VACCINES AND DIAGNOSTIC ASSAYS FOR HAEMOPHILUS INFLUENZAE

Abstract: Peptides and proteins related to an epitope comprising an outer membrane protein of Haemophilus influenzae are described. The peptides and proteins can be prepared by methods including novel and improved methos of purification from H. influenzae cultures, and by recombinant DNA and chemical synthetic techniques. Additionally, recombinant vectors containing nucleotide sequences encoding PBOMP-1 and PBOMP-2 related peptides, proteins and fusion proteins are also described. Recombinant vectors include plasmid DNA and viral DNA such as human viruses, animal viruses, insect viruses and bacteriophages that direct the expression of the PBOMP-1 and PBOMP-2 related peptides, proteins, and fusion proteins in appropriate host cells. The peptides, proteins, fusion proteins and viruses both ''live'' and ''inactivated'' are used as immunogens in vaccine formulations to protect against H. influenzae infections. The peptides, proteins and fusion proteins are also used as reagents in immunoassays as well as to prepare immunoglobulins for passive immunization. Use of the nucleotide sequences encoding the PBOMP related peptides, proteins and fusion proteins in hybridization assays is also described.

Description: -1- VACCINES AND DIAGNOSTIC ASSAYS FOR HAEMOPHILUS INFLUENZAE 1 FIELD OF THE INVENTION The present invention relates to compositions and methods for the preparation of proteins and peptides associated with the outer membrane of Haemophilus influenzae. More particularly, the invention is directed to compositions and methods for preparation of proteins and peptides related to a class of outer membrane proteins of about 16000 daltons molecular weight of type b and non-typable H^ influenzae including PBOMP-1 and PBOMP-2. The proteins and peptides ar used as im unogens in vaccine formulations for active immunization and for the generation of antibodies for use in passive immunization and as reagents in diagnostic assays. The proteins and peptides can be obtained by novel improved methods of 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 PBOMP-1 and PBOMP-2 related proteins and peptides. The nucleotide sequences are used as reagents in nucleic acid hybridization assays. 2 BACKGROUND OF THE INVENTION 2.1 RECOMBINANT DNA TECHNOLOGY AND GENE EXPRESSION Recombinant DNA technology involves insertion of specific DNA sequences into a DNA vehicle (vector) to form a recombinant DNA molecule which is capable of replication in host cell. Generally, the inserted DNA sequence is foreign to the recipient DNA vehicle, i.e. , the inserted DNA sequenc and the DNA vector are derived from organisms which do not exchange genetic information in nature, or the inserted DNA sequence may be wholly or partially synthetically made. Several general methods have been developed which enable 5 construction of recombinant DNA molecules. For example, U.S. Pat. No. 4,237,224 to Cohen and Boyer describes production o such recombinant plasmids using processes of cleavage with restriction enzymes and joining with DNA ligase by known methods of ligation. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture. Because of the general applicability of the techniques described therein, U.S. Pat. No. 4,237,224 is hereby incorporated by reference into the present specification. Another method for introducing recombinant DNA molecules into unicellular organisms is described by Collins and Hohn in U.S. Pat. No. 4,304,863 which is also incorporated herein by reference. This method utilizes a packagmg/transduction system with bacteriophage vectors (cosmids) . Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cellε infected with virus. Regardless of the method used for construction, the recombinant DNA molecule must be compatible with the host cell, i.e. , capable of autonomous replication in the host cell or stably integrated into one of the host cell's chromosomes. The recombinant DNA molecule or virus (e.g. , a vaccinia virus recombinant) should alεo have a marker function which allows the selection of the desired recombinant DNA molecule(s) or virus(es). In addition, if all of the proper replication, transcription and translatio signals are correctly arranged on the recombinant DNA molecule, the foreign gene will be properly expressed in th transformed bacterial cells, as iε the caεe with bacterial expreεsion plasmids, or in permissive cell lines infected with a recombinant virus or a recombinant plasmid carrying eucaryotic origin of replication. 5 Different genetic signalε and processing events control many levels of gene expresεion; for instance, DNA transcription and messenger RNA (mRNA) translation. Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA syntheεiε. The DNA εequences of eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system and further, procaryotic promotors are not recognized and do not function in eucaryotic cells. Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signalε which differ from thoεe of eucaryoteε. Efficient translation of 15 mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (SD) sequence on the mRNA. This sequence iε a εhort nucleotide εequence of mRNA that iε located before the start codon, uεually AUG, which encodeε the amino- terminal methionine of the protein. The SD εequenceε are n Λ complementary to the 3'-end of the 16S rRNA (riboεomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, 1979, Methods in Enzymology 68:473. 25 Many other factors complicate the expression of foreign genes in procaryotes even after the proper signals are inserted and appropriately positioned. One such factor is the presence of an active proteolytic system in E^_ coli and other bacteria. This protein-degrading system appears to 30selectively destroy ■■ abnormal" or foreign proteins. A tremendous utility, therefore, would be afforded by the development of a means to protect eucaryotic proteins expressed in bacteria from proteolytic degradation. One strategy is to construct hybrid genes in which the foreign 35 sequence is ligated in phase (i.e. , in the correct reading frame) with a procaryotic gene. Expression of this hybrid gene results in a fusion protein product (a protein that is a hybrid of procaryotic and foreign amino acid sequenceε) . Succeεsful expresεion of a cloned gene requireε . . . efficient tranεcription of DNA, translation of the mRNA and in some instances post-translational modification of the protein. Expression vectors have been used to express genes in a suitable host and to increase protein production. The cloned gene should be placed next to a strong promotor which is controllable so 'that transcription can be turned on whan necessary. Cellε can be grown to a high denεity and then the promotor can be induced to increaεe the number of transcripts. These, if efficiently translated will reεult in high yieldε of protein. Thiε is an especially valuable system if the foreign protein is deleterious to the host cell. 2.1.1 E. COLI AS A HOST SYSTEM FOR EXPRESSION Most plasmid cloning vectors commonly used in E^ coli are derivatives of ColEl-type repliconε (for additional information see Oka et al., 1979, Mol. Gen. Genet. 172;151- 159) . The ColEl plaεmidε are εtably maintained in E_^ coli strains as monomeric molecules with a copy number of about 15-20 copies per chromosome. Various levelε of expression o human and animal protein-products of foreign genes inserted into these plaεmidε have been obtained. However, very high expreεsion levels should be obtained in order for the system to become economically feasible to produce foreign protein products. One way to obtain large amounts of a given gene produc iε to clone a gene on a plasmid which has a very high copy number within the bacterial cell. In theory, by increasing the number of copies of a particular gene, mRNA levels shoul 5 alεo increase which should lead to increased production of the recombinant protein. 2.1.2. VACCINIA VIRUS AS AN EXPRESSION VECTOR Vaccinia virus may be used as a cloning and expression 5 vector. The vi.rus contai.nε a li.near double-stranded DNA genome of approximately 187 kb pairε which replicates within the cytoplasm of infected cells. These viruses contain a complete transcriptional enzyme system (including capping, methylating and polyadenylating enzymes) within the virus core which are necessary for virus infectivity. Vaccinia virus transcriptional regulatory sequences (promotors) allow for initiation of transcription by vaccinia RNA polymerase but not by eucaryotic RNA polymerase. Expression of foreign DNA in recombinant viruses 16 requires the fusion of vaccinia promotors to protein coding sequenceε of the foreign gene. Plaεmid vectorε, also called insertion vectors have been constructed to inεert the chimeric gene into vaccina virus. One type of insertion vector is composed of: (1) a vaccinia virus promotor 0 i.ncludi.ng the transcri.ptional i.ni.ti.ati.on si.te; (2) several unique restriction endonucleaεe cloning εites downstream from the transcriptional start site for insertion of foreign DNA fragmentε; (3) noneεεential vaccinia viruε DNA (εuch aε the TK gene) flanking the promotor and cloning sites which direct 25 insertion of the chimeric gene into the homologous nonesεential region of the viruε genome; and (4) a bacterial origin of replication and antibiotic reεiεtance marker for replication and selection in E. coli. Examples of εuch vectorε are deεcribed by MacKett (1984, J. Virol. : 857- 30 864). Recombinant viruses are produced by transfection of recombinant bacterial insertion plasmids containing the foreign gene into cells infected with vaccinia virus. Homologous recombination takeε place within the infected 35 cells and results in the insertion of the foreign gene into the viral genome. Recombinant viruses can be screened for and subsequently isolated using immunological techniques, DN plaque hybridization, or genetic selection. These vaccinia recombinantε retain their eεεential functions and infectivit and can be conεtructed to accommodate approximately 35 kb of foreign DNA. Expression of a foreign gene can be detected by enzymatic or immunological assays re.g. , immunoprecipitation enzyme-linked immunosorbent assay (ELISA) , radioimmunoasεay, or immunoblotting] . Additionally, naturally occurring membrane glycoproteins produced from recombinant vaccinia infected cells are glycosylated and may be transported to th cell surface. High expression levels can be obtained by using strong promotors or cloning multiple copies of a singl gene in appropriate vectors and suitable hostε. 2.1.3. BACULOVIRUS AS AN EXPRESSION VECTOR A baculoviruε, εuch as Autographica californica nucle polyhedrosis viruε (AcNPV) may also be used as a cloning or . expresεion vector. The infectious form of AcNPV is normall found in a viral occluεion. This structure is largely composed of polyhedrin peptide in which virus particles are embedded. Polyhedrin gene expression occurs very late in t infection cycle, after mature virus particles are formed. Therefore polyhedrin gene expression is a dispensible function, i.e. , non-occluded virus particles produced in th absence of polyhedrin gene expression are fully active and are capable of infecting cells in culture. According to European Patent Application Serial No. 84105841.5 by Smith . . . al. , a recombinant baculovirus expreεsion vector iε prepare by cleaving baculoviruε DNA to produce a fragment comprisin a polyhedrin gene or portion thereof, inserting this fragme into a cloning vehicle and thereafter inserting the gene to be expressed such that it is under control of the polyhedri -1- gene promotor. The recombinant transfer vector formed in this way is mixed with baculovirus helper DNA and used to transfect insect cells in culture to effect recombination and incorporation of the selected gene at the polyhedrin gene locus of the baculovirus genome. The resultant recombinant baculovirus is used to infect susceptible insectε or cultured inεect cellε. 2.2 HAEMOPHILUS INFLUENZAE AND DISEASE H. influenzae are divided into two groups. Those strains which posεess a known capsule are typed by the serological reaction of the capεule with reference antisera. Types a-f have been identified. Strains which fail to react with any of the reference antisera are known as non-typable. H. influenzae type b (Hib) iε the most frequent cause of neonatal meningitis and other invasive infections in the Unites States (Fraser et al., 1974, Am. J. Epidemiol. 100:29-34) . The major incidence of childhood meningitiε occurε between the ageε of one and five yearε. Sixty percent of thoεe meningitiε cases due to Hib occur in children under the age of two (Fraser et al., supra) . It is now well establiεhed that non-typable H. influenzae (Hi) alεo cauεe diseases including pneumonia, bacteremia, meningitis, postpartum εepεiε, and acute febrile tracheobronchitis in adults (Murphy et al., 1985, J. Infect. Diseaεes 152: 1300-1307) . Non-typable Hi are a frequent etiologic agent of otitis media in children and young adults, causing about 20 to 40% of all otitis media cases. Children may experience multiple infections due to the same organism since infection confers no long lasting immunity. Current therapy for chronic or repeated occurrences of otitis media includes administration of antibiotics ^ and insertion of tubes to drain the inner ear. Hi strains have also been implicated as a primary cause of sinuεitis (Cherry J.D. and J.P. Dudley, 1981, in Textbook of Pediatric Infectious Diεeases, Feigin — and Cherry eds., pp 103-105). Additionally, non-typable Hi cause neonatal sepsiε. Antiεerum produced againεt the capεular polyεaccharide of type b H. influenzae (Hib) which compriεes polyribosyl ribitol phosphate (PRP) , has been shown to be bactericidal and protective against Hib (Smith et al., 1973, Pediatrics £52:637-644; Anderson, et al., 1972, J. Clin. Inv. 51:31-38) . Anti-PRP antibody is ineffective against non-typable H. influenzae infections. 2.3 VACCINES AGAINST H. INFLUENZAE The ideal candidate for a Haemophilus vaccine would have three properties: a) it would be immunogenic in infants of 2-6 months (b) it would elicit an antibody which would protect against infections caused by typable and non-typable H. influenzae, and (c) it would elάcit antibody against a determinant found on the surface of all strains of H. influenzae. The currently availiable vaccines which protect agains Hib infections consist essentially of PRP, the type b capsular polysacchari.de. Puri.fi.ed PRP polysaccharide is immunogenic in children above 18 months of age, but does no elicit a protective antibody responεe in those younger than 18 months. In general, polysaccharideε have been εhown to poor immunogenε in children leεε than about 18 monthε of ag To address this problem, various laboratories have begun studieε in which PRP iε either chemically coupled to protein carrier molecule (Anderεon et al., 1985, Ped. Reε. 18_:252A) or mixed with protein molecules (Monji et al., 198 Infect. Immun. 51:865-871) and administered to animals or humans. Conjugation of PRP to protein has been shown to elicit an anti-PRP antibody responεe in human infants as young as 6 months, while a mixture of PRP with some protein has produced anti-PRP antibody in infant animals (Monji et al. , supra) . 5 — — Although the conjugate and admixture vaccine formulations address one difficulty of PRP vaccines, i.e. , their inability to protect infants younger than 18 monthε, they fail to addreεε another major problem of the PRP vaccine. Anti-PRP antibody iε ineffective against non- typable H. influenzae, which by definition lack the PRP capsule. Hence there is a long recognized need for a vaccine that will elicit a protective immune responεe in children of about 18 months and younger against both typable, including type b and non-typable H. influenzae. 0 One object of the present i.nvention is to provide a vaccine formulation that elicits a protective immune response againεt typable H^ influenzae including type b and non- typable H. influenzae in children under 6 months as well as in older children and adults. The approach of the present 15. . invention is to vaccinate with a protein or fragment thereof which is expoεed on the surface of Haemophilus. The best candidate iε an outer membrane protein (OMP) of H. influenzae. Outer membrane proteins are usually εurface exposed molecules. They are composed of protein which is 0normally i.mmunogeni.c i.n i.nfants, and they have been shown to be capable of eliciting protective antibody in other bacterial systems (Sugasawara et. al., 1983, Infect. Immun. £2:980-985) . In addition Hi and Hib εtrainε have been εhown to have 25 similar OMP profiles (Loeb and Smith, 1980, Infect. Immun. 3_ :709-717) . Antibody to an OMP of Haemophilus could be both bactericidal and opsonic much aε anti-PRP haε been εhown to be bactericidal and opsonic for Hib (Anderson et al., 1972, J. Clin, Invest. 5_l:31-38; Cates et al., 1985, Infect. 30 Immun. £8:183-189). An outer membrane protein haε the additional advantage of being common to Hi and Hib and could protect against both types of bacteria. 35 3 SUMMARY OF THE INVENTION The present invention is directed to peptides and proteins related to an outer membrane protein of about 16000 daltons molecular weight of Haemophilus influenzae identified by applicants and termed "Praxis Biologies Outer Membrane Protein-1" (PBOMP-1) and to an antigenically related outer membrane protein of about 16000 daltons molecular weight of Haemophilus influenzae also identified by applicants and termed "Praxis Biologies Outer Membrane Protein-2" (PBOMP-2) , aε well as the molecularly cloned genes or gene fragments whr.ch encode these peptides or proteins. The present invention is also directed to peptides and proteins related to a PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 fusion protein as well as the molecularly cloned genes or gene fragments which encode these peptides or proteins. In a specific embodiment of the invention, chemically synthesized PBOMP-1 or PBMOP-2 related peptides encompass an antigenic region(s) of PBOMP-1 or PBOMP-2 respectively. The peptides are conjugated to a protein carrier, resulting in the generation of an immunogenic peptide conjugate. The invention is also directed to a subεtantially pure PBOMP-1 obtained from H. influenzae uεing novel and improved methodε. The peptideε o proteinε of the present invention may be used as immunogenε in vaccine formulations for H^_ influenzae, or as reagents in diagnostic immunoaεsays for H. influenzae. The preεent invention iε alεo directed to methods for the molecular cloning of geneε or gene fragmentε encoding PBOMP-1 and PBOMP-2 related peptideε. Theεe molecularly cloned sequences can then be used in the further constructio of other vectors by recombinant DNA techniques, including expression vectors for the encoded peptide productε, or uεe in diagnostic assays for H^ influenzae based on nucleic acid hybridization, or in conεtruction of a εequence encoding a .PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 fusion protein. 5 The peptideε or proteinε of the preεent invention may be purified from H^_ influenzae, or produced using recombinant DNA techniques in any vector-host system, or synthesized by chemical methods. Accordingly, the invention iε alεo directed to the construction of novel DNA sequences and vectors including plasmid DNA, and viral DNA such as human viruseε, animal viruses, inεect viruεes, or bacteriophages which can be used to direct the expression of PBOMP-1 and PBOMP-2 related peptides or proteinε as well aε PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 related peptides or proteins in appropriate host cells from which the peptideε and proteins may be purified. Chemical methods for the synthesis of PBOMP-1 and PBOMP-2 related peptides and proteins are described. PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 and PBOMP-2: PBOMP-1 related peptideε and proteinε can be used as immunogens in subunit vaccine formulations for use againεt all pathogenic H. influenzae, including both type b and non-typable H. influenzae. PBOMP-1 and PBOMP-2 related proteinε or peptides for subunit vaccine preparations can be obtained by chemical synthesi.ε, puri.fi.cati.on from H^ influenzae or purification from recombinant expreεεion vector εyεtemε. PBOMP-1: PBOMP-2 and PBOMP-2: PBOMP-1 related proteins or peptides for subunit vaccine preparations can be obtained by purification from recombinant expresεion vector syεtems or by chemical synthesis. Alternatively, recombinant viruseε which produce the PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, or PBOMP-2: PBOMP-1 related peptideε or proteinε themεelveε or extractε of cellε infected with εuch recombinant viruεeε can be uεed aε immunogens in viral vaccine formulations. Since the PBOMP-1 or PBOMP-2 protein or PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 fusion protein will be recognized as "foreign" in the host animal, a humoral and possibly a cell-mediated immune response will be induced, directed against PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 respectively. In a properly prepared vaccine formulation, this should protect the host against subεequent H^ influenzae infectionε. Moreover, the present subunit vaccine formulations will be compatible with currently available PRP vaccines. The PBOMP-1-related and/or PBOMP-2 related sequences of the present invention can be used in human medical assayε. These include the use of the peptides and proteins of the present invention as reagents in immunoasεayε such aε ELISA tests and radioimmunoassayε which are useful as diagnostic tools for the detection of H. influenzae infection in blood samples, body fluid, tissues, etc. The PBOMP-1 encoding and/or PBOMP-2-encoding gene sequenceε can be used in DNA-DNA or DNA-RNA hybridization assayε for similar diagnostic detection of H^ influenzae. Additionally, these reagentε will provide a valuable tool in elucidating the mechanism of pathogenesiε of H^ influenzae. The preεent invention iε directed further to anti- PBOMP-1, anti-PBOMP-2, anti-PBOMP-1: PBOMP-2, and/or anti- PBOMP-2: PBOMP-1 monoclonal antibodieε which have uses in pasεive immunization regimes, and in diagnostic immunoasεayε. In one embodiment of the invention, monoclonal antibodieε ma be generated againεt PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, and/or PBOMP-2: PBOMP-1 related peptideε. 