H5、H7亚型流感多表位核酸疫苗的分子设计、构建及实验免疫研究
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摘要
流感是一种由流感病毒引发的传染病,患者以发热、粘膜充血、急性呼吸道症状为主。该病发病率高,破坏性强,从二十世纪三十年代人类发明流感疫苗至今,每年仍有25～50万人死于流感。季节性流感以H1、H3亚型为主,而禽流感H5、H7亚型也不断出现人类感染病例。流感病毒不断变异,随时都有可能进化成为大流行流感毒株,世界上很多国家都在研发新型流感疫苗以应对多种亚型流感大流行。
本研究利用生物信息学方法、免疫学知识,对H1、H3亚型流感病毒的T、B细胞表位进行预测与筛选,获得CTL表位3条:H1HA545-553、H3HA546-554、H3NP189-197,B和Th细胞表位4条:H1HA142~156、H1HA212~226、H3HA247~261、H3HA287~301。
结合已发表的表位,设计、合成CTL、B/Th多表位表达盒。以H5HA、H7HA、H1NP、多表位表达盒为主要目的基因,pVAX1为载体构建用来预防H5、H7、H1、H3亚型流感的多表位核酸疫苗。将所构建的疫苗载体转染BHK细胞,通过RT-PCR和IFA验证核酸疫苗抗原成分可以在真核细胞中成功表达。利用小鼠作为哺乳动物模型,对所构建的复合多表位核酸疫苗免疫原性进行研究,表明构建的核酸疫苗在诱导细胞免疫及体液免疫方面具有显著优势。
本实验的创新之处在于:1)依靠H5、H7亚型流感主要抗原成分,结合H1、H3抗原的功能表位构建了一种新型复合多表位核酸疫苗,目的是通过疫苗接种来预防H5、H7、H1、H3多种亚型流感病毒;2)预测多条H1、H3亚型流感功能表位,并将表位设计成能够发挥功能的多表位表达盒;3)通过小鼠实验免疫评价构建核酸疫苗的免疫原性。
Influenza is a highly infectious disease caused by frequently mutating influenza viruses. The major symptom are fever, mucous congestion and acute respiratory symptoms. This disease has a high incidence and characteristics of strong destruction. Thirties the twentieth century, mankind invented the flu vaccine. Up to now, 25 to 50 million people die annually worldwide of influenza epidemic outbreaks. According to the WHO, every year influenza viruses infected 10-20% of the total population. The greatest threat to mankind are primarily H1/H3/H5/H7 subtype.
Influenza vaccine can prevent infection and spread of the virus. The traditional vaccines proved safe and effective clinically include whole inactivated virus vaccine, live attenuated vaccine, split vaccine and subunit vaccine. But there are still some disadvantages can not be avoided, such as low level-immunization, high production costs and lack of immune monitoring and the key is that traditional vaccines can not effectively deal with the antigenic drift or antigen variation. So developing new influenza vaccine is the research focus in various countries all the time. DNA vaccine is a new technology originated in the nineties the twentieth century with its advantages stimulating the body to generate humoral and cellular immunity at the same time and it’s easily operate, build and safe. Pathogenic influenza virus antigens in the cells contains many conservative epitopes. To design and develop epitope-based multi-epitope vaccine, it can make up for the whole inactivated virus vaccine, attenuated live vaccine less than a single subtype of protection. Epitope is part of the immune system can generate immune response region, typically from 6-15 amino acid epitope or groups composed of carbon and water. It can be formed by the amino acid sequence of consecutive or discontinuous spatial three-dimensional structure of protein composition. Epitope vaccine based on defined epitopes, combined with some carriers such as the viral/ bacterial vectors or liposomes and virus-like particles.The body is immunitied after immunization against the infection. To epitope-based vaccines can efficiently use a variety of protective antigen of influenza viruses to reach the purpose of preventing a variety of virus subtypes. This study combined DNA vaccine and the advantages of epitope vaccines. We are researched on a multi-epitopes of influenza DNA vaccine. Details are as follows:
(1) Using bioinformatics methods, immunization and screening knowledge predict- ed H1, H3 subtypes of influenza virus T, B cell epitopes. We designed and synthesized CTL, B/Th multi-epitope expression cassette each one.
(2) This study was designed multi-CTL epitope expression cassette H5HA, H7HA series connected to the pVAX1 vector. Construction of H5, H7 influenza multi-epitope DNA vaccine. The B/Th multi-epitope gene expression cassette and H1NP series connected into the pVAX1 vector. Construction of NP multi-epitope DNA vaccine.
(3) The vaccine vector was transfected into BHK cells.By RT-PCR and IFA verify the expression of DNA vaccine antigen component of the various activity.
(4) Using mice as a mammalian model, the establishment of five immunization groups, two control group evaluate the ability which producted by complex multi-epitope DNA vaccine induced cellular immunity and humoral immune.
1 Forecasting H1, H3 subtypes of influenza virus T, B epitope and designed multi-epitope expression cassette.
(1) Forecasting H1, H3 subtypes of influenza virus T, B epitope
Influenza virus HA is the main component of the virus surface antigen. It can stimulate body to produce protective immunity. It is important to influenza vaccine target. The NP, M1 protein that is sequence conservation, can induce CTL immune response and immune protection. Because of its conservative design and more universal influenza vaccine, it is an important target antigen target. We predict and screen the H1, H3 subtypes of influenza virus structural proteins HA, NP, M1 T-cell epitopes, B cell epitopes by bioinformatics methods. It is obtained that the three CTL epitopes: H1HA 545~553, H3HA546~554, H3NP189~197, B and Th cell epitope 4: H1HA142~156, H1HA212~226, H3HA247~261, H3HA287~301.
(2) Multi-epitope expression cassette design.
Previous studies have shown the four CTL epitopes: H1HA344~353, H3NP380~393, H1NP1~9, H1M12~12; B cell epitope: H1HA344~357, H3HA91~108. Combined with their own forecast table phase, we design multi-CTL epitope expression cassette and B/Th epitope expression cassette. Two types of multi-epitope expression cassette are connected in series form. We used the unique connection which connect epitope to anthor epitope that protect the functional form to play. In the two expression cassettes were added in the Igκ-chain signal sequence. It can promote the potential energy of complex multi-table well into the functioning of endoplasmic reticulum region. PADRE sequence to optimize the use and enhance immunity. We named more than two epitope expression cassette for the CTL ep and B/Th ep. The design of multi-epitope amino acid sequence expression cassette restore the nucleic acid sequence..
2 Construction and identification of H5, H7 influenza multi-epitope DNA vaccine
(1) Construction of H5, H7 influenza multi-epitope DNA vaccine
We cloned influenza virus H5HA, H7HA, NP gene by molecular biology techniques. And the corresponding genes were PCR transformation. This was for constructing DNA vaccine. DNA vaccine is based on pVAX1 as vector. Construction of pVAX1-H5HA, pVAX1-H7HA, pVAX1-NP, pVAX1-H5HA-H7HA. On this basis, the multi-epitope expression box B/Th ep sequence transformed into pVAX1-NP gene of the vector, and the NP gene co-expression vector named as pVAX1-NP-B/Th ep; the multi-epitope expression Box CTL ep sequence transformed into pVAX1-H5HA-H7HA carrier, CTL ep sequence in the middle of H5HA, H7HA and the vector named pVAX1-H5HA-CTL ep-H7HA.
(2) Identification of H5, H7 influenza multi-epitope DNA vaccine
By restriction enzyme digestion and sequencing, it determin the constructed recombinant DNA vaccine including HA, NP, M1 sequence and nucleic acid sequence. This was selected strains of the same composition, consistent with the expected restrict- tion fragment sizes for further of the joint function of multi-epitope vaccine basis.
3 Expression of H5, H7 influenza multi-epitope DNA vaccine in BHK cells.
Using recombinant pVAX1-NP-B/Th ep, pVAX1-H5HA-CTL ep-H7HA transfer- cted BHK cells.We detected gene expression by RT-PCR and IFA. Through the different antibody tests,we found NP, CTL epitope expression cassette, B/Th multi-epitope expression cassette, H5HA, H7HA can be expressed in BHK cells, and it has activity. The results confirmed that the antigen components can be effectively expressed in eukaryotic cells.
4.Study on immunogenicity of H5, H7 influenza multi-epitope DNA vaccine
(1) Animal group, immune and sampling
Take 6-8 weeks old female seventy BALB/c mice, ten in each group, divided into five immunization groups, two control group. Respectively, 0d, 21d, 35d, immunization three.We got rat ophthalmic artery blood ten days after the third immunization.It was prepared to serum antibody. After the mice were killed and blood. We prepared sterile whichever spleen spleen cell suspension for detecting cellular immunity indexes in.
(2) Detection of H5, H7 multiple epitope DNA vaccine induced antibody and cellul- ar immune response.
Proved by indirect ELISA epitope vaccine immunization can stimulate the expression of antibodies and immune antigen alone group H5HA and H7HA quite specific ELISA antibody levels. Use of T lymphocyte transformation test, lymphocyte count category, IFN-γsecretion of the number of test data, this results show that the joint contains multiple epitopes that induced immune cells in the immune group optimal level. Proliferation of CD4+ T lymphocytes. H5 and H7 antigen used to stimulate T lymphocyte proliferation and IFN-γsecretion detected. Found that HA, NP components of the immune cells play a major role. Adding epitope improve the specificity of H3 subtype Stimulation Index, that some components have increased epitope immune function.
In summary, this study predicted H1, H3 epitope and then design CTL, B/Th epitope expression box. Based on the research front, building a H5, H7 subtypes of influenza virus multi-epitope DNA vaccine. DNA vaccines in inducing cellular immunity and humoral immunity has a significant advantage by immunogenicity. Derived from the H3 subtype of influenza virus epitope-specific cellular immune response has a significant advantage by immunogenicity. In this study, we developed a common influenza vaccine against H1, H3, H5, H7 subtypes of influenza virus and laid the foundation.
本研究利用生物信息学方法、免疫学知识,对H1、H3亚型流感病毒的T、B细胞表位进行预测与筛选,获得CTL表位3条:H1HA545-553、H3HA546-554、H3NP189-197,B和Th细胞表位4条:H1HA142~156、H1HA212~226、H3HA247~261、H3HA287~301。
结合已发表的表位,设计、合成CTL、B/Th多表位表达盒。以H5HA、H7HA、H1NP、多表位表达盒为主要目的基因,pVAX1为载体构建用来预防H5、H7、H1、H3亚型流感的多表位核酸疫苗。将所构建的疫苗载体转染BHK细胞,通过RT-PCR和IFA验证核酸疫苗抗原成分可以在真核细胞中成功表达。利用小鼠作为哺乳动物模型,对所构建的复合多表位核酸疫苗免疫原性进行研究,表明构建的核酸疫苗在诱导细胞免疫及体液免疫方面具有显著优势。
本实验的创新之处在于:1)依靠H5、H7亚型流感主要抗原成分,结合H1、H3抗原的功能表位构建了一种新型复合多表位核酸疫苗,目的是通过疫苗接种来预防H5、H7、H1、H3多种亚型流感病毒;2)预测多条H1、H3亚型流感功能表位,并将表位设计成能够发挥功能的多表位表达盒;3)通过小鼠实验免疫评价构建核酸疫苗的免疫原性。
Influenza is a highly infectious disease caused by frequently mutating influenza viruses. The major symptom are fever, mucous congestion and acute respiratory symptoms. This disease has a high incidence and characteristics of strong destruction. Thirties the twentieth century, mankind invented the flu vaccine. Up to now, 25 to 50 million people die annually worldwide of influenza epidemic outbreaks. According to the WHO, every year influenza viruses infected 10-20% of the total population. The greatest threat to mankind are primarily H1/H3/H5/H7 subtype.
Influenza vaccine can prevent infection and spread of the virus. The traditional vaccines proved safe and effective clinically include whole inactivated virus vaccine, live attenuated vaccine, split vaccine and subunit vaccine. But there are still some disadvantages can not be avoided, such as low level-immunization, high production costs and lack of immune monitoring and the key is that traditional vaccines can not effectively deal with the antigenic drift or antigen variation. So developing new influenza vaccine is the research focus in various countries all the time. DNA vaccine is a new technology originated in the nineties the twentieth century with its advantages stimulating the body to generate humoral and cellular immunity at the same time and it’s easily operate, build and safe. Pathogenic influenza virus antigens in the cells contains many conservative epitopes. To design and develop epitope-based multi-epitope vaccine, it can make up for the whole inactivated virus vaccine, attenuated live vaccine less than a single subtype of protection. Epitope is part of the immune system can generate immune response region, typically from 6-15 amino acid epitope or groups composed of carbon and water. It can be formed by the amino acid sequence of consecutive or discontinuous spatial three-dimensional structure of protein composition. Epitope vaccine based on defined epitopes, combined with some carriers such as the viral/ bacterial vectors or liposomes and virus-like particles.The body is immunitied after immunization against the infection. To epitope-based vaccines can efficiently use a variety of protective antigen of influenza viruses to reach the purpose of preventing a variety of virus subtypes. This study combined DNA vaccine and the advantages of epitope vaccines. We are researched on a multi-epitopes of influenza DNA vaccine. Details are as follows:
(1) Using bioinformatics methods, immunization and screening knowledge predict- ed H1, H3 subtypes of influenza virus T, B cell epitopes. We designed and synthesized CTL, B/Th multi-epitope expression cassette each one.
(2) This study was designed multi-CTL epitope expression cassette H5HA, H7HA series connected to the pVAX1 vector. Construction of H5, H7 influenza multi-epitope DNA vaccine. The B/Th multi-epitope gene expression cassette and H1NP series connected into the pVAX1 vector. Construction of NP multi-epitope DNA vaccine.
(3) The vaccine vector was transfected into BHK cells.By RT-PCR and IFA verify the expression of DNA vaccine antigen component of the various activity.
(4) Using mice as a mammalian model, the establishment of five immunization groups, two control group evaluate the ability which producted by complex multi-epitope DNA vaccine induced cellular immunity and humoral immune.
1 Forecasting H1, H3 subtypes of influenza virus T, B epitope and designed multi-epitope expression cassette.
(1) Forecasting H1, H3 subtypes of influenza virus T, B epitope
Influenza virus HA is the main component of the virus surface antigen. It can stimulate body to produce protective immunity. It is important to influenza vaccine target. The NP, M1 protein that is sequence conservation, can induce CTL immune response and immune protection. Because of its conservative design and more universal influenza vaccine, it is an important target antigen target. We predict and screen the H1, H3 subtypes of influenza virus structural proteins HA, NP, M1 T-cell epitopes, B cell epitopes by bioinformatics methods. It is obtained that the three CTL epitopes: H1HA 545~553, H3HA546~554, H3NP189~197, B and Th cell epitope 4: H1HA142~156, H1HA212~226, H3HA247~261, H3HA287~301.
(2) Multi-epitope expression cassette design.
Previous studies have shown the four CTL epitopes: H1HA344~353, H3NP380~393, H1NP1~9, H1M12~12; B cell epitope: H1HA344~357, H3HA91~108. Combined with their own forecast table phase, we design multi-CTL epitope expression cassette and B/Th epitope expression cassette. Two types of multi-epitope expression cassette are connected in series form. We used the unique connection which connect epitope to anthor epitope that protect the functional form to play. In the two expression cassettes were added in the Igκ-chain signal sequence. It can promote the potential energy of complex multi-table well into the functioning of endoplasmic reticulum region. PADRE sequence to optimize the use and enhance immunity. We named more than two epitope expression cassette for the CTL ep and B/Th ep. The design of multi-epitope amino acid sequence expression cassette restore the nucleic acid sequence..
2 Construction and identification of H5, H7 influenza multi-epitope DNA vaccine
(1) Construction of H5, H7 influenza multi-epitope DNA vaccine
We cloned influenza virus H5HA, H7HA, NP gene by molecular biology techniques. And the corresponding genes were PCR transformation. This was for constructing DNA vaccine. DNA vaccine is based on pVAX1 as vector. Construction of pVAX1-H5HA, pVAX1-H7HA, pVAX1-NP, pVAX1-H5HA-H7HA. On this basis, the multi-epitope expression box B/Th ep sequence transformed into pVAX1-NP gene of the vector, and the NP gene co-expression vector named as pVAX1-NP-B/Th ep; the multi-epitope expression Box CTL ep sequence transformed into pVAX1-H5HA-H7HA carrier, CTL ep sequence in the middle of H5HA, H7HA and the vector named pVAX1-H5HA-CTL ep-H7HA.
(2) Identification of H5, H7 influenza multi-epitope DNA vaccine
By restriction enzyme digestion and sequencing, it determin the constructed recombinant DNA vaccine including HA, NP, M1 sequence and nucleic acid sequence. This was selected strains of the same composition, consistent with the expected restrict- tion fragment sizes for further of the joint function of multi-epitope vaccine basis.
3 Expression of H5, H7 influenza multi-epitope DNA vaccine in BHK cells.
Using recombinant pVAX1-NP-B/Th ep, pVAX1-H5HA-CTL ep-H7HA transfer- cted BHK cells.We detected gene expression by RT-PCR and IFA. Through the different antibody tests,we found NP, CTL epitope expression cassette, B/Th multi-epitope expression cassette, H5HA, H7HA can be expressed in BHK cells, and it has activity. The results confirmed that the antigen components can be effectively expressed in eukaryotic cells.
4.Study on immunogenicity of H5, H7 influenza multi-epitope DNA vaccine
(1) Animal group, immune and sampling
Take 6-8 weeks old female seventy BALB/c mice, ten in each group, divided into five immunization groups, two control group. Respectively, 0d, 21d, 35d, immunization three.We got rat ophthalmic artery blood ten days after the third immunization.It was prepared to serum antibody. After the mice were killed and blood. We prepared sterile whichever spleen spleen cell suspension for detecting cellular immunity indexes in.
(2) Detection of H5, H7 multiple epitope DNA vaccine induced antibody and cellul- ar immune response.
Proved by indirect ELISA epitope vaccine immunization can stimulate the expression of antibodies and immune antigen alone group H5HA and H7HA quite specific ELISA antibody levels. Use of T lymphocyte transformation test, lymphocyte count category, IFN-γsecretion of the number of test data, this results show that the joint contains multiple epitopes that induced immune cells in the immune group optimal level. Proliferation of CD4+ T lymphocytes. H5 and H7 antigen used to stimulate T lymphocyte proliferation and IFN-γsecretion detected. Found that HA, NP components of the immune cells play a major role. Adding epitope improve the specificity of H3 subtype Stimulation Index, that some components have increased epitope immune function.
In summary, this study predicted H1, H3 epitope and then design CTL, B/Th epitope expression box. Based on the research front, building a H5, H7 subtypes of influenza virus multi-epitope DNA vaccine. DNA vaccines in inducing cellular immunity and humoral immunity has a significant advantage by immunogenicity. Derived from the H3 subtype of influenza virus epitope-specific cellular immune response has a significant advantage by immunogenicity. In this study, we developed a common influenza vaccine against H1, H3, H5, H7 subtypes of influenza virus and laid the foundation.
引文
[1]Chang-won and Lee yms. Avian influenza virus[J].Comparative Immunology, Micro- biology and Infectious Diseases, 2009, 32(4): 301-310.
[2]Subbarao K, Klimov A, Katz J, et al. Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness[J]. Science, 1998, 279(5349): 393-396.
[3]WHO.http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_12_21/en/index.html
[4]傅朝阳.人禽流感病原学与流行病学研究进展[J].应用预防医学, 2009, 15(2): 122- 125.
[5]蔡闯,钟南山.高致病性禽流感A(H5N1)[J].中国急救医学, 2008, 28(5): 463-465.
[6]WHO.http://www.who.int/csr/disease/avian_influenza/avian_faqs/zh/
[7]Perroncito CE. It typhoid epizootic in gallinaceous birds epizoozia tifoide nei gallinacei[J]. Torino:Annali Accademia Agricoltura, 1878, 21(1): 87-126.
[8]甘孟侯.禽流感(第2版)[M],北京:中国农业出版社: 2002.
[9]Joanne McManus. Avian flu returns to Hong Kong[J]. BMJ, 1999, 318(7191): 1098.
[10]Malik Peiris JS. Avian influenza virues in humans[J]. Rev Sci Tech, 2009,28 (1): 161-173.
[11]王敏,汤培洋,池慧,等.中国大陆H5N1亚型人禽流感流行病学分析[J].医学研究杂志, 2009, 38(4): 3-7.
[12]Wang H, Feng Z, Shu Y, et al. Probable limited person-to-person transmission of highly pathogenic avian influenza A (H5N1) virus in China.[J]. Lacet, 2008, 371 (9622): 1427-1434.
[13]Swayne DE and Halvorson DA. Diseases of poultry 11th[M]. Ames:Iowa State Univer- sity Press; 2003. p.135-160.
[14]Capua I and Alexander DJ. Avian influenza and human health[J]. Acta Trop, 2002, 83 (1): 1-6.
[15]Vaque-Rafart J. The threat of avian influenza human pandemic[J]. Med Clin (Barc), 2006, 126(5): 183-188.
[16]Lewis DB. Avian flu to human influenza[J]. Annu Rev Med, 2006, 57: 139-154.
[17]Roberts PC and Compans RW. Host cell dependence of viral morphology.Proc Natl Acad Sci USA.1998, 95(10): 5746-5751.
[18]Burleigh LM, Calder LJ, Skehel JJ, et al. Influenza a viruses with mutations in the M1 Helix six domain display a wide variety of morphological phenotype[J]. J Virol, 2005,79(2): 1262-1270.
[19]Lamb RA and Krug RM. Orthomyxoviridae: the viruses and their replication. in fields virology. vol 1[M]. Philadelphia: Lippincott Williams and Wilkins, 2001, 1487- 1531.
[20]Fouchier RA, Munster V, Wallensten A, et al. Characterization of a novel influenza Avirus hemagglutinin subtype (H16) obtained from black-headed gulls[J]. J Virol, 2005, 79: 2814-2822.
[21]Lamb RA and Choppin PW. The gene structure and replication of influenza virus[J]. Annu Rev Biochem, 1983, 52: 467-506.
[22]Nakajima K. Influenza virus genome structure and encoded proteins[J]. Japanese Journal of Clinical Medicine, 1997, 55(10): 2542-2546.
[23]Yasuda J, Nakada S, Kato A, et al.Molecular assembly of influenza virus: association of the NS2 protein with virion matrix[J]. Virology, 1993, 196(1): 249-255.
[24]Geiss G.K, SalvatoreM., Tumpey T.M, et al. Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells:the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution for pandemic influenza[J]. Proc Natl Acad Sci U S A, 2002, 99(1):10736-10741.
[25]Van Hoeven N, Pappas C, Belser JA, et al. Human HA and polymerase subunit PB2 proteins confer transmission of an avianinfluenza virus through the air[J]. Proc Natl Acad Sci U S A, 2009, 106(9): 3366-3371.
[26]Yamada S, Suzuki Y, Suzuki T, et al. Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors[J]. Nature, 2006, 444(7117): 378- 382.
[27]Tumpey TM, Maines TR, Van Hoeven N,et al. A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission[J]. Science, 2007, 315(5812): 655-659.
[28]Wang CC, Chen JR, Tseng YC, et al. Glycans on influenza hemagglutinin affect receptor binding and immune response[J]. Proc Natl Acad Sci U S A, 2009, 106 (43): 18137-18142
[29]Von Itzstein M, Wu WY, Kok GB, et al. Rational design of potent sialidase-based inhibitors of influenza virus replication[J]. Nature, 1993, 363(6428): 418-423.
[30]Gubareva LV, Kaiser L and Hayden FG. Influenza virus neuraminidase inhibitors[J]. Lancet, 2000, 355(9206): 827-835
[31]Matrosovich MN, Tatyana YM, Thomas Gray, et al. Neuraminidase is important for the initiation of influenza virusinfection in human airway epithelium[J]. J Virol, 2004, 78 (22): 12665-12667.
