People suffering from COVID-19 could have several different SARS-CoV-2 variants hidden away from the immune system in different parts of the body, finds new research published in Nature Communications by an international research team.
The study’s authors say that this may make complete clearance of the virus from the body of an infected person, by their own antibodies, or by therapeutic antibody treatments, much more difficult.
COVID-19 continues to sweep the globe causing hospitalisations and deaths, damaging communities and economies worldwide. Successive variants of concern (VoC), replaced the original virus from Wuhan, increasingly escaping immune protection offered by vaccination or antibody treatments.
In new research, comprising two studies published in parallel in Nature Communications, an international team led by Professor Imre Berger at the University of Bristol and Professor Joachim Spatz at the Max Planck Institute for Medical Research in Heidelberg , both Directors of the Max Planck Bristol Centre of Minimal Biology, show how the virus can evolve distinctly in different cell types, and adapt its immunity, in the same infected host.
The team sought to investigate the function of a tailor-made pocket in the SARS-CoV-2 spike protein in the infection cycle of the virus. The pocket, discovered by the Bristol team in an earlier breakthrough, played an essential role in viral infectivity.
“An incessant series of variants have completely replaced the original virus by now, with Omicron and Omicron 2 dominating worldwide.” said Professor Imre Berger. “We analyzed an early variant discovered in Bristol, BrisDelta.
It had changed its shape from the original virus, but the pocket we had discovered was there, unaltered.” Intriguingly, BrisDelta, presents as a small subpopulation in the samples taken from patients, but appears to infect certain cell-types better than the virus that dominated the first wave of infections.
Dr. Kapil Gupta, lead author of the BrisDelta study, explains: “Our results showed that one can have several different virus variants in one’s body. Some of these variants may use kidney or spleen cells as their niche to hide, while the body is busy defending against the dominant virus type. This could make it difficult for the infected patients to get rid of SARS-CoV-2 entirely.”
The team applied cutting-edge synthetic biology techniques, state-of-the-art imaging and cloud computing to decipher viral mechanisms at work. To understand the function of the pocket, the scientists built synthetic SARS-CoV-2 virions in the test tube, that are mimics of the virus but have a major advantage in that they are safe, as they do not multiply in human cells.
Using these artificial virions, they were able to study the exact mechanism of the pocket in viral infection. They demonstrated that upon binding of a fatty acid, the spike protein decorating the virions changed their shape. This switching ‘shape’ mechanism effectively cloaks the virus from the immune system.
Dr. Oskar Staufer, lead author of this study and joint member of the Max Planck Institute in Heidelberg and the Max Planck Centre in Bristol, explains: “By ‘ducking down’ of the spike protein upon binding of inflammatory fatty acids, the virus becomes less visible to the immune system. This could be a mechanism to avoid detection by the host and a strong immune response for a longer period of time and increase total infection efficiency.”
“It appears that this pocket, specifically built to recognize these fatty acids, gives SARS-CoV-2 an advantage inside the body of infected people, allowing it to multiply so fast. This could explain why it is there, in all variants, including Omicron” added Professor Berger.
“Intriguingly, the same feature also provides us with a unique opportunity to defeat the virus, exactly because it is so conserved – with a tailormade antiviral molecule that blocks the pocket.” Halo Therapeutics, a recent University of Bristol spin-out founded by the authors, pursues exactly this approach to develop pocket-binding pan-coronavirus antivirals.
This study aims to determine to what extend the mutations observed in large SARS-CoV-2 genome datasets can perturb the human cytotoxic response against this virus. This impact was studied in HLA class I molecules that practically cover the human population as a whole and, with special attention, to subsets with reduced SARS-CoV-2-ligand repertoires.
In general, human and pathogen variability can greatly influence the CD8+ response, which may affect the outcome of infection. Some combinations of HLA class I haplotypes and viral genomes appear to further offset the balance towards an insufficient cytotoxic response and, thus, a probable bad prognosis.
The surveillance of escape viral variants carried out in this study might therefore help to ameliorate enhanced susceptibility to COVID-19 in sub-populations by designing appropriate countermeasures.
The experimental evaluation of the immune response of every human allele associated to each viral variant is not feasible. Computational methods can facilitate this task and generate new, otherwise overlooked, hypotheses. Pioneering bioinformatic studies focused on predicting cytotoxic epitopes of a limited subset of common HLA alleles against the reference viral strain [12,19,22,25]. Former high-range reports have explored the epitope space of the virus with different purposes such as the assessment of the geographical prevalence of allele-peptide combinations  and the design of epitope-based vaccines .
However, SARS-CoV-2 has substantially evolved after more than a year of pandemic, resulting in a human-viral combination landscape of immense scale only approachable using automated techniques. To screen the genome cytotoxic dynamics, it is essential to estimate the mutations and the utilization of supertype concept and susceptible alleles, and the systemic analysis of mutation combinations in potentially emerging isolates.
Large epitope numbers were computationally predicted to be presented by most supertypes. Although all these supermotifs appeared mutated in at least one isolate, most of these mutations did not overcome the supermotif degeneracy. In most cases, the HLA binding affinity was reasonably maintained except from (i) residue substitutions in the second and C-terminal positions of the ligand core, amino acids that usually are anchor motifs; and (2) large deletions that fully removed the epitope.
For instance, the Spike-W152C mutation and deletions in the 6342–6432 range in ORF1ab removed several epitopes at the level of supermotifs, and were coupled to several other changes. Respect to the persistence of these escape mutations, point substitutions are likely less prone to impose a dramatic fitness although some extensive deletions have been also been shown to be compatible with infection and transmission [28,29]. Large deletions have been related to progressive adaptation to host and reduced virulence [30,31], but their middle-term stability should be analyzed case-to-case.
