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1.
Virol J ; 18(1): 87, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33910569

RESUMO

The emergence and rapid spread of the B.1.1.7 lineage (VOC-202012/01) SARS-CoV-2 variant has aroused global concern. The N501Y substitution is the only mutation in the interface between the RBD of B.1.1.7 and ACE2, raising concerns that its recognition by neutralizing antibodies may be affected. Here, we assessed the neutralizing activity and binding affinity of a panel of 12 monoclonal antibodies against the wild type and N501Y mutant SARS-CoV-2 pseudovirus and RBD protein, respectively. We found that the neutralization activity and binding affinity of most detected antibodies (10 out of 12) were unaffected, although the N501Y substitution decreased the neutralizing and binding activities of CB6 and increased that of BD-23. These findings could be of value in the development of therapeutic antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Afinidade de Anticorpos , Sítios de Ligação , Epitopos/imunologia , Células HEK293 , Humanos , Mutação , Testes de Neutralização , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
2.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800950

RESUMO

Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant α-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-α, agalsidase-ß and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r2 = 0.9063 and r2 = 0.8952, both p < 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-α, agalsidase-ß and Moss-AGAL (KD: 1.94 ± 0.11 µM, 2.46 ± 0.21 µM, and 1.33 ± 0.09 µM, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-α, agalsidase-ß and Moss-AGAL.


Assuntos
Anticorpos Neutralizantes/imunologia , Terapia de Reposição de Enzimas , Ensaio de Imunoadsorção Enzimática , Doença de Fabry/imunologia , alfa-Galactosidase/imunologia , alfa-N-Acetilgalactosaminidase/imunologia , Anticorpos Neutralizantes/biossíntese , Afinidade de Anticorpos , Relação Dose-Resposta Imunológica , Doença de Fabry/sangue , Doença de Fabry/tratamento farmacológico , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Masculino , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Padrões de Referência , alfa-Galactosidase/sangue , alfa-Galactosidase/uso terapêutico , alfa-N-Acetilgalactosaminidase/sangue , alfa-N-Acetilgalactosaminidase/uso terapêutico
4.
Sci Adv ; 7(10)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33674317

RESUMO

Limited knowledge exists on immune markers associated with disease severity or recovery in patients with coronavirus disease 2019 (COVID-19). Here, we elucidated longitudinal evolution of SARS-CoV-2 antibody repertoire in patients with acute COVID-19. Differential kinetics was observed for immunoglobulin M (IgM)/IgG/IgA epitope diversity, antibody binding, and affinity maturation in "severe" versus "mild" COVID-19 patients. IgG profile demonstrated immunodominant antigenic sequences encompassing fusion peptide and receptor binding domain (RBD) in patients with mild COVID-19 who recovered early compared with "fatal" COVID-19 patients. In patients with severe COVID-19, high-titer IgA were observed, primarily against RBD, especially in patients who succumbed to SARS-CoV-2 infection. The patients with mild COVID-19 showed marked increase in antibody affinity maturation to prefusion SARS-CoV-2 spike that associated with faster recovery from COVID-19. This study revealed antibody markers associated with disease severity and resolution of clinical disease that could inform development and evaluation of effective immune-based countermeasures against COVID-19.


Assuntos
Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Biomarcadores/sangue , /patologia , Índice de Gravidade de Doença , Afinidade de Anticorpos/imunologia , Formação de Anticorpos/imunologia , /virologia , Citocinas/sangue , Células HEK293 , Hospitalização , Humanos , Switching de Imunoglobulina , Cinética , Testes de Neutralização , Ligação Proteica , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Carga Viral
5.
J Mol Biol ; 433(10): 166956, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33775667

RESUMO

The Covid-19 pandemic is a centenarial global catastrophe. Similar events are likely to be recurring with more frequency in the future. The inability to control the virus' impact is caused by many factors, but the lack of a technology infrastructure to detect and impede the virus at an early stage are principal shortcomings. Using phage display mutagenesis, we have generated a cohort of high performance antibody fragments (Fabs) that can be used in a sensitive point of care (POC) assay and are potent inhibitors (IC50-0.5 nM) to viral entry into cells. The POC assay is based on a split-enzyme (ß-lactamase) complementation strategy that detects virus particles at low nM levels. We have shown that this assay is equally effective for detecting other viruses like Ebola and Zika. Importantly, its components can be freeze dried and stored, but becomes fully active when rehydrated.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , /isolamento & purificação , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Liofilização , Teste de Complementação Genética , Testes de Neutralização , Biblioteca de Peptídeos , Sistemas Automatizados de Assistência Junto ao Leito , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero , Internalização do Vírus , beta-Lactamases/metabolismo
6.
Int J Mol Sci ; 22(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652936

