Naturally Acquired Passive Protective Activity against Neisseria meningitidis Group C in the Absence of Serum Bactericidal Activity

The hallmark of immunity to meningococcal disease is a bactericidaltiter in serum of $≥$ 1:4 measured with human complement, but thisthreshold titer may underestimate the extent of protection.We used the infant rat model of meningococcal bacteremia tomeasure group C passive protective activity in serum samplesfrom 91 unimmunized adults living in California. A total of35 sera (38.5%) had passive protective activity. Sera with complement-mediatedbactericidal titers of $≥$ 1:4 were 3.4-fold more likely to conferprotection (89%) than nonbactericidal sera (26%; P < 0.0001).Thus, bactericidal titers of $≥$ 1:4 are a marker of protection,but this threshold lacks sensitivity for predicting protectiveactivity. We investigated the 73 sera with bactericidal titersof <1:4 to determine the basis of protective activity. The19 sera with protective activity had a higher geometric meangroup C anticapsular antibody concentration (0.72 µg/ml)than the 54 sera that lacked protective activity (0.16 µg/ml;P < 0.001). Thus, protective activity in the absence of bactericidalactivity was associated with higher concentrations of anticapsularantibodies, but not all sera with anticapsular antibodies conferredprotection. Of 18 nonbactericidal sera with anticapsular antibodyconcentrations between 0.31 and 0.99 µg/ml, the 11 serathat conferred protection had a higher mean antibody avidityconstant (21.9 nM^–1) than the 7 nonprotective sera (14.6nM^–1; P < 0.03). Thus, in sera with titers of <1:4,protective activity is associated with higher-avidity groupC anticapsular antibodies, which are present in concentrationsinsufficient to elicit complement-mediated bacteriolysis invitro but sufficient to confer protection in an in vivo bacteremiamodel.

INTRODUCTION

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Introduction

New multivalent meningococcal polysaccharide-protein conjugatevaccines are currently in development (5, 30) and will likelybe licensed in Europe and North America in the next few years(28). The low incidence of meningococcal disease in these populationsprecludes performing prospective randomized clinical trialsto determine the efficacy of these new vaccines. Vaccine efficacy,therefore, will be inferred from immunogenicity data (3), andvaccine effectiveness will be confirmed in subsequent postlicensurestudies (1), following a licensure pathway and monitoring strategiesadapted in the United Kingdom for the introduction of groupC meningococcal conjugate vaccines. There is a strong scientificbasis for inferring meningococcal vaccine efficacy from immunogenicitydata (3, 10, 11). However, the choice of in vitro assay conditionsand serologic endpoints for inferring protection against meningococcaldisease are topics of considerable recent debate (1, 3, 16,32). The reasons are complex but ultimately have to do withthe effects of potential disparities between in vitro antibodyfunctional assay conditions and in vivo host defenses.

Meningococci grown in vivo likely express different genes thanthose of bacteria grown in vitro (13). Also, when meningococciare grown in broth or agar, the choice of growth conditionsmay affect capsular production and/or the expression of differentsurface proteins or lipooligosaccharide structures (6, 22, 23,35), which in turn can affect the susceptibility of the bacterialcell to antibody binding and complement-mediated bacteriolysis.These factors may limit the interpretation of the results ofin vitro antibody functional studies. Members of our laboratoryrecently described an infant rat meningococcal bacteremia modelfor measuring antibody protective activity against group B orC strains (15, 25). Although meningococci are obligate humanpathogens with species-specific pathogenic mechanisms (17),the infant rat model permits the investigation of the protectiveactivity of antibodies in a setting where the organism is rapidlyreplicating in vivo.

In the present study, we used the infant rat model to investigatethe role of naturally acquired serum antibodies of human adultsin protection against group C meningococcal disease. Protectiveactivity in serum measured in vivo was related to the presenceor absence of group C complement-mediated bactericidal activitymeasured in vitro or to the concentrations and avidities ofgroup C anticapsular antibodies in serum. The results provideinsights into the antigenic targets of naturally acquired antibodiesconferring protection against group C Neisseria meningitidisand the extent to which measurements of serum bactericidal activitymay underestimate protective immunity.

