RESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, which caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), lead to a crisis with devastating disasters to global public economy and health. Several studies suggest that the SARS-CoV-2 nucleocapsid protein (N protein) is one of uppermost structural constituents of SARS-CoV-2 and is relatively conserved which could become a specific diagnostic marker. In this study, eight single domain antibodies recognized the N protein specifically which were named pN01-pN08 were screened using human phage display library. According to multiple sequence alignment and molecular docking analyses, the interaction mechanism between antibody and N protein was predicted. ELISA results indicated pN01-pN08 with high affinity to protein N. To improve their efficacy, two fusion proteins were prepared and their affinity was tested. These finding showed that fusion proteins had higher affinity than single domain antibodies and will be used as diagnosis for the pandemic of SARS-CoV-2.
Asunto(s)
Anticuerpos Antivirales , COVID-19 , Proteínas de la Nucleocápside de Coronavirus , Simulación del Acoplamiento Molecular , SARS-CoV-2 , Anticuerpos de Dominio Único , Humanos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , SARS-CoV-2/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/química , COVID-19/inmunología , COVID-19/diagnóstico , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos , Fosfoproteínas/inmunología , Fosfoproteínas/química , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Biblioteca de PéptidosRESUMEN
Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an 'all in one' solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.
Asunto(s)
Estreptavidina , Estreptavidina/química , Estreptavidina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos/metabolismo , Humanos , Biotinilación , Microscopía Fluorescente/métodos , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismoRESUMEN
American Tegumentary Leishmaniasis (ATL) is a disease of high severity and incidence in Brazil, in addition to being a worldwide concern in public health. Leishmania amazonensis is one of the etiological agents of ATL, and the inefficiency of control measures, associated with the high toxicity of the treatment and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present study proposes to elaborate a chimeric protein (rChiP), based on the fusion of multiple epitopes of CD4+/CD8+ T cells, identified in the immunoproteome of the parasites L. amazonensis and L. braziliensis. The designed chimeric protein was tested in the L. amazonensis murine model of infection using the following formulations: 25 µg of the rChiP in saline (rChiP group) and 25 µg of the rChiP plus 25 µg of MPLA-PHAD® (rChiP+MPLA group). After completing immunization, CD4+ and CD8+ T cells, stimulated with SLa-Antigen or rChiP, showed an increased production of nitric oxide and intracytoplasmic pro-inflammatory cytokines, in addition to the generation of central and effector memory T cells. rChiP and rChiP+MPLA formulations were able to promote an effective protection against L. amazonensis infection determined by a reduction in the development of skin lesions and lower parasitic burden. Reduction in the development of skin lesions and lower parasitic burden in the vaccinated groups were associated with an increase of nitrite, CD4+/CD8+IFN-γ+TNF-α+ and CD4+/CD8+CD44highCD62Lhigh/low T cells, IgGTotal, IgG2a, and lower rates of IgG1 and CD4+/CD8+IL-10+. This data suggests that proposed formulations could be considered potential tools to prevent ATL.
