A fowlpox virus vaccine vector with insertion sites in the terminal repeats: demonstration of its...

Veterinary Microbiology, 23 (1990) 305-316 305 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

A fowlpox virus vaccine vector with insertion sites in the terminal repeats: demonstration of its

efficacy using the fusion gene of Newcastle disease virus

M.E.G. Boursnell, P.F. Green, J.I.A. Campbell A. Deuter, R.W. Peters, F.M. Tomley, A.C.R. Samson 1, P.T. Emmerson 1 and M.M. Binns

Institute for Animal Health, Houghton Laboratory, Houghton, Huntingdon, Cambridgeshire (Great Britain)

~ Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne (Great Britain)

ABSTRACT

Boursnell, M.E.G., Green, P.F., Campbell, J.I.A., Deuter, A., Peters, R.W., Tomley, F.M., Samson, A.C.R., Emmerson, P.T. and Binns, M.M., 1990. A fowlpox virus vaccine vector with insertion sites in the terminal repeats: demonstration of its efficacy using the fusion gene of Newcastle disease virus. Vet. Microbiol., 23:305-316.

In this paper we report the development and testing ofa fowlpox virus vector system. Insertion sites in non-essential regions within the terminal inverted repeats of the virus have been characterised. Foreign genes inserted into these sites are shown to be present in two copies in the resultant recombinant virus. To test the potential use of this vector as a live vaccine the fusion gene of Newcastle disease virus (NDV) has been inserted into a vaccine strain of fowlpox virus, and inoculated into chickens. The experiments demonstrate the ability of the recombinant to protect chickens against challenge by a virulent strain of NDV and to elicit the formation of anti-fusion protein antibody.

INTRODUCTION

Since the discovery that the orthopox virus, vaccinia virus, could tolerate the insertion of non-poxvirus, or foreign, genes at various sites within its genome, and express those genes under the control of its own promoters (Pani- cali and Paoletti, 1982; Mackett et al., 1982), it has been widely used as an expression vector. The expression of genes from several pathogens and the demonstration of protection against challenge by the pathogen in model sys- tems (for review see Mackett and Smith, 1986 ) has led to the possibility of using vaccinia vectors as live recombinant vaccines in humans and in ani-

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306 M.E.G. BOURSNELL ET AL.

mals. However, the fact that serious side effects can be associated with the use of vaccinia as a vaccine (Lane et al., 1969 ) has meant that recombinant vaccines based on vaccinia have not yet been widely used in humans. In addition, the wide host range of vaccinia and the possibility of recombinant viruses spreading through animal populations has meant that recombinant vaccinia viruses have not been licensed for veterinary use. In the veterinary field atten- tion has recently been focussed on other poxviruses with a host range limited to the species in question, for example avipox viruses in poultry and capripox viruses in sheep and goats (Binns et al., 1986; Boyle and Coupar, 1988; Ger- shon and Black, 1989).

Fowlpox virus, an avipox virus, has a host range limited to avian species (Tripathy and Cunningham, 1984) and is hence non-infectious to humans and other mammals. Its use as a live vaccine vector in poultry has been demonstrated by expression of the avian influenza haemagglutinin gene and sub- sequent protection of birds against challenge by influenza virus (Taylor et al., 1988b ). In addit ion to its use in birds, there exists the fascinating possibility of its use as a mammal ian or even a human vaccine vector. Although fowlpox cannot cause a productive infection in mammals it has been shown that fowlpox virus expressing rabies virus glycoprotein can be used as a vaccine to protect mammals against rabies (Taylor et al., 1988a).

One of the most important pathogens of poultry worldwide is Newcastle disease virus (NDV), (Lancaster and Alexander, 1975 ). NDV, a member of the paramyxovirus group, possesses two major glycoprotein components of the virion envelope. These are the haemagglutinin-neuraminidase (HN) protein which allows virus at tachment to cells, and the fusion (F) protein which is involved in cell fusion and virus penetration. The F protein plays an important role in immuni ty to the disease (Meulemans et al., 1986) and this protein alone is sufficient to protect birds, as has been shown by expression of the F gene in vaccinia virus (Meulemans et al., 1988).

