Recombinant Measles Virus Research Articles

Reduced ability of hemagglutinin of the CAM-70 measles virus vaccine strain to use receptors CD46 and SLAM

The CAM-70 measles virus (MV) vaccine strain is currently used for vaccination against measles. We examined the fusion-inducing ability of the CAM-70 hemagglutinin (H) protein and found that it was impaired in both CD46- and signaling lymphocyte activation molecule (SLAM)-expressing cells. We also generated recombinant MVs possessing H genes derived from the CAM-70 strain. The CAM-70 H protein impaired viral growth in both CD46- and SLAM-expressing cells. In peripheral blood lymphocytes (PBL) and monocyte-derived dendritic cells (Mo-DC), the CAM-70 strain did not grow efficiently. Infection with recombinant MVs revealed that impaired growth of the CAM-70 strain was attributed to the H gene only partly in PBL and largely in Mo-DC. Thus, impaired fusion-inducing ability of the H protein may be one of the underlying molecular mechanisms resulting in the attenuation of the CAM-70 strain.

Recombinant measles viruses expressing single or multiple antigens of human immunodeficiency virus (HIV-1) induce cellular and humoral immune responses

Recombinant measles viruses (rMV) based on the live attenuated measles vaccine strain (MVb) expressing antigens of HIV-1 clade B were generated by reverse genetics. Recombinants expressing single or double antigens of HIV-1 (rMV-HIV) were genetically highly stable on human diploid cells. The production process of these viruses was essentially similar to the parental MV strain, yielding comparative end titers. Immunization of tg-mice by different regimens and formulations showed potent humoral and cellular immune responses against MV and HIV antigens. Recombinant MV-HIV expressing Gag protein conferred protective immunity in tg-mice after a high-dose pseudochallenge with recombinant vaccinia virus. In addition, rMV-HIV boosted anti-HIV antibodies, in the presence of pre-existing anti-vector antibodies.

Recombinant measles virus-HPV vaccine candidates for prevention of cervical carcinoma

Cervical cancer is mainly associated with HPV genotype 16 infection. Recombinant measles virus (rMV) expressing HPV genotype 16 L1 capsid protein was generated by construction of an antigenomic plasmid, followed by rescue using the human “helper” cell line 293-3-46. In cell cultures the recombinant MV-L1 virus replicated practically as efficiently as the standard attenuated MV established as commercial vaccine, devoid of the transgene. The high genetic stability of MVb2-L1 was confirmed by 10 serial viral transfers in cell culture. In transgenic mice expressing the MV receptor CD46 the recombinant induced strong humoral immune responses against both MV and HPV; the antibodies against L1 exhibited mainly neutralizing capacity. Our data suggest that MV is a promising vehicle for development of inexpensive and efficient vaccines protecting from HPV infection.

Measles studies in the macaque model.

Much of our current understanding of measles has come from experiments in non-human primates. In 1911, Goldberger and Anderson showed that macaques inoculated with filtered secretions from measles patients developed measles, thus demonstrating that the causative agent of this disease was a virus. Since then, different monkey species have been used for experimental measles virus infections. Moreover, infection studies in macaques demonstrated that serial passage of the virus in vivo and in vitro resulted in virus attenuation, providing the basis for all current live-attenuated measles vaccines. This chapter will review the macaque model for measles, with a focus on vaccination and immunopathogenesis studies conducted over the last 15 years. In addition, recent data are highlighted demonstrating that the application of a recombinant measles virus strain expressing enhanced green fluorescent protein dramatically increased the sensitivity of virus detection, both in living and sacrificed animals, allowing new approaches to old questions on measles vaccination and pathogenesis.

Protein Kinase PKR Mediates the Apoptosis Induction and Growth Restriction Phenotypes of C Protein-Deficient Measles Virus

