Prophylactic Vaccination and Intratumoral Boost with HER2-Expressing Oncolytic Herpes Simplex Virus Induces Robust and Persistent Immune Response against HER2-Positive Tumor Cells.

The development of effective cancer vaccines remains a significant challenge due to immune tolerance and limited clinical benefits. Oncolytic herpes simplex virus type 1 (oHSV-1) has shown promise as a cancer therapy, but efficacy is often limited in advanced cancers. In this study, we constructed and characterized a novel oHSV-1 virus (VG22401) expressing the human epidermal growth factor receptor 2 (HER2), a transmembrane glycoprotein overexpressed in many carcinomas. VG22401 exhibited efficient replication and HER2 payload expression in both human and mouse colorectal cancer cells. Mice immunized with VG22401 showed significant binding of serum anti-HER2 antibodies to HER2-expressing tumor cells, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, mice primed with VG22401 and intratumorally boosted with the same virus showed enhanced antitumor efficacy in a bilateral syngeneic HER2(+) tumor model, compared to HER2-null backbone virus. This effect was accompanied by the induction of anti-HER2 T cell responses. Our findings suggest that peripheral priming with HER2-expressing oHSV-1 followed by an intratumoral boost with the same virus can significantly enhance antitumor immunity and efficacy, presenting a promising strategy for cancer immunotherapy.

Targeting carcinoembryonic antigen-expressing tumors using a novel transcriptional and translational dual-regulated oncolytic herpes simplex virus type 1.

VG2025 is a recombinant oncolytic herpes simplex virus type 1 (HSV-1) that uses transcriptional and translational dual regulation (TTDR) of critical viral genes to enhance virus safety and promote tumor-specific virus replication without reducing virulence. The TTDR platform is based on transcriptional control of the essential HSV-1 immediate-early protein ICP27 using a tumor-specific carcinoembryonic antigen (CEA) promoter, coupled with translational control of the neurovirulence factor ICP34.5 using multiple microRNA (miR)-binding sites. VG2025 further incorporates IL-12 and the IL-15/IL-15 receptor alpha subunit complex to enhance the antitumor and immune stimulatory properties of oncolytic HSVs. The TTDR strategy was verified in vitro and shown to be highly selective. Strong in vivo antitumor efficacy was observed following both intratumoral and intravenous administration. Clear abscopal and immune memory effects were also evident, indicating a robust antitumor immune response. Gene expression profiling of treated tumors revealed increased immune cell infiltration and activation of multiple immune-signaling pathways when compared with the backbone virus. Absence of neurotoxicity was verified in mice and in rhesus monkeys. Taken together, the enhanced tumor clearance, excellent safety profile, and positive correlation between CEA levels and viral replication efficiency may provide an opportunity for using biomarker-based precision medicine in oncolytic virotherapy.

Pre-Existing HSV-1 Immunity Enhances Anticancer Efficacy of a Novel Immune-Stimulating Oncolytic Virus.

Oncolytic viruses (OVs) can specifically replicate in the host and cause cancer cell lysis while inducing an antitumor immune response. The aim of this study is to investigate the impact of either pre-existing immunity against herpes simplex virus type-1 (HSV-1) or multicycle treatment with OVs on anticancer efficacy of VG161, an HSV-1 OV in phase 2 clinical trial. VG161 efficacy was tested in CT26 mouse models by comparing the efficacy and immune response in naïve mice or in mice that were immunized with VG161. Moreover, VG161 efficacy in HLA-matched CD34+ humanized intrahepatic cholangiocarcinoma (ICC) patient-derived xenograft (PDX) models was also tested in multicycle treatment and was compared to standard chemotherapy for this type of cancer (gemcitabine). The HSV-1-immunized mice significantly inhibited tumor growth in VG161-treated mice compared to control naïve treated mice. RNA expression profiling and ELISPOT analyses indicated changes in the tumor's immune profile in the immunized and treated group compared to naïve and treated mice, as well as enhanced T cell function depicted by higher numbers of tumor specific lymphocytes, which was enhanced by immunization. In the ICC PDX model, repeated treatment of VG161 with 2 or 3 cycles seemed to increase the anticancer efficacy of VG161. In conclusion, the anticancer efficacy of VG161 can be enhanced by pre-immunization with HSV-1 and multicycle administration when the virus is given intratumorally, indicating that pre-existing antiviral immunity might enhance OV-induced antitumor immunity. Our results suggest potential clinical benefits of HSV-1-based OV therapy in HSV-1-seropositive patients and multicycle administration of VG161 for long-term maintenance treatment.

Induction of Durable Antitumor Response by a Novel Oncolytic Herpesvirus Expressing Multiple Immunomodulatory Transgenes.

Oncolytic virotherapy is a promising new tool for cancer treatment, but direct lytic destruction of tumor cells is not sufficient and must be accompanied by strong immune activation to elicit anti-tumor immunity. We report here the creation of a novel replication-competent recombinant oncolytic herpes simplex virus type 1 (VG161) that carries genes coding for IL-12, IL-15, and IL-15 receptor alpha subunit, along with a peptide fusion protein capable of disrupting PD-1/PD-L1 interactions. The VG161 virus replicates efficiently and exhibits robust cytotoxicity in multiple tumor cell lines. Moreover, the encoded cytokines and the PD-L1 blocking peptide work cooperatively to boost immune cell function. In vivo testing in syngeneic CT26 and A20 tumor models reveals superior efficacy when compared to a backbone virus that does not express exogenous genes. Intratumoral injection of VG161 induces abscopal responses in non-injected distal tumors and grants resistance to tumor re-challenge. The robust anti-tumor effect of VG161 is associated with T cell and NK cell tumor infiltration, expression of Th1 associated genes in the injection site, and increased frequency of splenic tumor-specific T cells. VG161 also displayed a superb safety profile in GLP acute and repeated injection toxicity studies performed using cynomolgus monkeys. Overall, we demonstrate that VG161 can induce robust oncolysis and stimulate a robust anti-tumor immune response without sacrificing safety.

Synchrotron X-Ray Microbeam Diffraction Measurements of Full Elastic Long Range Internal Strain and Stress Tensors in Commercial-Purity Aluminum Processed by Multiple Passes of Equal-Channel Angular Pressing.

Synchrotron X-ray microbeam diffraction was used to measure the full elastic long range internal strain and stress tensors of low dislocation density regions within the submicrometer grain/subgrain structure of equal-channel angular pressed (ECAP) AA1050 after 1, 2, and 8 passes. This is the first time that full tensors were measured in plastically deformed metals at this length scale. This work supplements previous studies that measured long range internal stresses (LRIS) in ECAP AA1050 of multiple passes, but only for a single direction. The maximum (most tensile or least compressive) principal elastic strain directions for the unloaded 1 pass sample for the grain/subgrain interiors align well with the pressing direction, and are more random for the 2 and 8 pass samples. The measurements reported here indicate that the local stresses and strains become increasingly isotropic (homogenized) with increasing ECAP passes using route BC. The average maximum (in magnitude) LRISs are -0.43 σa for 1 pass, -0.44 σa for 2 pass, and 0.14 σa for the 8 pass sample. These LRISs appear to be larger than LRISs reported by previous works (using single reflection measurements).

B7-H4.Ig inhibits the development of type 1 diabetes by regulating Th17 cells in NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease characterized by immunological destruction of insulin-producing pancreatic ß-cells and subsequent hyperglycemia. The non-obese diabetic (NOD) mouse strain spontaneously develops a disease similar to human T1D and is commonly used as an animal model for studying this disease. We have previously shown that the administration of B7-H4-immunoglobulin fusion protein (B7-H4.Ig), a newly identified T-cell co-inhibitory signaling molecule, blocks the onset of diabetes in NOD mice. However, the mechanism(s) by which B7-H4 protects NOD mice from T1D is not fully understood. IL-17 is a pro-inflammatory cytokine, produced by Th17 cells, that activates T cells and other immune cells to produce a variety of cytokines and chemokines. Increasing evidence has shown that therapeutic agents targeting the IL-17 molecule or directly inhibiting IL-17-producing cells regulate autoimmune diabetes in NOD mice, suggesting that IL-17 is involved in the pathogenesis of this disease. In this study, we investigate whether B7-H4.Ig treatment inhibits the generation of Th17 cells which subsequently decreases IL-17 production and prevents the onset of T1D in NOD mice. Pre-diabetic female NOD mice were injected intraperitoneally with control mouse IgG or B7-H4.Ig starting at 4 weeks of age for 12 weeks. Our data showed that the frequency of Th17 cells in B7-H4.Ig-treated mice was significantly decreased. In addition, our data showed that B7-H4.Ig-treated mice had decreased levels of pro-inflammatory cytokines and Th17-associated cytokines, and an increased level of the potent Th17 inhibitor IFN-γ. To further investigate the effect of B7-H4.Ig on differentiation of Th17 cells, we co-cultured splenocytes with Th17-polarizing cytokines in the absence or presence of B7-H4.Ig. Our results indicated that splenocytes, under the Th17 driving conditions in the presence of B7-H4.Ig, had significantly decreased the numbers of Th17 cells compared to cells co-cultured in the absence of B7-H4.Ig. Together, this study suggests that blocking the generation of Th17 cells with the administration of B7-H4.Ig effectively inhibits the development of T1D in NOD mice.

The quantitative assessment of the role played by basic amino acid clusters in the nuclear uptake of human ribosomal protein L7.

In this study, we used a multiple copy (EGFP)(3) reporter system to establish a numeric nuclear index system to assess the degree of nuclear import. The system was first validated by a FRAP assay, and then was applied to evaluate the essential and multifaceted nature of basic amino acid clusters during the nuclear import of ribosomal protein L7. The results indicate that the sequence context of the basic cluster determines the degree of nuclear import, and that the number of basic residues in the cluster is irrelevant; rather the position of the pertinent basic residues is crucial. Moreover, it also found that the type of carrier protein used by basic cluster has a great impact on the degree of nuclear import. In case of L7, importin ß2 or importin ß3 are preferentially used by clusters with a high import efficiency, notwithstanding that other importins are also used by clusters with a weaker level of nuclear import. Such a preferential usage of multiple basic clusters and importins to gain nuclear entry would seem to be a common practice among ribosomal proteins in order to ensure their full participation in high rate ribosome synthesis.

Endogenous expression of B7-H4 improves long-term murine islet allograft survival.

BACKGROUND: Allograft rejection is one of the main obstacles for islet transplantation. B7-H4 plays a key role in maintaining T-cell homeostasis by reducing T-cell proliferation and cytokine production. In this study, we investigated whether the endogenous expression of B7-H4 in ß cells from B7-H4 transgenic mice enhances islet allograft survival. METHODS: B7-H4 transgenic C57BL/6 (B6) mice (RIP.B7-H4) were developed by inserting the entire B7-H4 open reading frame under the rat insulin promoter (RIP). B7-H4 protein expression was examined by flow cytometric analysis and immunohistochemical staining. Islet allograft survival was investigated in streptozotocin-induced diabetic recipient BALB/c (H-2d) mice transplanted with 400 islets from RIP.B7-H4 (H-2b) mice under the kidney capsule. The recipient control group received islets from wild-type B6 donors. RESULTS: B7-H4 protein was significantly up-regulated in isolated islets from RIP.B7-H4 compared with wild-type B6 mice (56%±23% vs. 3%±1.2%). B7-H4 was coexpressed with insulin, but not glucagon, suggesting that B7-H4 is expressed in a ß-cell-specific manner. Recipient BALB/c mice transplanted with RIP.B7-H4 islets established euglycemia for 42.3±18.4 days (mean±SD; n=9) compared with controls at 23.1±7.8 days (mean±SD; n=12; P<0.004, log-rank test). CONCLUSIONS: The endogenous expression of B7-H4 in donor ß cells from transgenic mice prolongs islet allograft survival, confirming the negative role of B7-H4 in regulating alloreactive T-cell responses.

Blockade of both B7-H4 and CTLA-4 co-signaling pathways enhances mouse islet allograft survival.

Costimulation blockade is an effective way to prevent allograft rejection. In this study, we tested the efficacy of two negative co-signaling molecules in protecting islet allograft function. We used local expression of B7-H4 by adenoviral transduction of islets (Ad-B7-H4) and systemic administration of CTLA-4.Ig to investigate the outcomes of allograft survival. Five groups of streptozotocin-induced diabetic C57BL/6 mice received 400 islets each from BALB/c donors. The groups consisted of control (G1); CTLA-4.Ig (G2); Ad-LacZ (G3); Ad-B7-H4 (G4); and Ad-B7-H4 and CTLA-4.Ig combined (G5). G1 and G3 developed graft failure on average of two weeks. G2, G4 and G5 survived for 43.8 ± 34.8, 54.7 ± 31.2 and 77.8 ± 21.5 d, respectively. Activated T and B cells in the lymph nodes were significantly controlled by CTLA-4.Ig treatment. Significantly reduced infiltrates were also detected in the allografts of G2 compared with G1. By contrast, B7-H4 significantly inhibited Th1-associated IFN-gamma secretion in the early stage and increased Foxp3 (+) T cells in the long-term surviving allografts. Our study suggests that CTLA-4 and B7-H4 inhibit alloimmune responses through distinct mechanisms, and that combination therapy which activates two negative co-signaling pathways can further enhance islet allograft survival.

NKT cells are required for complete Freund's adjuvant-mediated protection from autoimmune diabetes.

Autoimmune diabetes in NOD mice can be prevented by application of Ags derived from Mycobacterium tuberculosis in the form of bacillus Calmette-Guérin or CFA. Disease protection by CFA is associated with a reduction in the numbers of pathogenic ß-cell specific, self-reactive CTLs, a phenomenon dependent on the presence and function of NK cells. However, the mechanisms by which NK cells are activated and recruited by heat-killed M. tuberculosis within CFA are unclear. In this study, we report that CFA-mediated NK cell activation and mobilization is dependent on CD1d expression. The administration of M. tuberculosis from CFA results in rapid NKT cell activation and IFN-γ secretion both in vitro and in vivo. CFA-induced NKT cell activation is intact in MyD88(-/-) mice suggesting that the mechanism is independent of TLR signaling. Furthermore, CD1d expression was found to be essential for both M. tuberculosis-triggered NKT cell activation and CFA-mediated protection of NOD mice from diabetes. Collectively, these findings reveal hitherto previously unidentified roles for NKT cells in the adjuvant-promoting effects of CFA on innate and adaptive immunity.

Natural killer cells from children with type 1 diabetes have defects in NKG2D-dependent function and signaling.

OBJECTIVE: Natural killer (NK) cells from NOD mice have numeric and functional abnormalities, and restoration of NK cell function prevents autoimmune diabetes in NOD mice. However, little is known about the number and function of NK cells in humans affected by type 1 diabetes. Therefore, we evaluated the phenotype and function of NK cells in a large cohort of type 1 diabetic children. RESEARCH DESIGN AND METHODS: Peripheral blood mononuclear blood cells were obtained from subjects whose duration of disease was between 6 months and 2 years. NK cells were characterized by flow cytometry, enzyme-linked immunosorbent spot assays, and cytotoxicity assays. Signaling through the activating NK cell receptor, NKG2D, was assessed by immunoblotting and reverse-phase phosphoprotein lysate microarray. RESULTS: NK cells from type 1 diabetic subjects were present at reduced cell numbers compared with age-matched, nondiabetic control subjects and had diminished responses to the cytokines interleukin (IL)-2 and IL-15. Analysis before and after IL-2 stimulation revealed that unlike NK cells from nondiabetic control subjects, NK cells from type 1 diabetic subjects failed to downregulate the NKG2D ligands, major histocompatibility complex class I-related chains A and B, upon activation. Moreover, type 1 diabetic NK cells also exhibited decreased NKG2D-dependent cytotoxicity and interferon-γ secretion. Finally, type 1 diabetic NK cells showed clear defects in NKG2D-mediated activation of the phosphoinositide 3-kinase-AKT pathway. CONCLUSIONS: These results are the first to demonstrate that type 1 diabetic subjects have aberrant signaling through the NKG2D receptor and suggest that NK cell dysfunction contributes to the autoimmune pathogenesis of type 1 diabetes.

Importin ß3 mediates the nuclear import of human ribosomal protein L7 through its interaction with the multifaceted basic clusters of L7.

We show that importin ß3 is essential for the nuclear import of L7. The import is mediated via the multifaceted basic amino acid clusters present in the NH(2)-region of L7, and is RanGTP-dependent. Using a (EGFP)(3) reporter system and a FRAP assay, the role the individual clusters play as a functional NLS has been characterized, and each cluster was found to exhibit a different rate of real time nuclear uptake. We assume that having such a multiple NLS may provide L7 with preferential nuclear uptake.

Critical role for IFN-gamma in natural killer cell-mediated protection from diabetes.

Autoimmune diabetes in nonobese diabetic (NOD) mice can be prevented by a single injection of complete Freund's adjuvant (CFA), but the mechanisms mediating protection remains unclear. We previously showed that NOD mice immunized with CFA have a markedly reduced incidence of diabetes that is associated with a significant decrease in the number of beta-cell-specific, autoreactive cytotoxic T lymphocytes and, furthermore, that the effect of CFA is mediated by natural killer (NK) cells. In this study, we report one mechanism by which NK cells regulate the onset of diabetes. Administration of CFA produced a rapid increase in NK cell frequency and function, including cytotoxicity and IFN-gamma secretion. By co-transferring NK cells from IFN-gamma-deficient (or wild-type) NOD mice and spleen cells from diabetic NOD mice to NOD/SCID recipients, we show that IFN-gamma secretion by NK cells significantly influences the effect of CFA protection. In contrast, NK cytotoxicity does not appear to participate in CFA-mediated protection from diabetes. Our findings demonstrate that NK cells mediate the protective effects of CFA through secretion of IFN-gamma.

Progression of spontaneous autoimmune diabetes is associated with a switch in the killing mechanism used by autoreactive CTL.

Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic beta-cells by T lymphocytes. Beta-cell death that is induced by autoreactive CTL in diabetes involves both Fas/Fas ligand (FasL)- and perforin/granzyme-mediated pathways, although their relative contributions during the progression of the disease remain unknown. We demonstrate here that despite the preferential use of the Fas/FasL pathway for cytolysis of beta-cell targets, transgenic beta-cell-specific CTL were able to kill targets via the perforin pathway when triggered by a higher affinity stimulus. In addition, we show that the killing mechanism used by islet-associated CD8(+) T cells from non-obese diabetic mice changed as the mice aged and correspondingly, with the stage of diabetes. These results provide direct evidence for age-related changes in the cytotoxic pathways used by diabetogenic T cells during the progression of autoimmune diabetes.

Regulation of autoimmune diabetes by complete Freund's adjuvant is mediated by NK cells.

Autoimmune (type 1) diabetes results from a loss of beta cells that is mediated by self-reactive T cells. Previous studies have shown that a single injection of CFA prevents diabetes in nonobese diabetic (NOD) mice, but the mechanism(s) of protection remain unknown. We show here that NOD mice immunized with CFA have a markedly reduced incidence of diabetes and that this reduced incidence is associated with a decrease in the number of beta cell-specific, autoreactive CTL. In addition, the adoptive transfer of diabetes into syngeneic NOD/SCID recipients was prevented by CFA immunization, and the protective effects of CFA were lost when cells expressing the NK cell marker, asialo GM1, were removed from both donor cells and recipient mice. Returning a population of CD3-DX5+ cells to the adoptive transfer restored the protective effects of CFA. Therefore, NK cells mediate the protective effects of CFA possibly through the down-regulation of autoreactive CTL and stimulation of NK cells represents a novel approach to the prevention of autoimmune diabetes.

Chronic active Epstein-Barr virus infection associated with low expression of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) on natural killer cells.

Chronic active Epstein-Barr virus (CAEBV) infection is an uncommon and severe manifestation of EBV infection that frequently leads to the development of natural killer (NK) or T cell lymphomas. We report here that NK cells derived from a boy with CAEBV showed decreased cytotoxic function and impaired interferon-gamma secretion against a variety of transformed target cell lines. In addition, NK cells from the patient lacked expression of the broadly expressed NK receptor, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). These data suggest that CAEBV is associated with low expression of LAIR-1 on NK cells.