Specificity of anti-Nef antibodies produced in mice immunized with DNA encoding the HIV-1 nef gene product

Mice immunized with plasmid DNA encoding Nef regulatory protein of human im munodeficiency virus type 1 developed high levels of anti-Nef antibodies. A fter 4 intramuscular injections of 100 mu g plasmid DNA, anti-Nef antibodie s reached titers up to 2 x 10(4). A significant specific antibody response was maintained for at least 16 months. Using a set of seven 31-66 mer synthetic peptides covering the entire seque nce of Nef, we analysed the specificity of ant-Nef antibodies. Interestingl y, specific antibodies produced in response to Nef expressing plasmid DNA d id not recognize the linear peptides except the long C-terminal peptide (aa 141-205) for 3 of the 10 sera. With anti-Nef antibodies produced in mice i mmunized with the protein Nef without any adjuvant, the same restraint epit ope binding was found. Only 3 of the 5 Nef positive sera reacted with the C -terminal peptide. This suggests that specific antibodies induced by plasmi d DNA as well as by the non-denatured protein recognize conformation-depend ent epitopes. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity patte rn. Interestingly, the analysis of immunoglobulin isotype profiles of antib odies generated by the different protocols of immunization showed that plas mid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 ant ibodies. (C) 1999 Elsevier Science Ltd. All rights reserved.