RESUMO
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.
Assuntos
Adjuvantes Imunológicos , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Epitopos de Linfócito T , Memória Imunológica , Vacinas contra Leishmaniose , Leishmaniose Cutânea , Animais , Leishmaniose Cutânea/prevenção & controle , Leishmaniose Cutânea/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Camundongos , Vacinas contra Leishmaniose/imunologia , Feminino , Adjuvantes Imunológicos/administração & dosagem , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/genética , Leishmania braziliensis/imunologia , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Anticorpos Antiprotozoários/imunologia , Citocinas/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Antígenos de Protozoários/imunologiaRESUMO
Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.
Assuntos
Toxinas Botulínicas Tipo A , Exocitose , Animais , Humanos , Ratos , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/isolamento & purificação , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Masculino , Sinaptossomos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/genética , Toxinas Botulínicas/química , Toxinas Botulínicas/isolamento & purificaçãoRESUMO
To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus.
Assuntos
Produtos do Gene gag , Vírus da Imunodeficiência Felina , Vírus da Imunodeficiência Símia , Montagem de Vírus , Dedos de Zinco , Animais , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Produtos do Gene gag/química , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Gatos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Linhagem Celular , Nucleocapsídeo/metabolismo , Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , FenótipoRESUMO
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.
Assuntos
Antígenos de Bactérias , Leptospirose , Proteínas Recombinantes de Fusão , Leptospirose/imunologia , Leptospirose/prevenção & controle , Animais , Camundongos , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/genética , Humanos , Anticorpos Antibacterianos/sangue , Leptospira/imunologia , Leptospira/genética , Biologia Computacional , Epitopos/imunologia , Epitopos/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/genética , Escherichia coli/genética , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genéticaRESUMO
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.
Assuntos
Anticorpos Antibacterianos , Vacinas Bacterianas , Leptospirose , Lipoproteínas , Animais , Leptospirose/prevenção & controle , Leptospirose/imunologia , Lipoproteínas/imunologia , Lipoproteínas/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/genética , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Cricetinae , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/genética , Adjuvantes Imunológicos/administração & dosagem , Imunoglobulina G/sangue , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/genética , Leptospira interrogans/imunologia , Leptospira interrogans/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Vacinação , Imunidade Humoral , Leptospira/imunologia , Leptospira/genética , Imunogenicidade da VacinaRESUMO
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.
Assuntos
Anticorpos Antiprotozoários , Antígenos de Protozoários , Vacinas Antimaláricas , Malária Vivax , Camundongos Endogâmicos BALB C , Plasmodium vivax , Proteínas de Protozoários , Animais , Plasmodium vivax/imunologia , Plasmodium vivax/genética , Camundongos , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/genética , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Anticorpos Antiprotozoários/imunologia , Vacinas Antimaláricas/imunologia , Feminino , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/genética , Modelos Animais de Doenças , Adjuvantes Imunológicos , Imunogenicidade da Vacina , Antígenos de SuperfícieRESUMO
Yellow fever (YF) is a disease caused by the homonymous flavivirus that can be prevented by a vaccine containing attenuated viruses. Since some individuals cannot receive this vaccine, the development of alternatives is desirable. Here, we developed a recombinant baculovirus (rBV) surface display platform utilizing a chimeric E-NS1 protein as a vaccine candidate. A pBacPAK9 vector containing the baculoviral GP64 signal peptide, the YFV prM, E, NS1 and the ectodomain of VSV-G sequences was synthesized. This transfer plasmid and the bAcGOZA bacmid were cotransfected into Sf9 cells, and an rBV-E-NS1 was obtained, which was characterized by PCR, WB, IFI and FACS analysis. Mice immunized with rBV-E-NS1 elicited a specific humoral and cellular immune response and were protected after YFV infection. In summary, we have developed an rBV that expresses YFV major antigen proteins on its surface, which opens new alternatives that can be tested in a mouse model.
Assuntos
Anticorpos Antivirais , Baculoviridae , Proteínas não Estruturais Virais , Febre Amarela , Vírus da Febre Amarela , Animais , Baculoviridae/genética , Baculoviridae/imunologia , Camundongos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Vírus da Febre Amarela/imunologia , Vírus da Febre Amarela/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/genética , Febre Amarela/prevenção & controle , Febre Amarela/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Células Sf9 , Vacinas Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Feminino , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/genética , Imunidade Celular , Camundongos Endogâmicos BALB C , Imunidade Humoral , Vetores Genéticos/genéticaRESUMO
The occlusion bodies of Autographa californica multiple nucleopolyhedrovirus are proteinaceous formations with significant biotechnological potential owing to their capacity to integrate foreign proteins through fusion with polyhedrin, their primary component. However, the strategy for successful heterologous protein inclusion still requires further refinement. In this study, we conducted a comparative assessment of various conditions to achieve the embedding of recombinant proteins within polyhedra. Two baculoviruses were constructed: AcPHGFP (polh+), with GFP as a fusion to wild type (wt) polyhedrin and AcΔPHGFP (polh+), with GFP fused to a fragment corresponding to amino acids 19 to 110 of polyhedrin. These baculoviruses were evaluated by infecting Sf9 cells and stably transformed Sf9, Sf9POLH, and Sf9POLHE44G cells. The stably transformed cells contributed another copy of wt or a mutant polyhedrin, respectively. Polyhedra of each type were isolated and characterized by classical methods. The fusion PHGFP showed more-efficient incorporation into polyhedra than ΔPHGFP in the three cell lines assayed. However, ΔPHGFP polyhedron yields were higher than those of PHGFP in Sf9 and Sf9POLH cells. Based on an integral analysis of the studied parameters, it can be concluded that, except for the AcΔPHGFP/Sf9POLHE44G combination, deficiencies in one factor can be offset by improved performance by another. The combinations AcPHGFP/Sf9POLHE44G and AcΔPHGFP/Sf9POLH stand out due to their high level of incorporation and the large number of recombinant polyhedra produced, respectively. Consequently, the choice between these approaches becomes dependent on the intended application.
Assuntos
Biotecnologia , Nucleopoliedrovírus , Spodoptera , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/metabolismo , Animais , Células Sf9 , Biotecnologia/métodos , Spodoptera/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Matriz de Corpos de Inclusão , Corpos de Oclusão Virais/metabolismo , Corpos de Oclusão Virais/genética , Linhagem Celular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Mycoplasma hyopneumoniae (M. hyopneumoniae) is a primary agent of porcine enzootic pneumonia, a disease that causes significant economic losses to pig farming worldwide. Commercial vaccines induce partial protection, evidencing the need for a new vaccine against M. hyopneumoniae. In our work, three chimeric proteins were constructed, composed of potentially immunogenic domains from M. hyopneumoniae proteins. We designed three chimeric proteins (Q1, Q2, and Q3) based on bioinformatics analysis that identified five potential proteins with immunogenic potential (MHP418, MHP372, MHP199, P97, and MHP0461). The chimeric proteins were inoculated in the murine model to evaluate the immune response. The mice vaccinated with the chimeras presented IgG and IgG1 against proteins of M. hyopneumoniae. There was induction of IgG in mice immunized with Q3 starting from 30 days post-vaccination, and groups Q1 and Q2 showed induction at 45 days. Mice of the group immunized with Q3 showed the production of IgA. In addition, the mice inoculated with chimeric proteins showed a proinflammatory cytokine response; Q1 demonstrated higher levels of TNF, IL-6, IL2, and IL-17. In contrast, animals immunized with Q2 showed an increase in the concentrations of TNF, IL-6, and IL-4, whereas those immunized with Q3 exhibited an increase in the concentrations of TNF, IL-6, IL-10, and IL-4. The results of the present study indicate that these three chimeric proteins can be used in future vaccine trials with swine because of the promising antigenicity.
Assuntos
Mycoplasma hyopneumoniae , Animais , Suínos , Camundongos , Mycoplasma hyopneumoniae/genética , Interleucina-4 , Interleucina-6 , Vacinas Bacterianas/genética , Imunoglobulina G , Proteínas Recombinantes de Fusão/genéticaRESUMO
In the last 20 years, various research groups have endeavored to develop recombinant vaccines against leptospirosis to overcome the limitations of commercially available bacterins. Numerous antigens and vaccine formulations have been tested thus far. However, the analysis of cellular response in these vaccine formulations is not commonly conducted, primarily due to the scarcity of supplies and kits for the hamster animal model. Our research group has already tested the Q1 antigen, a chimeric protein combining the immunogenic regions of LipL32, LemA, and LigANI, in recombinant subunit and BCG-vectored vaccines. In both strategies, 100 % of the hamsters were protected against clinical signs of leptospirosis. However, only the recombinant BCG-vectored vaccine provided protection against renal colonization. Thus, the objective of this study is to characterize the cellular immune response in hamsters immunized with different vaccine formulations based on the Q1 antigen through transcriptional analysis of cytokines. The hamsters were allocated into groups and vaccinated as follows: recombinant subunit (rQ1), recombinant BCG (rBCG:Q1), and saline and BCG Pasteur control vaccines. To assess the cellular response induced by the vaccines, we cultured and stimulated splenocytes, followed by RNA extraction from the cells and analysis of cytokines using real-time PCR. The results revealed that the recombinant subunit vaccine elicited a Th2-type response, characterized by the expression of cytokines IL-10, IL-1α, and TNF-α. This pattern closely resembles the cytokines expressed in severe cases of leptospirosis. On the other hand, the rBCG-vectored vaccine induced a Th1-type response with significant up-regulation of IFN-γ. These findings suggest the involvement of the cellular response and the IFN-γ mediated inflammatory response in the sterilizing immunity mediated by rBCG. Therefore, this study may assist future investigations in characterizing the cellular response in hamsters, aiming to elucidate the mechanisms of efficacy and establish potential correlates of protection.