Vaccines to Prevent Coccidioidomycosis: A Gene-Deletion Mutant of Coccidioides Posadasii as a Viable Candidate for Human Trials.

Coccidioidomycosis is an endemic fungal infection that is reported in up to 20,000 persons per year and has an economic impact close to $1.5 billion. Natural infection virtually always confers protection from future exposure, and this suggests that a preventative vaccine strategy is likely to succeed. We here review progress toward that objective. There has been ongoing research to discover a coccidioidal vaccine over the past seven decades, including one phase III clinical trial, but for reasons of either efficacy or feasibility, a safe and effective vaccine has not yet been developed. This review first summarizes the past research to develop a coccidioidal vaccine. It then details the evidence that supports a live, gene-deletion vaccine candidate as suitable for further development as both a veterinary and a human clinical product. Finally, a plausible vaccine development plan is described which would be applicable to this vaccine candidate and also useful to other future candidates. The public health and economic impact of coccidioidomycosis fully justifies a public private partnership for vaccine development, and the development of a vaccine for this orphan disease will likely require some degree of public funding.

The WOPR family protein Ryp1 is a key regulator of gene expression, development, and virulence in the thermally dimorphic fungal pathogen Coccidioides posadasii.

Coccidioides spp. are mammalian fungal pathogens endemic to the Southwestern US and other desert regions of Mexico, Central and South America, with the bulk of US infections occurring in California and Arizona. In the soil, Coccidioides grows in a hyphal form that differentiates into 3-5 micron asexual spores (arthroconidia). When arthroconidia are inhaled by mammals they undergo a unique developmental transition from polar hyphal growth to isotropic expansion with multiple rounds of nuclear division, prior to segmentation, forming large spherules filled with endospores. Very little is understood about the molecular basis of spherule formation. Here we characterize the role of the conserved transcription factor Ryp1 in Coccidioides development. We show that Coccidioides Δryp1 mutants have altered colony morphology under hypha-promoting conditions and are unable to form mature spherules under spherule-promoting conditions. We analyze the transcriptional profile of wild-type and Δryp1 mutant cells under hypha- and spherule-promoting conditions, thereby defining a set of hypha- or spherule-enriched transcripts ("morphology-regulated" genes) that are dependent on Ryp1 for their expression. Forty percent of morphology-regulated expression is Ryp1-dependent, indicating that Ryp1 plays a dual role in both hyphal and spherule development. Ryp1-dependent transcripts include key virulence factors such as SOWgp, which encodes the spherule outer wall glycoprotein. Concordant with its role in spherule development, we find that the Δryp1 mutant is completely avirulent in the mouse model of coccidioidomycosis, indicating that Ryp1-dependent pathways are essential for the ability of Coccidioides to cause disease. Vaccination of C57BL/6 mice with live Δryp1 spores does not provide any protection from lethal C. posadasii intranasal infection, consistent with our findings that the Δryp1 mutant fails to make mature spherules and likely does not express key antigens required for effective vaccination. Taken together, this work identifies the first transcription factor that drives mature spherulation and virulence in Coccidioides.

Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease.

Cassava (Manihot esculenta) is an important tropical subsistence crop that is severely affected by cassava brown streak disease (CBSD) in East Africa. The disease is caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Both have a (+)-sense single-stranded RNA genome with a 5' covalently-linked viral protein, which functionally resembles the cap structure of mRNA, binds to eukaryotic translation initiation factor 4E (eIF4E) or its analogues, and then enable the translation of viral genomic RNA in host cells. To characterize cassava eIF4Es and their potential role in CBSD tolerance and susceptibility, we cloned five eIF4E transcripts from cassava (accession TMS60444). Sequence analysis indicated that the cassava eIF4E family of proteins consisted of one eIF4E, two eIF(iso)4E, and two divergent copies of novel cap-binding proteins (nCBPs). Our data demonstrated experimentally the coding of these five genes as annotated in the published cassava genome and provided additional evidence for refined annotations. Illumina resequencing data of the five eIF4E genes were analyzed from 14 cassava lines tolerant or susceptible to CBSD. Abundant single nucleotide polymorphisms (SNP) and biallelic variations were observed in the eIF4E genes; however, most of the SNPs were located in the introns and non-coding regions of the exons. Association studies of non-synonymous SNPs revealed no significant association between any SNP of the five eIF4E genes and the tolerance or susceptibility to CBSD. However, two SNPs in two genes were weakly associated with the CBSD responses but had no direct causal-effect relationship. SNPs in an intergenic region upstream of eIF4E_me showed a surprising strong association with CBSD responses. Digital expression profile analysis showed differential expression of different eIF4E genes but no significant difference in gene expression was found between susceptible and tolerant cassava accessions despite the association of the intergenic SNPs with CBSD responses.

