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1.
Nat Commun ; 10(1): 1322, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30899022

RESUMO

Heparin-induced thrombocytopenia/thrombosis (HIT) is a serious immune reaction to heparins, characterized by thrombocytopenia and often severe thrombosis with high morbidity and mortality. HIT is mediated by IgG antibodies against heparin/platelet factor 4 antigenic complexes. These complexes are thought to activate platelets leading to thrombocytopenia and thrombosis. Here we show that HIT immune complexes induce NETosis via interaction with FcγRIIa on neutrophils and through neutrophil-platelet association. HIT immune complexes induce formation of thrombi containing neutrophils, extracellular DNA, citrullinated histone H3 and platelets in a microfluidics system and in vivo, while neutrophil depletion abolishes thrombus formation. Absence of PAD4 or PAD4 inhibition with GSK484 abrogates thrombus formation but not thrombocytopenia, suggesting they are induced by separate mechanisms. NETs markers and neutrophils undergoing NETosis are present in HIT patients. Our findings demonstrating the involvement of NETosis in thrombosis will modify the current concept of HIT pathogenesis and may lead to new therapeutic strategies.


Assuntos
Plaquetas/imunologia , Armadilhas Extracelulares/imunologia , Heparina/efeitos adversos , Neutrófilos/imunologia , Receptores de IgG/genética , Trombocitopenia/imunologia , Trombose/imunologia , Animais , Complexo Antígeno-Anticorpo/biossíntese , Plaquetas/efeitos dos fármacos , Citrulinação , Inibidores Enzimáticos/farmacologia , Armadilhas Extracelulares/química , Armadilhas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica , Histonas/genética , Histonas/imunologia , Humanos , Imunoglobulina G/biossíntese , Camundongos , Camundongos Transgênicos , Técnicas Analíticas Microfluídicas , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Neutrófilos/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/imunologia , Fator Plaquetário 4/genética , Fator Plaquetário 4/imunologia , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/imunologia , Receptores de IgG/imunologia , Transdução de Sinais , Trombocitopenia/induzido quimicamente , Trombocitopenia/patologia , Trombose/induzido quimicamente , Trombose/patologia , Trombose/prevenção & controle
2.
MAbs ; 11(3): 516-531, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30663541

RESUMO

We describe here the design, construction and validation of ALTHEA Gold Libraries™. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/JH (H3J) fragments. One IGHV gene provided a universal VH scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal VH scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries™ with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated KD values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C-80°C, demonstrating that ALTHEA Gold Libraries™ are a valuable source of specific, high affinity and highly stable antibodies.


Assuntos
Regiões Determinantes de Complementaridade , Biblioteca Gênica , Imunoglobulina G , Anticorpos de Cadeia Única , Regiões Determinantes de Complementaridade/biossíntese , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Leucócitos Mononucleares/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
3.
MAbs ; 11(3): 559-568, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30694096

RESUMO

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.


Assuntos
Anticorpos Monoclonais Murinos , Antígenos , Imunoglobulina A , Imunoglobulina G , Região Variável de Imunoglobulina , Proteínas Recombinantes de Fusão , Animais , Anticorpos Monoclonais Murinos/biossíntese , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/genética , Antígenos/biossíntese , Antígenos/química , Antígenos/genética , Células CHO , Cricetulus , Imunoglobulina A/biossíntese , Imunoglobulina A/química , Imunoglobulina A/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Camundongos , Peptídeos/química , Peptídeos/genética , Proteólise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Suínos
4.
Metab Eng ; 52: 315-323, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30610917

RESUMO

Escherichia coli (E. coli) is a promising platform for expression of full-length antibodies owing to its several advantages as a production host (fast growth, well characterized genetics, low manufacturing cost), however, low titers from shake flask (typically < 5 mg/L) has limited its use for production of research-grade material in antibody discovery programs. In this work, we used global transcriptional machinery engineering (gTME) with high throughput screening to increase the expression of full-length antibodies in E. coli. A library of E. coli mutants carrying mutations in the global sigma factor RpoD were generated and screened using the Bacterial Antibody Display (BAD) method for enhanced expression. RpoD mutants were isolated that resulted in full-length antibody titers of up to 130.7 ±â€¯6.6 mg/L of shake flask culture with chaperone co-expression. These results could be useful for production of several antibodies quickly in shake flasks for characterization (e.g. antigen binding, biological function) during the early discovery phase.


