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Use of an alternative open reading frame, potentially as a result of cellular stress, drives production of an unconventional insulin epitope that is recognized by cytotoxic T cells from individuals with type 1 diabetes; these T cells kill beta cells in vitro . Identification of epitopes that are recognized by diabetogenic T cells and cause selective beta cell destruction in type 1 diabetes (T1D) has focused on peptides originating from native beta cell proteins. Translational errors represent a major potential source of antigenic peptides to which central immune tolerance is lacking 1 , 2 . Here, we describe an alternative open reading frame within human insulin mRNA encoding a highly immunogenic polypeptide that is targeted by T cells in T1D patients. We show that cytotoxic T cells directed against the N-terminal peptide of this nonconventional product are present in the circulation of individuals diagnosed with T1D, and we provide direct evidence that such CD8 + T cells are capable of killing human beta cells and thereby may be diabetogenic. This study reveals a new source of nonconventional polypeptides that act as self-epitopes in clinical autoimmune disease.

Oncolytic adenovirus therapy is believed to be a promising way to treat cancer patients. To be able to target tumor cells with an oncolytic adenovirus, expression of the adenovirus receptor on the tumor cell is essential. Different adenovirus types bind to different receptors on the cell, of which the expression can vary between tumor types. Pre-existing neutralizing immunity to human adenovirus species C type 5 (HAdV-C5) has hampered its therapeutic efficacy in clinical trials, hence several adenoviral vectors from different species are currently being developed as a means to evade pre-existing immunity. Therefore, knowledge on the expression of appropriate adenovirus receptors on tumor cells is important. This could aid in determining which tumor types would benefit most from treatment with a certain oncolytic adenovirus type. This review provides an overview of the known receptors for human adenoviruses and how their expression on tumor cells might be differentially regulated compared to healthy tissue, before and after standardized anticancer treatments. Mechanisms behind the up- or downregulation of adenovirus receptor expression are discussed, which could be used to find new targets for combination therapy to enhance the efficacy of oncolytic adenovirus therapy. Additionally, the utility of the adenovirus receptors in oncolytic virotherapy is examined, including their role in viral spread, which might even surpass their function as primary entry receptors. Finally, future directions are offered regarding the selection of adenovirus types to be used in oncolytic adenovirus therapy in the fight against cancer.

Enhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.

ObjectivesTo understand the molecular features distinguishing anti-citrullinated protein antibodies (ACPA) from ‘conventional’ antibodies in rheumatoid arthritis (RA).MethodsSerum of ACPA-positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum, plasma and/or synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and analysed by mass spectrometry. Binding affinity to citrullinated antigens was measured by ELISA and imaging surface plasmon resonance using recombinant monoclonal ACPA with molecular modifications.ResultsIn all donor samples studied (n=24), ACPA-IgG exhibited a 10–20 kDa higher molecular weight compared with non-autoreactive IgG. This feature also distinguished ACPA-IgG from antibodies against recall antigens or other disease-specific autoantibodies. Structural analysis revealed that a high frequency of N-glycans in the (hyper)variable domains of ACPA is responsible for this observation. In line with their localisation, these N-glycans were found to modulate binding avidity of ACPA to citrullinated antigens.ConclusionsThe vast majority of ACPA-IgG harbour N-glycans in their variable domains. As N-linked glycosylation requires glycosylation consensus sites in the protein sequence and as these are lacking in the ‘germline-counterparts’ of identified variable domains, our data indicate that the N-glycosylation sites in ACPA variable domains have been introduced by somatic hypermutation. This finding also suggests that ACPA-hyperglycosylation confers a selective advantage to ACPA-producing B cells. This unique and completely novel feature of the citrulline-specific immune response in RA elucidates our understanding of the underlying B cell response.

Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin β1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance due to its genetic and cellular heterogeneity, dense stromal tissue, and immune‐suppressive tumour microenvironment. Oncolytic virotherapy has emerged as a new treatment modality which uses tumour‐specific viruses to eliminate cancerous cells. Non‐human primate adenoviruses of the human adenovirus B (HAdV‐B) species have demonstrated considerable lytic potential in human cancer cells as well as limited preexisting neutralizing immunity in humans. Previously, we have generated a new oncolytic derivative of the gorilla‐derived HAdV‐B AdV‐lumc007 named ‘GoraVir’. Here, we show that GoraVir displays oncolytic efficacy in pancreatic cancer cells and pancreatic‐cancer‐associated fibroblasts. Moreover, it retains its lytic potential in monoculture and co‐culture spheroids. In addition, we established the ubiquitously expressed complement receptor CD46 as the main entry receptor for GoraVir. Finally, a single intratumoural dose of GoraVir was shown to delay tumour growth in a BxPC‐3 xenograft model at 10 days post‐treatment. Collectively, these data demonstrate that the new gorilla‐derived oncolytic adenovirus is a potent oncolytic vector candidate that targets both pancreatic cancer cells and tumour‐adjacent stroma. Oncolytic adenovirus ‘GoraVir’ has strong lytic potential in both pancreatic cancer cells and cancer‐associated fibroblasts, which may be facilitated by its use of CD46 for viral entry. Moreover, GoraVir demonstrates superior oncolytic efficacy compared to HAdV‐C5 in spheroid cultures as well as in vivo. This shows that GoraVir exhibits unique oncolytic properties and seems a promising candidate for the treatment of pancreatic cancer.

