OBJECTIVETo construct human metapneumovirus (hMPV) DNA vaccines and evaluate the cellular and humoral immune response in mice.

METHODSFusion protein FdeltaTM (without transmembrane domain) gene and M gene of hMPV were amplified from cDNA by PCR, then DNA vaccines pcDNA3.1His-FdeltaTM and pcDNA3.1His-M were constructed to verify the expression of F and M protein by Western blotting and indirect immunofluorescent assay (IFA) respectively. Serum IgG and spleen cell CTL were detected with ELISA and ELISPOT assay after the BALB/c mice were immunized intramuscularly with the vaccines.

RESULTSThe candidate DNA vaccines could express FdeltaTM and M protein as detected with Western blotting and IFA. The IgG antibody titers of mice was 1:44 when immunized with pcDNA3.1His-FdeltaTM, but could increase to 1:64 when co-immunized with pcDNA3.1His-M. ELISPOT assay demonstrated that IFN-gamma-secreting effector T cells reached 42 +/- 8.9 in co-immunization group, higher than single vaccine pcDNA3.1His-FdeltaTM group (32 +/- 7.4).

CONCLUSIONDNA vaccine pcDNA3.1His-FdeltaTM could induce specific cellular and humoral immune responses, and the immune response could increase when co-immunization with pcDNA3.1His-M was carried out.

Animals ; Antibodies, Viral ; blood ; Female ; Humans ; Immunization ; Metapneumovirus ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Paramyxoviridae Infections ; immunology ; prevention & control ; virology ; Vaccines, DNA ; administration & dosage ; genetics ; immunology ; Viral Proteins ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

Adjuvants, Immunologic ; administration & dosage ; Administration, Intranasal ; Animals ; Antibodies, Viral ; blood ; Bacterial Toxins ; administration & dosage ; Capsid Proteins ; Enterotoxins ; administration & dosage ; Escherichia coli Proteins ; administration & dosage ; Female ; Hemagglutination Inhibition Tests ; Human papillomavirus 16 ; immunology ; Immunization ; Interferon-gamma ; biosynthesis ; Lymphocyte Activation ; Mice ; Mice, Inbred C57BL ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomavirus Vaccines ; Vaccines, DNA ; administration & dosage ; immunology ; Viral Vaccines ; administration & dosage ; immunology ; Virion ; immunology

More than 99% of cervical cancers have been associated with human papillomaviruses (HPVs), particularly HPV type 16. The clear association between HPV infection and cervical cancer indicates that HPV serves as an ideal target for development of preventive and therapeutic vaccines. Although the recently licensed preventive HPV vaccine, Gardasil, has been shown to be safe and capable of generating significant protection against specific HPV types, it does not have therapeutic effect against established HPV infections and HPV-associated lesions. Two HPV oncogenic proteins, E6 and E7, are consistently co-expressed in HPV-expressing cervical cancers and are important in the induction and maintenance of cellular transformation. Therefore, immunotherapy targeting E6 and/or E7 proteins may provide an opportunity to prevent and treat HPV-associated cervical malignancies. It has been established that T cell-mediated immunity is one of the most crucial components to defend against HPV infections and HPV-associated lesions. Therefore, effective therapeutic HPV vaccines should generate strong E6/E7-specific T cell-mediated immune responses. DNA vaccines have emerged as an attractive approach for antigen-specific T cell-mediated immunotherapy to combat cancers. Intradermal administration of DNA vaccines via a gene gun represents an efficient way to deliver DNA vaccines into professional antigen-presenting cells in vivo. Professional antigen-presenting cells, such as dendritic cells, are the most effective cells for priming antigen-specific T cells. Using the gene gun delivery system, we tested several DNA vaccines that employ intracellular targeting strategies for enhancing MHC class I and class II presentation of encoded model antigen HPV-16 E7. Furthermore, we have developed a strategy to prolong the life of DCs to enhance DNA vaccine potency. More recently, we have developed a strategy to generate antigen-specific CD4+ T cell immune responses to further enhance DNA vaccine potency. The impressive pre- clinical data generated from our studies have led to several HPV DNA vaccine clinical trials.
Female ; Humans ; Oncogene Proteins, Viral/genetics/immunology ; Papillomaviridae/*genetics/immunology ; Papillomavirus Infections/immunology/*prevention & control ; Papillomavirus Vaccines/*administration & dosage ; Repressor Proteins ; Uterine Cervical Neoplasms/*prevention & control ; Vaccines, DNA/*administration & dosage ; Viral Vaccines/administration & dosage

OBJECTIVESTo study the evolution of HBV quasispecies under the pressures of lamivudine (LAM) - entecavir (ETV) sequential therapy and its clinical significance.

