Mouse papillomavirus infections spread to cutaneous sites with progression to malignancy.

We report secondary cutaneous infections in the mouse papillomavirus (MmuPV1)/mouse model. Our previous study demonstrated that cutaneous MmuPV1 infection could spread to mucosal sites. Recently, we observed that mucosal infections could also spread to various cutaneous sites including the back, tail, muzzle and mammary tissues. The secondary site lesions were positive for viral DNA, viral capsid protein and viral particles as determined by in situ hybridization, immunohistochemistry and transmission electron microscopy analyses, respectively. We also demonstrated differential viral production and tumour growth at different secondarily infected skin sites. For example, fewer viral particles were detected in the least susceptible back tissues when compared with those in the infected muzzle and tail, although similar amounts of viral DNA were detected. Follow-up studies demonstrated that significantly lower amounts of viral DNA were packaged in the back lesions. Lavages harvested from the oral cavity and lower genital tracts were equally infectious at both cutaneous and mucosal sites, supporting the broad tissue tropism of this papillomavirus. Importantly, two secondary skin lesions on the forearms of two mice displayed a malignant phenotype at about 9.5 months post-primary infection. Therefore, MmuPV1 induces not only dysplasia at mucosal sites such as the vagina, anus and oral cavity but also skin carcinoma at cutaneous sites. These findings demonstrate that MmuPV1 mucosal infection can be spread to cutaneous sites and suggest that the model could serve a useful role in the study of the viral life cycle and pathogenesis of papillomavirus.

Efficient replication of Epstein-Barr virus in stratified epithelium in vitro.

Epstein-Barr virus is a ubiquitous human herpesvirus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva and must cross the oral mucosal epithelium before infecting B lymphocytes, where it establishes a life-long infection. The latter process is well understood because it can be studied in vitro, but our knowledge of infection of epithelial cells has been limited by the inability to infect epithelial cells readily in vitro or to generate cell lines from EBV-infected epithelial tumors. Because epithelium exists as a stratified tissue in vivo, organotypic cultures may serve as a better model of EBV in epithelium than monolayer cultures. Here, we demonstrate that EBV is able to infect organotypic cultures of epithelial cells to establish a predominantly productive infection in the suprabasal layers of stratified epithelium, similar to that seen with Kaposi's-associated herpesvirus. These cells did express latency-associated proteins in addition to productive-cycle proteins, but a population of cells that exclusively expressed latency-associated viral proteins could not be detected; however, an inability to infect the basal layer would be unlike other herpesviruses examined in organotypic cultures. Furthermore, infection did not induce cellular proliferation, as it does in B cells, but instead resulted in cytopathic effects more commonly associated with productive viral replication. These data suggest that infection of epithelial cells is an integral part of viral spread, which typically does not result in the immortalization or enhanced growth of infected epithelial cells but rather in efficient production of virus.

Tracking vaginal, anal and oral infection in a mouse papillomavirus infection model.

Noninvasive and practical techniques to longitudinally track viral infection are sought after in clinical practice. We report a proof-of-principle study to monitor the viral DNA copy number using a newly established mouse papillomavirus (MmuPV1) mucosal infection model. We hypothesized that viral presence could be identified and quantified by collecting lavage samples from cervicovaginal, anal and oral sites. Nude mice infected at these sites with infectious MmuPV1 were tracked for up to 23 weeks starting at 6 weeks post-infection. Viral DNA copy number was determined by SYBR Green Q-PCR analysis. In addition, we tracked viral DNA load through three complete oestrous cycles to pinpoint whether there was a correlation between the DNA load and the four stages of the oestrous cycle. Our results showed that high viral DNA copy number was reproducibly detected from both anal and cervicovaginal lavage samples. The infection and disease progression were further confirmed by histology, cytology, in situ hybridization, immunohistochemistry and transmission electron microscopy. Interestingly, the viral copy number fluctuated over the oestrous cycle, with the highest level at the oestrus stage, implying that multiple sampling might be necessary to provide a reliable diagnosis. Virus DNA was detected in oral lavage samples at a later time after infection. Lower viral DNA load was found in oral samples when compared with those in anal and vaginal tracts. To our knowledge, our study is the first in vivo study to sequentially monitor papillomavirus infection from mucosal anal, oral and vaginal tracts in a preclinical model.

