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The wild-type E6 and E7 genes of human papillomavirus type 16 (HPV16) can cooperate to immortalize normal human keratinocytes in culture. The E6 open reading frame of HPV16 and other HPV types highly associated with cervical cancer has the potential of encoding both full-length E6 and two truncated E6* proteins, the latter being generated via splicing within the E6 open reading frame portion of the E6-E7 polycistronic transcript. Those types, such as HPV6, that are infrequently associated with cervical carcinoma lack the splice site and encode only a full-length E6. We have now found that, in addition to cooperating with E7 to immortalize keratinocytes, HPV16 E6 can induce anchorage-independent growth in NIH 3T3 cells and trans-activate the adenovirus E2 promoter. HPV6 E6 was also able to trans-activate the adenovirus E2 promoter, although it was inactive in both cell transformation assays. An HPV16 splice site mutant which expressed only the full-length HPV16 E6 was active in all three assays, indicating that the E6* proteins are not required for these activities. The plasmid which encodes the E6* proteins was inactive and did not potentiate the activity of the HPV16 splice site mutant. The mutation that prevented splicing in E6-E7 mRNA severely reduced the level of E7 protein and increased E6 protein. Taken together, the results suggest that the primary function of the splice within E6 is to facilitate the translation of E7 and reduce translation of full-length E6, rather than to generate biologically active E6* proteins.

This review focuses on recent publications of clinical trials of two prophylactic human papillomavirus (HPV) vaccines: Gardasil® (Merck & Co., Inc., Whitehouse Station, NJ USA), a quadrivalent vaccine containing L1 virus-like particles (VLPs) of types -6, 11, 16, and 18, and Cervarix™ (GlaxoSmithKline Biologicals, Rixensart, Belgium), a bivalent vaccine containing VLPs of types -16 and 18. In efficacy trials involving young women, both vaccines produced outstanding efficacy against primary and secondary endpoints associated with the vaccine type HPVs and were highly and consistently immunogenic. Both had excellent safety records and, as expected, the most frequent vaccine-related adverse were mild to moderate injection site sequelae. No evidence of waning protection was observed after four years for endpoints examined ranging from incident infection to cervical intraepithelial neoplasia grade 3 associated with the vaccine type HPVs. Gardasil® was also highly efficacious at preventing vaginal/vulvar lesions and genital warts. However, neither vaccine demonstrated therapeutic efficacy against prevalent infections or lesions, regardless of the associated HPV type. Cervarix™ has shown limited cross-protection against infection with specific closely related types while preliminary results of limited cross-protection have been presented for Gardasil®. As expected, more limited efficacy was noted for both vaccines when women with prevalent infection were included or endpoints associated with any HPV type were evaluated. Immunological bridging trials involving adolescent girls and boys were also recently published. For both vaccines, serum VLP antibody levels in girls were non-inferior to those generated in young women and antibody response to Gardasil® was also non-inferior in boys. The results of these studies have led to the approval of Gardasil® and Cervarix™ by national regulatory agencies in a number of countries.

Previously, we reported that the oral administration of high molecular mass poly-gamma-glutamate (gamma-PGA) induced antitumor immunity but the mechanism underlying this antitumor activity was not understood. In the present study, we found that application of high molecular mass gamma-PGA induced secretion of tumor necrosis factor (TNF)-alpha from the bone-marrow-derived macrophages of wild type (C57BL/6 and C3H/HeN) and Toll-like receptor 2 knockout (TLR2(-/-)) mice, but not those of myeloid differentiation factor 88 knockout (MyD88(-/-)) and TLR4-defective mice (C3H/HeJ). Production of interferon (IFN)-gamma-inducible protein 10 (IP-10) in response to treatment with gamma-PGA was almost abolished in C3H/HeJ mice. In contrast to LPS, gamma-PGA induced productions of TNF-alpha and IP-10 could not be blocked by polymyxin B. Furthermore, gamma-PGA-induced interleukin-12 production was also impaired in immature dendritic cells (iDCs) from MyD88(-/-) and C3H/HeJ mice. Downregulation of MyD88 and TLR4 expression using small interfering RNA (siRNA) significantly inhibited gamma-PGA-induced TNF-alpha secretion from the RAW264.7 cells. Gamma-PGA-mediated intracellular signaling was markedly inhibited in C3H/HeJ cells. The antitumor effect of gamma-PGA was completely abrogated in C3H/HeJ mice compared with control mice (C3H/HeN) but significant antitumor effect was generated by the intratumoral administration of C3H/HeN mice-derived iDCs followed by 2,000 kDa gamma-PGA in C3H/HeJ. These findings strongly suggest that the antitumor activity of gamma-PGA is mediated by TLR4.

Cervical cytological screening, as first proposed by Papanicolaou, is one of the most successful cancer prevention strategies ever devised (1). Nevertheless, cervical cancer is the second or third most frequent cancer and cause of cancer deaths in women (2). This paradox has arisen because many of the world’s women either do not have access to an effective screening program or do not properly comply with an existing program (1). The problem of access is particularly acute in developing countries, where the high cost of screening limits access to only the wealthiest subset of women (3). Consequently, cervical cancer is the leading cause of cancer death in many developing countries (2). Countries with effective screening programs spend a substantial amount of health care financial resources on them. The cost of screening and follow-up of abnormal Papanicolaou (Pap) smears was recently estimated to cost more than $5 billion annually in the United States alone (National HPV & Cervical Cancer Prevention Resource Center; http://www.ashastd.org/hpvccrc/background.html). Thus cervical cancer prevention strategies that are both more accessible and acceptable to women need to be developed.

Persistent infection with the high-risk subset of genitotropic human papillomavirus (HPV) genotypes is a necessary cause of cervical cancer. Given the global burden of cervical cancer, a low-cost, broadly protective vaccine is needed. RG-1 is a cross-neutralizing and protective monoclonal antibody that recognizes residues 17–36 of HPV16 minor capsid protein L2. Because this epitope is highly conserved in divergent HPV types, we determined whether vaccination with HPV16 L2 17–36 peptide is broadly protective. The peptide was administered to BALB/c mice three times at monthly intervals, either alone or in the context of a synthetic lipopeptide vaccine candidate (P25-P2C-HPV) produced by linkage of the HPV peptide with a broadly recognized T helper epitope (P25) and the Toll-like receptor-2 (TLR2) ligand dipalmitoyl- S -glyceryl cysteine (P2C). In contrast to vaccination with the L2 17–36 peptide or P25-P2C alone, a potent L2-specific antibody response was generated to the P25-P2C-HPV lipopeptide when delivered either s.c. or intranasally. Sera from mice vaccinated with the P25-P2C-HPV lipopeptide neutralized not only HPV16 pseudovirions but also other evolutionarily divergent oncogenic genital (HPV18, HPV45) and cutaneous (HPV5, BPV1) types. The L2-specific antibody response depended on MHC class II, CD40, and MyD88 signaling. Additionally, vaccination with the P25-P2C-HPV lipopeptide protected mice from homologous challenge with HPV16 pseudovirions at cutaneous and genital sites and heterologous challenge with HPV45 pseudovirions. If provided in the appropriate context, therefore, HPV16 L2 17–36 might be used in a totally synthetic cross-protective HPV vaccine.

