Aug 26, 1976 - (RIA) technique was developed in our labora- tory to detect antibodies to rubella virus (4). The same methodology was further applied to.
Vol. 15, No. 3 Printed in U.S.A.
INFECTION AND IMMUNITY, Mar. 1977, p. 883-889 Copyright X 1977 American Society for Microbiology
Solid-Phase Radioimmunoassay of Human Immunoglobulin M and Immunoglobulin G Antibodies Against Herpes Simplex Virus Type 1 Capsid, Envelope, and Excreted Antigens KIRSTI 0. K. KALIMO,* REIJO J. MARTTILA, KAISA GRANFORS, AND MATTI K. VILJANEN Departments of Virology, Dermatology and Medical Microbiology, University of Turku, SF-20520 Turku 52, Finland Received for publication 26 August 1976
A solid-phase radioimmunoassay developed in our laboratory for detection of human viral immunoglobulin M (IgM) and IgG antibodies was applied to demonstrate human class-specific antibody response against capsid, envelope, and excreted antigens of herpes simplex virus type 1. In primary infections, a clear IgM and IgG antibody response was found predominantly against the envelope components, whereas the IgM and IgG antibodies to the capsid antigen appeared more slowly. Increasing IgG antibody titers to the excreted antigen were also found in primary infections, though appearing more slowly than antibodies to the other subunit antigens. The antibody response against capsid and envelope antigens was not type specific, whereas in primary infections IgG class antibodies against the excreted antigen showed distinct type specificity. In recurrent infections, no significant level of IgM class antibodies was demonstrated, but in the patients with a severe secondary herpes simplex virus infection a definite IgM class antibody response was found against the envelope antigen. In addition, during severe secondary infections the antibody response against the excreted antigen was enhanced. The host IgG antibody response in recurrent infections was directed against the envelope and excreted antigens, whereas the level of the capsid antibodies was relatively stable.
Antibody response to subunit antigens of herpes simplex virus (HSV) types 1 and 2, i.e., the envelope, capsid, and soluble antigens, has been studied by the complement fixation technique (6) and by an indirect hemagglutination test (la). The findings hitherto can be summarized as follows: (i) in primary herpetic infections, antibody response against the envelope antigen is predominant compared to that against the capsid antigen; (ii) in patients with recurrent herpetic infections, antibodies against the capsid antigen seem to increase; (iii) recurrent HSV infections seem to broaden heterotypic reactivity between type 1 and type 2 immune sera with the capsid and envelope antigens; (iv) antibodies against the soluble antigen can be readily detected in animal immune sera, but only rarely in human serum specimens; and (v) none of these antigens shows significant type specificity, though in antibody response against proteins excreted from HSV-infected cells, on the other hand, considerable type specificity was observed in animal sera (5). Recently, a sensitive radioimmunoassay
(RIA) technique was developed in our laboratory to detect antibodies to rubella virus (4). The same methodology was further applied to the detection of HSV antibodies (Kalimo et al., J. Immunol. Methods, in press) and measles virus antibodies (1) in human serum specimens. The advantages of the technique developed are high sensitivity and the possibility to demonstrate both immunoglobulin G (IgG) and IgM class antibodies. In the present study class-specific antibody responses detected by RIA in human sera against the crude and subunit antigens of HSV type 1 are described, and a possible diagnostic value of the antibody changes during primary and secondary HSV infections is discussed. MATERIALS AND METHODS Virus. For preparing all antigens the VR strains of HSV type 1 grown in Vero cells in 950-ml Roux bottles was used. Culture media. To prepare the crude, capsid, or envelope antigens, Eagle basal medium was supplemented with 0.2% bovine albumin, fraction V. Eagle basal medium without any supplements was used to produce the excreted antigen. 883
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c).
(12). After incubation for 1 h at 370C for IgG determination and for 16 h at room temperature for IgM determination, the radioactive solution was aspirated off and the balls were washed as above. The balls were removed into clean plastic tubes and the bound radioactivity was counted in a LKB Wallac 1280 gamma counter. A positive and negative control serum and a buffer blank, where serum was omitted, were included in each test. End-point titer values were obtained from the cpm (log,,,) versus dilution (log2) curve of each specimen. The end-point titer was taken to mean that serum dilution where the cpm values obtained were three times those of the negative serum in the IgG assay and two times the negative in the IgM assay. Titers were expressed as log2 values of reciprocals of the serum dilutions. Serum specimens. The serum specimens consisted of 64 specimens from 39 patients obtained from the Department of Dermatology and Department of Infectious Diseases at Turku University Hospital and from the routine material in our diagnostic laboratory. The specimens comprised the following. (i) Twenty-three specimens were from 10 patients with a primary HSV infection: 6 patients with clinically established diagnosis of HSV type 1 infection, and 4 patients with type 2 infection. (ii) Twenty specimens were from 15 patients with a history of several recurrent herpetic infections during the past year: 11 patients with a history of orofacial lesions, and 4 patients with genital lesions. (iii) Fifteen specimens were from 10 patients with a past history of herpetic infections, with the proviso that the last infection have been more than 1 year previously. In this group no patient gave a definite history of genital lesions. (iv) Six specimens were from 4 patients with a previous history of HSV infections and current severe secondary HSV infection: 1 patient with a serious recurrent stomatitis, 1 with meningitis, and 2 with a generalized eczema herpeticum. The pooled, positive control serum for the crude, capsid, and envelope antigen assays consisted of four convalescent phase specimens, and for the assay of the excreted antigen a large serum pool of one patient with several recurrent HSV infections was used. The negative control serum was pooled from one donor who did not have a history of HSV infections and did not show antibodies to any of the antigens tested in the HSV RIA. The specimens were stored at -200C until used.
RIA. The RIA used to detect class-specific HSV antibodies has been described previously in detail (Kalimo et al., in press). Briefly, fourfold serial serum dilutions were incubated in plastic tubes with the antigen-coated polystyrene balls. After incubation for 1 h at 37°C, the serum was aspirated off and the balls were washed twice with 5 ml of tap water. An aliquot of '25I-labeled anti-human IgG or IgM containing 30,000 cpm was added to each tube. The anti-human immunoglobulins were prepared in sheep. For immunization, pure IgG and IgM fractions were used. They were tested to be monospecific by immunodiffusion and immunoelectrophoresis
RESULTS RIA reactivity of different antigens. To establish the reactivity of the subunit antigens in the RIA, tests with positive and negative control sera were performed. The ratio of the radioactivity bound by the positive and by the negative serum, i.e., the binding ratio, in the IgG assay with all antigens was high, sometimes even as high as 30. There was, however, a distinct difference between different antigens regarding the binding ratios. When the balls
Crude antigen. Harvesting and purification of the crude antigen was done as previously described (Kalimo et al., in press). In brief, infected cells were harvested when a cytopathic effect (CPE) was evident in 75 to 100% of the cells and separated by lowspeed centrifugation. The cells were made 2% (vol/ vol) in Dulbecco phosphate-buffered saline and disrupted by freezing and thawing, followed by sonication. After low-speed centrifugation, the supernatant was collected and further centrifuged at 100,000 x g for 2 h. The pellet was run through a 10 to 60% (wt/vol) linear sucrose gradient at 100,000 x g for 2 h. The resulting band contained the viral material; the band was collected and sucrose was removed by an additional centrifugation. Capsid antigen. The preparation of the capsid antigen was carried out as described by Martin et al. (6). HSV-infected cells were harvested at 75% CPE into 2.5% Nonidet P-40 solution and sonicated. The suspension was filtered through 0.45- and 0.22,um membrane filters (Millex, Millipore S.A. 67120, Molsheim-France). The filtrate was layered onto a 30% (wt/vol) sucrose cushion and centrifuged at 100,000 x g for 1 h. The pellet contained the capsid antigen. Envelope antigen. The method of Martin et al. (6) was used. Infected cells were scraped into the maintenance medium at 75 to 100% CPE. After disruption by sonication, the suspension was centrifuged at 100,000 x g for 1 h. The pellet was treated with 50% diethyl ether for 30 min and then dialyzed against 0.85% NaCl buffered at pH 10.4 with 0.05 M glycine NaOH. The dialysate was layered onto a 35% (wt/vol) sucrose cushion and centrifuged for 1 h at 100,000 x g. The supernatant fluid containing the envelope antigen was collected and dialyzed against phosphate-buffered saline. Excreted antigen. The preparation of this antigen was carried out according to Kaplan et al. (5). The culture medium was decanted when the infected cells showed 75% CPE, usually 50 to 55 h postinfection. After low-speed centrifugation to remove the cellular debris, the supernatant was further centrifuged at 100,000 x g for 1 h to sediment the viral particles. The supernatant fluid was extensively dialyzed and considered as the excreted antigen. Electron microscopy. Specimens from the crude, capsid, and envelope antigens were prepared on carbon-coated Formvar grids and negatively stained with 2% phosphotungstic acid, pH 6.5, and examined with a JEM 100C electron microscope (Fig. la-
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FIG. 1. Electron micrographs ofHSV antigens used in the assays stained with phosphotungstic acid. Bar 100 nm. (a) Crude antigen, (b) capsid antigen, (c) envelope antigen.
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coated with the capsid antigen were used, the values of the negative sample in the dilutions from 1:8 to 1:4,096 were three to four times higher than the cpm values obtained with the envelope-antigen-coated balls. At higher dilutions the difference reduced to twofold. With the excreted antigen the cpm values for the negative serum were the same as or two times higher than those obtained with the envelope antigen. In the IgM assay the binding ratios between positive and negative samples were lower than in the IgG assay, reaching ratios from 4 to 8. As in the IgG assay, the capsid antigen gave a higher nonspecific background than the envelope antigen. IgM class antibodies against the excreted antigen were detected neither in the positive control serum nor in any of the sera tested. Antibody response in patients with a primary HSV infection. The development of IgM and IgG class antibodies in the serum specicpm
from the patients with a primary HSV infection against the crude and subunit antigens of HSV type 1 is presented in Table 1. In the specimens obtained within a week after the onset of symptoms, IgM RIA antibodies against the crude and envelope antigens were detected in four patients (no. 3, 4, 5, 7), whereas IgM RIA antibodies against the capsid antigen were detected in none of the acute stage specimens. In the convalescent-phase specimens, IgM RIA antibodies against the crude, capsid, and envelope antigens were demonstrated in each of the patients. In three specimens (patients no. 2, 5, 10), obtained more than 4 weeks after the onset of symptoms, IgM RIA antibodies against the capsid antigen had disappeared, and in all of these specimens a tendency for the IgM RIA envelope antibodies to decline was also demonstrated. The IgM class antibody response to the differmens
TABLE 1. HSV type 1 IgM RIA and IgG RIA antibody titers against crude, capsid, envelope, and excreted antigens in a series of serum specimens taken from 10 patients with a primary herpes simplex infection Titers (log2 values) Clinical diagnosis of patient
1. Gingivostomatitis
(AYge) 1
onset
2 14
Crude antigen
