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HLA class II-restricted presentation of cytoplasmic measles virus antigens to cytotoxic T cells.

To analyze the nature of the HLA class II-restricted cytotoxic T-lymphocyte (CTL) response to measles virus, murine fibroblasts were transfected with expressible cDNA clones for human HLA-DR antigen and for measles virus matrix or nucleocapsid proteins. DR-positive murine fibroblasts transfected with measles virus matrix or nucleocapsid genes were lysed by class II-restricted measles virus-specific CTL lines. Lysis was as efficient as with infected autologous B-cell lines, even though the measles virus cytoplasmic proteins were undetectable by antibodies in the transfected target cells. These results demonstrate that cytoplasmic viral antigens can be presented to CTL in the context of HLA class II antigens and that measles virus matrix and nucleocapsid proteins contribute to class II-restricted measles virus-specific CTL responses. These results also show that endogenously synthesized measles virus proteins can be efficiently presented by class II antigens. The implications of these findings for measles virus pathogenesis and for antigen processing are discussed.

Animals↗

Purification of measles virus with preservation of infectivity and antigenicity.

Measles virus Edmonston strain was purified by ultrafiltration followed by two successive sedimentations through sucrose. Purified virus retained infectivity and, when used as an immunogen, elicited high titred antibody to measles antigens by conventional serology. The measles preparations were examined by SDS-PAGE followed by staining. In addition, following PAGE, the purity of these preparations was assessed immunochemically using antisera directed to measles and host cell antigens. The results of these studies demonstrate the utility of the purification method for the preparation of milligram quantities of relatively pure measles virus.

Epitopes↗

Evidence of recombination among early-vaccination era measles virus strains.

BACKGROUND: The advent of live-attenuated vaccines against measles virus during the 1960'ies changed the circulation dynamics of the virus. Earlier the virus was indigenous to countries worldwide, but now it is mediated by a limited number of evolutionary lineages causing sporadic outbreaks/epidemics of measles or circulating in geographically restricted endemic areas of Africa, Asia and Europe. We expect that the evolutionary dynamics of measles virus has changed from a situation where a variety of genomic variants co-circulates in an epidemic with relatively high probabilities of co-infection of the individual to a situation where a co-infection with strains from evolutionary different lineages is unlikely. RESULTS: We performed an analysis of the partial sequences of the hemagglutinin gene of 18 measles virus strains collected in Denmark between 1965 and 1983 where vaccination was first initiated in 1987. The results were compared with those obtained with strains collected from other parts of the world after the initiation of vaccination in the given place. Intergenomic recombination among pre-/early-vaccination strains is suggested by 1) estimations of linkage disequilibrium between informative sites, 2) the decay of linkage disequilibrium with distance between informative sites and 3) a comparison of the expected number of homoplasies to the number of apparent homoplasies in the most parsimonious tree. No significant evidence of recombination could be demonstrated among strains circulating at present. CONCLUSION: We provide evidence that recombination can occur in measles virus and that it has had a detectable impact on sequence evolution of pre-vaccination samples. We were not able to detect recombination from present-day sequence surveys. We believe that the decreased rate of visible recombination may be explained by changed dynamics, since divergent strains do not meet very often in current epidemics that are often spawned by a single sequence type. Signs of pre-vaccination recombination events in the present-day sequences are not strong enough to be detectable.

Base Sequence↗

Antibody-mediated modification of encephalitis induced by hamster neurotropic measles virus.

The acute encephalopathy induced by hamster neurotropic (HNT) measles virus in BALB/c mice was abolished, and a subacute encephalitis was induced instead, when antibody to Edmonston (Ed) measles virus was administered three days after inoculation of HNT virus. Ascites fluid from 79XIC1, a monocolonal antibody to the hemaglutinin of strain Ed, had similar effects in vivo. However, other monoclonal antibodies to the hemagglutinin of Ed strain--79XVV17 and 80IIIB2--were ineffective in vivo at all doses tested. By viral neutralization, immunofluorescence, and radioimmunoprecipitation assays, it was shown that the hemagglutinin of HNT virus reacted with 79XIC1 antibody but lacked the epitopes recognized by 79XVV17 or 80IIIB2 antibody, a finding that explains their different reactivity in vivo. These observations also identified previously unrecognized antigenic differences between the hemagglutinin of Ed and HNT measles viruses.

