Vol. 55, No. 12INFECTION AND IMMUNITY, Dec. 1987, p. 2902-29110019-9567/87/122902-10$02.00/0Copyright © 1987, American Society for Microbiology

Antigenic Characterization of the Oligosaccharide Portion of theLipooligosaccharide of Nontypable Haemophilus influenzae

CHRISTIAN C. PATRICK,'t ALAN KIMURA,2t MARY ANN JACKSON,'§ LISA HERMANSTORFER,2ANTHONY HOOD,2 GEORGE H. McCRACKEN, JR.,' AND ERIC J. HANSEN2*

Department of Pediatrics, Southwestern Medical School,' and Department of Microbiology, Southwestern GraduateSchool ofBiomedical Sciences,2 University of Texas Health Science Center, Dallas, Texas 75235

Received 11 June 1987/Accepted 18 August 1987

Monoclonal antibodies (MAbs) directed against epitopes in the oligosaccharide portion of the lipooligosac-charide (LOS) of nontypable Haemophilus influenzae (NTHI) were used to characterize the LOS of thispathogen. Western blot (immunoblot) analysis with four LOS-specific MAbs and proteinase K-derived LOSpreparations from 69 NTHI strains allowed the classification of these strains into nine LOS antigenic groups.The use of these MAbs in a more sensitive colony blot radioimmunoassay system together with these sameNTHI strains identified 14 LOS antigenic groups. Extensive cross-reactivity was detected between the LOSepitopes of these NTHI strains and the LOS of H. influenzae type b. The epitopes recognized by these MAbswere not accessible to antibody on the surface of every strain. These LOS epitopes were also not stablyexpressed by NTHI growing in vitro; the observed frequency of LOS antigen variation ranged from 1 to 24%when large numbers of colonies of NTHI strains were screened for reactivity with the LOS-directed MAbs inthe colony blot radioimmunoassay. This LOS antigenic variation was sometimes associated with alterations inthe profile of the LOS molecule as resolved by sodium dodecyl sulfate-polyacrylamide gradient gel electropho-resis followed by staining with silver. These data indicate that considerable antigenic diversity exists amongNTHI strains with regard to the oligosaccharide epitopes in their LOS molecules.

Nontypable Haemophilus influenzae (NTHI) is being in-creasingly recognized as an important bacterial pathogen. Ithas been well established that this organism is second only tothe pneumococcus as an etiologic agent of bacterial otitismedia in infants and young children (30), and this organismis now known to cause sinusitis, bronchopneumonia, andbronchietasis in the pediatric population (32, 45, 52). NTHIalso has been shown to cause bacteremia and possiblyarthritis in neonates (9, 14, 26). Only in the past few years,however, has NTHI been shown to be a significant cause ofserious illness in adults. Several recent studies have indi-cated that NTHI is an important cause of nonbacteremicpneumonia in elderly or debilitated persons (5, 39) and thatNTHI is a major cause of invasive Haemophilus disease inadults (47, 53, 54).The emergence of NTHI as a major pathogen has given

impetus to efforts to identify factors associated with thepathogenicity and immunogenicity of this microorganism.The absence of a polysaccharide capsule on NTHI indicatesthat surface-exposed components of the NTHI outer mem-brane have the potential to interact directly with the hostboth in an active manner, as virulence factors, and in apassive fashion, as targets for antibodies which might beprotective against NTHI disease. The major constituents ofthe NTHI outer membrane are proteins and lipooligosac-charide (LOS). NTHI strains exhibit considerable diversityin their content of outer membrane proteins when thesemacromolecules are analyzed by sodium dodecyl sulfate

* Corresponding author.t Present address: Department of Pediatrics, Baylor College of

Medicine, Houston, TX 77030.t Present address: Praxis Biologics, Rochester, NY 14623.§ Present address: Division of Infectious Diseases, Children's

Mercy Hospital, Kansas City, MO 64108.

(SDS)-polyacrylamide gel electrophoresis (PAGE) (4, 31, 35)and characterized with serological techniques (33). In con-trast, relatively little is known about NTHI LOS except that,like the LOS of Neisseria gonorrhoeae (44), N. meningitidis(50), Bordetella pertussis (42), and H. influenzae type b (Hib)(8, 22), NTHI LOS appears to have no 0-antigen repeat unitbut consists of a lipid A moiety covalently coupled to astructure equivalent to the core oligosaccharide of entericlipopolysaccharide molecules (24).

Although the LOS of Hib has been well characterized bothbiochemically and antigenically (1, 2, 8, 24, 41) and has beenimplicated in the expression of virulence by this encapsu-lated pathogen (12, 28, 55), a paucity of similar data con-cerning NTHI LOS has hindered evaluation of the role ofthis amphipathic molecule as either a possible virulencefactor for NTHI or a target for protective antibodies. Char-acterization of the antigenic properties of NTHI LOS shouldfacilitate further investigation of the involvement of theseLOS molecules in both virulence and immunity. We utilizedmonoclonal antibody (MAb) technology to characterizeNTHI LOS and determined that (i) significant antigenicheterogeneity exists among strains of NTHI with regard toepitopes in the oligosaccharide region of their LOS mole-cules, (ii) significant commonality or cross-reactivity existsbetween the LOS of NTHI and that of Hib, (iii) the extent ofsurface exposure of a given LOS epitope can vary amongstrains of this pathogen, and (iv) these LOS epitopes are notstably expressed by NTHI strains growing in vitro.

MATERIALS AND METHODSBacterial strains and culture conditions. A total of 69 NTHI

strains were collected from 11 research centers across theUnited States. Thirty-three of these strains were obtainedfrom Children's Medical Center and Parkland Hospital,Dallas, Tex., by John D. Nelson and George H. McCracken,

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Jr. The other 36 strains were kindly provided by the follow-ing individuals: Peter Rice, Boston, Mass.; Melvin Marks,Oklahoma City, Okla.; William Feldman, Atlanta, Ga.;Barry Gray, Birmingham, Ala.; Timothy Murphy, Buffalo,N.Y.; Ram Yogev, Chicago, Ill.; Susan Gibson, Austin,Tex.; Katheryn Edwards, Nashville, Tenn.; Harry Wrightand Parvon Azimi, Los Angeles, Calif.; and Steven Berk,Johnson City, Tenn. These NTHI strains include isolatesfrom the middle ear, nasopharynx, blood, cerebrospinalfluid, and trachea. The 30 Hib strains used in this study were

described previously (15, 16).All strains were grown on brain heart infusion medium

supplemented with Levinthal base (BHIs) as described pre-

viously (17). NTHI strains were screened upon receipt forlack of reactivity with type-specific rabbit antisera to H.influenzae serotypes a through f (Burroughs-Wellcome Co.,Research Triangle, N.C.) in slide agglutination tests.MAbs. Lymphocyte hybridomas secreting MAbs were

produced by standard methods used routinely in this labo-ratory (43). Briefly, spleen cells from mice immunized withviable cells of NTHI strains AT200, BI104, TN100, TN103,BF103, and TN104 were fused with SP2/0-Agl4 plasmacy-toma cells, and the resultant hybridomas were screened forproduction of MAbs reactive with the LOS of the homolo-gous NTHI strain in a Western blot (immunoblot) system as

described below. MAbs directed against surface-exposedepitopes of NTHI LOS were identified through the use of theindirect antibody accessibility radioimmunoassay (RIA)(28). MAbs 12D9 and 4C4, which were raised against LOS ofHib strains, have been described in detail (16). All MAbswere used in the form of hybridoma culture supernatantfluids (43). MAbs 3D9 and 17E12 were raised against NTHILOS, as described in Results.

Colony blot RIA. The colony blot RIA described by Guliget al. (16) was used to screen NTHI and Hib strains for theirreactivity with LOS-directed MAbs. Each strain was used inthree independent experiments to determine its reactivitywith these MAbs.Measurement of LOS antigenic variation. Antigenic varia-

tion of NTHI LOS was assessed by using a modification ofthe colony blot RIA method to screen large numbers ofindividual colonies. Briefly, an exponential-phase broth cul-ture of the NTHI strain was diluted and spread on 20 BHIsagar plates at 150 to 250 CFU per plate. After overnightincubation at 37°C in an atmosphere containing 5% carbondioxide, the colonies on these plates were blotted onto filterpaper (Whatman no. 40, 82.5-mm diameter) and dried for 1 hat 37°C. Groups of five filters were probed with each of thefour LOS-directed MAbs (4C4, 12D9, 3D9, and 17E12) as

described previously (28). The colonies were allowed togrow again for 4 h, and the plates were then stored at 4°Cuntil LOS antigenic variants could be identified on theresultant autoradiographs from the colony blot RIA.SDS-PAGGE. LOS was resolved by SDS-polyacrylamide

gradient gel electrophoresis (PAGGE) (28) with a 4%(wt/vol) polyacrylamide stacking gel (2 cm in length) and a

separating gel (16 cm in length) consisting of a linear 10 to15% (wt/vol) polyacrylamide gradient containing a linear 0 to37.5% (vol/vol) glycerol gradient. Electrophoresis was car-

ried out at 4°C and 30 mA constant current until theassociated voltage increased to 400 V, at which time thecurrent was reduced to 20 mA. Electrophoresis was termi-nated when the tracking dye reached a point 1 cm above thebottom of the separating gel. LOS was visualized by thesilver stain method of Tsai and Frasch (51).

Preparation of NTHI LOS for SDS-PAGGE. Proteinase

K-digested whole cell lysates of NTHI were used as thesource of LOS for analysis by SDS-PAGGE. These lysateswere prepared by a modification of the method of Hitchcockand Brown (20). Bacteria grown on BHIs agar plates weresuspended in cold pH 7.2 phosphate-buffered saline (PBS) toa density of 4 x 109 CFU/ml. A 5-ml quantity of thissuspension was centrifuged at 7,000 x g for 20 min, washedonce with cold PBS, and then suspended in 1 ml of PBS. Thebacteria were solubilized by adding 0.5 ml of concentrateddigestion buffer composed of 0.1875 M Tris hydrochloride(pH 6.8), 6% (wt/vol) SDS, 30% (vol/vol) glycerol, and0.015% (wt/vol) pyronine Y tracking dye, followed by heat-ing at 100°C for 5 min. For LOS analysis, a 300-,ul portion ofthe lysate was treated with 250 ,ug of proteinase K (SigmaChemical Co., St. Louis, Mo.) for 1 h at 60°C. Thesepreparations were heated at 100°C for 5 min immediatelybefore electrophoresis. A 10- to 15-,u portion of the protein-ase K-treated material was used for SDS-PAGGE followedby silver staining (28).Western blot analysis. Immunoblotting of NTHI LOS was

accomplished as described previously for Hib LOS (28),with minor modifications. Briefly, the proteinase K-derivedLOS preparations (75 ,lp) were resolved by SDS-PAGE (27)in 12.5% (wt/vol) polyacrylamide separating gels andelectrophoretically transferred to nitrocellulose membranesby the method of Towbin et al. (49). Membranes were thenincubated in PBS containing 2.5% (wt/vol) bovine serumalbumin for 1 h to block nonspecific protein-binding sites onthe nitrocellulose. The membranes were next incubated for 6h at room temperature in a 50:50 mixture of PBS-bovineserum albumin and MAb. The membranes were then washedthree times (20 min each) in PBS and incubated overnight at4°C in a 1:2,000 dilution of goat anti-mouse immunoglobulinconjugated with horseradish peroxidase (Cappel Laborato-ries, Cochranville, Pa.) in PBS-bovine serum albumin. Themembrane was then washed three times in PBS as beforeand incubated with a 1:2,000 dilution of rabbit anti-goatimmunoglobulin coupled to horseradish peroxidase (CappelLaboratories) in PBS-bovine serum albumin. After threefinal washes in PBS, the Western blot was developed with4-chloro-1-naphthol as the enzyme substrate (19). Eachstrain was used in three independent Western blot experi-ments to determine its reactivitiy with the LOS-directedMAbs.Antibody accessibility RIA. The indirect antibody accessi-

bility RIA was used to assess cell surface exposure of theLOS determinants recognized by the MAbs (28). NTHI cellsgrown overnight on BHIs agar plates were suspended in coldPBS to a concentration of 2 x 109 CFU/ml. A 100-,ul volumeof this suspension was reacted with 500 ,ul of the MAb for 2h at 4°C with gentle rocking. After centrifugation at 12,000 x

g for 5 min, the pellet was washed once with PBS containing10% (vol/vol) heat-inactivated fetal calf serum (FCS) andthen suspended in 1 ml of this same buffer. To detect MAbsspecifically bound to epitopes on the cell surface, the bacte-rial suspension was incubated with 106 cpm of affinity-purified and radioiodinated goat anti-mouse immunoglobulin(specific activity, 108 cpm/,ug of protein) for 1 h at 4°C. Acarrier organism (ca. 109 CFU of an NTHI strain lackingreactivity with the MAb) was added to the suspension,which was then washed three times with 1-ml quantities ofPBS-FCS. The final pellet was suspended in 500 ,ul ofsolubilization buffer (17), and the amount of radioactivity(counts per minute of the radioiodinated probe) bound to thecells was determined in a gamma counter. A positive reac-tion (surface exposure of the epitope) was indicated by

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counts per minute at least sevenfold greater than that ob-tained with negative controls (NTHI strains lacking theepitope reactive with that MAb).The indirect antibody accessibility RIA was also used to

investigate the antigenic specificity of certain MAbs (3D9and 17E12). Various amounts of purified LOS from strainBI104 and oligosaccharide prepared from this LOS wereincubated with 25 ,ul of the LOS-directed MAbs and 300 jxl ofPBS-FCS for 1 h at room temperature. These mixtures werethen used directly in the antibody accessibility RIA, withthese preparations serving as the source of antibody asdescribed previously (16).

