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1.
Recurso na Internet em Português | LIS - Localizador de Informação em Saúde | ID: lis-47921

RESUMO

Novos testes diagnósticos aprovados pela OMS – acessíveis, confiáveis e realizáveis em qualquer lugar – podem transformar a resposta à COVID-19 na Região, permitindo que profissionais de saúde realizem testes precisos e rápidos, mesmo em comunidades remotas, afirmou nesta quarta-feira (14) a diretora da Organização Pan-Americana da Saúde (OPAS), Carissa F. Etienne.


Assuntos
Infecções por Coronavirus , América/epidemiologia , Antígenos/imunologia
2.
PLoS Biol ; 18(9): e3000821, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886672

RESUMO

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.


Assuntos
Anticorpos/química , Dissulfetos/isolamento & purificação , Domínios de Imunoglobulina , Fragmentos de Peptídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Linfócitos B/fisiologia , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dissulfetos/química , Dissulfetos/imunologia , Mapeamento de Epitopos/métodos , Humanos , Imunização , Domínios de Imunoglobulina/genética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/genética
3.
PLoS One ; 15(8): e0237404, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32776985

RESUMO

The cotton rat (Sigmodon hispidus) is an excellent small animal model for human respiratory viral infections such as human respiratory syncytial virus (RSV) and human metapneumovirus (HMPV). These respiratory viral infections, as well as other pulmonary inflammatory diseases such as asthma, are associated with lung mechanic disturbances. So far, the pathophysiological effects of viral infection and allergy on cotton rat lungs have not been measured, although this information might be an important tool to determine the efficacy of vaccine and drug candidates. To characterize pulmonary function in the cotton rat, we established forced oscillation technique in uninfected, RSV infected and HDM sensitized cotton rats, and characterized pulmonary inflammation, mucus production, pulmonary edema, and oxygenation. There was a gender difference after RSV infection, with females demonstrating airway hyper-responsiveness while males did not. Female cotton rats 2dpi had a mild increase in pulmonary edema (wet: dry weight ratios). At day 4 post infection, female cotton rats demonstrated mild pulmonary inflammation, no increase in mucus production or reduction in oxygenation. Pulmonary function was not significantly impaired after RSV infection. In contrast, cotton rats sensitized to HDM demonstrated airway hyper-responsiveness with a significant increase in pulmonary inflammation, increase in baseline tissue damping, and a decrease in baseline pulmonary compliance. In summary, we established baseline data for forced oscillation technique and other respiratory measures in the cotton rat and used it to analyze respiratory diseases in cotton rats.


Assuntos
Testes de Função Respiratória , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Animais , Antígenos/imunologia , Feminino , Complacência Pulmonar , Masculino , Pyroglyphidae/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Caracteres Sexuais , Sigmodontinae
4.
Biophys Chem ; 265: 106441, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32745829

RESUMO

The possibility of immobilizing a protein with antigenic properties on a solid support offers significant possibilities in the development of immunosensors and vaccine formulations. For both applications, the orientation of the antigen should ensure ready accessibility of the antibodies to the epitope. However, an experimental assessment of the orientational preferences necessarily proceeds through the preparation/isolation of the antigen, the immobilization on different surfaces and one or more biophysical characterization steps. To predict a priori whether favorable orientations can be achieved or not would allow one to select the most promising experimental routes, partly mitigating the time cost towards the final product. In this manuscript, we apply a simple computational model, based on united-residue modelling, to the prediction of the orientation of the receptor binding domain of the SARS-CoV-2 spike protein on surfaces commonly used in lateral-flow devices. These calculations can account for the experimental observation that direct immobilization on gold gives sufficient exposure of the epitope to obtain a response in immunochemical assays.


