Factor H–Igg Chimeric Proteins As a Therapeutic Approach Against the Gram-Positive Bacterial Pathogen Streptococcus Pyogenes

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Factor H−IgG Chimeric Proteins as a Therapeutic Approach against the Gram-Positive Bacterial Pathogen Streptococcus pyogenes This information is current as of September 29, 2021. Anna M. Blom, Michal Magda, Lisa Kohl, Jutamas Shaughnessy, John D. Lambris, Sanjay Ram and David Ermert J Immunol published online 30 October 2017 http://www.jimmunol.org/content/early/2017/10/28/jimmun Downloaded from ol.1700426 http://www.jimmunol.org/ Why The JI? Submit online.

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 30, 2017, doi:10.4049/jimmunol.1700426 The Journal of Immunology

Factor H–IgG Chimeric Proteins as a Therapeutic Approach against the Gram-Positive Bacterial Pathogen Streptococcus pyogenes

Anna M. Blom,*,1 Michal Magda,*,1 Lisa Kohl,*,1 Jutamas Shaughnessy,† John D. Lambris,‡ Sanjay Ram,† and David Ermert*,†

Bacteria can cause life-threatening infections, such as pneumonia, meningitis, or sepsis. Antibiotic therapy is a mainstay of treatment, although antimicrobial resistance has drastically increased over the years. Unfortunately, safe and effective vaccines against most pathogens have not yet been approved, and thus developing alternative treatments is important. We analyzed the efﬁciency of factor H (FH)6-7/Fc, a novel antibacterial immunotherapeutic protein against the Gram-positive bacterium Streptococcus pyogenes.

This protein is composed of two domains of complement inhibitor human FH (FH complement control protein modules 6 and 7) Downloaded from that bind to S. pyogenes, linked to the Fc region of IgG (FH6-7/Fc). FH6-7/Fc has previously been shown to enhance complement- dependent killing of, and facilitate bacterial clearance in, animal models of the Gram-negative pathogens Haemophilus inﬂuenzae and Neisseria meningitidis. We hypothesized that activation of complement by FH6-7/Fc on the surface of Gram-positive bacteria such as S. pyogenes will enable professional phagocytes to eliminate the pathogen. We found that FH6-7/Fc alleviated S. pyogenes– induced sepsis in a transgenic mouse model expressing human FH (S. pyogenes binds FH in a human-speciﬁc manner). Further-

more, FH6-7/Fc, which binds to protein H and selected M proteins, displaced FH from the bacterial surface, enhanced alternative http://www.jimmunol.org/ pathway activation, and reduced bacterial blood burden by opsonophagocytosis in a C3-dependent manner in an ex vivo human whole-blood model. In conclusion, FH-Fc chimeric proteins could serve as adjunctive treatments against multidrug-resistant bacterial infections. The Journal of Immunology, 2017, 199: 000–000.

treptococcus pyogenes, also known as group A Streptococcus, S. pyogenes is exclusively a human pathogen, which may in part is a Gram-positive bacterium that causes a wide spectrum be related to its ability to inhibit human complement by binding to S of diseases. Symptoms range from mild superficial skin the complement inhibitors FH and C4BP in a human-specific manner infections to invasive and life-threatening disease (1, 2). World- (4, 11, 12). Recently, we showed that recruitment of these human wide .700 million S. pyogenes infections occur annually with complement inhibitors is crucial for S. pyogenes virulence in the by guest on September 29, 2021 at least 663,000 new cases of invasive infections (3). To evade mouse model (13). the immune system, S. pyogenes binds a variety of serum pro- The complement system is a pivotal branch of innate immunity teins, including albumin, fibronectin, plasminogen, IgG, comple- against invading pathogens by marking them for removal following ment factor H (FH), and C4b-binding protein (C4BP) (4–10). opsonization with C3b (14). Complement is activated by surface- bound Abs and certain pathogen-associated molecular patterns, which initiate the protein cascade to eliminate bacteria, viruses, *Department of Translational Medicine, Medical Protein Chemistry, Lund Univer- sity, Skane˚ County Council, Malmo¨ 20502, Sweden; †Division of Infectious Diseases fungi, and non-self cells (15). Complement activation can occur and Immunology, University of Massachusetts Medical School, Worcester, MA via three pathways, namely the classical, lectin, and alternative 01605; and ‡Department of Pathology and Laboratory Medicine, University of Penn- sylvania, Philadelphia, PA 19104 pathways. All three pathways converge at the level of the C3 1A.M.B., M.M., and L.K. contributed equally to this work. convertase, resulting in C3b deposition on the target (16). In addition to C3b deposition that promotes opsonophagocytosis, com- ORCIDs: 0000-0002-1348-1734 (A.M.B.); 0000-0003-4857-0769 (M.M.); 0000- 0002-9370-5776 (J.D.L.); 0000-0003-4600-9070 (D.E.). plement activation also releases anaphylatoxins such as C3a and Received for publication March 22, 2017. Accepted for publication September 30, C5a to stimulate the immune system and initiate a response. The 2017. final step of complement activation results in formation of a lytic This work was supported by Swedish Research Council Grant 2016-01142, Swedish pore, called the membrane attack complex, which can directly lyse Government funds for clinical research (ALF), the Torsten So¨derberg Foundation, the Royal Physiographic Society of Lund, the Crafoord Foundation, the Knut and Alice Gram-negative bacteria and eukaryotic cells (17). Therefore, this Wallenberg Foundation, the Lars Hierta Memorial Foundation, the O¨ sterlund Foun- powerful, but potentially detrimental cascade has to be tightly dation, the Tore Nilsson Foundation, King Gustaf V’s 80-Year Fund, and National regulated to prevent unwanted host cell damage (16). In addition Institutes of Health/National Institute of Allergy and Infectious Diseases Grants AI114790, AI118161, AI132296, AI129106 (to S.R.), and AI 068730 (to J.D.L.). to several surface-bound complement regulators, C4BP and FH M.M. was supported by the University of Rzeszow (Biotechnology), Poland. are two of the major soluble complement inhibitors, which protect Address correspondence and reprint requests to Dr. David Ermert, Department of the body’s own cells from unwanted complement activation and Translational Medicine, Division of Medical Protein Chemistry, Lund University, The Wallenberg Laboratory, Floor 4, Inga Marie Nilsson’s Street, 53, Skane˚ Univer- killing (18, 19). sity Hospital, Malmo¨ 20502, Sweden. E-mail address: [email protected] Many bacteria have developed evasion strategies to prevent Abbreviations used in this article: a1AT, a1 antitrypsin; C4BP, C4b-binding protein; immune recognition. One strategy, also used by S. pyogenes as CHO, Chinese hamster ovary; FH, factor H; hFc, human Fc; MAC, membrane attack discussed above, is to recruit the host’s complement regulatory complex; mFc, mouse Fc; NHS, normal human serum. proteins FH and C4BP to the bacterial surface (20, 21). Ideally, Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 these two complement inhibitors should exclusively protect host

