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1.
Nat Commun ; 12(1): 1063, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594061

RESUMO

The most advanced P. falciparum circumsporozoite protein-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective antibodies and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. In this study, antibody apparent affinity correlates best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I ß- and Asn pseudo 310 turns; such insights can be incorporated into design of more effective immunogens and antibodies for passive immunization.


Assuntos
Anticorpos Antiprotozoários/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Sequências Repetitivas de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos/imunologia , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Cinética , Camundongos Endogâmicos C57BL , Modelos Moleculares , Parasitos/imunologia , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica
2.
Nat Commun ; 11(1): 3418, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647286

RESUMO

The emergence and spread of antiviral drug-resistant viruses have been a worldwide challenge and a great concern for patient care. We report A4 antibody specifically recognizing and binding to the mutant I223R/H275Y neuraminidase and prove the applicability of A4 antibody for direct detection of antiviral multidrug-resistant viruses in various sensing platforms, including naked-eye detection, surface-enhanced Raman scattering-based immunoassay, and lateral flow system. The development of the A4 antibody enables fast, simple, and reliable point-of-care assays of antiviral multidrug-resistant influenza viruses. In addition to current influenza virus infection testing methods that do not provide information on the antiviral drug-resistance of the virus, diagnostic tests for antiviral multidrug-resistant viruses will improve clinical judgment in the treatment of influenza virus infections, avoid the unnecessary prescription of ineffective drugs, and improve current therapies.


Assuntos
Anticorpos Antivirais/imunologia , Resistência a Múltiplos Medicamentos/imunologia , Farmacorresistência Viral/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Mutação/genética , Neuraminidase/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/química , Afinidade de Anticorpos/imunologia , Antígenos Virais/metabolismo , Líquidos Corporais/virologia , Análise Mutacional de DNA , Cães , Epitopos/química , Epitopos/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H3N2/enzimologia , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Imagem Óptica , Ligação Proteica , Análise Espectral Raman
3.
Sci Rep ; 10(1): 10895, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616763

RESUMO

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Afinidade de Anticorpos/imunologia , Betacoronavirus/química , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Antígenos de Histocompatibilidade/imunologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptidil Dipeptidase A/metabolismo , Filogenia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Vacinas Virais/metabolismo
4.
Nat Commun ; 11(1): 2251, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366817

RESUMO

The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV) in cell culture. This cross-neutralizing antibody targets a communal epitope on these viruses and may offer potential for prevention and treatment of COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Afinidade de Anticorpos/imunologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , Chlorocebus aethiops , Sequência Conservada , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reações Cruzadas/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Modelos Moleculares , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/imunologia , Receptores Virais/química , Receptores Virais/metabolismo , Vírus da SARS/química , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
5.
Int J Nanomedicine ; 15: 2197-2205, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32280214

RESUMO

Background: Glypican-3 (GPC3) is a newly identified target molecule for the early diagnosis of hepatocellular carcinoma (HCC), while targeted inhibition of GPC3 signaling may help to control the proliferation and metastasis of HCC cells. The purpose of this study was to prepare the anti-GPC3 nanobody and to investigate the affinity of the anti-GPC3 nanobodies in vitro and the anticancer effects on hepatocellular carcinoma in vivo. Methods: To screen for unknown anti-GPC3 antibodies, we constructed an antibody phage display library. After three rounds of panning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA). Further, the nanobody fusion protein was expressed in E. coli BL21 cells and purified by affinity chromatography. Competitive ELISA and flow cytometry were conducted to confirm the affinity of the anti-GPC3 nanobodies in vitro. The antitumor effects of VHHGPC3 were assessed in vivo. Results: The results showed that the nanobody VHHGPC3 had specific high-affinity binding to His-GPC3 antigen. Moreover, VHHGPC3 exhibited specific binding to commercial human GPC3 and recognized the surface GPC3 protein of the hepatoma cell line HepG2. Importantly, in vivo study showed that GPC3 nanobody suppresses the growth of HepG2 and improves the survival rate of tumor mice. Discussion: In summary, our new anti-GPC3 nanobody suggests a strong application potential for targeted therapy of liver cancer.