Assuntos
Betacoronavirus/metabolismo , Epitopos/química , Modelos Moleculares , Glicoproteína da Espícula de Coronavírus/metabolismo , Antígenos/química , Antígenos/imunologia , Antígenos/metabolismo , Epitopos/imunologia , Simulação de Acoplamento Molecular , Domínios Proteicos , Dióxido de Silício/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Propriedades de Superfície
5.
Biophys Chem ; 265: 106441, 2020 10.
Artigo em Inglês | MEDLINE | ID: covidwho-679608

RESUMO

The possibility of immobilizing a protein with antigenic properties on a solid support offers significant possibilities in the development of immunosensors and vaccine formulations. For both applications, the orientation of the antigen should ensure ready accessibility of the antibodies to the epitope. However, an experimental assessment of the orientational preferences necessarily proceeds through the preparation/isolation of the antigen, the immobilization on different surfaces and one or more biophysical characterization steps. To predict a priori whether favorable orientations can be achieved or not would allow one to select the most promising experimental routes, partly mitigating the time cost towards the final product. In this manuscript, we apply a simple computational model, based on united-residue modelling, to the prediction of the orientation of the receptor binding domain of the SARS-CoV-2 spike protein on surfaces commonly used in lateral-flow devices. These calculations can account for the experimental observation that direct immobilization on gold gives sufficient exposure of the epitope to obtain a response in immunochemical assays.


Assuntos
Betacoronavirus/metabolismo , Epitopos/química , Modelos Moleculares , Glicoproteína da Espícula de Coronavírus/metabolismo , Antígenos/química , Antígenos/imunologia , Antígenos/metabolismo , Epitopos/imunologia , Simulação de Acoplamento Molecular , Domínios Proteicos , Dióxido de Silício/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Propriedades de Superfície
6.
Nat Commun ; 11(1): 3734, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709894

RESUMO

Medullary thymic epithelial cells (mTEC) contribute to the development of T cell tolerance by expressing and presenting tissue-restricted antigens (TRA), so that developing T cells can assess the self-reactivity of their antigen receptors prior to leaving the thymus. mTEC are a heterogeneous population of cells that differentially express TRA. Whether mTEC subsets induce distinct autoreactive T cell fates remains unclear. Here, we establish bacterial artificial chromosome (BAC)-transgenic mouse lines with biased mTEClo or mTEChi expression of model antigens. The transgenic lines support negative selection of antigen-specific thymocytes depending on antigen dose. However, model antigen expression predominantly by mTEClo supports TCRαß+ CD8αα intraepithelial lymphocyte development; meanwhile, mTEChi-restricted expression preferentially induces Treg differentiation of antigen-specific cells in these models to impact control of infectious agents and tumor growth. In summary, our data suggest that mTEC subsets may have a function in directing distinct mechanisms of T cell tolerance.


Assuntos
Antígenos/imunologia , Diferenciação Celular/imunologia , Células Epiteliais/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos/metabolismo , Infecções Bacterianas , Medula Óssea , Linhagem Celular Tumoral , Feminino , Tolerância Imunológica , Linfonodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Linfócitos T/metabolismo , Timócitos/imunologia , Fatores de Transcrição/genética
7.
Anal Chem ; 92(16): 11305-11309, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32605363

RESUMO

The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Nucleocapsídeo/imunologia , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Anticorpos Antivirais/sangue , Antígenos/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Pandemias , Pneumonia Viral/virologia
8.
Proc Natl Acad Sci U S A ; 117(30): 17727-17736, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32665441

RESUMO

Erythrocytes naturally capture certain bacterial pathogens in circulation, kill them through oxidative stress, and present them to the antigen-presenting cells (APCs) in the spleen. By leveraging this innate immune function of erythrocytes, we developed erythrocyte-driven immune targeting (EDIT), which presents nanoparticles from the surface of erythrocytes to the APCs in the spleen. Antigenic nanoparticles were adsorbed on the erythrocyte surface. By engineering the number density of adsorbed nanoparticles, (i.e., the number of nanoparticles loaded per erythrocyte), they were predominantly delivered to the spleen rather than lungs, which is conventionally the target of erythrocyte-mediated delivery systems. Presentation of erythrocyte-delivered nanoparticles to the spleen led to improved antibody response against the antigen, higher central memory T cell response, and lower regulatory T cell response, compared with controls. Enhanced immune response slowed down tumor progression in a prophylaxis model. These findings suggest that EDIT is an effective strategy to enhance systemic immunity.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos/imunologia , Eritrócitos/imunologia , Imunização , Animais , Formação de Anticorpos/imunologia , Antígenos/química , Biomimética , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Feminino , Humanos , Camundongos , Nanopartículas , Baço/imunologia , Vacinação , Vacinas , Ensaios Antitumorais Modelo de Xenoenxerto
9.
PLoS One ; 15(7): e0236664, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722684

