Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, CanadaS7N 5E31
Qiagen GmbH, 40724 Hilden, Germany2
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 economically important pathogen in cattle and is commonly associated with the bovine respiratory disease complex and abortion (Yates, 1982 ). Moderately effective killed and modified-live BHV-1 vaccines are available, and a subunit vaccine using truncated glycoprotein D (tgD) has been developed (Harland et al., 1992
; van Drunen Littel-van den Hurk et al., 1993
). DNA immunization against BHV-1 has been demonstrated in cattle, and partial protection from challenge has been achieved (Braun et al., 1999
; Loehr et al., 2000
). However, there is a need to improve the efficacy of these vaccines. As an approach to augment the effectiveness of tgD-encoding plasmids as a vaccine, we have modified the vector backbone by adding a controlled number of CpG motifs. Here we describe the first use of CpG-enhanced vectors in a vaccine formulation against a pathogen that causes significant economic loss in the target species.
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Methods |
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Plasmid construction.
All enzymes used for cloning procedures were purchased from Amersham Pharmacia Biotech. Plasmid pMAS (Klinman et al., 2000 ; Krieg et al., 1998b
) (a gift from H. Davis, Coley Pharmaceutical Group, Ottawa, Canada) was modified by the introduction of specific CpG motifs based on the bovine immunostimulatory sequence (BIS) from ODN 2135 (Pontarollo et al., 2002
; Uwiera et al., 2001b
). Briefly, two complementary oligodeoxynucleotides were annealed to form a duplex containing eight CpG motifs with 5' protuding ends complementary to the restriction enzyme AvaII (Fig. 1a
). These duplexes were force-directional cloned by random ligation into the AvaII site of pMAS. The number of inserts per plasmid was verified by restriction enzyme digestion and sequencing. Plasmids pBIS40 (40 motifs) and pBIS88 (88 motifs) were selected and further modified by inserting intron A downstream of the human cytomegalovirus immediate-early (HCMV IE) promoter. Intron A was isolated from pCAN1 (Uwiera et al., 2001a
) using EagI and PstI (blunted with T4 DNA polymerase) and inserted into the pMAS and the CpG-modified vectors cut with EagI and EcoRV to create pMASIA, pBISIA40 and pBISIA88. Finally, the gene encoding tgD was isolated from pSLIAtgD (Braun et al., 1997
) by BglII digestion and inserted into the adapted vectors at the BglII site to create pMASIA-tgD, pBISIA40-tgD and pBISIA88-tgD. Transient expression of the [35S]methionine-labelled secreted tgD protein in COS-7 cells was confirmed by immunoprecipitation, followed by SDSPAGE (Laemmli, 1970
) and autoradiography, as previously described (van Drunen Littel-van den Hurk et al., 1984
).
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Immunizations.
BHV-1-seronegative calves (9 months old) were randomly sorted into four groups of seven animals and one group of five animals. The group with five animals received a commercial killed BHV-1 vaccine at the manufacturer's recommended dose (Triangle 3+Type II BVDV; Ayerst). The remaining calves received 500 µg of pMASIA-tgD, pBISIA40-tgD, pBISIA88-tgD or saline (control group) in a 500 µl volume administered intradermally using the Biojector 2000 needle-free injection system (Bioject). All animals were reimmunized after 28 days. The experiment was conducted in accordance with the guidelines provided by the Canadian Council on Animal Care.
Challenge and clinical observations.
Animals were challenged 20 days after secondary immunization, as previously described (Loehr et al., 2001a ). Briefly, each calf was exposed to a 4 min aerosol of 107 p.f.u./ml of BHV-1 strain 108 generated by a DeVilbis Nebulizer, model 65 (DeVilbis). For 10 days after challenge, blinded clinical examinations, rectal temperatures and body weights were recorded.
Sampling.
Sera were collected prior to primary and secondary immunization, before challenge and on days 4, 8 and 11 after challenge. Blood for the isolation of peripheral blood mononuclear cells (PBMCs) was collected 14 days after primary immunization, 14 days after secondary immunization and 11 days following challenge.