Assuntos
Formação de Anticorpos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Fator sigma/genética , RNA Polimerases Dirigidas por DNA/genética , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Humanos , Imunoglobulina G/biossíntese , Mutação/genética , Plasmídeos/genética , Transcriptoma
5.
Methods Mol Biol ; 1904: 431-454, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30539485

RESUMO

Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not possible with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated. Many of these are based on antibody fragments or on inclusion of non-antibody components. For some therapeutic applications, full-size, native IgG-like bsAbs may be the optimal format.To prepare bsAbs in IgG format, two challenges should be met. One is that each heavy chain will only pair with the heavy chain of the second specificity and that heavy chain homodimerization will be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and that pairing with the light chain of the second specificity will be prevented. The first solution to the first criterion (known as knobs into holes, KIH) was presented in 1996 by Genentech and additional solutions were presented more recently. However, until recently, out of >120 published formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested.Here, we present a protocol for preparing bsAbs in IgG format in transfected mammalian cells. For heavy chain dimerization we use KIH while as a solution for the second challenge-correct pairing of heavy and light chains of bispecific IgGs we present our "BIClonals" technology; an engineered (artificial) disulfide bond between the antibodies' variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL.During our studies of bsAbs we found that H-L chain pairing seems to be driven by VH-VL interfacial interactions that differ between different antibodies; hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs that suits every antibody sequence, making it necessary to carefully evaluate the optimal solution for each new antibody.


Assuntos
Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Linhagem Celular , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Engenharia de Proteínas , Transfecção
6.
J Pharm Biomed Anal ; 162: 91-100, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30227357

RESUMO

Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality. Here, we describe a platform of analytical methods to determine the composition of several metals in different sample matrices using an advanced automated Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These methods, validated to ICH Q2R1 regulatory validation parameters, were successfully applied to- (a) screen cell culture media; (b) determine changes in the metal concentration during cell growth in spinner flasks, and, (c) determine effect on the glycosylation pattern and homogeneity of an IgG3:κ produced from a murine-hybridoma cell line in bench-top parallel bioreactors due to a spike in copper and iron concentration. Our results show that maintenance of metal content in the cell culture media is critical for product consistency of the IgG3:κ produced.


Assuntos
Anticorpos Monoclonais/biossíntese , Cobre/metabolismo , Meios de Cultura/metabolismo , Glucuronidase/biossíntese , Imunoglobulina G/biossíntese , Cadeias kappa de Imunoglobulina/biossíntese , Ferro/metabolismo , Espectrometria de Massas/métodos , Animais , Anticorpos Monoclonais/genética , Reatores Biológicos , Células CHO , Proliferação de Células , Cricetulus , Glucuronidase/genética , Glicosilação , Hibridomas , Imunoglobulina G/genética , Cadeias kappa de Imunoglobulina/genética , Espectrometria de Massas/normas , Camundongos , Controle de Qualidade , Reprodutibilidade dos Testes , Fatores de Tempo , Transfecção
7.
Exp Parasitol ; 195: 1-7, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30266573

RESUMO

L-arginine (L-Arg), the precursor of nitric oxide (NO), plays multiple, important roles in nutrient metabolism and immune regulation. Hypoargininemia is one of the distinctive features of malaria patients in endemic areas. To understand the immunoregulatory function of L-Arg in malaria, we investigated the effects of L-Arg, pre- or/and post-treatment, on the cellular/humoral immune response during Plasmodium yoelii 17XL (P.y17XL) infection in DBA/2 mice. Populations of splenic CD4+T-bet+IFN-γ+ T cells (Th1), F4/80+ macrophages, CD4+GATA-3+IL-4+ T cells (Th2), B220+CD138+ plasmacytes and antibody-producing cells (IgG+/IgG1+-plasma cells) were assessed by flow cytometry. Pro-inflammatory cytokines and antibodies (IgG and IgG1) were quantified by immunoassays. We found that treatment with L-Arg significantly decreased parasitemia and shortened disease duration. Prophylactic treatment with L-Arg promotes an enhanced Th1 cell response during the early stages of P.y17XL infection, and treatment with L-Arg in the course of infection facilitates the later humoral immune response. Our findings suggest that treatment with L-Arg may decrease parasite burden and control the host's susceptibility to parasite synchronously by regulating host immune responses against P.y17XL, producing better outcomes for malaria infection. This implies that the supplementation of L-Arg may be a promising adjunctive therapy to reduce malaria-associated mortality in endemic areas.


