Protection against woodchuck hepatitis virus (WHV) infection by gene gun coimmunization with WHV core and interleukin-12

Abstract

Woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) are closely similar with respect to genomic organization, host antiviral responses, and pathobiology of the infection. T-cell immunity against viral nucleocapsid (HBcAg or WHcAg) has been shown to play a critical role in viral clearance and protection against infection. Here we show that vaccination of healthy woodchucks by gene gun bombardment with a plasmid coding for WHcAg (pCw) stimulates proliferation of WHcAg-specific T cells but that these cells do not produce significant levels of gamma interferon (IFN-gamma) upon antigen stimulation. In addition, animals vaccinated with pCw alone were not protected against WHV inoculation. In order to induce a Th1 cytokine response, another group of woodchucks was immunized with pCw together with another plasmid coding for woodchuck interleukin-12 (IL-12). These animals exhibited WHcAg-specific T-cell proliferation with high IFN-gamma production and were protected against challenge with WHV, showing no viremia or low-level transient viremia after WHV inoculation. In conclusion, gene gun immunization with WHV core generates a non-Th1 type of response which does not protect against experimental infection. However, steering the immune response to a Th1 cytokine profile by IL-12 coadministration achieves protective immunity. These data demonstrate a crucial role of Th1 responses in the control of hepadnavirus replication and suggest new approaches to inducing protection against HBV infection.

FIG. 1

Immune response against WHcAg following gene gun immunization with pCw (stippled bars) or pCw plus pIL12w (hatched bars). Nonimmunized woodchucks (shaded bars) served as controls. (A) Humoral immune response. (B) Production of IL-2 by woodchuck PBMC incubated in the presence of WHcAg. (C) IFN-γ mRNA levels (as determined by real-time quantitative RT-PCR) in woodchuck PBMC incubated in the presence of WHcAg. ∗, animal in which a second round of immunizations with pCw induced anti-WHcAg antibodies (titer = 90) and IL-2 production (SI = 21). ∗∗, healthy control animal that was not further infected or followed up.

FIG. 2

Evolution of viremia after challenge with WHV. (A) Control nonimmunized animals; (B) animals immunized with pCw, (C) woodchucks immunized with pCw plus pIL12w. Inoculation of WHV was performed at week 0. WHV DNA was quantified by real-time quantitative PCR. WHV DNA is expressed as genomic particles per microliter of serum.

FIG. 3

Anti-WHc (A) and anti-WHs (B) responses in woodchucks after challenge with 10¹⁰ WHV genomic particles. Symbols: ▵, nonvaccinated animals; ⧫, animals immunized with pCw alone; , animals immunized with pCw plus pIL12w. Mean values at each time point are shown.

FIG. 4

Cellular immunes response of woodchucks after challenge with 10¹⁰ WHV genomic particles at week 0. Data for nonimmunized woodchucks (WN1 and WN2) (A and D), woodchucks previously vaccinated with pCw (WC1, WC2, WC3, and WC4) (B and E), and woodchucks previously immunized with pCw plus pIL12w (WILC1, WILC2, WILC3, and WILC4) (C and F) are shown. Determinations of IL-2 production (A, B, and C) and IFN-γ mRNA levels (D, E, and F) in PBMC after stimulation with recombinant WHcAg were performed at different time points during follow-up after infection. IL-2 levels are expressed in terms of the SI. IFN-γ levels are expressed as the number of IFN-γ mRNA copies per copy of β-actin mRNA.