RESUMEN
The HIV-1 gp41 N-heptad repeat (NHR) region of the prehairpin intermediate, which is transiently exposed during HIV-1 viral membrane fusion, is a validated clinical target in humans and is inhibited by the Food and Drug Administration (FDA)-approved drug enfuvirtide. However, vaccine candidates targeting the NHR have yielded only modest neutralization activities in animals; this inhibition has been largely restricted to tier-1 viruses, which are most sensitive to neutralization by sera from HIV-1-infected individuals. Here, we show that the neutralization activity of the well-characterized NHR-targeting antibody D5 is potentiated >5,000-fold in TZM-bl cells expressing FcγRI compared with those without, resulting in neutralization of many tier-2 viruses (which are less susceptible to neutralization by sera from HIV-1-infected individuals and are the target of current antibody-based vaccine efforts). Further, antisera from guinea pigs immunized with the NHR-based vaccine candidate (ccIZN36)3 neutralized tier-2 viruses from multiple clades in an FcγRI-dependent manner. As FcγRI is expressed on macrophages and dendritic cells, which are present at mucosal surfaces and are implicated in the early establishment of HIV-1 infection following sexual transmission, these results may be important in the development of a prophylactic HIV-1 vaccine.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/tratamiento farmacológico , Receptores de IgG/inmunología , Secuencias Repetitivas de Aminoácido/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Afinidad de Anticuerpos , Cobayas , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/farmacología , Proteína gp41 de Envoltorio del VIH/genética , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Seropositividad para VIH/inmunología , VIH-1/efectos de los fármacos , VIH-1/inmunología , VIH-1/patogenicidad , Humanos , Sueros Inmunes/inmunología , Sueros Inmunes/farmacología , Inmunización , Inmunoglobulina G/inmunología , Secuencias Repetitivas de Aminoácido/inmunología , Internalización del Virus/efectos de los fármacosRESUMEN
The deadly symptoms of malaria occur as Plasmodium parasites replicate within blood cells. Members of several variant surface protein families are expressed on infected blood cell surfaces. Of these, the largest and most ubiquitous are the Plasmodium-interspersed repeat (PIR) proteins, with more than 1,000 variants in some genomes. Their functions are mysterious, but differential pir gene expression associates with acute or chronic infection in a mouse malaria model. The membership of the PIR superfamily, and whether the family includes Plasmodium falciparum variant surface proteins, such as RIFINs and STEVORs, is controversial. Here we reveal the structure of the extracellular domain of a PIR from Plasmodium chabaudi We use structure-guided sequence analysis and molecular modeling to show that this fold is found across PIR proteins from mouse- and human-infective malaria parasites. Moreover, we show that RIFINs and STEVORs are not PIRs. This study provides a structure-guided definition of the PIRs and a molecular framework to understand their evolution.
Asunto(s)
Plasmodium chabaudi/ultraestructura , Dominios Proteicos/inmunología , Proteínas Protozoarias/ultraestructura , Secuencias Repetitivas de Aminoácido/inmunología , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/ultraestructura , Dicroismo Circular , Genoma de Protozoos/genética , Humanos , Malaria/inmunología , Malaria/virología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/ultraestructura , Familia de Multigenes/genética , Familia de Multigenes/inmunología , Filogenia , Plasmodium chabaudi/genética , Plasmodium chabaudi/inmunología , Dominios Proteicos/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Secuencias Repetitivas de Aminoácido/genéticaRESUMEN
Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development in vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA)3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I ß-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP-Fab311 complex forming a highly organized helical structure. Together, these structural insights may aid in the design of a next-generation malaria vaccine.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/terapia , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/química , Antígenos de Protozoos/química , Antígenos de Protozoos/aislamiento & purificación , Antígenos de Protozoos/uso terapéutico , Ensayos Clínicos Fase II como Asunto , Cristalografía por Rayos X , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Humanos , Vacunas contra la Malaria/química , Vacunas contra la Malaria/uso terapéutico , Malaria Falciparum/inmunología , Ratones , Ratones Endogámicos C57BL , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/uso terapéutico , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/uso terapéutico , Secuencias Repetitivas de Aminoácido/inmunología , Relación Estructura-ActividadRESUMEN
