Vaccination of pigs with replication-defective adenovirus vectored vaccines: the example of pseudorabies

veterinary microbiology ELSEVIER

VeterinaryMicrobiology42 (1994) 205-215

Vaccination of pigs with replication-defective adenovirus vectored vaccines: the example of pseudorabies M. Adam a, M.F. Lepottier b, M. Eloit a,. " Unit~ de Gdndtique Mol~culaire, Gdndtique Virale, INRA, Ecole Nationale Vdtdrinaire d'Alfort, 94704 Maisons Alfort, France b CNEVA, Laboratoire Central de Recherches Avicole et Porcine, Station de Pathologie Porcine, BP 53, 22440 Ploufragan, France

Received 25 January 1994; accepted 12 April 1994

Abstract The efficacy of a recombinant human adenovirus type 5 expressing gD, one of the immunogenic glycoprotein of pseudorabies virus, was tested in pigs. Due to the deletion of the Ela gene, the recombinant virus is unable to replicate in non transcomplementing cells but is capable of eliciting an immune response against gp50 after inoculation into animals. The virus was formulated in a water/ oil/water emulsion, a strategy previously shown to enhance the immune response against the virusinduced gp50. Pigs of 18-25 kg were vaccinated twice and the recombinant virus was not isolated from nasal and rectal swabs taken after each injection of the vaccine. High levels of neutralizing antibodies were induced by the vaccination. Protection against a severe challenge was effective, as measured by growth performance (dG = 1.73), and reduction of the time of excretion of the challenge strain (mean time: 4.4 days for the vaccinated and 7.9 days for the control pigs). These results show that non replicating adenoviruses are able to induce a strong protective immune response against foreign genes in pigs, which may be of general interest for the design of pig vaccines. Keywords: Pseudorabies;Pig; Adenovirus;Adenovirusvector; Vaccine

I. Introduction Aujesky's disease is a major pathogen for swine which causes severe financial losses for the pig industry. The etiologic agent, pseudorabies virus (PRV) is an alphaherpesvirus which shares common properties with herpes simplex virus. * Correspondingauthor. 0378-1135/94/$07.00 © 1994ElsevierScienceB.V. All rights reserved SSD10378-1135(94)00065-5

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Some of the envelope glycoproteins have been shown to be involved in the immunogenicity of PRV. Among them, gp50 seems to be a major immunogen of the virus. Neutralizing antibodies against gp50 have been described (Wathen and Wathen, 1984; Eloit et al., 1988; Coe and Mengeling, 1990), and some of them were shown to block the penetration of the virus into cells (Eloit et al., 1990a). Vaccination of mice or pigs with purified or recombinant gp50 conferred a protection to the animals (Ishii et al., 1988; Marchioli et al., 1987). Vaccination with recombinant viral vectors harbouring the gp50 gene was effective (Eloit et al., 1990b; Marchioli et al., 1987; Riviere et al., 1992). A gp50- PRV strain was recently developed and induced a significant protective immune response in pigs, which was lower than that of the parental strain (Heffner et al., 1993 ). Nevertheless, comparison of antibody responses against the major immunogenic glycoproteins of PRV in vaccinated pigs showed that anti-gp50 antibodies were poorly correlated to protection, in contrast to glI and glII antibodies (Eloit et al., 1992). Finally, it is currently known that gp50 is necessary to infect target cells, but is dispensable for cell-to-cell spreading (Peeters et al., 1993; Raugh and Mettenleiter, 1991 ). In areas with high prevalence of PRV infection, vaccination is widely used to protect pigs against disease. It can also limit the spread of virulent strains between animals or herds, by decreasing the level of their excretion from vaccinated pigs (Pensaert et al., 1990; Vannier et al., 1991). Nevertheless, the use of live vaccines can lead, at least for the less attenuated strains, to dissemination of vaccine strains and, sometimes, to recombination between vaccine and/or wild strains leading to virulent phenotypes (Henderson et al., 1990; Henderson et al., 1991; Katz et al., 1990). Recombinant vectors based of type 2 (Ad2) or type 5 (Ad5) human adenoviruses have been developed either as replication competent (Prevec et al., 1989; Prevec et al., 1990; Prevec et al., 1991; Vernon et al., 1991) or as replication defective viruses (Eloit et al., 1990b; Ragot et al., 1993). While replication competent viruses demonstrated good evidence of efficacy for vaccination purposes, biosafety of such constructions remains to be assessed. On the other hand, recombinant defective viruses were constructed by deleting the E1A gene which is the first gene transcribed in the viral cycle. When E1A is lacking, the viral cycle is blocked in the early phase, and no replication of DNA occurs: no infectious particles are generated after infection of cells which do not provide the E1A gene, contributing to a high level of biosecurity. These types of viruses are currently being studied as viral vectors for gene therapy and for vaccination purposes. Some properties of these viruses are very promising (for a review, see Gerard and Meidell, 1993): first, adenoviruses are able to introduce foreign genes into a wide spectrum of cells including muscular, epithelial, neuronal and macrophage cells; secondly, viral DNA is maintained mainly as an episome for several months, the transduced gene being expressed during this time in such a way that a long lasting immune response can be expected; third, all mammal species which have been tested until this time including dogs, cattle (Prevec et al., 1989), pigs (this paper), mice, rabbits (Eloit et al., 1990b), chimpanzees (Ballay et al., 1987), cats and rats (unpublished results) develop an immune response against foreign genes carried by Ad5 or Ad2. Despite the safety advantages of replication defective viruses, the drawback of using such mutants is that high titres of virus must be injected into animals to elicit a strong immune response. We have recently shown that formulation in oil adjuvants of Ad-gp50, a replication defective Ad5 expressing the pseudorabies gp50 gene, dramatically increases its efficacy in

