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Frontiers in Pharmacology 2022Despite the power of antibiotics, bacterial infections remain a major killer, due to antibiotic resistance and hosts with dysregulated immune systems. We and others have...
Despite the power of antibiotics, bacterial infections remain a major killer, due to antibiotic resistance and hosts with dysregulated immune systems. We and others have been developing drug-loaded nanoparticles that home to the sites of infection and inflammation via engineered tropism for neutrophils, the first-responder leukocytes in bacterial infections. Here, we examined how a member of a broad class of neutrophil-tropic nanoparticles affects neutrophil behavior, specifically questioning whether the nanoparticles attenuate an important function, bacterial phagocytosis. We found these nanoparticles actually phagocytosis of non-opsonized bacteria, increasing it by ∼50%. We showed this augmentation of phagocytosis is likely co-opting an evolved response, as opsonized bacteria also augment phagocytosis of non-opsonized bacteria. Enhancing phagocytosis of non-opsonized bacteria may prove particularly beneficial in two clinical situations: in hypocomplementemic patients (meaning low levels of the main bacterial opsonins, complement proteins, seen in conditions such as neonatal sepsis and liver failure) or for bacteria that are largely resistant to complement opsonization (e.g., ). Additionally, we observe that; 1) prior treatment with bacteria augments neutrophil uptake of neutrophil-tropic nanoparticles; 2) neutrophil-tropic nanoparticles colocalize with bacteria inside of neutrophils. The observation that neutrophil-tropic nanoparticles enhance neutrophil phagocytosis and localize with bacteria inside neutrophils suggests that these nanoparticles will serve as useful carriers for drugs to ameliorate bacterial diseases.
PubMed: 35860017
DOI: 10.3389/fphar.2022.923814 -
Frontiers in Immunology 2018Intravenous immunoglobulins (IVIg) are used in the treatment of different autoimmune and inflammatory diseases, such as immune thrombocytopenia and hemolytic anemia. One...
Intravenous immunoglobulins (IVIg) are used in the treatment of different autoimmune and inflammatory diseases, such as immune thrombocytopenia and hemolytic anemia. One of the modes of action of IVIg is preventing phagocytosis of autoantibody-opsonized blood cells by saturation of the Fc-gamma receptors of macrophages in spleen and liver. IgG contains a fixed glycan, which is in most cases biantennary, at the asparagine residue at position 297 in the Fc tail. This glycan consists of a core structure of N-acetyl glucosamine (GlcNAc) and mannose groups, variably extended with core fucose, bisecting GlcNAc as well as terminal galactose and sialic acid. This structural glycan influences the binding affinity of IgG to Fc-gamma receptors. By glyco-engineering, we generated monoclonal IgG antibodies with different Fc-tail glycans and tested both their opsonizing and blocking capacity in a phagocytosis assay of IgG-opsonized erythrocytes with human monocyte-derived macrophages. In contrast to a lack of effect in opsono-phagocytosis, these IgG glycovariants differentially inhibited the uptake of opsonized erythrocytes. The level of bisecting GlcNAc and galactosylation had unexpectedly larger impact than core fucosylation, and suggest that targeted modifications different from the core fucose may well improve the immunomodulating efficacy of IVIg treatment.
Topics: Acetylglucosamine; Anemia, Hemolytic; Antibody Affinity; Autoantibodies; Cells, Cultured; Humans; Immunoglobulin G; Immunoglobulins, Intravenous; Macrophages; Phagocytosis; Protein Binding; Protein Engineering; Purpura, Thrombocytopenic, Idiopathic; Receptors, IgG; Structure-Activity Relationship
PubMed: 30405631
DOI: 10.3389/fimmu.2018.02442 -
Microbiology Spectrum Dec 2021The current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a...
The current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human ST3 pneumococcal capsular polysaccharide (PPS3) antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven PPS3-specific human monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutination , and four mediated both activities. Two humAbs, namely, C10 and C27, that used the same variable heavy (V) and light (V) chain domains (V3-9*01/V2-14*03) both altered ST3 gene expression ; however, C10 had fewer V somatic mutations, higher PPS3 affinity, and promoted ST3 opsonophagocytic and agglutinating activity, whereas C27 did not. In C57BL/6 mice, both humAbs reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. After performing V swaps, C10V/C27V exhibited reduced ST3 binding and agglutination, but C27V/C10V binding was unchanged. However, both humAbs lost the ability to reduce colonization when their light chains were replaced. Our findings associate the ability of PPS3-specific humAbs to reduce colonization with ST3 agglutination and opsonophagocytic activity, and reveal an unexpected role for the V in their functional activity and . These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3. Despite the global success of vaccination with pneumococcal conjugate vaccines, serotype 3 (ST3) pneumococcus remains a leading cause of morbidity and mortality. In comparison to other vaccine-included serotypes, the ST3 pneumococcal capsular polysaccharide (PPS3) induces a weaker opsonophagocytic response, which is considered a correlate of vaccine efficacy. Previous studies of mouse PPS3 monoclonal antibodies identified ST3 agglutination as a correlate of reduced ST3 nasopharyngeal colonization in mice; however, neither the agglutinating ability of human vaccine-elicited PPS3 antibodies nor their ability to prevent experimental murine nasopharyngeal colonization has been studied. We generated and analyzed the functional and efficacy of human vaccine-elicited PPS3 monoclonal antibodies and found that ST3 agglutination associated with antibody affinity, protection , and limited somatic mutations in the light chain variable region. These findings provide new insights that may inform the development of antibody-based therapies and next-generation vaccines for ST3.
Topics: Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Antibody Affinity; Bacterial Capsules; Cell Line; Female; HEK293 Cells; Humans; Immunoglobulin Fab Fragments; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Mice; Mice, Inbred C57BL; Nasopharynx; Opsonization; Pneumococcal Vaccines; Pneumonia, Pneumococcal; Polysaccharides, Bacterial; Serogroup; Single-Chain Antibodies; Streptococcus pneumoniae; Vaccine Efficacy
PubMed: 34756090
DOI: 10.1128/Spectrum.01446-21 -
Frontiers in Veterinary Science 2022Previous studies demonstrated that polyclonal antibodies against foot-and-mouth disease virus (FMDV) generated by vaccination can mediate immune functions not only...
Previous studies demonstrated that polyclonal antibodies against foot-and-mouth disease virus (FMDV) generated by vaccination can mediate immune functions not only through virus neutralization but also through promoting virus uptake by macrophages and dendritic cells that are otherwise resistant to FMDV infection. This causes abortive infections resulting in activation, enhanced antigen presentation but also cell death. Here we report the use of RAW264.7 cells representing a murine macrophage cells line to characterize opsonizing functions of a collection of monoclonal antibodies (mAbs) against FMDV O and A serotypes. We demonstrate that all neutralizing immunoglobulin G isotype mAbs are able to opsonize FMDV resulting in increased cell death of RAW264.7 cells. In contrast, neutralizing IgM antibodies did not possess this activity. Opsonization was observed with broader reactivity within the serotype when compared to neutralization. Importantly, the anti-O serotype D9 mAb reacting with the continuous epitope within the G-H loop of VP1 that contains the RGD binding site of FMDV, opsonized several FMDV serotypes despite its restricted neutralizing activity within the O serotype. Furthermore, by generating RAW264.7 cells expressing bovine CD32, an easy-to-use cell-based assay system to test for bovine antibody-dependent enhanced infection of FMDV was generated and tested with a collection of sera. The data indicate that opsonizing titers correlated better with vaccine dose when compared to neutralizing titers. On the other hand, neutralization and opsonization titers were similar predictive of protection. We conclude that low avidity interactions are sufficient to mediate Fcγ receptor-mediated immune functions that could contribute to protective immune responses against FMDV.
PubMed: 36311653
DOI: 10.3389/fvets.2022.1033276 -
Journal of Nanobiotechnology Oct 2023Nanoparticles represent one of the most important innovations in the medical field. Among nanocarriers, polymeric nanoparticles (PNPs) attracted much attention due to...
BACKGROUND
Nanoparticles represent one of the most important innovations in the medical field. Among nanocarriers, polymeric nanoparticles (PNPs) attracted much attention due to their biodegradability, biocompatibility, and capacity to increase efficacy and safety of encapsulated drugs. Another important improvement in the use of nanoparticles as delivery systems is the conjugation of a targeting agent that enables the nanoparticles to accumulate in a specific tissue. Despite these advantages, the clinical translation of therapeutic approaches based on nanoparticles is prevented by their interactions with blood proteins. In fact, the so-formed protein corona (PC) drastically alters the biological identity of the particles. Adsorbed activated proteins of the complement cascade play a pivotal role in the clearance of nanoparticles, making them more easily recognized by macrophages, leading to their rapid elimination from the bloodstream and limiting their efficacy. Since the mouse is the most used preclinical model for human disease, this work compared human and mouse PC formed on untargeted PNPs (uPNPs) and targeted PNPs (tPNPs), paying particular attention to complement activation.
RESULTS
Mouse and human serum proteins adsorbed differently to PNPs. The differences in the binding of mouse complement proteins are minimal, whereas human complement components strongly distinguish the two particles. This is probably due to the human origin of the Fc portion of the antibody used as targeting agent on tPNPs. tPNPs and uPNPs mainly activate complement via the classical and alternative pathways, respectively, but this pattern did not affect their binding and internalization in macrophages and only a limited consumption of the activity of the human complement system was documented.
CONCLUSIONS
The results clearly indicate the presence of complement proteins on PNPs surface but partially derived from an unspecific deposition rather than an effective complement activation. The presence of a targeting antibody favors the activation of the classical pathway, but its absence allows an increased activation of the alternative pathway. This results in similar opsonization of both PNPs and similar phagocytosis by macrophages, without an impairment of the activity of circulating complement system and, consequently, not enhancing the susceptibility to infection.
Topics: Humans; Mice; Animals; Protein Corona; Opsonization; Complement System Proteins; Antibodies; Polymers; Nanoparticles
PubMed: 37838659
DOI: 10.1186/s12951-023-02134-4 -
Frontiers in Immunology 2020poly-γ-D-glutamic acid (PGA) capsule is an essential virulent factor that helps the bacterial pathogen to escape host immunity. Like other encapsulated bacterial...
poly-γ-D-glutamic acid (PGA) capsule is an essential virulent factor that helps the bacterial pathogen to escape host immunity. Like other encapsulated bacterial species, the capsule may also inhibit complement-mediated clearance and ensure bacterial survival in the host. Previous reports suggest that spore proteins inhibit complement activation. However, the mechanism through which the capsule imparts a survival advantage to the active bacteria has not been demonstrated till date. Thus, to evaluate the role of the PGA capsule in evading host immunity, we have undertaken the present head-to-head comparative study of the phagocytosis and complement activation of non-encapsulated and encapsulated strains. The encapsulated virulent strain exhibited resistance toward complement-dependent and complement-independent bacterial phagocytosis by human macrophages. The non-encapsulated Sterne strain was highly susceptible to phagocytosis by THP-1 macrophages, after incubation with normal human serum (NHS), heat-inactivated serum, and serum-free media, thus indicating that the capsule inhibited both complement-dependent and complement-independent opsonic phagocytosis. An increased binding of C3b and its subsequent activation to C3c and C3dg, which functionally act as potent opsonins, were observed with the non-encapsulated Sterne strain compared with the encapsulated strain. Other known mediators of complement fixation, IgG, C-reactive protein (CRP), and serum amyloid P component (SAP), also bound more prominently with the non-encapsulated Sterne strain. Studies with complement pathway-specific, component-deficient serum demonstrated that the classical pathway was primarily involved in mediating C3b binding on the non-encapsulated bacteria. Both strains equally bound the complement regulatory proteins C4BP and factor H. Importantly, we demonstrated that the negative charge of the PGA capsule was responsible for the differential binding of the complement proteins between the non-encapsulated and encapsulated strains. At lower pH closer to the isoelectric point of PGA, the neutralization of the negative charge was associated with an increased binding of C3b and IgG with the encapsulated strain. Overall, our data have demonstrated that the capsule inhibits complement fixation and opsonization resulting in reduced phagocytosis by macrophages, thus allowing the bacterial pathogen to evade host immunity.
Topics: Anthrax; Antigens, Bacterial; Bacillus anthracis; Bacterial Capsules; Complement Activation; Complement C3b; Humans; Immune Evasion; Macrophages; Opsonin Proteins; Phagocytosis; Polyglutamic Acid; Protein Binding; THP-1 Cells; Virulence
PubMed: 32296419
DOI: 10.3389/fimmu.2020.00462 -
Infection and Immunity Aug 2016It is unclear whether naturally acquired immunity to Plasmodium falciparum results from the acquisition of antibodies to multiple, diverse antigens or to fewer, highly...
It is unclear whether naturally acquired immunity to Plasmodium falciparum results from the acquisition of antibodies to multiple, diverse antigens or to fewer, highly conserved antigens. Moreover, the specific antibody functions required for malaria immunity are unknown, and hence informative immunological assays are urgently needed to address these knowledge gaps and guide vaccine development. In this study, we investigated whether merozoite-opsonizing antibodies are associated with protection from malaria in a strain-specific or strain-transcending manner by using a novel field isolate and an immune plasma-matched cohort from Papua New Guinea with our validated assay of merozoite phagocytosis. Highly correlated opsonization responses were observed across the 15 parasite strains tested, as were strong associations with protection (composite phagocytosis score across all strains in children uninfected at baseline: hazard ratio of 0.15, 95% confidence interval of 0.04 to 0.63). Opsonizing antibodies had a strong strain-transcending component, and the opsonization of transgenic parasites deficient for MSP3, MSP6, MSPDBL1, or P. falciparum MSP1-19 (PfMSP1-19) was similar to that of wild-type parasites. We have provided the first evidence that merozoite opsonization is predominantly strain transcending, and the highly consistent associations with protection against diverse parasite strains strongly supports the use of merozoite opsonization as a correlate of immunity for field studies and vaccine trials. These results demonstrate that conserved domains within merozoite antigens targeted by opsonization generate strain-transcending immune responses and represent promising vaccine candidates.
Topics: Adolescent; Antibodies, Protozoan; Antigens, Protozoan; Child; Child, Preschool; Humans; Malaria, Falciparum; Merozoites; Opsonin Proteins; Patient Outcome Assessment; Phagocytosis; Plasmodium falciparum
PubMed: 27185785
DOI: 10.1128/IAI.00145-16 -
Microbiology Spectrum Aug 2023Mycobacterium abscessus, an intracellular nontuberculous mycobacterium, is considered the most pathogenic species among the group of rapidly growing mycobacteria. The...
Mycobacterium abscessus, an intracellular nontuberculous mycobacterium, is considered the most pathogenic species among the group of rapidly growing mycobacteria. The resistance of M. abscessus to the host innate response contributes to its pathogenicity in addition to several virulence factors. We have recently shown in that antimicrobial peptides (AMPs), whose production is induced by M. abscessus, are unable to control mycobacterial infection. This could be due to their inability to kill mycobacteria and/or the hidden location of the pathogen in phagocytic cells. Here, we demonstrate that the rapid internalization of M. abscessus by macrophages allows it to escape the AMP-mediated humoral response. By depleting phagocytes in AMP-deficient flies, we found that several AMPs were required for the control of extracellular M. abscessus. This was confirmed in the Tep4 opsonin-deficient flies, which we show can better control M. abscessus growth and have increased survival through overproduction of some AMPs, including Defensin. Furthermore, Defensin alone was sufficient to kill extracellular M. abscessus both and and control its infection. Collectively, our data support that Tep4-mediated opsonization of M. abscessus allows its escape and resistance toward the Defensin bactericidal action in . Mycobacterium abscessus, an opportunistic pathogen in cystic fibrosis patients, is the most pathogenic species among the fast-growing mycobacteria. How M. abscessus resists the host innate response before establishing an infection remains unclear. Using , we have recently demonstrated that M. abscessus resists the host innate response by surviving the cytotoxic lysis of the infected phagocytes and the induced antimicrobial peptides (AMPs), including Defensin. In this work, we demonstrate that M. abscessus resists the latter response by being rapidly internalized by phagocytes. Indeed, by combining and approaches, we show that Defensin is able to control extracellular M. abscessus infection through a direct bactericidal action. In conclusion, we report that M. abscessus escapes the host AMP-mediated humoral response by taking advantage of its internalization by the phagocytes.
Topics: Animals; Mycobacterium abscessus; Drosophila; Opsonization; Mycobacterium; Antimicrobial Peptides; Defensins; Mycobacterium Infections, Nontuberculous; Anti-Bacterial Agents
PubMed: 37260399
DOI: 10.1128/spectrum.00777-23 -
Nihon Ishinkin Gakkai Zasshi = Japanese... 2002Cryptococcus neoformans, a facultative intracellular pathogen of macrophages, is unique among medically important fungi in its possession of a polysaccharide capsule.... (Review)
Review
Cryptococcus neoformans, a facultative intracellular pathogen of macrophages, is unique among medically important fungi in its possession of a polysaccharide capsule. Capsule represents the organism's major virulence factor. In the absence of opsonins, binding of encapsulated C. neoformans to macrophages is minimal. Following incubation in serum, C. neoformans potently activates complement, resulting in surface deposition of the third component of complement. Macrophages bind and phagocytose opsonized C. neoformans via three major complement receptors (CR) for C3 fragments, designated CD35 (CR1), CD11b/CD18 (CR3), and CD11c/CD18 (CR4). Antibody in normal human serum generally lacks opsonic activity, although vaccination can elicit anticapsular antibodies that are opsonic. The major component of cryptococcal capsule, glucuronoxylomannan (GXM), is shed from the fungus and circulates in the blood and cerebrospinal fluid of patients with cryptococcosis. Cellular receptors defined for GXM include CD14, toll-like receptor-2, toll-like receptor-4, and CD18. GXM binding to macrophage receptors triggers activation of nuclear factor-kB, but not mitogen-activated protein kinases. This results in no proinflammatory gene expression or release. C. neoformans also secretes mannoproteins, which are recognized by mannose receptors as well as by mannose-binding lectin, perhaps in conjunction with CD14. Strategies directed at modulating how intact C. neoformans and its released components are recognized by phagocytes could lead to novel approaches to treating cryptococcosis
Topics: CD18 Antigens; Complement Activation; Cryptococcus neoformans; Drosophila Proteins; Humans; Lectins, C-Type; Lipopolysaccharide Receptors; Macrophages; Mannose Receptor; Mannose-Binding Lectin; Mannose-Binding Lectins; Membrane Glycoproteins; Phagocytosis; Polysaccharides; Receptors, Cell Surface; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; Virulence Factors
PubMed: 12145626
DOI: 10.3314/jjmm.43.133 -
Infection and Immunity Dec 2020Bubonic plague results when is deposited in the skin via the bite of an infected flea. Bacteria then traffic to the draining lymph node (dLN) where they replicate to...
Bubonic plague results when is deposited in the skin via the bite of an infected flea. Bacteria then traffic to the draining lymph node (dLN) where they replicate to large numbers. Without treatment, this infection can result in highly fatal septicemia. Several plague vaccine candidates are currently at various stages of development, but no licensed vaccine is available in the United States. Though polyclonal and monoclonal antibodies (Ab) can provide complete protection against bubonic plague in animal models, the mechanisms responsible for this antibody-mediated immunity (AMI) to remain poorly understood. Here, we examine the effects of Ab opsonization on interactions with phagocytes and Opsonization of with polyclonal antiserum modestly increased phagocytosis/killing by an oxidative burst of murine neutrophils Intravital microscopy (IVM) showed increased association of Ab-opsonized with neutrophils in the dermis in a mouse model of bubonic plague. IVM of popliteal LNs after intradermal (i.d.) injection of bacteria in the footpad revealed increased -neutrophil interactions and increased neutrophil crawling and extravasation in response to Ab-opsonized bacteria. Thus, despite only having a modest effect in assays, opsonizing Ab had a dramatic effect on -neutrophil interactions in the dermis and dLN very early after infection. These data shed new light on the importance of neutrophils in AMI to and may provide a new correlate of protection for evaluation of plague vaccine candidates.
Topics: Animals; Antibodies, Bacterial; Antibody-Dependent Cell Cytotoxicity; Complement System Proteins; Cytokines; Disease Models, Animal; Host-Pathogen Interactions; Immunity, Innate; Lymph Nodes; Mice; Neutrophils; Plague; Reactive Oxygen Species; Skin; Type III Secretion Systems; Yersinia pestis
PubMed: 33077628
DOI: 10.1128/IAI.00061-20