Identification of type-specific and cross-reactive neutralizing conformational epitopes on the major capsid protein of human papillomavirus type 31

Summary. The majority of the neutralizing epitopes of papillomaviruses (PV) are conformation-specific and have not been fully characterised. Studies have, to date, been limited to a few HPV types only. We analysed the epitopes on the major capsid protein (L1) of Human papillomavirus (HPV) type 31 using monoclonal antibodies (MAbs) generated against HPV-31 virus-like particles (VLPs). The type-specific MAbs against HPV-31 were all found to be neutralizing and recognized conformation-dependent epitopes. Two other MAbs directed against a conformational epitope were found to be cross-reactive with other HPV types, and one of them was found to be cross-neutralizing. Cross-reactive antibodies were further investigated using wild-type HPV-16 L1 VLPs and two mutants. The results obtained suggested the existence of a cross-neutralizing conformational epitope at the N-terminal part of the FG loop of the major capsid protein, and the other four cross-reactive MAbs recognized epitopes also located at the N-terminal part of the FG loop.     

Yeast coexpression of human papillomavirus types 6 and 16 capsid proteins

The L1 and L2 capsid proteins of animal and human papillomaviruses (HPVs) can self-assemble into virus-like particles (VLPs) that closely resemble native virions. The use of different animal models shows that VLPs can be very efficient at inducing a protective immune response. However, studies with infectious HPV virions and VLPs of different HPV types indicate that the immune response is predominantly type-specific. We have generated a diploid yeast strain that coexpresses the L1 and L2 capsid proteins of both HPV-6b and HPV-16, and we have purified fully assembled VLPs banding in a cesium chloride gradient at the expected density of 1.29-1.3 mg/ml. Experimental evidence strongly indicated that the four proteins coassembled into VLPs. Western blot analysis, using anti-HPV-6 and anti-HPV-16 L1-specific monoclonal antibodies and type-specific L2 antisera, demonstrated that all four proteins copurified. Most importantly, immunoprecipitation experiments, carried out using type-specific anti-L1 monoclonals and either total yeast cell extracts or purified VLPs, confirmed the interaction and the formation of covalent disulfide bonds between the two L1 proteins. Finally, HPV-6/16 VLPs administered to mice induced conformational antibodies against both L1 protein types. These results suggest that coexpression of different capsid proteins may provide new tools for the induction of antibodies directed against multiple HPV types.     

人乳头状瘤病毒16型结构蛋白L1和L2在大肠埃希菌 …

目的　研究人乳头状瘤病毒(HPV)主要结构蛋白L1(HPV16L1)和次要结构蛋白L2(HPV16L2)在原核系统的表达。方法　采用pET30a载体分别构建pET30aL1和pET30aL2质粒,并分别转入大肠埃希菌BL21菌株,经IPTG(异丙基硫代βD半乳糖苷)诱导,表达融合蛋白6×HisL1和6×HisL2,用SDSPAGE和Westernblot方法进行检测。结果　6×HisL1和6×HisL2融合蛋白在BL21菌中高效表达,约2～3mgml,其相对分子质量分别约为60000和97000;6×HisL1降解较6×HisL2多。结论　HPV16结构蛋白L1和L2能在原核表达系统高效表达;6×HisL2稳定性高于6×HisL1融合蛋白。     

Mucosal IgG and IgA responses to human papillomavirus type 16 capsid proteins in HPV16-infected women without visible pathology

Human papillomavirus type 16 (HPV16) may infect the cervical epithelium without producing pathological changes for a long time. To investigate if mucosal antibodies are induced in HPV16-infected women without visible pathology, cervical mucus from HPV16-infected patients with and without evident pathology, along with mucus from uninfected women were analyzed for the presence of mucosal IgG and secretory IgA (sIgA) antibodies to HPV16 capsid proteins by ELISA. sIgA and IgG antibodies were found in a significantly higher proportion of infected patients compared with uninfected women (p < 0.0001). sIgA antibodies were present in 13.1% of infected patients without visible pathology, the proportion of positivity increased to 27.0% in patients with visible pathology (p = 0.001). Mucosal IgG response was observed in 6.5% of patients without and 27.5% of patients with visible pathology (p = 0.00005). The antibody mean signal strength was significantly higher in patients with than in patients without pathological evidence (p < 0.005). In conclusion, both sIgA and IgG are found in patients without pathological signs of infection, however, the response increases significantly in patients with pathological evidence, suggesting that the appearance of these changes might be associated with a more vigorous antibody-mediated mucosal reaction.     

