Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma

Bacterial endotoxin adhesion to different types of orthodontic adhesives

Bacterial endotoxin (LPS) adhesion to orthodontic brackets is a known contributing factor to inflammation of the adjacent gingival tissues. Objective The aim of this study was to assess whether LPS adheres to orthodontic adhesive systems, comparing two commercial brands. Material and Methods Forty specimens were fabricated from Transbond XT and Light Bond composite and bonding agent components (n=10/component), then contaminated by immersion in a bacterial endotoxin solution. Contaminated and non-contaminated acrylic resin samples were used as positive and negative control groups, respectively. LPS quantification was performed by the Limulus Amebocyte Lysate QCL-1000™ test. Data obtained were scored and subjected to the Chi-square test using a significance level of 5%. Results There was endotoxin adhesion to all materials (p;0.05). There was no significant difference (p>;0.05) among commercial brands. Affinity of endotoxin was significantly greater for the bonding agents (p=0.0025). Conclusions LPS adhered to both orthodontic adhesive systems. Regardless of the brand, the endotoxin had higher affinity for the bonding agents than for the composites. There is no previous study assessing the affinity of LPS for orthodontic adhesive systems. This study revealed that LPS adheres to orthodontic adhesive systems. Therefore, additional care is recommended to orthodontic applications of these materials

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage

Abstract\ud \ud \ud \ud Background\ud \ud Vaccination of neonates is generally difficult due to the immaturity of the immune system and consequent higher susceptibility to tolerance induction. Genetic immunization has been described as an alternative to trigger a stronger immune response in neonates, including significant Th1 polarization. In this investigation we analysed the potential use of a genetic vaccine containing the heat shock protein (hsp65) from Mycobacterium leprae (pVAXhsp65) against tuberculosis (TB) in neonate mice. Aspects as antigen production, genomic integration and immunogenicity were evaluated.\ud \ud \ud \ud Methods\ud \ud Hsp65 message and genomic integration were evaluated by RT-PCR and Southern blot, respectively. Immunogenicity of pVAXhsp65 alone or combined with BCG was analysed by specific induction of antibodies and cytokines, both quantified by ELISA.\ud \ud \ud \ud Results\ud \ud This DNA vaccine was transcribed by muscular cells of neonate mice without integration into the cellular genome. Even though this vaccine was not strongly immunogenic when entirely administered (three doses) during early animal's life, it was not tolerogenic. In addition, pVAXhsp65 and BCG were equally able to prime newborn mice for a strong and mixed immune response (Th1 + Th2) to pVAXhsp65 boosters administered later, at the adult life.\ud \ud \ud \ud Conclusion\ud \ud These results suggest that pVAXhsp65 can be safely used as a priming stimulus in neonate animals in prime-boost similar strategies to control TB. However, priming with BCG or pVAXhsp65, directed the ensuing immune response triggered by an heterologous or homologous booster, to a mixed Th1/Th2 pattern of response. Measures as introduction of IL-12 or GM-CSF genes in the vaccine construct or even IL-4 neutralization, are probably required to increase the priming towards Th1 polarization to ensure control of tuberculosis infection.The authors are grateful to Secretaria da Saúde do Estado de São Paulo for providing BCG and to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) that supported this study with a grant (Proc. No. 03/06348-7).The authors are grateful to Secretaria da Saúde do Estado de São Paulo for providing BCG and to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) that supported this study with a grant (Proc. No. 03/063487)

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

Bacterial endotoxin adhesion to different types of orthodontic adhesives

Abstract Bacterial endotoxin (LPS) adhesion to orthodontic brackets is a known contributing factor to inflammation of the adjacent gingival tissues. Objective The aim of this study was to assess whether LPS adheres to orthodontic adhesive systems, comparing two commercial brands. Material and Methods Forty specimens were fabricated from Transbond XT and Light Bond composite and bonding agent components (n=10/component), then contaminated by immersion in a bacterial endotoxin solution. Contaminated and non-contaminated acrylic resin samples were used as positive and negative control groups, respectively. LPS quantification was performed by the Limulus Amebocyte Lysate QCL-1000™ test. Data obtained were scored and subjected to the Chi-square test using a significance level of 5%. Results There was endotoxin adhesion to all materials (p0.05). There was no significant difference (p>0.05) among commercial brands. Affinity of endotoxin was significantly greater for the bonding agents (p=0.0025). Conclusions LPS adhered to both orthodontic adhesive systems. Regardless of the brand, the endotoxin had higher affinity for the bonding agents than for the composites. There is no previous study assessing the affinity of LPS for orthodontic adhesive systems. This study revealed that LPS adheres to orthodontic adhesive systems. Therefore, additional care is recommended to orthodontic applications of these materials

IFN-gamma, IL-12, IL-10 and TNF-alpha in lung homogenates from immunized, infected mice and infected mice after 30 days of the infection

<p><b>Copyright information:</b></p><p>Taken from "Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming"</p><p>http://www.gvt-journal.com/content/5/1/7</p><p>Genetic Vaccines and Therapy 2007;5():7-7.</p><p>Published online 22 Aug 2007</p><p>PMCID:PMC2042972.</p><p></p> (A) IFN-gamma; (B) IL-12; (C) IL-10 and (D) TNF-alpha. (E) Correlation between CFU numbers and IFN-gamma production (F) Correlation between IFN-gamma and IL-10 production. Groups of 7 mice were immunized according table I and 15 days after the last immunization, they were challenged with H37Rv. After 30 days of infection, the lungs were removed and the cytokine production in lungs homogenates was analyzed. Bars represent the mean ± standard deviation. (A) ◆ BCGsc, BCGin, DNA-HSP65, BCGin/DNA and BCGsc/DNA vs Infected mice. * BCGin/DNA vs BCGsc, BCGin, and DNA-HSP65. (B) ◆ All immunized-infected mice vs Infected mice. * BCGin/DNA vs BCGsc, BCGin, DNA-HSP65 and BCGsc/DNA. ● BCGin vs BCGsc. (C) ◆ All immunized-infected mice vs Infected mice. * BCGin/DNA vs BCGsc, BCGin, DNA-HSP65. (D) ◆ All immunized-infected mice vs Infected mice. p < 0.05 was considered significant. Data are representative of two experiments

CD4/CD8cell numbers and expression of CD44CD62Lor CD44/CD62Lin the lungs of mice from the various experimental groups

<p><b>Copyright information:</b></p><p>Taken from "Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming"</p><p>http://www.gvt-journal.com/content/5/1/7</p><p>Genetic Vaccines and Therapy 2007;5():7-7.</p><p>Published online 22 Aug 2007</p><p>PMCID:PMC2042972.</p><p></p> (A) CD4/CD8cell numbers in the lungs. (B) Representative cell gating data showing the separation of effector cell populations. (C) CD44/CD62Lexpression on CD4and CD8lung cells. (D) CD44/CD62Lexpression on CD4and CD8lung cells. Groups of 7 mice were immunized according table I and 15 days after the last immunization, they were challenged with H37Rv. After 30 days of infection, the lungs were removed and analyzed by flow cytometry for cell population and expression of cell surface molecules. (A) ◆ All immunized-infected mice vs Infected mice. * BCGin/DNA vs BCGsc, BCGin and DNA-HSP65. (C) ◆ All immunized-infected mice vs Infected mice. * BCGin/DNA vs BCGin, BCGsc and DNA-HSP65.** BCGin/DNA vs BCGin. (D) ◆ All immunized-infected mice vs Infected mice. * BCGin/DNA vs BCGsc, BCGin, DNA-HSP65 and BCGsc/DNA. ** BCGin/DNA vs BCGsc. Bars represent the mean ± standard deviation. p < 0.05 was considered significant. Data are representative of two experiments

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage-0

<p><b>Copyright information:</b></p><p>Taken from "Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage"</p><p>http://www.gvt-journal.com/content/5/1/12</p><p>Genetic Vaccines and Therapy 2007;5():12-12.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2222600.</p><p></p>er intramuscular injection of 50 ug of pVAXhsp65. Total RNA (10 ug) isolated from each tissue was treated with DNase and subjected to RT-PCR amplification with specific primers. β-actin was amplified as an RNA quality control. All RT-PCR products were analysed by agarose gel electrophoresis and visualized by ethidium bromide staining. Similar results were observed in two animals analysed for each period. No products were seen (hsp65 and β-actin) when total RNA was subjected to PCR amplification in the absence of a previous reverse transcription

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage-3

<p><b>Copyright information:</b></p><p>Taken from "Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage"</p><p>http://www.gvt-journal.com/content/5/1/12</p><p>Genetic Vaccines and Therapy 2007;5():12-12.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2222600.</p><p></p>perimental groups were identified as DNA/DNA and BCG/DNA respectively. A non-immunized group and a group immunized with 3 pVAXhsp65 doses delivered at 5, 12 and 19-day-old were identified as control and neonate, respectively. Two weeks after last dose, the serum levels of IgG1 (a) and IgG2a (b) anti-hsp65 antibodies were evaluated by ELISA. Results represent the geometric mean ± SEM of 6 – 8 individually tested animals per group. *p < 0.05 in comparison to neonate group

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage-1

<p><b>Copyright information:</b></p><p>Taken from "Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage"</p><p>http://www.gvt-journal.com/content/5/1/12</p><p>Genetic Vaccines and Therapy 2007;5():12-12.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2222600.</p><p></p>a); IL-4 (b) and IL-5 (c) by splenic cells stimulated with ConA and serum levels of specific anti-hsp65 antibodies (d) were determined two weeks later. Results represent the geometric mean ± SEM of 4 to 8 individually tested animals per group. *p < 0.05 in comparison to vector group