NOVEL OPACITY ASSOCIATED PROTEINS, DNA ENCODING THE SAME, AND METHODS OF USE THEREOF

Abstract: Opacity associated proteins and DNA encoding the same are provided by the present invention. Opacity associated proteins are membrane proteins of H. influenzae which are useful as immunogens in vaccines against infection by H. influenzae. Recombinant cells transformed with DNA encoding opacity associated proteins and vectors encoding the same are also provided and are useful in and for the preparation of vaccines against H. influenzae infection. Antibodies against opacity associated proteins are also described. Methods H. influenzae in a sample and methods of immunizing mammals against H. influenzae are also provided.

Description: NOVEL OPACITY ASSOCIATED PROTEINS, DNA ENCODING THE SAME, AND METHODS OF USE THEREOF Field of the Invention This invention relates to compositions and methods for the preparation of opacity associated proteins and peptides such as Oap A and Oap B of Haemophilus influenzae. The proteins and peptides are used as immunogens in vaccine formulations for active immunization and for generation of antibodies for use in passive immunization. The proteins and peptides can be obtained by purification from H. influenzae or produced using either recombinant DNA or chemical synthetic methods. Additionally the invention relates to novel DNA sequences and vectors useful for directing expression of Oap A and Oap B proteins and peptides. Diagnostic assays and kits are also provided. Background of the Invention Haemophilus influenzae is a common resident on human mucosal surfaces, particularly the nasopharynx. Although it is usually in a commensal relationship with its host, it is also frequently identified in upper and lower respiratory tract infections of adults and children, in whom it remains a leading cause of bacterial disease such as bacterial meningitis, otitis media, epiglottitis and pneumonia. oxon and Wilson, Rev Infect Dis 13 (suppl) : 53183-53187 (1991) . H. influenzae infections have been traditionally treated with antibiotics. However, antibiotic resistant strains of pathogens have recently been reported, compromising antibiotic therapy Center for Disease Con trol , Morbidity and Mortality Weekly Rep., 23: 77, 1974. As a consequence, the common use of antibiotics to treat H. infl uenzae infections is questioned, and may be responsible in part for increasing frequency of resistance. Accordingly, new methods for the prevention and treatment of infection by H. infl uenzae are greatly desired. The use of bacterial and viral vaccines has been very successful in the prevention of infectious diseases in humans and other mammals. Antigenic components have been isolated and purified from H. infl uenzae type b and have been used as vaccines. For example, purified capsular polysaccharide material of Haemophilus influenzae type b has been used as a vaccine against the meningitis caused by this organism in humans (Parke et al . , J. Inf. Dis. 136 (Suppl.) : S51, 1977; Anderson et al . , J. Inf . Dis . , 136 (Suppl) : S63, 1977; Makela et al . , J " . Inf . Dis . 136 (Suppl) : S43, 1977) . However, until now there was not a vaccine which is effective against all types of Haemophilus influenzae infection. Accordingly, the need for an effective, safe vaccine against all Haemophilus influenzae strains is greatly desired. Objects of the Invention It an object of the invention to provide a vaccine formulation that elicits a protective immune response against typable and non-typable H. infl uenzae . It is another object of the invention to provide probes useful in diagnostic tests for the presence of H . influenzae infection. Summary of the Invention In accordance with the present invention are provided novel opacity proteins which are crucial to the ability of H. influenzae to colonize in the nasopharynyx of a host. Vaccine formulations encompassing at least one of the opacity proteins are provided by the present invention. In accordance with other aspects of the present invention, DNA sequences encoding opacity proteins of the present invention are provided. Said DNA sequences, encompassed in recombinant vectors, transformed into recombinant host cells and used in vaccine formuations, are also provided in accordance with the present invention. Monoclonal antibodies against opacity associated proteins of the present invention are also provided. In accordance with other aspects of the present invention diagnostic assays are provided for the detection of H. infl uenzae . In addition, kits comprising diagnostic probes or peptides for diagnostic assays are provided. Brief Description of the Figures Figure 1 is the DNA and putative amino acid sequence of Oap A and Oap B. Figure 2 is a bar graph showing the relationship between colony morphology and nasopharyngeal colonization. Arithmetic mean number of organisms of each phenotype in nasopharyngeal cultures on indicated days after inoculation are shown. Figure 3A is a graph showing nasopharyngeal colonization of the uncapsulated strain of H. influenzae, H3 (o) , strain H209 (oapA " ) (•) , strain H226 (corrected mutant) (D) and oapA: -.TnphoA * (■) . Figure 3B is a graph showing nasopharyngeal colonization of the capsulated strain of H. influenzae, Eagan (o) , Eagan oapA " mutant (•) and Eagan oapA: :TnphoA * (D) . Detailed Description The present invention generally relates to vaccines and their use for preventing, ameliorating or treating Haemophilus influenzae infections. Phenotypic variation appears to be an important mechanism by which bacterial pathogens adapt to varied environmental challenges encountered in distinct ecologic niches during colonization and infection. In some cases phenotypic variation is readily apparent as differences in colony morphology. Braun, "Bacterial Genetics" Philadelphia: WB Saunders 153-183 (1965) . In fact, variations in colonial morphology have provided important clues toward understanding the host-parasite interaction in H. influenzae and other mucosal pathogens. Braun, "Bacterial Genetics" Philadelphia: WB Saunders 153-183 (1965), Pittman, J. Exp Med 53 : 471-492 (1931) . For example, alterations in colony morphology of Neisseria gonorrhoeae and Neisseria eningi tidiε have been shown to correlate with the expression of a family of outer membrane proteins referred to as opacity proteins, which have been implicated in bacterial attachment to specific host cell types. Kupsch et al., EMBO J. 12 : 641-650 (1993) . Applicants have identified two variable opacity (Oap) membrane associated proteins which are expressed by H. influenzae and appear to be implicated in the ability of H. influenzae to colonize and thereby to cause infection. The