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  • We also demonstrated by ELISA ELISPOT and CTL assays

    2019-05-17

    We also demonstrated by ELISA, ELISPOT and CTL assays the capacity of alum to induce a Th1 immune response. The evaluation of the smoothened response against LALF32-51 peptide, E7 protein and LALF32-51-E7 fusion protein in immunized mice suggest that the antibodies response generated by the immunization is specific and preferentially against E7 protein. In the evaluation of IgG2c and IgG1 subclasses we detected levels of IgG2c higher than IgG1 in the serum of mice immunized with the protein alone and combined with alum or VSSP, suggesting a preferentially Th1 immune response. In our experiments to measure the cellular immune response, that is responsible for the anti-tumor effect, we detected an E7-specific IFN-γ-secreting CD8+ T cell response in the groups of mice immunized with LALF32-51-E7 + Al(OH)3 and LALF32-51-E7 + VSSP. This response to the CTL epitope E749-57 was similar in both groups and was greater with respect to the group immunized with the protein without adjuvant. However, the IFN-γ response to the stimulation with the T helper peptide E748-57 was higher in the group immunized with LALF32-51-E7 + VSSP suggesting a possible role of VSSP in activate the CTL response with the contribution of the T helper lymphocytes. The results obtained in the CTL assay shows the similar cytotoxic capacity of the effector cells derived from mice immunized with the protein combined with alum or VSSP adjuvants. Taken together the results obtained indicates that the use of alum as adjuvant combined with the fusion protein LALF32-51-E7 promote a cell-mediated immunity similar to that obtained with the combination of our antigen with a Th1 adjuvant model (VSSP). The differentiation of Th1 cells requires IL-12, which promotes IFN-γ secretion by T cells and NK cells [16] and is produced by dendritic cells in response to stimulation with pathogen-associated molecular patterns. Taking into account: (a) the report suggesting the inefficiency of alum as activators of Th1 responses is a consequence of its inhibitory effects on IL-12 production [4]; (b) other reports describing that combining alum with IL-12 enhance the Th1 responses to an antigen [5,6]; (c) the properties of LALF32-51 as cell penetrating and immunomodulatory peptide [8] and (d) the particulate-aggregate nature of the LALF32-51-E7 protein obtained in E. coli[9], we investigated if our promising results with LALF32-51-E7 + Al(OH)3 could be related with the secretion of IL-12 induced by LALF32-51 peptide. In this study we demonstrated that the immunization with LALF32-51-E7 alone and with LALF32-51-E7 + Al(OH)3 promotes the secretion of IL-12 p40 in tumor-bearing mice that could be associated with the immune-modulator properties of LALF32-51 peptide that induced the expression of IL-12 p40 and IFN-γ. Also, the Al(OH)3 adjuvant didn\'t suppress the expression of IL-12 induced by LALF32-51-E7 protein. The LALF32-51 peptide is a key component of the LALF32-51-E7 fusion protein, overcoming the poor immunogenicity of the E7 protein through their cell-penetrating capacity [8] and their immunomodulatory properties based on the induction of the Th1 cytokines IL-12 and IFN-γ. That\'s why the immunization with the LALF32-51-E7 fusion protein without adjuvant generates a potent smoothened CD8+ T cell and anti-tumor responses in mice and is one of the reasons why alum improve a cell-mediated immune response when is combined with LALF32-51-E7. The experiences obtained in humans with other protein-based therapeutic HPV vaccines has demonstrated the importance of including an adjuvant in the vaccine preparation to improve the immune response necessary in the treatment of immunocompromised individuals. Despite of the well known capacity of alum adjuvant to induce a humoral immune response [2,3] instead of the well known capacity of VSSP adjuvant as a powerful inducer of the activation of cytotoxic T lymphocytes to proteins [17], we demonstrated in this study that alum can also promote the activation of the cell-mediated immunity when is combined with our antigen.