VIROLOGY

183,703-710

Protection

(1991)

of Mice with Vaccinia Virus Recombinants

JOHN W. SUMNER,’ Division

MAKONNEN

of Viral and Rickettsial

That Express the Rabies Nucleoprotein

FEKADU, JOHN H. SHADDOCK, JOSEPH J. ESPOSITO,

Diseases.

Center

Received

for infectious January

Diseases,

17, 199 1; accepted

Centers April

for Disease 22.

Control,

AND Atlanta,

WILLIAM J. BELLINI Georgia

30333

199 1

The role of rabies virus nucleoprotein (N) in protection against rabies was examined with recombinant vaccinia viruses expressing the N of the Challenge Virus Standard strain. Two chimeric plasmids were constructed with the open reading frame of the N gene placed downstream of the vaccinia P,,, promoter (early/late class) or the vaccinia P,, promoter (late class), with each expression cassette flanked by vaccinia thymidine kinase (TK) sequences to enable marker rescue by TK insertional inactivation. Two recombinants were isolated that expressed the rabies N in infected cells as determined by radioimmunoprecipitation and immunofluoresence microscopy with an anti-N monoclonal antibody. Two groups of 25 ICR mice inoculated intradermally with the recombinants and challenged with 75 MFPLD,, of street rabies virus showed high survival ratios (22/25 and 21/25). Intramuscular inoculation, however, was not protective against 25 MFPLD,,. The intradermally vaccinated mice developed non-neutralizing antibodies against rabies N. 0 1991 Academic Press, Inc.

INTRODUCTION

istered in complete Freund’s adjuvant to mice and raccoons, have been reported to elicit a protective response against rabies (Dietzschold et al., 1987). We have begun to examine the role of the N protein in the protective immune response to rabies by constructing vaccinia-rabies N recombinants. The present report describes N protein expression under control of early/late or late vaccinia promoters and the analysis of the recombinant-expressed N. We also demonstrated the efficacy of the recombinants in protecting mice against lethal rabies challenge.

Rabies virus is the prototypic member of the Lyssavirus genus in the Rhabdoviridae family, containing about 12 kb of single-stranded, nonsegmented, negative-sense RNA that encodes five structural proteins. Three of these proteins, the nucleoprotein (N), phosphoprotein (variously called P, Ml, or NS), and the large polymerase protein (L), associate with viral genomic RNA to form a ribonucleoprotein (RNP). The viral matrix protein (M) and transmembrane-spanning glycoprotein (G) are associated with the virion lipid envelope that surrounds the RNP (Baer et al., 1990). Rabies virus-neutralizing antibodies are directed at G, and their levels correlate well with protection against rabies (Cox eta/., 1977). In addition, reports on influenza virus (Bennink ef al., 1987; McMichael et a/., 1986; Townsend el al., 1984; Yewdell et al., 1985), vesicular stomatitis virus (Puddington et al., 1986), and respiratory syncytial virus (Bangham et al., 1986; King et a/., 1987), for example, have indicated that internal virion proteins play crucial roles in evoking protective responses against infection. In particular, infection with these viruses showed cytotoxic lymphocyte (CTL) responses directed against the respective N and various other proteins of these viruses, suggesting that CTL response aids in the clearance of virus-infected cells. Furthermore, purified rabies virion RNPs admin-

Sequence EMBUGenBank ’ To whom dressed.

MATERIALS

AND

METHODS

Cells and viruses Mouse neuroblastoma cells were used to propagate the Kissling Challenge Virus Standard (CVS) strain of rabies virus (Wiktor et a/., 1977). Human TK-minus 143B cells (Rhim eta/., 1975) and monkey kidney CV-1 cells were used for production of the Copenhagen vaccinia virus rabies N recombinants described below. We also made Copenhagen vaccinia rabies G recombinants essentially as previously described, using TK insertion of rabies G coding sequences into the New York Board of Health strain of vaccinia virus (Esposito et al., 1987). Vaccinia viruses were purified from infected cells by sedimentation in sucrose gradients (Esposito et al., 1978). Rabies-N

data

from this article have been deposited with the Data Libraries under Accession No. M61047. correspondence and reprint requests should be ad-

cDNA repair

A cDNA clone of N sequences of CVS was kindly provided by 1. Obijeski and B. Holloway. In their experi703

0042.6822/91

$3.00

Copyright 0 1991 by Academic Press. Inc. All whts of reproduction I” any form resewed.

