Identification of B-Cell Epitopes on Virus-Like Particles of Cutaneous Alpha-Human Papillomaviruses

Human papillomavirus (PV) (HPV) types 2, 27, and 57 are closely related and, hence, represent a promising model system to study the correlation of phylogenetic relationship and immunological distinctiveness of PVs. These HPV types cause a large fraction of cutaneous warts occurring in immunocompromised patients. Therefore, they constitute a target for the development of virus-like particle (VLP)-based vaccines. However, the immunogenic structure of HPV type 2, 27, and 57 capsids has not been studied yet. Here we provide, for the first time, a characterization of the B-cell epitopes on VLPs of cutaneous alpha-HPVs using a panel of 94 monoclonal antibodies (MAbs) generated upon immunization with capsids from HPV types 2, 27, and 57. The MAbs generated were characterized regarding their reactivities with glutathione S-transferase-L1 fusion proteins from 18 different PV types, the nature of their recognized epitopes, their isotypes, and their ability to neutralize HPV type 2, 27, 57, or 16. In total, 33 of the 94 MAbs (35%) showed type-specific reactivity. All type-specific MAbs recognize linear epitopes, most of which map to the hypervariable surface loop regions of the L1 amino acid sequence. Four of the generated MAbs neutralized pseudovirions of the inoculated HPV type efficiently. All four MAbs recognized epitopes within the BC loop, which is required and sufficient for their neutralizing activity. Our data highlight the immunological distinctiveness of individual HPV types, even in comparison to their closest relatives, and they provide a basis for the development of VLP-based vaccines against cutaneous alpha-HPVs.Recently licensed prophylactic vaccines confer efficient protection against infections by human papillomavirus (PV) (HPV) types 16 and 18, thereby aiming to prevent approximately 70% of all cervical cancer cases (17, 39). These vaccines are composed of virus-like particles (VLPs), which spontaneously assemble from the major capsid protein L1 via 72 pentamers (capsomeres) as subunits (2, 23, 26).In the process of vaccine development, monoclonal antibodies (MAbs) proved to be valuable tools for the immunological analysis of recombinantly produced capsids and capsomeres (51) as well as for serological studies (25, 49, 56). Moreover, the identification and characterization of many neutralizing epitopes of HPV types 11 and 16 have been facilitated by the employment of MAbs (6, 11, 30-32, 41, 42, 55). Such epitopes to neutralizing antibodies are mostly conformation dependent, but a few neutralizing MAbs that recognize linear epitopes have also been generated (16, 18). Most neutralizing MAbs are HPV type specific due to the hypervariable nature of their respective epitopes, which typically reside in the surface-exposed loop regions of the L1 protein (10). In contrast, cross-reactive MAbs targeting rather conserved L1 epitopes are generally nonneutralizing.HPV types 2, 27, and 57 are the three members of Alphapapillomavirus species 4 (20). They are very closely related, and HPV types 2 and 27 hardly fulfill the requirement of more than 10% nucleotide variation in the L1 open reading frame to be classified as distinct types (8). Therefore, they represent a promising model system to study the immunological distinctiveness of closely related HPV types. Pathologically, HPV types 2, 27, and 57 infect primarily the cutaneous epithelia, thereby causing common skin warts, which often occur ubiquitously and confluently in immunocompromised patients (1, 24, 28). It is our long-term goal to develop a prophylactic L1 VLP-based vaccine to alleviate the burden provoked by HPV-induced skin lesions in these patients. However, to date, neither the structure nor the immunogenicity of HPV type 2, 27, and 57 capsids has been elucidated.The purpose of the present study was twofold. First, we sought to generate MAbs specific for HPV types 2, 27, and 57 as tools for type-specific diagnostic assays. Second, we aimed to exploit the generated MAbs for an investigation of the B-cell epitopes on capsids of HPV types 2, 27, and 57.     

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.     

Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

Papillomavirus capsids are composed of 72 pentamers reinforced through inter- and intrapentameric disulfide bonds. Recent research suggests that virus-like particles and pseudovirions (PsV) can undergo a redox-dependent conformational change involving disulfide interactions. We present here evidence that native virions exploit a tissue-spanning redox gradient that facilitates assembly events in the context of the complete papillomavirus life cycle. DNA encapsidation and infectivity titers are redox dependent in that they can be temporally modulated via treatment of organotypic cultures with oxidized glutathione. These data provide evidence that papillomavirus assembly and maturation is redox-dependent, utilizing multiple steps within both suprabasal and cornified layers.Human papillomaviruses (HPVs) exclusively infect cutaneous or mucosal epithelial tissues (14, 15, 30). HPV types that infect the mucosal epithelia can lead to the development of benign or malignant neoplasms, thus allowing for their categorization into low-risk or high-risk HPV types, respectively (14, 15, 30). A small subset of the more than 200 HPV types now identified are the causative agents of over 75% of all cervical cancers. HPV16 is the most prevalent type worldwide, found in ca. 50 to 62% of squamous cell carcinomas (14, 50).HPV16 virions contain a single, circular double-stranded DNA genome of ∼8 kb which associates with histones to form a chromatin-like structure. This minichromosome is packaged within a nonenveloped, icosahedral capsid composed of the major capsid protein L1 and the minor capsid protein L2. Similar to polyomaviruses, 72 capsomeres of L1 are geometrically arranged on a T=7 icosahedral lattice (2, 9, 17, 19, 36, 42). Recent cryoelectron microscopy images of HPV16 pseudovirions (PsV) suggest that L2 is arranged near the inner conical hollow of each L1 pentamer, although it is not known whether each L1 pentamer is occupied with a single L2 protein (5, 42).Due to technical constraints in the production of native HPV virions in organotypic culture, assembly studies of HPV particles have largely been restricted to the utilization of in vitro-derived particles such as virus-like particles (VLPs), PsV, and quasivirions (QV) (6, 12, 25, 40, 43). Recent research suggests that HPV and bovine papillomavirus PsV can undergo a redox-dependent conformational change that takes place over the course of many hours. This conformational change is characterized by resistance to proteolysis and chemical reduction and the appearance of a more orderly capsid structure via transmission electron microscopy (TEM) (7, 20).We present evidence that native virions, in the context of the complete papillomavirus life cycle, utilize a tissue-spanning redox gradient that facilitates multiple redox-dependent assembly and maturation events over the course of many days. We show that stability and specific infectivity of 20-day virions increases over 10-day virions, 20-day virions are more susceptible to neutralization than 10-day virions, and both viral DNA encapsidation and infectivity of HPV-infected tissues are redox dependent in that they can be manipulated via the treatment of organotypic tissues with oxidized glutathione (GSSG), which is concentration and temporally dependent.     

The Canine Papillomavirus E5 Protein Signals from the Endoplasmic Reticulum

The recently discovered Canis familiaris papillomavirus (PV) type 2 (CfPV2) provides a unique opportunity to study PV gene functions in vitro and in vivo. Unlike the previously characterized canine oral PV, CfPV2 contains an E5 open reading frame and is associated with progression to squamous cell carcinoma. In the current study, we have expressed and characterized the CfPV2-encoded E5 protein, a small, hydrophobic, 41-amino-acid polypeptide. We demonstrate that, similar to the E5 protein from high-risk human PV type 16, the CfPV2 E5 protein is localized in the endoplasmic reticulum (ER) and that its expression decreases keratinocyte proliferation and cell life span. E5 expression also increases the percentage of cells in the G₁ phase of the cell cycle, with a concomitant decrease in the percentage of cells in S phase. To identify a potential mechanism for E5-mediated growth inhibition from the ER, we developed a real-time PCR method to quantify the splicing of XBP1 mRNA as a measure of ER stress. We found that the CfPV2 E5 protein induced ER stress and that this, as well as the observed growth inhibition, is tempered significantly by coexpression of the CfPV2 E6 and E7 genes. It is possible that the spatial/temporal regulation of E6/E7 gene expression during keratinocyte differentiation might therefore modulate E5 activity and ER stress.Papillomaviruses (PVs) are a large group of DNA tumor viruses that infect differentiated cutaneous and mucosal epithelia in a wide variety of mammalian species. There are nearly 200 types of human PVs (HPVs) (61), some of which are termed high risk (e.g., HPV type 16 [HPV-16]) and have the potential to immortalize primary cells and facilitate malignant progression to cervical cancer (52). An estimated 20 million cases of HPV infection occur each year in the United States alone, and cervical cancer is the second most common cause of cancer deaths among women worldwide. In general, PV infections are species specific, making it impossible to study the in vivo life cycle of HPV and the roles of its encoded proteins in viral replication and tumorigenesis. However, a few animal models do exist and the canine oral PV (COPV) has been helpful in mimicking certain biological properties of the high-risk mucosatropic HPVs, leading to the development of highly effective prophylactic vaccines (39, 49, 56). Although COPV mimics the mucosal tropism of the high-risk HPVs, it rarely progresses to cancer and lacks one of the early viral genes that may play an important role in tumorigenesis, E5. Recently, a new canine PV (Canis familiaris PV type 2 [CfPV2]) was isolated from the footpads of dogs (43). Unlike COPV, CfPV2 induces epidermal tumors and, when persistent, these benign infections progress to squamous cell carcinoma and metastasize widely. CfPV2 also encodes an E5 protein. In general, PV E5 proteins are small hydrophobic oncoproteins that localize to the endoplasmic reticulum (ER) or Golgi membranes (11, 16) but have limited amino acid sequence homology. Numerous cellular binding partners have been described for HPV-16 E5 proteins, including the V-ATPase 16-kDa subunit (1, 16), the nuclear import protein karyopherin beta 3 (25), the ER-resident protein Bap31 (40), proteins involved in zinc transport (ZnT1, EVER1, and EVER2) (27, 35), erbB4 (24), and HLA I (2). The HPV-16 E5 protein alters signaling pathways, predominantly the epidermal growth factor receptor (EGFR) pathway (17, 21, 46, 58); induces koilocytosis in cooperation with the E6 protein (26); and alters the plasma membrane expression of caveolin (47), HLA (3), and ganglioside GM1 (47). The last two changes might explain the ability of HPV-16-infected cells to circumvent detection by the host immune response and initiate tumor formation (3, 4, 21, 36, 46, 47).To provide a foundation for future in vivo studies, we initiated a series of in vitro experiments to define the intracellular localization and biological activity of CfPV2 E5. The current study demonstrates that CfPV2 E5 exhibits several properties of the HPV-16 E5 protein, including ER localization and inhibition of cell proliferation. A novel finding is that CfPV2 E5 activates the ER stress-signaling pathway, which may explain some of E5''s growth-related activities.     

Human Papillomavirus Type 16 Infection of Human Keratinocytes Requires Clathrin and Caveolin-1 and Is Brefeldin A Sensitive

Human papillomavirus type 16 (HPV16) has been identified as being the most common etiological agent leading to cervical cancer. Despite having a clear understanding of the role of HPV16 in oncogenesis, details of how HPV16 traffics during infection are poorly understood. HPV16 has been determined to enter via clathrin-mediated endocytosis, but the subsequent steps of HPV16 infection remain unclear. There is emerging evidence that several viruses take advantage of cross talk between routes of endocytosis. Specifically, JCV and bovine papillomavirus type 1 have been shown to enter cells by clathrin-dependent endocytosis and then require caveolin-1-mediated trafficking for infection. In this paper, we show that HPV16 is dependent on caveolin-1 after clathrin-mediated endocytosis. We provide evidence for the first time that HPV16 infection is dependent on trafficking to the endoplasmic reticulum (ER). This novel trafficking may explain the requirement for the caveolar pathway in HPV16 infection because clathrin-mediated endocytosis typically does not lead to the ER. Our data indicate that the infectious route for HPV16 following clathrin-mediated entry is caveolin-1 and COPI dependent. An understanding of the steps involved in HPV16 sorting and trafficking opens up the possibility of developing novel approaches to interfere with HPV16 infection and reduce the burden of papillomavirus diseases including cervical cancer.Human papillomavirus (PV) type 16 (HPV16) is a member of the family Papillomaviridae, a group of double-stranded DNA (dsDNA) viruses with a tropism for squamous epithelia (70). Most PV infections result in benign lesions, although a subset of high-risk HPVs are capable of malignant transformation, resulting in various cancers including cervical carcinoma (21, 38). Infection with HPV16 is responsible for causing approximately half of the cases of invasive cervical cancer (7). In spite of the link between HPV16 and cervical cancer, the intracellular movement of HPV16 through target keratinocyte cells during infection has not been defined in detail.Viruses can enter into target cells by taking advantage of the cell''s natural endocytosis machinery (60). One of the best-characterized modes of internalization is by receptor-mediated, clathrin-dependent endocytosis. In this mode of entry, clathrin-coated pits internalize cargo into clathrin-coated vesicles, which are pinched from the plasma membrane by dynamin-2 in order to internalize (68). The process of clathrin-mediated endocytosis occurs rapidly, resulting in the delivery of cargo to early/sorting endosomes within seconds to minutes (23, 31). From the sorting endosome, most clathrin-dependent ligands are trafficked back to the plasma membrane in recycling endosomes or to lysosomes for degradation (35, 56). Another well-studied model of ligand entry is caveolin-1-mediated endocytosis. The caveolar pathway typically involves entry via cholesterol-rich caveolae at the plasma membrane, which deliver their contents to pH-neutral organelles known as caveosomes (44, 65). The delivery of cargo from caveosomes to the Golgi apparatus and the endoplasmic reticulum (ER) was demonstrated previously (44, 46, 50). The traffickings of cargo internalized via clathrin- and caveolin-1-mediated endocytosis were once thought to be separate; however, it is becoming evident that viruses including bovine PV type 1 (BPV1), JCV, HPV31, and BKV rely on both pathways depending on the stage of infection (29, 32, 50, 63).PV internalization is preceded by virion attachment to the extracellular matrix, followed by binding to heparan sulfate (14, 15, 25). The involvement of a secondary receptor has been suggested, putatively an alpha-6 integrin (24, 37). Postbinding, a conformational change in the PV capsid results in a furin cleavage event at the N terminus of the minor capsid protein L2, which has been suggested to play a role in the endosomal escape of the viral genome (19, 30, 52). An increasing body of evidence supports the entry of HPV16 by clathrin-mediated endocytosis (9, 27, 62). Electron microscopy of HPV16 infection in COS-7 cells demonstrated HPV16 pseudovirions in clathrin-coated vesicles 20 min after entry and within structures resembling endosomes by 1 h postentry (9). HPV16 infection of HaCaT keratinocyte, COS-7, and 293TT cells has been blocked by chlorpromazine, an inhibitor of the formation of clathrin-coated pits (9, 27, 62, 67). Importantly, those studies showed that two inhibitors of caveolin-1-mediated internalization, filipin and nystatin, did not interfere with HPV16 infection (9, 27, 62). Our laboratory demonstrated the importance of dynamin in HPV16 infection, presumably in the scission of clathrin-coated vesicles from the plasma membrane (1). Recently, a clathrin-, caveolin-, and dynamin-independent endocytosis of HPV16 was suggested, although the use of the HPV18-positive, heteroploid HeLa cell line calls into question the relevance of this finding to natural infection (64).In a previous study, we described the postentry trafficking of BPV1 from endosomes to caveolin-1-positive vesicles, similarly to a related nonenveloped dsDNA virus, JCV (32, 50). Our data demonstrated that the infectious route of BPV1 involved entry by clathrin-mediated endocytosis followed by transport to the caveolar pathway in order to traffic to the ER (32). We found that BPV1 infection was neutralized by an antibody that prevented viral particle transport to the ER (33). The movement of BPV1 from the endosome to the caveosome provides a possible explanation for why BPV1 trafficking is so slow compared to those of other ligands of clathrin-mediated endocytosis (20, 26). The kinetics of BPV1 and HPV16 entry were previously reported to be identical, and the coincident internalization of HPV16 and BPV1 virus-like particles (VLPs) showed colocalization between the VLPs during infection (20, 62). These data suggest that HPV16 and BPV1 infection may be occurring by a similar mechanism.Our goal in the present study was to determine the intracellular trafficking events leading to HPV16 infection. The use of reporter virion technology has allowed the production of high-titer HPV16 virions by a method previously shown to yield virions that are infectious in vivo (16). In this study, we used HPV16 reporter virions to study HPV16 infection in the spontaneously immortalized human HaCaT keratinocyte cell line. Our data show that the infectious route of HPV16 is from early endosomes to caveolin-1-positive vesicles and then to the ER. Using immunofluorescence and short hairpin RNA (shRNA) against caveolin-1, we demonstrate the importance of the caveolar pathway after HPV16 has been internalized. We show that HPV16 infection was blocked by inhibiting the formation of COPI transport vesicles, which function in trafficking between the ER and the Golgi apparatus and from caveosomes to the ER (5, 39). We provide evidence that after reaching the caveosome, HPV16 requires passage to the ER for successful infection, a trafficking event made possible by COPI vesicle-mediated movement from the caveosome to the ER.     

Papillomavirus Infection Requires γ Secretase

The mechanism by which papillomaviruses breach cellular membranes to deliver their genomic cargo to the nucleus is poorly understood. Here, we show that infection by a broad range of papillomavirus types requires the intramembrane protease γ secretase. The γ-secretase inhibitor (S,S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide (compound XXI) inhibits infection in vitro by all types of papillomavirus pseudovirions tested, with a 50% inhibitory concentration (IC₅₀) of 130 to 1,000 pM, regardless of reporter construct and without impacting cellular viability. Conversely, XXI does not inhibit in vitro infection by adenovirus or pseudovirions derived from the BK or Merkel cell polyomaviruses. Vaginal application of XXI prevents infection of the mouse genital tract by human papillomavirus type 16 (HPV16) pseudovirions. Nicastrin and presenilin-1 are essential components of the γ-secretase complex, and mouse embryo fibroblasts deficient in any one of these components were not infected by HPV16, whereas wild-type and β-secretase (BACE1)-deficient cells were susceptible. Neither the uptake of HPV16 into Lamp-1-positive perinuclear vesicles nor the disassembly of capsid to reveal both internal L1 and L2 epitopes and bromodeoxyuridine (BrdU)-labeled encapsidated DNA is dependent upon γ-secretase activity. However, blockade of γ-secretase activity by XXI prevents the BrdU-labeled DNA encapsidated by HPV16 from reaching the ND10 subnuclear domains. Since prior studies indicate that L2 is critical for endosomal escape and targeting of the viral DNA to ND10 and that γ secretase is located in endosomal membranes, our findings suggest that either L2 or an intracellular receptor are cleaved by γ secretase as papillomavirus escapes the endosome.The necessary causal association of persistent infection by an “oncogenic” type of human papillomavirus (HPV) with cervical cancer is firmly established (52, 53). HPV is the most prevalent sexually transmitted infection, and although the majority of patients clear their infection, HPV is directly responsible for 5% of all cancer deaths worldwide (30). HPV is also associated with multiple other anogenital cancers and oropharyngeal cancers.The life cycle of HPV is closely linked to epithelial differentiation within stratified squamous epithelia (16). Initial infection occurs within the undifferentiated proliferative basal cell layer in which only the viral early proteins are expressed, whereas production of the late proteins and, thus, progeny virus is restricted to the terminally differentiated suprabasal compartment (53). The exquisite dependence of virion production upon epithelial differentiation and lack of a rapid phenotype in culture can be circumvented by ectopic expression of the capsid proteins L1 and L2 in cells maintaining viral genome or reporter constructs as episomes, resulting in “quasivirions” or “pseudovirions,” respectively, whose infectivity can be readily and rapidly quantified in vitro or in vivo (6, 11, 35, 41).The completion of the entire papillomavirus life cycle is species specific. However, studies with bovine papillomavirus (BPV) in horses and hamsters, HPV pseudovirions in mouse challenge models, and quasivirions in rabbits suggest that virion internalization and delivery of the encapsidated DNA to the nucleus are promiscuous and that tropism is determined at a later stage of the life cycle (11, 27, 29, 39).Although significant progress has been made in understanding the HPV life cycle and virion structure, many of the molecular events of virus internalization and infection are poorly defined (43). Both the L1 (major) and L2 (minor) capsid proteins provide essential functions during infection (41) (8). L1 is sufficient to form empty capsids, termed virus-like particles (VLPs) (25), which bind to basement membrane and to the cell surface and which also form the basis of the licensed HPV vaccines (10). Glycosaminoglycans (GAGs), most notably heparan sulfate (HS), play a critical role in virion binding and infection, both in vitro and in the murine vaginal challenge model, although differences between HPV types and target cells in vitro have been described (14, 19, 20), for example, between HPV16 and HPV31 (4, 34, 42). Once bound to the basement membrane, the virions undergo a conformation change resulting in the surface display of the amino terminus of L2 and its cleavage by a proprotein convertase (PC), furin and/or PC5/PC6, and the transfer of virions to the cell surface (24). The uptake of the virions is apparently slow as late addition of neutralizing antibodies several hours after initial cell surface binding prevents infection in vitro (9). The endocytic mechanisms reported for various papillomavirus types are diverse, but furin cleavage of L2 and endosomal acidification are critical shared steps (15, 38). In a late endosomal compartment, the L1 capsid disassembles, releasing L2 associated with the previously encapsidated DNA to gain access to the nucleus by an unknown mechanism and to accumulate at the subnuclear domain, ND10 (13). Although L2 contains a C-terminal nuclear localization signal (17), entry to mitosis, which is associated with the dissolution of the nuclear membrane, is required for infection, suggesting that the complex with the viral nucleohistone core is unable pass through nuclear pores (36). It is unclear how the L2-genome complex escapes the endocytic compartment, but the carboxy terminus of L2 also contains both DNA binding and a membrane-destabilizing peptide (21).γ Secretase is an intramembranously cleaving protease (I-CliP) linked to Alzheimer''s disease through its cleavage of amyloid precursor protein (APP) (1). It is a multicomponent complex, and presenilin (PS) is the catalytic unit whose active site contains two aspartate residues. In addition to the nine-pass transmembrane protein PS, γ secretase requires nicastrin (NCT), anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 in an equimolar ratio for proteolytic activity (28). The subcellular localization of γ secretase is controversial but includes the endoplasmic reticulum (23), endosome (26), lysosome (31), and plasma membrane (37), all of which are subcellular locales possibly traversed by papillomavirus during infection (43).By analogy to the cleavage of L2 by furin that is critical for exit from the endosomes (38), we hypothesized that I-CLiP might contribute to papillomavirus infection. Here, we report that a γ-secretase inhibitor prevents HPV infection both in vitro and in the mouse vaginal challenge model and that cell lines lacking essential components of γ secretase are refractory to HPV infection.     

Analysis of Modified Human Papillomavirus Type 16 L1 Capsomeres: the Ability To Assemble into Larger Particles Correlates with Higher Immunogenicity

L1 capsomeres purified from Escherichia coli represent an economic alternative to the recently launched virus-like particle (VLP)-based prophylactic vaccines against infection with human papillomavirus types 16 and 18 (HPV-16 and HPV-18), which are causative agents of cervical cancer. It was recently reported that capsomeres are much less immunogenic than VLPs. Numerous modifications of the L1 protein leading to the formation of capsomeres but preventing capsid assembly have been described, such as the replacement of the cysteine residues that form capsid-stabilizing disulfide bonds or the deletion of helix 4. So far, the influence of these modifications on immunogenicity has not been thoroughly investigated. Here, we describe the purification of eight different HPV-16 L1 proteins as capsomeres from Escherichia coli. We compared them for yield, structure, and immunogenicity in mice. All L1 proteins formed almost identical pentameric structures yet differed strongly in their immunogenicity, especially regarding the humoral immune responses. Immunization of TLR4^−/− mice and DNA immunization by the same constructs confirmed that immunogenicity was independent of different degrees of contamination with copurifying immune-stimulatory molecules from E. coli. We hypothesize that immunogenicity correlates with the intrinsic ability of the capsomeres to assemble into larger particles, as only assembly-competent L1 proteins induced high antibody responses. One of the proteins (L1ΔN10) proved to be the most immunogenic, inducing antibody titers equivalent to those generated in response to VLPs. However, preassembly prior to injection did not increase immunogenicity. Our data suggest that certain L1 constructs can be used to produce highly immunogenic capsomeres in bacteria as economic alternatives to VLP-based formulations.Certain types of human papillomavirus (HPV) are the cause of cervical cancer, most frequently HPV types 16 and 18 (HPV-16 and HPV-18), which are responsible for about 50% and 20% of cases, respectively (8, 15, 16). Recently, two vaccines that prevent infection with HPV-16 and HPV-18 have been introduced to the market. These vaccines are based on the viral major structural protein L1, which can spontaneously self-assemble in vitro into empty virus-like particles (VLPs) that resemble the native virions in size and shape. VLPs have been shown to be highly immunogenic, as they can induce high titers of neutralizing antibodies (29, 30). HPV virions and VLPs consist of 72 L1 pentamers, also called capsomeres, which are arranged in an icosahedral T=7 particle lattice with a diameter of 55 nm. Cryo-electron microscopic analysis has revealed the presence of 60 hexavalent and 12 pentavalent capsomeres (4).Capsid assembly has been reported to be optimal at low pH (pH 5.4) and high ionic strength, whereas both high pH (pH 8.2) and the presence of reducing agents favor disassembly into capsomeres, the latter because the viral particles are stabilized by intercapsomeric disulfide bonds between two conserved cysteine residues at positions 175 and 428 (11, 35, 44). VLP formation is not affected by deletions of up to 9 amino acids (aa) from the N terminus and up to 34 aa from the C terminus of the L1 protein (11, 36). An N-terminally truncated L1 protein lacking 10 aa has been shown to assemble into particles consisting of 12 L1-pentamers with a T=1 lattice referred to as small VLPs (11, 12). Crystallographic analysis of the T=1 particles revealed that interpentameric contacts are established by hydrophobic interactions between the α-helices 2 and 3 of one capsomere and α-helix 4 of a neighboring capsomere (12). Consequently, a mutant L1 with helix 4 deleted formed homogenous capsomeres but failed in T=1 and T=7 particle assembly (7). Deletion of helices 2 and 3 impeded even pentamer formation, as a large fraction of the L1 protein was found to be insoluble, which suggests an essential role for these regions in L1 folding (7, 11).VLP-based prophylactic vaccines have been shown to induce high titers of neutralizing antibodies, which protect against virus challenge and associated diseases in humans (24, 31). However, due to the relatively high production and distribution costs of the vaccines—they are expressed in and purified from eukaryotic cells and require a cold chain for storage—they will probably be largely unavailable to developing countries, where more than 80% of all cervical cancer cases occur (1, 38, 46).L1 capsomeres represent a potentially lower cost alternative to VLPs, as they can be produced in large amounts from Escherichia coli and are considered more stable at room temperature (11, 34, 35). Capsomeres have been shown to induce high titers of neutralizing antibodies and T-cell responses upon oral, intranasal, and subcutaneous immunization and have also protected against viral challenge in the canine oral papillomavirus model (18, 19, 37, 42, 48, 53). Most of the immunization data for HPV capsomeres have been obtained from administration of full-length or N-terminally deleted (10 aa) wild-type L1 proteins (18, 37, 53). A recent report in which the L1 pentamers were derived from an L1 protein in which the conserved cysteines (aa 175 and 428) were replaced by alanines revealed that HPV-16 VLPs induce about 20- to 40-fold-higher humoral immune responses than capsomeres (47). The influence on immunogenicity of the other mutations and deletions of the L1 protein that prevent capsid assembly has so far not been studied in depth.In a comparative analysis of eight differently modified HPV-16 L1 proteins purified as capsomeres from E. coli, we now report that their potential to induce humoral immune responses in mice correlates with their ability to assemble into particles larger than capsomeres. One of the constructs, L1ΔN10, encoded for capsomeres that exhibited immunogenicity similar to that of VLPs.     

Abrogation of the Postmitotic Checkpoint Contributes to Polyploidization in Human Papillomavirus E7-Expressing Cells

High-risk types of human papillomavirus (HPV) are considered the major causative agents of cervical carcinoma. The transforming ability of HPV resides in the E6 and E7 oncogenes, yet the pathway to transformation is not well understood. Cells expressing the oncogene E7 from high-risk HPVs have a high incidence of polyploidy, which has been shown to occur as an early event in cervical carcinogenesis and predisposes the cells to aneuploidy. The mechanism through which E7 contributes to polyploidy is not known. It has been hypothesized that E7 induces polyploidy in response to mitotic stress by abrogating the mitotic spindle assembly checkpoint. It was also proposed that E7 may stimulate rereplication to induce polyploidy. We have tested these hypotheses by using human epithelial cells in which E7 expression induces a significant amount of polyploidy. We find that E7-expressing cells undergo normal mitoses with an intact spindle assembly checkpoint and that they are able to complete cytokinesis. Our results also exclude DNA rereplication as a major mechanism of polyploidization in E7-expressing cells upon microtubule disruption. Instead, we have shown that while normal cells arrest at the postmitotic checkpoint after adaptation to the spindle assembly checkpoint, E7-expressing cells replicate their DNA and propagate as polyploid cells. Thus, abrogation of the postmitotic checkpoint leads to polyploidy formation in E7-expressing human epithelial cells. Our results suggest that downregulation of pRb is important for E7 to induce polyploidy and abrogation of the postmitotic checkpoint.An important hallmark of human cancers is aneuploidy, the state in which a cell has extra or missing chromosomes (12, 25). Polyploidy is the state in which cells have more than two equal sets of chromosomes and is thought to be an early event in multistep carcinogenesis that can lead to aneuploidy (1, 24), as exemplified in Barrett''s esophagus (11). Polyploidy has recently been shown to occur as an early event in cervical carcinogenesis and to predispose the cells to aneuploidy (26). Other recent studies have shown that tetraploid but not diploid mouse or human cells induce tumor formation in mice (3, 9). These studies highlight the potential importance of polyploidy in carcinogenesis.The cellular mechanisms responsible for this polyploidy formation are as of yet undetermined, but several models have been proposed. First, abrogation of the spindle assembly checkpoint followed by cleavage failure may lead to polyploidy formation (36, 40). A second proposed model is rereplication, a process of multiple rounds of DNA replication without an intervening mitosis. Third, cells that adapt to the mitotic spindle checkpoint halt in a G₁-like state with 4C DNA content. Abrogation of this postmitotic checkpoint allows the cells to replicate their 4C DNA content, leading to polyploidy formation. This has been shown in cells that express the human papillomavirus type 16 (HPV-16) E6 oncogene that degrades p53 (21). Finally, cleavage failure, which yields binucleate cells with 4C DNA content, is also a potential mechanism for polyploidy formation (31).The postmitotic checkpoint becomes activated when cells with an intact spindle assembly checkpoint become arrested during mitosis for a prolonged period of time and eventually adapt to the checkpoint, exit mitosis without cleavage, and progress into a G₁-like state with 4C DNA content (19, 22). The cells are prevented from continuing through the cell cycle and replicating their DNA by a proposed p53- and pRb-dependent postmitotic checkpoint (18, 19).High-risk types of HPV (of which HPV-16 is the most prevalent) are commonly associated with lesions that can progress to cervical carcinoma, which is one of the leading causes of cancer death in women worldwide (42). The transforming properties of high-risk HPVs primarily reside in the E6 and E7 oncogenes (reviewed in reference 7). The ability of high-risk HPV E6 and E7 proteins to promote the degradation of p53 and pRb, respectively, has been suggested as a mechanism by which HPV induces cellular transformation (6, 30). Expression of the high-risk HPV E6 and E7 oncogenes in human keratinocytes leads to polyploidy, which is enhanced by DNA damage and by activation of the spindle checkpoint through microtubule disruption (15, 27, 37, 38).Previously, it was thought but not directly shown that high-risk E6 and E7 induce polyploidy in response to microtubule disruption by abrogating the spindle checkpoint and that degradation of the tumor suppressor p53 by E6 is the mechanism by which E6 accomplishes this polyploidy formation (27, 37, 38). Others have proposed that E7 may play a role in stimulating DNA rereplication that occurs prior to mitosis initiation and polyploidy formation (20). Our recent studies demonstrate that E6 does not affect the mitotic spindle checkpoint (21). Instead, E6 abrogates the postmitotic checkpoint to induce polyploidy after microtubule disruption. Interestingly, E6 mutant proteins defective in inducing p53 degradation also induce polyploidy (21). The mechanism by which HPV E7 induces polyploidy remains to be determined. In this study, we investigate these possible mechanisms through which HPV-16 E7 induces polyploidy formation.     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

The Ubiquitin-Specific Peptidase USP15 Regulates Human Papillomavirus Type 16 E6 Protein Stability

Proteomic identification of human papillomavirus type 16 (HPV16) E6-interacting proteins revealed several proteins involved in ubiquitin-mediated proteolysis. In addition to the well-characterized E6AP ubiquitin-protein ligase, a second HECT domain protein (HERC2) and a deubiquitylating enzyme (USP15) were identified by tandem affinity purification of HPV16 E6-associated proteins. This study focuses on the functional consequences of the interaction of E6 with USP15. Overexpression of USP15 resulted in increased levels of the E6 protein, and the small interfering RNA-mediated knockdown of USP15 decreased E6 protein levels. These results implicate USP15 directly in the regulation of E6 protein stability and suggest that ubiquitylated E6 could be a substrate for USP15 ubiquitin peptidase activity. It remains possible that E6 could affect the activity of USP15 on specific cellular substrates, a hypothesis that can be tested as more is learned about the substrates and pathways controlled by USP15.Human papillomaviruses (HPVs) are associated with several human cancers, most notably human cervical cancer, the second most common cancer among women worldwide (43). Papillomaviruses cause proliferative squamous epithelial lesions, and more than 100 HPV types have been described (14). The HPV types associated with mucosal squamous epithelial lesions have been further classified into high- or low-risk types based on the propensity for the lesions with which they are associated to progress to cancer. Among the high-risk HPV types, HPV type 16 (HPV16) and HPV18 account for approximately 70% of cervical cancers (43). The high-risk HPV types carry two genes, the E6 and E7 genes, which have oncogenic properties and are always expressed in HPV-positive cancers. E6 and E7 interfere with the p53 and retinoblastoma (pRB) tumor suppressor pathways, respectively, and contribute directly to cell cycle alterations, protection from apoptosis, and transformation (14). The dysregulated expression of the E6 and E7 oncoproteins is an important step in the progression from a preneoplastic stage to cancer in HPV-infected cells and is often a consequence of the integration of the viral genome into the host chromosome.The interaction between E6 and p53 is mediated by the E3 ubiquitin ligase E6AP (15). E6, p53, and E6AP form a complex in which E6 directs the ligase activity of E6AP to p53, thereby targeting p53 for ubiquitin-mediated degradation (36). E6, however, has a number of other cellular partners and other functions. For instance, the C terminus of the high-risk E6 protein contains a PDZ binding motif (20, 25) that mediates the interaction with several PDZ domain-containing proteins, including discs large (Dlg), Scribble (Scrib), the MAGI family of proteins, MUPP1, and PATJ (9, 10, 29). Some of these proteins are also targeted for degradation in an E6AP-dependent manner (22, 29). While the major mechanism of oncogenesis revolves around E6''s ability to inhibit the proapoptotic effects of p53, recent work involving the PDZ domain proteins indicates that these interactions are also important to the oncogenic potential of E6 (38, 41). Furthermore, E6 has been reported to bind a number of other cellular proteins, including but not limited to Bak, CBP/p300, c-Myc, E6TP1, hADA3, IRF3, MCM7, PTPH1, and TNF-R1 (7, 8, 17, 23, 24, 32, 35, 39, 40). The importance of the binding of several of these proteins with regard to the transformation or other functions of E6 remains to be established. E6 itself is thought to be targeted for degradation by an ubiquitin-proteasome pathway (18), although how E6 protein stability is regulated has not been well studied.Many of the E6 binding partners have been identified using purified bacterially expressed E6 fusion proteins and cell lysates from various cell types or using yeast two-hybrid screenings. While some of these interactions with E6 have been validated, the physiologic relevance of a number of proposed E6 targets remains undetermined. In an effort to identify E6-interacting proteins, perhaps under more physiologic conditions, we employed tandem affinity purification (TAP) using tagged HPV16 E6 stably expressed in the HPV16-positive cervical cancer cell line SiHa. We have discovered several new interacting proteins, including an interaction between E6 and the cellular deubiquitylating enzyme (DUB) USP15. USP15 is not targeted for degradation by E6, but we found that USP15 stabilizes E6 protein levels, suggesting that E6 may itself be a target for USP15 DUB activity.     

Nuclear Export of Human Papillomavirus Type 31 E1 Is Regulated by Cdk2 Phosphorylation and Required for Viral Genome Maintenance

The initiator protein E1 from human papillomavirus (HPV) is a helicase essential for replication of the viral genome. E1 contains three functional domains: a C-terminal enzymatic domain that has ATPase/helicase activity, a central DNA-binding domain that recognizes specific sequences in the origin of replication, and a N-terminal region necessary for viral DNA replication in vivo but dispensable in vitro. This N-terminal portion of E1 contains a conserved nuclear export signal (NES) whose function in the viral life cycle remains unclear. In this study, we provide evidence that nuclear export of HPV31 E1 is inhibited by cyclin E/A-Cdk2 phosphorylation of two serines residues, S92 and S106, located near and within the E1 NES, respectively. Using E1 mutant proteins that are confined to the nucleus, we determined that nuclear export of E1 is not essential for transient viral DNA replication but is important for the long-term maintenance of the HPV episome in undifferentiated keratinocytes. The findings that E1 nuclear export is not required for viral DNA replication but needed for genome maintenance over multiple cell divisions raised the possibility that continuous nuclear accumulation of E1 is detrimental to cellular growth. In support of this possibility, we observed that nuclear accumulation of E1 dramatically reduces cellular proliferation by delaying cell cycle progression in S phase. On the basis of these results, we propose that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect keratinocytes of the differentiating epithelium of the skin or mucosa (reviewed in references 4 and 63). Of more than 150 different HPV types identified thus far, about 25 infect the anogenital region (9). The low-risk types, such as HPV11 and HPV6, are associated with the development of genital warts, while the high-risk types, such as HPV16, -18, and -31, cause high-grade lesions that can progress to invasive cervical carcinoma (17, 38, 61).The HPV life cycle is coupled with the differentiation program that keratinocytes undergo in the epithelium. After infection of the basal cell layer of the epithelium, the virus establishes and maintains its genome as an extrachromosomal element (episome) in the nucleus of infected cells. While the viral episome is maintained at low levels in basal cells, its amplification to a high copy number is trigged in the upper layers of the epithelium by the action of the viral oncogenes E6 and E7 and the differentiation of the infected keratinocytes (reviewed in reference 21). Replication of the HPV genome relies on the viral proteins E1 and E2 and the host DNA replication machinery. Viral DNA replication is initiated by the binding of E2 to specific sites on the viral origin where it facilitates the recruitment and assembly of E1 into a double hexamer that is required to unwind DNA ahead of the bidirectional replication fork (3, 14, 15, 31, 33, 36, 43-45, 52, 60). In addition to its helicase activity, E1 interacts with several cellular replication factors, including polymerase α-primase, replication protein A (RPA), and topoisomerase I, to replicate the viral episome (5, 6, 19, 32, 35, 39).E1, which belongs to helicase superfamily III (SF3) (22, 26), can be divided into three functional regions. Its C-terminal domain has ATPase and helicase activity and can self-assemble into hexamers. It is also this domain that is contacted by E2 to recruit E1 at the origin (50, 57, 58). The middle portion of E1 encompasses the origin-binding domain (OBD) that binds and dimerizes on specific sequences in the origin (55, 56). We and others previously found that a fragment of E1 containing only the C-terminal enzymatic domain and the OBD is capable of supporting viral DNA replication in vitro but is inactive in vivo (2, 51). This suggested that the N-terminal region of E1 plays an essential regulatory function in vivo. As such, it has been shown for HPV11 E1 that this region contains a cyclin E/A-Cdk2 (cyclin-dependent kinase 2) binding motif (CBM), a bipartite nuclear localization signal (NLS) and an CRM1-dependent nuclear export signal (NES), which together regulate the nucleocytoplasmic shuttling of the protein (10, 30, 34). Specifically, it has been shown that phosphorylation of HPV11 E1 on three serine residues within its N-terminal region inhibits its nuclear export (10, 62). Interestingly, bovine papillomavirus (BPV) E1 was also shown to shuttle between the nucleus and the cytoplasm in a phosphorylation-dependent manner. In this case, however, Cdk2 phosphorylation was found to promote, rather than inhibit, the export of the viral helicase (24). This apparent discrepancy between HPV11 and BPV E1 prompted us to examine the regulation of a third E1 protein, specifically that of the high-risk HPV31.We report here that HPV31 E1 also shuttles between the nucleus and the cytoplasm through its conserved NLS and NES. We determined that nuclear export of HPV31 E1 is dependent on the CRM1 export pathway and is inhibited by Cdk2 phosphorylation of serines 92 and 106. We also found that nuclear export of E1 is not required for transient viral DNA replication and thus investigated its role in viral genome maintenance and amplification in immortalized keratinocytes. In contrast to the wild type (WT), a mutant genome carrying a defective E1 NES was poorly maintained and progressively lost upon cell division, indicating that nuclear export of E1 is required for long-term maintenance of the viral episome. Because nuclear export of E1 is not required for viral DNA replication per se but needed for episomal maintenance over several cell divisions, we investigated the possibility that continuous accumulation of E1 into the nucleus is detrimental to cellular proliferation. In support of this possibility, we found that the accumulation of E1 at high levels in the nucleus impedes cellular proliferation by delaying cell cycle progression in the S phase. In addition, we found that this delay was alleviated when nuclear export of E1 was increased. Altogether, these results suggest that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.     

Intradermal Vaccination with Influenza Virus-Like Particles by Using Microneedles Induces Protection Superior to That with Intramuscular Immunization

Influenza virus-like particles (VLPs) are a promising cell culture-based vaccine, and the skin is considered an attractive immunization site. In this study, we examined the immunogenicity and protective efficacy of influenza VLPs (H1N1 A/PR/8/34) after skin vaccination using vaccine dried on solid microneedle arrays. Coating of microneedles with influenza VLPs using an unstabilized formulation was found to decrease hemagglutinin (HA) activity, whereas inclusion of trehalose disaccharide preserved the HA activity of influenza VLP vaccines after microneedles were coated. Microneedle vaccination of mice in the skin with a single dose of stabilized influenza VLPs induced 100% protection against challenge infection with a high lethal dose. In contrast, unstabilized influenza VLPs, as well as intramuscularly injected vaccines, provided inferior immunity and only partial protection (≤40%). The stabilized microneedle vaccination group showed IgG2a levels that were 1 order of magnitude higher than those of other groups and had the lowest lung viral titers after challenge. Also, levels of recall immune responses, including hemagglutination inhibition titers, neutralizing antibodies, and antibody-secreting plasma cells, were significantly higher after skin vaccination with stabilized formulations. Therefore, our results indicate that HA stabilization, combined with vaccination via the skin using a vaccine formulated as a solid microneedle patch, confers protection superior to that with intramuscular injection and enables potential dose-sparing effects which are reflected by pronounced increases in rapid recall immune responses against influenza virus.Influenza is a major health threat among infectious diseases, posing a significant burden for public health worldwide. Over 200,000 hospitalizations and approximately 36,000 deaths are estimated to occur annually in the United States alone (48, 49). Vaccination is the most cost-effective measure for controlling influenza. However, the influenza vaccine needs to be updated and manufactured every year due to changes in circulating viral strains. Current influenza vaccines rely on egg substrate-based production, a lengthy process with limited capacity that can cause shortages in available vaccine supplies. The recent 2009 outbreak of H1N1 influenza virus is a good example of the urgent need to develop a more effective vaccine platform and vaccination method (38).Influenza virus-like particles (VLPs) have been suggested as a promising alternative candidate to current influenza vaccines. Influenza VLPs are noninfectious particles that mimic the virus in structure and morphology, can be produced using an egg-free cell culture system, and have been shown to be highly immunogenic, inducing protective immunity (9, 15, 19, 27, 35, 41, 42, 44). Most current vaccines are administered intramuscularly to humans in liquid formulations using hypodermic needles or syringes. Another strategy to meet the potential need for mass vaccination would be to develop an effective method for vaccine delivery to the skin (4, 8, 32, 50, 52). The skin is considered an important peripheral immune organ rich in potent immune-inducing cells, including Langerhans cells (LCs), dermal dendritic cells (DCs), and keratinocytes (5, 13, 14, 22). LCs and DCs residing in the epidermal and dermal layers of the skin have been shown to play an important role in antigen processing and presentation following skin immunization (1, 13, 14, 22). Intradermal (ID) vaccination delivering antigens to the dermal layer of the skin has been performed in many clinical studies and have demonstrated dose-sparing effects in some cases (4, 28, 29). Particularly, ID delivery of vaccines might be more effective in the elderly population (50), the highest risk group for influenza epidemics (49). However, ID delivery of vaccines using hypodermic needles is painful and needs highly trained medical personnel. In addition, more frequent local reactions at the injection site were observed after ID delivery. Therefore, a simple and effective approach for vaccination without using hypodermic needles would be highly desirable.To overcome the skin barrier of the outer layer of stratum corneum, solid microneedles were previously coated with inactivated influenza viruses and used to successfully deliver vaccines to the skin, which provided protection comparable to that with conventional intramuscular immunizations (32, 52). Other vaccines have also been delivered using microneedles (17, 17a), but VLPs have never been used this way before. Delivery of a powdered form of inactivated influenza vaccines to the skin has also been demonstrated using a high-speed jet delivery device (10). These previous studies used high doses of vaccines, possibly due to the instability of vaccines in dry formulations.Influenza hemagglutinin (HA) is responsible for attachment of the virus to sialic acid-containing receptors on target cells. However, it is not well understood how functional activity of HA affects the immunogenicity of influenza VLP vaccines. For the first time in this study, we investigated the effect of HA stability, immune responses, and protective efficacies of solid-microneedle VLP vaccines containing H1 HA as a major influenza viral component after delivery to the skin in comparison to results with intramuscular immunization. We found that the functional integrity of HA in influenza VLPs significantly influenced the immunological and protective outcomes for both microneedle and intramuscular vaccination. In addition, we have observed differential outcomes contributing to the protective immunity by the delivery of HA-stabilized VLPs to the skin in terms of the types of immune responses, recall antibody responses, and viral clearance at an early time point after challenge compared to those induced by intramuscular immunization.     

Env-Expressing Autologous T Lymphocytes Induce Neutralizing Antibody and Afford Marked Protection against Feline Immunodeficiency Virus

The envelope (Env) glycoproteins of HIV and other lentiviruses possess neutralization and other protective epitopes, yet all attempts to induce protective immunity using Env as the only immunogen have either failed or afforded minimal levels of protection. In a novel prime-boost approach, specific-pathogen-free cats were primed with a plasmid expressing Env of feline immunodeficiency virus (FIV) and feline granulocyte-macrophage colony-stimulating factor and then boosted with their own T lymphocytes transduced ex vivo to produce the same Env and interleukin 15 (3 × 10⁶ to 10 × 10⁶ viable cells/cat). After the boost, the vaccinees developed elevated immune responses, including virus-neutralizing antibodies (NA). Challenge with an ex vivo preparation of FIV readily infected all eight control cats (four mock vaccinated and four naïve) and produced a marked decline in the proportion of peripheral CD4 T cells. In contrast, five of seven vaccinees showed little or no traces of infection, and the remaining two had reduced viral loads and underwent no changes in proportions of CD4 T cells. Interestingly, the viral loads of the vaccinees were inversely correlated to the titers of NA. The findings support the concept that Env is a valuable immunogen but needs to be administered in a way that permits the expression of its full protective potential.Despite years of intense research, a truly protective AIDS vaccine is far away. Suboptimal immunogenicity, inadequate antigen presentation, and inappropriate immune system activation are believed to have contributed to these disappointing results. However, several lines of evidence suggest that the control or prevention of infection is possible. For example, despite repeated exposures, some individuals escape infection or delay disease progression after being infected (1, 14, 15). Furthermore, passively infused neutralizing antibodies (NA) (28, 42, 51) or endogenously expressed NA derivatives (29) have been shown to provide protection against intravenous simian immunodeficiency virus challenge. On the other hand, data from several vaccine experiments suggest that cellular immunity is an important factor for protection (6, 32). Therefore, while immune protection against human immunodeficiency virus (HIV) and other lentiviruses appears feasible, the strategies for eliciting it remain elusive.Because of its crucial role in viral replication and infectivity, the HIV envelope (Env) is an attractive immunogen and has been included in nearly all vaccine formulations tested so far (28, 30, 31). Env surface (SU) and transmembrane glycoproteins (gp) are actively targeted by the immune system (9, 10, 47), and Env-specific antibodies and cytotoxic T lymphocytes (CTLs) are produced early in infection. The appearance of these effectors also coincides with the decline of viremia during the acute phase of infection (30, 32). Individuals who control HIV infection in the absence of antiretroviral therapy have Env-specific NA and CTL responses that are effective against a wide spectrum of viral strains (14, 23, 35, 52, 60). At least some of the potentially protective epitopes in Env appear to interact with the cellular receptors during viral entry and are therefore highly conserved among isolates (31, 33, 39, 63). However, these epitopes have complex secondary and tertiary structures and are only transiently exposed by the structural changes that occur during the interaction between Env and its receptors (10, 11, 28). As a consequence, these epitopes are usually concealed from the immune system, and this may explain, at least in part, why Env-based vaccines have failed to show protective efficacy. Indeed, data from previous studies suggested that protection may be most effectively triggered by nascent viral proteins (22, 28, 30, 48, 62).We have conducted a proof-of-concept study to evaluate whether presenting Env to the immune system in a manner as close as possible to what occurs in the context of a natural infection may confer some protective advantage. The study was carried out with feline immunodeficiency virus (FIV), a lentivirus similar to HIV that establishes persistent infections and causes an AIDS-like disease in domestic cats. As far as it is understood, FIV evades immune surveillance through mechanisms similar to those exploited by HIV, and attempts to develop an effective FIV vaccine have met with difficulties similar to those encountered with AIDS vaccines (25, 37, 66). In particular, attempts to use FIV Env as a protective immunogen have repeatedly failed (13, 38, 58). Here we report the result of one experiment in which specific-pathogen-free (SPF) cats primed with a DNA immunogen encoding FIV Env and feline granulocyte-macrophage colony-stimulating factor (GM-CSF) and boosted with viable, autologous T lymphocytes ex vivo that were transduced to express Env and feline interleukin 15 (IL-15) showed a remarkable level of protection against challenge with ex vivo FIV. Consistent with recent findings indicating the importance of NA in controlling lentiviral infections (1, 59, 63), among the immunological parameters investigated, only the titers of NA correlated inversely with protection. Collectively, the findings support the notion that Env is a valuable vaccine immunogen but needs to be administered in a way that permits the expression of its full protective potential.