Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex
Reexamination Certificate
1999-09-29
2001-08-21
Graser, Jennifer E. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Conjugate or complex
C424S193100, C424S234100, C424S203100, C424S249100, C424S241100, C424S258100, C424S252100, C530S350000, C435S975000, C536S123100
Reexamination Certificate
active
06277379
ABSTRACT:
FIELD OF THE INVENTION
This invention provides a novel composition, Invaplex, comprising at least one invasin protein i.e. proteins essential for the process by which a bacterium enters a host cell, and lipopolysaccharide from invasive gram-negative bacteria. The composition of the present invention can be used as an adjuvant for vaccines, biochemical, or other substances, as a diagnostic tool, and as a vaccine against gram-negative bacterial infection.
INTRODUCTION
Bacillary dysentery is caused by members of the genus Shigella as well as enteroinvasive
Escherichia coli
(EIEC). Shigellosis is found in all parts of the world with developing countries accounting for the large majority of cases. A recent report in the Bulletin of the World Health Organization estimates that in developing countries, for children 0-4 years old, there were 113 million Shigella episodes per year and an additional 50 million cases per year in all other age groups. In industrialized countries it is estimated that there are approximately 1.5 million cases of shigellosis per year (Kotloff et al 1999,
WHO
77, 651-666). The rampant occurance of antibiotic resistance in Shigella spp. and the high incidence of this disease underscores the need for a vaccine against this human pathogen. However at the present time a vaccine is not commercially available for bacillary dysentery.
The pathogenesis of Shigella is attributed to this organism's ability to invade, reside, and replicate intracellularly within the colonic epithelium. The invasion of host cells by Shigella spp. is a complex multifactorial event in which many different bacterial proteins are involved. Many of the genes for key Shigella virulence proteins are encoded on a large 140 Mdal plasmid. Several of the plasmid encoded proteins called the invasion plasmid antigens (IpaA, IpaB, IpaC, and IpaD proteins) (Buysse et al., 1987,
J. Bacteriol.
169, 2561-2569) are essential virulence factors. Similar proteins, called Sip proteins, are made by members of the genus Salmonella (Kaniga et al, 1995,
J. Bacteriol.
95, 3965-3971). Upon contact or attachment to host cells, the Shigella invasins induce a phagocytic event which results in engulfment and internalization of the bacterium by the host cell. Recent reports have identified that IpaB and IpaC form a complex that can be found in the growth medium of Shigella cultures (Menard et al, 1998,
EMBO J
13, 5293-5302; Watari et al 1995,
EMBO J
14, 2461-2470). The components of this complex are involved in the invasion process, but the actual mechanisms have not been defined (Menard et al, 1994,
Cell
79:515-525). In addition, purified IpaC has been shown to bind to host cells and participate in the uptake of avirulent shigellae by host cells (Marquart et al.,
Infect Immun.
64:4182-4187, 1996). IpaB, IpaC and IpaD, along with LPS are known major antigens that infected individuals respond to after infection with shigellae (Li et al. 1993,
Scand. J. Infect. Dis.
25, 569-577; Oaks et al, 1986,
Infect. Immun.
53, 57-63; van DeVerg et al 1992,
J. Infect. Dis.
166, 158-161). Monkeys or humans infected with shigellae produce antibodies predominantly to IpaB and IpaC, and also produce antibodies at high frequencey to IpaA, IpaD and VirG (another plasmid encoded virulence protein involved in intercellular spreading) (Oaks et al, 1986, supra). It is not known if the immune response to the Shigella invasins or more specifically to the invasin complex is crucial to protective immunity.
There are no proven safe and effective vaccine for shigellosis, EIEC diarrhea or salmonellosis, although several living attenuated shigella vaccines are in human trials. Unfortunately, with the many serotypes of Shigella, and because immunity appears to be serotype specific, it would require a multitude of living vaccines to cover the spectrum of serotypes in nature. In addition, living attenuated vaccines are difficult to standardize.
Therefore, there is a need for a standardized vaccine against gram-negative bacteria which can be prepared from different serotypes of Shigella without the need for attenuation of each serotype.
SUMMARY
The present invention fulfills the needs described above. In this application is described a novel composition comprising at least one invasion protein, proteins essential for bacterial invasion of a host cell. The invasion protein(s) of the present invention are complexed with lipopolysaccharide (LPS). The complex of invasin protein(s) and LPS is in a native conformation and has been termed Invaplex. The Invaplex described below is not only effective as a vaccine against gram-negative bacterial infection but it can also serve as a mucosal adjuvant and a diagnostic tool for detecting antibody responses which correlates with protection against future infection.
Our initial experiments were aimed at isolating and purifying IpaC from a water extract of Shigella, a gram-negative bacteria. Usually, IpaC is extracted from growth culture medium. We chose to use the water extract, i.e. the solution resulting from incubating the bacteria with shaking in sterile water, because we hypothesized that the quantity of IpaC would be greater in such an extract. To our knowledge, no protein involved in the invasiveness of gram negative bacteris has been previously isolated from a water extract of gram-negative bacteria. To our suprise, when water extract was subjected to various separation techniques such as gel filtration and ion-exchange chromatography, we found that whenever we could detect detect IpaC from the water extract we also detected IpaB, IpaD and LPS in the same fractions. We proceeded to design a method to isolate this complex and characterize it. We have developed a method for purifying the invasin complex from intact invasive shigellae or enteroinvasive
E. coli
(see
FIG. 1
for general overview). Briefly, the Invaplex preparations are isolated from virulent, invasive shigellae. A crude mixture is extracted from the shigellae with water. The water extract consists of many proteins and lipopolysaccharide (LPS). The water extract material is then applied to a FPLC ion-exchange column which resolves two key protein peaks, called Invaplex (invasin complex) 24 and Invaplex 50. Fractions containing Invaplex 24 and Invaplex 50 are collected. We found that the complex was composed of many proteins, including IpaB, IpaC, IpaD in addition to LPS. The Invaplex 24 and Invaplex 50 preparations containing Ipa proteins and the LPS form a structure in a completely native configuration and environment.
If such a structure is used to immunize animals, it will lead to an immune response directed against a native structure presented by gram-negative bacteria during infection. Mice and guinea pigs immunized with the Invaplex preparations showed a marked serum IgA and IgG response to several different antigens (including the water extract antigen, IpaC and LPS) present in the Invaplex 24 and Invaplex 50 preparations. The two Invaplex preparations were similar in that they both primed the mucosal immune system, but differed in the specificity of the immune response generated. The animals were protected from challenge with gram-negative bacteria and immunization with either Invaplex. Animals immunized with either Invaplex showed no visible signs of distress or toxicity.
Therefore, the present invention relates to a purified composition, Invaplex, comprising invasin proteins in combination with LPS associated together to form a native structure. The Invaplex can be isolated from any invasive gram-negative bacteria such as Shigella, Salmonella or EIEC of different serotypes and combined to produce an effective vaccine against gram-negative bacteria.
Therefore, it is an object of the present invention to provide a vaccine against gram-negative bacteria comprising Invaplex from gram-negative bacteria in an amount effective to elicit protective antibodies in a subject to said bacteria; and a pharmaceutically acceptable diluent, carrier, or excipient.
It is another object of the present invention to provide a Shigella
Hartman Antoinette Berrong
Oaks Edwin V.
Turbyfill Kevin Ross
Arwine Elizabeth
Graser Jennifer E.
Harris Charles H.
The United States of America as represented by the Secretary of
LandOfFree
Use of purified invaplex from gram negative bacteria as a... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of purified invaplex from gram negative bacteria as a..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of purified invaplex from gram negative bacteria as a... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2472947