Towards ambient temperature-stable vaccines: the identification of thermally stabilizing liquid formulations for measles virus using an innovative high-throughput infectivity assay.

As a result of thermal instability, some live attenuated viral (LAV) vaccines lose substantial potency from the time of manufacture to the point of administration. Developing regions lacking extensive, reliable refrigeration ("cold-chain") infrastructure are particularly vulnerable to vaccine failure, which in turn increases the burden of disease. Development of a robust, infectivity-based high throughput screening process for identifying thermostable vaccine formulations offers significant promise for vaccine development across a wide variety of LAV products. Here we describe a system that incorporates thermal stability screening into formulation design using heat labile measles virus as a prototype. The screening of >11,000 unique formulations resulted in the identification of liquid formulations with marked improvement over those used in commercial monovalent measles vaccines, with <1.0 log loss of activity after incubation for 8h at 40°C. The approach was shown to be transferable to a second unrelated virus, and therefore offers significant promise towards the optimization of formulation for LAV vaccine products.

Combination of measles virus virotherapy and radiation therapy has synergistic activity in the treatment of glioblastoma multiforme.

PURPOSE: Glioblastoma multiforme is the most frequent primary brain tumor in adults and represents one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that an oncolytic measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) has significant antitumor activity against glioblastoma multiforme cell lines and xenografts. Radiation therapy (RT) represents one of the mainstays of glioma treatment. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas. EXPERIMENTAL DESIGN: 3-(4,5-Dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and clonogenic assays were used to test cytoxicity of the combination treatment in vivo. To examine the mechanism of synergy, one-step viral growth curves, terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays, and Western blot analyses were performed. In vivo assessment of synergistic antitumor activity was conducted in a U87 glioma model. RESULTS: MTS and clonogenic assays showed a strong synergistic interaction between MV-CEA and RT in glioblastoma multiforme cells including both primary and established glioma lines. Furthermore, significant antitumor efficacy was observed in vivo in a subcuteneous U87 xenograph model. There was significant prolongation of survival (P = 0.001) in the combination treatment group as compared with single modality- or control-treated animals. One-step viral growth curves showed increased viral burst size by up to 2 log in MV/RT combination-treated cells, as compared with single agent MV-CEA-treated glioma cells. Changes in CEA levels and expression of viral N and H protein were also consistent with increased viral production. Furthermore, TUNEL assays and Western blot analysis showed increase in apoptosis in MV/RT combination-treated cells. The pan-caspase inhibitor Z-VAD-FMK and the caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected glioma cells from MV-CEA/RT-induced cleavage of poly(ADP-ribose) polymerase (PARP), indicating that the apoptotic death in combination-treated cells is mostly mediated via the extrinsic caspase pathway. The Fas/Fas ligand interaction blocking antibody NOK-1 blocked MV/RT-induced PARP cleavage whereas the Fas agonistic antibody CH11 increased PARP cleavage in MV/RT combination-treated cells. Reverse transcription-PCR, fluorescence-activated cell sorting analysis and immunohistochemistry showed up-regulation of Fas in combination-treated tumor in vitro and in vivo cells. CONCLUSIONS: There is synergy between MV-CEA and RT in vitro and in vivo. The synergistic effect of the combination seems to be due to increase in viral burst size and increase in apoptotic cell death. This latter effect is mostly mediated via the extrinsic caspase-8 pathway, activated via increased signaling through the Fas death receptor pathway. These results could have translational implications in glioma therapy.

Interaction of measles virus vectors with Auger electron emitting radioisotopes.

A recombinant measles virus (MV) expressing the sodium iodide symporter (NIS) is being considered for therapy of advanced multiple myeloma. Auger electrons selectively damage cells in which the isotope decays. We hypothesized that the Auger electron emitting isotope 125I can be used to control viral proliferation. MV was engineered to express both carcinoembryonic antigen and NIS (MV-NICE). Cells were infected with MV-NICE and exposed to 125I with appropriate controls. MV-NICE replication in vitro is inhibited by the selective uptake of 125I by cells expressing NIS. Auger electron damage is partly mediated by free radicals and abrogated by glutathione. In myeloma xenografts, control of MV-NICE with 125I was not possible under the conditions of the experiment. MV-NICE does not replicate faster in the presence of radiation. Auger electron emitting isotopes effectively stop propagation of MV vectors expressing NIS in vitro. Additional work is necessary to translate these observations in vivo.

