CA1341574C - Plasmid for production of membrane protein, bacterium containing same, monoclonal antibody therefore, and method for the idenification of haemophilus influenzae - Google Patents

Abstract

A plasmid which contains a genetic code for an immunogenic portion which is conserved in many strains of nontypable Haemophilus influenzae and the bacterium containing this plasmid is disclosed. The immunogenic portion is preferably an epitope on an outer membrane protein of H. influenzae. A monoclonal antibody to the immunogenic portion and the hybridoma which will produce the monoclonal antibody is also included. The invention further includes a DNA probe constructed to correspond to the nucleic acids which code for the immunogenic portion. This probe may be labelled with a radioactive marker and may be used as a diagnostic tool to assay various clinical samples for the presence of H. influenzae.

Description

.. ... . . . _ I . . _ ..,_, a , . . _ . . .,..-.,.
PLASMID FOR PRODUCTION OF MEMBRANE
PROTEIN, BACTERIUM CONTAI NI NG SAME, MONOCLONAL ANTIBODY THEREFORE, AND
METHOD FOR THE IDENTIFICATION OF
HAEMOPHILUS INFLUENZAE
BACKGROUND OF THE INVENTION

Haemophilus influenzae type b has long been recognized as a frequent pathogen, particularly in infants and children, but only recently has nontypable H.
influenzae been recognized as an important pathogen. it is now well established that nontypable H. influenzae causes pneumonia, bacteremia, meningitis, postpartum sepsis, and acute febrile tracheobronchitis in adults. In addition, nontypable H. influenzae causes neonatal sepsis and is a frequent etiologic agent in acute otitis media in infants and children. Therefore, the importance of discovering a method to assay a clinical sample such as sputum, cerebral spinal fluid, blood and others for the presence of H. influenzae is clear.
The observation that nontypable Hs,influenzae causes serious infections in adults and children has stimulated interest in study of the pathogenesis and potential virulence factors associated with this bacterium. The ribitol capsule of H. influenzae type b is a virulence factor for the organism, and antibody to capsule protects the host by means of bactericidal and/or opsonizing actions. These observations have generated ,~... ~

';41 5 7 4 much investigation on the role of the oapaular polysaocharide in infection with H. influenzae type b and protection from these infections. However, nontypable H. influenzae lacks a polysaccharide capsule, and, aimilar to the outer membranes of other gram-negative bacteria, the outer membrane of H. influenzae is composed of outer membrane proteins (ONPs) and lipopoly-saccharide (LPS). Therefore, atudies of the relationship between virulence of nontypable H. influenzae and surface antigens focus on OMPs and LPS.

Analysis of OMPs of nontypable H. influenzae has shown that there are marked differences in OMP composition among strqins.
See e.g. Murphy et al, "A Subtyping System For Nontypable Haemophilus influenzae Based on Outer-membrane Proteins," J.
Infect. Dis, 1983, ~147:838-46f Barenkamp et al, "Outer Hembrane .
Protein and Biotype Analysia of Pathogenia NontyQable Haemophilus influenzae," Infect. Immun, 1982, 36s535-40= Loeb et al, "Outer Membrane Protein Composition in Disease Isolates of Haemophilus influenzae, Pathogenio and Epidemiological I,aoplications," Infect.
Immun, 198o, 30:709-17=
A subtyping syatem for nontypable H. influenzae based on the major OMPs has previously been developed. If a surfaoe exposed antigen (immunogen) which is conserved in all strains could be found, it would be an important tool in developing a method of identifying H. influenzae in clinioal apec rmena aa well as a vaccine against H. influenzae. It is therefore an objeot of this ci ~3 41514 invention to find a surface exposed antigen in both typable and nontypable H. influenzae which is conserved in all strains including typable H. influenzae such as type b which ia known to cause bacterial meningitis. It ia a further object of this invention to develop a means for predictably identifying such conserved surface exposed antigen. It is a further oboeot to develop a monoclonal antibody againat such a surface exposed antigen. A further objeot of the invention ia to develop a means for producing large quantities of such antigen and another object is to isolate and introduce the genetic sequenoe for such antigen into a novel plasmid and to cause expression -of such sequenoe in a bacteria such as E. coli to produce such antigen.

Another object of the invention is to construct a nucleic acid probe through the combination of the aurfaoe exposed antigen in tloth typable and nontypable H. influenzae which is oonserved in all strains and the monoolonal antibody which would be a diagnostic test for detecting H. influenzae.

BRIEF DESCRIPTION OF THE INYENTION

In accordance with the present invention there ie provided a plasmid containing a genetic code for an immunolkenio portion of a nontypable Haemophilus influenzae, whioh immunogenio portion is conserved in many strains of nontypable E. i=nfluenzae. The invention further includes a bacterium which contains said plasmid and will cause expression of said genetio sequence and ci 13 4 157'4 includes a monoclonal antibody to the immunogenic portion and further includes the hybridoma which will produce aaid monoclonal antibody.

The immunogenic portion may be and preferably is an epitope on an outer membrane protein of the H. influenzae and specifically may be and preferably is a 16,600-daltox outer membrane protein. The DNA sequence for the gene expreasing this 16,600-dalton outer membrane protein is believed to begin at nucleotide 125 and continues until nucleotide 526 of the cloned insert. The immunogenic portion may be produced in its pure state or as a part of a longer chain protein.

A diagnostic test for detecting I. influenzae in clinical samples comprises-a nucleic acid probe synthesized to correspond to the nucleic acids which code for the immunogenic portion conserved in many strains of nontypable I. influenzae. This probe may be labelled, for example, with a radioactive or any other suitable diagnostically recognizable marker.

DETAILED DESCRIPTION OF THE INVENTION

"Nontypable Haemophilus influenzae" as used herein, means H.
influenzae which lacks a polysaocharide capsule and which has an outer membrance comprising outer membrane proteins (OMPs) and also comprises lipopolysaccharides (LPS).

"immunogenic portion" means that portion which will result in an immunological antibody response in a host organism. Such portion may be considered an antigen.

- -~M

"Epitope" means that limited immunogenic portion which results in a specific immunological response.

In the drawings which form a part of this specification Figure 1 shows a Western blot assay with three lanes from the same gel;
Figure 2 is a Western blot assay depicting another experi-ment to assess whether the epitope recognized by antibody 7F3 is on the protein or LPS;
Figure 3 shows whole cell preparations of nontypable H.
influenzae strain 3524 assayed on the same gel and transferred to nitrocellulose paper;
Figure 4 shows the construction of the Haemophilus influenzae 1479 genomic library;
Figure 5 shows the methods used in screening the library.
Figure 6 shows the restriction map gene coding for the 16,600 dalton outer membrane protein.

~.

In accordance with the present invention, a mouse monoolonal antibody that recognizes an epitope on a 16,600-dalton outer membrane protein P6 was developed to nontypable Haemophilus influenzae . This epitope was present on all 115 isolates of H.
influenzae tested, including typable and nontypable strains.

Screening of 89 strains of other bacteria demonstrated that this epitope is a highly specific marker for H. influenzae because the epitope was absent in virtually all other baoterial species tested. Western blot assays were performed with two normal human serum samples and aonvalescent-phase serum from an adult with bacteremia due to nontypable H. influenzae. Antibody to the 16,600-dalton outer membrane protein was present in all three human serum samples.

Prototype strains of nontypable H. influenzae representing the eight OMP subtypes were obtained from our own collection.
See Murphy et al, supra. Strain 3524 was isolated from the sputum of a patient with chronic bronchitis at the Erie County Medical Center (Buffalo, NY). Dr. S. Berk (V.A. Medical Center, Mountain Home, 76enn) provided 14 strains of nontypable H.
infiuenzae from blood or transtraoheal aspirates. The remaining strains of nontypable H. influenzae were olinical isolates from 5a E

13 4~574 the Erie County Medical Center and the Buffalo V.A. Medical Center.

Dr. J. Ward (University of California at Los Angeles) provided 54 atrains of H. intluenzae. type b., The remaining strains of H. influenzae type b were olinioal, isolates from the Buffalo Children's Hobpital. Reference strains of other capsular serotypes of H. influenzae were obtained from the Centers for Disease Control (Atlanta). ' Cultures of Haemophilus paraphrophilua ATCC 29200, Haemophilus segnis ATCC 10977, HaemoEhilua par ainf:uenzae ATCC 7901 and 9276, Haemophilus aegyptious ATCC 11116, Hasmophilus parahemolytious ATCC 10014, nontypable H. influenzae ATCC 19418, Aotinobaoillus actinomycetemoomitans ATCC 29522, ATCC 29523, ATCC 29524, NCTC
9707, and NCTC 9710, aotinobaoillus e:gul*li ATCC 19392.
Actinobacillus seminia ATCC 15768, and Aotinobaoillua auis ATCC
15557 were provided by Dr. J. Zambon (School of Dentistry, State University of New York at Buffalo). Iaolates of all other species were provided by the olinioal microbiology liLboratory at the Erie County Medical Center.

The identity of strains of H. influenzae was oonfirmed by colonial morphology and growth requirement for hemin and nicotinamide adenine dinucleotide. Capsular aerotypes were determined by CIE with use of reference strains and antiaerum from the Centers for Disease Control', Hurphy et al, supra.

_~ :, ~341574 Strains were stored in Mueller-Hinton broth plus 10% glyoerol at -70 C.

