Coccidioidomycosis: Host Response and Vaccine DevelopmentRebecca A. Cox* and D. Mitchell Magee†
Department of Microbiology and Immunology, University of Texas HealthScience Center, San Antonio, Texas
INTRODUCTION .......................................................................................................................................................805HISTORICAL PERSPECTIVES...............................................................................................................................805BIOLOGY OF COCCIDIOIDES................................................................................................................................806
Ecology......................................................................................................................................................................806Phylogenetic Species ...............................................................................................................................................806Classification of Coccidioides as a Select Agent..................................................................................................808Critical Comments..................................................................................................................................................808
CLINICAL MANIFESTATIONS ..............................................................................................................................808Epidemiology ...........................................................................................................................................................808Categories of Disease .............................................................................................................................................809
Risk Factors for Severe, Disseminated Coccidioidomycosis .............................................................................810Genetically determined susceptibility ..............................................................................................................810Gender ..................................................................................................................................................................811Age ........................................................................................................................................................................811Pregnancy.............................................................................................................................................................811Immunocompromising diseases or conditions................................................................................................812
Critical Comments..................................................................................................................................................812HOST DEFENSES IN HUMANS.............................................................................................................................812
Critical Comments..................................................................................................................................................815MURINE MODEL OF COCCIDIOIDOMYCOSIS...............................................................................................816
Host Defenses in Experimentally Infected Mice ................................................................................................817Role of T lymphocytes in protective immunity ...............................................................................................817Role of cytokines in host defense .....................................................................................................................817Role of antibody in host defense ......................................................................................................................819
PBS extract of spherule cell walls ....................................................................................................................821
* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, The University of Texas Health Science Cen-ter at San Antonio, Texas Research Park, 15355 Lambda Dr., SanAntonio, TX 78245-3027. Phone: (210) 562-5037. Fax: (210) 562-5041.E-mail: [email protected].
† Present address: The Center for Biomedical Inventions, The Uni-versity of Texas Southwestern Medical Center, Dallas, TX 75390-9185.
Route of Immunization ..........................................................................................................................................832CURRENT AND FUTURE DIRECTIONS .............................................................................................................832REFERENCES ............................................................................................................................................................833
INTRODUCTION
Coccidioidomycosis (San Joaquin Valley fever) is a mycoticdisease caused by Coccidioides immitis (68, 98, 125, 214) andthe newly proposed phylogenetic species C. posadasii (116).The fungus propagates in soil in the semiarid regions of thesouthwestern United States, Mexico, and Central and SouthAmerica, in a region corresponding to the Lower Sonoran LifeZone. The saprobic phase is characterized by mycelia that giverise to infectious arthroconidia, which become aerosolizedwhen the soil is disturbed. Humans acquire the infection byinhalation of the arthroconidia, which differentiate into large,endosporulating spherules once they are in the host.
Coccidioides is a formidable pathogen, capable of causingprogressive pulmonary and/or disseminated disease in previ-ously healthy individuals. The disease presents a diverse clin-ical spectrum that includes inapparent infection, primary re-spiratory disease (usually with uncomplicated resolution),stabilized or progressive chronic pulmonary disease, and ex-trapulmonary dissemination which can be acute, chronic, orprogressive. The degree of severity varies considerably withineach category and depends, in part, on the dose of inhaledarthroconidia, the genetic predisposition of the host, and theirimmunologic status.
Between 25,000 and 100,000 new cases occur each year inthe areas of endemic infection in the United States, butmarked increases have occurred during sporadic epidemics(125, 248, 268). Among those who acquire primary infection,persons of African, Asian, and, to a lesser extent, Hispanicdescent are more likely to develop disseminated disease thanare Caucasians. This genetic predisposition, the geographicallylocalized areas of endemicity, and the resistance of personswho experienced a benign, self-limiting primary infection doc-ument the feasibility of developing a vaccine against Coccid-ioides (16).
Coccidioidomycosis is considered to be a reemerging diseaseowing to the dramatic increase in the number of cases duringthe past decade. Major outbreaks occurred in southern Cali-fornia in 1977 and late 1991 through 1994 (105, 221). A newresurgence is indicated by the increase in coccidioidomycosiscases during the past year in Arizona (50). These outbreaks
may be linked to climatic conditions, and the 1994 cases oc-curred after heavy rains, when the fungus propagated in thesoil, followed by hot, dry, and windy periods that resulted in theaerosolization of mycelium-derived arthroconidia. The result-ing high morbidity and mortality associated with these out-breaks prompted community and health-related organizationsto seek funding for intensifying efforts to develop a vaccine forcoccidioidomycosis. Financial support from the CaliforniaHealthCare Foundation, the State of California Department ofHealth Services, and the Valley Fever Research Foundationled to a coordinated research program involving investigatorslocated in California (Demosthenes Pappagianis and TheoKirkland), Arizona (John Galgiani), Texas (Rebecca Cox), andOhio (Garry Cole). New and fundamentally important discov-eries have emerged from these research studies, and it is rea-sonable to predict that a vaccine, composed of multiple immu-nogens, will be entered into phase I and II clinical trials withinthe near future.
It is the intent of this review to focus on progress in vaccinedevelopment and host responses that are crucial for protectiveimmunity.
HISTORICAL PERSPECTIVES
Coccidioidomycosis has been a recognized infection since1892 (235, 303). The first reported case was in an Argentiniansoldier who had exhibited skin lesions for 4 years; the causativeorganism was thought to be a protozoan. The first two cases inthe United States heralded the protean manifestations of thisinfection, with marked differences in clinical presentation be-tween the two patients (245). The first patient presented witha slowly progressing disease, which lead to his death approxi-mately 9 years after the first appearance of symptoms. In con-trast, the second patient presented with a rapidly progressingdisease leading to death within approximately 4 months of theonset of symptoms. The species name of Coccidioides wasproposed for the infectious agent, which was identified as thesame protozoan described by Posadas and Wernicke. Becauseof the differences in clinical presentation and lesion develop-ment, it was proposed that each of these initial U.S. cases wasinitiated by different species, and it was proposed that the
species name of immitis (meaning “not mild”) be used for thecausative organism of the first case and pyogenes be used forthe causative organism for the second case. The fungal natureof the organism was delineated four years later by Ophuls andMoffitt (209), who discovered that cultures of tissues alwaysyielded a mold and that when the mold was injected into theear vein of a rabbit, the rabbit developed “typical tubercle-like”nodules in the lungs, spleen, and kidneys. Microscopic exami-nation of the nodules revealed protozoan bodies and no my-celium. They further showed the organism was dimorphic, un-dergoing a change from a mold to the “protozoan” phase intissue and, conversely, sprouting hyphae when the tissue wasexamined in a coverslip preparation.
Until 1929, disseminated severe coccidioidomycosis, termedcoccidioidal granuloma, was the only recognized form of thedisease. However, a laboratory accident provided the first in-sight into milder forms of infection. Harold Chope, a 26-year-old Stanford University medical student who was doing re-search on Coccidioides in the laboratory of Ernest Dickson,accidentally opened a petri dish containing the mold form ofCoccidioides. Chope became ill within 9 days of the incident,with an acute pneumonia, with pleuritic pains, fever, cough,hemoptysis, and a 15-lb weight loss over an 8-day period (114).Four weeks later, erythematous nodules erupted on his shinsand endosporulating spherules were observed in the sputum. Adiagnosis of coccidioidal granuloma was made, and the prog-nosis was considered to be grave, but Chope soon recoveredcompletely. In 1937, Dickson presented Chope’s case and fouradditional cases of this newly recognized benign form of thisdisease to the annual meeting of the California Medical Asso-ciation. All five cases were characterized by acute pulmonarysymptoms and, with one exception, erythema nodosum. Dick-son’s paper, published in California and Western Medicine (93),was entitled “Valley Fever” of the San Joaquin Valley andfungus Coccidioides, and in it he stated that the cases ‘proveconclusively that fungus Coccidioides is sometimes the cause ofa symptom complex of acute illness identical with what hasbeen known locally in the San Joaquin Valley as “Valley Fe-ver.” ’ Dickson proposed the term “coccidioidomycosis” to in-clude all forms of infection by Coccidioides.
BIOLOGY OF COCCIDIOIDES
Ecology
Coccidioides is a haploid ascomycete classified in the familyOnygenaceae (order Onygenales), along with the human re-spiratory pathogens Histoplasma, Blastomyces, and Paracoccid-ioides (84, 211, 269). The fungus is dimorphic, having a sapro-bic phase characterized by mycelia that produce enterothallicarthroconidia and a parasitic phase characterized by endos-porulating spherules. The cytologic and ultrastructural detailsof the morphogenetic conversion have been described by sev-eral investigators (58,148, 163, 286). The arthroconidia, eachwith at least two nuclei, are derived by disarticulation of theseptate hyphae. This process involves sequential and coordi-nated events: arrest of apical growth, progressive septation ofthe hyphae, condensation of the cytoplasm in certain hyphalcompartments, autolysis of adjacent cells, and synthesis of newinner wall layer. Depending on the strain, arthroconidia are
typically barrel-shaped, measuring 2.5 to 4 �m in width and 3to 6 �m in length, and thus are small enough to reach thealveoli of the lungs when inhaled.
Early conversion of the arthroconidia into spherule-phasecells begins with isotropic growth characterized by a roundingup and swelling of the cells followed by synchronous nucleardivisions and segmentation, which is initiated by synchronized,centripetal growth of the spherule wall at multiple points (163).The central portion of the young spherule is occupied by avacuole. Progressive compartmentalization of the cytoplasmthat surrounds the vacuole gives rise to uninucleate compart-ments which round up and differentiate into endospores. Themature spherule measures 30 to 60 �m and can contain 200 to300 endospores. At maturity, the spherule ruptures, releasingthe endospores, which typically measure 2 to 4 �m in diameter.Each of these first-generation spherules is capable of develop-ing into a mature, endosporulating spherule, thereby repeatingthe parasitic phase cycle. Thus, at any given point in time, theinfected host is exposed to immature, mature, and rupturingspherules and newly released endospores, which differ quanti-tatively, if not qualitatively, in their cell wall and cytoplasmiccomposition.
An understanding of the regulatory events that underlie theinduction of morphogenetic conversion in Coccidioides is ru-dimentary at best. The development of a defined liquid me-dium by Converse (59–61) enabled studies to delineate theabsolute requirements for spherule induction and maintenancein vitro (29, 59, 60, 63). Increased temperature (between 34and 41°C) and CO2 concentration (10 to 20%) induce spheru-lation. The addition of a surface-active agent such as Tamol Nalso stimulates spherulation. At 41°C, 100% of arthroconidiaconvert into spherules, whereas at lower temperatures underthe same conditions, the arthroconidia give rise to hyphae. Thedifferentiation of arthroconidia into endosporulating spherulesin vitro appears to be identical to that observed in vivo (100)and, in both environments, typically takes between 72 and 96 h.
Phylogenetic Species
Until recently, C. immitis was the sole etiologic agent ofcoccidioidomycosis. Phylogenetic analyses using single-nucle-otide polymorphisms, genes, and microsatellites have showedthe existence of two genetically different C. immitis clades,California and non-California (116). The cumulative resultshave led to the proposal by Fisher et al. for a new speciesdesignation of C. immitis for the California clade and C. posa-dasii, in honor of Alejandro Posadas, who described the firstcase of coccidioidomycosis, for the non-California clade. Rec-ognition of two rather than a single species of Coccidioidescould have an impact on future studies regarding vaccine prep-aration and testing, as well as clinical, epidemiologic, and ge-netic studies of coccidioidomycosis. For this reason, this sec-tion will present details of the studies that led to the proposedspecies.
Molecular-genetic analyses of C. immitis strains began withthe use of restriction fragment length polymorphism to analyzeand compare the genomes of 14 isolates from California and 1isolate from Venezuela. In this study, Zimmermann et al. (326)demonstrated that C. immitis contains at least two subgroups.Of the 14 isolates, 2 isolates, including 1 of the standard lab-
oratory strains, Silveira, were placed in group I and the restwere placed in group II. There were no discernible differencesin the group I and II isolates in terms of the geographic regionwhere they were isolated or whether they were isolated from apatient with pulmonary or disseminated disease. The investi-gators noted, however, that group II isolates could be subdi-vided into additional subgroups. Subsequently, Burt et al. (32–34) identified polymorphic loci and examined 12 of thesepolymorphic loci in clinical isolates collected from 25 patientsin Bakersfield, Calif., 25 patients in Tucson, Ariz., and 20patients in San Antonio, Tex. Substantial genetic differentia-tion was observed between the isolates from California andthose from Arizona or Texas, with little to no gene flow. Therewas also a significantly reduced gene flow between isolatesfrom Arizona and Texas, but not as much as was observedbetween the isolates from these two states and those fromCalifornia. Koufopanou et al. (176, 177), in a comparison ofgene genealogies from 350- to 650-bp fragments of five nucleargenes, separated 17 isolates of Coccidioides that had beencollected from California, Texas, Arizona, Mexico, and Argen-tina into two strains: one from California and the other fromall other geographic locations. Strain Silveira was a notableexception in that it did not share polymorphisms with theCalifornia species, which corroborated the findings of Zimmer-mann et al. (326). Exceptions were also observed, since isolatesfrom three patients from California showed the non- Califor-nia genotype.
Fisher et al. (117, 119) identified seven microsatellite-con-taining loci for C. immitis, four of which were originally iso-lated from the California strains and three of which wereisolated from the non-California strains. Analyses of 20 clinicalisolates from the southwestern United States revealed that sixof the seven microsatellites showed nonoverlapping allele dis-tributions between the California and non-California strains.In a subsequent study, these investigators (118) examined 161isolates from the areas of endemic infection in California,Arizona, Texas, Mexico, and South America. Using nine mic-rosatellite-containing loci, the investigators confirmed that theisolates comprised two primary subgroups, i.e., the Californiaand non-California phylogenetic strains. Subclades were ob-served with divergence between isolates from Central Valley,Calif., and those from the rest of southern California, which isdelineated by the Tehachapi mountain range. Likewise, therewas phylogeographic divergence within the non-Californiaclade, with the Texas and South American isolates groupinginto a subclade.
Perhaps the strongest argument for the two species is thelack of evidence of genetic exchange between C. immitis and C.posadasii. It appears, however, that the vast majority of studiesperformed to date have been done with the same isolates of C.immitis, and at the time of this writing, only 167 strains havebeen examined. There appears to be considerable overlap inthe number of non-California stains (C. posadasii) in the Cal-ifornia group (C. immitis) and, conversely, the number of Cal-ifornia strains (C. immitis) in the non-California group (C.posadasii) (116). These collective results prompt the questionwhether posadasii should be used to designate a variety of C.immitis as opposed to a new species of Coccidioides.
As an argument against separation of the species, Pappagi-anis (213, 220) has emphasized that immunization with one C.
immitis strain provides protection against respiratory challengewith a phenotypically or genotypically different C. immitisstrain. Although differences in the virulence of C. immitisstrains have been documented, those differences do not corre-late with differences in their immunogenicity. For example,immunization of mice with viable arthroconidia of strain 46protected them from intranasal (i.n.) challenge with strain Sil-veira. Strains 46 and Silveira differ when analyzed by restrictionfragment length polymorphism (326) and by single-strain con-formational polymorphism analysis. Strain 46 is classified as aCalifornia strain and strain Silveira is classified as a non-Cal-ifornia strain by genotypic markers (116). Huppert et al. (146)showed that vaccination of mice with killed spherules of strainSilveira protected against i.n. challenge with strain 46, strainWoodville, and five other strains of C. immitis that were con-sidered to be phenotypically atypical. Further support thatimmunizing strains of C. immitis protect against challenge withother strains is evinced by the solid immunity of persons whorecovered from a primary benign coccidioidal pneumonia toexogenous reinfection. That is, such persons are likely to havebeen exposed to other strains of C. immitis as residents inregions of endemic infection.
While the differences in genotype between C. immitis and C.posadasii are strong, differences in phenotype are not remark-able and also argue against separation of the species. AlthoughC. posadasii was reported to grow more slowly on yeast extract-glucose agar medium with high salt concentration, there wasconsiderable overlap between the C. posadasii and C. immitisisolates, and hence the phenotype could not be used to distin-guish the two (116). Minor differences in amino acid sequenceof proteins of C. posadasii and C. immitis were reported byKoufopanou et al. (177) and Peng et al. (229), but as yet nodifferences have been detected in antigenicity, virulence, ormorphology of the two groups. To begin to answer this latterquestion, we collected 12 isolates of Coccidioides (7 C. posa-dasii strains and 5 C. immitis strains) to compare them in ananimal model of virulence (D. M. Magee and R. A. Cox,unpublished data). Since these isolates had been collectedfrom various investigators over time, they were passed throughmice before use in the present experiment. Groups of 10BALB/c mice were infected intranasally with each strain andthen monitored for survival over 45 days (Table 1). The anal-ysis of this initial experiment shows that there are three pat-terns of survival after challenge, showing high, intermediate,and low virulence. Strain Silveira, our standard laboratory iso-late, which is included with the proposed C. posadasii designa-tion, exhibited intermediate virulence, and mice challengedwith this isolate began to succumb on day 13, with a median50% survival on day 14. In contrast, strain RMSCC 1040 (fromthe Roche Molecular Services Culture Collection [RMSCC]),also in the C. posadasii designation, exhibited increased viru-lence, and mice challenged with this strain began to succumbon day 9, with a median 50% survival on day 10.5. Comparisonof the survival curves revealed that survival was significantlydecreased compared to that of mice infected with strain Sil-veira (P � 0.002, Mantel-Haenszel log-rank survival analysis).On the other extreme, strain RMSCC 1038, also in the C.posadasii designation, exhibited low virulence, with 100% sur-vival for over 45 days after challenge. The animals were sacri-ficed at that time, and all animals contained C. immitis in their
lungs; therefore, the increased survival is not due to failure ofinfection. Thus, while we have Coccidioides strains with differ-ent levels of virulence in mice, variation in virulence betweenthe proposed species designations was not demonstrable.
Classification of Coccidioides as a Select Agent
Coccidioides was classified as a select agent of bioterrorismin the Final Rule on Additional Requirements for FacilitiesTransferring or Receiving Select Agents in response to theU.S. Antiterrorism and Effective Death Penalty Act of 1996(94). This has been further refined; in the Federal Register on 23August 2002, the Department of Human and Health Servicesrequested comments regarding whether changes should bemade to the list of select agents. A recently issued InterimFinal Rule, effective as of 7 February 2003, includes C. immitisand C. posadasii as select agents (51, 90). The Select AgentRule was enacted to “establish a system of safeguards to befollowed when specific agents are transported; collect and pro-vide information concerning the location where certain poten-tially hazardous agents are transferred; track the acquisitionand transfer of these specific agents; and establish a process foralerting appropriate authorities if an unauthorized attempt ismade to acquire these agents.”