Toll-like receptors (TLRs) are important initiators in innate immune responses against pathogenic microbes such as viruses, intracellular bacteria or parasites. Although the innate immune system is designed to fight infectious pathogens, excessive activation of TLR signaling may lead to unwarranted inflammation with hazardous outcomes. Mechanisms of restraining excessive inflammation and controlling homeostasis for innate immunity are the focus of intense study. Here we showed that LRRC33, a novel member of leucine-rich repeat (LRR) protein family, plays a critical role in desensitizing TLR signaling. LRRC33 is TLR homolog that contains 17 putative LRRs in the extracellular region but lacks a cytoplasmic Toll/IL-1 receptor (TIR) domain. Expression of LRRC33 appears to be ubiquitous with high level of expression found in bone marrow, thymus, liver, lung, intestine and spleen. The LRRs of LRRC33 is required for the interaction with TLR and its inhibitory effect on NF-κB and AP-1 activation as well as cytokine production. Our study sheds new insight into the TLR signaling and inflammatory response in development and human diseases.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Inmunidad Innata , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas/química , Proteínas/fisiología , Receptores Toll-Like/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Inmunidad Innata/genética , Proteínas de Unión a TGF-beta Latente , Proteínas Repetidas Ricas en Leucina , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , FN-kappa B/genética , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Proteínas/genética , Proteínas Modificadoras de la Actividad de Receptores , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/fisiología , Células U937RESUMEN
Nucleoporin Nup98, an essential component of the nuclear pore complex, has multifunctional roles in nuclear functions including transcriptional regulation and nucleocytoplasmic transport. These functions mostly depend on a Gly-Leu-Phe-Gly (GLFG) sequence appearing repetitively in the N-terminal region of Nup98. As the GLFG sequence is well conserved among Nup98s from a wide variety of species including humans, yeasts, and ciliates such as Tetrahymena thermophila, a specific antibody that recognizes the GLFG sequence is expected to detect various Nup98s from a wide-range of species. To generate monoclonal antibodies specific to the GLFG repeat of Nup98, we used two synthetic polypeptides derived from the macronuclear Nup98 of T. thermophila as an antigen. We obtained two monoclonal antibodies (MAbs), 13C2 and 21A10, that recognize Nup98s in indirect immunofluorescence staining and Western blot analysis of T. thermophila. Peptide array analysis of these monoclonal antibodies located the position of their epitopes at or near GLFG residues: the epitope recognized by the 13C2 MAb is FGxxN (x being any amino acid), and the epitope recognized by the 21A10 MAb is GLF. As expected by their epitopes, these monoclonal antibodies also recognize Nup98 homologs expressed by human cells and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, indicating that 13C2 and 21A10 MAbs recognize Nup98 epitopes common to phylogenetically distinct organisms. Thus, these MAbs are useful in studying a wide variety of biological phenomena that involve Nup98, ranging from ciliate nuclear dimorphism to NUP98-related human leukemia.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/química , Proteínas de Complejo Poro Nuclear/inmunología , Proteínas Protozoarias/inmunología , Proteínas de Saccharomyces cerevisiae/inmunología , Proteínas de Schizosaccharomyces pombe/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Mapeo Epitopo , Técnica del Anticuerpo Fluorescente Indirecta , Células HeLa , Humanos , Hibridomas , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/química , Secuencias Repetitivas de Aminoácido/inmunología , Saccharomyces cerevisiae , Schizosaccharomyces , Tetrahymena thermophilaRESUMEN
The antigenic variation of influenza virus represents a major prevention problem. However, the ectodomain of the protein Matrix 2 (M2e) is nearly invariant in all human influenza A strains and has been considered as a promising candidate for a broadly protective vaccine because antibodies to M2e are protective in animal models. In this work we evaluated the possible use of Brucella abortus lumazine synthase protein (BLS), a highly immunogenic decameric protein, as a carrier of the M2e peptide. Chimeric proteins generated by the fusion of one or four in tandem copies of M2e to BLS were efficiently expressed in Escherichia coli and assembled in decameric subunits similarly to the wild type BLS enzyme, as demonstrated by the comparative circular dichroism spectra and size exclusion chromatography and static light scattering analysis. The M2e peptides were stably exposed at the ten N-terminal ends of each BLS molecule. Immunization of mice with purified chimeras carrying only one M2e (BLS-M2e) copy elicited a significant humoral immune response with the addition of different adjuvants. The fusion of four in tandem copies of the M2e peptide (BLS-4M2e) resulted in similar levels of humoral immune response but in the absence of adjuvant. Survival of mice challenged with live influenza virus was 100% after vaccination with BLS-4M2e adjuvanted with Iscomatrix(®) (P<0.001) and 80% when adjuvanted with alum (P<0.01), while the chimera alone protected 60% of the animals (P<0.05). The approach described in this study is intended as a contribution to the generation of universal influenza immunogens, through a simple production and purification process and using safe carriers that might eventually avoid the use of strong adjuvants.
