Immunization with C5a peptidase or peptidase-type III polysaccharide conjugate vaccines enhances clearance of group B Streptococci from lungs of infected mice

Abstract

Group B streptococci (GBS) are among the most common causes of life-threatening neonatal infections. Vaccine development since the late 1970s has focused on the capsular polysaccharides, but a safe, effective product is still not available. Our quest for a vaccine turned to the streptococcal C5a peptidase (SCPB). This surface protein is antigenically conserved across most if not all serotypes. A murine model was used to assess the impact of SCPB on clearance of GBS from the lungs of intranasally infected animals. Mutational inactivation of SCPB resulted in more-rapid clearance of streptococci from the lung. Immunization with recombinant SCPB alone or SCPB conjugated to type III capsular polysaccharide produced serotype-independent protection, which was evidenced by more-rapid clearance of the serotype VI strain from the lungs. Immunization of mice with tetanus toxoid-type III polysaccharide conjugate did not produce protection, confirming that protection induced by SCPB conjugates was independent of type III polysaccharide antigen. Histological evaluation of lungs from infected mice revealed that pathology in animals immunized with SCPB or SCPB conjugates was significantly less than that in animals immunized with a tetanus toxoid-polysaccharide conjugate. These experiments suggest that inclusion of C5a peptidase in a vaccine will both add another level to and broaden the spectrum of the protection of a polysaccharide vaccine.

FIG. 1.

Impact of SCPB on sensitivity to phagocytosis. (A) Phagocytosis of strains 090R and 090R(Del) by human whole blood. Data shown are from a single experiment but are representative of five independent experiments. (B) Association of strains 090R and 090R(Del) with PMNs in human whole blood. M1 bars represent PMNs with fluorescence greater than that of uninfected PMNs. Counts are the number of cells; a total of 10,000 cells were counted.

FIG. 2.

Clearance of strains 090R and 090R(Del) from adult mouse lungs 5 h postinfection. Data are from three independent experiments and are presented as geometric means (CFU per milligram of lung tissue). Experimental groups contained 11 mice. The differences between the results for strains 909R and 909R(Del) were significant (P = 0.02).

FIG. 3.

Immunization with SCPB enhances clearance from lungs. The clearance of serotype VI strain S2-02288 from mouse lungs was evaluated 5 h postinfection. Mice were immunized with TT or full-length SCPB. Twenty mice were in each experimental group. Data are from a single experiment but are representative of three independent experiments. Mice were euthanatized, and their lungs were harvested and homogenized in buffer. Homogenates were assayed for viable streptococci. The differences between the results for animals immunized with TT and SCPB were significant (P = 0.0004).

FIG. 4.

Immunization with SCPB and CpsIII speeds clearance of streptococci from lungs. Mice (20 mice per experimental group) were immunized with CpsIII-TT, CpsIII-SCPB, or CpsIII-SCPBw. Lungs were obtained from mice 5 h after infection with the serotype VI strain S2-02288. Data are from a single experiment but are representative of two experiments. The numbers of CFU per milligram of lung tissue are presented as geometric means. The differences between the results for animals immunized with SCPB-CpsIII and TT-CpsIII were significant (P = 0.02).

FIG. 5.

Immunization with SCPB reduced pathology in mouse lung. Mice were immunized with TT-CpsIII, SCPB-CpsIII, or SCPBw-CpsIII conjugate. One mouse from each experimental group was euthanatized 5 h after infection with serotype VI strain S2-02288. Lungs were removed, fixed, embedded in paraffin, and sectioned. Sections were either Gram stained (C) or stained with hematoxylin and eosin (A, B, D, E, and F). Representative sections from a mouse immunized with TT-CpsIII (A to C), a mouse immunized with SCPB-CpsIII (D and E), and an uninfected mouse immunized with SCPB-CpsIII (F) are shown. Magnifications, ×40 (A and D), ×200 (B, E, and F), and ×1,000 (C).