Lentiviral Vectors Targeted to MHC II Are Effective in Immunization

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؉ T lymphocytes are a mainstay of antitumoral immune responses, PTVs could be engineered for the transfer of specific tumor antigens provoking tailored antitumoral immunity. Therefore, PTVs can be used as safe and efficient alternatives to gene transfer vectors or live attenuated replicating vector platforms, avoiding genotoxicity or general toxicity in highly immunocompromised patients, respectively. Thereby, the potential for easy envelope exchange allows the circumventing of neutralizing antibodies, e.g., during repeated boost immunizations. V accination is the administration of one or more immunogens, the vaccine, into patients to trigger antigen-specific adaptive immune responses to prevent (prophylactic vaccination) or to treat (therapeutic vaccination) disease. Vaccines can be classified into several subtypes. Among these are live attenuated replicating vaccines, inactivated vaccines, subunit vaccines, DNA vaccines, or recombinant vector vaccines (for review, see reference 1). Wellknown live attenuated vaccines are, e.g., those against measles (2) or mumps (3). These vaccines replicate but have been attenuated to become apathogenic. The immune responses triggered by live attenuated vaccines are similar to those induced by the pathogenic form of the microbe (4, 5) and involve both the cellular and humoral arms of the immune system. However, attenuated replicating pathogens may carry an inherent risk of reversion to the parental virulent form by in vivo passaging during vaccination, as observed for the Sabin strain used as a polio vaccine (6), or may still be pathogenic in highly immunocompromised patients (7), depending on the respective degree of attenuation of the vaccine strains. On the other hand, inoculation of solely proteinaceous antigens (such as the hepatitis B virus vaccine [8]) or antigenencoding genes (as a DNA vaccine) is regarded as safe but relatively inefficient (9).As an alternative to such vaccines, the genes encoding an antigen can be transferred into cells and thereby presented to the immune system by using recombinant vaccine vectors. For that purpose, an attenuated vector is utilized as a carrier for the antigen-encoding sequences of another pathogen. Thereby, they are

Section: Generation and Characterization Of Lentiviral Protein Transfmentioning

confidence: 99%

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confidence: 99%

Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform and Induce a Strong Antigen-Specific Cytotoxic T Cell Response

Uhlig

Schülke

Scheuplein

et al. 2015

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“…Furthermore, the lentivectors' envelopes are well suited for engineering, enabling the design of targeted lentivectors. Several methods have been described to redirect lentivectors to specific antigen-presenting cells (9)(10)(11)(12). However, to our knowledge subset-specific delivery of transgenes has not been described.…”

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confidence: 99%

Targeting of Human Antigen-Presenting Cell Subsets

Goyvaerts

Dingemans

Groeve

et al. 2013

dAntigen-presenting cells are a heterogeneous group of cells that are characterized by their functional specialization. Consequently, targeting specific antigen-presenting cell subsets offers opportunities to induce distinct T cell responses. Here we report on the generation and use of nanobodies (Nbs) to target lentivectors specifically to human lymph node-resident myeloid dendritic cells, demonstrating that Nbs represent a powerful tool to redirect lentivectors to human antigen-presenting cell subsets. Since the discovery of dendritic cells (DCs) in 1973, these antigen-presenting cells have been at the center of attention in vaccine development (1). In the past decade, an extra dimension of complexity was introduced by revealing an unforeseen diversity of DC subsets with distinct functions and locations (2-4). Since DC subsets induce distinct immune responses, it might be beneficial to develop strategies to target particular DC subsets and as such exploit the immune system to its full potential (5).Numerous animal studies have proven the efficiency and safety of lentivectors (LVs) as vaccination moieties (6, 7). As lentivectors are intrinsically immunogenic, they deliver both antigens and activation signals to antigen-presenting cells (8). Furthermore, the lentivectors' envelopes are well suited for engineering, enabling the design of targeted lentivectors. Several methods have been described to redirect lentivectors to specific antigen-presenting cells (9-12). However, to our knowledge subset-specific delivery of transgenes has not been described.Targeting myeloid DCs could be advantageous as they are considered to be important mediators of antigen-specific immunity. They are able to induce proper and oriented stimulation of CD4 ϩ T helper 1 and CD8 ϩ cytotoxic T cells. In addition, targeting may reduce the risk of adverse reactions such as autoimmune responses or induction of tolerance due to transgene expression and presentation by non-antigen-presenting cells or tolerogenic DC subtypes. Finally, as myeloid DCs have a limited life span, their targeting should result in a natural clearance of the lentivector and as such in a reduction of the risk of insertional mutagenesis.We recently delivered a proof of concept on the use of nanobodies (Nbs) to target lentivectors to antigen-presenting cells (10). Nbs or V H H fragments are antibody fragments of about 12 to 25 kDa that are engineered from heavy-chain-only antibodies found in Camelidae. Because of their size and target affinity, they are of particular interest as targeting moieties. In the present study, we further refined the transduction profile of lentivectors, targeting them to human myeloid DCs using Nbs.Two Nb libraries, derived from peripheral blood lymphocytes of llamas that were immunized with immature or lipopolysaccharide-stimulated murine bone marrow-derived DCs, were at our disposal (13). These were screened for cross-reactivity with hu-

“…By truncating their cytoplasmic tails, lentiviral vectors (LVs) have been pseudotyped with the MV glycoproteins (F⌬30 and H mut ⌬18), and the MV-based retargeting system has been transferred to this important vector type for gene therapy applications (21,22). Thereby, the list of cell surface receptors used by the MV glycoproteins has been further extended to also include surface marker proteins relevant in immunology, hematology, and neurobiology (21,23,24). Usually, single-chain variable fragments (scFvs) derived from monoclonal antibodies or selected by phage display library screening have been used as targeting ligands (25).…”

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confidence: 99%

The Receptor Attachment Function of Measles Virus Hemagglutinin Can Be Replaced with an Autonomous Protein That Binds Her2/ neu While Maintaining Its Fusion-Helper Function

Rasbach

Abel²,

Münch³

et al. 2013

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cCell entry of enveloped viruses is initiated by attachment to the virus receptor followed by fusion between the virus and host cell membranes. Measles virus (MV) attachment to its receptor is mediated by the hemagglutinin (H), which is thought to produce conformational changes in the membrane fusion protein (F) that trigger insertion of its fusion peptide into the target cell membrane. Here, we uncoupled receptor attachment and the fusion-helper function of H by introducing Y481A, R533A, S548L, and F549S mutations into the viral attachment protein that made it blind to its normal receptors. An artificial receptor attachment protein specific for Her2/neu was incorporated into the membranes of pseudotyped lentivirus particles as a separate transmembrane protein along with the F protein. Surprisingly, these particles entered efficiently into Her2/neu-positive SK-OV-3 as well as CHO-Her2 cells. Cell entry was independent of endocytosis but strictly dependent on the presence of H. H-specific monoclonal antibodies, as well as a mutation in H interfering with H/F cooperation, blocked cell entry. The particles mediated stable and specific transfer of reporter genes into Her2/neu-positive human tumor cells also in vivo, while exhibiting improved infectivity and higher titers than Her2/neu-targeted vectors displaying the targeting domain on H. Extending the current model of MV cell entry, the data suggest that receptor binding of H is not required for its fusion-helper function but that particle-cell contact in general may be sufficient to induce the conformational changes in the H/F complex and activate membrane fusion.