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Effort to develop a vaccine to prevent infection of human immunodeficiency virus (HIV) have focused on the induction of neutralizing antibodies. In our previous study, we reported that chimeric gag-env virus-like particles (VLPs) induce neutralizing antibodies which block HIV infection. In addition to the neutralizing antibodies, the cytotoxic T-lymphocyte (CTL) response is considered to be another major immune defense mechanism required for recovery from many different viral infections. In the present study, we have constructed chimeric fusion proteins using HIV-2 gag precursor protein with (1) four neutralizing epitopes from HIV-1 gp160; (2) three tandem copies of consensus V3 domain, which have been derived from 245 different isolates of HIV-1 and carries both the principal neutralizing determinant (PND) and CTL epitopes; and (3) V3 domains from HIV-1IIIB, HIV-1MN, HIV-1RF, and HIV-1SF2. These chimeric fusion proteins were expressed in a large quantity within insect cells, and released as VLPs into the cell culture medium. The purified gag-env VLPs from all three constructs appear to be spherical particles similar to immature HIV but slightly larger than the gag VLPs. Immunoprecipitation analysis showed that the chimeric proteins were recognized not only by HIV-1 positive patient sera, but also by monoclonal and polyclonal antisera raised against V3 peptides of HIV-1IIIB, HIV-1MN, HIV-1RF, and the gp120 antiserum against HIV-1SF2. Balb/C mice immunized with these chimeric VLPs successfully induced CTL activity against V3 peptide-stimulated target cells. In addition, a high degree of cross-reactivity was observed among the four different strains of HIV-1 V3 domain, indicating that the tandem multiple consensus V3 peptide sequence carried by HIV-2 gag can be used as a potential HIV vaccine against various HIVs.

Hepatitis B virus (HBV) is one of the main pathogens of hepatitis and hepatocarcinoma. Human plasma-derived antibody to HBV is being used as a prophylactic for postexposure to HBV and liver transplantation currently. However, it is required to replace the plasma-derived anti-HBs antibody (Ab) to a recombinant antibody because of limited availability of human plasma with high anti-HBs Ab titer and possible contamination of human pathogens. We constructed an anti-HBs Ab-enriched phage-display library from peripheral blood B cells of vaccinated volunteers and the size of library was approximately 1.0 x 10(7). The library was panned against hepatitis B surface antigen (HBsAg) and five different clones were isolated. All five clones exhibited the same heavy chain sequence; in contrast, light-chain exhibited one lambda and four different kappa sequences. The Fabs were expressed soluble in E. coli and exhibited affinities of 2.1 x 10(8) approximately 7.7 x 10(8) M(-1).

PurposeTo evaluate a recently marketed commercial glycoprotein enzyme-linked immunosorbent assay (gpEIA) kit, the VaccZyme™ VZV gpEIA, for measuring the immunity of varicella-vaccinated children.Materials and MethodsWe investigated the accuracy and reproducibility of the VaccZyme™ VZV gpEIA kit for the detection of antibodies to VZV. We also examined the sensitivity, specificity, and correlation between antibody titers calculated with gpEIA versus fluorescent antibody to membrane antigen (FAMA) by using sera of 349 children, ranging from 1 to 6 years old.ResultsVaccZyme™ VZV gpEIA gave precise and reproducible intra- and inter-assay results. FAMA and gpEIA titers showed a linear correlation (Pearson correlation coefficient=0.987). The sensitivity and specificity of the VaccZyme™ gpEIA was 31.4% and 100%, respectively, when the guidelines of the gpEIA (<100 mIU/mL) and FAMA 1:4 were adopted as cutoff values. However, the maximum sensitivity and specificity were 88.9% and 95.1%, respectively, with the highest correlation (κ=0.840), if the cutoff values were set with gpEIA at 49.7 mIU/mL and FAMA 1:16.ConclusionThese results demonstrate that the VaccZyme™ VZV gpEIA kit gave precise and reproducible data for measuring antibody titer after varicella vaccination. The results also showed that the antibody titer calculated with the VaccZyme™ gpEIA kit strongly correlated with the FAMA titer. However, cutoff values should be re-optimized for the evaluation of vaccine immunity.

Eight monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) were characterized. Five clones are IgG(1), two clones are IgM and one clone is IgG(2b); all have kappa light chain. The affinities are in the range of 1.1 x 10(-7) approximately 2.4 x 10(-9) M; the affinities of two IgM clones could not be estimated because of their low enzyme-linked immunoadsorbent assay (ELISA) signal. Each clone was constructed as single-chain Fv (scFv) and expression was performed in E. coli. Four clones out of 8 could express scFv soluble to culture media and the expression was confirmed further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The nucleotide and amino acid sequences of V(H) and V(L) of four scFvs were deduced and their family and subgroup were analyzed. We found that the clones that do not express the scFv have aberrant kappa chain (incorrect V/J recombination or stop codon); in contrast, their heavy chain sequences proved correct. The E. coli-expressed scFvs showed 1.5 x 3.4-fold lower affinities (2.8 x 10(-8) approximately 3.6 x 10(-9) M) than those of hybridoma-derived parental antibodies except the one clone (C5), which exhibited approximately 10(-6) M of affinity.