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Background: Pneumococcal conjugate vaccines prevent invasive and noninvasive disease due to infection with vaccine serotypes. Pneumococcal conjugate vaccines also prevent nasopharyngeal acquisition of vaccine serotypes, although the mechanism is incompletely understood., Methods: An efficacy trial of a 7-valent pneumococcal conjugate vaccine was conducted on the Navajo and White Mountain Apache reservations, located in the Southwestern United States; group C meningococcal conjugate vaccine was the control vaccine. Infants were randomized to receive 7-valent pneumococcal conjugate vaccine or group C meningococcal conjugate vaccine at 2, 4, 6, and 12 months of age. Immunogenicity and nasopharyngeal colonization studies were nested in the efficacy trial. We analyzed the correlation between serotype-specific serum IgG concentration at 7 and 13 months of age and nasopharyngeal acquisition of disease at 12 and 18 months of age, respectively. We adjusted for potential confounders using multivariate logistic regression., Results: Among 203 subjects, we observed 60 acquisitions of vaccine-type pneumococci, including 19 acquisitions of serotype 19F (31.7%), and 17 acquisitions of serotype 23F (28.3%). Among recipients of 7-valent pneumococcal conjugate vaccine, increased serotype-specific serum IgG was associated with a reduction in nasopharyngeal acquisition of serotype 23F (relative risk, 0.53; 95% confidence interval, 0.31-0.93) but was not associated with a reduction in acquisition of serotype 19F (relative risk, 1.07; 95% confidence interval, 0.57-2.03). Among group C meningococcal conjugate vaccine recipients, serotype-specific serum IgG was not associated with a reduction in nasopharyngeal acquisition for either serotype., Conclusion: An increase in serum antibody concentration was associated with reduced acquisition of serotype 23F pneumococcus (but not with reduced acquisition of serotype 19F pneumococcus) among recipients of 7-valent pneumococcal conjugate vaccine. Differences in antibody concentration, in the functional characteristics of antibody, or in antibody kinetics during infancy may account for differences in carriage protection.

An enzyme-linked immunoassay (EIA) is described and evaluated which quantitates human antibodies to serotype specific S. pneumoniae polysaccharide (PnPs) in human sera. Based on the observations previously described by Koskela (1), native PnPs are used as coating antigens and sera are absorbed with a soluble pneumococcal absorbant material containing C-polysaccharide (CPs) to ensure measurement of serotype specific anti-PnPs antibodies. The robustness of this method was evaluated by ten laboratories using the same reagents, protocol, and five human serum samples. Reproducible antibody values were obtained for IgM, IgG, and IgA antibodies to five different PnPs serotypes, 3, 6B, 14, 19F, and 23F. The overall mean percent coefficients of variation in this interlaboratory study for all five selotype specific anti-PnPs determinations with the five coded sera were 30% for IgG, 3/% for IgM, and 36% for IgA. This assay can be standardized for quantitation of serotype specific anti-PnPs antibodies, allowing comparison of antibody values in vaccine trials evaluating pneumococcal vaccines.

Pneumococcal conjugate vaccines will eventually be licensed after favorable results from phase III efficacy trials. After licensure of a conjugate vaccine for invasive pneumococcal disease in infants, new conjugate vaccines will likely be licensed primarily on the basis of immunogenicity data rather than clinical efficacy. Analytical methods must therefore be developed, evaluated, and validated to compare immunogenicity results accurately within and between laboratories for different vaccines. At present no analytical technique is uniformly accepted and used in vaccine evaluation studies to determine the acceptable level of agreement between a laboratory result and the assigned value for a given serum sample. This multicenter study describes the magnitude of agreement among 12 laboratories quantifying an identical series of 48 pneumococcal serum specimens from 24 individuals (quality-control sera) by a consensus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) developed for this study. After provisional or trial antibody concentrations were assigned to the quality-control serum samples for this study, four methods for comparison of a series of laboratory-determined values with the assigned concentrations were evaluated. The percent error between assigned values and laboratory-determined concentrations proved to be the most informative of the four methods. We present guidelines that a laboratory may follow to analyze a series of quality-control sera to determine if it can reproduce the assigned antibody concentrations within an acceptable level of tolerance. While this study focused on a pneumococcal IgG ELISA, the methods that we describe are easily generalizable to other immunological assays.