Induction of the measles virus receptor SLAM (CD150) on monocytes.

Wild-type strains of measles virus (MV) isolated in B95a cells use the signalling lymphocyte activation molecule (SLAM; also known as CD150) as a cellular receptor, whereas the Edmonston strain and its derivative vaccine strains can use both SLAM and the ubiquitously expressed CD46 as receptors. Among the major target cells for MV, lymphocytes and dendritic cells are known to express SLAM after activation, but monocytes have been reported to be SLAM-negative. In this study, SLAM expression on monocytes was examined under different conditions. When freshly isolated from the peripheral blood, monocytes did not express SLAM on the cell surface. However, monocytes became SLAM-positive after incubation with phytohaemagglutinin, bacterial lipopolysaccharide or MV. Anti-SLAM monoclonal antibodies efficiently blocked infection of activated monocytes with a wild-type strain of MV. These results indicate that SLAM is readily induced and acts as a monocyte receptor for MV.

Disruption of Akt kinase activation is important for immunosuppression induced by measles virus.

Surface-contact-mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2-dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

Measles virus exploits dendritic cells to suppress CD4+ T-cell proliferation via expression of surface viral glycoproteins independently of T-cell trans-infection.

Dendritic cells (DC) have been proposed to play a pivotal role in transient immune suppression induced by measles virus (MV) infection. In the present study, we show that DC-induced suppression of T-cell proliferation was not mediated by IL-10 or IFNalpha/beta, which are released following infection of DC, but required cell contacts between MV-infected DC and T cells. Human sera containing neutralizing anti-MV antibodies, as well as anti-MV hemagglutinin (HA) or fusion protein (F) mAbs, were found (i) to reverse suppression and (ii) to restore DC allostimulatory capacity. Interestingly, DC-induced T-cell suppression was associated with both phenotypic and functional DC maturation, as demonstrated by IL-12 production and chemotaxis to MIP-3beta. These data suggest that MV infection turns on the maturation program of DC allowing migration to draining lymph nodes, where potent T-cell immune suppression might be achieved via cell surface expression of HA and F glycoproteins, independently of T cell trans-infection.

Measles virus-induced promotion of dendritic cell maturation by soluble mediators does not overcome the immunosuppressive activity of viral glycoproteins on the cell surface.

Measles virus (MV) infection promotes maturation of dendritic cells (DC), but also interferes with DC functions, and MV renders the DC inhibitory for T cell proliferation. We now describe that MV infection triggers the release of type I IFN from monocyte-derived DC (Mo-DC) which contributes to DC maturation. There is no evidence that soluble mediators are released interfering with the stimulatory activity of uninfected DC. Since inhibition of allogeneic T cell proliferation was unaffected by a fusion inhibitory peptide (Z-fFG), MV infection of T cells did not contribute to inhibition. Allogeneic T cell proliferation depended on the percentage of DC expressing MV F/H glycoproteins within the DC population and their surface expression levels, was induced upon addition of UV-inactivated MV to a mixed lymphocyte reaction stimulated by lipopolysaccharide-matured DC, and was not induced by DC infected with a recombinant MV encoding the ectodomain of vesicular stomatitis virus G protein (MG/FV) instead of the MV glycoproteins. Similarly, DC infected with MV, but not with MG/FV inhibited mitogen-induced proliferation of T cells. Thus, a dominant inhibitory signal is delivered to T cells by the MV glycoproteins on the surface of DC overcoming positive signals by co-stimulatory molecules promoted by maturation factors released from infected DC.

Failure of measles virus to activate nuclear factor-kappa B in neuronal cells: implications on the immune response to viral infections in the central nervous system.

