EDIII-Fc induces protective immune responses against the Zika virus in mice and rhesus macaque.

Zika virus can infect the fetus through the placental barrier, causing ZIKV congenital syndrome and even miscarriage, which can cause great harm to pregnant women and infants. Currently, there is no vaccine and drug available to combat the Zika virus. In this study, we designed a fusion protein named EDIII-Fc, including the EDIII region of Zika E protein and human IgG Fc fragment, and obtained 293T cells that stably secreted EDIII-Fc protein using the lentiviral expression system. Mice were immunized with the EDIII-Fc protein, and it was observed that viral replication was significantly inhibited in the immunized mice compared to non-immunized mice. In rhesus macaques, we found that EDIII-Fc effectively induce the secretion of neutralizing antibodies and T cell immunity. These experimental data provide valid data for further use of Zika virus E protein to prepare an effective, safe, affordable Zika vaccine.

Efficient Gene Transfer to Kidney Using a Lentiviral Vector Pseudotyped with Zika Virus Envelope Glycoprotein.

Gene therapy's entrance into clinical settings has made it an ever more attractive field of study for various diseases. However, relatively little progress has been made in targeting kidney diseases due to poor gene delivery efficiency in renal cells. The development of novel gene therapy vectors for medical intervention to treat kidney diseases is needed. In this study, we designed and produced a pseudotyped lentiviral vector with envelope glycoproteins of Zika virus (ZIKV), and evaluated its potential use in viral vector entry, neutralization assay, and gene delivery especially in the renal context. The lentiviral vector, simplified as ZIKV-E, is pseudotyped with Env/G-TC representing the transmembrane (TM) and cytoplasmic (CY) domains of Env replaced with the TM and CY domains of the glycoprotein (G) of the vesicular stomatitis virus. In vivo results show that ZIKV-E induced efficient transduction in tubular epithelial cells in mouse kidneys, demonstrating >100-fold higher expression of exogenous green fluorescent protein gene compared with that achieved by vesicular stomatitis virus G (VSV-G) protein pseudotyped lentiviral vector. The results also showed that the vector ZIKV-E transduced cells in a pH-independent manner and the transduction was inhibited by anti-ZIKV Env domain III antibodies. Results also show that ZIKV-E can be used as a surrogate for studies of ZIKV entry mechanisms and neutralization antibody assay. In all, this study successfully demonstrated a novel pseudotyped lentiviral vector ZIKV-E for inducing high transduction efficiency in renal tubular epithelial cells that could serve as a foundation for gene therapy for the treatment of inherited renal diseases in humans.

Construction of a eukaryotic expression system with stable and secretory expression of mycobacterium tuberculosis 38 kDa protein.

The 38 kDa protein is a major antigen of mycobacterium tuberculosis and has been widely used in TB serodiagnosis, due to its highly sensitivity and specificity. Here we attempt to establish a production platform of recombinant 38 kDa protein in mammalian cells and to evaluate the potential value of 38 kDa protein in TB serodiagnosis. The 38 kDa gene is synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-38 kDa-eGFP was packaged, titered, and then transduced into HEK 293 T cells. Recombinant cell lines were selected by limiting dilution. Supernatants were collected and purified by HisTrapTM HP column. Western blot showed a molecular weight of approximate 38 kDa in cell supernatants as expected. ELISA assay confirmed the immunological specificity of the obtained protein in the presence of MTB-infected human serum samples. In all, we have obtained a stable cell line with long-term and robust expression of secretory MTB 38 kDa protein, which may provide a promising candidate antigen for the development of TB serological diagnosis.

Zika Virus Envelope Protein induces G2/M Cell Cycle Arrest and Apoptosis via an Intrinsic Cell Death Signaling Pathway in Neuroendocrine PC12 Cells.

Clinical evidence suggests that there exists a strong correlation between Zika virus (ZIKV) infection and abnormal development of the nervous system. However, the underlying mechanisms remain to be elusive. In this study, recombinant lentiviral vectors coding for ZIKV structural proteins and truncations (prM-Env, M-Env and Env) were transduced into PC12 cells. Envelope (Env) overexpression induced significant inhibition of proliferation and triggered G2/M cell cycle arrest and apoptosis in PC12 cells. Flow cytometry and western blot analysis showed that the apoptosis was associated with up-regulation of both p53 and p21Cip1/Waf1 and down-regulation of cyclin B1. Presence of aberrant nuclei clusters were confirmed by immunofluorescence staining analysis. The data indicate that overexpression of prM-Env, M-Env or Env led to apoptosis via an intrinsic cell death signaling pathway that is dependent on the activation of caspase-9 and caspase-3 and accompanied by an increased ratio of Bax to Bcl-2 in transduced PC12 cells. In summary, our results suggest that ZIKV Env protein causes apoptosis in PC12 cells via an intrinsic cell death signaling pathway, which may contribute to ZIKV-induced abnormal development of the nervous system.

Construction and Identification of Recombinant HEK293T Cell Lines Expressing Non-structural Protein 1 of Zika Virus.

Background: Zika virus (ZIKV) infection has become a major public health problem all around the world. Early diagnosis of Zika infection is important for better management of the disease. Non-structural protein 1 (NS1) is a potential biomarker for ZIKV infections. The purpose of this study was to produce the ZIKV NS1 protein for establishing serological diagnostic methods for ZIKV. Methods: The cDNA fragment encoding a chimeric protein composed of murine Igκ signal peptide, NS1 and histidine tag was synthesized and cloned into the lentiviral expression vector pLV-eGFP. The resulting expression vector pLV-eGFP-ZIKV-NS1 was packaged and transduced into human embryonic kidney (HEK) 293T cells and clonal cell lines with NS1 gene were generated from the tranduced cells by limiting dilution. Over expressed recombination NS1 (rNS1) fusion protein was purified by nickel affinity chromatography. Mice immunization and enzyme-linked immunosorbent assay (ELISA) were carried out to evaluate the immunogenicity of rNS1. Results: Western blot analysis revealed that the reconstituted cells stably expressed and secreted high levels of approximately 45-kDa NS1, and no significant changes were observed in green fluorescent protein (GFP) fluorescence ratio and fluorescence intensity. The scanned gels showed that the purity of the purified rNS1 was 99.42%. BALB/c mice were then immunized with purified rNS1 and a high level of antibodies against NS1 was elicited in the mice. Conclusion: Overall, recombinant NS1 proteins were successfully purified and their antigenicity was assessed. Immunization of mice with recombinant proteins demonstrated the immunogenicity of the NS1 protein. Thus, the generated recombinant NS1 can be potentially used in the development of serological diagnostic methods for ZIKV.