Asunto(s)
Adyuvantes Inmunológicos , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Epítopos de Linfocito T , Memoria Inmunológica , Vacunas contra la Leishmaniasis , Leishmaniasis Cutánea , Animales , Leishmaniasis Cutánea/prevención & control , Leishmaniasis Cutánea/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Ratones , Vacunas contra la Leishmaniasis/inmunología , Femenino , Adyuvantes Inmunológicos/administración & dosificación , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Leishmania braziliensis/inmunología , Lípido A/análogos & derivados , Lípido A/inmunología , Anticuerpos Antiprotozoarios/inmunología , Citocinas/metabolismo , Citocinas/inmunología , Modelos Animales de Enfermedad , Antígenos de Protozoos/inmunologíaRESUMEN
Introduction: NK cells can mediate tumor cell killing by natural cytotoxicity and by antibody-dependent cell-mediated cytotoxicity (ADCC), an anti-tumor mechanism mediated through the IgG Fc receptor CD16A (FcγRIIIA). CD16A polymorphisms conferring increased affinity for IgG positively correlate with clinical outcomes during monoclonal antibody therapy for lymphoma, linking increased binding affinity with increased therapeutic potential via ADCC. We have previously reported on the FcγR fusion CD64/16A consisting of the extracellular region of CD64 (FcγRI), a high-affinity Fc receptor normally expressed by myeloid cells, and the transmembrane/cytoplasmic regions of CD16A, to create a highly potent and novel activating fusion receptor. Here, we evaluate the therapeutic potential of engineered induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing CD64/16A as an "off-the-shelf", antibody-armed cellular therapy product with multi-antigen targeting potential. Methods: iNK cells were generated from iPSCs engineered to express CD64/16A and an interleukin (IL)-15/IL-15Rα fusion (IL-15RF) protein for cytokine independence. iNK cells and peripheral blood NK cells were expanded using irradiated K562-mbIL21-41BBL feeder cells to examine in in vitro and in vivo assays using the Raji lymphoma cell line. ADCC was evaluated in real-time by IncuCyte assays and using a xenograft mouse model with high circulating levels of human IgG. Results: Our data show that CD64/16A expressing iNK cells can mediate potent anti-tumor activity against human B cell lymphoma. In particular, (i) under suboptimal conditions, including low antibody concentrations and low effector-to-target ratios, iNK-CD64/16A cells mediate ADCC, (ii) iNK-CD64/16A cells can be pre-loaded with tumor-targeting antibodies (arming) to elicit ADCC, (iii) armed iNK-CD64/16A cells can be repurposed with additional antibodies to target new tumor antigens, and (iv) cryopreserved, armed iNK-CD64/16A are capable of sustained ADCC in a tumor xenograft model under saturating levels of human IgG. Discussion: iNK-CD64/16A cells allow for a flexible use of antibodies (antibody arming and antibody targeting), and an "off-the-shelf" platform for multi-antigen recognition to overcome limitations of adoptive cell therapies expressing fixed antigen receptors leading to cancer relapse due to antigen escape variants.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos de Neoplasias , Células Madre Pluripotentes Inducidas , Células Asesinas Naturales , Linfoma , Receptores de IgG , Ensayos Antitumor por Modelo de Xenoinjerto , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Receptores de IgG/genética , Humanos , Animales , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones , Linfoma/terapia , Linfoma/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/inmunología , Antígenos de Neoplasias/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Línea Celular Tumoral , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Ratones SCIDRESUMEN
Synergistic factors can enhance the toxicity of Bt toxins and delay the development of Bt resistance. Previous research has demonstrated that a Helicoverpa armigera cadherin fragment (HaCad-TBR) increased the toxicity of Cry1Ac in Plutella xylostella larvae but did not have a synergistic effect on Cry1B, Cry1C, and Cry1F toxins. In this study, a fusion protein (HaCad-TBR-2D3 VL) derived from HaCad-TBR and a Bt Cry1-specific antibody peptide was expressed in Escherichia coli. The HaCad-TBR-2D3 VL enhanced Cry1Ac toxicity more efficiently in insects and Sf9 cells than HaCad-TBR and also significantly increased the toxicity of Cry1B, Cry1C, and Cry1F toxins in insects. Further investigation indicated that the improved stability in insect midguts and higher binding capacity with Bt toxins contributed to the enhanced synergism of HaCad-TBR-2D3 VL over HaCad-TBR. This study suggested that Bt antibody fragments can potentially broaden the synergistic range of Bt receptor fragments, providing a theoretical foundation for developing broad-spectrum synergists for other biopesticides.
