RESUMEN
The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.
Asunto(s)
COVID-19/inmunología , Inmunidad Humoral , Receptores de Reconocimiento de Patrones/inmunología , SARS-CoV-2/inmunología , Animales , Proteína C-Reactiva/inmunología , Proteína C-Reactiva/metabolismo , COVID-19/metabolismo , COVID-19/virología , Estudios de Casos y Controles , Chlorocebus aethiops , Activación de Complemento , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Femenino , Glicosilación , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Masculino , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/inmunología , Lectina de Unión a Manosa/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Polimorfismo Genético , Unión Proteica , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Componente Amiloide P Sérico/inmunología , Componente Amiloide P Sérico/metabolismo , Transducción de Señal , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células VeroRESUMEN
Complement pathways, traditionally regarded as separate entities in vitro, are increasingly noted for cross-communication and bypass mechanisms. Among these, the MBL/ficolin/CL-associated serine protease (MASP)-3, a component of lectin pathway pattern recognition molecules, has shown the ability to process critical substrates such as pro-factor D and insulin growth factor binding protein-5. Given shared features between lectin pathway pattern recognition molecules and C1q from the classical pathway, we hypothesized that C1q might be a viable in vivo binding partner for the MASPs. We used microscale thermophoresis, ELISA, and immunoprecipitation assays to detect C1q/MASP complexes and functionally assessed the complexes through enzymatic cleavage assays. C1q/MASP-3 complexes were detected in human serum and correlated well with MASP-3 serum levels in healthy individuals. The binding affinity between MASP-3 and C1q in vitro was in the nanomolar range, and the interaction was calcium-dependent, as demonstrated by their dissociation in the presence of EDTA. Furthermore, most of the circulating C1q-bound MASP-3 was activated. Based on immunoprecipitation, also C1q/MASP-2 complexes appeared to be present in serum. Finally, C1q/MASP-2 and C1q/MASP-3 in vitro complexes were able to cleave C4 and pro-factor D, respectively. Our study reveals the existence of C1q/MASP complexes in the circulation of healthy individuals, and both C1q/MASP-2 and C1q/MASP-3 complexes display proteolytic activity. Hence, this study uncovers a crosstalk route between complement pathways not previously described.
RESUMEN
Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway capable of interacting with collectin-10 (CL-10) and the MASPs to activate the complement cascade. Alternative splicing of the COLEC11 gene gives rise to two different isoforms found in serum (A and D). These isoforms vary in the length of their collagen-like region, which is involved in the stabilization of the trimeric subunit and the interaction with the MASPs. Here we aim at elucidating the biological differences of naturally occurring CL-11 isoforms A and D. We produced recombinant CL-11 as independent isoforms (CL-11A and CL-11D) and together with CL-10 (CL-10/11A, CL-10/11D). Both CL-11 isoforms associated with CL-10, but CL-11D did so to a lesser extent. CL-10/11 heterocomplexes were composed of trimeric subunits of CL-10 and CL-11, as opposed to CL-10 and CL-11 homotrimers. Heterocomplexes were more stable and migrated with higher apparent molecular weights. Immunoprecipitation of serum CL-11 and subsequent mass spectrometry analysis confirmed that native CL-11 circulates in the form of CL-10/11 heterocomplexes that associate with MASP-1, and MASP-3, but not necessarily MASP-2. Despite a shorter collagen region, CL-11D was capable to bind to the MASPs, suggesting that the missing exon 4 is not required for MASP association CL-11D had a reduced ligand binding compared to full-length CL-11A. Based on its reduced ability to oligomerize, form CL-10/11 heterocomplexes, and bind to ligands, we hypothesize that CL-11D may have a limited complement activation potential compared to full-length CL-11A.
