GTL001 and bivalent CyaA-based therapeutic vaccine strategies against human papillomavirus and other tumor-associated antigens induce effector and memory T-cell responses that inhibit tumor growth.

GTL001 is a bivalent therapeutic vaccine containing human papillomavirus (HPV) 16 and HPV18 E7 proteins inserted in the Bordetella pertussis adenylate cyclase (CyaA) vector intended to prevent cervical cancer in HPV-infected women with normal cervical cytology or mild abnormalities. To be effective, therapeutic cervical cancer vaccines should induce both a T cell-mediated effector response against HPV-infected cells and a robust CD8+ T-cell memory response to prevent potential later infection. We examined the ability of GTL001 and related bivalent CyaA-based vaccines to induce, in parallel, effector and memory CD8+ T-cell responses to both vaccine antigens. Intradermal vaccination of C57BL/6 mice with GTL001 adjuvanted with a TLR3 agonist (polyinosinic-polycytidylic acid) or a TLR7 agonist (topical 5% imiquimod cream) induced strong HPV16 E7-specific T-cell responses capable of eradicating HPV16 E7-expressing tumors. Tumor-free mice also had antigen-specific memory T-cell responses that protected them against a subsequent challenge with HPV18 E7-expressing tumor cells. In addition, vaccination with bivalent vaccines containing CyaA-HPV16 E7 and CyaA fused to a tumor-associated antigen (melanoma-specific antigen A3, MAGEA3) or to a non-viral, non-tumor antigen (ovalbumin) eradicated HPV16 E7-expressing tumors and protected against a later challenge with MAGEA3- and ovalbumin-expressing tumor cells, respectively. These results show that CyaA-based bivalent vaccines such as GTL001 can induce both therapeutic and prophylactic anti-tumor T-cell responses. The CyaA platform can be adapted to different antigens and adjuvants, and therefore may be useful for developing other therapeutic vaccines.

Improving administration regimens of CyaA-based vaccines using TRAP assays to detect antigen-specific CD8(+) T cells directly ex vivo.

Vaccination with recombinant adenylate cyclase of Bordetella pertussis (CyaA) carrying antigen is a promising approach to target antigen-presenting cells. We have used Trogocytosis Analysis Protocol (TRAP) assays to monitor immune responses raised by different vaccination regimens with recombinant CyaA carrying the ovalbumin antigen. We find that the intradermal, intramuscular or subcutaneous routes are all superior to intravenous injections, and actually lead to a sufficiently high frequency of reactive CTL to be detected and characterized directly ex vivo by TRAP assay or other standard assays. Finally, for all routes, we find a clear boosting effect upon re-injection of the vaccine.

Formulation of the adenylate cyclase toxin of Bordetella pertussis as protein-coated microcrystals.

Adenylate cyclase toxin (CyaA) is an important virulence factor of Bordetella pertussis, the causative agent of whooping cough, and, in its detoxified form, a potential component of acellular pertussis vaccines. This study reports the application of a novel technology, formulation of CyaA as protein-coated microcrystals (PCMC), to improve the performance of CyaA as a vaccine component. CyaA is normally stored in a high urea concentration to prevent aggregation and to maintain stability of the protein. The aim of the work was to stabilise CyaA on a crystalline support to create a dry powder that could be reconstituted in aqueous buffer, free of urea. CyaA, formulated as PCMC with microcrystals of dl-valine, retained full adenylate cyclase (AC) and cell invasive (cytotoxic) activities after solubilistion in urea buffer. After storage as a dry powder at 37 degrees C for 2 weeks, the AC activity recovered from the CyaA-PCMC was only marginally reduced when solubilised in urea buffer. No AC activity was detected after attempts to solubilise CyaA-PCMC in aqueous buffer alone, in the absence of urea. Inclusion of various ionic, non-ionic or zwitterionic detergents in the aqueous buffer had little effect on recovery of CyaA activities. However, preparation of PCMC with CyaA plus calmodulin (CaM) or bovine serum albumin (BSA) or with both proteins allowed restoration of AC and cytotoxic activities of CyaA upon solubilisation in aqueous buffer. Incorporation of BSA and CaM with CyaA allowed essentially full recovery of AC activity but lower recovery of cytotoxicity. CyaA-CaM-BSA-PCMC, after reconstitution in aqueous buffer, induced a strong serum IgG response to CyaA when injected subcutaneously into mice.

Adjuvant effects of adenylate cyclase toxin of Bordetella pertussis after intranasal immunisation of mice.

