181:1172-1175. predicting protective activity. We investigated the 73 sera with bactericidal titers of 1:4 to determine the basis of protective activity. The 19 sera with protective activity had a higher geometric mean group C anticapsular antibody concentration (0.72 g/ml) than the 54 sera that lacked protective activity (0.16 g/ml; 0.001). Thus, protective activity in the absence of bactericidal activity was associated with higher concentrations of anticapsular antibodies, but not all sera with anticapsular antibodies conferred protection. Of 18 nonbactericidal sera with anticapsular antibody concentrations between 0.31 and 0.99 g/ml, the 11 sera that conferred protection Afzelin had a higher mean antibody avidity constant (21.9 nM?1) than the 7 nonprotective sera (14.6 nM?1; 0.03). Thus, in sera with titers of 1:4, protective activity is associated with higher-avidity group C anticapsular antibodies, which are present in concentrations insufficient Afzelin to elicit complement-mediated bacteriolysis in vitro but sufficient to confer protection in an in vivo bacteremia model. New multivalent meningococcal polysaccharide-protein conjugate vaccines are currently in development (5, 30) and will likely BMP2 be licensed in Europe and North America in the next few years (28). The low incidence of meningococcal disease in these populations precludes performing prospective randomized clinical trials to determine the efficacy of these new vaccines. Vaccine efficacy, therefore, will be inferred from immunogenicity data (3), and vaccine effectiveness will be confirmed in subsequent postlicensure studies (1), following a licensure pathway and monitoring strategies adapted in the United Kingdom for the introduction of group C meningococcal conjugate vaccines. There is a strong scientific basis for inferring meningococcal vaccine efficacy from immunogenicity data (3, 10, 11). However, the choice of in vitro assay conditions and serologic endpoints for inferring protection against meningococcal disease are topics of considerable recent debate (1, 3, 16, 32). The reasons are complex but ultimately have to do with the effects of potential disparities between in vitro antibody functional assay conditions and in vivo host defenses. Meningococci grown in vivo likely express different genes than those of bacteria grown in vitro (13). Also, when meningococci are grown in broth or agar, the choice of growth conditions may affect capsular production and/or the expression of different surface proteins or lipooligosaccharide structures (6, 22, 23, 35), which in turn can affect the susceptibility of the bacterial cell to antibody binding and complement-mediated bacteriolysis. These factors may limit the interpretation of the results of in vitro antibody functional studies. Members of our laboratory recently described an infant rat meningococcal bacteremia model for measuring antibody protective activity against group B or C strains (15, 25). Although meningococci are obligate human pathogens with species-specific pathogenic mechanisms (17), the infant rat model permits the investigation of the protective activity of antibodies in a setting where the organism is rapidly replicating in vivo. In the present study, we used the infant rat model to investigate the role of naturally acquired serum antibodies of human adults in protection against group C meningococcal disease. Protective activity in serum measured in vivo was related to the presence or absence of group C complement-mediated bactericidal activity measured in vitro or to the concentrations and avidities of group C anticapsular antibodies in serum. The results provide insights into the antigenic targets of naturally Afzelin acquired antibodies conferring protection against group C and the extent to which measurements of serum bactericidal activity may underestimate protective immunity. MATERIALS AND METHODS Serum samples. We used a convenience sample of 91 stored preimmunization sera that had been obtained from healthy adults ranging in age from 18 to 58 years who were enrolled in meningococcal vaccine immunogenicity trials conducted at Children’s Hospital and Research Center at Oakland between 2001 and 2003. None of the subjects had been previously immunized with meningococcal vaccine. To preserve internal complement activity, the Afzelin blood was allowed to clot at room temperature for 30 min and centrifuged at 2,135 for 10 min at 4C. The sera were promptly separated, divided into 0.5-ml aliquots, and stored frozen at ?70C. Use of these sera for the present study was approved by the Institutional Review Board of Children’s Hospital and Research Center at Oakland. Serology. (i) Bactericidal assay. The test strain was strain 4243 (C:2a:P1.5,2), a member of the electrophoretic type 37 complex, sequence type 11 (http://www.mlst.net), expressing a polysaccharide capsule that is O acetylation positive (15). The organism was grown in Mueller-Hinton broth (with a starting group C strain 4243 cells (range in different experiments, 800 to 1 1,400 CFU/rat). Eighteen hours after the bacterial challenge, blood specimens were obtained by puncturing the heart with a.