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Recent studies show an important role for non-neutralizing anti-spike antibodies, including monoclonal antibodies (mAbs), in robustly protecting against SARS-CoV-2 infection. These mAbs use Fc-mediated functions such as complement activation, phagocytosis, and cellular cytotoxicity. There is an untapped potential for using non-neutralizing mAbs in durable antibody treatments; because of their available conserved epitopes, they may not be as sensitive to virus mutations as neutralizing mAbs. Here, we discuss evidence of non-neutralizing mAb-mediated protection against SARS-CoV-2 infection. We explore how non-neutralizing mAb Fc-mediated functions can be enhanced via novel antibody-engineering techniques. Important questions remain to be answered regarding the characteristics of protective non-neutralizing mAbs, including the models and assays used for study, the risks of ensuing detrimental inflammation, as well as the durability and mechanisms of protection.
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Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , SARS-CoV-2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , COVID-19/imunologia , Anticorpos Antivirais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Epitopos/imunologia , Fragmentos Fc das Imunoglobulinas/imunologiaRESUMO
Substantial evidence supports that Fc-mediated effector functions of anti-spike Abs contribute to anti-SARS-Cov-2 protection. We have previously shown that two non-neutralizing but opsonic mAbs targeting the receptor-binding domain and N-terminal domain (NTD), Ab81 and Ab94, respectively, are protective against lethal Wuhan SARS-CoV-2 infection in K18-hACE2 mice. In this article, we investigated whether these protective non-neutralizing Abs maintain Fc-mediated function and Ag binding against mutated SARS-CoV-2 variants. Ab81 and Ab94 retained their nanomolar affinity and Fc-mediated function toward Omicron and its subvariants, such as BA.2, BA.4, BA.5, XBB, XBB1.5, and BQ1.1. However, when encountering the more heavily mutated BA.2.86, Ab81 lost its function, whereas the 10 new mutations in the NTD did not affect Ab94. In vivo experiments with Ab94 in K18-hACE2 mice inoculated with a stringent dose of 100,000 PFU of the JN.1 variant revealed unexpected results. Surprisingly, this variant exhibited low disease manifestation in this animal model with no weight loss or death in the control group. Still, assessment of mice using a clinical scoring system showed better protection for Ab94-treated mice, indicating that Fc-mediated functions are still beneficial. Our work shows that a protective anti-receptor-binding domain non-neutralizing mAb lost reactivity when BA.2.86 emerged, whereas the anti-NTD mAb was still functional. Finally, this work adds new insight into the evolution of the SARS-CoV-2 virus by reporting that JN.1 is substantially less virulent in vivo than previous strains.
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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.
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COVID-19 , Fragmentos Fc das Imunoglobulinas , Imunoglobulina G , SARS-CoV-2 , Streptococcus pyogenes , Animais , Humanos , Camundongos , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos Endogâmicos BALB C , Fagocitose , Engenharia de Proteínas/métodos , SARS-CoV-2/imunologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/imunologiaRESUMO
The important bacterial pathogen Streptococcus pyogenes secretes IdeS (immunoglobulin G-degrading enzyme of S. pyogenes), a proteinase that cleaves human immunoglobulin G (IgG) antibodies in the hinge region resulting in Fc (fragment crystallizable) and F(ab')2 (fragment antigen-binding) fragments and protects the bacteria against phagocytic killing. Experiments with radiolabeled IdeS and flow cytometry demonstrated that IdeS binds to the surface of S. pyogenes, and the interaction was most prominent in conditions resembling those in the pharynx (acidic pH and low salt), the habitat for S. pyogenes. SpnA (S. pyogenes nuclease A) is a cell wall-anchored DNase. A dose-dependent interaction between purified SpnA and IdeS was demonstrated in slot binding and surface plasmon resonance spectroscopy experiments. Gel filtration showed that IdeS forms proteolytically active complexes with SpnA in solution, and super-resolution fluorescence microscopy revealed the presence of SpnA-IdeS complexes at the surface of S. pyogenes. Finally, specific IgG antibodies binding to S. pyogenes surface antigens were efficiently cleaved by surface-associated IdeS. IdeS is secreted by all S. pyogenes isolates and cleaves IgG antibodies with a unique degree of specificity and efficiency. These properties and the finding here that the proteinase is present and fully active at the bacterial surface in complex with SpnA implicate an important role for IdeS in S. pyogenes biology and pathogenesis.
