C. mice and to elicit protection against pneumococcal challenge (6, 24, 27, 29, 32). PspA has also been shown to elicit antibodies in humans that can passively Frentizole protect mice against pneumococcal sepsis (6). The PspA sequence is usually variable among pneumococcal strains, especially in the -helical N-terminal domain name, which is usually exposed around the bacterial surface (17, 18, 22). Based on sequence similarities, PspA sequences have been classified into three main families, with over 95% of strains belonging to either family 1 or family 2 (18, 34). Despite the variability in their -helical sequences (as much as 60% of amino acids differ between families), immunization with an individual family 1 PspA or family 2 PspA has been observed to elicit Frentizole antibodies that are protective against capsular group 6 or type 3 strains expressing serologically diverse PspAs (6, 21, 27). In previous studies it has been noted that pneumococcal strains of capsular types 2, 4, and 5 can be harder to protect against with PspA immunization than strains of other capsular types (9, 23, 24, 29, 32). Most of these prior studies were conducted before Frentizole PspA families had been described (18). In the case of the studies with the capsular type 4 strains, we know now that most were PspA family 2 and that the immunogens were generally PspA family 1 (9). In one Frentizole study, however, immunization with family 2 PspA from the capsular type 4 strain EF5668 gave solid protection against infection with the homologous strain and also against strains of capsular types 3 and 6 that expressed family 1 PspAs (21). More recently, when we immunized with fragments of family 2 PspA from the capsular type 4 strain EF3296, we observed statistically significant protection against EF3296, but seldom were all of the challenged mice completely guarded against fatal disease (29). In the present study, we have specifically investigated one of the PspA family 2 capsular type 4 strains and sought to determine Frentizole whether the difficulty of protecting against this strain, compared to type 3 strain WU2, could be eliminated by using a homologous immunogen or whether there might be something about the capsular type 4 strains’ genetic backgrounds that makes them difficult to protect against. To dissect the roles of the PspA family and the genetic background, two pairs of isogenic strains were prepared. One was BR93.1 (28), a variant of WU2 that makes TIGR4’s family 2 PspA instead of the family 1 PspA normally expressed by WU2. The other strain was BR6.1, a variant of TIGR4 that expresses family 1 PspA/Rx1 instead of TIGR4’s family 2 PspA. The ability of immunity to family 1 and 2 PspAs to protect against these strains was compared with protection against the parental strains WU2 and TIGR4. MATERIALS AND METHODS Reagents. Alexa Fluor 488-conjugated streptavidin was from Molecular Probes Inc. (Eugene, Oreg.). Alkaline phosphatase-conjugated streptavidin, biotin-conjugated goat anti-mouse, and biotin-conjugated goat anti-rabbit antibodies were from Southern Biotechnology Associates (Birmingham, Ala.). Protein markers and Ready gels were from Bio-Rad Laboratories (Hercules, Calif.). Monoclonal anti-PspA antibody (Xi126) was produced as described previously (22). Monoclonal antibody PC3.1 was elicited by immunization with PspA/EF3296 and was a gift from Aventis Inc., Toronto, Ontario, Canada. Bacteria. Bacterial strains and plasmids used are described in Table ?Table1.1. The pneumococcal strains were stored at ?80C in 12% Rabbit Polyclonal to FZD9 glycerol, transferred to blood agar plates, and incubated at 37C in a 5% CO2 atmosphere overnight. Colonies from blood agar were used to inoculate Todd-Hewitt medium made up of 0.5% yeast extract. Bacteria were harvested in early stationary phase at 1,500 for 15 min and suspended in 60 mM phosphate-buffered saline (PBS, pH 7.2). The bacterial concentration was estimated from the absorbance at 600 nm and confirmed by viable counts on blood agar. TABLE 1. Bacterial strains and plasmids TIGR4 was included because it is usually transformable and expresses a family 2 PspA whose -helical region is usually identical to that of PspA/EF3296 (18, 33). Cloned and expressed PspA fragments were already available from PspA/EF3296 (29). Strain WU2 was chosen as a transformable family 1 PspA capsular type 3 strain, which is usually readily guarded against by immunization with family 1 PspA/Rx1 (8). PspA/Rx1 was important to the study because we have used it more than any other PspA for immunization and challenge studies. The construction of the WU2 variant BR93.1, in which PspA/WU2 was replaced with PspA/TIGR4, has been described elsewhere (28). The TIGR4 variant expressing only family 1 PspA was.