BACKGROUND OF THE INVENTION

H. influenzae. a strict human pathogen, causes widespread and serious disease in both pediatric human populations (infants and children) and in adults and elderly. Pediatric diseases include mucosal surface diseases such as middle ear infections as well as invasive diseases such as meningitis, epiglottitis, septic arthritis and cellulitis. These diseases are leading causes of deafness and mental retardation and some of them can be fatal. While H. influenzae causes only about 30% of all otitis media, it is the most frequent cause of recurrent otitis media. Loss of hearing (and its accompanying retardation in learning) increases with the number of otitis episodes. Adult diseases are principally mucosal and include chronic bronchitis, sinusitis and pneumonia. H.influenzae is increasingly recognized as an important pathogen in adult and elderly populations.

Mucosal H.influenzae diseases are mainly caused by non-type b encapsulated and non-encapsulated (NT _b H.influenzae) strains of the organism and invasive H.influenzae diseases are almost exclusively caused by type b encapsulated strains (H.influenzae b). No vaccine exists for NT _b H.influenzae disease. A Type b capsular polysaccharide vaccine is available. The vaccine consisting of purified polyribitol phosphate (PRP), is partially effective but has several serious disadvantages. It is not antigenic or effective in the most susceptible group, that is children under the age of 18 months, and it is not completely effective in older children or in all populations. A new version of this vaccine has been recently licensed in which the polysaccharide is combined with a protein to enhance the polysaccharide's antigenicity. The conjugate vaccine appears more effective in younger populations. Neither of these vaccines gives any protection against mucosal H.influenzae diseases and neither of them prevents transmission of H.influenzae infection or colonization of the human nasopharynx.

SUMMARY OF THE INVENTION We have discovered four morphological and adhesion classes of pili on H.influenzae clinical isolates from different diseases, anatomical sites, ethnic groups and geographical areas. The principal pilus class, termed LKP pili, has been characterized in detail and evaluated as a purified pilus vaccine in a valid animal model of H.influenzae disease. LKP pili adhere to human erythrocytes, as do many

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of the pili used in other vaccines. Both NT _b H.influenzae and H.influenzae b strains can express LKP pili of the same serotypes, opening the possibility that one purified LKP pilus vaccine consisting of mixed serotypes can protect against several H.influenzae diseases. Although at least eight different serotypes of LKP pili have been found, only a restricted number of them occur frequently on clinical isolates.

Purified LKP pilus vaccines have been tested in the chinchilla model of otitis media. A single pilus vaccine was safe, antigenic, and protected against both nasopharyngeal colonization and middle ear disease. Excellent protection was obtained irrespective of whether inoculation of the bacteria was directly into the middle ear or into the nasopharynx, and whether inoculation was with the piliated phase or the nonpiliated phase of H.influenzae bacteria. When inoculation was with the nonpiliated phase, a rapid shift to the piliated phase occurred in the nasopharynges and middle ears of the animals. This observation is consistent with an important role for LKP pili in colonization and virulence. Pilus vaccine immunization completely eliminated piliated phase bacteria from both anatomical sites. Protection was shown to be LKP pilus type specific.

BRIEF DESCRIPTION OF THE DRAWINGS Figure i is a graph of proportion of positive cultures against time after nasal inoculation of Hemophilus influenzae into the bullae showing protection against bullar colonization in chinchillas by LKP pilus vaccine immunization. Intrabullar challenge.

Figure Jl is a graph of proportion of otitis media disease signs (middle ear effusion) against time after inoculation of Hemophilus influenzae into the bullae showing protection against otitis media in chinchillas by LKP pilus vaccine immunization. Intrabullar challenge.

Figure 111 is a graph of proportion of positive cultures against time after nasal instillation and barotrauma of Hemophilus influenzae showing nasal colonization in chinchillas protected by LKP pilus vaccine immunization. Nasopharyngeal/ barotrauma challenge.

