Enhanced avidity maturation of antibody to human immunodeficiency virus envelope: DNA vaccination with gp120-C3d fusion proteins

Abstract

DNA vaccination can elicit both humoral and cellular immune responses and can confer protection against several pathogens. However, DNA vaccines expressing the envelope (Env) protein of human immunodeficiency virus (HIV) have been relatively ineffective at generating high titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, we report that fusion of Env and the complement component, C3d, in a DNA vaccine, enhances the titers of antibody to Env. Plasmids were generated that expressed a secreted form of Env (sgp120) from three isolates of HIV and these same forms fused to three tandem copies of the murine homologue of C3d (sgp120-3C3d). Analyses of titers and avidity maturation of the raised antibody indicated that immunizations with each of the sgp120-3C3d-expressing DNAs accelerated both the onset and the avidity maturation of antibody to Env.

FIG. 1

Schematic representation of vector DNA vaccine constructs. (A) The pGA vector contains the cytomegalovirus immediate-early promoter (CMV-IE) plus intron A (IA) for initiating transcription of eukaryotic inserts and the bovine growth hormone polyadenylation terminator [BGH poly(A)] for termination of transcription. The vector also contains the Col E1 origin of replication for prokaryotic replication as well as the kanamycin resistance (Kan^r) gene for selection in antibiotic media. The lambda T0 terminator has been placed 3′ to the Kan^r to increase the stability of eukaryotic inserts. Inserts were cloned into the vector using the HindIII and BamHI restriction endonuclease sites. (B) The top schematic represents the wild-type, transmembrane form of the Env protein. The middle schematic represents the secreted gp120 form of the Env. The bottom schematic represents the sgp120-3C3d 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 HA and C3d and between each C3d repeat.

FIG. 2

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 the total volume was electrophoresed on a 10% polyacrylamide gel. Lane 1: pGA vector; lane 2: sgp120 (ADA)-DNA; lane 3: sgp120-(IIIB)-DNA; lane 4; sgp120-(89.6)-DNA; lane 5: sgp120-(ADA)-3C3d-DNA; lane 6: sgp120-(IIIB)-3C3d-DNA; lane 7: sgp120-(89.6)-3C3d-DNA.

FIG. 3

Anti-Env IgG raised by gene gun inoculation of DNAs expressing sgp120 proteins. Mice were primed at Day 0 and boosted at Weeks 4, 14, and 26. Sera were obtained from mice with vector (X), sgp120 (ADA)-DNA (□), sgp120-(IIIB)-DNA (○), sgp120-(89.6)-DNA (△), sgp120-(ADA)-3 C3d-DNA (■), sgp120-(IIIB)-3C3d-DNA (•), and sgp120-(89.6)-3C3d-DNA (▲). Sera collected at the indicated times from each group were pooled for determination of specific IgG levels by ELISA at a dilution of 1:100 via two different ELISA protocols. (A) 96-well plates were coated with recombinant gp120 protein-derived Chinese hamster ovary (CHO) cells expressing the HIV-1 isolate, IIIB. (B) 96-well plates were coated with supernatant containing sgp120s from 293T cells that were transiently transfected with sgp120 expression plasmids. Each ELISA plate was coated with the respective Env to detect antibodies from mice vaccinated with that particular sgp120, i.e., plates were coated with the ADA-Env in order to detect antibodies to Env in mice vaccinated with plasmids expressing sgp120-ADA proteins. Data are represented as the average of three individual assays. Preimmune sera from mice had no detectable specific IgG. End point dilution titers were conducted by diluting the sera until OD values reached background.

FIG. 4

Avidity of the anti-Env IgG raised by the IIIB Env-DNA vaccines. Sera were analyzed from Week 28 in an Env-specific NaSCN-displacement ELISA. Plates were with recombinant gp120-(IIIB). Sera were obtained from mice immunized with sgp120-(IIIB)-DNA (○) and sgp120-(IIIB)-3C3d-DNA (•). Assays used pooled serum samples from each mouse group at a dilution of 1:50 for sgp120-(IIIB) and 1:200 for sgp120-(IIIB)-3C3d. Data are representative of two independent experiments.