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700426 2 FH/Fc ALLEVIATES S. PYOGENES INFECTION cells, but not microbes from complement attack (22). However, assay included rabbit anti-human C4c (Dako), rabbit anti-human C3d bacteria “hijack” these C4BP and FH to their surface (23) to es- (Dako), and goat anti-rabbit IgG conjugated to HRP (Dako). The following cape opsonization and persist in the host (24). For example, Abs used for phagocytosis assay were purchased from BioLegend (unless stated otherwise): anti-human CD56 conjugated to allophycocyanin/Cy7, several bacteria, including S. pyogenes (25), bind FH via domains anti-human CD15 conjugated to PerCP/Cy5.5, anti-human CD19 conjugated 6–7 or 18–20 (reviewed in Ref. 26). Because the complement- to allophycocyanin/Cy7, anti-human MHC class II conjugated to Alexa inhibiting activity of FH is located in domains 1–4, FH bound Fluor 647, anti-human CD11c conjugated to PE, anti-human CD3 conju- to surfaces via domains 6–7 or 18–20 can effectively limit com- gated to allophycocyanin/Cy7, anti-human CD16 conjugated to PE/Cy7, anti-human CD14 conjugated to PE/Dazzle, human TruStain FcX, and plement activation (27). mouse anti–S. pyogenes (AbD Serotec) conjugated to Alexa Fluor 488 and Since the identification of penicillin nearly 90 y ago (28), infectious BV405. Ten micrograms per milliliter of the following Abs was used to diseases have been treated with various antibiotics. Unfortunately, block Fcg receptors (41): anti-human CD16 (no. 16-0166-85; Invitrogen), more and more bacteria exhibit resistance against common antibi- anti-human CD32 (no. LS-C187457; LSBio), and anti-human CD64 (no. 16- otics, and frequently against several antibiotic classes simulta- 0649-85; eBioscience). Compstatin CP40, which blocks C3 activation, was prepared at the University of Pennsylvania as described previously (42). Abs neously (29, 30), which may herald a “postantibiotic era.” New for Western blot analysis included anti-human C3a/C3a desArg (Hycult approaches for antibacterial therapies are urgently needed (31). Biotech) and anti-mouse Ig/HRP (Dako). To achieve this, we designed a chimeric protein consisting of Production of chimeric FH IgG proteins in CHO-S cells domains 6 and 7 of human FH, which are fused to the Fc domain of human IgG1 (FH6-7/human Fc [hFc]). Many pathogens recruit Chimeric proteins (FH6-7/18-20hIgG1, FH6-7/18-20mIgG2a) were expressed human FH via domains 6 and 7 to inhibit complement activation on in CHO cells and purified on protein A/G columns as described previously (34). Briefly, floating FreeStyle CHO-S cells (Life Technologies) at a Downloaded from their surfaces (32). FH6-7/hFc bound to a pathogen is expected to concentration of 1 3 106 cells/ml were transfected with plasmids encoding compete out serum FH from the microbial surface, which would fusion protein constructs in FreeStyle CHO expression medium (Thermo permit uninhibited alternative pathway amplification. Further- Fisher). Transfected floating FreeStyle CHO-S cells were incubated at more, the IgG1 Fc domain of the chimeric protein will activate the 37˚C and 8% CO2 under shaking at 125–130 rpm for up to 12 d. Every classical pathway of complement through interaction with C1q of second day cell medium containing the produced chimeric proteins was collected and stored at 220˚C for subsequent protein purification. Then, the C1 complex. Finally, surface-bound FH6-7/hFc also can be cells were resuspended in fresh FreeStyle CHO expression medium for recognized by Fc receptors (FcgR) on professional phagocytes to further incubation. Proteins within the collected cell supernatant were http://www.jimmunol.org/ initiate opsonophagocytosis. purified on a protein A/G column (GE Healthcare) at 4˚C. The elution of We have previously provided proof-of-concept of efficacy of FH/ the proteins was performed using glycine and 6 M guanidinium chloride. Eluates were transferred into Spectra pore membranes for dialysis in 13 Fc molecules in the treatment of Gram-negative bacterial infections PBS at 4˚C with three buffer exchanges during 24 h. Protein concentration caused by Haemophilus influenzae, Neisseria meningitidis, and was determined using a Cary 50 Bio photometer (Varian). Neisseria gonorrhoeae (33–35). In this study, we examine the efficacy of human FH-Fc chimeric proteins on Gram-positive FH and FH6-7/mFc fusion protein binding to protein H bacterial infections using the example of S. pyogenes. Purified proteins (protein M1, protein H, protein H fragments, and a-1 antitrypsin [a1AT]) were diluted in HEPES buffer (50 mM HEPES,

