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1.
Hum Vaccin ; 7 Suppl: 41-50, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21266846

RESUMEN

In 2009, a novel H1N1 swine influenza virus was isolated from infected humans in Mexico and the United States, and rapidly spread around the world. Another virus, a highly pathogenic avian influenza virus of the H5N1 subtype, identified by the World Health Organization as a potential pandemic threat in 1997, continues to be a significant risk. While vaccination is the preferred strategy for the prevention and control of influenza infections, the traditional egg-based approach to producing influenza vaccines does not provide sufficient capacity and adequate speed to satisfy global needs to combat newly emerging strains, seasonal or potentially pandemic. Significant efforts are underway to develop and implement new cell substrates with improved efficiency for influenza vaccine development and manufacturing. In recent years, plants have been used to produce recombinant proteins including subunit vaccines and antibodies. The main advantages of using plant systems for the production of vaccine antigens against influenza are their independence from pathogenic viruses, and cost and time efficiency. Here, we describe the large-scale production of recombinant hemagglutinin proteins from A/California/04/09 (H1N1) and A/Indonesia/05/05 (H5N1) strains of influenza virus in Nicotiana benthamiana plants, and their immunogenicity (serum hemagglutination inhibition and virus neutralizing antibodies), and safety in animal models. These results support the testing of these candidate vaccines in human volunteers and also the utility of our plant expression system for large-scale recombinant influenza vaccine production.


Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Plantas Modificadas Genéticamente/metabolismo , Animales , Anticuerpos Antivirales/sangre , Biotecnología/métodos , Hurones , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/efectos adversos , Vacunas contra la Influenza/genética , Gripe Humana/prevención & control , Ratones , Ratones Endogámicos BALB C , Plantas Modificadas Genéticamente/genética , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Tecnología Farmacéutica/métodos , Nicotiana/genética , Vacunas de Subunidad/efectos adversos , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología
2.
Hum Vaccin ; 7 Suppl: 191-8, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21266847

RESUMEN

Malaria is a serious and sometimes fatal mosquito-borne disease caused by a protozoan parasite. Each year, it is estimated that over one million people are killed by malaria, yet the disease is preventable and treatable. Developing vaccines against the parasite is a critical component in the fight against malaria and these vaccines can target different stages of the pathogen's life cycle. We are targeting sexual stage proteins of P. falciparum which are found on the surface of the parasite reproductive cells present in the mosquito gut. Antibodies against these proteins block the progression of the parasite's life cycle in the mosquito, and thus block transmission to the next human host. Transmission blocking vaccines are essential to the malaria eradication program to ease the disease burden at the population level. We have successfully produced multiple versions of the Pfs25 antigen in a plant virus-based transient expression system and have evaluated these vaccine candidates in an animal model. The targets are expressed in plants at a high level, are soluble and most importantly, generate strong transmission blocking activity as determined by a standard membrane feeding assay. These data demonstrate the feasibility of expressing Plasmodium antigens in a plant-based system for the economic production of a transmission blocking vaccine against malaria.


Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Transmisión de Enfermedad Infecciosa/prevención & control , Vacunas contra la Malaria/inmunología , Malaria Falciparum/transmisión , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Animales , Culicidae/parasitología , Culicidae/fisiología , Conducta Alimentaria , Vacunas contra la Malaria/administración & dosificación , Ratones , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Nicotiana
3.
Am J Trop Med Hyg ; 98(2): 420-431, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29231157

RESUMEN

Yellow fever (YF) is a viral disease transmitted by mosquitoes and endemic mostly in South America and Africa with 20-50% fatality. All current licensed YF vaccines, including YF-Vax® (Sanofi-Pasteur, Lyon, France) and 17DD-YFV (Bio-Manguinhos, Rio de Janeiro, Brazil), are based on live attenuated virus produced in hens' eggs and have been widely used. The YF vaccines are considered safe and highly effective. However, a recent increase in demand for YF vaccines and reports of rare cases of YF vaccine-associated fatal adverse events have provoked interest in developing a safer YF vaccine that can be easily scaled up to meet this increased global demand. To this point, we have engineered the YF virus envelope protein (YFE) and transiently expressed it in Nicotiana benthamiana as a stand-alone protein (YFE) or as fusion to the bacterial enzyme lichenase (YFE-LicKM). Immunogenicity and challenge studies in mice demonstrated that both YFE and YFE-LicKM elicited virus neutralizing (VN) antibodies and protected over 70% of mice from lethal challenge infection. Furthermore, these two YFE-based vaccine candidates induced VN antibody responses with high serum avidity in nonhuman primates and these VN antibody responses were further enhanced after challenge infection with the 17DD strain of YF virus. These results demonstrate partial protective efficacy in mice of YFE-based subunit vaccines expressed in N. benthamiana. However, their efficacy is inferior to that of the live attenuated 17DD vaccine, indicating that formulation development, such as incorporating a more suitable adjuvant, may be required for product development.


Asunto(s)
Modelos Animales de Enfermedad , Vacuna contra la Fiebre Amarilla/biosíntesis , Fiebre Amarilla/prevención & control , Animales , Ensayo de Immunospot Ligado a Enzimas/métodos , Humanos , Ratones/inmunología , Pruebas de Neutralización/métodos , Fiebre Amarilla/tratamiento farmacológico , Vacuna contra la Fiebre Amarilla/inmunología , Vacuna contra la Fiebre Amarilla/uso terapéutico , Virus de la Fiebre Amarilla/inmunología
4.
Cell Rep ; 22(7): 1798-1809, 2018 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-29444432

RESUMEN

Highly effective HIV-1-neutralizing antibodies could have utility in the prevention or treatment of HIV-1 infection. To improve the potency of 10E8, an antibody capable of near pan-HIV-1 neutralization, we engineered 10E8-surface mutants and screened for improved neutralization. Variants with the largest functional enhancements involved the addition of hydrophobic or positively charged residues, which were positioned to interact with viral membrane lipids or viral glycan-sialic acids, respectively. In both cases, the site of improvement was spatially separated from the region of antibody mediating molecular contact with the protein component of the antigen, thereby improving peripheral semi-specific interactions while maintaining unmodified dominant contacts responsible for broad recognition. The optimized 10E8 antibody, with mutations to phenylalanine and arginine, retained the extraordinary breadth of 10E8 but with ∼10-fold increased potency. We propose surface-matrix screening as a general method to improve antibodies, with improved semi-specific interactions between antibody and antigen enabling increased potency without compromising breadth.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Membrana Celular/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Semivida , Humanos , Pruebas de Neutralización , Polisacáridos/metabolismo , Unión Proteica
5.
J Biomed Mater Res A ; 104(1): 113-23, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26223817

RESUMEN

In this study, hydrophilic and hydrolytically degradable poly (ethylene glycol) (PEG) hydrogels were formed via Michael-type addition and employed for sustained delivery of a monoclonal antibody against the protective antigen of anthrax. Taking advantage of the PEG-induced precipitation of the antibody, burst release from the matrix was avoided. These hydrogels were able to release active antibodies in a controlled manner from 14 days to as long as 56 days in vitro by varying the polymer architectures and molecular weights of the precursors. Analysis of the secondary and tertiary structure and the in vitro activity of the released antibody showed that the encapsulation and release did not affect the protein conformation or functionality. The results suggest the promise for developing PEG-based carriers for sustained release of therapeutic antibodies against toxins in various applications.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Hidrogeles/química , Polietilenglicoles/química , Antígenos Bacterianos/química , Toxinas Bacterianas/química , Cromatografía en Gel , Dicroismo Circular , Preparaciones de Acción Retardada , Electroforesis en Gel de Poliacrilamida , Glicosilación , Hidrólisis , Polietilenglicoles/síntesis química , Conformación Proteica
6.
Biochim Biophys Acta ; 1699(1-2): 123-30, 2004 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-15158719

RESUMEN

Cyt1A is a cytolytic toxin produced by Bacillus thuringiensis var. israelensis. Due to its toxicity in vivo against mosquitoes and black flies, it is used as an environmentally friendly insecticide, although its mode of action is not completely understood. The toxin is membrane-active, but its membrane-bound conformation is unknown. In the absence of direct structural data, fluorescence spectroscopy was used to obtain indirect information on Cyt1A conformation changes in the environment mimicking the vicinity of the lipid membrane (lower pH and increased ionic strength). With decreasing pH, Cyt1A's surface hydrophobicity increased, which is consistent with an increased interaction with model membranes at low pH values, as observed previously. The pK(a) value of this conformation change is 4.4+/-0.1. Intrinsic tryptophan fluorescence decreased with decreasing pH, and the pK(a) value was the same as the one determined with synthetic probes. The protein has two types of hydrophobic binding sites, and at low pH these sites bind more probe molecules (bis-ANS) with a higher affinity than at pH 7.4. When bound to the lipid, the toxin exhibited conformation similar to the molten-globule state and showed some characteristics also observed at low pH. However, the conformation of the lipid-bound toxin did not depend on pH. Neutral salts like NaCl and KCl induced conformational changes at neutral pH, but not at low pH. These changes were most probably due to specific interactions of the salt ions with the charged amino acids on the protein surface rather than due to general effects such as Hofmeister and Debye-Hückel. Our results might contribute to elucidating the mode of action of Cyt1A, and perhaps also to improving the formulation of the insecticidal preparations.


