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1.
BMC Bioinformatics ; 22(1): 458, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34563132

RESUMO

BACKGROUND: Antibiotic resistance is a global health crisis. The adage that "prevention is better than cure" is especially true regarding antibiotic resistance because the resistance appears and spreads much faster than the production of new antibiotics. Vaccination is an important strategy to fight infectious agents; however, this strategy has not attracted sufficient attention in antibiotic resistance prevention. New Delhi metallo-beta-lactamase (NDM) confers resistance to many beta-lactamases, including important carbapenems like imipenem. Our goal in this study is to use an immunoinformatics approach to develop a vaccine that can elicit strong and specific immune responses against NDMs that prevent the development of antibiotic-resistant bacteria. RESULTS: In this study, 2194 NDM sequences were aligned to obtain a conserved sequence. One continuous B cell epitope and three T cell CD4+ epitopes were selected from NDMs conserved sequence. Epitope conservancy for B cell and HLA-DR, HLA-DQ, and HLA-DP epitopes was 100.00%, 99.82%, 99.41%, and 99.86%, respectively, and population coverage of MHC II epitopes for the world was 99.91%. Permutation of the four epitope fragments resulted in 24 different peptides, of which 6 peptides were selected after toxicity, allergenicity, and antigenicity assessment. After primary vaccine design, only one vaccine sequence with the highest similarity with discontinuous B cell epitope in NDMs was selected. The final vaccine can bind to various Toll-like receptors (TLRs). The prediction implied that the vaccine would be stable with a good half-life. An immune simulation performed by the C-IMMSIM server predicted that two doses of vaccine injection can induce a strong immune response to NDMs. Finally, the GC-Content of the vaccine was designed very similar to E. coli K12. CONCLUSIONS: In this study, immunoinformatics strategies were used to design a vaccine against different NDM variants that could produce an effective immune response against this antibiotic-resistant factor.


Assuntos
Epitopos de Linfócito T , Escherichia coli , Biologia Computacional , Simulação por Computador , Mapeamento de Epitopos , beta-Lactamases/genética
2.
Cell Rep ; 37(1): 109784, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34592170

RESUMO

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.


Assuntos
Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , SARS-CoV-2/química , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos , COVID-19/genética , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Microscopia Crioeletrônica , Mapeamento de Epitopos/métodos , Epitopos/química , Epitopos/imunologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/imunologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Células Vero
3.
Front Immunol ; 12: 692937, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34497604

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kills thousands of people worldwide every day, thus necessitating rapid development of countermeasures. Immunoinformatics analyses carried out here in search of immunodominant regions in recently identified SARS-CoV-2 unannotated open reading frames (uORFs) have identified eight linear B-cell, one conformational B-cell, 10 CD4+ T-cell, and 12 CD8+ T-cell promising epitopes. Among them, ORF9b B-cell and T-cell epitopes are the most promising followed by M.ext and ORF3c epitopes. ORF9b40-48 (CD8+ T-cell epitope) is found to be highly immunogenic and antigenic with the highest allele coverage. Furthermore, it has overlap with four potent CD4+ T-cell epitopes. Structure-based B-cell epitope prediction has identified ORF9b61-68 to be immunodominant, which partially overlaps with one of the linear B-cell epitopes (ORF9b65-69). ORF3c CD4+ T-cell epitopes (ORF3c2-16, ORF3c3-17, and ORF3c4-18) and linear B-cell epitope (ORF3c14-22) have also been identified as the candidate epitopes. Similarly, M.ext and 7a.iORF1 (overlap with M and ORF7a) proteins have promising immunogenic regions. By considering the level of antigen expression, four ORF9b and five M.ext epitopes are finally shortlisted as potent epitopes. Mutation analysis has further revealed that the shortlisted potent uORF epitopes are resistant to recurrent mutations. Additionally, four N-protein (expressed by canonical ORF) epitopes are found to be potent. Thus, SARS-CoV-2 uORF B-cell and T-cell epitopes identified here along with canonical ORF epitopes may aid in the design of a promising epitope-based polyvalent vaccine (when connected through appropriate linkers) against SARS-CoV-2. Such a vaccine can act as a bulwark against SARS-CoV-2, especially in the scenario of emergence of variants with recurring mutations in the spike protein.


