Due to our lack of knowledge as to what constitutes protective human immunity to S. aureus
, it has been difficult to use a rational approach to develop a vaccine. Given the high recurrence rates of S. aureus
infections, particularly in humans colonized with MRSA (4
), the usual means to identify potential effectors of humoral immunity — analysis of antibodies in sera from those convalescing from infection — may not be informative. Using the surface polysaccharides CP5, CP8, and PNAG as vaccines to induce OPK antibody would be a logical choice based on analogy to successful vaccines for other bacterial pathogens (24
Because there is no reliable source of human sera with OPKA to S. aureus
CP or PNAG antigens, we turned to vaccine-induce, antigen-specific animal antisera to study the potential for a synergistic effect between antibodies to S. aureus
CP and PNAG antigens to enhance protective immunity. Individual antisera from mice, rabbits, and goats had potent CP- and/or PNAG-specific OPKA, and the rabbit and goat antibodies had protective efficacy in mouse models of bacteremia and skin infections. Quite unexpectedly, these efficacies were lost when the antibodies to the CP and PNAG antigens were combined or coadministered. Mouse mAbs to CP antigens and a human mAb to PNAG also interfered with each other’s OPKA. The interference between antibodies to CP and PNAG antigens could be attributable to a specific binding interaction of these antibodies, with the most likely explanation being an idiotype–anti-idiotype binding. A similar type of antibody reactivity has been found in HIV-infected patients and suggested to contribute to AIDS-related pathogenesis and autoimmunity due to immune complexes (26
). The binding of antibody to CP and PNAG antigens appears to be mediated, in part, by electrostatic charges between these antibodies, which are raised to oppositely charged antigens, inasmuch as binding could be negated in the presence of 0.3 M NaCl. However, there is an additional specificity to the interaction of antibody to PNAG and staphylococcal CP since antibody raised to the negatively charged alginate antigen of P. aeruginosa
did not bind to the antibody to PNAG.
Expression of both CP and PNAG antigens on the S. aureus surface was not needed for interference, as the concurrent presence of antibodies to both of these antigens was sufficient to inhibit killing of CP-negative S. aureus or PNAG-producing E. coli. Combining antibodies to PNAG and CP antigens also resulted in decreased antibody binding and complement deposition onto the S. aureus surface. Addition of high levels (25–50 μg/ml) of either CP or PNAG antigens or adsorption with high levels of bacterial cells (approximately 1010 CFU) could relieve interference by inhibiting or adsorbing out the competitor antibody. When lower levels of S. aureus cells were used in opsonic assays (2 × 106 CFU) or challenge experiments (1 × 106 to 2 × 106 CFU), this relief did not occur. This is explained by the relative levels of antigens involved in these different settings. In the critical biologic assays — OPK and protection — there is likely insufficient CP or PNAG antigen present to neutralize enough interfering antibody, allowing effective OPK and protection to proceed. Overall, it appears that when antibodies to both CP and PNAG antigens are present at specific levels, there is little monospecific antibody free to bind to the bacterial surface and mediate bacterial killing, the main correlate of protective immunity.
When looking at interference in OPKA in human sera, we found that OPK antibody to S. aureus
surface polysaccharides was virtually absent from sera of normal humans or humans hospitalized with S. aureus
infections other than bacteremia. Even in this latter group of patients, the majority of serum samples from 22 patients with ongoing S. aureus
bacteremia (98 serum samples) plus 10 patients recovering from S. aureus
bacteremia (16 samples) had no OPKA greater than 30% (71 of 114 total samples) due to lack of antibody activity (30 samples) or interference between antibodies to CP and PNAG antigens (41 samples). Notably, the highest level of interference was found in sera from patients recovering from S. aureus
bacteremia, with 11 of 16 (69%) sera from 10 patients having this property. The lower percentage of sera from patients with ongoing bacteremia showing interference may reflect lack of sufficient time after infection for this activity to fully develop. Nine of the 10 patients recovering from S. aureus
bacteremia had at least one sample with either no OPKA or with interference, indicating that following S. aureus
bacteremia when patients actually produce OPK antibodies to surface polysaccharides, virtually no patient made a potentially protective OPK antibody response to these antigens. This is quite distinct from the infected human response to CPs of S. pneumoniae, Haemophilus influenzae
, and Neisseria meningitidis
, which was the basis for development of highly successful conjugate vaccines targeting the surface polysaccharides (24
). Twenty-one of the 32 (66%) bacteremic or convalescing S. aureus
–infected patients had at least one serum sample lacking OPKA due to interference based on the presence of antibodies to both CP and PNAG antigens. Overall, in both normal and S. aureus
–infected human sera, the vast majority of samples studied had surface polysaccharide–specific OPKA of 30% or less due to either lack of CP- or PNAG-specific antibody or interference between these two antibodies when both were present.
Notably, there were some human sera that did not exhibit interference, and even a handful showed augmentation of OPKA when antibodies to both CP and PNAG antigens were present. This indicates it may be feasible to use either active or passive immunization to the CP and PNAG antigens to elicit high serum levels of antigen-specific antibody that would not be subject to interference from responses to the heterologous antigen. However, two clinical trials for preventing bacteremia in hemodialysis patients using CP antigens conjugated to a carrier protein as a vaccine both failed to meet their primary end points (30
), although in one trial (30
) there was efficacy after 40 weeks, as determined by a post hoc analysis, but not at the predetermined 54-week endpoint. The loss of protection over the final 14 weeks was associated with a drop in antibody level to the CP antigens, which may have possibly resulted from development of interfering antibody to PNAG.
Overall, our finding of an interference in opsonic and protective activity between antibodies to PNAG and staphylococcal CP antigens have implications for immunity to S. aureus
, especially as related to vaccine development and insights into the high rates of recurrence of infection due to this pathogen. Clearly, vaccine components must be chosen carefully to avoid interference, which may be engendered not only by vaccine antigens but also by normal bacterial flora, many of which express PNAG antigens (32
). Additionally, the apparent lack of development of effective human immunity to S. aureus
in many situations might partly be explained by our finding both of a general lack of production of polysaccharide antigen–specific OPK antibody in most human sera and of interference between antibodies to CP and PNAG antigens that were present most commonly in sera of patients convalescing from S. aureus
bacteremia. Both the lack of effective opsonic antibody responses to S. aureus
surface polysaccharides and interference between antibodies to PNAG and CP antigens might contribute to the high recurrence rate of infections associated with this pathogen (4