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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

FH6,7/human Fc mediates human C3 deposition and complement-dependent killing of NTHi NT127. (A) FH6,7/Fc enhances C3 deposition on NT127. Wild-type strain NT127 (127 WT; left graph) and 127 ΔP5 (right graph) were incubated with human complement (C) alone, or C plus either FH6,7/Fc or FH18–20/Fc, each at a concentration of 20 μg/ml. Following incubation at 37°C for 30 min, C3 deposited on bacteria was measured by flow cytometry. “Control” represents the wild-type strain incubated in buffer alone (no C added). X-axis; fluorescence on a log10 scale; Y-axis, counts. Numbers alongside the histograms indicate the median fluorescence intensity of the entire bacterial population; the outline/shading of each value corresponds to that used for the histogram. One representative experiment of two reproducible repeats is shown. (B) FH6,7/Fc enhances C-dependent killing of NTHi NT127. Strain NT127 and its ΔP5 mutant were incubated with 20% C alone, or 20% C plus either FH6,7/Fc or FH18–20/Fc each at a final concentration of 20 μg/ml, and percent survival of bacteria (shown on the Y-axis) was measured in a serum bactericidal assay. Each bar represents the mean (range) of two separate experiment. ANOVA was used to compare the survival of each strain under the three incubation conditions. *P < 0.05; **P < 0.01.
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Figure 2: FH6,7/human Fc mediates human C3 deposition and complement-dependent killing of NTHi NT127. (A) FH6,7/Fc enhances C3 deposition on NT127. Wild-type strain NT127 (127 WT; left graph) and 127 ΔP5 (right graph) were incubated with human complement (C) alone, or C plus either FH6,7/Fc or FH18–20/Fc, each at a concentration of 20 μg/ml. Following incubation at 37°C for 30 min, C3 deposited on bacteria was measured by flow cytometry. “Control” represents the wild-type strain incubated in buffer alone (no C added). X-axis; fluorescence on a log10 scale; Y-axis, counts. Numbers alongside the histograms indicate the median fluorescence intensity of the entire bacterial population; the outline/shading of each value corresponds to that used for the histogram. One representative experiment of two reproducible repeats is shown. (B) FH6,7/Fc enhances C-dependent killing of NTHi NT127. Strain NT127 and its ΔP5 mutant were incubated with 20% C alone, or 20% C plus either FH6,7/Fc or FH18–20/Fc each at a final concentration of 20 μg/ml, and percent survival of bacteria (shown on the Y-axis) was measured in a serum bactericidal assay. Each bar represents the mean (range) of two separate experiment. ANOVA was used to compare the survival of each strain under the three incubation conditions. *P < 0.05; **P < 0.01.

Mentions: Because several medically important microbes bind to similar regions in FH, FH fragments fused to Fc have the potential to serve as anti-bacterial immunotherapeutics. We asked whether FH6,7/Fc could activate complement on NTHi NT127. As shown in Figure 2A, FH6,7/Fc enhanced C3 fragment deposition on the wild-type strain (gray histogram in the left panel) relative to C3 deposited on bacteria incubated either with complement alone (solid line) or complement plus the control protein FH18–20/Fc that bound only weakly to strain NT127 (dashed line). Compared to the wild-type strain, the P5 deletion mutant showed a smaller increase in C3 deposition in the presence of the FH/Fc's and the C3 deposition histograms in the presence of both FH/Fc molecules were similar.


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)

FH6,7/human Fc mediates human C3 deposition and complement-dependent killing of NTHi NT127. (A) FH6,7/Fc enhances C3 deposition on NT127. Wild-type strain NT127 (127 WT; left graph) and 127 ΔP5 (right graph) were incubated with human complement (C) alone, or C plus either FH6,7/Fc or FH18–20/Fc, each at a concentration of 20 μg/ml. Following incubation at 37°C for 30 min, C3 deposited on bacteria was measured by flow cytometry. “Control” represents the wild-type strain incubated in buffer alone (no C added). X-axis; fluorescence on a log10 scale; Y-axis, counts. Numbers alongside the histograms indicate the median fluorescence intensity of the entire bacterial population; the outline/shading of each value corresponds to that used for the histogram. One representative experiment of two reproducible repeats is shown. (B) FH6,7/Fc enhances C-dependent killing of NTHi NT127. Strain NT127 and its ΔP5 mutant were incubated with 20% C alone, or 20% C plus either FH6,7/Fc or FH18–20/Fc each at a final concentration of 20 μg/ml, and percent survival of bacteria (shown on the Y-axis) was measured in a serum bactericidal assay. Each bar represents the mean (range) of two separate experiment. ANOVA was used to compare the survival of each strain under the three incubation conditions. *P < 0.05; **P < 0.01.
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Figure 2: FH6,7/human Fc mediates human C3 deposition and complement-dependent killing of NTHi NT127. (A) FH6,7/Fc enhances C3 deposition on NT127. Wild-type strain NT127 (127 WT; left graph) and 127 ΔP5 (right graph) were incubated with human complement (C) alone, or C plus either FH6,7/Fc or FH18–20/Fc, each at a concentration of 20 μg/ml. Following incubation at 37°C for 30 min, C3 deposited on bacteria was measured by flow cytometry. “Control” represents the wild-type strain incubated in buffer alone (no C added). X-axis; fluorescence on a log10 scale; Y-axis, counts. Numbers alongside the histograms indicate the median fluorescence intensity of the entire bacterial population; the outline/shading of each value corresponds to that used for the histogram. One representative experiment of two reproducible repeats is shown. (B) FH6,7/Fc enhances C-dependent killing of NTHi NT127. Strain NT127 and its ΔP5 mutant were incubated with 20% C alone, or 20% C plus either FH6,7/Fc or FH18–20/Fc each at a final concentration of 20 μg/ml, and percent survival of bacteria (shown on the Y-axis) was measured in a serum bactericidal assay. Each bar represents the mean (range) of two separate experiment. ANOVA was used to compare the survival of each strain under the three incubation conditions. *P < 0.05; **P < 0.01.
Mentions: Because several medically important microbes bind to similar regions in FH, FH fragments fused to Fc have the potential to serve as anti-bacterial immunotherapeutics. We asked whether FH6,7/Fc could activate complement on NTHi NT127. As shown in Figure 2A, FH6,7/Fc enhanced C3 fragment deposition on the wild-type strain (gray histogram in the left panel) relative to C3 deposited on bacteria incubated either with complement alone (solid line) or complement plus the control protein FH18–20/Fc that bound only weakly to strain NT127 (dashed line). Compared to the wild-type strain, the P5 deletion mutant showed a smaller increase in C3 deposition in the presence of the FH/Fc's and the C3 deposition histograms in the presence of both FH/Fc molecules were similar.

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