Immunization with a vaccinia recombinant expressing the F protein protects Rabbits from challenge with a lethal dose of rinderpest virus

VIROLOGY 170,l l-l 8 (1989)

Immunization with a Vaccinia Recombinant Expressing the F Protein Protects Rabbits from Challenge with a Lethal Dose of Rinderpest Virus

THOMAS BARRETT,’ GRAHAM J. BELSHAM, SHAILA M. SUBBARAO,’ AND SHARON A. EVANS

AFRC Institute for Animal Health, Pirbright Laboratory, Waking, Surrey GU24 ONF, United Kingdom

Received October 3 1, 7988; accepted December 12, 1988

A cDNA clone containing the complete coding sequence of the rinderpest fusion protein (F) gene was inserted into the thymidine kinase gene of vaccinia virus (WR strain) under the control of the 7.5K early/late vaccinia virus promoter. All forms of the F protein, i.e., the glycosylated F0 precursor, the unglycosylated F, protein, and the glycosylated F2 protein, were detected in cells infected with the recombinant virus. Vaccination of rabbits with the recombinant virus induced antibodies which reacted in an ELISA system specific for rinderpest. The rabbit sera contained neutralizing antibodies against rinderpest virus and precipitated the F protein from lysates of rinderpest infected cells. Rabbits vaccinated with the recombinant rinderpest F gene vaccinia virus were protected from a lethal challenge with the lapinized Nakamura 3 strain of rinderpest virus. Variations in the severity of clinical symptoms correlated with the level of anti-F protein antibodies produced. o 1999Academic PWSS, hc.

INTRODUCTION

Rinderpest (or cattle plague) is one of the most im- portant veterinary problems in many parts of the world, particularly in Africa and Asia. The virus affects all artio- dactyl species and is a member of the morbillivirus ge- nus within the paramyxoviridae. There is only one sero- type of rinderpest virus although many strains have been isolated and some have been adapted to grow in different host species and cell types. This adaption does not alter the virus antigenicity. Although an effective live attenuated vaccine exists (Plowright, 1962) difficulties are encountered in its administration in countries with hot climates where the disease is en- demic because of the necessity to maintain a cold chain. Thus a more heat stable recombinant vaccine would be of great benefit.

The fusion(F) protein of paramyxoviruses is of crucial importance in allowing virus penetration and spread in an infected animal. It is produced in an inactive uncleaved form (F,) which must be cleaved by cellular proteases to yield the disulfide-linked F,/F* subunits to activate the fusion function of the protein (Choppin et a/., 1981). Vaccination of dogs with purified F protein of canine distemper virus (CDV) has been shown to protect dogs from challenge with the virus (Norrby et al., 1986; De Vries et al,, 1988). Effective vaccines against morbillivirus disease must induce an anti-F response, otherwise virus can spread from cell to cell even in the presence of neutralizing anti-hemagglutinin

I To whom requests for reprints should be addressed. ’ Permanent address: Indian Institute of Science, Bangalore, India,

560012.

antibody, and atypical disease symptoms are seen. Early inactivated vaccines to measles virus failed to protect from subsequent infection since the inactiva- tion process destroyed the immunogenicity of the F protein (Choppin and Scheid, 1980; Merz et al., 1980).

More recently vaccinia recombinants containing either the hemagglutinin or fusion protein genes of measles virus have been shown to protect mice from a lethal dose of measles by intracerebral inoculation (Dril- lien et al., 1988). In other paramyxoviruses similar protection with vaccinia recombinants has been found. Recombinants containing the F gene protein of respiratory syncytial virus (RSV) will cross-protect against both subtypes A and B whereas recombinants containing the G protein gene only protect against homologous challenge (Johnson eta/., 1987; Stott eta/., 1987). With parainfluenza viruses, however, complete protection has been reported with hemagglutinin gene recombinants and only partial protection with F gene recombinants (Ray et a/., 1988; Spriggs et a/., 1987).

In the case of rinderpest, an ideal small animal model exists in that rabbits can be infected and die with similar symptoms (fever, diarrhea, anorexia) to those seen in the natural hosts. We have constructed a rinderpest/ vaccinia recombinant virus containing the F gene of the RBOK vaccine strain of rinderpest (Plowright, 1962) and shown that it expresses authentic F protein in vitro. Rabbits vaccinated with this recombinant are protected from lethal challenge with the Nakamura 3 strain of rinderpest virus.

