RESUMEN
The influence of non-opsonized and opsonized S. aureus 2879M and E. coli 321 strains on the total strength of interaction between the endothelial cell and neutrophil during the docking process was studied using in vitro model of experimental septicemia. We observed a decrease in the force and work of adhesion between receptors of neutrophils and endothelial cells under the influence of non-opsonized strains and further decrease in the affinity of single interactions between cells under the influence of opsonized S. aureus, which was compensated by an increase in the number of contacts, as well as an increase in the force of adhesion under the influence of opsonized E. coli compared to non-opsonized bacteria, which remained below the control level, while adhesion work reaches the control level. Thus, opsonization of S. aureus aggravates the "immunological uncoupling" between neutrophils and endothelial cells, while opsonization of E. coli reduces the pathological effect compared to non-opsonized bacteria.
Asunto(s)
Células Endoteliales , Escherichia coli , Neutrófilos , Sepsis , Staphylococcus aureus , Neutrófilos/inmunología , Neutrófilos/metabolismo , Escherichia coli/inmunología , Staphylococcus aureus/inmunología , Staphylococcus aureus/patogenicidad , Sepsis/inmunología , Sepsis/microbiología , Sepsis/metabolismo , Sepsis/patología , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Humanos , Fagocitosis , Adhesión Celular/inmunología , Proteínas Opsoninas/metabolismo , Proteínas Opsoninas/inmunología , Adhesión Bacteriana , AnimalesRESUMEN
The bacterial pathogen Staphylococcus aureus responds to the host environment by increasing the thickness of its cell wall. However, the impact of cell wall thickening on susceptibility to host defenses is unclear. Using bacteria incubated in human serum, we show that host-induced increases in cell wall thickness led to a reduction in the exposure of bound antibody and complement and a corresponding reduction in phagocytosis and killing by neutrophils. The exposure of opsonins bound to protein antigens or lipoteichoic acid (LTA) was most significantly reduced, while opsonization by IgG against wall teichoic acid or peptidoglycan was largely unaffected. Partial digestion of accumulated cell wall using the enzyme lysostaphin restored opsonin exposure and promoted phagocytosis and killing. Concordantly, the antibiotic fosfomycin inhibited cell wall remodeling and maintained the full susceptibility of S. aureus to opsonophagocytic killing by neutrophils. These findings reveal that host-induced changes to the S. aureus cell wall reduce the ability of the immune system to detect and kill this pathogen through reduced exposure of protein- and LTA-bound opsonins. IMPORTANCE: Understanding how bacteria adapt to the host environment is critical in determining fundamental mechanisms of immune evasion, pathogenesis, and the identification of targets for new therapeutic approaches. Previous work demonstrated that Staphylococcus aureus remodels its cell envelope in response to host factors and we hypothesized that this may affect recognition by antibodies and thus killing by immune cells. As expected, incubation of S. aureus in human serum resulted in rapid binding of antibodies. However, as bacteria adapted to the serum, the increase in cell wall thickness resulted in a significant reduction in exposure of bound antibodies. This reduced antibody exposure, in turn, led to reduced killing by human neutrophils. Importantly, while antibodies bound to some cell surface structures became obscured, this was not the case for those bound to wall teichoic acid, which may have important implications for vaccine design.
Asunto(s)
Pared Celular , Neutrófilos , Proteínas Opsoninas , Fagocitosis , Staphylococcus aureus , Pared Celular/inmunología , Pared Celular/metabolismo , Humanos , Neutrófilos/inmunología , Neutrófilos/microbiología , Staphylococcus aureus/inmunología , Proteínas Opsoninas/metabolismo , Proteínas Opsoninas/inmunología , Opsonización/inmunología , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/sangre , Ácidos Teicoicos/metabolismo , Ácidos Teicoicos/inmunología , Evasión Inmune , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Interacciones Huésped-Patógeno/inmunologíaRESUMEN
BACKGROUND: Enterococcus faecium and Staphylococcus aureus are the Gram-positive pathogens of the ESKAPE group, known to represent a great threat to human health due to their high virulence and multiple resistances to antibiotics. Combined, enterococci and S. aureus account for 26% of healthcare-associated infections and are the most common organisms responsible for blood stream infections. We previously showed that the peptidyl-prolyl cis/trans isomerase (PPIase) PpiC of E. faecium elicits the production of specific, opsonic, and protective antibodies that are effective against several strains of E. faecium and E. faecalis. Due to the ubiquitous characteristics of PPIases and their essential function within Gram-positive cells, we hypothesized a potential cross-reactive effect of anti-PpiC antibodies. RESULTS: Opsonophagocytic assays combined with bioinformatics led to the identification of the foldase protein PrsA as a new potential vaccine antigen in S. aureus. We show that PrsA is a stable dimeric protein able to elicit opsonic antibodies against the S. aureus strain MW2, as well as cross-binding and cross-opsonic in several S. aureus, E. faecium and E. faecalis strains. CONCLUSIONS: Given the multiple antibiotic resistances S. aureus and enterococci present, finding preventive strategies is essential to fight those two nosocomial pathogens. The study shows the potential of PrsA as an antigen to use in vaccine formulation against the two dangerous Gram-positive ESKAPE bacteria. Our findings support the idea that PPIases should be further investigated as vaccine targets in the frame of pan-vaccinomics strategy.
Asunto(s)
Proteínas Bacterianas , Enterococcus faecalis , Enterococcus faecium , Isomerasa de Peptidilprolil , Staphylococcus aureus , Staphylococcus aureus/inmunología , Staphylococcus aureus/genética , Enterococcus faecium/inmunología , Enterococcus faecium/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/genética , Isomerasa de Peptidilprolil/inmunología , Isomerasa de Peptidilprolil/genética , Enterococcus faecalis/inmunología , Enterococcus faecalis/genética , Humanos , Infecciones por Bacterias Grampositivas/prevención & control , Infecciones por Bacterias Grampositivas/inmunología , Infecciones por Bacterias Grampositivas/microbiología , Vacunas Bacterianas/inmunología , Proteínas Opsoninas/inmunología , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/sangre , Animales , Reacciones Cruzadas , Ratones , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Fagocitosis , Infecciones Estafilocócicas/prevención & control , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiologíaRESUMEN
Introduction: Innate immunity is crucial to reducing parasite burden and contributing to survival in severe malaria. Monocytes are key actors in the innate response and, like macrophages, are plastic cells whose function and phenotype are regulated by the signals from the microenvironment. In the context of cerebral malaria (CM), monocyte response constitutes an important issue to understand. We previously demonstrated that decreased percentages of nonclassical monocytes were associated with death outcomes in CM children. In the current study, we postulated that monocyte phagocytosis function is impacted by the severity of malaria infection. Methods: To study this hypothesis, we compared the opsonic and nonopsonic phagocytosis capacity of circulant monocytes from Beninese children with uncomplicated malaria (UM) and CM. For the CM group, samples were obtained at inclusion (D0) and 3 and 30 days after treatment (D3, D30). The phagocytosis capacity of monocytes and their subsets was characterized by flow cytometry and transcriptional profiling by studying genes known for their functional implication in infected-red blood cell (iRBC) elimination or immune escape. Results: Our results confirm our hypothesis and highlight the higher capacity of nonclassical monocytes to phagocyte iRBC. We also confirm that a low number of nonclassical monocytes is associated with CM outcome when compared to UM, suggesting a mobilization of this subpopulation to the cerebral inflammatory site. Finally, our results suggest the implication of the inhibitory receptors LILRB1, LILRB2, and Tim3 in phagocytosis control. Discussion: Taken together, these data provide a better understanding of the interplay between monocytes and malaria infection in the pathogenicity of CM.
