Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant or stably-transformed bacterium encoding one or...
Reexamination Certificate
1998-07-24
2002-06-04
Schwartzman, Robert A. (Department: 1632)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant or stably-transformed bacterium encoding one or...
C424S093200, C424S184100, C435S252100, C435S252300, C435S252800, C435S320100
Reexamination Certificate
active
06399074
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates generally to vaccines for poultry and other birds and, more particularly, to vaccines for protecting poultry and other birds against infection by avian pathogenic gram-negative bacteria.
DESCRIPTION OF THE RELATED ART
Avian pathogenic
E. coli
(APEC) strains cause a number of related diseases in poultry and other birds, including air sacculitis, cellulitis, colibacillosis, coligranuloma, colisepticemia, Hjarre's disease, omphalitis, peritonitis, salpingitis, synovitis (Gross, W. B. in
Diseases of Poultry
, Calnek et al., eds., Iowa State University Press, p. 138-144, 1991; Messier et al.,
Avian Diseases
37:839-844, 1993). These diseases and other diseases caused by gram-negative avian pathogens can lead to increased rates of feed conversion carcass condemnation, or death of the animal, resulting in millions of dollars lost to the poultry industry each year. Norton, R. A.
Broiler Industry
, February 1998, pp. 28-32.
Contamination of poultry products by Salmonella is a significant source of Salmonella infection in humans, which causes gastroenteritis, and thus is a major public health concern. Oral administration of live cells from
S. typhimurium
strains having attenuating deletions in the cya and crp genes has been shown to provide excellent protection against wild-type Salmonella challenge in chickens (Hassan et al.,
Res. Microbiol.
141:839-850, 1990; Hassan et al.,
Infect. Immun.
62:5519-5527, 1994) and systems for stable expression of heterologous antigens in such strains have been developed (Hone et al.,
Microb. Pathog.
5:407-418, 1988; Strugnell, et al., Gene 88:57-63, 1990; Galan et al., Gene 94:29-35; 1990; Nakayama et al.,
Bio/Technology
6:693-697, 1995).
APEC strains are represented primarily by only a few serotypes, O1, O2, O35 and O78 (Cloud et al.,
Avian Dis.
29:1084-1093, 1985, Glantz et al.,
Avian Dis.
6:322-328, 1962; Gross, supra), while Salmonella serotypes most prevalent in poultry are in the B, C and D groups. O-serotypes of gram-negative bacteria such as
E. coli
and Salmonella are determined at the molecular level by the so-called O-antigen structure, also termed O-specific chain or O-polysaccharide (O-PS), which generally is comprised of varying lengths of polymerized identical sugar units anchored in the bacterial outer membrane as the outermost component of lipopolysaccharide (LPS) molecules (Helander et al., in
Molecular Biology and Biotechnology: A Comprehensive Desk Reference
, R. A. Meyers, ed., VCH Publishers, Inc., 1995).
Specific antibody against LPS O-antigen has been shown to be protective in mammalian models of extraintestinal
E. coli
infections in humans (Cryz et al.,
Vaccine
13;449-453, 1995; Pluschke et al.,
Infect. Immun.
49:365-370, 1985) and LPS O-antigen has been recognized as a protective antigen for other gram negative pathogens (Ding et al.,
J. Med. Microb.
31:95-102, 1990; Michetti et al.,
Infect. Immun.
60:1786-1792, 1992; Robbins et al.,
Clin. Infect Dis.
15:346-361, 1992). In addition, several research groups have reported using attenuated Salmonella and Salmonella-
E. coli
hybrids as vaccine delivery vehicles for O-antigens of several human pathogens, including
Shigella sonnei, Vibrio cholerae
, and
Pseudomonas aeruginosa.
(13lack et al.,
J. Infect. Dis.
155:1260-1265, 1987; Formal et al.,
Infect. Immun.
34:746-750, 1981; Pier et al.,
Infect. Immun.
63:2818-2825, 1995); (Morona et al., U.S. Pat. No. 5,110,588). However, until the work described herein, immunization of poultry with live, attenuated Salmonella expressing an APEC O-antigen had not been reported.
