Preclinical Efficacy of Clumping Factor A in Prevention of Staphylococcus aureus Infection

Abstract

Treatment of Staphylococcus aureus infections has become increasingly difficult because of the emergence of multidrug-resistant isolates. Development of a vaccine to prevent staphylococcal infections remains a priority. To determine whether clumping factor A (ClfA) is a good target protein for inclusion in a multivalent vaccine, we evaluated its efficacy in a variety of relevant staphylococcal infection models, challenging with different S. aureus strains. ClfA adsorbed to Alhydrogel and mixed with Sigma Adjuvant System was more immunogenic and stimulated a more robust Th17 response than ClfA administered with alum alone. ClfA immunization induced the production of functional antibodies in rabbits and mice that blocked S. aureus binding to fibrinogen and were opsonic for S. aureus strains that produced little or no capsular polysaccharide. Mice immunized with ClfA showed a modest reduction in the bacterial burden recovered from subcutaneous abscesses provoked by S. aureus USA300 strain LAC. In addition, the ClfA vaccine reduced lethality in a sepsis model following challenge with strain Newman, but not ST80. Vaccination with ClfA did not protect against surgical wound infection, renal abscess formation, or bacteremia. Passive immunization with antibodies to ClfA did not protect against staphylococcal bacteremia in mice or catheter-induced endocarditis in rats. Some enhancement of bacteremia was observed by ClfA immunization or passive administration of ClfA antibodies when mice were challenged by the intraperitoneal route. Although rodent models of staphylococcal infection have their limitations, our data do not support the inclusion of ClfA in an S. aureus multivalent vaccine.

Importance: Antibiotics are often ineffective in eradicating Staphylococcus aureus infections, and thus, a preventative vaccine is sorely needed. Two single-component vaccines and two immunoglobulin preparations failed to meet their designated endpoints in phase III clinical trials. Importantly, recipients of an S. aureus surface protein (iron surface determinant B) vaccine who developed a staphylococcal infection experienced a higher rate of multiorgan failure and mortality than placebo controls, raising safety concerns. Multicomponent S. aureus vaccines have now been generated, and several include surface protein clumping factor A (ClfA). We immunized mice with ClfA and generated a robust T cell response and serum antibodies that were functional in vitro. Nonetheless, ClfA was not protective in a number of relevant animal models of S. aureus infection, and high levels of ClfA antibodies enhanced bacteremia when mice were challenged with community-acquired methicillin-resistant S. aureus strains. Evidence supporting ClfA as a vaccine component is lacking.

FIG 1

ClfA-specific antibody levels in sera from mice immunized on days 0, 14, and 28 with various quantities of ClfAN123, ClfAN23, or BSA, as determined by ELISA at a 1:100 serum dilution. Solid lines depict mice immunized with vaccines adsorbed to alum and then mixed with SAS. Dashed lines depict mice immunized with vaccines adsorbed to alum only.

FIG 2

T cell response in mice immunized s.c. with ClfA or Tris-buffered saline (TBS) administered with alum (A, B, C, and D) or alum mixed with SAS (E, F, G, and H). Lymph node cells from immunized mice were stimulated with either 10 µg/ml ClfAN23 or medium alone for 6 days. IFN-γ (A and E) and IL-17 (B and F) in the culture supernatants were analyzed by ELISA. Antigen-specific responses by CD4⁺ lymph node cells were assessed by ex vivo restimulation followed by flow cytometric analyses of IFN-γ- and IL17-producing T cells. The data presented are pooled from six immunized mice in two independent experiments. Panels C, D, G, and H depict the fold change in the percentage of CD4⁺ T cells producing IFN-γ (C and G) or IL-17 (D and H) in mice vaccinated with TBS versus ClfAN23 and stimulated in vitro with 10 µg/ml ClfA.

FIG 3

Inhibition of S. aureus Newman Δspa mutant strain binding to immobilized Fg by rabbit anti-ClfAN23 IgG (α ClfA) (A) or mouse serum to ClfAN23 (B). Percent binding was calculated by dividing the signal of the antibody-treated S. aureus by the signal of untreated control bacteria.

FIG 4

Opsonic activity of antibodies to ClfA against S. aureus strains: acapsular USA300 strain LAC, Reynolds (CP8), CP8⁺ strain ST80, and CP5⁺ strain Newman. (A) Rabbit IgG to ClfAN23 or ClfAN123 showed similar opsonic activity against S. aureus LAC. The concentration (in micrograms per milliliter) of anti-ClfAN23 antibody (αClfAN23) or anti-ClfAN123 antibody is shown above the bars. no Ab, no antibody; SEM, standard error of the mean. (B) Rabbit IgG to ClfAN23 was opsonic for USA300 LAC, but it showed no opsonic activity against encapsulated S. aureus Reynolds (CP8), PS80, or Newman. IgG specific for CP8-Epa showed opsonic activity for the serotype 8 strains, while the control 2a-Epa antibodies showed no activity. (C) Threefold serial dilutions of sera from mice immunized with ClfAN23 showed opsonic activity for S. aureus that was inversely proportional to capsule production. The opsonic titers for strains LAC, ST80, Newman, and Reynolds (CP8) were 3,240, 360, 40, and <40, respectively. The data are pooled from at least three independent experiments.

FIG 5

Protective efficacy of ClfA in the mouse model of s.c. abscess formation. Mice were actively immunized with 2 µg ClfAN123, ClfAN23, or BSA adsorbed to alum and mixed with SAS. Two weeks after the third immunization, the animals were challenged s.c. with 4 × 10⁵ CFU of S. aureus LAC. (A to C) Number of CFU/abscess (A), abscess weight (B), and mouse weight change (C) were determined 2 days after challenge. The Kruskal-Wallis test showed a significant (P = 0.0306) difference among the three groups in panel A. The values for the BSA and ClfAN23 groups were significantly different (P  = 0.0369) by Dunn’s multiple-comparison posttest and are indicated by a bar and asterisk. Each symbol represents the value for an individual animal, and the median value for each group is indicated by a horizontal line. The lower limit by quantitative culture is indicated by a dotted line. Data are representative of two independent experiments.

FIG 6

Protective efficacy of ClfA immunization in the surgical wound infection model. Mice immunized with 30 µg ClfAN23, ClfAN123, or 2a-Epa (adsorbed with alum) were inoculated with 90 CFU of S. aureus Newman. (A and B) Bacterial burden (A) and weight change (B) were evaluated 3 days after bacterial challenge. Data are representative of three independent experiments. In a separate study, mice actively immunized with 2 µg ClfAN23 or BSA (mixed with alum and SAS) were challenged with 150 to 300 CFU of strain Newman. (C and D) The tissue bacterial burden (C) and weight change (D) were measured on day 3. k. Each symbol represents the value for an individual animal, and the median value for each group is indicated by a horizontal line. The lower limit by quantitative culture is indicated by a dotted line. The data are pooled from two independent experiments.

FIG 7

Protective efficacy of ClfA in mice challenged i.v. with ~10⁸ CFU of S. aureus Newman (8 mice/group) (A) or ST80 (8 to 16 mice/group) (B). Groups of mice were actively immunized with ClfAN23 or BSA mixed with alum plus SAS. Survival was monitored for 14 days, and the results were analyzed by the log rank test. Data in panel B are pooled from two independent experiments. **, P = 0.0048.

FIG 8

Protective efficacy of ClfA in mice challenged i.v. with S. aureus ST80 (2.7 × 10⁷ CFU/mouse) or LAC (1.3 × 10⁸ CFU/mouse). Groups of mice were actively immunized with 2 µg of ClfAN23 or BSA mixed with alum plus SAS. On day 3 after challenge, the bacterial burdens in the kidneys (A and D) and spleens (B and E) and mouse weight loss (C and G) were quantified. Horizontal lines represent group medians, and the lower limit of detection by culture is depicted by dotted lines. The P value for the median values for the two groups in panel A, determined by the Mann-Whitney test, was 0.0648.

FIG 9

Protective efficacy of active immunization with 2 µg ClfAN23 (mixed with alum plus SAS) in the murine bacteremia model. Mice were challenged i.p. with 1.5 × 10⁸ CFU of strain LAC (A) or 6 × 10⁷ CFU of strain ST80 (B). Quantitative blood culture were performed 1 to 1.5 h after challenge. Panel A shows data pooled from two independent experiments. The median values for the two groups in panel A were statistically different (P < 0.01) as indicated by the two asterisks.

FIG 10

Protective efficacy of passive immunization with antibodies to ClfA in the mouse bacteremia model. Mice passively immunized i.v. with 1 mg IgG purified by protein A affinity chromatography from rabbits immunized with ClfAN23 or 2a-Epa. (A to C) The following day, the animals were challenged i.p. with S. aureus LAC (A), ST80 (B), or Newman (C). Data presented in panels A and B are pooled from three and two independent experiments, respectively. In panel C, mice were challenged with the wild-type (wt) strain Newman or its isogenic ΔclfA mutant (DU5852). (D to G) Mice passively immunized with 1 mg IgG to ClfA or 2a-Epa were challenged i.v. with S. aureus LAC. Bacteremia was measured 4 h (D) and 3 days (E) after bacterial challenge, and the bacterial burden in the spleen (F) and kidneys (G) was measured 3 days after challenge. Values for the treatment group that are statistically significantly different from the value for the control group administered 2a-Epa IgG are indicated as follows: **, P < 0.01; ****, P < 0.0001.

FIG 11

Passive immunization with rabbit IgG specific for ClfAN23 or ClfAN123 failed to protect rats against infective endocarditis induced by S. aureus Newman. (A to D) Vegetation weight (A), number of CFU/vegetation (B), kidney bacterial burden (C), and survival (D) were assessed on day 3. (E and F) Bacteremia (E) and weight change (F) were analyzed at 24, 48, and 72 h after bacterial challenge. Horizontal lines represent group medians. The lower limit of detection by culture is indicated by a dotted line. The depicted data are pooled from three independent experiments.