Measles as a potential oncolytic virus

The use of replicating viruses for cancer therapy is attracting increasing interest. Numerous viruses are now being considered as potential cancer therapeutics, including the vaccine strain of measles virus (MV). The attenuated strain of measles readily lyses transformed cells, whilst replication and lysis are limited in normal human cells. It has a number of features which make it highly suitable for further development as an oncolytic agent, among them stability and a long history of safety in human use. These features are being combined with its ready potential for genetic manipulations to generate recombinant MVs with desirable therapeutic attributes. This review summarises the pre-clinical studies of the oncolytic efficacy of MV to date. Promising developments in MV engineering--such as re-targeting MV entry to specific cell types and enhancing its utility as a therapeutic agent by expression of non-viral proteins--as well as outstanding issues, such as the role of anti-MV immunity, are highlighted.     

Measles virus polypeptide synthesis in infected cells

The synthesis of measles virus polypeptides has been studied using an inhibitor of virus-induced cell fusion, carbobenzoxy-d-phenylalanyl-l-phenylalanyl-nitro-l-arginine (SV4814). Cells infected at a multiplicity of 10 fuse extensively by 17 hr and die shortly thereafter, making it difficult to detect viral polypeptides. Cells protected from fusion by SV4814 survive and continue to produce virus, and the synthesis of viral polypeptides can be followed for 4 days. The previously described measles virion polypeptides G, 2, NP, 5, and M have been identified in infected cells, and, in addition, a polypeptide (L) with a molecular weight of ～200,000 has been found in infected cells and in small amounts in virions. (New designations suggested for polypeptides G, 2, and 5 are H, P, and F₁, respectively.) A glycosylated polypeptide (F₀, MW ～62,000) has also been found in infected cells, but not in virions. This polypeptide is thought to be the precursor of two polypeptides which appear under pulse-chase conditions: F₁ (MW ～40,000), which is not glycosylated, and F₂, a small glycosylated polypeptide detected with [³H]glucosamine labeling. In addition to facilitating studies of measles virus polypeptide synthesis, the use of SV4814 has shown that cell fusion is the major factor in early cell death caused by measles virus, but that cell death ultimately ensues in the absence of cell fusion, indicating another mechanism of measles virus-induced cell damage.     

Measles virus genotyping by nucleotide-specific multiplex PCR

A simple genotyping method based on multiplex PCR has been developed to discriminate between all active measles virus (MV) clades and genotypes (A, B3.1, B3.2, C2, D2-D9, G2-G3, and H1-H2). The sequencing reaction was replaced by six multiplex PCRs: one to identify the clade and five to identify the respective genotype. Primers were sensitive to clade- and genotype-specific nucleotides and generated fragments of type-specific sizes that were analyzed by conventional agarose gel electrophoresis. On the basis of all published MV sequences, positive and negative predictive values of 99.2% and 98.6% were calculated. Variability in the primer binding sites, which could potentially reduce sensitivity, was very limited among published sequences. As new genotypes are described, additional specific primers can be included in the multiplex PCR with relative ease. Although sequencing remains the "gold standard," the present method should facilitate MV genotyping especially in developing countries and will therefore contribute to enhanced MV control and elimination strategies as recommended by the World Health Organization.     

Measles virus as an oncolytic vector platform

Viral vector systems are widely being used in the development of new genetic approaches for a variety of human diseases. Oncolytic viruses have shown great potential as cancer therapeutics. The ideal viral vector for cancer gene therapy eradicates a clinically significant fraction of malignant cells and leaves normal tissues unharmed. The Edmonston vaccine strain of measles virus is a replicating RNA virus which is characterized by its tumor selectivity and oncolysis. Its strong tumor suppressive potential combined with its excellent safety record as a viral vaccine makes it an optimal platform for oncolytic virotherapy of cancer. Recent advances in genetic engineering of measles virus allow insertion of therapeutic and diagnostic transgenes as well as complete retargeting of measles virus. These strategies resulted in the generation of recombinant measles viruses allowing non-invasive monitoring of viral replication and viral spread. The immune defense is a significant barrier for efficient viral gene therapy. Immune-evasive strategies have successfully been developed for measles virus enhancing its efficacy. This review gives an overview of measles virus as an anticancer agent; in particular, its use in oncologic virotherapy as well as new developments in targeting and immune evasive strategies.     

Measles virus nucleic acid sequences in human brain

We constructed a measles virus genomic recombinant DNA library, and used clones coding for portions of the viral P, M and H proteins to probe for measles virus nucleic acid sequences in post-mortem multiple sclerosis, SSPE and control brains. By dot blot hybridization, the probes detected measles virus nucleic acid sequences in as little as 3 nanograms of total RNA extracted from measles virus-infected cells and also in highly diluted RNA extracted from SSPE brain, but did not detect measles virus sequences in RNA extracted from 11 multiple sclerosis or 8 control brains, even at a 1 000-fold higher concentration of RNA. By in situ hybridization, these probes detected measles virus nucleic acid sequences in virtually every cell and the surrounding neuropile of SSPE brain, but again did not detect such sequences in multiple sclerosis or control brains. Our findings using these highly specific probes confirm that measles virus is found in SSPE brains and indicate that measles virus genome is unlikely to be present in multiple sclerosis or normal brains.     

