Characterization of immunologic properties of a second HLA-A2 epitope from a granule protease in CML patients and HLA-A2 transgenic mice.

The serine proteases, neutrophil elastase (HNE) and proteinase 3 (PR3), are aberrantly expressed in human myeloid leukemias. T-cell responses to these proteins have been correlated with remission in patients with chronic myeloid leukemia (CML). Human PR3/HNE-specific CD8(+) T cells predominantly recognize a nonameric HLA-A2-restricted T-cell epitope called PR1 which is conserved in both Ags. However, CML patients have CD8(+) T cells in peripheral blood recognizing an additional HLA-A2 epitope termed PR2. To assess immunologic properties of these Ags, novel recombinant vaccinia viruses (rVV) expressing PR3 and HNE were evaluated in HLA-A2 transgenic (Tg) mice (HHDII). Immunization of HHDII mice with rVV-PR3 elicited a robust PR3-specific CD8(+) T-cell response dominated by recognition of PR2, with minimal recognition of the PR1 epitope. This result was unexpected, because the PR2 peptide has been reported to bind poorly to HLA. To account for these findings, we proposed that HHDII mice negatively selected PR1-specific T cells because of the presence of this epitope within murine PR3 and HNE, leading to immunodominance of PR2-specific responses. PR2-specific splenocytes are cytotoxic to targets expressing naturally processed PR3, though PR1-specific splenocytes are not. We conclude that PR2 represents a functional T-cell epitope recognized in mice and human leukemia patients. These studies are registered at www.clinicaltrials.gov as NCT00716911.

Activation of the inflammasome and enhanced migration of microparticle-stimulated dendritic cells to the draining lymph node.

Porous silicon microparticles presenting pathogen-associated molecular patterns mimic pathogens, enhancing internalization of the microparticles and activation of antigen presenting dendritic cells. We demonstrate abundant uptake of microparticles bound by the TLR-4 ligands LPS and MPL by murine bone marrow-derived dendritic cells (BMDC). Labeled microparticles induce concentration-dependent production of IL-1ß, with inhibition by the caspase inhibitor Z-VAD-FMK supporting activation of the NLRP3-dependent inflammasome. Inoculation of BALB/c mice with ligand-bound microparticles induces a significant increase in circulating levels of IL-1ß, TNF-α, and IL-6. Stimulation of BMDC with ligand-bound microparticles increases surface expression of costimulatory and MHC molecules, and enhances migration of BMDC to the draining lymph node. LPS-microparticles stimulate in vivo C57BL/6 BMDC and OT-1 transgenic T cell interactions in the presence of OVA SIINFEKL peptide in lymph nodes, with intact nodes imaged using two-photon microscopy. Formation of in vivo and in vitro immunological synapses between BMDC, loaded with OVA peptide and LPS-microparticles, and OT-1 T cells are presented, as well as elevated intracellular interferon gamma levels in CD8(+) T cells stimulated by BMDC carrying peptide-loaded microparticles. In short, ligand-bound microparticles enhance (1) phagocytosis of microparticles; (2) BMDC inflammasome activation and upregulation of costimulatory and MHC molecules; (3) cellular migration of BMDC to lymphatic tissue; and (4) cellular interactions leading to T cell activation in the presence of antigen.

IFN-alpha enhances peptide vaccine-induced CD8+ T cell numbers, effector function, and antitumor activity.

Type I IFNs, including IFN-alpha, enhance Ag presentation and promote the expansion, survival, and effector function of CD8(+) CTL during viral infection. Because these are ideal characteristics for a vaccine adjuvant, we examined the efficacy and mechanism of exogenous IFN-alpha as an adjuvant for antimelanoma peptide vaccination. We studied the expansion of pmel-1 transgenic CD8(+) T cells specific for the gp100 melanocyte differentiation Ag after vaccination of mice with gp100(25-33) peptide in IFA. IFN-alpha synergized with peptide vaccination in a dose-dependent manner by boosting relative and absolute numbers of gp100-specific T cells that suppressed B16 melanoma growth. IFN-alpha dramatically increased the accumulation of gp100-specific, IFN-gamma-secreting, CD8(+) T cells in the tumor through reduced apoptosis and enhanced proliferation of Ag-specific CD8(+) T cells. IFN-alpha treatment also greatly increased the long-term maintenance of pmel-1 CD8(+) T cells with an effector memory phenotype, a process that required expression of IFN-alpha receptor on the T cells and IL-15 in the host. These results demonstrate the efficacy of IFN-alpha as an adjuvant for peptide vaccination, give insight into its mechanism of action, and provide a rationale for clinical trials in which vaccination is combined with standard-of-care IFN-alpha therapy for melanoma.

