Mutational spectra distinguish SARS-CoV-2 replication niches

Exposure to different mutagens leaves distinct mutational patterns that can allow prediction of pathogen replication niches (Ruis 2022). We therefore hypothesised that analysis of SARS-CoV-2 mutational spectra might show lineage-specific differences, dependant on the dominant site(s) of replication and onwards transmission, and could therefore rapidly infer virulence of emergent variants of concern (VOC; Konings 2021). Through mutational spectrum analysis, we found a significant reduction in G>T mutations in Omicron, which replicates in the upper respiratory tract (URT), compared to other lineages, which replicate in both upper and lower respiratory tracts (LRT). Mutational analysis of other viruses and bacteria indicates a robust, generalisable association of high G>T mutations with replication within the LRT. Monitoring G>T mutation rates over time, we found early separation of Omicron from Beta, Gamma and Delta, while the mutational burden in Alpha varied consistent with changes in transmission source as social restrictions were lifted. This supports the use of mutational spectra to infer niches of established and emergent pathogens.

Omicron sublineage BA.2.75.2 exhibits extensive escape from neutralising antibodies

Several sublineages of omicron have emerged with additional mutations that may afford further antibody evasion. Here, we characterise the sensitivity of emerging omicron sublineages BA.2.75.2, BA.4.6, and BA.2.10.4 to antibody-mediated neutralisation, and identify extensive escape by BA.2.75.2. BA.2.75.2 was resistant to neutralisation by Evusheld (tixagevimab + cilgavimab), but remained sensitive to bebtelovimab. In recent serum samples from blood donors in Stockholm, Sweden, BA.2.75.2 was neutralised, on average, at titers approximately 6.5-times lower than BA.5, making BA.2.75.2 the most neutralisation resistant variant evaluated to date. These data raise concerns that BA.2.75.2 may effectively evade humoral immunity in the population.

Evasion of neutralizing antibodies by Omicron sublineage BA.2.75

An emerging SARS-CoV-2 Omicron sublineage, BA.2.75, is increasing in frequency in India and has been detected in at least 15 countries as of 19 July 2022. Relative to BA.2, BA.2.75 carries nine additional mutations in spike. Here we report the sensitivity of the BA.2.75 spike to neutralization by a panel of clinically-relevant and pre-clinical monoclonal antibodies, as well as by serum from blood donated in Stockholm, Sweden, before and after the BA.1/BA.2 infection wave. BA.2.75 largely maintains sensitivity to bebtelovimab, despite a slight reduction in potency, and exhibits moderate susceptibility to tixagevimab and cilgavimab. For sera sampled both before and after the BA.1/BA.2 infection wave, BA.2.75 does not show significantly greater antibody evasion than the currently-dominating BA.5.

Continued Emergence and Evolution of Omicron in South Africa: New BA.4 and BA.5 lineages

South Africas fourth COVID-19 wave was driven predominantly by three lineages (BA.1, BA.2 and BA.3) of the SARS-CoV-2 Omicron variant of concern. We have now identified two new lineages, BA.4 and BA.5. The spike proteins of BA.4 and BA.5 are identical, and comparable to BA.2 except for the addition of 69-70del, L452R, F486V and the wild type amino acid at Q493. The 69-70 deletion in spike allows these lineages to be identified by the proxy marker of S-gene target failure with the TaqPath COVID-19 qPCR assay. BA.4 and BA.5 have rapidly replaced BA.2, reaching more than 50% of sequenced cases in South Africa from the first week of April 2022 onwards. Using a multinomial logistic regression model, we estimate growth advantages for BA.4 and BA.5 of 0.08 (95% CI: 0.07 - 0.09) and 0.12 (95% CI: 0.09 - 0.15) per day respectively over BA.2 in South Africa.