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Proteínas de Bactérias , Streptococcus pyogenes , Humanos , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G , Peptídeo Hidrolases , Streptococcus pyogenes/metabolismoRESUMO
Streptococcus pyogenes causes a multitude of local and systemic infections, the most common being pharyngitis in children. Recurrent pharyngeal infections are common and are thought to be due to the re-emergence of intracellular GAS upon completion of antibiotic treatment. The role of colonizing biofilm bacteria in this process is not fully clear. Here, live respiratory epithelial cells were inoculated with broth-grown or biofilm bacteria of different M-types, as well as with isogenic mutants lacking common virulence factors. All M-types tested adhered to and were internalized into epithelial cells. Interestingly, internalization and persistence of planktonic bacteria varied significantly between strains, whereas biofilm bacteria were internalized in similar and higher numbers, and all strains persisted beyond 44 hours, showing a more homogenous phenotype. The M3 protein, but not the M1 or M5 proteins, was required for optimal uptake and persistence of both planktonic and biofilm bacteria inside cells. Moreover, the high expression of capsule and SLO inhibited cellular uptake and capsule expression was required for intracellular survival. Streptolysin S was required for optimal uptake and persistence of M3 planktonic bacteria, whereas SpeB improved intracellular survival of biofilm bacteria. Microscopy of internalized bacteria showed that planktonic bacteria were internalized in lower numbers as individual or small clumps of bacteria in the cytoplasm, whereas GAS biofilm bacteria displayed a pattern of perinuclear localization of bacterial aggregates that affected actin structure. Using inhibitors targeting cellular uptake pathways, we confirmed that planktonic GAS mainly uses a clathrin-mediated uptake pathway that also required actin and dynamin. Clathrin was not involved in biofilm internalization, but internalization required actin rearrangement and PI3 kinase activity, possibly suggesting macropinocytosis. Together these results provide a better understanding of the potential mechanisms of uptake and survival of various phenotypes of GAS bacteria relevant for colonization and recurrent infection.
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Infecções Estreptocócicas , Streptococcus pyogenes , Humanos , Streptococcus pyogenes/genética , Sorogrupo , Virulência , Actinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Epiteliais/microbiologia , Biofilmes , Fatores de Virulência/metabolismo , Infecções Estreptocócicas/microbiologiaRESUMO
Phagocytosis is relevant for many research fields and is often measured as a functional outcome. However, accurate quantification can be challenging, and many researchers find it difficult to study in a robust manner. There are many ways to measure phagocytosis, but what is often overlooked is the importance of experimental design and how the analysis is planned and performed. Experimental factors like reaction volume, time, and phagocyte-prey concentrations often have a large impact on the outcome, as is the choice of detection strategy with different fluorescent or colorimetric labels of prey and phagocyte. By using dose-response curve principles for both experimental design and analysis, it is possible to increase the sensitivity and robustness, leading to accurate quantification of phagocytosis that is comparable across experiments and systems.Here, we describe how to quantify phagocytosis using flow cytometry with a robust, high-throughput, and easy-to-use approach. The prey is first fluorescently double stained, followed by optional opsonization before being introduced to the phagocyte in a wide range of ratios. After incubation, data is acquired through flow cytometry. It can be assessed on both the population and single-cell level of the phagocytes, separating adhesion and internalization. As an example, we provide an experimental protocol for studying phagocytosis of opsonized Streptococcus pyogenes using the THP-1 cell line. This approach is easily incorporated into most existing phagocytosis assays and allows for reproducible results with high sensitivity.
