The hinge-engineered IgG1-IgG3 hybrid subclass IgGh47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies.

Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3's Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGhxx. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh47 shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh47 has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh47 is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh47 versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh47 subclass in two distant biological systems provides new insights into antibody function.

Pathogen-driven degradation of endogenous and therapeutic antibodies during streptococcal infections.

Group A streptococcus (GAS) is a major bacterial pathogen responsible for both local and systemic infections in humans. The molecular mechanisms that contribute to disease heterogeneity remain poorly understood. Here we show that the transition from a local to a systemic GAS infection is paralleled by pathogen-driven alterations in IgG homeostasis. Using animal models and a combination of sensitive proteomics and glycoproteomics readouts, we documented the progressive accumulation of IgG cleavage products in plasma, due to extensive enzymatic degradation triggered by GAS infection in vivo. The level of IgG degradation was modulated by the route of pathogen inoculation, and mechanistically linked to the combined activities of the bacterial protease IdeS and the endoglycosidase EndoS, upregulated during infection. Importantly, we show that these virulence factors can alter the structure and function of exogenous therapeutic IgG in vivo. These results shed light on the role of bacterial virulence factors in shaping GAS pathogenesis, and potentially blunting the efficacy of antimicrobial therapies.

Exoproducts of the Most Common Achromobacter Species in Cystic Fibrosis Evoke Similar Inflammatory Responses In Vitro.

Achromobacter is a genus of Gram-negative rods, which can cause persistent airway infections in people with cystic fibrosis (CF). The knowledge about virulence and clinical implications of Achromobacter is still limited, and it is not fully established whether Achromobacter infections contribute to disease progression or if it is a marker of poor lung function. The most commonly reported Achromobacter species in CF is A. xylosoxidans. While other Achromobacter spp. are also identified in CF airways, the currently used Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) method in routine diagnostics cannot distinguish between species. Differences in virulence between Achromobacter species have consequently not been well studied. In this study, we compare phenotypes and proinflammatory properties of A. xylosoxidans, A. dolens, A. insuavis, and A. ruhlandii using in vitro models. Bacterial supernatants were used to stimulate CF bronchial epithelial cells and whole blood from healthy individuals. Supernatants from the well-characterized CF-pathogen Pseudomonas aeruginosa were included for comparison. Inflammatory mediators were analyzed with ELISA and leukocyte activation was assessed using flow cytometry. The four Achromobacter species differed in morphology seen in scanning electron microscopy (SEM), but there were no observed differences in swimming motility or biofilm formation. Exoproducts from all Achromobacter species except A. insuavis caused significant IL-6 and IL-8 secretion from CF lung epithelium. The cytokine release was equivalent or stronger than the response induced by P. aeruginosa. All Achromobacter species activated neutrophils and monocytes ex vivo in a lipopolysaccharide (LPS)-independent manner. Our results indicate that exoproducts of the four included Achromobacter species do not differ consistently in causing inflammatory responses, but they are equally or even more capable of inducing inflammation compared with the classical CF pathogen P. aeruginosa. IMPORTANCE Achromobacter xylosoxidans is an emerging pathogen among people with cystic fibrosis (CF). Current routine diagnostic methods are often unable to distinguish A. xylosoxidans from other Achromobacter species, and the clinical relevance of different species is still unknown. In this work, we show that four different Achromobacter species relevant to CF evoke similar inflammatory responses from airway epithelium and leukocytes in vitro, but they are all equally or even more proinflammatory compared to the classic CF-pathogen Pseudomonas aeruginosa. The results suggest that Achromobacter species are important airway pathogens in CF, and that all Achromobacter species are relevant to treat.

A pharmacoproteomic landscape of organotypic intervention responses in Gram-negative sepsis.

Sepsis is the major cause of mortality across intensive care units globally, yet details of accompanying pathological molecular events remain unclear. This knowledge gap has resulted in ineffective biomarker development and suboptimal treatment regimens to prevent and manage organ dysfunction/damage. Here, we used pharmacoproteomics to score time-dependent treatment impact in a murine Escherichia coli sepsis model after administering beta-lactam antibiotic meropenem (Mem) and/or the immunomodulatory glucocorticoid methylprednisolone (Gcc). Three distinct proteome response patterns were identified, which depended on the underlying proteotype for each organ. Gcc enhanced some positive proteome responses of Mem, including superior reduction of the inflammatory response in kidneys and partial restoration of sepsis-induced metabolic dysfunction. Mem introduced sepsis-independent perturbations in the mitochondrial proteome that Gcc counteracted. We provide a strategy for the quantitative and organotypic assessment of treatment effects of candidate therapies in relationship to dosing, timing, and potential synergistic intervention combinations during sepsis.

