Vaccinia-based oncolytic immunotherapy Pexastimogene Devacirepvec in patients with advanced hepatocellular carcinoma after sorafenib failure: a randomized multicenter Phase IIb trial (TRAVERSE).

Pexastimogene devacirepvec (Pexa-Vec) is a vaccinia virus-based oncolytic immunotherapy designed to preferentially replicate in and destroy tumor cells while stimulating anti-tumor immunity by expressing GM-CSF. An earlier randomized Phase IIa trial in predominantly sorafenib-naïve hepatocellular carcinoma (HCC) demonstrated an overall survival (OS) benefit. This randomized, open-label Phase IIb trial investigated whether Pexa-Vec plus Best Supportive Care (BSC) improved OS over BSC alone in HCC patients who failed sorafenib therapy (TRAVERSE). 129 patients were randomly assigned 2:1 to Pexa-Vec plus BSC vs. BSC alone. Pexa-Vec was given as a single intravenous (IV) infusion followed by up to 5 IT injections. The primary endpoint was OS. Secondary endpoints included overall response rate (RR), time to progression (TTP) and safety. A high drop-out rate in the control arm (63%) confounded assessment of response-based endpoints. Median OS (ITT) for Pexa-Vec plus BSC vs. BSC alone was 4.2 and 4.4 months, respectively (HR, 1.19, 95% CI: 0.78-1.80; p = .428). There was no difference between the two treatment arms in RR or TTP. Pexa-Vec was generally well-tolerated. The most frequent Grade 3 included pyrexia (8%) and hypotension (8%). Induction of immune responses to vaccinia antigens and HCC associated antigens were observed. Despite a tolerable safety profile and induction of T cell responses, Pexa-Vec did not improve OS as second-line therapy after sorafenib failure. The true potential of oncolytic viruses may lie in the treatment of patients with earlier disease stages which should be addressed in future studies. ClinicalTrials.gov: NCT01387555.

Retroviral insertional mutagenesis in telomerase-immortalized hepatocytes identifies RIPK4 as novel tumor suppressor in human hepatocarcinogenesis.

Carcinogenesis is a multistep process involving alterations in various cellular pathways. The critical genetic events driving the evolution of primary liver cancer, specifically hepatoblastoma and hepatocellular carcinoma (HCC), are still poorly understood. However, telomere stabilization is acknowledged as prerequisite for cancer progression in humans. In this project, human fetal hepatocytes were utilized as a cell culture model for untransformed, proliferating human liver cells, with telomerase activation as first oncogenic hit. To elucidate critical downstream genetic events driving further transformation of immortalized liver cells, we used retroviral insertional mutagenesis as an unbiased approach to induce genetic alterations. Following isolation of hyperproliferating, provirus-bearing cell clones, we monitored cancer-associated growth properties and characterized changes toward a malignant phenotype. Three transformed clones with the ability to form colonies in soft agar were expanded. As proof-of-principle for our experimental setup, we identified a transforming insertion on chromosome 8 within the pleiomorphic adenoma gene 1 (PLAG1), resulting in a 20-fold increase in PLAG1 expression. Upregulation of PLAG1 has already been described to promote human hepatoblastoma development. In a separate clone, a transforming insertion was detected in close proximity to the receptor-interacting serine-threonine kinase 4 (RIPK4) with an approximately eightfold suppression in RIPK4 expression. As validation for this currently unknown driver in hepatocarcinogenesis, we examined RIPK4 expression in human HCC samples and confirmed a significant suppression of RIPK4 in 80% of the samples. Furthermore, overexpression of RIPK4 in transformed human fetal hepatocytes resulted in an almost complete elimination of anchorage-independent growth. On the basis of these data, we propose RIPK4 as a novel putative tumor suppressor in human hepatocarcinogenesis.

Hepatic stem and progenitor cells in liver diseases and hepatocarcinogenesis.