METHODS Isolation and characterization of vaccinia recombinants expressing RPV F protein

A pUC13 plasmid containing an F gene-specific insert of approximately 2.4 kb was selected from a cDNA

11 0042.6822/89 $3.00 CopyrIght 0 1999 by Academic Press, Inc. All nghis of reproduction tn any form reserved.

12 BARRETT ET AL.

library made from polyadenylated RNA of the Plowright vaccine strain of RPV (Barrett eta/., manuscript in preparation). The cDNA insert was removed by double di- gestion with EcoRl and Smal, blunt-ended, and li- gated into the Smal site of the vaccinia transfer vector pGS20 (Mackett et a/., 1984). The SmallfcoRl fragment sequence contained the entire F coding region and approximately 300 nucleotides of 5’ noncoding sequence. It aligned with the published F sequence of the lapinized strain of RPV from nucleotide 291 to the end (Tsukiyama et al., 1988). Essentially the proce- dures of Mackett et a/. (1984) was used to obtain vaccinia virus recombinants. Briefly, 5.0 pg of the plasmid containing the F gene coding sequence of RPV (pVG24) and 2.0 pg of WRl vaccinia DNA were introduced as a calcium phosphate coprecipitate into WR vaccinia infected BSC40 ceils; from the virus yield TK- viruses were selected with bromodeoxyuridine (BUdr) at 25 pg/ml in HTK- 143 cells and screened using the Nal “quick blot procedure” (Gillespie and Bresser, 1983) for the presence of the F gene by hybridization to a 3zP-labeled F gene DNA probe. Positive isolates were plaque purified again in HTK-143 cells in the presence of BUdr and rescreened. Recombinants were passaged twice more in BSC40 cells to yield working stocks. To circumvent problems of cross-reactivity in subsequent ELISA studies with RPV antigen grown in Vero cells (derived like BSC40 cells from African green monkey kidney cells) virus for inoculation into rabbits was passaged again in BHK cells. Vaccinia DNA was prepared from wild-type or recombinant vaccinia infected BSC 40 cells using the method of Esposito et al, (1981).

For expression studies BSC40 cells (60-mm dishes) were infected where appropriate with wild-type vaccinia or recombinants at approximately 0.1 PFU/cell. After 20 hr c.p.e. was apparent and cells were labeled with [35S]methionine (50 &i) for l-2 hr in methionine-free medium. For carbohydrate-labeling studies [3H]glucosamine was added immediately after virus adsorption and incubation continued for 20 hr. In both cases cell extracts were prepared by solubilizing the cell sheet in 0.15 l\/I NaCI, 10 mfl/l Tris-HCI, pH 8.0, 1 m/l/l EDTA, 0.5% NP-40 (0.5 ml/dish). Insoluble material was removed by centrifugation at 10,000 g for 5 min and the extract either analyzed directly or stored at -70”. The extracts (50-100 ~1) were mixed with an equal volume of 0.5 M NaCI, 10 m/l/lTris-HCI, pH 7.5, 1 mh/f EDTA, 0.2% NP-40 and 5 ~1 rabbit hyperimmune rinderpest serum for immunoprecipitation. The mix- tures were incubated at room temperature for 1 hr, then 15 ~1 of washed Pansorbin (Calbiochem) was added and incubation continued for a further 20 min. The Pansorbin was pelleted by centrifugation at 10,000 g for 1 min, washed twice with 0.5 ml RIPA

buffer (150 mltl NaCI, 50 mM Tris-HCI, pH 7.5, 1% Triton, 1% DOC, 0.1% SDS) followed by one wash with TBS (140 mM NaCI, 20 mMTris-HCI, pH 7.5). The precipitated protein was solubilized by boiling in SDS sample buffer containing 100 mlVI dithiothreitol for 5 min and analyzed on 1 O*b polyacrylamide gels in the presence of SDS (Laemmli, 1970). Gels with [3H]glucosamine samples were prepared for fluorography by the method of Bonner and Lasky (1974) and the dried gels were exposed to preflashed Fuji RX X-ray film.

ELBA assays

Purified RPV for use as an ELISA antigen was prepared from the medium of virus (RPV RBOK strain) infected Vero cells when the cells were showing 90- 100% c.p.e. The medium was clarified by centrifugation at 2500 rpm for 15 min at 4”. The virus was then pelleted at 100,000 g for 1 hr, resuspended in PBS, and layered over a 65% (w/w) sucrose cushion in Tris- EDTA and centrifuged at 25,000 rpm in a Beckman SW27 rotor for 1 hr. The opalescent band at the inter- face was collected and layered on a 15-509/o (w/w) lin- ear tartrate gradient and centrifuged at 35,000 rpm in a Beckman SW 40 rotor for 4 hr. Fractions of 1 .O ml were collected and monitored at ODZeo and the peak fractions (density 1.24-l .25 gm/mI) pooled. Protein es- timations were carried out according to the method of Lowry et al. (1951). Plates were coated with 50 ~1 of virus, diluted to 4 pg/ml, per well in 96-well ELISA plates (Costar, Nunc) in 0.05 M sodium carbonate-bi- carbonate buffer, pH 9.5, at 37” for 1 hr or overnight at 4’. The coated plates were washed three times with PBST (PBS containing 0.05% Tween 20) and blocked using 1 O/O BSA in PBST for 1 hr at 37”. Twofold dilutions of the various antisera were carried out in the blocking buffer (50 PI/well) and the reaction was allowed to pro- ceed for 1 hr at 37”. The plates were then washed as above and 50 ~1 of swine anti-rabbit IgG peroxidase conjugate (Dakopats) was added at 1:2000 dilution. In- cubation was continued for 1 hr at 37” and the plates were washed as before. For color development 50 PI/ well of orthophenylene diamine-H,O, substrate was added in acetate buffer. After 30 min at room temperature the color reaction was stopped by the addition of 50 PI/well of 2.5 NI H,SO, and the OD at 492 nm read using a Titertec Multiscan ELISA reader. The titers were expressed as the reciprocal of the highest dilutions that gave an OD,,, value which was at least twice that of the corresponding preimmune serum.

Virus neutralization tests

Neutralization tests were performed in Vero ceils. Equal volumes (25 ~1) of two-fold dilutions of heat-inactivated (56”, 1 hr) serum and RPV (RBOK strain, 100

RINDERPEST F PROTEIN EXPRESSION 13

TCID 50) were mixed and incubated at 4“ overnight in 96-well tissue culture plates. Trypsinized Vero cells (50 ~1) resuspended at 1 05/ml in MEM containing 5% fetal calf serum were added to each well and the plates incubated at 37” for 3 days. Serum controls, cell controls, and virus (at 100, 10, and 1 TCID 50/well) controls were included on each plate. Development of c.p.e. was monitored by light microscopy and virus growth quantitated by an ELISA method on the fixed cells (10% formalin in PBS for 30 min) cells using rabbit anti-RPV hyperimmune serum and peroxidase-conju- gated protein A. The titers were expressed as the reciprocal of the highest serum dilution which neutralized 50% of virus infectivity (Scott et al., 1986).

Vaccination and challenge experiments

Rabbits used for vaccination were 2.0- to 2.5-kg out- bred females. Vaccination was carried out by intrader- mal inoculation at two sites with 0.1 ml/site of BHK cell grown vaccinia at 5 X 1 O6 PFU/ml. Two groups of four rabbits were vaccinated with either vaccinia-F recombinant w204A(rabbits GB7 to GBIO) orw204B (rabbits GBl 1 to GBl4). One group of two rabbits (GB15 and GB16) was vaccinated with a similar dose of vaccinia containing the bacterial chloramphenicol acetyltrans- ferase (CAT) gene inserted into the TK site (vv977D) as controls. Serum was taken before vaccination and at 2 and 4 weeks. The rabbits were then challenged iv with a l.O-ml dose of homogenized spleen material, prepared from a rabbit infected with the Nakamura 3 strain of rinderpest virus, sufficient to cause 95-l OOq/o fatality in unvaccinated rabbits (Scott et a/., 1986). The spleen material, stored as a freeze-dried extract, was reconsti- tuted in sterile PBS and diluted (1 O-fold) in sterile PBS to give the required inoculum. Temperature and clinical signs were monitored daily for 2 weeks, by which time all rabbits which had survived had recovered completely. Surviving rabbits which showed clinical symptoms (all except GB8 and GBl 0) during challenge lost, on average, 0.5 kg weight.

