JPH08505128A - Immunization against Neisseria gonorrhoeae and Neisseria meningitidis - Google Patents

Abstract

(57) Summary The present invention relates to a method of immunization against infection by a pathogenic Neisseria species. This method involves parenteral priming with a priming antigen, followed by oral immunization with gamma-irradiated protein III-deficient killed Neisserla gonorrhoeae 340 strain whole cells, or parenteral priming with a priming antigen. Administration followed by oral immunization with gamma-irradiated killed Class 4 protein-deficient Neisseria meningitidis whole cells is used.

Description

Detailed Description of the Invention           Neisseria gonorrhoeaeandNeisseria meningitidis                             Immunization against                                BACKGROUND OF THE INVENTION Field of the invention   The present inventionNeisseria gonorrhoeaeOrNeisseria meningitidisInfection by And a method of inducing immunity against. More particularly, the invention relates to parenteral ingredients First-time oral (PCP-oral) immunization method. Background technology   Neisseria gonorrhoeaeIs one of the most prevalent human epidemics in the United States and other countries. It is the causative bacterium of gonorrhea, a common infection. So farN.gonorrhoeaeTo many shares of No successful vaccine has been developed. Some vaccine preparations and Immunization methods have been proposed [US Pat. No. 4,443,431 (Buchanan et al.), 4,220 , 638 (Karkhanis et al.), 4,203,971 (Buchanan) and 4,681,761 (Mie tzner et al.), both effective in appropriate experimental models or in human trials Did not provide protection.   These patents include (1) cell surface antigenic complexes (Karkhanis et al., Buchanan); (2) Immunogenic fragments of pili proteins (Buchanan et al.); Or (3) Purification Claim any of the major iron regulatory proteins (MIRPs) (Meitzner et al.) It All of these The method in the patent describes parenteral administration of vaccine components. All of these patents For the purpose of cross-reactivity, the development of mucosal immunity; protein Blocking effect of antibodies against III (PIII) or poisons inherent in parenteral vaccines Does not handle sexual tasks effectively.   These factors are surface antigens on cells and human vacuum cells that use pilus proteins. Chin test shows no successful immune defense response against N. gonorrhoeae challenge, especially against heterologous strains Things (Boslego, J.W. and Deal, C.D., (1991),Vaccines and Immunother apy , (S.J.Cruz), pages 211-224, Pergamon Press, New York, Boslego et al. (199 1), Vaccine, 9: 154-162).   The state of the art of N. gonorrhoeae vaccine development is sophisticated, protein I (PI) and MIR The focus is on individual cell surface proteins such as P. But parenteral PI Despite many trials using vaccines, no protective response has been generated (Bo slego and Deal (1991), Gu1ati et al.,Neisseriae, 1991, pp. 229-234, Walter d e Guyter & Co., Berlin). Furthermore, it has shown potential as a vaccine agent for MIRP However, the data disclosed in Table 3 of the present specification and Tables 1, 2 and 4 of the present application are included. Parenteral administration of this protein alone produces a protective response to N. gonorrhoeae challenge. Indicates not to induce.   N.gonorrhoeaeSexually escapes the human immune mechanism very effectively Efficiently evolved as a disease Require new immunization strategies. Therefore, to prevent the spread of this infectious bacteria ,N.gonorrhoeaeThere is a great need for a vaccine against. Because of antibiotics This is because the development of multidrug resistance has made it more difficult to treat.   Related microorganismsNeisseria meningitidisIs the causative agent of sepsis and bacterial meningitis Is. The latter are the most commonly central nervous system infections that afflict infants and cause significant morbidity. Has morbidity and mortality.N.gonorrhoeaeUsed in the development of a vaccine against Many strategies that are similar toN.meninitidisTo produce a vaccine Used in trials, but lasted more extensively and longer, especially in early childhood There is a need for immunization protocols that provide protection against   The present invention addresses these needs:N.gonorrhoeaeandN.meningitidisFeeling Satisfy by providing a method that is effective in preventing dyeing. The present invention Is pathogenicNeisseriaIt provides a method of immunization against species, which is traditionally non-transgenic. The previous problem encountered by using the oral immunization method, namely the induction of "blocking" antibodies. The host responds to conformation-dependent epitopes found on intact microorganisms. Not responding, developing a long-lasting mucosal immune response, and due to endotoxin Solve the problems associated with toxicity due to pollution.                                