Long-Term Protective Immune Responses Induced by rBCG-RBD/rRBD Heterologous Prime/Boost Immunization Strategy: Fusion of RBD-Wuhan with LTB Adjuvant Induces Cross-Reactivity with SARS-CoV-2 Variant Omicron

Background/Objectives : SARS-CoV-2, the causative agent of COVID-19, has been responsible for more than seven million deaths worldwide since its emergence. The Bacillus Calmette-Guérin (BCG) vaccine, used for over 100 years to prevent tuberculosis, induces a Th1-prominent immune response that is important for protection against Mycobacterium tuberculosis , other mycobacteria, and intracellular pathogens. BCG has also been shown to induce innate immune memory and heterologous protection against non-related infections. Additionally, BCG has been used as a vector to express heterologous proteins, showing protective effects against various diseases, particularly respiratory viral infections, including SARS-CoV-2. In this report, we constructed two recombinant BCG strains as potential vaccine candidates based on the receptor-binding domain (RBD) of the Spike antigen: one expressing only the RBD protein (rBCG-RBD) and another expressing the RBD protein in fusion with the LTB ( Escherichia coli Labile Toxin subunit B) adjuvant (rBCG-LTB-RBD). Methods : We evaluated the induction of SARS-CoV-2-specific humoral and cellular immune responses using these vaccine candidates in a prime-boost strategy with a booster dose using the rRBD protein (produced in cell culture) and the Alum adjuvant. Antisera were evaluated for neutralization of the Wuhan and Omicron SARS-CoV-2 pseudotyped virus. Results : Either immunization scheme (rBCG-RBD/rRBD or rBCG-LTB-RBD/rRBD) induced high IgG antibody titers, with antibody neutralization against a Wuhan SARS-CoV-2 pseudotyped virus after 10 weeks. The antibody levels induced by rBCG-RBD/rRBD were maintained for up to 9 months. Interestingly, only the sera from mice receiving the prime-boost with rBCG-LTB-RBD/rRBD showed cross-reactive neutralization against the Omicron SARS-CoV-2 pseudotyped virus. Immunization with rBCG-RBD or rBCG-LTB-RBD and a rRBD booster dose promoted the induction of specific CD4+ and CD8+ T cells producing Th1/Th2 cytokines (IL-4, TNF-α and IFN-γ). Conclusions : Our study highlights the potential of the prime-boost immunization strategy using rBCG-RBD/rRBD to induce long-term immunity and rBCG-LTB-RBD/rRBD to induce cross-protection against different variants, both of which could serve as promising vaccine candidates.