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Development of antibody to measles virus polypeptides during complicated and uncomplicated measles virus infections.

Immune precipitation of 181 sera from 152 patients with natural measles was studied to determine the temporal course and frequency of antibody responses to nucleocapsid, fusion, hemagglutinin, and matrix proteins of measles virus. Large amounts of antibody to nucleocapsid protein developed in all patients by day one of the rash. Antibody to hemagglutinin and fusion proteins developed in all patients over the next 3 weeks, the former to high levels and the latter to low levels. Antibody to matrix protein developed to very low levels and was detectable in only 41% of the patients; this poor response to matrix protein was not correlated with the age of the patient or the acute neurological complications of measles.

Adolescent↗

Cotton rats (Sigmodon hispidus): an animal model to study the pathogenesis of measles virus infection.

Measles is still the most lethal infectious disease of infants worldwide. In spite of research efforts, two major problems associated with measles virus (MV) infection have not been resolved. One is the marked immune suppression leading to subsequent (often lethal) opportunistic infections and the second is waning of maternal antibodies which do not protect against wild type virus infection any longer, but impair vaccination. Monkeys are an animal model in which MV infection most closely resembles the human disease. The use of monkeys is restricted by ethical and financial reasons and their availability. A cost-effective alternative is the cotton rat (Sigmodon hispidus). Cotton rats are the only rodents which replicate measles virus in lung tissue after intranasal infection. Our research has shown that cotton rats are a valid model to study MV induced immune suppression and to test vaccine candidates. It is also useful for comparing various wild type measles virus strains as well as recombinant measles viruses.

Animals↗

Monoclonal antibodies against five structural components of measles virus. II. Characterization of five cell lines persistently infected with measles virus.

Groups of monoclonal antibodies against measles virus nucleoprotein (NP), phosphoprotein (P), matrix (M), hemagglutinin (H) and fusion (F) components were used for characterization of 5 persistently infected cell lines. In four of these lines (Lu106 carrier, MaSSPE, MaPi, HEpPi) all cells were infected but the cells mostly produced noninfectious virus products. The fifth line (HNT in vero cells) did not produce any infectious virus and only a fraction of the cells were infected in most passages. In agreement with earlier findings the virus strains showed marked variations in the M epitope pattern and also some variation in the H epitope pattern. In addition epitope variations were found in both NP and P protein, which contrasted with conserved antigen characteristics of these components in lytically replicating virus. Restriction of fusion in the persistent infections was studied further. HNT and Lu 106 cells showed selective quantitative restriction in F protein synthesis. Lu106 cells were found to contain distinct epitopic F species. In contrast MaSSPE cells produced readily detectable cleaved F protein and in addition extracellular virus products carried hemolytic activity. The fact that no cell fusion occurred was interpreted to be due to particular properties of the Ma 106 cells, a concept supported by the absence of fusion of these cells when infected with syncytiogenic measles virus. It is concluded that (a) under conditions of persistence of measles-virus without requirement for synthesis of complete virions a more pronounced variation in epitope characteristics of virus components is encountered than in lytic infections; and b) that persistence of measles virus shows individualistic characteristics which may reflect changes in the virus and/or innate properties of the host cells.

Animals↗

[Real time observation of binding of measles virus to Vero cells and neutralization of measles virus by human immunoglobulin using optical biosensor--a new real time diagnosis system for viral infections].

An optical biosensor to monitor molecular intractions was developed. This system made possible to observe reactions with a few minutes. We tried to apply this system for rapid diagnosis of viral infections. Measles virus propagated in our laboratory and crude rabbit anti-measles antiserum were used. A commercially available human immunoglobulin was used as a representative of patient sera. A crude rabbit anti-measles antiserum was fixed on the reaction surface of aminosilane coated cuvette, and specific binding of measles virus to this antibody was monitored. Specific purified IgG antibody was known to give as high sensitivity as the conventional culture method. But the sensitivity by crude antibody was found to be 1/10 in the sensitivity. This might be caused by the masked effects on specific antibody to the contaminated high amounts of non-specific proteins. None purified polyclonal antisera for most of the viruses are commercially available, and 10-20 times highly concentrated antibody solutions could be used for this titrations. Estimated anti-measles antibody titer of the commercial available human immunoglobulin by biosensor system was found to be the same as that by the conventional culture method. It was suggested that 1 X 10(3) virus particles/ml in the test solutions could be detectable and titerable by using commercially available specific anti-viral antibody in real time, and the viral infections could be diagnosed within an hour.

