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An Automated Strategy to Handle Antigenic Variability in Immunisation Protocols, Part I: Nanopore Sequencing of Infectious Agent Variants

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Gene, Drug, and Tissue Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2575))

Abstract

Infectious agents often challenge therapeutics, from antibiotics resistance to antigenic variability affecting inoculation measures. Over the last decades, genome sequencing arose as an important ally to address such challenges. In bacterial infection, whole-genome-sequencing (WGS) supports tracking pathogenic alterations affecting the human microbiome. In viral infection, the analysis of the relevant sequence of nucleotides helps with determining historical variants of a virus and elucidates details about infection clusters and their distribution. Additionally, genome sequencing is now an important step in inoculation protocols, isolating target genes to design more robust immunisation assays. Ultimately, genetic engineering has empowered repurposing at scale, allowing long-lasting repeating clinical trials to be automated within a much shorter time-frame, by adjusting existing protocols. This is particularly important during sanitary emergencies as the ones caused by the 2014 West African Ebola outbreak, the Zika virus rapid spread in both South and North America in 2015, followed by Asia in 2016, and the pandemic caused by the SARS-CoV-2, which has infected more than 187 million people and caused more than 4 million deaths, worldwide, as per July 2021 statistics. In this scenery, this chapter presents a novel fully automated strategy to handle antigenic variability in immunisation protocols. The methodology comprises of two major steps (1) nanopore sequencing of infectious agent variants – the focus is on the SARS-CoV-2 and its variants; followed by (2) mRNA vector design for immunotherapy. This chapter presents the nanopore sequencing step and Chapter 17 introduces a protocol for mRNA vector design.

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References

  1. Le VTM, Diep BA (2013) Selected insights from application of whole-genome sequencing for outbreak investigations. Curr Opin Crit Care 19:432–439

    Article  PubMed  PubMed Central  Google Scholar 

  2. Quainoo S, Coolen JPM, van Hijum SAFT et al (2017) Whole-genome sequencing of bacterial pathogens: the future of nosocomial outbreak analysis. Clin Microbiol Rev 30:1015–1063

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Reuter S, Ellington MJ, Cartwright EJP et al (2013) Rapid bacterial whole-genome sequencing to enhance diagnostic and public health microbiology. JAMA Intern Med 173:1397–1404. https://doi.org/10.1001/jamainternmed.2013.7734

    Article  PubMed  PubMed Central  Google Scholar 

  4. Roetzer A, Diel R, Kohl TA et al (2013) Whole genome sequencing versus traditional genoty** for investigation of a mycobacterium tuberculosis outbreak: a longitudinal molecular epidemiological study. PLoS Med 10:e1001387. https://doi.org/10.1371/journal.pmed.1001387

    Article  PubMed  PubMed Central  Google Scholar 

  5. Salipante SJ, SenGupta DJ, Cummings LA et al (2015) Application of whole-genome sequencing for bacterial strain ty** in molecular epidemiology. J Clin Microbiol 53:1072–1079. https://doi.org/10.1128/JCM.03385-14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Acinas SG, Sarma-Rupavtarm R, Klepac-Ceraj V, Polz MF (2005) PCR-induced sequence artifacts and bias: insights from comparison of two 16s rRNA clone libraries constructed from the same sample. Appl Environ Microbiol 71:8966–8969. https://doi.org/10.1128/AEM.71.12.8966-8969.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Andersen KG, Rambaut A, Lipkin WI et al (2020) The proximal origin of SARS-CoV-2. Nat Med 26:450–452

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chang CK, Hou MH, Chang CF et al (2014) The SARS coronavirus nucleocapsid protein – forms and functions. Antivir Res 103:39–50

    Article  CAS  PubMed  Google Scholar 

  9. Chen Z, Xu X, Wang Y et al (2019) DNA segments of African Swine Fever Virus detected for the first time in hard ticks from sheep and bovines. Syst Appl Acarol 24:59–80. https://doi.org/10.11158/saa.24.1.13

    Article  Google Scholar 

  10. Cowan D, Meyer Q, Stafford W et al (2005) Metagenomic gene discovery: past, present and future. Trends Biotechnol 23:321–329

    Article  CAS  PubMed  Google Scholar 

  11. Keller MW, Rambo-Martin BL, Wilson MM et al (2018) Direct RNA sequencing of the coding complete Influenza A virus genome. Sci Rep 8:14408. https://doi.org/10.1038/s41598-018-32615-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kemal KS, Reinis M, Weiser B, Burger H (2009) Methods for viral RNA isolation and PCR amplification for sequencing of near full-length HIV-1 genomes. Methods Mol Biol 485:3–14. https://doi.org/10.1007/978-1-59745-170-3_1

    Article  CAS  PubMed  Google Scholar 

  13. Khatoon Z, Figler B, Zhang H, Cheng F (2014) Introduction to RNA-seq and its applications to drug discovery and development. Drug Dev Res 75:324–330. https://doi.org/10.1002/ddr.21215

    Article  CAS  PubMed  Google Scholar 

  14. Kreuze JF, Perez A, Untiveros M et al (2009) Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388:1–7. https://doi.org/10.1016/j.virol.2009.03.024

    Article  CAS  PubMed  Google Scholar 

  15. Weng SF, Reps J, Kai J et al (2017) Can machine-learning improve cardiovascular risk prediction using routine clinical data? PLoS One 12:e0174944. https://doi.org/10.1371/journal.pone.0174944

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Maitra A, Sarkar MC, Raheja H et al (2020) Mutations in SARS-CoV-2 viral RNA identified in Eastern India: possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility. J Biosci 45:1–18. https://doi.org/10.1007/s12038-020-00046-1

    Article  CAS  Google Scholar 

  17. Nayak A, Tassetto M, Kunitomi M, Andino R (2013) RNA interference-mediated intrinsic antiviral immunity in invertebrates. Curr Top Microbiol Immunol 371:183–200. https://doi.org/10.1007/978-3-642-37765-5_7

    Article  CAS  PubMed  Google Scholar 

  18. Anderson EJ, Rouphael NG, Widge AT et al (2020) Safety and immunogenicity of SARS-CoV-2 mRNA-1273 vaccine in older adults. N Engl J Med 383:2427–2438. https://doi.org/10.1056/nejmoa2028436

    Article  CAS  PubMed  Google Scholar 

  19. Niu X, Sun Y, Chen Z et al (2017) Using small RNA-seq data to detect siRNA duplexes induced by plant viruses. Genes (Basel) 8:163. https://doi.org/10.3390/genes8060163

    Article  CAS  Google Scholar 

  20. Choi KY, Chung H, Min KH et al (2010) Self-assembled hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 31:106–114. https://doi.org/10.1016/j.biomaterials.2009.09.030

    Article  CAS  PubMed  Google Scholar 

  21. Paprotka T, Delviks-Frankenberry KA, Cingöz O et al (2011) Recombinant origin of the retrovirus XMRV. Science (80- ) 333:97–101. https://doi.org/10.1126/science.1205292

    Article  CAS  Google Scholar 

  22. Selitsky SR, Marron D, Hollern D et al (2020) Virus expression detection reveals RNA-sequencing contamination in TCGA. BMC Genomics 21:1–11. https://doi.org/10.1186/s12864-020-6483-6

    Article  CAS  Google Scholar 

  23. Srivastava S, Banu S, Singh P et al (2021) SARS-CoV-2 genomics: an Indian perspective on sequencing viral variants. J Biosci 46:1–14

    Article  Google Scholar 

  24. Su S, Wong G, Shi W et al (2016) Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends Microbiol 24:490–502

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Morris G, Arkadir D, Nevet A et al (2004) Coincident but distinct messages of midbrain dopamine and striatal tonically active neurons. Neuron 43:133–143. https://doi.org/10.1016/j.neuron.2004.06.012

    Article  CAS  PubMed  Google Scholar 

  26. Tang Q, Song Y, Shi M et al (2015) Inferring the hosts of coronavirus using dual statistical models based on nucleotide composition. Sci Rep 5:17155. https://doi.org/10.1038/srep17155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Xu X, Bei J, Xuan Y et al (2020) Full-length genome sequence of segmented RNA virus from ticks was obtained using small RNA sequencing data. BMC Genomics 21:1–8. https://doi.org/10.1186/s12864-020-07060-5