50 mM NaCl) and immobilized in microtiter plates (MaxiSorp Break- by guest on September 29, 2021 Materials and Methods Apart; Nunc) overnight at 4˚C. Next day, plates were washed three times Bacteria and cell lines with washing buffer (50 mM Tris [pH 8], 150 mM NaCl, 0.1% Tween 20) and unspecific binding was blocked with 5% Difco skim milk (Becton S. pyogenes strains AP1 (S. pyogenes strain 40/58, serotype M1) and AP18 Dickinson) diluted in wash buffer. 125I-FH and 125I-FH6-7/mFc were di- (strain 8/69, serotype M18), both from the World Health Organization luted in HEPES buffer and added in increasing amounts as indicated. After Collaborating Center for Reference and Research on Streptococci (Prague, overnight incubation at 4˚C, plates were washed using washing buffer and Czech Republic), MC25 (lacks M1 protein) (36), BM27.6 (lacks protein H radioactivity was measured using a Wizard2 gamma counter (PerkinElmer and M1) (37), and BMJ71 (lacks protein H and M1, SIC, and C5a pepti- Life Sciences). dase) (38) were grown in Todd–Hewitt broth medium (Oxoid) overnight at 37˚C, 5% CO2 without shaking. Mutant strains were cultured in medium Flow cytometry analysis of C9 deposition on bacteria supplemented with 150 mg/ml kanamycin (MC25), 1 mg/ml erythromycin (BM27.6), or 5 mg/ml tetracycline (BMJ71). Overnight bacteria cultures Harvested bacteria S. pyogenes AP1 or BM27.6 were incubated with 10% were diluted to OD600 of 0.1 in fresh medium and grown with same normal human serum and increasing amounts of FH6-7/hFc or FH18-20/ ++ conditions until exponential phase growth was reached at OD600 of 0.3– hFc, respectively, diluted in GVB buffer (5 mM veronal buffer [pH 7.3], 0.4. After centrifugation and washing with 13 PBS, bacteria were ready to 140 mM NaCl, 0.1% gelatin, 1 mM MgCl2, and 5 mM CaCl2) for 1 h at use. 37˚C, 5% CO2. After incubation, bacteria were washed with 0.5% BSA in FreeStyle Chinese hamster ovary (CHO)-S suspension cells (Life 13 PBS and stained for C9 (goat anti-human C9; CompTech) at 4˚C for Technologies) were used for expression of fusion proteins. Cells were 30 min. After washing with 13 PBS, primary Abs were detected with cultured in FreeStyle CHO expression medium (Thermo Fisher) supple- secondary anti-goat Abs conjugated to Alexa Fluor 647 (Jackson mented with 8 mM L-glutamine (HyClone) at 37˚C, 8% CO2, 130 rpm ImmunoResearch Laboratories). The amount of deposited C9 component shaking. bound to bacteria surface was measured using CyFlow space flow cytometer (Partec). Proteins and Abs Complement deposition Protein M1, protein H, and its fragments were expressed in Escherichia coli and purified on a human IgG column as described previously (5, 37, 39). Microtiter plates (MaxiSorp BreakApart; Nunc) were coated with FH6-7/ FH was purified from human plasma. Aggregated human IgG was obtained hFc (10 mg/ml), a1AT (10 mg/ml), and aggregated human IgG (classical by heat aggregation of Igs (Kiovig, Baxalta) at 65˚C. Zymosan and cy- pathway, 5 mg/ml; Kiovig, Baxalta) or zymosan (alternative pathway, 10 tochalasin D were purchased from Sigma-Aldrich. FH and FH6-7/mouse mg/ml, Z-4250; Sigma-Aldrich). The plates were incubated overnight at Fc (mFc) fusion protein were radioactively labeled using Iodo-Beads 4˚C and washed the next day three times with washing buffer (50 mM Tris (Pierce) and Na-125I (PerkinElmer Life Sciences) according to the manu- [pH 8], 150 mM NaCl, 0.1% Tween 20). Between every step, plates were facturers’ protocols. Abs used for flow cytometric analysis included goat washed thrice with washing buffer. Further unspecific binding was blocked anti-human C9 (CompTech), donkey F(ab9)2 anti-goat IgG conjugated to with 5% Difco skim milk (Becton Dickinson) diluted in wash buffer Alexa Fluor 647 (Jackson ImmunoResearch Laboratories), MRC OX-24 (classical pathway) or quenching buffer (3% fish gelatin, 50 mM Tris-HCl, anti-human FH conjugated to DyLight 650 (40), goat anti-mouse IgG 150 mM NaCl, 0.1% Tween 20 [pH 8]; alternative pathway). The next conjugated to Alexa Fluor 647 (Invitrogen), goat anti-human IgG conju- plate was incubated with increasing concentrations of normal human se- gated to Alexa Fluor 647 (Invitrogen), and goat anti-human IgG conju- rum diluted in GVB++ (5 mM veronal buffer [pH 7.3], 140 mM NaCl, 2+ gated to Alexa Fluor 488 (Invitrogen). Abs used for complement deposition 0.1% gelatin, 1 mM MgCl2, and 5 mM CaCl2; classical pathway) or Mg The Journal of Immunology 3

EGTA (2.5 mM veronal buffer [pH 7.3] containing 70 mM NaCl, 140 mM incubation, RBCs were lysed using RBC lysis/fixation solution (Bio- glucose, 0.1% gelatin, 7 mM MgCl2, and 10 mM EGTA; alternative Legend) with subsequent incubation for 15 min at room temperature. After pathway). Serum incubation time was 20 min for C3b, 15 min for C4b washing with PBS, cells were analyzed using Amnis FlowSight. (classical pathway), and 30 min for C3b (alternative pathway) at 37˚C. After incubation, deposited components were detected with primary anti- Surface plasmon resonance analysis C3d (Dako) (classical pathway, alternative pathway) and anti-C4d (Dako) The affinity between protein H and FH6-7/hFc was analyzed by surface (classical pathway) diluted in corresponding blocking buffer. Then, bound plasmon resonance using a Biacore 2000 (GE Healthcare). FH-IgG was primary Abs were detected with secondary anti-rabbit HRP-conjugated immobilized on a CM5 sensor chip (GE Healthcare) using standard amino Abs. Finally, samples were developed using OPD tablets (Kem-En-Tec coupling reaction to reach 2500 resonance units. Protein H diluted to 0.07– Diagnostics) and signal was measured at OD (Cary 50 MPR micro- 490 25 nM concentrations in running buffer (50 mM HEPES [pH 7.8] con- plate reader; Varian). taining 100 mM NaCl and 0.005% Tween 20) was injected at flow of 30 Hemolytic assay ml/s for 100 s and the dissociation was then followed for 300 s. The signal from the control surface was subtracted. In all experiments, two consec- For classical pathway activation, 150 ml of sheep erythrocytes (Hatunalab;˚ utive injections of 2 M NaCl, 100 mM HCl were used to remove bound corresponding to ∼3.75 3 108 RBCs) was washed three times with GVB++ ligands during a regeneration step. The BiaEvaluation 3.0 software (Bia- buffer (5 mM veronal buffer [pH 7.3], 140 mM NaCl, 0.1% gelatin, 1 mM core) was used to determine affinity constants using a Langmuir 1:1 in- ++ MgCl2,and5mMCaCl2) and incubated with Amboceptor (1:500 in GVB ; teraction model with drifting baseline. The experiment was performed two Dade Behring) for 20 min at 37˚C with 650 rpm shaking. After incubation, independent times. erythrocytes were washed three times and resuspended in GVB++.The suspension volume was adjusted in the way that 10 ml from erythrocyte stock Platelet aggregation assay lysed in 90 of H O resulted in OD of 1–1.2. For the alternative pathway, 2 410 Platelet aggregation was analyzed from three independent donors, as 150 ml of rabbit erythrocytes (Hatunalab)˚ was washed three times and sus- recommended by the International Society on Thrombosis and Haemostasis Downloaded from pended in Mg-EGTA buffer (2.5 mM veronal buffer [pH 7.3] containing (43). Briefly, plasma was purified from healthy volunteers and platelet-rich 70 mM NaCl, 140 mM glucose, 0.1% gelatin, 7 mM MgCl ,and10mM 2 plasma and platelet-poor plasma were prepared. After mixing platelet-poor EGTA). Then, the suspension volume was adjusted in the same way as for plasma and platelet-rich plasma together with 2 mM ADP (positive con- sheep erythrocytes. To assess hemolytic activity, sheep erythrocytes were trol), FH6-7/FH, or PBS (negative control), samples were analyzed in a incubated with increasing amounts of FH6-7/hFc diluted in GVB++ buffer for light transmission aggregometer. Additionally, one sample was left un- 20 min. After incubation, cells were washed and subsequently mixed with treated before analysis (spontaneous aggregation, additional negative con- 0.75% normal human serum (NHS) to allow complement activation for trol). OD was measured and plotted as percentage. Area under the curve 10 min. Remaining erythrocytes were pelleted and hemolysis was measured http://www.jimmunol.org/ analysis revealed the total amount of platelet aggregation. at OD410 (Cary 50 MPR microplate reader; Varian). Similarly, rabbit erythrocytes were incubated with increasing amounts of FH6-7/hFc fusion protein C3a Western blot diluted in Mg-EGTA buffer. After washing and mixing with 6% NHS, complement was allowed to be activated on erythrocytes for 15 min. Finally, Lepirudin-treated plasma was incubated either with 2 mg/ml zymosan erythrocytes were centrifuged and released hemoglobin was measured at particles, PBS, or 50 mg/ml FH6-7/hFc for 1 h at 37˚C with shaking. OD410. Particles were removed and supernatants were applied on a 15% SDS- All incubations were performed at 37˚C and 650 rpm shaking (Ther- polyacrylamide gel. Proteins were separated by SDS-PAGE, transferred moMixer; Eppendorf). Hemolytic assay using anti-CD59–treated human to a polyvinylidene difluoride membrane, and subsequently incubated with erythrocytes has been performed as described previously (35). mouse anti-human C3a followed by an incubation with goat anti-mouse Ig- HRP. The C3a signal was visualized using ECL (Millipore) and detection Binding assay on bacteria using FACS with a CCD camera (Chemi-Doc; Bio-Rad). Owing to a very strong signal by guest on September 29, 2021 in the positive control in the initial experiment, the zymosan-treated S. pyogenes strains AP1, MC25, BM27.6, and BMJ71 (1 3 107 CFU) in sample was diluted 10-fold prior to electrophoresis in the experiment GVB++ were coincubated with indicated concentrations of chimeric FH shown. proteins in the presence of 10% FCS at 37˚C, 5% CO2 for 1 h. Bacteria were harvested and washed with 13 PBS. Subsequently, bacteria were Animal survival studies stained with CellTrace Calcein Violet (Molecular Probes) and stained with goat anti-human IgG–Alexa Fluor 488 (Invitrogen; to detect FH–human All animals were housed and bred under specific pathogen-free conditions IgG1 proteins) or goat anti-mouse IgG–Alexa Fluor 647 (Invitrogen; to in the animal facility at the University of Massachusetts Medical School detect FH–mouse IgG2a proteins) for 30 min. Thereafter, bacteria were (Worcester, MA). washed twice with 13 PBS and resuspended in 13 PBS. Binding of fusion Two hours prior to infection, female and male animals were treated either proteins to bacterial surface was measured using CyFlow space flow with 50 mg of FH6-7/hFc or 50 mg of goat IgG i.p. Subsequently, via cytometer (Partec), and geometric mean fluorescence intensity was cal- lateral tail vein injection, animals were infected i.v. with 100 ml of bac- culated with FlowJo software (Tree Star). To assess serum FH binding, terial suspensions in PBS containing 1.4 3 108/ml S. pyogenes AP1. In- bacteria were similarly treated, but incubated in 10% NHS and stained jections were repeated on days 2, 4, and 6 (50 mg of protein per animal). with MRC OX-24 anti-human FH conjugated to DyLight 650. To identify All animals were closely monitored for signs of disease for up to 8 d; bacteria, CellTrace Violet–positive events were gated and further analyzed. gravely moribund mice were euthanized. Whole-blood killing assay Ethical statement FH6-7/hFc was added to lepirudin (Refludan; Celgene)-treated human Use of animals in this study was performed in strict accordance with the blood in different concentrations before infection with indicated amounts of recommendations in the Guide for the Care and Use of Laboratory different S. pyogenes strains. Complement activation was blocked at the Animals of the National Institutes of Health and the Swedish Animal level of C3 with 20 mM compstatin CP40. Fcg receptors were blocked with Welfare Act SFS1988:534. Experiments were approved by the Institu- a combination of 10 mg/ml each of anti-human CD16, CD32, and CD64 tional Animal Care and Use Committee at the University of Massachusetts that were added to whole blood and incubated for 20 min at 37˚C before Medical School. adding bacteria. Following the addition of bacteria, the blood was incubated on an end-over-end shaker at 37˚C, 5% CO2. At indicated time Statistical analysis points, 50 ml of the mixture was diluted serially in TBS and plated on Statistical analysis was performed using GraphPad Prism 6.0 and 7.0b blood agar plates. Blood agar plates were incubated overnight at 37˚C and software. To test for significance, we used either a one-way or two-way 5% CO to enumerate surviving S. pyogenes. 2 ANOVA analysis with a Bonferroni, Dunnett, or Tukey posttest or a , Phagocytosis assay using Amnis FlowSight Mantel–Cox test as indicated. A p value 0.05 was considered to be significant. Harvested bacteria were stained with CFSE and subsequently subcultured for 2 h to reduce the CFSE intensity per bacteria. Human blood (200 ml) was infected with 1 3 107 bacteria and incubated for 30 min at 37˚C and 5% Results CO2. Next, blood samples were treated with human TruStain FcX to block We analyzed binding of FH domains 6 and 7 coupled to human Fc receptors for 10 min on ice prior to addition of Abs. After 30 min IgG1 Fc (FH6-7/hFc; Fig. 1A) to S. pyogenes AP1 and its isogenic 4 FH/Fc ALLEVIATES S. PYOGENES INFECTION mutants MC25 (lacks the M protein), BM27.6 (lacks protein H), determine whether the Fc portion of FH6-7/hFc could activate and BMJ71 (lacks both M protein and protein H). FH6-7/hFc complement, we coated microtiter plates with the fusion protein, bound similarly to the parental wild-type strain AP1 and MC25 aggregated IgG as a positive control, and a1AT as a negative (Fig. 1B). Binding to the mutant BM27.6, as measured by fluo- protein control. Indeed, FH6-7/hFc increased both C3b (Fig. 2E) rescence intensity, was diminished almost 2-fold compared with as well as C4b deposition (Fig. 2F) on the plate similar to ag- the wild-type strain. Bacteria lacking both surface virulence gregated IgG, indicating that FH6-7/hFc activates the alternative factors, M/H proteins, BMJ71 showed binding barely above and classical pathways. To address the possibility that comple- background levels. These results indicate that FH6-7/hFc binds ment activation in solution may have contributed to complement to both M protein and protein H on S. pyogenes. Prior work has component deposition on “bystander” bacteria, we incubated suggested S. pyogenes also binds FH through domains18–20 (44, plasma for 60 min with either FH6-7/hFc, PBS, or zymosan 45). We observed binding of FH18-20/hFc to the wild-type AP1 particles and measured C3a generation as a measure of com- and, to a lesser extent, to MC25 (Fig. 1C). Both BM27.6 and plement activation. Plasma samples were subjected to Western BMJ71 did not bind FH18-20/hFc, suggesting that M protein and blot analysis for C3a (Fig. 2G). Zymosan strongly activated protein H may both contribute to binding of the C-terminal do- complement, resulting in high amounts of C3a production, mains of FH. whereas PBS and FH6-7/FH showed similar, but very weak Because S. pyogenes is known to bind human, but not mouse, signals. Taken together, FH6-7/hFc activates complement only IgG-Fc (39), we employed FH6-7 and FH18-20 fused to mouse when bound to the bacterial surface, but not when in solution. IgG2a to determine whether binding of FH6-7/hFc to AP1 is mediated mainly through FH or through human IgG-Fc domains FH binding site on S. pyogenes Downloaded from of the fusion proteins tested. As seen with FH6-7/hFc, AP1 and Initial binding experiments indicated that protein H and M protein MC25 bound FH6-7/mFc well, whereas BM27.6 and BMJ71 were both ligands for FH6-7 (Fig. 1B). To exclude the influence of bound reduced amounts or no FH6-7/mFc, respectively, indicating human IgG-Fc, we used 125I-labeled FH6-7/mFc to test binding of that binding of FH6-7/hFc occurred through the FH domains the FH domains to protein H, M1, and a1AT (Fig. 3A). FH6-7/ (Fig. 1D). However, we barely detected any binding of FH18-20/ mFc bound in a dose dependent-manner to protein H, slightly less