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Fagócitos , Fagocitose , Citometria de Fluxo/métodos , Fagócitos/fisiologia , Corantes , Streptococcus pyogenesRESUMO
Antibody binding to bacterial surfaces plays a crucial role in immunity, and a key characteristic of this protein-protein interaction is the binding affinity. Determining the affinity of an antibody binding to its antigen is the first step in predicting the function in a physiological environment where other competing protein interactions may be present. Antibody-antigen affinity is often evaluated with isolated proteins. It is informative to also be able to assess antibody binding to a bacterial surface where many antigens might be present, including multiple copies of the specific antigen the antibody recognizes, and in a context where the antigen might be in a more natural conformation. In this chapter, we present a flow cytometry-based assay to measure and calculate the cell surface binding affinity or avidity of any mono- or polyclonal antibody solution.
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Anticorpos , Antígenos , Citometria de Fluxo , Afinidade de AnticorposRESUMO
Light microscopy is a powerful single-cell technique that allows for quantitative spatial information at subcellular resolution. However, unlike flow cytometry and single-cell sequencing techniques, microscopy has issues achieving high-quality population-wide sample characterization while maintaining high resolution. Here, we present a general framework, data-driven microscopy (DDM) that uses real-time population-wide object characterization to enable data-driven high-fidelity imaging of relevant phenotypes based on the population context. DDM combines data-independent and data-dependent steps to synergistically enhance data acquired using different imaging modalities. As a proof of concept, we develop and apply DDM with plugins for improved high-content screening and live adaptive microscopy for cell migration and infection studies that capture events of interest, rare or common, with high precision and resolution. We propose that DDM can reduce human bias, increase reproducibility, and place single-cell characteristics in the context of the sample population when interpreting microscopy data, leading to an increase in overall data fidelity.
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Microscopia , Humanos , Microscopia/métodos , Reprodutibilidade dos TestesRESUMO
Antibodies play a central role in the immune defense against SARS-CoV-2. Emerging evidence has shown that nonneutralizing antibodies are important for immune defense through Fc-mediated effector functions. Antibody subclass is known to affect downstream Fc function. However, whether the antibody subclass plays a role in anti-SARS-CoV-2 immunity remains unclear. Here, we subclass-switched eight human IgG1 anti-spike monoclonal antibodies (mAbs) to the IgG3 subclass by exchanging their constant domains. The IgG3 mAbs exhibited altered avidities to the spike protein and more potent Fc-mediated phagocytosis and complement activation than their IgG1 counterparts. Moreover, combining mAbs into oligoclonal cocktails led to enhanced Fc- and complement receptor-mediated phagocytosis, superior to even the most potent single IgG3 mAb when compared at equivalent concentrations. Finally, in an in vivo model, we show that opsonic mAbs of both subclasses can be protective against a SARS-CoV-2 infection, despite the antibodies being nonneutralizing. Our results suggest that opsonic IgG3 oligoclonal cocktails are a promising idea to explore for therapy against SARS-CoV-2, its emerging variants, and potentially other viruses.
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COVID-19 , Imunoglobulina G , Humanos , Opsonização , SARS-CoV-2 , Fagocitose , Anticorpos Monoclonais/farmacologiaRESUMO
Group A streptococcus (GAS) is a highly adapted, human-specific pathogen that is known to manipulate the immune system through various mechanisms. GAS' M protein constitutes a primary target of the immune system due to its spatial configuration and dominance on the bacterial surface. Antibody responses targeting the M protein have been shown to favor the conserved C region. Such antibodies (Abs) circumvent antigenic escape and efficiently bind to various M types. The ability of GAS to bind to fibronectin (Fn), a high molecular weight glycoprotein of the extracellular matrix, has long been known to be essential for the pathogen's evolutionary success and fitness. However, some strains lack the ability to efficiently bind Fn. Instead, they have been found to additionally bind Fn via the A-B domains of their M proteins. Here, we show that human Abs can induce increased Fn-binding affinity in M proteins, likely by enhancing the weak A-B domain binding. We found that this enhanced Fn binding leads to a reduction in Ab-mediated phagocytosis, indicating that this constitutes a GAS immune escape mechanism. We could show that the Fc domain of Abs is necessary to trigger this phenomenon and that Ab flexibility may also play a key role. We, moreover, saw that our Abs could enhance Fn binding in 3 out of 5 emm type strains tested, belonging to different clades, making it likely that this is a more generalizable phenomenon. Together our results suggest a novel synergistic interplay of GAS and host proteins which ultimately benefits the bacterium.
