In this study, we developed a human fH transgenic mouse model with the intent to investigate the role of this complement down-regulator on infections unique to humans where fH has been proposed to play a role (27
). Human fH, whose amino acid sequence and structure varies from mouse fH, binds uniquely to Neisseria spp.; mouse fH does not. We carried this principle further and investigated the effect of immunization with the Neisseria meningitidis
outer membrane protein vaccine candidate, fHbp. To our knowledge, this study represents the first report of expression in a mouse of full-length human fH to permit fH direct binding, via short consensus repeat (SCR) sequences 6 and 7 (49
), to a vaccine component made from N. meningitidis
. Although the alternative pathway inhibitory action of human fH was not directly investigated in these studies, human fH is known to regulate the non-human alternative pathway of complement similar to its action on the respective human pathway (27
The respective serum antibody responses of the fH transgenic and wild-type mice to the control group C meningococcal conjugate vaccine were similar. These data indicated that the presence of the transgene and/or human fH in the transgenic mice did not impair serum IgG antibody responses to a control capsular polysaccharide or carrier protein antigen, or complement-mediated bactericidal activity of the elicited anticapsular antibodies. The effect of human fH on decreasing immunogenicity of the fHbp vaccine in the human fH transgenic mice, therefore, was specific for the fHbp vaccine antigen, and was observed in two independent studies. Moreover, there was an inverse correlation between the serum bactericidal antibody responses in the transgenic mice immunized with the vaccine that bound human fH and the serum human fH concentrations of individual mice (), which strengthened the case for a causal relationship between human fH and decreased fHbp vaccine immunogenicity. Finally, a mutant fHbp vaccine that did not bind fH, but which, importantly, retained immunogenicity in wild-type mice, elicited higher serum bactericidal antibody titers in transgenic mice than the fHbp vaccine that bound fH. Collectively, these data indicated that binding of the vaccine antigen to this complement protein, fH, impaired the development of protective antibody responses.
The possibility that a mutant fHbp molecule may be a superior vaccine candidate if it did not bind human fH had been suggested by Meri et al. (2
) and Schneider et al. (43
). Based on the crystal structure of fHbp binding to a fragment of fH, Schneider et al. identified two fHbp glutamate residues that were important for fH binding (at positions E218 and E239 based on the numbering of the mature fHbp beginning with the lipidated cysteine residue (22
)). Factor H binding was eliminated when alanine was substituted at these two positions (43
). In a subsequent study, an E218A/E239A mutant fHbp vaccine elicited lower serum bactericidal antibody responses in wild-type BALB/c and CD-1 mice compared to a native fHbp vaccine (30
), a finding that we confirmed in this report. As noted above, residues 218 and 239 are in the region of the fHbp molecule that is also important for eliciting bactericidal antibodies (44
). Thus, some mutations that eliminate fH binding may occur as a result of structural changes and/or alterations of electrostatic charges of the molecule that decrease protective antibody responses.
Several lines of evidence indicate that the fHbp R41S mutant vaccine described in the present study, which does not bind human fH, may be a superior fHbp vaccine for humans. In two studies in wild-type mice the respective serum bactericidal antibody responses elicited by the R41S mutant or wild-type fHbp vaccines were not significantly different. These data indicated that the epitopes required for eliciting bactericidal antibodies were preserved in the R41S mutant when tested in a mouse model in the absence of human fH binding to either vaccine. Second, there were no significant differences in the respective serum bactericidal antibody responses to the two fHbp vaccines in human fH transgenic mice with low human fH concentrations (), compared to elicitation of significantly higher serum bactericidal responses only by the mutant fHbp vaccine when serum human fH concentrations were high, but in a range present in many humans (). Third, despite having elicited similar serum IgG anti-fHbp titers in transgenic mice (), the R41S mutant fHbp vaccine elicited antibodies with greater fH blocking activity than antibodies elicited by the fHbp vaccine that bound fH (). We propose that antibodies elicited by the mutant “fHbp” vaccine that was no longer able to bind fH were directed at surface-exposed epitopes near the fH binding site, thereby inhibiting fH binding and contributing to higher serum bactericidal titers. In support of this hypothesis was a significant correlation between percent inhibition of fH binding to fHbp and serum bactericidal titer (r=0.69, P=0.004, ).
Collectively our data indicate that binding of the human complement regulatory protein, fH, to vaccine antigens can decrease protective antibody responses and that a mutant antigen freed of fH binding, in this case by replacing a large, positively charged amino acid (arginine, pI=11.15) with a smaller, more neutral one (serine, pI=5.68), may be potentially a superior immunogen in humans. Mutant fHbp vaccines that do not bind fH may also avoid the theoretical safety risk of exposure of the host to neo-antigens, in this case in the form of an fH-fHbp immune complex, that may elicit auto-reactive antibodies to fH or fH bound to cell surfaces. Our findings, therefore, may have broad implications in the development of vaccines against microbes that bind other host molecules as well.