Optimal induction of T-cell responses against hepatitis C virus E2 by antigen engineering in DNA immunization

Abstract

Although DNA immunization is a safe and efficient method for inducing cellular immune responses, it generates relatively weak and slow immune responses. Here, we investigated the effect of hepatitis C virus (HCV) antigen modifications on the induction of T-cell responses in DNA immunization. It is likely that the strength of T-cell responses has an inverse relationship with the length of the insert DNA. Interestingly, a mixture of several plasmids carrying each gene induced a higher level of T-cell responses than a single plasmid expressing a long polyprotein. Moreover, the presence of a transmembrane domain in HCV E2 resulted in stronger T-cell responses against E2 protein than its absence. Taken together, our results indicate that the tailored modifications of DNA-encoded antigens are capable of optimizing the induction of T-cell responses which is required for eliminating the cells chronically infected with highly variable viruses such as HCV and human immunodeficiency virus.

FIG. 1.

Schematic diagram of the HCV expression plasmids and their identification in vitro. (a) pTV2 vector was previously described (27). Corresponding amino acid positions in HCV polypeptides are indicated under each diagram. s, glycoprotein D signal sequence from herpes simplex virus type 1. (b) COS7 cells were transfected with the indicated plasmids and a luciferase-expressing plasmid as an internal control. Corresponding amounts of cell lysates were processed for sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis followed by Western blot analysis with anti-E2 monoclonal antibodies. The positions of molecular mass markers are indicated at the left of the panel; those of E2 and E2t are indicated on the right.

FIG. 2.

HCV E2-specific T-cell responses inversely correlated with insert lengths of DNA constructs. BALB/c mice were immunized intramuscularly with 100 μg of the indicated expression plasmids. Splenocytes were removed at 5 weeks after immunization and used for CTL and IFN-γ ELISPOT assays with CT26-hghE2t cells for stimulation. Plasmids containing intact core (a) or Δcore (b) were compared for their levels of induction of T-cell responses. Standard deviations are indicated as error bars. Data are representative of the results of one of three independent experiments. Statistical analysis was performed using Student's t test. P values between different groups of immunization were compared, and those of less than 0.01 were considered significant.

FIG. 3.

Effect of E2 expression level on the strength of T-cell responses against E2. Three different expression vectors were used to carry the same HCV structural gene, sΔST (a), and their E2 expression levels in COS7 as well as in C2C12 cells were compared by Western blot analysis (b). BALB/c mice were immunized intramuscularly at 0 and 4 weeks with 100 μg of the indicated expression plasmids, and IFN-γ ELISPOT and CTL responses were examined (c). Standard deviations are indicated as error bars. CMV, cytomegalovirus early promoter/enhancer; TPL, adenovirus tripartite leader; MCS, multicloning site; SV40 pA, simian virus 40 poly(A) signal; SV40 Enh, simian virus 40 enhancer; ITR, inverted terminal repeat of adeno-associated virus; bGH pA, bovine growth hormone poly(A) signal.

FIG. 4.

E2-specific T-cell response was not affected by the coadministration of separate plasmids expressing HCV nonstructural genes. (a) Plasmids pGX10-NS34 and pGX10-NS5, carrying nonstructural regions of HCV, were used together with pGX10-sΔST (100 μg of plasmids total, consisting of 33 μg of each plasmid with or without mock DNA) for immunization at 0 and 4 weeks. (b) At 3 weeks after booster injections were administered, an IFN-γ ELISPOT assay was performed using CT26-hghE2t cells for stimulation. Standard deviations are indicated as error bars. Mock, mock DNA.

FIG. 5.

Truncation of E2 TMD decreased the strength of E2-specific T-cell responses induced by DNA immunization. BALB/c mice were immunized intramuscularly with 100 μg of the indicated expression plasmids at 0 and 4 weeks. Splenocytes removed at 5 weeks after primary immunization or 3 weeks after boosting were used in IFN-γ ELISPOT assays after being stimulated either with a CT26-hghE2t or with an E2 peptide pool, respectively. Standard deviations are indicated as error bars. Data are representative of the results of three independent experiments.