RESUMO
Tembusu virus (TMUV) is a flavivirus responsible for panzootic outbreaks of severe egg-drop and fatal encephalitis of domestic waterfowl in China. Although TMUV can be attenuated by in vitro passaging, experimental evidence supporting the role of specific genetic changes in virulence attenuation is currently lacking. Here, we performed site-directed mutagenesis on five envelope (E) protein amino acid residues in accordance with the attenuated TMUV generated in our recent study. Our results showed that the Thr-to-Lys mutation of residue 367 in E protein (E367) plays a predominant role in viral cell adaptation and virulence attenuation in ducks compared with mutations in other residues. We further demonstrated that the positively charged basic amino acid substitution at E367 enhanced the viral binding affinity for glycosaminoglycans (GAGs) and reduced viremia levels and the efficiency of replication in major target organs in subcutaneously inoculated ducks. Interestingly, the T367K mutation increased viral neutralization sensitivity to the early immune sera. Together, our findings provide the first evidence that a basic amino acid substitution at E367 strongly impacts the in vitro and in vivo infection of TMUV.IMPORTANCE Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses in the production of domestic waterfowl since the virus was first recognized in China in 2010. To control TMUV infection, a live-attenuated vaccine candidate of TMUV was developed in our previous study, but the mechanisms of virulence attenuation are not fully understood. Here, we found that the Thr-to-Lys substitution at E367 is a crucial determinant of TMUV virulence attenuation in ducks. We demonstrated that the T367K mutation attenuates TMUV through reducing viral replication in the blood, brain, heart (ducklings), and ovaries. These data provide new insights into understanding the pathogenesis of TMUV and the rational development of novel TMUV vaccines.
Assuntos
Substituição de Aminoácidos , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/virologia , Flavivirus/genética , Proteínas do Envelope Viral/genética , Substituição de Aminoácidos/imunologia , Animais , Anticorpos Neutralizantes , Linhagem Celular , China/epidemiologia , Patos/virologia , Feminino , Infecções por Flavivirus/epidemiologia , Infecções por Flavivirus/patologia , Mutagênese Sítio-Dirigida , Mutação , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/mortalidade , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Carga Viral , Virulência , Replicação ViralRESUMO
Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Imunoterapia/métodos , Neoplasias Mamárias Experimentais/terapia , Receptor ErbB-2/imunologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Antígenos de Neoplasias/genética , Vacinas Anticâncer/genética , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral/transplante , Células Dendríticas/imunologia , Evolução Molecular , Feminino , Imunogenicidade da Vacina/genética , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Receptor ErbB-2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Tolerância a Antígenos Próprios/genética , Microambiente Tumoral/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêuticoRESUMO
Human herpesvirus 6A (HHV-6A) glycoprotein B (gB) is a glycoprotein consisting of 830 amino acids and is essential for the growth of the virus. Previously, we reported that a neutralizing monoclonal antibody (MAb) called 87-y-13 specifically reacts with HHV-6A gB, and we identified its epitope residue at asparagine (Asn) 347 on gB. In this study, we examined whether the epitope recognized by the neutralizing MAb is essential for HHV-6A infection. We constructed HHV-6A bacterial artificial chromosome (BAC) genomes harboring substitutions at Asn347, namely, HHV-6A BACgB(N347K) and HHV-6A BACgB(N347A). These mutant viruses could be reconstituted and propagated in the same manner as the wild type and their revertants, and MAb 87-y-13 could not inhibit infection by either mutant. In a cell-cell fusion assay, Asn at position 347 on gB was found to be nonessential for cell-cell fusion. In addition, in building an HHV-6A gB homology model, we found that the epitope of the neutralizing MAb is located on domain II of gB and is accessible to solvents. These results indicate that Asn at position 347, the linear epitope of the neutralizing MAb, does not affect HHV-6A infectivity.IMPORTANCE Glycoprotein B (gB) is one of the most conserved glycoproteins among all herpesviruses and is a key factor for virus entry. Therefore, antibodies targeted to gB may neutralize virus entry. Human herpesvirus 6A (HHV-6A) encodes gB, which is translated to a protein of about 830 amino acids (aa). Using a monoclonal antibody (MAb) for HHV-6A gB, which has a neutralizing linear epitope, we analyzed the role of its epitope residue, N347, in HHV-6A infectivity. Interestingly, this gB linear epitope residue, N347, was not essential for HHV-6A growth. By constructing a homology model of HHV-6A gB, we found that N347 was located in the region corresponding to domain II. Therefore, with regard to its neutralizing activity against HHV-6A infection, the epitope on gB might be exposed to solvents, suggesting that it might be a target of the immune system.
Assuntos
Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Herpesvirus Humano 6/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Fusão Celular , Linhagem Celular , Glicoproteínas/imunologia , Células HEK293 , Herpesviridae/química , Herpesviridae/imunologia , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/crescimento & desenvolvimento , Herpesvirus Humano 6/patogenicidade , Humanos , Proteína Cofatora de Membrana/metabolismo , Mutação , Testes de Neutralização , Domínios Proteicos/imunologia , Proteínas Recombinantes , Análise de Sequência de Proteína , Linfócitos T , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Internalização do VírusRESUMO
One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-ß) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-ß-inducing virus. IFN-ß induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells.IMPORTANCE The type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as high-level IFN-ß induction and strong inhibition of type I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes, as well as virus-host interactions during innate immunity.
Assuntos
Substituição de Aminoácidos/imunologia , Vírus Defeituosos/genética , Interferon beta/metabolismo , Nucleoproteínas/imunologia , Paramyxovirinae/genética , Paramyxovirinae/imunologia , Vírus Sendai/genética , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Proteína DEAD-box 58 , Vírus Defeituosos/imunologia , Feminino , Regulação da Expressão Gênica , Genoma Viral , Células HeLa , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/análise , Interferon Tipo I/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Nucleocapsídeo/metabolismo , Nucleoproteínas/genética , Infecções por Paramyxoviridae/imunologia , Infecções por Paramyxoviridae/virologia , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , RNA Viral/genética , Receptores Imunológicos , Vírus Sendai/imunologia , Replicação ViralRESUMO
Japanese encephalitis (JE) is a mosquito-borne viral disease. It is a global public health concern since it causes an acute encephalitis syndrome (AES). A large number of JE/AES cases are reported to occur in areas with established or developing JE vaccination program. Partial vaccine coverage and emergence of new variants of Japanese encephalitis virus (JEV) might be playing an important role. The envelope protein (E) of JEV is a major antigenic determinant and responsible for immunogenic responses as well as membrane fusion and virion assembly. In the present study, we have characterized the JEV live attenuated vaccine strain SA14-14-2 in baby hamster kidney cells (BHK-21). The vaccine strain showed enhanced replication following its passage in BHK-21 cells. Nucleotide sequence analysis of the E protein gene of the cell-culture adapted vaccine strain showed an important point mutation. The mutation in the E protein gene was identical to its wild-type parent strain SA14. This study suggests the possibility of reversion mutation and exaltation of vaccine strains following adaptation in the host cells.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/imunologia , Vacinas Atenuadas/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Linhagem Celular , Cricetinae , Encefalite Japonesa/imunologia , Encefalite Japonesa/prevenção & controle , Genes Virais/genética , Modelos Moleculares , Mutação , Análise de Sequência de Proteína , Proteínas do Envelope Viral/químicaRESUMO
Defining permissive and non-permissive mismatches for transplantation is a demanding challenge. Single mismatches at amino acid (AA) position 156 of human leucocyte antigen (HLA) class I have been described to alter the peptide motif, repertoire, or mode of peptide loading through differential interaction with the peptide-loading complex. Hence, a single mismatch can tip the balance and trigger an immunological reaction. HLA-B*35 subtypes have been described to evade the loading complex, 156 mismatch distinguishing B*35:01 and B*35:08 changes the binding groove sufficiently to alter the sequence features of the selected peptide repertoire. To understand the functional influences of residue 156 in B*35 variants, we analyzed the peptide binding profiles of HLA-B*35:01(156Leu), B*35:08(156Arg) and B*35:62(156Trp). The glycoprotein tapasin represents a target for immune evasions and functions within the multimeric peptide-loading complex to stabilize empty class I molecules and promote acquisition of high-affinity peptides. All three B*35 subtypes showed a tapasin-independent mode of peptide acquisition. HLA-B*35-restricted peptides of low- and high-binding affinities were recovered in the presence and absence of tapasin and subsequently sequenced utilizing mass spectrometry. The peptides derived from B*35 variants differ substantially in their features dependent on their mode of recruitment; all peptides were preferentially anchored by Pro at p2 and Tyr, Phe, Leu, or Lys at pΩ. However, the Trp at residue 156 altered the p2 motif to an Ala and restricted the pΩ to a Trp. Our results highlight the importance of understanding the impact of key micropolymorphism and how a single AA mismatch orchestrates the neighboring AAs.
Assuntos
Substituição de Aminoácidos/genética , Antígeno HLA-B35/genética , Peptídeos/genética , Polimorfismo Genético/imunologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Substituição de Aminoácidos/imunologia , Aminoácidos/genética , Aminoácidos/imunologia , Linhagem Celular , Retículo Endoplasmático/imunologia , Antígeno HLA-B35/imunologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Peptídeos/imunologia , Ligação Proteica/imunologiaRESUMO
UNLABELLED: Human enterovirus A71 (EV-A71) belongs to the Enterovirus A species in the Picornaviridae family. Several vaccines against EV-A71, a disease causing severe neurological complications or even death, are currently under development and being tested in clinical trials, and preventative vaccination programs are expected to start soon. To characterize the potential for antigenic change of EV-A71, we compared the sequences of two antigenically diverse genotype B4 and B5 strains of EV-A71 and identified substitutions at residues 98, 145, and 164 in the VP1 capsid protein as antigenic determinants. To examine the effects of these three substitutions on antigenicity, we constructed a series of recombinant viruses containing different mutation combinations at these three residues with a reverse genetics system and then investigated the molecular basis of antigenic changes with antigenic cartography. We found that a novel EV-A71 mutant, containing lysine, glutamine, and glutamic acid at the respective residues 98, 145, and 164 in the VP1 capsid protein, exhibited neutralization reduction against patients' antisera and substantially increased virus binding ability to human cells. These observations indicated that this low-neutralization-reactive EV-A71 VP1-98K/145Q/164E mutant potentially increases viral binding ability and that surveillance studies should look out for these mutants, which could compromise vaccine efficacy. IMPORTANCE: Emerging and reemerging EV-A71 viruses can cause severe neurological etiology, primarily affecting children, especially around Asia-Pacific countries. We identified a set of mutations in EV-A71 that both reduced neutralization activity against humoral immunity in antisera of patients and healthy adults and greatly increased the viral binding ability to cells. These findings provide important insights for EV-A71 antigenic determinants and emphasize the importance of continuous surveillance, especially after EV-A71 vaccination programs begin.
Assuntos
Variação Antigênica/imunologia , Proteínas do Capsídeo/imunologia , Enterovirus Humano A/imunologia , Infecções por Enterovirus/prevenção & controle , Epitopos/imunologia , Vacinas Virais/imunologia , Adulto , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Anticorpos Antivirais/sangue , Variação Antigênica/genética , Sequência de Bases , Evolução Biológica , Proteínas do Capsídeo/genética , Linhagem Celular Tumoral , Pré-Escolar , Enterovirus Humano A/classificação , Enterovirus Humano A/genética , Infecções por Enterovirus/imunologia , Mapeamento de Epitopos , Epitopos/genética , Humanos , Dados de Sequência MolecularRESUMO
To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the development of one or more antiviral drugs will be necessary. A set of five single-domain antibody fragments (variable parts of the heavy chain of a heavy-chain antibody [VHHs]) with an in vitro neutralizing activity against poliovirus type 1 was developed previously (B. Thys, L. Schotte, S. Muyldermans, U. Wernery, G. Hassanzadeh-Ghassabeh, and B. Rombaut, Antiviral Res 87:257-264, 2010, http://dx.doi.org/10.1016/j.antiviral.2010.05.012), and their mechanisms of action have been studied (L. Schotte, M. Strauss, B. Thys, H. Halewyck, D. J. Filman, M. Bostina, J. M. Hogle, and B. Rombaut, J Virol 88:4403-4413, 2014, http://dx.doi.org/10.1128/JVI.03402-13). In this study, neutralization escape mutants were selected for each VHH. Sequencing of the P1 region of the genome showed that amino acid substitutions are found in the four viral proteins of the capsid and that they are located both in proximity to the binding sites of the VHHs and in regions further away from the canyon and hidden beneath the surface. Characterization of the mutants demonstrated that they have single-cycle replication kinetics that are similar to those of their parental strain and that they are all drug (VHH) independent. Their resistant phenotypes are stable, as they do not regain full susceptibility to the VHH after passage over HeLa cells in the absence of VHH. They are all at least as stable as the parental strain against heat inactivation at 44°C, and three of them are even significantly (P < 0.05) more resistant to heat inactivation. The resistant variants all still can be neutralized by at least two other VHHs and retain full susceptibility to pirodavir and 35-1F4.
Assuntos
Anticorpos Neutralizantes/imunologia , Fragmentos de Imunoglobulinas/imunologia , Mutação/imunologia , Poliovirus/imunologia , Substituição de Aminoácidos/imunologia , Antivirais/farmacologia , Sítios de Ligação/imunologia , Proteínas do Capsídeo/imunologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Poliovirus/efeitos dos fármacos , Proteínas Virais/imunologiaRESUMO
T cell immunity directed against tumor-encoded amino acid substitutions occurs in some melanoma patients. This implicates missense mutations as a source of patient-specific neoantigens. However, a systematic evaluation of these putative neoantigens as targets of antitumor immunity is lacking. Moreover, it remains unknown whether vaccination can augment such responses. We found that a dendritic cell vaccine led to an increase in naturally occurring neoantigen-specific immunity and revealed previously undetected human leukocyte antigen (HLA) class I-restricted neoantigens in patients with advanced melanoma. The presentation of neoantigens by HLA-A*02:01 in human melanoma was confirmed by mass spectrometry. Vaccination promoted a diverse neoantigen-specific T cell receptor (TCR) repertoire in terms of both TCR-ß usage and clonal composition. Our results demonstrate that vaccination directed at tumor-encoded amino acid substitutions broadens the antigenic breadth and clonal diversity of antitumor immunity.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/uso terapêutico , Células Dendríticas/transplante , Antígeno HLA-A2/imunologia , Imunoterapia Ativa/métodos , Melanoma/terapia , Neoplasias Cutâneas/terapia , Linfócitos T/imunologia , Substituição de Aminoácidos/imunologia , Apresentação de Antígeno , Antígenos de Neoplasias/genética , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Antígeno HLA-A2/genética , Humanos , Melanoma/genética , Melanoma/imunologia , Monitorização Imunológica , Mutação , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologiaRESUMO
Signaling by immunoreceptors is often initiated by phosphorylation of cytosolic tyrosines, which then recruit effector molecules. In the case of MHC class I-specific inhibitory receptors, phosphorylation of cytosolic tyrosine residues within ITIMs results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work showed that signaling by an HLA-C-specific killer cell Ig-like receptor (KIR) is independent of signaling by activation receptors. It is not known how ITIM phosphorylation is initiated and regulated. In this article, we show that substitution of His-36 in the first Ig domain of KIR2DL1 with alanine (KIR2DL1-H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, as well as recruitment of tyrosine phosphatase SHP-1. Furthermore, substitution of His-36 with a similar bulky amino acid, phenylalanine, maintained the receptor in its unphosphorylated state, suggesting that steric hindrance by the His-36 side chain prevents constitutive KIR2DL1 self-association and ITIM phosphorylation. The equally strong phosphorylation of KIR2DL1 and KIR2DL1-H36A after inhibition of tyrosine phosphatase by pervanadate suggested that KIR2DL1-H36A is selectively protected from dephosphorylation. We propose that KIR phosphorylation is controlled by the accessibility of ITIM to tyrosine phosphatases and that KIR binding to HLA-C must override the hindrance that His-36 puts on KIR2DL1 self-association. Expression of KIR2DL1-H36A on NK cells led to stronger inhibition of lysis of HLA-C(+) target cells than did expression of wild-type KIR2DL1. These results revealed that ITIM phosphorylation is controlled by self-association of KIR and that His-36 serves as a gatekeeper to prevent unregulated signaling through KIR2DL1.
Assuntos
Substituição de Aminoácidos/imunologia , Antígenos HLA-C , Células Matadoras Naturais/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Receptores KIR2DL1 , Transdução de Sinais , Linhagem Celular , Antígenos HLA-C/genética , Antígenos HLA-C/imunologia , Humanos , Células Matadoras Naturais/citologia , Fosforilação/genética , Fosforilação/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Receptores KIR2DL1/genética , Receptores KIR2DL1/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Human cytomegalovirus (HCMV) infections are life-threating to people with a compromised or immature immune system. Upon adhesion, fusion of the virus envelope with the host cell is initiated. In this step, the viral glycoprotein gB is considered to represent the major fusogen. Here, we present for the first time structural data on the binding of an anti-herpes virus antibody and describe the atomic interactions between the antigenic domain Dom-II of HCMV gB and the Fab fragment of the human antibody SM5-1. The crystal structure shows that SM5-1 binds Dom-II almost exclusively via only two CDRs, namely light chain CDR L1 and a 22-residue-long heavy chain CDR H3. Two contiguous segments of Dom-II are targeted by SM5-1, and the combining site includes a hydrophobic pocket on the Dom-II surface that is only partially filled by CDR H3 residues. SM5-1 belongs to a series of sequence-homologous anti-HCMV gB monoclonal antibodies that were isolated from the same donor at a single time point and that represent different maturation states. Analysis of amino acid substitutions in these antibodies in combination with molecular dynamics simulations show that key contributors to the picomolar affinity of SM5-1 do not directly interact with the antigen but significantly reduce the flexibility of CDR H3 in the bound and unbound state of SM5-1 through intramolecular side chain interactions. Thus, these residues most likely alleviate unfavorable binding entropies associated with extra-long CDR H3s, and this might represent a common strategy during antibody maturation. Models of entire HCMV gB in different conformational states hint that SM5-1 neutralizes HCMV either by blocking the pre- to postfusion transition of gB or by precluding the interaction with additional effectors such as the gH/gL complex.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Citomegalovirus , Proteínas do Envelope Viral/química , Substituição de Aminoácidos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/química , Citomegalovirus/genética , Infecções por Citomegalovirus/diagnóstico , Humanos , Proteínas do Envelope Viral/genéticaRESUMO
Hepatitis B virus (HBV) infection affects more than 2 billion people throughout the world. Among them, more than 240 million have chronic infection. Every year, 0.5-1.2 million people die of chronic hepatitis B virus infection (CHBVI), and approximately 60% of liver cancers are related to CHBI and subsequent liver cirrhosis (LC). These HBVI-related diseases impose a considerable economic burden as well as morbidity on patients, families, and society. Family and twin studies have indicated that the host genetic constitution greatly influences the clinical outcomes of HBV infection. During the past several years, genome-wide association studies (GWAS) have identified susceptibility variants for various HBVI-related diseases. Of these variants, SNPs rs3077 and rs9277535 in HLA-DP on chromosome 6 show the strongest evidence for association with CHBVI and with viral clearance. However, whether there exists an association between HLA-DP variants and the progression of CHBVI remains to be determined. Thus, further study should focus not only on identifying more variants in HLA-DP that are associated with various HBVI-related diseases but also on characterizing any newly discovered functional variants at the molecular level. Further, given the complexity of CHBV infection and its progression, gene-gene and gene-environment interactions should also be taken into consideration. Moreover, because both smoking and alcohol affect HBV infection and progression, it is important to understand how these factors interact with genetics to influence HBV-related diseases.
Assuntos
Antígenos HLA-DP/genética , Antígenos HLA-DP/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/genética , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/genética , Cromossomos Humanos Par 6/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/imunologia , Humanos , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/genética , Polimorfismo de Nucleotídeo Único , Carga Viral/imunologiaRESUMO
The RV144 HIV-1 trial of the canary pox vector (ALVAC-HIV) plus the gp120 AIDSVAX B/E vaccine demonstrated an estimated efficacy of 31%, which correlated directly with antibodies to HIV-1 envelope variable regions 1 and 2 (V1-V2). Genetic analysis of trial viruses revealed increased vaccine efficacy against viruses matching the vaccine strain at V2 residue 169. Here, we isolated four V2 monoclonal antibodies from RV144 vaccinees that recognize residue 169, neutralize laboratory-adapted HIV-1, and mediate killing of field-isolate HIV-1-infected CD4(+) T cells. Crystal structures of two of the V2 antibodies demonstrated that residue 169 can exist within divergent helical and loop conformations, which contrasted dramatically with the ß strand conformation previously observed with a broadly neutralizing antibody PG9. Thus, RV144 vaccine-induced immune pressure appears to target a region that may be both sequence variable and structurally polymorphic. Variation may signal sites of HIV-1 envelope vulnerability, providing vaccine designers with new options.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica/imunologia , Conformação ProteicaRESUMO
Multiple epidemiologic studies demonstrate associations between chronic beryllium disease (CBD), beryllium sensitization (BeS), and HLA-DPB1 alleles with a glutamic acid residue at position 69 (E69). Results suggest that the less-frequent E69 variants (non-*0201/*0202 alleles) might be associated with greater risk of CBD. In this study, we sought to define specific E69-carrying alleles and their amino acid sequences in the DP peptide binding groove, as well as their relationship to CBD and BeS risk, using the largest case control study to date. We enrolled 502 BeS/CBD subjects and 653 beryllium-exposed controls from three beryllium industries who gave informed consent for participation. Non-Hispanic white cases and controls were frequency-matched by industry. HLA-DPB1 genotypes were determined using sequence-specific primer PCR. The E69 alleles were tested for association with disease individually and grouped by amino acid structure using logistic regression. The results show that CBD cases were more likely than controls to carry a non-*02 E69 allele than an *02 E69, with odds ratios (95% confidence interval) ranging from 3.1 (2.1-4.5) to 3.9 (2.6-5.9) (p < 0.0001). Polymorphic amino acids at positions 84 and 11 were associated with CBD: DD versus GG, 2.8 (1.8-4.6), p < 0.0001; GD versus GG, 2.1 (1.5-2.8), p < 0.0001; LL versus GG, 3.2 (1.8-5.6), p < 0.0001; GL versus GG, 2.8 (2.1-3.8), p < 0.0001. Similar results were found within the BeS group and CBD/BeS combined group. We conclude that the less frequent E69 alleles confer more risk for CBD than does *0201. Recent studies examining how the composition and structure of the binding pockets influence peptide binding in MHC genes, as well of studies showing the topology of the TCR to likely bind DPB1 preferentially, give plausible biological rationale for these findings.
Assuntos
Alelos , Beriliose/imunologia , Berílio/química , Cadeias beta de HLA-DP/química , Substituição de Aminoácidos/efeitos dos fármacos , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Beriliose/genética , Beriliose/patologia , Berílio/efeitos adversos , Estudos de Casos e Controles , Doença Crônica , Feminino , Cadeias beta de HLA-DP/genética , Humanos , Mediadores da Inflamação/efeitos adversos , Mediadores da Inflamação/química , Masculino , Polimorfismo Genético/imunologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/imunologia , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Eletricidade EstáticaRESUMO
The adipocyte-derived hormone leptin plays an important role in regulation of energy homeostasis and the innate immune response against bacterial infections. Leptin's actions are mediated by signaling events initiated by phosphorylation of tyrosine residues on the long form of the leptin receptor. We recently reported that disruption of leptin receptor-mediated STAT3 activation augmented host defense against pneumococcal pneumonia. In this report, we assessed leptin receptor-mediated ERK activation, a pathway that was ablated in the l/l mouse through a mutation of the tyrosine 985 residue in the leptin receptor, to determine its role in host defense against bacterial pneumonia in vivo and in alveolar macrophage (AM) antibacterial functions in vitro. l/l mice exhibited increased mortality and impaired pulmonary bacterial clearance after intratracheal challenge with Klebsiella pneumoniae. The synthesis of cysteinyl-leukotrienes was reduced and that of PGE(2) enhanced in AMs in vitro and the lungs of l/l mice after infection with K. pneumoniae in vivo. We also observed reduced phagocytosis and killing of K. pneumoniae in AMs from l/l mice that was associated with reduced reactive oxygen intermediate production in vitro. cAMP, known to suppress phagocytosis, bactericidal capacity, and reactive oxygen intermediate production, was also increased 2-fold in AMs from l/l mice. Pharmacologic blockade of PGE(2) synthesis reduced cAMP levels and overcame the defective phagocytosis and killing of bacteria in AMs from l/l mice in vitro. These results demonstrate that leptin receptor-mediated ERK activation plays an essential role in host defense against bacterial pneumonia and in leukocyte antibacterial effector functions.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Infecções por Klebsiella/imunologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Pneumonia Bacteriana/imunologia , Receptores para Leptina/antagonistas & inibidores , Receptores para Leptina/fisiologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Imunidade Inata/genética , Infecções por Klebsiella/patologia , Infecções por Klebsiella/prevenção & controle , Klebsiella pneumoniae/imunologia , Leucina/genética , Leucina/imunologia , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pneumonia Bacteriana/patologia , Pneumonia Bacteriana/prevenção & controle , Receptores para Leptina/deficiência , Tirosina/genética , Tirosina/imunologiaRESUMO
TCR of CD8 T cells recognizes peptides of 8-9 amino acids in length (epitope) complexed with MHC class I. Peptide ligands differing from an epitope by one or two amino acids are thought to modulate the immune response specific to that epitope. H60 is a minor histocompatibility antigen for which the specific CD8 T-cell response dominates during alloresponse after MHC-matched allogeneic transplantation. In the present study, we developed a transgenic mouse (designated H60H Tg) expressing a variant of H60, designated H60H, in which the arginine residue at position 4 of the H60 epitope sequence (LTFNYRNL) is replaced by a histidine residue (LTFHYRNL). Immunization of female C57BL/6 mice with splenocytes from male H60H Tg induced a CD8 T cell primary response and memory response after re-challenge. The response was CD4 help-dependent, demonstrating the potency of H60H as a cellular antigen. The response induced by the H60H cellular antigen was comparable to that induced by H60 in its peak magnitude and overall immune kinetics. H60H challenge recruited broadly diverse TCRs to the specific response, shaping a TCR repertoire different from that of the natural H60 epitope. However, some of the TCRs did overlap between the H60H- and H60-specific CD8 T cells, suggesting that H60H might modulate the H60-specific response. These results may provide a basis for the modulation of the H60-specific CD8 T-cell response.
Assuntos
Antígenos CD8/imunologia , Epitopos , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Antígenos CD8/genética , Epitopos/genética , Epitopos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor/genética , Peptídeos/imunologia , Transplante Homólogo/imunologiaRESUMO
The conserved domain of bacteria-derived flagellin coupling Toll-like receptor 5 (TLR5) activates NF-κB and MAPK signaling transductions, which subsequently regulate the transcription and expression of genes encoding immune mediators. However, whether the flagellin binding monoclonal antibody (MAb) obstructs TLR5-associated signaling is unclear. Here we report on the production and characterization of MAb 5G10 that specifically recognizes flagellin. The MAb 5G10 was produced by the hybridization of mouse myeloma cell SP2/0 with splenocyte from a flagellin immunized BALB/c mouse. We observed that deletion of the conserved amino acid residues 89-96 made flagellin lose its capacity for binding 5G10. Additionally, MAb 5G10 remarkably suppressed the expression of cytokine IL8 of Caco-2 cell by blocking the flagellin-TLR5 signaling. Furthermore, this MAb would be useful for cytosolic localization of flagellin and would facilitate the elucidation of the physiological function of specific pathogen-associated molecular patterns.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Flagelina/imunologia , Receptor 5 Toll-Like/imunologia , Receptor 5 Toll-Like/metabolismo , Substituição de Aminoácidos/imunologia , Animais , Células CACO-2 , Mapeamento de Epitopos , Feminino , Flagelina/antagonistas & inibidores , Humanos , Interleucina-8/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The identification of important amino acid substitutions associated with low survival in hematopoietic cell transplantation (HCT) is hampered by the large number of observed substitutions compared with the small number of patients available for analysis. Random forest analysis is designed to address these limitations. We studied 2107 HCT recipients with good or intermediate risk hematological malignancies to identify HLA class I amino acid substitutions associated with reduced survival at day 100 post transplant. Random forest analysis and traditional univariate and multivariate analyses were used. Random forest analysis identified amino acid substitutions in 33 positions that were associated with reduced 100 day survival, including HLA-A 9, 43, 62, 63, 76, 77, 95, 97, 114, 116, 152, 156, 166 and 167; HLA-B 97, 109, 116 and 156; and HLA-C 6, 9, 11, 14, 21, 66, 77, 80, 95, 97, 99, 116, 156, 163 and 173. In all 13 had been previously reported by other investigators using classical biostatistical approaches. Using the same data set, traditional multivariate logistic regression identified only five amino acid substitutions associated with lower day 100 survival. Random forest analysis is a novel statistical methodology for analysis of HLA mismatching and outcome studies, capable of identifying important amino acid substitutions missed by other methods.
Assuntos
Substituição de Aminoácidos/imunologia , Árvores de Decisões , Transplante de Células-Tronco Hematopoéticas/métodos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Doadores não Relacionados , Adulto , Feminino , Antígenos de Histocompatibilidade Classe I/biossíntese , Teste de Histocompatibilidade , Humanos , Masculino , Distribuição Aleatória , Análise de SobrevidaRESUMO
Position 45 represents a highly polymorphic residue within HLA class I alleles, which contacts the p2 position of bound peptides in 85% of the peptide-HLA structures analyzed, while the neighboring residues 41 and 46 are not involved in peptide binding. To investigate the influence of residue 45 at the functional level, we sequenced peptides eluted from recombinant HLA-B*44:08(41Ala/45Met/46Ala) molecules and compared their features with known peptides from B*44:02(41Thr/45Lys/46Glu). While HLA-B*44:02 has an anchor motif of E at the p2 anchor position, HLA-B*44:08 exhibits Q and L as anchor motif. The 45(Met/Lys) polymorphism contributes to the alteration in the peptide-binding motif and provides further evidence that mismatches at position 45 should be considered as nonpermissive in a transplantation setting.
Assuntos
Antígenos HLA-B/química , Antígenos HLA-B/genética , Peptídeos/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos/imunologia , Antígenos HLA-B/metabolismo , Histocompatibilidade/genética , Histocompatibilidade/imunologia , Humanos , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica/imunologia , Conformação ProteicaRESUMO
HLA-A*03:57 has two amino acid exchanges in exon 2 at adjacent positions of the peptide-binding groove.