| Breast cancer poses a major health risk for women in China and worldwide. Excluding cancers of the skin, it is the most frequently diagnosed cancer and the second leading cause of cancer death in women. Although recent years have seen an improvement in breast cancer detection and a few new therapeutic agents, not much progress has been made in the treatment of the disease. According to the results of a new study, an aggressive, difficult-to-treat form of breast cancer appears to be more common in young African-American and Hispanic women attributed to the effect of social pressure and environmental deterioration. Viral infectious disease is also a major health problem. Among viruses most frequently associated with cases in the U. S., the coxsackieviruses group B (CVB) which infect the myocardium and cause myocarditis and dilated cardiomyopathy, are major etiologic agents. Some research confirmed that the CVB induces the type 1 diabetes. Howecer, up to now, no effective drugs was available in treating cancer or viral infection. So, new types of drugs are badly needed to be developed for contributing the cancer and virus infection.Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN), as an innate immune system agonist through Toll-like receptor 9 (TLR9), have been extensively studied in recent years. Based on functional characteristics, CpG ODNs are divided into A, B and C classes. A-class CpG ODN induces plasmacytoid dendritic cells (pDC) to secret large amount of interferon-α(IFNα) and indirectly activates NK cells and facilitates the induction of CTLs. B-class CpG ODN stimulates the proliferation of B cells and activates them. C-class CpG ODN shares both of the properties of A-class and B-class CpG ODN. These immunstimulatory properties have been attracting the studies on developing CpG ODNs as monotherapeutic agents for the treatment of various cancers and virus infection. In mouse models, administration of B-class CpG ODNs was capable of bringing about rejection of glioblastoma, fibrosarcoma, B16 melanoma, 3LL lung carcinoma, colon carcinoma and breast cancer. In clinical trials, B-class CpG ODNs showed therapeutic potential in patients with recurrent glioblastoma, melanoma and advanced renal cell carcinoma. Also, accumulating clinical studies showed that CpG ODN can efficiently prevent virus infections. A phase I study in health volunteers showed that CpG ODN is safe and well tolerated over a wide dose range, there were also antiviral immune response such as IFN-αhad been detected. So, CpG ODNs manifest very promising potential in clinical applications and can be used as mono-therapeutics to treat cancers or virus infections.The early empirical structure-activity relationship studies showed that the existence and position of the stem-loop, the length of the stem and the location of CG motifs, in addition to primary structure, was critical for designing novel CpG ODNs'capable of inducing anti-tumor immunity. The stem-loop, the so called structure domain, in addition to provide additional resistant to ubiquitous nucleases, has also been shown to require for the IFN-αinducing properties of CpG ODN,which abolishment of the loop dramatically decreased the IFNαsecretion. However, the position was shown important that stem-loop at the 5'end resulted in loss of activity, which seemed an accessible 5'end is required for the TLR9 recognized. Furthermore, it had been demonstrated that elongated the length of the stem enhanced the IFN-αinductive capacity significantly. In other study, it was found that the active ODNs often have CG dinucleotides in or near the stem loop structure but the inactive ODNs did not. What's more, point mutations of the CG dinucleotides located within the loop of the ODNs reduce their ability severely. All this proved that the location of CG dinucleotides was important for the ODNs'stimulation.In the present study, we design novel ODNs based on the secondary structure which is predicted by the DNA'mfold web server', and more, there are three principle:1) the open read frame at the 5'end; 2) at least two stem-loop structure; 3) one or more CG within the predicted rigid loop. Follow this general idea, a series of ODNs were developed and screened by using the VSV-protection bioassay and 3H-thymidine incorporation assay. In the process, a batch of novel CpG ODNs were identified: 8 of them were B class CpG ODN, 6 of them were C class CpG ODN. And then, two of them, which named YWODN01 and YWODN02, with the advantage function to evaluate their effects on breast cancer and CVB.The contents of this paper are focused on the following parts:1. Designing and screening of novel CpG ODNIn order to develop novel CpG ODN, we have tried to design a series of CpG ODNs that display stem-loops when being analyzed for their secondary structures using'mfold web server'. In primary screening, proliferation of human PBMC and bioassay for IFN production in human PBMC were used as technical platforms to determine their functions. After the primary designing and screening, the sequences flanking and within the loop were adjusted and predicted again. After multiply predicting and adjusting cycles, up to now, more then 10 of CpG ODN were identified and two of them, which named YWODN01 and YWODN02, belonging to B class CpG ODN and C class CpG ODN respectively, with the advantage function to further analysis.2. Immunostimulatory properties of YWODN 01 and YWODN 022.1 Analyze the stimulatory effects of YWODN 01 and YWODN 02 on human PBMCFisrst, to evaluate their heterogeneous, the stimulatory effects of CpG ODN were tested by proliferation assay and VSV protection assay in human PBMC from 6 donors. In human PBMC proliferation assay, both of YWODN 01 and YWODN 02 could stimulate human PBMC to proliferate vigorously. The efficacy was stronger than that induced by 2216 and similar to that induced by 2006 and C274. In VSV protection assay, YWODN 02, but not YWODN 01, could stimulate human PBMC to produce efficient IFN with the similar efficacy, shown by 2216 and C274. Dose effect assay showed that YWODN 01 and YWODN 02 at 0.38μgml-1 began to induce obvious proliferation, YWODN 01 at 3-12μgml-1 induced the proliferative plateau as that induced by 2006 or C274, and that YWODN 02 at 12μg/ml induced the most vigorous proliferation. In VSV protection assay, YWODN 02 at 0.19μg/ml was sufficient to induce human PBMC to produce IFN to extent as that induced by the C274 at 1.5μg/ml or 2216 at 0.75μg/ml, and at dosage of 36μg/ml induced the highest production of the factor, which indicating that YWODN 02 was a robust IFN inducer comparatively. This data implies that YWODN 02 and YWODN 01 act as C-class and B-class CpG ODN of human use, respectively.2.2 Activation of immune cells induced by YWODN 01 and YWODN 02CD69 is an early marker of lymphoid cell activation. To identify types of the cells activated by CpG ODN, Human PBMC were stimulated by CpG ODNs (6μg/ml) for 18 h, stained with FITC-labeled anti-CD69 and PE-labeled anti-CD19, CD56, CD4 or CD14 mAbs and then analyzed on a FACS to observe the up-regulation of CD69. The flow cytometry analysis showed that both YWODN 01 and YWODN 02 up-regulated CD69 in CD19+, CD56+ and CD8+ cells, indicating that YWODN 01 and YWODN 02 could activate human B cells, NK cells and T cells. Noticeably, the proportion of CD69+ B and NK cells stimulated with YWODN 02 not YWODN 01 were much higher as compared to that stimulated by control CpG ODNs.Next, we observed the effect of CpG ODN on costimulatory factor of immune cells.Briefly, Human PBMC were stimulated by CpG ODNs (6μg/ml) for 18 h, stained with FITC-labeled anti-CD80, anti-CD86 and anti-HLA-DR mAbs and then analyzed on a FACS. The result showed that YWODN 01 and YWODN 02could upregulate the expression of CD80, CD86 and HLA-DR. All these results suggest that YWODN 01 and YWODN 02 can activate immune cells such as B cells, NK cell and monocytes cells.2.3 Analyze the stimulatory effects of YWODN 01 and YWODN 02 on mouse splenocytesBecause of the requirement for evaluating the anti-tumor efficacy of YWODN 01 and YWODN 02 in mouse model, we next tested whether they could also activate murine immune cells using proliferation assay, VSV protection assay and CD69-expression assay. In proliferation assay, splenocytes of BALB/c mice were stimulated by YWODN 01, YWODN 02 and control CpG ODN including 1585 (a mouse A-class CpG ODN), 1826 (a mouse B-class CpG ODN) and C274 and then tested for their incorporation of 3H-TdR. The result showed that YWODN 01 and YWODN 02 could stimulate the proliferation of the splenocytes to a level similar to that induced by C274 and 1826 and a much higher level than that induced by 1585 and medium control. In VSV protection assay, YWODN 02, but not YWODN 01, could stimulate mouse splenocytes to produce efficient IFN with the similar efficacy, shown by1585 and C274. In CD69-expression assay, mouse splenocytes were stained by fluorescence-labeled mAb after CpG ODN stimulation and analyzed by flow cytometry. The result demonstrated that all CpG ODN used in this assay could increase CD69 expression in CD19+ and panNK+ cells not in CD8+ cells. Comparatively, YWODN 02 was more powerful on activating CD19+ and panNK+ cells than C274 as well as YWODN 01. The results reveal that both of YWODN 01 and YWODN 02 are suitable to be used in mice for evaluating their anti-tumor effect.2.4 Effect of CG motifs of YWODN 01 on its stimulation of mouse splenocyte A panel of CpG ODN (YWODN 01 or YWODN 01 with point mutation) were synthesized based on the structure