MXPA97008481A - Acellular vaccines of pertussis and methods of preparation of the mis - Google Patents

Abstract

The present invention relates to acellular pertussis vaccines comprising purified toxin or toxoid thereof, filamentous haemagglutinin, pertactin and fimbrial agglutinogen formulated to confer protection to at least 70% of the members of a population at risk. The fimbrial agglutinogens can be prepared from a Bordetella layer, particularly the B. pertussis layer, by means of a multistage process that involves: the extraction of the fimbrial agglutinogens from the cell paste and the concentration and purification of the extracted material.

Description

ACETAL VACCINES OF PERTUSSIS AND METHODS OF PREPARATION THEREOF FIELD OF THE INVENTION The present invention relates to acellular pertussis vaccines, their components and their preparation.

REFERENCE WITH RELATED REQUESTS > This application is a continuation in part of co-pending United States patent application No. 08 / 501,743 filed July 12, 1995, which is a continuation in part of the co-pending United States patent application No. 08 / 433,646 filed on May 4, 1995.

BACKGROUND OF THE INVENTION Pertussis or pertussis is a severe infection of the upper respiratory tract, highly contagious, caused by Bordetella pertussis. The World Organization of Health estimates that there are 60 million cases of pertussis per year and 0.5 to 1 million associated deaths (ref.) Throughout this specification, various references will be pointed out to fully describe the state of the art to which the invention pertains.

Complete bibliographic information for each citation is at the end of the specification, immediately before the claims. The presentation of these references are incorporated herein by reference). In unvaccinated populations, the incidence rate of pertussis observed in children under 5 years of age is up to 80% (ref 2). Although pertussis is generally considered to be a childhood disease, there is increasing evidence of asymptomatic and clinical disease in adolescents and adults (refs 3, 4, and 5). The introduction of full cell vaccines composed of B organisms. pertuesis deactivated thermally and chemically, was in 1940, responsible for the dramatic reduction in the incidence of pertussis caused by B. pertussis. The efficacy rates of full-cell vaccines have been estimated up to 95% depending on the case definition (ref 6). While the infection with B. pertussis confers lifelong immunity, there is increasing evidence of reduced protection after immunization with whole cell vaccines (ref 3). Several reports cite a relationship between full-cell pertussis vaccine, reactogenicity, and serious side effects that lead to a decline in vaccine acceptance and consequent renewed epidemics (ref 7). More recently they have been developed pertussis vaccines with defined components.

Antigens for Defined Pertussis Vaccines Several acellular pertussis vaccines have been developed and include Bordetella pertussis antigens, Pertussis toxin (PT), filamentous haemagglutonin (FHA), 69 kDa outer membrane protein (pertactin) and fimbrial agglutinogens (see Table 1 below: Tables appear at the end of the specification).

Pertussis toxin Pertussis toxin is an exotoxin that is a member of the A / B family of bacterial toxins with ADP-ribosyltransferase activity (ref 8). The entity A of these toxins exhibits ADP-ribosyltransferase activity and the B portion mediates the binding of the toxin with host cell receptors and the translocation of A to its site of action. PT also facilitates the adhesion of B. pertussi s to the ciliated epithelial cells (ref 9) and also plays a role in the invasion of macrophages by B. pertussis (ref 10). All acellular pertussis vaccines have included PT, which has been proposed as an important virulence factor and a protective antigen (ref 11, 12). The natural infection with B. pertussis generates answers P511 both humoral and cell-mediated towards the PT (refs 13 to 17). Infants who have transplacentally derived anti-PT antibodies (refs. 16, 18) and human colostrum containing anti-PT antibodies were effective in the passive protection of mice against aerosol-transmitted infection (ref.19). A cell-mediated immune response (CMI) to PT subunits has been demonstrated after immunization with an acellular vaccine (ref 20) and a CMI response to PT has been generated after the whole cell vaccine (ref 13). ). PT chemically inactivated in whole cell or component vaccines is protective in animal models and in humans (ref 21). further, monoclonal antibodies specific for the SI subunit protect against B infection. pertuseis (refs. 22 and 23). The main pathophysiological effects of PT are due to its ADP-ribosyltransferase activity. The PT catalyzes the transfer of ADP-ribosome from NAD to the guanidine nucleotide binding protein Glf thus breaking the regulatory system of cell adenylate cyclase (ref 24). PT also prevents the migration of macrophages and lymphocytes to inflammation sites and interferes with neutrophil-mediated phagocytosis and bacterial extermination (ref 25). Several in vi tro and in vivo trials have been used to evaluate the enzymatic activity of SI and / or PT, including the ADP-ribosylation of bovine transducin (ref 26), the Chinese hamster ovary cell pool (CHO) (ref 27), histamine sensitization (ref. 28), leukocytosis and glycohydrolase NAD. When exposed to PT, CHO cells develop a characteristic grouped morphology. This phenomenon depends on the binding of PT and the subsequent translocation and ADP-ribosyltransferase activity of SI and, therefore, the cluster assay of CHO cells is widely used to test the integrity and toxicity of PT holotoxins.

I have filamentous agglutonin Filamentous hemagglutonin is a large non-toxic polypeptide (220 kDa) that mediates the binding of B. pertussis with the cililated cells of the upper respiratory tract during bacterial colonization (ref 9, 29). The natural infection induces anti-FH antibodies and cell-mediated immunity (refs 13, 15, 17, 30 and 31). Anti-FHA antibodies are found in human colostrum and are also transmitted through the placenta (refs 17, 18 and 19). Vaccination with acellular or whole cell pertussis vaccines generates anti-FHA antibodies and acellular vaccines containing FHA also induce a CMI response to FHA (refs 20, 32). FHA is an antigen protector in a mouse respiratory inoculation model after active or passive immunization (ref 33, 34). However, FHA alone does not protect in the intracerebral inoculation potency assay in mice (ref 28). 69 kDa External Membrane Protein (Pertactin) The 69 kDa protein is an outer membrane protein that was originally identified from B. bronchisepti ca (ref 35) It has been shown to be a protective antigen against B. bronchiseptica and subsequently was identified in both B. pertussis as in B. parapertussis The 69 kDa protein binds directly to the eukaryotic cells (ref 36) and the natural infection with B. pertussis induces a humoral anti-P.69 response (ref 14) and P.69 also induces a cell-mediated immune response (ref 17, 37, 38). Vaccination with whole cell or acellular vaccines induces anti-P.69 antibodies (ref 32, 39) and acellular vaccines induce CMI with P.69 (ref 39). Pertactin protects mice against aerosol inoculation with B. pertussis (ref 40) and in combination with FHA protects in the intracerebral inoculation test against B. pertussis (ref 41). Passive transfer of polyclonal and monoclonal anti-P.69 antibodies also protects mice against aerosol inoculation (ref 42).

Agglutinogens The serotypes of B. pertussis are defined by their fimbrias agglutinantes. The WHO recommends that full-cell vaccines include agglutinogen types 1, 2 and 3 (Aggs) since they are not protective in the transverse direction (ref 43). Agg 1 does not have fimbrias and is found in all strains B. pertussis while serotypes 2 and 3 of Aggs have fimbriae. The infection or natural immunization with acellular or whole cell vaccines induces the anti-Agg antibodies (ref 15, 32). A specific cell-mediated immune response can be generated in mice by Agg 2 and Agg 3 after the aerosol infection (ref 17). Aggs 2 and 3 are protective in mice against respiratory inoculation and human colostrum containing anti-agglutinogens will also protect in these assays (refs 19, 44, 45).

Acellular Vaccines The first acellular vaccine developed was the PT + FHA two-component vaccine (JNIH 6) by Sato et al. (ref 46). This vaccine was prepared by the co-purification of the PT and FHA antigens from the supernatant culture of B. pertussis, Tohama strain, followed by toxin with formalin. The acellular vaccines from different manufacturers and different compositions have been P511 successfully used to immunize Japanese children against pertussis since 1981, resulting in a considerable decrease in the incidence of the disease (ref 47). The JNIH 6 vaccine and a monocomponent PT toxoid vaccine (JNIH 7) were tested in a large clinical trial in Sweden in 1986. Initial results indicated lower efficiency than the reported efficiency of whole cell vaccines, but studies of Follow-up has shown that it is more effective against mild diseases diagnosed by serological methods (refs 48, 49, 50, 51). However, there was evidence of reversion to the toxicity of PT inactivated with formalin in these vaccines. These vaccines also protected against diseases and not against infections. A number of novel pertussis acellular vaccines are currently being evaluated including combinations of PT, FHA, P.69, and / or agglutinogens and these are listed in Table 1. Various chemical detoxification techniques have been used for PT , including inactivation with formalin (ref 46), glutaraldehyde (ref 52), hydrogen peroxide (ref 53), and tetranitromethane (ref 54). Thus, commercially available acellular pertussis vaccines currently can not contain adequate formulations of antigens P511 suitable in suitable immunogenic forms to achieve a desired level of efficacy in a human population susceptible to pertussis. It would be desirable to provide effective pertussis acellular vaccines containing selected relative amounts of selected antigens and methods for the production thereof.

SUMMARY OF THE INVENTION The present invention relates to preparations of acellular pertussis vaccine, components thereof, methods of preparation of these vaccines and their components, and methods of use thereof. In a further aspect of the invention, there is provided an immunogenic composition comprising the fimbrial agglutinogen preparation that is provided herein. The immunogenic composition can be formulated as a vaccine for in vivo use for the protection of a host immunized therewith from the disease caused by Bordetella and can comprise at least one other Bordetella antigen. The at least one other Bordetella antigen may be a filamentous hemagglutinin, the adenylate cyclase of the 69 kDa outer membrane protein, Bordetella lipooligosaccharide, outer membrane proteins and toxin P511 pertussis or a toxoid thereof, which includes the genetically detoxified analogs thereof. In a further aspect of the invention, the immunogenic composition provided herein may comprise at least one non-Bordetella immunogen. This non-Bordetella immunogen can be a diphtheria toxoid, tetanus toxoid, Haemophilus capsular polysaccharide, Haemophilus outer membrane protein, hepatitis B surface antigen, polio, mumps, measles and / or rubella. The immunogenic compositions provided herein may further comprise an adjuvant and that adjuvant may be aluminum phosphate, aluminum hydroxide, Quil A, QS21, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analogue, an octodecyl ester of an amino acid or a lipoprotein. According to one aspect of the present invention, a vaccine composition is provided to protect a human population at risk against a case of disease caused by infection with B. pertussis. which comprises pertussis toxoid, filamentous haemagglutinin, pertactin and agglutinogens in purified form, in relative amounts selected to confer protection to a degree of at least 70% of the members of the population at risk.

This vaccine composition can contain between about 5 and 30 μg of pertussis toxoid nitrogen, between about 5 and 30 μg of filamentous hemagglutinin nitrogen, between about 3 and 15 μg of pertactin nitrogen and between about 1 and 10 μg of nitrogen of agglutinogens. In a specific embodiment, the vaccines may comprise pertussis toxoid, filamentous haemagglutinin, the 69 kDa protein and Bordetella filamentous agglutinogens at a weight ratio of between about 10: 5: 5: 3 as provided by approximately 10 μg of the pertussis toxoid , approximately 5 μg of the filamentous hemagglutinin, approximately 5 μg of the 69 kDa protein and approximately 3 μg of the fimbrial agglutinogens in a single human dose. In a further particular embodiment, the vaccine may comprise the pertussis toxoid, filamentous haemagglutinin, 69 kDa protein and fimbrial agglutinogens in a weight ratio of between about 20: 20: 5: 3, as provided by approximately 20 μg of the pertussis toxoid , about 20 μg of the filamentous hemagglutinin, about 5 μg of the 69 kDa protein and about 3 μg of the fimbrial agglutinogens in a single human dose. Still in another particular modality, the vaccine may comprise pertussis toxoid, filamentous haemagglutinin, 69 kDa protein and fimbrial agglutinogens in a weight ratio of between about 20: 10: 10: 6 as provided by approximately 20 μg of the pertussis toxoid, about 10 μg of the filamentous hemagglutinin, about 10 μg of the 69 kDa protein and about 6 μg of the fimbrial agglutinogens in a single human dose. The degree of protection of the human population at risk that is achieved by the vaccine composition of this invention can be at least about 80%, preferably about 85%, for a case of spasmodic cough of at least duration 21 days and bacterial infection confirmed by culture. The degree of protection of the human population at risk can be at least 70% for a case of mild pertussis that has a cough of at least one day. The agglutinogen component of the vaccine preferably comprises fimbrial agglutinogen 2 (Agg 2) and fimbrial agglutinogen 3 (Agg 3) essentially free of agglutinogen 1. The weight ratio of Agg 2 to Agg 3 can be between approximately 1.5: 1 and 2. :1. The vaccine provided herein may be combined with tetanus toxoid and diphtheria toxoid to provide a DTP vaccine. In one embodiment, the vaccine contains approximately 15 Lfs of diphtheria toxoid and approximately 5 Lfs of tetanus toxoid. In addition, the vaccine may also comprise an adjuvant, particularly alum. In a further aspect of the present invention, there is provided a method for immunizing a human population at risk against the disease caused by B. pertussis infection, which comprises administering to the members of the human population at risk an effective amount of the composition of vaccine provided here, to confer protection in a degree of at least 70% of the members of the population at risk. The advantages of the present invention include an improved acellular pertussis vaccine composition with increased efficacy. The present invention further provides, in a further aspect, purified forms of pertussis toxin, filamentous haemagglutinin, pertactin and fimbrial agglutinogens of B. pertussis when used in the manufacture of a vaccine composition for administration to a population of humans at risk, to provide protection in an area of at least approximately 70% of the members of said population of humans at risk. For this use, it can be used in manufacturing P511 of a single dose for human of the vaccine composition of approximately 30 μg of pertactin nitrogen and from about 1 to about 10 μg of nitrogen from fimbrial agglutinogens. In particular, the vaccine composition as provided herein has been selected by the National Institute of Allergic and Infectious Diseases (NIAID) of the Government of the United States of America, for evaluation in a double blind human efficacy clinical trial , thus establishing for the specially experienced in this field, a sufficient basis that the compositions will be effective to a certain degree to avoid the declared disease (pertussis). The subject of the trial (it will be a vaccine as provided herein) has been subjected to the burden of proving to be reasonably predictive of its usefulness.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be further understood from the following detailed description and from the Examples in relation to the accompanying drawings, wherein: Figure 1 is a schematic flow diagram of a method for the isolation of a preparation of agglutinogen from a Bordetella strain.

P511 DETAILED DESCRIPTION OF THE INVENTION Referring to Figure 1, a flow diagram of a method for preparing an agglutinogen preparation from the Bordetella strain is illustrated. As seen in Figure 1, a Bordetella cell paste containing the agglutinogens, for example cell paste B. pertussis, is extracted, for example, with a buffer containing urea, such as 10 mM potassium phosphate, 150 mM NaCl and 4M urea, to selectively extract the agglutinogens from the cell paste and produce a first supernatant (spl). ) containing agglutinogens and a first residual precipitate (pptl) The first supernatant (spl) is separated from the first residual precipitate (pptl) for example by centrifugation. The residual precipitate (pptl) is discarded. The clarified supernatant (spl) can then be concentrated and filtered by dialysis against, for example, lOmM potassium phosphate / 150mM NaCl / 0.1% Triton X-100 using, for example, a NMWL membrane filter of 100 to 300 kDa. The first supernatant is then incubated at a temperature and for a time to produce a clarified supernatant (sp2) containing agglutinogens and a second precipitate is discarded (ppt2) containing non-agglutinogen type contaminants. Suitable temperatures include between approximately 50 ° C and 100 ° C, including P511 about 75 ° to about 85 ° C and suitable incubation times including between about 1 and 60 minutes. The clarified supernatant is then concentrated, for example, by the addition of polyethylene glycol of molecular weight of about 8000 (PEG 8000) to a final concentration of between about 4.5 ± 0.2% and stirring gently for a minimum of about 30 minutes to produce a third precipitate (ppt3) that can be collected by centrifugation. The remaining sp3 supernatant is discarded. This third precipitate (ppt3) is extracted, for example, with a buffer comprising lOmM potassium phosphate / 150mM NaCl to provide the solution containing crude fimbrial agglutinogen. Potassium phosphate 1M can be added to the crude fimbrial solution to make it approximately 100mM compared to potassium phosphate. Alternatively, the clarified supernatant of the thermally treated fimbrial agglutinogens can be purified without precipitation by gel filtration chromatography using a gel, for example Sepharose CL6B. The fimbrial agglutinogens in the crude solution are then purified by column chromatography, for example, by passing them through a PEI silica column, to produce the fimbrial agglutinogen preparation in the eluate.

P511 This eluent containing fimbrial agglutinogen can then be concentrated and filtered by dialysis for example against a buffer containing potassium phosphate lOmM / 150 150mM NaCl using a NMWL membrane of 100 to 300 kDa. The agglutinogen preparation can be sterilized by filtration through a membrane filter less than or equal to 0.22 μm, to provide the final purified fimbrial agglutinogen preparation containing fimbrial agglutinogen 2 and 3. A preparation of agglutinogen from an Bordeteila strain can comprising fimbrial agglutinogen 2 (Agg 2) and fimbrial agglutinogen 3 (Agg 3) essentially free of agglutinogen 1. The weight ratio of Agg 2 to Agg 3 may be between about 1.5: 1 and 2: 1. These fimbrial agglutinogen preparations can be produced by the method provided herein and described in detail before. The present invention also extends to immunogenic compositions (including vaccines) comprising the fimbrial agglutinogen preparations provided as described above. These vaccines contain other Bordetella immunogens that include filamentous hemagglutinins, the 69 kDa outer membrane protein and pertussis toxin or a toxoid thereof, including genetically detoxified analogs of PT as described, for example, in ref.

