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Vaccination of pigs with a recombinant porcine adenovirus expressing the gD gene from pseudorabies virus
Vaccine 19 (2001) 3752– 3758 www.elsevier.com/locate/vaccine
Vaccination of pigs with a recombinant porcine adenovirus expressing the gD gene from pseudorabies virus Jef M. Hammond a, Elisa S. Jansen a, Christopher J. Morrissy a, Brenda van der Heide a, Winsome V. Goff a, Mark M. Williamson a, Peter T. Hooper a, Lorne A. Babiuk b, Suresh K. Tikoo b, Michael A. Johnson a,* a
CSIRO, Li6estock Industries, Pri6ate Mail Bag 24, Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia b VIDO, 120 Veterinary Road, Uni6ersity of Saskatchewan, Saskatoon, Canada S7N 5E3 Received 8 December 2000; received in revised form 5 February 2001; accepted 19 February 2001
Abstract Five week old, commercially available large white pigs were vaccinated with either a single dose or two doses of a recombinant porcine adenovirus expressing the glycoprotein D gene from pseudorabies virus (PRV). Pigs were monitored for the development of serum neutralizing antibodies to PRV and challenged 3 weeks after final vaccination. Prior to challenge, pigs given 2 doses of the vaccine demonstrated boosted levels of antibody compared with those given a single dose, and all surviving pigs had increased neutralization titres over pre-challenge levels. Following challenge, pigs were monitored for clinical signs of disease, with blood and nasal swabs collected for virus isolation. All control animals became sick with elevated temperatures for 6 days post challenge, whereas; vaccinated animals displayed an increase in body temperature for only 2 – 3 days. Control pigs and those given a single dose all lost condition, but the group given 2 doses remained healthy. At postmortem, gross lesions of pneumonia only occurred in control animals and those given a single dose of vaccine. Histology carried out on the brains of all animals demonstrated a difference in severity of infection and frequency of immunohistochemical antigen detection between test animals, with control and single dose groups being most severely affected and pigs given 2 doses the least. Virus isolation studies demonstrated that no viraemia could be detected, but virus was found in nasal swabs from some animals in both groups of vaccinates following challenge. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Recombinant vaccine; Adenovirus; Pseudorabies; Aujeszky’s disease; Protection
1. Introduction The presence of porcine adenovirus (PAV) in swine is widespread, but infection generally does not cause clinical signs of disease [1]. The low pathogenicity of PAV in pigs and the proven efficacy of adenoviruses as vaccine and gene therapy vehicles has therefore led to our interest in the development of PAV as a vaccine vector. Previously, vaccination of pigs with a single dose of recombinant PAV (rPAV) expressing the gp55 gene from classical swine fever virus (CSFV) was shown to completely protect pigs against disease [2]. At least 7 * Corresponding author. Tel.: + 61-3-5227-5000; fax: +61-3-52275555. E-mail address: [email protected] (M.A. Johnson).
different serotypes of PAV have been characterized [3–7]. We have selected serotype 3 because of its excellent growth characteristics in tissue culture. Pseudorabies virus (PRV) is an alpha herpesvirus comprising a double stranded DNA genome of approximately 150 kbp, which causes the economically important and widespread Aujeszky’s disease (AJD) in pigs [8]. It is a highly neurotropic virus causing nervous and respiratory complications in pigs, the natural host, and in a variety of other animal species [9,10]. Clinical signs in pigs vary widely dependent upon age, virus strain and dose, with young pigs being the most severely affected [9]. Vaccination against AJD is widely practised with live attenuated or killed whole virus vaccines in common use (for review see Ref. [10]). Live AJD vaccines have the limitations of still being infectious with the possibil-
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J.M. Hammond et al. / Vaccine 19 (2001) 3752–3758
ity of reversion to virulence and/or latency, and they are able to replicate in other animal species [9,10]. The use of killed vaccines generally results in less effective protection than that obtained with live vaccines, hence, the use of live recombinant vaccines carrying individual PRV genes may provide a safe alternative to the attenuated live strains available. The glycoprotein D (gD) gene from PRV has been shown to protect pigs from AJD, when delivered as purified viral or recombinant protein [11,12] or in live vaccinia or human adenovirus vectors [13,14]. In order to eliminate the reversion and latency potential and attempt to improve both the efficacy and specificity of delivery of a PRV vaccine to pigs, the gD gene was inserted into the E3 region of PAV-3, and the resultant recombinant virus (rPAV-gD) used in a vaccine trial to determine protection from challenge with AJD.
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and nasal swabs taken. In addition, 3 pigs from each group were bled for virus isolation each day from day 3 post challenge (p.c.), so that each pig was bled every second day. All pigs were euthanased at the end of the experiment at 17 days p.c. or when showing severe clinical disease. Postmortem examinations were carried out with all pigs inspected for lesions of AJD. Spleen, lung, kidney, adrenal, liver, brain and trigeminal ganglion samples were taken for virus isolation and histology.
2.3. Pseudorabies 6irus antibody detection The presence of antibodies against PRV in pig serum was determined by SNT. SNT titres are expressed as the reciprocal of the serum dilution that neutralized 100 TCID50 of the ADV NZ strain in 50% of replicate cultures.
2. Materials and methods
2.4. Virus isolation
2.1. Cells and 6irus
Blood and nasal swab samples were tested on tissue culture for the presence of challenge virus. Pigs were bled every second day following challenge and whole blood and buffy coat cells were passaged twice in pig kidney cells (PK15) which were monitored for cytopathology. Nasal swabs were taken daily for 7 days p.c. from all pigs and passaged as above.
Construction of an rPAV containing the gD gene from PRV inserted into the E3 region of the PAV3 genome has been described previously [15]. Recombinant virus was cultured in primary pig kidney (1° PK) cells maintained in Earles modified Eagles medium supplemented with 2% foetal bovine serum and harvested when 90% of the cells were showing cytopathic effect.
