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1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 38(9): 842-847, 2022 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-36082715

RESUMO

Objective Mice were immunized with purified virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin) to prepare polyclonal antibody and identify specificity. Methods BALB/c mice were injected with duck tembusu virus to generate viperin in mouse brain by intracranial injection. Viperin gene, cloned from mouse brain tissue by reverse transcription PCR, was inserted into pGEX-6p-1 prokaryotic expression vector and transformed into E. coli Rosetta. The recombinant viperin protein was induced by isopropyl thiogalactoside (IPTG) and its solubility was analyzed. The protein was purified by potassium chloride (KCl) staining and gel cutting method. Polyclonal antibody was prepared by immunizing mice with purified recombinant viperin protein subcutaneously through abdomen, and the titer of polyclonal antibody was determined by indirect ELISA. Western blot analysis and indirect fluorescence assay (IFA) were used to detect the transient expression of viperin protein in BHK-21 cells to identify the specificity and sensitivity of the prepared polyclonal antibody against viperin protein. Results The mouse viperin gene was successfully cloned and the viperin protein was expressed. The titer of the prepared anti-viperin polyclonal antibody reached 1:25 600. The mouse anti-viperin polyclonal antibody could specifically recognize the transient expression of viperin protein in BHK-21 cells. Conclusion Mouse polyclonal antibody against viperin protein with high specificity and sensitivity was successfully prepared.


Assuntos
Escherichia coli , Proteína Viperina , Animais , Anticorpos , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Interferons , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética
2.
Monoclon Antib Immunodiagn Immunother ; 41(4): 221-227, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35917553

RESUMO

Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The overexpression of PDPN contributes to the malignant progression of tumors. Therefore, the development of anti-PDPN monoclonal antibodies (mAbs) to animals is essential to evaluate the pathogenesis and cellular functions. Using peptide immunization, we previously developed an anti-elephant PDPN (elePDPN) mAb, PMab-295, which is useful for flow cytometry, Western blotting, and immunohistochemistry. In this study, we determined the critical epitope of PMab-295 by enzyme-linked immunosorbent assay (ELISA). We performed ELISA with the alanine-substituted peptides of elePDPN extracellular domain (amino acids 38-51), and found that PMab-295 did not recognize the alanine-substituted peptides of M41A, P44A, and E47A. Furthermore, these peptides could not inhibit the recognition of PMab-295 to elePDPN-expressing cells by flow cytometry and immunohistochemistry. The results indicate that the binding epitope of PMab-295 includes Met41, Pro44, and Glu47 of elePDPN.


Assuntos
Anticorpos Monoclonais , Células Endoteliais , Alanina , Animais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos , Glicoproteínas de Membrana , Fatores de Transcrição
3.
Monoclon Antib Immunodiagn Immunother ; 41(4): 194-201, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35917562

RESUMO

Podoplanin (PDPN) is an essential marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) that can specifically recognize PDPN in immunohistochemistry are important to analyze the development of tissues and the pathogenesis of diseases, including cancers. We have developed anti-PDPN mAbs against many animal species; however, mAbs that can recognize elephant-derived membrane proteins and distinguish the specific cell types in immunohistochemistry are limited. In this study, a novel anti-elephant PDPN (elePDPN) mAb, PMab-295 (IgG1, kappa), was established using the peptide immunization method. PMab-295 recognized both elePDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous elePDPN-expressed LACF-NaNaI cells by flow cytometry and western blotting. Kinetic analyses using flow cytometry showed that the KD of PMab-295 for CHO/elePDPN was 1.5 × 10-8 M. Furthermore, PMab-295 detected elePDPN-expressing cells using immunohistochemistry. These results showed the usefulness of PMab-295 to investigate the molecular function of elePDPN and the pathogenesis of diseases.


