Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
1998-06-15
2003-09-02
Devi, S. (Department: 1645)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S184100, C424S261100, C424S258100, C424S257100, C424S234100, C424S200100
Reexamination Certificate
active
06613321
ABSTRACT:
The present invention relates to live attenuated gram-negative vaccine carrier strains which are useful for expression and delivery of heterologous O-antigens (O-PS) from gram-negative pathogens. Said strains are deficient in the expression of homologous O-PS due to a defined genetic modification, preferably a deletion, and, thus, capable of efficiently expressing a desired heterologous O-PS in such a way that it is covalently coupled either to homologous or heterologous LPS core lipid A. The present invention furthermore relates to live vaccine carrier strains containing a heterologous gene or a set of heterologous genes encoding O-PS. Preferably, said strains additionally contain genes necessary for the synthesis of complete smooth heterologous LPS. The present invention also relates to live vaccines comprising said strains, preferably for immunization against gram-negative enteric pathogens.
BACKGROUND OF THE INVENTION
Gram-negative enteric pathogens are the cause of a variety of diseases presenting with a broad spectrum of symptoms ranging from mild watery diarrhea to severe life-threatening symptoms such as fever, bloody diarrhea, perforation or ulceration of the stomach or intestine, alone or in combination. Examples of such diseases include typhoid fever, shigellosis, cholera, infections with enterotoxinogenic, enteropathogenic, and enterohemorragic
Escherichia coli
, and infections with
Heliobacter pylori
and
Campylobacter jejuni.
The first stage of the infectious process occurs at the mucosal surface within the digestive tract. Thus, interfering with this initial stage of infection prior to the onset of symptoms offers a particularly attractive approach. The most effective means by which to accomplish this would be to evoke a local protective immune response through the use of an orally administered vaccine (Mestecky, J. Clin. Immunol. 7 (1987), 265-276; McGhee and Kiyono, Infect. Agents Dis. 2 (1993), 55-73; Walker, Vaccine 12 (1994), 387-400). At present, 2 live oral attenuated vaccines against enteric disease have been licensed for human use these being the Ty21a strain of
Salmonella typhi
for the prevention of typhoid fever and the CVD103-HgR strain of
Vibrio cholerae
for the prevention of cholera (Germanier and Furer, J.Infect.Dis, 131 (1975), 553-558; Levine et al., Lancet ii (1998), 467-470).
There exists a large body of evidence indicating that protection against several enteric pathogens, such as
S. typhi, E. coli
, and Shigella species is associated with the induction of an immune response against cell surface components, specifically the O-antigen moiety of LPS, commonly referred to as O-polysaccharide (O-PS). For example, immunity to shigellosis, subsequent to recovery from either naturally-acquired or experimentally-induced disease is correlated with a substantial rise in serum serotype-specific anti-LPS antibodies (DuPont et al., J.Infect.Dis. 125 (1972), 5-11; DuPont et al., J.Infect.Dis. 12 (1972), 12-16; Herrington et al., Vaccine 8 (1990), 353-357). Furthermore, epidemiological studies have also found that protection against Shigella infections in the field was associated with increased levels of serum anti-LPS antibodies (Cohen et al. , J.Infect.Dis. 157 (1988), 1068-1071). High levels of serum antibodies against Shigella LPS can be detected among individuals residing in areas where such species of Shigella are endemic, presumably acquired by natural exposure and/or infection with these pathogens.
LPS is an essential constituent of the gram-negative outer membrane and may account for up to 70% of the cell surface components. LPS is composed of 3 regions: the innermost being lipid A which is embedded into the phospholipid outer membrane bilayer. The core polysaccharide is attached to the lipid A moiety usually via 2-keto,3-deoxyoctonate (KDO). The core is usually comprised of 5 to 7 sugars. To date, 7 types of core molecules have been identified within the Enterobacteriaceae family and have been named Ra, R1, R2, R3, R4, K-12, and B. Compared with the Enterobacteriaceae,
V. cholerae
possess an unusual core structure in that it contains fructose and a single KDO molecule in the inner core (Kondo et al., Carbohydrate Res. 231 (1992), 55-64). The biosynthesis of the LPS core is encoded by the rfa locus. Among the Enterobacteriaceae, the rfa and rfb loci appear to be unlinked. In contrast, some evidence exists to suggest a close linkage of at least part of these two loci for
V. cholerae
(Manning et al., p. 77-94. In
Vibrio cholerae
and
Cholera
: molecular to global perspectives (1994). Wachsmuth K., Blake, P. A., and Olsvik Y. (eds.). Washington, D.C.: American Society for Microbiology).
