Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of...
Reexamination Certificate
1994-09-21
2003-12-16
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
C435S259000, C435S822000, C435S821000, C436S543000, C436S547000, C424S249100, C424S250100, C424S234100, C530S412000
Reexamination Certificate
active
06664094
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to mutants of Neisseria useful for vaccine preparation. Specifically this invention relates to mutants of Neisseria containing mutations in a major outer membrane protein gene such that no immunologically functional polypeptides encoded by said gene are produced. More specifically, the invention relates to a mutant strain of
Neisseria gonorrhoeae
having a mutation of the PIII gene and to vaccines derived therefrom.
BACKGROUND OF THE INVENTION
The genus Neisseria includes two species of gram-negative pyogenic cocci that are pathogenic to man: the meningococcus,
Neisseria meningitidis
, the causative agent of cerebrospinal meningitis, also referred to meningococcal meningitis and the gonococcus,
Neisseria gonorrhoeae
, the causative agent of the venereal disease gonorrhea.
Neisseria gonorrhoeae
is an aerobic diplococcus that ferments glucose but not maltose, a characteristic useful in distinguishing the species from meningococci. Gonococci exhibit four colonial forms (T1-T4). Fresh isolates from clinical samples that retain their virulence grow as small colonies (T1 and T2). Repeated non-selective sub-culturing results in larger colonies (T3 and T4) which have been shown to be avirulent on inoculation in male volunteers.
Historically, the development of vaccines has been hampered by a number of technical problems inter alia, difficulty of cultivation, lack of a readily available and meaningful animal model, inability to classify the organisms by conventional serological techniques and the lack of protectiveness of whole bacterial cell vaccines when administered to humans.
Cell surface molecules are attractive candidates for vaccine compositions and have been extensively studied in bacteria in general and in Neisseria in particular.
Pilin-based Vaccines
Pili are proteinaceous, filamentous structures associated with the cell wall of infective strains of Neisseria. During infection the bacteria colonize the mucous membranes of the host; the attachment of the bacteria to the surface of the mucous membrane being mediated by the pili. Anti-pilin antibodies are thought to protect against infection by reacting with the pili and preventing the attachment of the bacterium to membrane target sites. Further the “coating action” of the antibody (i.e. opsonization) stimulates the removal of bacteria by phagocytic cells in the blood. U.S. Pat. Nos. 4,461,838 and 4,696,986 relate to crystalline and single rod structures derived from pili of Type 1 and Type 2
Neisseria gonorrhoeae
, methods for their preparation and the use of such material in vaccines.
One of the major shortcomings of pilin-based vaccines is the high antigenic variability associated with the pilin protein, thus antibodies raised against one strain will not necessarily react with any other strain. U.S. Pat. Nos., 4,584,195 and 4,622,223 and PCT application No. PCT/US85/00565 attempt to address this problem by employing as vaccine components, fragments of the pilin proteins which comprise conserved amino acid sequences, thus antisera raised thereto are characterized by a somewhat broader reactivity.
Protein I (PI) Vaccines
Protein I is found in the membranes of all gonococci and is usually present in the largest amount. The protein has been purified and when inserted into artificial bilayers acts as an anion-selective pore. There appear to be 14-20 serotypic PI variants, however only one PI is expressed by any one strain. Further certain PI variants have been associated specifically with pelvic inflammatory desease (P.I.D.) and other variants with disseminated gonococcal infection (D.G.I.). For these reasons PI has been used as a vaccine component,
U.S. Pat. Nos. 4,203,971 and 4,239,749 relate to methods of separating PI from outer membrane lipopolysaccharides which have been shown to have toxic properties and the use of the “detoxified” product as a vaccine. European Patent Application No. 83301813.8 points out some of the shortcomings of the above patents, such as low yields and low solubility, thus making the production of effective vaccines difficult. The European application discloses an improved purification protocol which results in a protein I preparation having a solubility of from 5 to 10 mg/ml. As described, the vaccine also contains small amounts of protein II and protein III as well as what is characterized as “trace” amounts of LPS (3.5-4.0%). As discussed in more detail below, these contaminants although present in only small amounts can have a significant adverse effect on the effectiveness of PI vaccines.
Protein II (PII) Vaccines
When observed with light directly reflected from the substage mirror of a colony microscope, gonococcal colonies vary in their opacity: some appear transparent like water droplets, others are opaque like ground glass, and other colonies are intermediate in appearance. Gonococci change from the transparent to the opaque phenotype (or vice versa) with great frequency, estimated to be 10
−3
per cell division. The change to the opaque form is accompanied by the acquisition of one or more additional outer membrane proteins in the molecular weight range of 24,000-30,000. These proteins exhibit heat-modifiable behavior, namely, their apparent molecular weight on SDS-PAGE changes, depending on the degree to which they have been heated prior to electrophoresis. The optical property of opacity is due to the fact that the gonococci stick to each other within the colony and these zones of adhesion between outer membranes are mediated by the opaque proteins. This indicates that protein II binds to some constituent on the neighboring cell; however, this receptor-ligand relationship has not been defined. It has been noted that if all the colonial variants of a single strain are carefully examined as many as six different opaque proteins can be distinguished.
Although it is known that protein II is readily accessible to antibodies, very little information is available on the antibacterial effects that protein II antibodies might have. The limited serological data available currently indicate that these proteins are serologically specific, and the degree of cross-reactivity between them in the native state is not clearly delineated. The high degree of variability of this class of proteins has also discouraged interest in their use in a vaccine. Definitive estimates of the potential of protein II vaccines cannot be made until a much clearer picture emerges on their role (or quite likely multiple roles) in pathogenesis.
Protein III (PIII) Vaccines
It has been shown that all gonococcal strains examined have an outer membrane protein that is not heat modifiable and that in the absence of a reducing agent migrates on SDS-PAGE with a molecular weight of 30,000, but with a molecular weight of 31,000 following reduction. This protein, named protein III, does not seem subject to variation, with all strains having an identical protein as judged by peptide analysis. A hybridoma has been cloned that produces and antibody reactive with protein III as it exists in the membrane, indicating that at least one antigenic determinant is exposed to the surface (Swanson, J. et al.,
Infect. Immunity
38: 668-672 (1982)). In vivo cross-linking studies have shown that protein III is closely associated with the trimeric complex of protein (McDade R. L., et al.,
J. Bacteriol
. 141: 1183-1191 (1980)).
The ubiquity and constancy of PIII initially made it an attractive candidate for vaccine, however, recent studies raise serious questions about the utility of PIII as a vaccine. For example, Rice, P. A. et al., (
J. Exp. Med
. 164: 1735-1748 (1986)) disclose that IgG antibodies directed against PIII act to block the killing of serum-resistant Neisseria by immune sera. As a result of eliciting these blocking antibodies, PIII quite paradoxically helps to protect the gonococcus from attack by antibodies to other surface antigens. Because it is technically very difficult to remove PIII from gonococcal membrane antigen preparations, vaccinations with PI results in, if anything,
Blake Milan Scott
Gotschlich Emil Claus
Koomey John Michael
Wetzler Lee Mark
Duffy Patricia A.
The Rockefeller University
White & Case LLP
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