Assuntos
Arginina/uso terapêutico , Malária/tratamento farmacológico , Plasmodium yoelii/imunologia , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Células Produtoras de Anticorpos/efeitos dos fármacos , Células Produtoras de Anticorpos/imunologia , Arginina/sangue , Citometria de Fluxo , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Interferon gama/metabolismo , Malária/imunologia , Malária/prevenção & controle , Camundongos , Camundongos Endogâmicos DBA , Óxido Nítrico/metabolismo , Parasitemia/tratamento farmacológico , Parasitemia/prevenção & controle , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/citologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia
8.
Exp Parasitol ; 194: 60-66, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30253134

RESUMO

Severe or lethal damages, caused by Toxoplasma gondii infection in congenital cases and immunocompromised patients implies the necessity for development of a vaccine and an appropriate adjuvant would be needed to elicit a protective Th1 biased-immune response. The adjuvant activity of propranolol was surveyed and compared with alum by immunization of BALB/c mice with protein components of T. gondii tachyzoites. Five groups of BALB/c mice were immunized with phosphate buffered saline (negative control), Toxoplasma lysate antigen (TLA), alum plus TLA, Propranolol plus TLA, and alum, propranolol and TLA. Immunization efficacy was evaluated by lymphocyte proliferation and DTH tests, challenge with live tachyzoites, IFN-γ production by spleen cells, serum TNF-α concentration and anti- Toxoplasma total IgG, IgG1 and IgG2a measurements. Mice of the PRP-TLA group induced significantly more IFN-γ and TNF-α production and lymphocyte proliferation than other groups. This group of mice also showed more anti-T. gondii IgG2a and DTH responses and showed a significantly increased survival time after challenge. These findings indicate that propranolol as an adjuvant in combination with TLA, may enhance cellular immunity against T. gondii.


Assuntos
Adjuvantes Imunológicos/normas , Imunização/normas , Propranolol/imunologia , Vacinas Protozoárias , Toxoplasma/imunologia , Toxoplasmose/prevenção & controle , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Proliferação de Células , Feminino , Hipersensibilidade Tardia/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Interferon gama/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologia , Taxa de Sobrevida , Toxoplasmose/imunologia , Toxoplasmose/mortalidade , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/imunologia
9.
Proc Natl Acad Sci U S A ; 115(34): 8615-8620, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30072430

RESUMO

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD ) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD , but not DNA-PKcs-/- , B cells. Meanwhile, the residual joints from DNA-PKcsKD/KD cells and the efficient Sµ-Sγ1 junctions from DNA-PKcs-/- B cells both display similar preferences for small (2-6 nt) microhomologies (MH). In DNA-PKcs-/- cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.


Assuntos
Linfócitos B/enzimologia , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Switching de Imunoglobulina/fisiologia , Imunoglobulina G/biossíntese , Proteínas Nucleares/metabolismo , Recombinação Genética/fisiologia , Animais , Linfócitos B/citologia , Linhagem Celular , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Imunoglobulina G/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética
10.
J Immunol Res ; 2018: 3479185, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30009186

RESUMO

Background: The use of hypoallergenic derivatives is considered beneficial to promote the safety and efficacy of allergen-specific immunotherapy. We aimed to assess the efficacy of reduced and alkylated (R/A) Pru p 3, a hypoallergenic folding variant of the major peach allergen, in subcutaneous immunotherapy (SCIT) using a murine model of peach allergy. Methods and Results: After sensitization with Pru p 3, BALB/c mice received SCIT with Pru p 3 or R/A Pru p 3 and were challenged with Pru p 3. SCIT with Pru p 3, but not with R/A Pru p 3, suppressed anaphylaxis upon the challenge significantly. SCIT with Pru p 3 did not suppress Pru p 3-specific IgE and IgG1 production, but enhanced IgG2a production. In contrast, SCIT with R/A Pru p 3 suppressed IgE and IgG1 production, but enhanced IgG2a production only moderately. The therapeutic efficacy of SCIT with Pru p 3 was associated with induction of IL-10 and IFN-γ. Conclusion: Hypoallergenic folding variant of Pru p 3 is not likely an efficacious therapeutic component in SCIT of peach allergy. The lower efficacy of R/A Pru p 3 might be attributed to poor antigenicity and/or weak stability due to its unfolded conformation.


Assuntos
Antígenos de Plantas/imunologia , Dessensibilização Imunológica/métodos , Proteínas de Plantas/imunologia , Anafilaxia/imunologia , Anafilaxia/prevenção & controle , Animais , Antígenos de Plantas/administração & dosagem , Modelos Animais de Doenças , Feminino , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/terapia , Imunoglobulina E/biossíntese , Imunoglobulina E/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Plantas/administração & dosagem , Prunus persica/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Células Th2/imunologia
11.
J Virol ; 92(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30021899

RESUMO

An effective human immunodeficiency virus (HIV) vaccine has yet to be developed, and defining immune correlates of protection against HIV infection is of paramount importance to inform future vaccine design. The complement system is a component of innate immunity that can directly lyse pathogens and shape adaptive immunity. To determine if complement lysis of simian immunodeficiency virus (SIV) and/or SIV-infected cells represents a protective immune correlate against SIV infection, sera from previously vaccinated and challenged rhesus macaques were analyzed for the induction of antibody-dependent complement-mediated lysis (ADCML). Importantly, the vaccine regimen, consisting of a replication-competent adenovirus type 5 host-range mutant SIV recombinant prime followed by a monomeric gp120 or oligomeric gp140 boost, resulted in overall delayed SIV acquisition only in females. Here, sera from all vaccinated animals induced ADCML of SIV and SIV-infected cells efficiently, regardless of sex. A modest correlation of SIV lysis with a reduced infection rate in males but not females, together with a reduced peak viremia in all animals boosted with gp140, suggested a potential for influencing protective efficacy. Gag-specific IgG and gp120-specific IgG and IgM correlated with SIV lysis in females, while Env-specific IgM correlated with SIV-infected cell lysis in males, indicating sex differences in vaccine-induced antibody characteristics and function. In fact, gp120/gp140-specific antibody functional correlates between antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis, and ADCML as well as the gp120-specific IgG glycan profiles and the corresponding ADCML correlations varied depending on the sex of the vaccinees. Overall, these data suggest that sex influences vaccine-induced antibody function, which should be considered in the design of globally effective HIV vaccines in the future.IMPORTANCE An HIV vaccine would thwart the spread of HIV infection and save millions of lives. Unfortunately, the immune responses conferring universal protection from HIV infection are poorly defined. The innate immune system, including the complement system, is an evolutionarily conserved, basic means of protection from infection. Complement can prevent infection by directly lysing incoming pathogens. We found that vaccination against SIV in rhesus macaques induces antibodies that are capable of directing complement lysis of SIV and SIV-infected cells in both sexes. We also found sex differences in vaccine-induced antibody species and their functions. Overall, our data suggest that sex affects vaccine-induced antibody characteristics and function and that males and females might require different immune responses to protect against HIV infection. This information could be used to generate highly effective HIV vaccines for both sexes in the future.


Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Proteínas do Sistema Complemento/imunologia , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Adenovirus dos Símios/genética , Adenovirus dos Símios/imunologia , Animais , Proteínas do Sistema Complemento/agonistas , Proteínas do Sistema Complemento/genética , Citotoxicidade Imunológica , Feminino , Regulação da Expressão Gênica , Produtos do Gene env/administração & dosagem , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Soros Imunes/química , Imunização Secundária/métodos , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Macaca mulatta , Masculino , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Vacinas contra a SAIDS/genética , Vacinas contra a SAIDS/imunologia , Fatores Sexuais , Transdução de Sinais , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas Sintéticas , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
12.
Vaccine ; 36(24): 3513-3521, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29739718

RESUMO

Staphylococcus aureus causes a chronic, contagious disease of the udder, or mastitis, in dairy cows. This infection is often refractory to antibiotic treatment, and has a significant economic impact on milk production worldwide. An effective vaccine to prevent S. aureus mastitis would improve animal health, reduce antibiotic dependence and inform human vaccine approaches. The iron-regulated surface determinant A (IsdA) and clumping factor A (ClfA) are conserved S. aureus extracellular-matrix adhesins and target vaccine antigens. Here we report the results of two bovine immunogenicity trials using purified IsdA and ClfA-cholera toxin A2/B chimeras (IsdA-CTA2/B and ClfA-CTA2/B). Cows were intranasally inoculated with IsdA-CTA2/B + ClfA-CTA2/B at dry off and followed for 70 days. Trial 1 utilized three groups with one or two booster doses at a total concentration of 600 or 900 µg. Trial 2 utilized two groups with one booster at a total concentration of 1200 µg. Humoral immune responses in serum and milk were examined by ELISA. Responses in serum were significant between groups and provide evidence of antigen-specific IgG induction after vaccination in both trials. Cellular proliferation was detected by flow cytometry using antigen-stimulated PBMCs from day 60 of Trial 2 and revealed an increase in CD4+ T cells from vaccinated cows. IsdA and ClfA stimulation induced IL-4 expression, but not IFN-γ or IL-17, in PBMCs from day 60 as determined by cytokine expression analysis. Opsonophagocytosis of S. aureus confirmed the functional in vitro activity of anti-IsdA antibodies from Trial 2 serum and milk. The vaccine was well tolerated and safe, and results support the potential of mucosally-delivered CTA2/B chimeras to protect cows from mastitis caused by S. aureus.


Assuntos
Anticorpos Antibacterianos/biossíntese , Mastite Bovina/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Infecções Estafilocócicas/prevenção & controle , Infecções Estafilocócicas/veterinária , Vacinas Antiestafilocócicas/biossíntese , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Bovinos , Proliferação de Células/efeitos dos fármacos , Toxina da Cólera/administração & dosagem , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Coagulase/administração & dosagem , Coagulase/genética , Coagulase/imunologia , Feminino , Expressão Gênica , Imunidade Humoral/efeitos dos fármacos , Imunização Secundária/métodos , Imunogenicidade da Vacina , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Interleucina-4/biossíntese , Interleucina-4/metabolismo , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/imunologia , Mastite Bovina/microbiologia , Leite/química , Leite/imunologia , Leite/microbiologia , Isoformas de Proteínas/administração & dosagem , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Vacinas Antiestafilocócicas/administração & dosagem , Vacinas Antiestafilocócicas/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade
13.
Vaccine ; 36(24): 3468-3476, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29739720

RESUMO

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic and zoonotic virus with a fatality rate in humans of over 35%. Although several vaccine candidates have been developed, there is still no clinically available vaccine for MERS-CoV. In this study, we developed two types of MERS-CoV vaccines: a recombinant adenovirus serotype 5 encoding the MERS-CoV spike gene (Ad5/MERS) and spike protein nanoparticles formulated with aluminum (alum) adjuvant. Next, we tested a heterologous prime-boost vaccine strategy, which compared priming with Ad5/MERS and boosting with spike protein nanoparticles and vice versa, with homologous prime-boost vaccination comprising priming and boosting with either spike protein nanoparticles or Ad5/MERS. Although both types of vaccine could induce specific immunoglobulin G against MERS-CoV, neutralizing antibodies against MERS-CoV were induced only by heterologous prime-boost immunization and homologous immunization with spike protein nanoparticles. Interestingly, Th1 cell activation was induced by immunization schedules including Ad5/MERS, but not by those including only spike protein nanoparticles. Heterologous prime-boost vaccination regimens including Ad5/MERS elicited simultaneous Th1 and Th2 responses, but homologous prime-boost regimens did not. Thus, heterologous prime-boost may induce longer-lasting immune responses against MERS-CoV because of an appropriate balance of Th1/Th2 responses. However, both heterologous prime-boost and homologous spike protein nanoparticles vaccinations could provide protection from MERS-CoV challenge in mice. Our results demonstrate that heterologous immunization by priming with Ad5/MERS and boosting with spike protein nanoparticles could be an efficient prophylactic strategy against MERS-CoV infection.


Assuntos
Adenovírus Humanos/imunologia , Infecções por Coronavirus/prevenção & controle , Imunização Secundária/métodos , Ativação Linfocitária/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/imunologia , Adenovírus Humanos/genética , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Esquemas de Imunização , Imunogenicidade da Vacina , Imunoglobulina G/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Nanopartículas/administração & dosagem , Nanopartículas/química , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/genética , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/virologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/virologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia
14.
Vaccine ; 36(25): 3701-3707, 2018 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-29748028

RESUMO

AIM: To determine if patients with untreated chronic lymphocytic leukemia (CLL) benefit from vaccination with a 13-valent pneumococcal conjugated vaccine (PCV13), Prevenar13®, compared to a 23-valent pneumococcal polysaccharide vaccine (PPSV23), Pneumovax®, in terms of immune response. BACKGROUND: Streptococcus pneumoniae causes substantial morbidity in patients with CLL, a group known to respond poorly to polysaccharide vaccines. Comparative studies with conjugated vaccines are lacking. METHODS: 128 treatment naïve CLL patients from eight hematology clinics in Sweden were randomized to vaccination with PCV13 (n = 63) or PPSV23 (n = 65) after stratification by IgG level and CLL clinical stage (Rai). Blood samples for evaluation of immune response were obtained at baseline, and at one and six months after vaccination. Analyses for each of the 12 pneumococcal serotypes common for PCV13 and PPSV23 were performed by opsonophagocytic assay (OPA) and enzyme-linked immunosorbent assay (ELISA). RESULTS: PCV13 elicited a superior immune response than PPSV23 in 10/12 serotypes one month after vaccination and in 5/12 serotypes six months after vaccination, measured as OPA geometric mean titers (GMTs). Geometric mean concentrations of serotype-specific IgG antibodies elicited by PCV13 as measured by ELISA, were higher than those elicited by PPSV23 in half of the common serotypes, both after one and six months. PPSV23 did not trigger a better immune response than PCV13 for any of the serotypes, regardless of analysis method or time point of analysis. Negative predictive factors for vaccination response were hypogammaglobulinemia and long disease duration. Both vaccines were well tolerated. CONCLUSIONS: In patients with previously untreated CLL, the efficacy of PCV13 in terms of immune response is superior to PPSV23 for most serotypes common for the two vaccines. We therefore propose that PCV13 should be included in vaccination programs against Streptococcus pneumoniae for CLL patients and administered as early as possible during the course of the disease.


Assuntos
Anticorpos Antibacterianos/biossíntese , Imunoglobulina G/biossíntese , Leucemia Linfocítica Crônica de Células B/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunogenicidade da Vacina , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Estudos Prospectivos , Distribuição Aleatória , Sorogrupo , Streptococcus pneumoniae/imunologia , Potência de Vacina , Vacinas Conjugadas
15.
Monoclon Antib Immunodiagn Immunother ; 37(2): 91-94, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29649375

RESUMO

Lung cancer is one of the leading causes of cancer-related deaths in the world. Regardless of the advances in lung cancer treatments, the prognosis is still poor. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is expressed in normal tissues, including the heart, pancreas, and breast. It is also found and used as a diagnostic marker in many cancers, such as renal, brain, breast, oral, and lung cancers. We previously developed specific and sensitive anti-PODXL monoclonal antibodies, PcMab-47 (mouse IgG1, kappa) and its mouse IgG2a-type (47-mG2a), both of which were suitable for immunohistochemical analyses of oral cancers. In this study, we investigated the utility of PcMab-47 and 47-mG2a for the immunohistochemical analyses of lung cancers. PcMab-47 stained 51/70 (72.9%) cases of lung cancer, whereas 47-mG2a stained 59/70 (84.3%) cases, indicating that the latter antibody is more sensitive and is useful for detecting PODXL in lung cancers.


Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Biomarcadores Tumorais/imunologia , Carcinoma Adenoescamoso/diagnóstico , Carcinoma de Células Pequenas/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Sialoglicoproteínas/imunologia , Adenocarcinoma Bronquioloalveolar/diagnóstico , Adenocarcinoma Bronquioloalveolar/imunologia , Adenocarcinoma Bronquioloalveolar/patologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Biomarcadores Tumorais/genética , Células CHO , Carcinoma Adenoescamoso/imunologia , Carcinoma Adenoescamoso/patologia , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/imunologia , Carcinoma Papilar/patologia , Carcinoma de Células Pequenas/imunologia , Carcinoma de Células Pequenas/patologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Cricetulus , Expressão Gênica , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Imuno-Histoquímica , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sialoglicoproteínas/genética , Análise Serial de Tecidos
16.
Nat Commun ; 9(1): 1421, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29650949

