Humoral immunity during primate lentivirus infection : evaluation of B cell apoptosis and engineering of virus specific antibodies

Abstract

The focus of this project was to evaluate the contribution of the humoral immunity in controlling infection with primate lentiviruses - an area which has implications both for vaccine design as well as for understanding of the pathogenesis of the disease. The first goal was to generate efficient neutralizing reagents which could be used for passive immunotherapy trials, preferably in the macaque system, and to investigate if peptide immunogens could elicit such antibodies. The second approach was to investigate neutralizing responses in vivo in relation to disease development and contribution of apoptotic cell death in the pathogenic process. In order to identify antigenic regions in the envelope glycoproteins of SIVsm thirty-eight peptides representing selected parts of the envelope were synthesized and used in site-directed ELISA with sera from experimentally infected macaques.

Four antibody-binding regions were identified, corresponding to the second variable region (V2; aa 170-196), the region homologous to V3 in HIV-I (aa 313-346), the carboxy terminus of gp 120 (aa 514-537) and the amino terminus of the transmembrane protein (aa 608-638). Serum reactivity to the V2 region was higher in long-term surviving monkeys than in animals with an early development of simian AIDS. Synthetic peptides did not, however, elicit virus neutralizing antibodies in immunized guinea-pigs. To further investigate if peptides can structurally mimic the epitope of an antibody, random peptide libraries displayed on filamentous phage were used to identify ligands of an HIV-I neutralizing monoclonal antibody. Two libraries, consisting of 9 mer and 15-mer peptides constrained by flanking cysteines, were used. Both the known binding motif, -GPGRA-, and an unrelated motif, -FRLLG-, were isolated and were shown to elicit virus neutralizing antibodies in vivo. SIV specific antibodies with neutralizing activity were cloned from a combinatorial antibody library. Functional Fab were expressed as chimeric macaque/human antibody molecules. Twelve chimeric clones reacting with the SIV envelope were isolated. Six of these clones showed a pronounced neutralizing activity against SIVsm with effects at concentrations of 0.01-0.1 ug/ml.

To solve the problem of low expression levels of recombinant Fab fragments in E. coli, a mammalian expression system was used for three HIV-l specific Fab clones. Antibody fragments expressed in mammalian cells showed increased virus neutralizing activity compared to the same Fab clones expressed in E. coli, underlining the inefficiency of procaryotic expression. No difference in HIV-l neutralizing capacity was detected between monovalent (Fab) and divalent (whole antibody) reagents expressed in CHO cells. Thus, according to these data, bivalency does not always confer improved neutralization efficacy. Apoptosis was investigated in CD4+ T cells and CD19+ B cells from HIV-l infected individuals and related to disease progression and the quality of the humoral immunity. B cells from HIV-l infected individuals were shown to be primed for apoptosis when cultured in vitro. Apoptosis in both cell types were inversely correlated with CD4 cell counts. Staining for cell surface markers demonstrated increased expression of Fas on T cells and Fas ligand on B cells from HIV-l infected individuals. To further investigate the relationship between apoptosis and disease development apoptosis was evaluated in HIV-l infected individuals with varying disease courses. Enhanced apoptosis in CD4+ T cells were observed in progressors whereas slow progressors had normal levels of apoptosis. Elevated levels of apoptosis in B cells were demonstrated in both progressors and slow progressors. Apoptosis in CD4+ T cells and CD19+ B cells correlated with viral load in plasma. Furthermore, low titers of neutralizing antibodies against primary HIV-I isolates were associated with higher levels of apoptosis in B cells.