plot created a slope that could be compared statistically among the vaccine candidates by analysis of differences in the rate of rise (log10 Concentration/Age). PcpA occurred prior to children attaining age 6 months old. PhtD, PcpA, and Ply elicit a synchronous natural acquisition of serum antibody in young children suggesting that a trivalent protein vaccine combining PhtD, PcpA, and PlyD1 would be less likely to display antigen competition when administered as a combination vaccine in young children. Keywords: generalized additive model, generalized additive mixed model, nasopharynx, pneumococcal histidine triad protein D, pneumococcal histidine triad protein E, pneumococcal choline binding protein A, pneumolysin, (capsular types may need to be targeted as well. Therefore, several groups are proceeding with study and development of pneumococcal vaccines based on conserved proteins expressed by protein virulence factors have been identified as vaccine candidates, including PsaA, PspC, PspA, PcpA, Ply, and SPP PhtD.7 Age-dependent natural antibody induction has been detected for these antigens in children after NP colonization and respiratory tract infections.8,9 Moreover, naturally acquired antibody increasing with age has been correlated with reduced progression from NP colonization to AOM.10,11 Our group has been investigating 5 pneumococcal proteins as possible ingredients to be included in a multi-component vaccine.12-18 We sought conserved proteins of with different functions during pathogenesis, including components that SPP could SPP elicit strong antibody responses to prevent adherence of to NP and lung epithelium while also inducing rapid innate immune cell activation. We reasoned a multi-component vaccine could be more efficacious than SPP a single-valent vaccine given the quick systemic dissemination of pneumococci during pathogenesis.16,17,19,20 The 5 proteins studied have been PhtD and PhtE (pneumococcal histidine triad proteins), PcpA (a choline binding protein), LytB (a murein hydrolase) and PlyD1 (a non-toxic pneumolysin derivative).21-25 The central focus of our research has been to examine serum and mucosal responses to the 5 protein vaccine candidates following natural NP colonization and AOM in young children. It may SPP be true that immunogens could be prepared in pure form and adjuvanted to stimulate an immune response in young children when natural exposure to the protein would not stimulate a response. However, natural priming and boosting of the immune system is of recognized importance in successful vaccination and in sustaining immunogenicity and protection from disease.19 Therefore, we hypothesized that among the antigens available, selection of those that were more immunogenic at the youngest ages enhances the chances of their success as vaccines. We have been particularly interested to Tead4 find a vaccine to prevent AOM caused by and specifically in young children who experience repeated, closely spaced AOM infections, termed otitis prone (OP) children, since AOM is by far the most frequent form of disease in children with an economic impact >$1 billion.26 OP children are defined as children with at least 3 AOM episodes in 6 months or 4 episodes in a 12-month time span.26-29 To meet the definition of stringently defined OP (sOP) children have every episode proven by a tympanocentesis-derived middle ear fluid positive culture of an otopathogen30 while non-otitis prone (NOP) children are those with 0 to 2 episodes of AOM per year.7,13-15 Our studies of relative immunogenicity in infants and toddlers following NP colonization and AOM in sOP children identified PhtD, PcpA and PlyD1 as the most immunogenic candidates among the 5 antigens tested to consider in a multi-component vaccine.12 Combining vaccine ingredients into a multi-component product can lead to diminished immunogenicity compared to immunogenicity elicited when the components are administered as single ingredients.31 mechanisms have been described to account for reduced immunogenicity in combination vaccines but common among the results has been a reduction in immunogenicity for the least immunogenic ingredient in a combination.31 Nevertheless, when targeting a disease-causing organism, having a multi-component vaccine can increase the overall effectiveness of the vaccine compared to a single vaccine antigen.31,32 Here we present a novel method of analysis of immunogenicity as a means to predict an increased likelihood of equivalent.