Vol. 65, No. 10
JOURNAL OF VIROLOGY, Oct. 1991, p. 5593-5596
0022-538X/91/105593-04$02.00/0 Copyright © 1991, American Society for Microbiology
Neutralizing Antibodies Induced by Recombinant Vaccinia Virus Expressing Varicella-Zoster Virus gpIV ABBAS VAFAI* AND WEINING YANGt
Department of Biomedical Sciences, University of Illinois College of Medicine, Rockford, Illinois 61107 Received 5 April 1991/Accepted 18 June 1991
Monoclonal antibodies generated against varicella-zoster virus (VZV) glycoprotein I (gpl) also recognize VZV gpIV (A. Vafai, Z. Wroblewska, R. Mahalingam, G. Cabirac, M. Wellish, M. Cisco, and D. Gilden, J. Virol. 62:2544-2551, 1988). To determine whether the virus-neutralizing activity of these antibodies belongs to gpl, gpIV, or both, the open reading frame encoding gpIV was inserted into the vaccinia virus genome. Immunoprecipitation of recombinant vaccinia virus-infected cells with anti-gpIV monoclonal antibody yielded synthesis and processing of gpIV similar to those expressed in VZV-infected cells. Antibodies raised against WgpIV in a rabbit recognized both native gpI and gpIV and neutralized VZV infectivity. In addition, antibodies raised against recombinant vaccinia virus carrying VZV gpl neutralized VZV infection. These results indicate a structural relationship between VZV gpI and gpIV and show that gpl and gpIV each induce virus-neutralizing antibody. Varicella-zoster virus (VZV) DNA encodes five glycoproteins, designated gpl through gpV, which are highly immunogenic and stimulate the immune response following a primary encounter with VZV (varicella) and viral reactivation (zoster) (5, 6, 16). VZV genes 67 and 68 are two adjacent genes which are located within the unique short sequences (Us) of the VZV genome and encode gpIV and gpI, respectively (6, 7, 9). DNA sequence analysis has shown two distinct open reading frames for the gpIV and gpl genes without any obvious amino acid sequence relatedness (6). In addition, the products of gpl and gpIV genes have been identified and characterized by monoclonal antibodies (MAbs) generated against these glycoproteins and antibodies to synthetic peptides derived from the predicted gpIV amino acid sequences (7, 9). gpl is the most abundant and immunogenic VZV glycoprotein and induces both complementdependent neutralizing antibodies and cell-mediated immunity (1, 2, 10, 12, 14, 15). However, MAbs prepared against gpl also recognize gpIV. This coprecipitation could result from either shared antigenic determinants (23) or physical interaction of the two proteins resulting in formation of heterodimers or heteromultimers. Because of this association, it is unknown whether the neutralizing activity of antibodies prepared against these glycoproteins belongs to gpl or gpIV or both. In this study, recombinant vaccinia viruses expressing either gpI or gpIV were produced and characterized in order to answer these questions. Construction and expression of recombinant vaccinia virus. The recombinant plasmid (pGEM-3) containing a 1,375-bp AvaI DNA fragment (spanning nucleotides 114385 to 115760 of the VZV genome) and containing the coding region of the gpIV gene (6, 23) was cleaved with the AvaI restriction enzyme. The AvaI fragment was extracted by electroelution and cloned at the SmaI site of insertion vector pSC11 (Fig. 1) as described previously (4, 23). The recombinant plasmid was designated pVVgpIV. The recombinant vaccinia virus was constructed by the method described previously (18) with minor modifications. Briefly, 143B thymidine kinase-
* Corresponding author. t Present address: Amgen Inc., Thousand Oaks, CA 91320.
negative (TK-) cells (18) were grown overnight in the absence of bromodeoxyuridine and were then infected with vaccinia virus (strain IHDJ) at a multiplicity of infection of 0.01 and incubated at 37°C for 90 min. The infected cells were then transfected with 30 ,ug of pVVgpIV DNA and 50 ,ug of Lipofectin reagent (GIBCO-BRL) according to the manufacturer's instructions. Cells were harvested 48 h after infection and transfection, and the resulting virus stock was passaged twice in TK- cells in the presence of 25 ,ug of bromodeoxyuridine per ml. Recombinant vaccinia virus was differentiated from spontaneous TK- virus by staining with Bluo-Gal (GIBCO-BRL) and was isolated by three cycles of plaque purification. The expression of recombinant vaccinia virus carrying VZV gpIV (designated VVgpIV) was analyzed by pulse-chase experiments with BSC-1 cells (23) infected with VVgpIV. Cells were infected with VVgpIV at a multiplicity of infection of 1.0; after a 22-h incubation period at 37°C, the cells were labeled with 300 ,uCi of [35S]methionine (specific activity, >1000 Ci/mmol; Amersham) per ml for 10 min in the absence or presence of 15 ,ug of tunicamycin per ml. The cells were either harvested or washed five times with serumfree medium, and the label was chased in normal medium for 120 min in the absence or presence of tunicamycin (15 ,ug/ml). The cells were then washed three times with cold phosphate-buffered saline and disrupted in 4 ml of lysis buffer (0.02 M sodium phosphate [pH 7.6], 0.1 M NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS]). The cell lysates were kept on ice for 2 h and centrifuged at 40,000 rpm in a Beckman SW60 rotor for 2 h at 5°C. The supernatants were immunoprecipitated with MAb G6, which recognizes VZV gpIV, and analyzed by SDS-10% polyacrylamide gel electrophoresis (PAGE) (23). MAb G6 was prepared according to procedures described previously (21-24). Figure 2 shows the expression and processing of VZV gpIV in VVgpIV-infected cells. A 50-kDa protein band was detected during a 10-min pulse period which was further processed to a mature form of gpIV (60 kDa) during a 120-min chase period. In the presence of tunicamycin, which inhibits the addition of N-linked oligosaccharides to native gpIV (23), a 36-kDa protein species was detected during the 5593
5594
J. VIROL.
NOTES IRL IRS US TRS
TRL
-TM + TM
-TM
U
U
P
P CI P C I P
P
+TM
C IP
-TM c
+TM
U
I
P
P C IP
W.
