Immunogenicity of herpes simplex virus glycoprotein gB-1-related protein produced in yeast

Immunogenicity of herpes simplex virus glycoprotein gB-l-related protein produced in yeast Yoichiro Kino *~, Chikateru Nozaki*, Hiroshi Nakatake*, Kyosuke Mizuno* and Ryoichi Mori t A protein related to glycoprotein B of herpes simplex virus type 1 (HSV-1) produced in yeast (ygB-1) was purified with an immunoadsorbent. The molecular weight of the purified ygB-1 as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis was 96000. Mice injected twice with ygB-1 adsorbed to alum developed ELISA antibody to ygB-1, neutralizing antibody to HSV-1 and a lymphoproliferative response to ygB-1 and HSV-1. The immunized mice were protected against intraperitoneal and corneal challenge with HSV-1. Latent infection in the trigeminal ganglia after corneal challenge was also inhibited by immunization with ygB-1. Guineapigs immunized with ygB-1 adsorbed to alum also developed ELISA antibody to ygB-1 and neutralizing antibody to both types of HSV. After the second dose, strong lymphoproliferative responses were seen upon stimulation with HSV-2. Animals were protected against intravaginal challenge with HSV type 2. Keywords:Herpes simplex: glucoprotein gB-1; immunogenicity; yeast

Introduction

models to determine the potential of ygB-1 as a constituent of an HSV component vaccine.

Although anti-herpes drugs such as acyclovir are available, herpes simplex virus (HSV) infections are a serious clinical problem. As a method of prophylaxis, development of an HSV vaccine seems to be very important. HSV contains four major glycoproteins I which seem to be target antigens in the immune response responsible for protection. Because of unfavourable characteristics of HSV, such as potential oncogenicity z and latency 3, a component vaccine consisting of the glycoprotein(s) appears to be the most desirable form of HSV vaccine. For that reason, the glycoproteins were purified from HSV-infected cells and their potentiality for use as an HSV component vaccine was indicated 4-8. As a possible constituent of an HSV vaccine gB was selected because it was an epitope(s) common to both types of HSV (Ref. 9). In our previous studies, we showed that mice are protected from HSV infection by passive immunization with monoclonal antibodies against gB-1 (Ref. 10) and active immunization with purified native gB-I (Ref. 6). It is difficult to obtain a large quantity of gB-1 by the conventional cell culture technique. It is also quite difficult to assure the absence of HSV D N A in a vaccine preparation. Recently we cloned gB-1 gene and found that it could be expressed in yeast cells 11. In this study we tested the immunogenicity of gB-l-related protein produced in yeast cells (ygB-l) in mouse and guinea-pig

Yeast cells and purification o f ygB-1 Yeast cells transformed with gB-1 gene (NaeI-BamHI fragment) were used as a source of ygB-1 (Ref. 11). The yeast cells cultured in a fermenter were collected by centrifugation, resuspended in phosphate buffered saline (PBS) containing 1% Triton X-100, and 1 mM phenyl methyl sulphonyl fluoride and disrupted by sonication. The lysate was clarified by centrifugation at 8000 rev min -~ for 30 min and the resulting supernatant was applied to an immunoadsorbent column to which a monoclonal antibody to gB-1 was conjugated. After overnight adsorption, the column was washed with PBS containing 1% Triton X-100 and the bound ygB-1 was eluted with 0.1 M diethylamine. After extensive dialysis, ygB-1 was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis ( S D S - P A G E ) and silver staining. Native gB-1 was also purified from a lystate of HEp-2 cells infected with HSV type 1 (HSV1), strain KOS, by the same method as above.

*The Chemo-Sero-Therapeutic Research Institute, 668 Okubo Shimizu, Kumamoto, 860 Japan. *Department of Virology, School of Medicine, Kyushu University, Fukuoka, Japan. *To whom correspondence should be addressed. (Received 10 August 1988; revised 5 October 1988)

Immunological methods ELISA antibody was measured by the micro ELISA method. A 96-well ELISA plate (Nunc, Roskilde, Denmark) was coated with 1 tzg of ygB-1 or 1 /zg of gD-1

0264-410X/89/020155-06$03.00 © 1989 Butterworth & Co. (Publishers) Ltd

Materials and methods

Animals Six-week-old female Balb/c mice and female Hartley guinea-pigs weighing 3 5 0 4 0 0 g were used throughout the study.