Asunto(s)
Proteínas Bacterianas/química , Toxinas Bacterianas/química , Endotoxinas/química , Insecticidas/química , Conformación Proteica , Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis , Sitios de Unión , Proteínas Hemolisinas , Concentración de Iones de Hidrógeno , Cinética , Concentración Osmolar , Espectrometría de Fluorescencia , Triptófano
7.
Hum Vaccin Immunother ; 9(3): 544-52, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23324615

RESUMEN

The potential use of Bacillus anthracis as a bioterrorism weapon threatens the security of populations globally, requiring the immediate availability of safe, efficient and easily delivered anthrax vaccine for mass vaccination. Extensive research efforts have been directed toward the development of recombinant subunit vaccines based on protective antigen (PA), the principal virulence factor of B. anthracis. Among the emerging technologies for the production of these vaccine antigens is our launch vector-based plant transient expression system. Using this system, we have successfully engineered, expressed, purified and characterized full-length PA (pp-PA83) in Nicotiana benthamiana plants using agroinfiltration. This plant-produced antigen elicited high toxin neutralizing antibody titers in mice and rabbits after two vaccine administrations with Alhydrogel. In addition, immunization with this vaccine candidate protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. The vaccine effects were dose-dependent and required the presence of Alhydrogel adjuvant. In addition, the vaccine antigen formulated with Alhydrogel was stable and retained immunogenicity after two-week storage at 4°C, the conditions intended for clinical use. These results support the testing of this vaccine candidate in human volunteers and the utility of our plant expression system for the production of a recombinant anthrax vaccine.


Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Aerosoles , Hidróxido de Aluminio/administración & dosificación , Animales , Carbunco/inmunología , Vacunas contra el Carbunco/administración & dosificación , Anticuerpos Antibacterianos/sangre , Anticuerpos Neutralizantes/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/aislamiento & purificación , Toxinas Bacterianas/genética , Toxinas Bacterianas/aislamiento & purificación , Modelos Animales de Enfermedad , Exposición por Inhalación , Ratones Endogámicos BALB C , Plantas Modificadas Genéticamente/genética , Conejos , Análisis de Supervivencia , Nicotiana/genética , Resultado del Tratamiento , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología
8.
Biochemistry ; 44(2): 589-97, 2005 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-15641784

RESUMEN

The cytolytic delta-endotoxin Cyt1A from Bacillus thuringiensis var. israelensis is used in commercial preparations of environmentally safe insecticides. The current hypothesis on its mode of action is that the toxin self-assembles into well-defined cation-selective channels or pores, which results in colloid-osmotic lysis of the cell. Recently, a new hypothesis has been put forward suggesting that Cyt1A rather nonspecifically aggregates on the membrane surface and acts in a detergent-like manner. To distinguish between these two hypotheses, we investigated whether in the presence of lipid Cyt1A self-assembles into stoichiometric oligomers, which are characteristic of pores or channels, or aggregates into nonstoichiometric complexes, which would support the detergent-like model. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that in the presence of lipid Cyt1A forms protein aggregates with a broad range of molecular weights, some being too large to enter the gel. Cyt1A tryptophan (Trp) fluorescence in the presence of lipid exhibited a decrease in anisotropy and quantum yield, but an unchanged lifetime, which is consistent with the presence of toxin aggregates in the membrane. Electrostatic interactions between the charged amino acid residues and the lipid headgroups are responsible for bringing the protein to the membrane surface, while hydrophobic and/or van der Waals interactions make the membrane binding irreversible. Fluorescence photobleaching recovery, a technique that measures the diffusion coefficient of fluorescently labeled particles, and epifluorescence microscopy revealed that upon addition of Cyt1A lipid vesicles were broken into smaller, faster diffusing objects. Since no change in size or morphology of the vesicles is expected when pores are formed in the osmotically equilibrated membranes, our results support the detergent-like mode of action of Cyt1A.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidad , Detergentes/toxicidad , Endotoxinas/química , Endotoxinas/toxicidad , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Electroforesis en Gel de Poliacrilamida , Endotoxinas/metabolismo , Polarización de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Proteínas Hemolisinas , Liposomas , Potenciales de la Membrana/efectos de los fármacos , Microscopía Fluorescente , Peso Molecular , Presión Osmótica/efectos de los fármacos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Unión Proteica/efectos de los fármacos , Electricidad Estática , Triptófano/metabolismo , Tirosina/metabolismo
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