Low efficacy of entecavir therapy in adefovir-refractory hepatitis B patients with prior lamivudine resistance.

We determined the virologic response, incidence of entecavir resistance, and evolution of lamivudine and adefovir-resistant mutants during entecavir (ETV) therapy in adefovir-refractory patients with prior lamivudine resistance. Forty adefovir-refractory chronic hepatitis B patients with prior lamivudine resistance who had received entecavir for > or = 6 months were included and monitored for virologic response and entecavir resistance. Ten per cent of patients achieved HBV DNA < 50 copies/mL by PCR after 24 weeks of ETV therapy, and an initial virologic response was observed in 12 of 40 patients (30%). Higher pretreatment ALT (P = 0.039) and the presence of the rtL180M mutation (P = 0.038) were associated with an initial virologic response. During a mean follow-up of 11.4 months, four patients (10%) experienced virologic breakthrough, while ETV-resistant mutants were detected in six patients (15%). YMDD and adefovir-resistant mutants were detected in 57 and 35% of patients at baseline, respectively. At 48 weeks of therapy, 96 and 4% of patients had YMDD and adefovir-resistant mutants, respectively. These data suggest an early development of ETV resistance and low antiviral response during ETV therapy in adefovir-refractory patients with prior lamivudine resistance.

A purified inactivated Japanese encephalitis virus vaccine made in Vero cells.

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

Nucleotide sequence of envelope protein of Japanese encephalitis virus SA14-14-2 adapted to vero cells.

Live attenuated Japanese encephalitis (JE) virus SA(14)-14-2 produced in primary dog kidney cells (PDK) was adapted to Vero cells. In an effort to gain insight into the molecular basis of the biological characteristics of the SA14-14-2(Vero) strain, the 1500 nucleotide sequence encoding the envelope (E) gene which possesses major neutralizing epitopes was determined and compared with the sequences of two other attenuated JE virus strains, SA14-14-2(PHK) and SA14-14-2(PDK). The amino acid sequence of the C-terminal region (a.a. 280-500) was found to be identical for all three strains, while the N-terminal region (a.a. 1-279) shows sequence variation. The distribution of mutations in the N-terminal region was nearly the same among the three attenuated strains, suggesting that the N-terminal sequences might be related with virus-host cell specificity. However, it was found that Lys and Val (a.a. 138 and 176, respectively), known to be responsible for attenuation, are still conserved in SA(14)-14-2(Vero). Animal testing showed that SA(14)-14-2(Vero) has an attenuation phenotype similar to that of the parent SA(14)-14-2(PDK) strain in mice.

Synthetic peptides of human papillomavirus type 18 E6 harboring HLA-A2.1 motif can induce peptide-specific cytotoxic T-cells from peripheral blood mononuclear cells of healthy donors.

To identify cytotoxic T-cell (CTL) epitopes against human papillomavirus type 18 (HPV 18) E6 protein that might be useful for developing peptide-based vaccine against HPV 18 infection, 18 peptides which possibly contain CTL epitopes were selected on the basis of previously described human leukocyte antigen (HLA)-A2.1-binding motif and chemically synthesized. In the binding assay of the synthetic peptides, 8 out of 18 synthetic peptides enhanced the expression of HLA-A2.1 molecules on T2 cell surface, which implies that these peptides were able to bind the HLA molecules. Those peptides having good binding affinity to HLA-A2.1 were tested for their ability to activate CTLs which were isolated from peripheral blood mononuclear cells (PBMCs) of healthy blood donors and to kill the target T2 cells pulsed with the same peptide. Five out of eight tested peptides activated CTLs and killed the target cells.

Analysis of the conformational change of recombinant human papilloma virus type 18 E7 protein induced by metal binding.

Human papillomavirus (HPV) type 18 E7 gene was isolated by polymerase chain reaction (PCR) amplification from tissues of Korean cervical cancer patients and cloned into a plasmid vector, pET-3a, for the expression of recombinant E7 protein (rE7) in Escherichia coli. The rE7 protein was purified to the homogeneity and its purity was confirmed by HPLC. The purified protein was analyzed for the metal-binding properties by UV spectroscopy and it was shown that two Cd2+ or Zn2+ ions bind to one E7 protein by the metal-sulfur ligand formation via two Cys-X-X-Cys motifs in E7 protein. When the change of intrinsic fluorescence of tryptophan residue was analyzed for rE7-Zn complex, the blue shift of emission wavelength and the decrease in maximum intensity of emission were observed compared with rE7. These results suggest that Zn(2+)-bound rE7 has undergone conformational change, in which a tryptophan residue located in the second Cys-X-X-Cys motif was moved into solvent-inaccessible or hydrophobic environment. The rE7-Zn complex was found to be resistant to chymotrypic digestion by comparing the digestion patterns of rE7. Therefore, we showed the folding status of HPV 18 E7 could be changed by metal binding resulting in a different conformation in which a tryptophan residue was driven into more hydrophobic environment and the resistancy to chymotryptic digestion was conferred.