Neutralization of human papillomavirus (HPV) pseudovirions: a novel and efficient approach to detect and characterize HPV neutralizing antibodies

The development of vaccines against human papillomaviruses (HPVs) has long been hampered by the inability to grow HPVs in tissue culture and the lack of an efficient neutralization assay. To date, less than 10% of more than 100 different HPV types can be grown in athymic and "SCID" mouse xenograft systems or raft culture systems. Recently, the in vitro generation of HPV pseudovirions and their use in neutralization assays were demonstrated. The major shortcomings of the current approaches to HPV neutralization are the lack of HPV virions for most types for the xenograft methods and the time-consuming and inefficient generation of infective pseudovirions for the latter methods, which precludes their use in large-scale HPV clinical trials or epidemiological studies. We describe here a novel and efficient approach to generating pseudovirions in which HPV virus-like particles (VLPs) are coupled to the beta-lactamase gene as a reporter. We show that it is not necessary to encapsidate the reporter gene constructs into the pseudovirions. Using sera from human volunteers immunized with HPV-11 VLPs expressed in yeast, we demonstrate that our novel neutralization assay compares favorably with the athymic mouse neutralization assay. Furthermore, our assay was used to define neutralizing monoclonal antibodies to HPV-6, which were previously unknown.     

Measurement of the humoral immune response following an incident human papillomavirus type 16 or 18 infection in young women by a pseudovirion-based neutralizing antibody assay

We have evaluated a neutralizing antibody assay which uses human papillomavirus (HPV) type 16 (HPV-16) and HPV-18 pseudovirions carrying a secretory alkaline phosphatase reporter gene and which can potentially measure functionally relevant HPV type-specific neutralizing antibodies. The reproducibility of the assay was excellent; for HPV-16, the intra- and interassay kappa values were 0.95 and 0.90, respectively; and for HPV-18, the corresponding values were 0.90 and 0.90. This assay was used to describe the kinetics of the neutralizing antibody response in a cohort of 42 young women who were recruited soon after first intercourse and who first tested positive for HPV-16 DNA or HPV-18 DNA, or both, during follow-up. Most women seroconverted following the first detection of type-specific HPV DNA and remained seropositive until the end of follow-up. Our findings are broadly consistent with those of two other cohort studies which have measured the serological response following an incident infection by using the technically simpler virus-like-particle-based enzyme-linked immunosorbent assay.     

Binding and neutralization efficiencies of monoclonal antibodies, Fab fragments, and scFv specific for L1 epitopes on the capsid of infectious HPV particles

We compared the neutralization abilities of individual monoclonal antibodies (MAb) of two large panels reactive with L1 epitopes of HPV-11 or HPV-16. Binding titers were compared using both L1-only VLPs and L1/L2 pseudovirions. While the VLPs were antigenically similar to the pseudovirions, clear differences in the surface exposure of some epitopes were evident with the HPV-16 particles. To determine whether all antibody binding events are equivalent in their neutralizing effect on infectious HPV virions or pseudovirions, the binding and neutralization titers for individual MAbs were used to calculate the relative neutralization efficiency for each antibody. HPV neutralization was achieved by all MAbs capable of strong binding to either linear or conformation-sensitive epitopes on pseudovirus particles. Our data suggest, however, that some L1 epitopes may be more neutralization-sensitive than other surface epitopes, in that successful infection can be blocked by varying degrees of epitope saturation. Additionally, the effective neutralization of virions by several monovalent Fab fragments and single-chain variable fragments (scFv) demonstrates that viral neutralization does not require HPV particle aggregation or L1 crosslinking. Identification of capsid protein structures rich in neutralization-sensitive epitopes may aid in the development of improved recombinant vaccines capable of eliciting effective and long-term antibody-mediated protection against multiple HPV types.     

