RESUMEN
Nervous necrosis virus (NNV) capsid protein plays an important role in producing viral particles without any genetic elements. Thus, NNV is a promising candidate for vaccine development and is widely used for constructing vaccines, including DNA, recombinant proteins, and virus-like particles (VLPs). Our study aimed to investigate the potential of NNV capsid protein (NNV) and NNV capsid protein fused to enhanced green fluorescent protein (NNV-EGFP) through VLP formation and whether their application can induce specific antibody responses against certain antigens. We focused on producing DNA and recombinant protein vaccines consisting of the genes for NNV, EGFP, and NNV-EGFP. The approach using NNV-EGFP allowed NNV to act as a carrier or inducer while EGFP was incorporated as part of the capsid protein, thereby enhancing the immune response. In vitro studies demonstrated that all DNA vaccines expressed in HINAE cells resulted in varying protein expression levels, with particularly low levels observed for pNNV and pNNV-EGFP. Consequently, structural proteins derived from HINAE cells could not be observed using transmission electron microscopy (TEM). In contrast, recombinant proteins of NNV and NNV-EGFP were expressed through the Escherichia coli expression system. TEM revealed that rNNV was assembled into VLPs with an approximate size of 30 nm, whereas rNNV-EGFP presented particles ranging from 10 nm to 50 nm in size. For the vaccination test, DNA vaccination marginally induced specific antibody responses in Japanese flounder compared to unvaccinated fish. Meanwhile, NNV and NNV-EGFP recombinant vaccines enhanced a greater anti-NNV antibody response than the others, whereas antibody responses against EGFP were also marginal. These results indicate that NNV capsid protein-based antigens, presenting as particles, play an important role in eliciting a specific anti-NNV antibody response and have the potential to improve fish immune responses.
Asunto(s)
Proteínas de la Cápside , Enfermedades de los Peces , Nodaviridae , Vacunas Virales , Animales , Nodaviridae/inmunología , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/prevención & control , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Infecciones por Virus ARN/veterinaria , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/prevención & control , Vacunas de ADN/inmunología , Vacunas de ADN/administración & dosificación , Desarrollo de Vacunas , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificaciónRESUMEN
Vaccines aim to efficiently and specifically activate the immune system via a cascade of antigen uptake, processing, and presentation by antigen-presenting cells (APCs) to CD4 and CD8 T cells, which in turn drive humoral and cellular immune responses. The specific formulation of vaccine carriers can not only shield the antigens from premature sequestering before reaching APCs but also favorably promote intracellular antigen presentation and processing. This study compares two different acid-degradable polymeric nanoparticles that are capable of encapsulating a moderately immunogenic antigen, GFP, at nearly full efficacy via electrostatic interactions or molecular affinity between His tag and Ni-NTA-conjugated monomners. This resulted in GFP-encapsulating NPs composed of ketal monomers and crosslinkers (KMX/GFP NPs) and NTA-conjugated ketal monomers and crosslinkers (NKMX/GFP NPs), respectively. Encapsulated GFP was found to be released more rapidly from NKMX/GFP NPs (electrostatic encapsulation) than from KMX/GFP NPs (affinity-driven encapsulation). In vivo vaccination studies demonstrated that while repeated injections of either NP formulation resulted in poorer generation of anti-GFP antibodies than injections of the GFP antigen itself, sequential injections of NPs and GFP as prime and booster vaccines, respectively, restored the humoral response. We proposed that NPs primarily assist APCs in antigen presentation by T cells, and B cells need to be further stimulated by free protein antigens to produce antibodies. The findings of this study suggest that the immune response can be modulated by varying the chemistry of vaccine carriers and the sequences of vaccination with free antigens and antigen-encapsulating NPs.
Asunto(s)
Antígenos , Nanopartículas , Polímeros , Nanopartículas/química , Animales , Polímeros/química , Ratones , Antígenos/inmunología , Antígenos/química , Vacunación , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/inmunología , Femenino , Ratones Endogámicos C57BL , Tamaño de la Partícula , Vacunas/inmunología , Vacunas/química , Vacunas/administración & dosificaciónRESUMEN
Response to protein complicates immunotherapy research.
Asunto(s)
Proteínas Fluorescentes Verdes/inmunología , Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , Animales , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Reproducibilidad de los Resultados , Linfocitos T/inmunologíaAsunto(s)
Proteínas Fluorescentes Verdes/inmunología , Inmunidad/inmunología , Metástasis de la Neoplasia/inmunología , Neoplasias/inmunología , Animales , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Metástasis de la Neoplasia/diagnóstico por imagen , Neoplasias/diagnóstico por imagen , Linfocitos T/inmunologíaRESUMEN
Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin-proteasome and autophagy-lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.
Asunto(s)
Epítopos/genética , Proteolisis , Ribonucleoproteínas/genética , Anticuerpos de Dominio Único/inmunología , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos/farmacología , Epítopos/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Proteínas Fluorescentes Verdes/farmacología , Células HEK293 , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/inmunología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/inmunología , Ribonucleoproteínas/inmunología , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/farmacología , Ubiquitina/genética , Ubiquitina/inmunologíaRESUMEN
Bivalent VHHs have been shown to display better functional affinity compared with their monovalent counterparts. Bivalency can be achieved either by inserting a hinge region between both VHHs units or by using modules that lead to dimerization. In this report, a small self-associating peptide originating from the tetramerization domain of p53 was developed as a tool for devicing nanobody dimerization. This E3 peptide was evaluated for the dimerization of an anti-eGFP nanobody (nano-eGFP-E3) whose activity was compared to a bivalent anti-eGFP constructed in tandem using GS rich linker. The benefit of bivalency in terms of avidity and specificity was assessed in different in vitro and in cellulo assays. In ELISA and SPR, the dimeric and tandem formats were nearly equivalent in terms of gain of avidity compared to the monovalent counterpart. However, in cellulo, the nano-eGFP-E3 construct showed its superiority over the tandem format in terms of specificity with a highest and better ratio signal-to-noise. All together, the E3 peptide provides a universal suitable tool for the construction of dimeric biomolecules, in particular antibody fragments with improved functional affinity.
Asunto(s)
Epítopos , Proteínas Fluorescentes Verdes/inmunología , Fragmentos de Péptidos/inmunología , Anticuerpos de Dominio Único/inmunología , Proteína p53 Supresora de Tumor/inmunología , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Mutación , Fragmentos de Péptidos/genética , Multimerización de Proteína , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Mu-opioid receptor (MOR) signaling regulates multiple neuronal pathways, including those involved in pain, reward, and respiration. To advance the understanding of MOR's roles in pain modulation, there is a need for high-throughput screening methods of opioids in vitro and high-resolution mapping of opioids in the brain. To fill this need, we designed and characterized a genetically encoded fluorescent reporter, called Single-chain Protein-based Opioid Transmission Indicator Tool for MOR (M-SPOTIT). M-SPOTIT represents a new and unique mechanism for fluorescent reporter design and can detect MOR activation, leaving a persistent green fluorescence mark for image analysis. M-SPOTIT showed an opioid-dependent signal to noise ratio (S/N) up to 12.5 and was able to detect as fast as a 30-second opioid exposure in HEK293T cell culture. Additionally, it showed an opioid-dependent S/N up to 4.6 in neuronal culture and detected fentanyl with an EC50 of 15â nM. M-SPOTIT will potentially be useful for high-throughput detection of opioids in cell cultures and cellular-resolution detection of opioids in vivo. M-SPOTIT's novel mechanism can be used as a platform to design other G-protein-coupled receptor-based sensors.
Asunto(s)
Analgésicos Opioides/análisis , Microscopía Fluorescente/métodos , Fentanilo/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Relación Señal-Ruido , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunologíaRESUMEN
GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.
Asunto(s)
Cromatografía en Gel , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/metabolismo , Nanotecnología , Péptidos/metabolismo , Anticuerpos de Dominio Único/inmunología , Animales , Microscopía por Crioelectrón , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/genética , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/inmunología , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Oryzias/genética , Oryzias/metabolismo , Péptidos/genética , Péptidos/inmunología , Estabilidad Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , Espectrometría de Fluorescencia , Temperatura , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismoRESUMEN
Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer.
Asunto(s)
Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Hibridomas/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/análisis , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hibridomas/citología , Isotipos de Inmunoglobulinas , Inmunoprecipitación , Ratones , Factores de TiempoRESUMEN
Genome-wide localization of chromatin and transcription regulators can be detected by a variety of techniques. Here, we describe a novel method 'greenCUT&RUN' for genome-wide profiling of transcription regulators, which has a very high sensitivity, resolution, accuracy and reproducibility, whilst assuring specificity. Our strategy begins with tagging of the protein of interest with GFP and utilizes a GFP-specific nanobody fused to MNase to profile genome-wide binding events. By using a GFP-nanobody the greenCUT&RUN approach eliminates antibody dependency and variability. Robust genomic profiles were obtained with greenCUT&RUN, which are accurate and unbiased towards open chromatin. By integrating greenCUT&RUN with nanobody-based affinity purification mass spectrometry, 'piggy-back' DNA binding events can be identified on a genomic scale. The unique design of greenCUT&RUN grants target protein flexibility and yields high resolution footprints. In addition, greenCUT&RUN allows rapid profiling of mutants of chromatin and transcription proteins. In conclusion, greenCUT&RUN is a widely applicable and versatile genome-mapping technique.
Asunto(s)
Genómica/métodos , Proteómica/métodos , Factores de Transcripción/metabolismo , Sitios de Unión , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Células HeLa , Humanos , Espectrometría de Masas , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Recombinantes de Fusión/análisis , Anticuerpos de Dominio Único , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismoRESUMEN
This protocol uses Nr4a1-GFP Nr4a3-Tocky mice to study T cell receptor (TCR) signaling using flow cytometry. It identifies the optimal mouse transgenic status and fluorochromes compatible with the dual reporter. This protocol has applications in TCR signaling, and we outline how to obtain high-quality datasets. It is not compatible with cellular fixation, and cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Jennings et al., 2020.
Asunto(s)
Citometría de Flujo , Proteínas Fluorescentes Verdes/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Proteínas Recombinantes de Fusión/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Transgénicos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.
Asunto(s)
Cromatina/inmunología , Epigénesis Genética/inmunología , Regulación de la Expresión Génica/inmunología , Regiones Promotoras Genéticas/inmunología , Anticuerpos de Dominio Único/inmunología , Algoritmos , Animales , Cromatina/genética , Silenciador del Gen/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Modelos Genéticos , Regiones Promotoras Genéticas/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Anticuerpos de Dominio Único/metabolismoRESUMEN
The nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D) was discovered by coincidence in the 1980s and has since been widely used in the investigation of T1D and diabetic complications. The current in vivo study was originally designed to prospectively assess whether hyperglycemia onset is associated with physical destruction or functional impairment of beta cells under inflammatory insult during T1D progression in diabetes-prone female NOD mice. Prediabetic 16- to 20-wk-old NOD mice were transplanted with green fluorescent protein (GFP)-expressing reporter islets in the anterior chamber of the eye (ACE) that were monitored longitudinally, in addition to glycemia, with and without immune modulation using anti-CD3 monoclonal antibody therapy. However, there was an early and vigorous immune reaction against the GFP-expressing beta cells that lead to their premature destruction independent of autoimmune T1D development in progressor mice that eventually became hyperglycemic. This immune reaction also occurred in nonprogressor NOD recipients. These findings showed a previously unknown reaction of NOD mice to GFP that prevented achieving the original goals of this study but highlighted a new feature of the NOD mice that should be considered when designing experiments using this model in T1D research.
Asunto(s)
Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 1/inmunología , Modelos Animales de Enfermedad , Animales , Femenino , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/inmunología , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Trasplante de Islotes Pancreáticos , Ratones , Ratones Endogámicos NOD , Estado Prediabético/inmunologíaRESUMEN
The affinity-directed protein missile (AdPROM) system utilizes specific polypeptide binders of intracellular proteins of interest (POIs) conjugated to an E3 ubiquitin ligase moiety to enable targeted proteolysis of the POI. However, a chemically tuneable AdPROM system is more desirable. Here, we use Halo-tag/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC) technology to develop a ligand-inducible AdPROM (L-AdPROM) system. When we express an L-AdPROM construct consisting of an anti-GFP nanobody conjugated to the Halo-tag, we achieve robust degradation of GFP-tagged POIs only upon treatment of cells with the HaloPROTAC. For GFP-tagged POIs, ULK1, FAM83D, and SGK3 were knocked in with a GFP-tag using CRISPR/Cas9. By substituting the anti-GFP nanobody for a monobody that binds H- and K-RAS, we achieve robust degradation of unmodified endogenous RAS proteins only in the presence of the HaloPROTAC. Through substitution of the polypeptide binder, the highly versatile L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.
