Recombinant proteins of a Pakistani strain of hepatitis E and their use in diagnostic methods and vaccines

Information

  • Patent Grant
  • 6207416
  • Patent Number
    6,207,416
  • Date Filed
    Wednesday, May 28, 1997
    27 years ago
  • Date Issued
    Tuesday, March 27, 2001
    23 years ago
Abstract
A strain of hepatitis E virus from Pakistan (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, is disclosed. The invention relates to the expression of the whole structural region of SAR-55, designated open reading frame 2 (ORF-2), in a eukaryotic expression system. The expressed protein is capable of forming HEV virus-like particles which can serve as an antigen in diagnostic immunoassays and as an immunogen or vaccine to protect against infection by hepatitis E.
Description




FIELD OF INVENTION




The invention is in the field of hepatitis virology. More specifically, this invention relates to recombinant proteins derived from an enterically transmitted strain of hepatitis E from Pakistan, SAR-55, and to diagnostic methods and vaccine applications which employ these proteins.




BACKGROUND OF INVENTION




Epidemics of hepatitis E, an enterically transmitted non-A/non-B hepatitis, have been reported in Asia, Africa and Central America (Balayan, M. S. (1987), Soviet Medical Reviews, Section E, Virology Reviews, Zhdanov, 0-V. M. (ed), Chur, Switzerland:


Harwood Academic Publishers


, vol. 2, 235-261; Purcell, R. G., et al. (1988) in Zuckerman, A. J. (ed), “Viral Hepatitis and Liver Disease”, New York: Alan R. Liss, 131-137; Bradley, D. W. (1990),


British Medical Bulletin,


46:442-461; Ticehurst, J. R. (1991) in Hollinger, F. B., Lemon, S. M., Margolis, H. S. (eds): “Viral Hepatitis and Liver Disease”, Williams and Wilkins, Baltimore, 501-513). Cases of sporadic hepatitis, presumed to be hepatitis E, account for up to 90° of reported hepatitis in countries where hepatitis E virus (HEV) is endemic. The need for development of a serological test for the detection of anti-HEV antibodies in the sera of infected individuals is widely recognized in the field, but the very low concentration of HEV excreted from infected humans or animals made it impossible to use such HEV as the source of antigen for serological tests and although limited success was reported in propagation of HEV in cell culture (Huang, R. T. et al. (1992),


J. Gen. Virol.,


73:1143-1148), cell culture is currently too inefficient to produce the amounts of antigen required for serological tests.




Recently, major efforts worldwide to identify viral genomic sequences associated with hepatitis E have resulted in the cloning of the genomes of a limited number of strains of HEV (Tam, A. W. et al. (1991),


Virology,


185:120-131; Tsarev, S. A. et al. (1992),


Proc. Natl. Acad. Sci. USA,


89:559-563; Fry, K. E. et al. (1992),


Virus Genes,


6:173-185). Analysis of the DNA sequences have led investigators to hypothesize that the HEV genome is organized into three open reading frames (ORFs) and to hypothesize that these ORFs encode intact HEV proteins.




A partial DNA sequence of the genome of an HEV strain from Burma (Myanmar) is disclosed in Reyes et al., 1990,


Science,


247:1335-1339. Tam et al., 1991, and Reyes et al., PCT Patent Application WO91/15603 published Oct. 17, 1991 disclose the complete nucleotide sequence and a deduced amino acid sequence of the Burma strain of HEV. These authors hypothesized that three forward open reading frames (ORFs) are contained within the sequence of this strain.




Ichikawa et al., 1991,


Microbiol. Immunol.,


35:535-543, discloses the isolation of a series of clones of 240-320 nucleotides in length upon the screening of a λgt11 expression library with sera from HEV-infected cynomolgus monkeys. The recombinant protein expressed by one clone was expressed in


E. coli


. This fusion protein is encoded by the 3′ region of ORF-2 of the Myanmar strain of HEV.




The expression of additional proteins encoded within the 3′ region of ORF-2 of a Mexican strain of HEV and of a Burmese strain of HEV is described in Yarbough et al., 1991


J. Virology,


65:5790-5797. This article describes the isolation of two CDNA clones derived from HEV. These clones encode the proteins in the 3′ region of ORF-2. The clones were expressed in


E. coli


as fusion proteins.




Purdy et al., 1992,


Archives of Virology,


123:335-349, and Favorov et al., 1992,


J. of Medical Virology,


36:246-250, disclose the expression of a larger ORF-2 protein fragment from the Burma strain in


E. coli


. These references, as well as those previously discussed, only disclose the expression of a portion of the ORF-2 gene using bacterial expression systems. Successful expression of the full-length ORF-2 protein has not been disclosed until the present invention.




Comparison of the genome organization and morphological structure of HEV to that of other viruses revealed that HEV is most closely related to the caliciviruses. Of interest, the structural proteins of caliciviruses are encoded by the 3′ portion of their genome (Neil, J. D. et al. (1991)


J. Virol.,


65:5440-5447; and Carter, M. J. et al. (1992),


J. Arch. Virol.,


122:223-235) and although there is no direct evidence that the 3′ terminal part of the HEV genome also encodes the structural proteins, expression of certain small portions of the 3′ genome region in bacterial cells resulted in production of proteins reactive with anti-HEV sera in ELISA and Western blots (Yarborough, et al., (1991); Ichikawa et al. (1991); Favorov et al. (1992) and Dawson, G. J. et al. (1992)


J. Virol. Meth;


38:175-186). However, the function of ORF-2 protein as a structural protein was not proven until the present invention.




The small proteins encoded by a portion of the ORF-2 gene have been used in immunoassay to detect antibodies to HEV in animal sera. The use of small bacterially expressed proteins as antigens in serological immunoassays has several potential drawbacks. First, the expression of these small proteins in bacterial cells often results in solubility problems and in non-specific cross-reactivity of patients' sera with


E. coli


proteins when crude


E. coli


lysates are used as antigens in immunoassays (Purdy et al. (1992)). Second, the use of Western blots as a first-line serological test for anti-HEV antibodies in routine epidemiology is impractical due to time and cost constraints. An ELISA using small-peptides derived from the 3′-terminal part of the HEV genome resulted in the detection of only 41% positives from known HEV-infected patients. Third, it has been shown that for many viruses, including Picornaviridae which is the closest family to the Caliciviridae, important antigenic and immunogenic epitopes are highly conformational (Lemon, S. M. et al. (1991), in Hollinger, F. B., Lemon, S. M., Margolis, H. S. (eds): “Viral Hepatitis and Liver Disease”, Williams and Wilkins, Baltimore, 20-24). For this reason, it is believed that expression in a eukaryotic system of a complete ORF encoding an intact HEV gene would result in production of a protein which could form HEV-virus-like particles. Such a complete ORF protein would have an immunological structure closer to that of native capsid protein(s) than would the above-noted smaller proteins which represent only portions of the structural proteins of HEV. Therefore, these complete ORF proteins would likely serve as a more representative antigen and a more efficient immunogen than the currently-used smaller proteins.




SUMMARY OF INVENTION




The present invention relates to an isolated and substantially pure preparation of a human hepatitis E viral strain SAR-55.




The invention also relates to an isolated and substantially pure preparation of the genomic RNA of the human hepatitis E viral strain SAR-55.




The invention further relates to the cDNA of the human hepatitis E viral strain SAR-55.




It is an object of this invention to provide synthetic nucleic acid sequences capable of directing production of recombinant HEV proteins, as well as equivalent natural nucleic acid sequences. Such natural nucleic acid sequences may be isolated from a cDNA or genomic library from which the gene capable of directing synthesis of the HEV proteins may be identified and isolated. For purpose of this application, nucleic acid sequence refers to RNA, DNA, cDNA or any synthetic variant thereof which encodes for protein.




The invention further relates to a method for detection of the hepatitis E virus in biological samples based on selective amplification of hepatitis E gene fragments utilizing primers derived from the SAR-55 cDNA.




The invention also relates to the use of single-stranded antisense poly-or oligonucleotides derived from the SAR-55 cDNA to inhibit the expression of hepatitis E genes.




The invention also relates to isolated and substantially purified HEV proteins and variants thereof encoded by the HEV genome of SAR-55 or encoded by synthetic nucleic acid sequences and in particular to recombinant proteins encoded by at least one complete open reading frame of HEV.




The invention also relates to the method of pre-paring recombinant HEV proteins derived from an HEV genomic sequence by cloning the nucleic acid and inserting the cDNA into an expression vector and expressing the recombinant protein in a host cell.




The invention also relates to the use of the resultant recombinant HEV proteins as diagnostic agents and as vaccines.




The present invention also encompasses methods of detecting antibodies specific for hepatitis E virus in biological samples. Such methods are useful for diagnosis of infection and disease caused by HEV, and for monitoring the progression of such disease. Such methods are also useful for monitoring the efficacy of therapeutic agents during the course of treatment of HEV infection and disease in a mammal.




This invention also relates to pharmaceutical compositions for use in prevention or treatment of Hepatitis E in a mammal.











DESCRIPTION OF FIGURES





FIG. 1

shows the recombinant vector used to express the complete ORF-2 protein of the genome of HEV strain SAR-55.





FIGS. 2A and 2B

are sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) in which cell lysates of insect cells infected with wild-type baculovirus or recombinant baculovirus (containing the gene encoding ORF-2) were either stained with Coomassie blue (A) or subjected to Western blotting with serum of an HEV-infected chimp (B). In both

FIGS. 2A and 2B

, lane 1 contains total cell lysate of noninfected SF-9 cells; lane 2 contains lysate of cells infected with wild-type baculovirus; lane 3 contains lysate of cells infected with recombinant baculovirus and lane 4 contains molecular weight markers.




FIGS.


3


A-


3


A′″ and


3


B show immunoelectron micrographs (IEM) of 30 and 20 nm virus-like particles respectively, which are formed as a result of the expression of ORF-2 protein in recombinant infected insect cells.





FIG. 4

shows the results of an ELISA using as the antigen, recombinant ORF-2 which was expressed from insect cells containing the gene encoding the complete ORF-2. Serum anti-HEV antibody levels were determined at various times following inoculation of cynomolgus monkeys with either the Mexican (Cyno-80A82, Cyno-9A97 and Cyno 83) or Pakistani (Cyno-374) strains of HEV.





FIGS. 5A-D

show the results of an ELISA using as the antigen, recombinant ORF-2 which was expressed from insect cells containing the gene encoding the complete ORF-2. Serum IgG or IgM anti-HEV levels were determined over time following inoculation of two chimpanzees with HEV.





FIGS. 6A-J

show a comparison of ELISA data obtained using as the antigen the recombinant complete ORF-2 protein derived from SAR-55 as the antigen vs. a recombinant partial ORF-2 protein derived from the Burma strain of HEV (Genelabs).





FIGS. 7A-J

show anti-HEV IgG ELISA and alanine aminotransferase (ALT) values for cynomolgus monkeys inoculated with ten-fold serial dilutions (indicated in parenthesis at the top of each panel) of a 10% fecal suspension of SAR-55 HEV. Recombinant antigens used in ELISA were: glutathione-S-transferase (GST); 3-2(M), a fusion of the 3-2 epitope [Yarbough et al., (1991) J. Virol, 65:5790-5797] and GST; SG3 (B), a fusion of 327 C-terminal amino acids of ORF-2 and GST [Yarbough et al., (1993): Assay Development of diagnostic tests for Hepatitis E in “International Symposium on Viral Hepatitis and Liver Disease. Scientific Program and Abstract Volume.” Tokyo:VHFL p. 87]; and a 55 kDa ORF-2 product directly expressed in insect cells.





FIGS. 8A-E

show anti-HEV IgM ELISA and ALT values for positive cynomolgus monkeys inoculated with ten-fold serial dilutions (indicated in parenthesis at the top of each panel) of the 10% fecal suspension of SAR-55 HEV. Recombinant antigens used in ELISA were: glutathione—S—transferase (GST); 3-2(M), a fusion of the 3-2 epitope [Yarbough et al., 1991] and (GST); SG3 (B), a fusion of 327 C-terminal amino acids of ORF-2 and GST [Yarbough et al., 1993]; and the 55 kDa ORF-2 product directly expressed in insect cells.





FIG. 9

shows an ethidium bromide stain of a 2% agarose gel on which PCR products produced from extracts of serial ten-fold dilutions (indicated at the top of each lane of the gel) of the 10% fecal suspension of the SAR-55 HEV were separated. The predicted length of the PCR products was about 640 base pairs and the column marked with an (M) contains DNA size markers.





FIG. 10

shows the pPIC9 vector used to express the complete ORF-2 protein or lower molecular weight fragments in yeast.











DETAILED DESCRIPTION OF INVENTION




The present invention relates to an isolated and substantially purified strain of hepatitis E virus (HEV) from Pakistan, SAR-55. The present invention also relates to the cloning of the viral genes encoding proteins of HEV and the expression of the recombinant proteins using an expression system. More specifically, the present invention relates to the cloning and expression of the open reading frames (ORF) of HEV derived from SAR-55.




The present invention relates to isolated HEV proteins. Preferably, the HEV proteins of the present invention are substantially homologous to, and most preferably biologically equivalent to, the native HEV proteins. By “biologically equivalent” as used throughout the specification and claims, it is meant that the compositions are capable of forming viral-like particles and are immunogenic. The HEV proteins of the present invention may also stimulate the production of protective antibodies upon injection into a mammal that would serve to protect the mammal upon challenge with a wild-type HEV. By “substantially homologous” as used throughout the ensuing specification and claims, is meant a degree of homology in the amino acid sequence to the native HEV proteins. Preferably the degree of homology is in excess of 70%, preferably in excess of 90%, with a particularly preferred group of proteins being in excess of 99% homologous with the native HEV proteins.




Preferred HEV proteins are those proteins that are encoded by the ORF genes. Of particular interest are proteins encoded by the ORF-2 gene of HEV and most particularly proteins encoded by the ORF-2 gene of the SAR-55 strain of HEV. Te preferred proteins of the present invention, encoded by the ORF-2 gene, form virus-like particles. The amino acid sequences of the ORF-1, ORF-2 and ORF-3 proteins are shown below as SEQ ID NO.: 1, SEQ ID NO.:2, and SEQ ID NO.: 3, respectively:













Met Glu Ala His Gln Phe Ile Lys Ala Pro Gly Ile Thr Thr Ala




(SEQ. ID NO.: 1)






1               5                   10                  15













Ile Glu Gln Ala Ala Leu Ala Ala Ala Asn Ser Ala Leu Ala Asn






                20                  25                  30













Ala Val Val Val Arg Pro Phe Leu Ser His Gln Gln Ile Glu Ile






                35                  40                  45













Leu Ile Asn Leu Met Gln Pro Arg Gln Leu Val Phe Arg Pro Glu






                50                  55                  60













Val Phe Trp Asn His Pro Ile Gln Arg Val Ile His Asn Glu Leu






                65                  70                  75













Glu Leu Tyr Cys Arg Ala Arg Ser Gly Arg Cys Leu Glu Ile Gly






                80                  85                  90













Ala His Pro Arg Ser Ile Asn Asp Asn Pro Asn Val Val His Arg






                95                  100                 105













Cys Phe Leu Arg Pro Ala Gly Arg Asp Val Gln Arg Trp Tyr Thr






                110                 115                 120













Ala Pro Thr Arg Gly Pro Ala Ala Asn Cys Arg Arg Ser Ala Leu






                125                 130                 135













Arg Gly Leu Pro Ala Ala Asp Arg Thr Tyr Cys Phe Asp Gly Phe






                140                 145                 150













Ser Gly Cys Asn Phe Pro Ala Glu Thr Gly Ile Ala Leu Tyr Ser






                155                 160                 165













Leu His Asp Met Ser Pro Ser Asp Val Ala Glu Ala Met Phe Arg






                170                 175                 180













His Gly Met Thr Arg Leu Tyr Ala Ala Leu His Leu Pro Pro Glu






                185                 190                 195













Val Leu Leu Pro Pro Gly Thr Tyr Arg Thr Ala Ser Tyr Leu Leu






                200                 205                 210













Ile His Asp Gly Arg Arg Val Val Val Thr Tyr Glu Gly Asp Thr






                215                 220                 225













Ser Ala Gly Tyr Asn His Asp Val Ser Asn Leu Arg Ser Trp Ile






                230                 235                 240













Arg Thr Thr Lys Val Thr Gly Asp His Pro Leu Val Ile Glu Arg






                245                 250                 255













Val Arg Ala Ile Gly Cys His Phe Val Leu Leu Leu Thr Ala Ala






                260                 265                 270













Pro Glu Pro Ser Pro Met Pro Tyr Val Pro Tyr Pro Arg Ser Thr






                275                  280                 285













Glu Val Tyr Val Arg Ser Ile Phe Gly Pro Gly Gly Thr Pro Ser






                290                 295                 300













Leu Phe Pro Thr Ser Cys Ser Thr Lys Ser Thr Phe His Ala Val






                305                 310                 315













Pro Ala His Ile Trp Asp Arg Leu Met Leu Phe Gly Ala Thr Leu






                320                 325                 330













Asp Asp Gln Ala Phe Cys Cys Ser Arg Leu Met Thr Tyr Leu Arg






                335                 340                 345













Gly Ile Ser Tyr Lys Val Thr Val Gly Thr Leu Val Ala Asn Glu






                350                 355                 360













Gly Trp Asn Ala Ser Glu Asp Ala Leu Thr Ala Val Ile Thr Ala






                365                 370                 375













Ala Tyr Leu Thr Ile Cys His Gln Arg Tyr Leu Arg Thr Gln Ala






                380                 385                 390













Ile Ser Lys Gly Met Arg Arg Leu Glu Arg Glu His Ala Gln Lys






                395                 400                 405













Phe Ile Thr Arg Leu Tyr Ser Trp Leu Phe Glu Lys Ser Gly Arg






                410                 415                 420













Asp Tyr Ile Pro Gly Arg Gln Leu Glu Phe Tyr Ala Gln Cys Arg






                425                 430                 435













Arg Trp Leu Ser Ala Gly Phe His Leu Asp Pro Arg Val Leu Val






                440                 445                 450













Phe Asp Glu Ser Ala Pro Cys His Cys Arg Thr Ala Ile Arg Lys






                455                 460                 465













Ala Val Ser Lys Phe Cys Cys Phe Met Lys Trp Leu Gly Gln Glu






                470                 475                 480













Cys Thr Cys Phe Leu Gln Pro Ala Glu Gly Val Val Gly Asp Gln






                485                 490                 495













Gly His Asp Asn Glu Ala Tyr Glu Gly Ser Asp Val Asp Pro Ala






                500                 505                 510













Glu Ser Ala Ile Ser Asp Ile Ser Gly Ser Tyr Val Val Pro Gly






                515                 520                 525













Thr Ala Leu Gln Pro Leu Tyr Gln Ala Leu Asp Leu Pro Ala Glu






                530                 535                 540













Ile Val Ala Arg Ala Gly Arg Leu Thr Ala Thr Val Lys Val Ser






                545                 550                 555













Gln Val Asp Gly Arg Ile Asp Cys Glu Thr Leu Leu Gly Asn Lys






                560                 565                 570













Thr Phe Arg Thr Ser Phe Val Asp Gly Ala Val Leu Glu Thr Asn






                575                 580                 585













Gly Pro Glu Arg His Asn Leu Ser Phe Asp Ala Ser Gln Ser Thr






                590                 595                 600













Met Ala Ala Gly Pro Phe Ser Leu Thr Tyr Ala Ala Ser Ala Ala






                605                 610                 615













Gly Leu Glu Val Arg Tyr Val Ala Ala Gly Leu Asp His Arg Ala






                620                 625                 630













Val Phe Ala Pro Gly Val Ser Pro Arg Ser Ala Pro Gly Glu Val






                635                 640                 645













Thr Ala Phe Cys Ser Ala Leu Tyr Arg Phe Asn Arg Glu Ala Gln






                650                 655                 660













Arg Leu Ser Leu Thr Gly Asn Phe Trp Phe His Pro Glu Gly Leu






                665                 670                 675













Leu Gly Pro Phe Ala Pro Phe Ser Pro Gly His Val Trp Glu Ser






                680                 685                 690













Ala Asn Pro Phe Cys Gly Glu Ser Thr Leu Tyr Thr Arg Thr Trp






                695                 700                 705













Ser Glu Val Asp Ala Val Pro Ser Pro Ala Gln Pro Asp Leu Gly






                710                 715                 720













Phe Thr Ser Glu Pro Ser Ile Pro Ser Arg Ala Ala Thr Pro Thr






                725                 730                 735













Pro Ala Ala Pro Leu Pro Pro Pro Ala Pro Asp Pro Ser Pro Thr






                740                 745                 750













Leu Ser Ala Pro Ala Arg Gly Glu Pro Ala Pro Gly Ala Thr Ala






                755                 760                 765













Arg Ala Pro Ala Ile Thr His Gln Thr Ala Arg His Arg Arg Leu






                770                 775                 780













Leu Phe Thr Tyr Pro Asp Gly Ser Lys Val Phe Ala Gly Ser Leu






                785                 790                 795













Phe Glu Ser Thr Cys Thr Trp Leu Val Asn Ala Ser Asn Val Asp






                800                 805                 810













His Arg Pro Gly Gly Gly Leu Cys His Ala Phe Tyr Gln Arg Tyr






                815                 820                 825













Pro Ala Ser Phe Asp Ala Ala Ser Phe Val Met Arg Asp Gly Ala






                830                 835                 840













Ala Ala Tyr Thr Leu Thr Pro Arg Pro Ile Ile His Ala Val Ala






                845                 850                 855













Pro Asp Tyr Arg Leu Glu His Asn Pro Lys Arg Leu Glu Ala Ala






                860                 865                 870













Tyr Arg Glu Thr Cys Ser Arg Leu Gly Thr Ala Ala Tyr Pro Leu






                875                 880                 885













Leu Gly Thr Gly Ile Tyr Gln Val Pro Ile Gly Pro Ser Phe Asp






                890                 895                 900













Ala Trp Glu Arg Asn His Arg Pro Gly Asp Glu Leu Tyr Leu Pro






                905                 910                 915













Glu Leu Ala Ala Arg Trp Phe Glu Ala Asn Arg Pro Thr Cys Pro






                920                 925                 930













Thr Leu Thr Ile Thr Glu Asp Val Ala Arg Thr Ala Asn Leu Ala






                935                 940                 945













Ile Glu Leu Asp Ser Ala Thr Asp Val Gly Arg Ala Cys Ala Gly






                950                 955                 960













Cys Arg Val Thr Pro Gly Val Val Gln Tyr Gln Phe Thr Ala Gly






                965                 970                 975













Val Pro Gly Ser Gly Lys Ser Arg Ser Ile Thr Gln Ala Asp Val






                980                 985                 990













Asp Val Val Val Val Pro Thr Arg Glu Leu Arg Asn Ala Trp Arg






                995                 1000               1005













Arg Arg Gly Phe Ala Ala Phe Thr Pro His Thr Ala Ala Arg Val






                1010                1015               1020













Thr Gln Gly Arg Arg Val Val Ile Asp Glu Ala Pro Ser Leu Pro






                1025                1030               1035













Pro His Leu Leu Leu Leu His Met Gln Arg Ala Ala Thr Val His






                1040                1045               1050













Leu Leu Gly Asp Pro Asn Gln Ile Pro Ala Ile Asp Phe Glu His






                1055                1060               1065













Ala Gly Leu Val Pro Ala Ile Arg Pro Asp Leu Ala Pro Thr Ser






                1070                1075               1080













Trp Trp His Val Thr His Arg Cys Pro Ala Asp Val Cys Glu Leu






                1080                1090               1095













Ile Arg Gly Ala Tyr Pro Met Ile Gln Thr Thr Ser Arg Val Leu






                1100                1105               1110













Arg Ser Leu Phe Trp Gly Glu Pro Ala Val Gly Gln Lys Leu Val






                1115                1120               1125













Phe Thr Gln Ala Ala Lys Ala Ala Asn Pro Gly Ser Val Thr Val






                1130                1135               1140













His Glu Ala Gln Gly Ala Thr Tyr Thr Glu Thr Thr Ile Ile Ala






                1145                1150               1155













Thr Ala Asp Ala Arg Gly Leu Ile Gln Ser Ser Arg Ala His Ala






                1160                1165               1170













Ile Val Ala Leu Thr Arg His Thr Glu Lys Cys Val Ile Ile Asp






                1175                1180               1185













Ala Pro Gly Leu Leu Arg Glu Val Gly Ile Ser Asp Ala Ile Val






                1190                1195               1200













Asn Asn Phe Phe Leu Ala Gly Gly Glu Ile Gly His Gln Arg Pro






                1205                1210               1215













Ser Val Ile Pro Arg Gly Asn Pro Asp Ala Asn Val Asp Thr Leu






                1220                1225               1230













Ala Ala Phe Pro Pro Ser Cys Glu Ile Ser Ala Phe His Glu Leu






                1235                1240               1245













Ala Glu Glu Leu Gly His Arg Pro Ala Pro Val Ala Ala Val Leu






                1250                1255               1260













Pro Pro Cys Pro Glu Leu Glu Gln Gly Leu Leu Tyr Leu Pro Gln






                1265                1270               1275













Glu Leu Thr Thr Cys Asp Ser Val Val Thr Phe Glu Leu Thr Asp






                1280                1285               1290













Ile Val His Cys Arg Met Ala Ala Pro Ser Gln Arg Lys Ala Val






                1295                1300               1305













Leu Ser Thr Leu Val Gly Arg Tyr Gly Arg Arg Thr Lys Leu Tyr






                1310                1315               1320













Asn Ala Ser His Ser Asp Val Arg Asp Ser Leu Ala Arg Phe Ile






                1325                1330               1335













Pro Ala Ile Gly Pro Val Gln Val Thr Thr Cys Glu Leu Tyr Glu






                1340                1345               1350













Leu Glu Glu Ala Met Val Glu Lys Gly Gln Asp Gly Ser Ala Val






                1355                1360               1365













Leu Glu Leu Asp Leu Cys Ser Arg Asp Val Ser Arg Ile Thr Phe






                1370                1375               1380













Phe Gln Lys Asp Cys Asn Lys Phe Thr Thr Gly Glu Thr Ile Ala






                1385                1390               1395













His Gly Lys Val Gly Gln Gly Ile Ser Ala Trp Ser Lys Thr Phe






                1400                1405               1410













Cys Ala Leu Phe Gly Pro Trp Phe Arg Ala Ile Glu Lys Ala Ile






                1415                1420               1425













Leu Ala Leu Leu Pro Gln Gly Val Phe Tyr Gly Asp Ala Phe Asp






                1430                1435               1440













Asp Thr Val Phe Ser Ala Ala Val Ala Ala Ala Lys Ala Ser Met






                1445                1450               1455













Val Phe Glu Asn Asp Phe Ser Glu Phe Asp Ser Thr Gln Asn Asn






                1460                1465               1470













Phe Ser Leu Gly Leu Glu Cys Ala Ile Met Glu Glu Cys Gly Met






                1475                1480               1485













Pro Gln Trp Leu Ile Arg Leu Tyr His Leu Ile Arg Ser Ala Trp






                1490                1495               1500













Ile Leu Gln Ala Pro Lys Glu Ser Leu Arg Gly Phe Trp Lys Lys






                1505                1510               1515













His Ser Gly Glu Pro Gly Thr Leu Leu Trp Asn Thr Val Trp Asn






                1520                1525               1530













Met Ala Val Ile Thr His Cys Tyr Asp Phe Arg Asp Leu Gln Val






                1535                1540               1545













Ala Ala Phe Lys Gly Asp Asp Ser Ile Val Leu Cys Ser Glu Tyr






                1550                1555               1560













Arg Gln Ser Pro Gly Ala Ala Vai Leu Ile Ala Gly Cys Gly Leu






                1565                1570               1575













Lys Leu Lys Val Asp Phe Arg Pro Ile Gly Leu Tyr Ala Gly Val






                1580                1585               1590













Val Val Ala Pro Gly Leu Gly Ala Leu Pro Asp Val Val Arg Phe






                1595                1600               1605













Ala Gly Arg Leu Thr Glu Lys Asn Trp Gly Pro Gly Pro Glu Arg






                1610                1615               1620













Ala Glu Gln Leu Arg Leu Ala Val Ser Asp Phe Leu Arg Lys Leu






                1625                1630               1635













Thr Asn Val Ala Gln Met Cys Val Asp Val Val Ser Arg Val Tyr






                1640                1645               1650













Gly Val Ser Pro Gly Leu Val His Asn Leu Ile Glu Met Leu Gln






                1655                1660                665













Ala Val Ala Asp Gly Lys Ala His Phe Thr Glu Ser Val Lys Pro






                1670                1675               1680













Val Leu Asp Leu Thr Asn Ser Ile Leu Cys Arg Val Giu






                1685                1690




















Met Arg Pro Arg Pro Ile Leu Leu Leu Leu Leu Met Phe Leu Pro




(SEQ. ID NO.: 2)






