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1.
APMIS ; 128(11): 593-602, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32870528

RESUMO

Induction of broad Th1 cellular immune responses and cytokines is crucial characteristics for vaccines against intracellular infections such as hepatitis C virus (HCV). Plants (especially oilseed tissues) and plant-immunomodulators (like oil bodies) offer cost-effective and scalable possibilities for the production of immunologically relevant and safe vaccine antigens and adjuvants, respectively. Herein, we provide data of the murine immunization by transgenic canola oilseed-derived HCV core protein (HCVcp) soluble extract (TSE) and Escherichia coli- derived rHCVcp in combination with Canola oil bodies (oil) compared to that of the Freund's (FA) adjuvant. Mice immunized by TSE+ oil developed both strong humeral (IgG) and Th1-biased cellular responses, manifested by high levels of IFN-γ and lower IgG1/IgG2a ratio and IL-4 secretion. Results of the intracellular cytokine staining indicated that TSE+ oil immunization in mice triggered both CD4+ and CD8+ T cells to release IFN-γ, while CD4+ cells were mostly triggered when FA was used. Analyses by qRT-PCR indicated that a combination of rHCVcp/TSE with oil body induced high levels of IL-10 cytokines compared to that of the FA adjuvant. These characteristics are important properties for the design of an HCV vaccine candidate and indicate the potential of Canola-derived antigen and oil bodies in addressing these concerns.


Assuntos
Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/prevenção & controle , Proteínas Recombinantes/administração & dosagem , Células Th1/efeitos dos fármacos , Proteínas do Core Viral/administração & dosagem , Vacinas contra Hepatite Viral/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Hepacivirus/imunologia , Hepacivirus/patogenicidade , Hepatite C Crônica/imunologia , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Imunidade Celular/efeitos dos fármacos , Imunoglobulina G/biossíntese , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-10/biossíntese , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Óleo de Brassica napus/administração & dosagem , Óleo de Brassica napus/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Células Th1/virologia , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/imunologia , Vacinas contra Hepatite Viral/biossíntese
2.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(4): 355-360, 2020 Aug 24.
Artigo em Chinês | MEDLINE | ID: mdl-32935508

RESUMO

OBJECTIVE: To investigate the biological properties of Schistosoma japonicum SjGrpE protein, and to express and purify the recombinant SjGrpE protein and test its immunogenicity. METHODS: The amino acid composition, molecular weight, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, localization, phosphorylation site, ubiquitination site, glycosylation site, secondary and tertiary structures and B cell epitopes of the SjGrpE protein were predicted using bioinformatics analyses. The SjGrpE gene was amplified using PCR assay using S. japonicum cDNA as a template, double enzyme-digested and linked to the pET28a vector to yield the recombinant plasmid pET28a-SjGrpE. The recombinant plasmid pET28a-SjGrpE was transformed into Escherichia coli BL21, and then IPTG was employed to induce the expression of the target protein, which was purified by nickel ion affinity chromatography. After mice were immunized with the recombinant SjGrpE protein, mouse sera were collected, and the polyclonal antibody against the SjGrpE protein was characterized. RESULTS: SjGrpE protein, which was identified as a hydrophilic protein, was predicted to have a molecular weight of approximately 24.3 kDa without transmembrane regions or signal peptides, and locate in the mitochondrion. SjGrpE protein contained 18 phosphorylation sites and 2 ubiquitination sites, but had no glycosylation sites. In addition, SjGrpE protein contained 5 B-cell epitopes. The full length of SjGrpE gene was approximately 660 bp. The recombinant pET28a-SjGrpE plasmid was successfully generated, and the recombinant SjGrpE protein was obtained following the affinity chromatography, which stimulated mice to secrete high-titer antibodies. CONCLUSIONS: The recombinant SjGrpE protein has been successfully prepared and this recombinant protein has a high immunogenicity, which provides a basis for evaluating its value as a vaccine candidate for S. japonicum infections.


