A live attenuated vaccine confers superior mucosal and systemic immunity to SARS-CoV-2 variants

Vaccines are a cornerstone in COVID-19 pandemic management. Here, we compare immune responses to and preclinical efficacy of the mRNA vaccine BNT162b2, an adenovirus-vectored spike vaccine, and the live-attenuated-virus vaccine candidate sCPD9 after single and double vaccination in Syrian hamsters. All regimens containing sCPD9 showed superior efficacy. The robust immunity elicited by sCPD9 was evident in a wide range of immune parameters after challenge with heterologous SARS-CoV-2 including rapid viral clearance, reduced tissue damage, fast differentiation of pre-plasmablasts, strong systemic and mucosal humoral responses, and rapid recall of memory T cells from lung tissue. Our results demonstrate that use of live-attenuated vaccines may offer advantages over available COVID-19 vaccines, specifically when applied as booster, and may provide a solution for containment of the COVID-19 pandemic.

Nucleocapsid 203 mutations enhance SARS-CoV-2 immune evasion

Previous work indicated that the nucleocapsid 203 mutation increase the virulence and transmission of the SARS-CoV-2 Alpha variant. However, Delta later outcompeted Alpha and other lineages, promoting a new wave of infections. Delta also possesses a nucleocapsid 203 mutation, R203M. Large-scale epidemiological analyses suggest a synergistic effect of the 203 mutation and the spike L452R mutation, associated with Delta expansion. Viral competition experiments demonstrate the synergistic effect in fitness and infectivity. More importantly, we found that the combination of R203M and L452R brings in a 3.2-fold decrease in neutralizing titers to the neutralizing serum relative to L452R-only virus. R203M/L452R show an increased fitness after the initiation of global vaccination programmes, possibly associated with the enhanced immune evasion. Another rapidly emerging variant Omicron also bears the 203 mutation. Thus, we proposed that nucleocapsid mutations play an essential role for the rise and predominance of variants in concern.

Insights into standards of care: dexamethasone and antibodies against COVID-19 in hamster models

RationaleIn face of the ongoing SARS-CoV-2 pandemic, effective and well-understood treatment options are still scarce. While vaccines have proven instrumental in fighting SARS-CoV-2, their efficacy is challenged by vaccine hesitancy, novel variants and short-lasting immunity. Therefore, understanding and optimization of therapeutic options remains essential. ObjectivesWe aimed at generating a deeper understanding on how currently used drugs, specifically dexamethasone and anti-SARS-CoV-2 antibodies, affect SARS-CoV-2 infection and host responses. Possible synergistic effects of both substances are investigated to evaluate combinatorial treatments. MethodsBy using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of treatment with either dexamethasone, anti-SARS-CoV-2 spike monoclonal antibody or a combination of both. scRNA sequencing was employed to reveal transcriptional response to treatment on a single cell level. Measurements and main resultsDexamethasone treatment resulted in similar or increased viral loads compared to controls. Anti-SARS-CoV-2 antibody treatment alone or combined with dexamethasone successfully reduced pulmonary viral burden. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe COVID-19-like disease model. Combination therapy showed additive benefits with both anti-viral and anti-inflammatory potency. Bulk and single-cell transcriptomic analyses confirmed dampened inflammatory cell recruitment into lungs upon dexamethasone treatment and identified a candidate subpopulation of neutrophils specifically responsive to dexamethasone. ConclusionsOur analyses i) confirm the anti-inflammatory properties and indicate possible modes of action for dexamethasone, ii) validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and iii) reveal synergistic effects of a combination therapy and can thus inform more effective COVID-19 therapies.

Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. The unconventionally secreted ORF8 recognizes the IL17RA receptor of macrophages and induces cytokine release. However, conventionally secreted ORF8 cannot bind to IL17RA due to N-linked glycosylation. Furthermore, we found that Yip1 interacting factor homolog B (YIF1B) is a channel protein that translocates unglycosylated ORF8 into vesicles for unconventional secretion. Blocking the unconventional secretion of ORF8 via a YIF1B knockout in hACE2 mice attenuates inflammation and yields delayed mortality following SARS-CoV-2 challenge.

Post-entry, spike-dependent replication advantage of B.1.1.7 and B.1.617.2 over B.1 SARS-CoV-2 in an ACE2-deficient human lung cell line

Epidemiological data demonstrate that SARS-CoV-2 variants of concern (VOC) B.1.1.7 and B.1.617.2 are more transmissible and infections are associated with a higher mortality than non-VOC virus infections. Phenotypic properties underlying their enhanced spread in the human population remain unknown. B.1.1.7 virus isolates displayed inferior or equivalent spread in most cell lines and primary cells compared to an ancestral B.1 SARS-CoV-2, and were outcompeted by the latter. Lower infectivity and delayed entry kinetics of B.1.1.7 viruses were accompanied by inefficient proteolytic processing of spike. B.1.1.7 viruses failed to escape from neutralizing antibodies, but slightly dampened induction of innate immunity. The bronchial cell line NCI-H1299 supported 24- and 595-fold increased growth of B.1.1.7 and B.1.617.2 viruses, respectively, in the absence of detectable ACE2 expression and in a spike-determined fashion. Superior spread in NCI-H1299 cells suggests that VOCs employ a distinct set of cellular cofactors that may be unavailable in standard cell lines.

De Novo Whole Genome Assembly of the Roborovski Dwarf Hamster (Phodopus roborovskii) Genome, an Animal Model for Severe/Critical COVID-19

The Roborovski dwarf hamster Phodopus roborovskii belongs to the Phodopus genus, one of seven within Cricetinae subfamily. Like other rodents such as mice, rats or ferrets, hamsters can be important animal models for a range of diseases. Whereas the Syrian hamster from the genus Mesocricetus is now widely used as a model for mild to moderate COVID-19, Roborovski dwarf hamster show a severe to lethal course of disease upon infection with the novel human coronavirus SARS-CoV-2.

In vitro efficacy of Artemisia extracts against SARS-CoV-2

Traditional medicines based on herbal extracts have been proposed as affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Teas and drinks containing extracts of Artemisia annua and Artemisia afra have been widely used in Africa in efforts to prevent and fight COVID-19 infections. We sought to study the ability of different A. annua and A. afra extracts and the Covid-Organics drink produced in Madagascar to inhibit SARS-CoV-2 and feline coronavirus (FCoV) replication in vitro. Several extracts as well as Covid-Organics inhibit SARS-CoV-2 and FCoV replication at concentrations that did not affect cell viability. It remains unclear whether peak plasma concentrations in humans can reach levels needed to inhibit viral replication following consumption of teas or Covid-Organics. Clinical studies are required to evaluate the utility of these drinks for COVID-19 prevention or treatment in patients.

Multi-specific DARPin(R) therapeutics demonstrate very high potency against mutated SARS-CoV-2 variants in vitro

SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced immunity and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron BA.1 and BA.2, as of February 2022. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19.

Longitudinal omics in Syrian hamsters integrated with human data unravel complexity of moderate immune responses to SARS-CoV-2

In COVID-19, the immune response largely determines disease severity and is key to therapeutic strategies. Cellular mechanisms contributing to inflammatory lung injury and tissue repair in SARS-CoV-2 infection, particularly endothelial cell involvement, remain ill-defined. We performed detailed spatiotemporal analyses of cellular and molecular processes in SARS-CoV-2 infected Syrian hamsters. Comparison of hamster single-cell sequencing and proteomics with data sets from COVID-19 patients demonstrated inter-species concordance of cellular and molecular host-pathogen interactions. In depth vascular and pulmonary compartment analyses (i) supported the hypothesis that monocyte-derived macrophages dominate inflammation, (ii) revealed endothelial inflammation status and T-cell attraction, and (iii) showed that CD4+ and CD8+ cytotoxic T-cell responses precede viral elimination. Using the Syrian hamster model of self-limited moderate COVID-19, we defined the specific roles of endothelial and epithelial cells, among other myeloid and non-myeloid lung cell subtypes, for determining the disease course.

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients by phage display is binding to the ACE2-RBD interface and is tolerant to known RBD mutations

The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed a sub nM IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody was demonstrated in the Syrian hamster and in the hACE2 mice model using a silenced human IgG1 Fc part. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD was solved at 2.0 [A] resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibtion of STE90-C11 is not blocked by many known RBD mutations including N439K, L452R, E484K or L452R+E484Q (emerging B.1.617). STE90-C11 derived human IgG1 with Fc{gamma}R silenced Fc (COR-101) is currently undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19. In BriefHuman antibodies were selected from convalescent COVID-19 patients using antibody phage display. The antibody STE90-C11 is neutralizing authentic SARS-CoV-2 virus in vitro and in vivo and the crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD revealed that this antibody is binding in the RBD-ACE2 interface. S1 binding of STE90-C11 and inhibition of ACE2 binding is not blocked by many known RBD mutations.