Vaccine, Vol. 7, April 1989

155

Immunogenicity of yeast-produced HSV gB- 1: Y. Kino et al. kDa

200

116 92

66

45

I

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3

Figure 1 SOS-PAGE analysis of purified ygB-1 and gB-1. ygB-1 and gB-1 purified by the immunoadaorbent were subjected to SDS-PAGE and stained with silver stain. Lane 1, molecular weight maker; lane 2, gB-1; lane 3, ygB-t

(purified with an immunoadsorbent) in sodium carbonate buffer p H 9.7 per well at 4°C overnight. After blocking with PBS-BSA at 37°C for 1 h, test serum at an appropriate dilution was added to the plate, which was incubated for 2 h at 37°C and then with anti-mouse or anti-guinea-pig IgG peroxidase conjugate for another 2 h at 370C. Finally, enzyme-substrate solution [0.05 M citric acid, 0.1 M dibasic sodium phosphate (pH 5.0) containing 0.04% orthophenylene diamine, 0.006% H202] was added. After 30-min incubation in the dark, the enzyme reaction was stopped with 0.5 M sulphuric acid and spectrophotometric readings were made at 492 rim. Between the reactions, the plate was washed five times with PBS-Tween. Neutralizing antibody was measured by the microplate method described by Hayashi et al. 12. The delayed type hypersensitivity ( D T H ) response in mice was measured by the increase in ear thickness. One day before challenge, five mice were challenged with heat-inactivated HSV-1 strain KOS ( l x 106 p.f.u. before inactivation) or 1/zg of ygB-1 intrtcutaneously in the ear pinae. Twenty-four hours after challenge, the ear thickness was measured with a dial thickness gauge (Mitutoyo Seiki, Tokyo, Japan). The D T H response in guinea-pigs was measured by a skin test. One day before challenge, the hair of the midflank was removed and heat-inactivated HSV type 2 (HSV-2) strain 8204 T N (a clinical isolate from a patient with herpes genitalis) (1 × 106 p.f.u, before inactivation) was inoculated intracutaneously as a challenge. Twentyfour hours after challenge, the longest and shortest diameters of erythema were measured. D T H value was expressed by the sum of the longest and shortest diameters. One day before challenge, lymphocytes were obtained from spleens of mice and from peripheral blood of guinea pigs. The lymphocytes were suspended in R P M I 1640 medium supplemented with 10% heat-inactivated horse serum (GIBCO Laboratories, Grand Island, N.Y.) and 0.2-ml quantities of the suspension (2 x 105 cells) were added to a 96-well flat-bottomed plate (Becton Dickinson Labware, Oxnard, Calif.). To triplicate wells, 10-p.l quantity of medium containing heat-inac156 Vaccine, Vol, 7, April 1989

tivated HSV-I strain KOS or HSV-2 strain 8204 T N (1 x 10 o p.f.u, before inactivation) or 1 t~g of ygB-I were added. To control wells 10/zl of medium only was added. After 5 days' incubation at 37°C in 5% CO2 in air, l/zCi of [aH]-thymidine (aH-TdR, New England Nuclear Corp, Boston, MA) was added to each well. After 16h incubation, incorporation of 3H-TdR was measured with a liquid scintillation counter and expressed as counts min -~. The results of lymphoproliferative responses were expressed by the stimulation index (SI) as follows: SI = incorporation of 3H-TdR with stimulation by HSV or ygB-1/incorporation of 3HT d R without stimulation. Ganglia of mice challenged in the corneas were tested for reactivation of virus as follows: the mice were killed and the bilateral trigeminal ganglia were removed from each mouse. The ganglia were cultured for one week in Dulbecco's modified Eagle minimal essential medium supplemented with 2% fetal calf serum. After 7 days' culture the ganglia were disrupted with sonication and centrifuged at 3000 rev min -~ for 10 min, and the reactivated virus in the supernatant was titrated on Veto cells.

Results

Purification of ygB-1 In this study, ygB-I was purified from the Triton X100-solubilized yeast lysate in a one-step process with immunoadsorbent to which a monoclonal antibody to gB-1 was conjugated. Native gB-1, as a control, was purified from HSV-l-infected HEp-2 cells. The purified ygB-1 and gB-1 were analysed by S D S - P A G E and silver staining (Figure 1). In the gB-1 lane the mature form of gB-1 and the precursor form of gB-I are seen, with molecular weight of 116 000 and 110 000 respectively. In the ygB-1 lane a single band with a molecular weight of 96000 is seen.

Immunization and challenge of Balbl c mice Six-week-old female Balb/c mice were injected twice with 2/~g of ygB-1 adsorbed to 50~g of alum gel subcutaneously in the rear foot pads on day 0 and day 14. Control mice were injected with alum gel only. One day before the second injection and challenge, serum was collected from five mice and ELISA antibody to ygB-1 and neutralizing antibody to HSV-1 were measured. Although neutralizing antibody was detected in only two mice, ELISA antibody was detected in all mice one day before the second dose. One day before challenge, all mice had developed strong ELISA and neutralizing antibody (Table 1). Table I

Development of ELISA and neutralizing antibodies in mice

Mouse no. 1 2 3 4 5 Alum alone (mean; n=5)

ELISA

Neutralization

T1

T2

"i"1

0.086 0.101 0.106 0.129 0.095 0.018

0.347 0.546 0.482 0.485 0.483 0.007

< 10 < 10 < 10 10 10 < 10

T2 40 80 160 80 80 < 10

Mice were injected twice with ygB-1 in the footpads. One day before the second dose (T1) and challenge (T2), serum was collected from the same mice and ELISA antibody to ygB-1 and neutralizing antibody to HSV-1 were measured. IELISA antibody titre is expressed by the absorbance at 492 nm at a 200-fold serum dilution

Immunogenicity of yeast-produced HSV gB-1: Y. Kino et al. Table 2

DTH and lymphoproliferative response in mice DTH

Injected

Lymphoproliferative

(10 -2 mm)

response

with

HSV-1

ygB-1

HSV-1

ygB-1

ygB-1

9.25 ± 3.5

7.4 ± 1.8

5.0 ± 1.2

6.0:1_ 2.0

Alum alone

3.60-]- 1.7

1.0± 1.0

1.0±0.5

1.2±0.8

One day before challenge, DTH was measured by the increase in ear thickness 24 h after challenge of the ear pinae with inactivated HSV-1 or ygB-1. One day before challenge, spleens were removed from five mice and the lymphoproliferative response upon stimulation with inactivated HSV-1 or ygB-1 was measured by incorporation of ~H-TdR into lymphocyte DNA and its value is expressed as SI + s.d.

but the lesions did not progress further and disappeared soon. After healing of the acute ocular lesion (day 60), the bilateral trigeminal ganglia were removed from each mouse and tested for reactivation of virus as described in Materials and methods. All the ganglia of mice injected with alum only harboured HSV and the titre of virus reactivated from the ganglia was very high. On the other hand, HSV could not be reactivated from most ganglia of the mice immunized with ygB-1. Some ganglia of mice immunized with ygB-1 were latently infected, but the titre of reactivated HSV was very low.

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Guinea-pigs were injectcd with 10/~g of ygB-I adsorbed to 100/~g of alum or with alum alone subcutaneously in the back on days 0 and 14. ELISA antibody to ygB-I and neutralizing antibody to both types of HSV were measured after injection. As was the case with Balb/c mice, ELISA antibody was detected in all guinea-pigs after the first dose, and reached a high level after the second dose (Table 4). High neutralizing antibody titres against both types of HSV developed after the booster injection (Table 4). The results of lymphoproliferative responses and skin tests are shown in Table 5. Guinea-pigs immunized with ygB-1 responded well to HSV-2 in both tests. On day 44, the guinea-pigs were challenged intravaginally with 1 x 104 p.f.u, of HSV-2 strain 8204 TN. After challenge, the extravaginal area was scored daily for lesions as described by Stanberry et al. 13. Score

(days)

Flgure 2 Efficacy of immunization with ygB-1 in protection against intraperitoneal challenge with HSV-I or HSV-2. Balblc mice were injected twice with I ~g of ygB-1 formulated with alum as described in the text. Ten days after the second dose the mice were challenged with I x I0 a p.f.u, of HSV-I or I x I0 e p.f.u, of HSV-2 intraperitoneally. Ten mice were used in each group, m, ygB-1-injected and HSV-l-challenged; E3, alum injected and HSV-I challenged; A, ygB-1-injected and HSV-2-challenged; A, alum-inlected and HSV-2-challenged

One day before challenge, D T H and lymphoproliferative responses were measured. As shown in Table 2, the mice immunized with ygB-1 showed a significant increase in ear thickness upon challenge with inactivated HSV-I or ygB-1. Splenocytes of mice immunized with ygB-1 also showed a good lymphoproliferative response against stimulation with inactivated HSV-1 or ygB-1. Ten days after the second dose, one group of mice was challenged intraperitoneally with 1 × 108 p.f.u, of HSV-1, strain K U (a clinical isolate from a patient with herpes labialis) or 1 × l06 p.f.u, of HSV-2 strain 8204 TN. In the case of intraperitoneal challenge, as shown in Figure 2, almost all mice injected with alum alone died within l0 days. However, no mice immunized with ygB1 died. Another group of mice was challenged with one drop of PBS containing HSV-I, strain K U (1 × 108 p.f.u. ml ~) in both corneas sacrified with a 26-guage needle. Five and 7 days later, the mice were observed microscopically for development of acute blepharitis and keratitis. As shown in Table 3, almost all mice injected with alum alone showed severe blepharitis and stromal keratitis. Some mice immunized with ygB-I developed mild hyperaemia of the eyelid and dendritic keratitis,

Table 3 Effect of immunization with ygB-1 on the course of corneal infection Infection Keratitis Blepharitis

Acute Latent

ygB-1

Alum alone

2/20 eyes a 6/20 eyes

19/20 eyes 16/20 eyes

4/14 ganglia (10'-102 p.f.u.)

14/14 ganglia (104-10. p.f.u.)

Balb/c mice injected twice with ygB-1 were inoculated with HSV-1 in both corneas. After challenge, the corneas were observed for development of keratitis (5 days after challenge) and blepharitis (7 days after challenge). On day 60, the trigeminal ganglia were removed from each mouse, and tested for reactivation of virus. aNo. positive/No, tested

Table 4 pigs

Development of ELISA and neutralizing antibodies in guineaNeutralization HSV-1 HSV-2

ELISA Guinea-pig No.

T1

1 2 3 4 5 6 7 8 9 10 11 Alum alone (mean; n = 18)

0.046 0.090 0.178 0.121 0,135 0.129 0.105 0.161 0.084 0.090 0.120 0.000

T2 0.431 0.555 0.533 0.369 0.439 0.488 0.470 0.597 0.312 0.249 0.398 0.000

T1

T2

T1

T2

<2 4 4 4 4 4 4 <2 <2 4 4 <2

64 128 128 128 128 64 64 64 32 32 64 <2

2 4 4 4 4 4 4 <2 <2 4 4 <2

64 128 64 64 128 64 64 128 32 32 32 <2

Guinea-pigs were injected twice with ygB-1 subcutaneously. One day before the second dose (T1) and challenge (T2), ELISA antibody to ygB-1 and neutralizing antibody to HSV-1 and 2 were measured. ELISA antibody is expressed as the absorbance at 495 nm at a 500-fold serum dilution

Vaccine, Vol. 7, April 1989

157

Immunogenicity of yeast-produced HSV gB- 1: Y. Kino et al. Table 5

Lymphoproliferative response and DTH in guinea-pigs Lymphoproliferative response

DTH

Guinea-pig No.

T1

T2

(mm)

1 2 3 4 5 6 7 8 9 10 11 Alum alone (mean; n = 18)

nd nd nd 2.9 1.5 1.8 0.5 3.4 2.6 0.3 4.4 0 85 + 0.5

24.7 11.9 10.9 7.4 10.9 10.8 7.9 11.4 9.8 24.9 12.8 1.7 4- 1.6

49 25 40 48 30 35 31 35 30 28 50 19 4- 3.6

Peripheral blood lymphocytes were collected from each guinea-pig by cardiac puncture one day before the second dose (T1) and challenge (T2). The lymphoproliferative response upon stimulation with inactivated HSV-2 was measured by incorporation of [=H]thymidine. The DTH response was investigated by measuring the diameter of erythema formed 24 h after intracutaneous challenge with HSV-2. The DTH value is expressed as the sum of the longest and shortest diameter of erythema

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Time after infection (days) Figure 3 Effect of immunization with ygB-1 on the development of extravaginal herpetic lesions in guinea-pigs. Guinea-pigs were injected twice with 10 #g of alum-precipitated ygB-1, and challenged with I x 10" p.f.u, of HSV-2 intravaginally. They were observed for the development of extravaginal lesions and scored as described in the text. II, ygB-1injected; C3, alum-injected

0 indicated no disease; 1, redness or swelling; 2, a few small vesicles; 3, several large vesicles; 4, several large ulcers with maceration. The profile of development of extravaginal lesions is shown in Figure 3. Of 18 guineapigs, 13 showed maximum score 4, two animals showed maximum score 3 and three animals showed no lesions in the control group. Eight severely infected guinea-pigs died with hind-limb paralysis. In the guinea-pigs injected with alum alone, swelling and redness of the extravaginal area was observed four days after infection. Severe vesiculation was observed six days after infection, and ulceration, urinary retention and maceration were observed from day 8. In the guinea-pigs immunized with ygB-1, in contrast, no lesions developed except in two animals out of ten which showed mild swelling and redness of the extravaginal area, but the lesions disappeared within several days. After healing of the primary lesions, the guinea-pigs were examined for recurrent lesions every day for 60 days after challenge. All guinea-pigs showing primary lesions, including immunized animals, developed recurrent lesions several times. In contrast, no recurrent lesions were observed in the guinea-pigs that had experienced no primary lesions. 158

Vaccine, Vol. 7, April 1989

Sixty days after challenge, serum were collected from each guinea-pig and anti-gD-I antibody was measured to detect possible inapparent infection. As seen in Table 6, guinea-pigs in which lesions developed had high antigD-I antibody titres. In contrast, almost all guinea-pigs without lesions had no antibody. Three guinea-pigs immunized with ygB-I and showing no lesions had a low level of anti-gD- 1 antibody.

Discussion It has been reported that gB-I can be effectively purified by an immunoadsorbent to which a monoclonal antibody to gB-I is conjugated 6. In the present study, ygB-1 from the Triton X-100-solubilized lysate was purified with the same immunoadsorbent. As shown in Figure 1, ygB-I was also effectively purified by the immunoadsorbent. The molecular weight of ygB-1 was slightly higher than expected from 'the nucleic acid sequences. The discrepancy is explained by glycosylation of ygB-l. By an endoglucosidase analysis, ygB-1 was sensitive to endoglycosidase H and F (data not shown). It has been shown that nude mice passively immunized with monoclonal antibodies against gB-I are protected from HSV-I infection 1°. The protection conferred by the passive immunization was stronger than that conferred by human immunoglobulin preparations 14. Furthermore, it was found that mice immunized with gB-I purified from HSV-l-infected Vero cells were protected from intraperitoneal challenge with HSV-1 and 2, and from acute corneal challenge and latent infection in the trigeminal ganglia 6. As to the potential of gB-I as an HSV vaccine, Chan 4 reported that mice immunized with purified gB-1 showed marked resistance to challenge with virulent HSV-1. Roberts et al. 8 also reported that immunization of m!ce with a mixture of purified gB-1 and its precursor pgB-1 induced a good level of neutralizing antibody which persisted for nine weeks and the mice survived virus challenge. In the sense of recombinant gB, Stanberry et al. Is reported that guinea-pigs immunized with gB-1 derived from Chinese hamster ovary cells were protected from the development of primary and recurrent lesions. From these findings gB-1 appears to be one of the candidates for a constituent of an HSV component vaccine. Table 6 Development of anti-gD-1 antibody in guinea-pigs challenge and development of recurrent lesions

ygB-l-immune

after

Alum-injected

No. of No. of GuineaMaximum recur- GuineaMaximum recurpig No. ELISA score rences pig No. ELISA score rences 1 2 3 4 5 6 7 8 9 10

0.068 0.117 0.197 0.090 0.031 0.067 0.397 0.053 0.306 0.147

0 0 0 0 0 0 1 0 1.5 0

0 0 0 0 0 0 2 0 3 0

1 2 3 4 5 6 7 8 9 10

0.614 0.019 0.579 0.837 0.027 0029 0.818 0.618 0.312 0.332

4 0 3 4 0 0 4 4 3 4

1 0 6 2 0 0 3 4 2 2

Two months after challenge, serum was collected from each guinea-pig and ELISA antibody to gD-1 was measured, The ELISA antibody titre is expressed as the absorbance at 492 nm at a 200-fold serum dilution. The guinea-pigs were examined for recurrent lesions every day for 60 days after challenge

I m m u n o g e n i c i t y o f y e a s t - p r o d u c e d H S V gB-1: Y. K i n o et al.

For HSV vaccine, safety is an absolute necessity as well as efficacy. In the case of gB-1 prepared by the conventional tissue culture technique, it is quite difficult to exclude the presence of viral D N A which has potential infectivity and oncogenicity. By using ygB-1 as a constituent of an HSV vaccine, it is possible to propose a safe vaccine. In the present study the efficacy of ygB-I in the mouse and guinea-pig model was investigated. In the mouse model, animals were well protected from acute intraperitoneal challenge with HSV-I or HSV-2 and corneal challenge with HSV-I and latent infection in the trigeminal ganglia, by the immunization with ygB-1. As immune responses of mice to HSV, production of neutralizing antibody, antibody-dependent complementmediated cytolysis (CDC), antibody-dependent cellmediated cytotoxicity (ADCC), D T H response and generation of cytotoxic T lymphocytes (CTL) have been known 16. The role of each immune response in protective immunity has been investigated by passive transfer of antibody or adoptive transfer of immune lymphocytes. By passive immunization studies with monoclonal antibodies against HSV glycoproteins t°'~7-~9, mice were protected against several routes of HSV infection. However, the mechanism of antibody-mediated protection is not clear. Although correlation between A D C C activity of monoclonal antibodies and their in vivo activity were reported 1°'~7, it was also reported that some monoclonal antibodies with no neutralizing, A D C C or C D C activity have a therapeutic effect in a passive immunization study 19. There seems to be several pathways in antibody-mediated protection. In the present study, mice and guinea-pigs immunized with ygB-1 developed good ELISA and neutralizing antibodies. Anti-ygB-1 antibody may be responsible for the protection in some way. However, a passive immunization study must be conducted to determine the exact role of antibody. Cell-mediated immunity is known to play a major role in the protection and recovery from HSV infection 2°'2~. In adoptive transfer studies in a murine model, protectivity was transferred by Thy-1 +,Lyt-1 +23 cells 22':3. In this study, the D T H response was elicited in mice and guinea-pigs by immunization with ygB-1. Schrier et a l l 4 showed that mice immunized with gC developed a D T H response and they were protected from HSV infection in the absence of detectable antibody. The authors postulated that the protective response was related to the D T H response. T - D T H cells induced by immunization with ygB-1 appear to play some role in the restriction of viral growth. As to CTL, Sethi et al. 25 showed that cyclophosphamide-treated mice were protected from lethal HSV infection by transfer of a herpes-specific cytotoxic T cell clone. From their data it is proposed that herpes-specific C T L play some role in the protective immunity. Although in the present study, we did not investigate whether C T L was generated by immunization with ygB-1, Blacklaws et al. 26 reported that HSV-specific C T L recognized gB expressed by an L cell line and that the cell line could induce CTL in mice. In our case, several immune responses were induced by immunization with ygB-1 and each immune response appears to play a role in the protection. Additional studies must be conducted to elucidate the precise mechanism(s) of protection conferred by immunization with ygB-1.

