Search

The ability to selectively target specific cell types is highly desirable for the in vivo application of viral vectors for the treatment of multiple diseases including cancer. Lentiviral vectors (LV) have the advantage of being easily pseudotyped allowing their tropism to be altered. LV can also be engineered to deliver therapeutic or cytotoxic genes directly to cancer cells. IL-13 receptor alpha 2 (IL-13Rα2) is overexpressed in many different tumor types, including glioma, sarcoma, kidney, breast and ovarian cancer, making it an attractive target for tumor therapy. We have previously shown that firefly luciferase-expressing LV pseudotyped with a truncated fusion (F) protein derived from measles virus (MV) and a tail-truncated, receptor-blind MV hemagglutinin (H) protein bearing IL-13 at the C-terminus were capable of transducing IL-13Rα2-positive U251 glioma cells when administered intratumorally. In addition, we have shown the ability of LV to be pseudotyped using the F and H proteins from the Tupaia paramyxovirus (TPMV). TPMV does not infect human cells, potentially making it a safer choice for pseudotyping LV for in vivo delivery. Similar to the MV H protein, the TPMV H protein can also display IL-13 and bind specifically to IL-13Rα2-positive tumor cells. We plan to confirm and extend these findings by examining the targeting of LV-MV-IL-13 and LV-TPMV-IL-13 in metastatic orthotopic models of breast cancer by intratumoral and intravenous administration. In addition, LV expressing HSV1 thymidine kinase (TK) were constructed and are being evaluated for antitumor efficacy.

The ability to selectively and efficiently target transgene delivery to specific cell types in vitro and in vivo remains one of the formidable challenges in gene therapy. Lentiviral vectors have several advantages that make them attractive as gene delivery vehicles and their tropism can be altered through pseudotyping, allowing transgene delivery to specific populations of cells. The human interleukin-13 receptor α2 (IL-13Rα2) is uniquely overexpressed in many different human tumors, making it an attractive target for cancer therapy. In this study, we examined whether IL-13Rα2-positive tumor cells can be specifically targeted with lentiviral vector pseudotypes containing a truncated fusion (F) protein derived from measles virus (MV) and a tail-truncated and receptor-blind MV hemagglutinin (H) protein bearing IL-13 at the C terminus. The retargeted lentiviral vector efficiently transduced cells that express high levels of IL-13Rα2, but not cells expressing low levels of IL-13Rα2 in vitro. In vivo, it specifically targeted IL-13Rα2-positive glioma cell xenografts in immunodeficient mice in the context of subcutaneous and intracranial glioma models. Similar lentiviral vectors may be developed for targeting other tumors expressing specific cell surface receptors.

Background The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. Methods To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. Results Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. Conclusion Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.

The availability of rapid and quantitative titration assays for retroviral vectors is important, especially in the context of clinical applications. In this report, we describe a novel assay to titrate lentiviral and gamma retroviral vectors. This rapid assay is based on protein fragment complementation involving the N-terminal (Bla1) and the C-terminal (Bla2) fragments of TEM-1 β-lactamase (BLAK). The Bla1 protein fragment is incorporated in the vector's envelope during vector production. Bla1-bearing vectors are titrated on Bla2-expressing cells. Upon transduction, Bla1 and Bla2 heterodimerize and restore BLAK's enzymatic function. The enzymatic activity of BLAK is quantified by flow cytometry using the green fluorescent CCF2/AM substrate, which is converted into a blue fluorescent product. The enzymatic conversion of the CCF2/AM substrate was found to be directly related to vector entry, as a neutralizing antibody completely blocked the conversion. The titers obtained using this rapid assay correlated well with the titers measured by functional transduction assays. The whole assay can be finished within 8 h. Thus, it is considerably less time consuming compared with other transduction-based titration assays for lentiviral and gamma retroviral vectors.

Ebolaviruses are the etiologic agents of severe viral hemorrhagic fevers in primates, including humans, and could be misused for the development of biological weapons. The ability to rapidly detect and differentiate these viruses is therefore crucial. Antibodies that can detect reliably the ebolavirus surface envelope glycoprotein GP1,2 or a truncated variant that is secreted from infected cells (sGP) are required for advanced development of diagnostic assays such as sandwich ELISAs or Western blots (WB). We used a GP1,2 peptide conserved among Bundibugyo, Ebola, Reston, Sudan, and Tai Forest viruses and a mucin-like domain-deleted Sudan virus GP1,2 (SudanGPΔMuc) to immunize mice or rabbits, and developed a panel of antibodies that either cross-react or are virus-specific. These antibodies detected full-length GP1,2 and sGP in different assays such as ELISA, FACS, or WB. In addition, some of the antibodies were shown to have potential clinical relevance, as they detected ebolavirus-infected cells by immunofluorescence assay and gave a specific increase in signal by sandwich ELISA against sera from mouse-adapted Ebola virus-infected mice over uninfected mouse sera. Rabbit anti-SudanGPΔMuc polyclonal antibody neutralized gammaretroviral particles pseudotyped with Sudan virus GP1,2, but not particles pseudotyped with other ebolavirus GP1,2. Together, our results suggest that this panel of antibodies may prove useful for both in vitro analyses of ebolavirus GP1,2, as well as analysis of clinically relevant samples.

We performed a prospective study to evaluate the intraoperative value of indocyanine green (ICG) video angiography in anterior circulation aneurysms. From January 2007 to April 2008, 42 patients with anterior circulation aneurysms who were to undergo aneurysm clipping were enrolled in the study. Intraoperative ICG video angiography was performed using a fluorescence microscope. After the operation, three-dimensional CT angiography (CTA), digital substraction angiography (DSA) and magnetic resonance angiography (MRA) were used to evaluate the use of intraoperative ICG video angiography. Of the 42 patients, on ICG video angiography after initial clip placement, neck remnants of the aneurysm were found in two patients, inadvertent clipping of branching vessels in one patient, and inadvertent clipping of perforating vessels in two patients. ICG video angiography after adjustment of the clip position showed a perfect residual elimination with no abnormal findings. Post-operative DSA, CTA and MRA results corresponded to the intraoperative ICG video angiography findings. Therefore, ICG video angiography is an important tool to monitor residual aneurysm, parent artery stenosis or perforating artery occlusion during intracranial aneurysm clipping.

Envelope glycoproteins (Envs) of retroviruses form trimers that mediate fusion between viral and cellular membranes and are the targets for neutralizing antibodies. Understanding in detail how Env trimers mediate membrane fusion, and how antibodies interfere with this process, is a fundamental problem in biology with practical implications for the development of antiviral drugs and vaccines. We investigated the stoichiometry of Env-mediated fusion and its inhibition by antibody by inserting an epitope from human immunodeficiency virus for a neutralizing antibody (2F5) into the surface (SU) or transmembrane (TM) protein of murine leukemia virus Env, along with point mutations that abrogate SU and TM function but complement one another. We transfected various combinations of these Env genes and investigated Env-mediated cell fusion and its inhibition by 2F5 antibody. Our results showed that heterotrimers with one functional SU molecule were fusion competent in complementation experiments and that one antibody molecule was sufficient to inactivate the fusion function of a trimer when its epitope was in functional SU or TM. 2F5 antibody could also neutralize trimers with the 2F5 epitope in nonfunctional SU or TM, but less efficiently.

Early stages of infection by the mouse polyomavirus have been studied using HeLa cells stably expressing small interfering RNA to protein disulfide isomerase (PDI). Infectibility measured by nuclear T antigen expression was reduced commensurately with the degree of PDI downregulation. Infectibility was restored by transfection with a plasmid expressing PDI but not with a control expressing catalytically inactive enzyme. Deconvolution microscopy using fluorescently labeled virus and cellular markers showed that virus reaches the endoplasmic reticulum (ER) normally in cells with reduced PDI but subsequently fails to exit the ER. Simian virus 40 infection was not inhibited in PDI-downregulated cells. The results are discussed in terms of structural differences between the two viruses and current knowledge of virus disassembly in the ER.

Background The N-heptad repeat region of the HIV-1 Transmembrane Envelope protein is a trimerization domain that forms part of a "six helix bundle" crucial to Envelope-mediated membrane fusion. N-heptad repeat peptides have been used as extracellular reagents to inhibit virus fusion. Results When expressed intracellularly with wild-type HIV-1 Envelope protein, the N-heptad repeat domain efficiently hetero-oligomerized with Envelope and trapped it in the endoplasmic reticulum or early Golgi, as indicated by lack of transport to the cell surface, absent proteolytic processing, and aberrant glycosylation. Conclusion Post-translational processing of HIV Envelope is very sensitive to an agent that binds to the N-heptad repeat during synthesis, suggesting that it might be possible to modify drugs that bind to this region to have transport-blocking properties.

The discovery that cell membranes are a mosaic of at least two domains (raft and nonraft) differing in lipid composition has led to an explosion of studies investigating the domain localization of a large number of individual proteins, including proteins involved in the cellular entry and egress of viruses and other pathogens. The driving force behind these studies is the assumption that the partitioning between domains has functional consequences, and will shed light on fundamental processes in cell biology such as signaling, trafficking, macromolecular assembly, regulation of protein interactions, and membrane fission and fusion.