A Recombinant Vesicular Stomatitis Virus Ebola Vaccine.

BACKGROUND: The worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate designed to prevent EVD. METHODS: We conducted two phase 1, placebo-controlled, double-blind, dose-escalation trials of an rVSV-based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV-ZEBOV vaccine (3 million plaque-forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed. RESULTS: The most common adverse events were injection-site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme-linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV-Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months. CONCLUSIONS: This Ebola vaccine candidate elicited anti-Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV∆G-ZEBOV-GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408 .).

Emergency postexposure vaccination with vesicular stomatitis virus-vectored Ebola vaccine after needlestick.

IMPORTANCE: Safe and effective vaccines and drugs are needed for the prevention and treatment of Ebola virus disease, including following a potentially high-risk exposure such as a needlestick. OBJECTIVE: To assess response to postexposure vaccination in a health care worker who was exposed to the Ebola virus. DESIGN AND SETTING: Case report of a physician who experienced a needlestick while working in an Ebola treatment unit in Sierra Leone on September 26, 2014. Medical evacuation to the United States was rapidly initiated. Given the concern about potentially lethal Ebola virus disease, the patient was offered, and provided his consent for, postexposure vaccination with an experimental vaccine available through an emergency Investigational New Drug application. He was vaccinated on September 28, 2014. INTERVENTIONS: The vaccine used was VSVΔG-ZEBOV, a replicating, attenuated, recombinant vesicular stomatitis virus (serotype Indiana) whose surface glycoprotein gene was replaced by the Zaire Ebola virus glycoprotein gene. This vaccine has entered a clinical trial for the prevention of Ebola in West Africa. RESULTS: The vaccine was administered 43 hours after the needlestick occurred. Fever and moderate to severe symptoms developed 12 hours after vaccination and diminished over 3 to 4 days. The real-time reverse transcription polymerase chain reaction results were transiently positive for vesicular stomatitis virus nucleoprotein gene and Ebola virus glycoprotein gene (both included in the vaccine) but consistently negative for Ebola virus nucleoprotein gene (not in the vaccine). Early postvaccination cytokine secretion and T lymphocyte and plasmablast activation were detected. Subsequently, Ebola virus glycoprotein-specific antibodies and T cells became detectable, but antibodies against Ebola viral matrix protein 40 (not in the vaccine) were not detected. CONCLUSIONS AND RELEVANCE: It is unknown if VSVΔG-ZEBOV is safe or effective for postexposure vaccination in humans who have experienced a high-risk occupational exposure to the Ebola virus, such as a needlestick. In this patient, postexposure vaccination with VSVΔG-ZEBOV induced a self-limited febrile syndrome that was associated with transient detection of the recombinant vesicular stomatitis vaccine virus in blood. Strong innate and Ebola-specific adaptive immune responses were detected after vaccination. The clinical syndrome and laboratory evidence were consistent with vaccination response, and no evidence of Ebola virus infection was detected.

U6 promoter-enhanced GlnUAG suppressor tRNA has higher suppression efficacy and can be stably expressed in 293 cells.

BACKGROUND: Almost one-third of all human genetic diseases are the result of nonsense mutations that can result in truncated proteins. Nonsense suppressor tRNAs (NSTs) were proposed as valuable tools for gene therapy of genetic diseases caused by premature termination codons (PTCs). Although various strategies have been adapted aiming to increase NST expression and efficacy, low suppression efficacies of NSTs and toxicity associated with stable expression of suppressor tRNAs have hampered the development of NST-mediated gene therapy. METHODS: We have employed the U6 promoter to enhance Gln-Amber suppressor tRNA (GlnUAG) expression and to increase PTC suppression in mammalian cells. In an attempt to study the toxic effects of NSTs, a stable 293 cell line constitutively expressing a U6 promoter-enhanced GlnUAG tRNA was established. To examine whether any proteomic changes occurred in cells that constitutively express suppressor tRNA, whole cell proteins from cells with and without any suppressor tRNA expression were analyzed. RESULTS: The data obtained suggest that U6 promoter-enhanced GlnUAG tRNAs have higher suppression efficacies than multimers of the same suppressor tRNA without a U6 promoter. Proteomic analysis of cells constitutively expressing the GlnUAG suppressor tRNA indicates that stable expression of NSTs may not lead to significant read through of normal cellular proteins. CONCLUSIONS: Because most tRNAs have cell-specific differential expression, this technique will enable the expression of different kinds of suppressor tRNAs in various cell types at high, functionally relevant levels. The techniques developed in the present study may contribute to the further development of suppressor tRNA-mediated gene therapy.

