Department of Virology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan1
Teijin Institute for Biomedical Research, Asahigaoka 4-3-2, Hino, Tokyo 191, Japan2
Author for correspondence: Kimiyasu Shiraki. Fax +81 76 434 5020. e-mail kshiraki{at}toyama-mpu.ac.jp
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Abstract |
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Human embryonic lung cells were grown and maintained in Eagles minimum essential medium supplemented with 10% and 2% foetal bovine serum (FBS), respectively. The VZV strains used were the Oka vaccine and Kawaguchi strains (Shiraki et al., 1982 ). Cell-free virus stocks were prepared by freezing and thawing followed by sonication in SPGC medium (PBS supplemented with 5% sucrose, 0·1% sodium glutamate and 10% FBS) (Shiraki & Takahashi, 1982
; Shiraki et al., 1982
, 1997
). The monoclonal antibodies used were clone 9 and clone 8 (Okuno et al., 1983
), which recognize gE and gB, respectively, and TI-57, which recognizes gH (Sugano et al., 1991
). Clone 9 and clone 8 are mouse monoclonal antibodies, and TI-57 is a human monoclonal antibody.
Glycoproteins were purified by affinity chromatography with monoclonal antibodies as described previously (Shiraki & Takahashi, 1982 ; Shiraki et al., 1997
; Sato et al., 1998
; Kamiyama et al., 2000
). Briefly, Oka varicella vaccine-infected cells were lysed, and the lysate was applied to affinity columns of gB, gE and gH prepared using monoclonal antibodies. The buffer in each purified fraction was replaced with PBS by ultrafiltration followed by removal of IgG with protein GSepharose 4F (Pharmacia). Purified glycoproteins were analysed by SDSPAGE and stained with silver according to the manufacturers instructions (Daiichi Kagaku).
The specificity of reactivity of gH:gL with anti-h-IgG was determined by ELISA. Individual wells of ELISA plates were coated with 0·5 µg of gE:gI, gB or gH:gL, followed by washing and blocking the wells. Then horseradish peroxidase-conjugated anti-whole h-IgG and antibodies specific for the ,
or
chain of h-IgG (Tago Immunochemicals) or the
chain of h-IgG (Dako and ICN) were used in the ELISA. 1-Step TMB Substrate (Pierce) was used as the enzyme substrate for determination of the enzyme reaction.
In order to examine the possibility of TI-57 contaminating the purified gH:gL preparation, two control experiments were performed. In the first, uninfected cell lysate was applied to an anti-gH column and eluted as mock gH:gL, and its reactivity with anti-h--IgG in the ELISA assay was compared with that of gH:gL. In the second, the reactivity of artificial mixtures of 500 ng/ml gH:gL and TI-57 at various concentrations (0·0011000 ng/ml) with anti-h-IgG
chain (anti-h-
-IgG) was tested in an ELISA assay.
As concanavalin A (ConA, Sigma) reacted with all three glycoproteins (data not shown), a ConA-captured ELISA was used to investigate the reactivity of anti-h--IgG with gE:gI, gB and gH:gL. The plates were coated with 0·5 µg of ConA and then coated with 1·0 µg of gE:gI, gB or gH:gL after washing and blocking the wells. Alkaline phosphatase-conjugated goat anti-h-
-IgG (Tago) was diluted in PBS and p-nitrophenyl phosphate was used as the enzyme substrate.
The interaction between anti-h--IgG and VZV glycoproteins was also investigated using a BIAcore test (Pharmacia Biosensor) based on surface plasmon resonance. The sensor tip was coated with anti-h-
-IgG (Biosource/Tago Immunochemicals) and analytes, including gH:gL, were applied. Prior to immobilization of anti-h-
-IgG, the carboxyl groups of the CM5 matrix were activated by derivatization with 50 mM N-hydroxysuccinimide mediated by treatment with 200 mM N-ethyl-N'-(dimethylaminopropyl)carbodiimide for 6 min. Anti-h-
-IgG (200 µg/ml) was dissolved in 10 mM sodium acetate, pH 4·5, and passed over the activated surface for 7 min at 5 µl/min. This method resulted in 600010000 resonance units of immobilized antibody. Non-covalently associated antibody was removed with 10 mM HEPES buffer (pH 7·4) containing 3·4 mM EDTA, 0·15 M sodium chloride and 0·05% Tween 20, and unreacted N-hydroxysuccinimide esters on the dextran surface were blocked by treatment with 1 M ethanolamine hydrochloride for 7 min. The analyte was then passed over the immobilized antibody and binding was quantified by measuring the mass increase on the matrix surface. The binding of the analyte to anti-h-
-IgG was monitored as a change in response during a specific injection time and was analysed by BIA evaluation software 3.0.
A neutralization test was performed based on a method described previously (Shiraki et al., 1982 ; Sugano et al., 1987
). Briefly, 100 p.f.u. of cell-free virus was pretreated with 125 µg/ml of anti-h-IgG (Dako) or SPGC for 1 h and then with various concentrations (010 µg/ml) of neutralizing TI-57 for another 1 h. The mixture was inoculated into cells and the number of plaques was counted. The neutralizing activity was expressed as the dilution necessary to reduce the number of plaques by 50% (ED50).
VZV gB, gE:gI and gH:gL were purified using affinity chromatography and analysed by SDSPAGE (data not shown). The molecular masses of glycoproteins were as reported previously (Shiraki et al., 1982 , 1997
; Sato et al., 1998
; Okuno et al., 1983
; Sugano et al., 1991
; Grose, 1990
).
The reactivity of the three glycoproteins with human antibodies was analysed by ELISA. There was a high level of background reactivity to gH:gL compared to gB or gE:gI, even after extensive blocking or changing the ratio of the first and second antibody combinations (data not shown). We subsequently examined the direct interaction between anti-h--IgG and gH:gL bound to the wells (Table 1a
). High levels of background activity were observed even using gH:gL without human sera and goat antibodies to the
,
and
chains of h-IgG were tested for the determination of the reactivity with gH:gL without human serum. The order of the reaction rates was
>
>
. In a separate experiment, the reactions of mock gH:gL and gH:gL with anti-h-
-IgG were compared in order to assess possible contamination with TI-57 (h-IgG). In contrast to gH:gL, mock gH:gL did not react with anti-h-
-IgG (Table 1b
). Thus gH:gL directly reacted with anti-h-
-IgG.
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The interaction of gH:gL or TI-57 with anti-h--IgG was characterized further using the BIAcore assay. Of the three glycoprotein complexes, gH:gL alone reacted with anti-h-
-IgG (Kd=2·16x10-7 M), whereas the Kd value of TI-57 was 4·45x10-10 M. Both reactivities were dose-dependent. As gH:gL was purified using h-IgG (TI-57), the possibility of contamination of gH:gL with TI-57 was examined in the ELISA and BIAcore assays by testing artificial mixtures of gH:gL and TI-57 as shown in Fig. 1
. More than 9·1% contamination of TI-57 in the purified gH:gL was detectable as a doseresponse reaction with anti-h-
-IgG under the baseline reactivity of gH:gL (9·1% and less of TI-57) as shown in Fig. 1(a)
. Therefore we postulated that the maximum level of contamination of gH:gL with TI-57 might be 9·1%. The artificial mixtures of TI-57 and gH:gL were also analysed for reactivity with anti-h-
-IgG in the BIAcore assay (Fig. 1b
). More than 0·25% contamination of TI-57 in the purified gH:gL was easily detected in the BIAcore assay, and the reaction profile and Kd value were similar to TI-57, and completely different from those of gH:gL alone. Thus 0·25% contamination of TI-57 was detectable in the BIAcore assay, whereas even 9·1% contamination was not detectable in the ELISA. The BIAcore assay was therefore more sensitive than the ELISA assay in detecting contaminating TI-57, and the Kd values of the mixtures were specific to contaminating TI-57 and different from gH:gL. This indicated that the baseline reactivity of gH:gL observed with 9·1% and less of TI-57 in the ELISA assay was the reactivity of gH:gL itself with anti-h-
-IgG and was not influenced by the presence of TI-57. These results excluded the possible contribution of contaminating TI-57 in gH:gL in the ELISA and BIAcore assays and confirmed specific reactivity between gH:gL with anti-h-
-IgG. The two control experiments using the mock gH:gL and the artificial mixtures of TI-57 and gH:gL excluded the possibility of the influence of contaminating TI-57 in gH:gL.
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Anti-h-IgG failed to neutralize the infectivity of VZV, suggesting that binding of anti-h-IgG to gH:gL does not inhibit the function of gH:gL, in contrast to that of neutralizing TI-57. Pretreatment of the virus with anti-h-IgG rendered it five times less susceptible to neutralization with TI-57. The interpretation of this finding is, however, not simple. Further experiments are needed to ascertain whether interaction of the virus particle with anti-h-IgG might interfere with the binding and subsequent neutralization by TI-57.
Amino acid sequence similarity between gH and the chain of h-IgG was not observed by analysis with the software Gene Works 2.5.1. Radiolabelled gH was not immunoprecipitated with anti-h-IgG (Dako) and protein GSepharose (data not shown). Although the nature of the binding was not clear, these results suggested that binding between anti-h-IgG and gH:gL might be conformational. If this reactivity between gH and anti-h-IgG is due to an immunological or antigenic similarity between gH and h-IgG, immune recognition of gH might be impaired in the infected hosts. This study suggests a new concept for potential immunological escape/evasion in VZV infection.
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Acknowledgments |
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References |
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Received 26 April 2000;
accepted 25 October 2000.