Measles virus: Induced transfer of biological properties

Summary Neurotropic mouse measles virus (MMV), lethal for suckling mice, infects HeLa cultures with cytopathic effect but no cell-free virus is released. Edmonston strain (Edm), non-pathogenic for suckling mice, infects HeLa cultures with cytopathic effect and release of cell-free virus.Simultaneous, or sequential, infection of cell culture with MMV followed by Edm results in release of cell-free virus pathogenic for suckling mice as long as 35 days after mixed infection. The product is more like a heterozygote or phenotypic mix than a stable genetic recombinant. Rescue of MMV from cell culture by means of Edm is a model for recovery of incomplete virus which may be applied to other agents.     

Measles virus receptors: SLAM and CD46

The success of vaccination against measles in developed countries has significantly reduced the incidence of measles-related morbidity and mortality. However, measles is still the leading cause of mortality in children from underdeveloped countries due to low vaccination coverage, high transmissibility of the measles virus as well as primary and secondary vaccine failure. As with any viral disease, the identification of the host molecule to which the measles virus binds and gains entry into the host cell is a major step in understanding the molecular pathology of the disease. Two cell surface receptors, CD46 and signaling lymphocyte-activation molecule (SLAM), have been identified as measles virus receptors. CD46 is ubiquitously expressed on all nucleated cells and acts as a receptor for the Edmonston strain and all vaccine strains derived from it. SLAM is selectively expressed on some T and B cells and is utilised by the Edmonston strain and wild-type strains that cannot use CD46 for cell entry. Understanding the structural and functional variations in measles virus receptors with regard to host response can facilitate the development of new vaccines as well as provide new insights into measles virus tropism and pathogenesis and, importantly, into possible mechanisms for vaccine non-response. Our review focuses on the structure of measles virus receptors, measles virus receptor function, isoforms and polymorphic forms.     

Measles virus gene expression in subacute sclerosing panencephalitis

RNA was extracted from the diseased brain of a case of human subacute sclerosing panencephalitis (SSPE) and analysed for the expression of measles-specific RNA. Measles virus-specific mRNAs were present, but the amount of matrix (M) protein mRNA was greatly reduced in comparison to lytically infected cells and phospho- (P) protein mRNA was hardly detectable whereas the level of the corresponding intermediate-sized (is-) RNA was greatly increased. RNA obtained from the human brain was also translated in vitro and measles virus nucleocapsid and P protein was produced. However, in marked contrast to control reactions M protein was not detected in the products formed by translation in vitro. These results indicate an impaired measles virus M protein mRNA synthesis in infected brain tissue.     

Measles virus meningoencephalitis. Immunofluorescence study of brains infected with virus mutants.

The localization of measles virus antigens in hamster brains inoculated with a temperature-sensitive mutant of measles virus r its revertant was studied by immunofluorescence. We found significantly less virus antigen in mutant-inoculated than in revertant-inoculated brains. In mutant-inoculated brains the virus antigens were very often found in the meninges. Possible mechanisms for the development of experimental hydrocephalus and meningoencephalitis are discussed.     

Measles virus and inactivated canine distemper virus induce incomplete immunity to canine distemper

Summary Pairs of specific pathogen free dogs were immunized with two injections of heat inactivated canine distemper virus (CDV) or one injection of a live CDV or live measles virus (MV) vaccine. Three unimmunized dogs were used as controls. All 9 dogs were challenged with virulent CDV (Snyder Hill strain). The three unimmunized dogs developed severe signs of disease with a lethal infection in one. The two dogs immunized with live CDV vaccine developed a strong humoral as well as cellular immune response after immunization and were protected against virus replication. Animals immunized with either inactivated CDV or modified live MV failed to develop a measurable cellular immune response after immunization and had a comparatively weak humoral immune response to distemper antigens. They showed mild signs of infection after challenge and responded with strong anamnestic cellular and humoral immunity. The measles vaccine immunized dogs had a moderate serum titer of measles hemolysin-inhibiting antibodies which, after exposure to distemper virus, was boosted to high levels. It is proposed that this response plays a role in the mitigation of the virulent distemper infection in these animals.With 1 Figure     

