A small step closer to the Holy Grail of DNA vaccines: undisputed clinical benefit in humans

A report of the DNA Vaccines 2008 meeting, Las Vegas, Nevada, USA, 9-11 December 2008

Pembuatan Aplikasi Sistem Informasi Persebaran Toko Batik Di Kota Pekalongan Berbasis Android

Pekalongan is located in the lowlands along the north coast of Java Island, at a height of approximately one meter above sea level. The geographical position of Pekalongan is between 6° 50\u27 42" to 6° 55\u27 44\u27\u27 North Latitude and 109° 37\u27 55\u27\u27to 109° 42\u27 19\u27\u27 East Longitude.Pekalongan area of about 42.25 km2 or approximately 0.14% from the area of Central Java Province.Pekalongan is known by the nickname city of Batik, because batik Pekalongan has distinctive and varied livery. Therefore, Pekalongan is espected to provide a practical and informative information for tourists, so it can be an attraction and increase the number of tourists coming to Pekalongan. This research uses of spatial and non spasial data in the form of batik store name, the owner\u27s name, phone number, address, and products sold with using the popularity of android smartphone as a platform system information.This application was developed using an SDK Andoidframework, java and PHP programming languages, and MySQL as base data, and Google Maps. The final result of this research is the Android application of the distribution of batik store in Pekalongan is accompanied by information (as has been mentioned above) from each store for easier searching the location of the batik stores in Pekalonga

Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell–BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS31073–1081 CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS31073 peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV

Development of vaccines and experimental models for chronic infections caused by the hepatitis C virus

The hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. It is estimated that HCV affects approximately 170 million people around the world. One feature of HCV infection is the high rate of viral persistence. The mechanism of viral persistence is largely unknown, although the high genetic variability is thought to play a key role. Today, no vaccine is available to prevent or cure HCV infections, albeit antiviral therapy is used quite effectively. This study aimed at developing new vaccines and new model systems to study HCV. We studied the HCV NS3 protein in detail since it performs key functions in the viral life cycle. These are unwinding and strand separation of the viral RNA and proteolytic processing of the precursor polyprotein. To obtain the complete protease we included the NS4A co-factor in our NS3-based vaccines. NS4A has been shown to enhance the stability of NS3 and to target the NS3/4A complex to intracellular membranes. The latter is most likely of importance for the formation of the replication complex. Also, the NS3 region has a limited genetic variability and several studies have now demonstrated that NS3-specific CD4+ and CD8+ T-cell responses are crucial for the resolution of HCV infections. Thus, several factors suggest that the NS3 region should be well suited for vaccine development. We could show that HCV NS3-based genetic vaccines effectively primed both humoral and cellular immune responses in mice. NS3/4A was shown to prime a Th1 CD4+ T-cell responses. The inclusion of NS4A in NS3-based vaccines primed antibody, CD4+, and CD8+ T-cell responses that were superior to those primed by NS3-gene alone. Thus, NS4A enhanced the immunogenicity of NS3. We could show that enhancement of the immunogenicity was most probably a result of the higher expression levels of NS3 generated by the inclusion of NS4A. We next tested if the overall immunogenicity of NS3/4A could be further enhanced by codon optimization or by mRNA amplification using the Semliki forest virus (SFV) replicon. The NS3 protein expression levels were further improved by either codon optimization and mRNA amplification. Subsequently, both these modifications enhanced the NS3-specific immune responses. One concern in development of genetic vaccines is that the gene displays unwanted properties when expressed in vivo. We therefore, generated a new transgenic mouse expressing the HCV NS3/4Aprotein in the liver. The protein expression was restricted to the liver to mimic the in vivo situation during a HCV infection. Protein expression was localized to the cytoplasm of the hepatocytes and displayed a similar staining pattern as seen in hepatocytes from HCV infected individuals. The intrahepatic protein expression did not cause overt liver damage, except for a slight enlargement of the liver. However, the NS3/4A-transgenic mice displayed less spontaneously appearing intrahepatic inflammatory foci, which are commonly found in laboratory mice. Thus, expression of NS3/4A-protein may affect the distribution of immune cells within the liver. The present studies demonstrate that NS3-based genetic vaccines that contain NS4A more effectively prime humoral and cellular immune responses against NS3. Intra-hepatic expression of NS3/4A did not cause any spontaneous liver disease or overt pathology suggesting that it safely can be used in genetic vaccines. Thus, the NS3/4A gene can safely activate immune responses that are similar to those found in humans who can clear HCV. The NS3/4A should therefore be a potential vaccine candidate against chronic HCV infections

Prospects and progress of DNA vaccines for treating hepatitis B

The hepatitis B virus (HBV) is a global cause of liver disease. The preventive HBV vaccine has effectively reduced the disease burden. However, an estimated 340 million chronic HBV cases are in need of treatment. Current standard therapy for chronic HBV blocks reversed transcription. As this therapy blocks viral maturation and not viral protein expression, any immune inhibition exerted by these proteins will remain throughout therapy. This may help to explain why these drugs rarely induce off-therapy responses. Albeit some restoration of immune function occurs during therapy, this is clearly insufficient to control replication. Central questions when considering therapeutic DNA vaccination as an addition to blocking virus production are as follows: what does one hope to achieve? What do we think is wrong and how can the vaccination correct this? We here discuss different scenarios with respect to the lack of success of tested DNA vaccines, and suggest strategies for improvement

