BackgroundProprotein convertase subtilisin/kexin type 9 (PSCK9) is secreted mainly from the liver and binds to the low-density lipoprotein receptor (LDLR), reducing LDLR availability and thus resulting in an increase in LDL-cholesterol. While the LDLR has been implicated in the cell entry process of the hepatitis C virus (HCV), overexpression of an artificial non-secreted, cell membrane-bound form of PCSK9 has also been shown to reduce surface expression of CD81, a major component of the HCV entry complex, leading to concerns that pharmacological inhibition of PCSK9 may increase susceptibility to HCV infection by increasing either CD81 or LDLR availability. Here, we evaluated effects of PCSK9 and PCSK9 blockade on CD81 levels and HCV entry with a physiologically relevant model using native secreted PCSK9 and a monoclonal antibody to PCSK9, alirocumab.Methods and ResultsFlow cytometry and Western blotting of human hepatocyte Huh-7 cells showed that, although LDLR levels were reduced when cells were exposed to increasing PCSK9 concentrations, there was no correlation between total or surface CD81 levels and the presence and amount of soluble PCSK9. Moreover, inhibiting PCSK9 with the monoclonal antibody alirocumab did not affect expression levels of CD81. In an in vitro model of HCV entry, addition of soluble PCSK9 or treatment with alirocumab had no effect on the ability of either lentiviral particles bearing the HCV glycoproteins or JFH-1 based cell culture virus to enter hepatocytes. Consistent with these in vitro findings, no differences were observed in hepatic CD81 levels using in vivo mouse models, including Pcsk9-/- mice compared with wild-type controls and hyperlipidemic mice homozygous for human Pcsk9 and heterozygous for Ldlr deletion, treated with either alirocumab or isotype control antibody.ConclusionThese results suggest that inhibition of PCSK9 with alirocumab has no effect on CD81 and does not result in increased susceptibility to HCV entry.

Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death 1 (PD-1) on virus-specific CD8+ T cells. PD-1 is induced upon T cell activation and its prolonged expression facilitates CD8+ T cell inhibitory signals when bound to its B7 family ligands, PD-L1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by antibodies may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1, in which virus-mediated upregulation induced a state of non-responsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1 after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. Additionally, we identify a critical role for the PI3K/Akt signaling pathway in PD-L1 upregulation of APC’s by HIV, as inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.

Alterations in N-linked glycosylation have long been associated with cancer but for the most part, the reasons why have remained poorly understood. Here we show that increased core fucosylation is associated with de-differentiation of primary hepatocytes and with the appearance of markers indicative of a transition of cells from an epithelial to a mesenchymal state. This increase in core fucosylation was associated with increased levels of two enzymes involved in α-1,6 linked fucosylation, GDP-mannose 4, 6-dehydratase (Gmds) and to a lesser extent fucosyltransferase 8 (Fut8). In addition, the activation of cancer-associated cellular signaling pathways in primary rat hepatocytes can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we show that increased levels of protein sialylation and α-1,6-linked core fucosylation are observed following activation of the β-catenin pathway. Activation of the Akt signaling pathway or induction of hypoxia also results in increased levels of fucosylation and sialylation. We believe that this knowledge will help in the better understanding of the genetic factors associated with altered glycosylation and may allow for the development of more clinically relevant biomarkers.

Challenges with the production and suboptimal immunogenicity of malaria vaccine candidates have slowed the development of a multiantigen vaccine. Attempting to resolve these issues, we focused on the use of highly immunogenic merozoite surface protein 8 (MSP8) as a vaccine carrier protein. Previously, we showed that a genetic fusion of the C-terminal 19-kDa fragment of merozoite surface protein 1 (MSP1) to MSP8 (MSP8) facilitated antigen production and folding and the induction of neutralizing antibodies to conformational B cell epitopes of MSP1 Here, using the MSP1/8 construct, we further optimized the recombinantMSP8 (rMSP8) carrier by the introduction of two cysteine-to-serine substitutions (CΔS) to improve the yield of the monomeric product. We then sought to test the broad applicability of this approach using the transmission-blocking vaccine candidate s25. The production of rs25-based vaccines has presented challenges. Antibodies directed against the four highly constrained epidermal growth factor (EGF)-like domains of s25 block sexual-stage development in mosquitoes. The sequence encoding matures25 was codon harmonized for expression in We produced a rs25-MSP8 fusion protein [rs25/8(CΔS)] as well as unfused, mature rs25. rs25 was purified with a modest yield but required the incorporation of refolding protocols to obtain a proper conformation. In comparison, chimeric rs25/8(CΔS) was expressed and easily purified, with the s25 domain bearing the proper conformation without renaturation. Both antigens were immunogenic in rabbits, inducing IgG that bound natives25 and exhibited potent transmission-reducing activity. These data further demonstrate the utility of MSP8 as a parasite-specific carrier protein to enhance the production of complex malaria vaccine targets.

Chikungunya virus (CHIKV) is an important emerging mosquito-borne alphavirus, indigenous to tropical Africa and Asia. It can cause epidemic fever and acute illness characterized by fever and arthralgias. The epidemic cycle of this infection is similar to dengue and urban yellow fever viral infections. The generation of an efficient vaccine against CHIKV is necessary to prevent and/or control the disease manifestations of the infection. In this report, we studied immune response against a CHIKV-envelope DNA vaccine (pEnv) and the role of the CHIKV nonstructural gene 2 (nsP2) as an adjuvant for the induction of protective immune responses in a relevant mouse challenge model. When injected with the CHIKV pEnv alone, 70% of the immunized mice survived CHIKV challenge, whereas when co-injected with pEnv + pnsP2, 90% of the mice survived viral challenge. Mice also exhibited a delayed onset signs of illness, and a marked decrease in morbidity, suggesting a nsP2 mediated adjuvant effect. Co-injection of the pnsP2 adjuvant with pEnv also qualitatively and quantitatively increased antigen specific neutralizing antibody responses compared to vaccination with pEnv alone. In sum, these novel data imply that the addition of nsP2 to the pEnv vaccine enhances anti-CHIKV-Env immune responses and maybe useful to include in future CHIKV clinical vaccination strategies.