| Athough vast progress has been made in treating the infectious diseases, virusinfection is still a major health problem. According to the data from China'sMinistry of Health, there are 120-million HBV carriers in China, nearly 10percent of its population, and 30-million of them have become active patients. Inaddition, a total of 38,000,000 cases of HCV occur in 1990's in China and manyof which will develop into recurring hepatitis, liver cirrhosis and hepatocellularcarcinoma. Also HIV, influenza virus is associated with tens of thousands ofdeaths per year. Foot and Mouth Disease (FMD) and avian influenza infection inanimals also have substantial economic impact.So far, it seems that recombinant interferons (IFN) is one of few effectivedrugs available in treating HBV, HCV infection and other virus-associateddiseases. However, IFN has a short half-life time and seems not effective inpatients with chronic HBV. Several side effects are also observed during thecourse of recombinant interferon treatment. So, new types of drugs are badlyneeded to be developed for controling the virus infection.CpG ODN are artificial oligodeoxynucleotides. Based on their stimulationeffects on human PBMC, CpG ODN can be divided into 3 types: CpG-A ODNs(also known as"type D"), CpG-B ODNs (also known as "type K") and CpG-CODNs. 2216, a published CpG ODN with the prototype sequence of CpG-AODNs, is a very potent inducer of IFN-αand IFN-γfrom pDC and NK cellsrespectively whereas stimulates weak proliferation of B cells and differentiationof plasmocytoid dendritic cells (pDC). 2006, a well-studied CpG ODN with theprototype sequence of CpG-B ODNs, is able to stimulate various functions of Bcells and induce maturation of DC, on the contrary, has little or no effects onpDC. CpG-C ODNs share the immune effects of both A-and B-Class CpG ODN.CpG ODN such as C274, M362, 2395 are all representatives of this class.In all, CpG ODN activate the cells of innate immune system, includingdendritic cells, macrophages, and natural killer (NK) cells, and consequentlystimulate the production of Th1 cytokines and chemokines, the up-regulation ofcostimulatory molecules, which facilitate the generation of adaptive immuneresponses. So, CpG ODN manifest very promising potential in clinicalapplications and can be used as adjuvants to enhance efficacies of variouscurrent vaccines, and as mono-therapeutics to treat cancers and allergies. Accumulating clinical studies show that CpG ODN can efficiently preventvirus infections. A phase Ia study in health volunteers show that CpG ODN issafe and well tolerated over a wide dose range. A Phase Ib study showed thatCpG ODN treatment could significantly reduce early viral burden and enhanceantiviral immune response in patients who had relapsed hepatitis C after or priorIFN-αtherapy. To identify CpG ODN with potential of clinical applications, we have beencarried out a large scale screening of novel CpG ODN. About 190 CpG ODNwere designed and screened by using the inhibition of cytopathic effect (CPE)bioassay and H-Thymidine incorporation assay. In this process, we successfully 3identified a batch of novel CpG ODN, among which 6 were CpG-A ODN, 8were CpG-B ODN and 9 were CpG-C ODN. To develop novel anti-virusmedicine, we selected CpG-302 (CpG-C ODN) and CpG-B4 (CpG-A ODN) forfurther analysis.The contents of this paper are focused on the following parts:1 Screening for optimized sequences with potent effects on human PBMC We synthesized more than 190 CpG ODN. By using the inhibition ofcytopathic effect (CPE) bioassay and H-Thymidine incorporation assay, we 3tested these CpG ODN for their capacity to stimulate human PBMC toproliferate and to secrete anti-virus substances. Up to now, we have identified abatch of novel CpG ODN including 6 CpG-A ODNs, 8 CpG-B ODNs and 9CpG-C ODNs. As reported, the effects of CpG ODN is species-specific, so we tested thescreened CpG ODN for their capacities to induce the proliferation and anti-virusactivity of mouse splenocytes, rhesus PBMC and porcine PBMC. Sequencescontaining 'GTCGTT'motif could stimulate proliferation of mouse splenocytes.CpG-667 and CpG-684, which contain 'GTCGTT'motif, could also stimulateporcine PBMC and rhesus PBMC to proliferate. These data indicated CpG ODNcontaining 'GTCGTT'motif had no species-specific in their function. However,as the anti-virus effects of the CpG ODN-conditioned supernatants wereconcerned, all the CpG ODN which had potent stimulatory effects on humanPBMC showed no effects on mouse splenocytes except CpG-B4 and CpG-1007,which also induced porcine and rhesus PBMC to secrete anti-virus cytokines.CpG-302 which could simulate the proliferation of PBMC and the productionof anti-virus substances from the PBMC was also tested for its heterogeneity bythe same methods described above. CpG-302 showed no stimulatory effects onmouse splenocytes and porcine PBMC. By comparation, CpG-302 failed tostimulte rhesus PBMC to proliferate, but could stimulate rhesus PBMC toproduce anti-virus factors. These findings suggest that the CpG-302 functions ina highly species-specific way.2 Analyze the stimulatory effects of CpG-302 on human PBMC Of the 9 CpG-C ODNs, CpG-302 showed the highest activity for humanPBMC and therefore was selected for further analysis.2.1 Time-and dose-dependent anti-VSV effects induced by CpG-302 To analyze the kinetics of CpG-302 in inducing anti-virus activity, thesupernatants of cultured human PBMC stimulated by CpG-302, 2216 orControl-302 (identical to CpG-302 except the CG is reversed) were collected atdifferent time point (0.5 h～96 h) and tested for their ability to inhibit thecytopathic effect (CPE) caused by VSV on VERO E6 cells. The results showedthat anti-VSV activity induced by CpG-302 appeared at 2 h, reached peak at 12 hand maintained a plateau from 12 to 96 h. Compared to 2216, CpG-302 hadsimilar effects in terms of the speed, level and maintained time of inducedanti-VSV activities. Control-302 couldn't induce human PBMC to produceanti-virus factors. To determine the optimal dosage of CpG ODN, human PBMC werestimulated with different doses of CpG-302, 2216 and Control-302 and thesupernatants were collected to test their abilities to protect Vero E6 cells fromVSV challenge. It was found that CpG-302 as little as 0.05μg/ml was sufficientto induce human PBMC to produce anti-virus factors, and at the dosage of 3～6μg/ml induced the highest production of the factors.2.2 Anti-influenza virus and anti-SARS-CoV activities of the supernatant of human PBMC stimulated by CpG-302 Next, we observed whether CpG-302 could induce activity against influenzavirus and SARS-CoV. The stimulated supernatants of human PBMC stimulatedby CpG-302, C274 (C-type control), 2006 (B-type control), 2216 (A-type control)and medium (negative control) were tested for its ability to inhibit the cytopathiceffect (CPE) caused by influenza virus on VERO E6 cells. The result showedthat CpG-302 induced similar anti-virus activity as 2216 but higher than C274.Anti-SARS-CoV activities of the series diluted supernatants of human PBMCstimulated with 25μg/ml, 12.5μg/ml, 6.25μg/ml and 3.13μg/ml CpG-302 werealso investigated. We found that supernatants stimulated by CpG-302 protectedVero E6 cells from SARS-CoV infection. Together, these data suggest thatCpG-302 can stimulate human PBMC to produce anti-virus factors.2.3 Heterogeneous response of human PBMC to CpG-302 The stimulated supernatants of human PBMC from different donorsstimulated by CpG-302 were tested for its ability to inhibit the cytopathic effect(CPE) caused by VSV on VERO E6 cells, and their capacity to stimulateproliferation of human PBMC from different donors were also evaluated. Wefound that the proliferation and anti-virus effects of human PBMC stimulated byCpG-302 were heterogeneous, and the harbored mechanisms needed to befurther explored.2.4 Analysis of cytokines induced by CpG-302 Cytokines in the supernatants of human PBMC stimulated by CpG-302 werequantified by ELISA Kits. CpG-302 could induce human PBMC to produce highlevel of IFN-αeven though it was lower than that stimulated by 2216. ButCpG-302, as well as 2006 and 2216, could stimulate human PBMC to secretcertain amount of IL-6, TNF-αand MIP-1βat similar level. IFN-γproductioninduced by CpG-302 or 2216 appeared higher level than that induced by 2006. Plasmacytoid dendritic cells (pDC) are the major cells to produce largeamount of IFN-αin response to viral infection. Human pDC were isolated andpurified from buffy coat and cultured with or without CpG-302 or 2216 in themedium containing different stimulators as indicated. The supernatants fromcultured human pDC were harvested and used for detection of IFN-αand IL-12.The result showed that CpG-302 as well as 2216 could stimulate human pDC toproduce large amount of IFN-αand certain amount of IL-12 when combinedwith CD40L.2.5 Anti-VSV activities of the supernatants before or after IFN-α neutralization Type I interferon (IFN-α/β) is the major anti-virus cytokines. To test whetheranti-virus activities of the supernatants due to the type I interferon induced byCpG-302, supernatants from human PBMC stimulated by CpG-302 wereneutralized by IFN-α, IFN-βand IFN-γpolyclonal neutralizing antibodies, andthe remaining anti-virus effect were tested by bio-assay as described.Neutralization of IFN-αcould partially weaken but could't fully abolish theanti-virus effect, whereas neutralizing IFN-βand IFN-γdid not influence theanti-virus activity, indicating that IFN-αdid contributed the anti-virus activityand except IFN-β, IFN-γother soluble factors were involved in.2.6 Activation of immune cells induced by CpG-302 Because of the unique properties of CpG-302 in anti-virus, we observedwhether the effect of CpG-302 on immune cells was different with other CpGODN. Human PBMC were stimulated with CpG-302, 2216 or 2006 for 48h,stained with FITC-conjugated CD40, CD80, CD83, CD86 and HLA-A2antibodies and then analyzed on a FACSCalibur. The result showed thatCpG-302 could upregulate the expression