Inhibition of the Aurora A kinase augments the anti-tumor efficacy of oncolytic measles virotherapy.

Oncolytic measles virus (MV) strains have demonstrated broad spectrum preclinical anti-tumor efficacy, including breast cancer. Aurora A kinase controls mitotic spindle formation and has a critical role in malignant transformation. We hypothesized that the Aurora A kinase inhibitor MLN8237 (alisertib) can increase MV oncolytic effect and efficacy by causing mitotic arrest. Alisertib enhanced MV oncolysis in vitro and significantly improved outcome in vivo against breast cancer xenografts. In a disseminated MDA-231-lu-P4 lung metastatic model, the MV/alisertib combination treatment markedly increased median survival to 82.5 days with 20% of the animals being long-term survivors versus 48 days median survival for the control animals. Similarly, in a pleural effusion model of advanced breast cancer, the MV/alisertib combination significantly improved outcome with a 74.5 day median survival versus the single agent groups (57 and 40 days, respectively). Increased viral gene expression and IL-24 upregulation were demonstrated, representing possible mechanisms for the observed increase in anti-tumor effect. Inhibiting Aurora A kinase with alisertib represents a novel approach to enhance MV-mediated oncolysis and antitumor effect. Both oncolytic MV strains and alisertib are currently tested in clinical trials, this study therefore provides the basis for translational applications of this combinatorial strategy in the treatment of patients with advanced breast cancer.

Measles Edmonston vaccine strain derivatives have potent oncolytic activity against osteosarcoma.

Osteosarcoma (OS) is the most common primary bone tumor affecting children and young adults, and development of metastatic disease is associated with poor prognosis. The purpose of this study was to evaluate the antitumor efficacy of virotherapy with engineered measles virus (MV) vaccine strains in the treatment of OS. Cell lines derived from pediatric patients with OS (HOS, MG63, 143B, KHOS-312H, U2-OS and SJSA1) were infected with MV expressing green fluorescent protein (MV-GFP) and MV-expressing sodium iodide symporter (MV-NIS) strains. Viral gene expression and cytotoxicity as defined by syncytial formation, cell death and eradication of cell monolayers were demonstrated. Findings were correlated with in vivo efficacy in subcutaneous, orthotopic (tibial bone) and lung metastatic OS xenografts treated with the MV derivative MV-NIS via the intratumoral or intravenous route. Following treatment, we observed decrease in tumor growth of subcutaneous xenografts (P=0.0374) and prolongation of survival in mice with orthotopic (P<0.0001) and pulmonary metastatic OS tumors (P=0.0207). Expression of the NIS transgene in MV-NIS infected tumors allowed for single photon emission computed tomography and positron emission tomography-computed tomography imaging of virus infected tumors in vivo. Our data support the translational potential of MV-based virotherapy approaches in the treatment of recurrent and metastatic OS.

Oncolytic measles virus strains have significant antitumor activity against glioma stem cells.

Glioblastoma (GBM) is the most common primary brain tumor in adults and has a dismal prognosis despite multimodality treatment. Given the resistance of glioma stem cells (GSC) to chemotherapy and radiation therapy, their eradication could prevent tumor recurrence. We sought to evaluate the antitumor activity of measles virus (MV) derivatives against GSC. We generated neurosphere cultures from patient-derived primary tumor GBM xenografts, and we characterized them for the GSC markers CD133, SOX2, Nestin, ATF5 and OLIG2. Using the MV-strains MV-GFP, MV-CEA and MV-NIS we demonstrated infection, viral replication and significant cytopathic effect in vitro against GSC lines. In tumorigenicity experiments, GBM44 GSC were infected with MV in vitro and subsequently implanted into the right caudate nucleus of nude mice: significant prolongation of survival in mice implanted with infected GSC was observed, compared with mock-infected controls (P=0.0483). In therapy experiments in GBM6 and GBM12 GSC xenograft models, there was significant prolongation of survival in MV-GFP-treated animals compared with inactivated virus-treated controls (GBM6 P=0.0021, GBM12 P=0.0416). Abundant syncytia and viral replication was demonstrated in tumors of MV-treated mice. Measles virus derivatives have significant antitumor activity against glioma-derived stem cells in vitro and in vivo.