Assuntos
Antígenos Virais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Sequência de Aminoácidos/genética , Antígenos Virais/genética , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/uso terapêutico , Biologia Computacional , Proteínas do Nucleocapsídeo de Coronavírus/genética , Desenho de Fármacos , Mapeamento de Epitopos , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Humanos , Fases de Leitura Aberta/genética , Fases de Leitura Aberta/imunologia , SARS-CoV-2/genética , Análise de Sequência de Proteína , Vacinas Combinadas/genética , Vacinas Combinadas/imunologia
4.
PLoS One ; 16(9): e0252849, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34499652

RESUMO

Reverse vaccinology is an evolving approach for improving vaccine effectiveness and minimizing adverse responses by limiting immunizations to critical epitopes. Towards this goal, we sought to identify immunogenic amino acid motifs and linear epitopes of the SARS-CoV-2 spike protein that elicit IgG in COVID-19 mRNA vaccine recipients. Paired pre/post vaccination samples from N = 20 healthy adults, and post-vaccine samples from an additional N = 13 individuals were used to immunoprecipitate IgG targets expressed by a bacterial display random peptide library, and preferentially recognized peptides were mapped to the spike primary sequence. The data identify several distinct amino acid motifs recognized by vaccine-induced IgG, a subset of those targeted by IgG from natural infection, which may mimic 3-dimensional conformation (mimotopes). Dominant linear epitopes were identified in the C-terminal domains of the S1 and S2 subunits (aa 558-569, 627-638, and 1148-1159) which have been previously associated with SARS-CoV-2 neutralization in vitro and demonstrate identity to bat coronavirus and SARS-CoV, but limited homology to non-pathogenic human coronavirus. The identified COVID-19 mRNA vaccine epitopes should be considered in the context of variants, immune escape and vaccine and therapy design moving forward.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Mapeamento de Epitopos , Motivos de Aminoácidos , Sequência de Aminoácidos , Infecções por Coronavirus/imunologia , Humanos , Imunoglobulina G/sangue , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
5.
Emerg Microbes Infect ; 10(1): 1931-1946, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34538222

RESUMO

Identification of relevant epitopes is crucial for the development of subunit peptide vaccines inducing neutralizing and cellular immunity against SARS-CoV-2. Our aim was the characterization of epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to generate a peptide vaccine. Epitope mapping using a panel of 10 amino acid overlapped 15-mer peptides covering region 401-515 from RBD did not identify linear epitopes when tested with sera from infected individuals or from RBD-immunized mice. However, immunization of mice with these 15-mer peptides identified four peptides located at region 446-480 that induced antibodies recognizing the peptides and RBD/S1 proteins. Immunization with peptide 446-480 from S protein formulated with Freund's adjuvant or with CpG oligodeoxinucleotide/Alum induced polyepitopic antibody responses in BALB/c and C56BL/6J mice, recognizing RBD (titres of 3 × 104-3 × 105, depending on the adjuvant) and displaying neutralizing capacity (80-95% inhibition capacity; p < 0.05) against SARS-CoV-2. Murine CD4 and CD8T-cell epitopes were identified in region 446-480 and vaccination experiments using HLA transgenic mice suggested the presence of multiple human T-cell epitopes. Antibodies induced by peptide 446-480 showed broad recognition of S proteins and S-derived peptides belonging to SARS-CoV-2 variants of concern. Importantly, vaccination with peptide 446-480 or with a cyclic version of peptide 446-488 containing a disulphide bridge between cysteines 480 and 488, protected humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2 (62.5 and 75% of protection; p < 0.01 and p < 0.001, respectively). This region could be the basis for a peptide vaccine or other vaccine platforms against Covid-19.


Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Imunidade Celular , Imunidade Humoral , SARS-CoV-2/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/normas , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B , Epitopos de Linfócito T/imunologia , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/imunologia
6.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445289

RESUMO

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.