Virus isolation and quantification.
Tampons were used to collect nasal fluid for virus titration 3 days before challenge and on days 2, 4, 6, 8, 10 and 12 after challenge. Virus secretion was determined by plaque assays in triplicate in 96-well plates (Nalge Nunc) with an antibody overlay as previously described (van Drunen Littel-van den Hurk et al., 1998 ).
Virus neutralization assays.
The virus neutralization titres in serum samples were determined as previously described and expressed as the highest reciprocal dilution of serum that resulted in a 50% reduction in p.f.u. relative to the virus control (van Drunen Littel-van den Hurk et al., 1990 ).
Lymphocyte proliferation assays.
Blood was collected into citratedextran and PBMCs were isolated on Ficoll-Plaque Plus (Amersham Pharmacia Biotech) as previously described (Loehr et al., 2000 ). PBMCs were cultured in 96-well plates (Nalge Nunc) at 3·5x106 cells/ml in 200 µl of MEM (Gibco-BRL) containing 10% (v/v) heat-inactivated FBS (Gibco-BRL), 2 mM L-glutamine (Gibco-BRL), 500 mg/ml gentamicin (Gibco-BRL), 1 mg/ml dexamethasone and 50 µM 2-mercaptoethanol (Bio-Rad), in triplicate wells containing medium alone or purified gD at 1 µg/ml. After 72 h in culture at 37 °C with 5% CO2, the cells were pulsed by the addition of 0·4 µCi [methyl-3H]thymidine per well (Amersham Pharmacia Biotech) and harvested 18 h later. Incorporation of [3H]thymidine was measured using a liquid scintillation counter (Beckman 1701). Stimulation indices (SI) were calculated as the quotient of the mean counts per minute of the gD-stimulated samples over that of the medium control.
IFN-
ELISPOT assays.
Nitrocellulose plates (Millipore) were coated overnight with a bovine IFN--specific monoclonal antibody (2-2-1) (Raggo et al., 2000
), washed and blocked for 1 h with medium. PBMCs in medium were dispensed at 106 cells per well in triplicate wells containing medium alone or gD at 0·4 µg/ml and incubated at 37 °C for 24 h. IFN-
-secreting cells were labelled with a rabbit serum specific for bovine IFN-
(lot 92-131) (Raggo et al., 2000
), followed by alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (H+L) (Kirkegaard and Perry Laboratories). Spots representing IFN-
-secreting cells were visualized with BCIPNBT substrate (Sigma). The number of INF-
-secreting cells/106 PBMCs was expressed as the difference between the number of spots in the gD-stimulated wells and the number of spots in the medium control wells.
ELISAs.
Immulon 2 microtitre plates (Dynatech Laboratories) were coated overnight with tgD at 0·05 µg per well and incubated with serially diluted serum samples. AP-conjugated goat anti-bovine IgG (H+L) (Kirkegaard and Perry Laboratories) was used to detect total IgG. Isotyping was performed by indirect ELISA using IgG1- and IgG2-specific monoclonal antibodies (provided by K. Neilson, Animal Disease Research Institute, Nepean, ON, Canada) followed by AP-conjugated rabbit anti-mouse IgG (Kirkegaard and Perry Laboratories). Conjugates were used at a final dilution of 1:10000 and the reactions were visualized with p-nitrophenyl phosphate (Sigma).
SDSPAGE and Western blotting.
An aliquot containing 60 µg of purified BHV-1 strain 108 was run in a single-lane SDSPAGE according to the Laemmli system (Laemmli, 1970 ), transferred to a nitrocellulose membrane as recommended by the manufacturer (Bio-Rad) and incubated overnight at 4 °C in 3% skimmed milk in Tris-buffered saline (TBS; 10 mM TrisHCl, pH 7·5, 170 mM NaCl). The membrane was mounted on to a slot blot apparatus (MiniPROTEAN II Multiscreen; Bio-Rad) and incubated for 2 h with pooled pre-challenge and post-challenge serum (diluted 1:50 in 1% skimmed milk in TBS) from each group. The membrane was washed three times in TBS and incubated for 1 h with AP-conjugated goat anti-bovine IgG (diluted 1:5000 in 1% skimmed milk in TBS). Seroreactive proteins were visualized using BCIPNBT.
