T-Cell Modulation of the Murine Antibody Response to Neisseria meningitidis Group A Capsular Polysaccharide

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1 NFECON AND MMUNY, Jn. 988, p /88/259-8$2./ Copyright C 988, Americn Society for Microbiology Vol. 56, No. -Cell Modultion of the Murine Antibody Response to Neisseri meningitidis Group A Cpsulr Polyscchride EUGENE MULLER*t AND MCHAEL A. APCELLA'2 Deprtment of Microbiology' nd Division of nfectious Disese,2 Stte University of New York t Bufflo, Bufflo, New York 425 Received 2 June 987/Accepted 6 October 987 -cell modultion of the ntibody response of BALB/c mice to group A meningococcl cpsulr polyscchride (PS) ws exmined by using n enzyme-linked immunosorbent ssy. An optiml dose (5,ug) of ntigen induced n immunoglobulin M (gm) response of short durtion; no gg or ga ntibody could be detected. he cpcity to produce serum ntibody begins t bout 3 weeks of ge. Concnvlin A (ConA) inhibited the mgnitude of the response by 4 to 6% when given t the time of immuniztion; it enhnced the response twoto eightfold when given 2 dys fter PS. -cell-medited suppression could be trnsferred to nive mice by injection of spleen cells from low-dose-primed mice. A secondry ntibody response could be induced by immuniztion with live meningococci. Here, the gm response ws 8- to -fold greter thn tht of mice given n optiml dose of PS; gg ntibody ginst group A PS incresed week fter immuniztion to levels tht were - to,-fold greter thn those of mice immunized with PS. he ntibody response could not be ugmented by multiple injections of PS; suppression occurred fter low-dose priming or hyperimmuniztion with PS. hese studies indicte tht the ntibody response to PS is not completely -cell independent; rther, it is inhibited nd mplified by cells. Meningococci continue to cuse serious endemic nd epidemic disese despite the development of vccines to serogroups A nd C (32). Protection ginst invsive diseses by these polyscchride (PS)-encpsulted orgnisms involves the production of nticpsulr ntibodies for the opsoniztion nd subsequent clernce of the orgnisms (5, 6). Currently vilble vccines re efficcious in preventing infection in dults nd older children. However, the purified meningococcl cpsulr PS vccines, s well s the PS of other bcteri, re poorly immunogenic in the ge group t risk for infections cused by encpsulted bcteri, i.e., children younger thn 2 yers of ge (4, 7, 33). hus, to improve the effectiveness of PS vccines, it is necessry to define those fctors which contribute to the development of such hyporesponsiveness. Much of our current knowledge of regultory mechnisms involved in the ntibody response to bcteril PS is bsed on studies of the ntibody response of mice to type 3 pneumococcl PS (); similr mechnisms hve been inferred in the ntibody response of mice to Hemophilus influenze type b PS (23), group C meningococcl PS (), nd dextrn (25). Humn ntibody responses to PS ntigens prllel those described for mice (7). Bcteril PS consist of repeting oligoscchride units nd re considered to be -cell-independent () ntigens, since helper cells re not essentil for the induction of ntibody synthesis (8). Although ntibody responses to ntigens do not require helper cells, the mgnitude of these responses re clerly regulted by cells (, 37, 38). Antibody responses to ntigens re restricted to short-lived immunoglobulin M (gm) ntibody; no significnt ugmenttion of the response is observed fter reimmuniztion (3, 8). Hence, prominent gol hs been to elicit long-lived ntibody responses to bcteril PS, s ob- * Corresponding uthor. t Present ddress: Chnning Lbortory, 8 Longwood Avenue, Boston, MA 25. served in ptients following disese cused by encpsulted orgnisms. he immunogenicity of meningococcl PS shows extreme diversity in dult humns nd experimentl nimls; the PS of groups A, C, nd B rnge from being good to extremely poor immunogens. o begin to investigte the possible resons for such responsiveness, we hve chrcterized the ntibody response to group A meningococcl PS in BALB/c mice by using n enzyme-linked immunosorbent ssy (ELSA). MAERALS AND MEHODS Mice. Mice were derived from breeding pirs of BALB/cByJ (Jckson Lbortory, Br Hrbor, Mine) mintined t the niml fcility of the Clinicl Center of the Stte University of New York t Bufflo. Antigens nd immuniztion procedures. Group A PS for ELSA ws prepred from strin Al (obtined from H. Schneider, Wlter Reed Army Hospitl, Wshington, D.C.) s previously described (8). Group A PS used to immunize mice ws kindly provided by the mnufcturer (Merck Shrp & Dohme, Rhwy, N.J.). Meningococci were grown overnight t 37 C nd 2 rpm in Mueller-Hinton broth (Difco Lbortories, Detroit, Mich.), diluted :2 in fresh broth, nd grown until the culture reched erly log phse (opticl density t 66 nm,.). t ws determined by inocultion of chocolte gr pltes with dilutions of such cultures tht there were bout 5 x 7 live meningococci per ml t log phse; for immuniztions, live orgnisms were precipitted from log-phse cultures by centrifugtion. Whole-cell vccine ws prepred from meningococci grown in broth which were precipitted by centrifugtion, wshed twice in 95% ethnol, nd then wshed once in cetone; this preprtion ws suspended in distilled wter nd lyophilized (34). he PS, whole-cell vccine, or live orgnisms were dministered intrperitonelly (i.p.) in volume of.5 ml of.85% (wt/vol) sline unless otherwise stted. Mice were exsngui- 259

2 26 MULLER AND APCELLA nted by crdic puncture fter chloroform nesthesi, nd the ser were stored t -2 C. rnsfer of low-dose suppression with cells. Mice were given subimmunogenic dose (.5,ug) i.p. of group A PS, nd 8 h lter their spleens were removed. A cell suspension ws prepred by perfusing the spleens with medium 99 (GBCO, Grnd slnd, N.Y.) with % (vol/vol) fetl bovine serum (GBCO). he cells were wshed by centrifugtion t 2 x g t 4 C for min nd then suspended to contin 7 nucleted cells per ml. o determine the type of cell responsible for the trnsfer of suppression, the splenocytes were treted for 45 min t 22 C with either mixture of :5 ffinity-purified rbbit nti-mouse gm nd gg (Litton Bionetics, Chrleston, S.C.) or :5 monoclonl rt nti-hy-.2 (superntnt of the hybridom 3-H2 [22], kindly provided by chiro Nkmur nd Michel Kirisits, Deprtment of Pthology, Stte University of New York t Bufflo), nd then were treted with : grose-dsorbed guine pig serum (GBCO) s source of complement for 3 min t 37 C. he cells were wshed by centrifugtion. At the time of immuniztion with 5,ug of group A PS, mice were injected intrvenously with 2 x 7 of either preprtion of cells in.2 ml of medium. Controls consisted of spleen cell suspensions from untreted mice which were treted with ntibodies nd complement. ELSA. Serum ntibodies specific for group A PS were detected by using n ELSA which ws performed s previously described (39). Flt-bottom, polystyrene microtiter pltes (Linbro/itertek; Flow Lbortories, nc., McLen, V.) were sensitized with.2 ml per well of PS conjugted to poly-l-lysine (9). Optimum coting occurred when PS ws conjugted to 24,-moleculr-weight poly- L-lysine (Sigm Chemicl Co., St. Louis, Mo.) t rtio (wt/wt) of ;.2, respectively. he conjugtion ws performed for 2 h t 4 C; the mteril then ws diluted to 25 p.g of PS per ml in sensitizing buffer (.5 M ris hydrochloride, 2 mm EDA,.3 M KCl, ph 8) nd llowed to dsorb to the pltes for h t 37 C nd then overnight t 22 C. Coted pltes were stored t 4 C for up to 2 weeks. Prior to use, the pltes were blocked with phosphte-buffered sline (PBS) contining 3% (wt/vol) geltin for h t 37 C. Ser to be tested were diluted serilly in wsh buffer nd PBS contining.5% (vol/vol) polysorbte (ween 2) nd then incubted in the pltes for 9 min t 37 C. Conjugte, got nti-mouse gm, gg, or ga linked to horserdish peroxidse (Kirkegrd & Perry Lbortories, nc., Githersburg, Md.) ws diluted :5, in PBS contining.5% (wt/vol) geltin nd then llowed to rect in the pltes for h t 37 C. Substrte buffer (.5 M citrte-phosphte buffer, ph 5, contining.3% H22 [wt/vol] nd.2% o-phenylene dimine [wt/vol]) ws incubted in the pltes t room temperture; fter 25 min, the rection ws stopped by dding 5 R of 4 N H2SO4. A495 ws mesured with model EL-37 EA reder (Bio-ek nstruments, nc., Burlington, Vt.). By using modifiction of the effective dose method of ELSA dt nlysis (24), the nti-ps serum ntibody responses of individul mice were expressed reltive to tht of pooled ser. he reference pool consisted of 87 mice which were treted with PS ccording to n optimlly immunogenic protocol, i.e., exsnguintion 5 dys fter i.p. injection of 5 jig of PS. he four to six different dilutions of the reference pool which best covered the close-to-liner portion of the sigmoidl dose-response curve (opticl density versus dilution) were selected nd used to construct reference line. hen, for ech serum smple, the dilutions which corresponded to the liner portion of the curve of the smple were NFEC. MMUN. compred with the reference line; i.e., the logrithmic distnce t the observed bsorbnce of ech dilution of the smple ws mesured to the corresponding vlue of the reference line. hus, the ntibody ctivity of ech smple ws expressed s reltive response, the ntilog of the rithmetic men of the mesured distnces (Fig. ). Sttistics. Student's t test (35) ws used to evlute the significnce of the differences observed between the reltive responses. Differences were considered to be significnt when probbility (P) vlues of <.5 were obtined. Other mterils. Horse nti-mouse lymphocyte serum (ALS) (lot 437; Microbiologicl Assocites, Bethesd, Md.) ws stored t -2 C. Concnvlin A (ConA; Sigm) ws stored t -2 C s solution in sline ( mg per ml). Mice were treted i.p. with.3 ml of ALS or 3 jig of ConA t the time of immuniztion with PS or 2 dys lter s previously described (4, 27). RESULS Dose-response reltionships. Mice were immunized with single i.p. injection of group A PS; the reltive ntibody response ws determined 5 dys lter (Fig. 2). he mgnitude of the specific gm response incresed s the dose incresed from.5 to 5 jig. Lrger doses resulted in decresed mounts of specific ntibody; hence, 5,ug ws considered to be the optiml dose for immuniztion, lthough the responses to doses of.25 to 25,ug were not significntly different. Similr dose-response reltionships were observed in mice immunized i.p. with killed, whole meningococci or intrvenously with PS; here, the optiml doses were 25 nd.5,ug, respectively (dt not shown). Furthermore, the mgnitude of the reltive gm responses to n optiml dose of immunogen were similr regrdless of the immunogen, either PS or killed orgnisms, or the route of immuniztion. he response on dy 5 to either PS or killed meningococci ws predominntly of the gm clss; gg or ga responses were not detected in immunized mice (dt not shown). Kinetics of ntibody response. Mice were immunized with n optiml dose (5,ug) of PS, nd the reltive ntibody response produced ws determined t vrious times fterwrds (Fig. 3). he mount of group A PS-specific gm ws mximl by dy 4 or 5; therefter, the level declined for 3 to 4 weeks until it pproched tht in unimmunized mice. he responses on dys 4 to 6 were not significntly different from ech other. gg or ga responses were not detected t ny time during this period. Furthermore, dministrtion of second optiml dose of PS 4 weeks fter the first tretment did not result in the development of immunologicl memory, i.e., the reltive gm, gg, nd ga responses t 5 dys fter the mice were boosted were the sme s ge-mtched mice given only one injection of the ntigen (P >.5) (dt not shown). Ontogeny of ntibody response. Dose responses 5 dys fter single injection of PS were determined in mice 2 to 8 weeks of ge (Fig. 4). he optimum immunizing dose ws found to be S,ug in ll ge groups considered. he mgnitude of the gm response incresed with ge. he gm ntibody responses of mice 2, 3, 4, nd 6 weeks old were 6,, 34, nd 94%, respectively, of those detected in mice 8 to 24 weeks old given n optiml dose of PS. Priming with low doses of PS. Mice were given mrginlly immunogenic dose (.,.5, or.,ug) of PS 2 to 3 dys prior to immuniztion with n optiml dose (5 jg). At 5 dys fter immuniztion, the mgnitude of the ntibody response

3 VOL. 56, 988 MENNGOCOCCAL A POLYSACCHARDE ANBODY RESPONSE 26 A 2. v B 2.- * v!/2 FL - * cc c / ' L f8 r 25 do 4 Reciprocl Serum Dilution ' Y FG.. An exmple of the reltionship of titrtion ELSA dt (A) to results clculted by the effective dose method (B). he men nd rnge of the reltive responses re shown for test ser with different mounts of nti-ps gm ntibodies. OD495, Opticl density t 495 nm. ws compred with tht of mice treted with only n optiml dose of PS (Fig. 5). Priming with.,.5, nd.,ug of PS decresed the response by 28, 52, nd 37%, respectively, reltive to tht elicited by single, optiml dose of PS. Furthermore, priming with 5,ug inhibited the response by 73%. he decresed responses due to priming with doses greter thn.,ug were significntly different (P <.25); the priming effect of.5,ug observed in experiment ws lso significntly different (P <.). nduction of suppression by trnsfer of low-dose-primed spleen cells. On the bsis of detiled studies of the kinetics of low-dose prlysis in the pneumococcl type 3 ntibody response (, 6, 26, 27), the following experiments were performed to demonstrte similr -cell-medited suppression in the nti-group A PS ntibody response. Mice were primed with subimmunogenic dose (.5 p,g) of PS, which elicited low-dose suppression (Fig. 5). Spleen cell suspensions were prepred 8 h lter from these mice nd then treted with either gm or gg ntibodies or monoclonl nti-hy-.2 ntibody plus complement before trnsfer to mice immunized with 5,ug of group A PS. he mgnitudes of the reltive gm nti-ps responses were determined 5 dys lter for those mice, s well s for immunized mice either not given cells or given nti-immunoglobulin-treted cells from unprimed mice. he dt from the first experiment (Fig. 6) show tht compred with the reltive ntibody response of mice not given cells, 48 to 57% decrese in the mgnitude of the response occurred in mice given 2 x ' primed cells (P <.) nd 33 to 6% decrese occurred when 2 x 7 nti-immunoglobulin-treted primed cells were given (P <.). he pretretment of primed spleen cells with ALS plus complement seemed to eliminte the suppressive cpcity of the trnsferred cells (P <.2). o clrify the nture of cells which medited the suppression induced by primed e. 2- x4j Y6.-V "r--a2 JA..5 -P * i so Do" (ug) FG. 2. Mgnitude of the serum gm response of mice immunized with different mounts of meningococcl group A PS. he vlues represent the mens + the stndrd errors for groups of three to eight similrly treted mice..