Assuntos
Carcinoma Hepatocelular/imunologia , Glipicanas/imunologia , Neoplasias Hepáticas/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Afinidade de Anticorpos/imunologia , Carcinoma Hepatocelular/patologia , Técnicas de Visualização da Superfície Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Células Hep G2 , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Neoplasias Hepáticas/patologia , Camundongos Nus , Análise de Sobrevida
6.
Sci Rep ; 10(1): 3229, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32094377

RESUMO

Diabetes mellitus (DM) patients are at an increased risk of complications following influenza-virus infection, seasonal vaccination (SV) is recommended. However, SV with trivalent influenza vaccine (TIV) can induce antibody and type-I interferon (IFN) responses, and the effect of anti-DM treatment on these responses is incompletely understood. We evaluated the antibody response and IFN-α expression in individuals with and without type 2 DM (T2DM) following SV, and examined the effects on anti-DM treatment. TIV elicited sero-protection in all groups, but antibody persistency was <8 months, except for the antibody response to B-antigens in non-DM. T2DM impaired the IgG avidity index, and T2DM showed a significantly decreased response against H1N1 and H3N2, in addition to delaying and reducing haemagglutination-inhibition persistency against influenza B-antigens in DM groups treated with metformin (Met-DM) or glibenclamide (GB-DM). Following TIV, the Met-DM and GB-DM groups exhibited reduced IFN-α expression upon stimulation with whole- and split-virion influenza vaccines. Suppression of IFN-α expression in the Met-DM group was associated with a reduction in the mechanistic target of rapamycin complex-1 pathway and impaired IgG avidity index. Thus, single-dose TIV each year might not be suitable for T2DM. Our data could aid the development of an efficacious influenza vaccine for T2DM.


Assuntos
Formação de Anticorpos/efeitos dos fármacos , Diabetes Mellitus Tipo 2/imunologia , Interferon-alfa/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metformina/farmacologia , Estações do Ano , Transdução de Sinais , Vacinação , Idoso , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos/imunologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Glibureto/farmacologia , Glibureto/uso terapêutico , Testes de Inibição da Hemaglutinação , Humanos , Imunoglobulina G/metabolismo , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Masculino , Metformina/uso terapêutico , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vírion/efeitos dos fármacos , Vírion/imunologia
7.
Mol Biol Rep ; 47(3): 2137-2147, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32080807

RESUMO

The aim of the study was to produce a single-domain antibody (nanobody) specific for endothelin receptor type B (EDNRB) which has high expression in melanoma. Cultured human melanoma cells were used as antigens to immunize alpacas. After antibody generation was verified in alpaca serum, total RNA was extracted from alpaca lymphocytes and the target VHH fragment was amplified by two-step PCR, cloned in the pCANTAB5E phagemid vector, and used to transform Escherichia coli TG1 cells to obtain a phage-display nanobody library, which was enriched by panning. The results indicated successful construction of a phage-display anti-human melanoma A375 nanobodies library with a size of 1.2 × 108/ml and insertion rate of 80%. After screening, eight positive clones of anti-EDNRB nanobodies were used to infect E. coli HB2151 for production of soluble nanobodies, which were identified by ELISA. Finally, we obtained a high-affinity anti-EDNRB nanobody, which consisted of 119 amino acids (molecular weight: 12.97 kDa) with 22 amino acids in CDR3 and had good affinity in vitro. The results suggest that the nanobody may be potentially used for the treatment of human melanoma.


Assuntos
Afinidade de Anticorpos , Antineoplásicos Imunológicos/farmacologia , Antagonistas do Receptor de Endotelina B/farmacologia , Receptor de Endotelina B/metabolismo , Anticorpos de Domínio Único/farmacologia , Afinidade de Anticorpos/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Técnicas de Visualização da Superfície Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Melanoma/tratamento farmacológico , Melanoma/imunologia , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica/imunologia , Receptor de Endotelina B/imunologia , Análise de Sequência de DNA , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação
8.
Sci Rep ; 10(1): 1194, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988343

RESUMO

Nimotuzumab is a humanized monoclonal antibody against the Epidermal Growth Factor Receptor with a long history of therapeutic use, recognizing an epitope different from the ones targeted by other antibodies against the same antigen. It is also distinguished by much less toxicity resulting in a better safety profile, which has been attributed to its lower affinity compared to these other antibodies. Nevertheless, the ideal affinity window for optimizing the balance between anti-tumor activity and toxic effects has not been determined. In the current work, the paratope of the phage-displayed nimotuzumab Fab fragment was evolved in vitro to obtain affinity-matured variants. Soft-randomization of heavy chain variable region CDRs and phage selection resulted in mutated variants with improved binding ability. Two recombinant antibodies were constructed using these variable regions, which kept the original fine epitope specificity and showed moderate affinity increases against the target (3-4-fold). Such differences were translated into a greatly enhanced inhibitory capacity upon ligand-induced receptor phosphorylation on tumor cells. The new antibodies, named K4 and K5, are valuable tools to explore the role of affinity in nimotuzumab biological properties, and could be used for applications requiring a fine-tuning of the balance between binding to tumor cells and healthy tissues.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Afinidade de Anticorpos/imunologia , Neoplasias/imunologia , Anticorpos Monoclonais Humanizados/metabolismo , Bacteriófagos/genética , Bacteriófagos/imunologia , Linhagem Celular Tumoral , Simulação por Computador , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Vetores Genéticos/genética , Humanos , Idiótipos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Neoplasias/patologia , Proteínas Recombinantes/imunologia , Transfecção
9.
Mol Med Rep ; 21(2): 759-767, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31974622

RESUMO

Thymic stromal lymphopoietin (TSLP) is a potentially important target for the treatment of asthma and malignancies. However, a fully human antibody reactive with TSLP is currently unavailable for clinical use. In a previous study, a human anti­TSLP­single­chain antibody variable fragment (anti­TSLP­scFv) 84 was selected by phage display from a constructed human scFv library. In the present study, a computer simulation method was developed using Discovery Studio 4.5 software, to increase the affinity of anti­TSLP­scFv­84. Specific primers were designed and mutated DNA sequences of anti­TSLP­scFvs were obtained by overlap extension PCR. The mutant scFvs were expressed in pLZ16 and affinity­enhanced anti­TSLP­scFv­M4 was screened using ELISA. However, in general the scFvs have low stability and short half­lives in vivo. Therefore, scFv­84 and scFv­M4 were inserted into eukaryotic expression vectors (pcDNA3.1­sp­Fc and PMH3EN­sp­Fc) and then transfected into 293F cells to express anti­TSLP­scFv­Fc. ELISA and western blotting results indicated the size of the anti­TSLP­scFv­Fc to be ~50 kDa. Binding of anti­TSLP­scFv­Fc­M4 to TSLP was enhanced compared with the pre­mutated scFv­Fc­84. The affinity of the mutated recombinant antibody was determined using the BIAcore technique and found to be ~10­fold greater than the pre­mutated antibody.