人16型乳头瘤病毒"假病毒"免疫保护作用的研究

目的评价构建的人16型乳头瘤病毒“假病毒”的免疫保护作用。方法利用杆状病毒表达系统在sf9昆虫细胞中表达组装了人16型乳头瘤病毒病毒样颗粒,将病毒样颗粒解聚后与真核表达质粒混合,重聚集成“假病毒”。用这种“假病毒”对小鼠进行免疫保护作用研究。结果小鼠经“假病毒”免疫后,可以在血清中检测到特异性的IgG,在阴道分泌物中检测到特异性的IgA,脾淋巴细胞可以检测特异性的CrrL活性。结论“假病毒”免疫能激活机体的免疫反应     

Induction of cross-neutralizing antibodies by sequential immunization with heterologous papillomavirus L1VLPs and its implications for HPV prophylactic vaccines

Sequential immunization with antigens from different strains of HIV-1, influenza viruses or dengue viruses induced cross-neutralizing antibodies and enhanced the antibody responses against previous antigens. The characteristics of neutralizing antibodies induced by sequential immunization with different types of human papillomavirus (HPV) L1 virus-like particles (L1VLPs) are unclear. In this study, mice were primed with one or two types (HPV-16 or HPV16/18) of L1VLPs, then boosted sequentially with HPV6/18/45/11/31/58 or HPV6/45/11/31/58 L1VLPs, and sera were analyzed with HPV pseudovirus-based neutralization assay. The results showed that neutralizing activities against earlier immunized vaccine types were enhanced gradually by subsequent immunizations, and low levels of neutralizing activities against nonvaccine types (HPV33/35/52/59/68) were also observed. After absorbing the immune sera with vaccine-type (HPV16/18/45) L1VLPs, neutralizing activities against tested priming and boosting types (HPV16/18/58) decreased significantly, and that against nonvaccine type (HPV-33) was also partially eliminated. Moreover, neutralizing activities against vaccine types (HPV16/58) were significantly reduced after absorbing with nonvaccine-type VLPs (HPV33/52). These data suggest that cross-neutralizing epitopes exist among different HPV L1VLPs. The cross-neutralizing activities against nonvaccine types and the enhanced neutralizing activities against earlier immunized vaccine types may result from sequential boosting with these cross-neutralizing epitopes. These observations support early vaccination with more types of L1VLPs derived from HPVs that cause a serious threat to the population.     

Human papillomavirus genotype spectrum in Czech women: correlation of HPV DNA presence with antibodies against HPV-16, 18, and 33 virus-like particles.

Because the biological spectrum of human papillomavirus (HPV) genotypes present in cervical cancer lesions varies according to the geographical region studied, and because little genotype information is available for Central and Eastern European countries, we studied the endemic HPV-genotype spectrum in cervical samples collected from women visiting gynaecological departments of selected hospitals in the Czech Republic. In a series of 389 samples, 171 (44.0%) were positive for HPV DNA using a consensus-primer polymerase chain reaction (PCR). Genotyping of the HPV PCR products was done using dot-blot hybridisation with type-specific oligonucleotide probes and thermocycle DNA sequencing. Twenty-two different HPV types were detected, HPV-16 being the most prevalent type irrespective of severity of the lesions (55.0%). Multiple HPV types were found in 16.4% of our HPV-DNA-positive samples. The prevalence of HPV infection was 23.0% in women with normal findings and 59.4% in patients with cervical neoplasia, and increased significantly with the severity of the disease: 52.9% in low-grade lesions, 58.0% in high-grade lesions, and 73.5% in cervical carcinomas (P for trend < .00001). In the sera of 191 subjects, 89 with normal findings and 102 with different forms of cervical neoplasia, the prevalence of HPV-specific IgG antibodies was tested by an enzyme-linked immunosorbent assay (ELISA) using virus-like particles (VLPs) of HPV-16, -18, and -33. Antibodies were significantly more prevalent in HPV-DNA-positive than in HPV-DNA-negative women and there was no association with age. In agreement with the results of HPV genotyping, antibodies reactive with HPV-16 VLPs were the most frequent and, moreover, their prevalence increased with the cervical lesion severity. About half of the subjects with smears in which either HPV-16 or HPV-33 DNA had been detected possessed antibodies reactive with homotypic VLPs. With HPV-18-DNA-positive subjects, however, fewer than 25% displayed homotypic antibodies. In general, subjects older than 30 years of age had antibodies reactive to HPV-specific VLPs more often than subjects younger than 30 years of age. In women with benign findings, the seropositivity to HPV-16, -18, and -33 VLPs increased with age, whereas in women with cervical neoplasia the seropositivity decreased with age.     