Integrations of the hepatitis B virus (HBV) and human papillomavirus (HPV) into the human telomerase reverse transcriptase (hTERT) gene in liver and cervical cancers.

Chronic infections with the hepatitis B virus (HBV) and high-risk human papillomaviruses (HPVs) are important risk factors for hepatocellular carcinoma (HCC) and cervical cancer (CC), respectively. HBV and HPV are DNA viruses that almost invariably integrate into the host genome in invasive tumors. The viral integration sites occur throughout the genome, leading to the presumption that there are no preferred sites of integration. A number of viral integrations have been shown to occur within the vicinity of important cancer-related genes. In studies of HBV-induced HCC and HPV-induced CC, we have identified two HBV and three HPV integrations into the human telomerase reverse transcriptase (hTERT) gene. Detailed characterization of the integrations revealed that four integrations occurred within the hTERT promoter and upstream region and the fifth integration occurred in intron 3 of the hTERT gene. None of the integrations altered the hTERT coding sequence and all resulted in juxtaposition of viral enhancers near hTERT, with potential activation of hTERT expression. Our work supports the hypothesis that the sites of oncogenic viral integration are nonrandom and that genes at the sites of viral integration may play important roles in carcinogenesis.

Electron optic aspects of a murine cytomegalovirus strain grown in R1 mouse kidney cell cultures.

The electron microscopic study of a mouse kidney cell line (R1) infected with a murine cytomegalovirus strain isolated from a micromammal captured in the area of endemic Balkan nephropathy demonstrated the presence of multiple types of virus particles, in different phases of replication. Nucleocapsids with a partially or almost totally empty centre were predominant. The small number of particles with an electron-dense core and double envelope points to a low yield of mature infectant particles.

Peculiarities of the virus-host cell relationship in a calf kidney cell line persistently infected with measles virus.

Persistent infection with measles virus established in a calf kidney cell line differed in some features from other measles virus carrier systems. After a variable life span, ranging from 50 to 1100 days, six of the chronically infected cell lines showed an evolution towards lytic infection. Only one of these lines, the K-2 cell line, exhibited marked morphological, growth and chromosomal alterations suggesting in vitro cellular transformation. The transformed K-2 cells released infectious measles virus with several modified biological parameters. Persistent infection was not due to the accumulation of thermosensitive mutants or defective particles. The role of the host-cell factor and of the selection of the virus population during its passage in RV cells are discussed.

Persistent measles virus infection in a calf kidney cell line. Note III. Cellular alterations in the selected chronically infected K-2 cell line.

Marked morphological, growth and chromosome alterations pointing to in vitro cellular transformation were made evident in a selected calf kidney cell line (K-2) chronically infected with measles virus. The transformed cell line released infectant virus and had no oncogenic potential for the hamster.

Stability of antimeasles vaccine under the influence of some physical and chemical factors.

A study on the stability of lyophilized and rehydrated measles vaccines under the influence of temperature and light was undertaken. After rehydration there was a considerable decrease in the potency of the vaccine, even when stored at +4 degrees C-- +8 DEGREES C and in the dark. Out of the several drugs tested, the commercially available drug AS was found to have a stabilizing effect on measles vaccine infectivity.

Pitfalls in the sensitivity of HBsAg detection.

A number of 1800 serum samples from apparently healthy subjects were tested by immunodiffusion (ID) and counterimmunoelectrophoresis (CIE) with a view to confirming the presence of HBsAg; results were uncertain in 21% of the cases. The enzyme immunoassay (ELISA kits) used to elucidate these uncertain reactions showed half of them to be negative. About 3% of the remaining sera, HBsAg-positive by ELISA, were positive by ID, but constantly negative by repeated CIE tests. Some possible explanations are suggested and the implications of such pitfalls in the interpretation of sero-epidemiological analyses are discussed.