Impfstoffwerk Dessau-Tornau GmbH, PO Box 214, 06855 Rosslau, Germany1
Institute for Epidemiological Diagnostics, Federal Research Centre for Virus Diseases of Animals, Seestrasse 55, 16868 Wusterhausen, Germany2
Micromun GmbH, Walther-Rathenau Strasse 49a, 17489 Greifswald, Germany3
Microbix Biosystems Inc., 341 Bering Avenue, Toronto, Ontario, CanadaM8Z 3A84
Author for correspondence: Ad Vos. Fax +49 34901 885797. e-mail ad.vos{at}idt-direct.de
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Abstract |
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Introduction |
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Methods |
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A specific intron sequence, present in plasmid pMLPsp1a, as described by Berkner & Sharp (1985) , is known to increase protein expression in multiple systems. It was cloned into pCA13-RG, downstream of the promoter region and before the RG gene. The intron sequence in pMLPsp1a contains the 5' splice-donor site from the Ad2 major late promoter (MLP) fused to the 3' acceptor site from an immunoglobulin light-chain gene. The primary sequence of the intron is as follows: 5' GAATTCAAAG GCGTCTAACC AGTCACAGTC GCAAGGTAG CTGAGCACCG TGGCGGGCGG CAGCGGGTG GCGGTCGGGGT TGTTTCTGGC GGAGGTGCTG CTGATGATGTAATTAAAGTA GGCGGTCTTG AGACGGCGGA TGGTCGAGGT GAGGTGTGGC AGGCTTGAGA TCTGGCCATA CACTTGAGTG ACAATGACAT CCACTTTGCC TTTCTCTCCA CAGGTGTCCA CTCCCAGGTC CAACCGGATC C 3'.
The intron was extracted from pMLPsp1a by PCR under standard conditions. The PCR product was restricted with EcoRI and ligated into similarly cut pCA13-RG to generate pCA13(int)-RG. Nucleotide orientation was confirmed by sequencing.
In order to produce recombinant human Ad5 bearing the HCMVintronERA cassette, pCA13(int)-RG was co-transfected with plasmid pBHGE3, which contains the right-hand end of Ad5 (rightward of E1), into 293 cells. 293 cells are a human kidney cell line that has been transformed with left-end regions of Ad5, including E1. Homologous recombination between pBHGE3 and pCA13(int)-RG results in the production of a viral genome that will replicate in 293 cells and produce progeny virus. 293 cells provide helper E1 function. Individual virus isolates are selected by plaque purification on immobilized 293 cell monolayers.
HCMVintronERA virus was propagated routinely in suspension culture using 293N3S as the host cell line. For cultivation, the suspension cells were placed in spinner flasks containing growth medium (MEM Eagle, Joklik modification, Sigma) with 10% foetal calf serum. Virus was harvested 3 days after infection of cells. HCMVintronERA virus was enumerated by TCID50 on 293N3S cells.
Animals.
All animals were without detectable levels of rabies virus-neutralizing antibodies (VNA) prior to vaccination, with the exception of the fox cubs born from rabies-immune vixens. Foxes were marked individually with electronic identifiers and juvenile specific-pathogen-free dogs (beagles) were identified individually by a tattoo (ear). All animal tests were conducted at the Experimental Animal Facility of Impfstoffwerk Dessau-Tornau GmbH (IDT). Animal experimentation was performed according to the German Animal Welfare Act of 25 May 1998. The experimental design of the tests, as required, was approved by the appropriate German authorities.
Rapid fluorescence focus inhibition test (RFFIT) assays
Rabies virus.
Prior to testing, sera were heat-inactivated at 56 °C for 30 min and then centrifuged at 926 g for 5 min. Serum samples were evaluated for rabies VNA by the RFFIT as described by Smith et al. (1973) , with the modifications described by Cox & Schneider (1976)
. The rabies VNA titres were converted to international units (IU). The international standard immunoglobulin (2nd human rabies immunoglobulin preparation, National Institute for Standards and Control, Potters Bar, UK), adjusted to 0·5 IU/ml, served as a positive control for these experiments. A level of 0·5 IU/ml is as an arbitrarily defined threshold indicative of protection against rabies infection (Anonymous, 1978
) and is used here as the threshold for positivity.
