Immunogenicity of an E1-deleted recombinant human adenovirus against rabies by different routes of administration.
ABSTRACT The immunogenic properties of an E1-deleted, human adenovirus type 5 (Ad5) vaccine virus with activity against rabies were examined in mice, foxes and dogs using different routes of administration. NMRI mice received 10(5.8), 10(5.3), 10(4.3), 10(3.3) and 10(2.3) TCID(50) by peroral or intramuscular (i.m.) administration. Furthermore, six mice received 10(5.8) TCID(50) intracerebrally (i.c.). The construct elicited marked seroconversion in mice after oral administration. Immunoreactivity in mice was even more pronounced i.m. and i.c. After direct oral administration (10(8.0) TCID(50)) in foxes, six of eight animals developed rabies virus-neutralizing antibodies (VNA). All foxes immunized by direct injection (10(7.7) TCID(50)) in the membrane of the jejunum were shown to seroconvert. Pre-existing immunity against canine adenovirus did not hinder the development of rabies VNA after oral application of the construct (10(8.0) TCID(50)). Fox cubs (24-29 days old) born from rabies-immune vixens were shown to develop very high levels of rabies VNA after i.m. administration (10(8.0) TCID(50)), indicating that the immunogenicity of the construct could surpass maternally transferred immunity. In dogs, the construct (10(8.0) TCID(50)) induced a very strong immune response after i.m. administration. However, no immune response was detectable in dogs after direct oral administration (10(8.3) TCID(50)) or after endoscopic deposition in the smaller intestine (10(8.0) TCID(50)). Hence, it must be concluded that the construct is not suitable for oral vaccination of dogs against rabies.
- SourceAvailable from: Sai V Vemula[Show abstract] [Hide abstract]
ABSTRACT: The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines.PLoS ONE 01/2012; 7(3):e33428. · 3.73 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Our previous studies indicated that recombinant rabies viruses (rRABV) expressing chemokines or cytokines (including GM-CSF) could enhance the immunogenicity by recruiting and/or activating dendritic cells (DC). In this study, bacterial flagellin was cloned into the RABV genome and recombinant virus LBNSE-Flagellin was rescued. To compare the immunogenicity of LBNSE-Flagellin with recombinant virus expressing GMCSF (LBNSE-GMCSF), mice were immunized with each of these rRABVs by intramuscular (i.m.) or oral route. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. The i.m.-immunized mice were bled at three weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with 50 LD50 challenge virus standard (CVS-24). Orally immunized mice were boosted after three weeks and then bled and challenged one week after the booster immunization. It was found that both LBNSE-GMCSF and LBNSE-Flagellin recruited/activated more DCs and B cells in the periphery, stimulated higher levels of adaptive immune responses (VNA), and protected more mice against challenge infection than the parent virus LBNSE in both the i.m. and the orally immunized groups. Together, these studies suggest that recombinant RABV expressing GM-CSF or flagellin are more immunogenic than the parent virus in both i.m. and oral immunizations.PLoS ONE 01/2013; 8(5):e63384. · 3.73 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: An Irkut virus (IRKV) was recently isolated from a bat in China. The protective ability of rabies biologics available in the Chinese market and experimental biologics were assessed in a hamster model against the rabies virus (RABV) and IRKV via pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) experiments. The results demonstrated that a single dose of rabies vaccine did not induce adequate protection against IRKV. However, routine PrEP with three doses of a vaccine induced complete protection against IRKV. Higher doses of rabies immune globulins and interferon α were required during PEP to protect hamsters against IRKV compared to RABV. Experimental recombinant vaccines containing IRKV glycoprotein induced more reliable protection against IRKV than against RABV. Those may be explained by limited cross-neutralization of these viruses (confirmed via in-vitro tests) in conjunction with antigenic distance between RABV and IRKV. These results indicate that development and evaluation of new biologics for PrEP and PEP are required to ensure sufficient protection against IRKV in China and other territories where this virus is present.Journal of clinical microbiology 08/2013; · 4.16 Impact Factor
Journal of General Virology (2001), 82, 2191–2197.
Printed in Great Britain
Immunogenicity of an E1-deleted recombinant human
adenovirus against rabies by different routes of administration
Ad Vos,1Andreas Neubert,1Elke Pommerening,1Thomas Mu ? ller,2Leopold Do ? hner,3
Larissa Neubert1and Kenneth Hughes4
1Impfstoffwerk Dessau-Tornau GmbH, PO Box 214, 06855 Rosslau, Germany
2Institute for Epidemiological Diagnostics, Federal Research Centre for Virus Diseases of Animals, Seestrasse 55, 16868 Wusterhausen,
3Micromun GmbH, Walther-Rathenau Strasse 49a, 17489 Greifswald, Germany
4Microbix Biosystems Inc., 341 Bering Avenue, Toronto, Ontario, Canada M8Z 3A8
The immunogenic properties of an E1-deleted, human adenovirus type 5 (Ad5) vaccine virus with
activity against rabies were examined in mice, foxes and dogs using different routes of
administration. NMRI mice received 105?8, 105?3, 104?3, 103?3and 102?3TCID50by peroral or
intramuscular (i.m.) administration. Furthermore, six mice received 105?8TCID50intracerebrally
(i.c.). The construct elicited marked seroconversion in mice after oral administration. Immuno-
reactivity in mice was even more pronounced i.m. and i.c. After direct oral administration (108?0
immunized by direct injection (107?7TCID50) in the membrane of the jejunum were shown to
seroconvert. Pre-existing immunity against canine adenovirus did not hinder the development of
rabies VNA after oral application of the construct (108?0TCID50). Fox cubs (24–29 days old) born
from rabies-immune vixens were shown to develop very high levels of rabies VNA after i.m.
administration (108?0TCID50), indicating that the immunogenicity of the construct could surpass
maternally transferred immunity. In dogs, the construct (108?0TCID50) induced a very strong
immune response after i.m. administration. However, no immune response was detectable in dogs
(108?0TCID50). Hence, it must be concluded that the construct is not suitable for oral vaccination of
dogs against rabies.
Oral vaccination of foxes (Vulpes vulpes) against rabies has
been developed as a preferred method to control, and
eventually to eradicate, vulpine rabies (Sto ? hr & Meslin,
1996; Mu ? ller & Schlu ? ter, 1998). This approach has also been
those that cannot be vaccinated by the parenteral route.
However, due to the close relationship between dogs and
humans, more stringent safety conditions are required for oral
vaccination of dogsagainst rabies (Wandeler, 2000). Presently,
no commercially available oral rabies vaccine is completely
without risk. In order to address this issue, new vaccine
Author for correspondence: Ad Vos.
Fax ?49 34901 885797. e-mail ad.vos?idt-direct.de
candidates have been under investigation. One such family of
candidates are recombinant human adenovirus type 5 (Ad5)
vectors that express the highly immunogenic rabies gly-
coproteinG (RG) (Yaroshet al., 1996; Xiang et al., 1996; Wang
et al., 1997). One of these prototype vaccines has already been
shown to induce protective immunity in animals upon
challenge with rabies virus (Prevec et al., 1990; Charlton et al.,
1992). However, the potential for in vivo replication of this E3-
inserted recombinant adenovirus is an undesirable attribute
from a safety perspective. In response to this shortcoming, a
replication-deficient, E1-deleted, human Ad5 vector has been
designated human Ad5 HCMV–intron–ERA, was evaluated
for its ability to induce an immune response by different routes
of administration in mice (Mus musculus), foxes and dogs. It
was also determined whether pre-existing canine adenovirus
0001-7747 ? 2001 SGM
A. Vos and othersA. Vos and others
(CAV)-specific immunity inhibited the development of rabies
Finally, the ability of the construct to surpass maternally
transferred immunity against rabies in young animals was
examined. The variability of construct immunogenicity in
terms of target species and route of administration was
? Vaccine virus. The vaccine virus was produced by a homologous
Biosystems Inc., Toronto, Canada). Briefly, cDNA for the ERA (Evelyn–
Rochitniki–Abelseth) strain RG was inserted into the XbaI site of plasmid
pCA13 just downstream of the forward-orientated HCMV immediate-
early promoter and ahead of the SV40 polyadenylation signal. This
generated plasmid pCA13-RG.
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 GCAAGGTA-
GG CTGAGCACCG TGGCGGGCGG CAGCGGGTG GCGGTC-
GGGGT TGTTTCTGGC GGAGGTGCTG CTGATGATGTAATT-
AAAGTA GGCGGTCTTG AGACGGCGGA TGGTCGAGGT GA-
GGTGTGGC 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 orien-
tation was confirmed by sequencing.
In order to produce recombinant human Ad5 bearing the HCMV–
intron–ERA 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
HCMV–intron–ERA 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. HCMV–intron–ERA virus was
enumerated by TCID??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 immuno-
globulin 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 inacti-
vated 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 recep-
tive Madin–Darby canine kidney (MDCK) cells for virus replication.
Four days later, CAV2-neutralizing antibody titres were evaluated ac-
cording to Spearman–Ka? rber, based on the CPE. A titre of 1:4 was
? ELISA. The objective of these tests was to detect both general
for human Ad5. For detection of the former, highly purified human Ad5
hexon protein was used. This protein was isolated by DEAE chromato-
graphy and crystallization (Do ? hner & Hudemann, 1972). The hexon
proteinis the most abundant adenovirusprotein (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. Student’s paired t-test was used to detect possible
intraspecific significance in absorbance differences between different
blood samples (Zo ? fel, 1988).
Study 1. Mice [peroral (p.o.), intramuscular (i.m.) and
Three groups of six mice were given 0?02 ml human Ad5
HCMV–intron–ERA (10??? TCID??) p.o., i.m. or i.c. Animals
that received the construct i.c. were anaesthetized by intra-
peritoneal (i.p.) injection with a 1:10 dilution of ketamine
hydrochloride in saline. The animals vaccinated p.o. drank the
construct voluntarily from a syringe. During a subsequent trial
in mice, four groups of 10 animals each received 0?02 ml of the
construct using different concentrations (10???, 10???, 10??? and
10??? TCID??). Five animals of each group received the
construct orally, the other five by the parenteral route (i.m.).
Recombinant adenovirus against rabiesRecombinant adenovirus against rabies
Table 1. Rabies VNA titres of mice inoculated with human Ad5 HCMV–intron–ERA by different routes of administration
Five or six animals were inoculated with the doses shown. Titres for individual animals are given in IU?ml, as determined by RFFIT. (–), No rabies VNA detectable (?0?5 IU?ml); ,
269?2, 226?3, 226?3, 269?3, 320?1, 269?2
67?3, 56?6, 56?6, 80?0, 190?3, 80?0
20?0, 20?0, 40?0, (–), 80?0
33?6, 5?0, 10?0, 8?4, Died*
(–), 56?5, 4?2, (–), 0?9
(–), (–), (–), (–), (–)
10?0, 7?1, 10?0, (–), 67?3, 5?9
47?6, 7?1, (–), 67?3, (–)
(–), 10?0, (–), (–), (–)
(–), (–), (–), (–), (–)
(–), (–), (–), (–), (–)
* Animal died 2 days post-vaccination; cause of death unknown, but unrelated to rabies.
Table 2. Rabies VNA titres of foxes inoculated orally with
108?33TCID50human Ad5 HCMV–intron–ERA
Titres are given in IU?ml, as determined by RFFIT. (–), No rabies
VNA detectable (?0?5 IU?ml); , not done.
Fox B0B1 B2 B3 B4
This time, the construct was applied orally with a perfusion
needle in the posterior part of the oral cavity of the animals.
For this purpose, the mice were anaesthetized in an ether jar
prior to administration of the construct. All mice were bled by
retro-orbital puncture and subsequently euthanized 55 or 60
days post-immunization. Blood samples were examined for
serum antibody titres against rabies by using RFFIT and
antibodies against the human Ad5 fibre and hexon proteins
The immune responses against rabies of mice inoculated
with human Ad5 HCMV–intron–ERA vaccine are shown in
Table 1. The highest levels of rabies VNA were observed in
themice inoculatedi.c.Theimmune responsein mice receiving
the human Ad5 HCMV–intron–ERA 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).
Study 2. Foxes (p.o.)
Eight juvenile foxes received 10??? TCID??human Ad5
HCMV–intron–ERA by direct oral instillation. Blood samples
werecollected 0 (B0), 21 (B1), 35 (B2), 70 (B3) and 95 (B4) days
post-vaccination. All blood samples were examined for the
presence of rabies virus- and CAV-neutralizing antibodies by
RFFIT. Detection of antibodies against the human Ad5 fibre
and hexon proteins was also performed by ELISA.
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-
neutralizingantibodies at any timeduring the study. The mean
absorbances against the fibre protein of serum samples B0 and
A. Vos and othersA. Vos and others
Table 3. CAV2 VNA titres of foxes pre-immunized with
Titres were determined by RFFIT. (–), No CAV2 VNA detectable
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
?2?43, d.f.?7, P?0?05).
Study 3. Foxes (intrajejunal)
Five cubs, 8 weeks old and born from rabies-naı?ve vixens,
received by injection 0?5 ml human Ad5 HCMV–intron–ERA
(10??? TCID??) in the membrane of the jejunum following
surgical exposure (laparotomy). For this purpose, the animals
were sedated with a ketamine–xylazine (10%?2%) solution.
