Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 39
Filtrar
2.
Emerg Microbes Infect ; 10(1): 1555-1573, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34304724

RESUMEN

To curb the pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), multiple platforms have been employed toward a safe and highly effective vaccine. Here, we develop a novel cell-based vaccine candidate, namely K562-S, by utilizing human cell K562 as a cellular carrier to display Spike (S) protein of SARS-CoV-2 on the membrane. Analogous to the traditional inactivated vaccine, K562-S cells can be propagated to a large scale by culturing and completely lose their viability after exposure to X-ray irradiation or formalin. We in turn demonstrated high immunogenicity of formalin-inactivated K562-S vaccine in both mouse and non-human primates and its protective efficacy in mice. In mice, immunization with inactivated K562-S vaccines can elicit potent neutralizing antibody (nAb) responses persisting longer than 5 months. We consequently showed in a hACE2 mouse model of SARS-CoV-2 infection that a two-shot vaccination with adjuvanted K562-S rendered greater than 3 log reduction in viral lung load and concomitant ameliorated lung pathology. Of importance, the administration of the same regimen in non-human primates was able to induce a neutralizing antibody titer averaging three-fold higher relative to human convalescent serum. These results together support the promise of K562-based, S-protein-expressing vaccines as a novel vaccination approach against SARS-CoV-2. Importantly, with a powerful capacity to carry external genes for cell-based vectors, this platform could rapidly generate two- and multiple-valent vaccines by incorporating SARS-CoV-2 mutants, SARS-CoV, or MERS-CoV.


Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , Inmunogenicidad Vacunal , SARS-CoV-2/inmunología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Animales Modificados Genéticamente , Vacunas contra la COVID-19/administración & dosificación , Femenino , Células HEK293 , Humanos , Células K562 , Macaca mulatta , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Primates , Organismos Libres de Patógenos Específicos , Glicoproteína de la Espiga del Coronavirus/administración & dosificación , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunación/métodos , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología
3.
Hum Gene Ther ; 32(19-20): 997-1003, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-33843251

RESUMEN

Advanced therapy medicinal products (ATMPs), such as gene therapies that consist of or contain genetically modified organisms (GMOs) need to comply with the European Union (EU) GMO legislation, as implemented in each EU Member State, before a clinical trial can commence. Complying with GMO requirements is complex, varies significantly across EU Member States and is leading to delays to clinical trials with ATMPs. Such delays and varying implementation of the GMO legislation makes the EU less attractive as a region to conduct clinical trials with investigational gene therapies. This is detrimental to EU patients, since their timely access to these transformative potentially curative medicines is delayed. Despite recent initiatives coordinated by the European Commission (EC) to facilitate and reduce discrepancies across the EU regarding the application of the GMO requirements, it remains particularly difficult to conduct multicenter clinical trials with ATMPs containing or consisting of GMOs involving several EU Member States. The recent decision for the EC to temporarily derogate potential coronavirus disease 2019 treatments and vaccines from some provisions of the GMO requirements was made on the basis of a clear recognition of such complexities and resulting delays to clinical development. The Alliance for Regenerative Medicine, the European Federation of Pharmaceutical Industries and Associations, and the European Association for Bioindustries call upon the EC, together with national competent authorities, to exempt ATMPs containing or consisting of GMOs from the GMO legislation. Such a simplification will eliminate the delays currently reported to occur when submitting environmental risk assessments and GMO applications to the national competent authorities. An exemption from GMO requirements will make the EU a more attractive region for clinical development of gene therapies and could accelerate European patients' access to these potentially life-saving medicines. Maintaining a system for GMO assessment that is different across countries may also prevent ATMPs from realizing the full benefits of a harmonized clinical trial approval process under the Clinical Trials Regulation. The undersigned organizations to this publication urge the EC to use its right of initiative to put forward a legislative proposal to exempt ATMPs in clinical development from the EU GMO legislation, within the timeframe proposed in the 2020 EU Pharmaceutical Strategy (by 2022). Implementation of a GMO exemption scheme before the end of the transition period for the Clinical Trial Regulation (the end of 2023) is important to avoid new Clinical Trial Application submissions for ATMPs under the Clinical Trial Regulation having to conduct the whole GMO assessment process in parallel. It is considered that ATMPs pose negligible risk to the environment. Such ATMPs include the following: human somatic cells modified ex vivo; recombinant virus-based vectors, including those containing genome editing nucleic acid sequences (which may also be delivered nonvirally); and bacterial vectors. Outside of controlled storage conditions, gene therapies cannot survive for any appreciable length of time. Upon clinical administration, any recombinant gene therapy viral vector particles that do not enter host cells are diluted within the body and if excreted are in such low multiplicity to no longer be viable or considered infectious to persons, animals, or living organisms within the environment. Any nucleic acids released into the environment are rapidly degraded.


