Antibodies to capsular polysaccharide and clumping factor A prevent mastitis and the emergence of unencapsulated and small-colony variants of Staphylococcus aureus in mice

Abstract

The pathogenesis of Staphylococcus aureus infections is influenced by multiple virulence factors that are expressed under variable conditions, and this has complicated the design of an effective vaccine. Clinical trials that targeted the capsule or clumping factor A (ClfA) failed to protect the recipients against staphylococcal infections. We passively immunized lactating mice with rabbit antibodies to S. aureus capsular polysaccharide (CP) serotype 5 (CP5) or CP8 or with monoclonal antibodies to ClfA. Mice immunized with antibodies to CP5 or CP8 or with ClfA had significantly reduced tissue bacterial burdens 4 days after intramammary challenge with encapsulated S. aureus strains. After several passages in mice passively immunized with CP-specific antiserum, increasing numbers of stable unencapsulated variants of S. aureus were cultured from the infected mammary glands. Greater numbers of these unencapsulated S. aureus variants than of the corresponding encapsulated parental strains were internalized in vitro in MAC-T bovine cells. Furthermore, small-colony variants (SCVs) were recovered from the infected mammary glands after several passages in mice passively immunized with CP-specific antiserum. A combination of antibodies effectively sterilized mammary glands in a significant number of passively immunized mice. More importantly, passive immunization with antibodies to both CP and ClfA fully inhibited the emergence of unencapsulated "escape mutants" and significantly reduced the appearance of SCVs. A vaccine formulation comprising CP conjugates plus a surface-associated protein adhesin may be more effective than either antigen alone for prevention of S. aureus infections.

FIG. 1.

Experimental regimen consisting of consecutive cycles of passive immunization with rabbit antiserum or MAbs followed by challenge with in vivo-passaged S. aureus. The capsule phenotype of colonies recovered from each infection cycle was assessed by a colony immunoblot method. IP, intraperitoneal.

FIG. 2.

Passive immunization with CP antibodies reduced the bacterial burden in the mammary glands of lactating mice. Each bar indicates the mean log₁₀ CFU/gland obtained in cycle 1, and the error bars indicate the standard errors of the means (six to eight mice/group). The dotted line indicates the limit of detection (0.7 log CFU/gland). *, significant difference in the number of CFU at 96 h after challenge of mice inoculated with immune or nonimmune serum. The levels of significance were as follows: for CP5-positive strain RA9, P < 0.001; for CP8-positive strain MBC212, P < 0.001; for Reynolds (CP5), P = 0.014; and for Reynolds (CP8), P = 0.001.

FIG. 3.

CP antiserum selects for NT S. aureus in the mammary glands of mice challenged with CP-positive S. aureus. Each symbols indicates the percentage of NT S. aureus variants obtained after a cycle of enrichment. (Upper panel) Strains RA9 (CP5) and MBC212 (CP8). (Lower panel) Strains Reynolds (CP5) and Reynolds (CP8).

FIG. 4.

Passive immunization with antibodies to CP5 and ClfA reduced the intramammary bacterial load 96 h after intramammary challenge. Each bar indicates the mean log₁₀ CFU S. aureus/gland, and the error bars indicate the standard errors of the means (six to eight mice/group). The dotted line indicates the limit of detection by culture (0.7 log CFU/gland). *, significant difference in the number of CFU at 96 h after challenge between mice inoculated with immune serum and the corresponding controls. The levels of significance were as follows: for mice passively immunized with ClfA MAbs versus the control, P < 0.01; for mice passively immunized with CP5 antiserum versus the control, P < 0.01; and for mice passively immunized with ClfA MAbs plus CP5 antiserum versus either control group, P < 0.001.

FIG. 5.

Emergence of NT S. aureus in the mammary glands of mice challenged with S. aureus strain RA9 (CP5 positive) did not occur in mice inoculated with ClfA MAbs (with or without CP5 antibodies) up to the 10th experimental cycle. Each symbol indicates the percentage of NT S. aureus colonies obtained after a cycle of enrichment.

FIG. 6.

Recovery of SCVs from the mammary glands of passively immunized mice during the enrichment experiment. The SCVs were isolated from mice treated with CP antibodies and challenged with bovine isolate RA9 or MBC212 (left panel) or human isogenic strain Reynolds (CP5) or Reynolds (CP8) (right panel). SCVs were recovered from the majority of mice inoculated with the bovine RA9 or MBC212 strain but from only one-half of the mice challenged with Reynolds (CP5) or Reynolds (CP8). SCVs were not detected in tissues from mice inoculated with nonimmune rabbit serum.

FIG. 7.

Passive immunization with CP5 antiserum, as shown in Fig. 1, resulted in emergence of S. aureus SCVs. Injection of ClfA MAbs plus CP5 antiserum significantly (P < 0.001) reduced the emergence of S. aureus SCVs in the mammary glands of mice challenged with S. aureus strain RA9 (CP5 positive) up to the 10th infection cycle. Passive immunization with ClfA MAbs prevented the emergence of S. aureus SCVs. Each symbol indicates the percentage of S. aureus SCV derivatives obtained for a cycle(s) of enrichment.