Target epitope and transplantation to select acceptor scaffolds. (A) Epitope for antibody 2F5. The HIV-1 virion (schematic based on ref. 40) employs numerous mechanisms of immune evasion to avoid recognition by neutralizing antibody. A potential site of vulnerability is recognized by antibody 2F5, depicted as an antigen-binding fragment (Fab) with heavy chain in blue and light chain in gray, which binds to residues 659–669 of gp41, depicted in red. The sequence of the HIV-1 MPER is from strain HxB2. (B) Shown are diverse structures of gp41 that have been previously determined (7–11, 14, 41), with residues 659–669 highlighted in red. (C) The 2F5-recognized conformation of the epitope shows essentially the same conformation in two different crystal lattices (12, 13). (D) Computational methods were used to transplant the 2F5 epitope into acceptor scaffolds, which were selected and further modified to present the gp41 epitope (red) in its 2F5-recognized conformation. Epitope scaffolds are drawn in gray as Cα-ribbons, with scaffold residues altered to accept transplantation highlighted in stick representation, and colored orange for ES1, yellow for ES2, green for ES3, blue for ES4, and purple for ES5. (E) Two potential measures of graft flexibility, recognition by sera generated by flexible representations of the epitope (vertical axis) and entropy of 2F5 recognition (horizontal axis), were found to correlate. Data points are colored according to epitope scaffold as in D, with gray for MPER peptide. (F) X-ray crystal structure of unbound ES2. The left image shows a Cα-ribbon of the unbound ES2 structure. The right graph shows rmsds for different residue ranges of the 2F5 epitope in the unbound ES2 structure (yellow) or in the initial ES2-computational model (gray), with both compared to the target 2F5-bound conformation of gp41. (G) Crystal structure of ES2 in complex with antibody 2F5. The left image shows a Cα-ribbon of the ES:2F5 complex, with the 2F5 Fab colored blue and gray and ES2 yellow. Coloring for the right graph is the same as in F.

Immununogencity of 2F5-epitope scaffolds. (A) Immunization scheme. A priori, it was unclear what factors would influence immunogenicity. We therefore utilized a highly redundant sparse matrix with three primary variables: type of epitope scaffold (ES1, orange; ES2, yellow; ES3, green; ES4, blue; ES5, purple); type of adjuvant (Alum/CpG, circles; ASO1B, squares); and the presence (closed symbols) or absence (open symbols) of linked T-help (“TH”; PADRE) (27). Twelve different immunization schemes were evaluated in guinea pigs, four animals per group, with sera sampled prior to immunizations (Pre) and after two (Post 2), four (Post 4), and six (Post 6) immunization cycles. (B) Overall titers. ELISA EC₅₀ values of polyclonal antibody responses against the entire scaffolds are shown, as assessed by binding of Pre, Post 2, and Post 4 serum time points to the whole scaffold. (C) Graft-specific titers. ELISA EC₅₀ values of polyclonal antibody responses against the 2F5-epitope portion of the epitope scaffolds are shown, as evaluated by binding of Pre, Post 2, and Post 4 serum time points to a 2F5-epitope peptide (individual responses are shown in Fig. S3). (D) Graft-specific titers (Post 2, circles and dashed black line; Post 4, triangles and solid black line) elicited by the epitope scaffolds (vertical axis) are compared with recognition of the epitope scaffolds by sera generated by the epitope when immunized in a flexible context (e.g., as free peptide or placed into a flexible loop) (horizontal axis). The overall fit is shown as a red line. (E) Graft-specific titers (Post 2, circles and dashed black line; Post 4, triangles and solid black line) elicited by the epitope scaffolds (vertical axis) are compared with the entropy of 2F5 recognition. Lower observed entropies are expected to result from interactions of 2F5 with more rigid grafts. The overall fit is shown as a red line. (F) Residue-by-residue interrogation of the elicited responses. Single alanine mutants were introduced into a collection of 2F5-epitope peptides spanning residues 658–670 (top left). The effects of these alanine mutants on antibody 2F5 binding were evaluated by ELISA, with changes to the central Asp-Lys-Trp tripeptide ablating binding and other residues displaying more muted responses (middle left, black line). The alanine mutants were also used to interrogate sera elicited by flexible and cyclized peptides (bottom left, dark brown and light brown, respectively), as well as against all Post 2, Post 4, and Post 6 sera (right panels; lines and symbols are colored based on scaffold coloring depicted in A; for Post 6, symbols are colored based on scaffolds used in the final two immunizations). The 2F5 alanine scan profile (black) is shown in all panels for comparison. (G) Optimal responses. Responses to the alanine-mutant epitope peptides were ranked by R-value of the response, as defined by the expression , where i is the residue position at which the MPER was mutated to alanine. Alum/CpG, linked T help, increasing number of immunizations, heterologous immunizations, and use of ES5 all biased toward reduced R-values (Fig. S3C and Table S5). Shown here are results from alanine-scanning for the top three responses along with corresponding R-values and p-values of the immunization schemes (p-values were obtained as described in SI Materials and Methods; because 58 different sera or grouped sera were analyzed, Bonferroni adjustments were calculated to account for multiple comparisons, with individual p-values from each serum comparison to 2F5 multiplied by a factor of 58). The 2F5 alanine scan profile (black) is shown in all panels for comparison.

Structure of an epitope-scaffold-elicited antibody. The epitope scaffolds developed here are mimics of the target template, chosen in this case to be the 2F5-recognized structure of gp41. Immunization teaches the immune system to make antibody “molds” capable of recognizing or inducing the desired conformation in the target template (even if the template is a flexible peptide, which would otherwise rarely assume the desired conformation). (A) Structure of ES5-ES1-elicited antibody, 11f10 (heavy chain, orange; light chain, purple), in complex with a peptide corresponding to residues 660–667 of gp41 (yellow). (B) Close-up of bound gp41 peptide in the 11f10 complex. Experimental electron density (2F_o-F_c, at 1σ contour) is shown in blue around the bound gp41 peptide (yellow). (C) Comparison of gp41-peptide conformations when bound by antibody 11f10 (yellow) or by template antibody 2F5 (salmon). (D) Electrostatic potential of ES5-ES1-elicited antibody 11f10 displayed at its molecular surface, with electronegative regions in red, electropositive regions in blue, and apolar regions in white. (E) Electrostatic potential of antibody 2F5 displayed at its molecular surface and colored as in D. (F) Antigen-combining surface of antibody 11f10, colored according to residue type (hydrophobic, green; polar, gray; positive, blue; negative, red). (G) Antigen-combining surface of template antibody 2F5, colored as in F. (H) Overlap of elicited and template antibodies (with light and heavy chains colored orange and purple for 11f10, respectively, and colored gray and blue for 2F5, repectively), aligned by superposition of all atoms of the bound gp41-peptides (residues 660–667). Bound gp41 peptides in D–H are colored as in C.