P511 68. These vaccines may include immunogens of the non-Bordetella type, which include diphtheria toxoid, tetanus toxoid, Haemophilus capsular polysaccharide, Haemophilus outer membrane protein, hepatitis B surface antigen, polio, mumps, measles, and rubella. Each of the Bordetella antigens is individually absorbed into the adjuvant (such as alum) to provide rapid and convenient production of vaccines containing the selected relative amounts of the antigen in the vaccines provided herein in order to provide protection in an extension of at least approximately 70% of the members of a population at risk, preferably of at least approximately 80% of that population. In the selected embodiments, the invention provides vaccines with the following characteristics (μg of proteins used here based on the results of the Kjeldahl test carried out on purified concentrates and expressed as μg of protein nitrogen), all of which are administered by intramuscular injection. : A formulation of the component pertussis vaccine combined with tetanus and diphtheria toxoids was designated CP10 / 5/5 / 3DT. Each human dose of 0.5 ml of P511 CP1o / 5/5 / 3DT was designed to contain approximately: μg Toxoid pertussis (PT) 5 μg Filamentous hemagglutonin (FHA) 5 μg Fimbrial agglutinogens 2 and 3 (FIMB) 3 μg outer membrane protein 69 kDa 15 Lf Diphtheria toxoid 5 Lf Tetanus toxoid 1.5 mg Aluminum 0.6% phosphate 2-phenoxyethanol, as a preservative A formulation of the component pertussis vaccine combined with tetanus and diphtheria toxoids was named CP2? / 20/5 / 3DT- Each human dose of 0.5 ml of CP20 / 20/5 / 3DT was formulated to contain approximately: μg Toxoid pertussis (PT) 205 μg Filamentous hemagglutonin (FHA) 5 μg Fimbrial agglutinogens 2 and 3 (FIMB) 3 μg outer membrane protein 69 kDa 15 Lf Diphtheria toxoid 5 Lf Tetanus toxoid 1.5 mg Aluminum phosphate 0.6% 2-phenoxyethanol, as a preservative Another formulation of the component pertussis vaccine combined with tetanus and diphtheria toxoids is P511 he named CP10 / 5 / 5DT. Each human dose of 0.5 ml of CP10 / 5 / 5DT was formulated to contain approximately: μg Toxoid pertussis (PT) 5 μg Filamentous hemagglutonin (FHA) 5 μg Fimbrial agglutinogens 2 and 3 (FIMB) 15 Lf Diphtheria toxoid 5 Lf Tetanus toxoid 1.5 mg Aluminum phosphate 0.6% 2-phenoxyethanol, as preservative A formulation of the component pertussis vaccine combined with diphtheria and tetanus toxoids was named CP2? /? O / lO / 6DT- Each 0.5 ml of human dose of CP2o / i? / LO / 6DT was formulated to contain approximately:r. 2o μg Toxoid pertussis (PT) 10 μg Filamentous hemagglutonin (FHA) 10 μg Fimbrial agglutinogens 2 and 3 (FIMB) 6 μg outer membrane protein 69 kDa 15 Lf Diphtheria toxoid 5 Lf Tetanus toxoid 1.5 mg Aluminum phosphate 0.6% 2-phenoxyethanol, as a preservative The other Bordetella immunogens, pertussis toxin (including genetically detoxified analogs) P511 of the same, as described, as for example, in Klein et al, U.S. Patent No. 5,085,862 assigned to the assignee hereof and incorporated herein by reference), FHA and the 69 kDa protein can be produced by a variety of methods such as those described below: Purification of PT PT can be isolated from the culture supernatant of a B strain. pertussis using conventional methods. For example, the method of Sekura et al (ref 55) can be used. PT is first isolated by absorbing the culture supernatant on a column containing the ligand gel matrix with dye, Affi-Gel Blue (Bio-Rad Laboratories, Richmond, CA). PT is eluted from this column by a high salt concentration, for example, 0.75 M magnesium chloride, and then the salt is removed, passed through a column of affinity matrix fetuin-Sepharose composed of fetuin linked to Sepharose activated with cyanogen bromide. The PT is eluted from the fetuin column using the 4M magnesium salt. Alternatively, the method of Irons et al (ref 56) can be used. The culture supernatant is absorbed on a Sepharose 4B column activated with CNBr to the P511 which is first covalently linked to haptoglobin. The PT binds to the absorber at a pH of 6.5 and is eluted from the column using a Tris 0. lM / 0.5 M NaCl buffer by a graduated change to a pH of 10. Alternatively, the method described in U.S. Patent No. 4,705,686 issued to Scott et al. On November 10, 1987 and mentioned herein by reference, may also be used. In this method, culture supernatants or B cell extracts. Pertussis are passed through a column of an anion exchange resin of sufficient capacity to adsorb the endotoxin but allow the Bordetella antigen to flow through it or separate from the endotoxin. Alternatively, PT can be purified using perlite chromatography, as described in EP Patent No. 336 736, assigned to the assignee herein and referred to by reference herein.

Detoxification of PT The PT is detoxified to remove unwanted activities that could cause lateral reactions in the final vaccine. Any of a variety of conventional chemical detoxifications can be used, for example treatment with formaldehyde, peroxide P511 hydrogen, tetranitro-methane or glutaraldehyde. For example, PT can be detoxified with glutaraldehyde using a procedure modification described in Muñoz et al (ref 57). In this detoxification process the purified PT is incubated in a solution containing 0.01 M phosphate buffered saline solution. The solution is brought to 0.05% with glutaraldehyde and the mixture is incubated at room temperature for two hours and then brought to 0.02 M of L-lysine. The mixture is further incubated for two hours at room temperature and then dialysed for two days against 0.01 M PBS. In a particular embodiment, the detoxification process of EP Patent No. 336 736 can be used. Briefly, PT can be detoxified with glutaraldehyde in the following way: PT purified in 75mM potassium phosphate at pH 8.0 containing 0.22M sodium chloride is diluted with an equal volume of glycerol at a protein concentration of approximately 50 to 400 μg / ml. The solution is heated to 37 ° C and detoxified by the addition of glutaraldehyde to a final concentration of 0.5% (w / v). The mixture is kept at 35 ° C for 4 hours and then aspartic acid (1.5 M) is added to a final concentration of 0.25 M. The mixture is incubated at room temperature for 1 hour and then filtered by dialysis with 10 volumes of phosphate P5X1 of 10 mM potassium at pH 8.0 containing 0.15M sodium chloride and 5% glycerol to reduce glycerol and to remove glutaraldehyde. The PT toxoid is filtered in sterile form through a 0.2 μM membrane. If the recombinant techniques are used to prepare a mutant PT molecule that does not show toxicity or shows little toxicity, for use as the toxoid molecule, chemical detoxification is not necessary.

Purification of FHA FHA can be purified from the culture supernatant essentially as described by Co ell et al (ref 58). Growth promoters, such as methylated beta-cyclodextrins, can be used to increase the yield of FHA in culture supernatants. The culture supernatant is applied to a hydroxylapatite column. The FHA is adsorbed on the column but the PT does not. The column is washed extensively with Triton X-100 to remove the endotoxin. The FHA is then eluted using 0.5M NaCl in 0.1M sodium phosphate and, if necessary, passed through a fetuin-Sepharose column to remove the residual PT. Further purification may involve passage through a Sepharose CL-6B column. Alternatively, FHA can be purified P511 using monoclonal antibodies to the antigen, where the antibodies are bound to an affinity column activated with CNBr (ref 59). Alternatively, the FHA can be purified using perlite chromatography as described in the above-mentioned EP 336 736.

Purification of the 69 kDa External Membrane Protein (pertactin) The 69 kDa outer membrane protein (69K or pertactin) can be recovered from bacterial cells by first inactivating the cells with a bacteriostatic agent, for example thimerosal, as described in published EP 484 621 and mentioned herein by reference. The inactivated cells are suspended in an aqueous medium, such as PBS (pH 7 to 8) and subjected to repeated extractions at elevated temperature (45 to 60 ° C) with subsequent cooling at room temperature or 4 ° C. The extractions release the 69K protein from the cells. The material containing the 69K protein is collected by precipitation and passed through an Affi-gel Blue column. The 69K protein is eluted with a high concentration of salt, for example 0.5M magnesium chloride. After dialysis, it is passed through a chromatofocusing support.

P511 Alternatively, the 69 kDa protein can be purified from the culture supernatant of a B culture. pertussis as described in the Application PCT published WO 91/15505, in the name of the assignee of the present and which is mentioned herein by reference. Other suitable methods of purification of the 69 kDa outer membrane protein from B. pertussis are described in U.S. Patent No. 5,276,142, issued to Gotto et al on January 4, 1984 and U.S. Patent No. 5,101,014, issued to Burns on March 31, 1992. Several tests were carried out. clinical trials in humans, as described herein, in order to establish the safety, absence of reactogenicity and usefulness of component vaccines for protection against pertussis. In particular, immune responses were obtained to each of the antigens contained in the vaccines (as shown, for example, in Table 3 below). A particular pertussis Cpi? / 5/5 / 3DT 'acellular vaccine was analyzed in a large double randomized, multi-center, placebo-controlled clinical trial in a high-risk human population to estimate the effectiveness of the pertussis vaccine typical The case definition for typical pertussis disease was: P511 Twenty-one or more days of spasmodic cough and either B. pertussis confirmed by culture, or serological evidence of specific infection with Bordetella indicated by an elevation of 100% IgG or IgA antibodies in ELISA against FHA or PT in paired sera or otherwise there is serological data, the child under study has been in contact with a case of B. pertussis confirmed by cultivation in the home with coughing within 28 days before or after the onset of cough in the child under study. The results of this study showed that CPi0 / 5/5 / 3DT is approximately 85% effective in avoiding pertussis as defined in the case definition for typical pertussis disease as described above. In the same study, a two-component acellular pertussis vaccine containing only PT and FHA was approximately 58% effective and a full-cell pertussis vaccine had an efficacy of approximately 48% (see following Table 4). In addition, the CP10 / 5/5 / 3DT vaccine prevented pertussis media defined as a cough of at least one day duration until an efficacy of approximately 77%. In particular, the profile of the P511 The immune response obtained was essentially the same as that obtained following immunization with whole-cell pertussis vaccines, which are reported to be highly effective against pertussis.

Preparation of the Vaccine and Use In this way, immunogenic compositions suitable for use as vaccines can be prepared from the Bordetella immunogens as disclosed herein. The vaccine triggers an immune response in a subject that produces antibodies that may be opsonizing or bactericidal. If the vaccinated subject is inoculated with B. pertussis, these antibodies bind to the bacterium and inactivate it. In addition, opsonizing or bactericidal antibodies can also provide protection by alternative mechanisms. Immunogenic compositions that include vaccines can be prepared as injectable solutions or as emulsions or liquid solutions. The Bordetella immunogens can be mixed with pharmaceutically acceptable excipients that are compatible with the immunogens. These excipients may include water, saline, dextrose, glycerol, ethanol, and combinations thereof. Immunogenic compositions and vaccines may also contain P511 auxiliary substances, for example wetting agents or emulsifiers, pH regulating agents, or adjuvants to improve the effectiveness thereof. Immunogenic compositions and vaccines can be administered parenterally, by subcutaneous or intramuscular injection. Immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation and in such an amount as to be therapeutically effective, as well as immunogenic and protective. The amount to be administered will depend on the subject being treated, including, for example, the ability of the individual's immune system to synthesize antibodies and, if necessary, to produce a cell-mediated immune response. The precise amounts of the active ingredient that are required to be administered depend on the judgment of the practicing physician. However, the range of suitable doses can be easily determined by an expert in this field and can be of the order of micrograms of the immunogens. Suitable regimens for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The doses may also depend on the route of administration and will vary according to the size of the host.

P511 The concentration of the immunogens in an immunogenic composition according to the invention is generally between about 1 and 95%. A vaccine containing antigenic material from only one pathogen is a monovalent vaccine. Vaccines containing antigenic material of various pathogens are combined vaccines and also belong to the present invention. These combined vaccines contain, for example, material from various pathogens or several strains of the same pathogen, or combinations of several pathogens. Immunogenicity can be significantly improved if the antigens are co-administered with adjuvants, commonly used as solutions of 0.005 to 0.5 percent in phosphate-buffered saline. The adjuvants improve the immunogenicity of the antigen but are not necessarily immunogenic by themselves. The adjuvants can act by retaining the antigen locally near the site of administration to produce a depot effect that facilitates a slow and sustained release of the antigen to the cells of the immune system. The adjuvants can also attract the cells of the immune system to an antigen deposit and stimulate these cells to elicit immune responses. Agents in a stimulant or adjuvant of this type have been used for many years to improve the P511 immune responses of hosts to, for example, vaccines. Intrinsic adjuvants such as lipopolysaccharides, are usually components of attenuated or killed bacteria that are used in vaccines. The extrinsic adjuvants are immunomodulators which are typically non-covalently bound to the antigen and are formulated to improve the immune responses of the host. In this form, adjuvants have been identified that improve the immune response to antigens administered parenterally. Some of these adjuvants are toxic, however, they can cause undesirable side effects, making them unsuitable for use in humans or in many animals. In fact, only aluminum hydroxide and aluminum phosphate (collectively referred to as alum) are routinely used as adjuvants in vaccines for humans and for veterinary use. The effectiveness of alum in increasing the antibody response to tetanus and diphtheria toxoids is well established and an HBsAg vaccine has been combined with alum as an adjuvant. While the utility of alum is well established for certain applications, it has its limitations. For example, alum is not effective for influenza vaccines and inconsistently generates a cell-mediated immune response. The antibodies generated by the antigens adjuvanted with alum are P511 mainly of the IgGl isotype in the mouse, which can not be optimal for protection by certain vaccine agents. A wide range of extrinsic adjuvants can elicit potent immune responses to antigens. These include saponins complexed with membrane protein antigens (immune stimulatory complexes), pluronic polymers with mineral oil, mycobacteria killed in mineral oil, Freund's complete adjuvants, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharides ( LPS), as well as lipid A, and liposomes. To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens are typically emulsified in adjuvants. Many adjuvants are toxic, induce granulomas, acute and chronic inflammation (complete Freund's adjuvant), FCA, cytolysis (saponins and Pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP). Although FCA is an excellent adjuvant and is widely used in research, its use in humans or in veterinary vaccines is not authorized due to its toxicity. The desirable characteristics of the ideal adjuvants include: P511 (1) lack of toxicity; (2) ability to stimulate a lasting immune response; (3) manufacturing simplicity and long-term storage stability; (4) ability to generate both CMI and HIR to antigens administered by different routes, if required; (5) synergy with other adjuvants; (6) selectivity interacting with antigen presenting cell populations (APC); (7) ability to specifically generate specific immune responses specific for Tjjl or T? J2 cells; Y (8) ability to selectively raise appropriate levels of antibody isotypes (eg, IgA) against antigens. U.S. Patent No. 4,855,283 issued to Lockhoff et al on August 8, 1989, which is mentioned herein by reference, shows glycolipid analogues including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which it is substituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants. Thus, Lockhoff et al, (U.S. Patent No. 4,855,283 and ref.27) reported that the N-glycolipid analogues that show P511 Structural similarities with glycolipids that occur naturally, such as glycophospholipids and glycoglycerolipids, are capable of generating strong immune responses in the herpes simplex virus vaccine and in the pseudorabies virus vaccine. Some glycolipids have been synthesized from long chain alkylamines and fatty acids and are directly linked to the sugars through the anomeric carbon atom, to resemble the functions of the lipid residues that occur naturally. U.S. Patent No. 4,258,029 issued to Moloney, assigned to the assignee of the present invention and incorporated herein by reference, shows that octadecyl tyrosine hydrochloride (OTH) functioned as an adjuvant when complexed with tetanus toxoid and vaccine of poliomyelitis virus type I, II and III inactivated with formalin. Also, Nixon-George et al. (ref 24), reported that the octadecyl esters of the aromatic amino acids complexed with a recombinant surface antigen of hepatitis B, improved the immune response of the host against the hepatitis B virus.

EXAMPLES The above disclosure describes in general the P511 present invention. The invention will be better understood by reviewing the following specific examples. These examples are described for purposes of illustration only and are not intended to limit the scope of the invention. Changes in the form and substitution of equivalents are contemplated as they may suggest or be convenient to the circumstances. Although specific terms have been employed herein, these terms are proposed in a descriptive sense and not for purposes of limitation. The methods of protein, fermentation and immunology biochemistry used in this disclosure, but not explicitly described in these Examples, are widely reported in the scientific literature and are suitable within the skill of those skilled in the art.

EXAMPLE 1 This example describes the culture of Bordetella pertussis.

Main progeny: Cultures of the main progeny of a Bordetella pertussis strain were conserved as freeze-dried progeny batches, between 2 ° C and 8 ° C.

P511 Work progeny: The freeze-dried culture was recovered in Hornibrook medium and used to sow Agar plates.

Bordet-Gengou (BGA). The Hornibrook medium has the following composition: COMPONENT per one liter Casein hydrosylate (treated with carbon) 10.0 g Nicotinic acid 0.001 g Calcium chloride 0.002 g Sodium chloride 5.0 g Magnesium chloride hexahydrate 0.025 g Potassium chloride 0.200 g Potassium dibasic phosphate 0.250 g Starch 1.0 g Distilled water 1.0 liters The pH was adjusted to 6.9 ± 0.1 with 1% sodium carbonate solution. The medium was distributed in tubes and sterilized by evaporation in the autoclave for 20 minutes and autoclaved for 20 minutes between 121 ° C to 124 ° C. The progeny were subcultured twice, first in BGA plates then in Pertussis Components Agar (CPA). The Pertussis Components Agar (CPA) has the following composition: P511 I was born 2 - 5 g KH2P04 ° -5 9 / KCl 0-2 g / L MgCl2 (M20) 6 0.1 g / L Base tris 1-5 g / L Casamino acids 10.0 g / L NaH glutamate 10.0 g / L HCl Conc. PH a 7.2 Agar 15.0 g / L Growth factors (CPGF) 10.0 mL / L The growth factors of Pertussis Components (CPGF) -100X have the following composition: L-cysteine-HCl 4.0 g / L • Niacin 0.4 g / L Ascorbic acid 40.0 g / L Glutathione, reduced 15.0 g / L Fe2S0, (H2o ) 7 1.0 g / L Dimethyl-β-cyclodextrin 100 g / L CaCl 2 (H20) 2 20 g / L The final culture was dispersed in the suspension buffer of the Pertussis progeny (CPSB), was distributed in aliquots of 2 to 4 ml and stored frozen from -60 ° C to -85 ° C. The shock absorber of the P511 Pertussis progeny (PSSB) has the following composition: Casamino acids 10 • ° / L Base tris 1-5 / Glycerol anhydrous 100 mL / L Con. HCl PH at 7-2 These glycerol suspensions provided the starting material for the preparation of the working progeny.

Cultivation Process: The propagation of the working progeny was carried out in Roux bottles of Pertussis Components Agar during 4 to 7 days from 34 ° C to 38 ° C. After this culture, the cells were washed completely of the agar with the Pertussis Component Broth (CPB). Samples were observed by Gram stain, for the purity and opacity of the culture. The cells were transferred to 4 liter conical flasks containing CPB and incubated at 34 ° C to 38 ° C for 20 to 26 hours with shaking. Samples were observed by staining and the purity of the culture was verified. The flasks were flooded and the suspension was used to sow two termendores that P511 contain CPB (volume of 10 liters starting at OD600 0.1-0.4) Progeny were grown to a final OD600 of 5.0 to 10.0. Samples were tested by Gram stain, for culture purity, by antigen-specific ELISA and for sterility.

EXAMPLE 2 This example describes the purification of antigens from the culture of Bordetella pertussis cells.

Production of the broth and cell concentrates: The bacterial suspension was cultivated in two production fermentors, from 34 ° C to 37 ° C for 35 to 50 hours. Samples are taken from the fermenters for the sterility test of the media. The suspension was fed to a centrifuge of continuous-flow truncated cone disks (12,000 x g) to separate the cells from the broth. The cells were collected to wait for the extraction of the fimbrial component. The clarified liquor was passed through a membrane filter = 0.22 μm. The filtered liquor was concentrated by ultrafiltration using the nominal molecular weight limit membrane (NMWL) of 10 to 30 kDa. The concentrate was stored to wait for P511 Separation and purification of the Pertussis toxin (PT). Filamentous hemagglutonin (FHA) and 69 kDa components (pertactin).

Separation of broth components: Broth components (69 kDa, PT and FHA) were separated and purified by perlite chromatography and selective elution steps, essentially as described in European Patent No. 336 736 and the application for PCT No. WO 91/15505, published by the applicants, described above. The specific purification operations carried out are described below.

Pertussis toxin (PT): The perlite column was washed with 50 M Tris, 50 mM Tris / 0.5% Triton X-100 and 50 M Tris buffers. The PT fraction was eluted from the perlite column with 50 mM Tris buffer / 0.12 M NaCl. The PT fraction from perlite chromatography was loaded onto a hydroxylapatite column and then washed with 30 mM potassium phosphate buffer. The PT was eluted with a potassium phosphate buffer of 75 mM / 225 M NaCl. The column was washed with 200 mM potassium phosphate / 0.6 M NaCl to obtain the fraction of FHA that was PSll discarded. Glycerol was added to the purified PT up to 50% and the mixture was stored at 2 ° C to 8 ° C until detoxification, within one week.

Filamentous hemagglutonin (FHA): The FHA fraction was eluted from the perlite column with 50 mM Tris / 0.6 M NaCl. The filamentous hemagglutinin was purified by chromatography on hydroxylapatite. The FHA fraction from the perlite column was loaded onto a hydroxylapatite column, then washed with 30 mM potassium phosphate containing 0.5% triton X-100, followed by 30 mM potassium phosphate buffer. The PT fraction was eluted with 85 mM potassium phosphate buffer and discarded. The FHA fraction was then eluted with 200 mM potassium phosphate / 0.6 M NaCl and stored at 2 ° C at 8 ° C until detoxification within one week. 69 kDa (pertactin): The concentrate of the broth was diluted with water for injection (WFI) to achieve a conductivity of 3 to 4 mS / cm and was loaded on a perlite column in a loading of 0. 5 to 3.5 mg of protein per ml of perlite. The eluate (fraction of the 69 kDa component) was concentrated by ultrafiltration using a NMWL membrane of 10 to 30 kDa.

P511 Ammonium sulfate was added to the concentrate of the eluate at 35% ± 3% (w / v) and the resulting mixture was stored at 2 ° C to 8 ° C for 4 + 2 days or centrifuged (7,000 x g) immediately. The excess supernatant was decanted and the precipitate was collected by centrifugation (7,000 x g). The pellet or pellet of 69 kDa was either stored frozen at -20 ° C to -30 ° C or dissolved in tris or phosphate buffer and used immediately. The 69 kDa outer membrane protein obtained by precipitation with 35% (w / v) ammonium sulfate from the concentrated perlite eluate was used for the purification. Ammonium sulfate (100 ± 5 g per liter) was added to the 69 kDa fraction and the mixture was stirred for at least 2 hours at 2 ° C to 8 ° C. The mixture was centrifuged (7,000 x g) to recover the supernatant. Ammonium sulfate (100 to 150 g per liter) was added to the supernatant and the mixture was stirred for at least 2 hours at 2 ° C to 8 ° C. The mixture was centrifuged (7,000 xg) to recover the pppy sediment, which was dissolved in 10 mM tris, pH 8. The ionic strength of the solution was adjusted to the equivalent of 10 mM Tris HCl (pH 8), which contains ammonium sulfate. 15 mM. The 69 kDa protein was applied to a hydroxylapatite column connected in tandem with a Q-Sepharose column. The 69 kDa protein was collected in the eluate, P511 it was cleaned from the columns by entrainment with 10 mM Tris, HCl (pH 8), containing 15 mM ammonium sulfate and met or pooled with the 69 kDa protein in the eluate. The combined 69 kDa protein was diafiltered with 6 to 10 volumes of 10 mM potassium phosphate (pH 8), containing 0.15 M NaCl in a NMWL membrane of 100 to 300 kDa. The ultrafiltrate was collected and the 69 kDa protein from the ultrafiltrate was concentrated. The 69 kDa protein was exchanged with solvent in 10 mM Tris, HCl (pH 8), and adsorbed on Q-Sepharose, washed with 10 mM Tris-HCL (pH 8) / 5 mM ammonium sulfate.

The 69 kDa protein was eluted with 50 mM potassium phosphate (pH 8). The 69 kDa protein was diafiltered with 6 to 10 volumes of 10 mM potassium phosphate (pH 8), which contains 0.15 M NaCl in a NMWL membrane of 10 to 30 kDa. The 69 kDa protein was sterilized by filtration through a << 0.22 μm. This sterile volume was stored at 2 ° C to 8 ° C and the adsorption was carried out in the space of three months.

Fimbrial agglutinogens: The agglutinogens were purified from the cell paste after separation of the broth. The cell paste was diluted to a volume fraction of 0.05 cells in a buffer containing potassium phosphate P511 M, 150 mM NaCl and 4 M urea and mixed for 30 minutes. The used cell was clarified by centrifugation (12,000 x g) was then concentrated and diafiltered against 10 mM potassium phosphate / 150 mM NaCl / 0.1% Triton X-100 using a NMWL membrane filter of 100 to 300 kDa. The concentrate was heat treated at 80 ° C for 30 minutes then clarified again by centrifugation (9,000 x g). PEG 8000 was added to the clarified supernatant at a final concentration of 4.5% ± 0.2% and stirred gently for a minimum of 30 minutes. The resulting precipitate was collected by centrifugation (17,000 x g) and the sediment or globule was extracted with 10 mM potassium phosphate buffer / 150 mM NaCl to provide a crude solution of fimbrial agglutinogens. The fimbrial agglutinogens were purified by passage over PEI silica. The crude solution was brought to 100 mM with respect to potassium phosphate, using 1 M potassium phosphate buffer and passed through the PEI silica column. The eluate from the columns was concentrated and diafiltered against 10 mM potassium phosphate buffer / 150 mM NaCl using a 100 to 300 kDa NMWL membrane filter. This sterile volume is stored at 2 ° C to 8 ° C and the adsorption was carried out in the space of three months. The preparation of fimbrial agglutinogens contained Agg 2 P511 fimbrial and Agg 3 fimbrial in a weight ratio of about 1.5 to about 2: 1 and found to be substantially free of Agg 1.

EXAMPLE 3: This example describes the toxoid conversion of Bordetella pertussis antigens, PT and FHA. The PT, prepared in pure form as described in Example 2, was converted to toxoid by adjusting the concentration of glutaraldehyde in the PT solution to 0.5% ± 0.1% and by incubating at 37 ° C ± 3 ° C for 4 hours . The reaction was achieved by adding L-aspartate at 0.21 ± 0.02 M. The mixture was then kept at room temperature for 1 ± 0.1 hours and then at 2 ° C to 8 ° C for 1 to 7 days. The resulting mixture was diafiltered with 10 mM potassium phosphate buffer / 0.15 M NaCl / 5% glycerol in a 30 kDa NMWL membrane filter and then sterilized by passage through a membrane filter < 0.22 μm. This sterile volume was stored at 2 ° C to 8 ° C and the adsorption was carried out in the space of three months. The FHA fraction, prepared in pure form as described in Example 2, was formed into a toxoid by adjusting the concentration of L-lysine and formaldehyde at 47 ± 5 mM and 0.24 ± 0.05%, respectively and at 35 ° C incubation. at 38 ° C P511 for 6 weeks. The mixture was then diafiltered against 10 M potassium phosphate / 0.5 mM NaCl using a 30 kDa NMWL membrane filter and sterilized by passage through a membrane filter. This sterile volume was stored at 2 ° C to 8 ° C and the adsorption was carried out in a space of three months.

EXAMPLE 4 This example describes the adsorption of the purified antigens of Bordetella pertuesis. For the individual adsorption of PT, FHA, Agg and 69 kDa on aluminum phosphate (alum), a stock solution of aluminum phosphate was prepared at a concentration of 18.75 ± 1 mg / ml. A suitable container was prepared and any of the antigens was aseptically distributed in the container. 2-Phenoxyethanol was aseptically added to produce a final concentration of 0.6% ± 0.1% v / v and stirred until homogeneous. The appropriate volume of aluminum phosphate was added aseptically in the container. An appropriate volume of distilled water was added, sterile to bring the final concentration to 3 mg of aluminum phosphate / ml. The containers were sealed and labeled and allowed to stir at room temperature for 4 days. The container was then stored to wait for the final formulation.

F511 EXAMPLE 5: This example describes the formulation of a pertussis component vaccine combined with diphtheria and tetanus toxoids. The antigens of B. pertussis prepared as described in the preceding examples were formulated with diphtheria and tetanus toxoids to provide several Pertussis component (CP) vaccines. The pertussis components were produced from Bordetella pertussis grown in submerged culture as described in detail in examples 1 to 4 above. After the termination of the culture, the culture broth and the bacterial cells were separated by centrifugation. Each antigen was purified individually. Pertussis toxin (PT) and filamentous hemagglutinin (FHA) were purified from the broth by sequential chromatography on perlite and hydroxylapatite. The PT was detoxified with glutaraldehyde and any residual PT (approximately 1%) present in the FHA fraction was detoxified with formaldehyde. Agglutinogens were prepared (2 + 3) (AGG) fimbrial from bacterial cells.

The cells were disrupted with urea and treated with heat, and the fimbrial agglutinogens were purified by precipitation with polyethylene glycol and chromatography on polyethylenimine silica. The protein component of P511 69 kDa (pertactin) was isolated from the eluate of the perlite chromatography step (Example 2) by precipitation with ammonium sulfate, and purified by sequential chromatography on hydroxylapatite and Q-Sepharose. All components were sterilized by filtration through a 0.22 μm membrane filter. The diphtheria toxoid was prepared from the culture of CoryneJac eriu? I diphtheriae, in submerged culture by normal methods. The production of the Diphtheria toxoid was divided into five stages, mainly maintenance of the working progeny, cultivation of Corynebac ter i um diphtheriae, collection of diphtheria toxin, detoxification of diphtheria toxin and concentration of Diphtheria toxoid.

Preparation of the Diphtheria Toxoid (I) Work Progeny The strain of Coryn ebacteri um diphtheriae was kept as a batch of progeny dried by freezing. The reconstituted progeny were grown on inclined Loeffler planes for 18 to 24 hours at 35 ° C ± 2 ° C, and then transferred to flasks of diphtheria medium. The culture was then tested for purity and Lf content. The remaining progeny were used to inoculate a fermentor.

P511 (II) Cultivation of Corynebac ter iu diphtheriae The culture was incubated at 35 ° C ± 2 ° C and stirred in the thermenter. Preset amounts of solutions of ferrous sulfate, calcium chloride and phosphate were added to the culture. The actual amounts of each solution (phosphate, ferrous sulfate, calcium chloride) were determined experimentally for each batch of the medium. The levels chosen are those that give the highest content of Lf. At the end of the cultivation cycle (from 30 to 50 hours), the cultures were shown for purity, and the content of Lf. The pH was adjusted with sodium bicarbonate, the culture was inactivated with 0.4% toluene for 1 hour at a maintained temperature of 35 ° C ± 2 ° C. A sterility test was then performed to confirm the absence of live C. diphtheriae.

(III) Collection of Diphtheria Toxin The cultures treated with toluene from one or several fermentors were grouped in a large tank. Sodium bicarbonate was added to approximately 0.12%, 0.25% carbon, and 23% ammonium sulfate, and the pH was tested. The mixture was stirred for about 30 minutes. Diatomaceous earth was added and the mixture was P511 pump up a deep filter. The filtrate was re-circulated until it became clear, then collected, and sampled for the Lf content test. Additional ammonium sulfate was added to the filtrate to give a 40% concentration. Diatomaceous earth was also added. This mixture was maintained for 3 to 4 days at 2 ° C to 8 ° C to allow the precipitate to settle. The precipitated toxin was collected and dissolved in 0.9% saline. The diatomaceous earth was removed by filtration and the toxin was dialysed against 0.9% saline to remove the ammonium sulfate. The dialyzed toxin was collected and sampled for the Lf content and the purity test.

(IV) Detoxification of diphtheria toxin Detoxification takes place immediately after dialysis. For detoxification, the toxin was diluted so that the final solution contained: a) diphtheria toxin at 1000 ± 10% Lf / ml b) 0.5% sodium bicarbonate c) 0.5% formalin d) L-monohydrochloride 0.9% lysine w / v The solution was brought to the volume with saline and the pH was adjusted to 7.6 ± 0.1. The toxoid was filtered through pads of diatomaceous earth filter, cellulose, and / or a membrane prefilter and a 0.2 μm membrane filter in the collection vessel and incubated for 5 to 7 weeks' at 34 ° C. A sample was removed for the toxicity test.

(V) Concentration of the purified toxoid The toxoids were combined, then concentrated by ultrafiltration, and collected in a suitable vessel. Tests were taken for the test of Lf content and purity. The preservative (2-phenoxyethanol) was added to give a final concentration of 0.375% and the pH was adjusted to 6.6 to 7.6. The toxoid was sterilized by filtration through a prefilter of a membrane filter of 0.2 μm (or equivalent) and was collected. The sterile toxoid was then sampled for the irreversibility of the Lf content of the toxoid, preservative content, purity (nitrogen content), sterility and toxicity test. The concentrated, sterile toxoid was stored at 2 ° C to 8 ° C until the final formulation.

Preparation of Tetanus Toxoid Tetanus toxoid (T) was prepared from Clostridiu tetani grown in submerged culture. The production of tetanus toxoid can be divided into five stages, mainly maintenance of the work progeny, Clostridimp tetani culture, tetanus toxin collection, detoxification of tetanus toxin and purification of tetanus toxoid.

(I) Work progeny The Clos tridi u tetani strain used in the production of the tetanus toxin for the toxoid conversion of tetanus was maintained in the lyophilized form in a batch of the progeny. The progeny were inoculated into the thioglycollate medium and allowed to grow for approximately 24 hours at 35 ° C ± 2 ° C. The sample was taken for the culture purity test.

(II) Culture of Clostridium um tetani The tetanus medium was distributed in a thermenator, treated with heat and cooled. The termenter was then seeded and the culture was allowed to grow for 4 to 9 days at 34 ° C ± 2 ° C. A sample was taken for the purity of the culture, and the content test Lf.

(III) Collection of Tetanus Toxin Tetanus toxin was separated by filtration through cellulose diatomaceous earth pads, and the clarified toxin was then sterilized by filter using membrane filters. The samples were taken for the Lf content and the sterility test. The toxin was concentrated by ultrafiltration, using a pore size of 30,000 daltons.

(IV) Detoxification of Tetanus Toxin The toxin was sampled for the Lf content test before detoxification. The concentrated toxin (475 to 525 Lf / ml) was detoxified by the addition of sodium bicarbonate at 0.5% w / v, 0.3% v / v formalin and 0.9% w / v L-lysine monohydrochloride, and to volume with saline solution. The pH was adjusted to 7.5 ± 0.1 and the mixture was incubated at 37 ° C for 20 to 30 days. Samples were taken for the sterility and toxicity test.

(V) Toxoid Purification Concentrated toxoid was sterilized through pre-filter, followed by 0.2 μm membrane filters. Samples were taken for sterility test and Lf content. The optimum concentration of ammonium sulfate was based on a "S" curve of fractionation determined from the toxoid samples. The first concentration was added to the toxoid (diluted to 1900-2100 P511 Lf / ml). The mixture was kept for at least 1 hour at 20 ° C to 25 ° C and the supernatant was collected and the precipitate containing the high molecular weight fraction was discarded. A second concentration of ammonium sulfate was added to the supernatant for the second fractionation, to remove impurities of low molecular weight. The mixture was kept for at least 2 hours at 20 ° C to 25 ° C and then kept at 2 ° C to 8 ° C for a maximum of three days. The precipitate, which represents the purified toxoid, was collected by centrifugation and filtration. The ammonium sulfate was removed from the purified toxoid by diafiltration, using Amicon ultrafiltration membranes (or equivalent) with PBS until no more ammonium sulfate could be detected in the toxoid solution. The pH was adjusted from 6.6 to 7.6, and 2-phenoxyethanol was added to give a final concentration of 0.375%. The toxoid was sterilized by membrane filtration, and samples were taken for the test (irreversibility of the toxoid, Lf content, pH, preservative content, purity, sterility and toxicity). A formulation of a pertussis component vaccine combined with diphtheria and tetanus toxoids was named CP10 / 5/5 / 3DT. Each human dose of 0.5 ml of P511 CP10 / 5/5 / 3DT was formulated to contain: 10 μg Toxoid pertussis (PT) 5 μg Filamentous hemagglutonin (FHA) 5 μg 2 and 3 fimbrial agglutinogens (FIMB) 3 μg External membrane protein of 69 kDa Lf Diphtheria toxoid 5 Lf Tetanus toxoid 1. 5 mg Aluminum phosphate 0. 6% 2 -Fenoxyethanol as preservative Another formulation of the pertussis component vaccine combined with diphtheria and tetanus toxoids was called CP10 / 5 / 5DT. Each human dose of 0.5 ml of CP10 / 5 / 5DT was formulated to contain: LO μg Pertussis toxoid (PT) 5 μg Filamentous hemagglutonin (FHA) 5 μg Agglutinogens 2 and 3 fimbrial (FIMB) L5 Lf Diphtheria toxoid 5 Lf Tetanus toxoid 1. 5 mg Aluminum phosphate 0. 6% 2-phenoxyethanol as preservative Another formulation of the pertussis component vaccine combined with diphtheria and tetanus toxoids is PSll called CP2o / 2? / 5 / 3DT- Each human dose of 0.5 ml of CP20 / 20/5 / 3DT was formulated to contain: 20 μg Pertussis toxoid (PT) 20 μg Filamentous hemagglutonin (FHA) 5 μg Agglutinogen 2 and 3 fimbrial (FIMB) 3 μg Outer membrane protein 69 kDa Lf Diphtheria toxoid 5 Lf Tetanus toxoid 1. 5 mg Aluminum phosphate 0. 6% 2-phenoxyethanol as preservative An additional formulation of pertussis component vaccine combined with diphtheria and tetanus toxoids was named CP20 / 10/10 / 6DT. Each human dose of 0.5 ml of CP2o /? O /? O / 6DT was formulated to contain: 20 μg Pertussis toxoid (PT) 10 μg Filamentous hemagglutonin (FHA) 10 μg Agglutinogens 2 and 3 fimbrial (FIMB) 6 μg Protein 69 kDa outer membrane Lf Diphtheria toxoid 5 Lf Tetanus toxoid 1. 5 mg Aluminum phosphate 0. 6% 2-phenoxyethanol as preservative P511 Example 6: This example describes the clinical assessment of the acellular vaccines of Pertussis components produced according to the invention. (a) Studies in adults Studies in adults and children aged 16 to 20 months indicated that multi-component vaccines containing fimbrial agglutinogens are safe (Table 2). A phase I clinical study was conducted in children 17 and 18 months of age in Calgary, Alberta with the five-component Pertussis vaccine (CP10 / 5/5 / 3DT) and the adverse reaction was reported. Thirty-three children received the vaccine and 35 additionally received the same vaccine without the 69 kDa protein component. Local reactions were rare. Systemic adverse reactions, consisting mainly of irritability, were present in approximately half of the study participants, without considering which vaccine was given. Significant increases in antibodies were measured for anti-Pt, anti-FHA, anti-agglutinogenic and anti-IgG antibodies of 69 kDa by enzyme immunoassay and anti-PT antibodies in the CHO cell neutralization test. No differences were detected in the antibody response in children who received the four components (CP10 / 5 / 5DT) or P511 five components (CP10 / 5/5 / 3DT) except in the anti-69 kDa antibody. Children who received the five component vaccine that contained the 69 kDa protein had a significantly higher level of anti-69 KDa antibodies after immunization. A dose-response study with the 4-component vaccine was attempted in Winnipeg, Manitoba, Canada. Two component vaccine formulations were used: CP10 / 5/5 / 3DT and CP20 /? O /? O / 6DT. A whole-cell DPT vaccine was also included as a control. This study was a double-blind study (or double blindness) in 91 infants from 17 to 18 months of age at the time of their booster pertussis dose. Both CP? O / 5/5 / 3DT and CP2o /? O /? O / 6DT were well tolerated by these children. No differences were shown in the number of children who had any local reactions, or systemic reactions after any of the component vaccines. In contrast, significantly more children who received the whole-cell vaccine had local and systemic reactions than those who received the CP2Q / IO / IO / GDT component vaccines. F511 Infant studies: Phase II: A study was conducted using the CP10 / 5/5 / 3DT vaccine in Calgary, Alberta and British Columbia, Canada. In this study, 432 infants received the pertussis component vaccine or the whole cell control vaccine, DPT at 2, 4 and 6 months of age. The CP10 / 5/5 / 3DT vaccine was well tolerated by these infants. Local reactions were less common with the component vaccine than with the whole-cell vaccine after each dose. A significant antibody response to all antigens was demonstrated after vaccination with the pertussis component vaccine. The recipients of the whole-cell vaccine had a vigorous antibody response to the fimbrial agglutinogens, D and T. At seven months, from 82% to 89% of the recipients of the component vaccine and 92% of the recipients of the vaccine. whole-cell vaccine had a four-fold increase or a greater increase with the antibody titer to the fimbrial agglutinogens. In contrast, the antibody response to FHA was 75% to 78% in the component vaccines compared to 31% of the whole cell receptors. A four-fold increase in the anti-69 kDa antibody occurred in 90% to 93% P511 of the recipients of the component vaccines and 75% of the complete cell receivers. A four-fold increase in antibodies against PT by enzyme immunoassay occurred in 40% to 49% of component vaccines in 32% of whole cell vaccines; a fourfold increase in PT antibodies by CHO neutralization was found in 55% to 69% of the component vaccines and in 6% of the whole cell vaccines (Table 2).

Phase IIB: CP2o / o / 5 / 3DT and CP? O /? O / 5 / 3DT vaccines in ur * were evaluated with random anonymity against a control of D15PT with a diphtheria content lower than 15 Lf in comparison to a formulation of 25 Lf of 100 infants of 2, 4 and 6 months of age. No differences in the proportions of the adverse reactions between the two component formulations were detected, both were significantly less reactogenic than the whole cell control. Higher titers of antibodies against PT were achieved by enzyme immunoassay and neutralization of CHO and FHA, in recipients of the CP2o / 2? / 5 / 3DT vaccine with an increased content of antigens. At 7 months, the geometric average anti-FHA rating was 95.0 in receivers of CP20 / 2o / 5 / 3DT, of 45.2 in receivers P511 of CP10 / 5/5 / 3DT were only 8.9 in D15PT receivers. The anti-PT titers were 133.3, 58.4 and 10.4 by the immunoassay and 82.4, 32.7 and 4.0 by the neutralization of CHO respectively (Table 2). This study demonstrated that the pertussis component vaccine combined with adsorbed diphtheria and tetanus toxoids, with increased antigen content, was safe and immunogenic in infants and that the increased content of antigens increased the immune response to the prepared antigens (PT and FHA) without an increase in reactogenicity.

NIAID North American Comparative Trial, PHASE II A Phase II study was conducted in the United States of America under the auspices of the National Institute of Allergic and Infectious Diseases (NIAID) as a prelude to an efficiency trial at Large scale acellular pertussis vaccines.

A vaccine of pertussis components of the invention in combination with diphtheria and tetanus toxoids adsorbed (CP? O / 5/5 / 3DT) was included in that trial along with 12 different acellular vaccines and 2 whole cell vaccines. Safety results were reported in 137 children immunized at 2, 4 and 6 months of age with the CP10 / 5/5 / 3DT component vaccine.

P511 As seen in previous studies, the component vaccine was found to be safe, low reactogenic and well tolerated by vaccines. At 7 months, the anti-PT antibody, the anti-FHA antibody, the anti-69 kDa antibody and the anti-agglutinogen fimbrial antibody were at levels higher than, or equivalent to, the levels achieved after the whole-cell vaccines. (ref 71 and Table 2). A double-blind study was conducted in which children were randomized to receive the formulation of either CP2o / 2? / 5 / 3DT or the CP10 / 5/5 / 3DT vaccine. A total of 2,050 infants were enrolled in the United States of America and Canada; 1961 infants finished the study. Both vaccine formulations were safe, low reactogenic and immunogenic in these infants. Immunogenicity was assessed in a subgroup of 292. There was an increase in antibodies to all antigens contained in the vaccine by both vaccine formulations. The formulation of CP20 / 20/5 / 3DT induced higher titers of antibodies against FHA but not against PT. The formulation of CP / 10/5/5 / 3DT produced higher titers against fimbrias and higher titers of agglutinogens. An additional safety and immunogenicity study was carried out in France. The design of the study was PSll Similar to the North American study, described above, except that the vaccines were administered at 2, 3, and 4 months of age. Local and systemic reactions were generally minor. The complete vaccine was well accepted by the French study participants who use this administration regimen.

A placebo-controlled efficacy trial of two acellular pertussis vaccines and a full-cell vaccine in 10,000 infants After the results of the North American, Phase II, NIAID comparative trial, a two-component and one five-component acellular vaccine was selected for a placebo-controlled, double-randomized, controlled efficacy trial conducted in several centers. The clinical trial was conducted in Sweden, where there is a high incidence of pertussis. The two-component vaccine contained glyceraldehyde and formalin-inactivated PT (25 μg), FHA treated with formalin (25 μg) and diphtheria toxoid at 17 Lf and tetanus toxoid 10 Lf. The five-component pertussis vaccine was CP10 / 5/5 / 3DT. For the trial, 10,000 infants, representing approximately half of infants in this age group in Sweden, were recruited from 14 study sites geographically defined by the use of the registry.

P511 birth. Children born in January and February of 1992 were randomized into a 3-group trial. After parental consent, two-thirds of the infants received one of the two preparations of diphtheria-tetanus-pertussis acellular at two, four and six months of age. The control group received only DT. In May 1992, a commercially available, licensed, whole-cell DTP vaccine was introduced, and children born in March through December 1992 were randomized into a 4-group trial. After parental consent, three quarters of the infants received one of three DTP preparations at two, four and six months of age. The control group received only DT. Each vaccine was administered to approximately 2,500 children. The vaccines were administered in three doses. The first dose was at 2 months of age and no more than 3 months of age. Subsequent doses were given at 8-week intervals. The vaccines were given by intramuscular injection. The children and their families were followed for 30 months. Without suspicion of pertussis, clinical data were collected, and verification of the laboratory sought by nasal aspirates for bacteriological culture and the P511 diagnosis by polymerase chain reaction (PCR). Samples of acute and convalescent blood were collected for serological diagnosis. Prior to this study, the degree of pertactin given by the vaccines of the pertussis components of the present invention in a human population at risk (particularly neonate) was unknown. In particular, the contribution of the various Bordetella components and their presence in pertussis vaccines in relative amounts related to the efficiency of vaccines was unknown. . The main purpose of the trial was to estimate the ability of acellular pertussis vaccines and the whole cell vaccine to protect against typical pertussis as compared to placebo. A secondary terminal point was to explore the efficiency of vaccines against confirmed infection of pertussis of varying severity. The effectiveness or efficiency of the vaccine is defined as the percentage reduction in the probability of contracting pertussis among the recipients of the vaccine in relation to unvaccinated children. The relative risk of pertussis in two vaccine groups is expressed as the ratio of the probability of disease in the two groups.

P511 The probability of contracting pertussis, also called attack rate, can be estimated in different ways. In the calculations of the sample size, the probability of finding pertussis in a given group of studies is estimated by the quotient between the number of children with pertussis and the children who remain in the study group at the end of the study follow-up. The efficiency of the component vaccine, CP10 / 5/5 / 3DT in this assay in the prevention of typical pertussis is shown in Table 4 and was approximately 85%. In the same assay, a two-component acellular pertussis vaccine containing only PT and FHA was approximately 58% efficient of a whole cell vaccine was approximately 48% efficient. The CP10 / 5/5 / 3DT was also effective in the prevention of mild pertussis at an estimated efficiency of approximately 77%.

SUMMARY OF THE INVENTION In summary of this disclosure, the present invention provides new preparations of Bordetella pertussis fimbrial agglutinogens and methods for their preparation. Fimbrial agglutinogens can be formulated with other Bordetella and not Bordetella antigens for P511 produce a number of multi-component pertussis vaccines. These vaccines are safe, non-reactogenic, immunogenic and protective in humans. Modifications are possible within the scope of this invention. eleven TABLE 1 Acellular pertussis O 0o vaccines a Inactivated with hydrogen peroxide, b Massachusetts Public Health Laboratories, c TNM, inactivated with tetranitromethane, Gl-inactivated with glutaraldehyde, and Fl, inactivated with formalin, f Center for Applied Microbiology and Research. • 0 01 TABLE 2 Responses of IqG antibody to pertussis antigens? to diphtheria and tetanus toxoids in adults and young children after immunization with placebo or acellular pertussis (AP) diphtheria-tetanus -pertussis (DTP) or toxoids The data is expressed as the geometric mean with confidence intervals of 95%. For pertussis toxoid filamentous hemagglutinin, agglutinogens, pertactin and tetanus toxoid and diphtheria, antibody units are expressed as ELISA units / nL. For the neutralizing assay of CHO cells the values reflect the reciprocal of the highest dilution showing 80% neutralization.

"O Ü1 TABLE 3. SEROLOGICAL RESULTS OF ACELLUlAR VACCINES OF PERTUSSIS IN INFANTS (2, 4, AND 6 MONTHS OF AGE) or CLI - Connaught Laboratories Incorporated, Mass. Massachusetts Public Swiftwater, Pennsylvania. Laboratories. CLL - Connaught Laboratories Limited, Lederle - Lederle Laboratories Inc Willowadale, Ontario TABLE 4 - PERFUSSIS ACELULAR VACCINE EFFICACY Vaccine Efficiency% A S P10 / 5/5 / 3DT 84-7 (80.3-88.5) 1 77 PT25.FHA25DT 58 (49.8-64.8) 1 DPT2 47.9 (37.1-56.9) 1 A: case definition: 21 days of spasmodic cough and positive culture B: case definition: average pertussis cough of at least one day Note 1: confidence limits Note 2: full-cell pertussis vaccine P511 REFERENCES .

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Zealey, G., Loosmore, S., Yacoob, R., Klein, M., Vaccine Research, Vol. 1, pp. 413-427.

Claims (13)

1. CLAIMS 1. A vaccine composition to protect a population of humans at risk, against a case of disease caused by infection with B. pertussis. The vaccine is characterized by a pertussis toxoid, filamentous haemagglutinin, pertactin and B. pertussis agglutinogens in purified form in relative amounts selected to provide protection in an extent of at least approximately 70% of the members of the population at risk. The vaccine of claim 1, characterized in that the pertussis toxoid is present in an amount of about 5 to about 30 μg of nitrogen, the filamentous haemagglutinin is present in an amount of about 5 to about 30 μg of nitrogen, Pertactin is present in an amount of about 3 to about 15 μg of nitrogen and the agglutinogens are present in an amount of about 1 to about 10 μg of nitrogen, in a single dose for human. The vaccine according to claim 2, which contains 10 μg of pertussis toxoid nitrogen, 5 μg of filamentous hemagglutinin nitrogen, 5 μg of P511 pertactin nitrogen and 3 μg of agglutinogen nitrogen, in a single dose for human. The vaccine according to claim 2, which contains 20 μg of pertussis toxoid nitrogen, 20 μg of filamentous hemagglutinin nitrogen, 5 μg of pertactin nitrogen and 3 μg of agglutinogen nitrogen, in a single dose for human. 5. The vaccine claimed in any of claims 1 to 4, characterized in that the degree of protection is at least about 80% in the case of pertussis with spasmodic cough of at least 21 days duration and with bacterial infection confirmed . 6. The vaccine claimed in any of claims 1 to 5, characterized in that the degree of protection is at least about 70% in the case of mild pertussis with cough of at least 1 day duration. The vaccine claimed in any of claims 1 to 4, characterized in that the degree of protection is approximately 85% for the case in which there is spasmodic cough of at least 21 days duration and with confirmed bacterial infection. 8. The vaccine claimed in any of claims 1 to 7, characterized in that the agglutinogen comprises fimbrial agglutinogen 2 (Agg 2) and P511 agglutinogen fimbrial 3 (Agg 3) substantially free of agglutinogen 1, 9. The vaccine claimed in claim 8, characterized in that the weight ratio of Agg 2 to Agg 3 is from about 1.5: 1 to about 2: 1. 10. The vaccine claimed in any of claims 9, further comprising tetanus toxoid and diphtheria toxoid, 11. The vaccine claimed in claim 10, characterized in that the diphtheria toxoid is present in an amount of about 15 Lfs and tetanus toxoid is present in an amount of about 5 Lfs, 12. The vaccine claimed in any of claims 1 to 11, further comprises an adjuvant. 13. The vaccine claimed in claim 12, characterized in that the adjuvant is alum. P5U