Coccidioidomycosis: host response and vaccine development

Abstract

Coccidioidomycosis is caused by the dimorphic fungi in the genus Coccidioides. These fungi live as mycelia in the soil of desert areas of the American Southwest, and when the infectious spores, the arthroconidia, are inhaled, they convert into the parasitic spherule/endospore phase. Most infections are mild, but these organisms are frank pathogens and can cause severe lethal disease in fully immunocompetent individuals. While there is increased risk of disseminated disease in certain racial groups and immunocompromised persons, the fact that there are hosts who contain the initial infection and exhibit long-term immunity to reinfection supports the hypothesis that a vaccine against these pathogens is feasible. Multiple studies have shown that protective immunity against primary disease is associated with T-helper 1 (Th-1)-associated immune responses. The single best vaccine in animal models, formalin-killed spherules (FKS), was tested in a human trial but was not found to be significantly protective. This result has prompted studies to better define immunodominant Coccidioides antigen with the thought that a subunit vaccine would be protective. These efforts have defined multiple candidates, but the single best individual immunogen is the protein termed antigen 2/proline-rich antigen (Ag2/PRA). Studies in multiple laboratories have shown that Ag2/PRA as both protein and genetic vaccines provides significant protection against mice challenged systemically with Coccidioides. Unfortunately, compared to the FKS vaccine, it is significantly less protective as measured by both assays of reduction in fungal CFU and assays of survival. The capacity of Ag2/PRA to induce only partial protection was emphasized when animals were challenged intranasally. Thus, there is a need to define new candidates to create a multivalent vaccine to increase the effectiveness of Ag2/PRA. Efforts of genomic screening using expression library immunization or bioinformatic approaches to identify new candidates have revealed at least two new protective proteins, expression library immunization antigen 1 (ELI-Ag1) and a beta-1,3-glucanosyltransferase (GEL-1). In addition, previously discovered antigens such as Coccidioides-specific antigen (CSA) should be evaluated in assays of protection. While studies have yet to be completed with combinations of the current candidates, the hypothesis is that with increased numbers of candidates in a multivalent vaccine, there will be increased protection. As the genome sequences of the two Coccidioides strains which are under way are completed and annotated, the effort to find new candidates can increase to provide a complete genomic scan for immunodominant proteins. Thus, much progress has been made in the discovery of subunit vaccine candidates against Coccidioides and there are several candidates showing modest levels of protection, but for complete protection against pulmonary challenge we need to continue the search for additional candidates.

FIG. 1.

Percent survival of BALB/c and DBA/2 mice on days 1 through 30 after i.n. infection with 10 arthroconidia. Each group consisted of ≥22 mice. Reprinted from reference with permission.

FIG. 2.

Footpad hypersensitivity responses in BALB/c and DBA/2 mice at 3-day intervals after i.n. infection with 10 arthroconidia. Bars depict means and standard errors (SE) obtained with groups of nine or more mice. Reprinted from reference with permission.

FIG. 3.

Levels of IFN-γ (A) and IL-4 (B) in homogenates of lung tissues obtained before and at various times after i.n. challenge. The bars depict means and standard errors obtained with groups of seven or more mice. Reprinted from reference with permission.

FIG. 4.

Therapeutic effects of rIFN-γ treatment of BALB/c mice. The bars depict means and standard errors of log₁₀ CFU per gram of lungs, livers, and spleens from groups of 13 mice treated with 10⁵ U of rIFN- γ or buffer alone at daily intervals beginning on the day before infection and continuing through 12 days postinfection. The mice were sacrificed 13 days after challenge. Reprinted from reference with permission.

FIG. 5.

Protective effect of rIL-12 against i.p. challenge in BALB/c mice. The bars depict the means and standard errors of log₁₀ CFU per gram of lungs, livers, and spleens obtained from groups of 9 or 10 BALB/c mice treated with 0.1 μg of rIL-12 or buffer alone beginning on the day before and continuing for 11 days after challenge. The mice were sacrificed 12 days after i.p. challenge. Reprinted from reference with permission.

FIG. 6.

(A) Survival of FKS-vaccinated and nonvaccinated BALB/c mice on days 1 through 30 after i.n. infection with 100 arthroconidia. Each group consisted of 15 mice. (B) Footpad hypersensitivity in FKS-vaccinated and nonvaccinated BALB/c and DBA/2 mice 15 days after i.n. infection with 10 arthroconidia. The bars depict means and standard errors (SE) obtained with groups of 15 mice. Reprinted from reference with permission.

FIG. 7.

Vaccine efficacy of 27K against i.n. challenge. Swiss Webster mice were immunized with 27K in alum, 27K alone, or alum alone and challenged with 5,000 arthroconidia via an i.n. route. Survival was monitored daily for 75 days post challenge. Reprinted from reference with permission.

FIG. 8.

Vaccine efficacy of 27K against i.n. challenge in susceptible mice. BALB/c mice were immunized with 27K in CFA/IFA or alum CFA/IFA alone and challenged with 30 arthroconidia via an i.n. route. Survival was monitored daily for 45 days after the challenge.

FIG. 9.

Nucleotide sequence and deduced amino acid sequence of cloned Ag2 cDNA. DNA base numbers are on the left, and amino acid numbers are on the right. The underlined amino acid region at the beginning of the N terminus of the Ag2 protein is the signal sequence. The doubly underlined amino acid region contains the tetrapeptide repeats. The underlined amino acid region at the C terminus is the GPI anchor. Reprinted from reference with permission.

FIG. 10.

Vaccine efficacy of rAg2, expressed as a GST fusion peptide, in BALB/c mice challenged by the i.p. route with 250 arthroconidia. Results are expressed as the numbers of CFU (means and standard errors) in the lungs, livers, and spleens on day 12 postinfection (A) and the percent survival in mice on days 1 through 30 postinfection (B). Reprinted from reference with permission.

FIG. 11.

Vaccine efficacy of pVR1012-Ag2 cDNA against i.p. challenge. BALB/c mice were immunized with pVR1012-Ag2 cDNA or pVR1012 alone and then challenged with 2,500 arthroconidia. (A) Fungal load, expressed as log₁₀ CFU/g of tissue, was determined 12 days after challenge. Bars depict means and standard errors obtained with groups of 20 mice. (B) Percent survival was determined on days 1 through 40 for groups of 11 mice. Reprinted from reference with permission.

FIG. 12.

Vaccine efficacy of pVR1012-Ag2 cDNA and FKS against i.n. challenge. BALB/c mice were immunized with pVR1012-Ag2 cDNA, pVR1012 alone, or FKS and then challenged with 50 arthroconidia via the i.n. route. Fungal load, expressed as log₁₀ CFU per gram of tissue, was determined 12 days after challenge. Reprinted from reference with permission.

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 or negative persons after in vitro stimulation with SOWgp58. (C) Structure of the SOWgp gene isolated from strain C735. (D) Expression of IFN-γ mRNA but not IL-5 or IL-10 mRNAs in spleen cells from SOWgp-immunized versus nonimmunized mice. (E) Ability of rSOWgp58 to induce sterilizing immunity in BALB/c mice. The mice were immunized with rSOWgp58 in IFA and then CFA and challenged with 50 arthroconidia via the i.p. route 42 days after the last immunization. Reprinted from reference with permission of the publisher.

FIG. 14.

Vaccine efficacy of rURE against i.p. challenge. BALB/c mice were immunized with rURE plus CpG-ODN and IFA or, for a control, 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 groups of 12 mice. Reprinted from reference with permission.

FIG. 15.

Identification of ELI-Ag1 as a vaccine candidate. BALB/c mice were immunized with individual clones from a pool of genes shown to protect mice from a systemic challenge of 2,500 arthroconidia. The screen started with a pool containing 100 genes from a cDNA library derived from endosporulating spherules of Coccidioides. The most protective clone, designated ELI-Ag1 (▴), is shown in comparison. Pool 7-3-5 (▪), from whence ELI Ag1 was derived, and the vector control (•) are depicted by black lines. The remaining nonprotective clones are depicted by gray circles. Reprinted from reference with permission of the publisher.