Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1998-06-16
2002-02-05
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C424S093600, C424S206100, C424S209100, C435S236000, C435S239000
Reexamination Certificate
active
06344354
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention, in the fields of virology and vaccine production, relates to replicated mammalian influenza viruses, grown in mammalian cell culture, which are suitable for use in mammalian influenza virus vaccine production. The replicated viruses are obtained from high growth strains of (i) reassortants between high growth master donor strains and clinical isolates, or (ii) passaged clinical isolates. The infected cell culture uses a low concentration range of trypsin (0.05-1.0 &mgr;g/ml), continuously present in the medium, to provide high titers of the replicated virus. The invention also relates to methods for making and using such replicated viruses, such as for vaccine compositions and for vaccination methods.
2. Related Art
For the past several decades, fertilized chicken eggs have been used as a host system to replicate human influenza viruses with infectivity titers sufficient for use in vaccine production. Clinical isolates of human influenza virus are taken from infected patients and are reasserted in embryonated chicken eggs with laboratory-adapted master strains of high-growth donor viruses. The purpose of this reassortment is to increase the yield of candidate vaccine strains achieved by recombining at least the HA and NA genes from the primary clinical isolate isolates, with the internal genes of the master strain donor viruses. The high growth reassortant vaccine strains must also not contaminated with genes coding for antigenic determinants of the laboratory adapted viruses. This provides high growth reassortants having antigenic determinants similar to those of the clinical isolates. (Robertson et al.,
Biologicals
20:213-220 (1992)). The reassorted influenza virus is then grown in embryonated chicken eggs, purified from virus-containing allantoic fluid of the eggs and subsequently inactivated for use as vaccines.
However, a large body of data now suggests that this is a problematic system because of the frequency of viral mutation in antigenic sites of the major virus glycoprotein, hemagglutinin (HA), during replication in the chicken eggs. Even a single passage of a human influenza virus isolate or reassortant in chicken eggs leads to the selection of viral variants that differ in their antigenic determinants from those of the original clinical isolates. For example, the cultivation of influenza A and B viruses in chicken eggs often leads to the selection by the host system of variants having antigenic and structural changes in the viral HA molecule, making the variants ineffective or significantly less effective when used in an influenza vaccine (Kodihalli et al.,
J. Virol
. 69:4888-4897 (1995); Gubareva et al.,
Virol
. 199:89-97 (1994); Katz & Webster,
J. Infect. Dis
. 160:191-198 (1989); Wood et al.,
Virol
. 171:214-221 (1989); Katz et al.,
Virology
156:386-395 (1987); Robertson et al.,
Virology
143:166-174 (1985)). In addition, the replicative properties of egg-grown viruses are not as consistent with natural infection as those of viruses grown in mammalian cells (Katz et al.,
J. Virol
. 64:1808-1811 (1990); Robertson et al.,
Virology
179:35-40 (1990)).
Additionally, embryonated chicken eggs have potentially serious limitations as a host system, e.g., due to the lack of reliable year-around supplies of high-quality eggs and the low susceptibility of summer eggs to influenza virus infection (Monto, et al.,
J. Clin. Microb
. 13:233-235 (1981)). Furthermore, the presence of adventitious agents in eggs can jeopardize the preparation of live-attenuated influenza virus vaccines. Adventitious agents are infectious contaminants (such as other viruses) in host systems that make them unsuitable or uncertifiable for use in vaccine production.
Cultured mammalian cells have also been used for virus replication and have been classified into at least two distinct groups. Primary diploid cells are those derived from intact tissue and have not been subcultivated. Continuous cell lines (CCLs) are cultured primary cells that replicate indefinitely and may be capable of growth in suspension culture. Haylick, in
Continuous Cell Lines as Substrates for Biologicals
, Arlington, Va., p. 2 (1988).
At present, many viral vaccines other than influenza are produced using primary trypsinized cells, including cells from monkey kidneys, and the kidneys of rabbits and hamsters. Primary diploid cell cultures have certain advantages such as easy preparation using simple media and bovine sera and sensitivity to a wide-range multiple viruses. However, primary diploid cells suffer from disadvantages, such as contamination by various adventitous agents, variable quality and sensitivity; and difficulty in obtaining suitable tissue for cultivation (e.g., monkey kidneys).
For example, primary diploid cell cultures obtained from monkey kidneys of wild animals usually contain endogenous viruses (Grachev, In Burgasov; ed., “
Guidance for the Production of Vaccines and Sera
.” Medicine, Moscow, p 176 (1978)). The number of adventitous agents depends on many factors, such as the methods of isolation, the cell systems used, the number of passages, the time of incubation and co-cultivation. The frequency of isolation of viruses from primary diploid cell cultures of monkey kidneys is directly proportional to the incubation period of the cells. Grachev, In
Zh. Microbiol. Epidemiol. Immunobiol
. 2:76 (1987).
In contrast, the advantages of using continuous cell lines are their retention of original antigenic characteristics of the infected virus, standardization, high susceptibility to variants of the same virus, and ability to be grown as a large mass of cells using microcarrier or suspension fermentor systems.
However, these advantages themselves do make such cell lines suitable for use in vaccine production. Mizrahi, ed.,
Viral Vaccines
, Wiley-Liss, New York (1990), pp. 39-67. For example, influenza A viruses isolated and passaged exclusively in mammalian cell cultures have been found in some cases to retain most or all of their original antigenic characteristics, a feature that would prove highly advantageous in vaccine production. (Katz et al.,
Virology
165:446-456 (1988); Robertson et al.,
Virology
179:35-40 (1990); Katz et al.,
J. Infect. Dis
. 160:191-198 (1989); Wood et al.,
Virology
171:214-221 (1989)).
However, mammalian primary diploid cell cultures present difficulties as a host system for vaccine production. This is due to problems such as contamination of the cell culture with adventitious agents, variable quality of the cells in the cell culture, different sensitivities of the cells to variants of the same virus, low virus titers and the high cost and difficulties in obtaining and preparing such cell cultures. In another example, although human diploid (MRC-5) cells can support the growth of influenza viruses, such systems have stringent growth media requirements making them suboptimal for large-scale production of influenza viruses for use in vaccines.
Furthermore, only MDCK cells, among the continuous cell lines tested, have been reported to support potentially sufficient growth and isolation of viruses (Frank et al.,
J. Clin. Microb
. 10:32-36 (1979); Schepetink & Kok,
J. Virol. Methods
42:241-250 (1993)). However, this line has been found to produce tumors and has thus not been certified for vaccine production, as not substantially free of adventitious agents.
Two other continuous cell lines—African green monkey kidney (Vero) cells and baby hamster kidney (BK-21)—are characterized, approved and certified by the World Health Organization (WHO) for production of human vaccines. However, Vero cells, while certified, were previously found unsuitable for large-scale production of human influenza virus vaccines. For example, the growth of influenza B in Vero cells was greatly restricted as compared to MDCK cells (Nakamura et al.,
J. Gen. Virol
. 56:199-202 (1981)). Additionally, attempts to use Vero cells to evaluate the rimantadine sensitivity of human H1N1 and H3N2 influenza A viruses gave ambiguous
Kaverin Nicolai V.
Webster Robert G.
Lankford , Jr. Leon B.
St. Jude Children's Research Hospital
Sterne Kessler Goldstein & Fox P.L.L.C.
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