<i>Coccidioides</i> growth cycle and study overview.

<p><i>Coccidioides</i> growth cycle in culture (<b>A</b>), total RNA was collected at 96 hours from hyphae and spherules, which were grown at 30°C and 39°C, respectively. Lifecycle in culture illustration adapted from Delgado <i>et al</i>, 2003 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041034#pone.0041034-Delgado1" target="_blank">[11]</a>. Samples were collected in biological triplicate and the results from <i>C. immitis</i> isolate RS and <i>C. posadasii</i> isolate C735 were compared (<b>B</b>).</p

Top 15 genes with significantly higher expression (up-regulated) in the saprobic phase.

*<p>Fold difference is the mean saprobic/parasitic-phase expression level in <i>C. immitis</i> and <i>C. posadasii</i>.</p

Genes of interest from previous studies.

*<p>Expression ratio: parasitic/saprobic expression. Ratios >1.0 indicate higher expression (up-regulation) in the parasitic phase and ratios <1.0 indicate higher expression (up-regulation) in the saprobic phase.</p>**<p>p-value <0.05.</p

Differentially expressed genes in <i>C. immitis</i> and <i>C. posadasii</i>.

<p>Venn diagrams showing the number of genes commonly differentially regulated in the saprobic vs. parasitic growth phases of <i>C. immitis</i> and <i>C. posadasii</i>.</p

mRNA expression levels of previously identified vaccine candidates [8].

*<p>Parasitic phase expression levels are normalized by library size and gene length.</p>**<p>Differential regulation: Up H: up-regulated in hyphae (saprobic phase), Up S: up-regulated in spherules (parasitic phase).</p

Top 15 genes with significantly higher expression (up-regulated) in the parasitic phase.

*<p>Fold difference is the mean parasitic/saprobic-phase expression level in <i>C. immitis</i> and <i>C. posadasii</i>.</p

Median-difference boxplots.

<p>Median-difference boxplots showing the interquartile range (box with median line) of individual counts from the median count for all genes. Plot “whiskers” extend 1.5 times the interquartile range; outlier points are not shown. Ci: <i>C. immitis</i>, Cp: <i>C. posadasii</i>, H: hyphae (saprobic phase), S: spherule (parasitic phase).</p

Novel adjuvant and vaccine delivery system for enhancement of protective immunity to coccidioidomycosis. (A) An agonist of human complement fragment C5a (EP67) bound to live cells of an attenuated vaccine strain of <i>Coccidioides</i> enhances T-cell immunity while downregulating inflammatory pathology.

<p>Hydration and dilation of live arthroconidia of the vaccine strain results in fracture of the hydrophobic outer conidial wall and exposure of lysine residues of the underlying inner wall that can conjugate with EP67 via S4BHyNic linkers (succinimidyl-4-benzoylhydrazino-nicotinamide). The concept is that the engineered conformational features of EP67 direct the conjugated vaccine to C5aR/CD88 receptors on DC and MΦ, but not neutrophils, and activate phagocytosis, antigen processing, and presentation by APCs while dampening persistent neutrophil-associated inflammation at infection sites <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003768#ppat.1003768-Hung2" target="_blank">[12]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003768#ppat.1003768-Morgan1" target="_blank">[13]</a>. <b>(B) A glucan particle (GP)-based subunit vaccine platform combines adjuvanicity with antigen delivery to induce robust and durable CD4⁺ and CD8⁺ T-cell responses to </b><b><i>Coccidioides</i></b><b> infection.</b> GPs are composed of a porous shell and hollow core. The protein vaccine is co-loaded with a carrier protein and interacted with yeast RNA within the core to form an antigen complex that is too large to diffuse out through the shell. Endo-Porter (EnP) peptides added to the antigen matrix enhance release of the CD8⁺ vaccine proteins to the cytoplasm of APCs after phagocytosis of GPs and promote processing and presentation of MHC I complexes, resulting in activation of protective CD8⁺ T cells. Between the antigen matrix and shell is a GP layer that can accommodate immune modulators (e.g., CpG, siRNA) that help to elicit a protective response to infection. Multiepitope constructs also may be coupled to the surface of GPs by addition of a pyridyl-S-cysteine peptide at the N-terminus (NPyr-Cys) of the vaccine protein. The C-terminus of GP surface-coupled vaccine proteins may be conjugated with EP67 as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003768#ppat-1003768-g001" target="_blank">Figure 1A</a>. EP67 directs the EBV to complement receptors and, although not yet tested, may further enhance Th17 immunity. CD4⁺ and CD8⁺ multiepitope vaccines are constructed using nonimmunogenic GPGPG or di-lysine (KK) linkers <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003768#ppat.1003768-Livingston1" target="_blank">[25]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003768#ppat.1003768-Yano1" target="_blank">[26]</a>. β-1,3-glucans and opsonic C3 deposited on the surface of GPs bind to Dectin-1 and complement receptors (CR), respectively, and induce phagocytosis by APCs (MΦ and DC) followed by antigen processing and peptide epitope display as MHC I or II complexes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003768#ppat.1003768-Hurtgen1" target="_blank">[17]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003768#ppat.1003768-Huang1" target="_blank">[19]</a>.</p