Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 42
Filtrar
1.
Methods Mol Biol ; 2197: 285-304, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32827144

RESUMEN

DNA vaccines have been used as a promising strategy for delivery of immunogenic and immunomodulatory molecules into the host cells. Although, there are some obstacles involving the capability of the plasmid vector to reach the cell nucleus in great number to promote the expected benefits. In order to improve the delivery and, consequently, increase the expression levels of the target proteins carried by DNA vaccines, alternative methodologies have been explored, including the use of non-pathogenic bacteria as delivery vectors to carry, deliver, and protect the DNA from degradation, enhancing plasmid expression.


Asunto(s)
ADN/genética , Vectores Genéticos/genética , Lactobacillales/genética , Plásmidos/genética , ADN/inmunología , ADN/aislamiento & purificación , Escherichia coli/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/inmunología , Vectores Genéticos/aislamiento & purificación , Humanos , Plásmidos/administración & dosificación , Plásmidos/inmunología , Plásmidos/aislamiento & purificación , Transfección , Transformación Bacteriana , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas de ADN/inmunología
2.
R. bras. Ci. avíc. ; 23(4): eRBCA-2021-1460, 2021. ilus, tab
Artículo en Inglés | VETINDEX | ID: vti-765868

RESUMEN

Salmonella enterica is a zoonotic pathogen transmitted to humans by the consumption of contaminated poultry products. Salmonella causes around 93 million cases of gastroenteritis and 155,000 deaths worldwide. A high number of multidrug-resistant Salmonella isolates has been found from different segments of poultry production, and it is often associated with horizontal antibiotic resistance gene transfer through mobile elements such as plasmids, integrons, and transposons. The emergence of antibiotic-resistant Salmonella has been associated with the misuse of antibiotics in animal production and it is classified as a highly important pathogen from public health due to its zoonotic character and its high dissemination ability. This study aimed to determine the antibiotic resistance associated with plasmids, and class 1 and 2 integrons from Salmonella Heidelberg isolates from healthy chickens in poultry farms from Santander, Colombia. 15 Salmonella Heidelberg isolates obtained from cloacal samples, were evaluated through endpoint PCR to determine the presence of plasmid-encoded resistance genes, and class 1 and 2 integrons. The bla CMY2 , strA and strB, sul1 and sul2, and tetB genes were found in all 15 Salmonella Heidelberg. The class 1 integron was present in 11 out of 15 isolates, harbored the gene cassette dfra7.The results indicate the presence of a high number of antibiotic resistance genes associated with plasmids and class 1 integrons in Salmonella Heidelberg strains isolated from poultry farms, resulting in a public health concern, both in humans and poultry production in Colombia.(AU)


Asunto(s)
Animales , Farmacorresistencia Microbiana , Plásmidos/inmunología , Integrones , Salmonella/aislamiento & purificación , Aves de Corral/microbiología
3.
Rev. bras. ciênc. avic ; 23(4): eRBCA, 2021. ilus, tab
Artículo en Inglés | VETINDEX | ID: biblio-1490898

RESUMEN

Salmonella enterica is a zoonotic pathogen transmitted to humans by the consumption of contaminated poultry products. Salmonella causes around 93 million cases of gastroenteritis and 155,000 deaths worldwide. A high number of multidrug-resistant Salmonella isolates has been found from different segments of poultry production, and it is often associated with horizontal antibiotic resistance gene transfer through mobile elements such as plasmids, integrons, and transposons. The emergence of antibiotic-resistant Salmonella has been associated with the misuse of antibiotics in animal production and it is classified as a highly important pathogen from public health due to its zoonotic character and its high dissemination ability. This study aimed to determine the antibiotic resistance associated with plasmids, and class 1 and 2 integrons from Salmonella Heidelberg isolates from healthy chickens in poultry farms from Santander, Colombia. 15 Salmonella Heidelberg isolates obtained from cloacal samples, were evaluated through endpoint PCR to determine the presence of plasmid-encoded resistance genes, and class 1 and 2 integrons. The bla CMY2 , strA and strB, sul1 and sul2, and tetB genes were found in all 15 Salmonella Heidelberg. The class 1 integron was present in 11 out of 15 isolates, harbored the gene cassette dfra7.The results indicate the presence of a high number of antibiotic resistance genes associated with plasmids and class 1 integrons in Salmonella Heidelberg strains isolated from poultry farms, resulting in a public health concern, both in humans and poultry production in Colombia.


Asunto(s)
Animales , Aves de Corral/microbiología , Integrones , Plásmidos/inmunología , Farmacorresistencia Microbiana , Salmonella/aislamiento & purificación
4.
Braz J Microbiol ; 50(1): 279-286, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30637649

RESUMEN

Porcine rotavirus (PoRV) and porcine epidemic diarrhea virus (PEDV) usually co-infect pigs in modern large-scale piggery, which both can cause severe diarrhea in newborn piglets and lead to significant economic losses to the pig industry. The VP7 protein is the main coat protein of PoRV, and the S protein is the main structural protein of PEDV, which are capable of inducing neutralizing antibodies in vivo. In this study, a DNA vaccine pPI-2.EGFP.VP7.S co-expressing VP7 protein of PoRV and S protein of PEDV was constructed. Six 8-week-old mice were immunized with the recombinant plasmid pPI-2.EGFP.VP7.S. The high humoral immune responses (virus specific antibody) and cellular immune responses (IFN-γ, IL-4, and spleen lymphocyte proliferation) were evaluated. The immune effect through intramuscular injection increased with plasmid dose when compared with subcutaneous injection. The immune-enhancing effect of IFN-α adjuvant was excellent compared with pig spleen transfer factor and IL-12 adjuvant. These results demonstrated that pPI-2.EGFP.VP7.S possess the immunological functions of the VP7 proteins of PoRV and S proteins of PEDV, indicating that pPI-2.EGFP.VP7.S is a candidate vaccine for porcine rotaviral infection (PoR) and porcine epidemic diarrhea (PED).


Asunto(s)
Antígenos Virales/inmunología , Proteínas de la Cápside/inmunología , Infecciones por Coronavirus/veterinaria , Plásmidos/inmunología , Infecciones por Rotavirus/veterinaria , Rotavirus/inmunología , Enfermedades de los Porcinos/prevención & control , Proteínas Virales de Fusión/inmunología , Vacunas Virales/inmunología , Animales , Antígenos Virales/administración & dosificación , Antígenos Virales/genética , Proteínas de la Cápside/administración & dosificación , Proteínas de la Cápside/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/virología , ADN Recombinante/administración & dosificación , ADN Recombinante/genética , ADN Recombinante/inmunología , Evaluación Preclínica de Medicamentos , Ratones , Plásmidos/administración & dosificación , Plásmidos/genética , Virus de la Diarrea Epidémica Porcina/genética , Virus de la Diarrea Epidémica Porcina/inmunología , Rotavirus/genética , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/prevención & control , Infecciones por Rotavirus/virología , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Proteínas Virales de Fusión/administración & dosificación , Proteínas Virales de Fusión/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genética
5.
Sci Rep ; 8(1): 11627, 2018 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-30072701

RESUMEN

There is no safe and efficacious vaccine against human leishmaniasis available and live attenuated vaccines have been used as a prophylactic alternative against the disease. In order to obtain an attenuated Leishmania parasite for vaccine purposes, we generated L. infantum KHARON1 (KH1) null mutants (ΔLikh1). This gene was previously associated with growth defects in L. mexicana. ΔLikh1 was obtained and confirmed by PCR, qPCR and Southern blot. We also generate a KH1 complemented line with the introduction of episomal copies of KH1. Although ΔLikh1 promastigote forms exhibited a growth pattern similar to the wild-type line, they differ in morphology without affecting parasite viability. L. infantum KH1-deficient amastigotes were unable to sustain experimental infection in macrophages, forming multinucleate cells which was confirmed by in vivo attenuation phenotype. The cell cycle analysis of ΔLikh1 amastigotes showed arrested cells at G2/M phase. ΔLikh1-immunized mice presented reduced parasite burden upon challenging with virulent L. infantum, when compared to naïve mice. An effect associated with increased Li SLA-specific IgG serum levels and IL-17 production. Thus, ΔLikh1 parasites present an infective-attenuated phenotype due to a cytokinesis defect, whereas it induces immunity against visceral leishmaniasis in mouse model, being a candidate for antileishmanial vaccine purposes.


Asunto(s)
Citocinesis , Leishmania infantum , Leishmaniasis Visceral , Mutación , Animales , Citocinesis/genética , Citocinesis/inmunología , Modelos Animales de Enfermedad , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Puntos de Control de la Fase G2 del Ciclo Celular/inmunología , Humanos , Leishmania infantum/genética , Leishmania infantum/crecimiento & desarrollo , Leishmania infantum/inmunología , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/metabolismo , Leishmaniasis Visceral/prevención & control , Puntos de Control de la Fase M del Ciclo Celular/genética , Puntos de Control de la Fase M del Ciclo Celular/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Plásmidos/genética , Plásmidos/inmunología , Plásmidos/metabolismo , Células THP-1
6.
Mem Inst Oswaldo Cruz ; 112(2): 123-130, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-28177046

RESUMEN

BACKGROUND: Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES: The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS: Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS: The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION: The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.


Asunto(s)
Antígenos Bacterianos/inmunología , Vacuna BCG/inmunología , Mycobacterium bovis/inmunología , Animales , Antígenos Bacterianos/metabolismo , Vacuna BCG/genética , Escherichia coli/genética , Femenino , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Ratones , Ratones Endogámicos BALB C , Mycobacterium bovis/genética , Plásmidos/genética , Plásmidos/inmunología
7.
Mem. Inst. Oswaldo Cruz ; 112(2): 123-130, Feb. 2017. tab, graf
Artículo en Inglés | LILACS | ID: biblio-841767

RESUMEN

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.


Asunto(s)
Animales , Femenino , Ratones , Plásmidos/genética , Plásmidos/inmunología , Vacuna BCG/genética , Vacuna BCG/inmunología , Vectores Genéticos/inmunología , Mycobacterium bovis/genética , Mycobacterium bovis/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Escherichia coli/genética , Vectores Genéticos , Ratones Endogámicos BALB C
8.
J Vector Borne Dis ; 54(4): 317-327, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29460861

RESUMEN

Background & objectives: The nature of the rickettsial antigens and the immune response generated by them, have been the subject of exhaustive research so that a suitable vaccine can be developed. Till date evaluations of Rickettsia rickettsii antigens that induce both humoral and cellular responses in animal models have only shown partial protection and short-term immunological memory. This study was aimed to evaluate the immune response induced by DNA plasmids generated from the OmpA and OmpB genes of R. rickettsii in peripheral blood mononuclear cells of rickettsial (sensitized) patients compared to healthy subjects. Methods: Plasmids OmpA-49, OmpB-15 and OmpB-24 were generated in the pVAX vector. Macrophages derived from the THP-1 cell line were transfected in vitro with the plasmids and were co-cultured with T-lymphocytes from sensitized subjects and healthy subjects to evaluate cell proliferation and cytokine production. Results: The OmpB-24 plasmid induced proliferative response in human lymphocytes, with production of IL-2, IFN-γ, IL-12p70, IL-6 and TNF-α, likely due to the presence of conserved epitopes among R. rickettsii, R. typhi and R. felis (differing from 1 to 3 amino acids) during the construction of the plasmids. Interpretation & conclusion: DNA sequences of rickettsial epitopes can be cloned into the pVAX vector. Constructed plasmids can generate a proliferative response and produce cytokines in vitro, in co-culture of transfected macrophages with sensitized human lymphocytes. Plasmid OmpB-24 proved to be the most immunogenic with respect to plasmids OmpA-49 and OmpB-15.


Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Rickettsia rickettsii/inmunología , Adolescente , Adulto , Anciano , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/farmacología , Citocinas/biosíntesis , Citocinas/efectos de los fármacos , Femenino , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Plásmidos/inmunología , Rickettsia rickettsii/química , Adulto Joven
9.
J Biomed Sci ; 23(1): 85, 2016 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-27903271

RESUMEN

BACKGROUND: The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serine-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work we evaluated the potential of the NS3 (protease domain) as a protective antigen by comparing the administration of a recombinant protein versus a DNA vaccine in the mouse model. RESULTS: BALB/c mice were immunized with the recombinant protein NS3-DEN3 via intraperitoneal and with plasmid pcDNA3/NS3-DEN3 intramuscularly and the immune response was evaluated. The activity of T lymphocytes was analyzed by the MTT assay, and cells of mice immunized with the recombinant protein showed no activity when stimulated with the homologous protein. However, cells from mice immunized with DNA, responded to stimulation with the recombinant protein. When the expression (RT-PCR) and cytokine production (ELISA) was evaluated in the splenocytes, different behavior depending on the type of immunization was observed, splenocytes of mice immunized with the recombinant protein expressed cytokines such as IL-4, IL-10 and produced high concentrations of IL-1, IL-6 and TNFα. Splenocytes from mice immunized with DNA expressed IL-2 and IFNγ and did not produce IL-6. In addition, immunization with the recombinant protein induced the production of antibodies that are detected up to a dilution 1:3200 by ELISA and Western blot assays, however, the serum of mice immunized with DNA presented no detectable antibody titers. CONCLUSION: The results obtained in this study show that administration of pcDNA3/NS3-DEN3 induces a favorable response in the activation of T lymphocytes with low production of specific antibodies against NS3-DEN3.


Asunto(s)
Virus del Dengue/inmunología , Inmunidad Celular , Inmunidad Humoral , Proteínas no Estructurales Virales/inmunología , Animales , Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Dengue/prevención & control , Dengue/virología , Virus del Dengue/patogenicidad , Humanos , Ratones , Plásmidos/administración & dosificación , Plásmidos/inmunología , ARN Helicasas/administración & dosificación , ARN Helicasas/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Serina Endopeptidasas/administración & dosificación , Serina Endopeptidasas/inmunología , Vacunación , Proteínas no Estructurales Virales/administración & dosificación
10.
Biomed Res Int ; 2013: 826570, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24163822

RESUMEN

The dog is considered the main domestic reservoir for Trypanosoma cruzi infection and a suitable experimental animal model to study the pathological changes during the course of Chagas disease (CD). Vaccine development is one of CD prevention methods to protect people at risk. Two plasmids containing genes encoding a trans-sialidase protein (TcSP) and an amastigote-specific glycoprotein (TcSSP4) were used as DNA vaccines in a canine model. Splenomegaly was not found in either of the recombinant plasmid-immunized groups; however, cardiomegaly was absent in animals immunized only with the plasmid containing the TcSSP4 gene. The inflammation of subendocardial and myocardial tissues was prevented only with the immunization with TcSSP4 gene. In conclusion, the vaccination with these genes has a partial protective effect on the enlargement of splenic and cardiac tissues during the chronic CD and on microscopic hearth damage, since both plasmids prevented splenomegaly but only one avoided cardiomegaly, and the lesions in heart tissue of dog immunized with plasmid containing the TcSSP4 gene covered only subepicardial tissue.


Asunto(s)
Enfermedad de Chagas/prevención & control , Plásmidos/farmacología , Proteínas Protozoarias/farmacología , Vacunas Antiprotozoos/farmacología , Trypanosoma cruzi/inmunología , Vacunas de ADN/farmacología , Animales , Enfermedad de Chagas/genética , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/patología , Modelos Animales de Enfermedad , Perros , Femenino , Humanos , Masculino , Miocardio/inmunología , Miocardio/patología , Plásmidos/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/genética , Vacunas Antiprotozoos/inmunología , Trypanosoma cruzi/genética , Vacunas de ADN/genética , Vacunas de ADN/inmunología
11.
Biol Res ; 43(1): 13-8, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-21157628

RESUMEN

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18 kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.


