Gene transfer using human papillomavirus pseudovirions varies according to virus genotype and requires cell surface heparan sulfate

Artificial viruses consisting of DNA plasmid packaged in vitro into virus-like particles (VLPs) are new vehicles for gene transfer. We therefore investigated the ability of nine human papillomavirus (HPV) VLPs to interact with heterologous DNA and transfer genes. HPV 16, 18, 31, 33, 39, 45, 58, 59, and 68 VLPs were able to bind heterologous DNA and to transfer genes into Cos-7 cells. Inhibition of gene transfer by preincubation of the pseudovirions with heparin confirmed that heparan sulfate on the cell surface plays a role as cell receptor for HPVs. As HPV neutralizing antibodies are mainly type-specific, gene transfer with different HPV pseudovirions offers the possibility of their sequential use in vivo for a greater efficacy.     

Development of AAVLP(HPV16/31L2) particles as broadly protective HPV vaccine candidate

The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17-36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2--simulating the high prevalence of AAV2 antibodies in the population--even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected na?ve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.     

Identification of neutralizing conformational epitopes on the human papillomavirus type 31 major capsid protein and functional implications

The aim of this study was to characterize the conformational neutralizing epitopes of the major capsid protein of human papillomavirus type 31. Analysis of the epitopes was performed by competitive epitope mapping using 15 anti‐HPV31 and by reactivity analysis using a HPV31 mutant with an insertion of a seven‐amino acid motif within the FG loop of the capsid protein. Fine mapping of neutralizing conformational epitopes on HPV L1 was analyzed by a new approach using a system displaying a combinatorial library of constrained peptides exposed on E. coli flagella. The findings demonstrate that the HPV31 FG loop is dense in neutralizing epitopes and suggest that HPV31 MAbs bind to overlapping but distinct epitopes on the central part of the FG loop, in agreement with the exposure of the FG loop on the surface of HPV VLPs, and thus confirming that neutralizing antibodies are mainly located on the tip of capsomeres. In addition, we identified a crossreacting and partially crossneutralizing conformational epitope on the relatively well conserved N‐terminal part of the FG loop. Moreover, our findings support the hypothesis that there is no correlation between neutralization and the ability of MAbs to inhibit VLP binding to heparan sulfate, and confirm that the blocking of virus attachment to the extracellular matrix is an important mechanism of neutralization.     

Identification of a human papillomavirus type 16-specific epitope on the C-terminal arm of the major capsid protein L1

To characterize epitopes on human papillomavirus (HPV) virus-like particles (VLPs), a panel of mutated HPV-16 VLPs was created. Each mutated VLP had residues substituted from HPV-31 or HPV-52 L1 sequences to the HPV-16 L1 backbone. Mutations were created on the HPV-31 and -52 L1 proteins to determine if HPV-16 type-specific recognition could be transferred. Correct folding of the mutated proteins was verified by resistance to trypsin digestion and by binding to one or more conformation-dependent monoclonal antibodies. Several of the antibodies tested were found to bind to regions already identified as being important for HPV VLP recognition (loops DE, EF, FG, and HI). Sequences at both ends of the long FG loop (amino acids 260 to 290) were required for both H16.V5 and H16.E70 reactivity. A new antibody-binding site was discovered on the C-terminal arm of L1 between positions 427 and 445. Recognition of these residues by the H16.U4 antibody suggests that this region is surface exposed and supports a recently proposed molecular model of HPV VLPs.     

Identification of human papillomavirus type 16 L1 surface loops required for neutralization by human sera

The variable surface loops on human papillomavirus (HPV) virions required for type-specific neutralization by human sera remain poorly defined. To determine which loops are required for neutralization, a series of hybrid virus-like particles (VLPs) were used to adsorb neutralizing activity from HPV type 16 (HPV16)-reactive human sera before being tested in an HPV16 pseudovirion neutralization assay. The hybrid VLPs used were composed of L1 sequences of either HPV16 or HPV31, on which one or two regions were replaced with homologous sequences from the other type. The regions chosen for substitution were the five known loops that form surface epitopes recognized by monoclonal antibodies and two additional variable regions between residues 400 and 450. Pretreatment of human sera, previously found to react to HPV16 VLPs in enzyme-linked immunosorbent assays, with wild-type HPV16 VLPs and hybrid VLPs that retained the neutralizing epitopes reduced or eliminated the ability of sera to inhibit pseudovirus infection in vitro. Surprisingly, substitution of a single loop often ablated the ability of VLPs to adsorb neutralizing antibodies from human sera. However, for all sera tested, multiple surface loops were found to be important for neutralizing activity. Three regions, defined by loops DE, FG, and HI, were most frequently identified as being essential for binding by neutralizing antibodies. These observations are consistent with the existence of multiple neutralizing epitopes on the HPV virion surface.     

Immunogenicity of Bivalent Human Papillomavirus DNA Vaccine Using Human Endogenous Retrovirus Envelope-Coated Baculoviral Vectors in Mice and Pigs

Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.     

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.     

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。     

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.     

Identification of two cross-neutralizing linear epitopes within the L1 major capsid protein of human papillomaviruses

The neutralizing activities of polyclonal antibodies and monoclonal antibodies (MAbs) obtained by immunization of mice with L1 virus-like particles (VLPs) were investigated by using pseudovirion infectivity assays for human papillomavirus type 16 (HPV-16), HPV-31, HPV-33, HPV-45, HPV-58, and HPV-59 to obtain a better definition of cross-neutralization between high-risk HPVs. In this study, we confirmed and extended previous studies indicating that most genital HPV genotypes represent separate serotypes, and the results suggest that the classification of serotypes is similar to that of genotypes. In addition, three cross-neutralizing MAbs were identified (HPV-16.J4, HPV-16.I23, and HPV-33.E12). MAb HPV-16.J4 recognized a conserved linear epitope located within the FG loop of the L1 protein, and HPV-16.I23 recognized another located within the DE loop. The results suggested that reactivity of MAb HPV-16.I23 to L1 protein is lost when leucine 152 of the HPV-16 L1 protein is replaced by phenylalanine. This confirmed the existence of linear epitopes within the L1 protein that induce neutralizing antibodies, and this is the first evidence that such linear epitopes induce cross-neutralization. However, the cross-neutralization induced by L1 VLPs represents less than 1% of the neutralizing activity induced by the dominant conformational epitopes, and it is questionable whether this is sufficient to offer cross-protection in vivo.     

Human papillomavirus type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice

We analyzed capsomeres of human papillomavirus type 16 (HPV16) consisting of the L1 major structural protein for their ability to trigger a cytotoxic T-cell (CTL) response. To this end, we immunized C57BL/6 mice and used the L1(165-173) peptide for ex vivo restimulation of splenocytes prior to analysis ((51)Cr release assay and enzyme-linked immunospot assay [ELISPOT]). This peptide was identified in this study as a D(b)-restricted naturally processed CTL epitope by HPV16 L1 sequence analysis, major histocompatibility complex class I binding, and (51)Cr release assays following immunization of C57BL/6 mice with HPV16 L1 virus-like particles (VLPs). HPV16 L1 capsomeres were obtained by purification of HPV16 L1 lacking 10 N-terminal amino acids after expression in Escherichia coli as a glutathione S-transferase fusion protein (GST-HPV16 L1 Delta N10). Sedimentation analysis revealed that the majority of the purified protein consisted of pentameric capsomeres, and assembled particles were not observed in minor contaminating higher-molecular-weight material. Subcutaneous (s.c.) as well as intranasal immunization of C57BL/6 mice with HPV16 L1 capsomeres triggered an L1-specific CTL response in a dose-dependent manner as measured by ELISPOT and (51)Cr release assay. Significant reduction of contaminating bacterial endotoxin (lipopolysaccharide) from the capsomere preparation did not diminish the immunogenicity. Antibody responses (serum and vaginal) were less robust under the experimental conditions employed. In addition, s.c. vaccination with HPV16 L1 capsomeres induced regression of established tumors expressing L1 determinants (C3 tumor cells). Our data demonstrate that capsomeres are potent inducers of CTL responses similar to completely assembled T=7 VLPs. This result is of potential relevance for the development of (combined prophylactic and therapeutic) HPV-specific vaccines, since capsomeres can be produced easily and also can be modified to incorporate heterologous sequences such as early HPV proteins.     

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     

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.     

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.     

Immunogenic assessment of plant‐produced human papillomavirus type 16 L1/L2 chimaeras

Cervical cancer is caused by infection with human papillomaviruses (HPV) and is a global concern, particularly in developing countries, which have ~80% of the burden. HPV L1 virus‐like particle (VLP) type–restricted vaccines prevent new infections and associated disease. However, their high cost has limited their application, and cytological screening programmes are still required to detect malignant lesions associated with the nonvaccine types. Thus, there is an urgent need for cheap second‐generation HPV vaccines that protect against multiple types. The objective of this study was to express novel HPV‐16 L1‐based chimaeras, containing cross‐protective epitopes from the L2 minor capsid protein, in tobacco plants. These L1/L2 chimaeras contained epitope sequences derived from HPV‐16 L2 amino acid 108–120, 56–81 or 17–36 substituted into the C‐terminal helix 4 (h4) region of L1 from amino acid 414. All chimaeras were expressed in Nicotiana benthamiana via an Agrobacterium‐mediated transient system and targeted to chloroplasts. The chimaeras were highly expressed with yields of ~1.2 g/kg plant tissue; however, they assembled differently, indicating that the length and nature of the L2 epitope affect VLP assembly. The chimaera containing L2 amino acids 108–120 was the most successful candidate vaccine. It assembled into small VLPs and elicited anti‐L1 and anti‐L2 responses in mice, and antisera neutralized homologous HPV‐16 and heterologous HPV‐52 pseudovirions. The other chimaeras predominantly assembled into capsomeres and other aggregates and elicited weaker humoral immune responses, demonstrating the importance of VLP assembly for the immunogenicity of candidate vaccines.     

The choice of resin-bound ligand affects the structure and immunogenicity of column-purified human papillomavirus type 16 virus-like particles

Cell growth conditions and purification methods are important in determining biopharmaceutical activity. However, in studies aimed at manufacturing virus-like particles (VLPs) for the purpose of creating a prophylactic vaccine and antigen for human papillomavirus (HPV), the effects of the presence of a resin-bound ligand during purification have never been investigated. In this study, we compared the structural integrity and immunogenicity of two kinds of VLPs derived from HPV type 16 (HPV16 VLPs): one VLP was purified by heparin chromatography (hHPV16 VLP) and the other by cation-exchange chromatography (cHPV16 VLP). The reactivity of anti-HPV16 neutralizing monoclonal antibodies (H16.V5 and H16.E70) towards hHPV16 VLP were significantly higher than the observed cHPV16 VLP reactivities, implying that hHPV16 VLP possesses a greater number of neutralizing epitopes and has a greater potential to elicit anti-HPV16 neutralizing antibodies. After the application of heparin chromatography, HPV16 VLP has a higher affinity for H16.V5 and H16.E70. This result indicates that heparin chromatography is valuable in selecting functional HPV16 VLPs. In regard to VLP immunogenicity, the anti-HPV16 L1 IgG and neutralizing antibody levels elicited by immunizations of mice with hHPV16 VLPs were higher than those elicited by cHPV16 VLP with and without adjuvant. Therefore, the ability of hHPV16 VLP to elicit humoral immune responses was superior to that of cHPV16 VLP. We conclude that the specific chromatographic technique employed for the purification of HPV16 VLPs is an important factor in determining the structural characteristics and immunogenicity of column-purified VLPs.     

人乳头瘤病毒18型病毒样颗粒在大肠杆菌中的表达及免疫原性分析

利用大肠杆菌表达系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,经过纯化和重组装过程获得HPV18病毒样颗粒(VLPs),研究其免疫原性和诱发中和抗体生成的水平。首先,提取HPV18的基因组DNA,通过PCR扩增获得HPV18 L1基因片段,将其插入pTrxFus表达载体,在大肠杆菌中可溶性表达HPV18 L1蛋白;其次,通过硫酸铵沉淀、离子交换层析和疏水相互作用层析获得高纯度的HPV18 L1蛋白,而后透析去除预先加入的还原剂DTT,使HPV18 L1蛋白自发组装成VLPs;最后,通过动态光散射技术和透射电子显微镜鉴定HPV18 VLPs的大小和形态,利用假病毒细胞中和实验评价HPV18 VLPs在实验动物体内的免疫原性和中和抗体生成水平。结果表明,HPV18L1蛋白可以在大肠杆菌表达系统中以可溶形式表达,经过纯化的HPV18 L1蛋白可以自发组装成为半径约为29.34nm、与HPV病毒外观相似的VLP。该VLPs在小鼠体内的中和抗体半数有效剂量为0.006μg,在兔及山羊体内诱导中和抗体滴度高达107。总之,本研究利用原核表达系统可简便高效地获得具有高度免疫原性的HPV18 VLPs,为HPV18...     

Targeting the vaginal mucosa with human papillomavirus pseudovirion vaccines delivering simian immunodeficiency virus DNA

The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV. We tested the immunogenicity of a vaccine that uses human papillomavirus (HPV)-based gene transfer vectors, also called pseudovirions (PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA induced Gag-specific Abs in serum and the vaginal tract, and T cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIV(mac251.) HPV PsV-based vehicles are immunogenic, which warrant further testing as vaccine candidates for HIV and may provide a useful model to evaluate the benefits and risks of inducing high levels of SIV-specific immune responses at mucosal sites prior to SIV infection.