Phase II trial of the IDO pathway inhibitor indoximod plus pembrolizumab for the treatment of patients with advanced melanoma.

BACKGROUND: The indoleamine 2,3-dioxygenase (IDO) pathway is a key counter-regulatory mechanism that, in cancer, is exploited by tumors to evade antitumor immunity. Indoximod is a small-molecule IDO pathway inhibitor that reverses the immunosuppressive effects of low tryptophan (Trp) and high kynurenine (Kyn) that result from IDO activity. In this study, indoximod was used in combination with a checkpoint inhibitor (CPI) pembrolizumab for the treatment for advanced melanoma. METHODS: Patients with advanced melanoma were enrolled in a single-arm phase II clinical trial evaluating the addition of indoximod to standard of care CPI approved for melanoma. Investigators administered their choice of CPI including pembrolizumab (P), nivolumab (N), or ipilimumab (I). Indoximod was administered continuously (1200 mg orally two times per day), with concurrent CPI dosed per US Food and Drug Administration (FDA)-approved label. RESULTS: Between July 2014 and July 2017, 131 patients were enrolled. (P) was used more frequently (n=114, 87%) per investigator's choice. The efficacy evaluable population consisted of 89 patients from the phase II cohort with non-ocular melanoma who received indoximod combined with (P).The objective response rate (ORR) for the evaluable population was 51% with confirmed complete response of 20% and disease control rate of 70%. Median progression-free survival was 12.4 months (95% CI 6.4 to 24.9). The ORR for Programmed Death-Ligand 1 (PD-L1)-positive patients was 70% compared with 46% for PD-L1-negative patients. The combination was well tolerated, and side effects were similar to what was expected from single agent (P). CONCLUSION: In this study, the combination of indoximod and (P) was well tolerated and showed antitumor efficacy that is worth further evaluation in selected patients with advanced melanoma.

Phase Ia study of the indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor navoximod (GDC-0919) in patients with recurrent advanced solid tumors.

BACKGROUND: Indoleamine-2,3-dioxygenase 1 (IDO1) catalyzes the oxidation of tryptophan into kynurenine and is partially responsible for acquired immune tolerance associated with cancer. The IDO1 small molecule inhibitor navoximod (GDC-0919, NLG-919) is active as a combination therapy in multiple tumor models. METHODS: This open-label Phase Ia study assessed safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary anti-tumor activity of navoximod in patients with recurrent/advanced solid tumors, administered as 50-800 mg BID on a 21/28 day and at 600 mg on a 28/28 day schedule. Plasma kynurenine and tryptophan were longitudinally evaluated and tumor assessments were performed. RESULTS: Patients (n = 22) received a median of 3 cycles of navoximod. No maximum tolerated dose was reached. One dose-limiting toxicity of Grade 4 lower gastrointestinal hemorrhage was reported. Adverse events (AEs) regardless of causality in ≥20% of patients included fatigue (59%), cough, decreased appetite, and pruritus (41% each), nausea (36%), and vomiting (27%). Grade ≥ 3 AEs occurred in 14/22 patients (64%), and were related to navoximod in two patients (9%). Navoximod was rapidly absorbed (Tmax ~ 1 h) and exhibited dose-proportional increases in exposure, with a half-life (t1/2 ~ 11 h) supportive of BID dosing. Navoximod transiently decreased plasma kynurenine from baseline levels with kinetics consistent with its half-life. Of efficacy-evaluable patients, 8 (36%) had stable disease and 10 (46%) had progressive disease. CONCLUSIONS: Navoximod was well-tolerated at doses up to 800 mg BID decreasing plasma kynurenine levels consistent with its half-life. Stable disease responses were observed. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02048709 .

Effective treatment of preexisting melanoma with whole cell vaccines expressing alpha(1,3)-galactosyl epitopes.