4 BRIEF DESCRIPTION OF THE FIGURES The present invention may be more fully understood by reference to the following detailed description and examples of specific embodiments as well as the appended figures in which: FIG. 1 represents sodium dodecylsulfate polyacrylamide 0 gel electrophoretic (SDS-PAGE) analyεiε of PBOMP-1. Sampleε and gelε were prepared aε deεcribed in Section 6.1. Lane A containε about 5 ug PBOMP-1. Lane B contains prestained low molecular weight (MW) standards: ovalbumin, alpha- chymotrypεinogen, beta-lactoglobulin, lyεozyme, bovine 5 trypεin inhibitor and insulin (A and B chains) . Relative MWs [in kilodaltonε (kd) ] are shown at the side. FIG. 2 (A and B) repreεentε reactivity of whole cell lysates of E. coli and H. influenzae with polyclonal anti- PBOMP-1 antibody and a monoclonal anti-PBOMP-1 antibody (Gl- 1) . In FIG. 2A, lysates were reacted with polyclonal anti- PBOMP-1 antibody. Lanes are as follows: (1) E. coli HB101; (2) E. coli JM83; (3) molecular weight standardε; (4) purified PBOMP-1 obtained from cultured H. influenzae cellε. In FIG. 2B, lyεateε were reacted with monoclonal anti-PBOMP-1 antibody. Laneε are as descr\bed in FIG. 2A. FIG. 3 repreεentε a reεtriction map of pGD103, a derivative of pLG339 (see Stoker et al., 1982, ^ Gene 18:335- 41) . FIG. 4 (A and B) representε mapε of pAA152 which comprises a 4.2 Kb fragment of H. influenzae DNA cloned into pGD103. A gene encoding PBOMP-1 iε localized to an 737 bp Bglll-BamHI fragment. FIG. 4A is a circular restriction map of pAA152. FIG. 4B illustrateε deletion analyεiε of the inεerted fragment of pAA152. The remaining H. influenzae DNA . . . . in the deletion derivativeε is denoted by black lines. PBOMP phenotype is noted at the right. FIG. 5 representε reactivity of whole cell lyεateε of E. coli JM83 containing pAA152 with individual monoclonal antibodieε which react with different epitopes of PBOMP-1. Lanes are as follows: (A) monoclonal antibody Gl-1; (b) monoclonal antibody G94-3; (C) monoclonal antibody G18-3; (D) monoclonal antibody 25-2; and (E) monoclonal antibody G2-3. FIG. 6 repreεentε autoradiographic analyεiε of DS410 minicellε containing recombinant plaεmidε pAA130 and pAA152. Molecular weight standards are noted at the left of the figure. Lanes represent: (A) DS410 (pAA130) ; (B) DS410 (pGD103) ; and (C) DS410 (pAA152) . The location of kanamycin aminoglycosidase is noted at the right of the figure. FIG. 7 (A and B) representε maps of pAA130 which comprises a 5.7 Kb fragment of H. influenzae DNA cloned into pGD103. FIG. 7A represents a circular reεtriction map of pAA130. FIG. 7B repreεentε deletion analysis of the H. influenzae inserted fragment of pAA130. Solid black lines denote remaining H^ influenzae DNA in the deletion deriva¬ tives. PBOMP phenotype is noted at the right. A gene encoding PBOMP-2 is localized to a 781 bp BstEII-Xmnl fragment. FIG. 8 represents reactivity of whole cell lysates of E. coli JM83 and E. coli JM83 containing pAA130 with polyclonal anti-PBOMP-1 antiserum. Lanes repreεent: (A) JM83 containing pAA130; (B) JM83 containing pAA130; (C) JM83; (D) JM83; (E) molecular weight εtandardε as displayed in kilodaltons on the right side of the FIG.; and (F) Hi S-2. FIG. 9 repreεentε the DNA sequencing strategy of the 737 bp insert fragment of pAA152 showing the origin, direction, and extent of sequence determined from the variou clones. The arrow at the bottom denotes the location of the major open reading frame (ORF) . FIG. 10 representε the nucleotide εequence of the 737 bp fragment which contains the PBOMP-1 gene. The predicted open reading frame (ORF) is shown by the underlined sequence and the direction of transcription indicated by the arrowhead. FIG. 11 representε the deduced ammo acid sequence of PBOMP-1. The nucleotide sequence is depicted on the upper line and the corresponding amino acid sequence below. The amino acid enclosed within the box represents the predicted N-terminal amino acid of the mature form of the protein. FIG. 12 represents alignment of the partial amino acid sequence of a peptide derived from PBOMP-1 (below) with a portion of the derived amino acid sequence of the PBOMP-1 gene (above) . Residues enclosed within boxes represent ismatcheε. FIG. 13 represents the εequencing εtrategy of the 789 bp BεtEII-XmnI fragment of pAA130 εhowing the origin, direction and extent of εequence determined from each clone. The arrow at the bottom denoteε the location of the major open reading frame (ORF) . FIG. 14 repreεentε the nucleotide εequence of the 789 bp BεtEII-XmnI fragment of pAA130 which containε the PBOMP-2 gene. The predicted ORF iε εhown by the underlined εequence. The direction of tranεcription is denoted by the arrowhead. The two bases designated "N" represent unknown nucleotides. FIG. 15 representε the deduced ammo acid εequence of PBOMP-2. The nucleotide εequence is depicted above and the corresponding amino acid sequence below. The residue enclosed within the box indicates the predicted N-terminal amino acid of the mature form of the protein. FIG. 16 representε a chromatogram, obtained uεing gaε liquid chromatography, of the fatty acidε of PBOMP-1. Nonadecanoic acid (C 19) waε included aε an internal εtandard. FIG. 17 repreεentε autoradiographic SDS-PAGE analyεiε of E_^ coli JM83 cellε contai.ni.ng recombi.nant plaεmidε pAA130 and pAA152 aε well aε control E _ coli JM83 cellε containing pGD103. Lanes represent: (1) pAA130; (2) pAA152 and (3) pGD103. The location of a band of about 15,000 daltons molecular weight is noted at the left of the figure. FIG. 18 (A and B) representε Weεtern blot gel analysis of whole cell lysateε of E^_ coli JM83 containing pAA130 or pAA152 in the preεence or abεence of globomycin. Molecular weight standards are noted at the left of FIG. 18 (A and B) . FIG. 18A represents lysateε of cellε containing pAA152 which contains the PBOMP-1 gene. Lanes represent: (1) globomycin absent; and (2) globomycin present. FIG. 18B represents lysateε of cells containing pAA130 which contains the PBOMP-2 gene. Lanes represent: (1) globomycin absent; and (2) globomycin present. FIG. 19 graphically illustrateε the antibody reεponεe obtained when a vaccine formulation compriεing PBOMP-1 (5.2 ug) was administered to human adults . FIG. 20 represents reactivity of whole cell lysates of E . coli JM101 or JM103 with monoclonal antibody G-204. Lanes represent: (A) JM103 containing pPX166; (B) JM103 containing pPX160; (C) JM101 containgin pUC19; (D) molecular weight standard displayed in kilodaltonε on the left side of the figure; and (E) native PBOMP-1 from H^ influenzae. FIG. 21 iε a εchematic repreεentation of the construction of plstsmids containing the PBOMP-1 protein coding εequence lacking the PBOMP-1 signal sequence. In plasmid pPX167, the PBOMP-1 gene lacking the signal sequence iε inserted downstream from the lac promoter. Plasmid pPX16 was constructed by cleaving the PBOMP-1 coding sequence in pPX167 at the BamHl site in the polylinker and cloning the resulting fragment into the BamHl site of plasmid pINIII- ompA3. Plasmid pPX168 contains a chimeric sequence coding for mature PBOMP-1 linked at the amino termiuns to the εigna εequence of E^_ coli omp A protein. FIG. 22 repreεents a chromatogram obtained uεing reverεe phaεe C-4 high performance liquid chromatography of the supernatant fraction of a cytoplasmic extract of E^ coli strain PR13 containing plasmid pPX167. FIG. 23A representε SDS-PAGE analysis of signal-less PBOMP-1 obtained from E^ coli PR13 containing plasmid pPX167 stained with Coomassie stain. Lanes represent: (1) cytoplasmic fraction; (2) DEAE eluate; and (3) reverse phase eluate. Molecular weight standards were run in the lane to the left of lane 1 and relative MWs (in kilodaltonε) are shown at the left side of the figure. FIG. 23B represents reactivity of the fractions with anti-PBOMP-1 monoclonal antibody. Lanes are as in FIG. 23A. FIG. 24 is a schematic representation of the conεtruction of plasmid pPX163 containing the entire coding 5 εequence of PBOMP-2 protein inεerted downεtream from the lac promoter. FIG. 25 repreεentε an SDS-PAGE analyεis of whole cell lysateε of E_^ coli JM103 containing pPX163 grown in the presence or absence of IPTG. Lanes represent: (1) molecular weight standards: Kilodaltons; (2) lysate of JM103 containing pPX163 grown without IPTG; and (3) as in Lane 2, grown in the presence of IPTG (5mM) for 4 hourε. Arrows εhow poεition of three PBOMP-2 reactive bandε induced by IPTG. FIG. 26 iε a schematic representation of the structure of PBOMP-1 repreεen'ts hydrophilic regions of the protein and representε reverse turns present in the secondary structure of the protein. The location and size of chemically synthesized PBOMP-1 related peptides within the PBOMP-1 sequenceε are εhown below. FIG. 27 repreεentε the ammo acid εequenceε of the five chemically synthesized PBOMP-1 related peptideε. FIG. 28 shows a map of epitopes on the PBOMP-1 protein recognized by monoclonal antibodies to PBOMP-1. FIG. 29 representε reactivity of whole cell lyεates of . infected E_ ; _ coli JM103 cells with the capsule deficient H. influenzae strain S2 (lane 4) ; or of the clinical non-typable H. influenzae strainε: 0045E (lane 5) , 1939 (lane 6) , HST31 (lane 7) , and Hib Eagan (lane 8) with anti-PBOMP-2 monoclonal antibody, 61-1. Molecular weight standards (kilodaltons) are shown in lane 2. The reactivities of PBOMP-2 and PBOMP-1 with anti-PBOMP-2 monoclonal antibody 61-1 are shown in lanes 1 and 3 respectively. "FIG. 30 is a schematic representation of the construction of a plasmid containing the PBOMP-2: PBOMP-1 fusion protein coding sequence. Plasmid pPX183 was constructed by separately digesting plasmids, pPXi63 and pPX167 with Seal to completion. The pPX163 Seal digest was further treated with Hindlll to yield a partial digest and the pPX167 Seal digest was completely digested with Hindlll. The 2.7 kb Seal-Hindlll fragment from pPX163 and the 1.-4 kb Seal-Hindlll fragment from pPX167 were isolated and gel purified. The two Seal-Hindlll fragments were subsequently ligated together. FIG. 31 is a schematic representation of the construction of plasmid pPX199 containing the PBOMP-2:PBOMP- fusion protein coding sequence. See text for details of the construction. FIG. 32 represents SDS-PAGE analysis of the fatty acylated PBOMP-2: PBOMP-1 fusion protein expressed by E. col . . — " cellε containing plaεmid pPX199. About 5 μg of the purified fuεion protein waε analyzed in a 15% gel (Lane 2) . Prestained low molecular weight standrds were run for comparison and estimation of molecular weight (Lane 1) . FIG. 33 is a schematic representation of the construction of plasmid pPX512 containing the PBOMP-2: PBOMP-1 fusion protein coding sequence lacking the PBOMP-2 signal sequence. In plasmid pPX512, the PBOMP-2 gene lackin the signal sequence is inserted downstream from the lac promoter. Plasmid pPX512 contains a chimeric sequence codin for mature PBOMP-2 sequence, except that the N-terminal cysteine is replaced by methionine, linked at the carboxy terminus to the PBOMP-1 sequence. See text for details of the construction. 5 DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to proteins and peptides related to epitopes of an approximately 16000 dalto molecular weight outer membrane protein of H^ influenzae, i.e. , PBOMP-1 and of a related approximately 16000 dalton molecular weight outer membrane protein of H. influenzae, i.e. , PBOMP-2. The invention is directed further to fusion proteins comprising epitopes of other important proteins of H. influenzae including IgA protease, fimbri and outer membrane proteinε. The preεent invention iε also directed 5 proteins and peptideε related to epitopes of a PBOMP-1: PBOMP-2 or a PBOMP-2: PBOMP-1 fusion protein. The apparent molecular weights as determined using SDS-PAGE reflect the total molecular weights of the mature (i.e. , proteolytically processed) forms, including any post-translational modification(s) (e.g. , fatty acylation, acetylation, etc.). The proteins and peptides of the invention can be produced using recombinant DNA methods or by chemical synthesis. Additionally, the PBOMP-1 and/or PBOMP-2 proteins and peptides of the invention can be obtained in substantially pure form from cultures of H. influenzae using novel and improved methods of isolation and purification. The PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, and PBOMP-2: PBOMP-1 proteins and peptides specifying epitopes of H^ influenzae can be used aε immunogens in various vaccine formulations to protect against infection with H^ influenzae, an etiological agent of bacterial meningitis, otitis media, epiglottitis, pneumonia, etc. • The vaccine formulations are effective against both H. influenzae typable strainε including typeε a, b, c, d, e, and f aε well as non-typable H^_ influenzae strains. The present invention further relates to the nucleotide sequence(ε) of the geneε encoding the PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, and PBOMP-2: PBOMP-1 proteinε as well aε the amino acid sequences of the PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, and PBOMP-2: PBOMP-1 proteins and polypeptide fragments thereof. According to one embodiment of the present invention, recombinant DNA techniques are used to inεert nucleotide sequences encoding PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 and PBOMP-2: PBOMP-1 epitopes into expresεion vectorε that will direct the expression of these εequenceε in appropriate hoεt cellε. Theεe expression vector host cell systems can be used to produce PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, and PBOMP-2: PBOMP-1, and related proteins and peptideε. The gene productε can be purified from cellε in culture and used aε immunogenε in εubunit vaccine formulationε. Alternatively, the amino acid sequence of PBOMP-1 and PBOMP-2 proteinε and peptideε may be deduced either (1) from the εubstantially pure PBOMP-1 protein iεolated from H^ influenzae aε taught herein or (2) from the H^ influenzae nucleotide εequenceε contained in recombinantε that expreεs immunogenic PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 related proteins and peptides. These proteins and peptides may then be chemically synthesized and used in synthetic subunit vaccine formulations. Where the expresεion vector that expreεεes the PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 sequence(ε) iε a recombinant virus, the virus itεelf may be uεed as a vaccine. Infectious recombinant viruseε that expreεε the PBOMP-1 and/or PBOMP-2 proteinε and peptideε and the PBOMP-1: PBOMP-2 and/or PBOMP-2: PBOMP-1 fusion proteins and peptides, and do not cause diseaεe in a 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 the PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 and/or PBOMP-2: PBOMP-1 proteins and peptides. The present invention is further directed to polyvalen antiserum and monoclonal antibody againεt PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, and/or PBOMP-2: PBOMP-1 aε well aε methods for use of such immunoglobulin for pasεive immunization, and diagnostic assays for H^_ influenzae. In a particular embodiment of the invention, monoclonal antibodies may be generated againεt PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 and/or PBOMP-2: PBOMP-1, related peptides. For the purpose of description, the method of the invention can be divided into the following stageε: (l) iεolation and purification of PBOMP-1 protein; (2) partial amino acid sequencing of PBOMP-1; (3) generation of PBOMP-l and/or PBOMP-2 related peptides which encompass an immunogenic region(ε) of PBOMP-1 or PBOMP-2 reεpectively; (4) molecular cloning of geneε or gene fragments encoding PBOMP-1 and PBOMP-2, and PBOMP-1: PBOMP-2 and/or PBOMP-2: PBOMP-1 fusion proteins including insertion of the genes or gene fragments into expresεion vectors and identification and purification of the recombinant gene products; (5) nucleotide εequencing of the geneε encoding PBOMP-1 and PBOMP-2; and (6) determination of the immunopotency of the PBOMP-1, PBOMP-2 PBOMP-1: PBOMP-2 and/or PBOMP-2: PBOMP-1 proteinε and related productε through production of antibodieε against purified and recombinant protein and peptide products. The method further encompaεseε (7) formulation of vaccineε and (8) diagnostic asεayε for detection of PBOMP-1 and PBOMP-2 genes or gene product (and hence H_^ influenzae) in sampleε of body fluids. 5.1 ISOLATION AND PURIFICATION OF PBOMP-1 In H. influenzae b Eagan and other strainε of H. influenzae, the outer membrane protein PBOMP-1 iε aεsociated with the outer membrane-cell wall complex. A neceεεary step . . . . . . m the purification of PBOMP-1 is the disruption of the bonds which keep the outer membrane proteins in tight asεociation with the outer membrane and cell wall. This can be accomplished by the novel and improved method of the invention which comprises the following two stages: (1) isolating a PBOMP-1 enriched mεoluble cell wall fraction from physically disrupted cells of H^ influenzae, and then (2) solubilizing PBOMP-1 from the cell wall fraction by heating in the presence of a detergent which iε suitable for adminiεtration to a human or digeεting the cell wall fraction with lysozyme either in the presence or abεence of detergent. The novel improved method of the preεent invention avoids the use of denaturants and reducing agents such as εodium dodecylεulfate and 2-mercaptoethanol (see Munson et al., 1984, Infect. Immun. 49:544-49) which might destroy important epitopes and which are not suitable components for vaccine formulations for administration to humans. 5.1.1 ISOLATION OF PBOMP-1 ENRICHED INSOLUBLE CELL WALL MATERIAL FROM H. INFLUENZAE A total cell membrane fraction may be obtained by differential sedimentation following disruption of H. influenzae cells by methods including but not limited to: sonication, grinding, by expulsion from a freneh press other homogenization device. The total membrane fraction may be further fractionated into inner and outer membranes by density gradient sedimentation or by differential solubilization of the inner membrane by certain detergents such as Triton X-100"' or N-lauroyl sarcosine, sodium salt (sarcosyl) . Outer membranes are preferably prepared by differential solubilization of inner membranes in 1% (W/V) sarcosyl in 10 mM HEPES-NaOH, pH 7.4. A subfraction enriche in PBOMP-1 can be produced by differential detergent extraction of other outer membrane-cell wall componentε. Thiε enrichment can be accompliεhed, for example, by εequential extraction of the outer membrane-cell wall comple (which remainε after Triton X-100™ or sarcosyl extraction as described above) with 1% octylglucoside, nonylglucoεide, zwittergent 3-14™, or zwittergent 3-16™, followed by extraction of the insoluble material with 1% sarcosyl, and then centrifugation to isolate the PBOMP-1 enriched insolubl material. 5.1.2 SOLUBILIZATION OF PBOMP-1 FROM THE PBOMP-1 ENRICHED INSOLUBLE CELL WALL MATERIAL - Solubilization of the PBOMP-1 from the outer membrane- cell wall complex can be achieved in several different ways using one of the following approaches or a combination thereof: (1) PBOMP-1 can be solubilized by extraction of th PBOMP-1 enriched fraction with one or any combination of 5 several detergents, including but not limited to deoxycholate, Triton X-100™, Tween 80, CHAPS, CHAPSO, dodecylmaltoside, zwittergent 3-14™, and zwittergent 3-16™, at 55"C - 60 β C for 1 hour; (2) PBOMP-1 can be solubilized by disruption of the cell wall in the PBOMP-1 enriched fraction with lysozyme, either in the preεence or absence of detergent. According to a preferred embodiment the detergent is selected from: deoxycholate and polyethoxylate sorbitan monooleate (Tween-80) . Alternatively, PBOMP-1 can be iεolated by extracting whole H. influenzae cellε, outer embrantiε or subfractions thereof with one or a combination of detergentε including but not limited to: Triton X-100™, εarcoεyl, octylglucoside, nonylglucoside, zwittergent 3-14™, or zwittergent 3-16™. This extraction could be performed at 55-60"C or at room temperature in an appropriate buffer system. After solubilization, further purification of the PBOMP-1 can be achieved by standard methods known in the art including but not limited to: ion exchange, molecular sieve, hydrophobic or reverse phase chromatography, affinity chromatography, chromatofocusing, lεoelectric focuεing and preparative electrophoreεiε. 5.2 CHARACTERIZATION OF PBOMP-1 BY AMINO ACID ANALYSIS AND SEQUENCING OF PBOMP PEPTIDES The PBOMP-1 obtained from H. influenzae can be characterized by amino acid analysis in combination with partial amino acid sequencing of peptide fragments. In order to mipimize the destruction and/or modification of amino acids in the purified protein it is preferable to hydrolyze the PBOMP-1 in methaneεulfonic acid containing tryptamine (Simpεon et al., 1970, J. Biol. Chem. 251:1936-40). In one experimental example of the present invention, such amino acid analysis was combined with amino acid sequencing of -tryptic peptide fragments of PBOMP-1 to characterize the novel peptide isolated aε described (see Section 6.1.1.). Difficulties experienced during initial attempts to sequence the PBOMP-1 by Edman chemistry suggested that the N-terminus of the Haemophilus outer membrane protein is blocked. Studieε by Braun (1970, Eur. J. Biochem : 38 7- 391) have shown that fatty acids are linked to the N-termina cysteinyl residue of Braun's lipoprotein of Escherichia coli. A palmityl moiety is amide-linked to the N-terminal cysteine; two additional fatty acidε are alεo attached to th same cysteinyl residue via a glyceryl group that forms a thioether bond in the E^ coli Braun lipoprotein. (Id.) Hence the PBOMP-1 obtained from H^ influenzae was further characterized by fatty acid analysiε. Such inveεtigation indicated that the N-terminal residueε of the outer membrane protein and peptideε of the preεent invention can have covalently attached fatty acid reεidueε. In one experimenta example of the present invention, such fatty acid analyεis revealed the presence of three major fatty acids, i.e., lauric acid, palmitic acid; and a derivative of palmitic acid. The acetylated proteins and peptideε of the preεent invention having a fatty acid moiety covalently attached may be of particular utility for vaccine formulationε againεt H. influenzae. 5.3 GENERATION OF PBOMP-1 AND/OR PBOMP-2 RELATED PEPTIDES PBOMP-1 and/or PBOMP-2 related peptideε which contain an antigenic or immunogenic region(ε) of PBOMP-1 or PBOMP-2 reεpectively may be generated by proteolytic cleavage of th entire PBOMP-1 or PBOMP-2 protein or chemical syntheεiε of peptide fragmentε. In the latter method, peptide fragments can be chemically synthesized, for example, using an automated peptide syntheεizer. PBOMP-1 or PBOMP-2 related peptideε of the present invention generated either by proteolytic digestion or 5 chemical syntheεiε may be purified by standard methods known in the art including but not limited to: high pressure liquid chromatography or column chromatography using ion exchange, molecular size, hydrophobic or reverεe-phase columns, affinity columns or chromatofocusing, isoelectric focusing or preparative gel electrophoresiε. The εequence of proteolytic digests of PBOMP-1 or PBOMP-2, or chemically syntheεized PBOMP-1 or PBOMP-2 are determined by techniqueε known in the art which include but are not limtied to an automated sequenator utilizing the Edman degradation procedure or the precedure of ammo acid analysis, which involves hydrolysiε of peptide bonds (Simpon et al., 1976, J. Biol. Chem. 251:1936-1940) . 5.4 MOLECULAR CLONING OF GENES OR GENE FRAGMENTS ENCODING PBOMP-1 AND PBOMP-2 5.4.1 ISOLATION OF GENES ENCODING PBOMP-1 AND RELATED PBOMPs A 16000 dalton molecular weight (MW) OMP has been detected, both by sodium dodecyl εulfate polyacrylamide gel electrophoreεiε (SDS-PAGE) and Weεtern Blot analyεiε in all H. influenzae strains tested (currently several hundred) . Monoclonal antibody data indicate that this protein iε highly conserved (Murphy et al., 1986, Infect. Immun. 54:774-49) . Thus, any H. influenzae strain could serve as the source for the PBOMP genes. Since many H_ influenzae strains contain no detectable plasmidε or inducible prophageε, the PBOMP geneε are probably chromoεomal. Accordingly, the firεt εtep in the molecular cloning of DNA sequences encoding PBOMPs is the isolation of such sequences from H^_ influenzae chromosomal DNA. Hereinafter, DNA encoding H^ influenzae genes will be referred to aε "Hi DNA", and DNA encoding PBOMPε εequenceε will be referred to as "PBOMP DNA". In order to generate Hi DNA fragmentε, the Hi DNA may be cleaved at εpecific εites using various restriction enzymes. Alternatively, one may use low concentrationε of DNAaεe I to fragment the DNA, or the DNA can be phyεically sheared, as for example, by sonication. The linear DNA fragments may then be separated according to size by standard techniques, including, but not _limited to: agarose and polyacrylamide gel electrophoresis, column chromatography (e.g. , molecular εieve or ion exchange chromatography) or velocity sedimentation in sucrose gradients. . . . Any restriction enzyme or combination of reεtriction enzymeε may be uεed to generate the Hi DNA fragment(ε) containing the PBOMP εequenceε provided the enzyme(ε) does not destroy the immunopotency of the PBOMP gene products. For example, the antigenic site of a protein can consist of from about 7 to about 14 ammo acids. Thus, a protein of th size of the PBOMP peptides may have many discrete antigenic sites and therefore, many partial PBOMP polypeptide gene sequenceε could code for an antigenic εite. Consequently many restriction enzyme combinations may be used to generate DNA fragments, which, when inserted into an appropriate vector are capable of directing the production of PBOMP specific amino acid sequences comprising different antigenic determinants. Once the DNA fragments are generated, identification o the specific DNA fragment containing the PBOMP gene may be accomplished in a number of ways. The DNA sequenceε containing the PBOMP geneε may be identified by hybridization of the Hi DNA fragments with a synthetic oligonucleotide probe. Redundant synthetic oligonucleotide probes are constructed based upon the amino acid sequence of peptide fragments of the PBOMP protein. Fo example, synthetic oligonucleotide probes can be prepared based upon the amino acid εequence of the εubεtantially pure PBOMP-1 protein iεolated from H. influenzae aε described in ~ Section 5.1. These synthetic probes can be radio-labeled with 32P-adenosme tπphoεphate and uεed to εcreen Hi DNA libraries for clones containing PBOMP-speeific gene sequenceε (see Anderson et al., 1983, Proc. Nat'l Acad. Sci. USA 80: 6838-42) . Alternatively, the PBOMP gene DNA may be identified and isolated after insertion into a cloning vector in a "shotgun" approach. A large number of vector-host systems known in the art may be used. Vector syεtemε may be either plasmids or modified viruses. Suitable cloning vectors include, but are not limited to the viral vectors such as lambda vector syεtem gtll, gt WES.tB, Charon 4, and plaεmid vectorε εuch aε pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl and other εimilar εyεtemε. The vector system must be compatible with the host cell used. Recombinant molecules can be introduced into cells via transformation, transfeetion or infection. When Hi DNA containing a PBOMP gene or gene fragment iε inserted into a cloning vector and used to transform appropriate host cells many copieε of the PBOMP gene or gene fragment can be generated. Thiε can be accompliεhed by ligating the Hi DNA fragment into a cloning vector which haε complementary coheεive termini. If, however, the complementary reεtriction sites are not present, the ends of the DNA molecules 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 (linkers) onto the DNA termini. These ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction εite recognition sequences. For example, according to the DNA modification procedure of Maniatis, (see Maniatis et al., 1982, Molecular Cloning, Cold Spring Harbor Laboratory, pp. 107-114) sheared DNA is treated with a restriction methylaεe (for example_, M. EcoRI) and ligated to εynthetic DNA linkerε which encode a restriction site for that enzyme. The DNA is then treated with restriction endonuclease to cleave the terminal linkers (but not the modified internal restriction sites) and ligated to the appropriate vector arms. In an alternative method, the cleaved vector and PBOMP DNA fragment may be modified by homopolymeric tailing. Identification of a cloned PBOMP gene can be accomplished by establiεhing a chromoεomal gene bank of Hi in . . a vector εyεte and screening individual clones for the production of PBOMP-1 or PBOMP-1 related protein by any of the methods described herein, including, but not limited to specific reaction with polyclonal or monoclonal antibodies against PBOMPs. 