U.S. patent application number 10/165868 was filed with the patent office on 2003-07-31 for hepatitis e virus vaccine and method.
This patent application is currently assigned to GeneLabs Technologies, Inc.. Invention is credited to Bradley, Daniel W., Krawczynski, Krzysztof Z., Purdy, Michael A., Reyes, Gregory R., Tam, Albert W., Twu, Jr-Shin, Yarbough, Patrice O..
Application Number | 20030143241 10/165868 |
Document ID | / |
Family ID | 27124631 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030143241 |
Kind Code |
A1 |
Reyes, Gregory R. ; et
al. |
July 31, 2003 |
Hepatitis E virus vaccine and method
Abstract
Antigen and antibody vaccine composition effective in preventing
hepatitis E virus (HEV) infection are disclosed. The antigen
composition includes a peptide corresponding to a carboxyl terminal
end region of the capsid protein encoded by the second open reading
frame 2 of the HEV genome. The composition is effective in
preventing HEV infection after vaccination. The antibody
composition contains an antibody effective to block HEV infection
of human primary hepatocytes in culture.
Inventors: |
Reyes, Gregory R.; (Madison,
NJ) ; Bradley, Daniel W.; (Lawrenceville, GA)
; Twu, Jr-Shin; (Daly City, CA) ; Purdy, Michael
A.; (Atlanta, GA) ; Tam, Albert W.; (San
Francisco, CA) ; Krawczynski, Krzysztof Z.;
(Norcross, GA) ; Yarbough, Patrice O.; (Union
City, CA) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Assignee: |
GeneLabs Technologies, Inc.
|
Family ID: |
27124631 |
Appl. No.: |
10/165868 |
Filed: |
June 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10165868 |
Jun 6, 2002 |
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07870985 |
Apr 20, 1992 |
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6455492 |
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07870985 |
Apr 20, 1992 |
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07822335 |
Jan 17, 1992 |
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07822335 |
Jan 17, 1992 |
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07505888 |
Apr 5, 1990 |
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07505888 |
Apr 5, 1990 |
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07420921 |
Oct 13, 1989 |
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07420921 |
Oct 13, 1989 |
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07367486 |
Jun 16, 1989 |
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07367486 |
Jun 16, 1989 |
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07336672 |
Apr 11, 1989 |
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07336672 |
Apr 11, 1989 |
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07208997 |
Jun 17, 1988 |
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Current U.S.
Class: |
424/189.1 |
Current CPC
Class: |
C07K 2319/23 20130101;
C07K 16/10 20130101; A61P 31/20 20180101; A61P 31/12 20180101; A61K
38/00 20130101; A61P 1/16 20180101; C12N 2770/28122 20130101; C07K
14/005 20130101; C07K 2319/61 20130101; C12N 2770/28022 20130101;
C07K 2319/40 20130101; A61K 39/00 20130101; A61P 31/14 20180101;
C07K 2319/00 20130101; G01N 33/5767 20130101 |
Class at
Publication: |
424/189.1 |
International
Class: |
A61K 039/29 |
Claims
It is claimed:
1. A vaccine composition used in immunizing an individual against
hepatitis E virus (HEV) comprising, in a pharmacologically
acceptable carrier, a peptide containing the C-terminal 42 amino
acids of the capsid protein encoded by the second open reading
frame of the HEV genome.
2. The composition of claim 1, wherein the peptide includes the
amino acid sequence identified by one of the following sequences:
(i) Sequence ID No. 13 (ii) Sequence ID No. 14, and (iii)
internally consistent variations between Sequence ID Nos. 13 and
14.
3. The composition of claim 1, wherein the peptide includes the
amino acid sequence identified by one of the following sequences:
(i) Sequence ID No. 15 (ii) Sequence ID No. 16, and (iii)
internally consistent variations between Sequence ID Nos. 15 and
16.
4. The composition of claim 1, wherein the peptide includes the
amino acid sequence identified by one of the following sequences:
(i) Sequence ID No. 17 (ii) Sequence ID No. 18, and (iii)
internally consistent variations between Sequence ID Nos. 17 and
18.
5. The composition of claim 1, wherein the peptide includes the
amino acid sequence identified by one of the following sequences:
(i) Sequence ID No. 19 (ii) Sequence ID No. 20, and (iii)
internally consistent variations between Sequence ID Nos. 19 and
20.
6. The composition of claim 1, wherein the peptide includes the
amino acid sequence identified by one of the following sequences:
(i) Sequence ID No. 21 (ii) Sequence ID No. 22, and (iii)
internally consistent variations between Sequence ID Nos. 21 and
22.
7. The composition of claim 1, wherein the peptide antigen is
covalently coupled to a carrier protein.
8. A method of inhibiting infection of an individual by hepatitis E
virus, comprising administering to the subject, by intramuscular
injection, the vaccine composition of claim 1.
9. The method of claim 7, wherein the peptide in the vaccine
composition includes the amino acid sequence identified by one of
the following sequences: (i) Sequence ID No. 13 (ii) Sequence ID
No. 14, (iii) internally consistent variations between Sequence ID
Nos. 13 and 14, (iv) Sequence ID No. 15 (v) Sequence ID No. 16,
(vi) internally consistent variations between Sequence ID Nos. 15
and 16, (vii) Sequence ID No. 17 (viii) Sequence ID No. 18, (ix)
internally consistent variations between Sequence ID Nos. 17 and
18, (x) Sequence ID No. 19 (xi) Sequence ID No. 20, and (xii)
Internally consistent variations between Sequence ID Nos. 19 and
20, (xiii) Sequence ID No. 21 (xiv) Sequence ID No. 22, and (xv)
Internally consistent variations between Sequence ID Nos. 21 and
22.
10. The method of claim 9, wherein the peptide in the vaccine
composition includes the amino acid sequence identified by one of
the following sequences: (vii) Sequence ID No. 17 (viii) Sequence
ID No. 18, and (ix) internally consistent variations between
Sequence ID Nos. 17 and 18.
11. The method of claim 10, wherein the peptide in the vaccine
includes the amino acid sequence identified by Sequence ID No.
17.
12. A vaccine composition containing antibodies capable of
neutralizing hepatitis E virus (HEV) infection, as evidenced by the
ability of the composition to block HEV infection of primary human
hepatocyte cells in culture.
13. The vaccine composition of claim 12, which contains an antibody
which is immunoreactive with a peptide containing one of the
sequences: (i) Sequence ID No. 13 (ii) Sequence ID No. 14, (iii)
internally consistent variations between Sequence ID Nos. 13 and
14, (iv) Sequence ID No. 15 (v) Sequence ID No. 16, (vi) internally
consistent variations between Sequence ID Nos. 15 and 16, (vii)
Sequence ID No. 17 (viii) Sequence ID No. 18, (ix) internally
consistent variations between Sequence ID Nos. 17 and 18, (x)
Sequence ID No. 19 (xi) Sequence ID No. 20, and (xii) Internally
consistent variations between Sequence ID Nos. 19 and 20, (xiii)
Sequence ID No. 21 (xiv) Sequence ID No. 22, and (xv) Internally
consistent variations between Sequence ID Nos. 21 and 22.
14. The composition of claim 13, wherein the antibody in the
composition is immunoreactive with a peptide containing the
sequence identified by (i) Sequence ID No. 13 (ii) Sequence ID No.
14, or (iii) internally consistent variations between Sequence ID
Nos. 13 and 14, and (iv) Sequence ID No. 15 (v) Sequence ID No. 16,
or (vi) internally consistent variations between Sequence ID Nos.
15 and 16, Sequence ID No. 13.
15. A method of preventing or treating HEV infection in an
individual, comprising administering to the subject, by parenteral
injection, the vaccine composition of claim 13.
16. The method of claim 15, wherein the antibody composition
includes an antibody which is immunoreactive against a peptide
having the amino acid sequence identified by one of the following
sequences: (vii) Sequence ID No. 17 (viii) Sequence ID No. 18, and
(ix) internally consistent variations between Sequence ID Nos. 17
and 18.
17. The method of claim 16, wherein the antibody composition
contains an antibody which is immunoreactive with a peptide
containing one of the sequences: (i) Sequence ID No. 13 (ii)
Sequence ID No. 14, or (iii) internally consistent variations
between Sequence ID Nos. 13 and 14, and (vii) Sequence ID No. 15
(viii) Sequence ID No. 16, (ix) internally consistent variations
between Sequence ID Nos. 15 and 16.
18. The method of 17, wherein the antibody in the composition is
immunoreactive with a peptide containing the sequence identified by
Sequence ID No. 15.
19. The method of claim 15, wherein the antibody composition
includes an antibody which is immunoreactive against a peptide
having the amino acid sequence identified by one of the following
sequences: (xiii) Sequence ID No. 21 (xiv) Sequence ID No. 22, and
(xv) Internally consistent variations between Sequence ID Nos. 21
and 22.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 07/882,335, filed Jan. 17, 1992, which is a
continuation-in-part of application Ser. No. 07/505,888, filed Apr.
5, 1990, which is a continuation-in-part of U.S. application Ser.
No. 420,921, filed Oct. 13, 1989, which is a continuation-in-part
of U.S. application Ser. No. 367,486, filed Jun. 16, 1989, which is
a continuation-in-part of U.S. Application Serial No. 336,672,
filed Apr. 11, 1989, which is a continuation-in-part of U.S.
application Ser. No. 208,997, filed Jun. 17, 1988, all of which are
herein incorporated by reference.