The hyperacute immune response in humans is a potent mechanism of xenograft rejection mediated by complement-fixing natural antibodies recognizing alpha(1,3)-galactosyl epitopes (alphaGal) not present on human cells. We exploited this immune mechanism to create a whole cell cancer vaccine to treat melanoma tumors. B16 melanoma vaccines genetically engineered to express alphaGal epitopes (B16alphaGal) effectively treated preexisting s.c. and pulmonary alphaGal-negative melanoma (B16Null) tumors in the alpha(1,3)-galactosyltransferase knockout mouse model. T cells from mice vaccinated with B16alphaGal recognized B16Null melanoma cells measured by detection of intracellular tumor necrosis factor-alpha. We showed successful adoptive transfer of immunity to recipient mice bearing lung melanoma metastasis. Mice receiving lymphocytes from donors previously immunized with B16alphaGal had reduced pulmonary metastases. The transfer of lymphocytes from mice vaccinated with control vaccine had no effect in the pulmonary metastasis burden. This study unequivocally establishes for the first time efficacy in the treatment of preexisting melanoma tumors using whole cell vaccines expressing alphaGal epitopes. Vaccination with B16alphagal induced strong long-lasting cell-mediated antitumor immunity extended to B16Null. These data formed the basis for the testing of this therapeutic strategy in human clinical trials currently under way.

Addition of αGal HyperAcute™ technology to recombinant avian influenza vaccines induces strong low-dose antibody responses.

Highly pathogenic avian influenza represents a severe public health threat. Over the last decade, the demand for highly efficacious vaccines against avian influenza viruses has grown, especially after the 2013 H7N9 outbreak in China that resulted in over 600 human cases with over 200 deaths. Currently, there are several H5N1 and H7N9 influenza vaccines in clinical trials, all of which employ traditional oil-in-water adjuvants due to the poor immunogenicity of avian influenza virus antigens. In this study, we developed potent recombinant avian influenza vaccine candidates using HyperAcute™ Technology, which takes advantage of naturally-acquired anti-αGal immunity in humans. We successfully generated αGal-positive recombinant protein and virus-like particle vaccine candidates of H5N1 and H7N9 influenza strains using either biological or our novel CarboLink chemical αGal modification techniques. Strikingly, two doses of 100 ng αGal-modified vaccine, with no traditional adjuvant, was able to induce a much stronger humoral response in αGT BALB/c knockout mice (the only experimental system readily available for testing αGal in vivo) than unmodified vaccines even at 10-fold higher dose (1000 ng/dose). Our data strongly suggest that αGal modification significantly enhances the humoral immunogenicity of the recombinant influenza vaccine candidates. Use of αGal HyperAcute™ technology allows significant dose-sparing while retaining desired immunogenicity. Our success in the development of highly potent H5N1 and H7N9 vaccine candidates demonstrated the potential of αGal HyperAcute™ technology for the development of vaccines against other infectious diseases.

Safety and immunogenicity of the rVSV∆G-ZEBOV-GP Ebola virus vaccine candidate in healthy adults: a phase 1b randomised, multicentre, double-blind, placebo-controlled, dose-response study.