Distinct antigenic properties of the SARS-CoV-2 Omicron lineages BA.4 and BA.5

Over the course of the pandemic variants have arisen at a steady rate. The most recent variants to emerge, BA.4 and BA.5, form part of the Omicron lineage and were first found in Southern Africa where they are driving the current wave of infection. In this report, we perform an in-depth characterisation of the antigenicity of the BA.4/BA.5 Spike protein by comparing sera collected post-vaccination, post-BA.1 or BA.2 infection, or post breakthrough infection of vaccinated individuals with the Omicron variant. In addition, we assess sensitivity to neutralisation by commonly used therapeutic monoclonal antibodies. We find sera collected post-vaccination have a similar ability to neutralise BA.1, BA.2 and BA.4/BA.5. In contrast, in the absence of vaccination, prior infection with BA.2 or, in particular, BA.1 results in an antibody response that neutralises BA.4/BA.5 poorly. Breakthrough infection with Omicron in vaccinees leads to a broad neutralising response against the new variants. The sensitivity of BA.4/BA.5 to neutralisation by therapeutic monoclonal antibodies was similar to that of BA.2. These data suggest BA.4/BA.5 are antigenically distinct from BA.1 and, to a lesser extent, BA.2. The enhanced breadth of neutralisation observed following breakthrough infection with Omicron suggests that vaccination with heterologous or multivalent antigens may represent viable strategies for the development of cross-neutralising antibody responses.

Omicron breakthrough infections in vaccinated or previously infected hamsters

The second and third years of the SARS-CoV-2 pandemic have been marked by the repeated emergence and replacement of variants with genetic and phenotypic distance from the ancestral strains, the most recent examples being Delta and Omicron. Here we describe a hamster contact exposure challenge model to assess protection conferred by vaccination or prior infection against re-infection. We found that 2-doses of self-amplifying RNA vaccine based on the ancestral spike ameliorated weight loss following Delta infection and decreased viral loads, but had minimal effect on Omicron/BA.1 infection. Prior infection with ancestral or Alpha variant was partially protective against Omicron/BA.1 infection, whereas all animals previously infected with Delta and exposed to Omicron became infected, although shed less virus. We further tested whether prior infection with Omicron/BA.1 protected from re-infection with Delta or Omicron/BA.2. Omicron/BA.1 was protective against Omicron/BA.2, but not Delta reinfection, again showing Delta and Omicron have a very large antigenic distance. Indeed, cross-neutralisation assays with human antisera from otherwise immunonaive individuals (unvaccinated and no known prior infection), confirmed a large antigenic distance between Delta and Omicron. Prior vaccination followed by Omicron or Delta breakthrough infection led to a higher degree of cross-reactivity to all tested variants. To conclude, cohorts whose only immune experience of COVID is Omicron/BA.1 infection may be particularly vulnerable to future circulation of Delta or Delta-like derivatives. In contrast, repeated exposure to antigenically distinct spikes, via infection and or vaccination drives a more cross-reactive immune response, both in hamsters and people. One Sentence SummaryInfection with the Delta and Omicron SARS-CoV-2 variants do not provide cross-protective immunity against reinfection with one another in hamsters.

Recurrent SARS-CoV-2 Mutations in Immunodeficient Patients

Long-term SARS-CoV-2 infections in immunodeficient patients are an important source of variation for the virus but are understudied. Many case studies have been published which describe one or a small number of long-term infected individuals but no study has combined these sequences into a cohesive dataset. This work aims to rectify this and study the genomics of this patient group through a combination of literature searches as well as identifying new case series directly from the COG-UK dataset. The spike gene receptor binding domain (RBD) and N-terminal domains (NTD) were identified as mutation hotspots. Numerous mutations associated with variants of concern were observed to emerge recurrently. Additionally a mutation in the envelope gene, - T30I was determined to be the most recurrent frequently occurring mutation arising in persistent infections. A high proportion of recurrent mutations in immunodeficient individuals are associated with ACE2 affinity, immune escape, or viral packaging optimisation. There is an apparent selective pressure for mutations which aid intra-host transmission or persistence which are often different to mutations which aid inter-host transmission, although the fact that multiple recurrent de novo mutations are considered defining for variants of concern strongly indicates that this potential source of novel variants should not be discounted.

The SARS-CoV-2 variant, Omicron, shows rapid replication in human primary nasal epithelial cultures and efficiently uses the endosomal route of entry.