Hybrid papillomavirus L1 molecules assemble into virus-like particles that reconstitute conformational epitopes and induce neutralizing antibodies to distinct HPV types

Human papillomavirus (HPV) hybrid virus-like particles (VLPs) were prepared using complementary regions of the major capsid L1 proteins of HPV-11 and -16. These hybrid L1 proteins were tested for assembly into VLPs, for presentation and mapping of conformational neutralizing epitopes, and as immunogens in rabbits and mice. Two small noncontiguous hypervariable regions of HPV-16 L1, when replaced into the HPV-11 L1 backbone, produced an assembly-positive hybrid L1 which was recognized by the type-specific, conformationally dependent HPV-16 neutralizing monoclonal antibody (N-MAb) H16.V5. Several new N-MAbs that were generated following immunization of mice with wild-type HPV-16 L1 VLPs also recognized this reconstructed VLP, demonstrating that these two hypervariable regions collectively constituted an immunodominant epitope. When a set of hybrid VLPs was tested as immunogens in rabbits, antibodies to both HPV-11 and -16 wild-type L1 VLPs were obtained. One of the hybrid VLPs containing hypervariable FG and HI loops of HPV-16 L1 replaced into an HPV-11 L1 background provoked neutralizing activity against both HPV-11 and HPV-16. In addition, conformationally dependent and type-specific MAbs to both HPV-11 and HPV-16 L1 VLP were obtained from mice immunized with hybrid L1 VLPs. These data indicated that hybrid L1 proteins can be constructed that retain VLP-assembly properties, retain type-specific conformational neutralizing epitopes, can map noncontiguous regions of L1 which constitute type-specific conformational neutralizing epitopes recognized by N-MAbs, and trigger polyclonal antibodies which can neutralize antigenically unrelated HPV types.     

Cell mediated immunity induced in mice by HPV 16 L1 virus-like particles

Recombinant human papillomavirus (HPV) type 16 L1 virus-like particles (VLPs) expressed in the baculovirus system were used to investigate the cellular immune response to human papillomavirus type 16. The cell-mediated immune response was evaluated through immunization of mice with HPV 16 L1 virus-like particles using a lymphoproliferation assay and cytokine production and cytometric analysis of lymphocyte subsets. A significant proliferative response was observed which was associated with secretion of both interferon-γ and interleukin-2. FACS analysis of splenic lymphocytes revealed that CD8⁺T-cells were increased in the immunized mice. These results demonstrate that HPV 16 L1 VLPs induce a T-cell response characterized by a Th1 profile and confirm that the HPV 16 VLP is a reasonable candidate for vaccine development.     

Detection and differentiation of human papillomavirus genotypes HPV-6 and HPV-11 by FRET-based real-time PCR

A real-time PCR (RT-PCR) assay was developed based on fluorescence resonance energy transfer (FRET) hybridization probe technology, allowing very sensitive and specific detection of HPV-6 and HPV-11, reliable differentiation of HPV-6 and HPV-11, as well as prototypic and non-prototypic HPV-6 genomic variants, in a single PCR reaction. The primers and probe were designed on the basis of multiple alignments of 74 HPV-6 E2 gene sequences and 20 HPV-11 E2 gene sequences. Testing on defined plasmid standards showed that the RT-PCR allowed simple and reliable identification of HPV-6 and HPV-11 using type specific amplification followed by probe-specific post-amplification dissociation analysis. Sensitivity, assessed by probit analysis at a 95% detection level, was 42.9, 43.4, and 25.3 DNA copies per assay for prototypic and non-prototypic HPV-6 variants and HPV-11, respectively. The results obtained by the developed assay on 51 HPV DNA-negative samples and 149 HPV DNA-positive samples, including 81 HPV-6 positive samples (19 prototypic and 62 non-prototypic HPV-6 variants), 28 HPV-11 positive samples, 10 samples of HPV-44 and HPV-74 (the closest relatives of HPV-6 and HPV-11) and 30 samples of 15 other important alpha HPV, showed complete agreement with those obtained with the INNO-LiPA human papillomavirus (HPV) Genotyping Assay and HPV-6 E2 and E6 gene sequencing.     

NHPV16 VLP vaccine induces human antibodies that neutralize divergent variants of HPV16

Genital HPV genotypes are generally distinct serotypes, but whether variants within a genotype can represent serologic subtypes is unclear. In this study we used serum from human volunteers vaccinated with HPV16 L1 VLPs from variant 114K, to examine cross-neutralization of variants from each of the five major phylogenetic branches of HPV16. Recombinant Semliki Forest virus-derived pseudovirions for each variant were generated and combined with serum from vaccines, and the mixture was monitored for infectivity in a standard C127 cell focal transformation assay. Sera from all 10 VLP-immunized individuals had neutralizing activity against each of the variant pseudovirions. For each of the sera, variant titers differed by only fourfold or less from the median titer. Therefore, from a vaccine perspective, HPV16 variants belong to a single serotype. Vaccination with HPV16 114K L1 VLPs generates antibodies that should confer a similar degree of protection against all known phylogenetic branches of HPV16.     

Genital human papillomavirus genotyping by HPV oligonucleotide microarray in Korean commercial sex workers

Because of the diversity in human papillomavirus (HPV) distribution, according to the population and region, detailed investigations of HPV genotypes are important in designing more effective HPV vaccines for any given country. HPV DNA oligonucleotide microarray was used to investigate the distribution of HPV genotypes among commercial sex workers. The prevalence of HPV in Korean commercial sex workers was 47%, with HPV-16 and HPV-51 as the dominant genotypes. HPV subtypes in 148 commercial sex workers comprised 70 with one genotype, 42 with two genotypes, 17 with three genotypes, and 19 with four or more genotypes. HPV-40, the most dominant low-risk genotype, was not detected in single-infection commercial sex workers. All women with multiple infections of low-risk genotypes had the HPV-40 genotype. This molecular epidemiological study of genital HPV will be useful for the development of a favorable strategy to prevent the spread of this potentially serious infection.     

Induction of productive human papillomavirus type 11 life cycle in epithelial cells grown in organotypic raft cultures

The study of the human papillomavirus (HPV) life cycle was hampered for more than 50 years by the lack of a conventional cell culture system for propagating HPV. Considerable progress has been made in the production of several HPV types using either organotypic rafts or human epithelial xenografts in immunocompromised mice. In this study, we demonstrated episomal maintenance of HPV-11 DNA in N-Tert cells. HPV-11 episomal DNA containing cell populations grown in raft culture showed induction of the productive viral life cycle. HPV-11 DNA amplification and viral capsid antigen synthesis were detected in differentiated layers of epithelia. The viruses generated were able to infect keratinocytes in vitro, which indicate that viruses generated were infectious. The demonstration of the productive HPV-11 life cycle in raft culture from cloned HPV-11 DNA will facilitate genetic analyses of viral gene functions that was not possible using the human xenograft athymic mouse model.     

The L1 Major Capsid Protein of Human Papillomavirus Type 16 Variants Affects Yield of Virus-Like Particles Produced in an Insect Cell Expression System

The L1 major capsid proteins of six human papillomavirus type 16 (HPV-16) strains were expressed in insect cells by using recombinant baculoviruses. Virus-like particles (VLPs) which appeared similar to empty virions were identified by electron microscopy for all HPV strains investigated. However, the yield of VLPs produced varied in a range from 1 to 79 depending on the HPV-16 strain. The L1 proteins of these strains differed by up to 15 amino acids from the L1 protein of the prototype HPV-16 strain. Mutations in the amino acid region from residues 83 to 97 seemed to affect the level of expression of the L1 protein. These results are important when considering the development of HPV vaccines and serological tests. They indicate that strains inducing high levels of VLP production must be selected for the development of vaccines. Moreover, the L1 proteins of all strains investigated were able to bind with DNA. We also investigated the seroreactivities of VLPs derived from three different HPV-16 strains from Algeria, Senegal, and the Philippines by testing sera from women from 11 countries in immunoglobulin G-specific enzyme-linked immunosorbent assays. We observed a strong correlation between the reactivities of the three different VLP variants, independent of the geographical origin of the sera investigated. These results indicate that the three strains investigated are serologically cross-reactive despite the fact that their L1 proteins differ in 14 amino acids and suggest that VLPs derived from only one HPV-16 strain could be sufficient for the development of an HPV-16 vaccine and anti-HPV-16 tests.     

高危型HPV感染与食管鳞癌组织中HLA-G表达的相关性研究

目的研究人乳头瘤状病毒（HPV）在新乡地区食管鳞癌、癌旁组织中的感染情况和人类白细胞抗原系统G（HLA-G）表达的关系。方法采用基因芯片技术检测114例食管鳞癌组织及癌旁食管正常组织中HPV亚型感染情况,免疫组化SP法检测HLA-G的表达。结果 114例食管鳞癌组织及癌旁食管正常组织中HPV感染阳性率分别为63.2%和6.0%,HLA-G的检出率分别为51.8%、0%,差异均有统计学意义（P〈0.05）;食管鳞癌存在HPV16、18和52三种亚型感染,其中HPV16感染率最高,HPV52感染率最低。结论新乡地区食管鳞癌组织以HPV16、18和52亚型感染为主;HPV感染和HLA-G蛋白表达与食管鳞癌分化程度正相关;HPV感染食管鳞癌组织与HLA-G蛋白表达存在一定的相关性,可能提示与食管鳞癌发生有较密切关系。     

HPV infections and tonsillar carcinoma

Since human papillomavirus (HPV) was first linked to laryngeal/oral carcinomas in 1983, several studies have confirmed its causal role in a subgroup of upper aerodigestive tract tumours. Of the non-genital cancers, tonsillar carcinomas (TCs) have the strongest association with HPV. By the end of 2002, 432 TCs had been analysed for HPV DNA. Overall detection rate was 51%, with HPV-16 being the most prevalent (84%). The original proposal that HPV-33 would be the most frequent HPV in TCs has not been confirmed, being present in only 4.6% of cases. HPV copy numbers are similar to those found in genital carcinomas (10-300 copies/cell), although HPV is mainly episomal in TC. The importance of this observation is unclear, although a role for subepithelial proliferative lymphatic tissue has been speculated. Patients with HPV-16 positive tumours have better overall and disease specific survival than HPV negative patients. They are also younger and the association with conventional risk factors-smoking and drinking-is less significant than in HPV negative patients. Thus, recent data suggest a distinct pattern for HPV-16 positive TCs.     

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6–8 kb. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Single-cell cytokine analysis allows detection of cervical T-cell responses against human papillomavirus type 16 L1 in women infected with genital HPV

Specific types of human papillomavirus (HPV) are known to play a causal role in the development of cervical cancer, with human papillomavirus type 16 (HPV-16) identified as the predominant type. Despite this, little is known about cervical immune responses to this pathogen. The aim of this study was to assess the feasibility of cervical cytobrush sampling and single-cell cytokine staining to investigate cervical lymphocyte-specific cytokine responses to HPV-16 antigens. Of eighteen women recruited into the study, five were HPV DNA positive at the cervix (current exposure) and a further five had circulating antibodies to HPV-16 (previous exposure). Cervical lymphocytes, isolated from the five HPV DNA-positive women, two HPV DNA-negative controls, and one woman with circulating HPV-16 antibodies were assessed for HPV-specific responses using intracellular staining for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). We demonstrate that both CD4(+) and CD8(+) cervical T lymphocytes, harvested from noninfected and infected subjects, produce these cytokines in response to nonspecific stimulation. However, antigen-specific (HPV-16 L1) IFN-gamma production by CD4(+) and CD8(+) cervical T lymphocytes is only detectable in women exposed currently or previously to HPV-16. This is the first time that antigen-specific cytokine responses of mucosal lymphocytes, obtained from a site of HPV infection, have been demonstrated. This finding clearly illustrates the use of intracellular cytokine staining for investigation of low precursor frequency single-cell antigen-specific responses in lymphocytes harvested from mucosal sites with HPV infection.     

Detection of human papillomavirus types 45 and 51 by type-specific polymerase chain reaction

Human papillomavirus (HPV) types 45 and 51 are both considered as high risk types for the development of human cervical cancer. To optimize the detection of these two types in clinical samples, HPV-45 and HPV-51 specific primers were designed to amplify respectively a 141bp and a 266bp fragment from the L1 gene by polymerase chain reaction (PCR). The sensitivity and the specificity of these two PCR reactions were determined using varying amounts of HPV DNA containing plasmids and negative and positive controls. Overall, the sensitivity for the HPV-45 plasmid DNA is 10fg, while for HPV-51 the sensitivity is 1fg. This is equivalent to approximately 100 and 10 HPV genome copies per PCR reaction, respectively.     

Modification of human papillomavirus-like particle vaccine by insertion of the cross-reactive L2-epitopes

Infection with human papillomavirus 16 (HPV16), which is one of the 15 types of HPV causally associated with cervical cancer and accounts for 50% of the cases, can be prevented in a type-specific manner by an HPV16 virus-like particle (VLP) vaccine comprised of particles of the L1 protein alone. We attempted to modify the VLP vaccine by inserting the HPV16 L2-peptides including cross-neutralization epitopes into the L1 polypeptide. The chimeric L1 had, between L1 amino acids (aa) 430 and 433, the L2 sequence of aa 18-38, 56-75, or 96-115 (with the replacements of S at aa 101 and T at aa 112 with L and S, respectively). The three chimeric L1s were each expressed from the recombinant baculovirus in insect Sf9 cells, and the resultant VLPs were characterized. The chimeric VLPs were shown to present the L2-peptides on their surface. By immunizing rabbits with the VLPs, it was shown that they retained capability to induce the antibody neutralizing HPV16 and acquired capability to elicit antibodies cross-neutralizing the infectious HPV18, 31, 52, and 58 pseudovirions. Although the cross-neutralizing titers were lower than the type-specific neutralizing titer, the results suggest that the chimeric VLPs have potential to serve as a vaccine candidate for a broad spectrum of high-risk HPVs.     

Role of DC-SIGN in the activation of dendritic cells by HPV-16 L1 virus-like particle vaccine

Dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN), a specific C-type lectin expressed on DC, binds and transmits different pathogens to susceptible cells. In the present study, we examined the role of DC-SIGN in the capture of human papillomavirus (HPV) pseudovirions and activation of DC. We demonstrate that HPV virus-like particles (VLP) bind to DC-SIGN expressed on transfected Raji cells and that antibodies against DC-SIGN block this interaction. DC take up VLP, which activate expression of costimulatory markers and cytokines/chemokines. Although our results indicate that DC-SIGN is not the major receptor for VLP in DC, this interaction contributes to the activation of DC surface antigens (HLA class I) and of various cytokines/chemokines, particularly TNF-alpha, IL-6, and RANTES. Induction of these markers in DC by VLP was significantly abrogated when binding to DC-SIGN was blocked by anti-DC-SIGN antibodies. These results suggest that DC-SIGN has a functional role in DC activation induced by HPV-16 L1-VLP, and thus highlight new aspects of DC interactions with HPV VLP.