D. Mitchell Magee

Coccidioidomycosis: Host Response and Vaccine Development

SUMMARY 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 β-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.

Identification of a protective antigen of Coccidioides immitis by expression library immunization.

Coccidioides immitis is a fungal pathogen of humans and is classified as a Select Agent. We have identified a new potential vaccine candidate for this pathogen using cDNA expression library immunization (ELI). A C. immitis spherule-phase cDNA library containing 800-1000 genes was divided into 10 pools and each was tested for its protective capacity in BALB/c mice against intraperitoneal challenge with 2500 arthroconidia of this dimorphic fungus. The most protective pool, designated Pool 7, was fractionated into five sublibraries, each containing 60 genes, and of these, only Pool 7-3 induced a significant level of protection in mice. Fractionation of Pool 7-3 into six sublibraries, each with 10 genes, yielded a protective fraction, designated Pool 7-3-5. Subsequent fraction of the latter pool into 10 sublibraries, each with one clone, yielded a clone designated 7-3-5-5 that was highly protective. Clone 7-3-5-5 was sequenced and found to contain a 672bp ORF encoding a 224 amino acid protein having a 19 amino acid signal sequence on the N-terminus and a 15 amino acid C-terminal GPI anchor site. The 7-3-5-5 clone, designated ELI-Antigen 1 (ELI-Ag1), showed partial homology with a hypothetical protein from Neurospora crassa. This is the first study to identify a protective antigen from a fungus using ELI, and it is also the first report in which sequential fractionation of an expression library successfully identified a single protective gene.

Role of Signal Sequence in Vaccine-Induced Protection against Experimental Coccidioidomycosis

ABSTRACT The vaccine efficacy of the gene sequence encoding the signal peptide of the antigen known as antigen 2 or proline-rich antigen (Ag2/PRA), an immunodominant antigen present in the cell wall of the fungal pathogen Coccidioides immitis, was investigated in a murine model of coccidioidomycosis. Expression plasmids for Ag2/PRA(1-18) DNA (signal sequence), Ag2/PRA(19-194) DNA (lacking the signal sequence), and Ag2/PRA(1-194) DNA (full length) were inserted in the pVR1012 vector, and the constructs were used to vaccinate the highly susceptible BALB/c mouse strain. Immunization with the signal gene sequence significantly reduced the fungal burden in the lungs and spleens of mice 12 days after intraperitoneal challenge with a lethal dose of 2,500 C. immitis arthroconidia, to a level comparable to the protection induced in mice immunized with the full-length Ag2/PRA(1-194) DNA. The Ag2/PRA(19-194) gene protected mice but to a significantly lower level than the signal sequence or the full-length Ag2 gene. The immunizing capacity of Ag2/PRA(1-18) was not attributable to a nonspecific immunostimulatory effect of DNA, as evidenced by the fact that mice immunized with a frameshift mutation of Ag2/PRA(1-18) were not protected against challenge. Furthermore, a synthetic peptide corresponding to the translated sequence of Ag2/PRA(1-18) DNA protected mice, albeit at a lower level than the Ag2/PRA(1-18) DNA vaccine. The protection induced with the signal gene vaccine correlated with the production of gamma interferon when splenocytes from Ag2/PRA(1-18)-immunized mice were stimulated with recombinant full-length Ag2 and was not associated with the production of anti-Coccidioides immunoglobulin G antibody. This is the first study to establish that a signal peptide sequence alone, administered as a gene vaccine or synthetic peptide, can induce protective immunity against a microbial pathogen.

Coccidioides immitis antigen 2: Analysis of gene and protein

Antigen 2 is a glycosylated protein present in the cell walls of the dimorphic fungus Coccidioides immitis. Using oligodeoxyribonucleotide (oligo) primers based on the sequences of Ag2 cDNA, the gene encoding Ag2 was cloned from genomic DNA derived from the mycelial phase of C. immitis by PCR. Nucleotide (nt) sequence analyses showed a 582 base pair (bp) ORF disrupted by two introns which are 78 bp and 101 bp long. The deduced primary translation product consists of 194 amino acids (aa), contains an N-terminal putative signal sequence to allow transport into the endoplasmic reticulum, and a C-terminal putative signal sequence to enable a GPI anchor addition. Putative GPI anchor/cleavage site and O-glycosylation sites, as well as phosphorylation and myristoylation sites are also present. On the basis of these analyses, we predict that a prepro-Ag2 undergoes a post-translational modification to yield the mature glycosylated Ag2 protein which is anchored on the extracellular plasma membrane of mycelial and spherule-phase cells.

Mycobacterial Membrane Vesicles Administered Systemically in Mice Induce a Protective Immune Response to Surface Compartments of Mycobacterium tuberculosis

ABSTRACT Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471–1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis. IMPORTANCE This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation. This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation.