2.2. Pig experiment Twelve commercially available 4– 5 week old, outbred, large white pigs were housed in the high security animal facility at the Australian Animal Health Laboratory. Six were given 2 doses of rPAV-gD with dose one at day 0 followed by a booster dose on day 25 post primary vaccination. The second group of 6 were given a single dose of rPAV-gD on day 25, so that challenge could be carried out simultaneously on both groups at day 24 post final vaccination. Each vaccination was given by subcutaneous injection comprising 2 ml of clarified tissue culture supernatant containing 1× 105.4 TCID50/ml of recombinant virus. Following vaccination, pigs were monitored for clinical signs and temperatures taken for 3 days. Pigs were bled weekly and serum tested for the presence of antibodies to PRV by serum neutralization test (SNT). On day 49 post primary vaccination (24 days post boost) all pigs were challenged intranasally with 7× 106 PRV New Zealand (NZ) strain. Three clean, age matched control pigs were brought in 2 days prior to challenge. Following challenge, all pigs were monitored daily for clinical signs of disease, rectal temperatures recorded
2.5. General condition of pigs Prior to termination, the condition of pigs was scored with 1 indicating very poor and 5 indicating very good condition. The differences in condition observed were compared using a student’s T-test.
2.6. Pathology Following termination, each pig was subjected to a postmortem examination. A general inspection was made of all major organs and the severity of pneumonia in the lungs scored as 0 (none), to 3 (severe signs). The differences in the severity of pneumonia observed were compared using a student’s T-test.
2.7. Histology Immunohistochemical examinations of all tissues were carried out on unmarked samples, where the pathologist had no prior knowledge of their identifications. Brains and lungs were fixed in 10% neutral buffered formalin. Transverse sections, trimmed out of the brains that highlighted cerebrum, midbrain, cerebellum and brain stem, were paraffin-embedded and stained with haematoxylin and eosin using conventional methods. For immunohistochemistry, formalin-fixed
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paraffin-embedded tissues were tested by an immunoperoxidase test for ADV antigen. Prior to the test, each section was trypsinised for 20 min (0.1% trypsin in 0.1% CaCl2 solution at pH 7.8). The specific anti-ADV goat antiserum was visualized by the action of biotinylated anti-mouse, rabbit and goat antibody and streptavidin–horseradish peroxidase conjugate (Dako Lab Peroxidase Kit, Dako Corporation, CA) on 3-amino-9ethylcarbazole. Pretreatment with hydrogen peroxide before the final reaction reduced the activity of nonspecific peroxidase and all tissues were counter-stained with haematoxylin.
3. Results
3.1. Temperatures and clinical obser6ations post 6accination No clinical signs or increased temperatures were observed following vaccination with rPAV-gD.
3.2. Temperatures and clinical obser6ations post challenge with pseudorabies 6irus Rectal temperatures of each pig were measured daily following PRV challenge and the mean temperatures for each group of pigs are shown in Fig. 1.
Control group: The mean temperature for the control group of pigs rose toE 40°C for 6 days p.c. (Fig. 1.) with all pigs showing clinical signs of AJD including; loss of appetite, slight diarrhoea and nervous signs comprising abnormal posture and an unsteady gait. However, although pigs lost condition, these signs were transitory and were not severe enough to warrant euthanasia. Single dose group: The mean temperature for the group of 6 pigs given a single dose of rPAV-gD rose to E 40°C for 2 days p.c. (Fig. 1.). Pigs 4 and 6 showed severe clinical signs of AJD and pig 4 was euthanased on day 6 p.c. following 4 days of pyrexia. Pig 6 lost condition but recovered and survived until termination of the experiment. The 4 remaining animals suffered a transient loss of appetite and slight diarrhoea resulting in loss of condition, but survived until termination of the experiment. Two dose group: The mean temperature for the group of 6 pigs given two doses of rPAV-gD rose to E 40°C for 3 days p.c. (Fig. 1.). Pigs 2 and 12 showed slight depression for 1 day p.c. and pig 12 also had slight diarrhoea on that day. However, neither lost any condition and survived until termination of the experiment. The 4 remaining pigs in the two dose group showed no clinical signs following AJD challenge and continued to thrive until termination of the experiment.
Fig. 1. Temperatures of pigs p.c. with PRV. Pigs were vaccinated with one or two doses of rPAV-gD and challenged with PRV. Rectal temperatures of each pig were measured daily following challenge and the mean temperatures for each group of pigs including standard error bars are shown. Single dose (2), two doses (D) and control groups ( ).
J.M. Hammond et al. / Vaccine 19 (2001) 3752–3758
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Fig. 2. SNT for PRV antibodies. All pigs were bled at various intervals post primary vaccination and p.c. and monitored for the presence of serum neutralizing antibodies to PRV by SNT. Pigs given two doses of rPAV-gD were vaccinated on day 0 and boosted on day 25. Pigs given a single dose were vaccinated on the equivalent of day 25 for the two dose group. All pigs were challenged on day 24 post final vaccination. Day 25 (a), day 32 ( ), day 40 (b), day 49 () and final bleed ( ). The negative cut off of 1/4 (- - -), euthanased ( +).
3.3. Serum neutralization test for pseudorabies 6irus antibodies At various intervals post primary vaccination and p.c., all pigs were bled and monitored for the presence of serum antibodies to PRV by SNT. None of the pigs in the control group had detectable antibodies to PRV prior to challenge (Fig. 2.). However, following challenge, they developed titres of 1/ 128, 1/180 and 1/45, respectively. Two of the pigs given a single dose of rPAV-gD had SNT titres \ 1/4 (considered background level) before challenge, with 5 out of 6 animals surviving challenge and developing titres from 1/90 up to \1/512. Pig number 4 did not develop any antibodies to PRV and was euthanased with severe clinical AJD on day 6 p.c. Five out of 6 pigs given 2 doses of rPAV-gD developed SNT titres of between 1/6 and 1/11 before challenge with one pig (number 8) having no detectable titre. However, all survived challenge and had final titres of between 1/45 and \ 1/512 at termination of the experiment on day 17.
3.4. Virus isolation Following challenge no virus was detected in either the whole blood or white blood cell fraction from any
pig, including controls. In contrast, virus was re-isolated from nasal swabs from a single pig in the control group on 2 days p.c., and from one pig in the single dose group again for 2 days p.c. Three pigs in the two dose group were positive for virus in nasal swabs for up to 5 days p.c.
3.5. General condition of pigs Prior to termination, the condition of pigs was scored from 1, indicating very poor to 5, indicating good/healthy condition (Table 1). The control pigs were all in poor condition with a mean score of 2.0. Pigs in the single dose group showed variation in condition with 3 out of 6 in average condition with scores of 3, 2 out of 6 in poor condition scoring 2 and 1 out of 6 very poor (1) which was euthanased with severe disease. The mean score for the single dose group was 2.3. The group of pigs given 2 doses of rPAV-gD were all of uniform appearance, with 5 out of 6 in very good condition scoring 5 and 1out of 6 in good condition with a score of 4, giving a mean score of 4.8. The final condition of the pigs given two doses of vaccine was significantly improved over the control and single dose groups, where PB 0.01 (Table 1).
