Respiratory sequelae of COVID-19: pulmonary and extrapulmonary origins, and approaches to clinical care and rehabilitation.

Although the exact prevalence of post-COVID-19 condition (also known as long COVID) is unknown, more than a third of patients with COVID-19 develop symptoms that persist for more than 3 months after SARS-CoV-2 infection. These sequelae are highly heterogeneous in nature and adversely affect multiple biological systems, although breathlessness is a frequently cited symptom. Specific pulmonary sequelae, including pulmonary fibrosis and thromboembolic disease, need careful assessment and might require particular investigations and treatments. COVID-19 outcomes in people with pre-existing respiratory conditions vary according to the nature and severity of the respiratory disease and how well it is controlled. Extrapulmonary complications such as reduced exercise tolerance and frailty might contribute to breathlessness in post-COVID-19 condition. Non-pharmacological therapeutic options, including adapted pulmonary rehabilitation programmes and physiotherapy techniques for breathing management, might help to attenuate breathlessness in people with post-COVID-19 condition. Further research is needed to understand the origins and course of respiratory symptoms and to develop effective therapeutic and rehabilitative strategies.

An ACAT inhibitor suppresses SARS-CoV-2 replication and boosts antiviral T cell activity.

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 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 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314.

ChAdOx1 nCoV-19 vaccination generates spike-specific CD8+ T cells in aged mice.

Effective vaccines have reduced the morbidity and mortality caused by severe acute respiratory syndrome coronavirus-2 infection; 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 titers are suboptimal, 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.

Tfh cells and the germinal center are required for memory B cell formation & humoral immunity after ChAdOx1 nCoV-19 vaccination.

Emergence from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been facilitated by the rollout of effective vaccines. Successful vaccines generate high-affinity plasma blasts and long-lived protective memory B cells. Here, we show a requirement for T follicular helper (Tfh) cells and the germinal center reaction for optimal serum antibody and memory B cell formation after ChAdOx1 nCoV-19 vaccination. We found that Tfh cells play an important role in expanding antigen-specific B cells while identifying Tfh-cell-dependent and -independent memory B cell subsets. Upon secondary vaccination, germinal center B cells generated during primary immunizations can be recalled as germinal center B cells again. Likewise, primary immunization GC-Tfh cells can be recalled as either Tfh or Th1 cells, highlighting the pluripotent nature of Tfh cell memory. This study demonstrates that ChAdOx1 nCoV-19-induced germinal centers are a critical source of humoral immunity.

Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development.

NSP14 is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both activities are essential for the viral life cycle and may be targeted for anti-viral therapeutics. NSP14 forms a complex with NSP10, and this interaction enhances the nuclease but not the methyltransferase activity. We have determined the structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 Å resolution. Comparisons with NSP14/NSP10 complexes reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. We have determined the structure of NSP14 in complex with cap analogue 7MeGpppG, and observe conformational changes within a SAM/SAH interacting loop that plays a key role in viral mRNA capping offering new insights into MTase activity. We perform an X-ray fragment screen on NSP14, revealing 72 hits bound to sites of inhibition in the ExoN and MTase domains. These fragments serve as excellent starting point tools for structure guided development of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.

ChAdOx1 nCoV-19 (AZD1222) or nCoV-19-Beta (AZD2816) protect Syrian hamsters against Beta Delta and Omicron variants.

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 has been shown to have 74% vaccine efficacy against symptomatic disease in clinical trials. However, variants of concern (VoCs) have been detected, with substitutions that are associated with a reduction in virus neutralizing antibody titer. Updating vaccines to include S proteins of VoCs may be beneficial, even though current real-world data is suggesting good efficacy following boosting with vaccines encoding the ancestral S protein. Using the Syrian hamster model, we evaluate 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. Minimal to no viral sgRNA could be detected in lungs of vaccinated animals obtained at 3- or 5- days post inoculation, in contrast to lungs of control animals. In Omicron-challenged hamsters, a single dose of AZD2816 or AZD1222 reduced virus shedding. Thus, these vaccination regimens are protective against the Beta, Delta, and Omicron VoCs in the hamster model.

Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected patients.

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a 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 or vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike mutants harbouring single and combinatorial amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing neutralisation. Moreover, introducing the 484 K 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 future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

Neutralizing antibody activity against 21 SARS-CoV-2 variants in older adults vaccinated with BNT162b2.