Assuntos
Anticorpos Monoclonais , Antineoplásicos Imunológicos , Animais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Células Endoteliais , Glicoproteínas de Membrana , Fatores de Transcrição
4.
Chembiochem ; 23(18): e202200303, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-35893479

RESUMO

Antibodies recognize their cognate antigens with high affinity and specificity, but the prediction of binding sites on the antigen (epitope) corresponding to a specific antibody remains a challenging problem. To address this problem, we developed AbAdapt, a pipeline that integrates antibody and antigen structural modeling with rigid docking in order to derive antibody-antigen specific features for epitope prediction. In this study, we systematically assessed the impact of integrating the state-of-the-art protein modeling method AlphaFold with the AbAdapt pipeline. By incorporating more accurate antibody models, we observed improvement in docking, paratope prediction, and prediction of antibody-specific epitopes. We further applied AbAdapt-AF in an anti-receptor binding domain (RBD) antibody complex benchmark and found AbAdapt-AF outperformed three alternative docking methods. Also, AbAdapt-AF demonstrated higher epitope prediction accuracy than other tested epitope prediction tools in the anti-RBD antibody complex benchmark. We anticipate that AbAdapt-AF will facilitate prediction of antigen-antibody interactions in a wide range of applications.


Assuntos
Anticorpos , Antígenos , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Epitopos/química
5.
Sci Rep ; 12(1): 12555, 2022 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-35869245

RESUMO

Antibodies recognize protein antigens with exquisite specificity in a complex aqueous environment, where interfacial waters are an integral part of the antibody-protein complex interfaces. In this work, we elucidate, with computational analyses, the principles governing the antibodies' specificity and affinity towards their cognate protein antigens in the presence of explicit interfacial waters. Experimentally, in four model antibody-protein complexes, we compared the contributions of the interaction types in antibody-protein antigen complex interfaces with the antibody variants selected from phage-displayed synthetic antibody libraries. Evidently, the specific interactions involving a subset of aromatic CDR (complementarity determining region) residues largely form the predominant determinant underlying the specificity of the antibody-protein complexes in nature. The interfacial direct/water-mediated hydrogen bonds accompanying the CDR aromatic interactions are optimized locally but contribute little in determining the epitope location. The results provide insights into the phenomenon that natural antibodies with limited sequence and structural variations in an antibody repertoire can recognize seemingly unlimited protein antigens. Our work suggests guidelines in designing functional artificial antibody repertoires with practical applications in developing novel antibody-based therapeutics and diagnostics for treating and preventing human diseases.


Assuntos
Aminoácidos , Regiões Determinantes de Complementaridade , Afinidade de Anticorpos , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo , Antígenos , Regiões Determinantes de Complementaridade/química , Humanos , Proteínas
6.
Monoclon Antib Immunodiagn Immunother ; 41(3): 163-169, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35666546

RESUMO

Chinese hamster (Cricetulus griseus) and golden hamster (Mesocricetus auratus) are important animal models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, which affect several organs, including respiratory tract, lung, and kidney. Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The development of anti-PDPN monoclonal antibodies (mAbs) for these animals is essential to evaluate the pathogenesis by SARS-CoV-2 infections. Using the Cell-Based Immunization and Screening method, we previously developed an anti-Chinese hamster PDPN (ChamPDPN) mAb, PMab-281 (mouse IgG3, kappa), and further changed its subclass into IgG2a (281-mG2a-f), both of which can recognize not only ChamPDPN but also golden hamster PDPN (GhamPDPN) by flow cytometry and immunohistochemistry. In this study, we examined the critical epitope of 281-mG2a-f, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with peptides derived from ChamPDPN and GhamPDPN extracellular domain, and found that 281-mG2a-f reacted with the peptides, which commonly possess the KIPFEELxT sequence. Next, we analyzed the reaction with the alanine-substituted mutants, and revealed that 281-mG2a-f did not recognize the alanine-substituted peptides of I75A, F77A, and E79A of ChamPDPN. Furthermore, these peptides could not inhibit the recognition of 281-mG2a-f to ChamPDPN-expressing cells by flow cytometry. The results indicate that the binding epitope of 281-mG2a-f includes Ile75, Phe77, and Glu79 of ChamPDPN, which are shared with GhamPDPN.


