlungs (102.5 to 103.6; P � 0.04) (Fig. 5B). None of thevaccinated BALB/c mice had detectable CFU in theirspleen, while the control mice typically had counts of 103 to104 CFU (not shown). Although not statistically significant,

the results suggested that mice vaccinated with a total of7.5 � 104 spores showed the highest degree of pathogenclearance from infected lungs. This same number of sporesof the attenuated strain was used to vaccinate C57BL/6 micefor evaluation of survival and clearance of the pathogenafter intranasal challenge (Fig. 5C and D). All vaccinatedmice survived to at least 75 days postchallenge, and themean number of CFU in their lungs at this time was signif-icantly lower than the number of CFU in the lungs of controlmice sacrificed at 15 days postchallenge (P � 0.001).

The live attenuated vaccine elicits moderate reactogenicityat sites of immunization. A formalin-fixed, mature spherule(FKS) vaccine was previously evaluated in mice (30) andhumans (34) for its ability to protect against coccidioido-mycosis. The human clinical trial was terminated, in partbecause of severe reactogenicity at sites of immunization.The unacceptably high level of inflammatory response tothis same FKS vaccine, consisting of mature spherules of theparental isolate of C. posadasii (isolate C735), is shown atsites of subcutaneous immunization of BALB/c mice at 5days after completion of the vaccination protocol (Fig. 6Aand B). A comparable degree of reactogenicity was observedafter vaccination with an equal number of formalin-killedspherules of the attenuated strain (not shown). In contrast,vaccination with live spores of the attenuated strain elicited

FIG. 4. (A) Survival plots of BALB/c mice challenged intranasallywith 50 viable spores of the C. posadasii parental isolate (C735) versusthose of the three mutant strains. Only mice challenged with thects2/ard1/cts3� strain survived to the time of sacrifice. (B) Survivalplots of two groups of BALB/c mice challenged intranasally with either50 viable spores of the parental strain (C735) or 1,000 viable spores ofthe cts2/ard1/cts3� strain. Also shown is a plot of CFU of the mutantstrain detected in dilution plate cultures of lung and spleen homoge-nates of the latter group of BALB/c mice sacrificed at 60 days post-challenge. Both the lungs and spleen of all mice were cleared of thesterile mutant.

FIG. 5. (A and B) Representative evaluations of the protectiveefficacy of subcutaneous vaccination of BALB/c or C57BL/6 mice,respectively, with saline suspensions of different total numbers of via-ble spores isolated from the attenuated strain versus immunizationwith saline alone. The mice were challenged with 50 to 70 viable sporesof the virulent C. posadasii parental isolate (C735). The statisticalsignificance (P value) of the difference between the survival plots forthe vaccinated versus nonvaccinated mice is shown. (C and D) Plots ofCFU of the challenge isolate (C735) detected in dilution plate culturesof lung homogenates of vaccinated mice are shown as described forpanels A and B. Control mice were immunized with saline alone andsacrificed at 15 days postchallenge. All mice vaccinated with indicatednumbers of spores of the attenuated strain were sacrificed at 75 dayspostchallenge. The median CFU values (log10) are indicated in panelsC and D by horizontal lines. No statistically significant difference inCFU was observed for BALB/c mice vaccinated with different totalnumbers of spores.

3202 XUE ET AL. INFECT. IMMUN.

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

a moderate inflammatory response, as revealed by slightswelling at sites of immunization (arrow in Fig. 6C) and thelower concentration of neutrophils observed in paraffin sec-tions of skin biopsy specimens compared to results for theFKS-vaccinated mice (Fig. 6D). We examined cultured ho-mogenates of skin biopsy specimens, lungs, and spleens ob-tained from BALB/c mice which were immunized with atotal of 7.5 � 104 spores of the attenuated strain and thenchallenged with a potentially lethal inoculum of the parentalstrain by the intranasal route. The results indicated a pro-longed viability of the vaccine strain at sites of immunizationin the skin but an absence of the hygromycin-resistant, at-tenuated strain in the lungs and spleen. We detected a rangeof CFU (101.0 to 103.8) in skin biopsy specimens of 40% ofthe vaccinated mice at 15 days postchallenge and persistenceof the attenuated strain in 20% of mice at 75 days afterchallenge (CFU range, 101.2 to 102.2).

