Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) are important candidates for an alternative vaccine against pneumococcal infections. linear epitopes of PspA were not protective, larger overlapping fragments made up of 100 amino acids of PspA of strain Rx1 were constructed (fragments 1 to 7, numbered from your N terminus) to permit the mapping of antibodies with conformational epitopes not represented in the peptide arrays. Antibodies from mice immunized with fragments 1, 2, 4, and 5 were capable of binding onto the surface of pneumococci and mediating protection against a lethal challenge. The fact that immunization of mice with 100-amino-acid fragments located at the more conserved N-terminal region of PspA (fragments 1 and 2) induced protection against a pneumococcal challenge indicates that this induction of antibodies against conformational epitopes present at this region may be important in strategies for inducing broad protection against pneumococci. INTRODUCTION is usually a major cause of morbidity and mortality due to pneumonia, meningitis, and bacteremia. It has been estimated that 14.5 million episodes of serious illnesses caused by pneumococci occurred worldwide in 2000, leading to the death of 826,000 children <5 years of age. Infections caused by pneumococci were therefore responsible for 11% of all deaths in this age group (1). The capsular polysaccharide (PS) Quizartinib is the most well-known virulence factor of (pneumococcal serine-rich repeat protein) (30). It was therefore proposed that these antigens are really important and are involved in evasion of the immune system, but their use as a vaccine could lead to a replacement phenomenon similar to that observed with PS-conjugated vaccines. Thus, the choice of PspA and PspC molecules capable of inducing antibodies with broad cross-reactivities is Quizartinib essential. We previously showed that PspA from clade 4 (PspA4), PspA from clade 5 (PspA5), and PspC from group 3 (PspC3) induced antibodies that acknowledged the majority of the pneumococcal clinical isolates tested (31,C33). Alternatively, cross-reactive immunogenic epitopes present in PspA and PspC can be selected to compose a multiepitope protein vaccine. In this work, we used a screening method with peptide arrays made up of 15-mer peptides covering the entire sequence of different PspAs and PspC3 to analyze sera immune to these antigens. We previously used a similar method Quizartinib to compare sera from mice immunized with one PspA variant as a recombinant protein and as a DNA vaccine (34). This technique has also been used successfully to screen epitopes of antigens from other pathogens (35, 36). The present study tested sera from mice immunized with several variants of PspA and PspC3 to identify the most immunogenic epitopes. We also evaluated the protective capacity of antibodies against immunogenic linear epitopes. We localized the PspA regions capable of inducing protective immunity Quizartinib using 100-amino-acid fragments of PspA to permit the detection of conformational epitopes. Our findings have important implications for vaccine development, providing insight into the protective capacity of antibodies Quizartinib against both linear and conformational epitopes of PspA. MATERIALS AND METHODS Expression of recombinant proteins. The plasmids for the expression of PspAs (from your mature N terminus to the proline-rich region) from clade 1 (PspA1; strain 435/96), clade 2 (PspA2; strain 371/00), clade 3 (PspA3; strain 259/98), clade 4 (PspA4; strain 255/00), Smad4 clade 5 (PspA5; strain 122/02) (31, 32), and PspC group 3 (PspC3; strain 491/00) (37) were previously constructed in our laboratory. and its fragments (fragments 1 to 7) were amplified by PCR from strain Rx1 and cloned into the pAE vector (38) for expression in with an N-terminal histidine tag, using the primers outlined in Table S1 in the supplemental material, generating pAE-(1,011 bp), pAE-(300 bp), pAE-(300.