Blood samples were collected 0 (B0), 21 (B1) and 49 (B2) days
post-vaccinationand examinedfor the presenceof rabies VNA
All five foxes had detectable levels of rabies VNA. Sera
GMT for the vaccinated foxes were 32?2 (B1) and 11?9 (B2)
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 10??? TCID??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 10??? TCID??human Ad5
HCMV–intron–ERA vaccine by direct oral instillation. Blood
samples were taken 28 (B2) and 55 (B3) days after adminis-
tration 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 adminis-
tration 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
Study 5. Fox cubs (i.m.)
In order to investigate whether maternally transferred
immunitytorabies viruswouldimpairthe immuneresponse to
RG upon vaccination with the human Ad5 HCMV–intron–
ERA construct, 13 cubs born from four rabies-immune vixens
were given the construct i.m. The vixens had been vaccinated
orally with SAD (Street Alabama Dufferin) B19 just before
mating. SAD B19 is a live-modified rabies virus vaccine, used
extensively for oral vaccination of wildlife (Schneider & Cox,
(mean 24?3 days), were bled (B0) and subsequently injected
i.m. with 10??? TCID??human Ad5 HCMV–intron–ERA.
Blood samples were collected 14 (B1), 21 (B2), 28 (B3) and 47
(B4) days post-vaccination and examined for the presence of
rabies VNA (RFFIT).
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 10??? TCID??human Ad5 HCMV–
intron–ERA 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
10??? TCID??, 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
route. The other four orally vaccinated dogs showed no
detectable immune response against these proteins (Fig. 1).
The four dogs that remained seronegative after secondary
oral administration received 10??? TCID??
HCMV–intron–ERA by direct intestinal instillation (endo-
scopic deposition) in the duodenum. The animals were
anaesthetized with a ketamine–xylazine (10%?2%) solution;
pre-medication was with atropine–diazepam. Blood samples
Recombinant adenovirus against rabiesRecombinant adenovirus against rabies
Fig. 1. Absorbances of serum samples (B0, B2–B5) from dogs inoculated
with 108?33and 108?0TCID50human Ad5 HCMV–intron–ERA during
primary and secondary vaccination against the human Ad5 hexon (a) and
fibre (b) proteins, as determined by ELISA. Samples were included from
dogs that received the construct p.o. during both primary and secondary
vaccinations (?) and from dogs that received the construct respectively
p.o. and i.m. during primary and secondary vaccinations (?).
Blood samples were examined by RFFIT for the presence of
rabies (B0–B8) and CAV (B0–B5) VNA. Antibody levels
against the human Ad5 fibre and hexon proteins were also
determined by ELISA (B0–B5).
VNA after endoscopic deposition of the HCMV–intron–ERA
construct in the duodenum.
An effective yet safe rabies vaccine is needed for the oral
immunization of a variety of wild animals, as well as the oral
vaccination of (stray) dogs. Human adenoviruses have been
studied intensively as virus vectors for vaccine delivery and
gene therapy applications (for reviews, see Klonjkowski et al.,
1999; Russell, 2000), and have been shown to induce
protective responses after oral administration (Fooks et al.,
1998). The adenovirus genome has been well characterized,
vaccine vectors. This application is supported further by the
widehostrangeof human adenoviruses and the stabilityof the
by routine culture techniques.
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 HCMV–
intron–ERA 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 recom-
binant, 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 HCMV–intron–ERA. 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.
Thereasonfor theabsence ofRCAinthe HCMV–intron–ERA
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
frequencies in the order of 10−?–10−? after five serial passages
(M. Moore and K. Hughes, unpublished).
In contrast to studies with similar replication-defective
humanAd5 constructs (Prevec et al.,1990; Xiang &Ertl,1999),
mice developed serum antibody to rabies upon oral adminis-
tration of human Ad5 HCMV–intron–ERA. 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-naı?ve foxes, six of eight animals did
seroconvert after direct oral administration. However, the
immune response was lower than that in foxes vaccinated
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
HCMV–intron–ERAconstruct toelicitan immune response to
rabies. We have shown previously that cubs whelped by
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 (Mu ? ller et al., 2001). The rabies-specific VNA
response to the human Ad5 HCMV–intron–ERA 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 naı?ve
vixens (Mu ? ller et al., 2001). This demonstrates that maternal