The type 1 diabetes resistance locus B10 Idd9.3 mediates impaired B-cell lymphopoiesis and implicates microRNA-34a in diabetes protection.

NOD.B10 Idd9.3 mice are congenic for the insulin-dependent diabetes (Idd) Idd9.3 locus, which confers significant type 1 diabetes (T1D) protection and encodes 19 genes, including microRNA (miR)-34a, from T1D-resistant C57BL/10 mice. B cells have been shown to play a critical role in the priming of autoantigen-specific CD4(+) T cells in T1D pathogenesis in non-obese diabetic (NOD) mice. We show that early B-cell development is impaired in NOD.B10 Idd9.3 mice, resulting in the profound reduction of transitional and mature splenic B cells as compared with NOD mice. Molecular analysis revealed that miR-34a expression was significantly higher in B-cell progenitors and marginal zone B cells from NOD.B10 Idd9.3 mice than in NOD mice. Furthermore, miR-34a expression in these cell populations inversely correlated with levels of Foxp1, an essential regulator of B-cell lymphopoiesis, which is directly repressed by miR-34a. In addition, we show that islet-specific CD4(+) T cells proliferated inefficiently when primed by NOD.B10 Idd9.3 B cells in vitro or in response to endogenous autoantigen in NOD.B10 Idd9.3 mice. Thus, Idd9.3-encoded miR-34a is a likely candidate in negatively regulating B-cell lymphopoiesis, which may contribute to inefficient expansion of islet-specific CD4(+) T cells and to T1D protection in NOD.B10 Idd9.3 mice.

Protection from tumor recurrence following adoptive immunotherapy varies with host conditioning regimen despite initial regression of autochthonous murine brain tumors.

Adoptive T cell transfer (ACT) has achieved clinical success in treating established cancer, particularly in combination with lymphodepleting regimens. Our group previously demonstrated that ACT following whole-body irradiation (WBI) promotes high-level T cell accumulation, regression of established brain tumors, and long-term protection from tumor recurrence in a mouse model of SV40 T antigen-induced choroid plexus tumors. Here we asked whether an approach that can promote strong donor T-cell responses in the absence of WBI might also produce this dramatic and durable tumor elimination following ACT. Agonist anti-CD40 antibody can enhance antigen-specific CD8(+) T-cell responses and has shown clinical efficacy as a monotherapy in the setting of cancer. We show that anti-CD40 conditioning promotes rapid accumulation of tumor-specific donor CD8(+) T cells in the brain and regression of autochthonous T antigen-induced choroid plexus tumors, similar to WBI. Despite a significant increase in the lifespan, tumors eventually recurred in anti-CD40-conditioned mice coincident with loss of T-cell persistence from both the brain and lymphoid organs. Depletion of CD8(+) T cells from the peripheral lymphoid organs of WBI-conditioned recipients failed to promote tumor recurrence, but donor cells persisted in the brains long-term in CD8-depleted mice. These results demonstrate that anti-CD40 conditioning effectively enhances ACT-mediated acute elimination of autochthonous tumors, but suggest that mechanisms associated with WBI conditioning, such as the induction of long-lived T cells, may be critical for protection from tumor recurrence.

Secondary infections, expanded tissue tropism, and evidence for malignant potential in immunocompromised mice infected with Mus musculus papillomavirus 1 DNA and virus.

Papillomavirus disease poses a special challenge to people with compromised immune systems. Appropriate models to study infections in these individuals are lacking. We report here the development of a model that will help to address these deficiencies. The MmuPV1 genome was synthesized and used successfully to produce virus from DNA infections in immunocompromised mice. In these early studies, we have demonstrated both primary and secondary infections, expanded tissue tropism, and extensive dysplasia.

Mouse Papillomavirus L1 and L2 Are Dispensable for Viral Infection and Persistence at Both Cutaneous and Mucosal Tissues.

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.

Papillomavirus can be transmitted through the blood and produce infections in blood recipients: Evidence from two animal models.

Human papillomaviruses (HPV) contribute to most cervical cancers and are considered to be sexually transmitted. However, papillomaviruses are often found in cancers of internal organs, including the stomach, raising the question as to how the viruses gain access to these sites. A possible connection between blood transfusion and HPV-associated disease has not received much attention. Here we show, in rabbit and mouse models, that blood infected with papillomavirus yields infections at permissive sites with detectable viral DNA, RNA transcripts, and protein products. The rabbit skin tumours induced via blood infection displayed decreased expression of SLN, TAC1, MYH8, PGAM2, and APOBEC2 and increased expression of SDRC7, KRT16, S100A9, IL36G, and FABP9, as seen in tumours induced by local infections. Furthermore, we demonstrate that blood from infected mice can transmit the infection to uninfected animals. Finally, we demonstrate the presence of papillomavirus infections and virus-induced hyperplasia in the stomach tissues of animals infected via the blood. These results indicate that blood transmission could be another route for papillomavirus infection, implying that the human blood supply, which is not screened for papillomaviruses, could be a potential source of HPV infection as well as subsequent cancers in tissues not normally associated with the viruses.

Comparative analysis of fixation and embedding techniques for optimized histological preparation of zebrafish.

In recognition of the importance of zebrafish as a model organism for studying human disease, we have created zebrafish content for a web-based reference atlas of microanatomy for comparing histology and histopathology between model systems and with humans (http://bio-atlas.psu.edu). Fixation, decalcification, embedding, and sectioning of zebrafish were optimized to maximize section quality. A comparison of protocols involving six fixatives showed that 10% Neutral Buffered Formalin at 21°C for 24h yielded excellent results. Sectioning of juveniles and adults requires bone decalcification; EDTA at 0.35M produced effective decalcification in 21-day-old juveniles through adults (≥~3Months). To improve section plane consistency in sets of larvae, we have developed new array casting molds based on the outside contours of larvae derived from 3D microCT images. Tissue discontinuity in sections, a common barrier to creating quality sections of zebrafish, was minimized by processing and embedding the formalin-fixed zebrafish tissues in plasticized forms of paraffin wax, and by periodic hydration of the block surface in ice water between sets of sections. Optimal H&E (Hematoxylin and Eosin) staining was achieved through refinement of standard protocols. High quality slide scans produced from glass histology slides were digitally processed to maximize image quality, and experimental replicates posted as full slides as part of this publication. Modifications to tissue processing are still needed to eliminate the need for block surface hydration. The further addition of slide collections from other model systems and 3D tools for visualizing tissue architecture would greatly increase the utility of the digital atlas.

Recent advances in preclinical model systems for papillomaviruses.

Preclinical model systems to study multiple features of the papillomavirus life cycle have greatly aided our understanding of Human Papillomavirus (HPV) biology, disease progression and treatments. The challenge to studying HPV in hosts is that HPV along with most PVs are both species and tissue restricted. Thus, fundamental properties of HPV viral proteins can be assessed in specialized cell culture systems but host responses that involve innate immunity and host restriction factors requires preclinical surrogate models. Fortunately, there are several well-characterized and new animal models of papillomavirus infections that are available to the PV research community. Old models that continue to have value include canine, bovine and rabbit PV models and new rodent models are in place to better assess host-virus interactions. Questions arise as to the strengths and weaknesses of animal PV models for HPV disease and how accurately these preclinical models predict malignant progression, vaccine efficacy and therapeutic control of HPV-associated disease. In this review, we examine current preclinical models and highlight the strengths and weaknesses of the various models as well as provide an update on new opportunities to study the numerous unknowns that persist in the HPV research field.

The Mouse Papillomavirus Infection Model.

The mouse papillomavirus (MmuPV1) was first reported in 2011 and has since become a powerful research tool. Through collective efforts from different groups, significant progress has been made in the understanding of molecular, virological, and immunological mechanisms of MmuPV1 infections in both immunocompromised and immunocompetent hosts. This mouse papillomavirus provides, for the first time, the opportunity to study papillomavirus infections in the context of a small common laboratory animal for which abundant reagents are available and for which many strains exist. The model is a major step forward in the study of papillomavirus disease and pathology. In this review, we summarize studies using MmuPV1 over the past six years and share our perspectives on the value of this unique model system. Specifically, we discuss viral pathogenesis in cutaneous and mucosal tissues as well as in different mouse strains, immune responses to the virus, and local host-restricted factors that may be involved in MmuPV1 infections and associated disease progression.

Mouse papillomavirus infection persists in mucosal tissues of an immunocompetent mouse strain and progresses to cancer.

Mouse papillomavirus has shown broad tissue tropism in nude mice. Previous studies have tested cutaneous infections in different immunocompromised and immunocompetent mouse strains. In the current study, we examined mucosal infection in several immunocompetent and immunocompromised mouse strains. Viral DNA was monitored periodically by Q-PCR of lavage samples. Immunohistochemistry and in situ hybridization were used to determine viral capsid protein and viral DNA respectively. All athymic nude mouse strains showed active infections at both cutaneous and mucosal sites. Interestingly, NOD/SCID mice, which have a deficiency in T, B, and NK cells, showed minimal disease at cutaneous sites but developed persistent infection at the mucosal sites including those of the anogenital region and the oral cavity. Three strains of immunocompetent mice supported mucosal infections. Infections of the lower genital tract in heterozygous (immunocompetent) mice of the NU/J strain progressed to high grade dysplasia and to carcinoma in situ. Anti-MmuPV1 neutralizing antibodies were detected in the sera of all immunocompetent animals. Our findings demonstrate that the mucosae may be the preferred sites for this virus in mice. The mouse model is expected to be a valuable model for the study of mucosal papillomavirus disease, progression, and host immune control.

Protective cell-mediated immunity by DNA vaccination against Papillomavirus L1 capsid protein in the Cottontail Rabbit Papillomavirus model.

Papillomavirus major capsid protein L1 has successfully stimulated protective immunity against virus infection by induction of neutralizing antibodies in animal models and in clinical trials. However, the potential impact of L1-induced protective cell-mediated immune (CMI) responses is difficult to measure in vivo because of the coincidence of anti-L1 antibody. In this study, we tested the hypothesis that L1 could activate CMI, using the Cottontail Rabbit Papillomavirus (CRPV)-rabbit model. A unique property of this model is that infections can be initiated with viral DNA, thus bypassing all contributions to protection via neutralizing anti-L1 antibody. DNA vaccines containing either CRPV L1, or subfragments of L1 (amino-terminal two-thirds of L1 [L1N] and the carboxylterminal two-thirds of L1 [L1C]), were delivered intracutaneously into rabbits, using a gene gun. After three booster immunizations, the rabbits were challenged with several viral DNA constructs: wild-type CRPV, CRPV L1ATGko (an L1 ATG knockout mutation), and CRPV-ROPV hybrid (CRPV with a replacement L1 from Rabbit Oral Papillomavirus). Challenge of L1 DNA-vaccinated rabbits with wild-type CRPV resulted in significantly fewer papillomas when compared with challenge with CRPV L1ATGko DNA. Significantly smaller papillomas were found in CRPV L1-, L1N-, and L1C-vaccinated rabbits. In addition, rabbits vaccinated with either L1 or L1N grew significantly fewer and smaller papillomas when challenged with CRPV-ROPV hybrid DNA. Therefore, CRPV L1 DNA vaccination induced CMI responses to CRPV DNA infections that can contribute to protective immunity. Cross-protective immunity against CRPV L1 and ROPV L1 was elicited in these CRPV L1- and subfragment-vaccinated rabbits.

Mouse papillomavirus MmuPV1 infects oral mucosa and preferentially targets the base of the tongue.

In 2010, a new mouse papillomavirus, MmuPV1, was discovered in a colony of NMRI- Foxn1(nu)/Foxn1(nu) athymic mice in India. This finding was significant because it was the first papillomavirus to be found in a laboratory mouse. In this paper we report successful infections of both dorsal and ventral surfaces of the rostral tongues of outbred athymic nude mice. We also report the observation that the base of the tongue, the area of the tongue often targeted by cancer-associated high-risk papillomavirus infections in humans, is especially susceptible to infection. A suitable animal model for the study of oral papillomavirus infections, co-infections, and cancers has long been sought. The work presented here suggests that such a model is now at hand.

Characterization of immune responses during regression of rabbit oral papillomavirus infections.

Rabbit oral papillomavirus (ROPV) is a mucosatropic papillomavirus that causes small benign discrete papillomas within the oral cavity of domestic rabbits. The goal of this study was to characterize the immune cell infiltrate over the course of regression of oral papillomas. ROPV-infected oral tissues were harvested at various time points after infection and analyzed by immunohistochemistry for papilloma morphology, viral capsid proteins, and associated immune infiltrates. The results of this study indicated that the L1 and L2 viral capsid proteins were lost rapidly at a time that coincided with an inflammatory response from the rabbit. This inflammatory response began with a rapid rise in numbers of CD11c+ cells at early regression. CD11c+ cells continued to increase in frequency through mid-regression and remained the most-represented cell through late regression. The initial rise in CD11c+ cells was followed by an infiltrate containing increased numbers of activated T cells, including CD4+ and CD25+ cells, during mid-regression. Mid-regression coincided spatially with a loss of viral capsid stain, suggesting that immune cells or cytokines or both were playing a key role in clearance of the papillomas. CD8+ cells increased at the lowest rate and were at low levels in the papilloma epidermis even at mid-regression. All cell types decreased by late regression. CD11c+ and major histocompatibility class II+ cells were the last populations of cells to decrease in number.

A novel pre-clinical murine model to study the life cycle and progression of cervical and anal papillomavirus infections.

BACKGROUND: Papillomavirus disease and associated cancers remain a significant health burden in much of the world. The current protective vaccines, Gardasil and Cervarix, are expensive and not readily available to the underprivileged. In addition, the vaccines have not gained wide acceptance in the United States nor do they provide therapeutic value. Papillomaviruses are strictly species specific and thus human viruses cannot be studied in an animal host. An appropriate model for mucosal disease has long been sought. We chose to investigate whether the newly discovered mouse papillomavirus, MmuPV1, could infect mucosal tissues in Foxn1nu/Foxn1nu mice. METHODS: The vaginal and anal canals of Foxn1nu/Foxn1nu mice were gently abraded using Nonoxynol-9 and "Doctor's BrushPicks" and MmuPV1 was delivered into the vaginal tract or the anal canal. RESULTS: Productive vaginal, cervical and anal infections developed in all mice. Vaginal/cervical infections could be monitored by vaginal lavage. Dysplasias were evident in all animals. CONCLUSIONS: Anogenital tissues of a common laboratory mouse can be infected with a papillomavirus unique to that animal. This observation will pave the way for fundamental virological and immunological studies that have been challenging to carry out heretofore due to lack of a suitable model system.

A small molecule antagonist of the alpha(v)beta3 integrin suppresses MDA-MB-435 skeletal metastasis.

INTRODUCTION: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. MATERIALS AND METHODS: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. RESULTS AND CONCLUSIONS: IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.

Mucosally-derived HPV-40 can infect both human genital foreskin and cutaneous hand skin tissues grafted into athymic mice.

HPV-40 is a rare HPV type that has been detected only in genital mucosal tissues. This HPV type is very closely related to HPV-7, which has a predominantly cutaneous tissue tropism. We have shown, previously, that an isolate of HPV-40 (described here as HPV-40(Hershey) or HPV-40(H)) productively infected genital tissues. In this study, HPV-40(H) was tested for productive infection of cutaneous tissue. Fetal hand skin fragments were incubated with infectious HPV-40(H) and implanted subrenally into athymic mice. After 120 days, xenografts showed morphological changes consistent with HPV-40(H) infection and were HPV-40 DNA in situ positive and capsid antigen positive. The results demonstrated that hand skin can support HPV-40(H) infection thereby indicating that this viral type has the capacity to infect both genital mucosal and cutaneous tissues.

Vaccines and immunization against human papillomavirus.

Prophylactic and therapeutic immunization strategies are an effective method to control human papillomavirus (HPV)-associated diseases and cancers. Current protective virus-like particle and capsid-based vaccines are highly protective against vaccine-matched HPV types, and continued improvements in second-generation vaccines will lead to broader protection and cross-protection against the cancer-associated types. Increasing the effectiveness of broadly cross-protective L2-based immunogens will require adjuvants that activate innate immunity to thus enhance adaptive immunity. Therapeutic immunization strategies are needed to control and cure clinical disease and HPV-associated cancers. Significant advances in strategies to improve induction of cell-mediated immunity to HPV early (and capsid) proteins have been pretested in preclinical animal papillomavirus models. Several of these effective protocols have translated into successful therapeutic immune-mediated clearance of clinical lesions. Nevertheless, there are significant challenges in activating immunity to cancer-associated lesions due to various immune downregulatory events that are triggered by persistent HPV infections. A better understanding of immune responses to HPV lesions in situ is needed to optimize immune effector T cells that efficiently locate to sites of infection and which should lead to an effective immunotherapeutic management of this important human viral pathogen. The most effective immunization strategy may well require combination antiviral and immunotherapeutic treatments to achieve complete clearance of HPV infections and associated cancers.

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, E1peptide 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 mediate 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.