Fierer and Kirkland have questioned the rationale and jus-tification for classifying the fungus as a select agent (110).These investigators argue that (i) it would be relatively easy toisolate Coccidioides from the soil (as would also be the case forBacillus anthracis); (ii) most primary infections are benign; (iii)the incubation period (usually 10 to 16 days) is too long todisable a military unit; (iv) no vaccine is available to protectthose who are using Coccidioides as a biothreat agent; and (v)Coccidioides is not contagious. Although it is difficult to per-ceive Coccidioides itself as a biothreat, unless a very high in-oculum of the fungus could be delivered, one could visualizetransfected Coccidioides arthroconidia as vectors for deliveringbiothreat agent toxins, in which case the availability of a vac-cine becomes essential.
Critical Comments
The separation of the dimorphic Coccidioides genera intomultiple species is somewhat controversial, and to date, therehas not been any debate in the literature. While the geneticevidence is compelling, the biological relevance of the geneticvariability is not convincing. Clearly, much work needs to bedone to determine if there are differences in antigenic varia-tion and virulence between C. immitis and the proposed C.posadasii species. Until the biological evidence is firmly estab-lished, we propose that a variety designation, be used with C.immitis var. immitis and C. immitis var. posadasii. For the restof this review, we limit the nomenclature of this organism tothe genus level.
The inclusion of Coccidioides as a potential bioweapon, com-bined with the failure of the National Institutes of Health toinclude it on the Category A/B/C list for prioritization, mayimpede the research on this organism. The result is that thisorganism falls under the strict federal regulations but does notwarrant targeted research funding. The cumulative effect couldbe an overall reduction in the effort to understand the biologyand pathogenesis of Coccidioides.
CLINICAL MANIFESTATIONS
Epidemiology
The distribution of Coccidioides in nature has been estab-lished on the basis of skin testing with coccidioidin or spheru-lin, recognition of clinical cases, and ecologic investigationsduring epidemics. These results reveal that coccidioidomycosisis endemic in the Western Hemisphere, with the areas ofhighest endemicity being in southwestern United States andthe bordering regions of northern Mexico and with regions oflower endemicity in Central and South America (216, 217).More recently, areas of endemicity have been documented inthe Brazilian states of Piauı, Bahia, Ceara, and Maranhao(297). In the United States, areas of endemicity include thesouth central portion of Arizona, particularly Tucson andPhoenix, the southern one-third of California, notably the SanJoaquin Valley, southwestern Texas, and New Mexico. Thefungus is also found in scattered foci in southern Nevada andUtah (179). The regions of endemicity for coccidioidomycosiscorrespond to the Lower Sonoran Life Zone, and the distri-bution of the fungus in the soil is notoriously spotty (194). Thiszone is characterized by plants such as the creosote bush,mesquites, palo verde, and yuccas; a semiarid climate charac-terized by hot summers and few winter freezes; and a soil thatis highly alkaline. Cumulative evidence has documented anassociation between climatic conditions and outbreaks of coc-cidioidomycosis (170, 171, 192, 216, 217, 278). The funguspropagates as mycelia in moist soils, and when the soil dries,the arthroconidia form and become airborne as a result of theaction of wind or some other disturbance of the soil. Thehighest incidence of the disease occurs in late summer andearly fall, when the soil is the driest.
Coccidioidomycosis is considered to be a reemerging diseasebecause of the dramatic increase in the number of cases duringthe early part of the past decade (47, 52, 105, 157, 207, 219,260, 282). Between 1991 and 1994, there was a notable increaseof new cases in California, in particular in Kern and Tularecounties in the southern part of the San Joaquin Valley (219).
TABLE 1. Virulence comparison of various Coccidioides strains
Strain Proposedspecies
Challengedose (no. of
arthroconidia)
Median timeto death of
50% of mice(days)
Silveiraa C. posadasii 27 14634b C. posadasii 29 14735b C. posadasii 25 15RMSCC 1037c C. posadasii 34 10.5RMSCC 1038c C. posadasii 35 �45RMSCC 1040c C. posadasii 29 11.5RMSCC 3700c C. posadasii 35 �45RSd C. immitis 30 15RMSCC 2008c C. immitis 24 14RMSCC 2010c C. immitis 31 14.5RMSCC 2012c C. immitis 26 15.5RMSCC 2013c C. immitis 19 16
a Current laboratory strain.b Kindly provided by Garry Cole.c Kindly provided by John Taylor through Gina Koenig and the RMSCC.d Kindly provided by Theo Kirkland.
During that time, there were 8,435 reported cases in KernCounty, representing a sevenfold increase. This epidemic wasattributable to an unusually rainy spring in March 1991 andFebruary-March 1992 following a 5-year drought, the migra-tion of persons previously unexposed to Coccidioides into areasof endemicity in southern California, and new constructionprojects. In a review of medical records in Kern County by theCenters for Disease Control as Prevention, medical bills to-taled $45 million for hospitalization and outpatient care (283).A similar increase in coccidioidomycosis incidence occurred inArizona between 1990 and 1995, from 7.0 per 100,000 popu-lation in 1990 to 14.9 per 100,000 in 1995 (10). The increasewas thought to be attributable to the influx of older persons (65years or older), who were nonimmune upon arrival. There wasno apparent correlation between the increased number ofcases and climatic conditions. After a period of quiescence, thenumber of cases is increasing in Arizona, which may herald theonset of a new epidemic (50).
More frequent travel, both domestic and international, toareas of endemicity has contributed significantly to this in-crease (37, 46, 48, 49, 53, 92, 212, 300). In 2000, nearly 28million travelers visited Arizona, and of these, nearly 1 millionwere from abroad. Even greater numbers of travelers visitCalifornia and other areas of endemicity. In 2001, more than300 persons from 30 countries participated in the WorldChampionship of Model Airplane Flying in Lost Hills, Calif.,an area where the fungus is highly endemic (46). Cases ofcoccidioidomycosis in participants who returned to Australia,Finland, New Zealand, and the United Kingdom were re-ported. Between 1992 and 1997, New York State had a total of161 cases of coccidioidomycosis, all of which occurred in pa-tients who had traveled to a region of endemicity, mostly in thesouthwestern United States (53). The Cleveland Clinic had 23cases between 1980 and 1998 among patients who had traveledto an area where C. immitis was endemic (92). Travelers fromthe United States to areas of endemicity in Central and SouthAmerica have also acquired coccidioidal infection. In 1996,members of a church congregation from Washington Statetraveled to Tecate, Mexico, to assist with construction projects(37); 21 (17%) of the members were diagnosed with coccid-ioidomycosis following their return. A similar incident oc-curred with church members from Pennsylvania, who acquiredprimary infection after traveling to Hermosillo, Mexico, toassist in a church construction project (48).
Categories of Disease
Primary pulmonary infection. Since the early epidemiolog-ical studies by Smith et al. (274, 277, 279), it has become almostaxiomatic that fully 60% of primary infections with Coccid-ioides are asymptomatic, being evident only by conversion ofskin test reactivity. Notable exceptions have occurred duringoutbreaks during archeological excavations, constructionprojects, and military exercises (83, 180, 260, 282, 302, 307),where symptomatic infections have been documented in 90%or more of persons. This increased incidence of symptomaticinfection following primary exposure to arthroconidia is prob-ably attributable to exposure to an unusually high dose ofarthroconidia (37). Of the remaining 40% of cases, the major-ity of patients exhibit only mild flu-like symptoms with anincubation period of 10 to 16 days (which can range from 7 to
20 days). The most common symptoms include cough, fever,chest pain, headache, fatigue, chills, malaise, and anorexia.Chest radiographs typically show pulmonary infiltrates, whichmay be single or multiple and are most often hilar or basal inlocation. Hilar adenopathy and pleural effusion may also bepresent. A diffuse erythematous rash termed toxic erythemaoccurs in 10 to 30% of individuals, usually within the first fewdays of clinical illness, and usually disappears shortly thereaf-ter. This rash appears to be nonspecific, being thought to beassociated with an acute febrile illness, and it usually covers thetrunk and extremities.
Cutaneous infection. Coccidioidomycosis can be acquiredvia a percutaneous route. Most of these occur in laboratoryworkers as a result of a hypodermic injection of Coccidioides(99, 310, 312). Primary cutaneous coccidioidomycosis is char-acterized by a painful suppurative lesion at the site of inocu-lation, often with regional lymphadenopathy. Of the 18 casesreported as of 1977, all but 2 have remained localized (41).
Valley fever. Approximately 5% of all primary infectionsdevelop what is termed as the Valley fever complex, usuallycoincident with the development of delayed-type hypersensi-tivity reactions (99,114, 275). Erythema nodosum and ery-thema multiforme comprise the principal manifestations of thevalley fever complex and may be accompanied by arthralgias(desert rheumatism) and a mild conjunctivitis. These are con-sidered to be specific cutaneous lesions of acute coccidioid-omycosis, in contrast to the nonspecific toxic erythema notedabove, and are temporally associated with the acquisition ofdelayed-type hypersensitivity to Coccioides. Of the two syn-dromes, erythema nodosum is the more extensively studied.Initially, the lesions appear as bright reddish nodules, butwithin days they become livid red or purplish, and on healingthey appear as bruises. They are commonly limited to the lowerextremities. Erythema nodosum occurs most often in Cauca-sian females and has long been regarded to denote a goodprognosis, although exceptions have been documented (99,122, 157). Curiously, the predisposition of females to develop-ing erythema nodosum is not manifest before puberty. Arthri-tis involving the joints, most commonly the ankle and knee,develops in approximately one-third of persons with erythemanodosum and/or erythema multiforme. These clinical manifes-tations of primary coccidioidomycosis are thought to be attrib-utable to a hypersensitivity to the fungus, a concept that issupported by the hyperreactivity of the patient to skin testingwith coccidioidin. Fungal cells are not present in the lesions oferythema nodosum or multiforme, nor have they been dem-onstrated in coccidioidal arthritis or conjunctivitis; as yet, theunderlying basis of the valley fever complex has not beendetermined. Erythema nodosum in other diseases, notably tu-berculosis, leprosy, sarcoidosis, and autoimmune disorders, isconsidered to be a hypersensitivity response, and in manycases, circulating immune complexes, C3, and immunoglobulinG (IgG) are demonstrable in the walls of venules (242). Al-though circulating immune complexes, composed of anti-Coc-cidioides IgG and coccidioidal antigen(s), have been demon-strable in the sera of patients with coccidioidomycosis (81,317), studies have not been done to assess immune complexesin persons with valley fever complex.
Pulmonary coccidioidomycosis. Approximately 5% of per-sons with primary coccidioidomycosis develop persistent pul-
monary coccidioidomycosis, manifested by chronic progressivepneumonia, miliary disease, pulmonary nodules, or pulmonarycavitation (99, 311, 313, 314). Pulmonary nodules are usuallybenign but can become cavitary. A classic radiologic finding isthe presence of a “thin-walled cavity,” which typically fails toshow a surrounding tissue reaction. Although the latter is notpathognomonic, it is strongly suggestive of coccidioidomycosis(114). Most patients have only a single cavity, whereas in oth-ers the cavities are multiple or multilocular (276). In a study of211 cases, Hyde (149) reported that half of cavities eventuallyclose spontaneously, requiring neither surgery nor chemother-apy. Possible complications of cavitation include hemorrhage,secondary infection, progressive increase in size, and, if locatedperipherally, bronchopleural fistulae. A few patients developchronic progressive pulmonary involvement, with symptoms ofcough, weight loss, fever, hemoptysis, dyspnea, and chest painthat may persist for years. Radiographic results include inflam-matory infiltrates, biapical fibronodular lesions, and multiplecavities.
Peripheral blood eosinophilia has been found in many pa-tients with primary coccidioidomycosis and patients with dis-seminated disease (106, 114, 135, 259, 306). In one study, 66 of75 patients with acute symptomatic pulmonary coccidioidomy-cosis showed eosinophil counts ranging from 3 to 26% of thetotal peripheral blood count (135). However, one patient wasobserved to have a peripheral blood eosinophilia as high as89% (306). Peak eosinophilia generally occurs between thesecond and third weeks of clinical illness. Schermoly andHinthorn (259) reported a patient with both 48% eosinophiliain his peripheral blood and 91% eosinophilia in his cerebro-spinal fluid. The investigators further noted that a bone mar-row biopsy specimen showed a marked proliferation of eosin-ophils. The patient was treated with amphotericin B, and after2 weeks of therapy her eosinophil counts were normal. Thebasis for the increased eosinophilia is not known, but it doesnot appear to be associated with erythema multiforme or ery-thema nodosum.
Disseminated coccidioidomycosis. The early epidemiologi-cal studies by Smith et al. (277) established that approximately1% of patients with primary coccidioidomycosis developed dis-seminated disease. This incidence has been higher in morerecent studies. In an epidemiologic investigation of the out-break that occurred in Ventura County following the earth-quake in Northridge, Calif., in 1994, Schneider et al. (260)reported that 3.7% of patients developed disseminated dis-ease. Pappagianis and Einstein (221) reported a 4.2% inci-dence of dissemination as a result of the 1977 dust storm inCalifornia, and Pappagianis reported a 5.7% rate in militarypersonnel and their families at Lemore U.S. Naval Air Stationbetween 1961 and 1977 (216). Similar findings were observedin the epidemic of coccidioidomycosis in the San Joaquin Val-ley between September 1991 and January 1994 (157).
Dissemination, when it occurs, is usually an early event andmay occur in the absence of any clinical or radiographic evi-dence of previous pulmonary infection (99, 114, 122), althoughexceptions have been noted (252). The process may be acute,subacute, or chronic. Extrapulmonary spread may consist of asingle lesion in the skin and subcutaneous tissues, bone, me-ninges, lymph nodes, spleen, liver, kidneys, pleura, or virtuallyany part of the body, with the general exception of the gastro-
intestinal tract (122). If only a single lesion develops, unless itis in the meninges, prognosis is generally favorable. If dissem-ination is multifocal, the overall mortality rate is greater than50%.
Lesions in the skin and subcutaneous tissues occur in morethan 65% of cases of disseminated disease and may present assmall papular nodules, ulcerated nodules, or verrucous granu-loma. When bone is involved, complicated sinuses may formcommunications between the bone and the adjacent soft tissue,leading to a draining sinus through the skin. Sinus tracts alsooriginate in subcutaneous tissues and the viscera. Meningitisoccurs in 30 to 50% of cases of disseminated disease, and insome patients this is the only site of extrapulmonary disease. Inthe absence of treatment, the disease is invariably fatal, withdeath usually occurring within 2 years of primary infection.Acute miliary dissemination, in which seeding of the fungus isthought to occur early after primary infection, is also almostinvariably fatal, with death occurring within 3 to 4 months.
Risk Factors for Severe, Disseminated Coccidioidomycosis
Genetically determined susceptibility. In no other mycosis isthe racial predisposition toward developing severe, dissemi-nated disease more conclusive. Gifford et al. (134) were amongthe first to document the increased susceptibility of Filipinos,African Americans, and Mexican Americans to developing dis-seminated coccidioidomycosis. On the basis of the number ofcases occurring in Kern County, Calif., between 1901 and 1936,Filipinos were 176 times more likely to develop disseminateddisease than were Caucasians; African Americans and MexicanAmericans were, respectively, 14 and 3 times more likely todevelop dissemination than were Caucasians. Sievers (267)evaluated risk factors in the American Indian population re-siding in the Phoenix Area of the Indian Health Service. Dur-ing a 16-year investigation period from 1959 through 1974,both American Indians and Mexicans were three times morelikely to develop disseminated coccidioidomycosis than wereCaucasians. The mortality rate was also increased, by fivefold,in American Indians and Mexicans compared to Caucasians.Some investigators raised the argument that these differencescould be ascribed to occupational exposure or socioeconomicconditions (144, 265). The results of more recent outbreaks,however, wherein exposure was less likely to be biased byoccupation, have borne out a racial predisposition to dissem-ination (108, 217, 225). In analyses of cases occurring after theCalifornia dust storm in 1977, Pappagianis and Einstein (221)reported that 54% of African American patients and 38% ofAsians had disseminated disease, compared to only 11.2% ofwhites. A similar increase in incidence in African Americanand Asians was noted in studies of patients treated at the NavalHospital in Lemoore, Calif., following the same dust storm(308). Rosenstein et al. (248) conducted a population-basedstudy for coccidioidomycosis in Kern County, Calif., for theperiod from January 1995 through December 1996. The pa-tient population consisted of 380 subjects, divided into 270persons with mild primary coccidioidomycosis (designated thecase control group), 77 patients with severe pulmonary coccid-ioidomycosis as judged by radiologic findings and hospitaliza-tion, and 38 patients with disseminated coccidioidomycosis.The percentage of patients with disseminated disease was in-
creased in African Americans but not Asian or Hispanics. Noassociation was detected between severe pulmonary or dissem-inated disease and occupation or outdoor activities.
Although the genetic basis for this increased susceptibilityremains elusive, investigators have conducted studies to assessgenetic polymorphisms that may control or be associated withthe predisposition of certain ethnic populations to Coccidoides.Human leukocyte antigen (HLA) molecules are highly poly-morphic and present antigenic peptides to �/� T lymphocytes.Scheer et al. reported an increased phenotype frequency of theHLA-A9 and -B9 antigens in patients with disseminated coc-cidioidomycosis (M. Scheer, G. Opelz, P. Tarasaki, and W.Hewitt; Program Abstr. 13th Intersci Conf. Antimicrob.Agents Chemother., 1973). Persons with the ABO blood groupB phenotype have also been reported to be at risk for devel-oping disseminated disease (91). These findings are consistentwith, and may merely reflect, the increased phenotype frequen-cies of the HLA-A9 and blood group B in persons of Filipinoand Black descent (2, 204, 228).
In a case-control study of persons from Kern County, Louieet al. (191) compared HLA class II alleles and haplotypes andABO phenotypes in mild versus severe (disseminated) coccid-ioidomycosis. No differences were observed in the ABO bloodtypes for the Caucasians or African Americans with regard towhether their disease was mild. Among Hispanics, A and Bphenotypes were significantly more frequent in patients withdisseminated disease than those with mild, uncomplicated pul-monary disease. The investigators reported that the HLA classII DRB1*1301 allele marks a predisposition to severe dissem-inated disease in all patients, regardless of their ethnic or racialbackground. It bears comment that the incidence this allelewas increased in Caucasian, Hispanic, and African Americanpatients compared with the ethnicity-matched controls. Therewas not, however, an increase in the incidence of this allele inpatients with disseminated disease versus those with mild pul-monary disease within any of the ethnic groups, as would beexpected if the DRB1*1301 allele were truly associated with anincreased risk for dissemination. Identification of host geneticvariants that predispose persons to severe disseminated coc-cidioidomycosis would be of great value in vaccine develop-ment and should be pursued, particularly in light of the grow-ing availability of intragenic single-nucleotide polymorphismsfor mapping human genome sequence variations (250).
