Design and adjuvant formulation of a mutant HPV-E7 protein devoid of transforming/oncogenic properties but retaining high anti-tumor cellular activities as a candidate immunotherapeutic vaccine - Bioinformatics and in vivo analyses

Background & Aim Infection with Human papillomavirus (HPV) is major cause of the cervical carcinoma. Approved vaccines based on induction of antibodies (Abs) against HPV-L1 capsid protein provide prophylactic immunity against infection but no therapeutic effect on already infected individuals who might develop cancer. Alternatively, for anti-tumor immunotherapy purposes, induction of cellular immune responses against HPV oncogenic proteins, especially E7 is required. The oncogenic potential of HPV-E7 antigen (Ag) however, promoted us to search for a modified (mutated/deleted) form of this Ag with eliminated transforming properties but retained antigenicity. Methods, Results & Conclusion Herein, we first present application of several immune-informatics tools to evaluate the changes in the antigenicity, MHC binding/processing and cellular responses of the reported HPV16-E7 mutations responsible for its main transformation capabilities including del 21-24, C24G, L67R, C58G,C91G, M84S, V69A, L79A, QKP96-98EEA and their various combinations. Finally, the HPV16-E7 with triple substitutions of C24G/L67R/C91G (mE7) which showed the maximal antigenic similarity profile to that of the wild type was synthesized and inserted in pET28a vector for expression of protein in E.coli. SDS-PAGE and WB analyses confirmed the expression of the 17 kDa recombinant mE7 protein which was purified by NI-NTA chromatography using the vector encoded 6xHis-taq. The E.coli-derived Ag in combination with human compatible adjuvants including: Montanaide ISA 50 (M50), ISA 720 (M720), Pluronic acid (F127) in mixture with Immunostimulatory CpG ODN 1826 (mE7+M50/CpG; mE7+M720/CpG; mE7+F127/CpG, respectively) was evaluated in immunized C57BL/6 mice. Evaluation of the lymphocytes replication by the Brdu method and cytokine measurements by ELISA indicated that all groups developed significant immune responses, but mice immunized with mE7+M50/CpG showed the highest stimulation index (0.4) and INF-γ levels (150 pg/ml) comparable to those immunized by mE7+complete/incomplete Freund adjuvants (C/IFA). Accordingly, the IL-4 cytokine levels were almost similar and lower (20 pg/ml) for all groups indicating a Th1 polarization. Results of the tumor-challenge studies on TC1 cells in tumor bearing mice showed complete reduction/inhibition of tumor growth for mE7+M50/CpG immunized mice, indicating the potency of this formulation as a candidate immunotherapeutic vaccine against HPV infection-induced cancer. Infection with Human papillomavirus (HPV) is major cause of the cervical carcinoma. Approved vaccines based on induction of antibodies (Abs) against HPV-L1 capsid protein provide prophylactic immunity against infection but no therapeutic effect on already infected individuals who might develop cancer. Alternatively, for anti-tumor immunotherapy purposes, induction of cellular immune responses against HPV oncogenic proteins, especially E7 is required. The oncogenic potential of HPV-E7 antigen (Ag) however, promoted us to search for a modified (mutated/deleted) form of this Ag with eliminated transforming properties but retained antigenicity. Herein, we first present application of several immune-informatics tools to evaluate the changes in the antigenicity, MHC binding/processing and cellular responses of the reported HPV16-E7 mutations responsible for its main transformation capabilities including del 21-24, C24G, L67R, C58G,C91G, M84S, V69A, L79A, QKP96-98EEA and their various combinations. Finally, the HPV16-E7 with triple substitutions of C24G/L67R/C91G (mE7) which showed the maximal antigenic similarity profile to that of the wild type was synthesized and inserted in pET28a vector for expression of protein in E.coli. SDS-PAGE and WB analyses confirmed the expression of the 17 kDa recombinant mE7 protein which was purified by NI-NTA chromatography using the vector encoded 6xHis-taq. The E.coli-derived Ag in combination with human compatible adjuvants including: Montanaide ISA 50 (M50), ISA 720 (M720), Pluronic acid (F127) in mixture with Immunostimulatory CpG ODN 1826 (mE7+M50/CpG; mE7+M720/CpG; mE7+F127/CpG, respectively) was evaluated in immunized C57BL/6 mice. Evaluation of the lymphocytes replication by the Brdu method and cytokine measurements by ELISA indicated that all groups developed significant immune responses, but mice immunized with mE7+M50/CpG showed the highest stimulation index (0.4) and INF-γ levels (150 pg/ml) comparable to those immunized by mE7+complete/incomplete Freund adjuvants (C/IFA). Accordingly, the IL-4 cytokine levels were almost similar and lower (20 pg/ml) for all groups indicating a Th1 polarization. Results of the tumor-challenge studies on TC1 cells in tumor bearing mice showed complete reduction/inhibition of tumor growth for mE7+M50/CpG immunized mice, indicating the potency of this formulation as a candidate immunotherapeutic vaccine against HPV infection-induced cancer.