SARS-CoV-2 variants may threaten the effectiveness of vaccines and antivirals to mitigate serious COVID-19 disease. This is of most concern in clinically vulnerable groups such as older adults. We analysed 72 sera samples from 37 individuals, aged 70-89 years, vaccinated with two doses of BNT162b2 (Pfizer-BioNTech) 3 weeks apart, for neutralizing antibody responses to wildtype SARS-CoV-2. Between 3 and 20 weeks after the second vaccine dose, neutralizing antibody titres fell 4.9-fold to a median titre of 21.3 (neutralization dose 80%), with 21.6% of individuals having no detectable neutralizing antibodies at the later time point. Next, we examined neutralization of 21 distinct SARS-CoV-2 variant spike proteins with these sera, and confirmed substantial antigenic escape, especially for the Omicron (B.1.1.529, BA.1/BA.2), Beta (B.1.351), Delta (B.1.617.2), Theta (P.3), C.1.2 and B.1.638 spike variants. By combining pseudotype neutralization with specific receptor-binding domain (RBD) enzyme-linked immunosorbent assays, we showed that changes to position 484 in the spike RBD were mainly responsible for SARS-CoV-2 neutralizing antibody escape. Nineteen sera from the same individuals boosted with a third dose of BNT162b2 contained higher neutralizing antibody titres, providing cross-protection against Omicron BA.1 and BA.2. Despite SARS-CoV-2 immunity waning over time in older adults, booster vaccines can elicit broad neutralizing antibodies against a large number of SARS-CoV-2 variants in this clinically vulnerable cohort.

Porcine Respiratory Coronavirus as a Model for Acute Respiratory Coronavirus Disease

In the light of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we have developed a porcine respiratory coronavirus (PRCV) model for in depth mechanistic evaluation of the pathogenesis, virology and immune responses of this important family of viruses. Pigs are a large animal with similar physiology and immunology to humans and are a natural host for PRCV. Four PRCV strains were investigated and shown to induce different degrees of lung pathology. Importantly, although all four strains replicated equally well in porcine cell lines in vitro and in the upper respiratory tract in vivo, PRCV strains causing more severe lung pathology were also able to replicate in ex vivo tracheal organ cultures as well as in vivo in the trachea and lung. The time course of infection of PRCV 135, which caused the most severe pulmonary pathology, was investigated. Virus was shed from the upper respiratory tract until day 10 post infection, with infection of the respiratory mucosa, as well as olfactory and sustentacular cells, providing an excellent model to study upper respiratory tract disease in addition to the commonly known lower respiratory tract disease from PRCV. Infected animals made antibody and T cell responses that cross reacted with the four PRCV strains and Transmissible Gastroenteritis Virus. The antibody response was reproduced in vitro in organ cultures. Comparison of mechanisms of infection and immune control in pigs infected with PRCVs of differing pathogenicity with human data from SARS-CoV-2 infection and from our in vitro organ cultures, will enable key events in coronavirus infection and disease pathogenesis to be identified.

SARS-CoV-2 variants of concern alpha, beta, gamma and delta have extended ACE2 receptor host ranges.

Following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in PR 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 angiotensin-converting enzyme 2 (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 that we subsequently attributed to N501Y and E484K substitutions within the spike RBD.

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

BACKGROUND: 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. METHODS: 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). FINDINGS: 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]), an increase in binding and neutralising antibodies against Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) is observed following each additional dose. In addition, a strong and polyfunctional T cell response was measured all booster regimens. INTERPRETATION: Real world data is demonstrating that one or more doses of licensed SARS-CoV-2 vaccines confer reduced protection against hospitalisation and deaths caused by divergent VoC, including Omicron. Our data support the ongoing clinical development and testing of booster vaccines to increase immunity against highly mutated VoC. FUNDING: This research was funded by AstraZeneca with supporting funds from MRC and BBSRC.

Rising COVID-19 related acute pulmonary emboli but falling national chronic thromboembolic pulmonary hypertension referrals from a large national dataset.

The average rate of new #CTEPH referrals has dropped by 32% in the UK during the pandemic, despite the high incidence of #COVID19 related pulmonary emboli. There have been no recorded new cases of CTEPH caused by COVID-19. A prospective study is underway. https://bit.ly/37msP2G.

Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase.

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.

Successful use of CPAP in a pregnant patient with COVID-19 pneumonia.

A 35-year-old nurse, who was 27 weeks pregnant at the time, was admitted to hospital with a short history of cough, fever and worsening shortness of breath. Oral and nasopharyngeal swabs were positive for SARS-CoV-2 on real-time viral PCR. During her admission, her breathing further deteriorated and she developed type 1 respiratory failure. A decision was made to trial treatment with continuous positive airway pressure (CPAP) as a means of avoiding intubation. The patient tolerated this well and made rapid improvements on this therapy. She was quickly weaned off and fully recovered before being discharged home. This case highlights the potential for CPAP to be used as a means of avoiding mechanical ventilation and iatrogenic preterm birth in COVID-19 pneumonia in pregnancy. Furthermore, it highlights the need for robust evidence to support this treatment.

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

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate 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 show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.

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

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.