Antibody responses to serogroup B meningococcal outer membrane antigens after vaccination and infection

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0095-1137/88/081543-06$02.00/0

CopyrightC 1988, American Society for Microbiology

Antibody

Responses

to

Serogroup

B

Meningococcal

Outer

Membrane Antigens

after

Vaccination and Infection

EINAR

ROSENQVIST,1*

STIG HARTHUG,2 L. ODDVARFR0HOLM,' E. ARNE

H0IBY,1

KJELLB0VRE,3

ANDWENDELL D. ZOLLINGER4

National Institute of PublicHealthlandNorwegianDefence MicrobiologicalLaboratory,2 N-0462 Oslo 4, and

Kaptein W. Wilhelmsen ogFruesBakteriologiskeInstitutt, University of Oslo, Rikshospitalet, N-0027Oslo j,3 Norway,

and Walter ReedArmy Instituteof Research, Washington, D.C.200124 Received4February1988/Accepted 12 May1988

Antibodyresponsesof adultvolunteers givenavaccine containing meningococcal capsular polysaccharides

(serogroups A, C, Y, and W-135) noncovalently complexed with serotype 2b:P1.2 and 15:P1.16 outer membrane proteins havebeenstudied. Serawere analyzedbyenzyme-linkedimmunosorbentassaymethods

forimmunoglobulin G (IgG), IgM, and IgAantibodies andforbactericidal activities against the homologous strains. The vaccinationwasperformedas adouble-blindexperiment with47volunteers,ofwhom23received

theprotein-polysaccharide vaccine and 24 received the control preparation containing the polysaccharides only.Tenadditionalpersonsvolunteered for the protein-polysaccharidevaccine. Beforevaccination,carriers ofmeningococci had significantly higher levels of specific IgGand IgAand alsohigherbactericidal activities than noncarriers. At 2 weeks postvaccination wefound significant IgG and bactericidal antibody responses against both the 2b:P1.2 and 15:P1.16 strains in about 70% of the protein-polysaccharide vaccinees. The

immuneresponseinducedby diseasewascomparedwiththat induced by vaccinationby analyzingpairedsera

from 13 survivorsofserogroupB serotype15meningococcaldisease. We found that themeanspecific IgGlevel

inacute-phasesera was lower thanaverage inprevaccination serafrom thevaccinees but similar to that of

healthynoncarriers before vaccination. The convalescent-phaseserashowedIgGresponsessimilar to those of

thevaccinees, buttheIgM responsetodisease wassignificantly higherthan after vaccination. The immune

responsetodisease caused byserogroup B serotype 15 meningococciwas found by enzyme-linked

immuno-sorbentassayanalysistobe about thesamewithouter-membraneantigensfromaserotype2b strainasitwas

withantigensfroma serotype 15 strain.

The incidence of meningococcal disease (MCd) has

re-mained at epidemic levels in parts ofNorway for over 10

years. In the country as a whole, the incidence has varied between 5.6 and 9.0casesper100,000 peopleper year,with thehighest incidence (26.3)innorthernNorway in 1975 (1, 2). There have been no signs of decreasing incidence of disease in the country as a whole, and the need for a protective vaccine is obvious. The epidemic is mainly

causedbyserogroup Borganisms, mostoften thosecarrying

the serotype:subtype combinations 15:P1.16, NT:P1.16, or 15:- (10). At the moment, no vaccine against group B

meningococcaldisease is available, but immunization trials inanimals and humans withouter membraneproteinsfrom serotypes 2a and 2b complexed with capsular

polysaccha-rides haveprovided encouragingresults (3, 4, 8, 19, 21, 23).

We present results from a clinical vaccination trial in

Norwaywhere human volunteers have been immunized with

a new vaccine against serogroup B meningococci, and we

compare the serological responses of the vaccinees with thoseof MCdpatients. The vaccinewas composedofouter membrane proteins from both serotype 15:P1.16 and sero-type 2b:P1.2 meningococci, noncovalently complexed with

A, C,Y,andW-135capsularpolysaccharides. The

lipopoly-saccharide content in this vaccine was very low compared

with that in other experimental vaccines against group B meningococci.