CAV2.
A simple inhibition test (Mayr et al., 1977 ) was used to test sera for VNA against CAV2. Firstly, the serum samples were inactivated at 56 °C for 30 min. A virus suspension with a known titre was added to each serum dilution and the mixture was incubated at 37 °C for 60 min. This mixture was then inoculated on monolayers of receptive MadinDarby canine kidney (MDCK) cells for virus replication. Four days later, CAV2-neutralizing antibody titres were evaluated according to SpearmanKärber, based on the CPE. A titre of 1:4 was considered negative.
ELISA.
The objective of these tests was to detect both general adenovirus-reactive serum antibodies and those with particular specificity for human Ad5. For detection of the former, highly purified human Ad5 hexon protein was used. This protein was isolated by DEAE chromatography and crystallization (Döhner & Hudemann, 1972 ). The hexon protein is the most abundant adenovirus protein (Zabner et al., 1997
), and antibodies directed against this protein could also be the result of earlier CAV infection. The Ad5-specific ELISA was conducted with highly purified human Ad5 fibre antigen. Fibre antigen was isolated by anion exchange and immunoaffinity chromatography.
The ELISA was performed under standard conditions using hexon or fibre antigens, at 1 µg/ml, for coating microtitre plates. Peroxidase-labelled protein A was used as the indicator reagent. Human antibodies were used as the positive control. Dog, fox and human antibodies react similarly with protein A (Yamamoto et al., 1985 ). The absorbances of cut-off values at 450/620 nm for specific antibodies were in the range of 0·5. Adenoviruses are ubiquitous; hence, it is to be expected that animals have circulating antibodies against adenoviruses. Serum samples from mice were diluted 1:10 to allow the detection of low reactivity between IgG and protein A.
Statistics.
Students paired t-test was used to detect possible intraspecific significance in absorbance differences between different blood samples (Zöfel, 1988 ).
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Results |
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The immune responses against rabies of mice inoculated with human Ad5 HCMVintronERA vaccine are shown in Table 1. The highest levels of rabies VNA were observed in the mice inoculated i.c. The immune response in mice receiving the human Ad5 HCMVintronERA construct orally was inferior to mice immunized i.m. with the same dose. It was also observed that the seroconversion rate was dose-dependent. With the exception of three (of six) mice vaccinated i.c., none of the animals showed high levels of antibodies against the fibre and hexon protein (data not shown).
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Six of the eight foxes seroconverted (threshold 0·5 IU/ml). The geometric mean titres (GMT) of the sera of immunized foxes were 2·75 (B1), 2·13 (B2), 1·33 (B3) and 1·48 (B4) IU/ml (Table 2). None of the animals had detectable levels of CAV-neutralizing antibodies at any time during the study. The mean absorbances against the fibre protein of serum samples B0 and B2 were 0·296±0·071 and 0·415±0·112, respectively. For the hexon protein, the following mean absorbances for B0 and B2 were obtained: 0·779±0·361 and 1·439±0·724. The levels of antibodies against the hexon and fibre proteins of human Ad5 as indicated by absorbances were both significantly higher for B2 than for B0 (paired t-test, Thexon=-3·16, Tfibre=2·43, d.f.=7, P <0·05).
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All five foxes had detectable levels of rabies VNA. Sera GMT for the vaccinated foxes were 32·2 (B1) and 11·9 (B2) IU/ml.
Study 4. Foxes (p.o.) vaccinated against CAV2
The effect of pre-existing CAV-specific immunity on the immune response against rabies, induced by the construct, was investigated by administering 107·8 TCID50 CAV2 antigen (produced at IDT) i.m. to four adult foxes after a blood sample (B0) had been taken. Twenty-nine days later, the foxes were bled (B1) and subsequently received 108·0 TCID50 human Ad5 HCMVintronERA vaccine by direct oral instillation. Blood samples were taken 28 (B2) and 55 (B3) days after administration of the construct. The blood samples were examined for the presence of rabies and CAV VNA by RFFIT.
