Intranasal or airborne transmission-mediated delivery of an attenuated SARS-CoV-2 protects Syrian hamsters against new variants.

Detection of secretory antibodies in the airway is highly desirable when evaluating mucosal protection by vaccines against a respiratory virus, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that intranasal delivery of an attenuated SARS-CoV-2 (Nsp1-K164A/H165A) induces both mucosal and systemic IgA and IgG in male Syrian hamsters. Interestingly, either direct intranasal immunization or airborne transmission-mediated delivery of Nsp1-K164A/H165A in Syrian hamsters offers protection against heterologous challenge with variants of concern (VOCs) including Delta, Omicron BA.1, BA.2.12.1 and BA.5. Vaccinated animals show significant reduction in both tissue viral loads and lung inflammation. Similarly attenuated viruses bearing BA.1 and BA.5 spike boost variant-specific neutralizing antibodies in male mice that were first vaccinated with modified vaccinia virus Ankara vectors (MVA) expressing full-length WA1/2020 Spike protein. Together, these results demonstrate that our attenuated virus may be a promising nasal vaccine candidate for boosting mucosal immunity against future SARS-CoV-2 VOCs.

Intranasal delivery of a rationally attenuated SARS-CoV-2 is immunogenic and protective in Syrian hamsters.

Few live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are in pre-clinical or clinical development. We seek to attenuate SARS-CoV-2 (isolate WA1/2020) by removing the polybasic insert within the spike protein and the open reading frames (ORFs) 6-8, and by introducing mutations that abolish non-structural protein 1 (Nsp1)-mediated toxicity. The derived virus (WA1-ΔPRRA-ΔORF6-8-Nsp1K164A/H165A) replicates to 100- to 1000-fold-lower titers than the ancestral virus and induces little lung pathology in both K18-human ACE2 (hACE2) transgenic mice and Syrian hamsters. Immunofluorescence and transcriptomic analyses of infected hamsters confirm that three-pronged genetic modifications attenuate the proinflammatory pathways more than the removal of the polybasic cleavage site alone. Finally, intranasal administration of just 100 PFU of the WA1-ΔPRRA-ΔORF6-8-Nsp1K164A/H165A elicits robust antibody responses in Syrian hamsters and protects against SARS-CoV-2-induced weight loss and pneumonia. As a proof-of-concept study, we demonstrate that live but sufficiently attenuated SARS-CoV-2 vaccines may be attainable by rational design.

Long-term immunity in convalescent Syrian hamsters provides protection against new-variant SARS-CoV-2 infection of the lower but not upper respiratory tract.

COVID-19 vaccines provide high levels of protection against severe disease and hospitalization due to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection. Vaccination may be less effective in preventing shedding of infectious viruses from otherwise immune patients. In this study, we describe breakthrough infections and shedding of infectious viruses in convalescent hamsters without significant replication in the lower respiratory tract following reinfection by Alpha and Delta variants despite high levels of circulating antibodies in sera. Using convalescent hamsters with long-term immunity (up to 1 year) following infection by ancestral SARS-CoV-2, we can model aspects of recurring COVID-19 in the context of preexisting immunity.

MVA vector expression of SARS-CoV-2 spike protein and protection of adult Syrian hamsters against SARS-CoV-2 challenge.

Numerous vaccine candidates against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are under development. The majority of vaccine candidates to date are designed to induce immune responses against the viral spike (S) protein, although different forms of S antigen have been incorporated. To evaluate the yield and immunogenicity of different forms of S, we constructed modified vaccinia virus Ankara (MVA) vectors expressing full-length S (MVA-S), the RBD, and soluble S ectodomain and tested their immunogenicity in dose-ranging studies in mice. All three MVA vectors induced spike-specific immunoglobulin G after one subcutaneous immunization and serum titers were boosted following a second immunization. The MVA-S and MVA-ssM elicited the strongest neutralizing antibody responses. In assessing protective efficacy, MVA-S-immunized adult Syrian hamsters were challenged with SARS-CoV-2 (USA/WA1/2020). MVA-S-vaccinated hamsters exhibited less severe manifestations of atypical pneumocyte hyperplasia, hemorrhage, vasculitis, and especially consolidation, compared to control animals. They also displayed significant reductions in gross pathology scores and weight loss, and a moderate reduction in virus shedding was observed post challenge in nasal washes. There was evidence of reduced viral replication by in situ hybridization, although the reduction in viral RNA levels in lungs and nasal turbinates did not reach significance. Taken together, the data indicate that immunization with two doses of an MVA vector expressing SARS-CoV-2 S provides protection against a stringent SARS-CoV-2 challenge of adult Syrian hamsters, reaffirm the utility of this animal model for evaluating candidate SARS-CoV-2 vaccines, and demonstrate the value of an MVA platform in facilitating vaccine development against SARS-CoV-2.

SARS-CoV-2 infection induces protective immunity and limits transmission in Syrian hamsters.

A critical question in understanding the immunity to SARS-COV-2 is whether recovered patients are protected against re-challenge and transmission upon second exposure. We developed a Syrian hamster model in which intranasal inoculation of just 100 TCID50 virus caused viral pneumonia. Aged hamsters developed more severe disease and even succumbed to SARS-CoV-2 infection, representing the first lethal model using genetically unmodified laboratory animals. After initial viral clearance, the hamsters were re-challenged with 105 TCID50 SARS-CoV-2 and displayed more than 4 log reduction in median viral loads in both nasal washes and lungs in comparison to primary infections. Most importantly, re-challenged hamsters were unable to transmit virus to naïve hamsters, and this was accompanied by the presence of neutralizing antibodies. Altogether, these results show that SARS-CoV-2 infection induces protective immunity that not only prevents re-exposure but also limits transmission in hamsters. These findings may help guide public health policies and vaccine development and aid evaluation of effective vaccines against SARS-CoV-2.