Asunto(s)
COVID-19 , Alimentos Modificados Genéticamente , Legislación Médica , Estudios Multicéntricos como Asunto/legislación & jurisprudencia , Organismos Modificados Genéticamente , SARS-CoV-2 , Unión Europea , Humanos
4.
Terminología | DeCS - Descriptores en Ciencias de la Salud | ID: 031494

RESUMEN

A functional system which includes the organisms of a natural community together with their environment. (McGraw Hill Dictionary of Scientific and Technical Terms, 4th ed)


Un sistema funcional el cual incluye los organismos de una comunidad natural junto a su ambiente. (Traducción libre del original: McGraw Hill Dictionary of Scientific and Technical Terms, 4th ed) Unidad ecológica básica, formada por el ambiente viviente (biotopo) y de organismos animales y vegetables que interactúan como un ente funcional único (Material II - IDNDR, 1992) Hábitat: Ambiente en el cual se establece el intercambio inmediato entre los seres vivientes y los recursos que les son esenciales para cumplir con sus funciones vitales.


1) Sistema funcional que inclui os organismos de uma comunidade natural junto a seu ambiente. (Tradução livre do original: McGraw Hill Dictionary of Scientific and Technical Terms, 4th ed) 2)Sistema aberto integrado pelos organismos vivos (inclusive o homem) e os elementos não vivos de um setor ambiental definido no tempo e no espaço, cujas propriedades globais de funcionamento e autorregulação derivam da interação entre seus componentes, tanto pertencentes aos sistemas naturais como aqueles modificados ou organizados pelo próprio homem. 3) Complexo constituído pelo biótopo e pela biocenose (Material III - Ministério da Ação Social, Brasília, 1992) Habitat: Ambiente no qual se estabelece o intercâmbio imediato entre os seres viventes e os recursos que lhes são essenciais para cumprir com suas funções vitais.

5.
Cytotherapy ; 23(1): 10-11, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33082093

RESUMEN

Attempts to streamline environmental procedures for those products containing or consisting of genetically modified organisms (GMOs) among the European Union (EU) Member States are ongoing but still need to be further developed. These procedures can be complex, resource-intensive and time-consuming. Some candidate vaccines currently under development for COVID-19 include genetically modified viruses, which may be considered GMOs. Given the public health emergency caused by the COVID-19 outbreak, on July 15, 2020, the European Parliament approved a temporary derogation of the European environmental requirements to facilitate that those clinical trials with GMOs intended to treat or prevent COVID-19 can start as soon as possible in Europe. This measure has been very controversial, since it could entail risks to human health and the environment, and could be seen as unfair for other products targeting unmet medical needs. With the adoption of this measure, the bottlenecks and obstacles for the development of innovative GMO-based medicines in the EU that the environmental legislation entails have become even more evident.


Asunto(s)
Vacunas contra la COVID-19/genética , COVID-19/epidemiología , COVID-19/prevención & control , Ensayos Clínicos como Asunto/legislación & jurisprudencia , Organismos Modificados Genéticamente , Pandemias/prevención & control , SARS-CoV-2/genética , SARS-CoV-2/inmunología , COVID-19/virología , Europa (Continente)/epidemiología , Unión Europea , Humanos , Medición de Riesgo
6.
Methods Mol Biol ; 2225: 39-61, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33108656

RESUMEN

Vaccines are the most effective means to prevent infectious diseases, especially for viral infection. The key to an excellent antiviral vaccine is the ability to induce long-term protective immunity against a specific virus. Bacterial vaccine vectors have been used to impart protection against self, as well as heterologous antigens. One significant benefit of using live bacterial vaccine vectors is their ability to invade and colonize deep effector lymphoid tissues after mucosal delivery. The bacterium Salmonella is considered the best at this deep colonization. This is critically essential for inducing protective immunity. This chapter describes the methodology for developing genetically modified self-destructing Salmonella (GMS) vaccine delivery systems targeting influenza infection. Specifically, the methods covered include the procedures for the development of GMSs for protective antigen delivery to induce cellular immune responses and DNA vaccine delivery to induce systemic immunity against the influenza virus. These self-destructing GMS could be modified to provide effective biological containment for genetically engineered bacteria used for a diversity of purposes in addition to vaccines.