BACKGROUND: The 2014 Zaire Ebola virus outbreak highlighted the need for a safe, effective vaccine with a rapid onset of protection. We report the safety and immunogenicity of the recombinant vesicular stomatitis virus-Zaire Ebola virus envelope glycoprotein vaccine (rVSV∆G-ZEBOV-GP) across a 6 log10 dose range in two sequential cohorts. METHODS: In this phase 1b double-blind, placebo-controlled, dose-response study we enrolled and randomly assigned healthy adults (aged 18-61 years) at eight study sites in the USA to receive a single injection of vaccine or placebo, administered by intramuscular injection. In cohort 1, participants were assigned to receive 3 × 103, 3 × 104, 3 × 105, or 3 × 106 PFU doses of rVSV∆G-ZEBOV-GP or placebo. In cohort 2, participants were assigned to receive 3 × 106, 9 × 106, 2 × 107, or 1 × 108 PFU doses of rVSV∆G-ZEBOV-GP or placebo. Participants were centrally allocated by the study statistician to vaccine groups or placebo through computer-generated randomisation lists. The primary safety outcome was incidence of adverse events within 14 days in the modified intention-to-treat population (all randomly assigned participants who received vaccine or placebo), and the primary outcome for immunogenicity was IgG ELISA antibody titres at day 28 in the per-protocol population. Surveillance was enhanced for arthritis and dermatitis through to day 56. This study is registered with ClinicalTrials.gov, number NCT02314923. FINDINGS: Between Dec 26, 2014, and June 8, 2015, 513 participants were enrolled and randomly assigned; one was not immunised because of unsuccessful phlebotomy. In cohort 1, 256 participants received vaccine (3 × 103 [n=64], 3 × 104 [n=64], 3 × 105 [n=64], or 3 × 106 PFU [n=64]) and 74 received placebo. In cohort 2, 162 participants received vaccine (3 × 106 [n=20], 9 × 106 [n=47], 2 × 107 [n=47], or 1 × 108 PFU [n=48]) and 20 received placebo. Most adverse events occurred in the first day after vaccination, and were mild to moderate in intensity, of a short duration, and more frequent at high vaccine doses (9 × 106 PFU and greater). At the 2 × 107 PFU dose (used in phase 3 trials), the most common local adverse events versus placebo within the first 14 days were arm pain (57·4% [27 of 47] vs 7·4% [seven of 94]) and local tenderness (59·6% [28 of 47] vs 8·5% [eight of 94]). The most common systemic adverse events at the 2 × 107 PFU dose versus placebo, occurring in the first 14 days, were headache (46·8% [22 of 47] vs 27·7% [26 of 94]), fatigue (38·3% [18 of 47] vs 19·1% [18 of 94]), myalgia (34·0% [16 of 47] vs 10·6% [10 of 94]), subjective fever (29·8% [14 of 47] vs 2·1% [two of 94]), shivering or chills (27·7% [13 of 47] vs 7·4% [seven of 94]), sweats (23·4% [11 of 47] vs 3·2% [three of 94]), joint aches and pain (19·1% [nine of 47] vs 7·4% [seven of 94]), objective fever (14·9% [seven of 47] vs 1·1% [one of 94]), and joint tenderness or swelling (14·9% [seven of 47] vs 2·1% [two of 94]). Self-limited, post-vaccination arthritis occurred in 4·5% (19 of 418) of vaccinees (median onset 12·0 days [IQR 10-14]; median duration 8·0 days [6-15]) versus 3·2% (three of 94) of controls (median onset 15·0 days [6-20]; median duration 47·0 days [37-339]), with no apparent dose relationship. Post-vaccination dermatitis occurred in 5·7% (24 of 418) of vaccinees (median onset 9·0 days [IQR 2-12]; median duration 7·0 days [4-9]) versus 3·2% (three of 94) of controls (median onset 5·0 days [3-53]; median duration 33·0 days [5-370]). A low-level, transient, dose-dependent viraemia occurred in concert with early reactogenicity. Antibody responses were observed in most participants by day 14. IgG and neutralising antibody titres were dose-related (p=0·0003 for IgG ELISA and p<0·0001 for the 60% plaque-reduction neutralisation test [PRNT60] by linear trend). On day 28 at the 2 × 107 PFU dose, the geometric mean IgG ELISA endpoint titre was 1624 (95% CI 1146-2302) and seroconversion was 95·7% (95% CI 85·5-98·8); the geometric mean neutralising antibody titre by PRNT60 was 250 (176-355) and seroconversion was 95·7% (85·5-98·8). These robust immunological responses were sustained for 1 year. INTERPRETATION: rVSV∆G-ZEBOV-GP was well tolerated and stimulated a rapid onset of binding and neutralising antibodies, which were maintained through to day 360. The immunogenicity results support selection of the 2 × 107 PFU dose. FUNDING: Biomedical Advanced Research and Development Authority, US Department of Health and Human Services.

Immunity to the alpha(1,3)galactosyl epitope provides protection in mice challenged with colon cancer cells expressing alpha(1,3)galactosyl-transferase: a novel suicide gene for cancer gene therapy.

