-
Quantitative Determination of the Human Immune
Response to Immunization with Meningococcal Vaccines
EMIL C. GOTSCHLICH, MicHEL REY, RENETRiAU, and KENNmrmJ.
SPARKSFrom The Rockefeller University, NewYork 10021, Faculte Mixte
de Medicineet de Pharmacie, Dakar, Senegal, and Institut Merieux,
Lyon, France
A B S T R A C T Radioactive antigen binding tests havebeen
developed to measure quantitatively the antibodyresponse of 167
adults, 84 children, and 51 infants toseveral different
preparations of group A and group Cmeningococcal polysaccharides.
Almost all the adultsinjected responded and the geometric mean
responseswere approximately 15 /Ag/ml of antibody protein
inindividuals vaccinated subcutaneously with two prepara-tions of
group A vaccine. The geometric mean antibodyconcentration after
immunization with two preparationsof group C vaccine was
approximately 35 Ag/ml. Mostchildren aged 7 yr responded to
immunization with twogroup A vaccines, and their mean response was
onlyslightly less than that seen in adults. There was no
dif-ference between the subcutaneous and the intradermalroute if
both were given with jet gun. The majorityof infants aged 6-13
months responded to a preparationof group A vaccine and the
geometric mean titer wasapproximately 1.2 ug/ml. Adults, children,
and infantsresponded significantly less to a preparation of groupA
polysaccharide which was of low molceular weight.
INTRODUCTIONCurrently there are under development two
meningo-coccal vaccines based upon the use of high molecularweight
group-specific polysaccharides. Immunization ofmilitary recruits
with the group C polysaccharide hasbeen shown to be effective in
preventing meningococcaldisease (1), and in lowering the rate of
acquisition ofthe nasopharyngeal carrier stage by group C
meningo-cocci (2). Such information has not yet been obtainedfor
the group A vaccine, in part because these strainsare exceedingly
rare in the U. S. military population.The efficacy of this vaccine
will probably need to betested in the African "Meningitis belt"
where diseaseis caused primarily by group A organisms (3).
Received for publication 12 July 1971.
Administration of either the group A or the groupC vaccine to
adults induces the formation of antibodiesbelonging to the three
major immunoglobulin classeswhich have both bactericidal (4) and
opsonic proper-ties (5). To date quantitative data is available on
theresponse of children.' There is no published data on theimmune
response of infants to these vaccines. Inasmuchas this age group
stands to benefit most from meningo-coccal immunoprophylaxis (6),
quantitative data oftheir serological response is vital.
The present report will describe the immune responseof adults,
children, and infants to immunization withseveral preparations of
meningococcal polysaccharidesmeasured by means of quantitative
radioactive antigenbinding tests (7).
METHODSAntigens. Group A meningococcal vaccines lots A-5,
A-7, V-1, and group C meningococcal vaccines lots C-6 andC-7
were prepared as described by Gotschlich, Liu, andArtenstein (8).
The group A polysaccharide contained inlots V-4 and V-5 was
prepared by a modified procedure,employing cold phenol extraction
to remove protein con-tamination.2 A-5 and C-6 were prepared at the
Walter ReedArmy Institute of Research; lots A-7 and C-7 by E.
R.Squibb & Sons, New York; and lots V-1, V-4, and V-5by
Institut Merieux in Lyons, France. The molecular sizeof the
different preparations was assayed by gel filtrationon Sepharose 4B
(Pharmacia Fine Chemicals, Inc., Piscata-way, N. J.). The void
volume of the column was deter-mined with Blue Dextran (Pharmacia
Fine Chemicals, Inc.,Piscataway, Nr J.) and the total volume with
tritiatedwater (New England Nuclear Corp., Boston, Mass.).
Thedistribution coefficient (Kd8) was calculated from the
peakelution volume of the major. peak of the polysaccharide (9).The
column was monitored either by determination ofphosphorus by the
method of Chen, Toribara, and Warner(10) in the case of the group A
polysaccharide, or by theresorcinol method of Svennerholm (11) in
the case of the
'Manuscript in preparation.2 Manuscript in
preparation.'Abbreviation used in this paper: Kd, distribution
coef-
ficient.
