Enhancement of antibodies to the human immunodeficiency virus type 1 envelope by using the molecular adjuvant C3d

Abstract

DNA vaccines expressing the envelope (Env) protein of the human immunodeficiency virus have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, the murine and human homologues of the complement component, C3d, were used in a DNA vaccine to enhance the titers of antibody to Env. Initially, plasmids expressing a secreted form of Env (sgp120) fused to one, two, or three copies of the murine homologue of C3d (mC3d) were constructed. Mice were inoculated with four vaccinations of DNA or two DNA vaccinations, followed by two boosts of affinity-purified gp120 protein. Analyses of titers demonstrated that multiple copies of mC3d coupled to sgp120 induced long-lasting, high-titer anti-Env antibody. Priming mice with sgp120-mC3d-DNA, followed by inoculation of purified gp120 protein, elicited the strongest antibody titers; however, the avidity maturation of the antibody was accelerated in the mice inoculated with sgp120-mC3d(3)-DNA. In addition, DNAs expressing sgp120 fused to three copies of the human homologue of C3d (hC3d(3)) efficiently enhanced the anti-Env antibody in rabbits. Lastly, antisera from both mice and rabbits vaccinated with DNA expressing sgp120-C3d(3) elicited higher titers of neutralizing antibody than did nonfused forms of Env. These results indicate that C3d, conjugated to sgp120, enhances the antibody responses to Env compared to non-C3d fused forms of Env, and this approach may be one way to overcome the poor ability of DNA vaccines to generate antibodies to Env.

FIG. 1.

Schematic representation of vector DNA vaccine constructs. (A) The pTR600 vector contains the cytomegalovirus immediate-early promoter plus intron A for initiating transcription of eukaryotic inserts and the bovine growth hormone polyadenylation terminator (BGH PolyA) for the termination of transcription. The vector also contains the ColE1 origin of replication for prokaryotic replication, as well as the kanamycin resistance (Kan^r) gene for selection in antibiotic media. Inserts were cloned into the vector by using the NheI and BamHI restriction endonuclease sites directly 3′ to the tpA sequence. (B) The first schematic represents the wild-type, transmembrane form of the Env protein. The second schematic represents the secreted gp120 form of the Env. The third schematic represents the sgp120-C3d₁ construct used as a vaccine insert. The fourth schematic represents the sgp120-C3d₂ construct used as a vaccine insert. The fifth schematic represents the sgp120-C3d₃ construct used as a vaccine insert. Linkers composed of two repeats of four glycines and a serine [(G₄S)₂] were fused at the junctures of Env and C3d and between each C3d repeat.

FIG. 2.

Western hybridization to detect expression of vaccine constructs in vitro. Human embryonic kidney cells (293T) were transfected with 2 μg of each vaccine plasmid. Supernatant was collected, and 1.5% of total volume was subjected to electrophoresis on a 10% polyacrylamide gel. Each blot was probed with HIV-Ig followed by anti-human IgG conjugated to horseradish peroxidase. Proteins were detected by using a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-human antiserum and enhanced chemiluminescence. (A) Lane 1, molecular mass marker; lane 2, sgp120_IIIB-DNA; lane 3, sgp120_IIIB-mC3d₁-DNA; lane 4, sgp120_IIIB-mC3d₂-DNA; lane 5, sgp120_IIIB-mC3d₃-DNA. (B) Lane 1, molecular mass marker; lane 2, sgp120_IIIB-DNA; lane 3, sgp120_IIIB-mC3d₃-DNA; lane 4, sgp120_ADA-DNA; lane 5, sgp120_ADA-mC3d₃-DNA; lane 6, sgp120_89.6-DNA; lane 7, sgp120_89.6-mC3d₃-DNA.

FIG. 3.

Anti-Env IgG raised by DNAs expressing sgp120 proteins in gene gun-vaccinated mice. (A) Mice were primed with DNA at day 0 and boosted at weeks 6, 12, and 18. Sera were obtained from mice at weeks 0, 6, 8, 12, 14, 18, and 20. Symbols: —, vector; ♦, sgp120_IIIB-DNA; •, sgp120_IIIB-mC3d₁-DNA; ▴, sgp120_IIIB-mC3d₂-DNA; ▪, sgp120_IIIB-mC3d₃-DNA. (B) Mice were primed with DNA at day 0 and 6 and then were boosted with sgp120_IIIB at weeks 12 and 18. (C) Mice were primed with DNA at day 0 and boosted at weeks 6, 12, and 18. Sera were obtained from mice at weeks 0, 6, 8, 12, 14, 18, and 20. Symbols: —, vector; ○, sgp120_89.6-DNA; ▿, sgp120_ADA-DNA; □,sgp120_IIIB-DNA; •, sgp120_89.6-mC3d₃-DNA; ▾, sgp120_ADA-mC3d₃-DNA; ▪, sgp120_IIIB-mC3d₃-DNA; tpA-mC3d₃. Sera collected at the indicated times from each mouse were assayed for specific IgG levels by ELISA. Then, 96-well plates were coated with recombinant gp120 protein-derived CHO cells expressing the HIV-1 isolate, IIIB. Data are averages for 5 to 10 mice. Preimmune sera from mice had no detectable specific IgG. Endpoint dilution titers were conducted by diluting the sera until OD values reached background levels. Titers for sera that had background titers at the minimum dilution tested were less than 1:100 (<100).

