Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes

<p>Abstract</p> <p>Background</p> <p>The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally.</p> <p>Results</p> <p>We developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 μg of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-γ and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 μg).</p> <p>Conclusion</p> <p>Our objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.</p

Correlation of the physicochemical and structural properties of pDNA/cationic liposome complexes with their in vitro transfection

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R±) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R± = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R± is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R± to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R± value. Interestingly, for R± = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R±) in the system. This information is used to explain the transfection differences observed at an R± = 9 as compared to R± = 3 and 6. Close to the isoneutrality region (R± = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process28311153511545FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

Correlation Of The Physicochemical And Structural Properties Of Pdna/cationic Liposome Complexes With Their In Vitro Transfection.

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R(±)) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R(±) = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R(±) is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R(±) to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R(±) value. Interestingly, for R(±) = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R(±)) in the system. This information is used to explain the transfection differences observed at an R(±) = 9 as compared to R(±) = 3 and 6. Close to the isoneutrality region (R(±) = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process.2811535-4

High adhesion strength and hybrid irreversible/reversible full-PDMS microfluidic chips

To the best of our knowledge, this paper outlines for the first time high adhesion and hybrid irreversible/reversible microfluidic devices fully composed of polydimethylsiloxane (PDMS). These chips were fabricated by the sandwich bonding (SWB), a method that was recently deployed by our group. SWB offers simple, fast, and low cost operation requiring only a laboratory oven. The devices showed burst pressures of up to 4.5 MPa. This value is more than tenfold the pressures withstood by the full-PDMS chips described in literature. In terms of the reversible behavior, the ability for disassembling the chip slides is crucial in research and development stages, especially when the device integrates high-cost components or harsh cleaning steps are needed. Following successive steps of detachment and bonding, the channels still withstood high pressures of approximately 1.8 MPa. Finally, the emulsification of corn oil 4.0% w/w to polyglycerol polyricinoleate with 10.0 μmol L−1 rhodamine B aqueous solution was realized to show the relevance in enhancing the flow rate in microfluidics. Such experiment was conducted at total flow rates of 0.8–160.0 μL min−1. The decrease in size and polydispersity of the droplets was observed at increasing flow rates. Monodisperse emulsions were achieved only at 160.0 μL min−1951116123FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2014/24126-

Protective efficacy of different strategies employing Mycobacterium leprae heat-shock protein 65 against tuberculosis

Tuberculosis is a major threat to human health. The high disease burden remains unaffected and the appearance of extremely drug-resistant strains in different parts of the world argues in favor of the urgent need for a new effective vaccine. One of the promising candidates is heat-shock protein 65 when used as a genetic vaccine (DNAhsp65). Nonetheless, there are substantial data indicating that BCG, the only available anti-TB vaccine for clinical use, provides other important beneficial effects in immunized infants. Methods: We compared the protective efficacy of BCG and Hsp65 antigens in mice using different strategies: i) BCG, single dose subcutaneously; ii) naked DNAhsp65, four doses, intramuscularly; iii) liposomes containing DNAhsp65, single dose, intranasally; iv) microspheres containing DNAhsp65 or rHsp65, single dose, intramuscularly; and v) prime–boost with subcutaneous BCG and intramuscular DNAhsp65. Results: All the immunization protocols were able to protect mice against infection, with special benefits provided by DNAhsp65 in liposomes and prime–boost strategies. Conclusion: Among the immunization protocols tested, liposomes containing DNAhsp65 represent the most promising strategy for the development of a new anti-TB vaccine8912551264sem informaçãosem informaçã

Protective efficacy of different strategies employing Mycobacterium leprae heat-shock protein 65 against tuberculosis

Background: Tuberculosis is a major threat to human health. The high disease burden remains unaffected and the appearance of extremely drug-resistant strains in different parts of the world argues in favor of the urgent need for a new effective vaccine. One of the promising candidates is heat-shock protein 65 when used as a genetic vaccine (DNAhsp65). Nonetheless, there are substantial data indicating that BCG, the only available anti-TB vaccine for clinical use, provides other important beneficial effects in immunized infants. Methods: We compared the protective efficacy of BCG and Hsp65 antigens in mice using different strategies: i) BCG, single dose subcutaneously; ii) naked DNAhsp65, four doses, intramuscularly; iii) liposomes containing DNAhsp65, single dose, intranasally; iv) microspheres containing DNAhsp65 or rHsp65, single dose, intramuscularly; and v) prime-boost with subcutaneous BCG and intramuscular DNAhsp65. Results: All the immunization protocols were able to protect mice against infection, with special benefits provided by DNAhsp65 in liposomes and prime-boost strategies. Conclusion: Among the immunization protocols tested, liposomes containing DNAhsp65 represent the most promising strategy for the development of a new anti-TB vaccine

Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes-2

Osome (corresponding to 50 μg of DNA). Anti-hsp65 antibody levels were evaluated in mice serum by ELISA 30 days after the last immunization. ) IgG2a and ) IgG1 isotypes. Results are presented by mean ± SD of optical density. *p < 0.05 were considered significant when compared to saline group or their respective controls.<p><b>Copyright information:</b></p><p>Taken from "Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes"</p><p>http://www.biomedcentral.com/1471-2172/9/38</p><p>BMC Immunology 2008;9():38-38.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2500095.</p><p></p