Pseudotyping vesicular stomatitis virus with lymphocytic choriomeningitis virus glycoproteins enhances infectivity for glioma cells and minimizes neurotropism.

Vesicular stomatitis virus (VSV)-based oncolytic virotherapy has the potential to significantly improve the prognosis of aggressive malignancies such as brain cancer. However, VSV's inherent neurotoxicity has hindered clinical development so far. Given that this neurotropism is attributed to the glycoprotein VSV-G, VSV was pseudotyped with the nonneurotropic envelope glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP→VSV-GP). Compared to VSV, VSV-GP showed enhanced infectivity for brain cancer cells in vitro while sparing primary human and rat neurons in vitro and in vivo, respectively. In conclusion, VSV-GP has a much wider therapeutic window than VSV and is thus more suitable for clinical applications, especially in the brain.

Decreased tumor surveillance after adoptive T-cell therapy.

The effect of cancer immunotherapy on the endogenous immune response against tumors is largely unknown. Therefore, we studied immune responses against murine tumors expressing the glycoprotein (GP) and/or nucleoprotein of lymphocytic choriomeningitis virus (LCMV) with or without adoptive T-cell therapy. In nontreated animals, CTLs specific for different epitopes as well as LCMV-GP-specific antibodies contributed to tumor surveillance. Adoptive immunotherapy with monoclonal CTLs specific for LCMV-gp33 impaired the endogenous tumor-specific antibody and CTL response by targeting antigen cross-presenting cells. As a consequence and in contrast to expectations, immunotherapy enhanced tumor growth. Thus, for certain immunogenic tumors, a reduction of tumor-specific B- and T-cell responses and enhanced tumor growth may be an unwanted consequence of adoptive immunotherapy.

Normal brain cells contribute to the bystander effect in suicide gene therapy of malignant glioma.

PURPOSE: Lentiviral vectors pseudotyped with glycoproteins of the lymphocytic choriomeningitis virus (LCMV-GP) are promising candidates for gene therapy of malignant glioma, as they specifically and efficiently transduce glioma cells in vitro and in vivo. Here, we evaluated the therapeutic efficacy of LCMV-GP and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors. EXPERIMENTAL DESIGN: Therapeutic efficacy was tested for unmodified (9L) and DsRed-modified (9LDsRed) gliomas using the suicide gene thymidine kinase of the herpes simplex virus type 1 (HSV-1-tk). Positron emission tomography (PET) and magnetic resonance imaging were done to analyze transduction of tumors and monitor therapeutic outcome. RESULTS: LCMV-GP pseudotypes mediated a successful eradication of 9LDsRed tumors with 100% of long-term survivors. Before initiation of ganciclovir treatment, a strong HSV-1-tk expression within the tumor was detected by noninvasive PET using the tracer 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butyl]guanine. Therapeutic outcome was successfully monitored by magnetic resonance imaging and PET imaging and correlated with the histopathologic data. In the 9L model, LCMV-GP and VSV-G pseudotyped lentiviral vectors displayed similar therapeutic efficacy. Further studies revealed that normal brain cells transduced with VSV-G pseudotypes were not eliminated by ganciclovir treatment and contributed significantly to the bystander killing of tumor cells. CONCLUSIONS: Suicide gene transfer using pseudotyped lentiviral vectors was very effective in the treatment of rat glioma and therefore is an attractive therapeutic strategy also in human glioblastoma especially in conjunction with an imaging-guided approach. In addition, high selectivity of gene transfer to tumor cells may not always be desirable for therapeutic genes that exert a clear bystander effect.

Bystander killing of malignant glioma by bone marrow-derived tumor-infiltrating progenitor cells expressing a suicide gene.

