Increase of Plasma IL-12/p40 Ratio Induced by the Combined Therapy of DNA Vaccine and Lamivudine Correlates with Sustained Viremia Control in CHB Carriers

BACKGROUND: We previously reported that IFN-gamma producing T cell responses induced by the combined therapy of DNA vaccine and lamivudine for one year are important for the induction of sustained virological response (SVR). However, IFN-gamma production is not sufficient to predict sustained viremia control in chronic hepatitis B (CHB) carriers treated. METHODS: Twelve CHB carriers were intramuscularly immunized 12 times at a 4-week interval with 8 mg of HBV DNA vaccine during the standard lamivudine treatment (100 mg/daily/1 year). The level of cytokines during and after the combined therapy in plasma of all 12 CHB carriers treated was determined by each ELISA kit. Six out of 12 CHB carriers revisited the clinic, and their HBV DNA levels were examined. RESULTS: The combined therapy increased plasma IL-12 and IL-12/p40 ratio during the treatment (baseline vs. peak level: 41.8+/-8.3 vs. 163.1+/-29.2 pg/ml; p<0.01 and 0.96+/-0.25 vs. 3.58+/-0.86; p<0.01, espectively), and the peak level of plasma IL-12 and IL-12/p40 ratio was evoked at 6 to 10 months during the combined therapy. In particular, CHB carriers with SVR had two and three-fold higher level of the peak plasma IL-12 and plasma IL-12/p40 ratio than non-virological responders (NVRs), respectively (218.0+/-41.4 vs. 108.1+/-28.6 pg/ml; p=0.09 and 5.35+/-1.38 vs. 1.80+/-0.29; p<0.05, respectively), while p40 level was consistent during the combined therapy. In addition, there was no significant temporal correlation between the peak IL-12/p40 ratio and the elevation of serum alanine aminotransferase (ALT) in this study, contrast to IFN-alpha therapy which induced peak IL-12 level following ALT flares. CONCLUSION: Our results indicate that the combined therapy induces the increase of plasma IL-12 and IL-12/p40 ratio, which are associated with long-term SVR in CHB carriers.

Increase of Plasma IL-12/p40 Ratio Induced by the Combined Therapy of DNA Vaccine and Lamivudine Correlates with Sustained Viremia Control in CHB Carriers

BACKGROUND: We previously reported that IFN-gamma producing T cell responses induced by the combined therapy of DNA vaccine and lamivudine for one year are important for the induction of sustained virological response (SVR). However, IFN-gamma production is not sufficient to predict sustained viremia control in chronic hepatitis B (CHB) carriers treated. METHODS: Twelve CHB carriers were intramuscularly immunized 12 times at a 4-week interval with 8 mg of HBV DNA vaccine during the standard lamivudine treatment (100 mg/daily/1 year). The level of cytokines during and after the combined therapy in plasma of all 12 CHB carriers treated was determined by each ELISA kit. Six out of 12 CHB carriers revisited the clinic, and their HBV DNA levels were examined. RESULTS: The combined therapy increased plasma IL-12 and IL-12/p40 ratio during the treatment (baseline vs. peak level: 41.8+/-8.3 vs. 163.1+/-29.2 pg/ml; p<0.01 and 0.96+/-0.25 vs. 3.58+/-0.86; p<0.01, espectively), and the peak level of plasma IL-12 and IL-12/p40 ratio was evoked at 6 to 10 months during the combined therapy. In particular, CHB carriers with SVR had two and three-fold higher level of the peak plasma IL-12 and plasma IL-12/p40 ratio than non-virological responders (NVRs), respectively (218.0+/-41.4 vs. 108.1+/-28.6 pg/ml; p=0.09 and 5.35+/-1.38 vs. 1.80+/-0.29; p<0.05, respectively), while p40 level was consistent during the combined therapy. In addition, there was no significant temporal correlation between the peak IL-12/p40 ratio and the elevation of serum alanine aminotransferase (ALT) in this study, contrast to IFN-alpha therapy which induced peak IL-12 level following ALT flares. CONCLUSION: Our results indicate that the combined therapy induces the increase of plasma IL-12 and IL-12/p40 ratio, which are associated with long-term SVR in CHB carriers.

Increased in vivo immunological potency of HB-110, a novel therapeutic HBV DNA vaccine, by electroporation

Pulse-induced permeabilization of cellular membranes, generally referred to as electroporation (EP), has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids, for gene therapeutic applications, and this technique has been shown to result in improved immunogenicity. In this study, we assessed the utility of EP as a tool to improve the efficacy of HB-110, a novel therapeutic DNA vaccine against chronic hepatitis B, now in phase 1 of clinical study in South Korea. The potency of HB-110 in mice was shown to be improved by EP. The rapid onset of antigen expression and higher magnitude of humoral and cellular responses in electric pulse-treated mice revealed that EP may enable a substantial reduction in the dosage of DNA vaccine required to elicit a response similar in magnitude to that achievable via conventional administration. This study also showed that EP-based vaccination at 4-week-intervals elicited a cellular immune response which was about two-fold higher than the response elicited by conventional vaccination at 2-week intervals. These results may provide a rationale to reduce the clinical dose and increase the interval between the doses in the multidose vaccination schedule. Electric pulsing also elicited a more balanced immune response against four antigens expressed by HB-110: S, preS, Core, and Pol.

Enhancement of Antigen-specific Antibody and CD8(+) T Cell Responses by Codelivery of IL-12-encapsulated Microspheres in Protein and Peptide Vaccination

BACKGROUND: Although IL-12 has been widely accepted to play a central role in the control of pathogen infection, the use of recombinant IL-12 (rIL-12) as a vaccine adjuvant has been known to be ineffective because of its rapid clearance in the body. METHODS: To investigate the effect of sustained release of IL-12 in vivo in the peptide and protein vaccination models, rIL-12 was encapsulated into poly (DL-lactic-co-glycolic acid) (PLGA). RESULTS: We found that codelivery of IL-12-encapsulated microspheres (IL-12EM) could dramatically increase not only antibody responses, but also antigen-specific CD4(+) and CD8(+) T cell responses. Enhanced immune responses were shown to be correlated with protective immunity against influenza and respiratory syncytial virus (RSV) virus challenge. Interestingly, the enhancement of CD8(+) T cell response was not detectable when CD4(+) T cell knockout mice were subjected to vaccination, indicating that the enhancement of the CD8(+) T cell response by IL-12EM is dependent on CD4(+) T cell "help". CONCLUSION: Thus, IL-12EM could be applied as an adjuvant of protein and peptide vaccines to enhance protective immunity against virus infection.

Measurement of HIV-1-specific T Cell Immunity by Using Recombinant Gag Protein / 감염과화학요법

BACKGROUND: HIV-specific immunity, such as strong CD4+ helper and CD8+ cytotoxic T lymphocytes (CTL) responses, develops soon after HIV infection, but usually it can not control HIV replication which ultimately results in severe immune deficiency. HIV-specific CTLs, which are induced by HIV-specific CD4+ helper responses, are the key to cellular immune control of HIV. Measurement of HIV-1-specific CTLs using recombinant Gag protein may be very useful, because it is not restricted by HLA haplotype of the infected individual. MATERIALS AND METHODS: Enzyme-linked immunospot (ELISPOT) assays by using recombinant Gag protein were performed to evaluate HIV-1-specific gamma-interferon cellular responses of 25 HIV-1 infected Korean patients, who had been treated at least for the prior 12 months with highly active antiretroviral therapy at Catholic University Kangnam St. Mary's Hospital. RESULTS: The study group consisted of 25 chronically HIV-infected individuals with a median age of 51 years. The median CD4 counts were 556/mm3 (range:369-994/mm3) and HIV RNA titers were < 25 copies/mL (range: <25-180copies/mL). HIV-1-specific ELISPOT assay results range from 0 to 49 SFCs/2 x 10(5) PBMCs (median, 23.5 SFCs/2 x 10(5) PBMCs). CMV pp65-specific ELISPOT assay results range from 5 to 591 SFCs/2 x 10(5) PBMCs (median, 34 SFCs/2 x 10(5) PBMCs). There was no correlation between CD4 counts and HIV-1-specific SFCs measured by ELISPOT using recombinant protein. CONCLUSION: ELISPOT assays by using recombinant Gag protein may be considerable value in the assessment of cell-mediated immunity of HIV-1 infected patients.

Measurement of HIV-1-specific T Cell Immunity by Using Recombinant Gag Protein / 감염과화학요법

BACKGROUND: HIV-specific immunity, such as strong CD4+ helper and CD8+ cytotoxic T lymphocytes (CTL) responses, develops soon after HIV infection, but usually it can not control HIV replication which ultimately results in severe immune deficiency. HIV-specific CTLs, which are induced by HIV-specific CD4+ helper responses, are the key to cellular immune control of HIV. Measurement of HIV-1-specific CTLs using recombinant Gag protein may be very useful, because it is not restricted by HLA haplotype of the infected individual. MATERIALS AND METHODS: Enzyme-linked immunospot (ELISPOT) assays by using recombinant Gag protein were performed to evaluate HIV-1-specific gamma-interferon cellular responses of 25 HIV-1 infected Korean patients, who had been treated at least for the prior 12 months with highly active antiretroviral therapy at Catholic University Kangnam St. Mary's Hospital. RESULTS: The study group consisted of 25 chronically HIV-infected individuals with a median age of 51 years. The median CD4 counts were 556/mm3 (range:369-994/mm3) and HIV RNA titers were < 25 copies/mL (range: <25-180copies/mL). HIV-1-specific ELISPOT assay results range from 0 to 49 SFCs/2 x 10(5) PBMCs (median, 23.5 SFCs/2 x 10(5) PBMCs). CMV pp65-specific ELISPOT assay results range from 5 to 591 SFCs/2 x 10(5) PBMCs (median, 34 SFCs/2 x 10(5) PBMCs). There was no correlation between CD4 counts and HIV-1-specific SFCs measured by ELISPOT using recombinant protein. CONCLUSION: ELISPOT assays by using recombinant Gag protein may be considerable value in the assessment of cell-mediated immunity of HIV-1 infected patients.

Efficient Induction of Th1-type Immune Responses to Hepatitis B Virus Antigens by DNA Prime-Adenovirus Boost

BACKGROUND: Chronic infection with hepatitis B virus (HBV) affects about 350 million people worldwide, which have a high risk of development of cirrhosis and hepatocellular carcinoma. Treatment of chronic HBV infection relies on IFN-alpha or lamivudine. However, interferon-alpha is effective in only about 30% of patients. Also, the occurrence of escape mutations limits the usage of lamivudine. Therefore, the development and evaluation of new compounds or approaches are urgent. METHODS: We comparatively evaluated DNA and adenoviral vaccines expressing HBV antigens, either alone or in combined regimens, for their ability to elicit Th1-type immune responses in Balb/c mice which are believed to be suited to resolve HBV infection. The vaccines were tested with or without a genetically engineered IL-12 (mIL-12 N220L) which was shown to enhance sustained Th1-type immune responses in HCV E2 DNA vaccine. RESULTS: Considering the Th1-type cytokine secretion and the IgG2a titers, the strongest Th1-type immune response was elicited by the DNA prime-adenovirus boost regimen in the presence of mIL-12 N220L. In addition, the codelivery of mIL-12 N220L modulated differentially the immune responses by different vaccination regimens. CONCLUSION: Our results suggest that the DNA prime-adenovirus boost regimen in the presence of mIL-12 N220L may be the best candidate for HBV vaccine therapy of the regimens tested in this study and will be worthwhile being evaluated in chronic HBV patients.

HCV core protein promotes liver fibrogenesis via up-regulation of CTGF with TGF-beta1

Liver cirrhosis is one of the major complications of hepatitis C virus (HCV) infection, but the mechanisms underlying HCV-related fibrogenesis are still not clear. Although the roles of HCV core protein remain poorly understood, it is supposed to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to examine the role of HCV core protein on the hepatic fibrogenesis. We established an in vitro co-culture system with primary hepatic stellate cell (HSC) isolated from rats, and a stable HepG2-HCV core cell line which had been transfected with HCV core gene. The expressions of fibrosis-related molecules transforming growth factor beta1 (TGF-beta1), transforming growth factor b receptor II (TGF beta RII), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were analyzed via histological or molecular methods. In addition, the expression levels of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) from the co-cultured media were measured by zymogram and ELISA, respectively. The expressions of alpha-SMA, TGF-beta1, Col I, TGF beta RII and MMP-2 were significantly increased in the co-culture of stable HepG2-HCV core with HSC. Moreover, the significant increases of CTGF and TGF-beta1 in the HCV core-expressing cells were observed by either Northern or Western blot analysis. These results suggest that HCV core protein may contribute to the hepatic fibrogenesis via up-regulation of CTGF and TGF-beta1.

The Role of Hepatitis C Virus Core Protein on Liver Fibrogenesis: A Study Using an In Vitro Co-culture System / 대한소화기학회지

BACKGROUND/AIMS: The study of liver fibrogenesis by hepatitis C virus (HCV) has been limited due to the lack of an efficiency in vitro culture systems. In the present study, we investigated whether or not HCV core protein is directly related to liver fibrogenesis through stimulation of hepatic stellate cells (HSC). METHODS: Human and rat HSC were isolated and we established an in vitro co-culture system of a stable HepG2-HCV core cell line which was transfected with HCV core gene and primary HSC. We performed immunocytochemical staining and Western and Northern blot analysis in the stimulated HSC by HCV ocre protein to identify the expression of transforming growth factor beta1 (TGF-beta1), transforming growth factor beta receptor II (TGFbeta R II), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF). The expression of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) in the culture media were measured by zymogram and ELISA, respectively. RESULTS: The expression of TGF-beta1 and CTGF was significantly higher in the stable HepG2-HCV core cell line than in HepG2 cells. Furthermore, the makers related to fibrosis such as alpha-SMA, TGF-beta1, Col I, TGFRII and MMP-2 were highly experssed in the co-culture of stable HepG2-HCV core with HSC. CONCLUSIONS: HCV core protein may play a direct role in the fibrogenesis of chronic liver disease with HCV infection.