重组幽门螺杆菌尿素酶B亚单位疫苗（rHp）剂型及免疫途径的实验研究
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摘要
幽门螺杆菌(Helicobacter pylori，Hp)是定植于人胃粘膜的重要致病菌，全球感染率高达50％以上，与慢性胃炎、胃十二指肠溃疡及胃粘膜相关淋巴组织淋巴瘤的发生、发展密切相关，1994年世界卫生组织(WHO)将幽门螺杆菌定为Ⅰ类致癌因子。药物治疗Hp存在较大的局限性(药物毒副作用、费用昂贵、患者依从性差及耐药菌株不断增多等)，而疫苗接种可使人群获得持久、有效的免疫力，是从人群中消除Hp的有效措施之一。动物实验表明，重组尿素酶B亚单位(rUreB)辅以适当佐剂免疫动物后，不仅能够产生有效的保护性免疫反应，而且可清除体内已感染的Hp。
幽门螺杆菌在粘膜部位感染机体，研究证实，Hp疫苗经粘膜途径接种能产生有效的保护性免疫应答，发挥预防、治疗Hp感染的作用。根据共同粘膜免疫系统(common mucosal immunity system，CMIS)理论，在一个粘膜部位接种可以在其它远距离粘膜部位产生免疫反应，胃、鼻腔、直肠均可作为粘膜接种的途径。
鼻腔含有的蛋白水解酶较少，抗原不易被破坏，鼻腔免疫可以在多个粘膜部位产生sIgA反应，诱导与胃肠相关淋巴组织(GALT)形式上相似的粘膜免疫反应。直肠内无蛋白水解酶、环境温和，血管及淋巴丰富，直肠接种同样可在胃肠粘膜诱导免疫应答。然而，鼻腔及直肠均为管腔通道，液体药物停留时间较短，生物利用度低，用生物黏附剂卡泊波(carbopol)制备的凝胶，具有粘度高、生物粘附性好的特点，能延长药物与粘膜的接触时间。
目前，国内外对幽门螺杆菌疫苗的研究仅局限在口服免疫，剂型以溶
液或微球为主，众所周知，液体疫苗口服后易受到胃内低pH及胃蛋白酶
的破坏;微球虽然是疫苗载体研究的热点，但在制备过程中应用的有机溶
剂及高温操作会破坏疫苗的完整性，降低免疫效果.W/o/W型复乳以油膜
包裹药物，可以保护药物免受胃蛋白酶的破坏，并具有淋巴趋向性，而且
制备条件温和，不影响药物的稳定性，适合作为蛋白类药物的口服剂型。
本研究以rHP疫苗(含rUreB及佐剂LTB)为对象，研制了适合鼻腔、
直肠及口服免疫的剂型，考察了rHP疫苗经鼻腔、直肠及口服接种后的免
疫应答水平及保护效率，并探讨了免疫保护机制。
疫苗凝胶的粘度系数为8050厘泊，常温放置6个月凝胶没有明显变化，
抗原的性质不受卡伯波及粘膜吸收促进剂的影响，动物实验表明，与疫苗
液体相比，疫苗凝胶鼻腔及直肠接种产生的免疫应答显著增强(p<0.01)。
疫苗复乳的包封率为98.3%，粒径主要分布于2~10 pm之间，集中
于5一7pm，粘度为1432厘泊，体内分布实验显示，6h胃中的抗原浓度
仍很高，肠系膜淋巴结中的放射量24h最高，与胃液作用0.5~6小时复乳
中的抗原不受影响，口服免疫小鼠后明显提高了rHP疫苗的免疫应答水平。
rHP疫苗经鼻腔、直肠及口服免疫小鼠后，均在小鼠的血清、胃粘膜、
小肠粘膜冲洗液中产生了较强的特异性免疫应答，并产生了明显的抗HP
的免疫保护。ELISPOT抗体分泌细胞测定表明，三种途径免疫均可在脾脏
及PP结产生抗体分泌细胞，与特异性抗体的测定结果相对应，但不同途
径存在差别。细胞因子的RT一PCR试验证实，小鼠感染后胃组织主要有INF-
Y表达，无IL一4表达，表现为Thl反应，攻毒后未感染的免疫鼠胃组织中
则表达IL一4，表现为ThZ反应。三种免疫途径中鼻腔免疫剂量最小(10p
留只)，其次为直肠免疫(25 pg/只)，复乳50 pg/只。
结论:
1.制备了包裹效率高、稳定性好的rHP复乳，粒径大小适合抗原提呈
细胞的摄取，复乳能保护抗原免受胃消化酶破坏，剂量小，免疫效率高。
2.制备了生物粘附性强、性质稳定的r乃少凝胶剂，鼻腔及直肠免疫后
能明显提高粘膜部位的免疫应答。
3.鼻腔、直肠及口服免疫rHP疫苗均能诱导较强的粘膜免疫应答，产
生明显的抗枷的免疫保护，三种途径均可作为rHP疫苗的免疫途径。
诱导以
枷感染诱导以Thl反应为主要特征的免疫应答，而rHP疫苗主要
ThZ反应为主要特征的保护性免疫应答。
Helicobacter pylori planting in stomach mucosa is now recognized as the most widespread human pathogen. Approximately half of the world's population is infected. The infection of H. pylori is highly associated with chronic active gastritis, peptic ulcers, gastric adenocarcinoma and lymphoma of the mucosa-associated lymphoid tissue (MALT).In 1994,WHO ranked H. Pylori as I grade carcinogen. Current drug therapies are not practical for global control due to the high cost, problems with patients' compliance and the emergence antibiotic resistant strains. Therefore, Vaccination against H. pylori has been considered to control H. pylori infection. It has been proved that administration of oral urease B subunit antigens together with a mucosal adjuvant can not only induce a series of protective immune reaction but also sweep Hp from stomach of infected mice. So, recombinant vaccine of Hp (rHp} was used in this study.