1. FIELD OF INVENTION
[0002] This invention relates to antigen and antibody vaccine
compositions related to enterically transmitted nonA/nonB hepatitis
viral agent, also referred to herein as hepatitis E virus (HEV),
and to vaccine methods.
2. REFERENCES
[0003] Arankalle, V. A., et al., The Lancet, 550 (Mar. 12,
1988).
[0004] Bradley, D. W., et al., J Gen. Virol., 69:1 (1988).
[0005] Bradley, D. W. et al., Proc. Nat. Acad. Sci., USA, 84:6277
(1987).
[0006] Dieckmann, C. L., et al., J. Biol. Chem. 260:1513
(1985).
[0007] Engleman, E. G., et al., eds., Human Hybridomas and
Monoclonal Antibodies, Plenum Press, 1985.
[0008] Gravelle, C. R. et al., J. Infect. Diseases, 131:167
(1975).
[0009] Kane, M. A., et al., JAMA, 252:3140 (1984).
[0010] Khuroo, M. S., Am. J. Med., 48:818 (1980).
[0011] Khuroo, M. S., et al., Am. J. Med., 68:818 (1983).
[0012] Lanford, R. E., et al., In Vitro Cellular and Devel Biol, 25
(2):174 (1989).
[0013] Larrick, J. W. and Fry, K., Huam Antibod Hybrid, 2:172
(1991).
[0014] Maniatis, T., et al. Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory (1982).
[0015] Saiki, R. K., et al., Science, 239:487 (1988).
[0016] Seto, B., et al., Lancet, 11:941 (1984).
[0017] Sreenivasan, M. A., et al., J. Gen. Virol., 65:1005
(1984).
[0018] Tabor, E., et al., J. Infect. Dis., 140:789 (1979).
[0019] Tam, A., et al., Virology, 185:120 (1991).
[0020] Yarbough, P. O., J. Virology, 65(11):5790 (1991).
[0021] Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC
Press, Boca Raton, La., 1987.
3. BACKGROUND OF THE INVENTION
[0022] Enterically transmitted non-A/non-B hepatitis viral agent
(ET-NANB, also referred to herein as hepatitis E virus or HEV) is
the reported cause of hepatitis in several epidemics and sporadic
cases in Asia, Africa, Europe, Mexico, and the Indian subcontinent.
Infection is caused usually by water contaminated with feces,
although the virus may also spread by close physical contact. The
virus does not seem to cause chronic infection.
[0023] The viral etiology in HEV has been demonstrated by infection
of volunteers with pooled fecal isolates; immune electron
microscopy (IEM) studies have shown virus particles with 27-34 nm
diameters in stools from infected individuals. The virus particles
reacted with antibodies in serum from infected individuals from
geographically distinct regions, suggesting that a single viral
agent or class is responsible for the majority of HEV hepatitis
seen worldwide. No antibody reaction was seen in serum from
individuals infected with parenterally transmitted NANB virus (also
known as hepatitis C virus or HCV), indicating a different
specificity between the two NANB types.
[0024] In addition to serological differences, the two types of
NANB infection show distinct clinical differences. HEV is
characteristically an acute infection, often associated with fever
and arthralgia, and with portal inflammation and associated bile
stasis in liver biopsy specimens (Arankalle). Symptoms are usually
resolved within six weeks. HCV, by contrast, produces a chronic
infection in about 50% of the cases. Fever and arthralgia are
rarely seen, and inflammation has a predominantly parenchymal
distribution (Khuroo, 1980).
[0025] The course of HEV is generally uneventful in healthy
individuals, and the vast majority of those infected recover
without the chronic sequelae seen with HCV. One peculiar
epidemiologic feature of this disease, however, is the markedly
high mortality observed in pregnant women; this is reported in
numerous studies to be on the order of 10-20%. This finding has
been seen in a number of epidemiologic studies but at present
remains unexplained. Whether this reflects viral pathogenicity, the
lethal consequence of the interaction of virus and immune
suppressed (pregnant) host, or a reflection of the debilitated
prenatal health of a susceptible malnourished population remains to
be clarified.
[0026] The two viral agents can also be distinguished on the basis
of primate host susceptibility. HEV, but not HCV, can be
transmitted to cynomolgus monkeys. HCV is more readily transmitted
to chimpanzees than is HEV (Bradley, 1987).
[0027] In the earlier-filed parent applications, HEV clones, and
the sequence of the entire HEV genome sequence were disclosed. From
HEV clones, recombinant peptides derived from HEV genomic coding
region were produced.
4. SUMMARY OF THE INVENTION
[0028] In one aspect, the invention includes a peptide vaccine
composition for immunizing an individual against hepatitis E virus
(HEV). The composition includes a pharmacologically acceptable
carrier, and a peptide containing the C-terminal 42 amino acids of
the putative capsid protein encoded by the second open reading
frame of the HEV genome. The peptide preferably includes the amino
acid sequence identified by one of the following sequences:
[0029] (i) Sequence ID No. 13
[0030] (ii) Sequence ID No. 14,
[0031] (iii) internally consistent variations between Sequence ID
Nos. 13 and 14,
[0032] (iv) Sequence ID No. 15
[0033] (v) Sequence ID No. 16,
[0034] (vi) internally consistent variations between Sequence ID
Nos. 15 and 16,
[0035] (vii) Sequence ID No. 17
[0036] (viii) Sequence ID No. 18,
[0037] (ix) internally consistent variations between Sequence ID
Nos. 17 and 18,
[0038] (x) Sequence ID No. 19
[0039] (xi) Sequence ID No. 20, and
[0040] (xii) Internally consistent variations between Sequence ID
Nos. 19 and 20, and
[0041] (xiii) Sequence ID No. 21
[0042] (xiv) Sequence ID No. 22, and
[0043] (xv) Internally consistent variations between Sequence ID
Nos.21 and 22.
[0044] In a related aspect, the invention includes a method of
inhibiting infection of an individual by HEV, by administering to
the subject, by parenteral injection, such as intramuscular or
intravenous injection, the above peptide vaccine composition.
[0045] In another aspect, the invention includes an antibody
vaccine composition effective in neutralizing hepatitis E virus
(HEV) infection, as evidenced by the ability of the composition to
block HEV infection of primary human hepatocyte cells in
culture.
[0046] The antibody composition preferably contains an antibody
which is immunoreactive with a peptide containing one of the above
(i)-(xv) sequences, and preferably with a peptide corresponding to
sequences (i)-(iii), (iv-vi) and (vii-xv). In a related aspect, the
invention includes a method for preventing or treating HEV
infection in an individual, by administering to the subject, by
parenteral injection, the above antibody composition.
[0047] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 shows the HEV genome, the arrangement of open reading
frames in the genome, and the approximate coding regions for
peptides 406.3-2, GS3, and trpE-C2;
[0049] FIGS. 2A and 2B show the blood ALT levels observed after
infection of cynomolgus monkeys with a Burma-strain HEV stool
sample in animals which were previously immunized with a trpE-C2
HEV antigen (2A) or an alum control (2B);
[0050] FIGS. 3A and 3B show the blood ALT levels observed after
infection of cynomolgus monkeys with a Mexico-strain HEV stool
sample in animals which were previously immunized with the trpE-C2
HEV antigen (3A) or an alum control (3B);
[0051] FIG. 4 shows Southern blots of PCR-amplified RNA from
non-infected human primary hepatocytes (lane 4) and primary
hepatocytes infected with HEV for increasing times from 3 hours to
11 days (lanes 5-11);
[0052] FIG. 5 shows Southern blots of PCR-amplified RNA from
HEV-infected human primary hepatocytes in which the infective virus
is preincubated with normal pre-immune rabbit serum (lanes 1 and 3)
or rabbit antiserum against the HEV antigen HEV 406.3-2 (B) (lane
2) and HEV 406.4-2 (M) (lane 4);
[0053] FIG. 6 shows Southern blots of PCR-amplified RNA from
HEV-infected human primary hepatocytes preincubated with normal
human serum (lane 1) and one of a number of different HEV-positive
immune human sera (lanes 2-12);
[0054] FIG. 7 shows the nucleotide sequences of the HEV ORF2 and
ORF3 for Burma (upper line) and Mexico (lower line) strains of
HEV;
[0055] FIG. 8 shows the amino acid sequences of the ORF3 peptide
for Burma (upper line) and Mexico (lower line) strains of HEV;
and
[0056] FIG. 9 shows the amino acid sequences of the ORF2 protein
for the Burma (upper line) and Mexico (lower line) strains of
HEV.
[0057] FIG. 10 shows in panel A, the ethidium bromide stained gel
of DNA produced from PCR-amplified RNA. The RNA was from HEV
infected primary cynomolgus macaque hepatocytes in which the
infective virus HEV Burma was preincubated with normal preimmune
rabbit serum as shown in lanes 1 and 3; or with rabbit anti-serum
against HEV antigen 406.3-2(B) (lane 2), or with HEV
406.4-2(B)(lane 4); panel B shows Southern Blots of the materials
as described above in panel A for lanes 1-4.
