Veterinary Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Rd, Saskatoon, Saskatchewan, CanadaS7N 5E31
Author for correspondence: Sylvia van Drunen Littel-van den Hurk. Fax +1 306 966 7478. e-mail vandenhurk{at}sask.usask.ca
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Abstract |
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Introduction |
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Bovine herpesvirus-1 (BHV-1) is an important pathogen in cattle. The respiratory form of BHV-1, also referred to as infectious bovine rhinotracheitis (IBR) virus, manifests itself as rhinotracheitis and conjunctivitis. Illness may be prolonged or death may occur in animals with secondary bacterial infections (Yates, 1982 ). Single vaccination with modified live BHV-1 vaccines, according to the label claim and proven efficacy testing, is commonly used in calves on feedlot entry and in the past it appeared to control clinical disease (Van Donkersgoed & Babiuk, 1991
). However, recently outbreaks of IBR have been observed in feedlot calves a few months after entry in spite of vaccination on arrival with an MLV vaccine (Van Donkersgoed & Klassen, 1995
), which suggests that priming with a DNA vaccine might be beneficial. In support of this concept, when DNA encoding herpes simplex virus-2 glycoprotein D (gD) was used for priming followed by a protein boost, both antigen-specific antibodies and Th1-type cellular responses were enhanced in mice (Sin et al., 1999
).
Previously we have demonstrated that intradermal immunization with DNA encoding gD of BHV-1 into the hip of cattle results in partial protection from virus challenge. We also reported that long-lasting T-cell and antibody responses were induced by the DNA vaccine (Braun et al., 1999 ). In another study we immunized cattle with DNA encoding gB of BHV-1 using the gene gun either at the hip or at the genital mucosa, and found that mucosal immunization resulted in significantly stronger T-cell responses and antibody priming than intradermal delivery (Loehr et al., 2000
). The enhanced immune responses induced by this method and route of delivery, the easily accessible genital tract in cattle and the availability of MLV vaccines provides an excellent model to study DNA immunization as a method to prime immune responses to existing vaccines.
In this study, we used plasmids encoding BHV-1 gB and gD and delivered them by gene gun to the genital mucosa of cattle prior to immunization with an MLV vaccine. The immune responses induced were compared to those of an MLV-vaccinated group and an unvaccinated control group. This demonstrated that even though significantly enhanced T-cell responses were induced by priming with the DNA vaccine, there was no difference in antibody titres and, likewise, the levels of protection induced by the MLV vaccine alone and the DNA prime and MLV boost regimen were similar.
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Methods |
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Plasmids.
Plasmids pSLIAgB, pSLIAtgB, pSLIAgD and pSLIAtgD were constructed by cloning the genes encoding gB and gD, and the truncated versions of gB (tgB) (Li et al., 1996 ) and gD (tgD) (Tikoo et al., 1990
), into pSL301 (Invitrogen) as described previously (Braun et al., 1997
, 1998
). The truncated forms lack the transmembrane anchor and cytoplasmic domain and are therefore secreted from transfected cells. The plasmids were amplified in transformed Escherichia coli (DH5
strain) and purified using anion exchange resins (Qiagen). After the concentrations were determined, the plasmids were stored at -20 °C. The A260/280 ratios were typically 1·8 or higher. All plasmids were shown to be free of endotoxins with the Limulus amoebocyte lysate kit (Bio-Whittaker).
Preparation of gene gun bullets.
Bullets were prepared as recommended by the manufacturer. Each DNA preparation containing one of the four plasmids was coated separately onto 1·6 µm gold beads (Bio-Rad) to ensure only one kind of plasmid on each gold particle. Gold beads, 0·05 M spermidine and DNA were mixed, 1 M CaCl2 was added dropwise while vortexing and the mixture was left at room temperature for 10 min. Subsequently, the gold bead preparations coated with plasmid encoding full-length and truncated versions of each glycoprotein were combined in a 1:2 ratio, so that each shot would have a combination of plasmids encoding gD and tgD or gB and tgB. The gold beads were washed three times with 100% ethanol, suspended in polyvinylpyrrolidoneethanol solution and coated to the inside of Teflon tubing to be used with the Bio-Rad Helios gene gun.
