Three percent of the world's population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8(+) T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses.

PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9⌬C-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF(A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9⌬C bound to EGF(AB) H306Y , a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB) H306Y -PCSK9 interaction is pH-independent, LDLR H306Y binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9⌬C containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) 4 has recently emerged as a major regulator of low density lipoprotein (LDL) cholesterol levels in plasma and consequently as an important determinant of cardiovascular health in humans. The link between cardiovascular disease and PCSK9 was initially made following the discovery that the PCSK9 missense mutations, S127R and F216L (1), and later, D374Y (2), are associated with a form of autosomal dominant hypercholesterolemia, a risk factor for coronary heart disease. Subsequently, two PCSK9 nonsense mutations (Y142X and C679X) (3) and several missense mutations (R46L, G106R, N157K, G236S, R237W, L253F, N354I and A443T) (4 -6) have been found to be associated with hypocholesterolemia. Remarkable degrees of protection against coronary heart disease were observed in humans heterozygous for the mutations Y142X and C679X (88% reduced incidence) and by R46L (47% reduced incidence) (7). Consequently, PCSK9 represents a novel therapeutic target for the prevention of premature atherosclerosis and coronary heart disease, and an understanding of its mechanism of action is of significant medical interest.PCSK9 is the ninth member of the mammalian proprotein convertase family of serine proteases. The translated proprotein includes an N-terminal signal sequence directing its secretion (residues 1-30), a prodomain (residues 31-152), a catalytic domain (residues 153-451)...

Background/Aims: Hepatitis C virus (HCV) infection results in a high frequency of chronic disease. The aim of this study was to identify early prognostic markers of disease resolution by performing a comprehensive analysis of viral and host factors during the natural course of acute HCV infection. Methods: The clinical course of acute hepatitis C was determined in 34 consecutive patients. Epidemiological and virological parameters, as well as cell mediated immunity (CMI) and distribution of human leukocyte antigens (HLA) alleles were analysed. Results: Ten out of 34 patients experienced self-limiting infection, with most resolving patients showing fast kinetics of viral clearance: at least one negative HCV RNA test during this phase predicted a favourable outcome. Among other clinical epidemiological parameters measured, the self-limiting course was significantly associated with higher median peak bilirubin levels at the onset of disease, and with the female sex, but only the latter parameter was independently associated after multivariate analysis. No significant differences between self-limiting or chronic course were observed for the distribution of DRB1 and DQB1 alleles. HCV specific T cell response was more frequently detected during acute HCV infection, than in patients with chronic HCV disease. A significantly broader T cell response was found in patients with self-limiting infection than in those with chronic evolving acute hepatitis C. Conclusion:The results suggest that host related factors, in particular sex and CMI, play a crucial role in the spontaneous clearance of this virus. Most importantly, a negative HCV RNA test and broad CMI within the first month after onset of the symptoms represent very efficacious predictors of viral clearance and could thus be used as criteria in selecting candidates for early antiviral treatment.

PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of ␤-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9⌬C, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) 4 is a key regulator of plasma low density lipoprotein (LDL) cholesterol and has emerged as a promising target for prevention and treatment of coronary heart disease. A strong link between PCSK9, LDL, cholesterol, and coronary heart disease has been established by multiple laboratories. Human genetic studies demonstrated remarkable correlations between several nonsense or missense PCSK9 mutations with plasma LDL cholesterol levels and the risk of coronary heart disease. Thus, putative gain-or loss-of-function mutants were found to correlate with increased or reduced plasma LDL levels and cardiovascular risk, respectively (1-7). A recent genome-wide association study further bolstered the importance of PCSK9 by establishing a linkage between a single nucleotide polymorphism at a locus near PCSK9 with early-onset myocardial infarction (8).There is extensive evidence that plasma PCSK9 raises LDL cholesterol levels by binding to cell surface LDLR and targeting the receptor to lysosomes for degradation (9 -13). Accordingly, inhibition of PCSK9 by recombinant LDLR fragments (14 -16) or by mono-or polyclonal antibodies (17, 18) restored LDL cholesterol uptake in cells. Moreover, either RNAi targeting liver PCSK9 (19) or intravenous injection of a monoclonal antibody disrupting the PCSK9-LDLR inter...

Our data provide evidence of strong and multispecific T cell responses with a sustained ability to proliferate in response to antigen stimulation as reliable pharmacodynamic measures of a protective CMI during acute infection, and suggest that early impairment of proliferation may contribute to loss of T cell response and chronic HCV persistence.