RESUMO

Human cytomegalovirus (CMV) infection is widespread among adults (60-90%) and is usually undetected in healthy individuals without symptoms but can cause severe diseases in immunocompromised hosts. T-cell receptor (TCR)-like antibodies (Abs), which recognize complex antigens (peptide-MHC complex, pMHC) composed of MHC molecules with embedded short peptides derived from intracellular proteins, including pathogenic viral proteins, can serve as diagnostic and/or therapeutic agents. In this study, we aimed to engineer a TCR-like Ab specific for pMHC comprising a CMV pp65 protein-derived peptide (495NLVPMVATV503; hereafter, CMVpp65495-503) in complex with MHC-I molecule human leukocyte antigen (HLA)-A*02:01 (CMVpp65495-503/HLA-A*02:01) to increase affinity by sequential mutagenesis of complementarity-determining regions using yeast surface display technology. Compared with the parental Ab, the final generated Ab (C1-17) showed ~67-fold enhanced binding affinity (KD ≈ 5.2 nM) for the soluble pMHC, thereby detecting the cell surface-displayed CMVpp65495-503/HLA-A*02:01 complex with high sensitivity and exquisite specificity. Thus, the new high-affinity TCR-like Ab may be used for the detection and treatment of CMV infection.


Assuntos
Anticorpos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Antígenos HLA-A/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas da Matriz Viral/imunologia , Afinidade de Anticorpos , Linhagem Celular , Humanos , Peptídeos/imunologia
7.
Front Immunol ; 12: 637982, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33777030

RESUMO

A novel betacoronavirus (SARS-CoV-2) that causes severe pneumonia emerged through zoonosis in late 2019. The disease, referred to as COVID-19, has an alarming mortality rate and it is having a devastating effect on the global economy and public health systems. A safe, effective vaccine is urgently needed to halt this pandemic. In this study, immunogenicity of the receptor binding domain (RBD) of spike (S) glycoprotein was examined in mice. Animals were immunized with recombinant RBD antigen intraperitoneally using three different adjuvants (Zn-chitosan, Alhydrogel, and Adju-Phos), and antibody responses were followed for over 5 months. Results showed that potent neutralizing antibodies (nAbs) can be induced with 70% neutralization titer (NT70) of ~14,580 against live, infectious viruses. Although antigen-binding antibody titers decreased gradually over time, sufficiently protective levels of nAbs persisted (NT80 >2,430) over the 5-month observation period. Results also showed that adjuvants have profound effects on kinetics of nAb induction, total antibody titers, antibody avidity, antibody longevity, and B-cell epitopes targeted by the immune system. In conclusion, a recombinant subunit protein immunogen based on the RBD is a highly promising vaccine candidate. Continued evaluation of RBD immunogenicity using different adjuvants and vaccine regimens could further improve vaccine efficacy.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , /prevenção & controle , Imunização , Imunogenicidade da Vacina , Glicoproteína da Espícula de Coronavírus/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Afinidade de Anticorpos , /imunologia , /imunologia , Epitopos , Feminino , Interações Hospedeiro-Patógeno , Camundongos Endogâmicos BALB C , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de Tempo , Vacinas de Subunidades/imunologia , Vacinas de Subunidades/farmacologia
8.
JAMA Netw Open ; 4(3): e214302, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33749770

RESUMO

Importance: Accumulating evidence suggests that children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more likely to manifest mild symptoms and are at a lower risk of developing severe respiratory disease compared with adults. It remains unknown how the immune response in children differs from that of adolescents and adults. Objective: To investigate the association of age with the quantity and quality of SARS-CoV-2 antibody responses. Design, Setting, and Participants: This cross-sectional study used 31 426 SARS-CoV-2 antibody test results from pediatric and adult patients. Data were collected from a New York City hospital from April 9 to August 31, 2020. The semiquantitative immunoglobin (Ig) G levels were compared between 85 pediatric and 3648 adult patients. Further analysis of SARS-CoV-2 antibody profiles was performed on sera from 126 patients aged 1 to 24 years. Main Outcomes and Measures: SARS-CoV-2 antibody positivity rates and IgG levels were evaluated in patients from a wide range of age groups (1-102 years). SARS-CoV-2 IgG level, total antibody (TAb) level, surrogate neutralizing antibody (SNAb) activity, and antibody binding avidity were compared between children (aged 1-10 years), adolescents (aged 11-18 years), and young adults (aged 19-24 years). Results: Among 31 426 antibody test results (19 797 [63.0%] female patients), with 1194 pediatric patients (mean [SD] age, 11.0 [5.3] years) and 30 232 adult patients (mean [SD] age, 49.2 [17.1] years), the seroprevalence in the pediatric (197 [16.5%; 95% CI, 14.4%-18.7%]) and adult (5630 [18.6%; 95% CI, 18.2%-19.1%]) patient populations was similar. The SARS-CoV-2 IgG level showed a negative correlation with age in the pediatric population (r = -0.45, P < .001) and a moderate but positive correlation with age in adults (r = 0.24, P < .001). Patients aged 19 to 30 years exhibited the lowest IgG levels (eg, aged 25-30 years vs 1-10 years: 99 [44-180] relative fluorescence units [RFU] vs 443 [188-851] RFU). In the subset cohort aged 1 to 24 years, IgG, TAb, SNAb and avidity were negatively correlated with age (eg, IgG: r = -0.51; P < .001). Children exhibited higher median (IQR) IgG levels, TAb levels, and SNAb activity compared with adolescents (eg, IgG levels: 473 [233-656] RFU vs 191 [82-349] RFU; P < .001) and young adults (eg, IgG levels: 473 [233-656] RFU vs 85 [38-150] RFU; P < .001). Adolescents also exhibited higher median (IQR) TAb levels, IgG levels, and SNAb activity than young adults (eg, TAb levels: 961 [290-2074] RFU vs 370 [125-697]; P = .006). In addition, children had higher antibody binding avidity compared with young adults, but the difference was not significant. Conclusions and Relevance: The results of this study suggest that SARS-CoV-2 viral specific antibody response profiles are distinct in different age groups. Age-targeted strategies for disease screening and management as well as vaccine development may be warranted.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Afinidade de Anticorpos/imunologia , Formação de Anticorpos/imunologia , Fatores Etários , /epidemiologia , /métodos , Criança , Correlação de Dados , Estudos Transversais , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Cidade de Nova Iorque/epidemiologia , /isolamento & purificação
9.
Nat Commun ; 12(1): 1577, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707427

RESUMO

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.


Assuntos
/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , /imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , /química , Animais , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Afinidade de Anticorpos , Linhagem Celular , Chlorocebus aethiops , Biblioteca Gênica , Voluntários Saudáveis , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Modelos Moleculares , Mutação , Testes de Neutralização , Pandemias , Biblioteca de Peptídeos , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Glicoproteína da Espícula de Coronavírus/química , Células Vero
10.
PLoS Pathog ; 17(3): e1009328, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33657135

RESUMO

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.


Assuntos
/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Afinidade de Anticorpos , Sítios de Ligação , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética
12.
Nat Commun ; 12(1): 1063, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594061

RESUMO

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


Assuntos
Anticorpos Antiprotozoários/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Sequências Repetitivas de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos/imunologia , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Cinética , Camundongos Endogâmicos C57BL , Modelos Moleculares , Parasitos/imunologia , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica
13.
Viruses ; 13(2)2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33546482

RESUMO

European Association of the Study of the Liver (EASL) guidelines specify HEV RNA, as well as anti-HEV IgG and IgM as positive markers for acute HEV infection. HEV RNA assay sensitivity limitations may lead to false negative test results in patients with low levels of viremia. Moreover, anti-HEV IgM positivity is not a reliable indicator for distinguishing between acute and resolved infections given the ability of this antibody to persist several months after a resolved infection. Our study aims were to assess HEV IgG avidity for diagnosing acute and resolved infections, regardless of the anti-HEV IgM serostatus, and examine assay reliability when evaluating different genotype 3 (GT3) HEV subtypes. Patient serum samples (n = 104) were tested for HEV IgG avidity by utilizing the DIA.PRO kit on a DSX automated instrument. Among patients identified with acute HEV infections, 32 were infected with GT3: GT3c (n = 5), GT3e (n = 8), 3f (n = 17) and GT3-unsubtyped (n = 2). Avidity sensitivity was 91.2% and specificity was 100%. For patients with long-lasting anti-HEV IgM persistence, an Avidity Index >70% was observed. Thus, the DIA.PRO avidity assay may be utilized to distinguish between recently acquired and resolved HEV GT3 infections. However, for equivocal results (Avidity Index > 40-70%), HEV RNA molecular testing will be required to confirm a recent infection.


