The origins and molecular evolution of SARS-CoV-2 lineage B.1.1.7 in the UK (preprint)

The first SARS-CoV-2 variant of concern (VOC) to be designated was lineage B.1.1.7, later labelled by the World Health Organisation (WHO) as Alpha. Originating in early Autumn but discovered in December 2020, it spread rapidly and caused large waves of infections worldwide. The Alpha variant is notable for being defined by a long ancestral phylogenetic branch with an increased evolutionary rate, along which only two sequences have been sampled. Alpha genomes comprise a well-supported monophyletic clade within which the evolutionary rate is more typical of SARS-CoV-2. The Alpha epidemic continued to grow despite the continued restrictions on social mixing across the UK, and the imposition of new restrictions, in particular the English national lockdown in November 2020. While from a case-number perspective these interventions succeeded in reducing the absolute number of cases of SARS-CoV-2 in the UK, the impact of these non-pharmaceutical interventions was predominantly to drive the decline of those SARS-CoV-2 lineages that preceded Alpha. We investigate the only two sampled sequences that fall on the branch ancestral to Alpha. We find that one is likely to be a true intermediate sequence, providing information about the order of mutational events that led to Alpha. We explore alternate hypotheses that can explain how Alpha acquired a large number of mutations yet remained largely unobserved in a region of high genomic surveillance: an under-sampled geographical location, a non-human animal population, or a chronically-infected individual. We conclude that the last hypothesis provides the best explanation of the observed behaviour and dynamics of the variant, although we find that the individual need not be immunocompromised, as persistently-infected immunocompetent hosts also display a higher within-host rate of evolution. Finally, we compare the ancestral branches and mutation profiles of other VOCs to each other, and identify that Delta appears to be an outlier both in terms of the genomic locations of its defining mutations, and its lack of rapid evolutionary rate on the ancestral branch. As new variants, such as Omicron, continue to evolve (potentially through similar mechanisms) it remains important to investigate the origins of other variants to identify ways to potentially disrupt their evolution and emergence.

Emergence of immune escape at dominant SARS-CoV-2 killer T-cell epitope (preprint)

The adaptive immune system protects against infection via selection of specific antigen receptors on B-cells and T-cells. We studied the prevalent CD8 killer T-cell response mounted against SARS-CoV-2 Spike269-277 epitope YLQPRTFLL via the most frequent Human Leukocyte Antigen (HLA) class I worldwide, HLA A*02. The widespread Spike P272L mutation has arisen in five different SARS-CoV-2 lineages to date and was common in the B.1.177 lineage associated with establishing the second wave in Europe. The large CD8 T-cell response seen across a cohort of HLA A*02+ convalescent patients, comprising of over 120 different TCRs, failed to respond to the P272L variant suggesting that proline 272 dominates TCR contacts with this epitope. Additionally, sizable populations (0.01%-0.2%) of total CD8 T-cells from individuals vaccinated against SARS-CoV-2 stained with HLA A*02-YLQPRTFLL multimers but failed to bind to the P272L reagent. Viral escape at prevalent T-cell epitopes restricted by high frequency HLA may be particularly problematic when vaccine immunity is focussed on a single protein such as SARS-CoV-2 Spike and provides a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlights the urgent need for monitoring T-cell escape in new SARS-CoV-2 variants.

Localised community circulation of SARS-CoV-2 viruses with an increased accumulation of single nucleotide polymorphisms that adversely affect the sensitivity of real-time reverse transcription assays targeting Nucleocapsid protein. (preprint)

Currently the primary method for confirming acute SARS-CoV-2 infection is through the use of molecular assays that target highly conserved regions within the viral genome. Many, if not most of the diagnostic targets currently in use were produced early in the pandemic, using genomes sequenced and shared in early 2020. As viral diversity increases, mutations may arise in diagnostic target sites that have an impact on the performance of diagnostic tests. Here, we report on a local outbreak of SARS-CoV-2 which had gained an additional mutation at position 28890 of the nucleocapsid protein, on a background of pre-existing mutations at positions 28881, 28882, 28883 in one of the main circulating viral lineages in Wales at that time. The impact of this additional mutation had a statistically significant impact on the Ct value reported for the N gene target designed by the Chinese CDC and used in a number of commercial diagnostic products. Further investigation identified that, in viral genomes sequenced from Wales over the summer of 2020, the N gene had a higher rate of mutations in diagnostic target sites than other targets, with 115 issues identified affecting over 10% of all cases sequenced between February and the end of August 2020. In comparison an issue was identified for ORFab, the next most affected target, in less than 1.4% of cases over the same time period. This work emphasises the potential impact that mutations in diagnostic target sites can have on tracking local outbreaks, as well as demonstrating the value of genomics as a routine tool for identifying and explaining potential diagnostic primer issues as part of a laboratory quality management system. This work also indicates that with increasing genomic sequencing data availability, there is a need to re-evaluate the diagnostic targets that are in use for SARS-CoV-2 testing, to better target regions that are now demonstrated to be of lower variability.

Evaluating the effects of SARS-CoV-2 Spike mutation D614G on transmissibility and pathogenicity (preprint)

Global dispersal and increasing frequency of the SARS-CoV-2 Spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of Spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large data set, well represented by both Spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the Spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant.