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Antibody responses to capsular polysaccharide, lipopolysaccharide, and outer membrane in adults infected with Haemophilus influenzae type b

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JOURNALOFCLINICALMICROBIOLOGY, Dec. 1984, p. 1154-1158 0095-1137/84/121154-05$02.00/0

Copyright ©3 1984, American Society forMicrobiology

Antibody

Responses

to

Capsular

Polysaccharide,

Lipopolysaccharide,

and Outer Membrane in

Adults Infected with

Haemophilus influenzae Type b

T. LAGERGARD,1 O. NYLEN,2 T. SANDBERG,3 ANDB. TROLLFORS3

Departments ofMedical Microbiology,1 Otorhinolaryngology,2 and InfectiousDiseases,3 Universityof Goteborg,

Goteborg, Sweden

Received 9 July 1984/Accepted 11 September 1984

The antibody responsetoHaemophilus influenzaetypeb antigensas capsular polysaccharide,

lipopolysac-charide, andoutermembrane components wasstudied by enzyme-linked immunosorbent assayin 10 adults infected with this bacterium. Almost all patients had detectable amounts of immunoglobulins G and M

antibodies specific to capsular polysaccharide, lipopolysaccharide, or outer membrane in their first serum sample. A significant antibody response in one or more antibody subclasses to capsular polysaccharide,

lipopolysaccharide, andoutermembranewasnoted in9 of 10, 7of10, and 7of10 patients, respectively.The

occurrenceofH. influenzaetypebinfections in adultscannotbe completely explained eitherby the absenceof

antibodies against H. influenzae type b in theirserum or by the failureto develop specificantibodies tothe

capsuleorcertaincell wallcomponentsevenif these factors probablyareimportantinsome cases,e.g.,inone patientwho wassplenectomized.

Haemophilus influenzae type b infections occurs almost exclusively in children and arerarely seen inadults(7, 13).

However, during the last decade, several studies have drawn attention to H. influenzae infections in adults (6, 8, 10, 12, 14, 15). It is not possible to determine from the literature whether there has been a true increase in H. influenzae infections in adults or whether the large number ofpapers

reflects an increased awareness of the condition. The rare occurrence of these infections in adults has beenassociated

with the presence of protective antibodies against capsular

polysaccharide (CPS)typebinadultsera(7, 11)as aresult of

either previous exposure and immunological response to

these bacteria or exposure to other bacteria with antigens cross-reacting with polysaccharidetypeb(11).Eventhough it is known that most adults with H. influenzae infections have conditions predisposing them to infections, little is

known about theimmunology of infections caused by

capsu-lated H. influenzae.

The aim of the present study was to investigate the

preexisting antibody levels and the antibody response to

differentcapsular polysaccharide, lipopolysaccharide (LPS), and outer membrane (OM) antigens of H. influenzae in

adults infected with thisbacterium.

MATERIALS ANDMETHODS

Patients. Serum samples from10patientswith H.

influen-zaetypeb infections wereanalyzed. Clinicalcharacteristics

of the patients aresummarized in Table 1.

Antigens. CPS was prepared from H. influenzae type b

(strain RAB)aspreviously described (2). The concentrated

culture supernatant was precipitated with cetavlon andgel filtered through Sephadex 2-B. LPS froma

non-encapsulat-edmutantofH. influenzaetypeb(strain RAB)wasprepared

by extraction with hot phenolwater(1). AnOMpreparation ofthe aforementioned non-encapsulated mutant was made

aspreviously described (3).

Antibody determination. Enzyme-linked immunosorbent assaywasperformedonmicroplatescoated withCPS, LPS, andOM antigens in concentrations of30, 50to75, and 100

p.g/ml, respectively (1, 2).The serumsamplesweretested in

serial 10-fold dilutions, and antibodies were demonstrated

with alkaline phosphatase-coupled swine antibodies against human immunoglobulin to immunoglobulin G (IgG), IgM, andIgA (Orion, Helsinki, Finland); the antibody

concentra-tion in the serum was expressed as

-logl0

value of the

highest serum dilution showing an extinction value of 0.2

above the background. The titer assigned to each sample

was the mean of two determinations. A threefold (0.5

-logl0)

orgreaterincrease in titerwasdefinedassignificant.

