Transduction Of Dendritic Cells Research Articles

Induction of primary anti-HIV CD4 and CD8 T cell responses by dendritic cells transduced with self-inactivating lentiviral vectors

In this study, we demonstrate that a minimal self-inactivating (SIN) lentiviral vector (LV) that does not encode any human immunodeficiency virus (HIV) genes is able to induce HIV-specific CD4 and CD8 T cell responses after transduction of dendritic cells (DCs). The LV-DC-primed T cells displayed HIV-specific lytic degranulation, as illustrated by acquisition of CD107a/b expression on the cell surface and up-regulation of active caspase 3. HIV-specific cytotoxic T lymphocyte (CTL) response was consistently detected using different assays, and T cell receptors specific to three prominent HIV epitopes, SL9 (Gag peptide: SLYNTVATL), IV9 (Pol peptide: ILKEPVHGV), and MA10 (In peptide: MASDFNLPPV) were detected using HLA-A0201 peptide-tetramers. These results demonstrate that DCs transduced with the minimal SIN-LV can efficiently induce HIV-specific CD4 and CD8 T cell responses. Since LVs are popular gene transfer tools, our results have fundamental implications for future LV applications and DC vaccine development.

Intradermal Delivery of Adenoviral Type-35 Vectors Leads to High Efficiency Transduction of Mature, CD8+ T Cell-Stimulating Skin-Emigrated Dendritic Cells

Recombinant adenovirus (Ad) type 35 (rAd35) shows great promise as vaccine carrier with the advantage of low pre-existing immunity in human populations, in contrast to the more commonly used rAd5 vector. The rAd35 vector uses CD46 as a high-affinity receptor, which, unlike the rAd5 receptor, is expressed on human dendritic cells (DC), the most powerful APCs identified to date. In this study, we show that in contrast to rAd5, rAd35 infects migrated and mature CD83+ cutaneous DC with high efficiency (up to 80%), when delivered intradermally in an established human skin explant model. The high transduction efficiency is in line with high expression levels of CD46 detected on migratory cutaneous DC, which proved to be further increased upon intradermal administration of GM-CSF and IL-4. As compared with Ad5, these Ad35 infection characteristics translate into higher absolute numbers of skin-emigrated DC per explant that both express the transgene and are phenotypically mature. Finally, we demonstrate that upon intracutaneous delivery of a rAd35 vaccine encoding the circumsporozoite (CS) protein of Plasmodium falciparum, emigrated DC functionally express and process CS-derived epitopes and are capable of activating specific CD8+ effector T cells, as evidenced by activation of an HLA-A2-restricted CS-specific CD8+ T cell clone. Collectively, these data demonstrate the utility of rAd35 vectors for efficient in vivo human DC transduction.

Induction of Antigen-Specific Immune Responsesin VivoAfter Vaccination with Dendritic Cells Transduced with Adenoviral Vectors Encoding Hepatitis C Virus NS3

Dendritic cells (DC) are potent antigen-presenting cells that play a critical role in the initiation of immunity to viral infections. Direct transduction of DC appears to be the major pathway in vivo responsible for induction of antigen specific immune responses. The aim of this study was to explore the vaccine potential of DC transduced with adenoviral vectors encoding the HCV nonstructural protein 3 (AdNS3) compared to DC pulsed with recombinant NS3 (rNS3). Mice (Balb/c and C57BL/6 transgenic for HLA-A2.1) were immunized with DC based vaccines. After the immunization, antigen specific immune responses including humoral responses, cytokine secretion, and IFN-gamma-producing T cell responses were analyzed. In both strains of mice inoculated with DC transduced with an adenovirus, the generated NS3 specific antibody response and IFN-gamma-secreting T cell response were stronger than that generated by rNS3-pulsed DC. Analysis of the cytokine profiles revealed that immunization with AdNS3 transduced DC shifted the antigen specific immunity towards Th1 responses. DC transduced with AdNS3 are superior to DC pulsed with rNS3 in inducing vigorous humoral and Th1-type cellular responses against NS3. The results demonstrate for the first time the immunogenic potential of genetically modified DC by a prime and boost approach in eliciting a strong NS3-specific, cell-mediated, humoral immune response in both Balb/c mice and HLA-A2.1 transgenic mice.

