The ERG25 gene encoding the Candida albicans C-4 sterol methyl oxidase was cloned and sequenced by complementing a Saccharomyces cerevisiae erg25 mutant with a C. albicans genomic library. The Erg25p is comprised of 308 amino acids and shows 65 and 38% homology to the enzymes from S. cerevisiae and Homo sapiens, respectively. The protein contains three histidine clusters common to nonheme iron-binding enzymes and an endoplasmic reticulum retrieval signal as do the proteins from S. cerevisiae and humans. A temperature-sensitive (ts) conditional lethal mutation of the C. albicans ERG25 was isolated and expressed in S. cerevisiae. Sequence analysis of the ts mutant indicated an amino acid substitution within the region of the protein encompassed by the histidine clusters involved in iron binding. Results indicate that plasmid-borne conditional lethal mutants of target genes have potential use in the rescue of Candida mutations in genes that are essential for viability.

Background: Tumor specific antigen (TSA) identification in human cancer predicts response to immunotherapy and provides vaccine targets for precision medicine. In addition to neoantigens from somatic coding mutations, numerous non-mutated TSAs can elicit T-cell responses but are often overlooked by current methods. We present a method that accurately and comprehensively predict TSAs from RNAseq data regardless of mutation status. Methods: HLA-I genotypes were predicted with seq2HLA. RNAseq fastq files were translated into all possible peptides of length 8-11, and peptides with high expression in the tumor and comparatively low expression in normal were tested for their MHC-I binding potential with netMHCpan-4.0. We defined our predicted TSA by i) high expression in tumor samples, ii) low expression in normal samples, and iii) high predicted patient-specific MHC-I binding affinity. Results: We developed a novel pipeline for TSA prediction from RNAseq that is not limited to mutation-derived TSAs. This pipeline was used to predict all possible unique peptides size 8-11 on previously published murine and human lung and lymphoma tumors then validated on matched tumor and control lung adenocarcinoma (LUAD) samples. This pipeline is able to predict TSAs in MHC-I ligand-purified proteomics data with favorable performance to existing methods. Furthermore, neoantigens predicted by exomeSeq are typically poorly expressed at the RNA level, (28% of predicted neoantigens with >0 expression, mean of 15.6 reads/sample) and a fraction of them (47/6,928, 0.68%) are expressed in matched normal samples. Finally, a set of 6 TSAs are expressed in 22/39 (56%) of LUAD tumors and represent attractive vaccine targets. Conclusion: Direct quantification of RNAseq evidence of the potential peptidome in matched tumor and control RNAseq samples, via our novel pipeline, allows for exhaustive detection of TSAs. Citation Format: Michael Sharpnack, Travis Johnson, Robert Chalkley, Zhi Han, David Carbone, Kun Huang, Kai He. Exhaustive tumor specific antigen detection with RNAseq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 238.