Cottontail Rabbit Papillomavirus E7 Research Articles

RNAi-Mediated Knockdown of Cottontail Rabbit Papillomavirus Oncogenes Using Low-Toxicity Lipopolyplexes as a Paradigm to Treat Papillomavirus-Associated Cancers.

The cottontail rabbit papillomavirus (CRPV)-associated VX2 carcinoma of the New Zealand White rabbit serves as a model system for human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCCs). The aim of this study was to evaluate the tumor-inhibiting effect of RNAi-mediated knockdown of the CRPV oncogenes, E6 and E7, using siRNA-loaded lipopolyplexes (LPPs). VX2-carcinoma-derived cells were cultured for up to 150 passages. In addition, CRPV E6 and E7 oncogenes were transiently expressed in COS-7 cells. Efficiency and safety of LPPs were evaluated in both VX2 cells and the COS-7 cell line. Both of these in vitro CRPV systems were validated and characterized by fluorescence microscopy, Western blot, and RT-qPCR. Efficient knockdown of CRPV E6 and E7 was achieved in VX2 cells and COS-7 cells pretransfected with CRPV E6 and E7 expression vectors. Knockdown of CRPV oncogenes in VX2 cells resulted in reduced viability, migration, and proliferation and led to a G0/G1 block in the cell cycle. CRPV E6 and E7 siRNA-loaded LPPs could represent promising therapeutic agents serving as a paradigm for the treatment of papillomavirus-positive cancers and could be of value for the treatment of CRPV-associated diseases in the rabbit such as papillomas and cancers of the skin.

Cottontail rabbit papillomavirus E6 proteins: Interaction with MAML1 and modulation of the Notch signaling pathway

Animal models are necessary to study how cutaneous human papillomaviruses (HPVs) are associated with carcinogenesis. The cottontail rabbit papillomavirus (CRPV) induces papilloma in the -cutaneous skin of rabbits and serves as an established animal model for HPVlinked carcinogenesis where viral E6 proteins play crucial roles. Several studies have reported the dysregulation of the Notch signaling pathway by cutaneous beta HPV, bovine PV and mouse PV E6 via their association with Mastermind-like 1 protein (MAML1), thus interfering with cell proliferation and differentiation. However, the CRPV E6 gene encodes an elongated E6 protein (long E6, LE6) and an N-terminally truncated product (short E6, SE6) making it unique from other E6 proteins. Here, we describe the interaction between both CRPV E6 proteins and MAML1 and their ability to downregulate the Notch signaling pathway which could be a way CRPV infection induces carcinogenesis similar to beta HPV.

Cottontail Rabbit Papillomavirus E1 and E2 Proteins Mutually Influence Their Subcellular Localizations.

The papillomavirus (PV) E2 protein is a nuclear, sequence-specific DNA-binding protein that regulates transcription and nuclear retention of viral genomes. E2 also interacts with the viral E1 protein to replicate the viral genome. E2 residue K111 is highly conserved among PV and has been implicated in contributing to nuclear transport, transcription, and replication. Cottontail rabbit (Sylvilagus floridanus) PV (CRPV or SfPV1) E2 K111R, A, or Q mutations are transcription deficient and localized to the cytoplasm, comparable to other PV types. The addition of a nuclear localization signal (NLS) resulted in nuclear E2 K111 mutant proteins but did not restore transcriptional activation, and this is most likely due to an impaired binding to the cellular Brd4 protein. Surprisingly, coexpression of E1 with E2 K111 mutations resulted in their nuclear localization and, for K111A and R mutations, the activation of an E1/E2-dependent reporter construct. Interestingly, the nuclear localization of E2 K111Q mutant protein was independent from the presence of the conserved bipartite NLS in E1 and the direct interaction between E1 and E2. On the other hand, the cytoplasmic E1 NLS mutation could be targeted to the nucleus by wild-type E2, and this was dependent upon an interaction between E1 and E2. In summary, our studies have uncovered that E1 and E2 control each other's subcellular localization: direct binding of E2 to E1 can direct E1 to the nucleus independently from the E1 NLS, and E1 can direct E2 to the nucleus without an intact NLS or direct binding to E2.IMPORTANCE Papillomaviruses encode the DNA-binding E1 and E2 proteins, which form a complex and are essential for genome replication. Both proteins are targeted to the nucleus via nuclear localization signals. Our studies have uncovered that cytoplasmic mutant E1 or E2 proteins can be localized to the nucleus when E1 or E2 is also present. An interaction between E1 and E2 is necessary to target cytoplasmic E1 mutant proteins to the nucleus, but cytoplasmic E2 mutant proteins can be targeted to the nucleus without a direct interaction, which points to a novel function of E1.

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.

Influence of HPV16 E2 and its localisation on the expression of matrix metalloproteinase-9

Infection with the high-risk HPV types 16 and 18 is the major cause of cervical cancer and plays a role in the development of certain head and neck and skin cancers. We have previously demonstrated that the Early Protein 2 of the Cottontail Rabbit papillomavirus (CRPV), required for skin carcinogenesis in a rabbit model, is able to induce the expression of a matrix metalloproteinase (MMP-9); a protease known to play a key role in invasion and metastasis. However, as of now we do not understand the underlying mechanism of activation nor relevance for the human system. Here, we report that high-risk human papillomavirus HPV16 E2 similar to our previously reported results on CRPV E2 activates the human MMP-9 promoter predominantly via the MEK1-ERK1/2-AP-1-signaling pathway. In addition this activation is associated with a nuclear sub-localisation of HPV16-E2 suggesting a nuclear protein-protein or protein-DNA interaction of E2 as the underlying mechanism of activation.

CRPV Genomes with Synonymous Codon Optimizations in the CRPV E7 Gene Show Phenotypic Differences in Growth and Altered Immunity upon E7 Vaccination

Papillomaviruses use rare codons relative to their hosts. Recent studies have demonstrated that synonymous codon changes in viral genes can lead to increased protein production when the codons are matched to those of cells in which the protein is being expressed. We theorized that the immunogenicity of the virus would be enhanced by matching codons of selected viral genes to those of the host. We report here that synonymous codon changes in the E7 oncogene are tolerated in the context of the cottontail rabbit papillomavirus (CRPV) genome. Papilloma growth rates differ depending upon the changes made indicating that synonymous codons are not necessarily neutral. Immunization with wild type E7 DNA yielded significant protection from subsequent challenge by both wild type and codon-modified genomes. The reduction in growth was most dramatic with the genome containing the greatest number of synonymous codon changes.

The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19 ARF induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53.

Vesicular Stomatitis Virus-Based Therapeutic Vaccination Targeted to the E1, E2, E6, and E7 Proteins of Cottontail Rabbit Papillomavirus

Persistent human papillomavirus (HPV)-associated benign and malignant lesions are a major cause of morbidity and mortality worldwide. Vaccination against HPV early proteins could provide an effective means of treating individuals with established infections. Recombinant vesicular stomatitis virus (VSV) vectors have been used previously to elicit strong humoral and cellular immune responses and develop prophylactic vaccines. We have shown that VSV vectors also can be used to elicit therapeutic immunity in the cottontail rabbit papillomavirus (CRPV)-rabbit model of high-risk HPV infection. In the present study, three new VSV vectors expressing the CRPV E1, E2, or E7 protein were produced and compared to the previously generated VSV-E6 vector for therapeutic efficacy. To determine whether vaccine efficacy could be augmented by simultaneous vaccination against two CRPV proteins, the four vaccines were delivered individually and in all possible pairings to rabbits 1 week after CRPV infection. Control rabbits received the recombinant wild-type VSV vector or medium only. Cumulative papilloma volumes were computed for analysis of the data. The analyses showed that VSV-based vaccination against the E1, E2, E6, or E7 protein significantly reduced papilloma volumes relative to those of the controls. Furthermore, VSV-based CRPV vaccination cured all of the papillomas in 5 of 30 rabbits. Of the individual vaccines, VSV-E7 was the most effective. The VSV-E7 vaccine alone was the most effective, as it reduced cumulative papilloma volumes by 96.9% overall, relative to those of the controls, and ultimately eliminated all of the disease in all of the vaccinees. Vaccine pairing was not, however, found to be beneficial, suggesting antigenic competition between the coexpressed CRPV proteins. These preclinical results, obtained in a physiologically relevant animal model of HPV infection, demonstrate that VSV vectors deserve serious consideration for further development as therapeutic antitumor vaccines.

An HLA-A2.1-Transgenic Rabbit Model to Study Immunity to Papillomavirus Infection

We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8(+) T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition, vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy.

Association of cottontail rabbit papillomavirus E6 oncoproteins with the hDlg/SAP97 tumor suppressor

Papillomaviruses are small DNA viruses that infect epithelial tissues and cause warts. Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The E6 and E7 oncogenes are the only genes consistently expressed in HPV-positive cervical cancer cells. Cottontail rabbit papillomavirus (CRPV) induces papillomas and carcinomas on cottontail and domestic rabbits and provides an excellent animal model of HPV infection and vaccine development. CRPV encodes three transforming proteins; LE6, SE6, and E7. Each of these proteins is required for papilloma formation. Like HPV E7, the CRPV E7 protein binds to the tumor suppressor pRB. In contrast, unlike HPV E6, the CRPV E6 proteins do not bind the tumor suppressor p53. Although more than a dozen cellular proteins have been identified as HPV E6 interacting proteins, nothing is known about the cellular interacting proteins of CRPV E6s. Here we describe the association of CRPV E6s with hDlg/SAP97, the mammalian homolog of the Drosophila discs large tumor suppressor protein. HPV E6 has previously shown to bind and target hDlg/SAP97 for degradation. Our results demonstrate that both LE6 and SE6 interact with hDlg/SAP97, although their association does not lead to the degradation of hDlg/SAP97. The PDZ domains of hDlg were shown to be sufficient for interaction with CRPV E6 proteins while the C-terminus of CRPV E6 is essential for the interaction with hDlg. The association of hDlg with SE6 may be important but not sufficient for the transformation of NIH 3T3 cells by SE6. Importantly, a CRPV SE6 mutant defective for papilloma formation did not interact with hDlg. These results suggest that interaction with hDlg/SAP97 plays a role in the biological function of CRPV E6s.

Gene Profiling of Cottontail Rabbit Papillomavirus-Induced Carcinomas Identifies Upregulated Genes Directly Involved in Stroma Invasion as Shown by Small Interfering RNA-Mediated Gene Silencing

To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 zeta and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin beta, which are involved in cell motility and tissue invasion, as well as casein kinase 1 alpha, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin beta in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.

A transactivator function of cottontail rabbit papillomavirus e2 is essential for tumor induction in rabbits.

Infection of domestic rabbits with cottontail rabbit papillomavirus (CRPV) causes local papillomas which progress to carcinomas in more than 80% of cases. This animal model system therefore allows the identification of molecular mechanisms required for the induction and progression of epithelial tumors. The viral E2 protein stimulates both viral DNA replication and transcription, and these functions can be genetically separated. We introduced the respective mutations into CRPV E2 and found, in line with published data for other papillomavirus E2 proteins, that mutation of the highly conserved amino acid 37 or 73 resulted in replication-competent but transactivation-deficient E2 proteins, whereas E2 proteins with mutations at residue 39 were replication deficient and transactivation competent. The R37A, I73L, and I73A E2 mutants, showing a loss of transactivation function, and the R37K E2 mutant, which is still transactivation competent, were introduced into the whole genome of CRPV, which was then injected into the skin of rabbits. Strikingly, the ability to induce tumors within 6 weeks was abolished by each of the E2 mutations, in contrast to the tumor induction rate (93%) obtained with wild-type CRPV DNA. Two small papillomas induced by mutant E2 I73A CRPV DNA appeared as late as 12 or 24 weeks postinjection, were significantly smaller, and showed no further extension of growth. These data suggest that functionally conserved amino acids in the transactivation domain of E2 are also required for the induction and growth of epithelial tumors in rabbits infected with CRPV.

Ubiquitin-fused and/or multiple early genes from cottontail rabbit papillomavirus as DNA vaccines.

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection.

Intracutaneous DNA vaccination with the E8 gene of cottontail rabbit papillomavirus induces protective immunity against virus challenge in rabbits.

The cottontail rabbit papillomavirus (CRPV)-rabbit model has been used in several studies for testing prophylactic and therapeutic papillomavirus vaccines. Earlier observations had shown that the CRPV nonstructural genes E1, E2, and E6 induced strong to partial protective immunity against CRPV infection. In this study, we found that CRPV E8 immunization eliminated virus-induced papillomas in EIII/JC inbred rabbits (100%) and provided partial protection (55%) against virus challenge in outbred New Zealand White rabbits. CRPV-E8 is a small open reading frame, coding for a 50-amino-acid protein, that is colinear with the CRPV E6 gene and has features similar to those of the bovine papillomavirus and human papillomavirus E5 genes. Papillomas that grew on E8-vaccinated outbred rabbits were significantly smaller than those on vector-vaccinated rabbits (P < 0.01; t test). Delayed-type hypersensitivity skin tests showed that some of the E8-vaccinated rabbits had positive responses to E8-specific peptides.

Combination treatment with intralesional cidofovir and viral-DNA vaccination cures large cottontail rabbit papillomavirus-induced papillomas and reduces recurrences.

We used the cottontail rabbit papillomavirus (CRPV) New Zealand White rabbit model to test a combination treatment of large established papillomas with intralesional cidofovir and DNA vaccination to cure sites and reduce recurrences. Intralesional 1% (wt/vol) (0.036 M) cidofovir treatment of rabbit papillomas led to elimination, or "cure," of the papillomas over a 6- to 8-week treatment period (N. D. Christenson, M. D. Pickel, L. R. Budgeon, and J. W. Kreider, Antivir. Res. 48:131-142, 2000). However, recurrences at periods from 1 to 8 weeks after treatment cessation were observed at approximately 50% of cured sites. DNA vaccinations with CRPV E1, E2, E6, and E7 were initiated either after or at the time of intralesional treatments, and the recurrence rates were observed. When DNA vaccinations were started after intralesional cures, recurrence rates were similar to those of vector-vaccinated rabbits. A small proportion of recurrent sites subsequently regressed (4 out of 10, or 40%) in the vaccinated group versus no regression of recurrences in the vector-immunized group (0 out of 19, or 0%), indicating partial effectiveness. In contrast, when DNA vaccinations were conducted during intralesional treatments, a significant reduction of recurrences (from 10 out of 19, or 53%, of sites in vector-immunized rabbits to 3 out of 20, or 15%, of sites in viral-DNA-immunized rabbits) was observed. DNA vaccination without intralesional treatments had a minimal effect on preexisting papillomas. These data indicated that treatment with a combination of antiviral compounds and specific immune stimulation may lead to long-term cures of lesions without the ensuing problem of papilloma recurrence.

High and Low Levels of Cottontail Rabbit Papillomavirus E2 Protein Generate Opposite Effects on Gene Expression

The papillomavirus E2 protein plays an important role in viral transcriptional regulation and replication. We chose to study the cottontail rabbit papillomavirus (CRPV) E2 protein as a transcriptional regulator because of the availability of an animal model for papilloma formation, which may be relevant for human papillomavirus (HPV) infection and replication. We studied the effect of expression levels of E2 on the long control region, which contains transcriptional promoter and enhancer elements, and synthetic E2-dependent artificial promoters in which the E2 was the dominant factor in the transcriptional activation. These experiments indicated that high levels of E2 were inhibitory and low levels were stimulatory for transactivation. In addition, we showed that the complex formed between CRPV E2 and the cognate binding site was less stable than the complex formed between HPV E2 and the same cognate binding site. Furthermore, we showed that CRPV E2 binding to its transcriptional regulatory sequence was stabilized by other proteins such as E1, which produced increments in transcriptional activation of E2-dependent genes. The data may be used to define conditions in which the rabbit model can be used for the screening of drugs which are inhibitory to the HPV and CRPV replication and gene expression.

DNA vaccination prevents and/or delays carcinoma development of papillomavirus-induced skin papillomas on rabbits.

Malignant progression is a life-threatening consequence of human papillomavirus-associated lesions. In this study, we tested the efficacy of papillomavirus early-gene-based vaccines for prevention of carcinoma development of papillomavirus-induced skin papillomas on rabbits. Rabbit skin papillomas were initiated by infection with cottontail rabbit papillomavirus (CRPV). The papillomas were allowed to grow for 3 months without any treatment intervention. Rabbits were then immunized by gene gun-mediated intracutaneous administration of four DNA plasmids encoding CRPV E1, E2, E6, and E7 genes, respectively. All eight control rabbits receiving vector alone developed invasive carcinoma within 8 to 13 months. In contrast, only two of eight vaccinated rabbits developed carcinoma at 12 and 15 months, respectively. Papilloma growth was suppressed in the majority of vaccinated rabbits but not completely eradicated. These results indicate that gene gun-mediated immunization with papillomavirus early genes may be a promising strategy for prevention of malignant progression of human papillomavirus-associated lesions in humans.

Granulocyte-macrophage colony-stimulating factor priming plus papillomavirus E6 DNA vaccination: effects on papilloma formation and regression in the cottontail rabbit papillomavirus--rabbit model.

A cottontail rabbit papillomavirus (CRPV) E6 DNA vaccine that induces significant protection against CRPV challenge was used in a superior vaccination regimen in which the cutaneous sites of vaccination were primed with an expression vector encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that induces differentiation and local recruitment of professional antigen-presenting cells. This treatment induced a massive influx of major histocompatibility complex class II-positive cells. In a vaccination-challenge experiment, rabbit groups were treated by E6 DNA vaccination, GM-CSF DNA inoculation, or a combination of both treatments. After two immunizations, rabbits were challenged with CRPV at low, moderate, and high stringencies and monitored for papilloma formation. As expected, all clinical outcomes were monotonically related to the stringency of the viral challenge. The results demonstrate that GM-CSF priming greatly augmented the effects of CRPV E6 vaccination. First, challenge sites in control rabbits (at the moderate challenge stringency) had a 0% probability of remaining disease free, versus a 50% probability in E6-vaccinated rabbits, and whereas GM-CSF alone had no effect, the interaction between GM-CSF priming and E6 vaccination increased disease-free survival to 67%. Second, the incubation period before papilloma onset was lengthened by E6 DNA vaccination alone or to some extent by GM-CSF DNA inoculation alone, and the combination of treatments induced additive effects. Third, the rate of papilloma growth was reduced by E6 vaccination and, to a lesser extent, by GM-CSF treatment. In addition, the interaction between the E6 and GM-CSF treatments was synergistic and yielded more than a 99% reduction in papilloma volume. Finally, regression occurred among the papillomas that formed in rabbits treated with the E6 vaccine and/or with GM-CSF, with the highest regression frequency occurring in rabbits that received the combination treatment.

Immunization of rabbits with cottontail rabbit papillomavirus E1 and E2 genes: protective immunity induced by gene gun-mediated intracutaneous delivery but not by intramuscular injection

We previously demonstrated that gene gun-based intracutaneous vaccination of rabbits with a combination of, but not with individual papillomavirus E1, E2, E6 and E7 genes provided complete protection against cottontail rabbit papillomavirus (CRPV) infection. In the present study, we tested whether vaccination of inbred and outbred rabbits with a combination of CRPV E1 and E2 genes could provide complete protection against virus infection. In the first experiment, gene gun-based intracutaneous vaccination with E1 and E2 genes prevented papilloma formation in the majority of inbred rabbits and promoted systemic papilloma regression in one non-protected rabbit. In contrast, needle-mediated intramuscular injection of E1 and E2 genes did not prevent papilloma formation nor promoted systemic papilloma regression, indicating an absence of strong protective immunity. In the second experiment, six outbred rabbits were immunized by gene gun-based intracutaneous administration of the E1 and E2 genes. Prevention of papilloma formation or systemic papilloma regression was observed in three vaccinated rabbits. Papillomas persisted on the remaining three rabbits, but were significantly smaller than that on control rabbits. These results suggested that gene gun-based intracutaneous vaccination with the combination of papillomavirus E1 and E2 genes induced strong protective antivirus immunity but may be insufficient for complete protection in an outbred population.

Intramuscular injection of plasmid DNA encoding cottontail rabbit papillomavirus E1, E2, E6 and E7 induces T cell-mediated but not humoral immune responses in rabbits

To test the efficacy of genetic vaccination against papillomavirus infection, plasmid DNA encoding cottontail rabbit papillomavirus (CRPV) E1, E2, E6, E7 or without insert were intramuscularly injected into five groups of rabbits. Peripheral blood mononuclear cells (PBMCs) showed specific proliferation upon in vitro stimulation with E1, E2, E6 or E7 proteins in a majority of vaccinated rabbits but Western blot analysis did not detect antibodies specific for these viral proteins in rabbit serum. All rabbits grew papillomas after virus challenge and none of the rabbits showed systemic papilloma regression. These observations showed that intramuscular injection of plasmid DNA encoding CRPV E1, E2, E6 or E7 induced CD4 + T cell-mediated but not humoral immune responses, and did not result in the protection of rabbits from virus infection.