DETAILED DESCRIPTION OF THE INVENTION
[0058] I. Definitions
[0059] The terms defined below have the following meaning
herein:
[0060] 1. "Enterically transmitted non-A/non-B hepatitis viral
agent", "hepatitis E virus", or "HEV" means a virus, virus type, or
virus class which (1) causes water-borne, infectious hepatitis,
(ii) is transmissible in cynomolgus monkeys, (iii) is serologically
distinct from hepatitis A virus (HAV), hepatitis B virus (HBV),
hepatitis C virus (HCV), and hepatitis D virus, and (iv) includes a
genomic region which is homologous to the 1.33 kb cDNA insert in
plasmid pTZKF1(ET1.1) carried in E. coli strain BB4 identified by
ATCC deposit number 67717.
[0061] 2. Two nucleic acid fragments are "homologous" if they are
capable of hybridizing to one another under hybridization
conditions described in Maniatis et al., op. cit., pp. 320-323.
However, using the following wash conditions: 2.times. SCC, 0.1%
SDS, room temperature twice, 30 minutes each; then 2.times. SCC,
0.1% SDS, 50.degree. C. once, 30 minutes; then 2.times. SCC, room
temperature twice, 10 minutes each, homologous sequences can be
identified that contain at most about 25-30% basepair mismatches.
More preferably, homologous nucleic acid strands contain 15-25%
basepair mismatches, even more preferably 5-15% basepair
mismatches. These degrees of homology can be selected by using more
stringent wash conditions for identification of clones from gene
libraries (or other sources of genetic material), as is well known
in the art.
[0062] 3. Two amino acid sequences or two nucleotide sequences (in
an alternative definition for homology between two nucleotide
sequences) are considered homologous (as this term is preferably
used in this specification) if they have an alignment score of
>5 (in standard deviation units) using the program ALIGN with
the mutation gap matrix and a gap penalty of 6 or greater. See
Dayhoff, M. O., in Atlas of Protein Sequence and Structure (1972)
Vol. 5, National Biomedical Research Foundation, pp. 101-110, and
Supplement 2 to this volume, pp. 1-10. The two sequences (or parts
thereof, preferably at least 30 amino acids in length) are more
preferably homologous if their amino acids are greater than or
equal to 50% identical when optimally aligned using the ALIGN
program mentioned above.
[0063] 4. A DNA fragment is "derived from" an HEV viral agent if it
has the same or substantially the same basepair sequence as a
region of the viral agent genome.
[0064] 5. A protein is "derived from" an HEV viral agent if it is
encoded by an open reading frame of a DNA or RNA fragment derived
from an ET-NANB viral agent.
[0065] 6. In two or more known peptide sequences which are more
than about 70% homologous in amino acid sequence, a third amino
acid sequence will be "internally consistent with the known
sequences" if each amino acid in the third sequence is identical to
at least one of amino acids in the known sequences.
[0066] II. HEV Antigen Vaccine
[0067] This section describes methods for preparing and using an
HEV antigen vaccine effective, when injected intramuscularly
(i.m.), to prevent HEV infection.
[0068] A. HEV Genomic Sequences
[0069] HEV genomic clones, and sequences corresponding to the
entire HEV genome for different HEV strains were obtained according
to published methods (Tam, Yarbrough) and as described in the
parent applications referenced above. Briefly, RNA isolated from
the bile of a cynomolgus monkey having a known HEV infection was
cloned, as cDNA fragments, to form a fragment library, and the
library was screened by differential hybridization to radiolabeled
cDNAs from infected and non-infected bile sources.
[0070] The basepair sequence of cloned regions of the HEV fragments
in identified clones was determined by standard sequencing methods.
With reference to FIG. 1, HEV is a virus with an approximately 7.5
kilobase (kb) single-stranded and polyadenylated RNA genome of
positive-sense polarity. Three open reading frames (ORFs) have been
assigned to HEV as ORF1, encoding polypeptides with domains of the
RNA-directed RNA polymerase and a helicase, ORF2, encoding the
putative capsid protein of the virus, and ORF3.
[0071] The genomic organization of HEV assigns its non-structural
gene(s) at the 5' terminus with the structural gene(s) at the 3'
end. Two subgenomic polyadenlated transcripts of approximately 2.0
kb and 3.7 kb in sizes are detected in infected liver and
co-terminated at their 3' ends with the 7.5 kb full-length genomic
transcript. The genomic organization and expression strategy of HEV
suggest that it might be the prototype human pathogen for a new
class of RNA virus or perhaps a separate genus within the
Caliciviridae family.
[0072] The genomic and peptide sequences shown in FIG. 7 correspond
to the ORF-2 and ORF-3 regions of Burma (B) (upper lines) and
Mexico (M) strains (lower lines) of HEV. The bases indicated in the
middle lines represent conserved nucleotides. The numbering system
used in the comparison is based on the Burma sequence. The Burma
sequence has SEQ ID No. 1; and the Mexico sequence, SEQ ID No. 2.
The region corresponding to ORF2 has SEQ ID nos. 3 and 4 for the
Burma and Mexico strains, respectively. The region corresponding to
406.3-2 has SEQ ID Nos. 5 and 6 for the Burma and Mexico strains,
respectively. The region corresponding to SG3 has SEQ ID Nos. 7 and
8 for the Burma and Mexican strains, respectively. The region
corresponding to C2 has SEQ ID Nos. 9 and 10 for the Burma and
Mexico strains, respectively. The region corresponding to 406.4-2
has SEQ ID Nos. 11 and 12 for the Burma and Mexico strains,
respectively.
[0073] B. Recombinant Peptide Antigens
[0074] The amino acid sequences corresponding to the third and
second open reading frames of the Burma and Mexico strains of HEV
are given in FIGS. 8 and 9, respectively. The sequence listings
shown are as follows:
[0075] SEQ ID Nos. 13 and 14 correspond to the amino acid sequences
for the peptides 406.3-2 (B) and 406.3-2 (M), respectively. Each
peptide is a 42 amino acid peptide in the C-terminal end region of
capsid protein encoded by the ORF2, as indicated in the ORF2
sequence (FIG. 9).
[0076] SEQ ID Nos. 15 and 16 correspond to the amino acid sequences
for the peptides SG3 (B) and SG3 (M), respectively. Each peptide
includes the carboxyl 324 amino acids of the HEV capsid.
[0077] SEQ ID Nos. 17 and 18 correspond to the amino acid sequences
for the peptides C2 (B) and C2 (M), respectively. Each includes the
carboxyl 461 amino acids of the HEV protein.
[0078] SEQ ID Nos. 19 and 20 correspond to the amino acid sequences
for the entire putative capsid protein encoded by the Burma and
Mexico strain ORF2, respectively.
[0079] SEQ ID Nos. 21 and 22 correspond to the amino acid sequences
for the 406.4-2 (B) and 406.4-2 (M), respectively (FIG. 8). These
are 33 amino acid sequences encoded by the ORF3.
[0080] Also contemplated are sequences which are internally
consistent with the above specified sequences from different
strains of HEV antigens. These include Sequence ID No. 13; Sequence
ID No. 14, and internally consistent variations between Sequence ID
Nos. 13 and 14; Sequence ID No. 15; Sequence ID No. 16; and
internally consistent variations between Sequence ID Nos. 15 and
16; Sequence ID No. 17; Sequence ID No. 18; and internally
consistent variations between Sequence ID Nos. 17 and 18; Sequence
ID No. 19; Sequence ID No. 20; internally consistent variations
between Sequence ID Nos. 19 and 20; Sequence ID No. 21; Sequence ID
No. 22; internally consistent variations between Sequence ID Nos.
21 and 22.
[0081] For example, the HEV 406.3-2 antigens have the sequence
homology shown below for the Burma (B) and Mexico (M) strains. The
single dots in the sequence comparison indicate recognized
high-probability or "neutral" amino acid substitutions. The blank
spaces indicate a non-neutral substitution.
1 10 20 30 MEXICAN (SEQ ID NO.17) ANQPGHLAPLGEIRPSAPPLPPVADL-
PQPGLRR ::.:.: :::: .::::::::.:.:::: : :: BURMA (SEQ ID NO.18)
BtJP.NA(SEQ ID NO.18) ANPPDHSAPLGVTRPSAPPLPHVVD- LPQLGPRR 10 20
30
[0082] A sequence which is internally consistent with these two
sequences would have one of the sequences:
[0083] AN(Q/P)P(G/D)H(L/S)APLG(E/V)(I/T)RPSAPPLP(P/H)V(A/V)DLPQ
(P/L)G(L/P)RR, where X/Y means either amino acid X or amino acid
Y.
[0084] The ORF3 amino acid sequences, 124 amino acids in length,
for the Burma and Mexico strains have an 87.1% identity in the 124
amino acids. The ORF2 amino acid sequences, having 659 amino acids
of overlap, have a 93.0 identity in the 659 amino acids.
[0085] To prepare the 406.3-2 (M) peptide, the lambda gt11 406.3-2
described in Example 3 was subcloned into the glutathione
S-transferase vector PGEX to express the 3-2(M) antigen, as
detailed in Example 3, and in the Tam reference.
[0086] The 406.3-2(B) antigen can be prepared by PCR amplification
of the Burma SEQ ID No. 5 from above by PCR amplification of the
pBET1 plasmid (Tam). This plasmid contains a 2.3 kb insert covering
the ORF2 and ORF3 for Burma strain HEV sequence. The plasmid is
amplified by PCR amplification, using a 51 primer containing an
NcoI site and a 3' primer containing a BamHI site (Sakai). The
amplified fragment is inserted into the NcoI/BamHI site of a pGEX
vector, and expressed in an E. coli expression system as described
in Example 3.
[0087] The SG3(B) peptide was prepared by first amplifying the SEQ
ID No. 7 sequence with 5' EcoRI-NcoI and 3' BamHI linkers, using a
gt 10 phage BET1 clone plasmid containing the entire ORF2 and ORF3
regions of HEV (B). The amplified fragment was inserted into the
EcoRI/BamHI site of a Bluescript.TM. vector (Stratagene, San Diego,
Calif.), according to the manufacturer's instructions. After vector
propagation and harvesting, the cloned insert was released by
digestion with NcoI and BamHI, and gel purified. The purified
fragment was inserted into the NcoI/BamHI site of a pGEX vector,
and expressed in an E. coli expression system as described in
Example 3. The SG3(M) peptide can be prepared similarly, using the
SEQ ID No. 8 in place of the SEQ ID No. 7.
[0088] The C2 (B) peptide is prepared as described in Example 5.
Briefly, a gt10 phage BET1 plasmid was digested with EcoRI to
release the SEQ ID No. 10 C2 sequence, and this fragment was
inserted into a pATH10 trpE fusion vector, and the recombinant
fusion protein expressed in an E. coli host.
[0089] The C2 (M) peptide can be prepared, substantially as
described above, by PCR amplification of the SEQ ID No. 10, using a
5' primer containing an EcoRI site and a 3' primer containing a
BamHI site. The amplified fragment is inserted into the EcoRI/BamHI
site of a pGEX vector, and expressed in an E. coli expression
system as described in Example 3.
[0090] The capsid protein (B) was prepared substantially as
described above by PCR amplification of the SEQ ID No. 3, , from a
gt10 BET1 plasmid using a 5' primer containing an NcoI site and a
3' primer containing a BamHI site. The amplified fragment was
inserted into the NcoI/BamHI site of a pGEX vector, and expressed
in an E. coli expression system as described in Example 3. The
capsid protein (M) is similarly prepared.
[0091] To prepare the 406.4-2 (M) peptide, the lambda gt11 406.4-2
described in Example 3 was subcloned into the glutathione
S-transferase vector PGEX to express the 3-2(M) antigen, as
detailed in Example 3.
[0092] The 406.4-2(B) antigen can be prepared by PCR amplification
of the Burma SEQ ID No. 11 from above by PCR amplification, using a
5' primer containing an NcoI site and a 3' primer containing a
BamHI site. The amplified fragments is inserted into the NcoI/BamHI
site of a pGEX vector, and expressed in an E. coli expression
system as described in Example 3.
[0093] It will be appreciated that other HEV peptides containing
selected portions, and preferably C-terminal portions of the HEV
capsid protein containing the 406.3-2 sequence, can similarly be
prepared, using the HEV genomic-insert plasmids above, with
amplification of the desired sequences and cloning into a suitable
expression vector, as outlined above, and detailed in Examples 3
and 5.
[0094] The coding sequences used in producing the recombinant
peptides can be derived from the cloning vectors described above
and detailed elsewhere (Tam), or from synthetic nucleotide
synthesis using PCR slicing methods to join oligonucleotide
fragments, according to known methods, in building up nucleotide
sequences.
[0095] C. Mature Capsid Protein
[0096] HEV peptide antigens may also be obtained from purified HEV
virus propagated in primary hepatocytes obtained from primate
liver, preferably from human or cynomolgus monkey liver. Methods
for preparing primary primate hepatocytes for culture, and culture
medium conditions effective to preserve liver-specific functions
for extended periods in culture are detailed for human hepatocytes
in Example 1 below.
[0097] After 3 days of growth in culture, the cells are infected
with a pooled inoculum of HEV-infected cynomolgus monkey stool pool
(fourth passage), as detailed in Example 2. The presence and level
of propagating HEV virus in the cells can be measured by indirect
immunofluorescence. Where, for example, the primary cells are
cynomolgus cells, the cells can be immunoreacted with human HEV
anti-sera, followed by immunoreaction with rabbit anti-human IgG
antibodies.
[0098] Alternatively, the HEV virus can be detected and measured by
selective amplification methods involving initial cDNA formation,
and PCR amplification of HEV cDNA sequences by PCR amplification,
as detailed in Example 2.
[0099] Virus particles can be isolated from HEV infected human
hepatocytes in culture medium by pelleting the virus through a 30%
sucrose cushion by ultracentrifugation. The pelleted virus may be
further purified, if desired, by zonal centrifugation through a
10-40% sucrose gradient, combining peak virus fractions.
[0100] Other methods for separating virus particles from soluble
culture-medium components may be used. For example, clarified
culture medium can be passed through a size-exclusion matrix, to
separate soluble components by size exclusion.
[0101] Alternatively, the clarified culture medium can be passed
through an ultrafiltration membrane having a 10-20 nm pore size
capable of retaining virus particles, but passing solute
(non-particulate) culture medium components.
[0102] The present invention allows glycosylation and other
post-translation modifications in intact HEV capsid protein. Capsid
isolation from the viral particles can be carried out by standard
methods, such as ion exchange and size-exclusion chromatography,
and HPLC purification, after solubilization of the virus particles
in a solubilizing medium, such as a solution of a non-ionic
surfactant. The protein may be purified by affinity chromatography,
employing, for example, antibodies purified from anti-HEV
antisera.
[0103] D. Preparation of Vaccine Compositions
[0104] The recombinant or intact HEV capsid or capsid fragment
peptides (HEV capsid antigens) described above are incorporated
into a vaccine composition, according to known procedures, to
enhance the antigenicity of the injected antigens.
[0105] In one composition, the HEV antigen is covalently coupled to
a carrier protein, such as keyhole limpet hemocyanin, and injected
either in solution form or in combination with an adjuvant.
Alternatively, where the HEV antigen is prepared as part of a
fusion protein, the non-HEV moiety of the protein may serve as the
carrier protein.
[0106] The derivatized or fusion protein is carried in a
pharmaceutically acceptable carrier, such as in solution or in an
adjuvant, such as converted alum.
[0107] Alternatively, the free peptide itself, e.g., the HEV C2
peptide, may be formulated in alum or used without adjuvant. A
suitable adjuvanted vaccine has a preferred antigen concentration
of about 1 mg peptide antigen/mg alum, and not to exceed 80 mg of
alum per injection.
[0108] III. Antigen Vaccine Method
[0109] In a related aspect, the invention is directed to a method
of inhibiting infection of an individual by hepatitis E virus, by
administering to the subject, by parenteral injection, e.g.,
intramuscular or intravenous injection, the vaccine composition of
the invention.
[0110] Preferred vaccine compositions, for use in the method are
those in which the HEV antigen includes the sequence in the
peptides identified by:
[0111] Sequence ID No. 13; Sequence ID No. 14, and internally
consistent variations between Sequence ID Nos. 13 and 14; Sequence
ID No. 15; Sequence ID No. 16; and internally consistent variations
between Sequence ID Nos. 15 and 16; Sequence ID No. 17; Sequence ID
No. 18; and internally consistent variations between Sequence ID
Nos. 17 and 18; Sequence ID No. 19; Sequence ID No. 20; internally
consistent variations between Sequence ID Nos. 19 and 20; Sequence
ID No. 21; Sequence ID No. 22; internally consistent variations
between Sequence ID Nos. 21 and 22.
[0112] The antigen vaccine composition is preferably administered
intramuscularly in a series of inoculations, for example, two-three
injections given at four week intervals.
[0113] In the method detailed in Example 7, cynomolgus monkeys were
injected i.m. with the C2 fusion protein trpE-C2 (B), formulated in
a converted alum adjuvant or with no adjuvant. Four animals
received the alum plus trpE-C2 (B) antigen in two injections,
spaced one month apart. Two other animals received alum only on the
same vaccination schedule. None of the animals showed the presence
of any anti-HEV serum antibody 4 weeks after the second injection,
as judged by Western blotting using a fusionless C2 HEV antigen or
by a separate fluorescence antibody blocking assay.
[0114] At this stage, two of the four experimental animals received
a third inoculation of non-adjuvanted, insoluble trpE-C2 peptide
antigen. Four weeks later, these animals showed anti-HEV
antibodies, as evidenced by Western blots. These results suggest
that the trpE-C2 antigen may be more effective when administered in
the absence of alum, possibly because of alum-denaturation of the
antigen during the alum co-precipitation procedure.
[0115] One month after the final inoculation, the animals were
challenged with an intravenous injection of a third-passage human
stool previously shown to be highly infectious for HEV (Burma
strain) or with a Mexico-strain human HEV stool sample. At selected
intervals after inoculation, serum samples from the animals were
used to measure ALT (alanine transferase) levels, as an indication
of necrosis and hepatocellular degradation. Liver biopsy samples
were also assayed for the presence of HEV antigens by a direct
fluorescent antibody assay (FA).
[0116] FIG. 2A shows the change in liver ALT levels in the period
following infection with Burma-strain HEV virus, in one of the
animals which received a third dose of trpE-C2. As seen, there was
no evidence of elevated ALT levels in the 7 and {fraction (1/2)}
week period following infection. The liver biopsy samples also
showed no evidence of HEV antigen.