The Journal of Clinical Investigation Volume 51 1972 89
-
,, 2000
0
_ Ii oo - t
0 1 2 4 8 169g of antigen
FIGURE 1 Quantitative precipitin curves obtained with hu-man
sera drawn 2 wk after immunization with meningococ-cal
group-specific polysaccharide. The upper curve depictsreactions
with group C antigen; the lower two curves withgroup A antigen.
group C antigen. The column was calibrated by determiningthe Kd
of two preparations of group A polysaccharide. Themolecular weights
of these two preparations had been de-termined by ultracentrifugal
methpds and were respectively170,000 and 25,000.' The two
distribution coefficients wereplotted against the logarithm of
their molecular weightand the resulting line was used to estimate
the averagemolecular weight of the vaccine preparations (12).
Thissame procedure was used to obtain an estimate of the molec-ular
size of group C vaccine preparations.
Quantitative precipitation analyses. Five sera obtained2 or 3 wk
after immunization with group A antigen andsix sera obtained 2 wk
after immunization with group Cantigen were centrifuged at 70,000 g
for 60 min to floatthe lipids, and centrifuged at 35,000 g for 30
min to removeall insoluble protein. A precipitin curve was
constructed byadding to 1 ml portions of serum 100 tul of antigen
solutiondissolved in 0.2 M sodium EDTA pH 7.0 and containing1,000 U
penicillin and 1 mg of streptomycin. The quantityof antigen added
consisted of 1, 2, 4, 8, and 16 Ag. Thetest was carried out in
duplicate in 3-ml conical centrifugetubes which were sealed with
parafilm and stored for 1 wkat 4°C. The tubes were gently shaken
daily. The precipi-tates were sedimented by centrifugation for 30
min at 2,000g at 40C and washed twice with 2-ml volumes of cold
saline.The precipitates were dissolved in 500 Al of 0.1 N NaOHand
the protein concentration determined by the Folin re-action
employing an autoanalyzer (13) (Technicon Co.,Inc., Tarrytown, N.
Y.). The test was quantitated by refer-ence to a standard curve
determined simultaneously usingchromatographically purified human
gamma globulin ob-tained from Pentex Biochemical (Kankakee,
Ill.).
Radioactive antigen binding test zeith intrinsically
labeledantigens. Radioactive group A polysaccharide was preparedby
incubating group A meningococci, strain A-1, in Frantzmedium (14)
supplemented with 1 mc of "4C-labeled sodium
'Manuscript in preparation.
acetate (New England Nuclear Corp., Boston, Mass.).Group C
meningococci of strain C-11 were grown in thesame medium
supplemented with 25 mc of tritium labeledsodium acetate (New
England Nuclear Corp., Boston,Mass.). The polysaccharides were
purified by the usualmethod (8). The recovered group A
polysaccharide had aspecific activity of approximately 2,800
cpm/,ug and thegroup C had approximately 10,000 cpm/,ug.
The radioactive antigen binding test was performed byadding 100
ul of radioactive antigen (0.17 ,ug of group Apolysaccharide or 0.5
,ug of group C polysaccharide) to 100,ul of serum, and after
overnight refrigeration the globu-lins were precipitated by the
addition of 200 ul of 80%saturated (room temperature) ammonium
sulfate. After theprecipitate had been sedimented by centrifugation
for 60min at 2,000 g, 100 ,ul of the supernate was dissolved in
10ml of a liquid scintillation fluor consisting of Liquifluor(Pilot
Chemicals, Inc., Watertown, Mass.): Soluene (Pack-ard Instrument
Co., Inc., Downers Grove, Ill.) : toluene (42:100: 858). The
radioactivity was measured in a Beckmanliquid scintillation
counter, Model LS 133 (Beckman In-struments, Inc., Fullerton,
Calif.).
Radioactive antigen binding test with group A polysac-charide
labeled with ...I. In order to be able to iodinategroup A
polysaccharide, phenolic groups were inserted byactivating the
polysaccharide with cyanogen bromide (15)and letting it react with
tyramine.' Such polysaccharidecould be readily iodinated by the
method of Hunter, Green-wood, and Glover (16) and polysaccharide
with a specificactivity of approximately 1,000 cpm/ng was obtained.