Purification of LOS. LOS was purified from NTHI strainB1104 by the phenol-chloroform-petroleum ether method ofGalanos et al. (10). The purity of this LOS preparation wasassessed by SDS-PAGGE followed by the use of the silverstain and Western blot techniques. LOS-derived oligosac-charide was prepared by treatment of the purified LOS with1% acetic acid at 100°C for 1 h. Unhydrolyzed LOS and lipidA were removed by differential centrifugation, and thesupernatant fluid containing the oligosaccharide was neutral-ized with 1 N NaOH (2). The quantity of oligosaccharide inthis preparation was measured by the phenol-sulfuric acidmethod (7), using glucose as the standard. LOS purified fromHib strain DL42 (16) was employed as a negative control incertain experiments.Immune electron microscopy. Visualization of immune

complexes on NTHI cell surfaces was performed as de-scribed previously (21). Briefly, NTHI cells freshly grown onsolid medium were suspended to a density of approximately109 CFU/ml in PBS-FCS, pelleted by centrifugation, andsuspended in PBS-FCS. A 0.25-ml portion of the suspendedcells was mixed with 1.0 ml of MAb and was rotated at 4°Cfor 60 min. After three washes in PBS-FCS, the cells weresuspended in PBS-FCS (1.0 ml), and a suspension ofstaphylococcal protein A-colloidal gold particles (11) wasadded such that the color of the final mixture was light pink.This suspension was rotated at 4°C for 120 min, after whichthe cells were washed three times in PBS-FCS, once in PBS,and finally suspended in PBS (0.5 ml). Formaldehyde wasadded to a final concentration of 1.2%; after 10 min, the cellswere applied to carbon-coated grids (10 to 15 RI per grid).Excess liquid was removed with a wedge of filter paper, anda drop of 2% (wt/vol) neutral aqueous phosphotungstic acidwas applied to each grid. The phosphotungstic acid wasremoved with a wedge of filter paper, and a drop of distilledH20 was applied to each grid and then removed. The gridswere examined in a Philips 301 electron microscope at 60 kVaccelerating voltage and photographed at a magnification ofx 34,000.

RESULTS

Characterization of MAbs. Six different NTHI strains wereused to immunize mice for subsequent use in hybridomaproduction. Plasma cell hybridomas secreting MAbs reac-tive with the LOS of the homologous immunizing strain ofNTHI were identified in Western blot analysis. The numbersof hybridomas producing NTHI LOS-specific MAbs ob-tained with each different NTHI strain were as follows:AT200, 13; B1104, 17; TN100, 11; TN103, 10; BF103, 14; andTN104, 6.

Preliminary screening of these 71 MAbs against 25 NTHIstrains in the colony blot RIA system showed that theseMAbs could be divided into four different reactivity groups.One group of MAbs, as represented by MAb 3D9 (raised

50

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z

zm

20

10

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5 10 15 20 25ul OF INHIBITORY SUBSTANCE

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FIG. 1. Inhibition ofMAb binding to NTHI cells by purified LOSand LOS-derived oligosaccharide. The indirect antibody accessibil-ity RIA method was used to quantitate the binding of MAbs toNTHI cells as described in Materials and Methods. MAb 17E12 wasincubated alone or with purified LOS from NTHI strain B1104 orwith oligosaccharide derived from this LOS for 1 h before use in theantibody accessibility RIA with NTHI strain B1104. Purified LOSwas used at a concentration of 1 mg/ml. Oligosaccharide was used ata concentration of 2.5 mg/ml. Symbols: 0, LOS; 0, oligosac-charide; r1, LOS from Hib strain DL42 (negative control).

against strain AT200), reacted with a majority (67%) ofNTHI strains. The second group of MAbs, as exemplified byMAb 17E12 (raised against strain B1104), recognized 37% ofthese NTHI strains. Two LOS-specific MAbs (4C4 and12D9) raised against Hib LOS (16) were also included in thisscreening system. The third group of NTHI-directed MAbsreacted with 55% of the NTHI strains and had a reactivitypattern identical to that of MAb 4C4 with these NTHIstrains. The fourth group of NTHI LOS-directed MAbsrecognized 62% of these NTHI strains in a pattern identicalto that obtained with MAb 12D9. None of the 71 MAbs wasstrain specific.MAbs 12D9 and 4C4 have been shown to be directed

against epitopes in the oligosaccharide of Hib LOS (16);therefore these two well-characterized MAbs were used inplace of the NTHI-raised MAbs with identical strain reac-tivity patterns. The antigenic specificity of MAbs 3D9 and17E12 was investigated first by examining whether NTHILOS-derived oligosaccharide exerted an inhibitory effect onthe binding of these MAbs to whole cells of the homologousNTHI strain in the antibody accessibility RIA. Both of theseMAbs reacted with NTHI strain BI104 in the colony blotRIA, and thus LOS was purified from this NTHI strain.Acetic acid was used to hydrolyze this LOS into its lipid Aand oligosaccharide constituents, and the purified oligosac-charide was incubated with these MAbs before their use inthe antibody accessibility RIA. Both the purified B1104 LOSand the oligosaccharide derived from the BI104 LOS inhib-ited the binding of MAb 17E12 to its homologous targetstrain (BI104) (Fig. 1). This oligosaccharide preparation didnot inhibit the binding of a Hib LOS-directed MAb to itshomologous Hib strain, thus indicating that the inhibitoryeffect of this preparation on the binding of MAb 17E12 wasantigen specific (data not shown). The binding of MAb 3D9to whole cells of its homologous strain (AT200) was similarlyinhibited by the BI104 core oligosaccharide (data notshown).SDS-PAGGE and Western blot analyses were used to

confirm that these NTHI LOS-directed MAbs bound toepitopes in the oligosaccharide region ofNTHI LOS and that

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A B

FIG. 2. Western blot analysis of purified NTHI LOS and LOS-derived oligosaccharide. LOS from NTHI strain BI104 (5 p.g) andLOS-derived oligosaccharide (25 p.1) were resolved by SDS-PAGGE, transferred to nitrocellulose, and incubated with MAb17E12 for Western blot analysis. Lanes: A, LOS; B, oligosaccharide.

the purified oligosaccharide contained no intact LOS. Quan-tities of B1104 LOS and oligosaccharide which exhibitedsimilar degrees of inhibition in the antibody accessibilityRIA (Fig. 1) were resolved by SDS-PAGGE and transferredto nitrocellulose. It has been previously shown that intactNTHI LOS will enter the polyacrylamide gel but that theuncharged oligosaccharide does not and therefore theoligosaccharide cannot be detected in subsequent Westernblot analysis (2). The purified BI104 LOS sample yielded areadily detectable reaction in Western blot analysis whenprobed with MAb 17E12 (Fig. 2), whereas no reactivity wasevident in the lane which contained the oligosaccharide

TABLE 1. Antigenic grouping of NTHI strains based onreactivity with LOS-directed MAbs in Western blot analysis

LOS antigenic phenotype (na) NTHI strains

4C4+ 3D9- 12D9- 17E12- (13) ........SA102, DL401, B1106,N83-011E, N83-0106E,BF107, B1103, TN102,BF100, BF104,N82-0156E, DL204, 069

4C4- 3D9+ 12D9+ 17E12+ (4) .........DL322, OC201, DL304,B1104

4C4- 3D9+ 12D9+ 17E12- (23) ........DL203, N83-0133E,LA303, OC202, DL209,N83-002E, DL205,N83-0159E, N139,TN104, N1937, TN101,N82-045E, BF103,AT200, LA304, BF106,DL325, BO-2, N2111,BF102, N82-0127E,B1102

4C4+ 3D9- 12D9- 17E12+ (2) .........SA101, TN1004C4+ 3D9+ 12D9- 17E12- (2) .........N182, N904C4+ 3D9+ 12D9+ 17E12- (9) ......... SA104, BI101, B1105,

N185, DL329, DL240,DL321, B1100, DL208

4C4- 3D9+ 12D9- 17E12+ (1) .........LA3004C4- 3D9+ 12D9- 17E12- (2)..........067, N82-0306E4C4- 3D9- 12D9- 17E12- (13) .........CH203, N1955, CV102,

LA305, BF105, TN103,LA301, N10, TN105,BF101, LA302, N2114,N82-091E

a Number of bacterial strains with preceding antigenic characteristics.

sample. Identical results were obtained with MAb 3D9 (datanot shown).

