DTIC ADA263990: The Role of IgG Antibodies from Irradiated Cercaria-Immunized Rabbits in the Passive Transfer of Immunity to Schistosoma Mansoni-Infected Mice

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by  Defense Technical Information Center

AD-A263 990
SDT~IC
SELEAFv- (cid:127)I
______ MAY 101993 0
E
PUBLICATION REPORT
1735
13/93
TIlE ROLE OF IGG ANTIBODIES FROM IRRADIATED CERCARIA-IMMUNIZED RABBITS
IN TilE PASSIVE TRANSFER Of' IMMUNITY TO SCHISTOSOMA MANSONI-INFECTI3D MICE
BY
Beverly L. Mangold and David A. Dean
U.S. NAVAL MEDICAL RESEARCH UNIT NO. 3
(CAIRO, ARAB REPUBLIC OF EGYPT)
PSC 452, BOX 5000
FPO AE 09835-0007
93-10037
,les: S' e6 1$i iI j~lhlllliHtli~l~l
,4
.4m J_ Iroi. Med. Hvg., 47(6). 1992. pn. 821-429
Copyright 0 1992 by The Aerincan society of Tropical ModK-mo and Hylpfte
THE ROLE OF IgG ANTIBODIES FROM IRRADIATED
CERCARIA-IMMUNIZED RABBITS IN THE PASSIVE TRANSFER
OF IMMUNITY TO SCHISTOSOMA AL4NSONI-INFECTED MICE
BEVERLY L. MANGOLD AN) DAVID A. DEAN
ULS. Naval Medical Research Unt No. 3. cairo, Egypt
Abstract. Antibodies of the lgG subclass isolated from the sera of rabbits immunized
with cercariae subjected to 50 kilorads of gamma irradiation passively provided partial
immunity against Schistosoma mansoni challenge in C57BI/6J mice. These mice exhibited
reductions in adult worm burdens of 43--61% compared with recipients of normal rabbit
antibodies. Passively transferred lgG antibodies were most effective when given 4-7-days
postchallenge; theý, were less effective when given just before challenge, and were totally
ineffective when given 15 days postchallenge. It was also shown that the Fc portion of the
IgG molecule was important for passive transfer of immunity. Finally, we observed that
although some antibodies from irradiated cercaria-immunized rabbits recognized keyhole
limpet hemocyanin (KLH). these KLH cross-reacting antibodies were not necessary for
successful passive transfer of immunity. Antibodies from a KLH-immunized rabbit also
failed to passively protect mice.
Our laboratory has previously shown that par- compared with control rats. and naive rats re-
tial immunity against Schistosoma mansoni in- ceiving serum from KLH-immunized rats were
fection can be passively transferred to C57B1/6 partially protected (48% worm reduction) against
mice with lgG antibodies isolated from the se- an S. mansoni challenge.' In light of these stud-
rum of mice multiply immunized with cercariae ies, we investigated whether antibodies from ir-
irradiated with 50 kilorads of gamma irradia- radiated cercaria-immunized rabbits reacted with
tion.1 In addition, Bickle and others have dem- KLH and, if so. whether they had a role in pas-
onstrated the effectiveness of serum from irra- sive protection. We found that serum from ir-
diated cercaria-immunized rabbits in partially radiated cercaria-immunized rabbits contained
protecting mice against an S. mansoni challenge.' antibodies that recognized KLH. However, nei-
The use of rabbits as antibody donors has the ther these cross-reacting antibodies nor antibod-
advantage that large amounts of serum can be ies from a KLH-immunized rabbit were found
collected from a relatively small number of an- to be important in passive immunity in this mod-
imals, which can be repeatedly boosted. In the el.
current study, we attempted to confirm the ef-
fectiveness ofa heterologous passive transfer sys-
tem and to further elucidate the requirements of , ANt MErhODs
passive transfer of immunity. Our results con- Parasite
firm the finding of Bickle and others that serum
from irradiated cercaria-immunized rabbits can A Puerto Rican-derived strain of S. mansoni
passively protect mice. We also extend these maintained in Biomphalariag labrataw as used."
findings by demonstrating that 1) lgG antibodies
are specifically involved in protection, 2) the tim- Serum donors
ing of injection is crucial, and 3) the Fc portion
of the antibody molecule is necessary for passive Male New Zealand white (NZW) rabbits (Ha-
protection. zleton Research Animals, Denver, PA) -erved as
Gr-zych and others have shown that S. man- donors of serum. Rabbits were immunized by
soni shares a protective carbohydrate epitope with percutaneous exposure to cercariae subjected I
keyhole limpet hemocyanin (KLH); rats im- hr earlier to 50 kilorads (I, 227 rad/min) of gam-
munized with KLH exhibited reductions of 50- ma-radiation from a I'" Cs source. They received
75% in the number of adult worms recovered approximately 5,000 irradiated cercariae for the
821
822 MANGOLD AND DEAN
first immunization and 10,000-12,000 irradi- pressed as the percentage reduction in the num-
ated cercariae for subsequent immunizations, ber of adult worms recovered from immune an-
which were given three months apart. At each tibody recipients compared with normal zntibody
immunization, rabbits were anesthetized by in- recipients, and was calculated as previously de-
tramuscularinjection with a mixture ofketamine scribed.' The resistance data were analyzed sta-
(5C mg/kg of body weight; Vetalar; Parke-Davis. tistically by the two-tailed Student's t-test. P val-
Morris Plains, NJ) and acepromazine (0.5 mg/ ues less than 0.05 indicated significant differences
kg body weight; Ayerst. New York, NY). and among the groups compared.
exposed to half the irradiated cercariae on shaved
abdominal skin and half on one of the ears (right Preparationo f Fbab'), fragments
and left ears being alternately used as immuni-
zation sites). Blood was collected from the ear of Rabbit [gG was digested with pepsin (Sigma.
anesthetized rabbits (Innovar: 0.2 ml/kg: Pit- St. Louis. MO) according to standard proce-
man-Moore. Washington Crossing. NJ) three dures.' The sample was then fractionated on a
weeks after immunization and again at 5-6 weeks Protein A-Sepharose 43 column to remove any
after immunization. Serum was prepared as pre- undigested IgG. The non-bound fraction was
viously described.' Six immunized rabbits were concentrated by ultrafiltration (10,000 MW cut-
used as donors. Normal serum was collected from off filters: Amicon: Danvers MA). and then frac-
several age-, strain-, and sex-matched control tionated on a Sephadex G-50 column equili-
rabbits. The experiments reported herein were brated with 0.15 M phosphate-buffered saline
conducted according to the principles set forth (PBS). pH 7.2 and 0.02% sodium azide. The frag-
in the current edition of the Guide for the ('are ments were collected in the void volume, con-
and Use otfLaboratory'A .-nimals. Institute of Lab- centrated by ultrafiltration, dialyzed extensively
oratory Animal Resources. National Research against PBS. and stored at - 70"C until used.
Council. To facilitate testing of the effectiveness of pep-
sin digestion. I ml of rabbit IgG anti-sheep red
Antibody fractionation blood cell hemolysin (Cappel, West Chester, PA)
was added to 40 ml of serum from irradiated
The IgG portions of immune and normal rab- cercaria-immunized rabbits prior to isolation of
bit sera were fractionated by Protein A-Sephar- IgG by Protein A-Sepharose 4B chromatogra-
ose 4B affinity chromatography, as previously phy. Both undigested and pepsin-digested sera
described.' The whole immunoglobulin fraction were then tested for their ability to agglutinate
was prepared by precipitating serum twice with sheep red '"lood cells (SRBC) and to support
ammonium sulfate, pH 7.2 (50% final concen- complement-mediated lysis of SRBC.
tration).' Protein concentrations of purified im- Hemaggluttnation. Sheep red blood cells were
munoglobulins were determined spectrophoto- washed three times in physiologic saline and di-
metrically (assuming I optical density unit at 280 luted to a 1% suspension in veronal-buffered sa-
nm = 0.7 mgiml of purified immunoglobulins).' line (VBS), pH 7.4 (5 mM Na 5. 5-diethylbar-
bituric acid, 142 mM NaCI, 200 mM MgCl,
,4nalvsis of'resistance 6HO, 30 mM CaCI2) plus bovine serum albu-
min (BSA. crystalline: Calbiochem. San Diego,
Female C57B1/6 mice (Jackson Laboratory. Bar CA)) at a concentration of I mg/ml (VBS/BSA).
