Obtaining a Vaccine Candidate Against Tumors Expressing the E7 Antigen of Human Papillomavirus type 18

Background: Cervical cancer is the fourth most common cancer in women worldwide and was the leading cause of death among approximately 300 000 cases in 2018. The main risk factor for developing this disease is the persistent infection of high-risk types of human papillomavirus (HPV); specifically HPV-18 is recognized as one of the major contributors for adenocarcinoma and squamous cancer. There are three prophylactic vaccines to prevent infection by HPV, but these vaccines are not effective in infected people. On the other hand, despite the success of various types of therapeutic vaccine candidates in clinical trials, none of them is commercially available to treat women with HPV-related malignancies. As the methods used for obtaining those therapeutic candidates are awfully expensive, they could be inaccessible for developing countries. In this scenario, E7 antigen of HPV is considered and ideal target for developing therapeutic vaccines. In accordance with this, the aim of this work is to obtain a vaccine candidate with high levels of purity through a profitable process, for treating tumors expressing the E7 antigen of HPV-18.Results: We have obtained the novel therapeutic vaccine candidate CIGB550-E718 that comprises the fusion between an E7 mutein of HPV-18 and the cell-penetrating peptide with immunostimulatory properties CIGB550. The expression of the fusion protein evaluated using two strains of E. coli and 16 culture media with different composition enabled us to choose the best setting in which the interest protein accounted for approximately 16% of the total cellular protein. The best results were obtained using BL21(DE3) cells as host system grown in a culture medium containing M9 salt, casein hydrolysate and glycerol as a carbon source. The recombinant protein was purified by a single affinity chromatographic step up to 90% purity. Yields of 32 mg of the CIGB550-E718 per liter of culture were achieved from 2.5 L scale.Conclusions: These results are a promising and cost-effective approach for the future production and scale-up of the protein CIGB550-E718, which could be a therapeutic alternative for treating women with lesions associated to HPV-18 infection.

Identification and characterization of heteroclitic peptides in TCR-binding positions with improved HLA-binding efficacy

The antigenicity as well as the immunogenicity of tumor associated antigens (TAAs) may need to be potentiated in order to break the immunological tolerance. To this aim, heteroclitic peptides were designed introducing specific substitutions in the residue at position 4 (p4) binding to TCR. The effect of such modifications also on the affinity to the major histocompatibility class I (MHC-I) molecule was assessed. The Trp2 antigen, specific for the mouse melanoma B16F10 cells, as well as the HPV-E7 antigen, specific for the TC1 tumor cell lines, were used as models. Affinity of such heteroclitic peptides to HLA was predicted by bioinformatics tools and the most promising ones were validated by structural conformational and HLA binding analyses. Overall, we demonstrated that TAAs modified at the TCR-binding p4 residue are predicted to have higher affinity to MHC-I molecules. Experimental evaluation confirms the stronger binding, suggesting that this strategy may be very effective for designing new vaccines with improved antigenic efficacy.

An Oncolytic Adenovirus Encoding SA-4-1BBL Adjuvant Fused to HPV-16 E7 Antigen Produces a Specific Antitumor Effect in a Cancer Mouse Model

Human papillomaviruses (HPVs) are responsible for about 25% of cancer cases worldwide. HPV-16 E7 antigen is a tumor-associated antigen (TAA) commonly expressed in HPV-induced tumors; however, it has low immunogenicity. The interaction of 4-1BBL with its receptor induces pleiotropic effects on innate, adaptive, and regulatory immunity and, if fused to TAAs in DNA vaccines, can improve the antitumor response; however, there is low transfection and antitumor efficiency. Oncolytic virotherapy is promising for antitumor gene therapy as it can be selectively replicated in tumor cells, inducing cell lysis, and furthermore, tumor cell debris can be taken in by immune cells to potentiate antitumor responses. In this study, we expressed the immunomodulatory molecule SA-4-1BBL fused to E7 on an oncolytic adenovirus (OAd) system. In vitro infection of TC-1 tumor cells and NIH-3T3 non-tumor cells with SA/E7/4-1BBL OAd demonstrated that only tumor cells are selectively destroyed. Moreover, protein expression is targeted to the endoplasmic reticulum in both cell lines when a signal peptide (SP) is added. Finally, in an HPV-induced cancer murine model, the therapeutic oncolytic activity of OAd can be detected, and this can be improved when fused to E7 and SP.

Anti-viral Effects of Superpositively Charged Mutant of Green Fluorescent Protein

Background: Supercharged GFP proteins were known as effective carriers for delivery of macromolecules into eukaryotic cells as well as fluorescent fusion tags for in vitro and in vivo detection. Objective: Herein, anti-viral effects of +36 GFP and its anti-tumor effects were studied in vitro and in vivo, respectively. Methods: We evaluated anti-HIV, anti-HSV, and anti-HCV effects of +36 GFP in vitro using ELISA, and real time PCR as common techniques for their detection, respectively. Moreover, we assessed the role of +36 GFP for eliciting HPV-related anti-tumor effects in mice due to the lack of HPV replication in vitro. Results: Our data showed that +36 GFP efficiently enter the cells and augment the transfection rate of HPV16E7 antigen, as well. Furthermore, +36 GFP significantly reduced HCV, HIV and HSV replication up to 75%, 49% and 43% in HCV-infected Huh7.5 cells, HIV-infected Hela cells and HSV-infected Vero cells, respectively. On the other hand, mice immunization with +36 GFP complexed with HPV16 E7 antigen (+36GFP + E7) or fused to HPV16 E7 antigen (+36GFP-E7) elicited a higher Th1 cellular immune response with the predominant IgG2a, IgG2b, IFN-γ and Granzyme B levels than those induced by other groups. These regimens protected mice against TC- 1 tumor challenge (~ 67%) compared to E7 protein alone (~ 33%). These data suggested that +36 GFP can act as an anti-viral agent at certain dose due to its high efficiency in cell penetration in vitro and in vivo. Conclusion: Generally, +36 GFP targets viral replication in vitro as well as helps to suppress the growth of HPV-related tumors in vivo.