Summary of the invention   The present invention is protective against many different N. gonorrhoeae strainsN.go norrhoeae Effective method of inducing immunity toN.meningiti dis To provide an effective method of inducing immunity against.   In one embodiment, the invention providesN.gonorrhoeaeFor immunization against To do. This method involves the use of an initial anti-body in a pharmaceutically acceptable carrier for human subjects. Raw (eg, synthetic immunoadjuvant (eg, polyphosphazene)), or amino Acid sequence DDQTYSIPSLFV, QHQVYSIPSLFV, EHQVYSIPSLFV or ASVAGTNTGWGNK, Rui has a combination of these peptidesN.gonorrhoeaeFrom protein IB Synthetic peptide) enhances the immune response to an oral dose of the subsequently administered N. gonorrhoeae immunogen. It includes parenterally administering in an amount sufficient to strengthen. The oral immunogen isN.gono rrhoeae Pharmaceutically acceptable in sufficient quantity to induce resistance to infection by Reduced PIII-deficient mortality in carriersN.gonorrhoeaeIt consists of whole cells.   In another embodiment, the present invention isN.meningitidisImmunization method against. This method involves the administration of an initial antigen in a pharmaceutically acceptable carrier to a human subject, which is then administered. Parenterally in an amount sufficient to enhance the immune response to an oral dose of the given immunogen Administering. The oral immunogen isN.meningitidisResistance to infection by Protein class in a pharmaceutically acceptable carrier sufficient to induce sex 4-Death lossN.meningitidisIt consists of whole cells.   In a further embodiment, the invention relates to various kits. This kit consists of the initial antigen in a pharmaceutically acceptable carrier suitable for parenteral administration. In a pharmaceutically acceptable carrier suitable for oral administration It includes a second container having an oral component.   Various other objects and advantages of the invention will be apparent from the description below.                           Description of the preferred embodiment   The present invention is described in the following detailed specific embodiments and examples included herein. It can be more easily understood by reference to the above. Singular table used in claims Present may mean one or more.   The present inventionNeisseria gonorrhoeaeHow to protect patients from infection by The method provides the patient with a sufficient amount of pharmaceutically acceptable amount to induce an immune response. Parenteral administration of the initial antigen in a possible carrier, and then a protective dose Protein III-reduced mortalityNeisseria gonorrhoeaeOral administration of whole cells Includes. According to the method of the present invention, a human subject is initially treated with a synthetic immune adjuvant. Immunity with one or more (eg, polyphosphazene) or one or more N. gonorrhoeae PIs Primary immunization with any of the primary peptides.   The term "synthetic immune adjuvant" enhances the immune response and is soluble and soluble. From organic polymers containing inorganic components that impart other properties such as flexibility Means a drug. Examples of synthetic immune adjuvants are carbopol (B.F.Goodrich, Cincin nati, 0hio) and polyphosphazene (Virus Research Institute, Cambridge , Massachusetts). Polyphosphazene has a phosphorus atom in its skeletal structure. It is an organic polymer having. Examples of polyphosphazenes in the present invention are water-soluble An ionically cross-linked molecule formed with a molecular weight of 3-4 million, and It is not excreted when injected parenterally and is slowly hydrolyzed to give It becomes monium and phosphoric acid and is removed from the tissue.   Examples of immunogenic peptides have the following amino acid sequences or Containing peptides consisting essentially of: DDQTYSIPSLFV (SEQ ID NO: 1), EHQVYSIPSLFV (SEQ ID NO: 3), QHQYSIPSLFV (SEQ ID NO: 2) and ASVAGTNTGWGNK (SEQ ID NO: 4) ). The term "consisting essentially of" refers to the amino acid sequence disclosed, which is the immunization of this peptide. Has an amino acid sequence containing small substitutions, additions or deletions that does not negatively affect the originality Means peptide.   Immunogenic peptides can be prepared using standard methods known in the art, eg Pewter-based prediction algorithms and automated peptide synthesis (Rothbard and And Taylor) 1989, EMBO 7: 93-100) or synthesized. Peptide The immunogenicity of can be measured by the method described in Examples 4 and 5. Peptide Purity can be measured by analytical HPLC, and the presence of endotoxin indicates the presence of rabbit pyro. It can be measured by the Gen test.   The initial antigen is parenteral in an amount sufficient to induce an immune response that is responsible for the effect of the initial antigen administration. (Hosmalin et al.,J. Immunol.146: 1667-1673 (1991)). In the present invention The above synthetic peptides are orally administered to human subjects in a pharmaceutically acceptable carrier. Will be given to you. Suitable carriers for use in the present invention include, but are not limited to, pyros. Includes water without gen. Sterile solution or parenteral administration of initial challenge The suspension may be loaded with additives such as ethyl oleate or isopropyl myristate. Can be prepared in water and can be injected into, for example, subcutaneous or intramuscular tissue It   Initial antigen between 100-400 μg or about 1-5 μg / kg is given 3 times at 2 week intervals , Or a single human dose of 800 μg (10 μg / kg) can be given , Age and weight of the individual must be considered in determining the final dose. Alternatively, the priming antigen is microencapsulated with a natural or synthetic polymer. obtain. Those of ordinary skill in the art will appreciate that doses will be best determined by the practitioner depending on the individual patient. It can be understood that a single intramuscular injection of 800 μg of microencapsulated primary antigen It may be sufficient to produce the desired response.   The second step of the method of immunization of the present invention is to orally administer a protective amount of Neisseria gonorrhoeae antigen. Including PIII-deficient killing (eg, gamma irradiation)N.gonorrhoeaeall The cell (eg strain 340) may be a Neisseria gonorrhoeae antigen.N.gonorrhoeae340 strain is a mouse Or parenteral formaldehyde death reduction in guinea pig infection model High level of cross protection against different N. gonorrhoeae strains when used as a whole cell immunogen Have been shown to induce. PIII^-Mutants are larger due to their use This is preferable because the level of defense result can be obtained. Other PIII^-Mutant strain is Wetzler Method taught byJ.Exp.Med, 169: 2199-2209 (1989)) 340 strains of PIII^-The mutant strain By methods known in the artrmpObtained by insertional inactivation of the structural gene. Simple Simply put,ermC(Erythromycin resistance) gene was inactivated by insertion of the gene LoanrmpIncorporate the gene into the N. gonorrhoeae chromosome, PIII^-Generated a phenotype.   Oral administration of whole cell immunogens is too toxic to be an essential antigen that can be administered parenterally To be vaccinated. In addition, the oral route interferes with N. gonorrhoeae colonization. It may be more effective in stimulating the mucosal immunity required for 1 x 10⁹CFU and 8 × 10⁹Oral immunogens with CFU can be administered at weekly intervals for 10 weeks. Oral immunity Sensitization can begin as early as 2 weeks after parenteral initial challenge, or up to 4 weeks Can be delayed. This 2-4 week period between parenteral priming and oral immunization Seems to be the optimal period.   Suitable carriers of Neisseria gonorrhoeae antigens used for oral administration include fragrances, lubricants, suspensions and Or contains one or more substances that may also act as protective agents It Suitable solid carriers are calcium phosphate, calcium carbonate, stearic acid. Ma Gnesium, sugar, starch, gelatin, cellulose, carboxypolymethylene , Or cyclodextrin. Suitable liquid carrier Can be water, pharmaceutically acceptable oils, or a mixture of both. This liquid Also, buffering agents, preservatives, perfumes, viscosity or osmotic pressure adjusting agents, stabilizers or suspending agents. Other suitable pharmaceutical additives such as An example of a suitable liquid carrier is pH With or without various additives including carboxypolymethylene as a conditioning gel Including fresh water. N. gonorrhoeae antigen is a gut that releases the antigen to the intestine to avoid decomposition in the stomach. It may be contained in a solvent peel capsule.   