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mice (Ganne et al., 1993 ). We demonstrate in this paper that such an oil formulated vaccine confers to the target species, pigs, a high level of protection against challenge, measured by the clinical status of the animals and the level of excretion of the challenge strain.

2. Materials and methods 2.1. Viruses and cells

The construction of Ad-gp50 was previously described (Eloit et al., 1990b). Briefly, the gp50 gene of the NIA3 PRV strain under the control of the major late promoter (MLP) of Ad2 was inserted at the very left end of Ad5, into the E 1A gene. Virus stocks were amplified in 293 cells, a cell line which provides phenotypic complementation of the E1A gene (Graham et al., 1977). Virus stocks were titrated in the same cell line, and titres were expressed as TCIDso. 2.2. Vaccine preparation

Vaccine preparation and formulation in Montanide ISA 206 (Seppic), a water/oil/water emulsion, was described (Ganne et al., 1993). Infected cells were collected in medium ( a b o u t 10 6 cells per ml), disrupted by three cycles of freezing-thawing and clarified by centrifugation. As gp50 is mainly membrane associated in Ad-gp50 infected cells (Eloit et al., 1990b), virus stocks should contain very low amounts of gp50. In fact, no detectable gp50 could be found in viral stocks by western-blotting, with an upper limit of detection of 600 ng/ dose calibrated with purified glycoproteins from PRV of known concentration. Each vaccine dose contained 10 9.8 TCIDso of Ad-gp50 in a final volume of 2 ml. 2.3. Pig experiments

Sixteen SPF pigs, 18-25 kg at the time of the first injection of vaccine, were used. Eight pigs were kept as non-vaccinated controls (group A) and 8 pigs were vaccinated (group B). Pigs were vaccinated intramuscularly twice in the neck behind the ear, respectively 35 and 14 days before the challenge. Pigs were challenged with 2 ml in each nostril of the 75V 19 strain of PRV, which titrated 106.9 TCIDso/ml at the day of challenge. The challenge strain had previously been passaged only three times in pig kidney primary cells. Clinical signs and rectal temperatures were recorded daily and each pig was weighed weekly. Clinical protection was mainly assessed by the duration of hyperthermia and by growth performances. An index (DG) was calculated (Stellmann et al., 1989) to compare the growth of the pigs from the two groups in the first week after the challenge, as already reported (Vannier et al., 1991 ). Pigs were euthanised and necropsied three weeks after the challenge. 2.4. Isolation of Ad-gp5O from vaccinated animals

Excretion of Ad-gp50 from each vaccinated pig was checked: nasal and rectal swabs were taken during a 6 day period after each injection and two passages were made in 293 cells.

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2.5. Isolation of PRV from nasal swabs

Nasal swabs were taken from vaccinated and control pigs from DO (before the challenge) to D 10, and PRV was titrated as described (Vannier et al., 1991 ). Titres were expressed in TCIDso per 100 mg of mucus. 2.6. Titration of antibodies

For titration of PRV antibodies, a micro-neutralisation test using a one hour contact between serum and virus without adding complement was used as described (Vannier et al., 1991). Antibodies against Ad5 at the time of the challenge were titrated by ELISA. Micro plates (Linbro EIA microtitration) were coated with Ad5 purified by banding twice in CsC1, and 50 ng of virus diluted in 0.1 M Tris pH 9.6, NaC10.15 M, EDTA 0.01 M were used per well. Sera were serially diluted in dilution buffer (PBS pH 7.2, 0.05% Tween 20, 0.5% gelatin) and incubated 30 mn at 37°C. After three washes with distilled water plus 0.05% Tween 20, bound antibodies were revealed with protein A conjugated with biotin (Amersham) and streptavidin-peroxidase (Amersham) diluted in the same dilution buffer as the sera. The reaction was developed as previously described (Eloit et al., 1992). Titres were expressed by the last dilution which gave an absorbency greater than twice the average absorbency of the sera from the non-vaccinated pigs. Antibodies against gp50 were also titrated at the time of the challenge by ELISA by the same protocol, using purified envelope proteins of PRV as antigen (Eloit et al., 1990b).

