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1.
Mol Immunol ; 112: 274-282, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31226552

RESUMO

The viral peptides presentation by major histocompatibility complex class I (MHC I) molecules play a pivotal role in T-cell recognition and the subsequent virus clearance. This process is delicately adjusted by the variant residues of MHC I, especially the residues in the peptide binding groove (PBG). In a series of MHC I molecules, a salt bridge is formed above the N-terminus of the peptides. However, the potential impact of the salt bridge on peptide binding and T-cell receptor (TCR) recognition of MHC I, as well as the corresponding molecular basis, are still largely unknown. Herein, we determined the structures of HLA-B*4001 and H-2Kd in which two different types of salt bridges (Arg62-Glu163 or Arg66-Glu163) across the PBG were observed. Although the two salt bridges led to different conformation shifts of both the MHC I α helix and the peptides, binding of the peptides with the salt bridge residues was relatively conserved. Furthermore, through a series of in vitro and in vivo investigations, we found that MHC I mutations that disrupt the salt bridge alleviate peptide binding and can weaken the TCR recognition of MHC I-peptide complexes. Our study may provide key references for understanding MHC I-restricted peptide recognition by T-cells.


Assuntos
Apresentação do Antígeno/imunologia , Genes MHC Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Ligação Proteica/imunologia , Linfócitos T/imunologia , Animais , Sítios de Ligação/imunologia , Feminino , Antígenos HLA-B/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Conformação Proteica , Receptores de Antígenos de Linfócitos T/imunologia
2.
Virus Genes ; 55(3): 280-289, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30725444

RESUMO

Noroviruses are leading cause of acute gastroenteritis worldwide. In our previous study, we established an in vitro histo-blood group antigens (HBGAs) binding blockade assay against GII.3 Norovirus virus like particles (VLPs) with trypsin digestion. In this study, we characterized the blocking antibody binding site and epitope type (linear or conformational) by using hyperimmune sera produced against different antigens. VP1 from Jingzhou402 (GII.3, JZ402) strain was expressed by using pGEX-6p-1 expression vector and the insoluble proteins were purified for immunization in rabbit. Previously characterized chimeric VP1-assembled VLPs (GII.4-VP1/GII.3-P2) were used to immunize guinea pig. Peptides reactive with hyperimmune serum against VLPs derived from the VP1 of JZ402 strain were conjugated with BSA and used to immunize rabbits. Hyperimmune sera against above antigens and JZ402 and JZ403 strain-derived VLPs were used to compare their HBGAs blocking effects. Rabbit anti-GST-VP1 and BSA-peptide conjugated hyperimmune sera demonstrated no blocking effects against the binding of GII.3 and GII.4 NoV VLPs to salivary HBGAs. Guinea pig anti-GII.4-VP1/GII.3-P2 hyperimmune serum blocked the binding of trypsin cleaved GII.3 VLPs to salivary HBGAs with no or very weak blocking effects against the binding of GII.4 VLPs to salivary HBGAs. Our data indicated that HBGAs blocking antibodies primarily bound the P2 domain of GII.3 NoV VP1 and their binding epitopes were most probably conformation-dependent.


Assuntos
Antígenos de Grupos Sanguíneos/genética , Epitopos/genética , Gastroenterite/genética , Norovirus/genética , Animais , Anticorpos/genética , Anticorpos/imunologia , Sítios de Ligação/imunologia , Antígenos de Grupos Sanguíneos/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Gastroenterite/imunologia , Gastroenterite/virologia , Genótipo , Cobaias , Humanos , Norovirus/imunologia , Ligação Proteica , Coelhos , Transdução de Sinais/genética
3.
Theranostics ; 9(1): 210-231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30662563