1               5                   10                   15













Met Leu Pro Ala Pro Pro Pro Gly Gln Pro Ser Gly Arg Arg Arg






                20                  25                   30













Gly Arg Arg Ser Gly Gly Ser Gly Gly Gly Phe Trp Gly Asp Arg






                35                  40                   45













Val Asp Ser Gln Pro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn






                50                  55                   60













Pro Phe Ala Pro Asp Val Thr Ala Ala Ala Gly Ala Gly Pro Arg






                65                  70                   75













Val Arg Gln Pro Ala Arg Pro Leu Gly Ser Ala Trp Arg Asp Gln






                80                  85                   90













Ala Gln Arg Pro Ala Ala Ala Ser Arg Arg Arg Pro Thr Thr Ala






                95                  100                 105













Gly Ala Ala Pro Leu Thr Ala Val Ala Pro Ala His Asp Thr Pro






                110                 115                 120













Pro Val Pro Asp Val Asp Ser Arg Gly Ala Ile Leu Arg Arg Gln






                125                 130                 135













Tyr Asn Leu Ser Thr Ser Pro Leu Thr Ser Ser Val Ala Thr Gly






                140                 145                 150













Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu Ser Pro Leu Leu Pro






                155                 160                 165













Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala Thr Glu Ala Ser






                170                 175                 180













Asn Tyr Ala Gln Tyr Arg Val Ala Arg Ala Thr Ile Arg Tyr Arg






                185                 190                 195













Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser Ile Ser






                200                 205                 210













Phe Tyr Pro Gln Thr Thr Thr Thr Pro Thr Ser Val ABp Met Asn






                215                 220                 225













Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile






                230                 235                 240













Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn






                245                 250                 255













Gln Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu






                260                 265                 270













Ala Thr Ser Gly Glu Val Met Leu Cys Ile His Gly Ser Pro Val






                275                 280                 285













Asn Ser Tyr Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu






                290                 295                 300













Asp Phe Ala Leu Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn






                305                 310                 315













Thr Asn Thr Arg Val Ser Arg Tyr Ser Ser Thr Ala Arg His Arg






                320                 325                 330













Leu Arg Arg Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala






                335                 340                 345













Ala Thr Arg Phe Met Lys Asp Leu Tyr Phe Thr Ser Thr Asn Gly






                350                 355                 360













Val Gly Glu Ile Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu






                365                 370                 375













Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser






                380                 385                 390













Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn






                395                 400                 405













Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln






                410                 415                 420













Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp Leu Gly Glu






                425                 430                 435













Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp






                440                 445                 450













Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu






                455                 460                 465













Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr






                470                 475                 480













Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Val Ser






                485                 490                 495













Asp Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val






                500                 505                 510













Ala Arg Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro






                515                 520                 525













Leu Ser Thr Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro






                530                 535                 540













Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala






                545                 550                 555













Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Leu Leu






                560                 565                 570













Val Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr






                575                 580                 585













Thr Ser Leu Gly Ala Gly Pro Val Ser Ile Ser Ala Val Ala Val






                590                 595                 600













Leu Ala Pro His Ser Val Leu Ala Leu Leu Glu Asp Thr Met Asp






                605                 610                 615













Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys






                620                 625                 630













Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val Ala






                635                 640                 645













Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Leu






                650                 655                 660




















Met Asn Asn Met Ser Phe Ala Ala Pro Met Gly Ser Arg Pro Cys




(SEQ. ID NO.: 3)






1               5                    10                  15













Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys






                20                   25                  30













Cys Pro Arg His Arg Pro Val Ser Arg Leu Ala Ala Val Val Gly






                35                   40                  45













Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu






                50                   55                  60













Ile Leu Ser Pro Ser Gln Ser Pro Ile Phe Ile Gln Pro Thr Pro






                65                   70                  75













Ser Pro Pro Met Ser Pro Leu Arg Pro Gly Leu Asp Leu Val Phe






                80                   85                  90













Ala Asn Pro Pro Asp His Ser Ala Pro Leu Gly Val Thr Arg Pro






                95                  100                 105













Ser Ala Pro Pro Leu Pro His Val Val Asp Leu Pro Gln Leu Gly






                110                 115                 120













Pro Arg Arg











The three-letter abbreviations follow the conventional amino acid shorthand for the twenty naturally occurring amino acids.




The preferred recombinant HEV proteins consist of at least one ORF protein. Other recombinant proteins made up of more than one of the same or different ORF proteins may be made to alter the biological properties of the protein. It is contemplated that additions, substitutions or deletions of discrete amino acids or of discrete sequences of amino acids may enhance the biological activity of the HEV proteins.




The present invention is also a nucleic acid sequence which is capable of directing the production of the above-discussed HEV protein or proteins substantially homologous to the HEV proteins. This nucleic acid sequence, designated SAR-55, is set forth below as SEQ ID NO.: 4 and was deposited with the American Type Culture Collection (ATCC) on Sep. 17, 1992 (ATCC accession number 75302).



















AGGCAGACCA CATATGTGGT CGATGCCATG GAGGCCCATC




40














AGTTTATCAA GGCTCCTGGC ATCACTACTG CTATTGAGCA




80













GGCTGCTCTA GCAGCGGCCA ACTCTGCCCT TGCGAATGCT




120













GTGGTAGTTA GGCCTTTTCT CTCTCACCAG CAGATTGAGA




160













TCCTTATTAA CCTAATGCAA CCTCGCCAGC TTGTTTTCCG




200













CCCCGAGGTT TTCTGGAACC ATCCCATCCA GCGTGTTATC




240













CATAATGAGC TGGAGCTTTA CTGTCGCGCC CGCTCCGGCC




280













GCTGCCTCGA AATTGGTGCC CACCCCCGCT CAATAAATGA




320













CAATCCTAAT GTGGTCCACC GTTGCTTCCT CCGTCCTGCC




360













GGGCGTGATG TTCAGCGTTG GTATACTGCC CCTACCCGCG




400













GGCCGGCTGC TAATTGCCGG CGTTCCGCGC TGCGCGGGCT




440













CCCCGCTGCT GACCGCACTT ACTGCTTCGA CGGGTTTTCT




480













GGCTGTAACT TTCCCGCCGA GACGGGCATC GCCCTCTATT




520













CTCTCCATGA TATGTCACCA TCTGATGTCG CCGAGGCTAT




560













GTTCCGCCAT GGTATGACGC GGCTTTACGC TGCCCTCCAC




600













CTCCCGCCTG AGGTCCTGTT GCCCCCTGGC ACATACCGCA




640













CCGCGTCGTA CTTGCTGATC CATGACGGCA GGCGCGTTGT




680













GGTGACGTAT GAGGGTGACA CTAGTGCTGG TTATAACCAC




720













GATGTTTCCA ACCTGCGCTC CTGGATTAGA ACCACTAAGG




760













TTACCGGAGA CCACCCTCTC GTCATCGAGC GGGTTAGGGC




800













CATTGGCTGC CACTTTGTCC TTTTACTCAC GGCTGCTCCG




840













GAGCCATCAC CTATGCCCTA TGTCCCTTAC CCCCGGTCTA




880













CCGAGGTCTA TGTCCGATCG ATCTTCGGCC CGGGTGGCAC




920













CCCCTCCCTA TTTCCAACCT CATGCTCCAC CAAGTCGACC




960













TTCCATGCTG TCCCTGCCCA TATCTGGGAC CGTCTCATGT




1000













TGTTCGGGGC CACCCTAGAT GACCAAGCCT TTTGCTGCTC




1040













CCGCCTAATG ACTTACCTCC GCGGCATTAG CTACAAGGTT




1080













ACTGTGGGCA CCCTTGTTGC CAATGAAGGC TGGAACGCCT




1120













CTGAGGACGC TCTTACAGCT GTCATCACTG CCGCCTACCT




1160













TACCATCTGC CACCAGCGGT ACCTCCGCAC TCAGGCTATA




1200













TCTAAGGGGA TGCGTCGCCT GGAGCGGGAG CATGCTCAGA




1240













AGTTTATAAC ACGCCTCTAC AGTTGGCTCT TTGAGAAGTC




1280













CGGCCGTGAT TATATCCCCG GCCGTCAGTT GGAGTTCTAC




1320













GCTCAGTGTA GGCGCTGGCT CTCGGCCGGC TTTCATCTTG




1360













ACCCACGGGT GTTGGTTTTT GATGAGTCGG CCCCCTGCCA




1400













CTGTAGGACT GCGATTCGTA AGGCGGTCTC AAAGTTTTGC




1440













TGCTTTATGA AGTGGCTGGG CCAGGAGTGC ACCTGTTTTC




1480













TACAACCTGC AGAAGGCGTC GTTGGCGACC AGGGCCATGA




1520













CAACGAGGCC TATGAGGGGT CTGATGTTGA CCCTGCTGAA




1560













TCCGCTATTA GTGACATATC TGGGTCCTAC GTAGTCCCTG




1600













GCACTGCCCT CCAACCGCTT TACCAAGCCC TTGACCTCCC




1640













CGCTGAGATT GTGGCTCGTG CAGGCCGGCT GACCGCCACA




1680













GTAAAGGTCT CCCAGGTCGA CGGGCGGATC GATTGTGAGA




1720













CCCTTCTCGG TAATAAAACC TTCCGCACGT CGTTTGTTGA




1760













CGGGGCGGTT TTAGAGACTA ATGGCCCAGA GCGCCACAAT




1800













CTCTCTTTTG ATGCCAGTCA GAGCACTATG GCCGCCGGCC




1840













CTTTCAGTCT CACCTATGCC GCCTCTGCTG CTGGGCTGGA




1880













GGTGCGCTAT GTCGCCGCCG GGCTTGACCA CCGGGCGGTT




1920













TTTGCCCCCG GCGTTTCACC CCGGTCAGCC CCTGGCGAGG




1960













TCACCGCCTT CTGTTCTGCC CTATACAGGT TTAATCGCGA




2000













GGCCCAGCGC CTTTCGCTGA CCGGTAATTT TTGGTTCCAT




2040













CCTGAGGGGC TCCTTGGCCC CTTTGCCCCG TTTTCCCCCG




2080













GGCATGTTTG GGAGTCGGCT AATCCATTCT GTGGCGAGAG




2120













CACACTTTAC ACCCGCACTT GGTCGGAGGT TGATGCTGTT




2160













CCTAGTCCAG CCCAGCCCGA CTTAGGTTTT ACATCTGAGC




2200













CTTCTATACC TAGTAGGGCC GCCACACCTA CCCCGGCGGC




2240













CCCTCTACCC CCCCCTGCAC CGGATCCTTC CCCTACTCTC




2280













TCTGCTCCGG CGCGTGGTGA GCCGGCTCCT GGCGCTACCG




2320













CCAGGGCCCC AGCCATAACC CACCAGACGG CCCGGCATCG




2360













CCGCCTGCTC TTTACCTACC CGGATGGCTC TAAGGTGTTC




2400













GCCGGCTCGC TGTTTGAGTC GACATGTACC TGGCTCGTTA




2440













ACGCGTCTAA TGTTGACCAC CGCCCTGGCG GTGGGCTCTG




2480













TCATGCATTT TACCAGAGGT ACCCCGCCTC CTTTGATGCT




2520













GCCTCTTTTG TGATGCGCGA CGGCGCGGCC GCCTACACAT




2560













TAACCCCCCG GCCAATAATT CATGCCGTCG CTCCTGATTA




2600













TAGGTTGGAA CATAACCCAA AGAGGCTTGA GGCTGCCTAC




2640













CGGGAGACTT GCTCCCGCCT CGGTACCGCT GCATACCCAC




2680













TCCTCGGGAC CGGCATATAC CAGGTGCCGA TCGGTCCCAG




2720













TTTTGACGCC TGGGAGCGGA ATCACCGCCC CGGGGACGAG




2760













TTGTACCTTC CTGAGCTTGC TGCCAGATGG TTCGAGGCCA




2800













ATAGGCCGAC CTGCCCAACT CTCACTATAA CTGAGGATGT




2840













TGCGCGGACA GCAAATCTGG CTATCGAACT TGACTCAGCC




2880













ACAGACGTCG GCCGGGCCTG TGCCGGCTGT CGAGTCACCC




2920













CCGGCGTTGT GCAGTACCAG TTTACCGCAG GTGTGCCTGG




2960













ATCCGGCAAG TCCCGCTCTA TTACCCAAGC CGACGTGGAC




3000













GTTGTCGTGG TCCCGACCCG GGAGTTGCGT AATGCCTGGC




3040













GCCGCCGCGG CTTCGCTGCT TTCACCCCGC ACACTGCGGC




3080













TAGAGTCACC CAGGGGCGCC GGGTTGTCAT TGATGAGGCC




3120













CCGTCCCTTC CCCCTCATTT GCTGCTGCTC CACATGCAGC




3160













GGGCCGCCAC CGTCCACCTT CTTGGCGACC CGAATCAGAT




3200













CCCAGCCATC GATTTTGAGC ACGCCGGGCT CGTTCCCGCC




3240













ATCAGGCCCG ATTTGGCCCC CACCTCCTGG TGGCATGTTA




3280













CCCATCGCTG CCCTGCGGAT GTATGTGAGC TAATCCGCGG




3320













CGCATACCCT ATGATTCAGA CCACTAGTCG GGTCCTCCGG




3360













TCGTTGTTCT GGGGTGAGCC CGCCGTTGGG CAGAAGCTAG




3400













TGTTCACCCA GGCGGCTAAG GCCGCCAACC CCGGTTCAGT




3440













GACGGTCCAT GAGGCACAGG GCGCTACCTA CACAGAGACT




3480













ACCATCATTG CCACGGCAGA TGCTCGAGGC CTCATTCAGT




3520













CGTCCCGAGC TCATGCCATT GTTGCCTTGA CGCGCCACAC




3560













TGAGAAGTGC GTCATCATTG ACGCACCAGG CCTGCTTCGC




3600













GAGGTGGGCA TCTCCGATGC AATCGTTAAT AACTTTTTCC




3640













TTGCTGGTGG CGAAATTGGC CACCAGCGCC CATCTGTTAT




3680













CCCTCGCGGC AATCCTGACG CCAATGTTGA CACCTTGGCT




3720













GCCTTCCCGC CGTCTTGCCA GATTAGCGCC TTCCATCAGT




3760













TGGCTGAGGA GCTTGGCCAC AGACCTGCCC CTGTCGCGGC




3800













TGTTCTACCG CCCTGCCCTG AGCTTGAACA GGGCCTTCTC




3840













TACCTGCCCC AAGAACTCAC CACCTGTGAT AGTGTCGTAA




3880













CATTTGAATT AACAGATATT GTGCATTGTC GTATGGCCGC




3920













CCCGAGCCAG CGCAAGGCCG TGCTGTCCAC GCTCGTGGGC




3960













CGTTATGGCC GCCGCACAAA GCTCTACAAT GCCTCCCACT




4000













CTGATGTTCG CGACTCTCTC GCCCGTTTTA TCCCGGCCAT




4040













TGGCCCCGTA CAGGTTACAA CCTGTGAATT GTACGAGCTA




4080













GTGGAGGCCA TGGTCGAGAA GGGCCAGGAC GGCTCCGCCG




4120













TCCTTGAGCT CGACCTTTGT AGCCGCGACG TGTCCAGGAT




4160













CACCTTCTTC CAGAAAGATT GTAATAAATT CACCACGGGG




4200













GAGACCATCG CCCATGGTAA AGTGGGCCAG GGCATTTCGG




4240













CCTGGAGTAA GACCTTCTGT GCCCTTTTCG GCCCCTGGTT




4280













CCGTGCTATT GAGAAGGCTA TCCTGGCCCT GCTCCCTCAG




4320













GGTGTGTTTT ATGGGGATGC CTTTGATGAC ACCGTCTTCT




4360













CGGCGGCTGT GGCCGCAGCA AAGGCATCCA GAATGACTTT




4400













TCTGAGTTTG ATTCCACCCA GAATAATTTT TCCTTGGGCC




4440













TAGAGTGTGC TATTATGGAG GAGTGTGGGA TGCCGCAGTG




4480













GCTCATCCGC TTGTACCACC TTATAAGGTC TGCGTGGATT




4520













CTGCAGGCCC CGAAGGAGTC CCTGCGAGGG TTTTGGAAGA




4560













AACACTCCGG TGAGCCCGGC ACCCTTCTGT GGAATACTGT




4600













CTGGAACATG GCCGTTATCA CCCACTGTTA TGATTTCCGC




4640













GATCTGCAGG TGGCTGCCTT TAAAGGTGAT GATTCGATAG




4680













TGCTTTGCAG TGAGTACCGT CAGAGCCCAG GGGCTGCTGT




4720













CCTGATTGCT GGCTGTGGCC TAAAGTTGAA GGTGGATTTC




4760













CGTCCGATTG GTCTGTATGC AGGTGTTGTG GTGGCCCCCG




4800













GCCTTGGCGC GCTTCCTGAT GTCGTGCGCT TCGCCGGTCG




4840













GCTTACTGAG AAGAATTGGG GCCCTGGCCC CGAGCGGGCG




4880













GAGCAGCTCC GCCTCGCTGT GAGTGATTTT CTCCGCAAGC




4920













TCACGAATGT AGCTCAGATG TGTGTGGATG TTGTCTCTCG




4960













TGTTTATGGG GTTTCCCCTG GGCTCGTTCA TAACCTGATT




5000













GGCATGCTAC AGGCTGTTGC TGATGGCAAG GCTCATTTCA




5040













CTGAGTCAGT GAAGCCAGTG CTTGACCTGA CAAATTCAAT




5080













TCTGTGTCGG GTGGAATGAA TAACATGTCT TTTGCTGCGC




5120













CCATGGGTTC GCGACCATGC GCCCTCGGCC TATTTTGCTG




5160













TTGCTCCTCA TGTTTCTGCC TATGCTGCCC GCGCCACCGC




5200













CCGGTCAGCC GTCTGGCCGC CGTCGTGGGC GGCGCAGCGG




5240













CGGTTCCGGC GGTGGTTTCT GGGGTGACCG GGTTGATTCT




5280













CAGCCCTTCG CAATCCCCTA TATTCATCCA ACCAACCCCT




5320













TCGCCCCCGA TGTCACCGCT GCGGCCGGGG CTGGACCTCG




5360













TGTTCGCCAA CCCGCCCGAC CACTCGGCTC CGCTTGGCGT




5400













GACCAGGCCC AGCGCCCCGC CGCTGCCTCA CGTCGTAGAC




5440













CTACCACAGC TGGGGCCGCG CCGCTAACCG CGGTCGCTCC




5480













GGCCCATGAC ACCCCGCCAG TGCCTGATGT TGACTCCCGC




5520













GGCGCCATCC TGCGCCGGCA GTATAACCTA TCAACATCTC




5560













CCCTCACCTC TTCCGTGGCC ACCGGCACAA ATTTGGTTCT




5600













TTACGCCGCT CCTCTTAGCC CGCTTCTACC CCTCCAGGAC




5640













GGCACCAATA CTCATATAAT GGCTACAGAA GCTTCTAATT




5680













ATGCCCAGTA CCGGGTTGCT CGTGCCACAA TTCGCTACCG




5720













CCCGCTGGTC CCCAACGCTG TTGGTGGCTA CGCTATCTCC




5760













ATTTCGTTCT GGCCACAGAC CACCACCACC CCGACGTCCG




5800













TTGACATGAA TTCAATAACC TCGACGGATG TCCGTATTTT




5840













AGTCCAGCCC GGCATAGCCT CCGAGCTTGT TATTCCAAGT




5880













GAGCGCCTAC ACTATCGCAA CCAAGGTTGG CGCTCTGTTG




5920













AGACCTCCGG GGTGGCGGAG GAGGAGGCCA CCTCTGGTCT




5960













TGTCATGCTC TGCATACATG GCTCACCTGT AAATTCTTAT




6000













ACTAATACAC CCTATACCGG TGCCCTCGGG CTGTTGGACT




6040













TTGCCCTCGA ACTTGAGTTC CGCAACCTCA CCCCCGGTAA




6080













TACCAATACG CGGGTCTCGC GTTACTCCAG CACTGCCCGT




6120













CACCGCCTTC GTCGCGGTGC AGATGGGACT GCCGAGCTCA




6160













CCACCACGGC TGCTACTCGC TTCATGAAGG ACCTCTATTT




6200













TACTAGTACT AATGGTGTTG GTGAGATCGG CCGCGGGATA




6240













GCGCTTACCC TGTTTAACCT TGCTGACACC CTGCTTGGCG




6280













GTCTACCGAC AGAATTGATT TCGTCGGCTG GTGGCCAGCT




6320













GTTCTACTCT CGCCCCGTCG TCTCAGCCAA TGGCGAGCCG




6360













ACTGTTAAGC TGTATACATC TGTGGAGAAT GCTCAGCAGG




6400













ATAAGGGTAT TGCAATCCCG CATGACATCG ACCTCGGGGA




6440













ATCCCGTGTA GTTATTCAGG ATTATGACAA CCAACATGAG




6480













CAGGACCGAC CGACACCTTC CCCAGCCCCA TCGCGTCCTT




6520













TTTCTGTCCT CCGAGCTAAC GATGTGCTTT GGCTTTCTCT




6560













CACCGCTGCC GAGTATGACC AGTCCACTTA CGGCTCTTCG




6600













ACCGGCCCAG TCTATGTCTC TGACTCTGTG ACCTTGGTTA




6640













ATGTTGCGAC CGGCGCGCAG GCCGTTGCCC GGTCACTCGA




6680













CTGGACCAAG GTCACACTTG ATGGTCGCCC CCTTTCCACC




6720













ATCCAGCAGT ATTCAAAGAC CTTCTTTGTC CTGCCGCTCC




6760













GCGGTAAGCT CTCCTTTTGG GAGGCAGGAA CTACTAAAGC




6800













CGGGTACCCT TATAATTATA ACACCACTGC TAGTGACCAA




6840













CTGCTCGTTG AGAATGCCGC TGGGCATCGG GTTGCTATTT




6880













CCACCTACAC TACTAGCCTG GGTGCTGGCC CCGTCTCTAT




6920













TTCCGCGGTT GCTGTTTTAG CCCCCCACTC TGTGCTAGCA




6960













TTGCTTGAGG ATACCATGGA CTACCCTGCC CGCGCCCATA




7000













CTTTCGATGA CTTCTGCCCG GAGTGCCGCC CCCTTGGCCT




7040













CCAGGGTTGT GCTTTTCAGT CTACTGTCGC TGAGCTTCAG




7080













CGCCTTAAGA TGAAGGTGGG TAAAACTCGG GAGTTATAGT




7120













TTATTTGCTT GTGCCCCCCT TCTTTCTGTT GCTTATTT




7168














The abbreviations used for the nucleotides are those standardly used in the art.




The sequence in one direction has been designated by convention as the “plus” sequence since it is the protein-encoding strand of RNA viruses and this is the sequence shown above as SEQ ID. NO.:4.




The deduced amino acid sequences of the open reading frames of SAR-55 have SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3. ORF-1 starts at nucleotide 28 of SEQ. ID NO. 4 and extends 5078 nucleotides; ORF-2 starts at nucleotide 5147 of SEQ. ID NO. 4 and extends 1979 nucleotides; and ORF-3 starts at nucleotide 5106 of SEQ. ID NO. 4 and extends 368 nucleotides.




Variations are contemplated in the DNA sequence which will result in a DNA sequence that is capable of directing production of analogs of the ORF-2 protein. By “analogs of the ORF-2 protein” as used throughout the specification and claims is meant a protein having an amino acid sequence substantially identical to a sequence specifically shown herein where one or more of the residues shown in the sequences presented herein have been substituted with a biologically equivalent residue such that the resultant protein (i.e. the “analog”) is capable of forming viral particles and is immunogenic. It should be noted that the DNA sequence set forth above represents a preferred embodiment of the present invention. Due to the degeneracy of the genetic code, it is to be understood that numerous choices of nucleotides may be made that will lead to a DNA sequence capable of directing production of the instant ORF proteins or their analogs. As such, DNA sequences which are functionally equivalent to the sequences set forth above or which are functionally equivalent to sequences that would direct production of analogs of the ORF proteins produced pursuant to the amino acid sequence set forth above, are intended to be encompassed within the present invention.




The present invention relates to a method for detecting the hepatitis E virus in biological samples based on selective amplification of hepatitis E gene fragments. Preferably, this method utilizes a pair of single-stranded primers derived from non-homologous regions of opposite strands of a DNA duplex fragment, which in turn is derived from a hepatitis E virus whose genome contains a region homologous to the SAR-55 sequence shown in SEQ ID No.: 4. These primers can be used in a method following the process for amplifying selected nucleic acid sequences as defined in U.S. Pat. No. 4,683,202.




The present invention also relates to the use of single-stranded antisense poly-or oligonucleotides derived from sequences homologous to the SAR-55 cDNA to inhibit the expression of hepatitis E genes. These anti-sense poly-or oligonucleotides can be either DNA or RNA. The targeted sequence is typically messenger RNA and more preferably, a signal sequence required for processing or translation of the RNA. The antisense poly-or oligonucleotides can be conjugated to a polycation such as polylysine as disclosed in Lemaitre, M. et al. (1989) Proc Natl Acad Sci USA 84:648-652; and this conjugate can be administered to a mammal in an amount sufficient to hybridize to and inhibit the function of the messenger RNA.




The present invention includes a recombinant DNA method for the manufacture of HEV proteins, preferably a protein composed of at least one ORF protein, most preferably at least one ORF-2 protein. The recombinant ORF protein may be composed of one ORF protein or a combination of the same or different ORF proteins. A natural or synthetic nucleic acid sequence may be used to direct production of the HEV proteins. In one embodiment of the invention, the method comprises:




(a) preparation of a nucleic acid sequence capable of directing a host organism to produce a protein of HEV;




(b) cloning the nucleic acid sequence into a vector capable of being transferred into and replicated in a host organism, such vector containing operational elements for the nucleic acid sequence;




(c) transferring the vector containing the nucleic acid and operational elements into a host organism capable of expressing the protein;




(d) culturing the host organism under conditions appropriate for amplification of the vector and expression of the protein; and




(e) harvesting the protein.




In another embodiment of the invention, the method for the recombinant DNA synthesis of a protein encoded by nucleic acids of HEV, preferably encoding by at least one ORF of HEV or a combination of the same or different ORF proteins, most preferably encoding at least one ORF-2 nucleic acid sequence, comprises:




(a) culturing a transformed or transfected host organism containing a nucleic acid sequence capable of directing the host organism to produce a protein, under conditions such that the protein is produced, said protein exhibiting substantial homology to a native HEV protein isolated from HEV having the amino acid sequence according to SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, or combinations thereof.




In one embodiment, the RNA sequence of the viral genome of HEV strain SAR-55 was isolated and cloned to cDNA as follows. Viral RNA is extracted from a biological sample collected from cynomolgus monkeys infected with SAR-55 and the viral RNA is then reverse transcribed and amplified by polymerase chain reaction using primers complementary to the plus or minus strands of the genome of a strain of HEV from Burma (Tam et al. (1991)) or the SAR-55 genome. The PCR fragments are subcloned into pBR322 or pGEM-32 and the double-stranded PCR fragments were sequenced.




The vectors contemplated for use in the present invention include any vectors into which a nucleic acid sequence as described above can be inserted, along with any preferred or required operational elements, and which vector can then be subsequently transferred into a host organism and replicated in such organism. Preferred vectors are those whose restriction sites have been well documented and which contain the operational elements preferred or required for transcription of the nucleic acid sequence.




The “operational elements” as discussed herein include at least one promoter, at least one operator, at least one leader sequence, at least one terminator codon, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the vector nucleic acid. In particular, it is contemplated that such vectors will contain at least one origin of replication recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid sequence.




In construction of the cloning vector of the present invention, it should additionally be noted that multiple copies of the nucleic acid sequence and its attendant operational elements may be inserted into each vector. In such an embodiment, the host organism would produce greater amounts per vector of the desired HEV protein. The number of multiple copies of the DNA sequence which may be inserted into the vector is limited only by the ability of the resultant vector due to its size, to be transferred into and replicated and transcribed in an appropriate host microorganism.




In another embodiment, restriction digest fragments containing a coding sequence for HEV proteins can be inserted into a suitable expression vector that functions in prokaryotic or eukaryotic cells. By suitable is meant that the vector is capable of carrying and expressing a complete nucleic acid sequence coding for HEV proteins, preferably at least one complete ORF protein. In the case or ORF-2, the expressed protein should form viral-like particles. Preferred expression vectors are those that function in a eukaryotic cell. Examples of such vectors include but are not limited to vectors useful for expression in yeast (e.g. pPIC9 vector-Invitrogen) vaccinia virus vectors, adenovirus or herpesviruses, preferably the baculovirus transfer vector, pBlueBac. Preferred vectors are p63-2, which contains the complete ORF-2 gene, and P59-4, which contains the complete ORF-3 and ORF-2 genes. These vectors were deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA on Sep. 10, 1992 and have accession numbers 75299 (P63-2) and 75300 (P59-4). Example 1 illustrates the cloning of the ORF-2 gene into pBlueBac to produce p63-2. This method includes digesting the genome of HEV strain SAR-55 with the restriction enzymes NruI and BglII, inserting a polylinker containing BlnI and BglII sites into the unique NheI site of the vector and inserting the NruI-BglII ORF-2 fragment in BlnI-BglII PBlueBac using an adapter.




In yet another embodiment, the selected recombinant expression vector may then be transfected into a suitable eukaryotic cell system for purposes of expressing the recombinant protein. Such eukaryotic cell systems include, but are not limited to, yeast, and cell lines such as HeLa, MRC-5 or Cv-1. One preferred eukaryotic cell system is SF9 insect cells. One preferred method involves use of the pBlueBac expression vector where the insect cell line SF9 is cotransfected with recombinant pBlueBac and AcMNPV baculovirus DNA by the Ca precipitation method.




The expressed recombinant protein may be detected by methods known in the art which include Coomassie blue staining and Western blotting using sera containing anti-HEV antibody as shown in Example 2. Another method is the detection of virus-like particles by immunoelectron microscopy as shown in Example 3.




In a further embodiment, the recombinant protein expressed by the SF9 cells can be obtained as a crude lysate or it can be purified by standard protein purification procedures known in the art which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity, and immunoaffinity chromatography and the like. In the case of immunoaffinity chromatography, the recombinant protein may be purified by passage through a column containing a resin which has bound thereto antibodies specific for the ORF protein. An example of a protocol for the purification of a recombinantly expressed HEV ORF protein is provided in Example 10.




In another embodiment, the expressed recombinant proteins of this invention can be used in immunoassays for diagnosing or prognosing hepatitis E in a mammal including but not limited to humans, chimpanzees, Old World monkeys, New World monkeys, other primates and the like. In a preferred embodiment, the immunoassay is useful in diagnosing hepatitis E infection in humans. Immunoassays using the HEV proteins, particularly the ORF proteins, and especially ORF 2 proteins, provide a highly specific, sensitive and reproducible method for diagnosing HEV infections, in contrast to immunoassays which utilize partial ORF proteins. Immunoassays of the present invention may be a radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay and the like. Standard techniques known in the art for ELISA are described in


Methods in Immunodiagnosis,


2nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, 1980 and Campbell et al.,


Methods of Immunology


, W. A. Benjamin, Inc., 1964, both of which are incorporated herein by reference. Such assays may be a direct, indirect, competitive, or noncompetitive immunoassay as described in the art. (Oellerich, M. 1984.


J.Clin. Chem. Clin


. BioChem. 22: 895-904) Biological samples appropriate for such detection assays include, but are not limited to, tissue biopsy extracts, whole blood, plasma, serum, cerebrospinal fluid, pleural fluid, urine and the like.




In one embodiment, test serum is reacted with a solid phase reagent having surface-bound recombinant HEV protein as an antigen, preferably an ORF protein or combination of different ORF proteins such as ORF-2 and ORF-3, ORF-l and ORF-3 and the like. More preferably, the HEV protein is an ORF-2 protein that forms virus-like particles. The solid surface reagent can be prepared by known techniques for attaching protein to solid support material. These attachment methods include non-specific adsorption of the protein to the support or covalent attachment of the protein to a reactive group on the support. After reaction of the antigen with anti-HEV antibody, unbound serum components are removed by washing and the antigen-antibody complex is reacted with a secondary antibody such as labelled anti-human antibody. The label may be an enzyme which is detected by incubating the solid support in the presence of a suitable fluorimetric or calorimetric reagent. Other detectable labels may also be used, such as radiolabels or colloidal gold, and the like.




In a preferred embodiment, protein expressed by the recombinant vector pBlueBac containing the entire ORF-2 sequence of SAR-55 is used as a specific binding agent to detect anti-HEV antibodies, preferably IgG or IgM antibodies. Examples 4 and 5 show the results of an ELISA in which the solid phase reagent has recombinant ORF-2 as the surface antigen. This protein, encoded by the entire ORF-2 nucleic acid sequence, is superior to the partial ORF-2 proteins, as it is reactive with more antisera from different primate species infected with HEV than are partial antigens of ORF-2. The protein of the present invention is also capable of detecting antibodies produced in response to different strains of HEV but does not detect antibodies produced in response to Hepatitis A, B, C or Hepatitis D.




The HEV protein and analogs may be prepared in the form of a kit, alone, or in combinations with other reagents such as secondary antibodies, for use in immunoassays.




The recombinant HEV proteins, preferably an ORF protein or combination of ORF proteins, more preferably an ORF-2 protein and substantially homologous proteins and analogs of the invention can be used as a vaccine to protect mammals against challenge with Hepatitis E. The vaccine, which acts as an immunogen, may be a cell, cell lysate from cells transfected with a recombinant expression vector or a culture supernatant containing the expressed protein. Alternatively, the immunogen is a partially or substantially purified recombinant protein. While it is possible for the immunogen to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation.




The formulations of the present invention, both for veterinary and for human use, comprise an immunogen as described above, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well-known in the pharmaceutical art.




All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.




Formulations suitable for intravenous intramuscular, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which are preferably isotonic with the blood of the recipient. Such formulations may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. These may be present in unit or multi-dose containers, for example, sealed ampoules or vials.




The formulations of the present invention may incorporate a stabilizer. Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of 0.11-10,000 parts by weight per part by weight of immunogen. If two or more stabilizers are to be used, their total amount is preferably within the range specified above. These stabilizers are used in aqueous solutions at the appropriate concentration and pH. The specific osmotic pressure of such aqueous solutions is generally in the range of 0.1-3.0 osmoles, preferably in the range of 0.8-1.2. The pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8. In formulating the immunogen of the present invention, anti-adsorption agent may be used.




Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved through the use of polymer to complex or absorb the proteins or their derivatives. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyester, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled-release preparations is to incorporate the proteins, protein analogs or their functional derivatives, into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.




When oral preparations are desired, the compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.




The proteins of the present invention may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition as described above.




Vaccination can be conducted by conventional methods. For example, the immunogen can be used in a suitable diluent such as saline or water, or complete or incomplete adjuvants. Further, the immunogen may or may not be bound to a carrier to make the protein immunogenic. Examples of such carrier molecules include but are not limited to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like. The immunogen can be administered by any route appropriate for antibody production such as intravenous, intraperitoneal, intramuscular, subcutaneous, and the like. The immunogen may be administered once or at periodic intervals until a significant titer of anti-HEV antibody is produced. The antibody may be detected in the serum using an immunoassay.




In yet another embodiment, the immunogen may be nucleic acid sequence capable of directing host organism synthesis of an HEV ORF protein. Such nucleic acid sequence may be inserted into a suitable expression vector by methods known to those skilled in the art. Expression vectors suitable for producing high efficiency gene transfer in vivo include, but are not limited to, retroviral, adenoviral and vaccinia viral vectors. Operational elements of such expression vectors are disclosed previously in the present specification and are known to one skilled in the art. Such expression vectors can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally or orally.




In an alternative embodiment, direct gene transfer may be accomplished via intramuscular injection of, for example, plasmid-based eukaryotic expression vectors containing a nucleic acid sequence capable of directing host organism synthesis HEC ORF protein(s). Such an approach has previously been utilized to produce the hepatitis B surface antigen in vivo and resulted in an antibody response to the surface antigen (Davis, H. L. et al. (1993)


Human Molecular Genetics,


2:1847-1851; see also Davis et al. (1993)


Human Gene Therapy,


4:151-159 and 733-740).




When the immunogen is a partially or substantially purified recombinant HEV ORF protein, dosages effective to elicit a protective antibody response against HEV range from about 2 μg to about 100 μg. A more preferred range is from about 5 μg to about 70 μg and a most preferred range is from about 10 μg to about 60 μg.




Dosages of HEV-ORF protein—encoding nucleic acid sequence effective to elicit a protective antibody response against HEV range from about 1 to about 5000 μg; a more preferred range being about 300 to about 1000 μg.




The expression vectors containing a nucleic acid sequence capable of directing host organism synthesis of an HEV ORF protein(s) may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition as described above.




The administration of the immunogen of the present invention may be for either a prophylactic or therapeutic purpose. When provided prophylactically, the immunogen is provided in advance of any exposure to HEV or in advance of any symptom due to HEV infection. The prophylactic administration of the immunogen serves to prevent or attenuate any subsequent infection of HEV in a mammal. When provided therapeutically, the immunogen is provided at (or shortly after) the onset of the infection or at the onset of any symptom of infection or disease caused by HEV. The therapeutic administration of the immunogen serves to attenuate the infection or disease.




A preferred embodiment is a vaccine prepared using recombinant ORF-2 protein expressed by the ORF-2 sequence of HEV strain SAR-55 and equivalents thereof. Since the recombinant ORF-2 protein has already been demonstrated to be reactive with a variety of HEV-positive sera, their utility in protecting against a variety of HEV strains is indicated.




In addition to use as a vaccine, the compositions can be used to prepare antibodies to HEV virus-like particles. The antibodies can be used directly as antiviral agents. To prepare antibodies, a host animal is immunized using the virus particles or, as appropriate, non-particle antigens native to the virus particle are bound to a carrier as described above for vaccines. The host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the virus particle. The gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art. The antibodies are substantially free of many of the adverse side effects which may be associated with other anti-viral agents such as drugs.




The antibody compositions can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the Fc portion of a foreign species antibody or using an antibody of the same species as the host animal, for example, the use of antibodies from human/human hybridomas. Humanized antibodies (i.e., nonimmunogenic in a human) may be produced, for example, by replacing an immunogenic portion of an antibody with a corresponding, but noninmunogenic portion (i.e., chimeric antibodies). Such chimeric antibodies may contain the reactive or antigen binding portion of an antibody from one species and the Fc portion of an antibody (nonimmunogenic) from a different species. Examples of chimeric antibodies, include but are not limited to, non-human mammal-human chimeras, rodent-human chimeras, murine-human and rat-human chimeras (Robinson et al., International Patent Application 184,187; Taniguchi M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al., 1987 Proc. Natl. Acad. Sci. USA 84:3439; Nishimura et al., 1987 Canc. Res. 47:999; Wood et al., 1985 Nature 314:446; Shaw et al., 1988 J. Natl. Cancer Inst. 80: 15553, all incorporated herein by reference).




General reviews of “humanized” chimeric antibodies are provided by Morrison S., 1985 Science 229:1202 and by Oi et al., 1986 BioTechniques 4:214.




Suitable “humanized” antibodies can be alternatively produced by CDR or CEA substitution (Jones et al., 1986 Nature 321:552; Verhoeyan et al., 1988 Science 239:1534; Biedleret al. 1988 J. Immunol. 141:4053, all incorporated herein by reference).




The antibodies or antigen binding fragments may also be produced by genetic engineering. The technology for expression of both heavy and light cain genes in


E. coli


is the subject the PCT patent applications; publication number WO 901443, WO901443, and WO 9014424 and in Huse et al., 1989 Science 246:1275-1281.




The antibodies can also be used as a means of enhancing the immune response. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation period of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells. Thus, antibodies reactive with the HEV virus particle can be passively administered alone or in conjunction with another anti-viral agent to a host infected with an HEV to enhance the effectiveness of an antiviral drug.




Alternatively, anti-HEV antibodies can be induced by administering anti-idiotype antibodies as immunogens. Conveniently, a purified anti-HEV antibody preparation prepared as described above is used to induce anti-idiotype antibody in a host animal. The composition is administered to the host animal in a suitable diluent. Following administration, usually repeated administration, the host produces anti-idiotype antibody. To eliminate an immunogenic response to the Fc region, antibodies produced by the same species as the host animal can be used or the FC region of the administered antibodies can be removed. Following induction of anti-idiotype antibody in the host animal, serum or plasma is removed to provide an antibody composition. The composition can be purified as described above for anti-HEV antibodies, or by affinity chromatography using anti-HEV antibodies bound to the affinity matrix. The anti-idiotype antibodies produced are similar in conformation to the authentic HEV-antigen and may be used to prepare an HEV vaccine rather than using an HEV particle antigen.




When used as a means of inducing anti-HEV virus antibodies in an animal, the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutaneously or the like in an effective concentration in a physiologically suitable diluent with or without adjuvant. One or more booster injections may be desirable.




The HEV derived proteins of the invention are also intended for use in producing antiserum designed for pre- or post-exposure prophylaxis. Here an HEV protein, or mixture of proteins is formulated with a suitable adjuvant and administered by injection to human volunteers, according to known methods for producing human antisera. Antibody response to the injected proteins is monitored, during a several-week period following immunization, by periodic serum sampling to detect the presence of anti-HEV serum antibodies, using an immunoassay as described herein.




The antiserum from immunized individuals may be administered as a pre-exposure prophylactic measure for individuals who are at risk of contracting infection. The antiserum is also useful in treating an individual post-exposure, analogous to the use of high titer antiserum against hepatitis B virus for post-exposure prophylaxis. Of course, those of skill in the art would readily understand that immune globulin (HEV immune globulin) purified from the antiserum of immunized individuals using standard techniques may be used as a pre-exposure prophylactic measure or in treating individuals post-exposure.




For both in vivo use of antibodies to HEV virus-like particles and proteins and anti-idiotype antibodies and diagnostic use, it may be preferable to use monoclonal antibodies. Monoclonal anti-virus particle antibodies or anti-idiotype antibodies can be produced as follows. The spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. (Goding, J. W. 1983. Monoclonal Antibodies: Principles and Practice, Pladermic Press, Inc., NY, N.Y., pp. 56-97). To produce a human-human hybridoma, a human lymphocyte donor is selected. A donor known to be infected with HEV (where infection has been shown for example by the presence of anti-virus antibodies in the blood or by virus culture) may serve as a suitable lymphocyte donor. Lymphocytes can be isolated from a peripheral blood sample or spleen cells may be used if the donor is subject to splenectomy. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas. Primary in vitro immunization with peptides can also be used in the generation of human monoclonal antibodies.




Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity. For monoclonal anti-virus particle antibodies, the antibodies must bind to HEV virus particles. For monoclonal anti-idiotype antibodies, the antibodies must bind to anti-virus particle antibodies. Cells producing antibodies of the desired specificity are selected.




The above described antibodies and antigen binding fragments thereof may be supplied in kit form alone, or as a pharmaceutical composition for in vivo use. The antibodies may be used for therapeutic uses, diagnostic use in immunoassays or as an immunoaffinity agent to purify ORF proteins as described herein.




MATERIAL




The materials used in the Examples were as follows:




Primates. Chimpanzee (Chimp) (


Pan troglodytes


). Old world monkeys: cynomolgus monkeys (Cyno) (


Macaca fascicularis


), rhesus monkeys (Rhesus) (


M. mulatta


), pigtail monkeys (PT) (


M. nemestrina


), and African green monkeys (AGM) (


Cercopithecus aethiops


). New World monkeys: mustached tamarins (Tam) (


Saguinus mystax


), squirrel monkeys (SQM) (


Saimiri sciureus


) and owl monkeys (OWL) (


Aotus trivigatus


). Primates were housed singly under conditions of biohazard containment. The housing, maintenance and care of the animals met or exceeded all requirements for primate husbandry.




Most animals were inoculated intravenously with HEV, strain SAR-55 contained in 0.5 ml of stool suspension diluted in fetal calf serum as described in Tsarev, S. A. et al. (1992),


Proc. Natl. Acad. Sci USA,


89:559-563; and Tsarev, S. A. et al. (1993),


J. Infect. Dis. (


167:1302-1306). Chimp-1313 and 1310 were inoculated with a pool of stools collected from 7 Pakistani hepatitis E patients.




Serum samples were collected approximately twice a week before and after inoculation. Levels of the liver enzymes serum alanine amino transferase (ALT), isocitrate dehydrogenase (ICD), and gamma glutamyl transferase (GGT) were assayed with commercially available tests (Medpath Inc., Rockville, Md.). Serologic tests were performed as described above.




EXAMPLE 1




Identification of the DNA Sequence of the Genome of HEV Strain SAR-55.




Preparation of Virus RNA Template for PCR. Bile from an HEV-infected cynomolgus monkey (10 μl), 20% (wt/vol) SDS (to a final concentration of 1%), proteinase K (10 mg/ml; to a final concentration of 1 mg/ml), 1 μl of tRNA (10 mg/ml), and 3 μl of 0.5 M EDTA were mixed in a final volume of 250 μl and incubated for 30 min. at 55° C. Total nucleic acids were extracted from bile twice with phenol/chloroform, 1:1 (vol/vol), at 65° C. and once with chloroform, then precipitated by ethanol, washed with 95% ethanol, and used for RT-PCR. RT-PCR amplification of HEV RNA from feces and especially from sera was more efficient when RNA was more extensively purified. Serum (100 μl) or a 10% fecal suspension (200 μl) was treated as above with proteinase K. After a 30-min incubation, 300 μl of CHAOS buffer (4.2 M guanidine thiocyanate/0.5 N-lauroylsarocosine/0.025 M Tris-HCL, pH 8.0) was added. Nucleic acids were extracted twice with phenol/chloroform at 65° C. followed by chloroform extraction at room temperature. Then 7.5 M ammonium acetate (225 μl) was added to the upper phase and nucleic acids were precipitated with 0.68 ml of 2-propanol. The pellet was dissolved in 300 ul CHAOS buffer and 100 ul of H


2


O was added. Chloroform extraction and 2-propanol precipitation were repeated. Nucleic acids were dissolved in water, precipitated with ethanol, washed with 95% ethanol, and used for RT-PCR.




Primers




Ninety-four primers, 21-40 nucleotides (nt) long, and complementary to plus or minus strands of the genome of a strain of HEV from Burma (BUR-121) (Tam, A. W. et al. (1991),


Virology,


185:120-131) or the SAR-55 genome were synthesized using an Applied Biosystems model 391 DNA synthesizer.




The sequences of these 94 primers are shown below starting with SEQ. ID NO. 5 and continuing to SEQ. ID NO. 98:




HEV Primer List


















ORF







Primer




Region




Seguence


























D 3042 B




1




ACATTTGAATTCACAGACAT




(SEQ. ID. NO. 5)








TGTGC






R 3043 B




1




ACACAGATCTGAGCTACATT




(SEQ. ID. NO. 6)








CGTGAG






D 3044 B




1




AAAGGGATCCATGGTGTTTG




(SEQ. ID. NO. 7)








AGAATGZ






R 3045 B




1




ACTCACTGCAGAGCACTATC




(SEQ. ID. NO. 8)








GAATC






R 261 S




1




CGGTAAACTGGTACTGCACA




(SEQ. ID. NO. 9)








AC






D 260 S




1




AAGTCCCGCTCTATTACCCA




(SEQ. ID. NO. 10)








AG






D 259 S




1




ACCCACGGGTGTTGGTTTTT




(SEQ. ID. NO. 11)








G






R 255 S




1




TTCTTGGGGCAGGTAGAGAA




(SEQ. ID. NO. 12)








G






R 254 S




2




TTATTGAATTCATGTCAACG




(SEQ. ID. NO. 13)








GACGTC






D 242 S




1




AATAATTCATGCCGTCGCTC




(SEQ. ID. NO. 14)








C






R 241 S




1




AAGCTCAGGAAGGTACAACT




(SEQ. ID. NO. 15)








C






R 231 S




1




AAATCGATGGCTGGGATCTG




(SEQ. ID. NO. 16)








ATTC






R 230 S




1




GAGGCATTGTAGAGCTTTGT




(SEQ. ID. NO. 17)








G






D 229 S




1




GATGTTGCACGGACAGCAAA




(SEQ. ID. NO. 18)








TC






D 228 S




1




ATCTCCGATGCAATCGTTAA




(SEQ. ID. NO. 19)








TAAC






D 227 B




1




TAATCCATTCTGTGGCGAGA




(SEQ. ID. NO. 20)








G






R 218 B




2




AAGTGTGACCTTGGTCCAGT




(SEQ. ID. NO. 21)








C






D 217 B




2




TTGCTCGTGCCACAATTCGC




(SEQ. ID. NO. 22)








TAC






D 211 B




1




CATTTCACTGAGTCAGTGAA




(SEQ. ID. NO. 23)








GZ






D 202 B




2




TAATTATAACACCACTGCTA




(SEQ. ID. NO. 24)








G






R 201 B




2




GATTGCAATACCCTTATCCT




(SEQ. ID. NO. 25)








G






R 200 S




1




ATTAAACCTGTATAGGGCAG




(SEQ. ID. NO. 26)








AAC






R 199 S




1




AAGTTCGATAGCCAGATTTG




(SEQ. ID. NO. 27)








C






R 198 S




2




TCATGTTGGTTGTCATAATC




(SEQ. ID. NO. 28)








C






R 193 B




1




GATGACGCACTTCTCAGTGT




(SEQ. ID. NO. 29)








G






R 192 B




1




AGAACAACGAACGGAGAAC




(SEQ. ID. NO. 30)






D 191 B




1




AGATCCCAGCCATCGACTTT




(SEQ. ID. NO. 31)








G






R 190 S




2




TAGTAGTGTAGGTGGAAATA




(SEQ. ID. NO. 32)








G






D 189 B




2




GTGTGGTTATTCAGGATTAT




(SEQ. ID. NO. 33)








G






D 188 B




2




ACTCTGTGACCTTGGTTAAT




(SEQ. ID. NO. 34)








G






R 187 S




2




AACTCAAGTTCGAGGGCAAA




(SEQ. ID. NO. 35)








G






D 186 S




2




CGCTTACCCTGTTTAACCTT




(SEQ. ID. NO. 36)








G






D 185 B




2,3




ATCCCCTATATTCATCCAAC




(SEQ. ID. NO. 37)








CAAC






D 184 S




2,3




CTCCTCATGTTTCTGCCTAT




(SEQ. ID; NC. 38)








G






R 181 S




2




GCCAGAACGAAATGGAGATA




(SEQ. ID. NO. 39)








GC






R 180 B




i




CTCAGACATAAAACCTAAGT




(SEQ. ID. NO. 40)








C






D 179 S




1




TGCCCTATACAGGTTTAATC




(SEQ. ID. NO. 41)








G




(SEQ. ID. NO. 42)






D 178 B




1




ACCGGCATATACCAGGTGC






D 177 B




2




ACATGGCTCACTCGTAAATT




(SEQ. ID. NO. 43)








C






R 174 B




1




AACATTAGACGCGTTAACGA




(SEQ. ID. NO. 44)








G






D 173 S




1




CTCTTTTGATGCCAGTCAGA




(SEQ. ID. NO. 45)








G






D 172 B




1




ACCTACCCGGATGGCTCTAA




(SEQ. ID. NO. 46)








GG






R 166 B




2




TATGGGAATTCGTGCCGTCC




(SEQ. ID. NO. 47)








TGAAG (EcoRI)






R 143 B




1




AGTGGGAGCAGTATACCAGC




(SEQ. ID. NO. 48)








G






D 141 B




1




CTGCTATTGAGCAGGCTGCT




(SEQ. ID. NO. 49)








C






R 142 S




1




GGGCCATTAGTCTCTAAAAC




(SEQ. ID. NO. 50)








C






D 135 B




1




GAGGTTTTCTGGAATCATC




(SEQ. ID. NO. 51)






R 134 B




1




GCATAGGTGAGACTG




(SEQ. ID. NO. 52)






R 133 B




1




AGTTACAGCCAGAAAACC




(SEQ. ID. NO. 53)






D 132 S




2,3




CCATGGATCCTCGGCCTATT




(SEQ. ID. NO. 54)








TTGCTGTTGCTCC (Bam HI)






D 131 B




5′NC




AGGCAGACCACATATGTG




(SEQ. ID. NO. 55)






R 119 B




1




GGTGCACTCCTGACCAAGCC




(SEQ. ID. NO. 56)






D 118 B




1




ATTGGCTGCCACTTTGTTC




(SEQ. ID. NO. 57)






R 117 B




1




ACCCTCATACGTCACCACAA




(SEQ. ID. NO. 58)








C






R 116 B




1




GCGGTGGACCACATTAGGAT




(SEQ. ID. NO. 59)








TATC






D 115 B




1




CATGATATGTCACCATCTG




(SEQ. ID. NO. 60)






D 114 B




1




GTCATCCATAACGAGCTGG




(SEQ. ID. NO. 61)






R 112 B




2




AGCGGAATTCGAGGGGCGGC




(SEQ. ID. NO. 62)








ATAAAGAACCAGG (EcoRI)






R 111 B




2




GCGCTGAATTCGGATCACAA




(SEQ. ID. NO. 63)








GCTCAGAGGCTATGCC








(EcoRI)






D 110 B




2




GTATAACGGATCCACATCTC




(SEQ. ID. NO. 64)








CCCTTACCTC (Bam HI)






D 109 B




2




TAACCTGGATCCTTATGCCG




(SEQ. ID. NO. 65)








CCCCTCTTAG (Bam HI)






D 108 B




1




AAATTGGATCCTGTGTCGGG




(SEQ. ID. NO. 66)








TGGAATGAATAACATGTC








(BamHI)






R 107 B




1




ATCGGCAGATCTGATAGAGC




(SEQ. ID. NO. 67)








GGGGACTTGCCGGATCC






D 101 B




2




TACCCTGCCCGCGCCCATAC




(SEQ. ID. NO. 68)








TTTTGATG






R 100 B




1




GGCTGAGATCTGGTTCGGGT




(SEQ. ID. NO. 69)








CGCCAAGAAGGTG (Bgl II)






R 99 B




2




TACAGATCTATACAACTTAA




(SEQ. ID. NO. 70)








CAGTCGG (Bgl II)






R 98 B




2




GCGGCAGATCTCACCGACAC




(SEQ. ID. NO. 71)








CATTAGTAC (Bgl II)






D 97 S




1




CCGTCGGATCCCAGGGGCTG




(SEQ. ID. NO. 72)








CTGTCCTG (Bam HI)






R 96 B




2




AAAGGAATTCAAGACCAGAG




(SEQ. ID. NO. 73)








GTAGCCTCCTC (EcoRI)






D 95 B




2




GTTGATATGAATTCAATAAC




(SEQ. ID. NO. 74)








CTCGACGG






R 94 B




3′NC




TTTGGATCCTCAGGGAGCGC




(SEQ. ID. NO. 75)








GGAACGCAGAAATGAG








(BamHI)






D 90 B




2




TCACTCGTGAATTCCTATAC




(SEQ. ID. NO. 76)








TAATAC (EcoRI)






R 89 B




3′NC




TTTGGATCCTCAGGGAGCGC




(SEQ. ID. NO. 77)








GGAACGCAGAAATG (BamHI)






R 88 B




1




TGATAGAGCGGGACTTGCCG




(SEQ. ID. NO. 78)








GATCC (BamHI)






R 8 7 B




1




TTGCATTAGGTTAATGAGGA




(SEQ. ID. NO. 79)








TCTC






D 86 B




1




ACCTGCTTCCTTCAGCCTGC




(SEQ. ID. NO. 80)








AGAAG






R 81 B




1




GCGGTGGATCCGCTCCCAGG




(SEQ. ID. NO. 81)








CGTCAAAAC (BamHI)






D 80 B




1




GGGCGGATCGAATTCGAGAC




(SEQ. ID. NO. 82)








CCTTCTTGG (EcoRI)






R 79 B




1




AGGATGGATCCATAAGTTAC




(SEQ. ID. NO. 83)








CGATCAG (BamHI)






D 78 B




1




GGCTGGAATTCCTCTGAGGA




(SEQ. ID. NO. 84)








CGCCCTCAC (EcoRI)






R 77 B




1




GCCGAAGATCTATCGGACAT




(SEQ. ID. NO. 85)








AGACCTC (Bgl II)






R 76 B




2




CAGACGACGGATCCCCTTGG




(SEQ. ID. NO. 86)








ATATAGCCTG (BamHI)






D 75 B




5′NC




GGCCGAATTCAGGCAGACCA




(SEQ. ID. NO. 87)








CATATGTGGTCGATGCCATG








(EcoRI)






D 72 B




1




GCAGGTGTGCCTGGATCCGG




(SEQ. ID. NO. 88)








CAAGT (BamHI)






R 71 B




1




GTTAGAATTCCGGCCCAGCT




(SEQ. ID. NO. 89)








GTGGTAGGTC (EcoRI)






D 63 B




1




CCGTCCGATTGGTCTGTATG




(SEQ. ID. NO. 90)








CAGG






D 61 B




1




TACCAGTTTACTGCAGGTGT




(SEQ. ID. NO. 91)








GC






D 60 B




1




CAAGCCGATGTGGACGTTGT




(SEQ. ID. NO. 92)








CG






R 59 B




2,3




GGCGCTGGGCCTGGTCACGC




(SEQ. ID. NO. 93)








CAAG






D 50 B




1




GCAGAAACTAGTGTTGACCC




(SEQ. ID. NO. 94)








AG






R 49 B




2




TAGGTCTACGACGTGAGGCA




(SEQ. ID. NO. 95)








AC






R 48 B




1




TACAATCTTTCAGGAAGAAG




(SEQ. ID. NO. 96)








G






R 47 B




1




CCCACACTCCTCCATAATAG




(SEQ. ID. NO. 97)








C






D 46 B




1




GATAGTGCTTTGCAGTGAGT




(SEQ. ID. NO. 98)








ACCG














The abbreviations to the left of the sequences represent the following: R and D refer to reverse and forward primers, respectively; B and S refer to sequences derived from the Burma-121 Strain of Hepatitis E and the SAR-55 Strain of Hepatitis E, respectively; 5′NC and 3′NC refer to 5 prime and 3 prime non-coding regions of the HEV genome, respectively; and 1, 2 and 3 refer to sequence derived from open reading frames 1, 2 or 3, respectively. The symbol ( ) to the right of some sequences shown indicates insertion of an artificial restriction site into these sequences.




For cloning of PCR fragments, EcoRI, BamHI, or BglII restriction sites preceded by 3-7 nt were added to the 5′ end of primers.




RT-PCR.




The usual 100-μl RT-PCR mixture contained template, 10 mM Tris-HCL (ph 8.4), 50 mM KCl, 2.5 mM MgCl


2


, all four dNTPs (each at 0.2 mM), 50 pmol of direct primer, 50 pmol of reverse primer, 40 units of RNasin (Promega), 16 units of avian myeloblastosis virus reverse transcriptase (Promega), 4 units of AmpliTaq (Cetus), under 100 μl of light mineral oil. The mixture was incubated 1 h at 42° C. and then amplified by 35 PCR cycles; 1 min at 94° C., 1 min at 45° C., and 1 min at 72° C. The PCR products were analyzed on 1% agarose gels.




Cloning of PCR Fragments




PCR fragments containing restriction sites at the ends were digested with EcoRI and BamHi or EcoRI and BglII restriction enzymes and cloned in EcoRI/BanmI-digested pBR322 or pGEM-3Z (Promega). Alternatively, PCR fragments were cloned into pCR1000 (Invitrogen) using the TA cloning kit (Invitrogen).




Sequencing of PCR Fragments and Plasmids




PCR fragments were excised from 1% agarose gels and purified by Geneclean (Bio 101, La Jolla, Calif.). Double-stranded PCR fragments were sequenced by using Sequenase (United States Biochemical) as described in Winship, P. R. (1984),


Nucleic Acids Rev.,


17:1266. Double-stranded plasmids purified through CsCl gradients were sequenced with a Sequenase kit (United States Biochemical).




Computer Analysis of Sequences Nucleotide sequences of HEV strains were compared using the Genetics Computer Group (Madison, Wis.) software package (Devereaux, J. et al. (1984),


Nucleic Acids Rev.,


12:387-395, version 7.5, on a VAX 8650 computer (at the National Cancer Institute, Frederick, Md.)).




EXAMPLE 2




Construction of a Recombinant Expression Vector, P63-2




A plasmid containing the complete ORF-2 of the genome of HEV strain SAR-55, Tsarev, S. A. et al. (1992),


Proc. Natl. Acad. Sci. USA,


89:559-563), was used to obtain a restriction fragment NruI-BglII. NruI cut the HEV CDNA five nucleotides upstream of the ATG initiation codon of ORF-2. An artificial Bgl II site previously had been placed at the 3′ end of HEV genome just before the poly A sequence (Tsarev, S. A. et al. (1992),


Proc. Natl. Acad. Sci. USA,


89:559-563). To insert this fragment into pBlueBac-Transfer vector (Invitrogen) a synthetic polylinker was introduced into the unique NheI site in the vector. This polylinker contained Bln I and Bgl II sites which are absent in both HEV cDNA and pBlueBac sequences. The NruI-BglII ORF-2 fragment was inserted in Bln I-BglII pBlueBac using an adapter as shown in FIG.


1


.




EXAMPLE 3




Expression of P63-2 in SF9 Insect Cells




p63-2 and AcMNPV baculovirus DNA (Invitrogen) were cotransfected into SF9 cells (Invitrogen) by the Ca precipitation method according to the Invitrogen protocol By following this protocol; the AcMNPV baculovirus DNA can produce a live intact baculovirus which can package p63-2 to form a recombinant baculovirus. This recombinant baculovirus was plaque-purified 4 times. The resulting recombinant baculovirus 63-2-IV-2 was used to infect SF9 cells.




SDS-PAGE and Western Blot




Insect cells were resuspended in loading buffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromphenol blue and 10% glycerol) and SDS-polyacrylamide gel electrophoresis was performed as described, Laemmli, U. K. (1970),


Nature,


227:680. Gels were stained with coomassie blue or proteins were electroblotted onto BA-85 nitrocellulose filters (Schleicher & Schuell). After transfer, nitrocellulose membranes were blocked in PBS containing 100 fetal calf serum and 0.5% gelatin. As a primary antibody, hyperimmune serum of chimpanzee-1313 diluted 1:1000 was used. As a secondary antibody, phosphatase-labeled affinity-purified goat antibody to human IgG (Kirkegaard & Perry Laboratories, Inc.) diluted 1:2000 was used. Filters were developed in Western blue stabilized substrate for alkaline phosphatase (Promega). All incubations were performed in blocking solution, and washes were with PBS with 0.05% Tween-20 (Sigma).




Expression of HEV ORF-2. The major protein synthesized in SF9 cells infected with recombinant baculovirus 63-2-IV-2 was a protein with an apparent molecular weight of 74 KD (

FIG. 2A

, lane 3). This size is a little larger than that predicted for the entire ORF-2 (71 KD). The size difference could be due to glycosylation of the protein since there is at least one potential site of glycosylation (Asn-Leu-Ser) in the N-terminal part. This protein was not detected in noninfected cells (

FIG. 2A

, lane 1) or in cells infected with wild-type nonrecombinant baculovirus (

FIG. 2A

, lane 2). In the latter case, the major protein detected was a polyhedron protein. When the same lysates were analyzed by Western blot (

FIG. 2B

) with serum of chimp-1313 (hyperimmunized with HEV), only proteins in the recombinant cell lysate reacted (lane 3) and the major band was again represented by a 74 KD protein (FIG.


2


B). Minor bands of about,25, 29, 35, 40-45 and 55-70 kDa present in the Coomassie-stained gel (

FIG. 2A

, lane 3) also reacted with serum in the Western blot (

FIG. 2B

, lane 3). Some of the bands having molecular weights higher than 74 KD result from different extents of glycosylation while the lower molecular weight bands could reflect processing and/or degradation. Serum drawn from Chimp-1313 prior to inoculation with HEV did not react with any of the proteins by Western blot.




EXAMPLE 4




Immunoelectron Microscopy of Recombinant Infected SF9 Cells




5×10


6


recombinant infected SF9 cells were sonicated in CsCl (1.30 g/ml) containing 10 mM Tris-HCl, pH 7.4, 0.3% sarcosyl and centrifuged 68 h, at 40,000 rpm (SW60Ti). 50 ul of the fraction, which had the highest ELISA response and a buoyant density of 1.30 g/ml was diluted in 1 ml PBS and 5 ul of chimp-1313 hyperimmune serum was added. The hyperimmune serum was prepared by rechallenging a previously infected chimp with a second strain of hepatitis E (Mexican HEV). Samples were incubated 1 h at room temperature and then overnight at 4° C. Immune complexes were precipitated using a SW60Ti rotor at 30,000 rpm, 4° C., 2 h. Pellets were resuspended in distilled water, negatively stained with 3% PTA, placed on carbon grids and examined at a magnification of 40,000 in an electron microscope EM-10, Carl Zeiss, Oberkochen, Germany.




Detection of VLPs




Cell lysates from insect cells infected with wild-type or recombinant baculovirus 63-2-IV-2 were fractionated by CsCl density centrifugation. When fractions of the CsCl gradient from the recombinant infected insect cells were incubated with Chimp-1313 hyperimmune serum, two kinds of virus-like particles (VLP) covered with antibody were observed in the fraction with buoyant density of 1.30 g/ml: first (FIGS.


3


A-


3


A′″), antibody covered individual particles that had a size (30 nm) and morphological structure suggestive of HEV, second (FIG.


3


B), antibody-coated aggregates of particles smaller than HEV (about 20 nm) but which otherwise resembled HEV. Direct EM showed the presence of a very heterogenous population of objects including some of 30 and 20 nm in diameter respectively, which looked like virus particles but, in the absence of bound antibody, could not be confirmed as HEV. A number of IEM experiments suggested that at least some of the protein(s) synthesized from the ORF-2 region of the HEV genome, had assembled into a particulate structure. It was observed that insect cells at a later stage of infection, when the proportion of smaller proteins was higher, consistently gave better results in ELISA. Therefore, unfractionated lysates of recombinant insect cells from a later stage of infection were used as antigen in ELISA in subsequent tests.




EXAMPLE 5




Detection by ELISA Based on Antigen from Insect Cells Expressing Complete ORF-2 of Anti-HEV Following Infection with Different Strains of HEV




5×10


6


SF9 cells infected with 63-2-IV-2 virus were resuspended in 1 ml of 10 mM Tris-HCl, pH 7.5, 0.15M NaCl then were frozen and thawed 3 times. 10 ul of this suspension was dissolved in 10 ml of carbonate buffer (pH 9.6) and used to cover one flexible microtiter assay plate (Falcon). Serum samples were diluted 1:20, 1:400 and 1:8000, or 1:100, 1:1000 and 1:10000. The same blocking and washing solutions as described for the Western blot were used in ELISA. As a secondary antibody, peroxidase-conjugated goat IgG fraction to human IgG or horse radish peroxidase-labelled goat anti-Old or anti-New World monkey immunoglobulin was used. The results were determined by measuring the optical density (O.D.) at 405 nm.




To determine if insect cell-derived antigen representing a Pakistani strain of HEV could detect anti-HEV antibody in cynomolgus monkeys infected with the Mexican strain of HEV, 3 monkeys were examined (FIG.


4


). Two monkeys cyno-80A82 and cyno-9A97, were infected with feces containing the Mexico '86 HEV strain (Ticehurst, J. et al. (1992),


J. Infect. Dis.,


165:835-845) and the third monkey cyno-83 was infected with a second passage of the same strain. As a control, serum samples from cyno-374, infected with the Pakistani HEV strain SAR-55, were tested in the same experiment. All 3 monkeys infected with the Mexican strain seroconverted to anti-HEV. Animals from the first passage seroconverted by week 15 and from the second passage by week 5. Interestingly, the highest anti-HEV titer among the 4 animals, was found in cyno-83, inoculated with the second passage of the Mexican strain. Cynos inoculated with the first passage of the Mexican strain developed the lowest titers while those inoculated with the first passage of the Pakistani strain developed intermediate titers.




EXAMPLE 6




Specificity of Anti-HEV ELISA Based on Antigen from Insect Cells Expressing Complete ORF-2




To estimate if the ELISA described here specifically detected anti-HEV to the exclusion of any other type of hepatitis related antibody, serum samples of chimps were analyzed, in sets of four, infected with the other known hepatitis viruses (Garci, P. et al. (1992),


J. Infect. Dis.,


165:1006-1011; Farci, P. et al. (1992),


Science


(in press); Ponzetto, A. et al. (1987)


J Infect. Dis.,


155: 72-77; Rizzetto; m.et al. (1981) Hepatology 1: 567-574; reference for chimps—1413, 1373, 1442, 1551 (HAV); and for chimps—982, 1442, 1420, 1410 (HBV); is unpublished data from Purcell et al) (Table 1). Samples of pre-inoculation and 5 week and 15 week post-inoculation sera were analyzed in HEV ELISA at serum dilutions of 1:100, 1:1000 and 1:10000. None of the sera from animals infected with HAV, HBV, HCV and HDV reacted in the ELISA for HEV antibody, but all 4 chimps inoculated with HEV developed the IgM and IgG classes of anti-HEV.












TABLE 1











Serological assay of anti-HEV antibody in chimpanzees infected with different






hepatitis viruses (Hepatitis A, B, C, D, E)

















week of










serocon-








version







inocu-




for ino-





weeks post-inoculation


















lated




culated




preserum




5




15




20/25





















chimp




virus




virus




IgG




IgM




IgG




IgM




IgG




IgM




IgG




IgM









Chimp-1413




HAV




5






































Chimp-1373




HAV




7




































Chimp-1442




HAV




5




































Chimp-1451




HAV




5




































Chimp-982




HBV




3




































Chimp-1442




HBV




7














































Chimp-1420




HBV




9




































Chimp-1410




HBV




5




































Chimp-51




HCV




10




































Chimp-502




HCV




12




































Chimp-105




HCV




28




































Chimp-793




HCV




13




































Chimp-904




HDV




8




































Chimp-814




HDV




7




































Chimp-800




HDV




10




































Chimp-29




HDV




10














































Chimp-1310




HEV




5














1:10,000




1:100




1:10,000











Chimp-1374




HEV




3














1:8000  




—*




1:8000  











Chimp-1375




HEV




3














1:8000  




1:400




1:400  











Chimp-1313




HEV1st°**




5














1:10,000




1:100




1:1000  











Chimp-1313




HEV2nd°**




0.5




1:100









1:10,000









1:10,000
















*Chimp-1374 was positive for IgM anti-HEV three and four weeks post-inoculation (see

FIG. 5

)










**Chimp-1313 was inoculated with HEV twice. 1st inoculation with pooled samples of 7 Pakistani patients. 2nd inoculation 45 months later with Mexican strain of HEV.













EXAMPLE 7




Determination of the Host Range of the SAR-55 Strain of HEV in Non-Human Primates




Different primate species were inoculated intravenously with a standard stool suspension of HEV and serial serum samples were collected to monitor for infection. Serum ALT levels were determined as an indicator of hepatitis while seroconversion was defined as a rise in anti-HEV. The results were compared with those obtained in cynomolgus monkeys and chimpanzees.




Both rhesus monkeys inoculated with HEV (Table 2) demonstrated very prominent peaks of alanine aminotransferase activity as well as a strong anti-HEV response. The peak of alanine aminotransferase activity was observed on day 35 for both animals, and seroconversion occurred on day 21. The maximum titer of anti-HEV was reached on day 29. Both African green monkeys used in this study (Table 2) developed increased alanine aminotransferase activity and anti-HEV. Although African green money 230 died 7 weeks after inoculation, proof of infection was obtained before that time. Peak alanine aminotransferase activity for monkey 74 exceeded the mean value of preinoculation sera by about three times and for monkey 230 by about five times. Peaks of alanine aminotransferase activity and seroconversion appeared simultaneously on days 28 and 21 in monkeys 74 and 230, respectively.












TABLE 2











Biochemical and serologic profiles of HEV






infection in eight primate species.













Anti-HEV IgG















Alanine aminotransferase (units/L)




Day first


















Preinoculation,






detected




Maximum






Animal




mean (SD)




Day




Value




(titer)




titer









Chimpanzee











1374




51(12)




27




114




 27(1:400)




1:8000






1375




41(14)




27




 89




 27(1:400)




1:8000






Cynomolgus monkey






 374*




46(20)




26




608




 19(1:400)




1:8000






 381*




94(19)




35




180




28(1:20)




1:8000






Rhesus monkey






 726




43(6) 




35




428




21(1:20)




1:8000






 938




29(10)




35




189




21(1:20)




1:8000






African green monkey






 74




72(21)




28




141




 28(1:400)




1:8000






 230




102(45) 




21




334




 21(1:8000)




1:8000






Pigtail macaque






 98




37(8) 




21




 47




 21(1:400)




1:8000






 99




41(6) 




28




 59




 21(1:400)




1:8000






Tamarin






 616




28(7) 




70




 41











 636




19(4) 




7, 56




 30











Squirrel monkey






 868




90(35)




40




355




41(1:20)




1:20 






 869




127(63) 




42




679




35(1:20)




1:20 






Owl monkey






 924




41(7) 




35




 97




21(1:20)




1:8000






 925




59(6) 




49, 91†




   78,199†




21(1:20)




1:8000











NOTE.










—, no anti-HEV detected.










*Previously studied using fragments of HEV proteins expressed in bacteria as antigen [18].










†Biomodal elevation of alanine aminotransferase.










SD = standard deviation.













Pigtail macaque 99 demonstrated an increase in alanine aminotransferase activity >3 SD above the mean value of preinoculation sera, while pigtail macaque 98 did not. However, both monkeys seroconverted on day 21 and the anti-HEV titers were equivalent to those of the chimpanzees and Old World monkeys. Because of the low peak alanine aminotransferase values in the pigtail macaques, the possibility of immunization instead of infection with HEV cannot be completely ruled out. However, immunization is unlikely for several reasons. First, immunization in either of 2 tamarins, which are only one-fourth as large as the pigtail macaques but received the same amount of inoculum was not observed. Second, it is well known that the amount of HEV excreted in feces is usually very small, and 0.5 mL of the 10% suspension of feces used in this study is unlikely to contain an amount of antigen sufficient to immunize an animal, especially when inoculated intraveneously.




Neither tamarin inoculated in this study had a significant rise in alanine aminotransferase activity or development of anti-HEV (Table 2). Therefore, these animals did not appear to be infected. The squirrel monkeys did develop anti-HEV, but at significantly lower levels than chimpanzees or Old World monkeys (Table 2). In addition, seroconversion occured later in other animals. Squirrel monkey 868 seroconverted on day 41 and 869 on day 35. The anti-HEV titer was not >1:20 at any time during >3 months of monitoring and clearly was waning in both animals after reaching a peak value on days 47-54. However, the increases in alanine aminotransferase activity were rather prominent in both animals and were temporally related to the time of seroconversion.




The owl monkeys responded to HEV infection about as well as the Old World monkey species (Table 2). Both owl monkeys seroconverted on day 21 and by day 28 the anti-HEV titer had reached a value of 1:8000. Alanine amino-transferase activity peaked on day 35 in owl monkey 924 but not until day 49 in monkey 925.




EXAMPLE 8




Detection of IgM and IgG Anti-HEV in Chimps




In both chimps, the serum ALT levels increased about 4 weeks post-inoculation (Table 2, FIG.


5


). Both chimps seroconverted at the time of ALT enzyme elevation or earlier (

FIG. 5A

, 5C). Levels of IgM anti-HEV also were determined for the chimps. In chimp-1374, the titer of IgM anti-HEV (

FIG. 5B

) was not as high as the IgG titer (

FIG. 5A

) and waned over two weeks. Although both IgG and IgM antibodies were first detected for this animal on day 20, the titer of IgM anti-HEV was the highest while the titer of IgG was the lowest on that day, but then rose and stayed approximately at the same level for more than three months. In chimp-1375, only IgM anti-HEV was detected on day 20 (FIG.


5


D). The titer was higher than in chimp-1374 and IgM anti-HEV was detected during the entire period of monitoring. IgG anti-HEV was first observed in this animal on day 27 (

FIG. 5C

) and remained at approximately the same level throughout the experiment.




EXAMPLE 9




Comparison of ELISA Based on Complete ORF-2 Protein Expressed in Insect Cells With That Based on Fragments of Structural Proteins Expressed in


E. coli






To estimate if expression of the complete ORF-2 region of the HEV genome in eukaryotic cells had any advantage over expression of fragments of structural proteins in


E. coli


, we used the former antigen in ELISA to retest cynomolgus monkey sera that had been analyzed earlier (Tsarev, S. A. et al. (1992),


Proc. Natl. Acad. Sci USA,


89:559-563; and Tsarev, S. A. et al. (1993)


J. Infect. Dis. (


167:1302-1306)), using the antigen fragments expressed in bacteria (Table 3).












TABLE 3











Comparison of ELISA based on antigen from insect






cells expressing complete ORF-2 with that based






on antigen from


E. coli


expressing fragments of






structural proteins















antigen derived from







antigen derived from




insect cells







bacterial cells




(Complete ORF-2)







(Portion of ORF-2)*




anti-HEV
















day anti-




first









HEV first




detected





max.






Cyno #




detected




day




titer




titer









Cyno-376




28




21




1:400




1:8000






Cyno-369




54




40




1:100




1:8000






Cyno-374




19




19




1:400




1:8000






Cyno-375




26




26




1:400




1:8000






Cyno-379




21




19




1:100




1:8000






Cyno-381




28




28




1:400




1:8000











*The sera were also tested with less sensitive ORF-3 antigen [. . .].










Tsarev, S.A. et al. (1993), J. Infect. Dis. (167:1302-1306)













For 3 of the 6 monkeys examined by ELISA, the antigen expressed in insect cells detected seroconversion earlier than the antigen expressed in


E. coli


. Using the insect cell-derived antigen, we were able to detect anti-HEV antibody in sera from all six monkeys at the highest dilution tested (1:8000). With


E. coli


-cell derived antigen (Burma Strain) no information about anti-HEV titers were obtained, since all sera were tested only at a dilution of 1:100 (Tsarev, SA et al (1992)


Proc. Nat. Acad. Sci. USA;


89:559-563; Tsarev et al. (1993)


J. Infect. Dis. (


167:1302-1306)).




In another study, hepatitis E virus, strain SAR-55 was serially diluted in 10-fold increments and the 10


−1


through 10


−5


dilutions were inoculated into pairs of cynomolgus monkeys to titer the virus. The serum ALT levels were measured to determine hepatitis and serum antibody to HEV was determined by the ELISA method of the present invention (data in figures) or by Genelab's ELISA (three ELISAS, each based on one of the antigens designated 4-2, 3-2 and 612 in Yarbrough et al. (


J. Virol


., (1991) 65:5790-5797) (data shown as positive (+) or negative (−) test at bottom of

FIGS. 6



a-g


). All samples were tested under code.




The ELISA method of the present invention detected seroconversion to IgG anti-HEV in all cynos inoculated and all dilutions of virus.




In contrast, Genelab's results were strikingly variable, as summarized below.














TABLE 4











ELISA of






Dilution





Present






of Virus




Genelab's ELISA




Invention











10


−1






did not test




positive






10


−2






positive for both animals,




positive







limited duration






10


−3






negative for both animals




positive






10


−4






Cyno 389: positive for IgM




positive







and IgG






10


−5






Cyno 383: negative




positive







Cyno 386: negative




positive







Cyno 385: positive




positive














Since Cyno 385 (10


−5


) was positive in ELISA tests both by Genelabs and the present invention, the 10


−4


(ten times more virus inoculated) and 10


−3


(100 times more virus inoculated) would also have been expected to be positive. The present invention scored them as positive in contrast to Genelab's ELISA test which missed both positives at 10


−3


and one at 10


−4


even though the ALT levels of Cyno 383 and 393 suggested active hepatitis. Therefore, the data support the advantages of the present ELISA method over the prior art methods of detecting antibodies to HEV.




EXAMPLE 10




Comparison Of ELISAs Based On Recombinant ORF-2 Antigens Consisting Of Either A 55 kDa Protein Expressed From The Complete ORF-2 Region Of The Pakistani SAR-55 Strain Of REV Or Of Shorter Regions Of ORF-2 Expressed As Fusion Proteins In Bacteria




As described in Example 3 and as shown in

FIGS. 2A and 2B

, a number of proteins of varying molecular weights are expressed in insect cells infected with the recombinant baculovirus containing the complete ORF-2. A protein with a molecular weight of approximately 55 kDa was partially purified from 5×10


8


SF-9 cells harvested seven days post-inoculation as follows: The infected cells were centrifuged, resuspended in 10 ml of 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, containing 40 μg/ml of phenylmethylsulfonyl fluoride (Sigma, St. Louis, Miss.), sonicated to disrupt the cells and the lysate was centrifuged at 90,000×g at 4+ C. for 30 min. The supernatant was loaded onto a DEAE-sepharose CL-6B (Pharmacia, Uppsala, Sweden) column equilibrated with 10 mM Tris-HCl (pH 8.0), 50 mM NaCl. The column was washed with loading buffer and the 55 kDa protein was eluted in 10 mM Tris-HCl (pH 8.0) 250 mM NaCl. Fractions containing the 55 kDa protein were combined and the protein was precipitated by addition of 3 g of (NH


4


)


2


S


4


to 10 ml of the protein solution. The protein pellet was dissolved in 10 mM Tris-HCl (pH 8.0), 50 mM NaCl. The 55 kDa protein was then used as the insect cell-expressed HEV antigen in ELISA in comparison testing against ELISAs based on either one of two HEV antigens expressed in bacteria, (3-2 (Mexico) (Goldsmith et al., (1992)


Lancet,


339:328-331) or SG3 (Burma) (Yarbough et al., (1993) Assay development of diagnostics tests for hepatitis E. In “International Symposium on Viral Hepatitis and Liver Disease. Scientific program and abstract volume.” Tokyo:VHFL, p 87, Abstract #687). These bacterial antigens were fusion proteins of the 26 kDa glutathione—S—transferase (GST) and either the antigenic sequence 3-2 (M) consisting of 42 amino acids located 6 amino acids upstream of the C-terminus of ORF-2 (Yarbough et al., (1991)


J. Virol.,


65:5790-5797) or the 327 C-terminal amino acids of ORF-2 (Yarbough et al., (1993)). The ELISAs were carried out as follows.




Sixty ng per well of the 55 kDa protein or 200 ng per well of the fusion antigens in carbonate buffer (pH 9.6) were incubated in wells of a polystyrene microtiter assay plate (Dynateck, S. Windham, Me.) for 2 h at 37° C. Plates were blocked with PBS containing 10% fetal calf serum and 0.5% gelatin. Serum samples from cynomolgus monkeys inoculated intravenously (note: cynos 387 and 392 were inoculated orally) with a dilution of feces containing the SAR-55 strain of HEV ranging from 10


−1


through 10


−8


as indicated in Table 5 and

FIGS. 7A-7J

and


8


A-


8


D were diluted 1:100 in blocking solution. Peroxidase-conjugated goat anti-human IgM (Zymed, San Francisco, Calif.) diluted 1:1000 or 1:2000, or peroxidase-labelled goat anti-human immunoglobulin diluted 1:1000 was used as the detector antibody.




In all of the ELISA tests except those for the two orally inoculated monkeys, cyno-387 and cyno-392, the 55 kDa and the fusion antigens were tested concurrently in the same laboratory so that the only variable was the antigen used. Criteria for scoring positive reactions in anti-HEV ELISA with the 55 kDa antigen were an optical density value a 0.2 and greater than twice that of a pre-inoculation serum sample for the same animal. In addition, since both antigens expressed in bacteria were fusion proteins with GST, the optical density of a sample tested with these antigens had to be 3 times higher than that obtained with non-fused GST in order to be considered positive (Goldsmith et al., (1992)).




RESULTS




Both cynomolgus monkeys (377, 378) inoculated with the 10


−1


dilution of the standard HEV fecal suspension had a pronounced increase in ALT activity at 4-5 weeks post-inoculation, indicative of hepatitis (Table 5, FIGS.


7


A and


7


B).












TABLE 5











Summary of biochemical and serological events occurring in cynomolgus monkeys






after inoculation with 10


−1


to 10


−8


dilutions of the standard stock of the SAR-55 HEV inoculum.














weeks post-inocu-


















Dilution




ALT





lation anti-HEV




weeks post-inoculation anti-HEV


















of viral




pre-inocu-






was detected with




was detected with fusion antigen



















stock




lation week




peak




peak




55 kDa antigen




IgG




IgM





















Cyno




inoculum




mean (SD)









value




(U/L)




IgG




IgM




SG3




3-2(M)




SG3




3-2(M)









377




10


−1






 76 (39)




5




264 




 4-15†




3-7 




4-10




4-5




3-4




3-5






378




10


−1






 50 (9)




4




285 




4-15































394




10


−2






 62 (14)




4




89




3-15




3-10









4-6
















395




10


−2






121 (21)




15 




314 




5-15































380




10


−3






 89 (20)




1




135 




 5-15*









6-15





















383




10


−3






 29 (8)




4




77




5-15




5-13


























389




10


−4






 60 (7)




15 




114 




6-15




6-8 


























393




10


−4






 41 (4)




5




87




6-15































385




10


−5






 59 (32)




7




56




11-15 














 7-15
















386




10


−5






 31 (4)




4




34




8-15




8-13


























397




10


−6






 60 (4)




8




94




































398




10


−6






 36 (3)




2




55




































399




10


−7






102 (16)




2




93




































400




10


−7






 57 (4)




9




188 




































403




10


−8






 33 (3)




2-3




49




































406




10


−8






 56 (4)




2




73




































387




10


−1


(oral)


§






 32 (4)




4




38














ND









ND











392




10


−1


(oral)


§






 49 (6)




3




70














ND









ND





















ALT mean and standard deviation (SD) values of pre-inoculation sera.















The experiment was terminated after 15 weeks.










*The OD values of pre-inoculation sera of Cyno-380, when tested by ELISA with 55 kDa antigen, were twice as high as the mean value of pre-inoculation sera for other cynomolgus monkeys.












§


All ELISA tests except for Cyno-387 and Cyno-392 were performed in the same experiments.










—not detected.










ND - not done.













All 3 antigens tested detected IgM anti-HEV in samples taken from cyno-377 3 weeks post-inoculation (Table 5, FIG.


8


A), but IgM anti-HEV was not detected in any samples from the second animal, cyno-378. IgG anti-HEV was detected in both animals with the 55 kda-based ELISA, but only in cyno-377 with the ELISA based on fusion antigens. Values of OD for IgG anti-HEV were significantly higher than those for IgM anti-HEV. ELISA values obtained with the 55 kDa antigen were also significantly higher than those obtained with either of the two fusion antigens (FIGS.


7


A and


7


B). The patterns of the OD values observed in ELISA with antigens from the two sources also differed significantly. In the case of ELISA based on the fusion antigens, positive signals reached a maximum shortly after seroconversion and then waned during the 15 weeks of the experiment. In ELISA based on the 55 kDa antigen, the positive signal reached a maximum shortly after seroconversion and remained at approximately the same high level throughout the experiment (15 weeks).




Elevation in ALT activities in both monkeys (394 and 395) inoculated with a 10


−2


dilution of the standard HEV stool suspension was significantly less pronounced at the expected time of hepatitis than in animals inoculated with the ten-fold higher dose (Table 5, FIGS.


7


C and


7


D). Cyno-395 actually had higher ALT activities prior to inoculation as well as at 15 weeks post-inoculation. The latter was probably not related to HEV infection. Weakly positive IgM anti-HEV was detected only in cyno-394 (

FIG. 8B

) and only with ELISA based on the 55 kDa antigen. Both animals were infected, however, since IgG anti-HEV seroconversion was detected in both animals. In cyno-394, anti-HEV IgG was first detected by the 55 kDa antigen at week 3 and one week later with the 3-2(M) antigen. The SG3 (B) antigen did not detect seroconversion in cyno-395 and anti-HEV IgG was detected only with the 55 kDa antigen. Anti-HEV tended to diminish in titer with time in this animal.




Cyno-380 and cyno-383 were inoculated with a 10


−3


dilution of the standard HEV fecal suspension (Table 5,

FIGS. 7E



7


F,


8


C). Cyno-380 had fluctuating ALT activities before and after inoculation; therefore, ALT levels could not be used to document hepatitis E in this animal. In Cyno-383, a slight rise of ALT activities was observed (FIG.


7


F), which was coincident with seroconversion and, therefore, might be due to mild hepatitis E. IgM Anti-HEV was not detected in sera from cyno-380 with any of the three antigens. Cyno-380 seroconverted for IgG anti-HEV when tested by ELISA with SG3 (B) but not with 3-2(M) antigen. This animal had preexisting IgG anti-HEV when tested with the 55 kDa antigen, but there was a large increase in IgG anti-HEV starting at week 5 (FIG.


7


E). Identification of preexisting antibody was reported earlier in sera from another cynomolgus monkey [Ticehurst et al., (1992)


J. Infect Dis.,


165:835-845; Tsarev et al., (1993)


J. Infect. Dis.,


168:369-378]. Seroconversion occured at the expected time but the levels of IgG anti-HEV in samples from cyno-383 remained low and detectable only with the 55 kDa antigen.




Cyno-389 and cyno-393 were inoculated with a 10


−4


dilution of the standard HEV fecal suspension (

FIGS. 7G

,


7


H,


8


D, Table 5). Neither animal had a significant rise in ALT activities, although the timing of a small but distinct ALT peak in sera of cyno-393 at week 5 (

FIG. 7H

) suggested borderline hepatitis. ELISA based on the SG3 (B) or 3-2(M) antigens scored both animals as negative for HEV infection. In contrast, the 55 kDa antigen detected IgM anti-HEV in sera of cyno-389 at weeks 6-8 post-inoculation (

FIG. 8D

) and IgG anti-HEV from week 6 through week 15 in both animals.




Neither animal inoculated with the 10


−5


dilution of the standard fecal suspension developed a noticeable rise in ALT activities (

FIGS. 7I

,


7


J, Table 5), but, in cyno-386, IgM and IgG anti-HEV were detected at weeks 8-13 and 8-15 respectively with the 55 kDa antigen (

FIGS. 7J

,


8


E). Cyno-385 anti-HEV IgG was detected with the 55 kDa and the 3-2(M) antigen but not with SG3 (B) antigen. In contrast to previous patterns, IgG anti-HEV was detected with a fusion antigen four weeks earlier and at higher levels than with the 55 kDa antigen.




None of the animals inoculated with dilutions of the standard HEV fecal suspension in the range of 10


−6


-10


−8


developed antibody to any of the three HEV antigens. Increased ALT activities were not observed in those animals, except for one rather prominent peak of ALT activity at week 9 in cyno-400 (Table 5). However, the absence of seroconversion in this animal indicated that this peak probably was not related to HEV infection.




With respect to the two cynomolgus monkeys (387 and 392) inoculated orally with the 10


−1


dilution of the 10% fecal suspension, neither monkey was infected since ALT levels did not rise and ELISA performed with the 3-2(M) and 55 kDa antigens did not detect seroconversion to HEV (Table 5).




Finally, serological evidence for HEV infection was found in all animals inoculated with decimal dilutions of the fecal suspension through 10


−5


; none of the animals receiving higher dilutions had such evidence. Prominent hepatitis, as defined by elevated ALT, was observed only in the two monkeys infected with the 10


−1


dilution. Significantly lower elevations of ALT activities were observed in some animals inoculated with higher dilutions of the fecal suspension while, in others, elevations were not found. Considered alone, these low ALT rises were not diagnostic of hepatitis. However, the coincidence of seroconversion and appearance of these ALT peaks suggests the presence of mild hepatitis in these animals. Anti-HEV IgG seroconversion was detected in all animals inoculated with dilutions of fecal suspension ranging from 10


−1


-10


−5


. A tendency toward lower levels of IgG anti-HEV and delayed seroconversion was observed in animals inoculated with higher dilutions of the stock.




In sum, the 55 kDa Pakistani ORF-2 antigen was more efficient than either the 3-2(M) or SG3 (B) antigen for detecting IgM and IgG anti-HEV in cynomolgus monkeys infected with the Pakistani strain of HEV. For example, for all animal sera except those from cyno-385, detection of IgG or IgM anti-HEV by ELISA was more efficient with the 55 kDa antigen than with either the 3-2(M) or SG3 antigen. ELISA with the 55 kDa antigen produced internally consistent and reproducible results, detecting IgG anti-HEV in all ten animals inoculated with a fecal dilution of 10


−5


or lower. The magnitude of ELISA signals also decreased as the inoculum was diluted. The fusion antigens did not produce consistent results between the pairs of animals. Only one of each pair of animals inoculated with the 10


−1


, 10


−2


, 10


−3


, or 10


−5


dilution showed seroconversion to IgG anti-HEV, and only a single seroconversion for IgM anti-HEV was detected with these antigens. Neither of the animals inoculated with the 10


−4


dilution of the original inoculum seroconverted to either of the two fusion antigens even though sera from one animal (cyno-393) had sustained high levels of anti-HEV IgG when assayed with the 55 kDa antigen. Although, as discussed above, ELISA for IgM anti-HEV was significantly less sensitive than ELISA for cynomolgus IgG anti-HEV, the 55 kDa antigen was able to detect anti-HEV IgM in more animals than the 3-2(M) or SG3 (B) antigen. In sum, a definitive conclusion about the infectious titer of the Pakistani viral inoculum used in this study could not be made with the combined data from the 3-2(M) and SG3 (B) based ELISA but could be made with data obtained with the 55 kDa Pakistani ELISA alone.




With respect to cyno-385, the difference in anti-HEV IgG detection between the two test results was four weeks. These data suggest the presence of a distinct epitope in the 3-2(M) antigen recognized by this animal that is absent in the larger 55 kDa and SG3 (B) antigens. When total insect cell lysate, which contained both complete ORF-2 (75 kDa) and 55 kDa proteins, was used as antigen to retest these samples, the results were the same as when 55 kDa was used alone. This finding suggests that the 55 kDa protein may not lack 3-2 epitope amino acids but rather that the conformation of the 3-2 epitope sequence differed among all three antigens used in this study. Finally, it is interesting to note that despite the fact that antigen SG3 (B) contained a longer portion of ORF-2 and included the entire sequence of epitope 3-2, it did not detect more positive sera than the 3-2(M) antigen.




EXAMPLE 11




Determination of the Infectious Titer of the HEV SAR-55 Viral Stock BY RT-PCR




Knowledge of the infectious titer of inocula is critical for interpretation of much of the data obtained in experimental infections of animal models. However, until now the infectious titer of an HEV viral stock has not been reported. Ten-fold dilutions of the fecal suspension containing the SAR-55 strain of HEV were extracted and RT-PCR amplification was performed as follows to determine the highest dilution in which HEV genomes could be detected. 200 ul of fecal suspension was mixed with 0.4 ml of 1.5M NaCl plus 15% polyethylene glycol (PEG) 8000 and kept overnite at 4° C. Pellets were collected by centrifugation for 3 minutes in a microcentrifuge (Beckman, Palo Alto, Calif.) at 16,000g and dissolved in 475 ul of solution containing 4.2M guanidine thiocyanate, 0.5% N-lauroylsarcosine, 0.25M TRIS-HCl (pH 8.0). 0.15 M dithiothreitol (DTT), and 1.0 μg of tRNA. Fifty microliters of 1M TRIS-HCl (pH 8.0), 100 mM EDTA, and 10% SDS was then added. The RNA was extracted twice with phenol-chloroform (1:1) at 65° C., followed by chloroform extraction at room temperature. To the upper phase, 250 μL of 7.5 M ammonium acetate was added, and nucleic acids were precipitated with 0.6 mL of 2-propanol, washed with 75% ethanol, washed with 100% ethanol, and used for reverse transcription (RT) PCR.




For detection of the HEV genome, two sets of nested primers were used that represented sequences from the 3′ region (ORF-2) of the SAR-55 genome. Primers for reverse transcription and the first PCR are shown as SEQ ID NO:99: GTATAACGGATCCACATCTCCCCTTACCTC and SEQ ID NO:100: TACAGATCTATACAACTTAACAGTCGG respectively. Primers for the second PCR are shown as SEQ ID NO: 101: GCGGCAGATCTCACCGACACCATTAGTAC and SEQ ID NO:102:




TAACCTGGATCCTTATGCCGCCCCTCTTAG respectively. The RNA pellet was dissolved in 20 μL of 0.05 M TRIS-HCl (pH 7.6), 0.06 M KCl, 0.01 M MgCl


2


, 0.001 M DTT, 40 units of RNasin (Promega Biotec, Madison, Wis.), 16 units of avian myeloblastosis virus reverse transcriptase (Promega Biotec), and 10 pmol of reverse primer and incubated 1 hour at 42° C. To 20 μL of reverse transcriptase mixture was added 100 μL of 0.01 M TRIS-HCl (pH 8.4), 0.05 M KCl, 0.0025 M MgCl


2


, 0.0002 M each dNTP, 50 pmol of direct primer, 50 pmol of reverse primer, and 4 units of AmpliTaq (Perkin-Elmer Cetus, Norwalk, Conn.) under 100 μL of light mineral oil. The HEV cDNA was amplified by 35 cycles of PCR:1 min at 94° C., 1 min at 55° C., 1 min at 72° C. The products of PCR were analyzed on 1- agarose gels. Then 5 μL of this mixture was used for the second round of amplification under the same conditions, except the extension time was increased to 3 min.




The RT-PCR products produced in all dilutions of the standard HEV feces in the range from 10


−1


to 10


−5


(

FIG. 9

) were separated on a 2% agarose gel and were detected by ethiduim bromide staining of the gel. A decrease in the amount of the specific PCR product at higher dilutions was observed and the highest dilution of the 10% fecal suspension in which the HEV genome was detected was 10


−5


. Therefore, taking into account the dilution factor, the HEV genome titer was approximately 10


6.7


per gram of feces.




In addition, only those dilutions that were shown by RT-PCR to contain the HEV genome were infectious for cynomolgus monkeys. Therefore, the infectivity titer of the standard fecal suspension and its genome titer as detected by RT-PCR were approximately the same. A similar correlation between RT-PCR and infectivity titer was found for one strain of hepatitis C virus [Cristiano et al., (1991)


Henatology,


14:51-55; Farci et al., (1991)


N. Enql. J. Med.,


25:98-104; Bukh et al., (1992);


Proc. Natl. Acad. Sci U.S.A.,


89:187-191)




EXAMPLE 12




Active Immunization Using The ORF-2 Protein As A Vaccine And Passive Immunization With Anti-HEV Positive Convalescent Plasma




Cynomolgus monkeys (


Macaca fascicularis


) that were HEV antibody negative (<1:10) in an ELISA based on the 55 kDa ORF-2 protein were individually housed under BL-2 biohazard containment and a suspension (in fetal bovine serum) of feces containing the Pakistani HEV strain SAR-55, diluted to contain 10,000 or 1,000 CID


50


, was used for intravenous inoculation of animals.




For active immunization studies, baculovirus recombinant-expressed 55 kDa ORF-2 protein was purified from 5×10


8


SF-9 cells harvested 7 days post-inoculation as described in Example 10. Three mg of the purified 55 kDa protein were precipitated with alum and eight cynomolgus monkeys were immunized by intramuscular injection with 0.5 ml of vaccine containing 50 pg of the alum-precipitated 55 kDa protein. Four monkeys received a single dose and four monkeys received two doses separated by four weeks. Primates were challenged intravenously with 1,000-10,000 CID


50


of HEV four weeks after the last immunization.




Four cynomolgus monkeys served as controls in the active immunization studies. Cyno-412 and 413 received one dose of placebo (0.5 ml of phosphate buffered saline) and cyno-397 and 849 received two doses of placebo. The control animals were challenged with 1,000-10,000 CID


50


of HEV.




For passive immunity studies, cyno-384 was infected with 0.5 ml of a 10% pooled stool suspension containing two Chinese HEV isolates, KS1-1987 and KS2-1987 and plasma was repeatedly collected from the animal during convalesence. (Yin et al. (1993)


J. Med. Virol.,


41:230-241;). Approximately 1% of the blood of cyno-396 and cyno-399 and 10% of the blood of cyno-401 and cyno-402 was replaced with convalescent plasma from cyno-384 having an HEV antibody titer of 1:10,000. Animals were challenged with 1000 CID


50


of HEV two days after infusion of the plasma. As a control, 10% of the blood of cyno-405 was replaced with anti-HEV negative plasma obtained from cyno-384 prior to infection with HEV. Cyno-405 was then challenged with 1000 CID


50


of HEV.




For both the passive and active immunization studies, percutaneous needle biopsies of the liver and samples of serum and feces were collected prior to inoculation and weekly for 15 weeks after inoculation. Sera were assayed for levels of alanine amino transferase (ALT) with commercially available tests (Metpath Inc., Rockville, Md.) and biochemical evidence of hepatitis was defined as a two-fold or greater increase in ALT. Liver biopsies were examined under code and the anti-HEV ELISA utilized was described in Example 10. RNA extraction and RT-PCR were performed as in Example 11 except that RNA from 100 μl of serum or from 100 μl of 10% fecal suspension was extracted with TRIzol Reagent (Gibco BRL, Gaithersburg, Md.) according to the manufacturer's protocol. For quantification, PCR positive serial sera or feces from each animal were combined and serially diluted in ten-fold increments in calf serum. One hundred μl of each dilution were used for RNA extraction and RT-PCR as described earlier in this Example. The PCR protocol used in this study could detect as few as 10 CID


50


of HEV per ml of serum and as few as 100 CID


50


per gram of feces.




Peak ALT values of weekly serum samples for 5 weeks prior to inoculation and for 15 weeks post-inoculation were expressed as ratios (post/pre) for each animal. The geometric mean of the ratios from the control group of animals was compared with that from the passively or actively immunized animals using the Simes test (Simes, R. J. (1986)


Biometrika,


73:751-754).




The durations of viremia and virus shedding in feces and the HEV genome titers in the control group of animals were compared with those in passively or actively immunized animals using the Wilcoxon test [Noether, G. (1967) in


Elements of nonparametric statistics


(John Wiley & Sons Inc., New York), pp. 31-36.]. The same test was used to compare the above parameters between passively and actively immunized animals.




For statistical analysis, serum samples that had <10 HEV genomes in 1 ml of serum were assigned a titer of 1:1 and fecal samples that had <100 HEV genomes in 1 g of feces were assigned a titer of 1:10.




RESULTS




Course of hepatitis E infection in nonimmunized animals.




In 3 of 5 nonimmunized animals that were challenged with HEV, biochemical evidence of hepatitis was documented by at least a two-fold increase in serum ALT values. In two animals, significant increases in ALT activity were not found. However, histopathological data documented hepatitis in all 5 animals as shown in Table 6.












TABLE 6











Histopathological, biochemical, serological, and virological profiles






of vaccinated and control animals challenged with HEV.
















Anti-HEV




Cumulative









positive




score of







plasma




histo-




HEV




HEV genome


















(%) or




pathology




Peak ALT value in U/L




antibody




serum




feces



















Animal #




55 kDA




(number of




(week)




titer at the




week de-




mean log


10






week de-




mean log


10






















and




protein




weeks de-




pre-




post-




time of




tected




titer per




tected




titer per






category




(μg)




tected)*.




inoculation




inoculation




challenge




(duration)




ml




(duration)




gram









control















405




0




10 + (8)




67 (0)




143 (9)




<1:10




1-11 (11)




3




1-11 (11)




5.7






412




0




2 + (1)




34 (0)




 45 (3)




<1:10




1-4 (4)




3




2-5 (4)




7






413




0




4 + (4)




44 (0)




261 (6)




<1:10




2-7 (6)




4.7




1-7 (7)




7






849




0




1 + (1)




79 (−2)




133 (2)




<1:10




1-4 (4)




3.7




1-4 (4)




7






397




0




3 + (3)




52 (−3)




139 (7)




<1:10




2-6 (5)




4.7




1-7 (7)




7






passive






IP











396




1%




1 + (1)‡




33 (0)




 53 (6)




 1:40




3-5 (3)




4




1-6 (6)




5.7






399




1%




0 (0)




69 (0)




 63 (11)




 1:40




2-4 (3)




3




1-4 (4)




4






401




10%




0 (0)




55 (0)




 45 (5)




1:200




3 (1)




3.6




1-3 (3)




5.7






402




10%




0 (0)




59 (0)




 35 (2)




1:200




4-6 (3)




1




2-6 (5)




5.7






active IP











003




50 μg




0 (0)




34 (−3)




 50 (6)




1:10,000




0




<1




2-4 (3)




3






009




50 μg




0 (0)




34 (−2)




 38 (6)




1:1,000




0




<1




0




<2






013


§






50 μg




0 (0)




44 (−3)




 36 (7)




1:100




0




<1




1-2 (2)




3






414




50 μg




0 (0)




65 (0)




 73 (8)




1:1,000




0




<1




2 (1)




2






398




2 × 50 μg




0 (0)




31 (0)




 41 (2)




1:10,000




0




<1




0




<2






407




2 × 50 μg




0 (0)




150 (0)




213 (4)




1:10,000




0




<1




0




<2











*Necro-inflammatory changes in the liver were rated as 1+, 2+, 3+, 4+ and the weekly scores were summed.















Immunoprophylaxis















Necro-inflammatory changes rated 1+ were detected during two weeks in cyno-396, however, they were consistent with viral hepatitis only during one week.












§


Cyno 013 died 9 weeks after challenge.













Necro-inflammatory changes ranged between 1+ and 2+ on a scale of 1+ to 4+ and were temporally associated with elevations of ALT activities in those animals with such elevations.




All control animals seroconverted to HEV 3-5 weeks post-challenge and developed maximum HEV antibody titers ranging from 1:1,000 to 1:32,000. There was a good correlation between the severity of infection, hepatitis, and the level of anti-HEV response. Cyno-405, which had the highest cumulative score for hepatitis, also had the longest period of viremia and viral excretion and the highest level of anti-HEV (Table 6). The duration of viral shedding in feces was the same as, or longer than, that of the viremia. For all of the control animals, titers of the HEV genome in serum were lower (10


−3


-10


−4.7


) than the titers in feces (10


−5.7


-10


−7


). In all five of these animals, viremia and virus shedding in feces were detected for 4-11 weeks and for an average of 4.2 weeks after seroconversion (range 2-9 weeks).




Passive immunization. Cyno-396 and 399, which had approximately 1% of their blood replaced with anti-HEV positive convalescent plasma, had an HEV antibody titer of 1:40 when it was determined two days post-transfusion (at the time of challenge) (Table 6). A two-fold fall in HEV antibody titer was observed in both animals 1 week post-transfusion and HEV antibodies fell below the detectable level (<1:10) by 2 weeks post-transfusion. Anti-HEV was again detected 5 weeks post-challenge in cyno-396 and 4 weeks post-challenge in cyno-399, indicating that infection with HEV had occurred. The maximum HEV antibody titer (1:8,000) was reached 9-10 weeks post-challenge. Neither cynomolgus monkey demonstrated a significant elevation of ALT activity after challenge. However, histologic evidence of hepatitis was detected in cyno-396 and the HEV genome was detected in serum and feces from both animals (Table 6).




Cyno-401 and 402 had approximately 10% of their blood replaced with convalescent plasma. Two days post-transfusion, at the time of challenge, the HEV antibody titer in both cynomolgus monkeys was 1:200 (Table 7).












TABLE 7











HEV antibody profiles in control and immunized cynomolgus monkeys.















HEV antibody





HEV antobody























max.





max.




max.




max.










titer





titer




titer




titer







HEV antibody




Passively




titer at




(week




Actively




(week




(week




(week





















titer (week




max.




immun-




the time




after




immun-




after 1st




after 2nd




after






Control




first de-




titer




ized




of chal-




chal-




ized




immuni-




immuni-




chal-






animals




tected)




(week)




animals




lenge




lenge)




animals




zation)




zation)




lenge)









cyno-405




1:80




1:32,000




cyno-396




1:40




1:8,000




cyno-003




1:10,000





1:10,000







(3)




(9)






(10)





(3)





(5)






cyno-412




1:100




1:10,000




cyno-399




1:40




1:8,000




cyno-009




1:10,000





1:10,000







(5)




(7)






(9)





(3)





(1)






cyno-413




1:100




1:10,000




cyno-401




1:200




1:4,000




cyno-013




1:100





1:10,000







(5)




(7)






(6)





(2)





(3)






cyno-849




1:100




1:1,000




cyno-402




1:200




1:80




cyno-414




1:1,000





1:1,000







(3)




(5)






(12)





(3)





(0)






cyno-397




1:100




1:10,000







cyno-398




1:1,000




1:10,000




1:10,000







(3)




(7)








(3)




(5)




(0)












cyno-407




1:1,000




1:10,000




1:10,000













(4)




(5)




(0)











Anti-HEV was detected continuously in both animals during the 15 weeks after challenge and reached a maximum titer of 1:4,000 in cyno-401 but only 1:80 in cyno-402.













of 1:4,000 in cyno-401 but only 1:80 in cyno-402. Biochemical and histologic analyses did not reveal hepatitis in either animal. However, in both animals, HEV viremia and fecal shedding of virus were observed indicating that infection had occurred (Table 6). Thus, passive immunoprophylaxis that achieved a higher titer of antibody protected cynomolgus monkeys against hepatitis after challenge with HEV.




Active immunization. Four primates immunized with one 50 μg dose of the 55 kDa protein developed antibody to the recombinant protein ranging in titer from 1:100 to 1:10,000 (Table 7). One (cyno 013) died of an anesthesia accident 9 weeks after challenge and is included in the analyses (Table 6). The four animals that received two doses of the antigen developed HEV antibodies with titers of 1:10,000. Two of the four monkeys died following intravenous challenge with HEV. This may have also been the result of an anesthesia accident but the exact etiology could not be determined. These two monkeys were deleted from further analyses. None of the 6 remaining animals developed abnormal ALT levels or histologic evidence of hepatitis following challenge (Table 6). Cynomolgus monkeys immunized with either 1 or 2 doses of the 55 kDa protein did not develop viremia. However, 3 of 4 animals that received one dose of the immunogen excreted virus in their feces. In contrast, virus shedding was not observed in either of the two challenged animals that had received two doses of the vaccine.




Most of the actively immunized animals developed higher HEV antibody titers than did passively immunized animals. However, cyno-013 had an HEV antibody titer of 1:100 at the time of challenge, compared with a titer of 1:200 in two animals immunized passively with anti-HEV plasma. Cyno-013, however, demonstrated greater protection against HEV infection than the passively immunized animals. Cyno-009, which had an HEV antibody titer of 1:1,000 at the time of challenge, was completely protected against hepatitis and HEV infection (Table 6). In contrast, cyno-003 was infected and shed HEV in feces, even though it had an HEV antibody titer of 1:10,000 at the time of challenge. However, neither hepatitis nor viremia was detected in this animal or in other cynomolgus monkeys that received one dose of immunogen and had HEV antibody titers of 1:10,000 or greater.




Comparison of course of HEV infection in control and immunized animals.




As measured by histopathology, all immunized animals, with the exception of one of the passively immunized monkeys, were protected against hepatitis after intravenous challenge with HEV. Comparison of mean values for severity of hepatitis and level of viral replication between the control group and the passively and actively immunized animals indicated that, in general, the severity of infection was inversely related to the HEV antibody titer at the time of challenge and diminished in the order: unimmunizedopassive immunization (1%)>passive immunization (10%)>active immunization (1 dose)>active immunization (2 doses) (Tables 6,8). However, the number of animals in each of the two subgroups of passively and actively immunized animals was not sufficient to permit statistical analysis. Therefore, statistical analysis was performed for combined passively immunized and combined actively immunized groups respectively in comparison with the combined control groups. The results of this analysis are presented in Table 8.












TABLE 3









Summary of mean values of HEV infection in control and immunized animals.










































*Geometric mean















Passive and active immunoprophylaxis










α- P < 0.01










β- P < 0.05










γ- not significant













and they show that the histopathology scores and duration of histologic changes in the control group were statistically different from those of passively or actively immunized animals. The higher post-/pre-inoculation ratios of peak ALT values in the control group were statistically significant when compared with those of the passively or actively immunized animals, indicating protection against biochemical manifestations of hepatitis in both groups of immunized animals. The duration of viremia and the titer of HEV in the feces were significantly lower in both groups of immunized animals than in the control group. Differences in the duration of virus shedding and titer of HEV in the serum, however, were not statistically different between the control group and the passively immunized group, although these parameters were significantly different when the control group was compared with the actively immunized group. Significant differences were also found between passively and actively immunized groups of animals for duration of viremia and fecal shedding as well as for HEV titers.




In sum, the results presented in Tables 6-8 show that both passively and actively acquired HEV antibodies protected cynomolgus monkeys against hepatitis following challenge with virulent HEV. Although all 5 nonimmunized cynomolgus monkeys developed histologic evidence of hepatitis when challenged with 1,000-10,000 CID


50


of SAR-55, both animals with passively acquired antibody titers of 1:200 were protected from hepatitis and one of two animals with an antibody titer as low as 1:40 also did not develop hepatitis.




However, it should be noted that actively immunized animals demonstrated complete protection against hepatitis and more effective resistance to HEV infection than did passively immunized animals. For example, in contrast to results obtained from the passively immunized animals, viremia was not detected in actively immunized animals after challenge with HEV. An HEV antibody titer as high as 1:10,000 could be achieved in cynomolgus monkeys after one or two immunizations with the recombinant 55 kDa protein. Although one monkey (013) developed a titer of 1:100 after active immunization, this level still prevented hepatitis and viremia.




The active immunization studies also demonstrated that while a single dose of vaccine prevented HEV viremia, viral shedding in feces was still detected. However, two doses of vaccine were observed to prevent all signs of hepatitis and HEV infection. These results thus suggest that a single dose of vaccine administered, for example, to individuals before foreign travel would protect them from hepatitis E in high risk environments.




Finally, it is noted that the results presented are very similar to results reported previously for passive and active immunoprophylaxis of nonhuman primates against hepatitis A: passive immunoprophylaxis prevented hepatitis but not infection whereas vaccination prevented not only hepatitis but infection with HAV as well (Purcell, R. H. et al. (1992)


Vaccine,


10:5148-5149). It is of interest that the study of immunoprophylaxis for HEV presented herein parallels the previous study of immunoprophylaxis against HAV, both in determination of the titer of antibody that protected (<1:100) and in outcome following intravenous challenge with virulent virus. Since other studies have demonstrated efficacy of comparable titers of passively and actively acquired anti-HAV in humans and have confirmed the predictive value of studies of primates in hepatitis research (Stapleton, J., et al. (1985)


Gastroenterology


89:637-642; Innis, B. L., et al. (1992)


Vaccine,


10: S159), it is therefore highly likely that these results in cynomolgus monkeys will be predictive of protection in humans.




EXAMPLE 13




Direct Expression In Yeast Of Complete ORF-2 Protein And Lower Molecular Weight Fragments




Four cDNA ORF-2 fragments coding for:




1. complete ORF-2 protein (aa 1-660, MW 70979), fragment 1778-1703. (where the fragment numbers refer to the primer numbers given below)




2. ORF-2 protein starting from 34th aa (aa 34-660, MW 67206), fragment 1779-1703.




3. ORF-2 protein starting from 96th aa (aa 96-660, MW 60782), fragment 1780-1703.




4. ORF-2 protein starting from 124th aa (aa 124-660, MW 58050), fragment 1781-1703.




were obtained using PCR by using plasmid P63-2 as template and the synthetic oligonucleotides shown below: SEQ ID NO.:103 (reverse primer #1703) GCACAACCTAGGTTACTATAACTCCCGAGTTTTACC, SEQ ID NO.:104 (direct primer #1778) GGGTTCCCTAGGATGCGCCCTCGGCCTATTTTG, SEQ ID NO.:105 (direct primer #1779) CGTGGGCCTAGGAGCGGCGGTTCCGGCGGTGGT, SEQ ID NO.:106 (direct primer #1780) GCTTGGCCTAGGCAGGCCCAGCGCCCCGCCGCT and SEQ ID NO.:107 (direct primer #1781) CCGCCACCTAGGGATGTTGACTCCCGCGGCGCC.




All sequences shown in SEQ ID NOs: 103-107 contain artificial sequence CCTAGG at their 5′ ends preceded by 4 nucleotides. The artificial sequence was a recognition site for Avr II (Bln I) restriction enzyme. Synthesized PCR fragments were cleaved with BlnI and cloned in the AvrII site of pPIC9 vector (

FIG. 10

) (Invitrogen). Correct orientation of the fragments was confirmed by restriction analysis, using asymmetric EcoRI site present in ORF-2 sequences and in the vector. Purified recombinant plasmids pPIC9-1778 (containing fragment 1778-1703); pPIC9-1779 (containing fragment 1779-1703); pPIC9-1780 (containing fragment 1780-1703) and pPIC9-1781 (containing fragment 1781-1730) were used for transformation of yeast spheroplast (Picha strain) according to Invitrogen protocol. Screening of recombinant clones and analysis of expression were performed using the same protocol. These expressed proteins may be used as immunogens in vaccines and as antigens in immunoassays as described in the present application. Finally, those of skill in the art would recognize that the vector and strain of yeast used in the above example could be replaced by other vectors (e.g. pHIL-Fl; Invitrogen) or strains of yeast (e.g.






Saccharomyces Cerevisiae


).




EXAMPLE 14




Purification and Amino Terminal Sequence Analysis of HEV ORF-2 Gene Products Synthesized in SF-9 Insect Cells Infected With Recombinant Baculovirus 63-2-IV-2




As described in Example 10, SF-9 cells were infected with recombinant baculovirus 63-2-IV-2 and harvested seven days post-inoculation. The predominant protein band present on SDS-PAGE of the insect cell lysate was approximately 55 kDa in molecular weight. Further purification of this 55 kDa band was accomplished by ion-exchange column chromatography using DEAE-sepharose with a 150-450 mM NaCl gradient. DEAE fractions were assayed for the presence of the 55 kDa band by SDS-PAGE followed by Coomassie blue staining. The peak fraction was then resolved by polyacrylamide gel electrophoresis in the absence of SDS into three bands of 55 kDa, 61 kDa and a band of intermediate molecular weight. Analysis of each protein band from the polyacrylamide gel by amino-terminal microprotein sequencing revealed that the 55 and 61 kDa proteins shared a unique N-terminus at Ala-112 of SEQ ID NO:2. It is believed that the size differences in the two ORF-2 cleavage products may reflect either different glycosylation patterns or a COOH-terminal cleavage of the larger product.




The third intermediate protein on the polyacrylamide gel was shown to be a baculovirus chitinase protein. The 55 and 61 kDa ORF-2 proteins were resolved into a single symmetrical peak fraction devoid of any chitinase by subjecting peak DEAE fractions to reverse phase HPLC using a micropore system with NaCl and acetonitrile solvents.




EXAMPLE 15




Direct Expression of 55 and 61 kDa Cleavace Products




A cDNA ORF-2 fragment coding for ORF-2 protein starting from the 112th amino acid (amino acids 112-660 of ORF-2) was obtained by PCR using plasmid p63-2 as the template. The cDNA fragment was then inserted into a PBlueBac-3Transfer vector at the BamHI-PstI site in the vector. SF9 insect cells are infected with the recombinant baculovirus generated from this vector and insect cell lysates are analyzed for the presence of the 55 and 61 kDa ORF-2 proteins by Coomassie blue staining of polyacrylamide gels. The directly expressed protein(s) may be used as immunogens in vaccines and as antigens in immunoassays as described herein.







107





1693 AMINO ACID RESIDUES


AMINO ACID


UNKNOWN


UNKNOWN




unknown



1
Met Glu Ala His Gln Phe Ile Lys Ala Pro Gly Ile Thr Thr Ala
1 5 10 15
Ile Glu Gln Ala Ala Leu Ala Ala Ala Asn Ser Ala Leu Ala Asn
20 25 30
Ala Val Val Val Arg Pro Phe Leu Ser His Gln Gln Ile Glu Ile
35 40 45
Leu Ile Asn Leu Met Gln Pro Arg Gln Leu Val Phe Arg Pro Glu
50 55 60
Val Phe Trp Asn His Pro Ile Gln Arg Val Ile His Asn Glu Leu
65 70 75
Glu Leu Tyr Cys Arg Ala Arg Ser Gly Arg Cys Leu Glu Ile Gly
80 85 90
Ala His Pro Arg Ser Ile Asn Asp Asn Pro Asn Val Val His Arg
95 100 105
Cys Phe Leu Arg Pro Ala Gly Arg Asp Val Gln Arg Trp Tyr Thr
110 115 120
Ala Pro Thr Arg Gly Pro Ala Ala Asn Cys Arg Arg Ser Ala Leu
125 130 135
Arg Gly Leu Pro Ala Ala Asp Arg Thr Tyr Cys Phe Asp Gly Phe
140 145 150
Ser Gly Cys Asn Phe Pro Ala Glu Thr Gly Ile Ala Leu Tyr Ser
155 160 165
Leu His Asp Met Ser Pro Ser Asp Val Ala Glu Ala Met Phe Arg
170 175 180
His Gly Met Thr Arg Leu Tyr Ala Ala Leu His Leu Pro Pro Glu
185 190 195
Val Leu Leu Pro Pro Gly Thr Tyr Arg Thr Ala Ser Tyr Leu Leu
200 205 210
Ile His Asp Gly Arg Arg Val Val Val Thr Tyr Glu Gly Asp Thr
215 220 225
Ser Ala Gly Tyr Asn His Asp Val Ser Asn Leu Arg Ser Trp Ile
230 235 240
Arg Thr Thr Lys Val Thr Gly Asp His Pro Leu Val Ile Glu Arg
245 250 255
Val Arg Ala Ile Gly Cys His Phe Val Leu Leu Leu Thr Ala Ala
260 265 270
Pro Glu Pro Ser Pro Met Pro Tyr Val Pro Tyr Pro Arg Ser Thr
275 280 285
Glu Val Tyr Val Arg Ser Ile Phe Gly Pro Gly Gly Thr Pro Ser
290 295 300
Leu Phe Pro Thr Ser Cys Ser Thr Lys Ser Thr Phe His Ala Val
305 310 315
Pro Ala His Ile Trp Asp Arg Leu Met Leu Phe Gly Ala Thr Leu
320 325 330
Asp Asp Gln Ala Phe Cys Cys Ser Arg Leu Met Thr Tyr Leu Arg
335 340 345
Gly Ile Ser Tyr Lys Val Thr Val Gly Thr Leu Val Ala Asn Glu
350 355 360
Gly Trp Asn Ala Ser Glu Asp Ala Leu Thr Ala Val Ile Thr Ala
365 370 375
Ala Tyr Leu Thr Ile Cys His Gln Arg Tyr Leu Arg Thr Gln Ala
380 385 390
Ile Ser Lys Gly Met Arg Arg Leu Glu Arg Glu His Ala Gln Lys
395 400 405
Phe Ile Thr Arg Leu Tyr Ser Trp Leu Phe Glu Lys Ser Gly Arg
410 415 420
Asp Tyr Ile Pro Gly Arg Gln Leu Glu Phe Tyr Ala Gln Cys Arg
425 430 435
Arg Trp Leu Ser Ala Gly Phe His Leu Asp Pro Arg Val Leu Val
440 445 450
Phe Asp Glu Ser Ala Pro Cys His Cys Arg Thr Ala Ile Arg Lys
455 460 465
Ala Val Ser Lys Phe Cys Cys Phe Met Lys Trp Leu Gly Gln Glu
470 475 480
Cys Thr Cys Phe Leu Gln Pro Ala Glu Gly Val Val Gly Asp Gln
485 490 495
Gly His Asp Asn Glu Ala Tyr Glu Gly Ser Asp Val Asp Pro Ala
500 505 510
Glu Ser Ala Ile Ser Asp Ile Ser Gly Ser Tyr Val Val Pro Gly
515 520 525
Thr Ala Leu Gln Pro Leu Tyr Gln Ala Leu Asp Leu Pro Ala Glu
530 535 540
Ile Val Ala Arg Ala Gly Arg Leu Thr Ala Thr Val Lys Val Ser
545 550 555
Gln Val Asp Gly Arg Ile Asp Cys Glu Thr Leu Leu Gly Asn Lys
560 565 570
Thr Phe Arg Thr Ser Phe Val Asp Gly Ala Val Leu Glu Thr Asn
575 580 585
Gly Pro Glu Arg His Asn Leu Ser Phe Asp Ala Ser Gln Ser Thr
590 595 600
Met Ala Ala Gly Pro Phe Ser Leu Thr Tyr Ala Ala Ser Ala Ala
605 610 615
Gly Leu Glu Val Arg Tyr Val Ala Ala Gly Leu Asp His Arg Ala
620 625 630
Val Phe Ala Pro Gly Val Ser Pro Arg Ser Ala Pro Gly Glu Val
635 640 645
Thr Ala Phe Cys Ser Ala Leu Tyr Arg Phe Asn Arg Glu Ala Gln
650 655 660
Arg Leu Ser Leu Thr Gly Asn Phe Trp Phe His Pro Glu Gly Leu
665 670 675
Leu Gly Pro Phe Ala Pro Phe Ser Pro Gly His Val Trp Glu Ser
680 685 690
Ala Asn Pro Phe Cys Gly Glu Ser Thr Leu Tyr Thr Arg Thr Trp
695 700 705
Ser Glu Val Asp Ala Val Pro Ser Pro Ala Gln Pro Asp Leu Gly
710 715 720
Phe Thr Ser Glu Pro Ser Ile Pro Ser Arg Ala Ala Thr Pro Thr
725 730 735
Pro Ala Ala Pro Leu Pro Pro Pro Ala Pro Asp Pro Ser Pro Thr
740 745 750
Leu Ser Ala Pro Ala Arg Gly Glu Pro Ala Pro Gly Ala Thr Ala
755 760 765
Arg Ala Pro Ala Ile Thr His Gln Thr Ala Arg His Arg Arg Leu
770 775 780
Leu Phe Thr Tyr Pro Asp Gly Ser Lys Val Phe Ala Gly Ser Leu
785 790 795
Phe Glu Ser Thr Cys Thr Trp Leu Val Asn Ala Ser Asn Val Asp
800 805 810
His Arg Pro Gly Gly Gly Leu Cys His Ala Phe Tyr Gln Arg Tyr
815 820 825
Pro Ala Ser Phe Asp Ala Ala Ser Phe Val Met Arg Asp Gly Ala
830 835 840
Ala Ala Tyr Thr Leu Thr Pro Arg Pro Ile Ile His Ala Val Ala
845 850 855
Pro Asp Tyr Arg Leu Glu His Asn Pro Lys Arg Leu Glu Ala Ala
860 865 870
Tyr Arg Glu Thr Cys Ser Arg Leu Gly Thr Ala Ala Tyr Pro Leu
875 880 885
Leu Gly Thr Gly Ile Tyr Gln Val Pro Ile Gly Pro Ser Phe Asp
890 895 900
Ala Trp Glu Arg Asn His Arg Pro Gly Asp Glu Leu Tyr Leu Pro
905 910 915
Glu Leu Ala Ala Arg Trp Phe Glu Ala Asn Arg Pro Thr Cys Pro
920 925 930
Thr Leu Thr Ile Thr Glu Asp Val Ala Arg Thr Ala Asn Leu Ala
935 940 945
Ile Glu Leu Asp Ser Ala Thr Asp Val Gly Arg Ala Cys Ala Gly
950 955 960
Cys Arg Val Thr Pro Gly Val Val Gln Tyr Gln Phe Thr Ala Gly
965 970 975
Val Pro Gly Ser Gly Lys Ser Arg Ser Ile Thr Gln Ala Asp Val
980 985 990
Asp Val Val Val Val Pro Thr Arg Glu Leu Arg Asn Ala Trp Arg
995 1000 1005
Arg Arg Gly Phe Ala Ala Phe Thr Pro His Thr Ala Ala Arg Val
1010 1015 1020
Thr Gln Gly Arg Arg Val Val Ile Asp Glu Ala Pro Ser Leu Pro
1025 1030 1035
Pro His Leu Leu Leu Leu His Met Gln Arg Ala Ala Thr Val His
1040 1045 1050
Leu Leu Gly Asp Pro Asn Gln Ile Pro Ala Ile Asp Phe Glu His
1055 1060 1065
Ala Gly Leu Val Pro Ala Ile Arg Pro Asp Leu Ala Pro Thr Ser
1070 1075 1080
Trp Trp His Val Thr His Arg Cys Pro Ala Asp Val Cys Glu Leu
1085 1090 1095
Ile Arg Gly Ala Tyr Pro Met Ile Gln Thr Thr Ser Arg Val Leu
1100 1105 1110
Arg Ser Leu Phe Trp Gly Glu Pro Ala Val Gly Gln Lys Leu Val
1115 1120 1125
Phe Thr Gln Ala Ala Lys Ala Ala Asn Pro Gly Ser Val Thr Val
1130 1135 1140
His Glu Ala Gln Gly Ala Thr Tyr Thr Glu Thr Thr Ile Ile Ala
1145 1150 1155
Thr Ala Asp Ala Arg Gly Leu Ile Gln Ser Ser Arg Ala His Ala
1160 1165 1170
Ile Val Ala Leu Thr Arg His Thr Glu Lys Cys Val Ile Ile Asp
1175 1180 1185
Ala Pro Gly Leu Leu Arg Glu Val Gly Ile Ser Asp Ala Ile Val
1190 1195 1200
Asn Asn Phe Phe Leu Ala Gly Gly Glu Ile Gly His Gln Arg Pro
1205 1210 1215
Ser Val Ile Pro Arg Gly Asn Pro Asp Ala Asn Val Asp Thr Leu
1220 1225 1230
Ala Ala Phe Pro Pro Ser Cys Glu Ile Ser Ala Phe His Glu Leu
1235 1240 1245
Ala Glu Glu Leu Gly His Arg Pro Ala Pro Val Ala Ala Val Leu
1250 1255 1260
Pro Pro Cys Pro Glu Leu Glu Gln Gly Leu Leu Tyr Leu Pro Gln
1265 1270 1275
Glu Leu Thr Thr Cys Asp Ser Val Val Thr Phe Glu Leu Thr Asp
1280 1285 1290
Ile Val His Cys Arg Met Ala Ala Pro Ser Gln Arg Lys Ala Val
1295 1300 1305
Leu Ser Thr Leu Val Gly Arg Tyr Gly Arg Arg Thr Lys Leu Tyr
1310 1315 1320
Asn Ala Ser His Ser Asp Val Arg Asp Ser Leu Ala Arg Phe Ile
1325 1330 1335
Pro Ala Ile Gly Pro Val Gln Val Thr Thr Cys Glu Leu Tyr Glu
1340 1345 1350
Leu Glu Glu Ala Met Val Glu Lys Gly Gln Asp Gly Ser Ala Val
1355 1360 1365
Leu Glu Leu Asp Leu Cys Ser Arg Asp Val Ser Arg Ile Thr Phe
1370 1375 1380
Phe Gln Lys Asp Cys Asn Lys Phe Thr Thr Gly Glu Thr Ile Ala
1385 1390 1395
His Gly Lys Val Gly Gln Gly Ile Ser Ala Trp Ser Lys Thr Phe
1400 1405 1410
Cys Ala Leu Phe Gly Pro Trp Phe Arg Ala Ile Glu Lys Ala Ile
1415 1420 1425
Leu Ala Leu Leu Pro Gln Gly Val Phe Tyr Gly Asp Ala Phe Asp
1430 1435 1440
Asp Thr Val Phe Ser Ala Ala Val Ala Ala Ala Lys Ala Ser Met
1445 1450 1455
Val Phe Glu Asn Asp Phe Ser Glu Phe Asp Ser Thr Gln Asn Asn
1460 1465 1470
Phe Ser Leu Gly Leu Glu Cys Ala Ile Met Glu Glu Cys Gly Met
1475 1480 1485
Pro Gln Trp Leu Ile Arg Leu Tyr His Leu Ile Arg Ser Ala Trp
1490 1495 1500
Ile Leu Gln Ala Pro Lys Glu Ser Leu Arg Gly Phe Trp Lys Lys
1505 1510 1515
His Ser Gly Glu Pro Gly Thr Leu Leu Trp Asn Thr Val Trp Asn
1520 1525 1530
Met Ala Val Ile Thr His Cys Tyr Asp Phe Arg Asp Leu Gln Val
1535 1540 1545
Ala Ala Phe Lys Gly Asp Asp Ser Ile Val Leu Cys Ser Glu Tyr
1550 1555 1560
Arg Gln Ser Pro Gly Ala Ala Val Leu Ile Ala Gly Cys Gly Leu
1565 1570 1575
Lys Leu Lys Val Asp Phe Arg Pro Ile Gly Leu Tyr Ala Gly Val
1580 1585 1590
Val Val Ala Pro Gly Leu Gly Ala Leu Pro Asp Val Val Arg Phe
1595 1600 1605
Ala Gly Arg Leu Thr Glu Lys Asn Trp Gly Pro Gly Pro Glu Arg
1610 1615 1620
Ala Glu Gln Leu Arg Leu Ala Val Ser Asp Phe Leu Arg Lys Leu
1625 1630 1635
Thr Asn Val Ala Gln Met Cys Val Asp Val Val Ser Arg Val Tyr
1640 1645 1650
Gly Val Ser Pro Gly Leu Val His Asn Leu Ile Glu Met Leu Gln
1655 1660 1665
Ala Val Ala Asp Gly Lys Ala His Phe Thr Glu Ser Val Lys Pro
1670 1675 1680
Val Leu Asp Leu Thr Asn Ser Ile Leu Cys Arg Val Glu
1685 1690






660 amino acid residues


amino acid


unknown


unknown




unknown



2
Met Arg Pro Arg Pro Ile Leu Leu Leu Leu Leu Met Phe Leu Pro
1 5 10 15
Met Leu Pro Ala Pro Pro Pro Gly Gln Pro Ser Gly Arg Arg Arg
20 25 30
Gly Arg Arg Ser Gly Gly Ser Gly Gly Gly Phe Trp Gly Asp Arg
35 40 45
Val Asp Ser Gln Pro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn
50 55 60
Pro Phe Ala Pro Asp Val Thr Ala Ala Ala Gly Ala Gly Pro Arg
65 70 75
Val Arg Gln Pro Ala Arg Pro Leu Gly Ser Ala Trp Arg Asp Gln
80 85 90
Ala Gln Arg Pro Ala Ala Ala Ser Arg Arg Arg Pro Thr Thr Ala
95 100 105
Gly Ala Ala Pro Leu Thr Ala Val Ala Pro Ala His Asp Thr Pro
110 115 120
Pro Val Pro Asp Val Asp Ser Arg Gly Ala Ile Leu Arg Arg Gln
125 130 135
Tyr Asn Leu Ser Thr Ser Pro Leu Thr Ser Ser Val Ala Thr Gly
140 145 150
Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu Ser Pro Leu Leu Pro
155 160 165
Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala Thr Glu Ala Ser
170 175 180
Asn Tyr Ala Gln Tyr Arg Val Ala Arg Ala Thr Ile Arg Tyr Arg
185 190 195
Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser Ile Ser
200 205 210
Phe Tyr Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met Asn
215 220 225
Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile
230 235 240
Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn
245 250 255
Gln Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu
260 265 270
Ala Thr Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val
275 280 285
Asn Ser Tyr Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu
290 295 300
Asp Phe Ala Leu Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn
305 310 315
Thr Asn Thr Arg Val Ser Arg Tyr Ser Ser Thr Ala Arg His Arg
320 325 330
Leu Arg Arg Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala
335 340 345
Ala Thr Arg Phe Met Lys Asp Leu Tyr Phe Thr Ser Thr Asn Gly
350 355 360
Val Gly Glu Ile Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu
365 370 375
Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser
380 385 390
Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn
395 400 405
Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln
410 415 420
Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp Leu Gly Glu
425 430 435
Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp
440 445 450
Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu
455 460 465
Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr
470 475 480
Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Val Ser
485 490 495
Asp Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val
500 505 510
Ala Arg Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro
515 520 525
Leu Ser Thr Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro
530 535 540
Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala
545 550 555
Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Leu Leu
560 565 570
Val Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr
575 580 585
Thr Ser Leu Gly Ala Gly Pro Val Ser Ile Ser Ala Val Ala Val
590 595 600
Leu Ala Pro His Ser Val Leu Ala Leu Leu Glu Asp Thr Met Asp
605 610 615
Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys
620 625 630
Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val Ala
635 640 645
Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Leu
650 655 660






123 amino acid residues


amino acid


unknown


unknown




unknown



3
Met Asn Asn Met Ser Phe Ala Ala Pro Met Gly Ser Arg Pro Cys
1 5 10 15
Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys
20 25 30
Cys Pro Arg His Arg Pro Val Ser Arg Leu Ala Ala Val Val Gly
35 40 45
Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu
50 55 60
Ile Leu Ser Pro Ser Gln Ser Pro Ile Phe Ile Gln Pro Thr Pro
65 70 75
Ser Pro Pro Met Ser Pro Leu Arg Pro Gly Leu Asp Leu Val Phe
80 85 90
Ala Asn Pro Pro Asp His Ser Ala Pro Leu Gly Val Thr Arg Pro
95 100 105
Ser Ala Pro Pro Leu Pro His Val Val Asp Leu Pro Gln Leu Gly
110 115 120
Pro Arg Arg






7168 base pairs


nucleic acid


single


linear




unknown



4
AGGCAGACCA CATATGTGGT CGATGCCATG GAGGCCCATC AGTTTATCAA 50
GGCTCCTGGC ATCACTACTG CTATTGAGCA GGCTGCTCTA GCAGCGGCCA 100
ACTCTGCCCT TGCGAATGCT GTGGTAGTTA GGCCTTTTCT CTCTCACCAG 150
CAGATTGAGA TCCTTATTAA CCTAATGCAA CCTCGCCAGC TTGTTTTCCG 200
CCCCGAGGTT TTCTGGAACC ATCCCATCCA GCGTGTTATC CATAATGAGC 250
TGGAGCTTTA CTGTCGCGCC CGCTCCGGCC GCTGCCTCGA AATTGGTGCC 300
CACCCCCGCT CAATAAATGA CAATCCTAAT GTGGTCCACC GTTGCTTCCT 350
CCGTCCTGCC GGGCGTGATG TTCAGCGTTG GTATACTGCC CCTACCCGCG 400
GGCCGGCTGC TAATTGCCGG CGTTCCGCGC TGCGCGGGCT CCCCGCTGCT 450
GACCGCACTT ACTGCTTCGA CGGGTTTTCT GGCTGTAACT TTCCCGCCGA 500
GACGGGCATC GCCCTCTATT CTCTCCATGA TATGTCACCA TCTGATGTCG 550
CCGAGGCTAT GTTCCGCCAT GGTATGACGC GGCTTTACGC TGCCCTCCAC 600
CTCCCGCCTG AGGTCCTGTT GCCCCCTGGC ACATACCGCA CCGCGTCGTA 650
CTTGCTGATC CATGACGGCA GGCGCGTTGT GGTGACGTAT GAGGGTGACA 700
CTAGTGCTGG TTATAACCAC GATGTTTCCA ACCTGCGCTC CTGGATTAGA 750
ACCACTAAGG TTACCGGAGA CCACCCTCTC GTCATCGAGC GGGTTAGGGC 800
CATTGGCTGC CACTTTGTCC TTTTACTCAC GGCTGCTCCG GAGCCATCAC 850
CTATGCCCTA TGTCCCTTAC CCCCGGTCTA CCGAGGTCTA TGTCCGATCG 900
ATCTTCGGCC CGGGTGGCAC CCCCTCCCTA TTTCCAACCT CATGCTCCAC 950
CAAGTCGACC TTCCATGCTG TCCCTGCCCA TATCTGGGAC CGTCTCATGT 1000
TGTTCGGGGC CACCCTAGAT GACCAAGCCT TTTGCTGCTC CCGCCTAATG 1050
ACTTACCTCC GCGGCATTAG CTACAAGGTT ACTGTGGGCA CCCTTGTTGC 1100
CAATGAAGGC TGGAACGCCT CTGAGGACGC TCTTACAGCT GTCATCACTG 1150
CCGCCTACCT TACCATCTGC CACCAGCGGT ACCTCCGCAC TCAGGCTATA 1200
TCTAAGGGGA TGCGTCGCCT GGAGCGGGAG CATGCTCAGA AGTTTATAAC 1250
ACGCCTCTAC AGTTGGCTCT TTGAGAAGTC CGGCCGTGAT TATATCCCCG 1300
GCCGTCAGTT GGAGTTCTAC GCTCAGTGTA GGCGCTGGCT CTCGGCCGGC 1350
TTTCATCTTG ACCCACGGGT GTTGGTTTTT GATGAGTCGG CCCCCTGCCA 1400
CTGTAGGACT GCGATTCGTA AGGCGGTCTC AAAGTTTTGC TGCTTTATGA 1450
AGTGGCTGGG CCAGGAGTGC ACCTGTTTTC TACAACCTGC AGAAGGCGTC 1500
GTTGGCGACC AGGGCCATGA CAACGAGGCC TATGAGGGGT CTGATGTTGA 1550
CCCTGCTGAA TCCGCTATTA GTGACATATC TGGGTCCTAC GTAGTCCCTG 1600
GCACTGCCCT CCAACCGCTT TACCAAGCCC TTGACCTCCC CGCTGAGATT 1650
GTGGCTCGTG CAGGCCGGCT GACCGCCACA GTAAAGGTCT CCCAGGTCGA 1700
CGGGCGGATC GATTGTGAGA CCCTTCTCGG TAATAAAACC TTCCGCACGT 1750
CGTTTGTTGA CGGGGCGGTT TTAGAGACTA ATGGCCCAGA GCGCCACAAT 1800
CTCTCTTTTG ATGCCAGTCA GAGCACTATG GCCGCCGGCC CTTTCAGTCT 1850
CACCTATGCC GCCTCTGCTG CTGGGCTGGA GGTGCGCTAT GTCGCCGCCG 1900
GGCTTGACCA CCGGGCGGTT TTTGCCCCCG GCGTTTCACC CCGGTCAGCC 1950
CCTGGCGAGG TCACCGCCTT CTGTTCTGCC CTATACAGGT TTAATCGCGA 2000
GGCCCAGCGC CTTTCGCTGA CCGGTAATTT TTGGTTCCAT CCTGAGGGGC 2050
TCCTTGGCCC CTTTGCCCCG TTTTCCCCCG GGCATGTTTG GGAGTCGGCT 2100
AATCCATTCT GTGGCGAGAG CACACTTTAC ACCCGCACTT GGTCGGAGGT 2150
TGATGCTGTT CCTAGTCCAG CCCAGCCCGA CTTAGGTTTT ACATCTGAGC 2200
CTTCTATACC TAGTAGGGCC GCCACACCTA CCCCGGCGGC CCCTCTACCC 2250
CCCCCTGCAC CGGATCCTTC CCCTACTCTC TCTGCTCCGG CGCGTGGTGA 2300
GCCGGCTCCT GGCGCTACCG CCAGGGCCCC AGCCATAACC CACCAGACGG 2350
CCCGGCATCG CCGCCTGCTC TTTACCTACC CGGATGGCTC TAAGGTGTTC 2400
GCCGGCTCGC TGTTTGAGTC GACATGTACC TGGCTCGTTA ACGCGTCTAA 2450
TGTTGACCAC CGCCCTGGCG GTGGGCTCTG TCATGCATTT TACCAGAGGT 2500
ACCCCGCCTC CTTTGATGCT GCCTCTTTTG TGATGCGCGA CGGCGCGGCC 2550
GCCTACACAT TAACCCCCCG GCCAATAATT CATGCCGTCG CTCCTGATTA 2600
TAGGTTGGAA CATAACCCAA AGAGGCTTGA GGCTGCCTAC CGGGAGACTT 2650
GCTCCCGCCT CGGTACCGCT GCATACCCAC TCCTCGGGAC CGGCATATAC 2700
CAGGTGCCGA TCGGTCCCAG TTTTGACGCC TGGGAGCGGA ATCACCGCCC 2750
CGGGGACGAG TTGTACCTTC CTGAGCTTGC TGCCAGATGG TTCGAGGCCA 2800
ATAGGCCGAC CTGCCCAACT CTCACTATAA CTGAGGATGT TGCGCGGACA 2850
GCAAATCTGG CTATCGAACT TGACTCAGCC ACAGACGTCG GCCGGGCCTG 2900
TGCCGGCTGT CGAGTCACCC CCGGCGTTGT GCAGTACCAG TTTACCGCAG 2950
GTGTGCCTGG ATCCGGCAAG TCCCGCTCTA TTACCCAAGC CGACGTGGAC 3000
GTTGTCGTGG TCCCGACCCG GGAGTTGCGT AATGCCTGGC GCCGCCGCGG 3050
CTTCGCTGCT TTCACCCCGC ACACTGCGGC TAGAGTCACC CAGGGGCGCC 3100
GGGTTGTCAT TGATGAGGCC CCGTCCCTTC CCCCTCATTT GCTGCTGCTC 3150
CACATGCAGC GGGCCGCCAC CGTCCACCTT CTTGGCGACC CGAATCAGAT 3200
CCCAGCCATC GATTTTGAGC ACGCCGGGCT CGTTCCCGCC ATCAGGCCCG 3250
ATTTGGCCCC CACCTCCTGG TGGCATGTTA CCCATCGCTG CCCTGCGGAT 3300
GTATGTGAGC TAATCCGCGG CGCATACCCT ATGATTCAGA CCACTAGTCG 3350
GGTCCTCCGG TCGTTGTTCT GGGGTGAGCC CGCCGTTGGG CAGAAGCTAG 3400
TGTTCACCCA GGCGGCTAAG GCCGCCAACC CCGGTTCAGT GACGGTCCAT 3450
GAGGCACAGG GCGCTACCTA CACAGAGACT ACCATCATTG CCACGGCAGA 3500
TGCTCGAGGC CTCATTCAGT CGTCCCGAGC TCATGCCATT GTTGCCTTGA 3550
CGCGCCACAC TGAGAAGTGC GTCATCATTG ACGCACCAGG CCTGCTTCGC 3600
GAGGTGGGCA TCTCCGATGC AATCGTTAAT AACTTTTTCC TTGCTGGTGG 3650
CGAAATTGGC CACCAGCGCC CATCTGTTAT CCCTCGCGGC AATCCTGACG 3700
CCAATGTTGA CACCTTGGCT GCCTTCCCGC CGTCTTGCCA GATTAGCGCC 3750
TTCCATCAGT TGGCTGAGGA GCTTGGCCAC AGACCTGCCC CTGTCGCGGC 3800
TGTTCTACCG CCCTGCCCTG AGCTTGAACA GGGCCTTCTC TACCTGCCCC 3850
AAGAACTCAC CACCTGTGAT AGTGTCGTAA CATTTGAATT AACAGATATT 3900
GTGCATTGTC GTATGGCCGC CCCGAGCCAG CGCAAGGCCG TGCTGTCCAC 3950
GCTCGTGGGC CGTTATGGCC GCCGCACAAA GCTCTACAAT GCCTCCCACT 4000
CTGATGTTCG CGACTCTCTC GCCCGTTTTA TCCCGGCCAT TGGCCCCGTA 4050
CAGGTTACAA CCTGTGAATT GTACGAGCTA GTGGAGGCCA TGGTCGAGAA 4100
GGGCCAGGAC GGCTCCGCCG TCCTTGAGCT CGACCTTTGT AGCCGCGACG 4150
TGTCCAGGAT CACCTTCTTC CAGAAAGATT GTAATAAATT CACCACGGGG 4200
GAGACCATCG CCCATGGTAA AGTGGGCCAG GGCATTTCGG CCTGGAGTAA 4250
GACCTTCTGT GCCCTTTTCG GCCCCTGGTT CCGTGCTATT GAGAAGGCTA 4300
TCCTGGCCCT GCTCCCTCAG GGTGTGTTTT ATGGGGATGC CTTTGATGAC 4350
ACCGTCTTCT CGGCGGCTGT GGCCGCAGCA AAGGCATCCA TGGTGTTCGA 4400
GAATGACTTT TCTGAGTTTG ATTCCACCCA GAATAATTTT TCCTTGGGCC 4450
TAGAGTGTGC TATTATGGAG GAGTGTGGGA TGCCGCAGTG GCTCATCCGC 4500
TTGTACCACC TTATAAGGTC TGCGTGGATT CTGCAGGCCC CGAAGGAGTC 4550
CCTGCGAGGG TTTTGGAAGA AACACTCCGG TGAGCCCGGC ACCCTTCTGT 4600
GGAATACTGT CTGGAACATG GCCGTTATCA CCCACTGTTA TGATTTCCGC 4650
GATCTGCAGG TGGCTGCCTT TAAAGGTGAT GATTCGATAG TGCTTTGCAG 4700
TGAGTACCGT CAGAGCCCAG GGGCTGCTGT CCTGATTGCT GGCTGTGGCC 4750
TAAAGTTGAA GGTGGATTTC CGTCCGATTG GTCTGTATGC AGGTGTTGTG 4800
GTGGCCCCCG GCCTTGGCGC GCTTCCTGAT GTCGTGCGCT TCGCCGGTCG 4850
GCTTACTGAG AAGAATTGGG GCCCTGGCCC CGAGCGGGCG GAGCAGCTCC 4900
GCCTCGCTGT GAGTGATTTT CTCCGCAAGC TCACGAATGT AGCTCAGATG 4950
TGTGTGGATG TTGTCTCTCG TGTTTATGGG GTTTCCCCTG GGCTCGTTCA 5000
TAACCTGATT GGCATGCTAC AGGCTGTTGC TGATGGCAAG GCTCATTTCA 5050
CTGAGTCAGT GAAGCCAGTG CTTGACCTGA CAAATTCAAT TCTGTGTCGG 5100
GTGGAATGAA TAACATGTCT TTTGCTGCGC CCATGGGTTC GCGACCATGC 5150
GCCCTCGGCC TATTTTGCTG TTGCTCCTCA TGTTTCTGCC TATGCTGCCC 5200
GCGCCACCGC CCGGTCAGCC GTCTGGCCGC CGTCGTGGGC GGCGCAGCGG 5250
CGGTTCCGGC GGTGGTTTCT GGGGTGACCG GGTTGATTCT CAGCCCTTCG 5300
CAATCCCCTA TATTCATCCA ACCAACCCCT TCGCCCCCGA TGTCACCGCT 5350
GCGGCCGGGG CTGGACCTCG TGTTCGCCAA CCCGCCCGAC CACTCGGCTC 5400
CGCTTGGCGT GACCAGGCCC AGCGCCCCGC CGCTGCCTCA CGTCGTAGAC 5450
CTACCACAGC TGGGGCCGCG CCGCTAACCG CGGTCGCTCC GGCCCATGAC 5500
ACCCCGCCAG TGCCTGATGT TGACTCCCGC GGCGCCATCC TGCGCCGGCA 5550
GTATAACCTA TCAACATCTC CCCTCACCTC TTCCGTGGCC ACCGGCACAA 5600
ATTTGGTTCT TTACGCCGCT CCTCTTAGCC CGCTTCTACC CCTCCAGGAC 5650
GGCACCAATA CTCATATAAT GGCTACAGAA GCTTCTAATT ATGCCCAGTA 5700
CCGGGTTGCT CGTGCCACAA TTCGCTACCG CCCGCTGGTC CCCAACGCTG 5750
TTGGTGGCTA CGCTATCTCC ATTTCGTTCT GGCCACAGAC CACCACCACC 5800
CCGACGTCCG TTGACATGAA TTCAATAACC TCGACGGATG TCCGTATTTT 5850
AGTCCAGCCC GGCATAGCCT CCGAGCTTGT TATTCCAAGT GAGCGCCTAC 5900
ACTATCGCAA CCAAGGTTGG CGCTCTGTTG AGACCTCCGG GGTGGCGGAG 5950
GAGGAGGCCA CCTCTGGTCT TGTCATGCTC TGCATACATG GCTCACCTGT 6000
AAATTCTTAT ACTAATACAC CCTATACCGG TGCCCTCGGG CTGTTGGACT 6050
TTGCCCTCGA ACTTGAGTTC CGCAACCTCA CCCCCGGTAA TACCAATACG 6100
CGGGTCTCGC GTTACTCCAG CACTGCCCGT CACCGCCTTC GTCGCGGTGC 6150
AGATGGGACT GCCGAGCTCA CCACCACGGC TGCTACTCGC TTCATGAAGG 6200
ACCTCTATTT TACTAGTACT AATGGTGTTG GTGAGATCGG CCGCGGGATA 6250
GCGCTTACCC TGTTTAACCT TGCTGACACC CTGCTTGGCG GTCTACCGAC 6300
AGAATTGATT TCGTCGGCTG GTGGCCAGCT GTTCTACTCT CGCCCCGTCG 6350
TCTCAGCCAA TGGCGAGCCG ACTGTTAAGC TGTATACATC TGTGGAGAAT 6400
GCTCAGCAGG ATAAGGGTAT TGCAATCCCG CATGACATCG ACCTCGGGGA 6450
ATCCCGTGTA GTTATTCAGG ATTATGACAA CCAACATGAG CAGGACCGAC 6500
CGACACCTTC CCCAGCCCCA TCGCGTCCTT TTTCTGTCCT CCGAGCTAAC 6550
GATGTGCTTT GGCTTTCTCT CACCGCTGCC GAGTATGACC AGTCCACTTA 6600
CGGCTCTTCG ACCGGCCCAG TCTATGTCTC TGACTCTGTG ACCTTGGTTA 6650
ATGTTGCGAC CGGCGCGCAG GCCGTTGCCC GGTCACTCGA CTGGACCAAG 6700
GTCACACTTG ATGGTCGCCC CCTTTCCACC ATCCAGCAGT ATTCAAAGAC 6750
CTTCTTTGTC CTGCCGCTCC GCGGTAAGCT CTCCTTTTGG GAGGCAGGAA 6800
CTACTAAAGC CGGGTACCCT TATAATTATA ACACCACTGC TAGTGACCAA 6850
CTGCTCGTTG AGAATGCCGC TGGGCATCGG GTTGCTATTT CCACCTACAC 6900
TACTAGCCTG GGTGCTGGCC CCGTCTCTAT TTCCGCGGTT GCTGTTTTAG 6950
CCCCCCACTC TGTGCTAGCA TTGCTTGAGG ATACCATGGA CTACCCTGCC 7000
CGCGCCCATA CTTTCGATGA CTTCTGCCCG GAGTGCCGCC CCCTTGGCCT 7050
CCAGGGTTGT GCTTTTCAGT CTACTGTCGC TGAGCTTCAG CGCCTTAAGA 7100
TGAAGGTGGG TAAAACTCGG GAGTTATAGT TTATTTGCTT GTGCCCCCCT 7150
TCTTTCTGTT GCTTATTT 7168






25 base pairs


nucleic acid


single


linear




unknown



5
ACATTTGAAT TCACAGACAT TGTGC 25






26 base pairs


nucleic acid


single


linear




unknown



6
ACACAGATCT GAGCTACATT CGTGAG 26






26 base pairs


nucleic acid


single


linear




unknown



7
AAAGGGATCC ATGGTGTTTG AGAATG 26






25 base pairs


nucleic acid


single


linear




unknown



8
ACTCACTGCA GAGCACTATC GAATC 25






22 base pairs


nucleic acid


single


linear




unknown



9
CGGTAAACTG GTACTGCACA AC 22






22 base pairs


nucleic acid


single


linear




unknown



10
AAGTCCCGCT CTATTACCCA AG 22






21 base pairs


nucleic acid


single


linear




unknown



11
ACCCACGGGT GTTGGTTTTT G 21






21 base pairs


nucleic acid


single


linear




unknown



12
TTCTTGGGGC AGGTAGAGAA G 21






26 base pairs


nucleic acid


single


linear




unknown



13
TTATTGAATT CATGTCAACG GACGTC 26






21 base pairs


nucleic acid


single


linear




unknown



14
AATAATTCAT GCCGTCGCTC C 21






21 base pairs


nucleic acid


single


linear




unknown



15
AAGCTCAGGA AGGTACAACT C 21






24 base pairs


nucleic acid


single


linear




unknown



16
AAATCGATGG CTGGGATCTG ATTC 24






21 base pairs


nucleic acid


single


linear




unknown



17
GAGGCATTGT AGAGCTTTGT G 21






22 base pairs


nucleic acid


single


linear




unknown



18
GATGTTGCAC GGACAGCAAA TC 22






24 base pairs


nucleic acid


single


linear




unknown



19
ATCTCCGATG CAATCGTTAA TAAC 24






21 base pairs


nucleic acid


single


linear




unknown



20
TAATCCATTC TGTGGCGAGA G 21






21 base pairs


nucleic acid


single


linear




unknown



21
AAGTGTGACC TTGGTCCAGT C 21






23 base pairs


nucleic acid


single


linear




unknown



22
TTGCTCGTGC CACAATTCGC TAC 23






21 base pairs


nucleic acid


single


linear




unknown



23
CATTTCACTG AGTCAGTGAA G 21






21 base pairs


nucleic acid


single


linear




unknown



24
TAATTATAAC ACCACTGCTA G 21






21 base pairs


nucleic acid


single


linear




unknown



25
GATTGCAATA CCCTTATCCT G 21






23 base pairs


nucleic acid


single


linear




unknown



26
ATTAAACCTG TATAGGGCAG AAC 23






21 base pairs


nucleic acid


single


linear




unknown



27
AAGTTCGATA GCCAGATTTG C 21






21 base pairs


nucleic acid


single


linear




unknown



28
TCATGTTGGT TGTCATAATC C 21






21 base pairs


nucleic acid


single


linear




unknown



29
GATGACGCAC TTCTCAGTGT G 21






19 base pairs


nucleic acid


single


linear




unknown



30
AGAACAACGA ACGGAGAAC 19






21 base pairs


nucleic acid


single


linear




unknown



31
AGATCCCAGC CATCGACTTT G 21






21 base pairs


nucleic acid


single


linear




unknown



32
TAGTAGTGTA GGTGGAAATA G 21






21 base pairs


nucleic acid


single


linear




unknown



33
GTGTGGTTAT TCAGGATTAT G 21






21 base pairs


nucleic acid


single


linear




unknown



34
ACTCTGTGAC CTTGGTTAAT G 21






21 base pairs


nucleic acid


single


linear




unknown



35
AACTCAAGTT CGAGGGCAAA G 21






21 base pairs


nucleic acid


single


linear




unknown



36
CGCTTACCCT GTTTAACCTT G 21






24 base pairs


nucleic acid


single


linear




unknown



37
ATCCCCTATA TTCATCCAAC CAAC 24






21 base pairs


nucleic acid


single


linear




unknown



38
CTCCTCATGT TTCTGCCTAT G 21






22 base pairs


nucleic acid


single


linear




unknown



39
GCCAGAACGA AATGGAGATA GC 22






21 base pairs


nucleic acid


single


linear




unknown



40
CTCAGACATA AAACCTAAGT C 21






21 base pairs


nucleic acid


single


linear




unknown



41
TGCCCTATAC AGGTTTAATC G 21






19 base pairs


nucleic acid


single


linear




unknown



42
ACCGGCATAT ACCAGGTGC 19






21 base pairs


nucleic acid


single


linear




unknown



43
ACATGGCTCA CTCGTAAATT C 21






21 base pairs


nucleic acid


single


linear




unknown



44
AACATTAGAC GCGTTAACGA G 21






21 base pairs


nucleic acid


single


linear




unknown



45
CTCTTTTGAT GCCAGTCAGA G 21






22 base pairs


nucleic acid


single


linear




unknown



46
ACCTACCCGG ATGGCTCTAA GG 22






25 base pairs


nucleic acid


single


linear




unknown



47
TATGGGAATT CGTGCCGTCC TGAAG 25






21 base pairs


nucleic acid


single


linear




unknown



48
AGTGGGAGCA GTATACCAGC G 21






21 base pairs


nucleic acid


single


linear




unknown



49
CTGCTATTGA GCAGGCTGCT C 21






21 base pairs


nucleic acid


single


linear




unknown



50
GGGCCATTAG TCTCTAAAAC C 21






19 base pairs


nucleic acid


single


linear




unknown



51
GAGGTTTTCT GGAATCATC 19






15 base pairs


nucleic acid


single


linear




unknown



52
GCATAGGTGA GACTG 15






18 base pairs


nucleic acid


single


linear




unknown



53
AGTTACAGCC AGAAAACC 18






33 base pairs


nucleic acid


single


linear




unknown



54
CCATGGATCC TCGGCCTATT TTGCTGTTGC TCC 33






18 base pairs


nucleic acid


single


linear




unknown



55
AGGCAGACCA CATATGTG 18






20 base pairs


nucleic acid


single


linear




unknown



56
GGTGCACTCC TGACCAAGCC 20






19 base pairs


nucleic acid


single


linear




unknown



57
ATTGGCTGCC ACTTTGTTC 19






21 base pairs


nucleic acid


single


linear




unknown



58
ACCCTCATAC GTCACCACAA C 21






24 base pairs


nucleic acid


single


linear




unknown



59
GCGGTGGACC ACATTAGGAT TATC 24






19 base pairs


nucleic acid


single


linear




unknown



60
CATGATATGT CACCATCTG 19






19 base pairs


nucleic acid


single


linear




unknown



61
GTCATCCATA ACGAGCTGG 19






33 base pairs


nucleic acid


single


linear




unknown



62
AGCGGAATTC GAGGGGCGGC ATAAAGAACC AGG 33






36 base pairs


nucleic acid


single


linear




unknown



63
GCGCTGAATT CGGATCACAA GCTCAGAGGC TATGCC 36






30 base pairs


nucleic acid


single


linear




unknown



64
GTATAACGGA TCCACATCTC CCCTTACCTC 30






30 base pairs


nucleic acid


single


linear




unknown



65
TAACCTGGAT CCTTATGCCG CCCCTCTTAG 30






38 base pairs


nucleic acid


single


linear




unknown



66
AAATTGGATC CTGTGTCGGG TGGAATGAAT AACATGTC 38






37 base pairs


nucleic acid


single


linear




unknown



67
ATCGGCAGAT CTGATAGAGC GGGGACTTGC CGGATCC 37






28 base pairs


nucleic acid


single


linear




unknown



68
TACCCTGCCC GCGCCCATAC TTTTGATG 28






33 base pairs


nucleic acid


single


linear




unknown



69
GGCTGAGATC TGGTTCGGGT CGCCAAGAAG GTG 33






27 base pairs


nucleic acid


single


linear




unknown



70
TACAGATCTA TACAACTTAA CAGTCGG 27






29 base pairs


nucleic acid


single


linear




unknown



71
GCGGCAGATC TCACCGACAC CATTAGTAC 29






28 base pairs


nucleic acid


single


linear




unknown



72
CCGTCGGATC CCAGGGGCTG CTGTCCTG 28






31 base pairs


nucleic acid


single


linear




unknown



73
AAAGGAATTC AAGACCAGAG GTAGCCTCCT C 31






28 base pairs


nucleic acid


single


linear




unknown



74
GTTGATATGA ATTCAATAAC CTCGACGG 28






36 base pairs


nucleic acid


single


linear




unknown



75
TTTGGATCCT CAGGGAGCGC GGAACGCAGA AATGAG 36






26 base pairs


nucleic acid


single


linear




unknown



76
TCACTCGTGA ATTCCTATAC TAATAC 26






34 base pairs


nucleic acid


single


linear




unknown



77
TTTGGATCCT CAGGGAGCGC GGAACGCAGA AATG 34






25 base pairs


nucleic acid


single


linear




unknown



78
TGATAGAGCG GGACTTGCCG GATCC 25






24 base pairs


nucleic acid


single


linear




unknown



79
TTGCATTAGG TTAATGAGGA TCTC 24






25 base pairs


nucleic acid


single


linear




unknown



80
ACCTGCTTCC TTCAGCCTGC AGAAG 25






29 base pairs


nucleic acid


single


linear




unknown



81
GCGGTGGATC CGCTCCCAGG CGTCAAAAC 29






29 base pairs


nucleic acid


single


linear




unknown



82
GGGCGGATCG AATTCGAGAC CCTTCTTGG 29






27 base pairs


nucleic acid


single


linear




unknown



83
AGGATGGATC CATAAGTTAC CGATCAG 27






29 base pairs


nucleic acid


single


linear




unknown



84
GGCTGGAATT CCTCTGAGGA CGCCCTCAC 29






27 base pairs


nucleic acid


single


linear




unknown



85
GCCGAAGATC TATCGGACAT AGACCTC 27






30 base pairs


nucleic acid


single


linear




unknown



86
CAGACGACGG ATCCCCTTGG ATATAGCCTG 30






40 base pairs


nucleic acid


single


linear




unknown



87
GGCCGAATTC AGGCAGACCA CATATGTGGT CGATGCCATG 40






25 base pairs


nucleic acid


single


linear




unknown



88
GCAGGTGTGC CTGGATCCGG CAAGT 25






30 base pairs


nucleic acid


single


linear




unknown



89
GTTAGAATTC CGGCCCAGCT GTGGTAGGTC 30






24 base pairs


nucleic acid


single


linear




unknown



90
CCGTCCGATT GGTCTGTATG CAGG 24






22 base pairs


nucleic acid


single


linear




unknown



91
TACCAGTTTA CTGCAGGTGT GC 22






22 base pairs


nucleic acid


single


linear




unknown



92
CAAGCCGATG TGGACGTTGT CG 22






24 base pairs


nucleic acid


single


linear




unknown



93
GGCGCTGGGC CTGGTCACGC CAAG 24






22 base pairs


nucleic acid


single


linear




unknown



94
GCAGAAACTA GTGTTGACCC AG 22






22 base pairs


nucleic acid


single


linear




unknown



95
TAGGTCTACG ACGTGAGGCA AC 22






21 base pairs


nucleic acid


single


linear




unknown



96
TACAATCTTT CAGGAAGAAG G 21






21 base pairs


nucleic acid


single


linear




unknown



97
CCCACACTCC TCCATAATAG C 21






24 base pairs


nucleic acid


single


linear




unknown



98
GATAGTGCTT TGCAGTGAGT ACCG 24






30 base pairs


nucleic acid


single


linear




unknown



99
GTATAACGGA TCCACATCTC CCCTTACCTC 30






27 base pairs


nucleic acid


single


linear




unknown



100
TACAGATCTA TACAACTTAA CAGTCGG 27






29 base pairs


nucleic acid


single


linear




unknown



101
GCGGCAGATC TCACCGACAC CATTAGTAC 29






30 base pairs


nucleic acid


single


linear




unknown



102
TAACCTGGAT CCTTATGCCG CCCCTCTTAG 30






36 base pairs


nucleic acid


single


linear




unknown



103
GCACAACCTA GGTTACTATA ACTCCCGAGT TTTACC 36






33 base pairs


nucleic acid


single


linear




unknown



104
GGGTTCCCTA GGATGCGCCC TCGGCCTATT TTG 33






33 base pairs


nucleic acid


single


linear




unknown



105
CGTGGGCCTA GGAGCGGCGG TTCCGGCGGT GGT 33






33 base pairs


nucleic acid


single


linear




unknown



106
GCTTGGCCTA GGCAGGCCCA GCGCCCCGCC GCT 33






33 base pairs


nucleic acid


single


linear




unknown



107
CCGCCACCTA GGGATGTTGA CTCCCGCGGC GCC 33







Claims
  • 1. A nucleic acid molecule consisting of nucleotides which encode amino acids 112-660 of a hepatitis E virus open reading frame 2 protein.
  • 2. A recombinant vector comprising the nucleic acid molecule of claim 1.
  • 3. A host cell transformed, transfected or infected in vitro with the expression vector of claim 2.
  • 4. The vector of claim 2, wherein said vector is a baculovirus vector.
  • 5. A host cell transformed, transfected or infected in vitro with the expression vector of claim 4.
  • 6. A method for producing a hepatitis E virus open-reading frame 2 protein comprising:(a) culturing a host cell containing a nucleic acid molecule consisting of nucleotides which encode amino acids 112-660 of a hepatitis E virus open reading frame 2 protein under conditions appropriate to cause expression of said protein; and (b) obtaining said expressed protein from the host cell.
  • 7. A nucleic acid molecule consisting of nucleotides which encode amino acids 112-660 of SEQ ID No. 2.
  • 8. A recombinant vector comprising the nucleic acid molecule of claim 7.
  • 9. A host cell transformed, transfected or infected in vitro with the expression vector of claim 8.
  • 10. The vector of claim 8, wherein said vector is a baculovirus vector.
  • 11. A host cell transformed, transfected or infected in vitro with the expression vector of claim 10.
  • 12. A method for producing a hepatitis E virus open-reading frame 2 protein comprising:(a) culturing a host cell containing a nucleic acid molecule consisting of nucleotides which encode amino acids 112-660 of SEQ ID NO:2 under conditions appropriate to cause expression of said protein; and (b) obtaining said expressed protein from the host cell.
Parent Case Info

This application is a national stage entry of PCT/US95/13102, filed Oct. 3, 1995, which was a continuation-in-part of U.S. application Ser. No. 08/316,765, filed Oct. 3, 1994, which was a continuation-in-part of U.S. application Ser. No. 07/947,263, filed Sep. 18, 1992, abandoned.

PCT Information
Filing Document Filing Date Country Kind 102e Date 371c Date
PCT/US95/13102 WO 00 5/28/1997 5/28/1997
Publishing Document Publishing Date Country Kind
WO96/10580 4/11/1996 WO A
US Referenced Citations (4)
Number Name Date Kind
5686239 Reyes et al. Nov 1997
5741490 Reyes et al. Apr 1998
5770689 Reyes et al. Jun 1998
5824649 Reyes et al. Oct 1998
Foreign Referenced Citations (7)
Number Date Country
WO9115603 Oct 1991 WO
WO9314208 Jul 1993 WO
WO9314116 Jul 1993 WO
WO9406913 Mar 1994 WO
WO9508632 Mar 1995 WO
WO9517501 Jun 1995 WO
WO9612807 May 1996 WO
Non-Patent Literature Citations (30)
Entry
He, J. et al. (1995) J. Clin. Microbiol, 33:3308-3311.
Panda et al. (1995) “An Indian Strain of Hepatitis E Virus (HEV): Cloning, Sequence, and Express of Structural Region and Antibody Responses in Sera from Individuals from an Area of High-Level HEV Endemicity” Journal of Clinical Microbiology, vol. 33, No. 10, p. 2653-2659.
Yin et al. (1994) A New Chinese Isolate of Hepatitis E Virus: Comparison with Strains Recovered from Different Geographical Regions, Virus Genes 9:1, 22-32.
Dawson et al., “Solid-Phase Enzyme-Linked Immunosorbent Assay For Hepatitis E Virus IgG and IgM Antibodies Utilizing Recombinant Antigens And Synthetic Peptides,” J. Virol. Methods, 38:175-186 (1992).
Uchida et al., “Hepatitis E Virus: cDNA Cloning and Expression, ” Microbiol. Immunol., 36:67-79 (1992).
Skidmore et al., “Hepatitis E Virus: The Cause Of A Waterbourne Hepatitis Outbreak,” J. Med. Virol., 37:58-60 (1992).
Saeed et al., “ELISA For Diagnosis Of Acute Sporadic Hepatitis E,” Lancet, 339:882 (1992).
Aye et al., “Complete Nucleotide Sequence Of A Hepatitis E Virus Isolated From The Xinjiang Epidemic (1986-1988) Of China,” Nucleic Acids Res., 20:3512 (1992).
Hyams et al., “Acute Sporadic Hepatitis E In Sundanese Children: Analysis Based On A New Western Blot Assay, ” J. Infect. Dis., 165:1001-1005 (1992).
Tsarev et al., “Characterization Of A Prototype Strain Of Hepatitis E Virus,” Proc. Nat. Acad. Sci., 89:559-563 (1992).
Yarbough et al., “Hepatitis E Virus: Identification Of Type-Common Epitopes,” J. Virol., 65:5790-5797 (1992).
Ichikawa et al., “Cloning and Expression of cDNAs From Enterically-Transmitted Non-A, Non-B Hepatitis Virus,” Microbiol. Immunol., 35:535-543 (1992).
Purdy et al., “Expression Of A Hepatitis E Virus (HEV)-trpE Fusion Protein Containing Epitopes Recognized By Antibodies In Sera From Human Cases And Experimentally Infected Primates,” Archives Virol., 123:335-349 (1992).
Favorov et al., Serologic Identification Of Hepatitis E Virus Infections In Epidemic And Endemic Settings, J. Med. Virol., 36:246-250 (1992).
Reyes et al., “Isolation Of A cDNA From The Virus Responsible For Enterically Transmitted Non-A, Non-B Hepatitis, ” Science, 247:1335-1339 (1992).
Tam et al., “Hepatitis E Virus (HEV): Molecular Cloning And Sequencing Of The Full-Length Viral Genome, ” Virology, 185:120-131 (1992).
Goldsmith et al., “Enzyme-Linked Immunosorbent Assay For Diagnosis Of Acute Sporadic Hepatitis E In Egyptian Children, ” Lancet, 339:328-331 (1992).
Fry et al., “Hepatitis E Virus (HEV): Strain Variation In The Nonstructural Gene Region Encoding Consensus Motifs For An RNA-Dependent RNA Polymerase And An ATP/GTP Binding Site,” Virus Genes, 6:173-185 (1992).
He, J. et al., “Expression and Diagnostic Utility of Hepatitis E Virus Putative Structural Proteins Expressed in Insect Cells,” J. Clin. Micro., 31:2167-2173 (1993).
Bryan J.P. et al., “Epidemic Hepatitis E In Pakistan: Patterns of Serologic Response and Evidence that Antibody to Hepatitis E Virus Protects Against Disease,” J. Infect. Dis., 170:571-21 (1994).
Purdy M.A. et al., “Preliminary Evidence that a trpE-HEV Fusion Protein Protects Cynomolgus Macaques Against Challenge With Wild-Type Hepatitis E Virus (HEV), ” J. Med. Virology, 41:90-94 (1993).
Tsarev S.A. et al., “ELISA for Antibody to Hepatitis E Virus (HEV) Based On Complete Open-Reading Frame-2 Protein Expressed in Insect Cells: Identification of HEV Infection in Primates,” J. Infect. Dis., 168:369-78 (1993).
Li, F. et al. Persistent and Transient Antibody Responses to Hepatitis E Virus Detected by Western Immunoblot Using Open Reading Frame 2 and 3 and Glutathione S-Transferase Fusion Proteins J. Clin. Microbiol, 32:2060-66 (1994).
Tsarev, S. et al., Variation In Course Of Hepatitis E In Experimentally Infected Cynomolgus Monkeys (1993) J. Infect. Dis., 167:1302-1306.
Tsarev, S. et al., Infectivity Titration Of A Prototype Strain Of Hepatitis E Virus Cynomolgus Monkeys (1994) J. Med. Virol., 43:135-142.
Carl et al, “Expression of Hepatitis E Virus Putative Structural Proteins in Recombinant Vaccinia Viruses, ” Clinical and Diagnostic Laboratory Immunology, 1:253-256 (1994).
Reyes et al. (1991) Gastroenterologia Japonica, 26:142-147.
Reyes et al. (1992) “Hepatitis E Virus (HEV): Epitope Mapping and Detection of Strain Variation”, Elsevier Science Publisher Shikata et al. eds., Chapter 43:237-245.
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Continuation in Parts (2)
Number Date Country
Parent 08/316765 Oct 1994 US
Child 08/809523 US
Parent 07/947263 Sep 1992 US
Child 08/316765 US