Measles virus induces cell-type specific changes in gene expression

Measles virus (MV) causes various responses including the induction of immune responses, transient immunosuppression and establishment of long-lasting immunity. To obtain a comprehensive view of the effects of MV infection on target cells, DNA microarray analyses of two different cell-types were performed. An epithelial (293SLAM; a 293 cell line stably expressing SLAM) and lymphoid (COBL-a) cell line were inoculated with purified wild-type MV. Microarray analyses revealed significant differences in the regulation of cellular gene expression between these two different cells. In 293SLAM cells, upregulation of genes involved in the antiviral response was rapidly induced; in the later stages of infection, this was followed by regulation of many genes across a broad range of functional categories. On the other hand, in COBL-a cells, only a limited set of gene expression profiles was modulated after MV infection. Since it was reported that V protein of MV inhibited the IFN signaling pathway, we performed a microarray analysis using V knockout MV to evaluate V protein's effect on cellular gene expression. The V knockout MV displayed a similar profile to that of parental MV. In particular, in COBL-a cells infected with the virus, no alteration of cellular gene expression, including IFN signaling, was observed. Furthermore, IFN signaling analyzed in vitro was completely suppressed by MV infection in the COBL-a cells. These results reveal that MV induces different cellular responses in a cell-type specific manner. Microarray analyses will provide us useful information about potential mechanisms of MV pathogenesis.     

Measles virus: immunomodulation and cell tropism as pathogenicity determinants

As important determinants of measles virus (MV) pathogenicity, the MV glycoproteins play a key role in conferring the cellular tropism of this virus, but also in modulating the activity of immunocompetent cells. Whereas all MV strains are able to use CD150 (SLAM) for binding and entry into target cells, only certain, mainly vaccine, strains, can use both CD46 and CD150. Both molecules are down-regulated from the cell surface and this is brought about by both infection and contact with the MV H protein of strains that are able to interact with these molecules. Whereas down-regulation of CD46 could be linked to enhanced sensitivity to complement-mediated lysis, and may thus represent an attenuation marker for vaccine strains, pathogenetic consequences of CD150 down-regulation are unknown as yet. Although the role of CD150 is not entirely clear, viruses containing a wild-type strain-derived H protein revealed a particular tropism for human dendritic cells in vitro, and replicated well in secondary lymphatic tissues of cotton rats where they were also able to cause immunosuppression, as documented by an impaired proliferative response of lymphocytes ex vivo. Most likely, inhibition of T cell expansion by these cells is brought about by another activity of the MV glycoprotein complex, namely by disrupting a pathway important for S-phase entry of T cells, by a mere surface contact.     

Measles virus infection in the placenta of monozygotic twins.

We report a case of monozygotic twins whose mother was infected with measles at 19 weeks' gestation. One of the twins died in utero at 32 weeks' gestation. The placenta of the stillbirth showed massive fibrin deposition, and some residual trophoblasts contained many inclusion bodies positive for measles virus antigen. Fetal organs and cells other than a few splenic lymphocytes showed no evidence of measles virus infection. The placenta of the surviving infant showed focal intervillous fibrin deposits, and only a few syncytiotrophoblasts were positive for measles virus antigen. At present, 7 months after the delivery, the surviving infant has not developed any sign of measles virus infection. Postpartum course of the mother has been uneventful, although high titers of serum anti-measles virus IgM persisted for 6 months after delivery. This case is informative in the following respects: the villous trophoblasts had diagnostic inclusion bodies and ultrastructural evidence of measles virus infection, the degree of viral involvement within the monochorionic placenta was uneven, both of the twins were virtually free from measles virus infection despite the marked involvement of the placenta, and measles virus infection had persisted in the monochorionic placenta for approximately 13 weeks.     

Measles virus L protein evidences elements of ancestral RNA polymerase

We have determined the nucleotide sequence of the measles virus (MV) L gene using a cDNA library encompassing the entire MV genome (J. Crowley et al. (1987) Intervirology, 28, 65-77). The L gene is 6639 nucleotides in length, and contains a single long open reading frame that could code for a protein of 247,611 kDa. Both the L gene and in particular the predicted L protein of MV bear substantial homology to their counterparts in Sendai virus and Newcastle disease virus, suggesting that the multifunctional nature of paramyxovirus L proteins imposes strong evolutionary constraints. The predicted MV L protein also contains distinct elements of a postulated ancestral RNA polymerase.     

Measles virus: Study of induced transfer of biological properties

Summary HeLa cells are non-permissive for the neurotropic suckling mouse strain of measles virus (MMV), but are permissive for cell-adapted Edmonston strain of measles virus (Edm). Fluorescence and electron microscopy demonstrated no membrane fluorescence and no membrane associated viral components, as well as characteristic lack of nuclear antigen after MMV infection of HeLa cells. This appearance differs markedly from the membrane, cytoplasmic and nuclear fluorescence after Edm infection of HeLa cells. Fluorescence microscopy demonstrates fusion of the two dissimilar syncytia after mixed infection. This suggests Edm envelopment of MMV nucleocapsids may be the means of MMV rescue in this system.With 6 FiguresPresented in part at the Annual Meeting of the American Society of Microbiology, Philadelphia, 28 April 1972.