INFECTION AND IMMUNITY, Apr. 1993, p. 1531-15370019-9567/93/041531-07$02.00/0

Antibodies to Haemophilus influenzae Type b OuterMembrane Proteins in Children with Epiglottitis or

Meningitis and in Healthy ControlsP. D. R. JOHNSON,* S. J. MACINNES, AND G. L. GILBERTt

Department ofMicrobiology and Infectious Diseases, Royal Children's Hospital,Melbourne, Victoria, Australia

Received 29 July 1992/Accepted 12 January 1993

The two most common manifestations of Haemophilus influenzae type b (Hib) infection in Westerncommunities are meningitis and epiglottitis. The role of antibodies against outer membrane proteins (OMP) inthe pathogenesis of these diseases was investigated by Western blotting (immunoblotting) with an OMP antigenprepared from a local Hib strain. Acute- and convalescent-phase serum samples from 25 children withepiglottitis and 20 with meningitis and single serum samples from 19 control children in the same age groupwere tested. Western blots were evaluated quantitatively by use of graphs generated from a densitometer. OMPantibody was detected in all sera from patients and controls. There was no significant difference between themean antibody level in acute-phase sera from children with meningitis (336 + 143 arbitrary units) and thosefrom children with epiglottitis (286 + 134 arbitrary units). However, the mean OMP antibody level in sera fromhealthy controls, with no known history of Hib disease, was significantly higher than that in sera from patientswith Hib disease within 2 days of admission to the hospital (patients [n = 35], 282 + 144; controls [n = 19],425 236; P = 0.007). The difference was due mainly to higher levels, in control sera, of antibody against fourproteins, one of which is either P1 or a comigrating protein of 49 kDa. The finding of higher levels of OMPantibody in healthy controls suggests a protective role for antibodies directed against one or more OMP. Thisinformation could be exploited in future vaccine development.

Haemophilus influenzae type b (Hib) causes invasivedisease in young children, who have not yet acquired anti-bodies to the polysaccharide capsule of Hib (1, 29). Menin-gitis and epiglottitis are the two most common manifesta-tions of Hib disease in Australian and other Westerncommunities (5, 9, 33). Although it is not known why a childdevelops epiglottitis instead of meningitis, children withepiglottitis tend to be older and to have lower-grade Hibbacteremia than those with meningitis (20, 39). This findingsuggests that patients with epiglottitis may be "partiallyimmune" at the time of their acute-phase infection and aretherefore better able to localize Hib, prevent prolongedhigh-grade bacteremia, and avoid infection of the centralnervous system. If this hypothesis were correct, patientswith epiglottitis would have higher levels of serum Hibantibody at the onset of infection than those with meningitis.Hib displays three types of antigen-capsular polysaccha-

ride, outer membrane proteins (OMP), and lipooligosaccha-ride. While antibody to capsular polysaccharide is the mostimportant determinant of host immunity, there is someevidence, mainly from animal models, that antibodies to oneor more OMP can also protect against invasive disease(10-15, 18, 19, 21, 23-27, 31).Using a semiquantitative Western blot assay, we mea-

sured levels of Hib OMP antibody in acute- and convales-cent-phase sera from patients with epiglottitis or meningitisand from healthy controls. We tested two hypotheses:firstly, that patients with meningitis have lower levels of HibOMP antibody than those with epiglottitis; and secondly,

* Corresponding author.t Present address: Department of Clinical Microbiology, Centre

for Infectious Diseases and Microbiology, Westmead Hospital,Westmead, New South Wales 2145, Australia.

that at the onset of their infection, patients with invasive Hibdisease have lower levels of Hib OMP antibody than healthycontrols.

MATERIALS AND METHODS

Patients and controls. Acute-phase sera were collectedduring hospitalization and convalescent-phase sera werecollected approximately 6 weeks after discharge from 45children who had invasive Hib disease and who were admit-ted to the Royal Children's Hospital between 1988 and 1990.Criteria for clinical case definition were as described previ-ously (9). Control sera were obtained from patients in thesame age group as those with Hib disease and whose bloodwas tested for cross-matching for miscellaneous surgicalprocedures during the 2 years of the study. Case recordswere reviewed for evidence of past or current Hib or otherinfectious disease. Sera collected from eight adults (labora-tory staff) and containing OMP antibody were pooled andused as positive control serum.

