Our results demonstrated that fusion peptides composed of a highly-immunogenic epitope and a poorly-immunoreactive tumor-associated antigen can effectively activate host immunity, and furthermore, using the prediction algorithm SYFPEITHI to enhance the affinity of antigen presentation, the resulting fusion peptides provoked even stronger immune responses. In addition, this study also provided new insight into tumor-specific immunity. By combining SARS-CoV epitope, a tumor unrelated antigenic fragment, with CEA, we finally demonstrated this could be a promising anti-cancer strategy through effectively increasing both Th1 and Th2 cytokines and decreasing tumor volume.
The DNA vaccine, though usually injected intramuscularly, can be administered orally in an animal model with bacteria, and it has been observed that some display preferential replication or preferential accumulation in the tumor microenvironment . Among all bacteria, S. typhimurium is one of the most utilized to carry therapeutic transgenes. Attenuation of S. typhimurium with partial gene modifications does not affect its ability to achieve high tumor/normal tissue ratios in mouse models, and the bacteria maintain their capacity to inhibit the growth of both subcutaneous tumors and lung metastatic diseases . Other modifications have also been reported; for example, the strain used in this study was attenuated aromatic acid-dependent (aro) S. typhimurium, which has been well-characterized as a carrier of various heterogeneous antigens [9, 10]. These vaccine strains are capable of colonizing the gut-associated lymphoid tissues (Peyer's patches) and secondary lymphatic tissues including the spleen and liver following oral administration in mice, eliciting mucosal, humoral and cell-mediated immune responses . Previous studies have proven the lipopolysaccharides on the Salmonella surface enable CD14+ monocytes to secrete TNF-α, IL-1 and IL-6 [12, 13]. Also S. Typhimurium could deliver directly into the cytosol of macrophages and dendritic cells resulting in vigorous antigen-specific CD8+ T cell priming and the induction of protective immunity against viruses, bacteria and tumors [14, 15].
Variations in the cytokine profiles arose after vaccination with pCEA-SARS-CoV. As a highly-immunogenic peptide, treatment with SARS-containing epitopes has elicited a striking increase in TNF-α expression as compared with the negative control and the CEA group (Figure 2a). TNF-α is a proinflammatory and Th1 cytokine, and as indicated by Austin and colleagues, TNF-α, IFN-γ, and IL-2 can be used to define cytotoxic T lymphocytes and Th1 effector populations . Reducing local expression of TNF-α was found to be associated with B16-F10 melanoma outgrowth in mice while s.c. administrating TNF-α significantly suppressed primary tumor growth of melanoma . IL-10, a typical Th2 cytokine, was enhanced in the pCEA-SARS-CoV group as compared with the negative control and the CEA group (Figure 2b). Though most often known as a Th2 cytokine, IL-10 is actually a pleiotropic cytokine with anti-inflammatory, immunosuppressive, and immunostimulatory properties , and exerts immunostimulatory effects on B cells, cytotoxic T-cell development and thymocytes . As indicated by Wogensen et al., expression of an IL-10 transgene by insulin-producing pancreatic cells led to the accelerated onset of diabetes in NOD mice [20, 21], with no inhibition of immune-mediated destruction of islets .
In the study of Moritani et al., NOD (Non-obese diabetic) mice expressing an IL-10 transgene in glucagon-producing pancreatic cells also developed accelerated diabetes . Consequently, the expression of IL-10 in our model may exert not only a Th2 effect but also a Th1 effect. In general, the CEA antigen only induces a Th2 response, whereas the addition of a SARS fragment helps to induce and enhance both Th1 and Th2 responses. Th1 is more famous for its antitumor effects, although Th2 responses have been reported in autologous tumors, T-cells from patients with indolent non-Hodgkin lymphomas frequently exhibiting an activated but apoptosis-prone phenotype . It may seem that the Th2 response might downregulate the effect of a Th1 response or contribute less to the antitumor activity, but Th2-dominated antitumor immunity has also been observed in some models. As reported by Chu et al. , their DNA vaccine, which comprised a modified core peptide of mucin1 (PDTRP) and a GM-CSF coding sequence at the C-terminus, induced better protection against tumor challenge, and that protection was correlated with type 2 immune responses manifested by an increased IgG1 to IgG2a antibody ratio and greater induction of GATA-3 and IL-4 mRNA than of T-bet and IFN-γ mRNA in spleen cells of vaccinated mice.
The enhancement of Th1 and Th2 in the SARS-fragment-containing treatment groups indeed displayed better immunoactivity and protective effects in CT26/pCEA-bearing mice. In comparison with the negative control group, no significant differences in tumor volume were observed in mice immunized with CEA alone, while the tumor volume was found to be smaller in the pCEA-SARS-CoV group in the protection and therapy assays (Figure 3 & 4). These results may indicate that enhancement of the affinity of antigen presentation by the prediction algorithm increased the immunogenicity of the CEA-SARS peptide.