* Correspondingauthor.

MATERIALS AND METHODS

Vaccinees. A total of 47Norwegiansoldiers(43male and4

female)volunteered for the vaccination trial. Ofthistotal,23

received one injection of the protein-polysaccharide

vac-cine, and 24 received the corresponding polysaccharide

control preparation in a double-blind manner. In addition, five medical students and five laboratory personnel volun-teered for the vaccine andwereincluded in thestudy. Nose and throat specimens werecollectedfrom all the vaccinees

weeklytodetectcarriage of meningococci and related

bac-teria. This samplingstarted 1 week before vaccination and ended 3 weeks after. A carrier is defined here as aperson from whommeningococciofany serogrouporserotypewere isolatedduringthisperiod.Theserumsamplesstudiedwere

collectedjustbefore and 16daysand 6 weeks after vaccina-tion.

Patients. Paired serumsamplesfrom 13patientssurviving serogroup B MCd were analyzed along with the vaccinee

serum. The meningococci isolated from 10 of the patients

wereclassifiedasserotypeB:15:P1.16; the remaining organ-isms were serotypes B:15:P1.15, B:15:-, and B:NT:P1.16. The firstsetofserumsamples wasdrawn duringthe first 2 daysafterhospitalization,and thesecondsetofsampleswas

drawn 1 weekto2 months later(average, 22.5 days).

Vaccine. Meningococcal capsular polysaccharides were

suppliedfrom ConnaughtLaboratories, Swiftwater, Pa., as

thecommerciallyavailable vaccine meningococcal

polysac-charide vaccine, groups A, C, Y, and W-135 combined

(ACYW polysaccharide vaccine). Multiple vials of vaccine

werereconstituted, pooled,andusedas abulkproductin the

preparation of the experimental vaccine. Members of the 1543

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94K 67K __

P1.16

JU 2 b

II - 15

30K_i

20K

1iK

ïa b d

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electropho-resisanalyses of theouter-membrane preparations usedasantigens

in ELISA. Lanes: a, standard proteins with known molecular

weights; b, 15:P1.16 (strain 44/76); c, 2b:P1.2 (strain 8047); d,

protein-polysaccharide vaccine. Molecular standards in kilodaltons (K)areindicated onthe left;serotypesand subtypesareindicated

ontheright.

controlgroupreceived 50,ug of eachpolysaccharide.

Outer-membrane proteins from the meningococcal strains 8047 (B: 2b:P1.2) and 44/76 (B:15:P1.16) were prepared and purified

byuseof the polar-ion detergent Empigen BB (19). One dose

of thevaccine contained 52 ,ug each ofserotypes2b:P1.2 and 15:P1.16 outer membrane proteins, 40 ,ug each of the four polysaccharides, 3.5 p.goflipopolysaccharide, and 1.2 ,ug of

nucleic acid. In addition, the vaccine contained 4.5 mg of

lactose per dose and was reconstituted in 0.85% saline.

About50% of the polysaccharide was bound to protein as

estimated by the elution profile on Sepharose CL-4B

com-pared with that of thepure polysaccharide mixture.

ELISA antigens. To prepare antigens for enzyme-linked

immunosorbent assay (ELISA), cultures of Neisseria

men-ingitidis (strains 8047 and 44/76) were grown overnight at

33°C in 1.4 liters ofdialyzed tryptic soybroth (Difco

Labo-ratories) in 2.8-liter Fernbach flaskson arotatoryshakerat 120rpm. Outer-membrane vesicleswereprepared by extrac-tion of thewetcell pellet for 2 hat45°C with5ml of0.2 M

lithium chloride-0.1 M sodiumacetatebuffer(pH 5.8)per g ofcells (5, 12). The protein concentrationsweredetermined by the Lowry technique, and sodium dodecyl sulfate-poly-acrylamide gel electrophoresis analyses of the productswere performed. The carbohydrate and lipid contents were ana-lyzed bygas-liquid chromatography.

