Antigenic characteristics of rhinovirus chimeras designed in silico for enhanced presentation of HIV-1 gp41 epitopes [corrected]

Abstract

The development of an effective AIDS vaccine remains the most promising long-term strategy to combat human immunodeficiency virus (HIV)/AIDS. Here, we report favorable antigenic characteristics of vaccine candidates isolated from a combinatorial library of human rhinoviruses displaying the ELDKWA epitope of the gp41 glycoprotein of HIV-1. The design principles of this library emerged from the application of molecular modeling calculations in conjunction with our knowledge of previously obtained ELDKWA-displaying chimeras, including knowledge of a chimera with one of the best 2F5-binding characteristics obtained to date. The molecular modeling calculations identified the energetic and structural factors affecting the ability of the epitope to assume conformations capable of fitting into the complementarity determining region of the ELDKWA-binding, broadly neutralizing human mAb 2F5. Individual viruses were isolated from the library following competitive immunoselection and were tested using ELISA and fluorescence quenching experiments. Dissociation constants obtained using both techniques revealed that some of the newly isolated chimeras bind 2F5 with greater affinity than previously identified chimeric rhinoviruses. Molecular dynamics simulations of two of these same chimeras confirmed that their HIV inserts were partially preorganized for binding, which is largely responsible for their corresponding gains in binding affinity. The study illustrates the utility of combining structure-based experiments with computational modeling approaches for improving the odds of selecting vaccine component designs with preferred antigenic characteristics. The results obtained also confirm the flexibility of HRV as a presentation vehicle for HIV epitopes and the potential of this platform for the development of vaccine components against AIDS.

Fig. 1

Competitive ELISA titers illustrating the competition of a 14-mer ELDKWA-containing peptide and a number of chimeric viruses for binding immobilized mAb 2F5. OD is the optical density (absorbance) and [peptide] is the concentration of the competitor peptide. Error bars represent the standard errors of the mean.

Fig. 2

Fluorescence intensity curve for the B1 construct, determined as a function of the 2F5 mAb concentration, used to calculate the equilibrium dissociation constants, K_d (as described in Materials and Methods). The non-linear fitting curve is shown in red: Q=(I – I₀)/(I_∞ – I₀). The corresponding curves for the C1 and 14-C40-1 constructs are similar in shape but shifted to higher concentrations of antibody, reflecting their lower affinities compared to B1. The goodness of fit is measured as χ² per degree of freedom (B1, 1.01; C1, 0.98; 14-C40-1, 0.8).

Fig. 3

Scatter plot of the C^α RMSD values from the ELDKWA motif from the REMD ensembles of the chimeric viruses in solution at 310 K with respect to the same motif of the peptide in the 1TJG crystal structure (x-axis) and in the 1LCX NMR structure (y-axis). The B1 and C1 structures display primarily β-turn conformations, and the 14-C40-1 structures display turn conformations that are less similar to a canonical β-turn conformation.

Fig. 3

Scatter plot of the C^α RMSD values from the ELDKWA motif from the REMD ensembles of the chimeric viruses in solution at 310 K with respect to the same motif of the peptide in the 1TJG crystal structure (x-axis) and in the 1LCX NMR structure (y-axis). The B1 and C1 structures display primarily β-turn conformations, and the 14-C40-1 structures display turn conformations that are less similar to a canonical β-turn conformation.

Fig. 4

A model of the B1 chimera bound to the 2F5 Fab. (a) One of the VP2 subunits of the protomeric unit is shown in green. The Fab has a ribbon representation, with the light chain shown in orange and the heavy chain shown in cyan. The DKW motif of the 2F5 epitope is shown in a stick representation. (b) Close-up view of the binding region of the modeled B1:2F5 Fab complex. Residues of the ELDKWA motif and residues of the 2F5 within 7 Å are shown in stick representations, and the rest are depicted as ribbons, with VP2 in green and the heavy chain, including the H3 region, and the light chain of 2F5 shown in cyan and orange, respectively. The HIV-1 residues are numbered according to their HIV-1 HxB2 amino acid numbers. (c) As (b) with the modeled conformation of the ELDKWA peptide (magenta) in the crystal structure (1TJG) superimposed.

Fig. 4

A model of the B1 chimera bound to the 2F5 Fab. (a) One of the VP2 subunits of the protomeric unit is shown in green. The Fab has a ribbon representation, with the light chain shown in orange and the heavy chain shown in cyan. The DKW motif of the 2F5 epitope is shown in a stick representation. (b) Close-up view of the binding region of the modeled B1:2F5 Fab complex. Residues of the ELDKWA motif and residues of the 2F5 within 7 Å are shown in stick representations, and the rest are depicted as ribbons, with VP2 in green and the heavy chain, including the H3 region, and the light chain of 2F5 shown in cyan and orange, respectively. The HIV-1 residues are numbered according to their HIV-1 HxB2 amino acid numbers. (c) As (b) with the modeled conformation of the ELDKWA peptide (magenta) in the crystal structure (1TJG) superimposed.

Fig. 5

The DNA oligonucleotides for the forward and reverse primers used to generate the new combinatorial library. N, equimolar fractions of A, G, C, and T; and S, equimolar fractions of C and G.