Figure IV is a graph of proportion of positive cultures against time after nasal instillation and barotrauma of Hemophilus influenzae showing bullar colonization in

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chinchillas protected by LKP pilus vaccine immunization. Nasopharyngeal/ barotrauma challenge.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The pili may be administered orally - say, in capsule form - or by injection - that is to say, subcutaneous, intradermal, or intramuscular injections. Where the mode of administration is by injection, since the pili may be insoluble, any pharmaceutically acceptable suspending medium may be employed. It has been found especially useful to employ phosphate buffer, suitably containing merthiolate, as the vehicle or suspending medium. It is preferred to use 0.0005-0.1 , most suitably 0.004M phosphate buffer, at ionic strength from 0.0005 to 0.1 u, and, suitably 0.01% merthiolate. The concentration of pili in the vehicle is not critical. The sole criterion of desirability being that the pili shall be sufficiently finely divided to provide a suspension which meets generally accepted standards of syringeability. A concentration of 1-30, preferably about 20 mg. of pilus protein per 10 ml. of suspending medium is especially suitable.

It is generally preferred to administer the vaccine composition in more than one dose separated by a predetermined time interval. This time interval is selected to permit the formation of an adequate titer of antibodies to the pili in the injected subject.

Since there are no local or systemic toxic effects engendered by the injection of vaccine, there appear to be no upper limits to the dosage administered. It has been found suitable, however, to administer between 1 and 100 micrograms of pili per kilogram of body weight, most suitably about 20 micrograms per kilogram of body weight per injection.

The foregoing amounts refer to total pilus protein. Thus, if 10 pilus types from each of the designated strains are used in a mixture, a certain measure of protection is provided by each pilus type in a vaccine containing at least one member of each pilus type.

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In vivo testing bv Intrabullar and Nasal Challenge Barotrauma Bacterial Isolates and Cultures A non-typable H.influenzae strain 86-0295 isolated from the middle ear of a child with acute otitis media were used in these experiments. Bacteria were grown overnight on Brain Heart Infusion Agar (DIFCO) supplemented with 10 ug. each of NAD (Sigma, N7004) and Hemin (Sigma H2250) (S-HBIA - Formula II) at 37°C with 80% relative humidity and 5% C0 ₂ . The original isolate contained no detectable (by hemagglutination and by Electron Microscopy) pilϊation and piliated phase bacteria were selected by hemabsorption (A Hemabsorption Method for Detection of Colonies of Hemophilus Influenzae Type b Expressing Fimbriae, Connor. E.M. and M.R. Loeb, Journal of Infectious Diseases. Vol. 148, pp. 855-860 (1983) (incorporated herein by reference). Aliquots of piliated (P ⁺ ) and non-piliated (P ^"} strain 86-0295 were stored at -70°C using 5.5% Dimethyl Sulfoxide (DMSO) in Tryptic Soy Broth (TSB) as crow- protectant.

Vaccine Preparation

Pilus vaccine was prepared from P ⁺ 86-0295 grown on S-BHIA in aluminum cookie trays by methods set forth below in Examples I thru III.

Experimental Animals and Vaccination

Adult chinchillas obtained from local ranges and examined by otoscopy and tympanometry to be free of prior ear diseases were used in the experiment. Each animal was identified by a number on a neck collar and on the external ear. A code assigning each animal as to vaccine or placebo was developed and kept by an uninterested colleague until all experimental observations were completed. One-half of the animals were assigned to the placebo (Phosphate Buffered Saline - PBS) group and the other half of the animals to the vaccine group. Each dose consisted of 0.5 ml. of the vaccine or the placebo contained in a syringe labeled only with the animal's identification number. Each animal was given 2 intramuscular injections 28 days apart.

Serological Assays immediately before the first immunization, 10 days after the booster injection and 14 days after challenge, 1 ml. of blood was taken from the heart of each animal. Serum samples were prepared by standard methods and antipilus antibody titers were determined by piliated bacteria agglutination assay (PBAA).