RESULTS

The antibody responses toCPS type b, LPS, and OM of the 10 adults studied are shown in Fig. 1, 2, and 3,

respec-tively.

All patients had detectable amounts of IgG and IgM

antibodies specifictoCPS of H. influenzae typeb (Fig. 1)in their firstserumsamples, whichwereobtainedwithin1to10

daysafteronsetofsymptoms. TheIgGandIgMtiters in the firstserumsamples rangedfrom 1.4to2.6 and from 1.2to2.3

-log10, respectively. IgA antibodiesweredetectedin 6of10 initial serum samples.

Asignificant antibodyresponseinoneor moresubclasses

was registered in all patients except one. Significant

in-creases in IgA, IgM, and IgG antibodies were seen in six, nine, and eight patients, respectively. The splenectomized patient (no. 3) failed to develop an increase in any of the threeantibodysubclasses. Twopatients, aged38to43years, withnoknownimmunodeficiencies(no. 5 and6), developed

no IgG antibodies but had pronounced increases in IgM

antibodies. The other seven patients, including no. 2 with

hypogammaglobulinemia, respondedwith bothIgMandIgG

antibodies.

The kinetics of the antibody response to CPS and the maximum levels achieved were highlyvariable (Fig. 1). In the twopatients with initial antibody response, in whom it

was possible to follow antibody levels for more than 3 months(no. 2and9),theIgGand IgMlevels remainedhigh during the whole observationperiodofca. 5 months.

The antibody responses to LPS are shown in Fig. 2. All

patients exceptone (no. 2)had detectable amountsof both 1154

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TABLE 1. Clinical characteristics of 10 patients with H. influenzaeinfections

Source

Patient Age Sex Predisposing condition Infectiousmanifestation of

no. (yr) isolatea

1 22 F Pregnant,week35 Meningitis, sinusitis Bi, Csf

2 29 F Hypogammaglobulinemia Epiglottitis Bi, Nph

3 29 F Splenectomized Pneumonia, septic shock Bi, Nph

4 31 F Epiglottitis BI

5 38 M (Mitral stenosis) Epiglottitis Bi

6 43 F Bronchiectasies Pneumonia Bi

7 43 F Meningitis Csf

8 46 M Sinusitis, septicaemia BI

9 57 F Chronicthyroiditis Pleuropneumonia, pericarditis Bi, P1

10 60 F Epiglottitis Nph

a

Bi,

Blood; Csf,cerebrospinal fluid;

P1,

pleuralfluid;Nph,nasopharyngealsmear.

1--* 60-0

26-6 7v..v IgA

30-0

8.--A

40-0 9o....o

5.--

10*

...*

1gM

1~~~~~~~~~~~~~

Ig

G

._.

.

.V.~~~~~~~~~~~~~~~~~~~.

.. .

0 20 40 60 80 100 160 18(

DAYS

FIG. 1. AntibodyresponsetotypebCPS in10patients withH. influenzaetypeb infection (patient numbers correspondtothosein Table

1). Day 0= onsetofsymptoms.

3- 1-<1

4-P

vE3

I

3-co

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1156 LAGERGARD ET AL.

Ig A v

lgM

IgG

V..~~~~~~~...

66.1~ ~ ~ ~ ~ ~ ~ L~

0 20 40

DAYS

FIG. 2. Antibodyresponseto typebLPSin 10patients withH.influenzaetypebinfection.Day0=onsetofsymptoms.See Fig. 1 for

def-inition of symbols.

IgGand IgMantibodies in their first serumsample. The IgG

andIgM antibodytitersrangedfrom 1.4to3.75 and from 1.0 to3.3

-logl0,

respectively. IgAantibodies weredetected in 8 of 10 serum samples.

Asignificant antibody increase inone or more subclasses

wasnoted in7of 10patients.Thethreepatientswho failedto

develop an antibody increase to LPS were no. 3,7, and 8.

