Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals.

The COVID-19 pandemic has revealed a range of disease phenotypes in infected patients with asymptomatic, mild, or severe clinical outcomes, but the mechanisms that determine such variable outcomes remain unresolved. In this study, we identified immunodominant CD8 T-cell epitopes in the spike antigen using a novel TCR-binding algorithm. The predicted epitopes induced robust T-cell activation in unexposed donors demonstrating pre-existing CD4 and CD8 T-cell immunity to SARS-CoV-2 antigen. The T-cell reactivity to the predicted epitopes was higher than the Spike-S1 and S2 peptide pools in the unexposed donors. A key finding of our study is that pre-existing T-cell immunity to SARS-CoV-2 is contributed by TCRs that recognize common viral antigens such as Influenza and CMV, even though the viral epitopes lack sequence identity to the SARS-CoV-2 epitopes. This finding is in contrast to multiple published studies in which pre-existing T-cell immunity is suggested to arise from shared epitopes between SARS-CoV-2 and other common cold-causing coronaviruses. However, our findings suggest that SARS-CoV-2 reactive T-cells are likely to be present in many individuals because of prior exposure to flu and CMV viruses.

Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.

Gallbladder cancer (GBC) is an aggressive gastrointestinal malignancy with no approved targeted therapy. Here, we analyze exomes (n = 160), transcriptomes (n = 115), and low pass whole genomes (n = 146) from 167 gallbladder cancers (GBCs) from patients in Korea, India and Chile. In addition, we also sequence samples from 39 GBC high-risk patients and detect evidence of early cancer-related genomic lesions. Among the several significantly mutated genes not previously linked to GBC are ETS domain genes ELF3 and EHF, CTNNB1, APC, NSD1, KAT8, STK11 and NFE2L2. A majority of ELF3 alterations are frame-shift mutations that result in several cancer-specific neoantigens that activate T-cells indicating that they are cancer vaccine candidates. In addition, we identify recurrent alterations in KEAP1/NFE2L2 and WNT pathway in GBC. Taken together, these define multiple targetable therapeutic interventions opportunities for GBC treatment and management.

A cancer vaccine approach for personalized treatment of Lynch Syndrome.

Lynch syndrome (LS) is a cancer predisposition disorder wherein patients have a 70-80% lifetime risk of developing colorectal cancers (CRC). Finding germline mutations in predisposing genes allows for risk assessment of CRC development. Here we report a germline heterozygous frame-shift mutation in the mismatch repair MLH1 gene which was identified in members of two unrelated LS families. Since defects in DNA mismatch repair genes generate frame-shift mutations giving rise to highly immunogenic neoepitopes, we postulated that vaccination with these mutant peptide antigens could offer promising treatment options to LS patients. To this end we performed whole-exome and RNA seq analysis on the blood and tumour samples from an LS-CRC patient, and used our proprietary neoepitope prioritization pipeline OncoPeptVAC to select peptides, and confirm their immunogenicity in an ex vivo CD8+ T cell activation assay. Three neoepitopes derived from the tumour of this patient elicited a potent CD8+ T cell response. Furthermore, analysis of the tumour-associated immune infiltrate revealed CD8+ T cells expressing low levels of activation markers, suggesting mechanisms of immune suppression at play in this relapsed tumour. Taken together, our study paves the way towards development of a cancer vaccine to treat or delay the onset/relapse of LS-CRC.

Isolation of precise plastid deletion mutants by homology-based excision: a resource for site-directed mutagenesis, multi-gene changes and high-throughput plastid transformation.

We describe a simple and efficient homology-based excision method to delete plastid genes. The procedure allows one or more adjacent plastid genes to be deleted without the retention of a marker gene. We used aadA-based transformation to duplicate a 649 bp region of plastid DNA corresponding to the atpB promoter region. Efficient recombination between atpB repeats deletes the intervening foreign genes and 1,984 bp of plastid DNA (co-ordinates 57,424-59,317) containing the rbcL gene. Only five foreign bases are present in DeltarbcL plants illustrating the precision of homology-based excision. Sequence analysis of non-functional rbcL-related sequences in DeltarbcL plants indicated an extra-plastidic origin. Mutant DeltarbcL plants were heterotrophic, pale-green and contained round plastids with reduced amounts of thylakoids. Restoration of autotrophy and leaf pigmentation following aadA-based transformation with the wild-type rbcL gene ruled out mutations in other genes. Excision and re-use of aadA shows that, despite the multiplicity of plastid genomes, homology-based excision ensures complete removal of functional aadA genes. Rescue of the DeltarbcL mutation and autotrophic growth stabilizes transgenic plastids in heteroplasmic transformants following antibiotic withdrawal, enhancing the overall efficiency of plastid transformation. Unlike the available set of homoplasmic knockout mutants in 25 plastid genes, the rbcL deletion mutant isolated here is readily transformed with the efficient aadA marker gene. This improvement in deletion design facilitates advanced studies that require the isolation of double mutants in distant plastid genes and the replacement of the deleted locus with site-directed mutant alleles and is not easily achieved using other methods.

The tobacco plastid accD gene is essential and is required for leaf development.

Angiosperm plastid genomes typically encode approximately 80 polypeptides, mainly specifying plastid-localized functions such as photosynthesis and gene expression. Plastid protein synthesis and expression of the plastid clpP1 gene are essential for development in tobacco, indicating the presence of one or more plastid genes whose influence extends beyond the plastid compartment. The plastid accD gene encodes the beta-carboxyl transferase subunit of acetyl-CoA carboxylase and is present in the plastids of most flowering plants, including non-photosynthetic parasitic plants. We replaced the wild-type accD gene with an aadA-disrupted mutant allele using homologous recombination. Persistent heteroplasmy in the presence of antibiotics indicated that the wild-type accD allele was essential. The phenotype of the accD knockout was revealed in plastid transformants grown in the absence of antibiotics. Leaves contained pale green sectors and lacked part or all of the leaf lamina due to arrested division or loss of cells. Abnormal structures were present in plastids found in mutant plants, indicating that accD might be required to maintain the plastid compartment. Loss of the plastid compartment would be expected to be lethal. These results provide genetic evidence showing the essential role of plastid ACCase in the pathway leading to the synthesis of products required for the extra-plastidic processes needed for leaf development.

Simple and efficient removal of marker genes from plastids by homologous recombination.

Removal of marker genes improves the design of transgenic plants. Homologous recombination between direct repeats provides a simple method for excising marker genes after transgenic cells and shoots have been isolated. Efficient implementation of the method requires high rates of homologous recombination relative to illegitimate recombination pathways. The procedure works well in plastids where homologous recombination predominates. Marker genes are flanked by engineered direct repeats. The number and length of direct repeats flanking a marker gene influence excision rate. Excision is automatic and loss of the marker gene is controlled by selection alone. After transgenic cells have been isolated selection is removed allowing loss of the marker gene. Excision is a unidirectional process resulting in the rapid accumulation of high levels of marker-free plastid genomes. Cytoplasmic sorting of marker-free plastids from marker-containing plastids leads to the isolation of marker free plants. Marker-free plants can be isolated following vegetative propagation or among the progeny of sexual crosses.