All four foxes developed detectable levels of CAV2 VNA after vaccination with CAV2 antigen and prior to administration of the construct (B0 and B1) (Table 3). All animals pre-immunized with CAV2 developed rabies VNA upon oral immunization with the construct. The GMT were 3·61 and 3·27 IU/ml at 28 (B2) and 55 (B3) days post-administration. These results indicate that antibodies to CAV2 did not inhibit the immune response to rabies after vaccination with the construct.
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The rabies VNA titres of the four vixens at the time of vaccination of the cubs were 1·81, 11·02, 11·88 and 5·12 IU/ml. All cubs born from these rabies-immune vixens developed rabies VNA upon immunization with the construct. The GMT of the sera of the cubs were 0·38 (B0), 31·08 (B1), 108·73 (B2) and 155·26 (B3) IU/ml.
Study 6. Dogs [p.o., i.m. and gastrointestinal (g.i.)]
Six dogs received 108·3 TCID50 human Ad5 HCMVintronERA by direct oral instillation. Blood samples were collected 0 (B0), 21 (B1), 35 (B2), and 118 (B3) days post-vaccination. After the first oral instillation, none of the animals seroconverted. Hence, vaccination was repeated using a different lot of vaccine virus. Vaccination was repeated with 108·0 TCID50, four animals by the oral route and two by parenteral (i.m.) administration. Dogs were bled 28 (B4) and 55 (B5) days after this second vaccination attempt.
The dogs that received the secondary vaccination by the oral route did not develop detectable levels of rabies VNA. Only the i.m.-inoculated animals seroconverted, with high levels of VNA [dog 3160, 99·00 (B4) and 160·89 (B5) IU/ml; dog 3161, 18·80 (B4) and 41·67 (B5) IU/ml]. Dogs 3160 and 3161 also showed a clear immune response against the hexon and fibre antigens after vaccination by the parenteral route. The other four orally vaccinated dogs showed no detectable immune response against these proteins (Fig. 1).
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None of the four dogs developed detectable levels of rabies VNA after endoscopic deposition of the HCMVintronERA construct in the duodenum.
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Discussion |
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The strong rabies immune response induced by replication-defective adenovirus recombinants that express RG and the absence of many of the safety risks inherent to multiplying agents led us to test the E1-deleted human Ad5 HCMVintronERA as a candidate for oral vaccination of carnivores against rabies. This replication-defective human Ad5 can only be propagated in cells that express the E1-region proteins exogenously and thereby complement the deletion in the viral genome. This virus is unable to replicate in cells that do not complement the deleted E1 region, but it is still able to infect these cells and to induce the synthesis of very high levels of foreign protein (RG). Hence, the potential spread of recombinant, replication-competent adenovirus (RCA) by horizontal transmission from vaccinated to unvaccinated animals, as mentioned by Hammond et al. (2000) , is not a concern with human Ad5 HCMVintronERA. However, the E1 deletion may not block the expression of virus genes completely following in vivo administration (Von Seggern & Nemerow, 1999
). The absence of RCA after serial passaging in 293 and 293N3S cells attests further to the safety of this virus vector. The reason for the absence of RCA in the HCMVintronERA virus preparation is not understood. All primary sequence elements required for homologous recombination have been confirmed as present by sequencing analysis. However, the absence of RCA is clearly linked to the presence of the intron moiety, as other recombinant rabies vaccines containing the HCMV promoter without the intron regularly produce RCA at frequencies in the order of 10-610-5 after five serial passages (M. Moore and K. Hughes, unpublished).
In contrast to studies with similar replication-defective human Ad5 constructs (Prevec et al., 1990 ; Xiang & Ertl, 1999
), mice developed serum antibody to rabies upon oral administration of human Ad5 HCMVintronERA. However, the immune response of mice vaccinated orally was inferior to that of animals vaccinated i.m. or i.c., and was clearly dose-dependent. In rabies-naïve foxes, six of eight animals did seroconvert after direct oral administration. However, the immune response was lower than that in foxes vaccinated orally with the live-modified virus vaccines SAD B19 and SAD P5/88, showing a sub-optimal immunization process (Neubert et al., 2001
; Schuster et al., 2001
).
As exposure to CAV is relatively common in wild and domestic canids (Garcelon et al., 1992 ; Laurenson et al., 1997
; Gese et al., 1997
; Truyen et al., 1998
; Cypher et al., 1998
; Spencer et al., 1999
), it is to be expected that many animals have developed VNA against CAV. However, pre-immunity to CAV did not impair the ability of the human Ad5 HCMVintronERA construct to elicit an immune response to rabies. We have shown previously that cubs whelped by rabies-immune vixens developed an impaired immune response upon oral immunization with the highly efficacious vaccine virus SAD B19. This inhibition of the immune response outlasted the presence of detectable levels of maternal antibodies (Müller et al., 2001
). The rabies-specific VNA response to the human Ad5 HCMVintronERA construct, after i.m. administration in cubs whelped by rabies-immune vixens, was significantly higher than the response elicited to the oral rabies virus vaccine SAD B19 in cubs born from naïve vixens (Müller et al., 2001
). This demonstrates that maternal immunity to rabies does not effect the immune response to RG, as presented by the human Ad5 recombinant vaccine. Therefore, the construct is highly suitable for neonatal immunization of dogs. Many human rabies cases are caused by bites from rabid puppies that are too young to be eligible for vaccination.
Dogs that received the human Ad5 HCMVintronERA construct by direct oral administration did not develop detectable levels of VNA against rabies. Furthermore, the construct did not elicit an immune response after g.i. immunization by endoscopic deposition. This supports the results of Papp et al. (1997) , who could not observe an immune response after g.i. immunization of cotton rats (Sigmodon hispidus) with another replication-defective human Ad5 expressing glycoprotein D of bovine herpesvirus-1. The relative inefficiency of mucosal delivery for adenovirus infection is striking, since adenoviruses naturally infect mucosal tissues and human epidemiological data suggest frequent adenovirus infection. Indeed, human Ad5 is a common pathogen that infects most humans at an early age (Xiang et al., 1996
; Wang et al., 1997
; Walters et al., 1999
). A likely explanation is that wild-type Ad5 infection requires a large initial inoculum (Walters et al., 1999
). This explains the relative inefficiency of replication-defective adenovirus as virus vectors for vaccine delivery when compared with similar recombinant RCA.
Entry of human adenoviruses into cells is a stepwise process. After binding to the fibre receptor (CAR; coxsackievirus/adenovirus receptor), interaction of penton base with integrins facilitates internalization via receptor-mediated endocytosis (Dmitriev et al., 2000 ). It has been suggested that the adenovirus fibre receptors are polarized to the basolateral plasma membrane of the epithelium, and the lack of fibre binding at the apical surface appears to be the rate-limiting step that explains insufficient adenovirus-mediated gene transfer (Walters et al., 1999
). According to Zabner et al. (1997)
, intentional injury of the epithelium increased the efficiency of gene transfer. However, in our study, the lumen epithelia in the intestines of three dogs were damaged (gastritis) and the animals still did not respond to the vaccine delivery. This suggests that the cells of the intestinal smooth muscle lack the appropriate cell-surface receptors for adenovirus attachment (Dmitriev et al., 2000
; Schmidt et al., 2000
). Surprisingly, direct injection of the construct in the membrane of the jejunum in foxes elicited a strong immune response. It seems that the inefficiency of recombinant replication-defective adenovirus vaccines in eliciting an immune response after oral delivery is partially species-dependent (Monteil et al., 2000
). Although human Ad5 HCMVintronERA was able to induce an immune response in mice after oral delivery, dogs could not be vaccinated against rabies by this route and foxes developed only relatively low VNA titres. It must, therefore, be concluded that, despite high levels of RG expression in vitro and significant VNA production by i.m. administration, the human Ad5 HCMVintronERA construct is not a suitable candidate for oral vaccination of the tested carnivores against rabies.
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Acknowledgments |
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References |
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Received 22 March 2001;
accepted 15 May 2001.