Assuntos
COVID-19 , Células Endoteliais , Alanina , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Mapeamento de Epitopos/métodos , Epitopos , Imunoglobulina G , Glicoproteínas de Membrana , Mesocricetus , Camundongos , SARS-CoV-2 , Fatores de Transcrição
7.
Nat Commun ; 13(1): 2951, 2022 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-35618731

RESUMO

The antibody response magnitude and kinetics may impact clinical severity, serological diagnosis and long-term protection of COVID-19, which may play a role in why children experience lower morbidity. We therefore tested samples from 122 children in Hong Kong with symptomatic (n = 78) and asymptomatic (n = 44) SARS-CoV-2 infections up to 200 days post infection, relative to 71 infected adults (symptomatic n = 61, and asymptomatic n = 10), and negative controls (n = 48). We assessed serum IgG antibodies to a 14-wide antigen panel of structural and accessory proteins by Luciferase Immuno-Precipitation System (LIPS) assay and circulating cytokines. Infected children have lower levels of Spike, Membrane, ORF3a, ORF7a, ORF7b antibodies, comparable ORF8 and elevated E-specific antibodies than adults. Combination of two unique antibody targets, ORF3d and ORF8, can accurately discriminate SARS-CoV-2 infection in children. Principal component analysis reveals distinct pediatric serological signatures, and the highest contribution to variance from adults are antibody responses to non-structural proteins ORF3d, NSP1, ORF3a and ORF8. From a diverse panel of cytokines that can modulate immune priming and relative inflammation, IL-8, MCP-1 and IL-6 correlate with the magnitude of pediatric antibody specificity and severity. Antibodies to SARS-CoV-2 internal proteins may become an important sero surveillance tool of infection with the roll-out of vaccines in the pediatric population.


Assuntos
COVID-19 , SARS-CoV-2 , Adulto , Especificidade de Anticorpos , Criança , Citocinas , Humanos , Imunoglobulina G
8.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 38(5): 452-459, 2022 May.
Artigo em Chinês | MEDLINE | ID: mdl-35603654

RESUMO

Objective To generate rabbit polyclonal antibody against mouse Tubby(Tub)-like protein 2 (TULP2) and detect the expression of TULP2 in mouse testis. Methods pET30a (+)-TULP2 and pET30(+)-TULP2-C recombinant plasmids were constructed by inserting TULP2 full-length gene fragment and TULP2-C gene fragment containing Tub domain into pET30a (+). pET30a (+)-TULP2 and pET30(+)-TULP2-C were transformed into E. coli BL21, and the prokaryotic protein expressions were induced with the supplementation of IPTG. The prokaryotic recombinant proteins were purified with His-Binding-resin, and denaturation was performed by adding urea with gradient concentration. Adult male New Zealand white rabbits were inoculated with recombinant TULP2 and TULP2-C proteins as immunogens to generate two kinds of TULP2 polyclonal antibodies. Titers of antibodies were detected by ELISA. The efficiency and specificity of antibodies were determined by Western blot and immunofluorescence (IF) staining. Results pET30a (+)-TULP2 and pET30a (+)-TULP2-C recombinant plasmids were constructed successfully, and the protein expressions of TULP2 and TULP2-C could be induced by adding IPTG. The titers of polyclonal antibodies were 1:1 000 000. Western blot and IF staining showed poor specificity of TULP2-C antibody. TULP2 antibody could specifically recognize the endogenous TULP2 protein in the testes of adult wild-type mice, and IF staining showed that TULP2 was expressed specifically in the round spermatids and elongating spermatids of mice. Conclusion A rabbit anti-mouse TULP2 polyclonal antibody is generated successfully using TULP2 full-length protein, which can be used for detecting TULP2 expression by Western blot and IF staining.


Assuntos
Anticorpos , Escherichia coli , Animais , Especificidade de Anticorpos , Western Blotting , Escherichia coli/genética , Isopropiltiogalactosídeo , Masculino , Camundongos , Coelhos
9.
Immunobiology ; 227(3): 152213, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35429697

RESUMO

Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have a pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity.


