Chronic hepatitis C virus (HCV) infection, with its cohort of life-threatening complications, affects more than 200 million persons worldwide and has a prevalence of more than 10% in certain countries. Preventive and therapeutic vaccines against HCV are thus much needed. Neutralizing antibodies (NAbs) are the foundation for successful disease prevention for most established vaccines. However, for viruses that cause chronic infection such as HIV or HCV, induction of broad NAbs from recombinant vaccines has remained elusive. We developed a vaccine platform specifically aimed at inducing NAbs based on pseudotyped virus-like particles (VLPs) made with retroviral Gag. We report that VLPs pseudotyped with E2 and/or E1 HCV envelope glycoproteins induced high-titer anti-E2 and/or anti-E1 antibodies, as well as NAbs, in both mouse and macaque. The NAbs, which were raised against HCV 1a, cross-neutralized the five other genotypes tested (1b, 2a, 2b, 4, and 5). Thus, the described VLP platform, which can be pseudotyped with a vast array of virus envelope glycoproteins, represents a new approach to viral vaccine development.

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ABSTRACTpim-1 and pim-3 encode serine/threonine kinases involved in the regulation of cell proliferation and apoptosis in response to cytokine stimulation. We analyzed the regulation of pim-1 and pim-3 by the leukemia inhibitory factor (LIF)/gp130/signal transducer and activator of transcription-3 (STAT3) pathway and the role of Pim-1 and Pim-3 kinases in mouse embryonic stem (ES) cell self-renewal. Making use of ES cells expressing a granulocyte colony-stimulating factor:gp130 chimeric receptor and a hormone-dependent signal transducer and activator of transcription-3 estrogen receptor (STAT3-ER T2 ), we showed that expression of pim-1 and pim-3 was upregulated by LIF/gp130-dependent signaling and the STAT3 transcription factor. ES cells overexpressing pim-1 and pim-3 had a greater capacity to self-renew and displayed a greater resistance to LIF starvation based on a clonal assay. In contrast, knockdown of pim-1 and pim-3 increased the rate of spontaneous differentiation in a self-renewal assay. Knockdown of pim-1 and pim-3 was also detrimental to the growth of undifferentiated ES cell colonies and increased the rate of apoptosis. These findings provide a novel role of Pim-1 and Pim-3 kinases in the control of self-renewal of ES cells. STEM CELLS 2007;25:2996 -3004 Disclosure of potential conflicts of interest is found at the end of this article.

Background:We compared viral subpopulations derived from humanized liver mouse model versus cell culture. Results:In vivo and in vitro produced particles show different biophysical properties and receptor usage. Conclusion:In vivo models allow functional investigations on how lipid metabolism and hepatic environment influence HCV entry. Significance: HCV produced from humanized liver mice may better reflect the characteristics of virus from HCV-infected patients.

The development of lentiviral vectors (LVs) for expression of a specific antibody can be achieved through the transduction of mature B-cells. This approach would provide a versatile tool for active immunotherapy strategies for infectious diseases or cancer, as well as for protein engineering. Here, we created a lentiviral expression system mimicking the natural production of these two distinct immunoglobulin isoforms. We designed a LV (FAM2-LV) expressing an anti-HCV-E2 surface glycoprotein antibody (AR3A) as a membrane-anchored Ig form or a soluble Ig form, depending on the B-cell maturation status. FAM2-LV induced high-level and functional membrane expression of the transgenic antibody in a nonsecretory B-cell line. In contrast, a plasma cell (PC) line transduced with FAM2-LV preferentially produced the secreted transgenic antibody. Similar results were obtained with primary B-cells transduced ex vivo. Most importantly, FAM2-LV transduced primary B-cells efficiently differentiated into PCs, which secreted the neutralizing anti-HCV E2 antibody upon adoptive transfer into immunodeficient NSG (NOD/SCIDγc(-/-)) recipient mice. Altogether, these results demonstrate that the conditional FAM2-LV allows preferential expression of the membrane-anchored form of an antiviral neutralizing antibody in B-cells and permits secretion of a soluble antibody following B-cell maturation into PCs in vivo.

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The LIM homeodomain transcription factor 1b (Lmx1b) is a key factor in the specification of the serotonergic neurotransmitter phenotype. Here, we explored the capacity of Lmx1b to direct differentiation of mouse embryonic stem (mES) cells into serotonergic neurons. mES cells stably expressing human Lmx1b were generated by lentiviral vector infection. Clones expressing Lmx1b at a low level showed increased neurogenesis and elevated production of neurons expressing serotonin, serotonin transporter, tryptophan hydroxylase 2, and transcription factor Pet1, the landmarks of serotonergic differentiation. To explore the role of Lmx1b in the specification of the serotonin neurotransmission phenotype further, a conditional system making use of a floxed inducible vector targeted into the ROSA26 locus and a hormone-dependent Cre recombinase was engineered. This novel strategy was tested with the reporter gene encoding human placental alkaline phosphatase, and demonstrated its capacity to drive transgene expression in nestin þ neural progenitors (NPs) and in Tuj1 þ neurons. When it was applied to inducible expression of human Lmx1b, it resulted in elevated expression of serotonergic markers. Treatment of neural precursors with the floor plate signal Sonic hedgehog further enhanced differentiation of Lmx1b-overexpressing NPs into neurons expressing 5-HT, serotonin transporter, tryptophan hydroxylase 2, and Pet1, when compared with Lmx1b-nonexpressing progenitors. Together, our results demonstrate the capacity of Lmx1b to specify a serotonin neurotransmitter phenotype when overexpressed in mES cell-derived NPs.

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.