RESUMO

BACKGROUND: Systemic lupus erythematosus (SLE) is a severe autoimmune disease in which immune tolerance defects drive production of pathogenic anti-nuclear autoantibodies. Anergic B cells are considered a potential source of these autoantibodies due to their autoreactivity and overrepresentation in SLE patients. Studies of lupus-prone mice have shown that genetic defects mediating autoimmunity can breach B cell anergy, but how this breach occurs with regards to endogenous nuclear antigen remains unclear. We investigated whether B and T cell defects in congenic mice (c1) derived from the lupus-prone New Zealand Black strain can breach tolerance to nuclear self-antigen in the presence of knock-in genes (Vκ8/3H9; dKI) that generate a ssDNA-reactive, anergic B cell population. METHODS: Flow cytometry was used to assess splenic B and T cells from 8-month-old c1 dKI mice and serum autoantibodies were measured by ELISA. dKI B cells stimulated in vitro with anti-IgM were assessed for proliferation and activation by examining CFSE decay and CD86. Cytokine-producing T cells were identified by flow cytometry following culture of dKI splenocytes with PMA and ionomycin. dKI B cells from 6-8-week-old mice were adoptively transferred into 4-month-old wild type recipients and assessed after 7 days via flow cytometry and immunofluorescence microscopy. RESULTS: c1 dKI mice exhibited B cell proliferation indicative of impaired anergy, but had attenuated autoantibodies and germinal centres compared to wild type littermates. This attenuation appeared to stem from a decrease in PD-1hi T helper cells in the dKI strains, as c1 dKI B cells were recruited to germinal centres when adoptively transferred into c1 wild type mice. CONCLUSION: Anergic, DNA-specific autoreactive B cells only seem to drive profound autoimmunity in the presence of concomitant defects in the T cell subsets that support high-affinity plasma cell production.


Assuntos
Anticorpos Antinucleares/genética , Anticorpos Antifosfolipídeos/genética , Antígenos/imunologia , Linfócitos B/imunologia , Anergia Clonal , Lúpus Eritematoso Sistêmico/imunologia , Animais , Linfócitos B/citologia , Proliferação de Células , DNA de Cadeia Simples/imunologia , Suscetibilidade a Doenças , Técnicas de Introdução de Genes , Lúpus Eritematoso Sistêmico/genética , Camundongos
10.
Anal Chem ; 92(16): 11305-11309, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: covidwho-733550

RESUMO

The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Nucleocapsídeo/imunologia , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Anticorpos Antivirais/sangue , Antígenos/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Pandemias , Pneumonia Viral/virologia
11.
Gene ; 756: 144911, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32574756

RESUMO

Enolase, a multifunctional glycolytic enzyme, is known to act as a plasminogen receptor in many species, involved in the pivotal processes such as motility, adhesion, invasion, growth, and differentiation of the parasites. Knowledge on the function of enolase from Dermanyssus gallinae is very limited. Here we report on the molecular cloning, enzymatic activity, tissue distribution and plasminogen binding activity of enolase from D. gallinae (DgENO). The full-length of cDNA was 1305 bp, specifying a peptide of 434 amino acids. Bioinformatics analysis showed that DgENO was highly conserved compared with a range of organisms, indicating the potentially similar functions in D. gallinae. A recombinant DgENO (rDgENO) protein was produced and characterized, it catalyzed the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate, the optimal pH was 7.5. Polyclonal antibodies were generated in mice and western blotting indicated that antiserum specifically recognized the native enolase in the somatic extracts from D. gallinae. Immunohistochemical staining of mite sections revealed that the distribution of DgENO was ubiquitous with high level in salivary gland, mite digestive tissues and fat bodies in D. gallinae. Expression level of DgENO was observed mostly in engorged adult mites. Moreover, ELISA binding assay showed that rDgENO could bind plasminogen, and lysine analog ε-aminocaproic acid significantly inhibited this binding activity, indicating that D. gallinae enolase is a receptor of plasminogen. The present study provided foundation for understanding of the biological functions of DgENO and its application in development of vaccines against D. gallinae.


