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)

Decreased production of TNF-alpha by lymph node cells indicates experimental autoimmune encephalomyelitis remission in Lewis rats

Experimental autoimmune encephalomyelitis (EAE) is mediated by CD4+ Th1 cells that mainly secrete IFN-γ and TNF-α, important cytokines in the pathophysiology of the disease. Spontaneous remission is, in part, attributed to the down regulation of IFN-γ and TNF-α by TGF-β. In the current paper, we compared weight, histopathology and immunological parameters during the acute and recovery phases of EAE to establish the best biomarker for clinical remission. Female Lewis rats were immunised with myelin basic protein (MBP) emulsified with complete Freund's adjuvant. Animals were evaluated daily for clinical score and weight prior to euthanisation. All immunised animals developed the expected characteristics of EAE during the acute phase, including significant weight loss and high clinical scores. Disease remission was associated with a significant reduction in clinical scores, although immunised rats did not regain their initial weight values. Brain inflammatory infiltrates were higher during the acute phase. During the remission phase, anti-myelin antibody levels increased, whereas TNF-α and IFN-γ production by lymph node cells cultured with MBP or concanavalin A, respectively, decreased. The most significant difference observed between the acute and recovery phases was in the induction of TNF-α levels in MBP-stimulated cultures. Therefore, the in vitro production of this cytokine could be used as a biomarker for EAE remission

Decreased production of TNF-alpha by lymph node cells indicates experimental autoimmune encephalomyelitis remission in Lewis rats

Experimental autoimmune encephalomyelitis (EAE) is mediated by CD4+ Th1 cells that mainly secrete IFN-γ and TNF-α, important cytokines in the pathophysiology of the disease. Spontaneous remission is, in part, attributed to the down regulation of IFN-γ and TNF-α by TGF-β. In the current paper, we compared weight, histopathology and immunological parameters during the acute and recovery phases of EAE to establish the best biomarker for clinical remission. Female Lewis rats were immunised with myelin basic protein (MBP) emulsified with complete Freund's adjuvant. Animals were evaluated daily for clinical score and weight prior to euthanisation. All immunised animals developed the expected characteristics of EAE during the acute phase, including significant weight loss and high clinical scores. Disease remission was associated with a significant reduction in clinical scores, although immunised rats did not regain their initial weight values. Brain inflammatory infiltrates were higher during the acute phase. During the remission phase, anti-myelin antibody levels increased, whereas TNF-α and IFN-γ production by lymph node cells cultured with MBP or concanavalin A, respectively, decreased. The most significant difference observed between the acute and recovery phases was in the induction of TNF-α levels in MBP-stimulated cultures. Therefore, the in vitro production of this cytokine could be used as a biomarker for EAE remission.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Decreased production of TNF-alpha by lymph node cells indicates experimental autoimmune encephalomyelitis remission in Lewis rats

Experimental autoimmune encephalomyelitis (EAE) is mediated by CD4+ Th1 cells that mainly secrete IFN-γ and TNF-α, important cytokines in the pathophysiology of the disease. Spontaneous remission is, in part, attributed to the down regulation of IFN-γ and TNF-α by TGF-β In the current paper, we compared weight, histopathology and immunological parameters during the acute and recovery phases of EAE to establish the best biomarker for clinical remission. Female Lewis rats were immunised with myelin basic protein (MBP) emulsified with complete Freund’s adjuvant. Animals were evaluated daily for clinical score and weight prior to euthanisation. All immunised animals developed the expected characteristics of EAE during the acute phase, including significant weight loss and high clinical scores. Disease remission was associated with a significant reduction in clinical scores, although immunised rats did not regain their initial weight values. Brain inflammatory infiltrates were higher during the acute phase. During the remission phase, anti-myelin antibody levels increased, whereas TNF-α and IFN-γ production by lymph node cells cultured with MBP or concanavalin A, respectively, decreased. The most significant difference observed between the acute and recovery phases was in the induction of TNF-α levels in MBP-stimulated cultures. Therefore, the in vitro production of this cytokine could be used as a biomarker for EAE remission

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

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