Misfolded ER proteins are retrotranslocated into the cytosol for degradation via the ubiquitin–proteasome system. The human cytomegalovirus protein US11 exploits this ER-associated protein degradation (ERAD) pathway to downregulate HLA class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. US11-mediated degradation of HLA class I has been instrumental in the identification of key components of mammalian ERAD, including Derlin-1, p97, VIMP and SEL1L. Despite this, the process governing retrotranslocation of the substrate is still poorly understood. Here using a high-coverage genome-wide shRNA library, we identify the uncharacterized protein TMEM129 and the ubiquitin-conjugating E2 enzyme UBE2J2 to be essential for US11-mediated HLA class I downregulation. TMEM129 is an unconventional C4C4-type RING finger E3 ubiquitin ligase that resides within a complex containing various other ERAD components, including Derlin-1, Derlin-2, VIMP and p97, indicating that TMEM129 is an integral part of the ER-resident dislocation complex mediating US11-induced HLA class I degradation. The human cytomegalovirus protein US11 downregulates host immune responses by redirecting HLA class I molecules for endoplasmic reticulum-associated protein degradation. Using a high-coverage genome-wide shRNA screen, the authors identify TMEM129 as an E3 ubiquitin ligase essential for this process.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance. Oncolytic viruses have emerged as a new treatment approach and convey their antitumor activity through lysis of cancer cells. The therapeutic efficacy of oncolytic viruses is largely dependent on the induction of immunogenic cell death (ICD) and the subsequent antitumor immune responses. However, the concurrent generation of antiviral immune responses may also limit the a virus’ therapeutic window. GoraVir is a new oncolytic adenovirus derived from the Human Adenovirus B (HAdV-B) isolate AdV-lumc007 which was isolated from a gorilla and has demonstrated excellent lytic activity in both in vitro and in vivo models of PDAC. In this study, we characterized the immunostimulatory profile of cancer cell death induced by GoraVir and the concerted cellular antiviral responses in three conventional pancreatic cancer cell lines. While GoraVir was shown to induce late apoptotic/necrotic cell death at earlier time points post infection than the human adenovirus type 5 (HAdV-C5), similar levels of ICD markers were expressed. Moreover, GoraVir was shown to induce ICD not dependent on STING expression and regardless of subsequent antiviral responses. Together, these data demonstrate that GoraVir is an excellent candidate for use in oncolytic virotherapy.

BackgroundT-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major barrier for efficacy in solid tumors. In this study, we exploited oncolytic reovirus as a strategy to enhance the efficacy of CD3-bsAbs in immune-silent solid tumors.MethodsThe mutant p53 and K-ras induced murine pancreatic cancer model KPC3 resembles human pancreatic ductal adenocarcinomas with a desmoplastic tumor microenvironment, low T-cell density and resistance to immunotherapy. Immune-competent KPC3 tumor-bearing mice were intratumorally injected with reovirus type 3 Dearing strain and the reovirus-induced changes in the tumor microenvironment and spleen were analyzed over time by NanoString analysis, quantitative RT-PCR and multicolor flow cytometry. The efficacy of reovirus in combination with systemically injected CD3-bsAbs was evaluated in immune-competent mice with established KPC3 or B16.F10 tumors, and in the close-to-patient human epidermal growth factor receptor 2 (HER2)+ breast cancer model BT474 engrafted in immunocompromised mice with human T cells as effector cells.ResultsReplication-competent reovirus induced an early interferon signature, followed by a strong influx of natural killer cells and CD8+ T cells, at the cost of FoxP3+ Tregs. Viral replication declined after 7 days and was associated with a systemic activation of lymphocytes and the emergence of intratumoral reovirus-specific CD8+ T cells. Although tumor-infiltrating T cells were mostly reovirus-specific and not tumor-specific, they served as non-exhausted effector cells for the subsequently systemically administered CD3-bsAbs. Combination treatment of reovirus and CD3-bsAbs led to the regression of large, established KPC3, B16.F10 and BT474 tumors. Reovirus as a preconditioning regimen performed significantly better than simultaneous or early administration of CD3-bsAbs. This combination treatment induced regressions of distant lesions that were not injected with reovirus, and systemic administration of both reovirus and CD3-bsAbs also led to tumor control. This suggests that this therapy might also be effective for metastatic disease.ConclusionsOncolytic reovirus administration represents an effective strategy to induce a local interferon response and strong T-cell influx, thereby sensitizing the tumor microenvironment for subsequent CD3-bsAb therapy. This combination therapy warrants further investigation in patients with non-inflamed solid tumors.

Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D) is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A) on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin) mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA) inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.