Previous findings have shown PNAG is an effective antigenic target for protecting animals against S. aureus
and S. epidermidis
infection via elicitation of polyclonal antibodies. Furthermore, there was a superiority in opsonic and protective properties of antibodies that bound well to the backbone epitopes on dPNAG compared with antibodies requiring acetate substituents to bind well to PNAG (18
). In this study we confirmed and extended these findings by showing that a fully human MAb with good binding activity to the backbone epitopes on the dPNAG antigen had better in vitro opsonic killing activity and was more protective in vivo than two other MAbs that bound less well to both native PNAG and dPNAG. Importantly, we found that the better binding of MAb F598 to PNAG and dPNAG was correlated with superior C3 deposition, opsonic killing, and protection, indicating the interrelatedness of these properties for protective antibodies for the PNAG antigen. These in vitro correlates of protective immunity can be used in experiments to help identify improved antibody-based therapeutics and potentially find additive or synergistic combinations of antibodies to target PNAG-expressing pathogens. Of note, we did not find any additive or synergistic activities when we combined the three MAbs studied here in all possible combinations and evaluated them in opsonic killing assays (C. Kelly-Quintos, unpublished observation).
To improve the biologic properties of the initial IgG2 MAbs, we switched isotypes for the cloned V regions to produce human IgG1 MAbs. Interestingly, switching the F598 MAb from IgG2 to the IgG1 subclass gave a modest improvement in C3 deposition, with a concomitant 25% to 30% increase in opsonic killing activity. The F628 IgG2 MAb had a good improvement in both of these characteristics when switched to the IgG1 isotype, but significantly increased killing was achieved only with the highest concentration of MAb tested (25 μg/ml). Of note, the IgG2 isotype of MAb F598 showed two to three times more opsonic killing activity than the IgG1 form of MAb F628 at concentrations of 3 to 12.5 μg/ml, consistent with the increased level of C3 deposition achieved by the IgG2 MAb F598. This result suggests MAb F598 may recognize a densely packed epitope on the PNAG molecule, as it has been shown that IgG2 antibodies have increased complement deposition and opsonic activity when epitope density is high, as is commonly found on carbohydrate molecules (1
). Although changing the F630 MAb from the IgG2 to the IgG1 form also increased complement deposition activity, this increase translated into only marginally better opsonophagocytic activity. Overall, converting the least opsonic F630 MAb into the IgG1 form did not improve the level of complement-fixing activity to that of MAbs F598 or F628, indicating a close relationship between the ability of these MAbs to deposit C3 onto the PNAG antigen and to mediate opsonic killing.
Even though it appeared that MAbs F598 and F628 had some partial epitope overlap, as revealed by competition binding experiments, the overall stronger binding of MAb F598 to the native PNAG and dPNAG antigens was likely a factor in its superior opsonic and protective properties. However, studies using human polyclonal antibodies produced in response to infection showed no effect of affinity on opsonic activity when comparing antibodies with specificity for dPNAG to those that bound optimally to native PNAG (14
). This suggests that good binding to dPNAG, and not overall binding to native PNAG, is an important factor for the most opsonic and protective antibodies to S. aureus
PNAG. Furthermore, when using diphtheria toxoid-conjugated PNAG or dPNAG antigens as active vaccines in animals, the native PNAG conjugate vaccine induced relatively low opsonic titers compared to the dPNAG conjugate vaccine, and all of the opsonic killing activity elicited by the native PNAG conjugate vaccine was inhibited by purified dPNAG antigen (18
). Thus, a consistent picture emerges showing that even when there are antibodies present that bind better to native PNAG versus dPNAG, the opsonically active ones are those that bind to dPNAG. The findings reported here for the relative antigen-binding and immune-effector activities of MAbs F598 and F628 are consistent with this overall conclusion.
Also, consistent with previous findings using polyclonal antibodies (18
), the dPNAG-binding MAb F598 had superior in vivo protective activity. The lowest dose of MAb F598 that we found was needed to achieve significant protection in the mice was ~10 mg/kg of body weight, which is quite comparable to the dose of the humanized MAb Palivizumab given clinically to infants for prophylaxis against respiratory syncytial virus infection. Palivizumab is administered to infants at a dose of 15 mg/kg (11
). Similarly, a humanized MAb to S. aureus
clumping factor A was used at 10 and 30 mg/kg to protect rabbits against consequences of S. aureus
) and this MAb has been evaluated in humans infused with doses of 2, 5, 10, or 20 mg/kg, with linear dose-response kinetics found for the final maximal serum concentration (31
). These results further indicate that our MAbs are effective in mice at doses comparable to those likely to be used in humans. Thus, while it may be possible to improve the affinity or other properties of MAb F598 to make it more potent, based on the in vivo protection studies of mice, it is already in the potency range that is used for MAbs targeted to preventing or adjunctively treating infections in humans.
A possible explanation for the molecular basis of the superior activity of antibodies that bind well to the backbone epitopes on PNAG was recently described (35
). Synthesis of PNAG is dependent on the protein products encoded by the genes in the intercellular adhesin (ica
) locus (5
). The protein encoded by the icaB
gene is responsible for deacetylation of PNAG. It was found that in-frame deletion of the icaB
gene in the ica
locus of both S. aureus
(N. Cerca, K. K. Jefferson, D. B. Pier, T. Maira-Litran, C. Kelly-Quintos, D. A. Goldmann, J. Azeredo, and G. B. Pier, unpublished data) and S. epidermidis
) (where PNAG was referred to as PIA) resulted in production of only fully acetylated PNAG, and this form of PNAG was not retained on the cell surface. Instead, fully acetylated PNAG was all released into the culture supernatant. In contrast, when icaB
was intact, most of the PNAG was retained on the cell surface and the overall level of acetylation was lower than that of the PNAG produced in the absence of functional IcaB (35
). Thus, some amount of deacetylation is required for surface retention of PNAG. Further work showed that overexpression of the IcaB protein in a wild-type strain of S. aureus
resulted in greater surface retention of PNAG and enhanced opsonophagocytic killing of dPNAG-specific antibodies without any effect on the opsonic killing activity of antibodies specific to native PNAG (N. Cerca, K. K. Jefferson, D. B. Pier, T. Maira-Litran, C. Kelly-Quintos, D. A. Goldmann, J. Azeredo, and G. B. Pier, unpublished data). These results indicate that dPNAG-specific antibodies may have an advantage over antibodies that require the presence of the acetate groups on PNAG to bind well to this antigen, based on the superior cell surface association of partially deacetylated PNAG. In addition, the reduction in acetylation that accompanies cell surface binding of PNAG would reduce the density of acetate-dependent epitopes and concomitant binding of antibodies requiring acetate for maximal binding, resulting in a less effective antibody, as was shown here.
Overall, the findings in this study complement previous results regarding the importance of epitope specificity in the human immune response to S. aureus PNAG. The results further support the conclusion that antibodies that bind well to the backbone of PNAG are superior in their ability to kill and protect against S. aureus infection compared with antibodies that require the acetate groups for maximal binding to PNAG. In addition, the fully human MAb to staphylococcal dPNAG has excellent in vitro properties that translate into high levels of in vivo protective efficacy. These are key preclinical findings supporting further development of this reagent as an immunotherapeutic for prophylaxis and possibly for adjunctive treatment of S. aureus infection.