Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system

High-titer, HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (< 1%), reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier, we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo, 3 rhesus macaques underwent transplantation with transduced, autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid, and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes, respectively, were observed 4 to 6 months after transplantation, consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models.     

Transfer of the HIV-1 cyclophilin-binding site to simian immunodeficiency virus from Macaca mulatta can confer both cyclosporin sensitivity and cyclosporin dependence

HIV-1 specifically incorporates the peptidyl prolyl isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). HIV-1 replication is inhibited by CsA as well as by nonimmunosuppressive CsA analogues that bind to CyPA and interfere with its virion association. In contrast, the related simian immunodeficiency virus SIV_mac, which does not interact with CyPA, is resistant to these compounds. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction between the active site of the enzyme and the capsid (CA) domain of the HIV-1 Gag polyprotein. We report here that the transfer of HIV-1 CA residues 86–93, which form part of an exposed loop, to the corresponding position in SIV_mac resulted in the efficient incorporation of CyPA and conferred an HIV-1-like sensitivity to a nonimmunosuppressive cyclosporin. HIV-1 CA residues 86–90 were also sufficient to transfer the ability to efficiently incorporate CyPA, provided that the length of the CyPA-binding loop was preserved. However, the resulting SIV_mac mutant required the presence of cyclosporin for efficient virus replication. The results indicate that the presence or absence of a type II tight turn adjacent to the primary CyPA-binding site determines whether CyPA incorporation enhances or inhibits viral replication. By demonstrating that CyPA-binding-site residues can induce cyclosporin sensitivity in a heterologous context, this study provides direct in vivo evidence that the exposed loop between helices IV and V of HIV-1 CA not merely constitutes a docking site for CyPA but is a functional target of this cellular protein.     

Stable transduction of quiescent CD34(+)CD38(-) human hematopoietic cells by HIV-1-based lentiviral vectors

We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34(+) cells (45.5% GFP+) and in CD34(+)CD38(-) cells in G0 (12.4% GFP+), 48 hr after transduction. However, GFP could also be detected short-term in CD34(+) cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34(+) cells. The MLV vector did not transduce more primitive, quiescent CD34(+)CD38(-) cells (n = 8). In contrast, stable transduction of CD34(+)CD38(-) cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 +/- 5.2%, n = 7). GFP expression in clones from single CD34(+)CD38(-) cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34(+) and CD34(+)CD38(-) cells (13.5 +/- 2.5%, n = 11 and 12.2 +/- 9.7%, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.     

Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos

The introduction of foreign genes into early mouse embryos and embryonic stem (ES) cells is invaluable for the analysis of gene function and regulation in the living animal. The use of vectors derived from retroviruses as gene transfer vehicles in this setting has had limited success because of silencing of transgene expression. Here, we show that vectors derived from lentiviruses, which are complex retroviruses, can efficiently deliver genes to murine ES cells and that transgene expression is stable during proliferation of undifferentiated ES cells. The transgene is expressed during differentiation of ES cells in vitro (embryoid bodies) and in vivo (teratomas). Transfer of lentivector-transduced ES cells into blastocysts resulted in chimeric animals that expressed the transgene in multiple tissues. Embryos derived from crossings of chimeric mice expressed the transgene, indicating successful germ-line transmission. Infection of murine preimplantation embryos at morula stage with lentiviral vectors resulted in stable transduction and expression of the transgene in mouse embryos and in newborn mice. Finally, human ES cells were transduced by lentiviral vectors and expressed the transgene over several passages. Thus, lentiviral vectors represent a significant improvement over oncoretroviral vectors used previously for gene transfer into murine ES cells and preimplantation embryos. Ability to transfer foreign genes into human ES cells has potential relevance for the development of gene and cell-based therapies.     

High-efficient lentiviral vector-mediated gene transfer into primary human NK cells

OBJECTIVE: The long-term transfection of genes into primary natural killer (NK) cells without disrupting normal cellular functions has been proven to be difficult with currently available gene-transfer methods. In this study, we establish a lentiviral vector-based technique for improved gene transfer into human NK cells in vitro and we report on high-efficient transduction of freshly isolated as well as cultured primary NK cells. METHODS: Freshly isolated or primary cultured human NK cells, as well as the human NK cell line YTS, were transduced with replication-incompetent human immunodeficiency virus (HIV)-based lentiviral vector bearing a GFP reporter gene or a gene of interest under the control of the elongation factor 1alpha (EF1alpha) promoter. Transduction efficiencies were monitored by flow cytometry. RESULTS: A long-term transgene expression was detected in up to 98% of YTS NK cells, whereas in freshly isolated or primary cultured NK cells exposed to interleukin (IL)-2 plus IL-12 upon infection, efficiency was in the range of 50% to 90%. Moreover, in freshly isolated quiescent NK cells a transfection efficiency of 18% to 20% was achieved without stimulation. Notably, no major phenotypic and functional modifications were observed in transduced cells with respect to control cells: the expression levels of activating receptors, CD69-antigen induction as well as cytotoxic function were unaffected. CONCLUSION: Results of our study demonstrate that NK cells can be efficiently transduced by lentiviral vectors.     

Human T lymphocytes transduced by lentiviral vectors in the absence of TCR activation maintain an intact immune competence

Gene transfer into T lymphocytes is currently being tested for the treatment of lymphohematologic disorders. We previously showed that suicide gene transfer into donor lymphocytes infused to treat leukemic relapse after allogeneic hematopoietic stem cell transplantation allowed control of graft-versus-host disease. However, the T-cell receptor (TCR) activation and sustained proliferation required for retroviral vector transduction may impair the half-life and immune competence of transduced cells and reduce graft-versus-leukemia activity. Thus, we tested lentiviral vectors (LVs) and stimulation with cytokines involved in antigen-independent T-cell homeostasis, such as interleukin 7 (IL-7), IL-2, and IL-15. Late-generation LVs transduced efficiently nonproliferating T cells that had progressed from G0 to the G1 phase of the cell cycle on cytokine treatment. Importantly, IL-2 and IL-7, but not IL-15, stimulation preserved physiologic CD4/CD8 and naive-memory ratios in transduced cells with only minor induction of some activation markers. Functional analysis of immune response to cytomegalovirus (CMV) showed that, although CMV-specific T cells were preserved by all conditions of transduction, proliferation and specific killing of autologous cells presenting a CMV epitope were higher for IL-2 and IL-7 than for IL-15. Thus, LV transduction of IL-2 or IL-7 prestimulated cells overcomes the limitations of retroviral vectors and may significantly improve the efficacy of T-cell-based gene therapy.     

Gene Delivery to Human B-Precursor Acute Lymphoblastic Leukemia Cells

Autologous leukemia cells engineered to express immune-stimulatingmolecules may be used to elicit antileukemia immune responses. Genedelivery to human B-precursor acute lymphoblastic leukemia (ALL) cellswas investigated using the enhanced green fluorescent protein (EGFP) asa reporter gene, measured by flow cytometry. Transfection of the Nalm-6and Reh B-precursor ALL leukemia cell lines with an expression plasmidwas investigated using lipofection, electroporation, and a polycationiccompound. Only the liposomal compound Cellfectin showed significantgene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Rehcells). Transduction with gibbon-ape leukemia virus pseudotyped Moloneymurine leukemia virus (MoMuLV)-based retrovirus vectors wasinvestigated in various settings. Cocultivation of ALL cell lines withpackaging cell lines showed the highest transduction efficiency forretroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3%to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bonemarrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to0.2% Reh cells), and in suspension with protamine sulfate (0.7% to3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of bothNalm-6 and Reh cells with human immunodeficiency virus-type 1 (HIV-1)-based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery intoprimary human B-precursor ALL cells from patients was then investigatedusing MoMuLV-based retrovirus vectors and HIV-1-based lentivirusvectors. Both vectors transduced the primary B-precursor ALL cells withhigh efficiencies. These studies may be applied for investigating genedelivery into primary human B-precursor ALL cells to be used forimmunotherapy.     

Vectors selected from adeno-associated viral display peptide libraries for leukemia cell-targeted cytotoxic gene therapy

