The antibody 2F5 is one of the few broadly neutralizing monoclonal antibodies against type 1 Human Immunodeficiency Virus (HIV-1). It recognizes a seven amino acid sequence, ELDKWAS, in gp41 of HIV-1. Previous studies have shown that immunogen-specific antibodies can be induced in an adjuvant-independent manner by directing protein immunogens to class II MHC expressing cells. Thus, in order to create an immunogen that recapitulates the native epitope of 2F5, a series of recombinant immunotargeting constructs were engineered, each incorporating the ELDKWAS sequence within the framework of an anti-class II major histocompatibility complex (MHC) antibody. Three sets of immunogens were constructed, with the ELDKWAS sequence incorporated at sites with different secondary structures. In the first set, designated H-HR1 to 5, the sequence was incorporated at helical sites. In the second, designated H-BT1 to 4, it was incorporated at beta-turn sites. In the third, designated L-CT and L-CT x 3, it was incorporated as minimally constrained C-terminal attachments. All constructs were found to retain class II MHC specificity. However, only three of the four H-BT's (H-BT1 to 3) and the two L-CT's were 2F5 reactive. None of the H-HR's showed any detectable 2F5 reactivity. This suggests that the native 2F5 epitope may be more beta-turn like than helical in conformation. Furthermore, the reactive H-BT's were found to bind 2F5 with 5- to 10-fold higher affinities than their unconstrained counterpart L-CT. When the H-BT's and L-CT's were used to immunize rabbits, immunogen-specific antibodies were induced with or without adjuvant. gp160 cross-reactive antibodies were also induced, with H-BT1 giving the highest gp160 titres among the H-BT's. The antibodies induced by H-BT1 were found to recognize the ELDKWAS sequence in a conformation that was not well mimicked by free ELDKWAS peptide in solution. Unfortunately, using in vitro pseudotyped HIV neutralization assays, virus neutralization by the antisera or the purified IgG samples was not detected. Further studies aimed at increasing the immunogenicity of the ELDKWAS sequence in the H-BT constructs or the engineering of alternative 2F5-reactive antigenic structures may lead to better understanding of the requirements of a vaccine capable of inducing 2F5-like neutralizing antibodies against HIV-1.