Human immunity to alpha(1,3)Galactosyl epitopes (alpha Gal) may provide the means for a successful cancer gene therapy that uses the immune system to identify and to destroy tumor cells expressing the suicide gene alpha(1,3)Galactosyltransferase (alpha GT). Innate antibody specific for cell surface alpha Gal constitutes a high percentage of circulating IgG and IgM immunoglobulins in humans and is the basis for complement-mediated hyperacute xenograft rejection and antibody-dependent cell-mediated cytotoxicity. In humans, the gene for alpha GT is mutated, and cells do not express the alpha Gal moiety. We hypothesized that human tumor cells induced to express the alpha Gal epitope would be killed by the hosts' innate immunity. Previous in vitro work by our group has demonstrated complement-mediated lysis of alpha Gal-transduced human tumor cells in culture by human serum. To induce antibodies to alpha Gal in this in vivo study, alpha GT knockout mice were used to determine whether immunization with alpha Gal could provide protection from challenge with alpha Gal-expressing murine MC38 colon cancer cells. Knockout mice were immunized either a single time, or twice, with rabbit RBC. Antibody titers to alpha Gal measured by indirect ELISA were significantly higher in mice immunized twice and approached the titers observed in human serum. Anti-alpha Gal antibodies were predominantly of the IgG1 and IgG3 subtype. Immunized knockout mice were challenged i.p. with varying doses of alpha Gal(+) MC38 colon carcinoma cells. Nonimmunized control groups consisting of alpha GT knockout mice, and wild-type C57BL/6 mice were challenged as well with MC38 cells. Immunized mice survived and exhibited slower tumor development in comparison to nonimmunized knockout and control mice. This study demonstrates, in vivo, the protective benefit of an immune response to the alpha Gal epitope. Our results provide a basis to pursue additional development of this cancer gene therapy strategy.

Iron chelators increase the resistance of Ataxia telangeictasia cells to oxidative stress.

Ataxia telangeictasia (A-T) is an autosomal recessive disorder characterized by immune dysfunction, genomic instability, chronic oxidative damage, and increased cancer incidence. Previously, desferal was found to increase the resistance of A-T, but not normal cells to exogenous oxidative stress in the colony forming-efficiency assay, suggesting that iron metabolism is dysregulated in A-T. Since desferal both chelates iron and modulates gene expression, we tested the effects of apoferritin and the iron chelating flavonoid quercetin on A-T cell colony-forming ability. We demonstrate that apoferritin and quercetin increase the ability of A-T cells to form colonies. We also show that labile iron levels are significantly elevated in Atm-deficient mouse sera compared to syngeniec wild type mice. Our findings support a role for labile iron acting as a Fenton catalyst in A-T, contributing to the chronic oxidative stress seen in this disease. Our findings further suggest that iron chelators might promote the survival of A-T cells and hence, individuals with A-T.

Desferrioxamine treatment increases the genomic stability of Ataxia-telangiectasia cells.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by genomic instability, chronic oxidative damage, and increased cancer incidence. Compared to normal cells, AT cells exhibit unusual sensitivity to exogenous oxidants, including t-butyl hydroperoxide (t-BOOH). Since ferritin releases labile iron under oxidative stress (which is chronic in AT) and labile iron mediates the toxic effects of t-butyl hydroperoxide, we hypothesized that chelation of intracellular labile iron would increase the genomic stability of AT cells, with and without exogenous oxidative stress. Here we report that desferrioxamine treatment increases the plating efficiency of AT, but not normal cells, in the colony forming-efficiency assay (a method often used to measure genomic stability). Additionally, desferrioxamine increases AT, but not normal cell resistance, to t-butyl hydroperoxide in this assay. Last, AT cells exhibit increased sensitivity to the toxic effects of FeCl(2) in the colony forming-efficiency assay and fail to demonstrate a FeCl(2)-induced G(2) checkpoint response when compared to normal cells. Our data indicates that: (1) chelation of labile iron increases genomic stability in AT cells, but not normal cells; and (2) AT cells exhibit deficits in their responses to iron toxicity. While preliminary, our findings suggest that AT might be, in part, a disorder of iron metabolism and treatment of individuals with AT with desferrioxamine might have clinical efficacy.

Pharmacological manipulation of ataxia-telangiectasia kinase activity as a treatment for Parkinson's disease.

Parkinson's disease (PD) is a major cause of morbidity and mortality among older individuals. Although the causes of Parkinson's disease are multifactorial, considerable evidence indicates that elevated labile iron in the substantia nigra pars compacta plays an important role in producing oxyradicals which subsequently damage nigro-striatal neurons. Based on this several researchers have suggested that blood-brain barrier crossing iron chelators might have clinical efficacy in treating PD. Work demonstrating that iron chelators protect nigro-striatal neurons in the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine-induced rodent PD models supports this hypothesis. Recently, we found that the ATM gene product (mutated in ataxia-telangiectasia, A-T), is required for cell survival and genomic stability maintenance following exposure to low labile iron concentrations. Iron chelators (desferal, quercetin, and apoferritin) also increase A-T cell genomic stability and viability, and activate ATM-dependent cellular events in normal cells. Additionally Atm-deficient mice exhibit a selective loss of dopaminergic nigro-striatal neurons. Based on this, we propose that iron chelators protect the substantia nigra pars compacta not only by chelating labile iron and reducing oxyradical formation, but also by inducing ATM activity, leading to increased oxidative stress resistance and DNA repair. Support for this hypothesis comes from the recent observation that the iron chelating flavonoid quercetin both directly activates ATM and protects neuronal cells from the toxic effects of the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Therefore since; (1) ATM is required for iron toxicity resistance, (2) iron chelators such as quercetin, desferal, and apoferritin induce ATM activity and/or ATM-dependent events, and (3), Atm-deficient mice preferentially lose dopaminergic nigro-striatal neurons, we propose that ATM activity has an important function in PD. Furthermore, pharmacological manipulation of ATM activity via iron chelation might have clinical efficacy in PD treatment.

Effective suppression of class I major histocompatibility complex expression by the US11 or ICP47 genes can be limited by cell type or interferon-gamma exposure.

An impediment encountered in many viral-based gene therapy clinical trials has been the rapid destruction of the transgene by the host's immune response. The processing and presentation of antigens through the class I major histocompatibility complex (MHC) pathway is the initial specific response to viral infection. Disruption of the class I MHC pathway by herpes simplex virus (HSV) or the human cytomegalovirus (HCMV) results in a decrease of the CD8(+) cytotoxic T lymphocyte (CTL) response and prolongs survival of infected cells in the host. Two viral immune suppression genes that interfere with the class I MHC presentation pathway, the HSV type I ICP47 gene and HCMV US11 gene, were cloned and each incorporated into a retroviral vector. HSV ICP47 and HCMV US11 transgenes were expressed in multiple cells lines and compared for their abilities to reduce antigen presentation on the cell surface by class I MHC. Retroviral supernatants were used to transduce human, canine, and rat cell lines. Fluorescence-activated cell sorter (FACS) analysis of US11- and ICP47-transduced cell lines demonstrated substantial reductions in class I MHC cell surface expression in most cell lines except in rodent cells where ICP47 is nonfunctional. The decrease in the level of class I MHC expression for ICP47 transduced cell lines ranged from 31-98% relative to negative controls. US11 decreased class I cell surface MHC by 67-96%. When both ICP47 and US11 are expressed in human cells, a further reduction of class I MHC was observed. Next, human A375 melanoma cells were tested to determine if the resulting reduction in cell surface class I MHC would reduce in vitro cytotoxicity by CTL. A375 cells expressing either ICP47 or US11 demonstrated a twofold to threefold reduction of specific lysis by primed CD8(+) CTL. These data clearly establish an ability to convey immune protection to human cells by viral genes. However, further analysis demonstrated that interferon (IFN)-gamma could reverse part or all of the downregulation of class I MHC induced by the ICP47 or US11 genes. The ICP47 and US11 genes, when expressed in target cells, decrease class I MHC presentation and as such might be used in strategies to create local immunosuppression against transgenes or allografts.

Antitumor effects on human melanoma xenografts of an amplicon vector transducing the herpes thymidine kinase gene followed by ganciclovir.

Herpes simplex virus type-1 (HSV-1) has been demonstrated as a potentially useful gene delivery vector for gene therapy due to its high efficiency of in vivo transduction. The helper virus-dependent, HSV- 1 amplicon vectors were developed for easier operation and their larger capacity. In this study, the herpes simplex virus type-1 thymidine kinase (HSVtk) gene was cloned into the pHE700 amplicon vector to make an HE7tk vector and used for in vivo gene delivery. Human melanoma xenografts were established in athymic nude mice. Tumors were injected directly with HE7tk vector alone, HE7tk vector followed by ganciclovir (GCV), or a pHE700 amplicon vector carrying a green fluorescent protein (HE7GFP) gene followed by GCV. Efficient HSVtk transgene expression was found in the tumor 3 days after injection. Animals transduced with HE7tk followed by GCV had minimal tumor growth (P < .01 ). Animals that received either HE7tk vector without GCV or HE7GFP vector with GCV had some reduction in tumor growth compared to animals that were injected with buffer only. These data indicate that replication-defective HSV-1 amplicon vectors can be used effectively to deliver transgenes into solid tumors in vivo.

The rat sodium iodide symporter gene permits more effective radioisotope concentration than the human sodium iodide symporter gene in human and rodent cancer cells.

Expression of the sodium iodide symporter (NIS) gene in tumor cells may provide a novel mechanism for treating cancer. The NIS mediates the normal physiological transport of iodide across the thyroid cell membrane. This mechanism of iodide uptake has been used to both diagnose and treat thyroid cancer. Tissue expression of the NIS is largely limited to the thyroid; therefore, expression of the NIS gene in cancer cells would allow for specific iodine uptake, radioisotope accumulation, and treatment. In this study, we directly compared the human and rat NIS (rNIS) for their ability to concentrate radioisotope into human and rodent cancer cells. Perchlorate-sensitive (125)I uptake in multiple cell lines was demonstrated following transduction with retroviral vectors expressing either the human or rNIS gene. Surprisingly, iodine uptake was consistently higher with the rNIS gene, up to 5-fold greater, when compared to the human gene, even within a variety of human tumor cell lines. This iodine uptake allowed for cell killing following (131)I treatment in NIS-transduced cells when assayed by in vitro clonogenic assays. These results demonstrate that the rNIS gene provides superior iodine uptake ability, and may be preferable for use in designing anticancer gene therapy approaches.

Combination immunotherapy for high-risk resected and metastatic melanoma patients.

BACKGROUND: Patients with advanced melanoma have a poor outcome. We hypothesize that combination immunotherapy can synergistically activate host immunity to generate an effective treatment for patients with high-risk, resected stage 3, recurrent, refractory, or stage 4 melanoma. METHODS: We conducted a phase 2 clinical trial of HyperAcute Melanoma (HAM) vaccine (NLG-12036, NewLink Genetics) combined with pegylated interferon (Sylatron, Merck). Trial design consisted of a 12-week regimen with the initial 4 weekly treatments consisting of HAM alone (intradermally) followed by 8 additional treatments of HAM plus Sylatron (subcutaneously, 6 µg/kg). Trial endpoint outcomes include clinical response, overall safety, and correlative findings for observed antitumor effect. RESULTS: Our cohort consisted of 25 patients with a median age of 60. Twenty-one patients completed the trial and 4 stopped because of progressive disease (PD). According to the Response Evaluation Criteria in Solid Tumors, of the 16 stage 4 patients, 2 had a complete response (CR), 1 had stable disease, and 4 had no evidence of disease (NED) after resection. For stage 2/3 patients, 3 of 9 remained NED, and the 1 stage 2C patient had slow PD with a single site resected and is currently NED. The median overall survival time was 29 months, with 60% of the patients surviving for >1 year. Of the 25 patients, 12 (48%) are still alive. All evaluable patients (21/21) seroconverted, developing autoimmune antibodies. Four of 25 patients developed vitiligo, correlating with 2 CR patients and 2 NED patients. CONCLUSION: Combination immunotherapy with HAM plus Sylatron shows clinical efficacy with tumor regression and concomitant immune activation. Optimization of dosing schedules and therapeutic efficacy should be further explored to enhance the benefit of this promising immunotherapeutic approach.

Cellular immunotherapy study of prostate cancer patients and resulting IgG responses to peptide epitopes predicted from prostate tumor-associated autoantigens.

The immunogenicity of a cellular immunotherapy using genetically modified vaccines to express α(1,3)galactosyl epitopes (αGal) was evaluated in advanced prostate cancer (PC) patients. In this dose escalation phase I study, we report safety, feasibility, and immunologic data of an immunotherapy composed of 2 human PC cell lines engineered to express αGal epitopes (HyperAcute-Prostate, HAP, NewLink Genetics). Eight patients received up to 12 biweekly vaccinations with HAP. Enrolled patients (aged range, 53-85 y) had American Joint Committee on Cancer stage IV, any T, any N, M1, Eastern Cooperative Oncology Group PS≤2, at least 1 prior hormonal treatment and <3 prior chemotherapies, adequate bone marrow and organ function, and albumin ≥3.0 g/dL. Serum IgG antibodies to synthetic peptides overexpressed in PC were determined by enzyme-linked immunosorbent assay. Results indicate that HAP immunotherapy induced humoral immune responses to autoantigens in 2 of 8 patients. These patients developed IgG antibody to multiple epitopes overexpressed in PC after immunization. These responding patients received higher doses of the immunotherapy suggesting a dose response. Two immunogenic proteins (prostate-specific membrane antigen, hepsin) belong to the extracellular molecules family participating in malignant cell invasion. Median overall survival for patients was 25.1 months with 1 patient surviving over 70 months with stable PSA and bone metastasis before expiring of other causes. Three of 8 patients showed PSA stabilization (>100 d). In conclusion, HAP immunotherapy induces IgG responses to epitopes from autoantigens overexpressed in PC suggesting dose-dependent effect. HAP represents a viable immunotherapy approach to induce immune responses against tumor cells and may provide clinical benefit with minimal toxicity.

IDO inhibits a tryptophan sufficiency signal that stimulates mTOR: A novel IDO effector pathway targeted by D-1-methyl-tryptophan.

Tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO) alters inflammation and favors T-cell tolerance in cancer, but the underlying molecular mechanisms remain poorly understood. The integrated stress response kinase GCN2, a sensor of uncharged tRNA that is activated by amino acid deprivation, is recognized as an important effector of the IDO pathway. However, in a mouse model of inflammatory carcinogenesis, ablation of Gcn2 did not promote resistance against tumor development like the absence of IDO does, implying the existence of additional cancer-relevant pathways that operate downstream of IDO. Addressing this gap in knowledge, we report that the IDO-mediated catabolism of tryptophan also inhibits the immunoregulatory kinases mTOR and PKC-Θ, along with the induction of autophagy. These effects were relieved specifically by tryptophan but also by the experimental agent 1-methyl-D-tryptophan (D-1MT, also known as NLG8189), the latter of which reversed the inhibitory signals generated by IDO with higher potency. Taken together, our results implicate mTOR and PKC-Θ in IDO-mediated immunosuppressive signaling, and they provide timely insights into the unique mechanism of action of D-1MT as compared with traditional biochemical inhibitors of IDO. These findings are important translationally, because they suggest broader clinical uses for D-1MT against cancers that overexpress any tryptophan catabolic enzyme (IDO, IDO2 or TDO). Moreover, they define mTOR and PKC-Θ as candidate pharmacodynamic markers for D-1MT responses in patients recruited to ongoing phase IB/II cancer trials, addressing a current clinical need.

Novel suspension cell-based vaccine production systems for Rift Valley fever virus-like particles.

Rift Valley fever virus (RVFV) is an arthropod-borne pathogen that often results in severe morbidity and mortality in both humans and livestock. As its geographic range continues to expand, it presents a real threat to naïve populations around the world by accidental introduction (e.g., the result of increased travel) or intentional release (e.g., a bioterror event). While there is a clear need for a safe and efficacious vaccine against this emerging and re-emerging pathogen, no FDA-approved vaccine is currently available. This need was addressed by the establishment of novel mammalian and insect suspension cell line systems for the efficient production of RVF virus-like particle (VLP)-based vaccine candidates. A direct comparison of the production of RVF VLPs in these systems was performed. Optimization and characterization resulted in a production platform suitable for scale-up. Furthermore, RVF VLP-based vaccines were tested in a lethal challenge model and showed full protection, demonstrating that RVF VLPs present promising RVFV vaccine candidates.

A replication-incompetent Rift Valley fever vaccine: chimeric virus-like particles protect mice and rats against lethal challenge.

Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases. Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins G(N) and G(C), nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates. Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate.