Attenuated hepatitis a virus vaccine which grows in MRC-5 cells

Abstract
A live hepatitis A virus (HAV) adapted to grow in MRC-5 cells is described, the HAV preferably characterized by suitable attenuation for effective vaccine administration to humans and animals without inactivation. Methods for adapting HAV to grow in MRC-5 cells, vaccine compositions comprising the attenuated HAV, and methods of vaccinating humans against HAV infection are also described.
Description




FIELD OF THE INVENTION




The present invention relates generally to the field of vaccinal compositions useful in the prophylaxis of hepatitis A. More specifically, the invention provides a novel live hepatitis A virus (HAV), and recombinant and chimeric HAVs, the genomes of which are modified from that of their parental strain HM-175 to provide them with the ability to propagate in MRC-5 cells and retain appropriate attenuation for use as live vaccines in humans and other primates.




BACKGROUND OF THE INVENTION




In the United States, hepatitis A virus is the cause of approximately 25% of all clinical hepatitis cases, accounting for approximately 150,000 such cases. Populations at high risk of acquiring hepatitis A in industrialized countries include the socially disadvantaged, medical personnel, military personnel, staff and adult contacts of children in day-care centers, male homosexuals, drug addicts, and travelers to endemic areas.




In developing countries, virtually the entire population is infected with hepatitis A virus at an early age. Much of this invention results in subclinical and inapparent infection, but, as countries improve their hygienic conditions, infection with hepatitis A virus occurs at progressively older ages, resulting in a higher proportion of clinical disease. Thus, there is a paradoxical increase in clinical hepatitis A as the overall rate of infection diminishes. To successfully immunize against hepatitis A in the United States and in other industrialized countries as well as in developing countries, it will be necessary to vaccinate the entire pediatric population. There will be an increasing need for hepatitis A vaccines in such countries for the foreseeable future.




Research in HAV vaccines has focused on inactivated, or killed, viruses. However, in vaccine, therapy there are several advantages to a live vaccine, rather than an inactivated, vaccine. With a live vaccine, one can use a lower dosage and smaller number of doses, because a live vaccine replicates in the vaccines to produce more antigen and can stimulate the immune system of the vaccinee to make both IgA and IgM. Inactivated vaccines, such as the Salk polio vaccine, which stimulates production of IgG only in vaccinees, do not protect against infection by ingested virus, only against disease.




A major obstacle to the development of live, attenuated vaccine has been the difficulty in adapting HAV to a cell line that supports rapid viral growth and is licensed for vaccine production. Wild type hepatitis A virus (HAV) grows poorly in cell culture.




U.S. Pat. Nos. 4,532,215 and 4,636,469 describe, respectively, a strain of HAV designated HM-175 initially isolated from human feces of a patient in Melbourne, Australia, and adapted to passage in vitro in African green monkey kidney (AGMK) culture cells and methods for obtaining same by serial passaging. U.S. Pat. No. 4,620,978 describes a vaccine employing the HAV HM-175, triply cloned in AGMK cell culture and attenuated. U.S. Pat. No. 4,894,228 describes HM-175 Pass 35, which differs from wild-type HM-175 by nucleotide changes in the genome, is attenuated for chimpanzees, elicits serum neutralizing antibodies, and is suitable for use as an attenuated HAV vaccine. It discloses the complete nucleotide sequence of HAV, strain HM-175/7. See, also, B. C. Ross et al.,


J. Gen. Virol.,


70:2805-2810 (1989); R. W. Jansen et al,


Virol.,


163:299-307 (1988); and Tedeschi et al,


J. Med. Virol.,


(in press). The disclosure of these patents and articles are incorporated by reference herein.




N. Fineschi et al,


J. Hepatol.,


13(4):S146-S151 (1991) describes an HAV isolate, LSH/S, which is a candidate for an inactivated vaccine. It was adapted to grow in human diploid MRC-5 cells, a preferred licensed cell for vaccine development. This document compares only a small part of its nucleotide sequence to that of wild-type HM-175.




Provost et al,


J. Med. Virol.,


20:165-175 (1986) described the F and F′ variants of the CR326 hepatitis A virus strain. While it is reported to be immunogenic in volunteer vaccinees, the F variant also caused abnormal serum ALT levels in a substantial proportion of individuals.




Another recent publication from this group of investigators has described further work with the F′ variant [K. Midthun et al,


J. Infect. Dis.,


163:735-739 (1991)]. They observed that the immunogenicity of the F′ vaccine product is dose dependent, i.e., a 10


7.3


TCID


50


evoked an antibody response in 100% of volunteers within 9 weeks after immunization whereas lower doses were immunogenic in a smaller percentage of volunteers, and anti-HAV was observed 4 to 6 months after immunization. Chinese investigators have recently described studies of a potential live attenuated hepatitis A vaccine prepared from the H2 strain of HAV [J. S. Mao et al,


J. Infect. Dis.,


159:621-624 (1989)]. Twelve volunteers received the vaccine by the subcutaneous route.




A live attenuated hepatitis A vaccine could have a significant impact on the eradication of the disease. It could be anticipated that a live attenuated vaccine which requires minimal purification and no adjuvant would be less costly than presently available inactivated hepatitis A vaccines.




There is a need in the art for methods and compositions for effective vaccination of humans and animals against hepatitis A.




SUMMARY OF THE INVENTION




In one aspect, the present invention provides a live hepatitis A virus adapted to growth in MRC-5 cells. This virus is preferably characterized by attenuation. The attenuated virus may be, recombinant or chimeric. More preferably, the HAV is characterized by suitable attenuation for effective vaccine administration to primates, preferably humans, without inactivation. The HAV may be characterized by containing one or more of fourteen specific nucleotides which differ from nucleotides in the same position in the genome of HAV HM-175, Pass 35.




In another aspect, the invention provides a vaccine useful for protecting humans or other primates against hepatitis A which vaccine contains at least one above-described HAV adapted to growth in MRC-5 cells. Preferably, the vaccine is effective in inducing a protective antibody response without adjuvant.




In still another aspect, the invention provides a method for protecting humans against hepatitis A virus infection which comprises administering to the human patient an effective amount of a vaccine composition of this invention.




In a further aspect, the invention provides a method for preparing a live HAV adapted to growth in MRC-5 cells by incorporating into a selected area of the genome of an HAV one or more of fourteen specific nucleotides. The HAV genome so modified is preferably HAV HM-175, Pass 35 or a related cell culture-adapted mutant.




In another embodiment, the HAV may be constructed using another HAV cDNA clone and inserting appropriate nucleotides into its genome. According to this method an attenuated, MRC-5-adapted HAV is provided without requiring further passaging in MRC-5 or other primate cell lines.




In still another aspect, the invention provides polynucleotide sequences encoding the recombinant or chimeric HAVs described above. Preferably these sequences are cDNAs useful as master seeds for vaccine preparation.




Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.











BRIEF DESCRIPTION OF THE DRAWINGS





FIG. 1

is a graph plotting anti-HAV antibody production vs. time (weeks) vs. ALT and ICD levels for the chimpanzee studies with Virus 4380 and subsequent challenge with wild-type HAV, as described in Example 1. These results were obtained from a chimpanzee that was infected with the attenuated HAV at time 0 and challenged with virulent virus at week 28.





FIG. 2

is a graph plotting anti-HAV antibody production vs. time (weeks) vs. ALT and ICD levels for the chimpanzee studies with Virus 4380 and subsequent challenge with wild-type HAV, as described in Example 1. The conditions were the same as for FIG.


1


.





FIG. 3

is a graph plotting anti-HAV antibody production vs. time (weeks) vs. ALT and ICD levels for the chimpanzee studies with Virus 4380 and subsequent challenge with wild-type HAV, as described in Example 1. The conditions were the same as for FIG.


1


.





FIG. 4

is a graph plotting anti-HAV antibody production vs. time (weeks) vs. ALT and ICD levels for the chimpanzee studies with Virus 4380 and subsequent challenge with wild-type HAV, as described in Example 1. These results were obtained from the chimpanzee that was not infected with the attenuated HAV, and therefore developed hepatitis following challenge with the virulent virus. The conditions were the same as for FIG.


1


.





FIG. 5

is a bar graph of endpoint dilutions of several of the chimeric viruses, Viruses #2, 3, 4, 5, and 6, listed in Table VI.











DETAILED DESCRIPTION OF THE INVENTION




The present invention provides hepatitis A virus (HAV) adapted to growth in the human fibroblast-like cell line, MRC-5, a cell substrate suitable for commercial production and licensing of inactivated and live, attenuated hepatitis A vaccines. In addition to such adapted HAVs, the invention provides a method for adapting a selected HAV to growth in that human cell line and preparing an MRC-5-adapted, attenuated HAV without passaging in other primate cells. The HAV of this invention and the preparative method also preferably provides the HAV with sufficient attenuation to enable its efficacy as a vaccine for humans and animals.




Although the prior art discloses other candidate vaccine strains of hepatitis A virus which have been adapted to growth in human diploid fibroblasts, the genetic changes in the virus genome necessary and sufficient for such adaptation have not been characterized. Thus, these strains cannot be manipulated in vitro to assure a reproducible and fully-characterized vaccine product.




The present invention is based on the wild-type HAV, strain HM-175, which is described in detail in the above-cited and incorporated art [Cohen et al.,


J. Virol.,


61:50-59 (1987); SEQ ID NO:1 and 2]. Briefly described, the wild type, infectious HAV HM-175 virus was previously adapted to growth in primary African green monkey kidney (AGMK) cells at 37° C. After 26 passages in AGMK, the virus was cloned three times in AGMK cells by serial dilution, then passage three more times to provide passage 32 (P-32). P-32 was found to be attenuated as described in R. A. Karron et al,


J. Infec. Dis.,


157:338-345 (1988).




The P-32 virus described above was passaged three more times in AGMK, and molecularly clones. The virus that was cloned was called P-35 and the full-length clone was referred to as pHAV/7. pHAV/7 is an infectious cDNA clone of the virus that can be maintained in a monoclonal state and amplified at will with diminished risk of spontaneous mutations. The resulting P-35 virus grew well in fetal rhesus monkey kidney (FRhK) cells and minimally in human fibroblastoid lung cells (MRC-5).




U.S. Pat. No. 4,894,228 and Cohen et al.,


Proc. Natl. Acad. Sci., USA,


84:2497-2501 (1987) provide the HAV nucleotide sequences of wild-type HAV strain HM-175 (see,

FIG. 1

of the patent; SEQ ID NO:1 and 2) and the nucleotide differences between HAV HM-175, Pass 35, clone pHAV/7 and the wild-type sequence [SEQ ID NO:1]. Thus, these documents, incorporated by reference, provide the sequence of pHAV/7, P-35. The nucleotide numbers used herein to which the mutations of this invention correspond (Tables I and VI below) are the nucleotide numbers assigned to positions of the wild-type sequence of

FIG. 1

[SEQ ID NO:1 and 2] from U.S. Pat. No. 4,894,228 containing the mutations for P-35. Note that the nucleotides deleted in P-35 are assigned the nucleotide position of the wild-type sequence [SEQ ID NO:1]. Thus, for example, nucleotide position 131 represents a nucleotide that was deleted between wild-type and P-35. The P-35 cDNA, i.e., HAV/HM-175/7, is on deposit at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. under Accession No. 67495, deposited Aug. 7, 1987. One of skill in the art can readily construct the nucleotide and amino acid sequences of P-35 by use of the above-cited art.




Thereafter the P-32 AGMK cell-adapted and attenuated virus was further manipulated to enable it to be adapted for growth in MRC-5 cells, so that it is available for large scale vaccine production. Passage 32 was double plaque cloned in MRC-5 to form Passage 37. Passage 37 was passaged once in MRC-5 of a selected clone 24-4-21. The resulting Passage 38 was passaged three times in MRC-5 cells, resulting in Passage 41, the master seed, designated 87J19. This master seed virus stock was also called virus 4380, and is referred to throughout this disclosure by the latter name.




Live attenuated virus HAV 4380, was deposited on Apr. 4, 1990 at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, 75724, Paris CEDEX 15 under Accession No. I-936. The deposited HAV 4380 virus has the nucleic acid sequence shown in SEQ ID NO:3.














TTCAAGAGGG GTCTCCGGGA ATTTCCGGAG TCCCTCTTGG AAGTCCATGG TGAGGGGACT




60














TGATACCTCA CCGCCGTTTG CCTAGGCTAT AGGCTAAATT TTCCCTTTCC CTTTTCCCTT




120













TCCCATTCCC TTTTGCTTGT AAATATTGAT TCCTGCAGGT TCAGGGTTCT TAAATCTGTT




180













TCTCTATAAG AACACTCATT TTCACGCTTT CTGTCTTCTT TCTTCCAGGG CTCTCCCCTT




240













GCCCTAGGCT CTGGCCGTTG CGCCCGGCGG GGTCAACTCC ATGATTAGCA TGGAGCTGTA




300













GGAGTCTAAA TTGGGGACAC AGATGTTTGG AACGTCACCT TGCAGTGTTA ACTTGGCTTT




360













CATGAATCTC TTTGATCTTC CACAAGGGGT AGGCTACGGG TGAAACCTCT TAGGCTAATA




420













CTTCTATGAA GAGATGCCTT GGATAGGGTA ACAGCGGCGG ATATTGGTGA GTTGTTAAGA




480













CAAAAACCAT TCAACGCCGG AGGACTGACT CTCATCCAGT GGATGCATTG AGTGGATTGA




540













CTGTCAGGGC TGTCTTTAGG CTTAATTCCA GACCTCTCTG TGCTTGGGGC AAACATCATT




600













TGGCCTTAAA TGGGATTCTG TGAGAGGGGA TCCCTCCATT AACAGCTGGA CTGTTCTTTG




660













GGGTCTTATG TGGTGTTTGC CGCTGAGGTA CTCAGGGGCA TTTAGGTTTT TCCTCATTCT




720













TAAATAATA  ATG AAC ATG TCT AGA CAA GGT ATT TTC CAG ACT GTT GGG AGT




771













GGT CTT GAC CAC ATC CTG TCT TTG GCA GAC ATT GAG GAA GAG CAA ATG




819













ATT CAA TCA GTT GAT AGG ACT GCA GTG ACT GGT GCT TCT TAT TTT ACT




867













TCT GTG GAT CAA TCT TCA GTT CAT ACA GCT GAG GTT GGA TCA CAC CAG




915













GTT GAA CCT TTG AGA ACC TCT GTT GAT AAA CCC GGT TCA AAG AGG ACT




963













CAG GGA GAG AAA TTT TTC TTG ATT CAT TCT GCA GAT TGG CTT ACT ACA




1011













CAT GCT CTT TTC CAT GAA GTT GCA AAA TTG GAT GTG GTG AAA TTA TTA




1059













TAC AAT GAG CAG TTT GCT GTT CAA GGG TTG TTG AGA TAC CAT ACA TAT




1107













GCA AGA TTT GGC ATT GAA ATT CAA GTT CAG ATA AAC CCT ACA CCT TTC




1155













CAA CAG GGG GGA TTG ATC TGT GCT ATG GTT CCT GGT GAC CAG AGC TAT




1203













GGT TCT ATA GCA TCA TTG ACT GTT TAT CCT CAT GGT TTG TTA AAT TGC




1251













AAT ATT AAC AAT GTG GTT AGA ATA AAG GTT CCA TTT ATT TAC ACA AGA




1299













GGT GCT TAC CAC TTT AAA GAT CCA CAA TAC CCA GTT TGG GAA TTG ACA




1347













ATT AGA GTT TGG TCA GAA TTA AAT ATT GGG ACA GGA ACT TCA GCT TAT




1395













ACT TCA CTC AAT GTT TTA GCT AGA TTT ACA GAT TTG GAG TTG CAT GGA




1443













TTA ACT CCT CTT TCT ACA CAA ATG ATG AGA AAT GAA TTT AGG GTC AGT




1491













ACT ACT GAG AAT GTG GTG AAT CTG TCA AAT TAT GAA GAT GCA AGA GCA




1539













AAG ATG TCT TTT GCT TTG GAT CAG GAA GAT TGG AAA TCT GAT CCG TCC




1587













CAG GGT GGT GGG ATC AAA ATT ACT CAT TTT ACT ACT TGG ACA TCT ATT




1635













CCA ACT TTG GCT GCT CAG TTT CCA TTT AAT GCT TCA GAC TCA GTT GGT




1683













CAA CAA ATT AAA GTT ATT CCA GTT GAC CCA TAT TTT TTC CAA ATG ACA




1731













AAT ACA AAT CCT GAC CAA AAA TGT ATA ACT GCT TTG GCT TCT ATT TGT




1779













CAG ATG TTT TGT TTT TGG AGA GGA GAT CTT GTC TTT GAT TTT CAA GTT




1827













TTT CCC ACC AAA TAT CAT TCA GGT AGA TTA CTG TTT TGT TTT GTT CCT




1875













GGC AAT GAG CTA ATA GAT GTT TCT GGA ATC ACA TTA AAG CAA GCA ACT




1923













ACT GCT CCT TGT GCA GTA ATG GAT ATT ACA GGA GTG CAG TCA ACT TTG




1971













AGA TTT CGT GTT CCC TGG ATT TCT GAC ACT CCT TAC AGA GTG AAC AGG




2019













TAT ACA AAG TCA GCA CAT CAG AAA GGT GAG TAC ACT GCC ATT GGG AAG




2067













CTT ATT GTG TAT TGT TAT AAC AGA TTG ACC TCT CCT TCT AAC GTT GCT




2115













TCC CAT GTC AGA GTG AAT GTT TAT CTT TCA GCA ATT AAC TTG GAA TGT




2163













TTT GCT CCT CTT TAT CAT GCT ATG GAT GTT ACT ACA CAA GTT GGA GAT




2211













GAT TCT GGA GGT TTT TCA ACA ACA GTT TCT ACA GAA CAG AAT GTT CCA




2259













GAT CCC CAA GTT GGT ATA ACA ACC ATG AAA GAT TTG AAA GGA AAA GCT




2307













AAC AGA GGG AAA ATG GAT GTT TCA GGA GTA CAA GCA CCT GTG GGA GCT




2355













ATC ACA ACA ATT GAG GAT CCA GTT TTA GCA AAG AAA GTA CCT GAG ACA




2403













TTT CCT GAA TTG AAA CCT GGA GAA TCC AGA CAT ACA TCA GAT CAT ATG




2451













TCC ATC TAC AAG TTT ATG GGA AGG TCT CAT TTC TTG TGC ACT TTT ACA




2499













TTC AAT TCA AAT AAT AAA GAG TAC ACA TTT CCT ATA ACC TTG TCT TCA




2547













ACC TCT AAT CCT CCT CAT GGT TTG CCA TCA ACA CTG AGG TGG TTT TTC




2595













AAC TTG TTT CAG TTG TAT AGA GGG CCT TTA GAT CTG ACA ATT ATT ATT




2643













ACA GGA GCA ACT GAT GTA GAT GGC ATG GCC TGG TTC ACT CCA GTA GGT




2691













CTT GCC GTT GAT ACT CCT TGG GTA GAG AAG GAG TCA GCT TTG TCT ATT




2739













GAC TAT AAA ACT GCT CTT GGA GCT GTC AGA TTT AAC ACA AGG AGA ACA




2787













GGG AAC ATT CAG ATT AGA TTA CCA TGG TAT TCT TAT TTA TAT GCT GTG




2835













TCT GGA GCA CTG GAT GGT TTG GGA GAC AAG ACA GAT TCT ACA TTT GGA




2883













TTG GTT TCT ATT CAG ATT GCA AAT TAC AAT CAT TCT GAT GAA TAC TTG




2931













TCT TTT AGT TGT TAT TTG TCT GTC ACA GAA CAA TCA GAG TTT TAT TTT




2979













CCC AGA GCT CCA TTG AAC TCA AAT GCC ATG TTA TCC ACT GTA ACA ATG




3027













ATG AGC AGA ATT GCA GCT GGA GAC TTG GAG TCA TCA GTG GAT GAT CCT




3075













AGA TCA GAG GAA GAT AAA AGA TTT GAG AGT CAT ATA GAA TGC AGG AAG




3123













CCA TAT AAA GAA CTG AGA TTA GAA GTT GGG AAA CAA AGA CTC AAG TAT




3171













GCT CAG GAA GAA TTG TCA AAT GAA GTA CTT CCA CCC CCT AGG AAA ATG




3219













AAG GGA CTG TTT TCA CAA GCC AAA ATT TCT CTT TTT TAT ACT GAG GAG




3267













CAT GAA ATA ATG AAG TTT TCC TGG AGA GGT GTG ACT GCT GAT ACT AGA




3315













GCT TTA AGG AGG TTT GGA TTC TCT TTG GCC GCA GGC AGA AGT GTG TGG




3363













ACT CTT GAA ATG GAT GCT GGG GTT CTT ACT GGG AGA CTG ATT AGA TTG




3411













AAT GAT GAG AAA TGG ACA GAA ATG AAG GAT GAC AAG ATT GTT TCA TTG




3459













ATT GAA AAG TTT ACA AGT AAC AAA TAT TGG TCC AAA GTG AAT TTC CCA




3507













CAT GGG ATG TTG GAT CTT GAA GAA ATT GCT GCC AAT TCT AAG GAT TTT




3555













CCT AAC ATG TCT GAA ACG GAT TTG TGT TTC TTG CTG CAT TGG TTA AAT




3603













CCA AAG AAA ATT AAT TTA GCA GAT AGA ATG CTT GGA TTG TCT GGA GTT




3651













CAG GAA ATT AAA GAA CAA GGT GTT GGA TTA ATA GCA GAG TGT AGA ACT




3699













TTC TTA GAT TCT ATT GCT GGA ACT TTA AAA TCT ATG ATG TTT GGA TTT




3747













CAT CAT TCT GTG ACT GTT GAA ATT ATA AAC ACT GTG CTC TGT TTT GTT




3795













AAG AGT GGA ATT TTG CTT TAT GTA ATA CAA CAA TTG AAT CAG GAT GAA




3843













CAT TCT CAC ATA ATT GGT TTG TTG AGA GTC ATG AAT TAT GTA GAT ATT




3891













GGT TGT TCA GTT ATT TCA TGT GCC AAA GTT TTT TCC AGA ATG CTG GAA




3939













ACA GTC TTT AAT TGG CAA ATG GAC TCC AGA ATG ATG GAG TTA AGG ACT




3987













CAG AGT TTT TCC AAC TGG TTA AGA GAT ATT TGT TCT GGG ATC ACC ATT




4035













TTC AAA AAC TTC AAG GAT GCA ATT TAT TGG CTT TAT ACA AAA TTA ATG




4083













GAC TTT TAT GAA GTG AAT TAT GGC AAG AAG AAG GAC ATT TTA AAT ATT




4131













CTT AAA GAT AAC CAA CAA AAA ATA GAG AAA GCC ATT GAG GAA GCC GAT




4179













AAA TTT TGC ATT TTG CAA ATC CAA GAT GTG GAA AAA TCT GAA CAG TAT




4227













CAG AAA GGG GTT GAC TTG ATA CAA AAA TTG AGA ACT GTT CAT TCA ATG




4275













GCT CAG GTT GAT CCA AAT TTA ATG GTT CAT TTG TCA CCT TTG AGA GAT




4323













TGT ATA GCA AGA GTT CAT CAG AAA CTT AAA AAC CTT GGA TCT ATA AAT




4371













CAG GCA ATG GTA ACG AGA TGT GAG CCA GTT GTT TGT TAT TTT TAT GGC




4419













AAA AGA GGG GGA GGA AAG AGC TTA ACA TCA ATT GCA TTG GCA ACC AAA




4467













ATT TGT AAA CAT TAT GGT GTT GAG CCT GAA AAG AAT ATC TAT ACT AAA




4515













CCT GTG GCT TCA GAT TAC TGG GAT GGA TAT AGT GGA CAA TTA GTT TGC




4563













ATC ATT GAT GAT ATT GGC CAA AAC ACA ACA GAT GAG GAT TGG TCA GAT




4611













TTT TGT CAG TTA GTG TCA GGA TGT CCT ATG AGA TTA AAC ATG GCC TCT




4659













CTT GAG GAG AAG GGT AGG CAT TTT TCT TCT CCT TTT ATA ATA GCA ACT




4707













TCA AAT TGG TCA AAT CCA AGT CCA AAA ACA GTT TAT GTT AAG GAA GCA




4755













ATT GAC CGC AGA CTC CAT TTC AAG GTT GAA GTT AAA CCT GCT TCA TTT




4803













TTC AAA AAT CCT CAC AAT GAT ATG TTG AAT GTT AAT TTA GCT AAA ACA




4851













AAT GAT GCA ATC AAA GAT ATG TCT TGT GTT GAT TTG ATA ATG GAT GGA




4899













CAT AAT GTT TCA TTG ATG GAT TTG CTC AGT TCT TTA GTC ATG ACA GTT




4947













GAA ATT AGA AAA CAA AAC ATG ACT GAA TTC ATG GAG TTG TGG TCT CAG




4995













GGA ATT TCA GAT GAT GAT AAT GAT AGT GCA GTA GCT GAG TTT TTC CAG




5043













TCT TTT CCA TCT GGT GAA CCA TCG AAC TCT AAA TTA TCT GGC TTT TTC




5091













CAA TCT GTT ACT AAT CAC AAG TGG GTT GCT GTG GGA GCT GCA GTT GGC




5139













GTT CTT GGA GTG CTC GTT GGA GGA TGG TTT GTG TAT AAG CAT TTC TCC




5187













CGC AAA GAG GAA GAA CCA ATC CCA GCT GAA GGG GTA TAT TAT GGT GTA




5235













ACT AAG CCC AAG CAA GTG ATT AAA TTA GAT GCA GAT CCA GTA GAA TCT




5283













CAG TCA ACT TTG GAA ATA GCA GGA CTG GTT AGG AAG AAC TTG GTT CAG




5331













TTT GGA GTT GGA GAG AAG AAT GGA TGT GTG AGA TGG GTT ATG AAT GCC




5379













TTG GGA GTG AAA GAT GAT TGG CTG CTT GTG CCT TCC CAT GCT TAT AAA




5427













TTT GAG AAA GAT TAT GAA ATG ATG GAG TTT TAT TTT AAT AGA GGT GGA




5475













ACT TAC TAT TCA ATT TCA GCT GGT AAT GTT GTT ATT CAA TCT TTG GAT




5523













GTG GGA TTC CAG GAT GTT GTT CTG ATG AAG GTT CCT ACA ATT CCT AAG




5571













TTT AGA GAT ATT ACT CAG CAT TTT ATT AAG AAA GGG GAT GTG CCT AGA




5619













GCT TTG AAT CGC CTG GCA ACA TTA GTG ACA ACT GTA AAT GGA ACC CCT




5667













ATG TTA ATT TCT GAG GGC CCA CTA AAG ATG GAA GAG AAA GCT ACT TAT




5715













GTT CAT AAG AAA AAT GAT GGT ACA TCA GTT GAT TTA ACT GTG GAT CAG




5763













GCA TGG AGA GGA AAA GGC GAA GGT CTT CCT GGA ATG TGT GGT GGG GCC




5811













TTG GTT TCA TCG AAT CAA TCT ATA CAG AAT GCA ATC TTG GGC ATC CAT




5859













GTT GCT GGA GGA AAT TCA ATT CTT GTT GCA AAA TTG GTT ACT CAA GAA




5907













ATG TTC CAA AAT ATT GAT AAG AAA ATT GAA AGT CAG AGA ATT ATG AAA




5955













GTG GAG TTT ACT CAG TGT TCA ATG AAT GTG GTC TCC AAA ACG CTT TTT




6003













AGA AAG AGT CCC ATT TAT CAT CAC ATT GAT AAA ACC ATG ATT AAT TTT




6051













CCT GCA GCT ATG CCC TTT TCT AAA GCT GAA ATT GAT CCA ATG GCT GTG




6099













ATG TTA TCT AAG TAT TCA TTA CCT ATT GTA GAA GAA CCA GAG AAT TAT




6147













AAA GAG GCT TCA ATT TTT TAT CAA AAT AAA ATA GTG GGT AAG ACT CAG




6195













TTA GTT GAT GAT TTT CTA GAT CTT GAT ATG GCC ATT ACA GGG GCC CCA




6243













GGA ATT GAT GCT ATC AAC ATG GAT TCA TCT CCT GGA TTT CCT TAT GTC




6291













CAG GAG AAG TTG ACC AAA AGA GAT TTA ATT TGG TTG GAT GAA AAT GGT




6339













TTA TTG CTG GGA GTT CAT CCA AGA TTG GCT CAG AGA ATC TTA TTC AAT




6387













ACT GTC ATG ATG GAA AAT TGT TCT GAT TTG GAT GTT GTT TTT ACA ACC




6435













TGT CCA AAA GAT GAA TTG AGA CCA TTA GAG AAA GTG TTG GAA TCA AAA




6483













ACA AGA GCT ATT GAT GCT TGT CCT CTG GAT TAC ACA ATT TTG TGC CGA




6531













ATG TAT TGG GGT CCA GCT ATT AGT TAT TTT CAT TTG AAT CCA GGT TTC




6579













CAT ACA GGT GTT GCT ATT GGC ATA GAT CCT GAT AGA CAG TGG GAT GAA




6627













TTA TTT AAA ACA ATG ATA AGA TTC GGA GAT GTT GGT CTT GAT TTA GAT




6675













TTC TCT GCT TTT GAT GCT AGT CTT AGT CCA TTT ATG ATT AGA GAA GCA




6723













GGT AGA ATC ATG AGT GAA CTA TCT GGA ACT CCA TCC CAT TTT GGC ACA




6771













GCT CTT ATC AAT ACT ATC ATT TAT TCC AAG CAT TTG CTG TAT AAC TGT




6819













TGT TAC CAT GTC TGT GGT TCA ATG CCC TCT GGG TCT CCT TGT ACA GCT




6867













TTG CTA AAT TCA ATT ATT AAT AAT GTC AAT TTG TAC TAT GTG TTT TCC




6915













AAG ATA TTT GGA AAG TCT CCA GTT TTC TTT TGT CAG GCT TTG AAG ATT




6963













CTC TGT TAT GGA GAT GAT GTT TTA ATA GTT TTC TCT CGA GAT GTT CAG




7011













ATT GAT AAT CTT GAT TTG ATT GGA CAA AAA ATT GTA GAT GAG TTT AAG




7059













AAA CTT GGC ATG ACA GCT ACT TCT GCT GAC AAG AAT GTA CCT CAG CTG




7107













AAA CCA GTT TCG GAA TTG ACT TTT CTC AAA AGA TCT TTC AAT TTG GTA




7155













GAG GAT AGA ATT AGA CCT GCA ATT TCG GAA AAA ACA ATT TGG TCT TTA




7203













ATA GCA TGG CAG AGA AGT AAC GCT GAG TTT GAG CAG AAT TTA GAA ATT




7251













GCT CAG TGG TTT GCT TTT ATG CAT GGC TAT GAG TTT TAT CAG AAA TTT




7299













TAT TAT TTT GTT CAG TCC TGT TTG GAG AAA GAG ATG ATA GAA TAC AGA




7347













CTT AAA TCT TAT GAT TGG TGG AGA ATG AGA TTT TAT GAC CAG TGT TTC




7395













ATT TGT GAC CTT TCA TGA TTTGTTTAAA CGAATTTTCT TAAAATTTCT




7443













GAGGTTTGTT TATTTCTTTT ATCAGTAAAT AAAAAAAAAA AAA




7486











This deposit was made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms and has not been publicly disseminated. HAV 4380 is a cell culture-adapted and attenuated strain of hepatitis A virus strain HM-175, adapted to growth in a human fibroblast cell line (MRC-5) suitable for vaccine development by incubation at a reduced temperature of 32-35° C. Growth of the virus is determined by detection of viral antigen in a serological assay. The adapted virus is purified by plaque-purification, using an accepted method (radioimmunofocus assay).




After a total of nine passages in MRC-5 cells at reduced temperature, the resultant virus was characterized for its biological characteristics in cell culture and in two primate species that are considered to be surrogates for man, i.e., marmosets and chimpanzees. See, e.g., Example 1 below. The HAV 4380 virus was found to be temperature-sensitive (i.e., only grew at reduced temperatures) in MRC-5 cells but was still capable of growing at 37° C. in primary African green monkey kidney cells. The virus was further attenuated in virulence, compared to the parent virus HM-175, P-32, when tested in chimpanzees and marmoset monkeys, in which species the virus replicated poorly or not at all. This reduced capacity for replication in primates was further confirmed in human volunteers, as described in Example 2.




A candidate inactivated hepatitis A vaccine was prepared from the HAV 4380 and demonstrated to be safe (i.e., it does not produce hepatitis or other serious adverse effects) and immunogenic in humans. It was also found to induce antibody production without adjuvant. HAV 4380, as it currently exists, grows well in a cell substrate suitable for commercial vaccine production. It also does not infect human beings when administered by the oral or intravenous route at doses of up to 10


7


tissue culture infectious doses, even when not inactivated. HAV 4380 is suitable for use as a live HAV vaccine in humans. However, as indicated in Example 2, vaccine 4380 is believed to be somewhat over-attenuated, because it is not infectious, which characteristic reduces its efficiency when used as an attenuated vaccine.




In order to produce other vaccine candidates which are maximized for desirable levels of attenuation and good growth in MRC-5 cells, the inventors discovered genetic changes that occurred in the genome of the MRC-5-adapted HAV 4380 virus that altered its growth characteristics and made it more suitable for vaccine production than the related AGMK-adapted virus HM-175, Passage 35. The discovery of the following mutations in the nucleotide sequences in 4380, when compared to HM-175 Pass 35 [Cohen et al, cited above; and U.S. Pat. No. 4,894,228,

FIG. 1

; permit the manipulation of the HAV genome by genetic engineering techniques.




Thus, knowledge of the genomic differences between the AGMK-adapted passages of HM-175 and the more attenuated 4380 permit the construction of chimeric viruses having the improved growth characteristics, i.e., rapid and efficient growth in MRC-5 cell culture, but with a level of attenuation of virulence for primate species, including man, that will permit the virus to replicate efficiently without producing hepatitis or other untoward effects. This invention permits the design of a chimeric HAV that can achieve the optimum characteristics for a candidate live-attenuated hepatitis A vaccine. Such a virus will also permit the design of preferred inactivated vaccine candidates, if desired. The present invention identifies the mutations that are believed to have occurred during adaptation to growth of the HM-175 HAV, passage 32, strain in MRC-5 cells. One or a combination of these mutations are responsible for MRC-5 cell adaptation and overattenuation in HAV 4380. The nucleotide sequence of the MRC-5 cell-adapted virus HAV 4380 was compared with that of the AGMK-adapted, HM-175 virus, passage 35, clone 7. Nucleotide consensus sequences were determined directly from polymerase chain reaction products.




The inventors have discovered that there are at least sixteen unique nucleotide differences between the pass-35 HM-175/7 virus and the MRC-5-adapted virus 4380. Table I lists these sixteen mutations by nucleotide differences and resulting amino acid (AA) differences, if any, acquired by the MRC-5 adapted virus HAV 4380. Note that the partial sequence of LHS/S HAV of Fineschi et al., cited above, overlaps with only the mutation observed at position 5145.




In the Table, A represents adenine, G represents guanine, C represents cytosine, and T represents thymine; Leu represents leucine, Phe represents phenylalanine, Ile represents isoleucine, Val represents valine, Ser represents serine, Lys represents lysine, Asn represents asparagine and Thr represents threonine.












TABLE I











Difference in Nucleotide Sequence of






MRC-5-Adapted Hepatitis A Virus:






Comparison with Sequence of HM-175/7 (P-35)















Nucleotide




Region of








Change




Genome




AA Change











591 A to G




5′ nc




NA







646 G to A




5′ nc




NA







669 C to T




5′ nc




NA







687 T to G




5′ nc




NA







2750 C to T




VP1




No change







3027 T to A




2A




Ser to Thr







3196 G to A




2A




Ser to Asn







3934 A to G




2B




Lys to Arg







4418 A to T




2C




Leu to Phe







4563 A to G




2C




Ile to Val







4643 A to T




2C




No change







5145 A to G




3A




Ile to Val







5745 A to T




3C




Thr to Ser







6908 T to C




3D




No change







7032 C to T




3D




No change







7255 A to T




3D




Asn to Ile















New HAV vaccine candidates are designed by introducing one or more of these nucleotides into an HAV at a nucleotide position homologous to the nucleotide position in the genomic sequence of the AGMK-adapted virus HM-175, Pass 35. These nucleotides identified in Table I may be introduced at analogous and/or homologous nucleotide positions to those of P-35 in the genomic sequences of other HAV strains and variants to produce a recombinant or chimeric HAV of this invention. By the phrase “analogous or homologous nucleotide position” is meant a nucleotide in an HAV other than HAV HM-175, Pass 35 which is present in the same viral region, e.g., 2C, 3D and the like, at a position in that region similar to that of the nucleotide of Table I. In other words, the nucleotide position may differ in position number due to deletions in other regions of the virus; but one of skill in the art can readily determine its functional similarity to the nucleotide position in HM-175, Pass 35.




While such nucleotide positions may not have the identical nucleotide position numbers corresponding to the wild-type HM-175 [SEQ ID NO:1], it is anticipated that these analogous and/or homologous positions can be readily identified to enable HAVs other than strain HM-175 derivatives to be modified to create novel HAVs according to this invention.




Similarly, the inventors are able to manipulate the genome of a progenitor or intermediate of HAV 4380 with resort to this knowledge and can thereby ‘reverse’ certain mutations in 4380 to create new chimeric HAV viruses. One or more of these nucleotides, or varying combinations thereof, can be incorporated, by chimera formation or oligonucleotide-directed mutagenesis, into an HAV strain, most readily the cDNA clone HAV/HM-175/7, to produce new viable virus which has acquired the ability to grow in MRC-5 cells. Other HM-175 HAV derivatives are available from the American Type Culture Collection under ATCC designation numbers VR 2089, VR 2090, VR 2091, VR 2092, VR 2093, VR 2097, VR 2098, and VR 2099. These and other HAVs may be employed to derive desired HAVs of this invention. Since there are indications that the MRC-5-adapted virus 4380 may be over-attenuated for humans, it is important to be able to remove or introduce selected mutations into HM-175. The construction of nine exemplary chimeric viruses containing one or more such mutations is described in detail in Example 3 below.




The mutagenic and genetic engineering techniques employed to construct chimeric or recombinant HAVs which incorporate one or more of these mutations are conventional and known to those of skill in the art [see, for example, Sambrook et al.,


Molecular Cloning. A Laboratory Manual,


2d edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)]. Other conventional techniques, including polymerase chain reactions and chemical synthetic techniques may also be used to design HAVs of this invention. Similarly, it is anticipated that homologous mutations may be made using other HM-175 passages. It also may be possible to adapt similar changes to HAV strains other than HM-175 by introducing these nucleotides into homologous regions.




Chimeric and recombinant viruses of this invention may be designed by application of similar techniques and selecting one or more different combinations of the nucleotides (mutations) appearing in Tables I and VI. For example, data from growth analyses of the chimeric viruses of Example 3 demonstrate that one or more of the four MRC-5 specific mutations in the 5′ non-coding region (mutations at nucleotide positions 591, 646, 669, and 687 of HM-175/7) and one or both of the MRC-5 specific mutations in the 2C region (mutations at nucleotide positions 4418 and 4643 of HM-175/7) may be desirable for optimal growth of the virus in MRC-5 cells. Other mutations may also be involved. Specific exemplary chimeric HAVs of this invention are characterized by the mutations in the genome of HAV HM-175/7 that appear in viruses designated #2 through #10 in Table VI of Example 3 below.




HAVs of this invention may be characterized by the presence of one or more of these nucleotides of Tables I or VI in analogous genomic positions of HAV HM-175 derivatives or other HAV strains. HAVs of this invention may also be characterized by two or more such nucleotides, where one nucleotide in the HAV parent strain is a guanine (G) at position 5145 of pHAV/7 or the analogous position of another HAV strain.




It is further anticipated that additional mutations may appear in a few regions of HAV that have yet to be sequenced. The mutations appearing in Table I may be incorporated in any combination, and/or with other mutations yet to be identified to construct a number of chimeric or recombinant HAVs with desired characteristics for use as live HAV vaccines.




Additional chimeras and recombinant viruses constructed by oligonucleotide-directed mutagenesis may be designed and evaluated for assessment of the individual effects of the mutations and combinations thereof on viral growth in MRC-5 cells and on adaptation to growth in selected cell culture. The attenuation phenotype of these chimeric viruses may be evaluated in marmosets or chimpanzees by techniques such as described below in Example 1 for HAV 4380.




Also provided by this invention are the polynucleotide sequences encoding the HAVs of this invention. Such polynucleotide sequences are preferably cDNA sequences, which can form a master seed for the HAV vaccine. A cDNA sequence of this invention comprises a DNA sequence encoding a selected HAV genome characterized by the presence of one or more of the nucleotides identified as the twelve mutations in Table I in any desired combination which imparts desired characteristics to the novel HAV. Such cDNAs may be obtained by conventional techniques known to those of skill in the art. See, e.g., Sambrook et al, cited above, and U.S. Pat. No. 4,894,228.




Thus, the present invention provides a live vaccine composition useful in protecting against HAV infection and a prophylactic method entailing administering to a primate, preferably a human, an effective amount of such a composition. This vaccine composition may contain one or more of the HAVs of the invention, including HAV 4380, as well as the chimeric and recombinant HAVs described herein. The vaccine composition may also contain mixtures of two or more of the HAVs, if desired.




A vaccinal composition may be formulated to contain a carrier or diluent and one or more of the HAVs of the invention. Suitable pharmaceutically acceptable carriers facilitate administration of the viruses but are physiologically inert and/or nonharmful. Carriers may be selected by one of skill in the art. Exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, and water. Additionally, the carrier or diluent may include a time delay material, such as glycerol monostearate or glycerol distearate alone or with a wax. In addition, slow release polymer formulations can be used.




Optionally, the vaccine composition may further contain preservatives, chemical stabilizers, other antigenic proteins, and conventional pharmaceutical ingredients. Suitable ingredients which may be used in a vaccinal composition in conjunction with the viruses include, for example, casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate, and dried milk. Typically, stabilizers, adjuvants, and preservatives are optimized to determine the best formulation for efficacy in the target human or animal. Suitable preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol.




A vaccine composition of this invention is most preferably produced without an adjuvant. However, where necessary, one or more of the above described vaccine component may be admixed or adsorbed with a conventional adjuvant. The adjuvant is used as a non-specific irritant to attract leukocytes or enhance an immune response. Such adjuvants include, among others, mineral oil and water, aluminum hydroxide, Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic plyois, muramyl dipeptide, killed Bordetella, saponins, and Quil A.




Alternatively, or in addition to the HAV of the invention, other agents useful in treating HAV infection, e.g., immunostimulatory agents, are expected to be useful in reducing and eliminating disease symptoms. The development of vaccine or therapeutic compositions containing these agents is within the skill of one of skill in the art in view of the teaching of this invention.




According to the method of the invention, a human or an animal may be vaccinated against HAV infection by administering an effective amount of a vaccine composition described above. An effective amount is defined as that amount of HAV vaccine capable of inducing protection in the vaccinee against HAV infection and/or against hepatitis. The vaccine may be administered by any suitable route. Such a composition may be administered parenterally, preferably intramuscularly or subcutaneously. However, it may also be formulated to be administered by any other suitable route, including orally.




Suitable effective amounts of the HAVs of this invention can be determined by one of skill in the art based upon the level of immune response desired. Such a composition may be administered once, and/or a booster may also be administered. However, suitable dosage adjustments may be made by the attending physician or veterinarian depending upon the age, sex, weight and general health of the human or animal patient.