Assuntos
Proteínas de Helminto , Proteínas Recombinantes , Schistosoma japonicum , Animais , Anticorpos Anti-Helmínticos/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/isolamento & purificação , Camundongos , Plasmídeos/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Schistosoma japonicum/genética , Schistosoma japonicum/metabolismo
3.
PLoS Negl Trop Dis ; 14(9): e0008518, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32915790

RESUMO

To eliminate schistosomiasis, appropriate diagnostic tests are required to monitor its prevalence and transmission, especially in the settings with low endemicity resulting from the consecutive mass drug administration. Antibodies that react with either crude soluble schistosome egg antigens or soluble worm antigen preparations have been used to monitor infection in low-prevalence regions. However, these detection methods cannot discriminate current and past infections and are cross-reactive with other parasites because both antigens contain numerous proteins and glycans from schistosomes, and standard preparations need maintenance of the life cycle of the schistosome. To evaluate the potential utility of nine recombinant Schistosoma mansoni proteins as single defined antigens for serological diagnosis, we monitored the kinetics of antibodies to each antigen during S. mansoni infection in mice before and after the treatment with praziquantel. C57BL/6 mice were infected with 50 cercariae. The levels of immunoglobulin G (IgG) raised against five recombinant antigens (RP26, sm31, sm32, GST, and LAP1) significantly increased as early as 2-4 weeks after infection and rapidly declined by 2 weeks after the treatment, whereas those raised against crude S. mansoni egg antigens or other antigens remained elevated long after the treatment. The IgG1 raised against RP26, sm31, and serpin decreased after the treatment with praziquantel, whereas the IgE raised against serpin declined strikingly after the treatment. This study clarifies the dynamics of the serological responses to recombinant S. mansoni proteins during infection and after the treatment with praziquantel and identifies several candidate antigens with potential utility in the monitoring and surveillance of schistosomiasis toward the elimination of schistosomiasis.


Assuntos
Praziquantel/farmacologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Animais , Anti-Helmínticos/farmacologia , Anticorpos Anti-Helmínticos , Antígenos de Helmintos/imunologia , Feminino , Proteínas de Helminto/imunologia , Imunoglobulina G , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/imunologia , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose mansoni/diagnóstico , Esquistossomose mansoni/tratamento farmacológico , Serpinas
4.
Rev Bras Parasitol Vet ; 29(3): e005820, 2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32756774

RESUMO

Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.


Assuntos
Proteínas de Bactérias , Doenças do Cão , Ehrlichia canis , Ehrlichiose/veterinária , Proteínas Recombinantes , Testes Sorológicos/veterinária , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Brasil , Linhagem Celular , Doenças do Cão/diagnóstico , Cães , Ehrlichia canis/genética , Ehrlichiose/diagnóstico , Escherichia coli/genética , Expressão Gênica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
5.
BMC Res Notes ; 13(1): 372, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32762746

RESUMO

OBJECTIVE: COVID19 has caused a global and ongoing pandemic. The need for population seroconversion data is apparent to monitor and respond to the pandemic. Using a lateral flow assay (LFA) testing platform, the seropositivity in 63 New York Blood Center (NYBC) Convelescent Plasma (CP) donor samples were evaluated for the presence of COVID19 specific IgG and IgM. RESULTS: CP donors showed diverse antibody result. Convalescent donor plasma contains SARS-CoV-2 specific antibodies. Weak antibody bands may identify low titer CP donors. LFA tests can identify antibody positive individuals that have recovered from COVID19. Confirming suspected cases using antibody detection could help inform the patient and the community as to the relative risk to future exposure and a better understanding of disease exposure.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Doadores de Sangue , Técnicas de Laboratório Clínico/métodos , Convalescença , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Pneumonia Viral/diagnóstico , Testes Imediatos , Glicoproteína da Espícula de Coronavírus/imunologia , Especificidade de Anticorpos , Infecções por Coronavirus/terapia , Coloide de Ouro , Humanos , Imunização Passiva , Plasma , Domínios Proteicos , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soroconversão
6.
PLoS One ; 15(8): e0236704, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790777

