Global variation in prior exposure shapes antibody neutralization profiles of SARS-CoV-2 variants up to BA.2.86 (preprint)

The highly mutated SARS-CoV-2 variant, BA.2.86, and its descendants are now the most frequently sequenced variants of SARS-CoV-2. We analyze antibody neutralization data from eight laboratories from the UK, USA, Denmark, and China, including two datasets assessing the effect of XBB.1.5 vaccines, to determine the effect of infection and vaccination history on neutralization of variants up to and including BA.2.86, and produce antibody landscapes to describe these neutralization profiles. We find evidence for lower levels of immune imprinting on pre-Omicron variants in sera collected from Denmark and China, which may be explained by lower levels of circulation of the ancestral variant in these countries, and the use of an inactivated virus vaccine in China.

ChAdOx1 nCoV-19 vaccination generates spike-specific CD8+ T cells in aged mice (preprint)

Effective vaccines have reduced SARS-CoV-2 morbidity and mortality; however, the elderly remain the most at risk. Understanding how vaccines generate protective immunity, and how these mechanisms change with age is key for informing future vaccine design. Cytotoxic CD8+ T cells are important for killing virally infected cells, and vaccines that induce antigen specific CD8+ T cells in addition to humoral immunity provide an extra layer of immune protection. This is particularly important in cases where antibody titres are sub-optimal, as can occur in older individuals. Here, we show that in aged mice, spike-epitope specific CD8+ T cells are generated in comparable numbers to younger animals after ChAdOx1 nCoV-19 vaccination, although phenotypic differences exist. This demonstrates that ChAdOx1 nCoV-19 elicits a good CD8+ T cell response in older bodies, but that typical age-associated features are evident on these vaccine reactive T cells.

An alpaca-derived nanobody neutralizes the SARS-CoV-2 omicron variant (preprint)

The SARS-CoV2 Omicron variant sub-lineages spread rapidly through the world, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for anti-SARS-CoV-2 agents that are effective against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production and potential for delivery via inhalation. Here, we characterize the RBD-specific nanobody W25, which we previously isolated from an alpaca, and show superior neutralization activity towards Omicron lineage BA.1 in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike surface glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. Furthermore, we show that W25 also binds the spike protein from the emerging, more infectious Omicron BA.2 lineage with picomolar affinity. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse prioritization of W25 for further clinical development.

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

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.

An ACAT inhibitor regulates SARS-CoV-2 replication and antiviral T cell activity (preprint)

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies have uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 entry and fusion independent of transmembrane protease serine 2 expression in multiple cell types. We also demonstrate a role for ACAT in regulating SARS-CoV-2 RNA replication in primary bronchial epithelial cells. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled in the acute phase of infection. Thus, re-purposing of available ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects.

Efficacy of ChAdOx1 vaccines against SARS-CoV-2 Variants of Concern Beta, Delta and Omicron in the Syrian hamster model (preprint)

ChAdOx1 nCoV-19 (AZD1222) is a replication-deficient simian adenovirus–vectored vaccine encoding the spike (S) protein of SARS-CoV-2, based on the first published full-length sequence (Wuhan-1). AZD1222 was shown to have 74% vaccine efficacy (VE) against symptomatic disease in clinical trials and over 2.5 billion doses of vaccine have been released for worldwide use. However, SARS-CoV-2 continues to circulate and consequently, variants of concern (VoCs) have been detected, with substitutions in the S protein that are associated with a reduction in virus neutralizing antibody titer. Updating vaccines to include S proteins of VoCs may be beneficial over boosting with vaccines encoding the ancestral S protein, even though current real-world data is suggesting good efficacy against hospitalization and death following boosting with vaccines encoding the ancestral S protein. Using the Syrian hamster model, we evaluated the effect of a single dose of AZD2816, encoding the S protein of the Beta VoC, and efficacy of AZD1222/AZD2816 as a heterologous primary series against challenge with the Beta or Delta variant. We then investigated the efficacy of a single dose of AZD2816 or AZD1222 against the Omicron VoC. As seen previously, minimal to no viral sgRNA could be detected in lungs of vaccinated animals obtained at 5 days post inoculation, in contrast to lungs of control animals. Thus, these vaccination regimens are protective against the Beta, Delta, and Omicron VoCs in the hamster model.

