BREAKING! Study Shows That SARS-CoV-2 Evolved To Evade T Cells Via The Debut Of The Mutation P272L Found In Several Lineages!

Researchers from the Cardiff University School of Medicine, Wales-UK, the Centre for Clinical Research at Royal Glamorgan Hospital, Wales-UK and the Public Health Wales NHS Trust-UK has found that the SARS-CoV-2 coronavirus has evolved to evade T Cells via the debut of the spike mutation P272L found in several lineages!


 
This new SARS-CoV-2 Spike mutation P272L emerged and transmitted in several viral lineages. Importantly, the residue 272 sits centrally in a frequently targeted, dominant killer T-cell epitope and it was found that the mutation P272L escaped from >180 T-cell receptors raised in cohorts of patients and vaccines!
 
The study team strongly advised the monitoring T-cell escape and including further proteins in future vaccines research and development.
 
The study team analyzed the prevalent cytotoxic CD8 T-cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein_269-277 epitope (sequence YLQPRTFLL) via the most frequent Human Leukocyte Antigen (HLA) class I worldwide, HLA A∗02.
 
Importantly, it was found that the spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘variants of concern’, was not recognized by the large CD8 T-cell response seen across cohorts of HLA A∗02+ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T-cell receptors.
 
Viral escape at prevalent T-cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 spike providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T-cell escape in new SARS-CoV-2 variants.
 
The study findings were published in the peer reviewed Journal: Cell.
https://www.cell.com/cell/fulltext/S0092-8674(22)00849-2
 
Existing evidence indicates that HLA-I-restricted CD8 ‘killer’ T-cells contribute to the control of SARSCoV-2 and the immunity to disease offered by the currently approved vaccines.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494270/
 
https://pubmed.ncbi.nlm.nih.gov/33497610/
 
Individual HLA-I alleles can be associated with increased likelihood of control or progression of disease with viruses such as HIV. The immune pressure of prevalent host CD8 T-cell responses against HIV results in selection of altered viral sequences and viral immune escape. Influenza virus is also known to gradually escape from CD8 T-cells with one study estimating that following the 1968 H3N2 Hong Kong flu pandemic the virus has lost a prominent CD8 T-cell epitope once every three years.
 
The study team was interested in whether there was evidence that SARS-CoV-2 might also escape from CD8 T-cells. Genome sequencing has shown extensive non-synonymous mutations occurred within the SARSCoV-2 genome during 2020. Some of these mutations alter viral fitness whi le others have been shown to diminish the binding of antibodies.
 
Corresponding author, Dr Andrew K. Sewell, a Professor at the Systems Immunology Research Institute at Cardiff University told Thailand Medical News, “So far, there has been limited study of T-cell escape and a broad brush-approach of examining T-cell recognition of key transmission variants failed to reveal any evidence that escape was occurring.”
 
A recent report described a patient on rituximab treatment for non-Hodgkin’s lymphoma who was infected with SARS-CoV-2 for over 300 days without detectable neutralizing antibodies.
https://www.researchsquare.com/article/rs-750741/v1
 
It was found that during this extended infection period, the virus from this patient formed a single unique clade within the B.1.1 lineage but gained over 30 non-synonymous mutations at a rate that substantially surpassed the evolutionary rate of SARS-CoV-2 in the general population suggesting prevalent intra-host viral adaptation.
 
Interestingly, many of these mutations reduced peptide binding to autologous HLA class I molecules and two were experimentally validated to escape from the patient’s CD8 T-cell response.
 
Yet another recent report demonstrated that mutations in the RBD that enhance ACE2 binding and viral infectivity concomitantly reduce T-cell recognition through HLA*24:02.
https://pubmed.ncbi.nlm.nih.gov/34171266/
 
A detailed study showed that some SARS-CoV-2 mutations in HLA-I-restricted epitopes resulted in lower binding to the HLA leading to reduced recognition by CD8 T-cells but there was little evidence of these mutations being disseminated.
https://pubmed.ncbi.nlm.nih.gov/33664060/
 
It was initially suggested that the L270F (YFQPRTFLL) Spike variant might be an escape mutant, but this variant was only seen six times throughout the study period, in six different locations and five different PANGO lineages.
 
From the study team’s data, this would suggest that while this mutation does arise, there is no evidence of spread of this variant following sporadic emergence across multiple different SARS-CoV-2 lineages.
 
A further study recently demonstrated the potential of SARSCoV-2 mutations to escape from CD8 T-cell responses to ORF3a and nucleocapsid.
https://pubmed.ncbi.nlm.nih.gov/34729465/
 
All these evidences demonstrate that SARS-CoV-2 can alter its sequence to escape from CD8 T-cells within a given host but there has been no proof of transmission of T-cell escape variants.
 
The study team set out to look for evidence for dissemination of CD8 T-cell escape in the wild. They reasoned that if escape variants were to disseminate then they would be most likely to first be seen in a prevalent response through a frequent HLA. HLA A*02 is frequently carried by all human populations except those of recent African ancestry where it is present at lower levels.
 
