Exposure to low infective doses of HCV induces cellular immune responses without consistently detectable viremia or seroconversion in chimpanzees

Abstract

In hepatitis C virus (HCV) infection, there is accumulating data suggesting the presence of cellular immune responses to HCV in exposed but seemingly uninfected populations. Some studies have suggested cross-reactive antigens rather than prior HCV exposure as the main reason for the immune responses. In this study we address this question by analyzing the immune response of chimpanzees that have been sequentially exposed to increasing doses of HCV virions. The level of viremia, as well as the immune responses to HCV at different times after virus inoculation, were examined. Our data indicate that HCV infective doses as low as 1-10 RNA (+) virions induce detectable cellular immune responses in chimpanzees without consistently detectable viremia or persistent seroconversion. However, increasing the infective doses of HCV to 100 RNA (+) virions overcame the low-inoculum-induced immune response and produced high-level viremia followed by seroconversion.

Fig. 1

Viral load and anti-HCV level after challenge with different doses of HCV. Sera from chimpanzees 251 (A) and chimpanzee 325 (B) were tested for anti-HCV (filled triangle) and viral load (filled circle) at different time points after challenge with HCV, using quantitative ELISA and PCR, respectively. The lower detection limit (LDL) of quantitative PCR is 250 copies/ml, and for qualitative TMA assay is 14 copies/ml at 50% detection limit. The lower detection limit of anti-HCV antibodies is 0.2 units/ml.

Fig. 2

Direct ex vivo IFN-γ ELISPOT for chimpanzees 251 and 325 after infection with different doses. PBLs from chimpanzee 251 (A) and chimpanzee 325 (B) were tested for anti-HCV cellular response using IFN-γ ELISPOT assay as described under Materials and methods. Cells from time 0 (before challenge) were infected with recombinant HCV-C-NS3-vaccinia (filled column), HCV-NS3-NS5 (unfilled column), or HCV-NS3 (hatched column) at an m.o.i. of 10:1 and used as stimulator cells. PBLs from different time points after challenge were incubated with the stimulator cells, and the number of IFN-γ-secreting cells (ISCs) was measured 18 h after in vitro stimulation. IFN-γ ELISPOT was done at all time points indicated in the graph. Sera from chimpanzees 251 and 325 were tested for viral load (line) using quantitative PCR as described in Fig. 1. The data represent average of two experiments. The cutoff value of ISCs (horizontal line) was calculated as described under Materials and methods as approximately 185.5 ISCs/10⁶. The average numbers of direct ex vivo ISCs in the presence of parent vaccinia were 78 ± 54 ISCs/10⁶ cells.

Fig. 3

Expanded ELISPOT for chimpanzees 251 and 325 after infection with different doses. Expanded PBLs from chimpanzee 251 (A) and chimpanzee 325 (B) were tested for anti-HCV cellular response using IFN-γ ELISPOT assay as described under Materials and methods. Briefly autologous PBLs cells (at time 0) were infected for 1 h with recombinant HCV-C-NS3-Alvac (filled column) at an m.o.i. of 10:1 and used as stimulator cells. Autologous PBLs from different time points after challenge were incubated with the stimulator cells for 1 week at 37°C and 5% CO₂. On day 7, IFN-γ ELISPOT assay was done as described in Fig. 2. The numbers of HCV-specific ISCs/10⁶ cells were plotted after subtracting the background (parental vaccinia virus alone). The cutoff value of ISCs (horizontal line) was calculated as described under Materials and methods as approximately 870.3 ISCs/10⁶ cells.

Fig. 4

TNF-α secretion from expanded ELISPOT for chimpanzees 251 and 325 after infection with different doses. The levels of TNF-α in the supernatant expanded PBLs from chimpanzee 251 (A) and chimpanzee 325 (B) were measured as previously described. TNF-α was expressed as pg/ml after subtracting the background (TNF-α secreted by vaccinia vector alone). The cutoff value was calculated as the level of TNF-α in the presence of vaccinia vector + 2 SD and was approximately 200 pg/ml.

Fig. 5

NS3-epitope mapping of effector cells from chimpanzees 251 after challenge with 1-100 RNA (+) virion molecules. PBLs from chimpanzee 251 were tested for cellular response to pools of 10-mer NS3 overlapping peptides using IFN-γ ELISPOT assay as described under Materials and methods. PBLs at time 0 (A), after challenge with an estimated one RNA (+) molecule (B), or after challenge with 10 (C), and 100 (D) RNA (+) molecules, were incubated with pools of ten 10-mer overlapping peptides of NS3 (HCV genotype 1b) at a concentration of 4 nM for each peptide. The numbers of IFN-γ-secreting cells (ISCs) were measured 18 h after in vitro stimulation. The numbers of HCV-specific ISCs/10⁶ cells were plotted after subtracting the background (media alone). The cutoff value of ISCs (horizontal line) was calculated as described under Materials and methods and estimated to be approximately 217 ISCs/10⁶.

Fig. 6

NS3-epitope mapping of effector cells from chimpanzees 325 after challenge with 1-100 RNA (+) virion molecules. PBLs from chimpanzee 325 were tested for cellular response to pools of 10-mer NS3 overlapping peptides using IFN-γ ELISPOT assay as described under Materials and methods. PBLs at time 0 (A), and after challenge with an estimated one RNA (+) molecule (B), or after challenge with 10 (C), and 100 (D) RNA (+) molecules, were incubated with pools of ten 10-mer overlapping peptides of NS3 (HCV genotype 1b) at a concentration of 4 nM for each peptide. The numbers of IFN-γ-secreting cells (ISCs) were measured 18 h after in vitro stimulation. The numbers of HCV-specific ISCs/10⁶ cells were plotted after subtracting the background (media alone). The cutoff value of ISCs (horizontal line) was calculated as described under Materials and methods and estimated to be approximately 217 ISCs/10⁶.

Fig. 7

Response of intrahepatic lymphocytes (IHLs) from chimpanzees 251 and 325 to HCV after challenge with different doses. IHLs prepared as described under Materials and methods, from chimpanzee 251 (A) or chimpanzee 325 (B), were tested for anti-HCV cellular response using direct ex vivo IFN-γ ELISPOT assay. Autologous PBLs from time 0 (before challenge) were infected with recombinant HCV-C-NS3-vaccinia (filled column), HCV-NS3-NS5-vaccinia (unfilled column), at an m.o.i. of 10:1 and used as stimulator cells. IHLs at 1, 6, and 12 months postchallenge were incubated with stimulator cells at 37°C and 5% CO₂. The numbers of direct ex vivo IFN-γ-secreting cells (ISCs) were measured 18 h after in vitro stimulation. The numbers of HCV-specific ISCs/10⁶ cells were plotted after subtracting the background (parental vaccinia virus alone). The cutoff value of direct ex vivo ISCs (horizontal line) was calculated as described under Materials and methods as approximately 185.5 ISCs/10⁶. The data represent the average of two experiments.