Alternatively, the N. gonorrhoeae antigen may be either a natural or synthetic polymer with a diameter of 4-8. It can be microencapsulated in μm microparticles, which remove intestinal or vaginal lymphoid tissue. Targeting and producing sustained release of antigen for up to 4 weeks (Eldridge et al.Cur.Topi csin Microbiol. and Immunol ., 146: 59-65 (1989); Oka et al.,Vaccine， 8: 573-5 76 (1990)).   Those skilled in the art will appreciate that a particular immunization schedule will be best tailored to the individual patient needs. It can be recognized that the effective oral regimen of the present invention is about 1 week apart. It may include 4-10 oral doses of N. gonorrhoeae antigen administered.   In another embodiment, the invention providesNeisseria menningitidisAgainst infection by A method of protecting a patient is provided that provides the patient with a pharmaceutically acceptable carrier. A parental amount of primary antigen sufficient to induce an immune response against the protein To The step of administering and then a protective amount of class 4 protein-deficient killingNeisseria meningitidis Orally administering the whole cells. Or wild-type death DestructionNeisseria meningitidisWhole cells can be administered as an oral immunogen.   According to the methods of the present invention, a human subject is initially treated with a synthetic immune adjuvant (eg, , Polyphosphazene) or one or more meningococcal class 2,3 tans It is primed with a synthetic peptide of protein. Initial antigen is effective for initial antigen administration In a pharmaceutically acceptable carrier in an amount sufficient to induce a fruit-bearing immune response. It is administered orally.   Immunogenic peptides can be prepared using standard methods known in the art, eg Pewter-based prediction algorithms and automated peptide synthesis (Rothbard and And Taylor) 1989, EMBO 7: 93-100) or synthesized. Peptide The immunogenicity of can be measured by the method described in Examples 4 and 5. Peptide Purity can be measured by analytical HPLC, and the presence of endotoxin indicates the presence of rabbit pyro. It can be measured by the Gen test.   The second step of the method of immunization of the present invention is to provide a protective amount of meningococcal antigen (eg S4 protein-killed (eg gamma irradiated)N.meningitidisThrough all cells Orally administering.   N.gonorrhoeaePreparation of synthetic peptides and microphones described for vaccines The encapsulation protocol isN.meningit idis It can be applied to vaccines as well.N.gonorrhoeaeParenteral and vaccination Examples of pharmaceutically acceptable carriers described for both oral administration are alsoN. meningitidis Can be extended to vaccines.   The method of the present invention overcomes some of the challenges common in the art. For example, a book Administration of the mutant cell line described in the invention,N.gonorrhoeaeDerived from PIII orN.menin gitidis The induction of blocking antibodies to the derived class 4 proteins may be reduced, This inhibits bactericidal antibodies against other cell surface antigens. In addition, conventional parenteral Unlike the method of dynamic immunization, the present invention uses a conformation-dependent enzyme on an intact organism. Provides an immunological response to the pitope. This is related to oral immunization This is achieved by using intact cells. Cell envelope antigen conformation Ion-dependent epitopes generate antibodies and other non-specific immune mechanisms Needed for, this can be preserved, and liquid gel, enteric peel capsules , Or microencapsulated suspensions without toxic effects, As a result, this antigen reaches the intestinal lymphoid tissue intact.   The present invention uses priming antigen as a primer and whole cells as an oral immunogen. It also addresses toxicity and cross-reactivity issues, especially in the induction of mucosal responses. To eliminate production of blocking antibody.   The experiments described in the parent application show that r-Fbp is orally on 340 WT cells. Although shown to be an effective parenteral priming antigen prior to immunization, the experiments of the present invention show that Liphosphazene or synthetic peptides are viable or irradiated PIII^-One of the cells Was shown to be substantially better as the primary antigen to be used before oral immunization (Tables 1 and 5).   How many synthetic peptides are used as parenteral priming antigens compared to the use of r-Fbp? 1) relatively small peptides can be synthesized and synthesized by biochemical methods. And can be purified to produce an endotoxin-free product; 2) this synthetic peptide. Have produced amino acid substitutions that can be evaluated for improved primary antigen sensitization capacity. 