Rad23 as a reciprocal agent for stimulating or repressing immune responses.

The proteasome degrades cellular proteins and provides peptides for major histocompatibility complex (MHC) class I molecules to drive CD8+ T-cell responses to kill intracellular pathogens. Rad23 plays a role in protein degradation by targeting polyubiquitinated substrates to the proteasome via an N-terminal ubiquitin-like (UbL) domain that binds the proteasome and two C-terminal ubiquitin-associated (UBA) domains that bind ubiquitinated proteins. We demonstrate here that fusion of Rad23 or its UBA domain to the green fluorescent protein (GFP) targets this antigen to the proteasome for increased degradation in mammalian cells and enhanced antigen-specific CD8+ T-cell responses in BALB/c mice. Conversely, we show that coexpression of unfused Rad23 with destabilized GFP inhibits degradation of the reporter protein and attenuates in vivo CD8+ T-cell responses. Rad23 therefore holds promise as a useful agent either to enhance or attenuate cellular immune responses to suit the reciprocal immunologic needs of both gene therapy and genetic vaccine applications.

Silencing of Tumor Necrosis Factor Receptor-1 in Human Lung Microvascular Endothelial Cells Using Particle Platforms for siRNA Delivery.

Acute lung injury (ALI) and its most severe manifestation, acute respiratory distress syndrome (ARDS), is a clinical syndrome defined by acute hypoxemic respiratory failure and bilateral pulmonary infiltrates consistent with edema. In-hospital mortality is 38.5% for AL, and 41.1% for ARDS. Activation of alveolar macrophages in the donor lung causes the release of pro-inflammatory chemokines and cytokines, such as TNF-α. To determine the relevance of TNF-α in disrupting bronchial endothelial cell function, we stimulated human THP-1 macrophages with lipopolysaccharide (LPS) and used the resulting cytokine-supplemented media to disrupt normal endothelial cell functions. Endothelial tube formation was disrupted in the presence of LPS-activated THP- 1 conditioned media, with reversal of the effect occurring in the presence of 0.1µg/ml Enbrel, indicating that TNF-α was the major serum component inhibiting endothelial tube formation. To facilitate lung conditioning, we tested liposomal and porous silicon (pSi) delivery systems for their ability to selectively silence TNFR1 using siRNA technology. Of the three types of liposomes tested, only cationic liposomes had substantial endothelial uptake, with human cells taking up 10-fold more liposomes than their pig counterparts; however, non-specific cellular activation prohibited their use as immunosuppressive agents. On the other hand, pSi microparticles enabled the accumulation of large amounts of siRNA in endothelial cells compared to standard transfection with Lipofectamine(®) LTX, in the absence of non-specific activation of endothelia. Silencing of TNFR1 decreased TNF-α mediated inhibition of endothelial tube formation, as well as TNF-α-induced upregulation of ICAM-1, VCAM, and E-selection in human lung microvascular endothelial cells.

HSP70 promoter-driven activation of gene expression for immunotherapy using gold nanorods and near infrared light.

Modulation of the cytokine milieu is one approach for vaccine development. However, therapy with pro-inflammatory cytokines, such as IL-12, is limited in practice due to adverse systemic effects. Spatially-restricted gene expression circumvents this problem by enabling localized amplification. Intracellular co-delivery of gold nanorods (AuNR) and a heat shock protein 70 (HSP70) promoter-driven expression vector enables gene expression in response to near infrared (NIR) light. AuNRs absorb the light, convert it into heat and thereby stimulate photothermal expression of the cytokine. As proof-of-concept, human HeLa and murine B16 cancer cells were transfected with a HSP70-Enhanced Green Fluorescent Protein (EGFP) plasmid and polyethylenimine (PEI)-conjugated AuNRs. Exposure to either 42 °C heat-shock or NIR light induced significant expression of the reporter gene. In vivo NIR driven expression of the reporter gene was confirmed at 6 and 24 h in mice bearing B16 melanoma tumors using in vivo imaging and flow-cytometric analysis. Overall, we demonstrate a novel opportunity for site-directed, heat-inducible expression of a gene based upon the NIR-absorbing properties of AuNRs and a HSP70 promoter-driven expression vector.

Activation of refractory T cell responses against hepatitis C virus core protein by ablation of interfering hydrophobic domains.

Hepatitis C virus (HCV) is the major pathogen of chronic hepatitis and liver disease, but currently there are no prophylactic HCV vaccines available. The HCV core protein-encoding sequence is among the most conserved genes in the HCV genome, making it a prime candidate for a component of a vaccine. The core protein localizes to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that is cotranslationally inserted into the ER membrane. Here we show that removal of the C-terminal hydrophobic region confers nuclear localization and enhances proteasomal degradation of the core protein in mammalian cells. This efficient protein proteolysis induces enhanced core-specific CD8(+) T cell responses in BALB/c mice immunized with plasmids expressing C-terminal deletions of the HCV core protein. These results suggest that more potent HCV vaccines can be achieved by targeting the core protein for proteasomal degradation by deletion of its C-terminal hydrophobic domain.

Maximizing antigen targeting to the proteasome for gene-based vaccines.

Wild-type or immunoevasive antigens can drive weak CD8+T-cell responses against both dominant and subdominant epitopes during gene-based vaccination. For many antigens, fusion to ubiquitin (Ub) to target them to the proteasome circumvents this problem. Although this procedure works in most cases, for one subset of antigens, Ub fusion does not improve immune responses. To determine why these failures occur, we have evaluated in detail the 'rules' for proteasome targeting that have been applied in mammalian vaccine studies, but that were actually defined in yeast systems. To do this, we fused a series of engineered Ub genes to green fluorescent protein (GFP) and tested their ability to target GFP to the proteasome for enhanced antigen processing and CD8+ T-cell responses. Here we demonstrate that Ub fusion mediates enhanced CD8+ responses by proteasome targeting rather than by enhancing protein translation. We also show that several of the yeast-defined Ub constructs failed to target the proteasome in mammalian cells and likewise failed to enhance transgene-specific CD8+ T-cell responses in mice. In contrast, when mammalian-optimized constructs were applied to target the influenza virus nucleoprotein, CD8+ responses were enhanced against its refractory subdominant epitope in mice. This work demonstrates that Ub fusion has efficacy to enhance CD8+ responses, especially against subdominant antigen epitopes, provided constructs are optimized for mammalian use.

Expression library immunization to discover and improve vaccine antigens.

Genetic immunization is a novel method for vaccination in which DNA is delivered into the host to drive both cellular and humoral immune responses against its protein product. While genetic immunization can be potent, it requires that one have, in hand, a gene that encodes a protective protein antigen. Therefore, for many diseases, one cannot make a genetic vaccine because no protective antigen is known or no gene for this antigen is available. This lack of candidate antigens and their genes is a considerable bottleneck in developing new vaccines against old infectious agents, new emerging pathogens, and bioweapons. To address this limitation, we developed expression library immunization (ELI) as a high-throughput technology to discover vaccine candidate genes at will, by using the immune system to screen the entire genome of a pathogen for vaccine candidate. To date, ELI has discovered new vaccine candidates from a diverse set of bacterial, fungal, and parasitic pathogens. In addition, the process of applying ELI to the genome of pathogens allows one to genetically re-engineer these antigens to convert immunoevasive pathogen proteins into immunostimulatory vaccine antigens. Therefore, ELI is a potent technology to discover new vaccines and also generate genomic vaccines with amplified, multivalent immunostimulatory capacities.