Asunto(s)
Ingeniería Genética/métodos , Inmunización/métodos , Vacunas contra la Influenza/genética , Gripe Humana/prevención & control , Salmonella typhimurium/genética , Vacunas de ADN/genética , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/genética , Antígenos Virales/inmunología , Femenino , Genes Letales , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Mucosa/efectos de los fármacos , Gripe Humana/inmunología , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Organismos Modificados Genéticamente , Plásmidos/química , Plásmidos/metabolismo , Salmonella typhimurium/inmunología , Transgenes
7.
Cuad. bioét ; 31(103): 387-401, sept.-dic. 2020.
Artículo en Español | IBECS | ID: ibc-200029

RESUMEN

La transgénesis constituye una parcela de la biotecnología consistente en la introducción de información genética no propia en el genoma de los seres vivos y al margen de los mecanismos del intercambio genético natural. Esto ha permitido abordar importantes aplicaciones en bacterias, animales y plantas con notables beneficios en las vertientes sanitaria, alimentaria y ambiental. Desde su origen, la obtención de los organismos modificados genéticamente (OMGs) suscitó una cierta polémica por la posible influencia negativa de estos organismos o de sus productos derivados para la salud y el medio-ambiente. Con el tiempo, las técnicas de modificación genética han mejorado dando paso a otras de mayor precisión, sencillez y seguridad. En la actualidad se utiliza ampliamente la técnica CRISPR-Cas9, que permite editar, modificar o eliminar secuencias específicas del ADN, con múltiples aplicaciones en los mismos campos de la transgénesis, pero con mayor simplicidad, seguridad y menor costo. En este trabajo, se presentan las principales técnicas, aplicaciones e implicaciones éticas de la utilización de estas técnicas y sus perspectivas en un mundo en continua evolución. Las bacterias para la obtención de productos de interés farmacológico, las nuevas variedades de plantas cultivadas de mayor producción, más resistencia a agentes limitantes de su crecimiento y mejor calidad nutricional y los animales domésticos modificados genéticamente, ofrecen un conjunto de ventajas necesarias para hacer frente a los desafíos globales que afectan a la vida de muchas personas en todo el mundo


Transgenesis is a parcel of biotechnology that allows the introduction of genetic information not pro-per to the genome of living beings, apart from the mechanisms of natural genetic exchange. This made possible to address important applications in bacteria, animals and plants with significant benefits in health, food and environmental aspects. Since its origin, the production of genetically modified organisms (GMOs) caused some controversy due to the possible negative influence of these organisms or their derived products on health and the environment. Over time, genetic modification techniques have renewed, giving way to others of greater precision, simplicity and safety. Currently the CRISPR-Cas9 technique is widely used, which allows to edit, modify or eliminate specific DNA sequences, with multiple applications in the same fields of transgenesis, but adding greater simplicity, security and lower cost. This work presents the main techniques, applications and ethical implications of using these methods and their perspectives in an ever-evolving world. The bacteria for obtaining products of pharmacological interest, new varieties of cultivated plants of higher production, more resistance to growth limiting agents and better nutritional quality and domestic animals modified genetically, offer a set of advantages needed to address the global challenges that affect the lives of many people around the world


Asunto(s)
Humanos , Técnicas de Transferencia de Gen/ética , Edición Génica/ética , Organismos Modificados Genéticamente , Factores de Riesgo , Bioética , Alimentos Modificados Genéticamente , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genoma/ética
8.
Med Sci (Paris) ; 36(8-9): 797-802, 2020.
Artículo en Francés | MEDLINE | ID: mdl-32755538

RESUMEN

SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2, which emerged in China at the end of 2019, is responsible for a global health crisis resulting in the confinement of more than 3 billion people worldwide and the sharp decline of the world economy. In this context, a race against the clock is launched in order to develop a treatment to stop the pandemic as soon as possible. A study published in Nature by the Volker Thiel team reports the development of reverse genetics for SARS-CoV-2 allowing them to recreate the virus in just a few weeks. The perspectives of this work are very interesting since it will allow the genetic manipulation of the virus and thus the development of precious tools which will be useful to fight the infection. Even though this approach represents a technological leap that will improve our knowledge of the virus, it also carries the germ of possible misuse and the creation of the virus for malicious purposes. The advantages and disadvantages of recreating SARS-CoV-2 in this pandemic period are discussed in this mini-synthesis.


Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/virología , Organismos Modificados Genéticamente , Pandemias , Neumonía Viral/virología , Genética Inversa/métodos , Betacoronavirus/patogenicidad , Derrame de Material Biológico , COVID-19 , Vacunas contra la COVID-19 , Cromosomas Artificiales de Levadura , Clonación Molecular/métodos , Coronaviridae/clasificación , Coronaviridae/genética , Coronaviridae/patogenicidad , Infecciones por Coronavirus/prevención & control , ADN Complementario/genética , Especificidad del Huésped , Humanos , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/patogenicidad , Pandemias/prevención & control , Neumonía Viral/prevención & control , ARN Viral/genética , Recombinación Genética , Riesgo , SARS-CoV-2 , Vacunas Virales
9.
Eur Rev Med Pharmacol Sci ; 24(8): 4558-4564, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32373995

RESUMEN

OBJECTIVE: SARS-CoV-2 is responsible for the present coronavirus pandemic and some suggestions were made about its possible artificial origin. We, therefore, compared SARS-CoV-2 with such known viruses that were prepared in the laboratory and other relevant natural strains to estimate their genetic relatedness. MATERIALS AND METHODS: BLAST and clustalW were used to identify and align viral sequences of SARS-CoV-2 to other animal coronaviruses (human, bat, mouse, pangolin) and related artificial constructs. Phylogenetics trees were then prepared using iTOL. RESULTS: Our study supports the notion that known artificial coronaviruses, including the chimeric SL-SHC014-MA15 synthesized in 2015, differ too much from SARS-CoV-2 to hypothesize an artificial origin of the latter. On the contrary, our data support the natural origin of the COVID-19 virus, likely derived from bats, possibly transferred to pangolins, before spreading to man. CONCLUSIONS: Speculations about the artificial origin of SARS-CoV-2 are most likely unfounded. On the contrary, when carefully handled, engineered organisms provide a unique opportunity to study biological systems in a controlled fashion. Biotechnology is a powerful tool to advance medical research and should not be abandoned because of irrational fears.


Asunto(s)
Betacoronavirus/clasificación , Biología Computacional , Filogenia , Secuenciación Completa del Genoma , Animales , COVID-19 , Quirópteros/virología , Infecciones por Coronavirus , Humanos , Ratones , Organismos Modificados Genéticamente , Pandemias , Neumonía Viral , ARN Viral/análisis , SARS-CoV-2 , Análisis de Secuencia de ARN
10.
Cell Host Microbe ; 27(5): 841-848.e3, 2020 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-32289263

RESUMEN

The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA. RNA transcribed from the full-genome cDNA was highly infectious after electroporation into cells, producing 2.9 × 106 plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectious-clone-derived SARS-CoV-2 (icSARS-CoV-2) exhibited similar plaque morphology, viral RNA profile, and replication kinetics. Additionally, icSARS-CoV-2 retained engineered molecular markers and did not acquire other mutations. We generated a stable mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) by introducing this reporter gene into ORF7 of the viral genome. icSARS-CoV-2-mNG was successfully used to evaluate the antiviral activities of interferon (IFN). Collectively, the reverse genetic system and reporter virus provide key reagents to study SARS-CoV-2 and develop countermeasures.


