A novel human monoclonal antibody potently neutralizes human adenovirus serotype 7 by primarily targeting the adenovirus hexon protein.

Human adenovirus serotype 7 (HAdV-7), belonging to species B, has caused severe lower respiratory tract diseases and even deaths recently. However, no adenovirus vaccine or therapeutic is available thus far. In this study, a HAdV-7-specific human monoclonal antibody (HMAb), 3-3E, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of HAdV-7-infected patients showed potent HAdV-7 neutralization activity. The results showed HMAb 3-3E only binds to the hexon protein of intact HAdV-7 or the recombinant hexon protein and it does not bind to other intact virion particles. This could mean the antibody recognizes a conformational epitope of the hexon protein. Further, HMAb 3-3E potently neutralized HAdV-7 in vitro at low concentrations. In vivo studies showed HMAb 3-3E protected from HAdV-7 infection in a murine model. Therefore, HMAb 3-3E is promising as a safe and effective prophylactic and therapeutic treatment for HAdV-7 infection.

miR-21-3p Regulates Influenza A Virus Replication by Targeting Histone Deacetylase-8.

Influenza A virus (IAV) is responsible for severe morbidity and mortality in animals and humans worldwide. miRNAs are a class of small noncoding single-stranded RNA molecules that can negatively regulate gene expression and play important roles in virus-host interaction. However, the roles of miRNAs in IAV infection are still not fully understood. Here, we profiled the cellular miRNAs of A549 cells infected with A/goose/Jilin/hb/2003 (H5N1) and a comparison A/Beijing/501/2009 (H1N1). miRNA microarray and quantitative PCR analysis showed that several miRNAs were differentially expressed in A549 cells during IAV infection. Subsequently, we demonstrated that IAV replication was essential for the regulation of these miRNAs, and bioinformatic analysis revealed that the targets of these miRNAs affected biological processes relevant to IAV replication. Specifically, miR-21-3p was found to be down-regulated in IAV-infected A549 cells and selected for further detailed analysis. Target prediction and functional study illustrated that miR-21-3p repressed the expression of HDAC8 by targeting its 3'UTR. Furthermore, we confirmed miR-21-3p could promote virus replication, which was similar to the result of knocking down HDAC8, indicating that miR-21-3p promoted IAV replication by suppressing HDAC8 expression. Altogether, our results suggest a potential host defense against IAV through down-regulation of miR-21-3p.

Potent Neutralization Ability of a Human Monoclonal Antibody Against Serotype 1 Dengue Virus.

The incidence of dengue virus (DENV) infections has been escalating in tropical and subtropical countries, but there are still no effective therapeutic options. In the present study, a DENV-1-specific human monoclonal antibody (HMAb), 1G5, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of dengue patients was found to have potent neutralization activity against serotype 1 DENV (DENV-1). Its neutralization activity against DENV-2 was not as strong, and it was almost absent for DENV-3 and DENV-4. The results showed that HMAb 1G5 only binds to the envelop protein of intact DENV-1 or the envelop protein under unheated and non-reducing conditions, and that it does not bind to recombinant envelope protein. This could mean that the antibody recognizes a conformational epitope of the envelope protein. Further, the findings showed that HMAb 1G5 potently neutralizes DENV-1 in both the pre- and post-attachment phases of the virus at low concentrations. In vivo studies showed that HMAb 1G5 provides protection from DENV-1 infection in a murine model. In addition, antibody-dependent enhancement that occurs at lower doses of the antibody was completely abrogated by the introduction of Leu-to-Ala mutations (1G5-LALA) or deletion of nine amino acids (1G5-9del) in the Fc region. Therefore, HMAb 1G5 shows promise as a safe and effective agent for prophylactic and therapeutic treatment of DENV-1 infection.

Improved synaptic and cognitive function in aged 3 × Tg-AD mice with reduced amyloid-ß after immunotherapy with a novel recombinant 6Aß15-TF chimeric vaccine.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder impairing memory and cognition. In this study, we describe the immunogenicity and protective efficacy of the novel recombinant 6Aß15-TF chimeric antigen as a subunit protein vaccine for AD. Recombinant 6Aß15-TF chimeric vaccine induced strong Aß-specific humoral immune responses without Aß-specific T cell immunity in C57/BL6 and 3 × Tg-AD mice at different ages. As an early immunotherapy model for AD, this vaccine induced high titers of long-lasting anti-Aß42 antibodies in aged 3 × Tg-AD mice, which led to improve behavioral performance and markedly reduced the levels of insoluble and soluble Aß and Aß oligomers. In agreement with these findings, immunotherapy with 6Aß15-TF prevented the Aß-induced decrease of presynaptic and postsynaptic proteins in aged 3 × Tg-AD mice. Our results suggest that this novel and highly immunogenic recombinant 6Aß15-TF chimeric vaccine provides neuroprotection in AD mice and can be considered an effective AD candidate vaccine.

