Production and characterization of recombinant human anti-HBs Fab antibodies.

Recombinant human Fab antibodies were generated with different reactivities against the hepatitis B virus surface (HBs) antigen. To isolate the antibodies, a method was used that combined transformation of human B cells by Epstein-Barr virus (EBV) infection with a primer-vector system developed for isolating DNA fragments of human Ig Fab portions. With this method, monoclonal and oligoclonal cell lines producing anti-HBs antibodies were established and three anti-HBs Fab antibodies were isolated from two of these cell lines. From analysis of affinity characteristics, immunohistochemical activity, and cytolysis activity, these three Fab antibodies were classified into three different groups. The first group had high affinity for HBs, the second had the ability to kill HBV-infected cells, and the third was applicable to immunohistochemical staining with HBV-infected cells. The combined effect of these antibodies was also investigated by complement-dependent cytotoxicity assay.

Cloning and expression of human anti-tumor necrosis factor-alpha monoclonal antibodies from Epstein-Barr virus transformed oligoclonal libraries.

Peripheral blood was obtained from a healthy human volunteer and transformed with Epstein-Barr virus (EBV). This produced an oligoclonal cell library in culture medium that was screened by ELISA for anti-human tumor necrosis factor-alpha (TNFalpha) activity. RNA from two positive clones was applied to RT-PCR using antibody-specific primers, and the light (kappa and lambda) and heavy chain genes (gamma and mu) were cloned into the plasmid vector pFab1-His2. The antibodies produced in Escherichia coli as Fab fragments were assayed for anti-TNFalpha activity utilizing ELISA. Two IgG1/kappa anti-TNFalpha antibodies and two IgM/kappa anti-TNFalpha antibodies were isolated. DNA sequence analysis showed that the VL and VH gene families of IgM and IgG were the same. Both the antibodies showed almost the same activity on ELISA-testing. Ten clones randomly selected from light (kappa and lambda) and heavy (gamma and mu) chain genes in the oligoclonal cell library 1D5 were sequenced, and each gene (kappa, lambda, gamma, and mu) was found to be composed of one to three different genes. These data support the conclusion that the cell clone is oligoclonal at the molecular level.

Bacterial expression of a human recombinant monoclonal antibody fab fragment against hepatitis B surface antigen.

The Fab fragment was cloned from the monoclonal cell line TAPC301-CL4, which was produced using the Epstein-Barr virus (EBV) transformation method. This cell line produces a human monoclonal antibody (CL4MAb) against the hepatitis B surface antigen (HBsAg). This MAb was shown to have hepatitis B virus (HBV) neutralizing activity in chimpanzees. The Fab fragment was produced by subjecting the heavy and light chain antibody genes of the TAPC301-CL4 cell line to reverse transcription-polymerase chain reaction, cloning the products in the plasmid vector pFab1-His2 and introducing the plasmid into bacteria. Sequence analyses of the CL4Fab fragment revealed that the light and heavy chains belong to the Vk3a and VH3 groups of the immunoglobulin (Ig) family, respectively. An enzyme-linked immunosorbent assay confirmed that specificity of the recombinant CL4Fab antibody against HBsAg was the same as that of the parental MAb. Flow cytometric analysis using PLC/PRF/5 (Alexander) cells, which express HBsAg, showed the reactivities of the CL4MAb and CL4Fab antibody were the same. These results suggest that the recombinant CL4Fab antibody produced by Escherichia coli using the new vector-primer system developed for human IgG Fab fragments has a very high affinity for the HBsAg and may be useful clinically. A source for generation of human MAb for human therapy with very stable and specific expression was thus produced by isolating antibodies from EBV-transformed cell lines.

Preparation of recombinant human monoclonal antibody Fab fragments specific for Entamoeba histolytica.

Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis.

Bacterial expression of a neutralizing mouse monoclonal antibody Fab fragment to a 150-kilodalton surface antigen of Entamoeba histolytica.

A mouse monoclonal antibody (MAb) (EH3015, IgG1 with a K light chain) prepared by hybridoma technology recognizes a 150-kD surface antigen of Entamoeba histolytica and inhibits adherence and cytotoxicity of the ameba to mammalian cells. The genes encoding the light chain and the Fd region of the heavy chain of the MAb were cloned and expressed in Escherichia coli. The plasmid used was designed for the expression of Fab with a hexa-histidine tag in the periplasmic space. Recombinant Fab fragments were purified and analyzed by an indirect immunofluorescence antibody test and Western immunoblot. The specificity of the recombinant Fab fragment was comparable with the parent whole IgG. In addition, the Fab fragments significantly inhibited the adherence of E. histolytica to erythrocytes. These results suggest that the production of a neutralizing MAb in Escherichia coli is practical and efficient with this expression system.

A micromethod for protein assay using a light microscope.

A micromethod which utilizes a protein-dye binding reaction on an acetate membrane and a light microscope with an automatic exposure instrument has been developed for measuring small amounts of protein from limited biological materials. After discoidal cellulose acetate membranes (2.0 mm diameter), which absorbed 0.5 microliter of protein solution containing 0.2-2.0 micrograms of protein, were stained with Coomassie Brilliant Blue G-250, they were examined by a light microscope with an automatic exposure instrument. A linear relation was confirmed between the intensity of dye binding to protein and exposure time under specific optical conditions.

Human monoclonal anti-HCMV neutralizing antibody from phage display libraries.

Human cytomegalovirus (HCMV) infection in immunocompromised patients causes considerable morbidity and mortality. Although ganciclovir prophylaxis reduces the incidence of HCMV disease, severe side effects raise serious problems. Thus, the development of new strategies for prophylaxis are clearly needed, and human monoclonal antibodies offer a potential alternative. We describe the cloning, using the phage display system, of a recombinant human Fab fragment against HCMV. A phage display library with 4 x 10(6) clones was panned three times against lysates of HCMV-infected cells, and screened by ELISA. Of six antigen-binding clones, one monoclonal antibody reacted strongly to HCMV. In immunostaining analysis, this Fab was able to stain HCMV-infected cells from 24 h post-infection (pi) through to 96 h pi, but not at 6 h pi. In the presence of cytosine arabinoside, HCMV-infected cells were not stained, even at 24 h pi. These results indicate that an HCMV protein that was recognized by the Fab was synthesized in the late phase of infection. In addition, this Fab exhibited neutralizing activity: at 1 microg/ml it reduced HCMV plaque formation by 50%. The Fab was able to neutralize three HCMV strains, but it did not neutralize HSV-1 or -2 infection.

[Neutralizing human antibody specific for human cytomegalovirus in E. coli expression system].

We isolated neutralizing human Fab fragment specific for human cytomegalovirus (HCMV) by phage display system. Fab libraries were constructed from peripheral lymphocyte of healthy individual. In several clones reacted for HCMV-infected HEL cells, one clone, designated 13-3, stained HCMV infected cells at 96 hrs post infection. It didn't react to cells at 6 hrs post infection or infected cells in the presence of AraC, meaning that 13-3 recognized protein synthesized at late times of infection. It also neutralized HCMV, Towne strain, efficiently. This neutralizing activity was specific for HCMV and no effect for HSV-1 and 2.

Use of a glycoprotein gB promoter for expression of genes inserted into the human cytomegalovirus genome.

We attempted to utilize the human cytomegalovirus (HCMV) as an expression vector by replacing the dispensable genes of the viral genome with foreign genes. The selection of a promoter to be fused to the foreign gene is important to achieve a high expression rate in the recombinant virus. We selected the glycoprotein B (gB) promoter of HCMV as a target of analysis because gB is one of the most abundantly synthesized components in cell culture. The gB promoter, fused to the E. coli lacZ gene, was introduced into the HCMV HindIII-O fragment region by homologous recombination. It was confirmed that the gB promoter-lacZ construct was inserted in the targeted site of HCMV. The expression of the lacZ gene in the recombinant virus infection was initiated 24 h after infection and increased until 120 h post infection. The lacZ gene expression was inhibited by the presence of cytosine arabinoside. These observations indicate that the expression of the lacZ gene is under the control of the late promoter of gB.

Purification and cDNA cloning of evolutionally conserved larval cuticle proteins of the silkworm, Bombyx mori.

A specific set of structural proteins termed larval cuticle proteins (LCPs) accumulates in integuments during larval development of the silkworm, Bombyx mori. Two major larval cuticle proteins, LCP17 and LCP22, were purified from the guanidine hydrochloride extract of the larval cuticle, and specific antibodies were raised against these proteins. Immunoblot analysis revealed that both LCPs are actively synthesized during larval intermolt stages, whereas the LCP17 epitope is also slightly but significantly detectable in pupal integuments. cDNA clones for LCPs were isolated by immunoscreening of the cDNA expression library constructed from larval epidermal mRNA. Predicted amino acid sequences of LCP17 and LCP22 are homologous to cuticle proteins from other insect species, including Manduca sexta, Drosophila melanogaster and Locusta migratoria. This fact suggests that these cuticle protein genes originated from a common ancestral gene and have been conserved during evolution. Northern blot hybridization demonstrated that the expression of LCP17 as well as LCP22 mRNA is controlled in a stage-specific manner in the epidermis of the final instar larvae, suggesting a common regulatory mechanism for transcription of these two intermolt genes.

Identification of mutation sites of a temperature-sensitive mutant of HCMV DNA polymerase activity.

The human cytomegalovirus (strain Towne) temperature-sensitive mutant ts 256 exhibits a virus specific DNA polymerase-negative phenotype. The position of the mutation of ts 256 was determined by three step marker-rescue assays to be within a 5.1 kb XbaI-BamHI fragment between map unit 0.33 and 0.35 in a HindIII-D fragment located in a long unique region. Nucleotide sequencing showed that the 5.1 kb fragment contained three open reading frames corresponding to those of the genes for UL52, UL53 and UL54 (DNA polymerase gene), respectively, of strain AD169. The functions of UL52 and UL53 are unknown. Comparison of the DNA sequences of the 5.1 kb fragments of the wild-type and ts 256 mutant revealed two base changes within UL53 and UL54, respectively, which result in amino acid substitutions. The mutation in the UL54 gene was located within a distinct conserved region VI common to alpha-like DNA polymerases, suggesting that this base change would be responsible for DNA negative phenotype.

Inducible expression of a foreign gene inserted into the human cytomegalovirus genome.

We previously described the insertion of a foreign gene into a non-essential region of human cytomegalovirus (HCMV) by homologous recombination. Here we report insertion of the Escherichia coli lacZ gene downstream of the mouse metallothionein promoter into the HindIII-O region of HCMV by replacement-type recombination. Expression of the lacZ gene in the recombinant was independent of viral growth, but dependent on induction by heavy metals. Of several metals tested for beta-galactosidase induction and also for their toxicity to HEL cells, Zn was found to be the most suitable for use as an inducer. In HEL cells infected with the recombinant in the presence of 50 microsM-Zn, beta-galactosidase activity was maximal 3 days after infection, and reached levels 27 times higher than the value obtained in the absence of Zn.

Expression of the immediate early antigens of human cytomegalovirus is responsible for virus proliferation; an intracellular immunization approach.

The purpose of this study was to determine the sequence within the immediate early (IE) region of human cytomegalovirus (HCMV) that is the most important in HCMV proliferation. Stable DNA-transfected VA/13 cell lines that express the immediate early antigens (IEA's) of HCMV were established and immunofluorescence staining with patients' antibodies revealed that the transfected DNAs were stable and able to express HCMV-IEA's. A cell line (VA/13-mt) that was transformed with a metallothionein (MT)-promoter was able to express IEA's constantly in 80-90% of cultured cells. Under this condition, VA/13-mt cells produced over 50 times the number of HCMV particles produced by parental VA/13 cells. However, another cell line (VA/13-PX) that was transformed with a fragment deleted at the region of EcoRI H of HCMV DNA did not produce as many HCMV particles as the parental VA/13 cells. These findings indicate that IEA's are responsible for virus proliferation, and that the products from a deleted gene of the IE region may contribute to intracellular immunization.

Evidence that the tyrosine kinase domain of a small fraction of epidermal growth factor receptor molecules is exposed on the outer surface of A431 cells.

Intact A431 cells were labeled with [gamma-32P]ATP. The major phosphorylation product of the ecto-kinase activity of A431 cells had the molecular mass of 170 kd and was identified as EGF receptor by specific immunoprecipitation. This phosphorylation was not stimulated by EGF added to the reaction buffer, but replacement of MgCl2 by MnCl2 in the buffer remarkably stimulated phosphorylation. An exogenous protein substrate, alpha-casein, was also phosphorylated by intact A431 cells. The analyses for phospho-amino acids of both EGF receptor and alpha-casein revealed that phosphorylation occurred mainly at phosphotyrosine residues. Tryptic phospho-peptides of the EGF receptor of intact A431 cells labeled with [gamma-32P]ATP were fractionated by HPLC. The elution patterns were essentially the same as that of the autophosphorylated EGF receptor, indicating that the phosphorylation sites of EGF receptor labeled in vivo with [gamma-32P]ATP are located in three tyrosine residues in the carboxyl terminus. These results indicate that the carboxyl-terminal tyrosine kinase domain of a small fraction of the EGF receptor molecules of an A431 cell is exposed on the outer surface of the cells.

Site-specific stable insertion into the human cytomegalovirus genome of a foreign gene under control of the SV40 promoter.

On the basis of a previous finding that the 7.8-kb HindIII-O fragment of the human cytomegalovirus strain Towne genome is nonessential for viral replication, we constructed a vector, pKM, that directs introduction of foreign genes by homologous recombination precisely replacing the O fragment. Using this vector, we constructed Towne-strain-derived recombinant virus in which a chimeric lacZ gene fused to the simian virus 40 promoter and a poly(A) signal were inserted in place of the O fragment. Two types of recombinants were obtained which carried the chimeric gene in opposite directions, beta-Galactosidase (beta Gal) was produced throughout the infection cycle in human embryonic lung cells infected with these recombinants, and the rate of its synthesis in the early stages of infection was comparable to that of synthesis of a 65-kDa viral glycoprotein, one of the abundantly produced viral proteins. The chimeric lacZ gene introduced was stable and no lacZ- revertants have been observed so far.

[Statistical analysis of nuclear medicine in the Higashi Nippon Gakuen University Dental Hospital].

The number of radionuclide in vivo tests carried out at the Higashi Nippon Gakuen University Dental Hospital was statistically analyzed and discussed. The subjects were all patients admitted to the Division of Nuclear Medicine during the seven years between 1981 and 1989. The total number of the patients included those introduced through medical facilities in Toubetsu-cho and others. The results of this analysis were as follows: 1) The proportion of the first time patients admitted to the Higashi Nippon Gakuen University Dental Hospital was only 11 percent of all patients. 2) Cases of radionuclide in vivo tests at the Higashi Nippon Gakuen University Dental Hospital decreased in number, annually. However the proportion of tumor scintigraphy and bone scintigraphy increased gradually.

Expression of human cytomegalovirus immediate early antigens is responsible for a novel chromatin structure of infected human embryonic lung cells.

Whereas human embryonic lung (HEL) cells displayed chromatin fibers composed of a repeat of conventional nucleosomes of 15 nm in diameter, human cytomegalovirus (HCMV) infection induced transient appearance of a novel chromatin structure composed of a repeat of large ellipsoids of 45-65 nm X 15-30 nm with linkers of 50-60 nm long and 6-7 nm thick. Essentially the same change in chromatin structure could be induced when uninfected HEL cell nuclei were incubated in vitro with a 0.4 M NaCl nuclear extract from HCMV-infected HEL cells expressing immediate early antigens (IEA's) or with a similar nuclear extract from NIH/3T3 cells constitutively expressing HCMV IEA's. The latter cell line was established by transformation of the mouse cells with a plasmid carrying the HCMV major immediate early and immediate early 2 genes. These results together with those of control experiments suggest that the expression of IEA's is directly or indirectly responsible for the appearance of the novel chromatin structure in HCMV-infected HEL cells.