Asunto(s)
Betacoronavirus/genética , Betacoronavirus/patogenicidad , Infecciones por Coronavirus/virología , ADN Complementario/genética , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/patogenicidad , Neumonía Viral/virología , Animales , Antivirales/uso terapéutico , COVID-19 , Chlorocebus aethiops , Células Clonales , Infecciones por Coronavirus/tratamiento farmacológico , Genes Reporteros/genética , Genoma Viral/genética , Interferones/uso terapéutico , Pandemias , Neumonía Viral/tratamiento farmacológico , ARN Viral/genética , SARS-CoV-2 , Células Vero/virología , Replicación Viral/fisiología
11.
Viruses ; 11(6)2019 06 14.
Artículo en Inglés | MEDLINE | ID: mdl-31207934

RESUMEN

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) induces reproductive disorders in sows and respiratory illnesses in growing pigs and is considered as one of the main pathogenic agents responsible for economic losses in the porcine industry worldwide. Modified live PRRSV vaccines (MLVs) are very effective vaccine types against homologous strains but they present only partial protection against heterologous viral variants. With the goal to induce broad and cross-protective immunity, we generated DNA vaccines encoding B and T antigens derived from a European subtype 1 strain that include T-cell epitope sequences known to be conserved across strains. These antigens were expressed either in a native form or in the form of vaccibodies targeted to the endocytic receptor XCR1 and CD11c expressed by different types of antigen-presenting cells (APCs). When delivered in skin with cationic nanoparticles and surface electroporation, multiple DNA vaccinations as a stand-alone regimen induced substantial antibody and T-cell responses, which were not promoted by targeting antigens to APCs. Interestingly, a DNA-MLV prime-boost strategy strongly enhanced the antibody response and broadened the T-cell responses over the one induced by MLV or DNA-only. The anti-nucleoprotein antibody response induced by the DNA-MLV prime-boost was clearly promoted by targeting the antigen to CD11c and XCR1, indicating a benefit of APC-targeting on the B-cell response. In conclusion, a DNA-MLV prime-boost strategy, by enhancing the potency and breadth of MLV vaccines, stands as a promising vaccine strategy to improve the control of PRRSV in infected herds.


Asunto(s)
Anticuerpos Antivirales/sangre , Esquemas de Inmunización , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Linfocitos T/inmunología , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Formación de Anticuerpos , Inmunidad Celular , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Porcinos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genética
12.
Acta Virol ; 63(1): 53-59, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30879313

RESUMEN

The avian adeno-associated virus (AAAV) is a replication-defective nonpathogenic virus that has been proved to be useful as a viral vector in gene delivery. In this study, the feasibility of AAAV for transgenic expression of duck hepatitis A virus (DHAV) VP3 structural protein and its ability to induce protective immunity in ducklings was assessed. The recombinant AAAV (rAAAV-VP3) expressing the VP3 protein was prepared by co-infection of Sf9 cells with recombinant baculovirus (rBac-VP3) containing VP3 gene flanked by inverted terminal repeats (ITRs) of AAAV and the other two recombinant baculovirus expressing AAAV functional and structural genes, respectively. The generation of rAAAV-VP3 was demonstrated by electron microscopy, immunofluorescence assay, and western blot analysis. One day old ducklings were inoculated with rAAAV-VP3 or commercial attenuated vaccine and then challenged with DHAV-1 strain SH two weeks post vaccination. Anti-DHAV-1 antibodies were detected in all vaccinated groups by ELISA, and the titers between the rAAAV-VP3 group and the attenuated vaccine group were not statistically significant. Real time RT-PCR analysis showed that the virus copy numbers in the livers of the PBS control group were significantly higher than that of the rAAAV-VP3 and attenuated vaccine groups. In conclusion, we demonstrated that the VP3 expression mediated by rAAAV in ducklings could induce protective immunity against DHAV challenge, and this could be a candidate vaccine for the control of duck viral hepatitis. Keywords: avian adeno-associated virus; duck hepatitis A virus; VP3 gene; immunogenicity.


Asunto(s)
Virus de la Hepatitis del Pato , Parvovirinae , Vacunas Virales , Animales , Anticuerpos Antivirales/sangre , Patos , Virus de la Hepatitis del Pato/genética , Virus de la Hepatitis del Pato/inmunología , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/inmunología , Parvovirinae/genética , Vacunas Atenuadas/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Vacunas Virales/inmunología
13.
Ticks Tick Borne Dis ; 10(3): 722-728, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30711475