This study was reviewed and supported by the EthicsCommittee of the Royal Children's Hospital.Antigen (OMP) preparation. We showed previously (6)

that 83% of Hib isolates in Melbourne belong to one OMPsubtype (subtype 1 in the typing system described by VanAlphen et al. [36]). A locally collected Hib strain (RCH#50),obtained from the cerebrospinal fluid of a child with Hibmeningitis and belonging to this subtype, was used toprepare antigen for Western blotting (immunoblotting) by amodification of the method of Barenkamp et al. (2).Hib isolate RCH#50 was streaked onto chocolate agar

plates and incubated for 24 h at 37°C in 10% CO2. Twenty-milliliter starter cultures containing brain heart infusion brothsupplemented with NAD (Sigma, St. Louis, Mo.) and hemin

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(Calbiochem-Behring, La Jolla, Calif.) were inoculated fromthe chocolate agar plates. After overnight incubation, cultureswere diluted in 200 ml of supplemented brain heart infusionbroth and shaken for 4 h at 37°C. Cells were harvested in themid-log growth phase by centrifugation at 1,700 x g, washed,and resuspended in 1.5 ml of 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer. Cellswere frozen (-70°C) and thawed before sonication (four 15-sbursts; Mullard sonicator) on ice. Cellular debris was re-moved by centrifugation at 1,700 x g for 20 min, and thesupernatant was transferred to Eppendorf tubes. Cell enve-lopes were sedimented at 11,600 x g for 30 min at 4°C in amicrocentrifuge. The pellet was resuspended in sodium laur-ylsarcosine (Sigma), and the insoluble fraction was washedand collected by centrifugation as described above. The pelletwas resuspended in 200 RI of reducing sample buffer (63 mMTris base, 10% glycerol, 2% sodium dodecyl sulfate [SDS],5% beta-mercaptoethanol [pH 6.8]) and boiled for 5 min.Antigen was divided into aliquots and stored at -70°C untilrequired.Western blotting. The same OMP antigen preparation was

used throughout. Antigen preparation (600 ,ul) containing 160,ug of protein per ml (Lowry micromethod; Sigma) wasloaded across a single wide well of an 11% Laemmli gel andresolved by SDS-polyacrylamide gel electrophoresis(PAGE). Protein molecular weight standards (Bio-Rad,Richmond, Calif.) were run on each gel. Protein was trans-ferred to 0.45-,um-pore-size nitrocellulose paper (Schleicher& Schuell, Dassell, Germany) by use of a semidry electro-phoretic transfer cell (Transblot SD; Bio-Rad). A section ofthe blot including the molecular weight standards wasstained for protein with Aurodye colloidal gold (Amersham,Little Chalfont, United Kingdom), and the remainder wasstored in 10 mM Tris-150 mM NaCl-0.05% Tween 20-5%skim milk-0.02% azide (TBST-milk) until use. The latter wasthen cut into strips, incubated at 37°C with test serum diluted1:100 in TBST-milk for 3 h, washed, and incubated with goatanti-human immunoglobulin G conjugated with alkalinephosphatase (Tago, Burlingame, Calif.), diluted 1:500 inTBST, for 30 min at 37°C. Antibody bands were visualizedwith bromochloroindolyl phosphate and Nitro Blue Tetra-zolium (Promega, Madison, Wis.) in alkaline phosphatasebuffer (pH 9.8). The reaction was stopped after 2 min withdistilled water, and the nitrocellulose paper strips wereallowed to dry.