Centrifugation enhances integrin-mediated transduction of dendritic cells by conventional and RGD-modified adenoviral vectors

The level of antigen loading can impact on the capacity for dendritic cells (DC) to activate T cell responses. Several different approaches to adenoviral (Ad)-based transduction were therefore assessed for their effect on both transgene expression and T cell activation. While a conventional E1(−)/E3Δ Ad vector (Ad/GFP) produced a concentration-dependent expression of GFP, a modified vector expressing Arginine–Glycine–Aspartic Acid (RGD) sequence on its fiber knob (Ad-RGD/GFP) enhanced transgene expression by 9–20-fold at each MOI. The addition of centrifugal force (2000× g) during DC transduction with Ad/GFP also increased expression up to 20-fold. However, combining centrifugation with the Ad-RGD/GFP vector produced no effect on transduction rate and only a 1.5- to 2-fold increase in GFP expression, suggesting overlapping mechanisms of action. Consistent with this, exogenous RGD peptide blocked transduction regardless of the vector used, or the addition of centrifugal force, and transduction was primarily limited to DC expressing the CD51 integrin receptor. Ad vectors expressing ovalbumin (OVA) were used to assess transduced DC for their capacity to activate OVA-specific T cells. We observed a significant relationship between transgene expression and the capacity for T cell activation regardless of whether transgene expression was increased by using a higher MOI, an RGD-modified vector, or by employing centrifugal force. Furthermore, combining these approaches produced synergistic effects on T cell activation. We conclude that RGD-modified vectors and centrifugation both enhance DC transduction by increasing entry via integrin receptors and that the capacity for T cell activation can be optimized by combining approaches to achieve the highest possible level of transgene expression.

Manipulation of dendritic cells for host defence against intracellular infections

Dendritic cells (DCs) are an important innate immune cell type which is the bridge between innate and adaptive immunity. Mounting experimental evidence suggests that manipulating DCs represents a powerful means to enhance host defence against intracellular infectious diseases. We have developed several strategies to manipulate DCs either in vivo or in vitro for the purpose of enhancing the effect of vaccination or immunotherapeutics. In vivo delivery of transgene encoding GM-CSF (granulocyte/macrophage colony-stimulating factor), a DC-activating cytokine, increases the number and activation status of DCs at various tissue sites and enhances antimicrobial immune responses in murine models. Co-expression or co-delivery of GM-CSF gene transfer vector with an antimicrobial vaccine enhances microbial antigen-specific T-cell responses and immune protection. Murine bone marrow-derived DCs are being manipulated in vitro and exploited as a vaccine delivery system. Transduction of DCs with a virus-vectored tuberculosis vaccine is a powerful way to activate T-cells in vivo. Such genetically modified DC vaccines can be administered either parenterally or mucosally via the respiratory tract.

Manipulation of dendritic cells for host defence against intracellular infections

Dendritic cells (DCs) are an important innate immune cell type which is the bridge between innate and adaptive immunity. Mounting experimental evidence suggests that manipulating DCs represents a powerful means to enhance host defence against intracellular infectious diseases. We have developed several strategies to manipulate DCs either in vivo or in vitro for the purpose of enhancing the effect of vaccination or immunotherapeutics. In vivo delivery of transgene encoding GM-CSF (granulocyte/macrophage colony-stimulating factor), a DC-activating cytokine, increases the number and activation status of DCs at various tissue sites and enhances antimicrobial immune responses in murine models. Co-expression or co-delivery of GM-CSF gene transfer vector with an antimicrobial vaccine enhances microbial antigen-specific T-cell responses and immune protection. Murine bone marrow-derived DCs are being manipulated in vitro and exploited as a vaccine delivery system. Transduction of DCs with a virus-vectored tuberculosis vaccine is a powerful way to activate T-cells in vivo. Such genetically modified DC vaccines can be administered either parenterally or mucosally via the respiratory tract.

627. Calcium Phopspate Precipitation of Adenovirus Enhances Gene Delivery into Human Dendritic Cells

Top of pageAbstract Mature dendritic cells (DC) are potent antigen presenting cells (APC) that have been used in vaccine studies as well as adoptive immunotherapy protocols. Adenovirus (Ad) vectors are frequently used to genetically modify DC; however gene transfer with recombinant adenovirus (serotype 5) into human DC is inefficient since DC do not express the cognate Ad5 receptor. Calcium phosphate (CaPi) precipitation of Ad has been shown to enhance gene transfer into murine DC and the aim of this study was to evaluate if CaPi precipitation enhances the gene delivery into human DC without interfering with their ability to activate antigen-specific T cells. Immature, monocyte derived DC were transduced with Ad5 or Ad5:CaPi complexes and gene expression was determined using GFP as a marker gene. CaPi precipitation of Ad5 increased DC gene transfer on average 35-fold when compared to unmodified Ad5. Ad5:CaPi complexes mediated gene delivery was similar in direct comparison to the optimal Ad for DC gene transfer (Ad5f35). Moreover, CaPi also increased the transduction efficiency of Ad5f35 at least 2-fold. CaPi transduction of immature DC did not reduce cell viability or their ability to be matured as judged by expression of CD83, and DR. To determine if Ad:CaPi treated DC can reactivate antigen-specific T cells, DC were incubated with an Ad5f35 vector encoding the subdominant Epstein Barr virus (EBV) antigens, latent membrane proteins (LMP) 2, which is expressed in EBV-positive malignancies. CaPi precipitation increased the expression of LMP2 in human DC 5|[ndash]|10 fold compared to virus alone, resulting in enhanced activation of LMP2-specific T cells in vitro. In conclusion, CaPi complexes increased gene transfer into human DC with both Ad5 and Ad5f35 vectors without changing their phenotype or their ability to activate antigen-specific T cells. Ad5:CaPi mediated transduction resulted in similar transduction as Ad5f35 vectors, which allows the use of existing Ad5 vector technology to genetically modify human DC.

Lentiviral vector expression of tumour antigens in dendritic cells as an immunotherapeutic strategy.

Therapeutic cancer vaccines need to stimulate a refractory immune system to make an effective anti-tumour response. We have explored the use of lentiviral vectors to deliver tumour antigen genes to dendritic cells (DC) as a possible mechanism of immune stimulation. Direct injection of a lentiviral vector encoding the melanoma antigen NY-ESO-1 in HLA-A2 transgenic mice primed NY-ESO-1-specific CD8+ cells that could be expanded by boosting with an NY-ESO-1 vaccinia virus. The expanded cells could kill NY-ESO-1(157-165) peptide-pulsed targets in vivo. In order to examine the priming step directly, we constructed another lentiviral vector expressing the melanoma antigen Melan-A (MART-1). Here we show that Melan-A protein is also efficiently expressed after transduction of human DC cultured from peripheral blood mononuclear cells. When these transduced DC are co-cultured with autologous naïve T cells, they cause the expansion of cells that recognise the HLA-A2 restricted Melan-A(27-35) epitope. The expanded cells are functional in that they release IFN-gamma upon antigen stimulation. Melan-A lentiviral vector transduced DC caused a similar level of naïve T-cell expansion to Melan-A(27-35) peptide-pulsed DC in four experiments using different HLA-A2 positive donors. These data suggest that a vaccine based either on DC transduced with a lentiviral vector ex vivo, or on direct lentiviral vector injection, should be assessed in a phase I clinical trial.

Enhanced transduction of mouse bone marrow-derived dendritic cells by repetitive infection with self-complementary adeno-associated virus 6 combined with immunostimulatory ligands

The potential of adeno-associated virus (AAV)-based vectors in human gene therapy is being explored for several diseases. Although sustained transgene expression and low vector-associated cellular immunity are attractive features of recombinant (r) AAV, the wider application of rAAV vectors encapsidated in serotype 2 capsid is hampered by poor transduction efficiency in many target tissues. These include ex vivo-generated dendritic cells (DC), which have demonstrated promising immunotherapeutic activity. We report here that efficient transduction of mouse bone marrow-derived DC can be achieved with self-complementary (sc) rAAV encapsidated in serotype 6 capsid. Sequential exposure of DC precursor cultures to IL-4 and GM-CSF with sc rAAV6 encoding the human tumor antigen, carcinoembryonic antigen (CEA), for 7 days followed by activation with CpG oligodeoxynucleotides (ODN) and anti-mouse CD40 antibody resulted in highly efficient transduction of DC. DC surface markers as determined by flow cytometry analysis of sc rAAV6-transduced DC were comparable to nontransduced DC. Efficiency of vector transduction and transgene expression were confirmed by immunostaining and real-time PCR. Microarray analysis of RNA from CpG ODN and CD40 antibody stimulated sc AAV6-transduced DC revealed upregulation of transcription factors and cytokines involved in immune activation and downregulation of inhibitory factors, suggesting a possible role of transcriptional activation in the observed effect. The adoptive transfer into syngeneic mice of the ex vivo-transduced and activated DC resulted in the development of CEA-specific antibody and T-helper 1-associated immune responses. Immunized mice also developed antibody to AAV6 capsid protein, which did not crossreact with AAV2 capsid protein. These studies demonstrate the potential utility of sc rAAV serotype 6-based vectors in transduction of DC for genetic vaccination approaches.

Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy

AbstractGenetic modification of dendritic-cell (DC) function is an attractive approach to treat disease, either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of DCs. Adenovirus (Ad), human immunodeficiency virus (HIV), equine anemia virus (EIAV), and Moloney murine leukemia virus (MMLV) all up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as, in the case of Ad and lentiviral vectors, inducing production of Th1 and proinflammatory cytokines. Following transduction there is activation of double-stranded (ds) RNA-triggered pathways resulting in interferon (IFN) α/β production. In addition, the function of virally infected DCs is altered; iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Viral transduction of mDCs results in up-regulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which down-regulates T-cell responsiveness. Inhibition of IDO restores the ability of mDCs to stimulate an MLR, indicating that IDO is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DCs.

849. Transduction of Mouse Dendritic Cells Is Enhanced by Repetitive Infection with Self-Complementary Adeno-Associated Virus 6 Combined with Immunostimulatory Ligands

The potential of adeno-associated virus (AAV)-based vectors in human gene therapy is being explored for several diseases. Although sustained transgene expression and low vector-associated cellular immunity are attractive features of recombinant (r) AAV, the wider application rAAV vectors encapsidated in serotype 2 capsid, the most commonly applied rAVV, is hampered by poor transduction efficiency in many target tissues. These include ex vivo generated dendritic cells (DC), which have demonstrated promising immunotherapeutic activity. We report here that efficient transduction of mouse bone marrow-derived DC can be achieved with self-complementary (sc) rAAV encapsidated in serotype 6 capsid. Sequential exposure of DC precursor cultures to IL-4 and GM-CSF with sc rAAV6 encoding the human tumor antigen, carcinoembryonic antigen (CEA), for seven days followed by activation with CpG oligodeoxynucleotides (ODN) and anti-mouse CD40 antibody resulted in highly efficient transduction of DC. DC surface markers as determined by flow cytometry analysis of sc rAAV6-transduced DC were comparable to non-transduced DC. Efficiency of vector transduction and transgene expression were confirmed by immunostaining and real-time PCR. Microarray analysis of RNA from CpG-ODN and CD40 antibody stimulated sc AAV6-transduced DC revealed upregulation of transcription factors and cytokines involved in immune activation and down-regulation of inhibitory factors, suggesting a possible role of transcriptional activation in the observed effect. The adoptive transfer into syngeneic mice of the ex vivo transduced and activated DC resulted in the development of CEA-specific antibody. Immunized mice also developed antibody to AAV-6 capsid protein which did not cross-react with AAV-2 capsid protein. These studies demonstrate the potential utility of sc rAAV serotype 6-based vectors in transduction of DC for genetic vaccination approaches.

981. Modulation of Human Dendritic Cell Function Following Viral Vectors: Implications for Gene Therapy in Vaccination

The use of genes to modify dendritic cell (DC) function is an attractive approach to treat many diseases; either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. While viral vectors are efficient at transducing DCs, their effect on the phenotype and function of DCs is less well characterised. We have investigated the effect of transduction with a variety of viral vectors. We show that adenovirus (Ad), human immunodeficiency virus (HIV), equine anaemia virus (EIAV) and Moloney murine leukemia virus (MMLV) can upregulate co-stimulatory molecules and MHC class II expression on DC, as well as inducing production of Th1 and proinflammatory cytokines. Following transduction with these vectors, there is upregulation of dsRNA-triggered pathways which result in IFN/ production, an established component of innate antiviral immunity. In addition, the function of virally-infected DCs is altered; the ability of iDCs to stimulate a mixed lymphocyte reaction (MLR) was increased and ability of mDCs to stimulate a MLR was decreased. We have shown that viral transduction of mDC results in upregulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which downregulates T cell responsiveness. Inhibition of the activity of IDO blocks the reduction in the MLR seen with mDC following viral transduction, indicating that this enzyme is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DC.

1130. A Single-Component CD40-Targeted Adenoviral Vector for Efficient Transduction and Activation of Cutaneous Dendritic Cells

Dendritic cell (DC)-based tumor vaccination usually involves the administration of ex vivo generated autologous DC. Transduction of DC by viral vectors in vivo has been proposed as a more standardized, and easily clinically applicable approach. Previously, we have employed retargeting approaches to route adenoviral (Ad) vectors via the CD40 pathway as a means to circumvent the paucity of the Ad5 virus's primary receptor (CAR), on the surface of DC and to selectively target DC in situ. CD40-targeted Ad-mediated transduction of DC has been successfully realized through the use of bispecific adapter proteins. With these CD40-targeted Ad complexes we have shown an enhanced and more selective transduction as well as a simultaneous CD40-dependent maturation of DC. However, these approaches required the production of several components followed by physical association to form targeted vector complexes. Evidently for those techniques the presence of an excess of free targeting ligands in analyzed samples leaves open questions about the causal component of DC activation and the ability of targeting ligands as integral components of viral particles to induce this process with similar efficiency. Direct incorporation of the TNF-like domain of human CD40L into the Ad capsid, preserving the targeting ability of the genetically modified Ad vector, now allows us to answer these questions and offers a single-component targeted vector for direct clinical application. In the present study, we evaluate the ability of this vector to deliver transgenes in a stringent human substrate system. We report the capacity of this CD40-targeted vector to infect, with high efficiency, cutaneous DC resident in human skin explants, while simultaneously inducing their activation and maturation. This latest generation of single-component, fully targeted vectors should make feasible the clinical testing of in vivo DC-targeted vaccines.

68. Development of a Novel Non-Viral DNA Vaccine-Delivering Gene into the Nucleus of Dendritic Cells

DNA vaccine has potential advantages exceeding the conventional vaccine. Recently, dendritic cells (DCs) attract attention as a target of DNA vaccine because DCs have a key role in initiating and controlling immune responses and are the most potent antigen-presenting cells (APCs). Currently, recombinant adenovirus (rAd) is used as the most efficient method for DC transduction. However, it is known that the DC transduction using rAd at a relatively high multiplicity of infection (MOI) interferes DC function and elicit strong immune response by presence of viral antigen. Therefore, the development of efficient non-viral gene vector is required to solve this problem.

533. Enhanced Transduction of Dendritic Cells by FCgRI-Targeted Adenovirus Vectorsγ

The production of an efficacious HIV-1 vaccine capable of inducing the humoral and cellular arms of the immune system continues to remain a major challenge in AIDS research. Dendritic cells (DC) are an attractive target for vaccine delivery because vaccine antigens presented to DC can potentially activate effector cells involved in immune defense. Adenovirus (Ad) has been widely used as a vector to express foreign proteins in a variety of cell types and tissues. However, this broad tropism represents a drawback when selective gene delivery is desired. In particular, inefficient gene delivery to antigen presenting cells (APC) is attributed to the absence of the Coxsackie and Adenovirus receptor (CAR). Based on the hypothesis that Ad vectors with improved infectivity of APC may be able to promote an enhanced immune response against a vectored antigen, our objective is to selectively deliver Ad vectors to APC by directing virus to the high affinity Fc-gamma Receptor I (FcgRI), constitutively expressed on DC. To this end, we constructed a bi-specific targeting molecule, H22sCAR, consisting of the extracellular domain of CAR (sCAR), a bacteriophage T4 fibritin trimerization domain, and a single-chain antibody molecule (H22) specific for the human FcgRI receptor. H22sCAR was produced in a baculovirus expression system, and was demonstrated to bind both the Ad fiber knob protein and cellular FcgRI. Ad vectors pre-complexed with H22sCAR enhanced the delivery of GFP and luciferase reporter genes to FcgRI-expressing cell lines, and to monocyte-derived DC, when compared with Ad alone or Ad targeted with an irrelevant control protein (Her2sCAR). These data support the hypothesis that FcgRI-targeted Ad vectors may enhance the delivery of target antigen to APC, and lend support to future studies on the use of FcgRI-targeted adenovirus vectors for HIV-1 vaccine development.

A Single-Component CD40-Targeted Adenovirus Vector Displays Highly Efficient Transduction and Activation of Dendritic Cells in a Human Skin Substrate System

Dendritic cell (DC) based tumor vaccination usually involves the administration of ex vivo generated autologous DC. Transduction of DC by viral vectors in vivo has been proposed as a more standardized and easily clinically applicable approach. Previously, we have reported that an Ad5 vector targeted to CD40 via genetic capsid incorporation of CD40L achieves selective transduction of DC in vitro. In the present study, we evaluate the ability of this vector to deliver transgenes in a stringent human substrate system. We report the capacity of this CD40-targeted vector to infect, with high efficiency, cutaneous DC resident in human skin explants, while simultaneously inducing their activation and maturation. This latest generation of single-component, fully targeted vectors should make feasible the clinical testing of in vivo DC-targeted vaccines.

In Vivo Transduction of Thymic Dendritic Cells with Adenovirus and Its Potential Use in Acute Inflammatory Diseases

Dendritic cells (DC) represent a potential target for gene therapy. In their ability to process antigens and present them to T cells, DC have been allocated a unique role as initiators of the immune response in both the innate and acquired immunity. Recent in vitro studies have showed the feasibility of DC transduction with adenoviral recombinants. In cancer therapy, targeting of DC with adenovirus has been proved to be effective in inhibiting tumour growth, as well as in reducing the number of tumour metastases. The aim of our study is to evaluate the feasibility of in vivo transduction of DC in a murine lymphocyte-rich compartment (thymus) as a potential treatment for acute inflammatory diseases. Nearly 50% of the total thymic DC were transduced with a first-generation adenoviral construct following intrathymic injection, and post-transductional inflammation was neglectable. Transduction of thymic cells with adenoviral recombinants was able to induce the expression of an intracellular protein (beta-galactosidase, green fluorescent protein), as well as the secretion of human interleukin-10, within the local compartment. Furthermore, this induction of the latter significantly decreased thymic apoptosis in the applied model of acute bacterial peritonitis (cecal ligation and puncture).

374 Transduction of dendritic cells with CD40-ligand recombinant adenovirus enhances the stimulatory capacity of immunologic effector cells against HCC-cells in vitro

374 Transduction of dendritic cells with CD40-ligand recombinant adenovirus enhances the stimulatory capacity of immunologic effector cells against HCC-cells in vitro

High efficiency transduction of dendritic cells by adenoviral vectors targeted to DC-SIGN

Dendritic cells (DCs) are a central element in the development of antigen-specific immune responses. The lack of a specific and efficient technique for the in vivo delivery of antigens to DCs remains a major obstacle limiting a vaccine’s ability to induce an effective immune response. The efficacy of adenoviral (Ad) vectors in this regard can be enhanced through alterations in vector tropism such that DC-targeted transduction is achieved. Here, the efficiency of DC transduction by Ad vectors retargeted to DC-specific ICAM-3 grabbing non-integrin (DC-SIGN) was studied and compared to that of Ad vectors retargeted through CD40. A comparable and significant enhancement of gene transfer to monocyte derived DCs (MDDCs) was accomplished by means of an Ad vector harboring the Fc-binding domain of Staphylococcus aureus protein A in combination with antibodies to DC-SIGN or to CD40 or with fused complexes of human Ig-Fc with their natural ligands, i.e. ICAM-3 or CD40L, respectively. Whereas CD40-targeted Ad transduction resulted in a more profound phenotypic DC maturation, DC-SIGN- and CD40-targeted Ad both induced similar levels of IL-12 secretion. These data demonstrate the usefulness of DC-SIGN as a DC-restricted targeting motif for Ad-mediated vaccination strategies.

Human Dendritic Cells Transduced with Herpes Simplex Virus Amplicons Encoding Human Immunodeficiency Virus Type 1 (HIV-1) gp120 Elicit Adaptive Immune Responses from Human Cells Engrafted into NOD/SCID Mice and Confer Partial Protection against HIV-1 Challenge

Small-animal models are needed to test human immunodeficiency virus (HIV) vaccine efficacy following viral challenge. To this end, we examined HIV-1-specific immune responses following immunization of nonobese diabetic-severe combined immunodeficient mice that were repopulated with human peripheral blood lymphocytes (hu-PBL-NOD/SCID mice). Autologous dendritic cells (DC) were transduced ex vivo with replication-defective, helper virus-free, herpes simplex virus type 1 (HSV-1) amplicons that expressed HIV-1 gp120 and were then injected into the hu-PBL-NOD/SCID mice. This resulted in primary HIV-1-specific humoral and cellular immune responses. Serum samples from vaccinated animals contained human immunoglobulin G that reacted with HIV-1 Env proteins by enzyme-linked immunosorbent assay and neutralized the infectivity of HIV-1 LAI and ADA strains. T cells isolated from the mice responded to viral antigens by producing gamma interferon when analyzed by enzyme-linked immunospot assay. Importantly, exposure of the vaccinated animals to infectious HIV-1 demonstrated partial protection against infectious HIV-1 challenge. This was reflected by a reduction in HIV-1(ADA) and by protection of the engrafted human CD4(+) T lymphocytes against HIV-1(LAI)-induced cytotoxicity. These data demonstrate that transduction of DC by HSV amplicon vectors expressing HIV-1 gp120 induce virus-specific immune responses in hu-PBL-NOD/SCID mice. This mouse model may be a useful tool to evaluate human immune responses and protection against viral infection following vaccination.