ABSTRACT
The nucleocapsid of the hepatitis B virus (HBV) is composed of 180 to 240 copies of the HBV core (HBc) protein. HBc antigen (HBcAg) capsids are extremely immunogenic and can activate naive B cells by cross-linking their surface receptors. The molecular basis for the interaction between HBcAg and naive B cells is not known. The functionality of this activation was evidenced in that low concentrations of HBcAg, but not the nonparticulate homologue HBV envelope antigen (HBeAg), could prime naive B cells to produce anti-HBc in vitro with splenocytes from HBcAg- and HBeAg-specific T-cell receptor transgenic mice. The frequency of these HBcAg-binding B cells was estimated by both hybridoma techniques and flow cytometry (B7-2 induction and direct HBcAg binding) to be approximately 4 to 8% of the B cells in a naive spleen. Cloning and sequence analysis of the immunoglobulin heavy- and light-chain variable (VH and VL) domains of seven primary HBcAg-binding hybridomas revealed that six (86%) were related to the murine and human VH1 germ line gene families and one was related to the murine VH3 family. By using synthetic peptides spanning three VH1 sequences, one VH3 sequence, and one VLkappaV sequence, a linear motif in the framework region 1 (FR1)complementarity-determining region 1 (CDR1) junction of the VH1 sequence was identified that bound HBcAg. Interestingly, the HBcAg-binding motif was present in the VL domain of the HBcAg-binding VH3-encoded antibody. Finally, two monoclonal antibodies containing linear HBcAg-binding motifs blocked HBcAg presentation by purified naive B cells to purified HBcAg-primed CD4(+) T cells. Thus, the ability of HBcAg to bind and activate a high frequency of naive B cells seems to be mediated through a linear motif present in the FR1-CDR1 junction of the heavy or light chain of the B-cell surface receptor.
Subject(s)
B-Lymphocytes/immunology , Hepatitis B Core Antigens/immunology , Receptors, Immunologic/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Antibodies, Viral/analysis , Antigen Presentation , Binding Sites, Antibody , CD4-Positive T-Lymphocytes/immunology , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Immunoglobulin M/analysis , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Mice, Transgenic , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/genetics , Peptide Fragments/immunology , Receptors, Immunologic/immunology , Sequence AlignmentABSTRACT
Comparison of the primary structures and theoretical prediction of the potential antigenic determinant of the deduced Fos proteins reveals the presence of a nonstructural and hydrophilic region juxtaposed to the leucine zipper and nonconserved among the Fos protein family. To develop monoclonal anti-peptide antibodies capable of distinguishing all Fos-proteins, synthetic peptides specific for the mentioned predicted region were synthesized manually by the "tea-bag" method. Immunization of Balb/c mice with fosB-related synthetic peptide BSA gave rise to mouse hybridoma cell line K21 (IgG1, kappa) secreting highly specific antibodies against corresponding human fosB protein. Fine mapping of the MAb K21 indicated that the minimal epitope essential for the recognition is the sequence GPGPLAE.
Subject(s)
Proto-Oncogene Proteins c-fos/immunology , Amino Acid Sequence , Animals , Antibody Specificity , Drug Resistance, Microbial/immunology , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Humans , Mice , Molecular Sequence Data , Peptide Fragments/immunologyABSTRACT
The construction of a mouse hybridoma FR52 secreting neutralizing monoclonal antibody specific for RNA bacteriophages fr, MS2 and GA is reported. The genes encoding the variable domains of the monoclonal antibody FR52 heavy and light chains were cloned and sequenced and the corresponding complementarity determining region (CDR) peptides were chemically synthesized. The CDR-peptides were tested for their ability to neutralize the activity of RNA phage fr and related RNA phages MS2 and GA. The CDR-derived peptides H2, L2 and L3 interacted with the fr phage particles and neutralized fr phage activity. Two of these peptides--H2 and L3 also had the ability to neutralize partly the activity of related bacteriophage MS2, but L1 and especially L3 neutralize the activity of the RNA phage GA. These results provide an excellent system for further antibody-antigen interaction studies and raise the possibility that simple CDR-peptides may serve as a new class of anti-viral molecules.
Subject(s)
Antibodies, Monoclonal/immunology , Immunoglobulin Variable Region/immunology , Peptide Fragments/immunology , RNA Phages/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Base Sequence , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Immunoglobulin Variable Region/chemistry , Mice , Mice, Inbred BALB C , Microscopy, Immunoelectron , Molecular Sequence Data , Neutralization TestsABSTRACT
We developed a typing assay for HIV-1 using subtype specific peptides corresponding to the five major subtypes of HIV-1 (A to E). In eight patients serologically subtyped as A (n = 1), B (n = 3), C (n = 3) and E (n = 1) phyllogenetic analysis of sequenced V3 domain DNA completely correlated to the peptide serotyping. Out of 106 HIV-1 seropositive samples of a diverse geographical origin 88 (83%) could be subtyped by the peptide assay. Five were of subtype A, 33 of subtype B, 48 of subtype C, one of subtype D, and one was of subtype E. Swedish patients were mainly of HIV-1 subtype B and Ethiopian patients were mainly of subtype C, confirming the performance of the assay. Furthermore, subtype specific antibodies may persist up to nine years in HIV-1 infected patients though sera close to AIDS diagnosis may be difficult to type.