The SARS-CoV-2 Omicron/BA.1 lineage emerged in late 2021 and rapidly displaced the Delta variant before being overtaken itself globally by, the Omicron/BA.2 lineage in early 2022. Here, we describe how Omicron BA.1 and BA.2 show a lower severity phenotype in a hamster model of pathogenicity which maps specifically to the spike gene. We further show that Omicron is attenuated in a lung cell line but replicates more rapidly, albeit to lower peak titres, in human primary nasal cells. This replication phenotype also maps to the spike gene. Omicron spike (including the emerging Omicron lineage BA.4) shows attenuated fusogenicity and a preference for cell entry via the endosomal route. We map the altered Omicron spike entry route and partially map the lower fusogenicity to the S2 domain, particularly the substitution N969K. Finally, we show that pseudovirus with Omicron spike, engineered in the S2 domain to confer a more Delta-like cell entry route retains the antigenic properties of Omicron. This shows a distinct separation between the genetic determinants of these two key Omicron phenotypes, raising the concerning possibility that future variants with large antigenic distance from currently circulating and vaccine strains will not necessarily display the lower intrinsic severity seen during Omicron infection.

Neutralising antibody activity against SARS-CoV-2 variants, including Omicron, in an elderly cohort vaccinated with BNT162b2

SARS-CoV-2 variants threaten the effectiveness of tools we have developed to mitigate against serious COVID-19. This is especially true in clinically vulnerable sections of society including the elderly. Using sera from BNT162b2 (Pfizer-BioNTech) vaccinated individuals aged between 70 and 89 (vaccinated with two doses 3-weeks apart) we examined the neutralising antibody (nAb) response to wildtype SARS-CoV-2. Between 3 and 20-weeks post 2nd dose, nAb titres dropped 4.9-fold to a median titre of 21.3 (ND80) with 21.6% of individuals having no detectable nAbs at the later time point. Experiments examining the neutralisation of twenty-one different SARS-CoV-2 variant spike proteins confirmed a significant potential for antigenic escape, especially for the Omicron (BA.1), Beta (B.1.351), Delta (B.1.617.2), Theta (P.3), C.1.2 and B.1.638 variants. Interestingly, however, the recently-emerged sub-lineage AY.4.2 was more efficiently neutralised than parental Delta pseudotypes. Combining pseudotype neutralisation with specific receptor binding domain (RBD) ELISAs we confirmed that changes to position 484 in the spike RBD were predominantly responsible for SARS-CoV-2 nAb escape, although the effect of spike mutations is both combinatorial and additive. Lastly, using sera from the same individuals boosted with a 3rd dose of BNT162b2 we showed that high overall levels of neutralising antibody titre can provide significant levels of cross-protection against Omicron. These data provide evidence that SARS-CoV-2 neutralising antibodies wane over time and that antigenically variable SARS-CoV-2 variants are circulating, highlighting the importance of ongoing surveillance and booster programmes. Furthermore, they provide important data to inform risk assessment of new SARS-CoV-2 variants, such as Omicron, as they emerge.

Context-specific emergence and growth of the SARS-CoV-2 Delta variant

The Delta variant of concern of SARS-CoV-2 has spread globally causing large outbreaks and resurgences of COVID-19 cases1-3. The emergence of Delta in the UK occurred on the background of a heterogeneous landscape of immunity and relaxation of non-pharmaceutical interventions4,5. Here we analyse 52,992 Delta genomes from England in combination with 93,649 global genomes to reconstruct the emergence of Delta, and quantify its introduction to and regional dissemination across England, in the context of changing travel and social restrictions. Through analysis of human movement, contact tracing, and virus genomic data, we find that the focus of geographic expansion of Delta shifted from India to a more global pattern in early May 2021. In England, Delta lineages were introduced >1,000 times and spread nationally as non-pharmaceutical interventions were relaxed. We find that hotel quarantine for travellers from India reduced onward transmission from importations; however the transmission chains that later dominated the Delta wave in England had been already seeded before restrictions were introduced. In England, increasing inter-regional travel drove Deltas nationwide dissemination, with some cities receiving >2,000 observable lineage introductions from other regions. Subsequently, increased levels of local population mixing, not the number of importations, was associated with faster relative growth of Delta. Among US states, we find that regions that previously experienced large waves also had faster Delta growth rates, and a model including interactions between immunity and human behaviour could accurately predict the rise of Delta there. Deltas invasion dynamics depended on fine scale spatial heterogeneity in immunity and contact patterns and our findings will inform optimal spatial interventions to reduce transmission of current and future VOCs such as Omicron.

SARS-CoV-2 variants of concern Alpha, Beta, Gamma and Delta have extended ACE2 receptor host-ranges

Following the emergence of SARS-CoV-2 in China in late 2019 a number of variants have emerged, with two of these - Alpha and Delta - subsequently growing to global prevalence. One characteristic of these variants are changes within the Spike protein, in particular the receptor binding domain (RBD). From a public health perspective these changes have important implications for increased transmissibility and immune escape; however, their presence could also modify the intrinsic host-range of the virus. Using viral pseudotyping we examined whether the variants of concern (VOCs) Alpha, Beta, Gamma and Delta have differing host ACE2 receptor usage patterns, focusing on a range of relevant mammalian ACE2 proteins. All four VOCs were able to overcome a previous restriction for mouse ACE2, with demonstrable differences also seen for individual VOCs with rat, ferret or civet ACE2 receptors, changes which we subsequently attribute to N501Y and E484K substitutions within the Spike RBD.

Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway.

SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.

The ChAdOx1 vectored vaccine, AZD2816, induces strong immunogenicity against SARS-CoV-2 B.1.351 and other variants of concern in preclinical studies.

There is an ongoing global effort to design, manufacture, and clinically assess vaccines against SARS-CoV-2. Over the course of the ongoing pandemic a number of new SARS-CoV-2 virus isolates or variants of concern (VoC) have been identified containing mutations in key proteins. In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine (AZD2816) which expresses the spike protein of the Beta VoC (B.1.351). We demonstrate that AZD2816 is immunogenic after a single dose. When AZD2816 is used as a booster dose in animals primed with a vaccine encoding the original spike protein (ChAdOx1 nCoV-19/ [AZD1222]), high titre binding and neutralising antibodies against Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) are induced. In addition, a strong and polyfunctional T cell response was measured in these booster regimens. These data support the ongoing clinical development and testing of this new variant vaccine.

The SARS-CoV-2 variants associated with infections in India, B.1.617, show enhanced spike cleavage by furin

The spike (S) glycoprotein of the SARS-CoV-2 virus that emerged in 2019 contained a suboptimal furin cleavage site at the S1/S2 junction with the sequence 681PRRAR/S686. This cleavage site is required for efficient airway replication, transmission, and pathogenicity of the virus. The B.1.617 lineage has recently emerged in India, coinciding with substantial disease burden across the country. Early evidence suggests that B.1.617.2 (a sublineage of B.1.617) is more highly transmissible than contemporary lineages. B.1.617 and its sublineages contain a constellation of S mutations including the substitution P681R predicted to further optimise this furin cleavage site. We provide experimental evidence that virus of the B.1.617 lineage has enhanced S cleavage, that enhanced processing of an expressed B.1.617 S protein in cells is due to P681R, and that this mutation enables more efficient cleavage of a peptide mimetic of the B.1.617 S1/S2 cleavage site by recombinant furin. Together, these data demonstrate viruses in this emerging lineage have enhanced S cleavage by furin which we hypothesise could be enhancing transmissibility and pathogenicity.