Asunto(s)
Brucella/genética , Portadores de Fármacos/administración & dosificación , Vacunas contra la Influenza/inmunología , Complejos Multienzimáticos/genética , Secuencias Repetitivas de Aminoácido/inmunología , Proteínas de la Matriz Viral/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Femenino , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Secuencias Repetitivas de Aminoácido/genética , Análisis de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas de la Matriz Viral/genéticaRESUMEN
Active and passive immunizations with Aß and Aß antibodies successfully reduced AD pathology and improved cognitive functions in an AD mouse model. However, human clinical trials of vaccination with synthetic Aß(AN1792), were halted due to brain inflammation, presumably induced by T cell-mediated immune response. In this study, we used Picha pastoris to produce a recombinant peptide vaccine, r4 × Aß15(recombinant 4 × Aß15), four tandem repeats of Aß(1-15) interlinked by spacers . Wild-type mice were injected subcutaneously with CFA/IFA as adjuvant. r4 × Aß15 vaccine elicited high titer anti-Aß antibodies which bound to Aß plaque in brain tissue from Tg2576 mouse. The antibody isotype was mainly IgG(1), indicating anti-inflammatory Th2 type. There was no splenocyte proliferation against Aß peptide, which indicates that the r4 × Aß15 vaccine does not induce Aß-specific T cellular immune response. Thus, r4 × Aß15 vaccine may be a safe and efficient vaccine for AD.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Fragmentos de Péptidos/inmunología , Pliegue de Proteína , Linfocitos T/inmunología , Vacunas de Subunidad/inmunología , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/inmunología , Encéfalo/patología , Modelos Animales de Enfermedad , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Pichia , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/metabolismoRESUMEN
Characteristic tau isoform composition of the insoluble fibrillar tau inclusions define tauopathies, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and frontotemporal dementia with parkinsonism linked to chromosome 17/frontotemporal lobar degeneration-tau (FTDP-17/FTLD-tau). Exon 10 splicing mutations in the tau gene, MAPT, in familial FTDP-17 cause elevation of tau isoforms with four microtubule-binding repeat domains (4R-tau) compared to those with three repeats (3R-tau). On the basis of two well-characterised monoclonal antibodies against 3R- and 4R-tau, we developed novel, sensitive immuno-PCR assays for measuring the trace amounts of these isoforms in CSF. This was with the aim of assessing if CSF tau isoform changes reflect the pathological changes in tau isoform homeostasis in the degenerative brain and if these would be relevant for differential clinical diagnosis. Initial analysis of clinical CSF samples of PSP (n = 46), corticobasal syndrome (CBS; n = 22), AD (n = 11), Parkinson's disease with dementia (PDD; n = 16) and 35 controls revealed selective decreases of immunoreactive 4R-tau in CSF of PSP and AD patients compared with controls, and lower 4R-tau levels in AD compared with PDD. These decreases could be related to the disease-specific conformational masking of the RD4-binding epitope because of abnormal folding and/or aggregation of the 4R-tau isoforms in tauopathies or increased sequestration of the 4R-tau isoforms in brain tau pathology.
