RESUMO
We describe the longitudinal kinetics of the serological response in COVID-19 recovered patients over a period of 14 months. The antibody kinetics in a cohort of 192 recovered patients, including 66 patients for whom follow-up serum samples were obtained at two to four clinic visits, revealed that RBD-specific antibodies decayed over the 14 months following the onset of symptoms. The decay rate was associated with the robustness of the response in that antibody levels that were initially highly elevated after the onset of symptoms subsequently decayed more rapidly. An exploration of the differences in the longitudinal kinetics between recovered patients and naïve vaccinees who had received two doses of the BNT162b2 vaccine showed a significantly faster decay in the naïve vaccinees, indicating that serological memory following natural infection is more robust than that following to vaccination. Our data highlighting the differences between serological memory induced by natural infection vs. vaccination contributed to the decision-making process in Israel regarding the necessity for a third vaccination dose.
Assuntos
COVID-19 , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , Humanos , Cinética , VacinaçãoRESUMO
MOTIVATION: A comprehensive characterization of the humoral response towards a specific antigen requires quantification of the B-cell receptor repertoire by next-generation sequencing (BCR-Seq), as well as the analysis of serum antibodies against this antigen, using proteomics. The proteomic analysis is challenging since it necessitates the mapping of antigen-specific peptides to individual B-cell clones. RESULTS: The PASA web server provides a robust computational platform for the analysis and integration of data obtained from proteomics of serum antibodies. PASA maps peptides derived from antibodies raised against a specific antigen to corresponding antibody sequences. It then analyzes and integrates proteomics and BCR-Seq data, thus providing a comprehensive characterization of the humoral response. The PASA web server is freely available at https://pasa.tau.ac.il and open to all users without a login requirement.
Assuntos
Anticorpos , Internet , Proteômica/métodos , Software , Animais , Anticorpos/sangue , Anticorpos/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Bases de Dados de Proteínas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , CamundongosRESUMO
It is predicted that the antibiotic resistance crisis will result in an annual death rate of 10 million people by the year 2050. To grapple with the challenges of the impending crisis, there is an urgent need for novel and rapid diagnostic tools. In this study, we developed a novel monoclonal antibody-named mAb-EspB-B7-that targets the EspB protein, a component within the bacterial type 3 secretion system (T3SS), which is mainly expressed in Gram-negative pathogens and is essential for bacterial infectivity. We found that mAb-EspB-B7 has high affinity and specificity toward recombinant and native EspB proteins; is stable over a range of pH levels, temperatures, and salt concentrations; and retains its functionality in human serum. We identified the epitope for mAb-EspB-B7 and validated it by competitive enzyme-linked immunosorbent assay (ELISA). Since this epitope is conserved across several T3SS-harboring pathogens, mAb-EspB-B7 holds great potential for development as an active component in precise and rapid diagnostic tools that can differentiate between commensal and pathogenic bacterial strains. To this end, we integrated the well-characterized monoclonal antibody into an electrochemical biosensor and demonstrated its high specificity and sensitivity capabilities in detecting pathogenic bacterial T3SS-associated antigens as well as intact bacteria. We foresee that in the near future it will be possible to design and develop a point-of-care biosensor with multiplexing capabilities for the detection of a panel of pathogenic bacteria.
Assuntos
Anticorpos Monoclonais/sangue , Técnicas Biossensoriais , Técnicas Eletroquímicas , Bactérias Gram-Negativas/genética , Testes Imediatos , Sistemas de Secreção Tipo III/sangue , Ensaio de Imunoadsorção Enzimática , Bactérias Gram-Negativas/patogenicidade , Humanos , Concentração de Íons de Hidrogênio , Temperatura , Sistemas de Secreção Tipo III/genéticaRESUMO
Antibodies are considered the hallmark of the adaptive immune system in that they mediate various key biological functions, such as direct neutralization and recruitment of effector immune cells to eliminate invading pathogens. Antibodies exhibit several unique properties, including high diversity (enabling binding to a wide range of targets), high specificity and structural integrity. These properties and the understanding that antibodies can be utilized in a wide range of applications have motivated the scientific community to develop new approaches for antibody repertoire analysis and rapid monoclonal antibody discovery. Today, antibodies are key modules in the pharmaceutical and diagnostic industries. By virtue of their high affinity and specificity to their targets and the availability of technologies to engineer different antibodies to a wide range of targets, antibodies have become the most promising natural biological molecules in a range of biotechnological applications, such as: highly specific and sensitive nanobiosensors for the diagnostics of different biomarkers; nanoparticle-based targeted drug delivery systems to certain cells or tissues; and nanomachines, which are nanoscale mechanical devices that enable energy conversion into precise mechanical motions in response to specific molecular inputs. In this review, we start by describing the unique properties of antibodies, how antibody diversity is generated, and the available technologies for antibody repertoire analysis and antibody discovery. Thereafter, we provide an overview of some antibody-based nanotechnologies and discuss novel and promising approaches for the application of antibodies in the nanotechnology field. Overall, we aim to bridge the knowledge gap between the nanotechnology and antibody engineering disciplines by demonstrating how technological advances in the antibody field can be leveraged to develop and/or enhance new technological approaches in the nanotechnology field.