In vitro efficacy of Artemisinin-based treatments against SARS-CoV-2

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Proof-of-concept for prophylactic efficacy of the extracts was obtained using a plaque-reduction assay in VeroE6 cells. Subsequent concentration-response studies using a high-throughput antiviral assay, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that pretreatment and treatment with extracts, artemisinin, and artesunate inhibited SARS-CoV-2 infection of VeroE6 cells. In treatment assays, artesunate (50% effective concentration (EC50): 7 g/mL) was more potent than the tested plant extracts (128-260 g/mL) or artemisinin (151 g/mL) and artemether (>179 g/mL), while generally EC50 in pretreatment assays were slightly higher. The selectivity index (SI), calculated based on treatment and cell viability assays, was highest for artemisinin (54), and roughly equal for the extracts (5-10), artesunate (6) and artemether (<7). Similar results were obtained in human hepatoma Huh7.5 cells. Peak plasma concentrations of artesunate exceeding EC50 values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.

Multi-species ELISA for the detection of antibodies against SARS-CoV-2 in animals

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of infected humans and hundreds of thousands of fatalities. As the novel disease - referred to as COVID-19 - unfolded, occasional anthropozoonotic infections of animals by owners or caretakers were reported in dogs, felid species and farmed mink. Further species were shown to be susceptible under experimental conditions. The extent of natural infections of animals, however, is still largely unknown. Serological methods will be useful tools for tracing SARS-CoV-2 infections in animals once test systems are validated for use in different species. Here, we developed an indirect multi-species ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2. The newly established ELISA was validated using 59 sera of infected or vaccinated animals including ferrets, raccoon dogs, hamsters, rabbits, chickens, cattle and a cat, and a total of 220 antibody-negative sera of the same animal species. Overall, a diagnostic specificity of 100.0% and sensitivity of 98.31% was achieved, and the functionality with every species included in this study could be demonstrated. Hence, a versatile and reliable ELISA protocol was established that enables high-throughput antibody detection in a broad range of animal species, which may be used for outbreak investigations, to assess the seroprevalence in susceptible species or to screen for reservoir or intermediate hosts.

A SARS-CoV-2 neutralizing antibody protects from lung pathology in a COVID-19 hamster model

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 [A] revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.

Age-dependent progression of SARS-CoV-2 infection in Syrian hamsters

In late 2019, an outbreak of a severe respiratory disease caused by an emerging coronavirus, SARS-CoV-2, resulted in high morbidity and mortality in infected humans1. Complete understanding of COVID-19, the multi-faceted disease caused by SARS-CoV-2, requires suitable small animal models, as does the development and evaluation of vaccines and antivirals2. Because age-dependent differences of COVID-19 were identified in humans3, we compared the course of SARS-CoV-2 infection in young and aged Syrian hamsters. We show that virus replication in the upper and lower respiratory tract was independent of the age of the animals. However, older hamsters exhibited more pronounced and consistent weight loss. In situ hybridization in the lungs identified viral RNA in bronchial epithelium, alveolar epithelial cells type I and II, and macrophages. Histopathology revealed clear age-dependent differences, with young hamsters launching earlier and stronger immune cell influx than aged hamsters. The latter developed conspicuous alveolar and perivascular edema, indicating vascular leakage. In contrast, we observed rapid lung recovery at day 14 after infection only in young hamsters. We propose that comparative assessment in young versus aged hamsters of SARS-CoV-2 vaccines and treatments may yield valuable information as this small-animal model appears to mirror age-dependent differences in human patients.

ACE2-Variants Indicate Potential SARS-CoV-2-Susceptibility in Animals: An Extensive Molecular Dynamics Study

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late 2019 and since evolved into a global threat with nearly 4.4 million infected people and over 290,000 confirmed deaths worldwide.1 SARS-CoV-2 is an enveloped virus presenting spike (S) glycoproteins on its outer surface. Binding of S to host cell angiotensin converting enzyme 2 (ACE2) is thought to be critical for cellular entry. The host range of the virus extends far beyond humans and non-human primates. Natural and experimental infections have confirmed high susceptibility of cats, ferrets, and hamsters, whereas dogs, mice, rats, pigs, and chickens seem refractory to SARS-CoV-2 infection. To investigate the reason for the variable susceptibility observed in different species, we have developed molecular descriptors to efficiently analyze our dynamic simulation models of complexes between SARS-CoV-2 S and ACE2. Based on our analyses we predict that: (i) the red squirrel is likely susceptible to SARS-CoV-2 infection and (ii) specific mutations in ACE2 of dogs, rats, and mice render them susceptible to SARS-CoV-2 infection.