Efficacy of Antigen 2/Proline-Rich Antigen cDNA-Transfected Dendritic Cells in Immunization of Mice against Coccidioides posadasii

Coccidioides posadasii causes coccidioidomycosis, or Valley fever, in the endemic regions of the Southwestern United States. The susceptibility to C. posadasii infection has been attributed to a decreased Th1 cellular response. APCs, especially dendritic cells (DCs), play an important role in the activation of Th1 response. In this study, we investigated the efficacy of a DC-based vaccine against C. posadasii in a mouse model of coccidioidomycosis. We intranasally immunized C57BL6 mice with syngeneic, bone marrow-derived DCs (JAWS II cells) transfected with a cDNA encoding the protective Coccidioides-Ag2/proline-rich Ag. The immunized mice were lethally challenged with C. posadasii through either an i.p. or intranasal route. Upon necropsy after 10 days of infection, fungal burden in lung and spleen of immunized mice was significantly reduced as compared with the control animals. The lung tissue homogenates of immunized animals showed higher levels of IFN-γ. Histologically, lung tissues of immunized mice were in better condition than the control mice. To further investigate, we studied the biodistribution and trafficking of injected DCs by nuclear imaging techniques. For this purpose, the transfected DCs were radiolabeled with 111In-oxime. Scintigraphic images showed that most of the label remained in the gastrointestinal tract. A significant amount was also observed in lung, but there were negligible circulating 111In label in blood. The results suggest that the DCs have a potent immunostimulatory activity, and immunization with DCs transfected with Ag2/proline-rich Ag-cDNA induces protective immunity against C. posadasii in C57BL6 mice.

High Density Diffusion-Free Nanowell Arrays

Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA) is a robust in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced interspot spacing. To address this limitation, we have developed an innovative platform using photolithographically etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8000 nanowell arrays. This is the highest density of individual proteins in nanovessels demonstrated on a single slide. We further present proof of principle results on ultrahigh density protein arrays capable of up to 24000 nanowells on a single slide.

Coccidioides posadasii infection alters the expression of pulmonary surfactant proteins (SP)-A and SP-D

BackgroundCoccidioidomycosis or Valley Fever is caused by Coccidioides in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in Coccidioides infection are not known.MethodsIn this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of Coccidioides posadasii infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of C. posadasii (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays.ResultsWe found that in lavage fluid samples of C. posadasii infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % (SEM 3.5, p < 0.001), 38 % (SEM 5.8, p < 0.001) and 4 % (SEM 1.3, p < 0.001) of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of C. posadasii protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens.ConclusionOur results suggest that the C. posadasii infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.

Role of B Cells in Vaccine-Induced Immunity against Coccidioidomycosis

ABSTRACT We investigated secondary immunity against coccidioidomycosis by using gene expression microarrays. Surprisingly, a high percentage of B-cell-related genes were associated with protective immunity. A functional confirmation of the importance of B cells against coccidioidomycosis was achieved by demonstrating that vaccination was not fully protective in B-cell-deficient MuMT mice.

Application of Immunosignatures for Diagnosis of Valley Fever

ABSTRACT Valley fever (VF) is difficult to diagnose, partly because the symptoms of VF are confounded with those of other community-acquired pneumonias. Confirmatory diagnostics detect IgM and IgG antibodies against coccidioidal antigens via immunodiffusion (ID). The false-negative rate can be as high as 50% to 70%, with 5% of symptomatic patients never showing detectable antibody levels. In this study, we tested whether the immunosignature diagnostic can resolve VF false negatives. An immunosignature is the pattern of antibody binding to random-sequence peptides on a peptide microarray. A 10,000-peptide microarray was first used to determine whether valley fever patients can be distinguished from 3 other cohorts with similar infections. After determining the VF-specific peptides, a small 96-peptide diagnostic array was created and tested. The performances of the 10,000-peptide array and the 96-peptide diagnostic array were compared to that of the ID diagnostic standard. The 10,000-peptide microarray classified the VF samples from the other 3 infections with 98% accuracy. It also classified VF false-negative patients with 100% sensitivity in a blinded test set versus 28% sensitivity for ID. The immunosignature microarray has potential for simultaneously distinguishing valley fever patients from those with other fungal or bacterial infections. The same 10,000-peptide array can diagnose VF false-negative patients with 100% sensitivity. The smaller 96-peptide diagnostic array was less specific for diagnosing false negatives. We conclude that the performance of the immunosignature diagnostic exceeds that of the existing standard, and the immunosignature can distinguish related infections and might be used in lieu of existing diagnostics.