RESUMEN

Babesia bovis, a tick-borne apicomplexan parasite responsible for bovine babesiosis has a complex life cycle including sexual development in its Rhipicephalus microplus vector. Understanding the molecular mechanisms involved in sexual development is essential for developing future-generation transmission blocking vaccines (TBVs) and/or non-transmissible attenuated live vaccines. The widely conserved members of the 6-Cys gene family likely play roles in the development of sexual stages of B. bovis, and are candidates for developing novel TBV. The recently defined sexual markers 6-CysA and 6-CysB of B. bovis are strain-conserved and exclusively surface-expressed in tick-stage parasites. However, the high level of sequence identity among the 6-Cys A and 6-Cys B proteins (52% identity), together with similar 6-Cys domain distribution and sub-cellular localization, are suggestive of redundant function. We hypothesized that disruption of both 6-CysA and 6-CysB in B. bovis would result in unaltered ability of the parasite to invade and grow in red blood cells (RBCs), with concomitant loss of the transmission phenotype. Taking advantage of their contiguous genome localization, we generated a double gene-knockout system to disrupt a 3287 bp region encompassing both 6-CysA and 6-CysB genes using a single transfection plasmid. The resulting red-fluorescent ΔAΔB 6-Cys B. bovis transgenic parasite line was able to grow continuously in bovine RBCs in vitro at a similar rate to wild-type parasites, demonstrating that the 6-CysA and 6-CysB genes are not required for the development of blood-stage parasites. This novel gene manipulation approach will allow future experiments aimed at determining the tick-transmission phenotype of parasites lacking tick-stage genes. Parasites deficient in genes required for sexual reproduction could be the foundation for genetically-defined, non-transmissible live vaccines against bovine babesiosis. Developing a non-tick transmissible live vaccine based on attenuated parasites unable to express critical 6-Cys genes and including a molecular vaccine marker could help reduce the burden of bovine babesiosis globally.


Asunto(s)
Babesia bovis/genética , Babesiosis/prevención & control , Enfermedades de los Bovinos/prevención & control , Genes Protozoarios , Organismos Modificados Genéticamente , Vacunas Antiprotozoos/genética , Animales , Bovinos , Enfermedades de los Bovinos/parasitología , Técnicas de Inactivación de Genes , Estadios del Ciclo de Vida/genética , Fenotipo , Transfección , Vacunas Atenuadas/genética
14.
Rev Sci Tech ; 37(1): 131-139, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30209423

RESUMEN

Proteins are involved in a majority of the biochemical events that take place in all living organisms. Protein synthesis is directed by genes. All genes contain two major DNA regions. The region containing the genetic message proper (the 'coding region') is preceded by a regulatory region ('the promoter'), which determines when and in which organs a given gene must produce the corresponding protein. The techniques of genetic engineering allow the association of the coding region from one gene with the regulatory region from another gene. The expression of these recombinant genes may be achieved in cultured cells, in transgenic animals or in plants. This leads to the production of the corresponding proteins, including pharmaceutical proteins. Milk from transgenic animals is one potential source of pharmaceutical proteins. To achieve this, the promoters from milk protein genes are bound to DNA fragments containing the coding region of the genes of interest. The desired proteins are then taken from the milk and purified. Two human pharmaceutical proteins are on the market and about 20 projects are in development. One of the proteins produced in milk, antithrombin III, is an anticoagulant and the other, human C1-esterase inhibitor, is an anti-inflammatory. Several human proteins have been produced in the egg white of transgenic chickens and one has been approved by the United States Federal Drug Administration. This process has also been used to modify antibodies in cows. The genes that code for antibodies in the cow were deleted and replaced by human antibody genes. These cows, immunised by various antigens, then secreted purely human antibodies in their blood. Antibodies from such cows were able to attenuate the effects of Ebola virus in human patients.


Asunto(s)
Animales Modificados Genéticamente , Mamíferos/metabolismo , Leche/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Animales , Productos Biológicos , Regulación de la Expresión Génica , Humanos , Mamíferos/genética , Preparaciones Farmacéuticas/metabolismo , Proteínas Recombinantes/química
15.
Am J Trop Med Hyg ; 95(2): 378-82, 2016 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-27296385