In this paper we present the characterisation and testing of a fowlpox virus vector system utilising non-essential insertion sites for foreign genes in the inverted terminal repeats of the virus. To test the usefulness of this vector we have constructed recombinants containing the gene for the fusion protein of the Beaudette C strain of NDV (Chambers et al., 1986 ). The resultant recombinant virus is used in vivo to investigate its efficacy as a vaccine against NDV.

MATERIALS AND METHODS

Virus and cells

The viruses used were FP9, which is a plaque-purified isolate of a tissue- culture adapted strain of fowlpox HP444 (Mayr and Malicki, 1966), and Px4.1 which is a plaque-purified variant of the Poxine vaccine strain (Du-

A FOWLPOX VIRUS VACCINE VECTOR 307

phar), passaged three times in tissue culture. Viruses were grown in chick embryo fibroblasts (CEFs) as described (Binns et al., 1987).

Construction of plasmid recombination vectors The sequences included in pB3ME, which is a clone of the terminal BamHI

fragment of the FP9 strain of fowlpox, which has had the tandem repeats removed (Campbell et al., 1989) are shown in Fig. 1. To aid insertion of DNA into the unique SpeI site of pB3ME (Fig. 1 ), a plasmid pEFLI6 was constructed by inserting the PvuII fragment from pUC 19 into the SpeI site of pB3ME. The fragment from pUC 19 confers on pB3ME a blue/white marker system for detection of recombinants on plates containing Xgal and has a unique SmaI site for insertion of promoter/gene cartridges.

0 4 5 6 6.3 kb t / / i i I J

ORF1 ORF2 ORF3

i t t - - - ] - - I I Tandem repeals

Sequences in pB3ME

Bglll Ncol Spel

(Bsln)

Ncol (Bsm)

BgI~

Fig. 1. Construction of clones used for recombinatigns. The scale at the top shows the 6.3 kilo- bases from the end of the fowlpox FP9 genome. ORFs 1, 2, and 3 represent the open reading frames found in this sequence. The sequences present in the clone pB3ME are shown, and the three unique restriction sites used. The plasmids pEFL2 and pEFF2 below show the position and orientation of the foreign genes inserted into pB3ME.


The lacZ gene from E. coli under the control of the vaccinia p7.5 promoter was excised from the plasmid pPG1 ,(a kind gift from Dr. Paul Gershon) by BamHI cleavage and inserted into the Bgl II site of pB3ME, the resultant plasmid being designated pEFL2 (Fig. 1 ). Equivalent plasmids pEFL6 and pEFL8 were constructed by insertion into the NcoI and SpeI sites.

The F gene of the Beaudette C strain of NDV in pUC 19 (Chambers et al., 1986) was subcloned by cutting with the restriction enzyme BamHI (which cuts 29 bases/upstream of the initiating ATG codon at the 5' end of the F gene) aad H/ndIII (which cuts approximately 820 bases downstream from the 3' end of the F gene) and inserting into pGS20 (Mackett et al., 1984) in order to place the F gene under the control of the p7.5 vaccinia promoter. The p7.5 promoter plus F gene was excised from this plasmid by using XbaI and NheI (which cuts 360 bases downstream of the F gene), end-repaired with Klenow and cloned into the unique SmaI site of pEFL 16. The resulting plasmid is called pEFF2 (Fig. 1 ).