Statistical analysis.
All data were analysed with the aid of Graphpad Prism 2.0. Differences between groups in the proliferation, neutralization, isotype ratio, ELISA and ELISPOT assays were examined by one-way analysis of variance (ANOVA) and Dunnett's test. Virus shedding, weights and temperatures were analysed by one-way ANOVA and Tukey's multiple comparison test.
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Results |
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Expression of tgD from these vectors was confirmed by transient transfection of COS-7 cells. The 35S-labelled tgD gene product, which is secreted into the culture medium, was visualized by SDSPAGE and autoradiography of immunoprecipitated supernatants. The molecular mass and relative gel mobility (Fig. 1d) of the tgD protein was consistent with previous reports (van Drunen Littel-van den Hurk et al., 1994
, 1997
).
Cellular immune responses
Antigen-specific proliferation elicited by immunization with plasmid-encoded tgD tended to be superior to that induced by the killed vaccine (Fig. 2a). The stimulation index (SI) was <5 in all groups 14 days after primary immunization. Secondary immunization enhanced the proliferative response by day 42 in the DNA-immunized groups in a CpG dose-dependent manner, correlating positively with the number of CpG motifs per plasmid. However, only the group that received pBISIA88-tgD displayed an SI that was significantly greater (P<0·05) than that of the saline control group and the group that received the killed vaccine. All groups exhibited an increased antigen-specific proliferative response following challenge, with the pBISIA88-tgD-immunized group having a significantly greater (P<0·05) SI than the saline and killed vaccine groups (Fig. 2a
).
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Humoral immune responses
The tgD-specific total serum IgG response to primary immunization was similar in all groups, with only the pMASIA-tgD- and pBISIA88-tgD-immunized groups having a significantly greater (P<0·01) titre than the control group (Fig. 3a). After secondary immunization, the groups immunized with plasmid encoding tgD had an antigen-specific titre significantly greater (P<0·05) than the titre of the saline control group when tested on day 48. The group that received the killed vaccine had the greatest increase (P<0·01) in tgD-specific titres relative to the saline control group after the secondary immunization. The IgG1:IgG2 ratio of tgD-specific serum antibody was determined to evaluate the type of response to immunization. When compared with all other immunized groups, the pBISIA88-tgD-immunized group had a significantly lower (P<0·05) IgG1:IgG2 ratio (Fig. 3c
).
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To evaluate the effectiveness of the anamnestic responses, the level of BHV-1 neutralizing antibodies in serum was assayed on days 0, 4, 8 and 11 after challenge (Fig. 3b). On the day of challenge, the pBISIA88-tgD- and killed-vaccine-immunized groups had a neutralizing antibody titre that was significantly greater (P<0·05) than that of the saline group. After 8 days, all vaccinated groups had significantly greater (P<0·05) neutralization titres than the saline control group, with the pBISIA88-tgD-immunized group having the greatest titre (P<0·01). By day 12, all immunized groups had a nearly equivalent neutralization titre, and titres remained significantly greater than that of the control group (P<0·01).
A Western blot was performed using pooled pre-challenge (day 48) and post-challenge (day 54) sera from each group to determine which BHV-1 proteins were seroreactive (Fig. 3d). The DNA-immunized groups developed a tgD-specific humoral response that detected both the monomeric (71 kDa) and dimeric (142 kDa) forms of the glycoprotein. In contrast, the group that received the killed vaccine developed a broad humoral immune response against several BHV-1 proteins in addition to gD (van Drunen Littel-van den Hurk & Babiuk, 1986
; Whitbeck et al., 1996
). After challenge, all immunized groups demonstrated an increase in the level of the antigen-specific humoral response.