Because of its physiological role and its central function in metabolism and homeostasis, the liver is exposed to an environment rich in toxins. In addition, the liver has to cope with various infectious pathogens, in particular hepatotropic viruses. Therefore, the liver needs efficient and highly regulated regeneration mechanisms. Under normal circumstances the liver shows a low rate of hepatocyte renewal but in the event of liver injury, for example, acute liver damage or drug intoxications, hepatocytes display a remarkable capacity to divide and to restore the liver parenchyma. Because of their enormous capability to regenerate the liver, which is unique among differentiated cells in human organs, hepatocytes function as stem cells. However, if the proliferation of hepatocytes is impaired, as in liver cirrhosis, a progenitor cell population is activated and serves as reserve compartment for liver restoration. Hepatic progenitor cells are bipotential and are located in the canals of Hering, the most peripheral branches of the biliary system. According to the current paradigm, hepatic progenitor cells drive liver regeneration in end-stage liver diseases, where hepatocytes become senescent and may therefore be a target cell population for carcinogenesis. In this review, we revisit landmark studies, summarize the current nomenclature, and discuss recent data elucidating the characteristics and the functional role of hepatic stem and progenitor cells in liver diseases and hepatocarcinogenesis.

Development of a constant surface pressure penetration langmuir balance based on axisymmetric drop shape analysis.

A new constant pressure pendant-drop penetration surface balance has been developed combining a pendant-drop surface balance, a rapid-subphase-exchange technique, and a fuzzy logic control algorithm. Beside the determination of insoluble monolayer compression-expansion isotherms, it allows performance of noninvasive kinetic studies of the adsorption of surfactants added to the new subphase onto the free surface and of the adsorption/penetration/reaction of the former onto/into/with surface layers, respectively. The interfacial pressure pi is a fundamental parameter in these studies: by working at constant pi one controls the height of the energy barrier to adsorption/penetration and can select different regimes and steps of the adsorption/penetration process. In our device a solution drop is formed at the tip of a coaxial double capillary, connected to a double microinjector. Drop profiles are extracted from digital drop micrographs and fitted to the equation of capillarity, yielding pi, the drop volume V, and the interfacial area A. pi is varied changing V (and hence A) with the microinjector. Control is based on a case-adaptable modulated fuzzy-logic PID algorithm able to maintain constant pi (or A) under a wide range of experimental conditions. The drop subphase liquid can be exchanged quantitatively by the coaxial capillaries. The adsorption/penetration/reaction kinetics at constant pi are then studied monitoring A(t), i.e., determining the relative area change necessary at each instant to compensate the pressure variation due to the interaction of the surfactant in the subsurface with the surface layer. A fully Windows-integrated program manages the whole setup. Examples of experimental protein adsorption and monolayer penetration kinetics are presented.

Cationic lipid polymerization as a novel approach for constructing new DNA delivery agents.

In vivo gene delivery mediated by cationic lipids is often compromised by aggregation due to complexation with proteins in the blood. To improve the stability of cationic lipid-DNA complexes, the present study aimed to develop a novel approach in which a poly(cationic lipid) (PCL) is utilized to form stable cationic polyplexes for gene transfection. Hydrogenation of the acrylamide analogue of betaAE-DMRI, the polymerizable precursor of PCL, provided a monomeric lipid derivative (MHL) which was used for direct comparison of corresponding lipoplex stability, toxicity, and transfection activity. Various formulations of cationic liposomes, such as MHL, MHL-cholesterol (Chol), PCL, PCL-Chol, DOTAP-Chol, and commercially available lipofectamine were generated and examined in this study. The new poly(cationic lipid) did not display any significant toxicity to rat hepatocytes or Hep G2 cells as indicated by an LDH leakage assay. Furthermore, PCL was significantly less toxic than MHL, DOTAP-Chol or lipofectamine. Suspensions of PCL were resistant to aggregation even after 24 h of exposure to solutions containing 50 and 100% fetal bovine serum (FBS). In contrast, suspensions of lipofectamine extensively aggregated after 24 h of exposure to 50% FBS. To examine the influence of lipid polymerization on gene transfer activity, liposome-mediated transfections of a luciferase vector (pGL3) were performed in Hep G2 and Alexander cell lines. The luciferase activity of the PCL formulations in Hep G2 cells were similar to those of the MHL, DOTAP-Chol and lipofectamine formulations, demonstrating that lipid polymerization does not compromise transfection activity. In comparison to the monomeric precursor MHL and to the industry transfection standards DOTAP and lipofectamine, the novel poly(cationic lipid) exhibited the lowest cytotoxicity, was the most resistant to serum-induced aggregation and had comparable transfection activity when coformulated with cholesterol. This novel polymerization approach for the development of stable and active polyplexes may prove a valuable alternative for in vivo gene delivery.

Porcine reproductive and respiratory syndrome virus (PRRSV): kinetics of infection in lymphatic organs and lung.

Pigs were infected by the oronasal route with European isolates of the porcine reproductive and respiratory syndrome virus (PRRSV; I10 and Cobbelsdorf). The kinetics of infection in lymphatic organs and the lung were analysed by immunofluorescence detection of virus antigen, re-isolation of the virus and reverse transcription--polymerase chain reaction (RT-PCR) for PRRSV-specific RNA. The kinetics of PRRSV infection proceeded in three phases, irrespective of the varying infestation of lymphatic organs within the first days post-infection (p.i.). First, an early acute infection of lymphatic organs developed within the first week and was characterized by a high number of antigen-positive macrophages. Second, a delayed acute infection of the lung was observed, which was most pronounced during the second and third week p.i. when a high number of infected alveolar macrophages was observed. The acute infection of lymphatic organs had resolved at this time. Infected cells in the lung were predominantly located in pneumonic lesions. Third, a persistent infection was demonstrated by RT-PCR and immunohistology when the experiments were terminated at day 49 p.i. The virus persisted in lymphatic organs, especially in the tonsils, and in the lung. At this stage, indications for a re-occurrence of acute infection were observed in restricted areas of the lung.

Coronavirus infection and demyelination. Development of inflammatory lesions in Lewis rats.

Coronavirus infections of rodents can cause diseases of the central nervous system characterised by inflammatory demyelination. The lesions mimick in many aspects the pathology of multiple sclerosis in humans and of other neurological diseases. As an animal model for demyelination, we studied the MHV-JHM induced encephalomyelitis of Lewis rats. The pathomorphological analysis revealed patterns of lesions which developed in stages. Infected oligodendrocytes were first destroyed by necrosis. Later stages were characterized by demyelinated plaques. In the center of plaques, no virus antigen was found and oligodendrocytes were mainly destroyed by apoptosis. At the edge of plaques, virus antigen was expressed in parallel to infiltrations consisting of lymphocytes and macrophages. The prevailing mechanisms leading to demyelination may change individually and during defined stages of the disease. The transcriptional expression of chemoattractants and other mediators of inflammation was studied by semiquantitative RT-PCR. Virus induced inflammatory demyelination was accompanied by high expression of a relatively novel cytokine, the endothelial monocyte activating polypeptide II (EMAP II). By immunocytochemistry, EMAP II was detected in parenchymal microglia located both within the lesions and in unaffected areas. Furthermore, the level of transcriptional expression of the regulatory calcium binding S100 proteins MRP8, MRP14 and CP10 was associated with inflammatory demyelination and expression of IFN gamma, IL-2, TNF alpha, and iNOS.

Arterivirus PRRSV. Experimental studies on the pathogenesis of respiratory disease.

Pigs were infected with the porcine respiratory and reproductive syndrome virus (PRRSV) by the oronasal route. We studied the development of histological lesions, sites of virus infection and of inflammatory infiltrates by quantitative evaluation of reactive cells. The animals developed a multifocal interstitial pneumonia. Clinical signs of pneumonia were observed from day 7 to 21. In the first stage, an acute alveolitis was found, which was characterised by a hyperplasia of type II pneumocytes within the septa and an accumulation of macrophages in the alveolar spaces. Within 2-4 days p.i., virus infected cells were prominent in lymphatic organs, but their number declined rapidly during the following days. In the following period, the number of virus antigen positive cells increased in the lung. An interesting discrepancy existed between the relatively small number of virus specific cells and the degree of intensive pneumonia. As a first step to analyse mechanisms leading to the induction of pneumonia, we studied transcriptional expression of cytokines and other immunomodulatory molecules by semiquantitative RT-PCR.

Coronavirus infection and demyelination. Sequence conservation of the S-gene during persistent infection of Lewis-rats.

Coronaviruses display a large phenotypic variability, which may be an important factor for diversification and selection. Previous studies have demonstrated that the S-protein is an essential determinant of virulence and pathogenicity. Therefore we studied the S-gene as an indicator molecule for selection processes employing two different MHV-JHM variants. First, Lewis-rats were infected with MHV-JHM-Pi, a variant that causes demyelinating disease after several weeks p.i. It was not possible to isolate infectious MHV-JHM-Pi from such rats, although viral proteins were expressed. The S-gene was rescued directly from brain tissue employing RT-PCR technology. The amplicons were sequenced in bulk or at the level of single clones. We detected no evidence for an increase of S-gene mutants during the length of time. Only few mutations were found at the clonal level. The changes were distributed throughout the analysed S-gene fragments without a predilection in their location. The frequency of mutation remained low within a range of 0.03 to 0.5 mutations per thousand nucleotides. As a second approach, we sequenced the S-genes of viruses isolated from brain tissue infected with MHV-JHM-ts43. Infection of adult Lewis rats with that mutant resulted several weeks to months p.i. in demyelinating encephalomyelitis. The S-gene of this virus contains an insertion of 423 bp in the S1 region, which is identical to a polymorphic region described for MHV-4. In contrast to JHM-Pi, infectious MHV-JHM-ts43 was readily to isolate from brain tissue. The S-gene sequences of virus isolated 45-106 days p.i. from diseased rats were identical with that of the input virus. These results show, that during a persistent infection of Lewis-rats the S-gene was highly conserved.

No evidence for quasispecies populations during persistence of the coronavirus mouse hepatitis virus JHM: sequence conservation within the surface glycoprotein gene S in Lewis rats.

The surface glycoprotein S (spike) of coronaviruses is believed to be an important determinant of virulence and displays extensive genetic polymorphism in cell culture isolates. This led us to consider whether the observed heterogeneity is reflected by a quasispecies distribution of mutated RNA molecules within the infected organ. Coronavirus infection of rodents is a useful model system for investigating the pathogenesis of virus-induced central nervous system (CNS) disease. Here, we investigated whether genetic changes in the S gene occurred during virus persistence in vivo. We analysed the variability of S gene sequences directly from the brain tissue of Lewis rats infected with the coronavirus mouse hepatitis virus (MHV) variant JHM-Pi using RT-PCR amplification methods. The S gene sequence displayed a remarkable genetic stability in vivo. No evidence for a quasispecies distribution was found by sequence analysis of amplified S gene fragments derived from the CNS of Lewis rats. Furthermore, the S gene also remained conserved under the selection pressure of a neutralizing antibody. Only a few mutations predicted to result in amino acid changes were detected in single clones. The changes were not represented in the consensus sequence. These results indicate that to retain functional proteins under the constraints of a persistent infection in vivo, conservation of sequence can be more important than heterogeneity.

Patterns of oligodendrocyte pathology in coronavirus-induced subacute demyelinating encephalomyelitis in the Lewis rat.

Intracerebral infection of rats with JHM coronavirus induces a chronic inflammatory demyelinating disease, which in many respects mimicks the pathology of multiple sclerosis. We investigated the patterns of demyelination and oligodendrocyte pathology in this model. In early stages of the disease infection of oligodendrocytes was associated with a downregulation of expression of mRNA for proteolipid protein in the absence of myelin destruction. When demyelinating lesions were formed infected oligodendrocytes were destroyed by necrosis, whereas oligodendrocytes that did not contain detectable virus antigen or RNA were in part dying by apoptosis. At this stage of the disease remyelination of the lesions was pronounced. At later stages after infection virus antigen was nearly completely cleared from the lesions. In spite of the lack of detectable virus, ongoing demyelination and unspecific tissue destruction occurred, and oligodendrocytes were mainly destroyed by apoptosis. These late lesions revealed only minimal central remyelination, but they were frequently repaired by Schwann cells. Our studies suggest that the mechanisms of myelin destruction in this model of virus-induced demyelination are complex and that the patterns of tissue damage may change during the course of the disease.

