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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of TA-CIN (HPV 16 L2E6E7) booster immunisation in vulval intraepithelial neoplasia patients previously vaccinated with TA-HPV (vaccinia virus encoding HPV 16/18 E6E7)

Heterologous prime-boost vaccination schedules employing TA-HPV, a vaccinia virus encoding HPV 16/18 E6 and E7, in combination with TA-CIN, an HPV 16 L2E6E7 fusion protein, may offer advantages over the use of either agent alone for the immunotherapy of human papillomavirus (HPV) type 16-associated vulval intraepithelial neoplasia (VIN). In the present study, 10 women with HPV 16-positive high grade VIN, previously primed with TA-HPV, received three booster immunisations with TA-CIN. All but one demonstrated HPV 16-specific proliferative T-cell and/or serological responses following vaccination. Three patients additionally showed lesion shrinkage or symptom relief, but no direct correlation between clinical and immunological responses was seen.     

Chimeric classical swine fever viruses containing envelope protein E(RNS) or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response

Three chimeric classical swine fever virus (CSFV)/bovine viral diarrhoea virus (BVDV) full-length DNA copies were constructed, based on the infectious DNA copy of the CSFV vaccine strain C. The antigenic region of E2 and/or the complete E(RNS) gene were replaced by the analogous sequence of BVDV II strain 5250. Viable chimeric virus Flc11, in which E(RNS) was replaced, was directly recovered from supernatant of SK6.T7 cells transfected with full-length DNA. Viable chimeric virus Flc9, in which E2 was replaced, resulted in recovery of virus only when SK6.T7 transfected cells were maintained for several passages. However, no virus could be recovered after replacement of both E(RNS) and E2, even after 10 cell passages. Both Flc9 and Flc11 grow in swine kidney cells (SK6), stably maintain their heterologous BVDV sequences and, as assessed by monoclonal antibody typing and radio-immunoprecipitation assays, express their heterologous proteins. Flc9 showed a slower growth rate on SK6 cells than Flc11 and wild-type Flc2 virus. Replacement of E(RNS) or E2 of C-strain-based chimeric viruses did not alter cell tropism compared to wild-type C-strain virus for SK6 and FBE cells. Both Flc9 and Flc11 induced E2 or E(RNS) antibodies, which could be discriminated from those induced after wild-type virus infection, even after repeated vaccination. Furthermore, pigs were completely protected against a lethal CSFV challenge. These results indicate the feasibility of introduction of marker antigens in a live-attenuated marker C-strain vaccine for CSFV.     

Enhanced immunogenicity of HPV16E7 accompanied by Gp96 as an adjuvant in two vaccination strategies

Human papillomavirus, particularly type 16 (HPV16) is present in more than 99% of cervical cancers. E7 is the major oncogenic protein produced in cervical cancer-associated HPV16. An efficient vaccine against viral infection requires induction of strong humoral and cellular responses against viral proteins. Heat shock proteins (HSPs) like Gp96 have been described as potent tumor vaccines in animal models and are currently studied in human clinical trials. In this study, we investigated the utility of HPV16 E7 along with Gp96 as an adjuvant in C57BL/6 mice model. We compared the level of humoral and cellular immune responses by E7+Gp96 co-injection as DNA/DNA and prime-boost (DNA/protein) immunization strategies. In prime-boost immunization strategies, we first immunized C57BL/6 mice with the complete open-reading frame of E7 and Gp96 (pcDNA-E7 and pcDNA-Gp96) and then boosted with rE7, rNT-gp96 (N-terminal extension of Gp96) and rCT-gp96 (C-terminal extension of Gp96) mixed with Montanide 720 in different formulations. The humoral immune responses against rE7 and the different truncated forms of rGp96 suggested a mixed Th1/Th2 response with high intensity toward Th2. Assessment of lymphoproliferative and cytokine responses against rE7 and the different fragments of Gp96, showed that DNA vaccination including E7 and Gp96 induced Th1 response. We concluded that co-delivery of naked DNA E7+Gp96 plasmid was immunologically more effective than E7 alone. Our study demonstrated that co-delivery of E7+Gp96 as DNA/DNA and E7+CT-gp96 as DNA/protein could be an effective approach to induce E7-specific immune responses as a potential vaccine candidate for cervical cancer.     

Preclinical safety evaluation of DNA vaccines encoding modified HPV16 E6 and E7

Persistent infection with high-risk human papillomaviruses (hrHPV) can result in the formation of anogenital cancers. As hrHPV proteins E6 and E7 are required for cancer initiation and maintenance, they are ideal targets for immunotherapeutic interventions. Previously, we have described the development of DNA vaccines for the induction of HPV16 E6 and E7 specific T cell immunity. These vaccines consist of 'gene-shuffled' (SH) versions of HPV16 E6 and E7 that were fused to Tetanus Toxin Fragment C domain 1 (TTFC) and were named TTFC-E6SH and TTFC-E7SH. Gene-shuffling was performed to avoid the risk of inducing malignant transformation at the vaccination site. Here, we describe the preclinical safety evaluation of these candidate vaccines by analysis of their transforming capacity in vitro using established murine fibroblasts (NIH 3T3 cells) and primary human foreskin keratinocytes (HFKs). We demonstrate that neither ectopic expression of TTFC-E6SH and TTFC-E7SH alone or in combination enabled NIH 3T3 cells to form colonies in soft agar. In contrast, expression of HPV16 E6WT and E7WT alone or in combination resulted in effective transformation. Similarly, retroviral transduction of HFKs from three independent donors with both TTFC-E6SH and TTFC-E7SH alone or in combination did not show any signs of immortalization. In contrast, the combined expression of E6WT and E7WT induced immortalization in HFKs from all donors. Based on these results we consider it justified to proceed to clinical evaluation of DNA vaccines encoding TTFC-E6SH and TTFC-E7SH in patients with HPV16 associated (pre)malignancies.     

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.     

Priming with DNA plasmids encoding the nucleocapsid protein and glycoprotein precursors from Rift Valley fever virus accelerates the immune responses induced by an attenuated vaccine in sheep

In this work we tested the ability of plasmid DNA constructs encoding structural Rift Valley fever virus (RVFV) antigens to induce specific immune responses in sheep. The sole immunization of DNA constructs encoding the glycoprotein precursor NSm/G2/G1 did not suffice to induce a detectable antibody response. In contrast, immunization of sheep with a plasmid vector encoding the viral nucleocapsid protein N elicited a potent and long lasting induction of antibodies but with low neutralizing titers. After DNA immunization, no antigen-specific proliferating cells were detected in sheep PBLs. Boosting with the attenuated vaccine strain MP12 was able to increase the levels of proliferating memory cell pools and induction of IFN-gamma in response to purified virus or recombinant proteins, particularly in sheep vaccinated with a combination of both plasmid constructs. These results open the possibility to exploit this strategy to improve the induction of immune responses against RVFV in sheep.     

Recombinant Semliki Forest virus vector exhibits potential for avian virus vaccine development

The Semliki Forest virus (SFV) expression system was evaluated as a basis for avian vaccine development. Initial studies indicated that 1-day-old specific pathogen-free (SPF) chicks were susceptible to infection with an infectious strain of SFV, producing SFV-specific antibodies but no clinical disease. One-day-old SPF chicks immunised intramuscularly with recombinant replication-defective SFV (rSFV) particles expressing the Escherichia coli (E. coli) lacZ reporter gene developed high titres of beta-gal- specific antibodies at 4 weeks p.i. after two inoculations. In contrast, significantly lower antibody levels were elicited in chicks immunised with a recombinant SFV-based DNA construct or a conventional CMV promoter-based DNA plasmid. rSFV particles encoding the protective VP2 protein or the VP2/VP4/VP3 polyprotein of infectious bursal disease virus (IBDV) were produced and the expressed antigens were characterised in cell culture. Proteins of the correct size were generated and found to react against a range of IBDV-specific monoclonal antibodies. Immunisation of 1-day-old SPF chicks with rSFV particles encoding the IBDV proteins resulted in specific antibodies being elicited in all birds, neutralising antibodies being induced in some but not all birds.     

A Semliki Forest virus replicon vectored DNA vaccine expressing the E2 glycoprotein of classical swine fever virus protects pigs from lethal challenge

Classical swine fever virus (CSFV) causes significant losses in pig industry in many countries in Asia and Europe. The E2 glycoprotein of CSFV is the main target for neutralizing antibodies. Recently, the replicon of alphaviruses, such as Semliki Forest virus (SFV), has been developed as replicative expression vectors for gene delivery. In this study, we constructed a plasmid DNA based on SFV replicon encoding the E2 glycoprotein of CSFV and evaluated its efficacy in rabbits and pigs. The results showed that the animals immunized with the DNA vaccine developed CSFV-specific neutralizing antibodies and were protected from virulent or lethal challenge. This demonstrates that the SFV replicon-derived DNA vaccine can be a potential marker vaccine against CSFV infections.     

Immunization with plasmid DNA encoding the integral membrane protein, Sm23, elicits a protective immune response against schistosome infection in mice.

Schistosomes are helminth parasites infecting at least 200 million people worldwide. In this study, we evaluated the feasibility of using a nucleic acid vaccine to induce protective immune responses to the Schistosoma mansoni integral membrane protein Sm23. C57BL/6 mice were immunized by intramuscular injection in three separate vaccination trials. ELISA and Western Blot analyses indicated that mice immunized with a DNA plasmid construct encoding Sm23 (Sm23-pcDNA) generated specific IgG for Sm23, while sera from mice immunized with the control pcDNA plasmid did not. The vaccine elicited IgG(2a), and IgG(1) antibody isotypes. We also tested the adjuvant activity of IL-12 and IL-4 on humoral responses to Sm23. Co-immunization with plasmid encoding IL-12 did not affect the level of anti-Sm23 IgG(2a), but did reduce the IgG(1) level. In contrast, co-injection with a plasmid encoding IL-4 significantly reduced the level of anti-Sm23 IgG(2a), while the level of IgG(1) was largely unchanged. Importantly, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection (21-44%, P<0.001-0.02). Co-administration of plasmids encoding either IL-12 or IL-4 did not significantly enhance this protective effect.     

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.     

Immunization with plasmid DNA encoding a truncated, secreted form of the bovine viral diarrhea virus E2 protein elicits strong humoral and cellular immune responses

The major protective antigen of bovine viral diarrhea virus (BVDV), the E2 protein, is cell-associated and not expressed on the cell surface. In this study we evaluated a DNA vaccine encoding various secreted versions of E2. In vitro analysis demonstrated that deletion of the transmembrane anchor and addition of the signal sequence of bovine herpesvirus-1 (BHV-1) (gDsDeltaE2) resulted in efficient secretion of E2 into the culture medium. In contrast, full-length E2, either without or with gDs (gDsE2), as well as truncated E2 without gDs (DeltaE2), remained entirely cell-associated. Mice immunized with plasmid encoding gDsDeltaE2 developed significantly higher IgG and virus neutralizing antibody titres compared to animals vaccinated with plasmid encoding E2, DeltaE2 or gDsE2. To optimize secretion of E2, the efficiency of gDs was compared with that of the tissue plasminogen activator signal (tPAs) sequence. In addition, the effect of the plasmid backbone was assessed by comparing two vectors. Four plasmids, pMASIA-gDsDeltaE2, pMASIA-tPAsDeltaE2, pSLKIA-gDsDeltaE2 and pSLKIA-tPAsDeltaE2, were constructed and administered intradermally to mice. The mice immunized with pMASIA-tPAsDeltaE2 developed the strongest and most balanced immune responses. Vaccination of cattle confirmed that pMASIA-tPAsDeltaE2 elicited both strong humoral and cellular immune responses and thus could be a candidate DNA vaccine against BVDV.