of YWODN 01 and were tested for their capacity to stimulate proliferation of mouse splenocyte. The results showed that point mutations of the CG located within the predicted stem-loop structures of YWODN 01 reduced their ability to activate mouse splenocyte more virogous, which point mutation the CG at the 5'end also inhibited its capacity. These data may indicated the CG with the stem-loop were necessary for its activity.2.5 The analysis of species-specific of CpG ODNAs reported, the effects of CpG ODN is species-specfic, so we tested the YWODN 01 and YWODN 02 for their capacities to induce the proliferation of splenocyte from rat, cattle, dog, rabbit, mouse and guinea pig and PBMC of swine. Briefly, the splenocyte or PBMC were isolated and stimulated with the indicated CpG ODN (6μg/ml) or PBS for 36h. The cells were tested for their proliferation by 3H-thymidine incorporation assay. These data show that both YWODN 01 and YWODN 02 could stimulate the proliferation of splenocyte from mouse, rat or guinea pig. However, YWODN 02 also showed vigorous stimulation effects on cattle, dog and swine. These findings suggest hat YWODN 02functions in a no specis-spiecfic way.3. The anti-breast cancer effect of YWODN 01 and YWODN 023.1 The direct cytotoxicity of CpG ODN on EMT6To rule out the possibility that YWODN 02 could directly induce the necrosis of tumor cells, EMT6 cells were stimulated with YWODN 02 in culture for 48 h and then counted by trepan blue staining. The result showed that there were no significant difference in cell numbers between YWODN 02 and PBS treated group. In YWODN 02 (0.1, 1, and 10μg/ml) groups, the cell numbers were 21416±2268; 22083±1233; 23583±2036, respectively. In PBS group, the cell number was 25666±1644. The data suggests that anti-tumor activity of YWODN 02 in mice is not due to its direct cytotoxicity.3.2 The CpG ODN displayed anti-breast cancer effect in miceAfter the in vitro screening, YWODN 01 and YWODN 02 were tested for their in vivo effects on inducing anti-tumor activity in mice inoculated with EMT6 cells, murine breast cancer cell line cells. From day 8 after tumor challenge, tumor-palpated mice were treated with YWODN 01, YWODN 02 or PBS by peritumoral injection for six times in one-day interval. In parallel, mice in a group were s.c. injected with YWODN 02 at tumor-draining lymph node (TDLN) area. The result showed that peritumoral injection of YWODN 01 and YWODN 02 could induce significant inhibition tumor growth. Surprisingly, TDLN administered YWODN 02 displayed the most significant inhibition on tumor growth. Furthermore, intramuscularly and peritoneally administered YWODN 02 was tested for its tumor inhibitory effect in the same model. The result demonstrated that intramuscular and peritoneal injection of YWODN 02 failed to induce significant growth inhibition of the tumors. These data indicate that TDLN-injection is an efficient way for CpG ODN to induce tumor inhibition.3.3 The anti-breast cance effect of YWODN 01 and YWODN 02Next, YWODN 01 and YWODN 02 were used to treat mice with palpable tumors from day 8 after tumor challenge by s.c. injection at the right inguinal TDLN area for six times in one-day interval. The result showed that both of YWODN 01 and YWODN 02 could significantly inhibit tumor growth but YWODN 02 showed much stronger effect than YWODN 01. There were 2 mice in YWODN 02-treated group, 9 mice in YWODN 01-treated group and 12 mice in PBS group bearing tumors exceeded 2000 mm3 in size by day 40. Interestingly, developed tumors were completely rejected in 4 out of 12 mice in YWODN 02-treated group. It was also found that both of YWODN 01 and YWODN 02 could prolong the life span of mice challenged with tumor cells, in which YWODN 02 was more significant than YWODN 01. The average survival time was 52 days in PBS treated mice. In contrast, the survival time was 60 days and 80 days in YWODN 01- and YWODN 02-treated mice, respectively. The data indicate that YWODN 02, a C-class CpG ODN, is more vigorous than YWODN 01, a B-class CpG ODN, on inducing anti-breast cancer effect in mice when administered by TDLN-injection.Based on the observation that TDLN-injection of YWODN 02 induced more potent anti-tumor effect, we further tested the dose-effect of YWODN 02 on tumor inhibition in mice. TDLN-injection of YWODN 02 at 6.2550μg per mouse could significantly inhibit the growth of EMT6 breast tumor in mice in a dose-dependent manner, whereas YWODN 02 at 3.12μg per mouse didn't display significant anti-tumor effect. The average survival of mice in PBS group was 54 days, and by day 65 after tumor challenged, 100% of the mice died. In contrast, by day 100 post tumor inoculation, 18%, 50%, 67%, 67% and 83% of the mice injected with YWODN 02 at doses of 3.12, 6.25, 12.5, 25 and 50μg were still alive respectively. The average survivals of mice after treated by YWODN 02 were 66 days in 3.12μg group, 81 days in group and 90, 88 and 94 days in 12.5, 25 and 50μg groups, respectively, indicating that YWODN 02 significantly prolongs the survival of breast cancer bearing mice. This result further confirmed that YWODN 02, as a C-class CpG ODN, can induce effective anti-tumor effect in mice even at a lower dose, such as 6.25μg per mouse.Upon the result that TDLN-injection of YWODN 02 eradicated the established tumors, we investigated whether YWODN 02 could induce similar inhibition of the tumor developed at distant sites. In one set of experiments, YWODN 02 was injected at either the TDLN area or its counterpart area in the opposite side of tumor. Interestingly, as the tumor sizes were significantly reduced by TDLN injection of YWODN 02, the tumor was also response to the treatment of YWODN 02 at area in the opposite side of tumor. Obviously, the TDLN area-injection of YWODN 02 induced more efficient tumor inhibition. Next, to examine whether TDLN-injection of YWODN 02 were also effective on curing larger tumor, the YWODN 02 therapy was started on day 12 when the average volume of tumors reached to 125 mm3 instead of on day 8 post tumor inoculation. The results showed that mice received TDLN-injection of YWODN 02 from day 12 still showed a significant inhibition in tumor growth, but the efficacy was less compared with that started from day 8.Next, we observed the effect of prophylactic use of YWODN 02 on tumor inhibition. On day 0, 2, 4, BALB/c mice were s.c. injected with 25μg and 50μg of YWODN 02 or PBS at right inguinal lymph node area. Immediately after the third YWODN 02 injection on day 4, the mice were inoculated with 5×105 EMT6 cells at right back near hind leg. The result showed that although the tumor growth in mice received 25μg of YWODN 02 was delayed compared with that in PBS group, 80% of mice developed tumors and finally died in the group. In contrast, in 50μg YWODN 02 group, only 20% mice developed tumors and 80% mice remained tumor free and were alive for more than 100 days post tumor challenge. On day 40 after tumor challenge, the average tumor weights were 3.92 g in PBS group, 1.48 g in 25μg YWODN 02 group and 0.44 g in 50μg YWODN 02 group, respectively. Together, these findings show that prophylactic injection of YWODN 02 can effectively induce growth inhibition of EMT6 tumor in mice. 3.4 YWODN 02-induced immune responses relevant to anti-breast cancer activity in miceTo understand the effect of YWODN 02 administered through TDLN injection in mice, BALB/c mice with established breast cancer were injected with 25μg of YWODN 02 or PBS as control at TDLN area for six times in one-day interval and sacrificed after 48 h of last injection. The lymph nodes from axillary and inguinal region and spleens were removed for observing their morphologic changes, and analyzing their total cell numbers and cell populations by flow cytometry. YWODN 02-treatment resulted in a significant increase in size of inguinal lymph nodes at injection region and spleens, whereas no obvious change in size of the lymph nodes away from the injection site. In parallel, total cell numbers approximately increased 350%-400% in inguinal lymph nodes at injection region and 100% in spleens in YWODN 02-treated mice compared with those in PBS-treated mice. To the cell populations, there was an average 4-fold increase in the number of CD4 or CD8 T cells, 15-fold increase in the number of CD11c DCs, and 20-fold increase in NK cells obtained from inguinal lymph nodes at injection region of YWODN 02-treated mice compared to those in PBS-treated mice. Thus, TDLN injection of YWODN 02 is likely to recruit and expand T cells, NK cells and DC in local lymph nodes, accompanying a systemic splenomegaly, which may all be involved in the anti-tumor activity induced by YWODN 02.To study the effects of YWODN 02 on tumor and infiltration of immune cells in the tumor, mice with established EMT6 tumors were treated with YWODN 02 or PBS through TDLN injection for six times in one-day interval. After 48 h of the last injection, tumors were removed for histological study by H-E staining and immune cell counting by flow cytometry. The result showed that a large area of necrosis located in periphery of tumors was observed in YWODN 02-treated mice but not in PBS-treated mice. In PBS-treated mice, tumor with well demarcation revealed complete cell shape with clear cytoplasm and typical nuclei. In contrast, the cell shape was completely destroyed in necrotic tumor tissue derived from YWODN 02-treated mice. To understand the reason of tumor necrosis in YWODN 02-treated mice, immune cells in tumor homogenates were counted by flow cytometry. The percentage of CD4+, CD8+, CD11c+ and NK cells in tumor homogenates from YWODN 02-treated mice were increased to 4-fold, 9-fold, 1.6-fold and 2-fold, respectively, compared with those from PBS-treated mice. It seemed that TDLN-injection of YWODN 02 triggered the migration of T cells, NK cells as well as DCs into tumor and the cells somehow mediated the growth inhibition and necrosis of tumor cells.To confirm whether or not YWODN 02 could also induce specific immune response in mice through TDLN injection, we selected 13 tumor-free mice from three separate experiments, which had undergone the first-round of YWODN 02 treatment from day 8 post-EMT6 cell inoculations, as models after living more than 100 days for observing specific anti-tumor immune response. The mice were divided into two groups and re-challenged with EMT6 cells (5×105 per mouse) same as that in the first challenged or CT26 colon carcinoma cells (2×105 per mouse), respectively, at right back near hind leg without YWODN 02 treatment. The tumor development was monitored further on. The result showed that no tumor was developed in 7 mice re-challenged with EMT6 cells but there were tumors developed in 5 of 6 mice re-challenged with CT26, indicating the YWODN 02 treatment could facilitate the tumor bearing mice to broke the immune tolerance to tumor cells and consequently establish tumor specific immune memory that is sufficient to protect the mice from re-challenge of the same tumor. Promisingly, this property of YWODN 02 might benefit tumor treatment in clinic setting, either in tumor bearing patients or in the patients received tumor removing operation.4. The effect of YWODN02 on CVBThe previous work of our laboratory showed that BW001 could induce human PBMC to produce anti-VSV, CVB and anti-SARS-CoV activity. First, a CVB/Hela protection assay was used to test its effect on CVB in vitro. Next, a mouse model was used to evaluate its effect on CVB in vivo.4.1 The effect of YWODN02 on CVB in vitroTo further investigate whether YWODN 02 could stimulate PBMC to produce anti-CVB activity, human PBMC were stimulated by CpG ODN for 36 h and the supernants were collected. And then, Hela cells were treated with the supernatants, followed by attacking with 10×TCID50 of CVB3. The result showed that YWODN 02 stimulated supernatant could significantly protect Hela cells from CVB3 infection.Dose effect assay showed that YWODN 02 at 0.19μg/ml was sufficient to induce human PBMC to produce IFN to extent as that induced by the 2216 at 0.75μg/ml, and at dosage of 36μg/ml induced the highest production of the factor, which indicating that YWODN 02 was a robust IFN inducer comparatively. This data implies that YWODN 02 could stimulate PBMC to produce anti-CVB activity.4.2 The effect of YWODN02 on CVB-infected miceTo test its anti-CVB effect in vitro, female Blab/c mice were infected with CVB3 (4×100 TCID50/0.2 mL) by intraperitoneal (i.p.) injection. From 1 hour after virus challenge, mice were treated with YWODN 02 or PBS by i.p. injection for six times in succession. Healthy control mice remained untreated. Body weight was monitored over a period of 9 days and at day 10, the mice were sacrificed for removing heart and heart tissue was histological evaluated by H-E staining. However, in vivo study showed that YWODN 02 reduced the body weight of CVB3-infected mice and aggravated the pathological changes in the myocardium.Taken together, based on their secondary structure, we designed and screened 50 CpG ODN with novel structures and successfully identified a batch of potent CpG ODN, including 10 B-class CpG ODNs or C-class CpG ODNs. The novel CpG ODN could be used as adjuvant for currently used vaccines and as monotherapeutics to treat cancer and infectious diseases.Among the novel CpG ODN we discovered, two of them, designated as YWODN 01 and YWODN 02 respectively, have been studied for their anti-tumor activities in mice inoculated with EMT6 breast cancer cells. We have found that YWODN 02, identified as a C-class CpG-ODN, displays more potent anti-tumor activity than YWODN01, identified as a B-class CpG-ODN. Inspiringly, after the tumor establishment, YWODN 02 injected through TDLN area induces dramatic retardation of tumor growth and complete tumor regression. This result suggests that YWODN 02, a C-class CpG ODN, may have the potential to be developed as a monotherapeutic medicament for the treatment of breast cancer through TDLN-injection.The data of CpG ODN in CVB-infected mice may indicated that when used for the treatment of viral-infected diseases in vivo, CpG ODN may function in a complicated way. These data indicated that its administration time, route and dosage, should be taken into full consideration.
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