Transforming complex immune disease through personalised medicine using nanopore technologies

Karen Sherwood and Paul Keown (Faculty of Medicine, University of British Columbia and Vancouver General Hospital) jointly presented their work on transforming complex immune disease through personalised medicine, on behalf of the Genome Canada Consortium.

Paul introduced the concept and impact of complex immune diseases, explaining how the immune system is fundamental in human health, with critical importance in oncology, infection, autoimmunity, and transplantation. However, immune diseases are complex and destructive, affecting 10% of the world’s population, with profound societal and economic impact. Current biological research is advancing our understanding of these diseases and aiding the development of novel diagnostic tools and therapies. Paul drew attention to the work of the Networks of Centres of Excellence, and the large scientific consortia that Canada has been involved in, such as the Genome Canada Consortium, which brings together over 70 principle investigators from 22 major universities. Such networks intend to link government, academia, healthcare, patients, and industry, ‘because these challenges are so immense and seldom solved by a single research unit’.

Immune complexities of transplantation

Paul introduced how transplantation has been a ‘tremendous success over the last 50 years’. However, success is short lived – few grafts survive beyond 10-20 years. Many grafts tend to fail within the first few years due to rejection, predominantly antibody mediated rejection (AMR) – the cause in ~60% of cases. In this phenomenon, antibodies are formed against epitopes expressed on the endothelial cells; this leads to blood vessel damage and ultimately tissue death from ischemia. The primary goal of the Genome Canada Transplant Consortium is to try and prevent such premature graft loss; this is based on four research pillars: epitope matching (HLA), immune monitoring, personalised immunotherapy, and evidence-based health policy to translate the scientific gains into societal benefits.

Regarding epitope matching of the extremely polymorphic HLA, Paul explained how they sequenced almost 5,000 subjects with short-read sequencing, and this is being validated by nanopore technology. He explained how there is an incredible amount of variety between individuals in terms of the number of HLA class I and class II alleles (n=206, n=155, respectively); however, if this is reduced to the sequences of the eplets (functional residues of epitopes that an antibody binds to), there is a ‘dramatic reduction in complexity’ (n=59, n=91, for class I and II eplets, respectively). There is a high rate of carriage of the eplets as opposed to the alleles, which provides ‘a very good opportunity for matching at the eplet level’. Paul showed a graph demonstrating the comparatively high benefit through HLA eplet matching. From a practical point of view, this should be achievable with the current transplant programmes and waiting lists.

Evaluation of Oxford Nanopore sequencing for clinical HLA typing

Karen delved deeper into how nanopore sequencing has been used in her and her team’s research into clinical HLA typing. She explained how using the power of nanopore technology, they have been able to develop a workflow for rapid HLA typing, for the context of clinical transplantation (Mosbruger et al. 2020. Human Immunology).

Amplicons were developed (lengths 2.5-7.5 kb) targeting the HLA class I and class II genes, with whole gene coverage for most HLA genes. Sequencing was then performed on the MinION (Guppy v3.2.10 fast basecalling). An ethnically diverse range (n=96) of individuals were analysed. Sequencing was performed between 6h and 12h, although sufficient reads for HLA assignment were achieved within 1 hr (from ~500 Mb of data). The average fragment length was ~10-fold longer than that sequenced on the short-read platform (~4,500 bp vs. ~450 bp), ‘which will make assignment and phasing much easier’.

Four-field typing was achieved for most of the 2,112 HLA alleles tested, although concordance was based on 2-field typing (the resolution that is clinically reported). For class I genes, 2-field assignment was correct in 98% of cases, and for class II genes, in 95% of cases. Although there were a few discrepancies, Karen wanted to point out that this didn’t prevent them from determining if alleles were immunogenically different or identical. Karen also described how often with short-read sequencing one allele may drop out, likely because of PCR bias. This occurred in four cases, but nanopore sequencing was able to detect the second allele in each of these cases.

Karen next presented the Vancouver Rapid Deceased Donor workflow for HLA typing, which has a 5h 20min turnaround time from blood box arrival, through DNA extraction, library prep, MinION sequencing, and HLA analysis, to final report. They are continuing to improve the workflow: by reducing amplification time or using hybrid capture instead of PCR; they have also validated 24 samples on the new R10.3 flow cells. Karen stated that they plan to use the Flongle for all deceased donors, as it is more suitable for single-use due to its reduced output and lower price. They are also testing the MinION Mk1C, which Karen said is particularly useful for on-call sample processing due to its faster basecalling.

T-cell receptor sequencing to identify repertoire clonality and diversity

Lastly Karen introduced another area of research for her team: T-cell receptor (TCR) sequencing, to identify clonality and diversity in the TCR repertoire. They are currently evaluating this with nanopore technology, using peripheral blood mononuclear cells (PBMCs) as starting material. The idea would be to observe the post-transplant T-cell repertoire and identify any expansion of donor-specific T-cell clones.

Conclusions

In conclusion, Karen stated that, for many transplant patients, getting post-transplant results back in a timely manner is crucial to allow early intervention, minimise graft rejection, and provide insight into changes in the recipient’s immune response, prior to full-blown symptomatic rejection. Rapid deceased donor sequencing for epitope matching also increases the accuracy of identifying a suitable recipient. Their ultimate goal is to establish epitope matching using nanopore technology across Canada in a ‘world first programme of this kind’. Karen stated that nanopore sequencing offers ‘tremendous potential for speed, depth, and flexibility in both discovery and delivery sciences, and is approaching the point of clinical application’.