704

SUMNER

ments, the cDNA had been synthesized by reverse transcription of viral poly(A) selected mRNA from rabies-infected BHK-21 cells. When we determined the nucleotide sequence of the inserted cDNA by the method of Maxam and Gilbert (1980) we found it lacked the first seven amino-terminal nucleotides of the N-coding region compared with the partial genomic RNA sequence reported for the same strain (Kurilla et al., 1984) and for the N sequence of a related rabies virus, the Pasteur strain (Tordo et a/., 1986). We also noted that the clone (pRabN) had about 70 bases at the 5’ end of the open reading frame (ORF) that were complementary to an internal region of N sequence. These extra nucleotides probably arose inadvertently during cDNA synthesis. We removed the 70 bases and repaired the 5’ end of the coding sequence with a synthetically prepared, double-stranded oligonucleotide fragment, 5’-AAlTCC ATG GAT GCC GAC AAG-3’. The oligonucleotide was designed to incorporate codons for the first five amino-terminal amino acids of N and add a 5’ overhanging EcoRl site to enable ligation into vaccinia shuttle vectors pGS62 and pKB3. The vector pGS62 utilizes the vaccinia early/late P,,, promoter (Mackett et al., 1984); pKB3 utilizes the vaccinia late P,, promoter (Esposito et a/., 1987). All routine cloning manipulations were performed essentially as described by Maniatis et a/. (1982). The correct insertion of the coding sequence was confirmed by restriction mapping of the chimeric plasmids and sequencing through the respective vaccinia promoter-N gene junctions (Maxam and Gilbert, 1980).

Screening

recombinant

viruses

Recombinants expressing rabies N were identified by immunoblotting cells infected with viral plaques formed under overlays containing bromodeoxyuridine (Mackett et a/., 1985). One recombinant based on pGS62RabN was designated VV,,B RabN, and one based on pKB3RabN was designated VV,, RabN. After each recombinant had been plaque-purified three times, the insertion of the rabies N cDNA was confirmed by Southern blotting (Esposito et al., 1988; Maniatis et al., 1982; Johnson et al., 1984).

ET AL.

cells infected with either of the VV RabN recombinants. lmmunoprecipitates were bound to staphylococcal protein A (ICN immunobiologicals, Lisle, IL) and pelleted by centrifugation (Kessler et a/., 1975). The pelleted proteins were denatured and then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970). Rabies N was also detected by indirect fluorescent antibody assay (IFA) of recombinantinfected 143B cells. For this procedure, infected cell monolayers were fixed with acetone and reacted with a monoclonal antibody against rabies N (Smith et al., 1984). Cells were then washed in phosphate-buffered saline (PBS) and reacted with a goat anti-mouse(IgG)-fluorescein isothiocyanate conjugate (Tago, Inc., Burlingame, CA). Mouse vaccination

and serologic

To establish the effective dose, 6-week-old ICR/cDS mice were intradermally injected with varying doses of a recombinant expressing N (VV,, RabN) or G (VV,, RabG). Unless indicated otherwise, mice were scarified on the buttocks and 1O7 PFU of gradient purified virus in 0.03 ml (PBS with 209/o sucrose) was applied for intradermal vaccination. Mice were challenged at 4 weeks postvaccination (p.v.) by footpad injection with Mexican dog 5951 (MD5951) street rabies virus (Fekadu et al., 1982). In another experiment, intramuscular and intradermal routes of vaccination with these recombinants were compared. Finally, groups of 25 to 28 ICRIcDS mice were intradermally vaccinated separately with VV,,E, RabN, VV,, RabN, VV,, RabG, or Copenhagen vaccinia and challenged as stated above. To monitor the development of antibody, mice selected from each group were earmarked prior to vaccination and bled from the periorbital sinus at 2 and 4 weeks p.v., and at 5 days postchallenge. Serum samples from these mice were tested for rabies VNA by the rapid fluorescent focus inhibition test (RFFIT) (Smith et a/., 1973), in which MD5951 virus was used as challenge virus. End-point titers were calculated by the method of Reed and Muench (Reed and Muench, 1938). Antibodies against rabies N were determined by IFA of acetone-fixed Spodoptera frugiperda cells that had been infected with a recombinant baculovirus expressing N of the CVS strain (Reid-Sandin et a/., 1990). Mouse sera were tested at the 1: 100 dilution.