While it is assumed that the increased susceptibility of Af-rican Americans, Asians, and Hispanics has an immunologe-netic basis, studies have not yet documented that supposition.If an immunologic basis exists, it does not appear to reside inan inherent inability of these persons to mount a cellular im-mune response to Coccidioides. Gifford and Catanzaro (133),in a comparison of Coccidioides antigen skin testing in patientswith active coccidioidomycosis, noted that although AfricanAmericans were more likely to have disseminated disease thanwere Caucasians, their skin test reactivity was comparable tothose among other ethnic groups. Along this same line, Wil-liams et al. (309) reported that African Americans and Filipi-nos acquired T-cell reactivity in response to vaccination withthe formalin-killed spherule (FKS) vaccine at levels compara-ble to that observed in Caucasians.
Gender. Males are reported to have a 3.5- to 5-fold-in-creased occurrence of disseminated coccidioidomycosis com-
pared to females (99, 217). Following the major 4-year epi-demic in San Joaquin Valley, Johnson et al. (157) studied 535patients, 25 of whom had disseminated disease, and found thatmale gender was a risk factor for dissemination when the datawere analyzed by univariate (P � 0.05) but not multivariateanalysis. However, Arsura et al. (13), in analyses of 536 casesin Kern County between September and December 1991,found that 76% of the 25 patients who had disseminated dis-ease were male, compared to 52% of males in the total pop-ulation; this was significant by chi-square analysis (P � 0.02;odds ratio, 2.9). It appears that males are more susceptiblethan females, but the differences may not be as high as notedin earlier studies.
Age. Sievers (266), in his study of disseminated coccidioid-omycosis in the American Indian population, found that chil-dren younger than 5 years and adults older than 50 years weresignificantly more susceptible than persons aged 6 through 49years. An increased susceptibility of older persons was ob-served in the outbreak in Ventura County following theNorthridge earthquake (260), in the 3-year coccidioidomycosisepidemic that started in San Joaquin Valley in 1991 (12, 157)as well as a subsequent follow-up covering the period fromJanuary 1995 through December 1996 (248), and in analyses ofcases of coccidioidomycosis in Arizona (182). In the last study,elderly persons who had recently relocated to Arizona ap-peared to be at the highest risk.
Pregnancy. Until recently, the increased susceptibility asso-ciated with pregnancy was considered to be unequivocal (99,136, 137, 238, 274). In areas of high endemicity, approximately0.1% of pregnancies have been complicated by coccidioidomy-cosis, with a resulting mortality rate of 88% (291). In a reviewof 65 women who had coccidioidomycosis during pregnancy,VanBergen et al. (291) reported that 37 (57%) developeddisseminated disease and, of these, 29 (78%) died. The laterthe gestation stage, the more likely it is that dissemination willoccur (217). These studies have prompted some to considerabortion or early delivery in gravid females with coccidioid-omycosis, in particular those in the third trimester of preg-nancy. More recent studies suggest that the risk of dissemina-tion and death in pregnancy have been overstated (15, 38, 293).Wack et al. (293) examined 47,120 pregnancies among threehealth care centers in Tucson, Ariz., during a 6-year period andreported that only 10 were complicated by coccidioidomycosis.None of these were fatal. The two women who developedfulminant, disseminated coccidioidomycosis were thought tohave acquired their coccidioidal infection during the third tri-mester. In a retrospective analysis of coccidioidomycosis inpregnant women during the California 1991 to 1994 outbreak,Caldwell et al. (38) reported that disseminated disease oc-curred in 3 (9%) of 32 patients. None of the patients died. Theinvestigators noted that although the 9% incidence of dissem-inated coccidioidomycosis in pregnant women was lower thanthat reported in earlier studies, it is higher than that observedin the general population and in females of reproductive age.One consistent finding is that the increased risk of developingdisseminated coccidioidomycosis occurs in women who ac-quired their primary infection during pregnancy and not inthose who had a previous coccidioidal infection (99).
It has been theorized that the high risk of severe coccidioid-omycosis in pregnant females is attributed to the immunosup-
pressive state that accompanies gestation. Another mechanismwas offered by Drutz et al. (101) and Powell et al. (236, 237),who reported that progesterone and 17 �-estradiol, at levelscomparable to those in the sera of pregnant females, stimulatethe growth of the spherule/endospore phase in vitro. While thisoffers an attractive and plausible explanation to account for theunique susceptibility of gravid females to coccidioidomycosis,there have been no reports of whether these hormones have astimulatory effect on fungal growth in vivo.
Immunocompromising diseases or conditions. Coccidioid-omycosis is a frequent complication for persons who are im-munologically compromised by human immunodeficiency virus(HIV) infection (3, 6, 115, 197, 315). A prospective studyconducted during a 4-year period in Phoenix and Tucson in-dicated that the risk of active coccidioidomycosis in HIV-in-fected persons ranged from 8% to 41% (6). It is not knownwhat percentage of HIV-infected persons with coccidioidomy-cosis have a primary infection with Coccidioides or reactivationof a previously quiescent infection as a consequence of theirpronounced immunodeficiency. In support of the former, Am-pel (3) did not observe any association between the length oftime the HIV-infected person resided in a coccidioidomycosis-endemic area and a history of a previous infection with thefungus. The course of coccidioidomycosis in HIV-infected per-sons can vary widely, ranging from a median survival of only 1month in patients with a diffuse bilateral reticulonodular infil-trates to several months or longer in persons who have unilat-eral focal pulmonary infiltrates (125).
Persons with other immunosuppressive diseases or condi-tions, such as Hodgkin’s disease, malignant neoplasms, andcollagen vascular disease, recipients of immunosuppressivedrug therapy, and organ transplant recipients also have a highrisk of dissemination of disease and death (30, 95, 292). Therehave been several reports of reactivation of previous coccid-ioidal infection in organ transplant recipients (30, 95, 292).
It is also possible that an acute bacterial or viral infectioncan reactivate a quiescent coccidioidal infection. To cite oneexample, coccidioidomycosis was diagnosed in a 48-year-oldCaucasian man who reactivated a prior benign coccidioidalinfection while in England after developing a group A, beta-hemolytic Streptococcus infection (307).
Critical Comments
Coccidioides is a true primary human pathogen, causing sig-nificant morbidity and mortality for those living in or visitingthe areas of endemicity. There are clear genetic influences thataffect the development of severe disseminated disease, but themechanisms of the genetic control of dissemination have notbeen characterized. A population-based study is needed todetermine genetic polymorphisms in patients with infections ofdifferent severities to delineate the gene(s) involved in genet-ically determined resistance to coccidioidomycosis.
HOST DEFENSES IN HUMANS
Innate Immunity
Polymorphonucleur leukocytes. Polymorphonucleur leuko-cytes (PMNL) comprise the earliest cellular influx to arthro-conidia (122, 256). This response may be attributable to che-
motactic components released by the fungus, as suggested byForbus and Bestebreurtje (122) in histologic studies of tissuesfrom coccidioidomycosis patients and subsequently confirmedby Galgiani et al. (129) in chemotactic assays using humanPMNL stimulated with coccidioidin or spherulin. The interac-tion of Coccidioides with PMNL has been examined usingPMNL from humans (100, 123, 126–130), rhesus macaques(23), dogs (301), and mice (31). Phagocytosis of arthroconidiais enhanced in the presence of immune serum (100, 123, 301).Ingestion of the arthroconidia is followed by a respiratory burst(31, 123), and, although the fungal cells are sensitive to theproducts of the respiratory burst (24, 127) and to cationicpeptides (defensins) (263), fewer than 20% of the arthro-conidia are killed by the encounter (23, 31, 100, 123). Indeed,some studies suggest that PMNL may promote the maturationof arthroconidia into endosporulating spherules (14, 128).
Transformation of arthroconidia into spherules renders thelatter impervious to phagocytosis and killing by PMNL (123,126), owing in part to the increased size of the spherules (60 to80 �m) relative to PMNL (12 �m). Although Galgiani (126)reported that PMNL appear to directly adhere to spherules invitro, Frey and Drutz (123) reported that spherules possess anextracellular fibrillar matrix that impedes their physical contactwith PMNL. Rupture of the spherules and release of the en-dospores triggers an influx of PMNL (122, 123). The newlyreleased endospores are encased in a fibrillar matrix, but overtime they become single cells that are readily phagocytized byPMNL. Ingestion of the endospores triggers an oxidative burst,albeit to a lesser extent than that induced by arthroconidia (23,123), and the level of intracellular killing is no more impressivethan that observed in the interaction of PMNL and arthro-conidia (23, 31, 123).
Monocytes/macrophages. Kashkin et al. (158) reported thatperitoneal macrophages from nonimmune guinea pigs phago-cytized but did not kill arthroconidia. The relative inefficacy ofnonimmune macrophages in killing Coccidioides was con-firmed by Beaman et al. (20–24). These investigators examinedthe in vitro interaction between arthroconidia and endosporeswith alveolar macrophages from nonimmune rhesus macaques,resident peritoneal macrophages from DBA/2 mice, and pe-ripheral blood monocytes from healthy, skin test-negative per-sons. Both arthroconidia and endospores are phagocytized bymonocytes/macrophages, but fewer than 1% of the phagocy-tized cells were killed. In contrast, Ampel and Galgiani (7)reported that peripheral blood monocytes from healthy, skintest-positive or -negative persons inhibited or killed arthro-conidia. The differences in the results of these studies could beattributable to differences in the methods used to quantifyfungal viability. Beaman et al. (22–24) used conventional as-says for determining fungal CFU, whereas Ampel and Galgiani(7) used a newly developed microtiter system for determiningfungal CFU (102) and a radiolabeled N-acetylglucosamine(precursor to chitin) uptake experiment to assess the inhibitionof fungal viability.
One mechanism that Coccidioides might use to survive in-tracellularly is the inhibition of phagosome-lysosome fusion(21, 24), a strategy used by many intracellular pathogens toevade the antimicrobial effects of phagocytes (11, 206, 232, 249,284, 305). Coincubation of monocytes/macrophages with im-mune T lymphocytes or gamma interferon (IFN-�) signifi-
cantly enhanced their anticoccidioidal activity (18, 19) (seebelow).
Natural killer cells. Natural killer (NK) cells are a majorcomponent of innate immunity. Under normal conditions, NKcells are confined primarily to the peripheral blood, spleen,and bone marrow, but they can migrate to sites of inflamma-tion in response to chemokines. On activation, NK cells secretecytokines, notably IFN-�, and chemokines that induce inflam-matory responses and control the growth of monocytes andgranulocytes (203). Before adaptive immunity has fully devel-oped, NK cells are thought to the main source of IFN-�, inresponse to macrophage-derived interleukin-12 (IL-12) andIL-18.
Petkus and Baum (231) reported that incubation of spher-ule/endospore-phase cells for 4 h with human peripheral bloodlymphocytes, depleted of monocytes/macrophages, signifi-cantly reduced the viability of the fungal cells. Incubation ofthe lymphocytes with Leu-11 (CD56) antibody, which binds theFc receptor of NK cells, and complement reduced the anticoc-cidioidal effect of the lymphocytes by 80%. Supernatants fromperipheral blood lymphocytes coincubated with K562 cells orCoccidioides were cytotoxic for Coccidioides, as judged by aninhibition of fungal CFU. These results suggest a direct cyto-toxicity of NK cells and NK-cell factor. However, becauseLeu-11 is not specific to NK cells, additional studies areneeded to confirm that NK cells are directly toxic to Coccid-ioides. One approach might be to utilize the NK-92 cell line(CDRL-2407; American Type Culture Collection), which is ahuman NK-cell line that is highly toxic to a broad range ofNK-cell targets (193, 289).
Dendritic cells. Dendritic cells (DCs) are potent antigen-presenting cells (APCs) and play a pivotal role in innate im-munity and adaptive immunity (190). On initial infection, pre-cursor DCs are recruited from the blood to inflammatory sites,where they transform to immature DCs. In the initial interac-tion, the pathogen binds to pattern recognition receptors, no-tably Toll-like receptors (TLR), which recognize structurallyconserved pathogen-associated microbial products. This initialrecognition and binding leads to the induction of proinflam-matory cytokines, which include tumor necrosis factor alpha(TNF-�), IL-1, IL-6, and IL-8.
Recent studies by Richards et al. (243) showed that a spher-ule-phase antigen, toluene spherule lysate (TSL), induced mat-uration of peripheral blood-derived DCs from healthy, nonim-mune subjects. DC maturation was demonstrated by increasedcell surface expression of HLA-DR, CD40, CD54, CD80,CD83, and CD86. The TSL-pulsed DCs also stimulated pro-liferation in allogeneic lymphocytes, to a greater level than didnonpulsed (immature) DCs, and they stimulated autologousnonadherent peripheral blood mononuclear cells to produceIFN-�. The potential immunotherapeutic use of DCs was es-tablished by Richards et al. (244), who showed that the anergydemonstrated by peripheral blood lymphocytes from patientswith disseminated coccidioidomycosis could be reversed by theaddition of DCs pulsed with coccidioidal antigen. Although thelatter studies were conducted in vitro, additional studies of therestoration of immunity by DC immunotherapy in animal mod-els could reveal a new avenue for adjunctive therapy in severecoccidioidomycosis.
Adaptive Immunity
Activation of immature DCs leads to their secretion of che-mokines such as CCL3 and CXCL8 and maturation of the DCsinto highly efficient APCs, which function to regulate T- andB-cell responses, a role in the immune response that distin-guishes DCs from other APCs such as macrophages and B cells(241). The antigen-bearing DCs travel from peripheral tissuevia afferent lymphatic channels to secondary lymphoid organs,such as the spleen and lymph nodes, and complete their mat-uration at these sites. The mature DCs lose their endocyticactivity by downregulating receptors that interact with antigen,and they upregulate major histocompatibility complex mole-cules; CD83; the costimulatory molecules such as CD40,CD58, CD80, and CD86; and the chemokine receptors CCR7and CXCR5 (45, 107, 253). The upregulation of the chemokinereceptors CCR7 and CXCR5 is strategic in that it effects thelocalization of DCs to appropriate sites within the lymph nodesand secondary lymphoid organs, where they interact with Tcells and B cells (251, 253). By producing cytokines that po-larize the Th response, the mature DCs effectively induce andorchestrate the adaptive immune response (107).
Beginning with the early studies by Smith et al. (276, 279–281), a profile of adaptive immune responses in persons withdifferent entities of coccidioidomycosis emerged. Persons withprimary, asymptomatic, or benign disease characteristicallyhave strong skin test reactivity to coccidioidin and low or non-demonstrable levels of anti-Coccidioides complement fixation(CF) antibody. The converse pattern develops in patients whodevelop severe, chronic, or progressive pulmonary or dissem-inated disease. Typically, these persons, in particular thosewith disease involving two or more organ systems (lungs, cen-tral nervous system, bones and/or joints), are hyporesponsiveor anergic to coccidioidal skin testing but have high levels ofanti-Coccidioides IgG antibody to the CF antigen. Recoveryfrom active disease, either spontaneous or in response to an-tifungal therapy, is in many patients associated with a reacqui-sition of T-cell reactivity to Coccidioides antigens and de-creased CF antibody titers. However, the responses of patientswith inactive disease do not coincide with those of persons whowere able to overcome their primary infection without conse-quence; instead, they tend to be intermediate between those ofthe latter patients and patients with active disseminated dis-ease.
Cellular immunity. (i) Cutaneous delayed-type hypersensi-tivity. The classical antigen preparation that was used in theearly skin test and serologic studies was coccidioidin. Thisantigen was prepared by Smith (276) as a soluble broth culturefiltrate of mycelial cells grown for 2 months in a syntheticasparagine-glycerol-salts medium. With the development of amedium for the cultivation of the spherule-endospore phase invitro (61), Levine et al. (184) produced an aqueous lysate ofspherules of strain Silveira that had been grown in Conversemedium and then incubated in distilled water for up to 40 daysat 34°C. The soluble aqueous lysate was designated spherulin(SPH) and used as a skin test antigen at a dose of 2.8 �g (UsualTest Strength), which corresponded to coccidioidin 1:100. Skintest reactivity persists in most persons who recover from pri-mary infection, and such persons are endowed with immunityto exogenous reinfection (274, 276, 279). The persistence of
coccidioidin reactivity in persons who have recovered fromtheir primary infection may be attributable to reexposure tothe fungus, as would probably occur in persons who reside inareas of endemicity, or to the presence of viable Coccidioidescells in calcified lesions (36). Although skin test reactivity per-sists in most persons, some gradually lose their coccidioidinsensitivity (215, 266). Whether this is accompanied by a loss ofresistance to Coccidioides is not known but is a question ofimmense importance since it may relate to long-term protec-tion in response to vaccination.
As many as 80% of persons who develop solitary pulmonarylesions manifest skin test reactivity to coccidioidin, whereasless than one-third of those who develop progressive or chronicpulmonary disease manifest reactivity (44, 82, 254, 274, 276,304). Skin test reactivity in persons with extrapulmonary dis-ease varies, depending on the extent of disease involvement.Approximately 70% of those who have a single extrapulmonarysite of involvement manifest reactivity to coccidioidin 1:100,whereas fewer than 30% of patients with multifocal disease arereactive to coccidioidin or SPH. Low or nondemonstrable skintest reactivity denotes a poor prognosis for recovery. Smith(274, 276) reported that 75% of patients who were skin testreactive to coccidioidin recovered from their disease whereasonly 17% of patients who were skin test negative recovered.
The specificity of the cutaneous anergy has been examinedin a number of studies (44, 82, 133, 274). In most patients, theanergy is specific to Coccidioides, as evidenced by skin testreactivity to a panel of recall antigens such as Candidin,mumps antigen, trichophyton, and streptodornase-streptoki-nase. The exceptions occur in patients who have severe dis-seminated disease involving multiple foci of infection. Approx-imately half of these patients fail to respond to recall antigens.In addition, Rea et al. (239, 240) reported that some patientsfail to respond to contact sensitization with dinitrochloroben-zene, a result that documents a pronounced suppression ofcell-mediated immune responsiveness.
(ii) Cytokine production. TNF-� is a cytokine produced by alarge variety of cells, including macrophages, DCs, CD4� andCD8� T cells, and B cells (27, 109, 285, 299, 320). Cumulativeevidence has established that TNF-� is responsible for many ofthe biological and physiological consequences of acute infec-tion, immunological reactions, and tissue injury (181). In ad-dition to its oncolytic activity and ability to induce cachexia,TNF-� can activate neutrophils, enhance the cytolytic activityof macrophages, augment NK-cell activity, promote T- andB-cell proliferation, and modulate endothelial cell surface an-tigens. TNF-� plays multiple roles in immune and pathologicresponses in tuberculosis (120, 132, 141, 162, 202, 246, 295).On one hand, TNF-� is required for the control of acuteinfection and the formation and maintenance of granulomas,but on the other hand, it has been implicated as a majorcomponent in host-mediated destruction of lung tissue.
Ampel (4) reported that autoclaved spherules and arthro-conidia of Coccidioides induced the production of TNF-� byadherent mononuclear cells from healthy human donors.TNF-� production was increased in cells from healthy, skintest-positive persons when the supernatants were assayed forcytotoxicity against the TNF-�-susceptible L929 cell line. Nodifferences were evident, however, when the supernatants wereassayed by an enzyme-linked immunosorbent assay (ELISA),
which, unlike the L929 assay, is specific for TNF-� (198).Dooley et al. (97) reported that both FKS and live spherulesinduced TNF-�, IL-1�, and IL-6 production by peripheralblood mononuclear cells and plastic-adherent monocytes/mac-rophages of healthy persons and coccidioidomycosis patients.The production of the proinflammatory cytokines was compa-rable in 15 healthy SPH skin test-positive subjects, 13 healthyskin-test negative persons, and 16 patients with activecoccidioidomycosis.