Antigenic characterization of NTHI strains. ProteinaseK-treated lysates of 69 NTHI strains were used in Westernblot analysis together with the LOS-directed MAbs 3D9,17E12, 4C4, and 12D9. A total of 56 (81%) of the NTHIstrains reacted with one or more of these MAbs. Classifica-tion of the NTHI strains into LOS antigenic groups based ontheir reactivities with these MAbs yielded nine different setsof strains, with a group of 13 strains representing a null setunreactive with any of these four MAbs (Table 1). Four ofthese LOS antigenic groups (4C4+ 3D9- 12D9- 17E12-;4C4- 3D9+ 12D9+ 17E12-; 4C4+ 3D9+ 12D9+ 17E12-; 4C4-3D9- 12D9- 17E12-) comprised the majority (58 of 69; 84%)of these NTHI strains. In contrast, four of the remaining fiveLOS antigenic groups had at most two strains each.

Figure 3 depicts the Western blot results obtained whenLOS preparations from representative strains from each ofthe nine LOS antigenic groups were probed with the fourdifferent MAbs. Figure 4 contains a gel in which these sameproteinase K-derived LOS preparations were resolved bySDS-PAGGE and stained with silver. A significant degree ofmicroheterogeneity was observed among the different anti-genic types of LOS, but there was no obvious correlationbetween the banding patterns and mobility characteristicsand the nine antigenic groups described above.These same 69 NTHI strains were also analyzed in the

A B C D E F G H I

4C4

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3D9

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12D9

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.....

17E12

FIG. 3. Western blot analysis of LOS from prototypic strainsrepresenting the nine LOS antigenic groups. Proteinase K-derivedLOS preparations from the nine NTHI strains were resolved bySDS-PAGE, transferred to nitrocellulose, and probed with MAbs4C4, 3D9, 12D9, and 17E12. Lanes: A, strain B1103; B, strainOC201; C, strain BF106; D, strain SA101; E, strain N182; F, strainDL208; G, strain LA300; H, strain 067; I, strain BF101.

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A B C D E F G H I

FIG. 4. SDS-PAGGE analysis of LOS from prototypic NTHIstrains representing the nine LOS antigenic groups as determined byWestern blot analysis. Proteinase K-derived LOS preparations wereresolved by SDS-PAGGE and stained with silver by the method ofTsai and Frasch (51). Lanes are as in Fig. 3.

colony blot RIA. It has previously been shown that some H.influenzae strains whose LOS fails to bind a particular MAbin Western blot analysis will bind this same MAb in both thecolony blot RIA and the antibody accessibility RIA (28). Thecolony blot RIA showed more extensive reactivity of thefour MAbs with these NTHI strains, such that 64 (93%) ofthe NTHI strains bound one or more of these antibodies.This expanded reactivity also resulted in different MAbreactivity patterns for some of the strains, such that the 69strains could be divided into 14 LOS antigenic groups (Table2). Again, a majority of these strains (52 of 69; 75%) weredivided among only a few (five) LOS antigenic groups, withsix other LOS antigenic groups having only one strain each.

Antigenic cross-reactivity between NTHI LOS and HibLOS. The MAbs originally raised against Hib LOS, 12D9and 4C4, reacted with 43 of 69 (62%) and 38 of 69 (55%)NTHI strains, respectively, in the colony blot RIA. Thisfinding suggested extensive sharing or cross-reactivity ofLOS epitopes between Hib and NTHI. Probing of 30 Hib

TABLE 2. Antigenic grouping of NTHI strains based onreactivity with LOS-directed MAbs in the colony blot RIA

LOS antigenic phenotype (n") NTHI strains

4C4+ 3D9+ 12D9+ 17E12- (10) ........SA102, N82-0127E,

N82-0156E, N83-0133E,B1100, BI101, Bl105,DL208, N83-0106E,N83-0159E

4C4- 3D9+ 12D9+ 17E12- (13) ........LA304, N2111, AT200,

BF106, CH203, DL205,TN101, LA303, N139,N185, N1937,N82-045E, TN104

4C4- 3D9- 12D9+ 17E12+ (1) .........N104C4- 3D9- 12D9- 17E12+ (1)..........N2114

4C4- 3D9+ 12D9- 17E12- (1)..........BF104

4C4+ 3D9- 12D9- 17E12+ (4) .........BF107, TN100, DL401,

LA3004C4+ 3D9+ 12D9- 17E12- (2) .........BF105, N90

4C4+ 3D9- 12D9- 17E12- (11) ........N83-0111E, BF100,

TN102, SA101, B1103,BI106, DL204, LA302,N182, TN105, BF101

4C4- 3D9+ 12D9- 17E12+ (1) .........067

4C4- 3D9- 12D9+ 17E12- (1)..........CV1024C4+ 3D9+ 12D9+ 17E12+ (10)........DL321, DL325, DL329,

N82-0306E, N83-002E,BI102, DL209, DL240,OC202, SA104

4C4- 3D9+ 12D9+ 17E12+ (8) .........DL322, B1104, BF103,BO-2, DL203, DL304,OC201, BF102

4C4+ 3D9+ 12D9- 17E12+ (1) .........069

4C4- 3D9- 12D9- 17E12- (5) ..........LA301, LA305, N1955,

N82-091E, TN103" Number of bacterial strains with preceding antigenic characteristics.

TABLE 3. Surface exposure of LOS epitopes on NTHI strains'

Reactivity in Reactivity inMAb and antibody MAb and antibody

NTHI strain accessi- NTHI strain accessi-bility RIAb bility RIA

4C4 12D9N83-0159E - CV102 +SA104 + N83-0106E -SAlQ1 - SA102BI101 - OC201 +B1106 + DL203 +DL209 - N139 +N182 + BO-2B1103 + BF102N82-0156E - DL325DL329 + LA303 +

3D9 17E12AT200 + BF103 -

OC201 + DL329 -

DL203 + 067 -

N139 + OC202 -

B1104 - SA104 -

N90 - DL322 +N83-0106E - B1104 +BF102 + TN100BO-2 - 069B1102 + OC201 +

aEach of these strains is reactive in the colony blot RIA with the specifiedMAb.bThe indirect antibody accessibility RIA was used to measure binding of

the MAbs to whole NTHI cells.

strains with MAbs 3D9 and 17E12 in the colony blot RIAreinforced this impression in that 9 of 30 (30%) of these Hibstrains reacted with each of these MAbs originally raisedagainst NTHI LOS.

