Yan-Peng Zheng

Administration of amyloid-β42 oligomer-specific monoclonal antibody improved memory performance in SAMP8 mice.

Amyloid-β peptide (Aβ) is recognized by many as the leading cause of Alzheimers disease (AD), and Aβ oligomers play a major role in the early-onset form of AD. Recently, the application of passive immunization targeting Aβ has been investigated as a potential method of AD immunotherapy. We used a strain of monoclonal antibody against Aβ42 oligomers, designated A8, as an Aβ inhibitor to suppress Aβ aggregation and Aβ-derived cell toxicity in vitro, and as a passive immunotherapy approach to treat SAMP8 (senescence accelerated mouse sub-line P8) mice, an animal model of AD, in vivo. First, our results showed that pre-incubation of A8 with Aβ oligomers inhibited both the maturation of Aβ fiber and Aβ oligomer toxicity on SH-SY5Y cells. Second, learning and memory was improved through intraperitoneal administration of A8 in SAMP8 mice. Third, Aβ pathology was ameliorated with decreased Aβ oligomers and phospho-tau levels in SAMP8 mice. Our data suggest that our monoclonal antibody A8 may be a candidate as a potential immunotherapeutic agent in AD.

On-column refolding purification of DT389-hIL13 recombinant protein expressed in Escherichia coli.

Protein refolding is a bottleneck in the production of therapeutic proteins from inclusion bodies. In recent years, several studies have described on-column refolding of recombinant proteins. DT389-hIL13 is a recombinant protein that targets the glioma. In our study, the recombinant protein DT389-hIL13 was expressed in Escherichia coli (E. coli). The isolated inclusion bodies were refolded using size exclusion chromatography (SEC) and further purified using anion exchange chromatography. Three different methods of SEC on-column refolding were studied. In vitro tests on U251 cells showed that the recombinant protein could effectively inhibit the proliferation of U251 cells, especially the protein refolded by urea and pH gradient method. The half-maximal inhibitory concentration (IC50) of 0.887 nM was achieved with this new method, unlike an IC50 of 11.4 nM achieved in the non-gradient method.

DNA vaccine encoding central conserved region of G protein induces Th1 predominant immune response and protection from RSV infection in mice

Human respiratory syncytial virus (RSV) can cause serious infection in the lower respiratory tract, especially in infants, young children, the elderly and the immunocompromised population worldwide. Previous study demonstrated the polypeptide (amino acids 148-198) of RSV attachment (G) glycoprotein, corresponding to the central conserved region and encompassing CX3C chemokine motif, could induce antibodies and protection from RSV challenge in mice [1,2]. In this study, we evaluated the immune efficacy of the recombinant DNA vaccine of pVAX1/3G148-198 encoding RSV G protein polypeptide. RSV specific serum IgG antibodies with neutralizing activity were stimulated following prime-boost immunization of pVAX1/3G148-198 intramuscularly, and the ratio of IgG2a/IgG1 was 4.93, indicating a Th1 biased immune response. After challenged intranasally with RSV Long, the vaccinated mice showed both decreased lung RSV titers, pulmonary inflammation and body weight loss. The results suggest that pVAX1/3G148-198 DNA vaccine may be an effective RSV vaccine candidate, and deserves further exploration.

Biomarkers of Alzheimer’s disease in body fluids

Various innovative diagnostic methods for Alzheimer’s disease (AD) have been developed in view of the increasing prevalence and consequences of later-life dementia. Biomarkers in cerebrospinal fluid (CSF) and blood for AD are primarily based on the detection of components derived from amyloid plaques and neurofibrillary tangles (NFTs). Published reports on CSF and blood biomarkers in AD indicate that although biomarkers in body fluids may be utilized in the clinical diagnosis of AD, there are no specific markers that permit accurate and reliable diagnosis of early-stage AD or the monitoring of disease progression.