RESUMO

Broadly neutralizing antibodies (bnAbs) targeting the receptor binding site (RBS) of hemagglutinin (HA) have potential for developing into powerful anti-influenza agents. Several previously reported influenza B bnAbs are nevertheless unable to neutralize a portion of influenza B virus variants. HA-specific bnAbs with hemagglutination inhibition (HI) activity may possess the ability to block virus entry directly. Polymeric IgM antibodies are expected to more effectively inhibit virus attachment and entry into target cells due to their higher avidity and/or steric hindrance. We therefore hypothesized that certain RBS-targeted IgM antibodies with strong cross-lineage HI activity might display broader and more potent antiviral activity against rapidly evolving influenza B viruses. Methods: In this study, we generated IgM and IgG bnAbs targeting the RBS of influenza B virus using the murine hybridoma technique. IgM and IgG versions of the same antibodies were then developed by isotype switching and characterized in subsequent in vitro and in vivo experiments. Results: Two IgM and two IgG bnAbs against influenza B virus HA were identified. Of these, one IgM subtype antibody, C7G6-IgM, showed strong HI and neutralization activities against all 20 representative influenza B strains tested, with higher potency and broader breadth of anti-influenza activity in vitro than the IgG subtype variant of itself, or other previously-reported influenza B bnAbs. Furthermore, C7G6-IgM conferred excellent cross-protection against distinct lineages of influenza B viruses in mice and ferrets, performing better than the anti-influenza drug oseltamivir, and showed an additive antiviral effect when administered in combination with oseltamivir. Mechanistically, C7G6-IgM potently inhibits infection with influenza B virus strains from different lineages by blocking viral entry. Conclusion: In summary, our study highlights the potential of IgM subtype antibodies in combatting pathogenic microbes. Moreover, C7G6-IgM is a promising candidate for the development of prophylactics or therapeutics against influenza B infection.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Sítios de Ligação/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina M/imunologia , Vírus da Influenza B/crescimento & desenvolvimento , Vírus da Influenza B/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Testes de Inibição da Hemaglutinação , Imunização Passiva/métodos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Imunoglobulina M/administração & dosagem , Imunoglobulina M/isolamento & purificação , Camundongos Endogâmicos BALB C , Testes de Neutralização , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/terapia , Resultado do Tratamento
4.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30602609

RESUMO

Human norovirus infections are a major disease burden. In this study, we analyzed three new norovirus-specific Nanobodies that interacted with the prototype human norovirus (i.e., genogroup I genotype 1 [GI.1]). We showed that the Nanobodies bound on the side (Nano-7 and Nano-62) and top (Nano-94) of the capsid-protruding (P) domain using X-ray crystallography. Nano-7 and Nano-62 bound at a similar region on the P domain, but the orientations of these two Nanobodies clashed with the shell (S) domain and neighboring P domains on intact particles. This finding suggested that the P domains on the particles should shift in order for Nano-7 and Nano-62 to bind to intact particles. Interestingly, both Nano-7 and Nano-94 were capable of blocking norovirus virus-like particles (VLPs) from binding to histo-blood group antigens (HBGAs), which are important cofactors for norovirus infection. Previously, we showed that the GI.1 HBGA pocket could be blocked with the soluble human milk oligosaccharide 2-fucosyllactose (2'FL). In the current study, we showed that a combined treatment of Nano-7 or Nano-94 with 2'FL enhanced the blocking potential with an additive (Nano-7) or synergistic (Nano-94) effect. We also found that GII Nanobodies with 2'FL also enhanced inhibition. The Nanobody inhibition likely occurred by different mechanisms, including particle aggregation or particle disassembly, whereas 2'FL blocked the HBGA binding site. Overall, these new data showed that the positive effect of the addition of 2'FL was not limited to a single mode of action of Nanobodies or to a single norovirus genogroup.IMPORTANCE The discovery of vulnerable regions on norovirus particles is instrumental in the development of effective inhibitors, particularly for GI noroviruses that are genetically diverse. Analysis of these GI.1-specific Nanobodies has shown that similar to GII norovirus particles, the GI particles have vulnerable regions. The only known cofactor region, the HBGA binding pocket, represents the main target for inhibition. With a combination treatment, i.e., the addition of Nano-7 or Nano-94 with 2'FL, the effect of inhibition was increased. Therefore, combination drug treatments might offer a better approach to combat norovirus infections, especially since the GI genotypes are highly diverse and are continually changing the capsid landscape, and few conserved epitopes have so far been identified.


Assuntos
Infecções por Caliciviridae/imunologia , Norovirus/imunologia , Anticorpos de Domínio Único/imunologia , Sítios de Ligação/imunologia , Antígenos de Grupos Sanguíneos/imunologia , Capsídeo/imunologia , Proteínas do Capsídeo/imunologia , Cristalografia por Raios X/métodos , Epitopos/imunologia , Escherichia coli/virologia , Ligação Proteica/imunologia
5.
J Virol ; 93(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30541855

RESUMO

Temporal changes in the GII.4 human norovirus capsid sequences occasionally result in the emergence of genetic variants capable of causing new epidemics. The persistence of GII.4 is believed to be associated with the recognition of numerous histo-blood group antigen (HBGA) types and antigenic drift. We found that one of the earliest known GII.4 isolates (in 1974) and a more recent epidemic GII.4 variant (in 2012) had varied norovirus-specific monoclonal antibody (MAb) reactivities but similar HBGA binding profiles. To better understand the binding interaction of one MAb (10E9) that had varied reactivity with these GII.4 variants, we determined the X-ray crystal structure of the NSW-2012 GII.4 P domain 10E9 Fab complex. We showed that the 10E9 Fab interacted with conserved and variable residues, which could be associated with antigenic drift. Interestingly, the 10E9 Fab binding pocket partially overlapped the HBGA pocket and had direct competition for conserved HBGA binding residues (i.e., Arg345 and Tyr444). Indeed, the 10E9 MAb blocked norovirus virus-like particles (VLPs) from binding to several sources of HBGAs. Moreover, the 10E9 antibody completely abolished virus replication in the human norovirus intestinal enteroid cell culture system. Our new findings provide the first direct evidence that competition for GII.4 HBGA binding residues and steric obstruction could lead to norovirus neutralization. On the other hand, the 10E9 MAb recognized residues flanking the HBGA pocket, which are often substituted as the virus evolves. This mechanism of antigenic drift likely influences herd immunity and impedes the possibility of acquiring broadly reactive HBGA-blocking antibodies.IMPORTANCE The emergence of new epidemic GII.4 norovirus variants is thought to be associated with changes in antigenicity and HBGA binding capacity. Here, we show that HBGA binding profiles remain unchanged between the 1974 and 2012 GII.4 variants, whereas these variants showed various levels of reactivity against a panel of GII.4 MAbs. We identified a MAb that bound at the HBGA pocket, blocked norovirus VLPs from binding to HBGAs, and neutralized norovirus virions in the cell culture system. Raised against a GII.4 2006 strain, this MAb was unreactive to a GII.4 1974 isolate but was able to neutralize the newer 2012 strain, which has important implications for vaccine design. Altogether, these new findings suggest that the amino acid variations surrounding the HBGA pocket lead to temporal changes in antigenicity without affecting the ability of GII.4 variants to bind HBGAs, which are known cofactors for infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Variação Antigênica/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Norovirus/imunologia , Sequência de Aminoácidos/genética , Variação Antigênica/genética , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Sítios de Ligação de Anticorpos/imunologia , Infecções por Caliciviridae/imunologia , Capsídeo/imunologia , Linhagem Celular , Cristalografia por Raios X , Humanos , Imunidade Coletiva/genética , Imunidade Coletiva/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Modelos Moleculares , Norovirus/genética , Conformação Proteica , Alinhamento de Sequência
6.
Immunol Invest ; 48(3): 222-241, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30081721

RESUMO

Two heavily O-glycosylated proteins and albumin co-purified with anti-α-galactoside (anti-Gal), the chief xenograft-rejecting antibody and anti-ß-glucan (ABG) antibody isolated from human plasma by affinity chromatography on respective ligand-bearing matrices. Both antibodies and O-glycoproteins co-purified with plasma albumin eluted from albumin-specific matrix. Using components of affinity-purified antibody samples separated by electrophoresis binding of either albumin or antibody to the affinity matrix of the other or binding of O-glycoprotein to either matrix was ruled out. Enzyme-linked immunoassay and ligand-induced fluorescence enhancement of fluorolabeled antibody showed that O-glycoproteins occupied sugar-binding sites of anti-Gal and ABG. Neither antibody recognized albumin. O-Glycoprotein-albumin complexes free in plasma, or released from antibodies by specific sugars, were captured on microwell-coated O-glycan-specific lectin jacalin and detected using labeled anti-albumin. We conclude that circulating anti-Gal and ABG form protein triplets in which either O-glycoprotein bridges between antibody and albumin by binding simultaneously to both. Bound albumin restricted O-glycoprotein occupation on antibodies enabling triplets to bind other ligands using spared binding sites. Free anti-Gal and ABG were undetectable in plasma. Jacalin treatment, but not de-O-glycosylation of O-glycoproteins abolished their recognition by anti-Gal or ABG indicating that antibodies recognized serine- and threonine-rich peptide sequences that underlie the O-glycans and are reported surrogate ligands for anti-Gal. The albumin- and antibody-binding O-glycoproteins AOP1 and AOP2 were single polypeptide proteins of size 107 kDa and 98 kDa, containing 54% and 51% carbohydrate respectively and conformed to no known plasma protein in properties. Prospects of triplet-mediated modulations in autologous tissues expressing antibody ligands are discussed.


Assuntos
Anticorpos/metabolismo , Galactosídeos/imunologia , Glucosídeos/imunologia , Glicoproteínas/metabolismo , Albumina Sérica Humana/metabolismo , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Sítios de Ligação/imunologia , Cromatografia de Afinidade/métodos , Glicoproteínas/imunologia , Glicoproteínas/isolamento & purificação , Glicosilação , Rejeição de Enxerto/sangue , Rejeição de Enxerto/imunologia , Humanos , Ligantes , Lectinas de Plantas/química , Ligação Proteica/imunologia , Albumina Sérica Humana/imunologia
7.
Biochem Biophys Res Commun ; 508(1): 46-51, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30470571

RESUMO

HIV-1 CRF01_AE viruses are highly prevalent in Southeast Asia. However, vulnerability sites in Env of CRF01_AE viruses have not been investigated sufficiently. We examined the sensitivity of CRF01_AE viruses from Japan and Vietnam, together with subtype B viruses from Japan, to neutralization and Fc-mediated signaling. Neutralization coverage of broadly neutralizing antibodies (bnAbs), 2G12 and b12, was significantly low against CRF01_AE viruses, compared with subtype B viruses. In contrast, the conventional antibody targeting the CD4 binding site (CD4bs), 49G2, showed better neutralization and Fc-mediated signaling activities against CRF01_AE viruses than subtype B viruses. Fc-mediated signaling activity of anti-CD4 induced (CD4i) antibody, 4E9C, was also detected against CRF01_AE viruses more than subtype B viruses. These results suggest that conventional antibodies against CD4bs and CD4i may play an important role in the control of CRF01_AE viruses.


Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação/imunologia , Antígenos CD4/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/genética , Humanos , Testes de Neutralização , Receptores de IgG/imunologia , Transdução de Sinais/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
8.
Sci Rep ; 8(1): 15031, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30302011

RESUMO

The HIV-1 envelope (Env) glycoprotein is the primary target of the humoral immune response and a critical vaccine candidate. However, Env is densely glycosylated and thereby substantially protected from neutralisation. Importantly, glycan N301 shields V3 loop and CD4 binding site epitopes from neutralising antibodies. Here, we use molecular dynamics techniques to evaluate the structural rearrangements that maintain the protective qualities of the glycan shield after the loss of glycan N301. We examined a naturally occurring subtype C isolate and its N301A mutant; the mutant not only remained protected against neutralising antibodies targeting underlying epitopes, but also exhibited an increased resistance to the VRC01 class of broadly neutralising antibodies. Analysis of this mutant revealed several glycans that were responsible, independently or through synergy, for the neutralisation resistance of the mutant. These data provide detailed insight into the glycan shield's ability to compensate for the loss of a glycan, as well as the cascade of glycan movements on a protomer, starting at the point mutation, that affects the integrity of an antibody epitope located at the edge of the diminishing effect. These results present key, previously overlooked, considerations for HIV-1 Env glycan research and related vaccine studies.


Assuntos
Proteína gp120 do Envelope de HIV/química , Infecções por HIV/virologia , HIV-1/genética , Polissacarídeos/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Sítios de Ligação/imunologia , Antígenos CD4/imunologia , Epitopos/imunologia , Glicosilação , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/genética , Soropositividade para HIV/imunologia , HIV-1/patogenicidade , Humanos , Imunidade Humoral , Polissacarídeos/imunologia , Conformação Proteica
9.
PLoS Pathog ; 14(8): e1007278, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30153309

RESUMO

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4ß7, a gut-homing receptor. Using both cell-surface expressed α4ß7 and a soluble α4ß7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4ß7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4ß7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4ß7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4ß7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4ß7. It includes the canonical LDV/I α4ß7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4ß7 interactions. These mAbs recognize conformations absent from the ß- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4ß7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.


Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/prevenção & controle , Integrinas/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/metabolismo , Animais , Anticorpos Monoclonais , Sítios de Ligação/imunologia , Linhagem Celular Tumoral , Epitopos/química , Epitopos/imunologia , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/imunologia , Macaca , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/imunologia , Vacinas contra a SAIDS/química , Vacinas contra a SAIDS/imunologia , Vacinas contra a SAIDS/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinação/métodos
10.
MAbs ; 10(6): 854-863, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29985753

RESUMO

Small bispecific antibodies (bsAbs) are important therapeutic molecules and represent the first bsAb format approved by the United States Food and Drug Administration. Diabody (Db), a small bsAb format, has four possible domain orders; we previously reported the differences in the expression levels and cancer growth inhibition effects upon rearranging the domain order of this format. However, there have been no comprehensive reports on domain rearrangements of bispecific single-chain Db (scDb) and tandem single-chain Fv (taFv), which are widely used bsAb formats. In this study, we designed all possible domain orders for scDb and taFv (each with eight variants) with identical Fv pairs and individually expressed all 16 variants using Escherichia coli, Pichia pastoris, and Brevibacillus choshinensis. Comprehensive investigations showed that the intrinsic functions of the variants were similar to each other, regardless of the expression host system, but expression levels varied depending on the format as well as on the host cell. Among the 16 variants, we found a promising candidate that exhibited high activity and productivity. Furthermore, we determined that B. choshinensis is an attractive expression host because of its secretory production of recombinant proteins.


Assuntos
Anticorpos Biespecíficos/imunologia , Sítios de Ligação/imunologia , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/imunologia , Anticorpos Biespecíficos/genética , Sítios de Ligação/genética , Brevibacillus/genética , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Variação Genética , Humanos , Pichia/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/genética
11.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29950421

RESUMO

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life in vivo; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics.IMPORTANCE Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the in vivo half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/química , Sítios de Ligação/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Epitopos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Ligação Proteica , Anticorpos de Domínio Único/economia , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/genética
12.
Immunity ; 48(4): 659-674.e6, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29669249

RESUMO

T cell receptor (TCR) stimulation of naive CD8+ T cells initiates reprogramming of cis-regulatory landscapes that specify effector and memory cytotoxic T lymphocyte (CTL) differentiation. We mapped regions of hyper-accessible chromatin in naive cells during TCR stimulation and discovered that the transcription factor (TF) Runx3 promoted accessibility to memory CTL-specific cis-regulatory regions before the first cell division and was essential for memory CTL differentiation. Runx3 was specifically required for accessibility to regions highly enriched with IRF, bZIP and Prdm1-like TF motifs, upregulation of TFs Irf4 and Blimp1, and activation of fundamental CTL attributes in early effector and memory precursor cells. Runx3 ensured that nascent CTLs differentiated into memory CTLs by preventing high expression of the TF T-bet, slowing effector cell proliferation, and repressing terminal CTL differentiation. Runx3 overexpression enhanced memory CTL differentiation during iterative infections. Thus, Runx3 governs chromatin accessibility during TCR stimulation and enforces the memory CTL developmental program.


Assuntos
Cromatina/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Memória Imunológica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Animais , Sítios de Ligação/imunologia , Diferenciação Celular/imunologia , Linhagem Celular , Proliferação de Células , Cricetinae , Ativação Enzimática/imunologia , Feminino , Humanos , Fatores Reguladores de Interferon/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo/biossíntese , Células Vero
13.
Mol Oral Microbiol ; 33(3): 249-256, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29498487

RESUMO

Treponema denticola is a proteolytic-anaerobic spirochete whose abundance in the subgingival crevice correlates with periodontal disease severity. Treponema denticola evades serum-mediated killing through the binding of factor H (FH), a negative regulator of the complement system. The T. denticolaFH receptor has been identified as FhbB, an 11.4kDa immunodominant lipoprotein. Three distinct subfamilies of FhbB proteins have been delineated and designated as FhbB1, FhbB2 and FhbB3. In this study we demonstrate that all FhbB variants bind human plasminogen (Plg). Competitive binding analyses revealed that FH and Plg do not compete for binding. Binding studies with FhbB135405 site-directed amino acid substitution mutants demonstrated that the interaction domains for FH and Plg on FhbB are separable. Inhibition of Plg-FhbB binding by ε-aminocaproic acid (a lysine analog) indicates that binding is mediated by electrostatic interactions that presumably occur with Lys binding sites contained within Plg "Kringle" domains 1, 2, 4 or 5. Similar to that demonstrated for FH, Plg can also serve as a substrate for the T. denticola protease, dentilisin. The in vivo consequences of dentilisin-mediated cleavage of Plg remained to be determined. The data presented demonstrate that FhbB is a multi-functional protein that may contribute to virulence through several mechanisms including immune evasion, manipulation of the host immune response, adherence or tissue invasion.


Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Plasminogênio/metabolismo , Treponema denticola/imunologia , Treponema denticola/metabolismo , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação/imunologia , Complemento C3b/metabolismo , Fator H do Complemento/genética , Humanos , Evasão da Resposta Imune/imunologia , Lipoproteínas/metabolismo , Modelos Moleculares , Peptídeo Hidrolases/metabolismo , Ligação Proteica/imunologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo
14.
JCI Insight ; 3(5)2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29515029

RESUMO

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs-specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades.


Assuntos
Anticorpos Neutralizantes/imunologia , Mapeamento de Epitopos/métodos , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Anticorpos Neutralizantes/metabolismo , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/sangue , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , Humanos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Mutação Puntual
15.
Vaccine ; 36(17): 2262-2272, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29571972

RESUMO

The end goal of HIV vaccine designing requires novel strategies to elicit a strong humoral and cell-mediated immune response. The emergence of drug resistance and the requirement of next line treatment necessitate the finding of the potential and immunogenic vaccine candidate. This study employed a novel immunoinformatics approach to design multi-epitope subunit vaccine against HIV infection. Here, we designed the subunit vaccine by the combination of CTL, HTL and BCL epitopes along with suitable adjuvant and linkers. Physiochemical characterization of subunit vaccine was assessed to ensure its thermostability, theoretical PI, and amphipathic behavior. In further assessment, subunit vaccine was found to be immunogenic with the capability to generate humoral and cell-mediated immune response. Further, homology modeling and refinement was performed and the refined modeled structure was used for molecular docking with the immune receptor (TLR-3) present on lymphocyte cells. Consequently, molecular dynamics simulation ensured the molecular interaction between TLR-3 and subunit vaccine candidate. Disulfide engineering was performed by placing the cysteine residues in the region of high mobility to enhance the vaccine stability. At last, in silico cloning was performed to warrant the translational efficiency and microbial expression of the designed vaccine.


Assuntos
Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Vacinas de Subunidades/imunologia , Sítios de Ligação/imunologia , Biologia Computacional/métodos , Humanos , Simulação de Acoplamento Molecular/métodos , Receptor 3 Toll-Like/imunologia
16.
J Immunol ; 200(7): 2280-2290, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29483359

RESUMO

Factor H related-protein 5 (CFHR5) is a surface-acting complement activator and variations in the CFHR5 gene are linked to CFHR glomerulonephritis. In this study, we show that FHR5 binds to laminin-521, the major constituent of the glomerular basement membrane, and to mesangial laminin-211. Furthermore, we identify malondialdehyde-acetaldehyde (MAA) epitopes, which are exposed on the surface of human necrotic cells (Homo sapiens), as new FHR5 ligands. Using a set of novel deletion fragments, we show that FHR5 binds to laminin-521, MAA epitopes, heparin, and human necrotic cells (HUVECs) via the middle region [short consensus repeats (SCRs) 5-7]. In contrast, surface-bound FHR5 contacts C3b via the C-terminal region (SCRs8-9). Thus, FHR5 uses separate domains for C3b binding and cell surface interaction. MAA epitopes serve as a complement-activating surface by recruiting FHR5. The complement activator FHR5 and the complement inhibitor factor H both bind to oxidation-specific MAA epitopes and FHR5 competes with factor H for binding. The C3 glomerulopathy-associated FHR21-2-FHR5 hybrid protein is more potent in MAA epitope binding and activation compared with wild-type FHR5. The implications of these results for pathology of CFHR glomerulonephritis are discussed. In conclusion, we identify laminins and oxidation-specific MAA epitopes as novel FHR5 ligands and show that the surface-binding site of FHR5 (SCRs5-7) is separated from the C3b binding site (SCRs8-9). Furthermore, FHR5 competes with factor H for binding to MAA epitopes and activates complement on these modified structures.


Assuntos
Acetaldeído/química , Complemento C3b/imunologia , Proteínas do Sistema Complemento/imunologia , Laminina/imunologia , Malondialdeído/química , Sítios de Ligação/imunologia , Células Cultivadas , Ativação do Complemento/imunologia , Fator H do Complemento/imunologia , Epitopos/imunologia , Glomerulonefrite/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Glomérulos Renais/patologia
17.
Cell Host Microbe ; 23(1): 101-109.e4, 2018 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-29324225

RESUMO

Since their first identification 50 years ago, marburgviruses have emerged several times, with 83%-90% lethality in the largest outbreaks. Although no vaccines or therapeutics are available for human use, the human antibody MR191 provides complete protection in non-human primates when delivered several days after inoculation of a lethal marburgvirus dose. The detailed neutralization mechanism of MR191 remains outstanding. Here we present a 3.2 Å crystal structure of MR191 complexed with a trimeric marburgvirus surface glycoprotein (GP). MR191 neutralizes by occupying the conserved receptor-binding site and competing with the host receptor Niemann-Pick C1. The structure illuminates previously disordered regions of GP including the stalk, fusion loop, CX6CC switch, and an N-terminal region of GP2 that wraps about the outside of GP1 to anchor a marburgvirus-specific "wing" antibody epitope. Virus escape mutations mapped far outside the MR191 receptor-binding site footprint suggest a role for these other regions in the GP quaternary structure.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Marburgvirus/imunologia , Receptores Virais/imunologia , Receptores Virais/ultraestrutura , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/ultraestrutura , Agrobacterium tumefaciens , Animais , Anticorpos Monoclonais/ultraestrutura , Sítios de Ligação/imunologia , Proteínas de Transporte/imunologia , Linhagem Celular , Cristalografia por Raios X , Drosophila melanogaster , Humanos , Marburgvirus/metabolismo , Glicoproteínas de Membrana/imunologia , Tabaco , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Ligação Viral
19.
Cell Immunol ; 322: 34-40, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28992949

RESUMO

In chickens, B cells develop in the bursa of Fabricius, a unique organ for B cell development. Most B cells will die within the bursa, mirroring cell losses seen in mammalian bone marrow as central tolerance is enforced at the transition to mature cells. B cell responses are shaped by a complex interplay of signals. Signals in addition to BCR that impact central tolerance have recently been described. We have been interested in chB6, a novel alloantigen on B cells in the chicken. chB6 is found in close proximity to the BCR and can trigger apoptosis after cross-linking by antibody. chB6 has two Ig domains, placing it within the CD2/SLAM family of molecules, but its cytoplasmic domain is unique. We have used a site-specific mutagenesis approach to show that an SH3 binding site in chB6 is required for the induction of apoptosis, suggesting parallels to CD2 signaling.


Assuntos
Linfócitos B/imunologia , Galinhas/imunologia , Domínios de Imunoglobulina/imunologia , Isoantígenos/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Domínios de Homologia de src/imunologia , Animais , Apoptose/imunologia , Linfócitos B/citologia , Sítios de Ligação/imunologia , Linhagem Celular , Transdução de Sinais/imunologia
20.
Immunity ; 47(4): 635-647.e6, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29045898

RESUMO

In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes.


Assuntos
Amiloide/imunologia , Proteínas de Transporte/imunologia , Proteínas de Drosophila/imunologia , NF-kappa B/imunologia , Receptores de Superfície Celular/imunologia , Transdução de Sinais/imunologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Amiloide/metabolismo , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Feminino , Expressão Gênica/imunologia , Masculino , Microscopia Confocal , Modelos Imunológicos , Mutação , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
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