Enhanced effects of DNA vaccine against botulinum neurotoxin serotype A by targeting antigen to dendritic cells.

As dendritic cells (DCs) play a critical role in priming antigen-specific immune responses, the efficacy of DNA vaccines may be enhanced by targeting the encoded antigen proteins to DCs. In this study, we constructed a DC-targeted DNA vaccine encoding the Hc domain of botulinum neurotoxin serotype A (AHc) fused with scDEC, a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Intramuscular injections of mice with the DC-targeted DNA vaccine (pVAX1-scDEC-AHc) stimulated more DCs to mature than the non-targeted DNA vaccine (pVAX1-SAHc) in the splenocytes. The DC-targeted DNA vaccine could induce more DCs maturation at the site of inoculation. The DC-targeted DNA vaccine induced stronger AHc-specific humoral immune responses, lymphocyte proliferative responses and protective potency against BoNT/A in mice than did pVAX1-SAHc. Moreover, the DC-targeting DNA vaccine provided effective protection after only two inoculations. In summary, these results showed that the DC-targeted fusion DNA vaccine could generate strong immunity, indicating that maturation of DCs induced by pVAX1-scDEC-AHc may be helpful for priming and boosting immune responses. Thus, we propose that the strategy of targeting antigen to DCs in vivo via DEC205 can enhance effectively the potency of DNA vaccines against BoNTs or other pathogens in an animal model.

The highly pathogenic H5N1 influenza A virus down-regulated several cellular MicroRNAs which target viral genome.

Higher and prolonged viral replication is critical for the increased pathogenesis of the highly pathogenic avian influenza (HPAI) subtype of H5N1 influenza A virus (IAV) over the lowly pathogenic H1N1 IAV strain. Recent studies highlighted the considerable roles of cellular miRNAs in host defence against viral infection. In this report, using a 3'UTR reporter system, we identified several putative miRNA target sites buried in the H5N1 virus genome. We found two miRNAs, miR-584-5p and miR-1249, that matched with the PB2 binding sequence. Moreover, we showed that these miRNAs dramatically down-regulated PB2 expression, and inhibited replication of H5N1 and H1N1 IAVs in A549 cells. Intriguingly, these miRNAs expression was differently regulated in A549 cells infected with the H5N1 and H1N1 viruses. Furthermore, transfection of miR-1249 inhibitor enhanced the PB2 expression and promoted the replication of H5N1 and H1N1 IAVs. These results suggest that H5N1 virus may have evolved a mechanism to escape host-mediated inhibition of viral replication through down-regulation of cellular miRNAs, which target its viral genome.

A novel recombinant 6Aß15-THc-C chimeric vaccine (rCV02) mitigates Alzheimer's disease-like pathology, cognitive decline and synaptic loss in aged 3 × Tg-AD mice.

Alzheimer's disease (AD) is a neurodegenerative disorder that impairs memory and cognition. Targeting amyloid-ß (Aß) may be currently the most promising immunotherapeutic strategy for AD. In this study, a recombinant chimeric 6Aß15-THc-C immunogen was formulated with alum adjuvant as a novel Aß B-cell epitope candidate vaccine (rCV02) for AD. We examined its efficacy in preventing the cognitive deficit and synaptic impairment in 3 × Tg-AD mice. Using a toxin-derived carrier protein, the rCV02 vaccine elicited robust Aß-specific antibodies that markedly reduced AD-like pathology and improved behavioral performance in 3 × Tg-AD mice. Along with the behavioral improvement in aged 3 × Tg-AD mice, rCV02 significantly decreased calpain activation concurrent with reduced soluble Aß or oligomeric forms of Aß, probably by preventing dynamin 1 and PSD-95 degradation. Our data support the hypothesis that reducing Aß levels in rCV02-immunized AD mice increases the levels of presynaptic dynamin 1 and postsynaptic PSD-95 allowing functional recovery of cognition. In conclusion, this novel and highly immunogenic rCV02 shows promise as a new candidate prophylactic vaccine for AD and may be useful for generating rapid and strong Aß-specific antibodies in AD patients with pre-existing memory Th cells generated after immunization with conventional tetanus toxoid vaccine.

A Novel Aß B-Cell Epitope Vaccine (rCV01) for Alzheimer's Disease Improved Synaptic and Cognitive Functions in 3 × Tg-AD Mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive amyloid-ß accumulation, loss of cognitive abilities, and synaptic alterations. Given the remarkable recovery of cognition in AD models of targeting-Aß immunotherapy, we sought to determine the molecular correlate(s) associated with improvement. We evaluated the efficacy of a recombinant chimeric 6Aß15-T antigen formulated with alum adjuvant as a novel Aß B-cell epitope vaccine (rCV01) in 3 × Tg-AD mice. rCV01 elicited robust Th2-polarized Aß-specific antibodies without autoimmune T cell responses in 3 × Tg-AD mice. The long-lasting anti-Aß42 antibodies were associated with markedly reduced AD-like pathology, enhanced synaptic function, and improved cognitive performance in aged 3 × Tg-AD mice. This is the first report to provide one hypothesis for the improved outcomes following vaccination is a reduction in the levels of active calpain in rCV01-immunized AD mice, which is likely attributable to preventing dynamin 1 and PSD-95 degradation allowing functional recovery of cognition. rCV01 is a highly immunogenic recombinant chimeric 6Aß15-T vaccine that shows clear neuroprotective properties in preclinical mouse models of AD and is a candidate for an effective AD vaccine.

A fully human monoclonal antibody with novel binding epitope and excellent neutralizing activity to multiple human IFN-α subtypes: A candidate therapy for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic, heterogeneous autoimmune disease short of effective therapeutic agents. A multitude of studies of SLE in the last decade have accentuated a central role of the interferon alpha (IFN-α) pathway in SLE pathogenesis. We report here a candidate therapeutic neutralizing antibody, AIA22, with a different binding epitope and discrepant neutralizing profile from the anti-multiple IFN-α subtype antibodies currently in clinical trials. AIA22 specifically interacts with multiple IFN-α subtypes, binds to the type I IFN receptor 2 (IFNAR2) recognition region of IFN-α (considered a novel antigen epitope), and effectively neutralizes the activity of almost all of the IFN-α subtypes (with the exception of IFN-α7) both in vitro and in vivo. Concurrently, structural modeling and computational design yielded a mutational antibody of AIA22, AIAmut, which exhibited substantially improved neutralizing activity to multiple IFN-α subtypes.

Intracellular delivery of artificial transcription factors fused to the protein transduction domain of HIV-1 Tat.

Protein transduction domains (PTDs), such as the TAT peptide derived from HIV Tat protein, may transduce macromolecules into cells. In the present study, the TAT peptide-fused artificial transcription factors (ATFs) were generated by fusion of the N-terminal TAT peptide with SV40 promoter-targeted three-fingered C2H2 zinc finger proteins and the KRAB transcriptional repression domain. The fusion proteins were then expressed in an E .coli system and purified by Ni-NTA affinity chromatography. The purified fusion proteins were tested on mammalian cell lines CHO DG44 and L929. TAT-ATF-S, which contains the zinc fingers that bind to the SV40 promoter with high specificity, exhibited the desired transcriptional repression activity to the reported genes, indicating the successful cellular delivery and desired conformation of TAT-ATF-S. Our study has provided a new strategy for intracellular ATF delivery.

CCCTC-binding factor controls its own nuclear transport via regulating the expression of importin 13.

CCCTC-binding factor (CTCF), a multivalent zinc-finger protein, is involved in different aspects of regulation including promoter activation or repression, gene silencing, chromatin insulation, gene imprinting, X-chromosome inactivation, cell growth or differentiation and tumor genesis. However, the molecular mechanisms of CTCF nuclear import remains unclear. In this study, we showed that the expression of CTCF influenced the intracellular distribution of itself, which might go through transport receptor - import 13 (IPO13). We further confirmed that there is a CTCF target site in ipo13 -774∼-573 bp promoter region and CTCF regulates the expression of IPO13. Besides, GST pull-down and Co-IP experiments demonstrated that CTCF interacts with IPO13. Immunofluorescence staining showed that IPO13 influenced intracellular distribution of CTCF. In all, we conclude that CTCF regulates the expression of IPO13, which, in turn, mediates the nuclear import of CTCF.

Analysis of Soluble protein complexes in Shigella flexneri reveals the influence of temperature on the amount of lipopolysaccharide.

Shigella flexneri, which is closely related to Escherichia coli, is the most common cause of the endemic form of shigellosis. In this study, 53 homomultimeric protein complexes and nine heteromultimeric protein complexes from S. flexneri 2a strain 2457T were separated and identified. Among these, three potential homomultimeric protein complexes had not been previously described. One complex, thought to be composed of 12 PhoN1 subunits, is a periplasmic protein with an unknown physiological role encoded on the virulence plasmid of S. flexneri. The abundance of the protein complexes was compared following growth at 37 or 30°C, and the abundance of three protein complexes (PyrB-PyrI, GlmS, and MglB) related to the synthesis of lipopolysaccharides (LPS) appeared to be temperature-dependent. Many studies have shown that LPS is essential to the virulence of S. flexneri. Here, we report the influence of temperature on the amount of LPS.

Creation of a six-fingered artificial transcription factor that represses the hepatitis B virus HBx gene integrated into a human hepatocellular carcinoma cell line.

Chronic hepatitis B virus (HBV) infection is an independent risk factor for the development of hepatocellular carcinoma (HCC). The HBV HBx gene is frequently identified as an integrant in the chromosomal DNA of patients with HCC. HBx encodes the X protein (HBx), a putative viral oncoprotein that affects transcriptional regulation of several cellular genes. Therefore, HBx may be an ideal target to impede the progression of HBV infection-related HCC. In this study, integrated HBx was transcriptionally downregulated using an artificial transcription factor (ATF). Two three-fingered Cys2-His2 zinc finger (ZF) motifs that specifically recognized two 9-bp DNA sequences regulating HBx expression were identified from a phage-display library. The ZF domains were linked into a six-fingered protein that specified an 18-bp DNA target in the Enhancer I region upstream of HBx. This DNA-binding domain was fused with a Krüppel-associated box (KRAB) transcriptional repression domain to produce an ATF designed to downregulate HBx integrated into the Hep3B HCC cell line. The ATF significantly repressed HBx in a luciferase reporter assay. Stably expressing the ATF in Hep3B cells resulted in significant growth arrest, whereas stably expressing the ATF in an HCC cell line lacking integrated HBx (HepG2) had virtually no effect. The targeted downregulation of integrated HBx is a promising novel approach to inhibiting the progression of HBV infection-related HCC.

The extremely high level expression of human serum albumin in the milk of transgenic mice.

The recombinant production of human serum albumin has been challenging due to the low unit price and huge amount needed, for the commercial production of rhSA at an economically feasible level, It will be well worth the effort to exploit new method for the extremely high level expression of rhSA. To this end, here a hybrid gene locus strategy was employed, a 37 Kb mWAP-hSA hybrid gene locus was constructed and used as mammary gland specific expression vector, in which the 3 Kb genomic coding sequence in the 24 Kb mouse whey acidic protein (mWAP) gene locus was substituted by the 16 Kb genomic coding sequence of human serum albumin (hSA), exactly from the start codon to the end codon. Corresponding transgenic mice were generated and rhSA was secreted into the milk at an extremely high level of 11.9 g/L. Our transgenic mice carrying the mWAP-hSA hybrid gene locus represent a model system for the cost-effective production of human serum albumin.

The high-level expression of human tissue plasminogen activator in the milk of transgenic mice with hybrid gene locus strategy.

Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin (hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8 g/l in mice milk. Here to demonstrate the broad application of this strategy, another 38.4 kb mWAP-htPA hybrid gene locus was constructed, in which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3 g/l. Our strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic animals.

The interaction between the PARP10 protein and the NS1 protein of H5N1 AIV and its effect on virus replication.

BACKGROUND: During the process that AIV infect hosts, the NS1 protein can act on hosts, change corresponding signal pathways, promote the translation of virus proteins and result in virus replication. RESULTS: In our study, we found that PARP domain and Glu-rich region of PARP10 interacted with NS1, and the presence of NS1 could induce PARP10 migrate from cytoplasm to nucleus. NS1 high expression could reduce the endogenous PARP10 expression. Cell cycle analysis showed that with inhibited PARP10 expression, NS1 could induce cell arrest in G2-M stage, and the percentage of cells in G2-M stage rise from the previous 10%-45%, consistent with the cell proliferation result. Plague forming unit measurement showed that inhibited PARP10 expression could help virus replication. CONCLUSIONS: In a word, our results showed that NS1 acts on host cells and PARP10 plays a regulating role in virus replication.

[Expression, purification, and bio-activity analysis of fusion protein HBx-EGFP-TLM].

Hepatitis B virus X protein (HBx) has various functions and plays a crucial role in the development of hepatocellular carcinoma (HCC). However, due to different transfection efficiency levels and experimental approaches, it is difficult to correlate the exact functions of HBx to HBV-associated HCC. In this study, we constructed two prokaryotic expression vectors, pGEX-HBx-EGFP-TLM and pGEX-EGFP-TLM, which expressed HBx-EGFP-TLM and EGFP-TLM fusion proteins respectively. Both vectors contained a coding sequence of TLM transduction motif derived from the PreS2-domain of Hepatitis B Virus surface antigens. In addition, EGFP was expressed as a reporter reflecting the transduction efficiency of TLM. The fusion protein HBx-EGFP-TLM or EGFP-TLM purified from Escherichia coli BL21(DE3) by AKTA Purifier system was incubated with AML12 and SMMC-7721 cells. Both Western blotting and laser confocal results indicated that the translocation motif TLM could lead HBx-EGFP and EGFP into the cytoplasm. Dual-Luciferase Reporter Assay revealed that the activity of mEZH2 promoter could be up-regulated by the recombinant HBx. In conclusion, we expressed a cell-permeable HBx, which could provide a new method to study the functions of HBx.

The NS1 protein of influenza A virus interacts with heat shock protein Hsp90 in human alveolar basal epithelial cells: implication for virus-induced apoptosis.

BACKGROUND: Our previous study showed that the NS1 protein of highly pathogenic avian influenza A virus H5N1 induced caspase-dependent apoptosis in human alveolar basal epithelial cells (A549), supporting its function as a proapoptotic factor during viral infection, but the mechanism is still unknown. RESULTS: To characterize the mechanism of NS1-induced apoptosis, we used a two-hybrid system to isolate the potential NS1-interacting partners in A549 cells. We found that heat shock protein 90 (Hsp90) was able to interact with the NS1 proteins derived from both H5N1 and H3N2 viruses, which was verified by co-immunoprecitation assays. Significantly, the NS1 expression in the A549 cells dramatically weakened the interaction between Apaf-1 and Hsp90 but enhanced its interaction with cytochrome c (Cyt c), suggesting that the competitive binding of NS1 to Hsp90 might promote the Apaf-1 to associate with Cyt c and thus facilitate the activation of caspase 9 and caspase 3. CONCLUSIONS: The present results demonstrate that NS1 protein of Influenza A Virus interacts with heat hock protein Hsp90 and meidates the apoptosis induced by influenza A virus through the caspase cascade.

Hepatitis B virus X protein regulates the mEZH2 promoter via the E2F1-binding site in AML12 cells.

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma (HCC) tissues and associated with hepatocarcinogenesis. However, the exact mechanism of EZH2 up-regulation in HCC has not been determined. In this study, we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein (HBx) in regulating the expression of mEZH2. Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner. To further investigate the mechanism of mEZH2 overexpression, the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid. The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid, and deleting the -486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%. The -486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found. Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells. These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor, thereby providing new epigenetic evidence for the carcinogenic effect of HBx.

UBE2W interacts with FANCL and regulates the monoubiquitination of Fanconi anemia protein FANCD2.

Fanconi anemia (FA) is a rare cancer-predisposing genetic disease mostly caused by improper regulation of the monoubiquitination of Fanconi anemia complementation group D2 (FANCD2). Genetic studies have indicated that ubiquitin conjugating enzyme UBE2T and HHR6 could regulate FANCD2 monoubiquitination through distinct mechanisms. However, the exact regulation mechanisms of FANCD2 monoubiquitination in response to different DNA damages remain unclear. Here we report that UBE2W, a new ubiquitin conjugating enzyme, could regulate FANCD2 monoubiquitination by mechanisms different from UBE2T or HHR6. Indeed, UBE2W exhibits ubiquitin conjugating enzyme activity and catalyzes the monoubiquitination of PHD domain of Fanconi anemia complementation group L (FANCL) in vitro. UBE2W binds to FANCL, and the PHD domain is both necessary and sufficient for this interaction in mammalian cells. In addition, over-expression of UBE2W in cells promotes the monoubiquitination of FANCD2 and down-regulated UBE2W markedly reduces the UV irradiation-induced but not MMC-induced FANCD2 monoubiquitination. These results indicate that UBE2W regulates FANCD2 monoubiquitination by mechanisms different from UBE2T and HRR6. It may provide an additional regulatory step in the activation of the FA pathway.