Similarly, suitable doses of the vaccine composition of the invention can be readily determined by one of skill in the art. The dosage can be adjusted depending upon the human patient or the animal species being treated, i.e. its weight, age, an general health.




The following examples illustrate the preferred methods for obtaining HAVs of the invention and using them as vaccine compositions. These examples are not intended to limit the scope thereof.




EXAMPLE 1




Test of MRC-5-Adapted HAB 4380 Vaccine in Marmosets and Chimpanzees




The attenuation of hepatitis A virus (HAV), strain HM-175, by serial passage in cell culture has previously been demonstrated. Following 32 passages in primary AGMK cells, the virus was completely attenuated for chimpanzees and almost completely attenuated for marmosets. Subsequently, according to this invention the virus was adapted to growth in MRC-5 cells and recloned by plaque purification.




HAV 4380 was prepared from Volunteer lot 87J19, passage level 41 of strain HM-175 HAV that was derived from previously characterized passage levels of the virus that have also been prepared as volunteer pools. Two such earlier passage pools were shown to be attenuated for chimpanzees and marmosets. However, neither was administered to volunteers because it was recognized that primary African green monkey kidney cells, the substrate for those volunteer pools, would not be available in sufficient quantities to produce an economically viable vaccine. Therefore, the virus was adapted to MRC-5 cells and further passaged to prepare volunteer lot 87J19 or HAV 4380.




The purpose of this experiment is to test the level of attenuation of this virus for marmosets and chimpanzees, prior to phase I trials in volunteers. Lot 87J19 was tested for safety and immunogenicity in four chimpanzees and four


Saquinus mystax


marmosets. Two additional marmosets served as uninoculated controls. The chimpanzees used in this study were bred and raised in captivity; the marmosets were wild-caught animals. An inoculum of 10


4


TCID


50


of candidate vaccine lot 87J19 was administered intravenously to each animal. Residual inoculum was frozen and the titer reconfirmed subsequently in two different laboratories.




According to the experimental protocol, marmosets identified by the arbitrary ID numbers 570, 572, 566, and 575 and chimpanzees identified by the arbitrary ID numbers 1300, 1333, 1309 and 1313 received an inoculum of HAV 4380 from Clone 25-4-21, Lot 87 J 19575, 17/11/87 at a dose and route of administration of 10


−3


dilution/1 ml/I.V. Marmosets No. 541 and 578 received a diluent at a dose and route of 1 ml/I.V.




A. Infection:




Three of four chimpanzees and one of four marmosets were infected, as determined by development of anti-HAV detectable by commercial radioimmunoassay (HAVAB, Abbot Laboratories, Chicago, Ill.). The chimpanzees seroconverted ten to eleven weeks following inoculation; the single marmoset seroconversion occurred eight weeks following inoculation. This marmoset subsequently died on week eleven of the study and another, noninfected, marmoset subsequently died on week fourteen, but neither death was attributable to the inoculum.




All three chimpanzees that seroconverted also developed IgM anti-Hav. Two of these, Chip 1309 and Chimp 1313, developed IgM anti-HAV on weeks ten and thirteen, respectively, when tested by the standard HAVAB-M (Abbott Laboratories, Chicago, Ill.) at a final serum dilution of 1:4,000. When sera were tested at a dilution of 1:40, Chimp 1313 and Chimp 1333 seroconverted at weeks nine and five, respectively. The HAVAB-M test is a capture assay utilization anti-human IgM and has not been standardized for use with sera from primates less closely related to man than to the chimpanzee. For this reason, the marmosets were not tested for IgM anti-HAV.




B. Biochemistry:




Biochemical evidence of hepatitis was monitored by weekly determinations of serum alanine amino transferase (ALT) and isocitric dehydrogenase (ICD). The former is the most reliable indirect means of diagnosing hepatitis in the chimpanzee and the latter is comparably sensitive for evaluating marmosets. None of the chimpanzees or marmosets had elevation of liver enzymers attributable to the inoculum. All values for chimpanzees were within normal limits. The only infected marmoset, number 582, had normal liver enzymes up to the time of its death. Marmosets 566, 570, and 578 each had one or more abnormal liver enzyme values, but the first two of these animals were not infected by the inoculum, as judged by failure to seroconvert, and the third was an uninoculated negative control.




Marmosets often have less stable liver enzyme values than chimpanzees, in part because they are, by nature, relatively fragile animals and because they are jungle-caught and therefore usually infected with a variety of endo- and ecto-parasites, including microfilaria.




C. Histology:




Histologic sections prepared from serial weekly liver biopsies obtained from the chimpanzees and marmosets were evaluated under code for histopathologic changes. Although some animals had a high base-line of histopathologic changes, none of the animals had evidence of histopathologic changes more severe than those seen in preinoculation biopsies. Equally important, there were no histologic changes that were temporally related to seroconversion in infected animals. The two marmosets that died were subjected to more extensive evaluation. Both animals had evidence of systemic disease that was probably etiologically related to their deaths, but histologic changes in the liver were diagnostic of chronic, not acute, disease and therefore not related to the inoculum.




A comparison of histopathologic changes observed in chimpanzees and marmosets with these various volunteer pools and wild-type virus was performed. See Tables II and III below.












TABLE II











HISTOPATHOLOGY: CHIMPANZEES














Liver Histopathology






















Range of







Inoculum*




# Inoc.




# Infected




#




Severity**











W.T.




4




4




4




±-3+







P-21




6




6




1




0-3+







P-32




6




6




0




0







MRC-5




4




3




0




0













*Dose: 10


3


-10


5


ID


50


IV











**Scale of 0-3+





















TABLE III











HISTOPATHOLOGY: MARMOSETS














Liver Histopathology






















Range of







Inoculum*




# Inoc.




# Infected




#




Severity**











W.T.




4




4




4




1+-2+







P-21




8




8




8




1+-3+







P-32




5




5




3




0-2+







MRC-5




4




1




0




0













*Dose: 10


3


-10


5


ID


50


IV











**Scale of 0-3+













Lot 87J19 appears to be more attenuated than the other volunteer pools or wild-type virus, based upon infectivity and severity of histopathologic changes.




D. Immunofluorescence:




Serial snap-frozen liver biopsies obtained from infected animals were evaluated for expression of viral antigen by immunofluorescence. Only one animal, Chimp 1313, was definitely but weakly positive for intrahepatic viral antigen. This animal was positive for only one week. These results were compared with those obtained in the previous study of other volunteer pools and wild-type virus. As seen in Tables IV and V, intrahepatic replication was further diminished in both chimpanzees and marmosets when compared with the AGMK-grown virus and wild-type virus.












TABLE IV











VIRAL REPLICATION IN THE LIVER:






MARMOSETS (IMMUNOFLUORESCENCE)




















Mean




Mean







Inoculum*




# Inoc.




# Infected




Peak




Duration (wks)











W.T.




4




4




2.5+




12.2







P-21




8




8




1+




3.5







P-32




5




5




<1+




2.4







MRC-5




4




1




0




0













*Dose: 10


3


-10


5


ID


50


I.V.





















TABLE V











VIRAL REPLICATION IN THE LIVER:






CHIMPANZEES (IMMUNOFLUORESCENCE)




















Mean




Mean







Inoculum*




# Inoc.




# Infected




Peak




Duration (wks)











W.T.




4




4




1+




1.8







P-21




6




6




<1+




0.5







P-32




6




6




<1+




0.6







MRC-5




4




3




<1+




0.3













*Dose: 10


3


-10


5


ID


50


I.V.













E. Protection:




Although the single infected marmoset on study died, all four chimpanzees were available for challenge with wild-type parent HAV to determine if the levels of anti-HAV present in infected animals were protective. Consequently, the three infected and one uninfected animals were challenged with approximately 10


3


chimpanzee infectious doses of wild-type HM-175 strain HAV (human stool suspension), administered intravenously (FIGS.


1


through


4


).




All three previously infected chimpanzees were protected against type A hepatitis, as measured by persistently normal serum enzyme values (FIGS.


1


through


3


). All three protected animals had an anamnestic antibody response to the challenge virus, suggesting that there was limited replication. In contrast, the previously uninfected chimpanzee developed high enzyme values diagnostic of hepatitis following challenge with wild-type virus (FIG.


4


). Thus, volunteer pool 87J19 produced an inapparent infection in chimpanzees that stimulated protection against subsequent challenge with virulent wild-type virus.




The results of these portions of the experiment demonstrate that volunteer pool 87J19 of HAV 4380, strain HM-175 (adapted to MRC-5 cells) was significantly more attenuated for chimpanzees and marmosets than its parent, HAV, strain HM-175 (AGMK, Pass-32). It is clear from these studies that HAV 4380, strain HM-175 volunteer pool 87J19, is highly attenuated for chimpanzees and marmosets which are accepted surrogates for man in the study of hepatitis A viruses.




EXAMPLE 2




Clinical Study of Volunteers




In this clinical trial, volunteers received increasing titers of the liver attenuated hepatitis A vaccine 4380, volunteer pool 87J19, which was previously tested in chimpanzees and marmosets as described in Example 1. These pre-clinical studies demonstrated that the vaccine was safe, immunogenic, and efficacious in experimental animal models.




Volunteers were admitted to a closed clinical ward at the United States Army Medical Research Institute of Infectious Diseases, For Detrick, Md. Eight volunteers received the live attenuated hepatitis A vaccine (1 ml) by the oral route in the following manner: two received a 10


4


TCID


50


dose, tow a 10


5


TCID


50


dose, two a 10


6


TCID


50


dose, and two a 10


7


TCID


50


dose. Six volunteers received the vaccine by the intramuscular route in the deltoid area in the following manner: 2 received a 10


5


TCID


50


dose, 2 a 10


6


TCID


50


dose and 2 a 10


7


TCID


50


dose.




Each volunteer remained on the ward for three days after immunization. Local or systemic side effects were monitored during the admission period and for 12 weeks following the immunization. Volunteers were asked to return at 6 and 12 months for serological follow-up.




Sera were obtained prior to immunization and once a week for the next 12 weeks. In volunteers who completed the appropriate follow-up time, sera were also obtained at 6 and 12 months after initial administration of vaccine. Serum specimens were tested for alanine aminotransferase (ALT) and antibody to hepatitis A. ALT was tested with a Kodak EKTA Chem 700XR analyzer (Rochester, N.Y.); normal values were 0 to 50 IU/ml. Antibody to hepatitis A was tested by four different methods, including a commercial radioimmunoassay (HAVAB, Abbott Laboratories, N. Chicago, Ill.). Second, an enzyme-linked immunoassay developed by SmithKline Beecham (SKB-ELISA), which was more sensitive than the standard HAVAB, in which a level of ≧20 milli-International Units (mIU) was considered positive. Selected sera were tested by the RIFIT (radioimmunofocus) assay for neutralizing antibody to hepatitis A. With this test, a serum titer of 23 1:10 was considered positive [S. M. Lemon et al,


J. Infect. Dis


., 148:1033-1039 (1983)]. Finally sera were tested for IgM anti-HAV by commercial radioimmunoassay (HAVAB-M, Abbott Laboratories, N. Chicago, Ill.).




Stools were collected from the volunteers two to three times per week for the first 12 weeks and were tested for the presence of hepatitis A virus by radioimmunoassay [R. H. Purcell et al,


J. Immunol


., 116:349-356 (1976)] and molecular biology techniques [J. Ticehurst et al,


J. Clin. Microbiol


., 25:1822-1829 (1987)].




All volunteers remained healthy during the follow-up period (14 weeks to one year). No systemic complaints were present immediately after immunization or during long-term follow-up. Serum alanine aminotransferase levels remained normal in all 14 individuals during the period of observation.




Antibody to hepatitis A was not observed in any of the eight volunteers who received the vaccine by the oral route or in the two volunteers who received the 10


5


TCID


50


dose by the intramuscular route. The four volunteers who received higher doses of vaccine (10


6


TCID


50


or 10


7


TCID


50


) all had detectable antibody by the SKB ELISA as early as 3 weeks after immunization. Detectable levels persisted for the 12 weeks of observation. Selected sera tested for neutralizing antibody had titers ranging from 1:10 to 1:40 in a volunteer who received a 10


6


dose and 1:40 to 1:2560 in a volunteer who received a 10


7


TCID


50


dose. The commercial HAVAB assay detected anti-HAV in only one of the volunteers, who received the 10


7


dose. IgM anti-HAV was not detected in any of the volunteers who received the vaccine orally. Sera from volunteers who received 10


7


TCID


50


I.M. had detectable IgM anti-HAV.




Stools from all volunteers who received the oral vaccine were negative for hepatitis A virus, while those from volunteers who had received the vaccine by intramuscular route are in the process of being tested.




Although only a small number of volunteers received the vaccine orally, it appeared that the vaccine is not immunogenic by this route. This is likely due to over-attenuation of the virus, although other causes, such as inactivation in the gastrointestinal tract or too small an inoculum, should be considered. The vaccine was safe and immunogenic by the intramuscular route at doses of 10


6


and 10


7


TCID


50


. The antibody response was prompt: anti-HAV was observed within 3 weeks of immunization, persisted during the period of observation, and did not diminish in titer. Such a response to one single inoculation of a preparation which lacked an adjuvant, is remarkable. If indeed, anti-HAV persists for a long time after one dose, the logistics of administration of this product would be much simpler and more successful than with present hepatitis A vaccines. The presence of IgM anti-HAV in volunteers who received 10


7


TCID


50


without evidence of hepatitis is suggestive of asymptomatic replication of the virus.




EXAMPLE 3




Construction of Chimeric Viruses




Several exemplary chimeric viruses were generated to evaluate the effect of several of the mutations of Table I on host range and/or attenuation in primates. The sequence of the MRC-5-adapted virus 4380 was obtained using reverse-transcriptase:polymerase chain reaction (RT:PCR) to amplify regions of the virus as cDNA prior to sequencing (hence T instead of U in Table VI below). Numbers 2-10 in Table VI designate chimeric viruses made by inserting mutations found in the MRC-5-adapted virus 4380 into the cDNA clone of pHAV/7 encoding the attenuated HM-175 virus, Pass 35, of Cohen et al.,


J. Virol


., 61:3035-3039 (1987). Mutations introduced by “chimera” means a portion of the 4380 virus genome was amplified by RT:PCR, digested with specific restriction enzymes and the fragment used to replace the homologous fragment in the cDNA clone pHAV/7. Mutations introduced by mutagenesis were inserted by oligonucleotide-directed mutagenesis of the cDNA clone pHAV/7 using the Amersham mutagenesis protocol.




The chimeric cDNAs were transcribed into RNA in vitro and the nucleic acids (both RNA and DNA) transfected into FRhK-4 cells to generate chimeric viruses. Quantification of chimeric virus growth for the exemplary chimeras was performed by slot-blot assay.




Table VI reports the results of the construction and testing of nine chimeric viruses. As used in Table VI, the following terms are defined: Cell culture refers to virus containing indicated mutations selected by growth in MRC-5 cells. Mutagenesis refers to oligonucleotide-directed mutagenesis of P-35 or HM-175 cDNA clones. A chimeric viral genome refers to the construction of a chimeric viral genome using portions of P-35 cDNA clone and PCR-generated fragments of the MRC-5 cell-adapted virus 4380. ND means that this study has not yet been performed. The + symbol refers to virus that has some growth in that cell type. The − symbol refers to virus that has little or no growth in that cell type. The two cell types employed to test the growth of the chimeric viruses are the human lung fibroblast-like cell line MRC-5 and fetal rhesus monkey kidney epithelial-like cell line, FRhK-4. Note that Virus #1 in the Table refers to MRC-5-adapted HAV 4380. Viruses #2 through 10 are chimeric viruses of this invention.












TABLE VI











Differences in Nucleotide Sequence






of MRC-5-Adapted Hepatitis A Virus:






Comparison with P-35 HM-175 Virus















Growth of






Nucleotide




Method




Mutated Virus






Differences




Mutation




in Cell Cultures














from P-35 HM-175




Introduced




FRhK-4




MRC-5









Virus #1 (MRC-5-adapted)




Cell Culture




+




+






591 A to G






646 G to A






669 C to T






687 T to G






2750 C to T






3027 T to A






3196 G to A






3934 A to G






4418 A to T






4563 A to G






4643 A to T






5145 A to G






5745 A to T






6908 T to C






7032 C to T






7255 A to T






Virus #2




Chimera




+




+






591 A to G






646 G to A






669 C to T






687 T to G






Virus #3




Chimera




+




+






124 C to T






131 d to T






132 d to T






133 d to T






134 d to G






152 G to A






203-207 d to T






591 A to G






646 G to A






669 C to T






687 T to G






Virus #4




Chimera




+




+






591 A to G






646 G to A






669 C to T






687 T to G






4418 A to T






4563 A to G






4643 A to T






Virus #5




Chimera




+




+






124 C to T






131 d to T






132 d to T






133 d to T






134 d to T






152 G to A






203-207 d to T






591 A to G






646 G to A






669 C to T






687 T to G






4418 A to T






4563 A to G






4643 A to T






Virus #6




Chimera




+











4418 A to T






4563 A to G






4643 A to T






Virus #7




Chimera Mutagenesis




+




ND






591 A to G






4418 A to T






4563 A to G






4643 A to T






Virus #8




Chimera Mutagenesis




+




ND






646 G to A






4418 A to T






4563 A to G






4643 A to T






Virus #9




Chimera Mutagenesis




+




ND






669 C to T






4418 A to T






4563 A to G






4643 A to T






Virus #10




Chimera Mutagenesis




+




ND






687 T to G






4418 A to T






4563 A to G






4643 A to T











d = Base at this position deleted in P-35 compared to wild-type













Introduction of four mutations found in the 5′ noncoding region, at nucleotide positions 591, 646, 669, and 687 of the P-35 genome, appear to be important for HAV host range in cell culture. They allow some growth of the transfected virus in MRC-5 cells, but do not account entirely for MRC-5 cell culture adaptation.




Introduction of three mutations, at nucleotides 4418, 4563 and 4643 in the 2C region of the MRC-5-adapted virus, with the 5′ mutations allow full growth in MRC-5 cells. Thus the four mutation in the 5′ noncoding region and the three mutations in the 2C region of the genome of the MRC-5 cell-adapted of this virus to efficient growth in MRC-5 cells. Introduction of only the three mutations in the 2 C region into the P-35 AGMK genome does not permit discernible growth of the transfected virus in MRC-5 cells.




EXAMPLE 4




Comparison of End Point Dilution of Chimeric Viruses in FRhK-4 Versus MRC-5 Cells




Chimeric viruses with the composition described in Table VI were serially diluted in ten-fold increments, and an equal aliquot of each dilution was plated onto FRhK-4 and MRC-5 cells. After 21 days incubation at 34.5° C. to permit virus growth, the cells were lysed by the addition of a buffer solution containing sodium dodecyl sulfate. The viral RNA was extracted with phenol and quantified by slot blot hybridization using a [


32p


]-labeled riboprobe specific for hepatitis A virus. A radioautograph of the slot blot obtained from the FRhK-4 cells and from the MRC-5 cells illustrates that the endpoint dilution of the MRC-5-adapted virus was the same in both cell lines, indicating that this virus can grow in either cell line. In contrast, the P35 HM-175 virus had an endpoint dilution of 10


−5


on FRhK-4 cells and <10


−1


on MRC-5 cells, demonstrating that this virus is unable to grow successfully on MRC-5 cells. As

FIG. 5

illustrates, virus #6 was most like the pass 35 virus while virus #4 was most like the MRC-5 adapted virus and viruses #2, 3, and 5 were intermediate. These results show that certain mutations from the MRC-5-adapted virus can be introduced into the pHAV/7 cDNA clone to generate new viruses which also have acquired the ability to grow in MRC-5 cells.




Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.







4




1


7493


DNA


WILD-TYPE HUMAN HEPATITIS A VIRUS, STRAIN HM-175




CDS




(735)..(7415)





1
ttcaagaggg gtctccggga atttccggag tccctcttgg aagtccatgg tgaggggact 60
tgatacctca ccgccgtttg cctaggctat aggctaaatt ttccctttcc cttttccctt 120
tcctattccc tttgttttgc ttgtaaatat taattcctgc aggttcaggg ttcttaaatc 180
tgtttctcta taagaacact catttttcac gctttctgtc ttctttcttc cagggctctc 240
cccttgccct aggctctggc cgttgcgccc ggcggggtca actccatgat tagcatggag 300
ctgtaggagt ctaaattggg gacacagatg tttggaacgt caccttgcag tgttaacttg 360
gctttcatga atctctttga tcttccacaa ggggtaggct acgggtgaaa cctcttaggc 420
taatacttct atgaagagat gccttggata gggtaacagc ggcggatatt ggtgagttgt 480
taagacaaaa accattcaac gccggaggac tgactctcat ccagtggatg cattgagtgg 540
attgactgtc agggctgtct ttaggcttaa ttccagacct ctctgtgctt agggcaaaca 600
tcatttggcc ttaaatggga ttctgtgaga ggggatccct ccattgacag ctggactgtt 660
ctttggggcc ttatgtggtg tttgcctctg aggtactcag gggcatttag gtttttcctc 720
attcttaaat aata atg aac atg tct aga caa ggt att ttc cag act gtt 770
Met Asn Met Ser Arg Gln Gly Ile Phe Gln Thr Val
1 5 10
ggg agt ggt ctt gac cac atc ctg tct ttg gca gac att gag gaa gag 818
Gly Ser Gly Leu Asp His Ile Leu Ser Leu Ala Asp Ile Glu Glu Glu
15 20 25
caa atg att caa tca gtt gat agg act gca gtg act ggt gct tct tat 866
Gln Met Ile Gln Ser Val Asp Arg Thr Ala Val Thr Gly Ala Ser Tyr
30 35 40
ttt act tct gtg gat caa tct tca gtt cat aca gct gag gtt gga tca 914
Phe Thr Ser Val Asp Gln Ser Ser Val His Thr Ala Glu Val Gly Ser
45 50 55 60
cac cag gtt gaa cct ttg aga acc tct gtt gat aaa ccc ggt tca aag 962
His Gln Val Glu Pro Leu Arg Thr Ser Val Asp Lys Pro Gly Ser Lys
65 70 75
aag act cag gga gag aaa ttt ttc ttg att cat tct gca gat tgg ctt 1010
Lys Thr Gln Gly Glu Lys Phe Phe Leu Ile His Ser Ala Asp Trp Leu
80 85 90
act aca cat gct ctt ttc cat gaa gtt gca aaa ttg gat gtg gtg aaa 1058
Thr Thr His Ala Leu Phe His Glu Val Ala Lys Leu Asp Val Val Lys
95 100 105
tta tta tac aat gag cag ttt gct gtt caa ggg ttg ttg aga tac cat 1106
Leu Leu Tyr Asn Glu Gln Phe Ala Val Gln Gly Leu Leu Arg Tyr His
110 115 120
aca tat gca aga ttt ggc att gaa att caa gtt cag ata aac cct aca 1154
Thr Tyr Ala Arg Phe Gly Ile Glu Ile Gln Val Gln Ile Asn Pro Thr
125 130 135 140
cct ttc caa cag ggg gga ttg atc tgt gct atg gtt cct ggt gac cag 1202
Pro Phe Gln Gln Gly Gly Leu Ile Cys Ala Met Val Pro Gly Asp Gln
145 150 155
agc tat ggt tct ata gca tca ttg act gtt tat cct cat ggt ttg tta 1250
Ser Tyr Gly Ser Ile Ala Ser Leu Thr Val Tyr Pro His Gly Leu Leu
160 165 170
aat tgc aat att aac aat gtg gtt aga ata aag gtt cca ttt att tac 1298
Asn Cys Asn Ile Asn Asn Val Val Arg Ile Lys Val Pro Phe Ile Tyr
175 180 185
aca aga ggt gct tac cac ttt aaa gat cca caa tac cca gtt tgg gaa 1346
Thr Arg Gly Ala Tyr His Phe Lys Asp Pro Gln Tyr Pro Val Trp Glu
190 195 200
ttg aca att aga gtt tgg tca gaa tta aat att ggg aca gga act tca 1394
Leu Thr Ile Arg Val Trp Ser Glu Leu Asn Ile Gly Thr Gly Thr Ser
205 210 215 220
gct tat act tca ctc aat gtt tta gct aga ttt aca gat ttg gag ttg 1442
Ala Tyr Thr Ser Leu Asn Val Leu Ala Arg Phe Thr Asp Leu Glu Leu
225 230 235
cat gga tta act cct ctt tct aca caa atg atg aga aat gaa ttt agg 1490
His Gly Leu Thr Pro Leu Ser Thr Gln Met Met Arg Asn Glu Phe Arg
240 245 250
gtc agt act act gag aat gtg gtg aat ctg tca aat tat gaa gat gca 1538
Val Ser Thr Thr Glu Asn Val Val Asn Leu Ser Asn Tyr Glu Asp Ala
255 260 265
aga gca aag atg tct ttt gct ttg gat cag gaa gat tgg aaa tct gat 1586
Arg Ala Lys Met Ser Phe Ala Leu Asp Gln Glu Asp Trp Lys Ser Asp
270 275 280
ccg tcc cag ggt ggt ggg atc aaa att act cat ttt act act tgg aca 1634
Pro Ser Gln Gly Gly Gly Ile Lys Ile Thr His Phe Thr Thr Trp Thr
285 290 295 300
tct att cca act ttg gct gct cag ttt cca ttt aat gct tca gac tca 1682
Ser Ile Pro Thr Leu Ala Ala Gln Phe Pro Phe Asn Ala Ser Asp Ser
305 310 315
gtt ggt caa caa att aaa gtt att cca gtt gac cca tat ttt ttc caa 1730
Val Gly Gln Gln Ile Lys Val Ile Pro Val Asp Pro Tyr Phe Phe Gln
320 325 330
atg aca aat acg aat cct gac caa aaa tgt ata act gct ttg gct tct 1778
Met Thr Asn Thr Asn Pro Asp Gln Lys Cys Ile Thr Ala Leu Ala Ser
335 340 345
att tgt cag atg ttt tgt ttt tgg aga gga gat ctt gtc ttt gat ttt 1826
Ile Cys Gln Met Phe Cys Phe Trp Arg Gly Asp Leu Val Phe Asp Phe
350 355 360
caa gtt ttt ccc acc aaa tat cat tca ggt aga tta ctg ttt tgt ttt 1874
Gln Val Phe Pro Thr Lys Tyr His Ser Gly Arg Leu Leu Phe Cys Phe
365 370 375 380
gtt cct ggc aat gag cta ata gat gtt tct gga atc aca tta aag caa 1922
Val Pro Gly Asn Glu Leu Ile Asp Val Ser Gly Ile Thr Leu Lys Gln
385 390 395
gca act act gct cct tgt gca gta atg gat att aca gga gtg cag tca 1970
Ala Thr Thr Ala Pro Cys Ala Val Met Asp Ile Thr Gly Val Gln Ser
400 405 410
act ttg aga ttt cgt gtt ccc tgg att tct gac act cct tac aga gtg 2018
Thr Leu Arg Phe Arg Val Pro Trp Ile Ser Asp Thr Pro Tyr Arg Val
415 420 425
aac agg tat aca aag tca gca cat cag aaa ggt gag tac act gcc att 2066
Asn Arg Tyr Thr Lys Ser Ala His Gln Lys Gly Glu Tyr Thr Ala Ile
430 435 440
ggg aag ctt att gtg tat tgt tat aac aga ttg acc tct cct tct aac 2114
Gly Lys Leu Ile Val Tyr Cys Tyr Asn Arg Leu Thr Ser Pro Ser Asn
445 450 455 460
gtt gct tcc cat gtc aga gtg aat gtt tat ctt tca gca att aac ttg 2162
Val Ala Ser His Val Arg Val Asn Val Tyr Leu Ser Ala Ile Asn Leu
465 470 475
gaa tgt ttt gct cct ctt tat cat gct atg gat gtt act aca caa gtt 2210
Glu Cys Phe Ala Pro Leu Tyr His Ala Met Asp Val Thr Thr Gln Val
480 485 490
gga gat gat tct gga ggt ttt tca aca aca gtt tct aca gaa cag aat 2258
Gly Asp Asp Ser Gly Gly Phe Ser Thr Thr Val Ser Thr Glu Gln Asn
495 500 505
gtt cca gat ccc caa gtt ggt ata aca acc atg aaa gat ttg aaa gga 2306
Val Pro Asp Pro Gln Val Gly Ile Thr Thr Met Lys Asp Leu Lys Gly
510 515 520
aaa gct aac aga ggg aaa atg gat gtt tca gga gta caa gca cct gtg 2354
Lys Ala Asn Arg Gly Lys Met Asp Val Ser Gly Val Gln Ala Pro Val
525 530 535 540
gga gct atc aca aca att gag gat cca gtt tta gca aag aaa gta cct 2402
Gly Ala Ile Thr Thr Ile Glu Asp Pro Val Leu Ala Lys Lys Val Pro
545 550 555
gag aca ttt cct gaa ttg aaa cct gga gaa tcc aga cat aca tca gat 2450
Glu Thr Phe Pro Glu Leu Lys Pro Gly Glu Ser Arg His Thr Ser Asp
560 565 570
cat atg tcc atc tac aag ttt atg gga agg tct cat ttc ttg tgc act 2498
His Met Ser Ile Tyr Lys Phe Met Gly Arg Ser His Phe Leu Cys Thr
575 580 585
ttt aca ttc aat tca aat aat aaa gag tac aca ttt cct ata acc ttg 2546
Phe Thr Phe Asn Ser Asn Asn Lys Glu Tyr Thr Phe Pro Ile Thr Leu
590 595 600
tct tca acc tct aat cct cct cat ggt ttg cca tca aca ctg agg tgg 2594
Ser Ser Thr Ser Asn Pro Pro His Gly Leu Pro Ser Thr Leu Arg Trp
605 610 615 620
ttt ttc aac ttg ttt cag ttg tat aga ggg cct tta gat ctg aca att 2642
Phe Phe Asn Leu Phe Gln Leu Tyr Arg Gly Pro Leu Asp Leu Thr Ile
625 630 635
att att aca gga gca act gat gta gat ggc atg gcc tgg ttc act cca 2690
Ile Ile Thr Gly Ala Thr Asp Val Asp Gly Met Ala Trp Phe Thr Pro
640 645 650
gta ggt ctt gcc gtt gat act cct tgg gta gag aag gag tca gct ttg 2738
Val Gly Leu Ala Val Asp Thr Pro Trp Val Glu Lys Glu Ser Ala Leu
655 660 665
tct att gac tac aaa act gct ctt gga gct gtc aga ttt aac aca agg 2786
Ser Ile Asp Tyr Lys Thr Ala Leu Gly Ala Val Arg Phe Asn Thr Arg
670 675 680
aga aca ggg aac att cag att aga tta cca tgg tat tct tat tta tat 2834
Arg Thr Gly Asn Ile Gln Ile Arg Leu Pro Trp Tyr Ser Tyr Leu Tyr
685 690 695 700
gct gtg tct gga gca ctg gat ggt ttg ggt gac aag aca gat tct aca 2882
Ala Val Ser Gly Ala Leu Asp Gly Leu Gly Asp Lys Thr Asp Ser Thr
705 710 715
ttt gga ttg gtt tct att cag att gca aat tac aat cat tct gat gaa 2930
Phe Gly Leu Val Ser Ile Gln Ile Ala Asn Tyr Asn His Ser Asp Glu
720 725 730
tac ttg tct ttt agt tgt tat ttg tct gtc aca gaa caa tca gag ttt 2978
Tyr Leu Ser Phe Ser Cys Tyr Leu Ser Val Thr Glu Gln Ser Glu Phe
735 740 745
tat ttt ccc aga gct cca ttg aac tca aat gcc atg tta tcc act gaa 3026
Tyr Phe Pro Arg Ala Pro Leu Asn Ser Asn Ala Met Leu Ser Thr Glu
750 755 760
tca atg atg agc aga att gca gct gga gac ttg gag tca tca gtg gat 3074
Ser Met Met Ser Arg Ile Ala Ala Gly Asp Leu Glu Ser Ser Val Asp
765 770 775 780
gat cct aga tca gag gaa gat aaa aga ttt gag agt cat ata gaa tgc 3122
Asp Pro Arg Ser Glu Glu Asp Lys Arg Phe Glu Ser His Ile Glu Cys
785 790 795
agg aag cca tat aaa gaa ctg aga tta gaa gtt ggg aaa caa aga ctc 3170
Arg Lys Pro Tyr Lys Glu Leu Arg Leu Glu Val Gly Lys Gln Arg Leu
800 805 810
aag tat gct cag gaa gaa ttg tca aat gaa gta ctt cca ccc cct agg 3218
Lys Tyr Ala Gln Glu Glu Leu Ser Asn Glu Val Leu Pro Pro Pro Arg
815 820 825
aaa atg aag gga ctg ttt tca caa gcc aaa att tct ctt ttt tat act 3266
Lys Met Lys Gly Leu Phe Ser Gln Ala Lys Ile Ser Leu Phe Tyr Thr
830 835 840
gag gag cat gaa ata atg aag ttt tcc tgg aga ggt gtg act gct gat 3314
Glu Glu His Glu Ile Met Lys Phe Ser Trp Arg Gly Val Thr Ala Asp
845 850 855 860
act aga gct tta agg agg ttt gga ttc tct ttg gcc gca ggc aga agt 3362
Thr Arg Ala Leu Arg Arg Phe Gly Phe Ser Leu Ala Ala Gly Arg Ser
865 870 875
gtg tgg act ctt gaa atg gat gct ggg gtt ctt act ggg aga ctg att 3410
Val Trp Thr Leu Glu Met Asp Ala Gly Val Leu Thr Gly Arg Leu Ile
880 885 890
aga ttg aat gat gag aaa tgg aca gaa atg aag gat gac aag att gtt 3458
Arg Leu Asn Asp Glu Lys Trp Thr Glu Met Lys Asp Asp Lys Ile Val
895 900 905
tca ttg att gaa aag ttt aca agt aac aaa tat tgg tcc aaa gtg aat 3506
Ser Leu Ile Glu Lys Phe Thr Ser Asn Lys Tyr Trp Ser Lys Val Asn
910 915 920
ttc cca cat ggg atg ttg gat ctt gaa gaa att gct gcc aat tct aag 3554
Phe Pro His Gly Met Leu Asp Leu Glu Glu Ile Ala Ala Asn Ser Lys
925 930 935 940
gat ttt cct aac atg tct gaa acg gat ttg tgt ttc ttg ctg cat tgg 3602
Asp Phe Pro Asn Met Ser Glu Thr Asp Leu Cys Phe Leu Leu His Trp
945 950 955
tta aat cca aag aaa att aat tta gca gat aga atg ctt gga ttg tct 3650
Leu Asn Pro Lys Lys Ile Asn Leu Ala Asp Arg Met Leu Gly Leu Ser
960 965 970
gga gtt cag gaa att aaa gaa caa ggt gtt gga tta ata gca gag tgt 3698
Gly Val Gln Glu Ile Lys Glu Gln Gly Val Gly Leu Ile Ala Glu Cys
975 980 985
aga act ttc tta gat tct att gct gga act tta aaa tct atg atg ttt 3746
Arg Thr Phe Leu Asp Ser Ile Ala Gly Thr Leu Lys Ser Met Met Phe
990 995 1000
gga ttt cat cat tct gtg act gtt gaa att ata aac act gtg ctc tgt 3794
Gly Phe His His Ser Val Thr Val Glu Ile Ile Asn Thr Val Leu Cys
1005 1010 1015 1020
ttt gtt aag agt gga att ttg ctt tat gta ata caa caa ttg aat cag 3842
Phe Val Lys Ser Gly Ile Leu Leu Tyr Val Ile Gln Gln Leu Asn Gln
1025 1030 1035
gat gaa cat tct cac ata att ggt ttg ttg aga gtc atg aat tat gca 3890
Asp Glu His Ser His Ile Ile Gly Leu Leu Arg Val Met Asn Tyr Ala
1040 1045 1050
gat att ggt tgt tca gtt att tca tgt ggc aaa gtt ttt tcc aaa atg 3938
Asp Ile Gly Cys Ser Val Ile Ser Cys Gly Lys Val Phe Ser Lys Met
1055 1060 1065
ctg gaa aca gtc ttt aat tgg caa atg gac tcc aga atg atg gag tta 3986
Leu Glu Thr Val Phe Asn Trp Gln Met Asp Ser Arg Met Met Glu Leu
1070 1075 1080
agg act cag agt ttt tcc aac tgg tta aga gat att tgt tct ggg atc 4034
Arg Thr Gln Ser Phe Ser Asn Trp Leu Arg Asp Ile Cys Ser Gly Ile
1085 1090 1095 1100
acc att ttt aaa aac ttc aag gat gca att tat tgg ctt tat aca aaa 4082
Thr Ile Phe Lys Asn Phe Lys Asp Ala Ile Tyr Trp Leu Tyr Thr Lys
1105 1110 1115
tta aag gac ttt tat gaa gtg aat tat ggc aag aag aag gac att tta 4130
Leu Lys Asp Phe Tyr Glu Val Asn Tyr Gly Lys Lys Lys Asp Ile Leu
1120 1125 1130
aat att ctt aaa gat aac caa caa aaa ata gag aaa gcc att gag gaa 4178
Asn Ile Leu Lys Asp Asn Gln Gln Lys Ile Glu Lys Ala Ile Glu Glu
1135 1140 1145
gcc gat gaa ttt tgc att ttg caa atc caa gat gtg gaa aaa ttt gaa 4226
Ala Asp Glu Phe Cys Ile Leu Gln Ile Gln Asp Val Glu Lys Phe Glu
1150 1155 1160
cag tat cag aaa ggg gtt gac ttg ata caa aaa ttg aga act gtt cat 4274
Gln Tyr Gln Lys Gly Val Asp Leu Ile Gln Lys Leu Arg Thr Val His
1165 1170 1175 1180
tca atg gct cag gtt gat cca aat tta atg gtt cat ttg tca cct ttg 4322
Ser Met Ala Gln Val Asp Pro Asn Leu Met Val His Leu Ser Pro Leu
1185 1190 1195
aga gat tgt ata gca aga gtt cat cag aaa ctt aaa aac ctt gga tct 4370
Arg Asp Cys Ile Ala Arg Val His Gln Lys Leu Lys Asn Leu Gly Ser
1200 1205 1210
ata aat cag gca atg gta acg aga tgt gag cca gtt gtt tgt tat tta 4418
Ile Asn Gln Ala Met Val Thr Arg Cys Glu Pro Val Val Cys Tyr Leu
1215 1220 1225
tat ggc aaa aga ggg gga gga aag agc tta aca tca att gca ttg gca 4466
Tyr Gly Lys Arg Gly Gly Gly Lys Ser Leu Thr Ser Ile Ala Leu Ala
1230 1235 1240
acc aaa att tgt aaa cat tat ggt gtt gag cct gaa aag aat atc tat 4514
Thr Lys Ile Cys Lys His Tyr Gly Val Glu Pro Glu Lys Asn Ile Tyr
1245 1250 1255 1260
act aaa cct gtg gct tca gat tac tgg gat gga tat agt gga caa tta 4562
Thr Lys Pro Val Ala Ser Asp Tyr Trp Asp Gly Tyr Ser Gly Gln Leu
1265 1270 1275
gtt tgc atc att gat gat att ggc caa aac aca aca gat gag gat tgg 4610
Val Cys Ile Ile Asp Asp Ile Gly Gln Asn Thr Thr Asp Glu Asp Trp
1280 1285 1290
tca gat ttt tgt cag tta gtg tca gga tgt cca atg aga tta aac atg 4658
Ser Asp Phe Cys Gln Leu Val Ser Gly Cys Pro Met Arg Leu Asn Met
1295 1300 1305
gcc tct ctt gag gag aag ggt agg cat ttt tct tct cct ttt ata ata 4706
Ala Ser Leu Glu Glu Lys Gly Arg His Phe Ser Ser Pro Phe Ile Ile
1310 1315 1320
gca act tca aat tgg tca aat cca agt cca aaa aca gtt tat gtt aag 4754
Ala Thr Ser Asn Trp Ser Asn Pro Ser Pro Lys Thr Val Tyr Val Lys
1325 1330 1335 1340
gaa gca att gac cgc aga ctc cat ttc aag gtt gaa gtt aaa cct gct 4802
Glu Ala Ile Asp Arg Arg Leu His Phe Lys Val Glu Val Lys Pro Ala
1345 1350 1355
tca ttt ttc aaa aat cct cac aat gat atg ttg aat gtt aat tta gct 4850
Ser Phe Phe Lys Asn Pro His Asn Asp Met Leu Asn Val Asn Leu Ala
1360 1365 1370
aaa aca aat gat gca atc aaa gat atg tct tgt gtt gat ttg ata atg 4898
Lys Thr Asn Asp Ala Ile Lys Asp Met Ser Cys Val Asp Leu Ile Met
1375 1380 1385
gat gga cat aat gtt tca ttg atg gat ttg ctc agt tct tta gtc atg 4946
Asp Gly His Asn Val Ser Leu Met Asp Leu Leu Ser Ser Leu Val Met
1390 1395 1400
aca gtt gaa att aga aaa caa aac atg act gaa ttc atg gag ttg tgg 4994
Thr Val Glu Ile Arg Lys Gln Asn Met Thr Glu Phe Met Glu Leu Trp
1405 1410 1415 1420
tct cag gga att tca gat gat gat aat gat agt gca gta gct gag ttt 5042
Ser Gln Gly Ile Ser Asp Asp Asp Asn Asp Ser Ala Val Ala Glu Phe
1425 1430 1435
ttc cag tct ttt cca tct ggt gaa cca tcg aac tct aaa tta tct ggc 5090
Phe Gln Ser Phe Pro Ser Gly Glu Pro Ser Asn Ser Lys Leu Ser Gly
1440 1445 1450
ttt ttc caa tct gtt act aat cac aag tgg gtt gct gtg gga gct gca 5138
Phe Phe Gln Ser Val Thr Asn His Lys Trp Val Ala Val Gly Ala Ala
1455 1460 1465
gtt ggc att ctt gga gtg ctc gtt gga gga tgg ttt gtg tat aag cat 5186
Val Gly Ile Leu Gly Val Leu Val Gly Gly Trp Phe Val Tyr Lys His
1470 1475 1480
ttc tcc cgc aaa gag gag gaa cca atc cca gct gaa ggg gta tat cat 5234
Phe Ser Arg Lys Glu Glu Glu Pro Ile Pro Ala Glu Gly Val Tyr His
1485 1490 1495 1500
ggt gta act aag ccc aag caa gtg att aaa tta gat gca gat cca gta 5282
Gly Val Thr Lys Pro Lys Gln Val Ile Lys Leu Asp Ala Asp Pro Val
1505 1510 1515
gaa tct cag tca act ttg gaa ata gca gga ctg gtt agg aag aac ttg 5330
Glu Ser Gln Ser Thr Leu Glu Ile Ala Gly Leu Val Arg Lys Asn Leu
1520 1525 1530
gtt cag ttt gga gtt gga gag aag aat gga tgt gtg aga tgg gtt atg 5378
Val Gln Phe Gly Val Gly Glu Lys Asn Gly Cys Val Arg Trp Val Met
1535 1540 1545
aat gcc ttg gga gtg aaa gat gat tgg ctg ctt gtg cct tcc cat gct 5426
Asn Ala Leu Gly Val Lys Asp Asp Trp Leu Leu Val Pro Ser His Ala
1550 1555 1560
tat aaa ttt gag aaa gat tat gaa atg atg gag ttt tat ttt aat aga 5474
Tyr Lys Phe Glu Lys Asp Tyr Glu Met Met Glu Phe Tyr Phe Asn Arg
1565 1570 1575 1580
ggt gga act tac tat tca att tca gct ggt aat gtt gtt att caa tct 5522
Gly Gly Thr Tyr Tyr Ser Ile Ser Ala Gly Asn Val Val Ile Gln Ser
1585 1590 1595
ttg gat gtg gga ttc cag gat gtt gtt ctg atg aag gtt cct aca att 5570
Leu Asp Val Gly Phe Gln Asp Val Val Leu Met Lys Val Pro Thr Ile
1600 1605 1610
cct aag ttt aga gat att act cag cat ttt att aag aaa ggg gat gtg 5618
Pro Lys Phe Arg Asp Ile Thr Gln His Phe Ile Lys Lys Gly Asp Val
1615 1620 1625
cct aga gct ttg aat cgc ctg gca aca tta gtg aca act gta aat gga 5666
Pro Arg Ala Leu Asn Arg Leu Ala Thr Leu Val Thr Thr Val Asn Gly
1630 1635 1640
acc cct atg tta att tct gag ggc cca cta aag atg gaa gag aaa gct 5714
Thr Pro Met Leu Ile Ser Glu Gly Pro Leu Lys Met Glu Glu Lys Ala
1645 1650 1655 1660
act tat gtt cat aag aaa aat gat ggt aca aca gtt gat tta act gtg 5762
Thr Tyr Val His Lys Lys Asn Asp Gly Thr Thr Val Asp Leu Thr Val
1665 1670 1675
gat cag gca tgg aga gga aaa ggc gaa ggt ctt cct gga atg tgt ggt 5810
Asp Gln Ala Trp Arg Gly Lys Gly Glu Gly Leu Pro Gly Met Cys Gly
1680 1685 1690
ggg gcc ttg gtt tca tcg aat caa tct ata cag aat gca atc ttg ggc 5858
Gly Ala Leu Val Ser Ser Asn Gln Ser Ile Gln Asn Ala Ile Leu Gly
1695 1700 1705
atc cat gtt gct gga gga aat tca att ctt gtt gca aaa ttg gtt act 5906
Ile His Val Ala Gly Gly Asn Ser Ile Leu Val Ala Lys Leu Val Thr
1710 1715 1720
caa gaa atg ttc caa aat att gat aag aaa att gaa agt cag aga att 5954
Gln Glu Met Phe Gln Asn Ile Asp Lys Lys Ile Glu Ser Gln Arg Ile
1725 1730 1735 1740
atg aaa gtg gag ttt act cag tgt tca atg aat gtg gtc tcc aaa acg 6002
Met Lys Val Glu Phe Thr Gln Cys Ser Met Asn Val Val Ser Lys Thr
1745 1750 1755
ctt ttt aga aag agt ccc att tat cat cac att gat aaa acc atg att 6050
Leu Phe Arg Lys Ser Pro Ile Tyr His His Ile Asp Lys Thr Met Ile
1760 1765 1770
aat ttt cct gca gct atg ccc ttt tct aaa gct gaa att gat cca atg 6098
Asn Phe Pro Ala Ala Met Pro Phe Ser Lys Ala Glu Ile Asp Pro Met
1775 1780 1785
gct gtg atg tta tct aag tat tca tta cct att gta gaa gaa cca gag 6146
Ala Val Met Leu Ser Lys Tyr Ser Leu Pro Ile Val Glu Glu Pro Glu
1790 1795 1800
gat tat aaa gag gct tca att ttt tat caa aat aaa ata gtg ggt aag 6194
Asp Tyr Lys Glu Ala Ser Ile Phe Tyr Gln Asn Lys Ile Val Gly Lys
1805 1810 1815 1820
act cag tta gtt gat gat ttt tta gat ctt gat atg gcc att aca ggg 6242
Thr Gln Leu Val Asp Asp Phe Leu Asp Leu Asp Met Ala Ile Thr Gly
1825 1830 1835
gcc cca gga att gat gct atc aac atg gat tca tct cct gga ttt cct 6290
Ala Pro Gly Ile Asp Ala Ile Asn Met Asp Ser Ser Pro Gly Phe Pro
1840 1845 1850
tat gtc cag gag aag ttg acc aaa aga gat tta att tgg ttg gat gaa 6338
Tyr Val Gln Glu Lys Leu Thr Lys Arg Asp Leu Ile Trp Leu Asp Glu
1855 1860 1865
aat ggt tta ttg ctg gga gtt cat cca aga ttg gct cag aga atc tta 6386
Asn Gly Leu Leu Leu Gly Val His Pro Arg Leu Ala Gln Arg Ile Leu
1870 1875 1880
ttc aat act gtc atg atg gaa aat tgt tct gat ttg gat gtt gtt ttt 6434
Phe Asn Thr Val Met Met Glu Asn Cys Ser Asp Leu Asp Val Val Phe
1885 1890 1895 1900
aca acc tgt cca aaa gat gaa ttg aga cca tta gag aaa gtg ttg gaa 6482
Thr Thr Cys Pro Lys Asp Glu Leu Arg Pro Leu Glu Lys Val Leu Glu
1905 1910 1915
tca aaa aca aga gct att gat gct tgt cct ctg gat tac tca att ttg 6530
Ser Lys Thr Arg Ala Ile Asp Ala Cys Pro Leu Asp Tyr Ser Ile Leu
1920 1925 1930
tgc cga atg tat tgg ggt cca gct att agt tat ttt cat ttg aat cca 6578
Cys Arg Met Tyr Trp Gly Pro Ala Ile Ser Tyr Phe His Leu Asn Pro
1935 1940 1945
ggt ttc cat aca ggt gtt gct att ggc ata gat cct gat aga cag tgg 6626
Gly Phe His Thr Gly Val Ala Ile Gly Ile Asp Pro Asp Arg Gln Trp
1950 1955 1960
gat gaa tta ttt aaa aca atg ata aga ttc gga gat gtt ggt ctt gat 6674
Asp Glu Leu Phe Lys Thr Met Ile Arg Phe Gly Asp Val Gly Leu Asp
1965 1970 1975 1980
tta gat ttc tct gct ttt gat gct agt ctt agt cca ttt atg att aga 6722
Leu Asp Phe Ser Ala Phe Asp Ala Ser Leu Ser Pro Phe Met Ile Arg
1985 1990 1995
gaa gca ggt aga atc atg agt gaa cta tct gga act cca tcc cat ttt 6770
Glu Ala Gly Arg Ile Met Ser Glu Leu Ser Gly Thr Pro Ser His Phe
2000 2005 2010
ggc aca gct ctt atc aat act atc att tat tcc aag cat ttg ctg tat 6818
Gly Thr Ala Leu Ile Asn Thr Ile Ile Tyr Ser Lys His Leu Leu Tyr
2015 2020 2025
aac tgt tgt tac cat gtc tgt ggt tca atg ccc tct ggg tct cct tgt 6866
Asn Cys Cys Tyr His Val Cys Gly Ser Met Pro Ser Gly Ser Pro Cys
2030 2035 2040
aca gct ttg cta aat tca att att aat aat gtc aat ttg tat tat gtg 6914
Thr Ala Leu Leu Asn Ser Ile Ile Asn Asn Val Asn Leu Tyr Tyr Val
2045 2050 2055 2060
ttt tcc aag ata ttt gga aag tct cca gtt ttc ttt tgt cag gct ttg 6962
Phe Ser Lys Ile Phe Gly Lys Ser Pro Val Phe Phe Cys Gln Ala Leu
2065 2070 2075
aag att ctc tgt tat gga gat gat gtt tta ata gtt ttc tct cga gat 7010
Lys Ile Leu Cys Tyr Gly Asp Asp Val Leu Ile Val Phe Ser Arg Asp
2080 2085 2090
gtt cag att gat aat ctt gat ttg att gga caa aaa att gta gat gag 7058
Val Gln Ile Asp Asn Leu Asp Leu Ile Gly Gln Lys Ile Val Asp Glu
2095 2100 2105
ttt aag aaa ctt ggc atg aca gct act tct gct gac aag aat gta cct 7106
Phe Lys Lys Leu Gly Met Thr Ala Thr Ser Ala Asp Lys Asn Val Pro
2110 2115 2120
cag ctg aaa cca gtt tcg gaa ttg act ttt ctc aaa aga tct ttc aat 7154
Gln Leu Lys Pro Val Ser Glu Leu Thr Phe Leu Lys Arg Ser Phe Asn
2125 2130 2135 2140
ttg gta gag gat aga att aga cct gca att tcg gaa aaa aca att tgg 7202
Leu Val Glu Asp Arg Ile Arg Pro Ala Ile Ser Glu Lys Thr Ile Trp
2145 2150 2155
tct tta ata gca tgg cag aga agt aac gct gag ttt gag cag aat tta 7250
Ser Leu Ile Ala Trp Gln Arg Ser Asn Ala Glu Phe Glu Gln Asn Leu
2160 2165 2170
gaa aat gct cag tgg ttt gct ttt atg cat ggc tat gag ttt tat cag 7298
Glu Asn Ala Gln Trp Phe Ala Phe Met His Gly Tyr Glu Phe Tyr Gln
2175 2180 2185
aaa ttt tat tat ttt gtt cag tcc tgt ttg gag aaa gag atg ata gaa 7346
Lys Phe Tyr Tyr Phe Val Gln Ser Cys Leu Glu Lys Glu Met Ile Glu
2190 2195 2200
tac aga ctt aaa tct tat gat tgg tgg aga atg aga ttt tat gac cag 7394
Tyr Arg Leu Lys Ser Tyr Asp Trp Trp Arg Met Arg Phe Tyr Asp Gln
2205 2210 2215 2220
tgt ttc att tgt gac ctt tca tgatttgttt aaacaaattt tcttaaaatt 7445
Cys Phe Ile Cys Asp Leu Ser
2225
tctgaggttt gtttatttct tttatcagta aataaaaaaa aaaaaaaa 7493




2


2227


PRT


WILD-TYPE HUMAN HEPATITIS A VIRUS, STRAIN HM-175



2
Met Asn Met Ser Arg Gln Gly Ile Phe Gln Thr Val Gly Ser Gly Leu
1 5 10 15
Asp His Ile Leu Ser Leu Ala Asp Ile Glu Glu Glu Gln Met Ile Gln
20 25 30
Ser Val Asp Arg Thr Ala Val Thr Gly Ala Ser Tyr Phe Thr Ser Val
35 40 45
Asp Gln Ser Ser Val His Thr Ala Glu Val Gly Ser His Gln Val Glu
50 55 60
Pro Leu Arg Thr Ser Val Asp Lys Pro Gly Ser Lys Lys Thr Gln Gly
65 70 75 80
Glu Lys Phe Phe Leu Ile His Ser Ala Asp Trp Leu Thr Thr His Ala
85 90 95
Leu Phe His Glu Val Ala Lys Leu Asp Val Val Lys Leu Leu Tyr Asn
100 105 110
Glu Gln Phe Ala Val Gln Gly Leu Leu Arg Tyr His Thr Tyr Ala Arg
115 120 125
Phe Gly Ile Glu Ile Gln Val Gln Ile Asn Pro Thr Pro Phe Gln Gln
130 135 140
Gly Gly Leu Ile Cys Ala Met Val Pro Gly Asp Gln Ser Tyr Gly Ser
145 150 155 160
Ile Ala Ser Leu Thr Val Tyr Pro His Gly Leu Leu Asn Cys Asn Ile
165 170 175
Asn Asn Val Val Arg Ile Lys Val Pro Phe Ile Tyr Thr Arg Gly Ala
180 185 190
Tyr His Phe Lys Asp Pro Gln Tyr Pro Val Trp Glu Leu Thr Ile Arg
195 200 205
Val Trp Ser Glu Leu Asn Ile Gly Thr Gly Thr Ser Ala Tyr Thr Ser
210 215 220
Leu Asn Val Leu Ala Arg Phe Thr Asp Leu Glu Leu His Gly Leu Thr
225 230 235 240
Pro Leu Ser Thr Gln Met Met Arg Asn Glu Phe Arg Val Ser Thr Thr
245 250 255
Glu Asn Val Val Asn Leu Ser Asn Tyr Glu Asp Ala Arg Ala Lys Met
260 265 270
Ser Phe Ala Leu Asp Gln Glu Asp Trp Lys Ser Asp Pro Ser Gln Gly
275 280 285
Gly Gly Ile Lys Ile Thr His Phe Thr Thr Trp Thr Ser Ile Pro Thr
290 295 300
Leu Ala Ala Gln Phe Pro Phe Asn Ala Ser Asp Ser Val Gly Gln Gln
305 310 315 320
Ile Lys Val Ile Pro Val Asp Pro Tyr Phe Phe Gln Met Thr Asn Thr
325 330 335
Asn Pro Asp Gln Lys Cys Ile Thr Ala Leu Ala Ser Ile Cys Gln Met
340 345 350
Phe Cys Phe Trp Arg Gly Asp Leu Val Phe Asp Phe Gln Val Phe Pro
355 360 365
Thr Lys Tyr His Ser Gly Arg Leu Leu Phe Cys Phe Val Pro Gly Asn
370 375 380
Glu Leu Ile Asp Val Ser Gly Ile Thr Leu Lys Gln Ala Thr Thr Ala
385 390 395 400
Pro Cys Ala Val Met Asp Ile Thr Gly Val Gln Ser Thr Leu Arg Phe
405 410 415
Arg Val Pro Trp Ile Ser Asp Thr Pro Tyr Arg Val Asn Arg Tyr Thr
420 425 430
Lys Ser Ala His Gln Lys Gly Glu Tyr Thr Ala Ile Gly Lys Leu Ile
435 440 445
Val Tyr Cys Tyr Asn Arg Leu Thr Ser Pro Ser Asn Val Ala Ser His
450 455 460
Val Arg Val Asn Val Tyr Leu Ser Ala Ile Asn Leu Glu Cys Phe Ala
465 470 475 480
Pro Leu Tyr His Ala Met Asp Val Thr Thr Gln Val Gly Asp Asp Ser
485 490 495
Gly Gly Phe Ser Thr Thr Val Ser Thr Glu Gln Asn Val Pro Asp Pro
500 505 510
Gln Val Gly Ile Thr Thr Met Lys Asp Leu Lys Gly Lys Ala Asn Arg
515 520 525
Gly Lys Met Asp Val Ser Gly Val Gln Ala Pro Val Gly Ala Ile Thr
530 535 540
Thr Ile Glu Asp Pro Val Leu Ala Lys Lys Val Pro Glu Thr Phe Pro
545 550 555 560
Glu Leu Lys Pro Gly Glu Ser Arg His Thr Ser Asp His Met Ser Ile
565 570 575
Tyr Lys Phe Met Gly Arg Ser His Phe Leu Cys Thr Phe Thr Phe Asn
580 585 590
Ser Asn Asn Lys Glu Tyr Thr Phe Pro Ile Thr Leu Ser Ser Thr Ser
595 600 605
Asn Pro Pro His Gly Leu Pro Ser Thr Leu Arg Trp Phe Phe Asn Leu
610 615 620
Phe Gln Leu Tyr Arg Gly Pro Leu Asp Leu Thr Ile Ile Ile Thr Gly
625 630 635 640
Ala Thr Asp Val Asp Gly Met Ala Trp Phe Thr Pro Val Gly Leu Ala
645 650 655
Val Asp Thr Pro Trp Val Glu Lys Glu Ser Ala Leu Ser Ile Asp Tyr
660 665 670
Lys Thr Ala Leu Gly Ala Val Arg Phe Asn Thr Arg Arg Thr Gly Asn
675 680 685
Ile Gln Ile Arg Leu Pro Trp Tyr Ser Tyr Leu Tyr Ala Val Ser Gly
690 695 700
Ala Leu Asp Gly Leu Gly Asp Lys Thr Asp Ser Thr Phe Gly Leu Val
705 710 715 720
Ser Ile Gln Ile Ala Asn Tyr Asn His Ser Asp Glu Tyr Leu Ser Phe
725 730 735
Ser Cys Tyr Leu Ser Val Thr Glu Gln Ser Glu Phe Tyr Phe Pro Arg
740 745 750
Ala Pro Leu Asn Ser Asn Ala Met Leu Ser Thr Glu Ser Met Met Ser
755 760 765
Arg Ile Ala Ala Gly Asp Leu Glu Ser Ser Val Asp Asp Pro Arg Ser
770 775 780
Glu Glu Asp Lys Arg Phe Glu Ser His Ile Glu Cys Arg Lys Pro Tyr
785 790 795 800
Lys Glu Leu Arg Leu Glu Val Gly Lys Gln Arg Leu Lys Tyr Ala Gln
805 810 815
Glu Glu Leu Ser Asn Glu Val Leu Pro Pro Pro Arg Lys Met Lys Gly
820 825 830
Leu Phe Ser Gln Ala Lys Ile Ser Leu Phe Tyr Thr Glu Glu His Glu
835 840 845
Ile Met Lys Phe Ser Trp Arg Gly Val Thr Ala Asp Thr Arg Ala Leu
850 855 860
Arg Arg Phe Gly Phe Ser Leu Ala Ala Gly Arg Ser Val Trp Thr Leu
865 870 875 880
Glu Met Asp Ala Gly Val Leu Thr Gly Arg Leu Ile Arg Leu Asn Asp
885 890 895
Glu Lys Trp Thr Glu Met Lys Asp Asp Lys Ile Val Ser Leu Ile Glu
900 905 910
Lys Phe Thr Ser Asn Lys Tyr Trp Ser Lys Val Asn Phe Pro His Gly
915 920 925
Met Leu Asp Leu Glu Glu Ile Ala Ala Asn Ser Lys Asp Phe Pro Asn
930 935 940
Met Ser Glu Thr Asp Leu Cys Phe Leu Leu His Trp Leu Asn Pro Lys
945 950 955 960
Lys Ile Asn Leu Ala Asp Arg Met Leu Gly Leu Ser Gly Val Gln Glu
965 970 975
Ile Lys Glu Gln Gly Val Gly Leu Ile Ala Glu Cys Arg Thr Phe Leu
980 985 990
Asp Ser Ile Ala Gly Thr Leu Lys Ser Met Met Phe Gly Phe His His
995 1000 1005
Ser Val Thr Val Glu Ile Ile Asn Thr Val Leu Cys Phe Val Lys Ser
1010 1015 1020
Gly Ile Leu Leu Tyr Val Ile Gln Gln Leu Asn Gln Asp Glu His Ser
1025 1030 1035 1040
His Ile Ile Gly Leu Leu Arg Val Met Asn Tyr Ala Asp Ile Gly Cys
1045 1050 1055
Ser Val Ile Ser Cys Gly Lys Val Phe Ser Lys Met Leu Glu Thr Val
1060 1065 1070
Phe Asn Trp Gln Met Asp Ser Arg Met Met Glu Leu Arg Thr Gln Ser
1075 1080 1085
Phe Ser Asn Trp Leu Arg Asp Ile Cys Ser Gly Ile Thr Ile Phe Lys
1090 1095 1100
Asn Phe Lys Asp Ala Ile Tyr Trp Leu Tyr Thr Lys Leu Lys Asp Phe
1105 1110 1115 1120
Tyr Glu Val Asn Tyr Gly Lys Lys Lys Asp Ile Leu Asn Ile Leu Lys
1125 1130 1135
Asp Asn Gln Gln Lys Ile Glu Lys Ala Ile Glu Glu Ala Asp Glu Phe
1140 1145 1150
Cys Ile Leu Gln Ile Gln Asp Val Glu Lys Phe Glu Gln Tyr Gln Lys
1155 1160 1165
Gly Val Asp Leu Ile Gln Lys Leu Arg Thr Val His Ser Met Ala Gln
1170 1175 1180
Val Asp Pro Asn Leu Met Val His Leu Ser Pro Leu Arg Asp Cys Ile
1185 1190 1195 1200
Ala Arg Val His Gln Lys Leu Lys Asn Leu Gly Ser Ile Asn Gln Ala
1205 1210 1215
Met Val Thr Arg Cys Glu Pro Val Val Cys Tyr Leu Tyr Gly Lys Arg
1220 1225 1230
Gly Gly Gly Lys Ser Leu Thr Ser Ile Ala Leu Ala Thr Lys Ile Cys
1235 1240 1245
Lys His Tyr Gly Val Glu Pro Glu Lys Asn Ile Tyr Thr Lys Pro Val
1250 1255 1260
Ala Ser Asp Tyr Trp Asp Gly Tyr Ser Gly Gln Leu Val Cys Ile Ile
1265 1270 1275 1280
Asp Asp Ile Gly Gln Asn Thr Thr Asp Glu Asp Trp Ser Asp Phe Cys
1285 1290 1295
Gln Leu Val Ser Gly Cys Pro Met Arg Leu Asn Met Ala Ser Leu Glu
1300 1305 1310
Glu Lys Gly Arg His Phe Ser Ser Pro Phe Ile Ile Ala Thr Ser Asn
1315 1320 1325
Trp Ser Asn Pro Ser Pro Lys Thr Val Tyr Val Lys Glu Ala Ile Asp
1330 1335 1340
Arg Arg Leu His Phe Lys Val Glu Val Lys Pro Ala Ser Phe Phe Lys
1345 1350 1355 1360
Asn Pro His Asn Asp Met Leu Asn Val Asn Leu Ala Lys Thr Asn Asp
1365 1370 1375
Ala Ile Lys Asp Met Ser Cys Val Asp Leu Ile Met Asp Gly His Asn
1380 1385 1390
Val Ser Leu Met Asp Leu Leu Ser Ser Leu Val Met Thr Val Glu Ile
1395 1400 1405
Arg Lys Gln Asn Met Thr Glu Phe Met Glu Leu Trp Ser Gln Gly Ile
1410 1415 1420
Ser Asp Asp Asp Asn Asp Ser Ala Val Ala Glu Phe Phe Gln Ser Phe
1425 1430 1435 1440
Pro Ser Gly Glu Pro Ser Asn Ser Lys Leu Ser Gly Phe Phe Gln Ser
1445 1450 1455
Val Thr Asn His Lys Trp Val Ala Val Gly Ala Ala Val Gly Ile Leu
1460 1465 1470
Gly Val Leu Val Gly Gly Trp Phe Val Tyr Lys His Phe Ser Arg Lys
1475 1480 1485
Glu Glu Glu Pro Ile Pro Ala Glu Gly Val Tyr His Gly Val Thr Lys
1490 1495 1500
Pro Lys Gln Val Ile Lys Leu Asp Ala Asp Pro Val Glu Ser Gln Ser
1505 1510 1515 1520
Thr Leu Glu Ile Ala Gly Leu Val Arg Lys Asn Leu Val Gln Phe Gly
1525 1530 1535
Val Gly Glu Lys Asn Gly Cys Val Arg Trp Val Met Asn Ala Leu Gly
1540 1545 1550
Val Lys Asp Asp Trp Leu Leu Val Pro Ser His Ala Tyr Lys Phe Glu
1555 1560 1565
Lys Asp Tyr Glu Met Met Glu Phe Tyr Phe Asn Arg Gly Gly Thr Tyr
1570 1575 1580
Tyr Ser Ile Ser Ala Gly Asn Val Val Ile Gln Ser Leu Asp Val Gly
1585 1590 1595 1600
Phe Gln Asp Val Val Leu Met Lys Val Pro Thr Ile Pro Lys Phe Arg
1605 1610 1615
Asp Ile Thr Gln His Phe Ile Lys Lys Gly Asp Val Pro Arg Ala Leu
1620 1625 1630
Asn Arg Leu Ala Thr Leu Val Thr Thr Val Asn Gly Thr Pro Met Leu
1635 1640 1645
Ile Ser Glu Gly Pro Leu Lys Met Glu Glu Lys Ala Thr Tyr Val His
1650 1655 1660
Lys Lys Asn Asp Gly Thr Thr Val Asp Leu Thr Val Asp Gln Ala Trp
1665 1670 1675 1680
Arg Gly Lys Gly Glu Gly Leu Pro Gly Met Cys Gly Gly Ala Leu Val
1685 1690 1695
Ser Ser Asn Gln Ser Ile Gln Asn Ala Ile Leu Gly Ile His Val Ala
1700 1705 1710
Gly Gly Asn Ser Ile Leu Val Ala Lys Leu Val Thr Gln Glu Met Phe
1715 1720 1725
Gln Asn Ile Asp Lys Lys Ile Glu Ser Gln Arg Ile Met Lys Val Glu
1730 1735 1740
Phe Thr Gln Cys Ser Met Asn Val Val Ser Lys Thr Leu Phe Arg Lys
1745 1750 1755 1760
Ser Pro Ile Tyr His His Ile Asp Lys Thr Met Ile Asn Phe Pro Ala
1765 1770 1775
Ala Met Pro Phe Ser Lys Ala Glu Ile Asp Pro Met Ala Val Met Leu
1780 1785 1790
Ser Lys Tyr Ser Leu Pro Ile Val Glu Glu Pro Glu Asp Tyr Lys Glu
1795 1800 1805
Ala Ser Ile Phe Tyr Gln Asn Lys Ile Val Gly Lys Thr Gln Leu Val
1810 1815 1820
Asp Asp Phe Leu Asp Leu Asp Met Ala Ile Thr Gly Ala Pro Gly Ile
1825 1830 1835 1840
Asp Ala Ile Asn Met Asp Ser Ser Pro Gly Phe Pro Tyr Val Gln Glu
1845 1850 1855
Lys Leu Thr Lys Arg Asp Leu Ile Trp Leu Asp Glu Asn Gly Leu Leu
1860 1865 1870
Leu Gly Val His Pro Arg Leu Ala Gln Arg Ile Leu Phe Asn Thr Val
1875 1880 1885
Met Met Glu Asn Cys Ser Asp Leu Asp Val Val Phe Thr Thr Cys Pro
1890 1895 1900
Lys Asp Glu Leu Arg Pro Leu Glu Lys Val Leu Glu Ser Lys Thr Arg
1905 1910 1915 1920
Ala Ile Asp Ala Cys Pro Leu Asp Tyr Ser Ile Leu Cys Arg Met Tyr
1925 1930 1935
Trp Gly Pro Ala Ile Ser Tyr Phe His Leu Asn Pro Gly Phe His Thr
1940 1945 1950
Gly Val Ala Ile Gly Ile Asp Pro Asp Arg Gln Trp Asp Glu Leu Phe
1955 1960 1965
Lys Thr Met Ile Arg Phe Gly Asp Val Gly Leu Asp Leu Asp Phe Ser
1970 1975 1980
Ala Phe Asp Ala Ser Leu Ser Pro Phe Met Ile Arg Glu Ala Gly Arg
1985 1990 1995 2000
Ile Met Ser Glu Leu Ser Gly Thr Pro Ser His Phe Gly Thr Ala Leu
2005 2010 2015
Ile Asn Thr Ile Ile Tyr Ser Lys His Leu Leu Tyr Asn Cys Cys Tyr
2020 2025 2030
His Val Cys Gly Ser Met Pro Ser Gly Ser Pro Cys Thr Ala Leu Leu
2035 2040 2045
Asn Ser Ile Ile Asn Asn Val Asn Leu Tyr Tyr Val Phe Ser Lys Ile
2050 2055 2060
Phe Gly Lys Ser Pro Val Phe Phe Cys Gln Ala Leu Lys Ile Leu Cys
1065 2070 2075 2080
Tyr Gly Asp Asp Val Leu Ile Val Phe Ser Arg Asp Val Gln Ile Asp
2085 2090 2095
Asn Leu Asp Leu Ile Gly Gln Lys Ile Val Asp Glu Phe Lys Lys Leu
2100 2105 2110
Gly Met Thr Ala Thr Ser Ala Asp Lys Asn Val Pro Gln Leu Lys Pro
2115 2120 2125
Val Ser Glu Leu Thr Phe Leu Lys Arg Ser Phe Asn Leu Val Glu Asp
2130 2135 2140
Arg Ile Arg Pro Ala Ile Ser Glu Lys Thr Ile Trp Ser Leu Ile Ala
2145 2150 2155 2160
Trp Gln Arg Ser Asn Ala Glu Phe Glu Gln Asn Leu Glu Asn Ala Gln
2165 2170 2175
Trp Phe Ala Phe Met His Gly Tyr Glu Phe Tyr Gln Lys Phe Tyr Tyr
2180 2185 2190
Phe Val Gln Ser Cys Leu Glu Lys Glu Met Ile Glu Tyr Arg Leu Lys
2195 2200 2205
Ser Tyr Asp Trp Trp Arg Met Arg Phe Tyr Asp Gln Cys Phe Ile Cys
2210 2215 2220
Asp Leu Ser
2225




3


7486


DNA


Attenuated HAV (4380), strain HM-175




CDS




(730)..(7410)





3
ttcaagaggg gtctccggga atttccggag tccctcttgg aagtccatgg tgaggggact 60
tgatacctca ccgccgtttg cctaggctat aggctaaatt ttccctttcc cttttccctt 120
tcccattccc ttttgcttgt aaatattgat tcctgcaggt tcagggttct taaatctgtt 180
tctctataag aacactcatt ttcacgcttt ctgtcttctt tcttccaggg ctctcccctt 240
gccctaggct ctggccgttg cgcccggcgg ggtcaactcc atgattagca tggagctgta 300
ggagtctaaa ttggggacac agatgtttgg aacgtcacct tgcagtgtta acttggcttt 360
catgaatctc tttgatcttc cacaaggggt aggctacggg tgaaacctct taggctaata 420
cttctatgaa gagatgcctt ggatagggta acagcggcgg atattggtga gttgttaaga 480
caaaaaccat tcaacgccgg aggactgact ctcatccagt ggatgcattg agtggattga 540
ctgtcagggc tgtctttagg cttaattcca gacctctctg tgcttggggc aaacatcatt 600
tggccttaaa tgggattctg tgagagggga tccctccatt aacagctgga ctgttctttg 660
gggtcttatg tggtgtttgc cgctgaggta ctcaggggca tttaggtttt tcctcattct 720
taaataata atg aac atg tct aga caa ggt att ttc cag act gtt ggg agt 771
Met Asn Met Ser Arg Gln Gly Ile Phe Gln Thr Val Gly Ser
1 5 10
ggt ctt gac cac atc ctg tct ttg gca gac att gag gaa gag caa atg 819
Gly Leu Asp His Ile Leu Ser Leu Ala Asp Ile Glu Glu Glu Gln Met
15 20 25 30
att caa tca gtt gat agg act gca gtg act ggt gct tct tat ttt act 867
Ile Gln Ser Val Asp Arg Thr Ala Val Thr Gly Ala Ser Tyr Phe Thr
35 40 45
tct gtg gat caa tct tca gtt cat aca gct gag gtt gga tca cac cag 915
Ser Val Asp Gln Ser Ser Val His Thr Ala Glu Val Gly Ser His Gln
50 55 60
gtt gaa cct ttg aga acc tct gtt gat aaa ccc ggt tca aag agg act 963
Val Glu Pro Leu Arg Thr Ser Val Asp Lys Pro Gly Ser Lys Arg Thr
65 70 75
cag gga gag aaa ttt ttc ttg att cat tct gca gat tgg ctt act aca 1011
Gln Gly Glu Lys Phe Phe Leu Ile His Ser Ala Asp Trp Leu Thr Thr
80 85 90
cat gct ctt ttc cat gaa gtt gca aaa ttg gat gtg gtg aaa tta tta 1059
His Ala Leu Phe His Glu Val Ala Lys Leu Asp Val Val Lys Leu Leu
95 100 105 110
tac aat gag cag ttt gct gtt caa ggg ttg ttg aga tac cat aca tat 1107
Tyr Asn Glu Gln Phe Ala Val Gln Gly Leu Leu Arg Tyr His Thr Tyr
115 120 125
gca aga ttt ggc att gaa att caa gtt cag ata aac cct aca cct ttc 1155
Ala Arg Phe Gly Ile Glu Ile Gln Val Gln Ile Asn Pro Thr Pro Phe
130 135 140
caa cag ggg gga ttg atc tgt gct atg gtt cct ggt gac cag agc tat 1203
Gln Gln Gly Gly Leu Ile Cys Ala Met Val Pro Gly Asp Gln Ser Tyr
145 150 155
ggt tct ata gca tca ttg act gtt tat cct cat ggt ttg tta aat tgc 1251
Gly Ser Ile Ala Ser Leu Thr Val Tyr Pro His Gly Leu Leu Asn Cys
160 165 170
aat att aac aat gtg gtt aga ata aag gtt cca ttt att tac aca aga 1299
Asn Ile Asn Asn Val Val Arg Ile Lys Val Pro Phe Ile Tyr Thr Arg
175 180 185 190
ggt gct tac cac ttt aaa gat cca caa tac cca gtt tgg gaa ttg aca 1347
Gly Ala Tyr His Phe Lys Asp Pro Gln Tyr Pro Val Trp Glu Leu Thr
195 200 205
att aga gtt tgg tca gaa tta aat att ggg aca gga act tca gct tat 1395
Ile Arg Val Trp Ser Glu Leu Asn Ile Gly Thr Gly Thr Ser Ala Tyr
210 215 220
act tca ctc aat gtt tta gct aga ttt aca gat ttg gag ttg cat gga 1443
Thr Ser Leu Asn Val Leu Ala Arg Phe Thr Asp Leu Glu Leu His Gly
225 230 235
tta act cct ctt tct aca caa atg atg aga aat gaa ttt agg gtc agt 1491
Leu Thr Pro Leu Ser Thr Gln Met Met Arg Asn Glu Phe Arg Val Ser
240 245 250
act act gag aat gtg gtg aat ctg tca aat tat gaa gat gca aga gca 1539
Thr Thr Glu Asn Val Val Asn Leu Ser Asn Tyr Glu Asp Ala Arg Ala
255 260 265 270
aag atg tct ttt gct ttg gat cag gaa gat tgg aaa tct gat ccg tcc 1587
Lys Met Ser Phe Ala Leu Asp Gln Glu Asp Trp Lys Ser Asp Pro Ser
275 280 285
cag ggt ggt ggg atc aaa att act cat ttt act act tgg aca tct att 1635
Gln Gly Gly Gly Ile Lys Ile Thr His Phe Thr Thr Trp Thr Ser Ile
290 295 300
cca act ttg gct gct cag ttt cca ttt aat gct tca gac tca gtt ggt 1683
Pro Thr Leu Ala Ala Gln Phe Pro Phe Asn Ala Ser Asp Ser Val Gly
305 310 315
caa caa att aaa gtt att cca gtt gac cca tat ttt ttc caa atg aca 1731
Gln Gln Ile Lys Val Ile Pro Val Asp Pro Tyr Phe Phe Gln Met Thr
320 325 330
aat aca aat cct gac caa aaa tgt ata act gct ttg gct tct att tgt 1779
Asn Thr Asn Pro Asp Gln Lys Cys Ile Thr Ala Leu Ala Ser Ile Cys
335 340 345 350
cag atg ttt tgt ttt tgg aga gga gat ctt gtc ttt gat ttt caa gtt 1827
Gln Met Phe Cys Phe Trp Arg Gly Asp Leu Val Phe Asp Phe Gln Val
355 360 365
ttt ccc acc aaa tat cat tca ggt aga tta ctg ttt tgt ttt gtt cct 1875
Phe Pro Thr Lys Tyr His Ser Gly Arg Leu Leu Phe Cys Phe Val Pro
370 375 380
ggc aat gag cta ata gat gtt tct gga atc aca tta aag caa gca act 1923
Gly Asn Glu Leu Ile Asp Val Ser Gly Ile Thr Leu Lys Gln Ala Thr
385 390 395
act gct cct tgt gca gta atg gat att aca gga gtg cag tca act ttg 1971
Thr Ala Pro Cys Ala Val Met Asp Ile Thr Gly Val Gln Ser Thr Leu
400 405 410
aga ttt cgt gtt ccc tgg att tct gac act cct tac aga gtg aac agg 2019
Arg Phe Arg Val Pro Trp Ile Ser Asp Thr Pro Tyr Arg Val Asn Arg
415 420 425 430
tat aca aag tca gca cat cag aaa ggt gag tac act gcc att ggg aag 2067
Tyr Thr Lys Ser Ala His Gln Lys Gly Glu Tyr Thr Ala Ile Gly Lys
435 440 445
ctt att gtg tat tgt tat aac aga ttg acc tct cct tct aac gtt gct 2115
Leu Ile Val Tyr Cys Tyr Asn Arg Leu Thr Ser Pro Ser Asn Val Ala
450 455 460
tcc cat gtc aga gtg aat gtt tat ctt tca gca att aac ttg gaa tgt 2163
Ser His Val Arg Val Asn Val Tyr Leu Ser Ala Ile Asn Leu Glu Cys
465 470 475
ttt gct cct ctt tat cat gct atg gat gtt act aca caa gtt gga gat 2211
Phe Ala Pro Leu Tyr His Ala Met Asp Val Thr Thr Gln Val Gly Asp
480 485 490
gat tct gga ggt ttt tca aca aca gtt tct aca gaa cag aat gtt cca 2259
Asp Ser Gly Gly Phe Ser Thr Thr Val Ser Thr Glu Gln Asn Val Pro
495 500 505 510
gat ccc caa gtt ggt ata aca acc atg aaa gat ttg aaa gga aaa gct 2307
Asp Pro Gln Val Gly Ile Thr Thr Met Lys Asp Leu Lys Gly Lys Ala
515 520 525
aac aga ggg aaa atg gat gtt tca gga gta caa gca cct gtg gga gct 2355
Asn Arg Gly Lys Met Asp Val Ser Gly Val Gln Ala Pro Val Gly Ala
530 535 540
atc aca aca att gag gat cca gtt tta gca aag aaa gta cct gag aca 2403
Ile Thr Thr Ile Glu Asp Pro Val Leu Ala Lys Lys Val Pro Glu Thr
545 550 555
ttt cct gaa ttg aaa cct gga gaa tcc aga cat aca tca gat cat atg 2451
Phe Pro Glu Leu Lys Pro Gly Glu Ser Arg His Thr Ser Asp His Met
560 565 570
tcc atc tac aag ttt atg gga agg tct cat ttc ttg tgc act ttt aca 2499
Ser Ile Tyr Lys Phe Met Gly Arg Ser His Phe Leu Cys Thr Phe Thr
575 580 585 590
ttc aat tca aat aat aaa gag tac aca ttt cct ata acc ttg tct tca 2547
Phe Asn Ser Asn Asn Lys Glu Tyr Thr Phe Pro Ile Thr Leu Ser Ser
595 600 605
acc tct aat cct cct cat ggt ttg cca tca aca ctg agg tgg ttt ttc 2595
Thr Ser Asn Pro Pro His Gly Leu Pro Ser Thr Leu Arg Trp Phe Phe
610 615 620
aac ttg ttt cag ttg tat aga ggg cct tta gat ctg aca att att att 2643
Asn Leu Phe Gln Leu Tyr Arg Gly Pro Leu Asp Leu Thr Ile Ile Ile
625 630 635
aca gga gca act gat gta gat ggc atg gcc tgg ttc act cca gta ggt 2691
Thr Gly Ala Thr Asp Val Asp Gly Met Ala Trp Phe Thr Pro Val Gly
640 645 650
ctt gcc gtt gat act cct tgg gta gag aag gag tca gct ttg tct att 2739
Leu Ala Val Asp Thr Pro Trp Val Glu Lys Glu Ser Ala Leu Ser Ile
655 660 665 670
gac tat aaa act gct ctt gga gct gtc aga ttt aac aca agg aga aca 2787
Asp Tyr Lys Thr Ala Leu Gly Ala Val Arg Phe Asn Thr Arg Arg Thr
675 680 685
ggg aac att cag att aga tta cca tgg tat tct tat tta tat gct gtg 2835
Gly Asn Ile Gln Ile Arg Leu Pro Trp Tyr Ser Tyr Leu Tyr Ala Val
690 695 700
tct gga gca ctg gat ggt ttg gga gac aag aca gat tct aca ttt gga 2883
Ser Gly Ala Leu Asp Gly Leu Gly Asp Lys Thr Asp Ser Thr Phe Gly
705 710 715
ttg gtt tct att cag att gca aat tac aat cat tct gat gaa tac ttg 2931
Leu Val Ser Ile Gln Ile Ala Asn Tyr Asn His Ser Asp Glu Tyr Leu
720 725 730
tct ttt agt tgt tat ttg tct gtc aca gaa caa tca gag ttt tat ttt 2979
Ser Phe Ser Cys Tyr Leu Ser Val Thr Glu Gln Ser Glu Phe Tyr Phe
735 740 745 750
ccc aga gct cca ttg aac tca aat gcc atg tta tcc act gta aca atg 3027
Pro Arg Ala Pro Leu Asn Ser Asn Ala Met Leu Ser Thr Val Thr Met
755 760 765
atg agc aga att gca gct gga gac ttg gag tca tca gtg gat gat cct 3075
Met Ser Arg Ile Ala Ala Gly Asp Leu Glu Ser Ser Val Asp Asp Pro
770 775 780
aga tca gag gaa gat aaa aga ttt gag agt cat ata gaa tgc agg aag 3123
Arg Ser Glu Glu Asp Lys Arg Phe Glu Ser His Ile Glu Cys Arg Lys
785 790 795
cca tat aaa gaa ctg aga tta gaa gtt ggg aaa caa aga ctc aag tat 3171
Pro Tyr Lys Glu Leu Arg Leu Glu Val Gly Lys Gln Arg Leu Lys Tyr
800 805 810
gct cag gaa gaa ttg tca aat gaa gta ctt cca ccc cct agg aaa atg 3219
Ala Gln Glu Glu Leu Ser Asn Glu Val Leu Pro Pro Pro Arg Lys Met
815 820 825 830
aag gga ctg ttt tca caa gcc aaa att tct ctt ttt tat act gag gag 3267
Lys Gly Leu Phe Ser Gln Ala Lys Ile Ser Leu Phe Tyr Thr Glu Glu
835 840 845
cat gaa ata atg aag ttt tcc tgg aga ggt gtg act gct gat act aga 3315
His Glu Ile Met Lys Phe Ser Trp Arg Gly Val Thr Ala Asp Thr Arg
850 855 860
gct tta agg agg ttt gga ttc tct ttg gcc gca ggc aga agt gtg tgg 3363
Ala Leu Arg Arg Phe Gly Phe Ser Leu Ala Ala Gly Arg Ser Val Trp
865 870 875
act ctt gaa atg gat gct ggg gtt ctt act ggg aga ctg att aga ttg 3411
Thr Leu Glu Met Asp Ala Gly Val Leu Thr Gly Arg Leu Ile Arg Leu
880 885 890
aat gat gag aaa tgg aca gaa atg aag gat gac aag att gtt tca ttg 3459
Asn Asp Glu Lys Trp Thr Glu Met Lys Asp Asp Lys Ile Val Ser Leu
895 900 905 910
att gaa aag ttt aca agt aac aaa tat tgg tcc aaa gtg aat ttc cca 3507
Ile Glu Lys Phe Thr Ser Asn Lys Tyr Trp Ser Lys Val Asn Phe Pro
915 920 925
cat ggg atg ttg gat ctt gaa gaa att gct gcc aat tct aag gat ttt 3555
His Gly Met Leu Asp Leu Glu Glu Ile Ala Ala Asn Ser Lys Asp Phe
930 935 940
cct aac atg tct gaa acg gat ttg tgt ttc ttg ctg cat tgg tta aat 3603
Pro Asn Met Ser Glu Thr Asp Leu Cys Phe Leu Leu His Trp Leu Asn
945 950 955
cca aag aaa att aat tta gca gat aga atg ctt gga ttg tct gga gtt 3651
Pro Lys Lys Ile Asn Leu Ala Asp Arg Met Leu Gly Leu Ser Gly Val
960 965 970
cag gaa att aaa gaa caa ggt gtt gga tta ata gca gag tgt aga act 3699
Gln Glu Ile Lys Glu Gln Gly Val Gly Leu Ile Ala Glu Cys Arg Thr
975 980 985 990
ttc tta gat tct att gct gga act tta aaa tct atg atg ttt gga ttt 3747
Phe Leu Asp Ser Ile Ala Gly Thr Leu Lys Ser Met Met Phe Gly Phe
995 1000 1005
cat cat tct gtg act gtt gaa att ata aac act gtg ctc tgt ttt gtt 3795
His His Ser Val Thr Val Glu Ile Ile Asn Thr Val Leu Cys Phe Val
1010 1015 1020
aag agt gga att ttg ctt tat gta ata caa caa ttg aat cag gat gaa 3843
Lys Ser Gly Ile Leu Leu Tyr Val Ile Gln Gln Leu Asn Gln Asp Glu
1025 1030 1035
cat tct cac ata att ggt ttg ttg aga gtc atg aat tat gta gat att 3891
His Ser His Ile Ile Gly Leu Leu Arg Val Met Asn Tyr Val Asp Ile
1040 1045 1050
ggt tgt tca gtt att tca tgt gcc aaa gtt ttt tcc aga atg ctg gaa 3939
Gly Cys Ser Val Ile Ser Cys Ala Lys Val Phe Ser Arg Met Leu Glu
1055 1060 1065 1070
aca gtc ttt aat tgg caa atg gac tcc aga atg atg gag tta agg act 3987
Thr Val Phe Asn Trp Gln Met Asp Ser Arg Met Met Glu Leu Arg Thr
1075 1080 1085
cag agt ttt tcc aac tgg tta aga gat att tgt tct ggg atc acc att 4035
Gln Ser Phe Ser Asn Trp Leu Arg Asp Ile Cys Ser Gly Ile Thr Ile
1090 1095 1100
ttc aaa aac ttc aag gat gca att tat tgg ctt tat aca aaa tta atg 4083
Phe Lys Asn Phe Lys Asp Ala Ile Tyr Trp Leu Tyr Thr Lys Leu Met
1105 1110 1115
gac ttt tat gaa gtg aat tat ggc aag aag aag gac att tta aat att 4131
Asp Phe Tyr Glu Val Asn Tyr Gly Lys Lys Lys Asp Ile Leu Asn Ile
1120 1125 1130
ctt aaa gat aac caa caa aaa ata gag aaa gcc att gag gaa gcc gat 4179
Leu Lys Asp Asn Gln Gln Lys Ile Glu Lys Ala Ile Glu Glu Ala Asp
1135 1140 1145 1150
aaa ttt tgc att ttg caa atc caa gat gtg gaa aaa tct gaa cag tat 4227
Lys Phe Cys Ile Leu Gln Ile Gln Asp Val Glu Lys Ser Glu Gln Tyr
1155 1160 1165
cag aaa ggg gtt gac ttg ata caa aaa ttg aga act gtt cat tca atg 4275
Gln Lys Gly Val Asp Leu Ile Gln Lys Leu Arg Thr Val His Ser Met
1170 1175 1180
gct cag gtt gat cca aat tta atg gtt cat ttg tca cct ttg aga gat 4323
Ala Gln Val Asp Pro Asn Leu Met Val His Leu Ser Pro Leu Arg Asp
1185 1190 1195
tgt ata gca aga gtt cat cag aaa ctt aaa aac ctt gga tct ata aat 4371
Cys Ile Ala Arg Val His Gln Lys Leu Lys Asn Leu Gly Ser Ile Asn
1200 1205 1210
cag gca atg gta acg aga tgt gag cca gtt gtt tgt tat ttt tat ggc 4419
Gln Ala Met Val Thr Arg Cys Glu Pro Val Val Cys Tyr Phe Tyr Gly
1215 1220 1225 1230
aaa aga ggg gga gga aag agc tta aca tca att gca ttg gca acc aaa 4467
Lys Arg Gly Gly Gly Lys Ser Leu Thr Ser Ile Ala Leu Ala Thr Lys
1235 1240 1245
att tgt aaa cat tat ggt gtt gag cct gaa aag aat atc tat act aaa 4515
Ile Cys Lys His Tyr Gly Val Glu Pro Glu Lys Asn Ile Tyr Thr Lys
1250 1255 1260
cct gtg gct tca gat tac tgg gat gga tat agt gga caa tta gtt tgc 4563
Pro Val Ala Ser Asp Tyr Trp Asp Gly Tyr Ser Gly Gln Leu Val Cys
1265 1270 1275
atc att gat gat att ggc caa aac aca aca gat gag gat tgg tca gat 4611
Ile Ile Asp Asp Ile Gly Gln Asn Thr Thr Asp Glu Asp Trp Ser Asp
1280 1285 1290
ttt tgt cag tta gtg tca gga tgt cct atg aga tta aac atg gcc tct 4659
Phe Cys Gln Leu Val Ser Gly Cys Pro Met Arg Leu Asn Met Ala Ser
1295 1300 1305 1310
ctt gag gag aag ggt agg cat ttt tct tct cct ttt ata ata gca act 4707
Leu Glu Glu Lys Gly Arg His Phe Ser Ser Pro Phe Ile Ile Ala Thr
1315 1320 1325
tca aat tgg tca aat cca agt cca aaa aca gtt tat gtt aag gaa gca 4755
Ser Asn Trp Ser Asn Pro Ser Pro Lys Thr Val Tyr Val Lys Glu Ala
1330 1335 1340
att gac cgc aga ctc cat ttc aag gtt gaa gtt aaa cct gct tca ttt 4803
Ile Asp Arg Arg Leu His Phe Lys Val Glu Val Lys Pro Ala Ser Phe
1345 1350 1355
ttc aaa aat cct cac aat gat atg ttg aat gtt aat tta gct aaa aca 4851
Phe Lys Asn Pro His Asn Asp Met Leu Asn Val Asn Leu Ala Lys Thr
1360 1365 1370
aat gat gca atc aaa gat atg tct tgt gtt gat ttg ata atg gat gga 4899
Asn Asp Ala Ile Lys Asp Met Ser Cys Val Asp Leu Ile Met Asp Gly
1375 1380 1385 1390
cat aat gtt tca ttg atg gat ttg ctc agt tct tta gtc atg aca gtt 4947
His Asn Val Ser Leu Met Asp Leu Leu Ser Ser Leu Val Met Thr Val
1395 1400 1405
gaa att aga aaa caa aac atg act gaa ttc atg gag ttg tgg tct cag 4995
Glu Ile Arg Lys Gln Asn Met Thr Glu Phe Met Glu Leu Trp Ser Gln
1410 1415 1420
gga att tca gat gat gat aat gat agt gca gta gct gag ttt ttc cag 5043
Gly Ile Ser Asp Asp Asp Asn Asp Ser Ala Val Ala Glu Phe Phe Gln
1425 1430 1435
tct ttt cca tct ggt gaa cca tcg aac tct aaa tta tct ggc ttt ttc 5091
Ser Phe Pro Ser Gly Glu Pro Ser Asn Ser Lys Leu Ser Gly Phe Phe
1440 1445 1450
caa tct gtt act aat cac aag tgg gtt gct gtg gga gct gca gtt ggc 5139
Gln Ser Val Thr Asn His Lys Trp Val Ala Val Gly Ala Ala Val Gly
1455 1460 1465 1470
gtt ctt gga gtg ctc gtt gga gga tgg ttt gtg tat aag cat ttc tcc 5187
Val Leu Gly Val Leu Val Gly Gly Trp Phe Val Tyr Lys His Phe Ser
1475 1480 1485
cgc aaa gag gaa gaa cca atc cca gct gaa ggg gta tat tat ggt gta 5235
Arg Lys Glu Glu Glu Pro Ile Pro Ala Glu Gly Val Tyr Tyr Gly Val
1490 1495 1500
act aag ccc aag caa gtg att aaa tta gat gca gat cca gta gaa tct 5283
Thr Lys Pro Lys Gln Val Ile Lys Leu Asp Ala Asp Pro Val Glu Ser
1505 1510 1515
cag tca act ttg gaa ata gca gga ctg gtt agg aag aac ttg gtt cag 5331
Gln Ser Thr Leu Glu Ile Ala Gly Leu Val Arg Lys Asn Leu Val Gln
1520 1525 1530
ttt gga gtt gga gag aag aat gga tgt gtg aga tgg gtt atg aat gcc 5379
Phe Gly Val Gly Glu Lys Asn Gly Cys Val Arg Trp Val Met Asn Ala
1535 1540 1545 1550
ttg gga gtg aaa gat gat tgg ctg ctt gtg cct tcc cat gct tat aaa 5427
Leu Gly Val Lys Asp Asp Trp Leu Leu Val Pro Ser His Ala Tyr Lys
1555 1560 1565
ttt gag aaa gat tat gaa atg atg gag ttt tat ttt aat aga ggt gga 5475
Phe Glu Lys Asp Tyr Glu Met Met Glu Phe Tyr Phe Asn Arg Gly Gly
1570 1575 1580
act tac tat tca att tca gct ggt aat gtt gtt att caa tct ttg gat 5523
Thr Tyr Tyr Ser Ile Ser Ala Gly Asn Val Val Ile Gln Ser Leu Asp
1585 1590 1595
gtg gga ttc cag gat gtt gtt ctg atg aag gtt cct aca att cct aag 5571
Val Gly Phe Gln Asp Val Val Leu Met Lys Val Pro Thr Ile Pro Lys
1600 1605 1610
ttt aga gat att act cag cat ttt att aag aaa ggg gat gtg cct aga 5619
Phe Arg Asp Ile Thr Gln His Phe Ile Lys Lys Gly Asp Val Pro Arg
1615 1620 1625 1630
gct ttg aat cgc ctg gca aca tta gtg aca act gta aat gga acc cct 5667
Ala Leu Asn Arg Leu Ala Thr Leu Val Thr Thr Val Asn Gly Thr Pro
1635 1640 1645
atg tta att tct gag ggc cca cta aag atg gaa gag aaa gct act tat 5715
Met Leu Ile Ser Glu Gly Pro Leu Lys Met Glu Glu Lys Ala Thr Tyr
1650 1655 1660
gtt cat aag aaa aat gat ggt aca tca gtt gat tta act gtg gat cag 5763
Val His Lys Lys Asn Asp Gly Thr Ser Val Asp Leu Thr Val Asp Gln
1665 1670 1675
gca tgg aga gga aaa ggc gaa ggt ctt cct gga atg tgt ggt ggg gcc 5811
Ala Trp Arg Gly Lys Gly Glu Gly Leu Pro Gly Met Cys Gly Gly Ala
1680 1685 1690
ttg gtt tca tcg aat caa tct ata cag aat gca atc ttg ggc atc cat 5859
Leu Val Ser Ser Asn Gln Ser Ile Gln Asn Ala Ile Leu Gly Ile His
1695 1700 1705 1710
gtt gct gga gga aat tca att ctt gtt gca aaa ttg gtt act caa gaa 5907
Val Ala Gly Gly Asn Ser Ile Leu Val Ala Lys Leu Val Thr Gln Glu
1715 1720 1725
atg ttc caa aat att gat aag aaa att gaa agt cag aga att atg aaa 5955
Met Phe Gln Asn Ile Asp Lys Lys Ile Glu Ser Gln Arg Ile Met Lys
1730 1735 1740
gtg gag ttt act cag tgt tca atg aat gtg gtc tcc aaa acg ctt ttt 6003
Val Glu Phe Thr Gln Cys Ser Met Asn Val Val Ser Lys Thr Leu Phe
1745 1750 1755
aga aag agt ccc att tat cat cac att gat aaa acc atg att aat ttt 6051
Arg Lys Ser Pro Ile Tyr His His Ile Asp Lys Thr Met Ile Asn Phe
1760 1765 1770
cct gca gct atg ccc ttt tct aaa gct gaa att gat cca atg gct gtg 6099
Pro Ala Ala Met Pro Phe Ser Lys Ala Glu Ile Asp Pro Met Ala Val
1775 1780 1785 1790
atg tta tct aag tat tca tta cct att gta gaa gaa cca gag aat tat 6147
Met Leu Ser Lys Tyr Ser Leu Pro Ile Val Glu Glu Pro Glu Asn Tyr
1795 1800 1805
aaa gag gct tca att ttt tat caa aat aaa ata gtg ggt aag act cag 6195
Lys Glu Ala Ser Ile Phe Tyr Gln Asn Lys Ile Val Gly Lys Thr Gln
1810 1815 1820
tta gtt gat gat ttt cta gat ctt gat atg gcc att aca ggg gcc cca 6243
Leu Val Asp Asp Phe Leu Asp Leu Asp Met Ala Ile Thr Gly Ala Pro
1825 1830 1835
gga att gat gct atc aac atg gat tca tct cct gga ttt cct tat gtc 6291
Gly Ile Asp Ala Ile Asn Met Asp Ser Ser Pro Gly Phe Pro Tyr Val
1840 1845 1850
cag gag aag ttg acc aaa aga gat tta att tgg ttg gat gaa aat ggt 6339
Gln Glu Lys Leu Thr Lys Arg Asp Leu Ile Trp Leu Asp Glu Asn Gly
1855 1860 1865 1870
tta ttg ctg gga gtt cat cca aga ttg gct cag aga atc tta ttc aat 6387
Leu Leu Leu Gly Val His Pro Arg Leu Ala Gln Arg Ile Leu Phe Asn
1875 1880 1885
act gtc atg atg gaa aat tgt tct gat ttg gat gtt gtt ttt aca acc 6435
Thr Val Met Met Glu Asn Cys Ser Asp Leu Asp Val Val Phe Thr Thr
1890 1895 1900
tgt cca aaa gat gaa ttg aga cca tta gag aaa gtg ttg gaa tca aaa 6483
Cys Pro Lys Asp Glu Leu Arg Pro Leu Glu Lys Val Leu Glu Ser Lys
1905 1910 1915
aca aga gct att gat gct tgt cct ctg gat tac aca att ttg tgc cga 6531
Thr Arg Ala Ile Asp Ala Cys Pro Leu Asp Tyr Thr Ile Leu Cys Arg
1920 1925 1930
atg tat tgg ggt cca gct att agt tat ttt cat ttg aat cca ggt ttc 6579
Met Tyr Trp Gly Pro Ala Ile Ser Tyr Phe His Leu Asn Pro Gly Phe
1935 1940 1945 1950
cat aca ggt gtt gct att ggc ata gat cct gat aga cag tgg gat gaa 6627
His Thr Gly Val Ala Ile Gly Ile Asp Pro Asp Arg Gln Trp Asp Glu
1955 1960 1965
tta ttt aaa aca atg ata aga ttc gga gat gtt ggt ctt gat tta gat 6675
Leu Phe Lys Thr Met Ile Arg Phe Gly Asp Val Gly Leu Asp Leu Asp
1970 1975 1980
ttc tct gct ttt gat gct agt ctt agt cca ttt atg att aga gaa gca 6723
Phe Ser Ala Phe Asp Ala Ser Leu Ser Pro Phe Met Ile Arg Glu Ala
1985 1990 1995
ggt aga atc atg agt gaa cta tct gga act cca tcc cat ttt ggc aca 6771
Gly Arg Ile Met Ser Glu Leu Ser Gly Thr Pro Ser His Phe Gly Thr
2000 2005 2010
gct ctt atc aat act atc att tat tcc aag cat ttg ctg tat aac tgt 6819
Ala Leu Ile Asn Thr Ile Ile Tyr Ser Lys His Leu Leu Tyr Asn Cys
2015 2020 2025 2030
tgt tac cat gtc tgt ggt tca atg ccc tct ggg tct cct tgt aca gct 6867
Cys Tyr His Val Cys Gly Ser Met Pro Ser Gly Ser Pro Cys Thr Ala
2035 2040 2045
ttg cta aat tca att att aat aat gtc aat ttg tac tat gtg ttt tcc 6915
Leu Leu Asn Ser Ile Ile Asn Asn Val Asn Leu Tyr Tyr Val Phe Ser
2050 2055 2060
aag ata ttt gga aag tct cca gtt ttc ttt tgt cag gct ttg aag att 6963
Lys Ile Phe Gly Lys Ser Pro Val Phe Phe Cys Gln Ala Leu Lys Ile
2065 2070 2075
ctc tgt tat gga gat gat gtt tta ata gtt ttc tct cga gat gtt cag 7011
Leu Cys Tyr Gly Asp Asp Val Leu Ile Val Phe Ser Arg Asp Val Gln
2080 2085 2090
att gat aat ctt gat ttg att gga caa aaa att gta gat gag ttt aag 7059
Ile Asp Asn Leu Asp Leu Ile Gly Gln Lys Ile Val Asp Glu Phe Lys
2095 2100 2105 2110
aaa ctt ggc atg aca gct act tct gct gac aag aat gta cct cag ctg 7107
Lys Leu Gly Met Thr Ala Thr Ser Ala Asp Lys Asn Val Pro Gln Leu
2115 2120 2125
aaa cca gtt tcg gaa ttg act ttt ctc aaa aga tct ttc aat ttg gta 7155
Lys Pro Val Ser Glu Leu Thr Phe Leu Lys Arg Ser Phe Asn Leu Val
2130 2135 2140
gag gat aga att aga cct gca att tcg gaa aaa aca att tgg tct tta 7203
Glu Asp Arg Ile Arg Pro Ala Ile Ser Glu Lys Thr Ile Trp Ser Leu
2145 2150 2155
ata gca tgg cag aga agt aac gct gag ttt gag cag aat tta gaa att 7251
Ile Ala Trp Gln Arg Ser Asn Ala Glu Phe Glu Gln Asn Leu Glu Ile
2160 2165 2170
gct cag tgg ttt gct ttt atg cat ggc tat gag ttt tat cag aaa ttt 7299
Ala Gln Trp Phe Ala Phe Met His Gly Tyr Glu Phe Tyr Gln Lys Phe
2175 2180 2185 2190
tat tat ttt gtt cag tcc tgt ttg gag aaa gag atg ata gaa tac aga 7347
Tyr Tyr Phe Val Gln Ser Cys Leu Glu Lys Glu Met Ile Glu Tyr Arg
2195 2200 2205
ctt aaa tct tat gat tgg tgg aga atg aga ttt tat gac cag tgt ttc 7395
Leu Lys Ser Tyr Asp Trp Trp Arg Met Arg Phe Tyr Asp Gln Cys Phe
2210 2215 2220
att tgt gac ctt tca tgatttgttt aaacgaattt tcttaaaatt tctgaggttt 7450
Ile Cys Asp Leu Ser
2225
gtttatttct tttatcagta aataaaaaaa aaaaaa 7486




4


2227


PRT


Attenuated (4380) HAV, strain HM-175



4
Met Asn Met Ser Arg Gln Gly Ile Phe Gln Thr Val Gly Ser Gly Leu
1 5 10 15
Asp His Ile Leu Ser Leu Ala Asp Ile Glu Glu Glu Gln Met Ile Gln
20 25 30
Ser Val Asp Arg Thr Ala Val Thr Gly Ala Ser Tyr Phe Thr Ser Val
35 40 45
Asp Gln Ser Ser Val His Thr Ala Glu Val Gly Ser His Gln Val Glu
50 55 60
Pro Leu Arg Thr Ser Val Asp Lys Pro Gly Ser Lys Arg Thr Gln Gly
65 70 75 80
Glu Lys Phe Phe Leu Ile His Ser Ala Asp Trp Leu Thr Thr His Ala
85 90 95
Leu Phe His Glu Val Ala Lys Leu Asp Val Val Lys Leu Leu Tyr Asn
100 105 110
Glu Gln Phe Ala Val Gln Gly Leu Leu Arg Tyr His Thr Tyr Ala Arg
115 120 125
Phe Gly Ile Glu Ile Gln Val Gln Ile Asn Pro Thr Pro Phe Gln Gln
130 135 140
Gly Gly Leu Ile Cys Ala Met Val Pro Gly Asp Gln Ser Tyr Gly Ser
145 150 155 160
Ile Ala Ser Leu Thr Val Tyr Pro His Gly Leu Leu Asn Cys Asn Ile
165 170 175
Asn Asn Val Val Arg Ile Lys Val Pro Phe Ile Tyr Thr Arg Gly Ala
180 185 190
Tyr His Phe Lys Asp Pro Gln Tyr Pro Val Trp Glu Leu Thr Ile Arg
195 200 205
Val Trp Ser Glu Leu Asn Ile Gly Thr Gly Thr Ser Ala Tyr Thr Ser
210 215 220
Leu Asn Val Leu Ala Arg Phe Thr Asp Leu Glu Leu His Gly Leu Thr
225 230 235 240
Pro Leu Ser Thr Gln Met Met Arg Asn Glu Phe Arg Val Ser Thr Thr
245 250 255
Glu Asn Val Val Asn Leu Ser Asn Tyr Glu Asp Ala Arg Ala Lys Met
260 265 270
Ser Phe Ala Leu Asp Gln Glu Asp Trp Lys Ser Asp Pro Ser Gln Gly
275 280 285
Gly Gly Ile Lys Ile Thr His Phe Thr Thr Trp Thr Ser Ile Pro Thr
290 295 300
Leu Ala Ala Gln Phe Pro Phe Asn Ala Ser Asp Ser Val Gly Gln Gln
305 310 315 320
Ile Lys Val Ile Pro Val Asp Pro Tyr Phe Phe Gln Met Thr Asn Thr
325 330 335
Asn Pro Asp Gln Lys Cys Ile Thr Ala Leu Ala Ser Ile Cys Gln Met
340 345 350
Phe Cys Phe Trp Arg Gly Asp Leu Val Phe Asp Phe Gln Val Phe Pro
355 360 365
Thr Lys Tyr His Ser Gly Arg Leu Leu Phe Cys Phe Val Pro Gly Asn
370 375 380
Glu Leu Ile Asp Val Ser Gly Ile Thr Leu Lys Gln Ala Thr Thr Ala
385 390 395 400
Pro Cys Ala Val Met Asp Ile Thr Gly Val Gln Ser Thr Leu Arg Phe
405 410 415
Arg Val Pro Trp Ile Ser Asp Thr Pro Tyr Arg Val Asn Arg Tyr Thr
420 425 430
Lys Ser Ala His Gln Lys Gly Glu Tyr Thr Ala Ile Gly Lys Leu Ile
435 440 445
Val Tyr Cys Tyr Asn Arg Leu Thr Ser Pro Ser Asn Val Ala Ser His
450 455 460
Val Arg Val Asn Val Tyr Leu Ser Ala Ile Asn Leu Glu Cys Phe Ala
465 470 475 480
Pro Leu Tyr His Ala Met Asp Val Thr Thr Gln Val Gly Asp Asp Ser
485 490 495
Gly Gly Phe Ser Thr Thr Val Ser Thr Glu Gln Asn Val Pro Asp Pro
500 505 510
Gln Val Gly Ile Thr Thr Met Lys Asp Leu Lys Gly Lys Ala Asn Arg
515 520 525
Gly Lys Met Asp Val Ser Gly Val Gln Ala Pro Val Gly Ala Ile Thr
530 535 540
Thr Ile Glu Asp Pro Val Leu Ala Lys Lys Val Pro Glu Thr Phe Pro
545 550 555 560
Glu Leu Lys Pro Gly Glu Ser Arg His Thr Ser Asp His Met Ser Ile
565 570 575
Tyr Lys Phe Met Gly Arg Ser His Phe Leu Cys Thr Phe Thr Phe Asn
580 585 590
Ser Asn Asn Lys Glu Tyr Thr Phe Pro Ile Thr Leu Ser Ser Thr Ser
595 600 605
Asn Pro Pro His Gly Leu Pro Ser Thr Leu Arg Trp Phe Phe Asn Leu
610 615 620
Phe Gln Leu Tyr Arg Gly Pro Leu Asp Leu Thr Ile Ile Ile Thr Gly
625 630 635 640
Ala Thr Asp Val Asp Gly Met Ala Trp Phe Thr Pro Val Gly Leu Ala
645 650 655
Val Asp Thr Pro Trp Val Glu Lys Glu Ser Ala Leu Ser Ile Asp Tyr
660 665 670
Lys Thr Ala Leu Gly Ala Val Arg Phe Asn Thr Arg Arg Thr Gly Asn
675 680 685
Ile Gln Ile Arg Leu Pro Trp Tyr Ser Tyr Leu Tyr Ala Val Ser Gly
690 695 700
Ala Leu Asp Gly Leu Gly Asp Lys Thr Asp Ser Thr Phe Gly Leu Val
705 710 715 720
Ser Ile Gln Ile Ala Asn Tyr Asn His Ser Asp Glu Tyr Leu Ser Phe
725 730 735
Ser Cys Tyr Leu Ser Val Thr Glu Gln Ser Glu Phe Tyr Phe Pro Arg
740 745 750
Ala Pro Leu Asn Ser Asn Ala Met Leu Ser Thr Val Thr Met Met Ser
755 760 765
Arg Ile Ala Ala Gly Asp Leu Glu Ser Ser Val Asp Asp Pro Arg Ser
770 775 780
Glu Glu Asp Lys Arg Phe Glu Ser His Ile Glu Cys Arg Lys Pro Tyr
785 790 795 800
Lys Glu Leu Arg Leu Glu Val Gly Lys Gln Arg Leu Lys Tyr Ala Gln
805 810 815
Glu Glu Leu Ser Asn Glu Val Leu Pro Pro Pro Arg Lys Met Lys Gly
820 825 830
Leu Phe Ser Gln Ala Lys Ile Ser Leu Phe Tyr Thr Glu Glu His Glu
835 840 845
Ile Met Lys Phe Ser Trp Arg Gly Val Thr Ala Asp Thr Arg Ala Leu
850 855 860
Arg Arg Phe Gly Phe Ser Leu Ala Ala Gly Arg Ser Val Trp Thr Leu
865 870 875 880
Glu Met Asp Ala Gly Val Leu Thr Gly Arg Leu Ile Arg Leu Asn Asp
885 890 895
Glu Lys Trp Thr Glu Met Lys Asp Asp Lys Ile Val Ser Leu Ile Glu
900 905 910
Lys Phe Thr Ser Asn Lys Tyr Trp Ser Lys Val Asn Phe Pro His Gly
915 920 925
Met Leu Asp Leu Glu Glu Ile Ala Ala Asn Ser Lys Asp Phe Pro Asn
930 935 940
Met Ser Glu Thr Asp Leu Cys Phe Leu Leu His Trp Leu Asn Pro Lys
945 950 955 960
Lys Ile Asn Leu Ala Asp Arg Met Leu Gly Leu Ser Gly Val Gln Glu
965 970 975
Ile Lys Glu Gln Gly Val Gly Leu Ile Ala Glu Cys Arg Thr Phe Leu
980 985 990
Asp Ser Ile Ala Gly Thr Leu Lys Ser Met Met Phe Gly Phe His His
995 1000 1005
Ser Val Thr Val Glu Ile Ile Asn Thr Val Leu Cys Phe Val Lys Ser
1010 1015 1020
Gly Ile Leu Leu Tyr Val Ile Gln Gln Leu Asn Gln Asp Glu His Ser
1025 1030 1035 1040
His Ile Ile Gly Leu Leu Arg Val Met Asn Tyr Val Asp Ile Gly Cys
1045 1050 1055
Ser Val Ile Ser Cys Ala Lys Val Phe Ser Arg Met Leu Glu Thr Val
1060 1065 1070
Phe Asn Trp Gln Met Asp Ser Arg Met Met Glu Leu Arg Thr Gln Ser
1075 1080 1085
Phe Ser Asn Trp Leu Arg Asp Ile Cys Ser Gly Ile Thr Ile Phe Lys
1090 1095 1100
Asn Phe Lys Asp Ala Ile Tyr Trp Leu Tyr Thr Lys Leu Met Asp Phe
1105 1110 1115 1120
Tyr Glu Val Asn Tyr Gly Lys Lys Lys Asp Ile Leu Asn Ile Leu Lys
1125 1130 1135
Asp Asn Gln Gln Lys Ile Glu Lys Ala Ile Glu Glu Ala Asp Lys Phe
1140 1145 1150
Cys Ile Leu Gln Ile Gln Asp Val Glu Lys Ser Glu Gln Tyr Gln Lys
1155 1160 1165
Gly Val Asp Leu Ile Gln Lys Leu Arg Thr Val His Ser Met Ala Gln
1170 1175 1180
Val Asp Pro Asn Leu Met Val His Leu Ser Pro Leu Arg Asp Cys Ile
1185 1190 1195 1200
Ala Arg Val His Gln Lys Leu Lys Asn Leu Gly Ser Ile Asn Gln Ala
1205 1210 1215
Met Val Thr Arg Cys Glu Pro Val Val Cys Tyr Phe Tyr Gly Lys Arg
1220 1225 1230
Gly Gly Gly Lys Ser Leu Thr Ser Ile Ala Leu Ala Thr Lys Ile Cys
1235 1240 1245
Lys His Tyr Gly Val Glu Pro Glu Lys Asn Ile Tyr Thr Lys Pro Val
1250 1255 1260
Ala Ser Asp Tyr Trp Asp Gly Tyr Ser Gly Gln Leu Val Cys Ile Ile
1265 1270 1275 1280
Asp Asp Ile Gly Gln Asn Thr Thr Asp Glu Asp Trp Ser Asp Phe Cys
1285 1290 1295
Gln Leu Val Ser Gly Cys Pro Met Arg Leu Asn Met Ala Ser Leu Glu
1300 1305 1310
Glu Lys Gly Arg His Phe Ser Ser Pro Phe Ile Ile Ala Thr Ser Asn
1315 1320 1325
Trp Ser Asn Pro Ser Pro Lys Thr Val Tyr Val Lys Glu Ala Ile Asp
1330 1335 1340
Arg Arg Leu His Phe Lys Val Glu Val Lys Pro Ala Ser Phe Phe Lys
1345 1350 1355 1360
Asn Pro His Asn Asp Met Leu Asn Val Asn Leu Ala Lys Thr Asn Asp
1365 1370 1375
Ala Ile Lys Asp Met Ser Cys Val Asp Leu Ile Met Asp Gly His Asn
1380 1385 1390
Val Ser Leu Met Asp Leu Leu Ser Ser Leu Val Met Thr Val Glu Ile
1395 1400 1405
Arg Lys Gln Asn Met Thr Glu Phe Met Glu Leu Trp Ser Gln Gly Ile
1410 1415 1420
Ser Asp Asp Asp Asn Asp Ser Ala Val Ala Glu Phe Phe Gln Ser Phe
1425 1430 1435 1440
Pro Ser Gly Glu Pro Ser Asn Ser Lys Leu Ser Gly Phe Phe Gln Ser
1445 1450 1455
Val Thr Asn His Lys Trp Val Ala Val Gly Ala Ala Val Gly Val Leu
1460 1465 1470
Gly Val Leu Val Gly Gly Trp Phe Val Tyr Lys His Phe Ser Arg Lys
1475 1480 1485
Glu Glu Glu Pro Ile Pro Ala Glu Gly Val Tyr Tyr Gly Val Thr Lys
1490 1495 1500
Pro Lys Gln Val Ile Lys Leu Asp Ala Asp Pro Val Glu Ser Gln Ser
1505 1510 1515 1520
Thr Leu Glu Ile Ala Gly Leu Val Arg Lys Asn Leu Val Gln Phe Gly
1525 1530 1535
Val Gly Glu Lys Asn Gly Cys Val Arg Trp Val Met Asn Ala Leu Gly
1540 1545 1550
Val Lys Asp Asp Trp Leu Leu Val Pro Ser His Ala Tyr Lys Phe Glu
1555 1560 1565
Lys Asp Tyr Glu Met Met Glu Phe Tyr Phe Asn Arg Gly Gly Thr Tyr
1570 1575 1580
Tyr Ser Ile Ser Ala Gly Asn Val Val Ile Gln Ser Leu Asp Val Gly
1585 1590 1595 1600
Phe Gln Asp Val Val Leu Met Lys Val Pro Thr Ile Pro Lys Phe Arg
1605 1610 1615
Asp Ile Thr Gln His Phe Ile Lys Lys Gly Asp Val Pro Arg Ala Leu
1620 1625 1630
Asn Arg Leu Ala Thr Leu Val Thr Thr Val Asn Gly Thr Pro Met Leu
1635 1640 1645
Ile Ser Glu Gly Pro Leu Lys Met Glu Glu Lys Ala Thr Tyr Val His
1650 1655 1660
Lys Lys Asn Asp Gly Thr Ser Val Asp Leu Thr Val Asp Gln Ala Trp
1665 1670 1675 1680
Arg Gly Lys Gly Glu Gly Leu Pro Gly Met Cys Gly Gly Ala Leu Val
1685 1690 1695
Ser Ser Asn Gln Ser Ile Gln Asn Ala Ile Leu Gly Ile His Val Ala
1700 1705 1710
Gly Gly Asn Ser Ile Leu Val Ala Lys Leu Val Thr Gln Glu Met Phe
1715 1720 1725
Gln Asn Ile Asp Lys Lys Ile Glu Ser Gln Arg Ile Met Lys Val Glu
1730 1735 1740
Phe Thr Gln Cys Ser Met Asn Val Val Ser Lys Thr Leu Phe Arg Lys
1745 1750 1755 1760
Ser Pro Ile Tyr His His Ile Asp Lys Thr Met Ile Asn Phe Pro Ala
1765 1770 1775
Ala Met Pro Phe Ser Lys Ala Glu Ile Asp Pro Met Ala Val Met Leu
1780 1785 1790
Ser Lys Tyr Ser Leu Pro Ile Val Glu Glu Pro Glu Asn Tyr Lys Glu
1795 1800 1805
Ala Ser Ile Phe Tyr Gln Asn Lys Ile Val Gly Lys Thr Gln Leu Val
1810 1815 1820
Asp Asp Phe Leu Asp Leu Asp Met Ala Ile Thr Gly Ala Pro Gly Ile
1825 1830 1835 1840
Asp Ala Ile Asn Met Asp Ser Ser Pro Gly Phe Pro Tyr Val Gln Glu
1845 1850 1855
Lys Leu Thr Lys Arg Asp Leu Ile Trp Leu Asp Glu Asn Gly Leu Leu
1860 1865 1870
Leu Gly Val His Pro Arg Leu Ala Gln Arg Ile Leu Phe Asn Thr Val
1875 1880 1885
Met Met Glu Asn Cys Ser Asp Leu Asp Val Val Phe Thr Thr Cys Pro
1890 1895 1900
Lys Asp Glu Leu Arg Pro Leu Glu Lys Val Leu Glu Ser Lys Thr Arg
1905 1910 1915 1920
Ala Ile Asp Ala Cys Pro Leu Asp Tyr Thr Ile Leu Cys Arg Met Tyr
1925 1930 1935
Trp Gly Pro Ala Ile Ser Tyr Phe His Leu Asn Pro Gly Phe His Thr
1940 1945 1950
Gly Val Ala Ile Gly Ile Asp Pro Asp Arg Gln Trp Asp Glu Leu Phe
1955 1960 1965
Lys Thr Met Ile Arg Phe Gly Asp Val Gly Leu Asp Leu Asp Phe Ser
1970 1975 1980
Ala Phe Asp Ala Ser Leu Ser Pro Phe Met Ile Arg Glu Ala Gly Arg
1985 1990 1995 2000
Ile Met Ser Glu Leu Ser Gly Thr Pro Ser His Phe Gly Thr Ala Leu
2005 2010 2015
Ile Asn Thr Ile Ile Tyr Ser Lys His Leu Leu Tyr Asn Cys Cys Tyr
2020 2025 2030
His Val Cys Gly Ser Met Pro Ser Gly Ser Pro Cys Thr Ala Leu Leu
2035 2040 2045
Asn Ser Ile Ile Asn Asn Val Asn Leu Tyr Tyr Val Phe Ser Lys Ile
2050 2055 2060
Phe Gly Lys Ser Pro Val Phe Phe Cys Gln Ala Leu Lys Ile Leu Cys
2065 2070 2075 2080
Tyr Gly Asp Asp Val Leu Ile Val Phe Ser Arg Asp Val Gln Ile Asp
2085 2090 2095
Asn Leu Asp Leu Ile Gly Gln Lys Ile Val Asp Glu Phe Lys Lys Leu
2100 2105 2110
Gly Met Thr Ala Thr Ser Ala Asp Lys Asn Val Pro Gln Leu Lys Pro
2115 2120 2125
Val Ser Glu Leu Thr Phe Leu Lys Arg Ser Phe Asn Leu Val Glu Asp
2130 2135 2140
Arg Ile Arg Pro Ala Ile Ser Glu Lys Thr Ile Trp Ser Leu Ile Ala
2145 2150 2155 2160
Trp Gln Arg Ser Asn Ala Glu Phe Glu Gln Asn Leu Glu Ile Ala Gln
2165 2170 2175
Trp Phe Ala Phe Met His Gly Tyr Glu Phe Tyr Gln Lys Phe Tyr Tyr
2180 2185 2190
Phe Val Gln Ser Cys Leu Glu Lys Glu Met Ile Glu Tyr Arg Leu Lys
2195 2200 2205
Ser Tyr Asp Trp Trp Arg Met Arg Phe Tyr Asp Gln Cys Phe Ile Cys
2210 2215 2220
Asp Leu Ser
2225






Claims
  • 1. A hepatitis A virus HAV 4380 having Institute Pasteur Accession No. I-936, where said virus is characterized by the ability to grow in MRC-5 cells.
  • 2. A vaccine composition useful for protecting humans or primates against hepatitis A, said vaccine comprising the hepatitis A virus of claim 1.
  • 3. A method for protecting a human or primate against hepatitis A, said method comprising administering the vaccine of claim 1 to said human or primate in an amount effective to protect said human or primate against hepatitis A.
  • 4. A hepatitis A virus encoded by a nucleic acid molecule having the nucleic acid sequence according to SEQ ID NO:3.
  • 5. A hepatitis A vaccine comprising the virus of claim 4.
  • 6. A method for protecting a human or primate against hepatitis A, said method comprising administering the vaccine of claim 5 to said human or primate in an amount effective to protect said human or primate against hepatitis A.
CROSS-REFERENCE TO RELATED APPLICATIONS

This is a 371 national phase filing of PCT/US93/08610, filed Sep. 17, 1993, which was a continuation-in-part of U.S. patent application Ser. No. 07/947,338, filed Sep. 18, 1992, now abandoned.

Government Interests

This invention was made with government support under certain Collaborative Research and Development Agreements awarded by the Department of Health and Human Services. The government has certain rights in this invention.

PCT Information
Filing Document Filing Date Country Kind 102e Date 371c Date
PCT/US93/08610 WO 00 4/17/1995 4/17/1995
Publishing Document Publishing Date Country Kind
WO94/06446 3/31/1994 WO A
US Referenced Citations (5)
Number Name Date Kind
4532215 Daemer et al. Jul 1985
4620978 Daemer et al. Nov 1986
4636469 Daemer et al. Jan 1987
4783407 Provost et al. Nov 1988
4894228 Purcell et al. Jan 1990
Foreign Referenced Citations (4)
Number Date Country
0323900 Jul 1989 EP
2398504 Mar 1979 FR
WOA9219268 Nov 1992 WO
WO9309139 May 1993 WO
Non-Patent Literature Citations (37)
Entry
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Accession M16632, GenEmbl database, Aug. 1987.
Provost EL., J. Med. Virol., 34(4): 227-231, 1991. (Ref BS).
Melnick JL., Prog. Med. Virol. Basel, Krager, 37:47-55, 1990 (Ref AAS).
Fineschi et al. J. Hepatol. 13(4):5146-5151, Apr. 1991 (Ref AU).
Cohen et al. J. Virol., 61(10): 3035-3039, Oct. 1987. (Ref BR).
B. Ross et al, “Nucleotide Sequence of High-Passage Hepatitis A Virus Strain HM175: Comparison with Wild-type and Cell Culture-adapted Strains”, J. Gen. Virol., 70:2805-2810 (Oct., 1989).
R. Jansen et al, “Complete Nucleotide Sequence of a Cell Culture-Adapted Variant of Hepatitis A Virus: Comparison with Wild-Type Virus with Restricted Capacity for in Vitro Replication”, Virol., 163:299-307 (1988).
V. Tedeschi et al, “Partial Characterization of Hepatitis A Viruses from Three Intermediate Passage Levels of a Series Resulting in Adaptation to Growth in Cell Culture and Attenuation of Virulence”, J. Med., Virol., 39(1):16-21 (Jan. 1993).
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P. Provost et al, “New Findings in Live, Attenuated Hepatitis A Vaccine Development”, J. Med. Virol., 20:165-175 (1986) [Provost I].
P. Provost et al, “New Findings in Live, Attenuated Hepatitis A Vaccine Development”, J. Med. Virol., 20:165-175 (1986) [Provost I].
K. Midthun et al, “Safety and Immunogenicity of a Live Attenuated Hepatitis A Virus Vaccine in Seronegative Volunteers”, J. Infect. Dis., 163:735-739 (Apr., 1991).
J. Mao et al, “Primary Study of Attenuated Live Hepatitis A Vaccine (H2 Strain) in Humans”, J. Infect. Dis.,159(4):621-624 (Apr., 1989).
I. Gust et al, “The Origin of the HM175 Strain of Hepatitis A Virus”, J. Infect. Dis., 151(2):365-366 (Feb., 1985).
F. Andre et al, “Inactivated Candidate Vaccines for Hepatitis A”, Prog. Med. Virol. Basel, Karger, 37:72-95 (1990).
R. Daemer et al, “Propagation of Human Hepatitis A Virus in African Green Monkey Kidney Cell Culture: Primary Isolation and Serial Passage”, Infect. Immun.,32:388-393 (Apr., 1981).
J. Melnick, “New Picornavirus Vaccines for Hepatitis A, and Lessons from the Control of Poliomyelitis by the Prototype Picornavirus Vaccines”, Prog. Med. Virol. Basel, Karger, 37:47-55 (1990).
R. Karron et al, “Studies of Prototype Live Hepatitis A Virus Vaccines in Primate Models”, J. Infect. Dis.,157(2):338-345 (Feb., 1988).
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J. Cohen et al, “Complete Nucleotide Sequence of Wild-Type Hepatitis A Virus: Comparison with Different Strains of Hepatitis A Virus and Other Picornaviruses”, J. Virol.,61(1):50-59 (Jan., 1987) [Cohen I].
J. Cohen et al, “Complete Nucleotide Sequence of an Attenuated Hepatitis A Virus: Comparison with Wild-Type Virus”, Proc. Natl. Acad. Sci. USA, 84(8):2497-2501 (Apr., 1987) [Cohen II].
S. Lemon et al, “Serum Neutralizing Antibody Response to Hepatitis A Virus”, J. Infect. Dis., 148(6):1033-1039 (Dec., 1983).
R. Purcell et al, “A Microtiter Solid-Phase Radioimmunoassay for Hepatitis A Antigen and Antibody”, J. Immunol., 116(2):349-356 (Feb., 1976).
J. Ticehurst et al, “Detection of Hepatitis A Virus by Extraction of Viral RNA and Molecular Hybridization”, J. Clin. Microbiol., 25(10):1822-1829 (Oct., 1987).
J. Cohen et al, “Hepatitis A Virus cDNA and its RNA Transcripts are Infectious in Cell Culture”, J. Virol.,61(10):3035-3039 (Oct., 1987) [Cohen III].
P. Provost et al, “Further Evaluation of a Live Hepatitis A Vaccine in Marmosets”, J. Med. Virol., 34(4):227-231 (Aug., 1991) [Provost II].
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Continuation in Parts (1)
Number Date Country
Parent 07/947338 Sep 1992 US
Child 08/397232 US