The live attenuated vaccine stimulates both Th1 and Th2pathways of immune response. Both clinical and murine stud-ies 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 essentialcomponents of a vaccine (5). A dominant Th2-type response,on the other hand, is not protective against Coccidioides infec-tion (31). The type and amount of cytokines detected inBALFs of vaccinated mice at increasing durations postchal-lenge have proved to be useful surrogates of the nature of thehost response to an intranasal challenge with the fungal patho-gen (53). In Fig. 7, we report the results of ELISAs of selectedcytokines detected in BALFs of BALB/c mice vaccinated withthe live, attenuated strain and sacrificed at 8, 25, and 75 dayspostchallenge. Infected control mice were immunized with sa-line alone, and all of these animals died within 12 to 28 dayspostchallenge. Normal mice (N) were included for determina-tion of baseline amounts of the cytokines in BALFs. Theamounts of both Th1-type cytokines (IL-5 and IL-10) andTh2-type cytokines (IL-12 and gamma interferon) were signif-icantly higher in vaccinated mice than in nonvaccinated mice at8 days postchallenge (P � 0.001) and showed a near-linearincrease in concentration over the 75-day period after intrana-

FIG. 6. Comparison of reactogenicity of the subcutaneously administered FKS vaccine (A and B) versus that of the live, attenuated vaccine (Cand D) in the abdominal region of BALB/c mice. Both the gross anatomy and histology of H&E-stained paraffin sections of skin biopsy specimensfrom vaccination sites showed striking differences in the levels of inflammatory response to these two vaccines. Bars in panels A to D represent4 mm, 0.5 mm, 4 mm, and 0.5 mm, respectively.

VOL. 77, 2009 LIVE GENETICALLY DEFINED COCCIDIOIDOMYCOSIS VACCINE 3203

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

sal challenge. However, the relative amounts of Th1-type cy-tokines were at least 2 to 15-fold higher than selected Th2-typecytokines at corresponding assay time points. Nonvaccinated,infected mice showed a high concentration of IL-6 in theirBALFs at 8 days postchallenge, in contrast to the vaccinated,infected mice, which revealed approximately threefold lessIL-6 after 8 days. The IL-6 concentration in the vaccinatedmice was sustained at 25 days and increased slightly at 75 dayspostchallenge.

Lung histopathology of vaccinated/infected mice providesevidence of a protective host response. Nonvaccinated BALB/cmice challenged intranasally with 50 viable arthroconidia ofthe virulent isolate of C. posadasii (C735) typically showed anintense suppurative response to lung infection within 1 to 3weeks postchallenge. The H&E-stained paraffin section of lungtissue from a nonvaccinated mouse at 18 days after challengein Fig. 8A shows multiple spherules, some of which have rup-tured and released their endospores (arrows). High concentra-tions of inflammatory cells are visible in association with thesespherules and appear to be directed to the site of rupture(arrow in Fig. 8B). In contrast, BALB/c mice which had beenvaccinated with the live, attenuated mutant showed an orga-nized response to infection (Fig. 8C and D). No lesions werevisible upon necropsy at 75 days postchallenge. Sections oflung tissue revealed well-differentiated granulomas, with ex-tensive fibrosis at their perimeter and minimal neutrophil in-volvement. No parasitic cells were observed outside the lumenof the granulomas. The majority of spherules detected withinthe granulomas appeared to be in preseptation or septationstages of development (Fig. 8E) rather than undergoing en-dosporulation.