METHODSConsecutive serum samples from 2 patients underwent LAM-ETV sequential therapy were extensively studied for HBV quasispecies composition and evolution, using PCR-cloning-sequencing method. Maximum likelihood trees were built to analyze the genetic relationship between representative sequences. Correlation between HBV quasispecies evolution and serological/virological data was analyzed to determined the clinical significance of the evolution of HBV quasispecies during prolonged nucleotide analog therapy.

RESULTSVirological breakthrough was observed in both patients. Patient I acquired sustained virological response after switching to ETV rescue therapy, whereas Patient II suffered from virological breakthrough after 72 weeks of ETV therapy. Each virological breakthrough was accompanied with the replacement of previous drug susceptible dominant quasispecies with a drug resistant variant, indicating a close correlation between quasispecies composition and drug susceptibility. The rtL180M+S202G+M204V triple mutant, which was most likely a descendant of the LAM resistant rtL180M+M204V variant, was closely correlated with ETV resistant in Patient II.

CONCLUSIONQuasispecies composition of HBV is closely correlated with nucleotide analog susceptibility. ETV resistant variant can emerge from a LAM resistant viral population. Dynamic monitoring of HBV quasispecies composition is of great importance during nucleotide analog therapy.

Antiviral Agents ; administration & dosage ; therapeutic use ; DNA, Viral ; genetics ; Drug Resistance, Viral ; Evolution, Molecular ; Guanine ; administration & dosage ; analogs & derivatives ; therapeutic use ; Hepatitis B ; drug therapy ; virology ; Hepatitis B virus ; drug effects ; genetics ; Humans ; Lamivudine ; administration & dosage ; therapeutic use

OBJECTIVEGanciclovir is a first line drug for treatment of cytomegalovirus (CMV) infection. This study aimed to compare the efficacy and side effects of relatively low and high doses of Ganciclovir in the treatment of neonatal congenital CMV infection.

METHODS37 neonates with congenital CMV infection were randomly assigned to high-dose (n = 19) and low-dose Ganciclovir groups (n = 18). The high-dose Ganciclovir group was injected with Ganciclovir of 7.5 mg/kg in the inducement phase and of 10 mg/kg in the maintaining phase. The low-dose Ganciclovir group was injected with Ganciclovir of 5 mg/kg in the inducement and the maintaining phases. The efficacy and side effects were observed in the two groups.

RESULTSThe results of different doses of GCV treatment of congenital CMV infection in symptomatic by the clinical symptoms were improved, high-dose treatment group CMV-IgM negative rate of 89.5%, CMV-DNA negative rate of 73.7%; low-dose treatment group CMV-IgM switch negative rate of 83.3%, CMV-DNA negative rate was 77.8%, no significant difference between the two groups. Low-dose GCV treatment of congenital CMV infection in newborns with symptomatic side effects than high dose GCV, the low dose group neutropenia, anemia, thrombocytopenia was lower than the high dose group, the difference was significant (P < 0.05).

CONCLUSIONLow- dose GCV treatment of symptomatic congenital CMV infection with high-doses of the same clinical efficacy, and less side effects than high-doses of GCV.

Antiviral Agents ; administration & dosage ; Cytomegalovirus Infections ; congenital ; drug therapy ; virology ; DNA, Viral ; blood ; Female ; Ganciclovir ; administration & dosage ; adverse effects ; Humans ; Infant, Newborn ; Male

OBJECTIVEGanciclovir is a first-line drug for treatment of cytomegalovirus (CMV) infection. However, some ganciclovir treatment-related side-effects can be found. This study aimed to compare the efficacy and side effects of relatively low and high doses of ganciclovir in the treatment of neonatal congenital CMV infection.