Assuntos
Anticorpos Anti-Hepatite/imunologia , Vírus da Hepatite E/imunologia , Hepatite E/diagnóstico , Hepatite E/imunologia , Imunoglobulina G/imunologia , Afinidade de Anticorpos , Feminino , Anticorpos Anti-Hepatite/sangue , Hepatite E/sangue , Hepatite E/virologia , Vírus da Hepatite E/genética , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Filogenia , RNA Viral/genética
14.
J Med Virol ; 93(5): 3092-3104, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33565617

RESUMO

The serological responses towards severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein, receptor-binding domain (RBD), and spike protein S1 are characterized by incomplete avidity maturation. Analysis with varying concentrations of urea allows to determine distinct differences in avidity maturation, though the total process remains at an unusually low level. Despite incomplete avidity maturation, this approach allows to define early and late stages of infection. It therefore can compensate for the recently described irregular kinetic patterns of immunoglobulin M and immunoglobulin G (IgG) directed towards SARS-CoV-2 antigens. The serological responses towards seasonal coronaviruses neither have a negative nor positive impact on SARS-CoV-2 serology in general. Avidity determination in combination with measurement of antibody titers and complexity of the immune response allows to clearly differentiate between IgG responses towards seasonal coronaviruses and SARS-CoV-2. Cross-reactions seem to occur with very low probability. They can be recognized by their pattern of response and through differential treatment with urea. As high avidity has been shown to be essential in several virus systems for the protective effect of neutralizing antibodies, it should be clarified whether high avidity of IgG directed towards RBD indicates protective immunity. If this is the case, monitoring of avidity should be part of the optimization of vaccination programs.


Assuntos
Afinidade de Anticorpos/fisiologia , /diagnóstico , Proteínas do Nucleocapsídeo/imunologia , Testes Sorológicos/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , /virologia , Humanos , Imunoglobulina G/fisiologia , Domínios Proteicos
15.
J Vis Exp ; (168)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33616097

RESUMO

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.


Assuntos
Afinidade de Anticorpos , Complexo Antígeno-Anticorpo/imunologia , Fotometria/métodos , Anticorpos/imunologia , Humanos , Imageamento Tridimensional , Peso Molecular , Distribuição Normal , Ligação Proteica , Software , Trombina/imunologia
16.
Gene ; 782: 145533, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33636291

RESUMO

BACKGROUND: Human papillomavirus is the most common sexually transmitted infection. It is associated with different cancers, mainly cervical cancer, which remains the fourth most frequent cancer among women worldwide; it is also related to anogenital (anus, vulvar, vagina, and penis) and oropharyngeal cancers. Vaccination against HPV infection is the major way of prevention, and it has demonstrated impressive efficacy in reducing cervical cancer incidence. Nowadays, all the licensed HPV recombinant vaccines were designed based on HPV major capsid L1 protein. However, some variations in the HPV L1 gene sequence may induce structural changes within the L1 protein, which may alter the affinity and interaction of monoclonal antibodies (MAbs) with L1 protein epitopes, and influence host immune response and recognition. Hence, the importance of accuracy in delineating epitopes relevant to vaccine design and defining genetic variations within antigenic regions in the L1 gene to predict its impact on prophylactic vaccine efficiency. The present review reports the sequence variations in HR-HPV L1 gene isolates from different countries around the world, which may help to understand the effect of HPV L1 gene variations on vaccine efficiency. METHODS: Research studies were retrieved from PubMed, Google Scholar, Science direct, and the National Center for Biotechnology Information (NCBI) database. A total of 31 articles describing genetic variations within the major capsid L1 gene and conducted in Africa, Europe, America and Asia were found. Only 26 studies conducted on HPV16, 18, 31, 33, 58, 45 and 52 which are the targets of HPV prophylactic vaccines, and which reported genetic variations within the L1 gene, were selected and evaluated in this review. FINDINGS: We found a total of 87, 49, 11, 7, 22, 3, and 17 non-synonymous single nucleotide polymorphisms (SNPs) within HPV16, HPV18, HPV31, HPV58, HPV45, and HPV52 L1 gene, respectively. Four mutations were frequently observed in HPV16 L1 sequences: T353P in the HI loop, H228D in the EF loop, T266A in the FG loop, and T292A in the FG loop. Two mutations in HPV58 L1 sequences: T375N in the HI loop and L150F in the DE loop. Three mutations in HPV33 L1 sequences: T56N in the BC loop, G133S in the DE loop, T266K in the FG loop. Other mutations were found in HPV18, HPV45, and HPV52 L1 sequences. Some were found in different countries, and others were specific to a given population. Furthermore, some variations were located on peptide binding epitopes and lead to a modification of epitopes, which may influence MAbs interactions. Others need further investigations due to the lack of studies. CONCLUSION: This study investigated the major capsid L1 genetic diversity of HPV16, 18, 31, 33, 58, 45, and 52 circulating in different populations around the world. Further investigations should be conducted to confirm their effect on immunogenicity and prophylactic vaccine efficiency.