BALB/c mice were immunized ip with 0.1 ml of 109 oells of nontypable H. influenzae strain 3524 on days 0 arZd 28. On day 32 after the initial immunization, aeleoted animals were killed with chloroform, their spleens were removed, and splenio lymphooytes were harvested by perfusion of splenio pulp with minimal essential medium.

To achieve hybridoma development by fusion of the donar spleen cells to the NS 1(nonseoreting varlant of the IgOl BA/c plasmacytome P3XAg8) plasmacytoma ceila (obtained from the Salk Institute of Biology [La Jolla, Calif] under National Cancer Institute contract N01-CB-23886), 35% poly.eti~ylsn~ glycol was used in a modifi'oa~ion of the prooedure :ot KenneLt, Cell 'Fuslon, Methods Enzymol, 1979. 58:345-359=, inebriafl F10~ 8p26e6 os11a were combined with' 106 NS-1 oells in m1a.iotal eesentlal medium with serum. The ceils were oentrifuge.d dL 170 g.fori 10 min at 25 C. All of the supernatant was removed, and the pellet was tapped to loosen it. Two-tenths milliliter dl~'j~~-~oD2~!et1l~''ferte;w-.'gtyooi 1,000 (Sigma Chemical Co., St. Louis) in minima2 essential medium without serum was added and the mixture was atrirred gently and left at 25 C for 8 min, with the last 3 min consisting of centrifugation at 500 g to pellet the cells. At the end of the original 8 min, 5 ml of minimum essential:medium (MEM) with serum was added and gently pipetted once to res.uspend the pellet. The .... 7 .

mixture was centrifuged at 250 g for 5 min at room temperature (25 C). All of the supernatant was removed. Five milliliters of complete minimal essential medium (medium with gluoose I4.5 mg/ml] and 20$ fetal bovine serum) was added eo resuspend the pellet. The mixture was transferred to a 25-m1 Erlenmyer flask containing the appropriate amount of complete minimal essential medium to obtain 3 x 105 plasmacytoma oells/ml. The oells were stirred gently and distributed in 0.05-ntl samples into miorotiter wells.

At 24 hr after the polyethylene glycol fusion, 0.05 ml of medium containing hypoxanthine (13.6 pg/ml), aminopterin 40.36 Ng/ml), and thymidine (3.87pg/ml) was added to each well. The microtiter plates were placed in a tissue attiture inoubator at 85x humidity in an4atmosphere of 5x C02 and 95x-room air. Freah medium containing hypoxanthine, aminopter.in,.a=nd thymidine was added on day 7, and plates w.ere oheokeds~'br mae~~s+~opia ~]saues .:,sytv,= ,r,= y .,:=. r~ . .
: . . . .
after day 10. The supernatant from all wslls'was tested for the presence of antibody with an ELISA (ens.'yme linked minimal absorbant assay).
ELISAs were performed in polyvinyl 96-well miorotiter plates (Dynatech, Alexandria, VA); 200 -vu1 volumes were used for each step. Wells were coated with a Qell envelope preparation (10 ug/mi) of nontypable H. influenzae strain 3524 prepared by the method of Johnston, "Immunobiology of Neiaseriagonorrhooae", American Society for Microbiology, 1978, 121-9. Plates were incubated at 37 C for 1 hr followed by overnight inoubation at 4 C. Wells were washed three times with PBS (phosphate buffered saline) plus 0.05% Tween 20R surfaotant between eaoh step.
Unbound sites on the plastic were blocked with 3% bovine serum albumin in PBS for 2 hr..at 37 C. Tissue culture supeNqatabts (or dilutions of mouse asoites fluid in subsequent expeMiments) containing monoolonal antibody were inoubated.,in the wells overnight at 4 C. Rabbit antibody to mouse 1g0.and IgM was then incubated for 2 hr at 37 C followed by p'rofseih A.-peroxi=dase for 2 hr at 37 C. Two hundred microlitera of aubsbrate was then added .
to each well. Substrate was prepared by dissolving 10 mg of 0-phenyl-enediamine in 1 ml of methanol and adding this solution to 99 ml of citrate-phosphate buffer, pH 5.0, plus 0.1 1 of 3%
H202. After the substrate was incubated for 45 min in the dark at room temperature, the reaction was stopped with 50p1 of 4 N
H2SO4. The 0D490 was measured. Each set of ELISAs was performed with a control in whioh NS-1 tissue culture supernatant or ascites fluid was used in plaoe of the monoalonal antibody being tested. On the basis of the results of ELISA sareening, selected clones were propagated by subsequent transfer to larger tissue culture wells. Large quantities of antibody were.produoed in tissue culture and by ip injeotion of. 105 hybridoma cells into pristane-primed BALB/c mice. The resulting asoitio -fluid was harvested in three to four weeks and tested forspeoifioity.

~3 4#514 The strains to be assayed were grown on Ohooolate agar (or other appropriate medium, depending on the speoies) overnight at 37 C in an atmosphere of 95% room air and 5% CO2. Cells from one plate were harvested by suspension in PBS and oentrifugation at 10,000 g for 20 min. The resulting pellet was suspended in enough PBS to allow the suspension to be drawn rinto a micropipette. One-tenth milliliter of the suspension of bacteria was added to 0.4 ml of sample buffer (-0.06 M Tris, 1.2% SDS, 1%
B-mercaptoethanol, and 11.9% glycerol) and heated for 5 min in a boiling water bdth. The resulting organisms are referred to as whole cell preparation. A 10 -.,pl drop of whole cell preparation was placed on a nitrocellulose sheet (Sohleic3her and Schve2l:, Ino:o Keeae, NH) and allowed to air-dry. The sheet waa -then pliaed in 3x :ge2dtin in buffer A (0.012 M Tris and 0.15 M< NaCl, pH 17=4) for 1 hr.
After the sheet was rinsed with buffer A, it was plaoed in an appropriate dilution of antibody and allowed to shake at room temperature overnight. The sheet was >~i;ed:.4-3Lh.bafter. A and placed in 1:3,000 dilution of protein A peroxidase' (Zymed Laboratories, San Francisco) and ahaken for 1 hr at room temperature. The sheet was rinsed and immersed in horseradiah peroxidase color development aolution (0.015% H20zi Bio-Rad, Richmond, Calif) for 45 min. Controls assayed on eaeh sheet included sample buffer (negative control). A negative result was _ recorded when the dot was no different from the background oolor, and a positive result was recorded when the dot turned purple-blue. About 90% of dot assays were unequivooally positive or negative. Those strains that yielded equivooal results in the dot assay were subjected to Western blot assay, Preparation=of LPS. Lipopolysacoharlde (LPS) .vas prepared from nontypable H. influenzae strain 3524 by,two msthodls. The first method was a modification of the phenol-water extraction method of Westphal and Jaan, "Baoteri~~";;~'' . =r~'. - . .
Methods in Carbohydrate Chemistry, 1965, 5036091. The second method was that of Hitchcock and Brown, Journal of Haateriology, 1983, 154:269-77. The latter method uses the enzyme proteinase K
(Boehringer Mannheim t3mbH, Mannheim, Federal Republic of Germany), which hydrolyzes proteins but has no effect on LPS.

Whole cell and LPS preparations were subjected to SDS-PAGE
(sodium dodecyl sulfate polyacryZamide ge1 eleotrophoresis) with either 11% .or 13.2% separating .gels, Murphy et,al, supra. When electrophoresis was oompleted, the g.l was placed with a nitrocellulose sheet that had been previo-usly-boiled:i-n distilled water, and the sheet was immersed in Q.3 H aodi.um citrate plus 3 M NaCl. Eleotrophoretic transfer was o-arrfed out in a Trans-B1otR oell (Bio-Rad) at 50 V for 90 min. The eleotrode buffer was 0.025 M Tris, pH 8.3, 0.192 M glycine, and 20% methanol. The nitrocellulose sheet was then treated exactly as described for the dot assay; it was blocked with 3% gelatin and inoubated . 11 ;3 415 74 sequentially with antibody 7F3, protein A-peroxidase, and substrate horseradish peroxidase color developer.

I radiolabeling of surface OMPs. Extrinsic labeling of - - --- ------- surface-exposed OMPs was accomplished with a laotoperoxidase-catalyzed radioiodination prooedure, Hansen et al, Infect. Immun.
1981, 32:1084-92.

The ELISA with outer membranes of nontypable H. inrluenzae strain 3524 coated on micr.otiter plates deadoaatr=atbd that the hybridoma designated 7F3 was produoing antibody 7F3 that recognized a determinant in the'outer membrane ~of the bacterium.
Gel immunodiffusion indicated that this antibody was of the.Ig(33 isotype. Figure 1 shows a Western blot that indioates that the determinant recognized by antibody 7F3 was on a pro-tein with a molecular size of '16,600 daltons; lane A showa molecular weight standards on the nitrocellulose sheet, and lane B shows the 16,600-dalton protein recognized by antibody 7F3 in a whole cell preparation of nontypable H. influenzae strain 3524.
Specifically, lane A shows molecular weight-standards transferred from a 13.2% gel; lane B shows a whole oell preparation of nontypable H. influenzae strain 3524 incubated with ahtibody 7F3, protein A-peroxidase, and peroxide subatratei and lane C is an autoradiograph of a whole cell preparation of nontypable H.
influenzae strain 3524 made from bacteria extrinsiaal2y labeled w i th 1251. All three lanes were from th ' . a$me ae1. Weatern blot assay done by this method in 25 strains of H. influenzae showed that antibody 7F3 recognized a determinant on this 16,600-dalton protein in every strain. Because the antibody recognized a determinant on a protein of identieal molecular size in multiple strains, we screened larger numbers of straina with use of a dot assay rather than Weatet~n blot.