MATERIALS AND METHODS

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Materials and Methods

Serum samples. We used a convenience sample of 91 stored preimmunization serathat had been obtained from healthy adults ranging in age from18 to 58 years who were enrolled in meningococcal vaccine immunogenicitytrials conducted at Children's Hospital and Research Centerat Oakland between 2001 and 2003. None of the subjects had beenpreviously immunized with meningococcal vaccine. To preserveinternal complement activity, the blood was allowed to clotat room temperature for 30 min and centrifuged at 2,135 x gfor 10 min at 4°C. The sera were promptly separated, dividedinto 0.5-ml aliquots, and stored frozen at –70°C.Use of these sera for the present study was approved by theInstitutional Review Board of Children's Hospital and ResearchCenter at Oakland.

Serology. (i) Bactericidal assay. The test strain was N. meningitidis strain 4243 (C:2a:P1.5,2),a member of the electrophoretic type 37 complex, sequence type11 (http://www.mlst.net), expressing a polysaccharide capsulethat is O acetylation positive (15). The organism was grownin Mueller-Hinton broth (with a starting A₆₂₀ of $~$ 0.1) for approximately2 h to an A₆₂₀ of $~$ 0.6. After the bacteria were washed twicein Dulbecco's buffer (pH 7.40) containing bovine serum albumin(Sigma, St. Louis, Mo.), approximately 300 to 400 CFU was addedto the reaction mixture. In the exogenous complement assay,the final 60-µl reaction mixture contained 20% (vol/vol)complement and serial twofold dilutions of test sera dilutedin Dulbecco's buffer. The human complement was serum from ahealthy adult with no detectable endogenous group C bactericidalactivity and no detectable group C anticapsular antibody asmeasured by a radioantigen binding assay (RABA; see below).The rabbit complement consisted of pooled infant rabbit serumthat lacked endogenous bactericidal activity (Cedarlane; Hornby,Ontario, Canada). The percent survival of bacteria after 60min of incubation in the reaction mixture compared to the numberof CFU per milliliter in the negative control serum at timezero was plotted against serum dilution. The bactericidal titerwas defined by the 50% intercept. The bactericidal assay usinginternal complement was performed as described above exceptthat we used single dilutions of test sera (final dilution of1:4) and did not add exogenous complement.

(ii) RABA. The concentrations of group C anticapsular antibodies were measuredby a RABA performed as previously described (15). For measurementof antibody avidity, we performed replicate assays using twodifferent concentrations of radiolabeled antigen (15). Avidityconstants (K_A) were calculated by comparing the fraction ofantigen bound at different antibody concentrations in the low-antigen-concentrationRABA to that of the total concentration of antibody determinedin the high-antigen-concentration RABA (15).

(iii) Passive protection. The animal model has been described previously (15, 26). Inbrief, 5- to 7-day-old pups from litters of outbred Wistar rats(Charles River, Raleigh, N.C.) were randomly redistributed tothe nursing mothers. At time zero, 100 µl of a 1:4 dilutionof serum was administered intraperitoneally (i.p.) (3 rats/serumsample). Two hours later, the animals were challenged i.p. with100 µl of washed, log-phase N. meningitidis group C strain4243 cells (range in different experiments, $~$ 800 to 1,400 CFU/rat).Eighteen hours after the bacterial challenge, blood specimenswere obtained by puncturing the heart with a syringe and needlecontaining approximately 25 U of heparin without preservative(American Pharmaceutical Partners, Inc., Los Angeles, Calif.).Aliquots of 1, 10, and 100 µl of blood were plated ontochocolate agar. The number of CFU per milliliter of blood wasdetermined after overnight incubation of the plates at 37°Cin 5% CO₂. Protection was defined as a 2-log decrease in thegeometric mean number of CFU/ml of blood in the triad of animalsgiven the test serum compared to that of a group of negativecontrol animals (geometric means, $~$ 100,000 to 544,000 CFU/mlin different experiments).

