Evaluation of a Pentavalent Conjugated Pneumococcal Vaccine in Toddlers

Pneumococcal disease is one of the 10 leading causes of death in the United States, with an overall incidence of pneumococcal infection in the US estimated at 21 to 43/100 000 population.1 Pneumococci have assumed a major role as a cause of childhood meningitis because of the decreased incidence of Haemophilus influenzae type b disease. Pneumococci are also isolated from about one-third of children with otitis media and cause sinusitis and pneumonia. Pneumococcal disease can be overwhelming in children with sickle cell disease, immunodeficiency, splenic dysfunction and other chronic illnesses.2, 3 Multiply drug-resistant pneumococci are being increasingly reported, and penicillin resistance among pneumococci is a worldwide problem.4, 5 In addition penicillin resistance is most frequent among pneumococcal serotypes 6, 14, 18, 19 and 23 which are associated with infections in young children. The emergence of resistance has intensified the need to develop more effective means to control pneumococcal infections. Efforts to develop pneumococcal vaccines have generally concentrated on generating immune responses to the pneumococcal capsular polysaccharide, because type-specific antibody to polysaccharide has opsonizing activity associated with protection in vitro.6 Conjugation of polysaccharide antigens to protein carriers represents a new approach to vaccine formulation that attempts to convert a T-independent antigen (polysaccharide) to a more immunogenic T-dependent antigen through addition of T-helper cell epitopes. This approach has been highly successful in the development of glycoconjugate vaccines for H. influenzae type b, which provide excellent protection against invasive disease in children by 4 to 6 months of age. Protein-conjugated pneumococcal vaccines should increase the response of infants and toddlers to the pneumococcal polysaccharides included. Åhman et al.7 evaluated the safety and immunogenicity of a three-dose sequence at 2, 4 and 6 months of age of a pentavalent (serotype 6B, 14, 18C, 19F and 23F) pneumococcal oligosaccharide-CRM197 conjugate vaccine (CRM197-OS) compared with unvaccinated controls in Finnish infants. The CRM197-OS vaccine was well-tolerated and induced IgG serum responses, especially after three doses. However, a concentration of ±1.0 μg/ml IgG antibody, protective in a chinchilla model,8 was achieved after two doses of vaccine for only one of five serotypes (18C) and after three doses for only two additional serotypes (14 and 19F). Two-year-olds respond better than infants to saccharide-protein conjugate vaccines; therefore we sought to evaluate this same CRM197-conjugated pentavalent oligosaccharide pneumococcal vaccine as a possible product for interim use in toddlers. Materials and methods.Study design. After we obtained parental written informed consent, healthy children between the ages of 21 and 27 months were enrolled in this study and then randomized to receive either 23-valent polysaccharide vaccine or pentavalent pneumococcal conjugate vaccine in a 1:2 ratio. Twenty children were assigned to receive a two 0.5-ml dose series of CRM197 pneumococcal oligosaccharide (CRM-OS) conjugate vaccine separated by 2 months, and 10 children were randomized to receive a single 0.5-ml dose of 23-valent polysaccharide vaccine intramuscularly. Investigators were blinded as to vaccine assignment groups to avoid bias in the event that parents contacted us regarding a reaction. The pneumococcal conjugate vaccine consisted of a pentavalent mixture containing 10 μg of carbohydrate antigen of each of the following serotypes: 6B, 14, 18C, 19F and 23. The oligosaccharides were conjugated to CRM197 by reductive amination at a ratio of between 0.5:1 and 1:1 by weight, so that the total protein content was ≤50 μg of CRM197 per dose; the antigens were absorbed to aluminum phosphate. Twenty-three-valent pneumococcal polysaccharide vaccine containing 25 μg of carbohydrate antigen/serotype was purchased commercially (Wyeth Lederle, PnuImune 23®). Adverse events were monitored by parents on a diary report card for 4 days after each injection. Parents were instructed to obtain rectal temperatures twice a day for 4 days after vaccination and to report fretfulness, sleepiness, crying, screaming or loss of appetite. In addition parents were asked to note the presence of redness or swelling at the site of injection and to grade tenderness on a 0, 1 or 2+ scale. Measurement of type-specific pneumococcal serum antibody. Sera were obtained before vaccination and 28 days after the last vaccination. Serum IgG antibody responses were assessed to the five serotypes in the conjugate vaccine by serotype-specific enzyme-linked immunosorbent assay compared with a standard. Samples from the same subject were run on the same plate. Corning No. 25801 enzyme immunoassay plates were used that consistently bound all of the serotypes (6B, 14, 6B, 18C, 19F and 23). Blocking plates with 1% bovine serum albumin with 0.1% (v/v) Tween 20 for 2 h at 21°C significantly reduced background activity. We did not subtract the optical density reading of a well without antigen from a well with antigen (blank subtraction) because it distorted the data. We found that antibody was more likely to stick nonspecifically in a well that was not treated with pneumococcal polysaccharide. In these assays cell wall polysaccharide is added to the diluent to reduce nonspecific activity. Serum diluted 1:500 in phosphate-buffered saline/0.1% Tween/1% bovine serum albumin with cell wall polysaccharide concentrations of 10 μg/ml reduced the signal to an optical density of <0.010. An internal standard from a pool of post-pneumococcal vaccine sera gave consistent slope over a wide range of dilutions under these conditions (1:200 to 1:10,000). Samples were run in duplicate; when variation in antibody quantitation was >10% we repeated the assay for verification. Our internal pooled control sera was calibrated against the Food and Drug Administration Serum 89-SF (from Carl Frasch, Ph.D.) and our results were adjusted to reflect the assigned IgG antibody content values of 89-SF. Statistics. Fold-increases were calculated from baseline and individuals with 4-fold or greater increases were defined as responders. Comparisons were made using Kruskal-Wallis rank sums for the fold increase and with corrected chi square tests for responder status. Results. There were no significant adverse events. Rates of complaints after vaccination are shown in Table 1. There was one dropout in the conjugate group after the first dose (the parent refused further phlebotomy). Rates of minor changes in behavior were similar in those receiving conjugate and polysaccharide vaccines. Local reactions at the injection site were mild and minor; however, approximately twice as many individuals experienced tenderness after conjugate vaccine than after polysaccharide vaccine (P not significant). None of these reactions was graded 2+ (cried when arm was moved) by parents. Reactions to the second dose of conjugate vaccine were no more frequent than to the first.TABLE 1: Systemic and local complaints of toddlers after receipt of conjugated or polysaccharide pneumococcal vaccines CRM-OS conjugate vaccine was more immunogenic than polysaccharide vaccine. CRM-OS induced an IgG antibody concentration >1.0 μg/ml for all five serotypes after two doses of vaccine (Table 2). Enhanced immunogenicity for the conjugate vaccine as measured by the mean fold increase and the proportion of subjects responding was particularly noticeable for the least immunogenic serotypes, types 6B and 14 (Table 2). Overall between 74 and 100% of subjects responded to two doses of conjugate vaccine. Pneumococcal conjugate vaccination generally produced higher antibody titers after the first dose than plain polysaccharide and further increases in antibody were seen after the second dose for four of the five serotypes. Serotype 18C conjugate induced a response to the first dose similar to that seen with the plain polysaccharide and no further rise after the second dose. The highest responses were observed to serotypes 18C and 23 and the lowest response to serotype 19F.TABLE 2: Response to pneumococcal polysaccharide (PS) vs. CRM-OS in 2-year-olds Discussion. The CRM-OS conjugate vaccine we studied was more immunogenic in toddlers than previously described in infants.7 Unconjugated types 6B, 14, 19F and 23F are generally poor immunogens in infants and toddlers, but types 14 and 19F are better than types 6B and 23F.9, 10 Unconjugated type 18C is a better immunogen in the plain polysaccharide vaccine than the other four included in the conjugate product evaluated here. The differences in immunogenicity of this CRM-OS-pneumococcal vaccine in infants7 compared with our observations in toddlers may reflect the maturation of the immune response to protein-polysaccharide vaccine constructs with age and/or enhanced priming for the protein carrier immune response of the conjugate because the toddlers all had received four doses of DTP vaccine before administration of the pneumococcal conjugate. Several factors may influence the immunogenicity of glycoconjugate vaccines including the specific carrier protein used, the degree of preexisting immunity to the protein carrier, the specific method of linkage used, the physical nature of the carbohydrate antigen as oligosaccharide or polysaccharide, the ratio of carbohydrate to protein present in the material, the presence of free polysaccharide in the vaccine and the use of adjuvants. Other factors that may influence immunogenicity and antibody quantitation of these pneumococcal conjugate vaccines are the populations studied, age when immunized and antibody assay methods. Several schemes have been used for the generation of conjugate vaccines, with coupling of pneumococcal antigen in the form of polysaccharide or oligosaccharide to a variety of protein carriers, including tetanus toxoid, diphtheria toxoid or CRM197 and the outer membrane protein complex of Neisseria meningitidis. Coupling has been done by reductive amination, use of a linker molecule or other means. In a recent study a type 23F polysaccharide-CRM197 conjugate was found to be more immunogenic in toddlers than the conjugate containing oligosaccharide, and a spacer molecule did not enhance responses.11 Preliminary results suggest that polysaccharide-CRM197 conjugates are more immunogenic than oligosaccharide-CRM197 conjugates in infants.12 The inclusion of additional serotypes (e.g. 4, 9V) in pneumococcal conjugate vaccines may be preferable to induce antibody to at least the seven most common serotypes causing pediatric disease. Thus although our clinical trial results with this CRM197 pentavalent pneumococcal oligosaccharide conjugate in toddlers is encouraging, future studies will evaluate vaccines with longer saccharides and more serotypes. Acknowledgments. Presented in part at the Society for Pediatric Research meeting, Washington, DC, May, 1996. Research was supported by Grant N01-AI-45248 from the National Institute of Allergy and Infectious Diseases. Dr. David Klein, National Institutes of Health, facilitated the conduct of this study. Michael E. Pichichero, M.D. Mark A. Shelly, M.D. John J. Treanor, M.D.