A targeted controlled force injection of genetic material in vivo

A general limitation in gene delivery is the cellular uptake in lager animals including humans. Several approaches have been tested including liposomes, micro-needles, in vivo electro-transfer, ballistic delivery, and needle-free delivery. All these techniques have individual limitations. One approach reproducibly delivering genetic material in muscle tissue in nonhuman primates is hydrodynamic injection, a forced injection of a volume equaling the volume of the tissue to be transfected thereby causing an increased local pressure resulting in an improved uptake of genetic material. We transferred the principle of hydrodynamic injection to a device, where a small injection volume can be delivered to a targeted tissue volume, termed in vivo intracellular injection (IVIN). The device is based on needle(s) with apertures along the needle shafts, where multiple needles can fix the tissue volume to be transfected. The apertures direct the injection from a central needle outward or inward to the centroid of a geometric arrangement thereby targeting the tissue to be transfected. With a controlled force, this results in a targeted injection with increased transfection efficiency. We here show that the IVIN technology reproducibly improved plasmid uptake and expression and the immunogenicity. The IVIN technology can be generally applied to a targeted delivery of genetic materials

Hepatitis C virus nonstructural 3/4A protein dampens inflammation and contributes to slow fibrosis progression during chronic fibrosis in vivo

HCV infection typically induces liver injury and inflammation, which appears to be responsible for the associated fibrogenesis. To date, the mechanism underlying the different rates of disease progression remains unclear. The aim of the study is to understand the possible role of the HCV non-structural (NS) 3/4A protein in the fibrosis progression. We used NS3/4A-expressing transgenic mice (NS3/4A-Tg) to accomplish the goals of the study. Different stages of liver fibrosis were induced in wild-type and NS3/4A-Tg mice by single carbon tetrachloride (acute) or multiple injections for 4 (intermediate) or 8 (chronic) weeks. Fibrotic parameters, inflammatory responses and hepatocyte turnover were extensively examined. Hepatic expression of HCV NS3/4A did not induce spontaneous liver damage. However, NS3/4A expression exerted contrasting effects during acute and chronic liver damage. During early fibrogenesis and intermediate fibrosis (4 weeks), NS3/4A-Tg mice exhibited enhanced liver damage whereas reduced fibrosis was observed in NS3/4A-Tg during chronic liver fibrosis (8 weeks). Furthermore, attenuated inflammation was observed in NS3/4A-Tg during chronic fibrosis with increase in M2 macrophages, hepatocyte proliferation, decreased hepatocyte apoptosis and decreased ductular reaction. In conclusion, during early fibrogenesis, HCV NS3/4A contributes to liver damage. While, during chronic liver fibrosis, NS3/4A dampens inflammation and induces hepatocyte regeneration thereby contributing to slow fibrosis progression to promote its survival or persistence

Hepatitis C Virus Nonstructural 3/4A Protein Dampens Inflammation and Contributes to Slow Fibrosis Progression during Chronic Fibrosis In Vivo

HCV infection typically induces liver injury and inflammation, which appears to be responsible for the associated fibrogenesis. To date, the mechanism underlying the different rates of disease progression remains unclear. The aim of the study is to understand the possible role of the HCV non-structural (NS) 3/4A protein in the fibrosis progression. We used NS3/4A-expressing transgenic mice (NS3/4A-Tg) to accomplish the goals of the study. Different stages of liver fibrosis were induced in wild-type and NS3/4A-Tg mice by single carbon tetrachloride (acute) or multiple injections for 4 (intermediate) or 8 (chronic) weeks. Fibrotic parameters, inflammatory responses and hepatocyte turnover were extensively examined. Hepatic expression of HCV NS3/4A did not induce spontaneous liver damage. However, NS3/4A expression exerted contrasting effects during acute and chronic liver damage. During early fibrogenesis and intermediate fibrosis (4 weeks), NS3/4A-Tg mice exhibited enhanced liver damage whereas reduced fibrosis was observed in NS3/4A-Tg during chronic liver fibrosis (8 weeks). Furthermore, attenuated inflammation was observed in NS3/4A-Tg during chronic fibrosis with increase in M2 macrophages, hepatocyte proliferation, decreased hepatocyte apoptosis and decreased ductular reaction. In conclusion, during early fibrogenesis, HCV NS3/4A contributes to liver damage. While, during chronic liver fibrosis, NS3/4A dampens inflammation and induces hepatocyte regeneration thereby contributing to slow fibrosis progression to promote its survival or persistenc

Hepatic stellate cells activation and proliferation in the livers of wild-type and NS3/4A-Tg mice after CCl₄ administration.

<p>Representative pictures of <b>(A)</b> desmin- and <b>(B)</b> α-SMA-stained liver sections of 4-weeks and 8-weeks olive-oil treated and CCl_<b>4</b>-treated wild-type and NS/4A-Tg mice. Scale bars, 200μm (<b>C</b>) quantitative analysis of desmin- and α-SMA stained liver sections. (D) mRNA expression of desmin and α-SMA (normalized with GAPDH) in the livers of wild-type and NS3/4A-Tg. For quantitative histological and mRNA analysis, groups were normalized to olive-oil treated wild-type mice. Bars represent mean ± SEM of n = 5 per group. *p<0.05; **p<0.01; #p<0.05; ##p<0.01; n.s. denotes non-significant.</p

Hepatocyte proliferation and apoptosis in wild-type and NS3/4A-Tg mice after CCl₄ administration.

<p><b>(A)</b> Representative pictures of Ki67-stained liver sections of 4-weeks and 8-weeks olive-oil treated and CCl_<b>4</b>-treated wild-type and NS3/4A-Tg mice. Scale bars, 200μm. Quantitative real-time PCR analysis (normalized with GAPDH) of <b>(B)</b> cyclin D1 and <b>(C)</b> Bax/Bcl2 ratio in the livers of wild-type and NS3/4A-Tg. For quantitative PCR analysis, groups were normalized to olive-oil treated wild-type mice. Bars represent mean ± SEM of n = 5 per group. *p<0.05; **p<0.01; #p<0.05; n.s. denotes non-significant.</p