A/Califonia/07/2009亚型猪流感冷适应减毒疫苗株的拯救及免疫效果评价
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摘要
甲型H1N1流感病毒是甲型(A型)流感病毒引起的严重威胁人类健康的一种急性呼吸道传染性疾病。疫苗和药物是控制和治疗该病的主要措施。但从长远看，疫苗仍是控制流感传播的主要手段。
流感疫苗有灭活和减毒两种，灭活流感疫苗据有较好的体液免疫效果，但细胞免疫、粘膜免疫效果较差。流感减毒活疫苗不但据有较好的体液免疫，而且细胞免疫及粘膜免疫效果也很好。近几年研究表明，细胞免疫及粘膜免疫在预防流感病毒的传播方面发挥着重要的作用，特别是激活机体的细胞免疫，能够增强疫苗对流感病毒的免疫保护效力。目前我国仅有灭活疫苗。因此，发展我国自主研制的流感减毒活疫苗就成为当前急需解决的问题。
本研究应用反向遗传学技术，以A/Ann Arbor/6/60ca (H2N2)型流感病毒做为骨架病毒，将其6个内部基因(PB1、PB2、NP、NA、M、NS)及WHO推荐的H1N1疫苗株-A/California/07/2009流感病毒HA、NA基因分别构建到双向表达载体pAD3000上，共转染MDCK～COS1细胞，得到冷适应、温度敏感、减毒的A/California/07/2009ca(CA/AA ca)感病毒。通过基因鉴定、抗体中和试验、IFA及病毒在MDCK细胞的培养等方法对重配病毒的生物学特性、型别、表型进行了鉴定。通过鸡胚培养建立了2级毒种库，并应用无菌试验、支原体检测、外源因子检测等方法证明重配病毒的毒种可以做为疫苗毒种应用。在此基础上，重配CA/AA ca流感病毒免疫的BALB/c小鼠及雪貂，应用TCID50、间接ELISA、MTT、HI等方法对CA/AA ca流感病毒进行了体液免疫、细胞免疫及粘膜免疫应答效果的评价以及对小鼠、雪貂的免疫保护效力的研究。
结果:
1病毒的拯救及鉴定：构建了pAD3000-HA和pAD3000-NA质粒，并与A/AnnArbor/6/60ca(H2N2)型流感病毒的6个内部骨架组成8质粒，应用反向遗传学技术拯救出的CA/AA ca病毒，经鉴定CA/AA ca病毒为A/California/07/2009亚型，并且具有冷适应性、温度敏感的表型。
2毒种库的鉴定及病毒纯化：建立了二级疫苗用CA/AA ca病毒种子库，其血凝效价均为1：256，经鉴定无细菌、支原体、外源因子污染。病毒接种时105倍稀释、稀释液为PBS、培养时间72h、培养温度33℃，并用蔗糖密度梯度离心的方法初步纯化了CA/AA ca流感病毒。
3CA/AA ca流感病毒免疫应答效果的研究：
（1）TPCK胰酶依赖性的检测应用TCID50检测BJ501病毒及CA/AA ca病毒的毒力，加入TPCK胰酶（0.8μg/mL）后，CA/AA ca流感病毒TCID50升高1.25，BJ501病毒TCID50无显著变化；经蔗糖密度梯度离心方法纯化流感病毒；纯化后CA/AA ca病毒TCID50为9.3，BJ501病毒TCID50为7.5；CA/AA ca病毒滴度免疫免疫BALB/c，免疫的最佳剂量为1×106TCID50/20μL。
（2）CA/AA ca病毒滴鼻免疫BALB/c小鼠，以A/California/07/2009流感灭活病毒肌肉注射为对照，A/California/07/2009流感灭活病毒HI效价显著高于CA/AAca病毒滴鼻组（p<0.05），而IgA显著低于滴鼻组（p<0.01）；灭活病毒肌肉注射组的IL-4水平显著高于滴鼻组（p<0.05），而IL-6水平两者差异不显著（p>0.05）；灭活病毒肌肉注射组IFN-γ水平显著低于CA/AA ca病毒组（p<0.01）。
（3）CA/AA ca病毒免疫小鼠，鼻、肺中检测到较低滴度的病毒滴度，4d后病毒消失。BJ501病毒在小鼠鼻、肺中检测到高滴度病毒，免疫后9d全部死亡。CA/AA ca流感病毒二免2周应用BJ501病毒攻毒，3d后鼻、肺、脾、肾、脑中未检测到病毒。小鼠存活率100%。
（4）CA/AA ca病毒、BJ501病毒滴鼻免疫雪貂，CA/AA ca病毒组雪貂未发生明显的生理变化，鼻腔3d后检测不到病毒的存在。BJ501病毒组出现感冒症状，并且1到7d鼻腔中均检测到较高的病毒滴度。CA/AA ca病毒组免疫3d后在雪貂的鼻、肺、脑中未检测到病毒的存在，BJ501病毒组在雪貂的鼻、肺、脑中检测到较高病毒滴度。CA/AA ca病毒免疫雪貂后处死采集血清，血清中的抗体对CA/AA ca病毒及BJ501病毒均有很好的中和效果，二者无显著性差异（p>0.05）, CA/AA ca病毒2免2周后免疫BJ501病毒，3d后在鼻、肺中仅检测到少量的病毒存在。
结论:
本试验成功拯救出的冷适应、温度敏感、减毒的CA/AA ca病毒。按照2010药典的要求建立了主种子库及工作种子库，并确定了病毒的培养温度、时间及接种浓度。应用蔗糖密度梯度离心的方法初步纯化了BJ501流感病毒及CA/AA ca流感病毒。
CA/AA ca流感病毒对小鼠无致病性，能够刺激小鼠产生较好的体液免疫、细胞免疫及粘膜免疫应答，并且CA/AA ca流感病毒对同亚型病毒具有较好的免疫保护效果；CA/AA ca流感病毒对雪貂无致病性，其抗体对同亚型病毒具有较好的中和作用。由此可见，CA/AA ca流感病毒是可以做为流感减毒疫苗的候选疫苗株。
H1N1influenza virus is a type influenza virus, which can cause acute respiratoryinfectious diseases and even death. Vaccines and drugs are the main measures to controlthese diseases. Compared with the medicines, vaccines are still the primary means tocontrol the spread of influenza due to those characteristics.
There are both inactivated and live attenuated influenza vaccines. Compared withinactivated influenza vaccines that only induce better humoral immunes, live attenuatedinfluenza vaccines induce not only good humoral immunes, but also better cellularimmunes and mucosal immunes. In recent years, studies have shown that cellular andmucosal immunes play important roles to prevent the spread of influenza viruses andcellular immunes enhance the vaccine protective effects. There are only inactivatedvaccines available in China. Therefore, It is an urgent problem to develop independentlylive attenuated influenza vaccines.
In this study, cold-adapted, temperature-sensitive, attenuated A/California/07/2009ca(CA/AA ca) influenza virus was obtained by reverse genetic technology. Six internal genes(PB1, PB2, NP, NA, M&NS) of the virus A/Ann Arbor/6/60ca (H2N2) influenza virusand two antigenic genes (HA&NA) of the WHO recommended H1N1vaccine strainA/California/07/2009were constructed to bi-directional expression vector pAD3000andco-transfected into MDCK+COS1cells. Biological characteristics, type, phenotype ofreassortant virus strain CA/AA ca were identified by sequencing, neutralization test, IFA.Virus seed stocks were produced in the chicken and proved qualified by sterility test,mycoplasma detection, exogenous factor detection, et al. Humoral, cellular, mucosalimmunes and protective efficacy of CA/AA ca influenza virus were evaluated in mice andferrets by TCID50, ELISA, MTT and HI.
The study was divided into three parts:
1.Rescue and identification of candidate vaccine virus strain: CA/AA ca influenzavirus was rescued by RG, identified by RT-PCR, HI, indirect immunofluorescence, electronmicroscopy and SDS-PAGE, which proved that it was A/California/07/2009subtypes withcold-adapted, temperature-sensitive phenotype.
2. Establishment, identification of seed lots and purification of this virus: Two seedbanks were established, with1:256hemagglutination titers and without bacteria, mycoplasma or exogenous factors. The virus was inoculated by105dilution into chickenembryos and incubated at33℃for72h. The virus was purified by the sucrose densitygradient centrifugation and assayed by HPLC, EM, TCID50, and so on. The CA/AA cavirus of high purification level were ultimately achieved.
3. Evaluation of the immune responses induced by the vaccine: the TCID50titer of CA/AA ca virus was increased by1.25with TPCK trypsin. The titer of purified CA/AA cavirus was9.3lg TCID50/20uL, and BJ5017.5lgTCID50/20uL. Optimal dosage of CA/AA ca virus was1×106TCID50/20uL.
Compared with HI titers of BALB/c mice controls that intramuscularly immunizedwith inactive A/California/07/2009virus, those of which intranasally immunized with CA/AA ca virus was lower (p<0.05) and IgA titer was higher (p<0.01), which indicated thatintranasal immunization can evoke higher mucosal immune responses, but lower humoralimmune responses. Compared with the controls, the IL-4and IFN-γ levels of live vaccinewere higher, but with IL-6level of no significance.
After BALB/c mice were immunized with CA/AA ca virus, the low titers of viruswere detected in mice nose and lungs within4days. The high titers of BJ501virus in micenoses and lungs were detected after BJ501challenged, with100%mortality rate in9dayspost infection. By2weeks after the second intranasal immunization, after BJ501virus waschallenged, the BJ501virus were not detected in the mice nose, lungs, spleen, kidney andbrain, with100%survival rate.
Ferrets were intranasally challenged with CA/AA ca and BJ501virus, respectively.There were no physiological changes in ferrets infected with CA/AA ca, but ferretsinfected with BJ501have clinical flu infection symptoms. The virus was detected in nose,lungs, brain at4days after challenge. Ferrets sera that immunized with CA/AA ca virusneutralized the CA/AA ca virus and BJ501, with no difference between the two viruses(p>0.05). BJ501virus was detected in the nose and lungs of CA/AA ca immunizedmice within3days after BJ501challenge.
Conclusion: We successfully rescued the cold-adapted, temperature-sensitive,attenuated the CA/AA ca virus, established master and working seed lots，determinedincubation temperature, time and inoculation concentration, and purified the CA/AA cavirus by Pharmacopoeia2010requirements.
Compared with A/California/07/2009virus, the results showed that CA/AA ca viruscan induce good humoral, cellular and mucosal immunes and better immune protection inmice, so do ferrets. In conclusion, CA/AA ca virus was an attenuated vaccine candidate strain.
流感疫苗有灭活和减毒两种，灭活流感疫苗据有较好的体液免疫效果，但细胞免疫、粘膜免疫效果较差。流感减毒活疫苗不但据有较好的体液免疫，而且细胞免疫及粘膜免疫效果也很好。近几年研究表明，细胞免疫及粘膜免疫在预防流感病毒的传播方面发挥着重要的作用，特别是激活机体的细胞免疫，能够增强疫苗对流感病毒的免疫保护效力。目前我国仅有灭活疫苗。因此，发展我国自主研制的流感减毒活疫苗就成为当前急需解决的问题。
本研究应用反向遗传学技术，以A/Ann Arbor/6/60ca (H2N2)型流感病毒做为骨架病毒，将其6个内部基因(PB1、PB2、NP、NA、M、NS)及WHO推荐的H1N1疫苗株-A/California/07/2009流感病毒HA、NA基因分别构建到双向表达载体pAD3000上，共转染MDCK～COS1细胞，得到冷适应、温度敏感、减毒的A/California/07/2009ca(CA/AA ca)感病毒。通过基因鉴定、抗体中和试验、IFA及病毒在MDCK细胞的培养等方法对重配病毒的生物学特性、型别、表型进行了鉴定。通过鸡胚培养建立了2级毒种库，并应用无菌试验、支原体检测、外源因子检测等方法证明重配病毒的毒种可以做为疫苗毒种应用。在此基础上，重配CA/AA ca流感病毒免疫的BALB/c小鼠及雪貂，应用TCID50、间接ELISA、MTT、HI等方法对CA/AA ca流感病毒进行了体液免疫、细胞免疫及粘膜免疫应答效果的评价以及对小鼠、雪貂的免疫保护效力的研究。
结果:
1病毒的拯救及鉴定：构建了pAD3000-HA和pAD3000-NA质粒，并与A/AnnArbor/6/60ca(H2N2)型流感病毒的6个内部骨架组成8质粒，应用反向遗传学技术拯救出的CA/AA ca病毒，经鉴定CA/AA ca病毒为A/California/07/2009亚型，并且具有冷适应性、温度敏感的表型。
2毒种库的鉴定及病毒纯化：建立了二级疫苗用CA/AA ca病毒种子库，其血凝效价均为1：256，经鉴定无细菌、支原体、外源因子污染。病毒接种时105倍稀释、稀释液为PBS、培养时间72h、培养温度33℃，并用蔗糖密度梯度离心的方法初步纯化了CA/AA ca流感病毒。
3CA/AA ca流感病毒免疫应答效果的研究：
（1）TPCK胰酶依赖性的检测应用TCID50检测BJ501病毒及CA/AA ca病毒的毒力，加入TPCK胰酶（0.8μg/mL）后，CA/AA ca流感病毒TCID50升高1.25，BJ501病毒TCID50无显著变化；经蔗糖密度梯度离心方法纯化流感病毒；纯化后CA/AA ca病毒TCID50为9.3，BJ501病毒TCID50为7.5；CA/AA ca病毒滴度免疫免疫BALB/c，免疫的最佳剂量为1×106TCID50/20μL。
（2）CA/AA ca病毒滴鼻免疫BALB/c小鼠，以A/California/07/2009流感灭活病毒肌肉注射为对照，A/California/07/2009流感灭活病毒HI效价显著高于CA/AAca病毒滴鼻组（p<0.05），而IgA显著低于滴鼻组（p<0.01）；灭活病毒肌肉注射组的IL-4水平显著高于滴鼻组（p<0.05），而IL-6水平两者差异不显著（p>0.05）；灭活病毒肌肉注射组IFN-γ水平显著低于CA/AA ca病毒组（p<0.01）。
（3）CA/AA ca病毒免疫小鼠，鼻、肺中检测到较低滴度的病毒滴度，4d后病毒消失。BJ501病毒在小鼠鼻、肺中检测到高滴度病毒，免疫后9d全部死亡。CA/AA ca流感病毒二免2周应用BJ501病毒攻毒，3d后鼻、肺、脾、肾、脑中未检测到病毒。小鼠存活率100%。
（4）CA/AA ca病毒、BJ501病毒滴鼻免疫雪貂，CA/AA ca病毒组雪貂未发生明显的生理变化，鼻腔3d后检测不到病毒的存在。BJ501病毒组出现感冒症状，并且1到7d鼻腔中均检测到较高的病毒滴度。CA/AA ca病毒组免疫3d后在雪貂的鼻、肺、脑中未检测到病毒的存在，BJ501病毒组在雪貂的鼻、肺、脑中检测到较高病毒滴度。CA/AA ca病毒免疫雪貂后处死采集血清，血清中的抗体对CA/AA ca病毒及BJ501病毒均有很好的中和效果，二者无显著性差异（p>0.05）, CA/AA ca病毒2免2周后免疫BJ501病毒，3d后在鼻、肺中仅检测到少量的病毒存在。
结论:
本试验成功拯救出的冷适应、温度敏感、减毒的CA/AA ca病毒。按照2010药典的要求建立了主种子库及工作种子库，并确定了病毒的培养温度、时间及接种浓度。应用蔗糖密度梯度离心的方法初步纯化了BJ501流感病毒及CA/AA ca流感病毒。
CA/AA ca流感病毒对小鼠无致病性，能够刺激小鼠产生较好的体液免疫、细胞免疫及粘膜免疫应答，并且CA/AA ca流感病毒对同亚型病毒具有较好的免疫保护效果；CA/AA ca流感病毒对雪貂无致病性，其抗体对同亚型病毒具有较好的中和作用。由此可见，CA/AA ca流感病毒是可以做为流感减毒疫苗的候选疫苗株。
H1N1influenza virus is a type influenza virus, which can cause acute respiratoryinfectious diseases and even death. Vaccines and drugs are the main measures to controlthese diseases. Compared with the medicines, vaccines are still the primary means tocontrol the spread of influenza due to those characteristics.
There are both inactivated and live attenuated influenza vaccines. Compared withinactivated influenza vaccines that only induce better humoral immunes, live attenuatedinfluenza vaccines induce not only good humoral immunes, but also better cellularimmunes and mucosal immunes. In recent years, studies have shown that cellular andmucosal immunes play important roles to prevent the spread of influenza viruses andcellular immunes enhance the vaccine protective effects. There are only inactivatedvaccines available in China. Therefore, It is an urgent problem to develop independentlylive attenuated influenza vaccines.
In this study, cold-adapted, temperature-sensitive, attenuated A/California/07/2009ca(CA/AA ca) influenza virus was obtained by reverse genetic technology. Six internal genes(PB1, PB2, NP, NA, M&NS) of the virus A/Ann Arbor/6/60ca (H2N2) influenza virusand two antigenic genes (HA&NA) of the WHO recommended H1N1vaccine strainA/California/07/2009were constructed to bi-directional expression vector pAD3000andco-transfected into MDCK+COS1cells. Biological characteristics, type, phenotype ofreassortant virus strain CA/AA ca were identified by sequencing, neutralization test, IFA.Virus seed stocks were produced in the chicken and proved qualified by sterility test,mycoplasma detection, exogenous factor detection, et al. Humoral, cellular, mucosalimmunes and protective efficacy of CA/AA ca influenza virus were evaluated in mice andferrets by TCID50, ELISA, MTT and HI.
The study was divided into three parts:
1.Rescue and identification of candidate vaccine virus strain: CA/AA ca influenzavirus was rescued by RG, identified by RT-PCR, HI, indirect immunofluorescence, electronmicroscopy and SDS-PAGE, which proved that it was A/California/07/2009subtypes withcold-adapted, temperature-sensitive phenotype.
2. Establishment, identification of seed lots and purification of this virus: Two seedbanks were established, with1:256hemagglutination titers and without bacteria, mycoplasma or exogenous factors. The virus was inoculated by105dilution into chickenembryos and incubated at33℃for72h. The virus was purified by the sucrose densitygradient centrifugation and assayed by HPLC, EM, TCID50, and so on. The CA/AA cavirus of high purification level were ultimately achieved.
3. Evaluation of the immune responses induced by the vaccine: the TCID50titer of CA/AA ca virus was increased by1.25with TPCK trypsin. The titer of purified CA/AA cavirus was9.3lg TCID50/20uL, and BJ5017.5lgTCID50/20uL. Optimal dosage of CA/AA ca virus was1×106TCID50/20uL.
Compared with HI titers of BALB/c mice controls that intramuscularly immunizedwith inactive A/California/07/2009virus, those of which intranasally immunized with CA/AA ca virus was lower (p<0.05) and IgA titer was higher (p<0.01), which indicated thatintranasal immunization can evoke higher mucosal immune responses, but lower humoralimmune responses. Compared with the controls, the IL-4and IFN-γ levels of live vaccinewere higher, but with IL-6level of no significance.
After BALB/c mice were immunized with CA/AA ca virus, the low titers of viruswere detected in mice nose and lungs within4days. The high titers of BJ501virus in micenoses and lungs were detected after BJ501challenged, with100%mortality rate in9dayspost infection. By2weeks after the second intranasal immunization, after BJ501virus waschallenged, the BJ501virus were not detected in the mice nose, lungs, spleen, kidney andbrain, with100%survival rate.
Ferrets were intranasally challenged with CA/AA ca and BJ501virus, respectively.There were no physiological changes in ferrets infected with CA/AA ca, but ferretsinfected with BJ501have clinical flu infection symptoms. The virus was detected in nose,lungs, brain at4days after challenge. Ferrets sera that immunized with CA/AA ca virusneutralized the CA/AA ca virus and BJ501, with no difference between the two viruses(p>0.05). BJ501virus was detected in the nose and lungs of CA/AA ca immunizedmice within3days after BJ501challenge.
Conclusion: We successfully rescued the cold-adapted, temperature-sensitive,attenuated the CA/AA ca virus, established master and working seed lots，determinedincubation temperature, time and inoculation concentration, and purified the CA/AA cavirus by Pharmacopoeia2010requirements.
Compared with A/California/07/2009virus, the results showed that CA/AA ca viruscan induce good humoral, cellular and mucosal immunes and better immune protection inmice, so do ferrets. In conclusion, CA/AA ca virus was an attenuated vaccine candidate strain.
引文
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11Centers for Disease Control and Prevention (CDC). Swine influenza A (H1N1) infectionin two children-Southern California, March-April2009. MMWR Morb Mortal Wkly Rep，2009,58(15):400-402
12Swine influenza A (H1N1) infection in two children Southern California, Marcha) April2009. MMWR Morb MortalWkly Rep，2009;58(15):400-402
13Cohen J, Enserink M. Swine flu. After delays, WHO agrees: the2009pandemic hasbegun.[J]. Science，2009,324(5934):1496-1507
14Pandemic (H1N1)2009，update112. August6,2010[cited; available from:a) http://www.who.int/csr/don/20100806/en/index.html]
15Munster VJ, de Wit E, van den Brand JM, et al. Pathogenesis and Transmission ofSwine-Origin2009A(H1N1) Influenza Virus in Ferrets[J]. Science,2009,325(5939)
1126-1177
16Maines TR, Jayaraman A, Belser JA, et al. Transmission and Pathogenesis ofSwine-Origin2009A(H1N1) Influenza Viruses in Ferrets and Mice [J]. Science,2009,325(5939):484-487
17Shinde V, Bridges CB, Uyeki TM, et al. Triple-reassortant swine influenza A (H1)in humans in the United States,2005～2009[J]. N Engl J Med,2009,360(25):2616-2625
18Lu X, Edwards LE, Desheva JA, et al.Cross-protective immunity in mice induced bylive-attenuated or inactivated vaccines against highly pathogenic influenza A (H5N1)viruses[J]. Vaccine，2006,24:6588-6593
19Ellebedy AH, Ducatez MF, Duan S, et al. Impact of prior seasonal influenza vaccinationand infectionon pandemic A(H1N1) influenza virus replication in ferrets[J]. Vaccine，2010，8:67-71
20Kobinger GP,Meunier I, PatelA, et al.Assessment of the efflcacy of commerciallyavailable and candidate vaccines against a pandemic H1N12009virus[J]. J InfectDis,2010,201:1000-1006
21Serum cross-reactive antibody response to a novel influenza A (H1N1) virus aftervaccination with seasonal influenza vaccine. MMWR Morb Mortal Wkly Rep，2009,58:521-524
22Collin N.Vaccine production capacity for seasonal and pandemic (H1N1)2009influenza[J].Vaccine,2009,27(38):5184-5186
23Grace LC,Ji YM, Elaine WL,et al.Comparison of a Live Attenuated2009H1N1Vaccinewith Seasonal Influenza Vaccines against2009Pandemic H1N1Virus Infection in Miceand Ferrets[J].Journal of Infectious Diseases,2011,203(7):930-936
24Garcia-Garcia L, Valdespino-Gomez JL, Lazcano-Ponce E, et al. Partial protectionof seasonal trivalent inactivated vaccine against novel pan-demic influenza A/H1N12009: case-control study inMexico City[J].BMJ，2009，339(10): b3928
25Hancock K, Veguilla V, Lu X, et al. Cross-reactive antibody responses to the2009pandemic H1N1influenza virus[J]. N Engl J Med，2009;361:1945-1952
26]Medlock J, Galvani AP. Optimizing influenza vaccine distribution[J]. Science,2009,325:1705-1708
27Theo JM， Irina VV， Harrie KL，et al. Master donor viruses A/Leningrad/134/17/57(H2N2) and B/USSR/60/69and derived reassortants used in live attenuated influenzavaccine (LAIV) do not display neurovirulent properties in a mouse model[J].Arch Virol,2010,155(9):1391-1399
28Belshe RB, Nichol KL, Black SB, et al. Safety,efflcacy and effectiveness of liveattenuated, cold-adapted influenza vaccine in an indicated population aged5～49years[J]. Clin Infect Dis,2004,39:920-927
29Nichol KL, Mendelman PM, Mallon KP, et al.Effectiveness of live attenuated intranasalinfluenza virus vaccine in healthy, working adults[J].JAMA,1999,282:137-144
30McMahonA W, Iskander J,Haber P,et al.Adverse events after inactivated influenzavaccination among children less than2years of age: analysis of reports fromthevaccine adverse event reporting system1993～2003[J]. Pediatrics,2005,115:453-460
31Izurieta HS, Haber P, Wise RP, et al. Adverse events reported followinglive,cold-adapted, intranasal influenza vaccine[J].JAMA,2005,294:2720-2725
32Belshe RB, Gruber WC. Safety efflcacy and effectiveness of cold-adapted, liveattenuated, trivalent intranasal influenza vaccine in adults and children[J]. PhilosTrans R Soc Lond Ser B,2001,356:1947-1951
33Petukhova G, Naikhin A, Chirkova T, et al. Comparative studies of local antibodyand cellular immune responses to influenza infection and vaccination with liveattenuated reassortant influenza vaccine(LAIV) utilizing a mouse nasal-associatedlymphoid tissue (NALT) separation method[J]. Vaccine,2009,27:2580-2587
34Buonagurio DA, Bechert TM, Yang CF, et al. Genetic stability of live, cold-adaptedinfluenza virus components of the FluMist/CAIV-T vaccine throughout themanufacturing process[J]. Vaccine,2006,24:2151-2160
35Hoffman E, Neumann G, Kawaoka Y, et al. A DNA transfection system for generationof influenza A virus from eight plasmids [J]. PNAS,2000,97(11):6108-61131
36Alexandrova GI, Maassab HF, Kendal AP, et al. Laboratory properties of cold-adaptedinfluenza B live vaccine strains developed in the US and USSR, and their B/AnnArbor/1/86cold-adapted reassortant vaccine candidates[J]. Vaccine,1990,8:61-64
37Ellebedy AH, Webby RJ.Influenza vaccines[J].Vaccine，2009,27(8)：65-68
38Ohmit SE, Victor JC, Rotthoff JR, et al. Prevention of antigenically driftedinfluenza by inactivated and live attenuated vaccines[J].N Engl J Med，2006,355:2513-2522
39Ohmit SE, Victor JC, Teich ER, et al. Prevention of symptomatic seasonal influenzain2005–2006by inactivated and live-attenuated vaccines[J]. J Infect Dis,2008,198:312-317
40Barry DW, Mayner RE, Hochstein HD, et al.Comparative trial of influenza vaccinesII. Adverse reactions in children and adults[J]. Am J Epidemiol,1976,104:47-59
41Beyer WE, Palache AM, de Jong JC, et al. Cold-adapted live influenza vaccine versusinactivated vaccine: systemic vaccine reactions, local and systemic antibodyresponse, and vaccine efflcacy. A meta-analysis[J]. Vaccine,2002,20:1340-1353
42Clements ML, O’Donnell S, Levine MM, et al. Dose response of A/Alaska/6/77(H3N2)cold-adapted reassortant vaccine virus in adult volunteers: role of local antibodyin resistance to infection with vaccine virus[J]. Infect Immun,1983,40:1044-1051
43Ambrose CS, Coelingh LCK. Current status of live attenuated influenza vaccine intheUnited States for seasonal and pandemic influenza[J]. InfluenzaOther RespiViruses,2008,2:193-202
44Govorkova EA, Kaverin NV, Gubareva LV, et al. Replication of influenza A virusesin a green monkey kidney continuous cell line(Vero)[J]. J Infect Dis,1995,172(1):250-253
45Edwards KM,Dupont WD,Westrich MK, et al.A randomized, controlled trial ofcold-adapted and inactivated vaccines for the prevention of influenza A disease[J].J Infect Dis,1994,169:68-76
46Steel J, Staeheli P, Mubareka S, et al. Transmission of pandemic H1N1influenzavirus and impact of prior exposure to seasonal strains or interferon treatment[J].J Virol,2009,84:21-26
47Luytjes W, Krystal M, Enami M, et al.Amplification, expression, and packaging ofa foreign gene by influenza virus[J].Cell,1989,59:1107-1113
48Enami M, Luytjes W, Krystal M, et al. Introduction of site-specific mutations intothe genome of influenza virus [J]. Proc Natl Acad Sci,1990,87:3802-3805
49Li S, Liu C, Klimov A, et al. Recombinant influenza A virus vaccines for the pathogenichuman A/Hong Kong/97(H5N1) viruses[J]. J Infect Dis1,1999,179:1132-1138
50Cong YL, Pu J, Liu QF, et al. Antigenic and genetic characterization of H9N2swineinfluenza viruses in China[J].J Gen Virol，2007，88:2035-2041
51Neumann G, Watanabe T, Ito H, et al. Generation of influenza A viruses entirely fromcloned cDNAs[J].Proc Natl Acad Sci,1999,96:9345-9350
52Fodor E, Devenish L, Engelhardt OG, et al. Rescue of influenzaA virus from recombinantDNA[J].J Virol,1999,73:9679-9682
53Smith GJ, Vijaykrishna D, Bahl J, et al. Origins and evolutionary genomics of the
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54Garten RJ, Davis CT, Russell CA, et al. Antigenic and genetic characteristics ofswine-origin2009A (H1N1)influenza viruses circulating in humans[J].Science,2009,10:197-201
55Murphy BR, Coelingh K, Principles underlying the development and use of live,attenuated cold-adapted influenza A and B virus vaccines[J]. Viral Immunol,2002,15:295-323
56Wareing MD, Tannock GA.Live attenuated vaccines against influenza; an historicalreview[J].Vaccine,2001,19:3320-3330
57国家药典委员会．中华人民共和国药典[M].第3版.中国医药出版社.（2010）
58Viviani L， Assael BM，Kerem E.Impact of the A (H1N1) pandemic influenza (season2009～2010) on patients with cystic fibrosis [J].Journal of CysticFibrosis.2011,10:370-376
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61Johnson PR, Feldman S, Thompson JM, et al. Immunity to influenza a virus infectionin young children: A comparison of natural infection, live cold-adapted andinactivated vaccines [J]. J Infect Dis,1986,154(1):1212-1261
62Romanova J, Katinger D, Ferko B, et al. Live cold-adapted influenza A vaccine producedin Vero cell line [J]. Virus Res,2004,103(122):1872-1931
63Cox RJ, Brokstad KA, Ogra P. Influenza virus: immunity and vaccination strategies.Comparison of the immune response to inactivated and live, attenuated influenzavaccines[J]. Scand J Immunol,2004,59(1):1-15
64Chen GL, Subbarao K. Live attenuated vaccines for pandemic influenza[J]. Curr TopMicrobiol Immunol,2009,333:109-132
65Poland GA. Vaccines against avian influenza—A race against time. N Engl J Med,2007,354:1411-1413
66Mendelman PM, Cordova J, Cho I. Safety, efficacy and effectiveness of the influenzavirus vaccine, trivalent, types A and B, live, cold-adapted (CAIV-T) in healthychildren and healthy adults[J]. Vaccine,2001,19:2221-2226
67Maassab HF, Bryant ML. The development of live attenuated cold-adapted influenzavirus vaccine for humans[J]. Rev Med Virol,1999,22:237-244
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11Centers for Disease Control and Prevention (CDC). Swine influenza A (H1N1) infectionin two children-Southern California, March-April2009. MMWR Morb Mortal Wkly Rep，2009,58(15):400-402
12Swine influenza A (H1N1) infection in two children Southern California, Marcha) April2009. MMWR Morb MortalWkly Rep，2009;58(15):400-402
13Cohen J, Enserink M. Swine flu. After delays, WHO agrees: the2009pandemic hasbegun.[J]. Science，2009,324(5934):1496-1507
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15Munster VJ, de Wit E, van den Brand JM, et al. Pathogenesis and Transmission ofSwine-Origin2009A(H1N1) Influenza Virus in Ferrets[J]. Science,2009,325(5939)
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16Maines TR, Jayaraman A, Belser JA, et al. Transmission and Pathogenesis ofSwine-Origin2009A(H1N1) Influenza Viruses in Ferrets and Mice [J]. Science,2009,325(5939):484-487
17Shinde V, Bridges CB, Uyeki TM, et al. Triple-reassortant swine influenza A (H1)in humans in the United States,2005～2009[J]. N Engl J Med,2009,360(25):2616-2625
18Lu X, Edwards LE, Desheva JA, et al.Cross-protective immunity in mice induced bylive-attenuated or inactivated vaccines against highly pathogenic influenza A (H5N1)viruses[J]. Vaccine，2006,24:6588-6593
19Ellebedy AH, Ducatez MF, Duan S, et al. Impact of prior seasonal influenza vaccinationand infectionon pandemic A(H1N1) influenza virus replication in ferrets[J]. Vaccine，2010，8:67-71
20Kobinger GP,Meunier I, PatelA, et al.Assessment of the efflcacy of commerciallyavailable and candidate vaccines against a pandemic H1N12009virus[J]. J InfectDis,2010,201:1000-1006
21Serum cross-reactive antibody response to a novel influenza A (H1N1) virus aftervaccination with seasonal influenza vaccine. MMWR Morb Mortal Wkly Rep，2009,58:521-524
22Collin N.Vaccine production capacity for seasonal and pandemic (H1N1)2009influenza[J].Vaccine,2009,27(38):5184-5186
23Grace LC,Ji YM, Elaine WL,et al.Comparison of a Live Attenuated2009H1N1Vaccinewith Seasonal Influenza Vaccines against2009Pandemic H1N1Virus Infection in Miceand Ferrets[J].Journal of Infectious Diseases,2011,203(7):930-936
24Garcia-Garcia L, Valdespino-Gomez JL, Lazcano-Ponce E, et al. Partial protectionof seasonal trivalent inactivated vaccine against novel pan-demic influenza A/H1N12009: case-control study inMexico City[J].BMJ，2009，339(10): b3928
25Hancock K, Veguilla V, Lu X, et al. Cross-reactive antibody responses to the2009pandemic H1N1influenza virus[J]. N Engl J Med，2009;361:1945-1952
26]Medlock J, Galvani AP. Optimizing influenza vaccine distribution[J]. Science,2009,325:1705-1708
27Theo JM， Irina VV， Harrie KL，et al. Master donor viruses A/Leningrad/134/17/57(H2N2) and B/USSR/60/69and derived reassortants used in live attenuated influenzavaccine (LAIV) do not display neurovirulent properties in a mouse model[J].Arch Virol,2010,155(9):1391-1399
28Belshe RB, Nichol KL, Black SB, et al. Safety,efflcacy and effectiveness of liveattenuated, cold-adapted influenza vaccine in an indicated population aged5～49years[J]. Clin Infect Dis,2004,39:920-927
29Nichol KL, Mendelman PM, Mallon KP, et al.Effectiveness of live attenuated intranasalinfluenza virus vaccine in healthy, working adults[J].JAMA,1999,282:137-144
30McMahonA W, Iskander J,Haber P,et al.Adverse events after inactivated influenzavaccination among children less than2years of age: analysis of reports fromthevaccine adverse event reporting system1993～2003[J]. Pediatrics,2005,115:453-460
31Izurieta HS, Haber P, Wise RP, et al. Adverse events reported followinglive,cold-adapted, intranasal influenza vaccine[J].JAMA,2005,294:2720-2725
32Belshe RB, Gruber WC. Safety efflcacy and effectiveness of cold-adapted, liveattenuated, trivalent intranasal influenza vaccine in adults and children[J]. PhilosTrans R Soc Lond Ser B,2001,356:1947-1951
33Petukhova G, Naikhin A, Chirkova T, et al. Comparative studies of local antibodyand cellular immune responses to influenza infection and vaccination with liveattenuated reassortant influenza vaccine(LAIV) utilizing a mouse nasal-associatedlymphoid tissue (NALT) separation method[J]. Vaccine,2009,27:2580-2587
34Buonagurio DA, Bechert TM, Yang CF, et al. Genetic stability of live, cold-adaptedinfluenza virus components of the FluMist/CAIV-T vaccine throughout themanufacturing process[J]. Vaccine,2006,24:2151-2160
35Hoffman E, Neumann G, Kawaoka Y, et al. A DNA transfection system for generationof influenza A virus from eight plasmids [J]. PNAS,2000,97(11):6108-61131
36Alexandrova GI, Maassab HF, Kendal AP, et al. Laboratory properties of cold-adaptedinfluenza B live vaccine strains developed in the US and USSR, and their B/AnnArbor/1/86cold-adapted reassortant vaccine candidates[J]. Vaccine,1990,8:61-64
37Ellebedy AH, Webby RJ.Influenza vaccines[J].Vaccine，2009,27(8)：65-68
38Ohmit SE, Victor JC, Rotthoff JR, et al. Prevention of antigenically driftedinfluenza by inactivated and live attenuated vaccines[J].N Engl J Med，2006,355:2513-2522
39Ohmit SE, Victor JC, Teich ER, et al. Prevention of symptomatic seasonal influenzain2005–2006by inactivated and live-attenuated vaccines[J]. J Infect Dis,2008,198:312-317
40Barry DW, Mayner RE, Hochstein HD, et al.Comparative trial of influenza vaccinesII. Adverse reactions in children and adults[J]. Am J Epidemiol,1976,104:47-59
41Beyer WE, Palache AM, de Jong JC, et al. Cold-adapted live influenza vaccine versusinactivated vaccine: systemic vaccine reactions, local and systemic antibodyresponse, and vaccine efflcacy. A meta-analysis[J]. Vaccine,2002,20:1340-1353
42Clements ML, O’Donnell S, Levine MM, et al. Dose response of A/Alaska/6/77(H3N2)cold-adapted reassortant vaccine virus in adult volunteers: role of local antibodyin resistance to infection with vaccine virus[J]. Infect Immun,1983,40:1044-1051
43Ambrose CS, Coelingh LCK. Current status of live attenuated influenza vaccine intheUnited States for seasonal and pandemic influenza[J]. InfluenzaOther RespiViruses,2008,2:193-202
44Govorkova EA, Kaverin NV, Gubareva LV, et al. Replication of influenza A virusesin a green monkey kidney continuous cell line(Vero)[J]. J Infect Dis,1995,172(1):250-253
45Edwards KM,Dupont WD,Westrich MK, et al.A randomized, controlled trial ofcold-adapted and inactivated vaccines for the prevention of influenza A disease[J].J Infect Dis,1994,169:68-76
46Steel J, Staeheli P, Mubareka S, et al. Transmission of pandemic H1N1influenzavirus and impact of prior exposure to seasonal strains or interferon treatment[J].J Virol,2009,84:21-26
47Luytjes W, Krystal M, Enami M, et al.Amplification, expression, and packaging ofa foreign gene by influenza virus[J].Cell,1989,59:1107-1113
48Enami M, Luytjes W, Krystal M, et al. Introduction of site-specific mutations intothe genome of influenza virus [J]. Proc Natl Acad Sci,1990,87:3802-3805
49Li S, Liu C, Klimov A, et al. Recombinant influenza A virus vaccines for the pathogenichuman A/Hong Kong/97(H5N1) viruses[J]. J Infect Dis1,1999,179:1132-1138
50Cong YL, Pu J, Liu QF, et al. Antigenic and genetic characterization of H9N2swineinfluenza viruses in China[J].J Gen Virol，2007，88:2035-2041
51Neumann G, Watanabe T, Ito H, et al. Generation of influenza A viruses entirely fromcloned cDNAs[J].Proc Natl Acad Sci,1999,96:9345-9350
52Fodor E, Devenish L, Engelhardt OG, et al. Rescue of influenzaA virus from recombinantDNA[J].J Virol,1999,73:9679-9682
53Smith GJ, Vijaykrishna D, Bahl J, et al. Origins and evolutionary genomics of the
2009swine-origin H1N1influenza A epidemic[J].Nature,2009,25:1122-1125
54Garten RJ, Davis CT, Russell CA, et al. Antigenic and genetic characteristics ofswine-origin2009A (H1N1)influenza viruses circulating in humans[J].Science,2009,10:197-201
55Murphy BR, Coelingh K, Principles underlying the development and use of live,attenuated cold-adapted influenza A and B virus vaccines[J]. Viral Immunol,2002,15:295-323
56Wareing MD, Tannock GA.Live attenuated vaccines against influenza; an historicalreview[J].Vaccine,2001,19:3320-3330
57国家药典委员会．中华人民共和国药典[M].第3版.中国医药出版社.（2010）
58Viviani L， Assael BM，Kerem E.Impact of the A (H1N1) pandemic influenza (season2009～2010) on patients with cystic fibrosis [J].Journal of CysticFibrosis.2011,10:370-376
59King JC, Stoddard JJ, Gaglani MJ, et al. Effectiveness of school-based influenzavaccination[J]. N Engl J Med,2006,355:2523-2532
60Poehling KA, Talbot HK, Williams JV, et al. Impact of school-based influenzaimmunization program on disease burden: comparison of two Tennessee counties[J].Vaccine,2009,27:2695-2700
61Johnson PR, Feldman S, Thompson JM, et al. Immunity to influenza a virus infectionin young children: A comparison of natural infection, live cold-adapted andinactivated vaccines [J]. J Infect Dis,1986,154(1):1212-1261
62Romanova J, Katinger D, Ferko B, et al. Live cold-adapted influenza A vaccine producedin Vero cell line [J]. Virus Res,2004,103(122):1872-1931
63Cox RJ, Brokstad KA, Ogra P. Influenza virus: immunity and vaccination strategies.Comparison of the immune response to inactivated and live, attenuated influenzavaccines[J]. Scand J Immunol,2004,59(1):1-15
64Chen GL, Subbarao K. Live attenuated vaccines for pandemic influenza[J]. Curr TopMicrobiol Immunol,2009,333:109-132
65Poland GA. Vaccines against avian influenza—A race against time. N Engl J Med,2007,354:1411-1413
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