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Comparative Study
. 2008 Aug;82(15):7369-78.
doi: 10.1128/JVI.00562-08. Epub 2008 May 21.

Improved induction of antibodies against key neutralizing epitopes by human immunodeficiency virus type 1 gp120 DNA prime-protein boost vaccination compared to gp120 protein-only vaccination

Michael Vaine  1 , Shixia Wang, Emma T Crooks, Pengfei Jiang, David C Montefiori, James Binley, Shan Lu
Affiliations
Comparative Study

Improved induction of antibodies against key neutralizing epitopes by human immunodeficiency virus type 1 gp120 DNA prime-protein boost vaccination compared to gp120 protein-only vaccination

Michael Vaine et al. J Virol. 2008 Aug.

Abstract

A major challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development is to elicit potent and broadly neutralizing antibodies that are effective against primary viral isolates. Previously, we showed that DNA prime-protein boost vaccination using HIV-1 gp120 antigens was more effective in eliciting neutralizing antibodies against primary HIV-1 isolates than was a recombinant gp120 protein-only vaccination approach. In the current study, we analyzed the difference in antibody specificities in rabbit sera elicited by these two immunization regimens using peptide enzyme-linked immunosorbent assay and a competitive virus capture assay. Our results indicate that a DNA prime-protein boost regimen is more effective than a protein-alone vaccination approach in inducing antibodies that target two key neutralizing domains: the V3 loop and the CD4 binding site. In particular, positive antibodies targeting several peptides that overlap with the known CD4 binding area were detected only in DNA-primed sera. Different profiles of antibody specificities provide insight into the mechanisms behind the elicitation of better neutralizing antibodies with the DNA prime-protein boost approach, and our results support the use of this approach to further optimize Env formulations for HIV vaccine development.

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Figures

FIG. 1.
FIG. 1.
Antibody responses elicited from immunization with JR-FL in a DNA prime-protein boost or protein-alone immunization regimen. (A) Anti-gp120 IgG endpoint binding titers as measured by ELISA. (B) NAb titers against JR-FL at 50% neutralization of the virus as seen in a PBMC-based neutralization assay. An * indicates that less than 50% neutralization was seen at a starting dilution of 1:10. (C) Serum recognition of linear peptides derived from the V1/V2 and V3 regions of JR-FL among DNA prime-protein boost and protein-alone rabbit sera. The lowest serum dilution tested was 1:100. (D) Virus capture competition assay using seven MAbs specific for domains on HIV-1 Env and sera from both DNA prime-protein-boosted and protein-immunization-alone animals. Antibody levels are reported as titers necessary to prevent 50% of virus binding. Data shown are average titers, with bars indicating standard deviations of data from rabbits in each group. An * indicates that no antibody competition was detected.
FIG. 2.
FIG. 2.
Immunization groups and pentavalent gp120 vaccine formulation. Rabbits received either DNA prime-protein boost or protein-alone immunizations. An * indicates the HIV-1 primary isolates from which the gp120 vaccine components were derived.
FIG. 3.
FIG. 3.
gp120-specific IgG titers in rabbits immunized with DNA prime-protein boost and protein-alone formulations against autologous antigens as measured by ELISA. Data are shown as geometric mean titers, with bars indicating standard deviations of data from two or four rabbits in the group immunized with protein alone or the group immunized with DNA plus protein, respectively. “Prebleed” and “Post Protein-2” indicate the sera collected at preimmunization and at 2 weeks after the second protein immunization, respectively.
FIG. 4.
FIG. 4.
gp120 peptide-specific IgG responses as measured by ELISA. The red and black curves indicate rabbit sera from DNA prime-protein boost or protein-alone immunization with polyvalent vaccine, respectively. Rabbit sera were normalized to equivalent gp120 binding titers. The gp120 peptides were linear overlapping peptides derived from the group M consensus gp120 sequence of HIV-1. Data shown are the average optical density (OD) values for two rabbits in the protein-alone group or four rabbits in the group immunized with DNA plus protein. Arrows indicate positive binding responses unique to the DNA-primed group against six different regions (i.e., no positive responses were observed in the protein-alone group). These six regions include the regions at p11, p30, p48 to p49, p56 to p57, p61 to p64, and p113 to p117.
FIG. 5.
FIG. 5.
Additional analysis of three highly reactive regions of gp120. (A) Recognition of linear peptides that contain CD4 or b12 contact residues by DNA-primed or protein-alone rabbit sera as measured by ELISA. Bars represent the average optical density (OD) values with standard deviations for two rabbits in the protein-alone group or four rabbits in the group immunized with DNA plus protein against each peptide listed on the right. (B) Locations of group M consensus linear peptides (blue) overlaid onto the crystal structure of JR-FL gp120 liganded with CD4 and MAb X5. CD4 contact residues are highlighted in red. (C) Locations of group M consensus linear peptides (blue) overlaid onto the crystal structure of JR-FL gp120 liganded with CD4 and MAb X5. b12 contact residues are highlighted in orange.
FIG. 6.
FIG. 6.
Polyvalent rabbit serum recognition of peptide sequences derived from the variable loops V1/V2, V3, V4, and V5 from individual gp120 proteins of the polyvalent vaccine components as measured by ELISA. Rabbits were immunized with DNA prime-protein boost (gray bars) or protein only (white bars). Peptides from gp120 A, B, C, and E were used in graphs A, B, C, and D, respectively. Bars represent the average optical density (OD) values for two rabbits in the protein-alone group or four rabbits receiving DNA plus protein against each peptide. Peptide sequences are shown in Table S2 in the supplemental material.
FIG. 7.
FIG. 7.
Detection of antibody specificities in DNA-primed or protein-alone polyvalent rabbit sera. This competitive virus capture assay used MAbs to study the specificities and titers of antibodies present in the polyvalent sera. Bars indicate the average dilutions of sera for two rabbits in the protein-alone group or four rabbits in the group receiving DNA plus protein, resulting in a 50% reduction in virus binding. Error bars indicate standard deviations within the group. Significance was calculated using a one-tailed Student's t test. *, P < 0.05.
FIG. 8.
FIG. 8.
Effect of V3 peptide adsorption on neutralization of HIV-1 isolate SF162. Clade B consensus overlapping peptides corresponding to the N-terminal strand and crown of the V3 loop were pooled and incubated with sera at 30 μg/ml prior to exposure to pseudotyped virus expressing Env from HIV-1 isolate SF162. Neutralization titers indicate the serum dilution that can achieve a 50% inhibition of virus infection. (A) Effect of peptide adsorption on the neutralizing activity of sera from a rabbit immunized with only a V3 fusion protein as a control. Error bars indicate standard deviations of data from a one-time multiwell neutralization assay. (B) Effect of V3 peptide adsorption on the neutralizing activity of individual rabbit sera from polyvalent DNA prime-protein boost (29-1, 29-2, 30-1, and 30-2) and protein-alone immunizations (33-1 and 33-2). Error bars indicate standard deviations from replicate experiments.
FIG. 9.
FIG. 9.
Effect of V3 peptide adsorption on neutralization of primary HIV-1 isolates. Clade B consensus overlapping peptides corresponding to the N-terminal strand and crown of the V3 loop were pooled and incubated with sera at 30 μg/ml prior to exposure to pseudotyped virus. Percent neutralization at a 1:10 dilution of rabbit sera is reported. “Protein Alone” and “DNA + Protein” indicate rabbits receiving polyvalent protein-alone (33-1 and 33-2) or DNA prime-protein boost (29-1, 29-2, 30-1, and 30-2) immunization, respectively. (A) Effect of V3 adsorption on neutralization of HIV-1 clade B virus SS1196. (B) Effect of V3 adsorption on neutralization of HIV-1 clade B virus SC422661.8. Error bars indicate standard deviations from replicate experiments.
FIG. 10.
FIG. 10.
Binding of rabbit immune sera to HIV-1 JR-FL Env trimers by BN-PAGE analysis. Sera from either DNA prime-protein boost immunization (29-1 and 30-1) or protein-only immunization (33-1 and 33-2) were incubated with solubilized trimers prior to being run on a BN-PAGE gel. Numbers below each lane indicate the percentage of unliganded trimer present in the gel based upon densitometry analysis.
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