Animals↗

Expression of defective measles virus genes in brain tissues of patients with subacute sclerosing panencephalitis.

The persistence of measles virus in selected areas of the brains of four patients with subacute sclerosing panencephalitis (SSPE) was characterized by immunohistological and biochemical techniques. The five measles virus structural proteins were never simultaneously detectable in any of the brain sections. Nucleocapsid proteins and phosphoproteins were found in every diseased brain area, whereas hemagglutinin protein was detected in two cases, fusion protein was detected in three cases, and matrix protein was detected in only one case. Also, it could be shown that the amounts of measles virus RNA in the brains differed from patient to patient and in the different regions investigated. In all patients, plus-strand RNAs specific for these five viral genes could be detected. However, the amounts of fusion and hemagglutinin mRNAs were low compared with the amounts in lytically infected cells. The presence of particular measles virus RNAs in SSPE-infected brains did not always correlate with mRNA activity. In in vitro translations, the matrix protein was produced in only one case, and the hemagglutinin protein was produced in none. These results indicate that measles virus persistence in SSPE is correlated with different defects of several genes which probably prevent assembly of viral particles in SSPE-infected brain tissue.

Adolescent↗

Role of CD8(+) lymphocytes in control and clearance of measles virus infection of rhesus monkeys.

The creation of an improved vaccine for global measles control will require an understanding of the immune mechanisms of measles virus containment. To assess the role of CD8(+) cytotoxic T lymphocytes in measles virus clearance, rhesus monkeys were depleted of CD8(+) lymphocytes by monoclonal anti-CD8 antibody infusion and challenged with wild-type measles virus. The CD8(+) lymphocyte-depleted animals exhibited a more extensive rash, higher viral loads at the peak of virus replication, and a longer duration of viremia than did the control antibody-treated animals. These findings indicate a central role for CD8(+) lymphocytes in the control of measles virus infections and the importance of eliciting a cell-mediated immune response in new measles vaccine strategies.

Animals↗

Replication of measles virus in continuous lymphoid cell lines.

The replication of measles virus was studied in a T cell-like line, MOLT-4, and a B cell-like line, Raji. Both cell lines supported measles virus replication. The infected lymphoid cells were assayed for infectious virus and expression of measles antigens at timed intervals up to 84 hr after inoculation. At time 0, after a 2-hr adsorption period at 4 degrees C, both cell lines produced a maximum of 3% infectious centers and up to 22% antigen-positive cells. Eventually all cells in both populations became infected during the experimental period. When Raji and MOLT-4 cells were infected under similar conditions 100% infection of cells was achieved 12 hr earlier in Raji than in MOLT-4. Increasing multiplicity of infection (MOI) from 1 to 10 made no significant change in the number of infected cells during the early part of the cycle. Spread of virus was largely due to released virus and not cell to cell transmission. Since only a small proportion of the population absorbed the virus at any one time, and eventually all cells were infected, it was concluded that the receptors for measles virus were expressed at some phase of the cell cycle.

Antigens, Viral↗

Activation of a latent measles virus infection in hamster cells.

The characteristics of infectious measles virus released from latently infected hamster embryo fibroblast cells are described. Low levels of virus were released spontaneously when the cultures were incubated at 37 C; this phenomenon was observed 19 passages after the cells had been exposed to the virus and has continued through cell passage 45. The virus yield could be significantly increased by cocultivation of the hamster cells with BSC-1 cells or incubation of the latently infected cells at 33.5 C rather than at 37 C. Measles virus released after cocultivation demonstrated increased cytopathology in cell culture and reduced temperature sensitivity when compared to the virus released at 33.5 C. After cell passage 45, there was an increase in spontaneous release of virus. However, the viruses recovered by cocultivation or temperature release after cell passage 45 were nearly identical. These observations suggest a possible mechanism for measles virus activation in cells latently infected with this virus.