5.4.2 INSERTION OF PBOMP GENES INTO EXPRESSION VECTORS The nucleotide sequences coding for PBOMPs or portionε thereof, are inεerted into an appropriate expreεsion vector, i.e. , a vector which contains the necessary elementε for the transcription and translation of the inserted protein-coding sequenceε. In a particular embodiment of the invention, the nucleotide sequences coding for both PBOMP-1 and PBOMP-2 or portions thereof are inserted into an appropriate expression vector as described in the previous sentence, infra. A variety of host-vector systems may be utilized to expresε th protein-encoding sequence(s) . Primarily the vector system must be compatible with the host cell used. Host-vector syεte ε include but are not limited to the following: bacteria tranεformed with bacteriophage DNA, plasmid DNA or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g. , vaccinia virus, adenovirus, etc.) ; insect cell systems infected with virus (e.g. , baculovirus) . The expresεion elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used. In order to obtain efficient expresεion of the gene (or a portion of the gene) , a promotor muεt be present in the expression vector. RNA polymerase normally bindε to the promotor and initiateε transcription of a gene or a group of linked genes and regulatory elements (called an operon) . Promotors vary in their "strength", i.e. , their ability to promote transcription. For the purpoεe of expreεεing a cloned gene, it iε desirable to use strong promotors in order to obtain a high level of transcription and, hence, expresεion of the gene. Depending upon the host cell syεtem utilized, any one of a number of suitable promotors may be used. For instance, when cloning in E. coli, its bacteriophages or plaεmidε, promotorε such as the lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the P R and P_ promotorε of coliphage lambda and otherε including but not limited to lacUV5, ompF, bla, lpp and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene. Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unleεs specifically induced. In certain operons the addition of specific inducerε is necessary for efficient transcription of the inserted DNA; for example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D- galactoεide) . A variety of other operonε, εuch aε trp, pro, etc., are under different controls. The trp operon is induced when tryptophan is absent in the growth media; and the P τ > promotor of lambda can be induced by an increase in temperature in host cells containing a temperature sensitive lambda represor, e.g. , CI857. In this way, greater than 95% of the promotor-directed transcription may be inhibited in uninduced cells. Thus, expresεion of the genetically , engineered PBOMP protein or peptide thereof may be controlled. Thiε iε important if the protein product of the cloned gene iε lethal or detrimental to hoεt cellε. In εuch caseε, tranεformants may be cultured under conditions such that the promotor is not induced, and when the cells reach a suitable density m the growth medium, the promotor can be induced for production of the protein. One such promotor/operator system is the so-called "tac" or trp-laπ promotor/operator syεte (Ruεεell and Bennett, 1982, Gene 20:231-243; DeBoer, European Patent ~ 7 Application, 67,540 filed May 18, 1982). Thiε hybrid promotor iε conεtructed by combining the -35 b.p. (-35 region) of the trp promotor and the -10 b.p. (-10 region or Pribnow box) of the lac promotor (the sequences of DNA which are the RNA polymerase binding site) . In addition to . . maintaining the εtrong promotor characteπεticε of the tryptophan promotor, tac iε alεo controlled by the lac repressor. When cloning in a eucaryotic host cell, enhancer sequences (e.g. , the 72 bp tandem repeat of SV40 DNA or the retroviral long terminal repeats or LTRs, etc.) may be inserted to increase transcriptional efficiency. Enhancer sequences are a set of eucaryotic DNA elements that appear t increase transcriptional efficiency in a manner relatively independent of their position and orientation with respect t a nearby gene. Unlike the classic promotor elements (e.g. , the polymerase binding site and the Goldberg-Hognesε "TATA" box) which must be located immediately 5" to the gene, enhancer sequences have a remarkable ability to function upstream from, within, or downstream from eucaryotic genes; therefore, the position of the enhancer sequence with respect to the inserted gene is less critical. Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These tranεcription and tranεlation initiation εignalε may vary in "εtrength" aε meaεured by the quantity of gene εpecific messenger RNA and protein synthesized, respectively. The DNA expresεion vector, which containε a promotor, may alεo contain any combination of various "strong" transcription and/or translation initiation signals. . . . . For instance, efficient translation in E^ coli requires a Shine-Dalgarno (SD) sequence about 7-9 baεeε 5' to the initiation codon (ATG) to provide a riboεome binding εite. Thuε, any SD-ATG combination that can be utilized by hoεt cell riboεomes may be employed. Such combinations include . but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E_^_ coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used. Any of the methods previously described for the insertion of DNA fragments into a vector may be used to ligate a promotor and other control elements into specific siteε within the vector. Accordingly, H^ influenzae genetic εequenceε containing those regions coding for the PBOMP proteins or peptides can be .ligated into an expression vector at a specific site in relation to the vector promotor and control elements so that when the recombinant DNA molecule is introduced into a hoεt cell the foreign genetic sequence can be expressed (i.e., . transcribed and translated) by the host cell. In a specific embodiment of the invention, regions coding for both PBOMP-1 and PBOMP-2 proteinε or peptideε can be ligated into an expreεsion vector. The relation or orientation of the PBOMP sequences to the vector promoter and control elements will determine whether the genetic εequence expreεεed iε a PBOMP- 1: PBOMP-2 or PBOMP-2: PBOMP-1 fuεion protein or peptide fragments, thereof. The recombinant DNA molecule may be introduced into appropriate host cells (including but not limited to bacteria, virus, yeast, mammalian cells or the like) by transformation, transduction or tranεfection (depending upon the vector/hoεt cell εystem) . Transformants are selected based upon the expression of one or more appropriate gene markers normally present in the vector, such as ampicillin resistance or tetracycline resistance in . . . pBR322, or thymidine kmase activity m eucaryotic host systems. Expression of such marker genes should indicate that the recombinant DNA molecule is intact and iε replicating. Expreεεion vectorε may be derived from cloning vectorε, which uεually contain a marker function. Such , . cloning vectorε may include, but are not limited to the following: SV40 and adenoviruε, vaccinia viruε vectors, insect viruseε εuch aε baculoviruεeε, yeaεt 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 plaεmid DNA vectorε such as pBR322, pAC105, pVA51, pACYC177, pKH47, pACYC184, pUBHO, pMB9, pBR325, Col El, pSClOl, pBR313, pML21, RSF2124, pCRl, RP4, pBR328 and the like. Transfer of drug resiεtance factorε between H. . . ~ influenzae and E^ coli via conjugation (Stuy, 1979, J. Bact. 139:520-529); and tranεformation (Mann, 1979, Plasmid 2 ^ :503- 505) and cloning of Haemophilus chromosomal geneε in E_^ coli (Mann et al., 1980, Gene 2 x 91 -112 ) indicate that at leaεt some genes can be efficiently expressed in both organismε; and that the basic mechanisms of transcriptional and translational control may be similar. In the particular embodiment in the examples of the present invention, an J^_ coli plasmid system was chosen as the expression vector. The invention, however, is not limited to the uεe of εuch E_ coli expreεεion vector. Genetic engineering techniqueε could alεo be uεed to further characterize and/or adapt the cloned gene. For example, site directed mutagenesis of the gene encoding a . . . . PBOMP protein could be used to identify regions of the protein responsible for generation of protective antibody responεeε. It could also be used to modify the protein in regions outεide the protective domainε, for example, to increaεe the solubility of the protein to allow easier ... .. purification. 5.4.3 IDENTIFICATION AND PURIFICATION OF THE EXPRESSED GENE PRODUCTS - Expression vectors containing foreign gene insertε can be identified by three general approacheε: (1) DNA-DNA hybridization using probes comprising sequenceε that are homologouε to the foreign inεerted gene; (2) preεence or abεence of "marker" gene functionε (e.g. , reεiεtance to antibiotics, transformation phenotype, thymidine kinase activity, etc.); and (3) expression of inserted sequences based on the physical, immunological or functional properties of the gene product. Once a recombinant which expresεeε a PBOMP gene iε identified, the gene product εhould be analyzed. Immunological analyεiε is especially important becauεe the ultimate goal iε to uεe the gene productε or recombinant viruεeε that express such products in vaccine formulations and/or aε antigenε in diagnoεtic immunoaεεayε. A variety of antiεera are available for analyzing immunoreactivity of the product, including, but not limited to polyvalent antiεera and monoclonal antibodieε deεcribed in Section 6.2., infra. Identification of the proteinε and peptideε of the invention requires that the PBOMP related protein or peptide be immunoreactive to a variety of antibodieε directed againεt PBOMP or itε analogε and derivativeε. The protein or peptide εhould be immunoreactive whether it reεultε from the expreεεion an entire PBOMP gene εequence, a portion of the gene sequence or from two or more gene sequences which are ligated to direct the production of fusion proteins. Thiε reactivity may be demonεtrated by εtandard immunological techniqueε, such as radio- immunoprecipitation, radioimmune competition, ELISA or immunoblots. Once the H^ influenzae PBOMP related protein lε identified, it may be iεolated and purified by εtandard methods including chromatography (e.g. , ion exchange, affinity, and sizing column chromatography) , centrifugation, differential solubility, or by any other standard techniques for the purification of proteins. Alternatively, once an immunoreactive H^ influenzae PBOMP related protein produced by a recombinant iε identified, the amino acid εequence of the immunoreactive protein can be deduced from the nucleotide εequence of the chimeric gene contained m the recombinant. Aε a reεult, the protein can be synthesized by standard chemical methods known in the art (e.g. , see Hunkapiller et al., 1984, Nature 310: 105-111) . In a particular embodiment of the present invention εuch peptideε, whether produced by recombinant DNA techniqueε or by chemical synthetic methods, include but are not limited to all or part of the amino acid εequenceε εubεtantially aε depicted in FIG. 11 and/or FIG. 15 including altered sequences in which functionally equivalent amino acid . . . residues are subεtituted for reεidues withm the sequence resulting in a silent change. For example, one or more amino acid reεidues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Subεtituteε for an amino acid within the εequence may be selected from other members of the claεs to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include glycine, alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acidε include εerine, threonine, cyεteine, tyroεine, aεparagine, and glutamine. The poεitively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acidε include aεpartic and glutamic acid. 5.5 NUCLEOTIDE SEQUENCING OF PBOMP GENES Once the fragmentε of DNA containing the PBOMP geneε have been identified, the actual nucleotide εequenceε of theεe genes can be ascertained by sequence analysiε of the DNA. The sequential order of the baεe pairε can be determined by either of two methodε, the method of Maxam and Gilbert (Maxam and Gilbert, 1980, Methodε in Enzymology, 5:49) or the dideoxy method (Sanger et al., 1977, Proc. Nat'l Acad. Sci. USA 7_4_:5463). The actual εtart and εtop εignalε of the PBOMP geneε can be ascertained by analyεiε of the nucleotide εequence for open reading frameε (Roεenberg et al., 1979, Ann. Rev. Genet. 3^:319). If more than one open reading frame iε found on a particular DNA fragment, the identity of the actual gene could be confirmed by comparing the predicted amino acid sequence of the gene product to the amino acid sequence of the PBOMP. The location of the proper reading frame may also be determined by use of gene fuεionε. 5.6 DETERMINATION OF IMMUNOPOTENCY OF PBOMPε AND PBOMP ASSOCIATED PEPTIDES Experience with antibodieε to the capsular polysaccharide of type b Haemophilus influenzae i.e. , PRP, shows that the ability of the antibodies to kill the bacteria in m vitro asεays and to protect against challenge with Hib in animal model syεtems is closely correlated with the ability to elicit a protective immune response in human infantε. Anti-PBOMP antibodies elicited in response to the PBOMP proteins and peptides of this invention which include but are not limited to PBOMP-1 and/or PBOMP-2 proteins and/or_PBOMP- 1: PBOMP-2 and PBOMP-2: PBOMP-1-fusion proteins can be tested using similar in vitro asεay εyεtemε and animal model εyεtem to demonstrate the ability to kill both Hi and Hib cells and to protect in animal model syεte ε from challenge with Hib. . . . . . In particular embo imentε of the invention, antibodieε may be elicited in reεponεe to chemically εyntheεized PBOMP peptides or PBOMP peptideε generated by proteolytic cleavage of the PBOMP protein. In a preferred embodiment, εuch PBOMP peptide fragmentε would be coupled to a protein carrier, e.g. thyroglobulin, bovine εeru albumin, diptheria toxin or detoxified toxin (toxoid) , tetanus toxin or toxoid, pseudomonas toxin or toxoid, stapholococcuε toxin or toxoid, pertuεsis toxin or toxoid, CRM ιg7 , etc. CRM g _ is distinguiεhed from native diphtheria toxin by a εingle amino acid. However CRM and native diphtheria toxin are immunologically indiεtinguiεhable. For a detailed deεcription of CRM.--, εee Patent No. 4,673,574, issued June 16, 1987, and referenceε incorporated therein. The reεultε from these syεtems εhould εhow a similar correlation with the potential of each of the PBOMPs to elicit a protective immune responεe and to serve in a vaccine for human infantε, children and adultε. An in vitro complement mediated bactericidal assay syεtem (Musher et al., 1983, Infect. Immun. 39:297-304; . Anderson et al., 1972, J. Clin. Invest. 5_l:31-38) which has been uεed previouεly for meaεuring bactericidal activity of antibodies of PRP and lipopolysaccharide (LPS) against H. influenzae could be used to determine whether or not antibody directed against a particular PBOMP peptide or fragment thereof has bactericidal activity against type b H. influenzae and non-typable H^ influenzae. These asεays can be performed against a relatively large number of clinical isolates of both types of bacteria to determine whether a broad range of strains are killed. See Sections 7.1, 9.4, and 10.5 (infra) for illustrative examples of such in vitro bactericidal asεayε. Further data on the ability of a PBOMP or fragment thereof to elicit a protective antibody reεponεe may be generated by uεe of the infant rat meningitiε model εystem (Smith et al., 1973, Infect. Immun. ;B:278-290). Infant rats challenged before the sixth day of life, with a suitable dose of H. influenzae type b develop bacteremia and a fatal meningitis similar to that seen in human infantε. If antibody which is bactericidal against a challenge strain is . . . . . used to paεεively immunize infant ratε 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 εyεtem. Paεεive protection againεt type b Haemophiluε meni.ngi.ti.ε could be demonεtrated by immun7izi.ng infant ratε with rabbit polyclonal anti-PBOMP antibody and εubεequently challenging the ratε with a lethal doεe of H. influenzae type b. See Section 7.2 (infra) for an illuεtrative example of εuch in vivo protective antibody reεponεe elicited by the proteins and peptides of the preεent invention. Data on the ability of antibody to a particular PBOMP to protect againεt Hi could be obtained uεing 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 Hi. An otitis media much like that seen in humans developε. Chinchillaε, which have been immunized, either actively with Hi OMP's, or passively with antibody directed against Hi OMP's are protected against aural challenge with Hi. (Barenkamp et al., supra) . This animal model system could be used to demonstrate the ability of antibody to a PBOMP to protect against Hi. It is possible to demonstrate that anti-PBOMP antibodies are capable of additive protection along with anti-PRP antibodies by use of the infant rat animal model. Anti-PBOMP-1 antibodies diluted to a point at which they no longer are capable of protecting infant rats against challenge with Hib, mixed with a similar dilution of anti-PRP antibodies, may show additive protection and thus prevent death of infant rats. This additive protection might be useful for a potential combination vaccine composed of PRP, or a fragment or conjugate thereof, and the PBOMP or a fragment thereof. 5.7 FORMULATION QF A VACCINE Many methods may be used to introduce the vaccine formulations described below into a human or animal. These include, but are not limited to: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes of administration. 5.7.1 SUBUNIT VACCINE FORMULATIONS One purpoεe of the present invention is to provide proteins or polypeptide fragments related to outer membrane proteins of H^ influenzae, PBOMPs including PBOMP-1, PBOMP-2 and related proteins and peptides as well as PBOMP-1: PBOMP- and PBOMP-2: PBOMP-1 fuεion proteins and related peptideε, which are uεed as immunogens in a subunit vaccine to protect against meningitis and other disease symptoms of H^ . . influenzae infections. Subunit vaccines comprise solely the relevant immunogenic material necessary to immunize a host. Vaccines made from genetically engineered immunogens, chemically synthesized immunogens and/or immunogens comprising authentic substantially pure H_^ influenzae PBOMPs iεolated aε deεcribed herein, which are capable of eliciting a protective immune reεponεe are particularly advantageouε becauεe there iε no riεk of infection of the recipientε. Thus, the PBOMP related protein or fragment thereof may be purified from recombinants that expreεε the PBOMP epitopeε. Such recombinants include any of the previously deεcribed bacterial tranεformantε, yeaεt tranεformantε, or cultured cellε infected with recombinant viruεeε that express the PBOMP epitopes (see Sections 5.4 and 5.5., supra) . Alternatively, the PBOMP related protein or peptide may be . chemically syntheεized. To thiε end, the ammo acid εequence of such a protein or peptide can be deduced from the nucleotide sequence of the gene which directs its expresεion (see Section 5.5., εupra) . In yet another alternative embodiment, the PBOMP related protein or peptide is isolated in εubεtantially pure form from cultureε of H_^ influenzae (see, for example, Section 5.1., supra) . Whether the immunogen iε purified from recombinantε or chemically εyntheεized, the final product iε adjuεted to an appropriate concentration and formulated with any suitable . . . vaccine adjuvant. Suitable adjuvants include, but are not limited to: surface active substances, e.g. , hexadecylamine, octadecylamine, octadecyl amino acid esterε, lysolecithin, dimethyl-dioctadecylammonium bromide, N, N-dicoctadecyl-N'- N-biε (2-hydroxyethyl-propane diamine) , methoxyhexadecylglycerol, and pluronic polyolε; plyamineε, e.g. , pyran, dextransulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g. , muramyl dipeptide, dimethylglycine, tuftsin; oil emulsionε; and mineral gelε, e.g. , aluminum hydroxide, aluminum phosphate, etc. The immunogen may also be incorporated into liposomeε, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation. In yet another embodiment of this mode of the invention, the PBOMP related protein or peptide is a hapten, i.e. , a molecule that iε 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 lmmunogenicity to the hapten coupled to it. The hapten-carrier may be formulated for use as a subunit vaccine. 5.7.2 VIRAL VACCINE FORMULATIONS ; ; . Another purpose of the present invention is to provide either a live recombinant viral vaccine or an inactivated recombinant viral vaccine which is used to protect against meningitiε and other diεeaεe εymptoms of HL_ influenzae. To this end, recombinant viruseε are prepared that expreεε PBOMP related epitopeε (εee Sectionε 5.4. and 5.5., supra) . Where the recombinant virus is infectious to the host to be immunized but does not cause disease, a live vaccine is preferred because multiplication in the hoεt leadε to a prolonged stimulus, therefore, conferring substantialy long- iasting immunity. The infectious reco.l.in._nt virus when introduced into a host can express the PBOMP related protein or polypeptide fragment from its chimeric gene and thereby elicit an immune responεe againεt H^ influenzae antigenε. In cases where such an immune reεponεe iε protective againεt 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 vitro (e.g. , tissue culture cells) and in vivo (e.g. , natural host animal) εystems. For instance, conventional methods for preparation and formulation of εmallpox vaccine may be adapted for the formulation of live recombinant viruε vaccine expreεεing a PBOMP related protein or polypeptide. Multivalent live virus vaccines can be prepared from a single or a few infectious recombinant viruses that expresε epitopeε of organiεmε that cause diseaεe in addition to the epitopeε of H^_ influenzae PBOMPs. For example, a vaccinia virus can be engineered to contain coding sequences for other epitopes in addition to those of H^ influenzae PBOMPs. Such a recombinant virus itεelf can be uεed aε the immunogen in a multivalent vaccine. Alternatively, a mixture of vaccinia or other viruses, each expressing a different gene encoding for differerent epitopes of PBOMPs and/or other epitopes of other diseaεe causing organiεmε can be formulated in a multivalent vaccine. Whether or not the recombinant viruε iε infectious to the host to be immunized, an inactivated virus vaccine formulation may 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 the PBOMP related protein or polypeptide 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 toget er. In either case, the inactivated recombinant virus or mixture of viruεeε εhould be formulated in a suitable adjuvant in order to enhance the immunological response to the antigens. Suitable adjuvants include, but are not limited to: surface active substances, e.g. , hexadecylamine, octadecyl amino acid esterε, octadecylamine, lysolecithin, dimethyl-dioctadecylam onium 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, tuftεin; oil emulεionε; and mineral gelε, e.g. , aluminum 5 hydroxide, aluminum phoεphate, etc. 5.7.3 PASSIVE IMMUNITY AND ANTI-IDIOTYPIC-ANTIBODIES Inεtead of actively immunizing with viral or subunit vaccines, it is possible to confer short-term protection to a 0 host by the administration of pre-formed antibody against an epitope of H^ influenzae. Thuε, 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 responεe to itε foreign immunogenic components. Such passive immunization could be used on an emergency basiε for immediate protection of unimmunized individualε expoεed to special risks, e.g. , young children exposed to contact with bacterial meningitis patients. Alternatively, these 0 antibodies can be uεed m the production of anti-idiotypic antibody, which in turn can be used as an antigen to stimulate an immune reεponεe against H_^ influenzae PBOMP epitopes. 5.8 DIAGNOSTIC ASSAYS Yet another purpose of the present invention is to provide reagents for use in diagnostic assays for the detection of PBOMP antigens (and hence H^ influenzae) in variouε body fluids of individuals suspected of H. influenzae 30. infection. 