Radioimmunoprecipitation and immunofluorescence microscopy Mouse anti-rabies hyperimmune serum was reacted with 35S-radiolabeled proteins from mouse neuroblastoma cells infected with rabies virus and from CV-1

testing

RESULTS Recombinant

viruses

The insertion of the promoter-rabies N cDNA cassette by homologous recombination through the flank-

VACCINIA-RABIES

NUCLEOPROTEIN

705

RECOMBINANT

gene probe with parental Copenhagen viral DNA fragments was observed (Fig. 1B, lane 1). The hybridization of N gene and TK gene probes to the ca. 7-kb fragments in the recombinants, coupled with the fact that the recombinants were TK-, suggested that appropriate insertional inactivation of the TK gene occurred and that a single copy of the rabies N gene had been successfully inserted in the TK locus of the parent virus. The ca. 7-kb HindIll fragment of VV,., RabN was slightly larger than that of VV,, RabN because the vaccinia early/late promoter sequence in pGS62 comprised 350 bases and the late promoter in pKB3 comprised 200 nucleotides (Esposito et al., 1987). Rabies N expression FIG. 1. Southern blot analyses of HindIll-digested vaccinia/rabies N recombinant DNAs. HindIll-digested genomic DNA from wild-type Copenhagen vaccinia virus (lanes l), recombinant W,,, RabN (lanes 2) and recombinant VV,, RabN (lanes 3) were electrophoretically separated in agarose (0.7%) gels and stained with ethidium bromide (A). The DNA fragments were bidirectionally transferred to nitrocellulose sheets and hybridized with either radiolabeled rabies N gene sequences (B) or radiolabeled vaccinia TK gene sequences (C). HindIll-digested X DNA and Haelll-digested phiX174 DNA were included in the far left lane of (A) as molecular size markers. The arrow in (A) indicates the position of the HindIll-J fragment of wild-type vaccinia DNA (lane 1).

ing TK sequences was confirmed by hybridization of vaccinia TK and rabies N-radiolabeled probes with Southern blots of HindIll-restricted DNA from vaccinia virus and the recombinants VV,,, RabN and W,, RabN (Fig. 1). The restriction digest pattern of the Copenhagen strain of vaccinia virus (lane l), VV,,, RabN (lane 2), and VV,, RabN (lane 3) is presented in Fig. 1A. Although the HindIll DNA digest patterns of recombinants VV,,, RabN and VV,, RabN were similar to that of parental Copenhagen vaccinia, both digest lacked the HindIll-J fragment (arrow) containing the TK gene. Instead, both recombinant DNA digest contained a unique HindIll fragment of approximately 7.0 kb, which was not observed in the wild-type digest. Radiolabeled TK gene probes specifically hybridized to the HindIll-J fragment of Copenhagen vaccinia DNA (Fig. 1 C, lane 1) and to larger HindIll fragments of the recombinant DNA digest which were identical in size with those found uniquely associated with the recombinant DNAs (Fig. 1C, lanes 2 and 3). Similar Hindlll-restricted DNA fragments from the recombinant viruses were detected when a radiolabeled rabies N gene probe was used in hybridization experiments (Fig. 1 B, lanes 2 and 3). In contrast, no hybridization of the N

Results of a time course study of rabies N synthesis in cells infected with W,,, RabN and VV,, RabN (Figs. 2A and 2B) were used to identify a protein, with both recombinants, that comigrated with authentic N immunoprecipitated from rabies virus-infected cells (lanes R). The relative abundance of the expressed N was increased about 1O-fold in W,, RabN infected cell lysates compared with VV,,, RabN-infected cell lysates, when the same number of cells were separately infected with equivalent PFU of virus per cell. Production of N in VV,,, RabN-infected cells was first detected at 1 to 2 hr postinfection (Fig. 2A); synthesis continued through 8 hr, the late time period examined. Synthesis of N in CV-1 cells infected with W,, RabN began at about 4 hr postinfection (Fig. 2B) with the P,, promoter driving expression. lmmunofluorescence

microscopy

of N

Figure 3 shows the immunofluorescent staining with anti-N monoclonal antibody of 1438 cells infected with either W,,, RabN (A) or VV,, RabN (B). By IFA, N was observed only in the cytoplasm of cells infected by the recombinants, and the appearance of the immunofluorescence was similar to that observed for N in rabies virus-infected cells. Use of a monoclonal antibody specific for rabies G showed no fluorescence in cells infected by the N recombinants (not shown). Protection

of mice

Having characterized expression of rabies N in cell culture, we next conducted mouse protection studies to determine whether expression of N in mice infected with the recombinants would protect against lethal rabies challenge. The number of rabies deaths in groups of mice given different intradermal doses of recombinants expressing either rabies N (W,, RabN) or G (W,, RabG) is presented in Table 1. A minimal dose of 1 O3

706

SUMNER

ET AL.

NS-

-

M-

R

1

2

3

4

5

6

7

8

wt

FIG. 2. Expression of rabies N protein in vaccinia recombinant-infected beled, immunoprecipitated rabies N from recombinant-infected CV-1 cells postinfection (lanes l-8). The lanes marked R and WT contain 35S-labeled, infected with the CVS strain of rabies virus (lane R) or CV-1 cells infected

PFU of VV,, RabG was determined to protect 100% of the mice challenged; however, a minimal dose of lo7 PFU of VV,, RabN was needed to fully protect mice against the same 37 MFPLD,, of rabies virus. Table 2 shows the results of an experiment comparing two routes of vaccination, All mice vaccinated intradermally with 10’ PFU of W,, RabN were protected against a challenge of 25 MFPLD,, of street virus, whereas, 90% of mice vaccinated intramuscularly with the same dose of W,, RabN died of rabies. Apparently, the protection with VV,, RabN depends on the route of vaccination. All mice vaccinated with 10’ PFU of VV,, RabG by either route were fully protected against challenge. Finally, groups of 25 to 28 ICR/cDS mice were intradermally inoculated with either VV,., RabN, VV,, RabN, Copenhagen vaccinia virus, or VV,, RabG (Table 3). Mice were examined for signs of rabies infection for 3 weeks after challenge with 75 MFPLD,, of street virus. All mice that received VV,, RabG were protected. Eighty-eight percent of mice vaccinated with VV,,, RabN and 84% of mice vaccinated with W,, RabN survived challenge; however, 93% of mice vaccinated with Copenhagen vaccinia virus and 100% of mice not vaccinated died of rabies.

R12345678 cells. Autoradiographs of SDS-polyacrylamide gels showing (VV,,, RabN in A; W,, RabN in B) pulse labeled and harvested immunoprecipitated proteins from either mouse neuroblastoma with wild-type Copenhagen vaccinia (lane WT).

%la1 to 8 hr cells

Sera from five or six mice from each group were tested by the RFFIT for VNA (anti-G) response and by IFA with baculovirus-expressed rabies N for anti-N antibody response. Only mice vaccinated with the VV,, RabG recombinant developed VNA before challenge, and a booster response was observed after challenge (Table 4). However, low levels of VNA were detected in blood samples taken 5 days after rabies challenge from mice vaccinated with either the W RabN recombinants or the Copenhagen vaccinia virus. The VNA titers induced by the challenge virus were quite similar in mice vaccinated with the N recombinants or the Copenhagen vaccinia virus. Mice vaccinated with the N recombinants clearly developed antibodies against N as determined by IFA. At 5 days postchallenge, antibodies to N were not detected in sera of mice in groups vaccinated with either W,, RabG or Copenhagen vaccinia virus. DISCUSSION The results of our studies in mice demonstrate that Copenhagen vaccinia rabies N recombinants given intradermally can induce a protective response against peripheral challenge with street rabies virus. The dose

VACCINIA-RABIES

FIG. 3. Rabies using

a monoclonal

N expressed antibody

in 1438 cells infected that reacts specifically

NUCLEOPROTEIN

with (A) W,,, RabN with rabies N.

of VV RabN needed to protect (10') was, however, approximately 10,000 times greater than the minimum effective dose of VV RabG (1 03). Previously, Dietzschold et al. (1987) reported that purified rabies virus RNPs can elicit a similar protective response, but we have extended those results by using vaccinia virus recombinants that express a single rabies virus protein, eliminating the possibility of a response to residual other rabies proteins that could occur in RNP preparations. In addition, Dietzschold e2 a/. (1987) reported an enhanced VNA response upon subsequent exposure to whole inactivated rabies virus in mice immu-

or (B) VV,,

RECOMBINANT

RabN

was

detected

by indirect

fluorescent

antibody

assay

nized with purified rabies RNP, and that RNP-protected animals had higher VNA titers than control animals 5 days after challenge with rabies street virus. The authors suggested that the enhanced VNA response induced by rabies RNP could be one of several mechanisms resulting in protection. However, we did not see such a response after mice vaccinated with W-RabN were challenged, suggesting that other factors may have contributed to the protective response. It is interesting to speculate what immunological mechanisms come into play in this protection. For example, the presence of VNA prior to infection is known to be protec-

708

SUMNER ET AL. TABLE 1

TABLE 3

.