Asunto(s)
Vacuna BCG/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Vectores Genéticos/genética , Leptospira interrogans/genética , Mycobacterium bovis/genética , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos/inmunología , Leptospira interrogans/inmunología , Lipoproteínas/genética , Lipoproteínas/inmunología , Mycobacterium bovis/inmunología , Plásmidos/genética , Plásmidos/inmunología
12.
Arch Virol ; 155(6): 847-56, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20390312

RESUMEN

A DENV-2 plasmid named pEII*EIII/NS1*,containing sequences encoding portions of the envelope protein that are potentially involved in the induction of neutralizing antibodies and a portion of the NS1 sequence that is involved in protection, is reported in this work. The synthesized subunit protein was recognized by human sera from infected patients and had the predicted size. The immunogenicity of this construct was evaluated using a mouse model in a prime-boost vaccination approach. The priming was performed using the plasmid pEII*EIII/NS1*, followed by a boost with recombinant full-length GST-E and GST-NS1 fusion proteins. The mice showed specific antibody responses to the E and NS1 proteins, as detected by ELISA, compared to the response of animals vaccinated with the parental plasmid. Interestingly, some animals had neutralizing antibodies. These results show that EII*, EIII and NS1* sequences could be considered for the design ofa recombinant subunit vaccine against dengue disease.


Asunto(s)
Anticuerpos Antivirales/sangre , Virus del Dengue/inmunología , Dengue/inmunología , Plásmidos/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas no Estructurales Virales/inmunología , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Cricetinae , Dengue/virología , Vacunas contra el Dengue/genética , Vacunas contra el Dengue/inmunología , Modelos Animales de Enfermedad , Humanos , Inmunización Secundaria , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Vacunación , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas no Estructurales Virales/genética
13.
Biol. Res ; 43(1): 13-18, 2010. ilus, graf
Artículo en Inglés | LILACS | ID: lil-548025

RESUMEN

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.


Asunto(s)
Animales , Cricetinae , Vacuna BCG/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Vectores Genéticos/genética , Leptospira interrogans/genética , Mycobacterium bovis/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos/inmunología , Leptospira interrogans/inmunología , Lipoproteínas/genética , Lipoproteínas/inmunología , Mycobacterium bovis/inmunología , Plásmidos/genética , Plásmidos/inmunología
14.
Vaccine ; 26(5): 677-85, 2008 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-18160187

RESUMEN

The gene Ldccys1 encoding a cysteine proteinase of 30 kDa from Leishmania (Leishmania) chagasi, as well as the recombinant cysteine proteinase rLdccys1, obtained by cloning and expression of the Ldccys1 gene in the pHIS vector, were used to evaluate their ability to induce immune protective responses in BALB/c mice against L. (L.) chagasi infection. Mice were immunized subcutaneously with rLdccys1 plus Bacille Calmette Guerin (BCG) or Propionibacterium acnes as adjuvants or intramuscularly with a plasmid carrying the Ldccys1 gene (Ldccys1/pcDNA3) and CpG ODN as the adjuvant, followed by a booster with rLdccys1 plus CpG ODN. Two weeks after immunization the animals were challenged with 1 x 10(7) amastigotes of L. (L.) chagasi. Both immunization protocols induced significant protection against L. (L.) chagasi infection as shown by a very low parasite load in the spleen of immunized mice compared to the non-immunized controls. However, DNA immunization was 10-fold more protective than immunization with the recombinant protein. Whereas rLdccys1 induced a significant secretion of IFN-gamma and nitric oxide (NO), animals immunized with the Ldccys1 gene increased the production of IgG2a antibodies, IFN-gamma and NO. These results indicated that protection triggered by the two immunization protocols was correlated to a predominant Th1 response.


Asunto(s)
Cisteína Endopeptidasas/inmunología , Genes Protozoarios/inmunología , Leishmania/inmunología , Vacunas contra la Leishmaniasis/administración & dosificación , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis/prevención & control , Plásmidos/administración & dosificación , Proteínas Protozoarias/inmunología , Vacunación , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antiprotozoarios/sangre , Células Cultivadas , Cisteína Endopeptidasas/biosíntesis , Cisteína Endopeptidasas/genética , Femenino , Esquemas de Inmunización , Inmunoglobulina G/sangre , Inyecciones Subcutáneas , Interferón gamma/metabolismo , Leishmaniasis/sangre , Ratones , Mycobacterium bovis/inmunología , Óxido Nítrico/metabolismo , Plásmidos/inmunología , Propionibacterium acnes/inmunología , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Bazo/inmunología , Bazo/parasitología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología
15.
Salvador; s.n; 2008. 134 p. ilus, tab, graf.
Tesis en Portugués | LILACS | ID: lil-540657