Asunto(s)
Empalme Alternativo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Isoformas de Proteínas/genética , Colágeno , Colectinas/genéticaRESUMEN
The complement system is a complex, tightly regulated protein cascade involved in pathogen defense and the pathogenesis of several diseases. Thus, the development of complement modulators has risen as a potential treatment for complement-driven inflammatory pathologies. The enzymatically inactive MAP-2 has been reported to inhibit the lectin pathway by competing with its homologous serine protease MASP-2. The membrane-bound complement inhibitor CD55 acts on the C3/C5 convertase level. Here, we fused MAP-2 to the four N-terminal domains of CD55 generating a targeted chimeric inhibitor to modulate complement activation at two different levels of the complement cascade. Its biological properties were compared in vitro with the parent molecules. While MAP-2 and CD55 alone showed a minor inhibition of the three complement pathways when co-incubated with serum (IC50MAP-2+CD55 1-4 = 60.98, 36.10, and 97.01 nM on the classical, lectin, and alternative pathways, respectively), MAP-2:CD551-4 demonstrated a potent inhibitory activity (IC50MAP-2:CD55 1-4 = 2.94, 1.76, and 12.86 nM, respectively). This inhibitory activity was substantially enhanced when pre-complexes were formed with the lectin pathway recognition molecule mannose-binding lectin (IC50MAP-2:CD55 1-4 = 0.14 nM). MAP-2:CD551-4 was also effective at protecting sensitized sheep erythrocytes in a classical hemolytic assay (CH50 = 13.35 nM). Finally, the chimeric inhibitor reduced neutrophil activation in full blood after stimulation with Aspergillus fumigatus conidia, as well as phagocytosis of conidia by isolated activated neutrophils. Our results demonstrate that MAP-2:CD551-4 is a potent complement inhibitor reinforcing the idea that engineered fusion proteins are a promising design strategy for identifying and developing drug candidates to treat complement-mediated diseases.
Asunto(s)
Activación de Complemento , Proteínas del Sistema Complemento , Animales , Ovinos , Antígenos CD55/farmacología , Lectinas/metabolismo , Factores de Transcripción , Inactivadores del Complemento , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismoRESUMEN
OBJECTIVES: To investigate the effect of COVID-19 mRNA revaccination (two doses) on the antibody response in patients with rheumatic diseases (RD) who were initial vaccine non-responders. Further, to examine if B-cell levels or T-cell responses before revaccination predicted seroconversion. METHODS: From a RD cohort vaccinated with the standard two-dose COVID-19 vaccinations, we enrolled cases without detectable antibody responses (n=17) and controls with detectable antibody response (n=29). Blood donors (n=32) were included as additional controls. Samples were collected before and six weeks after completed revaccination. Total antibodies and specific IgG, IgA, and IgM against SARS-CoV-2 spike protein, SARS-CoV-2 neutralising antibodies, and SARS-CoV-2 reacting CD4+ and CD8+ T-cells were measured before and after revaccination. B-cells (CD19+CD45+) were quantified before revaccination. RESULTS: Forty-seven percent of cases had detectable neutralising antibodies after revaccination. However, antibody levels were significantly lower than in controls and blood donors. Revaccination induced an antibody class switch in cases with a decrease in IgM and increase in IgG. No significant difference was observed in T-cell responses before and after revaccination between the three groups. Only 29% of cases had measurable B-cells compared to 100% of controls and blood donors. Fifty percent of revaccinated cases who seroconverted had measurable B-cells before revaccination. CONCLUSIONS: Forty-seven percent of initial non-responders seroconverted after two-dose revaccination but still had lower levels of SARS-CoV-2 antibodies compared with controls and blood donors. RD patients without a detectable serological response after the initial COVID-19 mRNA vaccine had a T-cell response similar to immunocompetent controls and blood donors.
