Induction of broad CD4+ and CD8+ T-cell responses and cross-neutralizing antibodies against hepatitis C virus by vaccination with Th1-adjuvanted polypeptides followed by defective alphaviral particles expressing envelope glycoproteins gpE1 and gpE2 and nonstructural proteins 3, 4, and 5

Abstract

Broad, multispecific CD4(+) and CD8(+) T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of these responses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins in various prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8(+) T-cell responses but low CD4(+) T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4(+) T helper responses but no CD8(+) T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4(+) T helper responses but no CD8(+) T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4(+) and CD8(+) T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.

FIG. 1.

Priming with E1E2/MF59/CpG followed by boosting with VEE/SIN-E1E2 stimulates strong, good CD4⁺ and CD8⁺ T-cell responses. BALB/c mice (n = 10) received three intramuscular injections (1, 2, 3) at 3-week intervals with the immunogens indicated. The spleen cells were harvested at 2 weeks after the last immunization and stimulated with 10 μg/ml HCV-specific peptides for ICS and fluorescence-activated cell sorter analysis as described in Materials and Methods. Twenty-mer overlapping peptide pools for the HCV strain 1a E1 region (E1 pool) and E2 region (E2 pool) and single E2 peptides specific for CD4⁺ T cells (CD4 E2 pep) and CD8⁺ T cells (CD8 E2 pep) were used for stimulation. The data are presented as the mean total percentage of CD8⁺ IFN-γ⁺ (A, B, and C) and CD4⁺ IFN-γ⁺ (D) cells in two pools (five mice per pool). Data from one representative experiment of two performed are shown. *, P < 0.05 compared with the medium control and the PBS-immunized group.

FIG. 2.

Prime/boost regimen with E1E2/MF59/CpG and VEE/SIN-E1E2 stimulated strong HCV-specific Th1 cytokine responses. Spleen cells from the immunized mice (immunogens as indicated) were stimulated with HCV-specific peptides for 24 h, and the supernatants were collected for detection of the cytokines IFN-γ, TNF-α, IL-5, and IL-10 with the Beadlyte Luminex mouse multicytokine detection system (Upstate, Charlottesville, VA). The data are presented as the means of pooled spleens from the same group (two pools per vaccine regimen, five spleens per pool). Data from one representative experiment of two performed are shown.

FIG. 3.

A prime/boost regimen using NS345Poly/IMX/CpG and VEE/SIN-NS345 is optimal for stimulating HCV-specific CD4⁺ (A) and CD8⁺ (B) T-cell responses. BALB/c mice in each test group (n = 10) received three injections (1, 2, 3) at 3-week intervals with immunogens as indicated. Intracellular cytokine staining was performed 2 weeks after the last injection, and the spleen cells were stimulated with 10 μg/ml HCV-specific peptides for ICS and fluorescence-activated cell sorter analysis as described in Materials and Methods. Twenty-mer overlapping peptide pools for the HCV strain 1a NS3 (NS3 pool), NS4 (NS4 pool), NS5A (NS5A pool), and NS5B (NS5B pool) regions; single peptides from the NS3 region (NS3-1 pep, NS3-2 pep, and NS3-3 pep) and NS5 (NS5B pep); and recombinant proteins SOD-C100 (NS34) and SOD-NS5 were used for stimulation. The data are presented as the means of pooled spleens from the same group (two pools per vaccine regimen, five spleens per pool). Data from one representative experiment of two performed are shown. *, P < 0.05 compared with the medium control and the PBS-immunized group. IMX, Iscomatrix.

FIG. 4.

Prime/boost regimen with NS345Poly/IMX/CpG and VEE/SIN-NS345 stimulates strong HCV-specific Th1 cytokine responses. Spleen cells from immunized mice (immunogens as indicated) were stimulated with HCV-specific peptides for 24 h, and the supernatants were collected for the detection of the cytokines IFN-γ, TNF-α, IL-5, and IL-10 with the Beadlyte Luminex mouse multicytokine detection system (Upstate, Charlottesville, VA). The data are presented as the means of pooled spleens from the same group (two pools per vaccine regimen, five spleens per pool). Data from one representative experiment of two performed are shown. IMX, Iscomatrix.

FIG. 5.

Induction of cross-neutralizing antibodies in vaccinated mice. (A) A prime/boost regimen with E1E2/MF59/CpG and VEE/SIN-E1E2 stimulates antibodies that block the binding of HCV gpE2 to CD81. BALB/c mice were immunized with immunogens as indicated. Mouse serum was collected 2 weeks after the last injection. The antibody titers are expressed as reciprocal values of serum dilutions which inhibit 50% of E2 binding to CD81. Serum samples from each individual mouse were measured. (B) Induction of antibodies that cross-neutralize the infectivity of JFH-1 2a HCVcc for Huh-7 cell lines. Serum samples collected as described for panel A were mixed at a 1:100 dilution with JFH-1 2a HCVcc and preincubated at 37°C for 1 h. The mixture was then applied to Huh-7 cells in triplicate, and the cell lysate was measured for luciferase activity at day 3 postinfection. The luciferase activity from mouse serum with PBS immunization was used as a control. The data are shown as percent inhibition of the luciferase activity from serum with different immunizations relative to the control. (C) A prime/boost regimen with E1E2/MF59/CpG and VEE/SIN-E1E2 stimulates cross-neutralizing antibodies against JFH-1 2a HCVcc infection of Huh-7 cells. Serum samples from immunized mice (antigens as indicated) were diluted 1:100 and mixed with JFH-1 2a HCVcc. The experiment was performed as described for panel B. The luciferase activity detectable with mouse serum following PBS immunization was used as a control. The data are shown as the percent inhibition of the luciferase activity from serum with different immunizations relative to the control. *, P < 0.05 relative to PBS immunization.

FIG. 6.

A combination prime/boost immunization regimen elicits broad cellular responses to HCV structural and nonstructural proteins, as well as cross-neutralizing antibodies. Groups of 10 BALB/c mice were immunized as indicated. For the combination immunization regimen, priming with E1E2/MF59/CpG and NS345Poly/IMX vaccines and boosting with VEE/SIN-E1E2 and VEE/SIN-NS345 were done individually to separate muscles for two different antigens at weeks 0, 3, and 6. At week 8, two pools of spleen cells were prepared (five spleens per pool) and stimulated with HCV-specific peptides prior to staining for intracellular IFN-γ and fluorescence-activated cell sorter analysis. The mean values obtained from the two pools are shown. Panels: A, CD4⁺ cells stimulated by E1 and E2 peptides; B, CD8⁺ cells stimulated by E1 and E2 peptides; C, CD4⁺ cells stimulated by NS3, -4, and -5 peptides; D, CD8⁺ cells stimulated by NS3, -4, and -5 peptides; E, cross-neutralizing antibody activity elicited against JFH-1 2a HCVcc. Serum obtained at week 8 was diluted 1:100 and preincubated with virus for 1 h at 37°C prior to infection of Huh-7 cells. Three days later, luciferase activity in cell lysates was determined. Data are expressed as percent inhibition (based on means from triplicate assays) relative to control mice immunized with PBS. *, P < 0.05 compared with the medium control and the PBS-immunized group. IMX, Iscomatrix.