RESUMO
Previously, we generated human monoclonal antibodies using peripheral blood mononuclear cells from an asymptomatic human immunodeficiency virus type 1 (HIV-1)-seropositive donor. One of these monoclonal antibodies (designated clone 3, CL3) recognized 10 amino acids (GCSGKLICTT) within the immunodominant region (cluster I) of the transmembrane envelope glycoprotein gp41 and neutralized infection of target cells with different laboratory isolates. Because the epitope recognized by CL3 has two cysteine residues that could potentially produce a disulfide loop in gp41, we analyzed binding of our monoclonal antibody to the cyclic and linear motif of the peptide sequence IWGCSGKLICTTAVP (residues 600-614). The CL3 antibody did not bind to the synthetic cyclic peptide but did recognize the linear form. Two polyclonal rabbit sera against both the linear and cyclic peptides were then generated. Both antisera bound to viral glycoproteins gp41 and gp160, but neither sera neutralized HIV-1 laboratory isolates. Using a set of alanine-substituted IWGCSGKLICTTAV peptides, we analyzed binding of polyclonal antisera and CL3. The profile of binding of polyclonal antisera to these peptides was different from that of CL3 to the same peptides. This suggests that CL3 recognized a unique neutralizable core epitope, which was not immunogenic in either the cyclic or the linear IWGCSGKLICTTAVP peptides used as immunogens in the rabbits.
Assuntos
Antígenos HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Epitopos Imunodominantes/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Linhagem Celular , Cisteína/imunologia , Cisteína/metabolismo , Dissulfetos/imunologia , Dissulfetos/metabolismo , Antígenos HIV/química , Proteína gp160 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/química , Soropositividade para HIV/imunologia , Soropositividade para HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Soros Imunes/imunologia , Epitopos Imunodominantes/química , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/imunologia , Coelhos , Replicação ViralRESUMO
A potential component that may be useful for passive immunotherapy for HIV-1 is human monoclonal antibodies (HumAbs) possessing potent anti-HIV-1 activity that is directed against conserved regions of the envelope glycoprotein. Such antibodies would, in principle, have the ability to neutralize diverse isolates of HIV-1. To develop such reagents, hybridomas were derived by initial Epstein Barr virus transformation of peripheral blood mononuclear cells (PBMCs) from an asymptomatic HIV-1 seropositive donor followed by fusion with heteromyelomas, and secreted anti-HIV-1 antibodies were further characterized. The specificity of one HumAb, designated as clone 3, was determined by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses that indicated reactivity to the transmembrane envelope glyco-protein gp41. Synthetic pentadecapeptides overlapping by 10 amino acids were utilized for epitope mapping of clone 3; a decapeptide GCSGKLICTT in the transmembrane gp41 was identified as the epitope. Clone 3 bound to SupT1 cells infected with HTLV-IIIB in fluorescent activated cell sorting analysis. In addition, in vitro biological assays demonstrated that clone 3 possessed neutralization reactivity against diverse laboratory isolates as well as an AZT-resistant isolate. Therefore, clone 3 reactivity defines a conserved neutralizable site on the HIV-1 transmembrane glycoprotein. Clone 3 and the conserved immunogenic epitope on gp41 could be useful in passive and active immunotherapy for the acquired immunodeficiency syndrome (AIDS).