    Article  CAS  Google Scholar 

  28. Xu X, Chen P, Wang J et al (2020) Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission. Sci China Life Sci 63:457–460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Aralaguppe SG, Siddik AB, Manickam A et al (2016) Multiplexed next-generation sequencing and de novo assembly to obtain near full-length HIV-1 genome from plasma virus. J Virol Methods 236:98–104. https://doi.org/10.1016/j.jviromet.2016.07.010

    Article  CAS  PubMed  Google Scholar 

  30. Zhu N, Zhang D, Wang W et al (2020) A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 382:727–733. https://doi.org/10.1056/nejmoa2001017

    Article  PubMed  PubMed Central  Google Scholar 

  31. Astuti I, Ysrafil (2020) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): an overview of viral structure and host response. Diabetes Metab Syndr Clin Res Rev 14:407–412. https://doi.org/10.1016/j.dsx.2020.04.020

    Article  Google Scholar 

  32. Azkur AK, Akdis M, Azkur D et al (2020) Immune response to SARS-CoV-2 and mechanisms of immunopathological changes in COVID-19. Allergy 75:1564–1581

    Article  CAS  PubMed  Google Scholar 

  33. Biswas N, Majumder P (2020) Analysis of RNA sequences of 3636 SARS-CoV-2 collected from 55 countries reveals selective sweep of one virus type. Indian J Med Res 151:450–458. https://doi.org/10.4103/ijmr.IJMR_1125_20

    Article  PubMed  PubMed Central  Google Scholar 

  34. Buclez PO, Dias Florencio G, Relizani K et al (2016) Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system. Mol Ther Methods Clin Dev 3:16035. https://doi.org/10.1038/mtm.2016.35

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Cantara WA, Olson ED, Musier-Forsyth K (2014) Progress and outlook in structural biology of large viral RNAs. Virus Res 193:24–38. https://doi.org/10.1016/j.virusres.2014.06.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Cao S, Strong MJ, Wang X et al (2015) High-throughput RNA sequencing-based virome analysis of 50 lymphoma cell lines from the Cancer Cell Line Encyclopedia project. J Virol 89:713–729. https://doi.org/10.1128/jvi.02570-14

    Article  PubMed  Google Scholar 

  37. Houldcroft CJ, Beale MA, Breuer J (2017) Clinical and biological insights from viral genome sequencing. Nat Rev Microbiol 15:183–192

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Rédei GP (2008) Sanger method of DNA sequencing. In: Encyclopedia of genetics, genomics, proteomics and informatics. Springer, Cham, pp 1755–1755

    Chapter  Google Scholar 

  39. Jankevics E (1998) DNA Sequencing. In: Basic cloning procedures. Springer, Berlin, Heidelberg, pp 22–42

    Chapter  Google Scholar 

  40. Kück U, Nowrousian M (2015) From Maxam-Gilbert and Sanger to the next generation sequencing. BIOspektrum 21:25–27. https://doi.org/10.1007/s12268-015-0529-3

    Article  CAS  Google Scholar 

  41. Elahi E, Ronaghi M (2004) Pyrosequencing: a tool for DNA sequencing analysis. Methods Mol Biol 255:211–219. https://doi.org/10.1385/1-59259-752-1:211

    Article  CAS  PubMed  Google Scholar 

  42. Philpott M, Watson J, Thakurta A et al (2021) Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq. Nat Biotechnol 39:1517–1520. https://doi.org/10.1038/s41587-021-00965-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Sun X, Song L, Yang W et al (2020) Nanopore sequencing and its clinical applications. In: Methods in molecular biology. Humana Press Inc., pp 13–32

    Google Scholar 

  44. Oude Munnink BB, Nieuwenhuijse DF, Stein M et al (2020) Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands. Nat Med 26:1405–1410. https://doi.org/10.1038/s41591-020-0997-y

    Article  CAS  PubMed  Google Scholar 

  45. SARS-CoV-2 coronavirus NGS reagents | IDT. https://eu.idtdna.com/pages/landing/coronavirus-research-reagents/ngs-assays. Accessed 6 Jul 2021

