A Novel Therapeutic and Prophylactic Vaccine (HVJ-Envelope / Hsp65 DNA + IL-12 DNA) against Tuberculosis Using the Cynomolgus Monkey Model

AbstractWe have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and –liposome (HSP65 + IL-12/HVJ). An IL-12 expression vector (IL-12DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine on the basis of C.F.U of number of TB, survival, an induction of the CD8 positive CTL activity and improvement of the histopathological tuberculosis lesions. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) (prolongation of survival time and the decrease in the number of TB in the lung) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. All monkeys in the control group (saline) died within 8 months, while 50% of monkeys in the HSP65+hIL-12/HVJ group survived more than 14 months post-infection (the termination period of the experiment). Furthermore, the BCG priming and HSP65 + IL-12/HVJ vaccine (booster) by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys from BCG Tokyo alone group were alive (33% survival). Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials

BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells

Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types, and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells.We have analyzed the ability for inducing differentiation of the heterodimer BMP-2/BMP-6 (BMP-2/6) compared to the homodimers BMP-2 or BMP-6, using human embryonic stem (hES) cells H9 as model system. When incubated in a medium with high concentration of basic fibroblastic growth factor (FGF2), 100 ng/ml of human recombinant BMPs induced morphological changes and differentiation of hES cells in 24 to 48 hours. After 5 days, expression of differentiation markers was induced and quantified by quantitative PCR (qPCR) and flow cytometry. BMP-2/6 exhibited stronger activity for the induction of the expression of trophectodermal (CDX2) and endodermal (SOX17, GATA4, AFP) markers than BMP-2 or BMP-6 homodimers. BMP-2/6 also induced the expression of BMPR2 gene more effectively than BMP-2 or BMP-6 when used at the same concentration and time. Moreover, the percentage of cells expressing the surface endodermal marker CXCR4 was also increased for the heterodimer when compared to both homodimers. BMP-2/6 was a more potent activator of Smad-dependent (SMAD1/5) and Smad-independent signaling (mitogen-activated protein kinases ERK and p38) than BMP-2 and BMP-6, and the activation of these pathways might play a role in its increased potency for inducing hES cell differentiation.Therefore, we conclude that BMP-2/6 is more potent than BMP-2 or BMP-6 for inducing differentiation of hES cells, and it can be used as a more powerful substitute of these BMPs in in vitro differentiation guidance