ABSTRACT
BackgroundSOBERANA 02 is a COVID-19 conjugate vaccine candidate based on SARS-CoV-2 recombinant RBD conjugated to tetanus toxoid. SOBERANA Plus antigen is dimeric-RBD. Here we report safety, reactogenicity and immunogenicity from phase I and IIa clinical trials using two-doses SOBERANA 02 (homologous protocol) and three-doses (homologous) or heterologous (with SOBERANA Plus) protocols. MethodWe performed an open-label, monocentric, sequential and adaptive phase I for evaluating safety, reactogenicity and exploring immunogenicity of SOBERANA 02 in two formulations (15 and 25 g) in 40 subjects, 19-59 years old. Phase IIa was open-label including 100 volunteers 19-80 years, receiving two doses of SOBERANA 02-25 g. In both trials, half of volunteers received a third dose of SOBERANA 02, half received a heterologous dose of SOBERANA Plus-50 g. Primary outcomes were safety and reactogenicity. The secondary outcome was vaccine immunogenicity evaluated by anti-RBD IgG ELISA, molecular neutralization test of RBD:hACE2 interaction, live-virus neutralization test and specific T-cells response. ResultsThe most frequent AE was local pain, other AEs had frequencies [≤] 5%. No serious related AEs were reported. Phase IIa confirmed the safety results in 60-80 years subjects. In phase-I SOBERANA 02-25{micro}g elicited higher immune response than SOBERANA 02-15 {micro}g; in consequence, the higher dose progressed to phase IIa. Phase IIa results confirmed the immunogenicity of SOBERANA 02-25 g even in 60-80 age range. Two doses of SOBERANA02-25 g elicited an immune response similar to that of the Cuban Convalescent Serum Panel; it was higher after both the homologous and heterologous third doses; the heterologous scheme showing a higher immunological response. ConclusionsSOBERANA 02 was safe and immunogenic in persons aged 19-80 years, eliciting neutralizing antibodies and specific T cell response. Highest immune responses were obtained in the heterologous three doses protocol. Trial registry: https://rpcec.sld.cu/trials/RPCEC00000340 and https://rpcec.sld.cu/trials/RPCEC00000347
ABSTRACT
Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage ([≥] 99 %) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.