5.8.1 IMMUNOASSAYS In one mode of this embodiment, the PBOMP related proteins and peptides of the present invention may be used as 35 antigenε in immunoaεεayε for the detection of H^ influenzae in variouε patient tiεsues and body fluids including, but not limited to: blood, spinal fluid, 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 asεays, "sandwich" asεayε, precipitin reactionε, gel diffuεion precipitin reactionε, immunodiffusion assayε, agglutination assays, fluorescent immunoassays, protein A immunoassayε and immunoelectrophoreεis asεayε, to name but a few. 5.8.2 NUCLEIC ACID HYBRIDIZATION ASSAY In another mode of thiε embodiment, the novel nucleotide sequence of the genes encoding the PBOMP related protein and peptides of the preεent invention may be used as probes in nucleic acid hybridization asεays for the detection of H_^ influenzae in various patient body fluids, including but not limited to: blood, εpinal fluid, εputum, etc. The nucleotide sequences of the present invention may be used in any nucleic acid hybridization assay system known in the art including, but not limited to: Southern blots (Southern, 1975, J. Mol. Biol. 9_8:508) ; Northern blots (Thomas et al., 1980, Proc. Nat'l Acad. Sci. USA 77:5201-05) ; colony blots (Grunstein et al., 1975, Proc. Nat'l Acad. Sci. USA 72:3961-65), etc. The following εeries of Examples are presented for purposes of illustration and not by way of limitation on the scope of the present invention. 6 EXAMPLES: ISOLATION AND CHARACTERIZATION OF NATURAL-AND RECOMBINANT DNA-DERIVED PBOMPs 6.1 ISOLATION, PURIFICATION AND ANALYSIS OF PBOMP-1 In one serieε of experimentε, PBOMP-1 εubεtantially free of other cell wall componentε waε obtained from H. influenzae as follows: H. influenzae Eagan was grown overnight on either brain heart infusion medium containing 10 ug/ml hemin and 1 ug/ml NAD (BHI-XV) or mMIC (modification of Herriott et al., 1970, J. Bacteriol. 101: 513-16) media. Following centifugation at 10,000 x g for 15 minutes at 4 β C, the supernatant was discarded into Rocal II™ disinfectant. The cell pellet was weighed and suspended in 10 mM HEPES-NaOH (pH 7.4), 1 mM EDTA, with a volume of buffer equal to about five times the wet weight of the cells. The cell suεpenεion waε then εonicated for 5 minuteε in an ice bath in 100 ml aliquotε with a Branεon Model 350 εonifier cell disruptor (Branson Sonic Power, Danbury, CT) at 60% power on a pulεe setting. Following sonication, the disrupted cell suspension was centrifuged at 10,000 x g for 5 minutes at 4"C to remove unbroken cells. The sedimented unbroken cells were then weighed and re-sonicated as before in a volume of 10 mM HEPES-NaOH pH 7.4, 1 mM EDTA, equivalent to about five times the wet weight of the unbroken cells. The total membrane fraction was obtained as a pellet following addition of sufficient NaCl to provide a final concentration of 0.5 M NaCl and ultracentrifugation of the broken cellular material at 100,000 x g for about 1 hour. An outer membrane-cell wall complex was then obtained by removing the inner membrane components from the total membrane fraction by repeated extraction of the total membrane fraction with 1% sarcosyl, in 10 mM HEPES-NaOH, pH 7.4. The insoluble residue containing the outer membrane cell wall fraction was isolated by centrifugation at 350,000 x g for 30 minutes, suspended in 50 mM Tris pH 8.0, 5 mM EDTA, and stored overnight at 4"C. A PBOMP-1 cell wall complex was isolated from the reεt of the other proteinε in the outer membrane fraction by sequential extraction of the outer membrane-cell wall fraction with octylglucoside (twice) , followed by sarcosyl (twice) . Both detergents were used at 1% (w/v) in 50 mM Tris, 5 mM EDTA, pH 8.0. Extractionε were carried out at room temperature (20°C) for 30 minuteε each. The mixture waε then centrifuged at 100,000 x g for 1 hour. The inεoluble, εedimented material remaining after extraction with octylglucoεide and εarcoεyl iε a PBOMP-1-cell wall complex. PBOMP-1 was solubilized by two methodε: (1) heating to 60°C for 1 hour in the preεence of one of εeveral detergentε; or (2) diεruption of the cell wall by lyεozyme digeεtion at 37"C for 1 hour in the preεence or abεence of detergent. Following either (1) or (2) , εoluble PBOMP-1 waε εeparated , . from insoluble material by centrifugation at 100,000 x g for 1 hour at 15 β C. In neither procedure (1) nor (2) was the particular detergent chosen critical for the solubilization. Indeed, all detergents tested to date (including: deoxycholate, Triton X-100™, Tween-80, CHAPS, CHAPSO, dodecylmaltoside, zwittergent 3-14™ and zwittergent 3-16™) are effective in the heat dependent εolubilzation aε well aε the lysozyme induced solubilization. Additionally, octylglucoside iε very effective in the lyεozyme induced εolubilizationε and was used routinely at 1% (w/v) final concentration. From 40 g wet weight cells, it was possible typically to isolate about 8 mg of PBOMP-1, subεtantially free from other cell wall components. This substantially pure PBOMP-1 preparation was analyzed in an SDS PAGE syεtem to determine the relative molecular weight of the reduced denatured form of thiε protein and to asseεε its purity (FIG. 1) . Samples were prepared for analysis by SDS-PAGE by adjusting them to 0.1 M Tris-HCl, pH 7.0, 25 mM dithiothreitol, and 2% SDS with 5X concentrated sample buffer, then heating for 5 minuteε at 100"C. Prior to electrophoresis all εampleε were adjuεted to 6% (w/v) εucroεe and 0.001% bromophenol blue. Moεt routine analyses were performed using the Bio-Rad Mini Protean Gel System (Redmond, CA) . Gels were 1.5 mm thick and the separating gel contained 15% acrylamide with an acrylamide to bis ratio of 30:0.8, 0.375 M Tris-HCl (pH 8.8) and 0.1% SDS. The stacking gel contained 4.8% acrylamide with the same ratio of acrylamide to bis, 125 mM Tris, HC1 (pH 7.0), and 0.1% SDS per gel. Following electrophoresis gels were stained for at least 1 hour with 0.125% (w/v) Coomasie blue in ethanol: acetic acid: water (5:i:5), then deεtained in the εame εolvent εyεtem without the blue dye. Pre-stained low molecular weight standards which included the following: ovalbumin, 43,000; alpha-chymotrypsinogen, 25,700; Beta-lactoglobulin, 18,400; lyεozyme, 14,300; -bovme trypεin inhibitor, 6,200; inεulin (A and B Chainε) , 2,300 and 3,400 (BRL, Betheεda, MD) were used to asεist in the determination of the relative molecular weight of the PBOMP-1. Further purification of PBOMP-1 can be achieved by standard methods such aε ion exchange chromatography, molecular εieving, hydrophobic or reserve phase chromatography, chromatofocuεing, gel electrophoreεiε and the like. 6.1.1 CHARACTERIZATION OF PBOMP-1 BY AMINO ACID COMPOSITION AND SEQUENCE Amino acid analyεiε waε performed according to the procedure of Simpεon et al., (1976, J. Biol. Chem. 251:1936- 1940). Hydrolyεiε waε accompliεhed by heating 0.5-1 mg of protein in 0.1 ml 4 N methane εulfonic acid under vacuum in a thick-walled εealed glaεε tube at 100"C for 22 hourε. The quantity of each amino acid iε obtained by compariεon of the areas under the various peaks with areas obtained using known quantities of standard amino acids. Results obtained are illuεtrated in Table 1. TABLE 1 a The ^ apparent molecular weight of PBOMP-1 waε 15,057 dal€ons. The total number of amino acid residues was 140. ' Initial attempts at sequencing the PBOMP-1 using Edman chemiεtry failed to yield satisfactory results because of a blocked N-terminal residue. In order to obtain partial amino acid sequence information, it has been necessary to enzymatically digest the 16,000 dalton molecular weight PBOMP , with proteolytic enzymes to obtain peptide fragments that are amenable to εequence analyεis. A proteolytic digest of the 16,000 dalton PBOMP-1 obtained using trypsin, at 27°C for 1 hour was separated by reverse phaεe high preεεure liquid chromatography (RP-HPLC) . using a C18 column. A large hydrophobic peptide peak (T9) was isolated and subsequently immobilized on a polybrene- coated glass fiber paper prior to the start of amino acid sequencing. The T9 peptide was sequenced by Edman degradation (Edman et al., 1967, Eur. J. Biochem. ].:80-91). Each cycle from the sequenator generated an anilinothiazolinone phenylthiohydantoin (PTH) -amino acid. Analyεis was performed on a reverse phaεe C18 HPLC cartridge column with a liquid chromatography εystem. The PTHs were eluted at room temperature wi.th a εodi.um acetate-acetoni.tπ.le gradient and detected at 270 nanometers with a variable UV wavelength detector. The sequence analysis of the T9 peptide is shown below: Tyr-Asn-Thr-Val-Tyr-Phe-Gly-Phe-Aεp-Lyε-Tyr-Asp-Ile-Thr-Gly- Phe-Tyr-Val-Thr-Ile-Asp-Ala-Aεp-Ala-Ala-Tyr-Leu-Asn-Ala-Thr- Pro-Ala-Ala The T9 peptide is very hydrophobic containing 8 aromatic amino acids (5 Tyr, 3 Phe) and 5 aliphatic side chain amino acids (1 Leu, 2 Val, 2 lie) . The tyrosine content of this peptide is high but is consistent with the total amino acid composition of PBOMP-1 (Table 1) . Additionally, PBOMP-1 is unusual in that it contains 13 tyrosineε, but no methionine or tryptophan. 6.1.2 CHARACTERIZATION OF PBOMP-1 BY FATTY ACID ANALYSIS As indicated in Section 6.1.1., initial attempts to sequence PBOMP-1 by Edman degradation did not yield satiεfactory reεultε becauεe of a blocked N-terminal residue. Fatty acid analysis of purified H^ influenzae PBOMP-1 was performed to investigate whether covalenty linked fatty acyl groups could be identified on the PBOMP-1 peptide. Prior to fatty acid analysiε, PBOMP-1 protein iεolated as described above in Section 6.1. was extracted exhaustively with a mixture of organic solventε, i.e., chloroform:methanol (2:1) and with deoxycholate detergent to remove any trace contaminantε of endogenouε lipidε, phoεpholipidε, etc. The denuded protein waε obtained either by acetone precipitation or by exhauεtive dialysis and dried by lyophilization. A known amount of nonadecanoic acid was added as an internal standard to the dried purified PBOMP-1 (1-3 mg) and the mixture was hydrolyzed with 200 ul of 4 N HC1 at 110°C for 4 hours under a nitrogen atmosphere. Such acid hydrolysiε releaεed amide- or eεter- linked fatty acidε. The hyrdolyεate, diluted to 2 ml with water, waε extracted three timeε with an equal volume of hexane. The combined hexane phaεe waε waεhed twice with an equal volume of εaline and then dried over εodium εulfate. The fatty acidε were converted into correεponding methyl eεterε with diazomethane (Schlenk, 1960, Anal. Chem. 3_2: 1412-1414) before injection into a Perkin Elmer Model 8500 gaε liquid chromatograph. Separation of fatty acid methyl esters waε performed on a SPB-1 fuεed εilica capillary column (Supelco, Inc., Belefonte, PA) . Reεultant peakε were identified by compariεon with known εtandardε. Reεultε obtained are illustrated in FIG. 16. As demonstrated in FIG. 16, three major fatty acids are asεociated with PBOMP-1, i.e., lauric acid (C12) , palmitic acid (C16) and a derivative of palmitic acid (C16') which remainε to be definitively identified. C16' iε perhapε a branched chain fatty acid having 16 carbon atomε. 6.2 PREPARATION OF ANTI-PBOMP-1 AND ANTI-PBOMP-2 ANTIBODIES 6.2.1 PREPARATION OF POLYCLONAL ANTI-PBOMP-1 AND ANTI-PBOMP-2 ANTISERUM Substantially pure PBOMP-1 was used as an immunogen to prepare anti-PBOMP-1 antibodies. Partially purified PBOMP-1 enriched fractions, prepared as described in Section 6.1, were electrophoresed on 15% SDS-PAGE gels at 35 mA constant current at 10 β C. The protein bands were fixed and stained as described in Section 6.1.1. PBOMP-1 bandε were exciεed from the gels and dialyzed against phosphate buffered εaline (PBS) (20 mM εodium phosphate, 150 mM NaCl, pH 7.4) until . . equilibrated. The acrylamide gel .fragments containing PBOMP-1 were minced by passing them through a 25 gauge needle in PBS. The fragmentε were injected intramuεcularly into New Zealand white rabbitε at multiple εiteε. Each rabbit received a total of approximately 20 ug of PBOMP-1. Rabbitε were reimmunized at two weekε and three weekε following the initial immunization. Animalε were bled one week following the last immunization and the serum collected. Animals were boosted with 20 ug of PBOMP-1 in acrylamide bimonthly to maintain high titers of anti-PBOMP-1 antibodies. Alternatively PBOMP-1, isolated as described in section 5.1, was mixed with incomplete Freund's adjuvant and emulsified. Rabbits were injected intramuscularly with approximately 20 ug of PBOMP-1 in Freund's adjuvant. Animals were reimmunized two weeks and three weeks following the initial immunization and bled one week following the last immunization. Substantially pure PBOMP-2 was used as an immunogen to prepare anti-PBOMP-2 antibodies. Partially purified PBOMP-2 enriched fractions, prepared as described in Section 6.1, were electrophoreεed on 15% SDS-PAGE gelε at 35 mA conεtant current at 10°C. The protein bandε were fixed and stained as described in Section 6.1.1. PBOMP-2 bandε were excised from the gels and dialyzed against phosphate buffered εaline PBS until equilibrated. The acrylamide gel fragmentε containing PBOMP-2 were minced by paεεing them through a 25 gauge needle in PBS. The minced PBOMP-2 gel fragmentε were mixed with complete Freund'ε adjuvant and emuεified. Rabbits were injected intramuscularly with approximately 10 ug of PBOMP-2 in Freund's adjuvant. Animals were boosted with 10 ug of the . . . same preparation in incomplete Freund's adjuvant four weeks after the primary inoculation. 6.2.2 PRODUCTION OF ANTI-PBOMP-1 AND ANTI-PBOMP-2 MONOCLONAL ANTIBODIES Hybridoma cell lines secreting antibodies to PBOMP-1 or PBOMP-2 were obtained by fusion of mouse myeloma cell line X63.Ag8.6543 with spleen cells obtained from a C57/B1 mouse immunized against H^ influenzae as follows: A female C57/B1 mouse was injected intraperitioneally four times over a period of two months with 1 x 10 H. influenzae εtrain S2 cellε. Three monthε later, the mouεe waε immunized with εubεtantially pure PBOMP-1 or PBOMP-2 isolated from an SDS- PAGE band aε described in Section 6.2.1. One month later, the mouse received an intravenous injection of total outer membranes from S2. Cell fusion waε performed on the fourth day post- intravenous injection by standard procedures common to those of εkill in the field (for example, Gefter et al., 1977, Somat. Cell. Genet 3_:231-36) . Hybridoma cell culture εupernatantε were εcreened by a standard ELISA using H. influenzae outer membrane proteinε as antigens. Aεsays were performed in 96 well polystyrene plates coated overnight at 4°C with OMPs. Plates were blocked with 40 mM Tris (pH 8.0), 150 mM NaCl, 5% nonfat dry milk (BLOTTO) (See Section 6.4.4) and waεhed with PBS/0.1% Tween-20. Culture εupernatantε diluted 1:10 in PBS/Tween-20 were added, incubated for 60 minuteε at 25°C, and waεhed as before. Bound antibodies were detected with alkaline phosphataεe-Goat F(ab') anti-mouεe (IgG, IgM) and the alkaline phoεphataεe εubstrate. Positive supernatants were then screened by dot blot analysiε with purified PBOMP-1, E_^ coli OMP'ε, and S2 lipopolyεaccharide (LPS) . Deεired hybridomaε were recloned by limiting dilution (McKearn, 1980, in Monoclonal Antibodies, Kennett, McKearn and Bechtol, eds. , Plenum Preεε, p. 374) and εcreened by 0 Weεtern blot with H b OMP'ε. Selected hybridomas were injected into Balb/c mice for growth as ascites by εtandard procedureε (Brodeur et al., 1984, J. Immunol. Meth. 71:265- 72) . Seventeen of the hybridomas generated by the fuεion of 15 mouεe myeloma cellε with εpleen cellε from C7/B1 mice immunized with PBOMP-2 were εcreened for their reactivity to PBOMP-2 uεing the εtandard ELISA aεεay. One of the hybridomaε, designated 61-1 was found to be specific for PBOMP-2. Western blot procedures described infra, in section 20 6.5.5. uεing 61-1 aε the primary antibody εhowed that PBOMP-2 or a PBOMP-2 like protein waε conserved and expresεed in the three clinical nontypable H^ influenzae εtrains 0045E, 1939, and HST31 (see FIG. 29). 25 6.3 REACTIVITY OF ANTI-PBOMP-1 AND ANTI-PBOMP-2 ANTIBODIES WITH E. COLI Western blot analysis of the reactivity of anti- PBOMP-1 and anti-PBOMP-2 antiserum waε performed as described in Section 6.4.4, infra. Ten microliters of an overnight 30bacterial culture lyεed in sample preparation buffer containing 2-Mercaptoethanol was applied to each lane of a 15% SDS-PAGE gel. After electrophoreεis and transfer to nitrocellulose, the blots were probed with 1:250 dilutions of rabbit polyclonal anti-PBOMP-1. Incubation with horseradish 35 peroxidaεe conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratorieε, Gaitherεburg, MD) showed that the anti- PBOMP-1 antisera recognized the PBOMP-1 in Haemophilus and an 18000 dalton protein in E^ coli (FIG. 2A) . In order to confirm that an epitope of the PBOMP-1 crosε-reactε with an 18000 dalton protein of E^ coli, monoclonal antibodies made against the PBOMP-1 were screened for reactivity against E^_ coli proteins. While-most of the monoclonals screened failed to react with E_^ coli, one clasε of monoclonalε, exemplified by monoclonal Gl-1, reacted εtrongly with the PBOMP-1 of Haemophilus and with an 18000 dalton protein in E^_ coli (FIG. 2B) . This demonstrateε that at leaεt one epitope present on the PBOMP-1 crosε-reacts with an epitope on an E_^_ coli protein. Thiε indicateε that an antiεerum againεt H. influenzae PBOMP-1 may alεo protect againεt some E_ coli infections. The anti-PBOMP-2 monoclonal antibody, 61-1, was found to crosε-react weakly with a protein of about 15000 daltonε from E_^ coli, but showed no detectable croεε-reactivity with PBOMP-1. 6.4 GENERAL PROCEDURES USED FOR PREPARATION OF RECOMBINANT PLASMIDS 6.4.1 CONDITIONS FOR RESTRICTION ENZYME DIGESTIONS Restriction endonucleases were purchaεed from BRL (Bethesda, MD.), IBI (New Haven, CT) , New England Biolabs (Beverly, MA) , or US Biochemical Corporation (Cleveland, OH) . Reεtriction enzyme digestions were carried out by suspending DNA in the appropriate restriction buffer, adding restriction endonuclease, and incubating for an appropriate period of time to enεure complete digeεtion. One unit of enzyme is defined as the amount required to completely digest 1.0 ug of phage lambda DNA in 1 hour in a total reaction mixture of 20 ul volume. Buffers used with the various enzymes are listed below: Low salt buffer used for Clal, Hpal, Hpall, and Kpnl digestionε consisted of: 10 mM Tris (pH 8.0), 10 mM MgCl 2 and 10 mM dithiothreitol (DTT) . Medium salt buffer used for Alul, Aval, EcoRII, EcoRV, Haell, Haelll, HincIII, Hindlll, Pstl, Sau3AI, Sphl, Sεtl, Sstll, Taql, and Xhol digestions consisted of: 50 mM Tris (pH 8.0), 10 mM MgCl 2 , 50 mM NaCl, and 10 mM DTT. High εalt buffer used for BamHl, EcoRI, Pvul, Sail and Xbal digestionε consisted of: 50 mM Tris (pH 8.0), 10 mM MgCl_, 150 mM NaCl and 10 mM DTT. 2 . . . The buffer used for S al digestions consisted of: 10 mM Triε (pH 8.0), 20 mM KCl, 10 mM MgCl 2 , and 10 mM DTT. The buffer uεed for Seal digestions was: lOOmM NaCl: 10 mM Tris HC1 (pH 7.4); 10 mM MgCl 2 and 1 mM DTT. All restriction digestions were carried out at 37"C except Taql, which was carried out at 60"C. 6.4.2 GEL PURIFICATION OF DNA FRAGMENTS After reεtriction enzyme digeεtionε, DNA fragments of varying sizeε were separated and purified using gel electophoresiε in low melting temperature agarose (FMC LGT agarose) using 50 mM Tris-acetate 1 mM EDTA buffer pH 7.8 at 10 volts/cm. Agarose concentrations varied from 0.8% to 1.5% depending on the size of fragments to be recovered. DNA bandε were viεualized by ethidium bromide fluoreεcence and cut out of the gel. DNA waε recovered by melting the agarose at 65 β C, adding 4 volumes of 0.65 M NaCl, 10 M Tris (pH 8.0), 1 mM EDTA to bring the mixture to a final concentration of 0.5 M NaCl, loading the DNA onto a NACS column (BRL, Bethesda, MD) equilibrated with 0.5 mM NaCl, 10 mM Tris pH 8.0, l mM EDTA (loading buffer), washing the column with 3-5 volumes of loading buffer, and eluting with 2-3 volumes 2 M Nacl, 10 mM Tris pH 8.0, 1 mM EDTA. The DNA eluate was diluted 1:1 with double distilled H_0 and precipitated with 3 volumes of ethanol. The pellet was waεhed with 70% ethanol, vacuum dried, and resuspended in 10 mM Tris-HCl buffer, pH 7.5 containing 1 mM EDTA (TE buffer). 6.4.3 DNA LIGATION All ligations were accomplished using T4 DNA ligase. T4 DNA ligase waε purchaεed from BRL (Bethesda, MD) , United States Biochemicals (Cleveland, OH) or Boehringer (Indianapoliε, IN) . One unit (U) of T4 DNA ligaεe is defined as the amount required to yield 50% ligation of Hindlll fragments of bacteriophage lambda DNA in 30 minuteε at 16 β C 10. m 20 ul volume ligaεe buffer at a 5'-DNA termini concentration of 0.12 uM (300 ug/ml). DNA ligationε were performed in ligase buffer consiεting of: 50 mM Triε (pH 7.5), 10 mM MgCl 2 , 10 mM DTT, 1 mM adenoεine triphoεphate) . Normally a DNA concentration ranging from 20-30 ug/ml, and a molar ratio of vector to inεert of 1:1 waε uεed. T4 DNA ligase was added at a ratio of 1 U per 20 ul reaction volume. Incubations were carried out for 18-24 hours. Temperatures used were 15°C for cohesive end ligations, and 22°C for blunt end ligations. If sufficient material was available, 20 ligations were checked by analyzing a portion of the reaction mixture by agarose gel electrophoresis. 6.4.4 PROTEIN IMMUNO BLOT ANALYSIS (WESTERN BLOT) Proteins were fixed to nitrocellulose sheets for immuno 5blot analysis by various techniques, depending on the particular application. Phage plaques were tranεferred from agar plateε by gently placing a εterile 8.1 cm diameter nitrocellulose disc onto the surface of a 10 cm diameter phage titer plate. The sheet was allowed to wet completely, 30 poεitionε were marked by punching through the filter with a εterile needle, and the filter waε lifted after two minutes. Colony blots were performed by transferring bacterial colonies to a nitrocellulose sheet, allowing the colonies to grow by placing the sheet (colony εide up) on nutrient agar 35 for 4 to 6 hourε, and expoεing the εheet to chloroform vapor for 30 minuteε to lyεe the colonies. Protein gel transfers were performed by placing an SDS-PAGE gel containing the protein mixture to be analyzed on a nitrocellulose sheet and applying horizontal electrophoresiε in a Hoeffer Tranεphor apparatuε at 0.5 A for 14 hours in 25 mM Tris 0.38M glycine pH 8.8 buffer. Once protein transfer was complete, filters were soaked in 50 mM Tris (pH 8.0) , 150 mM NaCl, 5% nonfat dry milk (BLOTTO) at 37 β C for one hour in all cases, except colony blots. When colony blotε were performed, the filters were soaked overnight at 4"C in BLOTTO containing 1 mg/ml lysozyme to digest cell debriε. Filterε were then abεorbed with a firεt antibody probe at an appropriate dilution (determined by trial and error) in BLOTTO for 3 hours at 37"C, waεhed three timeε for 15 minuteε with BLOTTO, abεorbed with horεeradiεh peroxidaεe conjugated second antibody (Kirkegaard and Perry, Gaithersburg, MD) at a dilution of 1:500 in BLOTTO for one hour at 37 β C and washed with BLOTTO three timeε for 15 minuteε. Filterε were placed in 50 mM Triε (pH 7.0), 150 mM NaCl, .01% hydrogen peroxide; and 0.06% 4-Chloro-l- naphthol (Sigma Chemical Co., St. Louiε, MO) in methanol waε added. If no blue color developed within 20 minuteε, the reaction waε considered negative. The reaction was stopped by transferring the filter to distilled water and blotting dry. 6.4.5 GENE FUSIONS Fusionε of a gene or gene fragment encoding a PBOMP protein or peptide thereof to another gene such as the gene encoding alkaline phosphataεe (PhoA) were carried out aε deεcribed by Manoil and Beckwith (1985, Proc. Nat'l Acad. Sci. USA 82:8129-8133) . Recombinant plasmids were introduced into an E^_ coli strain containing a deletion of the native PhoA gene and carrying a derivative of transposon Tn5 O (TnPhoA) which containε an alkaline phoεphataεe gene, which lackε both a promotor and a membrane transport signal sequence, inserted into the left terminal repeat of Tn5 on an F-prime plasmid. Hence, production of active alkaline phosphatase enzyme requireε transposition of TnPhoA such that the PhoA gene is fused in frame into an actively transcribed gene containing a membrane transport signal peptide. Such transpositions were detected by plating cells in the presence of 40 ug/ml 5-Bromo-4-Chloro-3-Indolyl Phoεphate (XP, Sigma Chemical Co. , St. Louiε, MO) . In the preεence of this dye, colonies producing active alkaline phosphataεe enzyme appear intenεely blue in color while colonieε which lack active alkaline phoεphataεe appear white. 6.4.6 DNA FILTER HYBRDIZATION ANALYSIS (SOUTHERN BLOT) DNA ~filter hybri ~ di. —zati :on analyεi :ε waε carri .ed out according to the procedure of Southern (1975, J. Mol Biol. 98: 508) . DNA to be analyzed by filter hybridization waε digeεted with appropriate reεtriction endonucleaεe(ε) and εeparated by agarose gel electrophoresis in 0.8% Agarose (SeaKem Portland, ME) uεing 90 mM Tπε-borate, 8 mM EDTA buffer and 10 voltε/cm. DNA in the gel waε denatured by εoaking the gel in 1.5 M NaCl/0.5 M NaOH for 1 hour and neutralized by εoaking in 1.5 M NaCl/1.0 M Tris-HCl for 1 hour. Denatured DNA was transferred to nitrocellulose filter paper by blotting. Following transfer of DNA, filter were washed with 6 X SSC (prepared by dilution from a 2OX SSC stock containing 175.5 g NaCl and 88.2 g Na citrate/liter) and air dried. DNA fragments were fixed to the filter by baking at 80°C for 2 hours under vacuum. . . . DNA hybridization probes were prepared by nick translation according to the procedure of Rigby et al., (1977, J. Mol. Biol., JL13: 237-244). DNA for the probe (1-2 ug) was dissolved in 100 ul nick-translation buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgSO , 10 mM DTT, 5 ug/ml bovine serum albumin, and 20 urn each dGTP, dCTP, and dTTP) . To this reaction mixture, 100 uCi of alpha 32P-dATP (Amersham, 2-3000 Ci/mmole) , 1.0 ng of deoxyribonucleaεe I (Sigma Chemical Co., St. Louiε, MO) and 10 ϋ E^ coli DNA polymerase I (Boehringer) were added and the mixture incubated at 15°C for 45 minutes. The reaction was stopped by the addition of EDTA to 50 mM and heating to 65"C for 10 minuteε to inactivate the enzymeε. The labeled DNA waε precipitated by addition of three volumes of ethanol and resuspended to 50 ul of 0.3 M ammonium acetate (NH L 4.OAc) . The sample waε loaded onto a 1 ml Biogel P-50 spin column equilibrated'with 0.3 M NH OAc and eluted by centrifugation at 500 x g for 5 minutes. The column was waεhed with 100 ul 0.3 M NH 4.OAc and the eluates combined and precipitated with three volumes of ethanol. The labelled DNA pellet was vacuum dried, resuεpended in TE buffer, radioactive incorporation measured in a Beckman (LS9000) scintillation counter by Cherenkov εcattering. For hybridization, filters with bound DNA were wetted with 6 x SSC and prehybridized with 6 X SSC/ 0.5% SDS/5X Denhardt's solution/100 ug/ml tRNA at 68 " C for 2 hours to block excess binding capacity of the filter (1 X Denhardt's solution is .02% Ficoll, 0.02% polyvinylpyrrolidone, .02% bovine serum albumin in water) . The hybridization reaction waε carried out in the εame buffer to which 0.01 M EDTA and 5-10,000,000 CPM (Cherenkov) labelled probe was added. The probe solution was heated to 90°C for 10 minutes prior to application to denature the DNA strandε, cooled to 68 β C, and incubated with the filter at 68 β C for 18-24 hours. After hybridization, filters were washed with several changes of 0.1 X SSC/0.5% SDS at 68°C in order to remove nonspecifically bound probe. Under the conditionε used, DNA homologies of greater than or equal to 90% would show positive binding of the DNA probe. Filters were air dried and expoεed on Kodak XAR film at -70 β C uεing Dupont CRONEX 'Lightning Plus' intenεifying screens. 6.5 CLONING THE PBOMP GENES OF H. INFLUENZAE The εource of H_^ influenzae chromoεomal DNA for cloning of the PBOMP geneε waε either I influenzae KW20b (HiKW20b) , a derivative of a non-encapεulated Rd εtain of Hi tranεformed to type b+ by DNA from εtrain b-Eagan (Moxon et al, 1984, Clin. Invest. 73 . : 29 ~ 306 ) or IL . influenzae S2, (Hi S2) , a spontaneous capsule-minus mutant of Hib Eagan. To generate a phage lambda library, chromosomal DNA from Hi was εheared to an average length of about 15000 baεe pairε (bp) , blunt ended by treatment with T4 DNA polymerase, . . . modified with EcoRI DNA methylase, ligated to synthetic EcoRI linkers, and cloned into the recombinant Lambda phage vector Charon 4. To generate a plasmid library chromosomal DNA of Hi S2 was partially cleaved with Sau3A, the 3-8 kilobase (kb) length reεtriction fragmentε thus generated were isolated, and ligated into plasmid vector pGD103 at the BamHl restriction site. Thiε plaεmid iε a derivative of pLG339 (See FIG. 3; see also Stoker et al., 1982, Gene 18:335-41) and iε carried in 6-8 copieε/cell. It alεo containε the lac Z-alpha peptide and polylmker region from plaεmid pUC8; and therefore, if tranεformed into an appropriate F^ coli εtrain (such as JM83) , allows selection of recombinant plasmids by screening for losε of the Lac+ phenotype. If cloned in the proper orientation, it is also posεible that a cloned gene which iε poorly expreεεed in IS^ coli could come under control of-the εtrong, regulated lac promotor. E_ coli containing recombinant plasmids were screened for production of PBOMPs using*a pooled mixture of monoclonal antibodies or polyclonal anti-PBOMP-1 antiserum. 6.5.1 CONSTRUCTION OF HI PLASMID LIBRARY It is possible that the PBOMP-1 protein is not expresεed on or iε incompatible with lambda phage. In order to test this we constructed a plasmid chromosomal library of Hi S2. Cloning of E^ coli OMP geneε on high copy number plaεmidε has been shown to be toxic (see, for example, Beck et al. , 1980, Nucleic Acid Reε. 8_:3011-3024) . In order to avoid thiε problem, we used the low copy number plasmid pGD103 (see FIG. 3). Chromosomal DNA from a Hi S2 was partially digested with restriction endonuclease Sau3A (BRL, Betheεda, MD) . Five hundred micrograms of DNA was digested in 5 ml restriction buffer with 50 units of Sau3A for 1 hour at 37"C. Fragments were separated by velocity sedimentation in a Beckman SW28 rotor on 10-40% εucroεe gradientε containing 10 mM Triε (pH 8.0), 1 mM EDTA, 1 M NaCl at 140,000 x g for 24 hourε. Two ml fractionε were collected and aliquots analyzed by agarose gel electrophoresis. Fractions containing restriction fragments of 3-8 Kb in length were pooled and concentrated in TE buffer. Plasmid pGD103 DNA was digested with BamHl endonuclease and treated with calf alkaline phosphatase (Boehringer, Indianapoliε, IN) (1 Unit/ug DNA, 37°C x 30 minuteε in reεtriction buffer). DNA waε purified from the reaction mixture by phenol extraction and ethanol . . precipitation and resuεpended in TE buffer. Since BamHl and Sau3A reεtriction enzymeε form coheεive endε, no further treatment of DNAε prior to ligation was necessary. About twenty-five ug each of Hi S2-Sau3A digested DNA and pGDl03/BamHl/CAP digested DNA were mixed in 500 ml ligation buffer containing 25 U T4-ligase (Boehringer, Indianapolis, IN) and incubated at 15 β C for 18 hours. A 20 ul aliquot of the reaction mixture was analyzed by agarose gel electrophoresiε in order to verify the ligation reaction (εtarting material waε run in an adjacent lane) . The ligation mixture waε then tranεformed into competent E^ coli JM83 (εe Maniatiε et al., 1982, Molecular Cloning, Cold Spring Harbor Laboratory, p. 250) incubated for 1 hour in LB-broth at 37 β C, and plated on LB-agar plateε containing 50 ug/ml kanamycin εulfate (Sigma Chemical Co., St. Louiε, MO) and 40 ug/ml 5-Bromo-4-chloro-3-indolyl-Beta-D-galactopy- ranoεide (X-gal, BRL Betheεda, MD) , and incubated 24 hours at 37"C. About 50% of the kanamycin resiεtant (kanR) colonieε which developed were white (Lac-) , indicating insertion of S2 DNA into the BamHl site in the lac region of pGD103 (Lac+ non-recombinants are blue) . Ten white colonies were selected at random, amplified, and shown to contain plasmids 4-8 Kb larger than pGD103 with insertionε at the vector BamHl site. One thousand five hundred and twenty-five white colonies were picked, amplified individually, and stored frozen at -70°C in LB broth containing 18% sterile glycerol in 96-well microtiter disheε. 6.5.2 CONSTRUCTION OF HIB LAMBDA GENE BANK High molecular weight chromosomal DNA from Hi KW20b waε suspended in TE buffer at a concentration of 200 ug/ml and sheared to an average length of 15000 bp by paεεage through a 25 gauge needle. Protruding endε were removed by treatment with T4 DNA polymeraεe in 50 mM Triε, (pH 8.8), 10 mM MgCl_, 20 mM (NH.),SO , 10 mM DTT, 50 uM dATP, dCTP, dGTP, and dTTP) at 37°C for 20 mi.nuteε. DNA waε then modi.fi.ed wi.th EcoRI DNA methylaεe (1 U/ug DNA) (BRL Betheεda, MD) , in 100 mM Triε (pH 8.0), 10 mM EDTA, 0.1 mM S-adenosyl-methionine) for 3 hours at 37°C. Methylation of DNA was verified by removing 1 ug of DNA from the reaction, mixing with 1 ug of unmodified lambda DNA and digesting in 20 uL of high salt restriction buffer with 5 units of EcoRI for 1 hour at 37 β C. Under these conditions, the modified Hi DNA was not digested, while the added lambda DNA was digested to completion. Twenty micrograms of modified Hi DNA was ligated to 1 ug chemically synthesized EcoRI linkers (BRL Bethesda, MD) in a 100 ul reaction mixture uεing T4 DNA ligase (5U) . After 18 hours, the reaction was stopped by heating to 60"C for 20 minutes, NaCl was added to a final concentration of 150 mM, and the mixture was digested with 10 U EcoRI for 6 hours. -62- Modified Hi DNA plus linker was separated from cleaved and unligated linkers by agarose gel electrophoresis as describe above. Prepared Hi DNA was mixed with the left and right EcoR fragments of lambda Charon 4 DNA at a 1:1:1 molar ratio and ligated with T4 DNA polymerase for 18 hours. The ligated DN mixture was packaged into Lambda phage particles using an in vitro packaging reaction. Five ug of ligated DNA in 4 ul H 2 was added to 7 ul of 20 mM Tris (pH 8) , 10 mM 2- mercaptoethanol (2-ME) , 3 mM MgCl2, 1 ul of 10 mM Tris, pH 7.5, 1 mM Spermidin re, 2 mM putrescine, 10 mM MgCl-_Λ, 1.5 mM ATP, 5 mM 2ME and 5 ul of sonic extract from E_^ coli BHB2690 (-1 imm 434, clts, b2.red3, Earn 15, Sam7) lysate (Hohn et al., 1977, Proc. Nat'l Acad. Sci. 7_4:3259). The reaction mixture was incubated at 22"C for 1 hour and packaged phages . were separated by centrifugation in a 3 M to 5 M CsCl [in 10 mM Tris (pH 7.5), 10 mM MgCl 2 , .02% gelatin (TMG buffer)] εtep gradient for 250,000 x g for 4 hourε in a BecJαnan SW50. rotor. Phage were removed from the interface and dialyzed against TMG. Titering of the phage thus prepared indicated library of 25-30,000 independent cloneε of the Hi genome had been generated. The phage library was amplified by plate amplification using E^_ coli KH802 as a phage host to yield 5 mull ooff pphhaa<ge suspension containing 10 —9 plaque forming units (PFU)/ml. 6.6 ISOLATION OF PBOMP GENES 6.6.1 ISOLATION OF A PBOMP GENE ENCODING A PROTEIN WHICH REACTS WITH MONOCLONAL ANTIBODIES AGAINST PBOMP-1 The Hi plasmid library was transferred to nitrocellulose sheets on LB-kanamycin (50 ug/ml) agar, grown for 24 hours at 37"C and analyzed by the colony blot procedure using a mixture of five non-competing monoclonal antibodieε to PBOMP-1 aε a probe. A clone which reacted to the mixed monoclonal probe waε iεolated and the plaεmid deεignated pAA152. FIG. 4 εhowε a reεtriction map of pAA152 which containε a 4.2 Kb Hi DNA inεert in vector pGD103. Weεtern blot analyεiε verified that clone pAA152 expreεεeε a 16000 dalton protein which was recognized by polyclonal anti-PBOMP-1 and also by pooled monoclonal antibody probes. Clone pAA152 was subεequently εhown to produce a protein which waε recognized by each individual monoclonal antibody uεed in the initial pool (FIG. 5) . The Sau3A insert in pAA152 was found to have regenerated the BamHl site of the polylinker region at one end of the insert. Deletions from this Ba Hl site to either the unique Bglll site or the unique Xbal site of the Hi DNA resulted in losε of expreεεion of the PBOMP detected on . . . Western blots. Deletions from the Hindi site of the polylinker to either the Xbal or Bglll siteε of the Hi insert DNA retained PBOMP expresεion (FIG. 4) . From theεe reεults, we conclude that the gene encoding this PBOMP lieε in the Bglll-BamHI 737 baεe pair fragment within the Hi DNA inεert . . . of pAA152. Analyεiε of minicellε (Achtman et al., 1979, Proc. Nat'l Acad. Sci. USA 7_6:4837-41) carrying pAA152 indicated that the cloned Hi DNA encodes two proteins of 16000 and 40,000 daltonε molecular weight reεpectively (FIG. 6) . Weεtern blotε of JM83 (pAA152) εhow that pooled monoclonal antibodieε raiεed againεt PBOMP-1 react with a 16000 dalton protein. No cross reaction iε apparent within the 40,000 dalton molecular weight region. 6.6.2 ISOLATION OF A PBOMP GENE ENCODING A PROTEIN WHICH REACTS WITH POLYCLONAL ANTI-PBOMP-1 ANTISERA The amplified phage library prepared aε deεcribed in εection 6.5.1. was diluted to 1-2000 PFU in one ml TMG and 50 9 ul of E. coli KH802 (5 x 10 cells/ml) were added. The mixture was incubated at 37 β C for 20 minutes and plated with 3 ml soft agar on agar plates containing NZYCM medium: 10 g NZ Amine A, 5.0 g NaCl, 2.0 g MgS04.7H 2 0, 5 g Yeast Extract, 1 g casamino acids (per liter) . Plates were incubated overnight, chilled to 4°C for 30-60 minutes, and plaques were 5 transferred to nitrocellulose. Filters were probed with polyclonal anti-PBOMP-1 aε deεcribed above in Section 6.4.4. Several poεitive plaqueε were detected in thiε manner. However, no poεitive plaqueε were detected when PBOMP-1 monoclonal antibodieε were uεed aε a probe. Positive plaques 0 were picked from the plate and amplified by growth in E_^ coli KH802. Clones were verified by SDS-PAGE gel/Western Blot analyεiε of phage lyεates. All positive clones expresεed a protein of apparent molecular weight 16000 daltonε which reacted with polyclonal antibody to PBOMP-1. Thiε protein was not present in control lyεateε of Charon 4 phage. In similar experiments, lysates from the positive cloneε failed to react with monoclonal antibodieε to PBOMP-1. One poεitive phage, deεignated lambda 16-3 waε selecte for further analysiε. This phage isolate was amplified by 0 growth m E^ coli KH802 in NZYCM broth, recovered by precipitation with 20% Polyethylene glycol 6000 and banded i cesium chloride equilibrium gradients (4 M CsCl in TMG, Beckman SW50.1 rotor, 300,000 x g for 24 hours). Phage DNA was isolated by treatment with 0.1% SDS and 20 ug/ml 5 proteinase K (Sigma Chemical Co., St. Louis, MO) at 55"C for 1 hour followed by extraction with phenol and ethanol precipitation. . e The lambda 16-3 DNA was digested with EcoRI and a partial physical map of the Hi chromosomal insert was 0 obtained. EcoRI fragments of the insert were isolated and subcloned into plasmid vector pGD103. Clones carrying fragments expressing the PBOMP-1 cross-reactive 16000 dalton protein were identified by Western blot transfer analysis of cell lysateε. One of these was designated pAA130. FIG. 7 vw represents a restriction map of this plasmid having an 5.7 Kb fragment from Hi DNA cloned into pGD103 plasmid. Monoclonal antibodies against PBOMP-1 did not react with the 16000 dalton protein expresεed from pAA130 (data not shown) . The protein expressed by this recombinant plasmid was recognized, however, by polyclonal anti-PBOMP-1 antisera (see FIG. 8 for example) . Analysiε of minicells (Achtman et al., supra) carrying pAA130 indicated that the cloned Hib DNA codes for proteins of apparent molecular weights of 16000 and 17000 daltons. . . . The labelled 16000 dalton protein was specifically immunoprecipitated by polyclonal anti-PBOMP-1 (data not shown) . Thus, plasmid pAA130 directs the expresεion of a 16000 dalton molecular weight PBOMP. An internal deletion generated by excision of DNA inserted between the unique Pstl site of the insert and the single Pstl in the polylinker did not affect the expresssion of the cross-reacting protein. The Xbal fragment of this plaεmid was deleted by a similar method and expresεion of the PBOMP-croεs reacting protein was retained (FIG. 7) . An . . additional deletion derivative of this plasmid was generated by religation of the two internal Bglll sites and this derivative alεo retained expreεεion of the PBOMP-croεε reactive protein. The 781 baεe pair BεtEII-XmnI fragment was cloned by isolating the fragment from a low melting point agarose gel, filling in the BstEII end with Klenow fragment of DNA Polymerase I and cloning the fragment into the Hindi site of pGD103. Weεtern blot analyεis using polyclonal anti-PBOMP-1 showed that this plaεmid retained expression of the 16000 dalton PBOMP. As with pAA130, the PBOMP produced from thiε plasmid failed to react with monoclonal antibodies to PBOMP-1. As an independent method of verifying the location of this PBOMP gene, the large EcoRI-PvuII fragment of pAA130 was ligated with the EcoRI-PvuII fragment of pLG339 to generate a new tetracycline resistant plasmid designated pAA136. This plasmid expreεεed the PBOMP aε verified by Western blots. This plasmid waε tranεformed into an E. coli εtrain with deletion of the chromoεomal alkaline phoεphataεe gene (phoA) , and carrying the tranεpoεible element TnPhoA. Three independent tranεpositions of the TnPhoA element into pAA136 which restored alkaline phosphataεe activity were isolated. The sites of the TnPhoA insertionε into pAA136 were determined using the unique Dral restriction site near the left terminal region, of TnPhoA and the Hindlll, BstEII, Xmnl, and Pstl sites of pAA136. All three insertions were determined to fall within the BstEII-Xmnl fragment of pAA136. All three TnPho insertionε were in the same orientation indicating that transcription of the PBOMP gene is directed from the BstEII εite towards the Xmnl εite in pAA136. All three TnPhoA tranεpoεitionε reεulted in loεs of the 16000 protein detected by polyclonal anti-PBOMP-1 antiserum as detected by Western blots. One fusion generated a new band on Western blots at 60000 dalton which waε detected by polyclonal anti-PBOMP-1 antiεerum. Thiε size is within the predicted range of fusion proteins that might be generated by fusion of alkaline phosphataεe (45000 daltonε MW) to a 16000 dalton MW protein. Reεtoration of PhoA activity in theεe transpositions verifies that the PBOMP protein contains a peptide signal for membrane transport; and hence, is probably a membrane protein. The TnPho fusionε were εequenced by εubcloning the junction between TnPhoA and the Hi cloned DNA εequenceε into M13. In all caεeε the PhoA coding εequenceε were determined to be in frame with the predicted open reading frame for the PBOMP-2 gene of pAA130 (see Section 6.7.2, infra) . -67- 6.7 DETERMINATION OF THE NUCLEOTIDE SEQUENCE OF PBOMP GENES 6.7.1 SEQUENCING STRATEGY FOR THE PBOMP GENE EXPRESSED BY pAA152 The nucleotide sequence of the PBOMP gene expressed by pAA152 was obtained by dideoxynucleotide sequencing (Sanger et al., 1978, Proc. Nat'l Acad. Sci USA 7 :5463-5467) of the 737 bp Bglll-BamHI fragment of pAA152, after subcloning into single stranded phages of the M13 family, i.e. , M13 mplδ and mpl9. The location and direction of εequenceε determined from theεe εubcloneε iε εhown in FIG. 9. The complete nucleotide εequence of the Bglll-BamHI fragment iε εhown in FIG. 10. The 737 Bglll-BamHI fragment of pAA152 containε a εingle open reading frame (ORF) coding for a polypeptide of 153 amino acids (FIG. 11) . The amino acid composition of the PBOMP gene determined from the DNA sequence closely matches the amino acid composition of the PBOMP-1 purified protein (see Tables 1 and 2) . The apparent molecular weight of mature PBOMP-1 was 14,23 daltons. The number of amino acid residues was 134. The apparent molecular weight of mature PBOMP-2 was 13,461. The number of amino acid residues was 136. In addition, the PBOMP-1 gene has an internal peptide sequence (AA 48-81) in which (30/33) amino acids align with the amino acid sequence of the T9 internal peptide of PBOMP- if an allowance is made for the Leu-68 reεidue which iε absent from the sequence of the T9 peptide (FIG. 12) . The amino terminal region of PBOMP-1 also contains an amino acid sequence which shows similarities with other membrane transport peptide sequenceε (Watson, 1984, Nucleic Acids Res 12:5145-5164). From these data and from the monoclonal — antibody binding data, we conclude that this gene encodes the PBOMP-1 protein. 6.7.2 SEQUENCING STRATEGY FOR THE PBOMP GENE OF pAA130 • *** The nucleotide sequence of the PBOMP gene of pAA130 was determined by dideoxynucleotide sequencing (Sanger, et al., supra) of the 789 base pair BεtEII-XmnI fragment of pAA130 after subcloning into M13 mpl8 and mpl9 phage. These recombinant phage are designated M18001 and M19001 respectively. The universal 17 baεe oligonucleotide sequencing primer (New England Biolabs) was used to determine the sequence from both endε of the BstEII-Xmnl fragment (see FIG. 13) . Two additional oligonucleotides were synthesized and used as primers for dideoxynucleotide sequencing (M18PRI, 5M19PR2). All other εequencing primerε were made at Praxis Biologies, Rochester, N.Y. on an Applied Biosyεtemε 380 B DNA synthesizer. The primers were made on a 1 umole controlled pore glass column with beta-cyanoethyl phosphate protecting group chemiεtry. The yield of oligonucleotide waε εufficiently pure to allow the uεe of the primers directly from the column without further purification. The two synthetic oligonucleotide primers bind to sequenceε approximately 200 nucleotides in from each end of the fragment as shown in FIG. 13. The total 789 bp DNA sequence of the BεtEII-XmnI fragment of pAA130 iε εhown in FIG. 14. The ORF is underlined as shown in FIG. 15. Thus ORF encodes a polypeptide of 154 amino acids. The amino terminal 18 residue peptide resembles a membrane transport signal sequence determined for other proteins (Watson, 1984, supra) . in addition, sequence data from the TnPhoA fusions in pAA130 demonstrated that all three transpositions were into the reading frame of the 154 amino acid polypeptide. The amino acid composition of the proposed mature gene product as deduced from the DNA sequence of the ORF of pAA130 5 -70- differs significantly from that determined by amino acid analysiε of purified PBOMP-1 (Tableε 1 and 2) . No εignificant homology waε found when the amino acid sequence of the PBOMP gene of pAA130 was compared to that of the tryptic peptide T9 from purified PBOMP-1 protein. In addition, although the product encoded by this gene is recognized by polyclonal anti-PBOMP-1 antisera, it is not recognized by monoclonal antibodies to PBOMP-1. From these observationε, it is clear that the Hi gene expressed by pAA130 is not the gene for PBOMP-1. Thus the PBOMP-gene encoded by pAA130 was designated PBOMP-2. 6.8. CHARACTERIZATION OF PBOMPs EXPRESSED BY RECOMBINANT E. COLI AS LIPOPROTEINS As demonstrated in Section 6.1.2., PBOMP-1 isolated from H_^ influenzae has covalently attached fatty acids, including lauric acid, palmitic acid and a derivative of palmitic acid, and hence can be classified as a bacterial lipoprotein. The following experiments were performed to investigate whether PBOMPs expreεεed by recombinant E_^ coli alεo exist as lipoproteins. Two different in vivo methodε were uεed to verify the lipoprotein nature of the expreεεed PBOMPε. In one series of experiments, cells containing recombinant plasmids expresεing PBOMPs were cultured in the presence of a radioactively labelled fatty acid. Under such conditions, any lipoprotein formed containing the covalently attached fatty acid will be specifically radiolabeled. E. coli JM83 cells containing either plasmid pAA152 expressing PBOMP-1 or plasmid pAA130 expressing PBOMP-2 were grown for 2 hours in M9-minimal medium containing 50 uCi/ml 14 C-palmitic acid. Whole cell lysates (10,000 trichloroacetic acid precipitable cpm/lane) were electrophoresed on 15% SDS-PAGE gels at 35 mA constant current. The gels were impregnated with sodium salieylate ( x gel volume x 1 M εolution for 20 minutes) , dried, and exposed at -70°C on XAR-5 film (Eastman Kodak Company, Rochester, N.Y.). Whole cell lysates of normal E^_ coli JM83 cells similarly cultured were run as controls. Results obtained are illustrated in FIG. 17. As demonstrated in FIG. 17, a radiolabeled protein of about 15,000 daltons was observed in lysates of E^ coli containing pAA152 and pAA130 which was not observed in lysates of control 12^ coli cells. Thus, the PBOMP-1 and the PBOMP-2 expressed by the recombinant E. coli were . . . specifically radiolabeled. In another series of experiments, cells containing recombinant plasmids expressing PBOMPs were cultured in the presence of globomycin, an antibiotic known to specifically block processing of all known bacterial liproproteins by inhibition of the liproprotein signal peptidase (Inukai et al., 1978, J. Antibiotics (Tokyo) 31:1203-1205) . E. coli JM83 cells containing either pAA152 or pAA130 were cultured in the presence of 25 ug/ml globomycin (obtained from Dr. M. Inouye, Robert Woods Johnson Medical Dental School, Piεcataway, N.J.) for 1 hour. Similar cultureε untreated with globomycin εerved as controls. Whole cell lysateε were electrophoreεed on 15% SDS-PAGE gelε, tranεferred to nitrocelluloεe, and probed with polyclonal anti-PBOMP-1 antiεera. Results obtained are illustrated in FIG. 18. As demonstrated in FIG. 18, in lysates of globmycin- treated cells expreεεing either PBOMP-1 or PBOMP-2, an approximately 16,500 dalton band was observed which waε not detected in lysates of control or untreated cells. BaεecL on results " obtained in both seπeε of in vivo experiments, the PBOMP-1 and PBOMP-2 proteins expressed by recombinant E^ coli containing PBOMP genes are lipoproteins. 7 EFFICACY OF PBOMP-1 AND PBOMP-2 SUBUNIT VACCINES 7.1 BACTERICIDAL ACTIVITY OF ANTI- SERA INDUCED BY PBOMP-1 AND PBOMP-2 Anti-PBOMP-1 polyclonal rabbit antiεera, prepared aε deεcribed in Section 6.2., were examined for their ability to kill Hib and Hi in an in vitro complement mediated bactericidal aεsay syεtem (εee Muεher et al., 1983, Infect. Immun. 3_9_:297-304; Anderson et al., 1972, J. Clin. Invest. 51:31-38). Sources of complement used for the assay system were either pre-collostral calf serum (PCCS) or normal rabbit serum (NRS) which had been absorbed previously with a non- typable Hi strain, S2, to remove any pre-existing anti- Haemophiluε antibodieε. The PCCS waε uεed undiluted and the NRS was used at a dilution of 1:4 for Hib and 1:8 for non- typable Hi. All dilutions were prepared using phosphate- buffered saline [20 mM phosphate buffer (pH 7.4), 0.15 M NaCl containing 0.15 mM MgCl 2 and 0.5 mM CaCl 2 (PCM)]. Bacterial strainε to be teεted were grown in BHI-XV until they reached a concentration of 1 x 10 9 cellε/ml aε measured by optical density at 490 mm. Bacteria were diluted to a final concentration of 1250 cells/20 ul in PCM. Twenty microliters of an appropriate antibody dilution in PCM was mixed with 20 ul of complement source on ice in wells of a 24 well microtiter plate (Costar) . The microtiter plate was removed from ice and 20 ul of test diluted bacteria were added to each well. Wells containing no antibody served as negative controls. After 30 minutes incubation at 37 β C, 800 ul of BHI-XV, containing 0.75% agar at 56 β C, were added to each well and allowed to solidify at room temperature. The plates were incubated overnight at 37 β C and read the next day. The BC titer of an antiεera waε read aε the reciprocal of the highest dilution capable of killing 50% of the test bacteria as compared to non-antibody control wells. ■ 73- The anti-PBOMP-1 was tested for bactericidal (BC) activity against several Hib clinical and laboratory isolates and the resultε shown in Table 3. TABLE 3 BC ACTIVITY OF ANTI-PBOMP-1 ANTISERA AGAINST LABORATORY & CLINICAL STRAINS OF HAEMOPHILUS INFLUENZAE LABORATORY STRAINS KILLED BY ANTI-PBOMP-1 H. influenzae type a HST-1 .a/ H. influenzae type c HST-5 +/ _*/ H. influenzae type b HST-3 + H. influenzae type b HST-10 + H. influenzae type b HST-12 + N.T. H. influenzae S-2 + N.T. CLINICAL STRAINS N.T. H. influenzae HST-31 + N.T. H. influenzae HST-35 + HIB CLINICAL STRAINS 112 strains tested 112 strainε killed + = greater than 50% killing of teεt bacteria +/~ - approximately 50% of test bacteria survived As can be seen from Table 3, anti-PBOMP-1 antibody had BC activity againεt a wide variety of clinical iεolateε both typable (e.g. a, b, c) and non-typable H. influenzae εtrainε. One hundred and twelve out of 112 Hib clinical isolates were killed by anti-PBOMP-1 antisera. These strainε were isolated in the Southwestern U.S., the Northeastern U.S. and Weεtern Canada. In order to eliminate the poεsibility that the killing was the result of anti-LPS antibody, the BC assay was performed with 200 ng of LPS from the Hib strain uεed to prepare the immunogen in each well. Reεultε of these experiments are shown in Table 4. TABLE 4 BACTERICIDAL ACTIVITY OF ANTISERA ABSORBED WITH LPS ANTISERUM LPS TEST BACTERIA TITER PBOMP-1 + N.T. H^_ influenzae 40 N.T. H^_ influenzae 160 Hib Eagan 40 Hib Eagan 40 PRP-CRM N.T. H^ influenzae 10 N.T. H_^ influenzae 10 Hib Eagan 400 Hib Eagan 800 ZERO or 200 ng Hib Eagan LPS used per well Expresεed as reciprocal of highest dilution of antisera showing 50% bacterial survival The LPS lowered the titer of the anti-PRP two-fold, the titer of the anti-PBOMP-1 against Hi four-fold and the titer of anti-PBOMP-1 against Hib not at all. While the LPS reduced the BC activity of anti-PBOMP-1, it did not eliminate it. Some of the observed reduction was undoubtably the result of anti-complementary activity of the LPS, as demonstrated by the reduction of the anti-PRP BC titer. The anti-PBOMP-2 antibody prepared as described in Section 6.2, was tested for bactericidal (BC) activity against the Haemophilus Influenzae S2 strain and the resultε are shown in Table 5. Sera was collected prior to inoculation and 7 and 10 weeks after initial immunization. 5 As shown below, the BC acti.vi.ty of anti.-PBOMP-2 anti.sera was found to increase significantly 7 weekε after inoculation. No further increaεe waε observed ~ 10 weeks after inoculation. 10 15 20 7.2 INFANT RAT PROTECTION FROM H. INFLUENZAE Infant rat protection studies were performed according to Smith et. al. (1973, Infec. Immun. : 278-90. Sprague- Dawley infant rats were pasεively immunized with 0.1 ml of varying dilutionε of rabbit antiεera in PCM by 2 5 intraperitoneal inoculation on day four of life. Eighteen hourε poεt-immunization, the ratε were challenged intraperitoneally with 10 4-106 Hi.b cellε in 0.1 ml of PCM. Survival of challenged ratε at 72 hours post-infection indicated protection. Reεultε of theεe experimentε are shown g n in Table 6. 35 -76- 5 0 5 -77- The results in Table 6 indicate that infant rats are protected against challenge with a fatal dose of Hib by passively transferred anti-PBOMP-1 antibody. The additional clinical Hib strainε which were used as challenge strains in the infant rat meningitis model to demonstrate protection that anti-PBOMP-1 protects against heterologous Hib clinical isolates. To determine whether anti-PBOMP-1 blocks the protective effects of anti-PRP or has additive effects, infant rats were pasεively immunized'with anti-PRP and anti-PBOMP-1 diluted beyond their protective end points. Upon challenge with Hib, the antisera together were able to protect high dilutions than either one above (Table 7) . These results shown in Table 7 indicate that anti-PBOMP-1 antibody and anti-PRP antibody do not interfere with each other and are capable of giving additive protection the infant rat meningitis model. TABLE 7 ANTI-PBOMP-1 + ANTI-PRP INFANT STUDIES SERA INJECTED ANTI-PBOMP-1 (l:100) a + + a Polyclonal rabbit anti PBOMP-1 diluted in PCM. b Polyclonal rabbit anti-PRP:CRM ιg _ conjugate. c Infant Sprague-Dawley rats surviving at 72 hours post- challenge. -78- 7.3 IMMUNOGENICITY OF PBOMP-1 IN HUMAN ADULTS Six human adult volunteers received two vaccinations with vaccine formulations comprising PBOMP-1 isolated from H. influenzae as described in Section 6.1., (5.2 ug PBOMP-1 without alum) at one month intervals, except for one individual who received only a single vaccination. Blood samples were obtained just prior to the initial vaccination and at monthly intervals thereafter. The specific antibody 0 response of vaccinated adults was evaluated by measurement of antibody titers to H^ influenzae PBOMP (ELISA) , to diphtheria toxoid (ELISA) (see, Engvall et al., 1972, J. Immunol. 109:129-135 for general description of ELISA assayε) and to Hib PRP polysaccharide (Farr-type RIA) (see, Farr, 1958, J. 15 Infect. Dis. 103:239-262 for description of Farr-type RIA). Results obtained in the PBOMP-1 ELISA assay are illustrated in FIG. 19. As demonstrated in FIG. 19, three of the six individuals showed a significant rise in antibody titer to '- BOMP-1. This rise in antibody titer was highly specific to the PBOMP-1 employed aε an immunogen aε no significant change was observed in the titer of antibody specific to either diptheria toxoid or Hib PRP (results not shown) . 7.4 BACTERICIDAL ACTIVITY OF ANTISERA INDUCED BY SIGNAL-LESS PBOMP-1 A recombinant signal-leεε PBOMP-1 (herein designated rPBOMP-1) obtained from E^ coli PR13 cells containing plasmi pPX167 as described infra in Section 8.1 was used as an 3.mmunogen to immunize white New Zealand rabbits. The rPBOMP-1 was either emulsified in incomplete Freund's adjuvant (IFA) or bound to aluminum hydroxide. rPBOMP-1 was bound to alum by mixing rPBOMP-1 at a concentration of 500 ug/ml in 0.85% saline with alum at a concentration of 500 35 ug/ml at a 1:1 ratio. The mixture was shaken at 37°C for 3 hours and the alum pelleted by centrifugation. Supernatent was assayed by BCA protein assay (Pierce Chem. Co., Chicago, IL) for unbound protein. None was detected. Alum was reεuεpended in phyεiological saline at 500 ug/ml. rPBOMP-1 was emulεified in IFA (Difco) in a 1:1 ratio. Animalε were immunized intramuεcularly with 50 ug of either preparation at 4 week intervals. Animals were bled at weeks 0, 6, and before each immunization. The anti-rPBOMP-1 polyclonal rabbit antisera obtained 0 . . . . . . . . were examined for the ability to kill Hi in an in vitro complement mediated bactericidal assay system as described supra in Section 7.1. Antisera were tested from juεt prior to the first immunization, week 0, and two weeks after a second immunization, week 6. The bactericidal (BC) activity 5 of the antisera were compared with .that of antisera made againεt PBOMP-1 iεolated from H^ influenzae (deεignated "native PBOMP-1") . The test bacterium was nontypable H. influenzae strain S-2. Results are shown in Table 8. 0 TABLE 8 BC ACTIVITY OF ANTISERA AGAINST rPBOMP-l 3 Antiserum Againεt Antiserum Native PBOMP-1 rPBOMP-1 (IFA) rPBOMP-1(Alum) 5 Week 0 1:5 1:5 1:10 Week 6 NT 1:160 1:80 Hyperimmune c 1:160 NT NT <£ Test organism: nontypable H^ influenzae S-2. For text for details of immunizationε employed to obtain antisera Hyperimmune antiserum was made against multiple doses of native PBOMP-1. W The BC titer of an antisera waε read aε the reciprocal of the higheεt dilution capable of killing 50% of the teεt bacteria as compared to non-antibody control wells. While both rabbits had low levels of BC activity prior to immunization, titers of 1:5 and 1:10, the BC of the sera 5 . . . . . . . obtained at week 6 had significant increases BC activity. The rabbit immunized with rPBOMP-1 in IFA had a titer of 1:160 and the rabbit immunized with rPBOMP-1 on-alum had a titer of 1:80. Hyperimmune antiserum was obtained after the rabbit received multiple doseε of native PBOMP-1. The ■ S,yperimmune rabbit anti-PBOMP-1 serum had a titer of 1:160. These results clearly indicate that the rPBOMP-1 is capable of eliciting antibody which recognizes the native PBOMP-1 in Haemophilus and is biologically active in the bactericidal assay system. 15 8 NOVEL PLASMIDS FOR ENHANCED EXPRESSION OF PBOMPs IN E. COLI 8.1 ENHANCED EXPRESSION OF PBOMP-1 IN E. COLI 20 The PBOMP-1 protein is expressed from the 737 bp BamHI-Bglll fragment of pAA152, presumably under control of its native promoter. The sequence contains a good consensus ribosome binding site and initiation codon of the PBOMP-1 gene. While PBOMP-1 expressed in E^_ coli with plasmids 25_ontaining this fragment was easily detected by Western blot analysis, the amount of such protein produced was lesε than 1% of cell protein, i.e. , leεε than the amount of PBOMP-1 made in H^ influenzae cells containing the native gene. As an initial attempt to produce higher levels of 3φB0MP-l in E^ coli, the cloned gene was placed under the control of promoterε lac and lambda P L known to yield high protein production. Promoterε were linked to the BεtNI site upstream of the PBOMP-1 initiation codon (FIG. 4A) . Cleavage 35 -81- at this site removes the native PBOMP-1 promoter but leaves the ribosome binding site intact. These constructionε were carried out aε followε. Firεt, the 739 bp BamHI-Bglll fragment of pAA152 carrying the PBOMP-1 gene was cloned into the BamHl site of lac promoter of plasmid pUC19. One clone carrying the PBOMP-1 gene in the same orientation as the lac promoter was designated pPX160. Expreεsion of PBOMP-1 from pPX160 in E^ coli JM103 was under regulation of the native promoter not under lac regulation; apparently due to a transcription termination signal in the '9 40 bp between the Bglll site and the tranεlation initiation codon of PBOMP-1. Plasmid pPX160 was then cleaved with BstNI, which cleaves between the PBOMP-1 initiation codon and the consensus translation initiation site of the gene, but leaves the ribosome binding site attached to the coding 15 . . . region. The ends were filled in with E^ coli DNA polymerase I (Klenow fragment) , and the linearized plasmid was cleaved with BamHl. The 577 bp BstNI-Bglll fragment carrying the promotorless PBOMP-1 gene was ligated to pUC19 cleaved with Hindi and BamHl. The resulting plasmid designated pPX166 w0as found by Western blot to express PBOMP-1 under regulation of the lac promoter in E^_ coli JM103. When PBOMP-1 was expresεed from lac or P τ promoters in E. coli JM103 or HB101 strain, only low levels of PBOMP-l were expresεed. Weεtern blot analysis of lysates from these c5ellε with monoclonal antibody G-204, which binds to the amino terminal 20 amino acids of mature PBOMP-1, demonstrated the presence of a 4000-5000 dalton peptide recognized by this monoclonal antibody (FIG. 20) . In cells expressing PBOMP-1 under regulation of the induced P τ promoter, greater than 90% 0 . . . . of the G-204 binding activity was in this presumed breakdown product, indicating PBOMP-1 expressed at- high levels in E. coli is unstable. Plasmids pPX160 and pPX166 were transformed into several E. coli strainε containing mutationε reported to 5 -82- stabilize foreign proteins. Theεe include ATP-dependent proteaεe (Ion "" ) mutationε, heat shock response (htp) , and an mRNA-stabilizing mutation (pnp) . In addition, εince processing of PBOMP-1 as a lipoprotein may enhance its stability, the plaεmidε were placed in an E^_ coli strain . . . . . lacking the major native E^ coli lipoprotein, the murem lipoprotein (lpp " ~) . In all strainε tested, the PBOMP-1 breakdown product recognized by monoclonal antibody G-204 was present at about the same level. Hence, it appears that breakdown of PBOMP-1 in E. coli is due to some other . . . . . unidentified activity. As a εecond approach, a modified PBOMP-1 gene waε created by removing the native εignal εequence of the gene. Such a construction offers two potential advantages over native PBOMP-1 protein. First, the εignal-leεs PBOMP-1 may not be transported to the membrane, and hence, toxicity effects due to overexpression of PBOMP-1 may be lessened. Second, since it is not modified with the extremely hydrophobic lipid groups, signal-leεε PBOMP-1 may not require uεe of detergents for isolation or storage in εolution. 0 Conεtruction of a signal-less form of PBOMP-1 is illuεtrated in FIG. 21. Aε shown in FIG. 21, the PBOMP-1 gene from plasmid pPX160 was cleaved at codon 19 with Alul restriction endonuclease. The resulting fragment was ligated to the Smal restriction site within the pUC19 polylinker 5region. The resulting gene expressed a hybrid protein containing all of the amino acids sequence of native PBOMP-1 plus an additional 18 amino acids from the pUC19 polylinker region * at the amino terminus. This plasmid was designated pPX167. 0 As εhown further in FIG. 21, a εecond recombinant plaεmid eεpressing PBOMP-1 was derived from plasmid pPX167 by cleaving at the BamHl site in the polylinker and cloning the fragment into the BamHl site of plasmid pINIII-ompA3. The resulting plasmid pPX168 containε a hybrid gene which encodeε mature PBOMP-1 linked at the amino terminus to the signal sequence of E^ coli OMP A protein. Thiε hybrid product iε processed through the membrane via the OMP A εignal sequence to generate a mature PBOMP-1 lacking lipoprotein modification and containing eight additional amino acids at its amino terminus. Plasmids pPX167 and pPX168 were transformed into E. coli JM103 and tested for recombinant PBOMP-1 synthesis. By SDS-PAGE Western blot analysis, both plasmidε were εhown to encode proteins which were recognized by polyclonal and monoclonal anti-PBOMP-1 antisera. The modified PBOMP-1 synthesized from pPX167 was inducible with isopropylthio- beta-d-galactopyranoεide (IPTG) , waε located in the cell cytoplaεm, and waε soluble in the absence of detergents. The modified PBOMP-1 waε not detectable by Coomaεsie blue 5 . . . staining of whole cell lyεates from IPTG-mduced cells; and comprised less than 1% of total cell protein. The modified PBOMP-1 εyntheεized from pPX168 waε alεo inducible with IPTG (under control of the hybrid lilac promoter) , waε excreted into the medium, and waε alεo εoluble in the abεence of detergentε. When fully i.nduced, thi.ε modi.fi.ed PBOMP-1 waε produced at a level of approximately 1 to 2 mg of cellε. Plasmids pPX167 and pPX168 were also tested in a variety of E_^ coli strainε for levelε of expreεεion. The moεt εuccessful combination tested was the pPX167 chimeric . plasmid transformed into E^ coli PR13, a strain containing the mRNA stabilizing mutation pnp. In this strain, recombinant PBOMP-1 is expresεed under control of the lac promoter at about 2-3% of total cell protein after lac induction. This recombinant PBOMP-1 is expressed as a cytoplasmic fusion protein containing about 17 amino acids from the lac alpha-peptide and multiple cloning sequence fused to the amino terminus of the PBOMP-1 gene. 5 8.1.1 PURIFICATION AND CHARACTERIZA- TION OF SIGNAL-LESS PBOMP-1 The following experiments were performed to purify and characterize the signal-lesε PBOMP-1 (deεignated herein rPBOMP-1) . 5 Iεolation of cytoplaεmic extract. Cellε from overnight cultureε of E^ coli PR13 containing pPX167 grown in Luria broth at 37°c were pelleted at 4°C in a GSA roter at 8,000 rpm for 10 minutes. Pellets were washed in 1/10 volume of 10 mM Tris-HCl, pH 7.5 and reεuspended in 1/100 volume of QLO mM Tris, pH 7.5 containing 10 ug/ml DNase and 10 ug/ml RNaεe. Cellε were disrupted by either sonication at 40W for 10 minutes on ice or by three passages through a French pressure cell at 20,000 psi. Unbroken cells were removed by centrifugation at 10,000 rpm in an SS-34 rotor at 4°C for 10 5jιinutes. Total cell membranes were removed by centrifugation at 55,000 rpm in a 60Ti rotor for 30 minutes at 4°C. Membranes were diεcarded and the supernatent retained. DEAE Fractionation of cytoplasmic extract. The supernatant waε paεsed over an ion exchange DEAE-Biogel A 0(Biorad Laboratories, Richmond, CA) column equilibrated with 10 mM Tris, pH 7.5. Esεentially all proteinε were bound to the column under theεe conditionε. Bound rPBOMP-1 and other proteins were eluted stepwise with 10 mM Tris, pH 7.5 containing 80 mM NaCl. All proteins eluted by this buffer 5rere pooled and concentrated by precipitation with 60% saturated ammonium sulfate at 4°C. The pellet was collected by centrifugation and diεsolved in 10 mM Tris, pH 7.5 followed by dialysis against the same buffer to remove any residual ammonium εulfate. 0 Reverεe phaεe chromatography of rPBOMP-1. The rPBOMP-1 containing εupernatant waε run over a 4.6 x 15 mm C-4 reverse phase HPLC column after dilution into a buffer containing 0.1% trifluoroacetic acid (TFA) in dH.O. The column was run at a flow rate of 2 ml/min using the same 35 buffer. Most of the proteinε bound to the column under these conditions. Bound rPBOMP-1 was eluted as a single peak by 0 to 95% acetonitrile gradient in 0.1% TFA over 20 minutes. The large peak containing rPBOMP-1 (see FIG. 22, peak 1) waε collected, and fractions pooled and concentrated by evaporation of the solvent. Dried rPBOMP-1 was rediεsolved in dH 2 0. SDS-PAGE was performed as described above herein, on the cytoplasmic extract, the DEAE eluate and the reverse phase eluate obtained from the E. coli PR13 cells containing 0 . ~ plasmid pPX167. After electrophoresis, the gels were stained with Coomassie brilliant blue stain for about 2 hours. Western blot analyεiε uεing monoclonal antibody GD204 was also performed. Results obtained are illustrated in FIG. 23 A and B. 15 . . As shown in FIG. 23A, rPBOMP-1 is the major protein present in the cytoplasmic extract of cells of E^ coli PR13 containing plasmid pPX167 when stained with Coomassie stain (FIG. 23A, lane 1) . As estimated by Western blot reactivity, greater than 95% of the rPBOMP-1 is located in the 0cytoplasmic extract. Since no detergent was used in preparing the cytoplasmic extract, the results indicate that the rPBOMP-1 obtained from those cells is soluble in 10 mM Tris-Hcl, pH 7.5 without detergent. This represents a departure from the PBOMP-1 obtained from Hib cells as a 'xipoprotein. Preliminary experiments uεing the cytoplaεmic extract indicated that rPBOMP-1 obtained from E. coli PR13 cells containing pPX167 may exist in aqueous solutions as a complex either with itself or with other cytoplasmic proteins. When 0 gel filtration chromatography using acrylamide-agarose polymerε or Sephadex beadε waε performed uεing thiε rPBOMP-1, the rPBOMP-1 eluted at an apparent molecular mass of greater than 100,000 daltons (data not shown). Ion exchange chromatography using DEAE-Biogel A (Biorad Laboratories, 5 Richmond, CA) showed that rPBOMP-1 did not elute as a single peak at a particular salt concentration. The rPBOMP-1 eluted over a range of NaCl concentrations from approximately 20 mM to 75 mM in 10 mM Tris, pH 7.5. While the eluted rPBOMP-1 was one of many eluted proteins in the 80 mM NaCl wash, a significant number of cytoplasmic proteinε remained bound to the column at salt concentrations greater than 80 mM. Thuε, DEAE chromatography aε deεcribed above waε uεed aε a preliminary εtep to remove a εignificant number of contaminating proteinε from the rPBOMP-1. Aε shown in FIG. 23A DEAE chromatography greatly reduced the number of proteins found in the cytoplasmic material (FIG. 23A, lane 2) and did not alter the reactivity of rPBOMP-1 with monoclonal antibody (FIG. 23B, Lane 2) . The amino acid sequence of rPBOMP-1 indicates that it sould be a relatively hydrophobic protein. While it waε soluble in the cytoplasmic extract without detergents, it was expected that the rPBOMP-1 would be more hydrophobic than most of the cytoplasmic proteins. The DEAE eluate containing the rPBOMP-1 was thuε chromatographed on a C-4 hydrophobic 0i.nteraction column aε described above and eluted using a gradient of increasing acetonitrile. In thiε system, more hydrophobic proteins should be bound more tightly to the column and thuε elute in higher concentrationε of acetonitrile. It waε thought that the rPBOMP-1 would bind 5more tightly than most of the contaminating proteins and elute after most of them. When the rPBOMP-1 containing fraction was chromatographed on a C-4 column, the PBOMP-1 was found to be the major peak eluted from the column (FIG. 21) and surpriεingly it eluted firεt at the loweεt concentration 0 of acetonitrile. Aε εhown in FIG. 23A, lane 3, the rPBOMP-1 peak was pure as determined by Coomasεie brilliant blue R-250 staining of 10 ug of protein after SDS-PAGE. The peak showed two bands, a major and a minor band, on Coomaεεie staining, both of which reacted with monoclonal antibody to PBOMP-1 which recognizeε the amino terminal 20 amino acidε of PBOMP-1 (FIG. 23B, lane 3). The minor band, approximately 2000 kilodaltons smaller than full size rPBOMP-1 was also present in whole cell extracts of PR13(pPX167) indicating that the smaller 5 protein is not a by-product of the purification process. After concentration by evaporatin of the solvent, the purified rPBOMP-1 was soluble in aqueous solvents without detergent. Immunization of rabbits . New Zealand white rabbits 0 were immunized with rPBOMP-1 obtained from E^ coli PR13 cells containing pPX167 either emulsified in incomplete Freunds adjuvant or bound to aluminum hydroxide. rPBOMP-1 waε bound to alum by mixing rPBOMP-1 at a concentration of 500 ug/ml in 0.85% εaline with alum at a concentration of 500 ug/ml at a 15 1:1 ratio. The mixture was shaken at 37°C for 3 hours and the alum pelleted by centrifugation. Supernatent was aεsayed by BCA protein assay (Pierce Chem. Co, Chicago, IL) for unbound protein. None was detected. Alum was resuεpended in phyεiological εaline at 500 ug/ml. rPBOMP-1 waε emulsified 0i.n i.ncomplete Freund's adj.uvant (Di.fco) i.n a 1:1 ratio. Animals were immunized intramuscularly with 50 ug of either preparation at 4 week intervals. Animals were bled at weeks 0, 6, and before each immunization. Sera were asεayed for anti-PBOMP-1 and anti-rPBOMP-1 antibody uεing ELISA and blot analyεeε. Immunization with the recombinant PBOMP-1 elicited antibody titerε againεt recombinant PBOMP-1 and PBOMP-1 obtained from H_^ influenzae (deεignated native PBOMP-1) aε determined by ELISA assays. Resultε are illustrated in Table °9. 5 ELISA-titer representε the reciprocal of the higheεt dilution of antiεerum yielding twice the background level. Aε shown in Table 9, immunization of animals with rPBOMP-1 in either alum or IFA elicited significant titers of antibody reactive with both rPBOMP-1 and native PBOMP-1 at 6 weeks post-immunization. 0 8.2 ENHANCED EXPRESSION OF PBOMP-2 IN E. COLI PBOMP-2 is also expreεεed at low levelε (less than 1% of cell protein) from plasmid pAA130. Expreεεion of PBOMP-2 is apparently under control of its native promoter. In order to improve the yield of PBOMP-2, the PBOMP-2 5jene in pAA130 was cleaved at a unique Sspl restriction εite 5 bases 5' to the PBOMP-2 initiation codon and ligated to the Smal site of pUC19 (FIG. 24) . The ligation results in a hybrid^open reading frame (ORF) containing the entire PBOMP-2 ORF (including the signal sequence) plus 18 codons from pUC19 Qmd 2 codons from the gene fusion site. The protein product of this fusion gene has a predicted molecular weight of 17,999 daltons; and is expresεed under regulation of lac promoter. Thiε plaεmid iε deεignated pPX163. 5 When plasmid pPX163 was tranεformed into E^ coli JM103 and induced with IPTG, a protein of 17,500 daltonε apparent molecular weight was expressed. In addition, a protein doublet at 15,000 daltons molecular weight was observed. All three proteins were recognized by anti-PBOMP-1 antisera on Western Blot (FIG. 25) . By Coomasεie blue εtaining of SDS- PAGE, the three formε of recombinant PBOMP-2 compriεed approximately 15% of total cell protein after lac induction (FIG. 25, Lane 3). The three formε of recombinant PBOMP-2 expreεεed from pPX163 have been isolated and N-terminal peptide analysis reveals that: 1. The 17,500 dalton apparent MW band is the predicted PUC19/PBOMP-2 hybrid protein; 2. The upper 15,000 dalton MW band starts at the first methionine residue of the PBOMP-2 signal sequence (i.e. the initiation codon of the PBOMP-2 ORF) ; and is apparently due to reinitiation of translation at this point; and 3. The lower 15,000 dalton MW band is blocked to N- terminal, analyεiε and can be labeled with 14C- palmitate. Hence, thiε band consistε of lipoprotein proceεεed PBOMP-2. 9 MAPPING OF EPITOPE DETERMINANTS OF PBOMP-1 9.1 SYNTHETIC AND CLEAVAGE PEPTIDES Most antibodies recognize antigenic determinants on proteins that are conformation-specific, formed by residueε adjacent in space but not in primary sequence. A small proportion of antibodies recognize linear portions of the protein molecule. These linear or continuous epitopes, usually consist of about 5-10 amino acids and protrude from the protein surface, forming the corner or bends of the molecule and asεuming reverse turns or loop structures. For the most part, continuous epitopes are hydrophilic and εurface acceεεible. Computer algorithmε of Hopp and Wood (1981, Proc Natl. Acad. Sci. USA 78_:3824-3828) and Chou and Faεman (1978, Ann. Rev. Biochem. 47_:251-276) were uεed to identify εeveral hydrophilic regionε (indicated by cross- hatched rectangular boxes in FIG. 26) of PBOMP-1 (amino acid sequence iε εhown in FIG. 11) that contained reverεe turnε (indicated by εolid arrows in FIG. 26) , each based on a tetrapeptide moving average. This computer search revealed hydrophilic turns predominating near the amino and carboxyl- terminal regions of PBOMP-1. 0 Five peptides, P1-P5, corresponding to the two end termini were selected for chemical synthesiε. Peptide PI correεpondε to the N-terminal reεidueε 1-20 of the mature protein (εee FIG. 26 and 27) . The remaining four peptideε, P2-P5, are a neεted εeries from the C-terminal end of PBOMP- 15 . . . 1; the longeεt of theεe is P5, a 38 ammo acid sequence containing residueε 97-134 of the mature protein. Synthetic peptideε were prepared by the εolid phaεe method of Merrifield (1964, J. Am. Chem. Soc. 85_:2149-2154) utilizing εymmetric anhydride coupling chemistry except for the amino 0 . . acids arg n e, asparagme, and glutamme, for which 1- hydroxybenotriazole esters were uεed. The synthesiε waε carried out with an Applied Bioεyεtems (Model 430A) automated peptide synthesizer. Each amino acid was double-coupled, resulting in stepwiεe yieldε of greater than 99.5% aε indicated by the ninhydrin test. Peptides were cleaved from the reεin by anhydrouε hydrogen fluoride. Proteolytic fragmentε of PBOMP-1 were obtained by digesting with a variety of proteases. EndoLys-C digestion resulted in the generation of a large 63 amino acid peptide 30 (residues 1-63) of the mature protein. Tyr 87-Glu 121 waε obtained from a digest with Staphylococcus V8 protease and Gly 18-Arg 85 was derived from an endoArg-C digestion. Both chemically synthesized peptides and proteolytic fragments were analyzed by reverse-phase HPLC on a Vydac C4 35 column using an acetonitrile/water gradient with 0.1% trifluoracetic acid (TFA) . In all cases, the purity of the synthetic peptideε exceeded 95% aε confirmed by amino acid εequencing. 5 9.2 MONOCLONAL ANTIBODIES (Mabs) Monoclonal antibodies were produced and purified as described in Section 6.2.2. Briefly, hybridoma cell lines secreting antibodies to PBOMP-1 were obtained by fusion of mouse myeloma cell line, X63. Ag8,6543 with spleen cells 0 obtained from a C57/B1 mouεe immunized with PBOMP-1. Deεired hybridomas were recloned by limiting dilution and screened for reactivity for PBOMP-1 by Western Blot. Selected hybridomas were injected into Balb/c mice for growth as ascites. 5 A total of 43 hybridoma cloneε εecreting anti-PBOMP-1 antibodieε were obtained from the fuεion experimentε. Using competitive ELISA, we demonstrated that extensive croεs- competition existε among Mabε, thus indicating that they recognized overlapping or very close antigenic determinants. abs were categorized according to their reactivity profiles into seven groups. Representative Mabs from these groupε were uεed in further experimentε, to determine and define uεeful contiguouε epitopeε for vaccine formulations. 5 9.3 EPITOPE MAPPING WITH Mabs 9.3.1 REACTIVITY OF SYNTHETIC PEPTIDES VIA DIRECT ELISA ASSAYS A direct ELISA asεay waε uεed to determine the binding ( fcf Mabε to εynthetic peptideε of PBOMP-1. Aε indicated in Table 10, only two Mabε, G204-2 and G194-3, reacted directly with the PI peptide. Theεe two Mabε apparently, recognize the εame epitope εince they competed with each other in competition binding experimentε (data not shown) . The 5 remaining Mabs did not react with any of the peptides under the conditions used to coat the microtiter plates. 5 0 5 TABLE 10 DOT BLOT ASSAY TO DETERMINE BINDING OF MAB TO SYNTHETIC PEPTIDES SEGMENTS OF PBOMP-1 PEPTIDE G SERIES MAB 196-3 204-2 216-3 219-3 212-6 214-3 194-3 190-8 56-5 130-3 PI CYS1-TYR20 ++ P5 LYS97-TYR134 P4 LYS103-TYR134 P3 LYS113-TYR134 P2 ASP120-TYR134 9.3.2 INHIBITORY ACTIVITY OF SYNTHETIC PEPTIDES The relative abilitieε of the synthetic peptides to inhibit the binding of the various Mabs to PBOMP-1, coated on the microtiter plates, are shown in Table 11. All of the 5 . . monoclonal antibodies used m the competition ELISA (see Section 6.2.2 for detailed description of the procedure) reacted with PBOMP-1. Peptide PI was 100% effective in inhibiting the binding of Mabs G204-2 and G194-3 to native PBOMP-1. Both of these Mabs also reacted directly with the ■ °P 1 peptide (see above) . Thus, the epitope(ε) recognized by Mabε G204-2 and G194-3 lies within the first twenty amino acids of PBOMP-1. The binding of Mabs G196-3, G212-6, G214-3, and G190-8 to PBOMP-1 was effectively inhibited (100%) by all of the C- rermini peptides, P2-P5 (Table 11) . These four Mabs must be recognizing an epitope(ε) contained within the P2 peptide, a 15 amino acid εequence εpanning Aεpl20-Tyrl3 . Both Mabs G187-1 and G216-3 were strongly inhibited from binding to PBOMP-1 coated on plate by the P4 and P5 peptides (Table 11) . On the other hand, both the P3 and P2 peptides were minimally effective in inhibiting the binding of theεe two Mabε to PBOMP-1. These inhibition data indicated that the 10 amino acid region (K-L-G-T-V-S-Y-G-E- E-K, residues 103-113 of the mature protein) contains an 25 %pitope recognized by Mabs G187-1 and G216-3. It is posεible that thiε 10 amino acid region may contain two sites since Mabs 216-3 and G187-1 are from two non-croεεreacting groupε. 30 35 9.3.3 REACTIVITY OF Mabε WITH PROTEOLYTICALLY CLEAVED PBMOP-1 FRAGMENTS PBOMP-1 peptide fragments generated by proteolytic cleavage were tested for reactivity with a limited number of 5 anti-PBOMP-1 Mabε by direct dot blot and Weεtern immunoblot analyεis. A large 63 amino acid peptide (residues 1-63 of the mature protein) obtained from endoLys-C digestion, reacted with Mab G204-2. This observation was not surprising since the N-terminal 20 amino acid sequence is contained 0 within this cleavage peptide. A peptide, Tyr87-Glul21, obtained from a V8 digest, reacted with Mab G216-3. This Mab has been shown (see above) to recognize a 10 amino acid sequence, reεidueε 103-113 of the mature prote n, which iε encompaεεed by thiε V8 peptide. Finally, Mab G219-3, which 15 did not bind with any of the εynthetic peptideε, waε εhown to react with a 68 amino acid peptide, Glyl8-Arg85, derived from an endoArg-C digestion. In summary, these studieε involving the binding of Mabε to εynthetic peptideε and peptides generated by proteolytic 0 digestion revealed the presence of four continuous antigenic regionε of PBOMP-1. A εchematic diagram representing the mapping of epitopes recognized by Mabs is depicted in FIG. 28. Two of the four determinants were localized at the chain termini, i.e, the N-terminal residueε 1-20 and the C-terminal 25120-134 of the mature protein. The third antigenic region, recognized by Mab G216-3 and G187-1, was localized to a small amino acid sequence (10 amino acids) spanning residues 103- 113 of the mature protein. Finally, a rather large antigenic region, recognized by Mab G219-3, is encompasεed by reεidueε 30Glyl8-Arg85. Since the latter peptide is quite large, the epitope could likely be conformation-dependent, since attempts so far to narrow it down have resulted in losε of antigenicity. 35 9.4 FUNCTIONAL ACTIVITY OF MABS The functional activity of the Mabs were tested in a bactericidal assay against H^ influenzae strain S2. The complement used waε precolloεtral calf εerum (PCCS) adεorbed 2X with washed S2 whole cellε. Bacteria were grown overnight in brain heart infuεion (BHI) broth supplemented with hemin at 10 ug/ml and NAD at 2 ug/ml (BHIXV) , then diluted 1:15 in fresh broth and incubated at 37 β C with aeration. Cells were grown to an OD at 490 nm of 0.9 (approximately 10 CFU/ml) . Bacteria were diluted 40,000 fold by a serieε of dilution in εterile phosphate-buffered saline containing 0.15 mM CaCl 2 , 0.5 mM MgCl 2 and containing 0.5% bovine serum albumin (PCMA) . The final dilution was done in PCMA with PCCS at a 4:1 ratio. Aεciteε fluid waε waεhed in PCMA by firεt diluting ten fold in the buffer then concentrating back to original volume by using a Centricon microconcentration unit with a 10,000 cut-off membrane. Monoclonal mouse antiserum was reacted with εaturated ammonium εulfate at 35% final concentration at 4°C overnight. The sample was centrifuged for 10 minutes at 4 β C in an Eppendorf table-top centrifuge. The supernatant was discarded and the pellet was resuεpended in 10 timeε the original εerum volume of PCMA. The sample was restored to original volume using a centricon unit with a 10,000 cut-off membrane. The sample waε waεhed four more timeε to remove any ammonium εulfate. Fifteen microliterε of the serum sample were placed in the first well of a sterile 96 well U-bottom microtiter plate held on ice. Two-fold serial dilutions using PCMA were done in the remaining wells. The plateε were removed from the ice and 15 ul of the cell/complement εuεpension were added to the serum in the wells. The plates were incubated at 37"C for 45 minutes. A 10 ul sample from each well was spread on a BHIXV plate was incubated overnight at 37°C. The bactericidal titer was reported as the reciprocal of the highest dilution capable of reducing the number of CFU's by 50% compared to a nonantibody containing control. The resultε of theεe bactericidal aεsays are εhown in Table 12. Of the ten anti-PBOMP Mabs tested, G102-5, G56-5, G216-3, and G204-2 were positive for bactericidal activity. An unrelated viral Mab showed no activity. Interestingly, two of the four defined antigenic determinants of PBOMP-1, N-terminal residueε 1-20 and 103-113 of the mature protein, were εeparately recognized by two bactericidal Mabs, namely G204-2 and G216-3, respectively. The bactericidal activity of these two Mabs supports the contention that these two regionε are surface-exposed on the bacteria. Surprisingly, preliminary inhibition data suggeεted that Mab G204-2 exhibited a εtronger reactivity to the εynthetic N-terminal peptide than to native PBOMP-1. This is in marked contraεt to the uεually low antigenic activity aεεociated with εynthetic peptides, when measured with antibodies to whole protein. Obviously, the free N-terminal peptide in solution was mimicking the structure of this peptide εequence in the mature PBOMP-1 molecule and may be εterically more acceεsible for antibody bonding. In addition to Mabs G204-2 and G216-3, two other Mabs, G102-5 and G56-5, have been found to be bactericidal to Haemophilus organismε. Since they did not react with εynthetic or cleavage peptideε, theεe bactericidal Mabε were thought to be recognizing discontinuous (or conformational) epitopes. TABLE 12 BACTERICIDAL ACTIVITY OF ANTI-PBOMP-1 MONOCLONAL ANTIBODIES AGAINST NONTYPABLE H. INFLUENZAE STRAIN S2 & Mabs G102 G56-5 G216-3 G146 G204-2 G125-6 G196-3 G64 G25-2 5 G219-3 G130-3 L7-4 Conditions used for the bactericidal aεεay are deεcribed in the text. Of the eleven anti-PBOMP-1 Mabε, four were poεitive for bactericidal aεεay againεt a nontypable Haemophilus S2 strain. An unrelated viral monoclonal showed no activity. 10 INDUCTION OF A BIOLOGICALLY FUNCTIONAL ANTIBOD BY A CONJUGATE OF A SYNTHETIC N- TERMINAL PEPTIDE OF PBOMP-1 10.1 CHEMICAL CHARACTERIZATION OF PEPTIDE-PROTEIN CONJUGATES The peptide εelected for εyntheεis in this study corresponded to the N-terminal 20 amino acid sequence, Cysl- Tyr20(Pl) of the mature PBOMP-1 using the procedures described in Section 9.1. As also described in Section 9.1., the peptide was purified by reverεe-phaεe HPLC. The final purity (95%) of the synthetic peptides was confirmed by direct amino acid seqencing with an Applied Biosyεtem Model 477A protein sequenator. Conjugateε were prepared using MBS (3-maleimidobenzoic acid N-hydroxysuccinimide eεter, Pierce Chemical Co., Rockport, IL) aε the cross-linking reagent (Liu et al., 1979, Biochemistry 18:690-697). The conjugation procesε proceeded in a stepwise manner. First, the epsilon amino groups of surface lysinyl reεidueε of BSA (or thyroglobulin) were N- acylated with an exceεs of MBS. The coupling reaction was carried out at room temperature in phosphate-buffered saline, pH 7.3, by mixing the protein carrier with MBS at a molar ratio of 1:100. The reaction mixture was desalted over Sephadex-G-50 to remove exceεs MBS reagents. Second, the peptide PI, bearing an amino terminal cysteinyl residue, was . . . . reduced with sodium borohydride to ensure a reactive sulfhydryl group. Third, conjugation was achieved by adding the reduced peptide to the solution containing the BSA-MBS or thyroglobulin-MBS derivative and allowing the covalent reaction to take place overnight. Dialysiε was performed to remove excess unreacted peptide prior to freeze-drying. Covalency was demonstrated by a shift in molecular weight on SDS gelε and by Weεtern blot analyεiε. The shift in molecular weight corresponded to 7 moleculeε of peptide per BSA molecule and 25 moleculeε of peptideε per thyroglobulin molecule. Moreover, the conjugate bands reacted with both anti-PBOMP-1 and anti-peptide antisera on a Western blot. To enhance immunogenicity, the N-terminal peptide was fatty*acid acylated with palmityl groups according to the method of Hopp (1984, Mol. Immuno. ].: 13-16). Esεentially, the peptide waε elongated at the ammo terminus with di- glycine spacer followed by a lysyl residue. Both the alpha- and epsilon-amino groups of N-terminal lysine were acylated with palmitic acidε by the εymmetric anhydride coupling procedure. Completeness of the fatty acid acylation was O verified by (1) a negative ninhydrin test indicating complete derivitization of lysyl amino groupε, (2) refractorineεε of the peptide toward amino acid εequencing due to blocked N- terminus, and (3) longer HPLC elution time using a reverse- phase column due to increased hydrophobicity. 10.2 PREPARATION OF POLYCLONAL ANTI-PBOMP-1 N-TERMINAL (N-l-20) PEPTIDE ANTISERUM Swisε-Webεter mice which were at leaεt 6 weekε old were obtained from Taconic Farmε (Germantown, NY) . Groups of 5 mice were pre-bled and vaccinated intramuscularly with one or 10 ug of a (N-l-20) carrier peptide conjugate, with and without complete Freund's adjuvant (CFA) . Animals were boosted at week 4 with the same doεe of vaccine uεed for priming. Mice primed with CFA were booεted with conjugate in incomplete Fueund'ε adjuvant (IFA) . Mice were bled at weekε 4 and 6. Rabbitε obtained from Taconic Farmε (Germantown, NY) were immunized with 10 ug peptide-conjugate and bled according to the εame vaccination εchedule aε deεcribed above. 10.3 PREPARATION OF MONOCLONAL ANTIBODIES TO PBOMP-1 N-TERMINAL (N-l-20) PEPTIDE Hybridoma cell lines secreting antibodies to the PBOMP-1 N-terminal peptide-carrier conjugate were obtained by the fusion of the mouse myeloma cell line, X63.Ag8.6543 with spleen cellε obtained from a C57/B1 mouse immunized with (N- 1-20)-carrier conjugate as described in Section 6.2.2. Desired hybridomas were recloned by limiting dilution (see Section 6.2.2. for a detailed description of procedure) and screened for reactivity with (N-l-20)-carrier conjugate by Western Blot. Selected hybridomas were injected in Balb/c mice for growth as ascites by standard procedures (Brodeur et al. , supra) . 10.4 IMMUNOGENICITY OF PBOMP-1 N-TERMINAL (N-l-20) PEPTIDE-CARRIER CONJUGATE The immunogenicity of a PBOMP-1 N-terminal (N-l-20) peptide conjugate was examined in one study. Mice and rabbits were immunized εubcutaneously with 10 ug of the (N- 5 1-20) peptide-carrier conjugate (or PBOMP-1) emulsified in CFA, followed 4 weeks later with a booεter injection in IFA. Animalε were bled 14 dayε after the laεt injection. Anti-(N-l-20) peptide activity of the animal antisera was determined by the ELISA method as described in Section 106.2.2., supra. Briefly, 96 well polystyrene plates (NUNC Intermed,_ Thousand Oakε, CA) were coated with PBOMP-1 or a (N-l-20)-peptide carrier conjugate, i.e., (N-l-20)-peptide- BSA, at a concentration of 1 ug/ml in PBS. Plateε were incubated overnight at 37 ° C in humidified chamber. Pre- 15vaccination εerum waε alwayε used as a negative control and a polyclonal anti-PBOMP-1 serum was uεed as a positive control. Alkaline phosphatase coupled to IgG anti-mouεe antiεera waε uεed aε εecondary antibody. The reaction waε developed with p-nitrophenylphosphate at 1 mg/ml in diethanolamine buffer. 20optical density (OD) at 410 and 690 nm was read using a Bio- Tek 310 Autoreader. Results are presented in Table 13. 25 30 35 TABLE 13 ANTIBODY RESPONSE TO N-TERMINAL PEPTIDE AS MEASURED BY ELISA MOUSE ANTI-(N-l-20) SERA 5 (N-l-20) :BSA PBOMP-1 BSA Week 0 <1:100 1:400 <1:500 Week 6 1:1,024,000 1:512,000 <_L:4000 RABBIT ANTI-(N-l-20) SERA #1 Week 0 1:400 <l:1000 <1:100 #2 Week 0 1:200 <1:1000 <1:100 #1 Week 6 1:256,000 1:32,000 <1:100 #2 Week 6 1:4,096,000 1:512,000 <1:100 MOUSE ANTI-PBOMP-1 SERA & —; Week 0 <1:1Q0 <1:100 <1:500 Week 6 1:256,000 1:512,000 <1:500 The mouεe εera are pooled sera from five animals. The ELISA results showed that (N-l-20)-carrier conjugate waε highly effective in eliciting mouεe and rabbit anti-peptide antibodieε having the ability to recognize the peptide (attached to BSA carrier) and native PBOMP-1 (see Table 13). Mice immunized with (N-l-20)-carrier conjugate, gave a high anti-peptide titer responεe of 1:1,024,000. The mouse anti-peptide antibodies alεo strongly recognized the parent PBOMP-1 in the ELISA at a 1:512,000. These anti- peptide antibodies appear to be specific to the N-termmal peptide since only negligible amounts of these antibodies reacted with BSA. The maximum rabbit anti-peptide titer waε obtained with Rabbit #2 at 1:4,096,000. Rabbit #1 alεo had a strong anti- peptide titer of 1:256,000. Both the rabbit anti-peptide sera were capable of recognizing native PBOMP-1 as seen by the 32-fold and 512-fold increase in titers compared to the preimmune titers of Rabbit #1 and Rabbit #2, respectively. Interestingly, antiprotein antibodies were raised to PBOMP-1 that reacted with the N-terminal peptide at greater than 1:256,000 ELISA titer, thus suggesting that the N-terminal epitope is immunodominant. The SDS/Western blot technique waε also used to evaluate the mouse antipeptide sera raised to the (N-l-20)- carrier conjugate. The antipeptide antibody and the mouse anti-PBOMP-1 polyclonal sera were obεerved to react with both conjugateε: peptide-BSA and peptide-thyroglobulin, and alεo with native PBOMP-1 on immunoblotε. The Weεtern blot reεults also revealed that little, if any, antibodies were made to the MBS-spacer since the antipeptide sera did not react with a control conjugate consisting of 'nonsense' peptide (of an equivalent size) linked to BSA through the MBS spacer. The free peptide (10 ug doεe) waε not immunogenic, even when adminiεtered with FCA. Contrary to other findings (Hopp, 1984, Mol. Immuno. 1.:13-16), the palmityl-peptide conjugate was also not immunogenic in mice. In order to elicit a useful antibody response, the N-terminal peptide was coupled to a protein carrier, e.g., thyroglobulin. 10.5 FUNCTIONAL ASSAYS: BACTERICIDAL ACTIVITY OF ANTI-(N-l-20) PEPTIDE ANTIBODIES The bactericidal activity (BC) of mouse anti-(N-l-20) peptide antisera against H^ influenzae S2 strain was measured using procedures deεcribed in Section 9.4, εupra. T e results are presented in Table 14. TABLE 14 BACTERICIDAL ACTIVITY OF ANTIPEPTIDE ANTIBODIES TO N-TERMINAL PEPTIDE OF PBOMP-1 BC Titer a MICE IMMUNIZED WITH Free (N-l-20) peptide Peptide-carrier Conjugate Native PBOMP-1 RABBITS IMMUNIZED WITH PEPTIDE-CARRIER CONJUGATE Rabbit 1 1/320 1/2560 Rabbit 2 1/320 1/1250 i Bactericidal activity was measured against a nontypable H. influenzae strain S2. The mouse anti-(N-l-20) peptide-conjugate antibodies were capable of killing S2 organisms at a dilution of 1/40, compared to mouse anti-PBOMP-1 polyclonal sera at 1/ 80. As a control group, mice immunized with free peptide (unconjugated, administered in CFA) did not yield an effective bactericidal antisera (Table 14) . In fact, no significant (greater than two-fold) immune responεe to the N-terminal (N-l-20) peptide waε obεerved in the control group. The bactericidal activities of the two individual rabbit anti-(N-l-20) peptide antisera are also shown in Table 14. Even though the two preimmune sera poεsesεed bacteridical activity, the post immunization sera were more efficient in mediating complement-dependent killing of H. influenzae. Rabbit #1 gave a bactericidal (BC) titer of 1/2560, representing an eight fold rise over itε preimmune level; whereas, Rabbit #2 gave a titer of 1/1280, representing a 4-fold rise. We expected rabbit #2 antisera, which had the higher anti-(N-l-20) titer, to have the higher BC activity; however, rabbit #1 antiserum exhibits better killing effect. 11. ISOLATION AND CHARACTERIZATION OF THE RECOMBINANT-DNA DERIVED PBOMP-2: PBOMP-1 FUSION PROTEIN 11.1. CONSTRUCTION OF PLASMID pPX183 FOR THE EXPRESSION OF PBOMP-2: PBOMP-1 IN E. COLI The construction of plasmid pPX183 for the expression of the PBOMP-2: PBOMP-1 fusion protein under the control of the lac promoter is described below and represented diagramatically in FIG. 30. DNA fragments including the PBOMP-1 and PBOMP-2 genes were derived from plasmids pPX167 and pPX163 respectively. The isolation of each of these plasmidε is described in Sections 8.1 and 8.2, supra. Purified DNA fragments of plasmids pPX163 and pPX167 were each separately digested wit Seal to completion. The pPX163 digest was then further treated with Hindlll to yield a partial digest. A resulting Seal-Hindlll fragment of 2.7 Kb, including the origin of replication, the lac promoter, and the PBOMP-2 gene was isolated from an agarose gel as described in Section 6.4.2., . . . supra. A 1.4 Kb Scal-Hmdlll fragment including the PBOMP-1 gene and regulatory sequences was iεolated in a similar fashion from a complete Seal-Hindlll digest of pPX167. The two isolated Seal-Hindlll fragments were then ligated together using procedures described in Section 6.4.3. . . . ^ Selection of ampicillm resistant transformantε ensured the reformation of an active 0-lactamase gene (interrupted by th Seal site) . This procedure yielded a 4.1 Kb plaεmid which was designated pPX183. This plasmid encodes a PBOMP-2: PBOMP-1 fuεion protein with the two PBOMP geneε ligated in frame aε εhown in FIG. 30. 11.2. EXPRESSION OF THE PBOMP-2: PBOMP-1 FUSION PROTEIN Plaεmid pPX183 was transformed into E. coli JM103 and the resulting strain was teεted for production of a recombinant fuεion protein under lac regulation. Coomaεεie Blue stained SDS-polyacrylamide gels of total cell extracts of IPTG-induced JM103 cells containing plaεmid pPX183 revealed a new protein of about 30,000 daltons molecular weight which comprised 1-2% of total cell protein. Western blot analyεiε showed that this recombinant fusion protein reacted with monoclonal antibodies specific for PBOMP-1 and also with those specific for PBOMP-2. Thus the fusion protein product encoded by plaεmid pPX183 carrieε epitopeε of both PBOMP-1 and PBOMP-2. 11.3. CONSTRUCTION OF PLASMID pPX199 EXPRESSING PBOMP-2: PBOMP-1 FUSION PROTEIN Although plaεmid pPX183 encodeε a hybrid PBOMP-1/ PBOMP-2 protein, i.e. a fuεion protein, the protein expreεsed by this plasmid contains additional amino acids encoded by the pUC19 vector. Specifically, the nucleotide sequence of pPX183 contains 18 additional codons at the 5' end from pPX163 and 13 additional codonε at the fusion junction - 31 codons derived from the pUC19 vector. A new fusion gene lacking most of this excesε information waε constructed as followε. Firεt, a derivative of pAA130 containing the PBOMP-2 gene waε digested with Sspl, which cleaves 5 nucleotides 5' to the initiation codon of PBOMP-2, and then with Hindlll which cleaves the PBOMP-2 gene at codon 148, i.e., 6 codons from the 3' end of the gene. Sequence analyεiε showed that this fragment could be ligated into plaεmid pUC8 and digeεted with Smal and Hindlll to generate a recombinant PBOMP-2 gene with seven codonε fuεed to the 5' end of the "native" gene and six amino acids missing at the 3' end. A plasmid was produced in this manner and designated pPX195 (see FIG. 31) . Next, plasmid pPX167 containing the PBOMP-1 gene (see Section 8.1, supra) was modified in the following manner: the BamHl site at th 3' end of the gene was removed by partial digestion with BamHl and religated by treatment with E. coli DNA polymerase I (Klenow fragment) . The resulting plasmid designated pX188 was verified by restriction analysis and production of rPBOMP-1 (see FIG. 31) . The 5' end of the . . rPBOMP-1 gene of pPX188 was modified by removal of the Hindlll - BamHl fragment from the pUC19 polylinker region and insertion of a synthetic oligonucleotide having Hindlll and BamHl εticky endε and the εix 3' codons of PBOMP-2. . This construction was verified by DNA sequence analyεiε and the plaεmid deεignated pPX198. Plaεmids pPX195 and pPX198 were digested with Hindlll and Seal and the fragments carrying the PBOMP-2 and PBOMP-1 genes respectively were isolated and ligated together. The reεulting plaεmid waε designated pX199 (FIG. 31) . Plasmid pX199 expresεeε a PBOMP-2: PBOMP-1 fusion gene with seven additional codons (from pUC8) at the 5' end and two at the fusion junction. The fusion protein has an apparent molecular weight of about 32000 daltons by SDS-PAGE (see FIG. 32) and is recognized on Western blots by polyclonal antisera against PBOMP-1, PBOMP-2, PBOMP-2 Mab 61-1 (See Section 6.2.2) and a variety of PBOMP-1 Mabs. 11.4. EXPRESSION OF SIGNAL-LESS PBOMP-2: PBOMP-1 FUSION PROTEIN PBOMP-2: PBOMP-1 fusion protein gene of pPX199 encodes the PBOMP-2 signal peptide, and the resulting fusion protein is modified as a lipoprotein and tightly associated with the outer membrane of E. coli cells expressing it. This fusion protein is difficult to separate from the membrane and poorly soluble in aqueous solventε. Conεtruction of a plaεmid which expresseε a εignal-leεε PBOMP-2: PBOMP-1 fuεion protein iε illustrated in FIG. 33. First, a signal-leεε form of the PBOMP-2 gene waε constructed in the following manner: The 660 bp EcoRI/Sall restriction fragment of plasmid pPX163 encoding the PBOMP-2 protein was isolated and cleaved at the unique Hphl site at nucleotide 91 of the gene and the 545 bp Hphl/EcoRI fragment encoding the carboxyterminal 125 amino acids of PBOMP-2 was isolated. 10 . . . . The following synthetic oligonucleotide linker was then constructed to reεtore a modified 5' end of the PBOMP-2 gene: Xbal SD box Ncol EcoRv Hphl • j5 CTAGAATAAAGGAAACAAACCATGGCAAATACTGATATCTTCAGCGGTGATGTTTATA TTATTTCCTTTGTTTGGTACCGTTTATGACTATAGAAGTCGCCACTACAAATATC Met The linker waε deεigned to create a modified PBOMP-2 gene with the following features: 0 (l) Absence of the signal peptide encoding sequences, (2) Restoration of the entire mature PBOMP-2 sequence, except the N-terminal cysteine residue (replaced by methionine) , (3) A new and unique Ncol restriction site at the 5 initiation codon, (4) A wobble base change to generate a new and unique EcoRV restriction site at nucleotide 12 of the coding region, (5) A new translation initiation site (SD box) , and 0 (6) An upstream in frame stop codon (TAA) to prevent lac translational readthrough. The linker was mixed at a 1:1:1 molar ratio with the Hphl/EcoRI fragment from pPX163 and pUC19 DNA cleaved with Xbal and EcoRI and ligated. The ligation mixture waε 5 tranεformed into E. coli DH5 lacl q and AmpR colonieε were screened for PBOMP-2 production with Mab 61-1. Several positive colonies were found and the construction waε verified by restriction and DNA sequence analysis. One of the isolateε was designated pPX510. Plasmid pPX510 encodes a 14000 apparent dalton molecular weight protein by SDS-PAGE which was recognized by both polyclonal anti-PBOMP-2 sera and anti-PBOMP-2 Mab 61-1. Expression of the signal-lesε recombinant PBOMP-2 waε under lac regulation, and fully induced cellε expreεsed the — construction aε ~5%. of total cell protein. A signal-lesε fuεion gene was then constructed from pPX510 and pPX198 in a manner analogous to the construction of pPX199 (see Section 11.3, supra) . Briefly, the two plasmids were digested with Hindlll and Seal and the PBOMP-1 ~ and PBOMP-2 fragments were ligated together. The resulting plasmid was transformed into E^ coli DH 5α (BRL, Gaithersburg, MD) and was designated pPX512. The signal-less fusion protein expresεed by pPX512 had an apparent molecular weight of 30000 daltonε by SDS-PAGE and was recognized by polyclonal antisera against both PBOMP-1 and PBOMP-2. The fusion protein was also recognized by PBOMP-2 MAb 61-1 and all PBOMP-1 Mabs tested by Western blot analysis. 12. EFFICACY OF FUSION PROTEIN SUBUNIT VACCINES 12.1. PREPARATION OF ANTI-PBOMP-2: PBOMP-1 ANTIBODIES Substantially pure PBOMP-2: PBOMP-1 was isolated from an SDS-PAGE gel of cell extracts of E^ coli JM103 containing pPX183 (see Section 11.1, supra) . The position of the fusio protein band on the preparative SDS-PAGE gel was deduced by Coomassie Blue staining of stripε cut from the edges of the gel. Slices containing the fusion protein were soaked in Ge Elution Buffer (0.2 M NaCl, 0.05 M Tris, pH 8.0, 0.1% SDS an 0.1 mM EDTA) overnight at room temperature with gentle agitation. Extracted protein was eεtimated by a Lowry Protein aεεay with BSA aε εtandard. Two female New Zealand White rabbitε were used in an initial immunogenicity study. Prebleeds (at week 0) were obtained prior to vaccination aε a εource of preimmune εera. Fifty microgramε of εubεtantially pure PBOMP-2: PBOMP-1 fuεion protein (adjuεted to a volume of 100 μl) were mixed with an equal volume of CFA and injected intramuscularly into the rabbits at multiple sites. Blood sampleε were collected 3 weekε later and the rabbits were reimmunized at week 4 with . . . . 50 μg of fusion protein emulsified in IFA. Blood εampleε were then collected at week 7 and εera prepared for ELISA and bactericidal aεεayε. Reεultε of the ELISA and bactericidal activity aεεayε are preεented in Table 15. TABLE 15 RESPONSE OF RABBIT POLYCLONAL ANTI-PBOMP-2: PBOMP-1 ANTISERA Increase in ELISA titer Immunization with the recombinant fuεion protein -- PBOMP-2: PBOMP-1 elicited antibody reεponεes to both PBOMP-1 and PBOMP-2 as determined in ELISA asεayε and theεe antiεera had bactericidal activity against the unencapsulated H. influenzae strain S2 (Table 15) . These resultε show that PBOMP-2: PBOMP-1 fusion protein is highly effective in provoking an antibody response against both PBOMP-1 and PBOMP-2 and that this immunogenic reεponεe iε biologically functional in a bactericidal aεεay. In another εeries of experiments, adult chinchillaε 0 were uεed as a test system to demonstrate further the immunogenicity and efficacy of the PBOMP-1: PBOMP-2 fusion protein as a subunit vaccine formulation. The chinchilla serves as a model animal syεtem becauεe this animal when infected with H. influenzae develops an otitiε media much 15 like that seen mr humans. (See, Barenkamp et al., 1986, Infect. Immun. 5_2:572-78) . As indicated in Section 11.3, the PBOMP-2: PBOMP-1 fuεion protein expreεsed by E . coli JM103 containing pPX199 is modified as a lipoprotein and is tightly asεociated with 0 the outer membrane of the E. coli hoεt cells. Substantially pure PBOMP-2: PBOMP-1 was isolated from E. coli JM103 containing pPX199 by the following method entailing differential detergent extraction of the inner and outer membrane proteinε of the E. coli hoεt cellε. Briefly, the 25. ~ . inner membrane proteins of E. coli were extracted using 1% εarcoεyl in 10 mM Triε-HCl pH 8, 5 mM EDTA leaving an unεoluble outer membrane-cell wall complex. * subfraction enriched in PBOMP-2: PBOMP-l fusion protein was obtained by differential detergent extraction of 30 other outer membrane-cell wall components. The outer membrane proteins were extracted from the sarcosyl insoluble outer membrane cell wall components by εequential extraction with 1% Zwittergent 3-12™, 1% Zwittergent 3-14 in 10 mM Triε-HCl pH 8, 5 mM EDTA, 1% deoxycholate at 60 β C. The 35 PBOMP-2: PBOMP-1 fusion protein enriched inεoluble material waε obtained following centrifugation. The PBOMP-2: PBOMP-1 fuεion protein waε εolubilized by extraction of the PBOMP-2: BOMP-1 enriched fraction with 1% Zwittergent 3-14, 50 mM Tris-HCl, 5 mM EDTA, pH 8.0, 6 M urea at 80°C for 45 minutes. After solubilization, the urea was removed from the extracted fusion protein by dialysiε against the same buffer without urea at room temperature overnight. The apparent purity of the solubilized fusion protein was in the range of 75-80%. Approximately 25 mg of protein . was solubilized from ~-10-20 g wet weight fusion protein. The major protein was identified as the intact fusion protein. The major lower MW protein also was derived from the fusion protein since it contained the N-terminal region of the PBOMP-1 protein fused to the PBOMP-2 protein, as determined . . . by epitope analysiε. The C-termmal 2/3 of the PBOMP-1 waε lost from this protein. This may have been the result of proteolytic degradation, poεεibly during induction, or to premature termination of tranεlation. The purity of thiε preparation iε εhown in FIG. 32. Five adult chmchillaε were uεed m one animal experiment. Preebleeds (at day 0) obtained via cardiac puncture prior to vaccination were used as a source of pre¬ immune sera. Twenty five micrograms of substantially pure PBOMP-2: PBOMP-1 fusion protein, obtained from E. coli cells containing plasmid pPX199 as deεcribed above, abεorbed onto alum waε injected intramuεcularly into the chinchillaε. Blood samples were collected thirty days later and the animals were re-immunized at day 30 with 25 μg of purified fusion protein on alum. Blood sampleε were again collected via cardiac puncture at day 60. Sera waε prepared for ELISA and bactericidal aεεayε. ELISA aεεayε were performed aε described above using as antigen either substantially purified PBOMP-1 (Anti-PBOMP-1) or a recombinant form of PBOMP-2 (anti-PBOMP-2) . The bactericidal activity of the -114- serum sampleε waε teεted aε deεcribed above in Section 7.1 using H. influenzae P860295, a clinical non-typable strain obtained from Dr. Charles Blueεtone, Children's Hospital of Pittsburgh, PA. Results of the ELISA and bactericidal activity assays are presented in Table 16. TABLE 16 RESPONSE OF CHINCHILLA ANTI-PBOMP-2: PBOMP-1 ANTISERA See text for details of immunizations. Bleeds were taken at day 0 (Pre); at day 30 (Poεt 1°) and at day 60 (Poεt 2°). Bactericidal activity againεt H. influenzae P860295. Immunization with 25 μg of the fusion protein PBOMP-2: PBOMP-1 elicited one of the highest titers of BC activity ever observed in our laboratory against an outer membrane of Haemophilus. Together with the high level of activity observed in the ELISA asεays against both PBOMP-1 and PBOMP- 2, the results in Table 16 show that the PBOMP-2: PBOMP-1 fusion protein is εurprisingly highly effective in provoking an antibody responεe against both PBOMP-1 and PBOMP-2 in chinchillas. These resultε further demonstrate that the fusion protein elicits an immune response which is biologically active in a bactericidal assay against a clinical strain of H. influenzae. 12.2. PBOMP-2: PBOMP-1 FUSION PROTEIN IN COMBINATION WITH E PROTEIN INDUCES ANTI-HAEMOPHILUS ANTIBODIES 5 The E protein is another outer membrane protein of H. influenzae having a molecular weight of about 28,000 daltons. E protein existε aε a lipoprotein in aεsociation with other outer membrane proteins of H. influenzae. 10 E protein from H. influenzae was obtained in substantially pure form as deεcribed in U.S. Patent application Serial No. 07/320,971 filed March 9, 1989. Briefly, cell envelopeε were iεolated from Hib strain Eagan cells grown on either brain heart infusion medium containing 15 io μg/ml hemin and 1 μg/ml NAD (BHI/XV) or mMIC (modification of Herriortt et al., 1970 J. Bacteriol., 101: 513-516 media) . Cellε were harveεted from liquid cultureε by centrifugation at 10,000 x g, 4 β C for 10 minutes. The cell pellet was weighed and resuεpended in 10 mM HEPES-NaOH (pH 207.4), 1 mM EDTA, with a volume equal to five times the wet weight of the cells. The cellε were disrupted using a Gaulin homogenizer. The diεrupted cell εuεpenεion waε centrifuged at 10,000 x g for 5 minuteε at 4°C to remove unbroken cells and large debris. The supernatant fraction was saved and 25NaCl added to 0.5 M. Cell membranes were pelleted by centrifugation at 100,000 x g for about 1 h at 4 β C. An outer membrane-cell wall complex was obtained by removing the inner membranes from the total membrane fraction by repeated extraction of the total membrane fraction with 2% 30Triton X-100 in 10 mM HEPES-NaOH, 1 mM MgCl 2 , pH.7.4. The inεoluble reεidue containing the outer membrane-cell wall complex waε pelleted by centrifugation at 350,000 x g for 30 minuteε at 4°C. Thiέ complex was then resuεpended in 50 mM Tris-HCl, 5 mM Na 2 EDTA, pH 8 and stored 4°C. 35 Contaminating proteins were soluilized from H. influenzae cell envelopeε by differential detergent extraction as follows. Cell envelopes prepared as described above were sequentially extracted twice with 1% εarcoεyl in 50 mM Tris-HCl, 5 mM Na.EDTA, pH 8. Thiε extraction waε repeated three timeε. The εolubilized protein E-containing fractionε were pooled and pasεed through a DEAE column equilibrated with 50 mM Triε-HCl, 5 mM Na.EDTA, pH 8. The E protein was not retained under these conditions but the major protein contaminants were retained. The fall-through . . . fractions containing E protein were then passed over a hydroxylapatite column which had been equilibrated with 50mM Tris-HCl, pH 8. The E protein was retained under these conditions. The hydroxylapatite column with the adsorbed E protein was then washed with one column volume of 50 mM . Tris-HCl, pH 8. The E protein waε eluted from the hydroxylapatite with 1% Zwittergent 3-14™ in 0.3 M dibaεic phosphate, pH 8. Fractions containing protein E were pooled, concentrated by diafiltration, and precipitated with ethanol. The precipitated E protein was then solubilized again by . differential detergent extraction. The precipitate was first extracted with 1% octylglucoside in 50 mM Tris-HCl, pH 8 and the insoluble E protein remained in the precipitate. The protein E was then solubilized with 1% Zwittergent 3-14™ in 50 mM Tris-HCl, 5 mM Na.EDTA, pH 8. E Protein prepared as desribed above is substantially pure and essentially free of endotoxin. Twenty-five micrograms of the εubεtantially pure E protein waε mixed with 25 μg of substantially pure PBOMP-2: PBOMP-1 fusion protein obtained as deεcribed in Section 12.1, supra, diluted into 0.85% saline and absorbed with alum. Adult chinchillas were injected intramuscularly with a aliquot of the mixture containing 25 μg of PBOMP-2: PBOMP-1 fusion protein and 25 μg of E protein. Blood samples were collected at day 0 immediately prior to the first immunization, at day 30 prior to the second immunization, and at day 60. ELISA asεayε were performed aε deεcribed above uεing PBOMP-2: PBOMP-1 or E protein aε antigen. Bactericidal aεεayε were performed aε above uεing H. influenzae P860295 aε the test strain. Resultε of the ELISA and bactericidal 5 aεεayε are tabulated in Table 17. TABLE 17 RESPONSE OF CHINCHILLA 0 ANTI-PBOMP-2: PBOMP-1 ANTI-E ANTISERA ELISA Titer See text for detailε of immunizationε. 0 BBlleeeeddss wweerree ttaakkeenn aatt ddaayy 0 (Pre); at day 30 (Post 1°) and at day 60 (Post 2°). Bactericidal activity against H. influenzae P860295. 5 Resultε of the ELISA studies show that the response to E protein was very high, achieving levels comparable to that of the PBOMP-2 portion of the fusion protein. No blocking effectε were observed between E protein and fusion protein. Biologic activity of the antisera was measured using the BC Q assay. Twenty-five μg of E elicited one of the highest BC titers our laboratory has ever seen with antibodies against OMPs of Haemophiluε. The BC titer was greater than that of the chinchillas which received only fuεion protein. While it 5 is not possible to state that the BC activity of the mixed vaccine antisera exhibited a synergistic or additive effect, the activity of the mixture iε aε effective or better than that of the fusion alone. These resultε strongly support th use of a mixture of the fusion protein in combination with the purified E protein as a vaccine against Haemophilus. 13. DEPOSIT OF MICROORGANISMS The following E^ coli εtrainε carrying the liεted plaεmids have been deposited with the Agricultural Reεearch Culture Collection (NRRL) , Peoria, IL. and have been aεεigne the following accession numbers: E. Coli Strain Plasmid Accession Number pAA152 B-18155 pAA130 B-18154 pGD103 B-18153 pPX163 B-18285 pPX167 B-18286 pPX168 B-18287 pPX183 B-18377 pPX199 B-18530 pPX512 B-18531 The present invention is not to be limited in scope b the microorganisms deposited since the deposited embodiment is intended as a single illustration of one aspect of the invention and the many microorganisms which are functionall equivalent are within the scope of the present invention. Indeed, various modifications of the invention in addition those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. It is also to be understood that all base pair sizes given for nucleotides are approximate and are used for purposes of description. Intarnatlonal Application No: PCT/ MICROORGANISMS Optional Shaat In connection with lha mlerβσrganlam ralarrad to on papa ..L J.Ju . , Hna—V.— - ol Ih* daacrtptlon * A. IDINTIFICATION OF DII»0»IT » Fuithar depoalle ar* Identified on an additional ahaal [24« Name si depoaltery Inatltullan • Agricultural Research Culture Collection Arfdr*aι ol dβpβttlβfγ intlHultoπ (Including ^••Ut ••*• a d country) * 1815 North University Street Peoria , IL 61604 U. S .A. Oa of rfβpββll * Aeetatlon Number * June 17 , 1988 B-18377 m. ADDITIONAL INDICATION* < (laava blank II not applleabla). Thla Inlar atlon la eonllnuad on a aapaiata atlaehad ahaal Q C. Of BIQNATCD *TAT(* FOR WHICH INDICATION* ARI MAOI • (If lha Indication, ai* net lor all daalonatad Stalaa) D. SIP AR ATC FURNISHINO OF INDICATIONS • (laava blank II not applleabla) Tha Indlcallsna lltlad balow will ba aubmllted Io lha Intarnallonat ■ureau latar • (Spaelly Ih* peneral naluia •• lha Indlcallona e.o-, • ' Aceaialoπ Number ot Depoalt ") I. QH Thla ahaal «aa received with lha I ainallanal applleallon whan fllad (Io ba ehecked by lha rece ntt Omee) (AulhDfliad Officer) PI Tha dala ol receipt (liorn lha applicant) by lha International Buiaau ■• (Authorlted OIDear) Form PCT.RO/IK (Januaiy HID gricultural Research Address of Depository: 1815 North University Street Peoria, IL 61604 U.S.A. Date of Deposit Accession Number December 24, 1986 B-18153 December 24, 1986 B-18154 December 24, 1986 B-18155 December 9, 1987 B-18285 December 9, 1987 B-18286 December 9, 1987 B-18287 August 10, 1989 B-18530 August 10, 1989 B-18531

Claims: WHAT IS CLAIMED IS; 1. A substantially pure antigenic peptide or protein having an epitope of Haemophilus influenzae, in whi the peptide or protein is an approximately 30000 dalton molecular weight fusion protein PBOMP-1: POMPB-2 or any antigenic portion thereof containing a portion of both PBOMP-1 and PBOMP-2. 2. The peptide or protein according to claim 1 havin an amino acid sequence comprising (a) the amino acid sequen substantially as depicted in FIG. 11 from amino acid residu 20-153 or any antigenic portion thereof and (b) the amino acid sequence substantially as depicted in FIG. 15 from ami acid residue 19-154 or any antigenic portion thereof. 3. The peptide or protein according to claim 1 havin an amino acid sequence comprising (a) the amino acid sequen substantially as depicted in FIG. 11 from amino acid residu 20-39 or any antigenic portion thereof and (b) the amino ac sequence substantially as depicted in FIG. 15 from amino ac residue 19-154 or any antigenic portion thereof. 4. A substantially pure antigenic peptide or protein having an epitope of Haemophilus influenzae, in which the peptide or protein is an approximately 30000 dalton molecul weight fusion protein PBOMP-2: PBOMP-1 or any antigenic portion thereof containing a portion of both PBOMP-2 and PBOMP-1. 5. The peptide or protein according to claim 4 havin an amino acid sequence comprising (a) the amino acid sequen substantially as depicted in FIG. 15 from amino acid residu 20-153 or any antigenic portion thereof and (b) the amino acid sequence substantially as depicted in FIG. 11 from ami acid residue 19-154 or any antigenic portion thereof. 6. The peptide or protein according to claim 4 havi an amino acid sequence comprising (a) the amino acid seque substantially as depicted in FIG. 15 from amino acid resid 20-39 or any antigenic portion thereof and (b) the amino a sequence substantially as depicted in FIG. 11 from amino a residue 19-154 or any antigenic portion thereof. 7. A peptide or protein having one or more antigeni determinants of an Haemophilus influenzae outer membrane protein produced by an Escherichia coli bacterium deposite with the NRRL and assigned accession No. B-18377 and mutan recombinants and genetically engineered derivatives thereo 8. A peptide or protein having one or more antigeni determinants of an Haemophilus influenzae outer membrane protein produced by an Escherichia coli bacterium deposite with the NRRL and assigned accession No. B-18530 and mutan recombinants and genetically engineered derivatives thereo 9. A peptide or protein having one or more antigeni determinants of an Haemophilus influenzae outer membrane protein produced by an Escherichia coli bacterium deposite with the NRRL and assigned accession No. B-18531 and mutan recombinants and genetically engineered derivatives thereo 10. A subunit vaccine formulation in which the immunogen comprises an effective amount of the peptide or protein of claim 1, 2, 3, 4, 5, 6, 7, 8 or 9 mixed with a pharmaceutical carrier. 11. A recombinant vector comprising a DNA sequence coding for an antigenic determinant of an Haemophilus influenzae fusion protein having (a) an amino acid sequence substantially as depicted in FIG. 11 or any portion thereof comprising at least seven contiguous amino acids, said amin acid sequence containing one or more antigenic determinants of an Haemophilus influenzae PBOMP-1 protein and (b) an ami acid sequence substantially as depicted in FIG. 15 or any portion thereof comprising at least seven contiguous amino acids, said amino acid sequence containing one or more antigenic determinants of an Haemophilus influenzae PBOMP-2 protein. 12. The recombinant vector according to claim 11 in which the vector is pPX183 or a mutant, recombinant or genetically engineered derivative thereof. 13. The recombinant vector according to claim 11, in which the vector is pPX199 or a mutant, recombinant or genetically engineered derivative thereof. 14. The recombinant vector according to claim 11, in which the vector is pPX512 or a mutant, recombinant or genetically engineered derivative thereof. 15. A bacterium containing the recombinant vector of claim 11, comprising an Escherichia coli bacterium deposite with the NRRL and assigned the accession No. B-18377 and mutants, recombinants and genetically engineered derivative thereof. 16. A bacterium containing the recombinant vector of claim 11, comprising an Escherichia coli bacterium deposite with the NRRL and assigned the accession No. B-18530 and mutants, recombinants and genetically engineered derivative thereof. 17. A bacterium containing the recombinant vector of claim 11, , comprising an Escherichia coli bacterium deposited with the NRRL and assigned the accession No. B- 18531 and mutants, recombinants and genetically engineered derivatives thereof. 18. A method for protection of a human against infection by typable or non-typable Haemophilus influenzae, comprising: administering to a human an effective amount of the subunit vaccine according to claim 10. 19. A method for protection of a human against typab or non-typable Haemophilus influenzae comprising: administering to a human an effective amount of a polyclona or monoclonal antibody against an outer membrane protein of Haemophilus influenzae, in which the antibody is specific f an antigenic determinant of a Haemophilus influenzae PBOMP- PBOMP-2 fusion protein. 20. A method for protection of a human against typab or non-typable Haemophilus influenzae, comprising: administering to a human an effective amount of a polyclona or monoclonal antibody against an outer membrane protein of Haemophilus influenzae, in which antibody is specific for a antigenic determinant of a Haemophilus influenzae PBOMP-2: PBOMP-1 fusion protein. 21. A method for the detection of Haemophilus influenzae in a sample of tissue or body fluids, comprising using the peptide or protein according to claim 1, 2, 3, 4, 5, 6, 7, 8 or 9 as an antigen in an assay system selected from the group consisting of an enzyme-linked imunosorbent assay, a radioimmunoassay, a gel diffusion precipitin reaction assay, an immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay a an immunoelectrophoresis assay; and detecting any reaction with the antigen, in which any reaction indicates the presence of Haemophilus influenzae in the sample. 22. A method for the detection of Haemophilus influenzae in a sample of tissue or body fluids, comprising: using a nucleotide sequence of a gene encoding a peptide or protein related to a fusion protein of Haemophilus influenza which comprises (a) an amino acid sequence as depicted in FIG. 11 or a fragment thereof and (b) an amino acid sequence as depicted in FIG. 15 or a fragment thereof as a probe in a nucleotide acid hybridization assay and detecting any reaction with the probe, in which any reaction indicates the presence of Haemophilus influenzae in the sample. 23. A peptide or protein having an amino acid sequenc comprising the amino acid sequence as depicted in FIG. 11 from amino acid residue 20 to 39 or any antigenic portion thereof. 24. A peptide or protein having an amino acid sequenc comprising the amino acid sequence as depicted in FIG. 15 from amino acid residue 20 to 39 or any antigenic portion thereof.

Inventors: ANILIONIS ALGIS, DEICH ROBERT A, GREEN BRUCE A, SEID ROBERT C JR, ZLOTNICK GARY W, ANILIONIS, ALGIS, DEICH, ROBERT, A., GREEN, BRUCE, A., SEID, ROBERT, C., JR., ZLOTNICK, GARY, W.
Applicants: PRAXIS BIOLOG INC, PRAXIS BIOLOGICS, INC.
Application year: 1989
Main classification: A61K35
IPC codes: A61K38, A61K39, C07K1, C07K7, C07K14, C07K16, C07K19, C12N15, C12P21, C12Q1, C12R1, G01N33