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1.
Cell Rep ; 36(10): 109679, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34464610

RESUMO

A wide range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing monoclonal antibodies (mAbs) have been reported, most of which target the spike glycoprotein. Therapeutic implementation of these antibodies has been challenged by emerging SARS-CoV-2 variants harboring mutated spike versions. Consequently, re-assessment of previously identified mAbs is of high priority. Four previously selected mAbs targeting non-overlapping epitopes are now evaluated for binding potency to mutated RBD versions, reported to mediate escape from antibody neutralization. In vitro neutralization potencies of these mAbs, and two NTD-specific mAbs, are evaluated against two frequent SARS-CoV-2 variants of concern, the B.1.1.7 Alpha and the B.1.351 Beta. Furthermore, we demonstrate therapeutic potential of three selected mAbs by treatment of K18-human angiotensin-converting enzyme 2 (hACE2) transgenic mice 2 days post-infection with each virus variant. Thus, despite the accumulation of spike mutations, the highly potent MD65 and BL6 mAbs retain their ability to bind the prevalent viral mutants, effectively protecting against B.1.1.7 and B.1.351 variants.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Afinidade de Anticorpos , COVID-19/terapia , COVID-19/virologia , Epitopos/genética , Epitopos/imunologia , Humanos , Imunização Passiva , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Testes de Neutralização , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Resultado do Tratamento
2.
Nature ; 596(7871): 276-280, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34237773

RESUMO

The SARS-CoV-2 B.1.617 lineage was identified in October 2020 in India1-5. Since then, it has become dominant in some regions of India and in the UK, and has spread to many other countries6. The lineage includes three main subtypes (B1.617.1, B.1.617.2 and B.1.617.3), which contain diverse mutations in the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein that may increase the immune evasion potential of these variants. B.1.617.2-also termed the Delta variant-is believed to spread faster than other variants. Here we isolated an infectious strain of the Delta variant from an individual with COVID-19 who had returned to France from India. We examined the sensitivity of this strain to monoclonal antibodies and to antibodies present in sera from individuals who had recovered from COVID-19 (hereafter referred to as convalescent individuals) or who had received a COVID-19 vaccine, and then compared this strain with other strains of SARS-CoV-2. The Delta variant was resistant to neutralization by some anti-NTD and anti-RBD monoclonal antibodies, including bamlanivimab, and these antibodies showed impaired binding to the spike protein. Sera collected from convalescent individuals up to 12 months after the onset of symptoms were fourfold less potent against the Delta variant relative to the Alpha variant (B.1.1.7). Sera from individuals who had received one dose of the Pfizer or the AstraZeneca vaccine had a barely discernible inhibitory effect on the Delta variant. Administration of two doses of the vaccine generated a neutralizing response in 95% of individuals, with titres three- to fivefold lower against the Delta variant than against the Alpha variant. Thus, the spread of the Delta variant is associated with an escape from antibodies that target non-RBD and RBD epitopes of the spike protein.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , COVID-19/virologia , Convalescença , Evasão da Resposta Imune/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/epidemiologia , Vacinas contra COVID-19/administração & dosagem , Epitopos/química , Epitopos/genética , Epitopos/imunologia , França , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
3.
Nat Immunol ; 22(8): 1030-1041, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34312544

RESUMO

T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional and transcriptional differences between memory and exhausted antigen-specific CD8+ T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation toward a memory-like profile. However, functionally, the cells showed little improvement, and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, the duration of T cell stimulation impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for the development of functional T cell memory.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Memória Imunológica/imunologia , Antivirais/uso terapêutico , Diferenciação Celular/imunologia , Epitopos/genética , Hepatite C Crônica/tratamento farmacológico , Humanos , Fenótipo
4.
MAbs ; 13(1): 1953683, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34313527

