大肠杆菌来源的人乳头瘤病毒11型病毒样颗粒的制备及其免疫原性

摘要：【目的】 利用大肠杆菌表达系统制备人乳头瘤病毒11型病毒样颗粒（HPV11 VLPs）,并对其免疫原性和所诱导中和抗体的型交叉反应性进行研究。 【方法】 在大肠杆菌ER2566中非融合表达HPV11-L1蛋白,并通过离子交换层析,疏水相互作用层析其进行纯化。纯化后的HPV11-L1经体外组装形成病毒样颗粒,通过动态光散射,透射电镜检测其形态,并通过多种HPV型别假病毒中和实验评价HPV11 VLPs的免疫原性及型交叉反应性。 【结果】 HPV11-L1蛋白在大肠杆菌中可以以可溶形式表达。经过硫酸铵沉     

人乳头瘤病毒52型病毒样颗粒制备及其生物活性

[目的]利用大肠杆菌系统表达人乳头瘤病毒52型(HPV52) L1蛋白,并获得HPV52病毒样颗粒(VLPs)。[方法]优化并合成HPV52L1基因,构建p ET-30a-52L1表达载体粒,转化大肠杆菌E. coli BL21 StarTM(DE3)。经IPTG诱导表达,阳离子交换层析纯化,SDS-PAGE和Western Blotting鉴定,BALB/c小鼠免疫2次,初次后4w检测免疫血清中HPV52中和抗体滴度。[结果]获得纯度90%的HPV52L1蛋白,将获得的HPV52L1蛋白进行解组装和重组装形成VLPs,动态光散射观察到粒径约为70nm,透射电镜观察到50～60nm的均一VLPs,中和抗体滴度达25600。[结论]大肠杆菌系统表达人乳头瘤病毒52型(HPV52) L1蛋白,并经过一步柱层析纯化获得纯度达90%的L1蛋白,经解组装重组装获得粒径大小为50～60nm的VLPs,具有良好免疫原性。     

人乳头瘤病毒18型L1蛋白在大肠埃希菌中的可溶性表达及病毒样颗粒的形成

目的利用大肠埃希菌系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,纯化和重组装获得HPV18病毒样颗粒(VLPs),为进一步研制HPV18基因工程疫苗奠定基础。方法首先按大肠埃希菌密码子偏好进行HPV18L1全基因合成,经PCR扩增出截短的HPV18L1基因,构建重组表达载体PET30a-L1,通过优化表达在大肠埃希菌BL21中可溶性表达L1蛋白,其次采用硫酸铵沉淀、离子交换层析、疏水层析后,获得高纯度的的L1蛋白,再通过解聚和重聚获得VLPs。结果全基因优化并截短的HPV18L1蛋白在大肠埃希菌系统中以可溶形式表达,纯化后的蛋白纯度达到90%以上,电镜下观察到直径为60 nm的VLPs颗粒。结论利用大肠埃希菌系统可溶性表达非融合HPV18L1蛋白,并获得均一的VLPs颗粒,为疫苗的开发奠定基础。     

人乳头瘤病毒16型病毒样颗粒的制备及其免疫原性研究

利用PCR技术从HPV16阳性阴道分泌物标本中获得HPV16 L1基因片段,并将其插入表达载体pTO-T7中,构建重组表达质粒pTO-T7-HPV16-L1;以该重组质粒转化大肠杆菌ER2566并表达HPV16 L1蛋白;所表达的HPV16 L1蛋白经过硫酸铵沉淀、离子交换层析和疏水相互作用层析等纯化步骤后,HPV16 L1纯度达到98%以上,并可在体外装配为直径50nm的病毒样颗粒;动物免疫原性研究结果显示,该病毒样颗粒可诱导高滴度的针对HPV16的中和抗体。上述研究结果表明通过大肠杆菌表达系统制备的HPV16病毒样颗粒具有纯度高,与天然病毒颗粒形态高度相似的特点,并具有高度免疫原性,可以应用于HPV16病毒样颗粒的结构功能研究及HPV16疫苗研发等领域。     

猪圆环病毒2型病毒样颗粒的高效组装技术研究*

目的:探索猪圆环病毒2型(PCV2)病毒样颗粒(VLPs)的高效组装技术,提高VLPs的稳定性。方法:利用大肠杆菌表达PCV2 Cap蛋白自组装为VLPs,分析不同离子强度下VLPs的稳定性。利用切向流技术添加尿素,降低pH,可使VLPs解组装,利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。结果:PCV2 Cap蛋白自组装VLPs在150mmol/L NaCl下稳定性较差,而在500mmol/L NaCl下可提高VLPs的稳定性,但仍较易发生聚集,核酸含量均较高。在150mmol/L NaCl、300mmol/L尿素和pH 5.5的缓冲体系条件下,能够使VLPs解组装。经25%~50%饱和硫酸铵(V/V)分级沉淀粗纯,阴离子交换层析500mmol/L NaCl下洗脱获得精纯Cap蛋白,蛋白质纯度≥95%,并能够有效去除核酸。通过切向流技术去除体系中的尿素,并将NaCl浓度提高至1mol/L、pH提高至8.0,改变蛋白质表面静电荷分布,实现VLPs的高效、均一再组装,组装效率≥99%,回收率为65.85%,并明显提高VLPs的稳定性,能够稳定保存6个月以上。结论:利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。     

乙肝核心抗原病毒样颗粒呈现HPV 16L1抗原表位及特异抗体诱导

目的:构建呈现HPV 16L1抗原表位的病毒样颗粒(VLPs),为新型HPV疫苗及特异抗体制备提供新的思路。方法:将编码HPV 16L1抗原表位QPLGVGISGHPLLNKLDDTE寡聚核苷酸片段克隆于HBc Ag基因编码第78、79位氨基酸序列之间,重组质粒转化大肠杆菌DH5α。重组蛋白经IPTG诱导后以SDS-PAGE分析表达情况,并以Western blot鉴定重组蛋白中HPV 16L1表位的免疫反应性。菌体超声破碎后经硫酸铵盐析法和蔗糖密度梯度离心进行纯化,并经凝胶层析Sepharose G25脱盐,最后以电子显微镜及高效液相(HPLC)凝胶过滤色谱鉴定VLPs的存在并分析纯度。纯化的病毒样颗粒分别于0周、2周、4周经皮下注射免疫BALB/c小鼠,以Western blot分析血清特异识别L1蛋白的能力。结果:HBc Ag/L1肽嵌合蛋白获得成功表达,能够被商业化L1抗体特异识别,经密度梯度超速离心及HPLC分析显示其与HBc Ag行为一致,电子显微镜观察进一步确定其以HBc Ag病毒样颗粒形式存在。VLPs免疫小鼠获得的抗血清能够特异识别酵母表达的重组L1蛋白。结论:HBc Ag VLPs成功呈现HPV16L1蛋白并有效激发特异抗体应答。     

人乳头瘤病毒18型L1蛋白的包涵体和可溶性表达及纯化

目的:原核表达系统表达人乳头瘤病毒18型(HPV18)L1蛋白,建立包涵体和可溶性表达的L1蛋白的纯化方法。方法:构建重组表达质粒p GEX-4T-1-HPV18 L1,在大肠杆菌BL21中以包涵体和可溶性方式表达HPV18 L1蛋白。通过超声波破碎菌体、洗涤包涵体、碱变性、透析复性和谷胱甘肽(GST)琼脂糖凝胶4B亲和层析纯化包涵体蛋白;在菌体中加入三磷酸腺苷(ATP)和3.5 mol/L尿素孵育后,GST 4B亲和层析纯化可溶性蛋白,凝血酶酶切。SDS-PAGE和Western印迹鉴定表达和纯化产物。结果:SDS-PAGE结果表明,HPV18 L1蛋白以包涵体和可溶性方式在大肠杆菌BL21内高效表达,均产生相对分子质量约为86 000的HPV18 L1-GST融合蛋白。Western印迹结果显示,包涵体纯化后获得的融合蛋白降解条带较多;而可溶性蛋白纯化后获得的融合蛋白未降解,凝血酶酶切后得到HPV18 L1蛋白,可与HPV18 L1蛋白单克隆抗体结合。结论:采用原核系统表达了HPV18 L1-GST融合蛋白,分别建立了包涵体和可溶性蛋白的纯化方法,获得HPV18 L1蛋白,为其进一步应用奠定了基础。     

原核表达系统制备HPV58L1 VLP可诱发高滴度且持久的中和抗体

目的:采用大肠杆菌表达系统制备人乳头瘤病毒58型(human papillomavirus type 58,HPV58)病毒样颗粒(virus-like particle,VLP)疫苗。方法:合成法获得HPV58 L1大肠杆菌密码子优化基因,构建HPV58 L1重组原核表达质粒mpET22b/HPV58 L1,检测其在BL21(DE3)中表达水平,饱和硫酸铵沉淀加阳离子交换层析法纯化蛋白后进行动态光散射(dynamic light scatter,DLS)分析。小鼠免疫后,检测免疫血清针对HPV58假病毒的中和抗体水平。结果:HPV58 L1蛋白在BL21(DE3)细胞中大部分以可溶形式表达,纯化获得的HPV58 L1蛋白可组装成水动力学直径约为74 nm的VLP。0.5μg的HPV58 L1 VLP可诱发小鼠产生高滴度的HPV58特异性中和抗体,可维持至少20周。结论:原核表达系统制备的HPV58 L1 VLP可诱发高滴度且持久的中和抗体,可用于成本低的HPV58疫苗的研究。     

A型塞内卡病毒的病毒样颗粒的制备及其免疫原性分析

为了研制A型塞内卡病毒 (Senecavirus A,SVA) 的病毒样颗粒 (Virus-like particles,VLPs) 疫苗,以SVA田间流行毒株CH-FJ-2017结构蛋白基因序列为研究对象,构建了能够同时表达SVA的3种结构蛋白VP0、VP1和VP3的单个原核重组表达质粒pET28a-SVA-VP031。通过大肠杆菌Escherichia coli表达、亲和层析纯化和体外自组装,获得SVA VLPs。透射电子显微镜鉴定显示,SVA的3种结构蛋白在体外能够自组装成直径约25–30 nm的VLPs,并且动物免疫试验结果表明,该VLPs能够有效刺激豚鼠产生高水平的抗原特异性中和抗体。上述研究结果为SVA VLPs疫苗的研制奠定了基础。     

HPV16 L2肽段偶联AP205 VLP的原核表达及免疫原性分析

目的:将编码HPV16衣壳蛋白L2的65～71、112～120免疫优势表位连接到RNA噬菌体衣壳蛋白AP205氮端,组装形成病毒样颗粒,通过在大肠杆菌中实现表达及纯化,对其免疫原性进行研究。方法:合成编码AP205衣壳蛋白基因和HPV16 L2的65～71、112～120位氨基酸表位的基因序列,PCR连接并克隆至pET30a(+)原核表达载体,构建重组表达质粒pET30-AP205-HPV16ΔL2,转化大肠杆菌BL21(DH3)感受态细胞,IPTG诱导表达。表达蛋白经凝胶层析纯化及SDS-PAGE、Western blot等理化性质检测,免疫接种ICR小鼠,通过间接ELISA法检测其免疫原性。结果:成功构建重组表达质粒,重组蛋白在大肠杆菌中以可溶性表达,透射电镜观察可见典型病毒样颗粒,该VLP在动物实验中表现出较好的免疫原性。结论:成功将HPV16 L2表位偶联AP205以形成VLP,在大肠杆菌中实现可溶性表达。     

A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2

Background

Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin.

Methodology/Principal Findings

L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV.

Conclusion/Significance

VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.     

Study of cell-mediated response in mice by HPV16 L1 virus-like particles expressed in Saccharomyces cerevisiae

The first vaccine against human papillomaviruses (HPV) formulated with HPV16 L1 virus-like particles (VLPs) produced in yeast was approved by the FDA in June 2006. Nevertheless, there have been few studies of the immunogenicity in mice of VLPs. In this study, we evaluated the cell-mediated immune response to VLPs produced in Saccharomyces cerevisiae. After immunization of mice with HPV16 L1 VLPs, we measured splenocytes proliferation and the levels of IFNgamma, IL2, IL4, and IL5. Splenocytes proliferation was significantly increased and a mixed Th1/Th2 response was indicated. IgG subtype immunoresponses were strongly induced and IgG1 titers were higher than those of IgG2a.     

Enhancement of vaccine potency by fusing modified LTK63 into human papillomavirus type 16 chimeric virus-like particles

To enhance the immunogenicity of human papillomavirus 16 (HPV 16) virus-like particles (VLPs), the modified adjuvant, mLTK63, was fused to the C-terminus of HPV 16 L2 protein. Coexpression of HPV 16 L1 and L2-mLTK63 proteins in insect cells led to the efficient assembly of HPV 16 L1/L2-mLTK63 chimeric VLPs (cVLPs), which combined the antigen and adjuvant as a unit. Compared with HPV 16 L1/L2 VLPs, the HPV 16 L1/L2-mLTK63 cVLPs had similar structural biology characteristics and binding activities with the cell surface receptors and HPV 16-specific neutralizing monoclonal antibodies. Intramuscular immunization of BALB/c mice with the HPV 16 L1/L2-mLTK63 cVLPs could induce higher titers of HPV 16-specific long-lasting neutralizing serum antibodies and stronger splenocyte proliferation, Th1- and Th2-type cytokines and CD4(+) Th responses than HPV 16 L1/L2 VLPs. The results suggested that it is possible to enhance the immunogenicity of HPV VLP vaccines via a strategy of fusing effective adjuvant protein into cVLPs.     

VLPs Displaying a Single L2 Epitope Induce Broadly Cross-Neutralizing Antibodies against Human Papillomavirus

Background

Virus-like Particles (VLPs) display can be used to increase the immunogenicity of heterologous antigens. Here, we report the use of a bacteriophage MS2-based VLP display platform to develop a monovalent vaccine targeting a broadly neutralizing epitope in the minor capsid protein human papillomavirus (HPV) that provides broad protection from diverse HPV types in a mouse pseudovirus infection model.

Methodology/Principal Findings

Peptides spanning a previously described cross-neutralizing epitope from HPV type 16 were genetically inserted at the N-terminus of MS2 bacteriophage coat protein. Three of the four recombinant L2-coat proteins assembled into VLPs. L2-VLPs elicited high-titer anti-L2 antibodies in mice, similar to recombinant VLPs that we had previously made in which the L2 peptide was displayed on a surface-exposed loop on VLPs of a related bacteriophage, PP7. Somewhat surprisingly, L2-MS2 VLPs elicited antibodies that were much more broadly cross-reactive with L2 peptides from diverse HPV isolates than L2-PP7 VLPs. Similarly, mice immunized with L2-MS2 VLPs were protected from genital and cutaneous infection by highly diverse HPV pseudovirus types.

Conclusion/Significance

We show that peptides can be displayed in a highly immunogenic fashion at the N-terminus of MS2 coat protein VLPs. A VLP-based vaccine targeting HPV L2 elicits broadly cross-reactive and cross-protective antibodies to heterologous HPV types. L2-VLPs could serve as the basis of a broadly protective second generation HPV vaccine.     

Papillomavirus capsid mutation to escape dendritic cell-dependent innate immunity in cervical cancer

Infection with oncogenic human papillomaviruses (HPVs), typified by HPV type 16 (HPV16), is a necessary cause of cervical cancer. Prophylactic vaccination with HPV16 L1 virus-like particles (VLPs) provides immunity. HPV16 VLPs activate dendritic cells and a potent neutralizing immunoglobulin G (IgG) response, yet many cervical cancer patients fail to generate detectable VLP-specific IgG. Therefore, we examined the role of the innate recognition of HPV16 L1 in VLP-induced immune responses and its evasion during carcinogenesis. Nonconservative mutations within HPV16 L1 have been described in isolates from cervical cancer and its precursor, high-grade cervical intraepithelial neoplasia (CIN). We determined the effect of mutations in L1 upon in vitro self-assembly into VLPs and their influence upon the induction of innate and adaptive immune responses in mice. Several nonconservative mutations in HPV16 L1 isolated from high-grade CIN or cervical carcinoma prevent self-assembly of L1 VLPs. Intact VLPs, but not assembly-defective L1, activate dendritic cells to produce proinflammatory factors, such as alpha interferon, that play a critical role in inducing adaptive immunity. Indeed, effective induction of L1-specific IgG1 and IgG2a was dependent upon intact VLP structure. Dendritic cell activation and production of virus-specific neutralizing IgG by VLPs requires MyD88-dependent signaling, although the L1 structure that initiates MyD88-mediated signaling is distinct from the neutralizing epitopes. We conclude that innate recognition of the intact L1 VLP structure via MyD88 is critical in the induction of high-titer neutralizing IgG. Tumor progression is associated with genetic instability and L1 mutants. Selection for assembly-deficient L1 mutations suggests the evasion of MyD88-dependent immune control during cervical carcinogenesis.     

Crystal structures of four types of human papillomavirus L1 capsid proteins: understanding the specificity of neutralizing monoclonal antibodies

Human papillomaviruses (HPVs) are known etiologic agents of cervical cancer. Vaccines that contain virus-like particles (VLPs) made of L1 capsid protein from several high risk HPV types have proven to be effective against HPV infections. Raising high levels of neutralizing antibodies against each HPV type is believed to be the primary mechanism of protection, gained by vaccination. Antibodies elicited by a particular HPV type are highly specific to that particular HPV type and show little or no cross-reactivity between HPV types. With an intention to understand the interplay between the L1 structure of different HPV types and the type specificity of neutralizing antibodies, we have prepared the L1 pentamers of four different HPV types, HPV11, HPV16, HPV18, and HPV35. The pentamers only bind the type-specific neutralizing monoclonal antibodies (NmAbs) that are raised against the VLP of the corresponding HPV type, implying that the surface loop structures of the pentamers from each type are distinctive and functionally active as VLPs in terms of antibody binding. We have determined the crystal structures of all four L1 pentamers, and their comparisons revealed characteristic conformational differences of the surface loops that contain the known epitopes for the NmAbs. On the basis of these distinct surface loop structures, we have provided a molecular explanation for the type specificity of NmAbs against HPV infection.