Common Neutralization Epitope in Minor Capsid Protein L2 of Human Papillomavirus Types 16 and 6

Studies of virus neutralization by antibody are a prerequisite for development of a prophylactic vaccine strategy against human papillomaviruses (HPVs). Using HPV16 and -6 pseudovirions capable of inducing beta-galactosidase in infected monkey COS-1 cells, we examined the neutralizing activity of mouse monoclonal antibodies (MAbs) that recognize surface epitopes in HPV16 minor capsid protein L2. Two MAbs binding to a synthetic peptide with the HPV16 L2 sequence of amino acids (aa) 108 to 120 were found to inhibit pseudoinfections with HPV16 as well as HPV6. Antisera raised by immunizing BALB/c mice with the synthetic peptide had a cross-neutralizing activity similar to that of the MAb. The data indicate that HPV16 and -6 have a common cross-neutralization epitope (located within aa 108 to 120 of L2 in HPV16), suggesting that this epitope may be shared by other genital HPVs.     

Direct Binding of Retromer to Human Papillomavirus Type 16 Minor Capsid Protein L2 Mediates Endosome Exit during Viral Infection

Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.     

Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.     

Naturally Occurring Major and Minor Capsid Protein Variants of Human Papillomavirus 45 (HPV45): Differential Recognition by Cross-Neutralizing Antibodies Generated by HPV Vaccines

We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of human papillomavirus genotype 45 (HPV45). Pseudoviruses (PsVs) representing HPV45 sublineages A1, A2, A3, B1, and B2 exhibited comparable particle-to-infectivity ratios and morphologies but demonstrated both increased (A2, A3, and B1) and decreased (B2) sensitivities to cross-neutralization by HPV vaccine antibodies compared to that of the A1 sublineage. Mutant PsVs identified HI loop residue 357 as being critical for conferring this differential sensitivity.     

The Merkel Cell Polyomavirus Minor Capsid Protein

The surface of polyomavirus virions is composed of pentameric knobs of the major capsid protein, VP1. In previously studied polyomavirus species, such as SV40, two interior capsid proteins, VP2 and VP3, emerge from the virion to play important roles during the infectious entry process. Translation of the VP3 protein initiates at a highly conserved Met-Ala-Leu motif within the VP2 open reading frame. Phylogenetic analyses indicate that Merkel cell polyomavirus (MCV or MCPyV) is a member of a divergent clade of polyomaviruses that lack the conserved VP3 N-terminal motif. Consistent with this observation, we show that VP3 is not detectable in MCV-infected cells, VP3 is not found in native MCV virions, and mutation of possible alternative VP3-initiating methionine codons did not significantly affect MCV infectivity in culture. In contrast, VP2 knockout resulted in a >100-fold decrease in native MCV infectivity, despite normal virion assembly, viral DNA packaging, and cell attachment. Although pseudovirus-based experiments confirmed that VP2 plays an essential role for infection of some cell lines, other cell lines were readily transduced by pseudovirions lacking VP2. In cell lines where VP2 was needed for efficient infectious entry, the presence of a conserved myristoyl modification on the N-terminus of VP2 was important for its function. The results show that a single minor capsid protein, VP2, facilitates a post-attachment stage of MCV infectious entry into some, but not all, cell types.     

人乳头瘤病毒E2蛋白及其相关疫苗

人乳头瘤病毒（human papilloma virus,HPV）感染在全球范围内颇为常见,其与肛门生殖器疣、生殖器肿瘤的发生关系密切。研究发现,乳头瘤的形成与HPVE2蛋白密不可分,该蛋白质涉及到病毒生命周期的各个阶段,与病毒的有丝分裂、其他早期蛋白的转录及细胞凋亡有关。近年来,各国学者利用E2蛋白的特性研制出E2相关疫苗,分别使用不同的重组病毒来传输E2,或是使用纯化的E2蛋白或E2融合蛋白,运输至体内的HPV转化细胞和／或HPV感染细胞中,以期达到防治HPV感染相关疾病的目的。     