LPS O-antigen made by
E. coli
and Salmonella bacteria is comprised of lipid A, an R-core oligosaccharide, and the O-specific polysaccharide (O-PS), which are covalently linked in that order. Sugiyama et al.,
J. Bacteriol.
173:55-58, 1991. In
S. typhimurium
, synthesis of the R-core moiety is directed by the rfa locus and certain housekeeping genes such as galE, galU, and pgi, while O-PS synthesis is directed by the rfb gene cluster, which encodes enzymes involved in biosynthesis of the monomer sugar unit, and the rfc gene, which encodes the O-antigen polymerase responsible for the polymerization of the sugar unit into a high molecular weight polysaccharide chain. Sugiyama et al., supra.
One group investigating the genes required for synthesis of LPS O-antigen in
E. coli
O9 introduced a plasmid containing the rfb locus from
E. coli
O9 into
S. typhimurium
wild-type and mutant strains with defects in the rfb, rfc, or rfe loci and reported that the wild-type strain containing the plasmid expressed LPS specific for both
E. coli
O9 and
S. typhimurium
on the cell surface, while the rfc mutant was expressed only O9-specific LPS.
E. coli
O-antigen was also synthesized in the
S. typhimurium
rfb mutant but not in the rfe mutant. Sugiyama et al., supra. This group concluded that gene products of the
S. typhimurium
rfa and
E. coli
O9 rfb loci can cooperate to synthesize
E. coli
O9-antigen on the R-core of
S. typhimurium.
However, this group did not report whether any of these recombinant
S. typhimurium
constructs could grow within an animal host or generate a protective host immune response against wild-type
E. coli
O9 or
S. typhimurium
. Accordingly, a need exists for a bivalent vaccine to control Salmonella and
E. coli
infection in poultry. Such a vaccine would simultaneously benefit the public health and reduce the costs of poultry production.
SUMMARY OF THE INVENTION
In one embodiment the present invention is directed to a vaccine that protects birds against infection by an avian pathogenic gram-negative (AP
G-N
) microbe. The vaccine comprises live cells of a recombinant Salmonella strain expressing O-antigen of the AP
G-N
microbe due to integration into the Salmonella chromosome of the rfb gene cluster and the rfc gene of the AP
G-N
(hereinafter used interchangeably with AP
G-N
rfb/rfc gene cluster). The recombinant Salmonella strain, which is an attenuated mutant of a virulent Salmonella strain, does not express O-antigen of the virulent Salmonella strain due to a mutation in the Salmonella rfb gene cluster and/or in the Salmonella rfc gene. In a preferred embodiment, the AP
G-N
microbe is an APEC strain and the AP
G-N
rfb/rfc gene cluster is an APEC rfb/rfc gene cluster.
This recombinant Salmonella strain has other features that make it particularly useful as a vaccine in poultry and other birds. First, the vaccine can be formulated for oral administration and oral vaccines are known to stimulate the gut associated lymphoid tissue (GALT), including mucosal, humoral and cellular immune responses. Oral, live vaccines also cost less to produce and are easier to administer in the field than injectable vaccines. Second, the lack of expression of LPS O-antigen specific for the carrier strain avoids any interference that the carrier LPS O-antigen might have on expression of the AP
G-N
O-antigen or its recognition by the vaccine recipient's immune system. Third, the recombinant Salmonella strain can protect against both AP
G-N
microbes and the parental Salmonella strain because it expresses other cell-surface antigens of the parental Salmonella strain. In addition, for vaccines expressing O-antigen from an APEC strain, use of Salmonella rather than
E. coli
as the carrier bacteria should provide a more vigorous immune response against the APEC O-antigen because while
S. enterica
subspecies persist in the spleen and bursa of Fabricius,
E. coli
does not effectively invade these lymphoid tissues or is quickly killed even if occasionally successful in entering them.
In some embodiments, the recombinant Salmonella strain used in the vaccine also contains a recombinant polynucleotide encoding a desired gene product. A preferred gene product is an antigen from an avian pathogenic gram-positive (AP
G-P
) microbe or from a eukaryotic avian pathogen.
In another embodiment, the invention provides a multivalent vaccine for immunizing birds against at
Beckerleg Anne Marie S.
Howell & Haferkamp
Megan Health, Inc.
Schwartzman Robert A.
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