Clinical response to BHV-1 challenge
On the day of challenge, and for the next 12 days, all animals were sampled and monitored to evaluate the protection achieved by immunization. Nasal secretions were collected on days 0, 2, 4, 6, 8, 10 and 12 and assayed for BHV-1 shedding (Fig. 4a). Virus shedding was greatest in the saline-treated group and lasted for at least 10 days. The killed-vaccine- and pBISIA88-tgD-treated groups had a significantly lower (P<0·05) level of shedding than the saline control group on day 6, and by day 8 all the immunized groups displayed significantly lower (P<0·05) shedding than the control group.
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Another clinical symptom used to measure the host response to challenge was the change in body temperature. By day 2 after challenge, a temperature increase was measured in the saline- and DNA-immunized groups that was significantly greater (P<0·05) than that in the killed-vaccine-immunized group (Fig. 4c). As expected, the saline control group had the greatest and longest temperature increase. After 10 days, the temperatures of all animals had returned to normal.
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Discussion |
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There are two essential features that all expression vectors used for vaccination must have. The first is the adjuvant properties of the plasmid backbone as determined by the balance of immunostimulatory and inhibitory CpG motifs (Klinman et al., 2000 ; Krieg et al., 1998b
). Plasmid pMAS was used as the starting material because it has undergone extensive modification to remove over 50 inhibitory CpG motifs and because it contains a specific site designed for the directional insertion of DNA fragments (Klinman et al., 2000
; Krieg et al., 1998b
). For this study, multiple copies of the pan-activating 5' GTCGTT 3' motif were inserted because it has been shown to be immunostimulatory in several species (Pontarollo et al., 2002
; Rankin et al., 2001
).
The second feature is a transcriptional unit able to express the antigen-encoding gene at high levels. The most commonly used expression cassette is the HCMV IE promoter with the bovine growth hormone polyadenylation signal. This expression system was further optimized by the addition of the HCMV intron A downstream of the IE promoter. Intron A has been shown to enhance the antibody response in cattle immunized with tgD-encoding plasmid (Braun et al., 1997 ). Furthermore, intron A is essential for the expression of genes from RNA viruses and bacteria that are not normally transcribed in the nucleus of a eukaryotic cell (van Drunen Littel-van den Hurk et al., 1999
). Overall, addition of intron A and the enrichment for immunostimulatory CpG motifs should make pMASIA and the pBISIA vectors a robust DNA vaccination system for numerous pathogens in many host species.
The role played by the plasmid backbone in the induction of an immune response to DNA vaccination is not well defined. It has been suggested that CpG motifs are essential for developing an effective immune response following vaccination via the intradermal route (Sato et al., 1996 ). In that study, mice administered a vector expressing
-galactosidase that had one or two additional palindromic 5' AACGTT 3' motifs had stronger humoral and cellular immune responses than vectors without the motifs. This study also suggested that the increased immune response induced by the adjuvant properties of CpG motifs is, to some extent, counter-balanced by down-regulation of gene expression by the increased expression of pro-inflammatory cytokines. Krieg et al. (1998a
) also suggested that down-regulation of the HCMV IE promoter was responsible for an increase, plateau and drop in antibody titres in mice immunized with vectors containing 0, 16 or 50 CpG motifs per plasmid. Nevertheless, in that study the CTL activity in splenocytes was positively correlated with the number of CpG motifs per plasmid.
The size of the immunized animal may have a significant bearing on the outcome of DNA immunization. In the mouse experiments described above, the amount of plasmid, and therefore the number of CpG motifs, administered per body weight is far in excess of what we have used in cattle. This could explain why we observed that the number of CpG motifs per plasmid had no significant effect on the total tgD-specific antibody titre before challenge. However, there was a dramatic decrease in the ratio of IgG1:IgG2 antibodies in the group immunized with pBISIA88-tgD. Therefore, the additional CpG motifs had a significant qualitative effect on the humoral response, but this was not observed until the CpG content of the vector exceeded 40 motifs. This suggests that a critical threshold of CpG motifs may be required to significantly influence the IgG isotype ratio in large animals such as cattle, while there is a more CpG dose-dependant correlation with cellular immune responses.
Prior to and following challenge, the host response in the pBISIA40-tgD-vaccinated group was lower than expected. This trend could be seen in the lymphocyte proliferative response (Fig. 2a), virus neutralization titres (Fig. 3b
) and in the intensity of the staining of gD in the Western blot (Fig. 3d
). However, it was especially evident in the total gD-specific IgG titres on day 56, where the pBISIA40-tgD treated group was significantly lower than the pMASIA-tgD- and pBISIA88-tgD-treated groups (Fig. 3a
). One possible explanation for this observation is that although each plasmid expressed tgD at similar levels in vitro (Fig. 1d
), there is evidence that the HCMV promoter on plasmids with 50 CpG motifs is down-regulated in mice due to the increase in proinflammatory cytokines induced by CpG motifs (Krieg et al., 1998a
; Sato et al., 1996
). Therefore, it is plausible that pBISIA40-tgD is also affected by down-regulation in cattle when compared with pMASIA-tgD, and that this is overcome by additional CpG motifs present in pBISIA88-tgD.
Intradermal delivery of plasmid-encoded tgD has previously been shown to be an effective route for vaccination in cattle (Braun et al., 1997 ). It is possible that the titres attained in this study are approaching the upper limit for this model system. Increasing the CpG content of the plasmid backbone in combination with approaches such as electroporation, vector targeting and antigen targeting could be used to increase antibody titres beyond those shown here.
Despite the augmentation of the cellular immune response and virus neutralization titres, the CpG-enhanced plasmids did not offer enhanced protection from virus challenge when compared with the killed vaccine. This can be explained in part by the choice of disease model. It has been established that even though a cellular response to infection is important, prevention from BHV-1 infection is more positively correlated with a strong antibody response (Babiuk et al., 1995 ; Loehr et al., 2000
, 2001b
). Thus, even though the CpG-enhanced vectors did augment the cellular immune response, this did not provide additional protection from challenge. Other important bovine pathogens, such as bovine viral diarrhoea virus, bovine respiratory syncytial virus and Mycobacterium bovis, require strong cellular immune responses for recovery (Buddle, 2001
; Rhodes et al., 1999
; Taylor et al., 1995
). Therefore, CpG-enhanced DNA vaccine vectors could be effective in preventing these infectious diseases.
In addition to the disease model chosen, it is important to clarify that the killed vaccine used in this study carries several BHV-1 antigens in addition to gD, the lone antigen in the DNA vaccine formulations. Indeed, a Western blot revealed that both pre- and post-challenge serum from the DNA-vaccinated groups specifically recognized gD, while the group that received the killed vaccine recognized several BHV-1 proteins (Fig. 4d). BHV-1 glycoproteins gB and gC have been shown to be protective in BHV-1 challenge models (Babiuk et al., 1987
). Therefore, we concluded that the additional protective antigens in the killed vaccine formulation probably contributed to the protective immunity demonstrated here. Plasmids with engineered genes encoding immunodominant epitopes from several BHV-1 antigens could be used to induce a broader, and perhaps more protective, immune response with DNA immunization.
In conclusion, we have shown that CpG-enhanced vectors are able to augment the cellular immune responses in a bovine disease challenge model. This work confirms previously published observations that enhanced cellular immunity to BHV-1 is not correlated with protection unless a strong humoral response is induced as well. These data lay the foundations for future studies that will investigate the efficacy of DNA immunization and CpG-enhanced vectors for use in other large animals and humans. Overall, these experiments provide valuable information for the design of better DNA-based vaccines.
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Acknowledgments |
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Published as VIDO's Journal Series no. 314.
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Received 6 May 2002;
accepted 23 July 2002.