RESUMO

The hepatitis B virus (HBV) envelope is composed of a lipid bilayer and three glycoproteins, referred to as the large (L), middle (M), and small (S) hepatitis B virus surface antigens (HBsAg). S protein constitutes the major portion of the viral envelope and an even greater proportion of subviral particles (SVP) that circulate in the blood. Recombinant S proteins are currently used as a preventive vaccine, while plasma fractions isolated from vaccinated people, referred to as hepatitis B immune globulin (HBIG), are used for short-term prophylaxis. Here, we characterized a recombinant human IgG1 type anti-S antibody named Lenvervimab regarding its binding property to a variety of cloned S antigens. Immunochemical data showed an overall consistent avidity of the antibody to S antigens of most viral genotypes distributed worldwide. Further, antibody binding was not affected by the mutations in the antigenic 'a' determinant found in many clinical variants, including the immune escape mutant G145R. In addition, mutations in the S gene sequence that confer drug resistance to the viral polymerase did not interfere with the antibody binding. These results support for a preventive use of the antibody against HBV infection.


Assuntos
Anticorpos Anti-Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Imunoglobulinas/imunologia , Sequência de Aminoácidos , Reações Antígeno-Anticorpo , Linhagem Celular , Farmacorresistência Viral , Genótipo , Células Hep G2 , Hepatite B/patologia , Hepatite B/virologia , Anticorpos Anti-Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
7.
Biomed Environ Sci ; 33(7): 493-501, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32807268

RESUMO

Objective: The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP Sc in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification. Methods: We prepared a PrP-specific polyclonal antibody (pAb P54) in a PRNP-knockout mouse model via immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays. Results: Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP C from healthy rodents and humans, and pathological PrP Sc in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP C and PrP Sc observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei. Conclusion: The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.


Assuntos
Anticorpos/imunologia , Imunização , Proteínas PrPC/imunologia , Proteínas PrPSc/imunologia , Proteínas Priônicas/imunologia , Animais , Western Blotting , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Proteínas Recombinantes/imunologia
8.
Exp Parasitol ; 216: 107944, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32619431

RESUMO

Evaluation of the murine isotype antibodies is essential in subunit vaccine development because inbred mouse strains with diverse genetic backgrounds respond different to recombinant proteins. In this regard, the main goal of this study was to measuring and comparing the profile of IgG isotype responses in C57BL/6 mice. For this purpose, the extracellular region of plasmodium vivax thrombospondin-related adhesive protein (PvTRAP) gene was expressed in Escherichia coli Rosetta (DE3)-pET23a. Then, the recombinant PvTRAP alone or emulsified with Freund's complete adjuvant were applied for immunization of the C57BL/6 mice. The role of antibodies and cellular immune responses induced by recombinant PvTRAP were evaluated. The results showed the level of anti-rPvTRAP IgG2c was significantly higher than IgG2a in the groups that received rPvTRAP alone (mean OD490 = 0.798 ± 0.12 and 0.39 ± 0.1, respectively) and emulsified with CFA/IFA (mean OD490 = 1.48 ± 0.07 and 0.605 ± 0.13, respectively; P < 0.05, independent sample t-test). Additionally, the immunized mice with rPvTRAP and rPvTRAP + CFA/IFA had an intermediate-avidity IgG2a antibody but high-avidity IgG2c antibody as well as the mean of serum antibody titers results exhibited that in both rPvTRAP and rPvTRAP + CFA/IFA mouse groups, IgG2a end-point titer (1:3200 and 1:25,600, respectively) was noteworthy lower than IgG2c (1:25,600 and 1:102,400, respectively). Moreover, the results revealed the eliciting significant levels of IFN-γ (P < 0.05, independent sample t-test) and no detectable level of IL-4 in the mouse groups received rPvTRAP alone and emulsified with CFA/IFA as compared to the mouse control groups. In general, our results showed that for correctly interpreting of Th1 immune responses in C57BL/6 mouse strain it is critical to measure IgG2c instead of IgG2a along with IFN-γ.


