Immunogenicity of a 23-valent pneumococcal polysaccharide vaccine in Brazilian elderly

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1 Brzilin Journl of Medicl nd Biologicl Reserch (2005) 38: Immunogenicity of pneumococcl vccine ISSN X 251 Immunogenicity of 23-vlent pneumococcl polyscchride vccine in Brzilin elderly V. Simonsen 1, A.P. Brndão 2,3, M.C.C. Brndileone 1, T.I. Yr 4, J.L. Di Fbio 5, M.H. Lopes 6 nd W. Jcob Filho 6 1 Seção de Bcteriologi, Instituto Adolfo Lutz, São Pulo, SP, Brsil 2 Fundção Oswldo Cruz, Rio de Jneiro, RJ, Brsil 3 Fundção Ncionl de Súde, São Pulo, SP, Brsil 4 Instituto Butntn, São Pulo, SP, Brsil 5 Orgnizção Pnmericn de Súde, Wshington, DC, USA 6 Fculdde de Medicin, Universidde de São Pulo, São Pulo, SP, Brsil Correspondence V. Simonsen Avenid Dr. Arnldo, 355, 9º ndr São Pulo, SP Brsil E-mil: vsimonsen@il.sp.gov.br Reserch supported by the Adolfo Lutz Institute, Brzil, nd the Pn-Americn Helth Orgniztion. Publiction supported by FAPESP. Received Mrch 29, 2004 Accepted Jnury 3, 2005 Abstrct Serum ntibodies specific for the cpsulr polyscchrides of Streptococcus pneumonie provide protection ginst invsive pneumococcl infection. In Brzil, this vccine hs been used for people over 65 yers with clinicl risk to develop pneumococcl infection since We evluted the immune response of 102 elderly subjects (75.5% femles nd 24.5% mles) with men ge of 71 yers, nd 19 young helthy dults (63.2% femles nd 36.8% mles) with men ge of 27 yers. The elderly study group consisted of outptients who received follow-up cre in the Geritric Deprtment of Generl Hospitl, Fculty of Medicine, University of São Pulo. None hd cute illness t the time of vccintion. Both groups were immunized with one intr-deltoid injection with 0.5 ml of 23-vlent pneumococcl polyscchride vccine. The totl IgG specific ntibody concentrtions to cpsulr polyscchrides 1, 3, 5, 6B, 8, nd 14 were determined ginst pre- nd 1-month post-vccintion ser. All smples were nlyzed ccording to the second-genertion pneumococcl polyscchride ELISA protocol. We observed tht the pneumococcl polyscchride vccine evoked consistent ntibody increse for serotypes 1, 5, 6B, 8, nd 14 (geometric men concentrtion increse of 2.46 in the elderly nd 2.84 in the young dults). Otherwise, we observed no increse in ntibody concentrtion for serotype 3 in both groups. Key words Streptococcus pneumonie Vccine Pneumococcl polyscchride vccine Immune response Elderly subjects Introduction Streptococcus pneumonie is n importnt bcteril pthogen of children nd dults worldwide, cusing infection of the respirtory trct, bcteremi, nd meningitis. Morbidity nd mortlity resulting from pneumococcl infection re high mong the elderly, who re t higher risk for invsive pneumococcl infections thn the generl popultion (1). Diseses cused by influenz nd pneumococci, tken together, re the fifth leding cuse of deth for persons ged 65 yers nd older (2). The nnul incidence of invsive pneumococcl disese hs been estimted to be 50 cses per 100,000 persons mong the

2 252 V. Simonsen et l. elderly in developed countries, wheres in Ltin Americ nd other developing countries the incidence is unknown (1,3). Becuse of the high risk of morbidity nd mortlity ssocited with respirtory infections in the elderly, the World Helth Orgniztion recommends pneumococcl nd influenz vccintion in this ge group. The 23- vlent pneumococcl polyscchride vccine is bout 50-80% effective in preventing invsive pneumococcl disese mong older persons (4). In Brzil, this vccine ws introduced in ntionl cmpign initited in 1999, nd is dministered to institutionlized elderly subjects nd to those recently hospitlized becuse of pneumoni (5,6). The im of the present study ws to ssess the immune response of elderly persons immunized with the 23-vlent pneumococcl vccine. Specific ntibodies to six pneumococcl cpsulr ntigens tht re mong the mjor invsive serotypes for this ge group were mesured. In ddition, we compred the immune response of this