RESUMEN

Hamsters têm sido utilizados como modelos experimentais visando compreender os mecanismos de respostas imunes contra espécies de Leishmania do complexo donovani. Estes modelos são capazes de reproduzir muitas das manifestações clínicas da leishmaniose visceral humana. Estudos recentes demonstraram que a imunização de hamsters com plasmídeos codificantes para proteínas salivares (LJM19) de Lutzomyia longipalpis, vetor de L. chagasi, bem como antígenos parasitários (KMPl1) protege hamsters contra um desafio letal com Leishmania chagasi. Neste trabalho, hamsters foram utilizados para avaliar o efeito protetor contra uma infecção por L. chagasi utilizando imunização com os plasmídeos que codificam as proteínas LJM19 e KMP11 administrados em conjunto. A imunização com os plasmídeos induziu a produção de IFN-y nos linfonodos drenantes do local das imunizações quando os animais foram avaliados 7, 14 e 21 dias após a última imunização. Uma vez imunizados e desafiados com L. chagasi mais saliva do vetor, os animais mostraram maiores relações (...) nos linfonodos drenantes quando mensuradas 7 e 14 dias após o desafio. Quando avaliados 2 e 5 meses após o desafio, os animais imunizados mostraram menores cargas parasitárias no baço e no fígado e maiores relações (...) no baço 2 meses após o desafio. Além disso, os hamsters imunizados apresentaram maior conservação da arquitetura histológica do baço e do fígado nos tempos avaliados e não desenvolveram distúrbios hematológicos quando comparados com animais controles sadios.


Contudo, efeito protetor adicional pela imunização com os diferentes plasmídeos administrados em conjunto não foi observado em relação às imunizações com os plasmídeos separados. Comparações entre rotas de administração de plasmídeos foram estudadas utilizando as vias intradérmica e intramuscular. Os grupos de animais que receberam a imunização intradérmica apresentaram uma proteção mais prolongada quando comparados aos animais imunizados intramuscularmente. Estes resultados mostram que apesar da combinação de plasmídeos não induzir maior proteção que os plasmídeos separados, a via de imunização intradérmica pode conferir uma proteção mais duradoura quando comparada com a imunização pela via intramuscular.


Asunto(s)
Animales , Ratas , Leishmaniasis Visceral/prevención & control , Proteínas y Péptidos Salivales/inmunología , Psychodidae/inmunología , Experimentación Animal , Cricetinae/inmunología , Modelos Animales de Enfermedad , Inmunización , Insectos Vectores , Factores Protectores , Plásmidos/inmunología
16.
Vaccine ; 25(46): 7962-71, 2007 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-17942199

RESUMEN

Vaccination of dogs, the domestic reservoir of Leishmania infantum, is the best method for controlling zoonotic visceral leishmaniasis. This strategy would reduce the incidence of disease in both the canine and, indirectly, the human population. Different vaccination approaches have been investigated against canine leishmaniasis (CaL) but to date there is only one licensed vaccine against this disease in dogs, in Brazil. DNA immunization is a promising method for inducing both humoral and cellular immune responses against this parasitic disease. Here, we report the results of a multiantigenic plasmid DNA vaccine encoding KMPII, TRYP, LACK and GP63 L. infantum antigens against experimentally induced CaL. Twelve dogs were randomly assigned to two groups receiving, at a 15 days interval, either four doses of plasmid DNA or similar injections of PBS. After vaccination, dogs were intravenously challenged with 5 x 10(7) promastigotes of L. infantum. The vaccine showed to be safe and well-tolerated. Neither cellular immune response nor antibodies directed against whole Leishmania antigen were detected after immunization in vaccinated dogs, although anti-LACK-specific antibodies were sporadically detected in two vaccinated dogs before challenge, thus suggesting that antigens were indeed expressed. A delay in the development of detectable specific immune response and parasite multiplication in vaccinated dogs was observed after challenge. Nevertheless, the multiantigenic Leishmania DNA vaccine was unable to induce protection against parasite dissemination or disease. This study emphasizes the need to strengthen DNA vaccines in order to obtain effective immune responses in models other than the murine.