Harbor, ME) were age-matched in each experi- Rabbit IgG and pepsin-generated F(ab') prep-
2
ment. but overall, groups of mice were between arations were diluted in VBS/BSA to an initial
six- and 10-weeks old when challenged. Re- concentration of I mg/ml. Twenty-five microl-
strained, unanesthetized mice were challenged iter aliquots of antibodies were serially diluted
percutaneously by a I-hr tail exposure to ap- two-fold in VBS/BSA in the wells of round-hot-
proximately 150 cercariae (exact numbers are tom microhemagglutination plates, and 25 JAIo f
given in the text). 1%S RBC was added to each well. The plates
Adult worms were recovered from killed an- were sealed and incubated for I hr at room tem-
imals six weeks after challenge infection by a perature. The titers reported are the highest di-
modification6 of the hepatic portal perfusion lutions to give clear agglutination of SRBC.
technique.' Complement-medlated lysis of SRDC. Rabbit
Resistance to challenge infection was ex- antibody fractions and SRBC were diluted in
PASSIVE TRANSFER Of IMMUNITY TO SWANSONI 823
TABLE I
Passive transfer to miCe of protection against Schistosoma mansoni ink~cuio w~ith lgG isolated frc,ým irradiated
cercaria-jinmunized rabbits
Worm, bu~rdenm. eain zSD
Experiment' Antibody (no. 01 Mice) protectionP
I-80.2 ± 11,3(17)-
Normal lgG 84.7 ± 16.9 (6) -
Immune lgG (rabbit M2) 46.3 t 10.3 (7) 45.3 <0.00 1
Immune IgG (rabbit M6) 33.0 ±t 7.1 (6) 61.0 -0.001
Immune lgG (rabbit M8) 34.7 ± 7.2 (7) 59.0 .-O.00l
2 Normal lgG 65.7 ± 2.6 (12) -
Immune lgG (pool of M2. 29.5 ± 4.2 (6) 55.1 -0001
M6. -and 148)
All mice rmeewd an injection of protein A-ponfied lgG equivalent to i ml of serum on dav 4 and day 7 posichalletp. laWs oib tained from
normal rabbits or rabbits bled three weeksa fter a third immunization with 50-kilorad arradiaied-cercartae and was admitnitsered bý intravenous
(experiment 1) or intrapensoneal (experiment 2) injecion. Mice werec dullentlrd wiih 157 )scnpenmen(te of 135 lexpeflmeni 2) peietrativilcercanae
VBS/BSA and added to the plates. as above. In rabbit received 100 uig of KLH in adjuv ant dis-
addition. 50 sdl of guinea pig complement (Cap- tributed equally into three sites: I) intramuscular
pei) diluted 1:20 in VBS/BSA or VBS!BSA alone injection in the right hind quarter. 2) intramus-
was added to the wells. The plates were sealed cular injection in the left hind quarter. and 3)
and incubated I hr at 37*C. The titers reported subcutaneous injection in the neck area. Im-
are the highest dilution to give complete lysis of munizahions were given two months apart. Se-
SRBC. rum for transfer was collected two weeks after
the second immunization.
EnzYme, linked immunosorbent assaY (ELISA)
Keyhole limpet hemocyanin (Megathura cren.-. fiiYchoaorp~
ulate-, MW = 3-7 x 106, Calbiochem) was used Keyhole limpet hemocyanin (70 mg) was cou-
to coat polystyrene flat-bottom 96-well micro- pled to CNBr-activated Sepharose 4B (4 g of
titer plates (Immunlon 2; Dynatech Laborato- powder- Pharinacia. Piscataway, NJ) according
ries, Alexandria. VA) at a concentration of 0.2 to protocol provided by the manufacturer.
pg/well. A standard ELISA protccol" was used The absorption of irradiated cercaria-immu-
with the following modifications: washing buffer nized rabbit serum was performed as follows. An
(36 mnM boric acid, 158 mM NaCI. 0.05% Tween- affinity column of KLH-Sepharose was equili-
20; 0.0 1% thimterosal, pH 8) was used in place brated with 0. 14 M sodium phosphate buffer. pH
ofPBS and all antibodies were diluted in washing 8.0. Immune rabbit serum was passed slowly over
buffer plus 0.5% BSA. Horse radish peroxidase- the column, and the unbound fraction was pre-
conjugated goat anti-rabbit IgG (1:4.000 dilu- cipitated with ammonium sulfate. pH 7.2.' ex-
tion-, Tago, Inc., Burlingame, CA) was used as tensively dialyzed against PBS. and stored at
secondary antibody. The plates were developed - 70*C until used. Unabsorbed immune and nor-
with ABTS substrate (Kirkegaard and Perry. mal sera were precipitated with ammonium sul-
Gaithersburg, MD) and read after 5 min with a fate by the same procedure.
Micro ELISA Reader MR-580 (Dynatech Lab-
oratories) at an optical density of 405 nm.REUT
Rabbit immunization with KLH In the initial experiments, lgG antibodies iso-
lated from rabbit sera collected three weeks after
A NZW male rabbit was immunized twice with the third immunization with irradiated cercariae
KLH in Ribi adjuvant (0.25 mg of monophos- were tested for their ability to transfer passive
phoryl lipid A, 0. 25 Ing of trehalose dimycolate, protection against S. mansoni infection to recip-
0.25 mg of cell wall skeleton, 0.02 mil. of hexa- ient mice (Table 1). In both experiments. mice
methyl-tetracosahexane, and 0.002 mil of Tween received two injections of [gG. at four days and
80/injection; Ribi ImmunoChemn Res Inc., seven days postchallengc. These times were se-
Hamilton. MT). For each immunization, the lected because previous studies with homologous
824 MANGOLD AND DFAN
70
6
50o
~50
E 40
.£ 230
.5 30
SU210' no. no 20
0 0
-I +1 +4 +7 +15 i- ISh 5d 1.5h Sd
Day of injection Dom: 30 "q 0.36 mt
FIGURE 1. Percent reduction in worm burdens in FIGURE 2. Effect of dose and time of injection of
mice receiving 4 mg (closed bar) or I mg (open bar) rabbit immunoglobulin on transfer of resistance Mice
of IgG from rabbits bled three weeks after the fourth received ammonium sulfate-precipitated total tim-
immunization with irradiated cercariae of Schistosonla munoglobulins from rabbits immunized wi!h irradi-
monsoni. Worm burdens were compared with those of ated cercanae of Schistosorna mansoni or from un-
mice receiving lgG from unimmunized rabbits. Values immunized normal rabbits. Values are the mean -
are the mean + SD. The reductions at days + 1, +4, SD. The reductions at - 1.5 hours (h) and 5 dais (d)
and +7 at both doses and at day -1 at a dose of 4 mg at a dose of 3 mg and at 5 d at a dose of0.38 mgwere
ml were highly significant (P < 0.001). ns = not sig- highly significant (P < 0.001). ns = not significant (P
nificant (P > 0.05). > 0.05).
serum transfer to mouse recipients had shown there was no statistical difference among the re-
that serum administered around the time of ductions seen in the mice receiving antibodies
schistosome lung-phase migration transferred on days -1. 1. 4, and 7.
optimal levels of protection.' In the first exper- A slightly different pattern was observed when
iment, IgG fractions isolated from three rabbits the amount of IgG was reduced four-fold. In this
were tested separately. Mouse recipients of IgG case, the levels of protection transferred when
from all three rabbits exhibited highly significant the immune IgG was administered on days 1.4.
reductions in adult worm burdens compared with and 7 were all significant. but were approxi-
recipients of IgG from normal rabbits or no IgG: mately one-third lower (P < 0.05) than those
levels of protection transferred were 45.3%. observed on the same days with the higher dose
59.0%, and 61.0%, respectively. In the second of IgG. In contrast. I mg of immune lgG mouse
experiment, IgG isolated from these same rabbits given on day - I no longer transferred a sign if-
was pooled and retested. A comparable level of icant level of protection. Irrespective ofthe dose,
protection (55.1%) was observed. Although not immune IgG given 15 days postchallenge was
formally tested in the same experiment, it would totally ineffective in transferring protection.
appear that the intravenous and intraperitoneal The effect of time and dose of Ig transfer was
routes of IgG injection are equally effective in
transferring protection. TAm.. 2
Hemagglutinattona nd complement lysis titers
Effect of time and dose of IgG transfer on
protection Xim - Comr.
16G. Undr .tort
The transfer efficiency of a single injection of Anti-Sm plus anti-SRBC 240 160
IgG (4 mg/mouse or I mg/mouse) administered Pepsin-digested
at various times relative to the time of challenge anti-Sm plus anti-SRBC 240 < 10
infection was examined (Figure 1). When the Anti-Sm <15 <10
higher dose of IgG was used. significant levels of Igo fi aa rabb mnpiy immminued wiitbim naid SoW(cid:127)o*W~
mm aenc_eftmh. w nuid wlvntbiS. dibo ft040 VaI-
between 55% and 6 1% reduction in worm burden unieof ta iab(cid:127)n, t IsO mn-,shp mdW ood cedb eumaem -a--
SRlCk wwn imind bap rmqim A-Se iinr4BammoatyhwnisWUpy.
(P < 0.001) were passively transferred if the an- p., d*pasie o.r,raWhh(cid:127) (cid:127)-.aa ndm mue,-use Mn-
tibodies were administered anytime from the day w mdM etof 5RB
prior to challenge to seven days postchallenge; Redpromi ofbabm dilution wi(cid:127),r n wnpku pmuo f SRC.
PASSIVE TRANSFER OF IMMUNITY TO S. MANSONI 825
TAaLE 3
Comparison of efficacy of intact IgG and F(ab').fragments in passively trarsternngp rotection
Antibody Woam burden. mean :r So
EiLenment tatst(cid:127)ret (io. of mic) % proiie P
I None 76.3 111 .9(12)
Normal igG 71.5_ 10.3(10) -
Immune lgG 42.5 t 7.2 (10) 43.1 <0.001
Normal F(ab'): 77.2 ± 8,8 (5) -
Immune F(ab'): 76.1 ± 16.0(7) -1.0 >0.05
2 None 62.3 ± 10.6 (9)
Normal IgG 60.7 ± 15.0(6) -
Immune lgG 34.3 ± 10.3 (8) 44.4 <0.001
Immune F(ab'). 66.8 ± 9.8 (6) -8.3 >0.05
Mice were infected with 165 (experiment I) or 148 fexperiment 2) penetrating cercaruae.
t Immune denotes that original senumw as collected from rabbits three weeksa fter the fifth immunization with irradiated cernanra. Each mouse
received an intvra(cid:127)ertntcal Injection o" 4.5 mg ot arntbodies, (cid:127)n days 4 and 7 postinfec|ion
t Calculated using the average worm burden of all control mice (no antibody and normal antibody recipients?, which was 74 7 -1 10 7 (man
SD) for expenment I and 61.7 t 12.0 for experiment 2.
retested in a second experiment (Figure 2). In valent antigen binding activity) and to support
this instance, mice received 3.0 or 0.38 mg of complement-mediated lysis of SRBC (an indi-
total Ig 1.5 hr prior to challenge infection or five cator of a functional Fc) (Table 2). Undigested
days postchallenge. Significantly more protection IgG was able to both agglutinate SRBC and sup-
was transferred at both doses if the immuno- port complement-mediated lysis. In contrast.
globulin was administered five days postchal- pepsin-digested material was able to agglutinate
lenge rather than near the time of challenge (P SRBC as efficiently as intact IgG. but could not
< 0.01 for 3 mg and P < 0.05 for 0.38 mg). support complement-mediated lysis. this being
Thus, in both experiments it would appear that consistent with F(ab') activity. Thus. pepsin di-
2
when the timing of the Ig administration coin- gestion appears to have successfully cleaved off
cided with the lung phase of schistosome migra- the Fc portion of the IgG while maintaining
tion, the level of protection passively transferred F(ab') activity.
2
was as high as. and generally higher than, when We examined the abilities of intact lgG and
the Ig was administered at the time of challenge. F(ab'), fragments to passively protect mice (Ta-
ble 3). In two experiments. it was observed that
Comparison of transfer efficiency of IgG versus mice receiving intact IgG from immunized rab-
pepsin-generatedF (ab') fragments bits had a highly significant (43-44%) reduction
2
in the number of adult worms recovered relative
The transfer efficiency of pepsin-generated to recipients of IgG from normal rabbits or to
F(ab'), fragments was compared with that of in- mice receiving no IgG. In contrast, mice receiv-
tact IgG molecules to determine whether the Fc ing F(ab')h fragments from immunized rabbits
portion of the antibody molecule was necessary exhibited no reductions in worm burden. Thus,
to interact with some component(s) of the effec- it would appear that the Fc portion of the IgG
tor system for immune elimination of the schis- molecule is necessary for successful passive
tosomes, or alternatively, whether binding by the transfer of protection.
divalent antigen binding portion of the molecule
was sufficient.
To facilitate testing of the effectiveness of pep- Evaluation ofanti-KLH activity in sera of
sin digestion, serum from irradiated cercaria- irradiatedc ercaria-immunizedr abbits
immunized rabbits was combined with a small
amount of rabbit IgG anti-SRBC hemolysin pri- In light of the evidence that S. mansoni shares
or to isolation of IgG by Protein A-Sepharose 4B a protective carbohydrate epitope with KLH,3
affinity chromatography and subsequent pepsin we investigated whether antibodies from irra-
digestion. Both undigested and pepsin-digested diated cercaria-immunized rabbits contained
preparations were tested for their ability to ag- anti-KLH activity. Results from an ELISA using
glutinate SRBC (an indicator of functional di- KLH as the antigen indicated that irradiated cer-
826 MANGOLD AND DEAN
1.2 -Efficatct of rabbit anti-kevihole hmper IwmotVanin
(KLII) Ig in passivetv protecting mice from Schis-
E 10 -tosonia mansoni iflfeaiofl
o VWormb urden.
0.8 -~ Antibody transferred meass SD (no of micei
ZNone 72.5 t 13-0 (13)
o. 6 Normal Ig 71,8 ± 6.7 48)
Anti-KLH Ig 72.A ± 14-200)
0.4 *Rabbit MU10 as immunized two times with 100)m e of KLH in Ribi
0.a4ln - -) adjusant: immunizaations wemf fisei iao months apart
Serum was coliected two weeksa fter the secondim munization and totLal
0 02 i wasis olated by ammonium sulfate precipittiaon, Mice received f0 mg
o.2 o( Ig subcutaneously one day and four days posiinfection wiih 157 cei-
carule.
162 54 15 6 2 The above experiment demonstrated that the
Rabbit Ig (jug) anti-KLH activity in irradiated cercarta-im-
FIGURE 3. Titration of antibodies against keyholc munized rabbit serum is not necessary f'or pas-
limpet heniocyanin (KLl-) as measured byi an enzyme- sive transfer of protection. It did not. however.
