EP0751956A1 - $i(PASTEURELLACEAE) ANTIGENS AND RELATED VACCINES - Google Patents

$i(PASTEURELLACEAE) ANTIGENS AND RELATED VACCINES

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Publication number
EP0751956A1
EP0751956A1 EP95910704A EP95910704A EP0751956A1 EP 0751956 A1 EP0751956 A1 EP 0751956A1 EP 95910704 A EP95910704 A EP 95910704A EP 95910704 A EP95910704 A EP 95910704A EP 0751956 A1 EP0751956 A1 EP 0751956A1
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EP
European Patent Office
Prior art keywords
antigens
pasteurella multocida
bacteria
approximately
kilodaltons
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP95910704A
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German (de)
French (fr)
Inventor
Robert Gerard Ankenbauer
Krishnaswamy Iyengar Dayalu
Wanda Kay Isaacson
Thomas James Kaufman
Wumin Li
Nancy Ellen Pfeiffer
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Pfizer Inc
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Pfizer Inc
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Publication date
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Publication of EP0751956A1 publication Critical patent/EP0751956A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • Animal vaccines designed to protect against pneumonias caused by Pasteurellaceae are generally produced from inactivated whole bacteria or extracts of bacterial cultures.
  • the protective potential of potassium thiocyanate extracts of culture-grown Pasteurella multocida have been investigated in mice, chickens, cattle and rabbits. These extracts contained protein, hyaluronic acid, lipopolysaccharide, DNA and RNA, making interpretation of the protective component difficult. Although some cross- protection has been observed, protection was mainly against homologous challenge.
  • Antibodies specific for polypeptides of approximately 153 kD, 179 kD, 192 kD and 204 kD recovered from in vivo- grown bacteria that were detergent insoluble have also been demonstrated to provide passive cross-protection against heterologous challenge in poultry (Wang CL et al. Abstract #32, p. 8, Conference of Research Workers in Animal Diseases, November 9-10, 1992, Chicago, IL) .
  • This antiserum was adsorbed against culture grown bacteria to remove antibodies that reacted with antigens in the culture grown preparation.
  • Induction of the pheST operon (which encodes two subunits of phenylalanyl-tRNA synthetase) is believed to be a response to the depletion of a charged tRNA, indicating starvation for the aromatic amino acid phenylalanine.
  • Pasteurella multocida isolates of turkey origin cultured in media containing iron chelators express novel outer membrane proteins that are not synthesized in standard, enriched culture media.
  • novel proteins are the cross- protective factors observed in in vivo-grown bacteria.
  • This relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown bacteria was investigated by comparison of their protein profiles by SDS-PAGE.
  • Antigens from the Pasteurella multocida and Actinobacillus pleuropneumoniae isolated directly from the pleural cavities of infected swine or from a minimal medium formulation have now been identified. These antigens are proteins which are up-regulated during infection in a host animal and are not observed during culture in standard, enriched media. It has now been found that these antigens are up-regulated in minimal medium formulations. However, these antigens are absent or only weakly expressed during in vitro cultivation in standard, enriched media. An immune response to these newly identified antigens invokes protection against heterologous challenge. Therefore, these antigens are useful in the production of an effective vaccine providing cross-protection between multiple isolates of the same species.
  • An object of the present invention is to provide antigens of the Pasteurella , Actinobacillus and Haemophilus species of bacteria capable of being up-regulated during infection in a host animal and in minimal medium formulations which provide protection against infections caused by these species.
  • Another object of the present invention is to provide vaccines comprising antigens of the Pasteurella , Actinobacillus and Haemophilus species of bacteria capable of being up-regulated during infection in a host animal and in minimal medium formulations which provide protection against infections caused by these species.
  • Yet another object of the present invention is to provide a method of immunizing healthy animals against infections caused by Pasteurella , Actinobacillus and
  • Haemophilus species of bacteria which comprises administering to a healthy animal an effective amount of a -6-
  • vaccine comprising antigens of the Pasteurella , Actinobacillus and Haemophilus species of bacteria capable of being up-regulated during infection in a host animal and in minimal medium formulations which provide protection against infections caused by these species.
  • Figure l is a graph depicting a passive immunity study in mice. All mice received 0.5 ml, i.p. , of an immune serum followed four hours later by 100 to 200 CFU of virulent Pasteurella multocida 16926.
  • immune serum was collected from a pig following a primary infection with P. multocida isolate 8261.
  • Group A (-) serum is a 1:10 dilution of this immune serum;
  • Group B ( )serum is a 1:10 dilution of this immune serum adsorbed with detergent solubilized, standard, enriched media grown isolate 8261; and
  • Group C is preimmune serum diluted 1:10.
  • FIG. IB immune serum was collected from a pig infected with isolate 8261 followed by a secondary exposure to P. multocida isolate 16926.
  • Group D (— —) serum is a 1:10 dilution of this immune serum
  • Group E (— ⁇ • —) serum is a 1:10 dilution of this immune serum adsorbed with detergent solubilized, standard, enriched media grown isolate 8261
  • Figure 2 is a graph depicting a second passive immunity study in mice. All mice received 0.5 ml, i.p. of an immune serum followed four hours later by 100 to 200 CFU of virulent P . multocida 16929. Immune serum was collected from a pig 7 days following a secondary exposure to P. multocida isolate 16926.
  • Group 1 (— —) is preimmune serum diluted 1:10
  • Group 2 (—) is immune serum diluted 1:10;
  • Group 3 is immune serum, treated with detergent and purified, diluted 1:10;
  • Group 4 is immune serum, adsorbed with detergent-solubilized bacteria and purified, diluted 1:10; and
  • Group 5 is lung washing fluids collected 18 hours following secondary exposure to isolate 16926.
  • the Pasteurellaceae family of bacteria contains species of the genera Pasteurella , Actinobacillus , and Haemophilus . Recent work on the phylogeny of the Pasteurellaceae family confirmed the grouping of these three genera into this family (Dewhirst et al. (1992) J . Bacteriol . 174:2002-2013).
  • the various species within the Pasteurellaceae family fall into four large clusters, each cluster containing species of three different genera. Examples of species within this family include, but are not limited to, the animal pathogens P . multocida , A. pleuropneumonia , P. haemolytica , H. somnus , and A. suis .
  • Pasteurellaceae infections in animals result in symptoms similar to those resulting from virulent septic pneumonia. Death is generally due to endotoxic shock and respiratory failure. High mortality rates can occur with the acute form of these infections, however, subacute and chronic forms which result in pleuritis are more common. Treatment of field infections is difficult and often unsuccessful due to widespread antibiotic resistance. Therefore, it is preferred to prevent the infection in animals through use of a vaccine. There has been difficulty, however, in achieving a vaccine which will provide protection against different isolates of a species of bacteria within the Pasteurellaceae family.
  • the present invention provides antigens from the Pasteurella , Actinobacillus and Haemophilus species of bacteria that are up-regulated during infection in a host animal or in minimal medium formulations but which are absent or only weakly expressed during in vitro cultivation of the bacteria in a standard, enriched media, said antigens being capable of providing protection against an infection by these species of bacteria.
  • These antigens provide the basis for effective vaccines designed to cross-protect animals from infection by multiple isolates of one of the Pasteurella , Actinobacillus or Haemophilus species of bacteria.
  • host animals for these bacteria include, but not limited to, swine, bovine, ovine, avian and equine species.
  • minimal medium formulations it is meant a culture media specifically designed to provide a sufficient concentration of nutrients to bacteria to allow for synthesis of antigens up-regulated in vivo in a host while removing all undefined components.
  • standard, enriched laboratory media are designed to allow optimal growth of bacteria and thus include an excessive levels of nutrients when compared to the minimal growth requirements for each species.
  • HP Heamophilus Pleuropneumoniae
  • Undefined components in these media such as yeast extract, tryptone, peptone or brain heart infusion all contain high levels of complexed nitrogen, amino acids, vitamins, iron and other minerals which provide an excellent nitrogen source " and general nutritional supplement so that little metabolic demand is placed on the bacteria.
  • culture media used in the present invention were designed to supply the bacteria with the minimum level of essential nutrients necessary to support growth, thus mimicking the environment encountered when bacteria invade the host organism.
  • Components of the minimal media used in the present invention comprise basal salts (elemental requirements) , carbon sources, special nutritional requirements of the Pasteurellaceae , and nonessential optimizing supplements.
  • basal salts include, but are not limited to, potassium phosphate, potassium sulfate, magnesium chloride, ammonium chloride, calcium chloride and sodium chloride.
  • elemental requirements include, but are not limited to, potassium, sulfur, phosphorus, sodium, chloride, and calcium.
  • carbon sources include, but are not limited to, glycerol and lactic acid. Glucose, galactose, fructose, mannose, sucrose, mannitol, and sorbitol can also be utilized by the Pasteurellaceae . However, because of the fermentative type of metabolism of these organisms, acid can be produced during catabolism of sugars resulting in a lower yield of bacterial cells in the culture.
  • non- fermentable carbohydrates glycerol and lactic acid which do not lead to acid accumulation in cultures, is preferred.
  • members of the Pasteurellaceae are not prototrophic in that they are unable to grow in a mineral salts medium with a single carbon source, special nutritional additives are required.
  • these species require organic nitrogen sources and may require several amino acids, B vitamins, 0-nicotinamide, adenine nucleotides, or protoporphyrin and its conjugates.
  • the minimal medium may comprise arginine, aspartic acid, cystine, gluta ic acid, glycine, leucine, lysine, methionine, serine, tyrosine, inosine, uracil, hypoxanthine, thiamine, pantothenate, and nicotihamide.
  • protoporphyrin or its conjugates can also be added as needed. The addition of several components were also found to enhance the cell yield of P. multocida and A. pleuropneumoniae .
  • non-essential optimizing supplements include a buffer such as HEPES to increase buffering capacity, a nitrogen source such as glutamic acid and Casamino acid (a semi-defined acid hydrolysate of casein, Difco Laboratories Ltd, West Molesey, Surrey KT8 OSE U.K.), and an organic sulfur source such as L-cysteine.
  • these minimal media formulations contains no iron, zinc, copper, manganese or cobalt salts or complex biological materials containing these minerals.
  • the sole source for these minerals is the trace amounts which may be present as trace contaminants in the various other defined components or water.
  • the laboratory analyses of trace minerals in these minimal media formulations are provided in the following table.
  • Pasteurellaceae species such as Pasteurella multocida of
  • Actinobacillus pleuropneumoniae bacteria is administered to a host animal, preferably a pig, by injection into the pleural cavity.
  • the host animal is euthanized approximately 12 to 18 hours later and the pleural fluids are collected. Large cellular debris is removed from the fluids, and in vivo- grown bacteria are recovered by centrifugation. The resulting bacterial pellet is washed several times in buffer and centrifuged. The bacterial pellets are then resuspended in buffer and stored at -70°C.
  • Pasteurellaceae bacteria such as P. multocida or A. pleuropneumoniae are grown in a minimal medium formulation. Cultured bacteria were centrifuged to concentrate the bacteria and remove media components. Bacterial pellets were resuspended in PBS to an optical density of approximately 3.0 and frozen at -70°C until analyzed. Proteins expressed by the in vivo-grown bacteria or bacteria grown in a minimal medium formulation are separated by gel electrophoresis. Those proteins which are strongly expressed can then be identified by transferring part of the gel to nitrocellulose for Western blot analysis using immune serum that has been adsorbed against in vitro-grown bacteria in a standard, enriched media or using antibody recovered from the local site of infection.
  • strongly expressed refers to proteins up-regulated or expressed during infection in a host animal or in bacteria grown in a minimal medium formulation which are absent or only weakly expressed during conventional in vitro cultivation in standard laboratory medium.
  • weakly expressed refers to expression in amounts so minor that the antigen is incapable of providing protection against a Pasteurellaceae infection.
  • Conventional in vitro cultivation refers to bacteria inoculated into a standard, enriched medium such as complete Heamophilus Pleuropneumoniae (HP) medium containing supplements. The inoculate is incubated at 37°C for several hours, preferably in a shaking incubator.
  • the bacteria are centrifuged at 10,000 x g to remove culture medium and resuspended in sterile PBS (10 mM phosphate, 0.87% NaCl, pH 7.2) prior to use.
  • sterile PBS 10 mM phosphate, 0.87% NaCl, pH 7.2
  • At least eleven antigens were identified from the Western blot analysis of in vivo grown bacteria that are absent from Pasteurella multocida grown in vitro using in a standard, enriched media.
  • Western blot analysis of bacteria grown in vitro in a minimal medium formulation demonstrated that the antigenic profile of bacterial proteins produced in this minimal medium formulation was identical to the antigenic profile produced in a host animal infected by the bacteria.
  • the corresponding protein bands had molecular weights of approximately 115 kD, 109 kD, 96 kD, 89 kD, 79 kD, 62 kD, 56 kD, 53 kD, 45 kD, 34 kD and 29 kD.
  • the corresponding protein bands for each antigen are then excised from the gel, re-isolated by gel-electrophoresis and transferred onto sequence membranes for N'-terminal amino acid sequencing.
  • the N'-terminal amino acid sequence of a 34 kD antigen is as follows:
  • N'-terminal amino acid sequence of 29 kD antigen is as follows:
  • Lys Phe Lys Val Gin lie Ala XXX XXX XXX Gin Asp He Asn Gin Tyr Tyr Ala Gly Asp Ala Ala Phe Val (SEQ ID NO: 3)
  • SEQ ID NO: 3 The ability of these antigens to invoke a protective immune response against Pasteurellaceae was verified in passive transfer experiments.
  • Antibodies to the bacteria were isolated from infected pigs. Those antibodies that were rendered specific for the antigens of the present invention by adsorption were administered to mice. These mice were then challenged with a virulent heterologous isolate of Pasteurella multocida . Control mice not receiving the antibodies developed infections while those receiving the antibodies did not.
  • antibodies developed as an immune response against these antigens provided cross protection against heterologous isolates of Pasteurella multocida .
  • vaccines were prepared from P. multocida cultured in standard, enriched media containing yeast extract and from bacteria cultured in a minimal medium formulation.
  • the vaccine prepared from bacteria cultured in standard, enriched media was unable to protect mice from either homologous or heterologous challenge of P. multocida .
  • mice immunized with the vaccine prepared from bacteria cultured in a minimal medium formulation had much better survival rates.
  • Pasteurella multocida antigens which are capable of being up-regulated during infection in a host animal and in a minimal media formulation which provide protection against a Pasteurella infection.
  • a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 115 kD.
  • a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 109 kilodaltons.
  • a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 96 kilodaltons.
  • a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 89 kilodaltons. In yet another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 79 kilodaltons. In yet another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 62 kilodaltons. In yet another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 56 kilodaltons.
  • a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 53 kilodaltons. In yet another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 45 kilodaltons. In a preferred embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 29 kilodaltons and an N'-terminal amino acid sequence comprising SEQ ID NO: 2.
  • a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 29 kilodaltons and an N'- terminal amino acid sequence comprising SEQ ID NO: 3. It is also preferred that a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 34 kilodaltons and an N'-terminal amino acid sequence comprising SEQ ID NO: 1.
  • Antigens up-regulated during infection in a host animal and in a minimal media formulation but not in bacteria grown in vitro in a standard, enriched media were also identified for isolates of Actinobacillus pleuropneumoniae .
  • A. pleuropneumoniae is member of the Pasteurellaceae family which exists in the most distinct phylogenetic cluster from that of P . multocida .
  • the most prominent antigenic difference between the in vivo or minimal media bacteria and the bacteria cultured in a standard, enriched media was the presence of additional bands of approximately 60 kD to 65 kD in the in vivo and minimal media preparations.
  • the antigens of Pasteurellaceae can be produced recombinantly using techniques well-known to those skilled in the art or induced in culture via genetic manipulation.
  • Antigens of the present invention may be incorporated into a vaccine and administered to a healthy animal in an effective amount to protect against infection by bacteria of the Pasteurellaceae family. Upon administration, the antigens in the vaccine will invoke an immune response resulting in the healthy animal forming antibodies against the bacteria. "Effective amount” refers to that amount of vaccine which invokes in an animal an immune response sufficient to result in production of antibodies to the antigens. The animal will then be protected from any subsequent exposure to an isolate of Pasteurellaceae .
  • Pasteurella multocida comprises at least one antigen having a molecular weight of 34 kD and an N'-terminal amino acid sequence comprising SEQ ID NO: 1 or a molecular weight of 29 kD and an N'-terminal amino acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 3.
  • the antigen can be dissolved or suspended in any pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include, but are not limited to, normal isotonic saline, standard 5% dextrose in water or water, preferably adjuvanted.
  • adjuvants include, but are not limited to, Quil A, Alhydrogel, and Quil A and 5% Alhydrogel in tissue culture media.
  • the vaccine can be administered subcutaneously, intramuscularly, intraperitoneally, intravitreally, orally. intranasally or by suppository at doses ranging from approximately 1 to 100 ⁇ g/dose.
  • the antigens of the present invention produced in vivo or in bacteria grown in vitro in a minimal media formulation, recombinantly or via genetic manipulation or under specialized culture conditions can be added to whole culture grown bacteria to produce an effective vaccine. Addition of these antigens to the culture grown bacteria increase the efficacy of the resulting vaccine.
  • Example 1 Bacterial isolates and growth conditions Pasteurella multocida isolates 8261 and 16926 were field isolates received from the Iowa State Veterinary Diagnostic laboratory. Both isolates were serotype 3A.
  • bacteria were inoculated into Heamophilus Pleuropneumoniae (HP) medium (Gibco, Grand Island, NY) containing supplements, and incubated for 6 hours at 37°C in a shaking incubator. The bacteria were centrifuged at 10,000 x g to remove culture medium and resuspended in sterile PBS (10 mM phosphate, 0.87% NaCl, pH 7.2).
  • HP Heamophilus Pleuropneumoniae
  • Example 2 Convalescent sera and antibody from immune, challenged pigs
  • the pigs were allowed to convalesce for a period of between 10 days to one month and then treated with antibiotics to clear any residual infection. Following an additional period of one to two months, the pigs were exposed to a heterologous isolate of PmA (8261 or 16926) by the transthoracic route (1.5 - 2.5 x 10" CFU/ml). The pigs were euthanized at day 0, 1, 2, 3, 4, or 7. Serum samples were collected from the pigs at the time of primary infection, one month following primary infection, at the time of second infection, and at the time of euthanasia.
  • Lung washings were recovered from the pigs at the time of euthanasia to obtain antibody from the local site of infection.
  • the lungs and trachea were rinsed with approximately 100 ml of sterile PBS.
  • the lungs were massaged, and then fluids containing the antibody were poured into sterile tubes.
  • the fluids were centrifuged at 250 x g for 20 minutes at 4°C to remove large cellular debris and red blood cells.
  • Example 3 Adsorption of antibody preparations with detergent-solubilized or whole Pasteurella multocida
  • Antibodies that recognize in vitro grown bacterial antigens were removed by adsorption against either whole bacteria or detergent-solubilized antigen.
  • Fresh culture grown bacteria (isolate 8261 or 16926) were obtained for the whole bacteria adsorptions.
  • Approximately 10 ml of bacteria (3 x 10 9 CFU/ml) were pelleted by centrifugation at 11,000 x g for 40 minutes at 4°C (Beckman microfuge, Beckman Instruments, Palo Alto, CA) . The supernatant was removed and 0.1 ml of antiserum added. The mixture was incubated overnight at 4°C while being gently agitated.
  • the adsorbed material was centrifuged at 11,000 x g for 40 minutes and the supernatant was removed and added into a fresh bacteria pellet. The final supernatant was collected and stored at -20°C for Western blot analysis.
  • the culture grown bacteria was solubilized in a solution containing 0.062 M Tris, 0.069 M SDS and 1.09 M glycerol, pH 7.0.
  • the antigen preparation was then boiled for 10 minutes at 100°C to solubilize the bacteria. After the solubilized antigen had cooled, it was mixed with an equal volume of antibody and incubated overnight at 4°C. The mixture was centrifuged at 20,000 x g for 40 minutes to pellet the precipitated antibody-antigen complexes. The final supernatant was collected and stored at -20°C.
  • Antibody preparations used for the passive immunity study in mice were partially purified using ammonium sulfate. Serum samples were diluted 1:3 in sterile PBS. A solution of saturated ammonium sulfate was diluted to 90% of saturation and then added drop-wise to the diluted serum until a volume equivalent to the diluted serum was added, resulting in a 45% ammonium sulfate precipitation of the antibody. This solution was incubated on ice for 1 hour. The mixture was centrifuged at 10,000 x g for 40 minutes at 4°C to pellet the ammonium sulfate precipitate. The supernatant was discarded and the pellet was resuspended to the initial serum volume using sterile PBS. The ammonium sulfate precipitation was repeated to further purify the antibody. Following resuspension of the antibody, the solution was dialyzed against three changes of PBS using a 12,000 " MW cutoff membrane tubing.
  • Proteins from PmA were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrotransfer and immunoblotting. Proteins from PmA isolates 8261 and 16926, both from in vivo and in vitro grown bacteria, were boiled for 10 minutes with 5X reducing buffer (Pierce, Rockford, IL) . The proteins were then separated on 10% SDS gels at a concentration of 16 ⁇ g per lane. After electrophoresis (20 n ⁇ constant amperage) , proteins were transferred on blotting membrane (Immobilon, Millipore, Bedford, MA) overnight at 4°C (30 volts, constant voltage) .
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • the membrane was blocked with 10% non-fat dry milk in 10 mM Tris, 0.9% NaCl, pH 7.2 (Tris-saline) and strips were further incubated with appropriate dilutions of absorbed antiserum for 1 hour at room temperature. Subsequently, strips were rinsed 3 times with Tris-saline containing 0.1% Triton X-100 and further incubated 1 hour with alkaline phosphatase conjugated goat ⁇ -swine IgG (H+L) (Kirkegaard and Perry, Gaithersburg, MD) at a 1: 1000 dilution.
  • At least eleven protein bands with molecular weights of approximately 115 kD, 109 kD, 96 kD, 89 kD, 79 kD, 62 kD, 56 kD, 53 kD, 45 kD, 34 kD and 29 kD were identified from in vivo grown bacteria that were absent from or poorly expressed by culture grown PmA.
  • the molecular weights of proteins identified to be unique or up-regulated under in vivo growth conditions were estimated by Whole Band Analysis using the Biolmage Computer
  • All antibody preparations used in the passive immunity experiments were generated in swine against a primary infection with P. multocida isolate 8261, and in some cases were followed by a second infection with isolate 16926. Serum collected prior to the primary infection was used as a negative control for the passive immunity experiments. Convalescent serum from pig 104 was collected either at 30 days following the primary infection or at 7 days following the secondary exposure, and lung washings from pig 103 were collected 18 hours following the secondary infection. A secondary antibody response would be expected following re- exposure with the second strain. However, the secondary response should be limited to antigens that are common between the two Pasteurella multocida isolates.
  • the serum antibody preparations were adsorbed with detergent-solubilized bacteria and purified by ammonium sulfate precipitation as described in Example 4.
  • the partially purified antibody preparations were standardized by volume to represent a 1:10 dilution of the original serum sample. Lung washings were not adsorbed or purified.
  • mice were injected by the intraperitoneal route with 0.5 ml of the antibody preparations. Four- hours later the mice were infected with isolate 16926 at a rate of 100-200
  • mice Both the antisera collected after the primary challenge and the antisera collected after the secondary challenge passively protected mice against the 16926 challenge (9 of 10 and 8 of 10 mice survived at least ten days post infection, respectively) . These antisera were also adsorbed with detergent treated culture grown bacteria (8261) . Following adsorption, the antisera lost some of their protective capacity, but the death rate in these groups were still lower than in the control group that received preimmune serum.
  • mice given immune serum had better survival rates than that of the control mice that received preimmune serum.
  • the group given non-treated immune serum exhibited the highest survival rate (9 out of 10 mice survived the ten day challenge period) .
  • the antisera that had been detergent treated and purified provided intermediate protection. In these groups mice began dying between 6 and 10 days following challenge. No difference in survival time or mortality was seen between the mice that received detergent treated and purified antiserum versus the mice that received antiserum that had been adsorbed with detergent-solubilized culture grown bacteria and then purified.
  • Preimmune serum did not afford any passive protection in this model. All mice in this group died between 1 and 3 days post challenge. While the preimmune pig serum reacted with a few bacterial proteins in a Western blot analysis, the reactions were weak (1:10 dilution). This would be expected since the pigs used in these studies were not specific-pathogen free. Although they may have had some exposure to Pasteurella multocida or other pathogens with cross-reactive antigens, they were susceptible to Pa ⁇ teurella multocida at the time of primary infection, and did develop clinical disease following challenge.
  • Antibody recovered from the lung of an immune pig that had been originally infected with isolate 8261 and then exposed to a heterologous isolate of Pasteurella multocida was also tested for its ability to passively protect mice.
  • Antibody was recovered from the lung 18 hours after a secondary transthoracic exposure to Pasteurella multocida . This antibody recovered from the local site of exposure would have direct contact with invading bacteria.
  • Vascular leakage of specific antibody from the circulation into the extracellular environment of the lung represents an important immune mechanism that contributes to the protection seen in immune pigs following secondary exposure. Partial protection was seen in these mice, when compared to the control group. Five mice died within the first 1 to 3 days after challenge, but the remaining 5 mice lived an additional 4 to 5 days.
  • Bacteria P. multocida recovered from the pleural cavities of acutely infected pigs were isolated as described in Example 1. Isolate 8261 was also grown in culture as described in Example 1. Both preparations were inactivated with 0.3% formalin and adjusted to a concentration of 1 x 10 9 colony forming units (CFU)/ml. Each preparation was adjuvanted with a mineral oil emulsion containing 5% aluminum hydroxide gel. Five-fold and twenty- five-f ⁇ ld dilutions of the two vaccines were prepared by diluting the original vaccine in adjuvant. All vaccine preparations were administered by intraperitoneal injection into CF1 mice. Mice were vaccinated twice at a three week interval with a 0.1 ml dose.
  • CFU colony forming units
  • mice were challenged with 50 to 100 CFU of virulent Pasteurella multocida isolates 8261 or 16926 and observed for 15 days. As shown in Table 2 below, when the highest concentrations of vaccine were used, both preparations protected mice from both homologous and heterologous challenge. However, when less concentrated vaccines were used, only the vaccine produced from in vivo grown bacteria was able to protect the mice against virulent challenge. Eight of ten mice vaccinated with 1 x 10 7 CFU equivalents of in vivo antigens were protected against homologous challenge, and seven of ten mice were protected against heterologous challenge.
  • mice vaccinated with the least concentrated cultured bacterial antigens survived homologous challenge and only three of ten mice vaccinated with cultured bacterial antigens survived heterologous challenge. None of the ten non-vaccinated mice challenged with isolate 8261 survived, and only one of ten non- accinated mice challenged with isolate 19629 survived.
  • This test of active immunity in mice demonstrated that the addition of antigens that are up-regulated by in vivo growth produced a vaccine that was between 5 and 25 times as effective as a vaccine produced from bacteria grown in a standard, enriched media.
  • NA is representative of not applicable.
  • proteins from in vivo grown bacteria isolate 8261 were separated on 10% SDS gels. Western blots were performed simultaneously to identify the correct protein bands. Then, protein bands were excised from the gels, electroeluted and transferred to a PVDF membrane (ProBlott, Applied Biosystems) . Subsequently, protein bands (40-50 pmol each) were stained with 0.1 % Coomassie blue R-250 (Bio-Rad, Hercules, CA) . Individual protein bands were excised from the membrane for amino acid sequencing. Sequencing was performed on an Applied Biosystems Model A vapor phase protein sequencer at the Biotechnology Instrumentation Facility, University of California, Riverside, CA.
  • Balb C mice were immunized with in vivo grown P. multocida as described in Example 8, and then reimmunized with in vivo bacteria solubilized in SDS. Spleens from immunized mice were fused with SP2/0 myeloma cells to produce antibody-secreting hybridomas.
  • a hybridoma cell secreting antibody specific for the 109 kD protein was cloned -twice by limiting dilution and designated as Mab PMA 3-1. The resulting monoclonal antibody was specific for the 109 kD protein produced by in vivo grown bacteria and did not react with bacteria grown in complete media.
  • a second monoclonal antibody was selected based on the ability to bind the 29 kD protein from in vivo grown P. mul tocida .
  • Western blot analysis of in vivo grown and cultured bacteria demonstrated strong binding to the 29 kD protein of in vivo grown bacteria, and very little reactivity to culture grown bacteria.
  • This monoclonal antibody producing cell was cloned by limiting dilution and designated Mab PMA 3-21.
  • Example 12 Active Protection of Mice by Vaccine Produced from bacteria grown in minimal medium formulation P .
  • multocida isolate 8261 was cultured in a minimal medium formulation as described in Example 11.
  • Bacteria were also cultured in a standard, enriched media as described in Example 1 or harvested directly from the pleural cavities of infected swine as described in Example 1. All preparations were inactivated with 0.3% formalin with constant stirring for 24 hours at 37° C. The inactivated bacteria were diluted to a pre-inactivation cell count of 1 x 10 9 CFU/ml.
  • the bacterial suspensions were adjuvanted in a squalene emulsion containing Quil A and TDM.
  • mice female CF1, Charles River Laboratories
  • mice received two vaccinations three weeks apart and were challenged with either 380 LD S0 (50% lethal dose) of P. multocida 8261 or 209 LD 50 of isolate 16926 two weeks following the second vaccination.
  • mice immunized with vaccines prepared from in vivo grown bacteria or from bacteria cultured in either of the minimal media formulations had much better survival rates. These survival rates ranged from seven out of ten to complete protection (ten of ten mice) .
  • Example 13 Induction of "in vivo-like" antigens of Actinobacillus pleuropneumoniae
  • Bacteria were recovered from the pleural infusions of pigs that died or were euthanized following acute infection with Actinobacillus pleuropneumoniae . Fluids were collected into bottles containing glass beads to remove fibrin clots and then transferred to centrifuge bottles. Red blood cells and other cellular debris were removed by centrifugation at 900 x g for 15 minutes at 4°C. Bacteria were then recovered from the supernatant fluid by centrifugation at 14,000 x g for 15 minutes at 4°C. The bacterial pellet was resuspended in phosphate buffered saline (PBS) and washed three times by centrifugation and resuspension as above. The final bacterial pellet was resuspended in PBS to an optical density of approximately 3.0 and frozen at -70°C until analyzed.
  • PBS phosphate buffered saline
  • Actinobacillus pleuropneumoniae were also cultured in defined media #1, defined media #3 and complete HP media (see Table 3) . All media contained 10 mM nicotinamide adenine dinucleotide (NAD) . Cultured bacteria were centrifuged as above to concentrate the bacteria and remove media components. Bacterial pellets were resuspended in PBS to an optical density of approximately 3.0 and frozen at -70 "C until analyzed.
  • NAD nicotinamide adenine dinucleotide

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Abstract

Antigens of the Pasteurella, Actinobacillus and Haemophilus species of bacteria capable of being up-regulated during infection in a host animal and in minimal medium formulations which provide protection against infections caused by these species are disclosed. Vaccine compositions containing antigens of the Pasteurella, Actinobacillus and Haemophilus species of bacteria are also provided along with methods of immunizing animals against infections by these species.

Description

PASTEORELLACEAE ANTIGENS AND RELATED VACCINES Background of the Invention
Animal vaccines designed to protect against pneumonias caused by Pasteurellaceae are generally produced from inactivated whole bacteria or extracts of bacterial cultures. The protective potential of potassium thiocyanate extracts of culture-grown Pasteurella multocida have been investigated in mice, chickens, cattle and rabbits. These extracts contained protein, hyaluronic acid, lipopolysaccharide, DNA and RNA, making interpretation of the protective component difficult. Although some cross- protection has been observed, protection was mainly against homologous challenge.
The immunogenic outer membrane proteins expressed by a rabbit isolate of Pasteurella multocida grown in culture have also been investigated. The major antibody response appeared to be directed against outer membrane polypeptides having molecular masses of 27 kD, 37.5 kD, 49.5 kD, 58.7 kD and 64.4 kD (Lu et al. (1988) Infect Immun 56:1532-1537). Further work demonstrated that a monoclonal antibody specific for the 37.5 kD protein could passively protect mice and rabbits from challenge, if the isolate used for challenge expressed the antigenic determinant recognized by the monoclonal antibody. However, not all isolates tested expressed the antigenic determinant (Lu et al. (1991) Infect Immun 59:172-180).
Most investigations that concern the cross-protective capacity of Pasteurella multocida Type A have used serotypes and isolates that infect poultry. Cross- protective antiserum made in turkeys by inoculating inactivated in vivo grown bacteria was used for passive immunization,, and results showed this antiserum passively protected young turkeys against heterologous challenge (Rimler RB (1987) Avian Diseases 31:884-887). In an attempt to determine the nature of these cross-protection factors in Pasteurella multocida , investigators have shown that complete lysis or partial solubilization of in vivo grown bacteria followed by gradient centrifugation resulted in a fraction that could provide partial protection against homologous and heterologous challenge. Four protein bands with molecular weights of approximately 74 kD, 65 kD, 39 kD and 30 kD were suggested as being responsible for cross- protection (Ri ler RB et al. (1989) Avian Diseases 33:258-263). Additional studies have shown that organisms recovered from bacteremic birds contain outer membrane proteins of similar sizes. However, these polypeptides were not detected in bacteria of the same isolate cultured in a standard, enriched media.
Antibodies specific for polypeptides of approximately 153 kD, 179 kD, 192 kD and 204 kD recovered from in vivo- grown bacteria that were detergent insoluble have also been demonstrated to provide passive cross-protection against heterologous challenge in poultry (Wang CL et al. Abstract #32, p. 8, Conference of Research Workers in Animal Diseases, November 9-10, 1992, Chicago, IL) . This antiserum was adsorbed against culture grown bacteria to remove antibodies that reacted with antigens in the culture grown preparation. These studies suggested that the regulation of protein expression in Pasteurella multocida may depend upon the local environment, and antigens that may be important immunogens might not be expressed during in vitro growth. These antigens may only be up-regulated during the infection process as the organism invades its natural host.
The use of minimal medium for the production of in vivo- expressed antigens is based on the idea that bacteria grown in a mammalian host experience nutrient deprivation. Numerous investigators have reported that certain auxotrophic mutants of pathogenic bacterial species are avirulent. Mutants with defects in the biosynthesis of purines, aspartic acid, p-aminobenzoic acid, aromatic amino acids, diaminopi elic acid, arginine and pyri idines have been found to be avirulent in such disparate species as Salmonella typhi (Brown, R.F. and Stocker B.A.D. (1987) Infect .' Immun . 55:892-898), Bacillus anthracis (Ivanovics et al. (1968) J . Gen . Microbiol . 53:147-162), Escherichia coli (Kwaga et al. (1994) Infect . Immun . 62:3766-3772), Pasteurella multocida (Homchampa et al. (1992) Molec Microbiol . 6:3585-3593) and Yersinia enterocol it ica (O'Gaora et al. (1990) Microb . Pathogenesiε 9:105-116). All of these reports suggest that mammalian hosts stringently limit the availability of essential nutrients to bacteria. These results also suggest that bacteria must activate numerous biosynthetic pathways to replicate inside a host and cause a disease. In vivo expression technology (IVET) , a methodology which selects for bacterial genes that are specifically induced in host tissues, has provided evidence of a nutritionally-exacting environment in a host (Mahan et al. (1993) Science 259:686-688; Mahan et al. (1995) Proc. Natl . Acad . Sci . USA 92:669-673). IVET studies have demonstrated that the Salmonella typhimurium carAB and pheST genes are specifically induced in vivo . Expression of the carAB operon results in the increased biosynthesis of arginine and pyrimidines. Induction of the pheST operon (which encodes two subunits of phenylalanyl-tRNA synthetase) is believed to be a response to the depletion of a charged tRNA, indicating starvation for the aromatic amino acid phenylalanine.
The in vivo activation of microbial biosynthetic pathways provides essential nutrients to bacteria which they are unable to acquire from the host. However, essential mineral requirements cannot be produced biosynthetically and therefore must be obtained from the host. Among these minerals are calcium, magnesium, iron, zinc, copper, manganese and cobalt. The inability to biosynthesize these mineral requirements puts bacteria into a severe nutritional crisis. Metal ion transport has been best studied in iron acquisition. Since bacteria are unable to biosynthesize their own iron, iron restriction places bacteria into a severe nutritional crisis. Under such iron deprivation, nearly"all bacterial species induce several distinct systems involved in iron acquisition (Cox, CD., "Importance of iron in bacterial virulence", T.J. Beveridge and R.J. Doyle (eds.), Metal Ions and Bacteria , John Wiley & Sons, Inc., New York, N.Y., 1989, p. 207-246). Most of these iron- acquisition systems include outer membrane proteins specifically expressed in response to iron starvation. In addition to forcing bacteria to biosynthesize many of their own essential growth factors, the mammalian iron-withholding defense system requires bacteria to induce numerous systems for the acquisition of this essential nutrient from the in vivo environment.
For example, Pasteurella multocida isolates of turkey origin cultured in media containing iron chelators, express novel outer membrane proteins that are not synthesized in standard, enriched culture media. Several investigators have suggested that these novel proteins are the cross- protective factors observed in in vivo-grown bacteria. This relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown bacteria was investigated by comparison of their protein profiles by SDS-PAGE. These analyses demonstrated that several proteins identified from bacteria grown in media containing an iron chelator were similar to those expressed by in vivo-grown bacteria isolated from turkeys, but were absent from bacteria grown in standard, enriched media. These proteins had molecular masses of 76 kD, 89 kD, and 94 kD (Choi KH et al. (1989) Am J Vet Res 50:676-683; Choi-Kim et al. (1991) Vet Microbiology 28:75-92; Donachle, W. , UK Patent Application 2 202 851 A) .
Other researchers have examined cell-free culture filtrates from iron restricted cultures. These filtrates contain secreted antigens, rather than membrane associated antigens. Cell-free culture filtrates from either standard, enriched or iron restricted medium protected against homologous challenge in turkeys, but did not protect against heterologous challenge.
Antigens from the Pasteurella multocida and Actinobacillus pleuropneumoniae isolated directly from the pleural cavities of infected swine or from a minimal medium formulation have now been identified. These antigens are proteins which are up-regulated during infection in a host animal and are not observed during culture in standard, enriched media. It has now been found that these antigens are up-regulated in minimal medium formulations. However, these antigens are absent or only weakly expressed during in vitro cultivation in standard, enriched media. An immune response to these newly identified antigens invokes protection against heterologous challenge. Therefore, these antigens are useful in the production of an effective vaccine providing cross-protection between multiple isolates of the same species.
Summary of the Invention
An object of the present invention is to provide antigens of the Pasteurella , Actinobacillus and Haemophilus species of bacteria capable of being up-regulated during infection in a host animal and in minimal medium formulations which provide protection against infections caused by these species. Another object of the present invention is to provide vaccines comprising antigens of the Pasteurella , Actinobacillus and Haemophilus species of bacteria capable of being up-regulated during infection in a host animal and in minimal medium formulations which provide protection against infections caused by these species.
Yet another object of the present invention is to provide a method of immunizing healthy animals against infections caused by Pasteurella , Actinobacillus and
Haemophilus species of bacteria which comprises administering to a healthy animal an effective amount of a -6-
vaccine comprising antigens of the Pasteurella , Actinobacillus and Haemophilus species of bacteria capable of being up-regulated during infection in a host animal and in minimal medium formulations which provide protection against infections caused by these species.
Brief Description of Figures
Figure l is a graph depicting a passive immunity study in mice. All mice received 0.5 ml, i.p. , of an immune serum followed four hours later by 100 to 200 CFU of virulent Pasteurella multocida 16926.
In Figure 1A immune serum was collected from a pig following a primary infection with P. multocida isolate 8261. Group A (-) serum is a 1:10 dilution of this immune serum; Group B ( )serum is a 1:10 dilution of this immune serum adsorbed with detergent solubilized, standard, enriched media grown isolate 8261; and Group C (...) is preimmune serum diluted 1:10.
In Figure IB immune serum was collected from a pig infected with isolate 8261 followed by a secondary exposure to P. multocida isolate 16926. Group D (— —) serum is a 1:10 dilution of this immune serum; Group E (—■ •—) serum is a 1:10 dilution of this immune serum adsorbed with detergent solubilized, standard, enriched media grown isolate 8261; and Group C (-=) is preimmune serum diluted 1:10. Figure 2 is a graph depicting a second passive immunity study in mice. All mice received 0.5 ml, i.p. of an immune serum followed four hours later by 100 to 200 CFU of virulent P . multocida 16929. Immune serum was collected from a pig 7 days following a secondary exposure to P. multocida isolate 16926. Group 1 (— —) is preimmune serum diluted 1:10; Group 2 (—) is immune serum diluted 1:10;
Group 3 ( ) is immune serum, treated with detergent and purified, diluted 1:10; Group 4 (...) is immune serum, adsorbed with detergent-solubilized bacteria and purified, diluted 1:10; and Group 5 ( ) is lung washing fluids collected 18 hours following secondary exposure to isolate 16926. "
Detailed Description of the Invention
The Pasteurellaceae family of bacteria contains species of the genera Pasteurella , Actinobacillus , and Haemophilus . Recent work on the phylogeny of the Pasteurellaceae family confirmed the grouping of these three genera into this family (Dewhirst et al. (1992) J . Bacteriol . 174:2002-2013). The various species within the Pasteurellaceae family fall into four large clusters, each cluster containing species of three different genera. Examples of species within this family include, but are not limited to, the animal pathogens P . multocida , A. pleuropneumonia , P. haemolytica , H. somnus , and A. suis . Pasteurellaceae infections in animals result in symptoms similar to those resulting from virulent septic pneumonia. Death is generally due to endotoxic shock and respiratory failure. High mortality rates can occur with the acute form of these infections, however, subacute and chronic forms which result in pleuritis are more common. Treatment of field infections is difficult and often unsuccessful due to widespread antibiotic resistance. Therefore, it is preferred to prevent the infection in animals through use of a vaccine. There has been difficulty, however, in achieving a vaccine which will provide protection against different isolates of a species of bacteria within the Pasteurellaceae family.
Cross-protection against different isolates of a species of bacteria within the Pasteurellaceae family seems to be dependent upon the ability of the host to mount an immune response against bacterial proteins exclusively expressed under the influence of microenvironmental conditions encountered during infection. Most vaccines designed to protect swine against pneumonias caused by these bacteria are prepared from inactivated whole bacteria. However, these vaccines only provide protection against homologous isolates or serotypes (serotyping based on capsular or lipopolysaccharide type) . In contrast, it was found that pigs that recover from an active infection' are generally quite resistant to reinfection, regardless of the isolate or serotype. In addition, it is difficult to incorporate crude bacterial products into large combination vaccines because of the potential of untoward systemic and site reactions that may result from immunization with combinations of crude bacterial products. The present invention provides antigens from the Pasteurella , Actinobacillus and Haemophilus species of bacteria that are up-regulated during infection in a host animal or in minimal medium formulations but which are absent or only weakly expressed during in vitro cultivation of the bacteria in a standard, enriched media, said antigens being capable of providing protection against an infection by these species of bacteria. These antigens provide the basis for effective vaccines designed to cross-protect animals from infection by multiple isolates of one of the Pasteurella , Actinobacillus or Haemophilus species of bacteria.
Examples of host animals for these bacteria include, but not limited to, swine, bovine, ovine, avian and equine species. By "minimal medium formulations" it is meant a culture media specifically designed to provide a sufficient concentration of nutrients to bacteria to allow for synthesis of antigens up-regulated in vivo in a host while removing all undefined components. In contrast, standard, enriched laboratory media are designed to allow optimal growth of bacteria and thus include an excessive levels of nutrients when compared to the minimal growth requirements for each species. Complete Heamophilus Pleuropneumoniae (HP) medium is an example of a standard, enriched medium. Undefined components in these media such as yeast extract, tryptone, peptone or brain heart infusion all contain high levels of complexed nitrogen, amino acids, vitamins, iron and other minerals which provide an excellent nitrogen source "and general nutritional supplement so that little metabolic demand is placed on the bacteria. In contrast, culture media used in the present invention were designed to supply the bacteria with the minimum level of essential nutrients necessary to support growth, thus mimicking the environment encountered when bacteria invade the host organism. Components of the minimal media used in the present invention comprise basal salts (elemental requirements) , carbon sources, special nutritional requirements of the Pasteurellaceae , and nonessential optimizing supplements. Examples of basal salts include, but are not limited to, potassium phosphate, potassium sulfate, magnesium chloride, ammonium chloride, calcium chloride and sodium chloride. Examples of elemental requirements include, but are not limited to, potassium, sulfur, phosphorus, sodium, chloride, and calcium. Examples of carbon sources include, but are not limited to, glycerol and lactic acid. Glucose, galactose, fructose, mannose, sucrose, mannitol, and sorbitol can also be utilized by the Pasteurellaceae . However, because of the fermentative type of metabolism of these organisms, acid can be produced during catabolism of sugars resulting in a lower yield of bacterial cells in the culture. Thus, use of the non- fermentable carbohydrates glycerol and lactic acid, which do not lead to acid accumulation in cultures, is preferred. In addition, since members of the Pasteurellaceae are not prototrophic in that they are unable to grow in a mineral salts medium with a single carbon source, special nutritional additives are required. For example, these species require organic nitrogen sources and may require several amino acids, B vitamins, 0-nicotinamide, adenine nucleotides, or protoporphyrin and its conjugates. To satisfy these requirements, the minimal medium may comprise arginine, aspartic acid, cystine, gluta ic acid, glycine, leucine, lysine, methionine, serine, tyrosine, inosine, uracil, hypoxanthine, thiamine, pantothenate, and nicotihamide. For the propagation of Actinobacillus and Haemophilus species β-NAD, protoporphyrin or its conjugates can also be added as needed. The addition of several components were also found to enhance the cell yield of P. multocida and A. pleuropneumoniae . These non-essential optimizing supplements include a buffer such as HEPES to increase buffering capacity, a nitrogen source such as glutamic acid and Casamino acid (a semi-defined acid hydrolysate of casein, Difco Laboratories Ltd, West Molesey, Surrey KT8 OSE U.K.), and an organic sulfur source such as L-cysteine. In addition, these minimal media formulations contains no iron, zinc, copper, manganese or cobalt salts or complex biological materials containing these minerals. The sole source for these minerals is the trace amounts which may be present as trace contaminants in the various other defined components or water. The laboratory analyses of trace minerals in these minimal media formulations are provided in the following table.
If) In one embodiment of the present invention, a cultured
Pasteurellaceae species such as Pasteurella multocida of
Actinobacillus pleuropneumoniae bacteria is administered to a host animal, preferably a pig, by injection into the pleural cavity.
The host animal is euthanized approximately 12 to 18 hours later and the pleural fluids are collected. Large cellular debris is removed from the fluids, and in vivo- grown bacteria are recovered by centrifugation. The resulting bacterial pellet is washed several times in buffer and centrifuged. The bacterial pellets are then resuspended in buffer and stored at -70°C.
In another embodiment Pasteurellaceae bacteria such as P. multocida or A. pleuropneumoniae are grown in a minimal medium formulation. Cultured bacteria were centrifuged to concentrate the bacteria and remove media components. Bacterial pellets were resuspended in PBS to an optical density of approximately 3.0 and frozen at -70°C until analyzed. Proteins expressed by the in vivo-grown bacteria or bacteria grown in a minimal medium formulation are separated by gel electrophoresis. Those proteins which are strongly expressed can then be identified by transferring part of the gel to nitrocellulose for Western blot analysis using immune serum that has been adsorbed against in vitro-grown bacteria in a standard, enriched media or using antibody recovered from the local site of infection. The term "strongly expressed" refers to proteins up-regulated or expressed during infection in a host animal or in bacteria grown in a minimal medium formulation which are absent or only weakly expressed during conventional in vitro cultivation in standard laboratory medium. The term "weakly expressed" refers to expression in amounts so minor that the antigen is incapable of providing protection against a Pasteurellaceae infection. Conventional in vitro cultivation refers to bacteria inoculated into a standard, enriched medium such as complete Heamophilus Pleuropneumoniae (HP) medium containing supplements. The inoculate is incubated at 37°C for several hours, preferably in a shaking incubator. The bacteria are centrifuged at 10,000 x g to remove culture medium and resuspended in sterile PBS (10 mM phosphate, 0.87% NaCl, pH 7.2) prior to use. At least eleven antigens were identified from the Western blot analysis of in vivo grown bacteria that are absent from Pasteurella multocida grown in vitro using in a standard, enriched media. Western blot analysis of bacteria grown in vitro in a minimal medium formulation demonstrated that the antigenic profile of bacterial proteins produced in this minimal medium formulation was identical to the antigenic profile produced in a host animal infected by the bacteria. The corresponding protein bands had molecular weights of approximately 115 kD, 109 kD, 96 kD, 89 kD, 79 kD, 62 kD, 56 kD, 53 kD, 45 kD, 34 kD and 29 kD.
The corresponding protein bands for each antigen are then excised from the gel, re-isolated by gel-electrophoresis and transferred onto sequence membranes for N'-terminal amino acid sequencing. The N'-terminal amino acid sequence of a 34 kD antigen is as follows:
Ala Thr Val Tyr Asn Gin Asp Gly Thr Lys Val Asp Val Asn Gly Ser Val Arg Leu Leu Leu Lys Gly Glu Lys Asp Pro Arg Arg Asp Leu Met Met Asn Gly (SEQ ID NO: 1)
The N'-terminal amino acid sequence of 29 kD antigen is as follows:
Ala Asp Tyr Asp Leu Lys Phe Gly Met Val Ala Gly Pro Ser Ala
Asn Asn Val Lys Ala Val Glu Phe lie Ala (SEQ ID NO: 2) The N'-terminal amino acid sequence of a second 29 kD antigen is as follows:
Lys Phe Lys Val Gin lie Ala XXX XXX XXX XXX Gin Asp He Asn Gin Tyr Tyr Ala Gly Asp Ala Ala Phe Val (SEQ ID NO: 3) The ability of these antigens to invoke a protective immune response against Pasteurellaceae was verified in passive transfer experiments. Antibodies to the bacteria were isolated from infected pigs. Those antibodies that were rendered specific for the antigens of the present invention by adsorption were administered to mice. These mice were then challenged with a virulent heterologous isolate of Pasteurella multocida . Control mice not receiving the antibodies developed infections while those receiving the antibodies did not. Thus, antibodies developed as an immune response against these antigens provided cross protection against heterologous isolates of Pasteurella multocida . In additional experiments, vaccines were prepared from P. multocida cultured in standard, enriched media containing yeast extract and from bacteria cultured in a minimal medium formulation. The vaccine prepared from bacteria cultured in standard, enriched media was unable to protect mice from either homologous or heterologous challenge of P. multocida . In contrast, mice immunized with the vaccine prepared from bacteria cultured in a minimal medium formulation had much better survival rates. These experiments demonstrate that growth of bacteria under conditions that limit the availability of nutrients and trace elements such as iron, copper, zinc, manganese or cobalt result in the expression of antigens normally up-regulated by the host environment and that they play an important role in protection. The ability to produce these antigens by growth in minimal media results in the ability to produce more efficacious vaccines than are currently available.
In the present invention, Pasteurella multocida antigens, which are capable of being up-regulated during infection in a host animal and in a minimal media formulation which provide protection against a Pasteurella infection, are identified. In one embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 115 kD. In another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 109 kilodaltons. In yet another embodiment. a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 96 kilodaltons. In yet another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 89 kilodaltons. In yet another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 79 kilodaltons. In yet another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 62 kilodaltons. In yet another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 56 kilodaltons. In yet another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 53 kilodaltons. In yet another embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 45 kilodaltons. In a preferred embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 29 kilodaltons and an N'-terminal amino acid sequence comprising SEQ ID NO: 2. In a second preferred embodiment, a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 29 kilodaltons and an N'- terminal amino acid sequence comprising SEQ ID NO: 3. It is also preferred that a Pasteurella multocida antigen has a molecular weight, as determined by gel electrophoresis, of approximately 34 kilodaltons and an N'-terminal amino acid sequence comprising SEQ ID NO: 1.
Antigens up-regulated during infection in a host animal and in a minimal media formulation but not in bacteria grown in vitro in a standard, enriched media were also identified for isolates of Actinobacillus pleuropneumoniae . A. pleuropneumoniae is member of the Pasteurellaceae family which exists in the most distinct phylogenetic cluster from that of P . multocida . The most prominent antigenic difference between the in vivo or minimal media bacteria and the bacteria cultured in a standard, enriched media was the presence of additional bands of approximately 60 kD to 65 kD in the in vivo and minimal media preparations.
In addition to being produced by bacteria grown in vivo in a host animal or in minimal medium formulations, the antigens of Pasteurellaceae can be produced recombinantly using techniques well-known to those skilled in the art or induced in culture via genetic manipulation.
Antigens of the present invention may be incorporated into a vaccine and administered to a healthy animal in an effective amount to protect against infection by bacteria of the Pasteurellaceae family. Upon administration, the antigens in the vaccine will invoke an immune response resulting in the healthy animal forming antibodies against the bacteria. "Effective amount" refers to that amount of vaccine which invokes in an animal an immune response sufficient to result in production of antibodies to the antigens. The animal will then be protected from any subsequent exposure to an isolate of Pasteurellaceae .
In a preferred embodiment a vaccine for the Pasteurellaceae bacteria Pasteurella multocida comprises at least one antigen having a molecular weight of 34 kD and an N'-terminal amino acid sequence comprising SEQ ID NO: 1 or a molecular weight of 29 kD and an N'-terminal amino acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 3. The antigen can be dissolved or suspended in any pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include, but are not limited to, normal isotonic saline, standard 5% dextrose in water or water, preferably adjuvanted. Examples of adjuvants include, but are not limited to, Quil A, Alhydrogel, and Quil A and 5% Alhydrogel in tissue culture media. The vaccine can be administered subcutaneously, intramuscularly, intraperitoneally, intravitreally, orally. intranasally or by suppository at doses ranging from approximately 1 to 100 μg/dose.
In another embodiment, the antigens of the present invention produced in vivo or in bacteria grown in vitro in a minimal media formulation, recombinantly or via genetic manipulation or under specialized culture conditions can be added to whole culture grown bacteria to produce an effective vaccine. Addition of these antigens to the culture grown bacteria increase the efficacy of the resulting vaccine.
This invention is further illustrated by the following nonlimiting examples.
EXAMPLES
Example 1: Bacterial isolates and growth conditions Pasteurella multocida isolates 8261 and 16926 were field isolates received from the Iowa State Veterinary Diagnostic laboratory. Both isolates were serotype 3A. For conventional in vitro growth, bacteria were inoculated into Heamophilus Pleuropneumoniae (HP) medium (Gibco, Grand Island, NY) containing supplements, and incubated for 6 hours at 37°C in a shaking incubator. The bacteria were centrifuged at 10,000 x g to remove culture medium and resuspended in sterile PBS (10 mM phosphate, 0.87% NaCl, pH 7.2). For in vivo growth, 1 ml of cultured bacteria at a concentration of 2 x 108 CFU/ml were administered to pigs by transthoracic injection into the diaphragmatic lobe. Pigs were euthanized 16 hours later and in vivo-grown bacteria were recovered from the pleural fluids. The pleural fluids were centrifuged at 250 x g to remove large cellular debris, and then in vivo-grown bacteria were recovered by centrifugation at 10,000 x g for 40 minutes at 4°C. The bacterial pellet was washed three times with sterile PBS by centrifugation as above. Bacterial pellets were resuspended in PBS and stored at -70°C. Protein concentrations from both in vivo and in vitro grown Isolates were determined using the BCA Protein Assay (Pierce, Rockford, IL.) in accordance with the manufacturer's instructions.
Example 2: Convalescent sera and antibody from immune, challenged pigs
Three groups of 6-8 week old pigs were infected with 5 to 10 x 106 colony forming units (CFU) /ml of PmA isolate
16926 or 8261 by transthoracic challenge. The pigs were allowed to convalesce for a period of between 10 days to one month and then treated with antibiotics to clear any residual infection. Following an additional period of one to two months, the pigs were exposed to a heterologous isolate of PmA (8261 or 16926) by the transthoracic route (1.5 - 2.5 x 10" CFU/ml). The pigs were euthanized at day 0, 1, 2, 3, 4, or 7. Serum samples were collected from the pigs at the time of primary infection, one month following primary infection, at the time of second infection, and at the time of euthanasia. Lung washings were recovered from the pigs at the time of euthanasia to obtain antibody from the local site of infection. To recover antibody, the lungs and trachea were rinsed with approximately 100 ml of sterile PBS. The lungs were massaged, and then fluids containing the antibody were poured into sterile tubes. The fluids were centrifuged at 250 x g for 20 minutes at 4°C to remove large cellular debris and red blood cells.
Example 3: Adsorption of antibody preparations with detergent-solubilized or whole Pasteurella multocida
Antibodies that recognize in vitro grown bacterial antigens were removed by adsorption against either whole bacteria or detergent-solubilized antigen. Fresh culture grown bacteria (isolate 8261 or 16926) were obtained for the whole bacteria adsorptions. Approximately 10 ml of bacteria (3 x 109 CFU/ml) were pelleted by centrifugation at 11,000 x g for 40 minutes at 4°C (Beckman microfuge, Beckman Instruments, Palo Alto, CA) . The supernatant was removed and 0.1 ml of antiserum added. The mixture was incubated overnight at 4°C while being gently agitated. Following incubation, the adsorbed material was centrifuged at 11,000 x g for 40 minutes and the supernatant was removed and added into a fresh bacteria pellet. The final supernatant was collected and stored at -20°C for Western blot analysis. For detergent solubilization, the culture grown bacteria was solubilized in a solution containing 0.062 M Tris, 0.069 M SDS and 1.09 M glycerol, pH 7.0. The antigen preparation was then boiled for 10 minutes at 100°C to solubilize the bacteria. After the solubilized antigen had cooled, it was mixed with an equal volume of antibody and incubated overnight at 4°C. The mixture was centrifuged at 20,000 x g for 40 minutes to pellet the precipitated antibody-antigen complexes. The final supernatant was collected and stored at -20°C.
Example 4: Purification of antibody preparations using ammonium sulfate precipitation
Antibody preparations used for the passive immunity study in mice were partially purified using ammonium sulfate. Serum samples were diluted 1:3 in sterile PBS. A solution of saturated ammonium sulfate was diluted to 90% of saturation and then added drop-wise to the diluted serum until a volume equivalent to the diluted serum was added, resulting in a 45% ammonium sulfate precipitation of the antibody. This solution was incubated on ice for 1 hour. The mixture was centrifuged at 10,000 x g for 40 minutes at 4°C to pellet the ammonium sulfate precipitate. The supernatant was discarded and the pellet was resuspended to the initial serum volume using sterile PBS. The ammonium sulfate precipitation was repeated to further purify the antibody. Following resuspension of the antibody, the solution was dialyzed against three changes of PBS using a 12,000 "MW cutoff membrane tubing.
Example 5: SDS-PAGE and Western blot analysis
Proteins from PmA were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrotransfer and immunoblotting. Proteins from PmA isolates 8261 and 16926, both from in vivo and in vitro grown bacteria, were boiled for 10 minutes with 5X reducing buffer (Pierce, Rockford, IL) . The proteins were then separated on 10% SDS gels at a concentration of 16 μg per lane. After electrophoresis (20 nλ constant amperage) , proteins were transferred on blotting membrane (Immobilon, Millipore, Bedford, MA) overnight at 4°C (30 volts, constant voltage) . The membrane was blocked with 10% non-fat dry milk in 10 mM Tris, 0.9% NaCl, pH 7.2 (Tris-saline) and strips were further incubated with appropriate dilutions of absorbed antiserum for 1 hour at room temperature. Subsequently, strips were rinsed 3 times with Tris-saline containing 0.1% Triton X-100 and further incubated 1 hour with alkaline phosphatase conjugated goat α-swine IgG (H+L) (Kirkegaard and Perry, Gaithersburg, MD) at a 1: 1000 dilution. After 3 washings with IX Tris-saline Triton-X (each for 5 minutes) , the strips were immersed in 5-bromo-4-chloro-3- indolyl-phosphate (BICP) at a concentration of 0.21 g/1 and nitroblue tetrazolium (NBT) at a concentration of 0.42 g/1 in an organic base/Tris buffer (Kirkegaard and Perry, Gaithersburg, MD) for 10 min. The strips were rinsed in distilled water to stop color development. Proteins that were strongly expressed by in vivo grown PmA isolates 8261 and 16926 were identified by Western blot analysis using immune serum that had been exhaustively adsorbed against culture-grown bacteria. At least eleven protein bands with molecular weights of approximately 115 kD, 109 kD, 96 kD, 89 kD, 79 kD, 62 kD, 56 kD, 53 kD, 45 kD, 34 kD and 29 kD were identified from in vivo grown bacteria that were absent from or poorly expressed by culture grown PmA.
Using non-adsorbed sera, the differences in band profiles could not be distinguished between the in vivo and culture grown antigens. This indicates that the majority of the antibody response mounted against an infection with Pasteurella multocida is specific for antigens that are expressed either when the bacteria are cultured in vitro or when the bacteria are recovered from their natural host. In contrast, the majority of antibodies that are not removed by adsorption with cultured bacteria react only with in vivo bacteria recovered from the host.
Example 6: Determination of Molecular Weights of Identified Proteins
The molecular weights of proteins identified to be unique or up-regulated under in vivo growth conditions were estimated by Whole Band Analysis using the Biolmage Computer
System (Biolmage/Millipore, Ann Arbor, MI) . The weights were estimated for the identified bands based on known molecular weights markers. Both Rainbow colored protein molecular weight markers (Amersham Life Science, Arlington Heights, IL) and Bio-Rad SDS-PAGE broad range molecular weight standards (Bio-Rad Laboratories, Hercules, CA) stained with Coomassie blue were used as the standards of comparison.
Example 7: Passive Immunity in Mice
All antibody preparations used in the passive immunity experiments were generated in swine against a primary infection with P. multocida isolate 8261, and in some cases were followed by a second infection with isolate 16926. Serum collected prior to the primary infection was used as a negative control for the passive immunity experiments. Convalescent serum from pig 104 was collected either at 30 days following the primary infection or at 7 days following the secondary exposure, and lung washings from pig 103 were collected 18 hours following the secondary infection. A secondary antibody response would be expected following re- exposure with the second strain. However, the secondary response should be limited to antigens that are common between the two Pasteurella multocida isolates.
The serum antibody preparations were adsorbed with detergent-solubilized bacteria and purified by ammonium sulfate precipitation as described in Example 4. The partially purified antibody preparations were standardized by volume to represent a 1:10 dilution of the original serum sample. Lung washings were not adsorbed or purified.
Mice were injected by the intraperitoneal route with 0.5 ml of the antibody preparations. Four- hours later the mice were infected with isolate 16926 at a rate of 100-200
CFU/mouse. Mice were observed for 10 days following challenge for clinical signs and mortality.
Both the antisera collected after the primary challenge and the antisera collected after the secondary challenge passively protected mice against the 16926 challenge (9 of 10 and 8 of 10 mice survived at least ten days post infection, respectively) . These antisera were also adsorbed with detergent treated culture grown bacteria (8261) . Following adsorption, the antisera lost some of their protective capacity, but the death rate in these groups were still lower than in the control group that received preimmune serum.
A second passive immunity study was performed to verify the protective capacity of adsorbed antisera. Serum collected following the secondary infection was used in this study. All groups of mice given immune serum had better survival rates than that of the control mice that received preimmune serum. The group given non-treated immune serum exhibited the highest survival rate (9 out of 10 mice survived the ten day challenge period) . The antisera that had been detergent treated and purified provided intermediate protection. In these groups mice began dying between 6 and 10 days following challenge. No difference in survival time or mortality was seen between the mice that received detergent treated and purified antiserum versus the mice that received antiserum that had been adsorbed with detergent-solubilized culture grown bacteria and then purified. Deaths occurring in these groups suggest that either the total quantity of specific antibody was reduced during purification, or that the half-life of the antibody was shortened by the detergent treatment or purification. In either case, the loss appeared to be non-specific. Western blot analysis of the antibody preparations used in these experiments demonstrated that purification in the absence of adsorption did not remove antibodies that were common to culture grown and in vivo grown bacteria and that most reactivity to culture grown bacteria was removed by the adsorption process. Adsorption of the immune serum was performed using isolate 8261. However, Western blot analysis demonstrated that the majority of reactivity against culture-grown isolate 16926 was also removed and that the remaining reactivity against the in vivo bands is seen with both the 8261 and 16926 isolates. These observations suggest that the antibodies that remained following adsorption are specific for in vivo grown bacterial antigens and that these antibodies are largely responsible for protection against challenge.
Preimmune serum did not afford any passive protection in this model. All mice in this group died between 1 and 3 days post challenge. While the preimmune pig serum reacted with a few bacterial proteins in a Western blot analysis, the reactions were weak (1:10 dilution). This would be expected since the pigs used in these studies were not specific-pathogen free. Although they may have had some exposure to Pasteurella multocida or other pathogens with cross-reactive antigens, they were susceptible to Paεteurella multocida at the time of primary infection, and did develop clinical disease following challenge.
Antibody recovered from the lung of an immune pig that had been originally infected with isolate 8261 and then exposed to a heterologous isolate of Pasteurella multocida (isolate 16926) was also tested for its ability to passively protect mice. Antibody was recovered from the lung 18 hours after a secondary transthoracic exposure to Pasteurella multocida . This antibody recovered from the local site of exposure would have direct contact with invading bacteria. Vascular leakage of specific antibody from the circulation into the extracellular environment of the lung represents an important immune mechanism that contributes to the protection seen in immune pigs following secondary exposure. Partial protection was seen in these mice, when compared to the control group. Five mice died within the first 1 to 3 days after challenge, but the remaining 5 mice lived an additional 4 to 5 days. Coomassie staining of the proteins contained in the lung washing material suggested that the majority of protein was immunoglobulin. However, Western blot examination of the lung washing material indicated that the specific antibody concentrations found in lung washings were considerably lower than those recovered from serum. Thus the decreased level of protection seen in these mice can be explained by the decreased concentration of antibody that was recovered from the lung.
Example 8: Active Protection against P. multocida in Mice
Bacteria P. multocida (isolate 8261) recovered from the pleural cavities of acutely infected pigs were isolated as described in Example 1. Isolate 8261 was also grown in culture as described in Example 1. Both preparations were inactivated with 0.3% formalin and adjusted to a concentration of 1 x 109 colony forming units (CFU)/ml. Each preparation was adjuvanted with a mineral oil emulsion containing 5% aluminum hydroxide gel. Five-fold and twenty- five-fσld dilutions of the two vaccines were prepared by diluting the original vaccine in adjuvant. All vaccine preparations were administered by intraperitoneal injection into CF1 mice. Mice were vaccinated twice at a three week interval with a 0.1 ml dose.
All mice were challenged with 50 to 100 CFU of virulent Pasteurella multocida isolates 8261 or 16926 and observed for 15 days. As shown in Table 2 below, when the highest concentrations of vaccine were used, both preparations protected mice from both homologous and heterologous challenge. However, when less concentrated vaccines were used, only the vaccine produced from in vivo grown bacteria was able to protect the mice against virulent challenge. Eight of ten mice vaccinated with 1 x 107 CFU equivalents of in vivo antigens were protected against homologous challenge, and seven of ten mice were protected against heterologous challenge. In contrast, zero of ten mice vaccinated with the least concentrated cultured bacterial antigens survived homologous challenge and only three of ten mice vaccinated with cultured bacterial antigens survived heterologous challenge. None of the ten non-vaccinated mice challenged with isolate 8261 survived, and only one of ten non- accinated mice challenged with isolate 19629 survived. This test of active immunity in mice demonstrated that the addition of antigens that are up-regulated by in vivo growth produced a vaccine that was between 5 and 25 times as effective as a vaccine produced from bacteria grown in a standard, enriched media. TABLE 2
Number of Surviving Mice at each Vaccine Dose
Vaccine Challenge (1 x 10" CFU) 2 x 107 CFU 5 X 10β CFU in vivo 8261 8261 9 7 8
Cultured 8261 8261 9 2 0
None 8261 0 NAb NA in vivo 8261 16926 10 8 7
Cultured 8261 16926 10 5 3
None 16926 1 NA NA
NA is representative of not applicable.
Example 9: Partial amino acid sequencing of proteins
To determine the N'-terminal amino acid sequence of the proteins identified from in vivo grown bacteria, proteins from in vivo grown bacteria isolate 8261 were separated on 10% SDS gels. Western blots were performed simultaneously to identify the correct protein bands. Then, protein bands were excised from the gels, electroeluted and transferred to a PVDF membrane (ProBlott, Applied Biosystems) . Subsequently, protein bands (40-50 pmol each) were stained with 0.1 % Coomassie blue R-250 (Bio-Rad, Hercules, CA) . Individual protein bands were excised from the membrane for amino acid sequencing. Sequencing was performed on an Applied Biosystems Model A vapor phase protein sequencer at the Biotechnology Instrumentation Facility, University of California, Riverside, CA.
Example 10: Production of monoclonal antibodies specific for antigens
Balb C mice were immunized with in vivo grown P. multocida as described in Example 8, and then reimmunized with in vivo bacteria solubilized in SDS. Spleens from immunized mice were fused with SP2/0 myeloma cells to produce antibody-secreting hybridomas. A hybridoma cell secreting antibody specific for the 109 kD protein was cloned -twice by limiting dilution and designated as Mab PMA 3-1. The resulting monoclonal antibody was specific for the 109 kD protein produced by in vivo grown bacteria and did not react with bacteria grown in complete media.
A second monoclonal antibody was selected based on the ability to bind the 29 kD protein from in vivo grown P. mul tocida . Western blot analysis of in vivo grown and cultured bacteria demonstrated strong binding to the 29 kD protein of in vivo grown bacteria, and very little reactivity to culture grown bacteria. This monoclonal antibody producing cell was cloned by limiting dilution and designated Mab PMA 3-21.
Example 11: Expression of Antigens in Minimal Medium Formulation
Culture conditions were designed to supply P. multocida with the minimum level of essential nutrients necessary to support growth, thus mimicking the environment that might be encountered when bacteria invade the host organism. Formulations of minimal medium are shown in Table 3.
TABLE 3
COMPONENT MEDIUM #1 MEDIUM #2 MEDIUM #3
CARBON SOURCE glycerol 40 mM 40 mM 40 mM sodium lactate 20 mM 20 mM 20 mM
BUFFER
HEPES 50 mM 50 mM 50 mM
Amino Acids
L-arginine•HC10 0.0300% 0.0300% 0.0300%
L-aspartic acid 0.0500% 0.0500% 0.0500%
L-cystine•2HC1 0.0260% 0.0260% 0.0260%
L-cysteineΗCl, 0.0790% anhydrous COMPONENT MEDIUM #1 MEDIUM #2 MEDIUM #3
L-glutamic acid 0.1300% 0.1300% 0.1300%
L-glutamic acid'Na 0.1870% 0.1870% glycine 0.0020% 0.0020% 0.0020%
L-leucine 0.0300% 0.0300% 0.0300%
L-lysineΗCl 0.0062% 0.0062% 0.0062%
L-methionine 0.0100% 0.0100% 0.0100%
L-serine 0.0100% 0.0100% 0.0100%
L-tyrosine•2Na•2H,0 0.0290% 0.0290% 0.0290%
SALTS sodium chloride 0.0058% 0.0058% 0.0058% potassium sulfate 0.1000% 0.1000% 0.1000% potassium phosphate 0.2656% 0.2656% 0.2656% dibasic potassium phosphate 0.2720% 0.2720% 0.2720% monobasic magnesium chloride 0.0187% 0.0187% 0.0187% anhydrous
EDTA tetrasodium 0.0004% 0.0004% 0.0004% salt ammonium chloride 0.0220% 0.0220% 0.0220% calcium chloride 0.0022% 0.0022% 0.0022% anhydrous
OTHER COMPONENTS nicotinamide 50 μM 50 μM 50 μM
Casamino acids 0.2000% 0.2000% inosine 0.2000% 0.2000% 0.2000% uracil 0.0100% 0.0100% 0.0100% sodium hypoxanthine 0.0023% 0.0023% 0.0023% thiamineΗCL 0.0004% 0.0004% 0.0004%
D-Ca(II) 0.0004% 0.0004% 0.0004% pantothenate Growth of P. multocida in this media resulted in production of the "same antigens as produced in vivo . Western blot analysis demonstrated that the antigenic profile of bacterial proteins produced in this minimal medium formulation was identical to the antigenic profile of in vivo grown bacteria as described in Example 5.
Example 12: Active Protection of Mice by Vaccine Produced from bacteria grown in minimal medium formulation P . multocida isolate 8261 was cultured in a minimal medium formulation as described in Example 11. Bacteria were also cultured in a standard, enriched media as described in Example 1 or harvested directly from the pleural cavities of infected swine as described in Example 1. All preparations were inactivated with 0.3% formalin with constant stirring for 24 hours at 37° C. The inactivated bacteria were diluted to a pre-inactivation cell count of 1 x 109CFU/ml. The bacterial suspensions were adjuvanted in a squalene emulsion containing Quil A and TDM. Mice (female CF1, Charles River Laboratories) were vaccinated with a 0.1 ml dose by the intraperitoneal route in the lower right quadrant of the abdomen. Mice received two vaccinations three weeks apart and were challenged with either 380 LDS0 (50% lethal dose) of P. multocida 8261 or 209 LD50 of isolate 16926 two weeks following the second vaccination.
In this experiment, the vaccine prepared from P. multocida cultured in a standard, enriched media containing yeast extract was unable to protect mice from either homologous (8261) or heterologous (16926) challenge. Only one of ten mice survived the 8261 challenge, and only five mice survived the 16926 challenge. In contrast, mice immunized with vaccines prepared from in vivo grown bacteria or from bacteria cultured in either of the minimal media formulations had much better survival rates. These survival rates ranged from seven out of ten to complete protection (ten of ten mice) .
TABLE 4 Number of Surviving Mice (n = 10)
VACCINE CHALLENGE WITH CHALLENGE WITH FORMULATION 8261 16926 in vivo grown 8261 7 8
Standard, enriched 1 5 media
Minimal media #1 8 9
Minimal media #2 7 10
Example 13: Induction of "in vivo-like" antigens of Actinobacillus pleuropneumoniae
Bacteria were recovered from the pleural infusions of pigs that died or were euthanized following acute infection with Actinobacillus pleuropneumoniae . Fluids were collected into bottles containing glass beads to remove fibrin clots and then transferred to centrifuge bottles. Red blood cells and other cellular debris were removed by centrifugation at 900 x g for 15 minutes at 4°C. Bacteria were then recovered from the supernatant fluid by centrifugation at 14,000 x g for 15 minutes at 4°C. The bacterial pellet was resuspended in phosphate buffered saline (PBS) and washed three times by centrifugation and resuspension as above. The final bacterial pellet was resuspended in PBS to an optical density of approximately 3.0 and frozen at -70°C until analyzed.
The same isolates of Actinobacillus pleuropneumoniae were also cultured in defined media #1, defined media #3 and complete HP media (see Table 3) . All media contained 10 mM nicotinamide adenine dinucleotide (NAD) . Cultured bacteria were centrifuged as above to concentrate the bacteria and remove media components. Bacterial pellets were resuspended in PBS to an optical density of approximately 3.0 and frozen at -70 "C until analyzed.
Western blot analyses were performed as described in Example 5. Convalescent antiserum collected from a pig that had been experimentally infected with A. pleuropneumoniae (Serotype 5) eight weeks previously was used to develop the immunoblot. The most prominent antigenic difference between the in vivo or defined media bacteria and the bacteria cultured in complete HP was the presence of additional bands of approximately 60 kD to 65 kD in the in vivo and minimal media preparations. Three different serotypes of Actinobacillus pleuropneumoniae (serotypes 1, 5 and 7) were grown in defined media and in complete HP media in order to confirm these differences. In each case, several additional bands were present in the defined media preparation that were not present in the HP grown extract. These antigens were also detected in in vivo preparation from the respective serotype.
Similar bacterial preparations from serotype 5 were probed with an antiserum that was specific for a transferrin binding protein that is known to be up-regulated by iron chelation and thought to function in acquiring complexed iron during in vivo growth (Deneer and Potter, Infection and Immunity (1989) 57(3) :798-804) . A heavy band at approximately 60 kD was detected in the lanes containing in vivo bacteria and in lanes containing bacteria grown in defined media, but not in lanes containing the bacteria grown in complete HP media. This finding confirms that the up-regulation of proteins seen during growth of Actinobacillus pleuropneumoniae within the host also occurs when these bacteria are cultured in defined media. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Pfeiffer, Nancy;
Ankenbauer, Robert; Dayalu, Krishnaswamy Iyengar; Isaacson, Wanda Kay; Kaufman, Thomas James; Li, Wumin (APPLICANTS FOR UNITED STATES OF AMERICA ONLY)
Pfizer Inc. (APPLICANT FOR ALL OTHER COUNTRIES)
(ii) TITLE OF INVENTION: Pasteurellaceae Antigens and
Related Vaccines
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Pfizer Inc. , Patent Dept.
(B) STREET: 235 East 42nd Street, 20th Floor
(C) CITY: New York
(D) STATE: New York
(E) COUNTRY: U.S.A.
(F) ZIP: 10017-5755 (V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: N/A
(B) FILING DATE: Herewith
(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/216,202
(B) FILING DATE: March 22, 1994 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Lorraine B. Ling
(B) REGISTRATION NUMBER: 35,251
(C) REFERENCE/DOCKET NUMBER: PC9064A (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 573-2030
(B) TELEFAX: (212) 573-1939 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ala Thr Val Tyr Asn Gin Asp Gly Thr Lys Val Asp Val Asn Gly15
5 10 15
Ser Val Arg Leu Leu Leu Lys Gly Glu Lys Asp Pro Arg Arg Asp30
20 25 30
Leu Met Met Asn Gly 35
35 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: Amino Acid (D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ala Asp Tyr Asp Leu Lys Phe Gly Met Val Ala Gly Pro Ser Alal5
5 10 15
Asn Asn Val Lys Ala Val Glu Phe He Ala 25
20 25
(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: Amino Acid (D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Lys Phe Lys Val Gin He Ala XXX XXX XXX XXX Gin Asp He Asnl5
5 10 15
Gin Tyr Tyr Ala Gly Asp Ala Ala Phe Val 25 20 25

Claims

What is claimed is:
1. Antigens of the Pasteurella , Actinobacillus and Haemophilus species of bacteria capable of being up- regulated during infection in a host animal and in minimal medium formulations which provide protection against infections caused by these species.
2. Antigens of claim 1 comprising Pasteurella multocida antigens.
3. A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 115 kilodaltons.
4. A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 109 kilodaltons.
5. A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 96 kilodaltons.
6. A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 89 kilodaltons.
7. A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 79 kilodaltons.
8. A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 62 kilodaltons.
9. A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 56 kilodaltons.
10. A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 53 kilodaltons.
11. A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 45 kilodaltons.
12. ' A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 29 kilodaltons and an N'-terminal amino acid sequence comprising SEQ ID NO:2.
13. A Pasteurella multocida antigen of claim 2 having a molecular weight, as determined by gel electrophoresis, of approximately 29 kilodaltons and an N'-terminal amino acid sequence comprising SEQ ID NO:3.
14. A Pasteurella multocida antigen having a molecular weight, as determined by gel electrophoresis, of approximately 34 kilodaltons and an N'-terminal amino acid sequence comprising SEQ ID NO: 1.
15. Pasteurellaceae antigens of claim l comprising Actinobacillus pleuropneumoniae antigens.
16. A vaccine for prevention of an infection by the Pasteurella , Actinobacillus and Haemophilus species of bacteria comprising antigens of Pasteurella , Actinobacillus and Haemophilus species of bacteria which are capable of being up-regulated during infection in a host animal and in a minimal medium formulation.
17. The vaccine of claim 16 wherein the antigens comprise Pasteurella multocida antigens.
18. The vaccine of claim 17 wherein at least one Pasteurella multocida antigen has a molecular weight of approximately 29 kilodaltons and an N'-terminal amino acid sequence comprising SEQ ID NO:2.
19. The vaccine of claim 17 wherein at least one Pasteurella multocida antigen is selected from a group consisting of antigens having molecular weights of approximately 115, 109, 96, 89, 79, 62, 56, 53, and 45 kilodaltons.
20. A vaccine for prevention of an infection by Pasteurella multocida comprising Pasteurella multocida antigens wherein at least one Pasteurella multocida antigen has a molecular weight of approximately 34 kilodaltons and an N'-terminal amino acid sequence comprising SEQ ID NO:l.
21. The vaccine of claim 16 wherein the Pasteurellaceae antigens comprise Actinobacillus pleuropneumoniae antigens.
22. A method of immunizing healthy animals against infections caused by Pasteurella, Actinobacillus and
Haemophilus species of bacteria comprising administering to a healthy animal an effective amount of a vaccine comprising antigens of the Pasteurella , Actinobacillus and Haemophilus species of bacteria capable of being up-regulated during infection in a host animal and in minimal medium formulations which provide protection against infections caused by these species. -39-
23. The method of claim 22 wherein the Pasteurellaceae infection comprises a Pasteurella multocida infection and the animal is administered an effective amount of a vaccine comprising Pasteurella multocida antigens.
24. The method of claim 23 wherein at least one Pasteurella multocida antigen of the vaccine has a molecular weight of approximately 29 kilodaltons and a N'-terminal amino acid sequence comprising SEQ ID NO: 2.
25. The method of claim 23 wherein at least one Pasteurella multocida antigen of the vaccine has a molecular weight of approximately 29 kilodaltons and a N'-terminal amino acid sequence comprising SEQ ID NO: 3.
26. The method of claim 23 wherein at least one Pasteurella multocida antigen of the vaccine is selected from a group consisting of antigens having molecular weights of approximately 115, 109, 96, 89, 79, 62, 56, 53, and 45 kilodaltons.
27. A method of immunizing healthy animals against infections caused by Pasteurella multocida comprising administering to a healthy animal an effective amount of a vaccine comprising antigens of Pasteurella multocida. wherein at least one Pasteurella multocida antigen of the vaccine has a molecular weight of approximately 34 T/IB95/00185
-40-
kilodaltons and a N'-terminal amino acid sequence comprising SEQ ID NO: 1.
28. The method of claim 22 wherein the Pasteurellaceae infection comprises a Actinobacillus pleuropneumoniae infection and the animal is administered an effective amount of a vaccine comprising Actinobacillus pleuropneumoniae antigens.
29. A method of growing species of bacteria from the Pasteurellaceae family in a minimal media formulation so that antigens normally up-regulated during growth of bacteria in a host are expressed.
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NZ322415A (en) * 1995-12-01 2000-02-28 Andrew Allan Potter Transferrin binding proteins (TbpA, TbpB) of pasteurella haemolytica and vaccines containing them
US6610506B1 (en) 1995-12-01 2003-08-26 University Technologies International, Inc. Transferrin binding proteins of Pasteurella haemolytica and vaccines containing same
CA2206575A1 (en) * 1996-05-31 1997-11-30 Akzo Nobel Nv Live attenuated rtx-producing bacteria of the family pasteurellaceae
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US4957739A (en) * 1987-08-13 1990-09-18 Board Of Regents, The University Of Texas System Pharmaceutical compositions of a 105 kD P. Haemolytica derived antigen useful for treatment of Shipping Fever
DE3882781T3 (en) * 1987-03-24 2000-04-06 British Tech Group Pasteurella vaccine.
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