Assuntos
Anticorpos/fisiologia , Bioengenharia , Biotecnologia , Nanotecnologia , Animais , Técnicas Biossensoriais , Sistemas de Liberação de Medicamentos , HumanosRESUMO
Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT(+) serum IgG repertoire comprises â¼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with Kd â¼ 10(-8)-10(-10) M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3(-)CD14(-)CD19(+)CD27(++)CD38(++)CD20(-)TT(+)) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the Kd). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.
Assuntos
Anticorpos Antibacterianos/biossíntese , Toxoide Tetânico/administração & dosagem , Sequência de Aminoácidos , Anticorpos Antibacterianos/química , Linfócitos B/imunologia , Cromatografia Líquida , Humanos , Imunofenotipagem , Dados de Sequência Molecular , Espectrometria de Massas em TandemRESUMO
We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V(H) CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V(H) clonotypes. Of these 34 CDRH3s, 12 account for â¼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.
Assuntos
Anticorpos Monoclonais/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Cromatografia de Afinidade , Regiões Determinantes de Complementaridade , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Espectrometria de Massas , Dados de Sequência Molecular , Proteômica , CoelhosRESUMO
Characterizing the in vivo dynamics of the polyclonal antibody repertoire in serum, such as that which might arise in response to stimulation with an antigen, is difficult due to the presence of many highly similar immunoglobulin proteins, each specified by distinct B lymphocytes. These challenges have precluded the use of conventional mass spectrometry for antibody identification based on peptide mass spectral matches to a genomic reference database. Recently, progress has been made using bottom-up analysis of serum antibodies by nanoflow liquid chromatography/high-resolution tandem mass spectrometry combined with a sample-specific antibody sequence database generated by high-throughput sequencing of individual B cell immunoglobulin variable domains (V genes). Here, we describe how intrinsic features of antibody primary structure, most notably the interspersed segments of variable and conserved amino acid sequences, generate recurring patterns in the corresponding peptide mass spectra of V gene peptides, greatly complicating the assignment of correct sequences to mass spectral data. We show that the standard method of decoy-based error modeling fails to account for the error introduced by these highly similar sequences, leading to a significant underestimation of the false discovery rate. Because of these effects, antibody-derived peptide mass spectra require increased stringency in their interpretation. The use of filters based on the mean precursor ion mass accuracy of peptide-spectrum matches is shown to be particularly effective in distinguishing between "true" and "false" identifications. These findings highlight important caveats associated with the use of standard database search and error-modeling methods with nonstandard data sets and custom sequence databases.
Assuntos
Anticorpos Monoclonais/sangue , Proteômica/métodos , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Cromatografia Líquida de Alta Pressão , Cisteína/química , Humanos , Dados de Sequência Molecular , Coelhos , Espectrometria de Massas em TandemRESUMO
The use of protein crystals as a source of nanoscale biotemplates has attracted growing interest in recent years owing to their inherent internal order. As these crystals are vulnerable to environmental changes, potential applications require their stabilization by chemical crosslinking. We have previously shown that such intermolecular chemical crosslinking reactions occurring within protein crystals are not random events, but start at preferred crosslinking sites imposed by the alignment of protein molecules and their packing within the crystalline lattice. Here we propose a new working hypothesis and demonstrate its feasibility in enabling us to extricate homogeneous populations of single protein molecules that display chemical point mutations or of dimers that show homogeneous chemical crosslinking, and that have the potential for isolation of higher structures. Characterization of the crosslinking mechanism and its end products opens the way to the potential retrieval of such specific modified/intermolecular crosslinked products simply by effecting partial crosslinking at identified preferred sites, followed by time-controlled arrest of the crosslinking reaction and dissolution of the crystals by medium exchange complemented by chromatographic purification.