Two of these patients (no. 7and 8) hadhighantibody titers

already in their first serum sample; the third patient was

splenectomized.

The antibodyresponse to OM is shown in Fig. 3. In the

first serum sample, antibodies ofIgG, IgM, and IgA

sub-classesweredetectable in all 10patients. The IgG andIgM

titers ranged from 2.2 to 3.5 and from 1.1 to 3.2 logl0, respectively. Asignificantantibodyresponseinone or more

subclasses was noted in 7 of 10 patients. The patients who didnot respondwere no. 3, 5, and 8.

The patterns of the antibody response to OM and,

espe-cially, toLPS were highlyvariable (Fig. 2 and3).

DISCUSSION

Thisstudy examined theantibodyresponsetoCPS, LPS,

andOM in adults withinvasive H. influenzaetype b infec-tions. Allbutoneof the 10patients studied had, in their first serum sample, levelsofspecific IgG and IgM antibodiesto CPS comparable to levels seen in healthy adults (1). Even

thoughtheserumantibody levels before thestartofinfection

are notknown,thetimebetweenonsetofsymptomsand the

first serumsamplewasinmostcases soshort that itmaybe

assumed that the patients developed their infection despite preexisting specific IgM and IgG antibodies. The only

pa-tientwithlowinitial antibodieswas asplenectomized wom-an withverylowIgM antibodies but with IgG antibodiesof

2.1 -log10on day5ofinfection, which is close tothemean

level inhealthyadults(1). This splenectomized woman was

also the only patient who failed to develop a significant

antibody increase to CPS in any of the three antibody

subclasses. It is wellknownfrom other studies that

splenec-1

4

0)

0-co

°73

cc

w

2 CO)

-i

w 1* 4.

1

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ANTIBODY RESPONSES IN ADULTS INFECTED WITH H. INFLUENZAE 1157

4.

3.

2

0

r) 0

3-co w

2-C') w

1

4-3.

2

0 20

1g

A

Ig

M

0.. ...0

I

N;;...

Ee~~~~~~~~~~...

A

--..

0-0

,

,

.

.

.

}

w/

o~~~~~~~~~~1

G

A

X-

~~~~~~~~~~~~~~~~~~~~~...

~~~~~po

~ ~ ~

..:

40 60 80 100 160 180

DAYS

FIG. 3. Antibodyresponse to type b OM in 10patients withH.influenzaetype binfection.Day0=onsetof symptoms. See Fig. 1 for defi-nition of symbols.

tomized patients sometimes

respond

poorly

to

polysaccha-rides of another encapsulated

organism,

Streptococcus pneumoniae (16), and that

they

are

highly susceptible

to

infections with this

organism

(14). Splenectomized patients

arealsoat risk of

becoming

infected withH.

influenzae (4),

and the absence ofan antibody response in our splenecto-mized patient suggests thatthis

susceptibility

mightbe due

to an inability to develop protective antibodies after

expo-sure.

Two middle-aged patients with no known

immunodefi-ciencies failed to develop a

significant

increase in

IgG

antibodiesagainsttype b

polysaccharide,

butthey both had pronouncedincreases in IgMand IgAantibodies. Theirage makes itunlikelythat they shouldnotpreviously have been exposedtoH. influenzaetype b. Sincetheyfailedtodevelop IgGantibodies, whichareresponsible forlastingimmunity,

oneexplanationfor thedevelopmentofH.influenzaetypeb infection in some adults might be an inability of certain individualstodevelop protective IgG antibodies after infec-tion. This explanation, however, cannot be valid for all adults withH. influenzaetypebeinfections, since sixofthe patients studied here developed their invasive infections despitenormal levels ofspecific IgGand IgMantibodiesand

all ofthese adults werecapable ofresponding to the infection with further antibody increases. In such patients, other explanations for the development of invasive H. influenzae type b infectionsmust be considered.