Papillomaviruses are a family of nonenveloped DNA tumor viruses. Some sexually transmitted human papillomavirus (HPV) types, including HPV type 16 (HPV16), cause cancer of the uterine cervix. Papillomaviruses encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein, L2, is not required for capsid formation, it is thought to participate in encapsidation of the viral genome and plays a number of essential roles in the viral infectious entry pathway. The abundance of L2 and its arrangement within the virion remain unclear. To address these questions, we developed methods for serial propagation of infectious HPV16 capsids (pseudoviruses) in cultured human cell lines. Biochemical analysis of capsid preparations produced using various methods showed that up to 72 molecules of L2 can be incorporated per capsid. Cryoelectron microscopy and image reconstruction analysis of purified capsids revealed an icosahedrally ordered L2-specific density beneath the axial lumen of each L1 capsomer. The relatively close proximity of these L2 density buttons to one another raised the possibility of homotypic L2 interactions within assembled virions. The concept that the N and C termini of neighboring L2 molecules can be closely apposed within the capsid was supported using bimolecular fluorescence complementation or “split GFP” technology. This structural information should facilitate investigation of L2 function during the assembly and entry phases of the papillomavirus life cycle.

To determine the cellular pathways induced by an HPV-16 L1 VLP vaccine, we compared the gene expression signature in L1 VLP-stimulated PBMC from 19 vaccine and 7 placebo recipients before vaccinati...

The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30–60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing., Author Summary To replicate, viruses have to enter into host cells. Since they have no means of locomotion, they rely entirely on cellular transport systems to access the cellular compartments where replication occurs. Following individual virus particles by video microscopy, we found that human papillomavirus type 16, the main causative agent of cervical cancer, bound to long finger-like protrusions of cells. There, they were transported from the periphery to the cell body. The transport was mediated by a process termed actin retrograde flow, where viruses bound to cell surface molecules hooked up to filamentuos actin and were dragged along with the actin-like items on a transport belt. Entry into the cell occured at the cell body. The results raised the interesting possibility that viruses use retrograde flow when they infect wounded epidermal keratinocytes, where finger-like protrusions of cells are abundant.

Some genital human papillomavirus (HPV) types, such as 16 and 18, are highly associated with malignant cervical tumors while others, such as HPV 6, are only rarely found in these malignancies. The E7 oncoproteins of HPV 6, 16 and 18 each have a 17 amino acid region with striking homology to adenovirus E1a and SV40 LT. E1a, LT and the E7 oncoprotein of HPV16 all bind the cellular Rb protein in vitro, and for E1a and LT this region of homology contains sequences essential for interaction with Rb. We have now found that in HPV 16 E7 this region (amino acids 21-37) contains two separate biochemical activities, each of which contributes to E7-mediated transformation. Rb binding was localized to the N terminus of this region, while the C terminus was shown to serve as a substrate for casein kinase (CK) II, which phosphorylated serine-31 and serine-32. Replacement of the two serines by non-phosphorylatable amino acids led to a reduction in transforming activity and abolished phosphorylation but did not affect Rb binding. Rb binding and CK II phosphorylation were also examined for the E7 proteins of HPV 6 and HPV 18. HPV 16 and 18 E7 bound similar amounts of Rb, but HPV 6 E7 consistently bound less. Phosphorylation rates also varied, with HPV 18 E7 being 2-fold faster than HPV 16 E7, which in turn was 2-fold faster than HPV 6 E7. We conclude that Rb binding and phosphorylation of E7 by CKII are independent activities which are required for efficient transformation by E7 and that these activities correlate directly with the relative oncogenic potential of these viruses.

Human papillomavirus (HPV) vaccines based on L1 virus-like particles (VLPs) can prevent HPV-induced genital neoplasias, the precursors of cervical cancer. However, most cervical cancers occur in developing countries, where the implementation of expensive vaccines requiring multiple injections will be difficult. A live Salmonella -based vaccine could be a lower-cost alternative. We previously demonstrated that high HPV type 16 (HPV16)-neutralizing titers are induced after a single oral immunization of mice with attenuated Salmonella enterica serovar Typhimurium strains expressing a codon-optimized version of HPV16 L1 (L1S). To allow the testing of this type of vaccine in women, we constructed a new L1-expressing plasmid, kanL1S, and tested kanL1S recombinants of three Salmonella enterica serovar Typhi vaccine strains shown to be safe in humans, i.e., Ty21a, the actual licensed typhoid vaccine, and two highly immunogenic typhoid vaccine candidates, Ty800 and CVD908- htrA . In an intranasal mouse model of Salmonella serovar Typhi infection, Ty21a kanL1S was unique in inducing HPV16-neutralizing antibodies in serum and genital secretions, while anti- Salmonella responses were similar to those against the parental Ty21a vaccine. Electron microscopy examination of Ty21a kanL1S lysates showed that L1 assembled in capsomers and capsomer aggregates but not well-ordered VLPs. Comparison to the neutralizing antibody response induced by purified HPV16 L1 VLP immunizations in mice suggests that Ty21a kanL1S may be an effective prophylactic HPV vaccine. Ty21a has been widely used against typhoid fever in humans with a remarkable safety record. These finds encourage clinical testing of Ty21a kanL1S as a combined typhoid fever/cervical cancer vaccine with the potential for worldwide application.

To determine the systemic cytokine pattern induced by vaccination with human papillomavirus (HPV) L1 virus-like particles (VLP), we analyzed 22 different cytokines in culture supernatants of L1 VLP-stimulated peripheral blood mononuclear cells from vaccine ( n = 19) and placebo ( n = 7) recipients at months 0 and 2 after vaccination, using a multiplex cytokine bead array. In vaccine recipients, incubation with L1 VLP in vitro led to a statistically significant increase in production of Th1 (granulocyte-macrophage colony-stimulating factor, interleukin-2 [IL-2], gamma interferon; P < 0.0007) and Th2 (IL-4, IL-5, IL-10, IL-13; P < 0.0017) cytokines and the chemokine IP-10 ( P = 0.0021) at month 2 after immunization, compared to levels seen prior to vaccination. These responses were not seen in placebo recipients. Cytokine and neutralizing antibody responses to vaccination followed the same pattern, with the highest antibody responses seen for subjects with higher cytokine responses. Cytokine profiling studies using samples from efficacy trials may provide important information about discriminators of long-term protection against HPV.

Background: Previous reports from the Costa Rica Vaccine Trial demonstrated strong vaccine efficacy against HPV16/18 at the cervical, anal, and oral regions separately. However, the combined “woman-level” vaccine efficacy against infections at all three anatomic sites has not been examined in women with and without previous HPV16/18 exposure. Methods: Women aged 18-25 from the Costa Rica Vaccine Trial were randomized to be vaccinated with the HPV16/18 Vaccine (Cervarix) or a Hepatitis A vaccine at enrollment. Cervical samples were collected at every annual visit, while oral and anal samples were collected only at the four year follow-up visit. Samples were tested for alpha mucosal HPV DNA types utilizing the SPF10 PCR-DEIA-LiPA25 version 1 method. An event in the multi-site woman-level vaccine efficacy analysis (n = 4,186) was defined as a women with prevalent HPV16/18 DNA at the cervical, anal, or oral regions. Vaccine efficacies (VEs) and 95% confidence intervals (95%CIs) were computed for one-time detection of HPV16/18 in the cervical, anal, and oral regions in this intention-to-treat analysis. Results: Four years following initial vaccination, the combined multi-site woman-level vaccine efficacy against HPV16/18 infections was 64.8%, 95%CI = 54.8-72.8. Multi-site woman-level efficacy was stronger among women without evidence of previous HPV exposure (HPV16/18 seronegative and cervical HPV16/18 DNA negative at enrollment): VE = 83.1%, 95%CI = 72.6-89.6, but was also demonstrated among women with evidence of previous HPV16/18 exposure (HPV16/18 seropositive and cervical HPV16/18 DNA negative at baseline): VE = 49.6%, 95%CI = 2.7-73.9. Further supporting the partial protection of the vaccine in previously HPV16/18-exposed women, we observed a particularly strong vaccine efficacy against HPV16/18 at more than one anatomic site (VE = 91.4%, 95%CI = 81.4-96.6). Indeed, HPV16/18-infected women were significantly less likely to be HPV16/18-infected at two or more anatomic sites in the HPV vaccine arm than the control arm (6 of 81 (7%) vs. 70 of 230 (30%), p Discussion: This is the first study to present a combined multi-site woman-level HPV16/18 vaccine efficacy. This study found strong multi-site efficacy among those not previously exposed to cervical HPV16/18, but also suggests the vaccine may provide some protection against HPV16/18 at multiple anatomic sites among women previously exposed to HPV16/18. If confirmed, the partial protection against cervical, anal, and/or oral HPV16/18 in women previously exposed to HPV16/18 could be considered in HPV vaccination catch-up program decision-making. Citation Format: Daniel C. Beachler, Aimee R. Kreimer, Mark Schiffman, Rolando Herrero, Sholom Wacholder, Ana Cecilia Rodriguez, Douglas R. Lowy, Carolina Porras, John T. Schiller, Silvia Jimenez, Linda Struijk, John Schussler, Allan Hildesheim, Paula Gonzalez, Costa Rica Vaccine Trial Group. Efficacy of the HPV16/18 vaccine against cervical, anal, and oral HPV infection among women with and without previous HPV16/18 exposure. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4680. doi:10.1158/1538-7445.AM2015-4680