Mie Scatter and Interfacial Tension Based Real-Time Quantification of Colloidal Emulsion Nucleic Acid Amplification.

This work demonstrates for the first time rapid, real-time Mie scatter sensing of colloidal emulsion nucleic acid amplification directly from emulsion droplets. Loop-mediated isothermal amplification is used in this study, and, to our knowledge, has not previously been used in a colloidal emulsion platform. Interfacial tension values (γ) associated with bulk protein adsorption and denaturation at the oil-water interface exhibit characteristic changes in the absence or presence of amplification. In the presence of target and amplicon, emulsions maintain a constant 300-400 nm diameter, whereas emulsions formed with no target control show a rapid decrease in droplet diameter to <100 nm over the first 20 min of incubation. This method is validated using whole bacteria (Staphylococcus aureus MSSA and Escherichia coli O157:H7) and whole virus (Potato virus Y and Zika virus) samples suspended in water, buffer, or serum-like matrices. Short-term formation of colloidal emulsion is quantified via 60° scatter monitoring, where the initial slope of scattering intensity is utilized to confirm target amplification in less than 5 min. The unique benefits of this method render it more cost-effective and field-deployable than existing methods, while being adaptable to a multitude of targets, sample matrices, and nucleic acid amplification tests.

A Coccidioides posadasii CPS1 Deletion Mutant Is Avirulent and Protects Mice from Lethal Infection.

The CPS1 gene was identified as a virulence factor in the maize pathogen Cochliobolus heterostrophus Hypothesizing that the homologous gene in Coccidioides posadasii could be important for virulence, we created a Δcps1 deletion mutant which was unable to cause disease in three strains of mice (C57BL/6, BALB/c, or the severely immunodeficient NOD-scid,γc(null) [NSG]). Only a single colony was recovered from 1 of 60 C57BL/6 mice following intranasal infections of up to 4,400 spores. Following administration of very high doses (10,000 to 2.5 × 10(7) spores) to NSG and BALB/c mice, spherules were observed in lung sections at time points from day 3 to day 10 postinfection, but nearly all appeared degraded with infrequent endosporulation. Although the role of CPS1 in virulence is not understood, phenotypic alterations and transcription differences of at least 33 genes in the Δcps1 strain versus C. posadasii is consistent with both metabolic and regulatory functions for the gene. The in vitro phenotype of the Δcps1 strain showed slower growth of mycelia with delayed and lower spore production than C. posadasii, and in vitro spherules were smaller. Vaccination of C57BL/6 or BALB/c mice with live Δcps1 spores either intranasally, intraperitoneally, or subcutaneously resulted in over 95% survival with mean residual lung fungal burdens of <1,000 CFU from an otherwise lethal C. posadasii intranasal infection. Considering its apparently complete attenuation of virulence and the high degree of resistance to C. posadasii infection when used as a vaccine, the Δcps1 strain is a promising vaccine candidate for preventing coccidioidomycosis in humans or other animals.

Comparative genomic analyses of the human fungal pathogens Coccidioides and their relatives.

While most Ascomycetes tend to associate principally with plants, the dimorphic fungi Coccidioides immitis and Coccidioides posadasii are primary pathogens of immunocompetent mammals, including humans. Infection results from environmental exposure to Coccidiodies, which is believed to grow as a soil saprophyte in arid deserts. To investigate hypotheses about the life history and evolution of Coccidioides, the genomes of several Onygenales, including C. immitis and C. posadasii; a close, nonpathogenic relative, Uncinocarpus reesii; and a more diverged pathogenic fungus, Histoplasma capsulatum, were sequenced and compared with those of 13 more distantly related Ascomycetes. This analysis identified increases and decreases in gene family size associated with a host/substrate shift from plants to animals in the Onygenales. In addition, comparison among Onygenales genomes revealed evolutionary changes in Coccidioides that may underlie its infectious phenotype, the identification of which may facilitate improved treatment and prevention of coccidioidomycosis. Overall, the results suggest that Coccidioides species are not soil saprophytes, but that they have evolved to remain associated with their dead animal hosts in soil, and that Coccidioides metabolism genes, membrane-related proteins, and putatively antigenic compounds have evolved in response to interaction with an animal host.

Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae.

Towards the goal of disrupting all genes in the genome of Magnaporthe oryzae and identifying their function, a collection of >55,000 random insertion lines of M. oryzae strain 70-15 were generated. All strains were screened to identify genes involved in growth rate, conidiation, pigmentation, auxotrophy, and pathogenicity. Here, we provide a description of the high throughput transformation and analysis pipeline used to create our library. Transformed lines were generated either by CaCl(2)/PEG treatment of protoplasts with DNA or by Agrobacterium tumefaciens-mediated transformation (ATMT). We describe the optimization of both approaches and compare their efficiency. ATMT was found to be a more reproducible method, resulting in predominantly single copy insertions, and its efficiency was high with up to 0.3% of conidia being transformed. The phenotypic data is accessible via a public database called MGOS and all strains are publicly available. This represents the most comprehensive insertional mutagenesis analysis of a fungal pathogen.

Genomic and population analyses of the mating type loci in Coccidioides species reveal evidence for sexual reproduction and gene acquisition.

Coccidioides species, the fungi responsible for the valley fever disease, are known to reproduce asexually through the production of arthroconidia that are the infectious propagules. The possible role of sexual reproduction in the survival and dispersal of these pathogens is unexplored. To determine the potential for mating of Coccidioides, we analyzed genome sequences and identified mating type loci characteristic of heterothallic ascomycetes. Coccidioides strains contain either a MAT1-1 or a MAT1-2 idiomorph, which is 8.1 or 9 kb in length, respectively, the longest reported for any ascomycete species. These idiomorphs contain four or five genes, respectively, more than are present in the MAT loci of most ascomycetes. Along with their cDNA structures, we determined that all genes in the MAT loci are transcribed. Two genes frequently found in common sequences flanking MAT idiomorphs, APN2 and COX13, are within the MAT loci in Coccidioides, but the MAT1-1 and MAT1-2 copies have diverged dramatically from each other. Data indicate that the acquisition of these genes in the MAT loci occurred prior to the separation of Coccidioides from Uncinocarpus reesii. An analysis of 436 Coccidioides isolates from patients and the environment indicates that in both Coccidioides immitis and C. posadasii, there is a 1:1 distribution of MAT loci, as would be expected for sexually reproducing species. In addition, an analysis of isolates obtained from 11 soil samples demonstrated that at three sampling sites, strains of both mating types were present, indicating that compatible strains were in close proximity in the environment.

Coccidioides posadasii contains single chitin synthase genes corresponding to classes I to VII.

Coccidioides posadasii is a dimorphic fungal pathogen of humans and other mammals. The switch between saprobic and parasitic growth involves synthesis of new cell walls of which chitin is a significant component. To determine whether particular subsets of chitin synthases (CHSes) are responsible for production of chitin at different stages of differentiation, we have isolated six CHS genes from this fungus. They correspond, together with another reported CHS gene, to single members of the seven defined classes of chitin synthases (classes I-VII). Using Real-Time RT-PCR we show their pattern of expression during morphogenesis. CpCHS2, CpCHS3, and CpCHS6 are preferentially expressed during the saprobic phase, while CpCHS1 and CpCHS4 are more highly expressed during the parasitic phase. CpCHS5 and CpCHS7 expression is similar in both saprobic and parasitic phases. Because C. posadasii contains single members of the seven classes of CHSes found in fungi, it is a good model to investigate the putatively different roles of these genes in fungal growth and differentiation.

Coccidioides posadasii contains a single 1,3-beta-glucan synthase gene that appears to be essential for growth.

1,3-beta-Glucan synthase is responsible for the synthesis of beta-glucan, an essential cell wall structural component in most fungi. We sought to determine whether Coccidioides posadasii possesses genes homologous to known fungal FKS genes that encode the catalytic subunit of 1,3-beta-glucan synthase. A single gene, designated FKS1, was identified, and examination of its predicted protein product showed a high degree of conservation with Fks proteins from other filamentous fungi. FKS1 is expressed at similar levels in mycelia and early spherulating cultures, and expression decreases as the spherules mature. We used Agrobacterium-mediated transformation to create strains that harbor DeltaFKS1::hygB, a null allele of FKS1, and hypothesize that Fks1p function is essential, due to our inability to purify this allele away from a complementing wild-type FKS1 allele in a heterokaryotic strain. The heterokaryon appears normal with respect to growth rate and arthroconidium production; however, microscopic examination of strains with DeltaFKS1::hygB alleles revealed abnormal swelling of hyphal elements.