Studies have also been done to assess the production of theTh1-asociated cytokines IL-2 and IFN-�. In a study of 20healthy subjects who were skin test positive to SPH and 15healthy, skin-test negative persons, Ampel et al. (5) showedthat peripheral blood mononuclear cells from skin test-positivebut not skin test-negative donors secreted both IL-2 and IFN-�in response to in vitro stimulation with a toluene-induced ly-sate of spherules, designated TSL (Table 2). Comparisons ofcytokine production, lymphocyte proliferation, and skin testreactivity revealed that there was a tendency toward a directcorrelation, but the heterogeneity of responses precluded asignificant correlation. Of note, lymphocytes from five subjectswho were skin test positive to SPH were essentially nonrespon-sive to TSL in vitro. The basis for this unexpected deviationbetween in vivo and in vitro T-cell responses is not known and,as noted by the investigators, is unlikely to be attributable todifferences in the antigenic composition of SPH and TSL,given that the other 15 skin test-positive subjects were reactiveto both preparations. An ensuing study by Corry et al. (66)compared the production of the Th1 cytokines IFN-� andIL-12 and the Th2 cytokines IL-4 and IL-10 by lymphocytesfrom healthy, SPH skin test-positive and -negative subjects andpatients with active pulmonary or disseminated coccidioidomy-cosis. The results established that IFN-� production was sig-nificantly lower in cells from the patients with disseminateddisease than in those from healthy, skin test-positive persons.By contrast, lymphocytes from patients with pulmonary diseasesecreted levels that were comparable to those of healthy, SPH-reactive donors. The study groups did not differ in their pro-duction of the Th1 cytokine IL-12 or the Th2 cytokines IL-4 orIL-10. Additional studies are clearly needed to further examinethe production of Th1 and Th2 cytokines in coccidioidomyco-sis, with emphasis on relating cytokine responses to clinicalprogression or regression. It would be at least as important toaddress the question whether genetically determined suscepti-bility is associated with, and perhaps attributable to, an inabil-
TABLE 2. Percentage of CD3� lymphocytes producing intracellularIFN-� after incubation with tissue culture medium alone, IL-12, the
Coccidioides antigen TSL, or TSL plus IL-12a
Subject (n)% of lymphocytes producing IFN-� after incubation with:
ity to maintain Th1 responses, as has been shown in the murinemodel (discussed below).
Ampel et al. (5, 8, 9) recently analyzed cytokine responses ofperipheral blood from healthy immune and nonimmune per-sons and patients with active coccidioidomycosis by using flowcytometry. Incubation of the peripheral blood specimens withthe Coccidioides antigen T27K induced CD3� T cells to pro-duce IFN-�. Of the CD3� T cells from immune donors, 0.43%were positive for intracellular IFN-�, compared to 0.01% ofthe CD3� T cells from nonimmune donors, 0.11% of theCD3� T cells from patients with pulmonary disease, and 0.09%of the CD3� T cells from patients with disseminated disease.Ampel et al. (9) subsequently quantified the expression ofCD69, a glycoprotein that is expressed by activated T cells andNK cells (270). Using flow cytometry, the percentage of CD3blood lymphocytes expressing CD69 in blood specimens incu-bated with and without T27K was determined. After subtract-ing the background level of CD69 expression, i.e., on non-stimulated blood lymphocytes, the mean fluorescenceintensities of CD69 expression on CD3 lymphocytes fromhealthy immune subjects and patients with active disease weresignificantly increased compared to those on cells fromhealthy, nonimmune persons. There was no difference in themean fluorescence intensity of CD69 expression on CD3 lym-phocytes from the 20 healthy immune donors and 70 patientswith active disease. However, within the patient group, thosewith pulmonary disease showed a significantly higher meanfluorescence intensity of CD69 expression in response to T27Kthan did those with disseminated disease. The investigatorsobserved a significant direct association between the meanfluorescence intensity of CD69 on CD3 lymphocytes and theproduction of the cytokines IFN-�, IL-2, and TNF-�.
(iii) Cytokine activation of monocytes. Beaman and Pappa-gianis (24) reported that human peripheral blood monocytesphagocytized but did not kill Coccidioides arthroconidia orendospores and that the lack of killing was associated with aninhibition of phagosome-lysosome fusion by the fungal cells.When monocytes were incubated in the presence of lympho-cytes from immune persons, there was a significant increase inphagosome-lysosome fusion and killing of the fungal cells.Incubation of the monocytes with recombinant human IFN-�or recombinant TNF-� augmented the fungicidal capabilitiesof the monocytes (18). The mechanism by which IFN-� orTNF-� activate human monocytes to an anti-Coccidioides levelis not known, but in studies of human alveolar macrophagesfrom tuberculosis patients, IFN-� and TNF-� activate the mac-rophages to generate nitric oxide and related reactive nitrogenintermediates via nitric oxide synthase, using L-arginine as thesubstrate (296).
Humoral immunity. (i) Antibodies. Chronic or progressivecoccidioidomycosis is associated with a polyclonal B-lympho-cyte activation, as evidenced by elevated levels of IgG, IgA,and IgE in serum (39, 69, 70, 226). Antibodies reactive withcoccidioidal antigens have been demonstrable within each ofthese Ig classes. Serum IgG levels directly correlate with dis-ease involvement, being highest in patients with multifocalinvolvement. The serum IgA level is elevated in approximately20% of patients, being manifested most often in patients withchronic pulmonary disease (69). To our knowledge, secretoryIgA levels have not been reported. Hyperproduction of IgE
would be consistent with a Th2 response and has been dem-onstrated in approximately 23% of patients with active disease,with the highest incidence occurring in patients with dissemi-nated disease and, within this group, in patients who havedisease involving two or more organ systems (for example,lungs, bones and/or joints, skin, and central nervous system)(69). Anti-Coccidioides IgE was demonstrable in most patientswith elevated IgE levels; however, IgE reactivity was also de-monstrable against common allergens, such as bermuda grassand ragweed. Longitudinal studies of coccidioidomycosis pa-tients with excessive IgE levels revealed that, in most patients,IgE production diminished to normal or near normal levelsafter clinical remission, suggesting that IgE hyperproduction isa consequence of the disease. This interpretation is countered,however, by the report that atopic persons are at greater risk ofdeveloping symptomatic coccidioidomycosis than are personswho are nonatopic (96).
(ii) Immune complexes. Circulating C1q-binding immunecomplexes have been detected in sera from coccidioidomycosispatients and shown to correlate with disease severity (81, 317).Whereas 33% of sera from patients with disease involving asingle organ system had elevated immune complex levels, 67%of sera from patients with disseminated multifocal diseaseshowed circulating immune complexes. Analyses of immunecomplexes in serum from a patient with severe disseminateddisease revealed Coccidioides antigen, C1q and anti-Coccid-ioides IgG antibody.
The role, if any, of immune complexes in the immunopatho-genesis of coccidioidomycosis is not known. Investigators re-ported suppression of lymphocyte proliferation responseswhen lymphocytes from healthy coccidioidin skin test-positivepersons were assayed in the presence of patient sera and,conversely, augmentation of the responses of patient lympho-cytes when assayed in sera from healthy subjects versus autol-ogous serum (80, 208). Immunoaffinity chromatography of pa-tient sera with Staphylococcus protein A ablated thesuppressive effect of the sera, a result that would be consistentwith suppression by antibody, alone or complexed with anti-gen. However, addition of immune complexes formed in vitro(by the addition of coccidioidin to a serum sample with highlevels of anti-Coccidioides IgG) to cultured mononuclear cellsfrom healthy, coccidioidin skin test-positive persons did notsuppress their proliferation response to coccidioidin (80).These results, taken together, argue against suppression byimmune complexes and raise the question whether the sup-pression observed with patient sera was merely attributable tothe neutralization of coccidioidin in such a manner that it wasnot available to stimulate lymphocytes.
Critical Comments
Protection against human coccidioidomycosis has been con-vincingly related to induction of Th1-associated immune re-sponses. The cumulative response includes processing and pre-sentation of critical antigens by macrophages and/or DCs,leading to the induction of T cells to produce IFN-� and otherTh1-associated cytokines. These cytokines, in turn, provide thesignals for recruiting and activating immune effector cells. Thework by Richards et al. provides the best hope for developingan immunotherapy for patients with progressive disseminated
disease. If the reversal of T-cell anergy, as shown is in vitroassays utilizing DCs pulsed with Coccidioides antigens, can beextrapolated and expanded, there will be a significant increasein the therapeutic armamentarium.
MURINE MODEL OF COCCIDIOIDOMYCOSIS
The vast majority of studies of Coccidioides vaccines havebeen conducted using the murine model. The focus on thismodel has been based largely on the relatively low expense ofusing mice compared with that of using larger animal modelsand on the availability of immunologic and molecular reagentsto delineate the host response to the fungus. Also, the avail-ability of inbred mouse strains, which differ in their suscepti-bility to Coccidioides, is immensely important in evaluating theprotective capacity of Coccidioides vaccines.
Despite the utility of the mouse model, there are someconspicuous differences in the course of the disease in miceand humans. First, the disease disseminates to the spleen andliver in both the relatively resistant DBA/2 mouse strain andthe highly susceptible BALB/c mouse strain after pulmonarychallenge with only 10 arthroconidia (77). This is in contrast tolow incidence of dissemination in humans, except in personsexposed to a high infectious dose. Second, in contrast to thedirect correlation between CF antibody and disease severity inhuman coccidioidomycosis, infected (nonimmunized) mice donot have detectable CF antibody levels (26, 175). On the otherhand, similarities have been demonstrated in the correlationbetween resistance and the production of Th1 cytokines in themurine and human models and, conversely, between suscepti-bility and the production of Th2 cytokines. The extent to whichthe murine model can be used to validate the potential ofvaccine candidates for human disease simply is not known atthis time. Inbred mouse strains also provide a model for study-ing genetically determined susceptibility to Coccidioides. Stud-ies by Kirkland and Fierer (165, 166) compared the suscepti-bility of inbred mouse strains to intraperitoneal (i.p.) infectionwith gradient doses of Coccidioides arthroconidia and estab-lished that BALB/cAnN mice were the most susceptiblewhereas DBA/2N mice were the most resistant (Table 3). C3H/HeN mice were of intermediate susceptibility. Differences inthe susceptibility of BALB/c and DBA/2 mice were also de-monstrable when the challenge was performed via the i.n.route (77) (Fig. 1). On the basis that both the susceptible
BALB/c and resistant DBA/2 mouse strains are of the H-2d
haplotype, susceptibility is not controlled primarily by the H-2locus. A cross between BALB/c and DBA/2 mice yielded an F1
progeny that was resistant to challenge, whereas the progeniesof matings between susceptible strains were susceptible.Hence, resistance is a dominant phenotype.
Analysis of a backcross between (BALB/c DBA/2)F1 BALB/c yielded a 1:1 distribution of susceptible and resistantoffspring, suggesting that a single gene accounts for the differ-ence in resistance (164). This gene was given the designationCmS and, on the basis of the relative susceptibilities of thevarious inbred mouse strains, differs from the Bcg-Ity-Lsh gene,designated NRAMP (natural resistance- associated macro-phage protein), that controls natural resistance to Salmonellaenterica serovar Typhimurium, Leishmania donovani, Mycobac-terium species, and other intracellular pathogens (35). TheCmS gene is not phenotypically expressed until 10 to 12 daysafter i.p. infection, as evidenced by the presence of comparablenumbers of fungal CFU in BALB/c and DBA/c mice 10 daysafter i.p. (164) or i.n. (77) challenge. Thereafter, the fungalCFU in the susceptible BALB/c mice are significantly in-creased compared to the resistant DBA/2 strain.
Fierer et al. (113) used a set of 26 recombinant inbred (RI)mouse lines derived from a cross between susceptible B6 miceand resistant DBA/2 mice to map the genes responsible forresistance to this fungus. Each of the 26 RI lines has either theB6 or the DBA/2 allele at every genetic locus; hence, analysisof resistance provides a means of searching for linkage. Com-parisons of the fungal load in the lungs of the recombinantinbred mice after i.p. infection showed that 4 of the 26 RI lineshad a fungal load that was greater than 10 times the load foundin the susceptible B6 parent; 5 had less than 10% of the fungalload found in the resistant DBA/2 strain. Similar results wereobtained in comparisons of fungal load in the spleens. Thesedata indicate that resistance is a polygenic trait and may belinked to two unlinked loci, as opposed to their earlier reportthat a single gene, CmS, determines resistance (164). One ofthe resistance loci is on chromosome 4 near the Lv gene (ami-nolevulinate dehydratase); the other locus is on chromosome 6near Tnfr1 (tumor necrosis factor receptor 1). A backcross
FIG. 1. Percent survival of BALB/c and DBA/2 mice on days 1through 30 after i.n. infection with 10 arthroconidia. Each group con-sisted of �22 mice. Reprinted from reference 77 with permission.
TABLE 3. Lethality of Coccidioides for inbred mouse strainschallenged by an i.p. routea
a Reprinted from reference 166 with permission.b LD50, 50% lethal dose; SE, standard error.c Significantly different from all other strains (P � 0.01) but not significantly
between (B6 DBA/2)F1 B6 mice showed that 87% of theprogeny that were heterozygous for both loci were resistantwhereas only 25% of the progeny that were resistant werehomozygous at both loci. Mice that were heterozygous at onlyone of the two loci showed intermediate resistance, whichwould be consistent with the additive effect of the two loci. Theinvestigators also found a direct correlation between expres-sion of IL-10 mRNA and increased fungal load in the 26 RIlines, suggesting that the gene that influences the IL-10 re-sponse may be linked near Lv and Tnfr1 on chromosomes 4and 6, respectively. In an ensuing study, Fierer et al. (112)compared the susceptibility of C57BL/6 and C57BL/10 mousestrains, which are nearly congenic for the Lv locus. At 14 daysafter i.p. challenge with 500 arthroconidia, 79% of the B6 micewere dead, compared to only 8% of the B10 mice. However,there was no difference in the survival rate of B6 and B10 miceby day 28. The investigators concluded from these results thata gene near the Lv locus is involved in the early, innate re-sponse to Coccidioides infection. The Lv locus is not the onlylocus implicated in the resistance to Coccidioides, however,since BXD 29, which is one of the RI lines derived from a crossbetween B6 and DBA/2 mice, is resistant to Coccidioides eventhough this line is B6 at Lv. B6 mice were also found toproduce a 100-fold-lower level of IL-10 than did B10 mice,which argues against the earlier supposition that a locus nearLv was responsible for modulating IL-10 production. That is, ifIL-10 production were modulated by a locus near Lv, then B6mice would have produced increased levels of IL-10 comparedwith B10 mice.
Host Defenses in Experimentally Infected Mice
Role of T lymphocytes in protective immunity. Unequivocalevidence from studies of murine models has established thatcellular immunity is crucial to host defense against Coccid-ioides. A series of early investigations established that neonatalor adult thymectomized mice and congenitally athymicBALB/c mice are highly susceptible to challenge with Coccid-ioides compared with their T-cell-sufficient counterparts (25,54, 140). Immunization of DBA/2 mice with the FKS vaccineinduced protection against challenge, which could be adop-tively transferred via splenic T cells but not via B cells or serumfrom immunized mice (25, 26). Depletion of T cells from theimmune spleen cells ablated protective transfer (25). Morerecently, adoptive transfer of the protective immunity byspleen cells from FKS-immunized DBA/2 mice was shown tobe dependent primarily on CD4� T cells, but optimal transferwas achieved with both CD4� and CD8� T cells (79).
One mechanism by which immune T cells function as effec-tors of protective immunity is by activating macrophages toinhibit the growth of arthroconidia and endospores (20, 21).Murine alveolar or peritoneal macrophages effectively phago-cytized arthroconidia and endospores in vitro, but the cellswere not killed. Rather, within 24 to 30 h, the phagocytizedendospores began to develop into spherules and the arthro-conidia germinated and formed hyphae. When the macro-phages were coincubated with immune lymphocytes, 37% ofthe phagocytized arthroconidia and 25% of phagocytized en-dospores were killed (21). The immune lymphocytes alone didnot kill arthroconidia or endospores. Rather, the effect of
immune lymphocytes was to activate the antifungal activity ofthe macrophages by increasing phagosome-lysosome fusion.Whereas phagosome-lysosome fusion occurred with only 13%of phagocytized spores when macrophages were cultured aloneor in the presence of nonimmune lymphocytes, it increased to61% when the macrophages were cocultured with immunelymphocytes. Increased phagosome-lysosome fusion was di-rectly correlated with antifungal activity. Depletion of T cells inthe immune lymphocyte preparation decreased phagosome-lysosome fusion to 26%. It was found that activation of mac-rophages occurred via a soluble factor produced by immune Tcells. This soluble factor was most probably IFN-�, since anti-body to murine IFN-� reduced the activation of macrophagesby immune lymphocytes and since macrophage activation waseffected by the addition of IFN-�.
The differences in the hereditary patterns of disease severityin human and murine coccidioidomycosis, coupled with theprotective role of cellular immunity in host defense againstCoccidioides, suggests an immunologic basis for geneticallydetermined susceptibility to this fungus. To address this pos-sibility, Cox et al. (77) infected BALB/c and DBA/2 mice with10 arthroconidia of Coccidioides strain Silveira via an i.n. in-stillation and, at 3 day intervals, footpad tested separate groupsof mice from each strain. Both mouse strains mounted a sig-nificant footpad hypersensitivity to coccidioidin by day 9postinfection, and this hypersensitivity increased in magnitudeby day 12 (Fig. 2). Thereafter, the susceptible BALB/c micedeveloped anergy whereas delayed-type hypersensitivity per-sisted in the resistant DBA/2 mice. Footpad responses werenot measured beyond day 18 because of the excessive mortalityin the BALB/c mouse group.
Role of cytokines in host defense. Since cytokines are criticalfor the induction and expression of protective immunity, Coxand Magee (78) examined the in vivo production of the proin-flammatory cytokines TNF-�, IL-1�, and IL-6 in BALB/c andDBA/2 mice at various times after pulmonary challenge. The
FIG. 2. Footpad hypersensitivity responses in BALB/c and DBA/2mice at 3-day intervals after i.n. infection with 10 arthroconidia. Barsdepict means and standard errors (SE) obtained with groups of nine ormore mice. Reprinted from reference 77 with permission.
levels of these cytokines in lung homogenates of the two mousestrains were essentially comparable in the two mouse strainswhen measured by ELISAs and PCR for cytokine mRNA. Anensuing study was conducted to examine the induction andexpression of IFN-� and IL-4 during active coccidioidomycosisin BALB/c and DBA/2 mice (196). Quantitation of cytokines inlung homogenates obtained from groups of mice at 3-day in-tervals after pulmonary challenge revealed that the resistantDBA/2 mice produced increased levels of the Th1-associatedcytokine IFN-� as early as day 6 postinfection whereas IFN-�was not detected in the lungs of BALB/c mice until day 9postinfection (Fig. 3A). Although the levels of IFN-� increasedin both strains thereafter, the magnitude of the response wassignificantly greater in the resistant mouse strain on days 12and 15 postinfection. A reciprocal pattern was observed in thekinetics of the Th2-associated IL-4 cytokine production (Fig.3B). IL-4 levels were significantly elevated in lung homoge-nates from BALB/c mice 9 days after pulmonary challenge, atwhich time DBA/2 mice showed only a modestly elevated IL-4level. Thereafter, on days 12 and 15, both strains showed in-creased and comparable levels of IL-4 in lung tissues.