Assuntos
Anticorpos Monoclonais , Imunoglobulina G , Especificidade de Anticorpos , Heme , Humanos , Imunoglobulina A , Íons
10.
Monoclon Antib Immunodiagn Immunother ; 41(2): 101-109, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35471053

RESUMO

Ferrets (Mustela putorius furo) have been used as small animal models to investigate severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infections. Pathological analyses of these tissue samples, including those of the lung, are, therefore, essential to understand the pathogenesis of SARS-CoVs and evaluate the action of therapeutic monoclonal antibodies (mAbs) against this disease. However, mAbs that recognize ferret-derived proteins and distinguish between specific cell types, such as lung epithelial cells, are limited. Podoplanin (PDPN) has been identified as an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-ferret PDPN (ferPDPN) mAb PMab-292 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. PMab-292 recognized ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells by flow cytometry and Western blotting. The kinetic analysis using flow cytometry showed that the KD of PMab-292 for CHO/ferPDPN was 3.4 × 10-8 M. Furthermore, PMab-292 detected lung type I alveolar epithelial cells, lymphatic endothelial cells, and glomerular/Bowman's capsule in the kidney using immunohistochemistry. Hence, these results propose the usefulness of PMab-292 in analyzing ferret-derived tissues for SARS-CoV-2 research.


Assuntos
Antineoplásicos Imunológicos , COVID-19 , Vírus da SARS , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Células Endoteliais , Mapeamento de Epitopos/métodos , Furões , Cinética , Glicoproteínas de Membrana/genética , Camundongos , SARS-CoV-2 , Fatores de Transcrição
11.
Methods Mol Biol ; 2491: 217-233, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35482193

RESUMO

High-throughput protein selection methods are a cornerstone for protein engineering and pharmaceutical development. Traditional high-throughput selection strategies rely largely on recombinant antigen to generate target specificity. Though effective, this selection strategy can be limited by soluble target quality, particularly in the case of recombinant extracellular domains of transmembrane proteins. Recent advances in cell-based selection techniques provide new opportunities for improving the outcomes of ligand selection campaigns but can introduce technical challenges in maintaining antigen specificity due to the heterogeneity of biomacromolecule expression on the mammalian cell surface. Here, we describe a combination technique using recombinant antigen to "train" library target specificity followed by cell panning selections to ensure that isolated ligands bind cell-expressed target, as well as a facile microscopy technique for assessing target specificity on a clonal basis without the need to produce soluble ligand.


Assuntos
Biblioteca de Peptídeos , Engenharia de Proteínas , Animais , Especificidade de Anticorpos , Antígenos , Ligantes , Mamíferos , Engenharia de Proteínas/métodos
12.
Proc Natl Acad Sci U S A ; 119(10): e2117034119, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35235454

RESUMO

Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease of the central nervous system (CNS) with a high socioeconomic relevance. The pathophysiology of MS, which is both complex and incompletely understood, is believed to be influenced by various environmental determinants, including diet. Since the 1990s, a correlation between the consumption of bovine milk products and MS prevalence has been debated. Here, we show that C57BL/6 mice immunized with bovine casein developed severe spinal cord pathology, in particular, demyelination, which was associated with the deposition of immunoglobulin G. Furthermore, we observed binding of serum from casein-immunized mice to mouse oligodendrocytes in CNS tissue sections and in culture where casein-specific antibodies induced complement-dependent pathology. We subsequently identified myelin-associated glycoprotein (MAG) as a cross-reactive antigenic target. The results obtained from the mouse model were complemented by clinical data showing that serum samples from patients with MS contained significantly higher B cell and antibody reactivity to bovine casein than those from patients with other neurologic diseases. This reactivity correlated with the B cell response to a mixture of CNS antigens and could again be attributed to MAG reactivity. While we acknowledge disease heterogeneity among individuals with MS, we believe that consumption of cow's milk in a subset of patients with MS who have experienced a previous loss of tolerance to bovine casein may aggravate the disease. Our data suggest that patients with antibodies to bovine casein might benefit from restricting dairy products from their diet.