RESUMO

The global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in widespread social and economic disruption. Effective interventions are urgently needed for the prevention and treatment of COVID-19. Neutralizing monoclonal antibodies (mAbs) have demonstrated their prophylactic and therapeutic efficacy against SARS-CoV-2, and several have been granted authorization for emergency use. Here, we discover and characterize a fully human cross-reactive mAb, MW06, which binds to both SARS-CoV-2 and SARS-CoV spike receptor-binding domain (RBD) and disrupts their interaction with angiotensin-converting enzyme 2 (ACE2) receptors. Potential neutralization activity of MW06 was observed against both SARS-CoV-2 and SARS-CoV in different assays. The complex structure determination and epitope alignment of SARS-CoV-2 RBD/MW06 revealed that the epitope recognized by MW06 is highly conserved among SARS-related coronavirus strains, indicating the potential broad neutralization activity of MW06. In in vitro assays, no antibody-dependent enhancement (ADE) of SARS-CoV-2 infection was observed for MW06. In addition, MW06 recognizes a different epitope from MW05, which shows high neutralization activity and has been in a Phase 2 clinical trial, supporting the development of the cocktail of MW05 and MW06 to prevent against future escaping variants. MW06 alone and the cocktail show good effects in preventing escape mutations, including a series of variants of concern, B.1.1.7, P.1, B.1.351, and B.1.617.1. These findings suggest that MW06 recognizes a conserved epitope on SARS-CoV-2, which provides insights for the development of a universal antibody-based therapy against SARS-related coronavirus and emerging variant strains, and may be an effective anti-SARS-CoV-2 agent.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Vírus da SARS/imunologia , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/uso terapêutico , Anticorpos Facilitadores , COVID-19/tratamento farmacológico , COVID-19/terapia , Sequência Conservada , Reações Cruzadas , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Modelos Moleculares , Testes de Neutralização , Pandemias , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/química , Vírus da SARS/genética , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
5.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206009

RESUMO

Toll-like receptor (TLR) signaling plays a critical role in the induction and progression of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, experimental autoimmune encephalitis, type 1 diabetes mellitus and neurodegenerative diseases. Deciphering antigen recognition by antibodies provides insights and defines the mechanism of action into the progression of immune responses. Multiple strategies, including phage display and hybridoma technologies, have been used to enhance the affinity of antibodies for their respective epitopes. Here, we investigate the TLR4 antibody-binding epitope by computational-driven approach. We demonstrate that three important residues, i.e., Y328, N329, and K349 of TLR4 antibody binding epitope identified upon in silico mutagenesis, affect not only the interaction and binding affinity of antibody but also influence the structural integrity of TLR4. Furthermore, we predict a novel epitope at the TLR4-MD2 interface which can be targeted and explored for therapeutic antibodies and small molecules. This technique provides an in-depth insight into antibody-antigen interactions at the resolution and will be beneficial for the development of new monoclonal antibodies. Computational techniques, if coupled with experimental methods, will shorten the duration of rational design and development of antibody therapeutics.


Assuntos
Anticorpos Monoclonais/imunologia , Artrite Reumatoide/imunologia , Encefalite/imunologia , Epitopos/genética , Doença de Hashimoto/imunologia , Doenças Neurodegenerativas/imunologia , Receptor 4 Toll-Like/genética , Sequência de Aminoácidos/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Técnicas de Visualização da Superfície Celular , Encefalite/genética , Encefalite/patologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Doença de Hashimoto/genética , Doença de Hashimoto/patologia , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Ligação Proteica/genética , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/imunologia
6.
Biosens Bioelectron ; 191: 113419, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34144470

RESUMO

Rational selection of predicted peptides to be employed as templates in molecular imprinting was carried out for the heat-denatured non-structural protein 1 (NS1) of dengue virus (DENV). Conservation analysis among 301 sequences of Brazilian isolates of DENV and zika virus (ZIKV) NS1 was carried out by UniProtKB, and peptide selection was based on in silico data of the conservational, structural and immunogenic properties of the sequences. The selected peptide (from dengue 1 NS1) was synthesized and employed as a template in the electropolymerization of polyaminophenol-imprinted films on the surface of carbon screen-printed electrodes. Heat denaturation of the protein was carried out prior to analysis, in order to expose its internal hidden epitopes. After removal of the template, the molecularly imprinted cavities were able to rebind to the whole denatured protein as determined by electrochemical impedance spectroscopy. This label-free sensor was efficient to distinguish the NS1 of DENV from the NS1 of ZIKV. Additionally, the sensor was also selective for dengue NS1, in comparison with human serum immunoglobulin G and human serum albumin. Additionally, the device was able to detect the DENV NS1 at concentrations from 50 to 200 µg L-1 (RSD below 5.04%, r = 0.9678) in diluted human serum samples. The calculated LOD and LOQ were, respectively, 29.3 and 88.7 µg L-1 and each sensor could be used for six sequential cycles with the same performance.


