This study tested whether the exogenous antigen, the SARS fragment, could induce and enhance the vaccine efficacy of a low immunogenous TAA construct, the CEA fragment, in our animal model. The immunotherapy was realized by orally immunizing Balb/c mice with transformed Salmonella typhimurium. Cytokine profile and tumor volume were monitored to understand the effect of the DNA vaccine. Moreover, mutations were introduced on the SARS fragment to enhance the affinity between the MHC molecule and the epitope. Serial accumulation of mutations was designed to compare the protective effect of each construct (m1, m2, m3) with the original fragment (SARS). Mutation affinity was calculated by the Internet software, SYFPEITHI. Unlike other immunotherapies which put an emphasis on TAAs alone, we have set up a platform where a universal antigen is expressed to enhance the immunogenicity of the TAA construct. The concept of utilizing mutations on TAAs for better immunogenicity is applied to the universal antigen. By doing so, we intended to power the strength of the SARS fragment without mutating individual TAA.
The Internet software, SYFPEITHI, has been widely utilized as an epitope prediction algorithm. Pavlenko (Pavlenko, Leder et al. 2005) has proved that epitope specificity of the CTLs was determined by their reactivity against a panel of C-terminus truncated or mutated PSA proteins and use of bioinformatical prediction with the SYFPEITHI algorithm. In the study of Neumann et al. (Neumann, Wagner et al. 2005), the SYFPEITHI algorithm was used to select peptides with a high binding affinity to major histocompatibility complex class 2 (MHC 2) molecules. The pentadecamer epitope p635-649 induced specific CD4+ T-cell responses that were shown to be restricted by HLA-DRB1*1401. The responses could be blocked by preincubation of T cells with anti-CD4 and antigen-presenting cells with anti-HLA-DR, respectively, proving the HLA-DR-restricted presentation of p635-649 and a
CD4+ T-cell-mediated effector response.
The credibility of SYFPEITHI can be justified by the comparison with other software.
For example, the transmembrane protein, HM1.24, expressed on the terminal differentiated B cells was scanned for immunogenic peptides using the HLAbinding prediction software SYFPEITHI and BIMAS. Of eight nona-peptides with the highest probability of binding to HLA-A2, the HM1.24 aa22-30 peptide (LLLGIGILV) showed the most frequent activation of CD8+ T cells in healthy volunteers. Moreover, antigen recognition by the HM1.24 aa22-30-specific CD8+ T cells was HLA-A22restricted (Hundemer, Schmidt et al. 2006).
Other researches have also utilized the software, SYFPEITHI, BIMAS, or Rankpep, to predict the possible epitopes that can elicit the immune responses (Gomez-Nunez, Pinilla-Ibarz et al.
2006; Molinier-Frenkel, Popa et al. 2006).
We have compared the predicted result with a docking software released by BioXJEM (Yang 2004; Yang 2005). Docking reveals that the sequence of WYVW within the SARS epitope (WYVWLGFIA) predicted by SYFPEITHI makes a large steric hindrance for binding.
However, the modifications of the tail part, LGFIA to LGTII (in m2 and m3), can form a better interaction with the MHC molecule at docking, which may partially explain why the CT26/CEA killing is more conspicuous in the m2 and the m3 groups.
To understand the different effect of the DNA vaccine quantitatively, 2 x 106 /well splenocytes in each group were stimulated by either CT26/CEA soup (specific stimulation) or CT26 (non-specific stimulation) soup for 24 hr. TNF-α expression was not up-regulated in the CEA group compared with the negative group after CT26/CEA or CT26 soup stimulation.
However, the SARS-containing groups had a striking increase in TNF-α expression when compared with the negative and the CEA groups. TNF-α is a proinflammatory and Th1 cytokine. As indicated by Austin et al., TNF-α, IFN-γ, and IL-2 can define cytotoxic T lymphocytes and Th1 effector populations (Austin, Ozawa et al. 1999). Yet, IFN-γ was not significantly increased and IL-2 was undetectable in our experiment.
IL-4, a typical Th2 cytokine, was enhanced in the CEA group compared to the negative group. Yet, it was decreased in the groups with an additional SARS fragment, whether mutated or not. IL-10, however, was raised in the SARS, m1, m2, and m3 groups but not in the CEA group. Though most often it is known as a Th2 cytokine, IL-10 is actually a pleiotropic cytokine with anti-inflammatory, immunosuppressive, immunostimulatory properties (Moore, de Waal Malefyt et al. 2001). It exerts immunostimulatory effects on B cells, cytotoxic T cell development and thymocytes (Conti, Kempuraj et al. 2003). As indicated by Wogensen et al., expression of an IL-10 transgene by insulin-producing pancreatic cells led to an accelerated onset of diabetes in NOD mice (Wogensen, Huang et al.
1993; Wogensen, Lee et al. 1994), with no inhibition of immune-mediated destruction of islets (Lee, Wogensen et al. 1994). In the study of Moritani et al., NOD mice expressing an IL-10 transgene in glucagon-producing pancreatic cells also developed accelerated diabetes (Moritani, Yoshimoto et al. 1994). Consequently, the expression of IL-10 in the model may exert not only a Th2 effect but a Th1 effect as well.
IL-12, though a Th1 cytokine, was slightly up-regulated in the CEA group but not in the SARS group when compared with the negative control after CT26/CEA soup stimulation. It was even more down-regulated in the mutation groups. IL-12 is mostly secreted by macrophages and DCs. The possible mechanism may lie in the fact that the increase in IL-10 in the SARS, m1, m2, and m3 groups exerts an inhibitory effect on macrophages so as to inhibit Th1 activation by blocking macrophage IL-12 synthesis. In the study of Lang et al., an overproduction of inflammatory cytokines and development of chronic inflammatory diseases have been shown in IL-10 gene-deficient mice (Lang, Rutschman et al. 2002).
In general, the CEA antigen only induces a Th2 response whereas the addition of a SARS fragment helps induce and enhance both Th1 and Th2 responses. Th1 is more famous for its antitumor effect. For instance, IL-27 has been proved to possess antiangiogenic and antitumor activities in the B16F10 model. The poorly immunogenic murine melanoma
B16F10 tumors were engineered to overexpress single-chain IL-27 (B16F10 + IL-27).
B16F10 + IL-27 cells exerted antitumor activity against not only s.c. tumor but also experimental pulmonary metastasis (Shimizu, Shimamura et al. 2006). On the other hand, the Th2 response has been reported in autologous tumor, where T cells from patients with indolent non-Hodgkin lymphomas frequently showed an activated but apoptosis-prone phenotype (Anichini, Mortarini et al. 2006). It may seem that the Th2 response may down regulate the effect of a Th1 response or contribute less to the antitumor activity, but it has also been observed in some models where a Th2-dominated antitumor immunity has occurred. As reported by Chu Y et al. (Chu, Xia et al. 2006), their DNA vaccine which comprised a modified core peptide of mucin1 (PDTRP) and GM-CSF coding sequence at the C-terminus induced better protection against tumor challenge. The protection is correlated with the type 2 immune responses manifested by an increased IgG1 to IgG2a antibody ratio and a greater induction of GATA-3 and IL-4 mRNA than that of T-bet and IFN-gamma mRNA in spleen cells from vaccinated mice.
Synergistic antitumor effect of both Th1 and Th2 cytokines has also been detected. As indicated by Lopez et al. (Lopez, Adris et al. 2005), the combination of autologous inactivated tumor cells expressing IL-12 and IL-10 induced tumor remission in 50-70% of mice harboring large established colon or mammary tumors and spontaneous lung metastases, with the consequent establishment of an antitumor immune memory. The production of IFN-gamma and IL-4 by spleen cells and the development of tumor-specific IgG1 and IgG2a Abs indicate that each cytokine stimulated its own Th pathway and that both arms were actively engaged in the antitumor effect. The study of IL-21 and IL-15 by Nakano et al.
(Nakano, Kishida et al. 2006) provides further evidence for the cellular and humoral responses to tumor cells. IL-21 induced significant elevation of head and neck squamous cell carcinoma-specific CTL activity, while IL-21 and IL-15 augmented NK activity in an additive manner. IL-21 gene transfer also promoted the production of tumor-specific IgG..
Not only does the in vitro cytokine assay reveal an enhanced Th1 and Th2 responses in the SARS-containing groups, but also the in vivo cytokine assay shows the activation of Th1 and Th2 cytokines. On the contrary, CEA alone could not stimulate immune cells to secrete cytokines in vivo. The in vivo assay reveals the total amount of cytokines within an animal, which is the outcome of immunization, suggesting the mechanism of the protection of our DNA vaccine.
The enhancement of Th1 and Th2 in the SARS-containing groups indeed displayed a better protective effect on the animal models. Tumor volume was monitored for 26 days.
When compared with the negative group, mice immunized with CEA alone did not show any significant difference in the protection assay and in the therapy assay. However, the SARS and m3 groups had the smallest tumor volume in the protection assay. The m1 construct also provided enough protection for mice, but the m2 construct did not seem to be able to sufficiently suppress tumor growth.
It was expected that the m2 construct should have a higher antitumor activity than the m1 construct. However, the protection assay reveals that it did not. Some reasons are given to explain the phenomenon. First of all, as introduced in 1.3.3, beside the binding affinity between the epitope and the MHC molecule, there exists another binding affinity between the epitope-MHC complex and the TCR. Some researches have demonstrated that such an affinity may influence the activation of cytotoxic T cells as well (Slansky, Rattis et al. 2000). Second, only nonamers were chosen as the epitopes in the software calculation. But other sequence length may also be generated in the animal. It is not known if such a nonamer calculation is the best outcome in vivo. Furthermore, there are other MHC alleles to accommodate a variety of epitopes in a mouse. For example, besides the H2-K form, H2-D and H2-L belong to the MHC class I molecules as well. Therefore, it is beyond our control to generate an epitope mutation that induces the best immunity in vivo. Some other unknown mechanisms may undermine the protective effect of such an immune strategy since immunity itself is interlaced
to reach homeostasis.
The truncated CEA antigen successfully simulates a low immunogenous TAA, which is often the case in most tumor cells. It did not induce any favorable cytokines in vitro and in vivo to enhance either Th1 or Th2 responses. Its killing assay reveals that CEA alone could not specifically kill CT26/CEA and its innate immunity (YAC-1 killing) did not show any significant difference when compared with the negative control. However, SARS fusion not only induces but also enhances anti-CEA activity, as shown in the CT26/CEA specific killing.
Moreover, specific killing is proportional to the number of mutations, indicating that our mutations did work well in vitro. The antitumor activity of the m2 construct is limited in vivo due to the reasons provided above. But all in all, the foreign parental or mutated SARS fragments could enhance the anti-tumor efficacy of the tumor vaccine against endogenous tumor antigens. As a result, we provide a platform to enhance the adjuvant effect of the foreign peptide by computer prediction.
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