Multiepitope Polypeptide of the HIV-1 Envelope Induces Neutralizing Monoclonal Antibodies against V3 Loop

Abstract

A gene encoding a multiepitope polypeptide (MEP) has been synthesized. It contains the information for (1) an 11-amino acid (aa) epitope from the C1 region of gp120 of HIV-1 and (2) 3 epitopes of 15 amino acids each, from the central part of the V3 loop of isolates MN, SC, and WMJII. These four segments are linked by the short spacer peptide AGGGA. This gene was cloned in a plasmid vector and expressed in Escherichia coli as a fusion product with a 62-aa fragment of human IL-2. The recombinant protein TAB1 was purified by washed pellet procedures and reversed-phase HPLC. TAB1 was recognized in ELISAs by 25 of 27 sera from seropositive individuals. Mice were immunized and several hybridomas were obtained. Two of them secrete monoclonal antibodies that react with synthetic peptides from isolates MN, WMJI, WMJIII, and SC with an affinity constant in the range of 10(8) M-1. They also recognized peptides from isolates SF2 and WMJII, but at much lower affinity. The results obtained from peptide ELISAs indicate that the putative epitope recognized by these MAbs lies within the sequence IHIGPGRAFYT. Classic neutralization assays demonstrated that MAb 2C4 neutralizes 50% of the MN isolate at 0.6 micrograms/ml but fails to neutralize IIIB and SF2 strains. The presence of antibodies directed against every one of the component peptides in the sera of rabbits immunized with TAB1 was also documented.

Immunogenicity comparison of a multi-antigenic peptide bearing V3 sequences of the human immunodeficiency virus type 1 with TAB9 protein in mice

“…Up to now, the strategy of MEP has been used to induce antibody-mediated responses against HIV [11]. For this purpose, the MAP has been widely used against diverse diseases such as hepatitis B virus [5], human malaria [28], blue tongue virus [29], and HIV-1 [8].…”

“…The multi-epitope polypeptides (MEP) concept as vaccine against HIV has been developed by Duarte et al [11,12]. This alternative was designed to generate a broadly reactive and neutralizing antibody response against the third variable region (V3 loop) of HIV type 1 (HIV-1).…”

Induction of a Strong HIV-Specific CD8+T Cell Response in Mice Using a Fowlpox Virus Vector Expressing an HIV-1 Multi-CTL-Epitope Polypeptide

“…TAB9-specific antibody titres in the sera of immunized mice were measured by ELISA, using a method based on that previously described (69). Briefly, 96-well polystyrene plates (Polysorp, Nunc) were coated with recombinant TAB9 protein, produced in E. coli (29), by incubation with 100 mL/well of TAB9 at 10 mg/mL in ELISA coating buffer (50 mM Na 2 CO 3 /NaHCO 3 pH 9.6) overnight at 4°C. The following day, nonspecific binding sites were blocked by incubation with 100 mL/well of 5% non-fat dry milk in PBS plus 0.5% Tween 20 for 1 h at 37°C.…”

“…We have developed multi-epitope polypeptides, which include sequences from different HIV-1 isolates, in an effort to induce broadly cross-reactive immune responses to HIV (29,58). In previous studies, we designed a DNA fragment encoding the central region of the V3 loop (15 amino acids [aa]) of gp120 from HIV-1 isolates LR150, JY1, RF, MN, BRVA and IIIB, fused to a sequence encoding the N-terminal part of the p64K protein from Neisseria meningitidis.…”