Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-12-23
2001-06-19
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S253400, C536S127000, C536S123000, C424S244100, C424S234100
Reexamination Certificate
active
06248570
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for extracting and isolating capsular polysaccharides (CPS) from both gram-negative and gram-positive bacteria. The extracted polysaccharides are useful for producing vaccines comprising the polysaccharides alone or conjugated to proteins.
BACKGROUND OF THE INVENTION
Bacterial infections caused by gram-positive bacteria such as Streptococcus, Staphylococcus, Enterococcus, Bacillus, Corynebacterium, Listeria, Erysipelothrix, and Clostridium and by gram-negative bacteria such as Haemophilus, Shigella,
Vibrio cholerae
, Neisseria and certain types of
Escherichia coli
cause serious morbidity throughout the world. This, coupled with the emerging resistance shown by bacteria to antibiotics, indicates the need for the development of bacterial vaccines. For example, streptococci are a large and varied genus of gram-positive bacteria which have been ordered into several groups based on the antigenicity and structure of their cell wall polysaccharide (26, 27). Two of these groups have been associated with serious human infections. The group A streptococci cause a variety of infectious disorders including “strep throat”, rheumatic fever, streptococcal impetigo, and sepsis.
Group B streptococci were not known as human pathogens in standard medical textbooks until the early 1970's. Since that time, studies have shown that group B streptococci are important perinatal pathogens in the United States as well as developing countries (37). Systemic group B streptococcal infections during the first two months of life affect approximately three out of every 1000 births (12), resulting in 11,000 cases annually in the United States. These infections cause symptoms of congenital pneumonia, sepsis, and meningitis. A substantial number of these infants die or have permanent neurological sequelae. Furthermore, group B streptococcal infections may be implicated in the high pregnancy-related morbidity which occurs in nearly 50,000 women annually. Others at risk from group B streptococcal infections are those who have an altered immune response, either congenitally, chemotherapeutically, or by other means.
Group B streptococci can be further classified into several different types based on the bacteria's capsular polysaccharide. Types Ia, lb, II, III, IV, V, VI, VII, and VIII account for most of the pathogenicity due to group B infection, with group B streptococci types Ia, Ib, II, III, and V representing over 90% of all reported cases. The structure of each of these various type polysaccharides has been characterized (19-22, 44). Similar to findings with many other human bacterial pathogens, capsular polysaccharides of group B streptococci, when used in vaccines, may provide effective protection against infections with these bacteria. See 4, 6, 24, 29, 30, 42, 43, 45.
Gram-negative bacteria are also a significant cause of disease. Until the recent development and use of polysaccharide-proteinvaccines directed against Haemophilus influenzae type b bacteria (Hib), Hib bacterial infections were responsible for many cases of mental retardation in infants. N. menigitidis and
E. coli
K1 infections are responsible for neonatal meningitis. Strains of gram-negative bacteria,
E. coli
, have been linked to serious illness including death from eating meat tainted with
E. coli
strains.
Large-scale production of capsular polysaccharide vaccines, and capsular polysaccharide conjugate vaccines, requires adequate supplies of purified capsular polysaccharides. Prior art methods (40, 42) for isolating capsular polysaccharides from bacterial cells rely on treatment of cells with the enzyme mutanolysin. Mutanolysin cleaves the bacterial cell wall which frees the cellular components. This procedure involves treating cell lysates with additional enzymes to remove proteins and nucleic acids and purification by differential precipitation and chromatography. More efficient, higher yielding and simpler means of obtaining purified capsular polysaccharides are desirable.
SUMMARY OF THE INVENTION
This invention provides a method for extracting capsular polysaccharides (CPS) from the cellular components of both gram-negative and gram-positive bacteria. The CPS can be extracted according to this invention from either bacterial supernatants or bacterial cells by hydrolysis of the base labile bond that connects the CPS to other cellular components. An advantage of the extraction procedure provided by this invention is that the extracted CPS are largely intact.
Another embodiment of this invention provides a method for obtaining purified capsular polysaccharide by deacetylating a percentage of the N-acetyl groups of the CPS during base extraction to facilitate separation of the CPS from other cellular components. A percentage of the acetyl groups can be reintroduced to afford purified CPS having the same repeat unit structure with respect to the N-acetyl groups as native polysaccharide, or, alternatively, acylation with modified alkyl groups can be used to obtain modified CPS.
In a preferred embodiment, the CPS are extracted from group B streptococci (GBS). In a most preferred embodiment the CPS are extracted from GBS types Ia, Ib, II, III, V and VIII.
In another preferred embodiment, the CPS are extracted from
S. pneumoniae
. In a most preferred embodiment the CPS are extracted from
S. pneumoniae
types III, IV and XIV.
In another preferred embodiment, the CPS are extracted from Neisseria or Escherichia bacteria. In a most preferred embodiment the CPS are extracted from
Neisseria meningitidis
types B, C, Y or W135 or
Escherichia coli
K1 .
Purification of capsular polysaccharides from either bacterial supernatants or bacterial cells according to this invention has the following advantages over other methods: (a) simplicity (a minimal number of steps), (b) efficiency (high yield and purity), (c) safety (e.g., reduction or elimination of the use of flammable organic solvents), and (d) general applicability to all gram-negative and gram-positive bacteria.
The method according to the invention comprises treatment of a concentrated extract and/or isolated bacterial cells with a basic solution. In addition to extracting the CPS, the base extraction also causes deacetylation of N-acetyl groups. The extent of the deacetylation may be varied by adjusting the reaction conditions. The extracted CPS are then separated from the cellular components to obtain the CPS preferably by chromatographic separation. Some or most of the acetyl groups may be reintroduced to obtain CPS or modified CPS. Final purification of the CPS may be achieved by gel-permeation chromatography. In a further embodiment, the invention provides novel, optionally modified CPS as a result of the basic extraction conditions which are suitable for use as vaccines or conjugate vaccines.
It is an embodiment of this invention to provide a method for producing substantially pure CPS which are capable of eliciting the production in mammals of antibodies that are bactericidal and protect the animals against infection.
It is another embodiment of this invention to use these CPS in vaccines, either alone or conjugated to a polypeptide, to protect humans or animals against infection, typically by that strain of bacteria from which the CPS was isolated. In certain cases the polysaccharide used with this invention may induce production of antibodies which are cross-reactive with other pathogenic bacteria thereby producing protection against infection by these other bacteria.
It is an objective of this invention to provide a method for isolating capsular polysaccharides from both gram-negative and gram-positive cellular components contained in either gram-negative or gram-positive bacterial supemates or gram-negative or gram-positive bacterial cells. These capsular polysaccharides can then be used as vaccines or bound to polypeptides to form conjugate molecules which are useful as vaccines.
REFERENCES:
patent: 3577527 (1971-05-01), Edwards
patent: 4356170 (1982-10-01), Jennings et al.
patent: 4413057 (1983-1
Blake Milan
Michon Francis
Baxter International Inc.
Morgan & Finnegan L.L.P.
Prats Francisco
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