Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/095—Neisseria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants

Definitions

• the present invention relates to a method for inducing immunity to infection by Neisseria gonorrhoeae or Neisseria meningitidis.
• the present invention relates to a parenteral component priming-oral (PCP-oral) immunization method.
• Neisseria gonorrhoeae is the causative agent of gonorrhea, an extremely common infection in humans, prevalent in the United States as well as in other countries.
• no successful vaccine against the numerous strains of N. gonorrhoeae has been developed.
• While several vaccine preparations and immunization methods have been proposed [U.S. Patent Nos. 4,443,431 (Buchanan et al.), 4,220,638 (Karkhanis et al.), 4,203,971 (Buchanan) and 4,681,761 (Mietzner et al.)], none have provided effective protection in appropriate experimental models or in human trials.
• MIRP Magnetic adsorption protein
• N. gonorrhoeae has efficiently evolved as a sexually transmitted disease which eludes the human body's immune mechanisms quite effectively and requires new immunization strategies.
• a vaccine against N. gonorrhoeae in order to prevent the spread of this infectious agent, for which treatment is becoming more difficult due to the development of multiple resistance to antibiotics.
• Neisseria meningitidis is a causative agent of septicemia and bacterial meningitis. The latter is a central nervous system infection most commonly afflicting small children with significant morbidity and mortality.
• numerous strategies, similar to those used in development of vaccines against N. gonorrhoeae, have been employed in attempts to produce an effective N. meningitidis vaccine, there remains a need for an immunization protocol which affords broader and more long lasting protection, especially in early childhood.
• This invention satisfies these needs by providing a method which is effective in preventing infection by N. gonorrhoeae and N. meningitidis.
• the present invention provides a method of immunization against pathogenic Neisseria species which overcomes problems previously encountered by using conventional parenteral immunization methods, i.e., induction of "blocking" antibodies, failure of the host to respond to conformation epitopes found on intact micro-organisms, development of a long-lasting mucosal immune response and problems associated with toxicity due to contamination with endotoxin.
• the present invention provides an effective method of inducing immunity in humans against N. gonorrhoeae that is protective against a number of different gonococcal strains and an effective method of inducing immunity in humans against N. meningitidis.
• the present invention provides a method of immunization against N. gonorrhoeae.
• the method comprises parenteral administration of a priming antigen (e.g. a synthetic immunoadjuvant, for example, polyphosphazene, or a synthetic peptide from N. gonorrhoeae Protein IB with the amino acid sequence DDQTYSIPSLFV, QHQVYSIPSLFV, EHQVYSIPSLFV or ASVAGT ⁇ TGWG ⁇ K, or a combination of these peptides) in a pharmaceutically acceptable carrier to a human subject, in an amount sufficient to enhance the immune response to subsequently administered oral doses of a gonococcal immunogen.
• the oral immunogen consists of PIII- deficient, killed whole cells of N. gonorrhoeae in a pharmaceutically acceptable carrier, in an amount sufficient to induce resistance to infection with N. gonorrhoeae.
• the present invention relates to a method of immunization against N. meningitidis.
• the method comprises parenteral administration of a priming antigen in a pharmaceutically acceptable carrier to a human subject in an amount sufficient to enhance an immune response to subsequently administered oral doses of an immunogen.
• the oral immunogen consists of killed protein class 4-deficient whole cells of N. meningitidis in a pharmaceutically acceptable carrier, in an amount sufficient to induce resistance to infection with N. meningitidis.
• kits comprise a first container with the priming antigen in a pharmaceutically acceptable carrier suitable for parenteral administration and a second container with the oral component in a pharmaceutically acceptable carrier suitable for oral administration.
• the present invention provides a method of protecting a patient against infection by Neisseria gonorrhoeae comprising the steps of parenterally administering to the patient a priming antigen in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response and subsequently orally administering a protective amount of a Protein Ill-deficient killed whole cell of Neisseria gonorrhoeae.
• a human subject is first primed with either a synthetic immunoadjuvant (e.g. polyphosphazene) or one or more immunogenic peptides of gonococcal PI.
• synthetic immunoadjuvant an agent which enhances an immune response and consists of an organic polymer containing inorganic moieties which confer additional properties, such as solubility and flexibility.