popultion with tht of young helthy dults vccinted t the sme time. Mteril nd Methods Study group The study group consisted of 102 elderly outptients who received follow-up cre in the Geritric Deprtment of the Generl Hospitl, Fculty of Medicine, University of São Pulo. The study ws pproved by the Ethics Committee nd every prticipnt signed the written informed consent. Seventy-seven subjects were femles (75.5%) with men ge of 71 yers (rnge: 61 to 87 yers) nd 25 were mles (24.5%) with men ge of 72 yers (rnge: 60 to 95 yers). All outptients hd stble chronic illnesses nd were monitored in primry cre clinic. None hd cute illness t the time of vccintion or hd previous vccintion ginst S. pneumonie. Control group The control group consisted of 19 helthy young dults with men ge of 27 yers (rnge: 22 to 38 yers). In this group there were femles (63.2%) nd 7 mles (36.8%). None hd history of pneumoni or previous vccintion with the 23-vlent vccine. Immuniztion nd serum collection Ech subject (N = ) received one intr-deltoid injection with 0.5 ml of the sme lot of pneumococcl vccine contining 25 µg of the following serotypes: 1 to 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, nd 33F. Blood for serum nlysis ws obtined prior to immuniztion nd 1 month fter immuniztion. Serum smples were stored t -70ºC until the time for ssy for cpsule pneumococcl polyscchride-specific IgG. Enzyme immunossy (ELISA) The level of pneumococcl cpsule specific IgG ws mesured with n interntionl reference ELISA method (7,8). Briefly, in this second-genertion ELISA, micropltes (Mxisorb, Nunc, Roskilde, Denmrk) were coted with 100 µl of solution of pneumococcl polyscchride (ATCC, Rockville, MD, USA). The pltes were incubted t 37ºC in moist chmber for 5 h. The subjects were evluted for specific ntibodies to six pneumococcl cpsulr polyscchrides (serotypes 1, 3, 5, 6B, 8, nd 14). All serum smples were bsorbed with purified pneumococcl cell wll polyscchride (C- PS) vilble from the Sttens Seruminstitute (Copenhgen, Denmrk) prior to the ELISA mesurements. Ser were tested in duplicte nd t two-fold seril dilutions. Specific ntibodies were detected using polyclonl-peroxidse conjugte nti-humn IgG gmmchin specific rised in got.

3 Immunogenicity of pneumococcl vccine 253 Antibody concentrtions re reported s µg/ml clculted on the bsis of the officilly ssigned IgG concentrtions for 89-SF reference serum. Antibody concentrtions were clculted by comprison of the prend post-vccintion ser with 89-SF using four-prmeter logistic ELISA progrm (9). Two dditionl serum smples known to contin high nd low levels of pneumococcl cpsule specific ntibodies were used s controls to ensure ssy reproducibility. Sttisticl nlysis Differences in log-trnsformed concentrtions of cpsule specific IgG ntibodies between pre- nd post-vccintion ser of both groups were clculted by nlysis of vrince for repeted mesures. Correltion coefficients were clculted by the method of Person for linerity. Differences between groups in IgG increses nd fold increses were determined by the Person chi-squre nd two-tiled Fisher exct tests. All P vlues were two-tiled nd the level of significnce ws set t P Sttistics were clculted with the use of the Sttisticl Softwre for Windows (Stt Corportion, College Sttion, TX, USA). Results Prior to immuniztion, elderly nd young dults hd pprecible levels of specific IgG ntibodies to the six serotypes nlyzed. Both groups showed wide vrition in serum concentrtion of type-specific totl IgG. As n exmple, the bsolute concentrtion of serotype 14 specific ntibody rnged from 2.3 to 0.2 µg/ml in the elderly nd 2.1 to 36.0 µg/ml in the young dults. The geometric men concentrtion (GMC) of the specific ntibodies to the six serotypes in prevccintion ser vried mong ll subjects, rnging from 2.5 to 10.8 µg/ml in the elderly nd from 1.7 to 9.8 µg/ml in the young dults (Figure 1). Before vccintion, the GMC of type-specific ntibody to polyscchride types 3, 5, nd 6B ws significntly higher