Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1

Abstract

The lack of a cell culture system to support hepatitis C virus (HCV) replication has hampered studies of this frequent cause of chronic liver disease. However, pseudotyped retroviral particles (pp) bearing the HCV envelope glycoproteins have provided a different approach to HCV studies. We used genotype 1a pp to detect neutralizing antibodies (NtAb) in eight chimpanzees and four humans infected with 1a strains, and developed pp of genotypes 2a, 3a, 4a, 5a, and 6a to study crossreactivity. NtAb was detected in one of four chimpanzees and none of three humans with acute resolving infection, suggesting that NtAb is not required for HCV clearance. NtAb were detected at high titer in two of four chimpanzees and, in Patient H, all with persistent infection; responses paralleled humoral responses to envelope 1 and 2 proteins and, in some cases, correlate also with antibodies to the hypervariable region 1, previously thought to be the primary site of neutralization. NtAb raised during 1a infections could neutralize HCVpp of genotypes 4a, 5a, and 6a but had only limited reactivity against 2a and 3a. The detection of high-titer NtAb with cross-genotype reactivity has important implications for the development of active and passive immune-prophylaxis strategies against HCV. Finally, we found that HCVpp infectivity was enhanced by human or chimpanzee sera; apolipoprotein C1 alone or as a component of high-density lipoproteins caused this enhancement. Future studies of the in vivo role of apolipoprotein C1 might provide additional insights into the infection process of HCV.

Fig. 1.

Humoral immune response in one chimpanzee (Ch1422, strain HC-TN) with acute resolving HCV infection that developed NtAb. Qualitative detection of HCV RNA in RT-nested PCR is indicated (on top) by red bars. Detection of antibody to core and nonstructural proteins by using the anti-HCV 2.0 assay is indicated (on top) by green bars. The shaded area indicates HCV genome titers as determined by Roche Monitor 2.0; values below the detection limit of ≈500 genome equivalents per ml are shown as 0. The blue, green, and orange lines and the red bars correspond to levels of anti-E1, anti-E2, anti-HVR1, and NtAb, respectively. Percentage of neutralization was calculated by comparison with a preinoculation sample from the same animal. Neutralization values ≤1 are indicated as equal to 1.

Fig. 2.

Humoral immune response in two chronically HCV-infected chimpanzees that developed NtAb. (A) Ch1530, strain H77. (B) Ch1581, strain HC-TN. See also Fig. 1 legend.

Fig. 3.

Humoral immune response in Patient H, who developed a chronic HCV infection with genotype 1a (strain H77). Because the patient had been vaccinated against smallpox, we could not test for anti-E1 (see Materials and Methods). Because of suboptimal storage of samples, most genome titers could not be determined. However, recent samples contained ≈10⁶ genome copies per ml, which is ≈1.5 logs lower than the peak titer during the acute infection. Samples were not available between week 33 and year 2. See also Fig. 1 legend.

Fig. 4.

Interassay reproducibility of neutralization assay. Result of four independent neutralization tests with sera obtained from Ch1494, Ch1530, and Patient H at the indicated weeks/years after infection. Results are expressed as the average of percentages of neutralization ± SD relative to incubation with the corresponding preinoculation serum for chimpanzees and with three normal human sera for Patient H.

Fig. 5.

Cross-neutralization of different HCVpp genotypes with sera obtained from Patient H at the indicated time points. pp were from genotype 1a (H77), 2a (J6CF), 3a (S52), 4a (ED43), 5a (SA13), and 6a (HK). The percentage of neutralization of the genotype 2a pp with serum from patient H taken at year 2 ranged from 41 to 52 in repeat assays.

Fig. 6.

Infectivity-enhancing activity of sera from different animal species. Shown are the results of a single neutralization assay of ppH77(1a). Percentage of enhancement was calculated by comparison with pp infection without preincubation with the indicated serum.

Fig. 7.

Influence of apolipoprotein on ppH77(1a) infection of Huh-7 cells. A normal human serum sample was tested in the same assay. 1× represents the concentration of serum usually used in pp assay (1:50) or concentration of apolipoprotein in normal sera at 1:50 dilution. Pseudotyped virus was preincubated with serum or apolipoprotein for 45 min at room temperature before infection of Huh-7 cells.

Fig. 8.

Influence of lipoproteins on ppH77(1a) infection of Huh-7 cells. See also Fig. 7 legend.