RESULTS

Construction and selection of vaccinia recombinants

The complete coding sequence of the F gene of rinderpest RBOK vaccine strain was inserted into the Smal site of plasmid pGS20 and introduced into vaccinia-infected cells with WR strain vaccinia DNA using calcium phosphate precipitation as described under Methods. Recombinant TK-viruses were selected with BUdr and screened for the presence of the F gene by hybridization with labeled F gene cDNA. Two strongly hybridizing vaccinia recombinants w204A and w204B

123 4 123 4

H- RP”.F-

I - RPV-F J- K- L--

FIG. 1. Analysis of wild-type and recombinant vaccinia virus DNA. Each DNA sample was run in duplicate. The left panel was hybridized with 32P-labeled wild-type vaccinia DNA. The right panel was hybridized with 3zP-labeled RPV F cDNA. (1) Wild-type vaccinia DNA; (2) recombinant w204A DNA; (3) recombinant w204B DNA: (4) RPV F- specific plasmid DNA.

were selected for further analysis. To check that the F gene of RPV was inserted into the correct site of vaccinia virus, HindIll digests of wild-type and two inde- pendently picked vaccinia-rinderpest F recombinant clones (w204A and w204B) were separated on a 1% agarose/TBE gel. Southern blots were performed on duplicate samples and hybridized either with 32P-labeled vaccinia DNA or with 32P-labeled RPV F cDNA. As can be seen from Fig. 1, the HindIll J fragment is altered in the recombinants and a novel band migrating between the H and I fragments, consistent with an insert of approximately 2 kb, is observed. This is the only fragment which hybridized with the RPV F probe.

Expression of rinderpest F glycoprotein in cells infected with the vaccinia-rinderpest F recombinant virus

To check that the RPV F recombinants were capable of expressing authentic F protein, BSC40 cells were infected with the two vaccinia-F recombinant clones (w204A and w204B) and labeled with [35S]methionine. The labeled proteins were immunoprecipitated using a rabbit hyperimmune serum to rinderpest. Control cells were infected with wild-type virus. As can be seen from Fig. 2 the serum precipitated proteins which migrated in positions expected for the F. and F, proteins. It is difficult to visualize the F2 portion of the protein using methionine label and so the F2 protein was visualized by labeling with [3H]glucosamine. A protein of the correct size for F2 was precipitated with hyperimmune anti-RPV serum specifically from cells infected with w204A or w204B. Very little F. protein is detectable under these conditions because of the long period required for glucosamine labeling. No glycosylated bands were precipitated from uninfected or wild-type vaccinia infected cells (see Fig. 2B).

Pulse-chase experiments were performed with [35S]methionine-labeled cells to demonstrate that the

14 BARRE-I-I- ET AL.

1234 5

92.5

69

46

30

92.5

69

46

30

FIG. 2. Analysis of proteins synthesized in wild-type and recombinant vaccinia virus infections. BSC40 cells were infected with vaccinia viruses and labeled with either [35S]methionine or [3H]glucosamine as described under Methods. Labeled proteins immunoprecipitated with rabbit anti- RPV hyperimmune serum were separated on 10% polyacrylamide gels. The positions of the molecular weight markers and the FD/F,/Fp proteins are indicated. (A) Proteins labeled with [35S]methionine; (B) proteins labeled with [3H]glucosamine. (1) Uninfected BSC40 cells; (2) cells infected with wild-type virus; (3) cells infected with recombinant W204A; (4) cells infected with recombinant VV204B; (5) [‘4C]labeled protein molecular weight markers (Amersham, UK).

F0 protein was correctly processed to F, and F2. As can be seen from Fig. 3 this was the case; F, increased in intensity with increasing time of chase. In addition, when dithiothreitol was omitted from the gel loading buffer the F, protein band disappeared and a slower migrating band similar, but not identical, in weight to F0 was seen (see Fig. 3). This difference is probably due to the fact that unprocessed F0 may have a conformation different from the processed F,/F2 disulfide-linked subunits. It is not clear why F,, as opposed to FI/F2, is not also apparent in the absence of reducing agent. It may be due to the fact that the FO, and possibly some of F,/ F2 also, aggregates in the absence of reducing agent and migrates further up the gel.