Papillomavirus Assembly Requires Trimerization of the Major Capsid Protein by Disulfides between Two Highly Conserved Cysteines

We have used viruslike particles (VLPs) of human papillomaviruses to study the structure and assembly of the viral capsid. We demonstrate that mutation of either of two highly conserved cysteines of the major capsid protein L1 to serine completely prevents the assembly of VLPs but not of capsomers, whereas mutation of all other cysteines leaves VLP assembly unaffected. These two cysteines form intercapsomeric disulfides yielding an L1 trimer. Trimerization comprises about half of the L1 molecules in VLPs but all L1 molecules in complete virions. We suggest that trimerization of L1 is indispensable for the stabilization of intercapsomeric contacts in papillomavirus capsids.     

In Vitro Dynamic Visualization Analysis of Fluorescently Labeled Minor Capsid Protein IX and Core Protein V by Simultaneous Detection

Oncolytic adenoviruses represent a promising therapeutic medicine for human cancer therapy, but successful translation into human clinical trials requires careful evaluation of their viral characteristics. While the function of adenovirus proteins has been analyzed in detail, the dynamics of adenovirus infection remain largely unknown due to technological constraints that prevent adequate tracking of adenovirus particles after infection. Fluorescence labeling of adenoviral particles is one new strategy designed to directly analyze the dynamic processes of viral infection in virus-host cell interactions. We hypothesized that the double labeling of an adenovirus with fluorescent proteins would allow us to properly analyze intracellular viruses and the fate of viral proteins in a live analysis of an adenovirus as compared to single labeling. Thus, we generated a fluorescently labeled adenovirus with both a red fluorescent minor capsid protein IX (pIX) [pIX monomeric red fluorescent protein 1 (mRFP1)] and a green fluorescent minor core protein V (pV) [pV enhanced green fluorescent protein (EGFP)], resulting in Ad5-IX-mRFP1-E3-V-EGFP. The fluorescent signals for pIX-mRFP1 and pV-EGFP were detected within 10 min in living cells. However, a growth curve analysis of Ad5-IX-mRFP1-E3-V-EGFP showed an approximately 150-fold reduced production of the viral progeny at 48 h postinfection as compared to adenovirus type 5. Interestingly, pIX-mRFP1 and pV-EGFP were initially localized in the cytoplasm and nucleolus, respectively, at 18 h postinfection. These proteins were observed in the nucleus during the late stage of infection, and relocalization of the proteins was observed in an adenoviral-replication-dependent manner. These results indicate that simultaneous detection of adenoviruses using dual-fluorescent proteins is suitable for real-time analysis, including identification of infected cells and monitoring of viral spread, which will be required for a complete evaluation of oncolytic adenoviruses.     

人乳头瘤病毒2(HPV-2)变异株的突变E2蛋白转录抑制作用的研究

皮肤寻常疣的发生与多种基因型别HPV的感染密切相关.本研究利用PCR方法对1例临床罕见的寻常疣患者感染的HPV-2毒株LCR及E2基因序列进行扩增、测序,分别构建含HPV-2变异株及原毒株LCR的重组CAT基因报导质粒pBLCAT-LCR和表达突变及野生型E2蛋白的重组真核表达质粒pcDNA3.1-E2,通过瞬时转染HeLa细胞,研究变异株启动子活性及突变E2蛋白的转录抑制作用.结果显示,患者感染的HPV-2变异株LCR及E2基因均存在多处点突变.变异株早期启动子活性明显高于原毒株;突变的E2蛋白转录抑制作用较野生型E2蛋白显著降低;变异株LCR上E2结合位点核苷酸的突变明显降低E2蛋白对病毒早期启动子的抑制作用.提示HPV-2变异株启动子活性增强及突变E2蛋白转录抑制作用的降低与这一罕见巨大寻常疣临床表型之间存在着重要的联系.     

