- Email: [email protected]
Immune response to varicella-zoster virus glycoproteins in guinea pigs infected with Oka varicella vaccine
Vaccine, Vol. 16, No. 13, pp. 1263-1269, 1996 0 1996 Elsevier Science Ltd. All riahts reserved Printed in-Great Britain 0264-41 OX/96 $19+0.00
PII: SO264-410X(98)00035-S
Immune response to varicella-zoster virus glycoproteins in guinea pigs infected with Oka varicella vaccine Hitoshi Sate”, Seiji Kageyama*, Masami Imakita”f, Mitsuru Ida*, Jun-ichi Yamamura*, Masahiko Kurokawa” and Kimiyasu Shiraki*$ A varicella skin test antigen has been developed based on the induction of delayed-type hypersensitivity to varicella-zoster virus (VZV), and has been used to evaluate the immune status to V’T!’ The authors have purified gB, gE:gI and gH:gL and examined their cutaneous reactivity in guinea pigs infected with Oka varicella vaccine. The cutaneous reaction to each glycoprotein was observed and the maturation process of cutaneous reaction was examined in infected guinea pigs. Cutaneous reaction to gH:gL, a major target of virus-neutralizing antibody, appeared first on day 3 among three glycoprotein complexes and the reaction to gE:gI, the most abundant glycoprotein, became strongest three weeks after infection. The earliest recognition of gH:gL may contribute to minimizing the spread of viral infection. Thus, the skin test may be a suitable marker to assess the cell-mediated immunity in varicella, including vaccine recipients and zostel; i,lzrelation to the immune status of glycoproteins. 0 1997 Elsevier Science Ltd. All rights reserved Keywords: delayed type hypersensitivity: VZV glycoproteins; Oka varicella vaccine
Varicella-zoster virus (VZV) infection causes clinical consequences for the general population as well as for the expanding number of immunocompromised hosts with primary immune defects, cancer, organ transplants, and acquired immunodeficiency syndrome’-‘. Oka varicella vaccine has been safely used to confer cell-mediated immunity and humoral immunity to individuals435. immunocompromised and healthy Immunity, especially cell-mediated immunity, plays a critical role in limiting .the morbidity of infection6- . In addition, booster augmentation of cell-mediated immune response can be achieved with Oka varicella vaccine’“-‘2. A varicella skin test antigen has been developed for diagnostic purposes to assess cellmediated immunity to \7ZV and can successfully distinguish the susceptibility from the resistance to VZV infection’“-‘h. Varicella, skin test antigen and its biological potency have been characterized in guinea pigs infected with Oka varicella vaccine’7-‘9. Specificity of the varicella skin test to VZV has been confirmed in *Department of Virology, Toyama Medical and Pharmaceutical University, 2630 Sugitani Toyama 930-01, Japan. TDivision of Pathology, National Cardiovascular Center, Osaka, Japan. *Author to whom all correspondence should be addressed. Tel: (81) 764 34 2281; Fax: (81) 764 34 5020; E-mail: [email protected]. A pat-l of the information in this manuscript has been presented at the 21st International Herpesvirus Workshop in July, 1996 at DeKalb, IL (abstract number 376). (Received 27 October 1997; revised vlsrsion received 3 February 1998; accepted 3 February 1998)
guinea pigs infected with HSV and VZV as well as in children with a history of recent HSV infection who developed varicella, althoub292yZV and HSV share a common antigenic epitope . Guinea pigs infected with Oka varicella vaccine show positive skin tests to varicella skin test antigen as delayed type hypersensitivity (DTH) and the lymphocyte proliferation response to viral antigen as well as the neutralizing antibody response’“. VZV glycoproteins, gB, gE:gI and gH:gL, are present on the surface of both VZV particles and infected cells, and contain epitopes that stimulate VZV-speci fit cellular humoral immune and responses”-‘4. The function of each glycoprotein is not as clear as that of other herpesviruses but its importance in the immune response or infection in vitro has been well documented22~‘“~25-‘7.Varicella skin test antigen that is clinically used contains VZV glycoproteins. The authors have purified gB, gE:gI and gH:gL, and examined their cutaneous reactivity representing DTH in guinea pigs infected with Oka varicella vaccine. Positive skin tests to each glycoprotein were observed in infected guinea pigs. Moreover, the the maturation process of the cutaneous reaction was investigated. Interestingly, the cutaneous reaction to gH:gL appeared first among three glycoprotein complexes and the reaction to gE:gI became strongest later on in guinea pigs. Thus, the authors have characterized the immune response to these three glycoprotein complexes in guinea pigs infected with Oka varicella vaccine, and discussed applying glycoproteins
Vaccine
1998 Volume
16 Number
13
1263
Immune response to VZV glycoproteins to the skin test as an immunological
in guinea pigs: H. Sato et al.
marker for VZV
infection. MATERIALS AND METHODS
vig. The cutaneous reaction was assessed at 8. 24 and is”h after injection, and its size was expresseh by the following formula: (Area) =
71x (long diameter) x (short diameter) 4
Virus and cells
The Oka varicella vaccine was supplied from the Research Foundation for Microbial Diseases of Osaka university, Suita, Japan, or prepared as cell-free virus as described previously20~28~29. The Oka varicella vaccine strain was used to immunize guinea pigs and for the preparation of the viral antigen. Human embryonic lung cells were grown and maintained in Eagle’s minimum essential medium supplemented with 10 and 2% fetal bovine serum, respectively, and used to propagate the virus. Antiserum
The antiserum used in Western blot analysis was anti-VZV guinea pig serum which was produced by immunizing guinea pigs with VZV-infected guinea pig embryonic fibroblasts mixed with Freund’s complete adjuvant3’. Mouse monoclonal antibodies, clone 8 and clone 9, and human monoclonal antibody TI-57 (Teijin, Hino, respectiv~~~~~~1,3~recognize gB, gE and gH, Purification of glycoproteins chromatography
by affinity column
Glycoproteins were purified from Oka varicella vaccine-infected cells by affinity column chromatography usin monoclonal antibodies as reported previously25,28.3. Briefly, infected cells were lysed in the lysing buffer (20 mM Tris-HCl (pH 8.0), 1% Triton X-100) and centrifuged at 7000 rpm for 10 min and at 27000 rpm for 1 h. The supernatant was sequentially applied to the affinity columns coupled with monoclonal antibodies against gB, gE or gH according to the manufacturer’s instructions (ImmunoPure IgG Orientation Kit, Pierce, IL). Glycoproteins were eluted by 0.2 M glycine-HCl buffer (pH 2.9) after extensive washing of the column with the lysing buffer and phosphate-buffered saline (PBS: pH 7.4). The elutes were immediately neutralized with 2 M Tris-HCl (pH 8.0) and the buffer was replaced to PBS by ultrafiltration (Centricon, Amicon, MA) followed by the removal of IgG with Protein G Sepharose 4F (Pharmacia, Uppsala, Sweden). The purified glycoprotein complexes were used for the skin test, the enzyme-linked immunosorbent assay (ELISA), and the lymphocyte proliferation assay. VZV infection
and skin tests in infected guinea pigs
Female guinea pigs (Yodo strain) weighing about 280 g (Japan Rabbit Center, Ibaraki, Japan) were used for infection with Oka varicella vaccine’ -19. Guinea pigs were infected with 2500-6000 plaque-forming units of Oka varicella vaccine subcutaneously, and 0.1 ml of gB, gE:gI and gH:gL as well as the varicella skin test antigen was injected intradermally on the back of guinea pigs after removal of hair on days 1, 3, 7, 14 and 21 after infection. VZV-crude antigen was prepared according to the method of Kamiya et aE.13. The skin test was performed only once for each guinea
1264
Vaccine 1998 Volume 16 Number 13
Serological
assay
Serum was harvested from VZV-infected or uninfected guinea pigs under ether anesthesia at the last inspection of the cutaneous reaction. 10 pg ml- ’ of gB, gE:gI or gH:gL was applied to the wells of the ELISA plate. Sera were diluted a hundred times with PBS containing 2% skim milk, and applied to the wells to titrate the antibody to VZV crude antigen, control antigen, gB, gE:gI and gH:gL in the ELISAz4. Then, peroxidase-conjugated goat IgG fraction to guinea pig IgG (whole molecule: Cappel, NC) was distributed to the wells and the reaction was visualized according to the manufacturer’s instructions (HAT-EIA, DENKA SEIKEN, Tokyo, Japan). The optical density (OD) values were measured at 450 nm. Lymphocyte
proliferation
assay
The skin test and the lymphocyte proliferation assay were performed with three guinea pigs three weeks after infection with Oka varicella vaccine. Mononuclear cells were prepared from the spleens of guinea pigs after centrifugation on the Ficoll-Hypaque gradient. Guinea pig mononuclear cells were incubated in RPM1 medium supplemented with 10% guinea pig serum and 2-mercaptoethanol (5 x lo-’ M) in 96-well microplates. The mononuclear cells at the concentration of 2 x 105/100 ~1 per well in triplicate were incubated with serially diluted VZV crude antigen, uninfected antigen, gB, gE:gI, gH:gL or PBS. Purified glycoprotein complexes were used at concentrations of 1000, 333, 111 and 37 ng per 100 ~1 per well. After 5 days incubation of mononuclear cells from guinea pigs at 37”C, the cells were labeled with 5 &i [3H]thymidine (64.3 Ci mmol-‘) per well for 18 h and harvested onto filter paper, and the radioactivi? was determined in a liquid scintillation counter2’,22’3,35. The mean radioactivity from triplicate cultures was used to determine the ratio of that in the antigen-stimulated cultures to that in the appropriate control cultures. Its largest ratio was used as the stimulation index (S.I.). Statistical
analysis
The statistical significance of mined by the repeated measure (the repeated measure ANOVA). 0.05 was considered as statistically
the data was deteranalysis of variance A P value less than significant.
RESULTS Purification
of gB, gE:gI and gH:gL
VZV glycoproteins were purified by affinity column chromatography coupled with monoclonal antibodies and were analyzed by SDS-PAGE and Western blot using anti-VZV guinea pig serum as shown in Figure 1. The results were consistent with those reported previously*‘. Briefly, the molecular weights of gB and gE:gI visualized by Coomassie brilliant blue staining
Immune response to VZV glycoproteins
Affinity
purified
in guinea pigs: H. Sato et al.
gB, gE:gl, and gH:gL
192K 192K 127K 127K
73K 73K
43K
43K Figure 1 Purified gB, gE:gl and gH:gL were subjected using anti-VZV serum.
to SDS-PAGE
with those and Western blot were consistent reported20.25.3’.36.37. However gH was not detected by Western blot because of loss of conformational antigenicity27.3R,39. The purified fractions of gE and gH contained the amino acid sequences of g1 and gL, respectively. Therefore gE and gH existed as gE:gI and gH:gL complexes, respelctively40-42. Cutaneous reaction and antibody titer of infected guinea pigs Figure 2 shows the dose-response profile of the
cutaneous reaction to glycoprotein complexes (gE:gI, gB and gH:gL) in three guinea pigs three weeks after
and analyzed
by Coomassie
brilliant blue staining and Western blot
infection. The size of the cutaneous reaction to three glycoprotein complexes increased dose-dependently. No cutaneous reaction was induced by either antigen in uninfected guinea pigs. The cutaneous reaction was strongest against gE:gI among three glycoprotein complexes at 10 and 20 @g per dose (P~0.05 by the repeated measure ANOVA). The antibody response to three glycoprotein complexes was assessed by the ELISA as shown in Figure 3. Poor antibody response to gH:gL in the ELISA was consistent with the results reported by Keller et ~1.~~and may be caused by the difficulty in detecting antibody to gH:gL in the ELISA system, possibly because of the loss of conformational antigenicity of gH’734’. 48 h
8 Ih
I
-n-gE:gI --+gB
--+-gH:gL
* T
*
* I Ti T
Dose of gl ycoprotei
n complexes
@g/dose)
Figure 2 Dose-response of cutaneous reaction to purified gB, gE:gl and gH:gL at 8, 24 and 48 h after their inoculation in guinea pigs three weeks after infection. The size of cutaneous reaction is expressed as the mean and positive SD of three guinea pigs. *P
Vaccine 1998 Volume 16 Number 13
1265
hmune
response to VZV glycoprofeins in guinea pigs: Ii. Sat0 et al.
Lymphocyte proliferation assay in guinea pigs infected with Oka varicella vaccine and the controls Table 1 shows the lymphocyte proliferation assay of the spleen cells from guinea pigs three weeks after infection with Oka varicella vaccine. Specificity of the response to various antigens was expressed by the S.I. Infected, but not uninfected, guinea pigs responded to various antigens in the cutaneous reaction, the lymphocyte proliferation assay and antibody production. Both the cutaneous reaction and the S.I. in lymphocyte proliferation assays were strongest against gE:gI among three glycoprotein complexes. Although the S.I. values of various antigen preparations were larger in infected
1.4
-
A A
1 -
B
1.2
0 E
6 In -ICI m $
0.8
-
0.6
-
.
•I gE:gl
.
A@
.
0 @-W-
0.4-
0.2
Q I -
o-
- 0.2
infected
uninfected
gui nea pigs Figure 3 Antibody response to gB, gE:gl and gH:gL in guinea pigs three weeks after VZV infection determined at 1:lOO serum dilution by the ELISA. OD, optical density. Table 1
Immune response to VZV antigen or glycoprotein
gB
gE:gl
gH:gL
Appearance of cutaneous reaction and antibody to glycoprotein complexes in guinea pigs after infection The authors examined cutaneous reaction and antibody response to glycoproteins in guinea pigs at different time points after infection. As all glycoprotein complexes induced the cutaneous reaction enough to be detectable at a dose of 10 pg as shown in Figure 2, this dose was used to evaluate the appearance of the cutaneous reaction in the infected guinea pigs. Figure 4(a) shows the time course of appearance of the cutaneous reaction to gB, gE:gI and gH:gL in guinea pigs infected with Oka varicella vaccine. No cutaneous reaction was observed in uninfected and infected guinea pigs on day 1 after infection. Positive cutaneous reaction to gH:gL appeared first among three glycoprotein complexes on day 3 after infection. To confirm this result, the authors repeated the two additional experiments. Figure 5 shows the results of the skin test on day 3 after infection in three independent experiments. The data of the first experiment were the same as those on day 3 in Figure 4(a). The cutaneous reaction to gH:gL was significantly strongest among those of three glycoprotein complexes in four infected guinea pigs in the second and third experiments (P~0.05 or P ~0.01 by the repeated measure ANOVA). Three glycoprotein complexes induced the strong cutaneous reaction on days 7 and 14, but the cutaneous reaction to gE:gI was not as prominent in these days as that observed three weeks after infection (Figure 2). The cutaneous reaction to VZV crude antigen was smaller than that to gH:gL on day 3 but became larger on day 7 and after (data not shown). Thus, the cutaneous reaction to gH:gL was most sensitive in the early phase of infection and the reaction to gE:gI became strongest three weeks after infection. Antibodies to gE:gI and gB became detectable on day 9 after infection and then increased in titer. Antibody response to gE:gI rose earlier than the response to gB. Antibody titer to gE:gI and gB was high but, in contrast, the antibody titer to gH:gL was in gunea pigs infected with Oka varicella vaccine
VZV-infected
Guinea pigs
vzv
complexes
guinea pigs than in uninfected guinea pigs, they were not as large as expected from the results with their cutaneous reaction.
cutaneous reaction= stimulation index antibody titer cutaneous reaction stimulation index antibody titer cutaneous reaction stimulation index antibody titer cutaneous reaction stimulation index antibody titer
1
2
3
1
56.5/38.5b 4.81 0.272’ 63.6144.0 1.40 0.520 132.7177.7 1.91 0.671 63.6144.0 1.49 0.024
38.5128.3 13.6 0.335 70.7156.5 5.97 0.598 188.4l86.4 7.24 0.698 56.5130.5 4.24 0.021
37.7138.5 2.95 0.100 33.0133.0 1.37 0.061 63.6150.2 1 .a9 0.109 50.2133.0 1.04 0.000
_I_”
aGuinea pigs were infected with Oka varicella vaccine and three weeks inoculation of skin test antigen. 10 pg per dose of glycoprotein was used. bArea size (mm*) of cutaneous reaction. “Less than 5 mm in diameter. dOptical density at 450 nm in ELISA.
1266
uninfected
Vaccine 1998 Volume 16 Number 13
later cutaneous
2
3
__ I
1.25 0.008
-I-
0.98
1.39 0.011
0.013
-I-
-I-
-I-
1.06 ~ 0.003
1 .Ol - 0.002 __ I 1.25 0.002
1.30 - 0.003 __ I
-I-
__ I
-I1.15 -0.002
-I1.00 0.000 reaction
1.06 0.004
was determined
1.35 0.006 1.25 0.006
at 24 h/48 h after
Immune response to VZV glycoproteins in guinea pigs: H. Sat0 et al. not, or barely, detected at any time in ELISA (Figure
vaccine, and the maturation of the cutaneous reaction was characterized in infected guinea pigs. Antibody to gH:gL neutralizes viral infectivity and inhibits the cellto-cell spread of infection and polykaryon formation’7.“6,44,45, and purified native gH:gL interferes with virus adsorption in vitro2’. Therefore, gH:gL is one of the major targets of the immune reaction in infected humans and guinea pigs. Positive skin tests to gH:gL first appeared in guinea pigs infected with Oka varicella vaccine. This is reasonable because the earliest recognition of gH:gL among glycoproteins may be the most beneficial for the host to minimize the
4(b))-
DISCUSSION Guinea pigs infected with Oka varicella vaccine are a suitable model to characterize skin test antigen with respect to cell-mediated immunity to VZV’7-19. In this study, skin tests with glycoprotein complexes were performed in guinea pigs infected with Oka varicella
a 100
n
gE:gl
n gB 0
gH:gL
-I-
14
7
Day 3 Days after
infection
-b 1.2 -
E
&
0.8 -
+
gE:gl
-
!3B
-f
gH:gL
Day 3
Day 5 Days
after
Da& 9
Day 16
infection
Figure 4 Appearance of cutaneous reaction and antibody response to glycoprotein complexes in guinea pigs after infection. (a) Cutaneous reaction to 10 pg per dose of each glycoprotein complex in three guinea pigs on days 3, 7 and 14 after WV infection. The size of cutaneous reaction is expressed as the mean and positive SD of three guinea pigs. (b) Antibody response to gB, gE:gl and gH:gL determined at 1 :lOO serum dilution by the ELBA in guinea pigs on days 3, 5, 9 and 16 after VZV infection. The data points are expressed as the means k SDS of three guinea pigs. OD, optical density.
Vaccine 1998 Volume 16 Number 13
1267
Immune response to VZV glycoproteins
in guinea pigs: H. Sato et al.
spread of viral infection. As the cutaneous reaction appears four days after vaccination in vaccinees46, the early appearance of the cutaneous reaction is not surprising, but the earliest response to gH:gL was interesting with respect to its biological importance. The strong cutaneous reaction to gE:gI was observed in infected guinea pigs and this is also reasonable from the most abundant and immunogenic nature of gE:gI among three glycoprotein complexes’3340. Thus, the cutaneous reaction to glycoprotein complexes was well correlated with their function or biological importance. The guinea pigs produced antibodies to gB and gE:gI after infection with Oka varicella vaccine. Antibody response to gB, gE:gl and VZV crude antigen in guinea pigs was detectable on day 9 and later, which is similar to the results in children with chickenpox or in children vaccinated with Oka varicella vaccine27.46. Antibodies to gH:gL were barely detectable in guinea pigs by ELISA. These results are consistent with the reports by Keller et ~1.~~. The authors have evaluated the relationship of the immunological responses among the skin test, the antibody response, and the lymphocyte proliferation assay to three glycoprotein complexes in guinea pigs infected with Oka varicella vaccine. Infected guinea pigs responded to each glycoprotein complex but the 1 st Ex.
8.
1 gE:gl 60
n @
1
40
I
T
20
-
0 I__._&
0 gH:gL
-
I&*
:
80 :Znd Ex.
5
60: 40:
z0 L
a
R
20 y
ACKNOWLEDGEMENTS
0 3rd
80
60
magnitude of their response was diverse. Their response to glycoprotein complexes was not as strong in terms of the S.I. as expected from the cutaneous reaction. The cutaneous reaction was more sensitive in detecting cell-mediated immune response to glycoproteins and VZV antigen than the lymphocyte proliferation assay. The cutaneous reaction may be mediated by the macrophages, Langerhans cells and dendritic cells in the skin. They may release interleukin 12 by the stimulation of VZV antigen or purified glycoproteins and can activate Thl cells, resulting in the induction of DTH4’. VZV grows in the skin and therefore the immune response mediated by cutaneous macrophages might contribute to the induction of the strong cutaneous reaction in the skin. This indicates that the cutaneous reaction mediated by the cutaneous macrophage may be more appropriate to assess cell-mediated immune response than the lymphocyte proliferation cells or peripheral blood assay using spleen mononuclear cells, because cutaneous macrophages are the natural effector cells in one of the major target tissues of VZV infection. The cutaneous reaction to the varicella skin test antigen is undetectable or weak in the acute phase of zoster and becomes stronger in the convalescent phase over time4’. Considering this close association of the cutaneous reaction with recovery from zoster, it is important to further clarify the relationship of the impairment in the cutaneous reactivity to specific glycoprotein in zoster patients. Varicella skin tests have been applied to screen people with low cell-mediated immunity to VZV by the lymphocyte proliferation assay or the skin test, and Oka varicella vaccine has been applied to boost it to prevent zoster’0-12”6. Thus, the skin test to VZV glycoproteins may be the most suitable marker to assess the cell- mediated immunity in varicella including vaccinees and to understand the pathogenesis of zoster in relation to the immune status to glycoproteins.
I
Ex. *
The authors thank the Research Foundation for Microbial Diseases of Osaka university for supplying Oka varicella vaccine, and Teijin institute for Biomedical Research for monoclonal antibody (TI-57).
*
*
Tll
REFERENCES
Time after
i nnocul ati on of skin
test
antigen
Figure 5 Cutaneous response to glycoprotein complexes on day 3 after VZV infection in three independent experiments. The first experiment was performed in three guinea pigs infected with Oka varicella vaccine. This figure is the same with Figure 4(a) on day 3. The second and third experiments were performed in four guinea pigs infected with Oka varicella vaccine. The virus and glycoprotein complexes used were the same lots in all the experiments. Cutaneous reactivity to gH:gL was significantly stronger than that to gB and gE:gl in both experiments (PcO.05 by the repeated measure ANOVA).
1268
Vaccine 1998 Volume 16 Number 13
Grose, C. and Giller, Ft. Varicella-zoster infection and immunization in the healthy and the immunocompromised hosts. Crit. Rev. Oncol. Hematol. 1988, 8, 27-64. Straus, SE., Ostrove, J.M. and Inchauspe, G. et al. Varicellazoster virus infections: biology, natural history, treatment, and prevention. Ann. Inter. Med. 1988, 108, 221-237. Arvin, A. Varicella-zoster virus. In: Fields Virology (Eds Fields, B.N., Knipe, D.M., Howley, P.M. et a/.). 3rd edn., Vol. 2, Lippincott-Raven, Philadelphia, New York, 1996, pp. 2547-2585. Takahashi, M., Hayakawa, Y., Shiraki, K., Yamanishi, K., Asano, Y. and Ozaki, T. Attenuation and laboratory markers of the Oka-strain varicella-zoster virus. Postgrad. Med. J. 1985, 4, 37-46. Gershon, A.A., LaRussa, P., Hardy, I., Steinberg, S. and Silverstein, S. Varicella vaccine: the American experience. J.
Immune response to VZV glycoproteins in guinea pigs: H. Sato et al. 6
7 8
9
IO
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Infect. Dis. 1992, 166, S63-S68. Baba, K., Yabuuchi, H., Takahashi, M. and Ogra, P.L. Increased incidence of herpes zoster in normal children infected with varicella zoster virus during infancy: communitybased follow-up study. .I! Pediatr. 1986, 109, 372-377. Dworsky, M., Whitley, R. and Alford, C. Herpes zoster in early infancy. Am. J. Dis. Chikl. 1980, 134, 233-235. Patterson, L.E., Butler. K.M. and Edwards, M.S. Clinical herpes zoster shortly following primary varicella in two HIV-infected children. C/in. Pediatr. 1989, 26, 354. Hayward, A. and Herberger, M. Lymphocytes responses to varicella-zoster virus in ,the elderly. J. C/in. Immunol. 1987, 7, 174-I 78. Hayward, A., Levin, M., Wolf, W., Angelova, G. and Gilden, D. Varicella-zoster virus-specific immunity after herpes zoster. J. infect. Dis. 1991, 163, 873-875. Berger, R., Luescher, D. and Just, M. Enhancement of varicella-zoster-specific immune responses in the elderly by boosting with varicella vaccine. J. Infect. Dis. 1984, 149, 647. Watson, B., Rothstein, Ei. and Bernstein, H. et a/. Safety and cellular and humoral immune responses of a booster dose of varicella vaccine 6 years after primary immunization. J. Infect. Dis. 1995, 172, 217-219. Kamiya, H., Ihara, T. and Hattori, A. et al. Diagnostic skin test reactions with varicella virus antigen and clinical application of the test. J. Infect. Dis. 1977, 136, 784-788. Asano, Y., Shiraki, K., Takahashi, M., Nagai, H., Ozaki, T. and Yazaki, T. Soluble skin test antigen of varicella-zoster virus prepared from the fluicl of infected cultures. J. Infect. Dis. 1981,143,684-692. LaRussa, P., Steinberg, S.P., Seeman, M.D. and Gershon, A.A. Determination of immunity to varicella-zoster virus by means of an intradermal skin test. J. Infect. Dis. 1985, 152, 869-875. Takahashi, M., Iketani, T. and Sasada, K. et a/. Immunization of the elderly and patients with collagen vascular diseases with live varicella vaccine and use of varicella skin antigen. J. Infect. Dis. 1992, 166, S!S8-S62. Shiraki, K., Yamanishi, K. and Takahashi, M. Biologic and immunologic characterization of the soluble skin test antigen of varicella-zoster virus. ,I. Infect. Dis. 1984, 149, 501-504. Shiraki, K., Yamanishi. K., Takahashi, M. and Dohi, Y. Delayed-type hypersensitivity and in vitro lymphocyte response in guinea pigs immunized with a live varicella vaccine. Biken. J. 1984, 27, 19-22. Baba, K., Shiraki, K. ant Kanesaki, T. et al. Specificity of skin test with varicella-zoster virus antigen in varicella-zoster and herpes simplex virus infections. J. C/in. Microbial. 1987, 25, 2193-2196. Shiraki, K., Okuno, T., Yamanishi, K. and Takahashi, M. Polypeptides of varicella-zoster virus (VZV) and immunological relationship of VZV and herpes simplex virus. J. Gen. Viral. 1982, 61, 225-269. Diaz, P.S., Smith, S., Hunter, E. and An/in, A.M. Immunity to whole varicella-zoster virus antigen and glycoproteins I and ~170, relation to the irmmunizing regimen of live attenuated varicella vaccine. J. /nfec:t. Dis. 1988, 158, 1245-l 252. Giller, R.H., Winistorfer, !j. and Grose, C. Cellular and humoral immunity to varicella zoster virus glycoproteins in immune and susceptible human subjects. J. Infect. Dis. 1989, 160, 919-928. LaRussa, P.S., Gershon, A.A., Steinberg, S.P. and Chartrand, S.A. Antibodies to varicella-zoster virus glycoproteins I, II, and Ill in leukemic and healthy children. J. infect Dis. 1990, 162, 627-633. Brunell, P.A., Novelli, V.M., Keller, P.M. and Ellis, R.W. Antibodies to the three major glycoproteins of varicella-zoster virus, search for the relevant host immune response. J. Infect. Dis. 1987, 156,430-435. Shiraki, K., Sato, H. anI1 Yamamura, J. et a/. Functions of purified gB, gE, gl, and gH, gL, and their sialyl residues in varicella-zoster virus i’unction. Arch. Viral. 1997, 142, 2295-2301. Atvin, A.M. Cell-mediated immunity to varicella-zoster virus. J. infect Dis. 1992, 166, S35-S41.
27 28
29
30 31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47 48
Grose, C. and Litwin, V. Immunology of the varicella-zoster virus glycoproteins. J. fnfect. Dis. 1988, 157, 877-881. Shiraki, K. and Takahashi, M. Virus particles and glycoprotein excreted from cultured cells infected with varicella-zoster virus (VZV). J. Gen. Viral. 1982, 61, 271-275. Shiraki, K., Ochiai, H. and Matsui, S. et a/. Processing of hepatitis B virus surface antigen expressed by recombinant Oka varicella vaccine virus. J. Gen. Viral. 1992, 73, 1401-1407. Shiraki, K. and Hyman, R.W. The immediate early proteins of varicella-zoster virus. Virology 1987, 156, 423-426. Okuno, T., Yamanishi, K., Shiraki, K. and Takahashi, M. Synthesis and processing of glycoproteins of varicella-zoster virus (VZV) as studied with monoclonal antibodies to VZV antigens. Viroiogy 1983, 129, 357-368. Sugano, T., Matsumoto, Y., Miyamoto, C. and Masuho, Y. Hybridomas producing human monoclonal antibodies against varicella-zoster virus. Eur. J. Immunol. 1987, 17, 359-364. Shiraki, K., Ogino, T., Yamanishi, K. and Takahashi, M. lmmunochemical characterization of pyrimidine kinase induced by varicella-zoster virus. J. Gen. Viral. 1985, 66, 221-229. Atvin, A.M., Solem, SM., Koropchak, CM., Kinney-Thomas, E. and Paryani, S.G. Humoral and cellular immunity to varicella-zoster virus glycoprotein and to a gpl non-glycosylated protein, ~170, in the strain 2 guinea-pig. J. Gen. Viral. 1987, 68, 2449-2454. Bogger-Goren, S., Bernstein, J.M., Gershon, A.A. and Ogra, P.L. Mucosal cell-mediated immunity to varicella zoster virus, role in protection against disease. J. Pediafr. 1984, 105, 195-199. Sugano, T., Tomiyama, T. and Matsumoto, Y. et a/. A human monoclonal antibody against varicella-zoster virus glycoprotein Ill. J. Gen. Viral. 1991, 72, 2065-2073. Grose, C. Glycoproteins encoded by varicella-zoster virus, biosynthesis, phosphorylation, and intracellular trafficking. Ann. Rev. Microbial. 1990, 44, 59-80. Montalvo, E.A. and Grose, C. Neutralization epitope of varicella zoster virus on native viral glycoprotein gpl18 (VZV glycoprotein gplll). Virology 1986, 149, 230-241. Forghani, B., Dupuis, K. and Schmidt, N.J. Epitopes functional in neutralization of varicella-zoster virus. J. C/in. Microbial. 1990, 28, 2500-2506. Cohen, J.I. and Straus, S.E. Varicella-zoster virus and its replication. In: Fields Virology (Eds Fields, B.N., Knipe, D.M., Howley, P.M. et a/.). 3rd edn., Vol. 2, Lippincott-Raven, Philadelphia, New York, 1996, pp. 25252545. Duus, K.M., Hatfield, C. and Grose, C. Cell surface expression and fusion by the varicella-zoster virus gH, gL glycoprotein complex, analysis by laser scanning confocal microscopy. Virology 1995, 210, 429-440. Forghani, B., Ni, L. and Grose, C. Neutralization epitope of the varicella-zoster virus gH, gL glycoprotein complex. Virology 1994, 199, 458-462. Keller, P.M., Lonergan, K., Neff, B.J., Morton, D.A. and Ellis, R.W. Purification of individual varicella-zoster virus (Vzv) glycoproteins gpl, gpll, and gplll and their use in ELISA for detection of VZV glycoprotein-specific antibodies. J. Viral. Methods 1986,14,177-188. Ito, M., Mizutani, K. and Kamiya, T. et a/. Inhibition of varicella-zoster virus (VZV) glycoprotein expression by a human monoclonal antibody against VZV glycoprotein Ill. J. Infect. Dis. 1993, 168, 1256-1259. Keller, P.M., Davison, A.J., Lowe, R.S., Riemen, M.W. and Ellis, R.W. Identification and sequence of the gene encoding gplll, a major glycoprotein of varicella-zoster virus. Virology 1987, 157,526-533. Baba, K., Yabuuchi, H., Takahashi, M., Gershon, A.A. and Ogra, P.L. Seroepidemiologic behavior of varicella zoster virus infection in a semiclosed community after introduction of VZV vaccine. J. Pediatr. 1984, 105, 712-716. Abul, K.A., Kenneth, M.M. and Sher, A. Functional diversity of helper T lymphocytes. Nature 1996, 383,787-793. Hata, S. Skin test with varicella-zoster virus antigen on herpes zoster patients. Arch. Dermatol. Res. 1980, 268, 65-70.
Vaccine 1998 Volume 16 Number 13
1269
PII: SO264-410X(98)00035-S
Immune response to varicella-zoster virus glycoproteins in guinea pigs infected with Oka varicella vaccine Hitoshi Sate”, Seiji Kageyama*, Masami Imakita”f, Mitsuru Ida*, Jun-ichi Yamamura*, Masahiko Kurokawa” and Kimiyasu Shiraki*$ A varicella skin test antigen has been developed based on the induction of delayed-type hypersensitivity to varicella-zoster virus (VZV), and has been used to evaluate the immune status to V’T!’ The authors have purified gB, gE:gI and gH:gL and examined their cutaneous reactivity in guinea pigs infected with Oka varicella vaccine. The cutaneous reaction to each glycoprotein was observed and the maturation process of cutaneous reaction was examined in infected guinea pigs. Cutaneous reaction to gH:gL, a major target of virus-neutralizing antibody, appeared first on day 3 among three glycoprotein complexes and the reaction to gE:gI, the most abundant glycoprotein, became strongest three weeks after infection. The earliest recognition of gH:gL may contribute to minimizing the spread of viral infection. Thus, the skin test may be a suitable marker to assess the cell-mediated immunity in varicella, including vaccine recipients and zostel; i,lzrelation to the immune status of glycoproteins. 0 1997 Elsevier Science Ltd. All rights reserved Keywords: delayed type hypersensitivity: VZV glycoproteins; Oka varicella vaccine
Varicella-zoster virus (VZV) infection causes clinical consequences for the general population as well as for the expanding number of immunocompromised hosts with primary immune defects, cancer, organ transplants, and acquired immunodeficiency syndrome’-‘. Oka varicella vaccine has been safely used to confer cell-mediated immunity and humoral immunity to individuals435. immunocompromised and healthy Immunity, especially cell-mediated immunity, plays a critical role in limiting .the morbidity of infection6- . In addition, booster augmentation of cell-mediated immune response can be achieved with Oka varicella vaccine’“-‘2. A varicella skin test antigen has been developed for diagnostic purposes to assess cellmediated immunity to \7ZV and can successfully distinguish the susceptibility from the resistance to VZV infection’“-‘h. Varicella, skin test antigen and its biological potency have been characterized in guinea pigs infected with Oka varicella vaccine’7-‘9. Specificity of the varicella skin test to VZV has been confirmed in *Department of Virology, Toyama Medical and Pharmaceutical University, 2630 Sugitani Toyama 930-01, Japan. TDivision of Pathology, National Cardiovascular Center, Osaka, Japan. *Author to whom all correspondence should be addressed. Tel: (81) 764 34 2281; Fax: (81) 764 34 5020; E-mail: [email protected]. A pat-l of the information in this manuscript has been presented at the 21st International Herpesvirus Workshop in July, 1996 at DeKalb, IL (abstract number 376). (Received 27 October 1997; revised vlsrsion received 3 February 1998; accepted 3 February 1998)
guinea pigs infected with HSV and VZV as well as in children with a history of recent HSV infection who developed varicella, althoub292yZV and HSV share a common antigenic epitope . Guinea pigs infected with Oka varicella vaccine show positive skin tests to varicella skin test antigen as delayed type hypersensitivity (DTH) and the lymphocyte proliferation response to viral antigen as well as the neutralizing antibody response’“. VZV glycoproteins, gB, gE:gI and gH:gL, are present on the surface of both VZV particles and infected cells, and contain epitopes that stimulate VZV-speci fit cellular humoral immune and responses”-‘4. The function of each glycoprotein is not as clear as that of other herpesviruses but its importance in the immune response or infection in vitro has been well documented22~‘“~25-‘7.Varicella skin test antigen that is clinically used contains VZV glycoproteins. The authors have purified gB, gE:gI and gH:gL, and examined their cutaneous reactivity representing DTH in guinea pigs infected with Oka varicella vaccine. Positive skin tests to each glycoprotein were observed in infected guinea pigs. Moreover, the the maturation process of the cutaneous reaction was investigated. Interestingly, the cutaneous reaction to gH:gL appeared first among three glycoprotein complexes and the reaction to gE:gI became strongest later on in guinea pigs. Thus, the authors have characterized the immune response to these three glycoprotein complexes in guinea pigs infected with Oka varicella vaccine, and discussed applying glycoproteins
Vaccine
1998 Volume
16 Number
13
1263
Immune response to VZV glycoproteins to the skin test as an immunological
in guinea pigs: H. Sato et al.
marker for VZV
infection. MATERIALS AND METHODS
vig. The cutaneous reaction was assessed at 8. 24 and is”h after injection, and its size was expresseh by the following formula: (Area) =
71x (long diameter) x (short diameter) 4
Virus and cells
The Oka varicella vaccine was supplied from the Research Foundation for Microbial Diseases of Osaka university, Suita, Japan, or prepared as cell-free virus as described previously20~28~29. The Oka varicella vaccine strain was used to immunize guinea pigs and for the preparation of the viral antigen. Human embryonic lung cells were grown and maintained in Eagle’s minimum essential medium supplemented with 10 and 2% fetal bovine serum, respectively, and used to propagate the virus. Antiserum
The antiserum used in Western blot analysis was anti-VZV guinea pig serum which was produced by immunizing guinea pigs with VZV-infected guinea pig embryonic fibroblasts mixed with Freund’s complete adjuvant3’. Mouse monoclonal antibodies, clone 8 and clone 9, and human monoclonal antibody TI-57 (Teijin, Hino, respectiv~~~~~~1,3~recognize gB, gE and gH, Purification of glycoproteins chromatography
by affinity column
Glycoproteins were purified from Oka varicella vaccine-infected cells by affinity column chromatography usin monoclonal antibodies as reported previously25,28.3. Briefly, infected cells were lysed in the lysing buffer (20 mM Tris-HCl (pH 8.0), 1% Triton X-100) and centrifuged at 7000 rpm for 10 min and at 27000 rpm for 1 h. The supernatant was sequentially applied to the affinity columns coupled with monoclonal antibodies against gB, gE or gH according to the manufacturer’s instructions (ImmunoPure IgG Orientation Kit, Pierce, IL). Glycoproteins were eluted by 0.2 M glycine-HCl buffer (pH 2.9) after extensive washing of the column with the lysing buffer and phosphate-buffered saline (PBS: pH 7.4). The elutes were immediately neutralized with 2 M Tris-HCl (pH 8.0) and the buffer was replaced to PBS by ultrafiltration (Centricon, Amicon, MA) followed by the removal of IgG with Protein G Sepharose 4F (Pharmacia, Uppsala, Sweden). The purified glycoprotein complexes were used for the skin test, the enzyme-linked immunosorbent assay (ELISA), and the lymphocyte proliferation assay. VZV infection
and skin tests in infected guinea pigs
Female guinea pigs (Yodo strain) weighing about 280 g (Japan Rabbit Center, Ibaraki, Japan) were used for infection with Oka varicella vaccine’ -19. Guinea pigs were infected with 2500-6000 plaque-forming units of Oka varicella vaccine subcutaneously, and 0.1 ml of gB, gE:gI and gH:gL as well as the varicella skin test antigen was injected intradermally on the back of guinea pigs after removal of hair on days 1, 3, 7, 14 and 21 after infection. VZV-crude antigen was prepared according to the method of Kamiya et aE.13. The skin test was performed only once for each guinea
1264
Vaccine 1998 Volume 16 Number 13
Serological
assay
Serum was harvested from VZV-infected or uninfected guinea pigs under ether anesthesia at the last inspection of the cutaneous reaction. 10 pg ml- ’ of gB, gE:gI or gH:gL was applied to the wells of the ELISA plate. Sera were diluted a hundred times with PBS containing 2% skim milk, and applied to the wells to titrate the antibody to VZV crude antigen, control antigen, gB, gE:gI and gH:gL in the ELISAz4. Then, peroxidase-conjugated goat IgG fraction to guinea pig IgG (whole molecule: Cappel, NC) was distributed to the wells and the reaction was visualized according to the manufacturer’s instructions (HAT-EIA, DENKA SEIKEN, Tokyo, Japan). The optical density (OD) values were measured at 450 nm. Lymphocyte
proliferation
assay
The skin test and the lymphocyte proliferation assay were performed with three guinea pigs three weeks after infection with Oka varicella vaccine. Mononuclear cells were prepared from the spleens of guinea pigs after centrifugation on the Ficoll-Hypaque gradient. Guinea pig mononuclear cells were incubated in RPM1 medium supplemented with 10% guinea pig serum and 2-mercaptoethanol (5 x lo-’ M) in 96-well microplates. The mononuclear cells at the concentration of 2 x 105/100 ~1 per well in triplicate were incubated with serially diluted VZV crude antigen, uninfected antigen, gB, gE:gI, gH:gL or PBS. Purified glycoprotein complexes were used at concentrations of 1000, 333, 111 and 37 ng per 100 ~1 per well. After 5 days incubation of mononuclear cells from guinea pigs at 37”C, the cells were labeled with 5 &i [3H]thymidine (64.3 Ci mmol-‘) per well for 18 h and harvested onto filter paper, and the radioactivi? was determined in a liquid scintillation counter2’,22’3,35. The mean radioactivity from triplicate cultures was used to determine the ratio of that in the antigen-stimulated cultures to that in the appropriate control cultures. Its largest ratio was used as the stimulation index (S.I.). Statistical
analysis
The statistical significance of mined by the repeated measure (the repeated measure ANOVA). 0.05 was considered as statistically
the data was deteranalysis of variance A P value less than significant.
RESULTS Purification
of gB, gE:gI and gH:gL
VZV glycoproteins were purified by affinity column chromatography coupled with monoclonal antibodies and were analyzed by SDS-PAGE and Western blot using anti-VZV guinea pig serum as shown in Figure 1. The results were consistent with those reported previously*‘. Briefly, the molecular weights of gB and gE:gI visualized by Coomassie brilliant blue staining
Immune response to VZV glycoproteins
Affinity
purified
in guinea pigs: H. Sato et al.
gB, gE:gl, and gH:gL
192K 192K 127K 127K
73K 73K
43K
43K Figure 1 Purified gB, gE:gl and gH:gL were subjected using anti-VZV serum.
to SDS-PAGE
with those and Western blot were consistent reported20.25.3’.36.37. However gH was not detected by Western blot because of loss of conformational antigenicity27.3R,39. The purified fractions of gE and gH contained the amino acid sequences of g1 and gL, respectively. Therefore gE and gH existed as gE:gI and gH:gL complexes, respelctively40-42. Cutaneous reaction and antibody titer of infected guinea pigs Figure 2 shows the dose-response profile of the
cutaneous reaction to glycoprotein complexes (gE:gI, gB and gH:gL) in three guinea pigs three weeks after
and analyzed
by Coomassie
brilliant blue staining and Western blot
infection. The size of the cutaneous reaction to three glycoprotein complexes increased dose-dependently. No cutaneous reaction was induced by either antigen in uninfected guinea pigs. The cutaneous reaction was strongest against gE:gI among three glycoprotein complexes at 10 and 20 @g per dose (P~0.05 by the repeated measure ANOVA). The antibody response to three glycoprotein complexes was assessed by the ELISA as shown in Figure 3. Poor antibody response to gH:gL in the ELISA was consistent with the results reported by Keller et ~1.~~and may be caused by the difficulty in detecting antibody to gH:gL in the ELISA system, possibly because of the loss of conformational antigenicity of gH’734’. 48 h
8 Ih
I
-n-gE:gI --+gB
--+-gH:gL
* T
*
* I Ti T
Dose of gl ycoprotei
n complexes
@g/dose)
Figure 2 Dose-response of cutaneous reaction to purified gB, gE:gl and gH:gL at 8, 24 and 48 h after their inoculation in guinea pigs three weeks after infection. The size of cutaneous reaction is expressed as the mean and positive SD of three guinea pigs. *P
Vaccine 1998 Volume 16 Number 13
1265
hmune
response to VZV glycoprofeins in guinea pigs: Ii. Sat0 et al.
Lymphocyte proliferation assay in guinea pigs infected with Oka varicella vaccine and the controls Table 1 shows the lymphocyte proliferation assay of the spleen cells from guinea pigs three weeks after infection with Oka varicella vaccine. Specificity of the response to various antigens was expressed by the S.I. Infected, but not uninfected, guinea pigs responded to various antigens in the cutaneous reaction, the lymphocyte proliferation assay and antibody production. Both the cutaneous reaction and the S.I. in lymphocyte proliferation assays were strongest against gE:gI among three glycoprotein complexes. Although the S.I. values of various antigen preparations were larger in infected
1.4
-
A A
1 -
B
1.2
0 E
6 In -ICI m $
0.8
-
0.6
-
.
•I gE:gl
.
A@
.
0 @-W-
0.4-
0.2
Q I -
o-
- 0.2
infected
uninfected
gui nea pigs Figure 3 Antibody response to gB, gE:gl and gH:gL in guinea pigs three weeks after VZV infection determined at 1:lOO serum dilution by the ELISA. OD, optical density. Table 1
Immune response to VZV antigen or glycoprotein
gB
gE:gl
gH:gL
Appearance of cutaneous reaction and antibody to glycoprotein complexes in guinea pigs after infection The authors examined cutaneous reaction and antibody response to glycoproteins in guinea pigs at different time points after infection. As all glycoprotein complexes induced the cutaneous reaction enough to be detectable at a dose of 10 pg as shown in Figure 2, this dose was used to evaluate the appearance of the cutaneous reaction in the infected guinea pigs. Figure 4(a) shows the time course of appearance of the cutaneous reaction to gB, gE:gI and gH:gL in guinea pigs infected with Oka varicella vaccine. No cutaneous reaction was observed in uninfected and infected guinea pigs on day 1 after infection. Positive cutaneous reaction to gH:gL appeared first among three glycoprotein complexes on day 3 after infection. To confirm this result, the authors repeated the two additional experiments. Figure 5 shows the results of the skin test on day 3 after infection in three independent experiments. The data of the first experiment were the same as those on day 3 in Figure 4(a). The cutaneous reaction to gH:gL was significantly strongest among those of three glycoprotein complexes in four infected guinea pigs in the second and third experiments (P~0.05 or P ~0.01 by the repeated measure ANOVA). Three glycoprotein complexes induced the strong cutaneous reaction on days 7 and 14, but the cutaneous reaction to gE:gI was not as prominent in these days as that observed three weeks after infection (Figure 2). The cutaneous reaction to VZV crude antigen was smaller than that to gH:gL on day 3 but became larger on day 7 and after (data not shown). Thus, the cutaneous reaction to gH:gL was most sensitive in the early phase of infection and the reaction to gE:gI became strongest three weeks after infection. Antibodies to gE:gI and gB became detectable on day 9 after infection and then increased in titer. Antibody response to gE:gI rose earlier than the response to gB. Antibody titer to gE:gI and gB was high but, in contrast, the antibody titer to gH:gL was in gunea pigs infected with Oka varicella vaccine
VZV-infected
Guinea pigs
vzv
complexes
guinea pigs than in uninfected guinea pigs, they were not as large as expected from the results with their cutaneous reaction.
cutaneous reaction= stimulation index antibody titer cutaneous reaction stimulation index antibody titer cutaneous reaction stimulation index antibody titer cutaneous reaction stimulation index antibody titer
1
2
3
1
56.5/38.5b 4.81 0.272’ 63.6144.0 1.40 0.520 132.7177.7 1.91 0.671 63.6144.0 1.49 0.024
38.5128.3 13.6 0.335 70.7156.5 5.97 0.598 188.4l86.4 7.24 0.698 56.5130.5 4.24 0.021
37.7138.5 2.95 0.100 33.0133.0 1.37 0.061 63.6150.2 1 .a9 0.109 50.2133.0 1.04 0.000
_I_”
aGuinea pigs were infected with Oka varicella vaccine and three weeks inoculation of skin test antigen. 10 pg per dose of glycoprotein was used. bArea size (mm*) of cutaneous reaction. “Less than 5 mm in diameter. dOptical density at 450 nm in ELISA.
1266
uninfected
Vaccine 1998 Volume 16 Number 13
later cutaneous
2
3
__ I
1.25 0.008
-I-
0.98
1.39 0.011
0.013
-I-
-I-
-I-
1.06 ~ 0.003
1 .Ol - 0.002 __ I 1.25 0.002
1.30 - 0.003 __ I
-I-
__ I
-I1.15 -0.002
-I1.00 0.000 reaction
1.06 0.004
was determined
1.35 0.006 1.25 0.006
at 24 h/48 h after
Immune response to VZV glycoproteins in guinea pigs: H. Sat0 et al. not, or barely, detected at any time in ELISA (Figure
vaccine, and the maturation of the cutaneous reaction was characterized in infected guinea pigs. Antibody to gH:gL neutralizes viral infectivity and inhibits the cellto-cell spread of infection and polykaryon formation’7.“6,44,45, and purified native gH:gL interferes with virus adsorption in vitro2’. Therefore, gH:gL is one of the major targets of the immune reaction in infected humans and guinea pigs. Positive skin tests to gH:gL first appeared in guinea pigs infected with Oka varicella vaccine. This is reasonable because the earliest recognition of gH:gL among glycoproteins may be the most beneficial for the host to minimize the
4(b))-
DISCUSSION Guinea pigs infected with Oka varicella vaccine are a suitable model to characterize skin test antigen with respect to cell-mediated immunity to VZV’7-19. In this study, skin tests with glycoprotein complexes were performed in guinea pigs infected with Oka varicella
a 100
n
gE:gl
n gB 0
gH:gL
-I-
14
7
Day 3 Days after
infection
-b 1.2 -
E
&
0.8 -
+
gE:gl
-
!3B
-f
gH:gL
Day 3
Day 5 Days
after
Da& 9
Day 16
infection
Figure 4 Appearance of cutaneous reaction and antibody response to glycoprotein complexes in guinea pigs after infection. (a) Cutaneous reaction to 10 pg per dose of each glycoprotein complex in three guinea pigs on days 3, 7 and 14 after WV infection. The size of cutaneous reaction is expressed as the mean and positive SD of three guinea pigs. (b) Antibody response to gB, gE:gl and gH:gL determined at 1 :lOO serum dilution by the ELBA in guinea pigs on days 3, 5, 9 and 16 after VZV infection. The data points are expressed as the means k SDS of three guinea pigs. OD, optical density.
Vaccine 1998 Volume 16 Number 13
1267
Immune response to VZV glycoproteins
in guinea pigs: H. Sato et al.
spread of viral infection. As the cutaneous reaction appears four days after vaccination in vaccinees46, the early appearance of the cutaneous reaction is not surprising, but the earliest response to gH:gL was interesting with respect to its biological importance. The strong cutaneous reaction to gE:gI was observed in infected guinea pigs and this is also reasonable from the most abundant and immunogenic nature of gE:gI among three glycoprotein complexes’3340. Thus, the cutaneous reaction to glycoprotein complexes was well correlated with their function or biological importance. The guinea pigs produced antibodies to gB and gE:gI after infection with Oka varicella vaccine. Antibody response to gB, gE:gl and VZV crude antigen in guinea pigs was detectable on day 9 and later, which is similar to the results in children with chickenpox or in children vaccinated with Oka varicella vaccine27.46. Antibodies to gH:gL were barely detectable in guinea pigs by ELISA. These results are consistent with the reports by Keller et ~1.~~. The authors have evaluated the relationship of the immunological responses among the skin test, the antibody response, and the lymphocyte proliferation assay to three glycoprotein complexes in guinea pigs infected with Oka varicella vaccine. Infected guinea pigs responded to each glycoprotein complex but the 1 st Ex.
8.
1 gE:gl 60
n @
1
40
I
T
20
-
0 I__._&
0 gH:gL
-
I&*
:
80 :Znd Ex.
5
60: 40:
z0 L
a
R
20 y
ACKNOWLEDGEMENTS
0 3rd
80
60
magnitude of their response was diverse. Their response to glycoprotein complexes was not as strong in terms of the S.I. as expected from the cutaneous reaction. The cutaneous reaction was more sensitive in detecting cell-mediated immune response to glycoproteins and VZV antigen than the lymphocyte proliferation assay. The cutaneous reaction may be mediated by the macrophages, Langerhans cells and dendritic cells in the skin. They may release interleukin 12 by the stimulation of VZV antigen or purified glycoproteins and can activate Thl cells, resulting in the induction of DTH4’. VZV grows in the skin and therefore the immune response mediated by cutaneous macrophages might contribute to the induction of the strong cutaneous reaction in the skin. This indicates that the cutaneous reaction mediated by the cutaneous macrophage may be more appropriate to assess cell-mediated immune response than the lymphocyte proliferation cells or peripheral blood assay using spleen mononuclear cells, because cutaneous macrophages are the natural effector cells in one of the major target tissues of VZV infection. The cutaneous reaction to the varicella skin test antigen is undetectable or weak in the acute phase of zoster and becomes stronger in the convalescent phase over time4’. Considering this close association of the cutaneous reaction with recovery from zoster, it is important to further clarify the relationship of the impairment in the cutaneous reactivity to specific glycoprotein in zoster patients. Varicella skin tests have been applied to screen people with low cell-mediated immunity to VZV by the lymphocyte proliferation assay or the skin test, and Oka varicella vaccine has been applied to boost it to prevent zoster’0-12”6. Thus, the skin test to VZV glycoproteins may be the most suitable marker to assess the cell- mediated immunity in varicella including vaccinees and to understand the pathogenesis of zoster in relation to the immune status to glycoproteins.
I
Ex. *
The authors thank the Research Foundation for Microbial Diseases of Osaka university for supplying Oka varicella vaccine, and Teijin institute for Biomedical Research for monoclonal antibody (TI-57).
*
*
Tll
REFERENCES
Time after
i nnocul ati on of skin
test
antigen
Figure 5 Cutaneous response to glycoprotein complexes on day 3 after VZV infection in three independent experiments. The first experiment was performed in three guinea pigs infected with Oka varicella vaccine. This figure is the same with Figure 4(a) on day 3. The second and third experiments were performed in four guinea pigs infected with Oka varicella vaccine. The virus and glycoprotein complexes used were the same lots in all the experiments. Cutaneous reactivity to gH:gL was significantly stronger than that to gB and gE:gl in both experiments (PcO.05 by the repeated measure ANOVA).
1268
Vaccine 1998 Volume 16 Number 13
Grose, C. and Giller, Ft. Varicella-zoster infection and immunization in the healthy and the immunocompromised hosts. Crit. Rev. Oncol. Hematol. 1988, 8, 27-64. Straus, SE., Ostrove, J.M. and Inchauspe, G. et al. Varicellazoster virus infections: biology, natural history, treatment, and prevention. Ann. Inter. Med. 1988, 108, 221-237. Arvin, A. Varicella-zoster virus. In: Fields Virology (Eds Fields, B.N., Knipe, D.M., Howley, P.M. et a/.). 3rd edn., Vol. 2, Lippincott-Raven, Philadelphia, New York, 1996, pp. 2547-2585. Takahashi, M., Hayakawa, Y., Shiraki, K., Yamanishi, K., Asano, Y. and Ozaki, T. Attenuation and laboratory markers of the Oka-strain varicella-zoster virus. Postgrad. Med. J. 1985, 4, 37-46. Gershon, A.A., LaRussa, P., Hardy, I., Steinberg, S. and Silverstein, S. Varicella vaccine: the American experience. J.
Immune response to VZV glycoproteins in guinea pigs: H. Sato et al. 6
7 8
9
IO
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Infect. Dis. 1992, 166, S63-S68. Baba, K., Yabuuchi, H., Takahashi, M. and Ogra, P.L. Increased incidence of herpes zoster in normal children infected with varicella zoster virus during infancy: communitybased follow-up study. .I! Pediatr. 1986, 109, 372-377. Dworsky, M., Whitley, R. and Alford, C. Herpes zoster in early infancy. Am. J. Dis. Chikl. 1980, 134, 233-235. Patterson, L.E., Butler. K.M. and Edwards, M.S. Clinical herpes zoster shortly following primary varicella in two HIV-infected children. C/in. Pediatr. 1989, 26, 354. Hayward, A. and Herberger, M. Lymphocytes responses to varicella-zoster virus in ,the elderly. J. C/in. Immunol. 1987, 7, 174-I 78. Hayward, A., Levin, M., Wolf, W., Angelova, G. and Gilden, D. Varicella-zoster virus-specific immunity after herpes zoster. J. infect. Dis. 1991, 163, 873-875. Berger, R., Luescher, D. and Just, M. Enhancement of varicella-zoster-specific immune responses in the elderly by boosting with varicella vaccine. J. Infect. Dis. 1984, 149, 647. Watson, B., Rothstein, Ei. and Bernstein, H. et a/. Safety and cellular and humoral immune responses of a booster dose of varicella vaccine 6 years after primary immunization. J. Infect. Dis. 1995, 172, 217-219. Kamiya, H., Ihara, T. and Hattori, A. et al. Diagnostic skin test reactions with varicella virus antigen and clinical application of the test. J. Infect. Dis. 1977, 136, 784-788. Asano, Y., Shiraki, K., Takahashi, M., Nagai, H., Ozaki, T. and Yazaki, T. Soluble skin test antigen of varicella-zoster virus prepared from the fluicl of infected cultures. J. Infect. Dis. 1981,143,684-692. LaRussa, P., Steinberg, S.P., Seeman, M.D. and Gershon, A.A. Determination of immunity to varicella-zoster virus by means of an intradermal skin test. J. Infect. Dis. 1985, 152, 869-875. Takahashi, M., Iketani, T. and Sasada, K. et a/. Immunization of the elderly and patients with collagen vascular diseases with live varicella vaccine and use of varicella skin antigen. J. Infect. Dis. 1992, 166, S!S8-S62. Shiraki, K., Yamanishi, K. and Takahashi, M. Biologic and immunologic characterization of the soluble skin test antigen of varicella-zoster virus. ,I. Infect. Dis. 1984, 149, 501-504. Shiraki, K., Yamanishi. K., Takahashi, M. and Dohi, Y. Delayed-type hypersensitivity and in vitro lymphocyte response in guinea pigs immunized with a live varicella vaccine. Biken. J. 1984, 27, 19-22. Baba, K., Shiraki, K. ant Kanesaki, T. et al. Specificity of skin test with varicella-zoster virus antigen in varicella-zoster and herpes simplex virus infections. J. C/in. Microbial. 1987, 25, 2193-2196. Shiraki, K., Okuno, T., Yamanishi, K. and Takahashi, M. Polypeptides of varicella-zoster virus (VZV) and immunological relationship of VZV and herpes simplex virus. J. Gen. Viral. 1982, 61, 225-269. Diaz, P.S., Smith, S., Hunter, E. and An/in, A.M. Immunity to whole varicella-zoster virus antigen and glycoproteins I and ~170, relation to the irmmunizing regimen of live attenuated varicella vaccine. J. /nfec:t. Dis. 1988, 158, 1245-l 252. Giller, R.H., Winistorfer, !j. and Grose, C. Cellular and humoral immunity to varicella zoster virus glycoproteins in immune and susceptible human subjects. J. Infect. Dis. 1989, 160, 919-928. LaRussa, P.S., Gershon, A.A., Steinberg, S.P. and Chartrand, S.A. Antibodies to varicella-zoster virus glycoproteins I, II, and Ill in leukemic and healthy children. J. infect Dis. 1990, 162, 627-633. Brunell, P.A., Novelli, V.M., Keller, P.M. and Ellis, R.W. Antibodies to the three major glycoproteins of varicella-zoster virus, search for the relevant host immune response. J. Infect. Dis. 1987, 156,430-435. Shiraki, K., Sato, H. anI1 Yamamura, J. et a/. Functions of purified gB, gE, gl, and gH, gL, and their sialyl residues in varicella-zoster virus i’unction. Arch. Viral. 1997, 142, 2295-2301. Atvin, A.M. Cell-mediated immunity to varicella-zoster virus. J. infect Dis. 1992, 166, S35-S41.
27 28
29
30 31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47 48
Grose, C. and Litwin, V. Immunology of the varicella-zoster virus glycoproteins. J. fnfect. Dis. 1988, 157, 877-881. Shiraki, K. and Takahashi, M. Virus particles and glycoprotein excreted from cultured cells infected with varicella-zoster virus (VZV). J. Gen. Viral. 1982, 61, 271-275. Shiraki, K., Ochiai, H. and Matsui, S. et a/. Processing of hepatitis B virus surface antigen expressed by recombinant Oka varicella vaccine virus. J. Gen. Viral. 1992, 73, 1401-1407. Shiraki, K. and Hyman, R.W. The immediate early proteins of varicella-zoster virus. Virology 1987, 156, 423-426. Okuno, T., Yamanishi, K., Shiraki, K. and Takahashi, M. Synthesis and processing of glycoproteins of varicella-zoster virus (VZV) as studied with monoclonal antibodies to VZV antigens. Viroiogy 1983, 129, 357-368. Sugano, T., Matsumoto, Y., Miyamoto, C. and Masuho, Y. Hybridomas producing human monoclonal antibodies against varicella-zoster virus. Eur. J. Immunol. 1987, 17, 359-364. Shiraki, K., Ogino, T., Yamanishi, K. and Takahashi, M. lmmunochemical characterization of pyrimidine kinase induced by varicella-zoster virus. J. Gen. Viral. 1985, 66, 221-229. Atvin, A.M., Solem, SM., Koropchak, CM., Kinney-Thomas, E. and Paryani, S.G. Humoral and cellular immunity to varicella-zoster virus glycoprotein and to a gpl non-glycosylated protein, ~170, in the strain 2 guinea-pig. J. Gen. Viral. 1987, 68, 2449-2454. Bogger-Goren, S., Bernstein, J.M., Gershon, A.A. and Ogra, P.L. Mucosal cell-mediated immunity to varicella zoster virus, role in protection against disease. J. Pediafr. 1984, 105, 195-199. Sugano, T., Tomiyama, T. and Matsumoto, Y. et a/. A human monoclonal antibody against varicella-zoster virus glycoprotein Ill. J. Gen. Viral. 1991, 72, 2065-2073. Grose, C. Glycoproteins encoded by varicella-zoster virus, biosynthesis, phosphorylation, and intracellular trafficking. Ann. Rev. Microbial. 1990, 44, 59-80. Montalvo, E.A. and Grose, C. Neutralization epitope of varicella zoster virus on native viral glycoprotein gpl18 (VZV glycoprotein gplll). Virology 1986, 149, 230-241. Forghani, B., Dupuis, K. and Schmidt, N.J. Epitopes functional in neutralization of varicella-zoster virus. J. C/in. Microbial. 1990, 28, 2500-2506. Cohen, J.I. and Straus, S.E. Varicella-zoster virus and its replication. In: Fields Virology (Eds Fields, B.N., Knipe, D.M., Howley, P.M. et a/.). 3rd edn., Vol. 2, Lippincott-Raven, Philadelphia, New York, 1996, pp. 25252545. Duus, K.M., Hatfield, C. and Grose, C. Cell surface expression and fusion by the varicella-zoster virus gH, gL glycoprotein complex, analysis by laser scanning confocal microscopy. Virology 1995, 210, 429-440. Forghani, B., Ni, L. and Grose, C. Neutralization epitope of the varicella-zoster virus gH, gL glycoprotein complex. Virology 1994, 199, 458-462. Keller, P.M., Lonergan, K., Neff, B.J., Morton, D.A. and Ellis, R.W. Purification of individual varicella-zoster virus (Vzv) glycoproteins gpl, gpll, and gplll and their use in ELISA for detection of VZV glycoprotein-specific antibodies. J. Viral. Methods 1986,14,177-188. Ito, M., Mizutani, K. and Kamiya, T. et a/. Inhibition of varicella-zoster virus (VZV) glycoprotein expression by a human monoclonal antibody against VZV glycoprotein Ill. J. Infect. Dis. 1993, 168, 1256-1259. Keller, P.M., Davison, A.J., Lowe, R.S., Riemen, M.W. and Ellis, R.W. Identification and sequence of the gene encoding gplll, a major glycoprotein of varicella-zoster virus. Virology 1987, 157,526-533. Baba, K., Yabuuchi, H., Takahashi, M., Gershon, A.A. and Ogra, P.L. Seroepidemiologic behavior of varicella zoster virus infection in a semiclosed community after introduction of VZV vaccine. J. Pediatr. 1984, 105, 712-716. Abul, K.A., Kenneth, M.M. and Sher, A. Functional diversity of helper T lymphocytes. Nature 1996, 383,787-793. Hata, S. Skin test with varicella-zoster virus antigen on herpes zoster patients. Arch. Dermatol. Res. 1980, 268, 65-70.
Vaccine 1998 Volume 16 Number 13
1269