Relative roles of genetic background and variation in PspA in the ability of antibodies to PspA to protect against capsular type 3 and 4 strains of Streptococcus pneumoniae

Abstract

Pneumococcal surface protein A (PspA) is able to elicit antibodies in mice and humans that can protect mice against fatal infection with Streptococcus pneumoniae. It has been observed that immunization with a single family 1 PspA can protect mice against infections with capsular type 3 or 6B strains expressing PspA family 1 or 2. However, several studies have shown that immunity to PspA is less efficacious against several capsular type 4 strains than against strains of capsular types 3, 6A, and 6B. To determine whether the greater difficulty in protecting against capsular type 4 strains resulted from differences in their PspAs or from differences in their genetic backgrounds, we performed protection experiments using four different challenge strains: a capsular type 3 strain expressing a family 1 PspA (WU2), a capsular type 4 strain expressing a family 2 PspA (TIGR4), and genetically engineered variants of WU2 and TIGR4 expressing each other's PspAs. Prior to infection, the mice were immunized with recombinant family 1 or family 2 PspA. The results revealed that much of the difficulty in protecting against capsular type 4 strains was eliminated when mice were immunized with a homologous PspA of the same PspA family. However, regardless of which PspA the strains expressed, those on the TIGR4 background were about twice as hard to protect against as WU2 strains expressing the same PspA based on the efficacy rates seen in our experiments. These results point out the importance of including more than one PspA in any PspA vaccines developed for human use.

FIG. 1.

PspA reactivity in pneumococcal strains. Bacterial lysates were run in sodium dodecyl sulfate-12% polyacrylamide gels and immunoblotted using monoclonal anti-PspA antibodies reactive with PspA/Rx1 (Xi126) or PspA/EF3296 (PC3.1). These two monoclonal antibodies were chosen because neither reacts with nonhomologous PspA. The samples run were EF3296 lysate (3296), TIGR4 lysate (TIGR), BR93.1 lysate (93.1), BR6.1 lysate (6.1), and WU2 lysate (WU2). The left panel shows the pattern of reactivities for the strains with Xi126 antibodies, and the right panel shows reactivities with PC3.1. EF3296, TIGR4, and BR93.1 (WU2 expressing PspA/TIGR4) lysates all showed reactivity with PC3.1 antibodies but did not react with Xi126, showing that the strains expressed only family 2 PspA. WU2 and BR6.1 (TIGR4 expressing PspA/Rx1) lysates reacted only with Xi126 antibodies, and no reactivity with PC3.1 antibodies was detected, showing that these strains expressed only the family 1 PspA. Numbers at left are molecular masses in kilodaltons.

FIG. 2.

Flow cytometric analysis of PspA expression. S. pneumoniae TIGR4, BR93.1, WU2, and BR6.1 were treated with anti-PspA Xi126 or PC3.1 antibodies and counterstained with biotinylated anti-mouse antibodies and Alexa Fluor 488-conjugated streptavidin. Binding was quantitated by flow cytometry. Dotted trace, streptavidin-alone-treated bacteria; gray trace, binding of Xi126 antibodies; black trace, binding of PC3.1 antibodies. Numerals above the black and gray traces indicate the levels of binding as the fluorescent signal of the sample divided by the fluorescent signal of the streptavidin control. TIGR4 and BR93.1 (WU2 expressing PspA/TIGR4) showed no binding of Xi126 antibodies but showed strong and similar levels of binding of PC3.1, indicating that the strains expressed only PspA/TIGR4. WU2 and BR6.1 (TIGR4 expressing PspA/Rx1) showed no binding with PC3.1 antibodies but showed strong and similar levels of binding with Xi126, indicating that they expressed only family 1 PspA.

FIG. 3.

Protection against pneumococcal infection after immunization with PspA/EF3296 fragments. Each point represents the results of a single mouse. P values were calculated from comparison of days to death between immunized and nonimmunized groups by the Mann-Whitney U test. No P values were given if the value was ≥0.05. The only exception is the value marked by an asterisk, which was >0.05 by the Mann-Whitney test. The value provided was calculated by Student's t test, which, when valid (as was the case here), can be more sensitive than the Mann-Whitney U test. (A) CBA/N mice were immunized with either SW111 or HR108. After a booster immunization, the mice were infected with 250 CFU of TIGR4, 240 CFU of BR93.1, or 300 CFU of WU2. (B) BALB/c mice were immunized with either SW111 or HR108. After a booster immunization, the mice were infected with 1.4 × 10⁶ CFU of TIGR4, 1.7 × 10⁶ CFU of BR93.1, or 1.0 × 10⁶ CFU of WU2. Immunization with SW111 and HR108 protected some mice against TIGR4. Although these results were not statistically significant, they may be real, since pooling the data for SW111 and HR108 immunizations showed significance at P = 0.0011.

FIG. 4.

Protection against pneumococcal infection after immunization with PspA/Rx1 fragment JAS218. Each point represents the results of a single mouse. P values were calculated from comparison of days to death between immunized and nonimmunized groups by the Mann-Whitney U test. No P values were given if the value was ≥0.05. The only exception is the value marked by an asterisk, which was 0.032 when comparison of live and dead mice was conducted with the Fisher exact test. This difference was not significant by the Mann-Whitney U test. The value provided was calculated by comparison of live with dead mice. (A) CBA/N mice were immunized with JAS218. After a booster immunization, the mice were infected with 270 CFU of TIGR4, 320 CFU of WU2, or 270 CFU of BR6.1. (B) BALB/c mice were immunized with JAS218. After a booster immunization, the mice were infected with 2.5 × 10⁶ CFU of TIGR4, 1.0 × 10⁶ CFU of WU2, or 1.1 × 10⁶ CFU of BR6.1.