Coronavirus-induced encephalomyelitis: balance between protection and immune pathology depends on the immunization schedule with spike protein S.

The neurotropic mouse hepatitis virus MHV-JHM induces central nervous system (CNS) demyelination in Lewis rats that pathologically resembles CNS lesions in multiple sclerosis. The mechanisms of MHV-JHM-induced demyelination remain unclear and several studies have implicated the role of the immune response in this process. We have shown previously that protective immunity against MHV-JHM-induced encephalomyelitis was induced by immunization with a vaccinia virus (VV) recombinant expressing MHV-JHM S-protein (VV-S). Here, we present evidence that the time of MHV-JHM challenge after immunization with VV-S plays a critical role in protective immunity. The induction of virus-neutralizing S-protein-specific antibodies prior to the MHV-JHM challenge modulates the disease process and a subacute encephalomyelitis based on a persistent virus infection developed. Typical pathological alterations were lesions of inflammatory demyelination. In addition, the results indicate that after seroconversion, CD8+ T cells were no longer essential for virus elimination in contrast to their role in protection during acute encephalomyelitis.

Location of antibody epitopes within the mouse hepatitis virus nucleocapsid protein.

Thirteen monoclonal antibodies (Mab) specific for the nucleocapsid (N) protein of mouse hepatitis virus were mapped using a panel of carboxy-terminal N protein truncations expressed by recombinant vaccinia viruses. All of the Mab recognized both native protein and full-length N protein expressed in this vector by both Western blot and enzyme-linked immunoabsorbent assays (ELISA), indicating that they recognized linear epitopes. The results obtained by both Western blot and ELISA for binding to the truncated N proteins coincide for seven of the Mab tested. The linear epitopes recognized localize to four domains dispersed between amino acids 171 and 196, 231 and 277, and 374 and 455. The epitopes for six Mab were localized to domains comprising 29 amino acids or less as determined by ELISA. Seven Mab showed different reactivity patterns in Western blot versus ELISA, suggesting binding may be influenced by local conformation. Therefore, the fine specificity of these Mab could not be determined with certainty. These data represent the first determination of antibody binding domains within the mouse hepatitis virus N protein which forms the viral helical nucleocapsids and appears to perform a number of regulatory functions during virus replication.

Induction of protective immunity against coronavirus-induced encephalomyelitis: evidence for an important role of CD8+ T cells in vivo.

Coronavirus MHV-JHM infections of rats provide useful models to study the pathogenesis of virus-induced central nervous system disease. To analyze the role of the immune response against defined MHV-JHM antigens, we tested the protective efficacy of vaccinia virus (VV) recombinants expressing either the nucleocapsid (N) or the spike (S) protein. A strong protection was mediated in animals by immunization with recombinant VV encoding a wild-type S protein (VV-Swildtype), whereas VV recombinant expressing a mutant S354CR protein (VV-S354CR) had no protective effect. Recombinant VV encoding N protein (VV-N) induces a humoral and a CD4+ T cell response, but did not prevent acute disease regardless of the immunization protocol. In these experiments, challenge with an otherwise lethal dose of MHV-JHM was performed prior to the induction of virus-neutralizing antibodies and studies with the anti-CD8+ monoclonal antibody. MRC OX8 showed that elimination of the CD8+ subset of T cells abrogates the protective effect. This result indicates that CD8+ T cells primed by recombinant VV expressing wild-type S protein are a primary mechanism of immunological defense against MHV-JHM infection in rats.

An immunodominant CD4+ T cell site on the nucleocapsid protein of murine coronavirus contributes to protection against encephalomyelitis.