Asunto(s)
Proteolisis , Anticuerpos de Cadena Única/metabolismo , Marcadores de Afinidad , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Técnicas de Sustitución del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Péptidos/química , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Ubiquitinación , Proteínas ras/metabolismoRESUMEN
BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (PAN, PαAg, PHsp60, PBlaF* and PL5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: PAN was the weakest in both Mycobacterium smegmatis and BCG and PBlaF* was higher than PHsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that PBlaF* and PHsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the PL5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.
Asunto(s)
Vacuna BCG/inmunología , Escherichia coli/inmunología , Vectores Genéticos/inmunología , Proteínas Fluorescentes Verdes/inmunología , Mycobacterium bovis/inmunología , Mycobacterium smegmatis/inmunología , Animales , Escherichia coli/genética , Femenino , Vectores Genéticos/genética , Ratones , Ratones Endogámicos BALB CRESUMEN
Variable domains of heavy chains of camelid heavy-chain antibodies (VHHs) are known as nanobodies. Nanobodies are approximately 15 kDa in size with high affinity to their antigens. They can be easily manipulated and produced in microorganisms. In this study, an alpaca was immunized with purified green fluorescence protein (GFP) and a VHH library from lymphocytes of the immunized alpaca was constructed with a capacity of 6.7 × 107. The library was biopanned against GFP by phage display technique and four unique DNA sequences coding for anti-GFP nanobodies were identified by enzyme-linked immunosorbent assay, named a12, e6, d5, and b9. The four DNA sequences were then cloned into pADL-10b-6×His or pBAD24-Flag-6×His for expression in bacteria. Purified A12, E6, D5, and B9 were demonstrated to bind GFP specifically both in vitro by enzyme-linked immunosorbent assay and native-PAGE analysis and in vivo by immunofluorescence and immunoprecipitation. Taken together, our results demonstrate that anti-GFP nanobodies are successfully selected from the immune library, are produced in bacteria, and are available for basic research.Key Points⢠Four different GFP binders were successfully obtained from an immune VHH library.⢠The four GFP binders were successfully purified from bacteria. ⢠Purified GFP binders can bind GFP both in vitro and in vivo and are ready for use in basic research.
Asunto(s)
Camélidos del Nuevo Mundo/inmunología , Proteínas Fluorescentes Verdes/inmunología , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/genética , Animales , Bacterias/genética , Sitios de Unión de Anticuerpos , Camelus , Técnicas de Visualización de Superficie Celular , Ensayo de Inmunoadsorción Enzimática , Biblioteca de PéptidosRESUMEN
Early and robust T cell responses have been associated with survival from Lassa fever (LF), but the Lassa virus-specific memory responses have not been well characterized. Regions within the virus surface glycoprotein (GPC) and nucleoprotein (NP) are the main targets of the Lassa virus-specific T cell responses, but, to date, only a few T cell epitopes within these proteins have been identified. We identified GPC and NP regions containing T cell epitopes and HLA haplotypes from LF survivors and used predictive HLA-binding algorithms to identify putative epitopes, which were then experimentally tested using autologous survivor samples. We identified 12 CD8-positive (CD8+) T cell epitopes, including epitopes common to both Nigerian and Sierra Leonean survivors. These data should be useful for the identification of dominant Lassa virus-specific T cell responses in Lassa fever survivors and vaccinated individuals as well as for designing vaccines that elicit cell-mediated immunity.IMPORTANCE The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/química , Fiebre de Lassa/inmunología , Virus Lassa/inmunología , Nucleoproteínas/inmunología , Proteínas del Envoltorio Viral/inmunología , Adolescente , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/virología , Niño , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Haplotipos , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Sueros Inmunes/análisis , Memoria Inmunológica , Fiebre de Lassa/genética , Fiebre de Lassa/patología , Virus Lassa/patogenicidad , Masculino , Nigeria , Nucleoproteínas/genética , Sierra Leona , Sobrevivientes , Proteínas del Envoltorio Viral/genética , Adulto JovenRESUMEN
BACKGROUND: Atopic dermatitis skin lesions demonstrate increased expression of IL-25 by keratinocytes and increased numbers of type 2 innate lymphoid cells (ILC2s) that express high levels of IL-25 receptor (IL-25R). IL-13 is expressed in atopic dermatitis skin lesions and plays an important role in pathogenesis of the disease. OBJECTIVE: Our aim was to determine the role of IL-25 and ILC2s in a mouse model of antigen-driven allergic skin inflammation. METHODS: Wild-type mice; mice that express an Il13-driven enhanced green fluorescent protein; and mice that lack IL-25R, IL-25 in keratinocytes, or IL-13 or IL-25R in ILC2s were subjected to acute or chronic epicutaneous sensitization with ovalbumin. Sensitized skin was examined by histology for epidermal thickening. Cellular infiltrates were analyzed for surface markers and intracellular expression of enhanced green fluorescent protein by flow cytometry. Gene expression was quantitated by RT quantitative PCR. RESULT: In both acute and chronic antigen-driven allergic skin inflammation, signaling by keratinocyte-derived IL-25 in ILC2s is important for epidermal hyperplasia, dermal infiltration by CD4+ T cells, and cutaneous expression of Il13 and the IL-13-dependent TH2-cell-attracting chemokines Cc17 and Ccl22. ILCs are the major source of IL-13 in acutely sensitized mouse skin, whereas T cells are its major source in chronically sensitized mouse skin. CONCLUSION: ILC2 activation by IL-25 is essential for IL-13 expression at sites of allergic skin inflammation.
Asunto(s)
Hipersensibilidad/inmunología , Inflamación/inmunología , Interleucina-13/inmunología , Interleucinas/inmunología , Queratinocitos/inmunología , Linfocitos/inmunología , Piel/inmunología , Alérgenos/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Dermatitis Atópica/inmunología , Femenino , Expresión Génica/inmunología , Proteínas Fluorescentes Verdes/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Células Th2/inmunologíaRESUMEN
BACKGROUND: The use of biomaterials has been expanded to improve the characteristics of vaccines. Recently we have identified that the peptide PH(1-110) from polyhedrin self-aggregates and incorporates foreign proteins to form particles. We have proposed that this peptide can be used as an antigen carrying system for vaccines. However, the immune response generated by the antigen fused to the peptide has not been fully characterized. In addition, the adjuvant effect and thermostability of the particles has not been evaluated. RESULTS: In the present study we demonstrate the use of a system developed to generate nano and microparticles carrying as a fusion protein peptides or proteins of interest to be used as vaccines. These particles are purified easily by centrifugation. Immunization of animals with the particles in the absence of adjuvant result in a robust and long-lasting immune response. Proteins contained inside the particles are maintained for over 1 year at ambient temperature, preserving their immunological properties. CONCLUSION: The rapid and efficient production of the particles in addition to the robust immune response they generate position this system as an excellent method for the rapid response against emerging diseases. The thermostability conferred by the particle system facilitates the distribution of the vaccines in developing countries or areas with no electricity.
Asunto(s)
Antígenos/inmunología , Inmunoglobulinas/metabolismo , Proteínas de la Matriz de Cuerpos de Oclusión/química , Péptidos/química , Vacunas/inmunología , Animales , Antígenos/química , Estabilidad de Medicamentos , Femenino , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/inmunología , Inmunización , Ratones , Nanopartículas , Tamaño de la Partícula , Agregado de Proteínas , Proteínas Recombinantes de Fusión/inmunología , Termodinámica , Vacunas/químicaRESUMEN
In the epididymis, prevention of autoimmune responses against spermatozoa and simultaneous protection against pathogens is important for male fertility. We have previously shown that mononuclear phagocytes (MPs) are located either in the epididymal interstitium or in close proximity to the epithelium. In the initial segments (IS), these 'intraepithelial' MPs extend slender luminal-reaching projections between epithelial cells. In this study, we performed an in-depth characterisation of MPs isolated from IS, caput-corpus and cauda epididymis of CX3CR1EGFP+/- mice that express EGFP in these cells. Flow cytometry analysis revealed region-specific subsets of MPs that express combinations of markers traditionally described in 'dendritic cells' or 'macrophages'. RNA sequencing identified distinct transcriptomic signatures in MPs from each region and revealed specific genes involved in inflammatory and anti-inflammatory responses, phagosomal activity and antigen processing and presentation. Functional fluorescent in vivo labelling assays showed that higher percentages of CX3CR1+ MPs that captured and processed antigens were detected in the IS compared to other regions. Confocal microscopy showed that in the IS, caput and corpus, circulatory antigens were internalised and processed by interstitial and intraepithelial MPs. However, in the cauda only interstitial MPs internalised and processed antigens, while intraepithelial MPs did not take up antigens, indicating that all antigens have been captured before they reached the epithelial lining. Cauda MPs may thus confer a stronger protection against blood-borne pathogens compared to proximal regions. By identifying immunoregulatory mechanisms in the epididymis, our study may lead to new therapies for male infertility and epididymitis and identify potential targets for immunocontraception.