Comparative immunogenicity of subtype a Human Immunodeficiency Virus type 1 envelope exhibiting differential exposure of conserved neutralization epitopes

Abstract

Development of broadly cross-reactive neutralizing antibodies (NAbs) remains a major goal of HIV-1 vaccine development, but most candidate envelope immunogens have had limited ability to cross-neutralize heterologous strains. To evaluate the immunogenicity of subtype A variants of HIV-1, rabbits were immunized with pairs of closely related subtype A envelopes from the same individual. In each immunogen pair, one variant was readily neutralized by a variety of monoclonal antibodies and plasma antibodies, while the other was neutralization resistant, suggesting differences in the exposures of key epitopes. The breadth of the antibody response was evaluated against subtype A, B, C, and D variants of HIV-1. The specificity of the immunogen-derived neutralizing antibody response was also compared to that of the infected individuals from whom these variants were cloned. None of the immunogens produced broad neutralizing antibodies in immunized animals, and most of the neutralizing antibodies were directed to the variable loops, particularly the V3 loop. No detectable antibodies to either of the potentially exposed conserved epitopes, the membrane proximal external region, or the CD4 binding site were found with immunized rabbits. In contrast, relatively little of the neutralizing activity within the plasma samples of the infected individuals was directed to linear epitopes within the variable loops. These data indicate that immunogens designed to expose conserved regions did not enhance generation of broadly neutralizing antibodies in comparison with the immunogens that failed to expose those regions using this immunization approach.

FIG. 1.

Analysis of Q461d1^S gp140 used for immunizations. (A) SDS-PAGE analysis of final preparation of Q461d1^S gp140 from the GNA capture and DEAE and CHAP columns. Lane 1 contains molecular weight standards, lane 2 the concentrated DEAE flowthrough, and lane 3 the final concentrated protein. The purified Q461d1^S gp140 protein is indicated by an arrow. The sizes of the molecular weight markers (in thousands) are indicated on the left. (B) Binding of purified gp140 subtype A to CD4 as determined by a high-pressure liquid chromatography (HPLC)-based assay. The bottom line represents the protein obtained after the GNA column, and the top line represents purified protein after all three steps. The trimer and monomer peaks are marked. (C) Summary of neutralization characteristics of all four HIV-1 subtype A Env variants used in the immunizations, adapted from reference . The pseudovirus is shown in the far left column. IC₅₀ values for plasma sample (left) and monoclonal antibodies (right) are displayed. The autologous plasma samples were taken 3.7 ypi for subject Q461 and 2.6 ypi for subject Q168. The Kenya pool was derived by pooling plasma from 30 HIV-1-infected individuals in Kenya and has been described previously (6).

FIG. 2.

Homologous and heterologous neutralizing antibody responses against pseudotyped viruses made with the four env variants used as the basis for immunogen design. The ability of a 1:20 dilution of rabbit serum samples to neutralize the pseudoviruses shown at the top following DNA priming and gp140 boosting is indicated along the y axis. Each point represents the immune response by an individual rabbit, with six rabbits per immunogen. The immunogen is indicated along the bottom x axis. Homologous responses are highlighted by the gray bars. Fifty percent neutralization is indicated by the dotted horizontal line.

FIG. 3.

Contribution of antibodies to the V1, V2, and V3 loops to the neutralizing potential of plasma from HIV-1-infected subjects and serum samples from rabbits immunized against the Q461d1^S pseudovirus. (A) Amino acid alignment of the V1V2 loops (top) and V3 loops (bottom) for the four Env variants used as immunogens is shown. The location of the 20-mer peptides used for peptide competition neutralization assays are indicated by the brackets above the sequence; the sequences were made to match the Q461e2^R variant, which is identical to the Q461d1^S sequence, except of a single amino acid within the V2 C crown. Dashes indicate that the same amino acid was present at that position, and dots indicate a deletion of an amino acid at that position. (B) Example of representative neutralization curves depicting a decrease in neutralization by preincubation of serum samples from Q461d1^S-immunized rabbits with V3 N-terminal, but not V2 C-crown, peptide. (C) Example of representative neutralization curves depicting minimal reduction in neutralization by preincubation of plasma from subject Q461 at 3.7 ypi with V2 C-crown and V3 N-terminal peptides. (D) The plasma or serum sample indicated in the far left column were preincubated with the peptide indicated along the top before the addition of the Q461d1 pseudovirus in a TZM-bl neutralization assay. The percentage of contribution of the indicated peptide to the neutralizing activity of that serum or plasma sample is indicated in the table. Increasing contribution to neutralizing activity is indicated by darker shaded boxes. Unshaded boxes indicate <5% contribution of that epitope to neutralizing activity.

FIG. 4.

Contribution of antibodies to the CD4 binding site plasma from subject Q461. (A) Plasma from subject Q461 at 3.7 ypi was preincubated with magnetic beads coated with bovine serum albumin (BSA) (squares), wild-type SF162 gp120 (diamonds), or SF162gp120 containing mutations in the CD4 binding site (circles). The beads were removed, and the depleted plasma samples were tested for their ability to bind to wild-type SF162 gp120 (closed symbols) or CD4 binding site mutant gp120 (open symbols). The absorbance at 450 nm is shown on the y axis, with the serum dilution on the x axis. (B) Antibodies eluted from the wild-type SF162 gp120 beads (squares) or the CD4 binding site mutant (circles) were tested for their ability to bind to wild-type gp120 protein (closed) or gp120bs mutant protein (open). The absorbance at 450 nm is shown on the y axis, with the concentration of antibody on the y axis. (C) Plasma (represented in panel A) that was nondepleted (squares), depleted with wild-type SF162 gp120 protein (closed circles), or depleted with beads coated with CD4 binding site mutant gp120 protein (open triangles) were tested for their ability to neutralize SF162 pseudovirus. The percentage of neutralization is shown on the x axis, with the plasma dilution on the y axis. (D) The antibodies eluted from the Wt-gp120 or BS-mut gp120 beads, represented in panel B, were tested for their ability to neutralize the SF162 pseudovirus. (E) Ability of depleted plasma to neutralize Q461d1^S pseudovirus, described in the legend for panel C. (F) Ability of antibodies eluted from Wt- or BS-mut-coated beads to neutralize Q461d1^S pseudovirus, as described in the legend for panel D. All results are representative of two independent experiments; contribution of neutralization was calculated according to the AUC, as described in Materials and Methods.

FIG. 5.

Contribution of binding antibodies to the CD4 binding site within serum samples of rabbits immunized with the Q461d1^S or Q461e2^R Envs. Rabbit serum samples from Q461d1^S-immunized animals (A) or Q461e2^R-immunized animals (B) at a 1:10 dilution were preincubated with magnetic beads coated with BSA (squares), wild-type SF162 gp120 (triangles), or SF162gp120 containing mutations in the CD4 binding site (circles). The beads were removed, and the depleted plasmas were tested for their ability to bind to wild-type SF162 gp120 (closed symbols) or CD4 binding site mutant gp120 (open symbols). The absorbance at 450 nm is shown on the y axis, with the serum dilution on the x axis.