RESUMO

Acute allergic symptoms are caused by allergen-induced crosslinking of allergen-specific immunoglobulin E (IgE) bound to Fc-epsilon receptors on effector cells. Desensitization with allergen-specific immunotherapy (SIT) has been used for over a century, but the dominant protective mechanism remains unclear. One consistent observation is increased allergen-specific IgG, thought to competitively block allergen binding to IgE. Here we show that the blocking potency of the IgG response to Cat-SIT is heterogeneous. Next, using two potent, pre-selected allergen-blocking monoclonal IgG antibodies against the immunodominant cat allergen Fel d 1, we demonstrate that increasing the IgG/IgE ratio reduces the allergic response in mice and in cat-allergic patients: a single dose of blocking IgG reduces clinical symptoms in response to nasal provocation (ANCOVA, p = 0.0003), with a magnitude observed at day 8 similar to that reported with years of conventional SIT. This study suggests that simply augmenting the blocking IgG/IgE ratio may reverse allergy.


Assuntos
Anticorpos Monoclonais/farmacologia , Dessensibilização Imunológica/métodos , Glicoproteínas/imunologia , Hipersensibilidade/terapia , Imunoglobulina G/farmacologia , Receptores de IgE/imunologia , Adolescente , Adulto , Alérgenos/administração & dosagem , Alérgenos/imunologia , Alérgenos/isolamento & purificação , Pelo Animal/química , Pelo Animal/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Ligação Competitiva , Gatos , Misturas Complexas/química , Misturas Complexas/imunologia , Modelos Animais de Doenças , Feminino , Glicoproteínas/administração & dosagem , Glicoproteínas/isolamento & purificação , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/fisiopatologia , Imunoglobulina E/química , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Imunoglobulina G/biossíntese , Masculino , Camundongos , Pessoa de Meia-Idade , Ligação Proteica/efeitos dos fármacos , Receptores de IgE/química , Receptores de IgE/metabolismo
17.
Med Sci Monit ; 24: 1962-1969, 2018 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-29611536

RESUMO

BACKGROUND This study aimed to investigate the therapeutic effect of low, medium, and high concentrations of medical ozone on trauma-induced lumbar disc herniation. MATERIAL AND METHODS A total of 80 patients were included and were grouped into a control group, a low medical ozone (20 µg/ml) group, a medium medical ozone (40 µg/ml) group, and a high medical ozone (60 µg/ml) group. The CT scan and enzyme-linked immunosorbent assay (ELISA) were used to detect IL-6 level, SOD activity, IgM, and IgG levels upon admission and at 6 and 12 months after follow-up. The area under the ROC curve (AUC) was calculated for visual analogue scale (VAS) and efficiency rate. RESULTS All patients showed disc retraction at 6- and 12-month follow-up; while patients in the medium medical ozone (40 µg/ml) group showed the greatest disc retraction rate. The IL-6, IgM, IgG, and VAS levels significantly decreased while SOD activity increased among all groups over time (p<0.05). The AUCIL-6, AUCIgG, AUCIgM, and AUCSOD was closest to 1 in the medium medical ozone (40 µg/ml) group compared with other groups (p<0.01), with the highest efficacy at 6 (35%) and 12 (85%) months during follow-up. CONCLUSIONS Low concentrations of medical ozone (20 µg/ml and 40 µg/ml) reduced the serum IL-6, IgG, and IgM expression, presenting as analgesic and anti-inflammatory effects, while high concentrations of medical ozone (60 µg/ml) increased the serum IL-6, IgG, IgM expression, presenting as pain and pro-inflammatory effects. The medical ozone concentration of 40 µg/ml showed the optimal treatment efficacy.


Assuntos
Deslocamento do Disco Intervertebral/terapia , Ozônio/administração & dosagem , Adulto , Idoso , Feminino , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Interleucina-6/sangue , Deslocamento do Disco Intervertebral/sangue , Dor Lombar/sangue , Dor Lombar/terapia , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/fisiopatologia , Masculino , Pessoa de Meia-Idade , Oxigênio/uso terapêutico , Medição da Dor , Superóxido Dismutase/sangue , Tomografia Computadorizada por Raios X , Resultado do Tratamento
18.
Exp Parasitol ; 188: 73-78, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29626423

RESUMO

We constructed a new plasmid pIRESneo/ROP18/PLP1 that was injected intramuscularly into Kunming mice to evaluate its immune efficacy. The immunized mice exhibited significantly increased serum IgG2a levels, lymphocyte counts and Th1-type cytokine (IL-2, IL-12 and IFN-γ) levels. Moreover, the immunized mice exhibited longer survival times (44.7 ± 2.1 days for ROP18/PLP1 and 47.2 ± 2.9 days for ROP18/PLP1 + IL-18) and lower brain cyst burden (68.9% for ROP18/PLP1 and 72.4% for ROP18/PLP1 + IL-18) than control mice after T. gondii challenge. Our results demonstrate that the multiple-gene DNA vaccine including both ROP18 and PLP1 elicits greater protection against T. gondii challenge and stronger immunogenicity than single-gene vaccines.


Assuntos
Proteína Proteolipídica de Mielina/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Vacinas Protozoárias , Toxoplasma/imunologia , Toxoplasmose Animal/prevenção & controle , Vacinas de DNA , Animais , Anticorpos Antiprotozoários/biossíntese , Encéfalo/parasitologia , Citocinas/análise , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Imunoglobulina G/análise , Imunoglobulina G/biossíntese , Injeções Intramusculares , Camundongos , Proteína Proteolipídica de Mielina/genética , Plasmídeos , Proteínas Serina-Treonina Quinases/genética , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/imunologia , Vacinas Protozoárias/normas , Organismos Livres de Patógenos Específicos , Baço/citologia , Baço/imunologia , Análise de Sobrevida , Toxoplasmose Animal/mortalidade , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas de DNA/normas
19.
Int Immunopharmacol ; 59: 295-300, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29677631

RESUMO

Murine hybridoma cells can produce monoclonal antibody (MAb) and the production of these antibodies in culture and peritoneum can be affected by different factors, including stimulants, inhibitors and supplements. Among these factors, the impact of micronutrients on the production of MAbs by mouse hybridoma cells has not fully been explored. In this study the murine hybridoma cells, M3C5, were cultured and treated with different concentrations of ATRA and DHA, alone, in combinations, and at different time of exposure. Then, changes in the production of MAb in culture medium were evaluated using ELISA. The hybridoma cells after single and combined treatment with ATRA, DHA and vehicles were IP injected to Balb/c mice and the changes in production of MAb in ascites were determined by ELISA. The results showed that single and combined treatment of ATRA and DHA elevated the production of MAb by hybridoma cells in both in vivo and in vitro. The production of MAb following in vitro single treatment with 1 µM of ATRA and 10 µM of DHA for 2 days was significantly increased. The in vitro effects of ATRA on increase of MAb production was obtained more than DHA. The MAb productions in combined treatment with 0.5 µΜ of ATRA plus 5 µΜ of DHA were significantly increased in in vivo and in vitro. However, the effect of DHA was obtained more significant in in vivo conditions. The results of this study showed for the first time that in vitro and in vivo treatments of ATRA and DHA could increase the production of MAb in mouse M3C5 hybridoma cells.


Assuntos
Anticorpos Monoclonais Murinos/biossíntese , Ácidos Docosa-Hexaenoicos/farmacologia , Imunoglobulina G/biossíntese , Tretinoína/farmacologia , Animais , Líquido Ascítico/metabolismo , Gonadotropina Coriônica/imunologia , Hibridomas , Masculino , Camundongos Endogâmicos BALB C
20.
Monoclon Antib Immunodiagn Immunother ; 37(2): 110-115, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29608408

RESUMO

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L1Mab-13 (IgG1, kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L1Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L1Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L1Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.


Assuntos
Anticorpos Monoclonais/química , Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Imunoglobulina G/química , Neoplasias Pulmonares/diagnóstico , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/imunologia , Células CHO , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Cricetulus , Citometria de Fluxo , Expressão Gênica , Células HEK293 , Humanos , Imunização/métodos , Imunoglobulina G/biossíntese , Imunoglobulina G/isolamento & purificação , Imuno-Histoquímica , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neuroglia/imunologia , Neuroglia/patologia , Neuroglia/transplante , Prognóstico
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