mFc to the bacteria (Fig. 1E). These results indicate that FH18-20/ to M1, but not to the negative control a1AT. http://www.jimmunol.org/ hFc bound almost exclusively through the human IgG-Fc part of Because we identified protein H as one of the major ligands for the protein, whereas FH6-7 presumably binds via both domains, FH6-7/hFc, we sought to identify the binding site on protein H for that is, FH6-7 as well as hIgG1-Fc. Using surface plasmon reso- FH domains 6 and 7 (Fig. 3B). We found that full-length protein H nance analysis, we estimated the binding affinity of protein H to bound most FH6-7/mFc, whereas protein H fragments Δ267–376 29 FH6-7/hFc to be KD of 5.2 3 10 M (Fig. 1F) and FH6-7/mFc to and Δ246–376 (see Fig. 3B, insert) still bound significantly 29 be KD of 3.7 3 10 M (Fig.1G), indicating a strong interaction more FH6-7/mFc than did the irrelevant protein control a1AT, for both chimeric proteins. yet markedly decreased compared with full-length protein H (Fig. 3B). Thus, we concluded that the binding site for FH6-7/mFc FH6-7/hFc competes out serum FH from the bacterial surface is located in domains C (mainly C3) and D of protein H. by guest on September 29, 2021 and increases complement attack Similarly, we used 125I-radiolabeled FH to compare binding to Next, we tested whether FH fusion proteins were able to compete M1 and protein H. Similar to FH6-7/mFc, we found that FH binds out serum FH on the surface of S. pyogenes. Therefore, we in- both protein H as well as M protein (Fig. 3C). Using the protein cubated the bacteria in 5% NHS that contained increasing con- H fragments, we detected significant binding of 125I-FH to full- centrations of FH6-7/hFc (Fig. 2A). Even 2.5 mg/ml FH6-7/hFc length protein H and fragments Δ267–376 and Δ246–376 as well was sufficient to completely block FH binding to the bacterial as Δ145–376. This binding pattern suggests that full-length FH surface of AP1 and MC25. BM27.6 and BMJ71 did not bind any binds to domains C and D, as well as domain B, of protein H FH from serum. In contrast, FH18-20/hFc did not displace any (Fig. 3D). serum FH from AP1 or MC25 (Fig. 2B). Even high concentrations (150 mg/ml) of FH18-20/hFc had no effect on serum FH binding FH6-7 does not affect hemolysis or platelet aggregation (data not shown). These data provide further evidence for binding Unwanted displacement of FH from tissues and cells could be of FH6-7/hFc via the FH domains, in contrast to binding of FH18- detrimental for the body. Therefore, we asked whether FH6-7/hFc 20/hFc via the Fc domain. affected hemolysis of erythrocytes. To determine whether FH6-7/ We hypothesized that FH-IgG binding to S. pyogenes would hFc augmented classical pathway activation, we sensitized sheep increase complement attack through enhanced classical pathway erythrocytes with Abs and subsequently incubated these with FH6- activation and opsonization by the Fc part of the chimeric pro- 7/hFc. Before addition of serum, cells were washed to assess the tein, and also by preventing FH-mediated inhibition of comple- function of only erythrocyte-bound fusion protein. We did not ment activation. To confirm our hypothesis, we analyzed C9 detect any difference in hemolytic activity at any tested FH6-7/hFc deposition on S. pyogenes AP1 from 10% NHS in the presence of concentration. Similarly, we used nonsensitized sheep erythrocytes increasing amounts of the chimeric proteins; multiple C9 mol- to identify whether FH6-7/hFc could directly sensitize the eryth- ecules participate in the formation of the membrane attack rocytes. Again, there was no increase in hemolysis compared with complex (MAC), which is the final stage in complement acti- background hemolysis (Fig. 3E, left side). These data show that vation. We found that FH6-7 significantly increased C9 deposi- FH6-7/hFc had no effect on classical pathway–mediated hemo- tion on AP1 compared with NHS alone (Fig. 2C). In contrast, lysis. To analyze the effect of the fusion protein on the alternative FH18-20/hFc did not increase C9 deposition on the bacterial pathway, we used rabbit erythrocytes, which were incubated with surface. As expected, neither fusion protein had any impact on increasing amounts of FH6-7/hFc. Erythrocytes were washed to BM27.6 (Fig. 2D), because this mutant does not bind any FH remove unbound FH6-7/hFc. As expected, the fusion protein had domains. Consistent with uninhibited complement activation on no influence on the hemolysis of rabbit erythrocytes, suggesting its surface, high MAC formation occurred on the surface of that it does not compete with FH in this experimental system BM27.6 and was not altered by addition of a fusion protein. To (Fig. 3E, right side). The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. FH domains 6–7 but not 18–20 bind to S. pyogenes AP1 proteins H and M. (A) Schematic representation of FH6-7/Fc and FH18-20/Fc. Human FH domains were fused to Fc of either human IgG1 or mouse IgG2a. Binding of the different fusion proteins to S. pyogenes AP1 and isogenic mutants lacking M protein (MC25), protein H (BM27.6), or both M protein and protein H (BMJ71) was tested using ﬂow cytometry. (B) Binding of FH6-7/ hFc to S. pyogenes AP1 and isogenic mutants lacking either protein H and/or M protein. (C) FH18-20/hFc binds only to bacteria expressing protein H (AP1 and MC25) but not the two mutants lacking protein H (BM27.6 and BMJ71). (D) FH6-7/mFc binds predominantly to AP1 and MC25, both expressing protein H. (E) FH18-20/mFc does not bind to any of the tested bacteria. Biacore analysis revealed very strong binding between protein H and FH6-7/hFc 29 29 or FH6-7/mFc with KD of 5.2 3 10 M(F) and KD of 3.7 3 10 M(G), respectively. Means (6SD) from at least three independent experiments are shown (B–E).

FH is the major protective molecule on human erythrocytes contrast, FH18-20/Fc, which is known to induce hemolysis, served where CD59 function is blocked, for example with anti-CD59 Abs as a positive control (35). Taken together, our experiments show (46). FH6-7/hFc did not induce hemolysis of anti-CD59–treated that FH6-7/hFc does not cause complement-dependent lysis of human RBCs (Fig. 3F) at any of the tested concentrations. In erythrocytes. 6 FH/Fc ALLEVIATES S. PYOGENES INFECTION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. FH6-7/hFc competes out serum FH and activates complement. Bacteria were incubated in serum in increasing amounts of FH6-7/hFc (A)or FH18-20/Fc (B) and analyzed for binding of serum FH. (C) Flow cytometry was used to measure surface-bound C9, the ﬁnal step of complement activation. FH18-20 did not increase C9 on AP1. (D) Complement C9 deposition was not affected by FH6-7/hFc or FH18-20/hFc on BM27.6 that does not bind either molecule. FH6-7/hFc immobilized on microtiter wells activates the classical and lectin pathways as measured by C3b (E) and C4b (F) deposition. (G) Plasma was incubated with FH6-7/hFc, PBS, or zymosan and analyzed for C3a generation as a sign of complement activation. Means (6SD) from at least three independent experiments are shown. Statistical signiﬁcance of differences was calculated using a two-way ANOVA with a Dunnett posttest [overall p value in (C) was .0.0001], *p , 0.05, **p , 0.01, ****p , 0.0001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Identiﬁcation of binding sites for FH on S. pyogenes AP1. (A) Both protein H and M bind FH6-7/mFc. Increasing amounts of 125I-FH6-7/ mFc were incubated with immobilized protein M1, protein H, and as a a1AT (negative control). (B) The binding site for FH domains 6–7 is mainly located in domains C3 and D of protein H (see insert). Immobilized full-length protein H as well as the indicated fragments of protein H were incubated with 125I-FH6-7/hFc, (C) Full-length FH binds to M protein and protein H. 125I-FH was tested for binding to immobilized proteins H and M1. (D) Full-length FH also binds to protein H via domains C3 and D. (E) FH6-7/hFc does not alter hemolysis of erythrocytes through classical or alternative pathways. (F) Human anti-CD59–treated RBCs are lysed by FH18-20/Fc, but not by FH6-7/Fc. (G) FH6-7/Fc does not induce platelet aggregation. Statistical signiﬁcance of differences was calculated using a one-way ANOVAwith a Dunnett posttest [overall p value in (B) and (D) was p , 0.0001] or a Bonferroni posttest [overall p value in (G) was ,0.0001], *p , 0.05, ***p , 0.001, ****p , 0.0001.

To verify that FH6-7/hFc does not affect coagulation, we per- untreated sample was used to measure spontaneous activation. A formed a platelet aggregation assay (Fig. 3G). Serum from three comparison of the area under the curves of the different reaction healthy individuals was analyzed by either adding FH6-7/hFc, mixtures revealed that FH6-7/hFc did not activate platelets beyond PBS as a negative control, or ADP as a positive control. A fourth levels seen with PBS and “spontaneous activation” tubes. 8 FH/Fc ALLEVIATES S. PYOGENES INFECTION

FIGURE 4. PMNs show increased uptake of bacteria in the presence of FH6-7/hFc. Nonopsonized S. pyogenes AP1 was incubated in human blood in the presence and absence of FH6-7/hFc. Leukocytes were stained and analyzed using Amnis FlowSight technology. (A) PMNs with ingested bacteria were

discriminated from extracellular bacteria. (B) FH6-7/hFc significantly increased phagocytosis rate of bacteria. PMNs treated with cytochalasin D served as a Downloaded from negative control. Means from five individual donors (indicated by color) are shown. Statistical significance of differences was calculated using a matched one-way ANOVA with a Tukey posttest [overall p value in (B) was ,0.0063], **p , 0.01.

Bacterial uptake in human blood increases upon addition of expected, bacteria unable to bind FH6-7/hFc were not affected and FH6-7/hFc CFU counts were similar in treated and untreated samples at all http://www.jimmunol.org/ We infected human blood from five healthy individuals ex vivo time points (Fig. 5C, 5D). This shows that FH6-7/hFc is effective with S. pyogenes AP1 and subsequently stained for professional only against bacteria that bind FH via domains 6–7. phagocytes to measure uptake of bacteria. Using Amnis FlowSight Complement activation is required for opsonophagocytic technology, we discriminated between bacterial uptake and ad- killing by FH6-7/hFc hesion (Fig. 4A). We excluded all cells with extracellular bacteria We sought to delineate the mechanism whereby FH6-7/hFc in- (Fig. 4A, “excluded cells” no. 78784 and no. 81540) and analyzed creases uptake and killing of group A streptococci. Because phago- the CFSE signal in the PMN population. We observed PMNS cytosis is mainly mediated either via Fcg receptors or complement without phagocytosed bacteria (Fig. 4A, no. 8074 and no. 33332) opsonization, we performed a blood killing assay in the presence as well as PMNS that phagocytosed one (no. 16415), two (no. of FH6-7/hFc where Fcg receptor, complement C3 activation, or by guest on September 29, 2021 40855), three (no. 83010), or more than three (no. 84420) bacteria, both were blocked (Fig. 5E). Blocking opsonization with C3 judged by the CFSE signal originating from the bacteria. fragments using compstatin led to a significant increase of bac- In total, 40.4% of all neutrophils in blood ingested S. pyogenes terial survival (∼1 log10 decrease in CFU at each time point in the absence of FH6-7/hFc (Fig. 4B, individual donors color tested). Surprisingly, blocking Fcg receptors alone did not change matched). Upon addition of the fusion protein, the phagocytosis bacterial blood burden, suggesting that complement activation was rate increased in the individual samples between 6.4 and 16.4– essential for FH6-7/hFc-mediated opsonophagocytic killing in 52% on average. Addition of cytochalasin D completely blocked whole human blood. phagocytosis and confirmed that we had measured bacterial uptake and not merely adhesion to neutrophils. FH6-7/hFc alleviates experimental S. pyogenes sepsis in mice To show in vivo efficiency of the fusion protein, we infected human FH6-7/hFc prevents bacterial growth in blood FH transgenic animals with 1.4 3 107 S. pyogenes AP1. S. pyogenes To assess the effect of FH6-7/hFc on bacteria during sepsis, we binds only human, but not mouse, FH. Therefore, use of human infected human blood with S. pyogenes AP1 in the presence or FH transgenic mice provided a complement-inhibiting barrier that absence of FH6-7/hFc. Bacterial growth was analyzed during a FH6-7/hFc may encounter in humans. Animals treated with 50 mg period of 3 h (Fig. 5A). In the presence of 50 mg/ml FH6-7/hFc of FH6-7/hFc every other day showed significantly reduced (red bars), bacterial burden was reduced by .60% within the first mortality compared with the control group receiving control goat 60 min. During the next 2 h the bacterial numbers remained stable IgG (Fig. 6). Four out of ten animals treated with the fusion (static) whereas untreated bacteria replicated impressively; after protein survived the infection, whereas all animals receiving 3 h, the difference between the untreated sample and the samples control goat IgG died within 5 d. These data provide evidence that treated with 50 mg/ml FH6-7/hFc was .1.5 log10. Treatment with FH6-7/hFc reduces bacterial burden in blood and increases sur- 10 mg/ml FH6-7/hFc (gray bars) showed significantly reduced vival of S. pyogenes–infected animals. bacterial burden compared with untreated controls, but the de- Taken together, our data suggest that FH6-7/hFc blocks FH crease was not as pronounced as in samples with 50 mg/ml (red binding to bacteria, leading to increased complement deposition, bars). In fact, almost 1 log10 bacterial growth occurred during 3 h enhanced phagocytosis, and impaired bacterial survival (Fig. 7). in the presence of the low dose. Similarly, blood infected with S. pyogenes AP18 and treated with FH6-7/hFc showed similar Discussion reduction in bacterial burden over time. The effect was even more In this study, we demonstrate efficacy of a FH-IgG fusion protein pronounced using a low level (10 mg/ml) of FH6-7/hFc. The against S. pyogenes infections, thus expanding their utility against difference between treated and untreated blood was ∼2 log10 Gram-positive bacteria that bind FH. FH-Fc chimeric proteins bacteria/ml at 2 or 3 h after addition of bacteria (Fig. 5B). As could be further developed as a new class of antibacterial proteins, The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Complement is important for FH6-7/hFc to reduce bacterial blood burden. Human blood was infected with 2 3 105 CFU/ml S. pyogenes AP1 (A) or AP18 (B), 2 3 106 CFU/ml S. pyogenes BM27.6 (C), or 2 3 107 CFU/ml S. pyogenes BMJ71 (D) treated with indicated amounts of FH6-7/hFc and incubated for up to 3 h. FH6-7/hFc significantly reduces the number of bacteria compared with untreated samples for both bacterial strains binding the fusion protein [AP1 (A) and AP18 (B)]. FH6-7/hFc had no influence on S. pyogenes BM27.6 (C) or BMJ71 (D), because both strains do not bind the fusion proteins. (E) Human blood treated with 20 mg/ml FH6-7Fc was infected with 2 3 105 CFU/ml S. pyogenes AP1. Prior to infection, samples were pretreated with compstatin and/or anti-Fcg receptors as indicated. Means (6SD) from at least three independent experiments are shown. Statistical significance of differences was calculated using two-way ANOVA with a Dunnett posttest [overall p value in (A), (B), and (E) was ,0.0001, in (C) p = 0.8247 and (D) p = 0.4248], *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. which activate complement to opsonize their targets for elimina- FH6-7/hFc has already been proven efficacious against tion by phagocytes or, in case of Gram-negative bacteria, directly N. meningitidis (34) and H. influenzae (33), whereas a derivative lyse them by MAC. of FH18-20/Fc that contains a D to G mutation at position 1119 in 10 FH/Fc ALLEVIATES S. PYOGENES INFECTION domain 19 was effective against N. gonorrhoeae (35). Of note, all 19–20) are distinct from the domains responsible for complement three bacterial species belong to the group of Gram-negative bac- inhibition (domains 1–4). Thus, FH6-7/hFc lacks any complement teria, which are generally susceptible to complement-mediated lysis inhibiting activity, prevents binding of functional host FH to the (17, 47). To achieve MAC-mediated lysis of Gram-negative bac- pathogen, and also activates complement through Fc. Given that teria, the chimeric FH-Fc needs to bind to bacteria to displace FH several pathogens bind FH through a similar region in FH, it raises from the bacterial surface and or activate the classical pathway the possibility that FH6-7/hFc may possess activity against several through Fc. However, complement alone cannot directly lyse other microbes (32). Gram-positive bacteria due to their thick cell wall (48). Instead, To our surprise, we found that in addition to protein H, the M1 complement-mediated opsonization of the bacteria with iC3b is protein from S. pyogenes AP1 also bound FH6-7-IgG. Protein H, crucial for efficient phagocytosis. We showed that the chimeric but not the M1 protein, has been described to bind FH (13, 55, 56). protein bound to the bacterial surface and competed out FH, in- Interestingly, FH seems to have more than one binding site: do- creased complement activation, led to a significant increase of phago- mains C and D, similar to FH6-7/Fc and presumably the N-terminal cytosis by PMNs, reduced bacterial burden in human blood ex part of domain B. Strain AP1 used in this study expresses both vivo, and significantly improved survival of mice in a S. pyogenes protein H and M1, whereas the previous study employed a different sepsis model. M1-expressing strain, S. pyogenes SF370. It is possible that dif- Many pathogens have developed the ability to recruit complement ferences in the M proteins between the strains may account for inhibitors such as FH and C4BP to their surface to evade complement differences in FH binding to these S. pyogenes strains. (reviewed in Refs. 20, 32). FH and C4BP tightly regulate comple- S. pyogenes is known to bind human, but not mouse, IgG (39, ment activation by accelerating the decay of the C3 convertase and 57). Thus, we used FH/Fc constructs that contained mouse IgG-Fc Downloaded from serving as cofactors for factor I–mediated inactivation of C3b (49– in this study to show that binding of FH6-7/hFc occurred specifically 52). Complement activation is dampened on pathogens “coated” via the FH domains. In contrast, FH18-20/hFc bound S. pyogenes with these inhibitors and thus are protected from opsonophagocy- exclusively through human IgG domains, thus explaining why tosis (44). Blocking the binding of complement inhibitors renders FH18-20 did not compete out serum FH and did not enhance the bacteria again susceptible to complement and would allow the complement activation on the bacteria. Although we cannot ex-

immune system to eliminate the invaders. FH-IgG chimeric protein clude the possibility that a proportion of FH6-7/hFc may also bind http://www.jimmunol.org/ acts by competing out serum FH bound to those bacteria and also to the bacteria via the human IgG part, we have shown that even a activates the classical pathway through its Fc region (33–35). low amount (∼50 nM) of FH6-7/hFc is able to effectively compete Similar to at least 10 other bacterial species (26), most strains of out FH completely. S. pyogenes recruit serum FH through domains 6 and 7 to their Another advantage of FH6-7/hFc is that drug resistance, if it were surface to protect themselves from complement attack (53, 54). to occur, would lead to a loss of binding of endogenous FH and FH- The regions in FH that interact with microbes (domains 5–7 and like protein 1 that both possess complement inhibiting function and thereby protect the bacteria. Loss of FH/FH-like protein 1 binding would lead to a considerable decrease of bacterial fitness in vivo because the resulting increase in C3 fragment deposition would by guest on September 29, 2021 lead to bacterial clearance by phagocytes (13, 53). In comparison with Gram-negative organisms, Gram-positive bacteria cannot be lysed through complement-mediated pore formation, although MAC can still be detected on their surface (58). Although the role of MAC associated with Gram-positive bacteria is unclear, a recent study showed that MAC can be transferred from the surface of an opsonized particle to the macrophage plasma membrane and initiate the NLRP3 inflammasome, resulting in caspase-1 activation and IL-1b and IL-18 secretion (59). Therefore, the mode of action of FH6-7/hFc on S. pyogenes must differ from the previously described mechanisms for Neisseria and Haemophilus. If bacteria cannot be directly lysed by complement, professional phagocytes must clear the microbes employing either extracellular killing mechanisms or intracellular degradation after phagocytosis. We showed that FH/Fc increased opsonization and following opsonophagocytosis. This was presumably caused on the one hand by displacement of FH and concomitant loss of protection from complement deposition, and on the other hand the additional activation of the classical complement pathway through the IgG1-Fc domains of our chimeric protein (60). In addition to C3b and iC3b, the Fc domain of the IgG1 backbone engages the Fcg receptor on professional phagocytes (61). Fcg receptor en- gagement alone does not mediate killing of S. pyogenes, whereas complement-driven opsonization is the most important pathway to FIGURE 6. FH6-7/hFc increases survival in experimental S. pyogenes clear the bacteria (62). Using compstatin CP40 we confirmed this sepsis. FH6-7/hFc–treated human FH transgenic mice show reduced mortality in an experimental i.v. S. pyogenes sepsis model. Human FH result in untreated human full blood as well as in the presence of transgenic mice (n = 10 per group) were treated with either FH6-7/hFc or FH6-7/Fc. Despite FH6-7/Fc binding to bacteria via FH domains goat IgG (control that does not bind S. pyogenes) i.p. with the dosing and presenting the hIgG1-Fc, blocking Fcg receptors (CD16, CD32, schedule shown on the x-axis. Statistical significance of differences was and CD64) had no influence on S. pyogenes killing. Compstatin in calculated using a Mantel–Cox analysis. turn promoted bacterial survival by blocking complement activation The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/

FIGURE 7. Graphical summary mechanism of action of FH-IgG against S. pyogenes. Bacteria recruit complement inhibitors to their surface and thus dampen complement activation. There is also less anaphylatoxin release, which combined with low C3 fragment deposition on the bacteria, does not adequately recruit or engage phagocytes, which results in bacterial replication and disease (upper panel). FH-IgG, however, diverts full-length, functional FH from the bacterial surface. The resulting complement activation releases abundant amounts of anaphylatoxins that attract professional phagocytes, which engage and engulf bacteria through surface-bound C3 fragments (lower panel). The Fc part of the molecule also activates complement and engages phagocytes via Fcg receptors, which results in downstream signaling and bacterial killing in phagolysosomes.

at the level of C3. Of note, although compstatin prevents C3b Disclosures by guest on September 29, 2021 deposition on the bacteria, at the same time it allows for increased S.R. and J.S. are inventors on a patent application in the area of FH/Fc ther- C4b deposition (63), which may serve as an opsonin by binding apeutics. J.D.L. is the inventor of patents and/or patent applications that CR1 (64). The potential relevance of this enhanced C4b deposition describe the use of complement inhibitors for therapeutic purposes; the is still not clear. Noncomplement bactericidal effects of serum, founder of Amyndas Pharmaceuticals, which is developing complement in- such as transferrin, antimicrobial peptides, and enzymes, also hibitors (i.e., next generation compstatins) for clinical applications; and the contribute to bacterial killing. Further work is needed to delineate inventor of the compstatin technology licensed to Apellis Pharmaceuticals the exact mechanism of action of FH6-7/Fc on Gram-positive (i.e., 4(1MeW)7W/POT-4/APL-1 and its PEGylated derivatives). The other authors have no financial conflicts of interest. bacteria. Taken together, the FH6-7/hFc mediates opsonophagocytosis through two mechanisms: 1) activating complement via C1q–IgG1 interaction, and 2) blocking FH-dependent complement References inhibition (see figure 2 in Ref. 26). 1. Bisno, A. L., and D. L. Stevens. 1996. Streptococcal infections of skin and soft It is urgent to develop novel approaches to cope with antibiotic- tissues. N. Engl. J. Med. 334: 240–245. 2. Nowak, R. 1994. Flesh-eating bacteria: not new, but still worrisome. Science resistant bacterial infections, which pose a major threat to human 264: 1665. health globally. The Centers for Disease Control as well as the World 3. Carapetis, J. R., A. C. Steer, E. K. Mulholland, and M. Weber. 2005. The global burden of group A streptococcal diseases. Lancet Infect. Dis. 5: 685–694. Health Organization have listed 15 pathogens as concerning, serious, 4. Horstmann, R. D., H. J. Sievertsen, J. Knobloch, and V. A. Fischetti. 1988. or urgent threats that need close monitoring, preventive activities, or Antiphagocytic activity of streptococcal M protein: selective binding of com- even urgent and aggressive actions to counteract their antibiotic plement control protein factor H. Proc. Natl. Acad. Sci. USA 85: 1657–1661. 5. Frick, I. M., P. Akesson, J. Cooney, U. Sjo¨bring, K. H. Schmidt, H. Gomi, resistance threat (29, 65). Among those 15 different bacterial species, S. Hattori, C. Tagawa, F. Kishimoto, and L. Bjo¨rck. 1994. Protein H—a surface 9areknowntobindFH,ofwhichatleast7employdomains5–7 protein of Streptococcus pyogenes with separate binding sites for IgG and al- for binding (26, 32). Because FH6-7/hFc especially targets those bumin. Mol. Microbiol. 12: 143–151. 6. Frick, I. M., K. L. Crossin, G. M. Edelman, and L. Bjo¨rck. 1995. Protein H—a binding sites, we think that the chimeric protein could be a valuable bacterial surface protein with affinity for both immunoglobulin and fibronectin adjunctive immunotherapeutic against antibiotic-resistant bacterial type III domains. EMBO J. 14: 1674–1679. pathogens. Although FH/Fc fusion proteins have shown promise in 7. Ermert, D., A. Weckel, V. Agarwal, I. M. Frick, L. Bjo¨rck, and A. M. Blom. 2013. Binding of complement inhibitor C4b-binding protein to a highly virulent preclinical models, we acknowledge that further work needs to be Streptococcus pyogenes M1 strain is mediated by protein H and enhances ad- undertaken to establish their safety and stability for use in humans. hesion to and invasion of endothelial cells. J. Biol. Chem. 288: 32172–32183. 8. Thern, A., L. Stenberg, B. Dahlba¨ck, and G. Lindahl. 1995. Ig-binding surface proteins of Streptococcus pyogenes also bind human C4b-binding protein Acknowledgments (C4BP), a regulatory component of the complement system. J. Immunol. 154: We thank Nancy Nowak and Bo Zhang for expert help with breeding and 375–386. 9. Carlsson, F., C. Sandin, and G. Lindahl. 2005. Human fibrinogen bound to caring for mice and the Center for Thrombosis and Haemostasis, Skane˚ Streptococcus pyogenes M protein inhibits complement deposition via the University Hospital, Malmo¨, Sweden for help with the coagulation assays. classical pathway. Mol. Microbiol. 56: 28–39. 12 FH/Fc ALLEVIATES S. PYOGENES INFECTION

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