Adouble label technique was used in order to be able to dothe
binding tests on very small quantities of sera. The prin-ciple of
this method was to add 1 ,uc of 'Na per milliliterof antigen
solution as a volume marker. To 10 jul of serumwas added 10 IA of
antigen solution and after overnightequilibration, 20 ,l of 80%
saturated (room temperature)ammonium sulfate was added and the
precipitate sedimentedby centrifugation. An arbitrary portion of
the supernatewas drawn off and discarded, taking care not to
disturb theprecipitates. This precipitate with a variable volume
ofsupernate overlying it was counted in a Model 4233, twochannel
gamma spectrometer (Nuclear Chicago, Des Plaines,Ill.) with one
channel set to count the sodium and the otherchannel the iodine.
The iodine count represented the totalamount of antigen left in the
tube, both that bound andthat present in the remaining supernate;
the sodium countindicated the volume of supernate left. With these
data itis possible to calculate the per cent of added antigen
boundto antibody (17).
Three antigen concentrations containing approximately4.0, 0.4,
and 0.04 ,ug of radioactive antigen per milliliterwere routinely
used. All sera were heat inactivated for 30min at 56°C and, when
called for, were diluted in heat in-activated fetal calf serum. All
volumetric measurementswere done with Eppendorf pipets (Brinkmann
Instruments,Inc., Westbury, N. Y.) and the test was carried out in
dis-posable Microfuge tubes, and centrifuged in a Microfuge(Beckman
Instruments, Inc., Fullerton, Calif.).
Statistical methods. Geometric mean antibody concentra-tions
were calculated by taking an arithmetic average of theper cent
binding. Inasmuch as the degree of binding islinearly related to
the logarithm of the antibody concen-tration, this yields a
geometric average of the antibody con-centrations. The significance
of differences in mean antibodyconcentration were tested by the
Student t test.
6 Manuscript in preparation.
90 E. C. Gotschlich, M. Rey, R. Triau, and K. J. Sparks
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Human sera. Dr. M. S. Artenstein kindly provided thesera of
military recruits vaccinated with preparation A-7,C-6, and C-7. The
source of the sera on which quantitativeprecipitation studies were
performed, as well as the seraobtained from recruits injected with
A-5, has been describedbefore (2, 4). All other sera were obtained
by personnel ofthe Department of Infectious Diseases of the School
ofMedicine of Dakar, Senegal. All subjects were volunteers,or
informed consent was obtained from their legal guard-ians. They
were observed clinically for- 48 hr and rectaltemperatures were
obtained before, and at 6, 24, and 48 hrafter vaccination on all
individuals except the military re-cruits. No significant pyrexic
responses were seen and onlyminor local reactions lasting for 24
hour were noted. APed-o-jet gun (Ver.nitron Corporation,
Farmingdale, N. Y.)was used in some of the studies and was adjusted
to injecta volume of 0.5 ml subcutaneously, or 0.25 ml
intradermally.Serum was obtained from bleedings performed 2 wk
afterimmunization. Both the infants and the children were ingood
health. The ages of the infants ranged from 6-13months with more
than half of them aged 9 months or less.
RESULTSQuantitative determination of antibody by precipita-
tion and by radioactive antigen binding capacity. Oneof the
simplest methods for the quantitative determina-tion of antibodies
is the radioactive antigen bindingtest developed by Farr (7).
Inasmuch as it measuresthe primary interaction of antibody with
antigen, it isaffected only by the concentration of antibody and
bythe average affinity of this antibody. The results of
theradioactive antigen binding test have usually been ex-pressed as
the dilution of a serum which will bind acertain amount of antigen
(antigen binding capacity,ABC) (7). However, because of the data
available inthe literature on the human immune response to
thepneumococcal polysaccharides (19) in terms of micro-grams of
precipitating antibody, it was thought desirableto express the
results obtained with the radioactive anti-gen binding test in
terms of antibody concentration. Torelate antigen binding capacity
to antibody concentration,
TABLE IPrecipitating Antibody in Sera from Volunteers
Immunized
with Meningococcal Polysaccharides
Precipitating antibody tomeningococcal polysaccharide
ImmunizedSerum with Group A Group C
#g/ml*W. C. B. C Not done 199I. G. A and C 38 38J. S. A and C
16.5 43.5M. S. A. A and C 47 401E.C.G. AandC 140 127J. W. A and C
85 36
*,ug/ml of antibody protein.