Animals↗

A functional measles virus replication and transcription machinery encoded by the vaccinia virus genome.

Measles virus encodes three proteins required for the encapsidation, transcription and replication of viral genomes. The genes for these proteins have been inserted into the vaccinia virus genome together with the gene for the bacteriophage T7 RNA polymerase. Cells infected with this recombinant virus were able to encapsidate, transcribe and replicate a CAT gene positioned in the negative polarity behind a T7 promoter and flanked by measles virus genomic termini. Inhibition of the accumulation of the nucleocapsid proteins by actinomycin D led to an increase in CAT expression. Thus the measles virus polymerase activity, encoded by the vaccinia genome, was regulated by the level of measles proteins just as the authentic polymerase. The recombinant vaccinia described in this study could be useful for the production of measles virus-like particles encoding foreign genes and employed in vaccination or gene therapy strategies.

Bacteriophage T7↗

Gene transfer into neurons from hippocampal slices: comparison of recombinant Semliki Forest Virus, adenovirus, adeno-associated virus, lentivirus, and measles virus.

Viral vectors are useful for transferring genes into neurons. Here, we characterized recombinant Semliki Forest virus (SFV), adenovirus type 5 (Ad5), adeno-associated virus type 2 (AAV), lentivirus, and measles virus (MV) by their expression of green fluorescent protein (GFP) in rat hippocampal slice cultures. SFV infected more neurons (>90% of all GFP-positive cells) than AAV, lentivirus, and MV (71, 69, and 62%, respectively), whereas no infected neurons were identified with Ad5. AAV-mediated GFP expression was neuron-specific when the platelet-derived growth factor beta-chain promoter rather than cytomegalovirus promoter was used. Transgene expression occurred rapidly but transiently for SFV, increased slowly but remained stable with AAV and lentivirus, and was fast with MV. Resting membrane potential and conductance, action potentials, firing accommodation, and H-current appeared normal in infected CA1 pyramidal cells. Thus, SFV is useful for short-term and AAV and lentivirus for long-term transduction of hippocampal slices, while MV constitutes a novel vector.

Adenoviridae↗

A chimeric human-bovine parainfluenza virus type 3 expressing measles virus hemagglutinin is attenuated for replication but is still immunogenic in rhesus monkeys.

The chimeric recombinant virus rHPIV3-N(B), a version of human parainfluenza virus type 3 (HPIV3) that is attenuated due to the presence of the bovine PIV3 nucleocapsid (N) protein open reading frame (ORF) in place of the HPIV3 ORF, was modified to encode the measles virus hemagglutinin (HA) inserted as an additional, supernumerary gene between the HPIV3 P and M genes. This recombinant, designated rHPIV3-N(B)HA, replicated like its attenuated rHPIV3-N(B) parent virus in vitro and in the upper and lower respiratory tracts of rhesus monkeys, indicating that the insertion of the measles virus HA did not further attenuate rHPIV3-N(B) in vitro or in vivo. Monkeys immunized with rHPIV3-N(B)HA developed a vigorous immune response to both measles virus and HPIV3, with serum antibody titers to both measles virus (neutralizing antibody) and HPIV3 (hemagglutination inhibiting antibody) of over 1:500. An attenuated HPIV3 expressing a major protective antigen of measles virus provides a method for immunization against measles by the intranasal route, a route that has been shown with HPIV3 and respiratory syncytial virus vaccines to be relatively refractory to the neutralizing and immunosuppressive effects of maternally derived virus-specific serum antibodies. It should now be possible to induce a protective immune response against measles virus in 6-month-old infants, an age group that in developing areas of the world is not responsive to the current measles virus vaccine.

Animals↗

Human cellular immune response to measles virus polypeptides.

Measles virus polypeptides were separated by polyacrylamide gel electrophoresis and electroeluted from gel sections. The antigenicity of the polypeptides was determined by enzyme-linked immunosorbent assays. The ability of these measles virus antigens to stimulate lymphoproliferation was measured in both high- and low-responder individuals. In contrast to the low-responder lymphocytes which did not proliferate when stimulated with measles virus antigens, the high-responder lymphocytes proliferated when challenged with hemagglutinin, nucleocapsid-associated phosphoprotein, nucleocapsid protein, matrix protein, and fusion protein.