The outermost portion of the LPS molecule is composed of the O-PS which consists of repeating saccharide units of variable length (Luderitz et al., Curr. Top. Membr. Trans. 17 (1982), 79-151; Raetz, Annu. Rev. Biochem. 59 (1990) 129-170). The O-PS region of the LPS molecule confers serospecificity to the bacteria. The LPS molecule interacts closely with other molecules expressed on the outer membrane surface such as porins and other outer membrane proteins (OMP), which determine the permeability of the outer membrane. It is known that the assembly of OMP as well as secretion of proteins from the cell is affected by mutations in the LPS of
E. coli
(Laird et al., J. Bacteriol. 176 (1994), 2259-2204; Stanley et al., Mol. Microbiol. 10 (1993) 781-787).
Serospecificity is conferred not only by the sugars present in the O-PS but also by their chemical linkage and sequence (Lüderitz et al., Curr. Top. Membr. Trans. 17 (1982), 79-151). Therefore, the O-PS is highly variable between gram-negative bacterial species whereas the core polysaccharide is relatively constant within a given species or genera (Lüderitz et al., Curr. Topics in Membranes and Transport 17 (1982), 79-151; Jansson et al., Eur.J.Biochem. 115 (1981), 571-577). For example, the genus Shigella includes a total of 47 known serotypes divided among the 4 predominant pathogenic species which are
S. dysenteriae
(subgroup A, 12 serotypes),
S. flexneri
(subgroup B, 13 serotypes)
S. boydii
(serogroup C, 18 serotypes) and
S. sonnei
(subgroup D; 1 serotype) (Ewing, In: Ewing W H, ed. Edwards and Ewing's identification of Enterobacteriaceae fourth edition. New York: Elsevier Sci. Publish. Comp. (1986), 135-172). For example, in
S. sonnei
, the O-PS consists of a repeated disaccharide unit with 2 unusual sugars, 2-amino-2-deoxy-L-alturonic acid linked to 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose by a 1,4 linkage (Kenne et al., Carbohydrate Res. 78 (1980), 119-126). In contrast, the O-PS of serotype 1 of
S. dysenteriae
(which is the most common cause of dysentery) is composed of repeating blocks of rhamnose-rhamnose-galactose-N-acetylglucosamine (Ewing and Lindberg, In: Bergan T. (ed) Methods in microbiology vol.14. , Academic Press, London, pp. 113-142). The O-PS of
V. cholerae
01 is comprised of 17-18 perosamine subunits each of which is acylated with 3-deoxy-L-glycero-tetronic acid. Quinovosamine has also been found in low concentrations but its location within the O-PS of
V. cholerae
01 is unknown (Redmond, FEBS Lett. 50 (1975), 147-149; Kenne et al., Carbohydrate Res. 100 (1982), 341-349).
The enzymes involved in the biosynthesis of enterobacterial O-PS are coded for by the rfb locus. In the case of Shigella species, an additional gene, termed rfc, encodes the O-PS polymerase which functions to polymerize the individual repeat units into chains of varying length. In most Shigella species, the rfb/rfc loci are located on the chromosome (Klena and Schnaitman, Microbiol. Rev. 57 (1993), 655-682). However, in some species of Shigella, all or part of the rfb locus is located on a plasmid episome (Maurelli and Sansonetti, Ann. Rev. Microbiol. 42 (1988), 127-150). An additional gene, termed rfe, which is involved in the synthesis of the enterobacterial common antigen (ECA) is also required for O-PS synthesis in Salmonella species of the O-antigen groups C1 and L (Kuhn et al., FEMS Mic
Cryz Stanley J.
Favre Didier
Viret Jean-Francois
Devi S.
Swiss Serum and Vaccine Institute Berne
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