RESUMO
SARS-CoV-2 has evolved as several variants. Immunization to boost the Ab response to Spike antigens is effective, but similar vaccines could not enhance Ab efficacy enough. Effective Ab responses against the human ACE2 (hACE2)-mediated infection of the emerging SARS-CoV-2 variants are needed. We identified closed linear epitopes of the SARS-CoV-2 Spike molecule that induced neutralizing Abs (nAbs) against both S1-RBD, responsible for attachment to hACE2, and S2-HR1/2, in convalescents and vaccine recipients. They inhibited a pseudo-virus infection mediated by the hACE2 pathway. The epitope sequences included epitopes #7 (aa411-432), #11 (aa459-480) and #111 (aa1144-1161), in S1-RBD and S2-HR2. Epitope #111 was conserved in Wuhan and variant strains, whereas #7 and #11 were conserved in Wuhan carried mutations K417N and S477N/T478K in Omicron BA.4/5. These mutations were recognized by the original epitope-specific Abs. These epitopes in RBD and HR2 neither contained, nor overlapped with, those responsible for the antibody-dependent enhancement of the SARS-CoV-2 infection. The sublingual administration of multiple epitope-conjugated antigens increased the IgG and IgA Abs specific to the neutralizing epitopes in mice pre-immunized subcutaneously. The findings indicated that S1-RBD and S2-HR2 epitopes were responsible for pseudo-virus SARS-CoV-2 infections and that sublingual boosts with multiple epitope-conjugated antigens could enhance the protection by nAbs of IgG and IgA against infection by a wide range of variants.
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 , SorogrupoRESUMO
M2 protein, a highly conserved protein of influenza A virus (IAV), plays an important role in virus particle uncoating, assembly, and budding. In the present study, eight monoclonal antibodies (mAbs) against the M2 protein of the H3N2 IAV strain were generated with recombinant truncated M2 protein or BSA-coupled M2 peptides as immunogens. The linear epitopes recognized by the mAbs were defined by IFA and peptide ELISA. The results showed that mAb 10F4 recognized an epitope located in the N-terminal 6-12 amino acids of the M2 peptide, and the mAbs 10D9, 1E2, 4B5, and 5G10 recognized the epitopes located in the C-terminal 62-77 amino acids of the M2 peptide. Importantly, mAb 10D9 recognized the M2 protein of H1-H13 IAV subtypes, which stained M2 protein located on the membrane of host cells and could be applied in immunoprecipitation and immunohistochemistry assays. The mAb 10D9 which recognizes the universal M2 epitope of IAVs will be a useful tool for studies on the function of IAV M2 protein and for the development of vaccines or detection methods for IAV infection.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Animais , Mapeamento de Epitopos , Epitopos/imunologia , Escherichia coli/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologiaRESUMO
Malignant transformations are often associated with aberrant glycosylation processes that lead to the expression of new carbohydrate antigens at the surface of tumor cells. Of these carbohydrate antigens, the Tn antigen is particularly highly expressed in many carcinomas, especially in breast carcinoma. We designed MAG-Tn3, a fully synthetic vaccine based on three consecutive Tn moieties that are O-linked to a CD4+ T cell epitope, to induce anti-Tn antibody responses that could be helpful for therapeutic vaccination against cancer. To ensure broad coverage within the human population, the tetanus toxoid-derived peptide TT830-844 was selected as a T-helper epitope because it can bind to various HLA-DRB molecules. We showed that the MAG-Tn3 vaccine, which was formulated with the GSK proprietary immunostimulant AS15 and designed for human cancer therapy, is able to induce an anti-Tn antibody response in mice of various H-2 haplotypes, and this response correlates with the ability to induce a specific T cell response against the TT830-844 peptide. The universality of the TT830-844 peptide was extended to new H-2 and HLA-DRB molecules that were capable of binding this T cell epitope. Finally, the MAG-Tn3 vaccine was able to induce anti-Tn antibody responses in cynomolgus monkeys, which targeted Tn-expressing tumor cells and mediated tumor cell death both in vitro and in vivo. Thus, MAG-Tn3 is a highly promising anticancer vaccine that is currently under evaluation in a phase I clinical trial.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/imunologia , Glicoproteína Associada a Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Toxoide Tetânico/imunologia , Sequência de Aminoácidos , Animais , Feminino , Antígenos H-2/genética , Cadeias HLA-DRB1/imunologia , Haplótipos , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Vacinação , Vacinas Sintéticas/imunologiaRESUMO
The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.