Assuntos
Estabilidade Proteica , Proteínas/química , CristalizaçãoRESUMO
Diarrheagenic E. coli infections, commonly treated with ß-lactam antibiotics, contribute to antibiotic resistance - a pressing public health concern. Rapid monitoring of pathogen antibiotic resistance is vital to combat antimicrobial spread. Current bacterial diagnosis methods identify pathogens or determine antibiotic resistance separately, necessitating multiple assays. There is an urgent need for tools that simultaneously identify infectious agents and their antibiotic resistance at the point of care (POC). We developed an integrated electrochemical chip-based biosensor for detecting enteropathogenic E. coli (EPEC), a major neonatal diarrheal pathogen, using an antibody against a virulence marker, termed EspB, and the ß-lactam resistance marker, ß-lactamase. A dual-channel microfabricated chip, bio-functionalized with a specific EspB monoclonal antibody, and nitrocefin, a ß -lactamase substrate was utilized. The chip facilitated electrochemical impedance spectroscopy (EIS)-based detection of EspB antigen and EspB-expressing bacteria. For ß-lactam resistance profiling, a second channel enabled differential-pulse voltammetric (DPV) measurement of hydrolyzed nitrocefin. EIS-based detection of EspB antigen was calibrated (LOD: 4.3 ng/mL ±1 and LOQ: 13.0 ng/mL ±3) as well as DPV-based detection of the antibiotic resistance marker, ß-lactamase (LOD: 3.6 ng/mL ±1.65 and LOQ: 10 ng/mL ±4). The integrated EIS and DPV biosensor was employed for the simultaneous detection of EspB-expressing and ß-lactamase-producing bacteria. The combined readout from both channels allowed the distinction between antibiotic-resistant and -sensitive pathogenic bacteria. The integrated electrochemical biosensor successfully achieved simultaneous, rapid detection of double positive EspB- and ß-lactamase-expressing bacteria. Such distinction enabled by a portable device within a short assay time and a simplified sample preparation, may be highly valuable in mitigating the spread of AMR. This new diagnostic tool holds promise for the development of POC devices in clinical diagnosis.
Assuntos
Técnicas Biossensoriais , beta-Lactamases , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Humanos , beta-Lactamases/metabolismo , Infecções por Escherichia coli/microbiologia , Escherichia coli Enteropatogênica/isolamento & purificação , Escherichia coli Enteropatogênica/patogenicidade , Escherichia coli Enteropatogênica/efeitos dos fármacos , Espectroscopia Dielétrica/instrumentação , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Desenho de Equipamento , Proteínas de Escherichia coli , Antibacterianos/farmacologia , CefalosporinasRESUMO
Despite tremendous inroads in the development of more sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies for mass spectrometry-based proteomics, there remains a significant need for enhancing the selectivity of MS/MS-based workflows for streamlined analysis of complex biological mixtures. Here, a novel LC-MS/MS platform based on 351 nm ultraviolet photodissociation (UVPD) is presented for the selective analysis of cysteine-peptide subsets in complex protein digests. Cysteine-selective UVPD is mediated through the site-specific conjugation of reduced cysteine residues with a 351 nm active chromogenic Alexa Fluor 350 (AF350) maleimide tag. Only peptides containing the AF350 chromophore undergo photodissociation into extensive arrays of b- and y-type fragment ions, thus providing a facile means for differentiating cysteine-peptide targets from convoluting peptide backgrounds. With the use of this approach in addition to strategic proteolysis, the selective analysis of diagnostic heavy-chain complementarity determining regions (CDRs) of single-chain antibody (scAb) fragments is demonstrated.
Assuntos
Cromatografia Líquida/métodos , Regiões Determinantes de Complementaridade/análise , Cisteína/química , Imunoglobulina G/análise , Fragmentos de Peptídeos/análise , Proteômica , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Antígenos/imunologia , Antígenos/metabolismo , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Processos Fotoquímicos , Homologia de Sequência de Aminoácidos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Espectrofotometria Ultravioleta , Fluxo de TrabalhoRESUMO
Antibody libraries came into existence 30 years ago when the accumulating sequence data of immunoglobulin genes and the advent of PCR technology made it possible to clone antibody gene repertoires. Phage display (most common) and additional display and screening technologies were applied to pan out desired binding specificities from antibody libraries. As other antibody discovery tools, phage display is not an off-the-shelf technology and not offered as a kit but rather requires experience and expertise for making it indeed very useful.Next-generation sequencing (NGS) coupled with bioinformatics is a powerful tool for analyzing large amount of DNA sequence output of the panning. Here, we demonstrate how NGS analysis of phage biopanning (phage-Seq) of complex antibody libraries can facilitate the antibody discovery process and provide insights regarding the biopanning process (see Fig. 1).
Assuntos
Bacteriófagos , Anticorpos de Cadeia Única , Humanos , Anticorpos de Cadeia Única/genética , Genes de Imunoglobulinas , Sequenciamento de Nucleotídeos em Larga Escala , BioprospecçãoRESUMO
BACKGROUND: The human milk antibody response following maternal immunization with the BNT162b2 mRNA vaccine is important for the protection of the infant during infancy. The vaccine-specific antibody response is still unclear at different stages of human milk production, as are the effects of maternal immunization timing on the robustness of the antibody response. OBJECTIVES: The study aimed to assess the antibody response (IgG/IgA/IgM) during various lactation stages and identify the best vaccination timing during pregnancy. METHODS: A prospective cohort study of 73 postpartum women who were administered the BNT162b2 COVID-19 mRNA vaccine during the second or third trimester of pregnancy were recruited. Statistical comparison was conducted using 16 human milk samples from a prepandemic control group. RESULTS: Excluding 11 women, the study included 62 lactating women who were administered the mRNA vaccine during the second or third trimester of pregnancy. A total of 149 samples of human milk were collected at different lactation stages. Our findings reveal that colostrum exhibits significantly higher levels of IgG (95% confidence interval [CI]: 1.3, 9.0; P = 0.023), IgA (95% CI: 55.98, 100.2; P = 0.0034), and IgM (95% CI: 0.03, 0.62; P < 0.0001) compared with mature milk IgG (95% CI: 0.25, 0.43), IgA (95% CI: 9.65, 13.74), IgM (95% CI: 0.03, 0.04). The timing of maternal immunization affected the antibody response. The level of IgA in mature milk was higher when immunization occurred in the second trimester (95% CI: 11.14, 19.66; P = 0.006) than in the third trimester (95% CI: 7.16, 11.49). Conversely, IgG levels in mature milk were higher when immunization occurred during the third trimester (95% CI: 0.36, 0.65; P < 0.0001) than in the second trimester (95% CI: 0.09, 0.38). CONCLUSIONS: Our study provides evidence that administering the mRNA vaccine to pregnant women during the second trimester increases vaccine-specific IgA levels during lactation. Considering the significance of human milk IgA in mucosal tissues and its prevalence throughout lactation, it is reasonable to recommend maternal immunization with the BNT162b2 mRNA vaccine during the second trimester. This trial was registered at the Helsinki Committee of the Tel Aviv Medical Center as clinical trial number 0172-TLV.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Imunoglobulina A , Leite Humano , Feminino , Humanos , Lactente , Gravidez , Anticorpos Antivirais , Vacina BNT162 , COVID-19/prevenção & controle , Imunização , Imunoglobulina G , Imunoglobulina M , Lactação , Leite Humano/imunologia , Segundo Trimestre da Gravidez , Estudos Prospectivos , VacinaçãoRESUMO
Protein crystals are routinely prepared for the elucidation of protein structure by X-ray crystallography. These crystals present an highly accurate periodical array of protein molecules with accompanying highly ordered porosity made of interconnected voids. The permeability of the porous protein crystals to a wide range of solutes has recently triggered attempts to explore their potential application as biotemplates by a controlled "filling" process for the fabrication of novel, nano-structured composite materials. Gaining control of the porosity of a given protein crystal may lead to the preparation of a series of "biotemplates" enabling different 'filler'/protein content ratios, resulting in different nanostructured composites. One way to gain such control is to produce a series of polymorphic forms of a given "parent-protein" crystal. As protein packing throughout crystallization is primarily dominated by the chemical composition of the surface of protein molecules and its impact on protein-protein interactions, modification of residues exposed on the surface will affect protein packing, leading to modified porosity. Here we propose to provide influence on the porosity of protein crystals for biotemplating by pre-crystallization chemical modification of lysine residues exposed on protein's surface. The feasibility of this approach was demonstrated by the serial application of chemical "modifiers" leading to protein derivatives exhibiting altered porosity by affecting protein "packing" throughout protein crystallization. Screening of a series of modifying agents for lysine modification of hen egg white lysozyme revealed that pre-crystallization modification preserving their positive charge did not affect crystal porosity, while modification resulting in their conversion to negatively charged groups induced dramatic change in protein crystal's packing and porosity. Furthermore, we demonstrate that chemical modification of lysine residues affecting modified protein packing may be simultaneously performed with the crystallization process: aldehydes generating Schiff base formation with protein's lysine residues readily affected modified protein packing, resulting in altered porosity. Our results demonstrate the feasibility of the use of site directed chemical modifications for the generation of a series of protein crystal exhibiting different porosities for biotemplating, all derived from one "parent" protein.
