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Development of cervical cancer is associated with persistent infections by high-risk human papillomavirus (HPV). Although current HPV L1-based prophylactic vaccines prevent infection, they do not help to eliminate prevalent infections or lesions. Our aims were (i) to generate a vaccine combining prophylactic and therapeutic properties by producing chimeric capsomers after fusion of the L1 protein to different fragments of E2 from HPV 16, and (ii) to evaluate their capacity to generate an antitumoral cellular response, while conserving L1 neutralizing epitopes. Chimeric proteins were produced in Escherichia coli and purified by glutathione S-transferase (GST)-affinity chromatography. Their structure was characterized using size exclusion chromatography, sucrose gradient centrifugation, electron microscopy, and anti-L1 enzyme-linked immunosorbent assay. All chimeric proteins form capsomers and heterogeneous aggregates. One, containing part of the carboxy-terminal domain of E2 and its hinge region (L1Δ+E2H/NC, aa 206-307), conserved the neutralizing epitope H16.V5. We then evaluated the capacity of this chimeric protein to induce a cytotoxic T-cell response against HPV 16 E2. In (51)Cr release cytotoxicity assays, splenocytes from C57BL/6 immunized mice recognized and lysed TC-1/E2 cells, which express and present endogenously processed E2 peptides. Moreover, this E2-specific cytotoxic response inhibited the growth of tumors of TC-1/E2 cells in mice. Finally, we identified an epitope (aa 292-301) of E2 involved in this cytotoxic response. We conclude that the L1Δ+E2H/NC chimeric protein produced in bacteria can be an effective and economically interesting candidate for a combined prophylactic and therapeutic vaccine that could help eliminating HPV16-positive low-grade cervical lesions and persistent viral infections, thus preventing the development of lesions and, at the same time, the establishment of new infections.

Recently, two prophylactic vaccines against the most significant oncogenic human papillomaviruses (HPV; 16 and 18) became available that efficiently protect against persistent HPV infection and cancer precursors. However, clinical trials performed with these vaccines did not provide evidence that they would influence the natural history of prevalent HPV infections, that is, their eventual malignant progression. Because, even under the optimistic assumption of high vaccine coverage, a significant reduction of cancer incidence can only be expected after two decades, there is a need for immune therapeutic strategies to be offered to persistently infected individuals who do not benefit from the prophylactic vaccines. Here, we describe the reasons for failure of most of the published approaches to HPV-specific therapies, highlight promising developments and present our view for future developments.

The potential as prophylactic vaccines of L1-based particles from cutaneous genus alpha human papillomavirus (HPV) types has not been assessed so far. However, there is a high medical need for such vaccines since HPV-induced skin warts represent a major burden for children and for immunocompromised adults, such as organ transplant recipients. In this study, we have examined the immunogenicity of capsomeres and virus-like particles (VLPs) from HPV types 2, 27, and 57, the most frequent causative agents of skin warts. Immunization of mice induced immune responses resembling those observed upon vaccination with HPV 16 L1-based antigens. The antibody responses were cross-reactive but type-restricted in their neutralizing capacities. Application of adjuvant led to an enhanced potential to neutralize the respective immunogen type but did not improve cross-neutralization. Vaccination with capsomeres and VLPs from all four analyzed HPV types induced robust IFNγ-associated T-cell activation. Immunization with mixed VLPs from HPV types 2, 27, and 57 triggered an antibody response similar to that after single-type immunization and capable of efficiently neutralizing all three types. Our results imply that vaccination with combinations of VLPs from cutaneous HPV types constitutes a promising strategy to prevent HPV-induced skin lesions.

Human papillomavirus (HPV) L1 self-assembles into virus-like particles (VLPs), which are the basis for the two commercially available prophylactic vaccines. For one of them (Cervarix®) HPV 16 and 18 VLPs are being produced in insect cells using the baculovirus expression system. However, due to low yield, production of VLPs remains challenging for certain other PV types. Here we report that employment of a modified baculovirus-based (MultiBac) expression system (Berger, I., Fitzgerald, D. J., and Richmond, T. J. (2004). Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol. 22(12), 1583–7) permits substantially improved VLP production of several PV types up to 40-fold. Highest VLP yields were achieved when two copies of the L1 gene were expressed from independently controlled cassettes. We have evaluated the production of HPV 57 L1 VLPs by the MultiBac system in more detail. Whereas the level of the HPV 57 L1 protein was only slightly increased in comparison to the standard protocol we monitored a strongly enhanced yield of HPV 57 VLPs. Our results imply that a critical concentration of L1 within the producer cell is required for efficient VLPs assembly. We show evidence that in addition a dominant negative factor in conventionally produced recombinant baculoviruses contributes to differences in VLP yield. This phenomenon might be attributable to the absence of the viral cysteine protease V-CATH in the modified baculovirus system. We anticipate that use of the MultiBac expression system will facilitate capsid production for papillomaviruses and thereby enable the generation of vaccines against infections by many of the as yet untargeted HPV types.

HPV 16 L1 capsomeres purified from Escherichia coli represent a promising and potentially cost-effective alternative to the recently licensed VLP-based vaccines for the prevention of cervical cancer. However, recombinant protein preparations from bacteria always bear the risk of contaminating endotoxins which are highly toxic in humans and therefore have to be eliminated from vaccine preparations. In this study, we measured the LPS concentration at various stages of the purification of HPV 16 L1 from E. coli and determined that it enhances the immunogenicity of HPV 16 VLPs and capsomeres. We confirmed the immunogenicity of the L1 capsomeres in TLR4(-/-) mice without the enhancing effect of the LPS and then elaborated a suitable protocol using Triton X-114 phase separation for the removal of LPS without any significant protein loss or influence on the structural integrity of the particles. The LPS-free capsomeres purified from E. coli induced neutralizing L1-specific antibodies. Our results demonstrate the excellent potential of capsomeres as an economically interesting alternative vaccine to prevent cervical cancer that could be made available in developing countries.

Epidemiologische Untersuchungen deuteten schon lange darauf hin, dass Zervixkarzinome als Folge einer durch Geschlechtsverkehr ubertragenen Infektion entstehen. Papillomviren des Menschen (HPV) wurden als verantwortliche Agentien vorgeschlagen, da Mitglieder der Papillomvirus-Familie bei Tieren Tumore auslosen konnen und HPV-Infektionen im Genitaltrakt des Menschen haufig vorkommen. Verschiedene Befunde – Entdeckung der Vielfalt der Papillomviren, regelmasiger Nachweis der Genome bestimmter, sog. Hochrisiko-HPV-Typen in Tumorbiopsien, Verstandnis des Mechanismus der Zelltransformation, sowie informative epidemiologische Studien – fuhrten schlieslich zum Konsens, dass Infektionen mit diesen Viren Zervixkarzinome und andere Krebsformen des Menschen verursachen konnen. Diese Kenntnis fuhrte letztendlich zur Entwicklung von Impfstoffen als Mittel der Krebspravention. Since a long time epidemiologic studies suggested a sexually transmitted infectious agent as cause of cervical cancer. Human papillomaviruses (HPV) were considered as causative agents since members of the papillomavirus family can induce tumors in animals, and genital infections with papillomaviruses in man are common. Several discoveries ranging from the identification of the plurality of HPVs, the regular finding of genomes of certain, so called high-risk HPV types within tumor biopsies, insights into the transforming mechanism and informative epidemiological surveys finally led to the conclusion within the scientific community of a causative link between these viruses and cervical cancer as well as other human malignancies. This awareness triggered the development of HPV-specific vaccines as means of cancer prevention.

This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette containing sequences of 2 diagnostic primer sets for foot-and-mouth disease virus (FMDV) and a set for swine vesicular disease virus (SVDV) was engineered into a full-length cDNA clone containing the RNA-2 segment of Cowpea Mosaic Virus (CPMV). After co-inoculation with a plasmid that expressed CPMV RNA-1, recombinant virus particles were rescued from cowpea plants (Vigna unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants, the particles are thermostable, RNase resistant and simple purification of RNA-2 containing capsids yields a preparation which is non-infectious.

The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas.

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.

L1 capsomeres purified fromEscherichia colirepresent 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 fromEscherichia 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 fromE. 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.