Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants.

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.

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Déclaration de conflit d'intérêts

Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sergio Andre Rodriguez-Aponte, Neil Chandra Dalvie, and J. Christopher Love have filed a patent related to the RBD-L452K-F490W (RBD-J) sequence. J. Christopher Love has interests in Sunflower Therapeutics PBC, Honeycomb Biotechnologies, OneCyte Biotechnologies, QuantumCyte, and Repligen. J.C.L.’s interests are reviewed and managed under MIT’s policies for potential conflicts of interest. Harish D. Rao, Meghraj P. Rajurkar, Rakesh R. Lothe, Umesh S. Shaligram, Saurabh Batwal, Rahul Chandrasekaran, Gaurav Nagar are employees of Serum Institute of India Pvt. Ltd. Sumi Biswas is an employee of SpyBiotech Limited.