Journal of Virological Methods, 46 (1994) 231-250 0 1994 Elsevier Science B.V.

All rights reserved / 0166-0934/94/$07.00 Journal of

VIRMET 01606 Virological Methods

Enzyme immunoassay for the detection of antibody to hepatitis E virus based on synthetic peptides

M.O. Favorova’b, Y.E. Khudyakov”,b, H.A. Fieldsa,*, N.S. Khudyakova”, N. Padhye”, M.J. Alter”, E. Masta, L. Polish”, T.L. Yashinab,

D.M. Yarashevab, G.G. Onischenkob and H.S. Margolis”

‘Hepatitis Branch, Division of Viral and Rickettsial Diseases, National Center of Infection Disease, Centers for Disease Control and Prevention, Atlanta, GA 30333 (USA) bThe D.I. Ivanovsky Institute

of Virology, Russian Academy of Medical Science, Moscow (Russia)

(Accepted 29 August 1993)

Summary

Five synthetic peptides were prepared based on the nucleotide sequence of open reading frames 2 and 3 encoded in the hepatitis E virus (HEV) genome and were used to develop an enzyme immunoassay (EIA) for the detection of anti-HEV activity in sera. Three different approaches were employed to ascertain the optimal preparation of these peptides as an immunodiagnostic reagent, including (1) a mixture of unconjugated peptides, (2) conjugating individual peptides to bovine serum albumin (BSA) followed by mixing each conjugate at various concentrations, and (3) mixing the peptides before conjugation with BSA to create an artificial antigen complex. The third method was superior in discriminating anti-HEV activity in sera previously tested by Western blot (WB). A frequency distribution of optical density values demonstrated that the peptide-based EIA was able to readily discriminate anti-HEV positive sera from sera devoid of anti-HEV activity. To confirm anti- HEV activity a neutralization test was developed using a mixture of 5 unconjugated peptides. With the exception of sera containing high levels of anti-HEV activity, all sera were neutralized greater than 50%. Strong sera required a higher dilution before a 50% neutralization was achieved. The sensitivity of the WB compared to EIA was 89.5% with and overall concordance of 94.8%. The peptide-EIA was used to determine anti-HEV activity in sera collected from various populations worldwide. In six outbreaks of ET-NANB hepatitis in various geographic regions, anti-HEV activity was demonstrated in 78-100% of cases. The peptide-EIA also detected anti-HEV

Corresponding author.

SSDI 0166-0934(93)E0122-I

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activity in 14 out of 14 follow-up sera obtained 4-6 months after onset of disease and in 2 of 2 of these patients 5 yr after the acute episode. Anti-HEV activity was found in 8.5% of sera obtain from a healthy population residing in an HEV endemic region and 0.5% in two non-endemic regions (P<O.OOl). These data demonstrate that a synthetic peptide-based EIA is sensitive for detecting anti-HEV activity in the sera of patients with acute hepatitis E, convalescents, and among healthy individuals.

Hepatitis E virus; Enzyme immunoassay; Synthetic peptides

Introduction

Since the mid-1950s many large waterborne outbreaks of enterically- transmitted non-A, non-B (ET-NANB) hepatitis with high rates of morbidity and mortality have been reported primarily in the northern hemisphere tropical regions of the world (Wong, 1980; Balayan et al., 1983; Bradley et al., 1987, 1990; Purcell et al., 1988; Zuckerman et al., 1990). The first reported outbreak of ET-NANB hepatitis occurred in New Delhi, India in 1955 (Wong et al., 1980). Until recently, the diagnosis of ET-NANB hepatitis remained a diagnosis of exclusion after serologically excluding other viral hepatitides. Subsequently, 27-32 nm virus-like particles were identified in stools collected from acutely infected individuals using immune electron microscopy (IEM) (Bradley et al., 1987). Anti-HEV activity was demonstrated in sera from experimentally infected animals and hepatitis E patients by immunofluorescent microscopy (IF) (Krawczynski et al., 1989).

In 1990, Reyes et al. reported the isolation of a partial cDNA clone from the virus responsible for ET-NANB hepatitis, and named the newly identified agent the hepatitis E virus (HEV). This clone from a Burma HEV isolate hybridized with cDNA from live other geographically distinct isolates. These lindings in combination with IEM and IF strongly suggested that a single agent was responsible for the majority of ET-NANB hepatitis infections seen worldwide (Purcell et al., 1981; Bradley et al., 1988; Krawczynski et al., 1989).

The HEV genome has been characterized as an RNA positive strand virus, composed of 3 open reading frames (ORF), with the non-structural region located at the S-end and the structural genes located at the 3’-end. A recombinant chimeric protein (C2) containing the N-terminal region of trypE (37 kDa) and the C-terminal half (46.8 kDa) of the polypeptide encoded by ORF2 has been used for the development of a WB test to detect IgG and IgM class antibodies to HEV (Tam et al., 1991; Favorov et al., 1992). Recombinant expressed proteins from ORF2 and ORF3 have also been utilized in the development of a specific enzyme immunoassay (EIA) for the detection of anti- HEV activity (Yarbough et al., 1991; Goldsmith et al., 1992; Lok et al., 1992).

Using synthetic peptides HEV-specific antigenic epitopes have been localized

239

on both ORF2 and ORF3 (Khudyakov et al., 1993). These findings provided the foundation for the development of a peptide-based EIA for the detection of anti-HEV activity. Herein, we describe the methods used for the development of this test, results of testing specimens from patients involved in a variety of geographically distinct ET-NANB outbreaks, distribution of anti-HEV activity among healthy persons residing in endemic and non-endemic regions, and a comparison between EIA and WB.

Materials and Methods

Specimens

Acute sera were obtained from 484 patients ages 1 to 67 yr who were involved in outbreaks of ET-NANB hepatitis between 1984 and 1992 residing in 6 geographically distinct regions of the world: Turkmenistan in 1985 (Favorov et al., 1986; n = 75) Somalia in 1986 (Polish, personal communica- tion, y1= 12), Mexico in 1987 (Velazquez et al., 1990; n= lOl), Kirgizstan in 1988 (Favorov et al., 1990; n=80), Tajikistan in 1990 (Iarasheva et al., 1991; y1= 64) and Kenya in 1991 (Mast et al., 1992; n = 151). Convalescent sera (n = 14) were also obtained l-72 mth following hospital discharge from 14 patients in Turkmenistan between 1985-199 1.

The following control sera were tested: 98 patients with acute viral hepatitis A,B,C or D; 28 patients with chronic HDV and HCV infections; 9 patients with CMV, EBV, HIV 1, toxoplasmosis or rubella; and 2 patients with high levels of C-reactive protein.

Sera obtained from healthy populations in one HEV-endemic and 2 non- endemic regions of the world were also analyzed: 934 specimens from Tajikistan (Iarasheva et al., 1991); 1721 sera from the Ukraine where outbreaks of hepatitis E never were registered. These two collections of samples were obtained from voluntary donors, from healthy children during an immunization program, from healthy pregnant woman, and from patients hospitalized because of trauma. In addition, 200 specimens from the staff of the institution for the developmentally disabled in Montana State collected during on HAV-infection investigation were also studied.

For development of the peptide-based EIA, a panel of 95 sera was assembled composed of 45 acute-phase specimens (l-10 days after onset of jaundice) obtained from outbreak involved patients in Tajikistan, 14 convalescent-phase specimens (obtained 4-6 month after onset of jaundice), and 36 sera obtained from healthy blood donors residing in a non-endemic region (Moscow, Russia). All acute-phase and convalescent-phase sera tested positive by WB, while all sera from blood donors were negative by WB.

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Synthetic peptides

The amino acid sequences, locations and secondary structure of the immunoreactive peptides used for the EIA are presented in Table 1. Peptides were synthesized by FMOC-chemistry (Barany and Merrifield, 1980) on an ABI Model 430A automated peptide synthesizer (Applied Biosystems, Inc., Foster City, CA) or on an ACT Model MPS 350 multiple peptide synthesizer (Advanced Chemtech, Louisville, KY) according to the manufacturer’s protocols. Peptides were characterized by amino acid analysis, high performance liquid chromatography, and capillary electrophoresis. Additional irrelevant amino acids were added to the C-terminus of peptides 3, 5, and 28 during synthesis to improve the yield.

Conjugation of synthetic peptides to BSA

Synthetic peptides (Table lA,B,C) were conjugated to BSA using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Pierce Chemical Co., Rockford, IL) according to the method of Bauminger and Wilchek et al., 1980. Briefly, 10 mg of each individual synthetic peptide (Table 2B) or a mixture of peptides (Table 2C) was dissolved in distilled water to achieve a linal peptide concentration of 1 mg/ml. EDC crystals were added to the peptide solution to a final concentration of 10 mg/ml and the pH was adjusted to 5.0. The reaction mixture was incubated at room temperature for 5 min and BSA was added to yield a concentration of approximately 1 mg of synthetic peptide to each 0.5 mg of BSA. The mixture was further incubated at room temperature for 4 h. The conjugation reaction was stopped by adding sodium acetate, pH 4.2, to a final concentration of 100 mM and the reaction mixture was incubated at room temperature for an additional 1 h. The peptide-BSA conjugate was

TABLE I

Location and primary structure of peptides used in the peptide-based enzyme immunoassay for the detection of anti-HEV activity

ORF” Peptide no.

2 11 12 13 22 23

3 2 3 5 6

28 29

“Open reading frame.

Amino acid position

3 19-340 422431 442460 63 l-648 64 l-660

3140 63-76 91-l 10

105-123 91-l 10

105Sl23

Primary structure

RVSRYSSTARHRLRRGADGTAE DKGIAIPHDIDLGESR DYDNQHEQDRPTPSPAPSR RPLGLQGCAFQSTVAELQ QSTVAELQRLKMKVGKTREL

CPRHRPVSRL SPSQSPIFIQPTPSG ANPPDHSAPLGVTRPSAPPLA PSAPPLPHVVDLPQLGPRR ANQPGHLAPLGEIRPSAPPLA PSAPPLPPVADLPQPGLRR

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TABLE 2

Comparison of different mixtures of peptides for the detection of anti-HEV

A: unconjugated peptides

Mixture no. Composition of mixture Western Blot Pos. Western Blot Neg. Sera IV(%)” Sera IVY

Peptide no. Concentration’ (n = 36) Acute Convalescent (n = 45) (n= 14)

I 2 1 3 1 5 1 6 I

28 1 38(84) 2(l4) 8(22) 29 1 12 1 22 1 23 1

2 5 2 6 2

II 2 28 2 38(84) 3(2l) 6(l7) 29 2 22 2 23 2

3 5 6

I1 23 28 29

IO IO 2 2 37(82) 2(l4) 7U9) 2 2

4 5 2 6 2 36(80) 5(36) 3(8)

23 2

5 5 IO 6 20

23 IO 11 5 41(91) 12(86) 3(8) 28 2 29 2 I2 1

‘Number and percentage of sera positive by peptide-based EIA. bNumber and percentage of sera negative by peptide-based EIA.

‘pg/ml. Table 2 continued on next page

separated from the remaining unconjugated peptides and other reaction products by dialysis against 0.01 M phosphate buffer saline (PBS), pH 7.2, overnight at 4°C. The peptide-BSA conjugate was stored in the same buffer at - 20°C.

242

TABLE 2

B. Peptides individually conjugated to bovine serum albumin”

Mixture no. Composition of mixture

Peptide no. Concentration’

Western Blot Pos. Western Blot Neg. Sera N(%)” Sera N(%)b

(n = 36)

1 5 6

28 29 22 23

Acute Convalescent (n = 45) (n=14)

40(89) 9(64) 3(8)

2 6 20 5 10

11 5 28 2 40(89) 9(64) 3(8) 29 2 12 2 22 1

3 6 20 23 10 36(80) 8(57) 2(6) 29 2

4 5 10 6 10

22 10 41(91) 9(64) 2(6) 23 15 29 5

5 5 10 6 10

22 10 43(96) 12(86) 13 5 23 20

z(6)

6 5 10 6 10

22 20 43(96) 12(86) 13 5 23 20

3(8)

1 5 8 6 14

13 4 44(97) 12(86) l(3) 22 4 23 20

“Footnotes same as in Table 2A. Table 2 continued on next page

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TABLE 2

C. Mixture of peptides conjugated to bovine serum albumin”

Mixture no. Composition of mixture Western Blot Pos. Western Blot Neg. Sera N(%)” Sera IVY

Peptide no. ConcentrationC (n = 36) Acute Convalescent (n = 45) (n= 14)

8 5 10 6 10

13 4 44(97) 12(86) l(3) 22 5 23 20

9 2

13 22 23

8 14 4 45(100) 13(93) O(0) 4

20

aFootnotes same as in Table 2A.

Buffers

Each specimen was diluted in normal goat serum buffer (NGS-Buf) composed of 0.01 M PBS, pH 7.2-7.4, containing 10% normal goat serum (NGS), 1% BSA, and 0.05% Tween 20.

Peptide-EIA

Immulon II EIA microtiter wells (Dynatech Laboratories, Inc. Chantilly, VA) were adsorbed with 105 ~1 of individual peptides or conjugated peptides overnight at room temperature. After adsorption, each well was washed 5 times with deionized HZ0 containing 0.5% Tween 20. Each specimen was then diluted in NGS-Buff and 100 ~1 of the diluted specimen was added to each well. Following an incubation period of 1 h at 37°C each well was washed 7 times, and 100 ~1 of a 1:40 000 dilution of affinity purified goat anti-human IgG, F(ab’)* fragment conjugated to horseradish peroxidase (Boehringer Man- nheim) was added to each well. The wells were then incubated for 1 h at 37°C washed 7 times, followed by the addition of 100 ~1 of substrate solution (o- phenylenediamine and H202). After incubation for 15-30 min at room temperature in the dark, the enzyme reaction was stopped with 50 ~1 of 1 N H2S04. The wells were read in an EIA reader set at an optical density (OD) of 493 nm.

The cutoff value for an initially reactive specimen was statistically determined based on a frequency distribution of 491 randomly selected anti- HEV WB positive and negative sera and was shown to be equal to 2.1 times the mean of negative controls. This value was applied to all subsequent assays.

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Neutralization assay

A neutralization test was developed to verify initial EIA positive reactions. Neutralization of anti-HEV activity was achieved by adding unconjugated synthetic peptides diluted in NGS-buffer to a concentration of 50 pg/ml (Neut- buf.) to the specimen. The composition of unconjugated peptides used for neutralization contained the same proportion of peptides as the mixture of BSA-conjugated peptides adsorbed to microtiter plates. Specimens were preincubated with neut-buf for 1 h at 37”C, before testing by EIA.

Results and Discussion

Synthetic peptides based on the nucleotide sequence of the Burma strain of HEV encoded in ORFZ and ORF3 were used individually for the identification of immunoreactive epitopes ~Khudyakov et al., 1993). Peptides containing important immunodiagnostic epitopes when tested against sera obtained from HEV infected individuals were selected for developing the peptide-EIA. Consequently, various mixtures of conjugated and unconjugated synthetic peptides (Tables 2A-2C) were adsorbed to microtiter EIA wells to ascertain their diagnostic significance to specifically detect IgG anti-HEV activity using the panel of 45 acute-phase, 14 convalescent-phase, and 36 healthy blood donor sera. Three different approaches were used.

In the first approach, mixtures of only unconjugated synthetic peptides in various concentrations were used. The immunoreactivity of each mixture was empirically determined, and mixture 5 yielded the highest sensitivity and specificity (Table 2A). The sensitivity of mixture 5 to detect anti-HEV activity was 41145 (91%) of acute-phase sera, and 12/14 (86%) of convalescent-phase sera. The specilicity of this mixture compared with WB was 92% with 3 specimens testing positive out of 36 among healthy blood donor sera. These 3 samples (8%) were considered false-positive reactions.

In the second approach, individual synthetic peptides were conjugated to BSA and mixed at various concentrations (Table ZB). Using this approach, the highest sensitivity and specificity was obtained with mixture 7. This mixture detected anti-HEV activity in 44/45 (98%) acute-phase sera and 12/14 (86%) convalescent-phase sera. The specificity of mixture 7 compared to WB was 97.2% (35/36) among healthy blood donor sera.

In the third approach the synthetic peptides used in mixture 7 were first mixed together and then conjugated to BSA (Table 2C). This method represented an attempt to express each epitope on each molecule of BSA in order to construct an artificial antigen complex that may approximate the immunoreactivity of the natural antigen. In addition, such a complex may allow for a more uniform distribution of antigenic epitopes on the microtiter well. With mixture 9, anti-HEV activity was detected in 45/45 (100%) acute- phase sera and 13/14 (93%) convalescent-phase sera. The specificity of mixture

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140 - negativezone (c mean plus 3.2 SD)

positive zone

(a mea” plus 6.3 SD)

0.4 0.8

Optical Density

1.2 1.8 2.0

Fig. 1. Frequency distribution of anti-HEV activity by peptide-based enzyme immunoassay using 491 sera tested previously by Western blot.

9 compared to WB was 100% (36/36) among healthy blood donors. To determine the utility of the mixture 9 peptide-EIA for discriminating

between positive and negative sera, 491 sera were tested at a 1: 100 dilution and a frequency distribution of the results are presented in Fig. 1. Three zones were defined: positive, negative, and indeterminate. Specimens with OD values below 0.07 (mean of OD values for the negative controls plus 3.2 SD of the mean) were considered to be in the negative zone, OD values equal to or greater than 0.07 and less than 0.1 were considered in the indeterminate zone, and the cutoff for the positive zone was equal or greater than 0.1 (mean of negative controls plus 6.3 SD). Only three (1.6%) specimens yielded indeterminate OD values, which were subsequently shown to be positive after retesting at a less dilution.

The peptide-EIA was compared to WB analysis using sera collected in a large outbreak of ET-NANB hepatitis that occurred in Kenya during 1991 (Mast et al., 1992). This collection was composed of 115 specimens from individuals who either presented with jaundice or reported as being jaundice within 6 months after reported illness onset. In addition, 211 non-case serum specimens were obtained from a cohort of persons residing in the same area. Of the 326 samples tested, 171 were identified by the peptide-EIA as anti-HEV positive, compared to 159 by WB analysis. Thus, the sensitivity of the WB compared to EIA was 89.5% with an overall concordance of 94.8%.

For confirmation of anti-HEV activity in sera detected initially by the peptide EIA, a neutralization test was developed. Individual synthetic unconjugated peptides used in the composition of mixture 9 were added to the specimen by incubating the diluted specimen at 37°C for 1 h before addition to wells containing adsorbed mixture 9. After testing various concentrations,

246

Number of patients Percent neutralization 21) _ .- 80

1 :o 2.b 2:5 >2’.5 ”

Optical Density

Number of patients m Mean percent neutralization

Fig. 2. Peptide-based enzyme immunoassay neutralization confirmation test by optical density activity using sera obtained from an outbreak of enterically transmitted non-A, non-B collected 1 to 20 days after

onset of jaundice.

the optimal concentration of each synthetic unconjugated peptide used as the neutralization agent was as follows: #23 (40 ,ug/ml), #6 (30 pg/ml), #5 (15 pg/ ml), #22 (10 pg/ml), #13 (10 pg/ml). The use of a neutralization test significantly lowered the false-positivity rate, especially in sera which have been improperly stored or which have undergone several freeze thaw cycles (data not shown) and in addition, this test is important for specimens in the indeterminate zone. The neutralizing agent should not be identical to the material adsorbed to the microtiter wells; otherwise, false-positive reactions would be incorrectly neutralized leading to confirming a truly negative specimen as being positive. Figure 2 presents the results of our neutralization test using 370 randomly selected sera which initially tested positive and yielded OD values from 0.1 to greater than 2.5. Sera were considered to be confirmed as positive when the OD was decreased by 50% following incubation with the neutralizing agent. With the exception of sera that had OD values greater than 2.5, all sera were confirmed as positive with mean neutralization activities ranging from between 63% to 78%. Sera that yielded OD values greater than 2.5 were retested at a higher dilution of 1:500 and were subsequently neutralized greater than 50%.

The final design of the peptide-EIA involved testing each specimen for anti- HEV activity simultaneously with and without neutralization at an initial dilution of 1:50. The first row contained sera diluted in NGS-buf and the second row contained diluted and neutralized sera so that the same sera were located in two wells in a vertical position. Initially reactive sera that yielded OD values greater then 2.0 and that were not neutralized were retested at a 1:lOO dilution and at a 1:500 dilution. All sera that yielded OD values greater than or equal to 2.1-times the mean of negative controls and not neutralized greater than 50% were subsequently retested at a 1:lO dilution. These sera were

241

TABLE 3

Frequency of anti-HEV activity among patients involved in outbreaks of enterically transmitted non-A, non-B hepatitis

Region (year of outbreak) No. of case Anti-HEV positive N(%)a

Tajikistan (1990) Somalia (1986) Turkmenistan (1985) Kirgizstan (1988) Kenya (1991) Mexico (1987)

Total

64 64(100) 12 12(100) 16 69(90.7) 80 70(87.5)

151 llS(78.1) 101 82(81.2)

484 415(86.7)

‘Number and percentage of sera positive by peptide-based EIA.

considered to be positive for anti-HEV activity if the OD values were reduced by 50% following neutralization.

Following the final design of the peptide-EIA, endpoint determinations were made on 33 randomly selected sera with OD values greater than 2.0. Thirty of these sera demonstrated an endpoint titer of 1: 1,000 to 1: 10 000. Three sera within this group demonstrated endpoint titers exceeding 1: 100 000.

The peptide-based EIA was used to detect anti-HEV activity (Table 3) in sera collected in various epidemiologic settings, Among 484 patients involved in 6 geographically distinct outbreaks of hepatitis E, anti-HEV activity was detected in 415 (85.7%). The proportion of anti-HEV activity among patients from these 6 outbreaks ranged from 78% to 100%. This variation may be related to possible antigenic differences of HEV in different regions of the world (Yarbough et al., 1991; Tsarev et al., 1992).

In 39 sera from patients with sporadic non-A, non-B, non-C (NABC) hepatitis from the USA (Alter et al., 1990) who also had no evidence of chronic liver disease, none had detectable anti-HEV activity. However, in another setting, anti-HEV activity was detected in 14 out of 14 patients with sporadic acute NABC hepatitis from Tajikistan, a region endemic for hepatitis E. Persistence of anti-HEV activity by peptide-EIA was demonstrated in two individuals from Turkmenistan 5 yr after an acute fulminant episode of HEV infection.

None of the 131 acute-phase sera positive for markers of HBV, hepatitis delta virus (HDV), HCV and other diseases (Table 4) demonstrated anti-HEV activity.

Sera obtained from healthy persons residing in an HEV endemic region (Tajiskistan), and sera from two HEV non-endemic areas (Montana, USA; Ukraine) were tested for detection of anti-HEV activity (Table 5). In Montana, one (0.5%) of 200 sera was positive for anti-HEV activity and remained positive when a follow-up specimen was obtained two years later. This patient had no history of travel to an endemic region and thus the origin of the HEV

248

TABLE 4

Anti-HEV activity by peptide-based enzyme immunoassay among control sera

Control Sera Number of Sera Anti-HEV positive

Acute hepatitis B Acute hepatitis A Convalescent hepatitis A Acute hepatitis D Acute and chronic hepatitis C Cytomegalovirus Epstein-Barr Virus C-reactive protein Human immunodeticiency-I Toxoplasmosis Rubella

38 20 12 25 : 25 0

1 2 8 2 0 2 0 2 0 2 0

Total 131 0

TABLE 5

Frequency of anti-HEV activity among various healthy populations residing in endemic and non- endemic regions of the world

Region Location No. of sera Anti-HEV positive N(%)”

Non-endemic Montana, U.S. 200 l(O.5) Non-endemic Ukraine 1721 9(0.5) Endemic Tajikistan 934 79(8.5)

‘Number and percentage of sera positive by peptide-based EIA.

infection remains unknown. In the Ukraine, another non-endemic region, 9 (0.5%) of 1721 sera demonstrated anti-HEV activity. These data demonstrate that among two geographically distinct HEV non-endemic regions of the world, the seroprevalence of anti-HEV was the same. However, of 934 sera collected from healthy individuals residing in an HEV endemic region (Tajikistan), 79 (8.5%) demonstrated anti-HEV activity indicating prior exposure to HEV.

The results of this study demonstrate the usefulness of a peptide-based EIA for the detection of anti-HEV activity in various epidemiologic settings, and the utility of this test to confirm the etiology of outbreaks of hepatitis E and sporadic cases of hepatitis E. This assay may also prove useful for determining the seroprevalence of prior HEV infections in both HEV endemic and non- endemic regions of the world.

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