OBJECTIVE: For acute myeloid leukemia (AML), gene therapy may be used to treat patients refractory to conventional chemotherapy. However, availability of vectors sufficiently and specifically transducing this cell type is very limited. METHOD: Here we report the selection of capsid-modified adeno-associated viral (AAV) vectors targeting Kasumi-1 AML cells by screening random AAV displayed peptide libraries. RESULTS: The peptide inserts of the enriched capsid mutants share a common sequence motif. The same motif was selected in an independent library screening on HL-60 AML cells. Recombinant targeted vectors displaying the selected peptides transduced the target leukemia cells they have been selected on up to 500-fold more efficiently compared to AAV vectors with control peptide inserts. One of the selected clones (NQVGSWS) also efficiently transduced all members of a panel of four other AML cell lines. Binding and blocking experiments showed that NQVGSWS binding to leukemia cells is independent of the wild-type AAV-2 receptor heparin sulfate proteoglycan. Transduction assays on a panel of hematopoietic and nonhematopoietic cell lines showed that the NQVGSWS capsid was able to overcome resistance to AAV transduction, especially in hematopoietic cancer cells, whereas normal peripheral blood mononuclear cells and CD34(+) hematopoietic progenitor cells were not transduced. CONCLUSIONS: Consequently, recombinant targeted NQVGSWS AAV vectors harboring a suicide gene conferred selective killing to Kasumi-1 cells, but not to control cells. This suggests that the AAV mutant selected here may be used as a tool to target therapeutic genes to AML cells.     

Chimeric gag-V3 virus-like particles of human immunodeficiency virus induce virus-neutralizing antibodies.

A 41-kDa unprocessed human immunodeficiency virus 2 (HIV-2) gag precursor protein that has a deletion of a portion of the viral protease assembles as virus-like particles by budding through the cytoplasmic membrane of recombinant baculovirus-infected insect cells. We have constructed six different combinations of chimeric genes by coupling the truncated HIV-2 gag gene to the neutralizing domain (V3) or the neutralizing and the CD4 binding domains (V3+CD4BD) of gp120 env gene sequences from HIV-1 or HIV-2. The env gene sequences were inserted either into the middle of the gag gene or at the 3' terminus of the gag gene. Virus-like particles were formed by chimeric gene products only when the env gene sequences were linked to the 3' terminus of the gag gene. Insertion of env gene sequence in the middle of the gag gene resulted in high-level chimeric gene expression but without the formation of virus-like particles. Three different chimeric genes [gag gene with HIV-1 V3 (1V3), gag gene with HIV-2 V3 (2V3), and gag gene with HIV-2 V3+CD4BD (2V3+CD4BD)] formed virus-like particles that were secreted into the cell culture medium. In contrast, the HIV-1 V3+CD4BD/HIV-2 gag construct did not form virus-like particles. The chimeric gag-env particles had spherical morphology and the size was slightly larger than that of the gag particles, but the chimeric particles were similar to the mature HIV particles. Western blot analysis showed that the gag-env chimeric proteins were recognized by antibodies in HIV-positive human serum and rabbit anti-gp120 serum. Rabbit anti-gag 1V3 and anti-gag 2V3 sera reacted with authentic gp120 of HIV-1 and HIV-2, respectively, and neutralized homologous HIV infectivity. Our results show that precursor gag protein has potential as a carrier for the presentation of foreign epitopes in good immunological context. The gag protein is highly immunogenic and has the ability to carry large foreign inserts; as such, it offers an attractive approach for HIV vaccine development.     

The presence of the Trim5alpha escape mutation H87Q in the capsid of late stage HIV-1 variants is preceded by a prolonged asymptomatic infection phase

BACKGROUND: Recently, the tripartite interaction motif 5alpha (Trim5alpha) has been identified as an inhibitory factor blocking infection of a broad range of retroviruses in a species-specific manner. In particular, HIV-1 replication can be efficiently blocked by Trim5alpha from Old World monkeys. The cyclophilin A binding region in the HIV-1 capsid is believed to be the viral determinant for Trim5alpha, and mutations in this region lift the restriction in simian cells. Human Trim5alpha is also able to inhibit HIV-1 replication in vitro, implying that Trim5alpha may contribute to host control of HIV-1 replication in vivo. METHODS: HIV-1 variants from participants of the Amsterdam cohort studies were analysed for Trim5alpha escape mutations in the capsid. Patients who harboured HIV-1 variants with Trim5alpha escape mutations were compared with patients who lacked such variants in terms of clinical course of infection. RESULTS: Trim5alpha escape mutants emerged in the late phase of infection and were ultimately present in 13.7% of HIV-1 infected individuals. Patients who developed Trim5alpha escape variants late in infection had a significantly lower set-point plasma viral RNA load and concomitantly a prolonged asymptomatic survival as compared to individuals who lacked Trim5alpha escape mutants. This protective effect was stronger in individuals who later developed X4 variants. In addition, X4-emergence was delayed in individuals who later developed Trim5alpha escape variants, compatible with suppression of viral replication. CONCLUSION: Our data are compatible with Trim5alpha-mediated suppression of viral replication, resulting in prolonged asymptomatic survival and ultimately the selection of Trim5alpha escape variants.     

Combined targeting of lentiviral vectors and positioning of transduced cells by magnetic nanoparticles

Targeting of viral vectors is a major challenge for in vivo gene delivery, especially after intravascular application. In addition, targeting of the endothelium itself would be of importance for gene-based therapies of vascular disease. Here, we used magnetic nanoparticles (MNPs) to combine cell transduction and positioning in the vascular system under clinically relevant, nonpermissive conditions, including hydrodynamic forces and hypothermia. The use of MNPs enhanced transduction efficiency of endothelial cells and enabled direct endothelial targeting of lentiviral vectors (LVs) by magnetic force, even in perfused vessels. In addition, application of external magnetic fields to mice significantly changed LV/MNP biodistribution in vivo. LV/MNP-transduced cells exhibited superparamagnetic behavior as measured by magnetorelaxometry, and they were efficiently retained by magnetic fields. The magnetic interactions were strong enough to position MNP-containing endothelial cells at the intima of vessels under physiological flow conditions. Importantly, magnetic positioning of MNP-labeled cells was also achieved in vivo in an injury model of the mouse carotid artery. Intravascular gene targeting can be combined with positioning of the transduced cells via nanomagnetic particles, thereby combining gene- and cell-based therapies.     

A genetically modified adenoviral vector exhibits enhanced gene transfer of human smooth muscle cells

Adenoviral vector-based gene therapy is a promising approach for the treatment of restenosis postangioplasty. However, a high concentration of adenoviral vector can cause cellular activation, damage, and an enhanced immune response. One approach to solving this problem is to increase gene transfer efficiency by directing adenoviral vector entry via an alternate receptor system. We have constructed an adenoviral vector, Av9LacZ, that encodes the beta-galactosidase gene and contains a chimeric fiber protein that redirects viral vector binding to the Ad3 adenoviral receptor on the host cell. We examined the ability of Av9LacZ to transduce primary human smooth muscle cells (SMC) and found that it showed a 10- to 15-fold higher transduction efficiency when compared to the prototypic adenoviral vector currently used for preclinical and clinical studies. While both vectors were able to transduce rabbit, pig and monkey SMCs, the genetically modified vector transduced human SMC with much higher efficiency. SMC obtained from the aorta, coronary, renal, popliteal and pulmonary arteries were all efficiently transduced by Av9LacZ. Consistent with the data obtained from cultured cells, Av9LacZ also transduced fresh human arterial tissues considerably more efficiently than Av1LacZ. We conclude that the large discrepancy between transduction of animal and human cells by conventional vectors supports a cautious extrapolation of the results of in vivo animal studies to man. Furthermore, the genetically modified AV9 vector may deliver better efficacy and studies in large animal models with this vector could be more predictive of therapeutic efficacy in the treatment of human restenosis.     

Characterization of human immunodeficiency virus type 1 Pr55gag membrane association in a cell-free system: requirement for a C-terminal domain.

Association of the human immunodeficiency virus type 1 (HIV-1) gag polyprotein precursor with cellular membranes is necessary for assembly of virions. We used in vitro synthesized HIV-1 gag to study its association with isolated cellular membranes. Rabbit reticulocyte lysates programmed with HIV-1 gag mRNA incorporated [35S]methionine and [3H]myristate into two predominant species of 55 kDa and 40 kDa. Radioimmunoprecipitation with HIV-1-specific antibodies suggested that the 55-kDa protein represented the polyprotein precursor (Pr55gag), while the 40-kDa protein was a mixture of N- or C-terminal truncations of the gag precursor. The Pr55gag protein bound to cellular membranes, while the 40-kDa mixed protein species did not. Membrane binding studies with C terminus-truncated and point mutants revealed that the seven-amino acid sequence located between the two Cys-His arrays in the nucleocapsid region was necessary for stable association to occur. Therefore, we propose that signals in addition to myristate are required for the membrane association of HIV-1 gag proteins and that these signals include a domain in the nucleocapsid protein.     

Intercellular delivery of a herpes simplex virus VP22 fusion protein from cells infected with lentiviral vectors

Effective gene therapy depends on the efficient transfer of therapeutic genes and their protein products to target cells. Lentiviral vectors appear promising for virus-mediated gene delivery and long-term expression in nondividing cells. The herpes simplex virus type 1 tegument protein VP22 has recently been shown to mediate intercellular transport of proteins, raising the possibility that it may be helpful in a setting where the global delivery of therapeutic proteins is desired. To investigate the effectiveness of lentiviral vectors to deliver genes encoding proteins fused to VP22, and to test whether the system is sufficiently potent to allow protein delivery from transduced cells in vitro and in vivo, fusion constructs of VP22 and the enhanced green fluorescent protein (EGFP) were prepared and delivered into target cells by using HIV-1-based lentiviral vectors. To follow the spread of VP22-EGFP to other cells, transduced COS-7 cells were coplated with a number of different cell types, including brain choroid plexus cells, human endothelial cells, H9 cells, and HeLa cells. We found that VP22-EGFP fusion proteins were transported from transduced cells to recipient cells and that such fusion proteins accumulated in the nucleus and in the cytoplasm of such cells. To determine the ability to deliver fusion proteins in vivo, we injected transduced H9 cells as well as the viral vector directly into the brain of mice. We present evidence that VP22-EGFP fusion proteins were transported effectively from lentivirus transduced cells in vivo. We also show that the VP22-EGFP fusion protein encoded by the lentivirus is transported between cells. Our data indicate that such fusion proteins are present in the nucleus and in the cytoplasm of neighboring cells. Therefore, lentiviral vectors may provide a potent biological system for delivering genes encoding therapeutic proteins fused to VP22.     

Lentiviral vectors pseudotyped with a modified RD114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and CD34+ cells derived from human and nonhuman primates

Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors, their interaction with the host immune system, and their host range. We have investigated the capacity of a panel of GPs of both retroviral (amphotropic murine leukemia virus [MLV-A]; gibbon ape leukemia virus [GALV]; RD114, feline endogenous virus) and nonretroviral (fowl plague virus [FPV]; Ebola virus [EboV]; vesicular stomatitis virus [VSV]; lymphocytic choriomeningitis virus [LCMV]) origins to pseudotype lentiviral vectors derived from simian immunodeficiency virus (SIVmac251). SIV vectors were efficiently pseudotyped with the FPV hemagglutinin, VSV-G, LCMV, and MLV-A GPs. In contrast, the GALV and RD114 GPs conferred much lower infectivity to the vectors. Capitalizing on the conservation of some structural features in the transmembrane domains and cytoplasmic tails of the incorporation-competent MLV-A GP and in RD114 and GALV GPs, we generated chimeric GPs encoding the extracellular and transmembrane domains of GALV or RD114 GPs fused to the cytoplasmic tail (designated TR) of MLV-A GP. Importantly, SIV-derived vectors pseudotyped with these GALV/TR and RD114/TR GP chimeras had significantly higher titers than vectors coated with the parental GPs. Additionally, RD114/TR-pseudotyped vectors were efficiently concentrated and were resistant to inactivation induced by the complement of both human and macaque sera, indicating that modified RD114 GP-pseudotyped lentiviral vectors may be of particular interest for in vivo gene transfer applications. Furthermore, as compared to vectors pseudotyped with other retroviral GPs or with VSV-G, RD114/TR-pseudotyped vectors showed augmented transduction of human and macaque primary blood lymphocytes and CD34+ cells.     

Multiple antigenic epitopes expressed on gag proteins, p26 and p15, of a human immunodeficiency virus (HIV) type 2 as defined with a library of monoclonal antibodies.

Thirty-two hybridoma clones producing monoclonal antibody (MAb) against HIV-2[GH-1] were established from mice immunized with NP-40-disrupted purified whole virus of a Ghanaian isolate of human immunodeficiency virus type 2 (HIV-2), strain HIV-2[GH-1]. Of these 32 MAbs, 20 reacted with p26 and the other MAbs recognized p15 of the HIV-2[GH-1] isolate. From the results of serological characterization by these MAbs, p26 and p15 were identified as capsid proteins and matrix protein, respectively, of HIV-2[GH-1] gag products. In addition to two gag proteins, a 55-kD protein corresponding to the primary translational product of gag gene and 39-kD protein corresponding to an intermediate product of cleavage of p55 were recognized by these MAbs in the lysate of HIV-2[GH-1]-infected cells. Moreover, these MAbs were used to analyze the number of antigenic epitopes on p26 and p15 of HIV-2[GH-1] isolate. The results of cross-reactivity with different HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates and competitive binding assay suggest that there are at least four and five antigenic epitopes in p26 and p15, respectively, of the HIV-2[GH-1] isolate. The biological activity of MAbs was studied by performing syncytium inhibition assay and infection inhibition assays. However, our MAbs could not inhibit syncytium formation and infection by cell-free virus.