Immunizations.
BHV-1-seronegative cows balanced by age and weight were randomly allocated to three groups. Seven animals served as negative controls (control group). Five animals (MLV group) were immunized with a modified live BHV-1 vaccine (Boehringer-Ingelheim) according to the manufacturers instructions 3 weeks before challenge. Five other animals (DNA-MLV group) were immunized by gene gun into the most caudal part of the vulva mucosa with plasmids encoding gB, tgB, gD and tgD at 12 and 8 weeks before challenge and subsequently with MLV vaccine 3 weeks before challenge. The conditions for delivery of plasmid were 300 p.s.i. of helium (ca. 2070 kPa), 0·25 mg gold and 1·25 µg of plasmid per shot. The total amount of plasmid delivered per immunization was 7·5 µg in six shots. Three of the six shots delivered per animal contained 0·417 µg of pSLIAgB and 0·833 µg of pSLIAtgB and the other three shots contained 0·417 µg of pSLIAgD and 0·833 µg of pSLIAtgD. All animals were housed under the same conditions in accordance with the guidelines of the Canadian Council on Animal Care.
Challenge and clinical observations.
Three weeks after immunization with MLV vaccine, each calf was exposed for 4 min to an aerosol of 107 p.f.u./ml of BHV-1 strain 108, which was generated by a DeVilbis nebulizer, model 65. On the day of challenge and for 10 days afterwards the calves were clinically examined each morning by the attending veterinarian, who was blind to the identities of the vaccine groups. Body weights and rectal temperatures were also measured daily during the clinical assessment.
Sampling.
Sera were collected at each immunization. Nasal tampons were used to obtain up to 5 ml of nasal fluid from all animals 6 days before challenge. Sera and nasal fluids were collected again on days 2, 4, 6, 8, 10, 13 and 17 post-challenge. Blood was collected into tubes with anticoagulant 1 day before and 9, 30 and 72 days after challenge.
Virus isolation.
Virus was recovered from the nasal fluids and quantified by plaque titration in microtitre plates with an antibody overlay as previously described (van Drunen Littel-van den Hurk et al., 1998 ).
ELISAs.
Polystyrene microtitre plates (Immulon 2, Dynatech Laboratories) were coated with 0·05 µg of tgD (Kowalski et al., 1993 ) or tgB (Li et al., 1996
) per well and incubated with serially diluted bovine sera, starting at 1:10 in threefold dilutions. Alkaline phosphatase-conjugated goat anti-bovine IgG (Kirkegaard and Perry Laboratories) at a dilution of 1:10000 was used to detect IgG, and biotin-labelled goat anti-bovine IgA (VMDR) at a dilution of 1:15000, followed by streptavidin alkaline phosphatase (Gibco-BRL) at a dilution of 1:3000, was used to detect IgA. The reaction was visualized with p-nitrophenyl phosphate (Sigma).
Virus neutralization assays.
The virus neutralization titres in the sera were determined as previously described (van Drunen Littel-van den Hurk et al., 1990 ). The titres were expressed as the highest dilution of antibody that caused a 50% reduction of plaques relative to the virus control.
Proliferation assays.
Bovine blood was collected into citrate-dextran and peripheral blood mononuclear cells (PBMCs) were isolated on Ficoll-Paque PLUS (Pharmacia). PBMCs were dispensed at 3·5x106 cells/ml of culture medium consisting of MEM (Gibco-BRL), 10% FBS (Sigma), 2 mM L-glutamine (Gibco-BRL), 500 mg/ml gentamycin, 5x10-5 M 2-mercaptoethanol and 1 mg/ml dexamethasone. Subsequently, 100 µl volumes were dispensed into the wells of microtitre plates. Purified gB or gD at 1 µg/ml was added in a 100 µl volume to triplicate wells. After 3 days in culture the cells were pulsed with [methyl-3H]-thymidine (Amersham) at a concentration of 0·4 µCi (14·8 kBq) per well. The cells were harvested 18 h later and thymidine uptake was measured by scintillation counting. Proliferative responses were calculated as the mean of triplicate wells and expressed as a stimulation index (mean counts per min in the presence of antigen/mean counts per min in the absence of antigen). The stimulation indices per group were calculated as the arithmetic average stimulation index.
ELISPOT assays.
PBMCs were cultured for 24 h in the presence of 1 µg of gB or gD, washed twice and resuspended to the appropriate concentration in culture medium. Nitrocellulose plates (Millipore) were coated for 2 h at room temperature with a bovine IFN--specific monoclonal antibody at a dilution of 1:400. Unbound antibody was removed and 100 µl of each cell suspension was added to triplicate wells. After an overnight incubation at 37 °C, the plates were incubated with rabbit serum specific for bovine IFN-
at a 1:100 dilution for 24 h at room temperature. Subsequently, the plates were incubated for 2 h at room temperature with biotinylated rat anti-rabbit IgG (Zymed), followed by streptavidinalkaline phosphatase (BIO/CAN Scientific), each at a 1:1000 dilution. The spots were visualized with substrate consisting of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) (Sigma), which was left on the plates for 1060 min at room temperature. The plates were washed in ddH2O and air-dried before counting the number of stained spots in the wells. IFN-
-secreting cells were expressed as the difference between the number of spots per 106 cells in antigen-stimulated wells and the number of spots per 106 cells in non-stimulated wells.
Statistical analyses.
All data were analysed with the aid of a statistical software program (Systat 7.0, SPSS Inc.). ELISA and virus neutralization data were transformed to normality by log transformation prior to performing the analysis. Differences between the groups were examined by performing one-way ANOVA and Tukey tests for ELISPOT counts and the two-way ANOVA and Tukey HSD multiple comparison for ELISA titres, virus neutralization titres, stimulation indices, temperatures, weights and virus shedding.
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Results |
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Discussion |
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Modulation of the immune response to a vectored or protein vaccine after initial DNA immunization has also been demonstrated for other infectious agents. Generally a DNA prime and protein boost regimen leads to a strongly increased T-cell response and better protection than other vaccination strategies. For example, when various methods for immunization with DNA and MVA containing different CTL epitopes were assessed, DNA prime and MVA boost was found to be the most potent protocol to enhance the induction of HIV-specific T-cells (Hanke et al., 1999 ). Intradermal DNA prime and MVA boost also induced good CD8+ T-cells and protection in malaria and influenza models in mice (Degano et al., 1999
). In another study, comparing eight different vaccination protocols in macaques, the most promising containment of HIV infection was again achieved by intradermal priming with DNA followed by a boost with a recombinant fowlpox virus, which resulted in neutralizing antibody-independent immunity (Robinson et al., 1999
). Different DNA prime and boost strategies using DNA, MVA and virus-like particles have also been compared for Plasmodium falciparum. The highest levels of CD8+ T-cells were obtained after DNA prime and MVA boost, which was also the only vaccination regime leading to 100% protection (Schneider et al., 1999
). Prime and boost vaccination strategies with DNA vaccines have also been performed for herpesvirus infections. DNA prime and protein boost with herpes simplex virus-2 gD (Sin et al., 1999
) or DNA prime and boost with recombinant baculovirus expressing equine herpesvirus-1 gD (Ruitenberg et al., 2000
) resulted in enhanced T-cell responses as well as increased antibody titres in a mouse model.
In contrast to the numerous reports on primary DNA immunization and secondary immunization with a vectored or protein vaccine, very few primeboost regimens have been reported using replication-competent organisms. One study demonstrated that DNA immunization with plasmid encoding the colonization factor I antigen of enterotoxogenic E. coli (CFA/I of ETEC) and two oral booster vaccinations with live recombinant Salmonella typhimurium expressing ETEC antigen had a synergistic effect that resulted in systemic IgG and mucosal IgA, which could not be attained by either immunization strategy alone (Lasaro et al., 1999 ). Because MLVs are widely used and yet have rarely been applied in primeboost strategies, we determined the effect of priming with a DNA vaccine on the immune responses induced by a replication-competent virus.
In our study, the DNA-MLV group developed a significant T-cell response prior to challenge, whereas essentially no T-cell activation could be observed in the MLV group. In contrast, the vaccinated groups developed similar levels of IgG in sera and nasal fluids, regardless of whether they were primed with a DNA vaccine or not. As intranasal challenge with BHV-1 following DNA immunization with BHV-1 gD or gB led to a significant increase in antibody levels (Braun et al., 1999 ; Loehr et al., 2000
), the DNA-MLV vaccination protocol was expected to reflect a similar situation. However, MLV is less virulent than the challenge strain of BHV-1, and it was delivered intramuscularly, which suggests that the level of virus replication and the route of delivery may influence the efficacy of the virus boost. Interestingly, with similar antibody titres in the MLV and DNA-MLV groups and little T-cell activation in the MLV group, this study suggests that the T-cell activation measured by lymphocyte proliferation and IFN-
production in the DNA-MLV group contributed marginally to the protection from BHV-1 infection. After BHV-1 challenge, there also was little difference between the gB- and gD-specific IgG and IgA titres of the DNA-MLV and MLV groups and, despite the significant difference in T-cell activation, there was no difference in recovery rate between the two vaccinated groups. These data show that the T-cell activation did not improve antibody production, protection or recovery from BHV-1 challenge as we had anticipated based on the results with challenge infection after DNA immunization (Braun et al., 1999
; Loehr et al., 2000
). Despite the contention that T-cell responses help recovery after BHV-1 infection (Turin et al., 1999
), this reveals that antibodies are necessary not only for protection, but also for recovery from BHV-1 infection (Babiuk et al., 1996
).
After challenge, T-cell activation in the DNA-MLV group lasted for at least 10 weeks, while the MLV group did not show any significant T-cell activation. This demonstrates not only the longevity of T-cell stimulation after DNA immunization, which has also been shown with several other immunization strategies (Haddad et al., 1999 ; Braun et al., 1999
; Johnson et al., 2000
; Hassett et al., 2000
), but more importantly it shows the potential to greatly increase the T-cell stimulation induced by live virus vaccination. This potential for further T-cell stimulation can be very valuable for vaccination against diseases where strong T-cell activation is required. For example, in infections with Listeria monocytogenes, where protective immunity is dependent on CD8+ T-lymphocytes and IFN-
secretion, a type 1 response induced by DNA vaccination by gene gun can result in protective immunity (Fensterle et al., 1999
). Other similar examples include HIV, malaria and influenza virus (Degano et al., 1999
; Johnson et al., 2000
).
A strong T-cell activation may be of great value not only for prevention, but also for treatment of disease, such as in tumour defence, where vaccination with DNA has been effective against mammary and lung tumors (Ohwada et al., 1999 ; Wei et al., 1999
; de Zoeten et al., 1999
), papilloma (Wei et al., 1999
; Chen et al., 1999
; Tan et al., 1999
; Ji et al., 1999
) and melanoma. For example, immunization with a plasmid encoding the melanoma antigen MAGE-1, which is expressed in several human tumours of various tissues, induced antibodies and CTL responses in mice (Park et al., 1999
) and thus could be a promising immune therapy for cancer. As shown in this as well as other studies, administration of protein in a replication-deficient or -competent vector could further increase the T-cell stimulation following priming with DNA, without concern about existing immunity against the vector. Since the development of immunity against the vector is one of the impediments to successful gene therapy, the combination of DNA prime and vector boost is a promising approach to inducing strong cellular immunity.
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Acknowledgments |
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Footnotes |
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c Present address: Coley Pharmaceutical GmbH, Elisabeth-Selbert-Strasse 9, D-40764 Langenfeld, Germany.
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References |
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Received 29 May 2001;
accepted 26 July 2001.