Electrophoresis, Polyacrylamide Gel↗

Effect of measles virus antibodies on a measles SSPE virus persistently infected C6 rat glioma cell line.

Maintenance of measles (SSPE-Lec) virus persistently infected C6 rat glioma cells in medium containing polyclonal measles antiserum resulted in the loss of detectable expression of all measles virus proteins. Removal of these cells from antiserum, however, led to a re-expression of virus proteins and the production of infectious virus. Cloning of antibody-modulated non-expressing cells in the presence of antiserum showed that re-expression of virus proteins was not due to an incomplete curing process following the addition of antiserum, as a large number of non-expressing cell clones developed the capacity to express measles virus antigen at different periods after removal of antiserum. Irradiation of persistently infected cells to give a non-growing culture showed that modulation was not mediated by a selection and outgrowth of a small percentage of non-expressing cells originally present in the culture. Antibody directed against C6 membrane proteins did not lead to modulation and it was also shown that only monoclonal antibodies with neutralizing activity could affect intracellular antigen expression. Immunoglobulin Fab fragments with neutralizing activity also had modulating activity. Although all modulated cell clones were more susceptible to homologous virus infection than control C6 cells, it was not possible to rescue any defective measles virus which may have been maintained in the culture.

Animals↗

Human histocompatibility determinants and virus antigens: effect of measles virus infection on HLA expression.

Histocompatibility antigens on the surface of human lymphoblastoid cells were quantified by a microadsorption technique. During the course of measles virus infection, no quantitative or qualitations in surface HLA antigens were observed. In contrast, infection with poliovirus type 1 or vesicular stomatitis virus, or treatment with puromycin (50 microgram/ml) resulted in a significant decrease in surface HLA. These experiments suggest that an inhibition of host protein synthesis rather than the insertion of virus-specificied antigens into the membrane results in a net decrease in amounts of this cell surface antigen. The HLA antigens also appear to be both functionally and structurally distinct from measles virus surface antigens. Pretreatment of cells with HLA-directed antibody did not prevent the infection of these cells by measles virus, thus HLA antigens appear unrelated to the measles virus receptor site on the plasma membrane. Electron microscopic studies revealed that measles virus maturation occurs at membrane sites devoid of demonstrable HLA. Furthermore, HLA antigens could not be detected on the surfaces of mature infectious virions.

Antibodies↗

Monoclonal antibodies against five structural components of measles virus. I. Characterization of antigenic determinants on nine strains of measles virus.

Monoclonal antibodies against five structural proteins of measles virus were used to determine the degree of antigenic variation within these proteins amongst nine strains of measles virus (four fresh wild-type isolates, two vaccine and two laboratory strains, and a strain derived from a case of subacute sclerosing panencephalitis) giving lytic infections in cell culture. The major surface proteins showed limited variations in their epitopes between the nine strains. No variations in the fusion (F) protein and only three variations in the hemagglutinin (H) protein epitopes were detected by radioimmune precipitation assay and other serological tests using a panel of 11 monoclonal antibodies against each protein. These antibody panels consisted of at least nine and six different binding groups for the H and F proteins, respectively. The two innermost proteins, the nucleocapsid and polymerase proteins, also appeared to be antigenically stable as no variation was detected between strains using in each case a panel of six hybridomas. In sharp contrast, the epitopes on the matrix (M) protein of different strains showed extensive variation in their reactivity with the nine anti-M monoclonal antibodies. The possible use of M protein epitopic markers in classification of measles virus strains is discussed.

Antibodies, Monoclonal↗

Salmonella vaccines secreting measles virus epitopes induce protective immune responses against measles virus encephalitis.

