Evaluation of a recombinant vaccinia virus containing pseudorabies (PR) virus glycoprotein genes gp50, gII, and gIII as a PR vaccine for pigs.

Abstract

Pigs vaccinated twice intramuscularly with a highly attenuated strain of vaccinia virus (NYVAC) containing gene inserts for pseudorabies virus (PRV) glycoproteins gp50, gII, and gIII produced neutralizing antibodies for PRV and were less clinically affected than were nonvaccinated pigs following oronasal exposure to virulent PRV. Also, following oronasal exposure to virulent PRV the duration of virulent virus shedding by pigs that had been vaccinated intramuscularly with the recombinant virus was statistically less (p < 0.05) than that of nonvaccinated pigs and like that of pigs vaccinated twice intramuscularly with inactivated PR vaccine. Intramuscular vaccination with the recombinant virus was compatible with the most commonly used differential diagnostic tests, namely those based on PRV glycoproteins gX and gI. Serum antibodies for these glycoproteins were absent from the sera of all pigs before and after vaccination with recombinant virus; whereas, they were present in the sera of all of the same pigs after they were exposed to virulent PRV. In contrast to the effectiveness of the recombinant virus administered intramuscularly, neither serum antibody nor clinical protection against PRV was detected when aliquots of the same recombinant virus preparation were administered either orally or intranasally. The latter finding suggests that recombinant virus replicates poorly, if at all, at these sites. If so, the dissemination of recombinant virus from vaccinated pigs to nonvaccinated pigs or other animals in contact seems unlikely.

Full text

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Arch Virol (1994) 134:259-269
_Archives
Vi rology
© Springer-Verlag t994
Printed in Austria
Evaluation of a recombinant vaccinia virus containing pseudorabies (PR)
virus glycoprotein genes gp50, glI, and glII as a PR vaccine for pigs
w. L. Mengeling, Susan L. Brockmeier, and K. M. Lager
Virology Swine Research Unit, National Animal Disease Center, USDA, Agricultural
Research Service, Ames, Iowa, U.S.A.
Accepted September 29, 1993
Summary. Pigs vaccinated twice intramuscularly with a highly attenuated
strain of vaccinia virus (NYVAC) containing gene inserts for pseudorabies virus
(PRV) glycoproteins gpS0, gII, and gIII produced neutralizing antibodies for
PRV and were less clinically affected than were nonvaccinated pigs following
oronasal exposure to virulent PRV. Also, following oronasal exposure to
virulent PRV the duration of virulent virus shedding by pigs that had been
vaccinated intramuscularly with the recombinant virus was statistically less
(p < 0.05) than that of nonvaccinated pigs and like that of pigs vaccinated twice
intramuscularly with inactivated PR vaccine. Intramuscular vaccination with
the recombinant virus was compatible with the most commonly used differential
diagnostic tests, namely those based on PRV glycoproteins gX and gI. Serum
antibodies for these glycoproteins were absent from the sera of all pigs before
and after vaccination with recombinant virus; whereas, they were present in
the sera of all of the same pigs after they were exposed to virulent PRV. In
contrast to the effectiveness of the recombinant virus administered intra-
muscularly, neither serum antibody nor clinical protection against PRV was
detected when aliquots of the same recombinant virus preparation were admin-
istered either orally or intranasally. The latter finding suggests that recombinant
virus replicates poorly, if at all, at these sites. If so, the dissemination of
recombinant virus from vaccinated pigs to nonvaccinated pigs or other animals
in contact seems unlikely.
Introduction
Pseudorabies (PR) is a contagious and sometimes fatal disease caused by a
herpesvirus, pseudorabies virus (PRV), of the subfamily Alphaherpesvirinae [1].
It affects several species of wild and domesticated animals but is most common
in pigs, which also serve as the major interepizootic reservoir and the primary
means for dissemination of the causative virus [2-4].