Neurons are postmitotic cells that foster virus persistence. These cells lack the HLA class I molecules required for clearance of infected cells. Previously, we showed that HLA class I is induced by measles virus (MV) on glial cells, which is primarily mediated by IFN-beta. In contrast, MV was unable to induce HLA class I or IFN-beta in neuronal cells. This failure was associated with lack of NF-kappa B binding to the positive regulatory domain II element of the IFN-beta promoter, which is essential for virus-induced IFN-beta gene activity. In this study, we demonstrate that the failure to activate NF-kappa B in neuronal cells is due to the inability of MV to induce phosphorylation and degradation of I kappa B, the inhibitor of NF-kappa B. In contrast, TNF-alpha induced degradation of I kappa B alpha in the neuronal cells, suggesting that failure to induce I kappa B alpha degradation is likely due to a defect in virus-mediated signaling rather than to a defect involving neuronal I kappa B alpha. Like MV, mumps virus and dsRNA failed to induce I kappa B alpha degradation in the neuronal cells, suggesting that this defect may be specific to viruses. Autophosphorylation of the dsRNA-dependent protein kinase, a kinase possibly involved in virus-mediated I kappa B alpha phosphorylation, was intact in both cell types. The failure of virus to induce I kappa B alpha phosphorylation and consequently to activate NF-kappa B in neuronal cells could explain the repression of IFN-beta and class I gene expression in virus-infected cells. These findings provide a potential mechanism for the ability of virus to persist in neurons and to escape immune surveillance.

Receptor (CD46)- and replication-mediated interleukin-6 induction by measles virus in human astrocytoma cells.

A major source of inflammatory cytokines in the measles virus (MV)-infected brain are astrocytes, which produce a variety of soluble mediators including interferons-alpha/beta (IFN-alpha/beta), interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6). Using the MV-strain Edmonston (ED) and the recombinant MV-strain MGV in which the MV-envelope proteins H and F have been replaced by the vesicular stomatitis virus (VSV) envelope protein G, we investigated IL-6 induction in human U-251 astrocytoma cells in the presence and absence of a MV-specific receptor (CD46) interaction. The CD46-MV interaction did not inhibit the induction of cytokines. Similar multiplicities of infection of MGV induced generally lower levels of IL-6 than MV-ED. UV-inactivated replication-incompetent MV-ED induced low levels of IL-6. In contrast, MGV did not induce IL-6 after inactivation with UV light, indicating that the MV-ED-receptor interaction or the uptake of viral particles by membrane fusion induced IL-6, whereas interaction with the VSV-G receptor and uptake of viral particles by endocytosis did not induce IL-6. Crosslink of the MV-receptor CD46 with antibodies and treatment of cells with purified viral glycoproteins led to the induction of small but significant amounts of IL-6. Our data suggest that triggering of CD46 and associated protein kinases can lead to the induction of low levels of IL-6, whereas the replication of the negative strand RNA virus constitutes the major stimulus leading to the synthesis of high levels of IL-6 in astrocytes.

T-cell independent IgM and enduring protective IgG antibodies induced by chimeric measles viruses.

B-cell activation depends on the intensity of B-cell receptor cross-linking. Studies of haptenated antigens and vesicular stomatitis virus (VSV) have demonstrated a correlation between antigen repetitiveness and the degree to which B-cell activation is independent of T cells. Here, we compare neutralizing antibody responses to inactivated VSV with those to two inactivated human pathogenic viruses: highly cytopathic poliovirus (PV) and poorly cytopathic measles virus (MV). The rigidly structured PV efficiently induced neutralizing IgM antibodies independent of T cells. In contrast, neutralizing antibodies to the pleomorphic MV were dependent on helper T cells. To test whether this resulted from the differences in virus structure or the capacity of MV to induce cell fusion and/or immunosuppression, we analyzed antibody responses to chimeric MV expressing VSV glycoprotein instead of MV fusion protein and hemagglutinin. IgM antibodies were independent of T cells; in addition, we found IgG responses dependent on T-cell help that were enduring and protective against lethal VSV infection. Because chimeric MV viruses look like MV ultrastructurally, we conclude that not only structural differences in the envelope but also the ability of MV to induce immunosuppression may limit its capacity to directly activate B cells. These findings are relevant for our understanding of B-cell activation by two prototypic human pathogenic viruses and for the design of new recombinant vaccines.

UV-inactivated measles virus stimulates human and mice naive lymphocytes to proliferate in vitro.

Ultra violet (UV) light-inactivated measles virus was used for the lymphocyte proliferation assay, where it caused consistently adult human peripheral blood monolymphocytes (PBML) proliferation which was not dependent on the measles virus complement fixation titre in the donor's sera. When the cord blood lymphocytes from newborn baby and spleen lymphocytes from unprimed Balb/c, C57bl and CBA mice were used in the proliferation assay, all the lymphocytes were stimulated by the UV-inactivated measles virus in a dose-dependent manner. These results suggest that: (1) lymphotropic measles virus can also activate lymphocytes in vitro, and (2) this activation appeared to be neither species- nor major histocompatibility complex (MHC)-restricted. These results may have important implications for measles vaccination and the understanding of the immunopathogenesis of measles virus infection.

Measles virus induction of human endothelial cell tissue factor procoagulant activity in vitro.

Measles virus infection of microvascular endothelium in vivo and ensuing endothelial cell activation may be important in the pathogenesis of subsequent inflammation in target organs. This study investigated the capacity of measles virus to induce procoagulant activity, in vitro, in endothelial cells isolated from human umbilical cord veins. Endothelial cells were infected with a clinical isolate of measles virus propagated in Vero cells. Cells were also incubated with bacterial lipopolysaccharide (10 micrograms/ml), herpes simplex virus type 1, cytomegalovirus or culture medium alone as positive and negative controls, respectively. Endothelial cell procoagulant activity was measured in a one-stage clotting assay. Measles virus stimulated both a time and dose-dependent endothelial cell procoagulant response by the induction of tissue factor synthesis, confirmed by both immunocytochemistry and its dependence on factor VII for activity. This activity was reduced by u.v.-irradiation of the virus. Infected cells were analysed by double immunofluorescent staining for both tissue factor and measles virus N-protein, and examined using confocal scanning laser microscopy. Cells expressing tissue factor were also positive for the measles virus N-protein. Low levels of interleukin-1 were detected in some viral inocula derived from measles virus-infected Vero cells, however neutralising antibody to interleukin-1 failed to inhibit the endothelial cell procoagulant response to measles virus, whereas it significantly reduced procoagulant activity induced in endothelial cells by recombinant interleukin-1. The capacity of measles virus to induce endothelial tissue factor in vitro, may be relevant to the thrombotic vasculopathy associated with measles virus infection in vivo.

Efficient MHC class II-restricted presentation of measles virus to T cells relies on its targeting to its cellular receptor human CD46 and involves an endosomal pathway.

The role of the measles virus (MV) receptor, human CD46, in the uptake of MV and antigen presentation by Major Histocompatibility Complex (MHC) class II molecules was investigated. Expression of CD46 in murine B cells resulted in cells highly efficient in capturing UV-inactivated MV particles and presenting both envelope hemagglutinin H and nucleoprotein N to specific T cell hybridomas. Although MV fuse with the plasma membrane of its target cells, presentation of both MV-H and -N was sensitive to inhibition by chloroquine but was not affected by a tripeptide which prevents virus-cell fusion. Whereas 50 microM of chloroquine was required to inhibit presentation of MV-H, purified H or soluble N, only a two-fold lower concentration was required to inhibit that of MV-N. This shows that some CD46-mediated captured MV particles are endocytosed, then disrupted and processed in an endosome/lysosome compartment.

Development and evaluation of the TD97 measles virus vaccine.

The TD97 strain vaccine virus was prepared from the Tanabe strain measles virus by low-temperature passages in primary cell cultures and ultraviolet (UV) mutagenesis. The TD97 strain exhibited the following characteristics: highly temperature sensitive, neither multiplying nor forming any plaques at 40 degrees C in Vero cells; genetically stable, maintaining high temperature sensitivity after ten successive passages in CE cells at 30 degrees C or 35 degrees C; and M proteins of this virus about 1 KD slower in mobility in SDS-PAGE than that of the Tanabe strain. The TD97 strain was further confirmed to be attenuated by an inoculation test into primate brain. In field trials, 752 healthy children were inoculated with a live virus vaccine prepared with this strain, and the following results were obtained: the seroconversion rate was 97% (517/533), and the average HI antibody titer was 2(5.2). An antibody-increasing effect was also observed in children who were initially seropositive. In children who seroconverted, the rates of fever were 15.7% (55/351) for 37.5 degrees C or higher and 4.0% (14/351) for 39 degrees C or higher. The rash rate was 7.7% (27/351), and the incidence of local reaction was 5.4% (19/351). The TD97 strain is thus considered to be suitable in use for an attenuated measles vaccine.

A study of human embryonic cells treated with UV-inactivated measles virus.

Some features of persistent infection were observed in human embryonic fibroblast cells (CLV21K cells) after single treatment with UV-inactivated measles virus. Morphological changes and chromosomal abnormalities were found. In subsequent passages of CLV21K cells, viral antigens were discovered by rosette haemadsorption, immunofluorescence tests and electron microscope or scanning electron microscope studies. No infectious virus was found.

Effect of measles-virus infection and interferon treatment on invasiveness of Shigella flexneri in HEp2-cell cultures.

The influence of measles-virus infection on the invasiveness of Shigella flexneri in HEp2-cell cultures was studied. Bacterial invasiveness was significantly enhanced in cell cultures incubated with virus before bacterial inoculation. This effect was a function of time after introduction of virus to the cell cultures and of the concentration of virus. The increase in bacterial invasiveness was observed before production of infectious virus particles and before a cytopathic effect was evident. A similar enhancement of invasiveness was demonstrated when cell cultures were pretreated with UV-inactivated measles virus. Pretreatment of cells with interferon did not influence invasiveness, although it reduced the effect of measles-virus infection.

Interferon induction by measles virus.

Interferon induction in BSC-1 cells by measles virus isolates previously found to be interferon inducing (IF+) or interferon noninducing (IF-) was studied in different cell types. Lack of induction by the IF- viruses was not due to an inhibitory factor since BSC-1 cells coinfected with IF+ and IF- stocks produced interferon levels similar to those infected with IF+ stocks alone. Various tests indicated that the phenotypic expression of interferon induction by these isolates was dependent on an infectious virus genome, the cell type tested, and the passage history of the virus. In contrast to the in vitro results, the IF- phenotype was stable in vivo, whereas passage of the IF+ virus resulted in selection against the IF+ phenotype. The phenotypic expression of interferon induction by measles virus was, therefore, controlled by complex interactions between virus and host cell factors exerted both in vivo and in vitro.

Immunogenicity of UV-inactivated measles virus.

By means of the antigen extinction limit test it was shown that a triple dose vaccination of guinea pigs with UV-inactivated measles virus gave better results, than a single dose vaccination which was proved by the very low immunogenicity index. For both vaccination schemes (single and triple) the immune response was only slightly influenced by a change of dose from 10(5) to 10(6) HadU50/ml or by the addition of aluminum adjuvant. In the antigen extinction limit test the antibody levels were determined by two methods (HIT and NT) the results of which were statistically equivalent. The UV-inactivated measles virus was also found to induce hemolysis-inhibiting antibodies.

Thermal and ultraviolet inactivation of plaque purified measles virus clones.

Three plaque-purified measles virus clones exhibited remarkable differences in their kinetics of inactivation only after heating at 45 degrees C and none after UV-irradiation. A hypothesis is discussed of the possible dependence, in fast inactivating populations, of thermolability on a relative incompleteness of the virions.