An optimally constrained V3 peptide is a better immunogen than its linear homolog or HIV-1 gp120

Abstract

Synthetic peptides offer an attractive option for development of a V3-directed vaccine. However, immunization with flexible linear peptides may result in an immune response to multiple conformations, many of which differ from the native conformation of the corresponding region in the protein. Here we show that optimization of the location of a disulfide bond in peptides constrained to mimic the beta-hairpin conformation of the V3, yields an immunogen that elicits a 30-fold stronger HIV-1 neutralizing response in rabbits compared with the homologous linear V3 peptide. The HIV-1 neutralizing response elicited by the optimally constrained peptide is also significantly stronger than that elicited by a gp120 construct in which the V3 is exposed. Neutralization of an HIV-1 strain that shares only 72% identity with the immunizing peptide was demonstrated. The most effective immunogen was also able to neutralize primary isolates that are more resistant to neutralization such as SS1196 and 6535.

Fig. 1

Chromatographic and Mass-Spectra (MS) evaluation of synthetic constrained immunogens. Analytical HPLC profile of crude linear (A), purified linear (B) and purified cyclic (C) C4-V3 N301C-G325C. ESI-MS spectrum of purified linear (D) and purified cyclic (E) C4-V3 N301C-G325C. The HPLC was run using a 10-60% acetonitrile/water gradient (containing 0.1%TFA) over 20 min; Column: Zorbax-Eclipse XDB- C8, 150×4.6 mm; Flow-rate: 1.0 mL/min; Detection at 220nm; Product R_t 11.799 min; The average molecular weight of the linear was 4798.1 while the average molecular weight of the cyclic was 4796.0.

Fig. 2

Binding of antibodies elicited by C4-V3 peptides or gp120 to the corresponding V3 peptide and gp120. A) Rabbit B707 serum, immunized with C4-V3L, B) Rabbit B963 serum, immunized with C4-V3_T303C-E322C, C) Rabbit B892 immunized with C4-V3_N301C-G325C and D) Rabbit B959 serum, immunized with gp120. Binding to the homologous peptide is shown in triangles (▲ post-immune, Δ pre-immune). Binding to gp120 in is shown in squares (■ post-immune, □ pre-immune). Y-axis represents OD at 650nm; X-axis represents the reciprocal of serum dilution. Standard deviation is for duplicates on plate.

Fig. 3

Relative cross reactivity of immune sera with gp120 and V3 peptide immunogens. The ratio between the immune-sera binding to gp120 and binding to the homologous peptide used for immunization is represented. The ratio is obtained by dividing the half maximal titer for gp120 by the half maximal titer to the V3 peptide used as immunogen. Shown is the average and standard deviation for each of the four rabbits immunized with each of the peptides. Peptide immunogens are listed by the positions replaced by cysteine according to Table1. P-value for one sample T-test for a hypothetical mean of 1 is shown above the histograms.

Fig. 4

Influence of peptide conformation on the binding of immune sera to a cyclic V3 peptide. The binding of the immune-sera to cyclic vs. reduced V3 _T303C/I323C is compared to binding to V3L for the C4V3L and C4-V3_T303C/I323C induced sera. Half-maximal binding titer to cyclic and reduced V3_T303C/I323C and to V3L with and without DTT was determined by ELISA for each V3_T303C/I323C and V3L immune serum. In each experiment the binding of the serum to V3_T303C/I323C in the cyclic (dark) or reduced state (light) was divided by the binding to V3L. Shown is the average and standard deviation for each of the four rabbits immune sera. P-value for two samples T-test for reduced vs. cyclic is shown.

Fig. 5

comparison of the neutralization of SF-162. GMT for serum dilution inflicting 50% inhibition (IC:50) of SF-162 is presented for the different immunogens used in the study.