Assuntos
Mapeamento de Epitopos , Imuno-Histoquímica/métodos , Proteínas do Nucleocapsídeo/imunologia , Vírus de Plantas/imunologia , América , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Epitopos/análise , Epitopos/química , Europa (Continente) , Imunoprecipitação , Vírus do Mosaico/química , Vírus do Mosaico/classificação , Vírus do Mosaico/imunologia , Proteínas do Nucleocapsídeo/química , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Vírus de Plantas/química , Vírus de Plantas/classificação , Potyvirus/química , Potyvirus/imunologia , Coloração e Rotulagem/métodos , Tospovirus/química , Tospovirus/classificação , Tospovirus/imunologia
7.
Int Immunopharmacol ; 99: 108020, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34426117

RESUMO

The spike protein of the SARS-CoV-2 virus is the foremost target for the designing of vaccines and therapeutic antibodies and also acts as a crucial antigen in the assessment of COVID-19 immune responses. The enveloped viruses; such as SARS-CoV-2, Human Immunodeficiency Virus-1 (HIV-1) and influenza, often hijack host-cell glycosylation pathways and influence pathobiology and immune selection. These glycan motifs can lead to either immune evasion or viral neutralization by the production of cross-reactive antibodies that can lead to antibody-dependent enhancement (ADE) of infection. Potential cross-protection from influenza vaccine has also been reported in COVID-19 infected individuals in several epidemiological studies recently; however, the scientific basis for these observations remains elusive. Herein, we show that the anti-SARS-CoV2 antibodies cross-reacts with the Hemagglutinin (HA) protein. This phenomenon is common to both the sera from convalescent SARS-CoV-2 donors and spike immunized mice, although these antibodies were unable to cross-neutralize, suggesting the presence of a non-neutralizing antibody response. Epitope mapping suggests that the cross-reactive antibodies are targeted towards glycan epitopes of the SARS-CoV-2 spike and HA. Overall, our findings address the cross-reactive responses, although non-neutralizing, elicited against RNA viruses and warrant further studies to investigate whether such non-neutralizing antibody responses can contribute to effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or ADE.


Assuntos
COVID-19/imunologia , Reações Cruzadas/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes , Reações Antígeno-Anticorpo , Sítios de Ligação de Anticorpos/imunologia , Técnicas de Cultura de Células , Chlorocebus aethiops , Cães , Mapeamento de Epitopos , Epitopos/imunologia , Glicosilação , Humanos , Vacinas contra Influenza/imunologia , Células Madin Darby de Rim Canino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , SARS-CoV-2/imunologia , Células Vero
8.
Vaccine ; 39(36): 5173-5186, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34353682

RESUMO

Zika virus (ZIKV) caused over two million human infections in more than 80 countries around 2015-2016. Current vaccines under development are mostly focused on inducing antibodies that despite capable of inhibiting the virus, may have the potential to trigger antibody dependent enhancement (ADE). T cell vaccines that do not induce antibodies targeting viral surface will unlikely cause ADE, but be capable of potentiating the effectiveness of an antibody-inducing vaccine. To develop such a protective T cell vaccine, we first examined the repertoire of antigen-specific T cells in immunocompetent mice that have been transiently infected by ZIKV. Through epitope mapping using 427 overlapping peptides spanning the entire length of ZIKV polyprotein, we discovered 27 immunodominant epitopes scattered throughout the virus on C, E, NS1-NS5 proteins. Among them, 8 were confirmed as CD4+ T cell epitopes, and 16 as CD8+ T cell epitopes, while 3 for both T cell subsets. From these 27 newly identified epitopes, the top 10 epitopes were selected to formulate three T cell vaccines comprised of either CD4+ T cell epitopes, or CD8+ T cell epitopes, or a mixture of both. Immunization with these T cell epitopes induced T cell-mediated cytotoxicity and cytokine production, and conferred varying degrees of protection against ZIKV challenge. Moreover, these new T cell vaccines also improved the protective efficacy of a neutralizing antibody-inducing recombinant E80 protein vaccine. Together, our results provided additional evidence in support of the protective role of ZIKV-specific CD4+ and CD8+ T cells, and laid foundation for future development of T cell vaccines for ZIKV.