A common TMPRSS2 variant protects against severe COVID-19

Infection with SARS-CoV-2 has a wide range of clinical presentations, from asymptomatic to life-threatening. Old age is the strongest factor associated with increased COVID19-related mortality, followed by sex and pre-existing conditions. The importance of genetic and immunological factors on COVID19 outcome is also starting to emerge, as demonstrated by population studies and the discovery of damaging variants in genes controlling type I IFN immunity and of autoantibodies that neutralize type I IFNs. The human protein transmembrane protease serine type 2 (TMPRSS2) plays a key role in SARS-CoV-2 infection, as it is required to activate the virus spike protein, facilitating entry into target cells. We focused on the only common TMPRSS2 non-synonymous variant predicted to be damaging (rs12329760), which has a minor allele frequency of [~]25% in the population. In a large population of SARS-CoV-2 positive patients, we show that this variant is associated with a reduced likelihood of developing severe COVID19 (OR 0.87, 95%CI:0.79-0.97, p=0.01). This association was stronger in homozygous individuals when compared to the general population (OR 0.65, 95%CI:0.50-0.84, p=1.3x10-3). We demonstrate in vitro that this variant, which causes the amino acid substitution valine to methionine, impacts the catalytic activity of TMPRSS2 and is less able to support SARS-CoV-2 spike-mediated entry into cells. TMPRSS2 rs12329760 is a common variant associated with a significantly decreased risk of severe COVID19. Further studies are needed to assess the expression of the TMPRSS2 across different age groups. Moreover, our results identify TMPRSS2 as a promising drug target, with a potential role for camostat mesilate, a drug approved for the treatment of chronic pancreatitis and postoperative reflux esophagitis, in the treatment of COVID19. Clinical trials are needed to confirm this.

Increased transmission of SARS-CoV-2 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary airway cells or antibody escape

Lineage B.1.1.7 (Variant of Concern 202012/01) is a new SARS-CoV-2 variant which was first sequenced in the UK in September 2020 before becoming the majority strain in the UK and spreading worldwide. The rapid spread of the B.1.1.7 variant results from increased transmissibility but the virological characteristics which underpin this advantage over other circulating strains remain unknown. Here, we demonstrate that there is no difference in viral replication between B.1.1.7 and other contemporaneous SARS-CoV-2 strains in primary human airway epithelial (HAE) cells. However, B.1.1.7 replication is disadvantaged in Vero cells potentially due to increased furin-mediated cleavage of its spike protein as a result of a P681H mutation directly adjacent to the S1/S2 cleavage site. In addition, we show that B.1.1.7 does not escape neutralisation by convalescent or post-vaccination sera. Thus, increased transmission of B.1.1.7 is not caused by increased replication, as measured on HAE cells, or escape from serological immunity.

Resistance of endothelial cells to SARS-CoV-2 infection in vitro

RationaleThe secondary thrombotic/vascular clinical syndrome of COVID-19 suggests that SARS-CoV-2 infects not only respiratory epithelium but also the endothelium activating thrombotic pathways, disrupting barrier function and allowing access of the virus to other organs of the body. However, a direct test of susceptibility to SARS-CoV-2 of authentic endothelial cell lines has not been performed. ObjectiveTo determine infectibility of primary endothelial cell lines with live SARS-CoV-2 and pseudoviruses expressing SARS-CoV-2 spike protein. Methods and ResultsExpression of ACE2 and BSG pathways genes was determined in three types of endothelial cells; blood outgrowth, lung microvascular and aortic endothelial cells. For comparison nasal epithelial cells, Vero E6 cells (primate kidney fibroblast cell line) and HEK 293T cells (human embryonic kidney cells) transfected with either ACE2 or BSG were used as controls. Endothelial and Vero E6 cells were treated with live SARS-CoV-2 virus for 1 hour and imaged at 24 and 72 hours post infection. Pseudoviruses containing SARS-CoV-2, Ebola and Vesicular Stomatis Virus glycoproteins were generated and added to endothelial cells and HEK 239Ts for 2 hours and infection measured using luminescence at 48 hours post infection. Compared to nasal epithelial cells, endothelial cells expressed low or undetectable levels of ACE2 and TMPRSS2 but comparable levels of BSG, PPIA and PPIB. Endothelial cells showed no susceptibility to live SARS-CoV-2 or SARS-CoV-2 pseudovirus (but showed susceptibility to Ebola and Vesicular Stomatitis Virus). Overexpression of ACE2 but not BSG in HEK 239T cells conferred SARS-CoV-2 pseudovirus entry. Endothelial cells primed with IL-1{beta} remained resistant to SARS-CoV-2. ConclusionEndothelial cells are resistant to infection with SARS-CoV-2 virus, in line with relatively low levels of ACE2 and TMPRSS2, suggesting that the vascular dysfunction and thrombosis seen in severe COVID-19 is a result of factors released by adjacent infected cells (e.g. epithelial cells) and/or circulating, systemic inflammatory mediators.

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells.

SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2 cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.