Faculté de Médecine Vétérinaire, Université de Montréal, Département de Pathologie et Microbiologie, Section Virologie, C.P. 5000, St-Hyacinthe, Québec, Canada, J2S 7C61
Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Québec, Canada, J1K 2R12
Institut de Recherches Cliniques de Montréal, Université de Montréal, Montréal, Canada3
Department of Veterinary Science and Biology/Microbiology, College of Agriculture and Biological Sciences, South Dakota State University, USA4
Author for correspondence: Brian Talbot.Fax +1 819 821 8049. e-mail btalbot{at}courrier.usherb.ca
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Abstract |
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Introduction |
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Commercially available inactivated and modified-live BVDV (MLV) vaccines have been extensively used for more than 30 years, but since their introduction the problem of BVDV-related infections appears to have become worse instead of better (Bolin, 1995 ). Both MLV and inactivated BVDV vaccines have significant shortcomings. MLV vaccines contain a limited antigen mass and require the opportunity to replicate in the host to establish significant immune activity. They are also susceptible to interference from maternal antibody in the system (Schultz, 1993
). Furthermore, MLV viruses are a potential source of in utero infections and/or immunosuppression (Liess et al., 1984
; Roth & Kaeberle, 1983
). MLV vaccines are produced in bovine cell cultures and thus have the potential to introduce other biological agents into herds as contaminants. Even if they provide a large preformed antigen mass, inactivated viruses are quite expensive to produce and susceptible to loss of important immunogenic activities during the process of inactivation.
The use of cloned viral genes holds great promise for the development of new vaccines to control BVDV. DNA vaccination can serve as an alternative to conventional immunization with MLV or inactivated vaccines to induce protection (Robinson et al., 1993 ; Sedegah et al., 1994
; Xiang et al., 1994
). Direct injection of plasmid DNA into animals offers several advantages over classical vaccine preparations and virus vectors for vaccination. Simple, rapid and inexpensive production of plasmid DNA, thermal stability of the plasmid product, and the potential for a long shelf-life of stabilized plasmid DNA are characteristics that make genetic vaccination very attractive for the next generation of vaccines against BVDV. This technology is insensitive to preformed antibody (Siegrist et al., 1997
) and DNA vaccines are capable of inducing both humoral and cellular immunity by providing access to newly synthesized antigen in both the MHC class I- and class II-restricted pathways (Donnelly et al., 1997
; Hasset & Whitton, 1996
). Most DNA vaccination studies have been carried out and designed in mice to determine the efficiency of DNA immunization. Our initial studies of DNA immunization with the E2 gene of BVDV in mice demonstrated the potential of the method (Harpin et al., 1997
).
In this report, we examined DNA vaccination for BVDV in cattle, the natural host. Vaccination was carried out by injecting calves with DNA encoding the BVDV major glycoprotein E2 either as naked plasmid dissolved in saline (N-DNA) or as plasmid entrapped in cationic liposome (L-DNA). The virus-specific neutralizing antibody and the virus-specific lymphocyte proliferation responses were studied. We also evaluated the response to challenge with the NY-1 strain of type 1 BVDV (produced by the NVSL and USDA-certified as a vaccine challenge strain).
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Methods |
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Plasmid DNA expression vector.
Cloning and expression of the BVDV E2 glycoprotein from a cytopathic type 1 strain (BVDV/NADL) yielding pcDNA/gp53 has been previously described (Harpin et al., 1997 ). The new gp53 expression vector used in this study and designated as pCMVigp53 (where i stands for intron) was constructed by replacing the region containing the cytomegalovirus (CMV) promoter in the pcDNA/gp53 vector with a BglIIHindIII fragment containing the CMV promoter and an intron from the pCI-neo plasmid (Promega). Plasmid pCMVintron was used as plasmid control. All plasmids used in this study were prepared according to the manufacturers directions using the Qiagen DNA purification kit. For use in vaccination, the DNA was dissolved in PBS at a final concentration of 1 mg/ml. The DNA aliquots were stored at -20 °C until the time of injection.
Entrapment of plasmid DNA into liposomes.
Liposomes were prepared as unilamellar vesicles containing the cationic lipid DOTAP as DOTAP/DOPE (1:1 molar ratio). DOTAP is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (Avanti Polar Lipids), and DOPE is the neutral lipid dioleoyl phosphatidylethanolamine (Avanti Polar Lipids). Briefly, a film made of an equimolar mixture of DOTAP and DOPE was first prepared by the standard hydration and re-hydration method (Bangham et al., 1965 ). The film was re-hydrated in PBS and sonicated for 30 s in a bath sonicator to form a clear liposomal suspension. Liposomes were stored at 4 °C until use. One day before the time of vaccination, a DNA:liposome ratio of 1:10 (w/w) was prepared as previously reported (Felgner et al., 1987
). For use in vaccination, the DNAliposome formulation was kept at 4 °C at a final concentration of 0·5 mg of plasmid DNA/ml. A total of 2 ml of the mixture (1 mg DNA) was injected per calf.
DNA immunization and challenge.
BVDV- and BVDV-antibody-negative cattle, 7 to 9 months of age, were used in this study. The animals were housed in an isolated barn and handled under strict precautions to prevent the introduction of BVDV. Calves were intramuscularly immunized in the thigh muscle. Three calves received 1 mg of pCMVigp53 in a liposome preparation (L-DNA), three calves received 1 mg of pCMVigp53 as naked DNA (N-DNA) and two calves received 1 mg of control plasmid pCMVintron. For the challenge study, two calves were added to serve as unvaccinated and unchallenged controls. Those two calves were housed separately from the challenged animals in order to prevent horizontal transmission of the virus. Booster injections were given at weeks 3 and 6. Blood samples were drawn from each calf every 2 weeks. On week 16 after vaccination, the animals were challenged intranasally with 1·2 ml of 107·9 TCID50/2 ml of BVDV/NY-1. A volume of 0·6 ml per nare was delivered dropwise to each DNA-vaccinated and mock-vaccinated animal. Clinical signs were observed daily from 3 days prior to challenge to day 14 post-challenge. During the challenge period, blood samples for antibody and lymphocyte proliferation assays were collected on days -3, 0, 2, 4, 6, 8, 10, 14 and 21. Nasal secretions were collected at weeks 0, 3, 5 and 8 during the vaccination phase and on day 21 post-challenge.
Virus neutralization assay.
The virus neutralization assays were conducted using heat-inactivated (30 min at 56 °C) samples (serum, nasal secretion). Two hundred TCID50 of BVDV (Singer or 125 strains) was pre-incubated with twofold dilutions of the heat-inactivated sample for 1 h at 37 °C. This mixture (50 µl) was then added to duplicate wells of microtitre plates containing 8090% confluent MDBK cells for 1 h at 37 °C. MEM (150 µl) was added to each well. The plates were incubated at 37 °C for 4 days. The reciprocal of the highest dilution that completely inhibited virus cytopathic effect in the two test wells was reported as the virus neutralization titre.
Proliferative response of bovine mononuclear cells.
Bovine leukocytes were enriched by centrifugation of peripheral blood to collect the buffy coat. The mononuclear (MN) cells were purified from the buffy coat by flotation on Histopaque 1.083 (Sigma), and suspended at a concentration of 3x106/ml in RPMI-1640 with 10% FBS, 2 mM sodium pyruvate, 2 mM glutamine and 50 µg/ml gentamicin. The MN cells were added to triplicate wells (6x105 cells per well) and incubated in the presence of live BVDV (Singer and 125 strains) at 5x104 particles per well in a final volume of 200 µl at 37 °C in 5% CO2. After 5 days, the wells were pulsed for 6 h with 0·2 µCi of [3H]thymidine (6·7 Ci/mM, ICN) and harvested with a Skatron semiautomatic cell harvester (Flow Laboratories). Stimulation index (SI) was calculated by the following formula: SI=average counts per minute in antigen-stimulated wells/average counts per minute in wells containing only cells with medium.
Statistical analysis.
The significance of differences observed under test conditions for cell proliferation was determined by ANOVA using the Statview+SE software (Abacus Concepts).
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Results |
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Characterization of the cellular immune response
Bovine MN cells were isolated and stimulated in vitro with live type 1 (Singer strain) or type 2 (strain 125) BVDV. Control wells were incubated with cells alone. The baseline response was established by using the average SI of the mock-vaccinated animals. Bovine MN cells from two N-DNA-vaccinated calves (1 and 3) exhibited an increase in proliferation following stimulation with the Singer strain compared to MN cells from mock-vaccinated calves at week 4 after vaccination (Fig. 1). This response slowly decreased during the remainder of the vaccination period. However, L-DNA-vaccinated calves did not show any measurable increase in MN cell response to the Singer strain of BVDV (Fig. 2
). No proliferative response was observed after stimulation with BVDV type 2 in any of the DNA-vaccinated calves (data not shown).
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Nasal secretion neutralizing antibodies to BVDV after challenge
There was no evidence of neutralizing antibodies to BVDV in nasal secretions from any of the animals during the vaccination period. Nevertheless, during the challenge period there was a significant seroneutralizing mucosal response in animals vaccinated with plasmid DNA pCMVigp53 (Table 2) as compared to the mock-vaccinated animals. Only DNA-vaccinated animals showed evidence of neutralizing antibody in nasal secretion after challenge. No mucosal responses (SN titre <4) were observed in the 3 weeks following challenge in mock-vaccinated animals or in unvaccinated, unchallenged control animals.
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Evidence of protection in DNA-vaccinated calves
Following challenge with a BVDV type 1 virus (BVDV/NY-1), the animals were assessed for evidence of disease on the basis of a composite clinical score (Table 4). Due to the small number of animals in the experiment, the results were assessed per animal rather than as an average (Fig. 3
). In mock-vaccinated animals, the symptoms occurred as two peaks at approximately day 4 and day 7. In N-DNA-vaccinated animals, the results show that calf N-DNA-3 had no signs of disease throughout the challenge period and appeared to be completely protected from disease. The remaining N-DNA-vaccinated animals showed reduced levels and duration of clinical signs compared with the controls, indicating partial protection from development of disease. L-DNA-vaccinated calves were unprotected and showed essentially identical patterns of disease severity and duration to the mock-vaccinated animals after challenge (data not shown).
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Discussion |
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We show here, for the first time, that a DNA vaccine encoding the BVDV (type 1) major glycoprotein E2 can not only induce both humoral and cellular immune responses, but also provide partial protection after challenge infection in cattle. This is not surprising since other groups have already used the BVDV E2 glycoprotein in vaccination trials. Bruschke et al. (1997) reported immunization of ewes with an E2-based subunit vaccine to prevent transplacental transmission of virus. This group showed that an E2 subunit vaccine prevented foetal infection after homologous challenge. Another report showed that vaccination of calves with a C-terminally truncated E2 (produced in baculovirus) provided limited protection from systemic infection and disease (Bolin & Ridpath, 1996
).
Only a few other DNA vaccines have been developed for protection of large animals, probably because of the technical and financial difficulty in using large enough numbers of animals. However, DNA vaccines against porcine viruses were shown to provide protection from disease in pigs (Pirzadeh & Dea, 1998 ; Gerdts et al., 1997
). In bovine species, DNA constructs encoding bovine herpesvirus-1 (BHV-1) glycoprotein D were shown to induce an immune response that partially protected cattle from respiratory disease (Cox et al., 1993
). As with our study, this research group also investigated the effects of different routes of DNA immunization and reported that intradermal delivery was more effective than an intramuscular injection in generating a protective response against BHV-1 as monitored by a reduction in clinical signs of disease (van Drunen Littel-van den Hurk et al., 1998
). In the present work, we chose to use intramuscular injection because this technique is already widely used and well accepted by practitioners in the field. However, in an attempt to investigate the effect of changes in DNA presentation, plasmid DNA was used as naked DNA (N-DNA) or was entrapped in cationic liposomes (L-DNA) which have previously shown some promise in genetic immunization studies of small animals (Donnelly et al., 1997
).
Overall, the results show that animals immunized with the plasmid pCMVigp53, as either L-DNA or N-DNA, developed an immune response against BVDV. However, there were significant differences between the responses to N-DNA and L-DNA. N-DNA-immunized animals showed variable levels of protection from disease after challenge. In contrast, the L-DNA-vaccinates were not protected. The small numbers (three) of animals preclude a quantitative evaluation of the protection but it is significant that in an out-bred population one N-DNA-vaccinated animal was completely protected and two showed limited protection. These observations are surprising when pre-challenge seroneutralizing results are taken into account. Until recently, it has been assumed that BVDV protection in cattle is almost exclusively due to the production of seroneutralizing antibodies. However, the L-DNA-immunized animals in this study all produced seroneutralizing antibodies in the vaccination period. Yet these animals were not protected. This is in contrast with the work of Bolin & Ridpath (1996) which reported that a vaccine-induced neutralizing antibody titre >>2 protected calves from clinical signs of disease induced by homologous virus challenge exposure. A similar examination of the results for N-DNA-immunized animals shows that two of the three calves (N-DNA-1 and -2) produced only a low level of seroneutralizing response during the vaccination period and the third one (N-DNA-3) had titres equivalent to those of the L-DNA vaccinates. This latter observation correlates well with the level of protection observed since the high titre animal (N-DNA-3) was the one that appeared completely protected. The lack of correlation between L-DNA and N-DNA protection and antibody titres is in some way resolved when the cell proliferation results are included in the scheme. Once again the results vary between animals and between treatment groups yet several distinct conclusions can be drawn. In general, the N-DNA-immunized animals showed higher levels of cell proliferation compared to the unvaccinated animals whereas the L-DNA vaccinates did not show this at all. The N-DNA animal (N-DNA-3) that demonstrated the highest cell proliferation results in addition to the high antibody titre also showed the highest level of protection to development of disease after virus exposure. All previous studies of E2 protection did not include lymphoproliferative studies. However, in a recent report, Cortese et al. (1998)
determined the efficacy of an MLV type-I isolate of BVDV vaccine in protecting calves from infection with a virulent type-II isolate, and showed that induction of a cellular immune response correlated with protection. Our own work using an inactivated BVDV vaccine has since confirmed this (Y. Chofry, B. G. Talbot, D. Hurley & Y. Elazhary, unpublished observation). Thus, it can be hypothesized in view of our results and from those of Cortese et al. (1998)
that, for maximum protection, the vaccine must induce not only a seroneutralizing antibody but also a cellular response. It is not known whether the lymphoproliferative response to the whole virus depends on E2, although we have determined that another viral protein, NS3, expressed in an adenovirus vector does not induce such an effect in mice (Elahi et al., 1999
).
The BVDV/E2 gene used in our DNA construct was cloned from the BVDV cytopathic type 1 NADL strain. In the field, several different genotypes of BVDV are circulating in the cattle population, thus we also measured the reactivity to a cytopathic type 2 strain (BVDV/125) to evaluate heterotypic immune responses induced by DNA vaccination. As demonstrated in Table 3, the challenge induced a strong memory response in the production of serum neutralizing antibodies (titres were 256-fold higher than mock vaccinates) to BVDV type 2. Our finding that a type 1 E2 gene will induce neutralizing antibody to type 2 virus is an interesting observation considering the high variability between the two E2 sequences. This is more understandable if one takes into account the fact that all E2-specific seroneutralizing antibodies obtained to date are configuration-specific. It is quite likely that despite sequence differences between the E2 of different strains, the configurations of some neutralizing epitopes are similar. An ideal BVDV vaccine should thus be able to induce antibodies capable of neutralizing a wide variety of virus genotypes in a heterotypic response if the antigen expresses an E2 consensus configuration.
BVDV infects cattle at mucosal surfaces, primarily the respiratory tract (Bolin, 1990 ). Thus, mucosal immunity is believed to play an important role in protection against BVDV infection. After virus challenge, we examined the development of BVDV-specific antibodies in nasal secretions. Three weeks after challenge, mucosal neutralizing antibodies were observed only in DNA-vaccinated animals. Significantly, these neutralizing antibodies were observed at similar titres after vaccination with both N-DNA and L-DNA. However, only N-DNA-vaccinated animals showed some level of protection. It should be noted that nasal virus-neutralizing antibodies do not normally appear in nasal secretions in a primary response until at least 4 weeks after infection. Thus, nasal secretion neutralizing antibodies are probably more important for secondary and memory responses. Their actual role in protection needs further investigation.
In summary, we have demonstrated that vaccination of cattle using a DNA construct encoding only the E2 protein of BVDV was able to induce both neutralizing antibody production and lymphocyte proliferation. It appeared that both humoral and cellular immune responses were implicated in the development of a protective response in cattle. This work shows the great potential of genetic vaccines for the control of BVDV.
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Acknowledgments |
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B.T. and Y.E. contributed equally to this work.
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References |
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Bolin, S. R. (1990). The current understanding about the pathogenesis and clinical forms of BVD. Veterinary Medicine 85, 1124-1132.
Bolin, S. R. (1995). Control of bovine viral diarrhea infection by use of vaccination. Veterinary Clinics of North America Food Animal Practice 11, 615-625.
Bolin, S. R. & Ridpath, J. F. (1996). Glycoprotein E2 of bovine viral diarrhea virus expressed in insect cells provides calves with limited protection from systemic infection and disease. Archives of Virology 141, 1463-1477.[Medline]
Bruschke, C. J., Moormann, R. J., van Oirschot, J. T. & van Rijn, P. A. (1997). A subunit based on glycoprotein E2 of bovine virus diarrhea induces fetal protection in sheep against homologous challenge. Vaccine 15, 1940-1945.[Medline]
Collett, M. S., Larson, R., Belzer, S. K. & Retzel, E. (1988). Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus. Virology 165, 200-208.[Medline]
Cortese, V. S., West, K. H., Hassard, L. E., Carman, S. & Ellis, J. A. (1998). Clinical and immunological responses of vaccinated and unvaccinated calves to infection with a virulent type-II isolate of bovine viral diarrhea virus. Journal of the American Veterinary Medical Association 1, 1312-1319.
Cox, G. J. M., Zamb, T. J. & Babiuk, L. A. (1993). Bovine herpesvirus 1: immune responses in mice and cattle injected with plasmid DNA. Journal of Virology 67, 5664-5667.[Abstract]
Donis, R. O. (1995). Molecular biology of bovine viral diarrhea virus and its interactions with the host. Veterinary Clinics of North America Food Animal Practice 11, 393-423.
Donis, R. O., Corapi, W. & Dubovi, E. J. (1988). Neutralizing monoclonal antibodies to bovine viral diarrhoea virus bind to the 56K to 58K glycoprotein. Journal of General Virology 69, 77-86.[Abstract]
Donnelly, J. J., Ulmer, J. B., Shiver, J. W. & Liu, M. A. (1997). DNA vaccines. Annual Review of Immunology 15, 617-648.[Medline]
Elahi, S. H., Shen, S.-H., Harpin, S., Talbot, B. G. & Elazhary, Y. (1999). Investigation of the immunological properties of the bovine viral diarrhea virus protein NS3 expressed by an adenovirus vector in mice. Archives of Virology 144, 1057-1070.[Medline]
Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M. & Danielsen, M. (1987). Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences, USA 84, 7413-7417.[Abstract]
Gerdts, V., Jöns, A., Makoschey, B., Visser, N. & Mettenleiter, T. C. (1997). Protection of pigs against Aujeszkys disease by DNA vaccination. Journal of General Virology 78, 2139-2146.[Abstract]
Harpin, S., Elahi, S. M., Cornaglia, E., Yolken, R. H. & Elazhary, Y. (1995). The 5'-untranslated region sequence of a potential new genotype of bovine viral diarrhea virus. Archives of Virology 140, 1285-1290.[Medline]
Harpin, S., Talbot, B., Mbikay, M. & Elazhary, Y. (1997). Immune response to vaccination with DNA encoding the bovine viral diarrhea virus major glycoprotein gp53 (E2). FEMS Microbiology Letters 146, 229-234.[Medline]
Hasset, D. & Whitton, J. L. (1996). DNA immunization. Trends in Microbiology 4, 307-312.[Medline]
Horzinek, M. C. (1991). Pestiviruses taxonomic perspectives. Archives of Virology Supplementum 3, 55-65.[Medline]
Liess, B., Orban, S., Frey, H. R., Trautwien, G., Wiefel, W. & Blindow, H. (1984). Studies on transplacental transmissibility of a bovine virus diarrhea (BVD) vaccine virus in cattle. Zentralblatt fur Veterinarmedizin Reihe B 31, 669-681.
Pellerin, C., Van Den Hurk, J., Lecomte, J. & Tijssen, P. (1994). Identification of a new group of bovine viral diarrhea virus strains associated with severe outbreak and high mortalities. Virology 203, 260-268.[Medline]
Pirzadeh, B. & Dea, S. (1998). Immune response in pigs vaccinated with plasmid DNA encoding ORF5 of porcine reproductive and respiratory syndrome virus. Journal of General Virology 79, 989-999.[Abstract]
Ridpath, J. F., Bolin, S. R. & Dubovi, E. J. (1994). Segregation of bovine viral diarrhea virus into genotypes. Virology 205, 66-74.[Medline]
Robinson, H. L., Hunt, L. A. & Webster, R. G. (1993). Protection against lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. accine 11, 957-960.
Roth, J. A. & Kaeberle, M. L. (1983). Suppression of neutrophil and lymphocyte function induced by a vaccinal strain of bovine viral diarrhea virus with or without administration of ACTH. American Journal of Veterinary Research 44, 2366-2372.[Medline]
Schultz, R. D. (1993). Certain factors to consider when designing a bovine vaccination program. In Proceedings of the 26th Annual Convention of the American Association of Bovine Practitioners, pp. 19-26. Edited by E. I. Williams. Stillwater, OK, USA: Frontier Printers..
Sedegah, M., Hedstrom, R., Hobart, P. & Hoffman, S. L. (1994). Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein. Proceedings of the National Academy of Sciences, USA 9, 9866-9870.
Siegrist, C. A. & Lambert, P. H. (1997). Immunization with DNA vaccines in early life: advantages and limitations as compared to conventional vaccines. Springer Seminars in Immunopathology 19, 233-243.[Medline]
van Drunen Littel-van den Hurk, S., Braun, R. P., Lewis, P. J., Karvonen, B. C., Baca-Estrada, M. E., Snider, M., McCartney, D., Watts, T. & Babiuk, L. A. (1998). Intradermal immunization with a bovine herpesvirus-1 DNA vaccine induces protective immunity in cattle. Journal of General Virology 79, 831-839.[Abstract]
Xiang, Z. Q., Spitalnik, S., Tran, M., Wunner, W. H., Cheng, J. & Ertl, H. C. J. (1994). Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus. Virology 199, 132-140.[Medline]
Received 22 March 1999;
accepted 2 August 1999.