Single Chain Variable Fragment Against Aβ Expressed in Baculovirus Inhibits Abeta Fibril Elongation and Promotes its Disaggregation

Alzheimer’s disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-β (Aβ) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aβ N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aβ fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aβ aggregation or disaggregation in vitro. Here, a model of cell-free Aβ “on-pathway” aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of “on-pathway” aggregation and Aβ fibril maturation. The effect of A8 scFv on Aβ fibrillogenesis was markedly more significant when administered at the start of the Aβ folding reaction. Furthermore, the results also showed that pre-formed Aβ fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aβ aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free “on-pathway” Aβ aggregation and will assist in the development of therapeutic strategies for AD.

Enhanced humoral and CD8 + T cell immunity in mice vaccinated by DNA vaccine against human respiratory syncytial virus through targeting the encoded F protein to dendritic cells

Abstract Human respiratory syncytial virus (RSV) is the most important cause of serious lower respiratory tract infection in infants, the elderly, and the immunocompromised population. There is no licensed vaccine against RSV until now. It has been reported that targeting antigen to DEC205, a phagocytosis receptor on dendritic cells (DCs), could induce enhanced CD4 + and CD8 + T cell responses in mice. To develop RSV DNA vaccine and target the encoded antigen protein to DCs, the ectodomain of fusion glycoprotein (sF, amino acids: 23–524) of RSV was fused with anti‐DEC205 single‐chain Fv fragment (scDEC) and designated scDECF. Following successful expression from the recombinant plasmid of pVAX1/scDECF, the recombinant protein of scDECF was found capable of specifically binding to DEC205 receptor on CHOmDEC205 cells, and facilitating uptake of RSV F by DC2.4 cells in vitro. Furthermore, the higher levels of RSV‐specific IgG antibody responses and neutralization antibody titers, as well as RSV F‐specific CD8 + T cell responses were induced in mice immunized intramuscularly by pVAX1/scDECF than by the control plasmid of pVAX1/scISOF encoding sF protein fused with isotype matched control single‐chain Fv fragment (scISO). Compared with pVAX1/scISOF, both the ratio of IgG2a/IgG1, > 1, and the enhanced IFN‐&ggr; cytokine were induced in mice following pVAX1/scDECF immunization, which exhibited a Th1 dominant response in pVAX1/scDECF vaccinated mice. Notably, the elevated efficiency of RSV F protein bound by DCs in vivo could also be observed in mice inoculated by pVAX1/scDECF. Collectively, these results demonstrate the enhanced IgG and CD8+ T cell immune responses have been induced successfully by DNA vaccine against RSV by targeting F antigen to DCs via the DEC205 receptor, and this DC‐targeting vaccine strategy merits further investigation. HighlightsscDECF encoded by pVAX1/scDECF could bind to DCs efficiently in vitro and in vivo.The raised humoral and CD8 + T cell responses were induced by pVAX1/scDECF.pVAX1/scDECF immunized mice displayed characteristic of Th1 biased immune response.It is a valuable exploration to strengthen the immunogenicity of RSV DNA vaccine.

Kinesin‐1 inhibits the aggregation of amyloid‐β peptide as detected by fluorescence cross‐correlation spectroscopy

Although the exact etiology and pathogenesis of Alzheimers disease (AD) are still unclear, amyloid‐β (Aβ) generated by the proteolytic processing of amyloid‐β precursor protein (APP) aggregate to form toxic amyloid species. Kinesin‐1 is the first identified ATP‐dependent axonal transport motor protein that has been proven to affect Aβ generation and deposition. In this paper, we applied dual‐color fluorescence cross‐correlation spectroscopy (DC‐FCCS) to investigate the direct interaction of Aβ with kinesin‐1 at the single‐molecule fluorescence level in vitro. The results showed that two kinds of enhanced green fluorescent protein (EGFP)‐tagged kinesin light‐chain subunits of kinesin‐1(KLCs), KLC‐E and E‐KLC inhibited the aggregation of Aβ over a period of time, providing additional insight into the mechanism of axonal transport deficits in AD.

A Built-In CpG Adjuvant in RSV F Protein DNA Vaccine Drives a Th1 Polarized and Enhanced Protective Immune Response

Human respiratory syncytial virus (RSV) is the most significant cause of acute lower respiratory infection in children. However, there is no licensed vaccine available. Here, we investigated the effect of five or 20 copies of C-Class of CpG ODN (CpG-C) motif incorporated into a plasmid DNA vaccine encoding RSV fusion (F) glycoprotein on the vaccine-induced immune response. The addition of CpG-C motif enhanced serum binding and virus-neutralizing antibody responses in BALB/c mice immunized with the DNA vaccines. Moreover, mice vaccinated with CpG-modified vaccines, especially with the higher 20 copies, resulted in an enhanced shift toward a Th1-biased antibody and T-cell response, a decrease in pulmonary pathology and virus replication, and a decrease in weight loss after RSV challenge. This study suggests that CpG-C motif, cloned into the backbone of DNA vaccine encoding RSV F glycoprotein, functions as a built-in adjuvant capable of improving the efficacy of DNA vaccine against RSV infection.

The effects of fluorescent labels on Aβ42 aggregation detected by fluorescence correlation spectroscopy

Fluorescence‐based methods are promising for measuring amyloid beta (Aβ) oligomers, given their capacity to analyse a sample at the single‐molecule level. As the attachment of fluorescent labels may influence the biochemical properties of the Aβ oligomers, the effects of fluorescent labels on Aβ oligomers must be evaluated. In this paper, we compared the impacts of five different fluorescent dyes on the aggregation of Aβ42 oligomers using fluorescence correlation spectroscopy (FCS). We found that fluorescent labels of BODIPY® FL‐C5 (BP), N‐hydroxysuccinimide rhodamine B ester (RB) and rhodamine B isothiocyanate (RITC) increased the propensity of labelled Aβ42 oligomers to aggregate, whereas 6‐(fluorescein‐5‐carboxamido) hexanoic acid succinimidyl ester (5‐SFX) and fluorescein 5(6)‐isothiocyanate (5(6)‐FITC) decreased the propensity of labelled Aβ42 oligomers to aggregate. This difference originated from the different electric charges and hydrophobicity of the fluorescent dyes. These results provide valuable information for establishing different aggregation models for Aβ42 oligomers in vitro using FCS.

Construction and Characterization of a Recombinant Human Respiratory Syncytial Virus Encoding Enhanced Green Fluorescence Protein for Antiviral Drug Screening Assay

Human respiratory syncytial virus (RSV) is the single most important cause of lower respiratory tract disease in infants and young children and a major viral agent responsible for respiratory tract disease in immunosuppressed individuals and the elderly, but no vaccines and antiviral drugs are available. Herein the recombinant RSV (rRSV) encoding enhanced green fluorescence protein (EGFP, rRSV-EGFP) was constructed and the potential for screening anti-RSV drugs was investigated. The recombinant plasmid of pBRATm-rRSV-EGFP, containing T7 transcription cassette composed of T7 promoter, RSV antigenomic cDNA with EGFP gene, HDV ribozyme (δ), and T7 terminator in the order of 5′ to 3′, was constructed and cotransfected into BHK/T7-9 cells together with helper plasmids encoding N, P, L, and M2-1 gene, respectively. The rescued rRSV-EGFP was confirmed by increasing expression of EGFP over blind passages and by RT-PCR. rRSV-EGFP was comparable to the other two recombinant RSVs encoding red fluorescent protein (RFP, rRSV-RFP) or luciferase (Luc, rRSV-Luc) in the growth kinetic, and there was a difference in sensitivity between them for screening anti-RSV agents based on infection of HEp-2 cells. The EGFP-encoding rRSV has been constructed and rescued successfully and has the potential for high-throughput anti-RSV drug screening in vitro.