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Journal of Clinical Microbiology, September 1999, p. 2863-2871, Vol. 37, No. 9
Hepatitis Branch,
Received 9 December 1998/Returned for modification 13 April
1999/Accepted 16 June 1999
The antigenic composition of the hepatitis E virus (HEV) protein
encoded by open reading frame 2 (ORF2) was determined by using
synthetic peptides. Three sets of overlapping 18-, 25-, and 30-mer
peptides, with each set spanning the entire ORF2 protein of the HEV
Burma strain, were synthesized. All synthetic peptides were tested by
enzyme immunoassay against a panel of 32 anti-HEV-positive serum
specimens obtained from acutely HEV-infected persons. Six antigenic
domains within the ORF2 protein were identified. Domains 1 and 6 located at the N and C termini of the ORF2 protein, respectively, contain strong immunoglobulin G (IgG) and IgM antigenic epitopes that
can be efficiently modeled with peptides of different sizes. In
contrast, antigenic epitopes identified within the two central domains
(3 and 4) were modeled more efficiently with 30-mer peptides than with
either 18- or 25-mers. Domain 2 located at amino acids (aa) 143 to 222 was modeled best with 25-mer peptides. A few 30-mer synthetic peptides
derived from domain 5 identified at aa 490 to 579 demonstrated strong
IgM antigenic reactivity. Several 30-mer synthetic peptides derived
from domains 1, 4, and 6 immunoreacted with IgG or IgM with more than
70% of anti-HEV-positive serum specimens. Thus, the results of this
study demonstrate the existence of six diagnostically relevant
antigenic domains within the HEV ORF2 protein.
Hepatitis E virus (HEV) is an agent
of enterically transmitted non-A, non-B hepatitis (6, 7, 35,
37), which is a serious problem in many developing countries
(6, 7, 41). The HEV genome is a single-stranded,
positive-sense RNA molecule of approximately 7.5 kb (35,
37). Three open reading frames (ORF) were identified within the
HEV genome (39): ORF1 encodes nonstructural proteins, ORF2
encodes the putative structural protein(s) (37, 39), and
ORF3 encodes a protein of unknown function.
The antigenic composition of HEV proteins has been examined with
synthetic peptides of different sizes (9, 18-21) and
recombinant proteins (24, 25, 33, 36, 43). One of the most
comprehensive studies was performed with overlapping 10-mer synthetic
peptides spanning the entire HEV ORF1-, ORF2-, and ORF3-encoded
proteins (18). This study found that the ORF1 protein
contains at least 12 antigenic regions that can be efficiently modeled
with short synthetic peptides. Only three antigenic regions were
identified within the HEV ORF2-encoded protein, at amino acids (aa) 25 to 38, 341 to 354, and 517 to 530. Each region was modeled with two overlapping synthetic peptides. Another antigenic region at aa 105 to
122, which was modeled with three overlapping peptides, was revealed
within the ORF3-encoded protein (18). Whereas no additional
studies have been conducted to further elaborate the antigenic
composition of the ORF1-encoded protein, antigenic epitopes from the
ORF2- and ORF3-encoded proteins have been extensively explored (9,
19-21, 24, 25, 33, 36, 43). The antigenic region within the ORF3
protein at aa 105 to 122, which was found with 10-mer peptides
(18), was also identified in other studies with synthetic
peptides of different sizes (9, 19) and recombinant proteins
(43). However, even though a thorough scan of the ORF3 protein with 10-mer peptides revealed just this single antigenic region
(18), two additional antigenic regions at aa 31 to 40 and 63 to 76 were identified within the ORF3 protein with synthetic peptides
of different sizes (19). In contrast to the ORF3 protein, only one of three antigenic epitopes found with 10-mer peptides within
the ORF2 protein at aa 517 to 530 (18) has been confirmed with peptides of different sizes (20). The other two
antigenic regions identified with 10-mer peptides were located at aa 25 to 38 and 341 to 354 (18). Strong antigenic reactivity was
recently associated with an approximately 100-aa N-terminal region of
the ORF2 protein (25), thus confirming the existence of a
strong antigenic region located at the N terminus. On the other hand, 10-mer peptides (18) could not confirm the existence of
strong antigenic epitopes at aa 319 to 340 (19), 394 to 470 (20), and 631 to 660 (9, 19, 43), identified with
synthetic peptides and recombinant proteins. Collectively, these
findings demonstrate the inconsistent modeling of some antigenic
epitopes from the HEV ORF2 and ORF3 proteins with synthetic peptides of
different sizes and with recombinant proteins.
The ORF2 protein expressed in the baculovirus expression system
(14, 40) or vaccinia expression system (8) or
fragments of this protein expressed in Escherichia coli
(10, 13, 24, 25, 27, 31, 33) have been used as important
diagnostic reagents for the development of diagnostic tests to detect
anti-HEV activity in serum specimens. Some of these proteins have also been identified as important vaccine candidates eliciting protective immunity in animal models (34, 41). An intensive analysis of
a large set of recombinant polypeptides containing different parts of
the HEV ORF2 protein clearly showed that the antigenic properties of
some fragments of this protein differ from the antigenic properties of
the whole protein. For example, some fragments could bind anti-HEV
antibodies from convalescent-phase serum specimens with a much higher
efficacy than that of the full-length protein expressed in the same
expression system (25), suggesting that ORF2 fragments may
be better suited as diagnostic reagents than the full-length protein.
In accordance with these findings, it was recently shown by using an in
vitro seroneutralization test that various HEV strains were neutralized
more efficiently with antibodies derived against ORF2 fragments
expressed in E. coli than with antibodies derived against an
ORF2 full-length protein expressed in the baculovirus expression system
(28). These findings suggest that the antigenic composition
of the HEV ORF2 protein is complex and that different antigenic
epitopes can be efficiently modeled with different fragments of this
protein as represented with either synthetic peptides or recombinant proteins.
In this paper, we report the identification of six antigenic domains
within the ORF2-encoded protein as modeled with three sets of
overlapping synthetic peptides of different sizes, with each set
spanning the entire ORF2 protein. The most diagnostically relevant
antigenic regions that were efficiently modeled with synthetic peptides
were found within domains 1, 4, and 6. The combination of only a few
peptides derived from these domains may be used to develop diagnostic
tests to detect anti-HEV activity in serum specimens obtained from
acutely infected patients.
Serum specimens.
All anti-HEV-positive serum specimens were
obtained from a collection reposited in the Hepatitis Branch, Division
of Viral and Rickettsial Diseases, National Center for Infectious
Diseases, Centers for Disease Control and Prevention, Atlanta, Ga. Ten
serum specimens were obtained from 10 acutely HEV-infected patients during an outbreak of HEV infection in central Asia (collected by
M. O. Favorov). Twenty-two anti-HEV-positive specimens were obtained from an outbreak of HEV infection in India from 22 persons with acute hepatitis E. Serum specimens were selected from these two
regions of the world because the HEV strains circulating in these
regions are the most homologous to the HEV Burma strain (2, 3,
30), whose sequence was used to design synthetic peptides
(39). Anti-HEV-negative serum specimens (n = 32) from normal blood donors were also obtained from a collection
reposited at the Centers for Disease Control and Prevention. All
specimens were initially tested for anti-HEV immunoglobulin G (IgG) and IgM activity by a commercially available kit (GeneLabs, Redwood City,
Calif.) and by the enzyme immunoassay (EIA) based on use of the HEV
mosaic antigen as previously described (12, 22).
Synthetic peptides.
Peptides were synthesized by
9-fluorenylmethoxycarbonyl chemistry (4) on an ACT model MPS
350 multiple-peptide synthesizer (Advanced Chemtech, Louisville, Ky.)
according to the manufacturer's protocols. After characterization by
amino acid analysis, high-performance liquid chromatography, and
capillary electrophoresis, peptides were characterized by EIA.
EIA.
Synthetic peptides (110 µl) at a concentration of 5 µg/ml in 0.1 M phosphate-buffered saline (PBS), pH 7.5, were adsorbed to microtiter wells (Immulon II; Dynatech Laboratories, Inc.) at room
temperature for 12 h. Serum was diluted 1:100 in PBS containing 0.1% Tween 20 and 10% normal goat serum (PBS-T). One hundred
microliters of diluted serum was added to each well and incubated for
1 h at 37°C. The binding of antibodies to the peptides was
identified with affinity-purified antibodies to human IgG or IgM
coupled to horseradish peroxidase (Boehringer Mannheim, Indianapolis, Ind.) by adding 100 µl of a 1:10,000 or 1:5,000 dilution,
respectively, in PBS-T and incubating for 1 h at 37°C. The
cutoff, expressed as a P/N ratio and equal to
3.0, was statistically established individually for each peptide as the
mean of the result with negative controls plus at least 3.5 standard
deviations above the mean, where P represents the optical
density at 493 nm (OD493) of anti-HEV-positive specimens
and N represents the OD of negative controls. Each serum specimen in every experiment was also tested with an irrelevant peptide
(no. 1546) with the sequence PMSMDTSDETSEGATFLSLS derived from a small ORF within the hepatitis G virus minus-sense RNA (26). As an additional criterion, the ratio between the
OD493 for each HEV peptide and the OD493 for
this irrelevant peptide found for each serum specimen was used. HEV
peptides were considered specifically immunoreactive with serum
specimens when this ratio was greater than 2.
Computer-assisted analysis.
Amino acid sequence analysis was
performed by using MegAlign and Protean programs from the Lasergene
software package (DNASTAR Inc., Madison, Wis.). Protein secondary
structure was predicted with the ALB program (32).
Design of synthetic peptides.
The sequence of the protein
encoded by ORF2 of the HEV Burma strain (39) was used to
design synthetic peptides. Three sets of overlapping peptides were
synthesized. Each set spanned almost the entire ORF2 protein without
gaps, with the exception of an 11-aa hydrophobic region at the extreme
N terminus (Fig. 1 to 3).
One set contained 80 18-mer peptides (Fig. 1H). A second set contained
72 25-mer peptides (Fig. 1E), and a third set contained 72 30-mer
peptides (Fig. 1B). Each set spanned the ORF2 protein in such a way
that every amino acid of this protein was represented an average of 2.2 times for 18-mers, 2.7 times for 25-mers, and 3.3 times for 30-mer
peptides. Within each set, peptides were distributed across the HEV
polypeptide chain in an almost uniform manner. However, some regions
were spanned with a higher density of peptides. These regions included
regions where antigenic epitopes had been found previously (9,
18-20, 43) or where antigenic epitopes could be predicted based
on hydropathic plots (23), backbone chain flexibility
(17), and protein secondary structure (32). Some
other regions, such as a region at aa 360 to 390, where a long
hydrophobic alpha-helix was predicted (Fig. 3B and D), were spanned
with a lower density of peptides (Fig. 1A).
Immunoreactivity of synthetic peptides with IgG anti-HEV
antibodies.
All synthetic peptides were tested for
immunoreactivity with IgG anti-HEV antibodies as described in Materials
and Methods. In general, 18-mer peptides were less immunoreactive than
either 25- or 30-mer peptides. Because of this observation, 18-mer
peptides were tested with only 10 anti-HEV-positive serum specimens,
whereas 25- and 30-mer peptides were tested with 32 anti-HEV-positive serum specimens. An analysis of the pattern of the antigenic
reactivities of all peptides allowed the identification of six
antigenic domains (Fig. 1A): domain 1 at aa 12 to 147, domain 2 at aa
143 to 222, domain 3 at aa 221 to 373, domain 4 at aa 381 to 504, domain 5 at aa 490 to 579, and domain 6 at aa 573 to 660 (Table
1).
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Antigenic Domains of the Open Reading Frame
2-Encoded Protein of Hepatitis E Virus
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
View larger version (78K):
[in a new window]
FIG. 1.
Antigenic reactivity of overlapping synthetic 30-, 25-, and 18-mer peptides with anti-HEV IgG antibodies. (A) Approximate
locations of six antigenic domains. (B) Vertical bars show the location
of the N-terminal amino acid of each synthesized peptide. (C, F, and J)
Charts demonstrating the number of serum specimens that are
immunoreactive with each peptide. (D, G, and K) Charts showing the
average P/N ratio (see Materials and Methods)
calculated for all serum specimens that are immunoreactive with each
synthetic peptide.
View larger version (55K):
[in a new window]
FIG. 2.
Antigenic reactivity of overlapping synthetic 30- and
25-mer peptides with anti-HEV IgM antibodies (for details see the
legend to Fig. 1).
View larger version (45K):
[in a new window]
FIG. 3.
Structural properties of the HEV ORF2 antigenic domains.
(A) Antigenic domains. (B) Predicted secondary structure
(32) with horizontal line representing random coil, open
boxes representing beta-turns, shaded boxes representing beta-sheets,
and black boxes representing alpha helices. (C) Plot of flexibility of
the protein chain (17), in which upward peaks show flexible
regions. (D) Hydrophilicity plot (23), in which upward peaks
show hydrophilic regions. (E) Profile of amino acid sequence
heterogeneity calculated for 10 known ORF2-encoded proteins (1, 2,
3, 5, 11, 15, 29, 30, 38, 39) by using a window of 40 aa sliding
along the polypeptide chain with a step of 10 aa; graph values were
calculated as the number of positions affected with amino acid
substitutions within each window across the entire ORF2 polypeptide.
TABLE 1.
Antigenic domains and regions within the HEV ORF2-encoded
protein as modeled with synthetic peptides immunoreactive with IgG
anti-HEV antibodies
The number of antigenic epitopes within each domain. Within N-terminal domain 1, immunoreactive 25- and 30-mer peptides spanned aa 12 to 138 and 12 to 147, respectively. These regions contained 4 to 5 nonoverlapping 25- or 30-mer peptides, with each peptide modeling at least one antigenic epitope. Four antigenic regions were modeled within this domain with 18-mer synthetic peptides (see above). Two of these regions at aa 32 to 68 and 89 to 125 are large enough to be spanned with two nonoverlapping peptides. Therefore, domain 1 may contain at least six antigenic epitopes (Table 1).
Domain 2 was modeled with two nonoverlapping 30-mer peptides (30-143 and 30-188) and five overlapping 25-mer peptides spanning aa 158 to 222, which is of sufficient size to be covered with two nonoverlapping 25-mers. This finding suggests that domain 2 may contain at least two antigenic epitopes. Within domain 3, only one 18-mer was immunoreactive. 25-mer peptides derived from this domain identified four antigenic regions, each of 25 to 34 aa. Therefore, these peptides model at least four antigenic epitopes. However, 30-mer peptides identified four antigenic regions (Table 1), one of which may be spanned with three nonoverlapping peptides. Thus, at least six antigenic epitopes may be modeled with synthetic peptides within this domain. The region of aa 390 to 438 within domain 4 was identified with six overlapping immunoreactive 18-mer peptides. The size of this region is sufficient to accommodate three 18-mer peptides overlapped by only 2 aa each, suggesting that this region may contain at least three antigenic epitopes. In addition, the use of 25-mer peptides identified another region at aa 436 to 465 that may contain one antigenic epitope. An additional antigenic epitope was identified with 30-mer peptides within the region of aa 475 to 504. Thus, domain 4 may contain at least five antigenic epitopes. Domain 5 was identified with only two overlapping 18-mer peptides and two 25-mer peptides representing two separate antigenic regions. Thus, this domain may contain at least two antigenic epitopes. C-terminal domain 6 contains at least five antigenic epitopes because 18-mer peptides identified three antigenic regions, one of which at aa 603 to 638 may be spanned with two nonoverlapping 18-mers, and one immunoreactive 30-mer spans aa 573 to 602 (Table 1), which is not covered with any immunoreactive 18-mer. Collectively, these data suggest that the entire HEV ORF2-encoded protein may contain a minimum of 26 antigenic epitopes.Immunoreactivity of synthetic peptides with IgM anti-HEV
antibodies.
All 25- and 30-mer synthetic peptides were tested for
IgM anti-HEV immunoreactivity as described in Materials and Methods. The set of 25-mer peptides was tested with 16 anti-HEV positive serum
specimens, whereas 30-mer peptides were tested with 23 anti-HEV-positive serum specimens. An analysis of the pattern of the
antigenic reactivities of these peptides allowed for the identification
of the very same six antigenic domains that were found when these
peptides were tested for IgG immunoreactivity (Fig. 2A and Table
2), although domain 2 was not as readily
discernible, especially when 30-mer peptides were tested, an
observation similar to that shown for IgG anti-HEV (Fig. 2C). Domain 2 was best identified when 25-mers were tested for IgG immunoreactivity
(Fig. 1F). Similarly, this domain was better identified with 25-mer
peptides when tested for IgM activity (Fig. 2F).
|
Antigenic epitopes of the HEV ORF2 protein. Various investigators have used synthetic peptides (9, 18-20) or recombinant proteins (24, 25, 43) to identify antigenic epitopes within the HEV ORF2-encoded protein. A recombinant protein containing the N-terminal region of approximately 100 aa was strongly immunoreactive (25). An analysis of the immunoreactivities of synthetic peptides derived from this region, however, yielded contradicting results. In one study (19) in which two peptides spanning sequences from aa 54 to 65 (12-mer peptide) and 84 to 101 (18-mer peptide) were tested, no antigenic reactivity was found. Two 18-mer peptides (18-56 and 18-82) described in the present paper, which are most related to the two peptides mentioned above, were also found to be antigenically inactive. On the other hand, a peptide scan using overlapping 10-mer peptides revealed only one antigenic region spanned with two peptides at aa 25 to 38 (18). The sequence from aa 29 to 34, shared by these two peptides, may play an essential role for this antigenic epitope. This sequence is also shared by immunoreactive 25- and 30-mer peptides (Fig. 1 and Table 1). However, the most IgG-immunoreactive peptides identified in this study within N-terminal domain 1 (aa 12 to 147) share sequences from aa 39 to 53 and 90 to 106 (Fig. 1), both of which are outside the region identified by peptide scanning (18). Of these two immunodominant regions within domain 1, only one region (aa 95 to 114) was associated with strong IgM immunoreactivity (Fig. 2). This region was spanned with one 30-mer peptide containing the sequence at aa 85 to 114 and one 25-mer peptide containing the sequence at aa 95 to 119 (Fig. 2).
No antigenic epitopes within domain 2 have been reported. However, within domain 3 two regions of antigenic reactivity were previously identified by using two overlapping 10-mer peptides at aa 341 to 354 (8) and one 20-mer peptide at aa 319 to 340 (20). In the present study, peptides overlapping these two regions were also found to be immunoreactive (Fig. 1 and 2). One of these regions was found to be strongly IgG immunoreactive by using a 30-mer peptide comprising the sequence from aa 309 to 338. This peptide immunoreacted with 12 of 32 serum specimens (Fig. 1C and D). However, two of the most IgM-immunoreactive 30-mer peptides from this domain were located elsewhere, at aa 242 to 271 and 282 to 311 (Fig. 2C). Domain 4 was previously identified with 20-mer synthetic peptides as one of the most immunodominant regions within the HEV ORF2 protein (20). Surprisingly, a recombinant protein comprising aa 394 to 470, which is a significant part of the domain 4 sequence (aa 381 to 504), was not immunoreactive (25). These contradicting observations suggest that antigenic epitopes within this region are highly conformation dependent and that they can be efficiently modeled only with peptides or protein fragments of specific sizes. Two antigenic regions within domain 5 at aa 515 to 530 (18, 20) and 546 to 589 (20) have been previously described. One of these regions (aa 515 to 530) was also reported as having nonspecific antigenic reactivity (20). In the present study, none of the synthetic peptides that overlapped this region was either specifically or nonspecifically immunoreactive. IgG antigenic epitopes within this domain could not be efficiently modeled with 30-mer peptides (Fig. 1C). The antigenic reactivity associated with the other region at aa 546 to 589 (20) was confirmed with only two overlapping 18-mer peptides spanning the region at aa 554 to 579 (Fig. 1J). 25- and 30-mer peptides derived from this domain were more immunoreactive with IgM anti-HEV (Fig. 2C and F) than with IgG antibodies (Fig. 1C and F). Antigenic epitopes derived from the extreme C terminus of the HEV ORF2 protein were found in the very first experiments on the identification of HEV antigenic epitopes (43). Later, this original observation was confirmed by several research groups (9, 19, 24, 25). This further substantiated this observation, namely, that the region from aa 631 to 660 contains strong IgG- and IgM-specific antigenic epitopes that can be efficiently modeled with synthetic peptides of different sizes (Fig. 1 and 2). In addition, synthetic peptides derived from the adjacent region at aa 573 to 630 are also strongly and broadly immunoreactive (Fig. 1 and 2). Computer-assisted analysis revealed 10 large hydrophilic regions within the HEV ORF2-encoded protein (Fig. 3D). When these hydrophilic regions were aligned with antigenic domains, each one contained at least one hydrophilic region. All domains are separated by broad hydrophobic regions, except at the border between domains 2 and 3 (Fig. 3A and D). Domains 1, 3, and 4 contain highly hydrophilic regions. In addition, domain 1 has a very high predicted backbone chain flexibility (Fig. 3C). Domains 3 and 4 are also quite flexible compared with domains 2 and 6, which have rather rigid backbone structure, and compared with domain 5, which has intermediate flexibility. Domains 1, 4, and 6 were the most immunoreactive domains modeled with synthetic peptides. Although antigenic epitopes tend to be located within highly hydrophilic and flexible regions (16), only domains 1 and 4 have high hydrophilicity and protein chain flexibility. Domain 6 is not very hydrophilic and, except for the extreme C-terminal region, has a very low backbone chain flexibility (Fig. 3C and D). The predicted secondary structure for the HEV ORF2 protein was aligned with the antigenic domains (Fig. 3B). This analysis revealed that (i) antigenic domain 1 is flanked by two alpha-helices and is mainly composed of random coil and beta-turn elements; (ii) domain 2 is a cluster of beta-sheet structures; (iii) domain 3 is alpha-helical with a long stretch of a random coil structure; (iv) domain 4 contains almost an equal mixture of random coil and beta-sheet structures with the centrally located extended random coil structure demonstrating the highest hydrophilicity and flexibility characteristics, which is true for a similar region within domain 3 (Fig. 3B, C, and D); (v) domain 5 is mainly represented with beta-turns and random coils flanked with beta-sheet structures; and (vi) the hydrophilic region of domain 6 comprises a mixture of random coils and beta-turns and contains one alpha-helix. The hydrophobic region of this domain is represented with a cluster of beta-sheets (Fig. 3B). Thus, each antigenic domain has a set of specific structural characteristics as predicted by the computer-assisted analysis. Finally, sequence heterogeneity along the HEV ORF2 protein was analyzed (Fig. 3E). This analysis revealed that domain 1 was the most heterogeneous. The sequence of the second strongest antigenic region at aa 90 to 106 was identified within this domain (Fig. 1) as very variable among different strains (Fig. 3E). Domain 6, the other strongly antigenic domain, is also variable. However, the most antigenically reactive region located at the extreme C terminus of the protein is rather conserved. Some sequence heterogeneity can also be found within domain 5, whereas sequences of domains 2, 3, and 4 from different HEV strains are conserved. Sequence heterogeneity of antigenic epitopes within domains 1 and 6 may affect the antigenic reactivities of these epitopes. A study of the influence of sequence variation on the antigenic properties of these antigenic regions is warranted. In conclusion, six antigenic domains within the ORF2-encoded protein were identified by using three sets of overlapping synthetic peptides of different sizes. It was shown that several ORF2 antigenic epitopes can be very efficiently modeled with synthetic peptides. The most diagnostically relevant antigenic regions that were reliably modeled with these peptides were found within domains 1, 4, and 6. The data presented in this paper strongly suggest that recombinant proteins and/or synthetic peptides derived from these domains may be used as diagnostic targets for the development of assays to detect anti-HEV activity in serum specimens obtained from acutely infected patients.| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Hepatitis Branch, MS A-33, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA 30333. Phone: (404) 639-2335. Fax: (404) 639-1563. E-mail: yek0{at}cdc.gov.
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Top
Abstract
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Materials and Methods
Results and Discussion
References
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Journal of Clinical Microbiology, September 1999, p. 2863-2871, Vol. 37, No. 9
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