DNA and putative amino acid sequences of OapA and OapB are set forth in Figure 1. An immune response to one or both of these membrane associated proteins can be elicited in order to prevent or limit colonization of H. infl uenzae in the nasopharynx. Such an immune response can be invoked by administration of substantially pure Oap A and Oap B protein as the antigenic component. Substantially pure, in accordance with some embodiments of the present invention is greater than about 50% purity. Greater than about 85% purity is preferred in some embodiments of the present invention and greater than about 90% is still more preferred. In some embodiments of the present invention greater than about 95% purity is preferred. The present invention is directed to opacity associated (Oap) proteins and peptides of H. influenzae . Specifically, the present invention is directed to Oap A and Oap B. The proteins and peptides of the invention can be produced using recombinant DNA method or by chemical synthesis. The Oap A and Oap B proteins and peptides can be used as immunogens in various vaccine formulations to protect against infection by H. influenzae, both typable and non-typable . The present invention further relates to the nucleotide sequences of DNA encoding Oap A and Oap B proteins as well as the amino acid sequences of the Oap A and Oap B proteins and polypeptide fragments thereof. According to one embodiment of the present invention, recombinant DNA techniques are used to insert nucleotide sequences encoding Oap A and/or Oap B into expression vectors that direct expression of the sequences in appropriate host cells. These expression vector-host cell systems can be used to produce H. influenzae opacity associated proteins, Oap A and Oap B and related proteins and peptides. The gene products can be purified from cells in culture and used as immunogens in vaccine formulations. Alternatively, the proteins can be chemically synthesized. Preferably proteins have sequences substantially homologous to the sequence set forth in SEQ ID NO: 2 and SEQ ID NO: 3. In preferred embodiments of the present invention proteins are at least 85% homologous. In still other preferred embodiments said proteins are at least 95% homologous. In yet other preferred embodiments said proteins are 100% homologous. Such proteins are used in synthetic vaccine formulations. Where the expression vector is a recombinant virus, the virus itself may be used as a vaccine. Infectious recombinant viruses that express one or both Oap A and Oap B proteins, and that do not cause disease in the host, can be used in live virus vaccine preparations to provide substantial immunity. Alternatively, inactivated virus vaccines can be prepared using "killed" recombinant viruses that express one or both Oap A and Oap B proteins. The present invention is further directed to polyvalent antiseru and monoclonal antibody against one or both Oap proteins, as well as methods for use of such immunoglobulins for passive immunization. Diagnostic assays for H. influenzae are also provided. The Oap gene, SEQ ID NO: 1 or a DNA sequence substantially homologous to SEQ ID NO: 1 can be prepared synthetically, or isolated from natural sources by methods known to those skilled in the art. Portions of the DNA sequence may also be prepared for use in some embodiments of the present invention. For example, probes may be prepared for diagnostic purposes. In preferred embodiments of the present invention the DNA sequence is 85% homologous to SEQ ID NO: 1. In still other preferred embodiments of the present invention the DNA sequence is 95% homologous to SEQ ID NO: 1. In yet other embodiments of the present invention the DNA sequence is 100% homologous to SEQ ID NO: 1. For some purposes it may be useful to substitute nucleotide analogs for naturally occuring nucleotides. For example, for use as nucleotide probes, nucleotide analogs such as nucleotides having 2' modification or backbone modifications may be preferred. Preparation of oligonucleotides comprising nucleotide analogs is known to those skilled in the art. For insertion of the Oap gene into an expression vector, a vector is selected having the necessary elements for transcription and translation of the inserted protein coding sequence. A variety of host-vector systems may be employed to express opacity associated proteins, as long as the vector is compatible with the host cell system selected. Expression systems useful in the present invention include, but are not limited to bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA, microorganisms such as yeast containing yeast vectors (e.g. Saccharomyces cerevi siae) , mammalian cell systems such as VERO, Hela and CHO cells infected with a vectors having viral control elements (e.g. vaccinia, adenovirus, simian virus 40 etc.) and insect cell systems (e.g. baculovirus) . Depending upon the expression system selected, any of a variety of suitable transcription and translation elements may be used. Promoter sequences which enhance levels of transcription are preferably selected. The promoter used will, however, depend upon the system selected. Commonly used procaryotic control sequences include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta- lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al . , Nature (1977) 198:1056) the tryptophan (trp) promoter system (Goeddel, et al. Nucleic Acids Res (1980) 8:4057) and the lambda derived P L promoter and Ν-gene ribosome binding site (Shi atake, et al . , Nature (1981) 292:128) . Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes. Additional promoters known in the art include the promoter for 3-phosphoglycerate kinase (Hitzeman, et al., J Biol Chem (1980) 255:2073), and those for other glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization (Holland, ibid) . Expression vectors for mammalian cell systems ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and late promoters from Simian Virus 40 (SV 40) Fiers, et al . , Nature (1978) 273:113) , or other viral promoters such as those derived from polyoma, Adenovirus 2, bovine papilloma virus, or avian sarcoma viruses. Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers in necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or isopropylthio-beta-D-galatoside (IPTG) . A variety of other operons, such as trp, pro, etc. are under different controls. Thus, expression of Oap proteins by an expression system can be controlled. This is especially of value in cases where the protein product is lethal or detrimental to the host cells. In such cases, cells may be cultured in conditions where the promoter is not induced in order to reach optimal cell density. Upon reaching optimal density, the promoter may be induced to express the gene of interest . When cloning in eucaryotic cells, enhancer sequences are useful to optimize expression. For example, a 72 bp tandem repeat of SV40 DNA or the retroviral long terminal repeats (or LTRs) etc. may be inserted to increase transcriptional efficiency. Position of the enhancer sequence has not been found to be critical. Accordingly, the enhancer sequence may be inserted upstream, downstream or within the gene of interest. Initiation signals are required for gene transcription and translation in procaryotic cells. For example, translation in E. Coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases from the initiation codon (ATG) to provide a ribosomal binding site. The gene of interest, and the elements described above, may be ligated, in appropriate orientation with the vector promoter and control elements, into the vector by methods known to those skilled in the art. For example, DNA can be ligated into a cloning vector which has complementary cohesive termini. If however, the complementary restriction sites are not present, the ends of the DNA molecule may be modified. Such modification includes producing blunt ends by digesting back single-stranded DNA termini or by filling the single stranded termini so that the ends can be blunt end ligated. Alternatively, any site desired may be produced by ligating nucleotide sequences onto the DNA termini. These ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction sites. The DNA is then treated with restriction enzymes to cleave the terminal linker (but not the modified internal restriction sites) and ligated to the appropriate vector arms. In an alternative methods, the cleaved vector and DNA fragment may be modified by homopolymer tailing. The vector is then introduced into the host cells. Depending upon the host cell used, transformation, transduction or transfection, is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described by Cohen, S.N. , Proc Natl Acad Sci (USA) (1972) 69:2110, or methods described in Molecular Cloning: A Laboratory Manual (1988) Cold Spring Harbor Press, can be used for procaryotes or other cells which contain substantial cell wall barriers. Infection with Agrrobacteriuin tumefaciens (Shaw, C. H., et al . , Gene (1983) 23:315) is believed useful for certain plant cells. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology (1978) 52:546 can be used. Transformations into yeast can be carried out according to the method of Van Solingen, P., et al . , J Bact (1977) 130:946 and Hsiao, C.L., et al., Broc Na tl Acad Sci (USA) (1979) 76:3829. Transformants can be selected based upon the expression of one or more appropriate gene markers normally present in the vector, such as ampicillin or tetracycline resistance in pBR322 or thymidine kinase activity in eucaryotic host systems. Expression vectors may be derived from cloning vectors, which usually contain a marker function. Such cloning vectors may include, but are not limited to the following: SV40 and adenovirus, vaccinia virus vectors, insect viruses such as baculoviruses, yeast vectors, bacteriophage vectors such as lambda gt-WES-lambda B, Charon 28, Charon 4A, lambda gt-1-lambda BC, lambda gt-1-lambda B, M13mp7, M13mp8, M13mp9, or plasmid DNA vectors such as pBR322, pAC105, pVA51, pACYC177, pKH47, pACYC184, pUBHO, pMB9, pBR325, Col El, pSClOl, pBR313, pML21, RSF2124, pCRl, RP4 , pBR328 and the like. The protein can be isolated using standard methods known to those skilled in the art. Such methods include, but are not limited to chromatography, centπfugation and differential solubilization. Opacity associated proteins such as Oap A and Oap B, whether prepared synthetically or by recombinant methods, are encompassed by the present invention. Oap A includes, but is not limited to the ammo acid sequence substantially as set forth in SEQ ID NO: 2. Oap B includes, but is not limited to the ammo acid sequence substantially as set forth in SEQ ID NO: 3. Both ammo acid sequences may include altered sequences in which functionally equivalent am o acid residues are substituted for residues within the sequence which do not affect the activity of the protein For example, one or more ammo acid residues may be substituted by another of similar polarity. At least 85% homology to the sequences set forth in SEQ ID NO: 2 and 3 are encompassed by the present invention. The ability of opacity associated proteins to elicit a protective immune response may be tested using in vitro assays and animal model systems, such as those used to test antibodies to the capsular polysaccharide of type b H . influenzae . These systems have been found to closely correlated with the ability to elicit a protective immune response in human infants. An m vi tro complement mediated bactericidal assay system, Musher et al . , 1983, Infect . Immun . 39:297-304; Anderson et al . , 1972, J. Cl m . Inves t . 51:31-38; which has been used previously for measuring bactericidal activity of antibodies for PRP and lipopolysaccharide (LPS) against H. influenzae could be used to determine whether or not antibody directed against an opacity associated protein or fragment thereof has bactericidal activity against type b H. influenzae and non-typable H. influenzae . These assays can be performed against a relatively large number of clinical isolates of both typable and non-typable bacteria to determine whether a broad range of strains are killed. Further data on the ability of opacity associated proteins, or fragments thereof, to elicit a protective antibody response may be generated by use of the infant rat meningitis model system. Smith et al . , 1973, Infect. Immuπ. 8:278-290. Infant rats challenged before the sixth day of life, with a suitable dose of H. infl uenzae type b develop bacteremia and a fatal meningitis similar to that seen in human infants. If antibody which is bactericidal against a challenge strain is used to passively immunize infant rats prior to challenge, then they are protected from meningitis and death. Antibodies directed against the current vaccine for type b Haemophilus , PRP, are protective in the infant rat model system. Passive protection against type b Haemophilus meningitis could be demonstrated by immunizing infant rats with rabbit polyclonal anti-Oap A and/or Oap B antibody and subsequently challenging the rats with a lethal dose of H. influenzae type b. Data on the ability of antibody for opacity associated proteins to protect against non-typable H. infl uenzae could be obtained using the chinchilla otitis media animal model system, Barenkamp et al . , 1986, Infect . Immun. 