Rabbit Papillomavirus Research Articles

Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17–36 epitopes

Vaccination with the minor capsid protein L2, notably the 17–36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17–36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum±MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17–36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant.

RNA sequencing analysis identifies novel spliced transcripts but does not indicate quantitative or qualitative changes of viral transcripts during progression of cottontail rabbit papillomavirus-induced tumours

Persistent infections with high-risk human papillomaviruses (HPVs) can result in the development of cancer of the cervix uteri and other malignancies. The underlying molecular mechanisms leading to the progression of HPV-induced lesions are, however, not well understood. Cottontail rabbit papillomavirus (CRPV) induces papillomas in domestic rabbits which progress at a very high rate to cancer. Using this model, we compared the transcriptional patterns of CRPV in papillomas and carcinomas by RNA sequencing (RNA-seq). The most abundant transcripts can encode E7, short E6 and E1^E4, followed by full-length E6, E2, E1 and E9^E2C. In addition, we identified two rare, novel splice junctions 7810/3714 and 1751/3065 in both papillomas and carcinomas which have been described for other papillomaviruses. Neither RNA-seq nor quantitative real-time PCR-based assays identified qualitative or quantitative changes of viral transcription between papillomas and carcinomas. In summary, our analyses confirmed that papillomaviruses have highly similar transcriptional patterns, but they do not suggest that changes in these patterns contribute to the progression of CRPV-induced tumours.

Comparisons of VLP-Based ELISA, Neutralization Assays with Native HPV, and Neutralization Assays with PsV in Detecting HPV Antibody Responses in HIV-Infected Women.

Human papillomavirus (HPV)-associated cancers are important public health problems in HIV-infected people. Assays based on HPV virus-like particles (VLP) and pseudoviruses (PsV) are commonly used to examine HPV antibody responses in HIV-infected people, but neutralization assays with native HPV have not been utilized and a comparison of these three assays is lacking. We evaluated the agreement of assays using VLP, native HPV and PsV in detecting HPV16 and 18 antibodies in HIV-infected women. The VLP-based ELISA (VLP-ELISA) was used to detect antibody responses to HPV16 and 18 and cottontail rabbit papillomavirus (CRPV) VLP antigens. Neutralization assays with native HPV (NA-HPV) and with PsV (NA-PsV) were conducted to examine HPV16 or 18 neutralizing antibodies. Intra class correlation coefficients (ICC) and kappa coefficients were used to assess the agreements of seropositivity between the assays. The seroprevalence detected by the VLP-ELISA, NA-HPV and NA-PsV in 94 HIV-infected women was 35%, 51% and 27% for HPV16 and 14%, 44% and 21% for HPV18. Cross-reactivity between HPV16 and HPV18 was 0.35, 0.04 and 0.33 (kappa coefficients) for the VLP-ELISA, NA-HPV and NA-PsV. The agreements of seropositivity between the three assays were low. Six women who were HPV16 DNA positive were seropositive by the NA-HPV but only two were HPV16 seropositive by the VLP-ELISA or NA-PsV. One HPV18 DNA positive woman was seropositive by all three assays. Repeated tests indicated excellent reproducibility of the NA-HPV. HPV serology results vary across different assays. The NA-HPV appears to be a sensitive and reliable approach in detecting natural HPV antibodies in HIV-infected women. The NA-HPV can be applied in both HPV natural history studies and vaccine studies in HIV-infected people.

The Viral Etiology of Skin Cancer

The concept that viruses may be etiological agents of cancers is as old as the discovery of viruses themselves. In 1908, 3 years before Peyton Rous was passaging what later became to be known as Rous Sarcoma Virus in chickens, two Danish scientists, Ellerman and Bang, were characterizing a transmissible filtrate that reproducibly caused leukemia in chickens. These findings were received with harsh skepticism, and the scientific community did not universally accept the concept that tumors could be caused by transmissible agents. Richard Shope, a colleague of Peyton Rous at the Rockefeller Institute, identified an infectious agent that infected cottontail rabbits. It caused cutaneous papillomas that could grow to be quite large and which may be the basis of sightings of the mystical and ravenous ‘‘Jackelope’’ of southwestern American lore. Shope later collaborated with Rous to demonstrate that exposure of these papillomas to coal tar or infection of a host that does not support viral replication caused malignant progression to skin cancers. This infectious agent, the cottontail rabbit papillomavirus or Sylvilagus floridanus Papillomavirus 1, was the first virus linked to a cancer in a mammalian host (Javier and Butel, 2008; Moore and Chang, 2010) (Figure 1).