The role of IFN-� in host resistance to Coccidioides wasdetermined by pretreating the susceptible BALB/c mice withmurine recombinant IFN-� (rIFN-�) beginning on the daybefore challenge, again on the day of challenge, and then atdaily intervals for 12 days. As shown in Fig. 4, rIFN-�-treated
BALB/c mice were protected against i.p. challenge. Con-versely, neutralization of endogenous IFN-� in the resistantDBA/2 mouse strain by administering anti-IFN-� effected asignificant decrease in their ability to control the fungus afteri.n. or i.p. challenge. These data establish that IFN-� plays apivotal role in resistance to Coccidioides whereas IL-4 down-regulates protective immunity against this fungus. Since IL-12is one cytokine that can act early during host defenses topromote the differentiation of cytokine production towardIFN-�, Magee and Cox (195) examined the effect of adminis-tering rIL-12 to BALB/c mice. Treatment of mice with rIL-12beginning on the day before challenge and at daily intervals for11 days after i.p. challenge significantly protected the treatedmice compared with the ontreated mice (Fig. 5). The protec-tive efficacy of rIL-12 was associated with a shift in the expres-sion of Th1- and Th2-associated cytokines, as evidenced by thefinding that IFN-� expression predominated in the lungs ofIL-12-treated infected BALB/c mice whereas IL-4 predomi-nated in lungs of nontreated infected mice. As a complemen-tary approach to assessing the immunoprotective role of IL-12,endogenous IL-12 in the resistant DBA/2 mice was neutralizedby treatment with rat anti-mouse IL-12 monoclonal antibodybeginning on the day before and continuing on days 3 and 6after i.p. challenge with arthroconidia. Rat IgG was used as acontrol. Treatment of the resistant DBA/c mice with rat anti-IL-12 treated rendered them significantly more susceptible tothe disease. Two further comments are relevant to these stud-ies. First, rIFN-� and rIL-12 protected BALB/c mice againsti.p. challenge but had no effect when the mice were challengedvia the i.n. route. Second, rIFN-� and rIL-12 immunotherapywas effective in ameliorating the course of disease in BALB/cmice if the cytokines were administered starting 1 day prior toinfection and continuing daily for the first 11 days after chal-lenge, but there was no increase in resistance if immunother-apy was initiated after day 5 post-challenge (R. A. Cox andD. M. Magee, unpublished data).
To determine if altering the method of delivery of IL-12might protect against pulmonary challenge, Jiang et al. (152)
FIG. 3. Levels of IFN-� (A) and IL-4 (B) in homogenates of lungtissues obtained before and at various times after i.n. challenge. Thebars depict means and standard errors obtained with groups of sevenor more mice. Reprinted from reference 196 with permission.
FIG. 4. Therapeutic effects of rIFN-� treatment of BALB/c mice.The bars depict means and standard errors of log10 CFU per gram oflungs, livers, and spleens from groups of 13 mice treated with 105 U ofrIFN- � or buffer alone at daily intervals beginning on the day beforeinfection and continuing through 12 days postinfection. The mice weresacrificed 13 days after challenge. Reprinted from reference 196 withpermission.
constructed a single-chain IL-12 retroviral construct and ex-pressed the construct in the BALB/c-derived J774 cell line.Treatment of BALB/c mice with the IL-12 cDNA-transducedJ774 cells inhibited Coccidioides growth in tissues from micechallenged by the i.n. route, as evidenced by significant reduc-tions in the fungal load in the lungs, livers, and spleens at day12 postinfection compared to the loads in recipients of non-transduced J774 cells. Whereas the recipients of nontrans-duced J774 cells contained 200 pg of IFN-� per 100 mg of lungtissue, recipients of IL-12-transduced J774 cells contained1,300 pg of IFN-� per 100 mg.
Other studies have corroborated the increased production ofTh2 cytokines in susceptible inbred mouse strains. Fierer et al.(111) reported that BALB/c, C57BL/6, and CAST/Ei strainsinfected with Coccidioides express more IL-10 and IL-4 mRNAthan do resistant strains. Of interest, IL-10 knockout mice ona C57BL/6 background were as resistant to Coccidioides aswere DBA/2 mice, which is consistent with a role of IL-10 insuppression of cellular immune responses.
Role of antibody in host defense. Little attention has beengiven to the role of antibody in the protection of Coccidioidessince Kong et al. (175) reported that passive transfer of serumfrom mice vaccinated with FKS did not protect recipients. Tothe contrary, they suggested that the serum may have pro-moted the disease. Beaman et al. (25, 26) extended that findingto show that neither serum nor B cells from immune micetransferred protection against challenge in mice. Beaman et al.(26) also showed that preincubation of arthroconidia with se-rum from immune mice failed to neutralize the infectivity ofthe arthrospores. Additional data that argue against a protec-tive role of antibody were obtained in a study by Segal andCatanzaro (262), who showed that the susceptible C57BL/6mouse produced high titers of antibody against coccidioidinafter intramuscular (i.m.) injection of Coccidioides arthro-conidia whereas the resistant A/J strain did not. While the
evidence for antibody-mediated protection in coccidioidomy-cosis is not strong, it is possible that there are protective anti-bodies that could be identified by using monoclonal antibodytechnology, as has been described for Cryptococcus neoformans(42, 205, 233). Additional studies need to be performed toidentify potential protective antibodies in coccidioidomycosis.
Critical Comments
The murine model is the best-studied animal model of coc-cidioidomycosis and has provided corroboration of the basicprotective immune mechanisms with those seen in humans.The advantage of inbred mouse strains is that they providemodels for assessing genetic susceptibility against progressivedisease. However, this model fails to faithfully reflect humandisease in that all mouse strains, regardless of the level ofgenetic susceptibility, experience disseminated disease earlyafter pulmonary challenge. While the advantages of the mousemodel include a wealth of immunological reagents and theavailability of mice with selective gene deletions, addition ofanimal models that more accurately reflect human diseasewould be of benefit.
VACCINE CANDIDATES
A large body of evidence documents the feasibility of devel-oping a vaccine for coccidioidomycosis. First, persons whorecover from a benign or asymptomatic infection with Coccid-ioides are resistant to exogenous reinfection (114, 213, 220,277, 279); hence, the fungus has immunizing capacity. Second,the fungus is geographically restricted, thereby delineating theareas of potential infection. Third, the target population is welldefined and includes persons who are genetically predisposedto developing disseminated disease and persons who have ahigh probability of exposure by virtue of their occupation.
Viable Cells
Rixford and Gilchrist (245), in 1896, reported that experi-mental cutaneous infection of a dog with the exudate of lesionsfrom a coccidioidomycosis patient induced immunity to cuta-neous challenge. The immunizing capacity of a sublethal in-fection was subsequently confirmed by several groups of inves-tigators (62, 65, 224, 227). Converse and Besemer (62)reported that vaccinating cynomolgous monkeys by subcutane-ous (s.c.) injection of 101 to 108 viable arthroconidia protectedthe monkeys against respiratory challenge with approximately7,000 viable arthroconidia, as judged by their healthy appear-ance throughout a 4-month observation period, normal chestX rays, only a minor histopathologic changes at autopsy, andnegative lung cultures in 80% of the animals.
The use of viable cells posed a major limitation, however, inthat organisms persisted at the site of vaccination and notinfrequently were demonstrable at distal sites, even when thevaccine was administered by the s.c. route (65, 173). In aneffort to mitigate the risk of disease from vaccination with liveCoccidioides cells, Pappagianis et al. (224) vaccinated mice byusing a riboflavin-requiring auxotrophic mutant created byFoley et al. (121) by X irradiation of Coccidioides strain Sil-veira. The mutant strain engendered immunity to challenge
FIG. 5. Protective effect of rIL-12 against i.p. challenge in BALB/cmice. The bars depict the means and standard errors of log10 CFU pergram of lungs, livers, and spleens obtained from groups of 9 or 10BALB/c mice treated with 0.1 �g of rIL-12 or buffer alone beginningon the day before and continuing for 11 days after challenge. The micewere sacrificed 12 days after i.p. challenge. Reprinted from reference195 with permission.
but reverted in vivo to a prototrophic state that was accompa-nied by a reacquisition of virulence. Similar findings were ob-tained with a cobalt-irradiated mutant of Coccidioides and withspherules that had been attenuated by in vitro passage (174).Efforts to reduce the risks imposed by viable attenuated vac-cines included prevaccination with killed organisms (62) andtreatment of vaccinated mice with amphotericin B (43). Bothapproaches ameliorated the disease but were not applicable inhumans because of the potential adverse consequences.
Nonviable Cells
Owing to the lack of a suitable medium for culturing spher-ules, most of the early work with nonviable vaccines was con-ducted with mycelium-phase cells (64, 124, 173, 185, 186).Following the development of a medium for culturing spher-ules (61), Levine and coworkers (173, 185) compared the vac-cine capacity of the parasitic and saprobic forms of Coccid-ioides. Mice vaccinated i.m. with 1.5 mg of FKS survived an i.n.challenge dose of 104 arthroconidia, whereas only 50% ofthose vaccinated with killed mycelia survived challenge withCoccidioides. Immunogenicity of the parasite-phase cells in-creased with maturation, with killed spherules being more pro-tective than endospores. Whether these differences are attrib-uted to qualitative or quantitative differences in immunogensthat comprise the morphogenetic phases of Coccidioides is notknown.
The effect of the dose, schedule, and route of immunizationwith killed spherules was delineated in studies by Levine et al.(187) using the murine model. Mice (NAMRU strain) immu-nized with 2 to 2.7 mg of FKS were more resistant to i.n.challenge than were mice given lower doses. Administration ofthe vaccine in three or more injections over a period of 7 daysengendered a higher level of protection than did a single in-jection, and protection was enhanced when the vaccine wasgiven at multiple sites (187). Although demonstrable levels ofimmunity were established as early as 7 days after the lastvaccination, protection was more pronounced at 21 to 30 days.The route of vaccine delivery proved to be a major determi-nant of protective efficacy. Both the i.m. and s.c. routes wereeffective in eliciting protection against i.n. challenge, whereasimmunization via an i.n. route was less protective and intrave-nous (i.v.) immunization offered the least protection. Indeed,i.v. injection of 120 �g of killed spherules prior to, concomitantwith, or within 35 days of i.m. vaccination with killed spherulesdiminished the protective efficacy of the latter. On the basis ofthe foregoing studies, studies of the murine model have used2.1 mg of the FKS vaccine, administered in three i.m. injectionsof 0.7 mg at weekly intervals. Using this protocol, we examinedthe efficacy of the FKS vaccine in the genetically susceptibleBALB/c mouse strain (77). As shown in Fig. 6A, BALB/c micewere protected against pulmonary challenge with 30 arthro-conidia of Coccidioides strain Silveira. Also, in contrast to theanergy that develops in nonimmunized BALB/c mice (Fig. 2),FKS-vaccinated BALB/c mice were able to maintain their foot-pad hypersensitivity response to coccidioidin at a level compa-rable to that is vaccinated or nonvaccinated DBA/2 mice (Fig.6B).
Levine et al. (188) evaluated the FKS vaccine in cynomol-gous monkeys. Nine female monkeys, with an average weight
of 1.8 kg, were given 1.5 mg of FKS s.c. on day 1, 1.5 mg i.m.on day 11, 3 mg s.c. on day 28, and 3 mg i.m. on day 54. Ninecontrol monkeys were treated with corresponding volumes ofsaline. The monkeys were challenged on day 107 (53 days afterthe completion of the vaccination or placebo immunization)with arthroconidia of strain Silveira. The arthroconidia weresuspended in physiologic saline at a concentration of 5 103
viable particles per ml, and a cloud was generated from thesuspension by using an atomizer in a modified Hendersonapparatus. The calculated dose was 200 arthroconidia per an-imal, except for one of the vaccinated monkeys who wasthought to have been exposed to 400 arthroconidia. During the265-day observation period, five of the control monkeys died(days 39, 44, 63, 116, and 244) whereas only one of the vacci-nated monkeys died of the disease. The latter monkey was theone that had inadvertently received 400 spores. One othervaccinated monkey was killed accidentally during cardiac punc-ture on day 63 postinfection. Survivors on day 262 after chal-lenge were skin tested with coccidioidin. Two of seven vacci-nated monkeys and all four of the controls were skin testnegative. It was noted that the two vaccinated monkeys hadshown sensitivity after vaccination and before challenge;hence, the subsequent negative skin test reactivity representedan acquired anergy. CF antibody titers were increased in bothvaccinated and control monkeys, and the median titers were
FIG. 6. (A) Survival of FKS-vaccinated and nonvaccinated BALB/cmice on days 1 through 30 after i.n. infection with 100 arthroconidia.Each group consisted of 15 mice. (B) Footpad hypersensitivity inFKS-vaccinated and nonvaccinated BALB/c and DBA/2 mice 15 daysafter i.n. infection with 10 arthroconidia. The bars depict means andstandard errors (SE) obtained with groups of 15 mice. Reprinted fromreference 77 with permission.
similar in the two groups. At necropsy, both vaccinated andcontrol monkeys showed pulmonary and disseminated involve-ment, but the disease was judged to be less severe in thevaccinated monkeys than in the control monkeys.
The efficacy of the FKS vaccine in the murine and monkeymodels, coupled with the severity of coccidioidomycosis inpatients with disseminated disease, prompted clinical trials toassess its toxicity and potential use in humans. Recipients of atotal of 2.7 mg of killed spherules, given i.m. in one to threeinjections, showed localized tenderness or induration at thesite(s) of injection but otherwise tolerated the vaccine well.Doses greater than 10 mg produced marked and unacceptablelevels of toxicity, an effect that could be attributable to theinduction of proinflammatory cytokines by the FKS (4, 273).Based on these and other trials (309), a regimen of three dosesof 1.75 mg each was chosen for use. Approximately 7% ofrecipients showed adverse reactions, ranging from local inflam-mation to systemic toxicity. Skin test conversions to coccidioi-din and/or spherulin occurred in approximately half of vaccinerecipients and generally persisted for longer than 6 months;16% showed seroconversion with reactivity to CF, tube preci-pitin (TP), or both (309). It is noteworthy that no differenceswere observed in the immunologic responses of Caucasians,African Americans, or Filipinos.
The protective efficacy of the spherule vaccine was evaluatedin a double-blind multicenter study between 1980 and 1985(218, 223). A total of 2,867 healthy persons who were skin testnegative to both coccidioidin and spherulin were randomizedinto placebo and vaccine groups. The vaccine recipients weregiven three injections of 1.75 mg of the FKS; the placebo groupwas given saline injections. After a 5-year follow-up period, nosignificant difference was observed in the incidence or severityof coccidioidal cases that developed in the two groups of sub-jects (218). The incidence of coccidioidomycosis was low whilethis study was being conducted, with only 21 cases occurring inthe two study groups; this low incidence probably contributedto the lack of detectable vaccine-induced protection. Given themarked increase in infections that occurred during 1991 andinto 1994, it would be of great interest to retrospectively de-termine if subjects enrolled in the FKS vaccine study mighthave acquired coccidioidomycosis during the epidemic and, ifso, whether differences exist in the incidence of the disease inthose who received the vaccine and those who received theplacebo vaccine.
It has been suggested that the ineffectiveness of the FKSvaccine in the clinical trials may be attributable to the low dose ofthe vaccine used, owing to its toxicity (218, 220). On a body weightbasis, the quantity of the FKS vaccine that was used in the humantrials was less than 0.1% of the dose needed to immunize mice.These discouraging results obtained with the FKS vaccine in hu-mans prompted investigators to seek subcellular vaccines. On thebasis that Kong et al. (172) reported that the protective capacityof killed spherules resided primarily in their cell walls, most in-vestigators have focused on the identification and isolation ofimmunoprotective cell wall components.
Cell-Derived Antigens
Antigens, either native extracts, recombinant proteins, orgenes, have been reported to induce protection and could be
candidates for a univalent or, most probably, multivalent vac-cine. A summary of the major results obtained with leadingvaccine candidates is presented in Table 4, which focuses onthe strain of mice, with BALB/c being the most geneticallysusceptible; the route of challenge, with pulmonary challengebeing the most relevant and rigorous; the number of arthro-conidia; and whether protection was measured as a reductionin CFU or increased survival or both.
PBS extract of spherule cell walls. Extraction of an immu-noprotective component(s) from cell walls of spherules wasfirst reported by Pappagianis et al. (222). These investigatorsisolated the cell walls by mechanical disruption of spherules ofstrain Silveira. Incubation of the cell walls for 5 days withconstant agitation in phosphate-buffered saline (PBS) contain-ing 2% chloroform as a preservative released a wall fractionwhich, when admixed with complete Freund’s adjuvant (CFA)or alum, protected NAMRU mice against pulmonary chal-lenge with 103 arthroconidia. The immunoprotective cell wallfraction contained some fragments, and, when the latter wereremoved by centrifugation, the opalescent supernatant wasprotective. It was not determined if the immunizing compo-nent(s) was soluble or present as a colloid; as of this writing,there have been no further reports of the spherule wall-derivedPBS extract.
27K. On the basis that FKS protects experimental-animalmodels against pulmonary challenge with Coccidioides, Zim-mermann et al. (325) sought to isolate the protective immu-nogen(s) directly from FKS. The whole killed spherules ofstrain Silveira were mechanically disrupted, and the superna-tant obtained after centrifugation of the lysate at 27,000 gwas designated 27K. When suspended in saline to a concen-tration of 3.5 mg/ml, 27K was slightly opalescent, which isreminiscent of the PBS wall fraction discussed above. Threeweekly immunizations of outbred Swiss-Webster mice with 1mg of the 27K vaccine with alum induced strong protectiveimmunity against i.n. challenge with 5,000 arthroconidia (P 0.003) and 15,000 arthroconidia (P 0.04), as judged by sur-vival over a 13-week period after challenge (Fig. 7). Of partic-ular importance, the immunity induced by the 27K vaccine wasequal to that induced by the FKS vaccine. In a one study, wehave assessed the protective capacity of the 27K vaccine ininbred mice (Magee and Cox, unpublished). Groups of suscep-tible BALB/c mice were immunized three times at weeklyintervals with 250 �g of the 27K vaccine in Freund’s adjuvant.CFA was used for the first immunization, and incompleteFreund’s adjuvant (IFA) was used for the last two injections.The animals were challenged i.n. with 30 arthroconidia 2 weeksafter the last immunization and sacrificed 12 days after chal-lenge for determination of total fungal CFU in the lungs andspleens. The results show that there was greater than a 2 logand 3 log reduction in fungal CFU in the lungs and spleens,respectively, in 27K-immunized mice compared to adjuvant-immunized mice (P � 0.001) (Fig. 8). The cumulative results ofall the reports to date show that the 27K vaccine provides thebest protection by a subcellular fraction of Coccidioides.
It is curious that the high level of protection engendered bythe 27K vaccine against pulmonary challenge was achievedusing alum, since this adjuvant enhances antibody but notTh-cell responses. It is even more curious that the vaccinealone, without alum adjuvant, induced protection, albeit at a
lower level than that provided by 27K in alum. Perhaps thereis a particulate component(s) in the 27K vaccine which servesas an antigen depot for presentation and processing by APCs.
Ag2/PRA. In 1975, Ward et al. (298) isolated an alkali-solu-ble, water-soluble fraction from trypsin-treated cell walls ofCoccidioides mycelia and showed that the cell wall extractelicited delayed-type footpad hypersensitivity response in mice
immunized with Coccidioides. Lecara et al. (183) showed thatthe C-ASWS extract from mycelium-phase cells induced pro-tective immunity in mice. Immunization of DBA/2 mice with atotal of 0.5 mg of the mycelium-derived C-ASWS in CFAafforded a significant degree of protection against i.p. chal-lenge with 1,500 arthroconidia of strain Silveira. Mice chal-lenged with 50 arthroconidia by an i.n. route were also pro-tected. Of 24 DBA/2 mice vaccinated with a total of 1 mg ofC-ASWS in CFA, 20 (83%) were significantly protected, com-
FIG. 7. Vaccine efficacy of 27K against i.n. challenge. Swiss Web-ster mice were immunized with 27K in alum, 27K alone, or alum aloneand challenged with 5,000 arthroconidia via an i.n. route. Survival wasmonitored daily for 75 days post challenge. Reprinted from reference325 with permission.
FIG. 8. Vaccine efficacy of 27K against i.n. challenge in susceptiblemice. BALB/c mice were immunized with 27K in CFA/IFA or alumCFA/IFA alone and challenged with 30 arthroconidia via an i.n. route.Survival was monitored daily for 45 days after the challenge.
pared to the survival of 14 (56%) of 25 mice given CFA alone(P � 0.05). The C-ASWS extract also protected DBA/2 miceagainst i.n. challenge with 500 arthroconidia, with a 53% sur-vival on day 30 postinfection versus 30% in control mice (P �0.01). Protection was not obtained against challenge with 1,500arthroconidia. Subsequent analyses showed that the spherule-derived C-ASWS was more protective than the mycelium-de-rived extract and that while the mycelium- and spherule-de-rived C-ASWS extracts protected DBA/2 mice against i.n.challenge, neither induced protective immunity against i.n.challenge in the highly susceptible BALB/c mice (Cox andMagee, unpublished). In addition to its protective capacity,C-ASWS was shown to be reactive in eliciting in vitro lympho-cyte proliferation responses in healthy, coccidioidin skin test-positive persons and patients with various stages of coccidioid-omycosis (75, 82). C-ASWS was also shown to react with TPantibody (74) and anti-Coccidioides IgG and IgE antibodies(69).
Antigenic analyses of C-ASWS by two-dimensional immu-noelectrophoresis (2D-IEP) revealed that it is a high-molecu-lar-weight polysaccharide-protein complex containing a poly-meric antigen which has antigenic identity to the coccidioidinprecipitinogen that Huppert et al. (147) designated as antigen2 (Ag2) (71). The extract also contained a second componentthat showed an incomplete precipitin antigen in 2D-IEP. Thelatter was subsequently identified as the TP antigen (71). Solid-phase immunoadsorption of coccidioidin with goat antiserumagainst C-ASWS affected the adsorption of Ag2 and traceamounts of the TP antigen. The desorbed; Ag2-enriched frac-tion elicited significant footpad hypersensitivity responses inCoccidioides-infected mice, to a level comparable to the re-sponses elicited by coccidioidin (67). Attempts to purify Ag2from the polysaccharide-enriched TP antigen were unsuccess-ful. Rather, both antigens coeluted on lectin affinity, molecu-lar-sieve, and ion-exchange columns, suggesting that the twoantigens are physically or chemically complexed (67, 71, 74).Studies were undertaken, therefore, to clone the gene thatencodes Ag2. Using a polyclonal goat antiserum preparedagainst the Ag2-enriched solid-phase immunoadsorption frac-tion and a monoclonal antibody directed against Ag2 (76) toidentify Ag2-positive clones, Zhu et al. (324; Y. Zhu, C. Yang,D. M. Magae, and R. A. Cox, Abstr. Third Natl. Inst. AllergyInfect. Dis. Workshop Med. Mycol., 1993.) cloned a 1,255-bpgene encoding Ag2 (Fig. 9). The cloned Ag2 cDNA encodes a194-amino-acid protein having an 18-amino-acid N-terminalsignal sequence and a C-terminal glycosylphosphatidylinositol(GPI) anchor. The cloned Ag2 gene encodes a product thatcontains a region with 10 tetrapeptide repeats TXX�T, whereX is alanine, histidine, or glutamic acid and X� is alanine,valine, or glutamic acid. Whereas no N-glycosylation sites werepredicted, several O-glycosylation sites were predicted within aThr-rich region (Thr97-Thr165). In an ensuing study, Zhu et al.(323) cloned the Ag2 gene from genomic DNA and showedthat it contains two introns, 78 and 101 bp long.
The cloned Ag2 cDNA was expressed in Escherichia coli as aglutathione S-transferase (GST) fusion peptide and shown toelicit footpad hypersensitivity tests of FKS-immunized mice ata level comparable to that of C-ASWS (324). For determina-tions of vaccine efficacy, Jiang et al. (154) immunized BALB/cmice with three weekly injections of 100 �g of recombinant
Ag2 (rAg2) fusion protein. The first immunization was given inRIBI 730 adjuvant via an i.m. route; the two subsequent im-munizations were given in RIBI 700 adjuvant via an s.c. route,and the mice were challenged i.p. with 250 arthroconidia ofstrain Silveira 1 month after the last immunization. The rAg2fusion protein induced a significant reduction in the fungalload in the livers and spleens by day 12 postinfection (Fig. 10A)but did not increase survival compared to that control of miceimmunized with the GST peptide alone (Fig. 10B). The lowprotective efficacy of the rAg2 peptide, expressed and isolatedfrom E. coli, was thought to be attributable to a lack of post-translational modification(s) required for optimal presentationand/or processing to APCs. Since genetic immunization pro-vides a means of producing native antigen in vivo and has beendocumented to be highly effective in inducing protective cel-lular and humoral immune responses (261), studies were doneto evaluate genetic immunization with Ag2 cDNA ligated tothe plasmid pVR1012 (154). The pVR1012 DNA plasmid vec-tor (Vical, Inc.) contains a kanamycin resistance gene and thehuman cytomegalovirus promoter and lacks a signal (leader)sequence. BALB/c mice were immunized by three weekly i.m.immunizations each with 50 �g of Ag2 cDNA-pVR1012 andchallenged i.p. with Coccioides. As shown in Fig. 11A, vacci-nated BALB/mice showed a 2-log or greater reduction in fun-gal load on day 12 after infection with 2,500 arthroconidia, adose 10-fold greater than that used in protection studies ofrAg2. The decreased fungal load in the lungs, livers, andspleens was accompanied by increased survival, with 20 of 20mice vaccinated with Ag2 cDNA-pVR1012 surviving a for 40-day period postinfection, compared to the survival of only 1(9%) of 20 mice vaccinated with plasmid pVR1012 alone (Fig.11B). Protection was associated with the induction of footpadhypersensitivity and the production of IFN-�. Genetic immu-nization with Ag2 cDNA-pVR1012 reduced the fungal load inthe livers and spleens of BALB/c mice challenged with 50arthroconidia via an i.n. instillation but did not reduce thefungal load in the lungs (Fig. 12) or decrease mortality. In ourexperience, failure to protect at the lung level predicts failure
FIG. 9. Nucleotide sequence and deduced amino acid sequence ofcloned Ag2 cDNA. DNA base numbers are on the left, and amino acidnumbers are on the right. The underlined amino acid region at thebeginning of the N terminus of the Ag2 protein is the signal sequence.The doubly underlined amino acid region contains the tetrapeptiderepeats. The underlined amino acid region at the C terminus is theGPI anchor. Reprinted from reference 324 with permission.
to prolong survival in vaccinated mice (Magee and Cox, un-published). In contrast to the results obtained with Ag2 cDNA,mice vaccinated with the FKS vaccine were strongly protectedagainst i.n. challenge. The latter is an important finding, sinceit establishes that BALB/c mice can be protected against i.n.challenge.
Jiang et al. (151) reported that administration of Ag2 cDNA-pVR1012 with single-chain IL-12 cDNA (p40-L-p35), eachsubcloned into the pVR1012 plasmid, significantly enhancedprotection against i.p. challenge with 2,500 arthroconidia overthat conferred by Ag2 cDNA alone. The enhanced protectionwas associated with increased IFN-� production, at levelshigher than those induced in mice vaccinated with Ag2-pVR1012 or IL-12-pVR1012 alone. Analyses of the anti-Coc-cidioides IgG isotype response revealed a predominant IgG1response, which is associated with a Th2 response, and a lowIgG2a response. While it is recognized that antibody isotyperesponses may vary during the course of immunization andinfection, owing to the changing dynamics of Th1 and Th2responses, the predominance of IgG1 both before and afterchallenge is paradoxical, particularly in the absence of detect-able IL-4, and merits further study. When mice immunizedwith Ag2 pVR1012 and IL-12 cDNA were challenged with 50arthroconidia via the i.n. route and evaluated for fungal load
12 days later, there was a significant decrease in the fungal loadin the spleens and livers but not in the lungs.
Recently, Jiang et al. (153) reported that the signal sequenceencoded in the first 18 amino acids of the translated Ag2 cDNA
FIG. 10. Vaccine efficacy of rAg2, expressed as a GST fusion pep-tide, in BALB/c mice challenged by the i.p. route with 250 arthro-conidia. Results are expressed as the numbers of CFU (means andstandard errors) in the lungs, livers, and spleens on day 12 postinfec-tion (A) and the percent survival in mice on days 1 through 30 postin-fection (B). Reprinted from reference 154 with permission.
FIG. 11. Vaccine efficacy of pVR1012-Ag2 cDNA against i.p. chal-lenge. BALB/c mice were immunized with pVR1012-Ag2 cDNA orpVR1012 alone and then challenged with 2,500 arthroconidia.(A) Fungal load, expressed as log10 CFU/g of tissue, was determined12 days after challenge. Bars depict means and standard errors ob-tained with groups of 20 mice. (B) Percent survival was determined ondays 1 through 40 for groups of 11 mice. Reprinted from reference 154with permission.
FIG. 12. Vaccine efficacy of pVR1012-Ag2 cDNA and FKS againsti.n. challenge. BALB/c mice were immunized with pVR1012-Ag2cDNA, pVR1012 alone, or FKS and then challenged with 50 arthro-conidia via the i.n. route. Fungal load, expressed as log10 CFU pergram of tissue, was determined 12 days after challenge. Reprintedfrom reference 154 with permission.
protected mice against i.p. challenge with 2,500 arthroconidia,as measured by fungal load and survival. The level of protec-tion induced by the signal sequence was lower than that in-duced by the full-length Ag2(1–194) cDNA, but the differencewas not statistically significant. A synthetic peptide corre-sponding to the Ag2 cDNA signal sequence also induced pro-tection, albeit at a lower level. The protection induced by thesignal sequence cDNA was not attributable to a nonspecificimmunopotentiation form nucleotide sequences, as establish-ing by the finding that immunization of BALB/c mice with aframeshift mutation of the Ag2(1–18) cDNA, created withPCR by deleting the first nucleotide in the second codon, didnot induce protection. The protection induced by the signalsequence vaccine correlated with an IFN-� response and wasnot associated with the production of anti-Coccidioides anti-body. The lack of an antibody response to Ag2(1–18) cDNA isconsistent with an earlier report showing that a syntheticAg2(1–18) peptide was not detected by hyperimmune goatanticoccidioidin or anti-Ag2 antisera or sera from patients withactive coccidioidomycosis (321). The Ag2(19–194) cDNA con-struct, which is devoid of the signal sequence, induced protec-tion, but to a significantly lower level than the signal sequenceor the full-length Ag2(1–194) cDNA.
In addition to its T-cell reactivity and vaccine capacity, therAg2 fusion peptide showed reactivity with sera of patientswith active coccidioidomycosis. Using PCR-generated Ag2truncations to identify the B-cell reactive epitopes, Zhu et al.(321) established that rAg2 peptide expressed both linear andconformational B-cell-reactive epitopes localized to a domaincomposed of amino acids 19 to 96 [Ag2(19–96)]. Truncationsdesigned to identify epitopes within the Ag2(19–96) domainyielded fragments that were either nonreactive [Ag2(62–194),Ag2(19–61), and Ag2(49–79)] or showed reduced reactivity[Ag2(19–70)] when assayed by ELISA and immunoblotting.When assayed against sera from 28 patients with active coc-cidioidomycosis, 79% of the sera showed reactivity to therAg2(19–96) fusion peptide. No reactivity was demonstrablewith sera from histoplasmosis or blastomycosis patients.
In 1991, Dugger et al. (103) deglycosylated TSL by usinganhydrous fluoride and then isolated a 33-kDa peptide fromthe deglycosylated antigen by molecular sieve chromatography.The 33-kDa peptide showed antigenic identity to the anodalprecipitin leg of Ag2 when assayed by 2D-IEP in tandem withcoccidioidin and was reactive with an IgG2a monoclonal anti-body specific to Ag2 (76). The purified 33-kDa antigen elicitedproliferation in peripheral blood mononuclear cells of healthy,skin test-positive persons and reacted with sera from coccid-ioidomycosis patients (131). Amino acid analysis of the 33-kDapeptide revealed an unusually high proline (17%) and threo-nine (15%) content. Immunoelectron microscopy using affin-ity-purified human antibodies revealed that the antigen waslocalized to the inner cell wall and cleavage planes of devel-oping spherules. In endospore preparations, the 33-kDa anti-gen appeared to be exposed on the wall surface and the inter-connecting glycocalyx (131).
Almost contemporaneously with the cloning of Ag2 by Zhuet al. (323, 324; Zhu et al., Abstr. Third NIAID Workshop,1995), Dugger et al. (104) cloned the gene that encodes the33-kDa peptide by screening a spherule/endospore phaselambda ZAP cDNA expression library with goat anti-33-kDa
antiserum. Nucleic acid sequence analyses revealed a 582-bpopen reading frame (ORF) encoding a 194-amino-acid proteinwith a predicted molecular mass of 19.5 kDa. The deducedamino acid composition showed a high proline and threoninecontent (12.9 and 11.3 mol%, respectively), and, on that basis,the 33-kDa peptide was given the designation “proline-richantigen” (PRA).
Kirkland et al. (167) ligated the gene encoding PRA intopET32a and expressed the construct in E. coli as a histidine-tagged recombinant protein. After removal of the histidine tagby thrombin cleavage, the rPRA elicited a T-cell proliferationin lymph node T cells from mice immunized with recombinantPRA (rPRA) and a weak but statistically significant responsein mice immunized with FKS or live attenuated Coccidioidesstrain 95-291 (294). Immunization of BALB/c mice with 5 �gof rPRA in IFA, followed by CFA, protected the mice againsti.p. challenge with 50 arthroconidia of Coccidioides (R.S.strain), as judged by a reduced fungal load in the lungs andspleens compared to the fungal load in mice immunized withCFA alone. Abuodeh et al. (1) extended these studies to acomparison of the vaccine efficacy of rPRA and PRA cDNA.BALB/c and C57BL/6 mice, which are less susceptible to Coc-cidioides than are BALB/c mice (164, 166), were immunizedwith 5 �g of rPRA emulsified in MPL adjuvant (see “Adju-vants” below) and then given a booster injection 4 weeks later.The mice were challenge i.p. using 50 arthroconidia forBALB/c mice and 500 arthroconidia for C57BL/6 mice. TherPRA in MPL adjuvant induced protection against i.p. chal-lenge in both BALB/c and C57BL/6 mice, as judged by areduced fungal load compared to that in mice given salinealone. It is noted that the appropriate control group for thisexperiment would have been mice given the MPL adjuvantalone, as opposed to saline, since adjuvant alone can increasethe host response.
For genetic vaccination, BALB/c and C57BL/6 mice wereimmunized i.m. with 100 �g of PRA cDNA ligated topVR1020. This DNA vector is similar to pVR1012, except thatit contains the tissue plasminogen activator signal peptide up-stream of the BamHI cloning site. At 4 weeks after the firstimmunization, the BALB/c and C57BL/6 mice were boosted;they were challenged 4 weeks later with 100 and 500 arthro-conidia, respectively, via an i.p. route (1). When the DNAvaccine was used, there was a reduction in fungal CFU in micevaccinated with the PRA cDNA-pVR1020 construct comparedwith that in mice vaccinated with saline alone. However, andmost importantly, there was not a significant difference in thefungal load in the lungs of BALB/c mice vaccinated with PRAcDNA-pVR1020 and those vaccinated with the pVR1020 vec-tor alone, the latter being a more appropriate control thansaline. In contrast to the results obtained with the highly sus-ceptible BALB/c mice, immunization of C57BL/6 mice withthe PRA cDNA-pVR1020 construct significantly reduced thefungal load in the lungs compared to that in mice given thevector alone (P 0.017). Splenocytes from both BALB/c andC57BL/6 mice immunized with the recombinant or PRA genevaccine showed a proliferative response to rPRA and secretedIFN-� in vitro in response to rPRA. IL-4 production was notobserved. The predominant IgG isotype response to PRA wasIgG1, which is consistent with the findings by Jiang et al. (151)
and raises the question why the antibody response is a Th2rather than a Th1 response.
Whereas the preceding studies evaluated the protective ca-pacity of rPRA and the PRA gene vaccine against i.p. chal-lenge, Shubitz et al. (264) examined the protective capacity ofrPRA in BALB/c and C57BL/6 mice challenged by i.n. infec-tion. BALB/c and C57BL/6 mice, vaccinated s.c. with 1 or 5 �gof rPRA in monophosphoryl lipid A (MPL-AF [see “Adju-vants” below]) and boosted 4 weeks later, were protectedagainst i.n. challenge with 7 arthroconidia but not 10 or morearthroconidia. Whereas 20 (59%) of 34 BALB/c mice immu-nized with rPRA in MPL-AF survived infection, only 4 (17%)of 24 mice immunized with the MPL-AF adjuvant alone sur-vived (P � 0.005). The investigators noted, however, that noneof the survivors in either the PRA-vaccinated or adjuvant con-trol group showed evidence of infection at necropsy, whichraises the question whether they had been infected. This is animportant point, since even the FKS vaccine does not inducesterilizing immunity against pulmonary challenge with Coccid-ioides arthroconidia (325; Cox and Magee, unpublished). Stud-ies with the more resistant C57BL/6 strain showed that rPRA-vaccinated mice were resistant to i.n. challenge with as many as145 arthroconidia, which is a 21-fold-increased dose comparedto that given the BALB/c mice. Of particular interest, theinvestigators reported that i.n. immunization was much moreprotective than s.c. immunization. This is an important finding,with implications for vaccine trials in humans.