Antibodies to other components of H. influenzae are probablyconsiderably less important forprotective immuni-tythan are antibodies to CPS. It has been shown in children thatantibodies to LPS do not confer protection (9). It has been suggested, however, that antibodies to components other than CPS might contribute to the immunity, since naturally acquired antibodies to CPS offer protection at lower serum levels than antibodies induced by a purified CPS vaccine (5). The results of the present study indicate that invasive infections in adults with H. influenzae type b can occurin the presence ofserum antibodies against both LPS and OM. The majority of our adult patients infected with this organism were also capable of producing serum antibodies ofdifferent subclasses to both of these bacterial components aswellas to CPS.

The occurrence of invasive H.

influenzae

type binfections inadultstherefore cannot be completely explained by either the absence of previous exposure to the organism or by a failure of theindividualsaffected todevelop specific

antibod-VOL. 20,1984

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1158 LAGERGARD ET AL.

iesto thecapsuleorcertain cell wallstructures,eventhough these factorsprobablyareofimportance in some cases.

LITERATURE CITED

1. Dahlberg, T. 1981. Quantitation of antibodies to Haemophilus influenzae type b in humans by enzyme-linked immunosorbent assay. J. Clin. Microbiol. 13:1080-1087.

2. Dahlberg, T., and P. Branefors. 1980. Enzyme-linked immuno-sorbentassay for titration of Haemophilus influenzae capsular and0 antigen antibodies. J. Clin. Microbiol. 12:185-192. 3. Dahlberg-Lagergard, T. 1982. Target antigens for bactericidal

and opsonsizing antibodies to Haemophilus influenzae. Acta Pathol. Microbiol. Scand. Sect. C 90:209-216.

4. Ferguson, A. 1982. Hazards of hypospleenism. Br. Mol. J. 285:1375-1376.

5. Kayhty, H., H. Peltole, V. Karanko, and H. Makala. 1983. The protective level of serum antibodies to the capsular polysaccha-ride of Haemophilus influenzae type b. J. Infect. Dis. 147:1100-1105.

6. Levin,D.C., M. I. Schwarz, R. A. Matthay, and F. M. LaForce. 1977. Bacteremic Haemophilusinfluenzaepneumonia inadults. Am.J. Med. 62:219-224.

7. Norden, C. W. 1974. Haemophilus influenzae in adults. Med. Clin. North. Am.622:1037-1039.

8. Sandberg, T., and T. Dahlberg. 1981. Meningitis and

septicae-mia due toHaemophilus influenzaetype b in pregnancy. Br. Med. J. 282:946.

9. Shenep, J. L., R. S. Munson Jr, and D. M. Granoff. 1982. Humanantibody responsestolipopolysaccharide after

meningi-tis due to Haemophilus influenzae type b. J. Infect. Dis. 145:181-190.

10. Simon, H. B., F. S. Southwick, R. C. Moellering, and E. Sherman. 1980. Haemophilusinfluenzaeinhospitalisedadults: currentperspectives. Am.J. Med. 69:219-226.

11. Solorotovsky, M.,and M.Lynn. 1978.Haemophilusinfluenzae:

immunology and immunoprotection. Crit. Rev. Microbiol. 61:11-32.

12. Spagnulo, P. J., J. J. ElIner, P. I. Lerner, M. C. McHenry, F. Flatauer, P. Rosenberg, and M. S. Rosenthal. 1982. Haemophi-lus influenzae meningitis: the spectrum of disease in adults. Medicine 61:74-85.

13. Turk, D. C., and I. R. May. 1967. Haemophilus influenzae: its clinical importance. English University Press, London. 14. Wallace R.J.,D. M.Musher,and R.R. Martin.1978.

Haemo-philus influenzae pneumonia in adults. Am. J. Med. 64:87-93. 15. Wallace,R.J.,D. M.Musher,E.J.Septimus, J.E.McGowan,

F. J. Quinones, K. Wiss, P. H. Wance, andP.A. Trier. 1981. Haemophilus influenzae infections in adults:characterization of strains by serotypes, biotypes and P-lactamase production. J. Infect. Dis. 144:101-106.

16. Wara, D. W. 1981. Host defence against Streptococcus pneu-moniae: therole ofthe spleen. Rev. Infect. Dis. 3:299-309.

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