Phenotypic Characterization of Acoustically Enriched Extracellular Vesicles from Pathogen-Activated Platelets.

Extracellular vesicles (EVs) are derived from the membrane of platelets and released into the circulation upon activation or injury. Analogous to the parent cell, platelet-derived EVs play an important role in hemostasis and immune responses by transfer of bioactive cargo from the parent cells. Platelet activation and release of EVs increase in several pathological inflammatory diseases, such as sepsis. We have previously reported that the M1 protein released from the bacterial pathogen Streptococcus pyogenes directly mediates platelet activation. In this study, EVs were isolated from these pathogen-activated platelets using acoustic trapping, and their inflammation phenotype was characterized using quantitative mass spectrometry-based proteomics and cell-based models of inflammation. We determined that M1 protein mediated release of platelet-derived EVs that contained the M1 protein. The isolated EVs derived from pathogen-activated platelets contained a similar protein cargo to those from physiologically activated platelets (thrombin) and included platelet membrane proteins, granule proteins, cytoskeletal proteins, coagulation factors, and immune mediators. Immunomodulatory cargo, complement proteins, and IgG3 were significantly enriched in EVs isolated from M1 protein-stimulated platelets. Acoustically enriched EVs were functionally intact and exhibited pro-inflammatory effects on addition to blood, including platelet-neutrophil complex formation, neutrophil activation, and cytokine release. Collectively, our findings reveal novel aspects of pathogen-mediated platelet activation during invasive streptococcal infection.

A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein mediates immune function.

Group A streptococci have evolved multiple strategies to evade human antibodies, making it challenging to create effective vaccines or antibody treatments. Here, we have generated antibodies derived from the memory B cells of an individual who had successfully cleared a group A streptococcal infection. The antibodies bind with high affinity in the central region of the surface-bound M protein. Such antibodies are typically non-opsonic. However, one antibody could effectively promote vital immune functions, including phagocytosis and in vivo protection. Remarkably, this antibody primarily interacts through a bivalent dual-Fab cis mode, where the Fabs bind to two distinct epitopes in the M protein. The dual-Fab cis-binding phenomenon is conserved across different groups of M types. In contrast, other antibodies binding with normal single-Fab mode to the same region cannot bypass the M protein's virulent effects. A broadly binding, protective monoclonal antibody could be a candidate for anti-streptococcal therapy. Our findings highlight the concept of dual-Fab cis binding as a means to access conserved, and normally non-opsonic regions, regions for protective antibody targeting.

Platelet Activation and Aggregation Induced by Streptococcus bovis/Streptococcus equinus Complex.

Streptococcus bovis/Streptococcus equinus complex (SBSEC) is a common cause of infective endocarditis (IE). For IE-pathogens, the capacity to activate and aggregate platelets is believed to be an important virulence mechanism. While the interactions between bacteria and platelets have been described in detail for many Gram-positive pathogens, little research has been carried out with SBSEC in this respect. Twenty-six isolates of the four most common species and subspecies of SBSEC identified in bacteremia were collected, and interactions with platelets were investigated in platelet rich plasma (PRP) from three donors. Aggregation was studied using light-transmission aggregometry and platelet activation using flow cytometry detecting surface upregulation of CD62P. Platelets and serum were treated with different inhibitors to determine mechanisms involved in platelet aggregation and activation. Twenty-two of 26 isolates induced aggregation in at least one donor, and four isolates induced aggregation in all three donors. In PRP from donor 1, isolate SL1 induced a rapid aggregation with a median time of 70 s to reach 50% aggregation. Blockade of the platelet Fc-receptor or enzymatic cleavage of IgG abolished platelet activation and aggregation. The capacity for bacteria-induced platelet aggregation was also shown to be transferable between donors through serum. SBSEC mediates platelet aggregation in an IgG and IgG-Fc-receptor dependent manner. Bacterial activation of platelets through this pathway is common for many bacteria causing IE and could be a potential therapeutic target for the prevention and treatment of this infection. IMPORTANCE The capacity of bacteria to activate and aggregate platelets is believed to contribute to the pathogenesis of IE. The Streptococcus bovis/Streptococcus equinus complex (SBSEC) contains known IE-pathogens, but there is limited research on the different subspecies ability to interact with platelets and what signaling pathways are involved. This study reports that 22 of 26 tested isolates of different subspecies within SBSEC can induce aggregation, and that aggregation is host dependent. The Fc-IgG-receptor pathway was shown essential for platelet activation and aggregation. To the best of our knowledge, this is the first study that reports on platelet interactions of SBSEC-isolates other than Streptococcus gallolyticus subspecies gallolyticus as well as the first study to report of mechanisms of platelet interaction of SBSEC-isolates. It adds SBSEC to a group of bacteria that activate and aggregate platelets via the platelet Fc-receptor. This could be a potential therapeutic target for prevention of IE.