To determine whether the protein recognized by anti0ody 7F3 could be extrinsically labeled, we labeled nontypable H.
influenzae strain 3524 with 1251. The proteins were subjected to SDS-PAGE and transferred to a nitrocellulose sheet. One lane was exposed to x-ray film, and one lane was inoubated with,7F3, protein A-peroxidase eonjugate, and substrate. Fl.gure 1 shows that the band recognized by antibody 7F3 (lane B) oorresponds to an 125I-labeled baAd (lane C).

To assess further whether the epitope reQognized by antibody 7F3 was on a protein or on LPS, we performed two additional experiments. An ELISA was performed as described above in which some welis were coated with a cell envelope preparation of nontypable H. influenzae atrain 3524 and other .wells were ooated with LPS prepared from nontypable H. infZuenzae strain 3524 by the phenol-water method Westphal et a1,-,;.:su1pra. ; Antibody', 7F3 was reactive 'with a cell envelope preparation (OD, 0.375) that contained OMPs and LPS, Johnston et al, suprao. but was nonreactive with LPS (OD, 0.062). This finding indioates that the epitope recognized by antibody 7F3 resides on an OMP.

Figure 2 is a Western blot assay depicting another experiment designed to assess whether the epitope recognized by antibody 7F3 is on a protein or LPS. The lanes marked A contain LPS prepared by proteinase K lysis of oells of strain 3524, Hitchcock et al, J. Bacteriol, 1983, 154:269-7T, the lanes marked D contains LPS of .1train 3524 prepared by the phenol-water t method, Westphal et al, supra, and the lanes marked C oontain a whole cell preparation of strain 3524. All aampled were assayed on the same gel and tranaferred to the same nitrooellulose sheet.
Figure 2, left, was inoubated with antibody 7Z3 (ascites fluid dilution, 1:500), and figure 2, right,was Inoubated -iith antibody 3D2 (asoites fluid dilution,, 1:500), a monoolonal antibody that recognizes the lipid A portion of H. influenzae LPS. Antibody 7F3 does not bind to either of the LPS
preparations and binds only to a band 'with a molecular weight of 16,600 in the whole cell preparation. This observation demonstrates that antibody 7F3 recognizes an epitope on a protein and not on LPS. Specifically, Figure 2 shows a Western blot assay from a 13.2% gel: (left) inoubation with antibody 7F3 and (right) incubation with antibody 3D2, which reoognizes an epitope on the lipid A of H. influenzae. The lanes marked A oontain LPS of.
nontypable H. influenzae strain 3524 prepared by lyais of oella with proteinase K, the lanes marked B bontain phenol-water prepared LPS of strain 3524, and the lanes marked C contain a whole cell preparation of st'rain 3524. Noleoular weight standards are noted on the right.

Studies were performed to determine the specieaspeaifioity of the antigen recognized by antfk:Cidy ::7.F3. Yhole : aell preparations of 115 isolates of H. ih:lluenzae' were e=tudied by either dot assay or Western blot assay.: =included 73 .<.., .
type b, 37 nontypable; and 1 each of Lyp:es.;a and.;a-f. All 115 wo,.
strains of H. influenzae oontdined th4=,-.epi''Lo0e recognieed 'by antibody 7F3, a result indicating that this epitope ia a common antigen among strains of H. intluenzae.

Sixty isolates of various baoterial apeoies were studied to determine whether this epitope is present in bacteria other than H. influenzae. 'AlA 60 of these atraine laaked the detersina-nt recognized by antibody 7F3 (table 1)..

~3 41574 Table 1 Bacterium No. tested No. positive Gram-negative Esaherichia coli 10 to Actinobaeillus speoies 10 0 Proteus species T 0 Pseudomonas species 5 0 Klebsiella speaies 4 0 Serratia speaies 4 0.Enterobacter oloaaae 1 0 Morganella "morganii 1 0 Neisseria gonorrhoeae 6 0 Neisseria species 2 0 Gram-positive Staphylococcus aureus 5 0 Staphylococcus species 2 0 Viridans streptoooooi 1 0 Streptococous faecalis 1 0 Diphtheroids 1 0 Total 60- 0 ;3-41514 Twenty-nine strains of Haemophilus species other than H.
influenzae were studied. Twenty-five ot these isolates lacked the 7F3 epitope (table 2). Two strains of ~ parahemo2;'ticus contained the determinant. In addition, one strain of H.
paraphrophilus and one of H. aegyptious contained a 20,000-dalton protein that was recognized by antibody 7F3.

Table 2 - .' Species No. tested No, positive ~

H. parainfluenzae 24 0 H. parahemolytiaus 2 H. paraphrophilus 1e H. s-egnis 0 H. aegypticus 1 1*
Total 21 * In the Western blot assay, antibody 7F3 reoognized a 20,000-dalton protein in these strains.

;3 41574 Human serum antibody. Human serum was tested for the presence of antibody to the 16,600-dalton OMP by Western blot assay. Figure 3 shows whole cell preparations of nontypable H.
influenzae strain 3524 that were assayed on the same gel and transferred to nitrooellulose paperf lane A was inoubated with 7F3 ascitea fluid and shows a single band oorresponding to the 16,600-dalton protein, lanes B and C were inoubated with two different samples of normal human serum,.and-lane D was incubated with serum obtained from an.. adult 17 days;~:a~ter;~~aoter,emia;.due- to nontypable H. influenzae. All three samples af human serum Jhave antibody to the 16,600-dalton OMP that contains the determinant recognized by antibody 7F3. The DNA sequenoe for the gene expressing this 16,600-dalton outer membrane protein is believed to begin at nucleotide 125 and eontinues until nuoleotide 526.
This amino acid sequence is inoluded as a portion of the insert.
The restriction map of this portion of the sequence is shown in Figure 6.
The gene is believed to have the following sequence:

;3 41574 Amino Acid DNA Sequence CCCAAGTAAAATTTnCAGCT[CAGTCrCATA.,~TPAACTAAATAAAAAAC'lC1TTCAOGAGAAATCTA
1 ssssa c~t .~sn lys p!~ val lys ser leu leu val ala gly ser val ala ala leu 11TG AAC AN1 TTr GPT 11AA TCA TTA 'rTA GTT (>CA GGT 'rCT (,'TA CL~'T lxA TTA

= tve ala ata c-ys ser-ser sQr aan asn asp ala.a2a,gly asn gly sla la gin ;'lt; c3Cr ~rC'r ACr TOC TCT MC AAC GAT w1' GCA Q= AA? a'.'r (X.T OCT rJa thr ;ihe gly gly tyr ser val ala asp leu'gln-gln arg tyr asn thrival .~r rrr L= GGA TAC TC'r GTT GCT GAT CCrr CAA G1A CGT TAe AAC ACC CTA

tyr plye gly ,?he asp lys tyr asp i,le thr.gly qltu'ty,r val qln ile leu T,-%T 'P'CT caGT 'PrT GAT AAA TAC GAC A'!C ACC GCP CAA TAC CTT CA11 ATC '!'rA
asp ala his ala, ala tyr leu asn ala thr pro ala ala lys'-val leu,val GAT C;:G CAC CiCA C~A TAT TTA AAT Cr-A AQG: a:A t3;."1' ClC'r AAA CrA TTA GTA
3e0;
glu gly asn thr asp glu arg gly thr prd"sflu ty;-asn..ile ala leu gly :AN OGr AAT ACT cAT GU OGT GCT ACh C'J1 GA4 'rAC : uC A7C GrA K"i'A (~GA
gln arg arg alfa asp ala val lys gly tyr leci ala gly lys 91y val asp CAA OGT aGT C1CA GAT CrA CTT AAA GGTT TAT TTA MA CaGT AAA OCT CTT d-T
. 400 ala gly lys leu gly thr val ser tyr gly glu glu lys pro ala val leu Wr (2;T AAA TTA = ACA GTA TCr TAC Wr GAA G'AA AAA a.'t' C1CA GTA TTA
gly his asp glu ala ala tyr ser lys asn arg arg ala val leu ala tyr Cr;T CAC GAT GAA C1G"r WA TAT ZCT AAA AAC CGT dGT (rA GTG TTA C1CG TAC

-termination TAA TZ~'t'T~GTATP1ti.'TAAT TCX'ATZT'PTTA'tTOC,A CITI-sermc ArCG'ITPfa'rAATTTAACCAATTAC~CT'K'AAAGAATGAATTTA7'l1CT'TT~ATTCTAAI~ATAAA'IC~G'G

'rTATCATTAACTCA'!'GACA+CAC'r00GTG67rT AGA AA'iC0C'T

TOGTCGAAwT"POGAA TTT~ ~'~
GCtrPOCr AAT'lCX+I'0(~C'i'CGpiGAGTI'OC'~0 CrA0Ca1T'PCT

TACC.'G

IE

It is worthy of note that this band is among the most prominent recognized by antibody in human serum.

