A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.

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 beta-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 PCSK9DeltaC, 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.

Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants.

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 PCSK9DeltaC-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 PCSK9DeltaC 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 PCSK9DeltaC 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.

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates.

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.

A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.

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.

A novel adenovirus type 6 (Ad6)-based hepatitis C virus vector that overcomes preexisting anti-ad5 immunity and induces potent and broad cellular immune responses in rhesus macaques.

Success in resolving hepatitis C virus (HCV) infection has been correlated to vigorous, multispecific, and sustained CD8(+) T-cell response in humans and chimpanzees. The efficacy of inducing T-cell-mediated immunity by recombinant serotype 5 adenovirus vector has been proven in many animal models of infectious diseases, but its immunogenicity can be negatively influenced by preexisting immunity against the vector itself. To evaluate the less prevalent adenovirus serotype 6 (Ad6) as an alternative vector for and HCV vaccine development, we have generated serotype 5 and 6 adenoviral vectors directing expression of the nonstructural region of HCV (MRKAd5-NSmut and MRKAd6-NSmut). Immunogenicity studies in mice showed that the two vectors induced comparable T-cell responses but that only MRKAd6-NSmut was not suppressed in the presence of anti-Ad5 immunity. In contrast, preexisting anti-Ad5 immunity dramatically blunted the immunogenicity of the serotype 5-based HCV vector. Furthermore, MRKAd6-NSmut showed equivalent potency, breadth, and longevity of HCV-specific T-cell responses in rhesus macaques as the corresponding Ad5-based vector over a wide range of doses and was capable of boosting DNA-primed animals even if administered at low doses. These data support the use of the MRKAd6-NSmut for anti-HCV immunotherapy and, more generally, for the Ad6 serotype as a better genetic vaccine vehicle than Ad5.