Immune response to vaccinia-RPV F recombinants in rabbits

Four rabbits were inoculated intradermally with either of the recombinants w204A or w204B and 2 rabbits with vaccinia recombinant w977D, which expresses the bacterial gene for chloramphenicol acetyl- transferase, as a control. Blood samples were taken before inoculation and at 2 and 4 weeks postvaccination. The ability of these sera to react in an ELISA test

using purified RPV as an antigen was determined and the results are given in Table 1. A great variation in response was seen ranging from a high of 2.5 x lo4 in rabbit GBlO to levels barely greater than background in rabbit GB7. However, in the case of the latter animal the preinoculation ELISA reactivity was high, due to some nonspecific reaction, and this may have ob- scured a low level response. The 4 rabbits vaccinated with clone w204A (GB7-GBlO) developed scabs at the site of inoculation of the virus, as did those (GB15- GB16) vaccinated with the control vaccinia-CAT recombinant w977D. The 4 rabbits vaccinated with clone w204B (GB 1 1 -GB 14) showed only a slight red- dening at the site of inoculation but did not develop scabs. Nevertheless in both groups of vaccinia-F recombinants titers of >l O3 were obtained in the ELISA test (see Table 1). To determine whether the response measured by the rinderpest ELISA in the sera of vaccinia-F recombinant vaccinees was biologically significant microneutralization tests were carried out using Vero cells infected with RPV. All sera showing a titer of >3 X 1 O3 in the ELISA test showed some ability to neutral- ize the virus. A significantly higher titer was obtained from the sera of rabbits GB8 and GBl 0. No detectable


I 2 3 4 5 6 7 8 9101112 13

200

92.5

69

46

30

FIG. 3. Pulse-chase labeling of proteins synthesized in recombinant vaccinia infections. BSC40 cells were infected with VV204A and labeled for 1 hr with [35S]methionine at 20 hr postinfection. At the end of this period, the label medium was removed and the infection continued for various times in the presence of excess cold methionine. Samples were either boiled in the presence (l-6) or absence (7-l 2) of dithiothreitol in the gel loading buffer. Samples were separated on 10% polyacrylamide gels. (1, 7) Uninfected control BSC40 cells; (2, 8) 0-hr chase; (3, 9) I-hr chase: (4, 10) 2-hr chase: (5, 11) 3-hr chase; (6, 12) 4-hr chase; (13) protein molecular weight markers.

neutralization was obtained with either preinoculation sera or with postinoculation sera from the control w977D-vaccinated animals (seeTable 2). The ability of the vaccinated rabbit sera to precipitate the F protein from 35S-labeled infected cell lysates also correlated with titers in the ELISA assays. Figure 4 shows the re- sult of an immunoprecipitation with the 4-week postinoculation sera from all 10 rabbits in the experiment and

TABLE 1

RPV ELISA TITERS IN RABBITS VACCINATED WITH VACCINIA VIRUS RECOMBINANTS

Rabbit Immune seruma Recombinants

GB7 160 vv204A GB8 12,800 w204A GB9 800 w204A GBlO 25,600 w204A GBll 160 w204B GE12 3,200 ~2046 GB13 3,200 w204B GB14 6,400 w204B GB15 160 w977D GB16 80 w977D

‘The titers are expressed as the reciprocal of the highest dilutions that gave an OD492 reading which was at least twice that of the corresponding preimmune serum.

TABLE 2

VIRUS NEUTRALIZATION TITERS IN SERAOF RABBITS VACCINATED WITH VACCINIA VIRUS RECOMBINANTS’

Rabbit Prechallenge* Postchallenget Recombinant

GB7 - 2560 w204A GB8 64 320 w204A GB9 16 >2560 w204A GBlO 256-320 320 w204A GBll - >2560 ~2040 GB12 - >2560 w204B GB13 2 >2560 ~2040 GB14 <2 22560 w204B GB15 - + w977D GB16 - + ~9770

a Titers are expressed as the reciprocal dilution of heat-inactivated serum neutralizing 50% of input virus (i.e., 50 TCID 50 of RPV, RBOK strain). Where no neutralizing activity was detected it is indicated by (-), a(+) indicates that the animals died.

* 4 weeks after vaccination. t 1 week after challenge with lapinised RPV.

with the pre- and postinoculation sera of a rabbit from an independent experiment (TBQ). In all postinoculation sera from vaccinia-F recombinants the F, protein can be clearly seen migrating slightly faster than the cellular actin band. A corresponding band was absent in the controls vaccinated with the w977D virus and in the preinoculation sera, see tracks 10 to 12, Fig. 4. The serafrom 2 rabbits GB8 and GBl 0, which had the highest ELISA and neutralization titers, precipitated the F protein very efficiently and both F2 and a small amount of uncleaved F. could be seen in these precipitates (see tracks 3 and 5, Fig. 4).

Response of vaccinated rabbits to challenge with rinderpest virus

Four weeks after vaccination with the vaccinia recombinants the rabbits were challenged with a dose of Nakamura 3 lapinized virus, which is lethal in 959/o of cases (Scott eta/., 1986). Temperatures were recorded daily and clinical signs such as anorexia, diarrhea, and lethargy were noted. A sharp temperature rise oc- curred at Day 2 and lasted to Day 5 in all rabbits except GB8 and GB 10 which showed no clinical signs of infection. Diarrhea was noted in all, except rabbits GB8 and GBlO, at 3 days postchallenge. Both control animals vaccinated with w977D died on Day 4 postchallenge and showed severe clinical symptoms. By Day 5 all w- F-vaccinated rabbits had recovered and their temperatures had returned to normal. Rabbits GB8 and GBIO which had the highest neutralization and ELISA titers were completely protected and did not even show a temperature response (see Fig. 5). All surviving rabbits, except GBl 0 where the neutralization titer was so high

16 BARRETT ET AL.

123 45 6 7 8 9 10 11 12 13 14 13

69

46

-

- HA N F 0

FIG. 4. Precipitation of [35S]methionine-labeled RPV-infected cell lysates with sera from rabbits vaccinated with recombinant vaccinia viruses. Equal amounts of lysate (25 ~1) were precipitated with 5 ~1 of the respective sera as described under Methods. The sera were collected at 4 weeks postvaccination, just prior to virus challenge. The washed precipitates were boiled in gel loading buffer containing dithiothreitol and separated on a 12.5% polyacrylamide gel. (1) Molecular weight markers; (2-9) samples precipitated with sera from rabbits GB7 to GB14; (10, 11) samples precipitated with sera from control rabbits GB15 and GBl6; (12, 13) samples precipitated with pre- and postimmune sera from rabbit TB9 from an independent vaccination with W204A; (14) sample precipitated with RPV hyperimmune serum; (15) shorter exposure of track 14.

that it prevented any virus replication, were successfully infected with the challenge virus as shown by a significant increase in the virus neutralization titers to rinderpest at 2 weeks after challenge (see Table 2).

DISCUSSION

Vaccinia virus recombinants containing glycoprotein genes from several paramyxoviruses have been produced and used successfully to protect animals from challenge with infectious virus (Tartaglia and Paoletti, 1988). We have inserted the fusion protein gene of RPV into the TK gene of vaccinia under the control of the ~7.5 promoter and shown the production of authentic glycosylated RPV F protein. In the present study we have also shown that it is possible to protect rabbits from a lethal dose of RPV using only the fusion protein gene of RPV in the recombinant. This agrees with studies using purified F protein either alone or in associa- tion with immune stimulating complexes (ISCOMS) to

protect against CDV and measles (De Vries et a/., 1988; Varsanyi et al., 1987; Norrby et al., 1986).

Vaccinia-measles virus recombinants containing either the F or H surface glycoprotein genes have also been shown to protect mice from a lethal intracerebral inoculation of measles virus (Drillien et a/., 1988). We have also shown that the degree of protection corre- sponds with the strength of the immune response of individual rabbits to the fusion protein of RPV. Those demonstrating the highest titers in both ELISA and neutralization tests were completely protected (rabbits GB8 and GBl 0), and in one case (rabbit GBl 0) the level of neutralizing antibody was sufficient to completely prevent virus replication in the challenged animal. Since all w-F-vaccinated rabbits were protected to some extent, those which did not elicit detectable neutralizing antibodies may have been protected by some mechanism other than neutralization, perhaps a cell- mediated immune response. In all rabbits, apart from GBlO, vaccinated with a vaccinia-F recombinant, the


GB18

I , 1 1 I I 0 1 2 3 4 5 6 7 6

Days post challenge

FIG. 5. Temperature response in vaccinated rabbits to challenge with lapinized RPV. Temperatures were monitored daily from the time of challenge. Three groups of rabbits were challenged. Group 1 (rabbits GE7-GBl O): vaccinated with VV204A. Group 2 (rabbits GBl l- GBl4): vaccinated with W204B. Group 3 (rabbits GB15 and GB16): vaccinated with VV977D.

levels of neutralizing antibody was higher 2 weeks after challenge indicating that the virus had grown but not sufficiently to kill them.

