CpG ODN对口蹄疫重组蛋白疫苗增效作用的研究
摘要
口蹄疫病毒可以引起偶蹄动物急性传染性接触性疾病,牛、羊、猪等家畜都是对此病毒敏感,其中牛最为易感。接种口蹄疫疫苗是预防和治疗口蹄疫的重要方法,通常在疫苗中加入佐剂来增强疫苗的免疫活性。
CpG ODN是一种包含非甲基化的胞嘧啶和鸟嘌呤核苷酸(CpG基序)的寡聚脱氧核苷酸,能够激活机体免疫系统,可以分为A型、B型、C型、N型四种类型,其中B型和C型都能通过刺激B细胞增生,并促使B细胞分泌多种细胞因子。
为了研究CpG ODN能否成为重组口蹄疫病毒疫苗的分子佐剂,我们采用重组牛Asia1型口蹄疫病毒A10蛋白疫苗,研究了自行设计的CpG 02的分子佐剂功能,结果表明:CpG 02能够增效重组A10蛋白疫苗免疫作用,CpG 02与重组A10蛋白在剂量上有配比相关性。
CpG 02对于A10蛋白疫苗有分子佐剂的效果,但能否作为A10蛋白疫苗理想的分子佐剂还有待进一步探讨。
Foot and mouth disease (FMD) is an acute, febrile, highly contagious diseasescaused by foot and mouth disease virus (FMDV). The disease mainly affects domesticcloven-hoofed animals, including cattle, swine, sheep as well as more than 70species of wild animals, characterized by fever, lameness, and vesicular lesions on thetongue, feet, snout and teats.The transmission of the disease is mainly through respiratory route by aerosolized viruses. FMD dose not kill animals usually but sickensinfected animals and severely reduces the production of meat and mike, resulting ineconomic disaster. FMDV is the type species of the Aphthovirus genus of thePicornaviridae family. By electron microscopy, the FMD virion appears to be a roundparticle with a smooth surface and the diameter of the virion is about 25 nm. Sevenserotypes (A, O, C, Asia1, and South African Territories 1, 2, and 3) have beenidentified. In China, the main popular serotypes are O, A, C and ZB (Yunnan Baoshantype).
Vaccination is the mian method of prevention of FMD. The killed FMD vaccinehas been successful in reducing the number of disease outbreaks in many parts of theworld. However, there are several disadvantages associated with the use of the killedvaccines, such as the requirement of a cold-chain to preserve vaccine stability and therisk of viral release during vaccine production. With the development ofbiotechnology, new vaccines were developed, such as genetic engineering subunitvaccines, synthetic peptide vaccines, DNA vaccines and live vector vaccine.Due tothe weak immunogenicity, the newly development vaccines require the appropriateadjuvants to boost their immunogenicity. For the present,the most widely adjuvantused in human and animal is the aluminum adjuvant. However, alum salts arerelatively weak adjuvants and rarely induce cellular immune responses. Highaluminium levels in the body predominately affect the brain, causing fatalneurological syndrome and dialysis-associated dementia .For the limitations of thealuminum adjuvant, the new adjuvants such as carbohydrate, bacterial derivatives, CpG ODN and other adjuvants are highlyexpected.
It has been reported that mycobacterial DNA had adjuvant activity, and thisadjuvant activity was correlated with a higher content of CpG motifs present inbacterial nucleic acids. Synthetic oligodeoxynucleotides (ODN) containingunmethylated CpG motifs act as immune adjuvants, accelerating and boostingantigen-specific immune responses. CpG motifs promote the induction of Th1 andpro-inflammatory cytokines and support the maturation/activation of professionalantigen presenting cells (particularly plasmacytoid dendritic cells).Co-administeringCpG ODN with a variety of vaccines has improved the resultant humoral and/orcellular immune responses. CpG ODN has been obtained good results in theanti-bacterial, viral, anti-tumor as vaccine adjuvants.
Recombinant A10 protein designed in our lab.is one of the bovine vaccinesagainst foot and mouth disease designed in our lab. In order to enhance theimmunological effect of the A10 protein, we immunized guinea pigs withrecombinant A10 protein vaccine with CpG ODN and Montanide ISA 206. Thisarticle was divided in to the following parts:
1. Screening the optimum CpG ODN
In order to screen the CpG OND which has the optimum adjuvant effect for therecombinant A10 protein, we chose the different CpG ODN designed in our lab tostimulate splenocytes of three guinea-pigs respectively and detect the effects of CpGODN on proliferation of guinea-pig splenocytes by MTTassay. We found CpG 02 hadthe optimum effet to stimulate the proliferation of guinea-pig splenocytes, so wechose the CpG 02 as adjuvant of the recombinant A10 protein and immuneguinea-pigs to observe the adjuvant effect of CpG 02.
2. Purityand concentration analysis of CpG 02
In order to prepare vaccine, we analysed the concentration and purity of CpG 02to test whether this batch of CpG 02 degraded or not. From the figure of 20%denaturing PAGE, we can see that there was no degradation band and the CpG 02 wasclear. So the purity of CpG 02 met the experiment requirement. We measuredconcentration of CpG 02 by using ultraviolet spectrophotometer, and theconcentration was 0.5mg/ml.
3. Concentration, purity and molecular weight analysis of recombinant A10protein.
Firstly, we measured concentration of the A10 protein by lowry assay and themeasured result was 2.73 mg/ml. Subsequently non-denaturing SDS-PAGE anddenaturing SDS-PAGE electrophoresis were carried out for A10 protein, we analysedthe purity of A10 protein was 91.64% and the molecular weight was 38.25 KD byGIS gel image processing system. From the foregoing, this batch of A10 protein canbe used as protein vaccine for animal immunization.
4.The enhancement of CpG ODN onA10 protein.
Firstly, we measured the antibody of guinea-pigs in different time points byELISA. According to the ELISA results, when the bovine Asia1 Inactived FMDV ascoating antigen, the antibodies of guinea-pigs in A10-100 ?g + CpG 02 group wasobviouslyhigher than the other adjuvant groups and the A10-200 ?g group (except forinactivated FMDV group), the results showed that when CpG 02 with certain dose ofA10 protein immuned guinea-pigs, CpG 02 had the optimum adjuvant effet. WhenA10 protein as coating antigen,the antibodies of guinea-pigs in A10-100 ?g + CpG 02group were obviously higher than the other groups (especially in 91 day aferimmunization), the results showed that CpG 02 could enhance and prolong A10protein-Specific Immune Response.
Next,we measured the antibody of guinea-pigs from -2 day to 91 day afterimmunization by ELISA. According to the ELISA results,when the bovine Asia1Inactived FMDV as coating antigen,the antibody of guinea-pigs in A10-100 g +CpG 02 group was obviously higher than A10-200 ?g group(P<0.05) (except forinactivated FMDV group) and the antibody still rased on day 91 afterimmunization.The antibody of other groups reached to the highest level on andrespectively decreased to the foot on day 42 and 72.When A10 protein as coatingantigen,the antibody of guinea-pigs in adjuvant groups were higher than theA10-200 g group and the antibody in A10-100 ?g + CpG 02 group washighest.Observation on Immunological efficacy of CpG 02 adjuvant A10 proteinAgainstFMDV,CpG 02 have adjuvant effect toA10 protein.
The conclusions are got from the results analysis in this paper as following: whenCpG 02 is used with A10 protein,CpG 02 can enhance immunogenicity of A10protein and A10 protein-Specific Immune Response. To exert the optimum adjuvanteffect, CpG 02 shoud be used with the proper dosage ofA10 protein(100 ?g).When changed the proper dosage of A10 protein,CpG 02 does not effectively exertadjuvant effect,and even inhibits immunogenicity of vaccine protein.
CpG ODN是一种包含非甲基化的胞嘧啶和鸟嘌呤核苷酸(CpG基序)的寡聚脱氧核苷酸,能够激活机体免疫系统,可以分为A型、B型、C型、N型四种类型,其中B型和C型都能通过刺激B细胞增生,并促使B细胞分泌多种细胞因子。
为了研究CpG ODN能否成为重组口蹄疫病毒疫苗的分子佐剂,我们采用重组牛Asia1型口蹄疫病毒A10蛋白疫苗,研究了自行设计的CpG 02的分子佐剂功能,结果表明:CpG 02能够增效重组A10蛋白疫苗免疫作用,CpG 02与重组A10蛋白在剂量上有配比相关性。
CpG 02对于A10蛋白疫苗有分子佐剂的效果,但能否作为A10蛋白疫苗理想的分子佐剂还有待进一步探讨。
Foot and mouth disease (FMD) is an acute, febrile, highly contagious diseasescaused by foot and mouth disease virus (FMDV). The disease mainly affects domesticcloven-hoofed animals, including cattle, swine, sheep as well as more than 70species of wild animals, characterized by fever, lameness, and vesicular lesions on thetongue, feet, snout and teats.The transmission of the disease is mainly through respiratory route by aerosolized viruses. FMD dose not kill animals usually but sickensinfected animals and severely reduces the production of meat and mike, resulting ineconomic disaster. FMDV is the type species of the Aphthovirus genus of thePicornaviridae family. By electron microscopy, the FMD virion appears to be a roundparticle with a smooth surface and the diameter of the virion is about 25 nm. Sevenserotypes (A, O, C, Asia1, and South African Territories 1, 2, and 3) have beenidentified. In China, the main popular serotypes are O, A, C and ZB (Yunnan Baoshantype).
Vaccination is the mian method of prevention of FMD. The killed FMD vaccinehas been successful in reducing the number of disease outbreaks in many parts of theworld. However, there are several disadvantages associated with the use of the killedvaccines, such as the requirement of a cold-chain to preserve vaccine stability and therisk of viral release during vaccine production. With the development ofbiotechnology, new vaccines were developed, such as genetic engineering subunitvaccines, synthetic peptide vaccines, DNA vaccines and live vector vaccine.Due tothe weak immunogenicity, the newly development vaccines require the appropriateadjuvants to boost their immunogenicity. For the present,the most widely adjuvantused in human and animal is the aluminum adjuvant. However, alum salts arerelatively weak adjuvants and rarely induce cellular immune responses. Highaluminium levels in the body predominately affect the brain, causing fatalneurological syndrome and dialysis-associated dementia .For the limitations of thealuminum adjuvant, the new adjuvants such as carbohydrate, bacterial derivatives, CpG ODN and other adjuvants are highlyexpected.
It has been reported that mycobacterial DNA had adjuvant activity, and thisadjuvant activity was correlated with a higher content of CpG motifs present inbacterial nucleic acids. Synthetic oligodeoxynucleotides (ODN) containingunmethylated CpG motifs act as immune adjuvants, accelerating and boostingantigen-specific immune responses. CpG motifs promote the induction of Th1 andpro-inflammatory cytokines and support the maturation/activation of professionalantigen presenting cells (particularly plasmacytoid dendritic cells).Co-administeringCpG ODN with a variety of vaccines has improved the resultant humoral and/orcellular immune responses. CpG ODN has been obtained good results in theanti-bacterial, viral, anti-tumor as vaccine adjuvants.
Recombinant A10 protein designed in our lab.is one of the bovine vaccinesagainst foot and mouth disease designed in our lab. In order to enhance theimmunological effect of the A10 protein, we immunized guinea pigs withrecombinant A10 protein vaccine with CpG ODN and Montanide ISA 206. Thisarticle was divided in to the following parts:
1. Screening the optimum CpG ODN
In order to screen the CpG OND which has the optimum adjuvant effect for therecombinant A10 protein, we chose the different CpG ODN designed in our lab tostimulate splenocytes of three guinea-pigs respectively and detect the effects of CpGODN on proliferation of guinea-pig splenocytes by MTTassay. We found CpG 02 hadthe optimum effet to stimulate the proliferation of guinea-pig splenocytes, so wechose the CpG 02 as adjuvant of the recombinant A10 protein and immuneguinea-pigs to observe the adjuvant effect of CpG 02.
2. Purityand concentration analysis of CpG 02
In order to prepare vaccine, we analysed the concentration and purity of CpG 02to test whether this batch of CpG 02 degraded or not. From the figure of 20%denaturing PAGE, we can see that there was no degradation band and the CpG 02 wasclear. So the purity of CpG 02 met the experiment requirement. We measuredconcentration of CpG 02 by using ultraviolet spectrophotometer, and theconcentration was 0.5mg/ml.
3. Concentration, purity and molecular weight analysis of recombinant A10protein.
Firstly, we measured concentration of the A10 protein by lowry assay and themeasured result was 2.73 mg/ml. Subsequently non-denaturing SDS-PAGE anddenaturing SDS-PAGE electrophoresis were carried out for A10 protein, we analysedthe purity of A10 protein was 91.64% and the molecular weight was 38.25 KD byGIS gel image processing system. From the foregoing, this batch of A10 protein canbe used as protein vaccine for animal immunization.
4.The enhancement of CpG ODN onA10 protein.
Firstly, we measured the antibody of guinea-pigs in different time points byELISA. According to the ELISA results, when the bovine Asia1 Inactived FMDV ascoating antigen, the antibodies of guinea-pigs in A10-100 ?g + CpG 02 group wasobviouslyhigher than the other adjuvant groups and the A10-200 ?g group (except forinactivated FMDV group), the results showed that when CpG 02 with certain dose ofA10 protein immuned guinea-pigs, CpG 02 had the optimum adjuvant effet. WhenA10 protein as coating antigen,the antibodies of guinea-pigs in A10-100 ?g + CpG 02group were obviously higher than the other groups (especially in 91 day aferimmunization), the results showed that CpG 02 could enhance and prolong A10protein-Specific Immune Response.
Next,we measured the antibody of guinea-pigs from -2 day to 91 day afterimmunization by ELISA. According to the ELISA results,when the bovine Asia1Inactived FMDV as coating antigen,the antibody of guinea-pigs in A10-100 g +CpG 02 group was obviously higher than A10-200 ?g group(P<0.05) (except forinactivated FMDV group) and the antibody still rased on day 91 afterimmunization.The antibody of other groups reached to the highest level on andrespectively decreased to the foot on day 42 and 72.When A10 protein as coatingantigen,the antibody of guinea-pigs in adjuvant groups were higher than theA10-200 g group and the antibody in A10-100 ?g + CpG 02 group washighest.Observation on Immunological efficacy of CpG 02 adjuvant A10 proteinAgainstFMDV,CpG 02 have adjuvant effect toA10 protein.
The conclusions are got from the results analysis in this paper as following: whenCpG 02 is used with A10 protein,CpG 02 can enhance immunogenicity of A10protein and A10 protein-Specific Immune Response. To exert the optimum adjuvanteffect, CpG 02 shoud be used with the proper dosage ofA10 protein(100 ?g).When changed the proper dosage of A10 protein,CpG 02 does not effectively exertadjuvant effect,and even inhibits immunogenicity of vaccine protein.
引文
[1] AK. Yi, et al. CpG DNA rescue of murine B lymphoma cells from anti-IgMinduced growth arrest and programmed cell death is associated with increasedexpression of c-myc and bcl-xl. J. Immunol, 1996, 157:4918–4925.
[2] Audibert FM, Lise LD. Adjuvants: current status, clinical perspectives and futureprospects. Immunol Today, 1993, 14: 281–284.
[3] Baylor NW, Egan W, Richman P. Aluminum salts in vaccinesUS perspective.Vaccine, 2002, 20(3): 18-23.
[4] Becker Y. A point of view: HIV-1/AIDS is an allergy but CpG ODN treatmentsmay inhibit virus replication and reactivate the adaptive immunity--hypothesisand implications.Virus Genes, 2005, 30(1): 127-31.
[5] Brewer JM, et al.J.In interleukin-4-deficient mice, alum not only generates Thelper 1 responses equivalent to Freund’s complete adjuvant, but continues toinduce T helper 2 cytokine production. Eur J. Immunol, 1996, 26: 2062–6.
[6] Cao X, et al.Interleukin 12 stimulates IFN-gamma-mediated inhibition oftumor-induced regulatory T-cell proliferation and enhances tumor clearance.Cancer Res, 2009, 1569(22): 8700-9.
[7] Cebon JS, et al.Quantitation of immune responses against the cancer testis antigenNY-ESO-1following vaccination with protein with and without ISCOMATRIXadjuvant. ASCO Meet Abstr , 2004, 22: 2518.
[8] Clarke BE, Newton SE, Carroll AR, et al. Improved immunogenicity of apeptide after fusion to hepatitis B core protein. Nature. 1987;330(6146):381-4.
[9] Cooper PD, McComb C, Steele EJ. The adjuvanticity of Algammulin,a newvaccine adjuvant. Vaccine, 1991, 9: 408–15.
[10] Cooper. CL, et al.CPG 7909, an immunostimulatory TLR9 agonistoligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: adouble-blind phase I/II study. J Clin Immunol, 2004, 24(6): 693-701.
[11] D.M. Klinman.CpG DNAas a vaccine adjuvant. Vaccines, 2003,2: 305–315.
[12] Dong L, Ito S, Ishii KJ, Klinman DM. Suppressive oligodeoxynucleotidesdelay the onset of glomerulonephritis and prolong survival in lupus-prone NZB xNZW mice.Arthritis Rheum, 2005, 52(2): 651-658.
[13] D. Steinberg, et al. Theoretical and experimentalapproaches to generalizedautoimmunity. Immunol Rev. 1990, 118: 129-63.
[14] Edelman R. Vaccine Adjuvants. Rev. Infect. Dis, 1980, 2: 370–83.
[15] Erik B Lindblad,et al.Aluminium compounds for use in vaccines,Immunol CellBiol, 2004,82(5): 497-505.
[16] Fonseca DM,et al.Mycobacterium tuberculosis culture filtrate proteins plus CpGOligodeoxynucleotides confer protection to Mycobacterium bovis BCG-primedmice byinhibiting interleukin-4 secretion.Infect Immun, 2009; 77(12): 5311-21. .
[17] G. Hartmann, et al.Oligodeoxynucleotides enhance LPS stimulated synthesis ofTNF: dependence on phosphorothioate modification and reversal by heparin. Mol.Med, 1996, 2: 429–438.
[18] Gouher A,Harris R.In~nasal vaccination with ISCOMATRIX adjuvantinfluenza vaccine[J].Vacine, 2003, 21(9-10): 946—949.
[19] Gramzinski RA, et al. Interleukin-12- and gamma interferon-dependentprotection against malaria conferred by CpG oligodeoxynucleotide in mice. InfectImmun, 2001, 69(3): 1643-1649.
[20] Gupta RK, Siber GR. Adjuvants for human vaccines-current status, problemsand future prospects. Vaccine, 1995, 13: 1263–1276.
[21] Hacker H, et al. Immune cell activation by bacterial CpG-DNA through myeloiddifferentiation marker 88 and tumor necrosis factor receptor-associated factor(TRAF) 6. J Exp Med, 2000, 192: 595–600.
[22] Hemmi H,et a1.A Toll—like receptor recognizes bacterial DNA.Nature, 2000,408(6813): 740-745.
[23] Hirunpetcharat C, et al. CpG oligodeoxynucleotide enhances immunity againstblood-stage malaria infection in mice parenterally immunized with ayeast-expressed 19 kDa carboxyl-terminal fragment of Plasmodium yoeliimerozoite surface protein-1 (MSP1(19)) formulated in oil-based Montanides.Vaccine, 2003, 21: 2923-32.
[24] Klinman DM, et al. CpG motifs expressed by bacterial DNA rapidly inducelymphocytes to secrete IL-6, IL-12 and IFNg. Proc Natl Acad Sci USA, 1996, 93:2879–2883.
[25] Klinman DM. CpG oligonucleotides accelerate and boost the immune responseelicited by AVA, the licensed anthrax vaccine. Expert Rev Vaccines, 2006, 5(3):365-9.
[26] Kramp WJ, Six HR, Kasel JA. Post-immunization clearance ofliposome-entrapped adenovirus type 5 hexon. Proc. Soc. Exp.Biol. Medical 1982; 169:55–9.
[27]Matthew P. Morrow and David B.Cytokines as adjuvants for improving anti-HIVresponses .WeinerAIDS, 2008, 22: 333–338.
[28] Murad YM, Clay TM. CpG oligodeoxynucleotides as TLR9 agonists:therapeutic applications in cancer.BioDrugs, 2009, 23(6): 361-75.
[29] Mullen GE,et al.Vaccine. Epub 2006 Jan 4. Enhancement of functional antibodyresponses to AMA1-C1/Alhydrogel, a Plasmodium falciparum malaria vaccine,with CpG oligodeoxynucleotide. 2006; 24(14): 2497-505.
[30] Nicholaou T, et al. Phase II trial of vaccination with full length NY-ESO-1/IMXin patients withadvanced malignant melanoma. ASCO Meet Abstr, 2006, 24(18S):2571.
[31] OttG, et a1. The adjuvant MF59:A 10 Year perspective[M].In:0Hagan DT.Vaccine Adjuvants,Prepatafion Methods and Research Protocols.New Jersey:Human Press, 2000, 211-228.
[32] Parant MA, et al. Immunostimulant activities of a lipophilic muramyl dipeptidederivative and of adesmuramyl peptidolipid analogue. Infect. Immun, 1980, 27:826–830.
[33] Podda A, Del Gudice G. MF59-adjuvanted vaccines:increased immunogenieitywith an optimal safetyprofile[J]. Exper Rev Vaccines, 2003, 2(2): 197-203.
[34] Pun PB, et al. Intranasal administration of peptide antigens of HIV with mucosaladjuvant CpG ODN coentrapped in microparticles enhances the mucosal andsystemic immune responses. Int Immunopharmacol, 2009, 9(4):468-477.
[35] Rankin R, et al. CpG motif identification for veterinary and laboratory speciesdemonstrates that sequence recognition is highly conserved. Antisense NucleicAcid Drug Dev 2001;11:333–340
[36] R.A. Zeuner, et al. Influence of stimulatory and suppressive DNA motifs on hostsusceptibilityto inflammatory arthritis. Arthritis Rheum, 2003, 48 : 1701–1707.
[37] Relyveld EH. Preparation and use of calcium phosphate adsorbed vaccines. Dev.Biol. Stand, 1986, 65: 131–6.
[38] Rothel JS, Wood PR, Seow HF, et al. Urea/DTT sulubilization of a recombinantTaenia ovis antigen 45W, expressed as a GST fusion protein results in enhancedprotective immune response to the 45W moiety. Vaccine. 1997;15(5):469-72.
[39] Sjolander A, Cox JC, Barr IG.SCOMs: an adjuvant withmultiple functions. J.Leukoc. Biol, 1998, 64: 713–723.
[40] Silva D, Cooper PD, Petrovsky N. The search for the ideal adjuvant;inulinderived adjuvants promote both Th1 and Th2 immune responses withminimal toxicity. Immunol. Cell Biol, 2004.
[41] Sugauchi F, et al. Vigorous hepatitis C virus-specific CD4+ and CD8+ T cellresponses induced by protein immunization in the presence of Montanide ISA720plus synthetic oligodeoxynucleotides containing immunostimulatorycytosine-guanine dinucleotide motifs. J Infect Dis, .2006, 193(4): 563-572.
[42] Tokunaga T, et al. Antitumor activity of deoxyribonucleic acid fraction fromMycobacterium bovis BCG. I. Isolation, physicochemical characterization, andantitumor activity. J Natl Cancer Inst, 1984, 72(4): 955-62.
[43] Tomai MA, Johnson AG. T cell and interferon-g involvement in the adjuvantaction of a detoxified endotoxin. Biol. Resp Modifiers, 1989, 8: 625–30.
[44] Valliet R. Improved technique for the preparation of water—in-oil emulsionscontaining protein antigens[J]. Biotechniques, 1996, 20(5): 797—800.
[45] Verthelyi D, et al. Human peripheral blood cells differentially recognize andrespond to two distinct CpG motifs. J Immunol, 2001, 166:2372–2377.
[46] Wack A, et al. Combination adjuvants for the induction of potent, long-lastingantibody and T-cell responses to influenza vaccine in mice. Vaccine, 2008, 26(4):552-561.
[47] Waters RV, Terrell TG, Jones GH. Uveitis induction in the rabbit bymuramyldipeptids. Infect. Immunol, 1986, 51: 816–25.
[48] Yamamoto S, et al. Unique palindromic sequences in synthetic oligonucleotidesare required to induce IFN and augment IFNmediated natural killer activity. JImmunol, 1992; 148:4072–4076.
[49] Yang C, et al. GLS/IL-12-modified Mycobacterium smegmatis as a novelanti-tuberculosis immunotherapeutic vaccine. Int J Tuberc Lung Dis, 2009; 13(11):1360-1366.
[50] Yang L, et al. Effect of a C-type CpG ODN on CVB3-infected mice. 2009, 25(2): 115-117.
[51]何萍,吕凤林,任建敏.铝佐剂机制及其纳米化前景[J].世界华人消化杂志,2003, 11 (11): 1764-1768.
[52]林树乾,张燕,杨少华,等.中药黄芪多糖的免疫佐剂作用[J].中国畜牧兽医, 2006, 33(5): 58-60.
[53]卢曾军,刘在新.口蹄疫病毒研究概况[J].中国兽医科技,2003, 33(4):69-74.
[54]李娜,孙志伟,俞炜源. CpG免疫刺激DNA序列及其在疫苗佐剂中的应用[J].生物技术通讯,2008,14 (94):572-575
[55]李伟.口蹄疫流行病学、病理变及防控措施[J].甘肃科技, 2009, 25( 2 ): 152-154.
[56]李瑶瑶,明恒强.我国免疫佐剂的研究现状[J].兽药与饲料添加剂, 2009,14(5): 11-14.
[57]沈永周,谢三星,魏建忠,等.免疫佐剂和免疫调节剂的研究近况[J].吉林畜牧兽医, 1996, 18(4): 24-26.
[58]向会耀,谭玉梅,向雅倩,等.蜂胶佐剂对E.coli多价灭活苗免疫小鼠效果的实验研究[J].细胞与分子免疫学杂志, 2008, 24(5): 535-535.
[59]扬增歧,赵余放,刘长富,等.兔瘟蜂胶佐荆灭活苗对家兔细胞免疫水平的影响[J].中国兽医科技, 1997, 27(6): 21—24.
[60]张红英,赵现敏,崔保安,等.板蓝根多糖对猪繁殖与呼吸综合征疫苗免疫猪T细胞亚群的影响[J].中国免疫学杂志, 2007, 23(2): 134—137.
[61]张敬哲.蜂胶的研究利用概况[J].特种经济动植物,2006(2):13-14.
[62]张中洋,王潇潇,郝春生,等.三种类型CpG-ODN作为蛋白亚单位疫苗佐剂的比较研究[J].中华微生物学和免疫学杂志, 2008, 28(4): 368—372。
[2] Audibert FM, Lise LD. Adjuvants: current status, clinical perspectives and futureprospects. Immunol Today, 1993, 14: 281–284.
[3] Baylor NW, Egan W, Richman P. Aluminum salts in vaccinesUS perspective.Vaccine, 2002, 20(3): 18-23.
[4] Becker Y. A point of view: HIV-1/AIDS is an allergy but CpG ODN treatmentsmay inhibit virus replication and reactivate the adaptive immunity--hypothesisand implications.Virus Genes, 2005, 30(1): 127-31.
[5] Brewer JM, et al.J.In interleukin-4-deficient mice, alum not only generates Thelper 1 responses equivalent to Freund’s complete adjuvant, but continues toinduce T helper 2 cytokine production. Eur J. Immunol, 1996, 26: 2062–6.
[6] Cao X, et al.Interleukin 12 stimulates IFN-gamma-mediated inhibition oftumor-induced regulatory T-cell proliferation and enhances tumor clearance.Cancer Res, 2009, 1569(22): 8700-9.
[7] Cebon JS, et al.Quantitation of immune responses against the cancer testis antigenNY-ESO-1following vaccination with protein with and without ISCOMATRIXadjuvant. ASCO Meet Abstr , 2004, 22: 2518.
[8] Clarke BE, Newton SE, Carroll AR, et al. Improved immunogenicity of apeptide after fusion to hepatitis B core protein. Nature. 1987;330(6146):381-4.
[9] Cooper PD, McComb C, Steele EJ. The adjuvanticity of Algammulin,a newvaccine adjuvant. Vaccine, 1991, 9: 408–15.
[10] Cooper. CL, et al.CPG 7909, an immunostimulatory TLR9 agonistoligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: adouble-blind phase I/II study. J Clin Immunol, 2004, 24(6): 693-701.
[11] D.M. Klinman.CpG DNAas a vaccine adjuvant. Vaccines, 2003,2: 305–315.
[12] Dong L, Ito S, Ishii KJ, Klinman DM. Suppressive oligodeoxynucleotidesdelay the onset of glomerulonephritis and prolong survival in lupus-prone NZB xNZW mice.Arthritis Rheum, 2005, 52(2): 651-658.
[13] D. Steinberg, et al. Theoretical and experimentalapproaches to generalizedautoimmunity. Immunol Rev. 1990, 118: 129-63.
[14] Edelman R. Vaccine Adjuvants. Rev. Infect. Dis, 1980, 2: 370–83.
[15] Erik B Lindblad,et al.Aluminium compounds for use in vaccines,Immunol CellBiol, 2004,82(5): 497-505.
[16] Fonseca DM,et al.Mycobacterium tuberculosis culture filtrate proteins plus CpGOligodeoxynucleotides confer protection to Mycobacterium bovis BCG-primedmice byinhibiting interleukin-4 secretion.Infect Immun, 2009; 77(12): 5311-21. .
[17] G. Hartmann, et al.Oligodeoxynucleotides enhance LPS stimulated synthesis ofTNF: dependence on phosphorothioate modification and reversal by heparin. Mol.Med, 1996, 2: 429–438.
[18] Gouher A,Harris R.In~nasal vaccination with ISCOMATRIX adjuvantinfluenza vaccine[J].Vacine, 2003, 21(9-10): 946—949.
[19] Gramzinski RA, et al. Interleukin-12- and gamma interferon-dependentprotection against malaria conferred by CpG oligodeoxynucleotide in mice. InfectImmun, 2001, 69(3): 1643-1649.
[20] Gupta RK, Siber GR. Adjuvants for human vaccines-current status, problemsand future prospects. Vaccine, 1995, 13: 1263–1276.
[21] Hacker H, et al. Immune cell activation by bacterial CpG-DNA through myeloiddifferentiation marker 88 and tumor necrosis factor receptor-associated factor(TRAF) 6. J Exp Med, 2000, 192: 595–600.
[22] Hemmi H,et a1.A Toll—like receptor recognizes bacterial DNA.Nature, 2000,408(6813): 740-745.
[23] Hirunpetcharat C, et al. CpG oligodeoxynucleotide enhances immunity againstblood-stage malaria infection in mice parenterally immunized with ayeast-expressed 19 kDa carboxyl-terminal fragment of Plasmodium yoeliimerozoite surface protein-1 (MSP1(19)) formulated in oil-based Montanides.Vaccine, 2003, 21: 2923-32.
[24] Klinman DM, et al. CpG motifs expressed by bacterial DNA rapidly inducelymphocytes to secrete IL-6, IL-12 and IFNg. Proc Natl Acad Sci USA, 1996, 93:2879–2883.
[25] Klinman DM. CpG oligonucleotides accelerate and boost the immune responseelicited by AVA, the licensed anthrax vaccine. Expert Rev Vaccines, 2006, 5(3):365-9.
[26] Kramp WJ, Six HR, Kasel JA. Post-immunization clearance ofliposome-entrapped adenovirus type 5 hexon. Proc. Soc. Exp.Biol. Medical 1982; 169:55–9.
[27]Matthew P. Morrow and David B.Cytokines as adjuvants for improving anti-HIVresponses .WeinerAIDS, 2008, 22: 333–338.
[28] Murad YM, Clay TM. CpG oligodeoxynucleotides as TLR9 agonists:therapeutic applications in cancer.BioDrugs, 2009, 23(6): 361-75.
[29] Mullen GE,et al.Vaccine. Epub 2006 Jan 4. Enhancement of functional antibodyresponses to AMA1-C1/Alhydrogel, a Plasmodium falciparum malaria vaccine,with CpG oligodeoxynucleotide. 2006; 24(14): 2497-505.
[30] Nicholaou T, et al. Phase II trial of vaccination with full length NY-ESO-1/IMXin patients withadvanced malignant melanoma. ASCO Meet Abstr, 2006, 24(18S):2571.
[31] OttG, et a1. The adjuvant MF59:A 10 Year perspective[M].In:0Hagan DT.Vaccine Adjuvants,Prepatafion Methods and Research Protocols.New Jersey:Human Press, 2000, 211-228.
[32] Parant MA, et al. Immunostimulant activities of a lipophilic muramyl dipeptidederivative and of adesmuramyl peptidolipid analogue. Infect. Immun, 1980, 27:826–830.
[33] Podda A, Del Gudice G. MF59-adjuvanted vaccines:increased immunogenieitywith an optimal safetyprofile[J]. Exper Rev Vaccines, 2003, 2(2): 197-203.
[34] Pun PB, et al. Intranasal administration of peptide antigens of HIV with mucosaladjuvant CpG ODN coentrapped in microparticles enhances the mucosal andsystemic immune responses. Int Immunopharmacol, 2009, 9(4):468-477.
[35] Rankin R, et al. CpG motif identification for veterinary and laboratory speciesdemonstrates that sequence recognition is highly conserved. Antisense NucleicAcid Drug Dev 2001;11:333–340
[36] R.A. Zeuner, et al. Influence of stimulatory and suppressive DNA motifs on hostsusceptibilityto inflammatory arthritis. Arthritis Rheum, 2003, 48 : 1701–1707.
[37] Relyveld EH. Preparation and use of calcium phosphate adsorbed vaccines. Dev.Biol. Stand, 1986, 65: 131–6.
[38] Rothel JS, Wood PR, Seow HF, et al. Urea/DTT sulubilization of a recombinantTaenia ovis antigen 45W, expressed as a GST fusion protein results in enhancedprotective immune response to the 45W moiety. Vaccine. 1997;15(5):469-72.
[39] Sjolander A, Cox JC, Barr IG.SCOMs: an adjuvant withmultiple functions. J.Leukoc. Biol, 1998, 64: 713–723.
[40] Silva D, Cooper PD, Petrovsky N. The search for the ideal adjuvant;inulinderived adjuvants promote both Th1 and Th2 immune responses withminimal toxicity. Immunol. Cell Biol, 2004.
[41] Sugauchi F, et al. Vigorous hepatitis C virus-specific CD4+ and CD8+ T cellresponses induced by protein immunization in the presence of Montanide ISA720plus synthetic oligodeoxynucleotides containing immunostimulatorycytosine-guanine dinucleotide motifs. J Infect Dis, .2006, 193(4): 563-572.
[42] Tokunaga T, et al. Antitumor activity of deoxyribonucleic acid fraction fromMycobacterium bovis BCG. I. Isolation, physicochemical characterization, andantitumor activity. J Natl Cancer Inst, 1984, 72(4): 955-62.
[43] Tomai MA, Johnson AG. T cell and interferon-g involvement in the adjuvantaction of a detoxified endotoxin. Biol. Resp Modifiers, 1989, 8: 625–30.
[44] Valliet R. Improved technique for the preparation of water—in-oil emulsionscontaining protein antigens[J]. Biotechniques, 1996, 20(5): 797—800.
[45] Verthelyi D, et al. Human peripheral blood cells differentially recognize andrespond to two distinct CpG motifs. J Immunol, 2001, 166:2372–2377.
[46] Wack A, et al. Combination adjuvants for the induction of potent, long-lastingantibody and T-cell responses to influenza vaccine in mice. Vaccine, 2008, 26(4):552-561.
[47] Waters RV, Terrell TG, Jones GH. Uveitis induction in the rabbit bymuramyldipeptids. Infect. Immunol, 1986, 51: 816–25.
[48] Yamamoto S, et al. Unique palindromic sequences in synthetic oligonucleotidesare required to induce IFN and augment IFNmediated natural killer activity. JImmunol, 1992; 148:4072–4076.
[49] Yang C, et al. GLS/IL-12-modified Mycobacterium smegmatis as a novelanti-tuberculosis immunotherapeutic vaccine. Int J Tuberc Lung Dis, 2009; 13(11):1360-1366.
[50] Yang L, et al. Effect of a C-type CpG ODN on CVB3-infected mice. 2009, 25(2): 115-117.
[51]何萍,吕凤林,任建敏.铝佐剂机制及其纳米化前景[J].世界华人消化杂志,2003, 11 (11): 1764-1768.
[52]林树乾,张燕,杨少华,等.中药黄芪多糖的免疫佐剂作用[J].中国畜牧兽医, 2006, 33(5): 58-60.
[53]卢曾军,刘在新.口蹄疫病毒研究概况[J].中国兽医科技,2003, 33(4):69-74.
[54]李娜,孙志伟,俞炜源. CpG免疫刺激DNA序列及其在疫苗佐剂中的应用[J].生物技术通讯,2008,14 (94):572-575
[55]李伟.口蹄疫流行病学、病理变及防控措施[J].甘肃科技, 2009, 25( 2 ): 152-154.
[56]李瑶瑶,明恒强.我国免疫佐剂的研究现状[J].兽药与饲料添加剂, 2009,14(5): 11-14.
[57]沈永周,谢三星,魏建忠,等.免疫佐剂和免疫调节剂的研究近况[J].吉林畜牧兽医, 1996, 18(4): 24-26.
[58]向会耀,谭玉梅,向雅倩,等.蜂胶佐剂对E.coli多价灭活苗免疫小鼠效果的实验研究[J].细胞与分子免疫学杂志, 2008, 24(5): 535-535.
[59]扬增歧,赵余放,刘长富,等.兔瘟蜂胶佐荆灭活苗对家兔细胞免疫水平的影响[J].中国兽医科技, 1997, 27(6): 21—24.
[60]张红英,赵现敏,崔保安,等.板蓝根多糖对猪繁殖与呼吸综合征疫苗免疫猪T细胞亚群的影响[J].中国免疫学杂志, 2007, 23(2): 134—137.
[61]张敬哲.蜂胶的研究利用概况[J].特种经济动植物,2006(2):13-14.
[62]张中洋,王潇潇,郝春生,等.三种类型CpG-ODN作为蛋白亚单位疫苗佐剂的比较研究[J].中华微生物学和免疫学杂志, 2008, 28(4): 368—372。
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |