Fusion Protein Comprising Factor H Domains 6 and 7 and Human IgG1 Fc as an Antibacterial Immunotherapeutic

Abstract

bThe emergence of antimicrobial resistance among several medically important pathogens represents a serious threat to human health globally and necessitates the development of novel therapeutics. Complement forms a key arm of innate immune defenses against invading pathogens. A mechanism of complement evasion employed by many pathogens is binding of complement inhibitors, including factor H (FH), a key downregulator of the alternative pathway. Most FH-binding bacteria engage FH through regions in FH spanned by domains 6 and 7 and/or 18 through 20. We created a chimeric protein that comprised human FH domains 6 and 7 fused to human IgG1 Fc (FH6,7/HuFc) and tested its activity as an immunotherapeutic against Neisseria meningitidis, which binds FH through domains 6 and 7. FH6,7/HuFc bound to meningococci and effectively blocked FH binding to bacteria. FH6,7/HuFc enhanced human C3 and C4 deposition and facilitated complement-mediated killing in a dose-responsive manner; complement activation and killing were classical pathway dependent. To investigate in vivo efficacy, infant Wistar rats were treated intraperitoneally (IP) with different doses of FH6,7/HuFc and challenged 2 h later with serogroup C strain 4243 given IP. At 8 to 9 h after the challenge, the FH6,7/HuFc-treated rats had >100-fold fewer CFU per ml of blood than control animals pretreated with phosphate-buffered saline (PBS) or FH18 -20/HuFc, which does not bind to meningococci (P < 0.0001). These data provide proof of concept of the utility of FH/Fc fusion proteins as anti-infective immunotherapeutics. Because many microbes share a common binding region(s) in FH, FH/Fc chimeric proteins may be a promising candidate for adjunctive therapy against drug-resistant pathogens.

Binding of Complement Factor H to PorB3 and NspA Enhances Resistance of Neisseria meningitidis to Anti-Factor H Binding Protein Bactericidal Activity

“…FH domain 6,7 binds to FHbp (35), to NspA (17), and to some PorB2 amino acid sequence variants (32). In previous studies, we found that recombinant fragments of FH domains 6 and 7 fused to mouse IgG2a or human IgG1 (FH6,7/Fc) bound with higher affinity than fulllength human FH (32,36). Therefore, we used a human FH6,7/ mouse Fc fragment and flow cytometry to probe binding of the double FHbp/NspA KO mutants.…”

Heterogeneity in Rhesus Macaque Complement Factor H Binding to Meningococcal Factor H Binding Protein (FHbp) Informs Selection of Primates To Assess Immunogenicity of FHbp-Based Vaccines

“…The recombinant fragments were similar to those described previously (21) except that mouse IgG2a Fc had been replaced by human IgG1 Fc (22). For inhibition of FH binding, the recombinant fragments (50 g/ml) in Dulbecco's PBS (DPBS) containing 1% bovine serum albumin (BSA) together with macaque serum (1:10 dilution) were incubated with bacteria for 30 min at room temperature.…”

A Novel Factor H–Fc Chimeric Immunotherapeutic Molecule against Neisseria gonorrhoeae

“…Binding of FH/Fc to bacteria and C3 fragments deposited on bacteria were measured by flow cytometry as described previously (35). Data were acquired on a LSRII flow cytometer and data were analyzed using FlowJo software.…”