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Infrared spectroscopic imaging is widely used for the visualization of biomolecule structures, and techniques such as optical photothermal infrared (OPTIR) microspectroscopy can achieve <500 nm spatial resolution. However, these approaches lack specificity for particular cell types and cell components and thus cannot be used as a stand-alone technique to assess their properties. Here, we have developed a novel tool, fluorescently guided optical photothermal infrared microspectroscopy, that simultaneously exploits epifluorescence imaging and OPTIR to perform fluorescently guided IR spectroscopic analysis. This novel approach exceeds the diffraction limit of infrared microscopy and allows structural analysis of specific proteins directly in tissue and single cells. Experiments described herein used epifluorescence to rapidly locate amyloid proteins in tissues or neuronal cultures, thus guiding OPTIR measurements to assess amyloid structures at the subcellular level. We believe that this new approach will be a valuable addition to infrared spectroscopy providing cellular specificity of measurements in complex systems for studies of structurally altered protein aggregates.
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Proteínas Amiloidogênicas , Espectrofotometria Infravermelho/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
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.
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Anticorpos Monoclonais , Antígenos de Bactérias , Humanos , Epitopos , FagocitoseRESUMO
The human pathogen Streptococcus pyogenes causes substantial morbidity and mortality. It is unclear if antibodies developed after infections with this pathogen are opsonic and if they are strain specific or more broadly protective. Here, we quantified the opsonic-antibody response following invasive S. pyogenes infection. Four patients with S. pyogenes bacteremia between 2018 and 2020 at Skåne University Hospital in Lund, Sweden, were prospectively enrolled. Acute- and convalescent-phase sera were obtained, and the S. pyogenes isolates were genome sequenced (emm118, emm85, and two emm1 isolates). Quantitative antibody binding and phagocytosis assays were used to evaluate isolate-dependent opsonic antibody function in response to infection. Antibody binding increased modestly against the infecting isolate and across emm types in convalescent- compared to acute-phase sera for all patients. For two patients, phagocytosis increased in convalescent-phase serum both for the infecting isolate and across types. The increase was only across types for one patient, and one had no improvement. No correlation to the clinical outcomes was observed. Invasive S. pyogenes infections result in a modestly increased antibody binding with differential opsonic capacity, both nonfunctional binding and broadly opsonic binding across types. These findings question the dogma that an invasive infection should lead to a strong type-specific antibody increase rather than a more modest but broadly reactive response, as seen in these patients. Furthermore, our results indicate that an increase in antibody titers might not be indicative of an opsonic response and highlight the importance of evaluating antibody function in S. pyogenes infections. IMPORTANCE The bacterium Streptococcus pyogenes is a common cause of both mild and severe human diseases resulting in substantial morbidity and mortality each year. No vaccines are available, and our understanding of the antibody response to this human pathogen is still incomplete. Here, we carefully analyzed the opsonic antibody response following invasive infection in four patients. Unexpectedly, the patients did not always generate opsonic antibodies against the specific infecting strain. Instead, we found that some patients could generate cross-opsonic antibodies, leading to phagocytosis of bacteria across strains. The emergence of cross-opsonic antibodies is likely important for long-term immunity against S. pyogenes. Our findings question the dogma that mostly strain-specific immunity is developed after infection and add to our overall understanding of how immunity to S. pyogenes can evolve.
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Bacteriemia , Infecções Estreptocócicas , Humanos , Infecções Estreptocócicas/microbiologia , Fagocitose , Streptococcus pyogenes/genética , Anticorpos Antibacterianos , Antígenos de Bactérias/genéticaRESUMO
The COVID-19 pandemic caught the world largely unprepared, including scientific and policy communities. On April 10-13, 2022, researchers across academia, industry, government, and nonprofit organizations met at the Keystone symposium "Lessons from the Pandemic: Responding to Emerging Zoonotic Viral Diseases" to discuss the successes and challenges of the COVID-19 pandemic and what lessons can be applied moving forward. Speakers focused on experiences not only from the COVID-19 pandemic but also from outbreaks of other pathogens, including the Ebola virus, Lassa virus, and Nipah virus. A general consensus was that investments made during the COVID-19 pandemic in infrastructure, collaborations, laboratory and manufacturing capacity, diagnostics, clinical trial networks, and regulatory enhancements-notably, in low-to-middle income countries-must be maintained and strengthened to enable quick, concerted responses to future threats, especially to zoonotic pathogens.
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COVID-19 , Ebolavirus , Humanos , Pandemias , COVID-19/epidemiologia , Surtos de DoençasRESUMO
The fetal-to-adult switch in hematopoietic stem cell (HSC) behavior is characterized by alterations in lineage output and entry into deep quiescence. Here we identify the emergence of megakaryocyte (Mk)-biased HSCs as an event coinciding with this developmental switch. Single-cell chromatin accessibility analysis reveals a ubiquitous acquisition of Mk lineage priming signatures in HSCs during the fetal-to-adult transition. These molecular changes functionally coincide with increased amplitude of early Mk differentiation events after acute inflammatory insult. Importantly, we identify LIN28B, known for its role in promoting fetal-like self-renewal, as an insulator against the establishment of an Mk-biased HSC pool. LIN28B protein is developmentally silenced in the third week of life, and its prolonged expression delays emergency platelet output in young adult mice. We propose that developmental regulation of Mk priming may represent a switch for HSCs to toggle between prioritizing self-renewal in the fetus and increased host protection in postnatal life.
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Sinais (Psicologia) , Megacariócitos , Animais , Camundongos , Megacariócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Plaquetas/metabolismo , HematopoeseRESUMO
Antibody binding to cell surface proteins plays a crucial role in immunity, and the location of an epitope can altogether determine the immunological outcome of a host-target interaction. Techniques available today for epitope identification are costly, time-consuming, and unsuited for high-throughput analysis. Fast and efficient screening of epitope location can be useful for the development of therapeutic monoclonal antibodies and vaccines. Cellular morphology typically varies, and antibodies often bind heterogeneously across a cell surface, making traditional particle-averaging strategies challenging for accurate native antibody localization. In the present work, we have developed a method, SiteLoc, for imaging-based molecular localization on cellular surface proteins. Nanometer-scale resolution is achieved through localization in one dimension, namely, the distance from a bound ligand to a reference surface. This is done by using topological image averaging. Our results show that this method is well suited for antibody binding site measurements on native cell surface morphology and that it can be applied to other molecular distance estimations as well.
Antibodies play a key role in the immune system. These proteins stick to harmful substances, such as bacteria and other disease-causing pathogens, marking them for destruction or blocking their attack. Antibodies are highly selective, and this ability has been used to target particular molecules in research, diagnostics and therapies. Typically, antibodies need to stick to a particular segment, or 'epitope', on the surface of a cell in order to trigger an immune response. Knowing where these regions are can help explain how these immune proteins work and aid the development of more effective drugs and diagnostic tools. One way to identify these sites is to measure the nano-distance between antibodies and other features on the cell surface. To do this, researchers take multiple images of the cell the antibody is attached to using light microscopy. Various statistical methods are then applied to create an 'average image' that has a higher resolution and can therefore be used to measure the distance between these two points more accurately. While this approach works on fixed shapes, like a perfect circle, it cannot handle human cells and bacteria which are less uniform and have more complex surfaces. Here, Kumra Ahnlide et al. have developed a new method called SiteLoc which can overcome this barrier. The method involves two fluorescent probes: one attached to a specific site on the cell's surface, and the other to the antibody or another molecule of interest. These two probes emit different colours when imaged with a fluorescent microscope. To cope with objects that have uneven surfaces, such as cells and bacteria, the two signals are transformed to 'follow' the same geometrical shape. The relative distance between them is then measured using statistical methods. Using this approach, Kumra Ahnlide et al. were able to identify epitopes on a bacterium, and measure distances on the surface of human red blood cells. The SiteLoc system could make it easier to develop antibody-based treatments and diagnostic tools. Furthermore, it could also be beneficial to the wider research community who could use it to probe other questions that require measuring nanoscale distances.