3) This peptide is highly cationic r-Fbp (pl 10.35). ) Is a more neutrally charged molecule and therefore in some types of autoimmune disease The effect on DNA binding suspected to be a factor appears to be minor; and 4) this page Petit PIII^-Better primed with N. gonorrhoeae, which is 340 WT cells May be better as an oral vaccine candidate than.   The use of polyphosphazene as a primed drug is 1) its water solubility, i Cross-linked structure and high molecular weight, which prevents excretion and slow addition It is hydrolyzed and removed from the tissue; 2) a common first shot against other oral vaccines Possibility as an antigenic substance; 3) Its parenteral administration in a relatively small dose (100 μg) Advantages due to immunoreactivity; and 4) lack of parenteral toxicity.   Dead cells and especially gamma-irradiated PIII^-Cells have some advantages as an oral immunogen 1) anti-PIII or "blocking" antibodies are 2) gamma-irradiation is PIII^-Oral immunization of mutant strain Can enhance the originality (Table 1); 3) Irradiated PIII^-Immunogen is live PIII^-Cell or other 3x higher level of chamber protection and significantly better than immunogen (P <.01) Inducing vaginal clearance (Table 1); and 4) gamma-irradiation is another chemically inactive Provides a convenient method of "in-container" sterilization without the toxic residues often left behind by sterilization methods To do. This results in a safer product of oral immunization.   N.gonorrhoeae The 340 WT strain was purchased on April 23, 1992 under the terms of the Budapest Treaty. American Type Culture Collection, 12301 Parklawn Drive, Rockville , Maryland 20852. 340 WT strain was given accession number ATCC 55320 . This strain may be made available upon issuance of a patent without restriction.   The invention is described in more detail in the following examples, which are for illustrative purposes only Many obvious modifications and variations will be apparent to those of ordinary skill in the art. Example   Example 1.Chambers of various combinations of parenteral priming antigen and oral components And vaginal clearance comparison .   To test different parenteral priming antigens and oral immunogens, More than 300 6-week-old female ICR outbred mice were treated with 15 μg / dose of recombinant iron-binding tantalum. Protein (r-Fbp) or synthetic peptide (amino acid sequence DDQTYSIPSLFV) at 20 μg / dose , SEQ ID NO: 1) was intramuscularly injected 7 times at weekly intervals. First antigen administration 2 weeks later, wild typeN.gonorrhoeae 340 strains (340 WT) or 340 strains of protein II I deletion mutant (340PIII^-Oral immunization with live bacterial cells or gamma-irradiated cells I made a sensitization.Escherichia coli I5 cell line as a control oral immunogen used. Once a week, 10 mice in 0.5 ml volume⁹Stomach with 10 doses including CFU The animals were immunized orally by feeding by feeding tube.   Two weeks before the last oral immunization, each mouse was surgically transferred to a subcutaneous culture chamber. Planted (Arko, R.J.,J. Infect. D1s., 129: 451-455 (1974)).   After 4 weeks, all groups of mice were graded with pathogenic 340 WT cell lines. Dose was administered. 50% of infectious dose (ID₅₀) From the graph for each group It was decided (Table 1).   In addition, a gonococcal vaginal clearance test was performed for each group. In the vagina of the mouse , 400,000 CFU of pathogenic 340 WT cell line and vaginal lavage 6 hours post infection Harvested and assayed by culture of N. gonorrhoeae (Table 1).   In the previous experiment, mice challenged with r-Fbp were orally challenged with 340 WT cells. After epidemic sensitization, protection is to a greater extent, while the synthetic peptide primes the primary antigen. 340PIII was administered to mice^-Greater degree after oral immunization with cells Well Was defended by. The data in Table 1 is for synthetic peptide prime antigen and 340 PIII.^-Cell oral immunity Compared to the r-Fbp primary antigen / 340 WT cell immunogen combination , Provided excellent chamber and vaginal protection. These data are Also, gamma irradiation 340 PIII^-Live cells 340 PIII^-A stronger immune response than cells Indicates that it has been induced.       Example 2.IgA and IgG antibody titers during vaginal lavage.   