The c3-v4 region is a major target of autologous neutralizing antibodies in human immunodeficiency virus type 1 subtype C infection

Abstract

The early autologous neutralizing antibody response in human immunodeficiency virus type 1 (HIV-1) subtype C infections is often characterized by high titers, but the response is type specific with little to no cross-neutralizing activity. The specificities of these early neutralizing antibodies are not known; however, the type specificity suggests that they may target the variable regions of the envelope. Here, we show that cross-reactive anti-V3 antibodies developed within 3 to 12 weeks in six individuals but did not mediate autologous neutralization. Using a series of chimeric viruses, we found that antibodies directed at the V1V2, V4, and V5 regions contributed to autologous neutralization in some individuals, with V1V2 playing a more substantial role. However, these antibodies did not account for the total neutralizing capacity of these sera against the early autologous virus. Antibodies directed against the C3-V4 region were involved in autologous neutralization in all four sera studied. In two sera, transfer of the C3-V4 region rendered the chimera as sensitive to antibody neutralization as the parental virus. Although the C3 region, which contains the highly variable alpha2-helix was not a direct target in most cases, it contributed to the formation of neutralization epitopes as substitution of this region resulted in neutralization resistance. These data suggest that the C3 and V4 regions combine to form important structural motifs and that epitopes in this region are major targets of the early autologous neutralizing response in HIV-1 subtype C infection.

FIG. 1.

(A) Development of anti-V3 binding antibodies in six individuals (absorbance at 405 nm [Abs₄₀₅] versus weeks postinfection). Binding of each serum to the autologous peptides is shown in black, with binding to heterologous peptides shown in gray. (B) Peptide adsorption neutralization assays. Sera were preincubated with autologous V3 peptides (black) or irrelevant peptides (gray) prior to performing neutralization assays against the autologous enrollment virus. Neutralization titer (ID₅₀) is plotted against the number of weeks p.i.

FIG. 2.

Entropy analysis of six amino acid Env (gp160) sequences. Regions of greatest variability (V1V2, C3, V4, and V5) are highlighted in gray. Above the entropy plot is a schematic depicting pairs of chimeric viruses constructed for this study.

FIG. 3.

Amino acid alignment of gp120 from the six parental envelope clones used to construct chimeric viruses. Regions exchanged between viruses are annotated, and within the C3 region, the α2-helix and β-14 and β-15 strands are shown. PNGS are highlighted in gray, and amino acids involved in CD4 binding (40) are indicated by asterisks.

FIG. 4.

Neutralization data for chimeric and parental viruses. ID₅₀ titers are shown on the y axis and the number of weeks p.i. are on the x axis. Solid lines indicate parent viruses; dotted lines indicate chimeras, with the color matching the backbone of the parental viruses. Each graph represents neutralization data using serum from a single individual, indicated above each column. Shaded boxes indicate transfer of sensitivity in chimeras. (A) Neutralization data for viruses chimeric for the V1V2, V4, and V5 regions. (B) Neutralization data for viruses chimeric for the C3 and C3-V4 regions.

FIG. 5.

Chimeras do not acquire a generally neutralization-sensitive phenotype. Summary of neutralization data for all chimeras and parental viruses tested against heterologous sera. Data are shown as neutralization titer (ID₅₀) and are color coded.

FIG. 6.

Helical wheel plot of the α2-helices of viruses from CAP45, CAP84, CAP63, and CAP88. Hydrophilic amino acid residues are shown in clear circles, and hydrophobic residues are shown in gray circles.