Formulation of cidofovir improves the anti-papillomaviral activity of topical treatments in the CRPV/rabbit model

Current topical treatments for papillomas use ablative, cytotoxic and immunomodulating strategies and reagents. However, the effectiveness of topical treatments using different formulations has not been examined in preclinical models or clinical trials. The purpose of this study was to determine whether formulation of the small molecule acyclic nucleoside, cidofovir (CDV), could lead to improved therapeutic endpoints following topical treatment of papillomas using the cottontail rabbit papillomavirus (CRPV)/rabbit model. Different formulations with a set dose of 1% cidofovir were tested to establish comparative data.The results demonstrated that anti-papilloma treatments with topical CDV were greatly enhanced when formulated versus unformulated. Best results were obtained with CDV formulated in cremophor, then in Carbomer 940, and then in DMSO. Further studies indicated that effective formulations led to complete cures of papillomas at dilutions less than 0.3% CDV. These studies together with previous observations demonstrated that unformulated CDV under the same treatment regime required doses of 2% to achieve cures demonstrating that much less compound can be used when properly formulated.

Antibody detection in tear samples as a surrogate to monitor host immunity against papillomavirus infections in vaccinated and naturally infected hosts.

Monitoring serum antibodies against natural infections or after immunizations has been a standard clinical diagnostic procedure. However, collecting blood samples requires trained personnel, and may cause discomfort and increase the risk of complications. In this study, we investigated whether tear samples could serve as a surrogate for serum samples to measure specific antibodies. A widely used preclinical cottontail rabbit papillomavirus (CRPV)/rabbit model has been a surrogate model for high-risk human papillomavirus (HPV) infections. New Zealand white rabbits, either naturally infected with CRPV or immunized with two clinically available HPV vaccines (Gardasil and Cervarix), were examined for antibody generation in both tear and serum samples. We demonstrated that antibodies were detectable in tears from both naturally infected as well as vaccinated animals. Overall, the antibody levels in tears were ~10-fold lower than those from the corresponding serum samples, but background noise was lower in tear samples. The isotypes of antibodies in tears were predominantly IgA and IgG. These findings showed clearly that tears could be a surrogate for serum samples for monitoring antibody responses. As collecting tears causes no discomfort and poses no risk to patients, it represents a novel and promising method for monitoring future HPV epidemiological studies as well as for use in clinical practice.

Low doses of flagellin-L2 multimer vaccines protect against challenge with diverse papillomavirus genotypes

Genetically modified bacterial flagellin (Fla), a Toll-like receptor-5 (TLR5) ligand, was evaluated as a fusion partner for human papillomavirus (HPV) L2-based immunogens in two animal challenge models; either cutaneous inoculation of rabbits with HPV ‘quasivirions’ containing cottontail rabbit papillomavirus (CRPV) genomes that induce warts, or intra-vaginal inoculation of mice with HPV ‘pseudovirions’ encapsidating a luciferase reporter plasmid and measurement of bioluminescence to determine infectivity. An Escherichia coli production system was developed for flagellin-L2 (Fla-L2) fusions containing either monomeric HPV-16 L2 a.a. 11(×11–200) or oligomeric L2 comprising a fusion of the a.a. 11–88 peptides of five (Fla∼5×11–88) or eight (Fla∼8×11–88) genital HPV types. Immunogenicity and bioactivity of Fla-L2 constructs were assessed using an in vitro neutralization and cell-based TLR-5 binding assay, respectively. Efficacy was evaluated following active immunization of rabbits or mice administered 3 intramuscular doses of Fla-L2 recombinants without exogenous adjuvant, followed by challenge. In addition, passive immunization studies of naïve rabbits with serial dilutions of pooled immune sera were used to determine End-Point Protection Titers (EPPT) for each formulation against a broader spectrum of HPV quasivirions. Efficacy was assessed for up to 10 weeks on the basis of wart volume induced following challenge and results compared to licensed L1-VLP vaccines (Gardasil and Cervarix). Following active immunization at doses as low as 1μg, Fla-L2 fusions afforded complete protection against infection (mice) and disease (rabbits) following either homologous or heterologous HPV challenge. Passive immunization with anti-L2 immune sera discriminated between the different vaccine candidates under evaluation, demonstrated the protective role of antibody and suggested the superiority of this oligomeric L2-TLR5 agonist fusion approach compared to L1-based vaccines in its ability to cross-protect against non-vaccine HPV types.