in the elderly thn in the young group. Among ll polyscchrides nlyzed, GMC to serotype 14 ws the highest in pre-vccintion ser of both ge groups (Figure 1). As group, more thn 90% of the elderly hd more thn 1 µg/ml IgG specific for serotypes 1, 3, 5, nd 8, nd ll of them hd t lest this concentrtion for serotypes 6B nd 14. In the young dult group, ll subjects hd 1 µg/ml IgG specific for serotypes 8 nd 14, nd bout 30% of them did not hve 1 µg/ml IgG ntibody to serotypes 3 nd 5. The young dult group presented the lowest percentge of subjects (68%) with n IgG concentrtion of less thn 1 µg/ml in pre-immuniztion ser ginst serotype 3 (Tble 1). Following immuniztion, ll subjects hd significnt increse in ntibody responses to the serotypes nlyzed, with the exception of type 6B for the young dult group nd type 3 for both groups (Figure 1). We lso observed wide vrition in the concentrtion of type-specific totl IgG elicited. For exmple, the bsolute concentrtion of serotype 14 specific ntibodies rnged from 4.7 Geometric men IgG concentrtion (µg/ml) Pre-vccintion (elderly) Post-vccintion (elderly) Pre-vccintion (young controls) Post-vccintion (young controls) b b 3 b b b B 8 14 Streptococcus pneumonie serotypes Figure 1. Geometric men concentrtion nd 95% confidence intervls of IgG specific for pneumococcl cpsulr polyscchride in serum smples from 102 elderly nd 19 young dults, pre- nd post-vccintion. P < 0.05 for pre-vccintion ser compred to postvccintion ser; b P < 0.05 for young dults compred to elderly subjects (nlyses of vrince for repeted mesures)..b

4 254 V. Simonsen et l. to µg/ml in the elderly nd from 3.6 to 1.7 µg/ml in the young dults. Specific ntibody GMC incresed fter vccintion by more thn 3 µg/ml for five of the six serotypes nlyzed in the elderly nd for four of the six serotypes in the young dults. Serotype 14 elicited the highest GMC nd the highest bsolute increse in totl ntibody concentrtions (more thn 20 µg/ml) for both groups (Figure 1). Hving estblished tht pneumococcl vccintion generted specific nd pprecible response ginst five serotypes in both ge groups, we determined whether the immune response of the elderly differed from tht of young people. In the elderly group, the post-vccintion GMCs were higher to serotypes 1, 3, 5, 6B, nd 14, with P < 0.05 for serotypes 3 nd 6B. On the other hnd, the younger controls developed significntly higher humorl response to serotype 8, lthough both groups hd similr bseline responses to this serotype (Figure 1). The fold increses in serotype-specific IgG ginst serotypes 1, 3, 5, 6B, nd 14 were comprble between the two groups (Tble 1). The young dults, however, hd significntly higher fold increse ginst serotype 8. The men increse in type-specific ntibody fter vccintion ws greter thn two-fold for serotypes 1, 5, 8, nd 14 in both groups. Serotypes 3 nd 6B elicited lower increses Tble 1. Geometric men concentrtion (GMC) nd increses of IgG ntibodies to 6 polyscchrides of Streptococcus pneumonie in elderly (N = 102) nd young dults (N = 19). GMC µg/ml (95% CI) % with IgG 1 µg % with FI 2 % with FI 4 % with IgG 1 µg increse Elderly Young Elderly Young Elderly Young Elderly Young Elderly Young dults dults dults dults dults Serotype 1 Pre-vccintion 3.5 ( ) 2.3 ( ) month 7.1 ( ) 5.7 ( ) b Fold increse Serotype 3 Pre-vccintion 2.6 ( ) 1.7 ( ) b b 1 month 2.7 ( ) 1.7 ( ) b b Fold increse Serotype 5 Pre-vccintion 2.5 ( ) 1.6 ( ) b b 1 month 7.5 ( ) 5.4 ( ) Fold increse Serotype 6B Pre-vccintion 4.4 ( ) 3.0 ( ) b month 8.2 ( ) 4.4 ( ) b b Fold increse Serotype 8 Pre-vccintion 2.5 ( ) 2.9 ( ) month 5.9 ( ) 10.6 ( ),b Fold increse b Serotype 14 Pre-vccintion 10.8 (9.3-.5) 9.8 ( ) month 33.4 ( ) 31.9 ( ) Fold increse FI = fold increse expressed s geometric mens of individully clculted increse. 95% CI = 95% confidence intervl. P < 0.05 for pre-vccintion vs one month. b P < 0.05 for elderly vs young dults (nlyses of vrince for repeted mesures nd Person chisqure or Fisher s exct test).

