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Research in Dr. Cole’s laboratory is focused both on development of human and veterinary vaccines against fungal diseases, and investigations of virulence mechanisms of medically-important fungi. Fungal infections of humans continue to escalate as the number of immunocompromised patients increases. Coccidioides is a human fungal pathogen that can also cause mild to fatal respiratory disease (coccidioidomycosis; San Joaquin Valley fever, desert rheumatism) in immunocompetent individuals. About 30 million people reside in the endemic regions of the United States (West Texas to Southern California), and over 350,000 military personnel are stationed in desert and semi-desert areas of the U.S. where Coccidioides is abundant in the soil.  Inhalation of Coccidioides spores causes symptomatic disease in more than 40% of infected individuals, and is responsible for escalating health care costs for long term antimicrobial treatment of patients who contract this mycosis. Although about 50% of the people exposed to Coccidioides may only experience mild discomfort and do not seek medical intervention, clinical evidence suggests that reactivation of the respiratory disease can occur months to years after the original insult.  Therefore, even people with mild symptoms of Coccidioides infection are at risk of contracting the respiratory disease later in life.  No person-to-person transmission of coccidioidomycosis is known to occur, except in rare cases of materno amesal-fetal transmission.

Rationale for Development of a Vaccine Against Valley Fever

Both clinical and experimental evidence have demonstrated that T-cell immunity is pivotal for effective immune protection against this respiratory disease, and antigen presentation to class II major histocompatibility (MHC II) complexes of CD4+ T lymphocytes is important.  The ability of the host to elicit a strong delayed-type hypersensitivity response to the pathogen is essential.  Two functionally distinct subsets of  T cells, T helper 1 (Th1) and Th2, have been identified and are distinguished by the kinds of cytokines they produce.  The latter are host proteins that help activate lymphocytes and contribute to “cross-talk” between the innate and acquired immune systems.  Th1 cells secrete cytokines that initiate and participate in cell-mediated immunity (CMI), while the Th2 subset of T lymphocytes secrete cytokines that stimulate B-cells to produce antibodies, activate mast cells and eosinophils, and may down-regulate cellular immune responses.  Current evidence in the literature suggests that a rising titer of antibody to Coccidioides antigen signals a poor prognosis of coccidioidal disease.  However, these clinical observations do not necessarily negate the possibility that protective antibodies are produced during the course of infection.  Antibody and complement undoubtedly provide opsonic activity, which enhances phagocytosis of the pathogen by neutrophils and macrophages.  Opsonins have been suggested to contribute to the activation and binding of dendritic cells to fungal pathogens, which results in enhanced antifungal activity.  However, dominance of either a Th1 or Th2 pathway of immune response to systemic fungal infections may lead to the inability of the host to control metastasis of the pathogen, result in an intense inflammation at sites of infection, and culminate in host tissue damage and exacerbation of disease.  It appears that regulation of an effective immune response to Coccidioides and generation of durable protection against coccidioidomycosis require the production of both Th1- and Th2-type cytokines and the establishment of balanced Th1/Th2-directed pathways of immunity. It has been suggested that the interactions of antibody-mediated immunity (AMI) and cell-mediated immunity (CMI) should be viewed in the context of an integrated immune response.  Recovery from symptomatic Coccidioides infection typically confers life-long immunity to reinfection, suggesting that vaccination against coccidioidomycosis is feasible.  No vaccine is currently available to protect humans against this respiratory disease.

A Recombinant Protein Vaccine

We have used an immunoproteomic/bioinformatics approach to the development of a recombinant protein vaccine against coccidioidomycosis. Our focus has been the identification of T-cell antigens, since both clinical and experimental evidence have demonstrated that T-cell immunity is pivotal for defense against this respiratory disease. The fungal cell wall has proved to be a reservoir of immunogenic proteins.  Extracts of the parasitic cell wall of Coccidioides have been shown to protect mice against pulmonary coccidioidomycosis. Immunoblots of electrophoretic separations of a detergent (Triton X-114) extract of the parasitic cell wall identified patient seroreactive proteins which were subsequently excised from the 2D-PAGE gel, trypsin digested, and sequenced by tandem mass spectrometry. The full-length genes which encode the dominant proteins in the immunoblots were identified using the Coccidioides posadasii genome database (www.tigr.org) combined with established methods of bioinformatics. The genes were cloned, expressed, and the recombinant proteins were shown to stimulate immune T cells in vitro. The deduced proteins were predicted to contain epitopes that bind to human major histocompatibility complex class II molecules using a TEPITOPE-based algorithm. Synthetic peptides corresponding to the predicted T-cell epitopes were shown to induce gamma interferon production by immune T lymphocytes . The T-cell-reactive antigens protected C57BL/6 mice against a potentially lethal pulmonary infection with Coccidioides posadasii . This immunoprteomic/bioinformatics approach to the identification of candidate vaccines against coccidioidomycosis has proved to be efficient and productive. The next step of these studies is to incorporate selected T-cell-reactive epitopes into a chimeric vaccine, and test its protective efficacy in transgenic mice which express human MHC II complexes. We argue that such an epitope-driven vaccine can simultaneously direct the host immune response to multiple dominant and subdominant epitopes and, thereby, induce robust and durable protection against coccidioidomycosis.