• synthetic immunoadjuvants are carbopol (B. F. Goodrich, Cincinnati, Ohio) and polyphosphazene (Virus Research Institute, Cambridge, Massachusetts).
• Polyphosphazene is an organic polymer with phosphorous atoms in its backbone structure.
• polyphosphazene in the present invention is a water soluble, ionically-crosslinked molecule that forms with molecular weight of 3—4 million and is thus not excreted when injected parenterally but is slowly hydrolyzed to ammonium phosphate and phosphoric acid to effect its removal from tissue.
• immunogenic peptides include those having or consisting essentially of the following amino acid sequences: DDQTYSIPSLFV (SEQ ID NO:l), EHQVYSIPSLFV (SEQ ID NO:3), QHQYSIPSLFV (SEQ ID NO:2) and ASVAGTNTGWGNK (SEQ ID NO:4).
• Consisting essentially of is meant a peptide having the amino acid sequence disclosed including minor substitutions, additions or deletions which do not negatively effect the immunogenicity of the peptide.
• Immunogenic peptides can be obtained or synthesized using standard methods known in the art, for example, by computer based predictive algorithms and automated peptide synthesis (Rothbard & Taylor, 1989, EMBO 7:93-100).
• the immunogenicity of the peptides can be determined according to the methods described in Examples 4 and 5.
• the purity of the peptides can be determined by analytical HPLC and the presence of endotoxin can be determined with a rabbit pyrogen test.
• the priming antigen is parenterally administered in an amount sufficient to induce an immune response which imparts a priming effect (Hosmalin et al, J. Immunol, 146:1667-1673 (1991)).
• the synthetic peptide is parenterally administered in a pharmaceutically acceptable carrier to human subjects.
• Suitable carriers for use in the present invention include, but are not limited to, pyrogen-free water.
• a sterile solution or suspension is prepared in water that may contain additives, such as ethyl oleate or isopropyl myristate, and can be injected, for example, into subcutaneous or intramuscular tissues.
• the priming antigen may be microencapsulated using natural or synthetic polymers. Although one skilled in the art will realize that dosages are best determined by the practicing physician, dependent on the individual patient, one intramuscular injection of 800 ⁇ g of the microencapsulated priming antigen can be sufficient to generate the desired response.
• the second step of the immunization method of the present invention involves orally administering a protective amount of a gonococcal antigen, for example, killed (e.g. gamma-irradiated) Pill-deficient whole cells of N. gonorrhoeae (for example, strain 340) can be the gonococcal antigen.
• N. gonorrhoeae strain 340 has been shown to induce a high level of cross-protection to different gonococcal strains when used as a formaldehyde-killed parenteral whole cell immunogen in a mouse or guinea pig infection model.
• PIII " mutants are preferred because their use results in a greater level of protection.
• a PIII " mutant of strain 340 was obtained by insertional inactivation of the rmp structural gene by methods known to the art, although it should be understood that other PIII " mutants can be generated by one skilled in the art using the method taught by Wetzler et al. (J. Exp. Med., 169:2199-2209 (1989)). Briefly, a cloned rmp gene, inactivated by insertion of a ermC (erythromycin resistance) gene, was integrated into the gonococcal chromosome, generating the PIII " phenotype. Oral administration of a whole cell immunogen permits vaccination with essential antigens which may be too toxic to be given parenterally.
• the oral route can be more effective in stimulating the mucosal immunity required to prevent gonococcal colonization.
• Between 1X10 9 and 8X10 9 CFU of the oral immunogen can be administered at one week intervals for ten weeks.
• Oral immunization can be initiated as early as two weeks after parenteral priming or may be delayed for up to four weeks. This two to four week period between parenteral priming and oral immunization appears to be an optimal time period.
• Suitable carriers for gonococcal antigens used for oral administration include one or more substances which may also act as flavoring agents, lubricants, suspending agents, or as protectants.
• Suitable solid carriers include calcium phosphate, calcium carbonate, magnesium stearate, sugars, starch, gelatin, cellulose, carboxypolymethylene, or cyclodextrans.
• Suitable liquid carriers may be water, pharmaceutically accepted oils, or a mixture of both.
• the liquid can also contain other suitable pharmaceutical additions such as buffers, preservatives, flavoring agents, viscosity or osmo-regulators, stabilizers or suspending agents.
• suitable liquid carriers include water with or without various additives, including carboxypolymethylene as a pH-regulated gel.
• the gonococcal antigen may be contained in enteric coated capsules that release antigens into the intestine to avoid gastric breakdown.
• the gonococcal antigen may be mic roencapsulated with either a natural or a synthetic polymer into microparticles 4—8 ⁇ m in diameter, which target intestinal or vaginal lymphoid tissues and produce a sustained release of antigen for up to four weeks (Eldridge et al, Cur. Topics in Microbiol and Immunol, 146:59-65 (1989); Oka et al, Vaccine, 8:573-576 (1990)).
• an effective oral regimen of the present invention can include as many as four to ten oral doses of gonococcal antigen administered at approximately one week intervals.
• the present invention provides a method of protecting a patient against infection by Neisseria meningitidis comprising the steps of parenterally administering to the patient a priming antigen in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response against the protein and subsequently orally administering a protective amount of a killed class 4 protein-deficient whole cell of Neisseria meningitidis.
• a priming antigen in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response against the protein
• a protective amount of a killed class 4 protein-deficient whole cell of Neisseria meningitidis Alternatively, the wild type killed whole cell of Neisseria meningitidis can be administered as the oral immunogen.
• a human subject is first primed with either a synthetic immunoadjuvant (e.g. polyphosphazene) or one or more synthetic peptides of meningococcal class 2,3 protein.
• the priming antigen is parenterally administered in a pharmaceutically acceptable carrier in an amount sufficient to induce an immune response which imparts a priming effect.
• Immunogenic peptides can be obtained or synthesized using standard methods known in the art, for example, by computer based predictive algorithms and automated peptide synthesis (Rothbard & Taylor, 1989, EMBO 7:93-100).
• the immunogenicity of the peptides can be determined according to the methods described in Examples 4 and 5.
• the purity of the peptides can be determined by analytical HPLC and the presence of endotoxin can be determined with a rabbit pyrogen test.
• the second step of the immunization method of the present invention involves orally administering a protective amount of a meningococcal antigen (for example, class 4 protein-deficient, killed (e.g. gamma-irradiated) whole cells of N. meningitidis).
• a meningococcal antigen for example, class 4 protein-deficient, killed (e.g. gamma-irradiated) whole cells of N. meningitidis.
• a meningococcal antigen for example, class 4 protein-deficient, killed (e.g. gamma-irradiated) whole cells of N. meningitidis.
• the administration of the mutant cells described in the present invention can reduce the induction of blocking antibodies to PIII from N gonorrhoeae or to class 4 protein from N meningitidis which inhibit the bactericidal antibodies directed against other cell surface antigens.
• the present invention provides for an immunologic response to conformational epitopes on intact organisms. This is achieved by using intact cells for oral immunization.
• conformational epitopes of cell envelope antigens necessary for generating antibodies and other non-specific immune mechanisms can be preserved and administered without toxic effects, in either a liquid gel, enteric coated capsule, or microencapsulated suspension, so that the antigens reach intestinal lymphoid tissues intact.
• the present invention employing a priming antigen as primer and a whole cell as an oral immunogen, also addresses toxicity and cross-reactivity issues, specifically directs induction of a mucosal response and eliminates the production of blocking antibodies.
• a synthetic peptide as a parenteral priming antigen offers several advantages over the use of r-Fbp: 1) the relatively small peptide can be synthesized and purified by biochemical methods to yield a product free of endotoxin; 2) the synthetic peptide is more amenable to making amino acid substitutions that can be evaluated for improved priming capabilities; 3) the peptide is a more neutrally charged molecule than is the highly cationic r-Fbp (pi 10.35) and is thus less likely to affect DNA binding which is suspected to be a factor in certain types of autoimmune disease; and 4) the peptide primes better for PIII " gonococci, which can be better as an oral vaccine candidate than the 340 WT cells.
• polyphosphazene as a priming agent is advantageous due to 1) its water-soluble, ionically-crosslinked structure and high molecular weight, which prevent excretion and allow for slow hydrolysis to effect its removal from tissue; 2) its potential as a universal primer for other oral vaccines; 3) its parenteral immunoactivity in relatively small doses (100 ⁇ g); and 4) its lack of parenteral toxicity.
• Killed cells and especially gamma-irradiated PIII " cells offer several advantages as an oral immunogen: 1) anti-PIII or "blocking" antibodies are not induced due to the failure of this protein to be expressed by the deletion mutant; 2) gamma irradiation can enhance the oral immunogenicity of the PIII " mutant (Table 1); 3) the irradiated PIII " immunogen elicits a three-fold higher level of chamber protection and significantly better (P ⁇ .01) vaginal clearance of gonococci compared to live PIII " cells or other immunogens (Table 1) ; and 4) gamma irradiation provides a convenient method of "in container” sterilization without toxic residues which are often left by other chemical methods of inactivation. This results in a safer product for oral immunization.
• N gonorrhoeae strain 340 WT was deposited on April 23, 1992 under the terms of the Budapest Treaty at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852. Strain 340 WT has been assigned accession number ATCC 55320. The strain will be made available, without limitation, on the issuance of a patent.
• Example 1 Comparison by chamber and vaginal clearance of various combinations of parenteral primers and oral components.
• mice were injected intramuscularly with either 15 ⁇ g/dose of recombinant iron-binding protein (r-Fbp) or with 20 ⁇ g/dose of a synthetic peptide (amino acid sequence DDQTYSIPSLFV, SEQ ID NO:l) seven times at weekly intervals.
• r-Fbp recombinant iron-binding protein
• SEQ ID NO:l synthetic peptide
• Parenteral priming was followed two weeks later by oral immunization with live or gamma- irradiated cells of N. gonorrhoeae strain 340 wild type (340 WT) or a protein III deletion mutant of strain 340 (340 PIII " ).
• Escherichia coli strain 15 cells were used as a control oral immunogen.
• Mice were orally immunized by giving ten weekly doses containing 10 9 CFU in a volume of 0.5 ml by means of a gastric feeding tube.
• mice Four weeks later, all groups of mice were given a graded dose challenge with virulent strain 340 WT cells. The infectious dose 50% (ID 50 ) was determined graphically for each group (Table 1). In addition, a gonococcal vaginal clearance test was performed with each group. Mice were inoculated intravaginally with 400,000 CFU of virulent strain 340 WT cells and vaginal wash fluids were collected 6h post- infection and assayed by culturing for gonococci (Table 1).
• mice were protected to a greater degree following oral immunization with 340 WT cells, while mice primed with synthetic peptide were protected to a greater degree following oral immunization with 340 PIII " cells.
• the data in Table 1 demonstrate that the combination of synthetic peptide primer and 340 PIII " cell oral immunogen provided superior chamber protection as well as vaginal protection in comparison with the r-Fbp primer/ 340 WT cell oral immunogen combination. These data also show that gamma-irradiated 340 PIII " cells elicited a stronger immune response than live 340 PIII " cells.
• Example 2 IgA and IgG antibody titers in vaginal washings.
• IgA and IgG antibody titers were measured in vaginal wash fluids two weeks prior to vaginal challenge with gonococci. Mice were parenterally primed with seven weekly injections of either r-Fbp at 15 ⁇ g/dose or synthetic peptide at 20 ⁇ g dose, followed by ten oral immunizations of 10 9 CFU at weekly intervals. Titers were determined by whole cell ELISA on plates coated with 340 WT cells.
• Example 3 Cross-reactive bactericidal activity of serum obtained from complement deficient mice.
• Sera from mice parenterally primed with either purified iron binding protein (Fbp) or 340 WT cells alone, orally immunized with 340 WT cells alone, or parenterally primed with Fbp followed by oral immunization with 340 WT cells were assayed for bactericidal activity against a variety of microorganisms.
• a serum aliquot of 10 ⁇ l was placed over agar plates inoculated with confluent numbers of the following test organisms: N. gonorrhoeae 340 WT, N. gonorrhoeae 11B, N. gonorrhoeae F62 and N.
• meningitidis group A meningitidis group A, and the percent of cells killed was calculated.
• Table 3 the parenteral primer/oral immunogen combination method of immunization induced greater cross-reactive bactericidal activity than immunization with either component alone. Also, this response was induced in outbred ICR mice, which are complement deficient, indicating that the bactericidal activity was not totally dependent upon the classical complement pathway.
• Example 4 Comparison of parenteral priming with peptide only and parenteral priming with peptide followed bv oral immunization with whole cells.
• mice A group of 154 six-week old, female, ICR outbred mice was injected intramuscularly with 5, 20 or 50 ⁇ g of a synthetic peptide composed of the amino acid sequence DDQTYSIPSLFV (SEQ ID ⁇ O:l) three, five or seven times at weekly intervals. In half of the mice, this parenteral priming was followed two weeks later by oral immunization with gamma-irradiated 340 PIII " cells. Orally immunized mice were given ten weekly doses containing 10 9 CFU in a volume of 0.5 ml by means of a gastric feeding tube. Two weeks before the last oral immunization, each mouse was surgically implanted with a subcutaneous culture chamber, as described in Example 1.
• mice Four weeks later, all mice were given a graded dose challenge with virulent strain 340 WT cells. The ID 50 was determined graphically for each group (Table 4). Mice primed with the synthetic peptide and orally immunized with gamma-irradiated 340 PIII " cells resisted greater numbers of gonococci on challenge than did mice primed with synthetic peptide alone or oral immunization alone.
• Example 5 Comparison of soluble polyphosphazene and a gonococcal synthetic peptide as single injection primers for oral immunization.
• mice A group of 63 six month old, female, ICR mice were injected intramuscularly with a combination of synthetic peptide (amino acid sequence DDQTYSIPSLFV, SEQ ID NO:l) and polyphosphazene or with either component alone in the doses listed in Table 5. This parenteral priming was followed four weeks later by oral immunization with gamma-irradiated 340 PIII " cells. Oral immunization consisted of ten weekly doses of 10 9 CFU in a volume of 0.5 ml administered by means of a gastric feeding tube. Two weeks before the last oral immunization, each mouse was surgically implanted with a subcutaneous culture chamber as described in Example 1.
• mice Four weeks later, all mice were challenged with graded doses of virulent 340 WT cells.
• the ID S0 was determined graphically for each group and the percent of mice infected was calculated after challenge doses of 700, 5,000 and 92,000 CFU (Table 5).
• the present invention also induced cross-reactive bactericidal activity (Table 3) against different gonococcal strains and protective immunity without the requirement for exogenous complement. Because the outbred ICR strain of mice used is deficient in the early complement component C2, the bactericidal antibodies and protection induced by parenteral immunization with whole gonococci in previous experiments required supplementation of the chamber fluid with an exogenous complement source in order to demonstrate this high level of immunity (Arko et al, J. Infect. Dis., 139:569-574 (1979)). These data show that the bactericidal activity demonstrated in these experiments was not totally dependent upon the classical complement pathway and that this invention can be more effective in providing protection at sites were complement components are limited (e.g., the urogenital tract).
• a regimen for administration of either the gonococcal or meningococcal vaccine to humans can include a single parenteral injection of up to 800 ⁇ g of priming antigen (e.g. polyphosphazene) followed four weeks later by oral administration of ten enteric coated capsules at one week intervals for ten weeks.
• Each capsule can contain 5X10 9 CFU of gamma-irradiated 340 PIII " gonococci.
• Such administration can result in protection from subsequent infection with the organism.
• Synthetic peptide DDQTYSIPSLFV injected at 20 ⁇ g per mouse for seven weekly doses. Same for recombinant iron binding protein (r-Fbp) at 15 ⁇ g/dose.
• ID 50 determined graphically from data obtained following graded dose challenges of 5— 10 mice per group. 2. Mice were orally immunized weekly with 10 9 CFU of 340 PIII " for ten weeks.
• Synthetic peptide DDQTYSIPSLFV injected intramuscularly in 0.1 ml volume of phosphate buffered saline, pH 7.4. 4. ID 50 of virgin control mice was 180 CFU.

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  • 1993-10-26 CA CA002147877A patent/CA2147877A1/en not_active Abandoned
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