In the present study we describe a live vaccine against measles virus (MV) infection on the basis of attenuated Salmonella typhimurium aroA secreting MV antigens via the Escherichia coli alpha-hemolysin secretion system. Two well-characterized MV epitopes, a B-cell epitope of the MV fusion protein (amino acids 404-414) and a T-cell epitope of the MV nucleocapsid protein (amino acids 79-99) were fused as single or repeating units to the C-terminal secretion signal of the E. coli hemolysin and expressed in secreted form by the attenuated S. typhimurium aroA SL7207. Immunization of MV-susceptible C3H mice revealed that S. typhimurium SL7207 secreting these antigens provoked a humoral and a cellular MV-specific immune response, respectively. Mice vaccinated orally with a combination of both recombinant S. typhimurium strains showed partial protection against a lethal MV encephalitis after intracerebral challenge with a rodent-adapted, neurotropic MV strain.

Administration, Oral↗

Interleukin-4 induced by measles virus and measles-derived peptides as measured by IL-4 receptor-blocking ELISA.

Interleukin-4 (IL-4) is a signature cytokine for T-helper 2 (Th2) type immune responses in humans. However, data on antigen-specific secretion of IL-4 is limited due to difficulties detecting IL-4. We evaluated an IL-4 receptor-blocking assay for the detection of secreted IL-4 in peripheral blood mononuclear cells (PBMC) stimulated in vitro with measles virus (MV) and MV-derived nucleoprotein (N) and phosphoprotein (P) peptides. We recruited 20 healthy subjects, previously immunized with two doses of measles-mumps-rubella-II (MMR-II) vaccine. We evaluated the cellular and humoral immune status of these study subjects by an in vitro lymphoproliferation assay and an enzyme-linked immunosorbent assay (ELISA), respectively. We analyzed the MV-induced and N and P peptide-induced IL-4 levels in PBMC culture supernatants. Using the IL-4 receptor blocking assay, 50% of the subjects were positive for secreted IL-4 in response to MV stimulation, and 5% and 23.1% of study subjects were positive for secreted IL-4 in response to MV-derived N and P peptides, respectively. In contrast, we did not find any positive secreted IL-4 response to MV using conventional ELISA without IL-4 receptor-blocking antibody in our optimization study. Further, we found very low frequencies of IL-4 secreting cells using an alternate ELISpot technique, accounting for only a 5% positive response to MV and no response to P peptide. We propose that the IL-4 receptor-blocking assay is an easy-to-adapt technique for screening antigen-specific immune responses in large-scale population-based studies.

Adolescent↗

The matrix proteins of neurovirulent subacute sclerosing panencephalitis virus and its acute measles virus progenitor are functionally different.

Persistence of measles virus in the brains of patients with subacute sclerosing panencephalitis (SSPE) is accompanied by changes in the viral matrix (M) protein. To understand the significance of these changes, cell culture and cell-free assays were developed to compare the functions of the M proteins of an SSPE virus Biken strain and its acute measles virus progenitor Nagahata strain. The Nagahata viral M protein is associated with the intracellular viral nucleocapsids and the plasma membrane, whereas the Biken viral M protein is localized mainly in the cytosol. The lack of M protein in the Biken viral nucleocapsids is due to a failure of the Biken M protein to bind to the viral nucleocapsids. The Biken M protein also fails to bind to the Nagahata viral nucleocapsids. Conversely, the Nagahata M protein can bind to the Biken viral nucleocapsids, although this association is not as stable at physiological salt concentration. These results offer concrete evidence that the M protein of an SSPE virus is functionally different from that of its progenitor acute measles virus.

Capsid↗

Characterization of measles viruses isolated after measles vaccination.

Seven measles virus (MV) strains were isolated from children who developed clinical signs of fever and rash 3-9 days after measles vaccination. The nucleotide sequence of the H gene, the molecular size of the H protein, the haemadsorption activity on African green monkey red blood cells, and antigenicity as determined by virus neutralization revealed that one strain was of the vaccine type and the remaining six were the wild virus type. Isolation of the virus directly from patients suspected of a vaccine-induced side-reaction and subsequent characterization of such isolated virus may be useful in differentiation between vaccine-induced side-reactions and natural measles.

Antibodies, Monoclonal↗

Leucocyte migration inhibition test as an index of immunological response to measles virus I. Effect of the environment and measles virus on migration of monkey leucocytes.

The parameters of migration inhibition test of leucocytes isolated both from the peripheral blood, spleen and lymph nodes of monkeys Cercopithecus aethiops were determined. Concentration of measles virus that does not produce nonspecific migration inhibition of leucocyte obtained from sero-negative animals was also defined.

Animals↗