Surface exposure of NTHI LOS epitopes. Each of the fourLOS-directed MAbs employed in the preceding experimentsbound to a cell surface-exposed LOS epitope on the homol-ogous NTHI or Hib strain. However, both Western blotanalysis and the colony blot RIA system detect antigenicreactivity involving both surface-exposed epitopes as well asthose epitopes which are buried in the cell envelope in wholeH. influenzae cells (15). For this reason, a different type ofRIA was used to determine whether the LOS epitopesrecognized by these MAbs in the two preceding test systemswere actually exposed on the surface of every NTHI strainand accessible to antibody.The indirect antibody accessibility RIA procedure uses

whole NTHI cells to measure binding of an antibody toepitopes exposed on the bacterial cell surface. Each of thefour MAbs was used in this procedure together with 10strains reactive with each MAb in the colony blot RIAsystem. MAb 17E12, originally raised against strain B1104,bound to the surface of this strain and to two other NTHIstrains (Table 3). MAb 3D9 bound to the surface of itshomologous parent strain (AT200) and to five other NTHIstrains. MAb 4C4 bound to the surface of five NTHI strains,as did MAb 12D9.Immune electron microscopy was used to confirm the

surface exposure of LOS epitopes in strains which werepositive for antibody binding in the antibody accessibilityRIA. Figure 5 shows two different NTHI strains which wereincubated with MAb 17E12 followed by exposure to proteinA-colloidal gold to detect surface-bound immunoglobulin Gantibodies (16, 21). Strain B1104, which was reactive with

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A -

FIG. 5. Binding of an LOS-directed MAb to the surface of NTHI

as detected by immune electron microscopy. Strains TN100 (A) and

B1104 (B), which both react with MAb 17E12 in the colony blot RIA,

were incubated with this MAb, and the resultant immune complexes

on the cell surface were visualized through the use of proteinA-colloidal gold particles and electron microscopy as described in

Materials and Methods.

this MAb in the antibody accessibility RIA (Table 3), also

bound this MAb to its surface in this immune electron

microscopy procedure, as evidenced by the electron-dense

gold particles covering the surface of these cells (Fig. SB3). In

contrast, strain TN100, which was reactive with this MAb in

the colony blot RIA (Table 2) but which did not bind this

antibody in the antibody accessibility RIA (Table 3), also did

not bind this MAb in this other system, as indicated by the

lack of colloidal gold particles on the TN100 cells (Fig. 5A).

Stability of LOS expression. Two different methods were

employed to determine whether the LOS epitopes recog-

nized by the four MAbs were stably expressed by NTHI.

The first experiment involved 10 strains of NTHI which were

passaged in vitro five times by the single colony isolation

method. Colony blots prepared from the original strains at

the beginning of this experiment and from the strains ob-

tained after in vitro passage were probed with MAbs 4C4,

3D9, 12D9, and 17E12. Of 10 strains, 9 had identical colony

blot reactivities before and after in vitro passage, whereas 1

strain (BI104), which was originally 4C4- 3D9+ 12D9+

17E12+ (Table 2), did not react with MAb 3D9 after in vitro

passage.

A more stringent method for examining LOS antigenicvariation involved the screening of large numbers (500 to1,200) of individual colonies for their reactivities with thefour MAbs. NTHI strains representing five different LOSantigenic groups (Table 4) were grown in BHIs broth,diluted, and plated on BHIs agar plates. The resultantcolonies were probed with the four MAbs in the colony blotRIA. All five strains analyzed in this manner exhibited LOSantigenic variation at frequencies ranging from 1 to 24%(Table 4). Both loss and acquisition of MAb reactivityoccurred among these strains. For example, 12% of thecolonies of stain BF101 (4C4+ 3D9- 12D9- 17E12-) did notbind MAb 4C4, whereas 9% of the colonies of this strainexhibited reactivity with MAb 3D9. Similarly, approxi-mately 8 to 9% of the colonies of strain LA300 (4C4+ 3D9-12D9-- 17E12+) lost reactivity with MAb 4C4 or 17E12,whereas 3 to 4% of these colonies bound MAb 3D9 or 12D9.LOS antigenic variation was detected regardless of thegrowth phase of the broth culture (data not shown).SDS-PAGGE analysis of LOS antigenic variants. Several

LOS antigenic variants of each strain described above werepurified by repeated single colony isolation. Proteinase K-derived LOS preparations from these antigenic variants andtheir respective parental strains were resolved by SDS-PAGGE and stained with silver (Fig. 6). These experimentsdetermined that LOS antigenic variation was often accom-panied by alterations in the SDS-PAGGE profile of the LOSmolecule. For example, all of the LOS antigenic variants ofstrain OC201 had LOS molecules which yielded SDS-PAGGE profiles different from that of the original parentalstrain (compare lane Al with lanes A2 through A4 in Fig. 6).In addition, two LOS antigenic variants with the samephenotype could have LOS molecules with different SDS-PAGGE profiles (compares lanes A2 and A3 in Fig. 6). Incontrast, some LOS antigenic variants of a given strain, asexemplified by strain BF101, did not have altered SDS-PAGGE profiles relative to the LOS of the original parentalstrain (compare lane Cl with lanes C2 and C3 in Fig. 6).

DISCUSSION

NTHI is an important concern in clinical medicine be-cause of the increased frequency of isolation of this organismfrom both pediatric and adult patients (5, 9, 14, 26, 30, 32, 37,39, 45, 47, 52-54). Although this relatively recent develop-ment may be due more to increased awareness and recogni-tion of NTHI as an important pathogen than to an actualincrease in infections caused by this organism (37, 39), thefact remains that this subset of H. influenzae organisms cancause disease in both the upper and lower respiratory tractas well as invasive infections. Although these organismshave in common the fact that they lack capsules detectablewith antisera to the polysaccharide capsules of H. influenzaeserotypes a to f, recent genetic evidence involving studies ofenzyme polymorphism indicates that each NTHI strain hasessentially unique metabolic enzyme characteristics, sug-gesting considerable genetic divergence among these strains(40).The ability to subtype NTHI strains is a prerequisite for

studying the epidemiology of NTHI carriage and disease andholds potential advantages for studies of both NTHI viru-lence mechanisms and immune response to this organism.However, perhaps because of the aforementioned variationin NTHI chromosomally encoded phenotypic markers,subtyping of NTHI strains has proven to be a fairly complex

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TABLE 4. LOS antigenic variation in NTHI strains"

StrainLOSantigeneNo.of colonies No. of colonies (%) reactive with the following MAbs:screened 4C4 3D9 12D9 17E12

OC201 4C4- 3D9+ 12D9+ 17E12+ 1,141 0 (0)1,226 933 (76)1,014 849 (84)842 683 (81)

BF101 4C4+ 3D9- 12D9- 17E12- 831 735 (88)802 76 (9)850 0 (0)555 0 (0)

N90 4C4+ 3D9+ 12D9- 17E12- 1,197 1,098 (92)1,031 1,021 (99)1,082 26 (2)754 70 (9)

BI101 4C4+ 3D9+ 12D9+ 17E12- 929 919 (99)978 957 (98)

1,085 922 (85)719 0 (0)

LA300 4C4+ 3D9- 12D9- 17E12+ 845 773 (92)826 24 (3)689 27 (4)635 575 (91)

a Reactivity with the MAbs was scored in the colony blot RIA.

