Efficacy of an unmodified bivalent mRNA vaccine against SARS-CoV-2 variants in female small animal models.

Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve control of the COVID-19 pandemic. We compare monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein in a transgenic mouse and a Wistar rat model. The blended low-dose bivalent mRNA vaccine contains half the mRNA of each respective monovalent vaccine, but induces comparable neutralizing antibody titres, enrichment of lung-resident memory CD8+ T cells, antigen-specific CD4+ and CD8+ responses, and protects transgenic female mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduces viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized female Wistar rats also contain neutralizing antibodies against the B.1.1.529 (Omicron BA.1 and BA.5) variants. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins are feasible approaches for extending the coverage of vaccines for emerging and co-circulating SARS-CoV-2 variants.

Next-generation diagnostics: virus capture facilitates a sensitive viral diagnosis for epizootic and zoonotic pathogens including SARS-CoV-2.

BACKGROUND: The detection of pathogens in clinical and environmental samples using high-throughput sequencing (HTS) is often hampered by large amounts of background information, which is especially true for viruses with small genomes. Enormous sequencing depth can be necessary to compile sufficient information for identification of a certain pathogen. Generic HTS combining with in-solution capture enrichment can markedly increase the sensitivity for virus detection in complex diagnostic samples. METHODS: A virus panel based on the principle of biotinylated RNA baits was developed for specific capture enrichment of epizootic and zoonotic viruses (VirBaits). The VirBaits set was supplemented by a SARS-CoV-2 predesigned bait set for testing recent SARS-CoV-2-positive samples. Libraries generated from complex samples were sequenced via generic HTS (without enrichment) and afterwards enriched with the VirBaits set. For validation, an internal proficiency test for emerging epizootic and zoonotic viruses (African swine fever virus, Ebolavirus, Marburgvirus, Nipah henipavirus, Rift Valley fever virus) was conducted. RESULTS: The VirBaits set consists of 177,471 RNA baits (80-mer) based on about 18,800 complete viral genomes targeting 35 epizootic and zoonotic viruses. In all tested samples, viruses with both DNA and RNA genomes were clearly enriched ranging from about 10-fold to 10,000-fold for viruses including distantly related viruses with at least 72% overall identity to viruses represented in the bait set. Viruses showing a lower overall identity (38% and 46%) to them were not enriched but could nonetheless be detected based on capturing conserved genome regions. The internal proficiency test supports the improved virus detection using the combination of HTS plus targeted enrichment but also points to the risk of cross-contamination between samples. CONCLUSIONS: The VirBaits approach showed a high diagnostic performance, also for distantly related viruses. The bait set is modular and expandable according to the favored diagnostics, health sector, or research question. The risk of cross-contamination needs to be taken into consideration. The application of the RNA-baits principle turned out to be user friendly, and even non-experts can easily use the VirBaits workflow. The rapid extension of the established VirBaits set adapted to actual outbreak events is possible as shown for SARS-CoV-2. Video abstract.

SARS-CoV-2 in fruit bats, ferrets, pigs, and chickens: an experimental transmission study.

BACKGROUND: In December, 2019, a novel zoonotic severe acute respiratory syndrome-related coronavirus emerged in China. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became pandemic within weeks and the number of human infections and severe cases is increasing. We aimed to investigate the susceptibilty of potential animal hosts and the risk of anthropozoonotic spill-over infections. METHODS: We intranasally inoculated nine fruit bats (Rousettus aegyptiacus), ferrets (Mustela putorius), pigs (Sus scrofa domesticus), and 17 chickens (Gallus gallus domesticus) with 105 TCID50 of a SARS-CoV-2 isolate per animal. Direct contact animals (n=3) were included 24 h after inoculation to test viral transmission. Animals were monitored for clinical signs and for virus shedding by nucleic acid extraction from nasal washes and rectal swabs (ferrets), oral swabs and pooled faeces samples (fruit bats), nasal and rectal swabs (pigs), or oropharyngeal and cloacal swabs (chickens) on days 2, 4, 8, 12, 16, and 21 after infection by quantitative RT-PCR (RT-qPCR). On days 4, 8, and 12, two inoculated animals (or three in the case of chickens) of each species were euthanised, and all remaining animals, including the contacts, were euthanised at day 21. All animals were subjected to autopsy and various tissues were collected for virus detection by RT-qPCR, histopathology immunohistochemistry, and in situ hybridisation. Presence of SARS-CoV-2 reactive antibodies was tested by indirect immunofluorescence assay and virus neutralisation test in samples collected before inoculation and at autopsy. FINDINGS: Pigs and chickens were not susceptible to SARS-CoV-2. All swabs, organ samples, and contact animals were negative for viral RNA, and none of the pigs or chickens seroconverted. Seven (78%) of nine fruit bats had a transient infection, with virus detectable by RT-qPCR, immunohistochemistry, and in situ hybridisation in the nasal cavity, associated with rhinitis. Viral RNA was also identified in the trachea, lung, and lung-associated lymphatic tissue in two animals euthanised at day 4. One of three contact bats became infected. More efficient virus replication but no clinical signs were observed in ferrets, with transmission to all three direct contact animals. Mild rhinitis was associated with viral antigen detection in the respiratory and olfactory epithelium. Prominent viral RNA loads of 0-104 viral genome copies per mL were detected in the upper respiratory tract of fruit bats and ferrets, and both species developed SARS-CoV-2-reactive antibodies reaching neutralising titres of up to 1/1024 after 21 days. INTERPRETATION: Pigs and chickens could not be infected intranasally by SARS-CoV-2, whereas fruit bats showed characteristics of a reservoir host. Virus replication in ferrets resembled a subclinical human infection with efficient spread. Ferrets might serve as a useful model for further studies-eg, testing vaccines or antivirals. FUNDING: German Federal Ministry of Food and Agriculture.