The murine coronavirus neurotropic strain JHM (MHV-JHM) nucleocapsid (N) protein induces a strong T-helper cell response in Lewis rats. It has been shown previously that N-specific CD4+ T cells can confer protection against acute disease upon transfer to otherwise lethally infected rats. To define the major antigenic regions that elicit this T cell response, truncated fragments of N protein were expressed from a bacterial expression vector and employed as T cell antigens. Lymphocytes from either MHV-JHM-infected or immunized rats were stimulated in culture with virus antigen, grown and tested for their specificity to the N protein fragments. The carboxy-terminally located C4-N fragment (95 amino acids) induced the most pronounced proliferative response irrespective of whether the lymphocyte culture was derived from immunized or MHV-JHM-infected rats. We established T cell lines specific for the truncated N protein fragments and tested their potential to mediate protection by transfer experiments. Only the T cell line C4-N and the T cell line specific for the full-length N protein were protective. By contrast, all truncated N protein fragments elicited a humoral immune response and contained antigenic sites recognized by antibodies from diseased rats.

Identification of an immunodominant linear neutralization domain on the S2 portion of the murine coronavirus spike glycoprotein and evidence that it forms part of complex tridimensional structure.

Numerous studies have demonstrated that the spike glycoprotein of coronaviruses bears major determinants of pathogenesis. To elucidate the antigenic structure of the protein, a panel of monoclonal antibodies was studied by competitive ELISA, and their reactivities were assayed against fragments of the murine coronavirus murine hepatitis virus strain A59 S gene expressed in prokaryotic vectors. An immunodominant linear domain was localized within the predicted stalk, S2, of the peplomer. It is recognized by several neutralizing antibodies. Other domains were also identified near the proteolytic cleavage site, in the predicted globular head, S1, and in another part of the stalk. Furthermore, competition results suggest that the immunodominant functional domain forms part of a complex three-dimensional structure. Surprisingly, some antibodies which have no antiviral biological activities were shown to bind the immunodominant neutralization domain.

Recombinant vaccinia viruses which express MHV-JHM proteins: protective immune response and the influence of vaccination on coronavirus-induced encephalomyelitis.

Vaccinia-virus (VV) recombinants encoding either the nucleocapsid (N) or the spike (S) protein of MHV-JHM were constructed to study the role of the immune response against defined coronavirus antigens. For the S-protein, a fusogenic (Sfus+) or non fusogenic variant (Sfus-) of the gene was inserted into the VV genome. A strong protection against acute encephalomyelitis (AE) was mediated in Lewis rats which were immunized by VV-Sfus+ and challenged with an otherwise lethal dose of MHV-JHM before the induction of S-specific IgG antibodies. By contrast, a VV recombinant encoding a variant non fusogenic S-protein or the N-protein was not capable conferring protection. In addition, we demonstrated that MHV-JHM S-specific IgG antibodies elicited before MHV-JHM challenge modulated the disease process, changing it from an acute disease to subacute demyelinating encephalomyelitis (SDE).

Coronavirus induced encephalomyelitis: an immunodominant CD4(+)-T cell site on the nucleocapsid protein contributes to protection.

In this communication we present clear evidence, that the N-protein of MHV-JHM contains immunodominant CD4+ T-cell sites. These sites were recognized by the immune system of virus infected Lewis rats. In previous investigations we have shown, that CD4+ T-cell lines with specificity for defined viral proteins can be selected from diseased Lewis rats and mediate protection, if transferred to otherwise lethally infected animals. To define regions of the N-protein, which are immunodominant for the T-cell response, we employed bacterially expressed N-protein and truncated subfragments of N as an antigen. We demonstrate, that T-cells from MHV-JHM infected, diseased Lewis rats recognized with high prevalence the carboxyterminal subfragment C4-N (95 aa) and to some extent the adjacent C3-N protein. The same results were obtained with T-cells derived from rats immunized with bacterially expressed N-protein or from animals vaccinated by a stable N-protein expressing vaccinia recombinant. Finally, transfer of CD4+ line T-cells to MHV-JHM infected rats specific for C4-N mediated protection against acute disease.