DISCUSSION

In this investigation, we have reported the first multiple genedisruption in Coccidioides, produced a genetically engineeredmutant of C. posadasii which retained viability but lost itscapacity to asexually reproduce within the host, and demon-strated that vaccination with this live, attenuated strain canprotect coccidioidal disease-vulnerable BALB/c mice againstdisseminated coccidioidomycosis. The disease-causing capacityof the vaccine strain was apparently eliminated as a result ofdisruption of two chitinase genes located on separate chromo-somes of Coccidioides. The experimental design used to gen-erate the sterile mutant was based on our understanding of themorphogenetic events of the parasitic cycle of C. posadasii.Results of light- and electron-microscopic studies of the para-sitic cycle in vitro and in vivo suggested that digestion of thechitin-rich septal wall complex of spherules is a pivotal eventwhich coincides with the initiation of endospore formation (3,4). It was reasonable to assume that chitinase activity played akey role in this morphogenetic process, but our discovery ofeight members of family 18 chitinases (21) in the C. posadasiigenome presented a challenge to validate this hypothesis.However, several features of the translated gene products sup-ported our choice of candidates worthy of further evaluation.The structural domains of Cts2 of C. posadasii and the previ-ously described Cts1 of S. cerevisiae (28) revealed striking sim-ilarities (37). The yeast chitinase was shown to be associatedwith the septal wall, and disruption of the gene which encodesthis enzyme resulted in a defect in separation of the maternaland daughter yeast cells. Results of phylogenetic analyses ofthe subfamily of Coccidioides chitinases suggested that Cts2, -3,and -4 were most closely related. Two classes of fungal chiti-nases have been proposed based upon their structural similar-ity 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 endochiti-nases that cleave the chitin chain randomly (44). Both Cts2 andCts4 were predicted to be secreted proteins based on the pres-ence of signal peptides at their N termini. Cts3 lacked a signalpeptide consensus sequence, suggesting that it is localized tothe spherule cytoplasm. Electron-microscopic studies de-scribed above indicated that the final stages of digestion of theseptal wall complex appear to involve chitinolytic activity in thematrix (residual spherule cytoplasm) surrounding the endo-spores. Both Cts2 and -3 showed the highest amino acid se-quence similarity to chitinases of Aspergillus, many of whichhave been reported to be cell wall associated and suggested toperform morphogenetic roles (20). Finally, only CTS2 andCTS3 showed significant increases in expression during thetransition period between spherule septation and initiation ofendosporulation, when elevated chitinase activity within thespherules was expected to occur. On the basis of these obser-vations, we decided to determine whether expression of eitherCTS2 or CTS3 was necessary for successful completion of theasexual reproductive process in C. posadasii.

Disruption of the CTS2 gene progressed without difficultyusing a method which is now well established in our laboratory(22, 40). On the other hand, disruption of the CTS3 gene wasproblematic because of an error in construction of the plasmidemployed in the knockout procedure. As a result of incomplete

FIG. 7. ELISAs of cytokines in BALFs of BALB/c mice which wereeither vaccinated with the live attenuated strain or not vaccinated.BALFs were obtained from vaccinated mice at 8, 25, and 75 dayspostchallenge. BALFs from nonvaccinated mice were obtained at 8days postchallenge only. Normal, untreated mice (N) of the same ageand gender were included for determination of baseline amounts ofeach selected cytokine in BALFs. Each BALF sample was analyzed intriplicate. The results are presented as mean values plus standarddeviations.

3204 XUE ET AL. INFECT. IMMUN.

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

annotation of the C. posadasii (C735) genome, the plasmid wasmistakenly designed to disrupt CTS3 as well as a contiguous,upstream gene, which was subsequently designated ARD1. Re-sults of Southern hybridization confirmed that disruption ofthe CTS2 and ARD1/CTS3 genes had occurred by homologousrecombination and that the two selected transformants werehomokaryotic. The two mutant strains, the cts2� and ard1/cts3� strains, were still able to endosporulate in vitro, although

both showed a significant delay in endospore formation com-pared to the parental strain. Nevertheless, BALB/c mice chal-lenged intranasally with 50 viable spores of either mutantstrain revealed survival plots which were nearly identical tothat of mice challenged with the same number of spores of theparental isolate. We suggest that the transformants in whicheither the CTS2 or CTS3 gene was disrupted were reciprocallycompensated by expression of the respective intact chitinase

FIG. 8. Comparative histopathology of coccidioidal infection in the lungs of nonvaccinated BALB/c mice (A and B) sacrificed at 18 dayspostchallenge versus results for BALB/c mice which had been vaccinated with the live, attenuated strain of C. posadasii and sacrificed at 75 dayspostchallenge (C to E). Both groups of mice were challenged intranasally with 50 viable spores of the C. posadasii parental isolate (C735). (A) Lunglesions with an abundance of inflammatory cells (encompassed by dotted line), most of which are adjacent to spherules (S) that had ruptured andpartially released their contents. (B) At higher magnification, a spherule which had undergone endosporulation and ruptured (arrow), with aconcentration of host inflammatory cells adjacent to the parasitic cell surface. (C and D) Representative sections of lungs of vaccinated mice at75 days postchallenge which show formation of well-differentiated granulomas. Few organisms or neutrophils were observed. Spherules which weredetected appeared to be in preseptation or septation stages of the parasitic cycle (arrow in panel E). Bars in panels A to E represent 130 �m, 60�m, 2 mm, 0.5 mm, and 40 �m, respectively.

