Therapeutic vaccination of rabbits with a ubiquitin-fused papillomavirus E1, E2, E6 and E7 DNA vaccine

Previously, we showed that intracutaneous vaccination of rabbits with DNA vectors encoding ubiquitin-fused versions of the cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, E6 and E7 protected against subsequent challenge with CRPV. Here, we tested the immunotherapeutic activity of a vaccine composed of the four CRPV DNA vectors (designated UbE1267) in rabbits. The results show that the UbE1267 DNA vaccine, relative to empty vector DNA, virtually eliminated papilloma growth in rabbits with subclinical infection and greatly reduced papilloma volumes in rabbits bearing papillomas at the time of vaccination. These results in a physiologically relevant animal model of high-risk human papillomavirus (HPV) infection indicate that DNA vaccines targeting the early papillomavirus proteins may have a role in the treatment of HPV-associated lesions in humans.     

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.     

Therapeutic efficacy of vesicular stomatitis virus-based E6 vaccination in rabbits

Millions of people worldwide are currently infected with human papillomaviruses (HPVs). A therapeutic HPV vaccine would have widespread applicability because HPV-associated lesions are difficult to treat and may progress to carcinoma. We developed three attenuated VSV recombinants expressing the cottontail rabbit papillomavirus (CRPV) early protein E6 for use as vaccines. In cultured cells, two vectors expressed different levels of the E6 protein, and one expressed a ubiquitin-E6 fusion protein. All three were tested for therapeutic efficacy in the cottontail rabbit papillomavirus (CRPV)-rabbit model. Mock vaccination had no effect on papilloma growth. In contrast, inoculation with any of the VSV-E6 vaccines reduced the rate of papilloma growth to as little as 24% the rate in the controls. In five experiments, these effects were achieved after a single immunization. Furthermore, complete papilloma regression occurred in some rabbits observed for 4 months. A VSV-based papillomavirus E6 vaccine could have significant advantages over other therapeutic HPV vaccine candidates described to date.     

GM-CSF enhances protective immunity to cottontail rabbit papillomavirus E8 genetic vaccination in rabbits

We have reported previously that cottontail rabbit papillomavirus (CRPV) E8 gene immunization induced strong protection against virus challenge. In this study, we primed E8 gene vaccination with mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF), a cytokine that induces differentiation and local recruitment of professional antigen-presenting cells. EIII/JC inbred rabbits were divided into four groups receiving vaccinations with the following constructs: mGM-CSF plus E8, mGM-CSF only, E8 only and vector only. After three immunizations at intervals of 3 weeks, rabbits were challenged with viral DNA at six scarified sites. Papillomas grew on all vaccinated rabbits 4 weeks after inoculation. At week 5, papillomas on four rabbits of mGM-CSF plus E8 and one of E8 only rabbits began to regress. At week 11, all the papillomas on rabbits in the GM-CSF plus E8 vaccination group regressed (regression rate = 100%); regression rates of the mGM-CSF only and E8 only vaccination groups were 50 and 25%, respectively. All papillomas on the vector immunized rabbits remained persistent until the end of the experiment (0%). Antibodies to mGM-CSF were detected in rabbit serum by Western blot. Rabbits vaccinated with E8 plus mGM-CSF or E8 only group had positive Delayed-type hypersensitivity (DTH) skin test to different E8 peptides. These results demonstrated that mGM-CSF could enhance the effects of E8 immunization in rabbits to CRPV infection through cell-mediated immune responses.     

Reversal of papilloma growth in rabbits therapeutically vaccinated against E6 with naked DNA and/or vesicular stomatitis virus vectors

Persistent infection with high-risk human papillomaviruses (HPVs) is the greatest risk factor for the development of HPV-associated cancers. In this study rabbits bearing persistent and potentially malignant papillomas were used to test the efficacy of vaccination with a recombinant DNA and/or vesicular stomatitis virus (VSV) targeting the cottontail rabbit papillomavirus (CRPV) E6 protein. Immune responses were primed with either vector and boosted twice with the homologous or heterologous E6 vector. Over the course of 18 weeks, E6 vaccination reduced papilloma volumes to one third the volume in the controls, and the rabbits boosted with an heterologous vector tended to mount stronger responses. Small and medium-sized papillomas responded significantly but only slightly better than large papillomas. Finally the initial papilloma burden per rabbit, ranging from <100 mm³ to >1000 mm³, was not prognostic of antitumor efficacy. In summary both E6 vaccines elicited significant therapeutic immunity, and their sequential use tended to be advantageous.     

Mucosally delivered peptides prime strong immunity in HLA-A2.1 transgenic rabbits

DNA vaccines delivered subcutaneously by gene-gun have generated strong protective and therapeutic immunity in rabbits. Recent studies have shown that peptides delivered by the mucosal routes also stimulate local and systemic immune responses. Since mucosal delivery is easier to administer and more cost-effective when compared to gene-gun delivery, we were interested to learn whether mucosally delivered peptides would prime protective immunity comparable to that of gene-gun-delivered DNA in rabbits. Our newly developed HLA-A2.1 transgenic rabbit model was used to test the hypothesis. We chose an HLA-A2.1 restricted cottontail rabbit papillomavirus (CRPV) E1 epitope (E1/303-311, MLQEKPFQL) for the peptide immunization studies because it provided complete protection when used as a DNA vaccine. Adjuvant has been widely used to boost immunity for vaccines. In this study, three adjuvants reported to be effective for rabbits (TT helper motif, PADRE and CpG2007) were tested with the peptide vaccine. Peptide alone or fused to TT helper or PADRE to create chimeric peptides was delivered by two mucosal routes (ocular and intranasal) together. Partial protection was found in HLA-A2.1 transgenic rabbits when peptide was delivered mucosally in the presence of adjuvant. When a subsequent booster of a half-dose of the corresponding DNA vaccine was delivered, complete protections were achieved. We conclude that mucosal peptide immunization can be combined with a single DNA vaccination to provide strong protective immunity in rabbits.     

Immunisation with recombinant BCG expressing the cottontail rabbit papillomavirus (CRPV) L1 gene provides protection from CRPV challenge

Recombinant Bacille Calmette-Guerin (rBCG) could potentially be the vaccine vehicle of choice to deliver foreign antigens from multiple pathogens. In this study we have used the cottontail rabbit papillomavirus (CRPV) rabbit model to provide a "proof of concept" that immunisation with rBCG expressing the CRPV major capsid protein, L1 (rBCG/CRPVL1), will protect outbred New Zealand White rabbits against CRPV challenge. Rabbits immunised with rBCG/CRPVL1 (10(7) cfu/ml) were protected 5 weeks post-CRPV challenge. Rabbits immunised with rBCG/CRPVL1 (10(5) cfu/ml) had papillomas, which were smaller and took longer to appear than the control rabbits. None of the negative control rabbits vaccinated with rBCG expressing an irrelevant gene or PBS were protected from CRPV challenge. Sera from rabbits immunised with rBCG/CRPVL1 (10(7) cfu/ml) were able to neutralise 54.5% of CRPV at serum dilutions of 1:200. These results provide evidence that BCG could potentially be used as a vaccine delivery vehicle for human papillomavirus proteins as a possible prophylactic vaccine.     

Protective immunity with an E1 multivalent epitope DNA vaccine against cottontail rabbit papillomavirus (CRPV) infection in an HLA-A2.1 transgenic rabbit model

Cottontail rabbit papillomavirus (CRPV)/rabbit model is widely used to study pathogenesis of papillomavirus infections and malignant tumor progression. Recently, we established HLA-A2.1 transgenic rabbit lines and demonstrated efficacy for the testing of immunogenicity of a well-known A2-resticted epitope (HPV16E7/82-90) [Hu J, Peng X, Schell TD, Budgeon LR, Cladel NM, Christensen ND. An HLA-A2.1-transgenic rabbit model to study immunity to papillomavirus infection. J Immunol 2006;177(11):8037-45]. In the present study, we screened five HLA-A2.1 restricted epitopes from CRPVE1 (selected using online MHCI epitope prediction software) and constructed a multivalent epitope DNA vaccine (CRPVE1ep1-5). CRPVE1ep1-5 and a control DNA vaccine (Ub3) were then delivered intracutaneously onto normal and HLA-A2.1 transgenic rabbits, respectively, by a helium-driven gene-gun delivery system. One, two or three immunizations were given to different groups of animals from both New Zealand White outbred and EIII/JC inbred genetic background. Two and three immunizations with CRPVE1ep1-5 DNA vaccine provided complete protection against viral DNA infection of HLA-A2.1 transgenic rabbits from both genetic backgrounds but not in the control-vaccinated groups. One immunization, however, failed to protect HLA-A2.1 transgenic rabbits against viral DNA infection. This study further demonstrated that the HLA-A2.1 transgenic rabbits can be used to test the immunogenicity of HLA-A2.1 restricted epitopes identified by MHCI epitope predication software.     

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.     

Protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing L2 capsid epitopes

Cottontail rabbit papillomavirus (CRPV) and rabbit oral papillomavirus (ROPV) represent distantly related, cutaneous and mucosal tissue tropic papillomaviruses respectively that can infect the same host. These two viruses were used to test the effectiveness of an L2 peptide-based vaccine (aa 94-122) that was delivered on the surface of recombinant tobacco mosaic virus (rTMV) particles. Groups of NZW rabbits received combinations of CRPVL2, ROPVL2 and CRPV+ROPVL2 rTMV vaccines, and were then challenged with infectious CRPV and ROPV. The rabbits developed antibodies that reacted to whole L2 protein and these sera were able to neutralize CRPV pseudovirions at half-maximal titers that were between 50 and 500. Rabbits receiving the CRPV L2 vaccine alone or in combination with ROPV L2 vaccines were completely protected against CRPV infections. Those rabbits vaccinated with the ROPV L2 vaccines showed a weak response in some rabbits against CRPV infection. These studies demonstrate that L2-based vaccines provide strong protection against experimental papillomavirus infection that is most likely based upon the induction of virus-neutralizing antibody. Notably, we observed some limited cross-protection induced by the L2 sequences tested in these vaccines. Finally, the study demonstrated that rTMV were excellent agents for the induction of strong protection in a pre-clinical disease model of papillomavirus infection.     

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.     

Glycoprotein E2 of classical swine fever virus expressed by baculovirus induces the protective immune responses in rabbits

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1 + 2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1 + 2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1 + 2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1 + 2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1 + 2.1E2 are promising candidate subunit vaccines against CSF.     

A novel, broad spectrum therapeutic HPV vaccine targeting the E7 proteins of HPV16, 18, 31, 45 and 52 that elicits potent E7-specific CD8T cell immunity and regression of large, established, E7-expressing TC-1 tumors

Persistent infection by high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, which remains one of the most common cancers among women worldwide. In addition, there is a growing appreciation that high risk HPVs are associated with a number of other cancers including anogenital cancers as well as a subset of head and neck cancers. Recently, prophylactic HPV vaccines targeting the two most prevalent high risk HPVs (HPV16 and HPV18) have been deployed in large-scale vaccination campaigns. However, the extent to which these prophylactic vaccines confer protection against other high risk HPV genotypes is largely unknown and prophylactic vaccines have been shown to be ineffective against pre-existing infection. Thus there continues to be an urgent need for effective therapeutic vaccines against HPV. The E7 protein of HPV16 has been widely studied as a target for therapeutic vaccines in HPV-associated cancer settings because HPV16 is the most prevalent of the high risk HPV genotypes. However, HPV16 accounts for only about 50% of cervical cancers and there are at least 15 other high risk HPVs that are known to be oncogenic. We have developed a novel, broad-spectrum, therapeutic vaccine (Pentarix) directed at the E7 proteins from five of the most prevalent high-risk genotypes of HPV worldwide (HPV16, 18, 31, 45 and 52) that together account for more than 80% of all HPV-associated cancers. Pentarix is a recombinant protein-based vaccine that elicits strong, multi-genotype specific CD8 T cell immunity when administered to mice in combination with adjuvants comprised of agonists of the TLR3 or TLR9 family of innate immune receptors. Furthermore, large, established E7-expressing TC-1 tumors undergo rapid and complete regression after therapeutic vaccination of mice with Pentarix. Together, these data suggest that Pentarix may be of clinical value for patients with E7-positive, HPV-associated precancerous lesions or malignant disease.     

Induction of antitumor immunity in vivo following delivery of a novel HPV-16 DNA vaccine encoding an E6/E7 fusion antigen

Human papillomavirus type 16 (HPV-16) infection is associated with a majority of cervical cancers and a significant proportion of head and neck cancers. Here, we describe a novel-engineered DNA vaccine that encodes a HPV-16 consensus E6/E7 fusion gene (pConE6E7) with the goal of increasing its antitumor cellular immunity. Compared to an early stage HPV-16 E7 DNA vaccine (pE7), this construct was up to five times more potent in driving E7-specific cellular immune responses. Prophylactic administration of this vaccine resulted in 100% protection against HPV E6 and E7-expressing tumors. Therapeutic studies indicated that vaccination with pConE6E7 prevented or delayed the growth of tumors. Moreover, immunization with pConE6E7 could also partially overcome immune tolerance in E6/E7 transgenic mice. Such DNA immunogens are interesting candidates for further study to investigate mechanisms of tumor immune rejection in vivo.     

Preclinical development of peptide vaccination combined with oncolytic MG1-E6E7 for HPV-associated cancer

Human papilloma virus (HPV)-associated cancer is a significant global health burden and despite the presence of viral transforming antigens within neoplastic cells, therapeutic vaccinations are ineffective for advanced disease. HPV positive TC1 cells are susceptible to viral oncolysis by MG1-E6E7, a custom designed oncolytic Maraba virus. Epitope mapping of mice vaccinated with MG1-E6E7 enabled the rational design of synthetic long peptide (SLP) vaccines against HPV16 and HPV18 antigens. SLPs were able to induce specific CD8+ immune responses and the magnitude of these responses significantly increased when boosted by MG1-E6E7. Logically designed vaccination induced multi-functional CD8+ T cells and provided complete sterilising immunity of mice challenged with TC1 cells. In mice bearing large HPV-positive tumours, SLP vaccination combined with MG1-E6E7 was able to clear tumours in 60% of mice and these mice were completely protected against a long term aggressive re-challenge with the TC1 tumour model. Combining conventional SLPs with the multi-functional oncolytic MG1-E6E7 represents a promising approach against advanced HPV positive neoplasia.     

Incorporation of RG1 epitope concatemers into a self-adjuvanting Flagellin-L2 vaccine broaden durable protection against cutaneous challenge with diverse human papillomavirus genotypes

AimTo achieve durable and broad protection against human papillomaviruses by vaccination with multimers of minor capsid antigen L2 using self-adjuvanting fusions with the toll-like receptor-5 (TLR5) ligand bacterial flagellin (Fla) instead of co-formulation with alum.MethodsFla fusions with L2 protective epitopes comprising residues 11-200, 11-88 and/or 17-38 of a single or multiple HPV types were produced in E. coli and their capacity to activate TLR5 signaling was assessed. Immunogenicity was evaluated serially following administration of 3 intramuscular doses of Fla-L2 multimer without exogenous adjuvant, followed by challenge 1, 3, 6 or 12 months later, and efficacy compared to vaccination with human doses of L1 VLP vaccines (Gardasil and Cervarix) or L2 multimer formulated in alum. Serum antibody responses were assessed by peptide ELISA, in vitro neutralization assays and passive transfer to naïve rabbits in which End-Point Protection Titers (EPPT) were determined using serial dilutions of pooled immune sera collected 1, 3, 6 or 12 months after completing active immunization. Efficacy was assessed by determining wart volume following concurrent challenge at different sites with HPV6/16/18/31/45/58 ‘quasivirions’ containing cottontail rabbit papillomavirus (CRPV) genomes.ResultsVaccination in the absence of exogenous adjuvant with Fla-HPV16 L2 11-200 fusion protein elicited durable protection against HPV16, but limited cross-protection against other HPV types. Peptide mapping data suggested the importance of the 17-38 aa region in conferring immunity. Indeed, addition of L2 residues 17-38 of HPV6/18/31/39/52 to a Fla-HPV16 L2 11-200 or 11-88 elicited broader protection via active or passive immunization, similar to that seen with vaccination with an alum-adjuvanted L2 multimer comprising the aa 11-88 peptides of five or eight genital HPV types.ConclusionsVaccination with flagellin fused L2 multimers provided lasting (>1 year) immunity without the need for an exogenous adjuvant. Inclusion of the L2 amino acid 17-38 region in such multi-HPV type fusions expanded the spectrum of protection.     

Protection from IBDV-induced bursal damage by a recombinant fowlpox vaccine, fpIBD1, is dependent on the titre of challenge virus and chicken genotype

Expression of the VP2 capsid protein of infectious bursal disease virus (IBDV) in an vaccine strain of fowlpox has produced an experimental recombinant vaccine, fpIBD1. Successful vaccination with fpIBD1 was dependent on the titre of challenge virus for high titres of challenge virus were able to overcome protection induced by fpIBD1 whereas challenge with a low titre of virus did not. The genotype of chicken also has an important effect on the outcome of challenge possibly as a result of the major histocompatability complex and its ability to present VP2-derived peptides to the immune system. It was not possible to protect the inbred white leghorn chicken strain, line 15I, from IBDV-induced bursal damage by vaccination with fpIBD1 even at the lowest titre of challenge virus used. All other inbred white leghorn chickens examined (line 6₁, C.B4 and C.B12) and outbred Rhode Island Red chickens were protected by fpIBD1. Protection by the fpIBD1 vaccine is induced in the absence of detectable serum antibodies, suggesting the possibility of a significant role for cell-mediated immunity in protection from IBDV challenge.     

Characterization of protective humoral and cellular immune responses against RHDV2 induced by a new vaccine based on recombinant baculovirus

Rabbit hemorrhagic disease (RHD) is a lethal disease in rabbits caused by RHD virus (RHDV). Protection is only possible through vaccination. A new virus variant (RHDV2) which emerged in 2010 in France differed from the classical RHDV1 variant in certain aspects and vaccines against RHDV1 induced limited cross protection only. In a previous study, we designed a recombinant baculovirus based RHDV2-VP1 vaccine, which provided a protective immunity in rabbits against RHDV2. In the present study this newly created vaccine is characterized with regard to onset and duration of protection, and possible cross protection against classical RHDV1. Furthermore, humoral and cellular immune mechanisms in vaccinated and infected rabbits were analyzed. In all experiments, the recombinant vaccine was compared to a conventional liver-based RHDV2 vaccine.The RHDV2-VP1 vaccine induced a protective immune response already seven days after single vaccination and fully protected for at least 14 months. A booster vaccination 21 days after the first had a negative influence on long-term protection. The cross protection provided by the RHDV2-VP1 vaccine against classical RHDV1 was limited since only 50% of vaccinated rabbits survived the infection. Conclusively, the new, baculovirus-based RHDV2-VP1 vaccine has the potential to protect rabbits against the infection with RHDV2, blocks completely the disease progression and prevents the spread of RHDV2 at the population level.     

Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes

An effective vaccine for treating human papillomavirus (HPV)-associated malignancies such as cervical cancer should elicit strong T cell-mediated immunity (CMI) against the E6 and/or E7 proteins necessary for the malignant state. We have developed Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) vaccines encoding the HPV16 E6 and E7 genes and tested their immunogenicity and antitumor efficacy. The E6 and E7 genes were fused to create one open reading frame and mutated at four or at five amino acid positions to inactivate their oncogenic potential. VRP encoding mutant or wild type E6 and E7 proteins elicited comparable cytotoxic T lymphocyte (CTL) responses to an immunodominant E7(49-57) epitope and generated comparable antitumor responses in several HPV16 E6(+)E7(+) tumor challenge models: protection from either C3 or TC-1 tumor challenge was observed in 100% of VRP-vaccinated mice. Eradication of C3 tumors was observed in approximately 90% of mice following therapeutic VRP vaccination. Eradication of HLF16 tumors lacking the E7(49-57) epitope was observed in 90% of human leukocyte antigen (HLA)-A(*)0201 transgenic mice following therapeutic VRP vaccination. Finally, the predicted inactivation of E6 and E7 oncogenic potential was confirmed by demonstrating normal levels of both p53 and retinoblastoma proteins in human mammary epithelial cells (MEC) infected with VRP expressing mutant E6 and E7 genes. These promising results support the continued development of mutant E6 and E7 VRP as safe and effective candidates for clinical evaluation against HPV-associated disease.