PROTECTION FROM RABIES CHALLENGE OF MICE VARYING DOSES OF VACCINIA VIRUS RECOUBINANTS RABIES N OR G

VACCINATED WITH EXPRESSING THE

PROTECTION OF MICE IMMUNIZED WITH VACCINIA RECOMBINANTS EXPRESSING THE RABIES VIRUS NUCLEOPROTEIN

Vaccine8

Mortality

W,,, RabN W,, RabN VV,, RabG W Copenhagen not vaccinated

3/25 4/25 0126 26J28 12112

Mortality Dosea

10’ 106 lo5

lo4 103 102

VV,, RabN

VV,, RabG

O/10 3110 2110 6/l 0

o/10 o/10 o/to o/10

9/10 lo/lo

o/10

a/to

a Recombinant viruses were administered intradermally by scarification Mice were challenged with 37 MFPLD,, of the MD5951 street rabies virus at 4 weeks p.v.

tive; this association has been noted after injection of whole virion vaccine (Bunn et al., 1984), a rabies neutralizing monoclonal antibody (P. Wunderli, personal communication), or rabies G recombinants of various types (Esposito et al., 1987, 1988; Wiktor et al., 1984). Yet protection is also noted in the absence of VNA (Dean et al., 1964; Sikes et al., 1971; Fekadu et al., 1985), indicating that other factors, such as cell-mediated immune responses, play a role in the protection against rabies. It may be that the presence of antibodies against rabies G prevents initial infection and thereby viral replication. In support of this assumption, we were unable to find antibodies to rabies N in the sera of VV RabG-vaccinated mice that were bled 2 weeks after rabies challenge, whereas most nonvaccinated control mice did produce antibodies to rabies N within 2 weeks after challenge (data not shown). In our previous studies with mice administered equal doses of VV RabG intradermally and intramuscularly,

TABLE 2 PROTECTION FROM RABIES CHALLENGE IN MICE VACCINATED INTRAMUSCULARLY OR INTFIADERMALLY WITH VACCINIAJRABIES RECOMBINANTS

Mortality Vaccine8

i.m.

i.d.

W,, RabN W,, RabG W Copenhagen

i a/20

O/l 9

O/20 18720

O/20 13120

a Mice were vaccinated with 10’ PFU of virus by intramuscular (i.m.) injection or intradermally (i.d.) by scarification. The mice were challenged 2 weeks p.v. by footpad injection of 25 MFPLD,, of Mexican dog 5951 street rabies virus.

* Mice received 10’ PFU of virus by scarification. Four weeks postvaccination mice were challenged with 75 MFPLD,, of Mexican dog 5951 street rabies.

the intradermal group had mean VNA titers approximately four times higher than the intramuscular group (Esposito el al., 1987). Remarkably, however, with W RabN the immunity was achieved after intradermal application, but not after intramuscular injection of an equal dose (although we might have obtained protection with higher intramuscular doses of W RabN). One possible explanation for this result is that Langerhans cells, found mainly in the skin but rarely in muscle, are involved in the presentation of antigens and thus may play an important part in the immune response to vaccinia (Nagao era/., 1976). In recent years, the amount of rabies vaccine needed to protect exposed persons has been reduced by administering only 10% of the conventional vaccine dose intradermally, indicating that vaccination with whole virion vaccine may also be more effective when administered intradermally than intramuscularly (Dreesen et al., 1982; Phanuphak et a/., 1987; Warrell et al., 1985). These observations raise important questions about the role of rabies N. What protective mechanisms are induced by rabies N protein that are not activated by rabies G? Will a more balanced immune response occur in animals vaccinated with recombinants expressing both N and G proteins, providing a longer duration of immunity and better protection against divergent strains? Obviously, immune responses occurring in animals vaccinated with VV RabG only, or VV RabN only, will be different than those in animals that receive whole virus vaccine. In a preliminary study, in which skunks were fed a combination of raccoon poxvirus-rabies N and raccoon poxvirus-rabies G recombinants, no increased protection was observed over those animals fed the raccoon poxvirus-rabies G recombinant alone (M. Fekadu et al., in preparation). Moreover, if recombinant vaccines expressing rabies N are used to aid medical practice, will they offer better protection against the rabies like viruses, for which current vaccines may not be efficacious? To this end, Dietzschold

VACCINIA-RABIES

NUCLEOPROTEIN

709

RECOMBINANT

TABLE 4 SERUM ANTIBODY RESPONSES IN MICE IMMUNIZED WITH VACCINIA VIRUS RECOMEIINANTVIRUSES THAT EXPRESS EITHER THE RABIES NUCLEOPRO~EIN OR GLYCOPROTEIN 4 weeksa postvaccination

2 weeks’ postvaccination

5 days”

postchallenge

Vaccine

RFFIT

IFA

RFFIT

IFA

RFFIT

IFA

VV (7.5) RabN VV (11) RabN W(ll)RabG W Copenhagen