SARS-CoV-2 RBD-Tetanus Toxoid Conjugate Vaccine Induces a Strong Neutralizing Immunity in Preclinical Studies

Abstract

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In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI–MS spectrum

“…The identification of the highly hydrophilic C-terminal peptides of these RBD proteins containing a His 6 tag and twelve histidine residues, an important aspect requested in the ICHQ6B guidelines, was always possible by applying the in-solution BFD while with the SD sample processing, the identification was achieved only in few cases. The results shown here support that in-solution BFD protocol in combination with ESI-MS analysis can be implemented successfully for the characterization of RBDs used as active pharmaceutical ingredients of SARS-CoV-2 subunit-based vaccines [31,57] including those derived from mutated variants of the virus [4,58,59].…”

“…Cysteinylation at Cys 538 has been reported by other authors [28,31], but to our knowledge, the other Cysmodifying groups (Table 3) have not previously been reported for recombinant RBDs. The species with Cys 538 modifications and O-glycoforms detected at protein level (Table 2) were further confirmed at tryptic peptide level by the in-solution BFD (Table 3).…”

“…2c) shows the most intense signal with molecular mass of 27,195.08 Da that agreed with the expected mass (27,195.46 Da) considering the N-deglycosylated monomer of RBD (319-541) -HEK_A 3 , cysteinylated and O-glycosylated with HexNAc:Hex:NeuAc 2 (Table 2). Other groups that also expressed RBD molecules in HEK-293 with an odd number of cysteine residues reported cysteinylation [28,31]. O-glycosylation has been reported for the native RBD of SARS-Cov-2 [32,33] as well as for several RBD versions expressed in mammalian cells [28,31].…”

“…Other groups that also expressed RBD molecules in HEK-293 with an odd number of cysteine residues reported cysteinylation [28,31]. O-glycosylation has been reported for the native RBD of SARS-Cov-2 [32,33] as well as for several RBD versions expressed in mammalian cells [28,31].…”

“…RBD (319-541) -HEK_A 3 (Table 1) expressed in HEK-293 T mammalian cell line has four disulfide bonds and a free cysteine residue (Cys 538 ) located towards the C-terminal region of the protein. The high reactivity of Cys 538 can be used for site-directed chemical conjugation to highly immunogenic carrier proteins such as tetanus toxoid [31].…”