制备流感疫苗的细胞基质研究进展

考虑到鸡胚生产流感疫苗的局限性,研究者们开始关注细胞培养疫苗生产技术.此文主要介绍了几种常用的细胞基质(如Vero、PER.C6、Madin-Darby狗肾细胞和其他动物细胞),阐述了利用细胞培养技术制备流感疫苗的方法及研究进展.相关研究显示细胞基质生产的流感疫苗与鸡胚生产的流感疫苗相比,效果相似或者更好.     

Vero细胞制备的流感疫苗诱导的体液与细胞免疫

作者将候选的经连续传代的哺乳动物细胞系 ( Vero)生产的三价流感全病毒疫苗与模拟商售的鸡胚培养疫苗 ,在 Balb/c小鼠中比较它们诱导体液免疫和细胞免疫的能力。　　将小鼠分为 Vero细胞全病毒疫苗组、模拟商售鸡胚全病毒疫苗组、鸡胚裂解疫苗组和鸡胚亚单位疫苗组 ,对照组接种含 <0 .0 1μg/ml的 Vero细胞或卵蛋白的模拟制品。初免后 4周疫苗组和对照组均给予相同剂量的加强接种。结果表明 ,各疫苗组都有较高的血凝抑制抗体滴度和高水平流感病毒特异性 Ig G,Ig G应答主要是 Ig G1和 Ig G2 a/Ig G2 b同型。不管是经 Vero细胞还是鸡胚…     

在6～18月龄儿童中评价流感减毒活疫苗的安全性、免疫原性及保护效果

本文报道用甲型流感病毒冷适应减毒株caA/川崎/9/86(H1N1)和caA/洛杉矶(H3N2)免疫儿童的结果,并对二价疫苗中毒株间的干扰作了初步探讨.将182名6～18月龄血清阴性的儿童以双盲法分为4组:H1N1组、H3N2组、H1N1及H3N2二价疫苗组和接种鸡胚尿囊液的对照组.试验用疫苗滴度均为10~(6.2)半数组织培养感染量(TCID_(50)).疫苗接种后密切观察1周:疫苗组儿童未见明显不良反应,轻微呼吸道症状及低热与对照组无显著性差异.鼻洗液的流感病毒分离率:免疫后6天H1N1组为16%,H3N2组为36%,二价疫苗组为55%;11天时H1N1组为0%,H3N2组为2%,二价疫苗组为8%.全部分离物经检验仍保留减毒特性而无毒力恢复迹象.疫苗接种后4周,H3N2组抗体阳转率达90%以上,抗体几何平均滴度(GMT)为7.2,均显著高于H1N1组.二价疫苗组的H3N2抗体阳转率与单价疫苗组无明显差别,但H1N1抗体仅为31%,明显低于单价疫苗组(83%).     

流感疫苗的研究进展

流感是我国每年秋冬季常见的呼吸道传染病。接种疫苗是防控流感最为有效的措施。目前,多种类型的流感疫苗正在研发之中或已上市,同时,流感疫苗规模化生产的基质也逐渐从鸡胚转为哺乳动物细胞。此文介绍了流感疫苗的研究进展以及疫苗生产基质的变迁。     

三种流感疫苗对H7N9禽流感病毒的免疫交叉反应

目的 评价季节性流感疫苗对H7 N9禽流感病毒的免疫效果.方法 使用3种季节性流感疫苗对受试者进行免疫接种,受试者于免疫前及免疫后21 d采集血样.应用微量血凝抑制试验(HI)对血清进行抗体检测.结果 在接种流感疫苗前,接种3种不同季节性流感疫苗的A、B、C组针对H7N9型毒株抗体几何平均滴度(GMT)均为血清1:5.0稀释阴性,保护抗体阳性率均为0.疫苗免疫后21 d,276例受试者中检测到6例受试者H7N9型毒株HI抗体结果 达到1:40阳转标准,阳转率为2.2%,A、B和C组各有2例(2.2%)、1例(1.1%)和3例(3.3%)H7N9型毒株HI抗体阳转.3组抗体阳转率差异无统计学意义(P=0.789).H7N9型抗体阳转率、GMT增长倍数、抗体保护率均未达到欧盟标准和美国食品药品管理局标准.结论 接种季节性流感疫苗不能为人群提供针对H7 N9禽流感病毒的免疫保护.     

甲型H1N1流感疫苗不良事件监测及评估

目的:分析甲型H1N1流感疫苗的接种情况,及时总结接种的监测与评估,为开展甲型H1N1流感防控提供参考。方法:对国内外2009年接种甲型H1N1流感疫苗的不良反应监测报告进行分析。结果:甲型H1N1流感疫苗被认为与季节性流感疫苗总体安全性近似。结论:在甲型H1N1流感大流行的背景下,接种该疫苗利大于弊。     

老年人接种甲型H1N1流感疫苗不良反应的临床观察

目的：比较季节性流感疫苗与甲型H1N1流感疫苗预防接种后的不良反应。方法：选择我部在前后两个阶段分别接种过季节性流感疫苗与甲型H1N1流感疫苗的离退休老干部及家属106例为观察对象,对观察对象分别在接种时、接种后30min、1h、2h、24h、48h、1周、2周、1个月进行不良反应观察,并统计比较观察结果。结果：季节性流感疫苗组和甲型H1N1流感疫苗组的不良反应均较轻微,且差异无统计学意义（P〉0.05）;季节性流感疫苗组发生常见不良反应4例（3.8%）,甲型H1N1流感疫苗组发生常见不良反应5例（4.7%）,两组均无少见、罕见及极为罕见的不良反应发生。结论：季节性流感疫苗与甲型H1N1流感疫苗的不良反应差异无统计学意义（P〉0.05）,老年人接种甲型H1N1流感疫苗是安全的。     

补充益生菌对接种流行性感冒疫苗后抗体滴度影响的Meta分析

目的:研究补充益生菌对接种流行性感冒(以下简称流感)疫苗后抗体滴度的影响。方法:计算机检索PubMed,EMBASE,Cochrane Central Register of Controlled Trials,MEDLINE,CBM,CNKI,收集补充益生菌对接种流感疫苗后抗体滴度影响的研究,用Review manager 5.0软件进行Meta分析。采用均数和95%置信区间(95%CI)评价补充益生菌对接种流感疫苗后抗体滴度的影响。结果:纳入7项随机对照临床研究,共计476例患者,Meta分析结果表明:补充益生菌可提高接种流感疫苗后甲型H1N1型流感抗体滴度(MD=4.710,95%CI:0.53~8.89,P<0.00001;I²=97%);补充益生菌可提高接种流感疫苗后甲型H3N2型流感抗体滴度(MD=16.86,95%CI:0.87~32.85,P<0.0001,I²=100%);补充益生菌可提高接种流感疫苗后乙型流感抗体滴度(MD=3.04,95%CI:-0.77~6.85,P<0.0001,I²=96%)。结论:补充益生菌可提高接种流感疫苗后甲型H1N1流感、甲型H3N2流感和乙型流感抗体滴度。     

联合酶联免疫的微量病毒中和法检测大流行流感疫苗血清中和抗体的可行性研究

目的:建立联合酶联免疫的微量病毒中和法(ELISA-MVN法),用于大流行流感疫苗流感病毒中和抗体效价的检测,并进行方法学验证。方法:采用4种羊抗流感病毒血清参考品,与人感染禽流感H5N1病毒疫苗株(A/Vietnam/1194/2004-NIBRG-14)病毒液,分别于37℃孵育18 h后,固定细胞,ELISA检测细胞内流感病毒核蛋白表达,确定其中和抗体效价,考察该方法的特异性;通过对高中低不同抗体滴度的血清样本的多次平行检测来评价该方法的重复性;通过ELISAMVN法和血凝抑制试验(HI试验)分别检测大流行流感疫苗接种者血清样本,比较2种方法的灵敏性和相关性。结果:ELISA-MVN结果显示羊抗H5N1的血清中和抗体检测滴度为5 120,其他3种血清的中和抗体滴度小于10,该方法特异性良好;采用ELISA-MVN法对3种不同滴度的血清进行重复检测,组内重复性试验的6次平行试验结果的RSD为3.5%。组间重复性5次结果,RSD为4.8%~8.6%。2种方法测定疫苗接种者血清样本抗体效价的结果具有显著相关性(P<0.001),相关系数r=0.932。ELISA-MVN法检测结果的几何平均滴度高于HI试验检测结果,该方法较HI试验在检测大流行流感疫苗血清样本灵敏度高。结论:ELISA-MVN可满足大流行流感疫苗血清学检测的要求,并可用于评价大流行流感疫苗的免疫效果。     

四价流感病毒裂解疫苗生产用毒种传代稳定性研究

目的  研究四价流感病毒裂解疫苗生产用毒种的传代稳定性,确保毒种适用于疫苗生产。方法  将WHO推荐的季节性流感疫苗甲型H1N1和H3N2、乙型BV和BY病毒株（原始种子批）在鸡胚中传代扩增,制备生产用主种子批和工作种子批毒种。按中国药典2015年版三部的要求对毒种进行检定,包括鉴别试验、病毒滴度和血凝滴度测定、外源微生物检查等。将毒种连续传10代,用反转录PCR扩增第2、3、4、5、10代病毒鸡胚尿囊液中的血凝素（hemagglutinin,HA）和神经氨酸酶（neuraminidase,NA）基因片段,并进行序列测定,分析传代过程中HA和NA的核苷酸和氨基酸序列同源性。结果  主种子批和工作种子批毒种的抗原性均与推荐的病毒株一致。各型病毒滴度均>6.5 lg半数鸡胚感染量/ml,血凝滴度均≥1：160。2个种子批的支原体和无菌检查结果均为阴性,主种子批外源性禽白血病病毒和禽腺病毒检查结果亦为阴性。毒种在鸡胚中连续传代后,各型病毒株HA和NA的核苷酸序列同源性均>99%,氨基酸序列同源性均为100%。结论  本研究的四价流感病毒裂解疫苗生产用毒种具有良好的传代稳定性,可用于疫苗生产。     

Modulation of immune response induced by co-administration of DNA vaccine encoding HBV surface antigen and HCV envelope antigen in BALB/c mice

Plasmid DNA vaccines encoding the hepatitis B virus (HBV) surface and hepatitis C virus (HCV) envelope antigens, respectively, were constructed, and attempt were made to find the possibility of a divalent vaccine against HBV and HCV. The expression of each plasmid in Cos-1 cells was confirmed using immunocytochemistry. To measure the induced immune response by these plasmids in vivo, female BALB/c mice were immunized intramuscularly with 100 microg of either both or just one of the plasmids. Anti-HBV and HCV-specific antibodies and related cytokines were evaluated to investigate the generation of both humoral and cellular immune responses. As a result, specific anti-HBV and anti-HCV serum antibodies from mice immunized with these plasmids were observed using immunoblot. The levels of IL-2 and RANTES showing a Th1 immune response were significantly increased, but there was no change in the level of IL-4 (Th2 immune response) in any of the immunized groups. Compared with each plasmid DNA vaccine, the combined vaccine elicited similar immune responses in both humoral and cell-mediated immunities. These results suggest that the combined DNA vaccine can induce not only comparable immunity experimentally without antigenic interference, but also humoral and Th1 dominant cellular immune responses. Therefore, they could serve as candidates for a simultaneous bivalent vaccine against HBV and HCV infections.     

Long-lasting protective immunity against H7N9 infection is induced by intramuscular or CpG-adjuvanted intranasal immunization with the split H7N9 vaccine

There is an urgent need for efficient vaccines against the highly pathogenic avian influenza A viral strain H7N9. The duration and intensity of the immune response to H7N9 critically impacts the epidemiology of influenza viral infection at the population level. However, the insufficient immunogenicity of H7N9 raises concerns about vaccine efficacy. In this study, we evaluated the impact of immunization routes and the adjuvant CpG on the immune response to a split H7N9 vaccine in mice. Determination of humoral and cellular responses to the vaccine revealed that after four vaccine doses, high titers of H7N9-specific serum IgG, determined by the influenza hemagglutination inhibition (HI) assay, were induced through the intramuscular (i.m.) route and lasted for at least 40 weeks. CpG-adjuvanted immunization increased the levels of long-lived IFN-γ⁺ T cells and raised the Th1-biased IgG2a/IgG1 response ratio. In addition, aside from mucosal IgA, CpG-adjuvanted intranasal (i.n.) immunization elicited serum IgG and cellular responses of a similar duration and intensity to CpG-adjuvanted i.m. immunization. Mouse challenge assays demonstrated that 24 weeks following i.m. immunization without CpG or CpG-adjuvanted immunization through the i.m. or i.n. routes, both offered a high level of protection against H7N9 infection. These results indicate that efficient long-term protection against H7N9 can be achieved via the optimization of vaccination strategies, such as immunization doses, routes, and adjuvants.     

Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological,cellular and protective immune responses

Avian influenza virus infection is a serious public health threat and preventive vaccination is the most cost-effective public health intervention strategy. Unfortunately, currently available unadjuvanted avian influenza vaccines are poorly immunogenic and alternative vaccine formulations and delivery strategies are in urgent need to reduce the high risk of avian influenza pandemics. Cationic polymers have been widely used as vectors for gene delivery in vitro and in vivo. In this study, we formulated H5N1 influenza vaccines with GenJet? or in vivo-jetPEI^®, and showed that these formulations significantly enhanced the immunogenicity of H5N1 vaccines and conferred protective immunity in a mouse model. Detailed analyses of adaptive immune responses revealed that both formulations induced mixed T_H1/T_H2 antigen-specific CD4 T-cell responses, antigen-specific cytotoxic CD8 T-cell and memory B-cell responses. Our findings suggest that cationic polymers merit future development as potential adjuvants for mucosal delivery of poorly immunogenic vaccines.     

Protective immunity elicited by a pseudotyped baculovirus-mediated bivalent H5N1 influenza vaccine

The development of novel H5N1 influenza vaccines to elicit a broad immune response is a priority in veterinary and human public health. In this report, a baculovirus vector was used to construct bivalent recombinant baculovirus vaccine encoding H5N1 influenza virus hemagglutinin proteins (BV-HAs) from clade 2.3.4 and clade 9 influenza viruses. Mice immunized with 5 × 10⁷ IFU BV-HAs developed significantly high levels of H5-specific neutralizing antibodies and cellular immunity that conferred 100% protection against infection with H5N1 influenza viruses. This study suggests that baculovirus-delivered multi-hemagglutinin proteins might serve as a candidate vaccine for the prevention of pre-pandemic and pandemic H5N1 influenza viruses.     

Influenza vaccine and adjuvant

Adjuvant is originated from the Latin word "adjuvare" which means "help" in English to enhance the immunological responses when given together with antigens. The beginning of adjuvant was mineral oil which enhanced the immune response when it was given with inactivated Salmonella typhimurium. Aluminium salt was used to precipitate diphtheria toxoid and increased level of antibody response was demonstrated when administered with alum-precipitated antigens. Since 1930, aluminium salt has been used as DTaP (diphtheria-tetanus-acellular pertussis vaccine) adjuvant. Many candidates were tested for adjuvant activity but only aluminum salt is allowed to use for human vaccines. New adjuvant MF59, oil-in-water emulsion type, was developed for influenza vaccine for elderly (Fluad) and series of AS adjuvant are used for hepatitis B, pandemic flue, and human papiloma virus vaccines. Oil-adjuvanted influenza pandemic vaccines induced higher antibody response than alum-adjuvanted vaccine with higher incidence of adverse events, especially for local reactions. Alum-adjuvanted whole virion inactivated H5N1 vaccine was developed in Japan, and it induced relatively well immune responses in adults. When it applied for children, febrile reaction was noted in approximately 60% of the subjects, with higher antibodies. Recent investigation on innate immunity demonstrates that adjuvant activity is initiated from the stimulation on innate immunity and/or inflammasome, resulting in cytokine induction and antigen uptake by monocytes and macrophages. The probable reason for high incidence of febrile reaction should be investigated to develop a safe and effective influenza vaccine.     

Local and Systemic Immune Responses to Intratracheal Instillation of Antigen and DNA Vaccines in Mice

PURPOSE: The purpose of this study was to determine the immunization efficacy of antigen and DNA vaccines after delivery to the lung, to assess the integrity of the pulmonary tissue after vaccination, and to elucidate mechanisms involved in the induction of immunity. METHODS: Ovalbumin, the plasmid encoding ovalbumin, the hepatitis B surface antigen (HBsAg), or plasmid encoding HBsAg were intratracheally instilled or injected in quadriceps in mice. The immune response and its Th polarization were analyzed over time. Markers of inflammation were measured in bronchoalveolar lavage, and lung histology was performed. The fate of ovalbumin following intratracheal instillation was studied. RESULTS: According to the vaccine, the pulmonary route produced stronger or equivalent humoral and cellular responses systemically and locally in the lung as compared to injection. The IgG subclasses and cytokine pattern indicate that the immunity was preferentially polarized toward the Th2 and Th1 type for antigen and DNA immunization, respectively. Ovalbumin penetrated the respiratory tissue and blood poorly after intratracheal instillation, suggesting that the immune response was triggered at airway surfaces. Overall, vaccines delivered to the lung did not induce any local sign of inflammation. CONCLUSIONS: Pulmonary administration of vaccines might be a promising alternative to conventional vaccination by injection.     

Pulmonary delivery of influenza vaccine formulations in cotton rats: site of deposition plays a minor role in the protective efficacy against clinical isolate of H1N1pdm virus

Administration of influenza vaccines to the lungs could be an attractive alternative to conventional parenteral administration. In this study, we investigated the deposition site of pulmonary delivered liquid and powder influenza vaccine formulations and its relation to their immunogenicity and protective efficacy. In vivo deposition studies in cotton rats revealed that, the powder formulation was mainly deposited in the trachea (?～?65%) whereas the liquid was homogenously distributed throughout the lungs (?～?96%). In addition, only 60% of the antigen in the powder formulation was deposited in the respiratory tract with respect to the liquid formulation. Immunogenicity studies showed that pulmonary delivered liquid and powder influenza formulations induced robust systemic and mucosal immune responses (significantly higher by liquids than by powders). When challenged with a clinical isolate of homologous H1N1pdm virus, all animals pulmonary administered with placebo had detectable virus in their lungs one day post challenge. In contrast, none of the vaccinated animals had detectable lung virus titers, except for two out of eight animals from the powder immunized group. Also, pulmonary vaccinated animals showed no or little signs of infection like increase in breathing frequency or weight loss upon challenge as compared to animals from the negative control group. In conclusion, immune responses induced by liquid formulation were significantly higher than responses induced by powder formulation, but the overall protective efficacy of both formulations was comparable. Thus, pulmonary immunization is capable of inducing protective immunity and the site of antigen deposition seems to be of minor relevance in inducing protection.     

375位医务人员接种甲流疫苗不良反应分析

目的:了解医务人员接种甲型H1N1流感裂解疫苗的不良反应(ADR)发生情况。方法:观察该医院375名医务工作者接种甲型H1N1流感裂解疫苗后的ADR,统计分析ADR发生率、出现时间、持续时间、转归等。结果:375例甲型H1N1流感裂解疫苗接种者中发生ADR10例,发生率2.7%。均为一般ADR,所有的ADR经对症治疗后均可治愈。结论:甲型H1N1流感裂解疫苗总体ADR以轻度ADR为主,均可治愈,疫苗的安全性较好。     

Protective efficacy of an H1N1 cold-adapted live vaccine against the 2009 pandemic H1N1, seasonal H1N1, and H5N1 influenza viruses in mice

Vaccination is a key strategy for preventing influenza virus infections. Here, we generated a reassortant virus (SC/AAca) containing the hemagglutinin and neuraminidase genes from a 2009 pandemic influenza virus A/Sichuan/1/2009 (H1N1) (SC/09) and six internal genes from the cold-adapted virus A/Ann Arbor/6/60 (H2N2) (AAca). The SC/AAca reassortant induced a sound humoral immune response and complete protection against homologous SC/09 virus challenge in mice after intranasal administration of an at least 10(6) 50% egg infectious dose (EID(50)) of SC/AAca. SC/AAca inoculation also induced significant CD4+ and CD8+ T cell responses and provided solid protection against heterologous H1N1 and H5N1 virus challenge. Our results suggest that this 2009 H1N1 live vaccine will provide protection against both 2009 pandemic and seasonal H1N1 virus infection and might reduce the severity of H5N1 virus infection in humans. The induction of cross-reactive virus-specific T cell responses may be an effective approach to develop universal influenza vaccines.     

Polycation-decorated PLA microspheres induce robust immune responses via commonly used parenteral administration routes

Recombinant viral subunit-based vaccines have gained increasing attention due to their enhanced safety over the classic live-attenuated or inactivated vaccines. The low immunogenicity of the subunit antigen alone, however, requires the addition of an adjuvant to induce immunity. Particulate-based delivery systems have great potential for developing new vaccine adjuvants, compared to traditional aluminum-based saline adjuvants. The physicochemical properties of particulate vaccines have been extensively investigated; however, few studies have focused on how the administration route of various adjuvant–antigen combinations impacts the efficacy of the immune response. Here, for the first time, the viral Hepatitis B surface antigen (HBsAg) was combined with aluminum-based or cationic-microsphere (MP) based adjuvants to investigate the characteristics of immune responses elicited after immunization via the subcutaneous, intramuscular, or intraperitoneal routes respectively. In vitro, the MP-based vaccine significantly increased dendritic cell (DC) activation with up-regulated CD40 and CD80 expression and IL-12 production compared to alum-based vaccine. After immunization, both MP and alum-based vaccines produced increased IgG titers in mice. The administration route of these vaccines did influenced immune responses. The MP-based vaccine delivered via the intramuscular route yielded the highest levels of the IgG2a isotype. The alum-based vaccine, delivered via the same route, produced an IgG1-dominated humoral immune response. Moreover, subcutaneous and intramuscular immunizations with MP-based vaccine augmented Granzyme B, Th1-type cytokines (IL-2, IL-12, and IFN-γ), and Th2 cytokine IL-4 secretions. These results demonstrate that MP-based vaccines have the capacity to induce higher cellular and humoral immune response especially via an intramuscular administration route than an alum-based vaccine.