Assuntos
Infecção por Zika virus , Zika virus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Mapeamento de Epitopos , Epitopos de Linfócito T , Epitopos Imunodominantes , Camundongos , Vacinas Sintéticas , Infecção por Zika virus/prevenção & controle
9.
Nat Commun ; 12(1): 4320, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262046

RESUMO

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Reações Cruzadas , Norovirus/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Variação Antigênica , Sítios de Ligação , Antígenos de Grupos Sanguíneos/metabolismo , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/metabolismo , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Cristalografia por Raios X , Mapeamento de Epitopos , Genótipo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Norovirus/genética , Ligação Proteica , Domínios Proteicos
10.
Anal Chem ; 93(34): 11669-11678, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34308633

RESUMO

Epitope mapping of antibodies (Abs) is crucial for understanding adaptive immunity, as well as studying the mode of action of therapeutic antibodies and vaccines. Especially insights into the binding of the entire polyclonal antibody population (pAb) raised upon vaccination would be of unique value to vaccine development. However, very few methods for epitope mapping can tolerate the complexity of a pAb sample. Here we show how hydrogen-deuterium exchange mass spectrometry (HDX-MS) can be used to map epitopes recognized by pAb samples. Our approach involves measuring the HDX of the antigen in absence or presence of varied amounts of pAbs, as well as dissociating additives. We apply the HDX-MS workflow to pAbs isolated from rabbit immunized with factor H-binding protein (fHbp), a Neisseria meningitidis vaccine antigen. We identify four immunogenic regions located on the N- and C-terminal region of fHbp and provide insights into the relative abundance and avidity of epitope binding Abs present in the sample. Overall, our results show that HDX-MS can provide a unique and relatively fast method for revealing the binding impact of the entire set of pAbs present in blood samples after vaccination. Such information provides a rare view into effective immunity and can guide the design of improved vaccines against viruses or bacteria.


Assuntos
Medição da Troca de Deutério , Espectrometria de Massa com Troca Hidrogênio-Deutério , Animais , Anticorpos Monoclonais , Deutério , Mapeamento de Epitopos , Espectrometria de Massas , Coelhos
11.
Clin Immunol ; 231: 108804, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34303849

RESUMO

In December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a novel variant of coronavirus has emerged from Wuhan in China and has created havoc impulses across the world with a larger number of fatalities. At the same time, studies are on roll to discover potent vaccine against it or repurposing of approved drugs which are widely adopted are under trial to eradicate the SARS-CoV-2 causing COVID-19 pandemic. Reports have also shown that there are asymptomatic carriers of COVID-19 disease who can transmit the disease to others too. However, the first line defense of the viral attack is body's strong and well-coordinated immune response producing excessive inflammatory innate reaction, thus impaired adaptive host immune defense which lead to death upon the malfunctioning. Considerable works are going on to establish the relation between immune parameters and viral replication that, might alter both the innate and adaptive immune system of COVID-19 patient by up riding a massive cytokines and chemokines secretion. This review mainly gives an account on how SARS-CoV-2 interacts with our immune system and how does our immune system responds to it, along with that drugs which are being used or can be used in fighting COVID-19 disease. The curative therapies as treatment for it have also been addressed in the perspective of adaptive immunity of the patients.


Assuntos
COVID-19/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Imunidade Adaptativa , COVID-19/prevenção & controle , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Mapeamento de Epitopos , Humanos , Imunidade Celular
12.
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
13.
J Gen Virol ; 102(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34280085

