Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214 mg/L in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over four weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax™, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.
RNase S is a complex consisting of two proteolytic fragments of RNase A: the S peptide (residues 1-20) and S protein (residues 21-124). RNase S and RNase A have very similar X-ray structures and enzymatic activities. Previous experiments have shown increased rates of hydrogen exchange and greater sensitivity to tryptic cleavage for RNase S relative to RNase A. It has therefore been asserted that the RNase S complex is considerably more dynamically flexible than RNase A. In the present study we examine the differences in the dynamics of RNase S and RNase A computationally, by MD simulations, and experimentally, using trypsin cleavage as a probe of dynamics. The fluctuations around the average solution structure during the simulation were analyzed by measuring the RMS deviation in coordinates. No significant differences between RNase S and RNase A dynamics were observed in the simulations. We were able to account for the apparent discrepancy between simulation and experiment by a simple model. According to this model, the experimentally observed differences in dynamics can be quantitatively explained by the small amounts of free S peptide and S protein that are present in equilibrium with the RNase S complex. Thus, folded RNase A and the RNase S complex have identical dynamic behavior, despite the presence of a break in polypeptide chain between residues 20 and 2 I in the latter molecule. This is in contrast to what has been widely believed for over 30 years about this important fragment complementation system. Keywords: dynamics; MD simulation; ribonuclease A: ribonuclease S; tryptic cleavage Fragment complementation systems are two or more fragments of a protein that can be reconstituted to form a complex with similar structure and activity to that of the native, uncleaved protein. Fragment complementation systems are a powerful tool in the study of protein folding and stability (Taniuchi et al., 1986). RNase S is one of the most well studied fragment complementation systems. It consists of S peptide (residues 1-20) and S protein (residues 21-124), two proteolytic fragments of the 124 amino acid protein RNase A. These fragments are obtained by cleavage of the peptide bond between Ala 20 and Ser 21 of RNase A by subtilisin. The two fragments can be reconstituted to give a noncovalently bound, active S peptide:S protein complex, RNase S (Richards & Vith-
A structure-based, sequence-design procedure is proposed in which one considers a set of decoy structures that compete significantly with the target structure in being low energy conformations. The decoy structures are chosen to have strong overlaps in contacts with the putative native state. The procedure allows the design of sequences with large and small stability gaps in a random-bond heteropolymer model in both two and three dimensions by an appropriate assignment of the contact energies to both the native and nonnative contacts. The design procedure is also successfully applied to the two-dimensional HP model.
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