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)

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-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-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

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-cl, interleukin-6, transforming growth factor-beta, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright (C) 2008 John Wiley & Sons, Ltd.Fundacao de Amparo Pesquisa do Estado de Sao Paulo (FAPESP)[02/07064-0]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Instituto do Milenio-Rede Brasileira de Pesquisa em Tuberculose (REDE-TB, Brazilian Tuberculosis Research Network