Dorothee von Laer

Oncoretrovirus and Lentivirus Vectors Pseudotyped with Lymphocytic Choriomeningitis Virus Glycoprotein: Generation, Concentration, and Broad Host Range

ABSTRACT Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.

Endoproteolytic Processing of the Lymphocytic Choriomeningitis Virus Glycoprotein by the Subtilase SKI-1/S1P

ABSTRACT The envelope glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) is posttranslationally cleaved into two subunits. We show here that this endoproteolytic processing is not required for transport to the cell surface but is essential for LCMV GP to mediate infectivity of pseudotyped retroviral vectors. By systematic mutational analysis of the LCMV GP cleavage site, we determined that the consensus motif R-(R/K/H)-L-(A/L/S/T/F)265 is essential for the endoproteolytic processing. In agreement with the identified consensus motif, we show that the cellular subtilase SKI-1/S1P cleaves LCMV GP.

Inhibition of Human Immunodeficiency Virus Type 1 Entry in Cells Expressing gp41-Derived Peptides

ABSTRACT As the limitations of antiretroviral drug therapy, such as toxicity and resistance, become evident, interest in alternative therapeutic approaches for human immunodeficiency virus (HIV) infection is growing. We developed the first gene therapeutic strategy targeting entry of a broad range of HIV type 1 (HIV-1) variants. Infection was inhibited at the level of membrane fusion by retroviral expression of a membrane-anchored peptide derived from the second heptad repeat of the HIV-1 gp41 transmembrane glycoprotein. To achieve maximal expression and antiviral activity, the peptide itself, the scaffold for presentation of the peptide on the cell surface, and the retroviral vector backbone were optimized. This optimized construct effectively inhibited virus replication in cell lines and primary blood lymphocytes. The membrane-anchored C-peptide was also shown to bind to free gp41 N peptides, suggesting that membrane-anchored antiviral C peptides have a mode of action similar to that of free gp41 C peptides. Preclinical toxicity and efficacy studies of this antiviral vector have been completed, and clinical trials are in preparation.

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.

CD271 antigen defines a subset of multipotent stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties

Background In vitro proliferative and differentiation potential of mesenchymal stromal cells generated from CD271+ bone marrow mononuclear cells (CD271-mesenchymal stromal cells) has been demonstrated in several earlier and recent reports. In the present study we focused, in addition to proliferative and differentiation potential, on in vitro and in vivo immunosuppressive and lymphohematopoietic engraftment-promoting potential of these mesenchymal stromal cells compared to bone marrow-derived mesenchymal stromal cells generated by plastic adherence (plastic adherence-mesenchymal stromal cells). Design and Methods We set up a series of experimental protocols in order to determine the phenotype of CD271-mesenchymal stromal cells, and their clonogenic, proliferative, differentiation and immunosuppressive potential. The potential of CD271-mesenchymal stromal cells to improve the engraftment of CD133+ hematopoietic stem cells at co-transplantation was evaluated in immunodeficient NOD/SCID-IL2Rγnull mice. Results In vitro studies demonstrated that CD271-mesenchymal stromal cells differentiate along adipogenic, osteogenic and chondrogenic lineages (trilineage potential), produce significantly higher levels of cytokines than plastic adherence-mesenchymal stromal cells, and significantly inhibit the proliferation of allogeneic T-lymphocytes in mixed lymphocyte reaction assays. Elevated levels of prostaglandin E2, but not nitric monoxide, mediated the majority of this immunosuppressive effect. In vivo studies showed that CD271-mesenchymal stromal cells promoted significantly greater lymphoid engraftment than did plastic adherence-mesenchymal stromal cells when co-transplanted with CD133+ hematopoietic stem cells at a ratio of 8:1 in immunodeficient NOD/SCID-IL2Rγnull mice. They induced a 10.4-fold increase in the number of T cells, a 2.5-fold increase in the number of NK cells, and a 3.6-fold increase in the number of B cells, indicating a major qualitative difference between these two mesenchymal stromal cell populations. Conclusions Our results indicate that CD271 antigen provides a versatile marker for prospective isolation and expansion of multipotent mesenchymal stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. The co-transplantation of such cells together with hematopoietic stem cells in patients with hematologic malignancies may prove valuable in the prevention of impaired/delayed T-cell recovery and graft-versus-host disease.

Membrane-Anchored Peptide Inhibits Human Immunodeficiency Virus Entry

ABSTRACT Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.

Positive selection of mC46-expressing CD4 + T cells and maintenance of virus specific immunity in a primate AIDS model

Despite continued progress in the development of novel antiretroviral therapies, it has become increasingly evident that drug-based treatments will not lead to a functional or sterilizing cure for HIV(+) patients. In 2009, an HIV(+) patient was effectively cured of HIV following allogeneic transplantation of hematopoietic stem cells (HSCs) from a CCR5(-/-) donor. The utility of this approach, however, is severely limited because of the difficulty in finding matched donors. Hence, we studied the potential of HIV-resistant stem cells in the autologous setting in a nonhuman primate AIDS model and incorporated a fusion inhibitor (mC46) as the means for developing infection-resistant cells. Pigtail macaques underwent identical transplants and Simian-Human Immunodeficiency Virus (SHIV) challenge procedures with the only variation between control and mC46 macaques being the inclusion of a fusion-inhibitor expression cassette. Following SHIV challenge, mC46 macaques, but not control macaques, showed a positive selection of gene-modified CD4(+) T cells in peripheral blood, gastrointestinal tract, and lymph nodes, accounting for >90% of the total CD4(+) T-cell population. mC46 macaques also maintained high frequencies of SHIV-specific, gene-modified CD4(+) T cells, an increase in nonmodified CD4(+) T cells, enhanced cytotoxic T lymphocyte function, and antibody responses. These data suggest that HSC protection may be a potential alternative to conventional antiretroviral therapy in patients with HIV/AIDS.

Protection of Stem Cell-Derived Lymphocytes in a Primate AIDS Gene Therapy Model after In Vivo Selection

Background There is currently no effective AIDS vaccine, emphasizing the importance of developing alternative therapies. Recently, a patient was successfully transplanted with allogeneic, naturally resistant CCR5-negative (CCR5Δ32) cells, setting the stage for transplantation of naturally resistant, or genetically modified stem cells as a viable therapy for AIDS. Hematopoietic stem cell (HSC) gene therapy using vectors that express various anti-HIV transgenes has also been attempted in clinical trials, but inefficient gene transfer in these studies has severely limited the potential of this approach. Here we evaluated HSC gene transfer of an anti-HIV vector in the pigtailed macaque (Macaca nemestrina) model, which closely models human transplantation. Methods and Findings We used lentiviral vectors that inhibited both HIV-1 and simian immunodeficiency virus (SIV)/HIV-1 (SHIV) chimera virus infection, and also expressed a P140K mutant methylguanine methyltransferase (MGMT) transgene to select gene-modified cells by adding chemotherapy drugs. Following transplantation and MGMT-mediated selection we demonstrated transgene expression in over 7% of stem-cell derived lymphocytes. The high marking levels allowed us to demonstrate protection from SHIV in lymphocytes derived from gene-modified macaque long-term repopulating cells that expressed an HIV-1 fusion inhibitor. We observed a statistically significant 4-fold increase of gene-modified cells after challenge of lymphocytes from one macaque that received stem cells transduced with an anti-HIV vector (p<0.02, Students t-test), but not in lymphocytes from a macaque that received a control vector. We also established a competitive repopulation assay in a second macaque for preclinical testing of promising anti-HIV vectors. The vectors we used were HIV-based and thus efficiently transduce human cells, and the transgenes we used target HIV-1 genes that are also in SHIV, so our findings can be rapidly translated to the clinic. Conclusions Here we demonstrate the ability to select protected HSC-derived lymphocytes in vivo in a clinically relevant nonhuman primate model of HIV/SHIV infection. This approach can now be evaluated in human clinical trials in AIDS lymphoma patients. In this patient setting, chemotherapy would not only kill malignant cells, but would also increase the number of MGMTP140K-expressing HIV-resistant cells. This approach should allow for high levels of HIV-protected cells in AIDS patients to evaluate AIDS gene therapy.

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

ABSTRACT 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.

Determinants of Human Immunodeficiency Virus Type 1 Resistance to Membrane-Anchored gp41-Derived Peptides

ABSTRACT The expression of a membrane-anchored gp41-derived peptide (M87) has been shown to confer protection from infection through human immunodeficiency virus type 1 (HIV-1) (Hildinger et al., J. Virol. 75:3038-3042, 2001). In an effort to characterize the mechanism of action of this membrane-anchored peptide in comparison to the soluble peptide T-20, we selected resistant variants of HIV-1NL4-3 and HIV-1BaL by serial virus passage using PM1 cells stably expressing peptide M87. Sequence analysis of the resistant isolates showed different patterns of selected point mutations in heptad repeat regions 1 and 2 (HR1 and HR2, respectively) for the two viruses analyzed. For HIV-1NL4-3 a single amino acid change at position 33 in HR1 (L33S) was selected, whereas for HIV-1BaL the majority of the sequences obtained showed two amino acid changes, one in HR1 and one in HR2 (I48V/N126K). In both selections the most important contiguous 3-amino-acid sequence, GIV, within HR1, associated with resistance to soluble T-20, was not changed. Site-directed mutagenesis studies confirmed the importance of the characterized point mutations to confer resistance to M87 as well as to soluble T-20 and T-649. Replication capacity and dual-color competition assays revealed that the double mutation I48V/N126K in HIV-1BaL results in a strong reduction of viral fitness, whereas the L33S mutation in HIV-1NL4-3 did enhance viral fitness compared to the respective parental viruses. However, the selected point mutations did not confer resistance to the more recently described optimized membrane-anchored fusion inhibitor M87o (Egelhofer et al., J. Virol. 78:568-575, 2004), strengthening the importance of this novel antiviral concept for gene therapy approaches.