3. Results 3.1. Vaccination with Ad-gp5O induces high levels of neutralizing antibodies

Neutralizing antibodies appeared between 15 and 21 days after the first injection (Fig. 1). At the 21st day, 6/8 pigs showed antibodies with neutralising titres between 2 and 16. After the second injection made 21 days after the first injection, all animals showed neutralising antibodies, with titres between 8 and 64 at the time of challenge. At the time of the challenge, vaccinated pigs showed ELISA titres against gp50 ranging from 1024 to 4096 (geometric mean: 2430). At the same day, the same animals possessed antibodies against Ad5 ranging from 1024 to 16384 (geometric mean: 2048). Non-vaccinated pigs were free of anti-Ad5 and anti-gp50 antibodies ( < 4). 3.2. Safety of the vaccine

Growth of the pigs after vaccination is depicted in Fig. 2. The mean weight at the time of the vaccination was 22 kg and 21.8 kg, respectively for control and vaccinated pigs. At the time of the challenge (DO), the mean dally weight gain was 940 g for the controls and 780 g for the vaccinates. So the growth of the vaccinated pigs was slower than that of the control pigs, and the mean weight of the animals at the date of the challenge was different (55.9 kg for the controls and 49.06 kg for the vaccinates). Nevertheless, no hyperthermia

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Fig. 2. Growthperformancesin the controlgroup (A) and the vaccinatedgroup (B). Pigswerechallengedat DO. Minimum, median and maximumvalues are depicted. 3.4. Clinical protection of pigs against the challenge All control pigs were severely affected and showed fever, prostration, anorexia, vomiting and dyspnea. Hyperthermia lasted for one week after the challenge (Table 1). One of the pigs showed intense nervous symptoms at D5 and dead at D6. At D10, another pig which had still an elevated temperature, revealed symptoms of prostration, nasal discharge and ataxia and was euthanised. In contrast the vaccinated pigs showed only mild (vomiting, transient prostration and, for one pig, coughing) or no symptoms. None of the animals died. Pig weights were recorded weekly before and after the challenge (Fig. 2). The mean daily weight gain at D7 (D21 ) was - 1031 (320) g for the controls and - 9 0 (770) g for the vaccinates. The mean weight loss at D7 was respectively 7 kg and 0.6 kg for the controls and the vaccinates. The calculated DG index was 1.73.

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Table 1 Hyperthermia after challenge in the control and the vaccinated group Results are expressed as the ratio of the numberof observations above the threshold/total numberof observations

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44/53 (83%) 13/53 (24%) 23/55 (41%) 7/55 (12%)

3/26 0/23 1/32 0/32

0/24 0/24 0/32 0/32

(11%) (0%) (3%) (0%)

(0%) (0%) (0%) (0%)

Necropsy examinations made three weeks after the challenge showed no pulmonary lesions in the vaccinated group, while one control pig demonstrated lesions o f pneumonia.

3.5. Reduction of the excretion of the challenge strain from vaccinated pigs Viral excretion o f PRV after the challenge was followed by taking nasal swabs from each pig. Results are summarized in Fig. 3. Excretion mean curves peaked at D3 or D4, respectively for the control and the vaccinated group, but reached the same m a x i m u m titre. In the 8A 7-

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control group, all animals excreted the virus at D7, and 3/8 were still positive at the end of the experiment (D10). In clear contrast, most of the vaccinated pigs did not shed the virus after D5, and all but one were shedding none or only small amounts of the virus (below 10 TCIDso per 100 mg mucus) after D7. The mean excretion time was 4.4 days for the vaccinated pigs and 7.9 days for the control group.

4. Discussion Defective adenovirus type 5, after amplification of virus stocks in 293 cells, are able to introduce foreign genes in a wide variety of cells, either in vitro or in vivo. It has been shown previously that such viruses are able to induce immune responses after inoculation in species that are permissive for the wild type virus (B allay et al., 1987; Ragot et al., 1993). Moreover, inoculation of adenovirus (wild type or E 1A - ) in non-permissive species like the mouse, led to the same results (Prevec et al., 1989; Eloit et al., 1990b). Different modes of administration are effective, including the subcutaneous (Ganne et al., 1993), intramuscular (Ballay et al., 1987), intranasal (Eloit et al., 1990b) or oral routes (Natuk et al., 1993). Recombinant adenovirus are able, not only to elicit antibodies against the foreign gene product, but also to induce cytotoxic cells (CTL) (Jacobs, 1993), and, finally, to protect against challenge (Eloit et al., 1990b; Ganne et al., 1993; Jacobs, 1993; Ragot et al., 1993). While a few models of viral diseases have already been studied, defective adenoviruses have still not been tested for a disease in its natural host, for which conventional live or inactivated vaccines exist. Aujeszky's disease is a good model, as many efforts have been made to develop effective vaccines, mainly with the objective of reducing shedding of wild strain virus from vaccinated pigs (Kimman, 1992). Particularly, several live strains have been oil formulated, with a clear improvement of efficacy when compared to non adjuvanted vaccines (Pensaert et al., 1990; Vannier et al., 1991 ). As we have used the same neutralization test as before (Vannier et al., 1991), and a challenge with the same strain and a higher dose than before ( 107.5 v s . 1 0 6 TCIDs0 per pig), useful comparisons can be made. Neutralizing titres after vaccination, obtained with a test using a one hour contact between serum and virus, can be considered as very high compared with those of conventional live vaccines, and comparable to those obtained with adjuvanted inactivated vaccines. Comparison with previously published results (Vannier et al., 1991 ) show that neutralizing antibody titres 14 days after the second injection were superior or similar to those obtained with nonadjuvanted live vaccines and to those of some oil formulated vaccines. Two oil formulated live strains which were used previously gave clearly better antibody responses. Protection against challenge, evaluated through growth performances, was effective but, in general, lower than that of previously tested live vaccines. Finally, the duration of shedding of the challenge strain was reduced in the same proportion previously noticed for oil formulated live vaccines, but, in contrast to these, the peak of excretion was not different to that of the control group. Results concerning protection and excretion can be in part explain by the more severe challenge which was used in the present study, and were clearly demonstrative of a good level of protection. Moreover, these results are probably underestimated because the growth performances before the challenge were lower in the vaccinated

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group than in the control one. The origin of the difference of growth between the two groups before challenge is unclear, and can be related to local pain due to the adjuvant. It is also important to keep in mind that such comparison of efficacy is made with conventional vaccines which express all immunogenic glycoproteins of PRV. On the contrary Ad-gp50 expresses only one of the glycoproteins of PRV. The use of adjuvants to enhance immune responses against live virus is rather unusual. While this approach has been successfully used for PRV vaccines, mechanisms of action are poorly understood. We have previously shown that oil formulation was also effective on Ad-gp50 which had been purified by banding twice in caesium chloride to avoid carrying gp50 from substrate cells, demonstrating that the adjuvant enhances the immune response against the virus-induced gp50. Nevertheless, small amounts of gp50 were present in the non-purified vaccine, with a concentration which was demonstrated to be unable to elicit alone a strong immune response and to protect mice. These very low amounts of free gp50 enhanced the immune response by comparison to that induced by the purified virus (Ganne et al., 1993). This can also be the case for oil formulated conventional live vaccines, which are not purified. On the other hand, pigs injected twice with 107 cells infected with a baculovirus expressing gp50, which were formulated in the same adjuvant as in the present experiment developed higher titres of neutralizing antibodies than those described in this paper; nevertheless, they were less protected against the same kind of challenge (A. Jestin, personnal communication, 1993). Taken together, these data show that oil formulation of the adenovirus-induced gp50 plays a pivotal role in the protection. Experiments with Adgp50 and other recently constructed adenoviruses, expressing either the/3-galactosidase or the luciferase gene, are currently in progress in our laboratory to better understand the mechanisms of action of oil formulation of recombinant adenoviruses. As Ad-gp50 showed evidence of efficacy similar to but still lower than some oil formulated live vaccines, it is tentative to speculate that simultaneous administration to pigs of vectored adenoviruses expressing other immunogenic proteins will enhance efficacy. Strong synergistic effects between PRV glycoproteins have already been demonstrated in other recombinant viruses (Riviere et al., 1992). If such an approach demonstrates its potency, this can lead to the achievement of highly effective but safe vaccines.

Acknowledgements We thank C. Houdayer, R. Cariolet, B. Beaurepaire (CNEVA-LCRAP, Ploufragan) for technical assistance and for management of the animals, and P. Vannier (CNEVA-LCRAP, Ploufragan) for helpful comments of the manuscript.

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