5 Immunogenicity of pneumococcl vccine 255 (<2) in both ge groups. Becuse GMC nd men fold increse my not ccurtely indicte differences in immune responses between the two popultions, we compred the proportion of elderly nd young dults who chieved t lest twofold nd four-fold increses in ntibody levels ginst the serotypes nlyzed. Vccintion produced two-fold rise in specific ntibody in 50% or more of ll subjects ginst serotypes 5, 8, nd 14, with comprble proportions of elderly nd young dult subjects. More young dults (68%) hd two-fold response to serotype 1 thn the elderly (40%). Although type 6B is considered to be poor immunogen (10), 38% of elderly subjects hd two-fold increses in specific ntibody fter vccintion nd only 16% of young subjects could set up this response. The proportion of subjects chieving four-fold response ws similr for the two ge groups: more thn 30% of ll subjects presented this increse for serotypes 5, nd 14 nd less thn 18% of ll subjects could set up four-fold response to serotypes 1 nd 6B. Although we observed considerble proportion of young dults (50%) tht set up four-fold rise to serotype 8, the response ws not significntly different between the two ge groups (Tble 1). As n rbitrry estimte of protective concentrtions of pneumococcl cpsulr ntibodies, we nlyzed the cut-off vlue of 1 µg/ml, s usully done for nticpsulr ntibodies to Hemophilus influenze type b (11,). After vccintion, lmost ll elderly subjects chieved t lest 1 µg/ml of totl IgG specific for ll six serotypes nlyzed. Among the young dults, we lso observed good response, ll of them chieving 1 µg/ml response to serotypes 1 nd 5. The lowest percentge (74%) ws observed in response to serotype 3 (Tble 1). When ll six serotypes were nlyzed, vccintion incresed the overll verge of the elderly with n ntibody concentrtion 1 µg/ml from 97 to 99% nd this increse vried from 88 to 95% in the young dult group. When the immune response ws ssessed for type-specific IgG increse 1 µg/ml fter vccintion, more thn 60% of ll elderly subjects showed this increse in response to ll serotypes, except type 3. In both groups, the lowest percentge of subjects with n IgG increse 1 µg/ml ws observed for serotype 3. A sttisticlly significnt difference between the two ge groups ws seen for serotype 6B, in response to which the young dults hd significnt lower percentge (37%) thn the elderly subjects (62%) who chieved this increse (Tble 1). We lso evluted the number of serotypes for which ech subject chieved n increse 1 µg/ml nd two-fold rise in IgG concentrtion fter vccintion (Figure 2). The elderly nd the young dults hd comprble frequencies of these immune response prmeters. None of the subjects chieved two-fold response to ll six serotypes simultneously, but 7% of the elderly hd n increse 1 µg/ml for the six ntigens. When five serotypes were nlyzed, 9% of the elderly nd 8% of the young dults chieved two-fold response, nd bout 25% of ll Percentge of subjects two-fold response Elderly subjects Young dults 1 µg/ml increse Number of Streptococcus pneumonie serotypes Figure 2. Immune response fter pneumococcl vccintion reported s two-fold increse or increse of 1 µg/ml or more of polyscchride-specific IgG ginst the indicted number of serotypes. No significnt differences were detected between elderly nd young dults (P > 0.05, chi-squre test).

6 256 V. Simonsen et l. Serotypes Age groups 3 elderly young 5 6B 8 14 elderly young elderly young elderly young elderly young Pre-vccintion subjects hd n increse 1 µg/ml. Among the elderly, 9 nd 2%, respectively, did not chieve two-fold response or n increse 1 µg/ml for ny of the six serotypes. In the younger group we did not detect null response. In n ttempt to determine if there ws reltionship between specific ntibody concentrtions for ny two serotypes, we performed ll pirwise comprisons of ntibody concentrtions for both ge groups, s suggested by Coughlin et l. (13). In Figure 3, we present the correltion coefficients for pre- nd post-immuniztion ser obtined for ech group. In pre-immuniztion ser, the comprison of ech elderly subject s ntibody level by cluster nlysis reveled considerble correltion (r 0.57) between the concentrtions of specific IgG for serotypes 1, 3, 5, 6B, nd 8. Serotype 14 correlted distinctly more poorly (r < 0.26) with the other serotypes. In the nlysis of postimmuniztion ser, the correltion coefficients decresed for ll pirwise comprisons of type-specific ntibody concentrtion, with the exception of serotype 14. We obtined similr results in the young dult group. Post-vccintion B B 8 Streptococcus pneumonise serotypes Ptterns for correltion coefficient rnges 0.2 >0.2 >0.4 >0.6 >0.8 Figure 3. Correltion coefficients for ntibody concentrtion between pirs of pneumococcl cpsulr polyscchrides in pre- nd post-vccintion ser from 102 elderly subjects nd 19 young dults. Degrees of correltions re indicted by shding ptterns. Discussion Protection fter immuniztion is thought to be dependent upon the production of circulting pneumococcl type-specific ntibodies tht ct lone or in concert with complement proteins to opsonize bcteri nd promote their phgocytosis. Antibodies specific for the cpsulr polyscchride of S. pneumonie re n importnt prt of the humn host defense, protecting ginst invsive pneumococcl disese. Consequently, the mesurement of ntibodies specific for pneumococcl polyscchrides is importnt in estimting vccine-induced immune protection. We mesured type-specific ntibodies ginst 5 pneumococcl polyscchride ntigens (1, 3, 5, 6B, nd 14) tht belong to the most common serotypes cusing invsive infections in the elderly in Brzil (14). Although serotype 8 is less common in Brzil, this ntigen ws included becuse, like serotypes 2, 9, nd 14, it hs reproducible immunogenicity s defined by two-fold increse in ntibody titers obtined for norml controls (15,16). All six serotypes nlyzed in the present study re included in the 23- vlent vccine. ELISA hs been widely used to mesure immunity to pneumococci nd the immunogenicity of pneumococcl vccines. Correltion between this method nd opsonophgocytic (functionl) ctivity hs been considered resonbly good in post-immuniztion serum smples from dults nd infnts (17-19). When we initited the immunossys in the lte 90 s we used the second-genertion stndrdiztion of pneumococcl ELISA. This protocol ws recommended by the World Helth Orgniztion t tht time nd ws extensively used by experts (7,8,20). The pneumococcl cpsulr polyscchride preprtions used in the present ELISA re contminted with common cell wll (C- PS), nd therefore ntibodies to C-PS should

7 Immunogenicity of pneumococcl vccine 257 be bsorbed to improve the specificity of the ELISA (21). However, recent reports hve shown tht the second-genertion ELISA ws found to hve insufficient specificity when serum smples from unimmunized subjects were nlyzed (13). Furthermore, severl investigtors reported tht, despite the C-PS bsorption, ntibodies cross-rective with severl types of pneumococcl polyscchrides re still mesured (17,18,22). As reported elsewhere (17,18,23), cross-rective ntibodies re found more often in the ser of non-immunized infnts nd dults thn in the ser of infnts or dults immunized with pneumococcl vccines, suggesting tht the mjority of the ntibodies induced by vccintion re polyscchride type or group specific. At the present time thirdgenertion ssy formt hs been dopted by experts (24) nd n dditionl step is performed for the removl of cross-rective ntibodies. This dditionl bsorption step, using 22F polyscchride ntigen, improves the correltion between ELISA nd opsonophgocytic ctivity, the two methodologies widely used to chrcterize the immune response (25). In our nlysis, the pre-vccintion ser of the mjority of elderly nd young dults hd detectble levels of ntibody for the six polyscchrides nlyzed in pre-vccintion ser. Considering the limittion of the ELISA used in our study, this mesurement my reflect ntibodies cross-rective with severl types of pneumococcl polyscchrides, ntibodies cquired during life s result of coloniztion, previous infection with S. pneumonie, or other cross-rective orgnisms. Nevertheless, the bsl nti-specific cpsulr polyscchride IgG concentrtions found in our study were in greement with previous reports on elderly people from industrilized countries (26) nlyzed by second-genertion ELISA. In generl, the elderly nd the young dults developed significnt men increse of ntibody levels for 5 of 6 nd 4 of 6 cpsulr polyscchrides, respectively. The present study demonstrted tht, t 1 month fter immuniztion, the ser of elderly persons hd levels of specific cpsulr ntibodies similr to those of helthy younger persons, s reported elsewhere (16,27). For both groups, serotypes 3 nd 6B elicited lower increses (<2) nd, by this criterion, these ntigens ppered to be less immunogenic in both groups (10,26). The finding tht most elderly subjects responded stisfctorily to the pneumococcl vccine grees with the results of other studies (27-29). It is still possible tht helthy elderly dults, such s those in our study, respond normlly to pneumococcl polyscchride ntigens, but tht their response is reduced with the ill effects of ging nd the increse of co-morbid conditions. As reported elsewhere (20), the response to certin polyscchrides my be reduced, with more mrked reduction in persons >85 yers of ge. In our study we found tht serotype 3 ws poorly immunogenic, eliciting the lowest bsolute IgG concentrtion nd fold increse in specific ntibodies for both groups. In similr serumconversion study, Rubbins et l. (26) observed tht serotypes 3 nd 4 ccount for mny of the vccine filures seen in clinicl trils, suggesting tht future vccines for use in the elderly must be improved for these ntigens. Although Sorensen et l. (30) noted tht subjects with high pre-immuniztion ntibody concentrtions tend to hve smller increses thn those with low pre-immuniztion concentrtions, we found tht serotype 14 ppered to be one of the most immunogenic by rise nd bsolute rise criteri, though this serotype hd the highest bseline level. These results were similr to those reported by others (,26). We lso observed tht serotypes 1, 5, nd 8 induced high level of humorl response for both groups. As lso noted by other investigtors (15,16), serotype 8 induced the highest fold increse in the young dult subjects. Actu-

8 258 V. Simonsen et l. lly, the concentrtion of nti-polyscchride 8 IgG in 89-SF serum hd not been ssigned t the time of our study, nd therefore we performed the cross-stndrdiztion method of Conception nd Frsch (31). The ssigned IgG ntibody vlue for serotype 8 defined in our study did not differ from tht found lter by Qutert et l. (32). We confirmed the usefulness of the cross-stndrdiztion method to clibrte unknown IgG concentrtions to pneumococcl serotypes. It hs been often stted tht norml ntibody response to vccintion should include t lest two-, three-, or four-fold increse in ntibody titer to the ntigens nlyzed. Trditionl criteri for the interprettion of the immune response to pneumococcl polyscchride hve been proposed bsed on the difference between the postnd pre-immuniztion ntibody concentrtions regrdless of the increse fter immuniztion (15,30). In the present study, the percentge of vccinees ttining two-fold rise in IgG ntibody ws similr for the two ge groups for 5 of the 6 serotypes nlyzed. Only for serotype 1 ws the percentge of elderly subjects tht chieved two-fold response lower thn tht of young dults. It should be noted tht some investigtors found lower percentge of institutionlized elderly subjects who responded to serotypes 6B nd 14 (20). When we compred the fourfold rise in specific IgG ntibody concentrtion for the two groups, the percentge of young dult nd elderly subjects ws similr for ll serotypes. In study of post-vccinl immune response in the elderly, Rubbins et l. (26) concluded tht only 3.7% of the elderly hd two-fold increses in ntibody for ll 23 serotypes, 48% hd two-fold increses for 15 or more serotypes, nd 80% responded to 6 or more vccinl serotypes. In our study we could not find in either group subjects who set up simultneously two-fold immune response ginst the 6 pneumococcl cpsulr ntigens nlyzed. Although some investigtors try to chrcterize the poor responders (16,26), we were not ble to identify them due the smll number of polyscchrides nlyzed. As reported elsewhere (13,26), we observed tht pre-vccintion levels of ntibodies to serotypes 1, 3, 5, 6B, nd 8 were considerbly correlted in the elderly. As discussed erlier, this event my be observed when the ssy is not specific, nd my be due to n ntigenic stimulus common to these serotypes, most likely cross-rective ntigen. On the other hnd, serotype 14 showed the lowest correltion coefficient, confirming the considerbly type-specific mesurement of ntibodies to serotype 14 by ELISA demonstrted in severl studies (13,17,22,23,25). However, fter vccintion, ll pirwise correltions showed consistent decrese, with the exception of serotype 14. These dt suggest n improvement in ntibody specificity to serotypes 1, 3, 5, 6B, nd 8. As reported by others (13,23,26), lower correltion coefficients for ntibody concentrtion with other serotypes could men the bsence of cross-rective ntigens. The increse of correltion for serotype 14 in post-vccinl ser my suggest n overll increse in totl IgG concentrtion. We observed similr correltions in the pre- nd post-vccintion ser of young dults. We confirmed tht pneumococcl vccintion rised the levels of ntibodies ginst five of the six pneumococcl serotypes in the elderly, nd elicited relevnt immune response in both ge groups. The 23-vlent polyscchride vccine my induce protective immune response ginst invsive pneumococcl diseses in the elderly popultion. Our study demonstrted tht the men increse in type-specific ntibody fter vccintion ws greter thn two-fold for serotypes 1, 5, 8, nd 14 in both groups, even though this clcultion ws influenced by bseline concentrtions obtined by secondgenertion ELISA. In fct, for five of six polyscchrides nlyzed, our study my

9 Immunogenicity of pneumococcl vccine 259 hve overestimted the IgG concentrtion in pre-immuniztion ser, since the second-genertion ELISA we performed my not be optiml for mesuring type-specific ntibodies, especilly in non-immune serum smples. Our lbortory is currently using the protocol recommended for the third-genertion pneumococcl ntibody ELISA ( / Acknowledgments We grtefully cknowledge the constructive criticism of Dr. Moon H. Nhm, University of Albm, Birminghm, USA, Dr. Crl E. Frsch, FDA, USA, for kindly providing reference serum 89-SF nd Dr. Luiz Jcintho d Silv, Coordenção dos Institutos de Pesquis, Secretri de Estdo d Súde de São Pulo, for the support of this publiction. References 1. Vlsich C (2001). Pneumococcl infection nd vccintion in the elderly. Vccine, 19: Mieczkowisk TA & Wilson SA (2002). Adult pneumococcl vccintion: review of physicin nd ptients. Vccine, 20: Fedson DS, Scott JA & Scott G (1999). The burden of pneumococcl disese mong dults in developed nd developing countries: wht is nd is not known. Vccine, 17: S11-S Fedson DS (1999). The clinicl effectiveness of pneumococcl vccintion: brief review. Vccine, 17: S85-S Toniolo-Neto J, Weckx LW, Hlker E, Lopes CH, de Menezes Succi RC, de Piv TM, Ishid MA & Forleo-Neto E (1999). Sfety of simultneous pneumococcl nd influenz vccintion in elderly ptients in Brzil. Drugs nd Aging, 15 (Suppl 1): Arnd CMSS (1999). Vcinção pr o idoso no no de Revist CIP - Ações em Súde, 3: Plikytis BD, Goldblt D, Frsch CE et l. (2000). An nlyticl model pplied to multicenter pneumococcl enzyme linked immunosorbent ssy study. Journl of Clinicl Microbiology, 38: Qutert SA, Kirch CS, Quckenbush LJ, Phipps DC, Strohmeyer S, Cimino CO, Skuse J & Mdore DV (1995). Assignment of weightbsed ntibody units to humn ntipneumococcl stndrd reference serum, lot 89-S. Clinicl nd Dignostic Lbortory Immunology, 2: Plikytis BD, Holder PF & Crlone GM (1995). Progrm ELISA for Windows User s Mnul, Version Centers for Disese Control nd Prevention, Atlnt, GA, USA. 10. Shelley MA, Jcoby H, Riley GJ, Grves BT, Pichichero M & Trenor JJ (1997). Comprison of pneumococcl polyscchride nd CRM- 197 conjugted pneumococcl oligoscchride vccines in young nd elderly dults. Infection nd Immunity, 65: Kyhty H, Ahmn H, Ronnberg PR, Tillikinen R & Eskol J (1995). Pneumococcl polyscchride-meningococcl outer membrne protein complex conjugte vccine is immunogenic in infnts nd children. Journl of Infectious Diseses, 172: Snkilmpi U, Honknen PO, Bloigu A, Herv E & Leinonen M (1996). Antibody response to pneumococcl cpsulr polyscchride vccine in the elderly. Journl of Infectious Diseses, 173: Coughlin RT, White AC, Anderson CA, Crlone GM, Klein DL & Trenor J (1998). Chrcteriztion of pneumococcl specific ntibodies in helthy unvccinted dults. Vccine, 16: Brndileone MC, Andrde AL, Di Fbio JL, Guerr ML & Austrin R (2003). Appropriteness of pneumococcl conjugte vccine in Brzil: potentil impct of ge nd clinicl dignosis, with emphsis on meningitis. Journl of Infectious Diseses, 187: Go ES & Blls ZK (1996). Anti-pneumococcl ntibody response in norml subjects: met nlysis. Journl of Allergy nd Clinicl Immunology, 98: Rubbins JB, Puri AKG, Loch J, Chrboneu D, McDonld R, Opstd N & Jnoff EM (1998). Mgnitude, durtion, qulity nd function of pneumococcl vccine responses in elderly dults. Journl of Infectious Diseses, 178: Soininen A, vn den Dobbelsteen G, Oomen L & Kyhty H (2002). Are the enzyme immunossys for ntibodies to pneumococcl cpsulr polyscchrides serotype specific? Clinicl nd Dignostic Lbortory Immunology, 7: Soininen A, Krpl M, Whlmn SL, Lehtonen H & Kyhty H (2002). Specificities nd opsonophgocytic ctivities of ntibodies to pneumococcl cpsulr polyscchrides in ser of unimmunized young children. Clinicl nd Dignostic Lbortory Immunology, 9: Romero-Steiner S, Libutti D, Pis LB, Dykes J, Anderson P, Whitin JC, Keyserling HL & Crlone GM (1997). Stndrdiztion of n opsonophgocytic ssy for the mesurement of functionl ntibody ctivity ginst Streptococcus pneumonie using differentited HL-60 cells. Clinicl nd Dignostic Lbortory Immunology, 4: Romero-Steiner S, Musher DM, Cetron MS, Pis LB, Groover JE, Fiore AE, Plykytis BD & Crlone G (1999). Reduction in functionl ntibody ctivity ginst Streptococcus pneumonie in vccinted elderly individuls highly correltes with decresed IgG ntibody vidity. Clinicl Infectious Diseses, 29: Koskell M (1987). Serum ntibodies to pneumococcl C polyscchride in children: response to cute pneumococcl otites medi or to vccintion. Peditric Infectious Disese, 6: Yu X, Sun Y, Frsch C, Conception N & Nhm MH (1999). Pneumococcl cpsulr polyscchride preprtions my contin non-c polyscchride contminnts tht re immunogenic. Clinicl nd Dignostic Lbortory Immunology, 6: Soininen A, Pursiinen H, Kilpi T & Kyhty H (2001). Nturl development of ntibodies to pneumococcl polyscchride depends on the serotype: ssocition with pneumococcl crrige nd cute otitis medi in young children. Journl of Infectious Diseses, 184: Wernette CM, Frsch CE, Mdore DV et l. (2003). Enzyme-linked immunosorbent ssy for quntifiction of humn ntibodies to

10 260 V. Simonsen et l. pneumococcl polyscchrides. Clinicl nd Dignostic Lbortory Immunology, 10: Conception N & Frsch C (2001). Pneumococcl type 22 F polyscchride bsorption improves the specificity of pneumococclpolyscchride enzyme-linked immunosorbent ssy. Clinicl nd Dignostic Lbortory Immunology, 8: Rubbins JB, Alter M, Loch J & Jnoff EN (1999). Determintion of ntibody responses of elderly dults to ll 23 cpsulr polyscchrides fter pneumococcl vccintion. Infection nd Immunity, 67: Musher DM, Groover JE, Grviss EA & Bughn RE (1996). Lck of ssocition between ging nd post vccintion levels of IgG ntibody cpsulr polyscchrides of Streptococcus pneumonie. Clinicl Infectious Diseses, 22: Musher DM, Groover JE, Rowlnd JM, Wtson DA, Struewing JB, Bughn RE & Mufson MA (1993). Antibody to cpsulr polyscchrides of S. pneumonie: prevlence, persistence nd response to revccintion. Clinicl Infectious Diseses, 17: Snkilmpi U, Honknen PO, Bloigu A & Leinonen M (1997). Persistence of pneumococcl cpsulr polyscchride vccine in the elderly. Journl of Infectious Diseses, 176: Sorensen RU, Leiv LE, Jvier III FC, Scerdote DM, Brdford N, Butler B, Gingrosso PA & Moore C (1998). Influence of ge on the response to S. pneumonie in ptients with recurrent infections nd norml immunoglobulin concentrtions. Journl of Allergy nd Clinicl Immunology, 102: Conception N & Frsch C (1998). Evlution of previously ssigned ntibody concentrtions in pneumococcl polyscchride reference serum 89-SF by the method of cross-stndrdiztion. Clinicl nd Dignostic Lbortory Immunology, 5: Qutert SA, Kirch C, Dikun K & Mdore DV (1998). Antibody units ssigned to nti-pneumococcl serum lot 89-S. In: Progrm nd Abstrcts of the Interntionl Symposium on Pneumococci nd Pneumococcl Disese, Helsinger, Denmrk. Streptococcus Unit t Sttens Serum Institute, Copenhgen, Denmrk, 1998: 67 (Abstrct P-28).

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