Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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RECOMBINANT EQUINE HERPESVIRUS TYPE 1 (EHV-1 ) COMPRISING A DYSFUNCTIONAL GENE
71 REGION
AND USE THEREOF AS A VACCINE
The present invention relates to a viral vaccine containing
an attenuated EHV-1 virus comprising a gene deletion in the
genome thereof, uses thereof and methods of treating EHV-1
related disease. In particular, the invention relates to a viral
vaccine composition for use against Equine herpesvirus type 1
( EHV-1 ) .
EHV-1 is a member of the subfamily alphaherpesvirinae and
is a significant viral pathogen of horses. Clinical problems
caused by EHV-1 include respiratory disease, abortion and
neurological disorders (Bryans J.T., and Allen, G.P., Kluwer
Academic Publishers, Norwell MA, 1989). As such, EHV-1 is
responsible for significant economic losses within the equine
industry.
The EHV-1 genome is a linear double-stranded DNA molecule
of approximately 150 kbp in size which can be divided into two
covalently linked components: the long and short regions. The
long region consists of an unique sequence (UL) flanked by a
small inverted repeat (IRS and TRL). The short region comprises
an unique sequence (Us) flanked by a large inverted repeat (IRS
and TRs) .
EHV-1 occurs as pathogenic and non-pathogenic strains and
recently, the complete DNA sequence of a pathogenic strain, Ab4,
has been determined and the sequence has been deposited with the
GenBank Library under Accession No. M 86664 (Telford, E.A.R. et
al., Virology 189, pp. 304-316 (1990)).
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The genome is 150,223 by in size and contains 81 open
reading frames predicted to encode polypeptides. The sizes of
its components are UL, 112,870 bp; TRL/IR~, 32 bp; U5, 11,861 bp;
and IRS/TR~, 12,714 bp. Interestingly, there are five genes, 1,
2, 67, 71, and 75, which have no homologues in any of the
herpesviruses sequenced to date; i.e. they are unique to EHV-1.
Each of the genes 1, 2, 67, 71 and 75 is believed to encode
a protein, however, the function of the individual proteins is
unclear. Recently it has been demonstrated that the EHV-1 gene
71 product is involved in adsorption/penetration of virus and
egress of virus from infected cell nuclei (Sun Y. et al. , Journal
of General Virology 77 pp. 493-500 (1996)).
The prior art does not teach or suggest the use of EHV-1
gene 71 deletion mutants comprising a dysfunctional gene 71
region in the manufacture and use of vaccines against EHV-1
related disease.
Control by vaccination of EHV-1 infection has been a long-
sought goal. Current EHV-1 vaccines comprise chemically
inactivated virus vaccines and modified live virus vaccines.
Inactivated vaccines generally induce a low level of immunity and
require additional immunisations and are expensive to produce.
The use of such vaccines carries with it the risk that some
infectious viral particles may survive the inactivation process
and cause disease after administration to the animal.
In general, attenuated live virus vaccines are preferred
because they evoke a longer-lasting immune response (often both
humoral and cellular) and are easier to produce. Live attenuated
EHV-1 vaccines are available which are based on live EHV-1 virus
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attenuated by serial passage of virulent strains in tissue
culture. However, serial passaging of virulent strains can give
rise to uncontrolled mutations of the viral genome, resulting in
a population of virus particles heterogeneous in their virulence
and immunising properties. It is also known that such EHV-1
attenuated live virus vaccines can revert to virulence resulting
in disease of the inoculated animals and the possible spread of
pathogen to other animals.
The present inventors have now identified a suitable strain
of live EHV-1 mutant virus comprising a dysfunctional region of
the EHV-1 genome located within the short unique region thereof,
which mutant may be used in a live EHV-1 vaccine formulation.
Specifically, the inventors have found that EHV-1 mutants
dysfunctional for production of a protein encoded by gene 71 can
be used in a live EHV-1 vaccine formulation. Such mutants are
shown to be substantially less virulent than wild type EHV-1
viruses. Furthermore, gene 71 has been found to be non-essential
for EHV-1 growth in cell culture (Sun Y. and Brown S.M. , Virology
199 pp. 448-452 (1994)). The inventors have also found that EHV-
1 viruses comprising dysfunctional gene 71 regions of their
genome are immunogenic. Such viruses are indicated for use as
components in vaccine formulations or therapeutic compositions
against EHV-1 infection. Accordingly, it is with EHV-1 viruses
w comprising a dysfunctional region located in the gene 71 protein
coding region, and in particular between nucleotides 129,096 and
131,489 of the native genome which the present invention is
concerned.
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Statement of Invention
A first aspect of the present invention provides a vaccine
formulation comprising a live recombinant EHV-1 virus modified
so as to contain a dysfunctional gene 71 region located within
the US region of the virus genome and a pharmaceutically
acceptable carrier.
A "dysfunctional gene 71 region" is one which is
substantially incapable of coding for the native polypeptide or
a functional equivalent. Thus, a "dysfunctional gene 71 regi9n"
means that the gene 71 region has been modified by deletion,
insertion or substitution (or other change in the DNA sequence
such as by rearrangement) such that the gene 71 region does not
express a native EHV-1 gene 71 polypeptide or a functionally
equivalent product thereof. It is known that EHV-1 gene 71
encodes a 797 amino acid polypeptide and that the peptide is an
O-linked 192 kDa glycoprotein (Sun, Y. et al., Journal of General
Virology 75, pp. 3117-3126 (1994)). Thus, vaccine formulations
comprising modified EHV-1 viruses of the invention may include
viruses modified in one or more ways via recombinant DNA
technology. Examples of the types of modifications which may be
made include:
(i) A deletion of the entire gene 71 from the genome of an
EHV-1 wild type virus. For example, a deletion of the nucleotide
sequence from the wild type EHV-1 genome between about nucleotide
129,096 to about nucleotide 131,489:
(ii) A deletion of a portion of gene 71 from the genome of
an EHV-1 wild type virus. A "portion of the gene 71" means a
deletion which is sufficient to render any polypeptide encoded
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by the gene 71 deletion mutant and expressed thereby
substantially incapable of a physiological activity similar to
that of the native polypeptide produced by wild type EHV-1. The
deletion may be between 50% and 1000 of the nucleotide sequence
located between about nucleotides 129,096 and 131,489 of the wild
type EHV-1 genome. The deletion may be from 70% to 1000 of the
gene 71 nucleotide sequence, or the deletion may be from about
70 o to 90% of the gene 71 nucleotide sequence, for example, about
80% of the gene 71 nucleotide sequence.
(iii) The deletion of the or a portion of gene 71 as
described in {i) and (ii) above will leave a "gap" in the EHV-1
genome corresponding to the gene 71 open reading frame (ORF) or
a portion thereof . A suitable gene or genes may be inserted into
the "gap" such as a marker gene. Suitable marker genes include
but are not restricted to enzyme marker genes, for example the
lac-Z gene from E.coli, antibiotic marker genes such as
hygromycin, neomycin and the like. Such marker genes are
commonly employed in the art. Generally, marker genes, if any,
which may be employed in a gene 71 deletion mutant of the
invention should be such so as to not cause substantial
deleterious or long lasting side-effects to a recipient animal.
In a preferment, the "gap" made by the deletion of the or
a portion of the gene 71 from a wild type EHV-1 virus is not
filled with a gene insert, the cut ends of the two pieces of the
genome being ligated together using conventional recombinant DNA
technology. The skilled addressee will appreciate that the term
"deletion mutant" encompasses those situations wherein the "gap"
left by the partial or total deletion of gene 71 may be filled
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with a gene insert, for example a marker gene or nonsense
nucleotide sequence (i.e. a sequence incapable of giving rise to
a protein or polypeptide product) or those situations wherein the
gap is not filled by a heterologous or other nucleotide sequence.
In such a case, the appropriate free ends of the two pieces of
the genome are ligated together.
(iv) The deletion within the gene 71 region may comprise
a deletion of a small number of nucleotides, for example 1, 2 or
more nucleotides. Such deletions can be achieved using
recombinant DNA technology. Thus, the translational ORF can be
altered resulting in the production of a protein which lacks the
physiological function of the gene 71 native polypeptide. The
skilled addressee will also appreciate that such deletions in the
translational ORF of gene 71 may also give rise to a
dysfunctional gene 71 which is incapable of coding for a whole
polypeptide, truncated peptide or even any peptide. Such
proteins, if produced, generally lack the physiological
functionality of the protein product of a normal gene 71 ORF.
(v) Nucleotide insertions can also be made in the EH~I-1
gene 71 region using recombinant DNA technology which gives rise
to dysfunctional gene 71 polypeptides substantially incapable of
functional activity. For example, stop codons may be inserted
into the gene 71 region, resulting in the production of non-
functional fragments of the polypeptide encoded by native gene
71.
The skilled addressee will appreciate that such nucleotide
insertions can be of any length from 1 or more nucleotides to a
number of nucleotides making up, for example, nonsense nucleotide
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sequences which can have the effect of altering the translational
ORF resulting in the non-production of a polypeptide or indeed,
the production of a protein lacking the physiological function
of the gene 71 native polypeptide. The skilled addressee will
also appreciate that such insertions in the translational ORF of
gene 71 may also give rise to a dysfunctional gene 71 which is
incapable of coding for a whole polypeptide, truncated peptide
or even any peptide. Such proteins, if produced, generally lack
physiological functionality.
Naturally, the skilled addressee will appreciate that gene
71 deletions and insertions from nvn-wild type EHV-1 viruses as
outlined above are encompassed by the present invention.
In a preferment there is provided a vaccine formulation
comprising a live recombinant attenuated immunogenic EHV-1 gene
71 deletion mutant virus and a pharmaceutically acceptable
carrier.
In a second aspect of the invention there is provided a
live, recombinant EHV-1 comprising a dysfunctional gene 71 region
for use as a vaccinating agent or in a vaccine formulation.
Preferably, there is provided a live, recombinant, attenuated
immunogenic EHV-1 gene 71 deletion mutant virus for use as a
vaccinating agent or in a vaccine formulation.
The live, recombinant EHV-1 may optionally include an
inserted gene positioned at the gene 71 locus in lieu of a
substantial portion of gene 71 or the whole of gene 71.
Generally, the vaccine or vaccine formulation is not used
on non-pregnant animals because it can give rise to
abortigenesis.
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In a third aspect of the invention there is provided the use
of a live, recombinant EHV-1 virus for producing antibodies or
cell mediated immunity to EHV-1 which comprises a dysfunctional
gene 71 region located within the US region of the virus genome
for the manufacture of an EHV-1 vaccine for the prophylaxis
and/or treatment of EHV-1 infection. Preferably, there is
provided use of a live, recombinant, attenuated immunogenic EHV-1
gene 71 deletion mutant virus for the manufacture of an EHV-1
vaccine for the prophylaxis and/or treatment of EHV-1 infecti9n.
Most preferably, the use is in horses.
In a fourth aspect of the invention there is provided a
method of treating animals which comprises administering thereto
a vaccine composition comprising a live, recombinant EHV-1 virus
modified so as to contain a dysfunctional gene 71 region located
within the US region of the virus genome to animals in need
thereof. Preferably, the animals are horses. Preferably still,
the method of treating animals comprises administering a vaccine
composition comprising a recombinant, live, attenuated,
immunogenic EHV-1 gene 71 deletion mutant virus to animals in
need thereof. Naturally, the vaccine formulation may be
formulated for administration by oral dosage (e. g. as an enteric
coated tablet), by parenteral injection or otherwise.
The invention also provides a process for preparing a live
modified EHV-1 virus vaccine, which process comprises admixing
a virus according to the invention with a suitable carrier or
adjuvant.
For the preparation of a live attenuated vaccine, standard
methodology may be used.
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The mode of administration of the vaccine of the invention
may be by any suitable route which delivers an immunoprotective
amount of the virus of the invention to the subject. However,
the vaccine is preferably administered parenterally via the
intramuscular or deep subcutaneous routes. Other modes of
administration may also be employed, where desired, such as oral
administration or via other parenteral routes, i.e.,
intradermally, intranasally, or intravenously.
Generally, the vaccine will usually be presented as a
pharmaceutical formulation including a carrier or excipient, for
example an injectable carrier such as saline or apyrogenic water.
The formulation may be prepared by conventional means.
The appropriate immunoprotective and non-toxic dose of such
a vaccine can be determined readily by those skilled in the art,
i.e., the appropriate immunoprotective and non-toxic amount of
the virus contained in the vaccine of this invention may be in
the range of the effective amounts of antigen in conventional
whole virus vaccines. It will be understood, however, that the
specific dose level for any particular recipient animal will
depend upon a variety of factors including age, general health,
and sex; the time of administration; the route of administration;
synergistic effects with any other drugs being administered; and
the degree of protection being sought. Of course, the
administration can be repeated at suitable intervals if
necessary.
Embodiments of the invention will now be illustrated by way
of the following Figures and Examples.
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FiQUre 1:
Schematic representation of the sequence arrangement of EHV-
1 DNA and plasmids constructed for gene 71: deletion and
substitution. Line 1, EHV-1 genome consisting of UL and US and
inverted repeat regions (IRS and TRs). Expanded cloned fragment:
5.8-kb BamHI/EcoRI fragment in pU71 (line 2). Line 3, location
and direction of genes. Line 4, sequence arrangement of
constructed deletion and substitution plasmid pD71 of gene 71
(line 4). Gaps flanked by solid lines represent deleted regi9ns
substituted by lacZ (solid boxes). Pertinent restriction sites:
Ba, Ec, EcoRI; Ms, Sg and Bam HI.
FiQUre 2:
Genome structure of the deletion and substitution mutant.
Restriction enzyme sites within the region of the genome
encompassing gene 71 are shown. The wild-type virus genome is
represented by line 1, and the deletion and substitution by line
2. Relevant fragments generated following digestion with Smal
are shown. Fragment sizes are given in kb. Pertinent retriction
sites Ms, Sm, Sg.
Figure 3:
Virus titres far mice inoculated with Ab4p.
FicTUre 4:
Virus titres for mice inoculated with ED 71.
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Figwre 5:
Virus titres for mice inoculated with ED 71 revertant.
Figwre 6:
Mean virus titres observed in challenged mice previously
immunised with RK cell lysate.
Fiqure 7:
Mean virus titres observed in challenged mice previously
immunised with Ab4p.
Figure 8:
Mean virus titres observed in challenged mice previously
immunised with ED 71.
Standard methods are as described in "Molecular Cloning -
A Laboratory Manual", Second Edition, Sambrook J. et al. Cold
Spring Harbor Laboratory Press 1989.
EXAMPLES SECTION 1
Methods
Cells and Virus
Baby hamster kidney clone 13 (BHK-21/C13; Macpherson I &
Stoker M.G. (1962) Virology 16 pp. 147-151) were grown as
previously described (Brown et al., 1973 J. Gen. Virol. 18 pp.
32-346). EHV-1 strain Ab4 was used as the wild-type strain in
this study. Stock preparation of virus at passage 13 was made
by low multiplicity infection in equine dermal NBL-6 cells
maintained in MEM with 1% fetal calf cerum. Mutant ED71 in which
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the gene 71 ORF was removed and replaced by the E.coli lacZ gene
and the revertant Re71 in which the deletion in ED71 was restored
have been previously described (Sun Y. and Brown S.M. (1994)
Virology 199 pp. 448-452; Sun Y. et al. (1994) J. Gen. Virol. 75
pp. 3117-3126).
Purification and Quantification of Virions
The procedure used was essentially as described by Szilagyi
J.F. and Cunningham C. (1992) J. Gen. Virol. 27 pp. 661-668 and
Sun et al. (1994) supra. BHK-21/C13 monolayers in roller bottles
were infected with virus at a multiplicity of infection (m.o.i.)
of 0.01 or 5 p.f.u. per cell. At either 72 hours post-infection
(p.i.) or 20 hours p.i. the supernatant was harvested and
centrifuged at 2500 r.p.m. for 20 minutes to remove the cell
debris. Supernatant virus was pelleted for 2 hours at 12000
r.p.m, and the pellet gently resuspended in 1 ml Eagle's medium
without phenol red and laid onto a 5 to 15o Ficoll gradient
before centrifuging at 12000 r.p.m. for 2 hours at 4°C. The
virion band collected by side puncture was diluted and pelleted
at 21000 r.p.m. for 2 hours at 4°C. The virion pellet was gently
resuspended in 200 ~,1 of Eagle's medium and stored at -70°C.
Infectivity was determined by titration on BHK-21/C13 cells. The
number of particles was determined by eletron microscopy. The
specific infectivities (particle p.f.u. ratio) of the purified
mutant and wild-type virus are presented in Table 1.
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Table 1: The Specific Infectivity (particle/p.f.u. ratio) of
ED71, and Wild-Type Virus EHV-1).
Virus Particle/p.f.u.ratio* Particle/p.f.u.
ratio ~
EHV-1 101.7/1 63.8/1
ED71 1440/1 2128/1
Re71 103.5/1 107.7/1
* Virus from 4x10 BHK-21/C13 cells infected with virus at an
m.o.i. of 0.01 p.f.u. per cell and harvested at 72 h.p.i.
.~ Purified virions from 4x10A BHK-21/C13 cells infected with
virus at an m.o.i. of 5 p.f.u./cell and harvested at 20 h.p.i.
Suitable gene 71 deletion mutant viruses of the invention
were prepared according to the teaching of Sun Y. and Brown S.M.
(supra).
Example I
Briefly, to clone the fragment which contains the gene 71,
equine dermal cells (NBL-6) were infected with EHV-1 strain Ab4
(Gibson J.S. et al. Arch. Virol. 124 pp. 351-366 (1992)) at 0.1
pfu/cell and the progeny virions were purified by centrifugation
on 5-55% (w/v) sucrose gradients as described by Dumas et a1. J.
Gen. Virol. 47 pp. 233-235 {1980)). EHV-1 Ab4 genomic DNA was
extracted from the purified virions and digested with a range of
restriction enzymes. A relevant fragment, for example, the 5.8-
kb BamHI/EcoRI fragment (residues 126,517 to 132,305) was cloned
into the vector pUCl9 so that a plasmid, pU71 containing the 5.8-
kb BamHI/EcoRI fragment inserted at BamHI/EcoRI sites, was
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constructed (Fig. 1). To construct a deletion plasmid, the
cloned plasmid was digested by restriction enzymes which cut at
unique sites to remove most of the coding sequence of gene 71.
The flanking sequences were relegated with complementary
synthetic oligonucleotides containing an unique Spe1 site to
allow insertion of the lacZ gene and an upstream in-frame stop
codon to prevent synthesis of a lacZ fusion protein. The lacZ
gene on a 4.1-kb Xba1 fragment from pFJ3 (Rixon F.J. and
McLauchlan J., J. Gen Virol. 71 pp. 2931-2939 (1990)) raas
inserted into the Spe1 site. lacZ was in the same orientation
as the gene transcript. The construct could encode only a very
short polypeptide of the remaining N-terminal amino acids of the
deleted gene. In this way, deletion plasmid pD71 with a deletion
from the Msc1 to the SgraAl site (residues 129,211 - 131,022) in
pU7l, was generated (Fig. 1).
Example 2
For generation of virus mutants, 1-2 ~.g of EHV-1 Ab4 DNA was
cotransfected into BHK21/C13 cells (MacPherson I. and Stoker M.G.
Virology 16 pp. 147-151 (1962)) with varying amounts of the
linearized deletion plasmid pD7l, (0.2 to 4 ~,g, an approximately
2- to 20-fold molar excess) in the presence of carrier calf
thymus DNA using the calcium phosphate precipitation/DMSO method
described by Stow N.D., and Wilkie N.M., J. Gen. Virol. 33 pp.
447-458 (1976). The cells were incubated at 37° in Eagle's
medium containing 5a newborn calf serum. When the c.p.e. was
widespread, the virus was harvested and titrated on BHK21/CI3
cells under methylcellulose. Two days after the infection, a
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further 2 ml of methylcellulose medium containing 0.7 mg/ml X-gal
was added to each plate. Individual blue plaques were isolated
for further rounds of plaque purification. A mutant with a lacZ
substitution was isolated: ED71 with a deletion of 1811 by from
the 2393 by gene 71 ORF. The deleted region of the mutant was
confirmed by Southern blotting with a probe of the 32P-labelled
deleted sequence. The structure of the virus mutant was
confirmed by Southern blotting and restriction enzyme digestion
of 3zP-labelled viral DNA prior to the preparation of virus stork.
The restriction enzyme digestion of 32P-labelled viral DNA is
represented diagrammatically in Fig. 2. Gene 71 lies within the
3.8-kb Smal fragment of wild-type viral DNA. Deletion of gene
71 and substitution by the lacZ gene resulted in the loss of the
3.8-kb fragment and the generation of a new larger fragment of
6.2-kb. The mutant had the expected genome structure, with no
other detectable differences from wild-type viral DNA.
Example 3
Growth characteristics of the mutant in tissue culture was
also investigated. Monolayers of BHK21/C13 cells were separately
infected at a multiplicity of infection (m.o. i. ) of 5 pfu/cell
and 0.01 pfu/cell with wild-type virus and the deletion and
substitution mutant ED71. The culture was harvested and virus
was released by sonication at intervals throughout a 72 hour
period. Virus titers were measured by plaque assay and the
growth patterns were compared with those of wild-type virus. The
plaque morphology of the mutants was not obviously different from
the wild-type virus plaques.
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Mutant ED71 grew more slowly and the final yield was reduced
by about 5-fold compared with that of wild-type virus. Similar
results were seen at high multiplicity (data not shown) , although
the reduction in the yield of the ED71 mutant was less than that
at low multiplicity. To determine whether the mutant was
temperature sensitive or had a host-range phenotype, they were
grown at a high m.o.i. of 5 pfu/cell in BHK21/C13 cells at
different temperatures ( 31 ° , 37 ° , and 38 . 5 °C) and
at 37 ° C in NBL-
6, Vero, HFL, and 3T6 cells. The cultures were harvested at 24
hour post-infection and progeny virus was titrated in BHK21/C13
cells. The ED71 mutant at 38°C grew 10-fold less well than at
31° and compared to wild-type virus at 38.5°C (data not shown).
The slightly impaired growth of the ED71 mutant was apparent in
NBL-6, Vero, HFL, and 3T6 as well as in BHK21/C13 cells. Thus
it is concluded that gene 71 is nonessential for EHV-1 growth in
cell culture.
Example 4: Infection Experiments: Mortality and Clinical Signs
Materials and Methods
Virus Strains
Wild-type and mutant viruses were grown either in RK cells
at the Department of Clinical Veterinary Medicine, Cambridge or
in BHK cells at the Institute of Virology, Glasgow. Wild-type
for primary infection experiments was EHV-1 strain Ab4p. Virus
used to challenge previously immunised mice was EHV-1 strain Ab4.
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Mouse Model
Female Balb/c mice were obtained at 3-4 weeks of age (Bantin
and Kingman, UK). Mice were inoculated intranasally under
isofluorane/oxygen anaesthesia.
Tissue Culture
RK cell monolayers were cultured in Eagle s Minimum
Essential Medium (EMEM) with Earle~s Salts with loo newborn calf
serum.
Virus Titration
Tissue samples obtained from three mice per group were
homogenised using an Ultraturrax motorised homogenises. Samples
were then sonicated in an ice-cold waterbath and centrifuged at
low speed to separate cellular debris. Ten-fold serial dilutions
of the supernatant were made and 100,1 of each dilution
inoculated onto confluent monolayers of RK cells, in duplicate.
Virus was allowed to adsorb to the cell sheet for 45 minutes
before all samples were overlayed with medium containing 4%
foetal calf serum and 2o carboxymethylcellulose. Plates were
incubated at 37 °C for about 3 days and then washed in sterile
phosphate buffered saline prior to fixing and staining with
crystal violet in 20% ethanol.
Experimental Protocol
At days 1, 3 and 5 post-infection groups of three mice were
euthanased with 0.15 ml of pentobarbitone sodium (Sagatal, Rhone
Merieux), tissues removed, placed in 1 ml of virus isolation
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medium, frozen at -70°C and then titrated for virus growth.
Tissue samples taken were lung, turbinates, olfactory bulb and
trigeminal ganglia. Clinical signs were monitored in a separate
group of mice from day 0 to day 8 post-infection. Blood samples
were taken on days 8, 16, 23 and 30 post-infection for
immunological tests. A group of surviving animals were then
challenged with a dose of 5x106pfu/mouse of EHV-1 strain Ab4.
Tissue samples were taken as above and clinical signs monitored.
Mortality and clinical results are shown in Table 2. Virus titre
results are shown in Figures 3 to 8 and Tables 4(a)-4(d)
inclusive.
Mortality and Clinical Signs
TABLE 2
Virus Mortality Clinical Signs*
Ab4 77~ Severe
ED71 8~ Mild
ED71 Rev 60~ Severe
* Clinical signs observed between Day 1 and Day 8 post-
infection.
Example 5: Immunology - ELISA
The protocol of Tewari D., et al (1994) Journal of Gen.
Virol. 75 pp. 1735-1741 was followed. Results are shown in Table
3.
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TABLE 3
ELISA
Acute Phase
Virus Day 8 p.i. Day 16 p.i. Day 23 p.i. Day 30 p.i.
ur t (C) 1:25 1:125 1:125 1:125
ED71 1:25 1:125 1:125 1:625
Post Challenge
Virus Day 3 Day 5 Day g
w/t (C) 1:125 1:625 1:3125
ED71 1:625 1:625 1:3125
TABLE 4(a)
Day +1 Post Challenge
Lung ..,
MEAN RANGE No. of +ve Log~oReduc-
Mice tion MEAN
Negative 4.3 4.6 3/3 -
control 4.0
Positive 2.6 3.3 3/3 1.7
control 2.0
Gene 2.9 4.2 2/3 1.4
Deletion <0.7
71
Turbinates
Negative 4.3 4.8 3/3 -
control 3.9
Positive 3.1 3.3 3/3 1.2
control 3.0
Gene 3.0 4.3 2/3 1.3
Deletion <0.7
71
Olfactorybulb
Negative 2.3 2.5 3/3 -
Control 2.0
Positive 1.3 1.6 3/3 1.0
Control 1.0
~
I
Gene 2.0 2.2 3/3 0.3
Deletion 1.7
71
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TABLE 4(b)
Day +3 Post Challenge
Lung
MEAN RANGE No. of +ve Log,QReduc-
Mice tion MEAN
Negative 4.9 5.2 3/3 -
Control 4.6
Positive 0.9 1.0 2/3 4.0
Control <0.7
Gene 0.8 0.9 1/3 4.1
Deletion 71 <0.7
Turbinates
Negative 4.6 5.2 3/3 -
Control 4.1
Positive <0.7 <0.7 0/3 >3.9
Control <0.7
Gene <0.7 <0.7 0/3 >3.9
Deletion 71 <0.7
Olfactor bulb
Negative 1.8 2.2 3/3 -
Control 1.4
Positive 0.8 0.9 1/3 1.0
Control <0.7
Gene 0.8 0.9 1/3 1.0
Deletion 71 <0.7
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TABLE 4(c)
Day +5 Post Challenge
Lung
MEAN RANGE No. of +ve Log~QReduc-
Mice tion MEAN
Negative <0.7 <0.? 0/3 -
Control <0.7
Positive <0.7 <0.'7 0/3 -
Control <0.7
Gene <0.7 <0.7 0/3 -
Deletion 71 <0.7
Turbinates
Negative 2.9 5.2 3/3 -
Control 4.1
Positive <0.7 <0.7 0/3 >2.2
Control <0.7
Gene <0.7 <0.7 0/3 >2.2
Deletion 71 <0.7
Olfactorybulb
Negative 0.75 2.2 1/3 -
Control 1.4
Positive <0.7 0.9 0/3 <0.05
Control <0.7
Gene <0.7 0.9 0/3 <0.05
Deletion 71 <0.7
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TABLE 4(d)
Day +8 Post Challenge
Lung
MEAN RANGE No. of +ve Log,oReduc-
Mice tion MEAN
Negative <0.7 <0.7 0/3 -
Control <0.7
Positive <0.7 <0.7 0/3 -
Control <0.7
Gene <0.7 <0.7 0/3 -
Deletion 71 <0.7
Turbinates
Negative <0.7 <0.7 0/3 -
Control <0.7
Positive <0.7 <0.7 0/3 -
Control <0.7
Gene <0.7 <0.7 0/3 -
Deletion 71 <0.7
Olfactorybulb
Negative <0.7 <0.7 0/3 -
Control <0.7
Positive <0.7 <0.7 0/3 -
Control <0.7
Gene <0.7 <0.7 0/3 -
Deletion 71 <0.7
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EXAMPLES SECTION 2
1. Experimental details
The trial was performed in pony colts using 3 animals per
group and two groups, one vaccinated and one not (control group) .