  46. Janus J (2020) Using whole genome sequencing to help combat COVID-19. https://www.phgfoundation.org/blog/wgs-to-combat-COVID-19. Accessed 6 Jul 2021

  47. Viral Genomes. https://www.ncbi.nlm.nih.gov/genome/viruses/. Accessed 6 Jul 2021

  48. Harrison AG, Lin T, Wang P (2020) Mechanisms of SARS-CoV-2 transmission and pathogenesis. Trends Immunol 41:1100–1115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Rodriguez-Morales AJ, Bonilla-Aldana DK, Balbin-Ramon GJ et al (2020) History is repeating itself: probable zoonotic spillover as the cause of the 2019 novel Coronavirus Epidemic. Infez Med 28(1):3–5. https://pubmed.ncbi.nlm.nih.gov/32009128/. Accessed 6 Jul 2021

    CAS  PubMed  Google Scholar 

  50. Variants: distribution of cases data, 20 May 2021 – GOV.UK. https://www.gov.uk/government/publications/covid-19-variants-genomically-confirmed-case-numbers/variants-distribution-of-cases-data. Accessed 6 Jul 2021

  51. Peeri NC, Shrestha N, Siddikur Rahman M et al (2021) The SARS, MERS and novel coronavirus (COVID-19) epidemics, the newest and biggest global health threats: what lessons have we learned? Int J Epidemiol 49:717–726

    Article  Google Scholar 

  52. Rodriguez-Morales AJ, Dhama K, Sharun K et al (2020) Susceptibility of felids to coronaviruses. Vet Rec 186:e21

    Article  PubMed  Google Scholar 

  53. Senapati S, Banerjee P, Bhagavatula S et al (2021) Contributions of human ACE2 and TMPRSS2 in determining host–pathogen interaction of COVID-19. J Genet 100:1–16

    Article  Google Scholar 

  54. COVID-19: epidemiology, virology and clinical features – GOV.UK. https://www.gov.uk/government/publications/wuhan-novel-coronavirus-background-information/wuhan-novel-coronavirus-epidemiology-virology-and-clinical-features. Accessed 7 Jul 2021

  55. Ni W, Yang X, Yang D et al (2020) Role of angiotensin-converting enzyme 2 (ACE2) in COVID-19. Crit Care 24:1–10

    Article  Google Scholar 

  56. Jaimes JA, Millet JK, Whittaker GR (2020) Proteolytic cleavage of the SARS-CoV-2 spike protein and the role of the novel S1/S2 site. iScience 23:101212. https://doi.org/10.1016/j.isci.2020.101212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. **a S, Lan Q, Su S et al (2020) The role of furin cleavage site in SARS-CoV-2 spike protein-mediated membrane fusion in the presence or absence of trypsin. Signal Transduct Target Ther 5:1–3

    Article  Google Scholar 

  58. Örd M, Faustova I, Loog M (2020) The sequence at Spike S1/S2 site enables cleavage by furin and phospho-regulation in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV. Sci Rep 10:1–10. https://doi.org/10.1038/s41598-020-74101-0

    Article  CAS  Google Scholar 

  59. Public Health England (2020) Understanding cycle threshold (Ct) in SARS-CoV-2 RT-PCR: a guide for health protection teams. Public Health England

    Google Scholar 

  60. Zhu Y, Li J, Pang Z (2021) Recent insights for the emerging COVID-19: drug discovery, therapeutic options and vaccine development. Asian J Pharm Sci 16(1):4–23

    Article  PubMed  Google Scholar 

  61. Gorbalenya AE, Baker SC, Baric RS et al (2020) The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5:536–544

    Article  Google Scholar 

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  1. Bioengineering Department, Imperial College London, London, UK

    Glaucia C. Pereira

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    Glaucia C. Pereira

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Pereira, G.C. (2023). An Automated Strategy to Handle Antigenic Variability in Immunisation Protocols, Part I: Nanopore Sequencing of Infectious Agent Variants. In: Pereira, G.C. (eds) Gene, Drug, and Tissue Engineering. Methods in Molecular Biology, vol 2575. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2716-7_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2716-7_16

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