Adult stem cells are promising cellular vehicles for therapy of malignant gliomas as they have the ability to migrate into these tumors and even track infiltrating tumor cells. However, their clinical use is limited by a low passaging capacity that impedes large-scale production. In the present study, a bone marrow-derived, highly proliferative subpopulation of mesenchymal stem cells (MSCs)-here termed bone marrow-derived tumor-infiltrating cells (BM-TICs)-was genetically modified for the treatment of malignant glioma. Upon injection into the tumor or the vicinity of the tumor, BM-TICs infiltrated solid parts as well as the border of rat 9L glioma. After intra-tumoral injection, BM-TICs expressing the thymidine kinase of herpes simplex virus (HSV-tk) and enhanced green fluorescent protein (BM-TIC-tk-GFP) were detected by non-invasive positron emission tomography (PET) using the tracer 9-[4-[(18)F]fluoro-3-hydroxymethyl)butyl]guanine ([(18)F]FHBG). A therapeutic effect was demonstrated in vitro and in vivo by BM-TICs expressing HSV-tk through bystander-mediated glioma cell killing. Therapeutic efficacy was monitored by PET as well as by magnetic resonance imaging (MRI) and strongly correlated with histological analysis. In conclusion, BM-TICs expressing a suicide gene were highly effective in the treatment of malignant glioma in a rat model and therefore hold great potential for the therapy of malignant brain tumors in humans.

Transfer of Autologous Gene-modified T Cells in HIV-infected Patients with Advanced Immunodeficiency and Drug-resistant Virus.

Drug toxicity and viral resistance limit the long-term efficacy of antiviral drug treatment for human immunodeficiency virus (HIV) infection. Thus, alternative therapies need to be explored. We tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an HIV entry-inhibitory peptide (maC46). Gene-modified autologous T cells were infused into ten HIV-infected patients with advanced disease and multidrug-resistant virus during anti-retroviral combination therapy. T-cell infusions were tolerated well, with no severe side effects. A significant increase of CD4 counts was observed after infusion. At the end of the 1-year follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified cells could be detected in peripheral blood, lymph nodes, and bone marrow throughout the 1-year follow-up, and marking levels correlated with the cell dose. No significant changes of viral load were observed during the first 4 months. Four of the seven patients who changed their antiviral drug regimen thereafter responded with a significant decline in plasma viral load. In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene marking, and may improve immune competence in HIV-infected patients with advanced disease and multidrug-resistant virus.

Selective transduction of malignant glioma by lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus glycoproteins.

Malignant gliomas are the most frequent primary brain tumors and have a dismal prognosis due to their infiltrative growth. Gene therapy using viral vectors represents an attractive alternative to conventional cancer therapies. In a previous study, we established lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus (LCMV) glycoproteins (GPs) and demonstrated transduction of human malignant glioma cells in culture. In the current approach, we compared the transduction efficacy of LCMV-GP- and vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral vectors for malignant glioma cells and normal brain cells in vitro and in vivo. LCMV-GP pseudotypes transduced almost exclusively astrocytes, whereas VSV-G pseudotypes infected neurons as well as astrocytes. LCMV-GP pseudotypes showed an efficient transduction of solid glioma parts and specific transduction of infiltrating tumor cells. In contrast, VSV-G-pseudotyped lentiviral vectors transduced only a few tumor cells in solid tumor parts and infected mostly normal brain cells in infiltrating tumor areas. In conclusion, lentiviral vectors pseudotyped with LCMV glycoproteins represent an attractive option for gene therapy of malignant glioma.

Remission of invasive, cancer stem-like glioblastoma xenografts using lentiviral vector-mediated suicide gene therapy.

BACKGROUND: Glioblastoma is the most frequent and most malignant primary brain tumor with a poor prognosis. The translation of therapeutic strategies for glioblastoma from the experimental phase into the clinic has been limited by insufficient animal models, which lack important features of human tumors. Lentiviral gene therapy is an attractive therapeutic option for human glioblastoma, which we validated in a clinically relevant animal model. METHODOLOGY/PRINCIPAL FINDINGS: We used a rodent xenograft model that recapitulates the invasive and angiogenic features of human glioblastoma to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors. Both, lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors very efficiently transduced human glioblastoma cells in vitro and in vivo. In contrast, pseudotyped gammaretroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer in vitro and in vivo. Both pseudotyped lentiviral vectors transduced cancer stem-like cells characterized by their CD133-, nestin- and SOX2-expression, the ability to form spheroids in neural stem cell medium and to express astrocytic and neuronal differentiation markers under serum conditions. In a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (HSV-1-tk) fused to eGFP, both lentiviral vectors mediated a complete remission of solid tumors as seen on MRI resulting in a highly significant survival benefit (p<0.001) compared to control groups. In all recurrent tumors, surviving eGFP-positive tumor cells were found, advocating prodrug application for several cycles to even enhance and prolong the therapeutic effect. CONCLUSIONS/SIGNIFICANCE: In conclusion, lentiviral pseudotyped vectors are promising candidates for gene therapy of glioma in patients. The inefficient gene delivery by gammaretroviral vectors is in line with the results obtained in clinical therapy for GBM and thus confirms the high reproducibility of the invasive glioma animal model for translational research.

Measurements of HCV neutralizing antibodies and of HCV-specific CD4+ and CD8+ cells using hepatitis C virus pseudo-particles (HCVpp).

BACKGROUND: For the medical management, it would be of great relevance to get a diagnostic marker predicting the outcome of infection. OBJECTIVES: For this purpose, the envelope antigens of the individual HCV strain in a patient was tested for their capacity to induce neutralizing antibodies and cytotoxic T lymphocytes. STUDY DESIGN: A system for the measurement of neutralizing antibodies as well as for the stimulation of a HCV-specific T-cell response using pseudo-typed HCV particles (HCVpp) was established. A report on results of a pilot study conducted with blood specimens of 19 chronically infected patients is also presented. RESULTS: Neutralization of HCVpp could be measured in nearly all HCV sero-positive patient samples. Nevertheless, in more than half of the patient samples (11/19), no HCV-specific CD4+ response was detectable. In addition, HCV-specific CD8+ response was measurable in most of the patients when HCVpp were used for T-cell stimulation. Although the same antigens (HCVpp) were used, there was no relevant correlation between neutralization titers and T-cell response. CONCLUSION: Our study shows that HCVpp are appropriate antigens for specific stimulation of lymphocytes as well as for the investigation of antibody neutralization activity.

Protein scaffold and expression level determine antiviral activity of membrane-anchored antiviral peptides.

Cell membrane-anchored (ma) antiviral peptides derived from the C-terminal heptad repeat of the HIV-1 transmembrane glycoprotein gp41 (C-peptides) and expressed from retroviral vectors were shown to efficiently inhibit HIV-1 entry into target cells. Here, we analyzed the influence of the vector backbone, the scaffold modules that anchor the peptide to the membrane and the length of the C-peptide on expression level and antiviral activity. In general, antiviral activity was determined primarily by the density of the C-peptide on the cell surface. By influencing expression levels, the scaffold elements indirectly also determined antiviral activity. Additional direct effects of the scaffold on antiviral activity were minor. At comparable expression levels, the elongated C-peptide (maC46) was found to be more potent than the shorter maC36. On the basis of these findings, a dose-response assay was established that quantifies antiviral activity relative to the expression level of the antiviral gene product. Taken together, these data demonstrate the importance of analyzing the efficacy of therapeutic genes relative to the dose of the gene product.

A retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells.

BACKGROUND: Early clinical trials for gene therapy of human gliomas with retroviral packaging cells (PC) have been hampered by low transduction efficacy and lack of dissemination of PC within the tumor. In the current approach, these issues have been addressed by creating a stable packaging cell line for retroviral vectors pseudotyped with glycoproteins of lymphocytic choriomeningitis virus (LCMV) based on tumor-infiltrating progenitor cells. METHODS: Tumor-infiltrating progenitor cells, which had been isolated from adult rat bone marrow (BM-TIC), were modified to stably express Gag-Pol proteins of moloney murine leukemia virus (Mo-MLV) and glycoproteins of LCMV. Packaging of a retroviral vector was measured by titration experiments on human fibroblast cells as well as on mouse and human glioma cell lines. Additionally, gene transfer was tested in a rat glioma model in vivo. RESULTS: The BM-TIC-derived packaging cell line (BM-TIPC) produced retroviral vectors with titers between 2-8 x 10(3) transducing units (TU)/ml. Extended culturing of BM-TIPC over several weeks and freezing/thawing of cells did not affect vector titers. No replication-competent retrovirus was released from BM-TIPC. In a rat glioma model, BM-TIPC infiltrated the tumors extensively and with high specificity. Moreover, BM-TIPC mediated transduction of glioma cells in vivo. CONCLUSION: This proof-of-principle study shows that primary adult progenitor cells with tumor-infiltrating capacity can be genetically modified to stably produce retroviral LCMV pseudotype vectors. These BM-TIPC may be a useful tool to enhance specificity and efficacy of gene transfer to gliomas in patients.

Signal peptide requirements for lymphocytic choriomeningitis virus glycoprotein C maturation and virus infectivity.

Insertion of the lymphocytic choriomeningitis virus (LCMV) precursor glycoprotein C (GP-C) into the membrane of the endoplasmic reticulum is mediated by an unusual signal peptide (SP(GP-C)). It is comprised of 58 amino acid residues and contains an extended hydrophilic N-terminal region, two hydrophobic regions, and a short C-terminal region. After cleavage by signal peptidase, SP(GP-C) accumulates in cells and virus particles. In the present study, we identified the LCMV SP(GP-C) as being an essential component of the GP complex and show that the different regions of SP(GP-C) are required for distinct steps in GP maturation and virus infectivity. More specifically, we show that one hydrophobic region of SP(GP-C) is sufficient for the membrane insertion of GP-C, while both hydrophobic regions are required for the processing and cell surface expression of the GPs. The N-terminal region of SP(GP-C), on the other hand, is essential for pseudoviral infection of target cells. Furthermore, we show that unmyristoylated SP(GP-C) exposes its N-terminal region to the exoplasmic side. This SP(GP-C) can promote GP-C maturation but is defective in pseudoviral infection. Myristoylation and topology of SP(GP-C) in the membrane may thus hold the key to an understanding of the role of SP(GP-C) in GP-C complex maturation and LCMV infectivity.

Transfer of autologous gene-modified T cells in HIV-infected patients with advanced immunodeficiency and drug-resistant virus.

Drug toxicity and viral resistance limit the long-term efficacy of antiviral drug treatment for human immunodeficiency virus (HIV) infection. Thus, alternative therapies need to be explored. We tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an HIV entry-inhibitory peptide (maC46). Gene-modified autologous T cells were infused into ten HIV-infected patients with advanced disease and multidrug-resistant virus during anti-retroviral combination therapy. T-cell infusions were tolerated well, with no severe side effects. A significant increase of CD4 counts was observed after infusion. At the end of the 1-year follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified cells could be detected in peripheral blood, lymph nodes, and bone marrow throughout the 1-year follow-up, and marking levels correlated with the cell dose. No significant changes of viral load were observed during the first 4 months. Four of the seven patients who changed their antiviral drug regimen thereafter responded with a significant decline in plasma viral load. In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene marking, and may improve immune competence in HIV-infected patients with advanced disease and multidrug-resistant virus.

Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus S protein.

The worldwide outbreak of severe acute respiratory syndrome (SARS) was shown to be associated with a novel coronavirus (CoV) now called SARS CoV. We report here the generation of SARS CoV S protein-pseudotyped murine leukemia virus (MLV) vector particles. The wild-type S protein pseudotyped MLV vectors, although at a low efficiency. Partial deletion of the cytoplasmic tail of S dramatically increased infectivity of pseudotypes, with titers only two- to threefold lower than those of pseudotypes generated in parallel with the vesicular stomatitis virus G protein. S-pseudotyped MLV particles were used to analyze viral tropism. MLV(SARS) pseudotypes and wild-type SARS CoV displayed similar cell types and tissue and host restrictions, indicating that the expression of a functional receptor is the major restraint in permissiveness to SARS CoV infection. Efficient gene transfer could be detected in Vero and CaCo2 cells, whereas the level of gene marking of 293T, HeLa, and HepG2 cells was only slightly above background levels. A cat cell line and a dog cell line were not susceptible. Interestingly, PK-15, a porcine kidney cell line, and primary porcine kidney cells were also highly permissive for SARS S pseudotypes and wild-type SARS CoV. This finding suggests that swine may be susceptible to SARS infection and may be a source for infection of humans. Taken together, these results indicate that MLV(SARS) pseudotypes are highly valuable for functional studies of viral tropism and entry and, in addition, can be a powerful tool for the development of therapeutic entry inhibitors without posing a biohazard to human beings.