task. Most studies on subtyping of NTHI strains haveinvolved methods previously found amenable for use withHib as well as with other pathogens. Barenkamp et al. (4)employed SDS-PAGE technology to characterize the outermembrane protein content of NTHI strains and found thatconsiderable diversity existed among these organisms interms of their outer membrane protein profiles. Similarly,Hansen et al. (18) reported that the outer membrane proteinsof NTHI strains appeared to be quite diverse in terms oftheir antigenic and immunogenic properties. Murphy et al.(35) later described an effective method for grouping NTHIstrains which used a simplified SDS-PAGE subtypingscheme involving outer membrane proteins, and more re-

Al A2 A3 A4

'Nwes.. OW" *00

B1 B2 B3 B4 B5

*:_"m2 ml

C1 C2 C3 DI D2 D3 D4 El E2 E3 E4

FIG. 6. SDS-PAGGE analysis of LOS from antigenic variants ofNTHI strains. Lanes: Al, OC201 (4C4- 3D9+ 12D9+ 17E12+); A2,12D9- variant of OC201; A3, 12D9- variant of OC201; A4, 17E12-variant of OC201; B1, LA300 (4C4+ 3D9- 12D9- 17E12+); B2, 4C4-variant of LA300; B3, 4C4- variant of LA300; B4, 3D9+ variant ofLA300; BS, 3D9+ variant of LA300; Cl, BF101 (4C4+ 3D9- 12D9-17E12-); C2, 3D9+ variant of BF101; C3, 17E12+ variant of BF101;Dl, N90 (4C4+ 3D9+ 12D9- 17E12-); D2, 4C4- variant of N90; D3,3D9- variant of N90; D4, 12D9+ variant of N90; El, BI101 (4C4+3D9+ 12D9+ 17E12-); E2, 4C4- variant of BI101; E3, 3D9- variantof Bl101; E4, 12D9- variant of BI101.

cently these workers devised a serological system forsubtyping NTHI strains based on the use of polyclonal rabbitsera to NTHI outer membrane proteins (33). It should benoted that even in this latter situation there was evidence ofantigenic heterogeneity among the outer membrane proteinsof strains in a given serogroup in their scheme (33), althoughmore recently these authors have identified an outer mem-brane protein which possesses a surface epitope common toall NTHI strains (34, 36).The fact that Hib strains can be subtyped according to

their LOS antigenic characteristics (16) prompted us toexamine the antigenic characteristics of NTHI LOS. Theprimary goal of this study was to provide a data base for usein subsequent studies of the pathogenesis and epidemiologyof diseases involving NTHI. Most previous investigations ofthe antigens of NTHI have concentrated primarily on func-tional studies involving the detection of bactericidal andopsonizing antibodies to this pathogen (3, 13, 25, 38, 46). Insome of these early studies, lack of appropriate technologyprecluded the characterization of the specificities of theseantibodies. More recently, two different groups have re-ported that antibodies to NTHI outer membrane proteins,rather than LOS, appear to be primarily responsible for thebactericidal or protective activities present in sera obtainedfrom humans convalescing from NTHI disease or fromanimals immunized with whole cells of this organism (3, 13,25). In contrast, antibody to NTHI LOS did not appear to beinvolved in these situations, because absorption of thesesera with purified NTHI LOS did not remove these bacteri-cidal or protective activities. Alternatively, the lack ofbactericidal or protective activity involving LOS-directedantibody in these studies could be due to a poor immuneresponse to this amphipathic molecule. In this regard, wehave shown that passive immunization of mice with a MAbto NTHI LOS markedly enhanced pulmonary clearance ofthis organism in an animal model system (E. J. Hansen,T. A. Loftus, and G. B. Toews, Program Abstr. 25th

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LOS OF NONTYPABLE H. INFLUENZAE 2909

Intersci. Conf. Antimicrob. Agents Chemother., abstr. no.282, 1985).

Information concerning the biochemistry and antigeniccharacteristics of NTHI LOS is still somewhat limited.Inzana (22) reported that the SDS-PAGE profiles of NTHILOS were not grossly dissimilar from those of Hib LOS,whereas Apicella et al. (2) reported that approximately halfof NTHI strains possess LOS with a lipid A moiety reactivewith a single MAb. More recently, Campagnari et al. (6)reported that a considerable degree of antigenic diversitywas present among the LOS molecules of NTHI strains. Theresults of these workers, obtained with polyclonal rabbitantisera, are confirmed by our MAb-based studies andreinforce the antigenic complexity of NTHI LOS. Thediversity of LOS phenotypes detected in both the previousstudy (6) and the present study indicate that subtyping ofNTHI strains by LOS antigenic phenotype is possible. ALOS-based subtyping system for NTHI has potential forapplication in epidemiological studies, although the sponta-neous LOS antigenic variation detected in the present studymay restrict somewhat the usefulness of such a subtypingscheme in field studies, as discussed below. Nonetheless,the ability to subtype accurately NTHI strains by LOSantigenic type will permit differentiation of strains by LOSphenotype and also should facilitate subsequent biochemicalanalysis of NTHI LOS structure.

It is also of interest to note that there exists considerablesharing or cross-reactivity of LOS oligosaccharide epitopesbetween NTHI and Hib strains. This finding complementsthe work of Apicella and colleagues, who reported that anMAb to H. influenzae lipid A reacted with approximatelyequal numbers of Hib and NTHI strains (2). These dataconcerning shared or common determinants in both theoligosaccharide and lipid A regions of NTHI and Hib LOSreaffirm the fact that these two different subsets of H.influenzae are probably derived from a common progenitorstrain (40).The MAbs used in this study were initially identified and

selected on the basis of their reactivity with surface-exposedepitopes in H. influenzae LOS. Therefore, it was somewhatsurprising to find that in some NTHI strains, which reactwith these same MAbs in antigen detection systems notrestricted to reactivity with surface antigens (i.e., colonyblot RIA, Western blot analysis), these LOS epitopes werenot accessible to antibody in their native state in wholeNTHI cells (Table 3). In contrast, our previous studies ofsurface-exposed epitopes of Hib outer membrane proteinsrevealed that a given epitope which is surface exposed onone Hib strain is also surface exposed on all other strainscarrying this epitope (27). The basis for this lack of antibodyaccessibility for certain LOS epitopes in these particularstrains ofNTHI is not known, but might possibly involve theinteraction of the LOS molecule with outer membraneproteins or other surface structures (33). Alternatively, it hasbeen shown that Hib strains phenotypically lose their abilityto bind LOS-directed polyclonal antibody to their surfaceafter incubation in serum (1, 23). Therefore, it is conceiv-able that these particular NTHI strains are constitutivelyexpressing this unusual phenotype, which reduces or elimi-nates the binding of antibody to surface-exposed LOS deter-minants. Studies involving this and other possible explana-tions for this phenomenon are currently in progress and willhopefully permit elucidation of the cause of this interestingvariation among NTHI strains in the surface exposure oftheir LOS epitopes.

This finding of variable surface exposure of NTHI LOS

epitopes is potentially important with regard to the interac-tion between NTHI and the immune defense system of thehost. Antibody to a given LOS epitope which is exposed onthe surface of one NTHI strain may not be able to bind toanother strain which possesses the same LOS epitope but ina location which is inaccessible to antibody. Such variationcould prevent LOS-directed antibodies from exerting anytype of protective effect against many NTHI strains.The instability of expression of LOS epitopes that was

observed in vitro in this study is similar to that recentlyfound with certain epitopes of Hib LOS (29). The molecularbasis for this phenomenon is unknown, but the fact that allfive randomly chosen NTHI strains exhibited LOS antigenicvariation suggests that most if not all strains of this organismmay undergo this type of antigenic variation, at least in vitro.This finding suggests that it may be difficult to utilizeconfidently NTHI LOS antigenic characteristics as pheno-typic strain markers in epidemiologic studies of NTHI dis-ease or carriage. Similarly, the fact that NTHI LOS anti-genic variation is often accompanied by changes in theSDS-PAGGE profile of the LOS (Fig. 6) may preclude theuse of these SDS-PAGGE profiles in epidemiologic studiesof NTHI disease. Which antigenic phenotype of LOS isexpressed in vivo by a given NTHI strain cannot be deter-mined from the available data, although certain LOS pheno-types may confer a selective advantage on Hib strains invivo (28). The availability of LOS antigenic variants of agiven NTHI strain now presents the opportunity for evalu-ating the effect(s) of changes in LOS phenotype on thesurvival of NTHI in vivo with appropriate animal models (3,25, 48). Future studies should indicate whether specificNTHI LOS phenotypes are important in vivo and whetherthe NTHI LOS phenotype affects the expression of viru-lence by this pathogen.

ACKNOWLEDGMENTS

We thank Robert Munford and Justin Radolf for their commentsconcerning this manuscript which was typed expertly by CindyBaselski.

This study was supported by Public Health Service grant A123366to E.J.H. from the National Institutes of Health.

LITERATURE CITED1. Anderson, P., A. Flesher, S. Shaw, A. L. Harding, and D. H.

Smith. 1980. Phenotypic and genetic variation in the suscepti-bility of Haemophilus influenzae type b to antibodies to somaticantigens. J. Clin. Invest. 65:885-891.

2. Apicella, M. A., K. L. Dudas, A. Campagnari, P. Rice, J. M.Mylotte, and T. F. Murphy. 1985. Antigenic heterogeneity oflipid A of Haemophilus influenzae. Infect. Immun. 50:9-14.

3. Barenkamp, S. J. 1986. Protection by serum antibodies inexperimental nontypable Haemophilus influenzae otitis media.Infect. Immun. 52:572-578.

4. Barenkamp, S. J., R. S. Munson, and D. M. Granoff. 1982.Outer membrane protein and biotype analysis of pathogenicnontypable Haemophilus influenzae. Infect. Immun. 36:535-540.

5. Berk, S. L., S. A. Holtsclaw, S. L. Weiner, and J. K. Smith.1982. Nontypable Haemophilus influenzae in the elderly. Arch.Intern. Med. 142:537-539.

6. Campagnari, A. A., M. R. Gupta, K. C. Dudas, T. F. Murphy,and M. A. Apicella. 1987. Antigenic diversity of lipooligosac-charides of nontypable Haemophilus influenzae. Infect. Immun.55:882-887.

7. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F.Smith. 1956. Colorimetric method for determination of sugarsand related substances. Anal. Chem. 28:350-357.

8. Flesher, A. R., and R. A. Insel. 1978. Characterization of

VOL. 55, 1987

on August 1, 2019 by guest

http://iai.asm.org/

Dow

nloaded from

2910 PATRICK ET AL.

lipopolysaccharide of Haemophilus influenzae. J. Infect. Dis.138:719-730.

9. Friesen, C. A., and C. T. Cho. 1986. Characteristic features ofneonatal sepsis due to Haemophilus influenzae. Rev. Infect.Dis. 8:777-780.

10. Galanos, C., 0. Luderitz, and 0. Westphal. 1969. A new methodfor the extraction of R lipopolysaccharides. Eur. J. Biochem.9:245-249.

11. Geohegan, W. C., and G. A. Ackerman. 1977. Adsorption ofhorseradish peroxidase, ovmucoid and immunoglobulin to col-loidal gold for the indirect detection of concanavalin A, wheatgerm agglutinin and goat anti-human immunoglobulin G on cellsurfaces at the electron microscopic level: a new method,theory, and applications. J. Histochem. Cytochem. 25:1187-1200.

12. Gilsdorf, J. R., and P. Ferrieri. 1986. Susceptibility of pheno-typic variants of Haemophilus influenzae type b to serumbactericidal activity: relationship to surface lipopolysaccharide.J. Infect. Dis. 153:223-231.

13. Gnehm, H. E., S. I. Pelton, S. Gulati, and P. A. Rice. 1985.Characterization of antigens from nontypable Haemophilusinfluenzae recognized by human bactericidal antibodies. J. Clin.Invest. 75:1645-1658.

14. Granoff, D. M., and G. A. Nankervis. 1975. Infectious arthritisin the neonate caused by Haemophilus influenzae. Am. J. Dis.Child. 129:730-733.

15. Gulig, P. A., and E. J. Hansen. 1985. Coprecipitation of lipo-polysaccharide and the 39,000-molecular-weight major outermembrane protein of Haemophilus influenzae type b by lipo-polysaccharide-directed monoclonal antibody. Infect. Immun.49:819-827.

16. Gulig, P. A., C. C. Patrick, L. Hermanstorfer, G. H. Mc-Cracken, Jr., and E. J. Hansen. 1987. Conservation of epitopesin the oligosaccharide portion of the lipooligosaccharide ofHaemophilus influenzae type b. Infect. Immun. 55:513-520.

17. Hansen, E. J., C. F. Frisch, R. L. McDade, Jr., and K. H.Johnston. 1981. Identification of immunogenic outer membraneproteins of Haemophilus inflienzae type b in the infant ratmodel system. Infect. Immun. 32:1084-1092.

18. Hansen, M. V., D. M. Musher, and R. E. Baughn. 1985. Outermembrane proteins of nontypable Haemophilus influenzae andreactivity of paired sera from infected patients with their homol-ogous isolates. Infect. Immun. 47:843-846.

19. Hawkes, R., E. Niday, and J. Gordon. 1982. A dot immunobind-ing assay for monoclonal and other antibodies. Anal. Biochem.119:142-147.

20. Hitchcock, P. J., and T. M. Brown. 1983. Morphological heter-ogeneity among Salmonella lipopolysaccharide chemotypes insilver-stained polyacrylamide gels. J. Bacteriol. 154:269-277.

21. Holmans, P. L., T. A. Loftus, and E. J. Hansen. 1985. Cloningand surface expression in Escherichia coli of a structural geneencoding a surface protein of Haemophilus influenzae type b.Infect. Immun. 50:236-242.

22. Inzana, T. J. 1983. Electrophoretic heterogeneity and interstrainvariation of the lipopolysaccharide of Haemophilus influenzae.J. Infect. Dis. 148:492-499.

23. Inzana, T. J., and P. Anderson. 1985. Serum factor-dependentresistance of Haemophilus influenzae type b to antibody tolipopolysaccharide. J. Infect. Dis. 151:869-877.

24. Inzana, T. J., W. E. Seifert, Jr., and R. P. Williams. 1985.Composition and antigenic activity of the oligosaccharide moi-ety of Haemophilus influenzae type b. Infect. Immun. 48:324-330.

25. Karasic, R. B., C. E. Trumpp, H. E. Gnehm, P. A. Rice, andS. I. Pelton. 1985. Modification of otitis media in chinchillasrechallenged with nontypable Haemophilius influenzae and sero-logical response to outer membrane antigens. J. Infect. Dis.151:273-278.

26. Khuri-Bulos, N., and K. McIntosh. 1975. Neonatal Haemophilusinfluenzae infection: report of eight cases and review of theliterature. Am. J. Dis. Child. 129:57-62.

27. Kimura, A., P. A. Gulig, G. H. McCracken, Jr., T. A. Loftus,and E. J. Hansen. 1985. A minor high-molecular-weight outermembrane protein of Haiemophilus inflienzae type b is a pro-

tective antigen. Infect. Immun. 47:253-259.28. Kimura, A., and E. J. Hansen. 1986. Antigenic and phenotypic

variations of Haemophilus influenzae type b lipopolysaccharideand their relationship to virulence. Infect. Immun. 51:69-79.

29. Kimura, A., C. C. Patrick, E. E. Miller, L. D. Cope, G. H.McCracken, Jr., and E. J. Hansen. 1987. Haemophilus influ-enzae type b lipooligosaccharide: stability of expression andassociation with virulence. Infect. Immun. 55:1979-1986.

30. Klein, J. 0. 1981. Microbiology and antimicrobial treatment ofotitis media. Ann. Otol. Rhinol. Laryngol. 90(Suppl. 84):30-42.

31. Loeb, M. R., and D. H. Smith. 1980. Outer membrane proteincomposition in disease isolates of Haemophilus influenzae:pathogenic and epidemiological implications. Infect. Immun.30:709-717.

32. May, J. R., N. C. Herrick, and D. Thompson. 1972. Bacterialinfection in cystic fibrosis. Arch. Dis. Child. 47:908-913.

33. Murphy, T. F., and M. A. Apicella. 1985. Antigenic heteroge-neity of outer membrane proteins of nontypable Haemophilusinfluenzae is a basis for a serotyping system. Infect. Immun.50:15-21.

34. Murphy, T. F., L. C. Bartos, A. A. Campagnari, M. B. Nelson,and M. A. Apicella. 1986. Antigenic characterization of the P6protein of nontypable Haemophilus influenzae. Infect. Immun.54:774-779.

35. Murphy, T. F., K. C. Dudas, J. M. Mylotte, and M. A. Apicella.1983. A subtyping system for nontypable Haemophilus influ-enzae based on outer membrane proteins. J. Infect. Dis.147:838-846.

36. Murphy, T. F., M. B. Nelson, K. C. Dudas, J. M. Mylotte, andM. A. Apicella. 1985. Identification of a specific epitope ofHaemophilus influenzae on a 16,600 dalton outer membraneprotein. J. Infect. Dis. 152:1300-1307.

37. Musher, D. M. 1983. Haemophilus influenzae infections. Hosp.Pract. 18:158-170.

38. Musher, D. M., M. Hague-Park, R. E. Baughn, R. J. Wallace,Jr., and B. Cowley. 1983. Opsonizing and bactericidal effects ofnormal human sera on nontypable Haemophilus influenzae.Infect. Immun. 39:297-304.

39. Musher, D. M., K. R. Kubitscheck, J. Crennan, and R. E.Baughn. 1983. Pneumonia and acute febrile tracheobronchitisdue to Haemophilus influenzae. Ann. Intern. Med. 99:444 450.

40. Musser, J. M., S. J. Barenkamp, D. M. Granoff, and R. K.Selander. 1986. Genetic relationships of serologically nontyp-able and serotype b strains of Haemophilus influenzae. Infect.Immun. 52:183-191.

41. Parr, T. R., Jr., and L. E. Bryan. 1984. Lipopolysaccharidecomposition of three strains of Haemophilus influenzae. Can. J.Microbiol. 30:1184-1187.

42. Peppler, M. S. 1983. Two physically and serologically distinctlipopolysaccharide profiles in strains of Bordetella pertussis andtheir phenotype variants. Infect. Immun. 43:224-232.

43. Robertson, S. M., C. F. Frisch, P. A. Gulig, J. R. Kettman,K. H. Johnston, and E. J. Hansen. 1982. Monoclonal antibodiesdirected against a cell surface-exposed outer membrane proteinof Haemophilus influenzae type b. Infect. Immun. 36:80-88.

44. Schneider, H., T. L. Hale, W. D. Zollinger, R. C. Seid, Jr.,C. A. Hammack, and J. M. Griffiss. 1984. Heterogeneity ofmolecular size and antigenic expression with lipooligosac-charides of individual strains of Neisseria gonorrhoreae andNeisseria meningitidis. Infect. Immun. 45:544-549.

45. Shann, F., G. Stephanie, D. Hazlett, M. Gratten, V. Linnemann,and R. Payne. 1984. Aetiology of pneumonia in children inGoroka Hospital, Papua, New Guinea. Lancet ii:537-538.

46. Shurin, P. A., S. I. Pelton, I. B. Tager, and D. L. Kasper. 1980.Bactericidal antibody and susceptibility to otitis media causedby nontypable strains of Haemophilus influenzae. J. Pediatr.97:364-369.

47. Simon, H. B., F. S. Southwick, R. L. Moellering, Jr., and E.Sherman. 1980. Haemophilus influenzae in hospitalized adults:current prospectives. Am. J. Med. 69:219-226.

48. Toews, G. B., S. Viroslav, D. A. Hart, and E. J. Hansen. 1984.Pulmonary clearance of encapsulated and unencapsulated Hae-mophilu.s influenzae strains. Infect. Immun. 45:437-442.

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http://iai.asm.org/

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LOS OF NONTYPABLE H. INFLUENZAE

49. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

50. Tsai, C. M., R. Boykins, and C. E. Frasch. 1983. Heterogeneityand variation among Neisseria meningitidis lipopolysaccha-rides. J. Bacteriol. 155:498-504.

51. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain fordetecting lipopolysaccharides in polyacrylamide gels. Anal.Biochem. 119:115-119.

52. Wald, E. R., G. J. Milmore, A. Bowen, J. Ledesma-Medina, N.Soloman, and C. D. Bluestone. Acute maxillary sinusitis inchildren. N. Engl. J. Med. 304:749-754.

53. Wallace, R. J., Jr., C. J. Baker, F. J. Quinones, D. G. Hoilis,R. E. Weaver, and K. Wiss. 1983. Nontypable Haemophilusinfluenzae (biotype 4) as a neonatal, maternal and genitalpathogen. Rev. Infect. Dis. 5:123-136.

54. Wallace, R. J., Jr., D. M. Musher, E. J. Septimus, J. E.McGowan, Jr., R. J. Quinones, K. Wiss, P. H. Vance, and P. A.Trier. 1981. Haemophilus influenzae infections in adults: char-acterization of strains by serotypes, biotypes, and P-lactamaseproduction. J. Infect. Dis. 144:101-106.

55. Zwahlen, A., L. G. Rubin, C. J. Connelly, T. J. Inzana, andE. R. Moxon. 1985. Alteration of the cell wall of Haemophilusinfluenzae type b by transformation with cloned DNA: associ-ation with attenuated virulence. J. Infect. Dis. 152:485-492.

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