175

Journal of Virological Methods, 38 (I 992) 175-I 86 0 1992 Elsevier Science Publishers B.V. / All rights reserved / Ol66-0934/92/$05.00 VIRMET 01344

Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides George J. Dawsona, Kurt H. Chau”, Carlos M. Cabal”, Patrice Yarboughb, Gregory R. Reyesb and Isa K. Mushahwar”

0.

aExperimental Biology Research, Abbott Laboratories, North Chicago, Illinois, USA and bMolecular Virology Department, Genelabs Incorporated, Redwood City, California, U.S.A. (Accepted

I7 January

1992)

Summary Four recombinant antigens representing two distinct antigenic domains from two different strains of hepatitis E virus (HEV), were used individually to develop four ELISAs designed to detect antibodies to HEV. Both IgG and IgM class antibodies to HEV were detected in 7 of 8 pedigreed serum/plasma from known outbreaks of HEV in Mexico, Burma, Somalia and Pakistan. In addition, specific HEV-antibodies were detected in cynomolgus macaques following inoculation with various HEV strains. Anti-HEV was also detected in 8 of 386 (2.1%) randomly selected American blood donors. Supplemental tests utilizing both synthetic peptides and specific blocking assays provided additional serologic data confirming the presence of anti-HEV. Similar prevalence studies on a limited number of available sera from other geographical regions (Alaska, Japan, Germany, New Zealand, Thailand and Mexico) confirmed the presence of anti-HEV in at least 1.1 to 7.6% of the specimens. Hepatitis E virus; Hepatitis E virus recombinant antigens; Hepatitis E virus synthetic peptides; Hepatitis E virus IgG and IgM antibodies

Correspondence to: George J. Dawson, Experimental Sheridan Road, North Chicago, IL 60064, U.S.A.

Biology Research, Abbott Laboratories,

1401

176

Introduction An endemic form of viral hepatitis has been noted as the cause of major epidemic outbreaks in developing countries (Bradley, 1990). This disease has a global distribution and has been variously referred to as waterborne, epidemic, or enterically transmitted non-A, non-B hepatitis (ET-NANBH). Although the fecal-oral route of transmission predominates, very limited person to person routes of exposure were also suggested in some epidemiologic studies (Velasquez et al., 1990). The disease has been documented as having a very high mortality (N 20%) in pregnant women who are infected during the third trimester of pregnancy (Bradley, 1990). Sporadic cases of ET-NANBH, as well as imported travel exposure, have been reported in developed countries (Skidmore, 199 1). Molecular cloning of the putative agent was hampered by the lack of a tissue culture system for virus propagation. However, using available animal models (Andjaparidze et al., 1986; Bradley et al., 1987; Krawczynski and Bradley, 1989) and a newly developed non-specific amplification procedure (Reyes et al., 1990) a unique cDNA clone (ET1.l) was identified from bile obtained from cynomolgus macaques infected with the Burmese strain of HEV. Successful confirmation of the viral origin of this clone led to the identification of similar sequences in human fecal specimens collected from ET-NANBH epidemics in Somalia, Tashkent, Borneo, Pakistan and Mexico (Reyes et al., 1990). cDNA libraries were also prepared from stool samples obtained during an ETNANBH epidemic in Mexico (Reyes et al., 1991). Immunoscreening of these libraries led to the identification of two cDNA clones which encode epitopes specific for HEV (Yarbough et al., 1991). The isolation and sequencing of a set of overlapping cDNA clones has led to the recognition that this form of hepatitis is caused by a novel virus unlike any of the other molecularly characterized agents of viral hepatitis (Tam et al., 1991). Various regions of the hepatitis E virus (HEV) genome have been cloned and expressed in E. coli as fusion proteins with glutathione-S-transferase (Skidmore, 1991). Four recombinant antigens, two derived from a Burmese (B) strain of HEV and two derived from a Mexican (M) strain, have been shown to contain antigenic sites recognized by individuals with previous exposure to HEV (Yarbough et al., 1991). The two antigens from the Mexican strain, namely, M 3-2 and M 4-2, correspond to amino acid sequences at the carboxy-terminus of the second open reading frame (ORF-2) and the third open reading frame (ORF-3), respectively. The two antigens from the Burmese strain, namely, B 3-2 and B 4-2, correspond to amino acid sequences at the carboxy-terminus of ORF-2 and ORF-3, respectively. Both, the M 3-2 and B 32 recombinant antigens are comprised of 42 amino acids from the carboxyterminus of ORF-2. The amino acid similarities between these two regions is 90.5% (Yarbough et al., 1991). The M 4-2 and B 4-2 are each comprised of 33 amino acids from the carboxy-terminus of ORF-3 and the amino acid similarity between them is 73.5% (Yarbough et al., 1991).

177

In the present study, the two recombinant antigens of the Mexican strain and the two antigens of the Burmese strain were utilized and compared individually as solid-phase antigens in enzyme-linked immunosorbent assays (ELISA) for the detection of both IgM and IgG to HEV in human and animal sera. In addition, supplemental ELISA procedures using synthetic HEV peptides and a blocking assay were developed and employed to support the results obtained with the recombinant antigens.

Materials and Methods Recombinant HEV antigens

The four recombinant HEV antigens (M 4-2, M 3-2, B 4-2 and B 3-2) described previously (see Introduction and Yarbough et at., 1991) were cloned and expressed in E. coli as fusion proteins with glutathione-S-transferase (GST) and supplied to us by Genelabs, Inc., Redwood City, California. Synthetic HEV peptides

Synthetic HEV peptides corresponding to the 33 contiguous amino acids of recombinant antigen B 4-2 and to the 42 contiguous amino acids of M 3-2 were prepared by standard solid-phase synthesis procedures (Barany and Merrifield, 1980) and are referred to as synthetic peptide sp-33 and sp-42, respectively. Cynomolgus macaque sera

Preimmune, acute and convalescent-phase sera collected from cynomolgus monkeys experimentally infected with the Burmese, Pakistani and Mexican strains of HEV (Bradley et al., 1987; Bradley et al., 1988) were kindly donated by Dr. Dan Bradley of the Centers for Disease Control (CDC), Atlanta, GA. Acute-phase sera were collected within a 2-wk period during the peak of alanine transaminase (ALT) activity. Convalescent-phase sera were collected between 39 and 115 days after the return of ALT activity to preinoculation levels. Human sera

A panel of human serum samples collected during ET-NANBH outbreaks in Burma, Mexico, Pakistan and Somalia (Bradley et al., 1987) were obtained from the CDC via Genelabs. Acute-phase sera were collected between 1 and 12 days after the onset of symptoms. Convalescent-phase sera were collected from asymptomatic individuals between 30 and 90 days after the onset of jaundice (Yarbough et al., 1991). Sera from volunteer blood donors were obtained from Southeastern Wisconsin. Other sera were obtained from in-house stored panels donated by a variety of American, European, Mexican and New Zealand collaborators.

178

ELISA for anti-HEV IgG and IgM antibodies Anti-HEV IgG Assay Serum anti-HEV was determined by solid-phase ELISA, similar in design to that used for HCV (Dawson et al., 1991) utilizing a variety of recombinant HEV antigens as solid-phase reagents. These antigens included M 4-2, M 3-2, B 4-2, B 3-2 and the corresponding B and M synthetic peptides, namely, sp33 and sp42. The sample to be tested was diluted 1:300 in sample diluent. Anti-HEV IgM assay Serum anti-HEV IgM was determined by solid-phase assays utilizing the individual recombinant HEV antigens as described above. The sample to be tested was run as described previously (Chau et al., 1991). 0 ther assays Synthetic peptides All specimens repeatedly reactive with the recombinant anti-HEV ELISA procedures were retested using solid-phase synthetic peptides corresponding to the amino acid sequences contained in the B and M 4-2 (~~33) and B and M 3-2 (~~42) recombinant antigens (Yarbough et al., 1991). Blocking assay A contirmatory assay was developed using blocking reagents (HEV recombinant antigens) to specifically inhibit anti-HEV from binding to the solid-phase. Polystyrene beads coated with synthetic peptide sp42 or sp33 were used as solid-phase antigen. Serum samples were diluted in a specimen diluent containing the blocking reagent (either recombinant antigen B 4-2 or M 3-2) to specitically inhibit antibodies directed against HEV epitopes from binding to the solid-phase, or in a specimen diluent containing the control reagent (recombinant HCV antigen (Dawson et al., 1991) which does not block anti-HEV from binding to the solid-phase. Specimens whose absorbance values were reduced by more than 50% by the blocking reagent were considered reactive.

Results Cut-off determination Specimens were obtained from individuals considered to be at low risk for infection with HEV and tested for both IgG and IgM anti-HEV with each of the four ELISA procedures. The specimens were obtained from 386 volunteer blood donors in Southeastern Wisconsin. The cut-offs for the assays were set at 6 SD from the mean absorbance value of the population. Specimens which were clear outliers with values greater than 10 times the normal population mean were not included in the calculation of standard deviation. The cut-off value was also expressed in terms of the ratio of the sample absorbance value to the negative control (S/N values) in Fig. 1. The distribution of absorbance values obtained for each of the four solid-phase lots coated with individual HEV recombinant antigens indicated that approximately 98-99% of the specimens

179

A

B

B4-2

ml

B3-2

250 200 e 150 zi k0 I! 50

0

: 035

S/N VALUE

D

M4-2

250

3.1

6.25 92S

12.25 15.25 ILLZS21.25 24.zS

S/N VALUE

M3-2

250 , 200-

6 E

lSO-

L 2 SO-

?n

;

;

I

,P

2425 0.25 3.25 6.25 9.7.5l2.2S IS.2518.2521sZ.1

S/N VALUE

O0:

3.25 6.25 9.X

l2.Z 15.225 1825 21.Z 24.25

S/N VALUE

Fig. 1. Distribution of (S/N) values expressed as the absorbance value of the sample (S) divided by the absorbance value of the negative control (N) obtained when testing a population of 386 volunteer blood donors for antibodies to HEV. Each specimen was tested for antibodies to each of four HEV recombinant antigens including A (B 4-2), B (B 3-2), C (M 4-2) and D (M 3-2). Specimens having S/N values above 6.0 were considered reactive in the B 4-2 and B 3-2 ELlSAs, while specimens with S/N values of 4.2 and above were considered as reactive in the M 4-2 and M 3-2 assays.

had absorbance values which were less than 6 SD from the population mean (Fig. 1). Reactive specimens were also assayed with solid-phase synthetic peptides (Table 1) and with the blocking assay as shown in Fig. 2. A total of 8 specimens from 386 (2.1%) volunteer donors were reactive with one or more synthetic peptides (Table 1) and their reactivity for these peptides could be specifically blocked using HEV recombinant proteins corresponding to the synthetic peptides (Fig. 2). Specificity

studies

In addition to the analyses of the volunteer blood donor sera from Southeastern Wisconsin, other specimens from patients and chimpanzees with antibodies against a variety of viruses and interfering globulins were tested by both anti-HEV IgG and IgM procedures. The high specificity of these

180

SPECIMEN

SPECIMEN

NUMBER

NUMBER

Fig. 2. Blocking assay S/N values obtained when testing 8 reactive specimens for antibodies to sp33 (B 4-2 synthetic peptide - Panel A) or sp42 (M 3-2 synthetic peptide - Panel B) in the presence of control antigen in solution denoted by lightly shaded columns, or the neutralization reagent in solution (HEV recombinant antigen) denoted by darkly shaded boxes.

procedures is demonstrated by examining the frequency distribution of antiHEV in a normal blood donor population (Fig. l), and also by the lack of cross-reactivity observed among specimens from various disease categories (Table 2). CDC panel Eight specimens (Table 3) obtained from various ET-NANBH outbreaks were tested for anti-HEV (IgM and IgG) using all available anti-HEV assays. One specimen (FVH-21 Burma) was negative by all assays. Four specimens were reactive for IgG class antibodies to all 4 recombinant antigens. Seven of 8 specimens were reactive with the B 4-2 epitope, however, only 6 (PAK-1, FVH8, SOM-19, SOM-20, FFI-4, and FFI-125) were confirmed with sp33. A Burmese specimen (B-IgG) was reactive only with B 4-2. Specimens reactive with both B 4-2 and sp33 assays were also found to be reactive with at least two additional HEV recombinant antigens. In comparison, by the IgM specific assay, 6 of 7 acute-phase sera reacted with B 4-2, 5 of 7 with B 3-2 and M 4-2, and 3 of 7 with M 3-2. TABLE I Specificity analyses of anti-HEV

reactive specimens from volunteer blood donors

Specimen

proteins

Recombinant Burma

60 96 174 199 242 265 354 353

Synthetic peptides Burma

Mexico

Mexico

4-2

3-2

4-2

3-2

sp33

sp44

sp33

sP44

+ + + + + +

+ + + + + + +

_ + + +

+ _ -

+ + + + + + +

+ _ + + -

+ + + + + + +

+ + + + +

181 TABLE 2 Lack of cross-reactivity categories

with anti-HEV

IgG and IgM ELISAs in sera positive for various disease Anti-HEV

Disease category

Acute hepatitis A Convalescent hepatitis A Acute hepatitis B Convalescent hepatitis B Anti-HBc reactive Acute NANB Rheumatoid factor’ ANA reactive Acute rubella Convalescent Norwalk virus’

No.

IgG

IgM

34 5 10 10 10

3 0 1 8

3 0 1 0 1

:; 14 5 4

02 0 0 0

8 0 8

’ IO-2000 IU/ml. * Chimpanzee (Wyatt et al., 1978). TABLE 3 Anti-HEV Serum

PAK-1

B-IgG FVH-8 FVH-21 SOM-19 SOM-20 FFI-4 FFI-125 Control

(IgM and IgG) detection in a CDC panel of human sera Source

Pakistan

Burma Burma Burma Somalia Somalia Mexico Mexico USA

Stage’

Acute

Conv. Acute Acute Acute Acute Acute Acute

Antibody

Reaction with solid-phase

antigen

B 4-2

B 3-2

B-sp33 B-sp42 M 4-2

IgM IgG

+

+

+

+

NT +

NT +

IgM IgG

-

-

+

-

NT -

IgM IgG

+

+

+

+

IgM IgG

-

_

_

-

IgM IgG

+

+

+

+

IgM IgG

+

+

+

-

IgM IgG

+

+

+

+

IgM IgG

+

+

+

+

-

-

-

-

IgM IgG

M 3-2

M-sp42

+

+

+

+

NT +

NT -

_

-

NT -

NT +

NT +

+ +

-

NT +

NT _

NT -

_

_ -

NT -

NT +

NT +

+ +

+ +

NT +

NT +

NT -

+ -

+ _

NT -

NT +

NT +

+ +

-

NT -

NT +

NT +

+ +

+ +

NT +

NT -

NT -

_

-

NT -

’ Acute-phase sera were collected between 1 and 12 days after the onset of HEV-related symptoms. Convalescent (Conv.)-phase sera were collected between 30 and 90 days after the onset of jaundice. NT (not tested).

182 TABLE 4 HEV recombinant protein and synthetic infected cynomolgus macaques Identification

Inoculum strain

peptides

Serum ‘Stage’

immunoreactivity

Reaction with Burma

Mexico

4-2 IgG C-24

c-49

c-111

Burma

Preimmune Acute-25 Convalescent-39 Convalescent-91 Preimmune Acute-38 Convalescent-68 Convalescent- 115

Pakistan

Preimmune Acute-26 Convalescent-SO Convalescent-61

Mexico

with sera of experimentally

3-2 -____ IgG IgM

IgM

-

-

R/P R/P -

1 -

-

_ -

pp-

R/P R/P

-

: -

4-2 IgG

IgM

_ _ -

_ _ _

R R

1 -

3-2 __IgG

IgM

-

_

R,! R

1 -

P P

1 -

p I P P --__ C, cynomolgus macaques; Stage, numbers designate days post-inoculation, acute phase sera were collected during peak alanine transaminase activities; R, reactive with recombinant protein; P, reactive with synthetic peptide; -, no reaction. -R R/P P-

R R R

R -

TABLE 5 Prevalence of anti-HEV

in different global populations. Total

No. Reactive

Reaction with one or more Mexico

Burma

Synthetic peptides

Recomb. antigens

4-2

3-2

4-2

3-2

No.

%

No.

%

Germany’

151

2

7

IO

6

11

7.3

3

2.0

Japan2 Mexico’ New Zealand’

100

5 4 0

8 3 I

3 5 I

8 6 3

8.0 9.1 3.4

3 5

3.0 7.6

z

0 5 1

1

I.1

Thailand4

100

2

1

14

4

15

15.0

7

7.0

United States: Alaska’ Wisconsin’

90 386

2 5

1 I

4 6

1

4 9

4.4 2.3

1 8

1.1 2.1

1

’ Volunteer blood donors. ’ 3 4 5

HTLV reactive donors and STD patients. Children sera (Hermon et al., 1984; Tobias et al., 1986). Villagers from rural regions obtained from Dr. K. Nelson (Johns Hopkins University, Baltimore). Hepatitis B carriers obtained from CDC, Alaska.

183

Cynomolgus

macaques

study

The immunoreactivity of sera obtained from experimentally infected cynomolgus macaques with both recombinant HEV proteins and synthetic peptides is shown in Table 4. Following their inoculation, all 3 animals produced antibodies reactive with one or more of the recombinant antigens or synthetic peptides. Some specimens were reactive with both recombinant antigens and synthetic peptides, while others were reactive for one or the other. Prevalence

of anti-HEV

in dijferent global populations

Sera of 982 individuals from different global populations were assayed for antibodies, via the four ELISA tests using the recombinant antigens. The number of specimens from various regions which were repeatedly reactive for one or more recombinant antigens ranged from 2.3 to 15.0% (Table 5). Approximately one-half of the reactive specimens were also reactive with synthetic peptides, thus providing supportive evidence for their positivity. While evidence for confirmed anti-HEV detection is low at l.l-3.0% in the U.S.A., Germany, Japan and New Zealand, the prevalence in Mexico (7.0%) and Thailand (7.6%) was high. Endpoint dilution studies

Eight seropositive serum specimens obtained from 4 different geographical areas were serially diluted in normal human serum devoid of anti-HEV and assayed by the 4 recombinant anti-HEV assays. The results expressed in reciprocal endpoint dilution are shown in Table 6. All 8 specimens were reactive with B 4-2, B 3-2 and M 4-2 assays. Only 5 of 8 specimens were reactive with the M 3-2 assay. For specimens nos. 69, 1, and 265, the highest endpoint dilutions were obtained with the B 4-2 assay at 153 600, 4800 and 2400, respectively. In comparison, for specimens nos. 5, 19 and 49, the highest endpoint dilutions were obtained by the M 3-2 assay.

TABLE 6 Reciprocal Specimen No. 1 5 19 69 29 49 265 363

endpoint

dilutions of HEV seropositive

Origin

Source

specimens

Reciprocal endpoint

Pakistan Pakistan Somalia Somalia

HEV HEV HEV HEV

Outbreak Outbreak Outbreak Outbreak

Mexico Mexico Wisconsin Wisconsin

Blood Blood Blood Blood

Donor Donor Donor Donor

dilution with

B 4-2 ~._.~ 4 800 I 200 4 800 153 600

B 3-2

M 4-2

M 3-2

600 300 600 4 800

600 300 300 2 400

< 300 9 600 19200 4 800

I 200 600 2 400 I 200

600 300 600 1200

I 200 300 I 200 2 400

300 2 400 < 300

Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides.

Four recombinant antigens representing two distinct antigenic domains from two different strains of hepatitis E virus (HEV), were used individually to...
830KB Sizes 0 Downloads 0 Views