of CD40, CD80, CD86 and HLA-A2. CD69 is an early marker of lymphoid cell activation. Human PBMCs werestimulated with different CpG ODN including CpG-302, Control-302, 2006 and2216, then stained with PE-conjugated anti-CD19, CD56, CD14, CD3 andFITC-labeled CD69 monoclonal antibodies and analyzed on a FACSCalibur.The results showed that activation of B cells (CD19+), NK cells (CD56+) andmonocytes (CD14+) stimulated by CpG-302 was much stronger than thatinduced by 2216 (A-type control). Control-302 couldn't activate immune cells,and all the sequences tested cannot acivate T cells (CD3+). In additon, CpG-302also enhanced the lytic activity of NK cells within PBMC and could stimulatepurified human B cells to proliferate. All these results suggest CpG-302 belongto CpG-C ODNs and can activate immune cells such as B cells, pDC, NK cells,monocytes etc.2.7 Effect of poly (G) and CG motifs of CpG-302 on its stimulation of human PBMC A panel of CpG ODN (CpG-3021～CpG-3030) were synthesized based on thestructure of CpG-302 and were tested for their capacity to simulate proliferationand to induce supernatant with anti-virus effects of human PBMC. The resultsshowed that the 6 guanines (G) at the 3'end could enhance CpG-302 capacity toinduce anti-virus effects, while inhibited its capacity to simulate proliferation ofhuman PBMC. Further, the 'TCG'motifs at 5'end and the palindromic sequencein CpG-302 were necessay for its activity.2.8 Direct inhibition of VSV on Vero E6 cells by CpG-302 Vero cells were seeded into 96-well flat-bottomed plates (3 ×104 cells/100μl/well) and cultured for 24 h to confluency. The cells were directlyincubated with CpG ODN and 10TCID50 (50% tissue culture infectious doses)of VSV for another 48h. The cytopathic effect caused by VSV was examined byusing crystal violet staining method. Anti-virus effect was expressed as ODvalues, which were correlated to the anti-virus effect of the stimulatedsupernatant. Of note, CpG-302, not 2006, 2216 or C274 could inhibit VSVinfection on Vero E6 cells. Further analysis showed that the anti-virus effect wasdependent on the phosphorothioated backbone and the sequence of CpG ODN.2.9 The function of CpG-302 related to scavenger receptor Scavenger receptor A (SR-A) can recognize a number of polyannions such asfucoidan, dextran sulfate (DS), polyG/polyI and play important role in innateimmunity. Also, it has some effects on cells'adhesion. CpG ODN is one kind ofpolyannions and may be recognized by SR-A. Our experiments also showedCpG-302 could inhibit cells adhesion to the bottom of 96-well flat-bottomedplates. So, we analyzed whether the stimulation effects of CpG-302 could beinhibited by DS, which did not induce any detectable cytokines and could notstimulate human PBMC to proliferate. Human PBMC were incubated with 30μg/ml dextran sulfate and 3μg/mlCpG-302, 2216, 2006, C274 for 48h, the supernatants were collected andassayed for their anti-virus effects as described above. PBMC proliferation wasalso assessed by H-thymidine incorporation assay. The effect of CpG ODN on 3stimulating human PBMC to produce interferon and the other anti-virus factorscould be completely inhibited by dextran sulfate. Also dextran sulfate couldinhibit PBMC proliferation stimulated by CpG-302, but failed to inhibit theproliferation stimulated by 2006 and C274. So, there are some differencesbetween C274 and CpG-302 in spite of their similarly stimulatory effects onhuman PBMC. C274 and CpG-302 have a different structure. There are 6 guanines at the 3'end of CpG-302. In order to elucidate whether the difference between C274 andCpG-302 is owed to the 6 guanines at the 3'end of CpG-302. The sequence(CpG-3026) identical to CpG-302 except there is no guanine at the 3'end wasused to stimulate human PBMC with or without dextran sulfate. The resultdemonstrated that the difference between C274 and CpG-302 was not owed tothe existence of the 6 guanines at the 3'end of CpG-302. Further, human PBMCwas cultured with dextran sulfate alone or with partial phosphorothioated302(Part-302) and unmodified 302(PD-302), and the result showed that dextransulfate could also inhibit the stimulatory effect of CpG ODN on inducing humanPBMC to produce anti-virus factors. All these result demonstrated that theinhibitory effect of dextran sulfate was dependent on the sequence structure, noton the phosphorothioated backbone or guanine at the 3'end of CpG ODN.3 Anti-virus effects of CpG-B4 Elegant studies have shown that CpG-A ODN could stimulate lymphocytes tosecrete type I interferons (IFN-α/β), IFN-γ,IL-12,IL-18 etc, enhance the antigenpresentation ability of antigen presenting cells (APC), activate innate immuneresponse, and facilitate the aquired immune response. So, CpG-A ODN may becompetent at combating virus infection. CpG-B4 is a novel CpG-A ODNidentified in the process of large-scale screening, which could stimulate humanPBMC and mouse splenocytes to secrete large amount of anti-virus factors.
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