It has been proved that vaccine administered by mucosa can produce effective immune response and prevent or cure Hp infection. In terms of CMIS, all mucosa such as gut, nasal and rectal routs can be used to inoculate vaccine. Intranasal immunization does not expose antigens to low pH and a broad range of degradative enzymes. So it can induce stronger and earlier IgA immune responses at almost all mucosal sites than oral route. The way induced by nasal route is similar to which gut associated lymphoid tissue(GALT) induced. There are rectal follicles but not protease in rectum. Rectal immunization can also induce mucosal immune responses in gastrointestinal tract.
However, liquid drugs administered by nasal cavity and rectum have a low bioavailability due to staying a short time. The gel prepared with carbopol has strong bio-adhesion, can prolong contact between drug and mucosa. It may be used in nasal and rectal immunization.
Now, studies on immunogenicity of Hp vaccine mainly focus on oral administration with liquid or microparticles. Whereas the vaccine solution administrated orally would be digested and decomposed by the low pH and pepsin. Though microparticle is a popular form, it has a series of significant problems such as low encapsulation rate and possibility of antigen denaturation as a consequence of expose to organic solvents and high temperature. Water-in-oil-in-water type multiple emulsion (w/o/w) in which drug is entrapped in oil droplet can protect drug from pepsin and has lymph taxis. So, it is suitable to protein drugs for oral.
In this study, two suitable preparation were made. Firstly, stable rHp gel (Viscid coefficient 8050 centipoise) was prepared. It did not destruct antigenic integrity and induced stronger immune response than rHp solution did after administered by intranasal route or rectal route. Secondly, rHp multiple emulsion(ME) was prepared in two-stage emulsification method. It has a high entrapment efficiency of 98.3%, particle size distributing within 5-7u m , Viscid coefficient of 1432 centipoise. Antigen was stable after Multiple emulsion treated with gastric juice for 0.5-6h. Study on distribution in vivo of ME revealed that ME could stay for a long time in stomach and that antigen concentration in mesentery was increased with time and reached peak at 24h. Antigen in ME was stable after treated with gastric juice for 6h. ME has significantly enhanced the immune efficiency of rHp.
