Cytotoxic antibody activity in measles and subacute sclerosing panencephalitis (SSPE) infection

Using the release of [⁵¹Cr] from measles virus carrier cells, cytotoxic antibodies were titrated in the sera and cerebrospinal fluid of subacute sclerosing panencephalitis (SSPE) patients and in the sera of individuals having recovered from natural measles virus infection. A similar range of titers was present in SSPE sera and in sera from individuals within three months of measles virus infection. Cerebrospinal fluid from SSPE patients and late measles sera contained lower cytotoxic antibody titers. An area of little cytotoxicity was present at low dilutions of some sera. Absorption of sera with the viral hemagglutinin produced by Tween-80 ether treatment of measles virus demonstrated the presence of cytotoxic antibodies directed against both the hemagglutinin and another antigen of measles virus.     

Western blot analyses of measles virus antibody in normal persons and in patients with multiple sclerosis, subacute sclerosing panencephalitis, or atypical measles.

A version of the Western blot was developed to detect serum antibodies against measles virus polypeptides. With this technique, a seroepidemiological survey of antibodies to the several measles virus proteins in diverse measles-related conditions was conducted. The sera were obtained from individuals with a recent or long-past history of natural measles, from persons with a history of immunization with live attenuated measles vaccine, and from patients with multiple sclerosis, subacute sclerosing panencephalitis, or atypical measles. The findings indicated that live attenuated measles vaccine elicits an antibody response qualitatively resembling that of a natural infection. In addition, multiple sclerosis patients made less antibody to the measles virus M protein than did individuals with a long-past history of natural measles. Thus, the immunological reaction of multiple sclerosis patients to measles virus is qualitatively, as well as quantitatively, different from that of normal persons. Finally, persons with subacute sclerosing panencephalitis and atypical measles mounted abnormally high antibody responses to measles virus polypeptides, in particular the P protein.     

Measles antibodies in patients with various types of measles infection

Summary Antibodies against measles antigens were titrated in the sera of 116 patients by means of complement-fixation and hemagglutination-inhibition tests; in the latter test, whole virions and Tween-ether split virions were used comparatively. The antibody titers were low in the 4 cases of measles encephalitis and in an important proportion of those cases of measles which were accompanied by mild fever. Most of the measles patients with sustained temperature had high antibody titers, as well as the 8 cases of modified measles which occurred in children previously vaccinated with inactivated virus. Sera from some of the latter cases differed from all the other sera studied by a high ratio of complement-fixation to hemagglutination-inhibition titers. In nearly all sera, the hemagglutination-inhibition titers were higher by a factor 4–8 when the split antigen was used instead of the whole virions, but this factor was increased to 50–62 in 5 of the 8 cases of subacute sclerosing panencephalitis (Van Bogaert's leucoencephalitis).     

Presence of neutralizing antibody to canine distemper virus in sera of patients with subacute sclerosing panencephalitis

Summary Sera collected from three patients with subacute sclerosing panencephalitis (SSPE) at various stages of the disease were demonstrated to contain remarkably high levels of neutralizing antibody to canine distemper virus in proportion to the well known high antibody titers against measles virus. In contrast, neutralizing antibody to canine distemper virus was detected only at low titer in sera of convalescents from natural measles, or of measles with or without atypical measles symptom following vaccination, as well as in sera of children vaccinated with live or killed measles vaccine. Anti-measles sera prepared in various experimental animals also contained neutralizing antibody to canine distemper virus only at low titer.The significance of these findings is discussed in relevance to the possible involvement of canine distemper virus in the pathogenesis of SSPE.     

Immunological study of the agent responsible for subacute sclerosing panencephalitis and biochemical characterization of measles antibody in subacute sclerosing panencephalitis

Antibody of restricted heterogeneity produced in high titers in response to a given antigen is commonly observed in patients affected with subacute sclerosing panencephalitis and is comparable to what is observed in rabbits hyperimmunized with bacterial vaccines. Subacute sclerosing panencephalitis immunoglobulins were isolated and partially sequenced. Their immunological activity was measured with measles virus, subacute sclerosing panencephalitis virus (LEC strain) and distemper virus.     

Do antibodies to myelin basic protein isolated from multiple sclerosis cross-react with measles and other common virus antigens?

Immunological activity to various antigens, including brain components, measles and other viruses, has been associated with IgG in multiple sclerosis (MS). One possible explanation for the presence of anti-viral antibodies and antibody to myelin basic protein (MBP) in MS patients is that there are antigenic determinants common to certain viruses and MBP. To assess this possibility, IgG from individual brains and sera from patients with MS, subacute sclerosing panencephalitis (SSPE) and controls was isolated by protein A and MBP-Sepharose affinity chromatography. Antibody to MBP was measured with a solid phase radioimmunoassay and antibody to measles and other viruses by immunofluorescence and/or complement fixation. Anti-MBP activity was detected in brain extracts and sera of all MS patients tested. In contrast to the low levels of antibody to MBP in control brains, high levels of anti-MBP antibodies were found in most of the normal sera. There was no correlation between the presence and levels of serum anti-measles antibodies and the anti-MBP activity. None of the anti-MBP antibodies affinity purified from brain and serum of MS patients reacted with any of the viruses tested, including measles. IgG purified from SSPE patients or from a rabbit hyperimmunized with measles antigen had no reactivity to MBP, despite high levels of anti-measles antibody. It is concluded that there is not direct link between the presence of antibody to MBP and antibody to measles and other viruses in MS patients.     

Identification of Different Measles Virus-Specific Antibodies in the Serum and Cerebrospinal Fluid from Patients with Subacute Sclerosing Panencephalitis and Multiple Sclerosis

Matched serum and cerebrospinal fluid (CSF) samples from eight cases of subacute sclerosing panencephalitis (SSPE) and 15 cases of multiple sclerosis (MS) were characterized in neutralization, hemolysis-inhibition (HLI), hemagglutination-inhibition (HI) with Tween 80—ether-treated antigen, complement-fixation (CF), and immunodiffusion tests. CF tests were carried out with crude virus material, purified nucleocapsids, and small particle hemagglutinin as antigens. A certain diversity in the relative content of antibodies against different virus products in various sera was found. There was a high degree of correlation between titers of neutralizing and HLI antibodies, but a less strict correlation between titers of HLI and HI antibodies. Serum samples from two cases of MS and one case of SSPE contained high titers of HLI and neutralizing antibodies in the presence of only low titers of HI antibodies demonstrable with Tween 80—ether-treated antigen. The major fraction of antibodies detected in CF and immunodiffusion tests reacted with nucleocapsids. There was a tendency of nucleocapsid CF antibody titers, as compared to neutralization and HLI antibody titers, to be higher in samples from patients with SSPE than from cases of MS. No significant differences were found between antibody titers recorded in neutralization, HLI, and HI tests carried out with two different measles virus strains, Edmonston and a strain (LEC) derived from a case of SSPE. Comparison of antibodies against measles virus products and, as a reference, against a group-specific vertex capsomer antigen of adenovirus in matched serum and CSF samples revealed a production of measles virus-specific antibodies within the central nervous system of all cases of SSPE and 8 out of 15 cases of MS.     

The significance of hemolysing-inhibiting antibodies in protection against measles

The occurence of hemagglutinating-inhibiting (HI), hemolysinginhibiting (HLI) and nucleocapsid complement fixing (NC-CF) antibodies in serum samples from individuals immunized with measles virus antigens under various conditions was analyzed. After regular measles infections all three kinds of antibodies were detectable and removal of HI antibodies by absorption with Tween 80 and ether treated virus material only caused an about 2-fold reduction of titers of HLI antibodies. Immunization with further attenuated measles vaccine also induced a production of antibodies detectable in the three different tests. However the response of non-HI HLI antibodies and NC-CF antibodies was relatively less pronounced than after regular measles. Immunization with three or four doses of Tween 80 ether treated inactivated measles vaccine caused a production of HI antibodies and in some cases NC-CF antibodies. Non-HI HLI antibodies were not detectable. The combined immunization with inactivated vaccine followed by further attenuated live vaccine caused the appearance of high titers of HI antibodies but in contrast to the situation of administration of only live vaccine non-HI HLI antibodies were not detectable. The significance of non-HI HLI antibodies in protection against disease also was indicated by observations on the reactions of individuals immunized with inactivated vaccine and subsequently exposed to wild measles virus. The non-HI HLI antibody response was poorer in the vaccinees who contracted a complicated infection as compared to those who had only a subclinical or a mitigated infection. An accentuated non-HI HLI antibody and NC-CF antibody response was seen in patients with subacute sclerosing panencephalitis. It is proposed that the failure of hitherto used inactivated measles vaccines is due to the absence of certain envelope antigens in the preparations which leads to a deficiency as concerns the capacity to induce a production of non-HI HLI antibodies.     

Expression of five viral antigens in cells infected with wild-type and SSPE strains of measles virus: Correlation with cytopathic effects and productivity of infections

Summary Cells infected with four strains (LEC, Biken, IP-3 and DR) of subacute sclerosing panencephalitis (SSPE) virus were compared with wild type measles virus (Edmonston) with respect to titers of extracellular virus, morphology of the cytopathic effect (CPE) and occurrence of different measles virus antigens within infected cells as determined by immune fluorescence. Murine monoclonal antibodies with specifities for the nucleocapsid (NP), polymerase (P), matrix (M), hemagglutinin (H), and fusion (F) proteins as well as specific hyperimmune sera prepared in rabbits against the NP, H and M proteins were used in immune fluorescence analyses of the various strains. All of the strains produced large amounts of NP and P. Only the NP antigen occurred in nuclei of cells. The Edmonston and LEC strains also showed bright fluorescence with the antibodies against the H, F, and M antigens. Immune fluorescent intensity was variably reduced in cells infected with the Biken, IP-3, and DR strains labelled with anti M, H, or F antibodies. The Biken strain produced moderate titers of extracellular virus and moderate amounts of M, H, and F antigens whereas the DR strain produced no extracellular virus and contained no detectable M or F and only trace amounts of H antigen. The IP-3 strain was intermediate both in antigen expression and in production of extracellular virus.     

Oligoclonal Measles Virus-Specific IgG Antibodies Isolated from Cerebrospinal Fluids, Brain Extracts, and Sera from Patients with Subacute Sclerosing Panencephalitis and Multiple Sclerosis

Measles virus-specific antibodies were isolated from sera, cerebrospinal fluids (CSF), and brain extracts of patients with subacute sclerosing panencephalitis (SSPE) and multiple sclerosis (MS) by absorption with measles antigens and subsequent acid elution of the antigen–antibody precipitates. Electrophoretically homogeneous measles antibodies were isolated from CSF or brain extracts in five patients with SSPE and in five out of seven patients with MS. Homogeneous IgG antibodies were also demonstrated in the sera from all SSPE patients and from three of the MS patients. The antibodies isolated from various control sera and from pooled CSF were electrophoretically heterogeneous. The results support the concept of a local synthesis in the nervous system of oligoclonal IgG antibodies to measles virus in all patients with SSPH and in some patients with MS. In SSPE, most or all oligoclonal IgG proteins of the CSF or brain carry measles antibody activities. In MS, only part of the oligoclonal IgG appears to be associated with measles antibody activity     

Immunoglobulin G subclass antibodies to measles virus in patients with subacute sclerosing panencephalitis or multiple sclerosis

Mouse monoclonal antibodies specific for human immunoglobulin G (IgG) subclasses and a sensitive immunoassay were used to evaluate the IgG subclass antibody response to measles virus antigens in cerebrospinal fluid and serum samples from 20 patients with subacute sclerosing panencephalitis (SSPE), 12 patients with multiple sclerosis (MS), and 11 controls with high measles virus antibody titers in serum. In patients with SSPE, measles virus-specific antibodies were found mainly in the IgG1 subclass and the IgG subclass distribution remained unchanged, irrespective of the clinical stage or duration of the disease. In patients with MS and in controls, measles virus activity was also associated mainly with IgG1. However, the activity was significantly lower than that found in patients with SSPE. The results suggest that there is no primary abnormality in humoral immune response to measles virus in patients with MS. The disproportionately high levels of the measles virus-specific IgG1 subclass found in patients with SSPE may be due to persistent antigenic stimulation or reflect a defect in immunoregulatory mechanisms in response to viral infection.     

Radioimmunoassay of measles virus antibodies in SSPE

A sensitive radioimmunological assay (RIA) was introduced for detection of measles virus IgG and IgM antibodies. The hyperimmune response to measles virus could be demonstrated more precisely by RIA than by haemagglutination inhibition (HI). The ratio between RIA and HI antibody titres was decidedly higher in sera and cerebrospinal fluids (CSF) of patients with subacute sclerosing panencephalitis (SSPE) than in those of other groups tested.     

Evidence for lack of synthesis of the M polypeptide of measles virus in brain cells in subacute sclerosing panencephalitis

The synthesis of measles virus polypeptides in cells in their fifth passage after removal from the brain of a patient with subacute sclerosing panencephalitis (SSPE) has been investigated using immunoprecipitation techniques. Although the other major virion polypeptides were found, synthesis of the M polypeptide was not detected. High titers of antibodies to the other virion proteins were found in the cerebrospinal fluid, as well as the serum, of a patient with SSPE, but only a trace of antibody to the M polypeptide was found at the lowest dilutions. These results, and the earlier finding of the lack of antibody to M in the sera of many patients with SSPE, support the previous suggestion that there is decreased synthesis of the M polypeptide in the cells of the brain in patients with SSPE.     

Measles antibodies detected by platelet aggregation and gel precipitation in patients with subacute sclerosing panencephalitis

Summary Two serological techniques previously developed in our laboratories, platelet aggregation (PA) and gel precipitation (GP), are introduced as methods for detecting measles antibodies in patients with subacute sclerosing panencephalitis (SSPE). In PA tests of 11 SSPE patients high serum titers and moderate cerebrospinal fluid (CSF) titers were found. In GP tests all the sera formed 3 to 6 lines and CSF 1 to 2 lines against measles antigens. In 6 out of 50 post-measles sera positive PA reactions were found and only weak GP reactions with 1 to 3 lines were seen in tests on 37 sera studied.In post-measles sera the greatest number of positive reactions in both tests were obtained when more than one month had elapsed from the onset of the disease. The results of PA and GP tests correlated well with the results of complement fixation tests in sera and CSF of patients with SSPE, but no clear correlation was observed in post-measles sera.     

A study of immunoglobulin M antibody to measles, canine distemper, and rinderpest viruses in sera of patients with subacute sclerosing panencephalitis.

Seven boys were studied who had the clinical features of subacute sclerosing panencephalitis (SSPE) and whose brain histology was consistent with SSPE. Measles antigen was detected in the seven brains by the direct fluorescent antibody method. Three out of the seven boys had in their sera measles specific immunoglobulin M (IgM) which was detected by the indirect fluorescent antibody method, and the cell receptors for it were acetone stable. A prozone effect was noted in the sera of two patients. The absorption of one patient's serum with Staphylococcus aureus to reduce the titre of immunoglobulin G (IgG) removed the prozone effect. Two of the boys who had high titres of measles specific IgM also had serum IgM which reacted with canine distemper virus antigen but the titres were eightfold lower. None of the boys had detectable rinderpest specific IgM in their sera.     