Evaluation of Positron Emission Tomographic Tracers for Imaging of Papillomavirus-Induced Tumors in Rabbits

In this study, simultaneous positron emission tomography (PET)/magnetic resonance (MR) imaging was employed to evaluate the feasibility of the PET tracers 2-deoxy-2-18F-fluoro-d-glucose (18F-FDG), 11C-choline, and 18F-fluorothymidine (18F-FLT) to detect papillomavirus-induced tumors in an established rabbit model system. The combined PET/MR allowed the analysis of tracer uptake of the tumors using the morphologic information acquired by MR. New Zealand White rabbits were infected with cottontail rabbit papillomavirus genomes and were imaged for up to 10 months with a simultaneous PET/MR system during the course of infection. The uptake characteristics of the PET tracers 11C-choline and 18F-FLT of tumors and reference tissues were examined relative to the clinical standard, 18F-FDG. Tracer biodistribution of various organs was measured by gamma-counting after the last PET scan and compared to the in vivo PET/MR 18F-FDG uptake. Increased tracer uptake was found 2 months postinfection in primary tumors with 18F-FDG and 11C-choline, whereas 18F-FLT failed to detect the tumors at all measured time points. Our data show that the PET tracer 18F-FDG is superior for imaging papillomavirus-induced tumors in rabbits compared to 11C-choline and 18F-FLT. However, 11C-choline imaging, which has previously been applied to detect various tumor entities in patients, appears to be an alternative to 18F-FDG.

Long-peptide therapeutic vaccination against CRPV-induced papillomas in HLA-A2.1 transgenic rabbits

Long peptide immunization is a promising strategy to clear established tumors. In the current study, we investigated the therapeutic effect of a naturally existing long peptide that contained two HLA-A2.1 restricted epitopes (CRPVE1/149–157 and CRPVE1/161–169) from cottontail rabbit papillomavirus (CRPV) E1 using our CRPV/HLA-A2.1 transgenic rabbit model. A universal Tetanus Toxin helper motif (TT helper) was tagged at either the N-terminus or the carboxyl-terminus of this long peptide and designated as TT-E1 peptide and E1 peptide-TT, respectively. Four groups of HLA-A2.1 transgenic rabbits were infected with wild type CRPV DNA. Three weeks post-infection, the rabbits were immunized four times with TT-E1 peptide, E1 peptide only, E1 peptide-TT or TT-control peptide with two-week intervals between immunizations. Tumor outgrowth was monitored and recorded weekly. After the third booster immunization, tumors on two of the four E1 peptide-TT immunized rabbits began to shrink. One animal from this group was free of tumors at the termination of the study. The mean papilloma size of E1 peptide-TT immunized rabbits was significantly smaller when compared with that of the three other groups (P<0.05, one way ANOVA analysis). It is interesting that E1 peptide-TT vaccination not only stimulated stronger T cell mediated immune responses but also stronger antibody generations. We conclude that the location of a TT helper motif tagged at the long peptide vaccine is critical for the outcome of therapeutic responses to persistent tumors in our HLA-A2.1 transgenic rabbit model.

Cottontail Rabbit Papillomavirus Infection in a Desert Cottontail (Sylvilagus audubonii) from Colorado, USA

A wild-caught desert cottontail rabbit (Sylvilagus audubonii) from Colorado was observed to have large, pedunculated, dark cutaneous lesions on its abdomen and cylindrical masses on its mouth. Morphologically, the masses were consistent with previous reports of virally induced papillomas. Subsequent DNA analysis indicated widespread infection with cottontail rabbit papillomavirus.

Developing Country Applications of Molecular Farming: Case Studies in South Africa and Argentina

Molecular farming is a technology that is very well suited to being applied in developing countries, given the reasonably high level of expertise in recombinant plant development in many centers. In addition, there is an urgent need for products such as inexpensive vaccines and therapeutics for livestock and for some human diseases - and especially those that do not occur or are rare in developed regions. South Africa and Argentina have been at the fore in this area among developing nations, as researchers have been able to use plants to produce experimental therapeutics such as nanoantibodies against rotavirus and vaccines against a wide variety of diseases, including Rabbit haemorrhagic disease virus, Foot and mouth disease virus, Bovine viral diarrhoea virus, bovine rotaviruses, Newcastle disease virus, rabbit and human papillomaviruses, Bluetongue virus, and Beak and feather disease virus of psittacines. A combination of fortuitous scientific expertise in both places, coupled with association with veterinary and human disease research centers, has enabled the growth of research groups that have managed to compete successfully with others in Europe and the USA and elsewhere, to advance this field. This review will cover relevant work from both South Africa and Argentina, as well as a discussion about the perspectives in this field for developing nations.

Francis Peyton Rous

This is a photograph of Peyton Rous (Francis Peyton Rous, 1879–1970), who in 1909–11 made 2 seminal discoveries that are now the foundation blocks of modern virology and oncology. First, he discovered that a malignant tumor (a sarcoma in chickens) was transmissible; this was the first transmissible solid tumor discovered. Second, he found that the tumor-inducing factor could be passed through a Berkefeld ultrafilter known to retain bacteria. In the context of the times, this finding proved that the agent was a virus, the first of its kind. The virus he discovered is now known as Rous sarcoma virus and has since been studied in many laboratories around the world. Rous was born in 1879 in Baltimore, Maryland, USA, and raised by his widowed mother, who persevered in supporting his education. He received bachelor of arts and doctoral degrees from The Johns Hopkins University in Baltimore in 1900 and 1905, respectively. After teaching pathology at the University of Michigan, Ann Arbor, and studying morbid anatomy at Friedrichstadt Municipal Hospital in Dresden, Germany, in 1909, he took a position at the Rockefeller Institute for Medical Research in New York, New York, where he spent the rest of his life. Rockefeller was the place in the United States where virology first emerged as a distinct medical science; from the beginning of his career, Rous was surrounded by great scientists. Rous’ entry into tumor virology was fortuitous; the founding director of the Rockefeller Institute, Simon Flexner, had been interested in oncology but wanted to redirect his own work toward polio, which was becoming a major problem. Rous was hired to continue Flexner’s research into oncology, a subject about which Rous at first knew nothing. The beginning of the story behind Rous’ claim to fame is remarkable: a woman came to the Rockefeller Institute with a barred Plymouth Rock hen that had a large tumor on its breast. Rous later wrote, “In this paper is reported the first avian tumor that has proved transplantable to other individuals. It is a spindle-celled sarcoma of a hen, which has thus far been propagated to the fourth generation….” In his research, he found that only closely related chickens were susceptible, but in these chickens, continuous passage of cell-free material led to tumors that grew quickly, were more malignant than usual, and produced widespread metastases. Rous continued to study the phenomenon he had begun to unravel for many years, but understanding the mechanistic bases for the complex natural history of Rous sarcoma virus and related viruses had to wait until modern molecular and cellular biologic technologies became available in the 1960s. In 1934, Rous’ Rockefeller Institute colleague, Richard E. Shope, asked him to examine warts on jackrabbits that had definitively been shown to be caused by an ultrafilterable virus. This virus was Shope papillomavirus (rabbit papillomavirus). When Rous confirmed that the warts were benign tumors, he was reinvigorated in his intent to unravel the mysteries of viral oncology. Over the next 30 years, Rous and his colleagues showed that the benign tumors could progress to malignant carcinomas and that chemical carcinogens could interact with the virus—further discoveries that formed building blocks for modern virology. Today, we recognize that ≈20% of human cancers worldwide have infectious etiologies, for which preventive measures such as vaccines have great promise. Rous’ colleagues, including the scientists Rene Dubos and Charles B. Huggins, lauded Rous’ personal and professional qualities, writing that he was gifted with supreme intellectual powers, unfailing integrity and honesty, a remarkably intuitive sense for the science itself, great perseverance and work ethic, and an enormous zest for life. One can imagine with wonder Rous in his laboratory and in the legendary lunchroom of the Rockefeller Institute. Rous was duly honored for his masterful work, winning the National Medal of Science and membership in the National Academy of Sciences, the American Philosophical Society, the Royal Society, and other prestigious organization. In 1966, when he was 87 years old, Rous was awarded the Nobel Prize in Medicine, an honor he shared with Charles Huggins. After 55 years, the longest “incubation period” in the history of the Nobel Prizes, Rous’ discovery had finally been recognized with this honor. In the end, proof that Rous was just ahead of his time might be found in the several additional Nobel Prizes awarded since 1966 to virologists who further unraveled viral oncology. Figure Francis Peyton Rous (1879–1970), pictured in 1923, at age 44, in his laboratory at the Rockefeller Institute for Medical Research, New York, NY, USA. Source: National Library of Medicine.

Synonymous codon changes in the oncogenes of the cottontail rabbit papillomavirus lead to increased oncogenicity and immunogenicity of the virus

Papillomaviruses use rare codons with respect to the host. The reasons for this are incompletely understood but among the hypotheses is the concept that rare codons result in low protein production and this allows the virus to escape immune surveillance. We changed rare codons in the oncogenes E6 and E7 of the cottontail rabbit papillomavirus to make them more mammalian-like and tested the mutant genomes in our in vivo animal model. While the amino acid sequences of the proteins remained unchanged, the oncogenic potential of some of the altered genomes increased dramatically. In addition, increased immunogenicity, as measured by spontaneous regression, was observed as the numbers of codon changes increased. This work suggests that codon usage may modify protein production in ways that influence disease outcome and that evaluation of synonymous codons should be included in the analysis of genetic variants of infectious agents and their association with disease.

HPV18 E1^E4 is assembled into aggresome-like compartment and involved in sequestration of viral oncoproteins.

Papillomavirus is the etiological agent for warts and several squamous carcinomas. Skin cancer induced by cottontail rabbit papillomavirus was the first animal model for virus-induced carcinogenesis. The target organ of the virus infection is stratified epithelium and virus replication is tightly regulated by the differentiation program of the host cell. E1^E4 protein is a viral gene product, and although it is considered to be involved in the control of virus replication, little is known about the biological role. We found that HPV18 E1^E4 was assembled into an aggresome-like compartment and was involved in sequestration of virus oncoproteins, which might contribute to the differentiation-dependent lifecycle of papillomavirus.

A recombinant cottontail rabbit papillomavirus genome for ectopic expression of genes in cells infected with virus in vivo

The objective of this study was to construct a cottontail rabbit papillomavirus (CRPV) genome that would co-express a gene of choice and the viral genome simultaneously. Using this construct, the effects of the ectopic expression of diverse viral or cellular genes on PV-infected cells can be examined to elucidate which genes are essential for tumor formation. CRPV-pLAIIdelXba1, which lacks the major portion of L2 (designated the XbaI fragment), has been previously shown to fully retain the ability to induce tumors, and this ability was confirmed in this study. Insertion of the XbaI fragment in an antisense orientation did not change the efficiency of tumor induction. An SV40 overexpression cassette that originated from pSG5 and contains a more diverse multiple cloning site (MCS) was cloned into CRPV-Xba1-mcs, a CRPV genome based on CRPV-pLAIIdelXba1 that contains an additional MCS inserted via XbaI digestion. Additionally, the L1 ATG initiation codon of this construct, designated CRPV-Xba1-oe-WT, was mutated to avoid unnecessary L1 protein expression, which produced the CRPV-Xba1-oe-L1mut construct. Injection of these constructs into two New Zealand White rabbits and monitoring of tumor growth for two to six months showed that CRPV-Xba1-oe-WT induced tumors at 1/10 and 1/10 of the injection sites in two animals, while the control injections in each rabbit induced tumors at 3/10 and 4/10 injection sites, respectively. However, CRPV-Xba1-oe-L1mut induced tumors at 3/10, 6/10, 7/12 and 11/12 sites in four injected animals, and the control injections induced tumor growth in these animals at 6/10, 10/10, 12/12 and 12/12 of the injected sites, respectively. Thus, CRPV-Xba1-oe-L1mut could potentially be used to conduct overexpression experiments in vivo that can be used to measure the negative or positive influences of ectopically expressed foreign or HPV genes on tumor growth.

Abstract PL04-04: Synthetic long peptide vaccines against established disease caused by high-risk human papillomavirus.

Abstract Therapeutic vaccination of persistent virus infections and associated diseases including (pre-)cancer so far has largely evaded success, mainly due to the fact that insufficiently consistent and robust effector T cell responses were induced by the commonly used vaccine constructs and formulations such as recombinant viruses and bacteria, recombinant proteins, DNA constructs, Dendritic Cell (DC) vaccines and exact HLA class I binding peptides (short peptides). Problems have been severe antigenic competion from vector sequences by recombinant viruses and bacteria, insufficiently powerful T cell generation by DNA constructs, insufficient homing to lymph nodes injected DC and antigen presentation of short peptides by non-professional antigen presenting cells in vivo, causing tolerance instead of immunity. Much more robust and consistent T cell responses can be obtained by vaccination with long (28–35 amino acid long) synthetic peptides. Such immunogens are more efficiently processed and presented than intact proteins by DC and only DC can efficiently perform this task. Moreover only concentrated antigen of choice is offered and antigenic competition therefore plays no role. In earlier work we showed that therapeutic vaccination with a synthetic long peptide (SLP®) vaccine mediated the eradication of established human papilloma virus type 16 (HPV16)-positive tumors in mice and controlled wart growth and latent virus infection in rabbits persistently infected with cottontail rabbit papilloma virus. Subsequent phase I/II studies with an HPV16 SLP® vaccine, consisting of 13 long peptides covering the HPV16 E6 and E7 antigens, in patients with advanced HPV16-positive cervical cancer, revealed that this vaccine was safe and highly immunogenic. We then tested the clinical efficacy of this HPV16 SLP® vaccine in HPV16-induced high grade vulvar intraepithelial neoplasia (VIN3), a premalignant epithelial disorder, spontaneous regression of which occurs in less than 2% of patients and in which recurrence after standard treatment is high. In a phase 2 trial, 20 women with VIN3 were vaccinated three times sc in the limbs with a mix of the HPV16 E6 and E7 synthetic long peptides formulated in Montanide ISA-51. The endpoints were objective clinical responses, defined as reduction of at least 50% in lesion size (partial response) or complete regressions, and HPV16-specific T-cell responses. The vaccine was safe. At 3 and 12 months after the last vaccination an objective response was observed in 12/20 (60%) and 15/19 (79%) patients respectively. Nine of them showed a complete and durable regression of the lesions at 12 months and at 24 months. The strength of the vaccine-induced HPV16-specific T-cell response was significantly higher in the group of patients with a complete regression of their lesions compared to non-responders. Patients with large lesions were less likely to experience a complete clinical response than patients with small lesions and we ascribe this to a larger proportion of vaccine induced HPV-specific regulatory cells in the patients with large lesions. In conclusion, treatment with the HPV16 SLP vaccine is clearly effective in patients with established VIN disease. The SLP platform lends itself for development of therapeutic vaccines against many other chronic infections and non-viral cancers. In patients with cancer, it is attractive to combine this type of vaccination with immunogenic forms of cancer chemotherapy and with immuno-modulatory drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL04-04.

Emerging Theme: Cellular PDZ Proteins as Common Targets of Pathogenic Viruses

More than a decade ago, three viral oncoproteins, adenovirus type 9 E4-ORF1, human T-lymphotropic virus type 1 Tax, and high-risk human papillomavirus E6, were found to encode a related carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with a select group of cellular PDZ proteins. Recent studies have shown that many other viruses also encode PBM-containing proteins that bind to cellular PDZ proteins. Interestingly, these recently recognized viruses include not only some with oncogenic potential (hepatitis B virus, rhesus papillomavirus, cottontail rabbit papillomavirus) but also many without this potential (influenza virus, Dengue virus, tick-borne encephalitis virus, rabies virus, severe acute respiratory syndrome coronavirus, human immunodeficiency virus). Examination of the cellular PDZ proteins that are targets of viral PBMs reveals that the viral proteins often interact with the same or similar types of PDZ proteins, most notably Dlg1 and other members of the membrane-associated guanylate kinase protein family, as well as Scribble. In addition, cellular PDZ protein targets of viral PBMs commonly control tight junction formation, cell polarity establishment, and apoptosis. These findings reveal a new theme in virology wherein many different virus families encode proteins that bind and perturb the function of cellular PDZ proteins. The inhibition or perturbation of the function of cellular PDZ proteins appears to be a widely used strategy for viruses to enhance their replication, disseminate in the host, and transmit to new hosts.

Can A Virus Cause Cancer: A Look Into The History And Significance Of Oncoviruses

C an A V irus C ause C ancer ? A L ook into the H istory and S ignificance of O ncoviruses Niema Rwazavian T he I mportance of O ncoviruses Cancer, a disease caused by unregulated cell growth, is often attributed to chemical carcinogens (e.g. tobacco), hormonal imbalances (e.g. high levels of estrogen), or genetics (e.g. breast cancer susceptibility gene 1). While cancer can originate from any number of sources, many people fail to recognize another important etiology: oncoviruses, or cancer-causing (van Epps 2005). Although many in the scientific community were unconvinced of the role of viruses in cancer, research on the subject nevertheless continued. In 1933, Richard Shope discovered the first mammalian oncovirus, cottontail rabbit papillomavirus (CRPV), which could infect cottontail rabbits, and in 1936, John Bittner discovered the mouse mammary tumor virus (MMTV), which could be transmitted from mothers to pups via breast milk (Javier and Butle 2008). By the 1960s, with the additional “…despite limited awareness, oncoviruses are discovery of the murine leukemia (MLV) in mice and the SV40 nevertheless important because they represent virus in rhesus monkeys, researchers virus over 17% of the global cancer burden.” began to acknowledge the possibility that viruses could be linked to human cancers as well. The growing concern viruses. Oncoviruses are often overlooked because over the possibility of human cancer viruses lead to current preventative measures against cancer focus the creation of the U.S. Special Virus Cancer Program on modifiable lifestyle risk factors, such as diet and in 1964, which was devoted to the search for human exercise. Despite limited awareness, oncoviruses oncoviruses. are nevertheless important because they represent over 17% of the global cancer burden (Parkin 2006). discovered the first mammalian oncovirus, cottontail Studies have demonstrated that if these pathogens rabbit papillomavirus (CRPV), which could infect were eliminated, there would be 23.6% fewer cases of cancer in developing countries and 7.7% in developed countries, leading to a Preduction of 1.5 million and 390,000 cases of cancer per year, respectively (Parkin 2006). Given their significant yet surprising role in human cancer, oncoviruses are considered to be the second-most important risk factor after tobacco consumption for cancer development in humans (zur Hausen 1991). Currently, six viruses have been proven to cause over ten types of cancer in humans (see Table T he H istory of O ncovirus R esearch The discovery of oncoviruses dates back to the early 20th century, when in 1908 Oluf Bang and Vilhelm Ellerman, researchers at the University of Copenhagen, demonstrated that leukemia could be induced in healthy chickens by treating them with a filterable extract containing the avian leukemia virus (Javier and Butle 2008). Despite their results, the discovery of the first known tumor virus was credited to Payton Rous, who proved that the Rous sarcoma virus caused cancer in Plymouth Rock chickens B S J Figure 1. An electron micrograph of two Epstein Barr Virus virons. The dark sphere in the middle contains the virus’ genome. cottontail rabbits, and in 1936, John Bittner discovered the mouse mammary tumor virus (MMTV), which could be transmitted from mothers to pups via breast B erkeley S cientific J ournal • I nfectious D isease • F all 2010 • V olume 14 • I ssue 1 • 19

Vaccine generated immunity targets an HPV16 E7 HLA-A2.1-restricted CD8+ T cell epitope relocated to an early gene or a late gene of the cottontail rabbit papillomavirus (CRPV) genome in HLA-A2.1 transgenic rabbits

The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well known and computer-predicted A2-restricted epitopes. In the current study we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope that has been relocated to positions within the CRPV E7 gene and the CRPV L2 gene. Epitope expression from both the E7 protein and the L2 protein resulted in increased protection against viral DNA challenge of the HLA-A2.1 transgenic rabbits as compared to control-vaccinated rabbit groups. These data indicate that proteins expressed at both early and late time points during a natural papillomavirus infection can be targeted by epitope-specific immunity and indicate this immunity is increased to early rather than late expressed proteins of papillomaviruses. This study also highlights the broad utility of the HLAA2.1 transgenic rabbit model for testing numerous immunological factors involved in vaccine generated protective immunity.

Abstract CN01-03: Immunotherapy of established lesions caused by high-risk HPV

Abstract Therapeutic vaccination with a synthetic long peptide (SLP@) vaccine mediated the eradication of established human papillomavirus type 16 (HPV16)-positive tumors in mice and controlled wart growth and latent virus infection in rabbits persistently infected with cottontail rabbit papillomavirus. Subsequent phase I/II studies with an HPV16 SLP@ vaccine, consisting of 13 long peptides covering the HPV16 E6 and E7 antigens, in patients with advanced HPV16-positive cervical cancer, revealed that this vaccine was safe and highly immunogenic. The purpose of the current study was to test the clinical efficacy of this HPV16 SLP@ vaccine in HPV16-induced high-grade vulvar intraepithelial neoplasia (VIN3), a premalignant epithelial disorder, spontaneous regression of which occurs in less than 2% of patients and in which recurrence after standard treatment is high. In a phase 2 trial, 20 women with VIN3 were vaccinated three times sc in the limbs with a mix of the HPV16 E6 and E7 synthetic long peptides formulated in Montanide ISA-51. The endpoints were objective clinical responses, defined as reduction of at least 50% in lesion size (partial response) or complete regressions, and HPV16-specific T-cell responses, determined before and after vaccination. The vaccine was safe, as no side effects exceeding CTC grade 2 were observed. At 3 and 12 months after the last vaccination an objective response was observed in 12/20 (60%) and 15/19 (79%) patients respectively. Nine of them showed a complete and durable regression of the lesions at 12 months and at 24 months. The strength of the vaccine-induced HPV16-specific T-cell response was significantly higher in the group of patients with a complete regression of their lesions as compared to nonresponders. This study shows that in women with VIN3 objective clinical responses can be achieved by therapeutic vaccination with synthetic long peptides that is able to induce effective HVP16-specific T-cell responses. Literature: 1. Kenter GG, Welters MJ, Valentijn AR, Lowik MJ, Berends-van der Meer DM, Vloon AP, Essahsah F, Fathers LM, Drijfhout JW, Offringa R,Wafelman AR, Oostendorp J, Fleuren GJ, Burg van der SH, Melief CJ. Vaccination against Human Papillomavirus 16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med. 2009 Nov 5;361(19):1838-47. 2. Melief CJ, van der Burg SH. Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines. Nat Rev Cancer. 2008 May;8(5):351-60. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN01-03.