Animals↗

Antibody-mediated cytotoxicity after measles virus infection.

Antibody cytotoxicity in the presence of complement to measles virus-infected cells was determined in sera of individuals at various stages of measles virus infection. A measles virus carrier cell line, which possessed viral antigens on the cell membrane, was employed as target cells. The cytotoxic effect was measured by 51-Cr release from the measles virus-infected cells. A microtiter method was used to determine the titer of antibody cytotoxicity. Cytotoxic antibodies could be detected in serum specimens from individuals after natural measles virus infection and vaccination. Sera from subacute sclerosing panencephalitis (SSPE) patients had only slightly higher titers than were found in sera obtained from individuals 1 week to 3 months after measles virus infection. Sera from children inoculated with measles virus vaccine lost their ability to react in hemagglutination inhibition, neutralization (NT) and cytotoxicity (CT) tests after absorption with the hemagglutinin from measles virus. SSPE sera treated in a similar manner retained their activity in NT and CT tests. Cytotoxic antibody was, therefore, shown to be directed against the hemagglutinin and some other antigen of measles virus.

Animals↗

No measles virus genetic information detected in multiple sclerosis--derived brains.

Measles virus nucleotide sequences were searched for in diseased and normal-appearing white matter from brains of four multiple sclerosis victims. At the level of sensitivity established, 2.5 to 7.8 measles virus genome-equivalents of RNA, or 4.9 genome-equivalents of putative DNA per diploid cell quantity of DNA, could have been detected. At these levels, no measles virus genetic information was found in any of the four brains.

Adult↗

Host DNA synthesis-suppressing factor in culture fluid of tissue cultures infected with measles virus.

Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated component which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither UV-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus.

Animals↗

The use of a tropism-modified measles virus in folate receptor-targeted virotherapy of ovarian cancer.

PURPOSE: Attenuated measles viruses are promising experimental anticancer agents currently being evaluated in a phase I dose escalation trial for ovarian cancer patients. Virus attachment, entry, and subsequent intercellular fusion between infected and uninfected neighboring cells are mediated via the two measles receptors (CD46 and SLAM). To minimize potential toxicity due to measles virus-associated immunosuppression and infection of nontarget tissues, we sought to develop an ovarian cancer exclusive fully retargeted measles virus. EXPERIMENTAL DESIGN AND RESULTS: Interactions of measles virus with its natural receptors were ablated, and a single-chain antibody (scFv) specific for alpha-folate receptor (FRalpha), a target overexpressed on 90% of nonmucinous ovarian cancer, was genetically engineered on the viral attachment protein (MV-alphaFR). Specificity of virus tropism was tested on tumor and normal cells. Biodistribution of measles virus infection was evaluated in measles-susceptible CD46 transgenic mice, whereas antitumor activity was monitored noninvasively by bioluminescence imaging in xenograft models. Tropism and fusogenic activity of MV-alphaFR was redirected exclusively to FRalpha without compromise to virus infectivity. In contrast to the parental virus, MV-alphaFR has no background infectivity on normal human cells. The antitumor activity of MV-alphaFR, as assessed by tumor volume reduction and overall survival increase, was equal to the parental virus in two models of human ovarian cancer (s.c. and i.p.). CONCLUSIONS: A FR-exclusive ovarian cancer targeted oncolytic virus was generated and shown to be therapeutically effective, thus introducing a new modality for FR targeting and a candidate measles virus for clinical testing.

Cancer Vaccines↗

Fatal measles virus infection in Japanese macaques (Macaca fuscata).

An outbreak of natural measles virus infection occurred in a group of Japanese macaques (Macaca fuscata). Over a period of 4 months, 12 of 53 Japanese macaques died following a 2-23-day history of anorexia, diarrhea, and dermatitis. The monkeys were kept in outdoor exhibits but had been moved temporarily into indoor caging and then transferred to new outdoor exhibits. Ten monkeys died while they were in temporary caging, and two monkeys died after they were moved to new outdoor exhibits. The diagnoses were made based on the results of histopathology, immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. Measles virus antigens were detected in the lung, stomach, skin, salivary gland, spleen, and lymph nodes. Tangled, tubular nucleocapsids compatible with paramyxovirus were noted in the lung tissue. As a result of immunosuppression following measles virus infection, various secondary infections including disseminated cytomegalovirus infection, adenoviral and bacterial pneumonia, and Candida albicans-associated gingivitis and esophagitis were noted. The primary infective source or the mode of infection could not be determined in this outbreak, but measles virus may have been transmitted to the monkeys from human visitors while the monkeys were on exhibit.

Animals↗

Intracerebral inoculation of rhesus monkeys with a strain of measles virus isolated from a case of subacute sclerosing panencephalitis.

Measles virus isolated from the brain of a patient with subacute sclerosing panencephalitis was injected intracerebrally (ic) into 34 rhesus monkeys. Groups of these animals were injected with measles antigen in Freund's complete adjuvant or treated by schedules used for suppression of the general or cell-mediated immune responsiveness. In another group of animals, experimental allergic encephalitis was induced parallel with measles infection. Measles virus was isolated from the brains of monkeys up to 13 days after ic inoculation. No virus was detected in the central nervous system after 3 to 4 weeks, the longest postinoculation period examined. It was concluded that the subacute sclerosing panencephalitis-derived virus either lost its neurotropic properties at the passage level at which it was used or that it submerged into a silent stage and escaped detection. Neither immunosuppression nor concomitant autoimmune encephalitis had an effect on the survival of measles virus in the central nervous system. The histology of the nervous tissue was basically normal except for characteristic lesions of experimental allergic encephalitis in animals receiving the respective treatment.

Animals↗

Marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus.

B95-8, an Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line, and its derivative B95a, capable of attachment to a substrate surface, were 10,000-fold more sensitive to measles virus present in clinical specimens than were Vero cells. B95-8 and B95a cells were thus thought to be useful host cells for the isolation of measles virus. Quantitation of measles virus present in clinical specimens showed that a large quantity of virus, exceeding 10(6) 50% tissue culture infective doses per ml of a nasal-swab eluate, is shed into secretions by patients with acute measles, consistent with the contagiousness of the disease. Measles viruses isolated in B95a cells differed in some biological properties from those adapted to Vero cells. First, the viruses isolated in B95a cells did replicate in Vero cells, but release into the fluid phase was less efficient than that of Vero cell-adapted viruses. Second, minor antigenic differences were found between virus strains isolated in B95a cells and those isolated in Vero cells from the same clinical specimens. Third, the viruses isolated and propagated in B95a cells caused clinical signs in experimentally infected monkeys resembling those of human measles. It was suspected that measles virus is subject to host cell-mediated selection and that the viruses grown in B95a cells are more representative of measles virus circulating among humans than are the viruses selected in Vero cells.

Animals↗

Binding of measles virus to membrane cofactor protein (CD46): importance of disulfide bonds and N-glycans for the receptor function.

Two cellular proteins, membrane cofactor protein (MCP) and moesin, were reported recently to be functionally associated with the initiation of a measles virus infection. We have analyzed the interaction of measles virus with cell surface proteins, using an overlay binding assay with cellular proteins immobilized on nitrocellulose. Among surface-biotinylated proteins from a human rectal tumor cell line (HRT), measles virus was able to bind only to a 67-kDa protein that was identified as MCP. The virus recognized different isoforms of MCP expressed from human (HRT and HeLa) and simian (Vero) cell lines. The binding of measles virus to MCP was abolished after cleavage of the disulfide bonds by reducing agents as well as after enzymatic release of N-linked oligosaccharides. By contrast, removal of sialic acid or O-linked oligosaccharides did not affect the recognition of MCP measles virus. These data indicate that the receptor determinant of MCP is dependent on a conformation of the protein that is maintained by disulfide bonds and N-glycans present in the complement binding domains. Our results are consistent with a role of MCP as primary attachment site for measles virus in the initial stage of an infection. The functional relationship between MCP and moesin in a measles virus infection is discussed.

Animals↗

On the relationship between measles virus and Alzheimer neurofibrillary tangles in subacute sclerosing panencephalitis.

We have studied the relationship between measles virus and the accumulation of abnormally phosphorylated tau (PHF-tau) in nine cases of subacute sclerosing panencephalitis (SSPE). By assessing the presence of viral intranuclear inclusions and neurofibrillary tangles (NFT) in each case, we found no correlation between presence and amount of measles virus and the numbers of neurons containing PHF-tau. Immunohistochemical double labeling in a case with long duration of disease and severe histopathologic change revealed no strict colocalization of measles virus antigen and PHF-tau throughout different brain regions. In areas containing both antigens, most neurons carrying measles virus did not have a tangle and vice versa, eventhough some colocalization beyond that expected by chance was observed in specific cortical areas. These results indicate that, although secondary to viral infection, NFT formation in SSPE is not restricted to cells carrying viral antigen. Conversely, measles virus infected cells do not necessarily accumulate PHF-tau. This lack of colocalization at the cellular level, throughout different brain areas and among different cases suggests that the formation of NFT in SSPE is not directly induced by the infectious agent. The formation of NFT in this disease appears to be elicited through a specific type of tissue damage and, thus, to be an epiphenomenon. This pathogenetic detail may be of interest for our understanding of the role of neurofibrillary degeneration in the pathogenesis of other more frequent neurodegenerative diseases with cytoskeletal pathology.

Adolescent↗

Cytokine production in vitro and the lymphoproliferative defect of natural measles virus infection.

In natural measles virus infection, evidence of intense immune system activation is present simultaneously with clinically relevant immune suppression. While evidence of activation is most prominent early in the disease, skin test responses and in vitro lymphoproliferation are depressed for weeks after the onset of the rash. It is not known whether the prolonged period of reduced immune responsiveness results from a single defect or a succession of different abnormalities. To gain further insight into measles-induced immune suppression we studied the production of soluble IL-2 receptor (sIL-2R), interferon-gamma (IFN-gamma), IL-1 beta, and tumor necrosis factor (TNF alpha) by peripheral blood mononuclear cells (PBMC) isolated from measles patients at various times after the onset of the rash. Studies included addition of supplemental recombinant IL-1 beta (rIL-1 beta) or recombinant IL-2 (rIL-2) or suppression of prostaglandin synthesis by indomethacin (IM). Proliferation in response to phytohemagglutin (PHA) was abnormal at all stages of disease. During the acute phase (first week after the onset of the rash) spontaneous production of sIL-2R was increased (76 +/- 54 vs. controls 4 +/- 4; P less than 0.03), suggesting in vivo T cell activation while PHA-induced sIL-2R was decreased (228 +/- 43 vs. control 582 +/- 127; P less than 0.002), suggesting that the capacity to produce IL-2 in response to mitogen was limited. Supplementation of PHA-stimulated cultures with rIL-2 improved but did not normalize both proliferation (58,600 +/- 4900 to 70,700 +/- 4400 vs. control 97,700 +/- 15,500; P less than 0.03) and sIL-2R levels (114 +/- 58 to 309 +/- 87 vs. control 582 +/- 127; P less than 0.003). Both spontaneous (25 +/- 18 vs. control 237 +/- 92; P less than 0.002) and PHA-induced (20 +/- 20 vs. control 604 +/- 129; P less than 0.004) TNF alpha levels were subnormal and were not improved with rIL-2, rIL-1 beta, or IM, suggesting a block in monocyte TNF alpha production. Spontaneous and PHA-induced IFN-gamma and IL-1 beta levels were normal. During the convalescent phase (greater than 2 weeks after the onset of the rash), spontaneous levels of sIL-2R were normal and PHA-induced levels were completely normalized with supplemental rIL-2 but proliferation remained below normal.(ABSTRACT TRUNCATED AT 400 WORDS)

CD4-CD8 Ratio↗