Assuntos
Imunoglobulina G/sangue , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Afinidade de Anticorpos , Dicroísmo Circular , Feminino , Imunofluorescência , Imunoglobulina G/classificação , Interferon gama/análise , Interleucina-4/análise , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/imunologia , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/imunologia
9.
Nat Commun ; 11(1): 3545, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669564

RESUMO

Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections.


Assuntos
Proteínas de Bactérias/imunologia , Proteção Cruzada/imunologia , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Aderência Bacteriana/imunologia , Linhagem Celular , Clonagem Molecular , Modelos Animais de Doenças , Feminino , Humanos , Imunogenicidade da Vacina , Camundongos , Testes de Neutralização , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Sorogrupo , Infecções Estreptocócicas/microbiologia , Vacinas Estreptocócicas/administração & dosagem , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia
10.
BMC Infect Dis ; 20(1): 493, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650739

RESUMO

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. Toxoplasmosis is asymptomatic in healthy individuals, whereas it may lead to death in immune suppressed or deficient patients. A vaccine against T. gondii is required to prevent consequences of the infection. The aim of this study is to generate a multivalent recombinant protein vaccine against T. gondii. METHODS: 49 previously discovered antigenic proteins of T gondii were evaluated by their expression level in E. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. Based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50 V. Humoral and cellular immune responses were determined by flow cytometry and ELISA. Vaccinated mice were challenged with T. gondii Ankara strain tachyzoites. RESULTS: In mice vaccinated with hexavalent vaccine, strong total IgG (P < 0.0001) and IgG2a (P < 0.001) responses were induced compared to controls, the ratio of CD4+ and CD8+ T lymphocytes secreting IFN-γ increased, and significantly higher extracellular IFN-γ secretion was achieved compared to the controls (P < 0.001). The survival time of the vaccinated mice increased to 8.38 ± 2.13 days which was significantly higher than controls (P < 0.01). CONCLUSIONS: Altogether, these results show that the hexavalent vaccine which is developed for the first time against T. gondii induced strong and balanced Th1 and Th2 immune responses as well as conferred significant protection against challenge with lethal toxoplasmosis in murine model.


Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas Protozoárias/farmacologia , Toxoplasmose/prevenção & controle , Vacinas de DNA/farmacologia , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Feminino , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunoglobulina G/sangue , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/genética , Vacinas Protozoárias/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Toxoplasma/patogenicidade , Toxoplasmose/imunologia , Vacinas de DNA/imunologia
11.
Euro Surveill ; 25(28)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32700671

RESUMO

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Animais , Betacoronavirus/genética , Western Blotting , Células COS , Chlorocebus aethiops , Sequência Conservada , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Genoma Viral , Camundongos , Pandemias , Peptidil Dipeptidase A/imunologia , Plasmídeos , Pneumonia Viral/genética , Proteínas Recombinantes/imunologia , Vírus da SARS/genética , Alinhamento de Sequência , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Transfecção , Células Vero , Integração Viral
12.
Arch Virol ; 165(9): 2065-2071, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32613291

RESUMO

Mink enteritis virus (MEV) is a parvovirus that causes acute enteritis in mink. The capsid protein VP2 of MEV is a major immunogenicity that is important for disease prevention. In this study, this protein was expressed in Spodoptera frugiperda 9 cells using a recombinant baculovirus system and was observed to self-assemble into virus-like particles (VLPs) with a high hemagglutination (HA) titer (1:216). A single-dose injection of VLPs (HA titer, 1:256) resulted in complete protection of mink against virulent MEV challenge for at least 180 days. These data suggest that these MEV VLPs could be used as a vaccine for the prevention of viral enteritis in mink.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Enterite Viral do Vison/prevenção & controle , Vírus da Enterite do Vison/imunologia , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas do Capsídeo/administração & dosagem , Expressão Gênica , Vison/imunologia , Vison/virologia , Enterite Viral do Vison/imunologia , Enterite Viral do Vison/virologia , Vírus da Enterite do Vison/genética , Vírus da Enterite do Vison/patogenicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Sf9 , Spodoptera , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência
13.
J Virol ; 94(19)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669327