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Overnight grown P ⁺ 86-0295 bacteria were harvested into formalinized (0.5%) PBS for use as stock test antigen (STA). Working test antigen was prepared by diluting with PBS from STA to a standardized concentration of 0.5 optical density (OD) units as determined in a 13 mm. glass tube at 540 nm. 50 ul. aliquots of the test serum were two-fold serially diluted in PBS in a 96-well flat bottom micro titer plate. The diluted serum was mixed with 50 ul. of the standardized test antigen and the test plate shaken at 150 rpm at room temperature for 20 minutes. The reactions were observed under a 20 x stereomicroscope. The titer of a test serum is defined as the reciprocal of the dilution factor under which a distinct bacterial agglutination can be observed.

Experimental Infection

10 days following the booster injection, each group of animals was randomly divided into two subgroups, equal in number by the vaccine code keeper. One subgroup of animals was infected with the P ⁺ bacteria and the other P ^"b acteria.

10 ml. of Brain Heart Infusion broth supplemented with 10 ul. per ml each of NAD and Hemin in a 15 x 160 mm. tube was inoculated with 20 ul. of either a P ⁺ or a P ^{" s} uspension. The liquid cultures were incubated unshaken at 37°C with 5% C0 ₂ and 80% relative humidity for about 17 hours. Such cultures routinely attained approximately 1 X 10 ⁹ colony forming units (cfu) per ml. Immediately before use. the cultures were diluted 1 :10 with sterile saline. This method was used for nasopharyngeal barotrauma challenge.

For intrabullar challenge, frozen aliquots of liquid cultures of predetermined colony forming units per ml. were thawed and diluted into phosphate buffered saline immediately before challenge.

Before infection, each animal was examined for prior middle ear infection.

Intrabullar Challenge.

0.1 ml. of the appropriate bacterial suspension was inoculated into the bulla through a small hole pierced in the bony covering of the bulla. The hole was then covered by suturing the skin closed. Samples of the bullar contents were removed

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subsequently through the same hole. The challenge dose was 10 colony-forming units per animal.

Nasal/Barotrauma Challenge 0.5 ml. of the appropriate bacterial suspension was delivered to the right naris using a 18 gauge catheter tube. The challenge dose was 10 ⁸ colony forming units/ml.

One day following nasal infection, nasal samples were collected on a chocolate agar plate from each animal by intranasal instillation of saline. Then, each animal was placed under barotrauma by placement of a needle into the epitympanic bulla and application of 25 mm Hg of negative pressure for 5 minutes. After barotrauma, the animals were returned to their cages.

Culture and Clinical Observations

Samples for culture and clinical observations were made 3, 6, 9 and 14 days following challenge according to Table 1 below:

Table 1

Clinical Observation Sample for Culture Dav Following (bilateral) Nasal Bullar

Barotrauma Otoscopv Tvmpanometrv Left Right

3 + + + +- 6 + + + +

9 + + + + +

14 + + + + +

The results of the intrabullar challenge are illustrated in Figures I and II. Figure

I data show that colonization of the chinchilla middle ear (bulla) can be effectively prevented by immunization with purified LKP pili. Figure II data show that the disease signs of otitis media can be effectively prevented by immunization with purified LK

P pili.

Serum Antibody Titers

Following primary and booster vaccination with the purified LKP pilus vaccine, 6 animals originally had no rise in piliated cell agglutination titer. 14 had titers in the

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2 to 16 range, 10 had titers in the 17 to 64 range and 5 had titers of greater than

100. No non-immunized animal had a detectable titer.

The results of the nasopharyngeal barotrauma challenge are illustrated in Figure III and IV. Figure IV data show that pilus immunization protects when the route of infection is the natural one via the nasopharynx. Large inocula (10 ⁸ colony- forming units) of bacteria are necessary to infect by this route. However, pilus immunization is highly effective in spite of the large challenge dose. Pilus immunization protected equally well whether the inoculation was in the piliated or nonpiliated phase. The nonpiliated inoculum rapidly shifted to the piliated phase in the nasopharynx in non-immunized animals. FIGURE III data show the rapid elimination of H.influenzae bacteria from the nasopharynx of pilus-immunized chinchillas. The significance of this result is great. The reservoir of H.influenzae bacteria causing disease is the human nasopharynx/throat (upper respiratory tract). H.influenzae infection is transmitted from one human respiratory tract to another. The H.influenzae bacteria that cause disease in an individual originate in that individual's upper respiratory tract. If pilus immunization can prevent transmission to and colonization of the upper respiratory tract, widespread use of a pilus vaccine could eradicate H.influenzae as a pathogen. Thus, our vaccine may provide epidemiological as well as individual disease control, and eventually eradicate H.influenzae disease.

