Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Patent
1992-07-31
1997-02-25
Mosher, Mary E.
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
4241991, 4242261, 536 2372, 4352351, 4353201, A61K 3929, A61K 39125, A61K 39285, C12N 701, C12N 1551
Patent
active
056056923
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the prevention of hepatitis and is particularly concerned with the production of new materials suitable for incorportion into a vaccine for the prevention of hepatitis A.
Infection with hepatitis A virus (HAV) remains an important cause of morbidity and occasionally mortality, both in countries where it is endemic and in the industrialised nations where it is normally sporadic. As sanitary conditions have been improving in developing countries, the prevelance of HAV seropositivity has been decreasing so creating a large pool of susceptible individuals. These individuals together with high risk groups such as day-care centre staff, parents and siblings of toddlers attending such centres, promiscuous homosexual men and military personnel and tourists in endemic areas would benefit from an effective HAV vaccine.
Hepatitis A virus is classified as a picornavirus with a single strand positive sense RNA genome coding for a single polyprotein which is subsequently processed into structural and nonstructural proteins. The structural proteins are divided into polypeptides, for example VP1, VP2, VP3 and VP4 which form the capsid polypeptides of the virus. There appears to be only one serotype and significant antigenic variation has not been recognised among different HAV strains.
Replication of the virus in tissue culture is slow and yields are poor, thus making the large scale production for vaccines difficult and expensive. Formalin inactivated (Provost et al, J. Med. Virol., 1986, 19, 23-31. Binn et al., J. Infect. DIS, 1986, 153, 749-756.) and live attenuated virus vaccines (Provost et al., Proc. Soc. Exp. Biol. Med, 1982, 170, 8-14. Karron et al., J. IneCt, Dis, 1988, 157, 338-345.) have been produced and shown to be protective both in primates and human volunteers. Problems of large scale production and stability of these vaccines in man need to be overcome and the safety and durability of immunity still have to be established. Moreover, one disadvantage of these vaccines is that 3 doses of vaccine are required to produce an adequate anti-HAV response.
A candidate sub-unit vaccine based on the VP1 structural polypeptide has been produced by recombinant DNA techniques in E. coli and used to immunize rabbits (Johnston et al., J. Infect. Dis, 1988, 157(6), 1203-1211). The resulting antibody reacted only with denatured VP1 and not with intact HAV, indicating that the conformational epitope had not been expressed by the E. Coli.
It has now been found that a valuable vaccine material can be produced by incorporating at least part of the HAV genome in a vaccinia virus, using recombinant DNA technology. The polypeptide expressed by the vaccinia virus is capable of evoking a protective immune response following only a single injection of the material.
According to one aspect of the invention there is provided an isolated polypeptide comprising a substantially antigenic part of at least one HAV epitope, free from infectious material.
The isolated HAV polypeptide may comprise all or part of VP1, either alone or together with VP3, or all or part of VP3 alone. Alternatively, the polypeptide may comprise all or part of VP1 together with VP4, VP2 and VP3, or at least two HAV structural proteins selected from VP1 to VP4 inclusive.
According to a second aspect of the invention there is provided a DNA molecule comprising a nucleotide sequence substantially coding for all or a portion of at least one HAV structural polypeptide.
The nucleotide sequence of the DNA molecule may code for, all or a part of HAV VP1, either alone or together with VP3, or all or part of VP3 alone. Alternatively the nucleotide sequence may code for all or part of VP1 together with VP4VP2 and VP3, or at least two HAV structural polypeptides selected from VP1 to VP4 inclusive.
The DNA molecule may also comprise a viral promotor operatively linked to the nucleotide sequence.
In yet a further aspect of the invention there is provided a virus genetically engineered to express all or a part of at least one HAV structural polypeptide.
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Karayiannis Peter
Thomas Howard C.
Imperial College of Science Technology and Medicine
Irons Edward S.
Mosher Mary E.
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