Asunto(s)
Inmunoensayo/métodos , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetitivas de Aminoácido , Tauopatías/metabolismo , Proteínas tau/líquido cefalorraquídeo , Anciano , Estudios de Cohortes , Homeostasis/genética , Homeostasis/inmunología , Humanos , Inmunoensayo/normas , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/normas , Isoformas de Proteínas/líquido cefalorraquídeo , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Reproducibilidad de los Resultados , Tauopatías/líquido cefalorraquídeo , Tauopatías/genética , Proteínas tau/genética , Proteínas tau/inmunologíaRESUMEN
A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 is a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Epítopos/inmunología , Vacunas Estreptocócicas/administración & dosificación , Vacunas Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Actividad Bactericida de la Sangre , Proteínas Portadoras/química , Proteínas Portadoras/genética , Dicroismo Circular , Secuencia Conservada , Epítopos/genética , Femenino , Ratones , Viabilidad Microbiana/efectos de los fármacos , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Vacunas Estreptocócicas/genética , Streptococcus pyogenes/genética , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to therapeutic development through the use of anti-sense strategies or small molecules targeting EBNA1 mRNA structure.
Asunto(s)
Secuencia de Aminoácidos/genética , Presentación de Antígeno/inmunología , Secuencia de Bases/genética , Antígenos Nucleares del Virus de Epstein-Barr , ARN Mensajero/genética , Presentación de Antígeno/genética , Línea Celular , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Mutación del Sistema de Lectura , Genes MHC Clase I , Células HEK293 , Humanos , Evasión Inmune , Datos de Secuencia Molecular , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Alineación de Secuencia , Linfocitos T Citotóxicos/inmunologíaRESUMEN
To develop a safe and efficient vaccine for the treatment of Alzheimer's disease, we constructed a novel adenovirus vaccine encoding ten repeats of Aß3-10 and CpG motif as an adjuvant and investigated the immune response in BALB/c mouse after intranasal inoculation with this vaccine. The Ad-10×Aß3-10-CpG induces an IgG1 predominant humoral response and production of IL-4 and IL-10 in splenocytes in vitro, indicating a Th2-polarized immune response. Stimulation of splenocytes with Aß3-10 peptide induces robust proliferation but not with full-length Aß42 peptide, demonstrating that Ad-10×Aß3-10-CpG does not induces Aß42 specific T cell immune response. The findings raise the possibility that the adenovirus vaccine Ad-10×Aß3-10-CpG could be a safe and effective alternative for immunotherapy in Alzheimer's disease.
Asunto(s)
Adenoviridae/inmunología , Péptidos beta-Amiloides/administración & dosificación , Inmunidad Celular , Secuencias Repetitivas de Aminoácido , Células Th2/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Adenoviridae/genética , Administración Intranasal , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/inmunología , Animales , Polaridad Celular/genética , Polaridad Celular/inmunología , Células Cultivadas , Vectores Genéticos/administración & dosificación , Vectores Genéticos/inmunología , Humanos , Inmunidad Celular/genética , Interleucina-10/biosíntesis , Interleucina-4/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Bazo/inmunología , Bazo/metabolismo , Células Th2/virología , Vacunas Virales/genéticaRESUMEN
Repetitive proteins (RP) of Trypanosoma cruzi are highly present in the parasite and are strongly recognized by sera from Chagas' disease patients. Flagelar Repetitive Antigen (FRA), which is expressed in all steps of the parasite life cycle, is the RP that displays the greatest number of aminoacids per repeat and has been indicated as one of the most suitable candidate for diagnostic test because of its high performance in immunoassays. Here we analyzed the influence of the number of repeats on the immunogenic and antigenic properties of the antigen. Recombinant proteins containing one, two, and four tandem repeats of FRA (FRA1, FRA2, and FRA4, respectively) were obtained and the immune response induced by an equal amount of repeats was evaluated in a mouse model. The reactivity of specific antibodies present in sera from patients naturally infected with T. cruzi was also assessed against FRA1, FRA2, and FRA4 proteins, and the relative avidity was analyzed. We determined that the number of repeats did not increase the humoral response against the antigen and this result was reproduced when the repeated motifs were alone or fused to a non-repetitive protein. By contrast, the binding affinity of specific human antibodies increases with the number of repeated motifs in FRA antigen. We then concluded that the high ability of FRA to be recognized by specific antibodies from infected individuals is mainly due to a favorable polyvalent interaction between the antigen and the antibodies. In accordance with experimental results, a 3D model was proposed and B epitope in FRA1, FRA2, and FRA4 were predicted.
Asunto(s)
Antígenos de Protozoos/inmunología , Proteínas Protozoarias/inmunología , Secuencias Repetitivas de Aminoácido/inmunología , Trypanosoma cruzi/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Afinidad de Anticuerpos , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Enfermedad de Chagas/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Sueros Inmunes/inmunología , Ratones , Conformación Molecular , Plásmidos , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetidas en Tándem/genética , Secuencias Repetidas en Tándem/inmunología , Trypanosoma cruzi/genéticaRESUMEN
Viruses are known to employ different strategies to manipulate the major histocompatibility (MHC) class I antigen presentation pathway to avoid recognition of the infected host cell by the immune system. However, viral control of antigen presentation via the processes that supply and select antigenic peptide precursors is yet relatively unknown. The Epstein-Barr virus (EBV)-encoded EBNA1 is expressed in all EBV-infected cells, but the immune system fails to detect and destroy EBV-carrying host cells. This immune evasion has been attributed to the capacity of a Gly-Ala repeat (GAr) within EBNA1 to inhibit MHC class I restricted antigen presentation. Here we demonstrate that suppression of mRNA translation initiation by the GAr in cis is sufficient and necessary to prevent presentation of antigenic peptides from mRNAs to which it is fused. Furthermore, we demonstrate a direct correlation between the rate of translation initiation and MHC class I antigen presentation from a certain mRNA. These results support the idea that mRNAs, and not the encoded full length proteins, are used for MHC class I restricted immune surveillance. This offers an additional view on the role of virus-mediated control of mRNA translation initiation and of the mechanisms that control MHC class I restricted antigen presentation in general.
Asunto(s)
Presentación de Antígeno/genética , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Herpesvirus Humano 4/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Evasión Inmune/genética , ARN Mensajero/metabolismo , Animales , Presentación de Antígeno/inmunología , Secuencia de Bases , Línea Celular Tumoral , Dipéptidos/química , Dipéptidos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/genética , Factores Eucarióticos de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/fisiología , Herpesvirus Humano 4/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Evasión Inmune/inmunología , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Ratones , Modelos Biológicos , Conformación de Ácido Nucleico , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/inmunología , ARN Mensajero/genética , Secuencias Repetitivas de Aminoácido/inmunología , Secuencias Repetitivas de Aminoácido/fisiologíaRESUMEN
Fibronectin-binding proteins A and B are multifunctional LPXTG staphylococcal adhesins, comprising an N-terminal region that binds fibrinogen and elastin, and a C-terminal domain that interacts with fibronectin. The C-terminal domain of fibronectin-binding protein A is organized into 11 tandem repeats, six of which bind the ligand with high affinity; other sites bind more weakly. Fibronectin-binding protein B has been postulated to harbor 10 rather than 11 repeats, but it contains the same number of high-affinity fibronectin-binding sites as fibronectin-binding protein A. In this study, we confirm this prediction and show that six of 10 sites bind with dissociation constants in the nanomolar range. We also found that the full-length repetitive region of fibronectin-binding protein B stimulated the production of a mAb (15E11) that binds with high affinity to an epitope shared by repeats 9 and 10 from both adhesins. With the use of truncated fragments of repeat 9 of fibronectin-binding protein A, we mapped the antibody epitope to the N-terminal segment and the fibronectin-binding site to the C-terminal segment of the repeat. The distinct localization of the 15E11 epitope and the fibronectin-binding site suggests that the interfering effect of the antibody might result from steric hindrance or a conformational change in the structure that reduces the accessibility of fibronectin to its binding determinant. The epitope is well exposed on the surface of staphylococcal cells, as determined by genetic analyses, fluorescence microscopy, and flow cytometry. When incubated with cells of Staphylococcus aureus strains, 15E11 inhibits attachment of bacteria to surface-coated fibronectin by almost 70%.
Asunto(s)
Adhesinas Bacterianas/inmunología , Anticuerpos Monoclonales/inmunología , Fibronectinas/metabolismo , Receptores Fc/inmunología , Proteína Estafilocócica A/inmunología , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos/inmunología , Adhesión Bacteriana/inmunología , Sitios de Unión , Unión Competitiva , Western Blotting , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Citometría de Flujo , Cinética , Ratones , Datos de Secuencia Molecular , Unión Proteica , Receptores Fc/genética , Receptores Fc/metabolismo , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMEN
The nucleotide-binding domain, leucine-rich repeat (NLR) proteins are a recently discovered family of intracellular pathogen and danger signal sensors. NLRs have emerged as important contributors to innate immunity in animals. The physiological impact of these genes is increasingly evident, underscored by the genetic association of variant family members with an array of inflammatory diseases. The association of mutations in NLR genes with autoinflammatory diseases indicates an important function of these genes in inflammation in vivo. This review summarizes the role of the inflammasome NLR proteins in innate immunity and inflammatory diseases and explores the possible utility of some of these NLRs as pharmacological targets.
Asunto(s)
Mediadores de Inflamación/metabolismo , Nucleótidos/metabolismo , Proteínas/metabolismo , Animales , Sistemas de Liberación de Medicamentos , Humanos , Inmunidad Innata/genética , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Proteínas Repetidas Ricas en Leucina , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Proteínas/genética , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunologíaRESUMEN
We describe a method for producing polyclonal antibodies against peptide antigen cytochrome P450 1A2 and 3A4 using a tandem repeat of the epitope region and incorporation of proline residue between the repeated sequences. An ELISA assay revealed more efficient generation of polyclonal antibodies to tandem repeat peptide antigens than mono-epitope peptides. The incorporation of proline residues further stimulated antibody production.
Asunto(s)
Citocromo P-450 CYP1A2/inmunología , Citocromo P-450 CYP3A/inmunología , Epítopos/inmunología , Prolina/fisiología , Secuencias Repetitivas de Aminoácido/inmunología , Secuencias Repetidas en Tándem , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos/fisiología , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP3A/química , Eficiencia , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/inmunología , Prolina/inmunología , Secuencias Repetidas en Tándem/fisiologíaRESUMEN
Ehrlichia chaffeensis and E. canis have a small subset of tandem repeat (TR)-containing protein orthologs, including p120/p140, which elicit strong antibody responses. The TR regions of these protein orthologs are immunoreactive, but the molecular characteristics of the p120/p140 epitopes have not been determined. In this study, the immunodeterminants of the E. chaffeensis p120 and E. canis p140 were identified and molecularly defined. Major antibody epitope-containing regions of both p120 and p140 were localized to the TR regions, which reacted strongly by Western immunoblotting with antibodies in sera from E. chaffeensis-infected dogs or patients and E. canis-infected dogs, respectively. Single continuous species-specific major epitopes within the E. chaffeensis p120 and E. canis p140 TRs were mapped to homologous surface-exposed glutamate/aspartate-rich regions (19 to 22 amino acids). In addition, minor cross-reactive epitopes were localized to homologous N- and C-terminal regions of p120 and p140. Furthermore, although the native and recombinant p120 and p140 proteins exhibited higher-than-predicted molecular masses, posttranslational modifications were not present on abnormally migrating p120 and p140 TR recombinant proteins as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Ehrlichia canis/inmunología , Ehrlichia chaffeensis/inmunología , Ehrlichiosis/inmunología , Mapeo Epitopo , Epítopos de Linfocito B , Secuencia de Aminoácidos , Animales , Reacciones Cruzadas , Enfermedades de los Perros/inmunología , Perros , Ehrlichiosis/veterinaria , Humanos , Datos de Secuencia Molecular , Secuencias Repetitivas de Aminoácido/inmunología , Alineación de SecuenciaRESUMEN
Herpesvirus saimiri (HVS) establishes a persistent infection in squirrel monkeys by maintaining its episome within T lymphocytes. The product of open reading frame 73 (ORF73) plays a key role in episomal maintenance and is the functional homologue of Epstein-Barr virus EBNA1 and Kaposi's sarcoma-associated herpesvirus LANA1 proteins. There is little sequence homology among these proteins, although all contain a central domain of repeating amino acids. The repeat domains of EBNA1 and LANA1 enhance the stability of these proteins and cause a retardation in self-protein synthesis, leading to poor recognition by CD8(+) cytotoxic T lymphocytes (CTL). The HVS ORF73 repeat domain is composed of a glutamic acid and glycine repeat linked to a glutamic acid and alanine repeat (EG-EA repeat). Here we show that the EG-EA repeat similarly causes a reduction in the recognition of ORF73 by CD8(+) CTL. However, deletion of the EG-EA repeat from HVS ORF73 had no affect on the stability of the protein or its rate of translation. In contrast, the presence of the EG-EA repeat was found to decrease the steady-state levels of ORF73 mRNA. The inhibitory properties of the EG-EA repeat were maintained when transferred to a heterologous protein, and manipulation of the repeat revealed that the motif EEAEEAEEE was sufficient to cause a reduction in recognition of ORF73 by CD8(+) CTL. Thus, the EG-EA repeat of HVS ORF73 plays a role in immune evasion but utilizes a mechanism distinct from that of the EBNA1 and LANA1 repeats.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Herpesvirus Saimiriino 2/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Proteínas Virales/biosíntesis , Proteínas Virales/inmunología , Línea Celular , Humanos , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Proteínas Virales/genéticaRESUMEN
The C-terminal repeating sequences of Clostridium difficile toxin A (designated ARU) are homologous to the carbohydrate-binding domain of streptococcal glucosyltransferases (GTFs) that were recently identified as potent modulins. To test the hypothesis that ARU might exert a similar biological activity on endothelial cells, recombinant ARU (rARU), which was noncytotoxic to cell cultures, was analyzed using human umbilical vein endothelial cells. The rARU could bind directly to endothelial cells in a serum- and calcium-dependent manner and induce the production of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein 1 in a dose-dependent manner. An oligosaccharide binding assay indicated that rARU, but not GTFC, binds preferentially to Lewis antigens and 3'HSO3-containing oligosaccharides. Binding of rARU to human endothelial or intestinal cells correlated directly with the expression of Lewis Y antigen. Bound rARU directly activated mitogen-activated protein kinases and the NF-kappaB signaling pathway in endothelial cells to release biologically active chemokines and adhesion molecules that promoted migration in a transwell assay and the adherence of polymorphonuclear and mononuclear cells to the endothelial cells. These results suggest that ARU may bind to multiple carbohydrate motifs to exert its biological activity on human endothelial cells.
Asunto(s)
Toxinas Bacterianas/inmunología , Quimiocinas/biosíntesis , Quimiotaxis de Leucocito , Clostridioides difficile/inmunología , Células Endoteliales/inmunología , Enterotoxinas/inmunología , Células Epiteliales/inmunología , Leucocitos/inmunología , Secuencias Repetitivas de Aminoácido/inmunología , Toxinas Bacterianas/genética , Línea Celular , Ensayos de Migración de Leucocitos , Clostridioides difficile/genética , Enterotoxinas/genética , Humanos , Antígenos del Grupo Sanguíneo de Lewis/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Distinct sequence differences within the repetitive elements (RepMP) of the Mycoplasma pneumoniae P1 adhesin are the only targets to discriminate patient isolates with molecular approaches into subtypes and variants. Since the P1 protein is also one of the most immunodominant proteins of the bacterium, the antigenic regions of the differing repetitive sequences might be of epidemiological significance for the observation of time-dependent outbreaks due to defined subtypes and variants of M. pneumoniae in the human population. By establishing a set of four subtype- and variant 2-specific recombinant proteins, we investigated the antigenicity of the variable P1 protein regions with sera of subtype- and variant-specific immunised animals and sera of M. pneumoniae-positive pneumonia patients. The results of the ELISA experiments confirmed the immunogenic character of the differing parts of the P1 adhesin and the occurrence of a specific immune response of the immunised animals. The detection of subtype- and variant-specific antibodies in the investigated sera strongly support the hypothesis of a selective immune response. It might be indicative for the partial protection of the host to a defined endemic or epidemic strain and therefore also the reason for a reduced protection against secondary infections with a differing subtype and variant of M. pneumoniae strains compared to the first contact.