Asunto(s)
Artritis Reumatoide , COVID-19 , Lupus Eritematoso Sistémico , Enfermedades Reumáticas , Glicoproteína de la Espiga del Coronavirus , Humanos , Vacunas contra la COVID-19 , Inmunización Secundaria , Seroconversión , COVID-19/prevención & control , SARS-CoV-2 , Vacunación , Anticuerpos Antivirales , Anticuerpos Neutralizantes , Inmunoglobulina G , Inmunoglobulina MRESUMEN
Tools to monitor SARS-CoV-2 transmission and immune responses are needed. We present a neutralization ELISA to determine the levels of Ab-mediated virus neutralization and a preclinical model of focused immunization strategy. The ELISA is strongly correlated with the elaborate plaque reduction neutralization test (ρ = 0.9231, p < 0.0001). The neutralization potency of convalescent sera strongly correlates to IgG titers against SARS-CoV-2 receptor-binding domain (RBD) and spike (ρ = 0.8291 and 0.8297, respectively; p < 0.0001) and to a lesser extent with the IgG titers against protein N (ρ = 0.6471, p < 0.0001). The preclinical vaccine NMRI mice models using RBD and full-length spike Ag as immunogens show a profound Ab neutralization capacity (IC50 = 1.9 × 104 to 2.6 × 104 and 3.9 × 103 to 5.2 × 103, respectively). Using a panel of novel high-affinity murine mAbs, we also show that a majority of the RBD-raised mAbs have inhibitory properties, whereas only a few of the spike-raised mAbs do. The ELISA-based viral neutralization test offers a time- and cost-effective alternative to the plaque reduction neutralization test. The immunization results indicate that vaccine strategies focused only on the RBD region may have advantages compared with the full spike.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Proteínas de la Nucleocápside de Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Pruebas de Neutralización/métodos , Receptores Virales/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , COVID-19/inmunología , COVID-19/terapia , Vacunas contra la COVID-19/inmunología , Humanos , Inmunización , Inmunización Pasiva , Inmunoglobulina A/sangre , Inmunoglobulina A/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Ratones , Dominios Proteicos/inmunología , Sueroterapia para COVID-19RESUMEN
Globally, the COVID-19 pandemic has had extreme consequences for the healthcare system and has led to calls for diagnostic tools to monitor and understand the transmission, pathogenesis, and epidemiology, as well as to evaluate future vaccination strategies. In this study, we have developed novel, to our knowledge, flexible ELISA-based assays for specific detection of human SARS-CoV-2 Abs against the receptor-binding domain, including an Ag sandwich ELISA relevant for large population screening and three isotype-specific assays for in-depth diagnostics. Their performance was evaluated in a cohort of 350 convalescent participants with previous COVID-19 infection, ranging from asymptomatic to critical cases. We mapped the Ab responses to different areas on protein N and S and showed that the IgM, A, and G Ab responses against receptor-binding domain are significantly correlated to the disease severity. These assays and the data generated from them are highly relevant for diagnostics and prognostics and contribute to the understanding of long-term COVID-19 immunity.
Asunto(s)
Anticuerpos Antivirales/inmunología , COVID-19/diagnóstico , COVID-19/inmunología , Convalecencia , SARS-CoV-2/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Neutralizantes/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/genética , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 from humans to animals has been reported for many domesticated species, including farmed minks. The identification of novel spike gene mutations appearing in minks has raised major concerns about potential immune evasion and challenges for the global vaccine strategy. One genetic variant, known as "cluster five," arose among farmed minks in Denmark and resulted in a complete shutdown of the world's largest mink production. However, the functional properties of this new variant are not established. Here we present functional data on the cluster-five variant, which contains a mutation resulting in a Y453F residue change in the receptor-binding domain (RBD) of the spike protein. Using an ELISA-based angiotensin-converting enzyme-2/RBD inhibition assay, we show that the Y453F variant does not decrease established humoral immunity from previously infected individuals or affect the neutralizing antibody response in a vaccine mouse model based on the original Wuhan strain RBD or spike as antigens. However, biolayer interferometry analysis demonstrates that it binds the human angiotensin-converting enzyme-2 receptor with a 4-fold higher affinity than the original strain, suggesting an enhanced transmission capacity and a possible challenge for viral control. These results also indicate that the rise in the frequency of the cluster-five variant in mink farms might be a result of the fitness advantage conferred by the receptor adaptation rather than evading immune responses.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , COVID-19/epidemiología , COVID-19/transmisión , Visón/virología , Pandemias , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genética , Sustitución de Aminoácidos , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , COVID-19/inmunología , Convalecencia , Dinamarca/epidemiología , Expresión Génica , Células HEK293 , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Sueros Inmunes/química , Inmunidad Innata , Modelos Moleculares , Mutación , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Internalización del VirusRESUMEN
C1q/TNF-related protein (CTRP) 6 is a member of the CTRP protein family associated with the regulation of cellular and endocrine processes. CTRP6 contains collagen and globular structures, resembling the pattern recognition molecules (PRMs) of the classical and lectin complement pathways. We expressed human CTRP6 in Chinese hamster ovary cells and investigated the binding to different putative ligands (acetylated BSA [AcBSA], zymosan, mannan, and LPS from Escherichia coli and Salmonella as well as to the monosaccharides l-fucose, d-mannose, N-acetylglucosamine, N-acetylgalactosamine, and galactose). Furthermore, we investigated the binding of CTRP6 to various Gram-negative bacteria as well as PRMs and enzymes of the lectin complement pathway. We found that CTRP6 bound to AcBSA and to a lesser extent to zymosan. Using EDTA as chelating agent, we observed an increased binding to AcBSA, zymosan and the two strains of LPS. We detected no binding to mannan and BSA. We identified l-fucose as a ligand for CTRP6 and that it bound to certain enteroaggregative Escherichia coli and Pseudomonas aeruginosa isolates, whereas to other bacterial isolates, no binding was observed. CTRP6 did not appear to interact directly with the activating enzymes of the lectin pathway; however, we could show the specific recruitment of collectin-11 and subsequent initiation of the complement cascade through deposition of C4. In conclusion, our results demonstrate the binding of CTRP6 to a variety of microbial and endogenous ligands identifying CTRP6 as a novel human lectin and PRM of importance for complement recognition and innate immunity.
Asunto(s)
Antígenos Bacterianos/metabolismo , Colágeno/metabolismo , Colectinas/metabolismo , Complemento C4/metabolismo , Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Animales , Antígenos Bacterianos/inmunología , Células CHO , Colágeno/genética , Colágeno/aislamiento & purificación , Activación de Complemento , Cricetulus , Escherichia coli/inmunología , Escherichia coli/metabolismo , Ligandos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The complement system constitutes an important part of the innate immune system. The collectins and the ficolins are soluble pattern recognition molecules that contribute to complement activation via the lectin pathway. During previous experiments with ficolin-2 and ficolin-3, we have observed that the molecules may interact. We therefore hypothesized the existence of stable ficolin-2/-3 heterocomplexes. We could demonstrate ficolin-2/-3 heterocomplexes in normal human serum and plasma by ELISA using Abs specific for ficolin-2 and ficolin-3. The formation of heteromeric protein complexes were validated by coimmunoprecipitation and Western blot analysis. When recombinant ficolin-2 and recombinant ficolin-3 were mixed, no complexes were formed. However, when coexpressing ficolin-2 and ficolin-3 in Chinese hamster ovary cells, we could detect ficolin-2/-3 heterocomplexes in the supernatant. Furthermore, we measured concentration of the ficolin-2/-3 heterocomplexes in arbitrary units in 94 healthy individuals. We also established the relationship between the concentrations of ficolin-2, ficolin-3, and the ficolin-2/-3 heterocomplexes. We observed that the concentration of the ficolin-2/-3 heterocomplex correlated significantly with ficolin-2 (ρ: 0.24, p < 0.018) and ficolin-3 concentrations (ρ: 0.46, p < 0.0001). In conclusion, we describe a novel protein complex between ficolin-2 and ficolin-3 present in serum and plasma, which might be of additional biological relevance apart from the native ficolin-2 and ficolin-3 molecules.
Asunto(s)
Lectinas/sangre , Animales , Células CHO , Línea Celular , Colectinas/metabolismo , Activación de Complemento/fisiología , Lectina de Unión a Manosa de la Vía del Complemento/fisiología , Proteínas del Sistema Complemento/metabolismo , Cricetulus , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Ratones , FicolinasRESUMEN
Dysregulation of the complement system is involved in the pathogenesis of several diseases, and its inhibition has been shown to be a feasible therapeutic option. Therefore, there is an interest in the development of complement modulators to treat complement activation-related inflammatory pathologies. Mannose-binding lectin (MBL)/ficolin/collectin-associated protein-1 (MAP-1) is a regulatory molecule of the lectin pathway (LP), whereas complement receptor 1 (CD35) and decay-accelerating factor (CD55) are membrane-anchored regulators with effects on the central effector molecule C3. In this study, we developed 2 novel soluble chimeric inhibitors by fusing MAP-1 to the 3 first domains of CD35 (CD351-3) or the 4 domains of CD55 (CD551-4) to modulate the complement cascade at 2 different stages. The constructs showed biologic properties similar to those of the parent molecules. In functional complement activation assays, the constructs were very efficient in inhibiting LP activation at the level of C3 and in the formation of terminal complement complex. This activity was enhanced when coincubated with recombinant LP recognition molecules MBL and ficolin-3. Recombinant MAP-1 fusion proteins, combined with recombinant LP recognition molecules to target sites of inflammation, represent a novel and effective therapeutic approach involving the initiation and the central and terminal effector functions of the complement cascade.-Pérez-Alós, L., Bayarri-Olmos, R., Skjoedt, M.-O., Garred, P. Combining MAP-1:CD35 or MAP-1:CD55 fusion proteins with pattern-recognition molecules as novel targeted modulators of the complement cascade.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Antígenos CD55 , Activación de Complemento/efectos de los fármacos , Complemento C3 , Receptores de Complemento 3b , Receptores de Reconocimiento de Patrones , Proteínas Recombinantes de Fusión , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/farmacología , Antígenos CD55/química , Antígenos CD55/genética , Antígenos CD55/farmacología , Células CHO , Complemento C3/química , Complemento C3/metabolismo , Cricetulus , Humanos , Receptores de Complemento 3b/química , Receptores de Complemento 3b/genética , Receptores de Reconocimiento de Patrones/química , Receptores de Reconocimiento de Patrones/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Mannose-binding lectin (MBL), collectin-10, collectin-11, and the ficolins (ficolin-1, ficolin-2, and ficolin-3) are soluble pattern recognition molecules in the lectin complement pathway. These proteins act as mediators of host defense and participate in maintenance of tissue homeostasis. They bind to conserved pathogen-specific structures and altered self-antigens and form complexes with the pentraxins to modulate innate immune functions. All molecules exhibit distinct expression in different tissue compartments, but all are found to a varying degree in the circulation. A common feature of these molecules is their ability to interact with a set of serine proteases named MASPs (MASP-1, MASP-2, and MASP-3). MASP-1 and -2 trigger the activation of the lectin pathway and MASP-3 may be involved in the activation of the alternative pathway of complement. Furthermore, MASPs mediate processes related to coagulation, bradykinin release, and endothelial and platelet activation. Variant alleles affecting expression and structure of the proteins have been associated with a variety of infectious and non-infectious diseases, most commonly as disease modifiers. Notably, the severe 3MC (Malpuech, Michels, Mingarelli, and Carnevale) embryonic development syndrome originates from rare mutations affecting either collectin-11 or MASP-3, indicating a broader functionality of the complement system than previously anticipated. This review summarizes the characteristics of the molecules in the lectin pathway.
Asunto(s)
Lectina de Unión a Manosa de la Vía del Complemento , Inmunidad Innata , Lectinas/metabolismo , Lectina de Unión a Manosa/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Animales , Coagulación Sanguínea , Colectinas/metabolismo , Homeostasis , Interacciones Huésped-Patógeno , Humanos , Activación Plaquetaria , FicolinasRESUMEN
Clinical immunity to malaria is associated with the acquisition of IgG specific for members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens on the surface of infected erythrocytes (IEs). The VAR2CSA subtype of PfEMP1 mediates IE binding in the placenta. VAR2CSA-specific IgG is normally acquired only after exposure to placental parasites. However, it was recently reported that men and children from Colombia often have high levels of functional VAR2CSA-specific IgG. This potentially undermines the current understanding of malaria immunity in pregnant women, and we thus conducted a study to assess further the levels of VAR2CSA-specific IgG in pregnant and nonpregnant Colombians. Plasma IgG against two full-length recombinant PfEMP1 proteins (one of the VAR2CSA type and one not) produced in baculovirus-transfected insect cells was detected frequently among Colombian men, children, and pregnant women with acute or previous malaria exposure. In contrast, IgG reactivity to a homologous full-length VAR2CSA-type protein expressed in Chinese hamster ovary (CHO) cells was low and infrequent among the Colombian plasma samples, as was reactivity to both corresponding native PfEMP1 proteins. Moreover, human and rabbit antibodies specific for Plasmodium vivax Duffy-binding protein (PvDBP), a protein with some homology to PfEMP1, did not react with VAR2CSA-type recombinant or native proteins, although the mouse monoclonal and PvDBP-specific antibody 3D10 was weakly reactive with recombinant proteins expressed in baculovirus-transfected insect cells. Our data indicate that the previously reported Colombian IgG reactivity to recombinant VAR2CSA is not malaria specific and that the acquisition of VAR2CSA-specific IgG is restricted to pregnancy, in Colombia and elsewhere.
Asunto(s)
Antígenos de Protozoos/inmunología , Reacciones Falso Positivas , Inmunoensayo/métodos , Inmunoglobulina G/sangre , Malaria Falciparum/inmunología , Malaria Vivax/inmunología , Complicaciones Infecciosas del Embarazo/inmunología , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antiprotozoarios/sangre , Niño , Preescolar , Colombia , Femenino , Glicosilación , Humanos , Masculino , Ratones , Persona de Mediana Edad , Embarazo , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Adulto JovenRESUMEN
Soluble defense collagens including the collectins play important roles in innate immunity. Recently, a new member of the collectin family named collectin-12 (CL-12 or CL-P1) has been identified. CL-12 is highly expressed in umbilical cord vascular endothelial cells as a transmembrane receptor and may recognize certain bacteria and fungi, leading to opsonophagocytosis. However, based on its structural and functional similarities with soluble collectins, we hypothesized the existence of a fluid-phase analog of CL-12 released from cells, which may function as a soluble pattern-recognition molecule. Using recombinant CL-12 full length or CL-12 extracellular domain, we determined the occurrence of soluble CL-12 shed from in vitro cultured cells. Western blot showed that soluble recombinant CL-12 migrated with a band corresponding to â¼ 120 kDa under reducing conditions, whereas under nonreducing conditions it presented multimeric assembly forms. Immunoprecipitation and Western blot analysis of human umbilical cord plasma enabled identification of a natural soluble form of CL-12 having an electrophoretic mobility pattern close to that of shed soluble recombinant CL-12. Soluble CL-12 could recognize Aspergillus fumigatus partially through the carbohydrate-recognition domain in a Ca(2+)-independent manner. This led to activation of the alternative pathway of complement exclusively via association with properdin on A. fumigatus as validated by detection of C3b deposition and formation of the terminal complement complex. These results demonstrate the existence of CL-12 in a soluble form and indicate a novel mechanism by which the alternative pathway of complement may be triggered directly by a soluble pattern-recognition molecule.
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Aspergillus fumigatus/inmunología , Colectinas/inmunología , Activación de Complemento/inmunología , Vía Alternativa del Complemento/inmunología , Receptores Depuradores/inmunología , Esporas Fúngicas/inmunología , Colectinas/sangre , Complemento C3b/inmunología , Femenino , Sangre Fetal , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inmunidad Innata/inmunología , Properdina/inmunología , Receptores Depuradores/sangreAsunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , COVID-19/prevención & control , ARN Mensajero/inmunología , SARS-CoV-2/inmunología , Formación de Anticuerpos , Vacuna BNT162 , COVID-19/epidemiología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/genética , Dinamarca , Personal de Salud , Humanos , Inmunogenicidad Vacunal , Inmunoglobulina G/inmunología , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , SARS-CoV-2/genéticaRESUMEN
Introduction: The complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated. Methods: To shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serumpurified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as sizeexclusion chromatography. Results: Protein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. Discussion: Clusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade.
Asunto(s)
Clusterina , Complemento C7 , Complemento C7/metabolismo , Proteínas del Sistema Complemento/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Activación de ComplementoRESUMEN
Acute SARS-CoV-2 infection triggers the generation of diverse and functional autoantibodies (AABs), even after mild cases. Persistently elevated autoantibodies have been found in some individuals with long COVID (LC). Using a >21,000 human protein array, we identified diverse AAB targets in LC patients that correlated with their symptoms. Elevated AABs to proteins in the nervous system were found in LC patients with neurocognitive and neurological symptoms. Purified Immunoglobulin G (IgG) samples from these individuals reacted with human pons tissue and were cross-reactive with mouse sciatic nerves, spinal cord, and meninges. Antibody reactivity to sciatic nerves and meninges correlated with patient-reported headache and disorientation. Passive transfer of IgG from patients to mice led to increased sensitivity and pain, mirroring patient-reported symptoms. Similarly, mice injected with IgG showed loss of balance and coordination, reflecting donor-reported dizziness. Our findings suggest that targeting AABs could benefit some LC patients.
RESUMEN
INTRODUCTION: The complement system anaphylatoxin C5a is a critical player in inflammation. By binding to complement C5a receptor 1 (C5aR1/CD88), C5a regulates many cellular functions, mainly as a potent pro-inflammatory inducer. We describe the generation and selection of a potent antagonistic C5aR1 mouse monoclonal antibody (mAb). METHODS: Initial C5aR1 hybridoma clone selection was performed with a cell-binding study in human whole blood. In-house C5aR1 mAb assessment for C5aR1 inhibition was done via the iLite® C5a assay. C5aR1 mAb specificity was investigated on C5aR1his- and C5aR2his-expressing Flp-In™-CHO cells. Physiological C5aR1 inhibition was assessed via a C5a-driven calcium flux assay and stimulation assay based on isolated polymorphonuclear leukocytes (PMNs) and a whole blood model stimulated with Escherichia coli. RESULTS: The supernatant of hybridoma clones targeting the N-terminal section of C5aR1 displayed efficient binding to C5aR1 in whole blood, which was confirmed for purified mAbs. The C5aR1 mAb 18-41-6 was selected following the assay of in-house C5aR1 mAbs via the iLite® C5a assay. The mAb 18-41-6 was specific for C5aR1. Full-size and/or F(ab')2 preparations of mAb 18-41-6 were found to efficiently abrogate C5a-induced calcium flux in neutrophils and to significantly reduce the upregulation of the activation markers CD11b (neutrophils, monocytes) and CD66b (neutrophils). CONCLUSION: Our results demonstrate that mAb 18-41-6 is a valuable tool for investigating the C5a-C5aR1 axis and a potential therapeutic candidate for inflammatory disease treatment.