Dix and Mills 5 reported that immunization of mice with purified gB-I or gB-2 had no prophylactic effect on HSV-2 infection in the footpads, whereas gD-1 or gD-2 conferred good protection. In the present study guineapigs immunized with ygB-1 were well protected from intravaginal infection with HSV-2, whereas guinea-pigs injected with alum alone developed severe lesions and 44% of them died. The results are similar to those obtained by Berman et al. 27 using cloned gD-l. Scriba 28 showed that guinea-pigs immunized with a crude extract from HSV-2-infected HEp-2 cells were protected from primary and recurrent infection. In that study, animals that developed no primary lesions showed no recurrent lesions, whereas animals with primary lesions (including immunized animals) experienced recurrent infections several times. According to the study of Stanberry et al.~3, extravaginal lesions are formed by retrogressive infection of the external skin with the virus which had once grown in the lumbosacral dorsal root ganglia. Since protected animals experienced no primary and recurrent infections and produced no or only a low.level of anti-gD-I ELISA antibody, virus growth in the inoculation site and the dorsal r o o t ganglia seemed to be restricted. The observations obtained in this study that mice and guinea-pigs were protected from primary, latent and recurrent infection show that ygB-1 has a potentiality as an HSV component vaccine. Recently, however, Meignier et al. 29 reported that immunization with an artificial mixture of gB, gC, gD and gE reduced the morbidity in mice and guinea-pigs but not in owl monkey. It remains to be determined whether our ygB-I is immunogenic in primates as it is in mice and guinea-pigs.

Acknowledgements The authors are grateful to Dr Mary Louise Robbins for reviewing the manuscript, and are also grateful for the superior technical and secretarial skills of Yukimi Fujimoto.

References 1 Spear, P.G. Glycoproteins specified by herpes simplex virus. In: The herpes viruses (Ed. Roizman, B.) Plenum Publishing Corp., New York, 1985, pp. 315-347 2 Duff, R. and Rapp, F. Oncogenic transformations of hamster cells after exposure to herpes simplex virus type 2. Nature NewBioL 1971,233, 48 3 Hill, T.J. Herpes simplex virus latency. In: The herpes viruses (Ed. Roizman, B.) Plenum Press Publishing Corp., New York, 1985, pp. 175-240 4 Chan, W.L. Protective immunization of mice with specific HSV-1 glycoproteins. Immunology 1983, 49,343 5 Dix, R.D. and Mills, J. Acute and latent herpes simplex virus neurological disease in mice immunized with purified virus-specific glycoproteins gB or gD. J. Gen. Virol. 1985, 17, 9 6 Kino, Y., Eto, T., Nishiyama, K., Ohtomo, N. and Mori, R. Immunogenicity of purified glycoprotein gB of herpes simplex virus. Arch. ViroL 1986, 89, 69 7 Long, D., Madara, T., Ponce de Leon, M., Cohen, M., Montgomery, P.D. and Eisenberg, R.J. Glycoprotein D protects mice against lethal challenge with herpes simplex virus types I and 2. Infect. Immun. 1984, 37, 761 8 Roberts, P.L., Duncan, B.E., Raybould, T.J.E. and Watson, D.H. Purification of herpes simplex virus glycoprotein B and C using monoclonal antibodies and their ability to protect mice against lethal challenge. J. Gen. ViroL 1985, 66, 1073 9 Showalter, S.D., Zweig, M. and Hampar, B. Monoclonal antibodies to herpes simplex virus type 1 protein, including the immediate-early protein ICP 4. Infect. Immun. 1981,34, 684 10 Kino, Y., Eto, T., Ohtomo, N., Hayashi, Y., Yamamoto, M. and Mori, R. Passive immunization of mice with monoclonal antibodies to glycoprotein gB of herpes simplex virus. MicrobioL ImmunoL 1985, 29, 143

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Immunogenicity

of yeast-produced

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