Assuntos
Anticorpos/imunologia , Afinidade de Anticorpos/imunologia , Citocinas/imunologia , Proteínas Recombinantes/imunologia , Aminoácidos/genética , Humanos , Simulação de Acoplamento Molecular , Mutação/genética , Reprodutibilidade dos Testes , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia
10.
Diabetes ; 69(3): 381-391, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31806623

RESUMO

ß-Cell antigen recognition by autoreactive T cells is essential in type 1 diabetes (T1D) pathogenesis. Recently, insulin hybrid peptides (HIPs) were identified as strong agonists for CD4 diabetogenic T cells. Here, using BDC2.5 transgenic and NOD mice, we investigated T-cell recognition of the HIP2.5 epitope, which is a fusion of insulin C-peptide and chromogranin A (ChgA) fragments, and compared it with the WE14 and ChgA29 -42 epitopes. We measured in situ two-dimensional affinity on individual live T cells from thymus, spleen, pancreatic lymph nodes, and islets before and after diabetes. Although preselection BDC2.5 thymocytes possess higher affinity than splenic BDC2.5 T cells for all three epitopes, peripheral splenic T cells maintained high affinity only to the HIP2.5 epitope. In polyclonal NOD mice, a high frequency (∼40%) of HIP2.5-specific islet T cells were identified at both prediabetic and diabetic stages comprising two distinct high- and low-affinity populations that differed in affinity by 100-fold. This high frequency of high- and low-affinity HIP2.5 T cells in the islets potentially represents a major risk factor in diabetes pathogenesis.


Assuntos
Peptídeo C/imunologia , Linfócitos T CD4-Positivos/imunologia , Cromogranina A/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Afinidade de Anticorpos/imunologia , Diabetes Mellitus Tipo 1/genética , Ilhotas Pancreáticas/citologia , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Baço/citologia , Linfócitos T/citologia , Linfócitos T/imunologia , Timócitos/citologia , Timócitos/imunologia , Timo/citologia
11.
J Immunol Methods ; 478: 112720, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31812660

RESUMO

BACKGROUND: Antibodies that target a single tumor antigen fail to cure stage IV cancer patients due to tumor heterogeneity and variable expression of antigen. Tumor cells with insufficient binding of antibody will not undergo antibody induced cytotoxicity. We describe targeting multiple tumor-specific antigens that resulted in homogeneous dense binding to mouse melanoma cells and significant tumor growth inhibition. METHODS: Surface-related tumor-specific mutations on B16-F10 cells were identified. Peptides containing the single amino acid mutation were synthesized for 9 different neoantigens. Rabbits were vaccinated with each of these peptides and high affinity polyclonal antibodies to each peptide were obtained. The 9 antibodies were combined as a cocktail and mice with implanted B16-F10 cells were treated with and without PD1 inhibitor. RESULTS: Even a single dose of the antibody cocktail inhibited tumor growth and prolonged survival. PD1 inhibitor alone had little effect on tumor growth. The antibody cocktail plus PD1 inhibition increased tumor response and 4 doses of the cocktail completely prevented tumor growth in 50% of the mice. Complete responses were durable. The complete responders were highly resistant to tumor re-challenge at 6 months. No adverse events were identified in the antibody treated mice. CONCLUSIONS: Multiple tumor-specific cell surface-related neoantigens were abundant in B16-F10 cells. Antibodies to 9 of these neoantigens had variable binding but when combined had dense homogeneous binding. Even one dose of this cocktail of 9 antibodies improved survival and when multiple doses were combined with PD1 inhibition 50% of the mice were rendered permanently tumor free.


Assuntos
Anticorpos/administração & dosagem , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/administração & dosagem , Melanoma Experimental/terapia , Neoplasias Cutâneas/terapia , Animais , Anticorpos/imunologia , Afinidade de Anticorpos/imunologia , Antineoplásicos Imunológicos/imunologia , Linhagem Celular Tumoral , Combinação de Medicamentos , Feminino , Humanos , Melanoma Experimental/imunologia , Camundongos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Coelhos , Neoplasias Cutâneas/imunologia
12.
Cell Rep ; 29(12): 3946-3957.e5, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31851925

RESUMO

Passive immunization (PI) with external antibodies has been used classically for rapid but temporary alleviation of disease. Transcending this role, recent studies have shown PI to induce lasting improvements in natural antibody production, suggesting that PI could become a powerful tool to engineer humoral responses. We propose a mechanism with which PI can alter the humoral response. Antigen-specific B cells evolve and get selected in germinal centers (GCs) on the basis of their ability to acquire antigen from antibody-antigen complexes presented in GCs. When external antibodies of high affinity for antigen are used, they form the majority of the complexes in GCs, letting only B cells with even higher affinities be selected. Using an in silico GC reaction model, we show that this mechanism explains the improved humoral responses following PI. The model also synthesizes several independent experimental observations, indicating the robustness of the mechanism, and proposes tunable handles to optimize PI.


Assuntos
Afinidade de Anticorpos/imunologia , Complexo Antígeno-Anticorpo/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunidade Humoral/imunologia , Imunização Passiva/métodos , Animais , Formação de Anticorpos , Simulação por Computador , Camundongos
13.
Sci Transl Med ; 11(520)2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776286

RESUMO

Nearly all chronic human infections are associated with alterations in the memory B cell (MBC) compartment, including a large expansion of CD19hiT-bethi MBC in the peripheral blood of HIV-infected individuals with chronic viremia. Despite their prevalence, it is unclear how these B cells arise and whether they contribute to the inefficiency of antibody-mediated immunity in chronic infectious diseases. We addressed these questions by characterizing T-bet-expressing B cells in lymph nodes (LN) and identifying a strong T-bet signature among HIV-specific MBC associated with poor immunologic outcome. Confocal microscopy and quantitative imaging revealed that T-bethi B cells in LN of HIV-infected chronically viremic individuals distinctly accumulated outside germinal centers (GC), which are critical for optimal antibody responses. In single-cell analyses, LN T-bethi B cells of HIV-infected individuals were almost exclusively found among CD19hi MBC and expressed reduced GC-homing receptors. Furthermore, HIV-specific B cells of infected individuals were enriched among LN CD19hiT-bethi MBC and displayed a distinct transcriptome, with features similar to CD19hiT-bethi MBC in blood and LN GC B cells (GCBC). LN CD19hiT-bethi MBC were also related to GCBC by B cell receptor (BCR)-based phylogenetic linkage but had lower BCR mutation frequencies and reduced HIV-neutralizing capacity, consistent with diminished participation in GC-mediated affinity selection. Thus, in the setting of chronic immune activation associated with HIV viremia, failure of HIV-specific B cells to enter or remain in GC may help explain the rarity of high-affinity protective antibodies.


Assuntos
Afinidade de Anticorpos/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Infecções por HIV/imunologia , Proteínas com Domínio T/metabolismo , Adulto , Anticorpos Neutralizantes/imunologia , Antígenos CD19/metabolismo , Citocinas/metabolismo , Feminino , Infecções por HIV/genética , Humanos , Memória Imunológica , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Fenótipo , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Transcriptoma/genética , Adulto Jovem
14.
Front Immunol ; 10: 2423, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31681310

RESUMO

Background: Optimal timing of gestational tetanus-diphtheria-acellular pertussis (Tdap) vaccination is not well-defined. No well-established specific anti-pertussis antibody level correlates with protection, suggesting the importance of antibody quality such as avidity. We aimed to determine the effect of timing of vaccination with Tdap in pregnancy on the avidity of cord anti-pertussis toxin (PT) immunoglobulin G (IgG). Methods: Prospective study of newborns in a tertiary hospital (Melbourne, Australia) born to women vaccinated with Tdap in pregnancy. Ammonium thiocyanate was used as a bond-breaking agent to measure the avidity of anti-PT IgG using concentrations between 0.25 M (to measure low avidity antibodies) and 3 M (to measure very high avidity antibodies). Anti-PT IgG levels achieved at each ammonium thiocyanate concentration in cord samples of women vaccinated during 28-32 weeks gestation (WG) vs. 33-36 WG, and women vaccinated 5-12 vs. 1-4 weeks prior to delivery were compared using t-tests. Results: Newborns of women vaccinated with Tdap during 28-32 WG (n = 43) had statistically significant higher concentrations of medium and high avidity anti-PT IgG compared with newborns of women vaccinated during 33-36 WG (n = 47), 11.6 IU/ml (95% CI, 8.8-15.2) IU/ml vs. 6.7 IU/ml (95% CI, 5.2-8.6) and 10.1 IU/ml (95% CI, 7.4-13.8) vs. 5.7 (95% CI, 3.6-8.9) IU/ml (p = 0.007 and p = 0.035), respectively. Newborns of women vaccinated 5-12 weeks before delivery (n = 64) had statistically significant higher concentrations of high and very high avidity anti-PT IgG compared with newborns of women vaccinated within 4 weeks before delivery (n = 25), 10.3 IU/mL (95% CI, 7.9-13.4) vs. 3.3 IU/mL (95% CI, 1.7-6.4), 12.6 IU/mL (95% CI, 9.4-16.9) vs. 4.3 IU/mL (95% CI, 2.2-8.5) (all p < 0.03), respectively. Conclusions: Quantification of levels of anti-PT IgG with different avidities demonstrated that pertussis vaccination 5-12 weeks before delivery was associated with higher anti-PT IgG avidity compared with vaccination within 4 weeks before delivery. Pertussis vaccination during 28-32 WG was associated with higher anti-PT IgG avidity compared with vaccination during 33-36 WG, supporting vaccination at 28-32 over 33-36 WG for optimal protection against pertussis in infancy.


Assuntos
Vacinas contra Difteria, Tétano e Coqueluche Acelular/administração & dosagem , Vacinas contra Difteria, Tétano e Coqueluche Acelular/imunologia , Tempo para o Tratamento , Vacinação , Coqueluche/prevenção & controle , Adulto , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Afinidade de Anticorpos/imunologia , Bordetella pertussis/imunologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Toxina Pertussis/imunologia , Gravidez , Proteômica/métodos , Fatores de Tempo , Coqueluche/imunologia
15.
Vaccine ; 37(49): 7280-7288, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31575492

RESUMO

BACKGROUND: In view of further reduction of HPV vaccination schedules, gaining more insight into humoral and cellular immune responses after a single HPV vaccine is of great interest. Therefore, these responses were evaluated after different doses of the bivalent (2v) HPV-vaccine in girls. METHODS: Blood was collected yearly up to seven years post-vaccination with one-, two- or three-doses of the 2vHPV vaccine (N = 890). HPV-type-specific IgG and IgA-antibody levels, IgG-isotypes and avidity indexes were measured by a virus-like-particle-based multiplex-immuno-assay for two vaccine and five non-vaccine HPV types. HPV-type-specific memory B-cell numbers- and T-cell cytokine responses were determined in a subpopulation. RESULTS: HPV-type-specific antibody concentrations were significantly lower in one- than in two- and three-dose vaccinated girls but remained stable over seven years. The lower antibody response coincided with reduced HPV-type-specific B- and T-cell responses. There were no differences in both the IgG subtypes and the avidity of the HPV16-specific antibodies between the groups. CONCLUSIONS: One-dose of the 2vHPV vaccine is immunogenic, but results in less B- and T-cell memory and considerable lower antibody responses when compared with more doses. Therefore, at least of some of girls receiving the one-dose of the vaccination might be at higher risk for waning immunity to HPV in the long-term.


Assuntos
Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Esquemas de Imunização , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/administração & dosagem , Linfócitos T/imunologia , Adolescente , Anticorpos Neutralizantes/sangue , Afinidade de Anticorpos/imunologia , Feminino , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Memória Imunológica/imunologia , Países Baixos , Vacinas contra Papillomavirus/imunologia , Vacinação , Adulto Jovem
16.
Front Immunol ; 10: 2112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31632387

RESUMO

Recently, agonistic autoantibodies (agAAb) activating the ß2-adrenergic receptor were detected in primary open-angle glaucoma (POAG) or ocular hypertension (OHT) patients and were linked to intraocular pressure (IOP) (1). The aim of the present study was to quantify ß2-agAAb in the sera of glaucoma suspects and patients with primary and secondary glaucoma. Patients with OHT (n = 33), pre-perimetric POAG (pre-POAG; n = 11), POAG (n = 28), and 11 secondary OAG (SOAG) underwent ophthalmological examinations including examinations with Octopus G1 perimetry and morphometry. Twenty-five healthy individuals served as controls. Serum-derived IgG samples were analyzed for ß2-agAAb using a functional bioassay. The beat-rate-increase of spontaneously beating cultured neonatal rat cardiomyocytes was monitored with 1.6 beats/15 s as cut-off. None of the sera of normal subjects showed ß2-agAAb. In POAG or OHT patients increased beating rates of 4.1 ± 2.2 beats/15 s, and 3.7 ± 2.8 beats/15 s were detected (p > 0.05). Glaucoma patients with (POAG) and without perimetric (pre-POAG) defects did not differ (pre-POAG 4.4 ± 2.6 beats/15 s, POAG 4.1 ± 2.0 beats/15 s, p > 0.05). Patients with SOAG yielded mean beating rates of 4.7 ± 1.7 beats/15 s (p > 0.05). ß2-agAAb were seen in 73% of OHT, 82% of pre-POAG, 82% of POAG, and 91% SOAG patients (p < 0.001). Clinical data did not correlate with beating rate (p > 0.05). The robust ß2-agAAb seropositivity in patients with OHT, pre-POAG, POAG, and SOAG suggest a primary common role for ß2-agAAb starting early in glaucoma pathophysiology and turned out to be a novel marker identifying all patients with increased IOP independent of glaucoma stage and entity.


Assuntos
Autoanticorpos/imunologia , Glaucoma/etiologia , Glaucoma/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Idoso , Afinidade de Anticorpos/imunologia , Feminino , Glaucoma/diagnóstico , Humanos , Pressão Intraocular , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo
17.
Cell Rep ; 29(5): 1066-1073.e5, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31665624

RESUMO

Broadly neutralizing antibodies are crucial for the control of many life-threatening viral infections like HIV, influenza, or hepatitis. Their induction is a prime goal in vaccine research. Using computer simulations, we identify strategies to promote the generation of broadly neutralizing antibodies in natural germinal center (GC) reactions. The simulations predict a feedback loop based on antibodies and memory B cells from previous GC reactions that promotes GCs to focus on new epitopes. Memory-derived or injected antibodies specific for immunodominant epitopes control epitope availability, suppress the participation of memory B cells in the GC reaction, and allow for the evolution of other B cells to affinity mature for hidden or rare epitopes. This defines a natural selection mechanism for GC B cells to concentrate on new epitopes rather than refine affinity to already-covered epitopes. This principle can be used for the design and testing of future therapies and vaccination protocols.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Centro Germinativo/imunologia , Epitopos Imunodominantes/imunologia , Injeções , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Simulação por Computador , Memória Imunológica , Modelos Imunológicos
18.
Lupus ; 28(12): 1387-1396, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570052

RESUMO

BACKGROUND: This study evaluated the diagnostic performances of total and high-avidity (HA) anti-dsDNA enzyme immunoassays (EIA) in Chinese systemic lupus erythematosus (SLE) patients. METHODS: A total of 410 serum samples from 217 SLE patients, 54 patients with other systemic autoimmune diseases, and 139 healthy subjects were tested on total and HA anti-dsDNA EIA, as well as three commercial in vitro diagnostic kits: BioPlex 2200 ANA Screen, Kallestad anti-dsDNA EIA, and Crithidia Lucilae IFA. The disease activities of SLE patients were assessed using the modified SLE Disease Activity Index. The diagnostic performances of each assay were analyzed using Analyse-it software. RESULTS: The diagnostic performances of the total and HA anti-dsDNA EIA kits were comparable to other commercially available in vitro diagnostic assays. Receiver operating characteristic curve analysis demonstrated an area under the curve ranging from 0.85 to 0.89, with the total anti-dsDNA kit demonstrating the highest sensitivity and the HA kit showing higher specificity. An overall agreement of >90% was observed between the total and HA anti-dsDNA EIA kits and commercially available quantitative anti-dsDNA kits. The ratio of HA to total anti-dsDNA antibody was significantly higher among SLE patients with active disease status and/or kidney damage. All assays exhibited a significant correlation with disease activity and multiple clinical manifestations. CONCLUSIONS: While the clinical performances of various anti-dsDNA assays showed adequate agreements, the BioPlex 2200 anti-dsDNA assay demonstrated the highest positive likelihood ratio and odds ratio. The HA anti-dsDNA EIA kit in association with the total anti-dsDNA kit provided superior performance in SLE diagnosis and monitoring disease activity.


Assuntos
Anticorpos Antinucleares/sangue , Afinidade de Anticorpos/imunologia , Autoanticorpos/sangue , Lúpus Eritematoso Sistêmico/imunologia , Adulto , Idoso , Grupo com Ancestrais do Continente Asiático/etnologia , Doenças Autoimunes/sangue , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/diagnóstico , Masculino , Pessoa de Meia-Idade , Kit de Reagentes para Diagnóstico/normas , Sensibilidade e Especificidade
19.
Front Immunol ; 10: 2115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31555299

RESUMO

The efficacy of T cells depends on their functional avidity, i. e., the strength of T cell interaction with cells presenting cognate antigen. The overall T cell response is composed of multiple T cell clonotypes, involving different T cell receptors and variable levels of functional avidity. Recently, it has been proposed that the presence of low avidity tumor antigen-specific CD8 T cells hinder their high avidity counterparts to protect from tumor growth. Here we analyzed human cytotoxic CD8 T cells specific for the melanoma antigen Melan-A/MART-1. We found that the presence of low avidity T cells did not result in reduced cytotoxicity of tumor cells, nor reduced cytokine production, by high avidity T cells. In vivo in NSG-HLA-A2 mice, the anti-tumor effect of high avidity T cells was similar in presence or absence of low avidity T cells. These data indicate that low avidity T cells are not hindering anti-tumor T cell responses, a finding that is reassuring because low avidity T cells are an integrated part of natural T cell responses.


Assuntos
Afinidade de Anticorpos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , Melanoma/imunologia , Animais , Citotoxicidade Imunológica/imunologia , Xenoenxertos , Humanos , Antígeno MART-1/imunologia , Camundongos , Células Tumorais Cultivadas
20.
Mol Immunol ; 114: 545-552, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31521018

RESUMO

Antibodies possessing high affinity and specificity are desired as therapeutic reagents and biosensor materials. Such antibodies are often obtained from immunized animals through the process referred to as affinity maturation where antibody affinity increases with time after immunization. Somatic hypermutation (SHM) was shown to be involved in this process; however, structural basis of affinity maturation has not well been understood yet. We analyzed the crystal structure of a high affinity anti-(4-hydroxy-3-nitrophenyl)acetyl antibody, C6, possessing Gly at position 95 of heavy chain and 17 amino acid replacements by SHM. Here, we discuss how the amino acid residues at position 95, introduced at a junction of VH and DH gene segments during gene-recombination, as well as those replaced by SHM contribute to increasing the affinity by comparing the C6 structure with that of a germline low affinity antibody, N1G9, possessing Tyr at position 95.


Assuntos
Anticorpos Monoclonais/química , Afinidade de Anticorpos/imunologia , Glicina/química , Cadeias Pesadas de Imunoglobulinas/química , Nitrofenóis/química , Sequência de Aminoácidos , Hipermutação Somática de Imunoglobulina/imunologia
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