Different methods of identifying new antigenic epitopes of human papillomavirus type 16 E6 and E7 proteins

Human papillomavirus (HPV) infection is the most common cause of sexually transmitted viral infection and is the main cause of cervical cancer. Identification of HPV T-cell epitopes would be instrumental not only in our understanding of the protective immune response but also in the development of vaccines and immunotherapies. In contrast to viruses which cause systemic infection, identification of HPV epitopes is technically challenging because HPV causes a localized mucosal infection and the frequency of pathogen-specific T lymphocytes in peripheral blood is expected to be low. Here we describe three new antigenic epitopes (E7 7-15 [TLHEYMLDL], E6 52-61 [FAFRDLCIVY], and E7 79-87 [LEDLLMGTL]) of HPV 16 E6 and E7 proteins which have oncogenic activities. E7 7-15 was identified among peptides previously shown to bind to human leukocyte antigen (HLA)-A2.1 molecule, but it was found likely to be restricted by the HLA-B48 molecule. E6 52-61 (likely to be restricted by HLA-B57) and E7 79-87 (likely to be restricted by HLA-B60) were detected, based on the magnitude of the T-cell immune responses, in another individual. In particular, T-cell clones specific for the E6 52-61 epitope were isolated effectively by magnetically selecting them based on gamma interferon secretion. This is an efficient method of identifying new epitopes of antigens for which the number of specific T lymphocytes in the circulation is expected to be small, and it should be widely applicable in identifying new T-cell epitopes.     

Mutations on the FG surface loop of human papillomavirus type 16 major capsid protein affect recognition by both type-specific neutralizing antibodies and cross-reactive antibodies

The aim of this study was to further characterize the conformational neutralizing epitopes present on the surface-exposed FG loop of human papillomavirus (HPV) type 16 L1 major capsid protein. We have generated previously two chimeric L1 proteins by insertion of a foreign peptide encoding an epitope of the hepatitis B core (HBc) antigen within the FG loop. In addition, three other chimeric L1 proteins were obtained by replacing three different FG loop sequences by the HBc motif and three others by point mutations. All these chimeric L1 proteins retained the ability to self-assemble into virus-like particles (VLPs), with the exception of the mutant with substitution of the L1 sequence 274-279 by the HBc motif. The eight chimeric VLPs were then analyzed for differential reactivity with a set of six HPV-16 and HPV-31 monoclonal antibodies that bound to conformational and linear epitopes. The binding patterns of these monoclonal antibodies confirmed that the FG loop contained or contributed to neutralizing conformational epitopes. The results obtained suggested that the H31.F7 antibody, an anti-HPV-31 cross-reacting and neutralizing antibody, recognized a conformational epitope situated before the 266-271 sequence. In addition, H16.E70 neutralizing antibody reactivity was reduced with L1 VLPs with an Asn to Ala point mutation at position 270, suggesting that Asn is a part of the epitope recognized by this antibody. This study contributes to the understanding of the antigenic structure of HPV-16 and -31 L1 proteins by confirming that the FG loop contributes to neutralizing epitopes and suggesting the existence of both type-specific and cross-reactive conformational epitopes within the FG loop.     