Asunto(s)
Malaria Cerebral , Monocitos , Fagocitosis , Humanos , Malaria Cerebral/inmunología , Malaria Cerebral/parasitología , Monocitos/inmunología , Masculino , Preescolar , Femenino , Niño , Lactante , Plasmodium falciparum/inmunología , Proteínas Opsoninas/metabolismo , Proteínas Opsoninas/inmunología , Eritrocitos/parasitología , Eritrocitos/inmunología , Inmunidad InnataRESUMEN
Introduction: The California purple sea urchin, Strongylocentrotus purpuratus, relies solely on an innate immune system to combat the many pathogens in the marine environment. One aspect of their molecular defenses is the SpTransformer (SpTrf) gene family that is upregulated in response to immune challenge. The gene sequences are highly variable both within and among animals and likely encode thousands of SpTrf isoforms within the sea urchin population. The native SpTrf proteins bind foreign targets and augment phagocytosis of a marine Vibrio. A recombinant (r)SpTrf-E1-Ec protein produced by E. coli also binds Vibrio but does not augment phagocytosis. Methods: To address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells. Results: The rSpTrf proteins are larger than expected, are glycosylated, and one dimerized irreversibly. Each rSpTrf protein cross-linked to inert magnetic beads (rSpTrf::beads) results in different levels of surface binding and phagocytosis by phagocytes. Initial analysis shows that significantly more rSpTrf::beads associate with cells compared to control BSA::beads. Binding specificity was verified by pre-incubating the rSpTrf::beads with antibodies, which reduces the association with phagocytes. The different rSpTrf::beads show significant differences for cell surface binding and phagocytosis by phagocytes. Furthermore, there are differences among the three distinct types of phagocytes that show specific vs. constitutive binding and phagocytosis. Conclusion: These findings illustrate the complexity and effectiveness of the sea urchin innate immune system driven by the natSpTrf proteins and the phagocyte cell populations that act to neutralize a wide range of foreign pathogens.
Asunto(s)
Fagocitos , Fagocitosis , Proteínas Recombinantes , Animales , Fagocitosis/inmunología , Fagocitos/inmunología , Fagocitos/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Unión Proteica , Strongylocentrotus purpuratus/inmunología , Strongylocentrotus purpuratus/genética , Inmunidad Innata , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Erizos de Mar/inmunología , Vibrio/inmunología , Proteínas Opsoninas/metabolismo , Proteínas Opsoninas/inmunologíaRESUMEN
Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca2+, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.
Asunto(s)
Péptidos Catiónicos Antimicrobianos , Proteínas de Artrópodos , Proteínas Opsoninas , Penaeidae/inmunología , Aglutinación , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/inmunología , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Proteínas de Artrópodos/farmacología , Bacterias/química , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/crecimiento & desarrollo , Epidermis/inmunología , Hemocitos/fisiología , Hepatopáncreas/inmunología , Proteínas Opsoninas/química , Proteínas Opsoninas/genética , Proteínas Opsoninas/inmunología , Proteínas Opsoninas/farmacología , Fagocitosis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologíaRESUMEN
The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Antígeno CD11b/inmunología , Proteínas del Sistema Complemento/inmunología , Células Dendríticas/inmunología , Receptores de Complemento/inmunología , Animales , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Células Cultivadas , Activación de Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Células Dendríticas/metabolismo , Dextranos/química , Portadores de Fármacos/química , Humanos , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Nanopartículas/química , Proteínas Opsoninas/inmunología , Proteínas Opsoninas/metabolismo , Fagocitosis/inmunología , Receptores de Complemento/metabolismoRESUMEN
Neutrophils play a key role in the human immune response to Staphylococcus aureus infections. These professional phagocytes rapidly migrate to the site of infection to engulf bacteria and destroy them via specialized intracellular killing mechanisms. Here we describe a robust and relatively high-throughput flow cytometry assay to quantify phagocytosis of S. aureus by human neutrophils. We show that effective phagocytic uptake of S. aureus is greatly enhanced by opsonization, i.e. the tagging of microbial surfaces with plasma-derived host proteins like antibodies and complement. Our rapid assay to monitor phagocytosis can be used to study neutrophil deficiencies and bacterial evasion, but also provides a powerful tool to assess the opsonic capacity of antibodies, either in the context of natural immune responses or immune therapies.
Asunto(s)
Técnicas Bacteriológicas , Citometría de Flujo , Neutrófilos/microbiología , Fagocitosis , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/patogenicidad , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/metabolismo , Células Cultivadas , Activación de Complemento , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Evasión Inmune , Neutrófilos/inmunología , Neutrófilos/metabolismo , Proteínas Opsoninas/inmunología , Proteínas Opsoninas/metabolismo , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/inmunología , Factores de TiempoRESUMEN
Understanding the effects mediated by a set of nanoparticle (NP)-bound host biomolecules, often indicated with the umbrella term of NP corona, is essential in nanomedicine, nanopharmacology, and nanotoxicology. Among the NP-adsorbed proteome, some factors mediate cell binding, endocytosis, and clearing by macrophages and other phagocytes (opsonins), while some others display few affinities for the cell surface (dysopsonins). The functional mapping of opsonins and dysopsonins is instrumental to design long-circulating and nanotoxicologically safe next-generation nanotheranostics. In this review, we critically analyze functional data identifying specific proteins with opsonin or dysopsonin properties. Special attention is dedicated to the following: (1) the simplicity or complexity of the NP proteome and its modulation, (2) the role of specific host proteins in mediating the stealth properties of uncoated or polymer-coated NPs, and (3) the ability of the innate immune system, and, in particular, of the complement proteins, to mediate NP clearance by phagocytes. Emerging species-specific peculiarities, differentiating humans from preclinical animal models (the murine especially), are highlighted throughout this overview. The operative definition of opsonin and dysopsonin and the measurement schemes to assess their in vitro efficacy is critically re-examined. This provides a shared and unbiased approach useful for NP opsonin and dysopsonin systematic identification.
Asunto(s)
Nanopartículas , Proteínas Opsoninas/inmunología , Nanomedicina Teranóstica , Animales , Proteínas Portadoras , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Guías como Asunto , Humanos , Inmunomodulación , Macrófagos/inmunología , Macrófagos/metabolismo , Nanopartículas/química , Proteínas Opsoninas/administración & dosificación , Proteínas Opsoninas/química , Fagocitos/inmunología , Fagocitos/metabolismo , Unión Proteica , Proteoma , Nanomedicina Teranóstica/métodosRESUMEN
HIV transmission via genital and colorectal mucosa are the most common routes of dissemination. Here, we explored the effects of free and complement-opsonized HIV on colorectal tissue. Initially, there was higher antiviral responses in the free HIV compared to complement-opsonized virus. The mucosal transcriptional response at 24 hr revealed the involvement of activated T cells, which was mirrored in cellular responses observed at 96 hr in isolated mucosal T cells. Further, HIV exposure led to skewing of T cell phenotypes predominantly to inflammatory CD4+ T cells, that is Th17 and Th1Th17 subsets. Of note, HIV exposure created an environment that altered the CD8+ T cell phenotype, for example expression of regulatory factors, especially when the virions were opsonized with complement factors. Our findings suggest that HIV-opsonization alters the activation and signaling pathways in the colorectal mucosa, which promotes viral establishment by creating an environment that stimulates mucosal T cell activation and inflammatory Th cells.
Asunto(s)
Activación de Complemento/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Mucosa Intestinal/inmunología , Activación de Linfocitos/inmunología , Adolescente , Adulto , Colon/inmunología , Colon/virología , Proteínas del Sistema Complemento/química , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Femenino , Humanos , Mucosa Intestinal/virología , Masculino , Proteínas Opsoninas/química , Proteínas Opsoninas/inmunología , Proteínas Opsoninas/metabolismo , Linfocitos T/inmunología , Adulto JovenRESUMEN
The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to opsono-phagocytic killing assays. An advantage of the opsono-adherence assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/inmunología , Anticuerpos Antibacterianos/inmunología , Bacillus anthracis/inmunología , Adhesión Bacteriana , Proteínas Opsoninas/inmunología , Animales , Carbunco/microbiología , Carbunco/prevención & control , Antígenos Bacterianos/inmunología , Fluoresceína-5-Isotiocianato/metabolismo , Fluorescencia , Humanos , Macrófagos/inmunología , Ratones , Primates/inmunología , Primates/microbiología , Células RAW 264.7RESUMEN
Quantification of complement deposition and subsequent neutrophil-mediated uptake provides an important way to assess the role of different bacterial factors in evasion of the host innate immune response. Here, we describe flow cytometry-based methods to allow quantification of deposition of the complement opsonin C3 on the bacterial surface and subsequent uptake by primary human neutrophils. The assays outlined below provide key methods to determine whether specific bacterial factors are involved in the evasion of complement-mediated immunity, using widely accessible reagents and equipment.
Asunto(s)
Citometría de Flujo/métodos , Neutrófilos/inmunología , Streptococcus pyogenes/inmunología , Complemento C3/inmunología , Humanos , Proteínas Opsoninas/inmunología , Fagocitosis/inmunologíaRESUMEN
Group A Streptococcus (GAS) is a major cause of global mortality, yet there are no licensed GAS vaccines. Vaccine progress has been hampered, in part, by a lack of standardized assays able to quantify antibody function in test antisera. The most widely used assay was developed over 50 years ago by Rebecca Lancefield and relies on human whole blood as a source of complement and neutrophils. Recently, an opsonophagocytic killing (OPK) assay has been developed for GAS by adapting the OPK methods utilized in Streptococcus pneumoniae vaccine testing. This assay uses dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60 cells) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in the Lancefield assays. This protocol outlines methods for performing a GAS OPK assay including titering test sera to generate an opsonic index. This in vitro assay could aid in selecting vaccine candidates by demonstrating whether candidate-induced antibodies lead to complement deposition and opsonophagocytic killing.
Asunto(s)
Proteínas Opsoninas/inmunología , Streptococcus pyogenes/inmunología , Vacunas/inmunología , Anticuerpos Antibacterianos/inmunología , Bioensayo , Proteínas del Sistema Complemento/inmunología , Células HL-60 , Humanos , Sueros Inmunes/inmunología , Neutrófilos/inmunología , Proteínas Opsoninas/metabolismo , Fagocitosis/inmunología , Vacunas Neumococicas , Infecciones Estreptocócicas/inmunologíaRESUMEN
Recent global advocacy efforts have highlighted the importance of development of a vaccine against group A Streptococcus (GAS). Combo5 is a non-M protein-based vaccine that provides protection against GAS skin infection in mice and reduces the severity of pharyngitis in nonhuman primates. However, Combo5 with the addition of aluminum hydroxide (alum) as an adjuvant failed to protect against invasive GAS infection of mice. Here, we show that formulation of Combo5 with adjuvants containing saponin QS21 significantly improves protective efficacy, even though all 7 adjuvants tested generated high antigen-specific IgG antibody titers, including alum. Detailed characterization of Combo5 formulated with SMQ adjuvant, a squalene-in-water emulsion containing a TLR4 agonist and QS21, showed significant differences from the results obtained with alum in IgG subclasses generated following immunization, with an absence of GAS opsonizing antibodies. SMQ, but not alum, generated strong interleukin-6 (IL-6), gamma interferon (IFN-γ), and tumor necrosis alpha (TNF-α) responses. This work highlights the importance of adjuvant selection for non-M protein-based GAS vaccines to optimize immune responses and protective efficacy.IMPORTANCE Availability of a group A Streptococcus vaccine remains an unmet public health need. Here, we tested different adjuvant formulations to improve the protective efficacy of non-M protein vaccine Combo5 in an invasive disease model. We show that novel adjuvants can dramatically shape the type of immune response developed following immunization with Combo5 and significantly improve protection. In addition, protection afforded by Combo5 is not mediated by opsonizing antibodies, believed to be the main correlate of protection against GAS infections. Overall, this report highlights the importance of adjuvant selection in raising protective immune responses against GAS invasive infection. Adjuvants that can provide a more balanced Th1/Th2-type response may be required to optimize protection of GAS vaccines, particularly those based on non-M protein antigens.
Asunto(s)
Inmunidad Celular , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/inmunología , Células TH1/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Animales , Anticuerpos Antibacterianos/inmunología , Citocinas/inmunología , Femenino , Inmunización , Inmunoglobulina G/inmunología , Interferón gamma/inmunología , Masculino , Ratones , Proteínas Opsoninas/inmunología , Vacunas Estreptocócicas/administración & dosificaciónRESUMEN
Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by antibody-mediated platelet destruction. Different mechanisms have been suggested to explain accelerated platelet clearance and impaired thrombopoiesis, but the pathophysiology of ITP has yet to be fully delineated. In this study, we tested 2 mouse models of immune-mediated thrombocytopenia using the rat anti-mouse GPIbα monoclonal antibody 5A7, generated in our laboratory. After a single IV administration of high-dose (2 mg/kg) 5A7, opsonized platelets were rapidly cleared from the circulation into the spleen and liver; this was associated with rapid upregulation of thrombopoietin (TPO) messenger RNA. In contrast, subcutaneous administration of low-dose 5A7 (0.08-0.16 mg/kg) every 3 days gradually lowered the platelet count; in this case, opsonized platelets were observed only in the spleen, and TPO levels remained unaltered. Interestingly, in both models, the 5A7 antibody was found on the surface of, as well as internalized to, bone marrow megakaryocytes. Consequently, platelets generated in the chronic phase of repeated subcutaneous 5A7 administration model showed reduced GPIbα membrane expression on their surface. Our findings indicate that evaluation of platelet surface GPIbα relative to platelet size may be a useful marker to support the diagnosis of anti-GPIbα antibody-induced ITP.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Púrpura Trombocitopénica Idiopática/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/toxicidad , Reacciones Antígeno-Anticuerpo , Plaquetas/inmunología , Modelos Animales de Enfermedad , Inyecciones Intravenosas , Inyecciones Subcutáneas , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Opsoninas/inmunología , Agregación Plaquetaria/inmunología , Complejo GPIb-IX de Glicoproteína Plaquetaria/antagonistas & inhibidores , Púrpura Trombocitopénica Idiopática/etiología , ARN Mensajero/biosíntesis , Ratas , Bazo/patología , Trombopoyetina/biosíntesis , Trombopoyetina/genética , Regulación hacia ArribaRESUMEN
Opsonins are soluble, extracellular proteins, released by activated immune cells, and when bound to a target cell, can induce phagocytes to phagocytose the target cell. There are three known classes of opsonin: antibodies, complement factors and secreted pattern recognition receptors, but these have limited access to the brain. We identify here two novel opsonins of bacteria, calreticulin, and galectin-3 (both lectins that can bind lipopolysaccharide), which were released by microglia (brain-resident macrophages) when activated by bacterial lipopolysaccharide. Calreticulin and galectin-3 both bound to Escherichia coli, and when bound increased phagocytosis of these bacteria by microglia. Furthermore, lipopolysaccharide-induced microglial phagocytosis of E. coli bacteria was partially inhibited by: sugars, an anti-calreticulin antibody, a blocker of the calreticulin phagocytic receptor LRP1, a blocker of the galectin-3 phagocytic receptor MerTK, or simply removing factors released from the microglia, indicating this phagocytosis is dependent on extracellular calreticulin and galectin-3. Thus, calreticulin and galectin-3 are opsonins, released by activated microglia to promote clearance of bacteria. This innate immune response of microglia may help clear bacterial infections of the brain.
Asunto(s)
Calreticulina/inmunología , Infecciones por Escherichia coli/inmunología , Galectina 3/inmunología , Microglía/inmunología , Proteínas Opsoninas/inmunología , Animales , Encéfalo/inmunología , Escherichia coli/inmunología , Inmunidad Innata/inmunología , Ratones , Fagocitosis/inmunología , RatasRESUMEN
Enhanced understanding of bio-nano interaction requires recognition of hidden factors such as protein corona, a layer of adsorbed protein around nano-systems. This study compares the biological identity and fingerprint profile of adsorbed proteins on PLGA-based nanoparticles through nano-liquid chromatography-tandem mass spectrometry. The total proteins identified in the corona of nanoparticles (NPs) with different in size, charge and compositions were classified based on molecular mass, isoelectric point and protein function. A higher abundance of complement proteins was observed in modified NPs with an increased size, while NPs with a positive surface charge exhibited the minimum adsorption for immunoglobulin proteins. A correlation of dysopsonin/opsonin ratio was found with cellular uptake of NPs exposed to two positive and negative Fc receptor cell lines. Although the higher abundance of dysopsonins such as apolipoproteins may cover the active sites of opsonins causing a lower uptake, the correlation of adsorbed dysopsonin/opsonin proteins on the NPs surface has an opposite trend with the intensity of cell uptake. Despite the reduced uptake of corona-coated NPs in comparison with pristine NPs, the dysopsonin/opsonin ratio controlled by the physicochemistry properties of NPs could potentially be used to tune up the cellular delivery of polymeric NPs.
Asunto(s)
Sistemas de Liberación de Medicamentos , Nanopartículas/química , Proteínas Opsoninas , Corona de Proteínas , Animales , Células CHO , Cricetulus , Humanos , Ratones , Proteínas Opsoninas/química , Proteínas Opsoninas/inmunología , Tamaño de la Partícula , Corona de Proteínas/química , Corona de Proteínas/inmunología , Células RAW 264.7RESUMEN
Vaccination against meningococcal serogroup B is recommended for patients with a complement deficiency; however, although immunogenicity in this patient group has been shown, efficacy has not yet been established. In this study, we collected serum from children with a complement deficiency in the alternative pathway or in late terminal pathway before and after vaccination with multi-component meningococcal serogroup B (MenB)-4C. MenB-4C is a multi-component, protein-based vaccine against MenB consisting of factor H-binding protein, Neisserial heparin-binding protein, Neisserial adhesion A and outer membrane vesicles containing Porin A. We assessed the vaccine immunogenicity and vaccine-mediated protection by a whole cell enzyme-linked immunosorbent assay with Neisseria meningitidis serogroup B strains H44/76, 5/99 and NZ98/254, which shows that vaccination induced antibody titers against meningococcus. We show that the classical serum bactericidal activity assay with exogenous serum indicates the presence of vaccine-induced antibodies and capacity to activate complement-mediated pathogen lysis. However, in children with a late terminal pathway deficiency, no complement-mediated pathogen lysis was observed when autologous serum was applied in the serum bactericidal activity assay, demonstrating a lack of serum bactericidal activity in children with complement deficiencies. However, MenB-4C vaccination still induced effective complement-dependent opsonophagocytic killing against N. meningitidis serogroup B in reconstituted whole blood with autologous serum from children with an alternative pathway or late terminal pathway deficiency. These findings support the recommendation to vaccinate all complement-deficient children against MenB.
Asunto(s)
Enfermedades por Deficiencia de Complemento Hereditario/inmunología , Meningitis Meningocócica/inmunología , Vacunas Meningococicas/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Proteínas Opsoninas/inmunología , Fagocitosis/inmunología , Adolescente , Adulto , Anticuerpos Antibacterianos/inmunología , Niño , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Femenino , Enfermedades por Deficiencia de Complemento Hereditario/microbiología , Enfermedades por Deficiencia de Complemento Hereditario/terapia , Humanos , Masculino , Meningitis Meningocócica/microbiología , Meningitis Meningocócica/terapia , Vacunas Meningococicas/administración & dosificación , Neisseria meningitidis Serogrupo B/fisiología , Proteínas Opsoninas/metabolismo , VacunaciónRESUMEN
Multidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here, we describe the development of two MAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide of Enterococcus faecalis, and the secreted antigen A (SagA), an immunogenic protein from Enterococcus faecium, are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while enzyme-linked immunosorbent assay and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process, two highly specific IgG1(κ) MAbs were obtained, one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both MAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing against E. faecalis type 2, and SagA.01 showed 40% killing against E. faecium 11231/6. In addition, both MAbs showed cross-reactivity toward other E. faecalis and E. faecium strains. The sequences from the variable regions of the heavy and light chains were reconstructed in expression vectors, and the activity of the MAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic MAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Farmacorresistencia Microbiana , Enterococcus faecalis/inmunología , Enterococcus faecium/inmunología , Inmunoterapia/métodos , Proteínas Opsoninas/inmunología , Animales , Antígenos Bacterianos/inmunología , Cápsulas Bacterianas/química , Ratones , Polisacáridos/inmunologíaRESUMEN
A fundamental challenge in medical microbiology is to characterize the dynamic protein-protein interaction networks formed at the host-pathogen interface. Here, we generate a quantitative interaction map between the significant human pathogen, Streptococcus pyogenes, and proteins from human saliva and plasma obtained via complementary affinity-purification and bacterial-surface centered enrichment strategies and quantitative mass spectrometry. Perturbation of the network using immunoglobulin protease cleavage, mixtures of different concentrations of saliva and plasma, and different S. pyogenes serotypes and their isogenic mutants, reveals how changing microenvironments alter the interconnectivity of the interaction map. The importance of host immunoglobulins for the interaction with human complement proteins is demonstrated and potential protective epitopes of importance for phagocytosis of S. pyogenes cells are localized. The interaction map confirms several previously described protein-protein interactions; however, it also reveals a multitude of additional interactions, with possible implications for host-pathogen interactions involving other bacterial species.