Virulence Mechanism of Coccidioides

Coccidioides is well equipped with mechanisms to withstand an attack by the sophisticated innate and acquired immune defense systems of the mammalian host. We have also demonstrated that Coccidioides can manipulate signaling of the inflammatory response to its advantage. A pivotal first step for in vivo survival if the microbial pathogen is to complete its first generation of parasitic cell development, which results in a 200 to 300-fold increase in the number of infectious cells (endospores) per inhaled spore (arthroconidium). Coccidioides alternates between an intracellular and extracellular relationship with phagocytes. Arthroconidia are engulfed but poorly killed by the host cells. Endospores enlarge rapidly to 60-120 micron diameter cells, and are then too large to be phagocytosed.

Evasion of Host Detection

A lipid-rich outer layer of the parasitic (spherule) cell wall (SOW) has been suggested to be a major virulence factor. As spherules mature, they synthesize and shed SOW components at their cell surface. The dominant antigenic polypeptide component of SOW is a glycoprotein (SOWgp), which both binds antibody and has been shown to induce a robust immune T-cell response in patients with confirmed coccidioidomycosis. A 47-amino acid repeat sequence within the SOWgp polypeptide contains B-cell-dominant epitopes, and as expected, Coccidioides-infected patients have high titers of antibody to this immunodominant antigen. Exposure of this antigen at the surface of endospores in patients with active disease would be expected to enhance opsonization and phagocytosis. Instead, the endosporse which emerge from ruptured spherules are devoid of SOWgp. Evidence has been provided that expression of an endosporulation stage-specific metalloproteinase (Mep1) is responsible for digestion of SOWgp and for the ability of the fragile endospores to evade host detection.

Modulation of Host Immune Response

Repeated exposure of host innate and acquired immune cells to SOWgp, as well as the products of Mep1 digestion of SOWgp presented during endosporulation, appear to induce a persistent, intense inflammatory response to infection. The latter results in host tissue damage and exacerbation of disease. B cells apparently play an important role in this process by functioning as antigen-presenting cells, and by secreting a cytokine (IL-10) which can down-regulates cell-mediated immunity. Since CMI plays an important role in host protection against Coccidioides, SOWgp-induced immunomodulation compromises host defenses and contributes to pathogen survival in vivo.

Urease Activity Contributes to Coccidioides Virulence

Urease activity during in vitro growth of Coccidioides has been shown to be partly responsible for production of intracellular ammonia released into the culture media, and contributes to the alkalinity of the external microenvironment. Enzymatically-active urease is released from the contents of mature spherules during the endosporulation phase of the parasitic cycle. The endospores, together with the urease and additional material which escape from the ruptured parasitic cells, elicit an intense inflammatory host response. Ammonia production by the spherules of Coccidioides is markedly increased by the availability of exogenous, host-derived urea found in relatively high concentration at sites of coccidioidal infection in the lungs of mice. Direct measurement of the pH at these infection sites revealed an alkaline microenvironment. Disruption of the urease gene of Coccidioides resulted in a marked reduction in the amount of ammonia secreted in vitro by the fungal cells. Intranasal inoculation of BALB/c mice, which are highly susceptible to coccidioidal infection, survived challenge with the urease mutant, revealed a well-organized granulomatous response to infection, and showed better clearance of the pathogen from lung tissue than animals challenged with the parental strain. Ammonia and ezymatically-active Coccidioides urease released from spherules during the parasitic cycle contribute to host tissue damage, which further exacerbates the severity of coccidioidal infection and enhances the virulence of this human respiratory pathogen. A summary of these virulence factors are presented in FIG. 1.

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