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 peptides corresponding to carboxyl terminal end regions of the second and third open reasing frames 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.
Description




FIELD OF THE INVENTION




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.




References




Arankalle, V. A., et al.,


The Lancet,


550 (Mar. 12, 1988).




Bradley, D. W., et al.,


J Gen. Virol.,


69:1 (1988).




Bradley, D. W. et al.,


Proc. Nat. Acad. Sci., USA,


84:6277 (1987).




Dieckmann, C. L., et al., J. Biol. Chem. 260:1513 (1985).




Engleman, E. G., et al., eds.,


Human Hybridomas and Monoclonal Antibodies


, Plenum Press, 1985.




Gravelle, C. R. et al.,


J. Infect. Diseases,


131:167 (1975).




Kane, M. A., et al.,


JAMA,


252:3140 (1984).




Khuroo, M. S.,


Am. J. Med.,


48:818 (1980).




Khuroo, M. S., et al.,


Am. J. Med.,


68:818 (1983).




Lanford, R. E., et al.,


In Vitro Cellular and Devel Biol,


25 (2):174 (1989).




Larrick, J. W. and Fry, K.,


Huam Antibod Hybrid,


2:172(1991).




Maniatis, T., et al.


Molecular Cloning: A Laboratory Manual


, Cold Spring Harbor Laboratory (1982).




Saiki, R. K., et al.,


Science,


239:487 (1988).




Seto, B., et al.,


Lancet,


11:941 (1984).




Sreenivasan, M. A., et al.,


J. Gen. Virol.,


65:1005 (1984).




Tabor, E., et al.,


J. Infect. Dis.,


140:789 (1979).




Tam, A., et al., Virology, 185:120 (1991).




Yarbough, P. O., J. Virology, 65(11):5790 (1991).




Zola, H.,


Monoclonal Antibodies: A Manual of Techniques


, CRC Press, Boca Raton, La., 1987.




BACKGROUND OF THE INVENTION




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.




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.




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).




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.




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).




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.




SUMMARY OF THE INVENTION




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:




(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, and




(xiii) Sequence ID No. 21




(xiv) Sequence ID No. 22, and




(xv) Internally consistent variations between Sequence ID Nos.21 and 22.




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.




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.




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.











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





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;





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);





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);





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);





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);





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);





FIG. 7

shows the nucleotide sequences of the HEV ORF2 and ORF3 for Burma (upper line) and Mexico (lower line) strains of HEV;





FIG. 8

shows the amino acid sequences of the ORF3 peptide for Burma (upper line) and Mexico (lower line) strains of HEV; and





FIG. 9

shows the amino acid sequences of the ORF2 protein for the Burma (upper line) and Mexico (lower line) strains of HEV.





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




I. Definitions




The terms defined below have the following meaning herein:




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.




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×SCC, 0.1% SDS, room temperature twice, 30 minutes each; then 2×SCC, 0.1% SDS, 50° C. once, 30 minutes; then 2×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.




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.




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.




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.




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.




II. HEV Antigen Vaccine




This section describes methods for preparing and using an HEV antigen vaccine effective, when injected intramuscularly (i.m.), to prevent HEV infection.




A. HEV Genomic Sequences




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.




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.




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.




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.




B. Recombinant Peptide Antigens




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:




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


).




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.




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.




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.




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.




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.




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.













                                           10         20        30







MEXICAN(SEQ ID NO.17)






ANQPGHLAPLGETRPSAPPLPPVADLPQPGLRR






  ::.:.: :::: .::::::::.:.:::: : :: BURMA (SEQ ID NO.18)






   ANPPDHSAPLGVTRPSAPPLPHVVDLPQLGPRR






          10       20        30











A sequence which is internally consistent with these two sequences would have one of the sequences:




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.




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.




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.




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 5′ 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.




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™ 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.




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.




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.




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.




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.




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.




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.




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.




C. Mature Capsid Protein




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.




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.




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.




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.




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.




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.




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.




D. Preparation of Vaccine Compositions




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.




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. The derivatized or fusion protein is carried in a pharmaceutically acceptable carrier, such as in solution or in an adjuvant, such as converted alum.




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.




III. Antigen Vaccine Method




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.




Preferred vaccine compositions, for use in the method are those in which the HEV antigen includes the sequence in the peptides identified by:




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.




The antigen vaccine composition is preferably administered intramuscularly in a series of inoculations, for example, two-three injections given at four week intervals.




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.




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.




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).





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 ½ week period following infection. The liver biopsy samples also showed no evidence of HEV antigen.





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.





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.





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.




Details of the vaccination method just reported are given in Example 5.




IV. Vaccine Composition




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.




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.




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.




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.




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.




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).




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).




The anti-HEV antibodies are formulated in a suitable solution for injection, typically by intramuscular, subcutaneous or intravenous route, to form the vaccine composition.




B. Neutralizing Activity of Anti-406.3-2 Antibodies 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.




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.




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.




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.




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.




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.




C. Neutralizing Activity of Anti-406.4-2(B) Antibody




D. Neutralizing HEV Antisera




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.




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.




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).














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)














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.















TABLE 1A











Serum




IgG




Neutralizing

















Samples




406.3-2 (M)




406.4-2 (B)




406.4-2 (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)

























TABLE 1A











Serum




IgG




Neutralizing

















Samples




406.3-2 (M)




406.4-2 (B)




406.4-2 (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)














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.




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.




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).
















TABLE 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




+++




+++




+














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.




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.




The antibody vaccine composition is used, according to another aspect of the invention, for preventing or treating HEV infection in humans.




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




Enzymes: DNAse I and alkaline phosphatase were obtained from Boehringer Mannheim Biochemicals (BMB, Indianapolis, IN); 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.).




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




A. Isolation of hepatocytes.




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


++


-, Mg


++


-free Hanks' balanced salt solution supplemented with 10 mM HEPES (pH7.4) 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 (pH7.4) and 100 U/ml collagenase (type I, Sigma Chemical Co., St. Louis, Mo.).




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 ×g 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 2×10 cells per 60-mm Primaria plates (Falcon) pre-coated with collagen (Collaborative Research).




Cultures were incubated at 37° C. in 5% CO


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 (pH7.4), 100 ug/ml gentamycin, as has been described (Lanford).




B. Detection of Liver-Specific Proteins.




Human hepatocyte cultures were maintained in serum-free medium for various period of time and labeled with [


35


S]-methionine for 24 hrs. The medium was adjusted to contain 1 mM PMSF, 1 mM EDTA, and 1% “NONIDET P-40” detergent (“NP-40”—an octylphenol-ethylene oxide condensate containing an average of 9 moles ethylene oxide per mole of phenol) 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° 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




A. HEV Infection of human hepatocytes.




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, pH7.4) for three times and changed to fresh SFM, which was changed at two day intervals thereafter.




B. Immunofluorescence staining assay.




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.




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-l% 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.




C. Reverse transcription/polymerase chain reaction (RT/PCR).




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.




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° C. for 5 minutes and subjected to reverse transcription at room temperature for 5 minutes and 42° 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×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° C. for 5 minutes to denature the MMLV-reverse transcriptase.




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×PCR buffer, overlayed with 50 ul of mineral oil, and subjected to 40 cycles of PCR in a Perkin-Elmer thermocycler (95° C.×1 minute; 52° C.×2 minutes; 72° C.×30 seconds). Ten microliters of the first-round PCR product then underwent another 40 cycles of nested PCR (95° C.×1 minute; 55° C.×2 minutes; 72° C.×30 seconds) in a total volume of 50 ul containing the internal PCR primers HEV3.2SF2 and HEV3.2SR2.




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 [


32


P-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 AntiqGns




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.




The fragments in the digest mixture were blunt-ended and ligated with EcoRI linkers. The resultant fragments were analyzed by electrophoresis (5-1OV/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° 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).




B. Cloning in an Expression Vector




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 lOX ligation buffer (above), 0.5 ml ligase (200 units), and distilled water to 5 ml. The mixture was incubated overnight at 14° C., followed by in vitro packaging, according to standard methods (Maniatis, pp. 256-268).




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).




C. Screening for HEV Recombinant Proteins




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.




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° C. The lawn was overlaid with a nitrocellulose sheet, causing transfer of expressed HEV recombinant protein from the plaques to the paper. The plate and filter were indexed for matching corresponding plate and filter positions.




The filter was washed twice in TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% “TWEEN 20” (a polyoxyethylenesorbitan monolaurate with a fatty acid composition of approximately 55% lauric acid, with a balance composed primarily of myristic, palmitic and steric acids), 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 TBST 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 NBT (50 mg/ml stock solution maintained at 4° C.) mixed with 16 ml BCIP (50 mg/ml stock solution maintained at 4° 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.




D. Screening Plating




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).




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.




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.












TABLE 4











Immunoreactivity of HEV Recombinant






Proteins: Human Sera

















Sera




Source




Stage


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.






































1


A = acute;










C = convalescent













E. Producing the 406.3-2 Antigen




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 3′ 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.




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.




F. Producing the 406.4-2 Antigen




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.




G. Preparing Antibodies




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




A. In vitro Infection




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


).




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 α-


32


P-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).




B. Neutralizing Activity of Antibody




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).




This result suggests that the HEV 3-2(M) but not HEV 4-2(M) or 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)




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.




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. 13) and HEV 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




A. Preparation of trpE-C2 peptide




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


DH5αF'host.




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° 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° 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° 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.




B. Preparation of Vaccine




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° 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.




A non-adjuvanted insoluble trpE-C2 peptide was prepared as above in section A.




C. Vaccination




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 μg 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. 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 μg trpE-C2 peptide each), and both animals showed anti-HEV by Western blotting 4 weeks later.




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

,


2


B, and


3


A, and


3


B, discussed above.




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.







22





2094 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



BURMA SEQUENCE, FIGURE 7



1
TGGAATGAAT AACATGTCTT TTGCTGCGCC CATGGGTTCG CGACCATGCG CCCTCGGCCT 60
ATTTTGTTGC TGCTCCTCAT GTTTTTGCCT ATGCTGCCCG CGCCACCGCC CGGTCAGCCG 120
TCTGGCCGCC GTCGTGGGCG GCGCAGCGGC GGTTCCGGCG GTGGTTTCTG GGGTGACCGG 180
GTTGATTCTC AGCCCTTCGC AATCCCCTAT ATTCATCCAA CCAACCCCTT CGCCCCCGAT 240
GTCACCGCTG CGGCCGGGGC TGGACCTCGT GTTCGCCAAC CCGCCCGACC ACTCGGCTCC 300
GCTTGGCGTG ACCAGGCCCA GCGCCCCGCC GTTGCCTCAC GTCGTAGACC TACCACAGCT 360
GGGGCCGCGC CGCTAACCGC GGTCGCTCCG GCCCATGACA CCCCGCCAGT GCCTGATGTC 420
GACTCCCGCG GCGCCATCTT GCGCCGGCAG TATAACCTAT CAACATCTCC CCTTACCTCT 480
TCCGTGGCCA CCGGCACTAA CCTGGTTCTT TATGCCGCCC CTCTTAGTCC GCTTTTACCC 540
CTTCAGGACG GCACCAATAC CCATATAATG GCCACGGAAG CTTCTAATTA TGCCCAGTAC 600
CGGGTTGCCC GTGCCACAAT CCGTTACCGC CCGCTGGTCC CCAATGCTGT CGGCGGTTAC 660
GCCATCTCCA TCTCATTCTG GCCACAGACC ACCACCACCC CGACGTCCGT TGATATGAAT 720
TCAATAACCT CGACGGATGT TCGTATTTTA GTCCAGCCCG GCATAGCCTC TGAGCTTGTG 780
ATCCCAAGTG AGCGCCTACA CTATCGTAAC CAAGGCTGGC GCTCCGTCGA GACCTCTGGG 840
GTGGCTGAGG AGGAGGCTAC CTCTGGTCTT GTTATGCTTT GCATACATGG CTCACTCGTA 900
AATTCCTATA CTAATACACC CTATACCGGT GCCCTCGGGC TGTTGGACTT TGCCCTTGAG 960
CTTGAGTTTC GCAACCTTAC CCCCGGTAAC ACCAATACGC GGGTCTCCCG TTATTCCAGC 1020
ACTGCTCGCC ACCGCCTTCG TCGCGGTGCG GACGGGACTG CCGAGCTCAC CACCACGGCT 1080
GCTACCCGCT TTATGAAGGA CCTCTATTTT ACTAGTACTA ATGGTGTCGG TGAGATCGGC 1140
CGCGGGATAG CCCTCACCCT GTTCAACCTT GCTGACACTC TGCTTGGCGG CCTGCCGACA 1200
GAATTGATTT CGTCGGCTGG TGGCCAGCTG TTCTACTCCC GTCCCGTTGT CTCAGCCAAT 1260
GGCGAGCCGA CTGTTAAGTT GTATACATCT GTAGAGAATG CTCAGCAGGA TAAGGGTATT 1320
GCAATCCCGC ATGACATTGA CCTCGGAGAA TCTCGTGTGG TTATTCAGGA TTATGATAAC 1380
CAACATGAAC AAGATCGGCC GACGCCTTCT CCAGCCCCAT CGCGCCCTTT CTCTGTCCTT 1440
CGAGCTAATG ATGTGCTTTG GCTCTCTCTC ACCGCTGCCG AGTATGACCA GTCCACTTAT 1500
GGCTCTTCGA CTGGCCCAGT TTATGTTTCT GACTCTGTGA CCTTGGTTAA TGTTGCGACC 1560
GGCGCGCAGG CCGTTGCCCG GTCGCTCGAT TGGACCAAGG TCACACTTGA CGGTCGCCCC 1620
CTCTCCACCA TCCAGCAGTA CTCGAAGACC TTCTTTGTCC TGCCGCTCCG CGGTAAGCTC 1680
TCTTTCTGGG AGGCAGGCAC AACTAAAGCC GGGTACCCTT ATAATTATAA CACCACTGCT 1740
AGCGACCAAC TGCTTGTCGA GAATGCCGCC GGGCACCGGG TCGCTATTTC CACTTACACC 1800
ACTAGCCTGG GTGCTGGTCC CGTCTCCATT TCTGCGGTTG CCGTTTTAGC CCCCCACTCT 1860
GCGCTAGCAT TGCTTGAGGA TACCTTGGAC TACCCTGCCC GCGCCCATAC TTTTGATGAT 1920
TTCTGCCCAG AGTGCCGCCC CCTTGGCCTT CAGGGCTGCG CTTTCCAGTC TACTGTCGCT 1980
GAGCTTCAGC GCCTTAAGAT GAAGGTGGGT AAAACTCGGG AGTTGTAGTT TATTTGCTTG 2040
TGCCCCCCTT CTTTCTGTTG CTTATTTCTC ATTTCTGCGT TCCGCGCTCC CTGA 2094