Specifically, Figure 3 shows a Western blot assay from a 13.2% gel. All four lanes contain a whole cell preparation of nontypable H. influenzae strain 3524 fromthe same gel, but each lane was incubated wi'th a different antiserum: lane A, antibody 7F3; lanes B and C, two different samples of normal human serum (dilution, 1:500); and lane D, serum obtained 17 days after bacteremia due to nontypable H. influenzae in an adult (dilution, 1:500). The incubation with antiserum was followed by inoubation with protein A-peroxidase and peroxide substrate. The arrows indicate that all three samples of human seruo oontarln sntibody to the 16,600-dalton OMP that oont-a-tns the fiF3 epi'tope.
molecular weight standards are indioated;~on theJeft.

In accordance with the invention; an 1903 mouse monoalonal r:>
antibody that rec.ognizes an epitope on a,16,600=dal;ton-:-OMP .on~ the ..., t. ,. .
surface of nontypable H. influenzae has been developed. This epitope is present in all 115 isolates of ai influenzae tested, including typable and nontypable strains. Screening of 60 strains of non-Haemophilus species demonstrated that the epitope is absent in all of'these bacteria. The epitope was absent in 24 strains of H. parainfluenzae but was:preaent in four to five strains of other Haemo hp ilus speoies (table 2). These species are unusual pathogens in humans. Therefore, from thb standpoint of clinically relevant isolates, antibody 7F3 is highly specific for H. influenzae.

This morioclonal antibody recognizing a common epitope that is highly specific for H. influenzae oan be useful aa a tool in the clinical microbiology laboratory. A rapid teat to confirm the identity of a cli'nical isolate as H. ee ;; .~i.n-t$uepgae<~Ctypable .or --~ . .r.~.,....,. . f nontypable) could be developed based on auoh a.n antibody. In order to construct a DNA probe to exploit this speoifio epitope, the DNA sequence of the gene encoding P6 is determined. Based on the DNA sequence, the amino acid sequence of the active P6 protein can be deduced. This information can be used to peeform what is known as epitope mapping.

Epitope mapping involves the construotion of a number of small peptides and testing these peptides for reactivity wAth monoclonal antibody 7F3. Since the eptitope reoognized by 7F3 is specific for H. influenzae, the corresponding peptide recognized by that antibody represents the speoific determinant on H.
influenzae. Once the amino acid sequenoir of the peptide is known, the DNA sequence of that segment'aan be deduoed. Since H.
influenzae contains the gene which eodea for this epitope, the bacterium is known to contain DNA which has a sequence corresponding to this sequence. A DNA probe oan, therefore, be constructed to correspond to the nucleic aeids which oode for the specific epitope on'P6. Once the probe is eonstrueted, it can be labelled, for example, with a radioactive member. This probe could then be used to assay a clinioal.sample suoh as aputum, cerebral spinal fluid, blood and others for the presenoe of H.
influenzae. This will be possible beoause the DNA probe,will bind to its complementary base pairs which are present in the genome of H. influenzae. Once this probe is oonstruoted, this approach would represent an advantage over the aurrent,twidely used method of demonstrating growth requirements for hemin and nicotinamide adenine dinuoleotide. An assay with a apeoific monoclonal antibody would yield resulta 24 hrs sooner.

OMPs and LPS are olosely associated on the outer membranes .
of gram-negative bacteria. This fact and the observation that the determinant recognized by antibody 7F3 is in the molecular weight range wherp LPS separates lead one to question whether this determinant is on a protein or.on LPS. Several lines of evidence indicate that the epitope recognized by antibody 7F3 is on a protein. First, staining with Coomaasie blue of SDS gels demonstrated the presence of a band reoognizad tiy antibody 7F3 at 16,600 daltons in all strains of H. influenzae. Because Coomassie blue stains protein but not LPS, this observation is presumptive evidence that antibody 7F3 reoognizes a protein determinant. Second, the configuration of the band on SDS-PAGE
and Western blot was typioal for proteini.LPS showed multiple bands that were generally less distinot than the band at whioh the antibody 7F3 epitope resides. 'This point is further corroborated by the observation that monoolonal antibodies that recognize LPS determinants showed the typical "LPS" pattern in Western blot assays of whole cell preparations, in contrast to the well-defined single band recognized by antibody M. Third, by ELISA, antibody 7F3 showed reaotivity with oell envelope preparations that oon.tain OMPs plus LPS, but the antibody showed no reactivity with isolated LPS. Finally, in the Weste6 blot assay (figure 2), antibody 7F3 recognized a band in a whole cell preparation but failed to reoognize determinants on LPS that was prepared by using two different methods. Taken together, these observations indicate that the epitope reoognized by antibody=7F3 resides on an OMP.

To assess whether the OMP oontaining the.antibody 7F3 epitope was surfacoe exposed, OMPs were labeled by using a lactoperoxidase-catalyzed radioiodination prooedure, Hansen et al, supra. Figure 1 shows that the protein containing the antibody 7F3 epitope is radiolabeled. Thia observation suggests that this 16,600-dalton OMP is surface expos.ed. For the purposes herein "surface exposed" or "outer membrane" means available for antibody binding.

The OMPs- of nontypable H. influenzae show substantial strain-to-strain variability, as demonstrated by SDS-PAaE
analysis. This variability in the major OMPs in the 32,000-42,000-dalton range is the basis of the subtyping system for nontypable H. influenzae, Murphy et al, supra. It is of interest that studies of OMPs of H. influenzae from three laboratories have independently noted the presence of a"16,000"-dalton OMP in all strains of H. influenzae studieds Murphy et .al, Barenkamp et al and Loeb et al, supra. It is this protein that contains the antigenic determinant recognized by antibody TF3. The present study indicates that the epitope reaog.bized by,antibo.dyOp'3 on this low-molecular-weight OMP is an antigen common to all strains of H. influenzae. Identifying common surface antigens among strains is useful from the point of view of vaccine development because immunization with a single common antigen might induce protection from diaease due to many strains. In addition, the observation that this 16,600-dalton protein has varied far less than other OMP-s in the oourse of evo=lution leads to the speculation that this protein serves an important tcihotion 7or the bacterium and that its function is closely related to conservation of its structure.

The outer membranes of gram-negative bacteria are im munologically important structures -'beoause of their accessibility to host defense meohaniams. Indeed, antibody to OMPs of H. influenzae type b are widely prevalent in adults and are detected in the serum of infants who are oonvalesoing from infections with H. influenzae. It has now been demonstrated that antibody to a 16,600-dalton OMP (p6) is present in human serum (figure 3). The presence of antibody to this OMP in normal human 2u serum suggests that the OMP is important with regard to the human antibody response to H. influenzae.

Several observations suggest that p6 is an important target in immunity to Haemophilus influenzaes 1) Antibody raised from p6 isolated from a type b strain protects in an infant rat model.

2) A monoolonal antibody, 7F3, directed against p6 blocks human bactericidal aotivity against Haemophilus influenzae (NtHi).

3) Depleting normal human eera of p6 by affinity chromatography resulted in reduced bacterioidal activity of~that sera for Haemophilus influenzae.

u) And, i~mmunopurified antibody to-p6 from human sera was ~
bactericidal.
This invention therefore includes the molecular cloning of p6 using H. influenzae as a source of bacterial chromosomal DNA, lambda gtll bacteriophage as the vector in oonstruotion of the genoinic library, pUC18 plasmid as the veotor u3ed in subaloning the gene to facilitate sequencing, and E. coli as the host strains. The results allow further analysis of the moieoular basis of both experimental and human immunity to p6 and permits large quantities of p6 to be produoed once it is approved for use in vaccine against Haemophilus influenzae.

Molecular Cloning of p6:

The 16,600 dalton protein, designated herein as p6, is therefore present in the outer membranes ot both typable and nontypable straina of Haemophilus influenzae and may be an important target in im munity to Haemophilus influenzae. The DNA
sequence for the gene expressing this 16,600-dalton outer membrane protein is'believed to begin at nuoleotide 125 and continues until nucleotide 526. In acoordance with this t invention p6 is cloned molecularly using a nontypable strain of Haemophilus intluenzae as a source of bacterial chromosomal DNA, lambda gtll bacteriophage as the vector in construction of the genomic library, pUC 18 plasmid as the vector in auboloning the gene to facilitate sequenoing, and E. coli as the host atrain.
The monoclonal ;Lntibodies previously disoussed and a polyolonal antiserum were used to soreen for expression of p6. A portion-of the genomic library was soreened re3iu2ting #n the detection of four positive reoombinants. One, olone e, appears to produoe a full length geneproduct expressed in high frequenay. The DNA
insert of this olone was used to subaloc-e the gene into a plasmid vector. An E. coli transformant, 7-9B, also appears to express a full length gene product. It is likely that transcription is initiated from the actual promoter of the p6 gene, since both clone 0 and transformant 7-9B express the gene product in both the uninduced and induced states. Isolating and sequencing the gene for p6 allows for further analysis of the molecular basis of both experimental and human immunity to p6.

More specifically, recombinent DNA teohology was used to clone the gene for the 16,600 dalton surface protein, p6, of nontypable Haemophilus influenzae (NtHi) into Esaheriahia coli.
Chromosomal DNA from a clinical isolate was shearedp ligated to lambda gtll arms and paokaged into phage heads. Four reoombinant phages were deteoted by screening with monoolonal antiboAies and a polyclonal antiserum. One, clone 0, was restricted with EooRt and ligated to plasmid vector pUC18 to faoi'litate sequencing.

coli carrying reoombinent.plasmf,ds were'sareen'ed resulting in-one positive, 7-9B. Both clone 0 and 7-9B produce a protein with an apparent moleoular weight equal to or similar, to native-p6 as determined by Western blot analyses. In soreening it was determined thatTtransoription and translat'ion of the Haemophilus lnfluenzae p6 gene(s) were not dependent on the lao operator 'and promoter of either vector. Using immunofluoreaoenoe, the recombinant gene produot's p6 epitopes could be localized on the surface of these E. coli and accessible to antiVody.

Haemophilus influenzae strain 1479 was grown at 37 degrees C
in brain heart infusion broth supplemented with hem (10yg/ml) and nicotinamide adenine dinucleotide (1 nag/ml).

The E. coli strain y1090 (r-m+) was used for the lytic growth of bacteriophage lambda gtll and strain JM83 as the host for the plamid pUC18. The E. coli strains were grown in L-broth (LB) or on LB agar with or without 50pg/ml of ampicillin, depending on the host strain. A more detailed deaoripti:oh for the use of the respective host strains oan be found elsewhere.
Young et al, Science 222: 778-782; Mersing Ree. DNA Tech. Bull.
2:43-48.

A pellet of Haemophilus influenzae 1479 cells from a 750m1 culture was resuspended in 10mis of 10mM HEPES butfer, pH 7.4.
To this mixture was added EDTA to a concentration of 5m M6d SDS
to a concentration of 0.5% w/v and then incubated =at 60 degrees C for 30 minutes. This lysate was then digested with 0.5m1 of pronase (10mg/ml) at 37 degrees C for 2 hours and then subjected to two phenol/CIAA extraotions followed by one CIAA extrac~ion.
Sodium chloride was added to a concentration of 0.2M to the aqueous phase, and DNA was precipitated with 2.5 volumes of chilled ethanol. Following precipitation in the oold, the DNA
was pelleted by centrifugation, resuspended in Tris-EDTA buffer, and treated with DNase-free RNase at a concentration of 0.1 mg/mi at 37 degrees C for 1 hour. Finally, the DNA was extracted with phenol/CIAA, precipitated with sodium chloride and ethanol, and pelleted by centrifugation. The DNA was resuspended in Tris-EDTA
buffer, measured for concentration by A260/A280 and =stored at 4 degrees C.
The phage library was soreened with monoolonal antibody 7F3.
Also used in screening was rabbit polyolonal antiserum produced by immunizations with solubiliz4d p6 preparations of Haemophilus influenzae strain 1808.

~341574 Construction of the Haemophilus influenza 1479 genomio library:
The strategies for the construction of the library, depioted in Figure 4, were essentially those described by Young and Davis, 1985, Vol. 7, pp 29-41, Genetic Engineering, Plenum Press, N.Y.
Haemophilus influenzae 1479 DNA was sheared by sonioation with one 10 second burst (output control setting at 2) to an average length of 2-4 kilobase pairs (kb). The degree of shear was monitored by agarose gel electrophoresis. The Eco R1 sites of 50ug of this sheared DNA were methylated using Eco R methylase.
=F
The ends of the methylated DNA were made fluah by the additidM of Klenow polymerase and deoxynualeotide triphosphates. Following this reaction and the addition of aodium aoetate to a concentration of' 0.3M, the DNA 'was preoipitated. After centrifugation, the pellet was resuspended in Tria-EDTA buffer.
The DNA was then ligated to Eoo R1 linkers (Bethesda Research Laboratories, Bethesda, Maryland) that had been phosphorylated.
The ligation reaction was terminated by heaLting the mixture to 70 degrees C and then the excess Eoo R1 linkera were digested using an excess of Eco 91. The methylated Haemophilus ieifluenza 1479 DNA blunt end-ligated to Eco R1 linkers was purified from excess linkers by passage over a gel filtration polumn (Biogel* p60, BIO
BAD laboratories, Richmond, CA) using a column buffer containing 10mM Tris pH 7.5, 100mM NaCl, 1mM EDTA. Fractions were monitored by A280 and agarose gel eleotrophoresis. Fractions * Trade mark ~3 41574 containing DNA of desired size range were pooled, and precipitated. The DNA was pelleted by centrifugation and resuspended in 4u1 of Tris-EDTA buffer. The DNA was ligated to 3ug of dephosphorylated lambda gtll arms (STRATAGENE*Cloning Systems, San Dieto, CA) in a total reaction volume of lOul. The ligation mixture was packaged using two packaging extracts according to the directions of the manufacturer (GigapackTM~
STRATAGENE Cloning Systema). P-aokaged phage wbre plated on E.
coli strain y1090 to determine the titer of plaque forming units and to determine the non-recombinant baokground by growth with IPTG and X-gal on LB + AMP plates. The library oontains approximately 1*.5X106 independent reoombinant olones with a background of less than or equal to 5%.

Screening the library:

Figure 5 depicts the methods used in screening. A portion of the y1090 plating stock, 0.2m1 of a y1090 pellet resuspended in 10 m M MgS04, was infected with 1.5X103 pfus of the lambda gtll library for each 85mm plate. d Following the adsorption incubation, the cells were mixed with LB-agarose butter, poured and spread evenly onto an LB + AMP plate. The plates were then incubated at 42 degrees C for 3 hours. Each plate was then overlaid with a dry nitrooellulose filter disk which had been saturated previously in 10m M IPTO. The plates were then incubated for 3 hours at 37 degrees C. Before removing the * Trade mark filters, the orientation was marked and the filters and respective plates were labelled. The filters were rinsed briefly with buffer A(0.01 M Tris, 0.15M NaCl, pH 7.4) and placed in 3%
gelatin in buffer A for 1 hour. After the filters were rinsed again with buffer A, they were incubated in a screening mixture of antibodies overnight at room temperature. The screening mixture was buffer A containing 7F3 ascites fluid, at titers of 1:1000. The monoclonal antibody used shared no crossreactivity with the E. coli host strains while anti-1808 antiserum required a working dilution of 1:10,000 to maintain sensitivity and .
specificity. The filters were rinsed with buffer A and placed in a 1:3000 dilution of protein A-peroxidase oonjugate and shaken for 1 hour at room, temperature. The filters were again rinsed with buffer A, then immersed in horoeradiah peroxidase color development solution (0.15% H202; BIO RAD, Richmond, CA) for 45 minutes. Plaques that appeared positive were removed from their respective plates, resuspended in 500).113, of SM buffer, and rescreened. Plaques that were positive in the rescreening were then rescreened again but against the individual=antibodies rather than the screening mixture.

Western blot analysis:

The Haemophilus influenza control, y1090 recombinants, and the molecular weight standards were prepared by heating at 100 degrees C for 5 minutes in a sample bufter containing 0.06M Tris pH 6.8, 1.2% SDS, 5% beta-mercaptoethanol, 11.9% glycerol and 0.003% bromophenol blue. The preparations were subjeoted to SDS-PAGE on a 15% separating gel. Gels were placed on a nitrocellulose sh,eet which had been previously boiled in distilled water and immersed in a 0.3M sodium citrate, 3M NaCl solution. Electrophoretie transfer was done using a TranaphorR
electrophoresis unit (Hoefer Scientific Instruments, San Francisco, CA) at 50 volts -for 90 minutes, in a buffer of 0.025M
Tris, pH 8.3, 0.192M glycine and 20% methanol. The blooking, with subsequent additions of antibody, conjugate, and substrate development was performed in the same manner as described ir! the plaque screening.

Subcloning into a l~ asmid vector:

A strategy was devised to facilitate sequencing. The DNA
insert (which consists of 867 residues), of a reoombinant phage expressing p6 epitopes (as determined by soreening), was subcloned into a plasmid vector. pUC18 was chosen as the plasmid vec'tor for suboloning for several reasons inoluding a means of selection, an inducible promotor, and an Eoo RI restriotion aite, features shared by phage cloning vector lambda gtll. The DNA of a recombinant phage was restricted with Eoo R1 and then ligated to pUC18 whioh had been restricted with Eco R1 and dephosphorylated using calf intestinal alkaline phosphatase. The ligation mixture was used to transform competent E. eoli atrain JM 83. Transformants were selected for by growth on LB + AMP
plates overlaid with IPTG and X-gal. White colonies, thought to represent JM 83 containing a plasmid plus insert, were individually picked and transferred to wells of microtiter plates containing L-broth + AMP + 10% glycerol. The plates were incubated overnight at 37 degrees C. A comb device was used to innoculate from the mierotiter plates onto nitrooellulose sheets, previously immersed in IPTO, overlaying LB + AMP' plates. The plates were incubated overnight at 37 degrees C, and then the nitrocellulose sheets were removed. The nitrocellulose was'hung for 15 minutes in a chamber containing chloroform vapors to =lyse the colonies, blocked in 3% gelatin containing 40pg/ml lysozyme and screened in the same way as the genomie library.

Results:

Screening the genomic library and characterization of recombinants.