Assuntos
Antígenos/imunologia , Ácaros/imunologia , Fosfopiruvato Hidratase/química , Vacinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Antígenos/química , Antígenos/genética , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Ácaros/enzimologia , Ácaros/genética , Ácaros/crescimento & desenvolvimento , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/genética , Plasminogênio/metabolismo , Alinhamento de Sequência
12.
Proc Natl Acad Sci U S A ; 117(23): 12969-12979, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32434911

RESUMO

CD5 is characterized as an inhibitory coreceptor with an important regulatory role during T cell development. The molecular mechanism by which CD5 operates has been puzzling and its function in mature T cells suggests promoting rather than repressing effects on immune responses. Here, we combined quantitative mass spectrometry and genetic studies to analyze the components and the activity of the CD5 signaling machinery in primary T cells. We found that T cell receptor (TCR) engagement induces the selective phosphorylation of CD5 tyrosine 429, which serves as a docking site for proteins with adaptor functions (c-Cbl, CIN85, CRKL), connecting CD5 to positive (PI3K) and negative (UBASH3A, SHIP1) regulators of TCR signaling. c-CBL acts as a coordinator in this complex enabling CD5 to synchronize positive and negative feedbacks on TCR signaling through the other components. Disruption of CD5 signalosome in mutant mice reveals that it modulates TCR signal outputs to selectively repress the transactivation of Foxp3 and limit the inopportune induction of peripherally induced regulatory T cells during immune responses against foreign antigen. Our findings bring insights into the paradigm of coreceptor signaling, suggesting that, in addition to providing dualistic enhancing or dampening inputs, coreceptors can engage concomitant stimulatory and inhibitory signaling events, which act together to promote specific functional outcomes.


Assuntos
Antígenos/imunologia , Antígenos CD5/metabolismo , Diferenciação Celular/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/fisiologia , Animais , Antígenos CD5/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica/imunologia , Ativação Linfocitária/genética , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Transdução de Sinais/genética , Transdução de Sinais/imunologia
13.
Int J Nanomedicine ; 15: 2685-2697, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32368049

RESUMO

Background: Nanocarriers could deliver significantly higher amounts of antigen to antigen-presenting cells (APCs), which have great potential to stimulate humoral and cellular response in cancer immunotherapy. Thereafter, silica solid nanosphere (SiO2) was prepared, and a model antigen (ovalbumin, OVA) was covalently conjugated on the surface of SiO2 to form nanovaccine (OVA@SiO2). And the application of OVA@SiO2 for cancer immunotherapy was evaluated. Materials and Methods: SiO2 solid nanosphere was prepared by the Stöber method, then successively aminated by aminopropyltriethoxysilane and activated with glutaraldehyde. OVA was covalently conjugated on the surface of activated SiO2 to obtain nanovaccine (OVA@SiO2). Dynamic light scattering, scanning electron microscope, and transmission electron microscope were conducted to identify the size distribution, zeta potential and morphology of OVA@SiO2. The OVA loading capacity was investigated by varying glutaraldehyde concentration. The biocompatibility of OVA@SiO2 to DC2.4 and RAW246.7 cells was evaluated by a Cell Counting Kit-8 assay. The uptake of OVA@SiO2 by DC2.4 and its internalization pathway were evaluated in the absence or presence of different inhibitors. The activation and maturation of bone marrow-derived DC cells by OVA@SiO2 were also investigated. Finally, the in vivo transport of OVA@SiO2 and its toxicity to organs were appraised. Results: All results indicated the successful covalent conjugation of OVA on the surface of SiO2. The as-prepared OVA@SiO2 possessed high antigen loading capacity, which had good biocompatibility to APCs and major organs. Besides, OVA@SiO2 facilitated antigen uptake by DC2.4 cells and its cytosolic release. Noteworthily, OVA@SiO2 significantly promoted the maturation of dendritic cells and up-regulation of cytokine secretion by co-administration of adjuvant CpG-ODN. Conclusion: The as-prepared SiO2 shows promising potential for use as an antigen delivery carrier.


Assuntos
Antígenos/metabolismo , Vacinas Anticâncer/farmacologia , Imunoterapia/métodos , Nanosferas/química , Ovalbumina/química , Adjuvantes Imunológicos/administração & dosagem , Animais , Apresentação do Antígeno , Antígenos/administração & dosagem , Antígenos/química , Antígenos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/química , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Nanosferas/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Ovalbumina/imunologia , Ovalbumina/farmacocinética , Células RAW 264.7 , Dióxido de Silício/química
14.
Chemistry ; 26(41): 8976-8982, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32428253

RESUMO

The addition of aluminum-based adjuvants in vaccines enhances the immune response to antigens. The strength of antigen adsorption on adjuvant gels is known to modulate vaccine efficacy. However, a detailed understanding of the mechanisms of interaction between aluminum gels and antigens is still missing. Herein, a new analytical approach based on dynamic nuclear polarization (DNP) enhanced NMR spectroscopy under magic angle spinning (MAS) is implemented to provide a molecular description of the antigen-adjuvant interface. This approach is demonstrated on hepatitis B surface antigen particles in combination with three aluminum gels obtained from different suppliers. Both noncovalent and covalent interactions between the phospholipids of the antigen particles and the surface of the aluminum gels are identified by using MAS DNP NMR 27 Al and 31 P correlation experiments. Although covalent interactions were detected for only one of the formulations, dipolar recoupling rotational echo adiabatic passage double resonance (REAPDOR) experiments reveal significant differences in the strength of weak interactions.


Assuntos
Adjuvantes Imunológicos/química , Alumínio/química , Antígenos/química , Vacinas/química , Adsorção , Antígenos/imunologia , Composição de Medicamentos , Espectroscopia de Ressonância Magnética/métodos , Vacinas/imunologia
15.
Curr Top Microbiol Immunol ; 426: 65-82, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32385532

RESUMO

The skin is the outermost organ of the body and is exposed to many kinds of external pathogens. To manage this, the skin contains multiple types of immune cells. To achieve sufficient induction of cutaneous adaptive immune responses, the antigen presentation/recognition in the skin is an essential process. Recent studies have expanded our knowledge of how T cells survey their cognate antigens in the skin. In addition, the formation of a lymphoid cluster, named inducible skin-associated lymphoid tissue (iSALT), has been reported during skin inflammation. Although iSALT may not be classified as a typical tertiary lymphoid organ, it provides specific antigen presentation sites in the skin. In this article, we provide an overview of the antigen presentation mechanism in the skin, with a focus on the development of iSALT and its function.


Assuntos
Imunidade Adaptativa , Tecido Linfoide/anatomia & histologia , Tecido Linfoide/imunologia , Pele/imunologia , Apresentação do Antígeno , Antígenos/imunologia , Humanos , Linfócitos T/imunologia
16.
PLoS One ; 15(4): e0231138, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32243477

RESUMO

In respect to the heterogeneity among influenza A virus strains and the shortcomings of current vaccination programs, there is a huge interest in the development of alternative vaccines that provide a broader and more long-lasting protection. Gene-based approaches are considered as promising candidates for such flu vaccines. In our study, innate signalling molecules from the RIG-I and the NALP3 pathways were evaluated as genetic adjuvants in intramuscular DNA immunizations. Plasmids encoding a constitutive active form of RIG-I (cRIG-I), IPS-1, IL-1ß, or IL-18 were co-administered with plasmids encoding the hemagglutinin and nucleoprotein derived from H1N1/Puerto Rico/8/1934 via electroporation in BALB/c mice. Immunogenicity was analysed in detail and efficacy was demonstrated in homologous and heterologous influenza challenge experiments. Although the biological activities of the adjuvants have been confirmed by in vitro reporter assays, their single or combined inclusion in the vaccine did not result in superior vaccine efficacy. With the exception of significantly increased levels of antigen-specific IgG1 after the co-administration of IL-1ß, there were only minor alterations concerning the immunogenicity. Since DNA electroporation alone induced substantial inflammation at the injection site, as demonstrated in this study using Mx2-Luc reporter mice, it might override the adjuvants´ contribution to the inflammatory microenvironment and thereby minimizes the influence on the immunogenicity. Taken together, the DNA immunization was protective against subsequent challenge infections but could not be further improved by the genetic adjuvants analysed in this study.


Assuntos
Adjuvantes Imunológicos/metabolismo , Imunidade Inata , Vacinas contra Influenza/imunologia , Transdução de Sinais , Vacinas de DNA/imunologia , Animais , Antígenos/imunologia , Linfócitos T CD8-Positivos , Bovinos , Linhagem Celular , Cães , Feminino , Imunidade Humoral , Imunização , Inflamação/patologia , Vírus da Influenza B/imunologia , Cinética , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae
17.
Nat Biotechnol ; 38(6): 715-721, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32231335

RESUMO

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100-1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450-900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.


Assuntos
Anticorpos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única , Animais , Antígenos/imunologia , Linfócitos B/imunologia , Vacinas Anticâncer/imunologia , DNA/análise , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoglobulina G/genética , Camundongos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
18.
Nat Immunol ; 21(4): 400-411, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32123373

RESUMO

Mucosal-associated invariant T (MAIT) cells are activated by microbial riboflavin-based metabolite antigens when presented by MR1. How modifications to the potent antigen 5-OP-RU affect presentation by MR1 and MAIT cell activation remains unclear. Here we design 20 derivatives, termed altered metabolite ligands (AMLs), to dissect the impact of different antigen components on the human MAIT-MR1 axis. Analysis of 11 crystal structures of MAIT T cell antigen receptor (TCR)-MR1-AML ternary complexes, along with biochemical and functional assays, shows that MR1 cell-surface upregulation is influenced by ribityl and non-ribityl components of the ligand and the hydrophobicity of the MR1-AML interface. The polar ribityl chain of the AML strongly influences MAIT cell activation potency through dynamic compensatory interactions within a MAIT TCR-MR1-AML interaction triad. We define the basis by which the MAIT TCR can differentially recognize AMLs, thereby providing insight into MAIT cell antigen specificity and potency.


Assuntos
Antígenos/imunologia , Células T Invariáveis Associadas à Mucosa/imunologia , Linhagem Celular Tumoral , Humanos , Células Jurkat , Ligantes , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Riboflavina/imunologia
19.
Immunity ; 52(3): 487-498.e6, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32155411

RESUMO

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.


Assuntos
Antígenos/imunologia , Butirofilinas/imunologia , Células Germinativas/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Antígenos/metabolismo , Antígenos CD/química , Antígenos CD/imunologia , Antígenos CD/metabolismo , Butirofilinas/química , Butirofilinas/metabolismo , Células CHO , Cricetinae , Cricetulus , Células Germinativas/metabolismo , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T gama-delta/química , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/metabolismo
20.
PLoS One ; 15(2): e0229042, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32097442

RESUMO

T cell anergy is known to be a crucial mechanism for various types of immune tolerance, including oral tolerance. The expression of several anergy-specific genes was reportedly up-regulated in anergic T cells, and played important roles in the cells. However, how the genes were up-regulated has not been understood. In this study, we comprehensively analyzed the altered gene expression and DNA methylation status in T cells tolerized by oral antigen in vivo. Our results showed that many genes were significantly up-regulated in the orally tolerized T cells, and most of the genes found in this study have not been reported previously as anergy related genes; for example, ribosomal protein L41 (FC = 3.54E06, p = 3.70E-09: Fisher's exact test; the same applies hereinafter) and CD52 (FC = 2.18E05, p = 3.44E-06). Furthermore, we showed that the DNA methylation statuses of many genes; for example, enoyl-coenzyme A delta isomerase 3 (FC = 3.62E-01, p = 3.01E-02) and leucine zipper protein 1 (FC = 4.80E-01, p = 3.25E-02), including the ones distinctly expressed in tolerized T cells; for example, latexin (FC = 3.85E03, p = 4.06E-02 for expression; FC = 7.75E-01, p = 4.13E-01 for DNA methylation) and small nuclear ribonucleoprotein polypeptide F (FC = 3.12E04, p = 4.46E-04 for expression; FC = 8.56E-01, p = 5.15E-01 for DNA methylation), changed during tolerization, suggesting that the distinct expression of some genes was epigenetically regulated in the tolerized T cells. This study would contribute to providing a novel clue to the fine understanding of the mechanism for T cell anergy and oral tolerance.


Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Linfócitos T/metabolismo , Administração Oral , Animais , Antígenos/administração & dosagem , Antígenos/imunologia , Biomarcadores , Ilhas de CpG , Citosina , Epigênese Genética , Perfilação da Expressão Gênica , Tolerância Imunológica , Ativação Linfocitária/imunologia , Camundongos , Transdução de Sinais , Linfócitos T/imunologia
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