Assuntos
Técnicas Biossensoriais , Vírus da Dengue , Dengue , Infecção por Zika virus , Zika virus , Anticorpos Antivirais , Dengue/diagnóstico , Vírus da Dengue/genética , Epitopos/genética , Temperatura Alta , Humanos , Proteínas não Estruturais Virais/genética , Zika virus/genética , Infecção por Zika virus/diagnóstico
7.
Vaccine ; 39(30): 4135-4143, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34116877

RESUMO

Hand-foot-and-mouth disease (HFMD) is an infectious disease of infants and young children frequently caused by the enterovirus A species, mainly enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In this study, we prepared the EV71 virus-like particle (EV71-VLP) and its chimeras using recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under CMV-IE promoter) proteins in Sf9 cells. EV71-VLP chimera ChiEV71(1E)-VLP or ChiEV71(4E)-VLP displayed single CA16 PEP71 epitope in VP1 or four conserved CA16 neutralizing epitopes (PEP71 in VP1, aa136-150 in VP2, aa176-190 in VP3 and aa48-62 in VP4) by substitution of the corresponding regions of EV71 structure proteins, respectively. In mice, EV71-VLP and its chimeras elicited similar EV71-specific IgG and neutralizing antibody (NAb) titers compared to inactivated EV71. Expectedly, vaccination of ChiEV71(1E)-VLP or ChiEV71(4E)-VLP resulted in significantly increased CA16-specific IgG and NAb production and improved cross-protection against CA16 infection compared to EV71-VLP. Interestingly, the VLPs induced potent cellular immune responses and significantly decreased Th2 type (IL-4 and IL-10) cytokines secretion in the splenocytes of immunized mice compared to inactivated EV71 or inactivated CA16. Neonatal mice born to dams immunized with the chimeric VLPs or neonatal mice passively transferred with sera of immunized mice were completely protected from lethal EV71 challenge and partially protected from lethal CA16 infection. Our study provides a novel bivalent or multivalent vaccine strategy to prevent EV71 and related-enterovirus infections.


Assuntos
Enterovirus Humano A , Enterovirus , Doença de Mão, Pé e Boca , Vacinas Virais , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Quimera , Proteção Cruzada , Enterovirus Humano A/genética , Epitopos/genética , Doença de Mão, Pé e Boca/prevenção & controle , Camundongos , Vacinas Virais/genética
8.
Structure ; 29(7): 655-663.e4, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34111408

RESUMO

Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively, show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of the spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented an immune response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies, such as 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.


Assuntos
Enzima de Conversão de Angiotensina 2/química , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Receptores Virais/química , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/genética , Receptores Virais/imunologia , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
9.
Nat Rev Microbiol ; 19(7): 409-424, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34075212

RESUMO

Although most mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome are expected to be either deleterious and swiftly purged or relatively neutral, a small proportion will affect functional properties and may alter infectivity, disease severity or interactions with host immunity. The emergence of SARS-CoV-2 in late 2019 was followed by a period of relative evolutionary stasis lasting about 11 months. Since late 2020, however, SARS-CoV-2 evolution has been characterized by the emergence of sets of mutations, in the context of 'variants of concern', that impact virus characteristics, including transmissibility and antigenicity, probably in response to the changing immune profile of the human population. There is emerging evidence of reduced neutralization of some SARS-CoV-2 variants by postvaccination serum; however, a greater understanding of correlates of protection is required to evaluate how this may impact vaccine effectiveness. Nonetheless, manufacturers are preparing platforms for a possible update of vaccine sequences, and it is crucial that surveillance of genetic and antigenic changes in the global virus population is done alongside experiments to elucidate the phenotypic impacts of mutations. In this Review, we summarize the literature on mutations of the SARS-CoV-2 spike protein, the primary antigen, focusing on their impacts on antigenicity and contextualizing them in the protein structure, and discuss them in the context of observed mutation frequencies in global sequence datasets.