Recent clinical trials in young women have shown that subunit vaccines based on human papillomavirus (HPV) 16 and HPV18 L1 virus-like particles are ∼100% effective in short-term prevention of persistent cervical infection and of cervical dysplasia by these major oncogenic types. These remarkable efficacy results, together with an excellent safety profile in thousands of vaccinated women, have led to the HPV prophylactic vaccine from one manufacturer having now been licensed for commercial use and the expectation that the vaccine from a second manufacturer will be approved in the near future. These vaccines seem to have great potential for reducing cervical cancer deaths and treatments to remove premalignant cervical lesions. However, before their public health effect can be fully estimated, several issues must be addressed. These include duration of protection, degree of cross-protection against nonvaccine types, efficacy in men, and vaccine availability to economically disadvantaged women. (Cancer Res 2006; 66(21): 10229-32)

Cervical cancer results from cervical infection by human papillomaviruses (HPV), especially HPV16. Intramuscular administrations of HPV16 virus-like particle (VLP) vaccines have been shown to induce strong neutralizing antibody responses and protect women against genital HPV16 infection and associated lesions. However, an alternative route of administration that avoids parenteral injection might facilitate vaccine implementation, particularly in developing countries which account for the majority of the worldwide cases of cervical cancer. In addition, inducing mucosal immunity could partially overcome the substantial variation in HPV16 antibodies at the cervix seen in ovulating women. Aerosol vaccination with HPV16 VLPs was previously shown to be immunogenic in mice and in women. Here, we examine whether exposure to other respiratory viral antigens may interfere with the HPV16 VLP-specific humoral response and whether two known mucosal adjuvants, CpG oligodeoxynucleotides and a natural non-toxic Escherichia coli heat-labile enterotoxin (HLT), can enhance the immunogenicity of airway immunization (nasal or aerosol-like) of mice with HPV16 VLPs. Our data show that HLT can significantly improve anti-VLP humoral responses in serum and mucosal secretions, as well as VLP-specific proliferative responses and IFN-gamma production by CD8 T cells, and that recent exposure to influenza surface antigens can diminish mucosal, but not systemic, antibody responses to the VLPs.

Certain sexually transmitted human papillomavirus (HPV) types are causally associated with the development of cervical cancer. Our recent development of high-titer HPV pseudoviruses has made it possible to perform high-throughput in vitro screens to identify HPV infection inhibitors. Comparison of a variety of compounds revealed that carrageenan, a type of sulfated polysaccharide extracted from red algae, is an extremely potent infection inhibitor for a broad range of sexually transmitted HPVs. Although carrageenan can inhibit herpes simplex viruses and some strains of HIV in vitro, genital HPVs are about a thousand-fold more susceptible, with 50% inhibitory doses in the low ng/ml range. Carrageenan acts primarily by preventing the binding of HPV virions to cells. This finding is consistent with the fact that carrageenan resembles heparan sulfate, an HPV cell-attachment factor. However, carrageenan is three orders of magnitude more potent than heparin, a form of cell-free heparan sulfate that has been regarded as a highly effective model HPV inhibitor. Carrageenan can also block HPV infection through a second, postattachment heparan sulfate–independent effect. Carrageenan is in widespread commercial use as a thickener in a variety of cosmetic and food products, ranging from sexual lubricants to infant feeding formulas. Some of these products block HPV infectivity in vitro, even when diluted a million-fold. Clinical trials are needed to determine whether carrageenan-based products are effective as topical microbicides against genital HPVs., Synopsis Sexually transmitted human papillomavirus (HPV) infections are very common. Although most HPV infections don't cause noticeable symptoms, persistent infection with some genital HPV types can lead to cervical cancer or other anal/genital cancers. Another subset of HPV types can cause genital warts. Recent studies have suggested that condoms are not highly effective in preventing HPV infection. Although HPV vaccines will soon become available, they probably will not protect against all genital HPV types and will be too expensive for use in the developing world. Inexpensive HPV-inhibitory compounds (known as topical microbicides) might be useful for blocking the spread of HPV. Using a newly developed cell culture–based HPV inhibition test, we have discovered that an inexpensive gelling agent called carrageenan is an unexpectedly potent HPV infection inhibitor. Carrageenan is also under investigation as a topical microbicide targeting HIV and herpes viruses, but it is a thousand times more effective against HPV in cell culture tests. Interestingly, carrageenan is used as a thickener in some commercially available sexual lubricants and lubricated condoms. Several of these commercial lubricant products are potent HPV inhibitors in our cell culture–infection system. Clinical trials are needed to determine the effectiveness of carrageenan as a topical microbicide against HPV.

Human papillomavirus (HPV) infection causes virtually all cases of cervical cancer, the second most common cause of death from cancer among women worldwide. This Review examines prophylactic HPV subunit vaccines based on the ability of the viral L1 capsid protein to form virus-like particles (VLPs) that induce high levels of neutralizing antibodies. Following preclinical research by laboratories in the nonprofit sector, Merck and GlaxoSmithKline are developing commercial versions of the vaccine. Both vaccines target HPV16 and HPV18, which account for approximately 70% of cervical cancer. The Merck vaccine also targets HPV6 and HPV11, which account for approximately 90% of external genital warts. The vaccines have an excellent safety profile, are highly immunogenic, and have conferred complete type-specific protection against persistent infection and associated lesions in fully vaccinated women. Unresolved issues include the most critical groups to vaccinate and when the vaccine's cost may be low enough for widespread implementation in the developing world, where 80% of cervical cancer occurs.

Each year, esophageal and gastric cancers cause more than 900,000 deaths worldwide. Human papilloma virus (HPV), especially type 16, has been suggested to have a role in the etiology of esophageal cancer, however, the results of previous seroepidemiological studies have not been consistent. We conducted a large prospective study to examine the association between serum antibodies to HPV 16, HPV 18 and HPV 73 and subsequent development of esophageal squamous cell carcinoma (ESCC), gastric cardia adenocarcinoma (GCA), and gastric noncardia adenocarcinoma (GNCA) in a high-risk population for these cancers in Linxian, China. Case and control subjects for this study were selected from the 29,584 participants of the Linxian General Population Trial. Prediagnostic serum samples from 99 cases of ESCC, 100 cases of GCA, 70 cases of GNCA, and 381 age- and sex- matched controls were selected for this study. The presence of antibodies to HPV virus-like particles was determined by type-specific enzyme-linked immunosorbent assays. Fewer than 15% of ESCC, GCA, or GNCA cases were positive for each HPV type, and no significant associations were found. The adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) for HPV 16 seropositivity and ESCC, GCA, and GNCA risk were 1.6 (0.8-3.3), 1.3 (0.6-2.8) and 0.4 (0.1-1.6), respectively. The comparable ORs (95% CIs) for HPV 18 were 1.0 (0.4-2.2), 0.9 (0.4-2.1) and 1.5 (0.6-3.4). For HPV 73, these figures were 1.3 (0.6-2.5), 1.2 (0.6-2.3) and 0.9 (0.4-2.1). The results of this study do not support a major role for HPV 16, HPV 18 and HPV 73 in the etiology of esophageal and gastric cancers in Linxian, China.

We identified the strategies to induce a CTL response to human papillomavirus (HPV) 16 E2 in HLA-A2 transgenic mice (AAD). A chimeric HPV16 virus-like particle (VLP) that includes full length HPV16 E7 and E2 (VLP-E7E2) was generated. The combination of E2 and E7 has the advantage that E2 is expressed in early dysplasia and neoplasia lesions, where E7 is expressed in more advance lesions. Since T cell response to E2 is less defined, we first evaluated the strategies to enhancing CD8(+) T cell responses to HPV E7, using different combinations of immune-modulators with VLP-E7E2. Data showed that the CTL response to E7 could be significantly enhanced by coinjection of GM-CSF and anti-CD40 antibodies with chimeric VLP-E7E2 without adjuvant. However, using the same combination, a low level of CD8(+) T cell response to E2 was detected. To enhance the CD8+ T cell response to E2, we analyzed T cell epitopes from E2 sequence. A heterogenous prime-boost with chimeric VLP-E7E2 and E2 peptides was performed. The data showed that the priming with chimeric VLP-E7E2, followed by boosting with E2 peptides, gave a better CTL response than 2 immunizations with E2 peptides. The enhanced immunity is due to the increase of CD11c(+) and CD11c(+) CD40(+) double positive dendritic cells in mice that received immune-modulators, GM-CSF and anti-CD40. Furthermore, the level of anti-L1 antibodies remains similar in mice immunized with chimeric VLP with/without immune-modulators. Thus, the data suggested that the chimeric VLP-E7E2 has a therapeutic potential for the treatment of HPV-associated CINs and cancer without diminishing VLPs potential as a prophylactic vaccine by inducing anti-L1 antibodies against free virus.

The finding that cervical cancer only occurs in women infected with specific, "high-risk" types of the human papillomavirus (HPV) has led to the development of novel, non-cytology-based cervical cancer prevention strategies. We now have sensitive molecular methods for detecting HPV that dramatically improve our ability to detect high-grade cervical cancer precursor lesions. Perhaps more importantly, prophylactic HPV vaccines have been developed that are protective against cervical cancer precursors caused by HPV 16 and 18. In the Spring of 2006, over 100 experts in HPV, cervical cancer screening, and vaccination worked together to define how best to incorporate HPV DNA testing and the HPV vaccines into cervical cancer prevention efforts. In this summary, we summarize the opinions of this expert group on how these advances can be introduced to provide the maximum benefit to women and to reduce the global burden of cervical cancer.

Papillomaviruses are internalized via clathrin-dependent endocytosis. However, the mechanism by which viral genomes pass endosomal membranes has not been elucidated. In this report we show that the minor capsid protein L2 is required for egress of viral genomes from endosomes but not for initial uptake and uncoating and that a 23-amino-acid peptide at the C terminus of L2 is necessary for this function. Pseudogenomes encapsidated by L1 and L2 lacking this peptide accumulated in vesicular compartments similar to that observed with L1-only viral particles, and these mutant pseudoviruses were noninfectious. This L2 peptide displayed strong membrane-disrupting activity, induced cytolysis of bacteria and eukaryotic cells in a pH-dependent manner, and permeabilized cells after exogenous addition. Fusions between green fluorescent protein and the L2 peptide integrated into cellular membranes like the wild type but not like C-terminal mutants of L2. Our data indicate that the L2 C terminus facilitates escape of viral genomes from the endocytic compartment and that this feature is conserved among papillomaviruses. Furthermore, the characteristic of this peptide differs from the classical virus-encoded membrane-penetrating peptides.

It has recently become possible to generate high-titer papillomavirus-based gene-transfer vectors. The vectors, also known as papillomavirus pseudoviruses (PsV), have been useful for studying papillomavirus assembly, entry, and neutralization, and may have future utility as laboratory gene-transfer tools or vaccine vehicles. This chapter outlines a simple method for production of PsV and their use in a high-throughput papillomavirus neutralization assay. The production method is based on transfection of a 293 cell line, 293TT, engineered to express high levels of SV40 large T antigen. The cells are co-transfected with codon-modified papillomavirus capsid genes, L1 and L2, together with a pseudogenome plasmid containing the SV40 origin of replication. Pseudogenome encapsidation within L1/L2 capsids is largely sequence independent, and plasmids entirely lacking PV sequences can be packaged efficiently, provided they are less than 8 kilobases in size. Non-infectious virus-like particles (VLPs) can also be produced after transfection of 293TT cells with L1 alone. Efficient purification of the PsV or VLPs is achieved by Optiprep (iodixanol) density gradient ultracentrifugation. Using these methods, it is possible to produce highly purified PsV with yields of at least 10(9) transducing units from a single 75-cm2 flask of cells. PsV encapsidating a secreted alkaline phosphatase (SEAP) reporter plasmid were used to develop a high-throughput in vitro neutralization assay in a 96-well plate format. Infection of 293TT cells is monitored by SEAP activity in the culture supernatant, using a highly sensitive chemiluminescent reporter system. Antibody-mediated PsV neutralization is detected by a reduction in SEAP activity. The neutralization assay has similar analytic sensitivity to, and higher specificity than, a standard VLP-based enzyme-linked immunosorbent assay (ELISA).

It has recently become possible to generate high-titer papillomavirus-based gene-transfer vectors. The vectors, also known as papillomavirus pseudoviruses (PsV), have been useful for studying papillomavirus assembly, entry, and neutralization, and may have future utility as laboratory gene-transfer tools or vaccine vehicles. This chapter outlines a simple method for production of PsV and their use in a high-throughput papillomavirus neutralization assay. The production method is based on transfection of a 293 cell line, 293TT, engineered to express high levels of SV40 large T antigen. The cells are co-transfected with codon-modified papillomavirus capsid genes, L1 and L2, together with a pseudogenome plasmid containing the SV40 origin of replication. Pseudogenome encapsidation within L1/L2 capsids is largely sequence independent, and plasmids entirely lacking PV sequences can be packaged efficiently, provided they are less than 8 kilobases in size. Non-infectious virus-like particles (VLPs) can also be produced after transfection of 293TT cells with L1 alone. Efficient purification of the PsV or VLPs is achieved by Optiprep (iodixanol) density gradient ultracentrifugation. Using these methods, it is possible to produce highly purified PsV with yields of at least 10(9) transducing units from a single 75-cm2 flask of cells. PsV encapsidating a secreted alkaline phosphatase (SEAP) reporter plasmid were used to develop a high-throughput in vitro neutralization assay in a 96-well plate format. Infection of 293TT cells is monitored by SEAP activity in the culture supernatant, using a highly sensitive chemiluminescent reporter system. Antibody-mediated PsV neutralization is detected by a reduction in SEAP activity. The neutralization assay has similar analytic sensitivity to, and higher specificity than, a standard VLP-based enzyme-linked immunosorbent assay (ELISA).

Vaccination with papillomavirus L2 has been shown to induce neutralizing antibodies that protect against homologous type infection and cross-neutralize a limited number of genital HPVs. Surprisingly, we found that antibodies to bovine papillomavirus (BPV1) L2 amino acids 1–88 induced similar titers of neutralizing antibodies against Human papillomavirus (HPV)16 and 18 and BPV1 pseudoviruses and also neutralized HPV11 native virions. These antibodies also neutralized each of the other pseudovirus types tested, HPV31, HPV6 and Cottontail rabbit papillomavirus (CRPV) pseudoviruses, albeit with lower titers. HPV16, HPV18, HPV31, HPV6 and CRPV L2 anti-sera also displayed some cross-neutralization, but the titers were lower and did not encompass all pseudoviruses tested. This study demonstrates the presence of broadly cross-neutralizing epitopes at the N-terminus of L2 that are shared by cutaneous and mucosal types and by types that infect divergent species. BPV1 L2 was exceptionally effective at inducing cross-neutralizing antibodies to these shared epitopes.

Objective : To study human papillomavirus type 16 in the aetiology of cervical carcinoma. Design : Within a cohort of 18814 Finnish women followed for up to 23 years a nested case-control study was conducted based on serological diagnosis of past infection with human papillomavirus type 16. Subjects : 72 women (27 with invasive carcinoma and 45 with in situ carcinoma) and 143 matched controls were identified during the follow up. Main outcome measure : Relative risk of cervical carcinoma in presence of IgG antibodies to human papillomavirus type 16. Results : After adjustment for smoking and for antibodies to various other agents of sexually transmitted disease, such as herpes simplex virus type 2 and Chlamydia trachomatis, the only significant association was with infection with human papillomavirus type 16 (odds ratio 12.5; 95% confidence interval 2.7 to 57, 2P Conclusion : This prospective study provides epidemiological evidence that infection with human papillomavirus type 16 confers an excess risk for subsequent development of cervical carcinoma. Key messages Key messages Human papillomavirus type 16 is the main micro-organism linked to the development of cervical cancer Prospective studies of infection with this virus and cervical cancer have not been reported because of ethical and clinical difficulties and because diagnosis of past infections with the virus has not been possible In this nested case-control study in over 18000 Finnish women who donated blood to a serum bank 25 years ago we were able to measure past infection with human papillomavirus type 16 with new serological tools The results show that infection with the virus confers an increased risk of developing cervical cancer

Cervical cancer results from cervical infection by human papillomaviruses (HPV), especially HPV16. Previous studies have shown that intramuscular vaccination of women with an HPV16 virus-like particle (VLP) vaccine induced a strong IgG response and protected against genital HPV16 infection. However, an alternative route of administration that avoids parenteral injection while inducing mucosal immunity might facilitate vaccine implementation in some settings, and partially overcome the substantial variation in HPV16 antibodies at the cervix seen in ovulating women. In this study, women were vaccinated with escalating doses of HPV16L1 VLPs via nasal nebulisation, bronchial aerosolisation, or a combination of intramuscular and aerosol vaccination. The alternative routes of vaccination were well tolerated and many of the volunteers who received aerosol vaccinations exhibited serum antibody titers that were comparable to those induced by intramuscular vaccination. A mucosal immune response was induced by aerosol vaccination as demonstrated by the induction of anti-HPV16 VLP IgA secreting cells in PBMC and SIgA in secretions. Our data suggest that aerosol administration of HPV VLPs may represent a potential alternative to parenteral injection.

Prophylactic vaccines against infectious diseases are among the most successful and cost effective public health interventions ever devised. They have led to the eradication of small pox and will likely soon lead to the eradication of polio. Prophylactic vaccination has also effectively controlled other infectious diseases, including rubella, mumps, tetanus, and diphtheria (Ulmer and Liu, 2002). Perhaps the most important implication of the establishment of sexually transmitted HPV infection as the central cause of cervical cancer (Bosch et al., 2002) is the potential for cervical cancer prevention through prophylactic vaccination against the oncogenic HPVs. This chapter will focus on the development of vaccines to prevent infection and neoplastic disease by oncogenic HPV types that infect the genital tract. However, similar vaccines are being developed to prevent infection by the non-oncogenic types that are largely responsible for genital warts.

Although the papillomavirus structural proteins, L1 and L2, can spontaneously coassemble to form virus-like particles, currently available methods for production of L1/L2 particles capable of transducing reporter plasmids into mammalian cells are technically demanding and relatively low-yield. In this report, we describe a simple 293 cell transfection method for efficient intracellular production of papillomaviral-based gene transfer vectors carrying reporter plasmids. Using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 as model papillomaviruses, we have developed a system for producing papillomaviral vector stocks with titers of several billion transducing units per milliliter. Production of these vectors requires both L1 and L2, and transduction can be prevented by papillomavirus-neutralizing antibodies. The stocks can be purified by an iodixanol (OptiPrep) gradient centrifugation procedure that is substantially more effective than standard cesium chloride gradient purification. Although earlier data had suggested a potential role for the viral early protein E2, we found that E2 protein expression did not enhance the intracellular production of BPV1 vectors. It was also possible to encapsidate reporter plasmids devoid of BPV1 DNA sequences. BPV1 vector production efficiency was significantly influenced by the size of the target plasmid being packaged. Use of 6-kb target plasmids resulted in BPV1 vector yields that were higher than those with target plasmids closer to the native 7.9-kb size of papillomavirus genomes. The results suggest that the intracellular assembly of papillomavirus structural proteins around heterologous reporter plasmids is surprisingly promiscuous and may be driven primarily by a size discrimination mechanism.

Background: In early-phase trials, a human papillomavirus 16 (HPV16) virus-like particle (VLP) vaccine has been shown to be well tolerated, immunogenic, and protective against HPV16 in women, most of whom were taking oral contraceptives. Previous studies have not determined whether HPV immunization results in specific antibody levels in the human genital tract or whether these levels might vary during contraceptive or ovulatory cycles. Therefore, we determined the levels of total and specific antibodies in the cervical secretions of women who had been immunized with HPV16 VLPs and examined the influence of the menstrual cycle and oral contraceptive use on these levels. Methods: Two groups of women were immunized, seven who were taking oral contraceptives and 11 who were ovulating. After seroconversion, serum and cervical secretions were collected twice weekly for 5 weeks. Total immunoglobulins (IgG and IgA) and vaccine-specific IgGs were determined by enzyme-linked immunosorbent assay. Nonparametric statistical analyses were used to determine the statistical significance of differences in IgG levels between groups, and correlations between serum- and cervical-specific IgG levels were determined by the Spearman correlation coefficient. Results: All participants developed detectable titers of anti-HPV16 VLP IgGs in their cervical secretions after immunization. The cervical titers of specific IgG and total IgGs and IgAs among participants in the contraceptive group were relatively constant throughout the contraceptive cycle. In contrast, the cervical titers of specific IgG and total IgGs and IgAs among participants in the ovulatory group varied during the menstrual cycle, being highest during the proliferative phase, decreasing approximately ninefold around ovulation, and increasing approximately threefold during the luteal phase. Serum- and cervical-specific IgG levels were correlated (r = .86) in women in the contraceptive group but not in women in the ovulatory group (r = .27). Conclusions: The relatively high titer of anti-HPV16 antibodies at the cervix is promising in terms of vaccine efficacy; however, the decrease in antibody titer around ovulation raises the possibility that the HPV16 VLP vaccine might be less effective during the peri-ovulatory phase

Infection with human papillomavirus (HPV) type 16 has been suggested to be a risk factor for squamous cell carcinoma of the head and neck (SCCHN) and to be more commonly associated with SCCHN occurring in the oropharynx and in never-smokers. We hypothesized that HPV-16 exposure, as evidenced by seropositivity, is a risk factor for SCCHN and may be of particular importance in never-smokers.To test this hypothesis, we conducted a hospital-based case-control study of 120 patients with SCCHN (60 never-smokers and 60 matched smokers) and 120 cancer-free matched controls. We compared the presence of HPV-16 antibodies in ever-smoker and never-smoker patients matched on age (+/-5 years), sex, and tumor site. Each patient was also matched with a corresponding ever-smoker or never-smoker cancer-free control on age (+/-5 years) and sex. Serum was collected from study subjects and assayed for IgG reactivity to HPV-16 L1 virus-like particles by using an ELISA.Forty-nine of the 120 case subjects (40.8%) but only 11 (9.2%) of the control subjects tested positive for HPV-16 antibodies (adjusted odds ratio, 6.69; 95% confidence interval, 3.01-14.90). Among cases, HPV-16 seropositivity was more common in those with oropharyngeal cancer (41 of 70, 58.6%) and poorly differentiated tumors (25 of 43, 58.1%). HPV-16 seropositivity was associated with a significantly increased risk of oropharyngeal cancer (adjusted odds ratio, 59.53; 95% confidence interval, 5.71-620.20). Whereas HPV-16 seropositivity was more common in never-smokers with SCCHN than in ever-smokers (43.3% versus 38.3%, respectively), this difference was not statistically significant.HPV-16 infection is associated with a significant increased risk for oropharyngeal cancer but not oral cavity cancer. Furthermore, HPV-16 infection does not appear to be more common in never-smokers than ever-smokers with SCCHN.