Assuntos
Cristalização , Lisina/química , Muramidase/química , Porosidade , Animais , Galinhas , NanoestruturasRESUMO
A mouse model supports the hypothesis that latent Epstein-Barr virus exacerbates the symptoms of rheumatoid arthritis.
Assuntos
Artrite Reumatoide , Infecções por Vírus Epstein-Barr , Animais , Herpesvirus Humano 4/genética , CamundongosRESUMO
The role of B cells in the tumor microenvironment (TME) has largely been under investigated, and data regarding the antibody repertoire encoded by B cells in the TME and the adjacent lymphoid organs are scarce. Here, we utilized B cell receptor high-throughput sequencing (BCR-Seq) to profile the antibody repertoire signature of tumor-infiltrating lymphocyte B cells (TIL-Bs) in comparison to B cells from three anatomic compartments in a mouse model of triple-negative breast cancer. We found that TIL-Bs exhibit distinct antibody repertoire measures, including high clonal polarization and elevated somatic hypermutation rates, suggesting a local antigen-driven B-cell response. Importantly, TIL-Bs were highly mutated but non-class switched, suggesting that class-switch recombination may be inhibited in the TME. Tracing the distribution of TIL-B clones across various compartments indicated that they migrate to and from the TME. The data thus suggests that antibody repertoire signatures can serve as indicators for identifying tumor-reactive B cells.
Assuntos
Diversidade de Anticorpos , Subpopulações de Linfócitos B/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Mamárias Experimentais/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Microambiente Tumoral/imunologia , Animais , Células Sanguíneas/imunologia , Medula Óssea/patologia , Linhagem Celular Tumoral/transplante , Movimento Celular , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Região Variável de Imunoglobulina/genética , Linfonodos/patologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Receptores de Antígenos de Linfócitos B/genética , Hipermutação Somática de Imunoglobulina , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The importance of breastmilk in postnatal life lies in the strong association between breastfeeding and the reduction in the risk of infection and infection-related infant mortality. However, data regarding the induction and dynamics of breastmilk antibodies following administration of the Pfizer-BioNTech BNT162b2 COVID-19 mRNA vaccine is scarce, as pregnant and lactating women were not included in the initial vaccine clinical trials. Here, we investigate the dynamics of the vaccine-specific antibody response in breastmilk and serum in a prospective cohort of ten lactating women who received two doses of the mRNA vaccine. We show that the antibody response is rapid and highly synchronized between breastmilk and serum, reaching stabilization 14 days after the second dose. The response in breastmilk includes both IgG and IgA with neutralization capacity.
Assuntos
Aleitamento Materno , Vacinas contra COVID-19/genética , RNA Mensageiro/sangue , Adulto , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/fisiologia , Vacina BNT162 , Feminino , Humanos , Leite/química , RNA Mensageiro/análise , Vacinas Sintéticas/uso terapêutico , Vacinas de mRNARESUMO
Antibodies are widely used in therapeutic, diagnostic, and research applications, and antibody derivatives such as F(ab')2 fragments are used when only a particular antibody region is required. F(ab')2 can be produced through antibody engineering, but some applications require F(ab')2 produced from an original formulated antibody or directly from a polyclonal antibody pool. The cysteine protease immunoglobulin-degrading enzyme (IdeS) from Streptococcus pyogenes digests immunoglobulin G (IgG) specifically and efficiently to produce F(ab')2 . Here we detail the production and purification of recombinant IdeS; its utilization to digest monoclonal or polyclonal antibodies to F(ab')2 fragments; and F(ab')2 purification through consecutive affinity chromatography steps. The resultant F(ab')2 exhibit high purity, retain antigen-binding functionality, and are readily utilizable in various downstream applications. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Production and purification of F(ab')2 fragments from monoclonal and polyclonal antibodies using IdeS Alternate Protocol: Purification of polyclonal antigen-specific F(ab')2 fragments from human serum or secretions Support Protocol: Production and purification of IdeS.