The measles virus (MV) accessory proteins V and C play important roles in MV replication and pathogenesis. Infection with recombinant MV lacking either V or C causes more cell death than infection with the parental vaccine-equivalent virus (MVvac), and C-deficient virus grows poorly relative to the parental virus. Here, we show that a major effector of the C phenotype is the RNA-dependent protein kinase PKR. Using human HeLa cells stably deficient in PKR as a result of RNA interference-mediated knockdown (PKR(kd) cells), we demonstrated that a reduction in PKR partially rescued the growth defect of C knockout (C(ko)) virus but had no effect on the growth of either wild-type (WT) or V knockout (V(ko)) virus. Increased growth of the C(ko) virus in PKR(kd) cells correlated with increased viral protein expression, while defective growth and decreased protein expression in PKR-sufficient cells correlated with increased phosphorylation of PKR and the alpha subunit of eukaryotic initiation factor 2. Furthermore, infection with WT, V(ko), or especially C(ko) virus caused significantly less apoptosis in PKR(kd) cells than in PKR-sufficient cells. Although apoptosis induced by C(ko) virus infection in PKR-sufficient cells was blocked by a caspase antagonist, the growth of C(ko) virus was not restored to the WT level by treatment with this pharmacologic inhibitor. Taken together, these results indicate that PKR plays an important antiviral role during MV infection but that the virus growth restriction by PKR is not dependent upon the induction of apoptosis. Furthermore, the results establish that a principal function of the MV C protein is to antagonize the proapoptotic and antiviral activities of PKR.

Measles vaccination: new strategies and formulations

Measles is a highly contagious viral disease. With 1 million deaths reported in 1996, measles was the leading cause of vaccine-preventable deaths. However, in recent years, significant progress has been made in measles control, reducing deaths attributed to measles to 454,000 in 2004 and 242,000 in 2006. The main strategy behind this reduction has been the improvement of vaccination coverage and implementation of a second opportunity for immunization with the live-attenuated measles vaccine. The Measles Initiative, a partnership between the American Red Cross, CDC, UNICEF, WHO and UN Foundation, has had a significant role in this achievement. Here, we provide an overview of old and new vaccination strategies, and discuss changes in the route of administration of the existing live-attenuated vaccine, the development of new-generation nonreplicating measles virus vaccine candidates and attempts to use recombinant measles virus as a vector for vaccination against other pathogens.

The Pathogenesis of Measles Revisited

Measles continues to be an important cause of childhood mortality in developing countries. The causative agent, measles virus (MV), is a member of the family Paramyxoviridae, genus Morbillivirus, and is spread via the respiratory route. MV was originally thought to enter the host by infecting epithelial cells of the respiratory tract, followed by viremia mediated by infected monocytes. However, neither of these cell types express signaling lymphocyte activation molecule (SLAM, CD150), which has been identified as the main receptor for wild-type MV. Measles has a relatively long incubation time, which makes it difficult to study the early stages of MV infection in humans. The animal models that best reflect the pathogenesis of measles are based on nonhuman primates. The use of recombinant MV strains expressing fluorescent proteins has greatly facilitated studies on viral tropism in macaques. These studies indicate that dendritic cells and lymphocytes expressing CD150 are the primary target cells for MV infection. At late stages of the infection MV also infects epithelial cells, despite the fact that these do not express CD150. Whether these cells express an as yet unidentified additional MV receptor remains unclear. On basis of these data it could be envisaged that dendritic cells are the first target cells for MV infection. These antigen-presenting cells may traffic the virus to the regional lymph nodes where they can transmit the virus to lymphocytes, which during viremia disseminate the virus throughout the body.

Heparin-like glycosaminoglycans prevent the infection of measles virus in SLAM-negative cell lines

The wide tissue tropism of the measles virus (MV) suggests that it involves ubiquitously expressed molecules. We have constructed a recombinant MV expressing the enhanced green fluorescent protein (EGFP) (rMV-EGFP) and demonstrated that the rMV-EGFP infected several cell types (HEK-293, HepG2, Hep3B, Huh7, and WRL68 cells) that do not express the human signalling lymphocyte activation molecule (SLAM), which is known as a cellular receptor for morbilliviruses. MV infection of HEK-293 and HepG2 cells was not inhibited in an infectivity–inhibition assay using an anti-SLAM monoclonal antibody, indicating that MV could infect cells without using SLAM. Soluble heparin (HP) inhibited the rMV-EGFP infectivity in SLAM-negative cell lines in a dose-dependent manner. Direct interaction between purified virions and HP was detected in a surface plasmon resonance assay. We also demonstrated that the hemagglutinin (H) protein, but not the fusion (F) protein is responsible for the interaction between the virions and HP. Taken together, our results suggest that HP-like glycosaminoglycans bind to the H protein of MV and play a key role in the infection of SLAM-negative cells.