linked immunosorbent assay. Microtiter wells were rule out the possibility that anti-KLH anti bodies
coated with 0.2 fig of KLH. The wells were incubated can passively protect. To test this, we hypenm-
with serial dilutions of immunoglobulin from unim- munized a rabbit against KLH to be used as a
munized rabbits (&Ž).f rom irradiated cercariae-im-
munized rabbits (6I). or KLH-absorbed immunoglob- source of antibodies for passive transfer. Serum
uilmn from irradiated cercariac-immunized rabbits (0), isolated from the donor rabbit was shown to have
a very high liter ofanti-KLH antibodies by ELISA
(i.e.. significant activity was still detected at a
caria-imniunized rabbit Ig does recognize an ep- serum dilution of 3 x 10Os). However. Ig isolated
itope on KLH (Figure 3). from this rabbit was unable to passively protect
Having identified an anti-KLH activity in im- mice against an S. 'nansoni infection (Table 5).
mune rabbit serum, we next attempted to dcicr-
mine whether this activity was associated with DISCUSSION
protection. To test this. we absorbed immune
rabbit Ig against KLH by affinity chromatogra- The results presented here demonstrate that
phy. The absorption was successful as judged by lgG antibodies obtained from sera of rabbits
ELISA analysis (Figure 3): absorbed Ig was in- multiply immunized with 50-kilorad-irradiated
distinguishable from normal 1g. Next, we pas- S. mansimn cercartae are able to passively trans-.
sively transferred both the absorbed and unab- fer high levels of resistance against an S. nwansoni
sorbed preparations to determine their protective challenge infection to naive mice (Table 1). The
a,.tivities. It was found that immune Ig absorbed timing of the passive transfer was shown to be
against KLH was as effective as unabsorbed irn- critical. Immune lgG antibodies administered
mune Ig int passively transferring protection (Ta- during the first week of infection were effective
ble 4). in transferring protection. while lgG given 15
TABL.E 4
Efficacy of keyhole limpet hemocyanin (KLH)-ab~orbed irradiatedc errarta-immunized rabbit Ig in passively
protecting mice from Schistosoma mansoni infection
Worm burden
Antibody transferred* ("ma.n t SD) %Protection
Normal ~73.7 ±4.7
Immune 32.1 ±3.8 56 <0 .00 1
KLH-absorbed immune 36.8 ±4.2 so <0.001
Mice received an uriitperitooeail injection of 5 me of lioul III an day 4 posichal~ (11m ice/srcep) Ig wif isolated by ammstoawmenu silhm
PPstetsuaon of setum from an umeimimuiad rabbit (nactual) or a rabbirt bind three weeksa fterth e senli immuiarlon with irradiated cetearwea
(immasset Immune IIIwas Male& I ? I d over a KLH-Sepbasoee 4B ooiwsno (KLIH-adeaottd imitsmoe).
PASSIVE TRANSFER OF IMMUNITY TO S. MANSONI 827
days postchallenge was totally ineffective (Figure or 5-6 days postchallenge (41.0 ± 9.3%. range
I). By 15 days postchallenge, schistosome mi- 31- 56%) (our calculations of averages). In a sim-
gration to the liver in mice is largely complete.9 ilar study, mice injected with 400 isg of Protein
Our previous parasite transfer studies have dem- A-purified IgG from 60-kilorad-irradiated cer-
onstrated that schistosomes are sensitive to im- caria-immunized NZW rabbits exhibited reduc-
mune-dependent elimination through the lung lions in adult worms of 21.6-28.4%.12
phase of migration, but become totally insensi- To evaluate the effector mechanisms involved
tive to immune elimination once they reach the in the passive protection of mice. we compared
liver.10 Thus, the inability of immune IgG to the ability of immune intact IgG antibodies and
"1
transfer protection on day 15 is probably due to pepsin-generated F(ab') fragments to transfer
2
the fact that at this time, the majority of schis- protection. It was found that removal of the Fc
tosomes have migrated from the lungs to the portion of the antibodies by pepsin digestion
liver, where they are no longer sensitive to im- completely abrogated the ability of these anti-
mitme elimination, bodies to confer passive protection. Thus, the
It was also observed that antibodies were less divalent antigen-binding portion of the IgG was
effective in transferring protection when admin- not sufficient to mediate protection. and it would
istered at the time of infection than when ad- appear that some effector mechanism(s) involv-
ministered during the lung phase of migration ing host effector components bearing Fc recep-
(Figures I and 2). This finding is consistent with tors is required for the immune elimination of
our previous passive transfer study, in which it schistosomes in this model. Examples of host
was shown that levels of protection transferred factors that bear Fc receptors and are often in'-
to mice with serum from irradiated cercaria-im- plicated in immune effector mechanisms are
munized mice were as good or better when the complement. polymorphonuclear leukocytes. and
serum was given seven days postchallenge com- macrophages. Complement may not be an irn-
pared with the day prior to challenge.' It is also portant effector system in mice actively "mmu-
consistent with our previous findings from au- nized with irradiated cercariae. however, since
toradiographic migration tracking9 and parasite 1) mice genetically deficient in C5 develop the
transfer'0 studies, which demonstrated that the same level of immunity as their congenetically
lung stage schistosomulum is the major target of C5 normal counterparts," and 2) the level of
immune-dependent elimination in irradiated immunity in irradiated cercaria-immunized mice
cercaria-immunized mice. is not affected by decomplementation with cobra
The observation that partial protection can be venom factor during the skin and/or lung phases
passively transferred to mice with IgG isolated of migration."4 Likewise, eosinophils appear not
from irradiated cercaria-immunized rabbits to be required in this model, since ablation of
compares favorably with our previous finding of eosinophil responses with an anti-interleukin-5
passive transfer to mice with homologous irra- (IL-5) neutralizing monoclonal antibody failed
diated cercaria-immunized mouse serum or lgG. I to effect the level of protective immunity in ir-
Indeed, the heterologous transfer system (rabbit radiated cercariae-immunized mice." There-
to mouse) gave consistently higher levels of pro- fore, if we assume that our heterologous (rabbit.
tection than the homologous system (mouse to to-mouse) passive transfer system is analogous
mouse). When optimal transfer conditions were to the actively immunized mouse model, then
used (i.e.. transfer during the first week of infec- the requirement for the Fc portion of IgG anti-
tion with 3 mg or more of immunoglobulins), body may depend on Fc interaction with host
the mean level of protection transferred with rab- components other than complement and eosin-
bit immunoglobulins in 11 experiments was 54 ophils.
± 1.9% (mean ± SEM) (range 43-61%). These The finding presented in this report that high
levels are comparable with those of Bickle and levels of protection can be passively transferred
others,2 who demonstrated that whole sera (ad- with IgO from rabbits multiply immunized with
sorbed against mouse red blood cells) from NZW irradiated cercariae, along with previous dem-
rabbits multiply immunized with irradiated cer- onstrations of passive transfer of protection with
cariae were able to transfer significant levels of serum or immunoglobulin-fractions from ira-
protection when transferred to mice around the munized mice,' 12.16 rats,'7-20 and rabbits,2 12
time of challenge (43.4 ± 4.0%, range 34-69%) passive transfer of protection with monoclonal
828 MANGOLD AND DEAN
antibodies.'2-a6 and ablation of immunity in Authors' address: Beverly L. Mangold and David A.
M-suppressed mice'3 indicate a role for antibody Dean. Schistosomiasis Research Laboratory. U S. Na-
in immunity intoo imsschuhinisstyoto ssoommiaassiis. . TThiiss dooeess nnoott v0a9l8 3M5e-d0i0c0al7 .Research Unit No. 3. PSC 452. FPO AE
preclude, however, that cell-mediated immune
responses are also important in irradiated cer- Reprint requests: Research Publications. U.S. Naval
caria-induced immunity. Involvement of such Medical Research Unit No, 3, PSC 452 Box 5000. FPO
responses is suggested by evidence of a role for AE 09835-0007.
L3T4* cells as determined by ablation experi-
ments.- 2 8 correlation of immunity in P/J and
P/N mice to interferon-gamma (IFN--y) produc- REFERENCES
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In vitro and in vivo effector function of rat lgG2a Acce-io.i For
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