A Bivalent Recombinant Mycobacterium bovis BCG Expressing the S1 Subunit of the Pertussis Toxin Induces a Polyfunctional CD4+ T Cell Immune Response.

BACKGROUND: A recombinant BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (rBCG-S1PT), previously constructed by our research group, demonstrated the ability to develop high protection in mouse models of pertussis challenge which correlated with the induction of a Th1 immune response pattern. The Th1 immune response induced by rBCG-S1PT treatment was also confirmed in the murine orthotopic bladder cancer model, in which the intravesical instillation of rBCG-S1PT resulted in an improved antitumor effect. Based on these observations, we hypothesize that the reengineering of the S1PT expression in BCG could increase the efficiency of the protective Th1 immune response in order to develop a new alternative of immunotherapy in bladder cancer treatment. OBJECTIVES: To construct rBCG strains expressing S1PT from extrachromosomal (rBCG-S1PT) and integrative vectors (rBCG-Sli), or their combination, generating the bivalent strain (rBCG-S1+S1i), and to evaluate the respective immunogenicity of rBCG strains in mice. METHODS: Mycobacterial plasmids were constructed by cloning the s1pt gene under integrative and extrachromosomal vectors and used to transform BCG, individually or in combination. Antigen expression and localization were confirmed by Western blot. Mice were immunized with wild-type BCG or the rBCG strains, and cytokines quantification and flow cytometry analysis were performed in splenocytes culture stimulated with mycobacterial-specific proteins. FINDINGS: S1PT expression was confirmed in all rBCG strains. The extrachromosomal vector directs S1PT to the cell wall-associated fraction, while the integrative vector directs its expression mainly to the intracellular fraction. Higher levels of IFN-γ were observed in the splenocytes culture from the group immunized with rBCG-S1i in comparison to BCG or rBCG-S1PT. rBCG-S1+S1i showed higher levels of CD4+ IFN-γ + and double-positive CD4+ IFN-γ + TNF-α + T cells. CONCLUSIONS: rBCG-S1+S1i was able to express the two forms of S1PT and elicited higher induction of polyfunctional CD4+ T cells, indicating enhanced immunogenicity and suggesting its use as immunotherapy for bladder cancer.

In vitro Evidence of Human Immune Responsiveness Shows the Improved Potential of a Recombinant BCG Strain for Bladder Cancer Treatment.

The live attenuated mycobacterial strain BCG, in use as vaccine against tuberculosis, is considered the gold standard for primary therapy of carcinoma in situ of the bladder. Despite its limitations, to date it has not been surpassed by any other treatment. Our group has developed a recombinant BCG strain expressing the detoxified S1 pertussis toxin (rBCG-S1PT) that proved more effective than wild type BCG (WT-BCG) in increasing survival time in an experimental mouse model of bladder cancer, due to the well-known adjuvant properties of pertussis toxin. Here, we investigated the capacity of rBCG-S1PT to stimulate human immune responses, in comparison to WT-BCG, using an in vitro stimulation assay based on human whole blood cells that allows for a comprehensive evaluation of leukocyte activation. Blood leukocytes stimulated with rBCG-S1PT produced increased levels of IL-6, IL-8, and IL-10 as compared to WT-BCG, but comparable levels of IL-1ß, IL-2, IFN-γ, and TNF-α. Stimulation of blood cells with the recombinant BCG strain also enhanced the expression of CD25 and CD69 on human CD4+ T cells. PBMC stimulated with rBCG-S1PT induced higher cytotoxicity to MB49 bladder cancer cells than WT-BCG-stimulated PBMC. These results suggest that the rBCG-S1PT strain is able to activate an immune response in human leukocytes that is higher than that induced by WT-BCG for parameters linked to better prognosis in bladder cancer (regulation of immune and early inflammatory responses), while fully comparable to WT-BCG for classical inflammatory parameters. This establishes rBCG-S1PT as a new highly effective candidate as immunotherapeutic agent against bladder cancer.