Peng et al. (230) generated overlapping subunits of PRA todetermine which region might express the protectiveepitope(s). Comparison of plasmids encoding PRA(1–106),PRA(27–106), PRA(90–151), or PRA(90–194) showed thatmice vaccinated with PRA(1–106) or PRA(27–106) had a sig-nificantly reduced-fungal load (P � 0.05) after i.p. challengewith 50 arthroconidia compared to mice immunized with vec-tor alone. Identical results were obtained when mice wereimmunized with recombinant proteins encoding the same PRAsubunits. Neither a plasmid nor a recombinant product encod-ing the signal sequence alone, which was shown by Jiang et al.(153) to be protective, was evaluated.
The genes encoding Ag2 and PRA have identical nucleicacid sequences and, for that reason, are now collectively des-ignated Ag2/PRA (153, 264). The combined studies discussedabove establish that rAg2/PRA will protect BALB/c miceagainst an i.p. challenge and a low dose given by the i.n. routewhen the rAg2/PRA is used with the MPL adjuvant. The moreresistant C57BL/6 strain can be protected against both routesof challenge. While the ability to protect C57BL/6 againstpulmonary challenge is an important and valuable finding, thelow level of protection in the genetically susceptible BALB/cmice leads to the question whether the Ag2/PRA vaccine willbe able to protect persons who are known to have a geneticpredisposition to severe, disseminated coccidioidomycosis.
T-cell-reactive protein. Cole et al. (56) isolated a water-soluble fraction from arthroconidia which had been stripped oftheir hydrophobic outer wall layer and showed that the solubleconidial wall fraction elicited lymphocyte proliferation inlymph node cells from (BALB/c DBA/2)F1 (CD2F1) micethat had been immunized with live spherules of the attenuatedstrain 95–271 and then boosted with FKS. When analyzed byadvancing-line immunoelectrophoresis with a reference coc-
cidioidin/burro anti-coccidioidin system that had been devel-oped by Huppert and et al. (147), at least 10 antigens wereidentified, notably Ag2 and an antigen designated AgCS.When analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), at least 14 bands were detected.The soluble conidial wall fraction showed reactivity in immu-nodiffusion assays for antibodies against the CF and TPantigens.
Kirkland et al. (169) subjected the soluble conidial wallfraction to SDS-PAGE and electrotransferred the bands tonitrocellulose membranes. The antigen-bearing nitrocellulosemembranes were cut into strips and tested for their reactivityin a T-cell proliferation assay using a soluble conidial wallfraction-specific murine T-cell line derived from lymph nodecells of immunized (BALB/c DBA/2)F1 mice. A 43- to 66-kDa region was found to be the most stimulatory. In concom-itant experiments, the investigators developed a lambda gt11mycelium-phase cDNA library and, using antiserum to thesoluble, conidial wall fraction, cloned a 200-bp cDNA insertthat encoded a protein which was shown to induce prolifera-tion in the soluble conidial wall fraction-specific T-cell line.This T-cell-reactive protein (TCRP) was cloned by Wyckoff etal. (316). The tcrP gene contains a 1,197-bp ORF that encodesa hydrophobic 45.2-kDa protein with 50% identity and 70%homology to a mammalian cytoplasmic enzyme, 4-hydroxyphe-nylpyruvate dioxygenase, which is involved in the degradationof phenylalanine to tyrosine, and a similar identity and homol-ogy to mammalian F antigen, which is a major epitope of themammalian liver. The gene does not contain a signal sequenceor GPI anchor site.
Kirkland et al. (168) ligated the tcrP gene with the pET28bplasmid and expressed the construct in E. coli. The 48-kDarecombinant fusion protein was used to induce anti-TCRPserum in rabbits, and experiments using the antiserum showedthat the antigen was expressed in the cytosolic fraction ofparasite-phase cells. The rTCRP stimulated proliferation andproduction of IFN-� in lymph node T cells from FKS-immu-nized BALB/c mice. However, the rTCRP was only weaklyprotective when evaluated for protective efficacy in rTCRP-immunized BALB/c mice that had been challenged by the i.p.route.
The relatively low protective capacity of rTCRP and its ho-mology to mammalian proteins argue against its use as a com-ponent of a vaccine against Coccidioides.
Spherule outer wall. When spherules are grown in liquidConverse medium, they shed a lipid-rich, membranous outerwall material which may be similar to the extracellular matrixdescribed by Frey and Drutz (123). Cole et al. (57) extractedthe spherule outer wall (SOW) fraction of Coccidioides strainsC634 and C735 by detergent extraction with the nonionic de-tergent N-octyl-D-glucopyranoside (OG). Tandem 2D-IEP ofthe OG-soluble fraction revealed the presence of Ag2, AgCS,and at least two other unidentified components. When assayedby SDS-PAGE, three Coomassie blue-stained polypeptidebands of 66, 58, and 14 kDa were detected. A fourth band,determined to have a size of 19 kDa, was not well stained. Inimmunoblot assays, the OG-soluble fraction showed two bandsthat were reactive with sera from coccidioidomycosis patients.These bands corresponded to the 66- and 19-kDa componentsobserved in SDS-PAGE. The isolated SOW and OG-soluble
fraction elicited proliferation in lymphocytes from immunized(BALB/c DBA/2)F1 mice, and in a separate study, SOW wasfound to protect BALB/c mice and C3H mice against i.p.challenge with 50 arthroconidia (168). The extract was alsoreactive with sera from coccidioidomycosis patients in anELISA and showed reactivity with the TP antigen in the IDTPassay (57).
Subsequently, Hung et al. (142) extracted the SOW materialof Coccidioides strain C735 with Triton X-114 and subjectedthe aqueous phase to cold-acetone precipitation. Neither Ag2nor AgCS was detected in the aqueous extract of the TritonX-114 fraction of SOW; instead, two major glycoproteins, withapproximate molecular masses of 58 and 82 kDa in SDS-PAGE, were detected. When the SOW preparation from Coc-cidioides strains C634 and Silveira were extracted with TritonX-114, the aqueous phase of the Triton X-114 extracts showedbands of 66 and 58 kDa, respectively. N-terminal amino acidsequencing showed that the bands all had identical sequences,and periodic acid-Schiff staining showed that all were glyco-peptides. Recombinant SOW glycoprotein (SOWgp) protectedBALB/c mice against i.p. infection, stimulated the proliferationof human peripheral mononuclear cells from healthy skin test-positive persons, and induced IFN-� production (Fig. 13). Im-munofluorescence using antibody prepared against the 58-kDa SOWgp of strain Silveira reacted with SOWgp of otherCoccidioides strains and, when used in immunofluorescencemicroscopy, detected SOWgp58 in spherules but not myceliumphase cells. The latter results indicate that the SOWgp is par-asite-phase specific. The SOWgp detected antibody in the seraof coccidioidomycosis patients and were also reactive with serafrom patients with histoplasmosis or blastomycosis, althoughthe heterologous reactions were weaker than those obtainedwith sera from coccidioidomycosis patients.
The gene that encodes SOWgp from strain C735 was clonedfrom a Coccidioides genomic library by using a PCR-generatedprobe (142, 163). The SOWgp gene contains an ORF of 1,266bp, which translates into a 422-amino-acid protein with a 20amino-acid-signal sequence on the N terminus and a GPI an-chor signal consensus sequence. The predicted molecular sizefor the translated mature protein is 39.5 kDa, which is muchsmaller than the 82 kDa for the native glycoprotein, a differ-ence thought to be attributable to the high proline content.When the cDNA sequences of the SOWgp genes from threeCoccidioides isolates were compared, differences were noted inthe number of proline-rich tandem repeats, which ranged fromfour to six. The SOWgp gene was expressed in E. coli, andantiserum to the rSOWgp was used in immunoblot analyses toexamine the expression of SOWgp during the growth of Coc-cidioides in vitro. The highest level of expression was observedin presegmented spherules, with lower expression observedduring spherule maturation and none detected in mycelium-phase cells. Northern hybridization confirmed that the SOWgpgene is parasite-phase specific (163).
Recombinant SOWgp was used to immunize BALB/c mice.Subcutaneous immunization with 15 �g of rSOWgp in IFA wasfollowed by two booster immunizations, given 2 and 4 weekslater in CFA. When challenged with 50 arthroconidia via thei.p. route and sacrificed 2 weeks later, 6 of the 10 mice vacci-nated with rSOWgp58 were reported to have cleared Coccid-ioides from the lungs, 2 of 10 mice had cleared the fungus from
the spleen, whereas none of the control mice immunized withbovine serum albumin in IFA/CFA had cleared their infection(Fig. 13). The rSOWgp-vaccinated BALB/c mice expressedIFN-� but not IL-5 or IL-10 mRNA in their spleen cells. Hunget al. (143) recently reported on the role of SOWgp as anadhesin to host extracellular matrix proteins (laminin � fi-bronectin � collagen) and a possible virulence factor inCoccidioides.
Although rSOW appeared to have potential as an immuno-protective antigen, no further studies of its vaccine capacityhave been reported. Rather, Cole (55) recently reported thatSOWgp probably will not play a useful role in vaccine devel-opment because it stimulates a biased Th2 pathway of immuneresponse, as indicated by cytokine analyses. This new informa-tion is difficult to interpret in light of the reported Th1 cytokineresponses and protection induced in SOWgp-vaccinatedBALB/c mice (163).
Urease. In vitro growth of mycelium- and spherule-phasecells of Coccidioides results in the release ammonia and aconcomitant increase in the pH of the culture medium (163),which may result from urea hydrolysis. It has been speculatedthat the enhanced production of ammonia by endospores in anacidic environment may be related to their ability to survivewithin the phagolysosome. Another hypothesis implicates ure-ase in the pathogenicity of Coccidioides on the basis that col-onization of host tissue by the fungus results in the formationof abscesses with an alkaline pH, which could compromise hostdefenses (163, 200).
Yu et al. (319) cloned the gene that encodes urease by usingdegenerate sense and antisense oligonucleotide primers de-signed on the basis of consensus sequences for URE genesfrom other organisms. The cloned Coccidioides URE gene iscomposed of a 2,517-bp ORF that encodes an 839-amino-acidprotein with a predicted molecular size of 91.5 kDa. The trans-lated URE protein showed a high sequence similarity to re-ported URE proteins of Cryptococcus neoformans, Schizosac-charomyces pombe, Canavalia ensiformis (jack bean),Helicobactor pylori, Klebsiella aerogenes, Bacillus pasteurii, andProteus mirabilis. Expression of the URE gene is highest duringthe endosporulation phase of the parasitic cycle (163).
BALB/c mice were immunized with rURE, together withsynthesized unmethylated CpG-containing oligonucleotides(CpG-ODN), which functions as a potent adjuvant in the in-duction of Th1 responses (discussed below), and IFA. Splenicand lymph node T cells from rURE-immunized, noninfectedmice proliferated in response to in vitro stimulation with rURE(189). Control mice, immunized with bovine serum albuminplus CpG-ODN and IFA, did not show a proliferative re-sponse. In vitro-stimulated T cells from mice immunized withrURE and CpG-ODN plus IFA-immunized mice also ex-pressed IFN-� and IL-2 mRNAs, but not the Th2-associatedcytokines IL-10, IL-5, and IL-4. Production of the Th1-associ-ated cytokines IFN-� and IL-2 was also demonstrable in lungsof immunized infected mice. BALB/c mice immunized withrURE plus CpG-ODN in IFA and then challenged by the i.p.route with 100 arthroconidia of strain C735 were significantlyprotected, with a 44% survival, in contrast to mice that wereimmunized with bovine serum albumin plus CpG-ODN inIFA, which all died. Protection was dose dependent, with sur-vival on day 40 post-challenge of only 25% of mice immunized
with 5 �g of rURE, in contrast to 44% of mice immunized with30 �g (189). It is particular noteworthy that sera from miceimmunized with rURE plus CpG-ODN in IFA showed a pre-dominant Th1-associated IgG2a antibody response when as-sayed 12 days after i.p. challenge. This is in contrast to thepredominant Th2-associated IgG1 response to Ag2/PRA, FKS,and SOWgp.
Immunization of BALB/c mice with the URE gene, ex-pressed by the pSecTag2A.URE plasmid construct, induced aneven higher level of protection, with survival of 10 (83%) of 12mice on day 40 after i.p. challenge with 100 arthroconidia,compared to survival of 2 (17%) of the 12 mice immunized
with the vector alone (Fig. 14). Moreover, 8 of the 10 survivorsimmunized with the URE cDNA showed sterile lungs andspleens whereas both of the control mice that survived thechallenge were culture positive for Coccidioides. As with therURE, mice immunized with the URE gene showed strongT-cell proliferation in vitro, in response to rURE, and pro-duced IFN-� and IL-2.
Native urease is a cytosolic enzyme (200) but is detected inbroth culture filtrates of the saprobic and parasitic phases after5 to 6 days of growth. The URE gene does not contain a signalsequence; hence, urease release most probably occurs whenthe fungal cells undergo autolysis. Mirbod (200) examined the
FIG. 13. Evaluation of SOW and SOWgp as candidate vaccine antigens. (A) Fungal load, expressed as log10 CFU, in lung tissue of SOW-immunized and nonimmunized BALB/c mice. (B) Proliferation response of peripheral blood mononuclear cells from healthy skin test-positive ornegative persons after in vitro stimulation with SOWgp58. (C) Structure of the SOWgp gene isolated from strain C735. (D) Expression of IFN-�mRNA but not IL-5 or IL-10 mRNAs in spleen cells from SOWgp-immunized versus nonimmunized mice. (E) Ability of rSOWgp58 to inducesterilizing immunity in BALB/c mice. The mice were immunized with rSOWgp58 in IFA and then CFA and challenged with 50 arthroconidia viathe i.p. route 42 days after the last immunization. Reprinted from reference 163 with permission of the publisher.
effect of the urease inhibitors, which included acetohydroxamicacid, boric acid, and thiourea, on the urease activity of thepurified enzyme. All of these inhibited urease activity. How-ever, when acetohydroxamic acid was added directly to freshmedia inoculated with mycelial mat obtained from 4-day-oldcultures in glucose-yeast extract, urease activity was decreasedbut ammonium production was only slightly decreased. Hence,urease activity does not account for all of the ammonia pro-duced by the fungus. Importantly, urease inhibitors did notalter the growth of the fungus. These results, coupled with thehigh sequence homology between Coccidioides urease and ure-ases from other sources, including eukaryotic ureases, generateskepticism that rURE or the URE DNA will be a safe andeffective vaccine.
HSP60. Thomas et al. (290) cloned and expressed the genethat encodes Coccidioides heat shock protein 60 (HSP60) byusing degenerate sense and antisense oligonucleotide primersdesigned on the basis of consensus sequences from HSPs fromHistoplasma capsulatum and Saccharomyces cerevisiae. Thecloned hsp60 gene has a 1,782-bp ORF which encodes a 594-amino-acid protein of 62.4 kDa. The latter has 78 to 83%sequence homology to HSP60 proteins from other fungi. Byusing antiserum raised against the rHSP60 as a probe in im-munoblots and immunofluorescence microscopy, HSP60 wasdetected in the cell wall and cytosolic fractions of parasiticcells. The translated protein showed a 72% identity to theHSP60 proteins of H. capsulatum, Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Homo sapiens.
In vitro T-cell proliferation assays of rHSP60-immunizedBALB/c mice revealed that the rHSP60 was a potent T-cellstimulant (290). Li et al. (189) examined the protective capac-ity of the rHSP60 in comparison with rURE, both adminis-tered with CpG-ODN and IFA. rHSP60 was much less effec-tive as a protective antigen, with only a 16% survival after i.p.challenge. Moreover, the rHSP60 fusion protein elicited a pre-dominant Th2 response in immunized infected BALB/c mice,
as judged by expression of IL-4, IL-5, and IL-10 mRNAs inlung homogenates.
Coccidioides-specific antigen. Early studies by Kaufman andStandard (159) established that Coccidioides produces a heat-stable exoantigen that is specific for this fungus. The exoanti-gen was demonstrable in culture filtrates of all Coccidioidesstrains examined and was not detected in extracts derived fromheterologous fungi, including such pathogens as Histoplasmacapsulatum and Blastomyces dermatitidis. Cox and Britt (73)purified the exoantigen and produced monospecific antiserum.When examined by line immunoelectrophoresis, the exoanti-gen was identified as antigen 11 in the CDN/burro anti-CDNreference system described by Huppert et al. (145) and waspresent in culture filtrates of both mycelia and spherules (72).In 1995, Pan and Cole (210) reported cloning the gene thatencodes this Coccidioides-specific antigen (CSA) and estab-lished that the antigen was a 19-kDa serine proteinase.
Recently, Yu et al. (J. Yu, L. F. Shubitz, T. Peng, K. Osborn,T. N. Kirkland, G. T. Cole, and J. N. Galgiani, Abstr. 103rdGen. Meet. Am. Soc. Microbiol. 2003, poster F-111, 2003)expressed the CSA gene in E. coli and reported that the re-combinant antigen administered with CpG in monophosphoryllipid-oil (MPL) protected C57BL/6 mice against i.p. challenge.More importantly, 1 �g of rCSA given with CpG/MPL alsoprotected C57BL/6 mice against i.n. challenge, with a 30%survival at 60 days postinfection. Coimmunization of the micewith 1 �g of rCSA and rAg2/PRA(1–106) increased survival to90%. Although this is a preliminary report, it provides evi-dence to support the contention that a multivalent recombi-nant vaccine would be more effective than a univalent vaccine.