FIG. 4.

Individual anti-Env IgG titer raised by DNAs expressing sgp120 fused to murine C3d. (A) 3. Mice were primed with DNA at day 0 and boosted at weeks 6, 12, and 18. Sera, collected at week 20, were assayed by ELISA for specific IgG, and the endpoint dilution titer was determined. (B) Mice were primed with DNA at days 0 and 6 and then boosted with sgp120_IIIB at weeks 12 and 18. Then, 96-well plates were coated with recombinant gp120 protein-derived CHO cells expressing the HIV-1 isolate, IIIB. Each datum point represents the endpoint dilution titer for an individual mouse. Preimmune sera from mice had no detectable specific IgG. Endpoint dilution titers were conducted by diluting the sera until the OD values reached background levels. The horizontal line in each lane represents the averaged titer for that group. Other symbols for panels A and B are as defined in the legend to Fig. 3.

FIG. 5.

Anti-Env IgG raised by intramuscular inoculation of DNAs expressing sgp120 fused to human C3d. Rabbits were primed with DNA at day 0; boosted at weeks 6, 12, and 18; and boosted with sgp120_IIIB protein at week 27. Sera were obtained from rabbits at weeks 0, 6, 8, 12, 14, 18, 20, 27, and 29. Symbols: —, vector; ▵, sgp120_89.6-DNA; ○, sgp120_JRFL-DNA; □, sgp120_IIIB-DNA; ▴, sgp120_89.6-hC3d₃-DNA; •, sgp120_JRFL-hC3d₃-DNA; ▪, sgp120_89.6-hC3d₃-DNA. Sera were collected at the indicated times from each rabbit and assayed for specific IgG levels by ELISA. Then, 96-well plates were coated with recombinant gp120 protein-derived CHO cells expressing the HIV-1 isolate, IIIB. Data are the averages for five rabbits. Preimmune sera from mice had no detectable specific IgG. Endpoint dilution titers were conducted by diluting the sera until the OD values reached background levels. (Inset) Expression of vaccine constructs in vitro. Human embryonic kidney cells (293T) were transfected with 2 μg of each vaccine plasmid. Supernatant was collected, and 1.5% of total volume was subjected to electrophoresis on a 10% polyacrylamide gel. Lanes: 1, molecular mass marker; 2, tpA-hC3d₃; 3, sgp120_IIIB-DNA; 4, sgp120_IIIB-hC3d₃-DNA; 5, sgp120_JRFL-DNA; 6, sgp120_JRFL-hC3d₃-DNA.

FIG. 6.

Western blot of anti-hC3d₃ antibodies in sera from vaccinated rabbit. Human embryonic kidney cells (293T) were transfected with 2 μg of each vaccine plasmid. Supernatant was collected, and 1.5% of the total volume was subjected to electrophoresis on a 10% polyacrylamide gel. Each blot was probed with antisera from rabbits vaccinated with sgp120_89.6-mC3d₃-DNA at the indicated time points. (A) Week 14 antisera; (B) week 20 antisera; (C) week 29 antisera; (D) week 29 antisera mixed with 250 μg of purified hC3d₃ protein. Lanes: Mock, cell supernatant only; hC3d₃, tpA-hC3d₃; gp120, sgp120_89.6.

FIG. 7.

Avidity of the anti-Env IgG raised by the IIIB Env-DNA vaccines. Sera were analyzed from week 20 in an Env-specific NaSCN-displacement ELISA. Plates were coated with recombinant gp120_IIIB. Sera were collected from sgp120_IIIB-DNA (▪), sgp120_IIIB-mC3d₁-DNA (○), sgp120_IIIB-mC3d₂-DNA (□), and sgp120_IIIB-mC3d₃-DNA (•). Assays used pooled serum samples from each mouse group that was normalized to the same OD (range, 1:200 to 1:400 for sgp120_IIIB and sgp120_IIIB-mC3d₁; range, 1:800 to 1:3,200 for sgp120_IIIB-mC3d₂-DNA and sgp120_IIIB-mC3d₃). Data are the averages of three independent experiments.