Cells infected with a cell-associated subacute sclerosing panencephalitis virus do not express M protein

Using immunoprecipitation techniques, we have analyzed measles-specific antibodies in the cerebrospinal fluids (CSF) as well as in sera from patients with subacute sclerosing panencephalitis (SSPE). We confirm previous reports that SSPE sera contain a relative lack of M protein-specific antibodies, and report that CSF also have antibodies specific for all measles polypeptides except the M protein. We have also found that Vero cells infected with one of the SSPE viruses used in this study, the cell-associated IP-3 strain, completely lack expression of detectable M protein. Thus, the lack of M protein-specific antibody in the CSF of SSPE patients may be the result of a lack of expression of this polypeptide in the cells of the central nervous system.     

Immunological studies of subacute measles encephalitis in ferrets: similarities to human subacute sclerosing panencephalitis.

Ferrets inoculated with subacute sclerosing panencephalitis virus strains D.R. and Biken developed a subacute encephalitis. Brain extracts, at neutral pH, from these ferrets showed high measles antibody titers, increased concentrations of immunoglobulin G (IgG), and higher IgG/albumin ratios than those of controls. Although the brain extracts of subacute encephalitic animals showed significant synthesis of measles-specific IgG (20 to 60% of the total IgG) within the central nervous system, the electrophoretic patterns of these extracts did not show oligoclonal bands in the gamma-globulin region. Brain residues from most ferrets with subacute encephalitis, when eluted at low pH, demonstrated the presence of bound measles-specific antibodies. Excluding the electrophoresis data, other results are identical to those seen in human subacute sclerosing panencephalitis, indicating that the subacute encephalitis in ferrets may serve as a model for human subacute sclerosing panencephalitis.     

Precipitation of measles virus proteins by immunoglobulin G fractions containing groups of oligoclonal bands isolated from sera of patients with subacute sclerosing panencephalitis.

Groups of oligoclonal immunoglobulin G (IgG) bands were isolated from sera of patients with subacute sclerosing panencephalitis by employing preparative isoelectric focusing. Six IgG fractions containing two to three oligoclonal bands with different isoelectric points were used to precipitate the proteins from Vero cells infected with measles virus. The results showed that all of the measles virus proteins except the M protein were precipitated by all of the IgG fractions and that the precipitation of viral proteins by the fractions containing groups of oligoclonal IgG showed slightly different patterns in some sera, whereas other sera showed no significant differences. The present study indicates that oligoclonal IgGs in subacute sclerosing panencephalitis sera are not specific to individual measles virus proteins.     

A study of immunoglobulin M antibody to measles,canine distemper and rinderpest viruses in sera of patients with subacute sclerosing panencephalitis

Seven boys were studied who had the clinical features of subacute sclerosing panencephalitis (SSPE) and whose brain histology was consistent with SSPE. Measles antigen was detected in the 7 brains by the direct fluorescent antibody method. Three out of the 7 boys had in their sera measles specific immunoglobulin M (IgM) which was detected by the indirect fluorescent antibody method and in 2 of these sera a prozone effect was noted. Two of the boys who had high titres of measles specific IgM also had serum IgM which reacted with canine distemper virus antigen, but the titres were 8 fold lower. None of the boys had detectable rinderpest specific IgM in their sera.Presented at the workshop on molecular and pathogenetic aspects of measles virus, 9./11. April 1974, Belfast, Northern Ireland.     

A rapid immunoperoxidase assay for determination of IgG antibodies to measles virus

A new indirect peroxidase antibody to membrane antigen (IPAMA) technique for the detection of IgG specific antibodies against measles virus is described. The technique utilizes as antigen measles-infected Vero cells dried on glass slides and stored at −70°C. Sera of 50 healthy medical students and laboratory workers and 24 sera of measles, encephalitis, and subacute sclerosing panencephalitis (SSPE) patients were checked by IPAMA and the results have been compared with the results obtained by the hemaglutination-inhibition (HI) test. There is good agreement between the results of both techniques as to the presence or absence of antibody in 48 out of the 50 tested. The advantages of the techniques are discussed.