GEL-1. With the advent of the genetic sequence of oneCoccidioides strain (C. posadasii, strain 735) being determinedat The Institute for Genomic Research (TIGR), it is possibleto utilize the predicted proteins to evaluate motifs that mightbe indicative of potential vaccine candidates. Delgado et al.(89) have recently reported the discovery of a new vaccinecandidate by using a strategy to identify proteins with GPIanchors to characterize potential cell wall-associated proteins.This line of experiments follows results by Kong et al. indicat-ing that the most protective subfraction of FKS was associatedwith the cell walls as opposed to the cytosol fraction (172).With this method, a �-1,3 glucanosyltransferase, termedGEL-1, was identified and cloned and tested for immunogeniccapacity. The predicted structure consisted of a 447-amino-acid protein with an 18-amino-acid signal peptide and a 34-amino-acid GPI anchor with two N-glycosylation sites. An E.coli-expressed recombinant protein was produced and used toimmunize BALB/c and C57Bl/6 mice with CpG oligonucleo-tides in IFA. Immunization with as little as 1 �g of rGel1protein protected BALB/c mice against i.p. challenge with 100arthroconidia. The protective capacity of rGel1 in BALB/cmice challenged with higher doses of arthroconidia was nottested, nor did the investigators determine whether rGel1 pro-tected BALB/c mice against the more rigorous pulmonaryroute of challenge. The latter test was conducted with the moreresistant C57BL/6 strain, however. Immunization of C57Bl/6with 1 �g of rGel-1 with CpG-ODN in IFA led to increasedsurvival after pulmonary challenge with 80 arthroconidia, incontrast to control mice immunized with PBS plus CpG in IFA.The investigators reported that rGel1 elicited IFN-� and a
FIG. 14. Vaccine efficacy of rURE against i.p. challenge. BALB/cmice were immunized with rURE plus CpG-ODN and IFA or, for acontrol, with bovine serum albumin (BSA) plus CpG-ODN and IFA.The mice were challenged with 100 arthroconidia via the i.p. route.Survival was determined on days 1 through 40 postinfection for groupsof 12 mice. Reprinted from reference 189 with permission.
predominant IgG2a antibody response, consistent with the in-duction of a protective Th1 pathway of immune response.
Coccidioides Gel1 shows a 90% similarity to the translatedsequence of Aspergillus fumigatus Gel1. Similarities were alsoobserved with glucanosyltransferase sequences of other fungi,included Candida albicans and C. glabrata. The CoccidioidesGel1 mRNA was expressed at the highest level during theendosporulation stage of the parasitic phase; by immunostain-ing, the protein was localized to the endospore cell surface,consistent with its presence in the cell wall.
ELI-Ag1. Our efforts at new vaccine candidate discoveryhave utilized expression library immunization (ELI) (17). Thisis a method for identifying genes within the genome of apathogen that are capable of inducing protective immunity inthe host. The procedure capitalizes on the facts that all theprotein antigens of a pathogen are encoded by the genomeand, second, that genetic immunization is a highly efficaciousmethod for inducing immune responses. The overall strategy isto divide the genome into sibling sublibraries, test each subli-brary for its ability to induce immunity, and then divide pro-tective sublibraries and repeat the process until the gene orgenes are identified. We modified the original process describeby Barry et al. and utilized RNA from Coccidioides spherulescultured in RPMI 1640 tissue culture medium to create acDNA library. To ensure that the results were not attributed toAg2/PRA, the initial unamplified library was plated at approx-imately 100 plaques per 150-mm plate and plaque lifts werescreened for the presence of Ag2/PRA-positive clones. Fromthe initial 15 100-plaque libraries, 3 contained Ag2/PRA andwere not included in further testing. The plaques from theremaining plates were amplified, excised into the pBKCMVplasmid vector, and stored for analysis. An initial series ofexperiments screened 10 of the initial pools, and 2 of thesepools provided significant protection against i.p. challenge.The pool providing the highest level of protection (59% sur-vival over a 35-day survival curve; P � 0.001) was subsequentlysubdivided and used to immunize mice with sequentiallysmaller pools of genes, leading to the discovery of a singlegene, termed ELI antigen 1 (ELI-Ag1) (150) (Fig. 15).
The deduced amino acid sequence of the ORF of the cDNAshowed that the protein, of unknown function or homology toother known proteins in GenBank, is a 224-amino-acid proteinwith an 18-amino-acid signal peptide and a 15-amino-acid GPIanchor. There are four potential O-glycosylation sites, four N-glycosylation sites, and a chitin binding domain between aminoacids 141 and 155. The cumulative data support the notion thatthis protein would be a cell wall-associated protein. The resultsin Fig. 15 show the survival curve of the fourth-level screen,representing mice immunized with the individual genes fromthe last protective pool of 10 genes. The highest level of pro-tection, equivalent to the complete pool of 10 genes, was pro-vided by the single clone encoding ELI-Ag1. To our knowl-edge, this was the first report of ELI leading to theidentification of a single protective gene for vaccination againsta fungal disease.
Studies are under way to express ELI-Ag1 in a eucaryoticexpression system and to test the recombinant protein for itsvaccine capacity, alone and in combination with other vaccinecandidates, in BALB/c mice challenged by a pulmonary route.
Studies are also needed to assess the host response toELI-Ag1.
Critical Comments
Of the vaccine candidates tested in the murine model, nonesurpass the FKS vaccine and only the 27K cellular extractapproaches the high level of protection of FKS in the mostsusceptible BALB/c mouse strain. We think that a vaccinecandidate should be taken to human clinical trials only after itis shown to protect the most susceptible mouse strain againstpulmonary challenge. Of the single-antigen candidates, Ag2/PRA is the most extensively studied and its protective capacityhas been reproduced in multiple laboratories. However, as arecombinant protein vaccine, Ag2/PRA-induced protectionhas been shown convincingly only against pulmonary challengein intermediately susceptible C57BL/6 mice. Other antigensare in the early phases of discovery and testing, and it is hopedthat new candidates will soon be proven in multiple laborato-ries to the level of that seen for Ag2/PRA. The current thoughtis that the next human coccidioidomycosis vaccine will be amultivalent composite of several immunodominant proteins.Therefore, new candidates need to be discovered. While DNA-based immunization is an efficient tool for vaccine discoveryand rapid testing, it is doubtful that a DNA-based vaccine willbe used against coccidioidomycosis. Thus, the eventual vaccineproduct will require the production of a recombinant pro-tein(s). In addition to the vaccine candidate(s) itself, the ad-juvant system used for immunization has a profound impact onthe protection observed (see below). It would also be of greatbenefit to observe a protective response in an animal modelother than the mouse. The cumulative results are that signifi-cant progress has been made for vaccine discovery againstCoccidioides but that much work is left before we can reliablyand reproducibly produce protection in the most susceptibleanimals.
FIG. 15. Identification of ELI-Ag1 as a vaccine candidate. BALB/cmice were immunized with individual clones from a pool of genesshown to protect mice from a systemic challenge of 2,500 arthro-conidia. The screen started with a pool containing 100 genes from acDNA library derived from endosporulating spherules of Coccidioides.The most protective clone, designated ELI-Ag1 (Œ), is shown in com-parison. Pool 7-3-5 (f), from whence ELI Ag1 was derived, and thevector control (●) are depicted by black lines. The remaining nonpro-tective clones are depicted by gray circles. Reprinted from reference150 with permission of the publisher.
Emphasis on vaccine development has shifted in recentyears from the use of live attenuated organisms and purifiedsubcellular components to the use of recombinant protein sub-units, synthetic peptides, protein polysaccharide conjugates,and plasmid DNA. These new-generation vaccines are likely tobe less reactogenic than traditional vaccines but are also lessimmunogenic. Given that a soluble protein (or peptide) orrecombinant peptide vaccine will not by itself induce cellularimmune responses, it is imperative that particular attention bepaid to selecting the optimal adjuvant for delivering the pep-tide to APCs. Consideration must be given to selecting anadjuvant that is approved, or under consideration for approval,for use in humans. Equally important, the vaccine must be ableto induce immunity against pulmonary challenge. The vastmajority of studies conducted with coccidioidal antigens orgene vaccines have been performed using the i.p. route. Whilethese experiments provide valuable information in regard toselecting potential vaccine candidates, the rigorous test will beto establish that protection is engendered against pulmonarychallenge.
Currently, the only adjuvants licensed for human use by theU.S. Food and Drug Administration are aluminum-based min-eral salts (generically called alum), namely, aluminum hydrox-ide and aluminum phosphate. Aluminum compounds prefer-entially skew the immune response toward a Th2 responsecharacterized by the secretion of IL-4 and IL-5 and the gen-eration of IgG1 and IgE. Th1 responses are required, however,for host defense against diseases that are dependent on cell-mediated immunity for protection, for example, coccidioid-omycosis, tuberculosis, malaria, HIV infection, leishmaniasis,and histoplasmosis. Although CFA has been a standard andeffective adjuvant for driving Th1 responses in experimental-animal models, it is reactogenic and thus is unacceptable foruse in humans. More recently developed Th1-type adjuvants,notably MPL, CpG-ODN, and QS-21, have not been licensedfor use in humans but have been evaluated in preclinical trialswith humans; in experimental-animal models, these adjuvantshave proved to be superior to alum and superior or compara-ble to CFA in the induction of humoral and cell-mediatedimmune responses (28, 40, 85, 156, 160, 201).
The identification of TLRs and their importance in innateand adaptive immunity have provided new insights into adju-vant design. Within minutes of recognizing pathogen-associ-ated molecular patterns, an array of cell types initiate defensemechanisms, including the production of reactive oxygen in-termediates and secretion of inflammatory cytokines, includingIL-1, TNF-�, IL-12, and IFN-�. These cytokines activate NKcells and also initiate a cascade of signals to cells of the adap-tive immune response. Pharmacological manipulation of in-nate immune responses might lead to more effective vaccines,as well as novel therapeutic strategies. While it is not within thescope of this review to present an in-depth discussion of adju-vants, a brief overview is provided of adjuvants that have effi-cacy in potentiating cellular immune responses and are cur-rently in, or close to being in, clinical trials with humans.
MPL adjuvant. The first microbial product discovered tobind TLRs was lipopolysaccharide (LPS) (234). Although LPSis a potent adjuvant for protein and carbohydrate antigens andinduces both humoral and cellular immunity, its toxicity pre-cludes its use in human vaccines (155). Investigators chemicallymodified lipid A by uncoupling its toxic effects while retainingits immunostimulatory effects (156). The resulting product,monophosphoryl lipid A (MLA), was further modified by hy-drolysis of the �-hydroxymyristoyl residue attached to the3-position of the reducing sugar, and the product was desig-nated MPL. This adjuvant has been administered to over12,000 persons and has resulted in minimal side effects. Evi-dence suggests that LPS, lipid A, MLA, and MPL bind TLR4(156, 234). Two formulations of MPL that have been used areMPL-SE, which is MPL, a naturally derived disaccharide ad-juvant of Salmonella enterica serovar Minnesota plus squalene,and MPL-AF, which is an aqueous micellar suspension of MPLdispersed in dipalmitoyl phosphatidylcholine. MPL-AF aug-ments humoral and cellular immune responses to vaccinesadministered by the i.n. route. In preclinical trials with over10,000 healthy persons, MPL was shown to have an acceptabletolerability and was effective in inducing IL-2, IFN-�, and cy-totoxic T cells (272). In a direct comparison of the vaccineefficacy of rAg2/PRA in MPL-SE versus rAg2/PRA in CFA,mice immunized with the former were significantly more pro-tected than mice immunized with the latter after i.n. challengewith 30 arthroconidia (unpublished data). Moreover, adminis-tration of rAg2/PRA in MPL-AF adjuvant significantly pro-tected BALB/c and C57BL/6 mice against pulmonary chal-lenge with Coccidioides (discussed above).
CpG-OGN. Microbial but not vertebrate DNA contains un-methylated cytosine-phosphate-guanine (CpG) dinucleotideswith specific flanking regions that are recognized by cells of theinnate immune system to allow the discrimination of pathogen-derived DNA from self DNA. In mice, the optimal stimulatoryCpG motif consists of two 5� purines and two 3� pyrimidines,with the most potent being 5�-GACGTT-3� (178). These motifsrapidly stimulate murine macrophages, DCs, and B cells tosecrete proinflammatory cytokines such as IL-1, TNF-�, IL-6,IL-12, and IL-18 and stimulate the IL-12 secondarily activatedmurine NK cells to secrete IFN-� and to have increased cyto-toxic activity. Recent studies have shown that unmethylatedCpG triggers innate immunity through TLR9 (139). Thus, onemechanism by which CpGs trigger Th1 responses is by bringingabout the maturation of immature DCs into mature APCs.
Although CpGs have been evaluated mainly in rodents, re-cent investigations by Hartmann et al. (138) showed that aCpG phosphorothioate ODN with a TpC dinucleotide at the 5�end followed by three 6-mer CpG motifs (5�-GTCGTT-3�)separated by TpT dinucleotides was most effective in stimulat-ing primate immune cells. Preclinical trials with humans haveshown impressive immunomodulatory effects and limited tox-icity with CpG-OGN-formulated cancer vaccines (40). An-other notable feature of CpG-OGN adjuvants is that they areuseful for delivering both recombinant (or native) antigens andgene vaccines. This provides a means of directly comparing thelevel of protection achieved by a given peptide vaccine versusits DNA. Thus far, the only published study using CpG-OGNsin studies of Coccidioides vaccine candidates was by Li et al.(189). As discussed above, both rURE and the URE gene
vaccine induced protective immunity when administered withCpG-OGN, but the level of immunity was significantly higherwith the gene vaccine. Unfortunately, the protective effect ofthe vaccine in CpG-OGN was not assessed by using a pulmo-nary challenge.
QS-21. QS-21 is a natural saponin adjuvant from the bark ofthe Chilean tree Quillaja saponaria. It is a water-soluble trit-erpene glycoside with amphiphilic character and can be mixedwith a soluble antigen, resulting in a fully soluble vaccine, or itcan be combined with emulsion or mineral salts adjuvants. Theadjuvant is highly effective in enhancing both Th1 cytokines(IL-2 and IFN-�), antibodies of the IgG2a isotype, and cyto-toxic T-lymphocyte (CTL) activity (161). Saponins intercalateinto cell membranes, which is thought to allow antigens to gainaccess to the cytoplasm for the induction of CTL. The capacityof QS-21 to induce CTL activity makes it an optimal candidatefor vaccines against pathogens that require a potent CTL re-sponse. Although CTL activity has not been demonstrated incoccidioidomycosis, the intracellular parasitism of macro-phages by Coccidioides endospores warrants studies to explorethis response.
QS-21 has been tested in more than 3,000 patients in 60clinical trials, with transient severe pain being noted in fourtrials where 200 �g of adjuvant was used but fewer side effectsat lower doses (272). Studies are under way to reduce the sideeffects by chemical alterations or reformulation. QS-7 is onesuch reformulation and has been shown to be active in stimu-lating IgG2a and CTL responses. ASO2 is a proprietary emul-sion containing MPL and QS-21; while it has been highlyeffective in clinical trials, it has several side effects, includingheadaches, myalgia, local pain, and systemic chills (160). Asyet, neither QS-21 nor ASO2 has been used in studies ofCoccidioides vaccines.
Microparticles. Microparticles composed of polylactide-co-glycolides (PLGs) are biodegradable and biocompatible poly-esters that have been used in humans as suture material andfor controlled release of drugs (272). PLG microparticles ap-pear to mediate their immunopotentiating effect as a conse-quence of their uptake by DCs and macrophages, and they canbe used with recombinant peptide or DNA vaccines. Anionicmicroparticles are used for the delivery of adsorbed proteins,whereas cationic microparticles are used for the delivery ofDNA vaccines alone or with CpG-OGN (271). These protein-or DNA-adsorbed microparticles have proven effective in in-ducting Th1 and CTL responses in mice, guinea pigs, andnonhuman primates. The biodegradation rate of PLG micro-spheres is a major advantage since it allows a single-dose im-munization (247).
Route of Immunization
Although most vaccines have been administered by an i.m.or s.c. route, administration by the mucosal route has a numberof important advantages. Immunization by the i.n. route is stillin exploratory stages but offers the advantage of targeted de-livery of the vaccine directly to lung cells, where the specializedbronchus-associated lymphoid tissue, the nasal-associated lym-phoid tissue, and the intraepithelial DCs within the lower air-ways would be directly exposed to the vaccine. Another impor-
tant advantage of mucosal delivery is that it avoids the use ofneedles, which is highly desirable for human vaccines.
While vaccines delivered by an i.n. route are effective ininducing antibody, oral or i.n. vaccines rarely stimulate effec-tive cellular immune responses. Since most adjuvants have anunacceptable level of toxicity for i.n. immunization, alternativedelivery systems are needed. One strategy that has promise isdelivery of the protein or DNA vaccine to the lung via i.n.instillation of the vaccine coupled to PLG microparticles. An-other strategy to optimize the stimulation of immunity at thelung level, as well as systemic immunity, has been to vaccinatevia an i.m. route and then use the i.n. route for boosting.
CURRENT AND FUTURE DIRECTIONS
Of all the fungal diseases, coccidioidomycosis is the bestsuited for the development and application of a vaccine. Thisis based on the geographically defined regions of endemicity,the well-defined target populations, and the immunizing ca-pacity of the fungus. The advances in technologies and appli-cation of genomics, proteomics, and bioinformatics, together withrapid throughput of identifying immunoreactive epitopes, willgreatly accelerate the achievement of this goal. While a numberof vaccine candidates have been identified and cloned, only the27K preparation induces protection against pulmonary challengeto the level achieved with the FKS vaccine. The 27K vaccine is,however, antigenically heterogeneous, and the protective compo-nent has yet to be identified. Therefore, there continues to be aneed to identify other vaccine candidates for use in a monovalentvaccine or, more probably, a multivalent component vaccine.
The genome of C. posadasii is currently being sequenced atTIGR and, at the time of this writing, has about an 8 cover-age (i.e., the sequence of the genome was covered to a theo-retical eight times) of the estimated 29-Mb of chromosomalDNA. There are approximately 6,000 predicted coding regionswithin the genome, and approximately 75% of the genes have asignificant match in the GenBank nonredundant protein database(T. Kirkland, personal communication). Bioinformatics tools willaccelerate the progress from genome sequencing to vaccine de-sign (86–88, 199, 257, 261). EpiMer and EpiMatrix are computer-driven pattern-matching algorithms that serve to identify T-cell-reactive epitopes. Conservatrix and BlastiMer permit theidentification of protein sequences for highly conserved regionsand homology to other known proteins (257). Although homol-ogy-based genomic analysis can yield misleading results in theassignment of gene function (255), it can narrow the list of puta-tive epitopes to be tested. By using this “epitope-driven vaccinedesign” (86), the epitopes can be screened directly for reactivitywith immune T cells and T-cell subsets to identify those to befurther evaluated in animal models.
Another approach to identifying potential vaccine candi-dates in the Coccidioides genome would be to clone the ORFsand evaluate their protective capacity. One way to avoid thiscolossal task would be to construct linear expression elements(287), which consist of PCR-amplified ORFs linked to a eu-caryotic promoter and terminator, and then evaluate the pro-tective efficacy of the constructs by using ELI (17, 288). An-other method would be to utilize a bioinformatic strategy tosearch for proteins with known immunogenic motifs or GPIanchors that would indicate that they were cell wall associated.
Once antigens have been identified by this strategy, studies canbe undertaken to evaluate their immunoreactivity and protec-tive capacity when administered to experimental-animal mod-els.
One potential problem that may impede vaccine develop-ment in coccidioidomycosis is the possible requirement forposttranslational modification for optimal presentation andprocessing. This may be circumvented by expressing the genesin a yeast system. However, in the case of Ag2/PRA whichcontains 3-O-methylmannose, the machinery for producingthis glycosylated peptide, in a manner corresponding to that ofthe native antigen, may be lacking. An attractive alternativestrategy would be to overexpress the gene in Coccidioides (55),using a tagged gene construct which will enable the translatedpeptide to be purified from other coccidioidal components.
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