Measles Viruses Possessing the Polymerase Protein Genes of the Edmonston Vaccine Strain Exhibit Attenuated Gene Expression and Growth in Cultured Cells and SLAM Knock-In Mice

Live attenuated vaccines against measles have been developed through adaptation of clinical isolates of measles virus (MV) in various cultured cells. Analyses using recombinant MVs with chimeric genomes between wild-type and Edmonston vaccine strains indicated that viruses possessing the polymerase protein genes of the Edmonston strain exhibited attenuated viral gene expression and growth in cultured cells as well as in mice expressing an MV receptor, signaling lymphocyte activation molecule, regardless of whether the virus genome had the wild-type or vaccine-type promoter sequence. These data demonstrate that the polymerase protein genes of the Edmonston strain contribute to its attenuated phenotype.

Advantages of using recombinant measles viruses expressing a fluorescent reporter gene with vibratome slice technology in experimental measles neuropathogenesis

In this study of experimental measles neuropathogenesis, the utility of enhanced green fluorescent protein (EGFP) as a sensitive indicator of measles virus (MV) cell-to-cell spread in the central nervous system (CNS) has been assessed in vibratome-cut brain slices to demonstrate the degree and mechanism of viral spread in the rodent CNS. Recombinant MVs expressing EGFP were visualized at different levels in 200-microm vibratome-cut brain sections from infected animals by confocal scanning laser microscopy (CSLM). Comparison was made with 7-microm microtome sections, stained for the N protein of measles by immunocytochemistry (ICC). The recombinant viruses were readily visualized in infected brain tissue, with no loss of neuropathogenicity. No difference was found in the sites of infection when MV infection was detected through EGFP fluorescence or by ICC. MV-infected cells were detected in the cerebral cortex, olfactory bulb and tract, hippocampus, thalamus, hypothalamus, ependyma and subventricular zone. However, the 200-microm vibratome-cut sections and confocal microscopy proved excellent for demonstrating virus distribution in neurites and for in-depth analysis of the extent of tract infection in the white matter of the cerebral hemispheres such as selective infection of the internal capsule and anterior commissure. The use of self-tracing recombinant MVs, viewed in thick vibratome-cut sections by CSLM, demonstrated that in experimental MV neuropathogenesis the infection is selective and spreads predominately by neurites using defined anatomical pathways.

Measles Virus Circumvents the Host Interferon Response by Different Actions of the C and V Proteins

Measles is an acute febrile infectious disease with high morbidity and mortality. The genome of measles virus (MV), the causative agent, encodes two accessory products, V and C proteins, that play important roles in MV virulence. The V but not the C protein of the IC-B strain (a well-characterized virulent strain of MV) has been shown to block the Jak/Stat signaling pathway and counteract the cellular interferon (IFN) response. We have recently shown that a recombinant IC-B strain that lacks C protein expression replicates poorly in certain cell lines, and its growth defect is related to translational inhibition and strong IFN induction. Here, we show that the V protein of the MV IC-B strain also blocks the IFN induction pathway mediated by the melanoma differentiation-associated gene 5 product, thus actively interfering with the host IFN response at two different steps. On the other hand, the C protein per se possesses no activity to block the IFN induction pathway. Our data indicate that the C protein acts as a regulator of viral RNA synthesis, thereby acting indirectly to suppress IFN induction. Since recombinant MVs with C protein defective in modulating viral RNA synthesis or lacking C protein expression strongly stimulate IFN production, in spite of V protein production, both the C and V proteins must be required for MV to fully circumvent the host IFN response.

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.

Development of a Novel Efficient Fluorescence-Based Plaque Reduction Microneutralization Assay for Measles Virus Immunity

The measurement of functional measles virus-specific neutralizing antibodies is of considerable interest for vaccine-related research. In this study, we developed and standardized a simple, rapid, highly sensitive, and reproducible fluorescence-based plaque reduction microneutralization (PRMN) assay with visual and automated readout, using a recombinant measles virus engineered to express enhanced green fluorescent protein. The assay is performed in micro format, requires less time to complete (2 versus 4 to 7 days), and is less labor-intensive and less costly than the classical plaque reduction neutralization (PRN) test, widely accepted as the "gold standard" in measles serology. Two available WHO international anti-measles virus standards and one in-house reference serum were used to develop and standardize the new assay. The mean PRMN values from repeated assays were found to be similar to those reported in the literature or assigned to the WHO standards by the classical PRN assay. For validation, we used three groups of low, moderate, and high measles virus vaccine responders' sera with moderate values of correlation in antibody levels (mIU/ml) between PRMN and the Dade Behring immunoglobulin G enzyme immunoassay (EIA). The PRMN assay was more sensitive at low antibody levels and more informative in terms of protection than this commercial EIA. In conclusion, we have developed and validated a sensitive and high-throughput measles virus-specific PRMN that can be readily used in large population-based measles studies.

