Christoph Springfeld

A ferret model of canine distemper virus virulence and immunosuppression.

ABSTRACT Canine distemper virus (CDV) infects many carnivores, including ferrets and dogs, and is the member of the Morbillivirus genus most easily amenable to experimentation in a homologous small-animal system. To gain insights into the determinants of CDV pathogenesis, we isolated a strain highly virulent for ferrets by repeated passaging in these animals. Sequence comparison of the genome of this strain with that of its highly attenuated precursor revealed 19 mutations distributed almost evenly in the six genes. We then recovered a virus from a cDNA copy of the virulent CDV strains consensus sequence by using a modified reverse genetics system based on B cells. We infected ferrets with this virus and showed that it fully retained virulence as measured by the timing of rash appearance, disease onset, and death. Body temperature, leukocyte number, lymphocyte proliferation activity, and cell-associated viremia also had similar kinetics. We then addressed the question of the relative importance of the envelope and other viral constituents for virulence. Viruses in which the envelope genes (matrix, fusion, and hemagglutinin) of the virulent strain were combined with the other genes of the attenuated strain caused severe rash and fever even if the disease onset was delayed. Viruses in which the nucleocapsid, polymerase, and phosphoprotein genes (coding also for the V and C proteins) of the virulent strain were combined with the envelope genes of the attenuated strain caused milder signs of disease. Thus, virulence-inducing mutations have accumulated throughout the genome.

Advanced-stage pancreatic cancer: therapy options

Pancreatic ductal adenocarcinoma is one of the most aggressive cancers, and surgical resection is a requirement for a potential cure. However, the majority of patients are diagnosed with advanced-stage disease, either metastatic (50%) or locally advanced cancer (30%). Although palliative chemotherapy is the standard of care for patients with metastatic disease, management of locally advanced adenocarcinoma is controversial. Several treatment options, including extended surgical resections, neoadjuvant therapy with subsequent resections, as well as palliative radiotherapy and/or chemotherapy, should be considered. However, there is little evidence available to support treatment options for locally advanced disease. As valid predictive biomarkers for stratification of therapy are not available today, future trials need to define the role of the different treatment options. This Review summarizes the current evidence and discusses available treatment options for both locally advanced and metastatic pancreatic adenocarcinoma.

Tumoral Immune Cell Exploitation in Colorectal Cancer Metastases Can Be Targeted Effectively by Anti-CCR5 Therapy in Cancer Patients

The immune response influences the clinical course of colorectal cancer (CRC). Analyzing the invasive margin of human CRC liver metastases, we identified a mechanism of immune cell exploitation by tumor cells. While two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10, CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5. CCR5 blockade in patient-derived functional in vitro organotypic culture models showed a macrophage repolarization with anti-tumoral effects. These anti-tumoral effects were then confirmed in a phase I trial with a CCR5 antagonist in patients with liver metastases of advanced refractory CRC. Mitigation of tumor-promoting inflammation within the tumor tissue and objective tumor responses in CRC were observed.

Lymphoma Chemovirotherapy: CD20-Targeted and Convertase-Armed Measles Virus Can Synergize with Fludarabine

Combination chemotherapy regimen incorporating CD20 antibodies are commonly used in the treatment of CD20-positive non-Hodgkins lymphoma (NHL). Fludarabine phosphate (F-araAMP), cyclophosphamide, and CD20 antibodies (Rituximab) constitute the FCR regimen for treating selected NHL, including aggressive mantle cell lymphoma (MCL). As an alternative to the CD20 antibody, we generated a CD20-targeted measles virus (MV)-based vector. This vector was also armed with the prodrug convertase purine nucleoside phosphorylase (PNP) that locally converts the active metabolite of F-araAMP to a highly diffusible substance capable of efficiently killing bystander cells. We showed in infected cells that early prodrug administration controls vector spread, whereas late administration enhances cell killing. Control of spread by early prodrug administration was also shown in an animal model: F-araAMP protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Enhanced oncolytic potency after extensive infection was shown in a Burkitts lymphoma xenograft model (Raji cells): After systemic vector inoculation, prodrug administration enhanced the therapeutic effect synergistically. In a MCL xenograft model (Granta 519 cells), intratumoral (i.t.) vector administration alone had high oncolytic efficacy: All mice experienced complete but temporary tumor regression, and survival was two to four times longer than that of untreated mice. Cells from MCL patients were shown to be sensitive to infection. Thus, synergy of F-araAMP with a PNP-armed and CD20-targeted MV was shown in one lymphoma therapy model after systemic vector inoculation.

Oncolytic Efficacy and Enhanced Safety of Measles Virus Activated by Tumor-Secreted Matrix Metalloproteinases

Cancer cells secrete matrix metalloproteinases (MMP) that degrade the extracellular matrix and are responsible for some hallmarks of malignant cancer. Many viruses, including a few currently used in oncolytic virotherapy clinical trials, depend on intracellular proteases to process their proteins and activate their particles. We show here for measles virus (MV) that particle activation can be made dependent of proteases secreted by cancer cells. The MV depends on the intracellular protease furin to process and activate its envelope fusion (F) protein. To make F protein activation cancer cell specific, we introduced hexameric sequences recognized by an MMP and identified the mutant proteins most effective in fusing MMP-expressing human fibrosarcoma cells (HT1080). We showed that an MMP inhibitor interferes with syncytia formation elicited by mutant F proteins and confirmed MMP-dependent cleavage by Edman degradation sequence analysis. We generated recombinant MVs expressing the modified F proteins in place of furin-activated F. These viruses spread only in cells secreting MMP. In nude mice, an MMP-activated MV retarded HT1080 xenograft growth as efficiently as the furin-activated MV vaccine strain. In MV-susceptible mice, the furin-activated virus caused lethal encephalitis upon intracerebral inoculation, whereas the MMP-activated did not. Thus, MV particle activation can be made dependent of proteases secreted by cancer cells, enhancing safety. This study opens the perspective of combining targeting at the particle activation, receptor recognition, and selective replication levels to improve the therapeutic index of MV and other viruses in ongoing clinical trials of oncolysis.

Locally Advanced Pancreatic Cancer: Neoadjuvant Therapy With Folfirinox Results in Resectability in 60% of the Patients.

Objective: For patients with locally advanced and unresectable pancreatic cancer (PDAC), neodadjuvant treatment and consecutive surgical exploration have been studied during the last decade with various neoadjuvant therapies including chemotherapy and combinations with radiation. Aim of the study was the evaluation of neoadjuvant therapy with a focus on Folfirinox. Methods: All consecutive patients undergoing surgery for PDAC after neoadjuvant treatment were analyzed (clinico-pathological characteristics, secondary resection rates, outcome). Patients receiving Folfirinox were compared with other treatment regimens. Results: Between December 2001 and June 2015, 575 patients received neoadjuvant treatment and were scheduled for resection after re-staging. A successful resection was achieved in 292 patients (50.8%). Resection rates following Folfirinox were 61% (76/125 patients) compared with 46% (150/322 patients) after gemcitabine and radiation, and 52% (66/128 patients) after other treatments (P = 0.026). Median overall survival was 15.3 months after resection vs 8.5 months after exploration alone (P < 0.0001). Subgroup median survival was 16.0 months (Folfirinox) vs 16.5 months (gemcitabine) and 14.5 months (others) with 3-year survival of 28.1%, 23.2%, and 19.7%, respectively (P = 0.8582). By multivariable analysis, Folfirinox was confirmed to be independently associated with a favorable prognosis. Conclusions: Folfirinox is a valuable treatment option in the neoadjuvant therapy of PDAC. From the present data, which represent the largest available study population to date, Folfirinox seems to be the most effective protocol resulting in a significantly better secondary resection rate and overall survival than other treatments. It should be considered in all patients fit for this regimen and consecutive surgical exploration.

Envelope-chimeric Entry-targeted Measles Virus Escapes Neutralization and Achieves Oncolysis

Measles virus (MV) is a promising vector for cancer therapy and multivalent vaccination, but high prevalence of pre-existing neutralizing antibodies may reduce therapeutic efficacy, particularly following systemic administration. MV has only one serotype, but here we show that its envelope glycoproteins can be exchanged with those of the closely related canine distemper virus (CDV), generating a chimeric virus capable of escaping neutralization. To target its entry, we displayed on the CDV attachment protein a single-chain antibody specific for a designated receptor. To enhance oncolytic efficacy we armed the virus with a prodrug convertase gene capable of locally activating chemotherapeutic prodrugs. The new virus achieved high titers, was genetically stable, and was resistant to neutralization by sera from both MV-immunized mice and MV-immune humans. The new virus targeted syngeneic murine tumor cells expressing the designated receptor implanted in immunocompetent mice, and synergized with a chemotherapeutic prodrug in a model of oncolysis. Importantly, the chimeric MV remained oncolytic when administered systemically even in the presence of anti-MV antibodies capable of abrogating the therapeutic efficacy of the parental, nonshielded MV. This work shows that targeting, arming, and shielding can be combined to generate a tumor-specific, neutralization-resistant virus that can synergize with chemotherapeutics.

Granulocyte-macrophage colony-stimulating factor-armed oncolytic measles virus is an effective therapeutic cancer vaccine.

Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased antitumor activity, and are currently under investigation in clinical phase 1 trials. Approaches with other viral vectors have shown that insertion of immunomodulatory transgenes enhances the therapeutic potency. In this study, we engineered MV for expression of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). For the first time, therapeutic efficacy and adaptive immune response in the context of MV oncolysis could be evaluated in the previously established immunocompetent murine colon adenocarcinoma model MC38cea. MC38cea cells express the human carcinoembryonic antigen (CEA), allowing for infection with retargeted MV. Intratumoral application of MV-GMCSF significantly delayed tumor progression and prolonged median overall survival compared with control virus-treated mice. Importantly, more than one-third of mice treated with MV-GMCSF showed complete tumor remission and rejected successive tumor reengraftment, demonstrating robust long-term protection. An enhanced cell-mediated tumor-specific immune response could be detected by lactate dehydrogenase assay and interferon-γ enzyme-linked immunospot assay. Furthermore, MV-GMCSF treatment correlated with increased abundance of tumor-infiltrating CD3(+) lymphocytes analyzed by quantitative microscopy of tumor sections. These findings underline the potential of oncolytic, GM-CSF-expressing MV as an effective therapeutic cancer vaccine actively recruiting adaptive immune responses for enhanced therapeutic impact and tumor elimination. Thus, the treatment benefit of this combined immunovirotherapy approach has direct implications for future clinical trials.

Broadly Neutralizing Immune Responses against Hepatitis C Virus Induced by Vectored Measles Viruses and a Recombinant Envelope Protein Booster

ABSTRACT Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.

Characterization of the Tupaia Rhabdovirus Genome Reveals a Long Open Reading Frame Overlapping with P and a Novel Gene Encoding a Small Hydrophobic Protein

ABSTRACT Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.