SARS-CoV-2 B.1.1.7 Infection of Syrian Hamster Does Not Cause More Severe Disease, and Naturally Acquired Immunity Confers Protection.

Epidemiological studies have revealed the emergence of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including the lineage B.1.1.7 that is rapidly replacing old variants. The B.1.1.7 variant has been linked to increased morbidity rates, transmissibility, and potentially mortality. To assess viral fitness in vivo and to address whether the B.1.1.7 variant is capable of immune escape, we conducted infection and reinfection studies in naive and convalescent Syrian hamsters (>10 months old). Nasal wash samples from hamsters infected by a B.1.1.7 variant exhibited slightly higher viral RNA levels but lower infectious titers than those from B.1 (G614) variant-infected hamsters, and the two variants induced comparable lung pathologies in hamsters. Despite a sporadic and transient low-level infection in the nasal cavity, convalescent hamsters that had recovered from a previous USA-WA1 isolate (D614) infection displayed no observable clinical signs or lung pathology following B.1.1.7 rechallenge. Altogether, our study did not find that the B.1.1.7 variant significantly differs from the B.1 variant in pathogenicity in Syrian hamsters and that a heterologous natural infection-induced immunity confers protection against a secondary challenge by the B1.1.7 variant. IMPORTANCE The rapid emergence of several variants of concern of SARS-CoV-2 calls for evaluations of viral fitness and pathogenicity in animal models in order to understand the mechanism of enhanced transmission and the possible increases in morbidity and mortality rates. Here, we demonstrated that immunity naturally acquired through a prior infection with the first-wave variant does confer nearly complete protection against the B.1.1.7 variant in Syrian hamsters upon reexposure. Strikingly, although the B.1.1.7 variant appears to replicate to a higher level in the nose than the ancestral B.1 variant, it does not induce more severe lung pathology in hamsters.

A method for detection of SARS-CoV-2 RNA in healthy human stool: a validation study.

BACKGROUND: Faecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool. METHODS: In this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at -80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing. FINDINGS: The lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at -80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at -80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p<0·0001), at 4°C storage in DNA/RNA Shield (from 28·5 [0·15] to 29·8 [0·09]; p=0·0019), and at ambient temperature in STAR buffer (from 30·4 [0·24] to 32·4 [0·62]; p=0·0083). 30 contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, SARS-CoV-2 RNA was detected using this assay in the stool and anal swab specimens of 11 of 23 individuals known to be positive for SARS-CoV-2. INTERPRETATION: This is a sensitive and reproducible assay for detection of SARS-CoV-2 RNA in human stool, with potential uses in faecal microbiota transplantation donor screening, sewage monitoring, and further research into the effects of faecal shedding on the epidemiology of the COVID-19 pandemic. FUNDING: National Institute of Allergy and Infectious Diseases, US National Institutes of Health; Center for Biologics Evaluation and Research, US Food and Drug Administration.

The PRRA insert at the S1/S2 site modulates cellular tropism of SARS-CoV-2 and ACE2 usage by the closely related Bat RaTG13.

Biochemical and structural analyses suggest that SARS-CoV-2 is well-adapted to infecting humans and the presence of four residues (PRRA) at the S1/S2 site within the spike (S) protein, which may lead to unexpected tissue or host tropism. Here we report that SARS-CoV-2 efficiently utilized ACE2 of 9 species to infect 293T cells. Similarly, pseudoviruses bearing S protein derived from either the bat RaTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. Removal of PRRA from SARS-CoV-2 S protein displayed distinct effects on pseudoviral entry into different cell types. Unexpectedly, insertion of PRRA into the RaTG13 S protein selectively abrogated the usage of horseshoe bat and pangolin ACE2 but enhanced the usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and RaTG13 S proteins.ImportanceThe four-residue insert (PRRA) at the boundary between the S1and S2 subunits of SARS-CoV-2 has been widely recognized since day 1 for its role in SARS-CoV-2 S protein processing and activation. As this PRRA insert is unique to SARS-CoV-2 among group b betacoronaviruses, it is thought to affect the tissue and species tropism of SARS-CoV-2. We compared the usage of 10 ACE2 orthologs and found that the presence of PRRA not only affects the cellular tropism of SARS-CoV-2 but also modulates the usage of ACE2 orthologs by the closely related bat RaTG13 S protein. The binding of pseudovirions carrying RaTG13 S with a PRRA insert to mouse ACE2 was nearly 2-fold higher than that of pseudovirions carrying RaTG13 S.

The G614 pandemic SARS-CoV-2 variant is not more pathogenic than the original D614 form in adult Syrian hamsters.

Dynamic tracking of variant frequencies among viruses circulating in the global pandemic has revealed the emergence and dominance of a D614G mutation in the SARS-CoV-2 spike protein. To address whether pandemic SARS-CoV-2 G614 variant has evolved to become more pathogenic, we infected adult hamsters (>10 months old) with two natural SARS-CoV-2 variants carrying either D614 or G614 spike protein to mimic infection of the adult/elderly human population. Hamsters infected by the two variants exhibited comparable viral loads and pathology in lung tissues as well as similar amounts of virus shed in nasal washes. Altogether, our study does not find that naturally circulating D614 and G614 SARS-CoV-2 variants differ significantly in pathogenicity in hamsters.