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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
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1.The effort to develop vaccines based on economically accessible technological platforms available by developing countries vaccine manufacturers is essential to extend the immunization to the whole world population and to achieve the desired herd immunity, necessary to end the COVID-19 pandemic. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed in yeast Pichia pastoris. The RBD was modified with addition of flexible N- and C-terminal amino acid extensions aimed to modulate the protein/protein interactions and facilitate protein purification. Fermentation with yeast extract culture medium yielded 30-40 mg/L. After purification by immobilized metal ion affinity chromatography and hydrophobic interaction chromatography, the RBD protein was characterized by mass-spectrometry, circular dichroism, and binding affinity to angiotensin-converting enzyme 2 (ACE2) receptor. The recombinant protein shows high antigenicity with convalescent human sera and also with sera from individuals vaccinated with the Pfizer-BioNTech mRNA or Sputnik V adenoviral-based vaccines. The RBD protein stimulates IFN{gamma}, IL-2, IL-6, IL-4, and TNF in mice secreting splenocytes from PBMC and lung CD3+ enriched cells. Immunogenicity studies with 50 {micro}g of the recombinant RBD formulated with alum, induce high levels of binding antibodies in mice and non-human primates, assessed by ELISA plates covered with RBD protein expressed in HEK293T cells. The mouse sera inhibited the RBD binding to ACE2 receptor in an in-vitro test and show neutralization of SARS-CoV-2 infection of Vero E6 cells. These data suggest that the RBD recombinant protein expressed in yeast P. pastoris is suitable as a vaccine candidate against COVID-19. HighlightsO_LIThe RBD protein (C-RBD-H6 PP) is expressed with high purity in P. pastoris. C_LIO_LIPhysico-chemical characterization confirms the right folding of the protein. C_LIO_LIThe recombinant protein shows high antigenicity with sera from convalescents. C_LIO_LIThe sera from animals inhibit the RBD-ACE2 binding and neutralize the virus. C_LIO_LIThe C-RBD-H6 protein stimulates IFN{gamma}, IL-2, IL-6, IL-4, and TNF in mice. C_LI