LENS: Landscape of Effective Neoantigens Software.

MOTIVATION: Elimination of cancer cells by T cells is a critical mechanism of anti-tumor immunity and cancer immunotherapy response. T cells recognize cancer cells by engagement of T cell receptors with peptide epitopes presented by major histocompatibility complex molecules on the cancer cell surface. Peptide epitopes can be derived from antigen proteins coded for by multiple genomic sources. Bioinformatics tools used to identify tumor-specific epitopes via analysis of DNA and RNA-sequencing data have largely focused on epitopes derived from somatic variants, though a smaller number have evaluated potential antigens from other genomic sources. RESULTS: We report here an open-source workflow utilizing the Nextflow DSL2 workflow manager, Landscape of Effective Neoantigens Software (LENS), which predicts tumor-specific and tumor-associated antigens from single nucleotide variants, insertions and deletions, fusion events, splice variants, cancer-testis antigens, overexpressed self-antigens, viruses, and endogenous retroviruses. The primary advantage of LENS is that it expands the breadth of genomic sources of discoverable tumor antigens using genomics data. Other advantages include modularity, extensibility, ease of use, and harmonization of relative expression level and immunogenicity prediction across multiple genomic sources. We present an analysis of 115 acute myeloid leukemia samples to demonstrate the utility of LENS. We expect LENS will be a valuable platform and resource for T cell epitope discovery bioinformatics, especially in cancers with few somatic variants where tumor-specific epitopes from alternative genomic sources are an elevated priority. AVAILABILITY AND IMPLEMENTATION: More information about LENS, including code, workflow documentation, and instructions, can be found at (https://gitlab.com/landscape-of-effective-neoantigens-software).

Associations of minor histocompatibility antigens with outcomes following allogeneic hematopoietic cell transplantation.

The role of minor histocompatibility antigens (mHAs) in mediating graft versus leukemia and graft versus host disease (GvHD) following allogeneic hematopoietic cell transplantation (alloHCT) is recognized but not well-characterized. By implementing improved methods for mHA prediction in two large patient cohorts, this study aimed to comprehensively explore the role of mHAs in alloHCT by analyzing whether (1) the number of predicted mHAs, or (2) individual mHAs are associated with clinical outcomes. The study population consisted of 2249 donor-recipient pairs treated for acute myeloid leukemia and myelodysplastic syndrome with alloHCT. A Cox proportional hazard model showed that patients with a class I mHA count greater than the population median had an increased hazard of GvHD mortality (hazard ratio [HR] = 1.39, 95% confidence interval [CI] = 1.01, 1.77, p = .046). Competing risk analyses identified the class I mHAs DLRCKYISL (GSTP), WEHGPTSLL (CRISPLD2), and STSPTTNVL (SERPINF2) were associated with increased GVHD mortality (HR = 2.84, 95% CI = 1.52, 5.31, p = .01), decreased leukemia-free survival (LFS) (HR = 1.94, 95% CI = 1.27, 2.95, p = .044), and increased disease-related mortality (DRM) (HR = 2.32, 95% CI = 1.5, 3.6, p = .008), respectively. One class II mHA YQEIAAIPSAGRERQ (TACC2) was associated with increased risk of treatment-related mortality (TRM) (HR = 3.05, 95% CI = 1.75, 5.31, p = .02). WEHGPTSLL and STSPTTNVL were both present within HLA haplotype B*40:01-C*03:04 and showed a positive dose-response relationship with increased all-cause mortality and DRM and decreased LFS, indicating these two mHAs contribute to the risk of mortality in an additive manner. Our study reports the first large-scale investigation of the associations of predicted mHA peptides with clinical outcomes following alloHCT.

The Efficacy of Using Patient-Derived Organoids to Predict Treatment Response in Colorectal Cancer.

Colorectal cancer is an important cause of morbidity and mortality worldwide. The current treatment landscape includes chemotherapy, targeted therapy, immunotherapy, radiotherapy, and surgery. A key challenge to improving patient outcomes is the significant inter-patient heterogeneity in treatment response. Tumour organoids derived from the patients' tumours via surgically resected or endoscopically biopsied tissue, have emerged as promising models for personalised medicine. This review synthesises the findings, to date, of studies which have explored the efficacy of ex vivo organoid sensitivity testing for predicting treatment response. Most studies have focused on predicting the response to standard-of-care radiotherapy and chemotherapy options. There is strong evidence to support organoid sensitivity testing of ionising radiation, 5-fluorouracil, and irinotecan, and to a lesser extent, oxaliplatin and TAS-102. Fewer studies have used organoids to identify patients who are likely to benefit from novel treatment options that otherwise remain in clinical trials. This review also summarises recent advancements in organoid culture to include non-epithelial components of the tumour microenvironment, to allow testing of immunotherapy and certain targeted therapy options. Overall, further prospective trials will support the implementation of organoid-based personalised medicine for colorectal cancer patients in the future.

Shared graft-versus-leukemia minor histocompatibility antigens in DISCOVeRY-BMT.

T-cell responses to minor histocompatibility antigens (mHAs) mediate graft-versus-leukemia (GVL) effects and graft-versus-host disease (GVHD) in allogeneic hematopoietic cell transplantation. Therapies that boost T-cell responses improve allogeneic hematopoietic cell transplant (alloHCT) efficacy but are limited by concurrent increases in the incidence and severity of GVHD. mHAs with expression restricted to hematopoietic tissue (GVL mHAs) are attractive targets for driving GVL without causing GVHD. Prior work to identify mHAs has focused on a small set of mHAs or population-level single-nucleotide polymorphism-association studies. We report the discovery of a large set of novel GVL mHAs based on predicted immunogenicity, tissue expression, and degree of sharing among donor-recipient pairs (DRPs) in the DISCOVeRY-BMT data set of 3231 alloHCT DRPs. The total number of predicted mHAs varied by HLA allele, and the total number and number of each class of mHA significantly differed by recipient genomic ancestry group. From the pool of predicted mHAs, we identified the smallest sets of GVL mHAs needed to cover 100% of DRPs with a given HLA allele. We used mass spectrometry to search for high-population frequency mHAs for 3 common HLA alleles. We validated 24 predicted novel GVL mHAs that are found cumulatively within 98.8%, 60.7%, and 78.9% of DRPs within DISCOVeRY-BMT that express HLA-A∗02:01, HLA-B∗35:01, and HLA-C∗07:02, respectively. We confirmed the immunogenicity of an example novel mHA via T-cell coculture with peptide-pulsed dendritic cells. This work demonstrates that the identification of shared mHAs is a feasible and promising technique for expanding mHA-targeting immunotherapeutics.

NeoSplice: a bioinformatics method for prediction of splice variant neoantigens.

Motivation: Splice variant neoantigens are a potential source of tumor-specific antigen (TSA) that are shared between patients in a variety of cancers, including acute myeloid leukemia. Current tools for genomic prediction of splice variant neoantigens demonstrate promise. However, many tools have not been well validated with simulated and/or wet lab approaches, with no studies published that have presented a targeted immunopeptidome mass spectrometry approach designed specifically for identification of predicted splice variant neoantigens. Results: In this study, we describe NeoSplice, a novel computational method for splice variant neoantigen prediction based on (i) prediction of tumor-specific k-mers from RNA-seq data, (ii) alignment of differentially expressed k-mers to the splice graph and (iii) inference of the variant transcript with MHC binding prediction. NeoSplice demonstrates high sensitivity and precision (>80% on average across all splice variant classes) through in silico simulated RNA-seq data. Through mass spectrometry analysis of the immunopeptidome of the K562.A2 cell line compared against a synthetic peptide reference of predicted splice variant neoantigens, we validated 4 of 37 predicted antigens corresponding to 3 of 17 unique splice junctions. Lastly, we provide a comparison of NeoSplice against other splice variant prediction tools described in the literature. NeoSplice provides a well-validated platform for prediction of TSA vaccine targets for future cancer antigen vaccine studies to evaluate the clinical efficacy of splice variant neoantigens. Availability and implementation: https://github.com/Benjamin-Vincent-Lab/NeoSplice. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Zebrafish Paralogs brd2a and brd2b Are Needed for Proper Circulatory, Excretory and Central Nervous System Formation and Act as Genetic Antagonists during Development.

Brd2 belongs to the BET family of epigenetic transcriptional co-regulators that act as adaptor-scaffolds for the assembly of chromatin-modifying complexes and other factors at target gene promoters. Brd2 is a protooncogene and candidate gene for juvenile myoclonic epilepsy in humans, a homeobox gene regulator in Drosophila, and a maternal-zygotic factor and cell death modulator that is necessary for normal development of the vertebrate central nervous system (CNS). As two copies of Brd2 exist in zebrafish, we use antisense morpholino knockdown to probe the role of paralog Brd2b, as a comparative study to Brd2a, the ortholog of human Brd2. A deficiency in either paralog results in excess cell death and dysmorphology of the CNS, whereas only Brd2b deficiency leads to loss of circulation and occlusion of the pronephric duct. Co-knockdown of both paralogs suppresses single morphant defects, while co-injection of morpholinos with paralogous RNA enhances them, suggesting novel genetic interaction with functional antagonism. Brd2 diversification includes paralog-specific RNA variants, a distinct localization of maternal factors, and shared and unique spatiotemporal expression, providing unique insight into the evolution and potential functions of this gene.

Landscape and selection of vaccine epitopes in SARS-CoV-2.

BACKGROUND: Early in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation. The rationale for this design was to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE). METHODS: We explored the set of computationally predicted SARS-CoV-2 HLA-I and HLA-II ligands, examining protein source, concurrent human/murine coverage, and population coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, sequence conservation, source protein abundance, and coverage of high frequency HLA alleles. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering for surface accessibility, sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites. RESULTS: From 58 initial candidates, three B cell epitope regions were identified. From 3730 (MHC-I) and 5045 (MHC-II) candidate ligands, 292 CD8+ and 284 CD4+ T cell epitopes were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we proposed a set of 22 SARS-CoV-2 vaccine peptides for use in subsequent murine studies. We curated a dataset of ~ 1000 observed T cell epitopes from convalescent COVID-19 patients across eight studies, showing 8/15 recurrent epitope regions to overlap with at least one of our candidate peptides. Of the 22 candidate vaccine peptides, 16 (n = 10 T cell epitope optimized; n = 6 B cell epitope optimized) were manually selected to decrease their degree of sequence overlap and then synthesized. The immunogenicity of the synthesized vaccine peptides was validated using ELISpot and ELISA following murine vaccination. Strong T cell responses were observed in 7/10 T cell epitope optimized peptides following vaccination. Humoral responses were deficient, likely due to the unrestricted conformational space inhabited by linear vaccine peptides. CONCLUSIONS: Overall, we find our selection process and vaccine formulation to be appropriate for identifying T cell epitopes and eliciting T cell responses against those epitopes. Further studies are needed to optimize prediction and induction of B cell responses, as well as study the protective capacity of predicted T and B cell epitopes.

Landscape and Selection of Vaccine Epitopes in SARS-CoV-2.

There is an urgent need for a vaccine with efficacy against SARS-CoV-2. We hypothesize that peptide vaccines containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation would drive both humoral and cellular immunity with high specificity, potentially avoiding undesired effects such as antibody-dependent enhancement (ADE). Additionally, such vaccines can be rapidly manufactured in a distributed manner. In this study, we combine computational prediction of T cell epitopes, recently published B cell epitope mapping studies, and epitope accessibility to select candidate peptide vaccines for SARS-CoV-2. We begin with an exploration of the space of possible T cell epitopes in SARS-CoV-2 with interrogation of predicted HLA-I and HLA-II ligands, overlap between predicted ligands, protein source, as well as concurrent human/murine coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, viral source protein abundance, sequence conservation, coverage of high frequency HLA alleles and co-localization of CD4+ and CD8+ T cell epitopes. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering to select regions with surface accessibility, high sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites. From 58 initial candidates, three B cell epitope regions were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we propose a set of SARS-CoV-2 vaccine peptides for use in subsequent murine studies and clinical trials.

Knockdown of epigenetic transcriptional co-regulator Brd2a disrupts apoptosis and proper formation of hindbrain and midbrain-hindbrain boundary (MHB) region in zebrafish.

Brd2 is a member of the bromodomain-extraterminal domain (BET) family of proteins and functions as an acetyl-histone-directed transcriptional co-regulator and recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. While Brd2 acts as a protooncogene in mammalian blood, developmental studies link it to regulation of neuronal apoptosis and epilepsy, and complete knockout of the gene is invariably embryonic lethal. In Drosophila, the Brd2 homolog acts as a maternal effect factor necessary for segment formation and identity and proper expression of homeotic loci, including Ultrabithorax and engrailed. To test the various roles attributed to Brd2 in a single developmental system representing a non-mammalian vertebrate, we conducted a phenotypic characterization of Brd2a deficient zebrafish embryos produced by morpholino knockdown and corroborated by Crispr-Cas9 disruption and small molecule inhibitor treatments. brd2aMO morphants exhibit reduced hindbrain with an ill-defined midbrain-hindbrain boundary (MHB) region; irregular notochord, neural tube, and somites; and abnormalities in ventral trunk and ventral nerve cord interneuron positioning. Using whole mount TUNEL and confocal microscopy, we uncover a significant decrease, then a dramatic increase, of p53-independent cell death at the start and end of segmentation, respectively. In contrast, using qualitative and quantitative analyses of BrdU incorporation, phosphohistone H3-tagging, and flow cytometry, we detect little effect of Brd2a knockdown on overall proliferation levels in embryos. RNA in situ hybridization shows reduced or absent expression of homeobox gene eng2a and paired box gene pax2a, in the hindbrain domain of the MHB region, and an overabundance of pax2a-positive kidney progenitors, in knockdowns. Together, these results suggest an evolutionarily conserved role for Brd2 in the proper formation and/or patterning of segmented tissues, including the vertebrate CNS, where it acts as a bi-modal regulator of apoptosis, and is necessary, directly or indirectly, for proper expression of genes that pattern the MHB and/or regulate differentiation in the anterior hindbrain.