Asunto(s)
Toxinas de Bacillus thuringiensis , Proteínas Bacterianas , Cadherinas , Endotoxinas , Proteínas Hemolisinas , Proteínas de Insectos , Larva , Mariposas Nocturnas , Proteínas Recombinantes de Fusión , Animales , Cadherinas/genética , Cadherinas/metabolismo , Cadherinas/inmunología , Cadherinas/química , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacología , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/genética , Endotoxinas/inmunología , Endotoxinas/química , Endotoxinas/farmacología , Endotoxinas/metabolismo , Endotoxinas/genética , Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/farmacología , Mariposas Nocturnas/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Insectos/inmunología , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/crecimiento & desarrollo , Péptidos/química , Péptidos/inmunología , Péptidos/farmacología , Anticuerpos/inmunología , Anticuerpos/química , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Insecticidas/química , Insecticidas/farmacología , Control Biológico de VectoresRESUMEN
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent pandemic have led to devastating public health and economic losses. The development of highly sensitive, rapid and inexpensive methods to detect and monitor coronaviruses is essential for family diagnosis, preventing infections, choosing treatments and programs and laying the technical groundwork for viral diagnosis. This study established one-step immunoassays for rapid and sensitive detection of SARS-CoV-2 by using a single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) or NanoLuc (NLuc) luciferase. First, a high-affinity scFv antibody specific to the SARS-CoV-2 nucleocapsid (N) protein was screened from hybridoma cells-derived and phage-displayed library. Next, prokaryotic expression of the scFv-AP and scFv-NLuc fusion proteins were induced, leading to excellent antibody binding properties and enzyme catalytic activities. The scFv-AP fusion had a detection limit of 3 pmol per assay and was used to produce eye-readable biosensor readouts. Moreover, the scFv-NLuc protein was applied in a highly sensitive luminescence immunoassay, achieving a detection limit lower than 0.1 pmol per assay. Therefore, the scFv-AP and scFv-NLuc fusion proteins can be applied for the rapid and simple diagnosis of SARS-CoV-2 to safeguard human health and provide guidance for the detection of other pathogenic viruses.
Asunto(s)
Fosfatasa Alcalina , COVID-19 , Luciferasas , SARS-CoV-2 , Anticuerpos de Cadena Única , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/química , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Fosfatasa Alcalina/química , Humanos , Luciferasas/química , Luciferasas/genética , COVID-19/diagnóstico , COVID-19/virología , Límite de Detección , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Biblioteca de Péptidos , Técnicas Biosensibles/métodos , Inmunoensayo/métodosRESUMEN
VP1, a major immunogenic protein of foot-and-mouth disease virus (FMDV), facilitates viral attachment and entry into host cells. VP1 possesses critical epitope sequences responsible for inducing neutralizing antibodies but its expression using Saccharomyces cerevisiae has been hampered despite evidence that the presence of VP1 does not negatively impact the yeast's biology. In this study, we fused proteins to enhance VP1 expression using S. cerevisiae. Among short P1 chimeras containing VP1 including VP3-VP1 and VP2-VP1, VP3-VP1 fusion proteins showed higher expression levels than VP2-VP1. We subsequently designed new fusion proteins, of which 20 amino acids of N-terminal VP3 fused with VP1-Co1 (referred to 20aaVP3-VP1-Co1) showed the highest expression level. Lowering the culture temperature from 30 °C to 20 °C further enhanced fusion protein production. The highest expression level of 20aaVP3-VP1-Co1 was estimated to be 7.7â¯mg/L, which is comparable to other heterologous proteins produced using our S. cerevisiae expression system. Oral administration of the cell expressing 20aaVP3-VP1-Co1 induced VP1-specific IgG and IgA responses in mice. The S. cerevisiae-expressed 20aaVP3-VP1-Co1 fusion protein induced a significant immune response to the FMDV structural epitope protein, which opens the possibility of an oral FMDV vaccine.
Asunto(s)
Anticuerpos Antivirales , Proteínas de la Cápside , Virus de la Fiebre Aftosa , Fiebre Aftosa , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae , Vacunas Virales , Animales , Virus de la Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Ratones , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Anticuerpos Antivirales/sangre , Fiebre Aftosa/prevención & control , Fiebre Aftosa/inmunología , Administración Oral , Inmunización , Femenino , Codón , Anticuerpos Neutralizantes/inmunología , Ratones Endogámicos BALB C , Inmunoglobulina ARESUMEN
Introduction: The clinical success of mRNA vaccine during the COVID-19 pandemic has inspired emerging approaches to elevate mRNA vaccine immunogenicity. Among them, antigen fusion protein designs for improved immune cell targeting have been shown to augment humoral immunity against small antigen targets. Methods: This research demonstrates that SARS-CoV-2 receptor binding domain (RBD) fusion with a minimalistic peptide segment of complement component 3b (C3b, residues 727-767) ligand can improve mRNA vaccine immunogenicity through antigen targeting to complement receptor 1 (CR1). We affirm vaccines' antigenicity and targeting ability towards specific receptors through Western blot and immunofluorescence assay. Furthermore, mice immunization studies help the investigation of the antibody responses. Results: Using SARS-CoV-2 Omicron RBD antigen, we compare mRNA vaccine formulations expressing RBD fusion protein with mouse C3b peptide (RBD-mC3), RBD fusion protein with mouse Fc (RBD-Fc), and wild-type RBD. Our results confirm the proper antigenicity and normal functionality of RBD-mC3. Upon validating comparable antigen expression by the different vaccine formulations, receptor-targeting capability of the fusion antigens is further confirmed. In mouse immunization studies, we show that while both RBD-mC3 and RBD-Fc elevate vaccine immunogenicity, RBD-mC3 leads to more sustained RBD-specific titers over the RBD-Fc design, presumably due to reduced antigenic diversion by the minimalistic targeting ligand. Conclusion: The study demonstrates a novel C3b-based antigen design strategy for immune cell targeting and mRNA vaccine enhancement.
Asunto(s)
Vacunas contra la COVID-19 , SARS-CoV-2 , Animales , Ratones , SARS-CoV-2/inmunología , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/farmacología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/química , Inmunogenicidad Vacunal , COVID-19/prevención & control , COVID-19/inmunología , Vacunas de ARNm , Femenino , Ratones Endogámicos BALB C , Humanos , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Novel engineered IL-2 agonists strive to increase the therapeutic window of aldesleukin (human IL-2) by increasing selectivity toward effector over regulatory T cells and reducing dose-limiting toxicities. Here we describe ANV419, an IL-2/anti-IL2 antibody fusion protein designed for selective IL-2 receptor ßγ (IL-2 Rßγ) activation by sterically hindering IL-2 from binding to IL-2 Rα. The fusion protein has an IL-2 connected to the light chain complementarity-determining region (CDR) domain of a humanized antibody that binds to IL-2 at the same epitope as IL-2 Rα. Optimization of the selectivity and pharmacological properties led to the selection of ANV419. ANV419 preferentially expands CD8+ T cells and natural killer (NK) cells over Tregs and can be safely administered at doses that elicit strong pharmacodynamic effects and efficacy in mouse tumor models. Its anti-tumor efficacy was enhanced when combined with programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) checkpoint inhibitors. ANV419 also enhances the NK cell killing capacity and increases tumor growth inhibition when used alongside trastuzumab in a Her-2+ xenograft mouse model. In cynomolgus monkeys, the estimated half-life of ANV419 is 24 h, and doses that induced sustained expansion of effector cells were well tolerated without the severe toxicities typically observed with high-dose IL-2. These data support the clinical development of ANV419 in solid tumors and hematological malignancies as monotherapy and in combination with checkpoint inhibitors or agents that induce antibody-dependent cellular cytotoxicity. ANV419 is currently in Phase 1/2 clinical development and may provide cancer patients with a wider therapeutic window than aldesleukin.
Asunto(s)
Linfocitos T CD8-positivos , Interleucina-2 , Células Asesinas Naturales , Proteínas Recombinantes de Fusión , Animales , Células Asesinas Naturales/inmunología , Humanos , Interleucina-2/inmunología , Linfocitos T CD8-positivos/inmunología , Ratones , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Inmunoterapia/métodos , Macaca fascicularis , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Neoplasias/inmunología , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , FemeninoRESUMEN
This study presents the design and fabrication of an innovative vaccine candidate targeting Pseudomonas aeruginosa (P. aeruginosa). The vaccine consists of gelatin nanoparticles (GNPs) encapsulating a chimeric protein (CP) derived from the ExoS and OprI proteins from P. aeruginosa. The physicochemical properties of the GNPs were assessed using dynamic light scattering (DLS) and electron microscopy. The toxicity, encapsulation efficacy, release profile, and effectiveness of CP-encapsulated GNPs (CP-GNPs) in an animal model were investigated. The resulting nanovaccine demonstrated uniform spherical particles with an average size of 135 nm and an encapsulation efficiency of 85 %. The release assay revealed that 23 % of the antigen was released from the CP-GNPs after 20 days. The GNPs did not exhibit any toxic effects on L929 cells in vitro. The formulation induced both systemic and mucosal antibody responses. Additionally, CP-GNPs stimulated cytokine responses, including IFN-γ, IL-4, and IL-17, indicating the induction of both humoral (Th2) and cellular (Th1) responses. The CP-encapsulated GNPs formulation effectively protected the mice lungs against experimental respiratory tract infection, reducing colony count and inflammation. These findings suggest that CP-GNPs hold promise as a potential strategy for preventing respiratory tract infections caused by P. aeruginosa. Further research is needed to explore its clinical application.
Asunto(s)
Gelatina , Nanopartículas , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Infecciones del Sistema Respiratorio , Animales , Gelatina/química , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/efectos de los fármacos , Nanopartículas/química , Infecciones por Pseudomonas/prevención & control , Infecciones por Pseudomonas/inmunología , Ratones , Infecciones del Sistema Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/inmunología , Citocinas/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/química , Femenino , Adyuvantes Inmunológicos/farmacología , Ratones Endogámicos BALB C , Línea CelularRESUMEN
Fasciolosis, caused by the liver fluke Fasciola gigantica, is a major parasitic disease that affects livestock and therefore causes significant economic losses in tropical countries. Although anthelminthic drugs can kill the parasite, drug-resistant liver fluke populations are increasing. In this study, a recombinant F. gigantica chimeric protein (rFgCHI) consisting of cathepsin L1H (FgCL1H), cathepsin B3 (FgCB3), and Saposin-like protein 1 (FgSAP1) was designed and expressed in Escherichia coli (BL21). The molecular weight of rFgCHI was 61â¯kDa. To study the antibody response, male BALB/c mice were immunized via the subcutaneous injection of rFgCHI combined with Quil A. Immunization with rFgCHI showed the induction of IgG1 and IgG2a with a higher IgG1 isotype level, indicating the potential of mixed Th1/Th2 immune responses, with Th2 predominating. However, the results showed high levels of IgG against the single proteins, except for rFgSAP1. Through Western blotting, mouse anti-rFgCHI polyclonal antibodies could be detected to the native proteins obtained from the parasite at all stages. Immunolocalization also revealed that the anti-rFgCHI antibodies could detect targeted antigens in the cecal epithelium of the parasite. These results demonstrated that rFgCHI is immunogenic to the mouse immune system and may potentially be a protein candidate for the development of a fasciolosis vaccine.
Asunto(s)
Anticuerpos Antihelmínticos , Fasciola , Proteínas del Helminto , Ratones Endogámicos BALB C , Animales , Fasciola/inmunología , Fasciola/genética , Ratones , Anticuerpos Antihelmínticos/sangre , Masculino , Proteínas del Helminto/inmunología , Proteínas del Helminto/genética , Fascioliasis/veterinaria , Fascioliasis/prevención & control , Fascioliasis/inmunología , Inmunoglobulina G/sangre , Antígenos Helmínticos/inmunología , Antígenos Helmínticos/genética , Inmunización/veterinaria , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Formación de AnticuerposRESUMEN
AIMS: Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in diagnosis and vaccines development. METHODS AND RESULTS: In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35 kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P < 0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P < 0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera. CONCLUSIONS: These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control.
Asunto(s)
Antígenos Bacterianos , Leptospirosis , Proteínas Recombinantes de Fusión , Leptospirosis/inmunología , Leptospirosis/prevención & control , Animales , Ratones , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Humanos , Anticuerpos Antibacterianos/sangre , Leptospira/inmunología , Leptospira/genética , Biología Computacional , Epítopos/inmunología , Epítopos/genética , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/genética , Escherichia coli/genética , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/genéticaRESUMEN
The available prophylactic vaccines for human papillomavirus (HPV) in the market are only effective against specific types of HPV, rendering them ineffective for other types of HPV infections. The objective of this research is to investigate the stability of the recombinant protein constructed, namely chimeric L1/L2 protein from HPV type 52, with improved cross-neutralization ability. The 3D model, predicted using Alphafold, Robetta, I-Tasser, and refined with Galaxy Refinement, is validated using Ramachandran plot analysis. The stability is verified through molecular dynamics simulations, considering parameters such as RMSD, RMSF, Rg, and SASA, where stable conditions are observed. The chimeric L1/L2 protein from HPV type 52 is purified using affinity chromatography, and the His-tag is cleaved using SUMO protease to obtain pure chimeric protein with the size of ~ 55 kDa. Western blot analysis confirms binding to anti-L1 HPV type 52 polyclonal antibody. The obtained vaccine candidate can be utilized as an effective prophylactic vaccine against HPV.
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Escherichia coli , Simulación de Dinámica Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/aislamiento & purificación , Proteínas de la Cápside/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Oncogénicas Virales/inmunología , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/aislamiento & purificación , Virus del Papiloma Humano , AlphapapillomavirusRESUMEN
INTRODUCTION: Shigellosis is a gastrointestinal disease causes high morbidity and mortality worldwide, however, there is no anti-Shigella vaccine. The use of antibiotics in shigellosis treatment exacerbates antibiotic resistance. Antibodies, particularly egg yolk antibody (IgY), offer a promising approach to address this challenge. This study aimed to investigate the prophylactic effect of IgY produced against a recombinant chimeric protein containing the immunogens IpaD, IpaB, StxB, and VirG from Shigella. METHODS: The chimeric protein, comprising IpaD, IpaB, StxB, and VirG, was expressed in E. coli BL21 and purified using the Ni-NTA column. Following immunization of chickens, IgY was extracted from egg yolk using the PEG-6000 method and analyzed through SDS-PAGE and ELISA techniques. Subsequently, the prophylactic efficacy of IgY was assessed by challenging of mice with 10 LD50 of S. dysenteriae and administering different concentrations of IgY (1.25, 2.5, 5, and 10â¯mg/kg) under various time conditions. RESULTS: The recombinant protein, weighing 82â¯kDa, was purified and confirmed by western blotting. The IgY concentration was determined as 9.5â¯mg/ml of egg yolk and the purity of the extracted IgY was over 90â¯%. The results of the ELISA showed that at least 19â¯ng of pure antibody identified recombinant protein and reacts with it. The challenge test employing IgY and Shigella demonstrated a direct correlation between the survival rate and antibody concentration, with increased concentrations leading to decreased mortality rates. Treatment of mice with 10â¯mg/kg IgY leads to 80â¯% survival of the mice against 10 LD50 S. dysenteriae. CONCLUSION: Our findings suggest that IgY may offer therapeutic potential in treating Shigella infections and combating antibiotic resistance.
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Pollos , Disentería Bacilar , Yema de Huevo , Inmunoglobulinas , Animales , Inmunoglobulinas/inmunología , Ratones , Yema de Huevo/inmunología , Disentería Bacilar/prevención & control , Disentería Bacilar/inmunología , Shigella/inmunología , Proteínas Bacterianas/inmunología , Proteínas Recombinantes/inmunología , Femenino , Anticuerpos Antibacterianos/inmunología , Ratones Endogámicos BALB C , Antígenos Bacterianos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
The hybridoma method for production of monoclonal antibodies has been a cornerstone of biomedical research for several decades. Here we convert the monoclonal antibody sequence from mouse-derived hybridomas into a "devilized" recombinant antibody with devil IgG heavy chain and IgK light chain. The chimeric recombinant antibody can be used in functional assays, immunotherapy, and to improve understanding of antibodies and Fc receptors in Tasmanian devils. The process can be readily modified for other species.
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Hibridomas , Inmunoglobulina G , Marsupiales , Animales , Ratones , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Hibridomas/inmunología , Marsupiales/inmunología , Marsupiales/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
This study aims to develop an effective bivalent subunit vaccine that is promising to prevent both porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV). The receptor-binding domains (RBDs) of PDCoV and PEDV were fused and cloned into the eukaryotic expression vector pCDNA3.1(+). The fusion protein PDCoV-RBD-PEDV-RBD (pdRBD-peRBD) was expressed by the ExpiCHOTM expression system and purified. Mice were immunized with the fusion protein at three different doses (10, 20, and 30 µg). The humoral immune response and cellular immune response induced by the fusion protein were evaluated by ELISA and flow cytometry. The neutralization titers of the serum of immunized mice against PDCoV and PEDV were determined by the microneutralization test. The results showed that high levels of IgG antibodies were induced in the three different dose groups after booster immunization, and there was no significant difference in the antibody level between different dose groups, indicating that the immunization dose of 10 µg could achieve the fine immune effect. The results of flow cytometry showed that the immunization groups demonstrated increased proportion of CD3+CD4+ T cells and decreased proportion of CD3+CD8+ T cells, which was consistent with the expectation about the humoral immune response induced by the subunit vaccine. At the same time, the levels of interleukin (IL)-2, IL-4, and interferon (IFN)-γ in the serum were determined. The results showed that the fusion protein induced both humoral immune effect and cellular immune response. The results of the neutralization test showed that the antibody induced by 10 µg fusion protein neutralized both PDCoV and PEDV in vitro, with the titers of 1:179.25 and 1:141.21, respectively. The above results suggested that the pdRBD-peRBD could induce a high level of humoral immune response at a dose of 10 µg, and the induced antibody could neutralize both PDCoV and PEDV. Therefore, the fusion protein pdRBD-peRBD is expected to be an effective subunit vaccine that can simultaneously prevent PDCoV and PEDV.
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Anticuerpos Antivirales , Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Proteínas Recombinantes de Fusión , Vacunas Virales , Animales , Virus de la Diarrea Epidémica Porcina/inmunología , Virus de la Diarrea Epidémica Porcina/genética , Ratones , Porcinos , Vacunas Virales/inmunología , Vacunas Virales/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Deltacoronavirus/inmunología , Deltacoronavirus/genética , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/genética , Ratones Endogámicos BALB C , Femenino , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Dominios Proteicos , Inmunogenicidad Vacunal , Inmunidad HumoralRESUMEN
This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo. The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD600=0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 â. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs (P=0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.
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Membrana Externa Bacteriana , Carcinoma Hepatocelular , Glipicanos , Neoplasias Hepáticas , Anticuerpos de Cadena Única , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/química , Animales , Ratones , Humanos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/inmunología , Células Hep G2 , Glipicanos/inmunología , Glipicanos/metabolismo , Glipicanos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Sistemas de Liberación de Medicamentos , Ratones DesnudosRESUMEN
BACKGROUND: The sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis. METHODOLOGY/PRINCIPAL FINDINGS: The coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest antigenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5α cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively. CONCLUSIONS/SIGNIFICANCE: The preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required.
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Antígenos Helmínticos , Ensayo de Inmunoadsorción Enzimática , Sensibilidad y Especificidad , Pruebas Serológicas , Strongyloides stercoralis , Estrongiloidiasis , Humanos , Estrongiloidiasis/diagnóstico , Estrongiloidiasis/inmunología , Animales , Pruebas Serológicas/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Strongyloides stercoralis/inmunología , Strongyloides stercoralis/genética , Antígenos Helmínticos/genética , Antígenos Helmínticos/inmunología , Anticuerpos Antihelmínticos/sangre , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Expresión GénicaRESUMEN
Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. KEY POINTS: ⢠Several T- and B-cell epitopes were identified in all the three proteins. ⢠Four different adjuvants were used in vaccine formulations. ⢠Immunization stimulated significant levels of IgG2/3 in vaccinated animals.
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Anticuerpos Antibacterianos , Vacunas Bacterianas , Leptospirosis , Lipoproteínas , Animales , Leptospirosis/prevención & control , Leptospirosis/inmunología , Lipoproteínas/inmunología , Lipoproteínas/genética , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/genética , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Cricetinae , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Adyuvantes Inmunológicos/administración & dosificación , Inmunoglobulina G/sangre , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/genética , Leptospira interrogans/inmunología , Leptospira interrogans/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Vacunación , Inmunidad Humoral , Leptospira/inmunología , Leptospira/genética , Inmunogenicidad VacunalRESUMEN
In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.