Epitope mapping of neutralizing monoclonal antibodies against duck hepatitis B virus.

In this article we report the first topological mapping of neutralizing epitopes of a hepadnavirus. Duck hepatitis B virus is the only hepadnavirus that can replicate and spread from cell to cell in tissue culture. As a result, it is possible to study hepadnaviral neutralization in vitro with this system. To accomplish this goal, we produced a library of monoclonal antibodies against duck hepatitis B virus and identified 12 neutralizing monoclonal antibodies by using an in vitro neutralization assay. The characteristics of six of the neutralizing monoclonal antibodies were further studied by epitope mapping. From the results of competitive binding studies, three distinct neutralizing epitopes were identified on the pre-S polypeptides and one was identified on the S polypeptide. Our findings suggest that antibodies to both the pre-S and S gene products of duck hepatitis B virus can neutralize viral infection in vitro. The pre-S gene product is at least as important as the S gene product in eliciting neutralizing antibodies.     

Properties of monoclonal antibodies interacting with determinants of hepatitis B virus surface antigen

Fourteen hybridoma clones have been isolated producing the monoclonal antibodies to the surface antigen of the hepatitis B virus (HBsAg). Monoclonal antibodies have been shown to react in high titres with HBsAg in the reactions of PHA, PH and ELISA. The specificity of monoclonal antibodies to two antigenic determinants has been found by the competitive solid phase ELISA technique. Monoclonal antibodies from nine clones react with one determinant while monoclonal antibodies from the rest five clones react with the other nonoverlapping determinant.     

Production of infectious hepatitis delta virus in vitro and neutralization with antibodies directed against hepatitis B virus pre-S antigens.

Hepatitis delta virus (HDV) particles were produced in Huh7 human hepatoma cells by transfection with cloned hepatitis B virus (HBV) DNA and HDV cDNA. The particles were characterized by their buoyant density, the presence of encapsidated viral RNA, and their ability to infect primary cultures of chimpanzee hepatocytes. Successful infection was evidenced by the appearance of increasing amounts of intracellular HDV RNA after exposure to particles. Infection was prevented when particles were incubated with antibodies directed against synthetic peptides specific for epitopes of the pre-S1 or pre-S2 domains of the HBV envelope proteins before exposure to hepatocytes. These data demonstrate that HDV particles produced in vitro are infectious and indicate (i) that infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) that epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.     

Preparation of monoclonal antibodies against duck hepatitis B core antigen and analysis of the monoclonal antibodies

Mice were immunized against duck hepatitis B virus core (DHBc) particles isolated from the liver of asymptomatic carrier ducks of duck hepatitis B virus (DHBV) by ultracentrifugation. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 12 clones of hybridoma cells secreting antibodies against DHBc (anti-DHBc) were isolated. According to the reactivity to core particles and core peptide obtained from DHBc particles treated with SDS-2ME, the 12 antibodies were classified into two groups. Two monoclonal antibodies reacted against both core particles and core peptide (B-type), the other ten monoclonal antibodies reacted against core particles but did not react against core peptide obtained from DHBc particles treated with SDS-2 ME. (A-type). Solid phase enzyme immuno assay (EIA) using these two types of antibodies could detect core antigenisity not only in the liver homogenate but also in the DHBV infected serum. Sucrose gradient analysis and gel filtration analysis revealed this DHBc antigenisity in the serum is not carried by core particles but carried by core peptide, equivalent to HBe antigen in the serum of Hepatitis B virus (HBV) carrier. This EIA may provide sensitive test monitoring both serum DHBe antigen levels and DHBc antigen levels in the liver during DHBV infection.     

Establishment of hybridomas secreting human monoclonal antibodies against tetanus toxin and hepatitis B virus surface antigen

Mouse-human heterohybrids (M X H) were constructed and compared with other cell lines (human or mouse) as parental cells to obtain hybrids secreting human monoclonal antibody (MoAb). One of the M X H lines, HM-5, was far superior to the others and useful for establishing hybrids secreting human MoAb. Using HM-5 as a parental cell line, we have obtained 2 hybrids secreting human anti-tetanus toxoid MoAb with neutralizing activity and a hybrid secreting human anti-hepatitis B virus surface antigen (HBsAg) MoAb which recognizes the a-determinant of HBsAg.     

Screen of multifunctional monoclonal antibodies against hepatitis B core virus-like particles

HBc-VLP can be used in an epitope presentation system to carry foreign epitopes and mimic live virus in order to study viral particle uptake, virion-mediated activation and antigen presentation by dendritic cells. In this study, a multifunctional mAb was produced using a novel research strategy. A truncated HBc-VLP bone vector with a special conformation was used as an immunogen and the target hybridoma cell lines were screened by a series of tests; including ELISA, Western blot, and cellular immunofluorescence based on the epitope presentation system. The screened monoclonal antibody was used to identify the HBc-VLP vector, a fusion HBc-VLP vaccine, and intracellular HBV capsids. The new strategy facilitated acquisition of the desired mAbs and will serve as a reference for other VLP-related research.     

Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen.

Several recently isolated monoclonal antibodies which reacted with simian virus 40 T antigens also reacted with proteins found in uninfected and untransformed cells. The proteins were different from each other, PAb419 reacting with a 35,000-molecular-weight protein, PAb427 reacting with a 75,000-molecular-weight phosphoprotein, PAb405 reacting with a 150,000-molecular-weight phosphoprotein, and PAb204 reacting with a 68,000-molecular-weight protein. It is suggested that although some of these cross-reactions may be fortuitous, they may, as an alternative, reflect similarities of shape and perhaps function between domains of the viral T antigen and the relevant host proteins.     

Properties and applications of new monoclonal antibodies raised against calf DNA polymerase alpha

A panel of 12 hybridoma cell lines secreting monoclonal antibodies against alpha-polymerase were prepared by fusion of mouse myeloma cells and spleen cells of a rat immunized with homogeneous calf thymus alpha-polymerase. Hybridomas were selected and cloned on the basis of immunobinding to pure alpha-polymerase in solid phase radioimmunoassay. Antibodies secreted by these cells eventually were purified in milligram quantities from ascites fluids. These antibodies, all of the rat immunoglobulin M class, cross-reacted with alpha-polymerases from calf and monkey cells as revealed by immunobinding in radioimmunoassay and by immunoprecipitation of DNA polymerase activity. The antibodies were not capable of neutralizing the enzyme activity. With the methods described these antibodies may be used to immunoprecipitate alpha-polymerase from crude extracts of mammalian cells and to measure levels of the enzyme protein.     

Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein

Identification of anti-hepatitis C virus (anti-HCV) human antibody clones with broad neutralizing activity is important for a better understanding of the interplay between the virus and host and for the design of an effective passive immunotherapy and an effective vaccine. We report the identification of a human monoclonal Fab (e137) able to bind the HCV E2 glycoprotein of all HCV genotypes but genotype 5. The results of antibody competition assays and testing the reactivity to alanine mutant E2 proteins confirmed that the e137 epitope includes residues (T416, W420, W529, G530, and D535) highly conserved across all HCV genotypes. Fab e137 neutralized HCV pseudoparticles bearing genotype 1a, 1b, and 4 E1-E2 proteins and to a lesser extent, genotype 2b. Fab e137 was also able to inhibit cell culture-grown HCV (genotype 2a). These data indicate that broadly cross-reacting and cross-neutralizing antibodies are generated during HCV infection.     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

Properties of monoclonal antibodies against glycoproteins of western equine encephalitis virus.

To analyze the biological activities of the alphavirus glycoproteins, eight different monoclonal antibodies against the two glycoproteins of western equine encephalitis virus were isolated. Five of the eight monoclonal antibodies were shown to be specific for E1 and three for E2 protein by an enzyme-linked immunosorbent assay and by radioimmunoprecipitation. Three of the five anti-E1 and all of the anti-E2 monoclonal antibodies inhibited hemagglutination by purified virions. One anti-E1 and two anti-E2 monoclonal antibodies possessed high virus-neutralizing activity.     

A human monoclonal antibody against small envelope protein of hepatitis B virus with potent neutralization effect

Hepatitis B virus (HBV) produces large (L), middle (M), and small (S) envelope proteins, alternatively referred to as hepatitis B surface antigen (HBsAg). Currently, yeast-derived S protein serves as the preventive vaccine, while hepatitis B immune globulin (HBIG) concentrated from pooled plasma of vaccine recipients is employed for post-exposure prophylaxis. However, only a small proportion of the antibodies in HBIG are HBV specific. In the present study, a human monoclonal anti-S antibody (G12) was developed, produced under GLP conditions, and subjected to a panel of functional assays. In vitro results demonstrated high affinity of G12 for the S protein (K_D = 7.56 nM). It reacted with envelope proteins of all 7 HBV genotypes tested (A-F, H) by immunofluorescent staining, and more than 97% of HBsAg-positive patient serum samples by enzyme-linked immunosorbent assay. G12 recognized a conformational epitope, although the exact sequence remains unknown. Strikingly, G12 was at least 1,000-fold more potent than HBIG in neutralizing HBV infectivity in both HepaRG cell line and HepG2 cells reconstituted with the HBV receptor. In a transgenic mouse model of HBV persistence, a single peritoneal injection of G12 markedly diminished serum HBsAg titers in all 7 mice, which was sustained for the observation period of 144 d in mice with low pre-treatment levels. While the therapeutic potential of G12 warrants further investigation using a large number of animals, G12 is a potent neutralizing human monoclonal antibody and a promising candidate to replace or supplement HBIG in the prevention of HBV infection.     

Stabilization of the amplification convertase of complement by monoclonal antibodies directed against human factor B

Three IgM mouse monoclonal antibodies (MoAb), 5B5-A, 2D2-B, and 5B12-A, were prepared by fusion of spleen cells from mice immunized with human B with the SP 2/0 myeloma cell line. They were assessed for their effect on cell-bound and fluid-phase amplification convertases of complement (C) with purified proteins in vitro. 5B5-A and 2D2-B were similar in their effects on cell-bound preformed C3bBb in that they bound to cell-bound C3bBb, stabilized the C3bBb convertase, and rendered the C3bBb convertase relatively resistant to the plasma protein H. These two MoAb were also able to enhance C3 consumption in vitro in reaction mixtures containing C3b, C3, B, and D. At the same time, they presumably stabilized the C3 convertase and caused relative sparing of B hemolytic activity in the reaction mixtures. In contrast, 5B12-A, which also bound to Bb and C3bBb, did not induce any stabilization, but rather caused accelerated decay of cell-bound C3bBb. These results indicate that MoAb against B can have C3NeF-like activity. On the other hand, not all MoAb against B have stabilizing activity on the C3bBb convertase.     

Species-specific recognition patterns of monoclonal antibodies directed against vimentin.

Two commercially available monoclonal antibodies raised against the intermediate filament protein vimentin were characterized concerning their species-specific reaction pattern on vertebrate cells. The antibody V9 exhibited extensive reactivity with vimentin of all mammalian species tested, but specifically did not detect vimentin in mouse cells and chicken fibroblasts. The antibody VIM 3B4 recognized vimentin in cells of chicken and most mammalian species, except for rodent species. Characterization of the binding site of VIM 3B4 on human vimentin by limited proteolysis and immunoblotting as well as by sequence comparison strongly suggested that the epitope is located in the coil 2 part of the vimentin rod domain. Site-directed mutagenesis of a mouse vimentin cDNA clone followed by in vivo expression showed that VIM 3B4 could detect rodent vimentin containing a single amino acid substitution (valine for leucine) at position 353 of the mouse vimentin sequence. Practical application for this finding was demonstrated by the unequivocal identification of a modified murine vimentin protein, distinct from the endogenous vimentin, in a cytoplasmic intermediate filament network in mouse skin fibroblasts transfected with a recombinant plasmid expression vector.     

Functional characterization of fingers subdomain-specific monoclonal antibodies inhibiting the hepatitis C virus RNA-dependent RNA polymerase

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), encoded by nonstructural protein 5B (NS5B), is absolutely essential for the viral replication. Here we describe the development, characterization, and functional properties of the panel of monoclonal antibodies (mAbs) and specifically describe the mechanism of action of two mAbs inhibiting the NS5B RdRp activity. These mAbs recognize and bind to distinct linear epitopes in the fingers subdomain of NS5B. The mAb 8B2 binds the N-terminal epitope of the NS5B and inhibits both primer-dependent and de novo RNA synthesis. mAb 8B2 selectively inhibits elongation of RNA chains and enhances the RNA template binding by NS5B. In contrast, mAb 7G8 binds the epitope that contains motif G conserved in viral RdRps and inhibits only primer-dependent RNA synthesis by specifically targeting the initiation of RNA synthesis, while not interfering with the binding of template RNA by NS5B. To reveal the importance of the residues of mAb 7G8 epitope for the initiation of RNA synthesis, we performed site-directed mutagenesis and extensively characterized the functionality of the HCV RdRp motif G. Comparison of the mutation effects in both in vitro primer-dependent RdRp assay and cellular transient replication assay suggested that mAb 7G8 epitope amino acid residues are involved in the interaction of template-primer or template with HCV RdRp. The data presented here allowed us to describe the functionality of the epitopes of mAbs 8B2 and 7G8 in the HCV RdRp activity and suggest that the epitopes recognized by these mAbs may be useful targets for antiviral drugs.     

Characterization of monoclonal antibodies directed against trail or trail receptors

A subset of tumour necrosis factor receptor family members is involved in death transducing signals and is, therefore, referred as the "death receptors." Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumour cells but only rarely in normal cells. Five distinct receptors have been described for TRAIL: TRAIL R1 (DR4), TRAIL R2 (DR5, TRICK), TRAIL R3 (TRID, DcR1), TRAIL R4 (TRUNDD, DcR2), and osteoprotegerin. In the Eighth International Workshop on Human Leukocyte Differentiation Antigens, 10 monoclonal antibodies (mAbs) reported to be specific for TRAIL or for TRAIL receptors were submitted. In the present study, the mAb specificity was determined by ELISA. Using these mAbs, investigation on the expression of TRAIL and TRAIL receptors was performed. Some of them were able to modulate TRAIL induced programmed cell death.     

TT病毒重组蛋白单克隆抗体的制备

采用杂交瘤技术,获得了4株稳定分泌抗TTV重组蛋白的单克隆抗体杂交瘤细胞株,1株属IgG2bλ链、1株属IgG1κ链、2株属IgG2aκ链。4株杂交瘤细胞培养上清液效价为1：80-1：1280,腹水效价为1：32万-1：160万。     

Characteristics of monoclonal antibodies against Lassa virus

Some properties of monoclonal antibodies to the Lassa virus have been characterized. The competitive immunoenzyme analysis has revealed the presence of at least three antigens in the Lassa virus nucleoprotein.     

Isolation and characterization of monoclonal antibodies directed against two subunits of rabbit poxvirus-associated, DNA-directed RNA polymerase.

A library of monoclonal antibodies directed against individual proteins of the rabbit poxvirus (RPV) virion within a complex immunogenic mixture has been generated through the use of in vivo and in vitro immunization regimens. The relative efficacies of the two procedures were compared. Based on immunoblot analysis, the in vitro immunization regimen led both to a wider variety of monoclonal antibodies to different proteins and to a larger number of antibodies directed against proteins of higher molecular weights. Each method, however, has advantages, and the two procedures appear to be complementary. A simple method to recognize antibodies directed against the virion DNA-directed RNA polymerase was developed. Monoclonal antibodies directed against two subunits (137 and 34 kilodaltons [kDa]) of the RNA polymerase were identified and used to study the biogenesis of the enzyme and to map the two corresponding genes within the viral genome by using an RPV DNA library cloned into the lambda gtll expression vector. Both proteins are synthesized late in the infectious cycle and are restricted totally to the cytoplasm. Preliminary mapping data place the genes encoding the 137-kDa protein within the HindIII H fragment, whereas the gene for the 34-kDa protein is located within the left most region of the HindIII A fragment.