COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette–Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.

This work was supported by the grants funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (grants 303044/2020-9, 401209/2020-2, and 465229/2014-0), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior grants 88887.506612/2020-00 and 88887.504421/2020-00, Fundação de Amparo à Pesquisa do Estado de São Paulo (grants 2017/24832-6 and 2023/02577-5), Pro-Reitoria de Pesquisa-Universidade de São Paulo (Pro-Reitoria de Pesquisa USP), Howard Hughes Medical Institute (grant 55007412) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais Rede Mineira de Imunobiologicos (grant REDE-00140-16).

The data presented in this article have been submitted to GenBank ( under accession number MT126808.1, the National Center for Biotechnology Information ( under accession number YP_009724390.1, and the National Center for Biotechnology ( under accession number YP_009724397.2.

The online version of this article contains supplemental material.

This content is only available via PDF.

Supplementary data