Human Papillomavirus Type 16 Pseudovirions with Few Point Mutations in L1 Major Capsid Protein FG Loop Could Escape Actual or Future Vaccination for Potential Use in Gene Therapy

HPV prophylactic vaccination based on VLPs was implemented 7 years ago and has now shown a high degree of efficiency to reduce HPV-induced lesions. Moreover, it was shown that HPV-derived virus-like particles or pseudovirions could be used as gene therapy vectors. As a consequence, characterization of the antigenic structure of HPV capsids is crucial for designing future HPV vaccines with better or broader efficacy and for the design of HPV-derived gene therapy vectors with reduced immunogenicity or vaccination escaping. In this study, we have generated 10 HPV16 FG loop L1 protein mutants and analyzed their ability to self-assemble into VLP, their immunogenicity, and their ability to transduce cells when used as pseudovirions. Most of the mutants had lost their ability to transduce cells at the exception of two chimeric HPV16/31 L1 protein FG loop mutants. Sera from mice immunized with HPV16 L1 wt VLPs very weakly neutralized pseudovirions derived from these two HPV16/31 L1 protein FG loop mutants. These findings suggest that only a few point substitutions within the FG loop are sufficient to generate a new serotype escaping vaccination. As a consequence, derived pseudovirions might be suitable as gene therapy vectors in vaccinated subjects.     

Chimeric L1-L2 Virus-Like Particles as Potential Broad-Spectrum Human Papillomavirus Vaccines

