| Abstract: | The broadly neutralizing antibody immunoglobulin G1 (IgG1) b12 binds to a conformationally conserved surface on the outer domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) glycoprotein. To develop outer domain proteins (ODs) that could be recognized selectively by CD4-binding-site (CD4-BS) antibodies, membrane-anchored ODs were generated from an HIV-1 clade B virus, TA1 R3A, which was highly sensitive to neutralization by the IgG1 b12 antibody. A 231-residue fragment of gp120 (residues 252 to 482) linked to transmembrane regions from CD4 showed b12 binding comparable to that of the native Env spike as measured by flow cytometry. Truncation of the β20-β21 hairpin (residues 422 to 436 to Gly-Gly) improved overall protein expression. Replacement of the immunodominant central 20 amino acids of the V3 loop (residues 302 to 323) with a basic hexapeptide (NTRGRR) increased b12 reactivity further. Surface calculations indicated that the ratio of b12 epitope to exposed immunogenic surface in the optimized OD increased to over 30%. This OD variant OD(GSL)(Δβ20-21)(hCD4-TM)] was recognized by b12 and another CD4-BS-reactive antibody, b13, but not by eight other CD4-BS antibodies with limited neutralization potency. Furthermore, optimized membrane-anchored OD selectively absorbed neutralizing activity from complex antisera and b12. Structurally designed membrane-anchored ODs represent candidate immunogens to elicit or to allow the detection of broadly neutralizing antibodies to the conserved site of CD4 binding on HIV-1 gp120.The human immunodeficiency virus type 1 (HIV-1) envelope is composed of surface gp120 and transmembrane gp41. Initial attempts to develop HIV vaccines through the induction of antibodies focused on recombinant gp120 glycoproteins. Two phase III clinical trials conducted in the United States and Thailand showed no protection from a gp120-based subunit vaccine against HIV infection, nor did the vaccine delay HIV-1 disease progression (11, 25). In addition, a phase II trial completed in Thailand with a live recombinant HIV-1 canarypox vaccine (vCP1452) in combination with a gp120 subunit protein did not stimulate a markedly improved immune response (28). The lack of efficacy was likely to be related to its failure to elicit broadly neutralizing antibodies (4, 10, 33).Several broadly neutralizing human monoclonal antibodies (MAbs) have been derived from infected individuals, including immunoglobulin G1 (IgG1) b12, 2G12, 2F5 and 4E10, which are directed against CD4-binding-site (BS), carbohydrate, and membrane-proximal regions of HIV Env (reviewed in reference 9). Among the most potent, the b12 antibody occludes the site of CD4 binding on gp120 and prevents virus attachment to CD4 on target cells (39). Other CD4-BS antibodies recognize epitopes on monomeric gp120 that overlap with b12 but lack the ability of b12 to neutralize primary HIV-1 isolates (5). An understanding of the specificity of b12 binding, neutralization, and protection should aid in the development of immunogens that induce neutralizing antibodies of a similar specificity.The structure of the b12-gp120 complex (39) shows that b12 binds to a conformationally conserved surface, which is centered around the CD4-binding loop on the outer domain of gp120. In the CD4-bound conformation of gp120, the CD4-binding loop or β15-strand makes antiparallel intermolecular hydrogen bonds to the C″ strand of CD4 (14). Overall, the outer domain of gp120 comprises 82% of the gp120 contact surface with b12, while most of the contacts outside of the outer domain have marginal importance (39). One exception, however, are contacts to the loop connecting the outer domain with the α5-helix of the inner domain (39), which appear to be important.Because it represents the smallest structural unit containing the b12 epitope, and therefore maximizes the b12-immunogenic surface relative to the overall surface, an outer domain-only immunogen with high b12 affinity represents an attractive immunogen. An outer domain construct (named OD1) was previously derived from HIV-1 strain YU2 gp120 and found to bind 2G12 and a number of anti-V3 antibodies (36); however, b12 binding to this construct was difficult to detect by enzyme-linked immunosorbent assay, probably due to an enhanced off rate (36, 39). A large, relatively flat interface exists between the inner and outer domains of gp120 in both CD4-bound and b12-bound conformations. We reasoned that the removal of the inner domain might partially destabilize it and decided to replace the inner domain with another polar surface, the cell membrane. We expressed outer domain proteins (ODs) in various membrane-anchored forms and tested their abilities to bind b12. An HIV-1 clade B R5 and X4 dual-tropic virus, R3A, was selected as a prototype (20). Laboratory-adapted virus strain R3A TA1 contains a truncated V1/V2 and a truncated V3 (named 9,9), maintains CCR5 tropism, and is highly sensitive to b12 neutralization (15, 23). We used available atomic-level structures to model an R3A gp120 core and to design truncations of flexible, potentially immunodominant structures, which emanate from OD, including the β20-β21 hairpin and the V3 loop. Thus, by using structure-based design to modify the OD form of R3A TA1, we attempted to remove strain-specific determinants, to enhance cell-surface expression, and to increase specific b12 binding compared to other native forms. |