Passive Immunization Bacterial Isolates and Cultures Same as NP-Barotrauma Experiment above.

Antiserum Preparation

Vaccines prepared by methods detailed (in NP-Barotrauma experiment) previously. Matured young New Zealand rabbits were immunized subcutaneously with 200 ug. pilus protein mixed with Freund's incomplete adjuvant. 3 injections were given at 4-week intervals. Two weeks after the last booster, animals were bled through the marginal ear vein and serum prepared by standard methods. The serum was titrated for antipilus antibodies using piliated bacteria agglutination as detailed previously. The preparation used in this experiment had an agglutination titer of 20,480.

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Experimental Animals. Immunization and Challenge

40 adult chinchillas were obtained from local ranches. Before immunization, each was examined for prior middle ear infection by otoscopy and tympanometry.

The animals were divided into 4 groups of 10 animals each, using a blind code. Code syringes containing 1 ml. of either undiluted hyperimmune antipilus serum, 1 ml. of 1 :10 diluted antipilus serum, 1 ml. of 1:100 diluted antipilus serum, or 1 ml. of non-immune serum. Each animal was given its coded injection intraperitoneally. One day following immunization, each animal was challenged into the right bulla with 10 cfu of piliated 86-0295.

Culture and Clinical Observations

Four and 8 days following bullar challenge, each animal was clinically observed by otoscopy and tympanometry. Samples for culture were taken through a surgically opened bullar by an alginate swab. Samples were streaked onto chocolate agar plates and incubated overnight at 37°C with 5% C0 ₂ and 80% relative humidity. Individual isolated colonies were picked using sterile filter paper strips and resuspended in a drop of PBS on a ring slide. A drop of appropriately diluted antipilus serum was added and the slide shaken (150 rpm) at room temperature for 15 minutes. Positive agglutination indicates the colony contained piliated bacteria.

The results are shown in Table 2. Table 2 shows that protection against H. influenzae colonization and otitis media disease can also be obtained when LKP pilus antibodies are administered passively. The antibodies may be produced in a different species than the one protected. This result is important because patients at high risk of disease or with an overwhelming H. influenzae infection could be given immediate protection by the injection of LKP pilus antibodies as a passive immunoprophylactic or as an immunotherapeutic.

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The following is a description of the general growth and purification procedures that were found to give acceptable yield and purity of protein. Exceptions to the general procedure are noted.

EXAMPLE I General Outline of purification of H. influenzae pili. Inoculum Preparation. Growth and Harvest

Frozen p ⁺ seed cultures of H. Influenzae CHP 86-1249b were thawed and plated on supplemented brain-heart infusion agar. Plating was performed 18-20 hours before the anticipated tray inoculation. The plates were incubated at 37°C, with 80% relative humidity and 5% C0 ₂ . The percentage of the colonies having hemagglutinating activity was assessed. Generally, ten individual colonies were picked and assayed. In all cases at least nine out of 10 colonies were HA ⁺ for human red cells.

Trays of GC base supplemented with DSF and hemin were inoculated with bacteria suspended in a culture transfer solution. The transfer solution was a potassium phosphate buffered saline solution containing 5mg/ml. beta-NAD. Bacteria were scraped from the plates with dacron swabs and suspended in the transfer solution until visibly turbid. Each tray was then inoculated with 2.5 ml. A glass spreader was used to distribute the inoculum evenly over the surface of the medium, inoculated trays were incubated for 20 hours at 37°C with 5% C0 ₂ and 80% relative humidity.

Growth was scraped from the agar using a metal scraper and 5-10 ml. of harvest buffer per tray. Since the cells were being washed before blending, the harvest buffer was at pH 5 to crystallize and recover any pili which had been shed

EXAMPLE

Blending and Initial Stages of Cycling

Each cell pellet from Example I, was resuspended in a volume of blending buffer such that the suspension volume was 150-200 ml., or about 5 ml. of buffer per gram of wet cell pellet. The pellet was not completely resuspended but instea

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d broken up sufficiently so that it could be removed from tne centrifuge bottle. Blending was performed using the large cup and unmodified blade assembly of th e Omni mixer. Each resuspended pellet was blended for 3 minutes at a speed of 10-11 k rpm. After blending, "depiliated" cells were removed by centrifugation at 15,300 x g. for 20 minutes. The supernatant was poured off and further clarified b y another centrifugation at 15,300 x g. for 20 minutes. The supernatant was pour ed off and the cell pellets discarded.

The first crystallization was performed by dialysis of the crude supernatant against a pH 5 acetate buffer. Crystalline pili, appearing as large chunky aggregates in the darkfield microscope, were sedimented for 60 minutes at 4°C and 22,100 x g.

The supernatant was poured off and discarded. The pellets were inverted over paper towels to drain briefly. This point marked the end of the first cycle.

Solubilϊzation of the pellets was performed by resuspension in 0.01 M caps buffer, at half the original crude volume. The pellets were broken up by the use of a rubber policeman and by drawing the pellets into and out of a 10 ml. pipette. T he pellets, brown in color, were then allowed to solubilize at 4°C with no stirring fo r several hours or overnight. The preparation was clarified by centrifugation at 22,100 x g for 60 minutes. The preparation was loaded into dialysis tubing then dialyzed against 50 mM sodium acetate buffer pH 5. Dialysis was performed in th e cold overnight. Pilus aggregates were then collected by centrifugation of the dialysis tubing contents. This point marked the end of the second cycle.

The third cycle was identical to the second, except that the volume of solubilϊ∑ing buffer used was half of that used in the second cycle.

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EXAMPLE III Final Stages of Cycling

After 3 cycles, the preparation still carried a faint yellow color, although it seemed fairly clean by SDS-PAGE. The following cycles were performed to remove additional impurities.

Crystalline pili were sedimented by centrifugation at 22,100 x g. Pili were solubilized in a pH 10.5 phosphate buffer containing 5 mM EDTA and 0.2% Triton X-100 (PBET). The same volume as in the previous cycle was used. Solubiiization was performed at 4°C with occasional mild stirring and was usually completed within 4 hours. Clarification was performed by centrifugation at 22,100 x g for 60 minutes. The supernatant was carefully poured off, leaving a clear gelatinous pellet.

Sodium chloride and polyethylene glycol were used as the crystallizing agent in the 4th cycle. However, to facilitate crystallization, it was necessary to first lower the pH from 10.5 to 7.5 - 8.0 by titration with HCI. The pili, still soluble at this stage, were crystallized by adding 5M sodium chloride to a final concentration of 0.5M and 30% PEG to a 3% final concentration. Streaming birefringence was visible immediately. The preparation was held at 4°C for 1 hour and then the crystalline pili sedimented by centrifugation at 22,100 x g for 60 minutes. This point marked the end of the 4th cycle.

The yellow supernatant was poured off and discarded and the pellets inverted to drain. Solubiiization was performed again in the PBET, followed by clarification by centrifugation at 22,100 x g for 60 minutes. Again, after removal of the supernatant, a clear gelatinous pellet remained. This point marked the end of the

5th cycle.

The 6th cycle was identical to the 5th, except that the pH 10.5 phosphate buffer used for solubiiization did not contain EDTA or Triton X-100.

The preparation is stored soluble, in the final pH 10.5 phosphate buffer.

EXAMPLE IV In accordance with the procedures of Examples I thru III. but in place of strain

CHP 86-1249. there were prepared all of the pili of the strains set forth in Table 3.

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Slight variations in solubility may occur. For example, LKP3 pili are less soluble at pH 6 than at pH 5. Also, LKP2 pili are less soluble at pH 4 than pH 5. However, all 8 types can be purified using the described procedure.

The following formulas were used in all of the foregoing standard preparations.