RESUMO

Pigs are susceptible to foot-and-mouth disease virus (FMDV), and the humoral immune response plays an essential role in protection against FMDV infection. However, little information is available about FMDV-specific mAbs derived from single B cells of pigs. This study aimed to determine the antigenic features of FMDV that are recognized by antibodies from pigs. Therefore, a panel of pig-derived mAbs against FMDV were developed using fluorescence-based single B cell antibody technology. Western blotting revealed that three of the antibodies (1C6, P2-7E and P2-8G) recognized conserved antigen epitopes on capsid protein VP2, and exhibited broad reactivity against both FMDV serotypes A and O. An alanine-substitution scanning assay and sequence conservation analysis elucidated that these porcine mAbs recognized two conserved epitopes on VP2: a linear epitope (2KKTEETTLL10) in the N terminus and a conformational epitope involving residues K63, H65, L66, F67, D68 and L81 on two ß-sheets (B-sheet and C-sheet) that depended on the integrity of VP2. Random parings of heavy and light chains of the IgGs confirmed that the heavy chain is predominantly involved in binding to antigen. The light chain of porcine IgG contributes to the binding affinity toward an antigen and may function as a support platform for antibody stability. In summary, this study is the first to reveal the conserved antigenic profile of FMDV recognized by porcine B cells and provides a novel method for analysing the antibody response against FMDV in its natural hosts (i.e. pigs) at the clonal level.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Vírus da Febre Aftosa/imunologia , Suínos/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Afinidade de Anticorpos , Antígenos Virais/imunologia , Linfócitos B/imunologia , Proteínas do Capsídeo/química , Mapeamento de Epitopos , Epitopos/imunologia , Vírus da Febre Aftosa/classificação , Genes de Cadeia Pesada de Imunoglobulina , Genes de Cadeia Leve de Imunoglobulina , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Sorogrupo
14.
Nat Commun ; 12(1): 3661, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135340

RESUMO

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC50) values as low as 9 × 10-14 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Apoferritinas/química , Disponibilidade Biológica , Mapeamento de Epitopos , Humanos , Imunoglobulina G/imunologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Engenharia de Proteínas/métodos , Subunidades Proteicas/química , Glicoproteína da Espícula de Coronavírus/imunologia , Distribuição Tecidual
15.
Int J Biol Macromol ; 183: 2376-2386, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34111485

RESUMO

Bovine pestivirus A and B, previously known as bovine viral diarrhea virus (BVDV)-1 and 2, respectively, are important pathogens of cattle worldwide, which causes significant economic losses. B-cell epitopes in BVDV glycoprotein E2 and nonstructural protein NS2/3 have been extensively identified. In this study, we screened a 12-mer phage display peptide library using commercial goat anti-BVDV serum, and identified a mimotope "LTPHKHHKHLHA" referred to as P3. With sequence alignment, a putative B-cell epitope "77ESRKKLEKALLA88" termed as P3-BVDV1/2 residing in BVDV core protein was identified. The synthesized peptides of both P3 and P3-BVDV1/2 show strong reactivity with BVDV serum in immune blot assay. Immunization of mice with these individual peptides leads to the production of antibody that cannot neutralize virus infectivity. Thus for the first time we identified a B-cell epitope, "77ESRKKLEKALLA88", in BVDV core protein. Interestingly, the epitope was highly conserved in Pestivirus A, B, C, D, as well as emerging Pestivirus E and I, but highly variable in Pestiviruses H, G, F, and J, as well as unclassified Pestivirus originated from non-ruminant animals. Whether this putative B-cell epitope is implicated in pestivirus pathogenesis or evolution needs further investigations once large numbers of isolates are available in the future.


Assuntos
Técnicas de Visualização da Superfície Celular , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Vírus da Diarreia Viral Bovina Tipo 2/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Biblioteca de Peptídeos , Proteínas do Core Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 1/patogenicidade , Vírus da Diarreia Viral Bovina Tipo 2/genética , Vírus da Diarreia Viral Bovina Tipo 2/patogenicidade , Cães , Epitopos de Linfócito B/administração & dosagem , Epitopos de Linfócito B/genética , Feminino , Imunização , Imunogenicidade da Vacina , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Mutação , Proteínas do Core Viral/administração & dosagem , Proteínas do Core Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
16.
Methods Mol Biol ; 2344: 107-117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34115355

RESUMO

Food allergy is becoming a great problem in industrialized countries. Thus, there is the need for a robust understanding of all aspects characterizing IgE response to allergens. The epitope mapping of B-cell epitopes has the potential to become a fundamental tool for food allergy diagnosis and prognosis and to lead to a better understanding of the pathogenesis. Using this approach, we have worked on epitope mapping of the most important plant food allergens identified in the Mediterranean area. The final aim of this study is to define the immune response regarding B epitopes and its clinical relevance in LTP allergy. This chapter describes the protocol to produce microarrays using a library of overlapping peptides corresponding to the primary sequences of allergenic lipid transfer proteins.


