Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2000-08-31
2002-07-23
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C435S236000, C435S237000, C435S235100, C424S093100, C536S023700
Reexamination Certificate
active
06423318
ABSTRACT:
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 disadvantages, 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 infection 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 vaccinee 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 wild-type HAV, designated HM-175 [see
FIG. 6
below; also SEQ ID NOS: 1 and 2], 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 the HAV strain, HM-175, Pass 35, passaged 35 times in AGMK, 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 clone 7 of the HAV, designated HM-175/7 or pHAV/7. See
FIG. 6
below; the nucleotide changes in pHAV/7 from wt HM-175 appear above the wt sequence; the amino acid changes in pHAV/7 from wt HM-175 appear below the wt amino acid. 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.,
39:16-22 (1993). The disclosures 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 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
73
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 also 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 or humans, without inactivation. The HAV may be characterized by containing one or more of sixteen specific nucleotides which differ from nucleotides in the same position in the genome of HAV HM-175, Pass 35 [SEQ ID NO: 3].
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 thirteen specific nucleotides. The HAV genome so modified is preferably HAV HM-175, Pass 35 [SEQ ID NO: 3] 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.
REFERENCES:
patent: 4532215 (1985-07-01), Daemer et al.
patent: 4620978 (1986-11-01), Daemer et al.
patent: 4636469 (1987-01-01), Daemer et al.
patent: 4894228 (1990-01-01), Purcell et al.
patent: A 0323900 (1989-07-01), None
patent: 2398504 (1978-03-01), None
patent: WO A9219268 (1992-11-01),
D'Hondt Eric
Emerson Suzanne U.
Funkhouser Ann W.
Purcell Robert H.
Li Bao-Qun
Morgan & Finnegan L.L.P.
Salimi Ali R.
The United States of America as represented by the Department of
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