Distinct Serotypes of Streptococcal M Proteins Mediate Fibrinogen-Dependent Platelet Activation and Proinflammatory Effects.

Sepsis is a life-threatening complication of infection that is characterized by a dysregulated inflammatory state and disturbed hemostasis. Platelets are the main regulators of hemostasis, and they also respond to inflammation. The human pathogen Streptococcus pyogenes can cause local infection that may progress to sepsis. There are more than 200 serotypes of S. pyogenes defined according to sequence variations in the M protein. The M1 serotype is among 10 serotypes that are predominant in invasive infection. M1 protein can be released from the surface and has previously been shown to generate platelet, neutrophil, and monocyte activation. The platelet-dependent proinflammatory effects of other serotypes of M protein associated with invasive infection (M3, M5, M28, M49, and M89) are now investigated using a combination of multiparameter flow cytometry, enzyme-linked immunosorbent assay (ELISA), aggregometry, and quantitative mass spectrometry. We demonstrate that only M1, M3, and M5 protein serotypes can bind fibrinogen in plasma and mediate fibrinogen- and IgG-dependent platelet activation and aggregation, release of granule proteins, upregulation of CD62P to the platelet surface, and complex formation with neutrophils and monocytes. Neutrophil and monocyte activation, determined as upregulation of surface CD11b, is also mediated by M1, M3, and M5 protein serotypes, while M28, M49, and M89 proteins failed to mediate activation of platelets or leukocytes. Collectively, our findings reveal novel aspects of the immunomodulatory role of fibrinogen acquisition and platelet activation during streptococcal infections.

Distinct phenotypes of platelet, monocyte, and neutrophil activation occur during the acute and convalescent phase of COVID-19.

SARS-CoV-2 has spread rapidly worldwide, causing the COVID-19 pandemic. Platelet activation and platelet-leukocyte complex formation are proposed to contribute to disease progression. Here, we report platelet and leukocyte activation during acute and convalescent COVID-19 in patients recruited between May-July 2020. Blood samples were analyzed by flow cytometry and ELISA using paired comparison between inclusion (day 0) and 28 days later. The majority of patients were mildly or moderately ill with significantly higher cytokine levels (IL-6 and IL-10) on day 0 as compared with day 28. Platelet activation and granule release were significantly higher on day 0 compared with day 28, as determined by ADP- or thrombin-induced surface CD62P expression, baseline released CD62P, and thrombin-induced platelet-monocyte complex formation. Monocyte activation and procoagulant status at baseline and post activation were heterogeneous but generally lower on day 0 compared with day 28. Baseline and thrombin- or fMLF-induced neutrophil activation and procoagulant status were significantly lower on day 0 compared with day 28. We demonstrate that during the acute phase of COVID-19 compared with the convalescent phase, platelets are more responsive while neutrophils are less responsive. COVID-19 is associated with thromboembolic events where platelet activation and interaction with leukocytes may play an important role.

The role of platelets in sepsis.

A State of the Art lecture titled "The role of platelets in sepsis" was presented at the ISTH congress in 2020. Sepsis is a life-threatening organ dysfunction caused by a dysregulated and multifaceted host response to infection. Platelets play a significant role in the coordinated immune response to infection and therefore in the inflammation and coagulation dysfunction that contributes to organ damage in sepsis. Thrombocytopenia has a high incidence in sepsis, and it is a marker of poor prognosis. The genesis of thrombocytopenia is likely multifactorial, and unraveling the involved molecular mechanisms will allow development of biomarkers of platelet function in sepsis. Such platelet biomarkers can facilitate study of antiplatelet interventions as immunomodulatory treatment in sepsis. Finally, relevant new data on this topic presented during the 2020 ISTH virtual congress are reviewed.

BPI-ANCA is expressed in the airways of cystic fibrosis patients and correlates to platelet numbers and Pseudomonas aeruginosa colonization.

