Journal of Virological Methods, 34 (1991) 57-70 0 1991 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/91/$03.50 ADONIS 016609349100414S
57
VIRMET 01210
Development and evaluation of an ELISA using secreted recombinant glycoprotein B for determination of IgG antibody to herpes simplex virus M. Grazia Revello’, Rita Gualandri2, Roberto Manservigi2 and Giuseppe Gerna’ ’ Virus Laboratory, Institute of Infectious Diseases, University of Pavia, IRCCS Policlinico S. Matteo, Pavia, Italy and 21nstitute of Microbiology, University of Ferrara, Ferrara, Italy (Accepted 16 April 1991)
Summary
An ELISA for the determination of IgG antibody to herpes simplex virus (HSV) was developed using a secreted recombinant HSV-1 glycobrotein B (gB1s) as a solid phase. The clinical validity of the ELISA was established by testing different groups of sera containing HSV-1, HSV-2, or mixed antibody, in parallel with gB-1s ELISA and conventional HSV- 1/HSV-2 ELISA. The new gB-1s ELISA detected HSV-l/HSV-2 antibody in sera from 48 subjects with either HSV-1 or HSV-2 past infection as well as in sera from 20 patients with primary infections by either serotype, in complete agreement with the results obtained using conventional ELISA. In 7 patients with HSV-1 encephalitis the kinetics of the gB-1s serum/cerebrospinal fluid antibody-titre ratio paralleled that of conventional ELISA over a period of time of up to 4 years. Acute and convalescent-phase sera from 28 patients with acute infections by human herpesviruses other than HSV did not show a significant cross-reactivity with gB-1s. In conclusion, gB-1s ELISA is a reliable assay for determination of HSV immune status as well as for diagnosis of both primary HSV-1 and HSV-2 infections and for diagnosis of HSV-1 encephalitis. Glycoprotein B; Herpes simplex virus; Recombinant DNA technology; IgG antibody
ELISA;
Correspondence to: Prof. Giuseppe Gema, Virus Laboratory, Institute of Infectious Diseases, University of Pavia, IRCCS Policlinico S. Matteo, 27100 Pavia, Italy.
58
Introduction Humoral immune response to herpes simplex virus (HSV) type 1 and type 2 infections is partly common and partly type-specific. The relative proportion of the two types of antibody varies according to the time interval after onset of infection, type of infecting HSV strain and preexisting antibody of either HSV type. That is the basis upon which diagnostic virology laboratories determine IgG antibody to HSV by testing human sera against both HSV-1 and HSV-2 antigens. Enzyme-linked immunosorbent assay (ELISA) represents the most widely used assay system for determination of the immune status to HSV. However, standardization of different batches of antigen is difficult to achieve and a control antigen is required as a specificity control of test results. On the other hand, type-specific antibody can only be determined by experienced laboratories using microneutralization procedures, while recently proposed methods based on the use of immunoaffnity-purified HSV glycoproteins, such as gG-1 and gG-2 (Lee et al., 1985, 1986), require procedures too cumbersome for general use in a diagnostic laboratory. In order to develop an enzyme immunoassay which could be standardized for determination of both the HSV immune status and diagnosis of acute HSV1 and HSV-2 infections, we used a secreted recombinant HSV-1 glycoprotein B (gB-1s) constitutively expressed in human cells transfected with a BK virus recombinant episomal vector (Manservigi et al., 1990a). It is known that gB is an essential glycoprotein of HSV and a major target for humoral and cellmediated immune response in the infected host (Corey and Spear, 1986) and has been shown to be a strong immunogen in eliciting neutralizing antibody (Glorioso et al., 1984; Dix and Mills, 1985; Manservigi et al., 1990a,b).This glycoprotein has been suggested as a promising candidate for the development of a subunit vaccine (Pack1 et al., 1987; Manservigi et al., 1988). While the mature and completely glycosylated form of gB-1 is 903 amino acids in length ahd 120 kDa in size (Campadelli-Fiume and Serafini-Cessi, 1985), gB-ls, which is deprived of the transmembrane portion of the molecule, is 690 amino acids long and 91.6 kDa in size (Manservigi et al., 1990a). In recent studies, gB-1s has been shown to protect mice against lethal and latent HSV-1 infections and to prevent CNS disease in rabbits, while reducing the severity of herpetic keratitis (Manservigi et al., 1990a,b). In the present study, the development of an ELISA using gB-1s as a solid phase is described. The test is capable of detecting small amounts of either HSV-1 or HSV-2 antibody in human sera from patients with remote, primary or recurrent HSV-l/HSV-2 infections as well as from patients with HSV encephalitis. The assay does not require handling of infectious material and may be performed in the absence of control antigen.
59
Material and Methods
Cloning and expression of gB-Is in human cells The gB-1 gene of HSV-1, strain F, deleted of 693 nucleotides encoding the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under the control of the Rous Sarcoma virus promoter in the episomal replicating vector pRP-RSV (Manservigi et al., 1990a). gB-1s was secreted into the culture medium of pRP-RSV-gB-Is-transformed 293 cells at a concentration of 0.1-0.2 pg/ml. The secreted product was identified in both radioimmunoprecipitation and ELISA by using monoclonal antibodies to HSV-1 gB (Manservigi et al., 1988) and gB1s developed in the laboratory. Using the same monoclonal antibodies, gB-1s was immunoaflinity-purified from 20 x concentrated (by ultrafiltration) and dialyzed cell culture medium. ELISA procedure Either purified or crude concentrated culture medium preparations of gB-1s were used as a solid phase in the ELISA. Following initial titration, the optimal concentration of gB-1s was found to be between 10 and 50 ng/well. Wells of 96well polystyrene microplates (M129A Dynatech, Plochingen, F.R.G.) were coated overnight at room temperature with the optimal dilution of gB-1s in 0.05 M sodium carbonate-bicarbonate buffer, pH 9.6. After washings, 100 ~1 of test sera, diluted 1:50 in phosphate-buffered saline (PBS) containing 1% foetal calf serum (FCS) and 0.1% Tween 20, were incubated for 2 h at 37°C. Plates were then washed with, PBS-Tween 20 and 100 ~1 of optimally diluted peroxidase-conjugated goat anti-human IgG (Cappel Laboratories, Cooper Biomedical, Malvern, PA, U.S.A.) were added to each well. Incubation was continued for 1 h at 37°C. After washing, enzymatic activity was detected by adding the substrate solution for 30 min at room temperature. The latter was prepared immediately before use by dissolving 3.4 mg of ortho-phenylenediamine in 10.0 ml of citrate phosphate buffer (0.05 M phosphate, 0.02 M citrate, pH 5.5) and then adding 40 ~1 of 3% H202. Absorbance values were recorded at 492 nm using a microtitre plate spectrophotometer (Titertek Multiskan, Flow Laboratories, Irvine, U.K.). The cut-off was set at 0.10 absorbance following testing of 39 HSV-negative sera. When required, quantitation of IgG antibody to gB-1s was carried out by constructing a calibration curve, which was obtained by plotting the mean absorbance value of each of five standard sera (containing different amounts of specific antibody) versus the amount of gB-ls-specific IgG expressed as antibody titre. In parallel, all sera were tested by conventional ELISA for HSV-l/HSV-2 IgG antibody determination, as previously reported (Revello and Gerna, 1988). In conventional ELISA, glycine-extracted HSV-1 and HSV-2 crude antigens as well as uninfected Vero cell control antigen were used. This assay was calibrated optimally to allow
60
typing of HSV antibody Gerna, 1988). Microneutralization
in human
sera, as reported
previously
(Revello
and
test
The microneutralization assay was carried out using growing Vero cells by testing serial twofold dilutions of heat-inactivated serum samples mixed with equal amounts of virus suspensions containing 100 50% tissue culture infectious doses of either HSV-1 or HSV-2 (Revello and Gerna, 1988). Corrected titres (tc) for each serum were calculated according to Stalder et al. (1975), and typing of HSV neutralizing antibody was achieved by calculating the tc difference between the titre to HSV-1 and that to HSV-2. Patients and sera The following groups of patients and sera were examined in parallel by gB- 1s ELISA and conventional HSV-l/HSV-2 ELISA: l 39 HSV antibody-negative sera from 39 healthy individuals; l 53 sequential sera from 14 patients with primary HSV-1 infection and 17 sequential sera from 3 patients with primary HSV-2 infection (in the absence of prior HSV-1 infection); l 48 sera from 48 patients, of which 23 contained HSV-1 antibody, 13 HSV-2 antibody and 12 dual HSV antibody, as determined previously by microneutralization; l acute- and convalescent-phase sera from 28 patients with acute infections caused by human herpesviruses other than HSV, i.e. varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6), and from 16 patients with primary HSV-1 infection; l 35 sequential paired serum-cerebrospinal fluid (CSF) samples from 7 patients with herpes simplex encephalitis. HSV isolation and typing Material from mucocutaneous lesions was collected by swabs, for inoculation of Vero and diploid cell cultures for virus isolation, and by scraping, for preparation of cell smears for direct virus identification and typing. HSV isolates were typed by indirect ELISA, as reported previously (Gerna et al., 1983), whereas direct typing on cell smears from clinical specimens was done by direct fluorescent antibody test, using in parallel fluorescein-labeled type-specific monoclonal antibodies both produced in our laboratory and available commercially (Syva Micro-Trak, Syva Company, Palo Alto, CA, U.S.A.).
61
Development of the indirect ELISA for determination of IgG antibody to gB-1s
Initially, the optimal concentration of gB- 1s to be used in the indirect ELISA for IgG antibody detection was determined. For this purpose, human sera with past and primary HSV-1 infections, containing only HSV-1 antibodies, were used. Low levels of HSV-1 antibody could be clearly detected when using a concentration of gB-1s of 10-15 ng/well. However, when early convalescentphase sera from primary HSV-2 infections in subjects with no prior experience of HSV-1 infection were tested, the amount of gB-is/well had to be increased to 40-50 ng in order to allow detection of low levels of early HSV-2 antibody. Afterwards, the cut-off was determined on 39 HSV-negative sera previously characterized by neutralization and ELISA. The mean Ad92 was 0.03 + 0.03. Thus, a cut-off of 0.10 was selected for further testing (0.03 + 2SD). Antibody response to gB-1s in primary HSV-1 and HSV-2 infections
All subsequent tests were run in parallel using the gB- 1s ELISA and the HSVl/HSV-2 ELISA. Initially, two monoclonal antibodies to gB and four to gB-1s a.0
_
1s
.
1.0
.
1.7
.
1.0
.
000 0
0 1s.
1.4
.
1.5
.
11
.
1.1
.
1.0
.
0.0
.
0
0
0
.
0 :
ao. f
a7
0 .
-
0
.
0.
.
0
Dae.
0 Eos. 5
00
.b
I--
O
0
0
0
C‘OJ. az.
Fig. 1. Absorbance values (A& in the gB-1s vs. HSV-I/HSV-2 ELISA of: 53 sera collected from 14 patients during the first month (0) of a primary HSV-1 infection or afterwards (0); 17 sera collected from 3 patients during the first month (m) of a primary HSV-2 infection or afterwards (0).
62
(shown in competitive binding assays to recognize different gB epitopes) were tested in both ELISAs. All reacted in the gB-1s ELISA with titres of 1O-5-1O-7, whereas none reacted at a dilution of 10e3 in the HSV-l/HSV-2 ELISA. Measurement of IgG antibody to gB-1s during primary HSV-1 infections was achieved by examining 53 sequential sera from 14 patients undergoing primary HSV- 1 infection. Among 34 sera collected within 30 days after onset of symptoms, 9 collected within the first 5 days were negative by both ELISA systems, 23 were concordant positive and 2 (collected 3 and 5 days after onset, respectively) were weak positive by gB-1s ELISA only (Fig. 1). Ad92 values measured using gB-1s ELISA and the HSV-l/HSV-2 ELISA were also grossly comparable, with the exception of 2 sera collected within 30 days and 2 sera collected afterwards, which gave significantly higher (greater than a fourfold difference in titre) reactivity in the gB-1s ELISA. Comparable results were observed when 17 sera from 3 patients undergoing primary HSV-2 infection (in the absence of prior HSV- 1 infection) were examined. Of 6 sera collected within 30 days, 5 were detected by both ELISAs and one only by the HSV-2 ELISA
.
5.8
.
5.7
.
33
.
3.5
.
a1
.
0 . .
. . . . . .
. . .
2.9
.
2.7
.
2.5
.
23
.
2.1
.
1.0
.
1.7
.
1.s
.
1.3
.
1.1
_
‘;.
0.2
_
f
0.7
.
z
03
.
0.3
.
0.1
.
5 s :
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oe
.
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.
e
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8
. .
.
0 0 0 0 0 0
. 8
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. .‘.
Am
WIT”
ttONOLOOW8
HW
ANTKILN
Fig. 2. Comparison of ELISA reactivities with gB-1s vs. HSV-l/HSV-2 crude antigens in 48 sera previously typed for HSV antibody by neutralization. Sera were divided into three groups containing: 23, HSV-1 (0); 13, HSV-2 (O), and 12, mixed (0) antibody, respectively. For sera containing mixed antibody, the higher Adp2 value in the HSV-l/HSV-2 ELISA was plotted.
63
with an A492 value close to cut-off. In addition, all 11 sera collected after 30 days were found positive by both systems. However, most late sera showed a tendency to give higher HSV-2 than gB-1s A492values (Fig. 1). Thus, although HSV-1 and HSV-2 gBs share a high degree of homology, at least some sera from primary HSV-1 infections seem to recognize gB-1s somewhat better than sera from HSV-2 infections. Determination of HSV immune status in patients with HSV-I, HSV-2 or mixed antibody The HSV immune status was determined by gB-1s ELISA and HSV-l/HSV2 ELISA in sera from 48 patients, in which HSV antibody type was previously determined by microneutralization. As shown in Fig. 2, all test sera were detected as positive by both ELISA systems. However, HSV-1 and mixed antibody sera gave A 492 values higher in the gB-1s ELISA as compared to HSV-l/HSV-2 ELISA, whereas most HSV-2 sera showed an equivalent reactivity between the two ELISAs. These data again suggest that HSV-1 antibody reacts more strongly with gB-1s than HSV-2 antibody. Study of the heterologous antibody response to gB-Is in patients with acute infections by HHVs other than HSV Since there are high levels of homology among gB genes of different human herpesviruses, we investigated the level of heterologous antibody response to 22 24
(MllAnv)
(CIIUAW)
(rnlllill)
( CIImAmv)
Fig. 3. Heterologous antibody response to gB-1s in acute and convalescent-phase sera from 28 patients with acute HHV infections other than HSV (6 with zoster, 5 with chickenpox, 10 with primary HCMV, 5 with primary EBV and 2 with primary HHV-6 infection). Homologous antibody response in acute and convalescent sera from 16 patients with primary HSV-1 infection is reported for comparison. Numbers inside columns indicate gB-Is-negative (shaded areas) or -positive (blank areas) sera. A, acute; C, convalescent serum.
64
gB-1s in acute and convalescent sera from 28 patients with acute infections by HHVs other than HSV (6 with zoster, 5 with chickenpox, 10 with primary HCMV infection, 5 with EBV infectious mononucleosis and 2 with primary HHV-6 infection). For comparison, acute and convalescent sera from 16 patients with primary HSV-1 infection were examined. Mean Ad92 values obtained in the gB-1s ELISA with different groups of sera are reported in Fig. 3. No significant difference (greater than a twofold dilution) in mean Ad92 between acute and convalescent sera was observed in any of the 5 groups of HSV-positive patients (where high gB-1s antibody titres reflected past HSV-1 infections), whereas seroconversion from seronegative to seropositive was found in all 16 TABLE 1 Heterologous Patient No.
Zoster RS A C R22 A C R25 : R26 2 R30 A C R31 A C Varicella 6806A A C 6163A A C 6251A f? 12198 A C 11968
antibody
response to gB-1s in 6 patients with zoster and 5 patients with varicella A 492
vzv
gB-1s (titre)
HSV-1 (titre)
1.12 2.28
2.00 (3,100) 1.99 (3,100)
1.28 (6,000) 1.12(3,500)
1.29 2.47
2.23 (4,000) 2.60 (7,000)
1.18(4,500)
0.75 1.97
2.52 (5,600) 2.64 (7,000)
0.94 (2,000) 1.04(2,500)
1.66 2.34
2.60 (7,000) 2.60 (7,000)
1.36 (8,000) 1.44 (10,000)
1.49 2.30
2.70 (7,000) 2.64 (7,000)
1.40 (9,500) 1.22 (7,000)
0.34 2.17
0.06 (< 50) 0.04 (< 50)
0.01 (
57
VIRMET 01210
Development and evaluation of an ELISA using secreted recombinant glycoprotein B for determination of IgG antibody to herpes simplex virus M. Grazia Revello’, Rita Gualandri2, Roberto Manservigi2 and Giuseppe Gerna’ ’ Virus Laboratory, Institute of Infectious Diseases, University of Pavia, IRCCS Policlinico S. Matteo, Pavia, Italy and 21nstitute of Microbiology, University of Ferrara, Ferrara, Italy (Accepted 16 April 1991)
Summary
An ELISA for the determination of IgG antibody to herpes simplex virus (HSV) was developed using a secreted recombinant HSV-1 glycobrotein B (gB1s) as a solid phase. The clinical validity of the ELISA was established by testing different groups of sera containing HSV-1, HSV-2, or mixed antibody, in parallel with gB-1s ELISA and conventional HSV- 1/HSV-2 ELISA. The new gB-1s ELISA detected HSV-l/HSV-2 antibody in sera from 48 subjects with either HSV-1 or HSV-2 past infection as well as in sera from 20 patients with primary infections by either serotype, in complete agreement with the results obtained using conventional ELISA. In 7 patients with HSV-1 encephalitis the kinetics of the gB-1s serum/cerebrospinal fluid antibody-titre ratio paralleled that of conventional ELISA over a period of time of up to 4 years. Acute and convalescent-phase sera from 28 patients with acute infections by human herpesviruses other than HSV did not show a significant cross-reactivity with gB-1s. In conclusion, gB-1s ELISA is a reliable assay for determination of HSV immune status as well as for diagnosis of both primary HSV-1 and HSV-2 infections and for diagnosis of HSV-1 encephalitis. Glycoprotein B; Herpes simplex virus; Recombinant DNA technology; IgG antibody
ELISA;
Correspondence to: Prof. Giuseppe Gema, Virus Laboratory, Institute of Infectious Diseases, University of Pavia, IRCCS Policlinico S. Matteo, 27100 Pavia, Italy.
58
Introduction Humoral immune response to herpes simplex virus (HSV) type 1 and type 2 infections is partly common and partly type-specific. The relative proportion of the two types of antibody varies according to the time interval after onset of infection, type of infecting HSV strain and preexisting antibody of either HSV type. That is the basis upon which diagnostic virology laboratories determine IgG antibody to HSV by testing human sera against both HSV-1 and HSV-2 antigens. Enzyme-linked immunosorbent assay (ELISA) represents the most widely used assay system for determination of the immune status to HSV. However, standardization of different batches of antigen is difficult to achieve and a control antigen is required as a specificity control of test results. On the other hand, type-specific antibody can only be determined by experienced laboratories using microneutralization procedures, while recently proposed methods based on the use of immunoaffnity-purified HSV glycoproteins, such as gG-1 and gG-2 (Lee et al., 1985, 1986), require procedures too cumbersome for general use in a diagnostic laboratory. In order to develop an enzyme immunoassay which could be standardized for determination of both the HSV immune status and diagnosis of acute HSV1 and HSV-2 infections, we used a secreted recombinant HSV-1 glycoprotein B (gB-1s) constitutively expressed in human cells transfected with a BK virus recombinant episomal vector (Manservigi et al., 1990a). It is known that gB is an essential glycoprotein of HSV and a major target for humoral and cellmediated immune response in the infected host (Corey and Spear, 1986) and has been shown to be a strong immunogen in eliciting neutralizing antibody (Glorioso et al., 1984; Dix and Mills, 1985; Manservigi et al., 1990a,b).This glycoprotein has been suggested as a promising candidate for the development of a subunit vaccine (Pack1 et al., 1987; Manservigi et al., 1988). While the mature and completely glycosylated form of gB-1 is 903 amino acids in length ahd 120 kDa in size (Campadelli-Fiume and Serafini-Cessi, 1985), gB-ls, which is deprived of the transmembrane portion of the molecule, is 690 amino acids long and 91.6 kDa in size (Manservigi et al., 1990a). In recent studies, gB-1s has been shown to protect mice against lethal and latent HSV-1 infections and to prevent CNS disease in rabbits, while reducing the severity of herpetic keratitis (Manservigi et al., 1990a,b). In the present study, the development of an ELISA using gB-1s as a solid phase is described. The test is capable of detecting small amounts of either HSV-1 or HSV-2 antibody in human sera from patients with remote, primary or recurrent HSV-l/HSV-2 infections as well as from patients with HSV encephalitis. The assay does not require handling of infectious material and may be performed in the absence of control antigen.
59
Material and Methods
Cloning and expression of gB-Is in human cells The gB-1 gene of HSV-1, strain F, deleted of 693 nucleotides encoding the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under the control of the Rous Sarcoma virus promoter in the episomal replicating vector pRP-RSV (Manservigi et al., 1990a). gB-1s was secreted into the culture medium of pRP-RSV-gB-Is-transformed 293 cells at a concentration of 0.1-0.2 pg/ml. The secreted product was identified in both radioimmunoprecipitation and ELISA by using monoclonal antibodies to HSV-1 gB (Manservigi et al., 1988) and gB1s developed in the laboratory. Using the same monoclonal antibodies, gB-1s was immunoaflinity-purified from 20 x concentrated (by ultrafiltration) and dialyzed cell culture medium. ELISA procedure Either purified or crude concentrated culture medium preparations of gB-1s were used as a solid phase in the ELISA. Following initial titration, the optimal concentration of gB-1s was found to be between 10 and 50 ng/well. Wells of 96well polystyrene microplates (M129A Dynatech, Plochingen, F.R.G.) were coated overnight at room temperature with the optimal dilution of gB-1s in 0.05 M sodium carbonate-bicarbonate buffer, pH 9.6. After washings, 100 ~1 of test sera, diluted 1:50 in phosphate-buffered saline (PBS) containing 1% foetal calf serum (FCS) and 0.1% Tween 20, were incubated for 2 h at 37°C. Plates were then washed with, PBS-Tween 20 and 100 ~1 of optimally diluted peroxidase-conjugated goat anti-human IgG (Cappel Laboratories, Cooper Biomedical, Malvern, PA, U.S.A.) were added to each well. Incubation was continued for 1 h at 37°C. After washing, enzymatic activity was detected by adding the substrate solution for 30 min at room temperature. The latter was prepared immediately before use by dissolving 3.4 mg of ortho-phenylenediamine in 10.0 ml of citrate phosphate buffer (0.05 M phosphate, 0.02 M citrate, pH 5.5) and then adding 40 ~1 of 3% H202. Absorbance values were recorded at 492 nm using a microtitre plate spectrophotometer (Titertek Multiskan, Flow Laboratories, Irvine, U.K.). The cut-off was set at 0.10 absorbance following testing of 39 HSV-negative sera. When required, quantitation of IgG antibody to gB-1s was carried out by constructing a calibration curve, which was obtained by plotting the mean absorbance value of each of five standard sera (containing different amounts of specific antibody) versus the amount of gB-ls-specific IgG expressed as antibody titre. In parallel, all sera were tested by conventional ELISA for HSV-l/HSV-2 IgG antibody determination, as previously reported (Revello and Gerna, 1988). In conventional ELISA, glycine-extracted HSV-1 and HSV-2 crude antigens as well as uninfected Vero cell control antigen were used. This assay was calibrated optimally to allow
60
typing of HSV antibody Gerna, 1988). Microneutralization
in human
sera, as reported
previously
(Revello
and
test
The microneutralization assay was carried out using growing Vero cells by testing serial twofold dilutions of heat-inactivated serum samples mixed with equal amounts of virus suspensions containing 100 50% tissue culture infectious doses of either HSV-1 or HSV-2 (Revello and Gerna, 1988). Corrected titres (tc) for each serum were calculated according to Stalder et al. (1975), and typing of HSV neutralizing antibody was achieved by calculating the tc difference between the titre to HSV-1 and that to HSV-2. Patients and sera The following groups of patients and sera were examined in parallel by gB- 1s ELISA and conventional HSV-l/HSV-2 ELISA: l 39 HSV antibody-negative sera from 39 healthy individuals; l 53 sequential sera from 14 patients with primary HSV-1 infection and 17 sequential sera from 3 patients with primary HSV-2 infection (in the absence of prior HSV-1 infection); l 48 sera from 48 patients, of which 23 contained HSV-1 antibody, 13 HSV-2 antibody and 12 dual HSV antibody, as determined previously by microneutralization; l acute- and convalescent-phase sera from 28 patients with acute infections caused by human herpesviruses other than HSV, i.e. varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6), and from 16 patients with primary HSV-1 infection; l 35 sequential paired serum-cerebrospinal fluid (CSF) samples from 7 patients with herpes simplex encephalitis. HSV isolation and typing Material from mucocutaneous lesions was collected by swabs, for inoculation of Vero and diploid cell cultures for virus isolation, and by scraping, for preparation of cell smears for direct virus identification and typing. HSV isolates were typed by indirect ELISA, as reported previously (Gerna et al., 1983), whereas direct typing on cell smears from clinical specimens was done by direct fluorescent antibody test, using in parallel fluorescein-labeled type-specific monoclonal antibodies both produced in our laboratory and available commercially (Syva Micro-Trak, Syva Company, Palo Alto, CA, U.S.A.).
61
Development of the indirect ELISA for determination of IgG antibody to gB-1s
Initially, the optimal concentration of gB- 1s to be used in the indirect ELISA for IgG antibody detection was determined. For this purpose, human sera with past and primary HSV-1 infections, containing only HSV-1 antibodies, were used. Low levels of HSV-1 antibody could be clearly detected when using a concentration of gB-1s of 10-15 ng/well. However, when early convalescentphase sera from primary HSV-2 infections in subjects with no prior experience of HSV-1 infection were tested, the amount of gB-is/well had to be increased to 40-50 ng in order to allow detection of low levels of early HSV-2 antibody. Afterwards, the cut-off was determined on 39 HSV-negative sera previously characterized by neutralization and ELISA. The mean Ad92 was 0.03 + 0.03. Thus, a cut-off of 0.10 was selected for further testing (0.03 + 2SD). Antibody response to gB-1s in primary HSV-1 and HSV-2 infections
All subsequent tests were run in parallel using the gB- 1s ELISA and the HSVl/HSV-2 ELISA. Initially, two monoclonal antibodies to gB and four to gB-1s a.0
_
1s
.
1.0
.
1.7
.
1.0
.
000 0
0 1s.
1.4
.
1.5
.
11
.
1.1
.
1.0
.
0.0
.
0
0
0
.
0 :
ao. f
a7
0 .
-
0
.
0.
.
0
Dae.
0 Eos. 5
00
.b
I--
O
0
0
0
C‘OJ. az.
Fig. 1. Absorbance values (A& in the gB-1s vs. HSV-I/HSV-2 ELISA of: 53 sera collected from 14 patients during the first month (0) of a primary HSV-1 infection or afterwards (0); 17 sera collected from 3 patients during the first month (m) of a primary HSV-2 infection or afterwards (0).
62
(shown in competitive binding assays to recognize different gB epitopes) were tested in both ELISAs. All reacted in the gB-1s ELISA with titres of 1O-5-1O-7, whereas none reacted at a dilution of 10e3 in the HSV-l/HSV-2 ELISA. Measurement of IgG antibody to gB-1s during primary HSV-1 infections was achieved by examining 53 sequential sera from 14 patients undergoing primary HSV- 1 infection. Among 34 sera collected within 30 days after onset of symptoms, 9 collected within the first 5 days were negative by both ELISA systems, 23 were concordant positive and 2 (collected 3 and 5 days after onset, respectively) were weak positive by gB-1s ELISA only (Fig. 1). Ad92 values measured using gB-1s ELISA and the HSV-l/HSV-2 ELISA were also grossly comparable, with the exception of 2 sera collected within 30 days and 2 sera collected afterwards, which gave significantly higher (greater than a fourfold difference in titre) reactivity in the gB-1s ELISA. Comparable results were observed when 17 sera from 3 patients undergoing primary HSV-2 infection (in the absence of prior HSV- 1 infection) were examined. Of 6 sera collected within 30 days, 5 were detected by both ELISAs and one only by the HSV-2 ELISA
.
5.8
.
5.7
.
33
.
3.5
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0 . .
. . . . . .
. . .
2.9
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.
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HW
ANTKILN
Fig. 2. Comparison of ELISA reactivities with gB-1s vs. HSV-l/HSV-2 crude antigens in 48 sera previously typed for HSV antibody by neutralization. Sera were divided into three groups containing: 23, HSV-1 (0); 13, HSV-2 (O), and 12, mixed (0) antibody, respectively. For sera containing mixed antibody, the higher Adp2 value in the HSV-l/HSV-2 ELISA was plotted.
63
with an A492 value close to cut-off. In addition, all 11 sera collected after 30 days were found positive by both systems. However, most late sera showed a tendency to give higher HSV-2 than gB-1s A492values (Fig. 1). Thus, although HSV-1 and HSV-2 gBs share a high degree of homology, at least some sera from primary HSV-1 infections seem to recognize gB-1s somewhat better than sera from HSV-2 infections. Determination of HSV immune status in patients with HSV-I, HSV-2 or mixed antibody The HSV immune status was determined by gB-1s ELISA and HSV-l/HSV2 ELISA in sera from 48 patients, in which HSV antibody type was previously determined by microneutralization. As shown in Fig. 2, all test sera were detected as positive by both ELISA systems. However, HSV-1 and mixed antibody sera gave A 492 values higher in the gB-1s ELISA as compared to HSV-l/HSV-2 ELISA, whereas most HSV-2 sera showed an equivalent reactivity between the two ELISAs. These data again suggest that HSV-1 antibody reacts more strongly with gB-1s than HSV-2 antibody. Study of the heterologous antibody response to gB-Is in patients with acute infections by HHVs other than HSV Since there are high levels of homology among gB genes of different human herpesviruses, we investigated the level of heterologous antibody response to 22 24
(MllAnv)
(CIIUAW)
(rnlllill)
( CIImAmv)
Fig. 3. Heterologous antibody response to gB-1s in acute and convalescent-phase sera from 28 patients with acute HHV infections other than HSV (6 with zoster, 5 with chickenpox, 10 with primary HCMV, 5 with primary EBV and 2 with primary HHV-6 infection). Homologous antibody response in acute and convalescent sera from 16 patients with primary HSV-1 infection is reported for comparison. Numbers inside columns indicate gB-Is-negative (shaded areas) or -positive (blank areas) sera. A, acute; C, convalescent serum.
64
gB-1s in acute and convalescent sera from 28 patients with acute infections by HHVs other than HSV (6 with zoster, 5 with chickenpox, 10 with primary HCMV infection, 5 with EBV infectious mononucleosis and 2 with primary HHV-6 infection). For comparison, acute and convalescent sera from 16 patients with primary HSV-1 infection were examined. Mean Ad92 values obtained in the gB-1s ELISA with different groups of sera are reported in Fig. 3. No significant difference (greater than a twofold dilution) in mean Ad92 between acute and convalescent sera was observed in any of the 5 groups of HSV-positive patients (where high gB-1s antibody titres reflected past HSV-1 infections), whereas seroconversion from seronegative to seropositive was found in all 16 TABLE 1 Heterologous Patient No.
Zoster RS A C R22 A C R25 : R26 2 R30 A C R31 A C Varicella 6806A A C 6163A A C 6251A f? 12198 A C 11968
antibody
response to gB-1s in 6 patients with zoster and 5 patients with varicella A 492
vzv
gB-1s (titre)
HSV-1 (titre)
1.12 2.28
2.00 (3,100) 1.99 (3,100)
1.28 (6,000) 1.12(3,500)
1.29 2.47
2.23 (4,000) 2.60 (7,000)
1.18(4,500)
0.75 1.97
2.52 (5,600) 2.64 (7,000)
0.94 (2,000) 1.04(2,500)
1.66 2.34
2.60 (7,000) 2.60 (7,000)
1.36 (8,000) 1.44 (10,000)
1.49 2.30
2.70 (7,000) 2.64 (7,000)
1.40 (9,500) 1.22 (7,000)
0.34 2.17
0.06 (< 50) 0.04 (< 50)
0.01 (