Chimeric human papillomavirus virus-like particles (HPV cVLPs), containing the HPV16 non-structural protein E7, are potent vaccines for inducing antigen-specific protective immunity against HPV-transformed tumors in animal models. Previous data demonstrated that the effectiveness of cytotoxic T lymphocyte (CTL) induction after repetitive vaccination with the same cVLP, and thus vaccine efficacy, is limited by the presence of neutralizing antibodies induced after the first application. Here, we determined if altering the route of vaccine delivery or incorporation of the target antigen into VLPs of a heterologous papillomavirus type could overcome inhibition of MHC class I antigen presentation by neutralizing antibodies, resulting in a boosting of CD8(+) T-cell responses against the incorporated antigen, HPV16 E7. Mucosal delivery of cVLPs resulted in detection of systemic E7-specific CD8(+) T cells, however, these routes were not able to bypass the inhibitory effect of circulating antibodies against homologous VLP types. In contrast, mice immunized and boosted with heterologous cVLPs containing HPV16 E7 showed a higher frequency of E7-specific T cells in vitro and displayed reduced tumor growth in a therapeutic setting compared to mice treated with homologous cVLPs. The data indicate that the use of different cVLP types for prime/boost regimens is a promising strategy to increase the efficacy and usefulness of cVLP-based vaccines for the treatment of cervical neoplasia.

Papillomavirus-like particles (VLPs) are potent inducers of humoral and cellular immune responses, making them attractive candidates for noninfectious viral subunit vaccines. To further our understanding of how VLPs activate the immune system, we have investigated their interaction with human myeloid antigen-presenting cells. We found that VLPs bound, with increasing density, to the cell surface of human monocytes, macrophages, and monocyte-derived dendritic cells (DCs). Interestingly, there was a negative correlation between binding intensity and CD83 expression in DCs, suggesting that the main receptor for binding of VLPs may be downregulated during maturation. Exposure to VLPs resulted in acute phenotypic activation of monocytes and DCs. Furthermore, VLPs rapidly induced production of inflammatory cytokines in monocytes, macrophages, and DCs, as assessed by intracellular cytokine staining. For each cell type, the patterns of interleukin-1beta, interleukin-12, tumor necrosis factor-alpha, and interleukin-6 production were distinct from the pattern induced by lipopolysaccharide (LPS), a bacterial activator of myeloid antigen-presenting cells. Our results indicate that VLPs target multiple cells of the immune system, which helps to account for VLPs being so effective in priming humoral and cellular immune responses even in the absence of adjuvant.

Immunization of mice with self-Ag arrayed on the surface of papillomavirus-like particles induces long-lasting high-titer IgG production by autoreactive B cells. In contrast, immunization with disorganized self-Ag linked to foreign Th epitopes induces weak autoantibody responses that are predominantly of the IgM isotype. In this study, we evaluated the structural correlates of autoantibody induction to determine the basis of these disparate observations, using a system in which mice were vaccinated with a fusion protein containing self (TNF-α) and foreign (streptavidin) components, conjugated to biotinylated virus-like particles (VLPs). Similar titers of autoantibodies to TNF-α were elicited using conjugated polyomavirus VLPs and papillomavirus VLPs, indicating that acute activation of dendritic cells by the Ag is not required. Strong autoantibody responses were also induced by conjugated papillomavirus capsid pentamers, indicating that a higher order particulate structure is also not required. However, a reduction of self-Ag density on VLP surfaces dramatically reduced the efficiency of IgG autoantibody induction. In contrast, the negative effects of reductions in foreign Ag density were limited and could be overcome by dosage and adjuvant. These data suggest that the immune system has evolved to differentially recognize closely spaced repetitive Ags and that the signals generated upon interactions with high-density self-Ags can overwhelm the normal mechanisms for B cell tolerance.

The causal association between papillomavirus (HPV) infection and cervical cancer has been demonstrated; the development of a prophylactic vaccine to protect against HPV infection may therefore reduce the incidence of this cancer worldwide. Noninfectious HPV-like particles (VLPs), composed of the L1 major capsid protein, are current candidate vaccines for prevention of HPV infection and cervical neoplasia. Although neutralizing antibodies have a pivotal role in the prevention of initial infection, cellular immune responses to HPV antigens may have an important role in viral clearance. A phase II trial was conducted to further evaluate the immunogenicity of a recombinant HPV-16 L1 VLP vaccine administered intramuscularly, without adjuvant, at 0, 1, and 6 months. Cell-mediated immune responses (lymphoproliferation and cytokine production) to HPV-16 L1 VLPs were evaluated in peripheral blood mononuclear cells (PBMCs) from 43 individuals receiving the L1 VLP vaccine and from 10 individuals receiving placebo. Vaccination resulted, at months 2 and 7 (i.e., 1 month after the second immunization and 1 month after third immunization, respectively), in increases in T cell-proliferative response to HPV-16 L1 VLPs (P

Measurement of antibodies to human papillomavirus (HPV) is complicated by many factors. Although enzyme-linked immunosorbent assays (ELISAs) that use virus-like particles (VLPs) have proved useful, the assays have, in general, had moderate sensitivities and low signal-to-noise ratios. To enhance the performance of the assay, a systematic investigation was undertaken to examine key variables used in ELISAs for the detection of antibodies to VLPs of HPV. Incorporation of two vinyl polymers, polyvinyl alcohol (molecular weight, 50,000) (PVA-50) and polyvinylpyrrolidone (molecular weight, 360,000) (PVP-360), was found to increase the sensitivity as well as the specificity of the assay for the detection of antibodies to VLPs of HPV. In particular, the addition of PVA-50 to the blocking solution reduced the amount of nonspecific binding of antibodies to VLPs and the microplate surface, whereas the addition of PVP-360 increased the sensitivity of antibody detection. The new ELISA demonstrated increased sensitivity and specificity for the detection of cervical HPV type 16 infection compared to those of a prototype assay with coded clinical serum samples from women with known cervicovaginal HPV infection status. It is anticipated that the enhanced ELISA conditions will have wide application to a large number of clinical diagnostic assays.

Chimeric human papillomavirus virus-like particles (HPV cVLPs) carrying HPV16 E7 protein are potent vaccines for inducing cell-mediated immunity (CMI) against HPV-induced tumors in animal models. We tested the hypothesis that virion-neutralizing antibodies generated during an initial vaccination might prevent effective boosting of CMI to the cVLPs. Mice with circulating HPV16-neutralizing antibodies, generated by direct immunization with wild-type VLPs or by passive transfer of hyperimmune anti-HPV16 VLP mouse sera, were subsequently vaccinated with HPV16 E7-containing cVLPs. Mice with preexisting neutralizing antibodies were not protected from HPV16 E7-positive TC-1 tumor challenge, compared to the protection seen in mice lacking these antibodies. Antibody-coated VLPs bound very inefficiently to receptor-positive cell lines, suggesting that one of the mechanisms of antibody interference is blocking of VLP binding to its receptor and thereby uptake of VLPs by antigen-presenting cells. Our results suggest that repetitive vaccination with a cVLP for induction of cellular immune responses to an incorporated antigen may be of limited effectiveness due to the presence of neutralizing antibodies against the capsid proteins induced after the first application. This limitation could potentially be overcome by boosting with cVLPs containing the same target antigen incorporated into other papillomavirus-type VLPs.

