Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1998-10-21
2003-12-02
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C424S213100, C424S184100
Reexamination Certificate
active
06656719
ABSTRACT:
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
Rotavirus Vaccines.
BACKGROUND OF THE INVENTION
Rotaviruses are the most common cause of acute infantile gastroenteritis. Rotavirus-induced disease is estimated to cause one million deaths of children under age two worldwide each year (Bishop 1993; Desselberger 1993). In the United States alone, approximately 200,000 hospitalizations and 150 deaths of children in this age cohort are attributable to rotavirus infection annually (for review, see Bishop 1993; Desselberger 1993). Rotavirus infections are so prevalent that nearly all children are seropositive by age three (Bishop 1993; Madeley 1993).
Natural rotavirus infection induces both humoral and cell mediated immune responses (Offit et al. 1993). Repeated infections with other serotypes produce only mild symptoms, if any (Bishop 1993). This evidence, together with the prevalence of infection and severe consequences of rotavirus diease has motivated rotavirus vaccine development. Several candidate vaccines, including those composed of live attenuated human, simian or bovine strains, reassortants of human and simian or bovine strains or recombinant subunits are in preclinical or clinical development.
One such vaccine, a bovine-human reassortant rotavirus adapted to replicate in Vero cells, induced protective immunity to severe disease in over 85% of vaccinees in recent clinical trials (Clark et al. 1990; Christy et al. 1993). Exemplary rotavirus reassortants and combinations thereof and their use in vaccines are found in U.S. Pat. No. 5,626,851, May 6, 1997, and in U.S. Pat. No. 5,750,109, May 12, 1998, both of which are incorporated herein by reference in their entireties.
Vero cells, a continuous African Green Monkey Kidney cell line, (Swanson et al. 1988) have been known and used in the art for the production of human viral vaccines including poliovirus and rabies virus for many years (Montagnon et al. 1981; Suntharasami et al. 1986; Montagnon 1989). The cells are well-characterized and have an excellent safety record. These cells can be propagated in static culture and in suspension culture as cell aggregates (Litwin 1992; Perusich et al. 1991) or on microcarriers (Clark and Hirtenstein 1981); the latter processes being more readily scalable.
Rotaviruses require a tryptic cleavage of one of the two major outer coat proteins, VP4, to efficiently infect Vero cells in vitro (Estes et al., 1979; Estes and Cohen 1989; Konno et al., 1993; Patton et al., 1993). Previously, the production of reassortant rotaviruses required that Vero cells first be grown in serum-containing medium, then washed to remove serum proteins that otherwise reduce infectivity, and finally infected in serum-free basal medium containing trypsin. To simplify the virus production process and to avoid problems associated with serum-containing processes (e.g. the risk of introducing adventitious agents and the potential for lot-to-lot variability), we developed defined, serum-free, low protein media that are devoid of animal-sourced proteins. These media, called LPKM-1, LPKM-2 and LPKM-3, support the growth of Vero cells in static and microcarrier cultures and also support the production of rotaviruses to levels approaching that of serum-containing media.
SUMMARY OF THE INVENTION
Defined serum-free, low protein media provided herein, supports 1) Vero cell growth for up to 20 passages, 2) Vero cell growth on microcarriers and 3) rotavirus production. We refer to media as LPKM. Maximum cell densities attained are 60-100% of that in serum-containing medium and the mean doubling time is equal to serum containing medium. Rotavirus titers achieved in LPKM-1 are 80-100% of the serum-containing microcarrier process. Since LPKM media contain no animal-sourced proteins, the problems associated with the serum-containing rotavirus production process (i.e. lengthy wash steps before infection, potential introduction of adventitious agents and lot-to-lot variability of serum) are avoided while maintaining nearly equivalent product titers.
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Distefano Daniel J.
Gould Sandra L.
Robinson David K.
Seamans T. Craig
Merck & Co. , Inc.
Smith Lynette R. F.
Tribble Jack L.
Yablonsky Michael D.
Zeman Robert A.
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