4 262 MULLER AND APCELLA NFEC. MMUN. c CL 2. A6 <% A - i :i ii~~~~~ ~ ~~~' ~ L.! Dy fter mmuniztion i R FG. 3. Kinetics of the serum gm response of mice immunized with 5,ug of meningococcl group A PS. R, Response of mice immunized on dy, reimmunized 3 to 4 weeks lter, nd bled 5 dys fter boosting. he vlues represent the mens ± the stndrd errors of groups of 3 to 32 mice. spleen cells, we obtined specific nti--cell regent, monoclonl nti-hy-.2 ntibody, which possessed complement-medited cytotoxic ctivity. n the second experiment, it ws shown tht the trnsferred suppression could be eliminted by prior tretment of the primed spleen cells with nti-hy-.2 ntibody plus complement (P >.5), wheres tretment with nti-immunoglobulin ntibodies plus complement produced degree of suppression comprble with tht produced by untreted primed cells (no cells versus ntiimmunoglobulin-treted cells, P <.). he trnsfer of 2 x 7 nti-immunoglobulin-treted cells from unprimed mice filed to influence the mgnitude of the reltive ntibody response of mice immunized with 5,ug of group A PS (P >.4). hese studies demonstrte tht primed spleen cells bering the hy-.2 phenotypic mrker, i.e., cells, medite the suppression observed.. 2F Y2 it 9 ẓ 9X 'D U.5. 5 Priming Dos.(ug) FG. 5. Effect of priming with mrginlly immunogenic or optiml dose of meningococcl group A PS 2 dys prior to immuniztion with 5,ug of PS. Symbols:, experiment ;, experiment 2. Effect of ConA or ALS on the ntibody response. Mice were given single i.p. injection of ConA or ALS t the time of immuniztion with n optiml dose of PS, nd the mgnitude of the nti-ps response ws determined 5 dys lter (Fig. 7). he results show tht ConA given t the time of immuniztion reduced the response by 4 to 6%, wheres tretment with ALS enhnced the response.6- to 3.5-fold. retment with ConA 2 dys fter immuniztion with PS cused.6- to 7.2-fold enhncement. he observed differences were significnt for ll three tretments (respectively, P <.2, P <., nd P <.). nduction of immunologicl memory to PS. Kinetic studies showed tht n gg response could not be detected 5 dys fter immuniztion with single, optiml dose of either PS or killed meningococci. However, the dministrtion of second dose of killed meningococci 3 weeks fter the first tretment elicited 6.7-fold increse in gg ntibody. An increse in specific gg ws not observed in mice similrly treted with two immunogenic doses of PS (dt not shown). Y/2[ *Z X cc z /6 ' FG. 4. Aqi S we Age of Mice (weeks) Mgnitude of the serum gm response of mice of vrious ges immunized with 5 (),.5 (-),.5 (A), nd (),ug of meningococcl group A PS. A A C S.4 m- n o : U NO CELLS N g N L. NORMAL CELLS g. ALS PRMED CELLS hy * FG. 6. Suppression of serum gm response to 5,ug of meningococcl group A PS by trnsfer of spleen cells from mice primed with.5 p.g of PS. Primed cells were not treted (N) or were treted with nti-lgm nd nti-gg ntibodies (g), ALS, or nti-hy-.2 ntibody (hy) plus complement prior to trnsfer. Symbols:, experiment ; *, experiment 2.

5 VOL. 56, 988 MENNGOCOCCAL A POLYSACCHARDE ANBODY RESPONSE Y2 C None C + Con A ALS Con A DyO DyO Dy2 Additionl retment FG. 7. Suppression or enhncement of the serum gm response to 5,ug of meningococcl group A PS by tretment with 3 jg of ConA or.3 ml of ALS. he immunogenicity of live meningococci ws exmined s follows. Mice were given n i.p. injection of 8 meningococci in.5 ml of sline. After 5 dys, the gm response ws similr to tht of mice given n optiml dose of either PS or killed orgnisms; however, fter 8 dys, the gg response ws eightfold greter. o determine the extent to which the PS-specific response could be enhnced, mice were hyperimmunized with live meningococci; 8 live orgnisms were dministered i.p. every 6 dys for up to 6 dys, nd ntibody responses were determined t vrious times (Fig. 8A). he gm response plteued by week 5; the mgnitude of the response reltive to single dose of PS ws 58- to 7-fold greter. he gg response ws more thn,-fold greter by week 9. As with untreted mice or those receiving single dose of PS, n ga response ws not detected. Hyperimmuniztion with PS, i.e., i.p. injection of n optiml dose (5,ug) every 6 dys for 3 dys, suppressed the gm response by 63% by the second tretment, nd this level of response ws mintined throughout the period of tretment (Fig. 8B). his response ws significntly different (P <.). Furthermore, no gg response ws observed during the tretments or in mice held for 3 dys fter the lst tretment. DSCUSSON Group A meningococci re frequent cuse of epidemic infections in developing ntions. Specific nti-ps ntibodies hve been found to confer immunity to diseses cused by meningococci of severl serogroups (5). Although vccines of purified, group-specific PS hve been vilble since the erly 97s, they hve only limited efficcy in the portion of the popultion most t risk, i.e., children younger thn 2 yers of ge. he present study ws designed to obtin informtion on some fctors which could influence responsiveness to these ntigens. he ntibody response of BALB/c mice to group A PS is dose relted. he kinetics of the gm response iinmice immunized with n optiml immunogenic dose (5 p,g) of group A PS is typicl of ntibody responses to PS of other bcteri (5, 8). he gm response to group A PS reched pek levels 4 to 5 dys fter immuniztion nd fell to Ad > r X OD' % 8 2 B * 6 l * ], E : o O S S * CP >} ' 6) Number of retments Number of retnents FG. 8. Kinetics of the serum gm () nd gg () responses of mice hyperimmunized with live group A meningococci (A) nd group A meningococcl PS (B). (6), Responses detected 3 dys fter six tretments. r _ * X -S

6 264 MULLER AND APCELLA bckground levels 3 to 4 weeks lter. Neither gg nor ga ntibody responses were detected. Administrtion of second optiml dose of PS 3 to 4 weeks fter the first dose resulted only in the induction of PS-specific gm. hese observtions suggest tht group A PS is similr to other ntigens (5, 8) in tht it does not elicit n nmnestic response. Although cells re not required for the induction of ntibody responses to ntigens, cells hve significnt influence on the mgnitude of such responses (4, 37, 38). On the bsis of extensive studies of the ntibody response of mice to pneumococcl type 3 PS (-6, 26, 27, 37, 38), we performed experiments to demonstrte the cellulr regultion of the ntibody response of mice to meningococcl group A PS. he negtive influence of cells ws suggested by the reduction of the nti-ps ntibody response which we observed in mice primed with PS 2 dys prior to immuniztion, s well s in mice given ConA t the time of immuniztion with PS. Furthermore, we performed cell trnsfer experiments to show tht the suppression induced by priming with subimmunogenic doses of PS ws medited by cells, i.e., suppression could be trnsferred with primed splenocytes nd suppression could be brogted by tretment of the spleen cells with nti-hy-.2 plus complement. Conversely, we observed tht the response ws enhnced if cells were inctivted t the time of immuniztion by tretment with ALS, which elimintes only ctivted cells (6, 27). Our observtions re consistent with the model of -cell-medited suppression proposed by Bker nd his co-workers. heir extensive studies of the ntibody response to pneumococcl type 3 PS demonstrted the kinetics of tht mode of suppression nd showed tht suppressor cells limit the extent to which ntigen-stimulted B cells proliferte nd/or differentite. Furthermore, we observed tht tretment with ConA 2 dys fter immuniztion with group A PS enhnced the nti-ps ntibody response. Agin, these observtions gree with the model of Bker nd his co-workers. His lbortory hs presented dt from cell trnsfer experiments which demonstrte tht mplifier cells re ctivted lter in the course of the response nd drive dditionl rounds of prolifertion of ntigen-stimulted B cells (2, 26). hus, we hve shown tht the ntibody response to group A PS, like tht to other ntigens, is regulted by two types of cells with opposing functions. We studied the ontogeny of the mouse ntibody response to group A PS to relte tht response to the hyporesponsiveness observed in humn infnts. he bility of mice to produce n gm response to group A PS incresed from 2 weeks of ge, nd by 6 weeks, the mgnitude of the response pproched tht of dult mice. Kinetic studies with pneumococcl type 3 PS hve demonstrted the ontogeny of the -cell subsets which regulte the responses to ntigens (3). n the mouse, -cell-medited suppression ppers to be fully developed by 2 weeks of ge. -cell mplifiction is miniml until week 4 nd does not rech dult levels until 8 to weeks of ge. Recently, nturl killer cells hve been implicted s nturl regultors, with suppressive ctivity, in the ntibody responses of wenling mice to pneumococcl types 3 nd 6 PS (2). Furthermore, the immturity of B-cell subpopultions, in terms of ntigen density nd the Lyb-5 mrker, correltes with the inbility of mice younger thn 3 or 4 weeks of ge to mount ntibody responses to mny ntigens (22, 3). Hence, the chrcteriztion of humn nti-ps ntibody responses s nlogous to the murine response (7) suggests tht the hyporesponsiveness of humn infnts my be due to the sme fctors responsible for poor NFEC. MMUN. responsiveness in neontl mice. As shown by our dt, the ntibody response of wenling mice to group A PS prllels tht of neontl humns; this indictes the potentil utility of the mouse model for studies of meningococcl PS vccines intended for humn infnts. he gol of vccintion is to chieve long-lsting protection ginst infectious diseses. Optimlly, immuniztion ginst encpsulted bcteri should result in the production of n nmnestic ntibody response to surfce ntigens of the orgnisms. Unfortuntely, the responses of young children to mny bcteril PS, ntigens, re not long lived. he ntibody response of children to group A PS is similr to the response we hve described in BALB/c mice; it is predominntly gm nd of short durtion (4, 7, 33). n both mice nd children, reimmuniztion with this PS elicits n ntibody response of similr mgnitude to the first response nd gm is predominnt. hus, the vccine does not elicit n nmnestic response in the ge group most t risk for meningococcl disese. Yet, mny dults produce gm, gg, nd ga in response to group A PS (9,, 5), suggesting tht prior sensitiztion with homologous or cross-recting ntigen cn elicit n nmnestic response, which is helper -cell dependent (D). n spite of its nture, group A PS, when ssocited with the bcteril cell surfce, hs components with D chrcteristics. Mice produce PS-specific gm nd gg in response to immuniztion with whole meningococci or with 5,ug of preprtion of group A PS contining lipooligoscchride,.6% (wt/wt) 2-keto-3-deoxyheptonic cid, nd less thn % (wt/wt) protein (dt not shown). Studies of the murine ntibody response to meningococcl group C PS indicte tht hydrophobic complexing of PS to outer membrne proteins is involved in the elicittion of gg B-cell memory (4). While the exct mechnism of the ugmenttion is unknown, it is ssumed tht the proteins ct s D crrier determinnts or enhnce the immunogenicity of PS through chnges in its conformtion. n ddition to the inbility of the form of PS to induce n cceptble level of immunity in children nd in portion of the dult popultion, immuniztion with pure PS ctivtes -cell-medited suppression. he level of suppression ssocited with single dose of group A PS cn be demonstrted fter the pproprite tretment with either ALS or ConA. he nti-group A PS response ws lso inhibited owing to priming with subimmunogenic PS or with optiml doses of PS 2 to 3 dys prior to immuniztion, nd this suppression could be trnsferred with primed cells. he extent of inhibition following priming ws limited s compred with tht elicited by low doses of pneumococcl type 3 PS, which is form of ntigen-specific suppression ctively medited by cells (2, 37). Contrry to the ntibody response induced by multiple tretments with live meningococci, hyperimmuniztion with group A PS inhibited the gm response nd did not elicit n gg response. Suppression ws observed fter the second tretment, nd it persisted throughout the durtion of hyperimmuniztion. Complexing of PS ppers to reduce the suppression ssocited with immuniztion with pure PS. he induction of gg memory cells following immuniztion with pneumococcl type 3 PS conjugted to D crrier is suppressed by the simultneous dministrtion of the pure PS (). Hence, owing to its nture, group A PS does not induce stisfctory immune response in the popultion most susceptible to meningococcl disese (4, 7, 33). his problem ssocited with the immune responses to ll bcteril PS hs led to studies of responsiveness to D forms of PS, either conjugtes or complexes of PS with outer mem-

7 VOL. 56, 988 MENNGOCOCCAL A POLYSACCHARDE ANBODY RESPONSE 265 brne structures, toxoids, or other proteins. he possibility of eliciting protective ntibodies through ltertion of the idiotypic network hs been demonstrted for D nd ntigens (3, 28, 36). With the intent of developing n internl imge-bsed vccine, we re currently exmining the effects of monoclonl nti-idiotypic ntibodies which re directed ginst monoclonl ntibodies to group A PS nd which stimulte n nti-ps response in the bsence of tretment with PS. ACKNOWLEDGMENS his work ws supported by grnt from the World Helth Orgniztion nd by Public Helth Service grnt Al 8384 from the Ntionl nstitute of Allergy nd nfectious Diseses. LERAURE CED. Bker, P. J., D. F. Amsbugh, P. W. Stshk, G. Cldes, nd B. Prescott. 98. Regultion of the ntibody response to pneumococcl polyscchride by thymus-derived cells. Rev. nfect. Dis. 3: Bker, P. J., D. F. Amsbugh, P. W. Stshk, G. Cldes, nd B. Prescott Direct evidence for the involvement of suppressor cells in the expression of low-dose prlysis to type pneumococcl polyscchride. J. mmunol. 28: Bker, P. J., H. C. Morse ll, S. S. Cross, P. W. Stshk, nd B. Prescott Mturtion of regultory fctors influencing mgnitude of ntibody response to cpsulr polyscchride of type Streptococcus pneumonie. J. nfect. Dis. 36(Suppl.): Bker, P. J., N. D. Reed, P. W. Stshk, D. F. Amsbugh, nd B. Prescott Regultion of the ntibody response to type pneumococcl polyscchride.. Nture of regultory cells. J. Exp. Med. 37: Bker, P. J., P. W. Stshk, D. F. Amsbugh, nd B. Prescott. 97. Chrcteriztion of the ntibody response to type pneumococcl polyscchride t the cellulr level.. Doseresponse studies nd the effect of prior immuniztion on the mgnitude of the ntibody response. mmunology 2: Bker, P. J., P. W. Stshk, D. F. Amsbugh, nd B. Prescott Regultion of the ntibody response to type pneumococcl polyscchride.. Mode of ction of thymus-derived suppressor cells. J. mmunol. 2: Brrett, D. J Humn immune responses to polyscchride ntigens: n nlysis of bcteril polyscchride vccines in infnts. Adv. Peditr. 32: Bsten, A., nd J. G. Howrd hymus independence. Contemp. op. mmunobiol. 2: Beuvery, E. C. 98. mmunistion ginst bcteril meningitis. J. nfect. 3(Suppl.): Beuvery, E. C., A. B. Leussink, R. W. Vn Delft, R. H. iesjem, nd J. Ngel mmunoglobulin M nd G ntibody responses nd persistence of these ntibodies in dults fter vccintion with combined meningococcl group A nd group C polyscchride vccine. nfect. mmun. 37: Beuvery, E. C., F. vn Rossum, nd J. Ngel Comprison of the induction of immunoglobulin M nd G ntibodies in mice with purified pneumococcl type 3 nd meningococcl group C polyscchrides nd their protein congugtes. nfect. mmun. 37: Brley-Mullen, H. 98. Antigen requirements for priming of gg producing B memory cells specific for type pneumococcl polyscchride. mmunology 4: Dreesmn, G. R., nd R. C. Kennedy Anti-idiotypic ntibodies: implictions of internl imge-bsed vccines for infectious diseses. J. nfect. Dis. 5: Gold, R., M. L. Lepow,. Goldschneider, nd E. C. Gotschlich mmune response of humn infnts to polyscchride vccines of groups A nd C Neisseri meningitidis. J. nfect. Dis. 36(Suppl.): Goldschneider,., E. C. Gotschlich, nd M. S. Artenstein Humn immunity to the menongococcus.. he role of humorl ntibodies. J. Exp. Med. 29: Goldschneider,., E. C. Gotschlich, nd M. S. Artenstein Humn immunity to the meningococcus.. Development of nturl immunity. J. Exp. Med. 29: Goldschneider,., M. L. Lepow, E. C. Gotschlich, F.. Muck, F. Bchl, nd M. Rndolph mmunogenicity of group A nd group C meningococcl polyscchrides in humn infnts. J. nfect. Dis. 28: Gotschlich, E. C.,. Y. Liu, nd M. S. Artenstein Humn immunity to the meningococcus.. Preprtion nd immunochemicl properties of the group A, group B, nd group C meningococcl polyscchrides. J. Exp. Med. 29: Gry, B. M ELSA methodology for polyscchride ntigens: protein coupling of polyscchrides for dsorption to plstic tubes. J. mmunol. Methods 28: Jones, J. M., D. F. Amsbugh, P. W. Stshk, B. Prescott, P. J. Bker, nd D. W. Alling Kinetics of the ntibody re sponse to type pneumococcl polyscchride.. Evidence tht suppressor cells function by inhibiting the recruitment nd prolifertion of ntibody-producing cells. J. mmunol. 6: Khter, M., J. Mci, C. Genye, nd J. Kpln Nturl killer cell regultion of ge-relted nd type-specific vrtions in ntibody responses to pneumococcl polyscchrides. J. Exp. Med. 64: Ledbetter, J. A., nd L. A. Herzenberg Xenogeneic monoclonl ntibodies to mouse differentition ntigens. mmunol. Rev. 47: Lee, C. J., F. G. Mlik, nd J. B. Robbins he regultion of the immune response of mice to Hemophilus influenze type b cpsulr polyscchride. mmunology 34: Leinikki, P. O., nd S. Pssil Quntittive, semiutomted, enzyme-linked immunosorbent ssy for virl ntibodies. J. nfect. Dis. 36(Suppl.): Leon, M. A., J. C. Chen, nd.-h. Kuo Regultory events in the immune response of mice to dextrn, p n J. A. Rudbch nd P. J. Bker (ed.), mmunology of bcteril polyscchrides. Elsevier/North-Hollnd Publishing Co., New York. 26. Mrkhm, R. B., N. D. Reed, P. W. Stshk, B. Prescott, D. F. Amsbugh, nd P. J. Bker Effect of concnvlin A on lymphocyte interctions involved in the ntibody response to type pneumococcl polyscchride.. Ability of suppressor cells to ct on both B cells nd mplifier cells to limit the mgnitude of the ntibody response. J. mmunol. 9: Mrkhm, R. B., P. W. Stshk, B. Prescott, D. F. Amsbugh, nd P. J. Bker Effect of concnvlin A on lymphocyte interctions involved in the ntibody response to type pneumococcl polyscchride.. Comprison of the suppression induced by con A nd low dose prlysis. J. mmunol. 8: McNmr, M. K., R. E. Wrd, nd H. Kohler Monoclonl idiotypic vccine ginst Streptococcus pneumonie infection. Science 226: Mond, J. J Use of lymphocyte regulted type 2 ntigens for the nlysis of responsiveness of Lyb5S nd Lyb5- B lymphocytes to lymphocyte derived fctors. mmunol. Rev. 64: Mond, J. J., S. Kessler, F. D. Finkelmn, W. E. Pul, nd. Scher. 98. Heterogeneity of expression on norml B cells, neontl B cells, nd on cells from B cell-defective CBA/N mice. J. mmunol. 24: Morse, H. C.,, B. Prescott, S. S. Cross, P. W. Stshk, nd P. J. Bker Regultion of the ntibody response to type pneumococcl polyscchride. V. Ontogeny of fctors influencing the mgnitude of the plque-forming cell response. J. mmunol. 6: Peltol, H Meningococcl disese: still with us. Rev. nfect. Dis. 5: Peltol, H., P. H. Mkel, H. Kyhty, H. Jousimier, E. Herv, H.

8 266 MULLER AND APCELLA NFEC. MMUN. Hlistrom, A. Sivonen,.-V. Renkonen,. Petly, V. Krnko, P. Ahvonen, nd S. Srn Clinicl efficcy of meningococcus group A cpsulr polyscchride vccine in children 3 months to 5 yers of ge. N. Engl. J. Med. 297: Rgppuoli, R Production nd chrcteriztion of high titer rbbit ntigonococcl R-type lipopolyscchride serum. Mikrobiologiy 5: Snedecor, G. W., nd W. G. Cochrn Sttisticl methods, 6th ed., p ow Stte University Press, Ames, ow. 36. Stein, K. E Network regultion of the immune response to bcteril polyscchride ntigens. Curr. op. Microbiol. mnmunol. 9: ylor, C. E., D. F. Amsbugh, P. W. Stshk, G. Cldes, B. Prescott, nd P. J. Bker Cell surfce ntigens nd other chrcteristics of cells regulting the ntibody response to type pneumococcl polyscchride. J. mmunol. 3: ylor, C. E., P. W. Stshk, J. Ching, W. M. Leiserson, G. Cldes, B. Prescott, nd P. J. Bker Chrcteristics of mplifier cells involved in the ntibody response to the cpsulr polyscchride of type Streptococcus pneumonie. J. mmunol. 32: Voller, A., D. Bidwell, nd A. Brtlett Microplte enzyme immunossys for the immunodignosis of virus infections, p n N. R. Rose nd H. Friedmn (ed.), Mnul of clinicl immunology, st ed. Americn Society for Microbiology, Wshington, D.C. 4. Zollinger, W. D., R. E. Mndreli, J. M. Grifiss, P. Altieri, nd S. Bermn Complex of meningococcl group B polyscchride nd type 2 outer membrne protein immunogenic in mn. J. Clin. nvest. 63: