Evaluation of Clumping Factor A Binding Region A in a Subunit Vaccine against Staphylococcus aureus-Induced Mastitis in Mice

The present study evaluated the potential of recombinant binding region A of clumping factor A (rClfA-A) to be an effective component of a vaccine against mastitis induced by Staphylococcus aureus in the mouse. rClfA-A and inactivated S. aureus were each emulsified in Freund''s adjuvant, mineral oil adjuvant, and Seppic adjuvant; phosphate-buffered saline was used as a control. Seven groups of 12 mice each were immunized intraperitoneally three times at 2-week intervals. The titers of IgG and subtypes thereof (IgG1 and IgG2a) in the rClfA-A-immunized group were more than 1,000-fold higher than those in the killed-bacteria-immunized group (P < 0.01). Of the three adjuvants used, mineral oil adjuvant induced the highest antibody levels for both antigens (P < 0.001). Furthermore, the anti-rClfA-A antibody capacities for bacterial adhesion and opsonizing phagocytosis were significantly greater in the rClfA-A-immunized group than in the killed-bacteria-immunized group (P < 0.05). Lactating mice immunized with either rClfA-A or inactivated vaccine were challenged with S. aureus via the intramammary route. The numbers of bacteria recovered from the murine mammary glands 24 h after inoculation were significantly lower in the rClfA-A group than in the killed-bacteria-immunized group (P < 0.001). Histologic examination of the mammary glands showed that rClfA-A immunization effectively preserved tissue integrity. Thus, rClfA-A emulsified in an oil adjuvant provides strong immune protection against S. aureus-induced mastitis in the mouse.Staphylococcus aureus is the most common etiologic agent of contagious bovine mastitis, which results in a decline in milk production, culling of the dairy cow, and increased treatment costs (1, 29). In addition, food-borne S. aureus has become a major public health concern owing to the rapid evolution of resistant lineages (6). A vaccine against S. aureus-induced mastitis in dairy cows would represent a safe and ideal means of preventing and controlling mastitis (27).S. aureus infection is a complex process that involves a series of events, resulting in malfunction or destruction of host tissues. Adherence of the microorganism to host tissues represents a critical first step. Nonadherent bacteria can be readily removed from the host by clearing mechanisms, such as peristalsis and excretion (3, 8). Thus, blocking bacterial adhesion to cells and colonization of the mucosal surface may be the most effective strategy for preventing S. aureus infection (19).S. aureus clumping factor A (ClfA), which is usually covalently anchored to the peptidoglycan of the bacterial cell wall, is an important adhesin and a critical virulence factor. ClfA mediates the binding of S. aureus to fibrinogen on the host cell surface and promotes bacterial invasion into host tissues. An S. aureus clfA mutant displayed reduced virulence in mice (16). Stutzmann et al. (26) showed that introduction of the clfA gene into a less virulent organism, such as Streptococcus gordonii, significantly increased its infective capacity in a mouse endocarditis model. Tuchscherr et al. (30) showed that the passive transfer of antibodies to ClfA protected mice against mastitis. As ClfA does not show genetic polymorphism, it appears to be a suitable component of a novel vaccine against S. aureus infection and the resultant mastitis (2, 23). Josefsson et al. (10) demonstrated that the severity of arthritis was markedly reduced in mice immunized with ClfA. A DNA vaccine that encodes ClfA, as well as the fibronectin-binding motifs of FnBP, delivered twice and boosted once with recombinant fibronectin-binding motifs and ClfA proteins provided partial protection to the mammary gland against staphylococcal mastitis and produced better postchallenge conditions in vaccinated heifers (25). However, a safety concern is that the introduced DNA may be integrated into the host cell chromosomes by insertional mutagenesis (5). To enhance the immune responses induced by ClfA, the potent cytokine interleukin-18 (IL-18) has been used as an adjuvant (32).The fragment of ClfA responsible for its activity lies within binding region A of ClfA (ClfA-A) (14). Hartford et al. (7) localized the fibrinogen-binding activity of ClfA to residues 221 to 559 of region A. Furthermore, the fibrinogen-binding sites P336 and Y338 of clumping factor A are crucial for S. aureus virulence (9). ClfA-A not only promotes bacterial binding to the cell surface but also camouflages S. aureus so as to inhibit phagocytosis (19). In addition, immunization with purified ClfA-A was found to protect against staphylococcus-mediated arthritis (10).In the present study, ClfA-A was expressed and subunit vaccines were prepared as several combinations of recombinant ClfA-A (rClfA-A) and various adjuvants, and these were then evaluated in a BALB/c mouse model of mastitis. The results indicate that a vaccine formulation composed of rClfA-A and an appropriate adjuvant was effective in the prevention of S. aureus-induced mastitis.