Assuntos
COVID-19/virologia , Evasão da Resposta Imune , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/classificação , Aminoácidos/química , Aminoácidos/genética , Variação Antigênica/genética , Variação Antigênica/fisiologia , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/transmissão , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/normas , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Evasão da Resposta Imune/genética , Mutação , Conformação Proteica , SARS-CoV-2/classificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
11.
Cell Rep ; 35(6): 109109, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33932326

RESUMO

It is unclear whether individuals with enormous diversity in B cell receptor repertoires are consistently able to mount effective antibody responses against SARS-CoV-2. We analyzed antibody responses in a cohort of 55 convalescent patients and isolated 54 potent neutralizing monoclonal antibodies (mAbs). While most of the mAbs target the angiotensin-converting enzyme 2 (ACE2) binding surface on the receptor binding domain (RBD) of SARS-CoV-2 spike protein, mAb 47D1 binds only to one side of the receptor binding surface on the RBD. Neutralization by 47D1 is achieved independent of interfering RBD-ACE2 binding. A crystal structure of the mAb-RBD complex shows that the IF motif at the tip of 47D1 CDR H2 interacts with a hydrophobic pocket in the RBD. Diverse immunoglobulin gene usage and convergent epitope targeting characterize neutralizing antibody responses to SARS-CoV-2, suggesting that vaccines that effectively present the receptor binding site on the RBD will likely elicit neutralizing antibody responses in a large fraction of the population.


Assuntos
Anticorpos Neutralizantes/genética , COVID-19/genética , Imunoglobulinas/genética , Adulto , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Sítios de Ligação/imunologia , COVID-19/imunologia , COVID-19/terapia , Epitopos/genética , Epitopos/imunologia , Feminino , Genes de Imunoglobulinas/genética , Variação Genética/genética , Humanos , Imunização Passiva/métodos , Imunoglobulinas/imunologia , Masculino , Pessoa de Meia-Idade , Peptidil Dipeptidase A/metabolismo , Ligação Proteica/imunologia , Domínios Proteicos/genética , Receptores Virais/imunologia , Receptores Virais/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade
12.
Microb Pathog ; 157: 104970, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34022362

RESUMO

Helicobacter Pylori, a Gram-negative bacterium in the human stomach, causes adenocarcinoma and MALT (mucosa-associated lymphoid tissue) lymphoma in addition to infection and gastric ulcer. With regard to Helicobacter Pylori prevalence rate and widespread, producing an effective vaccine against this bacterium appears reasonable and necessary. Today, vaccine design by immunoinformatics is a promising solution in vaccine field. In the present study, potential immunodominant CD4⁺ T cell epitopes of UreB, HpaA, and NapA antigens were selected with a focus on IFN-γ secretion inducing ability. After joining the selected epitopes with KK and GPGPG linkers, sequence of Melittin, the major active protein of honey bee venom, was put in C-terminal by DPRVPSS linker as adjuvant. After reverse translation and codon optimization, the designed vaccine was cloned into pET-23a vector. The final construct was estimated as antigenic (71 & 74%) and non-allergenic with molecular weight of 36.785KD. The instability index (II) and codon frequency distribution were predicted to be 26.5 and 92%, respectively. The pET-23a vector transformed to the E.coli BL21 (DE3) strain. The evaluation of expression by SDS-PAGE analysis showed that the optimized expression is in SOB medium 8 h after induction by 0.5 mM IPTG. Finally, purification was performed by Ni-NTA affinity chromatography and Western blot analysis validated the purified protein. Future research is needed to investigate the designed vaccine efficiency against H. pylori, and also it's potential as a gastric cancer-preventive candidate.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Vacinas Bacterianas/genética , Epitopos/genética , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/genética , Humanos , Meliteno
13.
Immunity ; 54(6): 1276-1289.e6, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33836142

RESUMO

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Epitopos/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , COVID-19/diagnóstico , Reações Cruzadas/imunologia , Epitopos/química , Epitopos/genética , Humanos , Modelos Moleculares , Mutação , Testes de Neutralização , Ligação Proteica/imunologia , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Relação Estrutura-Atividade
14.
J Med Virol ; 93(9): 5350-5357, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33913542

RESUMO

PARP14 and PARP9 play a key role in macrophage immune regulation. SARS-CoV-2 is an emerging viral disease that triggers hyper-inflammation known as a cytokine storm. In this study, using in silico tools, we hypothesize about the immunological phenomena of molecular mimicry between SARS-CoV-2 Nsp3 and the human PARP14 and PARP9. The results showed an epitope of SARS-CoV-2 Nsp3 protein that contains consensus sequences for both human PARP14 and PARP9 that are antigens for MHC Classes 1 and 2, which can potentially induce an immune response against human PARP14 and PARP9; while its depletion causes a hyper-inflammatory state in SARS-CoV-2 patients.


Assuntos
COVID-19/imunologia , Proteases Semelhantes à Papaína de Coronavírus/química , Síndrome da Liberação de Citocina/imunologia , Proteínas de Neoplasias/química , Poli(ADP-Ribose) Polimerases/química , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Sítios de Ligação , COVID-19/genética , COVID-19/patologia , COVID-19/virologia , Simulação por Computador , Sequência Consenso , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/imunologia , Síndrome da Liberação de Citocina/genética , Síndrome da Liberação de Citocina/patologia , Síndrome da Liberação de Citocina/virologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/virologia , Simulação de Acoplamento Molecular , Mimetismo Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/imunologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica
15.
Viruses ; 13(4)2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805417

RESUMO

Plant viruses can be genetically modified to generate chimeric virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as vaccine candidates. However, some factors may be especially significant in determining the properties of chimeras. In this study, peptides from various sources and of various lengths were inserted into the Bamboo mosaic virus-based (BaMV) vector CP N-terminus to examine the chimeras infecting and accumulating in plants. Interestingly, it was found that the two different strains Foot-and-mouth disease virus (FMDV) VP1 antigens with flexible linker peptides (77 or 82 amino acids) were directly expressed on the BaMV CP, and the chimeric particles self-assembled and continued to express FMDV antigens. The chimeric CP, when directly fused with a large foreign protein (117 amino acids), can self-fold into incomplete virus particles or disks. The physicochemical properties of heterologus peptides N-terminus, complex strand structures of heterologus peptides C-terminus and different flexible linker peptides, can affect the chimera accumulation. Based on these findings, using plant virus-based chimeras to express foreign proteins can increase their length limitations, and engineered plant-made CVP-based vaccines have increasing potential for further development as novel vaccines.


Assuntos
Antígenos Virais/genética , Proteínas do Capsídeo/genética , Potexvirus/genética , Antígenos Virais/imunologia , Epitopos/genética , Epitopos/imunologia , Vírus da Febre Aftosa/genética , Vírus de Plantas/imunologia , Potexvirus/imunologia , Vacinas Sintéticas/imunologia , Vírion/genética , Vírion/imunologia
16.
Mol Cell ; 81(10): 2246-2260.e12, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33861991

RESUMO

Exitron splicing (EIS) creates a cryptic intron (called an exitron) within a protein-coding exon to increase proteome diversity. EIS is poorly characterized, but emerging evidence suggests a role for EIS in cancer. Through a systematic investigation of EIS across 33 cancers from 9,599 tumor transcriptomes, we discovered that EIS affected 63% of human coding genes and that 95% of those events were tumor specific. Notably, we observed a mutually exclusive pattern between EIS and somatic mutations in their affected genes. Functionally, we discovered that EIS altered known and novel cancer driver genes for causing gain- or loss-of-function, which promotes tumor progression. Importantly, we identified EIS-derived neoepitopes that bind to major histocompatibility complex (MHC) class I or II. Analysis of clinical data from a clear cell renal cell carcinoma cohort revealed an association between EIS-derived neoantigen load and checkpoint inhibitor response. Our findings establish the importance of considering EIS alterations when nominating cancer driver events and neoantigens.


Assuntos
Epitopos/genética , Éxons/genética , Perfilação da Expressão Gênica , Íntrons/genética , Neoplasias/genética , Oncogenes , Splicing de RNA/genética , Sequência de Aminoácidos , Linhagem Celular , Estudos de Coortes , Humanos , Mutação/genética
17.
PLoS Pathog ; 17(4): e1009431, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33831133

RESUMO

Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH2-terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting.


Assuntos
COVID-19 , Epitopos , Imunidade Humoral , Mutação , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , COVID-19/genética , COVID-19/imunologia , Linhagem Celular , Epitopos/genética , Epitopos/imunologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
18.
Viruses ; 13(4)2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810465

RESUMO

Monoclonal antibodies represent an important avenue for COVID-19 therapy and are routinely used for rapid and accessible diagnosis of SARS-CoV-2 infection. The recent emergence of SARS-CoV-2 genetic variants emphasized the need to enlarge the repertoire of antibodies that target diverse epitopes, the combination of which may improve immune-diagnostics, augment the efficiency of the immunotherapy and prevent selection of escape-mutants. Antigen-specific controlled immunization of experimental animals may elicit antibody repertoires that significantly differ from those generated in the context of the immune response mounted in the course of disease. Accordingly, rabbits were immunized by several recombinant antigens representing distinct domains of the viral spike protein and monoclonal antibodies were isolated from single cells obtained by cell sorting. Characterization of a panel of successfully isolated anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies demonstrated that they exhibit high specificity and affinity profiles. Anti-RBD antibodies revealing significant neutralizing potency against SARS-CoV-2 in vitro were found to target at least three distinct epitopes. Epitope mapping established that two of these antibodies recognized a novel epitope located on the surface of the RBD. We suggest that the antibodies isolated in this study are useful for designing SARS-CoV-2 diagnosis and therapy approaches.


Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , COVID-19/virologia , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Testes de Neutralização , Coelhos , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
19.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 52(2): 194-201, 2021 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-33829691

RESUMO

Objective: To construct eukaryotic and prokaryotic recombinant vectors containing Pepck- Gp63 and to achieve protein expression by selecting the dominant epitope genes of Pepck and Gp63 of Leishmania infantum. Methods: The secondary structure and HLA epitopes of phosphoenolpyruvate carboxylase (PEPCK) were predicted by in silico analysis, and the dominant epitopes were picked out. According to the analysis results of glycoprotein of 63×10 3(GP63) epitopes identified by the same method in our laboratory, the dominant epitope genes of Pepck and Gp63 were used to construct pET32a- Pepck- Gp63 and pVAX1- Pepck- Gp63 by overlapping PCR and enzyme reaction. Then, for protein expression, the prokaryotic vectors were transfected into E.coil while the eukaryotic vectors were transfected into NIH3T3 cells by liposome transfection. Results: There were multiple dominant epitopes in Pepckand there were Pepck-Gp63 sequences in the polyclonal site of expression vector. The expression of Pepck-Gp63 in E.coil appeared in inclusion form and led to 74 kDa band in SDS-PAGE. The immunofluorescence results of NIH3T3 cells transfected by pVAX1- Pepck-Gp63 were positive. Conclusion: The recombinant prokaryotic expression plasmids pET32a- Pepck-Gp63 and eukaryotic expression plasmids pVAX1- P epck -Gp63 were successfully constructed, and it was shown that the recombinant plasmids were able to express the corresponding target proteins in E. coli and NIH3T3 cells, respectively, providing a preliminary experimental basis for the subsequent study of immunization strategies.


Assuntos
Leishmania infantum , Animais , Epitopos/genética , Escherichia coli/genética , Eucariotos , Vetores Genéticos/genética , Leishmania infantum/genética , Camundongos , Células NIH 3T3 , Fosfoenolpiruvato Carboxilase , Plasmídeos
20.
Immunity ; 54(4): 737-752.e10, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33740418

RESUMO

Acute myeloid leukemia (AML) has not benefited from innovative immunotherapies, mainly because of the lack of actionable immune targets. Using an original proteogenomic approach, we analyzed the major histocompatibility complex class I (MHC class I)-associated immunopeptidome of 19 primary AML samples and identified 58 tumor-specific antigens (TSAs). These TSAs bore no mutations and derived mainly (86%) from supposedly non-coding genomic regions. Two AML-specific aberrations were instrumental in the biogenesis of TSAs, intron retention, and epigenetic changes. Indeed, 48% of TSAs resulted from intron retention and translation, and their RNA expression correlated with mutations of epigenetic modifiers (e.g., DNMT3A). AML TSA-coding transcripts were highly shared among patients and were expressed in both blasts and leukemic stem cells. In AML patients, the predicted number of TSAs correlated with spontaneous expansion of cognate T cell receptor clonotypes, accumulation of activated cytotoxic T cells, immunoediting, and improved survival. These TSAs represent attractive targets for AML immunotherapy.


Assuntos
Epitopos/genética , Antígenos de Histocompatibilidade Classe I/genética , Leucemia Mieloide Aguda/genética , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular , Epigênese Genética/genética , Epigênese Genética/imunologia , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia/métodos , Leucemia Mieloide Aguda/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação/genética , Mutação/imunologia , Células-Tronco Neoplásicas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia
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