RESUMO

Infectious bronchitis (IB) caused by infectious bronchitis virus (IBV) is currently a major threat to chicken health, with multiple outbreaks being reported in the United States over the past decade. Modified live virus (MLV) vaccines used in the field can persist and provide the genetic material needed for recombination and emergence of novel IBV serotypes. Inactivated and subunit vaccines overcome some of the limitations of MLV with no risk of virulence reversion and emergence of new virulent serotypes. However, these vaccines are weakly immunogenic and poorly protective. There is an urgent need to develop more effective vaccines that can elicit a robust, long-lasting immune response. In this study, we evaluate a novel adjuvant system developed from Quil-A and chitosan (QAC) for the intranasal delivery of nucleic acid immunogens to improve protective efficacy. The QAC adjuvant system forms nanocarriers (<100 nm) that efficiently encapsulate nucleic acid cargo, exhibit sustained release of payload, and can stably transfect cells. Encapsulation of plasmid DNA vaccine expressing IBV nucleocapsid (N) protein by the QAC adjuvant system (pQAC-N) enhanced immunogenicity, as evidenced by robust induction of adaptive humoral and cellular immune responses postvaccination and postchallenge. Birds immunized with pQAC-N showed reduced clinical severity and viral shedding postchallenge on par with protection observed with current commercial vaccines without the associated safety concerns. Presented results indicate that the QAC adjuvant system can offer a safer alternative to the use of live vaccines against avian and other emerging coronaviruses.IMPORTANCE According to 2017 U.S. agriculture statistics, the combined value of production and sales from broilers, eggs, turkeys, and chicks was $42.8 billion. Of this number, broiler sales comprised 67% of the industry value, with the production of >50 billion pounds of chicken meat. The economic success of the poultry industry in the United States hinges on the extensive use of vaccines to control infectious bronchitis virus (IBV) and other poultry pathogens. The majority of vaccines currently licensed for poultry health include both modified live vaccine and inactivated pathogens. Despite their proven efficacy, modified live vaccine constructs take time to produce and could revert to virulence, which limits their safety. The significance of our research stems from the development of a safer and potent alternative mucosal vaccine to replace live vaccines against IBV and other emerging coronaviruses.


Assuntos
Bronquite/prevenção & controle , Infecções por Coronavirus/veterinária , Gammacoronavirus/imunologia , Membrana Mucosa/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas Virais/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Bronquite/virologia , Galinhas , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Modelos Animais de Doenças , Imunidade Celular , Imunização , Vírus da Bronquite Infecciosa/imunologia , Nucleocapsídeo/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/imunologia , Vacinas de DNA/imunologia , Carga Viral
14.
Nat Commun ; 11(1): 3677, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699279

RESUMO

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13+ follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.


Assuntos
Linfócitos B/imunologia , Microambiente Celular/imunologia , Quimiocina CXCL13/metabolismo , Células Dendríticas Foliculares/imunologia , Imunidade Adaptativa , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Linhagem Celular , Quimiocina CXCL13/imunologia , Simulação por Computador , Células Dendríticas Foliculares/citologia , Células Dendríticas Foliculares/metabolismo , Matriz Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Modelos Biológicos , Tonsila Palatina/citologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Estromais/imunologia , Células Estromais/metabolismo
15.
Virus Res ; 286: 198074, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32589897

