重组人乳头瘤病毒16型L1蛋白在昆虫细胞中的表达及条件优化研究

目的 利用昆虫细胞-杆状病毒表达系统表达人乳头瘤病毒16型(human papillomavirus 16,HPV16)L1蛋白,并确定含编码HPV16 LI蛋白基因杆状病毒的扩增及其相应目的 蛋白表达的最佳条件.方法 在不同感染复数(MOI)、不同时间条件下扩增病毒,以蚀斑试验检测病毒滴度,研究获得最高滴度病毒的最适条件;通过对相应蛋白进行表达条件的优化,以蛋白印迹法比较蛋白表达量,确定目的 蛋白表达的最佳条件.结果 确定了扩增含编码HPV16 L1蛋白基因杆状病毒及表达相应蛋白的最佳条件,即在sf9细胞系中,以MOI值为0.10接种病毒,扩增48 h后收获病毒;以细胞密度为(2-3)×106/ml,MOI值为10.0接种病毒,悬浮表达72 h收获目的 蛋白.蛋白印迹法检测结果证实,表达的目的 条带可与HPV16 LI蛋白的单克隆抗体发生反应.结论 建立了在昆虫细胞中表达HPV16 L1蛋白的最适条件,为下一步的纯化及免疫原性研究奠定了基础.     

无毒的霍乱毒素佐剂存在下16型人乳头瘤瘸毒壳粒鼻胶免疫诱生的阴道免疫球蛋白水平增加

人乳头瘤病毒（HPV）感染是引起宫颈癌的主要原因，其中最常见的是16型HPV。先前的研究显示，鼻腔免疫病毒样颗粒（VLP）和壳粒可诱导产生抗HPV主要核壳蛋白L1的体液免疫应答。德国Dell等对无毒的霍乱毒素佐剂能否提高壳粒鼻腔免疫小鼠血清和阴道中产生的HPV 16 L1特异性抗体和L1特异性CTL水平进行了研究。     

猴免疫缺陷病毒样颗粒鼻内免疫诱导全身及粘膜免疫应答

作者利用 Sf9昆虫细胞同时表达了猴免疫缺陷病毒 ( SIV)的核心蛋白 ( Gag)和包膜蛋白 ( Env) ,并形成了病毒样颗粒 ( VLP) ,采用连续蔗糖梯度离心法纯化出 SIV VLP。为研究 SIV VL P通过粘膜免疫途径能否诱导全身及粘膜免疫应答 ,作者选用 2 0 μg和2 0 0 μg两种免疫剂量、分别通过鼻内 ( i.n.)滴入和腹腔 ( i.p.)注射两种途径免疫BALB/c小鼠。 i.n.途径又分为用和不用佐剂组 ,佐剂为霍乱毒素 ( CT,每只小鼠每次免疫 1 0 μg)。采集各组小鼠的血液、唾液、阴道冲洗液 ,用 EL ISA法测定各类标本中特异性抗体水平。 SIV VLP2…     

人乳头瘤病毒及其病毒样颗粒的研究及应用

人乳头痛病毒( human papillomavirus,HPV)感染是导致尖锐湿疣、宫颈上皮瘤样病变和宫颈癌的主要原因.鉴于HPV的特殊扩增机制,目前尚不能通过体外培养的传统方法来获得病毒用于疫苗研究.HPV的主要衣壳蛋白L1在体外可自行组装成病毒样颗粒(virus-like particle,VLP),形成的VLP在实验动物和人体中均可诱生高滴度的保护性抗体,是理想的预防性疫苗组分.此文就HPV的流行病学、致病机制及其VLP的制备和应用作一综述.     

重组4-1BB配体对治疗性重组人乳头瘤病毒16型蛋白疫苗的佐剂作用

目的 研究重组4-1BB配体(recombinant 4-1BB ligand,r4-1BBL)作为佐剂对治疗性重组人乳头瘤病毒16型(human papillomavirus-16,HPV16)蛋白疫苗(HPV16蛋白疫苗)免疫效果的影响.方法 对4-1BBL胞外功能区进行密码子优化,并通过NdeⅠ和XhoⅠ酶切位点定向插入pET28a表达载体.将获得的重组表达载体转化至大肠埃希菌BL21 (DE3),并用异丙基-β-D-硫代半乳糖苷诱导重组蛋白表达.用蛋白质印迹法对重组蛋白进行鉴定,并用非还原性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和电子显微镜分析重组蛋白结构.用CCK-8试剂盒检测纯化r4-1BBL对小鼠T细胞体外增殖的影响.同时将r4-1BBL与HPV16蛋白疫苗联合免疫C57BL/6小鼠,用酶联免疫斑点试验检测小鼠的细胞免疫应答水平,观察r4-1BBL对HPV16蛋白疫苗抑制肿瘤生长的影响.结果 密码子优化的4-1BBL胞外功能区能在重组表达载体中表达,且表达产物同时以包涵体和可溶性形式存在.可溶性产物的结构主要为二聚体,电子显微镜下可以观察到类似亚单位的结构.纯化r4-1BBL可促进小鼠脾淋巴细胞的体外增殖.与单纯HPV16蛋白疫苗相比,r4-1BBL联合HPV16蛋白疫苗能诱导更强的特异性细胞免疫应答(t=3.525,P=0.024)和产生更明显的肿瘤生长抑制作用(t=2.534,P=0.021).结论 r4-1BBL能在小鼠中提高HPV16蛋白疫苗的免疫效果,具有作为HPV16蛋白疫苗佐剂的潜力.     

重组人乳头瘤病毒双价(16/18型)疫苗免疫原性评价方法的研究

目的:探讨重组人乳头瘤病毒双价(16/18型)候选疫苗免疫原性评价方法。方法:对以假病毒为基础的中和实验(Zs-Green法)测定血清中和滴度,以微球为基础建立的Luminex法测定中和滴度和ELISA法测定抗体滴度的3种方法进行相关性研究以及体内效价测定(ED50法)和体外相对效力测定(IVRP法)的相关性分析,同时对5种免疫原性的评价方法进行重复性验证。结果:ZsGreen法、Luminex法测定的中和滴度和ELISA法测定的抗体滴度检测结果表明:滴度值呈明显的剂量效应以及侯选疫苗具有良好的免疫原性。采用SPSS统计软件分析,对于HPV 16L1 ZsGreen法、Luminex法和ELISA法的相关系数分别为0.791,0.800,0.747(Spearman相关系数)和HPV 18L1的相关系数分别为0.734,0.625,0.634(Spearman相关系数)。并且ED50法和IVRP法具有一定的相关性。5种方法重复性验证的RSD分别为:5.3%(ZsGreen)、5.2%(Luminex)、8.0%(ELISA)、32.3%(ED50)、6.3%(IVRP)。结论:建立的HPV 16和HPV 18...     

人源热休克蛋白HSC70与HPV16型E7融合蛋白在大肠杆菌中的表达、纯化和活性检测

目的 克隆人热休克蛋白HSC70的N端具有ATPase活性的结构域(简称为A基因)与HPV 16型E7的融合基因,在大肠杆菌中表达,并纯化获得有生物活性的AE7融合蛋白.方法 利用PCR方法扩增获得A基因片段,插入原核表达载体pET29b中,再通过PCR方法扩增HPV 16型E7基因片段,插入A基因的3′端,构建原核表达质粒pET29b-AE7,并转化大肠杆菌BL21(DE3),表达产物经离子交换层析和金属螯合层析纯化后,在一定的剂量下皮下免疫小鼠,检测对HPV-16的E7基因感染的预防及治疗作用.结果 重组质粒pET29b-AE7经DNA序列测定确认.SDS-PAGE检测,表达产物分子量为66kD,大部分为包涵体.纯化后产物经SDS-PAGE电泳分析纯度>90%.纯化产物AE7在一定的剂量下皮下免疫小鼠,对HPV-16的E7基因的感染有预防及治疗作用.结论 AE7融合蛋白的获得为进一步临床研究奠定基础.     

基于HPV16的新型伪病毒对DNA特异性B细胞的杀伤研究

目的 构建基于人乳头状病毒(human papilloma virus,HPV)16型的新型伪病毒,并检测其对DNA特异性B细胞(R4A)的杀伤效应.方法 利用昆虫杆状病毒表达系统表达病毒蛋白,在体外变性与复性过程中将病毒蛋白包装B细胞特异性启动子IgK控制下的白喉毒素(Diphtheriatoxin,DT)A链(DT-A)基因表达型质粒DNA,形成伪病毒.转染R4A细胞,荧光显微镜和流式细胞仪检测其杀伤效应.酶联免疫法检测R4A细胞分泌抗ds-DNA抗体的滴度.结果 转染R4A细胞48 h后,荧光显微镜和流式细胞仪成功检测到新型伪病毒对R4A细胞的杀伤效应,R4A细胞分泌抗ds-DNA抗体的滴度明显降低.结论 基于HPV16的新型伪病毒能有效特异杀伤R4A细胞,并降低抗ds-DNA抗体的滴度,为系统性红斑狼疮的治疗提供了理想的策略.     

水痘-带状疱疹病毒与大肠杆菌肠毒素滴鼻接种小鼠的体液免疫应答

为了研究大肠杆菌肠毒素 (L T)作为粘膜佐剂加强水痘 -带状疱疹病毒 (VZV)疫苗的免疫效果 ,作者构建了含 L T基因及其突变体的质粒 ,将质粒转化大肠杆菌 ,纯化表达产物 ,得到了正常的 L T、只含有重组 B亚单位的 L T(r LTB)及 N端 1 1 2位由精氨酸突变为赖氨酸的无毒性 L T(E1 1 2 K)。分别将三种毒素各 1 0μg与 VZV减毒活疫苗 (Oka株 ) 2 0 0 PFU混匀后 ,2 0 μl滴鼻免疫小鼠。分一次免疫组和三次免疫组 (间隔 2周、2月或6月 )。末次免疫后 4周取血 ,用中和试验检测小鼠血清中的抗 VZV抗体滴度。纯化小鼠抗体 ,并用十二烷基硫…     

重组四价人乳头瘤病毒16/18/52/58型病毒样颗粒疫苗制剂工艺的优化

目的 考察不同制剂工艺流程下制备的重组四价人乳头瘤病毒（human papillomavirus,HPV）16/18/52/58型病毒样颗粒疫苗的抗原吸附效果、稳定性及免疫原性,根据实验结果筛选出最合适的制剂配方及工艺。方法  通过改变重组四价HPV疫苗的缓冲体系,以及氢氧化铝佐剂的含量、混合工艺、吸附方式与吸附条件,配制不同的实验疫苗,观察其外观,并分别进行蛋白质稳定性、抗原吸附率和小鼠体内半数有效剂量的检测。 结果 重组四价HPV疫苗原液与450 μg/ml氢氧化铝佐剂在pH6.5的组氨酸缓冲体系下混合后的实验疫苗,外观均一性良好,抗原蛋白稳定性最高,Tm=87.4 ℃,小鼠体内免疫原性较好;搅动吸附条件下的4型别HPV抗原吸附率明显高于静止吸附;铝佐剂和抗原蛋白的吸附与混合先后顺序,不同吸附比例、温度和时间对疫苗外观无显著影响,抗原吸附率均大于99.00%。结论 确定了重组四价HPV16/18/52/58型病毒样颗粒疫苗合适的制剂工艺条件。     

Comparison of the size distributions and immunogenicity of human papillomavirus type 16 L1 virus-like particles produced in insect and yeast cells

Insect and yeast cells are considered the expression systems of choice for producing virus-like particles (VLPs), and numerous types of VLPs have been produced in these systems. However, previous studies were restricted to identifying the characteristics of individual VLP preparations. No direct comparison of the structures and immunogenic properties of insect and yeast-derived VLPs has so far been made. In the present study, the size distribution and immunogenic properties of human papillomavirus type 16 (HPV16) L1 VLPs produced in Spodoptera frugipedra-9 insect cells and Saccharomyces cerevisiae were compared. The insect cell-derived VLPs were larger than the yeast ones (P?<?0.0001), with median sizes of 34 and 26 nm, respectively. In addition, the insect-derived VLPs appeared to be more diverse in size than the yeast-derived VLPs. Immunization of mice with 30 ng per dose of VLPs elicited 2.7- and 2.4-fold higher anti-HPV16 L1 IgG and anti-HPV16 neutralizing antibody titers than immunization with the same amounts of the yeast-derived VLPs after the 4th immunizations, respectively. Our results suggest that the choice of expression system critically affects the particle size and immunogenic property of HPV16 L1 VLPs.     

Effect of alternative aluminum adjuvants on the absorption and immunogenicity of HPV16 L1 VLPs in mice

Aluminum adjuvants are commonly used in prophylactic vaccines to enhance antigen immunogenicity through induction of high-titer antibody responses. Three major forms of aluminum adjuvants with substantially different physical and chemical properties have been described: aluminum phosphate (AlPO(4)), aluminum hydroxide (AlOH) and amorphous aluminum hydroxyphosphate sulfate (AAHS). Here we describe the effect of these different aluminum adjuvants on the formulation and subsequent immunogenicity in mice of virus-like particles (VLPs) consisting of the L1 protein of Human Papillomavirus (HPV) Type 16. Electron microscopy demonstrated that the physical appearance of the phosphate-containing aluminum adjuvants was markedly different from that of aluminum hydroxide. All three aluminum adjuvants were found to display unique surface charge profiles over a range of pH, while AAHS demonstrated the greatest inherent capacity for adsorption of L1 VLPs. These differences were associated with differences in immunogenicity: anti-HPV L1 VLP responses from mice immunized with AAHS-formulated HPV16 vaccine were substantially greater than those produced by mice immunized with the same antigen formulated with aluminum hydroxide. In addition, HPV L1 VLPs formulated on AAHS also induced a substantial interferon-gamma secreting T cell response to L1 peptides indicating the potential for an enhanced memory response to this antigen. These results indicate that the chemical composition of aluminum adjuvants can have a profound influence on the magnitude and quality of the immune response to HPV VLP vaccines.     

Expression and purification of human papillomavirus 18 L1 virus-like particle from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cervical cancer caused by human papillomavirus (HPV) might be successfully prevented by HPV vaccination and screening. HPV vaccination and HPV serology assays have been investigated using HPV virus-like particles (VLPs). In this study we produced HPV18 L1 VLPs in Saccharomyces cerevisiae and purified them. The HPV18 L1 gene was cloned into the yeast expression vector YEGα-HIR525, and transformed into Saccharomyces cerevisiae. Expression of HPV18 L1 protein was demonstrated by Western blotting. The HPV18 L1 protein was purified by ultracentrifugation, size-exclusion chromatography and cation-exchange chromatography, and was up to 95% pure. We showed by transmission electron microscopy that the purified protein self-assembled into VLPs. These findings should be useful for establishing vaccine efficacy as well as characterizing vaccine candidates, and may provide an international reference standard for HPV serology assays.     

AS04-adjuvanted human papillomavirus (HPV) types 16 and 18 vaccine (Cervarix®): a review of its use in the prevention of premalignant cervical lesions and cervical cancer causally related to certain oncogenic HPV types

The AS04-adjuvanted human papillomavirus (HPV) 16/18 vaccine (Cervarix?) is a noninfectious recombinant vaccine produced using purified virus-like particles (VLPs) that induce a strong immunogenic response eliciting high levels of anti-L1 VLP antibodies that persist at levels markedly greater than those observed with natural infection. The vaccine adjuvant (AS04) is composed of monophosphoryl-lipid A, which enhances cellular and humoral immune response, adsorbed to aluminium hydroxide. The vaccine is indicated for the prevention of premalignant cervical lesions and cervical cancer causally related to certain oncogenic HPV types in females aged ≥10 years. The AS04-adjuvanted HPV 16/18 vaccine administered in a three-dose schedule over 6 months elicits a high immunogenic response and is highly protective against cervical intraepithelial neoplasia and infection causally related to high-risk oncogenic HPV types. In well designed clinical trials in young women aged 15-25 years who were HPV 16/18 seronegative and DNA negative to 14 HPV high-risk types, high levels of immunogenicity and protection were sustained for follow-up periods of up to 8.4 years. High and persistent immunogenicity against infection with HPV 16/18 has also been demonstrated in older and younger females (aged 10-55 years) who were seronegative for vaccine HPV types. The AS04-adjuvanted HPV 16/18 vaccine elicited a greater immunogenic response than the quadrivalent HPV vaccine in women aged 18-45 years who were seronegative and DNA negative for HPV 16/18. The AS04-adjuvanted HPV 16/18 vaccine confers cross protection against certain non-vaccine, high-risk HPV types. A rapid and strong anamnestic humoral immune response was elicited following a fourth dose of the vaccine. The AS04-adjuvanted HPV 16/18 vaccine is generally well tolerated, and pharmacoeconomic analyses have demonstrated the potential for public health benefits and cost effectiveness when vaccination programmes are run in conjunction with screening programmes. Thus, the AS04-adjuvanted HPV 16/18 vaccine prevents cervical disease associated with certain oncogenic HPV types, thereby reducing the burden of premalignant cervical lesions and, very likely, cervical cancer.     

Humoral and cellular immune responses to airway immunization of mice with human papillomavirus type 16 virus-like particles and mucosal adjuvants

Cervical cancer results from cervical infection by human papillomaviruses (HPV), especially HPV16. Intramuscular administrations of HPV16 virus-like particle (VLP) vaccines have been shown to induce strong neutralizing antibody responses and protect women against genital HPV16 infection and associated lesions. However, an alternative route of administration that avoids parenteral injection might facilitate vaccine implementation, particularly in developing countries which account for the majority of the worldwide cases of cervical cancer. In addition, inducing mucosal immunity could partially overcome the substantial variation in HPV16 antibodies at the cervix seen in ovulating women. Aerosol vaccination with HPV16 VLPs was previously shown to be immunogenic in mice and in women. Here, we examine whether exposure to other respiratory viral antigens may interfere with the HPV16 VLP-specific humoral response and whether two known mucosal adjuvants, CpG oligodeoxynucleotides and a natural non-toxic Escherichia coli heat-labile enterotoxin (HLT), can enhance the immunogenicity of airway immunization (nasal or aerosol-like) of mice with HPV16 VLPs. Our data show that HLT can significantly improve anti-VLP humoral responses in serum and mucosal secretions, as well as VLP-specific proliferative responses and IFN-gamma production by CD8 T cells, and that recent exposure to influenza surface antigens can diminish mucosal, but not systemic, antibody responses to the VLPs.     

Antibodies from women immunized with Gardasil cross-neutralize HPV 45 pseudovirions

Human papillomavirus (HPV) types 6, 11, 16 and 18 L1 virus-like particles (VLPs) have been used to generate the prophylactic quadrivalent vaccine, Gardasil. There is a high degree of L1 homology between HPV types and it is likely that there is a substantial degree of surface exposed viral epitope similarity. An investigation of vaccine-induced antibody binding and neutralization was undertaken focusing on A7 species members, HPV 18 and 45. Polyclonal sera from Gardasil recipients and from HPV 18 L1 VLP recipients were evaluated. Vaccine-induced antibodies were found to cross-neutralize HPV 45 pseudovirions (PsV) in vitro. Examination of a panel of monoclonal antibodies made against L1 VLPs revealed the presence of conformational, neutralizing epitopes on the surface of VLPs that may be shared between HPV 18 and HPV 45. These data demonstrate that Gardasil(r) immunization induces antibodies capable of neutralizing HPV 18 PsV and HPV 45 PsV in vitro.     

Molecular and structural characterization of the L1 virus-like particles that are used as vaccine antigens in Cervarix™, the AS04-adjuvanted HPV-16 and -18 cervical cancer vaccine

Cervarix? is a prophylactic human papillomavirus (HPV)-16/18 vaccine developed for the prevention of cervical cancer. The vaccine antigens are HPV-16 and HPV-18 L1 virus-like particles (VLPs) made from baculovirus expression vector system (BEVS)-produced HPV-16 and HPV-18 L1 proteins, respectively. In this study, we demonstrate that truncation of the nuclear targeting and DNA binding signals at the C-terminus of the HPV-16 and HPV-18 L1 proteins prevented intranuclear formation of the VLPs in the host cells and led to cytoplasmic localization of the L1 proteins as shown by in situ immunogold detection and electron microscopy. Following purification, these L1 proteins were able to form VLPs. The characteristics of these HPV-16 and HPV-18 L1 VLPs were studied using various physicochemical and immunological techniques. Amino acid analysis, SDS-PAGE and western blotting demonstrated the high purity of the L1 proteins and batch-to-batch consistency. The structure of the VLPs was shown to be similar to that reported for the native virions, as evaluated by microscopic observations, protein tomography and disc centrifugation experiments. The presence of important conformation-dependent neutralizing epitopes, such as U4, V5 and J4, was confirmed by ELISA and surface plasmon resonance. Structural robustness and consistency among batches was also observed by differential scanning calorimetry and electron microscopy. Moreover, adsorption to aluminum was shown not to impair VLP structure. In conclusion, the BEVS-produced HPV-16 and HPV-18 L1 VLPs display key structural and immunological features, which contribute to the efficacy of Cervarix? vaccination.     

Disassembly and reassembly of yeast-derived recombinant human papillomavirus virus-like particles (HPV VLPs)

The human papillomavirus (HPV) virus-like particles (VLPs) produced by recombinant expression systems are promising candidate vaccine antigens for prevention of cervical cancers as well as genital warts. However, expression of HPV type 6, 11, and 16 L1 proteins in Saccharomyces cerevisiae yielded irregularly shaped, broadly distributed VLPs smaller in size (30-50 nm) than expected (60 nm). In this study, we demonstrate that these HPV VLPs can be disassembled into the constituent capsomers (L1 pentamers) by incubation at low ionic strength and elevated pH in the presence of relatively low concentration of reducing agents. Following the removal of reducing agents, lowering of pH and increasing of ionic strength, the capsomers spontaneously reassembled into homogenous, 60-nm VLPs characterized by significantly enhanced structural stability and improved immunogenicity. In order to achieve quantitative recovery of HPV VLPs, the disassembly/reassembly process was further optimized by use of high ionic strength (>0.5 M sodium chloride) to prevent aggregation of VLPs. The reassembled VLPs possess an architectural structure very similar to that of the natural HPV virus particles. This development illustrates how the natural, in vivo mechanisms facilitating cell entry and virus replication can be utilized to achieve an optimal, in vitro assembly state of yeast-expressed HPV VLPs.     

Analytical and Preformulation Characterization Studies of Human Papillomavirus Virus-Like Particles to Enable Quadrivalent Multi-Dose Vaccine Formulation Development

Introducing multi-dose formulations of Human Papillomavirus (HPV) vaccines will reduce costs and enable improved global vaccine coverage, especially in low- and middle-income countries. This work describes the development of key analytical methods later utilized for HPV vaccine multi-dose formulation development. First, down-selection of physicochemical methods suitable for multi-dose formulation development of four HPV (6, 11, 16, and 18) Virus-Like Particles (VLPs) adsorbed to an aluminum adjuvant (Alhydrogel®, AH) was performed. The four monovalent AH-adsorbed HPV VLPs were then characterized using these down-selected methods. Second, stability-indicating competitive ELISA assays were developed using HPV serotype-specific neutralizing mAbs, to monitor relative antibody binding profiles of the four AH-adsorbed VLPs during storage. Third, concentration-dependent preservative-induced destabilization of HPV16 VLPs was demonstrated by addition of eight preservatives found in parenterally administered pharmaceuticals and vaccines, as measured by ELISA, dynamic light scattering, and differential scanning calorimetry. Finally, preservative stability and effectiveness in the presence of vaccine components were evaluated using a combination of RP-UHPLC, a microbial growth inhibition assay, and a modified version of the European Pharmacopoeia assay (Ph. Eur. 5.1.3). Results are discussed in terms of analytical challenges encountered to identify and develop high-throughput methods that facilitate multi-dose formulation development of aluminum-adjuvanted protein-based vaccine candidates.