Proteome analysis of the HIV-1 Gag interactome

AbstractHuman immunodeficiency virus Gag drives assembly of virions in infected cells and interacts with host factors which facilitate or restrict viral replication. Although several Gag-binding proteins have been characterized, understanding of virus–host interactions remains incomplete. In a series of six affinity purification screens, we have identified protein candidates for interaction with HIV-1 Gag. Proteins previously found in virions or identified in siRNA screens for host factors influencing HIV-1 replication were recovered. Helicases, translation factors, cytoskeletal and motor proteins, factors involved in RNA degradation and RNA interference were enriched in the interaction data. Cellular networks of cytoskeleton, SR proteins and tRNA synthetases were identified. Most prominently, components of cytoplasmic RNA transport granules were co-purified with Gag. This study provides a survey of known Gag–host interactions and identifies novel Gag binding candidates. These factors are associated with distinct molecular functions and cellular pathways relevant in host–pathogen interactions

Virotherapy combined with anti-PD-1 transiently reshapes the tumor immune environment and induces anti-tumor immunity in a preclinical PDAC model

IntroductionPancreatic ductal adenocarcinoma (PDAC) is largely refractory to cancer immunotherapy with PD-1 immune checkpoint blockade (ICB). Oncolytic virotherapy has been shown to synergize with ICB. In this work, we investigated the combination of anti-PD-1 and oncolytic measles vaccine in an immunocompetent transplantable PDAC mouse model.MethodsWe characterized tumor-infiltrating T cells by immunohistochemistry, flow cytometry and T cell receptor sequencing. Further, we performed gene expression profiling of tumor samples at baseline, after treatment, and when tumors progressed. Moreover, we analyzed systemic anti-tumor and anti-viral immunity.ResultsCombination treatment significantly prolonged survival compared to monotherapies. Tumor-infiltrating immune cells were increased after virotherapy. Gene expression profiling revealed a unique, but transient signature of immune activation after combination treatment. However, systemic anti-tumor immunity was induced by virotherapy and remained detectable even when tumors progressed. Anti-PD-1 treatment did not impact anti-viral immunity.DiscussionOur results indicate that combined virotherapy and ICB induces anti-tumor immunity and reshapes the tumor immune environment. However, further refinement of this approach may be required to develop its full potential and achieve durable efficacy

A vector-encoded bispecific killer engager to harness virus-activated NK cells as anti-tumor effectors

Treatment with oncolytic measles vaccines (MV) elicits activation of immune cells, including natural killer (NK) cells. However, we found that MV-activated NK cells show only modest direct cytotoxic activity against tumor cells. To specifically direct NK cells towards tumor cells, we developed oncolytic measles vaccines encoding bispecific killer engagers (MV-BiKE) targeting CD16A on NK cells and carcinoembryonic antigen (CEA) as a model tumor antigen. MV-BiKE are only slightly attenuated compared to parental MV and mediate secretion of functional BiKE from infected tumor cells. We tested MV-BiKE activity in cocultures of colorectal or pancreatic cancer cells with primary human NK cells. MV-BiKE mediate expression of effector cytokines, degranulation and specific anti-tumor cytotoxicity by NK cells. Experiments with patient-derived pancreatic cancer cultures indicate that efficacy of MV-BiKE may vary between individual tumors with differential virus permissiveness. Remarkably, we confirmed MV-BiKE activity in primaryhuman colorectal carcinoma specimens with autochthonous tumor and NK cells.This study provides proof-of-concept for MV-BiKE as a novel immunovirotherapy to harness virus-activated NK cells as anti-tumor effectors. [Figure not available: see fulltext.]

Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors

Immune checkpoint inhibitors (ICIs) can block distinct receptors on T cells or tumor cells thus preventing T cell inactivation and tumor immune escape. While the clinical response to treatment with ICIs in cancer patients is impressive, this therapy is often associated with a number of immune-related adverse events. There is therefore a need to explore innovative strategies of tumor-specific delivery of ICIs. Delivery of therapeutic proteins on a genetic level can be accomplished with viral vectors including those derived from adeno-associated virus (AAV). Here, we assessed the tumor-targeted Her2-AAV, a receptor-targeted AAV vector binding to the tumor antigen Her2/neu for cell entry, as vehicle for ICI gene delivery. Initially, we packaged the coding sequence of a scFv-Fc fusion protein directed against mouse programmed cell death protein-1 (PD-1) into Her2-AAV. Upon transduction of Her2/neu+ RENCA cells, AAV-encoded αPD-1 was readily detectable in the cell culture supernatant and revealed specific binding to its target antigen. In vivo, in BALB/c mice bearing subcutaneous RENCA-Her2/neu tumors, Her2-AAV mediated specific gene delivery into tumor tissue upon intravenous administration as verified by luciferase gene transfer and in vivo imaging thus demonstrating unimpaired tumor-targeting by Her2-AAV vectors in immunocompetent animals. When delivering the αPD-1 gene, levels of ICI were similar in tumor tissue for Her2-AAV and AAV2 but substantially reduced in liver for Her2-AAV. When combined with chemotherapy a tendency for reduced progression of tumor growth was documented for Her2-AAV treated mice. To get closer to the clinical situation, AAV constructs that deliver the complete coding sequence of the therapeutic antibody nivolumab which is directed against human PD-1 were generated next. The AAV-Nivolumab constructs were expressed and released from transduced MDA-MB-453 cells in vitro and from RENCA-Her2/neu cells upon intratumoral as well as intravenous administration in vivo. Antibody processing and expression levels were further improved through optimization of construct design. In conclusion, we provide proof-of-principle for redirecting the biodistribution of ICIs from liver and serum to tumor tissue by the use of engineered AAV vectors. This strategy can be easily combined with other types of immunotherapeutic concepts

A non-controlled, single arm, open label, phase II study of intravenous and intratumoral administration of ParvOryx in patients with metastatic, inoperable pancreatic cancer: ParvOryx02 protocol

Background: Metastatic pancreatic cancer has a dismal prognosis, with a mean six-month progression-free survival of approximately 50% and a median survival of about 11 months. Despite intensive research, only slight improvements of clinical outcome could be achieved over the last decades. Hence, new and innovative therapeutic strategies are urgently required. ParvOryx is a drug product containing native parvovirus H-1 (H-1PV). Since H-1PV was shown to exert pronounced anti-neoplastic effects in pre-clinical models of pancreatic cancer, the drug appears to be a promising candidate for treatment of this malignancy. Methods: ParvOryx02 is a non-controlled, single arm, open label, dose-escalating, single center trial. In total seven patients with pancreatic cancer showing at least one hepatic metastasis are to be treated with escalating doses of ParvOryx according to the following schedule: i) 40% of the total dose infused intravenously in equal fractions on four consecutive days, ii) 60% of the total dose injected on a single occasion directly into the hepatic metastasis at varying intervals after intravenous infusions. The main eligibility criteria are: age ≥ 18 years, disease progression despite first-line chemotherapy, and at least one hepatic metastasis. Since it is the second trial within the drug development program, the study primarily explores safety and tolerability after further dose escalation of ParvOryx. The secondary objectives are related to the evaluation of certain aspects of anti-tumor activity and clinical efficacy of the drug. Discussion: This trial strongly contributes to the clinical development program of ParvOryx. The individual hazards for patients included in the current study and the environmental risks are addressed and counteracted adequately. Besides information on safety and tolerability of the treatment after further dose escalation, thorough evaluations of pharmacokinetics and intratumoral spread as well as proof-of-concept (PoC) in pancreatic cancer will be gained in the course of the trial. Trial registration: ClinicalTrials.gov-ID: NCT02653313, Registration date: Dec. 4th, 2015

A novel approach of homozygous haplotype sharing identifies candidate genes in autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heritable disorder of complex and heterogeneous aetiology. It is primarily characterized by altered cognitive ability including impaired language and communication skills and fundamental deficits in social reciprocity. Despite some notable successes in neuropsychiatric genetics, overall, the high heritability of ASD (~90%) remains poorly explained by common genetic risk variants. However, recent studies suggest that rare genomic variation, in particular copy number variation, may account for a significant proportion of the genetic basis of ASD. We present a large scale analysis to identify candidate genes which may contain low-frequency recessive variation contributing to ASD while taking into account the potential contribution of population differences to the genetic heterogeneity of ASD. Our strategy, homozygous haplotype (HH) mapping, aims to detect homozygous segments of identical haplotype structure that are shared at a higher frequency amongst ASD patients compared to parental controls. The analysis was performed on 1,402 Autism Genome Project trios genotyped for 1 million single nucleotide polymorphisms (SNPs). We identified 25 known and 1,218 novel ASD candidate genes in the discovery analysis including CADM2, ABHD14A, CHRFAM7A, GRIK2, GRM3, EPHA3, FGF10, KCND2, PDZK1, IMMP2L and FOXP2. Furthermore, 10 of the previously reported ASD genes and 300 of the novel candidates identified in the discovery analysis were replicated in an independent sample of 1,182 trios. Our results demonstrate that regions of HH are significantly enriched for previously reported ASD candidate genes and the observed association is independent of gene size (odds ratio 2.10). Our findings highlight the applicability of HH mapping in complex disorders such as ASD and offer an alternative approach to the analysis of genome-wide association data

Measles Virus as an Oncolytic Immunotherapy

Measles virus (MeV) preferentially replicates in malignant cells, leading to tumor lysis and priming of antitumor immunity. Live attenuated MeV vaccine strains are therefore under investigation as cancer therapeutics. The versatile MeV reverse genetics systems allows for engineering of advanced targeted, armed, and shielded oncolytic viral vectors. Therapeutic efficacy can further be enhanced by combination treatments. An emerging focus in this regard is combination immunotherapy, especially with immune checkpoint blockade. Despite challenges arising from antiviral immunity, availability of preclinical models, and GMP production, early clinical trials have demonstrated safety of oncolytic MeV and yielded promising efficacy data. Future clinical trials with engineered viruses, rational combination regimens, and comprehensive translational research programs will realize the potential of oncolytic immunotherapy