A viral etiology for cutaneous squamous cell carcinoma (cuSCC) has long been suspected, primarily on the basis of its dramatically increased incidence in immunocompromised individuals. In this issue, Arron and colleagues report a comprehensive hunt for viral gene transcription in cuSCC. Their findings show that it is very unlikely that any currently known virus is commonly responsible for the maintenance of this cancer.

One of the hurdles to effective cancer treatment is tumor specific targeting of drugs. We have observed, both in vitro and in vivo, that Human papillomavirus-derived nanoparticles (HPV-NPs) have an innate tropism for tumors. HPV's primary attachment factors in infection are heparan-sulfated proteoglycans (HSPG) and as tumors often upregulate HSPGs at the extracellular matrix, this may be a contributing factor for HPV's tumor tropic nature. Here we present data to support the use of HPV-NPs in the treatment of ovarian carcinoma and uveal melanoma with α-particle emitting radiation (227Th) and photodynamic therapy (PDT), respectively. We first explored in vivo tumor targeting using HPV-NPs delivering a plasmid expressing the luciferase gene. Luciferase signal was detected in SKOV-3 tumor bearing, but not control animals, and this signal was only present in tumor tissue as revealed by ex vivo imaging. This finding prompted us to explore the therapeutic effect of delivering 227Th. The agent AU-010 was constructed by conjugation of a chelator to the HPV-NPs. Th-227 was then used to radiolabel the conjugated HPV-NPs. Animals were injected with SKOV-3 tumor cells, and 10 days following implant, treatment was initiated with three weekly administrations of either AU-010 (100 nCi), free chelated-227Th (100 nCi), unlabeled HPV-NPs or saline. HPV-NP targeting of 227Th to tumors provided a significant survival advantage over the control arms, demonstrating the therapeutic potential of HPV-NPs. Currently, follow-up experiments are evaluating optimized chelators in additional animal models. We next explored the use of HPV-NPs in photodynamic therapy with the development of AU-011. AU-011 is an HPV-NP conjugated to ≈500 molecules of infrared dye, IR700 (LiCor), and when irradiated with 690 nm near-infrared light generates reactive oxygen species that are specifically and immediately lethal to dye bound cells. This technology was initially validated using Trastuzumab as a targeting agent (Mitsunaga et al, Nat Med 17(12):1685-91, 2011). We compared the cell binding activity of AU-011 and Trastuzumab-IR700 on SKOV-3 ovarian cancer cells in vitro and observed a higher binding capacity of AU-011. We next compared the killing activity of the two agents. Trastuzumab-IR700 was found to have an IC50 value of 1.6 nM when activated with 32 J of 690 nm light. By comparison, AU-011 was found to have an IC50 value of 5.6 pM when activated with 16 J of 690 nm light. Thus, AU-011 appeared to be considerably more potent than Trastuzumab-IR700. Finally, we extended these observations to a panel of uveal melanoma cell lines. We chose uveal melanoma because PDT with 690 nm light is FDA approved and used regularly in the ophthalmic clinic. When applied on the melanoma cell lines, AU-011 was found to show high cell binding and potent cell killing. Currently, proof-of-concept in vivo studies of AU-011 in uveal melanoma are underway, paving the way to clinical development for this indication. Citation Format: Rhonda Kines, Debaditya Bhattacharya, John R. MacDougall, Diane Milenic, Martin W. Brechbiel, Elisabet de los Pinos, John T. Schiller. Viral-based nanoparticles: a new therapeutic approach for anticancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1770. doi:10.1158/1538-7445.AM2014-1770

The immunocytes that regulate papillomavirus infection and lesion development in humans and animals remain largely undefined. We found that immunocompetent mice with varying H-2 haplotypes displayed asymptomatic skin infection that produced L1 when challenged with 6×1010 MusPV1 virions, the recently identified domestic mouse papillomavirus (also designated “MmuPV1”), but were uniformly resistant to MusPV1-induced papillomatosis. Broad immunosuppression with cyclosporin A resulted in variable induction of papillomas after experimental infection with a similar dose, from robust in Cr:ORL SENCAR to none in C57BL/6 mice, with lesional outgrowth correlating with early viral gene expression and partly with reported strain-specific susceptibility to chemical carcinogens, but not with H-2 haplotype. Challenge with 1×1012 virions in the absence of immunosuppression induced small transient papillomas in Cr:ORL SENCAR but not in C57BL/6 mice. Antibody-induced depletion of CD3+ T cells permitted efficient virus replication and papilloma formation in both strains, providing experimental proof for the crucial role of T cells in controlling papillomavirus infection and associated disease. In Cr:ORL SENCAR mice, immunodepletion of either CD4+ or CD8+ T cells was sufficient for efficient infection and papillomatosis, although deletion of one subset did not inhibit the recruitment of the other subset to the infected epithelium. Thus, the functional cooperation of CD4+ and CD8+ T cells is required to protect this strain. In contrast, C57BL/6 mice required depletion of both CD4+ and CD8+ T cells for infection and papillomatosis, and separate CD4 knock-out and CD8 knock-out C57BL/6 were also resistant. Thus, in C57BL/6 mice, either CD4+ or CD8+ T cell-independent mechanisms exist that can protect this particular strain from MusPV1-associated disease. These findings may help to explain the diversity of pathological outcomes in immunocompetent humans after infection with a specific human papillomavirus genotype., Author Summary Infection with papillomaviruses can cause benign warts (papillomas) on skin and mucosae of humans and animals but also malignancies, especially anogenital carcinomas and, in genetically predisposed or immunocompromised individuals, cutaneous squamous cell cancers. Control and clearance of these viruses are thought to be mediated by the cellular immune system, however experimental determination for the necessary cellular effector(s) is lacking. The recently identified mouse papillomavirus (MusPV1, also designated “MmuPV1”) is known to induce papilloma formation on skin of immunodeficient mice. However, its pathogenesis in immunocompetent mice is unclear. Our study shows that in an immunocompetent setting, MusPV1 generally causes asymptomatic skin infections, but no lesion outgrowth. Visible papillomas were consistently observed after profound immunosuppression in some, but not other, strains of mice. By selective removal of distinct cellular immune populations and employing genetically modified mice, we could show that T cells are pivotal for controlling MusPV1 infection and disease. We further show that surprising differences in the T cell subsets are required for protection in different strains of immunocompetent mice. This implies that unanticipated effector mechanisms can control virus infection and pathogenesis in specific genetic backgrounds. The findings may help to explain the wide of range of pathologic outcomes of infection by a specific human papillomavirus type in immunocompetent people.

Non-infectious human papillomavirus-like particles (VLP), composed of the L1 major capsid protein, are under active development as vaccines to prevent cervical cancer. They would presumably function primarily by generating virion-neutralising antibodies against the genital human papillomavirus (HPV) types that are the principal cause of most cervical cancers. Early phase clinical studies indicate that the VLP vaccines are well tolerated and able to consistently induce high titres of virus type-specific neutralising antibodies. Two types of second-generation VLP-based subunit vaccines with therapeutic implications, both related and unrelated to papillomavirus infection, are in preclinical development. One type seeks to induce cell-mediated immune responses, especially cytotoxic lymphocytes (CTL), against non-structural papillomavirus proteins, proteins of other viruses, or tumour associated antigens. The target antigen is incorporated into the VLPs as a fusion protein of L1 or the L2 minor capsid protein. In mouse models, this approach has generated potent CTL responses after low dose vaccination in the absence adjuvant. The second type of therapeutic VLP-based vaccine seeks to generate autoantibodies to self-antigens. The display of self polypeptides in the context of the highly ordered array of repetitive elements on the papillomavirus VLP surface abrogates the ability of the humoral immune system to functionally distinguish between foreign and self. High titre and high avidity auto-reactive IgG antibodies have been induced to both soluble (TNF-alpha) and cell surface (CCR5) central self-antigens. Vaccines based on this approach could potentially be effective alternatives to monoclonal antibody (mAb)-based therapies for a variety of disease targets.

Objective—To determine expression of a transforming gene (E5) of bovine papillomavirus in sarcoids, other tumors, and normal skin samples collected from horses with and without sarcoids. Sample Population—23 sarcoids and 6 samples of normal skin obtained from 16 horses with sarcoids, 2 samples of normal skin and 2 papillomas obtained from horses without sarcoids, and 1 papilloma obtained from a cow. Procedure—Protein was extracted from tissue samples collected from horses and incubated with agarose beads covalently coupled to Staphylococcus aureus protein A and an anti-E5 polyclonal antibody. Following incubation, proteins were eluted from the beads and electrophoresed on a 14% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The E5 protein was detected by use of western blot analysis, using a chemiluminescence detection system. Results—All 23 sarcoids had positive results for expression of E5 protein. Quantity of viral protein appeared to vary among sarcoids. All other tissues examined had negative results for E5 protein. Highest expression for E5 protein was observed in biologically aggressive fibroblastic variants of sarcoids, compared with expression in quiescent tumors. Conclusions and Clinical Relevance—This study documented that activation and expression of the E5 gene is evident in sarcoids obtained from horses. These data support the conclusion that infection with bovine papillomavirus is important in the initiation or progression of sarcoids in horses. Treatment strategies designed to increase immune recognition of virally infected cells are warranted. (Am J Vet Res 2001;62:1212–1217)

High avidity and long-lasting autoantibodies to a self-polypeptide (TNF-alpha) were generated after parenteral vaccination of mice with low doses of virus-like particle-based (VLP-based) vaccines that were constructed by linking mouse TNF-alpha peptides to the surface of papillomavirus VLPs. High-titer autoantibodies were induced with or without coadministration of potent conventional adjuvants, but were enhanced by coadministration of CFA. Compared with immunization with the fusion protein alone, attachment to VLPs increased autoantibody titers 1,000-fold. A comparison of Ab responses against the self (TNF-alpha) and foreign components of the fusion protein showed that VLP conjugation abrogated the ability of the humoral immune system to distinguish between self and foreign. Similar levels of IgM were detected to self and foreign epitopes regardless of the assembly state of the antigen, suggesting that conjugation of self-peptides to VLPs promotes survival or expansion of mature autoreactive B cells. In a mouse model, vaccination with conjugated particles inhibited development of type II collagen-induced arthritis. Together, these results suggest a potentially flexible method to efficiently generate autoantibodies against specific self-proteins that mediate arthritis and other diseases.

Human papillomavirus virus-like particles (HPV VLP) and chimeric VLP are immunogens that are able to elicit potent anti-viral/tumor B and T cell responses. To investigate the immunogenicity of VLP, we determined which cells of the immune system are able to bind HPV-16 VLP. VLP were found to bind very well to human and mouse immune cells that expressed markers of antigen-presenting cells (APC) such as MHC class II, CD80 and CD86, including dendritic cells, macrophages and B cells. mAb blocking studies identified Fc gamma RIII (CD16) as one of the molecules to which the VLP can bind both on immune cells and foreskin epithelium. However, transfection of a CD16(-) cell line with CD16 did not confer binding of VLP. Splenocytes from Fc gamma RIII knockout mice showed a 33% decrease in VLP binding overall and specifically to subsets of APC. These combined data support a role for CD16 as an accessory molecule in an HPV VLP-receptor complex, possibly contributing to the immunogenicity of HPV VLP.

BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.

Background Studies in animal models have shown that systemic immunization with a papillomavirus virus-like particle (VLP) vaccine composed of L1, a major structural viral protein, can confer protection against subsequent experimental challenge with the homologous virus. Here we report results of a double-blind, placebo-controlled, dose-escalation trial to evaluate the safety and immunogenicity of a human papillomavirus (HPV) type 16 (HPV16) L1 VLP vaccine in healthy adults. Methods Volunteers were given intramuscular injections with placebo or with 10- or 50-microg doses of HPV16 L1 VLP vaccine given without adjuvant or with alum or MF59 as adjuvants at 0, 1, and 4 months. All vaccine recipients were monitored for clinical signs and symptoms for 7 days after each inoculation. Immune responses were measured by an HPV16 L1 VLP-based enzyme-linked immunosorbent assay (ELISA) and by an HPV16 pseudovirion neutralization assay. The antibody titers were given as the reciprocals of the highest dilution showing positive reactivity in each assay. All statistical tests were two-sided. Results The prevaccination geometric mean ELISA titer for six seropositive individuals was 202 (range, 40--640). All vaccine formulations were well tolerated, and all subjects receiving vaccine seroconverted. Serum antibody responses at 1 month after the third injection were dose dependent in recipients of vaccine without adjuvant or with MF59 but were similar at both doses when alum was the adjuvant. With the higher dose, the geometric means of serum ELISA antibody titers (95% confidence intervals) to purified VLP 1 month after the third injection were as follows: 10,240 (1499 to 69 938) without adjuvant, 10,240 (1114 to 94 145) with MF59, and 2190 (838 to 5723) with alum. Responses of subjects within each group were similar. Neutralizing and ELISA antibody titers were highly correlated (Spearman correlation =.85), confirming that ELISA titers are valid proxies for neutralizing antibodies. Conclusions The HPV16 L1 VLP vaccine is well tolerated and is highly immunogenic even without adjuvant, with the majority of the recipients achieving serum antibody titers that were approximately 40-fold higher than what is observed in natural infection.

Papillomavirus-like particle (VLP)-based subunit vaccines have undergone rapid development over the past 8 years. Three types are being investigated. The most basic type is composed of only the L1 major capsid protein and is designed to prevent genital human papillomavirus (HPV) infection by inducing virus-neutralizing antibodies. On the basis of positive results in animal models, clinical trials of this type of vaccine for HPV16, and other types, are currently under way. Preliminary results have been encouraging in that systemic immunization with the L1 VLPs induced high serum titers of neutralizing antibodies without substantial adverse effects. The second type of vaccine incorporates other papillomavirus polypeptides into the VLPs as L1 or L2 fusion proteins. These chimeric VLPs are designed to increase the therapeutic potential of an HPV vaccine by inducing cell-mediated responses to nonstructural viral proteins, such as E7. Studies in mice indicate that these vaccines generate potent antitumor cytotoxic lymphocyte (CTL) responses while retaining the ability to induce high-titer neutralizing antibodies. It is likely that prophylactic and therapeutic clinical trials of chimeric VLPs will be initiated in the near future. The third type of VLP-based vaccine is designed to induce autoantibodies against central self-antigens by incorporating self-peptides into the outer surface of VLPs, a process that could have therapeutic potential in various disease settings unrelated to HPV infection. In a recent proof of concept study, a peptide from an external loop of mouse CCR5 protein was inserted into a neutralizing epitope of L1. In mice, the particles generated by this chimeric L1 were able to induce high titers of CCR5 antibodies that specifically recognized the surface of CCR5-transfected cells and blocked in vitro infection of an M-tropic human immunodeficiency virus strain.