Immunization with hepatitis C virus-like particles results in control of hepatitis C virus infection in chimpanzees

Abstract

Recombinant hepatitis C virus (HCV)-like particles (HCV-LPs) containing HCV structural proteins (core, E1, and E2) produced in insect cells resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice and baboons. Here, we present evidence on the immunogenicity and induction of protective immunity by HCV-LPs in chimpanzees. Chimpanzees (two in each group), were immunized with HCV-LPs or HCV-LPs plus AS01B adjuvant. After immunizations, all animals developed an HCV-specific immune response including IFN-gamma(+), IL-2(+), CD4(+), and CD8(+) T cell and proliferative lymphocyte responses against core, E1, and E2. Upon challenge with an infectious HCV inoculum, one chimpanzee developed transient viremia with low HCV RNA titers (10(3) to 10(4) copies per ml) in the third and fourth weeks after the challenge. The three other chimpanzees became infected with higher levels of viremia (10(4) to 10(5) copies per ml), but their viral levels became unquantifiable (<10(3) copies per ml) 10 weeks after the challenge. After the HCV challenge, all four chimpanzees demonstrated a significant increase in peripheral and intrahepatic T cell and proliferative responses against the HCV structural proteins. These T cell responses coincided with the fall in HCV RNA levels. Four naïve chimpanzees were infected with the same HCV inoculum, and three developed persistent infection with higher viremia in the range of 10(5) to 10(6) copies per ml. Our study suggests that HCV-LP immunization induces HCV-specific cellular immune responses that can control HCV challenge in the chimpanzee model.

Fig. 1.

Time course and magnitude of HCV-specific T cell immune responses during HCV-LP immunization and after challenge. Four chimpanzees (X0133, X0175, X0199, and X0212) were immunized with HCV-LPs four times over 32 weeks. Arrowheads indicate immunization times, and the arrow indicates the time of HCV-CG1b challenge at week 35 (week 0). (A) IFN-γ response against HCV core, E1/E2, and OLPs. (B) Lymphocyte proliferative responses against HCV core and E1/E2 proteins were assessed over 48 weeks. In each assay, 2 × 10⁵ cells were used. The T cell proliferative response is expressed as stimulation index. (C) IFN-γ responses against HCV nonstructural proteins NS3 helicase, NS4, and NS5A were determined over 48 weeks. NA, not analyzed. SFU, spot-forming unit.

Fig. 1.

Time course and magnitude of HCV-specific T cell immune responses during HCV-LP immunization and after challenge. Four chimpanzees (X0133, X0175, X0199, and X0212) were immunized with HCV-LPs four times over 32 weeks. Arrowheads indicate immunization times, and the arrow indicates the time of HCV-CG1b challenge at week 35 (week 0). (A) IFN-γ response against HCV core, E1/E2, and OLPs. (B) Lymphocyte proliferative responses against HCV core and E1/E2 proteins were assessed over 48 weeks. In each assay, 2 × 10⁵ cells were used. The T cell proliferative response is expressed as stimulation index. (C) IFN-γ responses against HCV nonstructural proteins NS3 helicase, NS4, and NS5A were determined over 48 weeks. NA, not analyzed. SFU, spot-forming unit.

Fig. 2.

CD4⁺ and CD8⁺ T cell responses in chimpanzees immunized with HCV-LPs. Chimpanzees were immunized with HCV-LPs four times. Two weeks after the fourth HCV-LP dose, CD4⁺ (A) and CD8⁺ (B) T cells were isolated and stimulated with recombinant core, E1/E2 proteins, and HCV-overlapping peptides as indicated. The HCV-specific IFN-γ SFUs are expressed as numbers of spots per 1 × 10⁵ cells.

Fig. 3.

The time course of HCV infection in HCV-LP-immunized chimpanzees. (A) Four chimpanzees were immunized four times over 32 weeks with HCV-LPs. Three weeks after the fourth dose (35/0), the chimpanzees were challenged with homologous HCV-CG1b strain. Arrowheads indicate immunization times and the arrow indicates the time of the HCV-CG1b challenge. HCV RNA titers and alanine transaminase (ALT) levels are shown. Black bars represent HCV RNA in log₁₀(copies per milliliter) (right ordinates) and the lined gray zone represents ALT levels in units/ml (left ordinates). Anti-HCV seroconversion is indicated as − or + at the top of each graph. Some of the samples (negative by PCR) were tested for HCV RNA by transcription-mediated amplification (TMA) and the results are shown. Follow-up samples beyond 1 year after the challenge were available for TMA testing in chimpanzees X0133 and X0199. (B) Comparison of HCV viremia between naïve and HCV-LP-immunized chimpanzees after the challenge. Previously, four control chimpanzees were infected with HCV-CG1b strain at a dose of 3–10 CID₅₀ (15, 32); the courses of HCV viremia are shown (Right). The viral titers were all converted to units per milliliter for ease of comparison because HCV quantification was done with different assays. For comparison, the viremia courses of the four HCV-LP-immunized chimpanzees are shown together (Left).

Fig. 4.

The magnitude of intrahepatic HCV-specific T cell proliferative responses in vaccinated chimpanzees. Intrahepatic lymphocytes were expanded from liver biopsies collected at multiple time points before immunization and after the challenge. The cells were stimulated with HCV core or E1/E2 recombinant proteins and proliferative responses were determined.