A deletion and point mutation study of the human papillomavirus type 16 major capsid gene

Recombinant human papillomavirus (HPV) virus-like particles (VLPs) made from the major capsid protein L1 are promising vaccine candidates for use as vaccines against genital and other HPV infections, and particularly against HPV-16. However, HPV-16 genotype variants have different binding affinities for neutralising mouse Mabs raised against HPV-16 L1 VLPs. This paper analyses, using a panel of well-characterised Mabs, the effects on the antigenicity of various C- and N-terminal deletants of HPV-16 L1 made in insect cells via recombinant baculovirus, of an A-->T mutation at residue 266 (A266T), and of a C-->G mutation at conserved position 428 (C428G). The effects of these changes on assembly of the variant L1s were studied by electron microscopy. Binding of Mab H16:E70 to A266T was reduced by almost half in comparison to wild type L1. Retention of the C-terminal region 428-483 was critical for the binding of conformation-specific Mabs (H16:V5, H16:E70, H16:U4 and H16:9A) whereas deletion of the nuclear localisation signal (NLS) or the C428G mutation or an N-terminal deletion (residues 2-9) did not affect the antigenicity. The N-terminal deletion resulted in a mixed population of 30 and 55nm VLPs, which differs from the same construct expressed in Escherichia coli, whereas pentamer aggregates resulted from deletion of the 428-465 region or the C428G mutation. The results have implications both for considering use of single-genotype HPV vaccines, and for design of novel second-generation vaccines.     

Type-specific human papillomavirus detection in cervical smears in Romania

Although Romania has one of the highest incidence of cervical cancer in Europe (30 new cases/100 000 women), little is known about the distribution of the human papillomaviruses (HPV) genotypes in this population. We seek to determine the distribution of HPV genotypes in women with normal and abnormal cervical cytology. We analyzed 460 cervical cytology specimens from women who self-referred to the gynecologic clinic. HPV was detected and genotyped using the commercially available INNOLiPA (INNOGENETICS NV) kit based on the reverse hybridization principle. HPV DNA was detected in 279 cases (60.7%) with a median age of 32.9 years. In HGSIL (High Grade Squamous Intraepithelial Lesion) cytology, the presence of HPV DNA was confirmed in 82.7% of cases. The most frequent high-risk genotype was HPV16, found in 32.6% of HPV-positive samples. The next common high-risk genotypes were HPV18, HPV31 and HPV51. Our findings on the distribution and frequency of the HPV genotypes in Romanian population confirmed the utility of the current available HPV vaccines, HPV16 and 18 being detected in 28.7% of cases in the investigated area.     

T-helper epitopes identified within the E6 transforming protein of cervical cancer-associated human papillomavirus type 16

The E6 oncoprotein of human papillomavirus type 16 (HPV16 E6) produced by tumor cells of HPV16-associated cervical carcinoma is poorly immunogenic in patients, but nonetheless is a tumor-specific antigen to which therapeutic vaccine strategies may be directed. To investigate the subunit immunogenicity of E6 protein at the T-helper cell level, we immunized mice with overlapping peptides spanning the entire 158 amino acid sequence. Two peptides recalled a proliferative response in lymph node cells (LNC) from C57BL/6 (H-2b)-immunized mice. One of these peptides also recalled proliferative responses in the context of 5/5 other major histocompatibility complex (MHC) class II haplotypes, indicating a "promiscuous" T-epitope. Minimal consensus motif analysis identified the epitopes as 60VYRDGNPYA68 and 98GYNKPLCDLL107. LNC from mice immunized with T-epitope proliferated in response to challenge with whole E6 protein. Immunization with E6 T-epitopes linked to B-epitopes of HPV16 E7 protein elicited specific antibody indicating that T-cells recognizing the T-epitopes provided cognate "help" for B-cells. LNC from mice co-immunized with E6 T-epitope and the major T-helper epitope of HPV16 E7 (48DRAHYNI54) proliferated comparably when challenged with the peptides individually indicating co-dominance of the two T-epitopes. The findings have implications for incorporation of E6 into a therapeutic vaccine.     