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Interferon beta/genética , NF-kappa B/genética , Proteínas do Nucleocapsídeo/genética , Proteínas Virais/genética , Betacoronavirus/imunologia , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Interferon beta/imunologia , Luciferases/genética , Luciferases/metabolismo , NF-kappa B/imunologia , Proteínas do Nucleocapsídeo/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Elementos de Resposta , Vírus Sendai/genética , Vírus Sendai/imunologia , Transdução de Sinais , Transfecção/métodos , Proteínas Virais/imunologia
16.
Mol Immunol ; 124: 161-171, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32585510

RESUMO

Leishmania infantum pyridoxal kinase (PK) protein was characterized after an immunoproteomics screening performed with the sera from patients suffering visceral leishmaniasis (VL). Since it was recognized by sera of mammalian hosts infected by a viscerotropic Leishmania species, PK could emerge as a new vaccine candidate against disease, due to its antigenicity and immunogenicity. In this context, in the present study, the effects of the immunization using PK were evaluated when administered as a DNA plasmid (pDNAA3/PK) or recombinant protein (rPK) plus saponin. The immune response elicited by both vaccination regimens reduced in significant levels the parasite load in spleen, liver, draining lymph nodes and bone marrow, being associated with the development of Th1-type immune response, which was characterized by high levels of IFN-γ, IL-12, GM-CSF, and specific IgG2a antibody, besides low production of IL-4, IL-10, and protein and parasite-specific IgG1 antibodies. CD8+ T cells were more important in the IFN-γ production in the pDNAA3/PK group, while CD4+ T cells contributed more significantly to production of this cytokine in the rPK/Saponin group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from VL patients after treatment and healthy individuals were stimulated with the protein. In conclusion, when administered either as a DNA plasmid or recombinant protein plus adjuvant, PK can direct the immune response towards a Th1-type immune profile, protecting mice against L. infantum challenge; therefore, it can be seen as a promising immunogen against human VL.


Assuntos
Antígenos de Protozoários/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Piridoxal Quinase/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Humanos , Leishmania infantum/imunologia , Camundongos , Proteínas Recombinantes/imunologia , Vacinas de DNA/imunologia
17.
PLoS One ; 15(6): e0231679, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559193

RESUMO

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.


Assuntos
Glicopeptídeos/análise , Anticorpos Anti-HIV/imunologia , HIV-1/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Adulto , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , Células CHO , Cricetinae , Cricetulus , Feminino , Glicosilação , Células HEK293 , Humanos , Pessoa de Meia-Idade , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto Jovem , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
18.
PLoS One ; 15(6): e0234043, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555593

RESUMO

Syphilis serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance and discordant results between tests make clinical decisions difficult. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. Our aim was to assess the varied from performance of T. pallidum-recombinant proteins TmpA, TpN17 and TpN47 for syphilis serodiagnosis. The proteins were evaluated using sera of 338 T. pallidum-negative, 173 T. pallidum-positive individuals and 209 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. In the liquid microarray analyses, the ROC curve varied from 99.0% for TmpA and TpN17 to 100% for TpN47. The sensitivity score yielded values of up to 90% for TpN17, 100% for TpN47 and 80.0% for TmpA. The lowest and highest specificity values were presented by TpN47 (91.9%) and TmpA antigens (100%), respectively. TpN47 showed the highest accuracy score (95.5%) among all the recombinant proteins assayed. For the ELISA, the ROC curve was 97.2%, 91.8% and 81.6% for TpN17, TmpA and TpN47, respectively. TpN17 and TmpA yielded a sensitivity of 69.9%, while TpN47 obtained a value of 53.8%. Specificity was almost 100% for all three proteins. No cross-reaction was observed for TpN17 in the serum samples from non-bacterial infections. Regarding leptospirosis-positive samples, cross-reactivity score was varied from 8.6 to 34.6%. This is most probably due to conservation of the epitopes in these proteins across bacteria. The use of recombinant proteins in immunoassays for syphilis diagnosis was showed provide greater reliability to results of the treponemal assays. Despite the low sensitivity, the proteins showed high diagnostic capacity due to the AUC values found. However, an improvement in sensitivity could be achieved when antigenic mixtures are evaluated.