Asunto(s)
Antígenos de Protozoos/inmunología , Enfermedades de los Perros/inmunología , Leishmania infantum/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/veterinaria , Vacunas de ADN/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Formación de Anticuerpos/inmunología , Antígenos de Protozoos/genética , Brasil , Enfermedades de los Perros/genética , Enfermedades de los Perros/prevención & control , Perros , Femenino , Humanos , Inmunidad Celular/inmunología , Inmunización , Leishmania infantum/genética , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/prevención & control , Ratones , Plásmidos/genética , Plásmidos/inmunología , Vacunación , Vacunas de ADN/genética , Zoonosis
17.
Int Microbiol ; 10(4): 261-9, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18228223

RESUMEN

Recombinant adenoviruses, poxviruses, and plasmid DNA vaccines encoding different hepatitis B virus (HBV)/murine cytomegalovirus (MCMV) protein chimeras were used to immunize mice. Processing of the chimeras resulted in presentation of a protective Ld/CD8+ T-cell epitope of the immediate early 1 protein pp89 (IE1 pp89) of MCMV to the immune system. Different levels of immunogenicity were observed depending on: (i) the type of viral vector used, (ii) whether the antigens were included in the cellular secretion pathway, and (iii) the location of the protective epitope within the chimeric protein. An adenovirus expressing a secretory HBV core protein with the MCMV epitope in its C-terminus induced the highest immune response. When the most immunogenic adenovirus and vaccinia virus were used in a heterologous prime-boost immunization protocol, even higher levels of epitope-specific T cells were obtained. Furthermore, responses were protective against a challenge with MCMV, inducing up to a 96% reduction of viral load in immunized animals, as determined by a sensitive real-time PCR assay. Together, these results confirmed previous observations of the efficient use of adenoviral and poxviral vectors in prime-boost protocols for immunization against diseases whose resolution depends on cellular immunity, as well as the aptness of correctly designed chimeric carrier proteins to facilitate this goal.


Asunto(s)
Infecciones por Citomegalovirus/prevención & control , Vectores Genéticos/inmunología , Virus de la Hepatitis B/inmunología , Proteínas Inmediatas-Precoces/metabolismo , Inmunización , Muromegalovirus/fisiología , Vacunas Virales/inmunología , Adenoviridae/genética , Animales , Línea Celular , Quimera , Vectores Genéticos/administración & dosificación , Virus de la Hepatitis B/genética , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/inmunología , Plásmidos/genética , Plásmidos/inmunología , Poxviridae/genética , Vacunación , Vacunas Virales/administración & dosificación
18.
Microbiol Immunol ; 50(4): 253-63, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16625047

RESUMEN

In the present work we investigated the role of killed Propionibacterium acnes or a soluble polysaccharide extracted from bacterium cell wall in modulated experimental immunization with plasmidial DNA. We used a plasmid, p154/13, containing a gene-encoding catalytic domain of Trypanosoma cruzi (T. cruzi) trans-sialidase. As previously described, immunization of BALB/c mice with p154/13 elicited humoral, cell-mediated and protective immune responses against T. cruzi infection. In this study we describe that both P. acnes and its soluble polysaccharide fraction have the ability to modulate the immune response elicited by p154/13. Treatment with these adjuvants enhanced specific trans-sialidase Th1 immune response, as revealed by a lower IgG1/IgG2a ratio and stronger in vitro IFN-gamma synthesis by CD4+ T cells. The most important fact was that treatment with P. acnes or its soluble polysaccharide fraction in the presence of p154/13 significantly reduced the peak of parasitemia observed 7 to 8 days after T. cruzi challenge. These data suggest that P. acnes or its soluble polysaccharide fraction may improve the protective potential of a DNA vaccine against experimental T. cruzi infection.


Asunto(s)
Enfermedad de Chagas/prevención & control , Propionibacterium acnes/inmunología , Vacunas Antiprotozoos/inmunología , Células TH1/inmunología , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología , Vacunas de ADN/inmunología , Animales , Especificidad de Anticuerpos , Enfermedad de Chagas/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas/sangre , Isotipos de Inmunoglobulinas/inmunología , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Neuraminidasa/genética , Neuraminidasa/inmunología , Parasitemia/inmunología , Parasitemia/parasitología , Parasitemia/prevención & control , Plásmidos/genética , Plásmidos/inmunología , Polisacáridos/inmunología , Polisacáridos/aislamiento & purificación , Propionibacterium acnes/química , Vacunas Antiprotozoos/genética , Bazo/inmunología , Células TH1/citología , Células Th2/citología , Células Th2/inmunología , Vacunas de ADN/genética
19.
Clin Diagn Lab Immunol ; 11(4): 711-9, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15242946

RESUMEN

A plasmid vector encoding the cholera toxin B subunit (pCtB) was evaluated as an intradermal genetic adjuvant for a model DNA vaccine expressing the human papillomavirus type 16 L1 capsid gene (p16L1) in mice. p16L1 was coadministered with plasmid pCtB or commercial polypeptide CtB as a positive control. Coadministration of pCtB induced a significant increment of specific anti-L1 immunoglobulin A (IgA) antibodies in cervical secretions (P < 0.05) and fecal extracts (P < 0.005). Additionally, coadministration of pCtB enhanced the production of interleukin-2 and gamma interferon by spleen cells but did not affect the production of interleukin-4, suggesting a Th1-type helper response. Furthermore, improved CD8+ T-cell-mediated cytotoxic activity was observed in mice vaccinated with the DNA vaccine with pCtB as an adjuvant. This adjuvant effect was comparable to that induced by the CtB polypeptide. These results indicate that intradermal coadministration of pCtB is an adequate means to enhance the mucosa-, Th1-, and CD8(+)-mediated cytotoxic responses induced by a DNA vaccine.


Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Toxina del Cólera/inmunología , Membrana Mucosa/inmunología , Vacunas de ADN/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Proteínas de la Cápside/inmunología , Moco del Cuello Uterino/inmunología , Toxina del Cólera/genética , Citocinas/biosíntesis , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos/inmunología , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina A/inmunología , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Inyecciones Intradérmicas , Ratones , Proteínas Oncogénicas Virales/inmunología , Papillomaviridae/inmunología , Plásmidos/inmunología , Células TH1/inmunología
20.
Vaccine ; 21(25-26): 4071-80, 2003 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-12922144

RESUMEN

Multiagent DNA vaccines for highly pathogenic organisms offer an attractive approach for preventing naturally occurring or deliberately introduced diseases. Few animal studies have compared the feasibility of combining unrelated gene vaccines. Here, we demonstrate that DNA vaccines to four dissimilar pathogens that are known biowarfare agents, Bacillus anthracis, Ebola (EBOV), Marburg (MARV), and Venezuelan equine encephalitis virus (VEEV), can elicit protective immunity in relevant animal models. In addition, a combination of all four vaccines is shown to be equally as effective as the individual vaccines for eliciting immune responses in a single animal species. These results demonstrate for the first time the potential of combined DNA vaccines for these agents and point to a possible method of rapid development of multiagent vaccines for disparate pathogens such as those that might be encountered in a biological attack.


Asunto(s)
Vacunas contra el Carbunco/inmunología , Ebolavirus/inmunología , Virus de la Encefalitis Equina Venezolana/inmunología , Marburgvirus/inmunología , Vacunas Virales/inmunología , Animales , Carbunco/inmunología , Carbunco/prevención & control , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antivirales/biosíntesis , Biolística , Encefalomielitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/prevención & control , Ensayo de Inmunoadsorción Enzimática , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Humanos , Macaca fascicularis , Enfermedad del Virus de Marburg/inmunología , Enfermedad del Virus de Marburg/prevención & control , Ratones , Ratones Endogámicos BALB C , Plásmidos/inmunología , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas Combinadas/inmunología , Vacunas de ADN/inmunología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA