# 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

## TL;DR

A new vaccine strategy using modified BCG and RBD proteins induces long-term immunity and cross-protection against SARS-CoV-2 variants like Omicron.

## Contribution

The fusion of RBD with LTB adjuvant in a BCG-based vaccine induces cross-reactive immunity against SARS-CoV-2 Omicron variant.

## Key findings

- Both rBCG-RBD/rRBD and rBCG-LTB-RBD/rRBD vaccine schemes induced high IgG antibody titers and neutralization against Wuhan SARS-CoV-2.
- Only rBCG-LTB-RBD/rRBD induced cross-reactive neutralization against Omicron SARS-CoV-2.
- The vaccines promoted Th1/Th2 cytokine-producing CD4+ and CD8+ T cell responses.

## Abstract

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.