Assuntos
Proteínas de Bactérias/imunologia , Proteínas Recombinantes/imunologia , Testes Sorológicos , Sífilis/diagnóstico , Treponema pallidum/imunologia , Reações Cruzadas , Sífilis/imunologia
19.
PLoS One ; 15(6): e0233695, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479551

RESUMO

Johne's disease (JD) is an infectious wasting condition of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) in domestic livestock of every country that has been investigated. Controlling JD is problematic due to the lack of sensitive, specific, efficient, and cost-effective diagnostic tests. A major challenge in the development of diagnostics like ELISA is the selection of an ideal antigen/(s) that is pathogen-specific and allows sensitive recognition. Therefore, the purpose of this study was to identify and use Mce-truncated protein-based ELISA assay for the diagnosis of MAP infection with high sensitivity and specificity. In silico epitope prediction by epitope mapping throughout the whole length of MAP2191 protein revealed that C-terminal portion of this protein presented potential T- and B-cell epitopes. Therefore, a novel Mce-truncated protein encoded by the selected region of MAP2191 gene was expressed, purified with Ni-NTA gel matrix and confirmed by SDS PAGE and western blot. A profiling ELISA assay was developed to evaluate sera from MAP infected and non-infected ruminant species for antibodies against Mce-truncated protein to infer the immunogenicity of this protein in the host. Using this Mce protein-based ELISA, 251 goats, 53 sheep, 117 buffaloes, and 33 cattle serum samples were screened and 49.4, 51.0, 69.2, and 54.6% animals, respectively, were found positive. Comparing with i-ELISA, the new Mce-based ELISA kit showed a relatively higher specificity but suffered from slightly reduced sensitivity. Mce-based ELISA excluded apparently false positive results of i-ELISA. Mce protein was found to be antigenic and Mce-ELISA test could be employed as a diagnostic test for JD in domestic livestock in view of the a relatively higher specificity and accuracy. The antigenic potential of Mce antigen can also be exploited for the development of a new vaccine for the control of MAP infection.


Assuntos
Doenças dos Bovinos/sangue , Mycobacterium avium/imunologia , Paratuberculose/sangue , Testes Sorológicos/veterinária , Doenças dos Ovinos/sangue , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/microbiologia , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Cabras , Mycobacterium avium/patogenicidade , Paratuberculose/diagnóstico , Paratuberculose/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/microbiologia
20.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(3): 264-270, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32389175

RESUMO

Objective To express E6 protein of human papillomavirus (HPV) type 16 in prokaryotic expression system and prepare its polyclonal antibody. Methods HPV16 E6 gene was obtained from Siha cells by PCR and cloned into pET21a(+) vector to construct the recombinant plasmid pET21a(+)/HPV16 E6 that was confirmed by sequencing. The recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3). The HPV16 E6-His tag recombinant protein was expressed after the induction of isopropyl beta-D-1-thiogalactopyranoside (IPTG), purified by Ni-NTA affinity chromatography, and then analyzed by Western blot analysis. The purified HPV16 E6 recombinant protein was used to immunize Japanese white rabbits to prepare polyclonal antibody. The titer of the serum polyclonal antibody was determined by ELISA. The specificity of the polyclonal antibody was analyzed by Western blotting and immunofluorescence. Results The recombinant plasmid pET21a(+)/HPV16 E6 was successfully constructed and confirmed by sequencing. After the recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3), the recombinant HPV16 E6 protein was expressed and purified by affinity chromatography. The polyclonal antibody at a titer of 1:40 000 was obtained by immunizing Japanese big-ear white rabbit with the purified recombinant HPV16 E6 protein, and its specificity was confirmed by Western blotting and immunofluorescence assay. Conclusion HPV16 E6 recombinant protein was successfully expressed and the rabbit polyclonal antibody against HPV16 E6 recombinant protein was prepared.


Assuntos
Anticorpos/imunologia , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/imunologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/imunologia , Animais , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Plasmídeos , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia
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