BACKGROUND: Autoantibodies to bactericidal/permeability-increasing protein (BPI), BPI-ANCA, are often present in serum of patients with cystic fibrosis (CF), and correlate to airway colonization with Pseudomonas aeruginosa. The aim of the study was to investigate if BPI-ANCA IgA is also present in the airways of CF patients, and if its presence correlates with neutrophil counts, platelets, and P. aeruginosa DNA in sputum. METHODS: BPI-ANCA IgA was quantified in serum and sputum samples from adult CF patients (n = 45) by ELISA. Sputum neutrophil counts, platelets, and platelet-neutrophil complexes were assessed by flow cytometry, and P. aeruginosa DNA was analysed with RT-PCR. RESULTS: Serum BPI-ANCA IgA was present in 44% of the study participants, and this group also had significantly enhanced BPI-ANCA levels in sputum compared to serum negative patients. Sputum levels of BPI-ANCA IgA correlated with P. aeruginosa DNA (r = 0.63, p = 0.0003) and platelet counts in sputum (r = 0.60, p = 0.0002). CONCLUSIONS: BPI-ANCA is expressed in the airways of CF patients and correlates with P. aeruginosa load and platelet counts, suggesting a link to airway inflammation and mucosal immunity.

Streptococcus pyogenes evades adaptive immunity through specific IgG glycan hydrolysis.

Streptococcus pyogenes (Group A streptococcus; GAS) is a human pathogen causing diseases from uncomplicated tonsillitis to life-threatening invasive infections. GAS secretes EndoS, an endoglycosidase that specifically cleaves the conserved N-glycan on IgG antibodies. In vitro, removal of this glycan impairs IgG effector functions, but its relevance to GAS infection in vivo is unclear. Using targeted mass spectrometry, we characterized the effects of EndoS on host IgG glycosylation during the course of infections in humans. Substantial IgG glycan hydrolysis occurred at the site of infection and systemically in the severe cases. We demonstrated decreased resistance to phagocytic killing of GAS lacking EndoS in vitro and decreased virulence in a mouse model of invasive infection. This is the first described example of specific bacterial IgG glycan hydrolysis during infection and thereby verifies the hypothesis that EndoS modifies antibodies in vivo. This mechanisms of immune evasion could have implications for treatment of severe GAS infections and for future efforts at vaccine development.

Complement Activation Occurs at the Surface of Platelets Activated by Streptococcal M1 Protein and This Results in Phagocytosis of Platelets.

Platelets circulate the bloodstream and principally maintain hemostasis. Disturbed hemostasis, a dysregulated inflammatory state, and a decreased platelet count are all hallmarks of severe invasive Streptococcus pyogenes infection, sepsis. We have previously demonstrated that the released M1 protein from S. pyogenes activates platelets, and this activation is dependent on the binding of M1 protein, fibrinogen, and M1-specific IgG to platelets in susceptible donors. In this study, we characterize the M1-associated protein interactions in human plasma and investigate the acquisition of proteins to the surface of activated platelets and the consequences for platelet immune function. Using quantitative mass spectrometry, M1 protein was determined to form a protein complex in plasma with statistically significant enrichment of fibrinogen, IgG3, and complement components, especially C1q. Using flow cytometry, these plasma proteins were also confirmed to be acquired to the platelet surface, resulting in complement activation on M1-activated human platelets. Furthermore, we demonstrated an increased phagocytosis of M1-activated platelets by monocytes, which was not observed with other physiological platelet agonists. This reveals a novel mechanism of complement activation during streptococcal sepsis, which contributes to the platelet consumption that occurs in sepsis.

Streptococcal inhibitor of complement (SIC) modulates fibrinolysis and enhances bacterial survival within fibrin clots.

Some strains of the bacterial pathogen Streptococcus pyogenes secrete protein SIC (streptococcal inhibitor of complement), including strains of the clinically relevant M1 serotype. SIC neutralizes the effect of a number of antimicrobial proteins/peptides and interferes with the function of the host complement system. Previous studies have shown that some S. pyogenes proteins bind and modulate coagulation and fibrinolysis factors, raising the possibility that SIC also may interfere with the activity of these factors. Here we show that SIC interacts with both human thrombin and plasminogen, key components of coagulation and fibrinolysis. We found that during clot formation, SIC binds fibrin through its central region and that SIC inhibits fibrinolysis by interacting with plasminogen. Flow cytometry results indicated that SIC and plasminogen bind simultaneously to S. pyogenes bacteria, and fluorescence microscopy revealed co-localization of the two proteins at the bacterial surface. As a consequence, SIC-expressing bacteria entrapped in clots inhibit fibrinolysis, leading to delayed bacterial escape from the clots as compared with mutant bacteria lacking SIC. Moreover, within the clots SIC-expressing bacteria were protected against killing. In an animal model of subcutaneous infection, SIC-expressing bacteria exhibited a delayed systemic spread. These results demonstrate that the bacterial protein SIC interferes with coagulation and fibrinolysis and thereby enhances bacterial survival, a finding that has significant implications for S. pyogenes virulence.