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3.6. Postmortem Following termination, each pig was subjected to a postmortem examination with a general inspection made of all major organs. Gross lesions were seen in the lungs but in no other organs or tissues. The presence of pneumonia in the lungs seen as dark red or variegated red/grey firm consolidation, was scored as 0 (no signs) to 3 (severe signs) (Table 1). Control pigs all showed severely consolidated lungs with scores of 3, 2 and 2 resulting in a mean score of 2.3. All pigs given a single dose of rPAV-gD also had severe lung infections with 5 out of 6 pigs scoring 3 and one pig scoring 2, giving a mean score of 2.8. In contrast, none of the pigs given two doses of the recombinant vaccine showed any signs of lung infection and all scored 0. The absence of pneumonia in the pigs given two doses of vaccine was significant when compared with the control and single dose groups, where P B0.01 (Table 1).
3.7. Histology Lesions in the brain were mostly those of perivascular cuffs of mononuclear cells and gliosis. In one case, pig no. 4, there was neuronal necrosis accompanied by intranuclear inclusion bodies. In the lungs, lesions confirmed as specific by immunoperoxidase staining were foci of mononuclear cells and thickening of alveTable 1 Assessment of pig conditiona Vaccine group
Pig number
Condition/5
Pneumonia/3
1 Dose
3 4b 5 6 7 10
2 1 3 2 3 3 2.3
3 3 3 3 2 3 2.8
1 2 8 9 11 12
5 5 5 4 5 5 4.8c
0 0 0 0 0 0 0c
C1 C2 C3
2 2 2 2
3 2 2 2.3
Mean 2 Doses
Mean Controls
Mean a
The overall condition of each pig was assessed and scored on a scale of 1 (poor) to 5 (very good) prior to termination. At postmortem, lungs were examined for disease pathology and scored for the presence of pneumonia from 0 (none) to 3 (severe). b Euthanased. c Score was significantly different from control group, where P0 0.01.
olar walls. Many pigs had intra-alveolar and intrabronchiolar masses of neutrophils, and peribronchiolar masses of lymphocytes. These were not associated with immunoperoxidase staining for PRV antigen, but their presence and intensity varied significantly between the groups. No evidence of PRV infection was detected in kidney and liver from any animal, and was found in only one spleen sample from a control pig (no. 21). Whereas, cerebrum, midbrain, brain stem and lung were found to be positive for the presence of PRV in a number of pigs. The assessment of histopathology and immunohistochemistry in these animals using a 0 (negative) to 4 (strong positive) rating is presented in Table 2. Control pigs were all positive for PRV lesions in the brain with scores up to 3 and lesions in the lung from between 1 and 4. Control pig 21 also scored 2 for lesions in both the spleen and draining lymph node (data not shown) and gave positive immunoperoxidase staining for the presence of PRV antigen in the brain. All pigs, which received a single dose of rPAV-gD, were positive for lesions in the brain with scores from 2–4. In addition, in pig 4, there were masses of lymphocytes in the tri-geminal ganglion and of plasma cells in the lymph node, and it was strongly positive for the presence of PRV antigen in the brain and lung. This pig developed severe clinical disease including paralysis of the hind limbs and was euthanased on day 6 p.c. In addition, 4 out of 6 pigs in the single dose group were positive for the presence of PRV lesions in the lung with scores from 1–4, and all 6 pigs showed positive immunoperoxidase staining for PRV antigen in the brain and/or lung. The pigs which received 2 doses of vaccine showed low level scores of 1– 2 for lesions in the brain with 4 pigs scoring 1 and 2 pigs scoring 2. Four of the 6 pigs had only mild lesions in the lung with scores from 1–3. However, all pigs were negative for PRV antigen in both brain and lungs.
4. Discussion An rPAV expressing the PRV gD gene was administered to pigs and the level of protection from challenge investigated. Pigs given 2 doses were protected from disease, showed no significant loss of condition and had lower levels of brain and lung pathology compared with control pigs or those given a single dose. Previously we have demonstrated complete protection of pigs from lethal challenge with CSFV following a single vaccination with a rPAV expressing the major protective antigen gp55 [2]. That recombinant virus was constructed so that expression of gp55 was driven by the PAV major late promoter (MLP) and enhanced by the inclusion of the PAV tri-partite leader (TPL) sequence up-
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Table 2 Assessment of histopathology and immunohistochemistry of pigs challenged with pseudorabiesa Vaccine group
1 Dose
Pig
3 4b 5 6 7 10
Total Mean 2 Doses
Total Mean
Midbrain
Brain stem
Lung
His
IPX
His
IPX
His
IPX
His
IPX
2 4 1 1 2 2 12 2.0
0 4 1 0 1 0 6 1.0
2 3 2 0 1 1 9 1.5
0 0 0 0 0 0 0 0
1 3 2 0 0 2 8 1.3
0 0 0 0 0 1 1 0.17
4 3 0 0 1 3 11 1.7
2 3 0 1 2 0 8 1.3
1 2 8 9 11 12
1 0 1 0 2 1 5 0.83
0 0 0 0 0 0 0 0
0 1 1 0 1 1 4 0.67
0 0 0 0 0 0 0 0
2 1 1 1 1 0 6 1.0
0 0 0 0 0 0 0 0
1 0 2 0 1 3 7 1.2
0 0 0 0 0 0 0 0
21 22 23
2 1 2 5 1.67
2 0 0 2 0.67
1 2 1 4 1.3
0 0 0 0 0
1 3 1 5 1.67
0 0 0 0 0
4 2 1 7 2.3
0 0 0 0 0
Total Mean Controls
Cerebrum
a
Following termination, brain and lung samples from each pig were examined for the presence of PRV infection apparent as lesions and the presence of viral antigen using immunoperoxidase staining. His – assessment of severity of histopathology, IPX – assessment of intensity of staining in an indirect immunoperoxidase test for PRV antigen. These were 0 – no visible lesions and no staining, 1 – mild lesion (almost certainly subclinical) or one focus or occasional cells staining, 2 – mild but widespread lesion (almost certainly subclinical) or several foci of cells staining, 3 – well developed lesions and staining, 4 – severe lesions and very widespread staining. b Euthanased.
stream of the coding region. Additionally, insertion of gp55 was made into the right hand end of the PAV genome without deletion of viral DNA sequence. In contrast, the gD gene from PRV was inserted into the E3 region of PAV3 with the subsequent deletion of approximately 600 bp of PAV3 E3 DNA [15]. It has been reported that deletion of the E3 region of human adenovirus can alter the virulence of recombinant viruses compared with wild type [16,17]. For this reason, it was important to determine whether the recombinant virus caused any adverse effects in experimental animals by monitoring the clinical reactions of pigs, when first vaccinated with rPAV-gD. No clinical signs were observed following sub-cutaneous injection, demonstrating that insertion of gD into the partially deleted E3 region did not increase the virulence of recombinant virus over that of wild type PAV3, which causes a mild transitory diarrhoea (data not shown). An important consideration concerning the use of the rPAV-gD recombinant, is that expression of gD was solely dependent upon the E3 transcriptional unit, since no additional promoter or translational enhancer elements were included upstream of the gene in this construct. Therefore, it is probable that expression of gD may have been at a lower level than was achieved with gp55 in the previously tested rPAV-gp55 recombinant,
where expression of gp55 was driven by the MLP [2]. However, expression of gD in tissue culture cells infected with rPAV-gD was demonstrated using a monoclonal antibody specific to gD [15], and following vaccination of pigs with rPAV-gD, neutralizing antibodies against gD were detected, demonstrating expression of this gene in vivo. However, levels of antibody were found to vary between the two groups of vaccinated pigs, with only 2 of 6 pigs given a single dose having PRV neutralizing antibody titres above background prior to challenge, but with 5 of 6 pigs given 2 doses having detectable levels. This demonstrates an enhancement of the induction of PRV neutralizing antibody levels as a result of the booster dose. The 4 pigs in the single dose group that did not develop detectable antibody titres before challenge, presented with varying degrees of clinical signs p.c., with one pig showing severe disease resulting in euthanasia. However, one pig in the 2 dose group also failed to develop a detectable neutralizing antibody response before challenge, but did not develop disease, and remained healthy until termination. Thus, there appears to be no absolute correlation between the presence of detectable neutralizing antibodies to gD prior to challenge and protection from disease, suggesting that protection from PRV induced by live rPAV expressing gD may be dependent upon cell mediated immunity as well as neutralizing antibody.
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Following challenge, control pigs had elevated temperatures for 6 days before returning to normal levels and presented a variety of clinical signs. Pigs in the two vaccinated groups also had elevated temperatures, but for only 2–3 days, demonstrating a marked effect of the vaccine in reducing the period of fever. There was also a clear difference in the development and severity of clinical signs between the two groups of vaccinates, with those receiving only a single dose presenting with more severe disease and loss of condition compared with those given 2 doses. Pigs given a booster dose of vaccine maintained their condition immediately p.c. and continued to thrive until termination. Postmortem examinations also revealed that control pigs and those given a single dose of vaccine suffered severe pneumonia, whereas pigs given 2 doses did not. The severe pneumonias were not necessarily correlated with viral antigen detection and may have been associated with secondary bacterial infection or enhancement of previous mycoplasmal infection. There was a total absence of detectable antigen in any of the pigs given the booster dose of vaccine. No viraemia was found in any of the challenged pigs, including controls, but virus was re-isolated from nasal swabs taken from animals in every group, suggesting that virus shedding may still occur even in pigs protected from clinical disease, following two doses of vaccine. It is possible that a third dose of vaccine or the administration of a rPAV expressing the gD gene under the control of a stronger promoter such as the PAV MLP may help reduce or prevent virus shedding. In summary, we have demonstrated successful delivery of the gD gene of PRV to pigs utilizing a live rPAV vector. Different levels of protection based upon production of neutralizing antibodies, ability to thrive, presence of pneumonia and detection of viral induced pathology in the brains and lungs of challenged animals were observed. In the examination of brain and lung pathology, it seems that a single dose of vaccine may have exacerbated disease compared with controls. Two doses of recombinant vaccine protected pigs, with animals having increased neutralizing antibody levels before challenge, suffering no significant loss of condition, and showing decreased disease pathology as compared with controls and single dose groups. It is quite probable that protection elicited by this recombinant could be improved by increasing the level of expression of the gD gene by coupling it to the PAV MLP and TPL sequences, as were used in the CSFV protection experiments [2]. Further work is needed to determine the extent of protection offered by such a recombinant and examine the occurrence and duration of virus shedding, if any, in nasal secretions.
Acknowledgements The authors would like to thank Don Carlson for
expert assistance with animal experiments and Megan Braun for the preparation of histological and immunohistochemical slides.
References [1] Barker IK, Van Dreumel AA, Palmer N. Porcine adenovirus. In: Jubb KVF, Kennedy PC, Palmer N, editors. Pathology of domestic animals, vol. 2, 4th ed. London: Academic press, 1993. p. 182 – 3. [2] Hammond JM, Mccoy RJ, Jansen EJ, Morrissy CJ, Hodgson ALM, Johnson MA. Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55 (E2) gene protects pigs against classical swine fever. Vaccine 2000;18:1040 – 50. [3] Derbyshire JB, Clarke MC, Collins AP. Serological and pathogenicity studies with some unclassified porcine adenoviruses. J Comp Pathol 1975;85:437 – 43. [4] Hirahara T, Yasuhara H, Matsui O, Yamanaka M, Tanaka M, Fukuyama S, et al. Isolation of porcine adenovirus from the respiratory tract of pigs in Japan. Nippon Juigaku Zasshi 1990;52:407 – 9. [5] Tuboly T, Reddy PS, Nagy E, Derbyshire JB. Restriction endonuclease analysis and physical mapping of the genome of porcine adenovirus type 5. Virus Res 1995;37:49 – 54. [6] Kadoi K, Iwabuchi M, Satoh T, Katase T, Kawaji T, Morichi T. Adenovirus isolation from spleen lymphocytes of apparently healthy pigs. New Microbiol 1997;20:215 – 20. [7] Kadoi K. Beneficial use of inactivated porcine adenovirus vaccine and antibody response of young pigs. New Microbiol 1997;20:89 – 91. [8] Mettenleiter TC. Aujeszky’s disease (pseudorabies) virus: the virus and molecular pathogenesis – state of the art. Vet Res 2000;31:99 – 115. [9] Kluge JP, Beran GW, Hill HT, Platt KB. Pseudorabies (Aujeszky’s Disease). In: Leman AD, Straw BE, Mengeling WL, D’Allaire S, Taylor DJ, editors. Diseases of swine, 7th ed. Ames, IA: Iowa State University Press, 1992. p. 312 – 23. [10] Mulder WA, Pol JM, Gruys E, Jacobs L, De Jong MC, Peeters BP, et al. Pseudorabies virus infections in pigs. Role of viral proteins in virulence, pathogenesis and transmission. Vet Res 1997;28:1 – 17. [11] Ishii H, Kobayashi Y, Kuroki M, Kodama Y. Protection of mice from lethal infection with Aujeszky’s disease virus by immunization with purified gVI. J Gen Virol 1988;69:1411 – 4. [12] Mukamoto M, Watanabe I, Kobayashi Y, Icatlo FC Jr., Ishii H, Kodama Y. Immunogenicity in Aujeszky’s disease virus structural glycoprotein gVI (gp50) in swine. Vet Microbiol 1991;29:109 – 21. [13] Riviere M, Tartaglia J, Perkus ME, Norton EK, Bongermino CM, Lacoste F, et al. Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants. J Virol 1992;66:3424 – 34. [14] Adam M, Lepotier MF, Eloit M. Vaccination of pigs with replication-defective adenovirus vectored vaccines: the example of pseudorabies. Vet Micro 1994;42:205 – 15. [15] Reddy PS, Idamakanti N, Hyun BH, Tikoo SK, Babiuk LA. Development of porcine adenovirus-3 as an expression vector. J Gen Virol 1999;80:563 – 70. [16] Ginsberg HS, Lundholm-Beauchamp U, Horswood RL, Pernis B, Wold WS, Chanock RM, et al. Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proc Natl Acad Sci USA 1989;86:3823 – 7. [17] Ginsberg HS, Prince GA. The molecular basis of adenovirus pathogenesis. Infect Agents Dis 1994;3:1 – 8.
Vaccination of pigs with a recombinant porcine adenovirus expressing the gD gene from pseudorabies virus Jef M. Hammond a, Elisa S. Jansen a, Christopher J. Morrissy a, Brenda van der Heide a, Winsome V. Goff a, Mark M. Williamson a, Peter T. Hooper a, Lorne A. Babiuk b, Suresh K. Tikoo b, Michael A. Johnson a,* a
CSIRO, Li6estock Industries, Pri6ate Mail Bag 24, Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia b VIDO, 120 Veterinary Road, Uni6ersity of Saskatchewan, Saskatoon, Canada S7N 5E3 Received 8 December 2000; received in revised form 5 February 2001; accepted 19 February 2001
Abstract Five week old, commercially available large white pigs were vaccinated with either a single dose or two doses of a recombinant porcine adenovirus expressing the glycoprotein D gene from pseudorabies virus (PRV). Pigs were monitored for the development of serum neutralizing antibodies to PRV and challenged 3 weeks after final vaccination. Prior to challenge, pigs given 2 doses of the vaccine demonstrated boosted levels of antibody compared with those given a single dose, and all surviving pigs had increased neutralization titres over pre-challenge levels. Following challenge, pigs were monitored for clinical signs of disease, with blood and nasal swabs collected for virus isolation. All control animals became sick with elevated temperatures for 6 days post challenge, whereas; vaccinated animals displayed an increase in body temperature for only 2 – 3 days. Control pigs and those given a single dose all lost condition, but the group given 2 doses remained healthy. At postmortem, gross lesions of pneumonia only occurred in control animals and those given a single dose of vaccine. Histology carried out on the brains of all animals demonstrated a difference in severity of infection and frequency of immunohistochemical antigen detection between test animals, with control and single dose groups being most severely affected and pigs given 2 doses the least. Virus isolation studies demonstrated that no viraemia could be detected, but virus was found in nasal swabs from some animals in both groups of vaccinates following challenge. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Recombinant vaccine; Adenovirus; Pseudorabies; Aujeszky’s disease; Protection
1. Introduction The presence of porcine adenovirus (PAV) in swine is widespread, but infection generally does not cause clinical signs of disease [1]. The low pathogenicity of PAV in pigs and the proven efficacy of adenoviruses as vaccine and gene therapy vehicles has therefore led to our interest in the development of PAV as a vaccine vector. Previously, vaccination of pigs with a single dose of recombinant PAV (rPAV) expressing the gp55 gene from classical swine fever virus (CSFV) was shown to completely protect pigs against disease [2]. At least 7 * Corresponding author. Tel.: + 61-3-5227-5000; fax: +61-3-52275555. E-mail address: [email protected] (M.A. Johnson).
different serotypes of PAV have been characterized [3–7]. We have selected serotype 3 because of its excellent growth characteristics in tissue culture. Pseudorabies virus (PRV) is an alpha herpesvirus comprising a double stranded DNA genome of approximately 150 kbp, which causes the economically important and widespread Aujeszky’s disease (AJD) in pigs [8]. It is a highly neurotropic virus causing nervous and respiratory complications in pigs, the natural host, and in a variety of other animal species [9,10]. Clinical signs in pigs vary widely dependent upon age, virus strain and dose, with young pigs being the most severely affected [9]. Vaccination against AJD is widely practised with live attenuated or killed whole virus vaccines in common use (for review see Ref. [10]). Live AJD vaccines have the limitations of still being infectious with the possibil-
0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0264-410X(01)00084-6
J.M. Hammond et al. / Vaccine 19 (2001) 3752–3758
ity of reversion to virulence and/or latency, and they are able to replicate in other animal species [9,10]. The use of killed vaccines generally results in less effective protection than that obtained with live vaccines, hence, the use of live recombinant vaccines carrying individual PRV genes may provide a safe alternative to the attenuated live strains available. The glycoprotein D (gD) gene from PRV has been shown to protect pigs from AJD, when delivered as purified viral or recombinant protein [11,12] or in live vaccinia or human adenovirus vectors [13,14]. In order to eliminate the reversion and latency potential and attempt to improve both the efficacy and specificity of delivery of a PRV vaccine to pigs, the gD gene was inserted into the E3 region of PAV-3, and the resultant recombinant virus (rPAV-gD) used in a vaccine trial to determine protection from challenge with AJD.
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and nasal swabs taken. In addition, 3 pigs from each group were bled for virus isolation each day from day 3 post challenge (p.c.), so that each pig was bled every second day. All pigs were euthanased at the end of the experiment at 17 days p.c. or when showing severe clinical disease. Postmortem examinations were carried out with all pigs inspected for lesions of AJD. Spleen, lung, kidney, adrenal, liver, brain and trigeminal ganglion samples were taken for virus isolation and histology.
2.3. Pseudorabies 6irus antibody detection The presence of antibodies against PRV in pig serum was determined by SNT. SNT titres are expressed as the reciprocal of the serum dilution that neutralized 100 TCID50 of the ADV NZ strain in 50% of replicate cultures.
2. Materials and methods
2.4. Virus isolation
2.1. Cells and 6irus
Blood and nasal swab samples were tested on tissue culture for the presence of challenge virus. Pigs were bled every second day following challenge and whole blood and buffy coat cells were passaged twice in pig kidney cells (PK15) which were monitored for cytopathology. Nasal swabs were taken daily for 7 days p.c. from all pigs and passaged as above.
Construction of an rPAV containing the gD gene from PRV inserted into the E3 region of the PAV3 genome has been described previously [15]. Recombinant virus was cultured in primary pig kidney (1° PK) cells maintained in Earles modified Eagles medium supplemented with 2% foetal bovine serum and harvested when 90% of the cells were showing cytopathic effect.
2.2. Pig experiment Twelve commercially available 4– 5 week old, outbred, large white pigs were housed in the high security animal facility at the Australian Animal Health Laboratory. Six were given 2 doses of rPAV-gD with dose one at day 0 followed by a booster dose on day 25 post primary vaccination. The second group of 6 were given a single dose of rPAV-gD on day 25, so that challenge could be carried out simultaneously on both groups at day 24 post final vaccination. Each vaccination was given by subcutaneous injection comprising 2 ml of clarified tissue culture supernatant containing 1× 105.4 TCID50/ml of recombinant virus. Following vaccination, pigs were monitored for clinical signs and temperatures taken for 3 days. Pigs were bled weekly and serum tested for the presence of antibodies to PRV by serum neutralization test (SNT). On day 49 post primary vaccination (24 days post boost) all pigs were challenged intranasally with 7× 106 PRV New Zealand (NZ) strain. Three clean, age matched control pigs were brought in 2 days prior to challenge. Following challenge, all pigs were monitored daily for clinical signs of disease, rectal temperatures recorded
2.5. General condition of pigs Prior to termination, the condition of pigs was scored with 1 indicating very poor and 5 indicating very good condition. The differences in condition observed were compared using a student’s T-test.
2.6. Pathology Following termination, each pig was subjected to a postmortem examination. A general inspection was made of all major organs and the severity of pneumonia in the lungs scored as 0 (none), to 3 (severe signs). The differences in the severity of pneumonia observed were compared using a student’s T-test.
2.7. Histology Immunohistochemical examinations of all tissues were carried out on unmarked samples, where the pathologist had no prior knowledge of their identifications. Brains and lungs were fixed in 10% neutral buffered formalin. Transverse sections, trimmed out of the brains that highlighted cerebrum, midbrain, cerebellum and brain stem, were paraffin-embedded and stained with haematoxylin and eosin using conventional methods. For immunohistochemistry, formalin-fixed
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paraffin-embedded tissues were tested by an immunoperoxidase test for ADV antigen. Prior to the test, each section was trypsinised for 20 min (0.1% trypsin in 0.1% CaCl2 solution at pH 7.8). The specific anti-ADV goat antiserum was visualized by the action of biotinylated anti-mouse, rabbit and goat antibody and streptavidin–horseradish peroxidase conjugate (Dako Lab Peroxidase Kit, Dako Corporation, CA) on 3-amino-9ethylcarbazole. Pretreatment with hydrogen peroxide before the final reaction reduced the activity of nonspecific peroxidase and all tissues were counter-stained with haematoxylin.
3. Results
3.1. Temperatures and clinical obser6ations post 6accination No clinical signs or increased temperatures were observed following vaccination with rPAV-gD.
3.2. Temperatures and clinical obser6ations post challenge with pseudorabies 6irus Rectal temperatures of each pig were measured daily following PRV challenge and the mean temperatures for each group of pigs are shown in Fig. 1.
Control group: The mean temperature for the control group of pigs rose toE 40°C for 6 days p.c. (Fig. 1.) with all pigs showing clinical signs of AJD including; loss of appetite, slight diarrhoea and nervous signs comprising abnormal posture and an unsteady gait. However, although pigs lost condition, these signs were transitory and were not severe enough to warrant euthanasia. Single dose group: The mean temperature for the group of 6 pigs given a single dose of rPAV-gD rose to E 40°C for 2 days p.c. (Fig. 1.). Pigs 4 and 6 showed severe clinical signs of AJD and pig 4 was euthanased on day 6 p.c. following 4 days of pyrexia. Pig 6 lost condition but recovered and survived until termination of the experiment. The 4 remaining animals suffered a transient loss of appetite and slight diarrhoea resulting in loss of condition, but survived until termination of the experiment. Two dose group: The mean temperature for the group of 6 pigs given two doses of rPAV-gD rose to E 40°C for 3 days p.c. (Fig. 1.). Pigs 2 and 12 showed slight depression for 1 day p.c. and pig 12 also had slight diarrhoea on that day. However, neither lost any condition and survived until termination of the experiment. The 4 remaining pigs in the two dose group showed no clinical signs following AJD challenge and continued to thrive until termination of the experiment.
Fig. 1. Temperatures of pigs p.c. with PRV. Pigs were vaccinated with one or two doses of rPAV-gD and challenged with PRV. Rectal temperatures of each pig were measured daily following challenge and the mean temperatures for each group of pigs including standard error bars are shown. Single dose (2), two doses (D) and control groups ( ).
J.M. Hammond et al. / Vaccine 19 (2001) 3752–3758
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Fig. 2. SNT for PRV antibodies. All pigs were bled at various intervals post primary vaccination and p.c. and monitored for the presence of serum neutralizing antibodies to PRV by SNT. Pigs given two doses of rPAV-gD were vaccinated on day 0 and boosted on day 25. Pigs given a single dose were vaccinated on the equivalent of day 25 for the two dose group. All pigs were challenged on day 24 post final vaccination. Day 25 (a), day 32 ( ), day 40 (b), day 49 () and final bleed ( ). The negative cut off of 1/4 (- - -), euthanased ( +).
3.3. Serum neutralization test for pseudorabies 6irus antibodies At various intervals post primary vaccination and p.c., all pigs were bled and monitored for the presence of serum antibodies to PRV by SNT. None of the pigs in the control group had detectable antibodies to PRV prior to challenge (Fig. 2.). However, following challenge, they developed titres of 1/ 128, 1/180 and 1/45, respectively. Two of the pigs given a single dose of rPAV-gD had SNT titres \ 1/4 (considered background level) before challenge, with 5 out of 6 animals surviving challenge and developing titres from 1/90 up to \1/512. Pig number 4 did not develop any antibodies to PRV and was euthanased with severe clinical AJD on day 6 p.c. Five out of 6 pigs given 2 doses of rPAV-gD developed SNT titres of between 1/6 and 1/11 before challenge with one pig (number 8) having no detectable titre. However, all survived challenge and had final titres of between 1/45 and \ 1/512 at termination of the experiment on day 17.
3.4. Virus isolation Following challenge no virus was detected in either the whole blood or white blood cell fraction from any
pig, including controls. In contrast, virus was re-isolated from nasal swabs from a single pig in the control group on 2 days p.c., and from one pig in the single dose group again for 2 days p.c. Three pigs in the two dose group were positive for virus in nasal swabs for up to 5 days p.c.
3.5. General condition of pigs Prior to termination, the condition of pigs was scored from 1, indicating very poor to 5, indicating good/healthy condition (Table 1). The control pigs were all in poor condition with a mean score of 2.0. Pigs in the single dose group showed variation in condition with 3 out of 6 in average condition with scores of 3, 2 out of 6 in poor condition scoring 2 and 1 out of 6 very poor (1) which was euthanased with severe disease. The mean score for the single dose group was 2.3. The group of pigs given 2 doses of rPAV-gD were all of uniform appearance, with 5 out of 6 in very good condition scoring 5 and 1out of 6 in good condition with a score of 4, giving a mean score of 4.8. The final condition of the pigs given two doses of vaccine was significantly improved over the control and single dose groups, where PB 0.01 (Table 1).
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3.6. Postmortem Following termination, each pig was subjected to a postmortem examination with a general inspection made of all major organs. Gross lesions were seen in the lungs but in no other organs or tissues. The presence of pneumonia in the lungs seen as dark red or variegated red/grey firm consolidation, was scored as 0 (no signs) to 3 (severe signs) (Table 1). Control pigs all showed severely consolidated lungs with scores of 3, 2 and 2 resulting in a mean score of 2.3. All pigs given a single dose of rPAV-gD also had severe lung infections with 5 out of 6 pigs scoring 3 and one pig scoring 2, giving a mean score of 2.8. In contrast, none of the pigs given two doses of the recombinant vaccine showed any signs of lung infection and all scored 0. The absence of pneumonia in the pigs given two doses of vaccine was significant when compared with the control and single dose groups, where P B0.01 (Table 1).
3.7. Histology Lesions in the brain were mostly those of perivascular cuffs of mononuclear cells and gliosis. In one case, pig no. 4, there was neuronal necrosis accompanied by intranuclear inclusion bodies. In the lungs, lesions confirmed as specific by immunoperoxidase staining were foci of mononuclear cells and thickening of alveTable 1 Assessment of pig conditiona Vaccine group
Pig number
Condition/5
Pneumonia/3
1 Dose
3 4b 5 6 7 10
2 1 3 2 3 3 2.3
3 3 3 3 2 3 2.8
1 2 8 9 11 12
5 5 5 4 5 5 4.8c
0 0 0 0 0 0 0c
C1 C2 C3
2 2 2 2
3 2 2 2.3
Mean 2 Doses
Mean Controls
Mean a
The overall condition of each pig was assessed and scored on a scale of 1 (poor) to 5 (very good) prior to termination. At postmortem, lungs were examined for disease pathology and scored for the presence of pneumonia from 0 (none) to 3 (severe). b Euthanased. c Score was significantly different from control group, where P0 0.01.
olar walls. Many pigs had intra-alveolar and intrabronchiolar masses of neutrophils, and peribronchiolar masses of lymphocytes. These were not associated with immunoperoxidase staining for PRV antigen, but their presence and intensity varied significantly between the groups. No evidence of PRV infection was detected in kidney and liver from any animal, and was found in only one spleen sample from a control pig (no. 21). Whereas, cerebrum, midbrain, brain stem and lung were found to be positive for the presence of PRV in a number of pigs. The assessment of histopathology and immunohistochemistry in these animals using a 0 (negative) to 4 (strong positive) rating is presented in Table 2. Control pigs were all positive for PRV lesions in the brain with scores up to 3 and lesions in the lung from between 1 and 4. Control pig 21 also scored 2 for lesions in both the spleen and draining lymph node (data not shown) and gave positive immunoperoxidase staining for the presence of PRV antigen in the brain. All pigs, which received a single dose of rPAV-gD, were positive for lesions in the brain with scores from 2–4. In addition, in pig 4, there were masses of lymphocytes in the tri-geminal ganglion and of plasma cells in the lymph node, and it was strongly positive for the presence of PRV antigen in the brain and lung. This pig developed severe clinical disease including paralysis of the hind limbs and was euthanased on day 6 p.c. In addition, 4 out of 6 pigs in the single dose group were positive for the presence of PRV lesions in the lung with scores from 1–4, and all 6 pigs showed positive immunoperoxidase staining for PRV antigen in the brain and/or lung. The pigs which received 2 doses of vaccine showed low level scores of 1– 2 for lesions in the brain with 4 pigs scoring 1 and 2 pigs scoring 2. Four of the 6 pigs had only mild lesions in the lung with scores from 1–3. However, all pigs were negative for PRV antigen in both brain and lungs.
4. Discussion An rPAV expressing the PRV gD gene was administered to pigs and the level of protection from challenge investigated. Pigs given 2 doses were protected from disease, showed no significant loss of condition and had lower levels of brain and lung pathology compared with control pigs or those given a single dose. Previously we have demonstrated complete protection of pigs from lethal challenge with CSFV following a single vaccination with a rPAV expressing the major protective antigen gp55 [2]. That recombinant virus was constructed so that expression of gp55 was driven by the PAV major late promoter (MLP) and enhanced by the inclusion of the PAV tri-partite leader (TPL) sequence up-
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Table 2 Assessment of histopathology and immunohistochemistry of pigs challenged with pseudorabiesa Vaccine group
1 Dose
Pig
3 4b 5 6 7 10
Total Mean 2 Doses
Total Mean
Midbrain
Brain stem
Lung
His
IPX
His
IPX
His
IPX
His
IPX
2 4 1 1 2 2 12 2.0
0 4 1 0 1 0 6 1.0
2 3 2 0 1 1 9 1.5
0 0 0 0 0 0 0 0
1 3 2 0 0 2 8 1.3
0 0 0 0 0 1 1 0.17
4 3 0 0 1 3 11 1.7
2 3 0 1 2 0 8 1.3
1 2 8 9 11 12
1 0 1 0 2 1 5 0.83
0 0 0 0 0 0 0 0
0 1 1 0 1 1 4 0.67
0 0 0 0 0 0 0 0
2 1 1 1 1 0 6 1.0
0 0 0 0 0 0 0 0
1 0 2 0 1 3 7 1.2
0 0 0 0 0 0 0 0
21 22 23
2 1 2 5 1.67
2 0 0 2 0.67
1 2 1 4 1.3
0 0 0 0 0
1 3 1 5 1.67
0 0 0 0 0
4 2 1 7 2.3
0 0 0 0 0
Total Mean Controls
Cerebrum
a
Following termination, brain and lung samples from each pig were examined for the presence of PRV infection apparent as lesions and the presence of viral antigen using immunoperoxidase staining. His – assessment of severity of histopathology, IPX – assessment of intensity of staining in an indirect immunoperoxidase test for PRV antigen. These were 0 – no visible lesions and no staining, 1 – mild lesion (almost certainly subclinical) or one focus or occasional cells staining, 2 – mild but widespread lesion (almost certainly subclinical) or several foci of cells staining, 3 – well developed lesions and staining, 4 – severe lesions and very widespread staining. b Euthanased.
stream of the coding region. Additionally, insertion of gp55 was made into the right hand end of the PAV genome without deletion of viral DNA sequence. In contrast, the gD gene from PRV was inserted into the E3 region of PAV3 with the subsequent deletion of approximately 600 bp of PAV3 E3 DNA [15]. It has been reported that deletion of the E3 region of human adenovirus can alter the virulence of recombinant viruses compared with wild type [16,17]. For this reason, it was important to determine whether the recombinant virus caused any adverse effects in experimental animals by monitoring the clinical reactions of pigs, when first vaccinated with rPAV-gD. No clinical signs were observed following sub-cutaneous injection, demonstrating that insertion of gD into the partially deleted E3 region did not increase the virulence of recombinant virus over that of wild type PAV3, which causes a mild transitory diarrhoea (data not shown). An important consideration concerning the use of the rPAV-gD recombinant, is that expression of gD was solely dependent upon the E3 transcriptional unit, since no additional promoter or translational enhancer elements were included upstream of the gene in this construct. Therefore, it is probable that expression of gD may have been at a lower level than was achieved with gp55 in the previously tested rPAV-gp55 recombinant,
where expression of gp55 was driven by the MLP [2]. However, expression of gD in tissue culture cells infected with rPAV-gD was demonstrated using a monoclonal antibody specific to gD [15], and following vaccination of pigs with rPAV-gD, neutralizing antibodies against gD were detected, demonstrating expression of this gene in vivo. However, levels of antibody were found to vary between the two groups of vaccinated pigs, with only 2 of 6 pigs given a single dose having PRV neutralizing antibody titres above background prior to challenge, but with 5 of 6 pigs given 2 doses having detectable levels. This demonstrates an enhancement of the induction of PRV neutralizing antibody levels as a result of the booster dose. The 4 pigs in the single dose group that did not develop detectable antibody titres before challenge, presented with varying degrees of clinical signs p.c., with one pig showing severe disease resulting in euthanasia. However, one pig in the 2 dose group also failed to develop a detectable neutralizing antibody response before challenge, but did not develop disease, and remained healthy until termination. Thus, there appears to be no absolute correlation between the presence of detectable neutralizing antibodies to gD prior to challenge and protection from disease, suggesting that protection from PRV induced by live rPAV expressing gD may be dependent upon cell mediated immunity as well as neutralizing antibody.
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Following challenge, control pigs had elevated temperatures for 6 days before returning to normal levels and presented a variety of clinical signs. Pigs in the two vaccinated groups also had elevated temperatures, but for only 2–3 days, demonstrating a marked effect of the vaccine in reducing the period of fever. There was also a clear difference in the development and severity of clinical signs between the two groups of vaccinates, with those receiving only a single dose presenting with more severe disease and loss of condition compared with those given 2 doses. Pigs given a booster dose of vaccine maintained their condition immediately p.c. and continued to thrive until termination. Postmortem examinations also revealed that control pigs and those given a single dose of vaccine suffered severe pneumonia, whereas pigs given 2 doses did not. The severe pneumonias were not necessarily correlated with viral antigen detection and may have been associated with secondary bacterial infection or enhancement of previous mycoplasmal infection. There was a total absence of detectable antigen in any of the pigs given the booster dose of vaccine. No viraemia was found in any of the challenged pigs, including controls, but virus was re-isolated from nasal swabs taken from animals in every group, suggesting that virus shedding may still occur even in pigs protected from clinical disease, following two doses of vaccine. It is possible that a third dose of vaccine or the administration of a rPAV expressing the gD gene under the control of a stronger promoter such as the PAV MLP may help reduce or prevent virus shedding. In summary, we have demonstrated successful delivery of the gD gene of PRV to pigs utilizing a live rPAV vector. Different levels of protection based upon production of neutralizing antibodies, ability to thrive, presence of pneumonia and detection of viral induced pathology in the brains and lungs of challenged animals were observed. In the examination of brain and lung pathology, it seems that a single dose of vaccine may have exacerbated disease compared with controls. Two doses of recombinant vaccine protected pigs, with animals having increased neutralizing antibody levels before challenge, suffering no significant loss of condition, and showing decreased disease pathology as compared with controls and single dose groups. It is quite probable that protection elicited by this recombinant could be improved by increasing the level of expression of the gD gene by coupling it to the PAV MLP and TPL sequences, as were used in the CSFV protection experiments [2]. Further work is needed to determine the extent of protection offered by such a recombinant and examine the occurrence and duration of virus shedding, if any, in nasal secretions.
Acknowledgements The authors would like to thank Don Carlson for
expert assistance with animal experiments and Megan Braun for the preparation of histological and immunohistochemical slides.
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