Assuntos
Anticorpos/imunologia , Caseínas/imunologia , Reações Cruzadas , Doenças Desmielinizantes/imunologia , Esclerose Múltipla/imunologia , Glicoproteína Associada a Mielina/imunologia , Animais , Especificidade de Anticorpos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Leite/imunologia
14.
Elife ; 112022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35289271

RESUMO

Preexisting antibodies to endemic coronaviruses (CoV) that cross-react with SARS-CoV-2 have the potential to influence the antibody response to COVID-19 vaccination and infection for better or worse. In this observational study of mucosal and systemic humoral immunity in acutely infected, convalescent, and vaccinated subjects, we tested for cross-reactivity against endemic CoV spike (S) protein at subdomain resolution. Elevated responses, particularly to the ß-CoV OC43, were observed in all natural infection cohorts tested and were correlated with the response to SARS-CoV-2. The kinetics of this response and isotypes involved suggest that infection boosts preexisting antibody lineages raised against prior endemic CoV exposure that cross-react. While further research is needed to discern whether this recalled response is desirable or detrimental, the boosted antibodies principally targeted the better-conserved S2 subdomain of the viral spike and were not associated with neutralization activity. In contrast, vaccination with a stabilized spike mRNA vaccine did not robustly boost cross-reactive antibodies, suggesting differing antigenicity and immunogenicity. In sum, this study provides evidence that antibodies targeting endemic CoV are robustly boosted in response to SARS-CoV-2 infection but not to vaccination with stabilized S, and that depending on conformation or other factors, the S2 subdomain of the spike protein triggers a rapidly recalled, IgG-dominated response that lacks neutralization activity.


Assuntos
Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Reações Cruzadas/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Especificidade de Anticorpos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Testes de Neutralização , Vacinação
15.
Int J Biol Macromol ; 206: 501-510, 2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-35245575

RESUMO

As a novel functional protein, juxtaposed with another zinc finger protein 1 (JAZF1) can regulate the growth and apoptosis through various pathways, and maintain the body's normal physiological metabolism. To explore the important role of JAZF1 in broiler ascites syndrome (BAS), we analysed the expression and distribution of the protein in poultry and mammal tissues based on the prepared polyclonal antibody. In this study, the recombinant plasmid PET32a-JAZF1 was constructed by TA cloning, subcloning and other technical methods, and the fusion protein His-JAZF1 was successfully expressed. After purification, His-JAZF1 was used as the antigen to prepare high-quality chicken-derived antibodies. Subsequently, the results showed that JAZF1 protein in broiler tissues could be specifically recognized by this antibody. Immunofluorescence showed that JAZF1 protein mainly exists in the cytoplasm of pulmonary artery, liver, kidney, heart and lung tissue cells of various animals. The expression of this protein was more obvious in broiler and duck tissues than in mammalian tissues. In addition, western blotting combined with immunofluorescence showed that BAS caused a significant decrease in JAZF1 protein in tissue cells. This effect further indicated that JAZF1 protein was closely related to the occurrence of BAS and provided a new entry point for the functional study of JAZF1 protein.


Assuntos
Ascite , Galinhas , Animais , Anticorpos , Especificidade de Anticorpos , Western Blotting , Mamíferos , Aves Domésticas , Síndrome , Fatores de Transcrição
16.
Toxins (Basel) ; 14(3)2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35324663

RESUMO

In view of the toxicological hazard and important applications in analgesics and cancer chemotherapeutics of αB-CTX, it is urgent to develop an accurate, effective and feasible immunoassay for the determination and analysis of αB-CTX in real samples. In this study, MBP-αB-CTX4 tandem fusion protein was used as an immunogen to elicit a strong immune response, and a hybridoma cell 5E4 secreting IgG2b against αB-CTX was successfully screened by hybridoma technology. The affinity of the purified 5E4 monoclonal antibody (mAb) was 1.02 × 108 L/mol, which showed high affinity and specificity to αB-CTX. Epitope 1 of αB-CTX is the major binding region for 5E4 mAb recongnization, and two amino acid residues (14L and 15F) in αB-CTX were critical sites for the interaction between αB-CTX and 5E4 mAb. Indirect competitive ELISA (ic-ELISA) based on 5E4 mAb was developed to detect and analyze αB-CTX in real samples, and the linear range of ic-ELISA to αB-CTX was 117-3798 ng/mL, with a limit of detection (LOD) of 81 ng/mL. All the above results indicated that the developed ic-ELISA had high accuracy and repeatability, and it could be applied for αB-CTX detection and drug analysis in real samples.


Assuntos
Anticorpos Monoclonais , Conotoxinas , Animais , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos , Camundongos , Camundongos Endogâmicos BALB C
17.
Science ; 375(6584): 1041-1047, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35143256

RESUMO

Heterologous prime-boost immunization strategies have the potential to augment COVID-19 vaccine efficacy. We longitudinally profiled severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-specific serological and memory B cell (MBC) responses in individuals who received either homologous (ChAdOx1:ChAdOx1) or heterologous (ChAdOx1:mRNA-1273) prime-boost vaccination. Heterologous messenger RNA (mRNA) booster immunization induced higher serum neutralizing antibody and MBC responses against SARS-CoV-2 variants of concern (VOCs) compared with that of homologous ChAdOx1 boosting. Specificity mapping of circulating B cells revealed that mRNA-1273 boost immunofocused ChAdOx1-primed responses onto epitopes expressed on prefusion-stabilized S. Monoclonal antibodies isolated from mRNA-1273-boosted participants displayed overall higher binding affinities and increased breadth of reactivity against VOCs relative to those isolated from ChAdOx1-boosted individuals. Overall, the results provide molecular insight into the enhanced quality of the B cell response induced after heterologous mRNA booster vaccination.


Assuntos
/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , /imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , /administração & dosagem , Adulto , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Feminino , Humanos , Esquemas de Imunização , Imunização Secundária , Imunogenicidade da Vacina , Masculino , Pessoa de Meia-Idade , Conformação Proteica , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química
18.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35210365

RESUMO

The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody-drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody's intended function in vivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities in vivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine's half-life, and activation of bispecific, T cell-engaging antibodies.


Assuntos
Anticorpos/metabolismo , Imunoconjugados/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Especificidade de Anticorpos , Humanos
19.
Cell ; 185(4): 614-629.e21, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35148840

RESUMO

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos Virais/imunologia , Candida albicans/química , Mananas/imunologia , Hidróxido de Alumínio/química , Animais , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Linfócitos B/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Chlorocebus aethiops , Epitopos/imunologia , Imunidade Inata , Imunização , Inflamação/patologia , Interferons/metabolismo , Lectinas Tipo C/metabolismo , Ligantes , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Linfonodos/imunologia , Linfonodos/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Seios Paranasais/metabolismo , Subunidades Proteicas/metabolismo , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Solubilidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Linfócitos T/imunologia , Fator de Transcrição RelB/metabolismo , Células Vero , beta-Glucanas/metabolismo
20.
Monoclon Antib Immunodiagn Immunother ; 41(1): 39-44, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35225666

RESUMO

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are required for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, sheep, and California sea lion PDPNs. However, no anti-Steller sea lion PDPN (stePDPN) antibody has been developed. Immunohistochemical analysis showed that an anti-California sea lion PDPN mAb (PMab-269) reacted with type I alveolar cells from the Steller sea lion lung, renal glomeruli and Bowman's capsules from kidney, and lymphatic endothelial cells from the colon, indicating that PMab-269 is useful for detecting stePDPN.


Assuntos
Anticorpos Monoclonais , Leões-Marinhos , Animais , Especificidade de Anticorpos , Células CHO , Bovinos , Cricetinae , Cricetulus , Cães , Células Endoteliais , Mapeamento de Epitopos , Cavalos , Glicoproteínas de Membrana , Camundongos , Coelhos , Ratos , Ovinos , Suínos
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