HLA A*02 is believed to be the most frequent HLA-I in the human population worldwide occurring in ~40% of individuals. HLA A*02 is hypothesized to have become so frequent in the population due to its success at presenting well-recognized epitopes from historically dangerous pathogens, a supposition consistent with this HLA having entered the population via interbreeding with Neanderthals and Denisovans after early humans migrated from Africa.
 
A past study used overlapping peptides from the entire SARS-CoV-2 proteome to identify that the most common HLA A*02-restricted responses in CP were to epitopes contained within peptides spanning residues 3,881-3900 of ORF1ab and 261-280 of Spike.
https://pubmed.ncbi.nlm.nih.gov/33128877/
 
The Spike epitope was narrowed down to residues 269-277; sequence YLQPRTFLL.
 
Another study confirmed the prevalence of CD8 T-cells that respond to YLQPRTFLL in CP that are absent in healthy donor blood taken before the spread of SARS-CoV-2.
https://pubmed.ncbi.nlm.nih.gov/33326767/
 
The study team confirmed the prevalence of responses to this epitope in their cohort where responses were observed in 12/13 HLA A*02+ CP tested and all 7/7 SARS-CoV-2 vaccinees including those who had received only the first dose of a double dose schedule.
 
The study team focused their attention on nonsynonymous mutations in the sequence encoding YLQPRTFLL. While at least 73 different amino acid changes have occurred in this region, 33 were seen in more than six sequenced cases in the period from the beginning of the pandemic to January 1st, 2022.
 
The occurrences of mutations in this region that were basal to phylogenetically grouped clusters of 100+ cases all occurred at position 272 with the P272L mutation occurring in over 10,500 sequenced cases as of January 1st 2022.
 
Interestingly, over the course of the pandemic to date the P272L variant has arisen independently in at least 10 different PANGO lineages during the study period but was most prevalent in the B.1.177 lineage that played a key role in establishing the ‘second wave’ of SARS-CoV-2 in the UK and Europe during the Autumn of 2020. P272L was the 4th most common variant seen in the B.1.177 lineage and its descendants and in the top 15 Spike mutations observed globally to January 31st, 2021. Twelve of thirteen of the study team’s HLA A*02+ CP cohort tested made a response to the YLQPRTFLL epitope; a response comprising >120 different TCRs in total for nine of the patients. Remarkably, no response was seen to the P272L variant.


                                                                                                      Graphical Abstract

This study finding was confirmed using four CP-derived T-cell clones that included TCR alpha or beta chains that have previously been described in other CP cohorts by independent research groups.
 
Responses to the YLQPRTFLL epitope in a cohort of HLA A*02+ healthy donors who had been vaccinated against SARS-CoV-2 but had never had symptoms or a positive test since January 2020 ranged from 0.01-0.2% of CD8 T-cells by peptide-HLA staining.
 
However, all of the T-cells raised against the YLQPRTFLL epitope across the vaccinee cohort (>50 TCRs) engaged the P272L very poorly and did not stain with P272L peptide-HLA tetramers or react to P272L peptide.
 
The fact that >175 different TCRs from CP and vaccinees raised against the YLQPRTFLL epitope all fail to see the P272L variant suggests that either the Proline at position 272 must form a major focus of TCR contact that a Leucine residue cannot compensate for, or that substitution with Leucine at this position must interfere with a commonly shared TCR binding mode (or both).
 
Comparison of the atomic structures of HLA A*02:01-YLQPRTFLL and HLA A*02:01-YLQLRTFLL showed a largely preserved fold, but with local geometry alterations at the epitope residues 3 to 5 (Spike residues 271-273) which presumably form the majority of contacts with TCRs.
 
In order to study the interaction between a TCR and HLA A*02:01-YLQPRTFLL the study team attempted to make soluble forms of the TCRs from the four T-cell clones they generated from CP. They were successful in manufacturing and crystallizing the YLQ36 TCR in complex with HLA A*02:01-YLQPRTFLL. The structure of this complex at 3.0 Å showed a canonical mode of TCR binding with a crossing angle of 51º.
 
As the YLQ36 T-cell failed to recognize the P272L variant or stain with P272L tetramers, the study team examined the effect substitution of Proline 272 with Leucine might have by superposing the P272L variant peptide (PDB 7P3E) into the TCR complex with the Wuhan epitope. This model showed that the Leucine at position 272 in the variant would protrude within 1 Å of the YLQ36 CDR3α loop which forms many important contacts with both peptide and HLA. This would be much closer than the allowed VdW contact distance and would result in repulsive forces. Accommodation of the P272L variant by the YLQ36 TCR would require a movement of the CDR3α loop of at least 1.3 Å, lengthening or breaking >25 molecular bonds with the peptide and 4 bonds with the MHC, to potentially result in a substantial loss in binding affinity. However, the P272L mutant side chain may also adopt a rotamer that would render it clear of the TCR if no other conformational changes occur, though it is unclear whether such rotamers would be energetically favorable.
 
A detailed comparison of the Wuhan and P272L variant peptides may also suggest why the YLQ36 T-cell does not recognize the P272L variant. Structural analysis of HLA A*02:01-YLQLRTFLL indicates there is a lack of intra-peptide bonds present in the P272L peptide compared to HLA A*02:01-YLQPRTFLL.
 
This may be due to the greater flexibility of Leucine compared to Proline which would allow a reduction in the distance between the Gln271 and Arg273 Cα atoms, resulting in potential clashes between the Gln271 side chain and Gln271/Arg273 main chain which would be relieved by breaking any bonds formed. The lack of support in the P272L variant peptide resulting from a lack of intra-peptide bonds may cause the peptide to collapse under YLQ36 TCR approach, displacing the peptide and abolishing peptide:TCR interactions.
 
Although the study team’s structural data does suggest steric interference may be responsible for the lack of P272L variant recognition, other factors may also be responsible for this loss of recognition which would require further study to dissect.
 
Large reductions in TCR binding are expected to drastically reduce T-cell activation and are consistent with the study’s finding that HLA A*02:01- YLQPRTFLL-specific T-cells from CP or vaccinees fail to respond to physiological levels of the P272L variant.
 
SARS-CoV-2 variants have been rapidly emerging throughout the current pandemic with mutants that enhance transmissibility, evade host immunity or increase disease severity being of particular concern.
 
Current systems for identifying variants of public health concern involve risk assessments that identify mutations that are present, and then looks for mutations that have a known biological effect in order to assess their significance. While extensive amounts of work have been undertaken to predict and characterize the likely effects of mutations in Spike on antibody recognition, the same is not true for T-cells.
 
Current risk assessments that only consider antibody affinity and factors associated with transmissibility (e.g. mutations that improve cellular infectivity) are clearly incomplete. The range of potential effects of mutations that affect recognition by T-cells is significant, and urgently requires further study.
 
Importantly, it is clear that there was a significant SARS-CoV-2 outbreak that spanned Europe of a lineage carrying a mutation that the study team has shown has a detectable impact on T-cell recognition, which went unrecognized at the time. Ultimately, it is unclear whether SARS-CoV-2 incorporates enough genetic plasticity to allow it to escape from humoral immune responses.
 
Examining the study findings, it is clear that P272L in B.1.177 was on an upward trajectory in the autumn of 2020, but this lineage was ultimately displaced by B.1.1.7/Alpha, that had a much higher growth advantage due to a range of mutations selecting for cell entry and ACE2 binding. It is important to note that although P272L in B.1.177 was clearly increasing in frequency in late 2020, the case numbers concerned, despite being relatively large, would not be sufficient to detect a signal of a growth advantage compared to B.1.177 lineages lacking the P272L variant at the time.
 
When combined with the analysis of the impact of the P272L change characterized here, it is likely that P272L conferred some advantage, but it was also clear that at the time that advantage was not on the same scale as the selective advantage that was conferred by mutations increasing transmissibility in B.1.1.7/Alpha.
 
It is important to note, however, that as the pandemic progresses, and SARS-CoV-2 achieves reaches an optimal position with regards to ACE2 binding and cell entry, it is likely that other selective advantages (such as T-cell escape) will become more important in the mix of potential advantages that the virus could develop to enable it to persist as a human pathogen.
 
It is also instructive that P272L has continued to emerge on a local level since its first emergence in early 2020, including in all variants of concern identified to date. Amongst these, P272L emerged and showed local spread in both B.1.1.7/Alpha and B.1.617.2/Delta, including significant numbers of cases in Australia, Italy and the US.
 
Based upon these observations, the study team believes that P272L, and other similar mutations that will impact T-cell epitopes, will become increasingly important as a route for SARS-CoV-2 lineages to increase their competitiveness, particularly as global vaccination rates increase.
 
The study findings suggest that the 269-277 epitope of Spike is one region that should be monitored, and its impact considered as part of the development of next generation vaccines.
 
Importantly, the potential of T-cell escape presented by mutations in this region would argue for the inclusion of mutations in the 269-277 epitope of Spike in risk assessments by public health agencies going forward.
 
In conclusion, the study findings demonstrate that SARS-CoV-2 can readily alter its Spike protein via a single amino acid substitution so that it is not recognized by CD8 T-cells targeting the most prevalent epitope in Spike restricted by the most common HLA-I across the population.
 
Although it is not possible to directly attribute the emergence and propagation of the Spike P272L SARS-CoV-2 variant in parts of the world where HLA A*02:01 is frequently expressed to CD8 T-cell-mediated selection pressure, specific focusing of immune protection on a single protein (e.g. SARS-CoV-2 Spike favored by all currently approved vaccines is likely to enhance any tendency for escape at predominant T-cell epitopes like YLQPRTFLL. https://www.nature.com/articles/s41586-020-2798-3
 
The study findings demonstrates that mutations that evade immunodominant T-cell responses through population-frequent HLA can readily arise and disseminate, strongly suggests that it will be prudent to monitor such occurrences and to increase the breadth of next generation SARS-CoV-2 vaccines to incorporate other viral proteins.
 
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