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Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos , Membrana Celular/metabolismo , Mapeamento de Epitopos/métodos , Proteínas de Membrana/metabolismo , Sítios de Ligação , Membrana Celular/imunologia , Epitopos/metabolismo , Humanos , Ligantes , Proteínas de Membrana/imunologia , Modelos MolecularesRESUMO
Severe coronavirus disease 2019 (COVID-19) can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hallmark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe patients with COVID-19 reveal high levels of neutrophil extracellular trap (NET) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and lead to acute respiratory distress syndrome. Recombinant human DNase (Pulmozyme; Roche) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated five patients pwith COVID-19 resenting acute symptoms with clinically approved aerosolized Pulmozyme. No adverse reactions to the drug were seen, and improved oxygen saturation and recovery in all severely ill patients with COVID-19 was observed after therapy. Immunofluorescence and proteome analysis of sputum and blood plasma samples after treatment revealed a marked reduction of NETs and a set of statistically significant proteome changes that indicate reduction of hemorrhage, plasma leakage and inflammation in the airways, and reduced systemic inflammatory state in the blood plasma of patients. Taken together, the results indicate that NETs contribute to acute respiratory failure in COVID-19 and that degrading NETs may reduce dependency on external high-flow oxygen therapy in patients. Targeting NETs using recombinant human DNase may have significant therapeutic implications in COVID-19 disease and warrants further studies.
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Tratamento Farmacológico da COVID-19 , Desoxirribonuclease I/farmacologia , Armadilhas Extracelulares/metabolismo , Proteoma/análise , Idoso , Proteínas Sanguíneas/análise , COVID-19/metabolismo , COVID-19/terapia , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacologia , Índice de Gravidade de Doença , Escarro/efeitos dos fármacos , Escarro/metabolismo , Escarro/virologia , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/virologiaRESUMO
INTRODUCTION: Streptococcus dysgalactiae can cause severe recurrent infections. This study aimed to investigate antibody responses following S. dysgalactiae bacteraemia and possible development of protective immunity. MATERIALS AND METHODS: Patients with S. dysgalactiae bacteraemia in the county of Skåne between 2017 and 2018 were prospectively included. Acute and convalescent sera were obtained. All isolates were emm typed and enzyme-linked immunosorbent assay (ELISA) was utilised to analyse specific antibody responses to bacteria and antigens. Bactericidal- and phagocytosis assays were applied to further establish antibody function. RESULTS: Sixteen patients with S. dysgalactiae bacteraemia were included of whom one had recurrent episodes of bacteraemia. Using ELISA with S. dysgalactiae isolates and mutants, development of IgG antibodies was demonstrated in few patients. Type-specific antibodies were demonstrated in one patient when recombinant M proteins as antigens, were applied. The type-specific serum mediated a small increase in phagocytosis but did not facilitate increased killing of the S. dysgalactiae isolate, carrying that M protein, in blood or by phagocytic cells. CONCLUSION: S. dysgalactiae bacteraemia sometimes results in increased levels of antibodies to the infecting pathogen. We did not find evidence that these antibodies are effectively opsonising. Apparent failure to produce opsonising antibodies might partially explain why S. dysgalactiae can cause recurrent invasive infections in the same host.
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Many bacteria can interfere with how antibodies bind to their surfaces. This bacterial antibody targeting makes it challenging to predict the immunological function of bacteria-associated antibodies. The M and M-like proteins of group A streptococci (GAS) exhibit IgGFc-binding regions, which they use to reverse IgG binding orientation depending on the host environment. Unraveling the mechanism behind these binding characteristics may identify conditions under which bound IgG can drive an efficient immune response. Here, we have developed a biophysical model for describing these complex protein-antibody interactions. We show how the model can be used as a tool for studying the binding behavior of various IgG samples to M protein by performing in silico simulations and correlating this data with experimental measurements. Besides its use for mechanistic understanding, this model could potentially be used as a tool to aid in the development of antibody treatments. We illustrate this by simulating how IgG binding to GAS in serum is altered as specified amounts of monoclonal or pooled IgG is added. Phagocytosis experiments link this altered antibody binding to a physiological function and demonstrate that it is possible to predict the effect of an IgG treatment with our model. Our study gives a mechanistic understanding of bacterial antibody targeting and provides a tool for predicting the effect of antibody treatments in the presence of bacteria with IgG-modulating surface proteins.