To determine whether immunization by the method of the invention produces a mucosal immune response , IgA and IgG antibody titers in vaginal lavage fluid were measured 2 weeks before vaginal challenge with N. gonorrhoeae did. Mice received either 15 μg / dose r-Fbp or 20 μg / dose synthetic peptide. Were parenterally primed with 7 injections per week and then 10⁹History of CFU Oral immunogen was administered 10 times at weekly intervals. Whole cells on plates coated with 340 WT cells The titer was determined by vesicle ELISA.   The data in Table 2 are for synthetic peptide prime antigen and 340 PIII.^-Cell combinations, and And r-Fbp priming antigen and 340 WT cells combined with IgA and 340 WT cells. We show that it induces high titer production of both IgG antibodies in the vagina.       Example 3.Cross-reactive bactericidal activity of sera obtained from complement-deficient mice.   Purified iron-binding protein (Fbp) or 340 WT cells, either alone Serum from a mouse parenterally primed by itself, orally with 340 WT cells alone Sera derived from mice immunized with Escherichia coli or Fbp were parenterally primed Sera from mice immunized orally with 340 WT cells were then challenged with various microorganisms. Assayed for bactericidal activity. Aliquot 10 μl of serum into Place a confluent number of the product on the inoculated agar plate:N.gonorrhoeae 340 WT,N.gonorrhoeae 11B,N.gonorrhoeae F62, andN.meningitidis Agroup. Soshi The percentage of dead cells was calculated. As shown in Table 3, parenteral first immunization The combination method of multiple antigens / oral immunogens was used to immunize with either component alone. In comparison, it induced a greater cross-reactive bactericidal activity. This response also lacked complement. Induced in the missing outbred ICR mouse, which has bactericidal activity but classical complement pathway Shows that it did not depend on.       Example 4.Parenteral primary challenge only with peptides and oral with whole cells Comparison with parenteral initial challenge with peptide with immunity .   340 PIII^-Set of synthetic peptide prime challenge for oral immunization with cells The combination was superior to the combination of r-Fbp primed antigen and 340 WT cells. Since it provided bar protection and vaginal protection (Table 1), synthetic peptide prime antigen and 340 PI II^-The combination with cells was further investigated for its potential as a vaccine .   A group of 154 6-week-old female ICR outbred mice was treated with the amino acid sequence DDQTYSIPSLFV ( 5 μg, 20 μg or 50 μg of synthetic peptide consisting of column number 1) at weekly intervals Three, five or seven intramuscular injections were made. This is the first time after 2 weeks with half of the mice 340 PIII which was gamma-irradiated following single challenge^-Cells were immunized orally. Orally free 10 in 0.5 ml volume in quarantine mice⁹Gastric nutrition with 10 doses once a week including CFU Given by tube. Two weeks before the last oral immunization, in each mouse, Subcutaneous culture chambers were surgically implanted as described.   After 4 weeks, all mice were challenged with the graded dose of pathogenic 340 WT cells. Was administered. ID₅₀Was determined from the graph for each group (Table 4). First in synthetic peptide Single dose and gamma irradiation 340 PIII^-Mice immunized orally with cells are Mice treated with the initial antigen or synthetic oral immunization alone. Resisted a greater number of Neisseria gonorrhoeae.   In addition, the data in Table 4 shows that the initial resistance with 50 μg synthetic peptides for 3 or 5 weeks was not significant. Original dose, then 340 PIII^-10 oral immunizations with cells, 7 weeks with r-Fbp Challenge with 340 WT cells followed by 10 oral immunizations Greater protection (greater ID than protection)₅₀) Is provided.       Example 5. Soluble Poly as a Single Injection Primer for Oral Immunization Comparison of phosphazene and N. gonorrhoeae synthetic peptides .   A group of 63 6-month-old female ICR mice was treated with a synthetic peptide (amino acid sequence DDQTYSIPS LFV, SEQ ID NO: 1) in combination with polyphosphazene, or either Was administered intramuscularly at the doses listed in Table 5 with the above ingredients alone. This parenteral primary antigen Γ-irradiated 340 PIII 4 weeks after administration^-Cells were immunized orally. Oral immunity Sensitization is 10 in 0.5 ml volume by gastric feeding tube⁹10 doses once a week including CFU It consisted of Two weeks before the last oral immunization, each mouse was described in Example 1. Subcutaneous culture chambers were surgically implanted as described.   Four weeks later, all mice were challenged with pathogenic 340 WT strain cells at a graded dose. I gave it. ID₅₀Was determined from the graph for each group, and 700, 5,000 and 92,000  After challenge with a CFU dose, the percentage of infected mice was calculated (Table 5).   Result: polyphosphazene (with or without peptide), synthetic polypeptide Or r-Fbp for parenteral primary challenge, followed by 340 WT cells. Or 340 PIII^-In mice induced by immunization with any of the cells The immune response was evaluated by the following measurements: 1) ID in subcutaneous chamber challenge₅₀ 2) vaginal clearance, 3) vaginal IgG and IgA antibody production, 4) cross-reactivity, And 5) bactericidal activity in the absence of complement.   These data are for either polyphosphazene or synthetic peptide prime antigens. Parenteral administration of 340 PIII^-Oral immunization of cells (into the subcutaneous chamber ID₅₀Level and challenge as measured by vaginal clearance of N. gonorrhoeae ) Shows that a strong immune response is obtained (Tables 1, 4 and 5).   Synthetic peptides and 340 PIII^-In combination with cells, both IgA and IgG antibodies in the vagina (Table 2), which is required to prevent N. gonorrhoeae colonization. It is important in providing mucosal immunity.   Polyphosphazene alone then 340 PIII^-Application of oral immunization with cells: 1 ) Combination of polyphosphazene and peptide, then 340 PIII^-Oral immunization (P <0.001), 2) Peptide alone, then 340 PIII^-Oral immunization (P <0.05), And 3) 340 PIII^-Significantly better antigen than oral immunization alone (P <0.01) Protected against dose infection.   The present invention also provides cross-reactive bactericidal activity (Table 3) and heterogeneity against different N. gonorrhoeae strains. Induced protective immunity that does not require complement. Outcrossing deficient in the early resting component C2 Since ICR mice were used, they were induced by parenteral immunization with whole Neisseria gonorrhoeae in the previous experiment. Bactericidal antibodies and defenses are needed to show this high level of immunity in order to Requires addition of chamber fluid with source (Arko et al.,J. Infect. Dis.， 139: 5 69-574 (1979)). These data show that the bactericidal activity shown in these experiments is classical. The present invention does not depend on the complement pathway at all, and the present invention is a site where complement components are restricted (eg, Urogenital tract) may be more effective in providing protection.                        Example 6. Human vaccination   Curing humans with either N. gonorrhoeae or N. meningitidis vaccine The method involves a single parenteral injection of up to 800 μg of the initial antigen (eg, polyphosphazene), It then included oral administration of enteric-coated capsules for 4 weeks, then at weekly intervals for 10 weeks. obtain. Each capsule is 5 × 10⁹CFU gamma irradiation 340 PIII^-It may include N. gonorrhoeae.   Such administration may protect against subsequent infection by this organism.   Throughout this application various publications are referenced. The disclosures of these publications are The body is hereby incorporated in order to more fully describe the state of the art to which this invention pertains. It is used as a reference.   The method of the present invention will be described with reference to the particular details of its particular embodiment. However, such details are to be considered as and as extensions included in the appended claims. To be considered limiting with respect to the scope of the invention, except as it is done Is not intended.                                 Sequence listing (1) General information:   (i) Applicant:     (A) Name: The Government of the United States of              United States, AS Represented By The Secretary     (B) Address: Suite 325 Executive Building 6011     (C) City: Rockville     (D) State: Maryland     (E) Country: United States     (F) Postal code (ZIP): 20852     (G) Telephone: (301)496-7056     (H) Telefax: (301)402-0220     (I) Telex: None   (ii) Title of invention:Neisseria gonorrhoeaeandNeisseria meningitidisAgainst Immunization to   (iii) Number of sequences: 4   (iv) Computer readout form:     (A) Medium type: Floppy disk     (B) Computer: For IBM PC compatibility     (C) Operating system: PC-DOS / MS-D0S     (D) Software: Patent-in Release # 1.0, Version # 1.25 (EP0)   (v) Current application data:     (A) Application number: undecided     (B) Application date: October 26, 1993   (vi) Information on prior application     (A) Application number: US07 / 965,916     (B) Application date: October 26, 1992 (2) Information of SEQ ID NO: 1:   (i) Features of the array:     (A) Length: 12 amino acids     (B) type: amino acid     (C) Number of chains: single chain     (D) Topology: linear   (ii) Sequence type: peptide   (xi) Sequence: SEQ ID NO: 1: (2) SEQ ID NO: 2 information:   (i) Features of the array:     (A) Length: 12 amino acids     (B) type: amino acid     (C) Number of chains: single chain     (D) Topology: linear   (ii) Sequence type: peptide   (xi) Sequence: SEQ ID NO: 2: (2) SEQ ID NO: 3 information:   (1) Features of array:     (A) Length: 12 amino acids     (B) type: amino acid     (C) Number of chains: single chain     (D) Topology: linear   (ii) Sequence type: peptide   (xi) Sequence: SEQ ID NO: 3: (2) SEQ ID NO: 4 information:   (i) Features of the array:     (A) Length: 13 amino acids     (B) type: amino acid     (C) Number of chains: single chain     (D) Topology: linear   (ii) Sequence type: peptide   (xi) Sequence: SEQ ID NO: 4:

─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Arco, Robert Jay.             United States of America Georgia 30307,             Tanta, N. E. ， Indiana             Avenue 1760 (72) Inventor Chen, Cheng Guen.             United States Georgia 30084, Ta             Kucker, Angels Lane 6718 (72) Inventor Morse, Stephen A.             United States Georgia 30341, Chi             Embry, Hearts Run 1703 (72) Inventor Trees, David             United States Georgia 30244, Ro             Lensville, Millstream Trail               1409

Claims (1)

[Claims] 1. Protein III-deficient killed Neisseria gonorrhoeae whole cells in a first container having an initial antigen in a pharmaceutically acceptable carrier suitable for parenteral administration and in a pharmaceutically acceptable carrier suitable for oral administration And a second container having a protective amount of killed whole cells selected from Class 4 protein-deficient killed Neisseria meningitidis whole cells. 2. 2. The kit of claim 1, wherein the priming antigen is an immunogenic peptide from Neisseria gonorrhoeae protein I and the killed whole cells are protein III-deficient killed Neisseria gonorrhoea e whole cells. 3. The kit according to claim 2, wherein the peptide consists essentially of the amino acid sequence QHQVYSIPSLFV defined in SEQ ID NO: 2 of the Sequence Listing. 4. The kit according to claim 2, wherein the peptide consists essentially of the amino acid sequence EHQVYSIPSLFV defined in SEQ ID NO: 3 of the Sequence Listing. 5. The kit according to claim 2, wherein the peptide consists essentially of the amino acid sequence DDQTYSIPSLFV defined in SEQ ID NO: 1 of the Sequence Listing. 6. The kit according to claim 2, wherein the peptide consists essentially of the amino acid sequence ASVAGTNTGWGN K defined in SEQ ID NO: 4 in the sequence listing. 7. 7. The kit according to any one of claims 1 to 6, wherein the killed whole cells are protein III-deficient killed Neisseria gonorrhoeae 340 strain whole cells deposited with the American Type Culture Collection under accession number AT CC 55320. 8. The kit according to claim 1, wherein the initial antigen is an immunogenic peptide derived from Neisseria meningitidis , and the dead whole cells are dead Neisseria meningitidis class 4 protein-deficient strain whole cells. 9. The kit according to claim 8, wherein the peptide is derived from Neisseria meningitidis class 2 protein protein. 10. The kit according to claim 8, wherein the peptide is derived from Neisseria meningitidis class 3 protein. 11. The kit according to claim 2, which comprises 11.1 or more types of peptides. 12. The kit according to claim 1, wherein the initial antigen is a synthetic immune adjuvant. 13. The kit according to claim 12, wherein the immunoadjuvant is polyphosphazene. 14. Use of protein III-deficient killed Neisseria gonorrhoea e whole cells for the manufacture of a medicament for oral administration for use in a human patient against infection by Neisseria gonorrhoeae, wherein the patient produces an immune response. Use, wherein the primary antigen is administered parenterally in an amount sufficient to induce. 15. Use of class 4 protein deficient killed Ne isseria meningitidis whole cells for the manufacture of a medicament for oral administration for use in protecting human patients against infection by Neisseria meningitidis , wherein said patient is Use of a priming antigen administered parenterally in an amount sufficient to induce an immune response. 16. Use according to claim 14, wherein the whole cells are whole cells as defined in claim 7.