Recombinant vaccines of this type would be of un- doubted value in field situations where it is difficult to maintain the cold chain necessary to preserve the infectivity of the present live virus vaccines. Recent studies have shown that the loss of TK activity associated with the insertion of foreign genes greatly reduces the pathogenicity of vaccinia in experimental animals (Buller et a/., 1985) and field trials with a vaccinia recombinant carrying the rabies virus glycoprotein gene have been initiated in Europe (Pastoret et al., 1988). Other studies where the growth factor gene of vaccinia has been deleted (Buller et al., 1988) or the gene for interleukin 2 has been introduced into the vector (Flex- ner eta/., 1987) also show that the pathogenicity of the virus can be reduced. These types of vaccinia vector or some other poxvirus, e.g., avipoxor capripox, vector (Binns et a/., 1988) which is not pathogenic for humans

may enable recombinant vaccines to be developed for general use.

Following submission of this manuscript Yilma et al. (1988) published the results of a successful protection of cattle against rinderpest infection with a rinderpest vaccinia recombinant virus. Similar studies carried out at our Institute confirmed that complete protection of cattle from lethal rinderpest challenge can be achieved with a single vaccination with a recombinant vaccinia virus expressing rinderpest F protein.

ACKNOWLEDGMENTS

We thank Drs. B. W. J. Mahyand C. Bostock for helpful discussions and critical reading of the manuscript, Miss Julia Brangwyn and Miss Lynette Goatley for excellent technical assistance, and Mr. Len Pul- len for carrying out the animal experimental work. This work was sup- ported by a grant from the Wellcome Trust (No. 14294/1.5). Dr. S. M. Subbarao was the recipient of a Rockefeller Foundation Biotechnology Career Fellowship.

REFERENCES

BINNS, M. M., TOMLEY, F. M., CAMPBELL, 1. and BOURSNELL, M. E. G. (1988). Comparison of a conserved region in fowlpox virus and vaccinia virus genomes and the translocation of the fowlpox thymidine kinase gene. J. Gen. Viral. 69, 1275-l 283.

BONNER, W. M., and I&KEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46,83-88.

BULLER, R. M. L., CHAKRABARTI, S., COOPER, J. A., TWARDZIK, D. R., and MOSS, B. (1988). Deletion of the vaccinia virus growth factor gene reduces virus virulence. J. Viral. 62, 866-874.

BULLER, R. M. L., SMITH, G. L., CREMER. K., NOEKINS, A. L., and Moss, B. (1985). Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase negative phenotype. Nature (London) 317, 813-815.

CHOPPIN. P. W., RICHARDSON, C. D., MERZ, D. C., HALL, W. W., and SCHEID. A. (1981). The functions and inhibition of the membrane glycoproteins of paramyxoviruses and myxoviruses and the role of measles virus M protein in subacute sclerosing panencephalitis. J. Infect. Dis. 143, 352-363.

CHOPPIN, P. W.. and SCHEID. A. (1980). The role of viral glycoproteins in adsorption, penetration and pathogenicity of viruses. Rev. ln- fee?. Dis. 2,40-61.

DE VRIES, P., UYTDEHAAG, F. G. C. M., and OSTERHAUS. A. D. M. E. (1988). Canine distemper virus (CDV) immune-stimulating complexes (Iscoms), protect dogs against CDV infection. J. Gen. Viral. 69,2071-2083.

DRILLIEN, R., SPEHNER, D., KIRN, A., GIRAUDON, P., BUCKLAND, R., WILD, F., and LECOCO, J.-P. (1988). Protection of mice from fatal measles encephalitis by vaccination with vaccinia virus recombinants en- coding either the haemagglutinin or the fusion protein. Proc. Nat/. Acad. Sci. USA 85,1252-l 256.

ESPOSITO, J., CONDIT, R., and OBIJESKI, J. (1981). The preparation of orthopoxvirus DNA. /. Viral. Methods 2, 175-l 79.

FLEXNER, C., H~~GIN, A., and Moss, B. (1987). Prevention of vaccinia virus infection in immunodeficient mice by vector-directed IL-2 expression. Nature (London) 330, 259-262.

GILLESPIE, D.. and BRESSER, J. (1983). mRNA immobilisation in Nal: Quick blots. Biotechniques 1, 184-l 92.

JOHNSON, P. R., OLMSTED. R. A., PRINCE, G. A., MURPHY, B. R., ALLING, D. W., WALSH, E. E., and COLLINS, P. L. (1987). Antigenic related- ness between glycoproteins of human respiratory syncytial virus

18 BARRETT ET AL.

subgroups A and B: Evaluation of the contributions of F and G glycoproteins to immunity. /. Viral. 61, 3163-3 166.

LAEMMLI, U. K. (1970). Cleavage of structural proteins during the as- sembly of the head of bacteriophage T4. Nature (London) 227, 680-685.

LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein estimation with folin phenol reagent. 1. Biol. Chem. 193,265-275.

MACKE-, M., SMITH, G., and Moss, B. (1984). Selection of infectious vaccinia recombinants expressing foreign genes. J. viral. 49,857- 864.

MERZ, D. C., SCHEID, A., and CHOPPIN, P. W. (1980). Importance of antibodies to the fusion glycoprotein of paramyxoviruses in the prevention and spread of infection. /. Exp. Med. 151, 275-288.

NORRBY, E., UTTER, G., ORVELL, C., and APPEL, M. (1986). Protection against canine distempervirus in dogs afterimmunisation with isolated fusion protein. /. l&o/. 58, 536-541.

PASTORET, P.-P.. BROCHIER, B., LANGUET, B., THOMAS, I., PAQUOT, A., BAUDUIN, B., KIENY, M. P., LECOCCI, J. P., DEBRUYN, J., COSTY. F., ANTOINE, H., and DESMETTRE, P. (1988). First field trial of fox vaccination against rabies using a vaccinia-rabies recombinant virus. Vet. Rec. 123,481-483.

PLOWRIGHT, W. (1962). The application of monolayer tissue culture techniques in rinderpest research. II, The use of attenuated culture virus as a vaccine for cattle. Bull. Off lnt. fpizoot. 57, 253-276.

PORTNER, A., SCROGGS, R. A., and NAEVE, C. W. (1987). The fusion glycoprotein of sendai virus: Sequence analysis of an epitope in- volved in fusion neutralisation. Virology 157, 556-559.

RAY, R., GLAZE, B., and COMPANS. R. (1988). Role of individual glycoproteins of human parainfluenza virus type 3 in the induction of a protective immune response. 1. viral. 62,783-787.

SCOTT, G. R., TAYLOR, W. P., and ROSSITER, P. B. (1986). “Manual for the Diagnosis of Rinderpest.” FAO, Rome.

SPRIGGS, M., MURPHY, B. R., PRINCE, G. A., OLMSTED, R. A., and COL- LINS, P. L. (1987). Expression of the F and HN glycoproteins of human parainfluenza virus type 3 by recombinantvaccinia viruses: Contributions of the individual proteins to host immunity. J. Viral. 61,3416-3423.

STOW, E. J., TAYLOR, G., BALL, L. A., ANDERSON, K., YOUNG, K.K.-Y., KING, A. M. Q., and WERTZ, G. W. (1987). Immune and histopatho- logical responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus. /. viral. 61, 3855-3861.

TARTAGLIA, J., and PAOLE~I, E. (1988). Recombinant vaccinia virus vaccines. Tibtech. 6, 43-46.

TSUKIYAMA. K., YOSHIKAWA, Y., ~~~YAMANOUCHI, K. (1988). Fusion glycoprotein (F) of rinderpest virus: Entire nucleotide sequence of the F mRNA, and several features of the F protein. virology 164, 523- 530.

VARSANYI, T. M., MOREIN, B., LOVE, A., and NORRBY, E. (1987). Protec- tion against lethal measles virus infection in mice by immune-stimulating complexes containing the haemagglutinin or fusion protein. J. Viral. 61,3896-3901.

YILMA, T., Hsu, D., JONES, L., OWENS, S., GRUBMAN, M., MEBUS, C., YAMANAKA, M., and DALE, B. (1988). Protection of cattle against rinderpest with vaccinia virus recombinants expressing the HA or Fgene. Science242, 1058-1061.

Documents

Immunization with a vaccinia recombinant expressing the F protein protects Rabbits from challenge with a lethal dose of rinderpest virus