A central question is whether escape mutations have longitudinally accumulated in genomes of individual isolates. If so, such emerging strains would have acquired, or be in the process of acquiring, enhanced capacity to infect individuals previously able to mount an effective cytotoxic response. However, the emergence of this challenging phenotype would not be expected after the examination of the genomic space of the virus carried out in this study.
Even the forward line of mutated variants in this respect only combined low numbers (<15%) of escape supermotifs of a given supertype. The remaining intact supermotifs, other HLA class I loci and heterozygosity should compensate escape mutations, provided that the pool of naïve lymphocyte is high enough and the innate-to-adaptive response priming correctly coordinated. Notably, the humoral and CD4+ responses would likely remain active and be sufficient in many cases.
Therefore, we conclude that the systemic nature of the immune response translates into most healthy subjects remaining competent to respond against variants. The only exception that moderately threatens supertype redundancy was the B27 supertype with isolates that convey evading mutations for up to 33% of these supermotifs. This supertype is common in many populations such, in particular, in Inuit , which may be exposed to “Northern America” isolates with disabled B27 supermotifs.
Importantly, our results support that VOCs and VUMs are essentially devoid of cytotoxic escape mutations. The only notable example is the epsilon strain, which carries the relevant spike-W152C mutation . Nevertheless, the emergence of alternative isolates that undergo the step-wise accumulation of genetic markers to achieve extended cytotoxic resistance should not be ruled out. This may be favored by considering the explosive expansion of the virus worldwide.
The mutational space would be reduced in practice due to potential antagonism between cytotoxic evasion pressure and structural-functional restrictions of proteins. However, a sizable fraction of the human population has been infected with the virus, which represents innumerable replication cycles and infection attempts. Some variants have been linked by other scientific groups to different clinical phenotypes such as increased mortality  and antibody escape .
Likewise, progressive mutation and recombination in SARS-CoV-2 may conceivably achieve a critical number of supermotif escape mutations that collectively constitute a selective advantage. Some identified isolates appeared to have experienced a higher-than-expected number of these changes over the genetic noise, and may have initiated the evasion-driven process.
On the other hand, according to our computational study, a worrisome scenario has already occurred for around ~10% of alleles able to bind a reduced number of ligands from the SARS-CoV-2 proteome. Among them, the A74, B82 and C18 allele families, with sub-Saharan African origin, and the C46 family, with Far East origin, excelled. Lost or debilitation of the cytotoxic response would make these individuals too dependent upon the humoral response, which can be inefficient during primary infection in some cases (1). This may be very relevant when these alleles are combined into the same haplotype, in particular when in homozygosis.
Underprotection may become exacerbated if these individuals become infected with these escape variants. Given their low epitope redundancy, a very few number of viral mutations, such as those identified in this study, may suffice to circumvent both the cytotoxic primary and memory T responses.
The geographical co-existence of viral variants that experience epitope switch respect to some HLA class I molecules and individuals expressing these alleles may exert immediate selective pressure. This may cause rampant dissemination of emergent strains in these niches with local clinical consequences.
Most isolates at great risk of achieving critical mutations to impair the CD8+ ligand repertoires in these families were found in “Northern America” where some African Americans and Asian subpopulations carried these alleles. Whether these immunotypes with further diminished SARS-CoV-2 ligandomes have undergone positive selection warrants massive local HLA genotyping and viral sequencing. Some of these alleles may be ancestrally specialized in single pathogens, but unable to be effective against international viral infections as reported for Dengue , HIV  and influenza .
Bioinformatic approaches suffer from intrinsic limitations. These include the possible application of biologically inappropriate thresholds and potentially low predictive performance. Furthermore, alleles considered in algorithms as much as the priceless genome sampling by the worldwide sequencing effort still represent an underestimation of biological variability.
Such obstacles were addressed in this study by: (i) utilizing a state-of-the-art algorithm that permits nearly universal fine-grained predictions; (ii) the application of stringent cutoffs that reflect the natural strictness of the ligand-HLA binding; (iii) the re-calculation of peptide binding affinity for each mutation; and (iv) the utilization of a large dataset of viral genomes and their corresponding metadata.
Mutations were stratified by occurrence, reduction of HLA-binding affinity and geographical dissemination. Thus, the integration of omic data and immunoinformatics in this study very likely capture, despite drawbacks, the principal trends that respond to the posed questions.
In conclusion, here we provide a complete repository of the predicted escape mutations in a recent NCBI genome sampling of SARS-CoV-2. Fortunately, accumulation of these mutations in single isolates does not appear close enough yet to be alarming at the global population level. However, isolates carrying mutations able to override limited CD8+ response in some alleles and haplotypes are already co-circulating with individuals carrying these HLA class I molecules.
Emerging SARS-CoV-2 variants may further increment the susceptibility of highly vulnerable communities and should be actively surveyed to coordinate appropriate countermeasures. In this respect, bioinformatic pipelines operating on a timely basis may play an irreplaceable role in the protection against this and other pandemic threats.
reference link : https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1009726
More information: Kapil Gupta et al, Structural insights in cell-type specific evolution of intra-host diversity by SARS-CoV-2, Nature Communications (2022). DOI: 10.1038/s41467-021-27881-6
Oskar Staufer et al, Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein, Nature Communications (2022). DOI: 10.1038/s41467-022-28446-x
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