[32]Hurt AC, Holien JK, Parker M, et al. Zanamivir-resistant influenza viruses with a novel neuraminidase mutation[J]. J Virol, 2009, 83(20): 10366-10373.
[33]Park JW and Jo WH. Infiltration of water molecules into the oseltamivir-binding site of H274Y neuraminidase mutant causes resistance to oseltamivir[J]. J Chem Inf Model, 2009, 49(12): 2735-2741.
[34]Huang IC, Li W, Sui J, Marasco W, et al. Influenza A virus neuraminidase limits viral superinfection[J]. J Virol, 2008, 82(10): 4834-4843.
[35]Kaiser J.A one-size-fits-all flu vaccine?[J]. Science, 2006, 312(5772): 380-382.
[36]Ye Q, Krug RM, Tao YJ. The mechanism by which influenza A virus nucleo-protein forms oligomers and binds RNA[J]. Nature, 2006, 444 (7122): 1078-1082.
[37]Wang P, Song W, Mok BW, et al. Nuclear factor 90 negatively regulates influenza virus replication by interacting with viral nucleoprotein[J]. J Virol, 2009, 83(16): 7850-7861.
[38]Zhirnov OP and Klenk HD. Alterations in caspase cleavage motifs of NP and M2 proteins attenuate virulence of a highly pathogenic avian influenza virus[J]. Virolog- y, 2009, 394(1): 57-63.
[39]Ali A, Avalos RT, Ponimaskin E, et al. Influenza virus assembly:effect of influen- zavirus glycoproteins on the membrane association of M1 protein[J]. J Virol, 2000, 74(18): 8709-8719.
[40]Zhang J, Li G, Liu X, et al.Influenza A virus M1 blocks the classical complement pathway through interacting with C1qA[J]. J Gen Virol, 2009, 90(11): 2751-2758.
[41]Bright RA, Carter DM, Crevar CJ et al. Cross-clade protective immune responses to influenzaviruses with H5N1 HA and NA elicited by an influenza virus-like particle [J]. PLoS One, 2008,3:e1501.
[42]Kollerova E and Betakova T. Influence of extracellular and cytoplasmic domains of M2 ion Channel of influenzaa virus on its activity[J]. Acta Virol, 2007, 51 (2): 119- 124.
[43]McCown MF and Pekosz A. Distinct domains of the influenza a virus M2 protein cytoplasmic tail mediatebinding to the M1 protein and facilitate infectious virus production[J]. J Virol, 2006, 80(16): 8178-8189.
[44]Sui HY, Zhao GY, Huang JD, et al. Small interfering RNA targeting m2 gene induces effective and long term inhibition of influenza A virus replication[J]. PLoS One, 2009, 4(5): e5671.
[45]Song MS, Pascua PN, Lee JH, et al. The polymerase acidic protein gene of influenza a virus contributes topathogenicity in a mouse model[J]. J Virol, 2009, 83(23): 12325-12335.
[46]Hara K, Schmidt FI, Crow M, et al. Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding[J]. JVirol, 2006, 80(16): 7789-7798.
[47]Obayashi E, Yoshida H, Kawai F, et al. The structural basis for an essential subunit interaction in influenza virus RNA polymerase[J]. Nature, 2008, 454(7208): 1127- 1131.
[48]Rao Z, Liu Y, Ge R, et al .Crystal structure of the polymerase PA(C)-PB1(N) compl- ex from an avian influenza H5N1 virus[J]. Nature, 2008, 454(7208): 1123-1126.
[49]Dias A, Bouvier D, Crépin T, et al. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit[J]. Nature, 2009, 458(7240): 914-918.
[50]Taubenberger JK, Reid AH, Lourens RM, et al. Characterization of the 1918 influenza virus polymerase genes[J]. Nature, 2005, 437(7060): 889-893.
[51]Conenello GM, Zamarin D, Perrone LA, et al. A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence[J]. PLoS Pathog, 2007, 3(10): 1414-1421.
[52]Basler CF and Aguilar PV. Progress in identifying virulence determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses[J]. Antiviral Res. 2008, 79(3): 166-178.
[53]Cheng C, Yao L,Chen A, et al. Inhibitory effect of small interfering RNA specific for a novel candidate target in PB1 gene of influenza A virus[J]. J Drug Target, 2009, 17 (2): 133-139.
[54]Van Hoeven N, Pappas C, Belser JA, et al. Human HA and polymerase subunit PB2 proteins confer transmission of an avian influenza virus through the air[J]. Proc Natl Acad Sci U S A, 2009, 106(9): 3366-3371.
[55]Manzoor R, Sakoda Y, Nomura N, et al. PB2 protein of a highly pathogenic avian influenza virus strain A/chicken/Yamaguchi/7/2004 (H5N1) determines its replica- tion potential in pigs[J]. J Virol, 2009, 83(4): 1572-1578.
[56]Bradel-Tretheway BG, Kelley Z, Chakraborty-Sett S, et al. The human H5N1 influe- nza A virus polymerase complex is active in vitro over a broad range of temper- atures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit[J]. J Gen Virol, 2008, 89(12): 2923-2932.
[57]Labadie K, Dos Santos Afonso E, Rameix-Welti MA, et al. Host-range deter- minants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells[J]. Virology. 2007, 362(2): 271-282.
[58]Haye K, Burmakina S, Moran T, et al. The NS1 protein of a human influenza virus inhibits type I interferon production and the induction of antiviral responses in primary human dendritic andrespiratory epithelial cells[J]. J Virol, 2009, 83(13): 6849-6862.
[59]Jiao P, Tian G, Li Y, et al. A single-amino-acid substitution in the NS1 protein changesthe pathogenicity of H5N1 avian influenza viruses in mice[J]. J Virol. 2008, 82(3): 1146-1154.
[60]Zhu Q, Yang H, Chen W, et al. A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens[J]. J Viro, 2008, 82(1): 220-228.
[61]Jackson D, Hossain MJ, Hickman D, et al. A new influenza virus virulence deter- minant: the NS1 protein four C-terminal residues modulate pathogenicity[J]. Proc Natl Acad Sci U S A, 2008, 105(11): 4381-4386.
[62]Webster RG., Bean WJ, Gorman OT, et al. Evolution and ecology of influenza A Viruses[J]. Microbiological Rev, 1992, 56(1): 152-179.
[63]Connor RJ, Kawaoka Y, Webster RG, et al. Receptor specificity inhuman, avian, and equine H2 and H3 influenza virus isolates[J]. Virology, 1994, 205(1): 17-23.
[64]Gambaryan AS, Tuzikov AB, Piskarev VE, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylgly- copolymers:non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6’-sialy(N-acetylactosamine) [J]. Virology, 1997, 232(1): 345-350.
[65]Ito T, Couceiro JN, Kelm S, et al. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential[J]. J Virol, 1998, 72(9): 7367-7373.
[66]Shortridge KF, Zhou NN, Guan Y, et al. Characterization of avian H5N1 influenza viruses from poultry in Hong Kong[J]. Virology, 1998, 252(2): 331-342.
[67]Peiris JSM, de Jong MD, Guan Y, et al. Avian influenza virus (H5N1): a threat to human health[J]. Clin Microbiol Rev, 2007, 20(2): 243-267.
[68]Okazaki K, Takada A, Ito T, et al. Precursor genes of future pandemic influenza viruses are perpetuated in ducks nesting in Siberia[J]. Arch Virol, 2000, 145(5): 885-893.
[69]De Jong JC, Claas EC, Osterhaus AD, et al. A pandemic warning[J]. Nature, 1997, 389(6651): 554.
[70]Christian Sandrock and Terra Kelly. Clinical review: Update of avian influenza A infections in humans[J]. Critical Care, 2007, 11:209 (doi:10.1186/cc5675)
[71]Wu WL, Chen Y, Wang P, et al. Antigenic profile of avian H5N1 viruses in Asia from 2002 to 2007[J]. J Virol, 2008, 82(4): 1798-1807
[72]Monto AS. The role of antivirals in the control of influenza[J]. Vaccine 2003, 21(16): 1796-1800.
[73]World Health Organization (2007) Clinical management of human infection with avian influenza (H5N1)virus.http://www.who.int/csr/disease/avian_influenza/ guide- lines/ClinicalManagement07.pdf
[74]Monto AS, Gunn RA, Bandyk MG, et al. Prevention of Russian influenza amantadine [J]. JAMA, 1979, 241(10): 1003-1007.
[75]Hayden FG, Osterhaus AD, Treanor JJ, et al. Efficacy and safety of the neural- minidase inhibitor zanamivir inthe treatment of influenza virus infections GG167 Influenza Study Group[J]. N Engl J Med, 1997(13), 337: 874-880.
[76]Bdel-Ghafar AN, Chotpitayasunondh T, Gao Z, et al. Update on avian influenzaA (H5N1) virus infection in humans[J]. N Engl J Med, 2008, 358(3): 261-273.
[77]Yen HL, Monto AS, Webster RG, et al. Virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic A/Vietnam/ 1203/04 influenza virus in mice[J]. J Infect Dis, 2005, 192(4): 665-672.
[78]Le QM, Kiso M, Someya K, et al. Avianflu:isolation of drug-resistant H5N1 virus [J]. Nature, 2005, 437(7062): 1108.
[79]Gubareva LV. Molecular mechanisms of influenza virus resistance to neuralmini- dase inhibitors[J]. Virus Res, 2004, 103(1-2): 199-203.
[80]Hanson BJ, Boon AC, Lim AP, et al. Passive immunoprophylaxisand therapy with humanized monoclonal antibody specific for influenza A H5 hemagglutinin in mice [J]. Respir Res, 2006, 14(7): 126.
[81]Francis T. The development of the 1943 vaccination study of the Commission on Influenza[J]. Am J Hyg, 1945, 42(1): 1-11.
[82]Cate TR, Rober B couch, Parker D, et al. Reactogenicity, immunogenicity, and antibody persistence in adults given inactivated influenza virus vaccines-1978[J]. Rev Infect Dis, 1983, 5(4): 737-747.
[83]Subbarao K, Chen H, Swayne D, et al. Evaluation of a genetically modified reassort- ant H5N1 influenza A virusvaccine candidate generated by plasmid based reverse genetics[J]. Virology , 2003, 305(1): 192-200.
[84]Baez M, Palese P and Kilbourne ED. Gene composition of high-yielding influenza vaccine strains obtained by recombination[J]. J Infect Dis, 1980, 141(3): 362-365.
[85]Wendy A, Keitel MD, Robert L, et al. Safety of high doses of influenza vaccine and effect on antibody responses in elderly persons[J]. Arch Intern Med, 2006, 166(10): 1121-1127.
[86]LoGrippo GA. Investigations of the use of beta propiolactone in virus inactivation [J]. Ann NY Acad Sci, 1960, 83(13): 578-594.
[87]Fodor E, Devenish L, Engelhardt OG, et al. Rescue of influenza A virus from recom- binant DNA[J]. J Virol ,1999, 73(11): 9679-9682.
[88]Hoffmann E, Neumann G, Kawaoka Y, et al. A DNA transfection system for generat- ion of influenza A virus from eight plasmids[J]. Proc Natl Acad Sci USA, 2000, 97(11): 6108-6113.
[89]Hoffmann E, Krauss S, Perez D, et al. Eight-plasmid system for rapid generation of influenza virus vaccines[J]. Vaccine, 2002, 20(25-26): 3165-3170.
[90]Massab HF. Adaptation and growth characteristics of influenza virus at 25 degrees C[J]. Nature, 1967, 213(5076): 612-614.
[91]Murphy B and Coelingh K. Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines[J]. Viral Immunol, 2002, 15(2): 295-323.
[92]Massab HF and Bryant ML. The development of live attenuated cold-adapted influe- nza virus vaccine for humans[J]. Rev Med Virol, 1999, 9(4): 237-244.
[93]Alexandrova G and Smorodintsev A. Obtaining of an additionally attenuated vaccin- eting cryophilic influenza strain[J]. Rev Rown Inframicrobiol, 1965, 2: 179-189.
[94]Garmashova L, Plezhaev F and Alexandrova G.. Cold-adapted A/Leningrad/134/ 47/ 57 (H2N2) strain, a specific donor of attenuation of live influenza vaccine for children, and recombinants based on its base[J]. Vopr Virusol, 1984(1), 29: 28-31.
[95]Hoare M, Levy MS, Bracewell DG, et al. Bioprocess engineering issues that would be faced in producing a DNA vaccine at up to 100 m3 fermentation scale for an influenza pandemic[J]. Biotechnol Prog, 2005, 21(6): 1577-1592.
[96]Cinatl J Jr, Michaelis M and Doerr HW. The threat of avian influenza A (H5N1).Part IV:Development of vaccines[J]. Med Microbiol Immunol, 2007, 196(4): 213-225.
[97]Deck RR, DeWitt CM, Donnelly JJ, et al. Characterization of humoral immune responses induced by an influenza hemagglutinin DNA vaccine[J]. Vaccine, 1997, 15(1): 71-78.
[98]Akbari O, Panjwani N, Garcia S, et al. DNA vaccination:transfection and activation of dendritic cells as key events for immunity[J]. J Exp Med, 1999, 189(1): 169-178.
[99]Gilkeson GS, Ruiz P, Howell D, et al. Induction of immune-mediated glomerulo- nephritis in normal mice immunized with bacterial DNA[J]. Clin Immunol Immuno- pathol, 1993, 68(3): 283-292.
[100]Mor G, Yamshchikov G, Sedegah M, et al. Induction of neonatal tolerance by plasmid DNA vaccination of mice[J]. J Clin Invest, 1996, 98(12): 2700-2705.
[101]Klinman DM, Sechler JM, Conover J, et al. Contribution of cells at the site of DNA vaccination to the generation of antigen-specific immunity and memory [J].J Immunol, 1998, 160(5): 2388-2392.
[102]Sedegah M, Weiss W,Sacci JB, et al. Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus[J]. J Immunol, 2000, 164(11): 5905-5912.
[103]Turnes CG, Aleixo JA, Monteiro AV, et al. DNA inoculation with a plasmid vectorcarrying the faeG adhesin gene of Escherichia coli K88ab induced immune responses in mice and pigs[J]. Vaccine, 1999, 17(15-16): 2089-2095.
[104]Garg S, Oran AE, Hon H, et al. The hybrid cytomegalovirus enhancer/chicken beta-actin promoter along with woodchuck hepatitis virus posttranscriptional regulatory element enhances the protective efficacy of DNA vaccines[J]. J Immunol, 2004, 173(1): 550-558.
[105]James CM, Abdad MY, Mansfield JP, et al. Differential activities of alpha/beta IFN subtypes against influenza virus in vivo and enhancement of specific immune responses in DNA vaccinated mice expressing haemagglutinin and nucleoprotein[J]. Vaccine, 2007, 25(10): 1856-1867.
[106]Sharpe M, Lynch D, Topham S, et al. Protection of mice from H5N1 influenza challenge by prophylactic DNA vaccination using particle mediated epidermal delivery[J]. Vaccine, 2007, 25(34): 6392-6398.
[107]Epstein SL, Kong WP, Misplon JA, et al.Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein[J]. Vaccine, 2005, 23(46-47): 5404-5410.
[108]Ozaki T, Yauchi M, Xin KQ, et al.Cross-reactive protection against influenza A virus by a topically applied DNA vaccine encoding M gene with adjuvant [J]. Viral Immunol, 2005, 18(2): 373-380.
[109]Deliyannis G, Boyle JS, Brady JL, et al.A fusion DNA vaccine that targets antigen- presenting cells increases protection from viral challenge[J]. Proc Natl Acad Sci USA, 2000, 97(12): 6676-6680.
[110]Hung CF, Cheng WF, Chai CY, et al. Improving vaccine potency through interce- llular spreading and enhanced MHC class I presentation of antigen[J]. J Immunol, 2001, 166(9): 5733-5740.
[111]Laddy DJ, Yan J, Corbitt N, et al. Immunogenicity of novel consensus-based DNA vaccines against avian influenza[J]. Vaccine, 2007, 25(16): 2984-2989.
[112]Wong CH, Huang YH, Cheng TJ, et al. A consensus–hemagglutinin-based DNA vaccine that protects mice against divergent H5N1 influen viruses[J]. Proc Natl Acad Sci U S A. 2008, 105(36): 13538-13543.
[113]Kodihalli S, Goto H, Kobasa DL, et al. DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice[J]. J Virol, 1999, 73(3): 2094-2098.
[114]Sandbulte MR, Jimenez GS, Boon AC, et al. Cross-reactive neuraminidase anti- bodies afford partial protection against H5N1 in mice and are present in unexposed humans[J]. PLoS Med, 2007, 4:e59.
[115]Epstein SL, Tumpey TM, Misplon JA, et al. DNA vaccine expressing conserved influenza virus proteins protective against H5N1 challenge infection in mice[J].Emerg Infect Dis, 2002, 8(8): 796-801.
[116]Kodihalli S, Kobasa DL and Webster RG.. Strategies for inducing protection against avian influenza A virus subtypes with DNA vaccines[J]. Vaccine, 2000, 18(23): 2592-2599.
[117]Souza AP, Haut L, Reyes-Sandoval A, et al. Recombinant viruses as vaccines against viral diseases[J]. Braz J Med Biol Res, 2005, 38(4): 509-522.
[118]Freeman AI, Zakay-Rones Z, Gomori JM, et al. Phase I/II trial of intravenous NDVHUJ oncolytic virus in recurrent glioblastoma multiforme[J]. Mol Ther, 2006, 13(1): 221-228.
[119]Laurie SA, Bell JC, Atkins HL, et al. A phase 1 clinical study of intravenous administration of PV701, an oncolytic virus, using two-step desensitization[J]. Clin Cancer Res, 2006, 12(8): 2555-2562.
[120]Park MS, Steel J, Garcia-Sastre A, et al. Engineered viral vaccine constructs with dual specificity: avian influenza and Newcastle disease[J]. Proc Natl Acad Sci USA, 2006, 103(21): 8203-8208.
[121]Veits J, Wiesner D, Fuchs W, et al. Newcastle disease virus expressing H5 hemagg- lutinin gene protects chickens against Newcastle disease and avian influenza[J]. Proc Natl Acad Sci USA, 2006, 103(21): 8197-8202.
[122]Roberts A and Rose JK .Redesign and genetic dissection of the rhabdoviruses[J]. Adv Virus Res, 1999, 53(1): 301-319.
[123]Clarke DK, Cooper D, Egan MA, et al. Recombinant vesicular stomatitis virus as an HIV-1 vaccine vector[J]. Springer Semin Immunopathol, 2006, 28(3): 239-253.
[124]Garcia-Sastre A. Palese P.Influenza virus vectors[J]. Biologicals, 1995, 23(2): 171- 178.
[125]Steel J, Burmakina SV, Thomas C, et al. A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus[J]. Vaccine, 2008, 26(4): 522-531.
[126]Lehrman S. Virus treatment questioned after gene therapy death[J]. Nature,1999, 401 (6753): 517-518.
[127]Marshall E. Gene therapy death prompts review of adenovirus vector[J]. Science, 1999, 286(5448): 2244-2245.
[128]Souza AP, Haut L, Reyes-Sandoval A, et al. Recombinant viruses as vaccines agai- nst viral diseases[J]. Braz J Med Biol Res, 2005, 38(4): 509-522.
[129]Steinbrook R. One step forward, two steps back-will there ever be an AIDS vacc- ine[J]. N Engl J Med, 2007, 357(26): 2653-2655.
[130]Davis NL, Brown KW and Johnston RE. A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge[J]. J Virol, 1996, 70(6):3781-3787.
[131]Charles PC, Brown KW, Davis NL, et al. Mucosal immunity induced by parenteral immunization with a live attenuated Venezuelan equine encephalitis virus vaccine candidate[J]. Virology, 1997, 228(2): 153-160.
[132]Davis NL, West A, Reap E, et al. Alphavirus replicon particles as candidate HIV vaccines[J]. IUBMB Life, 2002, 53(4-5): 209-211.
[133]Gherardi MM and Esteban M .Recombinant poxviruses as mucosal vaccine vectors [J]. J Gen Virol, 2005, 86(11): 2925-2936.
[134]Souza AP, Haut L, Reyes-Sandoval A, et al. Recombinant viruses as vaccines against viral diseases[J]. Braz J Med Biol Res, 2005, 38(4): 509-522.
[135]Vijaysri S, Jentarra G, Heck MC, et al. Vaccinia viruses with mutations in the E3L gene as potential replication-competent,attenuated vaccines:intranasal vaccination[J]. Vaccine, 2008, 26(5): 664-676.
[136]Zuniga A, Wang Z, Liniger M, et al. Attenuated measles virus as a vaccine vector [J]. Vaccine, 2007, 25(16): 2974-2983.
[137]Ernst WA, Kim HJ, Tumpey TM, et al. Protection against H1, H5, H6 and H9 influenza A infection with liposomal matrix 2 epitope vaccines[J]. Vaccine, 2006, 24(24): 5158 -5168.
[138]Liu W, Zou P, Chen YH, et al. Monoclonal antibodies recognizing EVETPIRN epitope of influenza A virus M2 protein could protect mice from lethal influenza A virus challenge[J]. Immunol Lett, 93(2-3): 131-136.
[139]Bui HH, Peters B, Assarsson E, et al. Ab and T cell epitopes of influenza A virus, knowledge and opportunities[J]. Proc Natl Acad Sci U S A, 2007, 104(1):246-251.
[140]马绍辉,李琦涵.多表位病毒疫苗的研究进展[J].国外医学(微生物学分册), 2002, 25(2): 5-6.
[141]Ben-Yedidia T and Arnon R. Epitope-based vaccine against influenza.[J].Expert Rev Vaccines, 2007, (6): 939-948.
[142]Adar Y, Singer Y, Levi R, et al. A universal epitope-based influenza vaccine and its efficacy against H5N1[J]. Vaccine, 2009, 27(15): 2099-2107.
[143]Baulcombe D and Hamilton A. A species of small antisense RNA in posttran scriptional gene silencing in plants[J]. Science, 1999, 286(5441): 950-952.
[144]Elbashir S, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature, 2001, 411 (6836): 494-498.
[145]Zhang W, Wang CY, Yang ST, et al. Inhibition of highly pathogenic avian influenza virus H5N1 replication by the small interfering RNA targeting polymerase A gene[J]. Biochem Biophys Res Commun, 2009, 390(3): 421-426.
[146]Lerner RA, Kashyap AK, Steel J, et al. Combinatorial antibody libraries from surviv- ors of the Turkish H5N1 avianinfluenza outbreak reveal virus neutralization strategies[J]. Proc Natl Acad Sci U S A., 2008, 105(16): 5986-5991.
[147]Ekiert DC, Bhabha G, Elsliger MA, et al. Antibody recognition of a highly con- served influenza virus epitope[J]. Science, 2009, 24(5924): 246-251.
[148]Chang-won and Lee yms. Avian influenza virus[J]. Comparative Immunology, Microbiology and Infectious Diseases, 2009, 32: 301-310.
[149]方钟,罗文新,夏宁邵.表位疫苗研究进展[J].中国生物工程杂志, 2007, 27(11): 86-91.
[150]李健.表位疫苗的研究进展[J].中国热带医学, 2007, 7(9): 1681-1684.
[151]T. Ben-Yedidia, Y.Adar and Y.Singer. A universal epitope-based influenza vaccine and its efficacy against H5N1[J]. Vaccine, 2009, 27(15): 2099-2107.
[152]黄艳新,鲍永利,李玉新.抗原表位预测的免疫信息学方法研究进展[J].中国免疫学杂志, 2008, 24(9): 857-961.
[153]南文龙,金宁一,鲁会军,等. H5N1亚型禽流感病毒血凝素Th和B细胞表位预测及抗原性分析[J].中国免疫学杂志, 2009, 7(25): 630-637.
[154]Glenn Y. I, Fikes J, Hermanson G, et al.Utilization of MHC classⅠtransgenic mice for development of minigene DNA vaccines encoding multiple HLA-restricted CTL epitopes[J]. J Immunol, 1999, 162(7): 3915-3925.
[155]Livingston B, Crimi C, Newman M, et al. A rational strategy to design multiepitope immunogens based on multiple the lymphocyte epitopes[J]. Immunol, 2002, 168: 5499-5500.
[156]Yano A, Onozuka A, Asahi-Ozaki Y, et al. An ingenious design for peptide vaccines [J]. Vaccine, 2005, 23: 2322-2326.
[157]Sant AJ, Richards KA, Chaves FA, et al. Direct ex vivo analyses of HLA-DR1 transgenic mice reveal an exceptionally broad pattern of immunodominance in the primary HLA-DR1-restricted CD4 T-cell response to influenza virus hemagglutinin [J]. J Virol, 2007, 81(14): 7608-7619.
[158]Lee A, Mackay I, Rowley M, et al. Measurement of antibody-producing capacity to flagellin in man. IV. Studies in autoimmune disease,allergy and after azathioprine treatmen[J]. Clin Exp Immunol, 1971, 9(4): 507-518.
[159]Carreno B, Koenig S, Coligan J, et al. The peptide binding specificity of HLA class I molecules is largely allele-specific and non-overlapping[J]. Mol Immunol, 1992, 29(9): 1131-1140.
[160]Hans-Georg R, Jutta Bachmann, Niels NE, et al. SYFPEITHI: database for MHC ligands and peptide motifs[J]. Immunogenetics, 1999, 50(3-4): 213-219.
[161]Hans D, Young P and Fairlie D. Current status of short synthetic peptides as vaccines[J]. Med Chem, 2006, 2(6): 627-646.
[162]Ben-Yedidia, Marcus H, Reisner Y, et al. Intranasal administration of peptide vaccine protects human/mouse radiation chimera from influenza infection[J]. Int Immunol, 1999, 11(7): 1043-1051.
[163]Immune Epitope Database.http://www.immuneepitope.org/
[164]Bui HH, Peters B, Assarsson E, et al. Ab and T cell epitopes of influenza A virus, knowledge and opportunities[J]. Proc Natl Acad Sci U S A., 2007, 104(1): 246-251.
[165]De Groot, Sbai H, Aubin CS, et al. Immunoinformatics:Mining genomes for vacci- ne components [J]. Immunol Cell Biol, 2002, 80(3): 255-269.
[166]张伯伟,赵磊,郭如华,等. DNA基因芯片在中华骨髓库HLA组织配型检测中的应用价值[J].中国卫生工程学, 2006, 5(4): 231-232.
[167]Parida R, Shaila MS, Mukherjee S, et al. Computational analysis of proteome of H5N1 avian influenza virus to define T cell epitopes with vaccine potential [J]. Vaccine, 2007, 25(43): 7530-7539.
[168]D'Amaro J, Houbiers JG, Drijfhout JW, et al. A computer program for predicting possible cytotoxic T lymphocyte epitopes based on HLA class I peptide-binding motifs[M]. Hum Immunol, 1995, 43(1): 13-18.
[169]Zhang GL, Khan AM, Srinivasan KN, et al. MULTIPRED:a computational system for prediction of promiscuous HLA binding peptides [J]. Nucleic Acids Res, 2005, 33 (Web Server issue): W172-179.
[170]Velders MP, Weijzen S, Eiben GL, et al. Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine[J]. Immunol, 2001, 166(9): 5366-5373.
[171]黄健,郭建巍,蔡美英.辅助性T细胞表位预测[J].微生物学免疫学进展, 2004, 2(2): 50-52.
[172]J.萨姆不鲁克, D. W.拉赛尔.分子克隆实验指南[M]:第三版黄培堂等译,科学出版社2002.
[173]Manzoli L, Schioppa F, Boccia A, et al. The efficacy of influenza vaccine for healthy children: a meta-analysis evaluating potential sources of variation in efficacy estimate including study quality[J]. Pediatr Infect Dis, 2007, 26(2): 97-106.
[174]Jefferson T, Rivetti D, Di Pietrantonj C, et al. Vaccines for preventing influenza in healthy adults[M]. Cochrane Database Syst Rev. 2007: 2.
[175]Ben-Yedidia T and Arnon R. Design of peptide and polypeptide vaccines[J]. Curr Opin Biotechnol, 1997, 8(4): 442-448.
[176]Keogh E, Fikes J, Southwood S. et al. Identification of new epitopes from fourdifferent tumor-associated antigens: recognition of naturally processed epitopes corre- lates with HLA-A*0201-binding affinity[J]. J Immunol, 2001, 167(2): 787-796.
[177]De Filette M, Min Jou W, Birkett A, et al. Universal influenza A vaccine: optimi- zation of M2-based constructs [J]. Virology, 2005, 337(1): 149-161.
[178]Saha S, Yoshida S, Ohba K, et al. A fused gene of nucleoprotein (NP) and herpes simplex virus genes (VP22) induces highly protective immunity against different subtypes of influenza virus [J]. Virology, 2006, 354(1): 48-57.
[179]吴玉章,刘茂昌,贾正才,等. HBV新型免疫原的设计、合成及免疫原性研究[J].第三军医大学学报, 2000, 22(10): 919-923.
[180]Hanke T, Neumann VC, Blanchard T K, et al. Effective induction of HIV-specific CTL by multi-epitope using gene gun in a combined vaccination regime [J]. Vaccine, 1999, 17(6): 589-596.
[181]徐道华,夏永鹏,邱宗荫等.宫颈癌治疗性多肽疫苗的分子设计及免疫学功能研究[J].中国免疫学杂志, 2006, 22(5): 944-947.
[182]Yang W, Jackson DC, Zeng Q, et al. Multi-epitope schistosome vaccine candidate tested for protective immunogenicity in mice [J]. Vaccine, 2001, 19(1): 103-113.
[183]Chinnasamy D, Milsom MD, Shaffer J, et al. Multicistronic lentiviral vectors contain- ing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI[J] .J Virol, 2006, 3: 14.
[184]Klump H, Schiedlmeier B, Vogt B, et al. Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy [J]. Gene Ther, 2001, 8(10): 811-817.
[185]Wang SX, Taaffe J, Parker C, et al. Hemagglutinin (HA) proteins from H1 and H3 serotypes of influenza A viruses require different antigen designs for the induction of optimal protective antibody responses as studied by codon-optimized HA DNA Vaccines [J]. J Virol, 2006, 80(23): 11628-11637.
[186]Theobald M, Ruppert T, Kuckelkorn U, et al. The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymph- ocytes specific for a flanking peptide epitope [J]. Exp Med, 1998, 188(6): 1017- 1028.
[187]Holzhutter HG, Frommel C and Kloetzel PM. A theoretical approach towards the identifycation of cleavage-determining amino acid motifs of the 20S proteasome [J]. Mol.Biol, 1999, 286(4): 1251-1265.
[188]Jimenze-Cavero M A. Molecular cloning,expressing and immunological analysis of capid precursor polupeptide(p1) from swing vesicular disease virus[J]. Virus Res, 1998, 57(5): 763-170.
[189]Yoshikawa T, Imazu S, Gao JQ, et al. Augmentation of antigen-specific immune Responses using DNA-fusogenic liposome vaccine[J]. Biochem Biophys Res Com-mun, 2004, 325(2): 500-505.
[190]Ulmer JB. Influenza DNA vaccines[J]. Vaccine, 2002, 20(1): 74-76.
[191]Sacher T, Jordan S, Mohr CA, et al. Conditional gene expression systems to study herpesvirus biology in vivo[J]. Med Microbiol Immunol, 2008, 197(2): 269-276.
[192]Chen C and OkayamaH. High efficiency transformation of mammalian cells by plasmid DNA.[J]. Mol Cell Biol, 1987, 7(8): 2745-2752.
[193]Awan A, Oliveri RS, Jensen PL, et al. Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions[J]. Methods Mol Biol, 2010, 584(1): 195-210.
[194]Beveridge WI. The chronicle of influenza epidemics [J]. Hist Philos Life Sci, 1991, 13(1): 223-234.
[195]WHO. http://www.who.int/zh/index.html.
[196]Palese P. Making better influenza virus vaccines[J]. Emerg Infect Dis, 2006, 12 (1): 61-65.
[197]Stohr K. Overview of the WHO Global Influenza Programme[J]. Dev Biol (Basel), 2003, 115(1): 3-8.
[198]Kemble G and Greenberg H. Novel generations of influenza vaccines [J]. Vaccine, 2003, 21(16): 1789-1795.
[199]Gerdil C. The annual production cycle for influenza vaccine [J]. Vaccine, 2003, 21 (16): 1776-1779.
[200]Fedson DS. Preparing for pandemic vaccination: an international policy agenda for vaccine development [J]. J Public Health Policy, 2005, 26(1): 4-29.
[201]Thomson SA, Sherritt MA, Medveczky J, et al. Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination[J]. Immuno, 1998, 160(4): 1717-1723.
[202]Li H, Ding J and Chen YH. Recombinant protein comprising multi-neutralizing epitopes induced high titer of antibodies against influenza A virus[J]. Immunobiol, 2003, 207 (5): 305-313.
[203]贾雷立,金宁一,鲁会军,等.流感复合多表位疫苗的设计及小鼠免疫研究[J].广西农业生物科学, 2006, 25(9): 209-210.
[204]陈义锋.多亚型多表位流感病毒核酸疫苗与重组鸡痘病毒构建及实验免疫研究[D].吉林省:吉林大学硕士论文. 2007.
[2]Subbarao K, Klimov A, Katz J, et al. Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness[J]. Science, 1998, 279(5349): 393-396.
[3]WHO.http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_12_21/en/index.html
[4]傅朝阳.人禽流感病原学与流行病学研究进展[J].应用预防医学, 2009, 15(2): 122- 125.
[5]蔡闯,钟南山.高致病性禽流感A(H5N1)[J].中国急救医学, 2008, 28(5): 463-465.
[6]WHO.http://www.who.int/csr/disease/avian_influenza/avian_faqs/zh/
[7]Perroncito CE. It typhoid epizootic in gallinaceous birds epizoozia tifoide nei gallinacei[J]. Torino:Annali Accademia Agricoltura, 1878, 21(1): 87-126.
[8]甘孟侯.禽流感(第2版)[M],北京:中国农业出版社: 2002.
[9]Joanne McManus. Avian flu returns to Hong Kong[J]. BMJ, 1999, 318(7191): 1098.
[10]Malik Peiris JS. Avian influenza virues in humans[J]. Rev Sci Tech, 2009,28 (1): 161-173.
[11]王敏,汤培洋,池慧,等.中国大陆H5N1亚型人禽流感流行病学分析[J].医学研究杂志, 2009, 38(4): 3-7.
[12]Wang H, Feng Z, Shu Y, et al. Probable limited person-to-person transmission of highly pathogenic avian influenza A (H5N1) virus in China.[J]. Lacet, 2008, 371 (9622): 1427-1434.
[13]Swayne DE and Halvorson DA. Diseases of poultry 11th[M]. Ames:Iowa State Univer- sity Press; 2003. p.135-160.
[14]Capua I and Alexander DJ. Avian influenza and human health[J]. Acta Trop, 2002, 83 (1): 1-6.
[15]Vaque-Rafart J. The threat of avian influenza human pandemic[J]. Med Clin (Barc), 2006, 126(5): 183-188.
[16]Lewis DB. Avian flu to human influenza[J]. Annu Rev Med, 2006, 57: 139-154.
[17]Roberts PC and Compans RW. Host cell dependence of viral morphology.Proc Natl Acad Sci USA.1998, 95(10): 5746-5751.
[18]Burleigh LM, Calder LJ, Skehel JJ, et al. Influenza a viruses with mutations in the M1 Helix six domain display a wide variety of morphological phenotype[J]. J Virol, 2005,79(2): 1262-1270.
[19]Lamb RA and Krug RM. Orthomyxoviridae: the viruses and their replication. in fields virology. vol 1[M]. Philadelphia: Lippincott Williams and Wilkins, 2001, 1487- 1531.
[20]Fouchier RA, Munster V, Wallensten A, et al. Characterization of a novel influenza Avirus hemagglutinin subtype (H16) obtained from black-headed gulls[J]. J Virol, 2005, 79: 2814-2822.
[21]Lamb RA and Choppin PW. The gene structure and replication of influenza virus[J]. Annu Rev Biochem, 1983, 52: 467-506.
[22]Nakajima K. Influenza virus genome structure and encoded proteins[J]. Japanese Journal of Clinical Medicine, 1997, 55(10): 2542-2546.
[23]Yasuda J, Nakada S, Kato A, et al.Molecular assembly of influenza virus: association of the NS2 protein with virion matrix[J]. Virology, 1993, 196(1): 249-255.
[24]Geiss G.K, SalvatoreM., Tumpey T.M, et al. Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells:the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution for pandemic influenza[J]. Proc Natl Acad Sci U S A, 2002, 99(1):10736-10741.
[25]Van Hoeven N, Pappas C, Belser JA, et al. Human HA and polymerase subunit PB2 proteins confer transmission of an avianinfluenza virus through the air[J]. Proc Natl Acad Sci U S A, 2009, 106(9): 3366-3371.
[26]Yamada S, Suzuki Y, Suzuki T, et al. Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors[J]. Nature, 2006, 444(7117): 378- 382.
[27]Tumpey TM, Maines TR, Van Hoeven N,et al. A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission[J]. Science, 2007, 315(5812): 655-659.
[28]Wang CC, Chen JR, Tseng YC, et al. Glycans on influenza hemagglutinin affect receptor binding and immune response[J]. Proc Natl Acad Sci U S A, 2009, 106 (43): 18137-18142
[29]Von Itzstein M, Wu WY, Kok GB, et al. Rational design of potent sialidase-based inhibitors of influenza virus replication[J]. Nature, 1993, 363(6428): 418-423.
[30]Gubareva LV, Kaiser L and Hayden FG. Influenza virus neuraminidase inhibitors[J]. Lancet, 2000, 355(9206): 827-835
[31]Matrosovich MN, Tatyana YM, Thomas Gray, et al. Neuraminidase is important for the initiation of influenza virusinfection in human airway epithelium[J]. J Virol, 2004, 78 (22): 12665-12667.
[32]Hurt AC, Holien JK, Parker M, et al. Zanamivir-resistant influenza viruses with a novel neuraminidase mutation[J]. J Virol, 2009, 83(20): 10366-10373.
[33]Park JW and Jo WH. Infiltration of water molecules into the oseltamivir-binding site of H274Y neuraminidase mutant causes resistance to oseltamivir[J]. J Chem Inf Model, 2009, 49(12): 2735-2741.
[34]Huang IC, Li W, Sui J, Marasco W, et al. Influenza A virus neuraminidase limits viral superinfection[J]. J Virol, 2008, 82(10): 4834-4843.
[35]Kaiser J.A one-size-fits-all flu vaccine?[J]. Science, 2006, 312(5772): 380-382.
[36]Ye Q, Krug RM, Tao YJ. The mechanism by which influenza A virus nucleo-protein forms oligomers and binds RNA[J]. Nature, 2006, 444 (7122): 1078-1082.
[37]Wang P, Song W, Mok BW, et al. Nuclear factor 90 negatively regulates influenza virus replication by interacting with viral nucleoprotein[J]. J Virol, 2009, 83(16): 7850-7861.
[38]Zhirnov OP and Klenk HD. Alterations in caspase cleavage motifs of NP and M2 proteins attenuate virulence of a highly pathogenic avian influenza virus[J]. Virolog- y, 2009, 394(1): 57-63.
[39]Ali A, Avalos RT, Ponimaskin E, et al. Influenza virus assembly:effect of influen- zavirus glycoproteins on the membrane association of M1 protein[J]. J Virol, 2000, 74(18): 8709-8719.
[40]Zhang J, Li G, Liu X, et al.Influenza A virus M1 blocks the classical complement pathway through interacting with C1qA[J]. J Gen Virol, 2009, 90(11): 2751-2758.
[41]Bright RA, Carter DM, Crevar CJ et al. Cross-clade protective immune responses to influenzaviruses with H5N1 HA and NA elicited by an influenza virus-like particle [J]. PLoS One, 2008,3:e1501.
[42]Kollerova E and Betakova T. Influence of extracellular and cytoplasmic domains of M2 ion Channel of influenzaa virus on its activity[J]. Acta Virol, 2007, 51 (2): 119- 124.
[43]McCown MF and Pekosz A. Distinct domains of the influenza a virus M2 protein cytoplasmic tail mediatebinding to the M1 protein and facilitate infectious virus production[J]. J Virol, 2006, 80(16): 8178-8189.
[44]Sui HY, Zhao GY, Huang JD, et al. Small interfering RNA targeting m2 gene induces effective and long term inhibition of influenza A virus replication[J]. PLoS One, 2009, 4(5): e5671.
[45]Song MS, Pascua PN, Lee JH, et al. The polymerase acidic protein gene of influenza a virus contributes topathogenicity in a mouse model[J]. J Virol, 2009, 83(23): 12325-12335.
[46]Hara K, Schmidt FI, Crow M, et al. Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding[J]. JVirol, 2006, 80(16): 7789-7798.
[47]Obayashi E, Yoshida H, Kawai F, et al. The structural basis for an essential subunit interaction in influenza virus RNA polymerase[J]. Nature, 2008, 454(7208): 1127- 1131.
[48]Rao Z, Liu Y, Ge R, et al .Crystal structure of the polymerase PA(C)-PB1(N) compl- ex from an avian influenza H5N1 virus[J]. Nature, 2008, 454(7208): 1123-1126.
[49]Dias A, Bouvier D, Crépin T, et al. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit[J]. Nature, 2009, 458(7240): 914-918.
[50]Taubenberger JK, Reid AH, Lourens RM, et al. Characterization of the 1918 influenza virus polymerase genes[J]. Nature, 2005, 437(7060): 889-893.
[51]Conenello GM, Zamarin D, Perrone LA, et al. A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence[J]. PLoS Pathog, 2007, 3(10): 1414-1421.
[52]Basler CF and Aguilar PV. Progress in identifying virulence determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses[J]. Antiviral Res. 2008, 79(3): 166-178.
[53]Cheng C, Yao L,Chen A, et al. Inhibitory effect of small interfering RNA specific for a novel candidate target in PB1 gene of influenza A virus[J]. J Drug Target, 2009, 17 (2): 133-139.
[54]Van Hoeven N, Pappas C, Belser JA, et al. Human HA and polymerase subunit PB2 proteins confer transmission of an avian influenza virus through the air[J]. Proc Natl Acad Sci U S A, 2009, 106(9): 3366-3371.
[55]Manzoor R, Sakoda Y, Nomura N, et al. PB2 protein of a highly pathogenic avian influenza virus strain A/chicken/Yamaguchi/7/2004 (H5N1) determines its replica- tion potential in pigs[J]. J Virol, 2009, 83(4): 1572-1578.
[56]Bradel-Tretheway BG, Kelley Z, Chakraborty-Sett S, et al. The human H5N1 influe- nza A virus polymerase complex is active in vitro over a broad range of temper- atures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit[J]. J Gen Virol, 2008, 89(12): 2923-2932.
[57]Labadie K, Dos Santos Afonso E, Rameix-Welti MA, et al. Host-range deter- minants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells[J]. Virology. 2007, 362(2): 271-282.
[58]Haye K, Burmakina S, Moran T, et al. The NS1 protein of a human influenza virus inhibits type I interferon production and the induction of antiviral responses in primary human dendritic andrespiratory epithelial cells[J]. J Virol, 2009, 83(13): 6849-6862.
[59]Jiao P, Tian G, Li Y, et al. A single-amino-acid substitution in the NS1 protein changesthe pathogenicity of H5N1 avian influenza viruses in mice[J]. J Virol. 2008, 82(3): 1146-1154.
[60]Zhu Q, Yang H, Chen W, et al. A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens[J]. J Viro, 2008, 82(1): 220-228.
[61]Jackson D, Hossain MJ, Hickman D, et al. A new influenza virus virulence deter- minant: the NS1 protein four C-terminal residues modulate pathogenicity[J]. Proc Natl Acad Sci U S A, 2008, 105(11): 4381-4386.
[62]Webster RG., Bean WJ, Gorman OT, et al. Evolution and ecology of influenza A Viruses[J]. Microbiological Rev, 1992, 56(1): 152-179.
[63]Connor RJ, Kawaoka Y, Webster RG, et al. Receptor specificity inhuman, avian, and equine H2 and H3 influenza virus isolates[J]. Virology, 1994, 205(1): 17-23.
[64]Gambaryan AS, Tuzikov AB, Piskarev VE, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylgly- copolymers:non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6’-sialy(N-acetylactosamine) [J]. Virology, 1997, 232(1): 345-350.
[65]Ito T, Couceiro JN, Kelm S, et al. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential[J]. J Virol, 1998, 72(9): 7367-7373.
[66]Shortridge KF, Zhou NN, Guan Y, et al. Characterization of avian H5N1 influenza viruses from poultry in Hong Kong[J]. Virology, 1998, 252(2): 331-342.
[67]Peiris JSM, de Jong MD, Guan Y, et al. Avian influenza virus (H5N1): a threat to human health[J]. Clin Microbiol Rev, 2007, 20(2): 243-267.
[68]Okazaki K, Takada A, Ito T, et al. Precursor genes of future pandemic influenza viruses are perpetuated in ducks nesting in Siberia[J]. Arch Virol, 2000, 145(5): 885-893.
[69]De Jong JC, Claas EC, Osterhaus AD, et al. A pandemic warning[J]. Nature, 1997, 389(6651): 554.
[70]Christian Sandrock and Terra Kelly. Clinical review: Update of avian influenza A infections in humans[J]. Critical Care, 2007, 11:209 (doi:10.1186/cc5675)
[71]Wu WL, Chen Y, Wang P, et al. Antigenic profile of avian H5N1 viruses in Asia from 2002 to 2007[J]. J Virol, 2008, 82(4): 1798-1807
[72]Monto AS. The role of antivirals in the control of influenza[J]. Vaccine 2003, 21(16): 1796-1800.
[73]World Health Organization (2007) Clinical management of human infection with avian influenza (H5N1)virus.http://www.who.int/csr/disease/avian_influenza/ guide- lines/ClinicalManagement07.pdf
[74]Monto AS, Gunn RA, Bandyk MG, et al. Prevention of Russian influenza amantadine [J]. JAMA, 1979, 241(10): 1003-1007.
[75]Hayden FG, Osterhaus AD, Treanor JJ, et al. Efficacy and safety of the neural- minidase inhibitor zanamivir inthe treatment of influenza virus infections GG167 Influenza Study Group[J]. N Engl J Med, 1997(13), 337: 874-880.
[76]Bdel-Ghafar AN, Chotpitayasunondh T, Gao Z, et al. Update on avian influenzaA (H5N1) virus infection in humans[J]. N Engl J Med, 2008, 358(3): 261-273.
[77]Yen HL, Monto AS, Webster RG, et al. Virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic A/Vietnam/ 1203/04 influenza virus in mice[J]. J Infect Dis, 2005, 192(4): 665-672.
[78]Le QM, Kiso M, Someya K, et al. Avianflu:isolation of drug-resistant H5N1 virus [J]. Nature, 2005, 437(7062): 1108.
[79]Gubareva LV. Molecular mechanisms of influenza virus resistance to neuralmini- dase inhibitors[J]. Virus Res, 2004, 103(1-2): 199-203.
[80]Hanson BJ, Boon AC, Lim AP, et al. Passive immunoprophylaxisand therapy with humanized monoclonal antibody specific for influenza A H5 hemagglutinin in mice [J]. Respir Res, 2006, 14(7): 126.
[81]Francis T. The development of the 1943 vaccination study of the Commission on Influenza[J]. Am J Hyg, 1945, 42(1): 1-11.
[82]Cate TR, Rober B couch, Parker D, et al. Reactogenicity, immunogenicity, and antibody persistence in adults given inactivated influenza virus vaccines-1978[J]. Rev Infect Dis, 1983, 5(4): 737-747.
[83]Subbarao K, Chen H, Swayne D, et al. Evaluation of a genetically modified reassort- ant H5N1 influenza A virusvaccine candidate generated by plasmid based reverse genetics[J]. Virology , 2003, 305(1): 192-200.
[84]Baez M, Palese P and Kilbourne ED. Gene composition of high-yielding influenza vaccine strains obtained by recombination[J]. J Infect Dis, 1980, 141(3): 362-365.
[85]Wendy A, Keitel MD, Robert L, et al. Safety of high doses of influenza vaccine and effect on antibody responses in elderly persons[J]. Arch Intern Med, 2006, 166(10): 1121-1127.
[86]LoGrippo GA. Investigations of the use of beta propiolactone in virus inactivation [J]. Ann NY Acad Sci, 1960, 83(13): 578-594.
[87]Fodor E, Devenish L, Engelhardt OG, et al. Rescue of influenza A virus from recom- binant DNA[J]. J Virol ,1999, 73(11): 9679-9682.
[88]Hoffmann E, Neumann G, Kawaoka Y, et al. A DNA transfection system for generat- ion of influenza A virus from eight plasmids[J]. Proc Natl Acad Sci USA, 2000, 97(11): 6108-6113.
[89]Hoffmann E, Krauss S, Perez D, et al. Eight-plasmid system for rapid generation of influenza virus vaccines[J]. Vaccine, 2002, 20(25-26): 3165-3170.
[90]Massab HF. Adaptation and growth characteristics of influenza virus at 25 degrees C[J]. Nature, 1967, 213(5076): 612-614.
[91]Murphy B and Coelingh K. Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines[J]. Viral Immunol, 2002, 15(2): 295-323.
[92]Massab HF and Bryant ML. The development of live attenuated cold-adapted influe- nza virus vaccine for humans[J]. Rev Med Virol, 1999, 9(4): 237-244.
[93]Alexandrova G and Smorodintsev A. Obtaining of an additionally attenuated vaccin- eting cryophilic influenza strain[J]. Rev Rown Inframicrobiol, 1965, 2: 179-189.
[94]Garmashova L, Plezhaev F and Alexandrova G.. Cold-adapted A/Leningrad/134/ 47/ 57 (H2N2) strain, a specific donor of attenuation of live influenza vaccine for children, and recombinants based on its base[J]. Vopr Virusol, 1984(1), 29: 28-31.
[95]Hoare M, Levy MS, Bracewell DG, et al. Bioprocess engineering issues that would be faced in producing a DNA vaccine at up to 100 m3 fermentation scale for an influenza pandemic[J]. Biotechnol Prog, 2005, 21(6): 1577-1592.
[96]Cinatl J Jr, Michaelis M and Doerr HW. The threat of avian influenza A (H5N1).Part IV:Development of vaccines[J]. Med Microbiol Immunol, 2007, 196(4): 213-225.
[97]Deck RR, DeWitt CM, Donnelly JJ, et al. Characterization of humoral immune responses induced by an influenza hemagglutinin DNA vaccine[J]. Vaccine, 1997, 15(1): 71-78.
[98]Akbari O, Panjwani N, Garcia S, et al. DNA vaccination:transfection and activation of dendritic cells as key events for immunity[J]. J Exp Med, 1999, 189(1): 169-178.
[99]Gilkeson GS, Ruiz P, Howell D, et al. Induction of immune-mediated glomerulo- nephritis in normal mice immunized with bacterial DNA[J]. Clin Immunol Immuno- pathol, 1993, 68(3): 283-292.
[100]Mor G, Yamshchikov G, Sedegah M, et al. Induction of neonatal tolerance by plasmid DNA vaccination of mice[J]. J Clin Invest, 1996, 98(12): 2700-2705.
[101]Klinman DM, Sechler JM, Conover J, et al. Contribution of cells at the site of DNA vaccination to the generation of antigen-specific immunity and memory [J].J Immunol, 1998, 160(5): 2388-2392.
[102]Sedegah M, Weiss W,Sacci JB, et al. Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus[J]. J Immunol, 2000, 164(11): 5905-5912.
[103]Turnes CG, Aleixo JA, Monteiro AV, et al. DNA inoculation with a plasmid vectorcarrying the faeG adhesin gene of Escherichia coli K88ab induced immune responses in mice and pigs[J]. Vaccine, 1999, 17(15-16): 2089-2095.
[104]Garg S, Oran AE, Hon H, et al. The hybrid cytomegalovirus enhancer/chicken beta-actin promoter along with woodchuck hepatitis virus posttranscriptional regulatory element enhances the protective efficacy of DNA vaccines[J]. J Immunol, 2004, 173(1): 550-558.
[105]James CM, Abdad MY, Mansfield JP, et al. Differential activities of alpha/beta IFN subtypes against influenza virus in vivo and enhancement of specific immune responses in DNA vaccinated mice expressing haemagglutinin and nucleoprotein[J]. Vaccine, 2007, 25(10): 1856-1867.
[106]Sharpe M, Lynch D, Topham S, et al. Protection of mice from H5N1 influenza challenge by prophylactic DNA vaccination using particle mediated epidermal delivery[J]. Vaccine, 2007, 25(34): 6392-6398.
[107]Epstein SL, Kong WP, Misplon JA, et al.Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein[J]. Vaccine, 2005, 23(46-47): 5404-5410.
[108]Ozaki T, Yauchi M, Xin KQ, et al.Cross-reactive protection against influenza A virus by a topically applied DNA vaccine encoding M gene with adjuvant [J]. Viral Immunol, 2005, 18(2): 373-380.
[109]Deliyannis G, Boyle JS, Brady JL, et al.A fusion DNA vaccine that targets antigen- presenting cells increases protection from viral challenge[J]. Proc Natl Acad Sci USA, 2000, 97(12): 6676-6680.
[110]Hung CF, Cheng WF, Chai CY, et al. Improving vaccine potency through interce- llular spreading and enhanced MHC class I presentation of antigen[J]. J Immunol, 2001, 166(9): 5733-5740.
[111]Laddy DJ, Yan J, Corbitt N, et al. Immunogenicity of novel consensus-based DNA vaccines against avian influenza[J]. Vaccine, 2007, 25(16): 2984-2989.
[112]Wong CH, Huang YH, Cheng TJ, et al. A consensus–hemagglutinin-based DNA vaccine that protects mice against divergent H5N1 influen viruses[J]. Proc Natl Acad Sci U S A. 2008, 105(36): 13538-13543.
[113]Kodihalli S, Goto H, Kobasa DL, et al. DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice[J]. J Virol, 1999, 73(3): 2094-2098.
[114]Sandbulte MR, Jimenez GS, Boon AC, et al. Cross-reactive neuraminidase anti- bodies afford partial protection against H5N1 in mice and are present in unexposed humans[J]. PLoS Med, 2007, 4:e59.
[115]Epstein SL, Tumpey TM, Misplon JA, et al. DNA vaccine expressing conserved influenza virus proteins protective against H5N1 challenge infection in mice[J].Emerg Infect Dis, 2002, 8(8): 796-801.
[116]Kodihalli S, Kobasa DL and Webster RG.. Strategies for inducing protection against avian influenza A virus subtypes with DNA vaccines[J]. Vaccine, 2000, 18(23): 2592-2599.
[117]Souza AP, Haut L, Reyes-Sandoval A, et al. Recombinant viruses as vaccines against viral diseases[J]. Braz J Med Biol Res, 2005, 38(4): 509-522.
[118]Freeman AI, Zakay-Rones Z, Gomori JM, et al. Phase I/II trial of intravenous NDVHUJ oncolytic virus in recurrent glioblastoma multiforme[J]. Mol Ther, 2006, 13(1): 221-228.
[119]Laurie SA, Bell JC, Atkins HL, et al. A phase 1 clinical study of intravenous administration of PV701, an oncolytic virus, using two-step desensitization[J]. Clin Cancer Res, 2006, 12(8): 2555-2562.
[120]Park MS, Steel J, Garcia-Sastre A, et al. Engineered viral vaccine constructs with dual specificity: avian influenza and Newcastle disease[J]. Proc Natl Acad Sci USA, 2006, 103(21): 8203-8208.
[121]Veits J, Wiesner D, Fuchs W, et al. Newcastle disease virus expressing H5 hemagg- lutinin gene protects chickens against Newcastle disease and avian influenza[J]. Proc Natl Acad Sci USA, 2006, 103(21): 8197-8202.
[122]Roberts A and Rose JK .Redesign and genetic dissection of the rhabdoviruses[J]. Adv Virus Res, 1999, 53(1): 301-319.
[123]Clarke DK, Cooper D, Egan MA, et al. Recombinant vesicular stomatitis virus as an HIV-1 vaccine vector[J]. Springer Semin Immunopathol, 2006, 28(3): 239-253.
[124]Garcia-Sastre A. Palese P.Influenza virus vectors[J]. Biologicals, 1995, 23(2): 171- 178.
[125]Steel J, Burmakina SV, Thomas C, et al. A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus[J]. Vaccine, 2008, 26(4): 522-531.
[126]Lehrman S. Virus treatment questioned after gene therapy death[J]. Nature,1999, 401 (6753): 517-518.
[127]Marshall E. Gene therapy death prompts review of adenovirus vector[J]. Science, 1999, 286(5448): 2244-2245.
[128]Souza AP, Haut L, Reyes-Sandoval A, et al. Recombinant viruses as vaccines agai- nst viral diseases[J]. Braz J Med Biol Res, 2005, 38(4): 509-522.
[129]Steinbrook R. One step forward, two steps back-will there ever be an AIDS vacc- ine[J]. N Engl J Med, 2007, 357(26): 2653-2655.
[130]Davis NL, Brown KW and Johnston RE. A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge[J]. J Virol, 1996, 70(6):3781-3787.
[131]Charles PC, Brown KW, Davis NL, et al. Mucosal immunity induced by parenteral immunization with a live attenuated Venezuelan equine encephalitis virus vaccine candidate[J]. Virology, 1997, 228(2): 153-160.
[132]Davis NL, West A, Reap E, et al. Alphavirus replicon particles as candidate HIV vaccines[J]. IUBMB Life, 2002, 53(4-5): 209-211.
[133]Gherardi MM and Esteban M .Recombinant poxviruses as mucosal vaccine vectors [J]. J Gen Virol, 2005, 86(11): 2925-2936.
[134]Souza AP, Haut L, Reyes-Sandoval A, et al. Recombinant viruses as vaccines against viral diseases[J]. Braz J Med Biol Res, 2005, 38(4): 509-522.
[135]Vijaysri S, Jentarra G, Heck MC, et al. Vaccinia viruses with mutations in the E3L gene as potential replication-competent,attenuated vaccines:intranasal vaccination[J]. Vaccine, 2008, 26(5): 664-676.
[136]Zuniga A, Wang Z, Liniger M, et al. Attenuated measles virus as a vaccine vector [J]. Vaccine, 2007, 25(16): 2974-2983.
[137]Ernst WA, Kim HJ, Tumpey TM, et al. Protection against H1, H5, H6 and H9 influenza A infection with liposomal matrix 2 epitope vaccines[J]. Vaccine, 2006, 24(24): 5158 -5168.
[138]Liu W, Zou P, Chen YH, et al. Monoclonal antibodies recognizing EVETPIRN epitope of influenza A virus M2 protein could protect mice from lethal influenza A virus challenge[J]. Immunol Lett, 93(2-3): 131-136.
[139]Bui HH, Peters B, Assarsson E, et al. Ab and T cell epitopes of influenza A virus, knowledge and opportunities[J]. Proc Natl Acad Sci U S A, 2007, 104(1):246-251.