E 1.800
c
o l.400-
C
c 1.0000~01-
00
0
0'
0 0.200J~
F0
I..
x
Fx
0
-x /ra
a
l0 30 50 70 90% Antigen binding
FIGURE 2 The antigen binding capacity of dilutions of fivesera
obtained from volunteers immunized with group Ameningococcal
polysaccharide. The per cent of added in-trinsically labeled (14C)
group A polysaccharide bound byantibody is plotted against the
logarithm of the antibodyconcentration as determined by
quantitative precipitation.Serum: 0, I. G.; E, E. C. G.; X, M. A.
S.; 0, J. W.;A, J. S.
the radioactive antigen binding capacity of several serawith
known content of precipitating antibody was deter-mined. Sera were
obtained 2 or 3 wk after intradermalimmunization of six volunteers.
One was immunizedwith group C vaccine only (W. C. B.) and the
otherswith both group A and group C polysaccharide. Theirimmune
response has been documented previously (4)by means of passive
hemagglutination, bactericidal ac-tivity, and immunofluorscence.
The concentration ofantibody was determined by quantitative
precipitationThree representative precipitin curves indicating
thehighest, the lowest, and an intermediate response aredepicted in
Fig. 1. The quantity of precipitating anti-body found in each serum
is summarized in Table I.
The capacity of these sera and dilutions thereof tobind
intrinsically labeled group A and group C antigenwas measured by
the method of Farr as indicated inthe Methods section and it was
found that there was alinear relationship between the per cent of
antigen boundand the logarithm of the antibody concentration.
Thislinear relationship appeared to be valid between thelimits of
10 and 90% antigen binding. The results areset forth in Figs. 2 and
3. The lines were drawn by themethod of least squares, and the
correlation coefficientswere 0.94 in both instances.
The immune response of adults. The antigen bindingcapacity of
sera obtained from 167 adults immunized withseveral different lots
of meningococcal vaccines weremeasured and the quantity of antibody
determined byreference to the calibration curves obtained with
the
Immune Response to Meningococcal Vaccines 91
L
-
E 1.800
CP
cxo 1.4000
0)D 1.000
c
0U
V0.2
g' 0.200-J
_
+ 0
I-a
0a
oIF+ 0
IF
0 30 50 70 90
% Antigen binding
FIGURE 3 The antigen binding capacity of dilutions of sixsera
obtained from volunteers immunized with group Cmeningococcal
polysaccharide. The per cent of added in-trinsically labeled (8H)
group C polysaccharide bound byantibody is plotted against the
logarithm of the antibody-concentration as determined by
quantitative precipitation.Serum: 0, J. W.; EO, W. C. B.; o, I. G.;
A, E. C. G.;+, J. S.; X, M. A. S.
standard antisera (Figs. 2 and 3). The results are setforth in
the scattergrams (Fig. 4) and summarized inTable II.
>8'84
C-6 S.C. C-7Before After Before
0 T~:5?o
70r_ ._
as60
>~ 50-oc 400
0
g' 30
a
-o
0
. 0
10
5
-
TABLE I IThe Antibody Responses of Adults Injected with
Various
Preparations of Group A and Group CMeningococcal
Polysaccharide
Geometric meanantibody
No., No., concentrationRoute of of of
administra- sub- fail- Pre-Vaccine Kd* tion j ects ures immune
Immune
pg/miC-6 0.30 s. c. (needle) 22 0 6.2 33.5C-7 0.27 s. c.
(needle) 19 0
-
S.C. V-4 I.D.After Before After
* *..
V-5 S.C. V-5Before After Before
-if
>3.0E 3.0
0.,* 2
0
.0a)
20
-0
*0
E 1.0- 0* 0.5a
.. 0
-
seemed the most suitable because of its simplicity andits
versatility in terms of sensitivity. The quantity ofantigen bound
by a serum is a function of the con-centration of antibody present
and the average affinityof this antibody. In this study the
radioactive antigenbinding technique was standardized by reference
toantisera with a known content of precipitating antibodyto be able
to convert the observed degree of antigen bind-ing to antibody
concentration. Strictly, this is correctonly in the instance where
the average association con-stant of the unknown serum and of the
standard serumare identical. This criticism, of course, also
applies to agreater or less extent to other immunological
techniquessuch as quantitative precipitation. To explore the
mag-nitude of the error introduced by ignoring differencesin
affinity of different antibodies, the antigen bindingof dilutions
of a number of sera with a known contentof precipitating antibody
was determined and the resultsare summarized in Figs. 2 and 3. All
the points tend tofall rather closely on the line derived by least
squaresas is evidenced by the high correlation coefficients
(r=0.94) obtained in both cases indicating that for thesesera the
antigen binding capacity was closely correlatedwith the amount of
antibody present as determined byquantitative precipitation. This
suggests that little erroris introduced by this treatment and that
the associationconstant of these adult sera do not differ widely
enoughto be noticeable in this system.
The conversion of antigen binding capacity to anti-body
concentration, aside from the potential errors dis-cussed above,
has two advantages. It allows the resultsobtained in this study to
be compared with the serologicaldata obtained by quantitative
precipitation on volun-teers immunized with several pneumococcal
polysac-charides. The other advantage is that the test is
stan-dardized by reference to antisera whose potency remainsstable
and which can readily be exchanged between dif-ferent
laboratories.
The response of 167 adults to vaccination with groupA or group C
meningococcal vaccines was measured. Itcan be concluded that the
vast majority of adults in-jected with group C vaccine or with
group A vaccine ofhigh molecular weight had immune responses
whichquantitatively were comparable to those seen in indi-viduals
immunized with various pneumococcal polysac-charides (18). The
responses to the group A vaccineapparently are lower than those
obtained with group Cvaccine. One preparation of group A vaccine,
lot V-1,because of improper storage before packaging, was
ofconsiderably lower molecular weight than the otherpreparations
tested. The immune response to this vac-cine was significantly
less, not only in adults but also inchildren and in infants. This
is in accord with data
obtained with various preparations of dextran of differ-ent
molecular weight (19).
The response of children to two lots of group A vac-cine of very
high molecular weight, V-4 and V-5, werestudied. The vaccine was
administered by jet gun eitherintradermally or subcutaneously. In
both instances the re-sponses were comparable to those observed in
adultsinjected subcutaneously by needle with vaccine lotV-4, and
there was no advantage to intradermal in-jection. These data do not
exclude the possibility thatintradermal injection by needle may
give a higherresponse than subcutaneous injection as suggested
bythe results obtained with adults, because there is con-siderable
leakage from an intradermal injection sitewhen performed with a jet
gun.
Infants were injected with lot V-1 and V-4 and theresponse to
the larger molecular weight vaccine was sig-nificantly higher. The
mean response of these infants,who ranged in age from 6 to 13
months, was approxi-mately tenfold lower than those observed in
adultsand children. The reasons for this lower response are
atpresent unknown. The simplest possibility is that theinfants are
having a primary immune response, whereasthe children are having an
anamnestic response. It istrue that the preimmune antibody levels
of the infantsare much lower than those seen in children and
thatalmost half had no detectable antibodies (less than 0.1/g/ml).
This thesis can easily be tested in future stud-ies by
reimmunization of infants and observing whetherthey then produce a
response akin to those seen inchildren or adults.
Controls consisting of infants not immunized a sec-ond time but
studied over a longer period of time wouldresolve the question
whether infants need more than2 wk to mount their maximal immune
response.
The crucial question is whether the immune responsethat the
infants did mount is adequate to protect themfrom meningococcal
disease. This will be answered de-finitively only by a large field
trial. Nevertheless, it isa fact that agammaglobulinemic children
are protectedagainst meningococcal disease by passive
immunopro-phylaxis. It can be calculated that with the usual
dose(0.7 mVkg body weight) of concentrated gammaglobu-lin, and
taking into account that the average concentra-tion of antibody in
adult sera is approximately 5 tg/ml,the concentration of
antipolysaccharide antibody in thepatient's blood stream would be
less than 1.0 /g/ml.This consideration suggests that the low
responses ofthe infants may be sufficient to be protective.
ACKNOWLEDGMENTSWe would like to thank Dr. Branko Cvjetanovic of
theWorld Health Organization, for making this study pos-sible and
for his help in the preparation of this report.
Immune Response to Meningococcal Vaccines 95
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Supported in part by the Commission on Acute Respira-tory
Diseases of the Armed Forces Epidemiological Boardunder Contract
No. DADA 17-70-C-0027 and by a grantfrom The World Health
Organization.
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96 E. C. Gotschlich, M. Rey, R. Triau, anid K. J. Sparks