Single Fusion Protein Research Articles

Immunization With a Bivalent Adenovirus-vectored Tuberculosis Vaccine Provides Markedly Improved Protection Over Its Monovalent Counterpart Against Pulmonary Tuberculosis

Recombinant virus-vectored vaccines hold great promise for tuberculosis (TB) vaccination strategies. However, there is a lack of side-by-side comparative investigations to dissect the functional differences and support the advantage of multivalent virus-vectored vaccine over its monovalent counterpart. We previously successfully developed a monovalent adenovirus (Ad)-vectored vaccine expressing Ag85a (AdAg85a) and demonstrated its superior protective efficacy in models of pulmonary TB. In this study, we have developed a bivalent Ad TB vaccine expressing Ag85a and TB10.4 antigens as a fusion protein (AdAg85a:TB10.4) and compared its T-cell-activating and immune protective efficacy with that by monovalent AdAg85a. A single intranasal (i.n.) administration of AdAg85a:TB10.4 induced robust T-cell responses toward the respective antigens within the airway lumen and spleen, although the level of Ag85a-specific T-cell responses in the airway lumen triggered by bivalent AdAg85a:TB10.4 was lower than that by its monovalent counterpart at earlier time points. Thus, a single i.n. delivery of AdAg85a:TB10.4 conferred a markedly improved and sustained level of protection in the lung against Mycobacterium tuberculosis (M.tb) challenge over that by AdAg85a or by conventional BCG immunization with similarly induced levels of protection in the spleen. Our results indicate a unique advantage of multivalent viral-vectored TB vaccines for immunization against pulmonary TB.

Mitochondrial outer and inner membrane fusion requires a modified carrier protein

In yeast, three proteins are essential for mitochondrial fusion. Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively. At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing. The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family. We show that Ugo1 is a modified member of this family, containing three transmembrane domains and existing as a dimer, a structure that is critical for the fusion function of Ugo1. Our functional analysis of Ugo1 indicates that it is required distinctly for both outer and inner membrane fusion after membrane tethering, indicating that it operates at the lipid-mixing step of fusion. This role is distinct from the fusion dynamin-related proteins and thus demonstrates that at each membrane, a single fusion protein is not sufficient to drive the lipid-mixing step, but instead, this step requires a more complex assembly of proteins.

Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity

A vaccine comprising human papillomavirus type 16 (HPV16) L2, E6 and E7 in a single tandem fusion protein (termed TA-CIN) has the potential advantages of both broad cross-protection against HPV transmission through induction of L2 antibodies able to cross neutralize different HPV types and of therapy by stimulating T cell responses targeting HPV16 early proteins. However, patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. Here we test TA-CIN formulated along with the adjuvant GPI-0100, a semi-synthetic quillaja saponin analog that was developed to promote both humoral and cellular immune responses. Subcutaneous administration to mice of TA-CIN (20μg) with 50μg GPI-0100, three times at biweekly intervals, elicited high titer HPV16 neutralizing serum antibody, robust neutralizing titers for other HPV16-related types, including HPV31 and HPV58, and neutralized to a lesser extent other genital mucosatropic papillomaviruses like HPV18, HPV45, HPV6 and HPV11. Notably, vaccination with TA-CIN in GPI-0100 protected mice from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP without adjuvant. Formulation of TA-CIN with GPI-0100 enhanced the production of E7-specific, interferon γ producing CD8+ T cell precursors by 20-fold. Vaccination with TA-CIN in GPI-0100 also completely prevented tumor growth after challenge with 5×104 HPV16-transformed TC-1 tumor cells, whereas vaccination with TA-CIN alone delayed tumor growth. Furthermore, three monthly vaccinations with 125μg of TA-CIN and 1000μg GPI-0100 were well tolerated by pigtail macaques and induced both HPV16 E6/E7-specific T cell responses and serum antibodies that neutralized all HPV types tested.

Flagellin-F1-V Fusion Protein Is an Effective Plague Vaccine in Mice and Two Species of Nonhuman Primates

A number of studies have clearly demonstrated that flagellin is a potent adjuvant that promotes robust immune responses when it is given with a protein antigen. In view of the potential biological and practical benefits of a recombinant protein vaccine composed of a single fusion protein containing flagellin and antigen, we have evaluated the efficacy of a fusion protein composed of flagellin and two protective antigens of Yersinia pestis (F1 and V) in eliciting protection against respiratory challenge with Y. pestis. Flagellin-F1-V was produced and purified in high yield under good manufacturing practices conditions. The fusion protein retains full Toll-like receptor 5-stimulating activity in vitro. Using a prime-boost immunization protocol, we found that flagellin-F1-V elicits robust antigen-specific humoral immunity in mice and two species of nonhuman primates. Immune mice were fully protected against intranasal challenge with 150 mean tolerated doses of Y. pestis CO92. In immune mice, the bacteria were completely cleared within 3 days after challenge. Flagellin-F1-V exhibited full stability for at least 297 days at 4 degrees C and at least 168 days at 25 degrees C. At between 29 and 84 days at 37 degrees C, the protein exhibited a loss of biological activity that appeared to be associated with a substantial change in protein diameter, possibly due to oligomerization. On the basis of our results, we believe that flagellin-F1-V is an outstanding candidate for evaluation in studies with humans.

Citations

Citations

Abstract A138: Vaccination with HPV 16 L2E6E7 with GPI-0100 adjuvant elicits protective humoral as well as cell-mediated immune responses

Abstract A138 L1 virus-like particle (VLP) vaccines provide effective protection against Human papillomavirus (HPV) type from which the L1 was derived via the induction of neutralizing antibodies. While partial protection can occur against very closely related types, it is generally type-restricted and necessitates highly multivalent L1 VLP vaccines to obtain broad coverage. The minor capsid protein, L2, is emerging as an attractive alternative cross-protective antigen. Unfortunately, neither L1 VLP nor L2-based vaccines are effective against pre-existing disease, suggesting the need to develop therapeutic vaccines. Unlike the capsid proteins, the E6 and E7 oncoproteins are both required to maintain and expressed throughout cervical intraepithelial neoplasia (CIN) and cancer. TA-CIN, a vaccine comprising the HPV16 L2, E6 and E7 in a single tandem fusion protein, attempts to combine the advantages of broad cross-protection against HPV transmission with therapeutic responses targeting HPV16 early proteins. Here we test TA-CIN formulated along with GPI-0100, a semi-synthetic quillaja saponin analog, that was developed to promote both humoral and cellular immune responses. TA-CIN administered subcutaneously to mice three times at monthly intervals (125μg of clinical-grade protein) with 50μg GMP-grade GPI-0100 was found to elicit high titer antibodies that effectively neutralized not only HPV16 but also other oncogenic HPV types including HPV18, HPV31, HPV45, HPV58. Similarly, vaccination of pigtail macaques (Macaca nemestrina) with TA-CIN (three doses of 125μg of TA-CIN with 1000μg GMP-grade GPI-0100 at monthly intervals) was well tolerated and induced serum antibodies that neutralized HPV16, HPV18, and HPV31 in vitro. Notably, vaccination of mice with TA-CIN protected them from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP. Combination of TA-CIN along with adjuvant GPI-0100 enhanced production of E7-specific, interferon gamma producing CD8+ T cell precursors by 20-fold. Vaccination with TA-CIN in adjuvant GPI-0100 completely prevented tumor growth after challenge with 5 x 104 HPV16-transformed TC-1 tumor cells. Protection of the mice immunized with TA-CIN plus GPI-0100 against TC-1 in a prophylactic setting was significantly more effective than TA-CIN alone or GPI-0100 alone. Patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. GPI-0100 has been used safely as an adjuvant in several human trials suggesting that the combination of TA-CIN and GPI-0100 warrants further study. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A138.

Activity of soluble OX40 ligand is enhanced by oligomerization and cell surface immobilization

OX40 ligand (OX40L) and OX40 are typical members of the tumor necrosis factor ligand family and the tumor necrosis factor receptor superfamily, respectively, and are involved in the costimulation and differentiation of T cells. Like other tumor necrosis factor ligands, OX40L is a type II transmembrane protein. Recombinant soluble human OX40L assembles into trimers and is practically inactive despite binding to OX40. However, oligomerization of soluble OX40L trimers by cross-linking with antibodies or by expression as a hexameric fusion protein strongly increased the activity of the ligand. Moreover, a fusion protein of OX40L with a single chain fragment recognizing the tumor stroma antigen fibroblast activation protein showed a cell surface antigen-dependent increase in the activity of the ligand domain of the molecule and thus mimicked the activity of membrane OX40L upon antigen binding. Trimeric single chain OX40L fusion proteins therefore represent a novel type of OX40L-derived immunostimulatory molecule with potentially reduced systemic side effects.

Both CD4+ and CD8+ T cells respond to antigens fused to anthrax lethal toxin.

The lethal toxin produced by Bacillus anthracis is a bipartite toxin in which the first protein, protective antigen (PA), transports the second protein, lethal factor, across the host cell membrane. We have previously shown that CD8(+) T-cell epitopes fused to a nontoxic derivative of lethal factor (LFn) are delivered into the host cell cytosol in a PA-dependent manner. Delivery of these antigens targets them to the intracellular major histocompatibility complex (MHC) class I processing and presentation pathway and leads to the stimulation of antigen-specific CD8(+) T cells in vivo. In this report, we describe the generation and characterization of LFn fusion proteins that include not only a CD8(+) T-cell epitope but also a CD4(+) T-cell epitope. We first show that these fusion proteins induce antigen-specific CD4(+) T-cell responses following incubation with dendritic cells in vitro or injection into mice. Stimulation of CD4(+) T cells by LFn fusion proteins does not require PA but is enhanced by PA in vitro. We also show that a single LFn fusion protein and PA can deliver antigen to both the MHC class II and the MHC class I pathways, resulting in the simultaneous induction of antigen-specific CD4(+) T cells and antigen-specific CD8(+) T cells in the same mouse. These results suggest that this toxin delivery system is capable of stimulating protective immune responses where effective immunization requires stimulation of both classes of T cells.

Coping with Viral Diversity in HIV Vaccine Design: A Response to Nickle et al.

Coping with Viral Diversity in HIV Vaccine Design: A Response to Nickle et al.

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in Escherichia coli

The immunocytokine scFvMEL/TNF is a fusion protein composed of tumor necrosis factor (TNF-alpha) and a single-chain Fv antibody scFvMEL targeting the melanoma-associated gp240 antigen. The fusion protein containing thioredoxin and a hexa-histidine tag was expressed in two Escherichia coli host cells, AD494 (DE3) pLysS and T7 Express I (q). The cell growth and expression level of target protein, His-tagged scFvMEL/TNF, were highly dependent on the induction temperature, inducer types and host strains. The ratio of insoluble to soluble target proteins was found to be controllable and could be minimized using cold shock conditions at less than 18 degrees C. The total productivity of soluble target protein was further improved by high cell density cultivation using a DO-STAT feeding strategy. The scFvMEL/TNF purified under their conditions was specifically cytotoxic to gp240-antigen positive melanoma A375-M cells as previously described.

A general computational model for predicting ribosomal frameshifts in genome sequences

Programmed ribosomal frameshifts are frequently used by RNA viruses to synthesize a single fusion protein from two or more overlapping open reading frames. We previously developed a program called FSFinder for predicting - 1 and + 1 frameshifts for Windows systems. With our new web application and web service called FSFinder2, users can predict frameshifts of any type from any web browser and operating system. We tested it on Shewanella oneidensis MR-1, for which exact frameshift sites are not known. FSFinder2 is the first program capable of finding frameshifts of general type and it is a powerful tool for predicting and analyzing genes that utilize frameshifts for their expression. FSFinder2 is available at http://wilab.inha.ac.kr/FSFinder.

PTD-hEGF Fusion Protein—Gene Expression and Function Analysis

To produce membrane-permeable human epidermal growth factor (hEGF), a pPTD-hEGF prokaryocyte expression vector was constructed and transformed into E. coli BL 21(DE3). The PTD-hEGF fusion protein was induced by 0.3 mmol/L of IPTG expressed in the form of inclusion body with an yield of 40% of the total protein in the cells, and then purified by Ni 2+-NTA affinity chromatography. The SDS-PAGE analysis showed a single fusion protein band with a molecular weight of 16 kD. The amino acid sequence was checked by MALDI-TOF-MS analysis. The genetic engineering PTD-hEGF fusion protein obviously promoted the proliferation and growth of the HEK-293 cells in vitro.

Improved Pharmacokinetics of Recombinant Bispecific Antibody Molecules by Fusion to Human Serum Albumin

Recombinant bispecific antibodies such as tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb) have shown to be able to retarget T lymphocytes to tumor cells, leading to their destruction. However, therapeutic efficacy is hampered by a short serum half-life of these small molecules having molecule masses of 50-60 kDa. Thus, improvement of the pharmacokinetic properties of small bispecific antibody formats is required to enhance efficacy in vivo. In this study, we generated several recombinant bispecific antibody-albumin fusion proteins and analyzed these molecules for biological activity and pharmacokinetic properties. Three recombinant antibody formats were produced by fusing two different scFv molecules, bispecific scDb or taFv molecules, respectively, to human serum albumin (HSA). These constructs (scFv(2)-HSA, scDb-HSA, taFv-HSA), directed against the tumor antigen carcinoembryonic antigen (CEA) and the T cell receptor complex molecule CD3, retained full binding capacity to both antigens compared with unfused scFv, scDb, and taFv molecules. Tumor antigen-specific retargeting and activation of T cells as monitored by interleukin-2 release was observed for scDb, scDb-HSA, taFv-HSA, and to a lesser extent for scFv(2)-HSA. T cell activation could be further enhanced by a target cell-specific costimulatory signal provided by a B7-DbCEA fusion protein. Furthermore, we could demonstrate that fusion to serum albumin strongly increases circulation time of recombinant bispecific antibodies. In addition, our comparative study indicates that single chain diabody-albumin fusion proteins seem to be the most promising format for further studying cytotoxic activities in vitro and in vivo.

Overexpression and purification of single zinc finger peptides of human zinc finger protein ZNF191

ZNF191, a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain structural information of zinc finger domain a convenient method for obtaining milligram quantities of each zinc finger peptide of ZNF191 is necessary. Here, we report an Escherichia coli expression system for rapid and high-level expression of zinc finger 3 and zinc finger 4 of ZNF191. The gene of zinc finger 3 or zinc finger 4 was cloned into pET31b vector to allow expression of single zinc finger peptide as a ketosteroid isomerase (KSI) fusion protein. The KSI–single zinc finger fusion protein was overexpressed in the form of inclusion body, which can be purified by washing several times using buffer solutions, and then be cleaved directly by cyanogen bromide to release single zinc finger peptide. The more than 20 mg/L yield of single zinc finger peptide was achieved with more than 95% purity by using YM ultrafiltration membranes. Circular dichroism spectra of these two single zinc finger peptides titrated with Zn 2+ ions demonstrate that they have different secondary structures.

Enhanced fluorescence resonance energy transfer immunoassay with improved sensitivity based on the Fab′-based immunoconjugates

Fluorescence resonance energy transfer (FRET) is a powerful technique to monitor protein–protein interaction. Recently, we developed homogeneous and noncompetitive immunoassay based on the enhanced FRET by leucine zipper interaction. Here we improved the assay by establishing a general method for preparation of the Fab′-based immunoconjugate. Anti-human serum albumin Fab′ numbers 11 and 13 were chemically conjugated with recombinant proteins consisting of thioredoxin, flexible linker, and green fluorescent protein color variant tethered with a leucine zipper motif. Compared with single chain antibody variable region-based fusion proteins prepared by the gene fusion method in our previous study, the resultant Fab′-based immunoconjugates accomplished an assay with nearly 10 times greater sensitivity. Furthermore, the conjugation method enabled us to apply the assay generally to measurement of another high-molecular weight antigen for which antibodies prepared for sandwich immunoassay are commercially available. Because of the facility and generality of the preparation method for the immunoconjugate, the assay is expected to be applied to many antigens that require rapid diagnosis and moderate measurement range.

Antitumor Activity of a Dual Cytokine/Single-chain Antibody Fusion Protein for Simultaneous Delivery of GM-CSF and IL-2 to Ep-CAM Expressing Tumor Cells

Cytokine targeting to tumor-associated antigens via antibody cytokine fusion proteins has demonstrated potent antitumor activity in numerous animal models and has led to the clinical development of 2 antibody-interleukin-2 (IL-2) fusion proteins. We previously reported on the construction and in vitro properties of a "dual" cytokine fusion protein for simultaneous targeted delivery of human granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-2 to human tumors. The fusion protein is based on a heterodimerized core structure formed by human CH1 and Ckappa domains (heterominibody) with C-terminally fused human cytokines and N-terminally fused single-chain antibody fragments specific for the tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM). For testing the antitumor activity in syngeneic mouse xenograft models, we developed "dual cytokine heterominibodies" with murine cytokines (mDCH). mDCH fusion proteins and, as controls, "single cytokine heterominibodies" (SCH) carrying either murine GM-CSF (mGM-CSF) or murine IL-2 (mIL-2) were constructed, of which all retained the specific activities of cytokines and binding to the Ep-CAM antigen on human Ep-CAM transfected mouse colon carcinoma CT26-KSA cells. Over a 5-day treatment course, DCH fusion proteins induced significant inhibition of established pulmonary CT26-KSA metastases in immune-competent Balb/c mice at low daily doses of 1 mug of fusion protein per mouse. However, with the tested dosing schemes, antitumor activity of mDCH was largely independent of cytokine targeting to tumors as demonstrated by a control protein with mutated Ep-CAM binding sites. Single cytokine fusion proteins mSCH-GM-CSF and mSCH-IL-2 showed similar antitumor activity as the dual cytokine fusion protein mDCH, indicating that GM-CSF and IL-2 in one molecule did not significantly synergize in tumor rejection under our experimental conditions. Our results seem to contradict the notion that IL-2 and GM-CSF can synergize in antitumor activity and that with conventional dose regimens, their specific targeting to tumors, as tested here with 2 antibodies of different affinities, enhances their antitumor activity.

Construction and evaluation of novel fusion proteins for targeted delivery of micro particles to cellulose surfaces

The use of IgG antibodies and fragments has been limited to specific sectors of the biotechnology industry due to the high cost of producing large batches of product necessary for alternative applications. A novel class of Camelid antibodies, known as V(HH) offer a more economical opportunity to meet a wider application in industry. In this study, we report the evaluation of four llama V(HH)-cellulose binding domain fusion proteins displaying varying formats of V(HH) and CBD domains. Proteins were characterized in a targeted particle delivery system as a method of delivering agents such as perfume to laundry in the wash cycle. Fusion proteins were shown to be stable at high pH and in the presence of a detergent base. They were also shown to bind effectively to both the designated antigen, the azo-dye reactive-red 6 (either conjugated to BSA or attached to coacervate microparticles), and cellulose. Binding strength differences were observed between the different fusion protein formats using surface plasmon resonance. The effect of key laundry ingredients was also studied. Combining the fusion proteins and particles into a delivery and deposition study generated clear microscopy evidence for bifunctionality. Confirmation of this was validated by GC-MS analysis of retained fragrance. This research, reporting the construction and characterization of a variety of fusion proteins, illustrates that the single multidomain fusion protein route offers a new technology for successful targeted delivery of encapsulated benefit agents. Furthermore, the potential to modify or select for proteins to recognize a wide range of surfaces is also possible.

Function, oligomerization and N-linked glycosylation of the Helicoverpa armigera single nucleopolyhedrovirus envelope fusion protein

In the family Baculoviridae, two distinct envelope fusion proteins are identified in budded virions (BVs). GP64 is the major envelope fusion protein of group I nucleopolyhedrovirus (NPV) BVs. An unrelated type of envelope fusion protein, named F, is encoded by group II NPVs. The genome of Helicoverpa armigera (Hear) NPV, a group II NPV of the single nucleocapsid or S type, also encodes an F-like protein: open reading frame 133 (Ha133). It was demonstrated by N-terminal sequencing of the major 59 kDa protein present in HearNPV BV that this protein is one of the two F subunits: F1 (transmembrane subunit of 59 kDa) and F2 (surface subunit of 20 kDa), both the result of cleavage by a proprotein convertase and disulfide-linked. The HearNPV F protein proved to be a functional analogue of GP64, as the infectivity of an AcMNPV gp64-deletion mutant was rescued by the introduction of the HearNPV F gene. It was also demonstrated by chemical cross-linking that HearNPV F is present in BVs as an oligomer whereby, unlike GP64, disulfide bonds are not involved. Deglycosylation assays indicated that both F1 and F2 possess N-linked glycans. However, when F was made in Hz2E5 cells, these glycans did not have an alpha-1-3 core fucose modification that usually occurs in insect cells. As alpha-1-3 core fucose is a major inducer of an allergic response in humans, the present observation makes the HearNPV-Hz2E5 system an attractive alternative for the production of recombinant glycoproteins for therapeutic use in humans.

Adding value to fusion proteins through covalent labelling

Recombinant proteins are often expressed with an additional polypeptide the role of which is to aid purification, detection or functional studies. Recently, the role of these tags has been extended to mediate the labelling of the protein of interest with chemically diverse compounds. These approaches are of importance for protein science as they allow proteins to be equipped with properties that cannot be genetically encoded and also enable the use of a single fusion protein for several different applications. Several new approaches have been developed for the covalent labelling of fusion proteins both in living cells and in vitro.

1131. Generation of Whole Cell Vaccines for Acute Myeloid Leukaemia by Lentivirus Mediated IL-2/CD80 Transduction

Despite recent treatment advances, the prognosis for the majority of AML patients remains bleak, particularly in the elderly, those with poor prognostic cytogenetics, and/or progression from myelodysplasia. We and others have previously shown that tumour cells engineered to express CD80 and IL-2 can stimulate T and NK cell responses against the unmodified tumour cells, and that vaccination with irradiated CD80/IL-2 expressing leukaemic cells can prolong survival in mouse models of leukaemia. In order to test the therapeutic potential of this approach, we have devised an efficient gene transfer protocol for primary human acute myeloid leukaemia blasts. Based on a previously established methodology for retroviral vectors, we show that disabled SIN-lentiviral vectors can be conjugated to streptavidin paramagnetic microparticles (PMP) after covalent linkage of biotin to 293T packaging cells. Biotin coated lentivirus is then captured and magnetically concentrated, resulting in up to 2600 fold titre enhancements after 100 fold volume reductions to achieve titres in excess of 109 cfu/ml for amphotropic or VSV-G pseudotypes. A self-inactivating lentiviral backbone, incorporating the HIV flap region and a myeloid efficient internal promoter, derived from spleen focus forming virus (SFFV), is used for the efficient co-expression of CD80 and IL-2 in primary AML blasts. These vectors express CD80 and IL-2 as a single fusion protein that incorporates the recognition sequence for a golgi/ER located ubiquitous endoprotease |[ndash]| furin between the CD80 and IL-2 moieties. This fusion protein is post-synthetically cleaved by furin to generate biologically active membrane anchored CD80 and secreted IL-2. Following a single round of infection of primary or established AML cells at an MOI of 10, between 40%-99% of AML blasts express CD80 and secrete 2-15 ng of IL-2/106/24hrs. The combined expression of IL-2 and CD80 enables the increased in vitro stimulation of allogeneic and autologous T cells obtained from AML patients, either before or following the induction of remission, with or without preceding bone marrow transplantation. The stimulated lymphocytes have a TH1 cytokine secretion profile and show evidence of specificity, as indicated by their increased proliferation in the presence of unmodified AML compared to remission bone marrow cells. The stimulated T cells also demonstrate cytotoxic activity against AML blasts. This data supports the notion that IL-2/CD80 whole cell vaccination could stimulate therapeutically useful immune responses in AML patients, particularly following chemotherapy induced remission and allogeneic bone marrow transplantation. We have recently been granted permission for the vaccination of poor prognosis AML patients with lentivirus mediated ex vivo transduced AML blasts expressing CD80 and IL-2.