In this study, high specific antibodies levels were induced in serum, gastric and intestinal mucosa of BALB/c mice immunized with rHp by oral, rectal and intranasal route. In the meantime, comparing with control group,
evident immune protection against Hp was induced. The dose is 10 μ g per mouse of intranasal immunization, 25 μ g per mouse of rectal immunization and 50 μ g per mouse of oral immunization.
Specific IgA and IgG antibody secreting cells detected by ELISPOT were discovered in spleen and Peyer'patch of mice immunized with rHp by three routes, which were corresponded with mucosal specific antibodies. RT-PCR of cytokine revealed that IL-4 was mainly expressed
幽门螺杆菌在粘膜部位感染机体，研究证实，Hp疫苗经粘膜途径接种能产生有效的保护性免疫应答，发挥预防、治疗Hp感染的作用。根据共同粘膜免疫系统(common mucosal immunity system，CMIS)理论，在一个粘膜部位接种可以在其它远距离粘膜部位产生免疫反应，胃、鼻腔、直肠均可作为粘膜接种的途径。
鼻腔含有的蛋白水解酶较少，抗原不易被破坏，鼻腔免疫可以在多个粘膜部位产生sIgA反应，诱导与胃肠相关淋巴组织(GALT)形式上相似的粘膜免疫反应。直肠内无蛋白水解酶、环境温和，血管及淋巴丰富，直肠接种同样可在胃肠粘膜诱导免疫应答。然而，鼻腔及直肠均为管腔通道，液体药物停留时间较短，生物利用度低，用生物黏附剂卡泊波(carbopol)制备的凝胶，具有粘度高、生物粘附性好的特点，能延长药物与粘膜的接触时间。
目前，国内外对幽门螺杆菌疫苗的研究仅局限在口服免疫，剂型以溶
液或微球为主，众所周知，液体疫苗口服后易受到胃内低pH及胃蛋白酶
的破坏;微球虽然是疫苗载体研究的热点，但在制备过程中应用的有机溶
剂及高温操作会破坏疫苗的完整性，降低免疫效果.W/o/W型复乳以油膜
包裹药物，可以保护药物免受胃蛋白酶的破坏，并具有淋巴趋向性，而且
制备条件温和，不影响药物的稳定性，适合作为蛋白类药物的口服剂型。
本研究以rHP疫苗(含rUreB及佐剂LTB)为对象，研制了适合鼻腔、
直肠及口服免疫的剂型，考察了rHP疫苗经鼻腔、直肠及口服接种后的免
疫应答水平及保护效率，并探讨了免疫保护机制。
疫苗凝胶的粘度系数为8050厘泊，常温放置6个月凝胶没有明显变化，
抗原的性质不受卡伯波及粘膜吸收促进剂的影响，动物实验表明，与疫苗
液体相比，疫苗凝胶鼻腔及直肠接种产生的免疫应答显著增强(p<0.01)。
疫苗复乳的包封率为98.3%，粒径主要分布于2~10 pm之间，集中
于5一7pm，粘度为1432厘泊，体内分布实验显示，6h胃中的抗原浓度
仍很高，肠系膜淋巴结中的放射量24h最高，与胃液作用0.5~6小时复乳
中的抗原不受影响，口服免疫小鼠后明显提高了rHP疫苗的免疫应答水平。
rHP疫苗经鼻腔、直肠及口服免疫小鼠后，均在小鼠的血清、胃粘膜、
小肠粘膜冲洗液中产生了较强的特异性免疫应答，并产生了明显的抗HP
的免疫保护。ELISPOT抗体分泌细胞测定表明，三种途径免疫均可在脾脏
及PP结产生抗体分泌细胞，与特异性抗体的测定结果相对应，但不同途
径存在差别。细胞因子的RT一PCR试验证实，小鼠感染后胃组织主要有INF-
Y表达，无IL一4表达，表现为Thl反应，攻毒后未感染的免疫鼠胃组织中
则表达IL一4，表现为ThZ反应。三种免疫途径中鼻腔免疫剂量最小(10p
留只)，其次为直肠免疫(25 pg/只)，复乳50 pg/只。
结论:
1.制备了包裹效率高、稳定性好的rHP复乳，粒径大小适合抗原提呈
细胞的摄取，复乳能保护抗原免受胃消化酶破坏，剂量小，免疫效率高。
2.制备了生物粘附性强、性质稳定的r乃少凝胶剂，鼻腔及直肠免疫后
能明显提高粘膜部位的免疫应答。
3.鼻腔、直肠及口服免疫rHP疫苗均能诱导较强的粘膜免疫应答，产
生明显的抗枷的免疫保护，三种途径均可作为rHP疫苗的免疫途径。
诱导以
枷感染诱导以Thl反应为主要特征的免疫应答，而rHP疫苗主要
ThZ反应为主要特征的保护性免疫应答。
Helicobacter pylori planting in stomach mucosa is now recognized as the most widespread human pathogen. Approximately half of the world's population is infected. The infection of H. pylori is highly associated with chronic active gastritis, peptic ulcers, gastric adenocarcinoma and lymphoma of the mucosa-associated lymphoid tissue (MALT).In 1994,WHO ranked H. Pylori as I grade carcinogen. Current drug therapies are not practical for global control due to the high cost, problems with patients' compliance and the emergence antibiotic resistant strains. Therefore, Vaccination against H. pylori has been considered to control H. pylori infection. It has been proved that administration of oral urease B subunit antigens together with a mucosal adjuvant can not only induce a series of protective immune reaction but also sweep Hp from stomach of infected mice. So, recombinant vaccine of Hp (rHp} was used in this study.
It has been proved that vaccine administered by mucosa can produce effective immune response and prevent or cure Hp infection. In terms of CMIS, all mucosa such as gut, nasal and rectal routs can be used to inoculate vaccine. Intranasal immunization does not expose antigens to low pH and a broad range of degradative enzymes. So it can induce stronger and earlier IgA immune responses at almost all mucosal sites than oral route. The way induced by nasal route is similar to which gut associated lymphoid tissue(GALT) induced. There are rectal follicles but not protease in rectum. Rectal immunization can also induce mucosal immune responses in gastrointestinal tract.
However, liquid drugs administered by nasal cavity and rectum have a low bioavailability due to staying a short time. The gel prepared with carbopol has strong bio-adhesion, can prolong contact between drug and mucosa. It may be used in nasal and rectal immunization.
Now, studies on immunogenicity of Hp vaccine mainly focus on oral administration with liquid or microparticles. Whereas the vaccine solution administrated orally would be digested and decomposed by the low pH and pepsin. Though microparticle is a popular form, it has a series of significant problems such as low encapsulation rate and possibility of antigen denaturation as a consequence of expose to organic solvents and high temperature. Water-in-oil-in-water type multiple emulsion (w/o/w) in which drug is entrapped in oil droplet can protect drug from pepsin and has lymph taxis. So, it is suitable to protein drugs for oral.
In this study, two suitable preparation were made. Firstly, stable rHp gel (Viscid coefficient 8050 centipoise) was prepared. It did not destruct antigenic integrity and induced stronger immune response than rHp solution did after administered by intranasal route or rectal route. Secondly, rHp multiple emulsion(ME) was prepared in two-stage emulsification method. It has a high entrapment efficiency of 98.3%, particle size distributing within 5-7u m , Viscid coefficient of 1432 centipoise. Antigen was stable after Multiple emulsion treated with gastric juice for 0.5-6h. Study on distribution in vivo of ME revealed that ME could stay for a long time in stomach and that antigen concentration in mesentery was increased with time and reached peak at 24h. Antigen in ME was stable after treated with gastric juice for 6h. ME has significantly enhanced the immune efficiency of rHp.
In this study, high specific antibodies levels were induced in serum, gastric and intestinal mucosa of BALB/c mice immunized with rHp by oral, rectal and intranasal route. In the meantime, comparing with control group,
evident immune protection against Hp was induced. The dose is 10 μ g per mouse of intranasal immunization, 25 μ g per mouse of rectal immunization and 50 μ g per mouse of oral immunization.
Specific IgA and IgG antibody secreting cells detected by ELISPOT were discovered in spleen and Peyer'patch of mice immunized with rHp by three routes, which were corresponded with mucosal specific antibodies. RT-PCR of cytokine revealed that IL-4 was mainly expressed
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38．毛世瑞，杨宏图，毕殿洲。提高药物鼻粘膜吸收的途径。中国新药杂志，1998，33(11)：641—644
39. Muguet V, Seiller. M, Barratt .G, et al. Formulation of shear rate sensitive emultiple emulsions[J].J Controlled Release,2001, 70,1-2:37-49
40. Tabata Y, Inoue Y, Ikada Y. Size effect on systemic and mucosal immune responses induced by oral administration of biodegradable microspheres[J]. Vaccine, 1996,14:1677
41. Nakaoka R,Tabata Y. Ikada Y. Enhanced antibody production through sustained release from biodegrable granules [J]. J Controlled Release, 1995, 37:215.
42. Offit PA, Khoury CA, Moser CA, et al. Enhancement of rotavirus immunogenicity by microencapsulation[J]. Virology, 1994, (1): 134
43. Hu KF, Ekstrom J, Merza M, et al. Induction of antibody responses in the common mucosal immune system by respiratory syncytical virus immunostimulating complexes [J], Med. Microbiol. Immunol .(Berl.), 1999,187(4):191-198
44. Barchfeld GL, Hessler AL, Chen M, et al. The adjuvants MF59 and
LT-K63 enhance the mucosal and systemic immunogenicity of subunit influenza vaccine administered intranasally in mice [J], Vaccine, 1999, 17(7-8): 695-704
45. Okochi H, Nakano, M. Preparation and evaluation of w/o/w type emulsions containing vancomycin [J] .Adv. Drug Deliv. Rev.2000, 45,1: 5—26
46. Khopade A.J, Nandakumar K.S, Jain N.K. Lectin-functionalized multiple emulsions for improved cancer therapy [J] J. Drug Target. 1998, (6): 285—292
47. Silva-Cunhad A, Chéronc M, Grossiordb J.L, et al. W/O/W multiple emulsions of insulin containing a protease inhibitor and an absorption enhancer: biological activity after oral administration to normal and diabetic rats[J]. Int. J. Pharm, 1998,169(1):33-44
48. Keenan J, Neal S, Allardyce R, et al. Serum derived IgG-mediaded immue exclusion as a mechanism of protection against H.pylori infection. Vaccine, 2002, 20:2981-2988
49. Guy B, Hessler C, Fourage S, et al. Systemic immunization with urease protect mice against Helicobacter polyri infection. Vaccine, 1998, 8:850-856
50. Marchetti M, Rossi M, Giannelli V. et al. Protection against Helicobacter pylori infection in mice by intragastric vaccination with Helicobacter pylori antigens is achieved using a non-toxic mutant of E. Coli heat-labile enterotoxin (LT) as adjuvant. Vaccine. 1998; 16(1): 33-37.
51. Fujihashi K, Kato H, van Ginkel FW, et al. A revisit of mucosal IgA immunity and oral tolerance [J]. Acta Odontol Scand, 2001,59(5):301-8
52．赖燕来，高英杰。用BA-ELISA斑点法检测痢疾杆菌免疫小鼠中的特异抗体分泌细胞(ASC)。中国免疫学杂志，1995，11(5)：280
53. Quiding JM, Ahlstedt I, Lindholm C, et al. Homing commitment of
lymphocytes activated in the human gastric and intestinal mucosa. Gut, 2001,49(4): 519-525
54. Goto T, Nishizono A, Fujioka T, et al. Local secretory immunoglobulin A and postimmunization gastritiss correlate with protection against Helicobacter pylori infection afer oral vaccineation of mice. Infect Immun, 1999,67(5):2531-2539
55．(美)F．奥斯伯，R．E．金斯顿，J．G．赛德曼，等著。精编分子生物学实验指南，科学出版社，1998
56. Davis S.S. Nasal vaccines. Adv. Drug Rev. 2001,51:21-42
57．舒翠莉，高英杰，彭虹，等。双价痢疾菌苗滴鼻免疫诱导不同粘膜部位的免疫反应。中国免疫杂志，2000，16(suppl)：25-28
58. Yanagita M,Hiroi T, Kitagaki N, et al. Nasopharyngeal associated lymphoreticular tissue (NALT) immunity: fimbriae-specific Th1 and Th2 cell-regulated IgA responses for the inhibition of bacterial attachment to epithelial cells and subsequent inflammatory cytokine production [J], J. Immunol, 1999, 162: 3559-3565
59. Hamajima K, Hoshino Y, Xin KQ, et al. Systemic and Mucosal Immune responses in Mice after rectal and vaginal immunization with HIV-DNA Vaccine. Clin Immunol 2002,120(1): 12-18
60. Quiding JM, Lonroth H, Ahlstedt I, et al. Human gastric B cell responses can be induced by intestinal immunisation. Gut, 2001,49(4):512-518
61. Wu HY, Nkolova EB, Beagly KW, et al. Induction of antibody -secreting cells and T-helper and memory cells in murine nasal lymphoid tissue [J], J Immunol, 1996, 88(4): 493-500.
62. Brandtzaeg P, Farstad IN, Haraldsen G. Regional specialization in the mucosal immune system: primed cells do not always home along the same track. Immunol. today, 1999(20): 267-277
63. Ferrero R L,Thiberge J M, Kansau I, et al. The GroES homolog of
helicobacter pylori confers protective immunity against mucosal infection in mice. Proc. Acad.Sci.USA, 1995, 92:6499-6503
64. Mohammadi M, Czinn S, RedlineR, et al.Helicobacter specific cell-mediated immune responses display a predominante Th1 phenotype and promote a delayed-type hypersensitivity response in the stomachs of mice. J Immunol, 1996, 156:4729-4738
65. Mohammadi M, Nedrud J, Redline R, et al. Murine CD4 T-cell response to Helicobacter infection Th1 cells enhance gastritis and Th2 cells reduce bacterial load[J].Gastroenterology, 1997, 113(6): 1848-1857
66. Cawbxaura JK, Jutila MA, Pascual KW. Nasal-associated lymphiod tissue: Phenotypic and functional evidence for the primary role of peripheral node addressin in naive lymphocyte adhesion to high endothelial venules in a mucosal site[J]. J Immunol, 1999,163:1382-1398
67. Schmausser B, Eck M, Greiner A,et al. Disparity between mucosal and serum IgA and IgG in Helicobacter pylori infection. Virchows Arch, 2002, 441(2): 143-7
68. Pappo J, Thomas JR, Kobok NS, et al.Effect of oral immunization with recombinat urease on murine Helicobacter feils gastitis. Infect Immun, 1995,63:1246-1252
69. Thomas JE, Austin S, Dale A, et al. Protection by human milk IgA against Helicobacter pylori infection in infancy. Lancer, 1993,342:121
70. Keenan J, Neal S, Allardyce, et al. Serum-derived IgG1-mediated immune exclusion as a mechanism of protection against H.pylory infection. Vaccine, 2002(20): 2981-2988
71. Ferrero, R.L, ThilbergeJ.M, Labigne A. Local immunoglobulin G antibodies, and not IgA, contribute to protcetive immunity against gastric H.felis infection in mice. Gut, 1996, 39(2):44
72. Ghiara P, Rossi M, Marchdetti M, et al. Therapeutic intragastic vaccination
against Helicobacter pylori in mice eradicates an otherwise chronic infection and confers protection against reinfection. Infect Immun, 1997, 65:4997-5002
73. Nedrud JG, Blanchard TG, Gottwein JM, et al. Systemic vaccination inducing either Th1 or Th2 immunity protects mice from challenge with H.pylori. Immunol Lett, 1999, 69(1):52
74. Romagnani S. The Th1/Th2 paradigm[J]. Immunol Today, 1997, 18(6):263-267
75. Weltzin R, Kleanthous H, Guirakhoo F, et al. Novel intranasal immunization techniques for antibody induction and protection of mice against gastric Helicobacter felis infection. Vaccine. 1997, 15(4):370-376
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36．黄静琳，陆锦芳，郭圣荣。生物粘附性聚丙烯酸类高分子在药剂中的应用，中国医药工业杂志，20001，32(2)：91
37．曲俊兵。复方环麻滴鼻凝胶剂的制备及质量控制，海峡药学，2001，13(3)；12—14
38．毛世瑞，杨宏图，毕殿洲。提高药物鼻粘膜吸收的途径。中国新药杂志，1998，33(11)：641—644
39. Muguet V, Seiller. M, Barratt .G, et al. Formulation of shear rate sensitive emultiple emulsions[J].J Controlled Release,2001, 70,1-2:37-49
40. Tabata Y, Inoue Y, Ikada Y. Size effect on systemic and mucosal immune responses induced by oral administration of biodegradable microspheres[J]. Vaccine, 1996,14:1677
41. Nakaoka R,Tabata Y. Ikada Y. Enhanced antibody production through sustained release from biodegrable granules [J]. J Controlled Release, 1995, 37:215.
42. Offit PA, Khoury CA, Moser CA, et al. Enhancement of rotavirus immunogenicity by microencapsulation[J]. Virology, 1994, (1): 134
43. Hu KF, Ekstrom J, Merza M, et al. Induction of antibody responses in the common mucosal immune system by respiratory syncytical virus immunostimulating complexes [J], Med. Microbiol. Immunol .(Berl.), 1999,187(4):191-198
44. Barchfeld GL, Hessler AL, Chen M, et al. The adjuvants MF59 and
LT-K63 enhance the mucosal and systemic immunogenicity of subunit influenza vaccine administered intranasally in mice [J], Vaccine, 1999, 17(7-8): 695-704
45. Okochi H, Nakano, M. Preparation and evaluation of w/o/w type emulsions containing vancomycin [J] .Adv. Drug Deliv. Rev.2000, 45,1: 5—26
46. Khopade A.J, Nandakumar K.S, Jain N.K. Lectin-functionalized multiple emulsions for improved cancer therapy [J] J. Drug Target. 1998, (6): 285—292
47. Silva-Cunhad A, Chéronc M, Grossiordb J.L, et al. W/O/W multiple emulsions of insulin containing a protease inhibitor and an absorption enhancer: biological activity after oral administration to normal and diabetic rats[J]. Int. J. Pharm, 1998,169(1):33-44
48. Keenan J, Neal S, Allardyce R, et al. Serum derived IgG-mediaded immue exclusion as a mechanism of protection against H.pylori infection. Vaccine, 2002, 20:2981-2988
49. Guy B, Hessler C, Fourage S, et al. Systemic immunization with urease protect mice against Helicobacter polyri infection. Vaccine, 1998, 8:850-856
50. Marchetti M, Rossi M, Giannelli V. et al. Protection against Helicobacter pylori infection in mice by intragastric vaccination with Helicobacter pylori antigens is achieved using a non-toxic mutant of E. Coli heat-labile enterotoxin (LT) as adjuvant. Vaccine. 1998; 16(1): 33-37.
51. Fujihashi K, Kato H, van Ginkel FW, et al. A revisit of mucosal IgA immunity and oral tolerance [J]. Acta Odontol Scand, 2001,59(5):301-8
52．赖燕来，高英杰。用BA-ELISA斑点法检测痢疾杆菌免疫小鼠中的特异抗体分泌细胞(ASC)。中国免疫学杂志，1995，11(5)：280
53. Quiding JM, Ahlstedt I, Lindholm C, et al. Homing commitment of
lymphocytes activated in the human gastric and intestinal mucosa. Gut, 2001,49(4): 519-525
54. Goto T, Nishizono A, Fujioka T, et al. Local secretory immunoglobulin A and postimmunization gastritiss correlate with protection against Helicobacter pylori infection afer oral vaccineation of mice. Infect Immun, 1999,67(5):2531-2539
55．(美)F．奥斯伯，R．E．金斯顿，J．G．赛德曼，等著。精编分子生物学实验指南，科学出版社，1998
56. Davis S.S. Nasal vaccines. Adv. Drug Rev. 2001,51:21-42
57．舒翠莉，高英杰，彭虹，等。双价痢疾菌苗滴鼻免疫诱导不同粘膜部位的免疫反应。中国免疫杂志，2000，16(suppl)：25-28
58. Yanagita M,Hiroi T, Kitagaki N, et al. Nasopharyngeal associated lymphoreticular tissue (NALT) immunity: fimbriae-specific Th1 and Th2 cell-regulated IgA responses for the inhibition of bacterial attachment to epithelial cells and subsequent inflammatory cytokine production [J], J. Immunol, 1999, 162: 3559-3565
59. Hamajima K, Hoshino Y, Xin KQ, et al. Systemic and Mucosal Immune responses in Mice after rectal and vaginal immunization with HIV-DNA Vaccine. Clin Immunol 2002,120(1): 12-18
60. Quiding JM, Lonroth H, Ahlstedt I, et al. Human gastric B cell responses can be induced by intestinal immunisation. Gut, 2001,49(4):512-518
61. Wu HY, Nkolova EB, Beagly KW, et al. Induction of antibody -secreting cells and T-helper and memory cells in murine nasal lymphoid tissue [J], J Immunol, 1996, 88(4): 493-500.
62. Brandtzaeg P, Farstad IN, Haraldsen G. Regional specialization in the mucosal immune system: primed cells do not always home along the same track. Immunol. today, 1999(20): 267-277
63. Ferrero R L,Thiberge J M, Kansau I, et al. The GroES homolog of
helicobacter pylori confers protective immunity against mucosal infection in mice. Proc. Acad.Sci.USA, 1995, 92:6499-6503
64. Mohammadi M, Czinn S, RedlineR, et al.Helicobacter specific cell-mediated immune responses display a predominante Th1 phenotype and promote a delayed-type hypersensitivity response in the stomachs of mice. J Immunol, 1996, 156:4729-4738
65. Mohammadi M, Nedrud J, Redline R, et al. Murine CD4 T-cell response to Helicobacter infection Th1 cells enhance gastritis and Th2 cells reduce bacterial load[J].Gastroenterology, 1997, 113(6): 1848-1857
66. Cawbxaura JK, Jutila MA, Pascual KW. Nasal-associated lymphiod tissue: Phenotypic and functional evidence for the primary role of peripheral node addressin in naive lymphocyte adhesion to high endothelial venules in a mucosal site[J]. J Immunol, 1999,163:1382-1398
67. Schmausser B, Eck M, Greiner A,et al. Disparity between mucosal and serum IgA and IgG in Helicobacter pylori infection. Virchows Arch, 2002, 441(2): 143-7
68. Pappo J, Thomas JR, Kobok NS, et al.Effect of oral immunization with recombinat urease on murine Helicobacter feils gastitis. Infect Immun, 1995,63:1246-1252
69. Thomas JE, Austin S, Dale A, et al. Protection by human milk IgA against Helicobacter pylori infection in infancy. Lancer, 1993,342:121
70. Keenan J, Neal S, Allardyce, et al. Serum-derived IgG1-mediated immune exclusion as a mechanism of protection against H.pylory infection. Vaccine, 2002(20): 2981-2988
71. Ferrero, R.L, ThilbergeJ.M, Labigne A. Local immunoglobulin G antibodies, and not IgA, contribute to protcetive immunity against gastric H.felis infection in mice. Gut, 1996, 39(2):44
72. Ghiara P, Rossi M, Marchdetti M, et al. Therapeutic intragastic vaccination
against Helicobacter pylori in mice eradicates an otherwise chronic infection and confers protection against reinfection. Infect Immun, 1997, 65:4997-5002
73. Nedrud JG, Blanchard TG, Gottwein JM, et al. Systemic vaccination inducing either Th1 or Th2 immunity protects mice from challenge with H.pylori. Immunol Lett, 1999, 69(1):52
74. Romagnani S. The Th1/Th2 paradigm[J]. Immunol Today, 1997, 18(6):263-267
75. Weltzin R, Kleanthous H, Guirakhoo F, et al. Novel intranasal immunization techniques for antibody induction and protection of mice against gastric Helicobacter felis infection. Vaccine. 1997, 15(4):370-376