Chikateru Nozaki

Production of Human Monoclonal and Polyclonal Antibodies in TransChromo Animals

We have developed TransChromo (TC) technology, which enables the introduction of megabase-sized segments of DNA into cells. We have used this approach to derive mice that carry megabases of human DNA by the use of a human chromosome fragment (HCF) as a vector. TC technology has been applied to the construction of the TC Mouse,trade mark which incorporates entire human immunoglobulin (hIg) loci. TC Mouse expresses a fully diverse repertoire of hIgs, including all the subclasses of IgGs (IgG1-G4). Immunization of the TC Mouse with various human antigens produced antibody responses comprised of human antibodies. Furthermore, it was possible to obtain hybridoma clones expressing fully human antibodies specific for the target human antigen. However, because of the instability of the Igkappa locus-bearing HCF2, the efficiency of hybridoma production was less than one-tenth of that observed in normal mice. An instant solution to this problem was to cross-breed the Kirin TC Mouse carrying the HCF14, which was stable in mouse cells, with the Medarex YAC-transgenic mouse carrying about 50% of the hIgVkappa gene segments as a region that is stably integrated into the mouse genome. The resulting mouse, dubbed the KM Mouse, performed as well as normal mice with regard to immune responsiveness and efficiency of hybridoma production. Another application of TC technology is the production of polyclonal antibodies in large animals such as chickens and cows. To test the efficacy of human polyclonal antibodies derived from TC animals, feasibility studies were performed using antisera and purified gamma-globulin from TC mice immunized with Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), or Japanese encephalitis virus (JEV). The TC mouse-derived antisera and gamma-globulin showed a much higher titer and efficacy in terms of the neutralizing activity of the pathogens in vitro and in vivo than either human serum or gamma-globulin prepared from human blood.

Molecular cloning and heterogeneity of the human hepatitis C virus (HCV) genome

The Japanese variant of the hepatitis C virus (HCV-N) genome, consisting of 9440 nucleotides in length, was cloned from a small amount (2 ml) of plasma from a single Japanese carrier by using RT-PCR and modified RT-PCR. The HCV-N genome has a long open reading frame that encodes a 3014 amino acid polyprotein with 340 and 57 bases of 5 and 3 non-coding sequences, respectively. HCV-N has a 4-amino-acid insertion in the NS5 region as compared to other HCV isolates, but this insertion is found to be very rare upon direct sequencing of that region. Comparative sequence analysis of all the complete and partial HCV sequences that were reported indicates that HCV can be subdivided into at least 4 groups. The HCV-N isolate has a high homology with HCV-J and HCV-BK (> 90%) and so belongs to group II, but shows less similarity to HCV-1 (> 78%, group I) and least to HC-J6 (> 67%, group III). Among these HCV isolates, the 5 non-coding region was the most conserved (> 93%) since it plays an important role in replication. The RT-PCR assay to detect HCV-RNA, using the primers deduced from this region, was very sensitive and specific. The putative core protein could become an important target for immunoassay because of a high degree of amino acid sequence similarity in that region. A high degree of diversity and a low similarity between each HCV isolate in the putative envelope protein play an important role in the chronicity of HCV infection and development of immunopreventive agents, such as immunoglobulin and vaccine for that infection.

Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells

Recombinant baculoviruses that produce a putative non-structural protein 1 (NS1) of hepatitis C virus (HCV), predicted to be the second envelope glycoprotein, were constructed. The recombinant NS1 protein (re-NS1) produced in infected insect cells was localized on the cell surface and was apparently glycosylated, because it was susceptible to treatment with both tunicamycin and N-glycanase. Furthermore, re-NS1 was effectively secreted into the culture supernatant when the putative NS1 signal peptide (SP) was replaced by the SP of rabies virus G protein, and the C-terminal hydrophobic region was eliminated. The secreted re-NS1 was tagged with six His residues at the C terminus and purified simply by native Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) affinity column chromatography. An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of HC using purified re-NS1. Anti-NS1 antibody (Ab) was detected in 55 of 60 patients (92%) with chronic HC liver diseases. Thus, this ELISA for Ab directed against HCV re-NS1 produced in insect cells is useful for the detection of chronic HC patients.

Immunogenicity of herpes simplex virus glycoprotein gB-1-related protein produced in yeast.

A protein related to glycoprotein B of herpes simplex virus type 1 (HSV-1) produced in yeast (ygB-1) was purified with an immunoadsorbent. The molecular weight of the purified ygB-1 as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis was 96,000. Mice injected twice with ygB-1 adsorbed to alum developed ELISA antibody to ygB-1, neutralizing antibody to HSV-1 and a lymphoproliferative response to ygB-1 and HSV-1. The immunized mice were protected against intraperitoneal and corneal challenge with HSV-1. Latent infection in the trigeminal ganglia after corneal challenge was also inhibited by immunization with ygB-1. Guinea-pigs pigs immunized with ygB-1 adsorbed to alum also developed ELISA antibody to to ygB-1 and neutralizing antibody to both types of HSV. After the second dose, strong lymphoproliferative responses were seen upon stimulation with HSV-2. Animals were protected against intravaginal challenge with HSV type 2.

Production of Transgenic Chimera Rabbit Fetuses Using Somatic Cell Nuclear Transfer

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.

Immunoreactive core peptides of hepatitis C virus produced in Escherichia coli and in vitro DNA amplification-restricted transcription-translation system

Three kinds of hepatitis C virus (HCV) core peptides were produced directly and efficiently in E. coli: 1-120 aa of the C region as NCC, 1-157 aa as NCCT and 1-190 aa as NCCL. These peptides were estimated to be 16, 22 and 24 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The processing to produce p22 core protein observed in insect cells and mammalian systems did not occur in E. coli. These peptides were similarly reactive with serum antibody from patients with hepatitis C. A mutant clone of NCC recombinant plasmid pKNCC4 was obtained, whose product, NCC4, was more stable in the E. coli lysate and was highly immunoreactive with sera of hepatitis C patients. This stable immunoreactive core peptide produced by pKNCC4 is useful for the detection of anti-HCV core antibody. Immunoreactive core peptides were also produced by DNA amplification-restricted transcription-translation. Five kinds of cDNA from C to E1 region were amplified and transcribed in vitro, and these five transcripts were then translated in vitro using rabbit reticulocyte lysate: 1-120 aa as 17 kDa of C1, 1-155 aa as 21 kDa of C2, 1-174 aa as 22 kDa of C3, 1-192 aa as 24 kDa of C4, and 1-213 aa as 26 kDa of C5. Cotranslational processing using microsomal membranes occurred in peptides C4 and C5 to produce p22 the same size as C3. These results indicate that the C-terminus of the mature core protein p22 may be generated at around aa 174 by cleavage with the signal peptidase.

Characterization of neutralizing mouse-human chimeric and shuffling antibodies against botulinum neurotoxin A

Mouse‐human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2–4, 2–5, 9–4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (VH) and light chains (VL) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO‐DG44 cells were transfected with these plasmids and mouse‐human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their VH or VL domains were constructed. A shuffling antibody (AC2494) that derived its VH and VL domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high‐potency neutralizing chimeric antibodies by selecting and shuffling VH and VL domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.

Expression and epitope analysis of hepatitis C virus NS1

Abstract A cDNA encoding putative NS1 of hepatitis C virus (HCV) was expressed in Escherichia coli . The recombinant product reacted with antibodies in sera from patients with hepatitis C. For epitope analysis, immunoreactivities of synthetic peptides deduced from the NS1 region were examined. Three peptides corresponding to amino acid residues 384–411, 514–550 and 639–667 reacted with antibodies in sera from patients with hepatitis C.