This study examined the ability of the adenylate cyclase toxin (CyaA) of Bordetella pertussis to act as a mucosal adjuvant for other antigens when co-administered by the intranasal route in mice. Two forms of CyaA were used: the cell-invasive, enzymically active form and a cell-invasive toxin lacking adenylate cyclase enzymic activity (CyaA*). Co-administration intranasally (i/n) of CyaA or CyaA* with ovalbumin (Ova) significantly enhanced (P<0.05) anti-Ova IgG and IgA antibody responses in the serum and anti-Ova IgA responses in lung and nasal secretions compared to those generated by immunisation i/n with Ova alone. The effects were greater with CyaA*. Administration of CyaA* with Ova induced priming of Ova-specific T cells in vivo to a greater extent than that obtained after immunisation with Ova alone. Co-administration of CyaA or CyaA* with pertactin (Prn) significantly enhanced (P<0.05) the serum anti-Prn IgG responses and immunisation with Prn and CyaA* significantly increased the anti-Prn IgA responses in the lungs compared with responses after immunisation with Prn alone. Immunisation i/n with Prn alone partially protected mice (P<0.05) against challenge i/n with B. pertussis. Co-administration of CyaA or CyaA* with pertactin (Prn) significantly increased protection (P<0.05) against challenge compared to that obtained with Prn alone. These effects were particularly apparent with CyaA* as the adjuvant.

Antigen targeting to CD11b allows efficient presentation of CD4+ and CD8+ T cell epitopes and in vivo Th1-polarized T cell priming.

Bordetella pertussis adenylate cyclase (CyaA) is an invasive bacterial toxin that delivers its N-terminal catalytic domain into the cytosol of eukaryotic cells bearing the alpha(M)beta(2) integrin (CD11b/CD18), such as myeloid dendritic cells. This allows use of engineered CyaA for targeted delivery of CD8(+) T cell epitopes into the MHC class I pathway of APC and induction of robust and protective cytotoxic responses. In this study, we demonstrate that CyaA can efficiently codeliver both a CD8(+) T cell epitope (OVA(257-264)) and a CD4(+) T cell epitope (MalE(100-114)) into, respectively, the conventional cytosolic or endocytic routes of processing of murine bone marrow-derived dendritic cells. Upon CyaA delivery, a strong potentiation of the MalE(100-114) CD4(+) T cell epitope presentation is observed as compared with the MalE protein, which depends on CyaA interaction with its CD11b receptor and its subsequent clathrin-mediated endocytosis. In vivo, CyaA induces strong and specific Th1 CD4(+) and CD8(+) T cell responses against, respectively, the MalE(100-114) and OVA(257-264) epitopes. These results underscore the potency of CyaA for design of new vaccines.

Adenylate cyclase toxin from Bordetella pertussis synergizes with lipopolysaccharide to promote innate interleukin-10 production and enhances the induction of Th2 and regulatory T cells.

Adenylate cyclase toxin (CyaA) from Bordetella pertussis can subvert host immune responses allowing bacterial colonization. Here we have examined its adjuvant and immunomodulatory properties and the possible contribution of lipopolysaccharide (LPS), known to be present in purified CyaA preparations. CyaA enhanced antigen-specific interleukin-5 (IL-5) and IL-10 production and immunoglobulin G1 antibodies to coadministered antigen in vivo. Antigen-specific CD4(+)-T-cell clones generated from mice immunized with antigen and CyaA had cytokine profiles characteristic of Th2 or type 1 regulatory T (Tr1) cells. Since innate immune cells direct the induction of T-cell subtypes, we examined the influence of CyaA on activation of dendritic cells (DC) and macrophages. CyaA significantly augmented LPS-induced IL-6 and IL-10 and inhibited LPS-driven tumor necrosis factor alpha and IL-12p70 production from bone marrow-derived DC and macrophages. CyaA also enhanced cell surface expression of CD80, CD86, and major histocompatibility class II on immature DC. The stimulatory activity of our CyaA preparation for IL-10 production and CD80, CD86, and major histocompatibility complex class II expression was attenuated following the addition of polymyxin B or with the use of DC from Toll-like receptor (TLR) 4-defective mice. However, treatment of DC with LPS alone at the concentration present in the CyaA preparation (0.2 ng/ml) failed to activate DC in vitro. Our findings demonstrate that activation of innate cells in vitro by CyaA is dependent on a second signal through a TLR and that CyaA can promote Th2/Tr1-cell responses by inhibiting IL-12 and promoting IL-10 production by DC and macrophages.