Background The rate at which COVID-19 has spread throughout the globe has been alarming. While the role of fomite transmission is not yet fully understood, precise data on the environmental stability of SARS-CoV-2 is required to determine the risks of fomite transmission from contaminated surfaces. Methods This study measured the survival rates of infectious SARS-CoV-2, suspended in a standard ASTM E2197 matrix, on several common surface types. All experiments were carried out in the dark, to negate any effects of UV light. Inoculated surfaces were incubated at 20 °C, 30 °C and 40 °C and sampled at various time points. Results Survival rates of SARS-CoV-2 were determined at different temperatures and D-values, Z-values and half-life were calculated. We obtained half lives of between 1.7 and 2.7 days at 20 °C, reducing to a few hours when temperature was elevated to 40 °C. With initial viral loads broadly equivalent to the highest titres excreted by infectious patients, viable virus was isolated for up to 28 days at 20 °C from common surfaces such as glass, stainless steel and both paper and polymer banknotes. Conversely, infectious virus survived less than 24 h at 40 °C on some surfaces. Conclusion These findings demonstrate SARS-CoV-2 can remain infectious for significantly longer time periods than generally considered possible. These results could be used to inform improved risk mitigation procedures to prevent the fomite spread of COVID-19.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80–100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25–250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.