C
v--
_,
'A 9582-
gp
60-
"
50-
US
a mD
45-
gpiv
Am -.j
36-
FIG. 1. Construction of a recombinant vaccinia virus insertion plasmid carrying the VZV gpIV gene. The VZV gpIV gene cloned in transcription vector pGEM-3 (23) was cleaved with restriction enzyme AvaI (A), electroeluted, blunt-ended, and cloned at the SmaI (S) site of vaccinia virus insertion vector pSC11 as described in the text. A physical map of VZV DNA and the location of gpIV on the viral genome are shown.
pulse and a 45-kDa protein was detected during the chase period. These results were in agreement with those reported earlier (19, 20, 23) and suggested that gpIV expressed in VVgpIV contains both 0-linked and N-linked oligosaccharides. Similar patterns of gpIV synthesis and processing were observed when VZV-infected cells were pulse-chased and immunoprecipitated with MAb G6, which is directed against VZV gpIV and also recognizes gpl (Fig. 2B). These results indicated that the expression of VZV gpIV in recombinant vaccinia virus was similar to the expression of native gpIV in VZV-infected cells. However, the mature form of gpIV expressed by VVgpIV was somewhat larger than the native gpIV, suggesting the possibility of further glycosylation of this glycoprotein when it is expressed in vaccinia virus. Antibody response to VVgpIV. Antibodies against VVgpIV were generated in a rabbit by immunization procedures described previously (21). Briefly, a New Zealand White female rabbit was immunized subcutaneously at multiple sites in the back and the hind legs with 107 PFU of VVgpIV. This immunization was followed by three weekly injections each consisting of 107 PFU. The animal was bled 7 days after the last injection, and the serum (designated RAnti-VVgpIV) was assayed by immunoprecipitation. Pulse-chase experiments and immunoprecipitation of VZV-infected cells revealed that RAnti-VVgpIV recognized the high-mannose form (50 kDa) and the mature form (60 kDa) of VZV gpIV (Fig. 2C). In addition, RAnti-VVgpIV reacted with precurTABLE 1. VZV neutralization test in the presence of complement Antibody
RAnti-VZV
RAnti-VVgpI RAnti-VVgpIV
NRSb
Target
No. of
protein(s)
plaques"'
Plaque reduction (%)
Viral proteins VZV gpl VZV gpIV
0 10 28 130
100 92.3 78.5 0
" Average number of plaques in duplicate wells of six-well plates, determined with a 1:10 dilution of each serum sample. b NRS, normal rabbit serum (serum obtained from nonimmunized animals).
a
A
I
B
a
I
-
c
FIG. 2. (A) Expression of a recombinant vaccinia virus carrying the VZV gpIV gene (VVgpIV). BSC-1 cells were infected with VVgpIV, and after 22 h, infected cells were pulse-labeled (P) with [35S]methionine (300 p.Ci/ml) for 10 min in the absence or presence of tunicamycin (15 ,ug/ml) (TM). The cells were either harvested or washed, and the label was chased (lanes C) for 2 h in the absence or presence of TM (15 p.g/ml). U, uninfected cells were pulsed for 10 min. The cell lysates were prepared and immunoprecipitated with MAb G6, which recognizes VZV gpIV. (B) Immunoprecipitation of VZV-infected cells with MAb G6. BSC-1 cells were infected with VZV, and after 72 h postinfection, the cells were pulse-chased as described for panel A, and the cell lysates were immunoprecipitated with MAb G6. (C) Immunoprecipitation of VZV-infected cells with antibodies against VVgpIV (RAnti-VVgpIV) generated in a rabbit. VZV-infected cells were pulsed for 10 min and chased for 2 h as described for panel, A and the cell lysates were immunoprecipitated with RAnti-VVgpIV. Samples were analyzed by SDS-PAGE (10% polyacrylamide) as described previously (23). The apparent sizes (in kilodaltons) of the precursor products of VZV gpl and gpIV are shown on the left. Lysozyme (14.3 kDa), P-lactoglobulin (18.4 kDa), a-chymotrypsinogen (25.7 kDa), ovalbumin (43.0 kDa), bovine serum albumin (68.0 kDa), phosphorylase B (97.4 kDa), and myosin (200.0 kDa) were used as internal size markers.
sor products (78, 82, 90, and 95 kDa) of VZV gpL. These results indicated that the native VZV gpIV was recognized by RAnti-VVgpIV. The results also supported our previous findings (23), suggesting the recognition of gpl and gpIV in VZV-infected cells by antibodies raised against either of these glycoproteins. RAnti-VVgpIV was also tested for VZV-neutralizing activity. Neutralization tests were performed by the constantvarying serum technique as described previously (21). The results (Table 1) showed a plaque reduction of 100% with a 1:10 dilution of antibodies raised against purified VZV virions (21) in the presence or absence of complement and a plaque reduction of 78.5% with a 1:10 dilution of RAntiVVgpIV only in the presence of complement. To determine whether VZV gpl alone is also capable of inducing virusneutralizing antibody response, antibodies against a recombinant vaccinia virus (designated VVgpI) carrying the VZV gpl gene were prepared in a rabbit (3). Antibodies against VVgpI were prepared as described above and designated RAnti-VVgpI. Neutralization tests using RAnti-VVgpI also indicated a plaque reduction of 92.3% only in the presence of complement. These results demonstrated that both VZV gpl and gpIV are capable of inducing complement-dependent neutralizing antibodies.
NOTES
VOL. 65, 1991
a b
ab6
ab
a b
u P
P C
5595
P C
* *.
ir
up
gi
gPp
_~ -
_
D A B C FIG. 3. Immunoprecipitation of recombinant vaccinia viruses expressing VZV gpl (VVgpI) and gpIV (VVgpIV) with MAbs. BSC-1 cells (105) were infected with VVgpl (A), VVgpIV (B), or VVgpI and VVgpIV (C) at a multiplicity of infection of 1.0, and 22 h postinfection, the cells were labeled with [35S]methionine for 1 h. Cell lysates were prepared and immunoprecipitated with MAb G6 (lanes a) and MAb Cl (lanes b), which are directed against VZV gpIV and gpl, respectively. (D) Cell lysates from VVgpI- and VVgpIV-infected cells were combined and immunoprecipitated with MAbs G6 and Cl.
Since antibodies to gpl and gpIV are cross-reactive in VZV-infected cells, the following experiments were carried out to determine whether (i) MAbs cross-react when gpl and gpIV are expressed separately by recombinant vaccinia viruses and (ii) RAnti-VVgpIV binds to purified gpI. BSC-1 cells were infected with either VVgpI, VVgpIV, or both VVgpI and VVgpIV at a multiplicity of infection of 1.0, and 22 h postinfection, the cells were pulse-labeled with [35S]methionine (200 ,uCi/ml) for 1 h. The cell lysates were then immunoprecipitated with MAbs Cl and G6, which are directed against VZV gpl and gpIV, respectively. SDS-PAGE showed only the recognition of MAb Cl by gpl and of MAb G6 by gpIV (Fig. 3A and B). However, when VVgpI and VVgpIV were expressed in the same culture, MAb Cl also recognized gpIV (Fig. 3C). This recognition was not detected when cell lysates from VVgpI and VVgpIV were combined before immunoprecipitation (Fig. 3D). These results further support the association of gpI and gpIV and suggest that the expression of both proteins is necessary for physical association of these glycoproteins. To determine whether RAnti-VVgpIV binds to gpl, immunoglobulin G was purified from tissue culture fluid of MAb Cl-producing hybridomas by using protein G-Sepharose Fast Flow (Pharmacia LKB Biotechnology) as described previously (22). Purified immunoglobulin G was coupled to CNBr-activated Sepharose 4B beads and used to purify gpl from VVgpI-infected cell lysates as described previously (26). The purified gpl was used to prepare a column containing gpl coupled to CNBr-activated Sepharose 4B beads. RAnti-VVgpIV and MAb G6 were applied (10 times each) to the column, and the flowthrough was used to immunoprecipitate VZV-infected cell lysates. The results showed the reactivity of flowthrough RAnti-VVgpIV and MAb G6 with native gpIV in VZV-infected cells (Fig. 4). These results indicated that although preadsorption of RAnti-VVgpIV and
4410-
A
-.-
B
FIG. 4. Immunoprecipitation of VZV-infected cells with preadsorbed anti-gpIV MAb G6 and rabbit anti-recombinant vaccinia virus antibody expressing VZV gpIV (RAnti-VVgpIV). VZV gplspecific immunoglobulin G was purified and used to purify gpl from VVgpI-infected cell lysates as described in the text. Columns containing gpl-bound CNBr-activated Sepharose 4B beads were prepared and used to absorb gpl-specific antibody molecules in MAb G6 and RAnti-VVgpIV. Flowthrough samples were used for immunoprecipitation of VZV-infected cells. BSC-1 cells were infected with VZV. After 72 h postinfection, the cells were pulsed (P) for 10 min and chased (C) for 2 h, and the cell lysates were immunoprecipitated with flowthrough samples of MAb G6 (A) and RAnti-VVgpIV (B). U, uninfected cells were pulsed for 10 min.
MAb G6 resulted in the binding of some antibody molecules to gpl, amounts of unbound gpIV-specific antibody molecules sufficient to bind and precipitate gpIV in VZV-infected cells were present in RAnti-VVgpIV and MAb G6. Taken together, the lack of MAb G6 binding to VVgpI and the
recognition of native VZV gpIV by RAnti-VVgpIV which had been preabsorbed to purified gpl suggest that precipitation of VZV gpl and gpIV with RAnti-VVgpIV could be due to the physical association of these glycoproteins or due to both physical association and shared antigenic sites on gpl and gpIV. Although the biological functions of VZV gpl and gpIV during VZV replication have not yet been defined, these glycoproteins appear to be the initial targets for the induction of the immune response following VZV infection or VZV vaccination (8, 11, 13, 17, 25). Availability of recombinant viruses expressing VZV glycoproteins (e.g., gpl, gpIV, or both) not only will allow identification of antigenic epitopes which are important in the induction of humoral and cellmediated immunity but also will allow preparation of purified gpI and gpIV. These glycoproteins may have potential application for immunization against VZV infection or for boosting the immune response following VZV vaccination. We thank D. Kilpatrick for helpful discussions. This work was supported by a Public Health Service Program Project grant from the National Institutes of Health (POlAG0734) and by a grant from the National Multiple Sclerosis Society
(RG2354-A).
5596
J. VIROL.
NOTES
REFERENCES 1. Arvin, A. M., E. Kinney-Thomas, K. Shriver, C. Grose, C. M. Koropchak, E. Scanton, A. E. Wittck, and P. S. Diaz. 1986. Immunity to varicella-zoster viral glycoproteins, gpl (gp 90/58) and gpIII (gp 118), and to a nonglycoslated protein p170. J. Immunol. 137:1346-1351. 2. Arvin, A. M., S. M. Solem, C. M. Koropchak, E. KinneyThomas, and S. G. Paryani. 1987. Humoral and cellular immunity to varicella-zoster virus glycoprotein gpl and a non-glycosylated protein, P170, in the strain 2 guinea-pig. J. Gen. Virol. 68:2449-2454. 3. Cabirac, G., D. Gilden, M. Wellish, and A. Vafai. 1988. Expression of varicella-zoster virus glycoprotein I in cells infected with a vaccinia virus recombinant virus. Virus Res. 10:205-214. 4. Chakrabarti, S., K. Brechling, and B. Moss. 1985. Vaccinia virus expression vector: coexpression of P-galactosidase produces visual screening of recombinant virus plaques. Mol. Cell. Biol. 5:3403-3409. 5. Davison, A. J., C. M. Edson, R. W. Ellis, B. Forghani, D. Gilden, C. Grose, P. M. Keller, A. Vafai, Z. Wroblewska, and K. Yamanishi. 1986. New common nomenclature for glycoprotein genes of varicella-zoster virus and their glycosylated products. J. Virol. 57:1195-1197. 6. Davison, A. J., and J. E. Scott. 1986. The complete DNA sequence of varicella-zoster virus. J. Gen. Virol. 67:1759-1816. 7. Davison, A. J., D. J. Waters, and C. M. Edson. 1985. Identification of the products of a varicella-zoster virus glycoprotein gene. J. Gen. Virol. 66:2237-2242. 8. Dubey, L., S. P. Steinberg, P. LaRussa, P. Oh, and A. A. Gershon. 1988. Western blot analysis of antibody to varicellazoster virus. J. Infect. Dis. 157:882-888. 9. Ellis, R. W., P. M. Keller, R. S. Lowe, and R. A. Zivin. 1985. Use of a bacterial expression vector to map the varicella-zoster virus major glycoprotein gene, gC. J. Virol. 53:81-88. 10. Forghani, B., K. W. Dupuis, and N. J. Schmidt. 1984. Varicellazoster viral glycoproteins analyzed with monoclonal antibodies. J. Virol. 52:55-62. 11. Giller, R. H., S. Winistorfer, and C. Grose. 1989. Cellular and humoral immunity to varicella zoster virus glycoproteins in immune and susceptible human subjects. J. Infect. Dis. 160: 919-928. 12. Grose, C., D. P. Edwards, W. E. Friedrichs, K. A. Weigle, and W. L. McGuire. 1983. Monoclonal antibodies against three major glycoproteins of varicella-zoster virus. Infect. Immun. 40:381-388. 13. Grose, C., and V. Litwin. 1988. Immunology of the varicellazoster virus glycoproteins. J. Infect. Dis. 157:877-881. 14. Ito, M., T. Ihara, C. Grose, and S. Starr. 1985. Human leukocytes kill varicella-zoster virus-infected fibroblasts in the pres-
15. 16.
17.
18.
19.
20.
21. 22.
23.
24.
25.
26.
ence of murine monoclonal antibodies to virus-specific glycoproteins. J. Virol. 54:98-103. Keller, P. M., B. J. Neff, and R. W. Ellis. 1984. Three major glycoprotein genes of varicella-zoster virus whose products have neutralization epitopes. J. Virol. 52:293-297. Kinchington, P. R., P. Ling, M. Pensiero, B. Moss, W. T. Ruyechan, and J. Hay. 1990. The glycoprotein products of varicella-zoster virus gene 14 and their defective accumulation in a vaccine strain (Oka). J. Virol. 64:4540-4548. LaRussa, P. S., A. A. Gershon, S. P. Steinberg, and S. A. Chartrand. 1990. Antibodies to varicella-zoster virus glycoproteins I, II, and III in leukemic and healthy children. J. Infect. Dis. 162:627-633. Mackett, M., G. L. Smith, and B. Moss. 1985. The construction and characterization of vaccinia virus recombinants expressing foreign genes, p. 191-211. In D. M. Glover (ed.), DNA cloning: a practical approach. IRL Press, Oxford. Montalvo, E. A., R. T. Parmley, and C. Grose. 1985. Structural analysis of the varicella-zoster virus gp98-gp62 complex: Posttranslational addition of N-linked and 0-linked oligosaccharide moieties. J. Virol. 53:761-770. Okuno, T., K. Yamanishi, K. Shiraki, and M. Takahashi. 1983. Synthesis and processing of glycoproteins of varicella-zoster virus (VZV) as studied with monoclonal antibodies to VZV antigens. Virology 129:357-368. Vafai, A., K. Jensen, and R. Kubo. 1989. Existence of similar antigenic-sites on varicella-zoster virus gpl and gpIV. Virus Res. 13:319-336. Vafai, A., Z. Wroblewska, and L. Graf. 1990. Antigenic crossreaction between a varicella-zoster virus nucleocapsid protein encoded by gene 40 and a herpes simplex virus nucleocapsid protein. Virus Res. 15:163-174. Vafai, A., Z. Wroblewska, R. Mahalingam, G. Cabirac, M. Wellish, M. Cisco, and D. Gilden. 1988. Recognition of similar epitopes on varicella-zoster virus gpl and gpIV by monoclonal antibodies. J. Virol. 62:2544-2551. Vafai, A., Z. Wroblewska, M. Wellish, M. Green, and D. Gilden. 1984. Analysis of three late varicella-zoster virus proteins, a 125,000-molecular-weight protein and gpl and gp3. J. Virol 52:953-959. Watson, B., P. M. Keller, R. W. Ellis, and S. F. Starr. 1990. Cell-mediated immune responses after immunization of healthy seronegative children with varicella vaccine: kinetics and specificity. J. Infect. Dis. 162:794-799. Wroblewska, Z., D. Gilden, M. Green, M. Devlin, and A. Vafai. 1985. Affinity purified varicella-zoster virus glycoprotein gpl/ gp3 stimulates the production of neutralizing antibody. J. Gen. Virol. 66:1795-1799.
JOURNAL OF VIROLOGY, Oct. 1991, p. 5593-5596
0022-538X/91/105593-04$02.00/0 Copyright © 1991, American Society for Microbiology
Neutralizing Antibodies Induced by Recombinant Vaccinia Virus Expressing Varicella-Zoster Virus gpIV ABBAS VAFAI* AND WEINING YANGt
Department of Biomedical Sciences, University of Illinois College of Medicine, Rockford, Illinois 61107 Received 5 April 1991/Accepted 18 June 1991
Monoclonal antibodies generated against varicella-zoster virus (VZV) glycoprotein I (gpl) also recognize VZV gpIV (A. Vafai, Z. Wroblewska, R. Mahalingam, G. Cabirac, M. Wellish, M. Cisco, and D. Gilden, J. Virol. 62:2544-2551, 1988). To determine whether the virus-neutralizing activity of these antibodies belongs to gpl, gpIV, or both, the open reading frame encoding gpIV was inserted into the vaccinia virus genome. Immunoprecipitation of recombinant vaccinia virus-infected cells with anti-gpIV monoclonal antibody yielded synthesis and processing of gpIV similar to those expressed in VZV-infected cells. Antibodies raised against WgpIV in a rabbit recognized both native gpI and gpIV and neutralized VZV infectivity. In addition, antibodies raised against recombinant vaccinia virus carrying VZV gpl neutralized VZV infection. These results indicate a structural relationship between VZV gpI and gpIV and show that gpl and gpIV each induce virus-neutralizing antibody. Varicella-zoster virus (VZV) DNA encodes five glycoproteins, designated gpl through gpV, which are highly immunogenic and stimulate the immune response following a primary encounter with VZV (varicella) and viral reactivation (zoster) (5, 6, 16). VZV genes 67 and 68 are two adjacent genes which are located within the unique short sequences (Us) of the VZV genome and encode gpIV and gpI, respectively (6, 7, 9). DNA sequence analysis has shown two distinct open reading frames for the gpIV and gpl genes without any obvious amino acid sequence relatedness (6). In addition, the products of gpl and gpIV genes have been identified and characterized by monoclonal antibodies (MAbs) generated against these glycoproteins and antibodies to synthetic peptides derived from the predicted gpIV amino acid sequences (7, 9). gpl is the most abundant and immunogenic VZV glycoprotein and induces both complementdependent neutralizing antibodies and cell-mediated immunity (1, 2, 10, 12, 14, 15). However, MAbs prepared against gpl also recognize gpIV. This coprecipitation could result from either shared antigenic determinants (23) or physical interaction of the two proteins resulting in formation of heterodimers or heteromultimers. Because of this association, it is unknown whether the neutralizing activity of antibodies prepared against these glycoproteins belongs to gpl or gpIV or both. In this study, recombinant vaccinia viruses expressing either gpI or gpIV were produced and characterized in order to answer these questions. Construction and expression of recombinant vaccinia virus. The recombinant plasmid (pGEM-3) containing a 1,375-bp AvaI DNA fragment (spanning nucleotides 114385 to 115760 of the VZV genome) and containing the coding region of the gpIV gene (6, 23) was cleaved with the AvaI restriction enzyme. The AvaI fragment was extracted by electroelution and cloned at the SmaI site of insertion vector pSC11 (Fig. 1) as described previously (4, 23). The recombinant plasmid was designated pVVgpIV. The recombinant vaccinia virus was constructed by the method described previously (18) with minor modifications. Briefly, 143B thymidine kinase-
* Corresponding author. t Present address: Amgen Inc., Thousand Oaks, CA 91320.
negative (TK-) cells (18) were grown overnight in the absence of bromodeoxyuridine and were then infected with vaccinia virus (strain IHDJ) at a multiplicity of infection of 0.01 and incubated at 37°C for 90 min. The infected cells were then transfected with 30 ,ug of pVVgpIV DNA and 50 ,ug of Lipofectin reagent (GIBCO-BRL) according to the manufacturer's instructions. Cells were harvested 48 h after infection and transfection, and the resulting virus stock was passaged twice in TK- cells in the presence of 25 ,ug of bromodeoxyuridine per ml. Recombinant vaccinia virus was differentiated from spontaneous TK- virus by staining with Bluo-Gal (GIBCO-BRL) and was isolated by three cycles of plaque purification. The expression of recombinant vaccinia virus carrying VZV gpIV (designated VVgpIV) was analyzed by pulse-chase experiments with BSC-1 cells (23) infected with VVgpIV. Cells were infected with VVgpIV at a multiplicity of infection of 1.0; after a 22-h incubation period at 37°C, the cells were labeled with 300 ,uCi of [35S]methionine (specific activity, >1000 Ci/mmol; Amersham) per ml for 10 min in the absence or presence of 15 ,ug of tunicamycin per ml. The cells were either harvested or washed five times with serumfree medium, and the label was chased in normal medium for 120 min in the absence or presence of tunicamycin (15 ,ug/ml). The cells were then washed three times with cold phosphate-buffered saline and disrupted in 4 ml of lysis buffer (0.02 M sodium phosphate [pH 7.6], 0.1 M NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS]). The cell lysates were kept on ice for 2 h and centrifuged at 40,000 rpm in a Beckman SW60 rotor for 2 h at 5°C. The supernatants were immunoprecipitated with MAb G6, which recognizes VZV gpIV, and analyzed by SDS-10% polyacrylamide gel electrophoresis (PAGE) (23). MAb G6 was prepared according to procedures described previously (21-24). Figure 2 shows the expression and processing of VZV gpIV in VVgpIV-infected cells. A 50-kDa protein band was detected during a 10-min pulse period which was further processed to a mature form of gpIV (60 kDa) during a 120-min chase period. In the presence of tunicamycin, which inhibits the addition of N-linked oligosaccharides to native gpIV (23), a 36-kDa protein species was detected during the 5593
5594
J. VIROL.
NOTES IRL IRS US TRS
TRL
-TM + TM
-TM
U
U
P
P CI P C I P
P
+TM
C IP
-TM c
+TM
U
I
P
P C IP
W.
C
v--
_,
'A 9582-
gp
60-
"
50-
US
a mD
45-
gpiv
Am -.j
36-
FIG. 1. Construction of a recombinant vaccinia virus insertion plasmid carrying the VZV gpIV gene. The VZV gpIV gene cloned in transcription vector pGEM-3 (23) was cleaved with restriction enzyme AvaI (A), electroeluted, blunt-ended, and cloned at the SmaI (S) site of vaccinia virus insertion vector pSC11 as described in the text. A physical map of VZV DNA and the location of gpIV on the viral genome are shown.
pulse and a 45-kDa protein was detected during the chase period. These results were in agreement with those reported earlier (19, 20, 23) and suggested that gpIV expressed in VVgpIV contains both 0-linked and N-linked oligosaccharides. Similar patterns of gpIV synthesis and processing were observed when VZV-infected cells were pulse-chased and immunoprecipitated with MAb G6, which is directed against VZV gpIV and also recognizes gpl (Fig. 2B). These results indicated that the expression of VZV gpIV in recombinant vaccinia virus was similar to the expression of native gpIV in VZV-infected cells. However, the mature form of gpIV expressed by VVgpIV was somewhat larger than the native gpIV, suggesting the possibility of further glycosylation of this glycoprotein when it is expressed in vaccinia virus. Antibody response to VVgpIV. Antibodies against VVgpIV were generated in a rabbit by immunization procedures described previously (21). Briefly, a New Zealand White female rabbit was immunized subcutaneously at multiple sites in the back and the hind legs with 107 PFU of VVgpIV. This immunization was followed by three weekly injections each consisting of 107 PFU. The animal was bled 7 days after the last injection, and the serum (designated RAnti-VVgpIV) was assayed by immunoprecipitation. Pulse-chase experiments and immunoprecipitation of VZV-infected cells revealed that RAnti-VVgpIV recognized the high-mannose form (50 kDa) and the mature form (60 kDa) of VZV gpIV (Fig. 2C). In addition, RAnti-VVgpIV reacted with precurTABLE 1. VZV neutralization test in the presence of complement Antibody
RAnti-VZV
RAnti-VVgpI RAnti-VVgpIV
NRSb
Target
No. of
protein(s)
plaques"'
Plaque reduction (%)
Viral proteins VZV gpl VZV gpIV
0 10 28 130
100 92.3 78.5 0
" Average number of plaques in duplicate wells of six-well plates, determined with a 1:10 dilution of each serum sample. b NRS, normal rabbit serum (serum obtained from nonimmunized animals).
a
A
I
B
a
I
-
c
FIG. 2. (A) Expression of a recombinant vaccinia virus carrying the VZV gpIV gene (VVgpIV). BSC-1 cells were infected with VVgpIV, and after 22 h, infected cells were pulse-labeled (P) with [35S]methionine (300 p.Ci/ml) for 10 min in the absence or presence of tunicamycin (15 ,ug/ml) (TM). The cells were either harvested or washed, and the label was chased (lanes C) for 2 h in the absence or presence of TM (15 p.g/ml). U, uninfected cells were pulsed for 10 min. The cell lysates were prepared and immunoprecipitated with MAb G6, which recognizes VZV gpIV. (B) Immunoprecipitation of VZV-infected cells with MAb G6. BSC-1 cells were infected with VZV, and after 72 h postinfection, the cells were pulse-chased as described for panel A, and the cell lysates were immunoprecipitated with MAb G6. (C) Immunoprecipitation of VZV-infected cells with antibodies against VVgpIV (RAnti-VVgpIV) generated in a rabbit. VZV-infected cells were pulsed for 10 min and chased for 2 h as described for panel, A and the cell lysates were immunoprecipitated with RAnti-VVgpIV. Samples were analyzed by SDS-PAGE (10% polyacrylamide) as described previously (23). The apparent sizes (in kilodaltons) of the precursor products of VZV gpl and gpIV are shown on the left. Lysozyme (14.3 kDa), P-lactoglobulin (18.4 kDa), a-chymotrypsinogen (25.7 kDa), ovalbumin (43.0 kDa), bovine serum albumin (68.0 kDa), phosphorylase B (97.4 kDa), and myosin (200.0 kDa) were used as internal size markers.
sor products (78, 82, 90, and 95 kDa) of VZV gpL. These results indicated that the native VZV gpIV was recognized by RAnti-VVgpIV. The results also supported our previous findings (23), suggesting the recognition of gpl and gpIV in VZV-infected cells by antibodies raised against either of these glycoproteins. RAnti-VVgpIV was also tested for VZV-neutralizing activity. Neutralization tests were performed by the constantvarying serum technique as described previously (21). The results (Table 1) showed a plaque reduction of 100% with a 1:10 dilution of antibodies raised against purified VZV virions (21) in the presence or absence of complement and a plaque reduction of 78.5% with a 1:10 dilution of RAntiVVgpIV only in the presence of complement. To determine whether VZV gpl alone is also capable of inducing virusneutralizing antibody response, antibodies against a recombinant vaccinia virus (designated VVgpI) carrying the VZV gpl gene were prepared in a rabbit (3). Antibodies against VVgpI were prepared as described above and designated RAnti-VVgpI. Neutralization tests using RAnti-VVgpI also indicated a plaque reduction of 92.3% only in the presence of complement. These results demonstrated that both VZV gpl and gpIV are capable of inducing complement-dependent neutralizing antibodies.
NOTES
VOL. 65, 1991
a b
ab6
ab
a b
u P
P C
5595
P C
* *.
ir
up
gi
gPp
_~ -
_
D A B C FIG. 3. Immunoprecipitation of recombinant vaccinia viruses expressing VZV gpl (VVgpI) and gpIV (VVgpIV) with MAbs. BSC-1 cells (105) were infected with VVgpl (A), VVgpIV (B), or VVgpI and VVgpIV (C) at a multiplicity of infection of 1.0, and 22 h postinfection, the cells were labeled with [35S]methionine for 1 h. Cell lysates were prepared and immunoprecipitated with MAb G6 (lanes a) and MAb Cl (lanes b), which are directed against VZV gpIV and gpl, respectively. (D) Cell lysates from VVgpI- and VVgpIV-infected cells were combined and immunoprecipitated with MAbs G6 and Cl.
Since antibodies to gpl and gpIV are cross-reactive in VZV-infected cells, the following experiments were carried out to determine whether (i) MAbs cross-react when gpl and gpIV are expressed separately by recombinant vaccinia viruses and (ii) RAnti-VVgpIV binds to purified gpI. BSC-1 cells were infected with either VVgpI, VVgpIV, or both VVgpI and VVgpIV at a multiplicity of infection of 1.0, and 22 h postinfection, the cells were pulse-labeled with [35S]methionine (200 ,uCi/ml) for 1 h. The cell lysates were then immunoprecipitated with MAbs Cl and G6, which are directed against VZV gpl and gpIV, respectively. SDS-PAGE showed only the recognition of MAb Cl by gpl and of MAb G6 by gpIV (Fig. 3A and B). However, when VVgpI and VVgpIV were expressed in the same culture, MAb Cl also recognized gpIV (Fig. 3C). This recognition was not detected when cell lysates from VVgpI and VVgpIV were combined before immunoprecipitation (Fig. 3D). These results further support the association of gpI and gpIV and suggest that the expression of both proteins is necessary for physical association of these glycoproteins. To determine whether RAnti-VVgpIV binds to gpl, immunoglobulin G was purified from tissue culture fluid of MAb Cl-producing hybridomas by using protein G-Sepharose Fast Flow (Pharmacia LKB Biotechnology) as described previously (22). Purified immunoglobulin G was coupled to CNBr-activated Sepharose 4B beads and used to purify gpl from VVgpI-infected cell lysates as described previously (26). The purified gpl was used to prepare a column containing gpl coupled to CNBr-activated Sepharose 4B beads. RAnti-VVgpIV and MAb G6 were applied (10 times each) to the column, and the flowthrough was used to immunoprecipitate VZV-infected cell lysates. The results showed the reactivity of flowthrough RAnti-VVgpIV and MAb G6 with native gpIV in VZV-infected cells (Fig. 4). These results indicated that although preadsorption of RAnti-VVgpIV and
4410-
A
-.-
B
FIG. 4. Immunoprecipitation of VZV-infected cells with preadsorbed anti-gpIV MAb G6 and rabbit anti-recombinant vaccinia virus antibody expressing VZV gpIV (RAnti-VVgpIV). VZV gplspecific immunoglobulin G was purified and used to purify gpl from VVgpI-infected cell lysates as described in the text. Columns containing gpl-bound CNBr-activated Sepharose 4B beads were prepared and used to absorb gpl-specific antibody molecules in MAb G6 and RAnti-VVgpIV. Flowthrough samples were used for immunoprecipitation of VZV-infected cells. BSC-1 cells were infected with VZV. After 72 h postinfection, the cells were pulsed (P) for 10 min and chased (C) for 2 h, and the cell lysates were immunoprecipitated with flowthrough samples of MAb G6 (A) and RAnti-VVgpIV (B). U, uninfected cells were pulsed for 10 min.
MAb G6 resulted in the binding of some antibody molecules to gpl, amounts of unbound gpIV-specific antibody molecules sufficient to bind and precipitate gpIV in VZV-infected cells were present in RAnti-VVgpIV and MAb G6. Taken together, the lack of MAb G6 binding to VVgpI and the
recognition of native VZV gpIV by RAnti-VVgpIV which had been preabsorbed to purified gpl suggest that precipitation of VZV gpl and gpIV with RAnti-VVgpIV could be due to the physical association of these glycoproteins or due to both physical association and shared antigenic sites on gpl and gpIV. Although the biological functions of VZV gpl and gpIV during VZV replication have not yet been defined, these glycoproteins appear to be the initial targets for the induction of the immune response following VZV infection or VZV vaccination (8, 11, 13, 17, 25). Availability of recombinant viruses expressing VZV glycoproteins (e.g., gpl, gpIV, or both) not only will allow identification of antigenic epitopes which are important in the induction of humoral and cellmediated immunity but also will allow preparation of purified gpI and gpIV. These glycoproteins may have potential application for immunization against VZV infection or for boosting the immune response following VZV vaccination. We thank D. Kilpatrick for helpful discussions. This work was supported by a Public Health Service Program Project grant from the National Institutes of Health (POlAG0734) and by a grant from the National Multiple Sclerosis Society
(RG2354-A).
5596
J. VIROL.
NOTES
REFERENCES 1. Arvin, A. M., E. Kinney-Thomas, K. Shriver, C. Grose, C. M. Koropchak, E. Scanton, A. E. Wittck, and P. S. Diaz. 1986. Immunity to varicella-zoster viral glycoproteins, gpl (gp 90/58) and gpIII (gp 118), and to a nonglycoslated protein p170. J. Immunol. 137:1346-1351. 2. Arvin, A. M., S. M. Solem, C. M. Koropchak, E. KinneyThomas, and S. G. Paryani. 1987. Humoral and cellular immunity to varicella-zoster virus glycoprotein gpl and a non-glycosylated protein, P170, in the strain 2 guinea-pig. J. Gen. Virol. 68:2449-2454. 3. Cabirac, G., D. Gilden, M. Wellish, and A. Vafai. 1988. Expression of varicella-zoster virus glycoprotein I in cells infected with a vaccinia virus recombinant virus. Virus Res. 10:205-214. 4. Chakrabarti, S., K. Brechling, and B. Moss. 1985. Vaccinia virus expression vector: coexpression of P-galactosidase produces visual screening of recombinant virus plaques. Mol. Cell. Biol. 5:3403-3409. 5. Davison, A. J., C. M. Edson, R. W. Ellis, B. Forghani, D. Gilden, C. Grose, P. M. Keller, A. Vafai, Z. Wroblewska, and K. Yamanishi. 1986. New common nomenclature for glycoprotein genes of varicella-zoster virus and their glycosylated products. J. Virol. 57:1195-1197. 6. Davison, A. J., and J. E. Scott. 1986. The complete DNA sequence of varicella-zoster virus. J. Gen. Virol. 67:1759-1816. 7. Davison, A. J., D. J. Waters, and C. M. Edson. 1985. Identification of the products of a varicella-zoster virus glycoprotein gene. J. Gen. Virol. 66:2237-2242. 8. Dubey, L., S. P. Steinberg, P. LaRussa, P. Oh, and A. A. Gershon. 1988. Western blot analysis of antibody to varicellazoster virus. J. Infect. Dis. 157:882-888. 9. Ellis, R. W., P. M. Keller, R. S. Lowe, and R. A. Zivin. 1985. Use of a bacterial expression vector to map the varicella-zoster virus major glycoprotein gene, gC. J. Virol. 53:81-88. 10. Forghani, B., K. W. Dupuis, and N. J. Schmidt. 1984. Varicellazoster viral glycoproteins analyzed with monoclonal antibodies. J. Virol. 52:55-62. 11. Giller, R. H., S. Winistorfer, and C. Grose. 1989. Cellular and humoral immunity to varicella zoster virus glycoproteins in immune and susceptible human subjects. J. Infect. Dis. 160: 919-928. 12. Grose, C., D. P. Edwards, W. E. Friedrichs, K. A. Weigle, and W. L. McGuire. 1983. Monoclonal antibodies against three major glycoproteins of varicella-zoster virus. Infect. Immun. 40:381-388. 13. Grose, C., and V. Litwin. 1988. Immunology of the varicellazoster virus glycoproteins. J. Infect. Dis. 157:877-881. 14. Ito, M., T. Ihara, C. Grose, and S. Starr. 1985. Human leukocytes kill varicella-zoster virus-infected fibroblasts in the pres-
15. 16.
17.
18.
19.
20.
21. 22.
23.
24.
25.
26.
ence of murine monoclonal antibodies to virus-specific glycoproteins. J. Virol. 54:98-103. Keller, P. M., B. J. Neff, and R. W. Ellis. 1984. Three major glycoprotein genes of varicella-zoster virus whose products have neutralization epitopes. J. Virol. 52:293-297. Kinchington, P. R., P. Ling, M. Pensiero, B. Moss, W. T. Ruyechan, and J. Hay. 1990. The glycoprotein products of varicella-zoster virus gene 14 and their defective accumulation in a vaccine strain (Oka). J. Virol. 64:4540-4548. LaRussa, P. S., A. A. Gershon, S. P. Steinberg, and S. A. Chartrand. 1990. Antibodies to varicella-zoster virus glycoproteins I, II, and III in leukemic and healthy children. J. Infect. Dis. 162:627-633. Mackett, M., G. L. Smith, and B. Moss. 1985. The construction and characterization of vaccinia virus recombinants expressing foreign genes, p. 191-211. In D. M. Glover (ed.), DNA cloning: a practical approach. IRL Press, Oxford. Montalvo, E. A., R. T. Parmley, and C. Grose. 1985. Structural analysis of the varicella-zoster virus gp98-gp62 complex: Posttranslational addition of N-linked and 0-linked oligosaccharide moieties. J. Virol. 53:761-770. Okuno, T., K. Yamanishi, K. Shiraki, and M. Takahashi. 1983. Synthesis and processing of glycoproteins of varicella-zoster virus (VZV) as studied with monoclonal antibodies to VZV antigens. Virology 129:357-368. Vafai, A., K. Jensen, and R. Kubo. 1989. Existence of similar antigenic-sites on varicella-zoster virus gpl and gpIV. Virus Res. 13:319-336. Vafai, A., Z. Wroblewska, and L. Graf. 1990. Antigenic crossreaction between a varicella-zoster virus nucleocapsid protein encoded by gene 40 and a herpes simplex virus nucleocapsid protein. Virus Res. 15:163-174. Vafai, A., Z. Wroblewska, R. Mahalingam, G. Cabirac, M. Wellish, M. Cisco, and D. Gilden. 1988. Recognition of similar epitopes on varicella-zoster virus gpl and gpIV by monoclonal antibodies. J. Virol. 62:2544-2551. Vafai, A., Z. Wroblewska, M. Wellish, M. Green, and D. Gilden. 1984. Analysis of three late varicella-zoster virus proteins, a 125,000-molecular-weight protein and gpl and gp3. J. Virol 52:953-959. Watson, B., P. M. Keller, R. W. Ellis, and S. F. Starr. 1990. Cell-mediated immune responses after immunization of healthy seronegative children with varicella vaccine: kinetics and specificity. J. Infect. Dis. 162:794-799. Wroblewska, Z., D. Gilden, M. Green, M. Devlin, and A. Vafai. 1985. Affinity purified varicella-zoster virus glycoprotein gpl/ gp3 stimulates the production of neutralizing antibody. J. Gen. Virol. 66:1795-1799.
