Shun'ichi Kuroda

Adjuvant-independent enhanced immune responses to recombinant herpes simplex virus type 1 glycoprotein D by fusion with biologically active interleukin-2.

A truncated herpes simplex virus (HSV) type 1 glycoprotein D (t-gD) gene was fused to the human interleukin-2 (IL-2) gene (t-gD-IL-2 gene) and introduced into mouse myeloma Sp2/0 cells. The gene product, t-gD-IL-2, secreted from the cells was immunoprecipitated with five monoclonal antibodies specific for native gD. Purified t-gD-IL-2 supported the growth of IL-2-dependent cells, with a specific activity almost comparable to that of recombinant human IL-2. Mice immunized with t-gD-IL-2 in an adjuvant-free form showed superior anti-HSV antibody responses, and were completely protected against HSV challenge, whereas immunization with t-gD adsorbed onto aluminum hydroxide (alum) partially failed to prevent the virus infection. The high immunogenicity of t-gD-IL-2 was due to the biological activity of the fused IL-2 rather than to a hapten-carrier effect of the IL-2 moiety, because mice primed with t-gD-IL-2 showed delayed-type hypersensitivity against stimulation with gD, but not against that with IL-2 antigen, and because a booster immunization with t-gD-IL-2 extensively augmented the response of anti-gD antibody, but not that of the anti-human IL-2 antibody. The serological half-life of IL-2 activity in mice injected with t-gD-IL-2 was prolonged to about four times that of rIL-2. However, when t-gD-IL-2 was co-administered with human albumin (HSA), the mouse anti-HSA antibody response was slightly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of protection level of anti-pre-S2 antibodies in humans immunized with a novel hepatitis B vaccine consisting of M (pre-S2 + S) protein particles (a third generation vaccine).

An enzyme-linked immunosorbent assay (ELISA) for anti-pre-S2 antibodies was developed by the use of both recombinant yeast-derived S and M (pre-S2 + S) protein particles as antigens. By this ELISA was determined the amount of both human and chimpanzee anti-pre-S2 antibodies produced by a new type of yeast-derived hepatitis B (HB) vaccine (TGP-943, subtype adr), which consists of modified M protein particles. In seven randomly selected human individuals who were vaccinated intramuscularly with 10 micrograms (as a protein) TGP-943 three times (0, 4th and 24th week), a detectable level of anti-pre-S2 antibodies was found to be rapidly elicited at 4th or 8th week after the first vaccination. The protective level of anti-pre-S2 antibodies in humans was tentatively assessed by comparing the anti-pre-S2 antibody titres in the vaccinated human individuals with that in chimpanzees which produced only anti-pre-S2 antibodies to tolerate well against the challenge by 1000 chimpanzee infectious units of HBV (subtype ayw). From this assessment, it was speculated that all human individuals tested had already acquired the protective level of anti-pre-S2 antibodies at 4th or 8th week after the first vaccination with TGP-943.

Protective efficacy of a novel hepatitis B vaccine consisting of M (pre-S2 + S) protein particles (a third generation vaccine).

The protective efficacy of a new type of yeast-derived hepatitis B (HB) vaccine (TGP-943, subtype adr), which was formulated from modified M (pre-S2 + S; P31) protein (M-P31c) particles, was investigated in chimpanzees. Animals were injected intramuscularly three times at 4-week intervals with doses of 10 or 40 micrograms (as a protein) of TGP-943. There were no significant differences in the immunogenicity of 10 micrograms compared to that of 40 micrograms of TGP-943 in terms of anti-S antibody response, while the induction and persistence of anti-pre-S2 antibodies seemed dose-related. Chimpanzees, vaccinated with 40 micrograms of TGP-943, produced anti-pre-S2 antibodies 2 weeks after the first injection, which appeared earlier than anti-HBs (S) antibodies. A maximum level of the anti-pre-S2 antibodies was reached 2 weeks after the second injection. Apart from immunization with TGP-943, chimpanzees injected with denatured TGP-943, consisting of 10 micrograms (as a protein) of non-particulate M-P31c antigen, produced anti-pre-S2 antibodies with a non-protecting level of anti-S antibodies (less than 10 mIU ml-1). Five weeks after the third injection, all animals were challenged intravenously with 1000 chimpanzee infectious units of HBV subtype (ayw) and were protected as confirmed by normal serological markers, no signs of infection in the sera and liver biopsies, and no detection of HBV-DNA by PCR method. No side effects from inoculation with TGP-943 or denatured TGP-943 were also encountered in any animals.

Identification of polymerized-albumin receptor domain in the pre-S2 region of hepatitis B virus surface antigen M protein.

The pre-S2-coding region in the hepatitis B virus surface antigen M (P31; pre-S2 + S) protein gene was modified to identify a polymerized-albumin receptor (PAR) domain by deleting restriction fragments or performing site-directed mutagenesis. The modified M protein genes (M-P31x; x = d, e, f, h and i) were cloned into the yeast generalized-expression vector pGLD 906-1 and expressed in Saccharomyces cerevisiae under the control of yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. The PAR activities of these gene products suggested that the PAR domain is located in the hydrophilic and highly conserved domain in the pre-S2 region (around Leu12 approximately Tyr21). Antibodies specific for a pre-S2 peptide (Phe8 approximately Pro34, subtype adr), which covers the PAR domain, were purified from sera of rabbits immunized with yeast-derived M protein particles having a natural PAR domain. Immune electron microscopy showed that the purified antibodies could aggregate HBV particles. Therefore, it was speculated that the PAR domain overlapped with the dominant virus-neutralizing and virus-protecting epitopes.

A supersensitive DOT-hybridization method: Rapid and quantitative detection of host-derived DNA in recombinant products at the one picogram level

We have developed a highly sensitive method of DNA dot-blotting hybridization to detect host-derived DNA (nuclear DNA and plasmid DNA) in a recombinant product. This method has two distinctive features compared to the conventional hybridization method: firstly, a highly specific radioactive probe is prepared by using ultrasonicated DNA, instead of untreated DNA, as a template for the oligo-labeling reaction; secondly, the signal to noise ratio is increased by the use of lambda phage DNA as non-homologous DNA. This method enabled us to detect host-derived DNA at the one picogram level without using a radioisotope of high specific activity and long exposure times.