Statistical analysis. The geometric means of the antibody concentrations or titerswere computed by exponentiation (base 10). For log transformation,samples below the lower limits of detection were assigned avalue of half of the lower limit (i.e., 1:2 for the bactericidaltiter and 0.05 µg/ml for group C anticapsular antibodyconcentration measured by RABA). The proportion of sera withbactericidal titers of $≥$ 1:4 (considered a protective titer [10])or $≥$ 1:128 when measured with rabbit complement (a titer previouslyshown to predict a bactericidal titer of $≥$ 1:4 when measured withhuman complement [32]) was computed. Differences found in theproportion of subjects in the respective groups were comparedby chi-square analysis. For determination of sensitivity andspecificity, sera with passive protective activity in the ratmodel were considered "true positives" of immunity to groupC meningococcal disease. Sensitivity (percent) was defined bythe equation (number of sera with positive serology/number ofprotective sera) x 100. Specificity (percent) was defined bythe equation (number of sera with negative serology/number ofsera that lacked protective activity) x 100.

RESULTS

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Results

Passive protective activity in infant rats. Fig. 1 shows the results of a representative experiment measuringthe kinetics and time course of bacteremia in 5-day-old infantrats challenged i.p. with group C strain 4243. The level ofbacteremia increased exponentially for $~$ 8 h, reaching >250,000CFU/ml. As shown in Table 1, pretreatment of the rats with humanserum 2 h before bacterial challenge can confer protection againstbacteremia. In the examples shown, a 1:26 dilution of a positivecontrol serum from an adult immunized with meningococcal polysaccharidevaccine conferred protection, as did 1:4 dilutions of preimmunizationsera from subjects 1 and 3 but not subjects 2 and 4. Of the91 preimmunization sera tested, 35 sera (38%) conferred passiveprotection in the infant bacteremia model and 56 sera (62%)showed no significant protective activity (<2-log decreasein the geometric mean number of CFU/ml compared to that of thenegative control animals). As summarized in Table 2, the demographicsof the groups whose sera did and did not confer protection weresimilar. The geometric means of the bactericidal titers andgroup C anticapsular antibody concentrations were significantlyhigher in the protective sera than in the nonprotective sera(P < 0.0001).

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FIG. 1. Kinetics of meningococcal bacteremia in infant rats challenged with N. meningitidis group C strain 4243. Five-day-old infant rats were challenged i.p. with $~$ 900 CFU. Blood cultures were obtained from groups of five animals at each time point. The number of CFU per milliliter of blood was determined by plating 100, 10, and 1 µl of blood. At 8 h, all animals had >250 CFU/µl, the smallest volume of blood cultures, and were assigned values of >250,000/ml. Therefore, a precise geometric (Geo.) mean and a 95% confidence interval (CI) could not be calculated. For blood obtained at 27 h, additional dilutions were plated (equivalent to 0.1 and 0.01 µl of blood) to permit a precise assignment of the number of CFU per milliliter for each animal. SD, standard deviation.

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TABLE 1. Passive protective activity of representative adult sera in infant rats challenged i.p. with N. meningitidis group C strain 4243^a

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TABLE 2. Demographic characteristics of the adults whose sera were used and antibody concentrations in serum in relation to passive protective activity^a