VOL. 77, 2009 LIVE GENETICALLY DEFINED COCCIDIOIDOMYCOSIS VACCINE 3205

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

gene, and a loss of ARD1 expression did not influence endo-sporulation of the ard1/cts3� mutant. To test whether a loss ofboth CTS2 and CTS3 expression was necessary to inhibit en-dosporulation, we used the cts2� mutant as the parental strainand repeated the ARD1/CTS3 disruption. The phenotype ofthe parasitic phase of this cts2/ard1/cts3� mutant was sterilespherules when the mutant was grown either in vitro or in vivo.Confirming that disruption of the two chitinase genes is re-sponsible for the observed phenotype necessitates generationof a revertant strain by complementation of the cts2/ard1/cts3�mutant with the wild-type CTS2 and CTS3 genes. Of relevanceto this study, however, is the observation that the cts2/ard1/cts3� strain was avirulent in BALB/c mice challenged intrana-sally with either 50 or 1,000 viable spores. Particularly intrigu-ing was that BALB/c mice totally cleared the cts2/ard1/cts3�mutant from their lungs and spleen after a potentially lethalchallenge with 1,000 viable spores of this attenuated strain. Onthe basis of these results, we considered using the geneticallyengineered avirulent strain of C. posadasii as a live vaccineagainst coccidioidal infection.

Vaccination of BALB/c and C57BL/6 mice was conducted bysubcutaneous immunization with viable spores of the attenu-ated strain suspended in saline. Three different vaccinationdoses were tested, and although results of survival and patho-gen clearance were not significantly different, a trend was sug-gested in which an initial immunization with 5 � 104 sporesfollowed 2 weeks later with a boost of 2.5 � 104 spores yieldedthe best results. Both vaccinated strains of mice appearedhealthy at 75 days postchallenge. This degree of protection ofthe two inbred strains of mice using the live vaccine was supe-rior to that with all recombinant protein vaccines against coc-cidioidomycosis reported to date (5, 7, 45). Most striking wasthe ability of the live vaccine to protect BALB/c mice, thestrain which is most susceptible to disseminated coccidioido-mycosis following an intranasal challenge (27).

Pappagianis (34) has pointed out that in most human casesof coccidioidomycosis, recovery from infection is followed byimmunity to exogenous reinfection. Recognition of this in-duced, protective immunity led to studies of the abilities of live,attenuated, and killed vaccines to protect mice, cynomolgousmonkeys, and humans against coccidioidal infection (30, 34).Prominent in this list of early experimental vaccines was aformulation consisting of formaldehyde-killed spherules(FKS), which was shown to protect BALB/c mice and wastested in a human double blind “phase 3” study conductedbetween 1980 and 1985 (34). Protective studies of mice hadshown that it was necessary for the FKS vaccine to consist ofmature spherules for optimal results. Intramuscular injectionof a total of 3.2 mg of the killed parasitic cells was claimed tobe tolerated using the murine model. On the other hand, weobserved an unacceptable level of reactogenicity in BALB/cmice following subcutaneous injections of a total of only 2.7 mgof the FKS vaccine, which required the sacrifice of these ani-mals prior to completion of the protection experiment. Inhumans, unfavorable local reactions necessitated adoption of1.75 mg/dose for the clinical trial (34), which may have beeninsufficient to mount a protective immune response to infec-tion. There was no statistically significant difference in theincidence of coccidioidomycosis between the vaccinated andplacebo groups of volunteers. An ideal vaccine should elicit

protection against infection with minimal reactogenicity andonly one or two doses of the immunogen (13). Our live, atten-uated vaccine showed transient reactogenicity at sites of im-munization of BALB/c mice over a period of 2 to 6 weeks aftercompletion of the vaccination protocol. The vaccine strainappeared to remain localized to sites of subcutaneous immu-nization. We suggest that immunization with the live sporesfollowed by their differentiation into full-size spherules re-sulted in associated dendritic cell maturation and activation asreported by others (12). We also propose that as a result of thepersistence of the live vaccine strain, progressive, acquiredimmunity against Coccidioides was effectively induced, and theimmune response was much better tolerated by the mamma-lian host than vaccination with FKS. We have not testedwhether a single dose of 7.5 � 104 spores of the attenuatedstrain affords the same level of protection to BALB/c mice asthe prime and boost vaccination protocol described in thisstudy. However, given how long the vaccine strain persists, it ispossible that a single immunization with the live, attenuatedstrain would suffice.