Analysis of results. After the completion of immunoblot-ting, there were 109 blots (19 controls and 45 acute- andconvalescent-phase pairs from patients), each showing up to20 bands. Because of the difficulty in interpreting thesequalitative data, a numerical assessment system was devel-oped.The numbering system for the Western blot bands was

devised by use of the adult pooled serum blot, a section ofthe blots stained for protein alone, and the positions ofmolecular weight standards. Twenty consistent bands repre-senting antibody to individual OMP were identified; 6 ofthese corresponded to the six major OMP identified byBarenkamp et al. (2).The Western blot strips were read on a logarithmic scale

by use of a densitometer (Quick Scan R&D model 1053;Helena Laboratories, Beaumont, Tex.), a device that pro-duces a graphic output representing the amount of incidentlight absorbed by a stained band on an electrophoresis gel orimmunoblot. The area under the whole curve generated bythe densitometer after reading of each Western blot strip wasassumed to be proportional to total OMP antibody, and the

area under the curve for a single band was assumed to beproportional to antibody directed against that particularOMP. To control for interassay variation, we includedpositive adult control pooled serum in each run to allowinterassay standardization. The densitometer positive gainwas set to 80% of maximum (log scale) by use of the bandrepresenting antibody to P6 and lipooligosaccharide on thepositive control blot for each assay.Because the widths of the bands on the Western blots

were similar, we analyzed the graph produced for each bandmathematically as if it were a rectangle with a width of 1unit. The area of the rectangle was therefore its height inmillimeters multiplied by 1. This assumption greatly simpli-fied analysis. A numerical value (the height of a graph peakin millimeters) was obtained for each band on each Westernblot and entered into a computer spread sheet and data base.The mean total OMP antibody levels in arbitrary units (logscale) for each of the following five groups were obtained:acute-phase epiglottitis (EA), acute-phase meningitis (MA),convalescent-phase epiglottitis, convalescent-phase menin-gitis (MC), and controls. Comparisons of mean individualOMP antibody levels were also made between groups.

Statistical methods. Continuous data were compared byuse of paired or unpaired Student's t test, analysis ofvariance, or linear regression as appropriate. Enumerativedata were analyzed by use of the chi-square statistic orFisher's exact test.

RESULTS

Comparison of groups. Acute- and convalescent-phaseserum pairs from 20 patients with meningitis and 25 withepiglottitis and single serum samples from 19 controls weretested.The groups were similar with respect to male/female ratio

(epiglottitis, 15:10; meningitis, 11:9; controls, 10:9), butthere were differences in age distribution. The age rangesand means ± standard deviations in months for each groupwere as follows: meningitis, 2 to 64, 20 ± 15; epiglottitis, 11to 98, 38 + 23; and controls, 6 to 57, 30 ± 15. The epiglottitispatients were significantly older than those with meningitis(P = 0.003), and the mean age of the controls was interme-diate. When results were pooled and analyzed for the effectof age, no correlation was found. Age adjustment of data wastherefore not performed.One control patient had a gram-negative urinary tract

infection at the time of serum collection, but otherwise nonehad any history of past or current Hib infection.OMP antibody assay. The coefficient of variation for

antibody directed to band 1 (a mixture of a 16-kDa proteinsynonymous with P6 and lipooligosaccharide) of the adultcontrol pooled serum was 21% in 13 assays. The intra-assayvariation (duplicate controls in the same assay) was 2% in 2assays. For reduction of error due to interassay variation,acute- and convalescent-phase serum pairs were run to-gether. The observed differences between control and acute-phase sera (see below) were confirmed by a repeat assay offive representative acute-phase serum samples and threecontrol serum samples (Fig. 1).

Total OMP antibody levels. Sample densitometer graphsgenerated from Western blots and protein stains of antigenare shown in Fig. 2. Results in arbitrary units (means ±standard deviations) for mean total OMP antibody levelswere as follows: MA, 324 + 191; MC, 366 ± 205; EA, 286 ±139; EC, 368 ± 143; and controls, 426 - 234. There was asignificant difference between EA and EC (P = 0.006) but

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acute-phase serum samples from patients with epiglottitis (E), andthe adult control pooled serum (Pool), assayed together. Ages inmonths (m) are indicated. The upper arrowhead indicates antibodyto a protein of 49 kDa (probably P1) and antibody to a 48-kDaprotein (immediately below the 49-kDa protein). The lower arrow-head indicates antibody to a minor OMP of approximately 34 kDaand present in two of three control samples and the pool but absentin acute-phase samples.

