Antigenicity of a candidate varicella-zoster virus glycoprotein subunit vaccine

Antigenicity of a candidate varicella-zoster virus glycoprotein subunit vaccine Abbas Vafai A 1642 bp DNA fragment, encoding the N-terminal region and 511 amino acid residues of varicella-zoster virus (VZV) gpI gene, was inserted into vaccinia virus genome. The expression of recombinant vaccinia virus (designated VVTgpI-511) yielded the synthesis of a 60 kDa protein species which was processed to a secretory 76 kDa polypeptide (designated TgpI-511). The antigenicity of this protein was examined by subcutaneous inoculation of one rabbit with 100 gg purified TgpI-511 in the Ribi adjuvant system. The animal was boosted 3 weeks after the initial inoculation and antisera were tested 7 days after the last injection by immunoprecipitation and neutralization tests. The results showed that rabbit antibodies to TgpI-511 (RAnti- TgpI-511) were reactive with purified TgpI-511 as well as native gpI in VZV-infected cells. In addition, TgpI-511 was capable of eliciting complement-dependent V Z V neutralizing antibodies. These results suggested that the purified preparation of TgpI-511 may have the potential to be used as a candidate V Z V subunit vaccine. Keywords: Varicella-zoster virus; subunit vaccine; truncated glycoprotein I

Infection with varicella-zoster virus (VZV) results in two distinct clinical manifestations: childhood chickenpox (varicella) and shingles (zoster). Varicella is the outcome of the primary encounter with VZV, whereas zoster is the result of VZV reactivation from latently infected sensory ganglia which occurs predominantly in ageing and immunocompromised individuals. There are an estimated 4 million cases of varicella and 1.2 million cases ofzoster each year in the United States 1-3. An attenuated VZV vaccine (Oka/Merck) is currently being tested in the United States for the prevention of varicella 4'5. Although this vaccine provides a high degree of protection against varicella, one concern regarding its use has been the duration of protection after immunization 6. In addition, similar to natural infection, varicella vaccine becomes latent in dorsal root ganglia and will reactivate to produce zoster 7'8. Therefore, there is a need for developing additional VZV vaccines (e.g. subunit vaccine) that are capable of eliciting immune responses to virus infection but that will not establish virus latency. Subunit vaccines have been prepared for the members of herpesvirus family (for a review see Ref. 9). Vaccine development for these viruses has centred mainly on viral glycoproteins, since these proteins are highly immunogenic and capable of inducing both humoral and cell-mediated immune responses following virus infection 9. VZV DNA encodes five glycoproteins designated gpI Department of Biomedical Sciences, University of Illinois College of Medicine, Rockford, IL 61107-1897, USA. (Received 15 June 1992; revised 22 September 1992; accepted 22 September 1992) 0264-410X/93/09/0937-04 ((} 1993Butterworth-HeinemannLtd

to gpV 1°'11. These glycoproteins are the major virus-specific components found on the viral envelope and VZV-infected cell membrane. Our initial studies indicated that recombinant vaccinia viruses expressing VZV glycoprotein I (gpI) or gplV are capable of inducing VZV-neutralizing antibodiesl3 and therefore have potential application as VZV subunit vaccines. However, since the use of recombinant vaccinia viruses as subunit vaccines has not yet been approved by the regulatory agencies, our efforts are concentrated on the construction of a secretory VZV glycoprotein which can be purified from tissue culture fluids of the infected cells. Since VZV gpI is the most abundant and immunogenic of VZV glycoproteins and is capable of inducing both humoral and cell-mediated immunity 1° we have considered this viral glycoprotein as a candidate VZV subunit vaccine. In this study, we expressed a secretory truncated VZV gpI with 511 amino acid residues and showed that the purified preparation of this glycoprotein was capable of inducing complement-dependent neutralizing antibodies in the rabbit. Expression of a secretory truncated VZV gpI in vaccinia virus For the construction of vaccinia recombinant, a 1642bp DNA fragment containing 2 b p from the pGEM-4 polylinker upstream from the SmaI site and 1640 bp VZV nucleotides (spanning nucleotides 115 712 to 117 352 of the VZV genome) was cleaved with KpnI and XmaIII, respectively, from p G E M recombinant plasmid carrying a blunt-ended VZV BglI DNA fragment

Vaccine, Vol. 11, Issue 9, 1993 937

Candidate varicella-zoster subunit vaccine: A. Vafai

(spanning nucleotides 115712 to 118181 of the VZV genome) and containing gpI open reading frame 11. The truncated gpI DNA, encoding the N-terminal region of gpI with 511 amino acid residues (designated TgpI-511 ), was blunt-ended and cloned at the SmaI site of p S C l l (EB1) plasmid (Figure 1) as described 12. The EB1 (a gift from E. Stephens) is a derivative of p S C l ! plasmid in which the p7.5 promotor has been replaced with the vaccinia virus pl 1 promotor (Figure 1). The recombinant p S C l l (EB1) plasmid carrying TgpI-511 was designated pVVTgpI-511. The TgpI-511 DNA was then inserted into the vaccinia virus genome as described13.14. The expression of the recombinant vaccinia virus was analysed by pulse-chase experiments. BSC-I cells were infected with VVTgpI-511 (1.0 p.f.u./cell) and after 22 h, the cells were pulse-labelled with 100/~Ci ml 1 of Tran 3SS-label (specific activity, >1000 Ci mmol 1; ICN Biomedicals, Inc.) for 60 rain. Cells were either harvested or washed five times with serum-free medium and the label was chased in serum-free medium for 2 h. Cells were washed three times with cold phosphate-buffered saline (PBS) and disrupted in 4 ml of lysis buffer (0.2 M sodium phosphate, pH 7.6, 0.1 M NaCI, 1% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS)). Lysates were kept on ice for 2 h and centrifuged at 40 000 rev rain-1 in a Beckman SW60 rotor for 2 h at 5c~C. Tissue culture fluids from cells which had been pulse chased were collected and centrifuged for 10 min in a microfuge. Supernatants were collected and an equal volume of lysis buffer was added to each sample. Supernatants from cell lysates and tissue culture fluids were immunoprecipitated with monoclonal antibodies

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Construction of a recombinant vaccinia virus insertion vector carrying a truncated VZV gpl gene. A 2.5kb Bgll DNA fragment (containing VZV gpl open reading frame), which had been cloned in pGEM-4 transcription vector (containing SP6 and T7 promotors), was cleaved with Kpnl and Xmalll, electroeluted, blunt-ended, and cloned at the Sinai (S) site of vaccinia virus insertion vector pSC11(EB1) as described in the text. The truncated VZV gpl DNA fragment (1640 bp) encodes the N-terminal region of gpl with 511 amino acid residues. A physical map of VZV DNA and the location of gpl on the viral genome

are shown

938

Vaccine, Vol. 11, Issue 9, 1993

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Figure 2 Expression of the truncated VAV gpl (Tgpl-511) by recombinant vaccinia virus. BSC-1 cells were infected (1.0 p.f.u./cell) with recombinant vaccinia virus expressing Tgpl-511 (VVTgpl-511). After 22 h, infected cells were pulse-labelled with Tran 35S-label (100 #Ci ml ') for 60 min and cells were either harvested or washed and the label was chased for 2 h. Cell lysates (CL) from pulse-labelled experiments and tissue culture fluids (TCF) from cells which had been pulse-chased were prepared and immunoprecipitated with gpl-specific monoclonal antibodies (mAb79.7, mAbC1, mAbG7) and mAbF8 and mAbF9 which are directed against VZV gpll and a 155 kDa VZV nucleocapsid protein, respectively. The samples were analysed by 9% SDS PAGE. The size (kDa) of precursor and processed form of Tgpl-511 are shown on the left

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(mAbs) prepared against VZV gpI (mAb79.7, mAbC1, mAbG7), gpII (mAbF8) and the 155kDa VZV nucleocapsid protein (mAbF9). The results showed the expression of a polypeptide with an apparent size of 60 kDa by VVTgpI-511 which was processed to a 76 kDa protein species during the 1 h labelling period (Fioure 2). Both 60 kDa and 76 kDa protein species were recognized by mAb79.7 and mAbC1 which react with the precursor products of VZV gp113'15 and mAbG7 which reacts with the maturei'orm of VZV gpI (Figure 2, lanes 1, 2 and 3). However, mAbF8 and mAbF9 which are directed against VZV gplI and VZV nucleocapsid protein, respectively, did not react with the precursor product of the truncated gpI (TgpI-511) as shown in Figure 2, lanes 4 and 5. To determine whether TgpI-511 was secreted from the cell, infected cells were pulse-labelled with Tran 35S-label for 1 h, washed with serum-free medium and incubated in serum-free medium for 2h. Tissue culture fluids were harvested and immunoprecipitated with mAbs as described above. The results demonstrated that the mature form of TgpI-511 was secreted from VVTgpI-511-infected cells and was recognized by VZV gpI-specific mAbs (Figure 2, lanes 6, 7, 8). These results indicated that the anchoring region of VZV gpI is located near or at the C-terminus of this glycoprotein. In addition, pulse chase experiments with VVTgpI-511-infected cell lysates and tissue culture fluids with gpI-specific mAbC1 resulted in: (1) the synthesis of a 56 kDa protein species in the presence of tunicamycin (which inhibits the addition of N-linked

Candidate varicella-zoster subunit vaccine: A. Vafai

oligosaccharides to native gpI) which was processed to a 63 kDa polypeptide (data not shown), suggesting that the TgpI-511 contains both N-linked and O-linked oligosaccharides; and (2) the secretion of TgpI-51 ! from the infected cells in the presence of absence of tunicamycin (data not shown), suggesting that N-linked glycosylation of this protein is not required for its release from the cell.

!

lmmunogenicity of truncated VZV gpI To examine the antigenicity of TgpI-511, rabbits were immunized with recombinant vaccinia virus expressing TgpI (VVTgpI-511) and with prufied preparation of TgpI-511. One rabbit was immunized with VVTgpI-511 ( 1 × 107 p.f.u.) as described 13. Five weeks after immunization, the rabbit was bled and the serum was tested by immunoprecipitation and neutralization assays. For immunization against purified truncated gpI, TgpI-511 was purified as follows. BSC-1 cells (2 × 109) were infected with VVTgpI-511 (1.0 p.f.u./cell) and at 22h postinfection, cells were washed once with Hank's balanced salt solution (HBSS) and incubated in HBSS for 3 h. Tissue culture fluids were harvested, centrifuged to remove cell debris, and the supernatants were concentrated tenfold by Amicon Centriprep-10. TgpI-511 was then purified by CNBr beads crosslinked to VZV gpI-specific IgG as described 13. TgpI-511 was further concentrated by Amicon Centricon-10 and resuspended in 20mM NaHzPO 4, pH 7.0. The purity of each protein preparation was examined by SDS-polyacrylamide gel electrophoresis (PAGE) as shown in Figure 3, lane 1. The specificity of the purified TgpI was tested by immunoprecipitation of TgpI-511 with gpI-specific mAbC1 (Figure 3, lane 3). This procedure yielded 10 pg purified TgpI-511 per 1 x 108 infected cells. One rabbit was immunized subcutaneously with 100 pg of purified TgpI-51! emulsified in Ribi adjuvant system (Ribi Immunochem. Research, Inc.) by the procedure described in the manufacturer's instructions. Three weeks after the initial injection, the animal was injected with the same antigen formulation and 7 days after the last injection, the animal was bled and the serum was tested by immunoprecipitation and neutralization tests. The results from immunoprecipitation studies showed that both VVTgpI-511 and purified TgpI-511 were capable of inducing antibody responses which were reactive with Tgpl-511 (Figure 3, lanes 2 and 4). In addition, pulse-chase experiments and immunoprecipitation of VZV-infected cells showed that the precursor products of VZV gpI were recognized by anti-TgpI-511 antibodies (data not shown). Neutralization tests were performed using the constant-varying serum technique in the presence or absence of complement as described 16. The results revealed plaque reductions of more than 80% with 1:10 and 1:100 dilutions of antibodies raised against VVTgpI-511 and TgpI-511, respectively, in the presence of complement (Table 1). Controls were provided by neutralization tests using non-immunized rabbit sera and rabbit antisera prepared against recombinant vaccinia viruses carrying VZV gpI and VZV gpIV a3. These results demonstrated that purified TgpI-511 is capable of inducing complementdependent neutralizing antibodies. The results also suggested that two injections of the purified TgpI-511 along with the Ribi adjuvant system were sufficient to mount an antibody response in rabbit that was reactive

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T g P l - 511 Figure 3 Immunoprecipitation of purified truncated VZV gpl with 511 amino acid residues (Tgpl-511). Tgpl-511 was purified (lane 1) from tissue culture fluids of VVTgpl-511-infected cells by CNBr crosslinked to gpl-specific IgG as described 13. Antibodies were raised in rabbit (RAnti-Tgpl) against Tgpl-511 and used to immunoprecipitate purified Tgpl-511 (lane 2). Purified Tgpl-511 was also immunoprecipitated with VZV gpl-specific mAbC1 (lane 3) and antibodies prepared in rabbit (RAnti-VVTgpl) against recombinant vaccinia virus expressing Tgpl-511 (lane 4). The samples were analysed by 9% SDS-PAGE. The size (kDa) of the mature form of Tgpl-511 is indicated on the left

with the native VZV gpI and was capable of neutralizing VZV infectivity. Although the neutralizing antibodies induced in animals may not always correlate with protection against virus infection, they are generally considered the most reliable predictor of an important immune response in vivo 9. However, in addition to stimulating humoral immunity, ideally cellular immunity must be stimulated, since this form of immunity is of greater importance in host defence against VZV. The ability of purified TgpI-511 to mount VZV-neutralizing antibody response suggests that this protein can serve as a candidate for a VZV subunit vaccine. Such a subunit vaccine is safe and

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939

Candidate Table 1

v a r i c e l l a - z o s t e r s u b u n i t v a c c i n e : A. Vafai

Varicella-zoster virus neutralization test a Number of plaques ~ (with complement)

Plaque reduction (%)

Number of plaques (without complement)

Plaque reduction (%)

Antibody

Target protein

1:10

1:100

1:1000

1:10

1:100

1:1000

1:10

1:100

1:1000

1:10

1:100

1:1000

RAnti-VVgpl c RAnti-VVgplV ~ RAnti-VVTgpl-511 ~ RAnti-pTgpl-511 ' NRS q

VZVgpl VZVgplV VZVTgpl-511 VZVTgpl-511

6.0 5.5 7.5 7.0 78.5 74.5

8.0 7.0 9.5 9.0 80.0 82.0

30.5 23.0 29.0 24.0 87.0 82.0

91.9 92.6 89.9 90.6

90.2 91.5 88.4 89.0 2.4

62.8 71.9 64.6 70.7

52.0 45.5 52.0 45.0 54.0 57.0

55.0 51.5 55.0 48.0 56.0 61.5

46.0 56.0 57.5 52.0 63.5 64.0

8.8 20.2 8.8 21.0 5.3

10.6 16.3 10.6 21.9 8.9

28.1 12.5 10.1 18.7 0.8

aNeutralization tests were performed by the constant-varying serum technique (dilutions: 1:10; 1:100; 1:1000) in the absence or presence of 0.2 ml guinea-pig complement (Cappel Research Products) per reaction as described previously1~ ~Average number of plaques in duplicate wells of six-well plates, determined with 1:10, 1:100 and 1:1000 dilutions of each serum ~RAnti-Wgpl, rabbit antibodies against recombinant vaccinia virus expressing VZV gpl ~ ~RRAnti-WgplV, rabbit antibodies against recombinant vaccinia virus expressing VZV gplV ~3 ~RAnti-VVTgpl-511, rabbit antibodies against recombinant vaccinia virus expressing a truncated VZV gpl with 511 amino acid residues 'RAnti-pTgpl-511, rabbit antibodies against purified truncated gpl with 511 amino acid residues ~NRS, normal rabbit serum obtained from a non-immunized rabbit

lacks DNA and infectious material. Therefore, unlike attenuated VZV vaccine, the subunit vaccine will not establish latency. The immunogenicity of this vaccine in animals provides the rationale for further testing in human clinical trials to determine whether such a subunit vaccine can provide protection from virus infection and whether this vaccine can prevent or reduce virus reactivation in the elderly who are at increased risk of developing herpes zoster.

5 6

7

8 9

ACKNOWLEDGEMENTS The authors are grateful to D. Kilpatrick for helpful discussions and to J. Javaherian for rabbit immunization. This work was supported by a Public Health Service Program Project grant from the National Institutes of Health (PO1 AG0734) and by a grant from the National Multiple Sclerosis Society (RG2354-A).

10

11 12

13

REFERENCES 1 2

3 4

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Gelb, L.D. Varicella-zoster virus. In: Fields Virology (Eds Knipe, D.M. et al.) Raven Press, New York, 1990, pp. 2011-2054 Guess, H.A., Broughton, D.D., Melton, L.J. III and Kurland, L.T. Population-based studies of varicella complications. Pediatrics 1986, 78 (Suppl.), 723-727 Preblud, S.R. Varicella: Complications and costs. Pediatrics 1986, 78 (Suppl.), 728 735 Gershon, A.A., Steinberg, S.P., LaRussa, P., Ferrar, A., Hammerschlag, M. and Gelb, L. Immunization of healthy adults with live attenuated varicella vaccine. J. Infect. Dis. 1988, 158, 132 137

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Starr, S.E. Status of varicella vaccine for healthy children. Pediatrics 1989, 84, 1097-1098 Johnson, C., Rome, L.P., Stancin, T. and Kumar, M.L. Humoral immunity and clinical reinfection following varicella vaccine in healthy children. Pediatrics 1989, 84, 418-421 Hardy, I., Gershon, A.A., Steinberg, S.P. and LaRussa, P. The incidence of zoster after immunization with live attenuated varicella vaccine: A study in children with leukemia. N. Engl. J. Med. 1991, 325, 1545 1550 Plotkin, S.A., Starr, S., Connor, K. and Morton, D. Zoster in normal children after varicella vaccine. J. Infect. Dis. 1989, 159, 1000 Collett, M.S. The development of biosynthetic vaccines. Adv. Vet. Sci. Comp. Med. 1989, 33, 109 172 Davison, A.J., Edson, C.M., Ellis, R.W., Forghani, B., Gilden, D., Grose, C. et al. New common nomenclature for glycoprotein genes of varicella-zoster virus and their glycosylated products. J. Virol 1986, 57, 1195 1197 Davison, A.J. and Scott, J.E. The complete DNA sequence of varicella-zoster virus. J. Gen. Virol. 1986, 67, 1759 1816 Chakrabarti, S., Brechling, K. and Moss, B. Vaccinia virus expression vector: Coexpression of fl-galactosidase produces visual screening of recombinant virus plaques. Mol. Cell. Biol. 1985, 5, 3403 3409 Vafai, A. and Yang, W. Neutralizing antibodies induced by recombinant vaccinia virus expressing varicella-zoster virus gplV. J. Virol. 1991, 65, 5593 5596 Mackett, M., Smith, G.L. and Moss, B. The construction and characterization of vaccinia virus recombinants expressing foreign genes. In: DNA Cloning: A Practical Approach (Ed. Glover, D.M.) IRL Press, Oxford, 1985, pp. 191 211 Vafai, A., Wroblewska, Z., Mahalingam, R., Cabirac, G., Wellish, M., Cisco, M. and Gilden, D. Recognition of similar epitopes on varicella-zoster virus gpl and gplV by monoclonal antibodies. J. Virol. 1988, 62, 2544-2551 Vafai, A., Jensen, K. and Kubo, R. Existence of similar antigenic-sites on varicella-zoster virus gpl and gplV. Virus Res. 1989,13, 319- 336