Analysis of cytokine production by vaccinated BALB/c miceat different times postchallenge suggested that stimulation ofboth Th1 and Th2 pathways of T-cell immunity had occurred.The Th1/Th2 dichotomy is based on evidence that Th1 cellssecrete cytokines that initiate and participate in cell-mediatedimmune responses while the Th2 subset of T lymphocytessecretes cytokines that stimulate B cells to produce antibodies,activate mast cells and eosinophils, and can downregulate cel-lular immune responses (5). Clinical studies of patients withcoccidioidomycosis have indicated that high pathogen-specificantibody titers are of little benefit since they typically correlatewith a poor prognosis (5–7). On the other hand, there is agrowing consensus that antibodies collaborate with phagocyticcells and T cells to reduce inflammation and polarize the Th1response against systemic mycoses (8, 18). Disseminated coc-cidioidomycosis in our BALB/c model typically correlated withearly and persistently high levels of production of the proin-flammatory cytokine IL-6. Mice vaccinated with the live, at-tenuated strain, on the other hand, showed a more than three-fold reduction in the IL-6 concentration in BALFs at 1 to 10weeks postchallenge. The histopathology of the nonvaccinatedversus vaccinated mice underscored this difference in host re-sponse. Vaccination with the attenuated strain significantlydampened host tissue damage, which in the nonvaccinatedBALB/c mice is associated with an intense, persistent inflam-matory response that most likely exacerbates the course ofdisease. Paraffin sections of infected lungs of control micetypically revealed an apparent migration of a large number ofinflammatory cells to mature spherules which had rupturedand released their contents. Thin sections of infected murinelung tissue have shown this same host-pathogen association(22). Vaccinated mice at 75 days postchallenge showed noevidence of such an intense inflammatory response but insteadhad produced well-differentiated granulomas which restrictedthe metastasis of the pathogen.

Wuthrich and coworkers (52) reported a genetically engi-neered, live, attenuated strain of Blastomyces dermatitidis thatconferred sterilizing immunity against lethal pulmonary infec-tion. Practical concerns raised in their study were whether themammalian host deficient in CD4� T cells, as can be the case

3206 XUE ET AL. INFECT. IMMUN.

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

for a patient with AIDS, would respond poorly to this vaccineor be at risk for adverse effects. Results of their investigationsshowed that protective immunity required the presence of �T lymphocytes but vaccine-induced immunity could beachieved in the absence of CD4� T cells, suggesting a role forCD8� T cells in a vaccinated, immunocompromised host. BothCD4� and CD8� T cells have also been shown to mediatelive-vaccine-induced immunity against Coccidioides infectionin mice (14). Additional studies are required to evaluatewhether our genetically engineered, live, attenuated strain ofC. posadasii can provide protection against coccidioidal infec-tion in a mammalian host which is depleted of CD4� T cells.

ACKNOWLEDGMENTS

Support for this study was provided by Public Health Service grantAI071118 from the National Institute of Allergy and Infectious Dis-eases (NIAID), National Institutes of Health. Additional support forthis project was provided by the California HealthCare Foundationand the Margaret Batts Tobin Foundation, San Antonio, TX.

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

REFERENCES

1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:3389–3402.

2. Centers for Disease Control and Prevention. 2009. Increase in coccidioido-mycosis—California 2000–2007. MMWR Morb. Mortal. Wkly. Rep. 58:105–132.

3. Cole, G. T., and C.-Y. Hung. 2001. The parasitic cell wall of Coccidioidesimmitis. Med. Mycol. 39(Suppl. 1):31–40.

4. Cole, G. T., D. Kruse, K. R. Seshan, S. Pan, S. J. Szaniszlo, J. Richardson,and B. Bian. 1993. Factors regulating morphogenesis in Coccidioides immitis,p. 191–212. In H. V. Bossche, F. C. Odds, and D. Kerridge (ed.), Dimorphicfungi in biology and medicine. Plenum Press, New York, NY.

5. Cole, G. T., J. Xue, C. N. Okeke, E. J. Tarcha, V. Basrur, R. A. Schaller, R. A.Herr, J.-J. Yu, and C.-Y. Hung. 2004. A vaccine against coccidioidomycosisis justified and attainable. Med. Mycol. 42:189–216.

6. Cox, R. A., and D. M. Magee. 1998. Protective immunity in coccidioidomy-cosis. Res. Immunol. 149:417–428.

7. Cox, R. A., and D. M. Magee. 2004. Coccidioidomycosis: host response andvaccine development. Clin. Microbiol. Rev. 17:804–839.

8. Cutler, J. E., G. S. Deepe, and B. S. Klein. 2007. Advances in combatingfungal diseases: vaccines on the threshold. Nat. Rev. 5:13–28.

9. Delgado, N., J. Xue, J.-J. Yu, C.-Y. Hung, and G. T. Cole. 2003. A recom-binant �-1,3-glucanosyltransferase homolog of Coccidioides posadasii pro-tects mice against coccidioidomycosis. Infect. Immun. 71:3010–3019.

10. d’Enfert, C., G. Weidner, P. C. Mol, and A. A. Brakhage. 1999. Transforma-tion systems of Aspergillus fumigatus: new tools to investigate fungal viru-lence. Contrib. Microbiol. 2:149–166.

11. Dereeper, A., V. Guignon, G. Blanc, S. Audic, S. Buffet, F. Chevenet, J. F.Dufayard, S. Guindon, V. Lefort, M. Lescot, J. M. Claverie, and O. Gascuel.2008. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nu-cleic Acids Res. 36:W465–W469.

12. Dionne, S. O., A. B. Podany, Y. W. Ruiz, N. M. Ampel, J. N. Galgiani, andD. F. Lake. 2006. Spherules derived from Coccidioides posadasii promotehuman dendritic cell maturation and activation. Infect. Immun. 74:2415–2422.

13. Ellis, R. W. 1999. New technologies for making vaccines, p. 881–901. In S. A.Plotkin and W. A. Orenstein (ed.), Vaccines, 3rd ed. W.B. Saunders, Phil-adelphia, PA.

14. Fierer, J., C. Waters, and L. Walls. 2006. Both CD4� and CD8� T cells canmediate vaccine-induced protection against Coccidioides immitis infection inmice. J. Infect. Dis. 193:1323–1331.

15. Fisher, M. C., G. L. Koenig, T. J. White, G. San-Blas, R. Negroni, I. G.Alvarez, B. Wanke, and J. W. Taylor. 2001. Biogeographic range expansioninto South America by Coccidioides immitis mirrors New World patterns ofhuman migration. Proc. Natl. Acad. Sci. USA 98:4558–4562.

16. Fisher, M. C., G. L. Koenig, T. J. White, and J. W. Taylor. 2002. Molecularand phenotypic description of Coccidioides posadasii sp. nov., previouslyrecognized as the non-California population of Coccidioides immitis. Myco-logia 94:73–84.

17. Flaherman, V. J., R. Hector, and G. W. Rutherford. 2007. Estimating severecoccidioidomycosis in California. Emerg. Infect. Dis. 13:1087–1090.

18. Guimaraes, A. J., S. Frases, F. J. Gomez, R. M. Zancope-Oliveira, and J. D.Nosanchuk. 2009. Monoclonal antibodies to heat shock protein 60 alter thepathogenesis of Histoplasma capsulatum. Infect. Immun. 77:1357–1367.

19. He, X., W. Huai, W. Guo, and B. Zhang. 2001. Cloning and expression ofD-arabitol dehydrogenase gene from Acetobacter suboxydans in Escherichiacoli. Wei Sheng Wu Hsueh Pao Acta Microbiol. Sin. 41:248–251.

20. Hearn, V. M., G. M. Escott, E. G. V. Evans, and D. J. Adams. 1998. Complexchitinolytic system of Aspergillus fumigatus. Microbios 93:85–104.

21. Henrissat, B. 1991. A classification of glycosyl hydrolases based on aminoacid sequence similarities. Biochem. J. 280:309–316.

22. Hung, C.-Y., K. R. Seshan, J.-J. Yu, R. Schaller, J. Xue, V. Basrur, M. J.Gardner, and G. T. Cole. 2005. A metalloproteinase of Coccidioides posa-dasii contributes to evasion of host detection. Infect. Immun. 73:6689–6703.

23. Hung, C.-Y., J.-J. Yu, P. F. Lehmann, and G. T. Cole. 2001. Cloning andexpression of the gene which encodes a tube precipitin antigen and wall-associated �-glucosidase of Coccidioides immitis. Infect. Immun. 69:2211–2222.

24. Jaques, A. K., T. Fukamizo, D. Hall, R. C. Barton, G. M. Escott, T. Parkin-son, C. A. Hitchcock, and D. J. Adams. 2003. Disruption of the gene encod-ing the ChiB1 chitinase of Aspergillus fumigatus and characterization of arecombinant gene product. Microbiology 149:2931–2939.

25. Karlsson, M., and J. Stenlid. 2008. Comparative evolutionary histories of thefungal chitinase gene family reveal non-random size expansions and contrac-tions due to adaptive natural selection. Evol. Bioinform. 4:47–60.

26. Kim, M. M., J. E. Blair, E. J. Carey, Q. Wu, and J. D. Smilack. 2009.Coccidioidal pneumonia, Phoenix, Arizona, USA, 2000–2004. Emerg. Infect.Dis. 15:397–401.

27. Kirkland, T. N., and J. Fierer. 1983. Inbred mouse strains differ in resistanceto lethal Coccidioides immitis infection. Infect. Immun. 40:912–916.

28. Kuranda, M. J., and P. W. Robbins. 1991. Chitinase is required for cellseparation during growth of Saccharomyces cerevisiae. J. Biol. Chem. 266:19758–19767.

29. Levine, H. B. 1961. Purification of the spherule-endospore phase of Coccid-ioides immitis. Sabouraudia 1:112–115.

30. Levine, H. B., Y.-C. M. Kong, and C. E. Smith. 1965. Immunization of miceof Coccidioides immitis: dose, regimen and spherulation stage of killed spher-ule vaccines. J. Immunol. 94:132–142.

31. Li, K., J.-J. Yu, C.-Y. Hung, P. F. Lehmann, and G. T. Cole. 2001. Recom-binant urease and urease DNA of Coccidioides immitis elicit an immuno-protective response against coccidioidomycosis in mice. Infect. Immun. 69:2878–2887.

32. Mirbod-Donovan, F., R. Schaller, C.-Y. Hung, J. Xue, U. Reichard, and G. T.Cole. 2006. Urease produced by Coccidioides posadasii contributes to thevirulence of this respiratory pathogen. Infect. Immun. 74:504–515.

33. Nakai, K., and P. Horton. 2007. Computational prediction of subcellularlocalization. Methods Mol. Biol. 390:429–466.

34. Pappagianis, D. 1993. Evaluation of the protective efficacy of the killedCoccidioides immitis spherule vaccine in humans. Am. Rev. Respir. Dis.148:656–660.

35. Pappagianis, D. 2001. Seeking a vaccine against Coccidioides immitis andserologic studies: expectations and realities. Fungal Genet. Biol. 32:1–9.

36. Parish, J. M., and J. E. Blair. 2008. Coccidioidomycosis. Mayo Clin. Proc.83:343–348.

37. Pishko, E. J., T. N. Kirkland, and G. T. Cole. 1995. Isolation and character-ization of two chitinase-encoding genes (cts1, cts2) from the fungus Coccid-ioides immitis. Gene 167:173–177.

38. Punt, P. J., R. P. Oliver, M. A. Dingemanse, P. H. Pouwels, and C. A. van denHondel. 1987. Transformation of Aspergillus based on the hygromycin Bresistance marker from Escherichia coli. Gene 56:117–124.

39. Punt, P. J., and C. A. van den Hondel. 1992. Transformation of filamentousfungi based on hygromycin B and phleomycin resistance markers. MethodsEnzymol. 216:447–457.

40. Reichard, U., C.-Y. Hung, P. W. Thomas, and G. T. Cole. 2000. Disruptionof the gene which encodes a serodiagnostic antigen and chitinase of thehuman fungal pathogen Coccidioides immitis. Infect. Immun. 68:5830–5838.

41. Rohe, M., J. Searle, A. C. Newton, and W. Knogge. 1996. Transformation ofthe plant pathogenic fungus, Rhynchosporium secalis. Curr. Genet. 29:587–590.

42. Saville, S. P., A. L. Lazzell, A. K. Chaturvedi, C. Monteagudo, and J. L.Lopez-Ribot. 2009. Efficacy of a genetically engineered Candida albicanstet-NRG1 strain as an experimental live attenuated vaccine against hematog-enously disseminated candidiasis. Clin. Vaccine Immunol. 16:430–432.

43. Sharpton, T. J., J. E. Stajich, S. D. Rounsley, M. J. Gardner, J. R. Wortman,V. S. Jordar, R. Maiti, C. D. Kodira, D. E. Neafsey, Q. Zeng, C.-Y. Hung, C.McMahan, A. Muszewska, M. Grynberg, M. A. Mandel, E. M. Kellner, B. M.Barker, J. N. Galgiani, M. J. Orbach, T. N. Kirkland, G. T. Cole, M. R.Henn, B. W. Birren, and J. W. Taylor. Comparative genomic analyses of thehuman fungal pathogens Coccidioides and their relatives. Genome Res., inpress.

44. Shoresh, M., and G. E. Harman. 2008. Genome-wide identification, expres-sion and chromosomal location of the genes encoding chitinolytic enzymes inZea mays. Mol. Genet. Genomics 280:173–185.

VOL. 77, 2009 LIVE GENETICALLY DEFINED COCCIDIOIDOMYCOSIS VACCINE 3207

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

45. Shubitz, L. F., J.-J. Yu, C.-Y. Hung, T. N. Kirkland, T. Peng, R. Perrill, J.Simons, J. Xue, R. A. Herr, G. T. Cole, and J. N. Galgiani. 2006. Improvedprotection of mice against lethal respiratory infection with Coccidioides posa-dasii using two recombinant antigens expressed as a single protein. Vaccine24:5904–5911.

46. Spinello, I. M., A. Munoz, and R. H. Johnson. 2008. Pulmonary coccidioido-mycosis. Semin. Respir. Crit. Care Med. 29:166–173.

47. Sunenshine, R. H., S. Anderson, L. Erhart, A. Vossbrink, P. C. Kelly, D.Engelthaler, and K. Komatsu. 2007. Public health surveillance for coccid-ioidomycosis in Arizona. Ann. N. Y. Acad. Sci. 1111:96–102.

48. Tarcha, E. J., V. Basrur, C.-Y. Hung, M. J. Gardner, and G. T. Cole. 2006.Multivalent recombinant protein vaccine against coccidioidomycosis. Infect.Immun. 74:5802–5813.

49. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G.Higgins. 1997. The CLUSTAL X windows interface: flexible strategies for

multiple sequence alignment aided by quality analysis tools. Nucleic AcidsRes. 24:4876–4882.

50. Valdivia, L., D. Nix, M. Wright, E. Lindberg, T. Fagan, D. Lieberman, T.Stoffer, N. M. Ampel, and J. N. Galgiani. 2006. Coccidioidomycosis as acommon cause of community-acquired pneumonia. Emerg. Infect. Dis. 12:958–962.

51. van Scheltinga, A. C. T., M. Hennig, and B. W. Dijkstra. 1996. The 1.8 Åresolution structure of hevamine, a plant chitinase/lysozyme, and analysis ofthe conserved sequence and structure motifs of glycosyl hydrolase family 18.J. Mol. Biol. 262:243–257.

52. Wuthrich, M., H. I. Filutowicz, T. Warner, and B. S. Klein. 2002. Requisiteelements in vaccine immunity to Blastomyces dermatitidis: plasticity uncoversvaccine potential in immune-deficient hosts. J. Immunol. 169:6969–6976.

53. Xue, J., C.-Y. Hung, J.-J. Yu, and G. T. Cole. 2005. Immune response ofvaccinated and non-vaccinated mice to Coccidioides posadasii infection. Vac-cine 23:3535–3544.

Editor: A. Casadevall

3208 XUE ET AL. INFECT. IMMUN.

on October 6, 2020 by guest

http://iai.asm.org/

Dow

nloaded from