Approximately 45,000 plaques were screened, the remainder of the iunamplified library being frozen in aliquots at -70 degrees C
in 7% DMSO. Four reactive clones, designated as lambda gtll-Haemophilus influenzae 1479 olones 0, P, 8, and 10, were found.
Efforts focused on clones 0 and P since they appear to express gene products that are in larger quantities than 8 or 10 and/or more closely resemble the conformation of the native protein, p6.

Plates containing clones 0 and P were oarefully scraped to ;3 4 1574 harvest protein for Western blot analysis. Western blots show that both clones 0 and P produce a protein that is the aame or similar in size to native p6. However, clone 0 produces the protein in larger quantities when compared to clone P.
Therefore, clone 0 was selected as the recombinant phage for the source of DNA to sUbalone into aplasmid vector..

Subcloning into a plasmid vector and characterization of the transformant.

One thousand tranaformants, from the same transformation, were screened before one, 7-9B, was found to be positive. To make certain that 7-9B was in pure oulture, positi,ve colonies were pi.cked, passaged and resoreened. Western.blota.ot 7-9B
grown on platea and in broth show that this reoombinant also produces a gene product that is of an apparent moleoular weight equal or si m i lar to native p6. The reooIDbinant pl.asm 3d isolated from 7-9B, was restricted using Eco R1 and subjeat to agarose gel electrophoresis to determine the insert size. Restriction analys is reveals the presence of a 2.5 kb DNA insert containing the p6 gene.

Discussion Using molecular cloning techniques, we produced both recombinant phages and reoombinant plasmid.containing the gene encoding for Haemophilus influenzae 1479 p6, a 16.6K dalton outer membrane protein. The protein can be described by the nucleic acid sequence between nucleie acid 125 and nuoleie acid 526, inclusively. Expression of the gene product by phage alone 0 and by recombinant plasmid 7-9B is independent of induotion of the B-galactasidase promoter. This finding, coupled with Western blot analyses revealing apparently no difference in eleetrophoretic mobilities between the gene products of olbne 0 and reeombinant 7-9B, provide evidence for the likelihood that the reooJbinant gene product is initiated from its own, the p6 gene's, constitutive promoter.

The epitopes of the 16.6K dalton protein are aooessible to monoclonal antibody 7F3 on the surface of E. ooli transformaqt 7-9B.

In summary, the gene which codes for p6 was cloned into E.
coli using a phag'e vector and plasmid veotor. The E. ao1i recombinants express the protein on-the surfaae in a form which is fully immunogenic.
Except as otherwise indicated all miorobiologioal strai'ns are generally available. All such strains as described herein are available from Dr. Timothy Murphy, Division of Infectious Diseases, State University of New York Cllnioal Center, 462 Grider Street, Buffalo, NY 14215. H. influenzae strains 3524 and 1479; E. coli strain JM83 containing plasmid 7-9B and hybridoma 7F3 are being deposited with the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, Maryland 20852 in accordance with"the Budapest Treaty.

The registration date of the deposits with ATCC are listed below:

ATCC Designation Deposit Numbers Date Nontypable H. influenzae, 3524 53599 3-18-87 Nontypable H. irifluenzae, 1479 53600 3-18-87 Escherichia coli, JM83 containing plasmid 7-9B 67358 3-18-87 Hybridoma cell line, 7F3 HB9625 1-14-88

Claims (16)

1. An epitope of a surface exposed outer membrane protein, said protein being isolated from Haemophilus influenzae and having a molecular size of from about 15,000 to about 17,000 daltons and said epitope constituting a portion of said protein and being recognized by a monoclonal antibody produced by hybridoma 7F3, A.T.C.C. Accession No. HB 9625.

2. The epitope of claim 1, wherein said protein is P6 having an approximate molecular size of 16,600 daltons.

3. The epitope of claim 2, wherein said epitope is conserved amongst typable and non-typable strains of Haemophilus influenzae.

4. An epitope of a surface exposed outer membrane protein, said protein being isolated from Haemophilus influenzae and having a molecular size from about 15,000 to 17,000 daltons and said epitope constituting a portion of said protein and being recognized by a monoclonal antibody produced by hybridoma 7F3, A.T.C.C. Accession No. HB 9625, wherein said outer membrane protein of Haemophilus influenzae has an amino acid sequence substantially similar to the sequence:

met asn lys phe val lys ser leu leu val ala gly ser val ala ala leu ala ala cys ser ser ser asn asn asp ala ala gly asn gly ala ala gln thr phe gly gly tyr ser val ala asp leu gin gln arg tyr asn thr val tyr phe gly phe asp lys tyr asp ile thr gly glu tyr val gin ile leu asp ala his ala ala tyr leu asn ala thr pro ala ala lys val leu val glu gly asn thr asp glu arg gly thr pro glu tyr asn ile ala leu gly gln arg arg ala asp ala val lys gly tyr leu ala gly lys gly val asp ala gly lys leu gly thr val ser tyr gly glu glu lys pro ala val leu gly his asp glu ala ala tyr ser lys asn arg arg ala val leu ala tyr.

5. An epitope of a surface exposed outer membrane protein, said protein being isolated from Haemophilus influenzae and having a molecular size from about 15,000 to 17,000 daltons and said epitope constituting a portion of said protein and being recognized by a monoclonal antibody produced by hybridoma 7F3, A.T.C.C. Accession No. HB 9625, wherein said outer membrane protein of Haemophilus influenzae is encoded by a nucleic acid sequence substantially similar to the sequence:
ATG AAC AAA TTT GTT AAA TCA TTA TTA GTT GCA GGT TCT GTA GCT GCA TTA
GCG GCT TGT AGT TCC TCT AAC AAC GAT GCT GCA GGC AAT GGT GCT GCT CAA
ACT TTT GGC GGA TAC TCT GTT GCT GAT CTT CAA CAA CGT TAC AAC ACC GTA
TAT TTT GGT TTT GAT AAA TAC GAC ATC ACC GGT GAA TAC GTT CAA ATC TTA
GAT GCG CAC GCA GCA TAT TTA AAT GCA ACG CCA GCT GCT AAA GTA TTA GTA
GAA GGT AAT ACT GAT GAA CGT GGT ACA CCA GAA TAC AAC ATC GCA TTA GGA
CAA CGT CGT GCA GAT GCA GTT AAA GGT TAT TTA GCA GGT AAA GGT GTT GAT
GCT GGT AAA TTA GGC ACA GTA TCT TAC GGT GAA GAA AAA CCT GCA GTA TTA
GGT CAC GAT GAA GCT GCA TAT TCT AAA AAC CGT CGT GCA GTG TTA GCG TAC
TTA.

6. A hybridoma which produces a monoclonal antibody to an epitope conserved amongst strains of Haemophilus influenzae, said epitope being localized on a Haemophilus influenzae outer membrane protein having an approximate molecular size of from about 15,000 to 17,000 daltons, said hybridoma designated as 7F3, A.T.C.C. Accession No. HB 9625.

7. A composition useful in the therapeutic treatment of disease due to typable or non-typable Haemophilus influenzae, said composition comprising the monoclonal antibody produced by the hybridoma according to claim 6 or a binding fragment or portion thereof, and a pharmaceutically acceptable carrier.

8. Use of the composition of claim 7 to passively protect a human against disease caused by typable or non-typable Haemophilus influenzae.

9. A method for the detection of Haemophilus influenzae in a sample of tissue or body fluids comprising:

a) obtaining a sample of tissue or body fluids as a source of antigen;

b) reacting the sample with purified monoclonal antibody produced from the hybridoma of claim 6, said monoclonal antibody labeled with a detectable marker and capable of binding to an outer membrane protein of Haemophilus influenzae, so as to form an antigen-labeled antibody complex; and c) assaying for antigen-labeled antibody complexes, 16. An Escherichia coli bacterium designated JM83, having A.T.C.C. Accession No. 67358 and containing the recombinant plasmid vector designated 7-9B.

17. A vaccine comprising protein produced by the bacterium of claim 15, said protein having at least one antigenic determinant from the outer membrane protein of Haemophilus influenzae, said antigenic determinant being recognized by a monoclonal antibody produced by hybridoma 7F3 having A.T.C.C. Accession No. HB 9625.

18. The vaccine of claim 17, further mixed with a pharmaceutical carrier.

19. Use of the vaccine according to claim 17 or 18 to protect a human against disease caused by typable or non-typable Haemophilus influenzae.

20. A vaccine comprising an immunogenic Haemophilus influenzae outer membrane protein, the outer membrane protein being encoded by a gene having a nucleotide sequence substantially similar to the sequence:

thereby detecting Haemophilus influenzae in said sample.

10. A recombinant vector comprising a DNA sequence encoding a substantially pure surface exposed outer membrane protein isolated from Haemophilus influenzae having an approximate molecular size of from about 15,000 to 17,000 daltons characterized by comprising an epitope recognized by a monoclonal antibody produced by hybridoma 7F3 having A.T.C.C. Accession No. HB 9625, said outer membrane protein of Haemophilus influenzae having an amino acid sequence substantially similar to the sequence:

met asn lys phe val lys ser leu leu val ala gly ser val ala ala leu ala ala cys ser ser ser asn asn asp ala ala gly asn gly ala ala gln thr phe gly gly tyr ser val ala asp leu gln gln arg tyr asn thr val tyr phe gly phe asp lys tyr asp ile thr gly glu tyr val gln ile leu asp ala his ala ala tyr leu asn ala thr pro ala ala lys val leu val glu gly asn thr asp glu arg gly thr pro glu tyr asn ile ala leu gly gln arg arg ala asp ala val lys gly tyr leu ala gly lys gly val asp ala gly lys leu gly thr val ser tyr gly glu glu lys pro ala val leu gly his asp glu ala ala tyr ser lys asn arg arg ala val leu ala tyr or an immunological peptide fragment thereof.