Assuntos
Proteínas de Transporte/imunologia , Mapeamento de Epitopos , Hipersensibilidade Alimentar/imunologia , Análise Serial de Proteínas , Humanos
17.
Methods Mol Biol ; 2344: 119-135, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34115356

RESUMO

Peptide microarrays have been used to study protein-protein interaction, enzyme-substrate profiling, epitope mapping, vaccine development, and immuno-profiling. Unlike proteins, peptides are cheap to produce, and can be produced in a high-throughput manner, in a reliable and consistent procedure that reduces batch-to-batch variability. All this provides the peptide microarrays a great potential in the development of new diagnostic tools. Noncontact printing, such as piezoelectric systems, results in a considerable advance in protein and peptide microarray production. In particular, they improve drop deposition, sample distribution, quality control, and flexibility in substrate deposition and eliminate cross-contamination and carryover. These features contribute to creating reproducible assays and generating more reliable data. Here we describe the methods and materials for epitope mapping of food allergens using peptide microarrays produced with a noncontact piezoelectric microarray printer.


Assuntos
Alérgenos/imunologia , Mapeamento de Epitopos , Hipersensibilidade Alimentar/imunologia , Análise Serial de Proteínas , Humanos
19.
Front Immunol ; 12: 658601, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995376

RESUMO

Antigen presentation by MHC-II proteins in the thymus is central to selection of CD4 T cells, but analysis of the full repertoire of presented peptides responsible for positive and negative selection is complicated by the low abundance of antigen presenting cells. A key challenge in analysis of limiting abundance immunopeptidomes by mass spectrometry is distinguishing true MHC-binding peptides from co-eluting non-specifically bound peptides present in the mixture eluted from immunoaffinity-purified MHC molecules. Herein we tested several approaches to minimize the impact of non-specific background peptides, including analyzing eluates from isotype-control antibody-conjugated beads, considering only peptides present in nested sets, and using predicted binding motif analysis to identify core epitopes. We evaluated these methods using well-understood human cell line samples, and then applied them to analysis of the I-Ab presented immunopeptidome of the thymus of C57BL/6 mice, comparing this to the more easily characterized splenic B cell and dendritic cell populations. We identified a total of 3473 unique peptides eluted from the various tissues, using a data dependent acquisition strategy with a false-discovery rate of <1%. The immunopeptidomes presented in thymus as compared to splenic B cells and DCs identified shared and tissue-specific epitopes. A broader length distribution was observed for peptides presented in the thymus as compared to splenic B cells or DCs. Detailed analysis of 61 differentially presented peptides indicated a wider distribution of I-Ab binding affinities in thymus as compared to splenic B cells. These results suggest different constraints on antigen processing and presentation pathways in central versus peripheral tissues.


Assuntos
Apresentação do Antígeno/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Peptídeos/imunologia , Timo/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular , Biologia Computacional/métodos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Mapeamento de Epitopos/métodos , Epitopos/química , Antígenos HLA-DR/química , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Ligação Proteica , Baço/imunologia , Baço/metabolismo , Timo/metabolismo
20.
Front Immunol ; 12: 658372, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33986749

RESUMO

Conventional immunoprecipitation/mass spectroscopy identification of HLA-restricted peptides remains the purview of specializing laboratories, due to the complexity of the methodology, and requires computational post-analysis to assign peptides to individual alleles when using pan-HLA antibodies. We have addressed these limitations with ARTEMIS: a simple, robust, and flexible platform for peptide discovery across ligandomes, optionally including specific proteins-of-interest, that combines novel, secreted HLA-I discovery reagents spanning multiple alleles, optimized lentiviral transduction, and streamlined affinity-tag purification to improve upon conventional methods. This platform fills a middle ground between existing techniques: sensitive and adaptable, but easy and affordable enough to be widely employed by general laboratories. We used ARTEMIS to catalog allele-specific ligandomes from HEK293 cells for seven classical HLA alleles and compared results across replicates, against computational predictions, and against high-quality conventional datasets. We also applied ARTEMIS to identify potentially useful, novel HLA-restricted peptide targets from oncovirus oncoproteins and tumor-associated antigens.


Assuntos
Mapeamento de Epitopos/métodos , Espectrometria de Massas/métodos , Peptídeos/química , Peptídeos/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , Fluxo de Trabalho
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