Glycoprotein targeting signals influence the distribution of measles virus envelope proteins and virus spread in lymphocytes

We previously demonstrated the presence of tyrosine-dependent motifs for specific sorting of two measles virus (MV) glycoproteins, H and F, to the basolateral surface in polarized epithelial cells. Targeted expression of the glycoproteins was found to be required for virus spread in epithelia via cell-to-cell fusion in vitro and in vivo. In the present study, recombinant MVs (rMVs) with substitutions of the critical tyrosines in the H and F cytoplasmic domains were used to determine whether the sorting signals also play a crucial role for MV replication and spread within lymphocytes, the main target cells of acute MV infection. Immunolocalization revealed that only standard glycoproteins are targeted specifically to the uropod of polarized lymphocytes and cluster on the surface of non-polarized lymphocytes. H and F proteins with tyrosine mutations did not accumulate in uropods, but were distributed homogeneously on the surface and did not colocalize markedly with the matrix (M) protein. Due to the defective interaction with the M protein, all mutant rMVs showed an enhanced fusion capacity, but only rMVs harbouring two mutated glycoproteins showed a marked decrease in virus release from infected lymphocytes. These results demonstrate clearly that the tyrosine-based targeting motifs in the MV glycoproteins are not only important in polarized epithelial cells, but are also active in lymphocytes, thus playing an important role in virus propagation in different key target cells during acute MV infection.

Induction of neutralising antibodies and cellular immune responses against SARS coronavirus by recombinant measles viruses

Live attenuated recombinant measles viruses (rMV) expressing a codon-optimised spike glycoprotein (S) or nucleocapsid protein (N) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were generated (rMV-S and rMV-N). Both recombinant viruses stably expressed the corresponding SARS-CoV proteins, grew to similar end titres as the parental strain and induced high antibody titres against MV and the vectored SARS-CoV antigens (S and N) in transgenic mice susceptible to measles infection. The antibodies induced by rMV-S had a high neutralising effect on SARS-CoV as well as on MV. Moreover, significant N-specific cellular immune responses were measured by IFN-γ ELISPOT assays. The pre-existence of anti-MV antibodies induced by the initial immunisation dose did not inhibit boost of anti-S and anti-N antibodies. Immunisations comprising a mixture of rMV-S and rMV-N induced immune responses similar in magnitude to that of vaccine components administered separately. These data support the suitability of MV as a bivalent candidate vaccine vector against MV and emerging viruses such as SARS-CoV.

Modeling of cancer virotherapy with recombinant measles viruses

The Edmonston vaccine strain of measles virus has potent and selective activity against a wide range of tumors. Tumor cells infected by this virus or genetically modified strains express viral proteins that allow them to fuse with neighboring cells to form syncytia that ultimately die. Moreover, infected cells may produce new virus particles that proceed to infect additional tumor cells. We present a model of tumor and virus interactions based on established biology and with proper accounting of the free virus population. The range of model parameters is estimated by fitting to available experimental data. The stability of equilibrium states corresponding to complete tumor eradication, therapy failure and partial tumor reduction is discussed. We use numerical simulations to explore conditions for which the model predicts successful therapy and tumor eradication. The model exhibits damped, as well as stable oscillations in a range of parameter values. These oscillatory states are organized by a Hopf bifurcation.

Targeting the Untargetable: Oncolytic Virotherapy for the Cancer Stem Cell

The biological and therapeutic importance of cancer stem cells has become increasingly clear over the past several years.1 The success or failure of cancer treatment approaches may be influenced greatly by the presence and treatment sensitivity of these cells. Cancer cures may therefore require effective targeting and destruction of the cancer stem cell population. For therapeutics to be effective against such cells, they must be effective against quiescent, apoptosis-resistant, and drug transporter–overexpressing cells. Further complicating this challenge, normal stem cells share many of these same features, to the extent that selectively targeting the cancer stem cell while leaving the normal stem cells unharmed is a difficult proposition. In this issue of Molecular Therapy, Eriksson and colleagues report proof-of-concept studies for the efficacy of oncolytic virotherapy against cancer stem cells.2

Predominant infection of CD150+ lymphocytes and dendritic cells during measles virus infection of macaques.

Measles virus (MV) is hypothesized to enter the host by infecting epithelial cells of the respiratory tract, followed by viremia mediated by infected monocytes. However, neither of these cell types express signaling lymphocyte activation molecule (CD150), which has been identified as the receptor for wild-type MV. We have infected rhesus and cynomolgus macaques with a recombinant MV strain expressing enhanced green fluorescent protein (EGFP); thus bringing together the optimal animal model for measles and a virus that can be detected with unprecedented sensitivity. Blood samples and broncho-alveolar lavages were collected every 3 d, and necropsies were performed upon euthanasia 9 or 15 d after infection. EGFP production by MV-infected cells was visualized macroscopically, in both living and sacrificed animals, and microscopically by confocal microscopy and FACS analysis. At the peak of viremia, EGFP fluorescence was detected in skin, respiratory and digestive tract, but most intensely in all lymphoid tissues. B- and T-lymphocytes expressing CD150 were the major target cells for MV infection. Highest percentages (up to 30%) of infected lymphocytes were detected in lymphoid tissues, and the virus preferentially targeted cells with a memory phenotype. Unexpectedly, circulating monocytes did not sustain productive MV infection. In peripheral tissues, large numbers of MV-infected CD11c+ MHC class-II+ myeloid dendritic cells were detected in conjunction with infected T-lymphocytes, suggesting transmission of MV between these cell types. Fluorescent imaging of MV infection in non-human primates demonstrated a crucial role for lymphocytes and dendritic cells in the pathogenesis of measles and measles-associated immunosuppression.

Two functionally linked amino acids in the stem 2 region of measles virus haemagglutinin determine infectivity and virulence in the rodent central nervous system

Rodent brain-adapted measles virus (MV) strains, such as CAM/RB and recombinant MVs based on the Edmonston strain containing the haemagglutinin (H) of CAM/RB, cause acute encephalitis after intracerebral infection of newborn rodents. We have demonstrated that rodent neurovirulence is modulated by two mutations at amino acid positions 195 and 200 in the H protein, one of these positions (200) being a potential glycosylation site. In order to analyse the effects of specific amino acids at these positions, we introduced a range of individual and combined mutations into the open reading frame of the H gene to generate a number of eukaryotic expression plasmids. The functionality of the mutant H proteins was assessed in transfected cells and by generating recombinant viruses. Interestingly, viruses caused acute encephalitis only if the amino acid Ser at position 200 was coupled with Gly at position 195, whereas viruses with single or combined mutations at these positions, including glycosylation at position 200, were attenuated. Neurovirulence was associated with virus spread and induction of neuronal apoptosis, whereas attenuated viruses failed to infect brain cells. Similar results were obtained by using primary brain-cell cultures. Our findings indicate that a structural alteration in the stem 2 region of the H protein at position 195 or 200 interferes with infectivity of rodent neurons, and suggest that the interaction of the viral attachment protein with cellular receptors on neurons is affected.

Immune synapses formed with measles virus-infected dendritic cells are unstable and fail to sustain T cell activation

Interaction with dendritic cells (DCs) is considered as central to immunosuppression induced by viruses, including measles virus (MV). Commonly, viral infection of DCs abrogates their ability to promote T cell expansion, yet underlying mechanisms at a cellular level are undefined. We found that MV-infected DCs only subtly differed from LPS-matured with regard to integrin activation, acquisition of a migratory phenotype and motility. Similarly, the organization of MV-DC/T cell interfaces was consistent with that of functional immune synapses with regard to CD3 clustering and MHC class II surface recruitment. The majority of MV-DC/T cell conjugates was, however, unstable and only promoted abortive T cell activation. Thus, MV-infected DCs retain activities required for initiating, but not sustaining T cell conjugation and activation. This is partially rescued if surface expression of the MV glycoproteins on DCs is abolished by infection with a recombinant MV encoding VSV G protein instead, indicating that these contribute directly to synapse destabilization and thereby act as effectors of T cell inhibition.