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

At the end of 2021 a new SARS-CoV-2 variant, Omicron, emerged and quickly spread across the world. It has been demonstrated that Omicrons high number of Spike mutations lead to partial immune evasion from even polyclonal antibody responses, allowing frequent re-infection and vaccine breakthroughs. However, it seems unlikely these antigenic differences alone explain its rapid growth; here we show Omicron replicates rapidly in human primary airway cultures, more so even than the previously dominant variant of concern, Delta. Omicron Spike continues to use human ACE2 as its primary receptor, to which it binds more strongly than other variants. Omicron Spike mediates enhanced entry into cells expressing several different animal ACE2s, including various domestic avian species, horseshoe bats and mice suggesting it has an increased propensity for reverse zoonosis and is more likely than previous variants to establish an animal reservoir of SARS-CoV-2. Unlike other SARS-CoV-2 variants, however, Omicron Spike has a diminished ability to induce syncytia formation. Furthermore, Omicron is capable of efficiently entering cells in a TMPRSS2-independent manner, via the endosomal route. We posit this enables Omicron to infect a greater number of cells in the respiratory epithelium, allowing it to be more infectious at lower exposure doses, and resulting in enhanced intrinsic transmissibility.

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

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 2 nd 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 3 rd 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.

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

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. (preprint)

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.

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

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.

Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected Patients (preprint)

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a large number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients as well as vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike chimeras and mutants harbouring single amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing how efficiently they are neutralised. Moreover, introducing the 484K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the immune-escape mutations found in SARS-CoV-2 VOCs might be driven by strong antibody pressures, and that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

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

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 that negatively impact the role of neutralising antibodies. In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine against the variant of concern B.1.351 (AZD2816). We demonstrate AZD2816 is immunogenic after a single dose and when used as a booster dose in animals primed with original vaccine AZD1222, we see no evidence of original antigenic sin but high titre antibodies against a number of variant spike proteins. In addition, neutralisation titres against B.1.351 (Beta), B.1.617.1 (Kappa) and B.1.617.2 (Delta), are induced in these boost regimens. These data support the ongoing clinical development and testing of this new variant vaccine.

Circadian regulation of SARS-CoV-2 infection in lung epithelial cells (preprint)

The COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract, via binding human angiotensin-converting enzyme (ACE2), and infection can result in pneumonia and acute respiratory distress syndrome. Circadian rhythms coordinate an organisms response to its environment and recent studies report a role for the circadian clock to regulate host susceptibility to virus infection. Influenza A infection of arhythmic mice, lacking the circadian component BMAL1, results in higher viral replication and elevated inflammatory responses leading to more severe bronchitis, highlighting the impact of circadian pathways in respiratory function. We demonstrate circadian regulation of ACE2 in lung epithelial cells and show that silencing BMAL1 or treatment with the synthetic REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry and RNA replication. Treating infected cells with SR9009 limits viral replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Our study suggests new approaches to understand and improve therapeutic targeting of COVID-19.

ChAdOx1 nCoV-19 protection against SARS-CoV-2 in rhesus macaque and ferret challenge models (preprint)

Vaccines against SARS-CoV-2 are urgently required. Here we report detailed immune profiling after ChAdOx1 nCoV-19 (AZD1222) and subsequent challenge in two animal models of SARS-CoV-2 mediated disease. We demonstrate in rhesus macaques the lung pathology caused by SARS-CoV-2 mediated pneumonia is reduced by prior vaccination with ChAdOx1 nCoV-19 which induced neutralising antibody responses after a single intramuscular administration. In a second animal model, ferrets, ChAdOx1 nCoV-19 reduced both virus shedding and lung pathology. Antibody titers were boosted by a second dose. Data from these challenge models and the detailed immune profiling, support the continued clinical evaluation of ChAdOx1 nCoV-19.

A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice (preprint)

The spread of SARS-CoV-2 has caused a global pandemic that has affected almost every aspect of human life. The development of an effective COVID-19 vaccine could limit the morbidity and mortality caused by infection, and may enable the relaxation of social distancing measures. Age is one of the most significant risk factors for poor health outcomes after SARS-CoV-2 infection, therefore it is desirable that any new vaccine candidates should elicit a robust immune response in older adults. Here, we test the immunogenicity of the adenoviral vectored vaccine ChAdOx1 nCoV-19 (AZD-1222) in aged mice. We find that a single dose of this vaccine induces cellular and humoral immunity in aged mice, but at a reduced magnitude than in younger adult mice. Furthermore, we report that a second dose enhances the immune response to this vaccine in aged mice, indicating that a primeboost strategy may be a rational approach to enhance immunogenicity in older persons.

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses (preprint)

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19 (preprint)

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins (preprint)

SARS-CoV-2 emerged in late 2019, leading to the COVID-19 pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus we demonstrate that, in addition to human ACE2, the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the twenty-two different hosts we investigated, ACE2 proteins from dog, cat and rabbit were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the three genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Interestingly, while SARS-CoV-2 pseudoparticle entry was inefficient in cells bearing the ACE2 receptor from bats or birds the live virus was still able to enter these cells, albeit with markedly lower efficiency. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock and wildlife.