RESUMEN

Whole parasite immunization strategies employing genetically attenuated parasites (GAP), which arrest during liver-stage development, have been applied successfully for induction of sterile malaria protection in rodents. Recently, we generated a Plasmodium berghei GAP-lacking expression of multidrug resistance-associated protein (MRP2) (PbΔmrp2) that was capable of partial schizogony in hepatocytes but showed complete growth arrest. Here, we investigated the protective efficacy after intravenous (IV) immunization of BALB/c and C57BL/6J mice with PbΔmrp2 sporozoites. Low-dose immunization using 400 PbΔmrp2 sporozoites induced 100% sterile protection in BALB/c mice after IV challenge with 10,000 wild-type sporozoites. In addition, almost full protection (90%) was obtained after three immunizations with 10,000 sporozoites in C57BL/6J mice. Parasite liver loads in nonprotected PbΔmrp2-challenged C57BL/6J mice were reduced by 86% ± 5% on average compared with naive control mice. The mid-to-late arresting PbΔmrp2 GAP was equipotent in induction of protective immunity to the early arresting PbΔb9Δslarp GAP. The combined data support a clear basis for further exploration of Plasmodium falciparum parasites lacking mrp2 as a suitable GAP vaccine candidate.


Asunto(s)
Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/inmunología , Parasitemia/prevención & control , Plasmodium berghei/inmunología , Proteínas Protozoarias/inmunología , Esporozoítos/inmunología , Animales , Relación Dosis-Respuesta Inmunológica , Femenino , Inmunización , Hígado/inmunología , Hígado/parasitología , Malaria/inmunología , Malaria/parasitología , Vacunas contra la Malaria/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/deficiencia , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Organismos Modificados Genéticamente/inmunología , Organismos Modificados Genéticamente/metabolismo , Parasitemia/inmunología , Parasitemia/parasitología , Plasmodium berghei/genética , Proteínas Protozoarias/genética , Esporozoítos/metabolismo , Vacunas Atenuadas
16.
PLoS Biol ; 13(11): e1002288, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26565995

RESUMEN

The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.


Asunto(s)
Retículo Endoplásmico/enzimología , Glicerofosfolípidos/metabolismo , Proteínas Protozoarias/metabolismo , Treonina/análogos & derivados , Toxoplasma/fisiología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Animales , Encéfalo/inmunología , Encéfalo/parasitología , Encéfalo/patología , Células Cultivadas , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Organismos Modificados Genéticamente/inmunología , Organismos Modificados Genéticamente/metabolismo , Enquistamiento de Parásito , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Vacunas Antiprotozoos/uso terapéutico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Piel/citología , Piel/inmunología , Piel/metabolismo , Piel/parasitología , Treonina/metabolismo , Toxoplasma/genética , Toxoplasma/inmunología , Toxoplasma/patogenicidad , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Toxoplasmosis/patología , Toxoplasmosis/prevención & control , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Vacunas Atenuadas/uso terapéutico , Virulencia
17.
Int J Surg ; 23(Pt B): 199-204, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26188183

RESUMEN

The limited availability of deceased human organs and cells for the purposes of clinical transplantation remains critical worldwide. Despite the increasing utilization of 'high-risk', 'marginal', or 'extended criteria' deceased donors, in the U.S. each day 30 patients either die or are removed from the waiting list because they become too sick to undergo organ transplantation. In certain other countries, where there is cultural resistance to deceased donation, e.g., Japan, the increased utilization of living donors, e.g., of a single kidney or partial liver, only very partially addresses the organ shortage. For transplants of tissues and cells, e.g., pancreatic islet transplantation for patients with diabetes, and corneal transplantation for patients with corneal blindness (whose numbers worldwide are potentially in the millions), allotransplantation will never prove a sufficient source. There is an urgent need for an alternative source of organs and cells. The pig could prove to be a satisfactory source, and clinical xenotransplantation using pig organs or cells, particularly with the advantages provided by genetic engineering to provide resistance to the human immune response, may resolve the organ shortage. The physiologic compatibilities and incompatibilities of the pig and the human are briefly reviewed.


Asunto(s)
Porcinos , Obtención de Tejidos y Órganos , Trasplante Heterólogo , Trasplantes/provisión & distribución , Animales , Animales Modificados Genéticamente , Trasplante de Córnea , Cultura , Ingeniería Genética , Humanos , Trasplante de Islotes Pancreáticos , Donadores Vivos , Porcinos/genética
18.
Braz. j. microbiol ; 45(4): 1117-1129, Oct.-Dec. 2014. tab
Artículo en Inglés | LILACS | ID: lil-741261

RESUMEN

Genetically attenuated microorganisms, pathogens, and some commensal bacteria can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system, while still offering good levels of safety. A key feature of these live vectors is their capacity to stimulate mucosal as well as humoral and/or cellular systemic immunity. This enables the use of different forms of vaccination to prevent pathogen colonization of mucosal tissues, the front door for many infectious agents. Furthermore, delivery of DNA vaccines and immune system stimulatory molecules, such as cytokines, can be achieved using these special carriers, whose adjuvant properties and, sometimes, invasive capacities enhance the immune response. More recently, the unique features and versatility of these vectors have also been exploited to develop anti-cancer vaccines, where tumor-associated antigens, cytokines, and DNA or RNA molecules are delivered. Different strategies and genetic tools are constantly being developed, increasing the antigenic potential of agents delivered by these systems, opening fresh perspectives for the deployment of vehicles for new purposes. Here we summarize the main characteristics of the different types of live bacterial vectors and discuss new applications of these delivery systems in the field of vaccinology.


Asunto(s)
Animales , Humanos , Vacunas Bacterianas/inmunología , Portadores de Fármacos , Infecciones Bacterianas/prevención & control , Vacunas Bacterianas/genética , Neoplasias/terapia , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/inmunología , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología
19.
Braz J Microbiol ; 45(4): 1117-29, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25763014

RESUMEN

Genetically attenuated microorganisms, pathogens, and some commensal bacteria can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system, while still offering good levels of safety. A key feature of these live vectors is their capacity to stimulate mucosal as well as humoral and/or cellular systemic immunity. This enables the use of different forms of vaccination to prevent pathogen colonization of mucosal tissues, the front door for many infectious agents. Furthermore, delivery of DNA vaccines and immune system stimulatory molecules, such as cytokines, can be achieved using these special carriers, whose adjuvant properties and, sometimes, invasive capacities enhance the immune response. More recently, the unique features and versatility of these vectors have also been exploited to develop anti-cancer vaccines, where tumor-associated antigens, cytokines, and DNA or RNA molecules are delivered. Different strategies and genetic tools are constantly being developed, increasing the antigenic potential of agents delivered by these systems, opening fresh perspectives for the deployment of vehicles for new purposes. Here we summarize the main characteristics of the different types of live bacterial vectors and discuss new applications of these delivery systems in the field of vaccinology.


Asunto(s)
Vacunas Bacterianas/inmunología , Portadores de Fármacos , Animales , Infecciones Bacterianas/prevención & control , Vacunas Bacterianas/genética , Humanos , Neoplasias/terapia , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/inmunología , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología
20.
Mol Ther ; 21(2): 485-92, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23247101

RESUMEN

The extracellular domain of M2 (M2e), a small ion channel membrane protein, is well conserved among different human influenza A virus strains. To improve the protective efficacy of M2e vaccines, we genetically engineered a tandem repeat of M2e epitope sequences (M2e5x) of human, swine, and avian origin influenza A viruses, which was expressed in a membrane-anchored form and incorporated in virus-like particles (VLPs). The M2e5x protein with the transmembrane domain of hemagglutinin (HA) was effectively incorporated into VLPs at a several 100-fold higher level than that on influenza virions. Intramuscular immunization with M2e5x VLP vaccines was highly effective in inducing M2e-specific antibodies reactive to different influenza viruses, mucosal and systemic immune responses, and cross-protection regardless of influenza virus subtypes in the absence of adjuvant. Importantly, immune sera were found to be sufficient for conferring protection in naive mice, which was long-lived and cross-protective. Thus, molecular designing and presenting M2e immunogens on VLPs provide a promising platform for developing universal influenza vaccines without using adjuvants.


Asunto(s)
Protección Cruzada , Inmunoglobulina G/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Proteínas de la Matriz Viral/genética , Virión/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Línea Celular , Reacciones Cruzadas , Modelos Animales de Enfermedad , Femenino , Humanos , Sueros Inmunes/genética , Sueros Inmunes/inmunología , Inmunoglobulina G/genética , Vacunas contra la Influenza/farmacología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Organismos Modificados Genéticamente , Spodoptera/genética , Secuencias Repetidas en Tándem , Proteínas de la Matriz Viral/inmunología , Virión/química
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...