CpG ODNs Enhance Vaccine Efficacy And Influence Viral Pathogenecity And Tumor Growth

Both viral infections and carcinoma are highly fatal diseases. Nearly seventy-five percent of infectious diseases are caused by virus. Hepatitis B virus, hepatitis C virus or human immunodeficiency virus can induce chronic infection with an increased risk of malignant cell transformation. Influenza virus and avian influenza virus easily emerge mutation or re-assortment and cause sustained pandemic. Furthermore, viral infections are associated with an increased risk of malignancy, autoallergic disease and allergy. Carcinoma is the second death reason in western countries and has become the first death reason in China according to the data from China's Ministry of Health in 2006. The incidences of lung cancer and breast cancer are the fastest increasing all over the world and displays increasing trend in young attributed to the effect of smoking and stress. CpG containing synthetic oligodeoxynucleotides (CpG ODN) can directly or indirectly activate immune system by recognizing toll like receptor 9 expressed major in B cells and plasmacytoid dendritic cells (pDC), induce the secretion of Th1-like cytokines, such as IFN, TNF-α,IL-6,IL-12,IL-18,IP-10, indicating that CpG ODN has the huge potential for anti-infective or anti-neoplastic applications.To develop highly effective adjuvant for viral vaccines, investigate the influences of CpG ODN on the influenza infection and tumor growth, we finished the following three part experiments: 1 CpG ODN as adjuvant for virus vaccinesViral infectious diseases are the most common contagious diseases and up to now there is no effective medical treatment for them. So, vaccine inoculation for prophylaxis of these kinds of diseases becomes extremly important. However, new types of vaccines, adjuvants or delivery systems are needed be developed for the following purposes: to improve the vaccine immunogenicity so that the vaccine could stimulate the earlier and higher antibody production, to enhance the vaccine ability of inducing Th2 response and to reduce the vaccine dose. To develop new type CpG ODN as virus vaccine adjuvant, we conducted the following experiments:1.1 Activation of human PBMCs and mouse splenocytes stimulated by optimized CpG ODNAccording to their structures and their activities in stimulating human peripheral blood mononuclear cells (hPBMCs) in vitro, CpG ODNs can be grouped into A-(also known as"type K"), B-(also known as"type K") or C-types. Structurally, the A type CpG ODN has phosphodiester-backbone (PO) palindrome sequence containing CpG motif and phosphorothioate (PS)–modified polyG at the 3′and 5′ends. The A type CpG ODN can directly stimulate high level production of IFNα/βand secondarily activate natural killer (NK) cells. Structurally, the B type CpG ODN has full PS-modified backbone containing one or more CpG motif(s), and has no polyG structure. The B type CpG ODN strongly induces B cell proliferation and pDC maturation, and also activates NK cells indirectly. The structure of C type CpG ODN is similar to that of the A type CpG ODN except its full phosphorothioate-modified backbone. The C type CpG ODN has immune properties intermediate between the A and B types, inducing both B cell activation and IFNαsecretion. All types of CpG ODN have adjuvant properties. However, because of degrading rapidly by nuclease digestion in vivo, the instability of PO backbone is a restriction on the clinical application of the A type CpG ODN. The stimulation of B cell proliferation and secretion of large amounts of Ig, IL-6 and IL-10 induced by the B type CpG ODN is stronger than that did by the C type CpG ODN, which may be a partial reason for the focus studies on B type CpG ODN as adjuvant. Furthermore, the recognition of CpG motifs is different in variant species: the optimal CpG motif is GACGTT for stimulating the proliferation of mouse splenocytes but GTCGTT for stimulating the proliferation of hPMBCs. Based on these knowledges, we designed a large scale of CpG ODNs and used the 3H-TdR incorporation assay for the screening of the B type CpG ODN. A CpG ODN, designated as BW006, outshined with stronger ability to stimulate the proliferation of both hPBMCs and mouse splenocytes. Furthermore, B cell activation induced by BW006 was detected on a FACSCalibur. The result showed that BW006 could significantly up-regulate CD69 expression on CD19+ cells in human PBMCs and mouse splenocytes. These data indicate that BW006 may be developed into a potential adjuvant for human use and its potential can be primarily evaluated in mice before human clinical trial since BW006 is able to stimulate both human and mouse immune cells especially B cells.1.2 Immunogenicity of inactivated rabies vaccine enhanced by BW006Up to now, the postexposure prophylaxis (PEP) with vaccine is still a mainstream treatment for the people attacked by rabies virus infected animals. Obviously, it is required to develop rabies vaccines that induce an earlier and higher level production of rabies virus neutralizing antibody (RVNAb). Traditionally, rabies vaccine is administered in five intramuscular shots on day 0, 3, 7, 14, and 28. Overall, it is required to find an optimal adjuvant that not only promotes the rabies vaccines to induce an earlier and stronger RVNAb response, but also reduces their immunization shots. To determine the enhancement of BW006 on inactivated rabies vaccine (IRV), we performed a series experiments in mice. The result showed that: BW006 enhanced IRV to induce an earlier and much higher production of RVNAb, and aluminum delayed the RVNAb production; BW006 co-operated with aluminum to enhance the immunogenicity of IRV; BW006 reduced the immunization shots of rabies vaccines; BW006 augmented the rabies vaccine with or without aluminum to induce effective protection of mice against rabies virus. The results indicate that BW006 has the potential as rabies vaccine adjuvant. The immunization strategy against rabies might be adjusted to one in which the first shot is administered with rabies vaccine plus BW006, and the later shots with rabies vaccine containing both aluminum and BW006.1.3 Immunogenicity of inactivated influenza vaccine enhanced by BW006Influenza virus easily causes sustained pandemic and vaccine supply is insufficient to meed the needs of potential pandemic mediated by the frequent re-assortment between two different virus strains or subtype strains. To determine the enhancement of BW006 on inactivated influenza vaccine, we performed a series of experiments in mice. The results showed that: BW006 improved earlier production of high level antibody induced by low dose of H3N2 antigen and prolonged production of high level antibody induced by high dose of H3N2 antigen; BW006 reduced H3N2 antigen doses to 1/8th dose and enhanced recalled proliferation of mouse splenocytes of mice immunized with H3N2 antigen stimulated by different influenza subtype strain. Furthermore, we identified the enhancement effect of BW006 on the inactivated influenza spilit vaccine. Similarly, BW006 reduced the full vaccine dose to 1/10th dose and promoted IgG2a production in mouse serum. In conclusion, the superiority of BW006 as influenza vaccine adjuvant is to reduce vaccine dose, increase cross-reactivity to different subtype strain and induce Th1 response.2 The influence of CpG ODN on the pathogenecity of influenza virus Based on the production of large amounts of anti-infective materials by CpG ODN stimulated immune cells, we conducted the in vivo experiments to observe the influence of CpG ODN on the acute infection caused by influenza virus. Influenza virus FM1 stain, which is safety for human, was used to infect mice. Body weight, survival rate, lung pathology, the levels of XOD and IL-12 in BALF were used to evaluate the immune effect of CpG ODN 1826 (1826), a prototype of B type CpG ODN in mouse. We unexpectedly found that 1826 inoculated before the influenza virus attack provided no protection for infected mice. On the contrary, 1826 aggravated the interstitial pneumonia, accelerated the body weight loss and promoted the death of infected mice, accompanished with the improved activity of xanthine oxidase and the high level IL-12 production in bronchoalveolar lavage fluid. The results suggest that CpG ODN seems to have side effects on self-limited flu-like acute infection, and the possible reason may be due to the synergy of CpG ODN and virus to stimulate the over secretion of inflammatory cytokines which damage body itself. Consequently, immunostimulating agents such as CpG ODN should be used cautiously in patients suffering from acute viral infection because of their potential to augment viral pathogenecity.3 The influence of CpG ODN on tumor growthThe complex pathogenesis of carcinoma is associated with the low cure rates and high recurrence rates of these kinds of diseases. Any single-target drugs are insufficient for the cancer therapy. Adherence properties of tumor cells have important effects on tumor growth, invasion, and