Production of fowlpox recombinants The plasmid constructs were allowed to recombine into fowlpox essentially

as has been described for vaccinia virus (Mackett and Smith, 1986 ). Briefly, 80% confluent monolayers of CEFs were infected with 3 plaque forming units (pfu)/cell of virus. Two hours post infection the recombination plasmid plus 1/2g of fowlpox DNA were transfected into the cells using the calcium phos- phate method. The cells were incubated for 4-5 days and then freeze-thawed to release virus. Virus from the recombinations was titrated on CEFs and then plaqued out on 10 cm Petri dishes at a concentration of virus producing about 1000 plaques per dish. For the lacZ construct the plaques were over- layed with agarose containing the colourigenic substrate Xgal. Recombinant plaques were identified by their blue colour, picked into 10 mM Tris pH 9.0, freeze-thawed three times to release the virus, and plaque-purified three times. For the F gene construct viral plaques were lifted onto nitrocellulose (Villa- real and Berg, 1977 ) and probed with radiolabelled probe specific for the F gene. Recombinant virus containing the F gene sequences was then isolated from the agarose-overlay by removing a plug of agarose using a Pasteur pi- pette. The plus was resuspended in 10 mM Tris pH 9.0, freeze-thawed three times and replaqued. Recombinant virus was identified again by probing, and this process was repeated three times until a plaque-purified virus was achieved.

Analysis of recornbinant virus DNA was extracted from purified virions of the recombinant virus and of

the parent virus as described by Binns et al. (1987). Restriction enzymes were used according to the manufacturer's recommendations, restricted fragments were analysed on 0.8% agarose gels and transferred to nitrocellulose by the method of Southern ( 1975 ).


In vivo experiments Fowlpox virus was inoculated intravenously ( 10 6 pfu/bird in 0.5 ml of 199-

medium) into Rhode Island Red chickens, or by the wing-web method (various doses in 50 #1 of 199-medium) into Light Sussex chickens. Challenge virus (NDV strain Herts 33 ) was grown and titrated in eggs and diluted to a concentration of 105 ELDso per 0.5 ml of Eagles MEM tissue culture medium.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting Purified NDV virions, NDV-infected or fowlpox-infected cells were boiled

for 2 min in SDS-PAGE sample buffer (Laemmli, 1970) containing 1% w/v SDS and no reducing agent, and subjected to electrophoresis in a 10% polyacrylamide gel. Western blotting and detection of bound antibody was as described (Samson, 1986 ) except that peroxidase-conjugated rabbit anti-chicken antibody was used to detect chicken antibodies. Monoclonal antibody U45 was a gift from Dr. D. Alexander and monoclonal antibody 14 was a gift from Dr. P.H. Russell.

RESULTS

Non-essential regions The terminal BamHI fragment of fowlpox strain FP9 contains three major

open reading frames designated ORF1, ORF2 and ORF3 (Campbell et al., 1989; see Fig. 1 ). In order to test whether any of these genes are non-essential, the E. coli lacZ gene under the control of the vaccinia p7.5 promotor was inserted into unique Bgl II, NcoI and SpeI sites in pB3ME (Fig. 1 ). Recom- binations were carried out using these plasmids as described in Materials and Methods. In each stable recombinant viruses expressing fl-galactosidase were isolated. Fig. 2a shows analysis of DNA from the recombinant virus with lacZ inserted into the Bgl II site (i.e. using plasmid pEFL2). The ethidium bro- mide stained gel shows that the 6.3 kilobase (kb) terminal BamHI fragment (a double band being present at both ends) is absent in the recombinant and is replaced by a 9.9 kb fragment containing the lacZ sequences, the length of the lacZ gene plus the promoter being approximately 3.6 kb. Because no parental-sized terminal fragment of 6.3 kb is detectable in the recombinant it can be deduced that the gene has been inserted at both ends and is therefore present in two copies. The Southern blot was probed with sequences specific for the lacZ gene and confirms that the inserted sequences are lacZ sequences.

Insertion ofF gene into recombinants We have characterised and tested separate late (Binns et al., 1989) and

early (Tomley et al., 1988 and unpublished results) fowlpox promoterS. However, because it is known that the choice of either early or late promoters can affect the stimulation of cell-mediated immunity (Coupar et al., 1986), for initial experiments on expression of pathogen genes we have used the vaccinia p7.5 promoter which acts at both early and late times in vaccinia (Coch-

3 1 0 M.E.G. BOURSNELL ET AL.

bp

2 3 1 3 0 b

9 4 1 6 J,.

6 5 5 7

4361 D,

2 3 2 2 ~

1353

1078 •

I 2 3 4

....

Fig. 2. (a) Analysis of viral DNA from a recombinant made with pEFL2. Tracks 1 and 2, BamHI restriction digests of the recombinant and parent viruses respectively; tracks 3 and 4, the same digests probed on Southern blots with sequences specific for the lacZ gene. (b) Western blot of infected cell lysates probed with mouse anti-fl-galactosidase antibodies. Bound antibody was detected with goat anti-mouse peroxidase conjugate. Track 1, cells infected with the fowlpox/ lacZ recombinant; track 2, cells infected with the parent FP9 fowlpox virus; track 3, uninfected cells; track 4, molecular weight markers.

ran et al., 1985) and in fowlpox (our results, unpublished). For insertion of the NDV F gene a plaque-purified isolate of the 'Poxine' vaccine strain of fowlpox was used (Px4.1). A plasmid construct, pEFF2, was made, consist- ing of the p7.5 promoter plus the F gene from the Beaudette C strain of NDV, inserted into the SpeI site in pB3ME (Fig. 1 ). This site is in the open reading frame ORF3 (Campbell et al., 1989 ) in the terminal repeat region.

After recombinations had been carried out as described in Materials and Methods, virus from the recombinat ion was plaqued out and probed. A few


plaques (approximately 0.1%) were identified as having F gene sequences and these were picked and subjected to several further rounds of purification, testing for F sequences at each stage. The Px4.1 strain of fowlpox has not been sequenced in the terminal regions as has the FP9 strain, so the exact arrange- ment of ORFs is not known. However, it is known that the terminal sequences are not identical because Px4.1 possesses a larger terminal BamHI fragment than does FP9 (data not shown). In spite of these differences, recombination with the FP9 plasmid was successful and a recombinant containing the F gene was isolated and designated A255.1. Viral DNA for A255.1 was digested with BamHI, Southern blots hybridised with an F gene specific radiolabelled probe and shown to contain F gene sequences (data not shown).

Analysis of expression of genes in vitro Monolayers of chick embryo fibroblasts (CEFs) infected with the recom-

binant viruses and the parent strains were lysed as described in Materials and Methods, separated by polyacrylamide gel electrophoresis and the proteins transferred to nitrocellulose (Samson, 1986 ). The proteins were probed with a mouse anti-fl-galactosidase antibody in the case of the lacZ recombinant, and monoclonal antibody specific for NDV F (monoclonal U45 ) in the case of the NDV F recombinant. The anti-fl-galactosidase antibody revealed a 116 kD protein, the correct size for fl-galactosidase (Fig. 2b). The monoclonal antibody to the F protein, however, could detect no production of F protein from the recombinant (Fig. 3). Purified NDV virions were run on the same gel and the F protein in these lanes can be seen clearly. In CEFs infected with NDV, a band of the correct size was also detected.

Analysis of protection of birds in vivo Three groups of four or six birds were inoculated at 2 weeks old as follows:

the first with 106 pfu /b i rd of the fowlpox/NDV F recombinant A255.1, the second with 106 pfu /b i rd of the FP9 strain of fowlpox as a fowlpox control, and the third was uninoculated. Two weeks later the birds were boosted with the same dose of fowlpox virus or the recombinant. After a further 6 weeks all birds were challenged intramuscularly with 105 ELDso of a virulent strain o f N D V (Herts 33 ) per bird. This challenge dose is that used in the standard test carried out by the Central Veterinary Laboratory, Weybridge, Great Brit- ain for evaluating vaccines. Table 1 shows the results of this challenge. In the groups of birds inoculated with the fowlpox control or uninoculated, all 12 birds were dead within 5 days, 4 /12 being dead within 3 days and 11/12 being dead within 4 days. In the group inoculated with the fowlpox/NDV F recombinant, all birds survived the challenge.

In a second experiment, groups of six or seven birds were inoculated, at 1 day of age, by the wing-web method with either the A255.1 fowlpox/F recombinant or the Px4.1 parental virus strain (Table 2). Each virus strain was


i 2 3 4 5 6 7 8 9

Fig. 3. Western blot of proteins separated on a non-reducing SDS-polyacrylamide gel, probed with anti-F monoclonal U45, bound antibody being detected with peroxidase-conjugated rabbit anti-mouse IgG antiserum. Tracks 1 and 11, purified NDV virions; tracks 2 and 3, uninfected CEFs at 24 and 48 h; tracks 4, 5 and 6, CEFs infected with the fowlpox/F recombinant A255.1 at 12, 24 and 48 h post infection; tracks 7 and 8, CEFs infected with fowlpox/lacZ recombinant at 24 and 48 h post infection; track 9, uninfected CEFs; track 10; CEFs infected with NDV. Track 12 shows an NDV virion preparation ink-stained as a marker.

TABLE1

Challenge of birds inoculated intravenously with the fowlpox/NDV F recombinant A255.1 and with controls

Inoculum Total number of birds dead

day 3 day 4 day 5

A255.1 0/4 0/4 0/4 Controls (FP9 or uninoculated) 4/12 11/12 12/12

inoculated into four groups of birds, at doses of 5 X 10 4, 5 X 103, 5 X 102 and 5 × 101 pfu/bird. Thus there were eight groups in all, with an additional group of uninoculated birds. Birds were inoculated at 1 day of age and challenged intramuscularly 26 days later as above. The results of this challenge are shown in Table 2. Uninoculated and control birds were not protected against challenge, all 38 birds being dead by 4 days. The birds inoculated with the fowl- p o x / N D V F recombinant were protected to a significant degree, 22 birds out of 28 surviving the challenge (79%). The dose of vaccine used appeared to have little effect on the levels of protection, although the lowest dose gave the best protection.


TABLE 2

Challenge of birds inoculated by the wing-web method at 1 day of age with the fowlpox/NDV F recombinant A255.1 and controls

Inoculum Viral dose

Total number of birds dead

day 3 day 4 day 5 day 6

A255.1 5 X 10 4 0/7 0/7 (F recombinant) 5X 103 0/7 0/7

5× 102 1/7 I /7 5×101 0/7 0/7

Px4.1 5× 10 4 6/7 7/7 (Fowlpox alone) 5X 10 3 3/6 6/6

5X 10 2 5/6 6/6 5X 101 1/5 5/5

Uninoculated - 4/7 7/7

2/7 2/7 2/7 2/7 2/7 2/7 0/7 0/7

Testing of sera from inoculated birds for antibody to the F protein Birds were bled prior to challenge with NDV. In the first experiment, sera

from birds inoculated with the fowlpox/F recombinant and from control birds, were used to probe western blots of SDS-PAGE separated proteins from purified NDV virions, to investigate whether antibodies to the F protein had been induced by the recombinant. These results are shown in Fig. 4. Sera from birds inoculated with the fowlpox/F recombinant contained antibody which bound to the F protein in purified NDV preparations. The U45 anti-F monoclonal antibody also bound to the F protein. No significant binding to the F protein was seen with sera from birds inoculated with fowlpox alone or sera from uninoculated birds, although some faint, presumably non-specific, binding could be seen at the 1 / 100 dilutions.