not between MA and MC (P = 0.18). EA and MA were notsignificantly different, but the trend was against our initialhypothesis, with MA being higher. The time from admissionto the hospital and the collection of acute-phase sera alsodiffered between the groups: MA, 2.8 + 2.3 days; and EA,0.7 + 0.8 days (P < 0.001). When total OMP antibody levelsin MA patients were subdivided on the basis of serumcollection time, there was a nonsignificant trend towardslater collection being associated with higher levels: MA, 0 to2 days [n = 11], 272 164; and MA, 3 to 7 days [n = 9], 387+ 211(P = 0.18). The mean for MA at 0 to 2 days was very closeto that for EA (286 + 139).There was no statistically significant correlation between

total OMP antibody levels and time of serum collection.However, when the four individual OMP antibody levelsthat differed the most between acute- and convalescent-phase sera were considered as a subgroup (48, 49, 55, and 85kDa), there was a significant relationship (r = 0.4; P =

0.013). The difference in sample collection times probablyexplains the observed difference between the two acute-phase serum groups. Therefore, for comparison with con-trols, all patient sera collected within 2 days of admission(n = 35) were analyzed together.When compared with healthy controls who had no known

history of Hib disease, Hib patients had significantly lower

mean levels of OMP antibody in acute-phase sera (controls,426 ± 235; Hib patients [n = 35], 282 ± 146; P = 0.008).During convalescence, a significant increase in total OMPantibody levels was observed for all Hib patients together(n = 35) (EA and MA, 281 + 146; EC and MC, 356 + 170;P = 0.003). The mean antibody levels for the control, EC,and MC groups and all convalescing patients grouped to-gether were not significantly different.

Antibodies against individual OMP. Results for each groupwere also obtained for antibody directed against each of 20individual OMP (Fig. 3). When the control sera were com-pared with the acute-phase sera from Hib patients (n = 35),antibodies against four OMPs with apparent molecularmasses of 34, 39, 48, and 49 kDa contributed most signifi-cantly (P < 0.005) to the difference in total antibody betweenthe two groups. When acute- and convalescent-phase serafrom Hib patients were compared, levels of antibodies to thesame 48- and 49-kDa OMP and two additional OMP (ca. 58and 88 kDa) showed the greatest increase in convalescent-phase sera (P < 0.007) (Fig. 4). Levels of antibodies to these58- and 88-kDa OMP were also higher in control sera than inacute-phase sera, but the difference was less significant(58-kDa OMP, control sera, 38 + 31 units; acute-phase sera,22 ± 25 units; P = 0.04; 88-kDa OMP: control sera, 28 ± 26units; acute-phase sera, 15 ± 16 units; P = 0.03).A comparison with OMP preparations from standard

isolates kindly supplied by L. Van Alphen and S. J. Baren-kamp established that the 49-kDa protein either is P1 ormigrates with P1. P1 is a major OMP identified in the OMPsubtyping system described by Barenkamp et al. (2).Age and OMP antibody. Patient age did not influence the

levels of total OMP antibody in acute-phase sera or thedegree of response in convalescent-phase sera. Controlpatients also failed to show any association between levels oftotal OMP antibody and age. However, when antibodies toindividual OMP were analyzed separately, control sera butnot acute-phase sera showed a significant positive correla-tion between age and levels of antibody against the 49-kDaOMP (control sera: r = 0.46; P = 0.05; acute-phase sera: r =0.002; P = 0.99).

Antigenicity of different OMP subtypes. Eighteen of 21(86%) available Hib isolates from the 25 patients withepiglottitis and 14 of 19 (74%) available isolates from the 20patients with meningitis were OMP subtype 1VA. No isolatewas available for OMP analysis from four patients withepiglottitis and one patient with meningitis. The eight non-1VA patient isolates, classified on the basis of the system ofBarenkamp et al. (2), were 14L (six isolates: four meningitisand two epiglottitis), 1L (epiglottitis), and 6U (meningitis).Mean ± standard deviation (arbitrary units) total acute- andconvalescent-phase serum OMP antibody levels for the eightpatients with non-1VA isolates were 216 ± 120 and 284 ±153, respectively (P = 0.013, paired t test). There was alsoan increase in the levels of antibody to the 49-kDa OMP inpatients with non-1VA isolates (acute-phase sera, 2 ± 3;convalescent-phase sera, 10 + 15; P = 0.1). These resultsdid not differ significantly from those obtained for patientswhose isolates were subtype 1VA.

Anti-Hib capsular antibody in acute-phase sera. Anti-Hibcapsular immunoglobulin G was measured in patient andcontrol sera by use of a capture enzyme immunoassay (16).The results were expressed in enzyme immunoassay units byreference to a positive control serum from a convalescingepiglottitis patient. The positive and negative controls usedin this assay were also tested for anticapsular immunoglob-ulin G by use of a standardized enzyme immunoassay (28) by

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FIG. 2. (Lower panel) SDS-PAGE of antigen after transfer to nitrocellulose paper and staining with Aurodye colloidal gold (protein stain).The corresponding densitometer graph is immediately above the antigen preparation. (Upper panel) Same antigen preparation after Westernblotting with adult control serum and the corresponding densitometer graph. Anti-Pl, anti-P2, and anti-P6 refer to antibodies to bands withapproximate apparent molecular masses of 49, 39, and 16 kDa, respectively. The antibodies recognised these named proteins or comigratingproteins within the same bands (see discussion). The band labelled anti-P6 includes antibody to lipooligosaccharide, which runs with the16-kDa OMP in SDS-PAGE. MW Stds., molecular weight standards (in thousands).

M. Hanlon, Children's Hospital, Camperdown, Sydney,New South Wales, Australia. The geometric means for thethree groups tested, EA, MA, and controls, were not signif-icantly different from each other (EA, 0.01 + 0.002; MA,0.012 + 0.004; controls, 0.008 + 0.003 ,ug of anticapsularimmunoglobulin G per ml; P = 0.6).When age and levels of anticapsular antibody were com-

pared, there was a trend towards a correlation betweenincreasing age and antibody levels in control sera but a muchweaker trend in acute-phase sera (controls: r = 0.4; slope,

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1.3; acute-phase sera: r = 0.2; slope, 0.33; P = 0.07 for thedifference between the slopes of the two regression lines).No correlation was found between levels of antibody to

the 49-kDa OMP and anticapsular antibody in the controls (r= 0.03; P = 0.92).

DISCUSSION

The system of Western blot quantitation described wasdeveloped because of the difficulty of interpreting over 2,000

16 19 26 28 30 31 33 34 37 3838.539 48 49 57 58 77 88 98 105

Approx. MWFIG. 3. Column graph showing mean + standard deviation OMP antibody levels for individual OMP in control (10) and acute-phase (U)

sera. The horizontal axis shows the approximate apparent molecular weight (MW) (in thousand) of the OMP against which antibody isdirected. The vertical axis shows arbitrary units on a log scale. Asterisks denote statistically significant differences at the 0.5% level. In thesubtyping system developed by Barenkamp et al. (2), the 49-, 39-, and 16-kDa OMP are equivalent to P1, P2, and P6, respectively. Antibodyto P6 includes antibody to lipooligosaccharide, which comigrates with the 16-kDa OMP in SDS-PAGE.

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FIG. 4. Western blots of acute- and convalescent (conv.)-phase serum pairs from six patients from whom an acute-phase serum samplewas collected within 2 days of admission to the hospital. The blots are displayed with antibodies to the higher-molecular-weight proteinstowards the top of the figure. Serum pairs were assayed together. Differences in band positions between patients are due to interassayvariations during electrophoresis. Arrows mark antibodies to three OMP with approximate molecular masses of 88, 58, and 49 kDa. Age inmonths (m) and diagnosis (epiglottitis [E] or meningitis or [M]) are indicated.

bands on 109 Western blots (45 acute- and convalescent-phase serum pairs and 19 controls) without a priori knowl-edge of what differences, if any, would be found. Assigningeach Western blot band a value representing its intensityallowed us to easily compare antibody levels for individualpatients and for individual OMP by use of a computer spreadsheet. Although Western blotting is not primarily a quanti-tative method, other investigators have performed densito-metric analyses of Western blots and found a quantitativerelationship between the area under the curve and theamount of antibody detected (7, 38).

Despite evidence that antibodies to certain OMP can

protect against experimental Hib infection, there are fewstudies concerning the role of OMP antibody in humaninfection. It has been suggested that anticapsular antibodyalone is probably not sufficient to explain natural immunityto Hib (17). However, most investigators have found (as wedid) that OMP antibody is universally detectable in theacute-phase sera of patients with Hib disease and concludedthat it is not protective (4, 37). In a previous study, it wasfound by Western blotting that significantly more controlsthan patients had antibody directed against an OMP of 40kDa (8). The identity of this 40-kDa protein is unclear.Our findings that control sera had significantly higher

levels of total OMP antibody than acute-phase sera and thatcontrols had the same low levels of anticapsular antibody as

children with invasive disease provide circumstantial evi-dence that OMP antibody is partially protective.An alternative explanation for the lower levels of OMP

antibody in acute-phase sera than in control sera is theabsorption of OMP antibody by living or lysed Hib. If suchwere the case, levels of antibody to all Hib OMP and Hibcapsule should be similarly affected. This idea is contrary toour findings that the levels of antibody to only selected OMPwere lower in patients than in controls and that anticapsularantibody did not differ between the two groups.The difference between acute-phase sera from Hib pa-

tients and controls was mainly due to antibodies directedagainst four OMP with approximate molecular masses of 34,39, 48, and 49 kDa. The 49-kDa protein is likely to be P1 onthe basis of the subtyping system developed by Barenkampet al. (2). It is also possible that the antibody is directedagainst a protein that comigrates with P1. The 48-kDaprotein appears as a band immediately below P1 on theWestern blots, does not correspond to a visible protein inSDS-PAGE, and may possibly be a dimorphic form of P1that retains antigenicity. The 49-kDa OMP is also one of thethree OMP associated with most of the increase in total OMPantibody levels in convalescent-phase sera. The 39-kDaprotein is probably P2. However, the abundance of P2 in theantigen preparation may have obscured a comigrating pro-tein subsequently revealed by immunoblotting. Denaturation

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of certain protein antigens, including Hib P2, sometimesresults from the relatively harsh conditions used in SDS-PAGE (14).

In common with other investigators (4, 22), we found norelationship between age and total acute- and convalescent-phase OMP antibody levels in children with invasive dis-ease. However, levels of antibody to the 49-kDa OMP didincrease with increasing age in control sera but not inacute-phase sera, a result raising the possibility that theacquisition of this antibody may contribute to the well-established age specificity of Hib disease. Furthermore, thelack of correlation between levels of anticapsular antibodyand antibody to the 49-kDa OMP in controls suggests thatthese antibodies are acquired independently. This findingfurther supports the possibility that this OMP antibody couldbe protective.OMP antibody may be strain specific (12, 31). However, in

the eight patients in this study who were not infected withthe common 1VA strain, significant increases in convales-cent-phase total OMP antibody levels were observed. Al-though the sample size was small, this result suggests thatthere are no major antigenic differences between the 1VAsubtype used as an antigen in this study and OMP subtypes14L, 6U, and 1L.Although the pathogenesis of epiglottitis is not under-

stood, several differences between children with epiglottitisand those with meningitis caused by Hib have been demon-strated. Children with epiglottitis tend to be older (5, 10, 33,39), to have lower-grade bacteremia (20), and to respondmore vigorously to Hib capsular antigen in convalescence(16, 39) and may have different genetic markers (39). Al-though there is evidence that some Hib subtypes are morelikely to cause meningitis than epiglottitis (34), this finding isnot a universal one (6, 35). In this study, 18 of 25 patientswith epiglottitis and 14 of 20 patients with meningitis hadisolates belonging to Hib OMP subtype 1VA.

It has been suggested that epiglottitis may be a form ofhyperresponsiveness to Hib, perhaps in a previously ex-posed child (3). In contrast, several studies have concludedthat children with meningitis may represent a subset of poorresponders to the capsular polysaccharide of Hib, evenwhen older than 24 months (10, 16, 30, 39). If the develop-ment of epiglottitis rather than meningitis were related topreexisting partial immunity, patients with epiglottitis shouldhave demonstrably higher levels of Hib antibody than thosewith meningitis. There appear to be no differences in thelevels of acute-phase anticapsular antibody (3, 5, 16). Thisstudy has shown that there are no observable differences inacute-phase OMP antibody levels between patients withepiglottitis and those with meningitis and that both groupsrespond equally well to OMP antigens. However, it ispossible that epitope denaturation resulting from SDS-PAGE may have prevented us from finding a real difference.The response to certain OMP appeared to be rapid,

developing within a few days of admission to the hospital.This observation was important in the analysis of results.Children with epiglottitis have a very rapid onset of symp-toms, recover quickly, and are hospitalized for only 3 to 4days in our community. Those with meningitis usually havea longer prodrome and spend 10 to 14 days in the hospital (9).Sera taken at different times during hospitalization may notbe representative of events early in the process of infection.Failure to take into account the timing of serum collectionmay lead to the erroneous conclusion that children with Hibmeningitis respond poorly to protein antigens (32).

In conclusion, we have been unable to prove our initial

hypothesis that patients with Hib epiglottitis are partiallyimmune at the onset of illness compared with those with Hibmeningitis. However, the finding that sera from healthycontrols have significantly higher levels of OMP antibodythan acute-phase sera from patients provides circumstantialevidence that OMP antibody may be protective in humans.Because of the success of current conjugate Hib vaccines, itis unlikely that this finding will contribute to the preventionof Hib disease. However, the possible protective role ofOMP antibody in Hib disease could be exploited in theprevention of other infections caused by encapsulated patho-gens.

ACKNOWLEDGMENTS

This study was supported by a project grant (to G.L.G.) and apostgraduate medical research scholarship (to P.D.R.J.) from theNational Health and Medical Research Council (Australia).We thank John Carlin and Ted Byrt of the Clinical Epidemiology

and Bio-statistics Unit, Royal Children's Hospital, for their assis-tance and advice with the statistical analysis and M. Hanlon and D.Isaacs of the Department of Immunology and Infectious Diseases,Children's Hospital, Camperdown, Sydney, New South Wales,Australia, for checking and quantitating the positive controls used inthe capsular enzyme immunoassay.

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2. Barenkamp, S. J., R. S. Munson, and D. M. Granoff. 1981.Subtyping isolates of Haemophilus influenzae type b by outermembrane protein profiles. J. Infect. Dis. 143:668-676.

3. Branefors-Helander, P., and P. H. Jeppsson. 1975. Acute epi-glottitis: a clinical, bacteriological and serological study. Scand.J. Infect. Dis. 7:103-111.

4. Claesson, B. A., T. Lagergard, B. Trollfors, L. Gotherfors, andU. Jodal. 1987. Serum antibody response to capsular polysac-charide, outer membrane and lipooligosaccharide in childrenwith invasive Haemophilus influenzae type b infections. J. Clin.Microbiol. 25:2339-2343.

5. Claesson, B. A., B. Trollfors, B. Ekstrom-Jodal, P.H. Jeppsson,T. Lagergard, 0. Nylen, and P. Rigner. 1984. Incidence andprognosis of acute epiglottitis in children in a Swedish region.Pediatr. Infect. Dis. J. 3:534-538.

6. Clements, D. A., S. J. MacInnes, and G. L. Gilbert. 1992. Outermembrane protein subtypes of Haemophilus influenzae type bcausing invasive disease in Australia, from 1988 to 1990. J. ClinMicrobiol. 30:1879-1881.

7. Dalmau, J., H. M. Furneaux, R. J. Gralla, M. G. Kris, and J. B.Posner. 1990. Detection of the anti-Hu antibody in the serum ofpatients with small cell lung cancer-a quantitative Western blotanalysis. Ann. Neurol. 27:544-552.

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