Platelet activation and aggregation by the opportunistic pathogen Cutibacterium (Propionibacterium) acnes.

Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction.

Increased platelet activation occurs in cystic fibrosis patients and correlates to clinical status.

Cystic fibrosis (CF) is an inflammatory lung disease. Platelets have an emerging role in inflammation, however previous studies of platelet activation in CF have generated conflicting results. In this study, we determined platelet function in CF patients and correlated platelet activation to establish clinical and laboratory parameters. Twenty-two patients, aged 20.7 to 54.4 (mean 34.0, SD 9.45) years and with a mean FEV1%pred (forced expiratory volume in one second, % of predicted) of 72 (SD 21.4, range 32-110) were recruited. A combination of platelet assays was used: platelet aggregation, platelet activation and platelet-leukocyte complex formation. Platelets from CF patients exhibited significantly increased aggregation when stimulated ex-vivo, a tendency towards increased platelet upregulation of CD62P, but no increase of GPIIb/IIIa activation (PAC-1). Platelet-monocyte complex (PMC) formation was significantly increased in CF patients compared to controls, while platelet-neutrophil complex formation was not. In the CF group, platelet aggregation correlates with levels of anti-neutrophil cytoplasmic antibodies (ANCA) with specificity for bactericidal/permeability-increasing protein (BPI), BPI-ANCA (r=0.56). The formation of PMCs correlates with lung function decline (1-FEV1%), CRP and BPI-ANCA (r=0.61, 0.55, 0.5). We therefore confirm the presence of increased platelet activation in CF patients, and determine that further evaluation of platelet activation in relation to prognostic factors in CF is warranted.

Determining Platelet Activation and Aggregation in Response to Bacteria.

Many pathogenic bacteria have been reported to interact with human platelets to mediate platelet activation and aggregation. The importance of these interactions to the immune response or pathogenesis of bacterial infection has not been clarified. It may therefore be valuable to assess platelet responses mediated by diverse strains of bacteria. Here, I describe a method to study platelet integrin activation and granule release using flow cytometry, and a complementary method to study platelet aggregation using a dedicated platelet aggregometer. The combination of these methods represents a rapid and cost-effective strategy to provide mechanistic insight on the type of platelet response mediated by the bacteria.

Measuring Antibody Orientation at the Bacterial Surface.

Many bacteria have the ability to interact with antibodies as a means to circumvent the immune response. This includes binding to the Fc portion of antibodies, effectively reversing the antibody orientation and thus decreasing the Fc-mediated immune signaling. Since antibody orientation at the bacterial surface has been shown to be important in human disease, it is valuable to be able to assess how antibodies are interacting with bacterial pathogens. Here, we describe a method to measure the proportion of human IgG that are bound via their Fc or Fabs to a bacterial surface. This is achieved by treating antibody-coated bacteria with the bacterial enzyme IdeS - which will cleave IgG into Fc and Fab fragments - and subsequently detect remaining fragments with fluorescent Fabs. The method is easy and fast, and the principle is most likely also applicable to other systems where distinguishing between antibody Fc and Fab binding is important.

The Dynamics of Platelet Activation during the Progression of Streptococcal Sepsis.

Platelets contribute to inflammation however, the role of platelet activation during the pathophysiological response to invasive bacterial infection and sepsis is not clear. Herein, we have investigated platelet activation in a mouse model of invasive Streptococcus pyogenes infection at 5, 12, and 18 hours post infection and correlated this to parameters of infection. The platelet population in ex-vivo blood samples showed no increased integrin activation or surface presentation of CD62P, however platelet-neutrophil complex formation and plasma levels of CD62P were increased during bacterial dissemination and the progression of sepsis, indicating that platelet activation had occurred in vivo. Platelet-neutrophil complex formation was the most discriminatory marker of platelet activation. Platelet-neutrophil complexes were increased above baseline levels during early sepsis but decreased to significantly lower levels than baseline during late sepsis. The removal of these complexes from the circulation coincided with a significant increase in organ damage and the accumulation of platelets in the liver sinusoids, suggesting that platelet activation in the circulation precedes accumulation of platelets in damaged organs. The results demonstrate that monitoring platelet activation using complementary methods may provide prognostic information during the pathogenesis of invasive S. pyogenes infection.