FORMULA 1 Hemin Stock 10 mg.ml. Hemin is dissolved at a concentration of 10 mg./ml. in 0.05N NaOH. The hemin is usually autoclaved to sterilize, but filter sterilization through a 0.2u. filter is possible if care is taken to thoroughly dissolve the hemin. The stock may be stored at 4°C for up to 1 week or at -20°C indefinitely.

(For 40 trays: 0.6 gm. hemin in 60 ml. 0.05N NaOH)

FORMULA II NAD Stock 5 mg./ml.

Beta-nicotinamide adenine dinucleotide is dissolved in a potassium phosphate buffered saline solution. The lab standard 20 x k ₂ /KH ₂ P0 ₄ media stock solution is diluted 1 :20 with 0.85% NaCI to make the appropriate volume of buffer.

The NAD is dissolved and then filter sterilized through a 0.2 u. filter. The stock may be stored at 4°C for up to 1 month or frozen at -20°C indefinitely.

FORMULA 111 Hemin-NAD Stock for Plate Media

Combine 1 part sterile Hemin Stock with 2 parts sterile NAD Stock. Dispense into freezing vials, 3.5 ml. per vial. Store frozen at -20°C. To use, thaw and add 3 ml./liter of sterile medium at no higher than 52°C. The medium will be 10 ug./ml. hemin, 10 ug./ml. NAD.

FORMULA IV Supplemented Brain-Heart Infusion Agar (S-BHIA) To a 2-liter flask, add: 1 stir bar 37 gm. DIFCO brain-heart infusion 02 gm. DIFCO Bacto agar

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1 L dH ₂ 0 (dH ₂ 0 is distilled water) Autoclave 25 minutes to sterilize. Cool to 50-52 _o C. With stirring, add 3 ml. of Hemin-NAD stock. Pour.

FORMULA V

DSF Supplement

Dextrose 600 gm.

1-Glutamine 15 gm. Ferric Nitrate 0.75 gm. dH ₂ 0 I.5 L

Dissolve by stirring with mild heating. Dispense into 1 liter bottles, 500 ml. /bottle. Autoclave 20 minutes to sterilize. Store at 4°C.

FORMULA VI GC Tray Medium

For each 4.0 L flask or 4 trays:

90 gm. DIFCO GC Base 2.5 L dH ₂ 0 Autoclave 40 minutes to sterilize. Add 25 ml. DSF supplement and 5 ml. sterile Hemin Stock, 10 mg./ml.

Swirl to mix, then pour. This medium should contain approximately 20 ug./ml. Hemin.

FORMULA VII Harvest Buffer — PBS, 0.15 u., pH 5.0

Sodium phosphate, monobasic monohydrate 5.6 gm.

Sodium chloride 3.2 gm. dH ₂ 0 800 ml. Adjust pH to 5.0, then bring to 1 L with dH ₂ 0.

FORMULA VIII Blending Buffer — TBS, pH 10-10.3

TRIS 12.1 gm.

Sodium Chloride 8.5 gm. Adjust pH to 10-10.3, then bring to 1 L with dH ₂ 0.

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FORMULA IX Dialysis Buffer — 50m M Acetate Buffer Sodium Acetate,

Anhydrous 17.23 gm. Glacial Acetic Acid 5.14 ml. d.H ₂ 0 5 L

Adjust pH to 5.0 with acetic acid, then bring to 6 L with d.H ₂ 0 (distilled H ₂ 0).

FORMULA X Solubilizing Buffer --- 0.01 M CAPS, pH 10.4

CAPS (3-[cyclohexylaminol]-1 -propane sulfonic acid)

2.21 gm. dH ₂ 0 800 ml.

Adjust pH to 10.4 with NaOH, then bring to 1 L with dH ₂ 0.

FORMULA XI Solubilizing Buffer — 0.1 u. Phosphate Buffer + 5m M EDTA + 0.2% Triton X-100 (PBET).

Sodium phosphate dibasic 7.92 gm.

Tertiary sodium phosphate ! 2H ₂ 0- 0.08 gm. Disodium ethylene- diaminetetraacetate 1.86 gm. Triton X-100 2 ml. dH ₂ 0

800 ml.

Titrate to pH 10.3-10.5 with NaOH. Bring to 1 L with dH ₂ 0.

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FORMULA XII Solubilizing Buffer — 0.1 u. Phosphate Buffer Sodium phosphate dibasic 7.92 gm. Tertiary sodium phosphate 12H ₂ 0 0.08 gm. dH ² 0 800 ml.

Titrate to pH 10.3-10.5. Bring to 1 L with dH ₂ 0.

FORMULA Xlll

5M Sodium Chloride

Sodium Chloride 292.2 gm. dH ₂ 0 1 L

Filter through a 0.45u. filter to sterilize.

FORMULA XIV Polyethylene Glycol, 30%

PEG-8000 (Fisher Carbowax) 300 gm. dH ₂ 0 700 ml.

Filter through a 0.45u. filter to sterilize.

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16

Table 2

Reverse Phase HPLC Amino Acid Compositions and Molecular Weights of Eight Hemophilus influenzae LKP Pilus Preparations

Strain and/or Preparation

Mix

Lot 001

LKPl,

86-0295 86-0295 86-0297 Εagan 86-1249b 86-1067 81-0384 LKP2, Lot 001 Lot 001 Lot 001 and

Amino acid (n,-) LKPl LKPl LKPl LKP3 LKP4 LKP2 LKP5 LKP3

Aspartic acid or Asparagine 45.8 4o.5 47.2 35.4 32.4 33.8 38.7 38.2

Glutamic acid or Glutamine 22.7 22.5 22.3 18.4 22.0 21.6 20.6 21.4

Serine 14.8 14.5 14.3 15.4 13.2 9.4 17.2 13.0

Glycine 16.2 15.6 15.5 16.6 16.8 19.3 22.1 17.8

Histidine 5.0 5.2 5.2 5.6 6.5 3.6 4.5 4.8

Arginine 2.7 2.3 2.3 1.1 4.5 8.5 5.8 4.4

Threonine 32.6 33.2 33.0 27.6 24.2 20.4 24.1 26.2

Alaniπe 23.3 23.5 23.4 17.7 17.7 22.0 24.1 21.3

Proline 5.3 5.3 4.3 3.7 6.5 4.0 6.4 3.7

Tvrosine 9.0 9.2 9.4 5.4 6.5 6.3 11.1 6.6

Valine 21.4 21.4 21.7 17.5 17.1 19.1 17.3 19.1

Mefhionine 2.0 2.0 1.8 1.8 3.3 0.8 1.8 1.8

1/2 Cystine 2.1 2.5 2.1 1.7 1.8 2.1 2.1 2.0

Isoleucine 9.8 9.7 9.7 7.3 9.5 11.4 12.3 9.6

Leucine 9.9 9.7 9.4 15.4 14.9 14.6 13.9 13.9

Fhenylalanine 8.7 8.5 8.6 9.5 8.5 4.3 7.1 7.3

Tryplophan 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Lvsine 18.8 18.3 19.7 16.8 16.6 16.0 15.7 16.9

Total (N) 250.1 249.9 249.9 216.9 .0 217.2 244.8 228.0

Daltons 26934.3 26937.0 27001.0 23223.6 24105.9 23263.7 26168.8 24460.5 μg/tube ⁷ - ^{8 5} - ⁹ - ⁶ -6 9.2 33.4 8.8 6.1 8.8

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Table 3

FREQUENCY OF LKP PILUS TYPES ON INDEPENDENT

CLINICAL ISOLATES OF HAEMOPHILUS INFLUENZAE

Purified Pilus Typing Serum (Rabbit. Unabsorbed

LKP1 LKP2 LKP3 LKP4 LKP5 LKP6 LKP7 LKP8 (LKP) (EM)

Pilus Disease (86-(81 - E a / (86- (81 - (86- (87- (FIN HA + P + @ Type Strain/Capsule Source 02950568 gan 1249 0384 0612 0297 48) #

LKP1 CB/86-0295.nt OM + JR/Hendrsn.b NP + KL/8418-1.b epigl + KL/8430-1.b men +

LKP2 CB/81-0568.e OM + CB/85-0911.e OM + CB/86-0779.e OM + CB/86-1067.e OM + + CB/86-1454.nt OM + + CB/87-0199.e OM + +

LKP3 JR/Eagan/b men + JR/1147/b epigl + JR/Madigan/b men + KL/8434-1/b epigl < + + KL\8440-1 /b epigl +

LKP4 CB/86-1269.nt OM + +

KL/8405-1/b men + +

KL/8408/b PC + +

KL/8410-1/b men + +

KL/8411-1/b SA +

KL/8414-1/b BC +

KL/8415-1/b SA +

KL/8417-1/b men +

KL/8419-1/b BC +

KL/8420-1/b men +

KL/8429/b men +

KL/8433/b men + +

KL/8436-1/b men +

KL/8448-1/b men +

KL/8453/b men +

KL/8501-1/b men +

KL/8502-1/b men +

KL/8503-1/b men +

KL/8504-1/b epigl +

KL/8505-1/B men +

KL/8507-1/b men +

LKP4 EH/AT102/b +

EH/CH100/b +

VCOLNYIO.b + +

EH/DV102/b + +

EH/NA100/b + +

EH/NO100/b +

EH/NO105/b +

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9/12460 1

FREQUENCY OF LKP PILUS TYPES ON INDEPENDENT

CLINICAL ISOLATES OF HAEMOPHILUS INFLUENZAE

Purified Pilus Tvpinα Serum (Rabbit. Unabsorbed' )

LKP1 LKP2 LKP3 LKP4 LKP5 LKP6 LKP7 LKP8 (LKP) (EM)

Pilus Disease !(86-(81 - E a / (86- (81 - (86- (87- (FIN HA + P+@

Type Strain/Capsule Source 02950568 gan 1249 0384 0612 0297 48) #

EH/DL166/b + +

EH/OC104/b + +

RM/P2047/b SA + +

RM/B5361/b men + +

RM/B5379/b BC + +

RM/P4488/b SA + + RM/B2917/b SA + + RM/B3813/b men + + RM/B1565/b men + + RW/82 men + +

RW/121 men + +

LKP5 CB/81-0384.n OM + + + CB/86-1814.nt OM + + + CB/87-0944.nt OM + + +

KL/8458-1/b SA + + +

LKP6 CB/86-0612.f OM + + +

CB/88-0349.nt Thr + + + LKP7 CB/87-0297/f OM LKP8 FIN 48 OM

Note: * OM, otitis media; NP. nasopharyngeal; epigl.. epigiottitis: men. meningitis.

PC, periorbital cellulitis; SA. septic arthritis; BC. buccal cellutitis.

# Hemagglutinastion of human erythrocytes.

@ LKP piliation by electron microscopy.

SUBSTITUTE SHEET

TabI c 4

Protection of Chinchillas Against Experimental

Otitis Media by The Passive Administration of

Homologous Antipilus Rabbit Serum

Intrabullar Challenge with 10 Colony-For ing Units.

Ant ibody Relβt i ve(2) Fract ion(3) Geometri c Fract i on(4 ) Percent Fract ion(5) Percent Fract i on{6) Dosage Serum with Mean Infected Prot ection Sympto- Prote t ion with Pili¬ Group Di lut ion C i rculat ing Agglutina¬ at 4 Days Against mβt ic at Again t ated Phase Ant ibody tion Titer Infection at Dflys Symptoms at t Days

(1) (1)

High 8/9 95 3/8 63X 3/8 63X 1/8

Medtum 1/10 8/8 99 3/8 63X 1/8 88X 0/8

D

Low 1/100 7/7 2/7 86X 3/7 57X 0/7

Control 0/10 10/10 OX 10/10 OX 10/10

I) The single βnimal infected with piliated phase cells was also the single βnimal with no circulating antibody.

(0 c ?) Anjmnts injected IP with 1ml serum; undiluted titer = 20,480.

3) Animals bled at time of challenge.

00 i. ) Determined by bullar culture. (0 H 5) Determined by direct observation of bulla.

6) Piliated phase bacteriβ determined by antiserum agglutination.

H c m ω

I m rπ