Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant or stably-transformed bacterium encoding one or...
Reexamination Certificate
1999-05-18
2002-05-07
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant or stably-transformed bacterium encoding one or...
C424S093200, C424S258100, C435S471000, C435S252300, C435S252800, C435S897000
Reexamination Certificate
active
06383496
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to attenuated microbes and, more particularly, to novel attenuated bacteria having an RpoS
+
phenotype for use as vaccines and delivery vehicles for genes and gene products and to methods for their preparation. This invention is particularly applicable to Salmonella such as
Salmonella enterica
serotype Typhi (also referred to as
Salmonella typhi
).
2. Description of the Related Art
Live attenuated Salmonella strains can serve as delivery vehicles for recombinant antigens or other proteins. As antigen carriers, the recombinant Salmonella have been shown to be useful in live vaccines (For review see Curtiss et al. in Essentials of
Musocal Immunology
, Kagnoff and Kiyono, Eds., Academic Press, San Diego, 1996, pp. 599-611; Doggett and Brown, in
Mucosal Vaccines
, Kiyono et al., Eds., Academic Press, San Diego, 1996 pp 105-118; see also Hopkins et al.
Infect Immun
. 63:3279-3286, 1995; Srinavasin et al
Vaccines
95, R. N. Chanock et al., Eds., Cold Spring Harbor Laboratory Press, Plainview, N.Y., p 273-280, 1995).
Ideally, live attenuated vaccine strains should possess a balance between the two properties of attenuation and immunogenicity. Such vaccine strains would not cause any disease or impair normal host physiology or growth, thus being attenuated, and at the same time be able to colonize the intestine and gut associated lymphoid tissue upon oral administration or other lymphoid organs upon administration by some other route so as to be immunogenic. As a practical matter, however, such an ideal balance has not been achieved (Curtiss, in
New Generation Vaccines
Woodrow and Levine, Eds., Marcel Dekker, Inc., New York, 1990, pp. 161-188). This may be a result of the almost exclusive focusing of efforts in Salmonella vaccine development on improving the attenuation component of strains rather than on producing strains with high immunogenicity.
Work directed toward achieving attenuation in microbes for use in vaccines has utilized attenuating mutations in biosynthetic genes, regulatory genes and/or genes involved in virulence. (See Doggett and Brown, supra). One such regulatory gene which has been mutated as a means for achieving attenuation has been the rpoS gene. The rpoS gene encodes an alternative sigma factor, RpoS, which is known to regulate the stationary phase expression of over 30 genes (for review, see Loewen and Hengge-Aronis,
Annu Rev Microbiol
48:53-80, 1994). The rpoS gene has been shown to contribute to the virulence of Salmonella enterica serotype Typhimurium (also referred to as
Salmonella typhimurium
) in mice by RpoS regulation of chromosomal as well as plasmid-borne genes (Fang et al.,
Proc Natl Acad Sci
89:11978-11982, 1992; Norel et al.,
FEBS Microbiol Lett
99:271-276, 1992; Kowarz et al.,
J Bacteriol
176:6852-6860, 1994). Similarly, RpoS is thought to contribute to the virulence of
Salmonella typhi
in humans by an action on chromosomal gene determinants of virulence, inasmuch as these microbes do not possess the virulence plasmid present in
S. typhimurium
(Robbe-Saule et al., FEMS Microbiol Let 126:171-176, 1995; Coynault et al.,
Mol Microbiol
22:149-160, 1996). Mutant rpoS
S. typhimurium
strains have been shown to be attenuated (Fang et al, supra) and capable of eliciting protective immunity in mice (Nickerson and Curtiss,
Abstracts of the
96
th General Meeting of the American Society for Microbiology
B-141:179, 1996; Coynault et al.,
Mol Microbiol
22:149-160, 1996). As a result, it has been suggested that rpoS mutants may be attractive candidates for the development of vaccines (Nickerson and Curtiss, supra).
Attenuated strains of
Salmonella typhi
have been used as human vaccines against typhoid fever as well as against heterologous antigens when used as recombinant antigen delivery vehicles (Forrest, in CRC Press Inc., 1994, pp. 59-80; Levine et al, in
New Generation Vaccines
Woodrow and Levine, Eds., Marcel Dekker, Inc., New York, 1990, pp. 269-287). These vaccines based upon Typhi strains have almost exclusively been derived from the Ty2 strain, in particular, Ty21a, which contains a galE mutation along with other mutations. Ty2 and its Ty21a derivative vaccine strain have been shown to be rpoS mutants and this mutation may account, at least in part, for the attenuation seen with Ty21a and with other vaccine strains derived from Ty2 presumably by the down regulation of chromosomal virulence genes controlled by the rpoS gene product. The Ty21a vaccine is typical of vaccines derived from Ty2 in that although being attenuated, the Ty21a vaccine has proven to have low vaccine efficacy, requiring three high doses of approximately 10
10
cfu to induce protective immunity in approximately two-thirds of the vaccinated individuals. (Forrest, supra). Thus, there remains a continuing need for Salmonella typhi strains which exhibit not only low virulence, but, also high immunogenicity for use in vaccines suitable for the delivery of a desired gene product to a host.
Other strains of
S. typhi
have been reported which may, however, have a functional rpoS gene although this was not appreciated at the time of the report. For example, human vaccines have been reported based upon the 27V and ISP1820 strains (Reitman,
J Infect Dis
117:101-107, 1967; Levine et al.,
J Infect Dis
133:424-429, 1976; Tacket et al.,
Infect Immun
60:536-541, 1992). Neither of these strains contained a recombinant gene nor were they used to deliver a recombinant gene in a vaccine composition.
In a report of recombinant rpoS
+
S. typhi
, Coynault et al. disclosed the construction of a Ty2 derivative containing a recombinant rpoS gene which gave the microbe an RpoS
+
phenotype. However, this Ty2 derivative was used only in a laboratory study and no additional recombinant gene was incorporated nor was there any teaching of the use of this derivative in a vaccine composition.
Finally, the
S. typhi
strains ISP1820 and ISP1822 (U.S. Pat. Nos. 5,387,744 and 5,294,441 and PCT application WO/9424291) and the
S. typhi
strain 531Ty (U.S. Pat. No. 4,837,151) have been used to construct derivative vaccine strains. Although the studies reported herein show ISP1820, ISP1822 and 531Ty to be RpoS
+
, this was not known at the time of these earlier publications. Furthermore, none of these references recognized the importance of the presence of a functional rpoS gene in achieving a high immunogenicity in a vaccine preparation. As a result, these references did not disclose the selection of vaccine strains based upon the presence of an RpoS
+
phenotype.
All references cited in this specification either supra or infra are hereby incorporated by reference. The discussion of the references herein is intended to summarize the assertions made by their authors and no admission is made as to the accuracy or pertinency of the cited references or that any reference is material to patentability.
SUMMARY OF THE INVENTION
In accordance with the present invention, the inventors herein have succeeded in discovering the critical importance of a functional rpoS gene in Salmonella vaccine strains in that the presence of a functional rpoS gene and an RpoS
+
phenotype confers upon the Salmonella the property of high immunogenicity. As a result, when the RpoS
+
phenotype is present with one or more inactivating mutations other than a mutation in an rpoS gene, which render the microbe attenuated, a new and advantageous balance of attenuation and high immunogenicity is achieved. This invention is particularly applicable to
S. typhi
based vaccines, however, it is also applicable to other Salmonella such as
S. paratyphi
A, B and C as well as to other serotypes of
S. enterica
such as Typhimurium, Enteritidis, Dublin and Choleraesuis. The invention is also applicable to other bacteria having an rpoS gene, or functional equivalent thereof, that can colonize human tissues, including Shigella,
E. coli
, and hybrids between such bacteria, such as Salmonella-Shigella
Curtiss III Roy
Nickerson Cheryl A.
Mosher Mary E.
Thompson & Coburn LLP
Washington University
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