Assuntos
Anticorpos Monoclonais/metabolismo , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/metabolismo , Animais , Antígenos/imunologia , Cromatografia de Afinidade , Cisteína Proteases/isolamento & purificação , Cisteína Proteases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Soro/química , Streptococcus pyogenes/enzimologiaRESUMO
Monoclonal antibodies (mAbs) are a crucial asset for human health and modern medicine, however, the repeated administration of mAbs can be highly immunogenic. Drug immunogenicity manifests in the generation of anti-drug antibodies (ADAs), and some mAbs show immunogenicity in up to 70% of patients. ADAs can alter a drug's pharmacokinetic and pharmacodynamic properties, reducing drug efficacy. In more severe cases, ADAs can neutralize the drug's therapeutic effects or cause severe adverse events to the patient. While some contributing factors to ADA formation are known, the molecular mechanisms of how therapeutic mAbs elicit ADAs are not completely clear. Accurate ADA detection is necessary to provide clinicians with sufficient information for patient monitoring and clinical intervention. However, ADA assays present unique challenges because both the analyte and antigen are antibodies, so most assays are cumbersome, costly, time consuming, and lack standardization. This review will discuss aspects related to ADA formation following mAb drug administration. First, we will provide an overview of the prevalence of ADA formation and the available diagnostic tools for their detection. Next, we will review studies that support possible molecular mechanisms causing the formation of ADA. Finally, we will summarize recent approaches used to decrease the propensity of mAbs to induce ADAs.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/efeitos adversos , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos , Anticorpos Anti-Idiotípicos/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/sangue , Especificidade de Anticorpos , Epitopos , Humanos , Imunoensaio , Fatores de RiscoRESUMO
HIV viremia can be controlled by chronic antiretroviral therapy. As a potentially single-shot alternative, B cells engineered by CRISPR/Cas9 to express anti-HIV broadly neutralizing antibodies (bNAbs) are capable of secreting high antibody titers. Here, we show that, upon immunization of mice, adoptively transferred engineered B cells home to germinal centers (GC) where they predominate over the endogenous response and differentiate into memory and plasma cells while undergoing class switch recombination (CSR). Immunization with a high affinity antigen increases accumulation in GCs and CSR rates. Boost immunization increases the rate of engineered B cells in GCs and antibody secretion, indicating memory retention. Finally, antibody sequences of engineered B cells in the spleen show patterns of clonal selection. Therefore, B cells can be engineered into what could be a living and evolving drug.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/genética , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Anti-HIV/genética , Memória Imunológica/genética , Vacinas contra a AIDS/genética , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Linfócitos B/fisiologia , Linfócitos B/transplante , Anticorpos Amplamente Neutralizantes/sangue , Anticorpos Amplamente Neutralizantes/imunologia , Engenharia Genética/métodos , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/imunologia , Imunização , Isotipos de Imunoglobulinas/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MutaçãoRESUMO
Bioinspired nano-scale biotemplating for the development of novel composite materials has recently culminated in several demonstrations of nano-structured hybrid materials. Protein crystals, routinely prepared for the elucidation of protein 3D structures by X-ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein "building blocks" in the crystal, a complementary 3D array of interconnected cavities--voids array, exhibiting highly ordered porosity is formed. The porous arrays of protein crystal may serve as a nano-structured, accurate biotemplate by a "filling" process. These cavities arrays are shaped by the mode of protein packing throughout the crystallization process. Here we propose and demonstrate feasibility of targeting site specific mutations to modify protein's surface to affect protein crystal packing, enabling the generation of a series of protein crystal "biotemplates" all originating from same parent protein. The selection of these modification sites was based on in silico analysis of protein-protein interface contact areas in the parent crystal. The model protein selected for this study was the N-terminal type II cohesin from the cellulosomal scaffold in ScaB subunit of Acetivibrio cellulolyticus and mutations were focused on lysine residues involved in protein packing as prime target. The impact of systematically mutating these lysine residues on protein packing and its resulting interconnected cavities array were found to be most significant when surface lysine residues were substituted to tryptophan residues. Our results demonstrate the feasibility of using pre-designed site directed mutations for the generation of a series of protein crystal biotemplates from a "parent" protein.