DISCUSSION

It can be seen from the data presented in this paper that non-essential regions in the terminal inverted repeats of fowlpox virus can be used as insertion sites to construct recombinants containing foreign gene sequences. Expression of fl-galactosidase from sites in three genes can be demonstrated in vitro, a protein of the correct size on SDS-polyacrylamide gels being detected by antibody to fl-galactosidase. Using cloned fowlpox DNA from the FP9 highly tissue-culture adapted strain of fowlpox, foreign genes can be inserted into these sites in FP9 or into equivalent sites in other strains of fowlpox such as the Poxine vaccine strain.

In order to test the usefulness of this vector system and these insertion sites, experiments using the fusion gene of NDV were undertaken. These results of


Fig. 4. Western blot of separated proteins from NDV virions run on a non-reducing SDS-polyacrylamide gel, probed with the following sera: tracks 1 and 12, blot rinse; tracks 2 and 9, no antibody control; tracks 3 and 4, sera from uninoculated birds at 1/100 and 1/1000; tracks 5 and 6, sera from fowlpox-inoculated birds at 1 / 100 and 1 / 1000; tracks 7 and 8, sera from birds inoculated with the fowlpox/F gene recombinant at 1 / 100 and 1 / 1000; track 10, anti-HN monoclonal 14 at 1/1000; track 11, anti-F monoclonal U45 at 1/1000. The outer tracks are ink- stained to show the position of the HN protein and the unreduced F1 plus F2 (FI,2) protein. Bound antibody was detected with peroxidase-conjugated rabbit anti-chicken IgG antiserum ~(tracks 1-8) and peroxidase-conjugated rabbit anti-mouse IgG antiserum (tracks 9-11 ).

these experiments showed that this vector system can be used successfully to protect chickens against challenge by a virulent strain of NDV. Birds inoculated intravenously at 2 weeks of age and boosted again at 4 weeks were 100% protected against challenge. The Poxine vaccine strain, from which Px4.1 is derived, is normally used in older birds, and has marked pathogenic effects when used at high doses in 1-day-old birds. Nevertheless, we were interested to see if protection could be achieved by inoculating 1-day-old birds, by the wing-web method, with a range of doses of the recombinant. Although the birds are not immunologically competent at this age, and wing-web inocula- tions of 1-day-old birds are technically difficult, this experiment also demonstrated good protection (79%), even at very low doses of the recombinant virus. Experiments are in progress to determine the op t imum vaccination reg- imen, using the Px4.1 and other strains of fowlpox.

Sera from birds inoculated with the fowlpox/F recombinant contained antibodies to the F protein. This demonstrates that expression of the F gene has occurred, even though expression has not been shown directly in vitro 'on western blots. The reason for the failure to detect the protein on western blots is not clear. It is known that the monoclonal antibody used can detect the F protein from NDV-infected cells on western blots but it is possible that in cells infected with the fowlpox recombinant fairly low levels of the F protein


accumulate, and that the detection system is not sensitive enough to detect these levels.

The demonstration that this fowlpox virus-based vaccine vector can successfully protect chickens against challenge opens the way for the use of such a recombinant fowlpox virus as a vaccine against NDV. Recombinant vaccines using the F gene could have several advantages over conventional vaccines. For example, vaccination with a recombinant fowlpox virus expressing the N D V F gene and not the HN gene would allow easy differentiation be- tween antibodies induced by the vaccine and those induced by infection with NDV. In addition, a recombinant poxvirus could be used as a multivalent vaccine, expressing genes that have been cloned from other poultry pathogens such as infectious bronchitis virus (Boursnell et al., 1989 ) and Marek's disease virus (Buckmaster et al., 1988).

ACKNOWLEDGEMENTS

We would like to thank Ian Foulds, Bridgette Britton, Martin Woods, Deb- bie Southee, Mark Elson, Christine Mason, Rachel Anderson and Mick Hug- gins for excellent technical assistance, and Jane Cook for much helpful advice and discussion.

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