The amino (N) terminus of the human papillomavirus (HPV) minor capsid protein L2 can induce low-titer, cross-neutralizing antibodies. The aim of this study was to improve immunogenicity of L2 peptides by surface display on highly ordered, self-assembled virus-like particles (VLP) of major capsid protein L1, and to more completely characterize neutralization epitopes of L2. Overlapping peptides comprising amino acids (aa) 2 to 22 (hereafter, chimera or peptide 2-22), 13 to 107, 18 to 31, 17 to 36, 35 to 75, 75 to 112, 115 to 154, 149 to 175, and 172 to 200 of HPV type 16 (HPV16) L2 were genetically engineered into the DE surface loop of bovine papillomavirus type 1 L1 VLP. Except for chimeras 35-75 and 13-107, recombinant fusion proteins assembled into VLP. Vaccination of rabbits with Freund''s adjuvanted native VLP induced higher L2-specific antibody titers than vaccination with corresponding sodium dodecyl sulfate-denatured proteins. Immune sera to epitopes within residues 13 to 154 neutralized HPV16 in pseudovirion neutralization assays, whereas chimera 17-36 induced additional cross-neutralization to divergent high-risk HPV18, -31, -45, -52, and -58; low-risk HPV11; and beta-type HPV5 (titers of 50 to 10,000). Aluminum hydroxide-monophosphoryl lipid A (Alum-MPL)-adjuvanted VLP induced similar patterns of neutralization in both rabbits and mice, albeit with 100-fold-lower titers than Freund''s adjuvant. Importantly, Alum-MPL-adjuvanted immunization with chimeric HPV16L1-HPV16L2 (peptide 17-36) VLP induced neutralization or cross-neutralization of HPV16, -18, -31, -45, -52, and -58; HPV6 and -11; and HPV5 (titers of 50 to 100,000). Immunization with HPV16 L1-HPV16 L2 (chimera 17-36) VLP in adjuvant applicable for human use induces broad-spectrum neutralizing antibodies against HPV types evolutionarily divergent to HPV16 and thus may protect against infection with mucosal high-risk, low-risk, and beta HPV types and associated disease.The more than 100 types of human papillomaviruses (HPV) identified to date (14) are the etiological agents of skin and mucosal papillomas or warts. Persistent infection with high-risk mucosal types, most often HPV type 16 (HPV16) and HPV18, causes cervical cancer, which constitutes the second leading fatal cancer in women worldwide, causing 274,000 deaths per year. Substantial morbidity results from other noncervical HPV-related conditions, such as anogenital warts or anal cancer (23).The development of current prophylactic papillomavirus vaccines was launched by observations that recombinantly expressed major capsid protein L1 self-assembles into virus-like particles (VLP). These empty viral capsids are composed of 360 L1 molecules and resemble native virions in both structure and immunogenicity, yet are nononcogenic and noninfectious. Moreover, VLP cannot replicate because the cells in which VLP are made contain only L1 and no other papillomavirus genes. Subunit VLP vaccines induce high-titer and type-restricted antibody responses to conformational L1 epitopes (12, 26, 39, 44). When applied to women prior to infection, available vaccines targeting the most prevalent high-risk types, HPV16 and HPV18, have demonstrated up to 100% efficacy against persistent infection and associated disease caused by the included types and thus are potentially able to prevent ∼70% of cervical high-grade dysplasias and probably cancers (22, 46). Therefore, use of currently licensed L1 vaccines necessitates continuation of cytological cervical screening of women. The prevention of 96% of cervical cancer would require immunity to seven high-risk HPV types (HPV16, -18, -31, -33, -45, -52, and -58) (32) and the development of more highly multivalent (and presumably costly) L1 VLP vaccines.The search for alternative broader-spectrum immunogens drew attention to the minor capsid protein L2, which is immunogenically subdominant in the context of coexpressed L1-L2 capsids (38). Immunization of animals with the amino (N)-terminal peptide of L2 demonstrated its ability to elicit low-titer neutralizing antibodies that protect against challenge with cognate papillomavirus types in vivo (16, 19), cross-neutralize heterologous types in vitro (25, 33, 38), and confer cross-protection in vivo (17).This study addresses two major issues that may further the development of L2-based broader-spectrum vaccines. First, the N terminus of L2 is more closely examined for potential neutralization epitopes, by incorporating peptides into papillomavirus VLP as peptide-presenting platforms (7, 21, 42). Moreover, we take advantage of the immunogenic characteristics of virion surfaces, such as the dense repetitive surface array of VLP, to induce strong and enduring immune responses to displayed L2 epitopes.     

mRNA Instability Elements in the Human Papillomavirus Type 16 L2 Coding Region

Human papillomavirus capsid proteins L1 and L2 are detected only in terminally differentiated cells, indicating that expression of the L1 and L2 genes is blocked in dividing cells. The results presented here establish that the human papillomavirus type 16 L2 coding region contains cis-acting inhibitory sequences. When placed downstream of a reporter gene, the human papillomavirus type 16 L2 sequence reduced both mRNA and protein levels in an orientation-dependent manner. Deletion analysis revealed that the L2 sequence contains two cis-acting inhibitory RNA regions. We identified an inhibitory region in the 5′-most 845 nucleotides of L2 that acted by reducing cytoplasmic mRNA stability and a second, weaker inhibitory region in the 3′ end of L2. In contrast, human papillomavirus type 1 L1 and L2 genes did not encode strong inhibitory sequences. This result is consistent with observations of high virus production in human papillomavirus type 1-infected tissue, whereas only low levels of human papillomavirus type 16 virions are detectable in infected epithelium. The presence of inhibitory sequences in the L1 and L2 mRNAs may aid the virus in avoiding the host immunosurveillance and in establishing persistent infections.     

Multivalent Human Papillomavirus L1 DNA Vaccination Utilizing Electroporation

Objectives

Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV) L1 and L2 DNA vaccination.

Methods

Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11−88 of 8 different HPV types (11−88×8) or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies.

Results

Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2) vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type.

Discussion

Multivalent vaccination via in vivo electroporation requires spatial separation of individual type L1 DNA vaccines.     

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.