2100 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



MEXICO, FIGURE 7



2
CTGAATGAAT AACATGTGGT TTGCTGCGCC CATGGGTTCG CCACCATGCG CCCTAGGCCT 60
CTTTTGCTGT TGTTCCTCTT GTTTCTGCCT ATGTTGCCCG CGCCACCGAC CGGTCAGCCG 120
TCTGGCCGCC GTCGTGGGCG GCGCAGCGGC GGTACCGGCG GTGGTTTCTG GGGTGACCGG 180
GTTGATTCTC AGCCCTTCGC AATCCCCTAT ATTCATCCAA CCAACCCCTT TGCCCCAGAC 240
GTTGCCGCTG CGTCCGGGTC TGGACCTCGC CTTCGCCAAC CAGCCCGGCC ACTTGGCTCC 300
ACTTGGCGAG ATCAGGCCCA GCGCCCCTCC GCTGCCTCCC GTCGCCGACC TGCCACAGCC 360
GGGGCTGCGG CGCTGACGGC TGTGGCGCCT GCCCATGACA CCTCACCCGT CCCGGACGTT 420
GATTCTCGCG GTGCAATTCT ACGCCGCCAG TATAATTTGT CTACTTCACC CCTGACATCC 480
TCTGTGGCCT CTGGCACTAA TTTAGTCCTG TATGCAGCCC CCCTTAATCC GCCTCTGCCG 540
CTGCAGGACG GTACTAATAC TCACATTATG GCCACAGAGG CCTCCAATTA TGCACAGTAC 600
CGGGTTGCCC GCGCTACTAT CCGTTACCGG CCCCTAGTGC CTAATGCAGT TGGAGGCTAT 660
GCTATATCCA TTTCTTTCTG GCCTCAAACA ACCACAACCC CTACATCTGT TGACATGAAT 720
TCCATTACTT CCACTGATGT CAGGATTCTT GTTCAACCTG GCATAGCATC TGAATTGGTC 780
ATCCCAAGCG AGCGCCTTCA CTACCGCAAT CAAGGTTGGC GCTCGGTTGA GACATCTGGT 840
GTTGCTGAGG AGGAAGCCAC CTCCGGTCTT GTCATGTTAT GCATACATGG CTCTCCAGTT 900
AACTCCTATA CCAATACCCC TTATACCGGT GCCCTTGGCT TACTGGACTT TGCCTTAGAG 960
CTTGAGTTTC GCAATCTCAC CACCTGTAAC ACCAATACAC GTGTGTCCCG TTACTCCAGC 1020
ACTGCTCGTC ACTCCGCCCG AGGGGCCGAC GGGACTGCGG AGCTGACCAC AACTGCAGCC 1080
ACCAGGTTCA TGAAAGATCT CCACTTTACC GGCCTTAATG GGGTAGGTGA AGTCGGCCGC 1140
GGGATAGCTC TAACATTACT TAACCTTGCT GACACGCTCC TCGGCGGGCT CCCGACAGAA 1200
TTAATTTCGT CGGCTGGCGG GCAACTGTTT TATTCCCGCC CGGTTGTCTC AGCCAATGGC 1260
GAGCCAACCG TGAAGCTCTA TACATCAGTG GAGAATGCTC AGCAGGATAA GGGTGTTGCT 1320
ATCCCCCACG ATATCGATCT TGGTGATTCG CGTGTGGTCA TTCAGGATTA TGACAACCAG 1380
CATGAGCAGG ATCGGCCCAC CCCGTCGCCT GCGCCATCTC GGCCTTTTTC TGTTCTCCGA 1440
GCAAATGATG TACTTTGGCT GTCCCTCACT GCAGCCGAGT ATGACCAGTC CACTTACGGG 1500
TCGTCAACTG GCCCGGTTTA TATCTCGGAC AGCGTGACTT TGGTGAATGT TGCGACTGGC 1560
GCGCAGGCCG TAGCCCGATC GCTTGACTGG TCCAAAGTCA CCCTCGACGG GCGGCCCCTC 1620
CCGACTGTTG AGCAATATTC CAAGACATTC TTTGTGCTCC CCCTTCGTGG CAAGCTCTCC 1680
TTTTGGGAGG CCGGCACAAC AAAAGCAGGT TATCCTTATA ATTATAATAC TACTGCTAGT 1740
GACCAGATTC TGATTGAAAA TGCTGCCGGC CATCGGGTCG CCATTTCAAC CTATACCACC 1800
AGGCTTGGGG CCGGTCCGGT CGCCATTTCT GCGGCCGCGG TTTTGGCTCC ACGCTCCGCC 1860
CTGGCTCTGC TGGAGGATAC TTTTGATTAT CCGGGGCGGG CGCACACATT TGATGACTTC 1920
TGCCCTGAAT GCCGCGCTTT AGGCCTCCAG GGTTGTGCTT TCCAGTCAAC TGTCGCTGAG 1980
CTCCAGCGCC TTAAAGTTAA GGTGGGTAAA ACTCGGGAGT TGTAGTTTAT TTGGCTGTGC 2040
CCACCTACTT ATATCTGCTG ATTTCCTTTA TTTCCTTTTT CTCGGTCCCG CGCTCCCTGA 2100






2049 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



ORF 2, BURMA, FIGURE 7



3
ATGCGCCCTC GGCCTATTTT GTTGCTGCTC CTCATGTTTT TGCCTATGCT GCCCGCGCCA 60
CCGCCCGGTC AGCCGTCTGG CCGCCGTCGT GGGCGGCGCA GCGGCGGTTC CGGCGGTGGT 120
TTCTGGGGTG ACCGGGTTGA TTCTCAGCCC TTCGCAATCC CCTATATTCA TCCAACCAAC 180
CCCTTCGCCC CCGATGTCAC CGCTGCGGCC GGGGCTGGAC CTCGTGTTCG CCAACCCGCC 240
CGACCACTCG GCTCCGCTTG GCGTGACCAG GCCCAGCGCC CCGCCGTTGC CTCACGTCGT 300
AGACCTACCA CAGCTGGGGC CGCGCCGCTA ACCGCGGTCG CTCCGGCCCA TGACACCCCG 360
CCAGTGCCTG ATGTCGACTC CCGCGGCGCC ATCTTGCGCC GGCAGTATAA CCTATCAACA 420
TCTCCCCTTA CCTCTTCCGT GGCCACCGGC ACTAACCTGG TTCTTTATGC CGCCCCTCTT 480
AGTCCGCTTT TACCCCTTCA GGACGGCACC AATACCCATA TAATGGCCAC GGAAGCTTCT 540
AATTATGCCC AGTACCGGGT TGCCCGTGCC ACAATCCGTT ACCGCCCGCT GGTCCCCAAT 600
GCTGTCGGCG GTTACGCCAT CTCCATCTCA TTCTGGCCAC AGACCACCAC CACCCCGACG 660
TCCGTTGATA TGAATTCAAT AACCTCGACG GATGTTCGTA TTTTAGTCCA GCCCGGCATA 720
GCCTCTGAGC TTGTGATCCC AAGTGAGCGC CTACACTATC GTAACCAAGG CTGGCGCTCC 780
GTCGAGACCT CTGGGGTGGC TGAGGAGGAG GCTACCTCTG GTCTTGTTAT GCTTTGCATA 840
CATGGCTCAC TCGTAAATTC CTATACTAAT ACACCCTATA CCGGTGCCCT CGGGCTGTTG 900
GACTTTGCCC TTGAGCTTGA GTTTCGCAAC CTTACCCCCG GTAACACCAA TACGCGGGTC 960
TCCCGTTATT CCAGCACTGC TCGCCACCGC CTTCGTCGCG GTGCGGACGG GACTGCCGAG 1020
CTCACCACCA CGGCTGCTAC CCGCTTTATG AAGGACCTCT ATTTTACTAG TACTAATGGT 1080
GTCGGTGAGA TCGGCCGCGG GATAGCCCTC ACCCTGTTCA ACCTTGCTGA CACTCTGCTT 1140
GGCGGCCTGC CGACAGAATT GATTTCGTCG GCTGGTGGCC AGCTGTTCTA CTCCCGTCCC 1200
GTTGTCTCAG CCAATGGCGA GCCGACTGTT AAGTTGTATA CATCTGTAGA GAATGCTCAG 1260
CAGGATAAGG GTATTGCAAT CCCGCATGAC ATTGACCTCG GAGAATCTCG TGTGGTTATT 1320
CAGGATTATG ATAACCAACA TGAACAAGAT CGGCCGACGC CTTCTCCAGC CCCATCGCGC 1380
CCTTTCTCTG TCCTTCGAGC TAATGATGTG CTTTGGCTCT CTCTCACCGC TGCCGAGTAT 1440
GACCAGTCCA CTTATGGCTC TTCGACTGGC CCAGTTTATG TTTCTGACTC TGTGACCTTG 1500
GTTAATGTTG CGACCGGCGC GCAGGCCGTT GCCCGGTCGC TCGATTGGAC CAAGGTCACA 1560
CTTGACGGTC GCCCCCTCTC CACCATCCAG CAGTACTCGA AGACCTTCTT TGTCCTGCCG 1620
CTCCGCGGTA AGCTCTCTTT CTGGGAGGCA GGCACAACTA AAGCCGGGTA CCCTTATAAT 1680
TATAACACCA CTGCTAGCGA CCAACTGCTT GTCGAGAATG CCGCCGGGCA CCGGGTCGCT 1740
ATTTCCACTT ACACCACTAG CCTGGGTGCT GGTCCCGTCT CCATTTCTGC GGTTGCCGTT 1800
TTAGCCCCCC ACTCTGCGCT AGCATTGCTT GAGGATACCT TGGACTACCC TGCCCGCGCC 1860
CATACTTTTG ATGATTTCTG CCCAGAGTGC CGCCCCCTTG GCCTTCAGGG CTGCGCTTTC 1920
CAGTCTACTG TCGCTGAGCT TCAGCGCCTT AAGATGAAGG TGGGTAAAAC TCGGGAGTTG 1980
TAGTTTATTT GCTTGTGCCC CCCTTCTTTC TGTTGCTTAT TTCTCATTTC TGCGTTCCGC 2040
GCTCCCTGA 2049






2055 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



ORF 2, MEXICO, FIGURE 7



4
ATGCGCCCTA GGCCTCTTTT GCTGTTGTTC CTCTTGTTTC TGCCTATGTT GCCCGCGCCA 60
CCGACCGGTC AGCCGTCTGG CCGCCGTCGT GGGCGGCGCA GCGGCGGTAC CGGCGGTGGT 120
TTCTGGGGTG ACCGGGTTGA TTCTCAGCCC TTCGCAATCC CCTATATTCA TCCAACCAAC 180
CCCTTTGCCC CAGACGTTGC CGCTGCGTCC GGGTCTGGAC CTCGCCTTCG CCAACCAGCC 240
CGGCCACTTG GCTCCACTTG GCGAGATCAG GCCCAGCGCC CCTCCGCTGC CTCCCGTCGC 300
CGACCTGCCA CAGCCGGGGC TGCGGCGCTG ACGGCTGTGG CGCCTGCCCA TGACACCTCA 360
CCCGTCCCGG ACGTTGATTC TCGCGGTGCA ATTCTACGCC GCCAGTATAA TTTGTCTACT 420
TCACCCCTGA CATCCTCTGT GGCCTCTGGC ACTAATTTAG TCCTGTATGC AGCCCCCCTT 480
AATCCGCCTC TGCCGCTGCA GGACGGTACT AATACTCACA TTATGGCCAC AGAGGCCTCC 540
AATTATGCAC AGTACCGGGT TGCCCGCGCT ACTATCCGTT ACCGGCCCCT AGTGCCTAAT 600
GCAGTTGGAG GCTATGCTAT ATCCATTTCT TTCTGGCCTC AAACAACCAC AACCCCTACA 660
TCTGTTGACA TGAATTCCAT TACTTCCACT GATGTCAGGA TTCTTGTTCA ACCTGGCATA 720
GCATCTGAAT TGGTCATCCC AAGCGAGCGC CTTCACTACC GCAATCAAGG TTGGCGCTCG 780
GTTGAGACAT CTGGTGTTGC TGAGGAGGAA GCCACCTCCG GTCTTGTCAT GTTATGCATA 840
CATGGCTCTC CAGTTAACTC CTATACCAAT ACCCCTTATA CCGGTGCCCT TGGCTTACTG 900
GACTTTGCCT TAGAGCTTGA GTTTCGCAAT CTCACCACCT GTAACACCAA TACACGTGTG 960
TCCCGTTACT CCAGCACTGC TCGTCACTCC GCCCGAGGGG CCGACGGGAC TGCGGAGCTG 1020
ACCACAACTG CAGCCACCAG GTTCATGAAA GATCTCCACT TTACCGGCCT TAATGGGGTA 1080
GGTGAAGTCG GCCGCGGGAT AGCTCTAACA TTACTTAACC TTGCTGACAC GCTCCTCGGC 1140
GGGCTCCCGA CAGAATTAAT TTCGTCGGCT GGCGGGCAAC TGTTTTATTC CCGCCCGGTT 1200
GTCTCAGCCA ATGGCGAGCC AACCGTGAAG CTCTATACAT CAGTGGAGAA TGCTCAGCAG 1260
GATAAGGGTG TTGCTATCCC CCACGATATC GATCTTGGTG ATTCGCGTGT GGTCATTCAG 1320
GATTATGACA ACCAGCATGA GCAGGATCGG CCCACCCCGT CGCCTGCGCC ATCTCGGCCT 1380
TTTTCTGTTC TCCGAGCAAA TGATGTACTT TGGCTGTCCC TCACTGCAGC CGAGTATGAC 1440
CAGTCCACTT ACGGGTCGTC AACTGGCCCG GTTTATATCT CGGACAGCGT GACTTTGGTG 1500
AATGTTGCGA CTGGCGCGCA GGCCGTAGCC CGATCGCTTG ACTGGTCCAA AGTCACCCTC 1560
GACGGGCGGC CCCTCCCGAC TGTTGAGCAA TATTCCAAGA CATTCTTTGT GCTCCCCCTT 1620
CGTGGCAAGC TCTCCTTTTG GGAGGCCGGC ACAACAAAAG CAGGTTATCC TTATAATTAT 1680
AATACTACTG CTAGTGACCA GATTCTGATT GAAAATGCTG CCGGCCATCG GGTCGCCATT 1740
TCAACCTATA CCACCAGGCT TGGGGCCGGT CCGGTCGCCA TTTCTGCGGC CGCGGTTTTG 1800
GCTCCACGCT CCGCCCTGGC TCTGCTGGAG GATACTTTTG ATTATCCGGG GCGGGCGCAC 1860
ACATTTGATG ACTTCTGCCC TGAATGCCGC GCTTTAGGCC TCCAGGGTTG TGCTTTCCAG 1920
TCAACTGTCG CTGAGCTCCA GCGCCTTAAA GTTAAGGTGG GTAAAACTCG GGAGTTGTAG 1980
TTTATTTGGC TGTGCCCACC TACTTATATC TGCTGATTTC CTTTATTTCC TTTTTCTCGG 2040
TCCCGCGCTC CCTGA 2055






147 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



406.3-2, BURMA, FIGURE 7



5
ACCTTGGACT ACCCTGCCCG CGCCCATACT TTTGATGATT TCTGCCCAGA GTGCCGCCCC 60
CTTGGCCTTC AGGGCTGCGC TTTCCAGTCT ACTGTCGCTG AGCTTCAGCG CCTTAAGATG 120
AAGGTGGGTA AAACTCGGGA GTTGTAG 147






147 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



406.3-2, MEXICO, FIGURE 7



6
ACTTTTGATT ATCCGGGGCG GGCGCACACA TTTGATGACT TCTGCCCTGA ATGCCGCGCT 60
TTAGGCCTCC AGGGTTGTGC TTTCCAGTCA ACTGTCGCTG AGCTCCAGCG CCTTAAAGTT 120
AAGGTGGGTA AAACTCGGGA GTTGTAG 147






984 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



SG3, BURMA, FIGURE 7



7
GGTGCGGACG GGACTGCCGA GCTCACCACC ACGGCTGCTA CCCGCTTTAT GAAGGACCTC 60
TATTTTACTA GTACTAATGG TGTCGGTGAG ATCGGCCGCG GGATAGCCCT CACCCTGTTC 120
AACCTTGCTG ACACTCTGCT TGGCGGCCTG CCGACAGAAT TGATTTCGTC GGCTGGTGGC 180
CAGCTGTTCT ACTCCCGTCC CGTTGTCTCA GCCAATGGCG AGCCGACTGT TAAGTTGTAT 240
ACATCTGTAG AGAATGCTCA GCAGGATAAG GGTATTGCAA TCCCGCATGA CATTGACCTC 300
GGAGAATCTC GTGTGGTTAT TCAGGATTAT GATAACCAAC ATGAACAAGA TCGGCCGACG 360
CCTTCTCCAG CCCCATCGCG CCCTTTCTCT GTCCTTCGAG CTAATGATGT GCTTTGGCTC 420
TCTCTCACCG CTGCCGAGTA TGACCAGTCC ACTTATGGCT CTTCGACTGG CCCAGTTTAT 480
GTTTCTGACT CTGTGACCTT GGTTAATGTT GCGACCGGCG CGCAGGCCGT TGCCCGGTCG 540
CTCGATTGGA CCAAGGTCAC ACTTGACGGT CGCCCCCTCT CCACCATCCA GCAGTACTCG 600
AAGACCTTCT TTGTCCTGCC GCTCCGCGGT AAGCTCTCTT TCTGGGAGGC AGGCACAACT 660
AAAGCCGGGT ACCCTTATAA TTATAACACC ACTGCTAGCG ACCAACTGCT TGTCGAGAAT 720
GCCGCCGGGC ACCGGGTCGC TATTTCCACT TACACCACTA GCCTGGGTGC TGGTCCCGTC 780
TCCATTTCTG CGGTTGCCGT TTTAGCCCCC CACTCTGCGC TAGCATTGCT TGAGGATACC 840
TTGGACTACC CTGCCCGCGC CCATACTTTT GATGATTTCT GCCCAGAGTG CCGCCCCCTT 900
GGCCTTCAGG GCTGCGCTTT CCAGTCTACT GTCGCTGAGC TTCAGCGCCT TAAGATGAAG 960
GTGGGTAAAA CTCGGGAGTT GTAG 984






981 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



SG3, MEXICO, FIGURE 7



8
GCCGACGGGA CTGCGGAGCT GACCACAACT GCAGCCACCA GGTTCATGAA AGATCTCCAC 60
TTTACCGGCC TTAATGGGGT AGGTGAAGTC GGCCGCGGGA TAGCTCTAAC ATTACTTAAC 120
CTTGCTGACA CGCTCCTCGG CGGGCTCCCG ACAGAATTAA TTTCGTCGGC TGGCGGGCAA 180
CTGTTTTATT CCCGCCCGGT TGTCTCAGCC AATGGCGAGC CAACCGTGAA GCTCTATACA 240
TCAGTGGAGA ATGCTCAGCA GGATAAGGGT GTTGCTATCC CCCACGATAT CGATCTTGGT 300
GATTCGCGTG TGGTCATTCA GGATTATGAC AACCAGCATG AGCAGGATCG GCCCACCCCG 360
TCGCCTGCGC CATCTCGGCC TTTTTCTGTT CTCCGAGCAA ATGATGTACT TTGGCTGTCC 420
CTCACTGCAG CCGAGTATGA CCAGTCCACT TACGGGTCGT CAACTGGCCC GGTTTATATC 480
TCGGACAGCG TGACTTTGGT GAATGTTGCG ACTGGCGCGC AGGCCGTAGC CCGATCGCTT 540
GACTGGTCCA AAGTCACCCT CGACGGGCGG CCCCTCCCGA CTGTTGAGCA ATATTCCAAG 600
ACATTCTTTG TGCTCCCCCT TCGTGGCAAG CTCTCCTTTT GGGAGGCCGG CACAACAAAA 660
GCAGGTTATC CTTATAATTA TAATACTACT GCTAGTGACC AGATTCTGAT TGAAAATGCT 720
GCCGGCCATC GGGTCGCCAT TTCAACCTAT ACCACCAGGC TTGGGGCCGG TCCGGTCGCC 780
ATTTCTGCGG CCGCGGTTTT GGCTCCACGC TCCGCCCTGG CTCTGCTGGA GGATACTTTT 840
GATTATCCGG GGCGGGCGCA CACATTTGAT GACTTCTGCC CTGAATGCCG CGCTTTAGGC 900
CTCCAGGGTT GTGCTTTCCA GTCAACTGTC GCTGAGCTCC AGCGCCTTAA AGTTAAGGTG 960
GGTAAAACTC GGGAGTTGTA G 981






1311 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



C2, BURMA, FIGURE 7



9
AATTCAATAA CCTCGACGGA TGTTCGTATT TTAGTCCAGC CCGGCATAGC CTCTGAGCTT 60
GTGATCCCAA GTGAGCGCCT ACACTATCGT AACCAAGGCT GGCGCTCCGT CGAGACCTCT 120
GGGGTGGCTG AGGAGGAGGC TACCTCTGGT CTTGTTATGC TTTGCATACA TGGCTCACTC 180
GTAAATTCCT ATACTAATAC ACCCTATACC GGTGCCCTCG GGCTGTTGGA CTTTGCCCTT 240
GAGCTTGAGT TTCGCAACCT TACCCCCGGT AACACCAATA CGCGGGTCTC CCGTTATTCC 300
AGCACTGCTC GCCACCGCCT TCGTCGCGGT GCGGACGGGA CTGCCGAGCT CACCACCACG 360
GCTGCTACCC GCTTTATGAA GGACCTCTAT TTTACTAGTA CTAATGGTGT CGGTGAGATC 420
GGCCGCGGGA TAGCCCTCAC CCTGTTCAAC CTTGCTGACA CTCTGCTTGG CGGCCTGCCG 480
ACAGAATTGA TTTCGTCGGC TGGTGGCCAG CTGTTCTACT CCCGTCCCGT TGTCTCAGCC 540
AATGGCGAGC CGACTGTTAA GTTGTATACA TCTGTAGAGA ATGCTCAGCA GGATAAGGGT 600
ATTGCAATCC CGCATGACAT TGACCTCGGA GAATCTCGTG TGGTTATTCA GGATTATGAT 660
AACCAACATG AACAAGATCG GCCGACGCCT TCTCCAGCCC CATCGCGCCC TTTCTCTGTC 720
CTTCGAGCTA ATGATGTGCT TTGGCTCTCT CTCACCGCTG CCGAGTATGA CCAGTCCACT 780
TATGGCTCTT CGACTGGCCC AGTTTATGTT TCTGACTCTG TGACCTTGGT TAATGTTGCG 840
ACCGGCGCGC AGGCCGTTGC CCGGTCGCTC GATTGGACCA AGGTCACACT TGACGGTCGC 900
CCCCTCTCCA CCATCCAGCA GTACTCGAAG ACCTTCTTTG TCCTGCCGCT CCGCGGTAAG 960
CTCTCTTTCT GGGAGGCAGG CACAACTAAA GCCGGGTACC CTTATAATTA TAACACCACT 1020
GCTAGCGACC AACTGCTTGT CGAGAATGCC GCCGGGCACC GGGTCGCTAT TTCCACTTAC 1080
ACCACTAGCC TGGGTGCTGG TCCCGTCTCC ATTTCTGCGG TTGCCGTTTT AGCCCCCCAC 1140
TCTGCGCTAG CATTGCTTGA GGATACCTTG GACTACCCTG CCCGCGCCCA TACTTTTGAT 1200
GATTTCTGCC CAGAGTGCCG CCCCCTTGGC CTTCAGGGCT GCGCTTTCCA GTCTACTGTC 1260
GCTGAGCTTC AGCGCCTTAA GATGAAGGTG GGTAAAACTC GGGAGTTGTA G 1311






1308 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



C2, MEXICO, FIGURE 7



10
AATTCCATTA CTTCCACTGA TGTCAGGATT CTTGTTCAAC CTGGCATAGC ATCTGAATTG 60
GTCATCCCAA GCGAGCGCCT TCACTACCGC AATCAAGGTT GGCGCTCGGT TGAGACATCT 120
GGTGTTGCTG AGGAGGAAGC CACCTCCGGT CTTGTCATGT TATGCATACA TGGCTCTCCA 180
GTTAACTCCT ATACCAATAC CCCTTATACC GGTGCCCTTG GCTTACTGGA CTTTGCCTTA 240
GAGCTTGAGT TTCGCAATCT CACCACCTGT AACACCAATA CACGTGTGTC CCGTTACTCC 300
AGCACTGCTC GTCACTCCGC CCGAGGGGCC GACGGGACTG CGGAGCTGAC CACAACTGCA 360
GCCACCAGGT TCATGAAAGA TCTCCACTTT ACCGGCCTTA ATGGGGTAGG TGAAGTCGGC 420
CGCGGGATAG CTCTAACATT ACTTAACCTT GCTGACACGC TCCTCGGCGG GCTCCCGACA 480
GAATTAATTT CGTCGGCTGG CGGGCAACTG TTTTATTCCC GCCCGGTTGT CTCAGCCAAT 540
GGCGAGCCAA CCGTGAAGCT CTATACATCA GTGGAGAATG CTCAGCAGGA TAAGGGTGTT 600
GCTATCCCCC ACGATATCGA TCTTGGTGAT TCGCGTGTGG TCATTCAGGA TTATGACAAC 660
CAGCATGAGC AGGATCGGCC CACCCCGTCG CCTGCGCCAT CTCGGCCTTT TTCTGTTCTC 720
CGAGCAAATG ATGTACTTTG GCTGTCCCTC ACTGCAGCCG AGTATGACCA GTCCACTTAC 780
GGGTCGTCAA CTGGCCCGGT TTATATCTCG GACAGCGTGA CTTTGGTGAA TGTTGCGACT 840
GGCGCGCAGG CCGTAGCCCG ATCGCTTGAC TGGTCCAAAG TCACCCTCGA CGGGCGGCCC 900
CTCCCGACTG TTGAGCAATA TTCCAAGACA TTCTTTGTGC TCCCCCTTCG TGGCAAGCTC 960
TCCTTTTGGG AGGCCGGCAC AACAAAAGCA GGTTATCCTT ATAATTATAA TACTACTGCT 1020
AGTGACCAGA TTCTGATTGA AAATGCTGCC GGCCATCGGG TCGCCATTTC AACCTATACC 1080
ACCAGGCTTG GGGCCGGTCC GGTCGCCATT TCTGCGGCCG CGGTTTTGGC TCCACGCTCC 1140
GCCCTGGCTC TGCTGGAGGA TACTTTTGAT TATCCGGGGC GGGCGCACAC ATTTGATGAC 1200
TTCTGCCCTG AATGCCGCGC TTTAGGCCTC CAGGGTTGTG CTTTCCAGTC AACTGTCGCT 1260
GAGCTCCAGC GCCTTAAAGT TAAGGTGGGT AAAACTCGGG AGTTGTAG 1308






102 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



406.4-2, BURMA, FIGURE 7



11
GCCAACCCGC CCGACCACTC GGCTCCGCTT GGCGTGACCA GGCCCAGCGC CCCGCCGTTG 60
CCTCACGTCG TAGACCTACC ACAGCTGGGG CCGCGCCGCT AA 102






102 base pairs


nucleic acid


double


linear




cDNA to mRNA



NO



406.4-2, MEXICO, FIGURE 7



12
GCCAACCAGC CCGGCCACTT GGCTCCACTT GGCGAGATCA GGCCCAGCGC CCCTCCGCTG 60
CCTCCCGTCG CCGACCTGCC ACAGCCGGGG CTGCGGCGCT GA 102






48 amino acids


amino acid


linear




protein



NO



406.3-2, BURMA, FIGURE 9



13
Thr Leu Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro
1 5 10 15
Glu Cys Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val
20 25 30
Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Leu
35 40 45






48 amino acids


amino acid


linear




protein



NO



406.3-2, MEXICO, FIGURE 9



14
Thr Phe Asp Tyr Pro Gly Arg Ala His Thr Phe Asp Asp Phe Cys Pro
1 5 10 15
Glu Cys Arg Ala Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val
20 25 30
Ala Glu Leu Gln Arg Leu Lys Val Lys Val Gly Lys Thr Arg Glu Leu
35 40 45






327 amino acids


amino acid


linear




protein



NO



SG3, BURMA, FIGURE 9



15
Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe
1 5 10 15
Met Lys Asp Leu Tyr Phe Thr Ser Thr Asn Gly Val Gly Glu Ile Gly
20 25 30
Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly
35 40 45
Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr
50 55 60
Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr
65 70 75 80
Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His
85 90 95
Asp Ile Asp Leu Gly Glu Ser Arg Val Val Ile Gln Asp Tyr Asp Asn
100 105 110
Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro
115 120 125
Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala
130 135 140
Ala Glu Tyr Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr
145 150 155 160
Val Ser Asp Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala
165 170 175
Val Ala Arg Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro
180 185 190
Leu Ser Thr Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu
195 200 205
Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr
210 215 220
Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Leu Leu Val Glu Asn
225 230 235 240
Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly
245 250 255
Ala Gly Pro Val Ser Ile Ser Ala Val Ala Val Leu Ala Pro His Ser
260 265 270
Ala Leu Ala Leu Leu Glu Asp Thr Leu Asp Tyr Pro Ala Arg Ala His
275 280 285
Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Pro Leu Gly Leu Gln Gly
290 295 300
Cys Ala Phe Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Met Lys
305 310 315 320
Val Gly Lys Thr Arg Glu Leu
325






327 amino acids


amino acid


linear




protein



NO



SG3, MEXICO, FIGURE 9



16
Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe
1 5 10 15
Met Lys Asp Leu His Phe Thr Gly Leu Asn Gly Val Gly Glu Val Gly
20 25 30
Arg Gly Ile Ala Leu Thr Leu Leu Asn Leu Ala Asp Thr Leu Leu Gly
35 40 45
Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr
50 55 60
Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr
65 70 75 80
Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Val Ala Ile Pro His
85 90 95
Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn
100 105 110
Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro
115 120 125
Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala
130 135 140
Ala Glu Tyr Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr
145 150 155 160
Ile Ser Asp Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala
165 170 175
Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro
180 185 190
Leu Pro Thr Val Glu Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu
195 200 205
Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr
210 215 220
Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn
225 230 235 240
Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Arg Leu Gly
245 250 255
Ala Gly Pro Val Ala Ile Ser Ala Ala Ala Val Leu Ala Pro Arg Ser
260 265 270
Ala Leu Ala Leu Leu Glu Asp Thr Phe Asp Tyr Pro Gly Arg Ala His
275 280 285
Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Ala Leu Gly Leu Gln Gly
290 295 300
Cys Ala Phe Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Val Lys
305 310 315 320
Val Gly Lys Thr Arg Glu Leu
325






436 amino acids


amino acid


linear




protein



NO



C2, BURMA, FIGURE 9



17
Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile
1 5 10 15
Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln
20 25 30
Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu Ala Thr
35 40 45
Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Leu Val Asn Ser Tyr
50 55 60
Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu
65 70 75 80
Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn Thr Asn Thr Arg Val
85 90 95
Ser Arg Tyr Ser Ser Thr Ala Arg His Arg Leu Arg Arg Gly Ala Asp
100 105 110
Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp
115 120 125
Leu Tyr Phe Thr Ser Thr Asn Gly Val Gly Glu Ile Gly Arg Gly Ile
130 135 140
Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro
145 150 155 160
Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro
165 170 175
Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val
180 185 190
Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp
195 200 205
Leu Gly Glu Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu
210 215 220
Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val
225 230 235 240
Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr
245 250 255
Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Val Ser Asp
260 265 270
Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg
275 280 285
Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro Leu Ser Thr
290 295 300
Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly Lys
305 310 315 320
Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn
325 330 335
Tyr Asn Thr Thr Ala Ser Asp Gln Leu Leu Val Glu Asn Ala Ala Gly
340 345 350
His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro
355 360 365
Val Ser Ile Ser Ala Val Ala Val Leu Ala Pro His Ser Ala Leu Ala
370 375 380
Leu Leu Glu Asp Thr Leu Asp Tyr Pro Ala Arg Ala His Thr Phe Asp
385 390 395 400
Asp Phe Cys Pro Glu Cys Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe
405 410 415
Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys
420 425 430
Thr Arg Glu Leu
435






435 amino acids


amino acid


linear




protein



NO



C2, MEXICO, FIGURE 9



18
Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile
1 5 10 15
Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln
20 25 30
Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu Ala Thr
35 40 45
Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val Asn Ser Tyr
50 55 60
Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu
65 70 75 80
Glu Leu Glu Phe Arg Asn Leu Thr Thr Cys Asn Thr Asn Thr Arg Val
85 90 95
Ser Arg Tyr Ser Ser Thr Ala Arg His Ser Ala Arg Gly Ala Asp Gly
100 105 110
Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu
115 120 125
His Phe Thr Gly Leu Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala
130 135 140
Leu Thr Leu Leu Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr
145 150 155 160
Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val
165 170 175
Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu
180 185 190
Asn Ala Gln Gln Asp Lys Gly Val Ala Ile Pro His Asp Ile Asp Leu
195 200 205
Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln
210 215 220
Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu
225 230 235 240
Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp
245 250 255
Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Ile Ser Asp Ser
260 265 270
Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser
275 280 285
Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Pro Thr Val
290 295 300
Glu Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly Lys Leu
305 310 315 320
Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr
325 330 335
Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His
340 345 350
Arg Val Ala Ile Ser Thr Tyr Thr Thr Arg Leu Gly Ala Gly Pro Val
355 360 365
Ala Ile Ser Ala Ala Ala Val Leu Ala Pro Arg Ser Ala Leu Ala Leu
370 375 380
Leu Glu Asp Thr Phe Asp Tyr Pro Gly Arg Ala His Thr Phe Asp Asp
385 390 395 400
Phe Cys Pro Glu Cys Arg Ala Leu Gly Leu Gln Gly Cys Ala Phe Gln
405 410 415
Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Val Lys Val Gly Lys Thr
420 425 430
Arg Glu Leu
435






660 amino acids


amino acid


linear




protein



NO



ORF 2, BURMA, FIGURE 9



19
Met Arg Pro Arg Pro Ile Leu Leu Leu Leu Leu Met Phe Leu Pro Met
1 5 10 15
Leu Pro Ala Pro Pro Pro Gly Gln Pro Ser Gly Arg Arg Arg Gly Arg
20 25 30
Arg Ser Gly Gly Ser Gly Gly Gly Phe Trp Gly Asp Arg Val Asp Ser
35 40 45
Gln Pro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn Pro Phe Ala Pro
50 55 60
Asp Val Thr Ala Ala Ala Gly Ala Gly Pro Arg Val Arg Gln Pro Ala
65 70 75 80
Arg Pro Leu Gly Ser Ala Trp Arg Asp Gln Ala Gln Arg Pro Ala Val
85 90 95
Ala Ser Arg Arg Arg Pro Thr Thr Ala Gly Ala Ala Pro Leu Thr Ala
100 105 110
Val Ala Pro Ala His Asp Thr Pro Pro Val Pro Asp Val Asp Ser Arg
115 120 125
Gly Ala Ile Leu Arg Arg Gln Tyr Asn Leu Ser Thr Ser Pro Leu Thr
130 135 140
Ser Ser Val Ala Thr Gly Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu
145 150 155 160
Ser Pro Leu Leu Pro Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala
165 170 175
Thr Glu Ala Ser Asn Tyr Ala Gln Tyr Arg Val Ala Arg Ala Thr Ile
180 185 190
Arg Tyr Arg Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser
195 200 205
Ile Ser Phe Trp Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met
210 215 220
Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile
225 230 235 240
Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln
245 250 255
Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu Ala Thr
260 265 270
Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Leu Val Asn Ser Tyr
275 280 285
Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu
290 295 300
Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn Thr Asn Thr Arg Val
305 310 315 320
Ser Arg Tyr Ser Ser Thr Ala Arg His Arg Leu Arg Arg Gly Ala Asp
325 330 335
Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp
340 345 350
Leu Tyr Phe Thr Ser Thr Asn Gly Val Gly Glu Ile Gly Arg Gly Ile
355 360 365
Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro
370 375 380
Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro
385 390 395 400
Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val
405 410 415
Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp
420 425 430
Leu Gly Glu Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu
435 440 445
Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val
450 455 460
Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr
465 470 475 480
Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Val Ser Asp
485 490 495
Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg
500 505 510
Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro Leu Ser Thr
515 520 525
Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly Lys
530 535 540
Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn
545 550 555 560
Tyr Asn Thr Thr Ala Ser Asp Gln Leu Leu Val Glu Asn Ala Ala Gly
565 570 575
His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro
580 585 590
Val Ser Ile Ser Ala Val Ala Val Leu Ala Pro His Ser Ala Leu Ala
595 600 605
Leu Leu Glu Asp Thr Leu Asp Tyr Pro Ala Arg Ala His Thr Phe Asp
610 615 620
Asp Phe Cys Pro Glu Cys Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe
625 630 635 640
Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys
645 650 655
Thr Arg Glu Leu
660






659 amino acids


amino acid


linear




protein



NO



ORF 2, MEXICO, FIGURE 9



20
Met Arg Pro Arg Pro Leu Leu Leu Leu Phe Leu Leu Phe Leu Pro Met
1 5 10 15
Leu Pro Ala Pro Pro Thr Gly Gln Pro Ser Gly Arg Arg Arg Gly Arg
20 25 30
Arg Ser Gly Gly Thr Gly Gly Gly Phe Trp Gly Asp Arg Val Asp Ser
35 40 45
Gln Pro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn Pro Phe Ala Pro
50 55 60
Asp Val Ala Ala Ala Ser Gly Ser Gly Pro Arg Leu Arg Gln Pro Ala
65 70 75 80
Arg Pro Leu Gly Ser Thr Trp Arg Asp Gln Ala Gln Arg Pro Ser Ala
85 90 95
Ala Ser Arg Arg Arg Pro Ala Thr Ala Gly Ala Ala Ala Leu Thr Ala
100 105 110
Val Ala Pro Ala His Asp Thr Ser Pro Val Pro Asp Val Asp Ser Arg
115 120 125
Gly Ala Ile Leu Arg Arg Gln Tyr Asn Leu Ser Thr Ser Pro Leu Thr
130 135 140
Ser Ser Val Ala Ser Gly Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu
145 150 155 160
Asn Pro Pro Leu Pro Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala
165 170 175
Thr Glu Ala Ser Asn Tyr Ala Gln Tyr Arg Val Ala Arg Ala Thr Ile
180 185 190
Arg Tyr Arg Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser
195 200 205
Ile Ser Phe Trp Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met
210 215 220
Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile
225 230 235 240
Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln
245 250 255
Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu Ala Thr
260 265 270
Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val Asn Ser Tyr
275 280 285
Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu
290 295 300
Glu Leu Glu Phe Arg Asn Leu Thr Thr Cys Asn Thr Asn Thr Arg Val
305 310 315 320
Ser Arg Tyr Ser Ser Thr Ala Arg His Ser Ala Arg Gly Ala Asp Gly
325 330 335
Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu
340 345 350
His Phe Thr Gly Leu Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala
355 360 365
Leu Thr Leu Leu Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr
370 375 380
Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val
385 390 395 400
Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu
405 410 415
Asn Ala Gln Gln Asp Lys Gly Val Ala Ile Pro His Asp Ile Asp Leu
420 425 430
Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln
435 440 445
Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu
450 455 460
Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp
465 470 475 480
Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Ile Ser Asp Ser
485 490 495
Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser
500 505 510
Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Pro Thr Val
515 520 525
Glu Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly Lys Leu
530 535 540
Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr
545 550 555 560
Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His
565 570 575
Arg Val Ala Ile Ser Thr Tyr Thr Thr Arg Leu Gly Ala Gly Pro Val
580 585 590
Ala Ile Ser Ala Ala Ala Val Leu Ala Pro Arg Ser Ala Leu Ala Leu
595 600 605
Leu Glu Asp Thr Phe Asp Tyr Pro Gly Arg Ala His Thr Phe Asp Asp
610 615 620
Phe Cys Pro Glu Cys Arg Ala Leu Gly Leu Gln Gly Cys Ala Phe Gln
625 630 635 640
Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Val Lys Val Gly Lys Thr
645 650 655
Arg Glu Leu






33 amino acids


amino acid


linear




protein



NO



406.4-2, BURMA, FIGURE 8



21
Ala Asn Pro Pro Asp His Ser Ala Pro Leu Gly Val Thr Arg Pro Ser
1 5 10 15
Ala Pro Pro Leu Pro His Val Val Asp Leu Pro Gln Leu Gly Pro Arg
20 25 30
Arg






33 amino acids


amino acid


linear




protein



NO



406.4-2, MEXICO, FIGURE 8



22
Ala Asn Gln Pro Gly His Leu Ala Pro Leu Gly Glu Ile Arg Pro Ser
1 5 10 15
Ala Pro Pro Leu Pro Pro Val Ala Asp Leu Pro Gln Pro Gly Leu Arg
20 25 30
Arg







Claims
  • 1. A method of preventing or treating Hepatitis E Virus (HEV) infection in an individual, comprisingadministering to the individual, by parenteral injection, a vaccine composition containing antibodies capable of neutralizing HEV infection, as evidenced by the ability of the composition to block HEV infection of primary human hepatocyte cells in culture, and where said composition contains an antibody which is immunoreactive with a peptide containing the C-terminal 48 amino acids of the capsid protein encoded by the second open reading frame of the HEV genome.
  • 2. A method of claim 1, where said vaccine 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, (iii) Sequence ID No. 15, (iv) Sequence ID No. 16, (v) Sequence ID No. 17, (vi) Sequence ID No. 18, (vii) Sequence ID No. 19, and (viii) Sequence ID No. 20.
  • 3. A method of claim 2, 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:(v) Sequence ID No. 17, or (vi) Sequence ID No. 18.
  • 4. A method of claim 2, wherein the antibody composition contains an antibody which is immunoreactive with a peptide containing one of the sequences:(i) Sequence ID No. 13, or (ii) Sequence ID No. 14,and one of sequences: (iii) Sequence ID No. 15, or (iv) Sequence ID No. 16.
  • 5. A method of claim 4, wherein the antibody in the composition is immunoreactive with a peptide containing the sequence identified by Sequence ID No. 15.
Parent Case Info

This application is a continuation-in-part of co-owned, U.S. application Ser. No. 07/822,335, filed Jan. 17, 1992, now abandoned, which is a continuation-in-part of application Ser. No. 07/505,888, filed Apr. 5, 1990, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 07/420,921, filed Oct. 13, 1989, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 07/367,486, filed Jun. 16, 1989, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 07/336,672, filed Apr. 11, 1989, now abandoned, which is a continuation-in-part of U.S. application Ser. No 07/208,997, filed Jun. 17, 1988, now abandoned, all of which are herein incorporated by reference.

US Referenced Citations (3)
Number Name Date Kind
4871659 Pillot Oct 1989 A
5202430 Brian et al. Apr 1993 A
5218099 Reyes et al. Jun 1993 A
Foreign Referenced Citations (2)
Number Date Country
4200388 Jul 1992 JP
9115603 Oct 1991 WO
Non-Patent Literature Citations (10)
Entry
Cohen, “Immunization”, p. 703-706, Bosie and Clinical Immunology, Stites et al., eds., 1982, Lange Medical Publications, Los Altos, California.*
Bradley et al., PN.A.S., U.S.A., vol. 84, pp. 6277-6281, Sep. 1987.*
Purdy, et al., Archives of Virology, vol. 123, pp. 335-349, 1992.*
Sequence Comparison—Computer Generated.*
Tam et al, Virology, 185:120-131 (1991).
Huang et al, Virology, :1-9 (1992).
Bradley et al, Proc. Natl. Acad. Sci. USA, 84:6277-6281 (1987).
Purdy et al, Arch Virol, 123:335-349 (1992).
Yarbough et al, J. Virology, 65:5790-5797 (1991).
Reyes et al, Reports, :1335-1339 (1990).
Continuation in Parts (6)
Number Date Country
Parent 07/822335 Jan 1992 US
Child 07/870985 US
Parent 07/505888 Apr 1990 US
Child 07/822335 US
Parent 07/420921 Oct 1989 US
Child 07/505888 US
Parent 07/367486 Jun 1989 US
Child 07/420921 US
Parent 07/336672 Apr 1989 US
Child 07/367486 US
Parent 07/208997 Jun 1988 US
Child 07/336672 US