Sotrovimab restores neutralisation against current Omicron subvariants in patients with blood cancer

Wu et al. report that patients with hematologic malignancies have reduced immunity against SARS-CoV-2 Omicron subvariants and Sotrovimab retains neutralizing capacity against all tested Omicron subvariants.

Strong peak immunogenicity but rapid antibody waning following third vaccine dose in older residents of care homes.

Third-dose coronavirus disease 2019 vaccines are being deployed widely but their efficacy has not been assessed adequately in vulnerable older people who exhibit suboptimal responses after primary vaccination series. This observational study, which was carried out by the VIVALDI study based in England, looked at spike-specific immune responses in 341 staff and residents in long-term care facilities who received an mRNA vaccine following dual primary series vaccination with BNT162b2 or ChAdOx1. Third-dose vaccination strongly increased antibody responses with preferential relative enhancement in older people and was required to elicit neutralization of Omicron. Cellular immune responses were also enhanced with strong cross-reactive recognition of Omicron. However, antibody titers fell 21-78% within 100 d after vaccine and 27% of participants developed a breakthrough Omicron infection. These findings reveal strong immunogenicity of a third vaccine in one of the most vulnerable population groups and endorse an approach for widespread delivery across this population. Ongoing assessment will be required to determine the stability of immune protection.

Antibody correlates of protection from SARS-CoV-2 reinfection prior to vaccination: A nested case-control within the SIREN study.

OBJECTIVES: To investigate serological differences between SARS-CoV-2 reinfection cases and contemporary controls, to identify antibody correlates of protection against reinfection. METHODS: We performed a case-control study, comparing reinfection cases with singly infected individuals pre-vaccination, matched by gender, age, region and timing of first infection. Serum samples were tested for anti-SARS-CoV-2 spike (anti-S), anti-SARS-CoV-2 nucleocapsid (anti-N), live virus microneutralisation (LV-N) and pseudovirus microneutralisation (PV-N). Results were analysed using fixed effect linear regression and fitted into conditional logistic regression models. RESULTS: We identified 23 cases and 92 controls. First infections occurred before November 2020; reinfections occurred before February 2021, pre-vaccination. Anti-S levels, LV-N and PV-N titres were significantly lower among cases; no difference was found for anti-N levels. Increasing anti-S levels were associated with reduced risk of reinfection (OR 0·63, CI 0·47-0·85), but no association for anti-N levels (OR 0·88, CI 0·73-1·05). Titres >40 were correlated with protection against reinfection for LV-N Wuhan (OR 0·02, CI 0·001-0·31) and LV-N Alpha (OR 0·07, CI 0·009-0·62). For PV-N, titres >100 were associated with protection against Wuhan (OR 0·14, CI 0·03-0·64) and Alpha (0·06, CI 0·008-0·40). CONCLUSIONS: Before vaccination, protection against SARS-CoV-2 reinfection was directly correlated with anti-S levels, PV-N and LV-N titres, but not with anti-N levels. Detectable LV-N titres were sufficient for protection, whilst PV-N titres >100 were required for a protective effect. TRIAL REGISTRATION NUMBER: ISRCTN11041050.

SARS-CoV-2 S2-targeted vaccination elicits broadly neutralizing antibodies.

Several variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged during the current coronavirus disease 2019 (COVID-19) pandemic. Although antibody cross-reactivity with the spike glycoproteins (S) of diverse coronaviruses, including endemic common cold coronaviruses (HCoVs), has been documented, it remains unclear whether such antibody responses, typically targeting the conserved S2 subunit, contribute to protection when induced by infection or through vaccination. Using a mouse model, we found that prior HCoV-OC43 S-targeted immunity primes neutralizing antibody responses to otherwise subimmunogenic SARS-CoV-2 S exposure and promotes S2-targeting antibody responses. Moreover, vaccination with SARS-CoV-2 S2 elicited antibodies in mice that neutralized diverse animal and human alphacoronaviruses and betacoronaviruses in vitro and provided a degree of protection against SARS-CoV-2 challenge in vivo. Last, in mice with a history of SARS-CoV-2 Wuhan-based S vaccination, further S2 vaccination induced broader neutralizing antibody response than booster Wuhan S vaccination, suggesting that it may prevent repertoire focusing caused by repeated homologous vaccination. These data establish the protective value of an S2-targeting vaccine and support the notion that S2 vaccination may better prepare the immune system to respond to the changing nature of the S1 subunit in SARS-CoV-2 variants of concern, as well as to future coronavirus zoonoses.

Infecting human hematopoietic stem and progenitor cells with SARS-CoV-2.

Determining how hematopoietic stem and progenitor cells (HSPCs) can be infected by viruses is necessary to understand and predict how the immune system will drive the host response. We present here a protocol to analyze the capacity of SARS-CoV-2 to infect different subsets of human HSPCs, inlcuding procedures for SARS-CoV-2 production and titration, isolation of human HSPCs from different sources (bone marrow, umbilical cord, or peripheral blood), and quantification of SARS-Cov-2 infection capacity by RT-qPCR and colony forming unit assay. For complete details on the use and execution of this protocol, please refer to Huerga Encabo et al. (2021).

SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay.

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP in 25 minutes that is robust, reliable, repeatable, sensitive, specific, and inexpensive.

SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity.

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo-electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite.

Author Correction: Scalable and robust SARS-CoV-2 testing in an academic center.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains.

Background: The degree of heterotypic immunity induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains is a major determinant of the spread of emerging variants and the success of vaccination campaigns, but remains incompletely understood. Methods: We examined the immunogenicity of SARS-CoV-2 variant B.1.1.7 (Alpha) that arose in the United Kingdom and spread globally. We determined titres of spike glycoprotein-binding antibodies and authentic virus neutralising antibodies induced by B.1.1.7 infection to infer homotypic and heterotypic immunity. Results: Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa variant B.1.351 (Beta) than of the infecting variant. The drop in cross-reactivity was significantly more pronounced following B.1.1.7 than parental strain infection. Conclusions: The results indicate that heterotypic immunity induced by SARS-CoV-2 variants is asymmetric. Funding: This work was supported by the Francis Crick Institute and the Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg.

An isoform of Dicer protects mammalian stem cells against multiple RNA viruses.

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase.

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease.

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome.

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease.

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease.

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease.

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.