METHODSOne hundred and sixty-seven neonates with congenital CMV infection were randomly assigned to high-dose (n=79) and low-dose ganciclovir groups (n=88). The high-dose ganciclovir group was injected with ganciclovir of 7.5 mg/kg in the inducement phase and of 10 mg/kg in the maintaining phase. The low-dose ganciclovir group was injected with ganciclovir of 5 mg/kg in the inducement and the maintaining phases. The efficacy and side effects were observed in the two groups.

RESULTSAfter treatment the clinical symptoms and signs were obviously improved in both groups. CMV-IgM became negative in 93.8% of neonates in the high-dose ganciclovir group and 93.1% of neonates in the low-dose ganciclovir group (P>0.05). CMV-DNA became negative in 80.8% of neonates in the high-dose ganciclovir group and in 86.7% in the low-dose ganciclovir group (P>0.05). The low-dose ganciclovir group had lower incidence of side effects than the high-dose ganciclovir group: vomiting 2.3% vs 11.4%; anemia 8.0% vs 20.3%; reduction of neutrophilic granulocytes 5.7% vs 16.5%; increase in platelet count 8.0% vs 18.9% (P<0.05).

CONCLUSIONSLow-dose ganciclovir has the same clinical efficacy to high-dose ganciclovir for treatment of neonatal congenital CMV infection, but fewer side effects occur in the low-dose group.

Antiviral Agents ; administration & dosage ; Cytomegalovirus Infections ; congenital ; drug therapy ; DNA, Viral ; analysis ; Dose-Response Relationship, Drug ; Female ; Ganciclovir ; administration & dosage ; adverse effects ; Humans ; Infant, Newborn ; Male

To investigate the adjuvant effect of granulocyte macrophage colony stimulating factor (GM-CSF) in Flaviviridae virus DNA vaccines. After DNA immunization, the antibody levels of serum from mice were detected by ELISA and indirect immunofluorescence assay. Co-immunization of GM-CSF suppressed the immune responses induced by DV1 and DV2 candidate vaccines whereas enhanced the immune response induced by HCV C and E1 DNA vaccines. As genetic adjuvant for DNA vaccines, GM-CSF might display complex diversity on the immune responses: an augmentation or suppression due to different immunogens. Therefore, GM-CSF should be used with some cautions in clinic.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Antibodies, Viral ; immunology ; DNA, Viral ; administration & dosage ; genetics ; immunology ; Dengue ; immunology ; prevention & control ; virology ; Dengue Vaccines ; administration & dosage ; genetics ; immunology ; Dengue Virus ; genetics ; immunology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; immunology ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

Hela is the cell line of adenocarcinoma of the uterine cervix, and human papillomavirus (HPV) 18 shows positive. We delivered siRNA with target specifically to HPV18 E7 mRNA into nude mice Hela tumor xenografts by nanopatch to inhibit the HPV gene expression, and further to study the superiority, the best action time and concentration of siRNA of using nanopatch to transfer siRNA in vivo. We designed siRNA that target specifically to HPV18 E7 mRNA (siE7) and checked the effect of siE7 in vitro. Tumor xenografts were transfected with siE7 and GenEscort III by nanopatch. Expression of HPV18 E7 mRNA and protein were detected 0 hours, 24 hours, 48 hours, 72 hours after transfection with PT-PCR and Western blot, and the best action time was analyzed using nanopatch to thansfect siRNA in vivo. We transfected GenEscort III and siE7 of Different concentration into tumor xenografts respectively by nanopatch and intraperitoneal injection. Expression of HPV18 E7 mRNA and protein was detected 72 hours after transfection by PT-PCR and Western blot, to analyze the best action concentration of siRNA and the superiority of using nanopatch to thansfect siRNA in vivo. The results proved that SiE7was efficient to inhibit expression of HPV18 E7 mRNA and to advance Hela apoptosis in vitro. SiE7 transfected by nanopatch into xenografts could inhibit effectively expression of HPV18 E7 mRNA and protein. The best action time and concentration of siRNA of using nanopatch to thansfect siRNA in vivo are 72 hour post-transfection and 2 micromol/L siE7. To compare intraperitoneal injection in delivering siRNA in vivo, the effect of nanopatch is very predominant. It can be well concluded that Nanopatch can effectively transfer siRNA in vivo, which can effectively inhibit the HPV gene expression.
Animals ; Apoptosis ; DNA-Binding Proteins ; genetics ; Female ; Gene Expression Regulation, Viral ; HeLa Cells ; Humans ; Mice ; Mice, Nude ; Nanostructures ; administration & dosage ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; RNA, Small Interfering ; administration & dosage ; Transfection ; Uterine Cervical Neoplasms ; therapy ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the relationship between the method of administration of lamivudine and the therapeutic effect of the treatment in patients with chronic hepatitis B virus (HBV) infection.

METHODSOne hundred and seventy-nine patients were given lamivudine 100 mg daily for 1 to 3 years. The relationships of the therapeutic effect and the early response, YMDD mutants and duration of treatment were analyzed.

RESULTSAlanine aminotransferase normalization rate, the negativity rate of HBV DNA and HBeAg, and HBeAg sero-conversion all were increased gradually with prolonged treatment. At the end of 1 year, HBV DNA negativity rate (57.0%) reached its peak, HBeAg negativity rate (39.7%) and HBeAg sero-conversion rate (16.8%) were higher than those at the end of 3 months (chi2 = 28.489, 33.238, 12.690, P<0.01). The lower the HBV DNA level was at the end of 3 months, the higher the HBV DNA negativity and HBeAg sero-conversion rates were at the end of 52 weeks and at the end of the 6 months follow-up. When the duration of treatment reached 1 year and 1.5 years, HBV DNA rebound rate in the patients (40.0% and 40.0% respectively) with HBeAg sero-conversion was obviously less (chi2 = 12.424, 10.237, P<0.01) than in those without sero-conversion (88.2% and 85.0% respectively).

CONCLUSIONLamivudine therapy for HBV infection is safe and effective. The optimal duration of treatment was 1.5 years. The early responders had better therapeutic effects. HBV DNA positivity persisting at the end of 3 months medication or no HBeAg sero-conversion in 1 year predicts poor therapeutic effects.

Adolescent ; Adult ; Antiviral Agents ; administration & dosage ; therapeutic use ; DNA, Viral ; Drug Resistance, Viral ; Female ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; Humans ; Lamivudine ; administration & dosage ; therapeutic use ; Male ; Middle Aged ; Treatment Outcome ; Young Adult

BACKGROUND/AIMS: To investigate the association between the baseline profiles and dynamics of hepatitis B virus (HBV) DNA polymerase gene mutations and the long-term virological response of lamivudine (LAM)-adefovir (ADV) combination therapy in patients with LAM-resistant chronic hepatitis B. METHODS: Seventy-five patients who received LAM-ADV combination therapy for more than 12 months were analyzed. Restriction fragment mass polymorphism assays were used to detect and monitor the dynamics of LAM- and ADV-resistant mutations. RESULTS: The median duration of LAM-ADV combination therapy was 26 months (range, 12 to 58 months). The baseline mutation profiles, rtM204I (p=0.992), rtM204I/V (p=0.177), and rtL180M (p=0.051), were not correlated with the cumulative virological response, and the baseline HBV DNA level (p=0.032) was the only independent predictive factor for cumulative virological response. Tests for LAM- and ADV-resistant mutations were performed in 12 suboptimal responders in weeks 48 and 96. The population of rtM204 mutants persisted or increased in 8 of 12 patients, and rtA181T mutants newly emerged as a minor population in four patients until 96 weeks. Nevertheless, the viral loads progressively decreased during rescue therapy, and these dynamics did not correlate with virological response. CONCLUSIONS: The baseline profile and dynamics of LAM-resistant mutations during LAM-ADV combination therapy are not associated with a virological response.
Adenine/administration & dosage/*analogs & derivatives/therapeutic use ; Adult ; Aged ; Aged, 80 and over ; Antiviral Agents/administration & dosage/*therapeutic use ; DNA-Directed DNA Polymerase/genetics ; Drug Resistance, Viral/genetics ; Drug Therapy, Combination ; Female ; Hepatitis B virus/*genetics ; Hepatitis B, Chronic/*drug therapy/virology ; Humans ; Lamivudine/administration & dosage/*therapeutic use ; Male ; Middle Aged ; Organophosphonates/administration & dosage/*therapeutic use ; Treatment Outcome ; Viral Load/drug effects ; Young Adult