Alfred Mayer Prince

Visualization of hepatitis C virions and putative defective interfering particles isolated from low-density lipoproteins

SUMMARY. Hepatitis C virus (HCV) in highly infectious sera has been shown to be predominantly associated with low‐density lipoproteins. To determine whether the association is specific to low‐density lipoproteins (LDL) or very low‐density lipoproteins (VLDL), we fractionated HCV‐containing plasma by a column chromatographic procedure known to separate these classes. Hepatitis C virus RNA detected by polymerase chain reaction (PCR) was associated primarily with the very low‐density (VLDL) fraction. However, it could not be ruled out that virus‐associated LDL may have eluted with this fraction. Hepatitis C virus virions isolated from sera having sufficient titre for visualization by electron microscopy are generally coated with antiviral antibodies, therefore we utilized the lipid association to isolate antibody‐free virions. Very low‐density lipoproteins were isolated by ultracentrifugal flotation and then treated with deoxycholate to release the virions. These were then isolated in a highly purified form by centrifugation in a sucrose gradient. The 1.10–1.11 gml‐1 region of the gradients contained 60–70 nm particles. Particles with similar surface structure but having a diameter of only 30–40 nm constituted about 30% of the total. The latter may represent defective interfering particles. The identity of both small and large particles with HCV virions and associated particles was confirmed by their trapping on grids by an anti‐HCV E2 monoclonal antibody, and by their aggregation by rabbit antiserum to an amino‐terminal peptide of E1. Thus, both E1 and E2 epitopes are displayed on the surface of intact HCV virions.

Characterization of the immune response against hepatitis C infection in recovered, and chronically infected chimpanzees

summary.u2002The immune response to hepatitis C virus (HCV) is believed to be critical in determining the outcome of the disease. In this study we have analysed epitope recognition, cytokine profile, and anti‐HCV antibody responses in chronically HCV‐infected and recovered chimpanzees. Quantitative measurement of anti‐HCV antibody in HCV‐infected chimpanzees revealed that the response in HCV‐ recovered chimpanzees peaked within 4–20u2003weeks. In contrast, the anti‐HCV antibody responses in chronically HCV infected chimpanzees did not peak until 100–200u2003weeks after infection, and decreased gradually thereafter. T cell proliferation assays measuring responses to pooled HCV proteins revealed significant increases in the 3H‐uptake during the early stages of infection in recovered chimpanzees in comparison to the chronically infected ones. Class I‐restricted epitopes of the core, and NS3 proteins of HCV were analysed using 9–10 mer overlapping peptides covering the core and NS3 proteins, and IFN‐γ ELISPOT technique. Our data indicated early and broad class‐I restricted core, and NS3 protein epitope recognitions in HCV‐recovered chimpanzees but not in chimpanzees that had been chronically infected. Additionally, dominant epitopes recognized early in infection (8u2003weeks) were no longer recognized later in infection (followed up to 64u2003weeks). Cytokines profiling revealed a 50‐fold increase in TNF‐α secretion in the supernatant of core‐specific CD8 memory cells of the chronically infected chimpanzees in comparison to the recovered ones. In summary, multiple parameters correlate with HCV recovery in chimpanzees.

Schistosoma infection inhibits cellular immune responses to core HCV peptides

Patients coinfected with hepatitis C virus (HCV) and the trematode, Schistosoma mansoni, have an increased incidence of viral persistence and accelerated fibrosis. To investigate immunological mechanisms responsible for this more aggressive natural history of HCV, the core HCV‐specific T‐cell responses were analysed in 44 donated blood units rejected because they had antibodies to HCV (anti‐HCV). Half also had anti‐S. mansoni antibodies, evidence of past or active infection. HCV‐specific ELISPOT responses were examined using pools of 180 overlapping 9‐mer peptides with offsets of one covering the core of HCV genotype 4a. Comparison of T‐cell responses in blood units positive for both anti‐HCV and anti‐Schistosoma antibodies with blood units positive only for anti‐HCV antibodies showed a significant decrease in core‐specific T‐cell IFN‐γ (505± 46 vs. 803 ± 66 ISC/106 cells, P < 0·001), IL‐4 (2 ± 108 vs. 641 ± 131 ISC/106 cells, P < 0·001), and IL‐10 (159 ± 105 vs. 466 ± 407 ISC/106 cells, P < 0·002) responses. In contrast, there was no significant difference in cell‐mediated immune response (CMI) to PHA mitogen between these two groups. Therefore, we concluded T cells from persons with anti‐Schistosoma have reduced IFN‐γ, IL‐4, and IL‐10 secreting HCV‐specific T‐cell responses. This may explain why Schistosoma coinfection increases persistence and severity of HCV infection.

Towards a new predictor of AIDS progression through the quantisation of HIV-1 DNA copies by PCR in HIV-infected individuals

Summary. The proviral copy (PVC) number in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)‐infected individuals was measured by a quantitative polymerase chain reaction (PCR) assay to determine over time the relation between the viral load and the evolution towards the disease in HIV‐infected people: 67 anti‐HIV‐1 positive individuals (60 stage II/III, 7 stage IV) were studied. The mean PVC number per 1.5 × 105 PBMCs in stage II/III individuals (14.4 ± 14.2) and in stage IV individuals (32.2 ± 22.9) was significantly different (P <0.02). PVC number was inversely correlated to the CD4 lymphocyte count (P <0.01). In the logistic regression, the PVC number was a better marker of evolution towards the disease than the CD4 lymphocyte count. The mean proportion of HIV‐infected PBMCs in stage IV individuals and in stage II/III individuals was 1/4606 and 1/10714, respectively.

Hepatitis C virus replication kinetics in chimpanzees with self-limited and chronic infections

Summary.u2002 The availability of molecular beacon‐based, real time polymerase chain reaction (PCR) and a semi‐automated sample extraction procedure have made it possible for us to retrospectively examine HCV replication kinetics in HCV naive chimpanzees infected during the past 20u2003years. We compared these in 17 animals that developed chronic infection, and in 21 that developed self‐limited infection. No differences were found in infecting dose, or replication kinetics in the acute phase between these two types of infection. An unanticipated finding was the fact that 10 of 17 animals developing chronic infection partially controlled virus replication for 48u2003±u200348u2003weeks after typical acute phase viraemia, and prior to development of chronic infection. Twenty‐nine out of 30 (29/30) sera, which were negative by quantitative PCR during the downregulated period, were, however, positive by the more sensitive Genprobe isothermal transcription‐mediated amplification (TMA) assay. Thus, downregulation was not complete. Ten animals showing self‐limited infection showed complete resolution of viraemia by TMA assay. Quasispecies analysis revealed that in all, except one case, the virus reappearing after downregulation was essentially identical to that of the originally infecting virus.