[0117] FIG. 2B shows ALT levels measured after HEV (B) infection of
a control animal (alum alone injections) which was infected
intravenously with the Burma strain HEV. The elevated ALT levels
indicate the level of infection which is expected in the absence of
vaccine protection. HEV antigen was also detected in the liver
biopsy samples. A similar result was observed in the animal which
received two injections of trpE-C2 alum composition, but not the
third alum-free vaccination, as described above.
[0118] FIG. 3A shows the change in liver ALT levels following
infection with Mexico-strain HEV virus, in one of the animals which
received a third dose of trpE-C2. Again, there was no evidence of
elevated ALT levels out to day 32 (The animal died of unrelated
causes at day 32). The liver biopsy samples also showed minimal
evidence of HEV antigen. This result demonstrates that an antigen
vaccine directed against one HEV strain can provide protective
immunity against other HEV strains.
[0119] FIG. 3B shows ALT levels measured after HEV infection of a
control animal (alum alone injections) which was infected
intravenously with the Mexico strain of HEV. High levels of
infection (ALT activity) were observed. A similar result was
observed in the animal which received two injections of trpE-C2
alum composition, but not the third alum-free vaccination, as
described above.
[0120] Details of the vaccination method just reported are given in
Example 5.
[0121] IV. Vaccine Composition
[0122] In another aspect, the invention includes an antibody
vaccine composition effective in neutralizing HEV infection, as
evidenced by the ability of the composition to block HEV infection
in HEV-infectable primary hepatocytes in culture. Two exemplary
primary cells are human and cynomolgus monkey cells.
[0123] The antibodies in the composition are preferably
immunoreactive with a peptide containing one of the sequences:
Sequence ID No. 13; Sequence ID No. 14, and internally consistent
variations between Sequence ID Nos. 13 and 14. As will be seen
below, antibodies prepared against the 406.3-2 antigen (M) are
effective to block HEV infection in human primary hepatocytes.
[0124] Antibodies which are immunoreactive with larger capsid
peptides or proteins containing the carboxy terminal of SEQ ID No.
13 or 14 are also preferred. These may include, specifically
Sequence ID No. 15; Sequence ID No. 16; and internally consistent
variations between Sequence ID Nos. 15 and 16. As will be seen
below, human sera which are effective to prevent HEV infection of
human primary hepatocyes are immunoreactive with the SG3 peptides
defined by these sequences.
[0125] Antibodies which are immunoreactive with the trpE-C2
peptides defined by Sequence ID No. 17; Sequence ID No. 18; and
internally consistent variations between Sequence ID Nos. 17 and 18
are also preferred, as are antibodies immunoreactive with the
entire capsid protein, as defined by Sequence ID No. 19; Sequence
ID No. 20; internally consistent variations between Sequence ID
Nos. 19 and 20; and antibodies that are immunoreactive with the
product of ORF3, as defined in part by Sequence ID No. 21; Sequence
ID No. 22; and internally consistent variations between Sequence ID
Nos 21 and 22.
[0126] The antibodies may be obtained as polyclonal antibodies from
antisera, prepared for example, by immunization of a suitable
animal, such as a rabbit or goat, with one of the HEV antigens
specified above. Alternatively, polyclonal antibodies may be
obtained from human or other primate HEV antisera. Anti-HEV
polyclonal antibodies from the antisera may be purified or
partially purified according to standard methods, such as used to
obtain partially purified serum IgG fractions (see, e.g.,
Antibodies: A laboratory Manual, 1988, Cold Springs Harbor Lab).
Alternatively anti-HEV antibodies can be obtained in purified form
by affinity chromatography, employing a solid support derivatized
with one of the capsid antigens described above.
[0127] In another embodiment, the antibodies are monoclonal
antibodies secreted by hybridoma cell lines. To prepare the
hybridoma cell lines, lymphocytes from an immunized animal,
preferably mouse or human, are immortalized with a suitable
immortalizing fusion partner, according to established methods
(e.g., Engleman, Zola).
[0128] Alternatively, human monoclonal antibodies may be produced
by recombinant methods, in which light and heavy human anti-HEV IgG
genes obtained from cultured lymphocytes are inserted into suitable
expression vectors, and used to co-infect a suitable host. Methods
for obtaining and cloning light and heavy genes from human
lymphocytes, and for expressing the cloned genes in a co-infected
host cell are known (larrick).
[0129] The anti-HEV antibodies are formulated in a suitable
solution for injection, typically by intramuscular, subcutaneous or
intravenous route, to form the vaccine composition.
[0130] B. Neutralizing Activity of Anti-406.3-2 Antibodies
[0131] To demonstrate the neutralizing capability of antibodies
prepared as above, antibodies against the 406.3-2 (B) antigen were
tested for their abilities to block HEV infection in human primary
hepatocytes in culture.
[0132] The primary hepatocytes were prepared and cultured according
to published procedures and as detailed in Example 1. The unique
culture conditions allow for long-term cell growth in culture
without loss of specialized hepatocyte function, as evidenced by
the cells' continued ability to make and secrete liver-specific
proteins, such as serum albumin, up to several months after initial
culturing, as described in Example 1.
[0133] The cultured cells were inoculated with either normal human
sera or a cynomolgus stool preparation. To demonstrate HEV
infection in the cells, the cells were examined on days 1-11 after
infection for the presence of HEV RNA, using a combination of
reverse transcriptase, to form cDNA's, and polymerase chain
reaction (PCR) to amplify HEV-specific cDNA. The amplified fragment
is expected to have a 551 basepair length. FIG. 4 shows Southern
blots of the amplified material, using an HEV ORF2 radiolabeled
probe for detecting amplified HEV sequence.
[0134] The results are shown in FIG. 4. Lanes 1-3 are controls.
Lane 4 is total amplified material from cells inoculated with
normal (non-infected) sera. Lanes 5-11 show amplified material 3
hours, 1 day, 3 days, 5 days, 7 days, 9 days, and 11 days after
infection with the cyno stool sample, respectively. The results
show that HEV propagated in human primary hepatocytes within one
day after initial infection (lane 6). There was a time-dependent
increase at the level of HEV replication up to 5 days post
infection (lanes 7 and 8), which appeared to decrease thereafter
(lanes 9-11). There was no evidence of HEV in total cellular RNA
isolated from uninfected primary cells.
[0135] Rabbit antisera against antigen peptides 406.3-2 (B) and
406.4-2 (M) and 406.4-2 (B) were prepared. As noted above, the
406.3-2 peptide is from the carboxy terminal end region of the HEV
capsid protein, and the 406.4-2 peptide, from the peptide encoded
by the HEV ORF3. Preimmune rabbit serum or rabbit antiserum against
one of HEV antigens was added to the cyno stool inoculum, at a 1:20
dilution, and the antibody was incubated with the viral
preparation. The antibody-treated stool sample was then used to
infect human primary hepatocytes. 14 days later, the cells were
examined for HEV infection by the RT/PCR/Southern blot method just
described, except employing primers which are expected to yield a
448 basepair amplified fragment.
[0136] The results are shown in FIG. 5. Lanes 1 and 3 in this
figure show amplified RNA from cells infected with cyno stool
sample previously incubated with human preimmune serum. The 448
basepair band in the figure indicates HEV infection. The second
lane corresponds to cells which were exposed to anti-406.3-2 (B)
rabbit antisera, and indicates virtually complete absence of HEV
infection. Lane 4 shows amplified material from cells exposed to
anti-406.4-2 (M) rabbit antisera. The antibody showed little or no
protective effect against HEV infection. However, as shown in
Example 5, both anti-406.3-2(B) and anti-406.4-2(B) were shown to
offer protective effect against HEV infection.
[0137] C. Neutralizing Activity of Anti-406.4-2(B) Antibody
[0138] D. Neutralizing HEV Antisera
[0139] Another source of neutralizing antibodies, in accordance
with the invention, is human HEV antisera which is characterized by
immunospecific reaction to the 406.3-2 antigen and the SG3 antigen,
both described above.
[0140] To examine the neutralizing antibody characteristics of
human HEV antisera, a panel of human antisera were tested for the
ability to block HEV infection of cultured hepatocytes,
substantially as described above. The ten HEV positive human
antisera are shown in Table 1 below, and are from patients who
developed HEV infection in India, Pakistan, and Mexico. The
antisera were not tested for strain type.
[0141] Briefly, cultured cells were exposed to HEV-positive cyno
stool treated with samel (Burma strain) treated with normal pooled
serum or HEV antiserum, and tested for the presence of HEV-specific
nucleic acid sequences, by PCR amplification and Southern blotting
with an HEV radiolabled probe. The Southern blots are shown in FIG.
6. The lane numbers of the 12 serum samples are given in
parentheses in Table 1 below. As seen from FIG. 6, and indicated in
Table 1, the antisera which were effective in neutralizing HEV were
India 10 (lane 2), India 18 (lane 3), India 210 (lane 5), India 265
(lane 8), Pak 143 (lane 9), and Pak 336 (lane 10). Other human
sera, however, showed very little (lane 11, Mex 387C) or no effect
(lane 4, India 29; lane 6, India 242; lane 7, India 259; lane 12,
Mex 387C[IgG]) in their ability to neutralize HEV infection. As a
negative control, the normal human serum pool revealed absolutely
no neutralizing activity against HEV (lane 1).
2 TABLE 1 Neutralizing Serum Clinical Activity normal (1) pooled -
India 10 (2) -- + India 18 (3) acute, import + India 29 (4) acute,
import - India 210 (5) acute + India 242 (6) acute, fulminant -
India 259 (7) acute, fulminant - India 265 (8) acute + Pak 143 (9)
acute + Pak 336 (10) acute + Mexico F387c (11) convalescent -
Mexico F387c (IgG) convalescent - (12)
[0142] Several of the human antisera were tested for their IgG and
IgM immunoreactivity to 406.3-2 (M), 406.4-2 (M) and 406.4-2 (B)
antigens noted above. Reaction with IgM antibodies tends to
indicate early-phase infection, whereas immunoreactivity with IgG
is indicative of both early and later stages of infection. Reaction
was measured in an ELISA test. The results are shown in Table 2A
and 2B, where a "+" sign indicates a positive reaction; numbers in
the table indicate dilution titre of IgG against the specific
recombinant protein indicated.
3 TABLE 1A IgG Serum 406.3-2 406.4-2 406.4-2 Neutralizing Samples
(M) (B) (M) Activity Clinical Normal - - - - Pooled Human Human
Serum India 18 + + + + acute, import India 29 - + - - acute, import
India 210 + + + + acute India 242 + + + - acute, fulminant India
259 + + + - acute, (500) (>5000) (2000) fulminant India 265 + +
+ + acute (>5000) (>5000) (1000)
[0143]
4 TABLE 1B Serum IgM Samples 406.3-2 (M) 406.4-2 (B) 406.4-2 (M)
Normal ND ND ND Human India 18 - - - India 29 - - - India 210 - - -
India 242 + + - India 259 + + - India 265 + + -
[0144] The data from the table indicates that those human antisera
capable of neutralizing were positive by an IgG ELISA for
antibodies to the HEV 3-2(M) epitope. India 29 was not positive for
IgG(s) to HEV 3-2(M) and did not neutralize HEV infection (lane 4).
Although India 242 and India 259 were positive for IgG(s) to HEV
406.3-2(M), they were also positive for IgM to HEV 406.3-2(M),
which is indicative of an early stage HEV infection. Therefore in
these particular samples, the levels of IgG(s) to HEV 3-2(M)
elicited might be sufficient to neutralize HEV infection of primary
human hepatocytes.
[0145] To further study the correlation of neutralizing activities
of sera of HEV-infected humans with immunoreactivities to HEV3-2
epitope, Western blotting analyses were performed on these human
serum samples, with the results shown in Table 3. As seen in this
table, India 18, India 265, and especially India 210, previously
shown to be neutralizing for HEV infection, were immunoreactive to
HEV406.3-2(M) in these Western blotting analyses and their
immunoreactivities correlated with their neutralizing
activities.
[0146] As a confirmation for the specific immunoreactivities of
these sera to HEV406.3-2(M), Western analyses were performed
against the fusion protein SG3 (B), which contains the 329
carboxy-terminal amino acids (nucleotides 6146-7129) of ORF-2 of
HEV Burma strain. The immunoreactivities of these sera against
HEV406.3-2(M) and SG3 [or HEV406.3-2(B)] were perfectly matched
(Table 3).
5TABLE 3 Serum 406.3-2 (M) 406.3-2 (M) SG3 Neutralizing Samples
ELISA Titre Western Blot Western Blot Activity Normal Human -- - -
- India 18 2000 ++ + + India 29 -- - - - India 210 100 ++ + + India
242 500 - - - India 259 500 .+-. - - India 265 5000 +++ +++ +
[0147] Thus, human HEV antisera which provide a suitable source of
neutralizing antibodies are those characterized by (a)
immunoreactivity with a 406.3-2 antigen, and (b) the SG3 antigen,
both as evidenced by immunoreactivity in a Western blot, i.e.,
where the antigen is in an exposed, accessible configuration.
[0148] More generally, a preferred vaccine composition of the
invention contains antibodies immunospecific against the 406.3-2
antigenic and against the SG3 antigenic peptide. The vaccine
composition includes the immunospecific antibodies in a suitable
carrier for parenteral injection.
[0149] The antibody vaccine composition is used, according to
another aspect of the invention, for preventing or treating HEV
infection in humans.
[0150] The following examples, which illustrate various methods and
compositions in the invention, are intended to illustrate, but not
limit the scope of the invention.
Materials
[0151] Enzymes: DNAse I and alkaline phosphatase were obtained from
Boehringer Mannheim Biochemicals (BMB, Indianapolis, Ind.); EcoRI,
EcoRI methylase, DNA ligase, and DNA Polymerase I, from New England
Biolabs (NEB, Beverly Mass.); and RNase A was obtained from Sigma
(St. Louis, Mo.).
[0152] Other reagents: EcoRI linkers were obtained from NEB; and
nitro blue tetrazolium (NBT), S-bromo-4-chloro-3-indolyl phosphate
(BCIP) S-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (Xgal) and
isopropyl B-D-thiogalactopyranoside (IPTG) were obtained from
Sigma. cDNA synthesis kit and random priming labeling kits are
available from Boehringer-Mannheim Biochemical (BMB, Indianapolis,
Ind.).
EXAMPLE 1
Human Primary Hepatocytes in Culture
[0153] A. Isolation of hepatocytes.
[0154] Hepatocytes were isolated from human liver obtained from
Stanford University Medical Center. The liver was either perfused
in situ or excised as a wedge for perfusion in laboratory. The
initial perfusion was performed for 10 minutes at 60 ml/min using
Ca.sup.++-, Mg.sup.-+-free Hanks' balanced salt solution
supplemented with 10 mM HEPES (pH 7.4) and 0.5 mM [ethylene
bis(oxyethylenenitrillo]-tetraacetic acid. Perfusion was continued
for additional 20 minutes using Williams' medium E (WME)
supplemented with 10 Mm HEPES (pH 7.4) and 100 U/ml collagenase
(type I, Sigma Chemical Co., St. Louis, Mo.).
[0155] After perfusion the liver capsule was removed using fine
forceps, and hepatocytes were dislodged by gentle shaking in
collagenase solution. The hepatocyte suspension was filtered
through several layers of gauze and mixed with an equal volume of
WMW containing 10% fetal bovine serum (FBS). Hepatocytes were
sedimented by centrifugation at 50 Xg for 5 minutes and resuspended
in WME containing 5% FBS. Hepatocytes were sedimented and
resuspended in the manner for 2 additional times. The final cell
preparation was further filtered through several layers of gauze
before examining for viability using trypan blue. The cells were
plated at a density of 2.times.10.sup.6 cells per 60-mm Primaria
plates (Falcon) pre-coated with collagen (Collaborative
Research).
[0156] Cultures were incubated at 37.degree. C. in 5% CO.sub.2 for
3 hours to allow attachment and the medium was changed to a
serum-free formulation and every 48 hrs thereafter. The serum-free
formulation was a WME-based medium supplemented with growth
factors, hormones, 10 mM HEPES (pH 7.4), 100 ug/ml gentamycin, as
has been described (Lanford).
[0157] B. Detection of Liver-Specific Proteins.
[0158] Human hepatocyte cultures were maintained in serum-free
medium for various period of time and labeled with
[.sup.35S]-methionine for 24 hrs. The medium was adjusted to
contain 1 mM PMSF, 1 mM EDTA, and 1% NP40. Antibodies specific for
the different plasma proteins were bound to protein A-agarose
beads, the beads were washed with PBS, and aliquots of the labeled
medium were incubated for 16 hrs at 4.degree. C. with the
antibody-bead complexes. The beads were washed 3 times with a
buffer containing 1% NP40, and immunoprecipitated proteins were
eluted with gel electrophoresis sample buffer containing 2% SDS and
2% 2-mercaptoethanol. Samples were analyzed by gradient SDS-PAGE (4
to 15%) and autoradiography.
EXAMPLE 2
In vitro HEV Infection of Primary Human Hepatocytes
[0159] A. HEV Infection of Human Hepatocytes.
[0160] The HEV-infected cynomolgus monkey #73 stool pool (fourth
passage) was used as an inoculum for infections of primary human
hepatocytes. Various amounts of inoculum was diluted in 1 ml of
serum-free medium (SFM) and applied to the culture during a 3 hr
incubation period. This solution was then supplemented with 2 ml of
fresh SFM and the entire mixture was incubated overnight. The next
day, cell monolayers were washed with WME (10 mM HEPES, pH 7.4) for
three times and changed to fresh SFM, which was changed at two day
intervals thereafter.
[0161] B. Immunofluorescence Staining Assay.
[0162] Primary cynomolgus monkey hepatocytes were isolated and
plated in tissue culture plates with collagen-coated coverslips as
described. Cells on coverslips were infected with either the
HEV-infected cynomolgus monkey #73 stool pool or the NIH normal
human serum three days after initial plating. The infections were
allowed to proceed for 2 weeks.
[0163] Cells on coverslips were fixed in 90% acetone at room
temperature for 1 minute. The coverslips were then air-dried. The
coverslips were blocked in 1% goat serum in PBS for 1 hour, washed
with PBS for three times, and incubated with a mixture of rabbit
antisera against HEV recombinant proteins 1L6, 4-2, and 6-1-4 at
room temperature for 3 hours. The coverslips were again washed with
PBS for 3 times and reacted with fluorescein
isothiocyanate-conjugated (FITC) goat anti-rabbit IgG(H+L) (Zymed)
diluted in PBS-1% goat serum for 30 minutes. After the coverslips
were washed with PBS for 3 times and air-dried, they were mounted
with FITC glycerol solution and examined under a fluorescent
microscope.
[0164] C. Reverse Transcription/Polymerase Chain Reaction
(RT/PCR).
[0165] HEV infection of primary cynomolgus macaque hepatocytes was
evaluated by RT/PCR assays. The primers for cDNA synthesis and PCR
were based on the nucleotide sequences of the full-length HEV cDNA
(A. Tam et al.). Primers HEV3.2SF1 (nt 6578-6597) and HEV3.2SF2 (nt
6650-6668) are of sense polarity from the ORF2 region of the viral
genome and HEV3.2SR1 (nt 7108-7127) and HEV3.2SR2 (nt 7078-7097)
are antisense primers within the region.
[0166] Following extraction of total cellular RNA from HEV-infected
cells using one-step guanidinium procedure or HEV-infected
supernatants according to the method of Sherker et al., aliquots of
RNA samples were heat-denatured at 95.degree. C. for 5 minutes and
subjected to reverse transcription at room temperature for 5
minutes and 42.degree. C. for 60 minutes using 200 units per
reaction of MMLV-reverse transcriptase (BRL) in a 20 ul reaction
volume containing 20 units of RNasin (Promega), 1.times. PCR buffer
(Perkin-Elmer Cetus), with a concentration of 1 mM each
deoxyribonucleotide (Perkin-Elmer Cetus), and 2.5 uM of HEV3.2SR1
primer. The reaction mixture was then heat-treated at 95.degree. C.
for 5 minutes to denature the MMLV-reverse transcriptase.
[0167] Ten microliters of the cDNA synthesis product was used for
PCR in a final volume of 50 ul with 0.5 uM HEV3.2SF1 primer, 1.25
units Taq DNA polymerase (AmpliTaq, Perkin-Elmer Cetus), and
1.times. PCR buffer, overlayed with 50 ul of mineral oil, and
subjected to 40 cycles of PCR in a Perkin-Elmer thermocycler
(95.degree. C..times.1 minute; 52.degree. C..times.2 minutes;
72.degree. C..times.30 seconds). Ten microliters of the first-round
PCR product then underwent another 40 cycles of nested PCR
(95.degree. C..times.1 minute; 55.degree. C..times.2 minutes;
72.degree. C..times.30 seconds) in a total volume of 50 ul
containing the internal PCR primers HEV3.2SF2 and HEV3.2SR2.
[0168] First- and second-round PCR products were subjected to
agarose electrophoresis, ethidium bromide stained and photographed
under UV light. The results are shown in FIG. 4, discussed above.
Southern transfer was performed and filters were hybridized with
[.sup.32P-dCTP]-labeled internal probe HEVORF2-7 exclusive of the
primers (nt 6782-6997), and autoradiography performed.
EXAMPLE 3
Preparation of 406.3-2 and 406.4-2 Antigens
[0169] A TZKF1 plasmid (ET1.1), ATCC deposit number 67717, was
digested with EcoRI to release the 1.33 kb HEV insert which was
purified from the linearized plasmid by gel electrophoresis. The
purified fragment was suspended in a standard digest buffer (0.5M
Tris HCl, pH 7.5; 1 mg/ml BSA; 10 mM MnC12) to a concentration of
about 1 mg/ml and digested with DNAse I at room temperature for
about 5 minutes. These reaction conditions were determined from a
prior calibration study, in which the incubation time required to
produce predominantly 100-300 basepair fragments was determined.
The material was extracted with phenol/chloroform before ethanol
precipitation.
[0170] The fragments in the digest mixture were blunt-ended and
ligated with EcoRI linkers. The resultant fragments were analyzed
by electrophoresis (5-10V/cm) on 1.2% agarose gel, using
PhiX174/HaeIII and lambda/HindIII size markers. The 100-300 bp
fraction was eluted onto NA45 strips (Schleicher and Schuell),
which were then placed into 1.5 ml microtubes with eluting solution
(1 M NaCl, 50 mM arginine, pH 9.0), and incubated at 67.degree. C.
for 30-60 minutes. The eluted DNA was phenol/chloroform extracted
and then precipitated with two volumes of ethanol. The pellet was
resuspended in 20 ml TE (0.01 M Tris HCl, pH 7.5, 0.001 M
EDTA).
[0171] B. Cloning in an Expression Vector
[0172] Lambda gt11 phage vector (Huynh) was obtained from Promega
Biotec (Madison, Wis.). This cloning vector has a unique EcoRI
cloning site 53 base pairs upstream from the beta-galactosidase
translation termination codon. The genomic fragments from above,
provided either directly from coding sequences 5) or after
amplification of cDNA, were introduced into the EcoRI site by
mixing 0.5-1.0 mg EcoRI-cleaved gt11, 0.3-3 ml of the above sized
fragments, 0.5 ml 10.times. ligation buffer (above), 0.5 ml ligase
(200 units), and distilled water to 5 ml. The mixture was incubated
overnight at 14.degree. C., followed by in vitro packaging,
according to standard methods (Maniatis, pp. 256-268).
[0173] The packaged phage were used to infect E. coli strain KM392,
obtained from Dr. Kevin Moore, DNAX (Palo Alto, Calif.).
Alternatively, E. coli strain Y1090, available from the American
Type Culture Collection (ATCC #37197), could be used. The infected
bacteria were plated and the resultant colonies were checked for
loss of beta-galactosidase activity-(clear plaques) in the presence
of X-gal using a standard X-gal substrate plaque assay method
(Maniatis). About 50% of the phage plaques showed loss of
beta-galactosidase enzyme activity (recombinants).
[0174] C. Screening for HEV Recombinant Proteins
[0175] HEV convalescent antiserum was obtained from patients
infected during documented HEV outbreaks in Mexico, Borneo,
Pakistan, Somalia, and Burma. The sera were immunoreactive with
VLPs in stool specimens from each of several other patients with
ETNANB hepatitis.
[0176] A lawn of E. coli KM392 cells infected with about 104 pfu of
the phage stock from above was prepared on a 150 mm plate and
incubated, inverted, for 5-8 hours at 37.degree. C. The lawn was
overlaid with a nitrocellulose sheet, causing transfer of expressed
HEV recombinant protein from the plagues to the paper. The plate
and filter were indexed for matching corresponding plate and filter
positions.
[0177] The filter was washed twice in TBST buffer (10 mM Tris, pH
8.0, 150 mM NaCl, 0.05% Tween 20), blocked with AIB (TBST buffer
with 1% gelatin), washed again in TBST, and incubated overnight
after addition of antiserum (diluted to 1:50 in AIB, 12-15
ml/plate). The sheet was washed twice in TEST and then contacted
with enzyme-labeled anti-human antibody to attach the labeled
antibody at filter sites containing antigen recognized by the
antiserum. After a final washing, the filter was developed in a
substrate medium containing 33 ml NET (50 mg/ml stock solution
maintained at 4.degree. C.) mixed with 16 ml BCIP (50 mg/ml stock
solution maintained at 4.degree. C.) in 5 ml of alkaline
phosphatase buffer (100 mM Tris, 9.5, 100 mM NaCl, 5 mM MgC12).
Purple color appeared at points of antigen production, as
recognized by the antiserum.
[0178] D. Screening Plating
[0179] The areas of antigen production determined in the previous
step were replated at about 100-200 pfu on an 82 mm plate. The
above steps, beginning with a 5-8 hour incubation, through NBT-BCIP
development, were repeated in order to plaque purify phage
secreting an antigen capable of reacting with the HEV antibody. The
identified plaques were picked and eluted in phage buffer
(Maniatis, p. 443).
[0180] Two subclones which were selected are the 406.3-2 and
406.4-2 clones whose sequences are set forth above. These sequences
were isolated from an amplified cDNA library derived from a Mexican
stool. Using the techniques described in this section, polypeptides
expressed by these clones have been tested for immunoreactivity
against a number of different human HEV-positive sera obtained from
sources around the world. As shown in Table 4 below, 8 sera
immunoreactive with the polypeptide expressed by the 406.4-2, and 6
sera immunoreacted with polypeptide expressed by the 406.3-2
clone.
[0181] For comparison, the Table also shows reactivity of the
various human sera with the non structural peptide Y2. Only one of
the sera reacted with the polypeptide expressed by this clone. No
immunoreactivity was seen for normal expression products of the
gt11 vector.
6TABLE 4 Immunoreactivity of HEV Recombinant Proteins: Human Sera
Sera Source Stage.sup.1 406.3-2 406.4-2 Y2 lgt11 FVH-21 Burma A - -
- - FVH-8 Burma A - + + - SOM-19 Somalia A + + - - SOM-20 Somalia A
+ + - - IM-35 Borneo A + + - - IM-36 Borneo A - - - - PAK-1
Pakistan A + + - - FFI-4 Mexico A + + - - FFI-125 Mexico A - + - -
F 387 IC Mexico C + + ND - Normal U.S.A. - - - - - .sup.1A = acute;
C = convalescent
[0182] Here Y2 represents a sequence encoded by the HEV sequence
157 basepair sequence from the first open reading frame of the HEV
genome.
[0183] E. Producing the 406.3-2 Antigen
[0184] The 406.3-2 gt11 plasmid from above was digested with EcoRI
and the released HEV fragment was amplified by PCR in the presence
of linkers which added an NcoI site at the 5' fragment end, and a
BamHI site at the 31 fragment end. The amplified material was
digested with NcoI and BamHI and inserted into the NcoI/BamHI site
of the glutathione S-transferase vector PGEX expression vector,
according to the manufacturer's instructions.
[0185] The PGEX plasmid was used to transform E. coli host cells,
and cells which were successfully transformed with the pGEX vector
were identified by immunofluorescence, using anti-HEV human
antisera.
[0186] F. Producing the 406.4-2 Antigen
[0187] The 406.4-2 gt11 plasmid from above was digested with EcoRI
and the released HEV fragment was amplified by PCR, and the
amplified fragment was inserted into the NcoI/BamHI site of the
pGEX expression vector, as above. Peptide expression of the 406.4-2
peptide was similar to that described for the 406.3.2 fusion
peptide.
[0188] G. Preparing Antibodies
[0189] The 406.3-2(M) and 406.4-2(M) fusion proteins, prepared as
above, were used to immunize rabbits to generate HEV-specific
antisera, according to standard procedures.
EXAMPLE 4
Neutralizing Activity of Anti-3.2(M) Antibody
[0190] A. In vitro Infection
[0191] To prove that primary human hepatocytes were permissive for
HEV infection and replication, cells were exposed to either normal
human serum (NIH normal human serum pool) or HEV-infected
cynomolgus macaque stool preparation (cyno#73). Fourteen days
postinfection, total cellular RNAs were prepared for
reverse-transcription (RT)/polymerase chain reaction (PCR) assays
to evaluate the infectability of primary human hepatocytes with
HEV. The results indicated that primary human hepatocytes were
capable of supporting HEV propagation (FIG. 4).
[0192] Although quantitative PCR was not applied, total cellular
RNA isolated from HEV-infected primary human hepatocytes would
indicate a high level of virus replication as suggested by the
extent of hybridization with the .alpha.-.sup.32P-dCTP labeled
HEV-specific probe (lane 5). There was no evidence of HEV in total
cellular RNA isolated from primary human hepatocytes treated with
normal human serum pool (lane 4). As negative controls for RT/PCR
assays, no carry-over or cross-contamination was detected (lanes 1,
2, and 3). The original HEV-infected cynomolgus macaque stool
(cyno#73) was served as a positive control in the RT/PCR assays
(lane 6).
[0193] B. Neutralizing Activity of Antibody
[0194] To examine the neutralizing activities of anti-3-2 (M),
-4-2-(M), each rabbit antiserum was used at a final dilution of
1:20 with the viral inoculum for HEV infection of primary human
hepatocytes. The diluted antibody and viral inoculum were incubated
together prior to infection of the cultured cells. Rabbit
anti-3-2(M) exhibited a high level of neutralizing activity against
HEV infection (FIG. 5, lane 2 versus lane 1). Very little
neutralizing activity was observed in rabbit anti-4-2 (M) (lane 4
versus lane 3).
[0195] This result suggests that the HEV 3-2(M) but not HEV 4-2(M)
4-2 (B) recombinant protein encoded a neutralizing epitope capable
of eliciting protective antibody or antibodies against HEV
infection. The fact that the Mexico clone 3-2(M) and the Burma
clone 3-2(B) share 90.5% homology at the amino acid level (79.8% at
the nucleotide level) suggested that antibody(ies) raised against
3-2(M) should cross-neutralize or cross-protect HEV of Mexico or
Burma strain from infecting permissive cells.
EXAMPLE 5
Neutralizing Activity of Anti-3-2(B) and Anti-4-2(B)
[0196] HEV type-common epitopes 3-2 and 4-2 of Burma (B) or Mexico
(M) strains were previously identified by screening high titer
lambda library for HEV-specific antigen-producing clones using
convalescent human serum F387-C. The lambda gt11 clones, 406.3-2
and 406.4-2, were characterized and subcloned to express as
betagalactosidase fusion proteins. These fusion proteins were
subsequently used to immunize rabbits to generate HEV-specific
antisera.
[0197] To examine the neutralizing activities of anti-3-2(B) and
anti-4-2(B), preimmune rabbit serum or rabbit anti-3-2(B) or
anti-4-2(B) antiserum was used at a final dilution of 1:20 with the
viral inoculum of Burma strain for HEV invection of primary
cynomolgus macaque hepatocytes. Both rabbit anti-3-2(B) (FIG. 10,
lane 2) and anti-4-2(B) (FIG. 10, lane 4) but not rabbit preimmune
serum (FIG. 10, lane 1 or lane 3) exhibited extraordinary levels of
neutralizing activity against HEV infection as indicated by RT/PCR
analysis (FIG. 10 panels A and B). This result indicated that both
HEV 3-2(B)(Sequence ID No. 21) and REV 4-2(B)(Sequence ID No. 22)
recombinant proteins encode a neutralizing epitope capable of
eliciting protective antibody or antibodies against HEV infection.
The neutralizing activity of anti-4-2(B) was previously not shown.
Therefore, in a cynomologus macaque hepatocyte system it has now
been shown that rabbit anti-4-2(B) antibody will neutralize HEV.
Thus, the HEV protein designated by sequence ID No 22 is suitable
as an immunogen against HEV.
EXAMPLE 6
Vaccine Protection Against HEV
[0198] A. Preparation of trpE-C2 Peptide
[0199] The PBET1 plasmid containing a 2.3 kb insert, corresponding
to the 1.8 kb 3' end portion of HEV has been described (Tam). The
plasmid was digested with EcoRI, releasing two HEV fragments having
sizes of about 600 bp and 1400 bp of which 1210 bp contain coding
sequence. The larger fragment was purified by electrophoresis and
inserted into the EcoRI site of the pATH10 trpE fusion vector,
obtained from T. J. Koerner et al. (Department of Microbiology,
UCLA). The recombinant vector was used to transform E. coli
DH5.alpha.F'host.
[0200] The recombinant typE-C2 fusion protein from pATH C2 was
isolated by a modification of the procedure of Dieckmann et al. The
bacterium containing the pATH C2 plasmid was grown overnight in
growth media containing tryptophane. Two ml of the overnight
culture was inoculated into 100 ml of fresh growth media and grown
at 37.degree. C. for an additional four hours. The bacterial broth
was added to one liter of fresh growth media without tryptophane
and allowed to grow at 30.degree. C. for 1.5 hours. Ten ml
indoleacrylic acid (1 mg/ml) was added and growth was continued for
an additional 5 to 10 hours at 30.degree. C. The bacterial cells
were collected by centrifugation. The cell pellet was resuspended
in a hypotonic solution containing lysozyme to degrade the
bacterial cell wall. Sodium chloride and the detergent NP-40 were
added to the suspension to cause hypertonic lysis of the cells. The
lysed cell solution was sonicated. The solution was centrifuged.
The resulting protein pellet was resuspended in about 5 ml of 10 mM
Tris pH 7.5 using a dounce homogenizer. Approximately 75% of the
protein in solution was composed of the trpE-C2 protein.
[0201] B. Preparation of Vaccine
[0202] Converted alum adjuvant was prepared according to standard
methods. Briefly, a 10 alum suspension was titrated to pH 6.6 with
1N NaOH, then stirred overnight at 4.degree. C. The suspension is
clarified by low-speed centrifugation, and the supernatant
decanted. A small amount of 0.9% NaCl+1:20,000 formalin was added
to each pellet, and suspended by vortexing. To prepare an antigen
vaccine composition, trpE-C2 fusion protein from above is added in
a 0.9% NaCl solution to a desired final antigen concentration.
[0203] A non-adjuvanted insoluble trpE-C2 peptide was prepared as
above in section A.
[0204] C. Vaccination
[0205] Six cynomolgus monkeys, designated 8901, 8902, 8903, 8910,
9902, and 9904, were used in the vaccination study. Four of the
monkeys, 8901, and 8902 8903, and 8910 were immunized by
intravenous injection with 1.0 ml of the alum adjuvanted-trpE-C2
composition (containing about 50 .mu.g of C2 peptide). The other
two animals received adjuvant only. One month later the six animals
were given a second vaccination, identical to the first.
[0206] 4 weeks after the second vaccination, saer from the animals
was tested for anti-HEV antibodies by Western blotting, using a
fusionless C2 protein. At this stage, animals 8901 and 8902 each
received a third vaccination with the non-adjuvanted, insoluble
trpE-C2 composition (a total IV dose of about 80 .mu.g trpE-C2
peptide each), and both animals showed anti-HEV by Western blotting
4 weeks later.
[0207] Animals 8901, 8903, and 9002 were each challenged IV with 1
ml each of a 10% third passage cyno stool (Burma strain) previously
shown to be highly infectious. Animals 8902, 8910, and 9004 were
each challenged IV with 1 ml of a proven infectious human stool
isolate, Mexican #14, known to cause severe disease in cynos and
moderate disease in chimpanzees. The results are shown in FIGS. 2A,
2B, and 3A, and 3B, discussed above.
[0208] While the invention has been described with reference to
particular embodiments, methods, construction and use, it will be
apparent to those skilled in the art that various changes and
modifications can be made without departing from the invention.
Sequence CWU 1
1
* * * * *