metastasis, so inhibition of tumor adherence becomes a new way to cure cancer. To study the capability of CpG ODN on the inhibition of tumor adherence, B16 cells were selected as tumor cell models for in vitro and in vivo experiments.3.1 The influence of CpG ODN on the tumor cell adherence in vitroFor the purpose of screening CpG ODN with the ability to inhibit tumor cell adherence, cell counting experiments were used. In the process, self-designed CpG ODN, named as BW001, outshined others because of its dose-dependent inhibitions of tumor cell adherence, spreading and growth in vitro. Furthermore, a panel of CpG ODN were synthesized based on the structure of BW001 and were tested for their capacity to inhibit tumor cell adherence. The result showed that more than 5 continuous guanines (G) and fully PS-modified backbone, but not CpG motif were the necessary structures for BW001's activity to inhibit cell adherence. Moreover, the activity of BW001 could be blocked by polycations (including poly-L-lysine, poly-L-arginine and poly-L-ornithine) and affected by FITC labeling, and could not influenced by dextran sulfate and chlorochine. These results indicate that the activity of BW001 on the inhibition of tumor cell adherence displays specific structure-dependence and is associated with polyanion character, but has no relationships with scavenger receptor or TLR9. The reduced inhibiton of FITC-BW001 may be due to the effect of FITC molecular on the formation of BW001 polymers.3.2 The influence of CpG ODN on the tumor cell adherence ex vivoTumor metastasis is a multistep process and dependeds in part on the interactions of tumor cells and extracellular matrix (ECM) or endothelial cells. To study the activity of BW001 on the inhibition of tumor metastasis, fibronetin and typeⅠcollagen were used to mimic ECM and human umbilical vein endothelial cells (HUVECs) were used to mimic angiogenesis. The results showed that BW001 could inhibit the tumor cell adherence on ECM or inhibit the conformation of ECM mimicked by fibronetin and typeⅠcollagen; BW001 could inhibit tumor cell adherence to HUVECs. The results indicate that BW001 may inhibit tumor metastasis through the inhibition of cell adherence on ECM or endothelial cells.Adherence to endothelium of distant organs may be partially responsible for the preferential metastatic patterns associated with differetent tumor types. Melanoma cells typically form tumor metastasis primarily in the lung. To observe the influence of BW001 on the lung metastasis of B16 cells, fresh cryostat sections of lung tissue were used to mimic lung microenviroment. The results showed that BW001 could inhibit the B16 cell adherence on lung tissue, incidating that BW001 may reduce the metastasis of tumor by the inhibition of tumor cell adherence on distant organs.3.3 The influence of BW001 on the tumorigenicity of B16 cell in miceTo study the effect of BW001 on the inhibition of tumor growth and metastasis in vivo, we established three kinds of tumor models in mice with intraperitoneal, subcutaneous or intravenous injection of B16 cells mixed with or without BW001. We found that the BW001 could significantly decrease the tumor colony formations in peritoneal cavity and prolong the survival of tumor-bearing mice, delay the growth of subcutaneous tumor and inhibit the lung disseminateion of B16 cells via tail vein injection. The activity of BW001 displays sequence-dependence and structure-dependence: CG reversed control for BW001 failed to prolong the survival of tumor-bearing mice as compared with solvent did, indicating sequence-specific is a necessary condition for BW001's activity. Moreover, the suppressing effect was weakened when polyG tail was removed or fully PS-modified backbone was changed to partially PS-modified backbone. Further study showed that the tumor inhibition activity of BW001 in these tumor models was induced by the direct interaction of BW001 and B16 cells and potential activation of immune system stimulated by BW001 may also participate in anti-tumor effects in peritoneal tumor model.Based on all these results, we conclude that:1 self-designed CpG ODN (BW006) may be a potential adjuvant for rabies vaccine and influenz vaccine. 2 Because of their potential to augment viral pathogenecity, immunostimulating agents such as CpG ODN should be used cautiously in patients suffering from acute viral infection.3 Self-designed CpG ODN (BW001) may be developed into novel anti-neoplastic medication with the ability of tumor cell inhibition.