Engineering a dual vaccine against COVID-19 and tuberculosis

Abstract

The COVID-19 pandemic, caused by SARS-CoV-2 virus, has been one of the top public health threats across the world over the past three years. Mycobacterium bovis BCG is currently the only licensed vaccine for tuberculosis, one of the deadliest infectious diseases in the world, that is caused by Mycobacterium tuberculosis. In the past decades, recombinant M.bovis BCG has been studied as a novel vaccine vector for other infectious diseases in humans besides tuberculosis, such as viral infections. In the current study, we generated a recombinant M. bovis BCG strain AspikeRBD that expresses a fusion protein consisting of M. tb Ag85A protein and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using synthetic biology technique. Our results show that the recombinant M. bovis BCG strain successfully expressed this fusion protein. Interestingly, the recombinant M. bovis BCG strain AspikeRBD significantly induced SARS-CoV-2 spike-specific T cell activation and IgG production in mice when compared to the parental M.bovis BCG strain, and was more potent than the recombinant M.bovis BCG strain expressing SARS-CoV-2 spike RBD alone. As expected, the recombinant M. bovis BCG strain AspikeRBD activated an increased number of M. tb Ag85A-specific IFNγ-releasing T cells and enhanced IgG production in mice when compared to the parental M.bovis BCG strain or the BCG strain expressing SARS-CoV-2 spike RBD alone. Taken together, our results indicate a potential application of the recombinant M. bovis BCG strain AspikeRBD as a novel dual vaccine against SARS-CoV-2 and M. tb in humans.

Keywords: COVID-19; Mycobacterium bovis BCG; dual vaccine; mice; tuberculosis.

Figure 1

Generation of recombinant M. bovis BCG strains AspikeRBD and SpikeRBD. (A) Structure of the SARS-CoV-2 spike protein. SS, signal sequence; NTD, N-terminal domain; RBD, receptor-binding domain; SD1, subdomain 1; SD2, subdomain 2; TM, transmembrane anchor; IC, intracellular tail; S1, S1 subunit; S2, S2 subunit. (B) Side view of the prefusion structure of the SARS-CoV-2 spike protein with a single RBD in the up conformation (cited from Wrapp et al., 2020). (C) Schematic of the AspikeRBD and SpikeRBD engineering.

Figure 2

Verification of recombinant M. bovis BCG strains. (A) PCR analysis of the generated plasmids. pMV-AspikeRBD, pMV261 containing the DNA fragment encoding the fusion protein Ag85A::SpikeRBD; pMV-SpikeRBD, pMV261 containing the DNA fragment encoding the RBD domain of the SARS-CoV-2 spike protein. Negative Control, no DNA template. (B) Western blot for recombinant M. bovis BCG strains expressing AspikeRBD or SpikeRBD. Wild-type M. bovis BCG was used as a negative control.

Figure 3

Spike-specific T-cell response in C57BL/6 mice immunized with recombinant M. bovis BCG strains. The number of antigen-specific IFNγ-releasing T-cells in the lung and spleen was determined by ELISPOT analysis in the presence of the recombinant SARS-CoV-2 spike full protein (A) or RBD (B). Data are mean ± SD (n=3/group) and representatives of two independent experiments. Mock, unimmunized. n.s., not statistically significant; *p < 0.05, **p < 0.01 and ***p < 0.001 by two-tailed Student’s t-test.

Figure 4

Titration of spike RBD-specific IgG in C57BL/6 mice immunized with recombinant M. bovis BCG strains. SARS-CoV-2 spike RBD-specific IgG endpoint titers were determined using mouse serum that was collected 4 weeks after M.bovis BCG immunization. Mean reciprocal dilutions are used as the endpoint titer (log10). Data shown are the mean ± SD (n=3/group) and representative of two independent experiments. Mock, unimmunized. **p < 0.01 by two-tailed Student’s t-test.

Figure 5

M. tb Ag85A-specific T-cell response in C57BL/6 mice immunized with recombinant M. bovis BCG strains. The number of antigen-specific IFNγ-releasing T-cells in the lung and spleen was determined by ELISPOT analysis in the presence of the M. tb Ag85A protein. Data are mean ± SD (n=3/group) and representatives of two independent experiments. Mock, unimmunized. n.s., not statistically significant; *p < 0.05, **p < 0.01 and ***p < 0.001 by two-tailed Student’s t test.

Figure 6

Titration of M. tb Ag85A-specific IgG in C57BL/6 mice immunized with recombinant M. bovis BCG strains. M. tb Ag85A-specific IgG endpoint titers were determined using mouse serum that was collected 4 weeks after M.bovis BCG immunization. Mean reciprocal dilutions are used as the endpoint titer (log10). Data shown are the mean ± SD (n=3/group) and representative of two independent experiments. Mock, unimmunized. n.s., not statistically significant; ***p < 0.001 by two-tailed Student’s t test.