ELISA technique. ELISA analyses were performed in

triplicate on microdilution plates as described previously

(12), with alkaline phosphatase-conjugated swine anti-human immunoglobulin G (IgG), IgM, and IgA antibodies. Asaninternalantibody standard,atwofold dilution series of a positive postvaccination serum was used in all

experi-ments. The meanvalue of theobservedoptical densitywas

transformed to arbitrary units per milliliter by a sigmoidal

standardcurve(logit-log transformation) calculated from the values of the reference serum (14). Initially, all serum

samples were analyzed in a 1:200 dilution. Samples with

optical density values of .0.9 maximumoptical densitywere

further diluted and reanalyzed. The amounts of IgG, IgM, and IgA specific against the two antigens in the positive referenceserumwerelateranalyzed by solid-phase

radioim-munoassay(20). ThisgaveIgG:IgM:IgA ratios of 20:1:1 and 12.5:1.5:1 for the 15:P1.16 and 2b:P1.2 antigens,

respec-tively. The IgG, IgM, and IgA values obtained by ELISA

were scaled according to these results. One scaled unit correspondsapproximately to 1 ,ug ofspecific antibody per

ml.

Bactericidalassay.Thebactericidal activities of the

vacci-nation sera were analyzed as described by Frasch and

Robbins (7) in duplicate against the two vaccine strains. A normal blood donorwith verylowlevels ofmeningococcal antibodies, as determined by ELISA, and no bactericidal

activity against the strains investigated servedas a

comple-TABLE 1. ELISAantibody level of vaccinees and patientsas afunction of time after vaccinationor onsetof meningococcal disease

Antigen Antibodylevelsin':

(strain) Immunoglobulin Study

group

Aetubod

Sevem

2

c:

(strain)

~~~~~~~~~~~~~~~~Serum

i Serum 2 Serum 3

2b:P1.2 (8047) IgG Polysacc. 5.6(4.4-7.2) 5.4(4.2-6.7) 6.4(5.1-6.4)

Polysacc-prot. 3.6(3.0-4.3) 20.6(16.2-25.6) 15.1(12.1-19.0) Patients 2.7(2.1-3.5) 13.9(9.8-19.4)

IgM Polysacc. 0.9(0.7-1.0) 0.9(0.7-1.0) 1.1(1.0-1.3)

Polysacc-prot. 1.1 (1.0-1.3) 1.4(1.3-1.6) 1.4(1.3-1.6) Patients 0.9(0.7-1.1) 2.2(1.9-2.7)

IgA Polysacc. 0.3 (0.2-0.4) 0.3 (0.2-0.4) 0.3(0.2-0.4)

Polysacc-prot. 0.2 (0.2-0.3) 0.4 (0.3-0.5) 0.3(0.3-0.4) Patients 0.3(0.2-0.3) 0.6(0.5-0.7)

15:P1.16 (44/76) IgG Polysacc. 2.5(2.1-3.1) 2.6(2.1-3.2) 2.9(2.3-3.8)

Poysacc-prot. 2.3(1.9-2.7) 11.3 (8.8-14.6) 8.7(6.9-11.1) Patients 1.9(1.7-2.2) 14.0(10.0-19.7)

IgM Polysacc. 0.5(0.4-0.6) 0.5(0.4-0.6) 0.6(0.5-0.7)

IgA Polysacc. 0.4(0.3-0.4) 0.4(0.3-0.4) 0.4(0.3-0.5)

"Forvaccinees,sera1, 2, and 3 were taken 0, 2 and 6 weeks aftervaccination, respectively.Forpatients,serum 1 wastaken between days 0 and 2 and serum 2 wastaken betweendays9and65(average,23days)afterhospitalization.

bPolysacc., persons vaccinated with ACYW polysaccharide vaccine (n = 23); polysacc-prot., persons vaccinated with polysaccharide-protein vaccine

ACYW2bl5-2(n= 33);andpatients, persons withgroupBserotype 15meningococcal disease(n= 13).

rGeometric meanunitspermilliliter.One unitcorrespondstoapproximately1,ug ofspecific antibody. Rangesare means+ onestandard error of the mean.

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TABLE 2. Geometric mean bactericidal titers against two different serogroup B N. meningitidis strains as a

function of time after vaccination

Strain Geometric mean titer at:

(serotype)

Vaccine'

WkO Wk2 Wk6

8047(2b:P1.2) Polysacc. 3.7 3.6 4.6

Polysacc-protein 2.4 14.9 11.0 44/76(15:P1.16) Polysacc. 2.0 2.0 2.0 Polysacc-protein 2.4 7.8 5.5 a Polysacc., ACYWpolysaccharide vaccine; polysacc-protein, polysaccha-ride-protein vaccineACYW2b15-2.

ment source. Titers are given as the highest serum dilution resulting in >50% killing of the inoculum.

Statistical methods. The statistical analyses of the data

were performed by use of the program package SPSSIPC+ (SPSS Inc., Chicago, Ill.). The ELISA results were

trans-formed to logarithmic values to calculate the geometrical

means. This gave an approximately normal distribution of thedata and allowed us to use standard statistical tests. The significance levels ofdifferences between groups were

ex-amined with the Student t test on the log-transformeddata. RESULTS

Antigen analyses. Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis analyses of the ELISA antigens are shown in Fig. 1. In strain 44/76, the class 1 (serosubtype

P1.16) and class 3 (serotype 15) outer membrane proteins predominated, with only small amounts of class 5 protein.

The antigen from strain 8047 contained mainly class 1

(serosubtypeP1.2) andclass2(serotype2b)proteinsbutalso agreater amount of class 5protein. Therelative amountof class 5 protein in theELISA antigen from strain 8047 was higher than in the vaccine (Fig. 1). The ratios of lipopoly-saccharidetoproteinin the ELISAantigenswereabout1:1,

andonlytracesofsialic acid couldbedetectedbygas-liquid chromatography. The vaccine contained the same major proteinsastheELISAantigens,but inaddition,someweak

bands corresponding to proteins with molecular sizes of about60,000 to 70,000 daltons were seen.

Prevaccination andearly-phase sera from patients. Tables 1 and 2 show thelevels ofantibodiesagainst theantigens 2b: P1.2 and 15:P1.16 in the groups examined. The observed

differences in prevaccination serabetween the polysaccha-ride-vaccinated control group and the

protein-polysaccha-ride vaccinees were not statistically significant (Student's t tests).

TABLE 3. Influence ofmeningococcalcarriageonELISA antibodylevelsbeforeandafter vaccination

ELISAantibody level for antigen (time): Immunoglobulin Carrier 2b:P1.2 15:P1.16

statUSa

Wk0 Wk 2 Wk 6 Wk 0 Wk 2 Wk 6

IgG - 2.3 15.9 11.5 1.4 7.3 5.8

+ 6.5 29.1 21.9 4.2 20.4 15.3

IgM - 1.1 1.4 1.3 0.6 0.9 0.8

+ 1.2 1.5 1.6 0.6 0.8 0.6

IgA - 0.2 0.3 0.2 0.3 0.5 0.4

+ 0.3 0.5 0.4 0.4 0.7 0.5

aSee Table 1,footnotec.

bOnly the 33 subjects whoreceived the polysaccharide-protein vaccine werestudied.+,Carriers ofN.meningitidis(n=14); -, noncarriers (n=19).

Amnong

the47military recruits in the trial, wefound that

25 carried meningococci, whereas only 2 of the 10 other

volunteers werecarriers. The carrierswere evenly

distrib-uted between the vaccine and the control groups. When carriers werecomparedwithnoncarriers(Table3), we found

significantly highervalues for IgGagainstboth serotype 15 andserotype 2bantigensin the carriers(P=0.007and0.009;

Student'sttests).Thisalsoappliedtobactericidal antibodies (P =0.006 and0.01)(Table 4).Asimilardifference was not demonstratedfor IgMandIgA.

Theacute-phase serum samples from the

meningococcal

patients showed lower levels of IgG

specific against

both antigensthan thesamplesfromvaccineeswhowerecarriers

(Tables 1and 3), and their

geometric

meanIgGlevelswere

close to those of the noncarriers. For

IgM

and

IgA,

the

differences between

patients

and thecarriers weresmall. Postvaccination and convalescent-phase sera. Two weeks after

vaccination,

a

significant

increaseinmean

IgG

antibod-iestobothELISA outer-membrane vesicle

antigens,

aswell

as in bactericidal

activity against

the

corresponding

men-ingococcal strains,

wasfound in the group

given

the

poly-saccharide-protein

vaccine

(Tables

1 and 2). With both

strains,

atleasta

doubling

inbactericidal titerwasshown in about 70% (23 of33) of the vaccinees. In ELISA

studies,

82% (27 of33) showedatleast a

doubling

of

IgG

antibodies with the2b:P1.2antigen,and70%(23 of 33) showedasimilar

resultwiththe15:P1.16

antigen.

Forthose whoreceivedthe

vaccine, the meanfoldriseof

IgG

wasabout 11times with

both the 15:P1.16 and 2b:P1.2

antigens.

The

IgA

levelsalso

increased

significantly

(3 to 4

times),

whereas the

IgM

response

proved

poor. In thecontrolgroup,whichwas

given

only

the ACYW

polysaccharides,

a

slight

increaseinmean

antibody activities was

observed,

but the differences

be-tweenweek 6 and week Owere not

statistically

significant.

The increase in

antibody

levelsafter

protein-polysaccha-ride vaccination was

substantially higher

among carriers

than noncarriers. Two weeks after

vaccination,

the mean

IgG

level in the serum

samples

taken from the vaccinated carrierswasabouttwo tothree times thatfoundin theserum takenfrom the noncarriers

(Table 3).

Themean

IgG

level in theserumtaken fromvaccinated noncarrierswasthenfound

to be abouttwicethatofthe serumtaken fromthe carriers before

vaccination,

and itwasfourtosixtimes

higher

than in the acute-phase sera from the

patients (Tables

1 and

3).

Apparently,

the vaccination had no effect on the carrier

state. Table 5 shows the

importance

of

preimmunization

antibodies forbactericidal

activity.

The increase in bacteri-cidal titer aftervaccination was

substantially higher

for the group with demonstrableantibodies before vaccinationthan

forthegroup without suchantibodies.Themeantiterratios

were,however,about thesamefor thetwogroups.Ofthe 57

healthy adults in this

study,

32

(56%)

had no measurable bactericidal

activity

against

the two vaccine strains before

TABLE 4. Influence ofmeningococcal

carnage

on serum

bactericidalactivitybeforeandafter vaccination Bactericidal titer for strain andserotype(time):

Carrier 8047(2b:Pl.2) 44/76(15:P1.16)

status ________________

Wk0 Wk 2 Wk 6 Wk 0 Wk2 Wk 6

- 1.4 7.9 6.0 1.4 4.9 3.4

+ 5.0 35.6 24.5 4.0 14.8 10.5

aOnlythe 33subjectswhoreceived thepolysaccaride-proteinvaccinewere

studied. +,Carriers of N.meningitidis (n= 14); -,noncarriers(n= 19).

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TABLE 5. Geometric mean bactericidal titers of pre- and postvaccinationserumsamplesfrom protein-polysaccharide

vaccineeswithout and withdetectableprevaccination bactericidalactivity

Titer of serum fromvaccinees:

Without With prevaccination

Time

bactericidal

activity

bactericidalactivity against strain: against strain:

8047 44/76 8047 44/76

WkO 1.0 1.0 9.8 7.3

Wk 2 7.6 3.8 43.3 24.2

Wk6 5.0 2.6 37.1 17.6

vaccination. Ofthe 33 vaccinees who received the

protein-polysaccharide vaccine, 20 subjects were without

prevacci-nation bactericidal titers. Ofthese, seven (35%) and eight

(40%) vaccinees did not respond to vaccination with

in-creased bactericidal activity against the serotype 2b or 15 strains, respectively. Ofthe 13 vaccinees who had

prevac-cination bactericidal titers, two (15%) did not respond

sig-nificantly to the serotype 2b strain and 3 (23%) did not respond significantly to the serotype 15 strain.

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Six weeks after vaccination, we observed a decline in

antibody levels among the protein-polysaccharide vacci-nees. Thé geometric mean IgG antibody level, determined by ELISA, wasreducedabout 25%compared with the peak value at 2 weeks. However, the mean IgG level still re-mained about fourtimes higherthanbefore vaccination.

In serum samples collected at any time, there was a significant positive correlation between the antibody re-sponses against the two strains. Two weeks after

vaccina-tion, the correlation coefficient (r) of the ELISA IgG

anti-body levels between the two outer membrane vesicle

antigens was -0.8 (Fig. 2a). The correlation between the

bactericidalassayswiththe twostrainswassomewhatlower (r = 0.6 to 0.7) (Fig. 2b). Six weeks after vaccination, we

found that ofthose subjects who had received the protein-polysaccharide vaccine, four had bactericidal activity

against the serotype 15 strain but not against the serotype2b strain; the reverse situation was observedin six vaccinees.

The correlation coefficients between bactericidal titers and IgG ELISA values with the corresponding antigens were also

relatively high

(r 0.7 to

0.8)

(Fig.

2c and

d),

whereas

only a weak positive correlation was observed between bactericidal titersand IgA. Nocorrelationwasdemonstrated betweenbactericidal activity and IgM.

9g

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Log Bactericidal titer against 44/76

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FIG. 2. Correlations between ELISA IgG values against 15:P1.16 and 2b:P1.2 antigens (r = 0.82) (a), bactericidal titers against

meningococcal strains 44/76 and 8047 (r=0.69)(b), ELISA IgG values andbactericidal titers against strain 8047 (2b:P1.2) (r=0.78) (c), and

ELISA IgG values and bactericidal titers against strain 44/76(14:P1.16) (r = 0.81) (d). All serum samples were collected 2 weeks after

vaccinationwith the protein-polysaccharide vaccine. Least-squares regressionlinesareshown.

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Theconvalescent-phase sera from the patients, collected

from 9 to 65days (average, 22.5 days) afterhospitalization,

also showed a substantial increase in antibodies compared with their acute-phase sera. The highest response was with IgG against the homologous serotype (15:P1.16). Although no patient was known to have been infected with

meningo-cocci of serotype 2a or 2b, we also observed a strong

increase in IgG antibodies against the 2b:P1.2 antigen. In contrast to the protein vaccinees, the convalescent group showed adistinct IgM increase, in particular to the serotype

15:P1.16 antigen. Also, the IgA response was higher in

patients than in vaccinees. One patient had negligible

anti-body response as determined by ELISA. Three months

later, he suffereda new MCd caused by the same serotype

(B:15:P1.16), still without developing a significant immune

response.

DISCUSSION

The protein-polysaccharide vaccine induced significant

immune responses against boththe2b:P1.2andthe15:P1.16

meningococcal strains in about70%of thevaccinees,judged

fromboth theELISAresultsandthebactericidal assay. In a previousvaccinationtrial witha B:2avaccine, we observed

85% seroconversion with both ELISA and bactericidalassay

(9). The B:2a vaccine was, however, given twice with a

4-weekintervalbetweeninoculations, indicatingthat

revac-cination is necessary to give a satisfactory response. It is also possible that the higher lipopolysaccharide content in the B:2a vaccine, ordifferences in minor protein composi-tion, could be of importance. Two vaccine doses and an

adjuvant should probably be tried to increase the serocon-version rate. Experiments in mice andhumans with alumi-numhydroxideandaluminum phosphate as adjuvantshave

beenpromising(8, 15, 17).

The observation that about 50% of the recruits were

carriers of meningococci is similar to our findings in a

previous studyinanotherNorwegianmilitarycamp(9). The meanprevaccination and

postvaccination

bactericidal titers and IgG antibody levels were significantly higher for the carriergroup thanforthenoncarriers, althoughmostcarrier isolateswerenongroupable and nontypable (Tables3and4).

Carriage may thus induce antibody responses directed

against both B:15 and B:2b strains. This observation is in

close agreement with the report of Reller et al. (13) that

colonization ofthenasopharynxes ofadults with nongroup-ablemeningococci, whichveryrarelycausedisease, canbe apotent stimulusto the production ofbactericidal

antibod-ies, not only against the carrier strain but also against selected disease-causing strains. Goldschneider et al.

(11)

found that 87% of men colonized with group B or C organisms developed bactericidal antibodies toone ormore

heterologous strains of pathogenic meningococci. These

observationssupport therole ofthecarrierstatein develop-ing and maintaindevelop-ing natural immunity to meningococcal disease.

For

evaluation

of vaccine efficiency, it seems particularly importantto studythe immuneresponse insubjectswithout measurable antibodies before vaccination. Although the

proportion

ofsuchvaccineeswhorespondedwaslowerthan that in the whole study, we found that 60 to 65% of them seroconverted after oneinjectionof the vaccine.

With thebactericidalassay with humancomplementused in this study,the

correlations

betweenELISA IgGand the

corresponding bactericidaltiters(Fig. 2c andd)were signif-icantly better than those observed previously with baby

rabbitcomplement (12). Thisis inagreement withthe report

ofZollinger and Mandrell (22).

Because the response against the individualantigen com-ponents has not been quantifiedseparately, acomparisonof the immunogenicity of the two serotype antigens must be interpreted with care. However, both pre- and

postimmuni-zation sera from thevaccineesshowed higher IgG antibody levels with the 2b:P1.2 antigen than with the 15:P1.16 antigen.We foundsimilarresultswith thebactericidal assay. These observations may indicate that (i) the serotype 15: P1.16 antigens are weaker immunogens than the 2b:P1.2 antigens in this vaccine,(ii)the15:P1.16antigens studied by

ELISA are less antigenic than 2b:P1.2 antigens, or(iii) the

15:P1.16 strainused inthe bactericidal assay is more

resis-tant toantibody-complement attackthan the2b:P1.2 strain.

The ELISA results indicate that infection with group B serotype 15 meningococci induced about the same IgG antibody levels as vaccination with a combined protein-polysaccharide vaccine. However, the high total antibody

responses observed in patients andcarriers couldhave been due in part to

lipopolysaccharide

and other antigens which

werecomponentsinthe ELISA antigens and not present in

the vaccine. Serum samples taken from MCd patients, all except one ofwhom wereinfected withserotype 15

strains,

showed strong responses in ELISA with both the 2b:PI.2 and

15:PI.16

antigenpreparations. Wedonotknow whether this is mainly due to immunological cross-reactions with commondeterminantsonthe serotype andsubtype

proteins,

or whether the cross-reaction is due to other common

antigens.

Immunoblotting

experiments with

postvaccination

sera from the B:2a vaccination trial have demonstrated cross-reactions within the class 1 and 5

proteins

in both serotypes 15:P1.16and 2a:P1.2(E. Wedege, personal

com-munication).

Six weeks after

vaccination,

the

IgG antibody

levels and bactericidal titers had declined 25% from the peak value

observed after2weeks.Thisis inagreementwithourresults from the B:2a vaccination trial (16). Preliminary data from

ELISA indicate that 6 months after vaccination, the mean

IgG level was reduced to aboutone-third ofthepeak value butstill remainedabout two to three times higher than the

prevaccination level. Although the antibody titers have

declined, it is quite possible that the immune system has beenprimed withinduction ofmemory cells which may be

rapidly activatedupon a newinfection.Theresultsobtained

with serum taken from thecarriersmay supportthis.

Exper-imental data show thatimmunologicalmemory, asmeasured by the degree ofantibody response to a challenge dose of

vaccine, progressivelyincreases withtime to amaximumat 6 months and thereafter tends to persist at a plateau (18).

However, long-termfollow-up ofthevaccineresponsemust

be

performed

to assess the

duration

ofthe antibodies and corresponding

protection.

Such studies will probably be

complicated by the influence ofnew carriage of meningo-cocci in the vaccinees duringthefollow-up

period.

ACKNOWLEDGMENTS

Wegratefully acknowledge C. E. Frasch, U.S. Food andDrug Administration, for use of his computer program for logit-log

transformations of theELISAdata; K. Bryn, National Instituteof Public Health, forgas-liquid chromatography analyses ofthe anti-gens; and E. Wedege, National Institute of Public Health, for immunoblotting experimentsandhelpful discussions.

on April 11, 2020 by guest

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