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Defining the Binding Region in Factor H to Develop a Therapeutic Factor H-Fc Fusion Protein against Non-Typeable Haemophilus influenzae.

Wong SM, Shaughnessy J, Ram S, Akerley BJ - Front Cell Infect Microbiol (2016)

Bottom Line: FH18-20/Fc bound weakly to three of the strains but did not promote complement dependent killing.FH6,7/Fc efficiently promoted binding of C3 to NTHi exposed to mouse serum, and intranasal delivery of FH6,7/Fc resulted in significantly enhanced clearance of NTHi from the lung.These results provide evidence for the potential utility of FH6,7/Fc as a therapeutic against NTHi lung infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Mississippi Medical Center Jackson, MS, USA.

ABSTRACT
Non-typeable Haemophilus influenzae (NTHi) cause a range of illnesses including otitis media, sinusitis, and exacerbation of chronic obstructive pulmonary disease, infections that contribute to the problem of antibiotic resistance and are themselves often intractable to standard antibiotic treatment regimens. We investigated a strategy to exploit binding of the complement inhibitor Factor H (FH) to NTHi as a functional target for an immunotherapeutic containing the NTHi binding domain of FH fused to the Fc domain of IgG1. Chimeric proteins containing the regions that most FH-binding bacteria use to engage human FH, domains 6 and 7 (FH6,7/Fc) and/or 18 through 20 (FH18-20/Fc), were evaluated for binding to NTHi. FH6,7/Fc bound strongly to each of seven NTHi clinical isolates tested and efficiently promoted complement-mediated killing by normal human serum. FH18-20/Fc bound weakly to three of the strains but did not promote complement dependent killing. Outer-membrane protein P5 has been implicated in FH binding by NTHi, and FH6,7/Fc binding was greatly diminished in five of seven P5 deficient isogenic mutant strains tested, implicating an alternative FH binding protein in some strains. Binding of FH18-20/Fc was decreased in the P5 mutant of one strain. A murine model was used to evaluate potential therapeutic application of FH6,7/Fc. FH6,7/Fc efficiently promoted binding of C3 to NTHi exposed to mouse serum, and intranasal delivery of FH6,7/Fc resulted in significantly enhanced clearance of NTHi from the lung. Moreover, a P5 deficient mutant was attenuated for survival in the lung model, suggesting that escape mutants lacking P5 would be less likely to replace strains susceptible to FH6,7/Fc. These results provide evidence for the potential utility of FH6,7/Fc as a therapeutic against NTHi lung infection. FH binding is a common property of many respiratory tract pathogens and FH/Fc chimeras may represent promising alternative or adjunctive therapeutics against such infections, which are often polymicrobial.

No MeSH data available.


Related in: MedlinePlus

Binding of FH6,7/Fc (upper panel) and FH18–20/Fc (lower panel) to six additional NTHi isolates and their P5 deletion mutants (ΔP5). Flow cytometry to measure the amount of binding of the two FH/Fc fusion molecules was carried out as described in Figure 1. Axes are as described in Figure 1. Numbers alongside the histograms indicate the median fluorescence intensity of the entire bacterial population; the number in the shaded box represents binding to the wild-type strain. Control reactions represent wild-type bacteria incubated with anti-human IgG FITC (no FH/Fc added). The median fluorescence for the controls was ~5 and has not been indicated in the figure for simplicity.
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Figure 3: Binding of FH6,7/Fc (upper panel) and FH18–20/Fc (lower panel) to six additional NTHi isolates and their P5 deletion mutants (ΔP5). Flow cytometry to measure the amount of binding of the two FH/Fc fusion molecules was carried out as described in Figure 1. Axes are as described in Figure 1. Numbers alongside the histograms indicate the median fluorescence intensity of the entire bacterial population; the number in the shaded box represents binding to the wild-type strain. Control reactions represent wild-type bacteria incubated with anti-human IgG FITC (no FH/Fc added). The median fluorescence for the controls was ~5 and has not been indicated in the figure for simplicity.

Mentions: To ascertain that binding and activity of FH6,7/Fc was not restricted to a single NTHi isolate, we studied six additional NTHi strains and their ΔP5 mutants. All wild-type NTHi strains bound FH6,7/Fc (Figure 3; gray shaded histograms in the upper panel). Rather surprisingly, the P5 deletion mutants of two isolates, 375 and PittG, continued to bind FH6,7/Fc, suggesting that these strains could bind to FH domains 6 and 7 through a molecule distinct from P5. It is worth noting that the P5 deletion mutant of 375 showed an increase in FH6,7/Fc binding compared to the parent strain (1.7- and 1.4-fold increase in fluorescence in two experiments performed).


Defining the Binding Region in Factor H to Develop a Therapeutic Factor H-Fc Fusion Protein against Non-Typeable Haemophilus influenzae.

Wong SM, Shaughnessy J, Ram S, Akerley BJ - Front Cell Infect Microbiol (2016)

Binding of FH6,7/Fc (upper panel) and FH18–20/Fc (lower panel) to six additional NTHi isolates and their P5 deletion mutants (ΔP5). Flow cytometry to measure the amount of binding of the two FH/Fc fusion molecules was carried out as described in Figure 1. Axes are as described in Figure 1. Numbers alongside the histograms indicate the median fluorescence intensity of the entire bacterial population; the number in the shaded box represents binding to the wild-type strain. Control reactions represent wild-type bacteria incubated with anti-human IgG FITC (no FH/Fc added). The median fluorescence for the controls was ~5 and has not been indicated in the figure for simplicity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829610&req=5

Figure 3: Binding of FH6,7/Fc (upper panel) and FH18–20/Fc (lower panel) to six additional NTHi isolates and their P5 deletion mutants (ΔP5). Flow cytometry to measure the amount of binding of the two FH/Fc fusion molecules was carried out as described in Figure 1. Axes are as described in Figure 1. Numbers alongside the histograms indicate the median fluorescence intensity of the entire bacterial population; the number in the shaded box represents binding to the wild-type strain. Control reactions represent wild-type bacteria incubated with anti-human IgG FITC (no FH/Fc added). The median fluorescence for the controls was ~5 and has not been indicated in the figure for simplicity.
Mentions: To ascertain that binding and activity of FH6,7/Fc was not restricted to a single NTHi isolate, we studied six additional NTHi strains and their ΔP5 mutants. All wild-type NTHi strains bound FH6,7/Fc (Figure 3; gray shaded histograms in the upper panel). Rather surprisingly, the P5 deletion mutants of two isolates, 375 and PittG, continued to bind FH6,7/Fc, suggesting that these strains could bind to FH domains 6 and 7 through a molecule distinct from P5. It is worth noting that the P5 deletion mutant of 375 showed an increase in FH6,7/Fc binding compared to the parent strain (1.7- and 1.4-fold increase in fluorescence in two experiments performed).

Bottom Line: FH18-20/Fc bound weakly to three of the strains but did not promote complement dependent killing.FH6,7/Fc efficiently promoted binding of C3 to NTHi exposed to mouse serum, and intranasal delivery of FH6,7/Fc resulted in significantly enhanced clearance of NTHi from the lung.These results provide evidence for the potential utility of FH6,7/Fc as a therapeutic against NTHi lung infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Mississippi Medical Center Jackson, MS, USA.

ABSTRACT
Non-typeable Haemophilus influenzae (NTHi) cause a range of illnesses including otitis media, sinusitis, and exacerbation of chronic obstructive pulmonary disease, infections that contribute to the problem of antibiotic resistance and are themselves often intractable to standard antibiotic treatment regimens. We investigated a strategy to exploit binding of the complement inhibitor Factor H (FH) to NTHi as a functional target for an immunotherapeutic containing the NTHi binding domain of FH fused to the Fc domain of IgG1. Chimeric proteins containing the regions that most FH-binding bacteria use to engage human FH, domains 6 and 7 (FH6,7/Fc) and/or 18 through 20 (FH18-20/Fc), were evaluated for binding to NTHi. FH6,7/Fc bound strongly to each of seven NTHi clinical isolates tested and efficiently promoted complement-mediated killing by normal human serum. FH18-20/Fc bound weakly to three of the strains but did not promote complement dependent killing. Outer-membrane protein P5 has been implicated in FH binding by NTHi, and FH6,7/Fc binding was greatly diminished in five of seven P5 deficient isogenic mutant strains tested, implicating an alternative FH binding protein in some strains. Binding of FH18-20/Fc was decreased in the P5 mutant of one strain. A murine model was used to evaluate potential therapeutic application of FH6,7/Fc. FH6,7/Fc efficiently promoted binding of C3 to NTHi exposed to mouse serum, and intranasal delivery of FH6,7/Fc resulted in significantly enhanced clearance of NTHi from the lung. Moreover, a P5 deficient mutant was attenuated for survival in the lung model, suggesting that escape mutants lacking P5 would be less likely to replace strains susceptible to FH6,7/Fc. These results provide evidence for the potential utility of FH6,7/Fc as a therapeutic against NTHi lung infection. FH binding is a common property of many respiratory tract pathogens and FH/Fc chimeras may represent promising alternative or adjunctive therapeutics against such infections, which are often polymicrobial.

No MeSH data available.


Related in: MedlinePlus