Relationship between serum complement-mediated bactericidal activity and passive protective activity in the infant rat model. Of the 91 sera, 73 sera (80.2%) had titers of <1:4, measuredwith exogenous human complement, and 18 sera (19.8%) had bactericidaltiters of $≥$ 1:4 (Table 3). We performed absorption studies on16 of the bactericidal sera using 25 µg of soluble groupC polysaccharide/ml, which was sufficient to give 100% inhibitionof high-titer bactericidal control sera from an adult immunizedwith meningococcal polysaccharide vaccine. Seven of the bactericidalsera (44%) were completely inhibited by the polysaccharide,and nine sera (66%) were not inhibited. Five of the sera thatwere not inhibited had low anticapsular antibody concentrationsmeasured by RABA (<0.33 µg/ml), a result consistentwith the bactericidal antibodies being directed at noncapsularantigens. The remaining four sera that were not inhibited hadanticapsular antibody concentrations of >1 µg/ml, andit is likely that both anticapsular and antinoncapsular antibodiescontributed to their bactericidal activities.

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TABLE 3. Serum protective activity in relation to bactericidal activity assayed with different complement sources^a

Sera with bactericidal titers of $≥$ 1:4 were 3.4-fold more likelyto confer passive protective activity against bacteremia inthe rat model (16 of 18 sera [88.9%]) than nonbactericidal sera(19 of 73 sera [26%]; P < 0.0001 by the chi-square test).As summarized in Table 3, the proportion of sera with bactericidaltiters between 1:4 and 1:6 that conferred protection (5 of 6)was not significantly different from that of sera with bactericidaltiters of $≥$ 1:20 (11 of 12). Note that there were no sera withtiters between 1:7 and 1:19 when sera were assayed with exogenoushuman complement.

Serum bactericidal activity also was measured with internalhuman complement, which was used in the Goldschneider et al.study (10), or exogenous rabbit complement, which because ofease of standardization is currently used by most laboratoriesto measure bactericidal titers (1, 3) but is known to give higherbactericidal titers than human complement (3, 32). Irrespectiveof the source of complement used, protective activity was morefrequently found in sera that were positive for bactericidalactivity (with a titer of $≥$ 1:4 with human complement or $≥$ 1:128with rabbit complement) than in sera that were negative forbactericidal activity (P < 0.001).

The specificities of sera with bactericidal titers of $≥$ 1:4 whenmeasured with internal or external human complement were 91and 96%, respectively, for passive protective activity (Table4). However, a titer positive for bactericidal activity wasnot very sensitive for passive protective activity (40 and 46%when measured with internal and external human complement, respectively),since 26 to 29% of sera with bactericidal titers of <1:4conferred protection in the animal model (Table 3). When bactericidaltiters were measured with exogenous infant rabbit complementinstead of human complement, a titer of $≥$ 1:128 was 60% sensitiveand 93% specific for protective activity (Table 4).

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TABLE 4. Sensitivities and specificities of serum bactericidal activities or different concentrations of group C anticapsular antibodies for passive protective activity in the infant rat bacteremia model^a

Relationship between serum anticapsular antibody concentrations and passive protective activity in infant rats. Of the 91 sera, 50 sera had group C anticapsular antibody concentrationsof $≤$ 0.3 µg/ml as measured by RABA, 21 sera had anticapsularantibody concentrations between 0.31 and 0.99 µg/ml, and20 sera had concentrations of $≥$ 1.0 µg/ml. The respectiveproportions of sera with protective activity in the infant ratmodel were 12, 62, and 80%. Thus, the proportion of sera withpassive protective activity increased with increasing groupC anticapsular antibody concentration (P < 0.0001 by thechi-square test for trend). The specificity and sensitivityof a serum anticapsular antibody concentration of $≥$ 1 µg/mlfor passive protective activity were 93 and 46%, respectively,values nearly identical to the specificity and sensitivity ofa bactericidal titer of $≥$ 1:4 measured with human complement (Table4). As expected, at lower concentrations of anticapsular antibodiesin serum, there was increased sensitivity but lower specificityfor passive protective activity (Table 4).

Of the 50 sera with anticapsular antibody concentrations of $≤$ 0.3 µg/ml, 6 sera (12%) conferred passive protection inthe animal model. Given these low anticapsular antibody concentrations,the protective antibodies in these sera were likely directedat noncapsular antigens. Of the 21 sera with anticapsular antibodyconcentrations between 0.31 and 0.99 µg/ml, 13 sera conferredpassive protection against bacteremia in the infant rat modeland 8 were not protective. The respective geometric mean anticapsularantibody concentrations of protective and nonprotective serawere not significantly different (0.59 and 0.52 µg/ml;P > 0.4). One possible explanation for why some sera withanticapsular antibody levels in this range were protective whileothers were not is a difference in anticapsular avidity, whichwas significantly higher in the 13 protective sera K_A (mean± standard error) of (22.1 ± 1.8 nM)^–1]than in the 8 nonprotective sera [K_A of (15.5 ± 1.9 nM)^–1;P = 0.02 by t test]. Although this difference in mean aviditymay seem too small to explain the difference in protective activitybetween the two groups, the results are consistent with previousdata on the magnitude of avidity differences between highlyprotective and poorly protective vaccine-induced group C anticapsularantibodies (12, 15).

Characterization of protective antibodies in sera with bactericidal titers of <1:4 measured with exogenous human complement. Of the 73 sera that lacked bactericidal activity when measuredwith exogenous human complement, 19 sera conferred passive protectionagainst bacteremia in the infant rat model and 54 sera did not.The group C anticapsular antibody concentrations of the individualsamples are shown graphically in Fig. 2A. The protective serahad, on average, 4.5-fold-higher group C anticapsular antibodyconcentrations than sera that lacked protective activity (geometricmeans of 0.72 and 0.16 µg/ml, respectively; P < 0.0001).The group C antibody avidity constants for protective and nonprotectivesera with anticapsular antibody concentrations of >0.3 µg/mlare shown in Fig. 2B (avidity could not be determined in sampleswith lower concentrations). For samples with anticapsular antibodyconcentrations between 0.31 and 0.99 µg/ml, the 11 protectivesera with bactericidal titers of <1:4 had on average higher-avidityanticapsular antibodies than did the 7 nonprotective sera [K_A(mean ± standard error) of (21.9 ± 2.0 nM)^–1versus (14.6 ± 2.0 nM)^–1; P < 0.03]. Althoughthere were no significant differences between the mean aviditiesof the protective and nonprotective sera with anticapsular antibodyconcentrations of $≥$ 1.0 µg/ml, there were only four nonprotectivesera in this group. One of these four sera had low avidity,and two sera with higher avidities had relatively high immunoglobulinA (IgA) anticapsular antibody concentrations measured by enzyme-linkedimmunosorbent assay (0.83 and 0.55 µg/ml), which couldexplain their lack of protective activity. We do not have anexplanation for the lack of protection of the remaining serumsample in this group. Nevertheless, taken together the datasuggest that most of the protective activity in sera with bactericidaltiters of <1:4 results from subbactericidal concentrationsof higher-avidity anticapsular antibodies.

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FIG. 2. Relation of anticapsular antibody to passive protective activity of sera with bactericidal titers of <1:4. (A) Anticapsular antibody concentrations measured by RABA. The geometric mean concentration was higher in sera with protective activity than in those that were not protective (0.72 versus 0.16 µg/ml; P < 0.0001). (B) Anticapsular avidity. In sera with anticapsular antibody concentrations between 0.31 and 0.99 µg/ml, the mean avidity of protective sera (closed circles) was higher than that of the nonprotective sera (open circles) (21.3 versus 14.6 nM^–1; P < 0.03). There were no significant differences between the mean avidities of the protective and nonprotective sera with anticapsular antibody concentrations of $≥$ 1.0 µg/ml (20.3 and 21.2 nM^–1; P > 0.9).

DISCUSSION

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Discussion

Studies of naturally acquired meningococcal antibodies in thepopulation have provided important insights into the immunologicbasis of meningococcal immunity (reviewed in reference 27).For example, adults have a much lower incidence of meningococcaldisease than children less than 4 years of age (28), which isthought to be a result of an age-related acquisition of serumantibodies stimulated by asymptomatic colonization by N. meningitidisstrains (11, 29) or cross-reacting bacteria (7, 37).

In their seminal study, Goldschneider et al. demonstrated theimportance of serum bactericidal antibodies in protection againstgroup C meningococcal disease during an epidemic among militaryrecruits in the 1960s (10). Group C bactericidal antibodieswere present in baseline sera of approximately 82% of the recruits.The subjects with detectable bactericidal antibodies in serumfrequently became carriers of the epidemic strain but did notdevelop meningococcal disease, while virtually all cases ofdisease occurred in the 18% of individuals whose baseline seralacked bactericidal activity (titers of <1:4 measured withhuman complement). Recruits who lacked bactericidal antibodiesand developed group C carriage had meningococcal disease attackrates as high as 38.5%.

Based on the Goldschneider et al. data, a bactericidal antibodytiter of 1:4 or greater when measured with human complementhas become an accepted surrogate of protective meningococcalimmunity. However, the interpretation of a bactericidal titerof <1:4 as an indication of susceptibility is more problematic,and some have questioned whether the proportion of persons withtiters below this threshold overestimates the size of the susceptiblepopulation (1).

In the present study, we used the infant rat bacteremia modelto investigate the basis of naturally acquired immunity to N.meningitidis group C. Although it is impossible to be sure thatprotective activity measured by this animal model is entirelyapplicable to human disease, we found that adult sera with bactericidaltiters of $≥$ 1:4 were more likely to confer passive protectiveactivity in the animal model than nonbactericidal sera (Table3). Several sera with bactericidal titers of 1:4 or greaterfailed to confer protection, a result which may reflect someof the limitations of testing the passive protective activityof human antibody in the presence of infant rat complement andrat phagocytic cells and in an animal model that is subjectto expected biologic variability. Nevertheless, the specificityof a bactericidal titer of 1:4 or greater for protective activityin the animal model was 91% when bactericidal activity was measuredwith internal human complement and 96% when measured with exogenoushuman complement (Table 4).

Approximately one-quarter of the nonbactericidal sera had passiveprotective activity in the animal model. These results are consistentwith the conclusions of the 1969 Goldschneider et al. studies(10, 11) that the presence of serum bactericidal activity isa reliable marker of protection against disease but that theabsence of serum bactericidal activity does not necessarilyimply susceptibility, since not all recruits who lacked serumbactericidal activity and who became colonized with the epidemicstrain developed invasive disease. Although not directly testedin our study, the most likely mechanism responsible for protectiveactivity in the absence of serum bactericidal activity is opsonization(21), which can result from antibody binding to the bacterialsurface and activation of C3b deposition without proceedingto bacteriolysis (36). Alternatively, there may be blockingof bactericidal antibodies in vitro, for example, by the presenceof non-complement-activating IgA antibodies (14). In the infantrat, the IgA antibodies may be diluted to a concentration thatdoes not block passive protective activity.

Protective antibodies to N. meningitidis group C strains canbe directed at a number of antigenic targets, including thecapsular polysaccharide or noncapsular antigens, such as PorA(18) or Opc (class 5 outer membrane proteins) (31). Based onabsorption studies of gamma globulin prepared from more than2,000 North American donors, Goldschneider et al. concludedthat the majority of naturally acquired group C serum bactericidalactivity was directed against the capsular polysaccharide (10),which is consistent with our absorption data on individual sera.Our results also showed a strong association between serum passiveprotective activity for the animal model and the presence ofhigher concentrations of naturally acquired group C anticapsularantibodies than anticapsular antibody concentrations in serathat failed to confer protection in the animal model (Table2 and Fig. 2A). Our data also underscore the importance of thequality of the anticapsular antibodies, since protective activitywas associated with higher-avidity anticapsular antibodies (Fig.2B).

In the Goldschneider et al. studies, 82% of military recruitshad group C meningococcal titers of $≥$ 1:4 (10, 11). In contrast,only 20% of unimmunized adults in the present study had bactericidaltiters of $≥$ 1:4 when measured with human complement (Table 3),which was the complement source used in the Goldschneider etal. studies. A study performed in British Columbia from 1991through 1993, which used group C strain C11 (the same strainas that used in the Goldschenider et al. studies), found thatonly 9.5% of adolescents, aged 13 to 19 years, had bactericidaltiters of >1:4 (24). Recent studies in the United Kingdomalso reported that only 10 to 30% of sera from unimmunized adultswere positive for group C bactericidal antibodies (19, 34).Taken together, the data suggest that the seroprevalence ofgroup C bactericidal activity has decreased in the populationsince the 1960s, when the Goldschneider et al. studies weredone. A decrease in the prevalence of serum bactericidal antibodiesin the adult population may explain the recent apparent increasein the rate of meningococcal disease in neonates in the UnitedStates (33), since newborns are thought to be protected fromdeveloping meningococcal disease by transplacentally acquiredmaternal antibodies (10, 28).

Group C polysaccharide-protein conjugate vaccines were introducedin the United Kingdom in the fall of 1999 and were followedsoon thereafter by a marked decrease in the incidence of groupC meningococcal disease (2). Estimates of vaccine efficacy are>90 percent in all age groups targeted for immunization (1).The high efficacy is particularly striking in infants and toddlers,the age groups at highest risk of acquiring meningococcal diseaseand in whom peak titers of bactericidal antibodies in serumpresent 1 month after vaccination decline to low or undetectablelevels by 7 to 9 months after vaccination (1). One of the principalfeatures of polysaccharide-protein conjugate vaccines is theirability to prime for immunological memory (4, 20). Another isavidity maturation of serum anticapsular antibodies over time(8, 9, 12). The authors of the United Kingdom study interpretedtheir data on the high group C meningococcal conjugate vaccineefficacy in the face of a low seroprevalence of group C bactericidaltiters as suggesting that continuing protection against groupC meningococcal disease is less dependent on the persistenceof serum bactericidal activity than the ability of immunizedpersons to mount memory antibody responses upon encounteringgroup C meningococci (34). However, based on our findings ofserum passive protective activity in the infant rat meningococcalbacteremia model in the absence of serum bactericidal activity,an equally plausible explanation for the high vaccine efficacyobserved in the United Kingdom is the persistence of vaccine-inducedhigh-avidity group C anticapsular antibodies at concentrationsthat are insufficient to elicit bactericidal activity but thatare sufficient to confer protection against disease.

ACKNOWLEDGMENTS

This work was supported by grants RO1 AI45642 and RO1 AI46464from the National Institutes of Allergy and Infectious Diseases,National Institutes of Health (NIH), and T32-HL007951 (traininggrant for J.A.W.) from the NIH. The serum samples were obtainedas part of vaccine trials conducted in the Pediatric ClinicalResearch Center of Children's Hospital and Research Center atOakland, supported by grant M01-RR01271 from National Centerfor Research Resources, NIH.

We thank Raffella Rossi, Patricia Zuno Mitchell, and Lisa Nelbachfor superb technical assistance when performing the serologicassays and Shannon Harris for help in performing the animalprotection experiments. We also thank Bridget Canty and BettyFlores for enrolling the subjects and obtaining the serum samplesduring the clinical trials. Trudy Murphy reviewed the manuscriptand gave helpful critical comments.

FOOTNOTES

* Corresponding author. Mailing address: 5700 Martin Luther King Jr. Way, Oakland, CA 94609. Phone: (510) 450-7640. Fax: (510) 450-7915. E-mail: dgranoff{at}chori.org.

Editor: D. L. Burns

REFERENCES

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