[140]马绍辉,李琦涵.多表位病毒疫苗的研究进展[J].国外医学(微生物学分册), 2002, 25(2): 5-6.
[141]Ben-Yedidia T and Arnon R. Epitope-based vaccine against influenza.[J].Expert Rev Vaccines, 2007, (6): 939-948.
[142]Adar Y, Singer Y, Levi R, et al. A universal epitope-based influenza vaccine and its efficacy against H5N1[J]. Vaccine, 2009, 27(15): 2099-2107.
[143]Baulcombe D and Hamilton A. A species of small antisense RNA in posttran scriptional gene silencing in plants[J]. Science, 1999, 286(5441): 950-952.
[144]Elbashir S, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature, 2001, 411 (6836): 494-498.
[145]Zhang W, Wang CY, Yang ST, et al. Inhibition of highly pathogenic avian influenza virus H5N1 replication by the small interfering RNA targeting polymerase A gene[J]. Biochem Biophys Res Commun, 2009, 390(3): 421-426.
[146]Lerner RA, Kashyap AK, Steel J, et al. Combinatorial antibody libraries from surviv- ors of the Turkish H5N1 avianinfluenza outbreak reveal virus neutralization strategies[J]. Proc Natl Acad Sci U S A., 2008, 105(16): 5986-5991.
[147]Ekiert DC, Bhabha G, Elsliger MA, et al. Antibody recognition of a highly con- served influenza virus epitope[J]. Science, 2009, 24(5924): 246-251.
[148]Chang-won and Lee yms. Avian influenza virus[J]. Comparative Immunology, Microbiology and Infectious Diseases, 2009, 32: 301-310.
[149]方钟,罗文新,夏宁邵.表位疫苗研究进展[J].中国生物工程杂志, 2007, 27(11): 86-91.
[150]李健.表位疫苗的研究进展[J].中国热带医学, 2007, 7(9): 1681-1684.
[151]T. Ben-Yedidia, Y.Adar and Y.Singer. A universal epitope-based influenza vaccine and its efficacy against H5N1[J]. Vaccine, 2009, 27(15): 2099-2107.
[152]黄艳新,鲍永利,李玉新.抗原表位预测的免疫信息学方法研究进展[J].中国免疫学杂志, 2008, 24(9): 857-961.
[153]南文龙,金宁一,鲁会军,等. H5N1亚型禽流感病毒血凝素Th和B细胞表位预测及抗原性分析[J].中国免疫学杂志, 2009, 7(25): 630-637.
[154]Glenn Y. I, Fikes J, Hermanson G, et al.Utilization of MHC classⅠtransgenic mice for development of minigene DNA vaccines encoding multiple HLA-restricted CTL epitopes[J]. J Immunol, 1999, 162(7): 3915-3925.
[155]Livingston B, Crimi C, Newman M, et al. A rational strategy to design multiepitope immunogens based on multiple the lymphocyte epitopes[J]. Immunol, 2002, 168: 5499-5500.
[156]Yano A, Onozuka A, Asahi-Ozaki Y, et al. An ingenious design for peptide vaccines [J]. Vaccine, 2005, 23: 2322-2326.
[157]Sant AJ, Richards KA, Chaves FA, et al. Direct ex vivo analyses of HLA-DR1 transgenic mice reveal an exceptionally broad pattern of immunodominance in the primary HLA-DR1-restricted CD4 T-cell response to influenza virus hemagglutinin [J]. J Virol, 2007, 81(14): 7608-7619.
[158]Lee A, Mackay I, Rowley M, et al. Measurement of antibody-producing capacity to flagellin in man. IV. Studies in autoimmune disease,allergy and after azathioprine treatmen[J]. Clin Exp Immunol, 1971, 9(4): 507-518.
[159]Carreno B, Koenig S, Coligan J, et al. The peptide binding specificity of HLA class I molecules is largely allele-specific and non-overlapping[J]. Mol Immunol, 1992, 29(9): 1131-1140.
[160]Hans-Georg R, Jutta Bachmann, Niels NE, et al. SYFPEITHI: database for MHC ligands and peptide motifs[J]. Immunogenetics, 1999, 50(3-4): 213-219.
[161]Hans D, Young P and Fairlie D. Current status of short synthetic peptides as vaccines[J]. Med Chem, 2006, 2(6): 627-646.
[162]Ben-Yedidia, Marcus H, Reisner Y, et al. Intranasal administration of peptide vaccine protects human/mouse radiation chimera from influenza infection[J]. Int Immunol, 1999, 11(7): 1043-1051.
[163]Immune Epitope Database.http://www.immuneepitope.org/
[164]Bui HH, Peters B, Assarsson E, et al. Ab and T cell epitopes of influenza A virus, knowledge and opportunities[J]. Proc Natl Acad Sci U S A., 2007, 104(1): 246-251.
[165]De Groot, Sbai H, Aubin CS, et al. Immunoinformatics:Mining genomes for vacci- ne components [J]. Immunol Cell Biol, 2002, 80(3): 255-269.
[166]张伯伟,赵磊,郭如华,等. DNA基因芯片在中华骨髓库HLA组织配型检测中的应用价值[J].中国卫生工程学, 2006, 5(4): 231-232.
[167]Parida R, Shaila MS, Mukherjee S, et al. Computational analysis of proteome of H5N1 avian influenza virus to define T cell epitopes with vaccine potential [J]. Vaccine, 2007, 25(43): 7530-7539.
[168]D'Amaro J, Houbiers JG, Drijfhout JW, et al. A computer program for predicting possible cytotoxic T lymphocyte epitopes based on HLA class I peptide-binding motifs[M]. Hum Immunol, 1995, 43(1): 13-18.
[169]Zhang GL, Khan AM, Srinivasan KN, et al. MULTIPRED:a computational system for prediction of promiscuous HLA binding peptides [J]. Nucleic Acids Res, 2005, 33 (Web Server issue): W172-179.
[170]Velders MP, Weijzen S, Eiben GL, et al. Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine[J]. Immunol, 2001, 166(9): 5366-5373.
[171]黄健,郭建巍,蔡美英.辅助性T细胞表位预测[J].微生物学免疫学进展, 2004, 2(2): 50-52.
[172]J.萨姆不鲁克, D. W.拉赛尔.分子克隆实验指南[M]:第三版黄培堂等译,科学出版社2002.
[173]Manzoli L, Schioppa F, Boccia A, et al. The efficacy of influenza vaccine for healthy children: a meta-analysis evaluating potential sources of variation in efficacy estimate including study quality[J]. Pediatr Infect Dis, 2007, 26(2): 97-106.
[174]Jefferson T, Rivetti D, Di Pietrantonj C, et al. Vaccines for preventing influenza in healthy adults[M]. Cochrane Database Syst Rev. 2007: 2.
[175]Ben-Yedidia T and Arnon R. Design of peptide and polypeptide vaccines[J]. Curr Opin Biotechnol, 1997, 8(4): 442-448.
[176]Keogh E, Fikes J, Southwood S. et al. Identification of new epitopes from fourdifferent tumor-associated antigens: recognition of naturally processed epitopes corre- lates with HLA-A*0201-binding affinity[J]. J Immunol, 2001, 167(2): 787-796.
[177]De Filette M, Min Jou W, Birkett A, et al. Universal influenza A vaccine: optimi- zation of M2-based constructs [J]. Virology, 2005, 337(1): 149-161.
[178]Saha S, Yoshida S, Ohba K, et al. A fused gene of nucleoprotein (NP) and herpes simplex virus genes (VP22) induces highly protective immunity against different subtypes of influenza virus [J]. Virology, 2006, 354(1): 48-57.
[179]吴玉章,刘茂昌,贾正才,等. HBV新型免疫原的设计、合成及免疫原性研究[J].第三军医大学学报, 2000, 22(10): 919-923.
[180]Hanke T, Neumann VC, Blanchard T K, et al. Effective induction of HIV-specific CTL by multi-epitope using gene gun in a combined vaccination regime [J]. Vaccine, 1999, 17(6): 589-596.
[181]徐道华,夏永鹏,邱宗荫等.宫颈癌治疗性多肽疫苗的分子设计及免疫学功能研究[J].中国免疫学杂志, 2006, 22(5): 944-947.
[182]Yang W, Jackson DC, Zeng Q, et al. Multi-epitope schistosome vaccine candidate tested for protective immunogenicity in mice [J]. Vaccine, 2001, 19(1): 103-113.
[183]Chinnasamy D, Milsom MD, Shaffer J, et al. Multicistronic lentiviral vectors contain- ing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI[J] .J Virol, 2006, 3: 14.
[184]Klump H, Schiedlmeier B, Vogt B, et al. Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy [J]. Gene Ther, 2001, 8(10): 811-817.
[185]Wang SX, Taaffe J, Parker C, et al. Hemagglutinin (HA) proteins from H1 and H3 serotypes of influenza A viruses require different antigen designs for the induction of optimal protective antibody responses as studied by codon-optimized HA DNA Vaccines [J]. J Virol, 2006, 80(23): 11628-11637.
[186]Theobald M, Ruppert T, Kuckelkorn U, et al. The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymph- ocytes specific for a flanking peptide epitope [J]. Exp Med, 1998, 188(6): 1017- 1028.
[187]Holzhutter HG, Frommel C and Kloetzel PM. A theoretical approach towards the identifycation of cleavage-determining amino acid motifs of the 20S proteasome [J]. Mol.Biol, 1999, 286(4): 1251-1265.
[188]Jimenze-Cavero M A. Molecular cloning,expressing and immunological analysis of capid precursor polupeptide(p1) from swing vesicular disease virus[J]. Virus Res, 1998, 57(5): 763-170.
[189]Yoshikawa T, Imazu S, Gao JQ, et al. Augmentation of antigen-specific immune Responses using DNA-fusogenic liposome vaccine[J]. Biochem Biophys Res Com-mun, 2004, 325(2): 500-505.
[190]Ulmer JB. Influenza DNA vaccines[J]. Vaccine, 2002, 20(1): 74-76.
[191]Sacher T, Jordan S, Mohr CA, et al. Conditional gene expression systems to study herpesvirus biology in vivo[J]. Med Microbiol Immunol, 2008, 197(2): 269-276.
[192]Chen C and OkayamaH. High efficiency transformation of mammalian cells by plasmid DNA.[J]. Mol Cell Biol, 1987, 7(8): 2745-2752.
[193]Awan A, Oliveri RS, Jensen PL, et al. Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions[J]. Methods Mol Biol, 2010, 584(1): 195-210.
[194]Beveridge WI. The chronicle of influenza epidemics [J]. Hist Philos Life Sci, 1991, 13(1): 223-234.
[195]WHO. http://www.who.int/zh/index.html.
[196]Palese P. Making better influenza virus vaccines[J]. Emerg Infect Dis, 2006, 12 (1): 61-65.
[197]Stohr K. Overview of the WHO Global Influenza Programme[J]. Dev Biol (Basel), 2003, 115(1): 3-8.
[198]Kemble G and Greenberg H. Novel generations of influenza vaccines [J]. Vaccine, 2003, 21(16): 1789-1795.
[199]Gerdil C. The annual production cycle for influenza vaccine [J]. Vaccine, 2003, 21 (16): 1776-1779.
[200]Fedson DS. Preparing for pandemic vaccination: an international policy agenda for vaccine development [J]. J Public Health Policy, 2005, 26(1): 4-29.
[201]Thomson SA, Sherritt MA, Medveczky J, et al. Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination[J]. Immuno, 1998, 160(4): 1717-1723.
[202]Li H, Ding J and Chen YH. Recombinant protein comprising multi-neutralizing epitopes induced high titer of antibodies against influenza A virus[J]. Immunobiol, 2003, 207 (5): 305-313.
[203]贾雷立,金宁一,鲁会军,等.流感复合多表位疫苗的设计及小鼠免疫研究[J].广西农业生物科学, 2006, 25(9): 209-210.
[204]陈义锋.多亚型多表位流感病毒核酸疫苗与重组鸡痘病毒构建及实验免疫研究[D].吉林省:吉林大学硕士论文. 2007.