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260 W.L. Mengeling et al.
Presently the control of PR of pigs is based largely on the use of vaccines
that contain either the entire complement of virus-coded proteins (inactivated
and attenuated virus vaccines) [2, 5] or selected viral glycoproteins (subunit
vaccine) [6]. All are effective in reducing the clinical effects of the disease.
Moreover, attenuated and inactivated vaccines prepared from deletion-mutant
strains of PRV, as well as subunit vaccines, can be used in conjunction with
appropriate diagnostic tests to identify pigs exposed to virulent, wild-type PRV.
Differentiation depends on the detection of antibodies for one or more viral
proteins associated only with virulent virus. The most commonly used tests
are based on glycoprotein I (gI) [7] or glycoprotein X (gX) [8] which are, respec-
tively, structural and nonstructural glycoproteins of PRV [1]. The primary
indication for differential testing is when vaccines are used in conjunction with
PR eradication programs (such as those now in progress in the United States
and Europe) because, regardless of vaccination history, all pigs that survive
exposure to virulent PRV are likely to become latently-infected carriers with
the potential for virus reactivation and shedding [9].
Recently another type of vaccine was developd by using a highly attenuated
strain (NYVAC) of vaccinia virus as a vector for selected genes of PRV [10].
Three constructs of this vector have already been tested for their immunogeni-
city in pigs [11]. They differed in that each contained a single gene for one of
the three glycoproteins of PRV that are believed to be important immunogens
involved in clinical protection, namely, gp50, gII, and gIII. Although the
gp50-vectored vaccine appeared to be the most effective, all three stimulated
a humoral, virus-neutralizing (VN) antibody response and some degree of
clinical protection.
In the study reported here we vaccinated pigs with the NYVAC vector
containing PRV genes for gp50, gII, and gIII to test the combined effect of
these antigens on immunity. We also compared the effects of administering the
recombinant vaccine intramuscularly (IM), intranasally, or orally, and investi-
gated the compatibility of NYVAC-vectored vaccination with two of the
differential diagnostic tests (gI and gX) most often used in conjunction with
the PR eradication program in the United States.
Materials and methods
Experimental design
Thirty-two 5-week-old pigs were purchased from a commercial herd that was free of
infection with PRV. On the day they were delivered to our facility they were weighed, ear
tagged, and allocated to five treatment groups (eight pigs for group I and six pigs/group
for groups 2 through 5), so that each group was comprised of pigs of about the same
weight distribution and, thus, the same average weight. Pigs of group 1 were kept as
nonvaccinated controls, whereas all pigs of groups 2 through 5 were vaccinated twice at
a 28-day interval. Pigs of group 2 were vaccinated (2 ml/dose) IM with inactivated PRV.
Pigs of groups 3 through 5 were vaccinated (2 ml/dose) IM (group 3), intranasally (group 4),
or orally (group 5) with the NYVAC strain of vaccinia vector containing genes for PRV
glycoproteins gp50, gII, and gIII. Infectivity titers and additional details relative to these

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Vaccinia-virus-vectored vaccine for pseudorabies 261
vaccines are presented in a subsequent section on "Vaccine preparation." The treatment
schedule was: day- 14 to day 0, all pigs were acclimated to our isolation facilities; day 0,
first vaccination; day 28, second vaccination; day 56, challenge-exposure, i.e., challenge of
immunity by oronasal exposure of each pig to 2 ml of virulent PRV containing 2.8 x 108
plaque forming units (PFU) of virus per ml; day 84, euthanasia by an overdose of barbiturate
and necropsy. A blood sample was collected from each pig on days 0, 28, 56, and 84, and
the corresponding sera were titered for VN antibodies for PRV. Sera collected on days 0
and 56 were also titered for VN antibodies for vaccinia virus (both the parent NYVAC
strain and the recombinant virus). Selected sera also were tested for antibodies for gI and
gX. All pigs were weighed at the time they were received on day- 14 and on days 0, 3, 5,
10, 14, 56, 59, 63, 66, 70, and 74. Body (rectal) temperatures were recorded for all pigs on
days 0, 3, 5, 10, 14, 56, 57 through 67, 70, and 74. Oropharyngeal swabs were collected
from all pigs on days 56, 58, 60, 63, 66, 70, and 74. Swabs were tested for the presence and
titer of PRV shedding.
Isolation and observation of pigs
Pigs were kept in four isolation rooms throughout the study. The arrangement from the
time they were received until 1 day after they were exposed to virulent PRV (an interval
of about 10 weeks, see above) was: room 1, six pigs of group 1 and all pigs of group 2;
room 2, two pigs of group 1 and all pigs of group 3; rooms 3 and 4, all pigs of groups 4
and 5, respectively. On the first day after exposure to virulent PRV, two pigs of group t,
room t, were moved to room 3, and two pigs of group 1, room 1, were moved to room
4, so that each isolation room thereafter contained two pigs of group 1 and all pigs of one
other treatment group. All pigs were observed daily throughout the experiment.
Virus neutralizing antibody titrations
Titers of serum VN antibodies for PRV and vaccinia virus were determined by methods
previously described [12].
Cell cultures
Bovine embryonic spleen (BESp) cells [13] were used to test oropharyngeal swabs for PRV
and a porcine kidney (PK-15) established cell line was used for all other laboratory
procedures involving PRV. Primary chicken embryo fibroblasts were used to propagate
the NYVAC strain of vaccinia virus for vaccine production and BESp cells were used for
all other laboratory procedures involving vaccinia virus. All cells were grown as stationary
monolayer cultures in medium that consisted of Eagle's minimal essential medium (EMEM)
supplemented with 0.5~ lactalbumin hydrolysate, gentamicin sulfate (50 gg/ml) and either
5~o fetal bovine serum (FBS) for PK-15 cells or 10~o FBS for all other cell types.
Pseudorabies viruses
The attenuated, commercially available, Tolvid strain of PRV containing deletions in its
thymidine kinase (TK) and gX genes [14] was used to prepare inactivated PRV vaccine.
The virulent Indiana-Funkhauser strain of PRV was used to challenge immunity of pigs
and to test sera for VN activity.

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262 W.L. Mengeling et al.
Vaccinia virus
The NYVAC strain of vaccinia virus was derived by genetically engineering the deletion
of putative virulence and host range genes from the Copenhagen strain of vaccinia virus
[10]. The NYVAC strain containing insertions of PRV genes for gp50, gII, and gIII was
used to prepare vaccinia-vectored PRV vaccine. The NYVAC strain, both with and without
PRV gene insertions, was used to test sera for VN activity.
Vaccine preparation
Inactivated PRV vaccine used in this study was prepared and evaluated previously [9].
The infectivity titer prior to inactivation was 7 x 108 PFU/ml. It was used undiluted except
for the addition of 0.! volume of aluminium hydroxide as an adjuvant.
The vaccinia-vectored PRV vaccine virus was prepared by infecting stationary, monolayer
cultures of primary chicken embryo fibroblast at a multiplicity of infection of about 0.1.
Two days later, when virus-induced cytopathic effects (CPE) were extensive, the cultures
were frozen (-80°C) and thawed twice and the cell culture medium was clarified by
centrifugation (500 x g, 15 min). The clarified cell culture medium was used undiluted as
vaccine. Its infectivity titer was 10 v median cell culture infective doses (CCIDso)/ml.
Expression of all three PRV gtycoproteins by the vaccinia vector was confirmed by
propagating an aliquot of the vaccine in BESp and porcine lung celt cultures on coverslips
in Leighton tubes and testing replicate, infected cultures by indirect immunofluorescence
microscopy against primary sera specific for gII, gIII, or gp50.
Differential diagnostic tests
Selected sera were tested for antibodies for PRV glycoproteins gI and gX using test kits
licensed for use in the PR eradication program in the United States (HerdChek: Anti-PRV-gI
and HerdChek: Anti-PRV-gpX, IDEXX Laboratories Inc., One IDEXX Drive, Westbrook,
ME, U.S.A.). All sera collected from pigs on days 56 and 84 were first tested for antibodies
for gI and gX in a regulatory laboratory where such testing is routinely performed
(Diagnostic Virology Laboratory, National Veterinary Services Laboratories, USDA,
Animal and Plant Health Inspection Service, Ames, IA, U.S.A.). After obtaining the results
of these tests we retested all of the same sera plus sera collected from the same pigs on
day 0 for the presence of antibodies for gX.
Oropharyngeal swab samples
Procedures for collection of oropharyngeal swab samples, for detection and titration of
associated PRV, and for statistical analyses of virus shedding among groups were as
previously described [9].
Results
After the first vaccination (days 0 through 28)
All pigs remained clinically normal during the 28 days immediately following
the first vaccination. Sera collected from pigs just before the first vaccination
(day 0) were free of VN antibody for both PRV and vaccinia virus. Those
collected from pigs of groups 1, 3, 4, and 5 at 28 days after the first vaccination
also were free of VN antibody for PRV, whereas all of those collected at the
same time from pigs of group 2 had VN antibody (Table 1).

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Vaccinia-virus-vectored vaccine for pseudorabies 263
Table 1. Virus neutralizing (VN) antibody titer.s for pseudorabies virus
(PRV) in sera obtained from pigs before and after vaccination and
challenge exposure
Group Vaccine Route of
vaccination
Days after 1st vaccination"
0 28 56 84
1
2
3
4
5
none
inactivated PRV intramuscular <2
vaccinia vector c intramuscular <2
vaccinia vector c oral
vaccinia vectoff intranasal
.... <2 b <2 <2 9.75
8.5 9.7 13.3
7.3 13.3
< 2
<2
<2
< 2
<2
< 2
<2
10.8
10.3
"Swine were vaccinated on days 0 and 28, and exposed to virulent PRV
on day 56
b Geometric mean titer of VN antibodies
c Recombinant vaccinia virus containing PRV glycoprotein genes gp50,
gII, and gIII
After the second vaccination (days 28 through 56)
All pigs remained clinically normal during the 28 days immediately following
the second vaccination. Sera collected from pigs of groups 1, 4, and 5 just before
their immunity was challenged at day 56 were free of VN antibody for PRV.
Conversely, sera collected at the same time from pigs of group 3 had VN
antibody for PRV, and those collected from pigs of group 2 had an increase
in VN antibody for PRV (Table 1). Sera collected from pigs of group 3 on day 56
(i.e. 28 days after the second dose of vaccinia-vectored vaccine) appeared to have
a low level of inhibitory activity for vaccinia virus in that the vaccinia-virus-
induced CPE usually developed slower at low serum dilutions than it did when
the virus was reacted with the same dilutions of prevaccination (day 0) sera from
the same pigs. However, the inhibitory activity was never sufficient to block
infectivity in all of the cultures even at the lowest dilution (1:2) of serum tested.
There was no apparent difference between the reactivity of sera with the NYVAC
strain of vaccinia virus either with or without PRV gene insertions.
After challenge of immunity with virulent PRV (days 56 through 84)
All pigs of all treatment groups had clinical signs (inappetence, listlessness,
respiratory distress, and in some cases incoordination) following oronasal
exposure to virulent PRV. In general, the severity of signs paralleled the increase
in body temperatures (Fig. 1). Pigs of groups 2 and 3 appeared less severely
affected than did those of groups t, 4, and 5. One pig of group 4 died 7 days
after challenge-exposure, whereas all others recovered. Pigs of groups 2 and
3 gained body weight during the 7 days immediately following challenge-
exposure, whereas those of groups 1, 4, and 5 lost weight during the same
interval (Fig. 2). All groups had gained weight by the 14th day after challenge-