The trial animals were selected on the basis that they had no or
low EHV-1 neutralising and EHV-1 complement fixing (CF)
antibodies. The experimental groups were kept in separate rooms
in isolation with filtered air in and out. Colts, 7, 15 and 20
were each vaccinated intranasally with 6.0 1og10 TCIDS~ of gene
71 deleted EHV-1 (ED71), in 2.Omls of MEM (Gibco) containing
neomycin (100~,g/ml), 2% y-irradiated foetal calf serum (FCS)
(Tissue Culture Services), giving l.Oml into each nostril.
Following vaccination both vaccinated test colts (internal
numbering 7, 15 and 20) and control colts (internal numbering 5,
8 and 16) were tested for virus replication in the upper
respiratory tract by taking nasal swabs daily for 2 weeks. All
six animals were bled at intervals and their sera tested for EHV-
1 neutralising and CF antibodies (Table 7). Intranasal challenge
infection with wild type strain AB-4 was conducted 51 days after
vaccination when colts in both groups were each given 6.0 log,
TCIDSO of AB-4 in 2.Omls of MEM medium supplemented with 2o FCS.
Following challenge the procedures performed were the same as
those after vaccination, ie. assessment of virus growth in the
upper respiratory tract (Table 5).
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TABLE 5
Experimental groups and procedures
Group Colt Vaccination Procedures afterChallenge &
No vaccination Procedures
Test 7,15,20 Intranasally All 6 colts All 6 colts
Group 6.0 log~p of
ED71 in 2.Omls(i) Nasal swabs (i) Nasal swabs
day 1-14 day 1-14
Intranasally
with 6.0 log,
TCID~ of wild
' type strain AB-4
and
(ii) Leukocyte
viraemia on
days 0,1,3,
5,7,9,11 &
13.
Control 5,8,16 None (control)
Group
2. Results
2.1 Replication of ED71 virus in the upper respiratory tract
Viruses were isolated from nasal swabs in MEM medium
supplemented as described above, following standard procedures.
Results of virus isolation from daily nasal swabs following
intranasal vaccination are given in Table 6. ED71 virus at low
titre (mostly below 3.0 log,, TCIDS~,/ml) was isolated from 2 of 3
vaccinated colts, on days 2 and 3 from colt 7, and days 1 to 5
from colt 15. No EHV-1 was recovered from control colts from
daily nasal swab samples over 14 days.
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2.2 Serological responses following vaccination
Sera were titrated fro virus neutralising (VN) and
complement fixing (CF) antibodies. Results of VN tests performed
according to the method of Thompson G.R., et al Equine Vet.
Journal Vol. 8 pp 58-65, for both post vaccination and challenge
are given in Table 7 and those for CF test performed according
to the method of Thompson et al supra (vaccination only) are
given in Table 8.
In the VN test against two different strains of EHV-1 namely
ED71 virus (parent strain AB-4) and M8 no significant differences
in titres were recorded. In the vaccinated group all three colts
were just detectably VN antibody positive at intranasal
vaccination. All three animals responded with significant (>_4-
fold rise) antibody response, Nos 7 & 20 by week four and No 15
by week two.
There was no VN antibody rise in the control animals until
after challenge. By the CF test against EHV-1 two (15 and 20)
of three colts showed a significant rise (>_4-fold rise) by week
two after vaccination; colt No 7 had high activity at vaccination
(Table 8). Control animals (5, 8 and 16) did not show
significant change in CF antibody titres.
In keeping with the virus isolation results, there was no
seroconversion in control animals indicating the absence of a
field infection or EHV-1 recrudescence.
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3. Challenge findings
3.1 Challenge virus replication in the upper respiratory tract
Virus isolation results from nasal swabs are given in Table
9. Virus at low titre (2.0 loge, TCIDSO/ml) isolated from only one
(no 7) of three colts on two occasions (day 1 and 2). This was
in marked contrast to the control colts (5, 8 and 16) from which
virus was recovered for 3 (no 5) to 5 to 6 days (Nos 8 and 16)
at much higher titres.
3.2 Viraemia due to the challenge virus
Challenge EHV-1 isolation from leukocytes is given in Table
10. There was no challenge virus detected in ED71 vaccinated
colts. In contrast all three control colts became viraemic
yielding, at peak between 12 to 200 infected leukocytes/2 x 10'
cells.
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TABLE 6
Vaccine virus replication in upper respiratory tract
Group Colt Virus
No isolated
(log,o
TCID~/ml)
from
nasal
swabs
following
intranasal
vaccination
(days)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
TEST 7 - - 2.7 s.5_ _ _ _ _ _ _ _ _ _ _
ED71
(
) 1S - 1.54.3 l.72.0 1.5_ _ _ _ _ _ _ _ _
20 - _ _ _ _ _ _ _ _ _ _ _ _ _ _
CONTROL 5 - - - - - - - - - - - _ _ _ _
a denotes no EHV-1 isolated in equine dermal cells from
4x200,1 of the lowest (10'') dilution of the nasal swab
titration.
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TABLE 7
Virus neutralising (VN) antibody responses
Group Colt Circulating
VN and
EHV-MBantibody'
to EHV-1-ED71
No
week week week weak week +7 week +
-1 0 +2 +4 challenge'10
vacb
TEST 7 8) 8 4) 8 l6, 16 32) 32 16, 32 32) 32
15 4) 4 4, 8 32) 64 64, 64 64) 64 64) 128
20 8, 8 8) 8 16, 16 32) 64 32, 32 32, 32
CONTROL S 4) 4 4, 4 4) 4 4, 4 4, 4 32, 32
8 <4, <4 <4, <4 <4, <4 <4) <4 <4) <4 32, 32
16 4, 4 4) 4 < 4) 4) 4 4, 4 32, 64
4
a VN tests were performed against ED71 virus (lefthand figures)
and EHV-1 M8 (righthand figures). Titres denote reciprocal of
serum dilution completely neutralising. 200 (ED71) to 316
(M8 ) TCIDS~ of EHV-1 .
b Vac denotes vaccination on week 0.
c Challenge on week +7.
TABLE 8
Complement fixing (CF) antibody responses to EHV-1
Group Colt Circulating
CF antibody
to EHV-1
(AB-4)
No
week -1 week 0 week +2 week +4
vac"
TEST 7 160 320 640 640
15 40 10 640 640
20 40 20 320 640
CONTROL 5 20 5 0 0
8 10 5 5 5
16 20 40 40 40
a vac denotes vaccination on week 0.
t
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TABLE 9
Challenge virus replication in the upper respiratory tract
Group Colt Virus
No isolated
(log,a
TCID~/ml)
from
nasal
swabs
following
intranasal
challenge
(days)
0 1 2 3 4 5 6 7 8 9 10 11 12 13
TEST 7 -' - 2.0 2.0 _ _ _ _ _ _ _ _ _ _
(ED71)
15 - _ _ _ _ _ _ _ _ . _ _ _ _
20 - _ _ _ _ _ _ _ . _ _ _ _ _
CONTROL 5 - 3.72.0 _ _ 2.7_ _ _ _ _ _
$ - 2.73.7 3.5 2.53.72.0 - _ _ _ _ _
16 - 4.54.3 3.~t3.72.5_ _ _ _ _ _ _ _
TABLE 10
Leukocyte viraemia following intranasal EHV-1 challenge
Group Colt Number
No of
ED71
virus
infected
leukocytes"/2X10'
cells
days
after
challenge
0 1 3 5 7 9 11 13
TEST 7 -b - _ _ _ _ _ _
(ED71)
15 - _ _ _ _ _ _ _
20 - _ _ _ _ _ _ _
CONTROL 5 - - - 12 5 2.5 - -
g _ _ _ 200 1.3 - _ _
16 - - - 20 2.5 2.5 - -
a Buffy coat cell layer from lOmls of citrated blood was
separated on a percoll cushion washed in phosphate buffered
saline and titrated in monolayers of equine dermal cells
(Animal Health Trust, Newmarket, Suffolk) using 8
monolayers/dilution and 3 tenfold dilutions (MEM, 10% 'y-
irradiated FCS supplemented with neomycin).
b No EHV-1 isolated after two serial passages at 37°C.