11. The recombinant vector of claim 10, in which the vector is 7-9B.

12. A recombinant plasmid vector designated 7-9B.

13. A bacterium containing the recombinant vector of claim 10.

14. A bacterium containing the recombinant vector of claim 11.

15. The bacterium of claim 14, wherein said bacterium is Escherichia coli isolate designated JM83, having A.T.C.C. Accession No. 67358.

ATG AAC AAA TTT GTT AAA TCA TTA TTA GTT GCA GGT TCT GTA GCT GCA TTA
GCG GCT TGT AGT TCC TCT AAC AAC GAT GCT GCA GGC AAT GGT GCT GCT CAA
ACT TTT GGC GGA TAC TCT GTT GCT GAT CTT CAA CAA CGT TAC AAC ACC GTA
TAT TTT GTT TTT GAT AAA TAC GAC ATC ACC GGT GAA TAC GTT CAA ATC TTA
GAT GCG CAC GCA GCA TAT TTA AAT GCA ACG CCA GCT GCT AAA GTA TTA GTA
GAA GGT AAT ACT GAT GAA CGT GGT ACA CCA GAA TAC AAC ATC GCA TTA GGA
CAA CGT CGT GCA GAT GCA GTT AAA GGT TAT TTA GCA GGT AAA GGT GTT GAT
GCT GGT AAA TTA GGC ACA GTA TCT TAC GGT GAA GAA AAA CCT GCA GTA TTA
GGT CAC GAT GAA GCT GCA TAT TCT AAA AAC CGT CGT GCA GTG TTA GCG TAC
TTA, said outer membrane protein being recognized by a monoclonal antibody produced by hybridoma 7F3 having A.T.C.C.
Accession No. HB 9625.

21. A compound useful in the therapeutic treatment of disease due to typable or non-typable Haemophilus influenzae, said compound comprising a monoclonal antibody produced by hybridoma 7F3, A.T.C.C. Accession No. HB 9625, said monoclonal antibody recognizing an epitope of a surface exposed outer membrane protein, said protein being isolated from Haemophilus influenzae and having a molecular weight size of from about 15,000 to about 17,000 daltons, said epitope constituting a portion of said protein.

22. The compound of claim 21, wherein said outer membrane protein is P6 having a molecular size of about 16,600 daltons and encoded by a gene having a nucleic acid sequence substantially similar to the sequence:

ATG AAC AAA TTT GTT AAA TCA TTA TTA GTT GCA GGT TCT GTA GCT GCA TTA
GCG GCT TGT AGT TCC TCT AAC AAC GAT GCT GCA GGC AAT GGT GCT GCT CAA
ACT TTT GGC GGA TAC TCT GTT GCT GAT CTT CAA CAA CGT TAC AAC ACC GTA
TAT TTT GTT TTT GAT AAA TAC GAC ATC ACC GGT GAA TAC GTT CAA ATC TTA
GAT GCG CAC GCA GCA TAT TTA AAT GCA ACG CCA GCT GCT AAA GTA TTA GTA
GAA GGT AAT ACT GAT GAA CGT GGT ACA CCA GAA TAC AAC ATC GCA TTA GGA
CAA CGT CGT GCA GAT GCA GTT AAA GGT TAT TTA GCA GGT AAA GGT GTT GAT
GCT GGT AAA TTA GGC ACA GTA TCT TAC GGT GAA GAA AAA CCT GCA GTA TTA
GGT CAC GAT GAA GCT GCA TAT TCT AAA AAC CGT CGT GCA GTG TTA GCG TAC
TTA.

23. A use of the compound according to any one of claims 21 or 22 to passively protect a human against disease caused by typable or non-typable Haemophilus influenzae.

24. A method for the detection of Haemophilus influenzae in a sample of tissue or body fluids comprising:

a) obtaining a sample of tissue or body fluids as a source of antigen;

b) reacting the sample with purified antiserum which specifically recognizes a Haemophilus influenzae outer membrane protein having a molecular size of from about 15,000 to about 17,000 daltons, or an immunogenic peptide fragment thereof, the outer membrane protein characterized by comprising an epitope recognized by a monoclonal antibody produced by hybridoma 7F3 having A.T.C.C. Accession No. HB 9625, said antiserum comprising an antibody labeled with a detectable marker and capable of binding to an outer membrane protein of Haemophilus influenzae, so as to form an antigen-labeled antibody complex; and c) assaying for antigen-labeled antibody complexes, thereby detecting Haemophilus influenzae in said sample.

25. The method of claim 24, wherein said outer membrane protein is P6 having a molecular size of about 16,600 daltons and encoded by a gene having a nucleic acid sequence substantially similar to the sequence:

ATG AAC AAA TTT GTT AAA TCA TTA TTA GTT GCA GGT TCT GTA GCT GCA TTA
GCG GCT TGT AGT TCC TCT AAC AAC GAT GCT GCA GGC AAT GGT GCT GCT CAA
ACT TTT GGC GGA TAC TCT GTT GCT GAT CTT CAA CAA CGT TAC AAC ACC GTA
TAT TTT GGT TTT GAT AAA TAC GAC ATC ACC GGT GAA TAC GTT CAA ATC TTA
GAT GCG CAC GCA GCA TAT TTA AAT GCA ACG CCA GCT GCT AAA GTA TTA GTA
GAA GGT AAT ACT GAT GAA CGT GGT ACA CCA GAA TAC AAC ATC GCA TTA GGA
CAA CGT CGT GCA GAT GCA GTT AAA GGT TAT TTA GCA GGT AAA GGT GTT GAT
GCT GGT AAA TTA GGC ACA GTA TCT TAC GGT GAA GAA AAA CCT GCA GTA TTA
GGT CAC GAT GAA GCT GCA TAT TCT AAA AAC CGT CGT GCA GTG TTA GCG TAC
TTA.

26. The method of claim 24, wherein said antiserum is a monoclonal antibody.

27. The method of claim 26, wherein said monoclonal antibody recognizes an antigenic determinant that is specific for typable and non-typable Haemophilus influenzae.

28. The method of claim 27, wherein said monoclonal antibody is produced by hybridoma cell line 7F3 having A.T.C.C.
Accession No. HB 9625.

29. A method for the detection of Haemophilus influenzae in a sample of tissue or body fluids comprising:
a) obtaining a sample of tissue or body fluids for use as a source of antibody directed to a Haemophilus influenzae outer membrane protein having a molecular size from about 15,000 to 17,000 daltons, or an immunogenic peptide fragment thereof, the outer membrane protein characterized by comprising an epitope recognized by a monoclonal antibody produced by hybridoma 7F3 having A.T.C.C. Accession No. HB 9625;
b) reacting said sample with an antigen comprised of the outer membrane protein of a) or an immunogenic peptide fragment thereof, said antigen labeled with a detectable marker and capable of binding to the antibody of a), so as to form a labeled antigen-antibody complex; and c) assaying for labeled antigen-antibody complexes thereby detecting Haemophilus influenzae in said sample.

30. The method of claim 29, wherein said outer membrane protein is P6 having a molecular size of about 16,600 daltons and encoded by a gene having a nucleic acid sequence substantially similar to the sequence:

ATG AAC AAA TTT GTT AAA TCA TTA TTA GTT GCA GGT TCT GTA GCT GCA TTA
GCG GCT TGT AGT TCC TCT AAC AAC GAT GCT GCA GGC AAT GGT GCT GCT CAA
ACT TTT GGC GGA TAC TCT GTT GCT GAT CTT CAA CAA CGT TAC AAC ACC GTA
TAT TTT GGT TTT GAT AAA TAC GAC ATC ACC GGT GAA TAC GTT CAA ATC TTA
GAT GCG CAC GCA GCA TAT TTA AAT GCA ACG CCA GCT GCT AAA GTA TTA GTA
GAA GGT AAT ACT GAT GAA CGT GGT ACA CCA GAA TAC AAC ATC GCA TTA GGA
CAA CGT CGT GCA GAT GCA GTT AAA GGT TAT TTA GCA GGT AAA GGT GTT GAT
GCT GGT AAA TTA GGC ACA GTA TCT TAC GGT GAA GAA AAA CCT GCA GTA TTA
GGT CAC GAT GAA GCT GCA TAT TCT AAA AAC CGT CGT GCA GTG TTA GCG TAC
TTA.

31. The method of claim 29, wherein said assay is selected from the group consisting of enzyme-immunoassay, enzyme-linked immunosorbent assay, radioimmunoassay, agglutination assay, immunofluorescence assay, and immunoprecipitin assay.

32. A method for the detection of Haemophilus influenzae comprising:
a) obtaining a sample of tissue, body fluid or secretion;
b) reacting the sample with a probe comprising a nucleotide sequence, wherein the nucleotide sequence is substantially similar to the sequence:

ATG AAC AAA TTT GTT AAA TCA TTA TTA GTT GCA GGT TCT GTA GCT GCA TTA
GCG GCT TGT AGT TCC TCT AAC AAC GAT GCT GCA GGC AAT GGT GCT GCT CAA
ACT TTT GGC GGA TAC TCT GTT GCT GAT CTT CAA CAA CGT TAC AAC ACC GTA
TAT TTT GGT TTT GAT AAA TAC GAC ATC ACC GGT GAA TAC GTT CAA ATC TTA
GAT GCG CAC GCA GCA TAT TTA AAT GCA ACG CCA GCT GCT AAA GTA TTA GTA
GAA GGT AAT ACT GAT GAA CGT GGT ACA CCA GAA TAC AAC ATC GCA TTA GGA
CAA CGT CGT GCA GAT GCA GTT AAA GGT TAT TTA GCA GGT AAA GGT GTT GAT
GCT GGT AAA TTA GGC ACA GTA TCT TAC GGT GAA GAA AAA CCT GCA GTA TTA
GGT CAC GAT GAA GCT GCA TAT TCT AAA AAC CGT CGT GCA GTG TTA GCG TAC
TTA, wherein said probe comprising a nucleotide sequence is synthesized to correspond to an immunogenic portion of a gene encoding a Haemophilus influenzae outer membrane protein having a molecular size of from about 15,000 to about 17,000 daltons, the outer membrane protein characterized by comprising an epitope recognized by a monoclonal antibody produced by hybridoma 7F3 having A.T.C.C. Accession No. HB
9625, said probe labeled with a detectable marker and capable of hybridizing to the immunogenic portion of the gene encoding the Haemophilus influenzae outer membrane protein so as to form a hybridized complex of the labeled probe and the immunogenic portion of the gene; and c) detecting the labeled probe of the hybridized complex, thereby detecting Haemophilus influenzae.

33. The method of claim 32, wherein said probe is comprised of a nucleotide sequence corresponding to a nucleic acid sequence capable of specifically binding to the P6 gene of Haemophilus influenzae.

34. The method of claim 33, wherein the detectable marker is selected from the group consisting of a radioactive marker, enzyme marker, fluorescent marker or a chemical marker.

35. A method for the detection of Haemophilus influenzae comprising:
a) obtaining a sample of tissue, body fluid or secretion;
b) reacting the sample with a probe having a nucleotide sequence complementary to a nucleotide sequence substantially similar to the sequence of an immunogenic portion of a gene encoding a Haemophilus influenzae outer membrane protein, the gene having the nucleotide sequence:

ATG AAC AAA TTT GTT AAA TCA TTA TTA GTT GCA GGT TCT GTA GCT GCA TTA
GCG GCT TGT AGT TCC TCT AAC AAC GAT GCT GCA GGC AAT GGT GCT GCT CAA
ACT TTT GGC GGA TAC TCT GTT GCT GAT CTT CAA CAA CGT TAC AAC ACC GTA
TAT TTT GGT TTT GAT AAA TAC GAC ATC ACC GGT GAA TAC GTT CAA ATC TTA
GAT GCG CAC GCA GCA TAT TTA AAT GCA ACG CCA GCT GCT AAA GTA TTA GTA
GAA GGT AAT ACT GAT GAA CGT GGT ACA CCA GAA TAC AAC ATC GCA TTA GGA
CAA CGT CGT GCA GAT GCA GTT AAA GGT TAT TTA GCA GGT AAA GGT GTT GAT
GCT GGT AAA TTA GGC ACA GTA TCT TAC GGT GAA GAA AAA CCT GCA GTA TTA
GGT CAC GAT GAA GCT GCA TAT TCT AAA AAC CGT CGT GCA GTG TTA GCG TAC
TTA, said probe labeled with a detectable marker and capable of hybridizing to the immunogenic portion of the gene encoding the Haemophilus influenzae outer membrane protein so as to form a hybridized complex of the labeled probe and the immunogenic portion of the gene; and c) detecting the labeled probe of the hybridized complex, thereby detecting Haemophilus influenzae.

36. A vaccine comprising an epitope of a surface exposed outer membrane protein, said protein being isolated from Haemophilus influenzae and having a molecular size of from about 15,000 to about 17,000 daltons and said epitope constituting a portion of said protein and being recognized by a monoclonal antibody produced by hybridoma 7F3, A.T.C.C. Accession No.
HB9625.

37. The vaccine of claim 36, further mixed with a pharmaceutical carrier.

38. The vaccine of claim 36, wherein the outer membrane protein is P6 having a molecular size of about

16,600 daltons and encoded by a nucleic acid sequence substantially similar to the sequence:

ATG AAC AAA TTT GTT AAA TCA TTA TTA GTT GCA GGT TCT GTA GCT GCA TTA
GCG GCT TGT AGT TCC TCT AAC AAC GAT GCT GCA GGC AAT GGT GCT GCT CAA
ACT TTT GGC GGA TAC TCT GTT GCT GAT CTT CAA CAA CGT TAC AAC ACC GTA
TAT TTT GGT TTT GAT AAA TAC GAC ATC ACC GGT GAA TAC GTT CAA ATC TTA
GAT GCG CAC GCA GCA TAT TTA AAT GCA ACG CCA GCT GCT AAA GTA TTA GTA
GAA GGT AAT ACT GAT GAA CGT GGT ACA CCA GAA TAC AAC ATC GCA TTA GGA
CAA CGT CGT GCA GAT GCA GTT AAA GGT TAT TTA GCA GGT AAA GGT GTT GAT
GCT GGT AAA TTA GGC ACA GTA TCT TAC GGT GAA GAA AAA CCT GCA GTA TTA
GGT CAC GAT GAA GCT GCA TAT TCT AAA AAC CGT CGT GCA GTG TTA GCG TAC
TTA.

39. The vaccine of claim 36, wherein the immunogenic peptide represents a portion of outer membrane protein P6 which is conserved among strains of Haemophilus influenzae.

40. A use of the vaccine according to any one of claims 36, 37, 38 and 39 to passively protect a human against disease caused by typable Haemophilus influenzae.

41. A use of a vaccine consisting of a viral or bacterial agent which has been genetically engineered to include the nucleic acid sequence of the gene encoding P6, wherein said nucleic acid sequence comprises:

ATG AAC AAA TTT GTT AAA TCA TTA TTA GTT GCA GGT TCT GTA GCT GCA TTA
GCG GCT TGT AGT TCC TCT AAC AAC GAT GCT GCA GGC AAT GGT GCT GCT CAA
ACT TTT GGC GGA TAC TCT GTT GCT GAT CTT CAA CAA CGT TAC AAC ACC GTA
TAT TTT GGT TTT GAT AAA TAC GAC ATC ACC GGT GAA TAC GTT CAA ATC TTA
GAT GCG CAC GCA GCA TAT TTA AAT GCA ACG CCA GCT GCT AAA GTA TTA GTA
GAA GGT AAT ACT GAT GAA CGT GGT ACA CCA GAA TAC AAC ATC GCA TTA GGA
CAA CGT CGT GCA GAT GCA GTT AAA GGT TAT TTA GCA GGT AAA GGT GTT GAT
GCT GGT AAA TTA GGC ACA GTA TCT TAC GGT GAA GAA AAA CCT GCA GTA TTA
GGT CAC GAT GAA GCT GCA TAT TCT AAA AAC CGT CGT GCA GTG TTA GCG TAC
TTA
or an immunogenic portion thereof, to protect a human against disease caused by typable Haemophilus influenzae.

42. A use of the composition of claim 7 for the production of a medicament to passively protect a human against disease caused by typable or non-typable Haemophilus influenzae.

43. A use of the compound according to claim 21 or 22 for the production of a medicament to passively protect a human against disease caused by typable Haemophilus influenzae.

44. A use of the vaccine according to any one of claims 36, 37, 38 and 39 to passively protect a human against disease caused by non-typable Haemophilus influenzae.

45. A use of a vaccine consisting of a viral or bacterial agent which has been genetically engineered to include the nucleic acid sequence of the gene encoding P6, wherein said nucleic acid sequence comprises:

ATG AAC AAA TTT GTT AAA TCA TTA TTA GTT GCA GGT TCT GTA GCT GCA TTA
GCG GCT TGT AGT TCC TCT AAC AAC GAT GCT GCA GGC AAT GGT GCT GCT CAA
ACT TTT GGC GGA TAC TCT GTT GCT GAT CTT CAA CAA CGT TAC AAC ACC GTA
TAT TTT GGT TTT GAT AAA TAC GAC ATC ACC GGT GAA TAC GTT CAA ATC TTA
GAT GCG CAC GCA GCA TAT TTA AAT GCA ACG CCA GCT GCT AAA GTA TTA GTA
GAA GGT AAT ACT GAT GAA CGT GGT ACA CCA GAA TAC AAC ATC GCA TTA GGA
CAA CGT CGT GCA GAT GCA GTT AAA GGT TAT TTA GCA GGT AAA GGT GTT GAT
GCT GGT AAA TTA GGC ACA GTA TCT TAC GGT GAA GAA AAA CCT GCA GTA TTA
GGT CAC GAT GAA GCT GCA TAT TCT AAA AAC CGT CGT GCA GTG TTA GCG TAC
TTA
or an immunogenic portion thereof, to protect a human against disease caused by non-typable Haemophilus influenzae.

46. A use of the compound according to claim 21 or 22 for the production of a medicament to passively protect a human against disease caused by non-typable Haemophilus influenzae.