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  • cyclic adenosine monophosphate E protein expressed in a plas


    E1 protein expressed in a plasmid encoding codon-optimized canine oral papillomavirus (COPV) led to therapeutic immunization of COPV-infected Beagle dogs [25], [26], which was better than that achieved with plasmids encoding E2 or E7 COPV. Moreover, E1 and E2 from cottontail rabbit papillomavirus (CRPV) induced strong immunity against infection with CRPV particles in rabbits [27], [28]. In both models, the adaptive immune system cleared virally-induced papillomas. Recently, it was found that immunization of macaques with E1/E2 sequences in an adenoviral vector induced antigen-specific T cell immunity against established primate papillomavirus (MfPV) infections [29]. Moreover, immunization of mice with HPV-16 E1 in a vaccinia vector induced specific CTL immune responses [30]. Protein cyclic adenosine monophosphate contain a variable number of T-cell-epitopes capable of binding to major histocompatibility complex class I and class II molecules (MHC-I and MHC-II) and are recognized by CD8+ CTLs or CD4 + T cells, respectively. However, purified soluble proteins generally fail to induce robust CTL responses [31] unless they are administered together with adjuvants of different chemical natures, which activate innate immunity and inflammation, leading to the induction of costimulatory signals which are critical for antigen-presenting cell (APC) [32] induction of T cell activation. In addition to being an adjuvant, α-galactosylceramide (α-GalCer) that increases the numbers of antigen-specific CD8+ T cells when co-administered with cyclic adenosine monophosphate antigens [33], [34], stimulates an NKT cell-dependent cell immune cascade involving dendritic cells, NK and T cells [35]. Moreover, α-GalCer appears to promote a better CTL immune response than other adjuvants, such as incomplete Freund’s adjuvant, IFN-α, poly (I:C) or TLR-9 agonists [33]. Its high homology among different HPV types makes the E1 protein a good candidate for a therapeutic vaccine against most HPV types in early HR-HPV infections and low-grade cervical intraepithelial lesions, where the viral genome is replicating and E1 expression is at its highest. As yet, it is not known whether antigen-specific CD8+ T cells are capable to eliminate HPV E1-expressing cells in-vivo. Here, we used a combination of HPV-E1 with the strong adjuvant α-GalCer to induce specific CD8+ T cell immune responses against HPV18-E1 expressing grafted cells. This combination induced E1-specific CD8+ T cells in mice, which correlated with a decrease in the growth of E1-expressing cells both in prophylactic and therapeutic approaches.
    Discussion Induction of cytotoxic T cell responses to viral antigens constitutes the main goal of therapeutic vaccination in chronic viral diseases, including those related to cancer, such as cervical cancer. In the current studies, we examined the role of an HPV-E1 protein fragment as an immunogen with therapeutic and/or prophylactic potential and capable of inducing HPV-E1-specific CD8+ cytotoxic T cells in a mouse model. For this, B16F0 murine melanoma cells were transfected with a codon-optimized E1244-550 plasmid, which, in contrast to the full-length HPV18-E1 reference sequence, was efficiently expressed, as reported for COPV E1 [26]. We found that mice inoculated with E1202-654 in combination with the potent adjuvant α-GalCer [33], [34], impaired the growth of B16-F0/E1244-550 bearing target cells in a therapeutic as well as in a prophylactic protocol. The resulting tumors in mice of the therapeutic protocol were smaller than in control mice, whereas in the prophylactic model four out of six mice did not develop any tumor at all. This indicates that vaccination with E1 + α-GalCer can induce a strong antitumor and potentially therapeutic response. As E1- bearing tumor growth inhibition was observed in the α-GalCer and E1202-654 + α-GalCer immunized groups, although inhibition was stronger in the E1202-654 + α-GalCer group, it was important to examine separately the ability of NK and CD8+ T cells from either mouse group ex-vivo. As expected, only mice immunized with E1202-654 + α-GalCer showed specific cytotoxic activity against B16-F0/E1244-550 cells, which is consistent with previous findings in mice immunized with Ankara vaccinia virus encoding E1 from HPV-16 [30]. We next measured an NK cell response in the α-GalCer treated mice from both protocols, which showed an NK cytotoxic activity, correlating with the unspecific antitumor effect observed in these groups. Moreover, NK cytotoxic activity was also detected in mice treated with E1202-654 + α-GalCer, indicating that the protective and therapeutic effects of E1202-654 + α-GalCer against HPV involve an adaptive branch represented by CD8+ CTLs plus an increase in NK cell activity, which acting together could contribute to a stronger anti-tumor response.