Assuntos
Proteínas do Capsídeo/genética , Variação Genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Vacinas contra Papillomavirus/imunologia , Animais , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Proteínas do Capsídeo/imunologia , Saúde Global , Humanos , Imunogenicidade da Vacina/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/fisiologia , Vacinas contra Papillomavirus/genética
17.
Nat Commun ; 12(1): 1221, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33619281

RESUMO

Hospitalized COVID-19 patients often present with a large spectrum of clinical symptoms. There is a critical need to better understand the immune responses to SARS-CoV-2 that lead to either resolution or exacerbation of the clinical disease. Here, we examine longitudinal plasma samples from hospitalized COVID-19 patients with differential clinical outcome. We perform immune-repertoire analysis including cytokine, hACE2-receptor inhibition, neutralization titers, antibody epitope repertoire, antibody kinetics, antibody isotype and antibody affinity maturation against the SARS-CoV-2 prefusion spike protein. Fatal cases demonstrate high plasma levels of IL-6, IL-8, TNFα, and MCP-1, and sustained high percentage of IgA-binding antibodies to prefusion spike compared with non-ICU survivors. Disease resolution in non-ICU and ICU patients associates with antibody binding to the receptor binding motif and fusion peptide, and antibody affinity maturation to SARS-CoV-2 prefusion spike protein. Here, we provide insight into the immune parameters associated with clinical disease severity and disease-resolution outcome in hospitalized patients that could inform development of vaccine/therapeutics against COVID-19.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos/imunologia , Imunoglobulina A/imunologia , /imunologia , Adulto , Idoso , /metabolismo , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , /virologia , Estudos de Coortes , Citocinas/sangue , Citocinas/imunologia , Citocinas/metabolismo , Epitopos/imunologia , Feminino , Hospitalização/estatística & dados numéricos , Humanos , Imunoglobulina A/sangue , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , /fisiologia , Glicoproteína da Espícula de Coronavírus/imunologia , Ressonância de Plasmônio de Superfície
18.
Sci Rep ; 11(1): 3318, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558635

RESUMO

Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with potential therapeutic applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of Nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos/genética , /imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , /imunologia , Animais , Camelídeos Americanos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Imunização , Masculino , Testes de Neutralização , Biblioteca de Peptídeos , Ligação Proteica/genética , /isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Transfecção
19.
Science ; 371(6530)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436526

RESUMO

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , /imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Antígenos Virais/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular , Microscopia Crioeletrônica , Epitopos , Humanos , Fusão de Membrana , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , /genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Replicação Viral
20.
Science ; 371(6531): 823-829, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33495307

RESUMO

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , /metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Sítios de Ligação , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Amplamente Neutralizantes/metabolismo , /terapia , Técnicas de Visualização da Superfície Celular , Evolução Molecular Direcionada , Epitopos/imunologia , Humanos , Imunização Passiva , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos Endogâmicos BALB C , Domínios Proteicos , Engenharia de Proteínas , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/terapia , Glicoproteína da Espícula de Coronavírus/metabolismo
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