人乳头瘤病毒16型晚期蛋白L1的表达及病毒样颗粒的装配

目的　在昆虫细胞中表达人乳头瘤病毒 16型 (HPV16 )哈尔滨地区分离株的晚期蛋白L1,并分离纯化由L1蛋白在细胞中自主组装成的病毒样颗粒 ,为HPV16预防性疫苗的研制奠定基础。方法　获得含有HPV16L1基因的重组杆状病毒并使目的蛋白L1在昆虫细胞中表达 ;对重组杆状病毒的扩增条件及L1蛋白的表达水平进行优化 ;电镜下观察昆虫细胞中晚期蛋白装配成病毒样颗粒的情况 ,经氯化铯密度梯度纯化病毒样颗粒 ,并经Westernblot进行验证。结果　获得了稳定表达L1蛋白的重组杆状病毒 ;以MOI值为 0 .2感染昆虫细胞可获得较高滴度的重组病毒 ,以MOI值为 10感染昆虫细胞可获得较高水平的L1蛋白表达 ;电镜下可观察到昆虫细胞核内直径为 5 5nm的病毒样颗粒 ;病毒样颗粒可通过氯化铯密度梯度离心获得。结论　获得人乳头瘤病毒哈尔滨地区分离株L1蛋白可在昆虫细胞中自主装配的病毒样颗粒并成功分离纯化。     

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     

Self-assembly of in vitro-translated human papillomavirus type 16 L1 capsid protein into virus-like particles and antigenic reactivity of the protein.

The human papillomavirus type 16 (HPV-16) L1 capsid protein is the major component of the HPV virion. We prepared L1 protein of HPV-16 in a cell-free system. The L1 gene was cloned in an expression plasmid and transcribed and translated in vitro in a rabbit reticulocyte lysate. The expressed protein had the molecular mass (55 kDa) expected for the L1 protein, and it assembled into virus-like particles that closely resembled papillomavirus virions. The protein retained conformational epitopes, as evidenced by its reactivity with monoclonal antibodies which recognize only intact viral particles. In radioimmunoprecipitation assays with sera from college women grouped by their genital tract HPV DNA status, high reactivity was found in 68% of HPV-16 DNA-positive women, in 23% of women with other HPVs, and in 19% of HPV-negative women. In comparison, none of the sera of children were reactive. The results of the radioimmunoprecipitation assays showed a significant correlation with results obtained with the same sera in an enzyme-linked immunosorbent assay with virus-like particles produced in baculovirus (chi-square test for linear trend, P = 0.0023). Although the amounts of L1 protein obtained are small, the ability to produce virus-like particles by in vitro translation may be useful in the study of virus assembly, virus binding, and the immunological response to HPV infection.     

Mapping of human serum-reactive epitopes in virus-like particles of human papillomavirus types 16 and 11

Most human antibodies against HPV16 can be blocked by the monoclonal antibody H16.V5. To investigate whether H16.V5 and human sera recognize similar epitopes, hybrid capsids containing different parts of HPV16 and HPV11 were evaluated for reactivity with human sera. The antibody responses among HPV 16-/HPV11+sera to HPV11 and to hybrid capsids containing the HPV11 C-terminus were strongly correlated. The antibody responses among HPV 16+/HPV11-sera to HPV16 and to a hybrid containing the HPV16 C-terminus were correlated and there was also reactivity with a hybrid containing the H16.V5 epitope in the HPV11 backbone. Several HPV16-/11- children's sera were reactive with hybrid capsids, implying that a native capsid structure is essential for serological specificity. For both HPV16 and HPV11, the major serologic reactivity was directed toward the C-terminal part of the protein and the H16.V5 binding site appeared to be a major serologically reactive epitope of HPV16.     

Humoral assays of human sera to disrupted and nondisrupted epitopes of human papillomavirus type 1.

The use of different assay systems and the disparity in results obtained has meant that we have little understanding about the role played by the humoral response during human papillomavirus (HPV) infection. Human antibody responses have so far appeared to be largely directed against the major capsid protein, L1. This protein possesses both type-specific and type-common antigenic determinants but it is not known which of these is important in vivo during the natural course of infection. In this study humoral responses of 83 individuals to purified HPV 1 virions were tested in three types of antibody assay. Western blot analysis detected antibodies in only eight of the serum samples, whereas an enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation assay using nondisrupted HPV 1 virions showed positive antibody reactivities for 71 and 64 individuals, respectively. We suggest from these results that the humoral response to L1 is mainly directed against native conformational epitopes present on the whole HPV 1 particle and that type-common epitopes are not largely involved. This was further demonstrated by the fact that when samples were tested in the same ELISA system using disrupted HPV 1 virions as the antigen instead of whole virus particles, the number of positive sera was reduced to 9 out of 83. We further conclude that humoral assays using antigenic material pertaining to disrupted HPV epitopes are of limited use, at least in the case of HPV type 1. There were no obvious correlations between the antibody assay results and clinical histories of wart infection except that a lower number of positive serum reactivities were found among the group of individuals claiming to have no past history of HPV infection.     

Identification of recombinant human papillomavirus type 16 variants

Intratypic diversity of human papillomavirus (HPV) genome is generally characterized by point mutation, insertion, and/or deletion. Using PCR-based cloning and sequencing, we detected concurrent infection with 8 HPV16 variants in a woman enrolled in the ASCUS-LSIL Triage Study. The European variant was the major variant; each of the 7 minor variants had partial DNA sequences identical to the European variant and another part identical to the African 2 variant. At a follow-up visit, only an HPV16 African 2 variant was detected. Results from the present study suggest presence of intratypic recombination of HPV genome in natural infection.     

Characterization of a cross-reactive linear epitope in human genogroup I and bovine genogroup III norovirus capsid proteins

The Southampton norovirus (SV) capsid protein was expressed as VLPs by recombinant baculoviruses in insect cells and was used to immunize mice for the production of monoclonal antibodies (mAbs). One mAb, CM54, showed broad cross-reactivity to genogroup I (GI) noroviruses, but was not reactive to GII capsid proteins. Interestingly mAb CM54 reacted to a bovine norovirus capsid protein. Immunoblot analysis indicated the binding site for CM54 was located in the shell domain between amino acid residues 102-225 of the SV capsid protein. The epitope was mapped to high resolution using a peptide array and was located to the sequence LEDVRN at amino acid residues 162-167. Alignment of norovirus capsid protein sequences confirmed the epitope sequence was common to particular groups of human and bovine noroviruses. Modeling of the epitope onto the recombinant NV capsid protein revealed it was located to the inner surface of the shell domain.     

Levels of immunoglobulin G antibodies against defined epitopes of the L1 and L2 capsid proteins of human papillomavirus type 6 are elevated in men with a history of condylomata acuminata.

Sera from 159 men attending the sexually transmitted disease clinic at Karolinska Hospital, Stockholm, Sweden, were analyzed for the presence of immunoglobulin A (IgA) and IgG antibodies to a panel of synthetic peptides derived from the E2, L1, and L2 regions of the human papillomavirus types 1 (HPV 1), 6, 8, 11, 16, 18, 31, and 33. The study subjects were divided into three groups: (i) asymptomatic men with no history of genital warts who served as controls, (ii) men with visible condylomata, and (iii) men who had previously been afflicted with condylomata. There were no significant differences in antibody titers for any of the HPV 6- or 11-derived peptides among patients with current condylomata and the controls. For the peptide from L1 of HPV 6, there was an increase in the IgG titers among men with previous condylomata compared with the titers for the controls (52% versus 27% seropositivity; P less than 0.05). Also, for the peptide from L2 of HPV 6, there was an increase in the IgG titers among men who had been afflicted with condylomata previously (P less than 0.05). Increased IgA antibody titers against an HPV 16-derived peptide and an HPV 18-derived peptide were also detected. For the peptides from L1 and L2 of HPV 6, the study was extended to an additional group of 127 males attending the sexually transmitted disease clinic at Huddinge Hospital in southern Stockholm. Again, significantly increased antibody levels were detected only for IgG and only among asymptomatic men with a history of condylomata (P < 0.01 for the L1 peptide and P < 0.05 for the L2 peptide). The results suggest that the IgG response against the late proteins of HPV 6 reflects mainly previous exposure to the virus rather than ongoing viral disease.     

Independent association of antibodies against human papillomavirus type 16 and E7 proteins with cervical cancer

The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac- and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti- antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti- and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma.