SUCCESSFUL MANUFACTURING OF CLINICAL-GRADE SARS-CoV-2 SPECIFIC T CELLS FOR ADOPTIVE CELL THERAPY

BackgroundAdoptive therapy with SARS-CoV-2 specific T cells for COVID-19 has not been reported. The feasibility of rapid clinical-grade manufacturing of virus-specific T cells from convalescent donors has not been demonstrated for this or prior pandemics. MethodsOne unit of whole blood was collected from each convalescent donor following standard blood bank practices. After the plasma was separated and stored separately, the leukocytes were stimulated using overlapping peptides of SARS-CoV-2, covering the immunodominant sequence domains of the S protein and the complete sequence of the N and M proteins. Thereaftesr, functionally reactive cells were enriched overnight using an automated device capturing IFN{gamma}-secreting cells. FindingsFrom 1x109 leukocytes, 0.56 to 1.16x106 IFN{gamma}+ T cells were produced from each of the first two donors. Most of the T cells (64% to 71%) were IFN{gamma}+, with preferential enrichment of CD56+ T cells, effector memory T cells, and effector memory RA+ T cells. TCRV{beta} spectratyping revealed oligoclonal distribution, with over-representation of subfamilies including V{beta}3, V{beta}16 and V{beta}17. With just two donors, the probability that a recipient in the same ethnic group would share at least one donor HLA allele or one haplotype could be as high as >90% and >30%, respectively. InterpretationsThis study is limited by small number of donors and absence of recipient data; however, crucial first proof-of-principle data are provided demonstrating the feasibility of clinical-grade production of SARS-CoV-2 specific T cells for urgent clinical use, conceivably with plasma therapy concurrently. Our data showing that virus-specific T cells can be detected easily after brief stimulation with SARS-CoV-2 specific peptides suggest that a parallel diagnostic assay can be developed alongside serology testing. FundingThe study was funded by a SingHealth Duke-NUS Academic Medicine COVID-19 Rapid Response Research Grant.

The RNA Pol II sub-complex hsRpb4/7 is required for viability of multiple human cell lines

The evolutionarily conserved RNA Polymerase II Rpb4/7 sub-complex has been thoroughly studied in yeast and impacts gene expression at multiple levels including transcription, mRNA processing and decay. In addition Rpb4/7 exerts differential effects on gene expression in yeast and Rpb4 is not obligatory for yeast (S. cerevisiae) survival. Specialised roles for human (hs) Rpb4/7 have not been extensively described and we have probed this question by depleting hsRpb4/7 in established human cell lines using RNA interference. We find that Rpb4/7 protein levels are inter-dependent and accordingly, the functional effects of depleting either protein are co-incident. hsRpb4/7 exhibits gene-specific effects and cells initially remain viable upon hsRpb4/7 depletion. However prolonged hsRpb4/7 depletion is cytotoxic in the range of cell lines tested. Protracted cell death occurs by an unknown mechanism and in some cases is accompanied by a pronounced elongated cell morphology. In conclusion we provide evidence for a gene-specific role of hsRpb4/7 in human cell viability.

A transcription assay for EWS oncoproteins in Xenopus oocytes

Aberrant chromosomal fusion of the Ewing's sarcoma oncogene (EWS) to several different cellular partners produces the Ewing's family of oncoproteins (EWS-fusion-proteins, EFPs) and associated tumors (EFTs). EFPs are potent transcriptional activators, dependent on the N-terminal region of EWS (the EWS-activation-domain, EAD) and this function is thought to be central to EFT oncogenesis and maintenance. Thus EFPs are promising therapeutic targets, but detailed molecular studies will be pivotal for exploring this potential. Such studies have so far largely been restricted to intact mammalian cells while recent evidence has indicated that a mammalian cell-free transcription system may not support bona fide EAD function. Therefore, the lack of manipulatable assays for the EAD presents a significant barrier to progress. Using Xenopus laevis oocytes we describe a plasmid-based micro-injection assay that supports efficient, bona fide EAD transcriptional activity and hence provides a new vehicle for molecular dissection of the EAD.