Objective: To determine the efficacy, safety and immunogenicity of the heptavalent CRM197 pneumococcal conjugate vaccine against invasive disease caused by vaccine serotypes and to determine the effectiveness of this vaccine against clinical episodes of otitis media., Methods: The Wyeth Lederle Heptavalent CRM197 (PCV) was given to infants at 2, 4, 6 and 12 to 15 months of age in a double blind trial; 37,868 children were randomly assigned 1:1 to receive either the pneumococcal conjugate vaccine or meningococcus type C CRM197 conjugate. The primary study outcome was invasive disease caused by vaccine serotype. Other outcomes included overall impact on invasive disease regardless of serotype, effectiveness against clinical otitis media visits and episodes, impact against frequent and severe otitis media and ventilatory tube placement. In addition the serotype-specific efficacy against otitis media was estimated in an analysis of spontaneously draining ears., Results: In the interim analysis in August, 1998, 17 of the 17 cases of invasive disease caused by vaccine serotype in fully vaccinated children and 5 of 5 of partially vaccinated cases occurred in the control group for a vaccine efficacy of 100%. Blinded case ascertainment was continued until April, 1999. As of that time 40 fully vaccinated cases of invasive disease caused by vaccine serotype had been identified, all but 1 in controls for an efficacy of 97.4% (95% confidence interval, 82.7 to 99.9%), and 52 cases, all but 3 in controls in the intent-to-treat analysis for an efficacy of 93.9% (95% confidence interval, 79.6 to 98.5%). There was no evidence of any increase of disease caused by nonvaccine serotypes. Efficacy for otitis media against visits, episodes, frequent otitis and ventilatory tube placement was 8.9, 7.0, 9.3 and 20.1% with P < 0.04 for all. In the analysis of spontaneously draining ears, serotype-specific effectiveness was 66.7%., Conclusion: This heptavalent pneumococcal conjugate appears to be highly effective in preventing invasive disease in young children and to have a significant impact on otitis media.

Objectives: The objectives of this study were (1) to determine the safety and immunogenicity of heptavalent pneumococcal CRM197 conjugate (PNCRM7) vaccine in infants and (2) to determine the effect of concurrent hepatitis B immunization during the primary series and the effect of concurrent diphtheria and tetanus toxoid and acellular pertussis [DTaP (ACEL-IMUNE)] and conjugate CRM197 Haemophilus influenzae type b [HbOC (HibTITER) immunization at time of the booster dose on the safety and immunogenicity of PNCRM7and these other concurrently administered vaccines., Methods: This was a randomized double-blinded study in 302 healthy infants in the Northern California Kaiser Permanente (NCKP) Health Plan. Infants received either PNCRM7 vaccine or meningococcal group C conjugate vaccine as a control at 2, 4 and 6 months of age and a booster at 12 to 15 months of age. Study design permitted the evaluation of immunology and safety of concurrent administration of routine vaccines. Antibody titers were determined on blood samples drawn before and 1 month after the primary series and the booster dose., Results: After the third dose of PNCRM7 geometric mean concentrations (GMCs) ranged from 1.01 for serotype 9V to 3.72 microg/ml for serotype 14. More than 90% of all subjects had a post-third dose titer of > or =0.15 microg/ml for all serotypes, and the percentage of infants with a post-third dose titer of > or =1.0 microg/ml ranged from 51% for type 9V to 89% for type 14. After the PNCRM7 booster dose, the GMCs of all seven serotypes increased significantly over both post-Dose 3 and pre-Dose 4 antibody levels. In the primary series there were no significant differences in GMCs of pneumococcal antibodies between the subjects given PN-CRM7 alone or concurrently with hepatitis B vaccine. At the toddler dose concurrent administration of PNCRM7 and DTaP and HbOC resulted in a near conventional threshold for statistical significance of a post-Dose 4 GMC for serotype 23F [alone 6.75 mirog/ml vs. concurrent 4.11 microg/ml (P = 0.057)] as well as significantly lower antibody GMCs for H. influenza polyribosylribitol phosphate, diphtheria toxoid, pertussis toxin and filamentous hemagglutinin. For all antigens there were no differences between study groups in defined antibody titers that are considered protective., Conclusion: We conclude that PNCRM7 vaccine was safe and immunogenic. When this vaccine was administered concurrently at the booster dose with DTaP and HbOC vaccines, lower antibody titers were noted for some of the antigens when compared with the antibody response when PNCRM7 was given separately. Because the GMCs of the booster responses were all generally high and all subjects achieved similar percentages above predefined antibody titers, these differences are probably not clinically significant.

Most animal species respond with high antibody levels to polysaccharide antigens after they have been covalently linked to a protein carrier, converting a T-cell independent to a T-cell dependent antigen. This chemical modification has enabled the development of glycoconjugate vaccines for Haemophilus influenzae type b, Neisseria meningitidis, and multivalent Streptococcus pneumoniae. This new generation of vaccines can be well characterized physically and chemically to ensure consistent vaccine manufacture. Such analytical tests provide an alternative to animal models for Quality Control Laboratories; biological models can be difficult and costly to develop and use on a routine basis. If animal tests are used, they need to be refined, defined, and validated for their intended purpose.

A phagocytosis assay for Streptococcus pneumoniae based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of S. pneumoniae grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (r = 0.98, P < 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 microliter per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.

Objective: To determine the safety and immunogenicity of heptavalent pneumococcal saccharide vaccine (serotypes 4, 6B, 9V, 14, 18C, 19F, 23F) individually conjugated to CRM197 (PNCRM7), administered at 2, 4, 6, and 12 to 15 months of age., Design: Two hundred twelve healthy 2-month-old infants were equally randomized to receive four consecutive doses of PNCRM7 or an investigational meningococcal group C conjugate vaccine, which served as a control. Concomitantly administered routine vaccines were oral polio vaccine and combined diphtheria toxoid, tetanus toxoid, and whole cell pertussis vaccine/Haemophilus influenzae type b vaccine consisting of capsular oligosaccharides conjugated to CRM197 (DTP/HbOC) at 2, 4, and 6 months, and either measles-mumps-rubella vaccine or HbOC at 12 to 15 months. Active safety surveillance was conducted for 3 days after each dose. Antibody concentrations to each of the 7 pneumococcal serotypes were measured by enzyme-linked immunosorbent assay prevaccination, after doses two and three, prebooster, and postbooster., Results: Significantly fewer children experienced local reactions at the PNCRM7 injection site than at the DTP/HbOC site. There was no increase in the incidence or severity of local reactions at the PNCRM7 site with increasing doses of vaccine. Mild to moderate postvaccination fever was common in both the PNCRM7 and control vaccine groups, however DTP/HbOC was administered concurrently. All 7 vaccine serotypes were immunogenic. The kinetics of the immune responses were serotype-specific. After three doses of PNCRM7, between 92% to 100% of children had >/=0.15 microg/mL of antibody, and 51% to 90% achieved a level of >/=1 microg/mL against specific serotypes. A booster dose of PNCRM7 resulted in a brisk anamnestic response to all 7 vaccine serotypes, demonstrating effective stimulation of T-cell memory by the primary series of vaccinations., Conclusion: Primary immunization followed by a booster dose of PNCRM7 seemed to be acceptably safe and resulted in significant rises in antibody to all 7 serotypes. Implications. Studies to assess vaccine efficacy of PNCRM7 for prevention of systemic disease, nasopharyngeal colonization, and acute otitis media are in progress. If PNCRM7 proves to be protective, there is the potential to prevent up to 85% of invasive pneumococcal disease occurring in US children.

Background: Streptococcus pneumoniae is the most common cause of invasive bacterial disease and otitis media in infants and young children. Licensed pneumococcal polysaccharide vaccines are not reliably immunogenic in children younger than 2 years of age; therefore pneumococcal glycoprotein conjugate vaccines are currently being evaluated for safety, immunogenicity and efficacy in various age groups., Methods: During a 12-month period we determined the kinetics of pneumococcal IgG antibody in 60 children who received primary immunization with one dose of bivalent (serotypes 6A and 23F) pneumococcal polysaccharide-CRM197 vaccines at 18 to 30 months of age. To assess immunologic priming a subgroup of 20 subjects received secondary immunization with pneumococcal polysaccharide vaccine, including serotypes 6B and 23F, at 11 to 20 months after primary immunization. Pneumococcal-specific IgG subclass distributions were also evaluated in the subgroup., Results: In the 12 months after primary immunization with glycoprotein conjugate vaccine, geometric mean pneumococcal IgG antibody concentrations to 6B and 23F serotypes remained stable. Pneumococcal polysaccharide vaccine induced a greater anamnestic response in children primed with glycoprotein conjugate vaccines (13- to 40-fold increases to geometric mean concentrations of 6 to 30 micrograms/ml for type 23F), than in those primed with polysaccharide (2- to 4-fold increases). A greater IgG response to pneumococcal serotype 23F than to 6B was observed with both primary and secondary immunization. The serotype-specific pneumococcal IgG antibody response was virtually restricted to the IgG1 subclass after primary immunization, but secondary immunization elicited antibodies of IgG1 and IgG2 subclasses., Conclusions: These glycoprotein conjugate vaccines appear to prime for anamnestic IgG antibody responses to subsequent immunization with polysaccharide vaccine, suggesting that the polysaccharide-CRM197 vaccine effectively induces a predominantly T cell-dependent immune response. The greater IgG response to 23F than to 6B indicates that pneumococcal serotype is a major determinant of immunogenicity of pneumococcal glycoprotein conjugate vaccines.

A human reference serum pool, lot 89-S, has been developed for use in quantitating concentrations of antibody to Streptococcus pneumoniae. Weight-based units have been assigned to antibodies to 11 pneumococcal polysaccharide (PnPs) serotypes (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) by using enzyme-linked immunosorbent assay methodology and a human standard reference serum, USNRP IS 1644. The experimentally derived assignments for anti-PnPs antibodies of the immunoglobulin G (IgG), IgM, and IgA isotypes in lot 89-S correlate well to the separately determined immunoglobulin assignment. These assignments for this antipneumococcal standard serum were used to quantitate IgG, IgM, and IgA isotype levels and the total immunoglobulin level in pediatric samples from a pneumococcal conjugate vaccine trial. The data indicate that these assignments may be used to assess levels of antibody to PnPs serotypes in human serum.

Because most childhood invasive pneumococcal disease occurs before the age of 2 years, the development of a pneumococcal vaccine that is immunogenic in infants is a priority. We assessed the safety and serum antibody responses to two dose levels of three bivalent pneumococcal capsular polysaccharide (CPS)-protein conjugate vaccines incorporating the poorly immunogenic serotypes 6A and 23F. The conjugate vaccines differed in CPS size and chemical linkage, but all used a nontoxic cross-reactive mutant diphtheria toxin (CRM197) as the protein carrier. 118 young children 18 to 30 months of age received a single immunization with one of the three glycoconjugates or with licensed pneumococcal vaccine. Sera were obtained before and 1 month after immunization and analyzed by enzyme-linked immunosorbent assay for serotype-specific antibody titers. The 23F CPS was more immunogenic than the 6A CPS in all vaccine formats. The most immunogenic 23F conjugate vaccine consisted of native CPS directly linked to the carrier protein; smaller CPS or the use of a six-carbon linker did not appear to enhance immunogenicity in these young children. Conjugation of two pneumococcal CPSs is associated with an increase in immunogenicity, and the characteristics of the CPS and of the CPS-protein linkage appear to influence the antibody response.