52:572-78. In this animal model, chinchillas are challenged by inoculation of the inner ear canal with non- typable H. influenzae . An otitis media much like that seen in humans develops. Chinchillas, which have been immunized, either actively with non-typable H. influenzae outermembrane proteins, or passively with antibody directed against non- typable H. influenzae outermembrane proteins are protected against aural challenge with the bacteria, Barenkamp et al . , supra. Specifically, immunization should inhibit colonization by H. influenzae . This animal model system could be used to demonstrate the ability of antibody to one or both opacity associated proteins to protect against non- typable H. infl uenzae and specifically to inhibit colonization of H. influenzae . Passive immunization during fetal development may also be studies using this model. For example, a female chinchilla may be immunized against H. influenzae prior to or during gestation. Thereafter, the response of her offspring to challenge by H. influenzae is observed. Specifically inhibition of colonization by H. infl uenzae can be analyzed. Vaccine formulations of the present invention are comprised of an opacity associated protein, such as Oap A and Oap B, whether recombinantly prepared, synthetically prepared, or isolated in substantially purified form in combination with any suitable vaccine adjuvant. Suitable adjuvants include, but are not limited to, surface active substances, e.g., hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl-dioctadecylammonium bromide, N, N-dicoctadecyl-N' -N-bis (2-hydroxyethyl-propane diamine) , methoxyhexadecylglycerol , and pluronic polyols; plyamines, e.g., pyran, dextransulfate, poly IC, polyacrylic acid, carbopol ; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; oil emulsions; and mineral gels, e.g., aluminum hydroxide and aluminum phosphate. The immunogen may also be incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation. In yet another embodiment of the invention, an opacity associated protein or fragments thereof, is a hapten, i.e., a molecule that is antigenic in that it reacts specifically or selectively with cognate antibodies, but is not immunogenic in that it cannot elicit an immune response. In such case, the hapten may be covalently bound to a carrier or immunogenic molecule; for example, a large protein such as protein serum albumin will confer immunogenicity to the hapten coupled to it . The hapten-carrier may be formulated for use as a subunit vaccine. Vaccine formulations of the present invention may also be prepared as combination vaccines which include additional vaccine formulations to achieve an additive immunogenic response thereby augmenting present immunization regimes. For example, vaccine formulations of the present invention may be prepared in combination with other vaccine formulations which protect against H. influenzae type b such as commercially available vaccines prepared from the purified capsule of H. influenzae type b polyribosylribitol phosphate (PRP) . In addition, the vaccine formulation may include formulation for targeting additional diseases such as diphtheria, Neisseria meningi tidis and tetanus toxoid. In accordance with some embodiments of the present invention are provided viral vaccines. Live recombinant viral vaccine or an inactivated recombinant viral vaccine are encompassed in aspects of the present invention. Recombinant viruses may be prepared as described above. Where the recombinant virus is infectious to the host to be immunized but does not cause disease, a live vaccine is preferred. The infectious recombinant virus when introduced into a host can express the opacity associated protein or proteins or polypeptide fragment thereof from its chimeric gene and thereby elicit an immune response against H. influenzae antigens. In cases where such an immune response is protective against subsequent H. influenzae infection, the live recombinant virus itself may be used in a preventative vaccine against H. influenzae infection. Production of such recombinant virus may involve both in vi tro (e.g., tissue culture cells) and in vivo (e.g., natural host animal) systems. For instance, conventional methods for preparation and formulation of smallpox vaccine may be adapted for the formulation of live recombinant virus vaccine expressing one or both opacity associated proteins or polypeptides. Multivalent live virus vaccines can be prepared from a single or a few infectious recombinant viruses that express epitopes of organisms that cause disease in addition to the epitopes of H. influenzae opacity associated proteins. For example, a vaccinia virus can be engineered to contain coding sequences for other epitopes in addition to those of H. influenzae opacity associated proteins. Such a recombinant virus itself can be used as the immunogen in a multivalent vaccine. Alternatively, a mixture of vaccinia or other viruses, each expressing a different gene encoding for different epitopes of opacity associated proteins and/or other epitopes of other disease causing organisms can be formulated in a multivalent vaccine. Inactivated virus vaccine formulations may also be prepared. Inactivated vaccines are "dead" in the sense that their infectivity has been destroyed, usually by chemical treatment (e.g., formaldehyde) . Ideally, the infectivity of the virus is destroyed without affecting the proteins which carry the immunogenicity of the virus. In order to prepare inactivated vaccines, large quantities of the recombinant virus expressing Oap proteins or polypeptides must be grown in culture to provide the necessary quantity of relevant antigens. A mixture of inactivated viruses which express different epitopes may be used for the formulation of "multivalent" vaccines. In certain instances, these "multivalent" inactivated vaccines may be preferable to live vaccine formulation because of potential difficulties with mutual interference of live viruses administered together. In either case, the inactivated recombinant virus or mixture of viruses should be formulated in a suitable adjuvant in order to enhance the immunological response to the antigens. In still other embodiments of the present invention it is possible to confer short-term protection to a host by the administration of pre-formed antibody against an epitope of H. infl uenzae . Thus, the vaccine formulations can be used to produce antibodies for use in passive immunotherapy. Human immunoglobulin is preferred in human medicine because a heterologous immunoglobulin may provoke an immune response to its foreign immunogenic components. Such passive immunization could be used on an emergency basis for immediate protection of unimmunized individuals exposed to special risks, e.g., young children exposed to contact with bacterial meningitis patients. Alternatively, these antibodies can be used in the production of anti-idiotypic antibody, which in turn can be used as an antigen to stimulate an immune response against H. influenzae Oap epitopes. Treatment regimes will vary with a patient's age, weight and condition. Immunization schedules are well known to those skilled in the art and will be easily modified for purposes of the present invention. For example a standard protocol for children provides for the admininstration of 2 primary doses of the vaccine followed by a booster at a later date . Diagnostic assays are also encompassed by the present invention. For example, the opacity associated proteins and peptides of the present invention may be used as antigens in immunoassays for the detection of H. influenzae in various patient tissues and body fluids including, but not limited to: blood, spinal fluid, urine and sputum, etc. The antigens of the present invention may be used in any immunoassay system known in the art including, but not limited to, radioimmunoassays, ELISA assays, "sandwich" assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays. Furthermore, novel nucleotide sequence of the gene encoding opacity associated proteins and peptides of the present invention may be used to prepare probes for use in nucleic acid hybridization assays for the detection of H. influenzae in various patient body fluids, including but not limited to: blood, spinal fluid, urine and sputum. The nucleotide sequences of the present invention may be used to prepare probes useful in any nucleic acid hybridization assay system known in the art including, but not limited to Southern blots (Southern, 1975, J. Mol . Biol . 98:508) ; Northern blots (Thomas et al . , 1980, Proc . Na t ' l Acad . Sci . USA 77:5201-05) ; colony blots (Grunstein et al . , 1975, Proc . Na t ' l Acad . Sci . USA 72:3961-65) and capture plate assays. Probes of the present invention may range in length from about 10 to about 200 nucleotides. In preferred embodiments of the present invention, probes are from about 15 to about 100 nucleotides in length. In still other embodiments of the present invention probes are from about 15 to about 25 nucleotides in length. Kits may be supplied comprising one or more probes which specifically hybridize to a gene coding for an opacity associated protein. Such probes may be appropriately labeled with detectable label or reagents for labeling purposes may be included. Kits comprising one or more peptides suitable for immunoassay systems described above are also encompassed in some aspects of the present invention. The following examples are illustrative but are not meant to be limiting of the present invention. Examples Example 1 Bacterial strains, media, and chemicals. H. infl uenzae strains used for the study are shown in Table 1. The strains were grown in brain-heart infusion (BHI) broth supplemented with 1.5% Files enrichment (Difco, Detroit) . BHI plates were solidified with 1% agar. Colony morphology was determined using a stereo zoom dissecting microscope with a substage mirror and 100-W halogen illuminator. Colony morphology was assessed after incubation of agar plates for 14-16 h at 37°C. Chemicals were purchased from Sigma (St. Louis ) unless otherwise specified. Strain Fuju 16K has a kanamycin resistance marker from Tn903 cloned into the Bg/II site in the major pilin gene. Gilsdorf, J.R., et al . , Infect Immunol . , 1990, 58, 1063-72. Chromosomal DNA from Fuju 16K was used to transform strain Eagan by the method of Herriot et al . Herriot et al . , J Bacteriol 101 : 517-524 (1970) to obtain kanamycin- resistant strain Eagan 16K. When appropriate H. infl uenzae strains were maintained on kanamycin (20 μg/mL) or erythromycin (10 μg/mL) . On gross examination with oblique illumination on translucent, solid media without magnification, colonies of H. influenzae Eagan of a clonal origin varied in appearance. Observation with transmitted light and magnification revealed three distinct colony morphologies that differed m color and opacity. The most transparent (T) colonies had a bluish hue, the intermediate (I) colonies appeared whitish, and the most opaque (0) forms had an orange tint. Example 2 Relationship between opacity and H. influenzae pathogenesis. The infant rat model of H. influenzae colonization and bacteremia, which mimics natural infection during childhood, was used to investigate the relationship between opacity phase variation and colonization. Five day old infant rats received intranasal inoculum of 10 5 colony- forming units (cfu) of H. influenzae . Serial washes of nasopharynx were cultured to determined quantity and phenotype of organisms colonizing nasal mucosa 1, 2, 5, and 12 days after inoculation. Animals received mutant strains Eagan 16K (E) or Fuju 16K (U) in which the major pilin gene had been inactivated by insertion of a kanamycin resistance marker. This was done to eliminate any unrecognized contribution of fimbriae which has been implicated as a factor in colonization; Weber et al, Infect Immun 59 : 4724- 4728 (1991) ; and to provide a means of selecting against contaminants in nasal washes. For each strain, 20 pups received inoculum of > 95% of single opacity phenotype (transparent [T] , intermediate [I] and opaque [O] as indicated) . Results are shown in Figure 2. The 0 phenotype was impaired in its ability to colonize the nasopharynx in contrast to the T and I variants. Of the few isolates obtained from nasal washes of pups receiving the O inoculum, all had either the T or I phenotype by 48 h after challenge (Figure 2 (O) ) . The T variant colonized the nasopharynx most efficiently, as assessed by the number of culture-positive pups and density of colonization (Figure 2 (T) ) . By 24 h after administration, most of the I inoculum had shifted in phenotype to the T form (Figure 2(D) . At 12 days after inoculation, at the time of weaning >98% of isolates were of the T phenotype regardless of the original phenotype of the inoculum. Results for both strains were similar. Further evidence that the T variant is best suited to survival in the nasopharynx comes from experiments in which a mixed inoculum consisting of equal numbers of Eagan 16K-T, -I, and -0 organisms were administered. Again, most colonies from nasopharyngeal washes were of the T phenotype by 24 h after inoculation, and no 0 variants were identified (data not shown) . Values are arithmetic mean number of cfu in 10 μl of nasal wash of 20 pups. These results suggest that H. influenzae type b can vary between two phenotypes, one adapted for nasopharyngeal colonization (transparent) and the other for systemic infection (opaque) . Example 3 Identification of Opaque Transformant H175. A spontaneous variant of H. influenzae strain Rd with an opaque phenotype, denoted H175, was obtained by screening a large number of colonies for altered morphology. It was found that chromosomal DNA from H175 was able to transform Rd to the opaque phenotype by the method of Herriot et al . , J. Bacteriol . , 101:517-524 (1970) . Example 4 Construction of a genomic library of H175 in XEMBL3. The genetic locus determining the expression of colony opacity was isolated as follows. Genomic DNA was prepared substantially as described by Hoiseth, et al . , Infect . Immun . , 49:389-395 (1985) . Chromosomal DNA from H175 (50μg) was partially digested with Sau3Al and size fractionated on a sucrose gradient. Sambrook, et al . , Mol ecular cloning: A labora tory manual . Cold Spring Harbor Press, Cold Spring Harbor, NY. Samples which contained fragments ranging in size from 9kB to 15kB were pooled, ethanol precipitated, and used for ligation into XEMBL3 arms cut with BamHI (Stratagene, La Jolla, CA) . The ligation mixture was then packaged and used to infect E . Coli strain XL-Blue MRA (P2) (Stratagene, La Jolla, CA) . DNA was prepared by the plate lysate method obtained from pools of XEMBL3 plaques. Sambrook, et al . , Molecular cloning: A labora tory manual . Cold Spring Harbor Press, Cold Spring Harbor, NY. When DNA from a pooled group of plaques was shown to possess tranforming activity to the opaque phenotype, it was further subdivided until a single clone was identified. Example 5 Cloning and Sequencing of the H. influenzae opacity locus. A single XEMBL3 clone with tranforming activity was mapped using various restriction endonucleases (New England Biolabs, Beverly, MA) . Fragments were subcloned into vector pACYC184 and transformed into frozen competent DH5α (supE44ΔlacU169 (08OlacZM15) hs R17recAlen AlgyrA96t i-lrelAl) and screened for tranforming activity. Plasmid DNA was isolated by the alkaline lysis method. Birnboim and Doly, Nucl ei c Acid Res . , 7:1513-1518 (1979) . These constructs were transformed into Rd and the kanamycin resistant transformants screened for opacity. Insertion of the marker into a EcoRI site resulted in a loss of the transparent phenotype. The region flanking the EcoRI site in PE195 was sequenced using M13 bacteriophage. Sanger, et al . , Proc . Na tl . Acad. Sci . USA, 74: 5463-5467 (1977) . The sequences were analyzed using the software package of the Genetics Computer Group. Databases were searched using National Center for Biotechnology blast program. The DNA sequence of oapA and oapB, with the putative amino acid sequences, are set forth in Figure 1. Example 6 The oapA Gene is Essential to Colonization. Using the infant rat model as described in Example 2, rats were inoculated with strains of mutant and wild type capsular (Eagan) and uncapsulated (H3) H. influenzae . Mutant strains were prepared substantially as described in Example 5 by transforming bacteria strains with a plasmid construct carrying the oapA mutation. As shown in Figure 3, capsular (Figure 3B) and uncapsulated (Figure 3A) wild type strains colonized the nasopharynyx while mutant strains lacking the oapA gene did not. The colonization ability of mutant strains was restored by addition of the oapA gene. Example 7 Colorimetric Assays Show that the OapA Protein is a Surface Protein. E.Coli hosts (kan r ) were transformed with a plasmid carrying the oapA gene. For detection purposes, a gene fusion was prepared by introducing a transposon carrying a gene for alkaline phosphatase (PhoA + Kan r ) and selecting for plasmids in which the PhoA gene was inserted in frame with the oapA gene. It was expected that for transformants in which the PhoA gene was in frame with the oapA gene, if oapA was expressed on the surface of the cell, then OapA + PhoA * transformants would be kanamycin resistant and would be blue in colorimetric assays. Blue colonies were observed, thereby establishing that opaA is expressed on the cell surface . Example 8 Colorimetric Assay shows that the OapA Protein is Expressed on the Surface of H. influenzae Cells. The construct from positive transformants from Example 7 were used to transform H. infl uenzae . H. infl uenzae transformants were positive for PhoA and kan r (as evidenced by blue colony formation) . Example 9 Western Blot Analysis Shows OapA Protein Expression in Both E. Coli and H. influenzae . Membrane proteins were divided into sarcosyl soluble and insoluble fractions by centrifugation and run on 10% separating SDS-PAGE 5% stacking gene with IX SDS-PAGE buffer at 152V. The proteins were transferred to nitrocellulose at lAmp for 1 hour. Western blots were also performed in accordance with standard protocols. The filter was incubated for 2 hours at 25°C with a 1:500 dilution of antibody recognizing PhoA (rabbit anti-BAP) in tris saline blotting buffer (TSBB) . Thereafter the filter was washed 5X in TSBB. The filter was incubated for 1 hour at 25°C with a secondary antibody (antl-rabbit antibody conjugate) at 1:3000 dilution m TSBB, washed 4 times in TSBB and dried. The western blot showed that oapA was expressed m the outermembrane (sarcosyl insoluble fraction) by both E. Coli and H. infl uenzae . Example 10 Immunoblotting shows that OapA is Expressed in the Outermembrane of H. influenzae . Bacterial cell membranes are comprised of an inner layer and an outer layer For purposes of eliciting a immunogenic response, it is generally accepted that proteins which are expressed on the outer membrane should be used as antigens since it has not yet been shown that antibodies can recognize proteins of the cell interior. To test whether oapA was expressed on the outermembrane, nitrocellulose membrane was applied to SBHI kan2r + 40 mL xP/40mL BHI plates streaked with two strains of H. influenzae , strain H238 (oapA PhoA') and strain H228 (oapAPhoA * ) . The membrane was washed 3X in TSBB. The filter was incubated for 2 hours at 25°C with a 1:1000 dilution of antibody recognizing PhoA (rabbit anti-BAP) in TSBB. Thereafter the filter was washed 5X m TSBB. The filter was incubated for 1 hour at 25°C with a secondary antibody (anti-rabbit antibody conjugate) at 1:300 dilution in TSBB, washed 4 times in TSBB and once alkaline phosphate buffer. Color was developed by adding 66μl nitroblue tetrazolium (NBT) X 33μl 5-bromo-4-chloro-3- mdoly-phosphate toluidme salt (Sigma, St. Louis, MO) to 10ml alkaline phosphate buffer at room temperature. Strain H238 exhibited blue color while Strain H228 did not. Therefore, the oapA protein is expressed on the outermembrane and is available to recognition by antibody SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Weiser, Jeffrey M. (ii) TITLE OF INVENTION: Vaccines for Haemophilis Influenza (iii) NUMBER CF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Wocdcock Washburn Kurtz Mackiewicz and Norris (B) STREET: One Liberty Place - 46th Floor (C) CITY: Philadelphia (D) STATE: PA (E) COUNTRY: U.S.A. (F) ZIP: 19103 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (3) COMPUTER: IBM FC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: 08/332,576 (B) FILING DATE: October 31, 1994 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Ralph, Rebecca L. (3) REGISTRATION NUMBER: 35,152 (C) REFERENCE/DOCKET NUMBER: CH-536 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 215-568-3100 (B) TELEFAX: 215-568-3439 (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2100 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: double (D! TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: < ?.) NAME/ E"- CDS (3) LOCATION: 1553..2005 - 25 - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 209..1492 (xii SEQUENCE DESCRIPTION: SEQ ID NO: 1 : AAATCTATAA AACCTTGCAA TGTCTGACTT CTGGCTATCT TGTTCCTAAA CTTGCACGAG 60 AAATTGCGGG CGAGOCAAAT AAGACTTTAT ACGCAGAATA AGATCCGATA AAATACACAT 120 AATTTTTTCG GGGGACTTTT C-AGGTTGTCA ATTTTGTGGG TAATTAATAT AAATAAGAAC ISO AAGCATCAGA G3CAATCCAG Tl.AATTCT ATG GAT AAA AAT CAA CAΛ TGA TCT 232 Mec Asp Lys Asn Gin Gin Ser Ser 1 5 CAA AAT GAA TTG GAT TTA GC-2 CTT AAT CAA GAG CCA ATT AC.A CCA AAG 230 Gin Asn Glu Leu Asp Leu Gly Leu Asn Gin Glu Fro He Tnr Pro Lys 10 " 15 20 Lys Tnr He Gin Pro Ser Ser Ser He Leu Gly Lys Ala Lvs G v Leu 25 30 35 40 TTT G C A ^ AAA AAT CAG GTG CAA ACT AAG TTT CAG CAA CGT AAA GAA 376 Phe Ala Lys Lys Asn H s Val Gin Tnr Asn Phe Gin Gin Arg Lys Glu 45 50 55 CGT ACT TTT GGC GAT TGA TGA ACG CAA GAA AAT GAT CGT TTA ATT CCG 424 Pro Thr Phe Gly Asp Ser Ser Tnr Gin Glu Asn Asp Pro Leu He Pro 60 65 70 AGT GAA AAT TTG AAA AAA GTG CAA AAG CGT GTT CTT CAA ACT TCT TCC 472 Ser Glu Asn Leu Lys Lys Val Gin Lys Pro Val Leu Gin Thr Ser Ser 7Ξ 50 ' 85 52: Tnr Glu Glα -Asn He Ser .Ala Val Aso Glu Glu He Ser Ala Glu Asn 9C 95 " 100 AAC GCG GAT GAA CCG GTG GAA AAG GGT GAA AAA CGT ATT TTA GCT CAA Asn Ala Asp Glu Pro Val Glu Lys Ala Glu Lys Pro He Leu Ala Gin 105 " 1C 115 120 ^ --i ------ . .-—-—-i .—.-.-. m. - .~ . ^.-. Lr^.-i A-- .-1 .--- .xx, , ___. Pro Glu lys Trp Fro Ala Lys ri s Arg Arg ...- _. _. _ _ _. __; ._.__. .-._. _ . G_. _. _. ^ 7 ^_:_. _. .-.. __ X ..A _.1 b _-. - -. _ _. _. _.._. be — Leu Pr'.r ;.;.=- .-.l lie ?ne Val Leu Val He Leu Leu He He Pr.e Pne 1 3 145 15 " Al L.._ L _■ - * --._ £"___: S--~ A-_> Cr ~ H G__.u ≤3__ T .-- Tnr Hr. Ser Λar. AGC AAT GAA GTT CCT GTG CAG TTC CAA TGG TTA GAT CAA AGT CAG CCA 760 Ser Asn Glu Val Pro Val Gin Phe Gin Ser Leu Asp Gin Ser Gin Pro 170 175 180 CTG GAA ACC ACG ATT TTA GAT AAT CCT CCT GCA CAA AAT CAA ATG GCT 808 Leu Glu Thr Thr He Leu Asp Asn Pro Pro Ala Gin Asn Gin Met Ala 135 190 195 200 GTA G.AA CAA GGT AAG C A TCT GAA TTT GCA CCA AAA GCA GAG GAA GCG 856 Val Glu Gin Ala Asn Gin Ser Glu Phe Ala Pro Lys Ala Glu Glu Ala 205 210 ' 215 GCG AAT AAT ACG ACG GCT CAA AAC CCA TTA GTA. GAA AAT GCG CCA ATG 904 Ala Asn As Thr Thr Ala Gin Asn Pro Leu Val Glu Asn Ala Pro Met 220 225 230 CAA CAA AAT GTT GTT CAA TCT CCA AGT CAA ATG CCA AAT GAA ATG GCT 952 Gin Gin Asn Val Val Gin Ser Pro Ser Gin Met Pro Asn Glu Met Ala 235 240 245 GCG GCA TGT GTT GCG CCT ATG CAA CCA GCT CAA GCG GAA CAG CCA AAA 1000 Ala Ala Ser Val Aia Pro Met Gin Pro Ala Gin Ala Glu Gin Pro Lvs 25C 255 260 GCA ACT GTG CCA GTT C G CCG ATG AAA AAA GCG GTA GAG CCA CAA CTT 104S Ala Tnr Val Pro Val Gin Pro Met Lys Lys Ala Val Glu Pro Gin Leu 265 270 275 280 GCG CAT AAA GAT ACA GTG AAA AAA GAA GTG AAA GTG GCG GAG AAA GCA 1096 Ala Kis Lys Asp Thr Val Lys Lys Glu Val Lys Val Ala Glu Lys Ala 285 " 290 " 295 CAG GCT CCA GCA AAA. GCA A.CG GAA CAA AAC GTC GCT AAA ACA GCA GGA 1144 Gin Ala Fro Aia Lys Ala Thr Glu Gin Asn Val Ala Lys Thr Ala Gly 300 305 310 AAC GC CCG ATT GTT GAA GCC AAA CCT GTT CAA GCT AAA AAA GAA AAG 1192 Asn Ala Pro He Val Glu Aia Lys Pro Val Gin Ala Lys Lys Glu Lys 313 320 325 AAA GTT CAA ATC GTT GAT GCA AAA CCT GTG AGT AAA TCT ACA GCT TCT 124C Lvs Val Gin He Val Asp Ala Lys Pro Val Ser Lys Ser Thr Ala Ser 33C - 335 ' 340 i. o' . - i O C Arg Leu Ser Ala Lys Thr Ls Tnr Val Pro Lys Gly Val Ser Leu Met 345 350 355 360 CAA CTC TTC CCT GA.T AA.T C A CTT AAT A.TT TCC GAT GTA AAC GCA. ATC 133c Gin Leu Phe Arg Asc Asn Gin Leu Asn He Ser Asp Val Asn Ala Met 365 370 375 ACC AAA GCC ACA GGC GCG GGA AAT GTT TTA ACT AGC TTT AAA TCT GGC 13=4 Ser Lvs Ala Tnr Civ .Ala C-iv Asn Val Leu Ser Ser Phe Lys Ser Gly 32: " " 385 3°0 GAT AAA. GTA ACG GTA TCT GTG AAT AAT CAA GGC CG.A GTA AAT GAA ATG 1432 Aso Lvs Val Tnr Val Ser Val A.s Asn Gin Gly A.rg Val Asn Glu Met ' ' ' 395 400 405 CCT TTA TCC AA.T GGT GCG CGT TTT GTG CGT CAG TCT GAT GGT TCT TAT 1430 Ar- Leu Ser Asn Gly .Ala Arg Pπe Val Arg Gin Ser Asp Gly Ser Tyr CAA TAT AAA AAA T.AATTTTTTC- CCCCAGTAAT CGGCTTTTTC TTTTT.AAGTT 1532 "r ._. " __) _. ' .= AA ACA TCT CT' ys Tnr Ser Le. c CC CTT TTA ATC ACT GCC TCT CTC- CAA. AT GCC- .AAT C-T.A ACA ACA CCT 1630 Ala L≤_ leu Met T r Gly Cys Val Gin .Asn .Ala Asn Val Tnr Tnr Pro _-_) ___ J 2D L.-_.-. ; ^_.-_-. .<-__.--_.—. -rv- - U_---. ^ _.-. ^.--_.-. .-*--!__-'. <U_. J r.-T... -rt__-_- i .— . iΛ U_r__A AΛΛ I M Gin .Ala Gin Lys Met Gin Val Glu Lys Val Asp Lys Aia Leu Gin Lys 30 35 40 GGC GAA GCT GAT CG.A TA.T TTA TGT CAA GAT C-.AT AG.A GTT GTT CGT GTT 1725 Gly Gl- Ala Asp Arg Tyr Leu Cys Gin Asp Asp Arg Val Val Arg Val 45 50 55 GTA CAC CCC ACG CAT AAA AAA. T.G AAA AAA AA.T TTG CA.T TAT GTT ACT 1774 Val :-_5 .Ala Tnr H s Lys Lys Tyr Lys Lys Asn Leu His Tyr Val Thr 2 55 70 GTC .ACT TTT CA GCC GTA. TC.A G A AAA. CTA. ACC TTA ATG -ATT TCT GAA. 1322 Val Tnr Pr.e Gin Gly Val Ser Glu Lys Leu Thr Leu Met He Ser Glu 75 3 ~ 85 SC CCT GAT 137_ Arg Asp CAT TTT .ACT A.CG GTA A_AA ACG .AAT CTC GGC G.AA ATT TTA GCA ACG CAA 1913 As- Pr.≤ Ser Tnr le_ L' Ξ T r Asn Leu Gly Glu He Leu Ala Tnr Gin (2) INFORMATION FOR SEQ ID NO:2 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 428 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 : Met Asp Lys Asn Gin Gin Ser Ser Gin Asn Glu Leu Asp Leu Gly Leu 1 " 5 10 15 Asn Gin Glu Pro He Thr Pro Lys Lys Thr He Gin Pro Ser Ser Ser 20 25 30 He Leu Giy Lys Ala Lys Gly Leu Phe Ala Lys Lys Asn His Val Gin 35 40 * 45 Thr Asn Phe Gin Gin Arg Lys Glu Pro Thr Phe Gly Asp Ser Ser Thr 50 55 60 Gin Glu Asn Asp Pro Leu He Pro Ser Glu Asn Leu Lys Lvs Val Gin 65 70 75 80 Lys Pro Val Leu Gin Thr Ser Ser Thr Glu Glu Asn He Ser Ala Val 85 SO 95 Asp Giu Glu He Ser Aia Glu Asn Asn Ala Asp Glu Pro Val Glu Lys 100 105 110 Ala Giu Lys Pro He Leu Ala Gin Pro Glu Lys Trp Lys He Leu Gin 115 120 ' " 125 Val Leu Pro Ala Lys His Arg Arg Leu Phe Met A.la He Phe Val Leu 130 135 140 Val He Leu Leu He He Phe Phe Ala Leu Lys Pro Ser Ser Asp Thr 145 150 155 " 160 Val Glu Ser Phe Thr Gin Ser Asn Ser Asn Glu Val Pro Val Gin Phe 165 170 175 Gin Ser Leu Asp Gin Ser Gin Pro Leu Glu Thr Thr He Leu Asp Asn 180 185 190 Pre Fro Aia Gin Asn Gin Met Ala Val Giu Gin Ala Asn Gin Ser Glu 195 200 205 Phe Ala Pro Lys Aia Glu Glu Ala Ala Asn A.sn Thr Thr Aia Gin Asn 21C ' 215 220 Pre Leu Val Glu Asn Aia Pro Met Gin Gin Asn Val Val Gin Ser Pre 225 23 235 2 : Ser Gin Met Pro Asn Glu Met Ala Ala Ala Ser Val Ala Pro Met Gin 245 250 255 Pro Aia Gin Aia Giu Gin Pro Lys Aia Thr Val Pro Val Gin Pro Met 250 265 270 Lys Lys Ala Val Glu Pro Gin Leu Aia H s Lys Asp Thr Val Lvs Lys 273 23: * 235 GH Val Lvs Val Aia Glu I • _ s Ala Gin Ala Pro Ala Lvs Ala Thr Glu OS: 295 300 Gin Asn " /al .Ala Lys Tnr Aia Gly Asn Ala Pro He Val Glu Ala Lys 305 310 315 320 Pre Val Gin Ala Lys Lvs Giu Lvs Lys Val Gin He Val ASD Ala Lys 225 33C " 335 Pro Val Ser Lys Ser Thr Aia Ser Arg Leu Ser Aia Lys Thr Le__ι Thr 340 345 ' 350 Val ?r: Lys Gly Val Ser Leu .Vet Gin Leu Phe Arc ASD Asn Gin Leu 335 360 " 365 Asn He Ser Asp Val Asn Ala Met Ser Lys Ala Thr Gly Ala Giv Asn 37: 375 ' 380 Val ^ Ser Ser Phe Lys Ser Gly Asp Lys Val Tnr Val Ser Val Asn 333 390 * * * 395 400 Asn Gin Gly Arg Val Asn Glu Met Arg Leu Ser Asn Giv Ala A.rq Phe 405 " 410 " 415 Val Arg C-ln Ser Asp Gly Ser Tyr Gin Tyr Lys Lys .". ~> ii; VTICN FOR SEQ ID KO:3 : SEQUENCE CHARACTERISTICS: A. LENGTH: 151 ammo acids (3) TYPE: ammo acid 1 2 ] TOPOLOGY: linear :? :0M: SEQ ID NO: Leu Me: Tnr Pro C-ln Gin _vs Met Leu Cys Gin Asp Asp Arg Val Val Arg Val Val His Ala Thr His Lys 50 55 60 Lvs Tyr Lys Lys Asn Leu His Tyr Val Thr Val Thr Phe Gin Gly Val 65 70 75 80 Ser Giu Lys Leu Thr Leu Met He Ser Glu Arg Gly Lys Asn Tyr Ala 85 90 95 Asn He Arc Trp Met Trp Gin Glu Arg Asp Aso Phe Ser Thr Leu Lys ICO ' 105 110 Thr Asn Leu Gly Giu He Leu Aia Thr Gin Cys Val Ser Gin Thr Ser 115 120 125 Giu Arg Leu Ser Asp A_ Asn A.rg Ala Lys Arg Thr Ser Thr Trp Ph€ 130 135 140 Cys Phe Ala Ser Ser Pro Leu 145 150

Claims: What is claimed is: 1. A substantially pure opacity associated protein. 2. A substantially pure opacity associated protein having the sequence substantially as set forth in SEQ ID NO: 2. 3. A substantially pure opacity associated protein having the sequence substantially as set forth in SEQ ID NO: 3. 4. An isolated DNA sequence coding for substantially pure opacity associated protein having a sequence substantially as set forth in SEQ ID NO: 1. 5. A protein produced from the DNA of claim 4. 6. Recombinant host cells transformed with the DNA encoding at least one opacity associated protein operably linked to regulatory control sequences, which cells express at least one opacity associated protein by said host cells . 7. An opacity associated protein produced by the cells of claim 6. 8. Recombinant host cells transformed with the DNA having a sequence substantially as set forth in SEQ ID NO: 1 operably linked to regulatory control sequences, which cells express at least one opacity associated protein. 9. An opacity associated protein produced by the cells of claim 8. 10. A recombinant vector comprising a DNA sequence encoding at least one opacity associated protein operably linked to regulatory control sequences compatible with an expression system. 11. The recombinant vector of claim 10 wherein the DNA sequence is substantially as set forth in SEQ ID NO: 1. 12. A monoclonal antibody which specifically binds to an opacity associated protein. 13. The monoclonal antibody of claim 12 where the opacity associated protein is selected from the group consisting of Oap A and Oap B. 14. A vaccine composition for the prevention of H. infl uenzae infection in a mammal comprising an immunogenically effective amount of at least one substantially pure opacity associated protein and a physiologically acceptable carrier. 15. The vaccine composition of claim 14 comprising at least one opacity associated protein having a sequence substantially homologous to at least one sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3. 16. A vaccine composition comprising recombinant cells transformed with DNA encoding at least one opacity associated protein, which cells express at least one opacity associated protein and a pharmaceutically acceptable carrier. 17. The vaccine composition of claim 16 wherein said recombinant cells are transformed with at a DNA sequence substantially homologous to the sequence set forth in SEQ ID NO: 1. 18. A vaccine composition comprising a monoclonal antibody which specifically recognizes an opacity associated protein and a pharmaceutically acceptable carrier. 19. A method of protecting a mammal against infection by H. infl uenzae which comprises inoculating said mammal with a vaccine composition comprising an immunogenically effective amount of at least one opacity associated protein. 20. The method of claim 19 wherein the vaccine composition comprises at least one opacity associated protein having an amino acid sequence substantially homologous to at least one sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3. 21. The method of claim 19 wherein the vaccine composition comprises at least one opacity associated protein encoded for by DNA having a sequence substantially homologous to the sequence set forth in SEQ ID NO: 1. 22. A method of protecting a mammal against infection by H. influenzae which comprises inoculating said mammal with a vaccine composition comprising recombinant cells transformed with DNA encoding at least one opacity associated protein, which cells express said opacity associated protein. 23. The method of claim 22 wherein the DNA has the sequence substantially homologous to the sequence set forth in SEQ ID NO: 1. 24. A method of detecting H. influenzae in a sample comprising contacting the sample with a nucle:.c acid probe which specifically hybridizes to a gene coding for an opacity associated protein and detecting the presence or absence of hybridization whereby detection of hybridization indicates the presence of H. influenzae in a sample. 25. A method of detecting H. influenzae in a sample comprising contacting the sample with an antibody which specifically binds to an opacity associated protein wherein the detection of binding indicates the presence of H. infl uenzae in a sample.

Inventors: WEISER JEFFREY N, WEISER, JEFFREY, N.
Applicants: PHILADELPHIA CHILDREN HOSPITAL, THE CHILDREN'S HOSPITAL OF PHILADELPHIA
Application year: 1995
Main classification: C07K1
IPC codes: A61K39, C07K14, C07K16, C12N1, C12N15, C12P21, C12Q1, C12R1, G01N33