Interpretation of the results of identification of measles virus antibodies in the serum and cerebrospinal fluid from patients with suspected subacute sclerosing panencephalitis (SSPE)]

Clinical diagnosis of subacute sclerosing panencephalitis+ (SSPE) requires laboratory confirmation relying on an evaluation of immune response in serum and in cerebrospinal fluid (CSF) to measle virus. In our study a comparison of antibody level for this virus by ELISA and haemagglutination inhibition test was made in materials derived from 1396 patients with SSPE, sclerosis multiplex, acute measles++ neuroinfections and other diseases of central nervous system (CNS). Statistical analysis permitted to settle criteria for differentiation of SSPE from remaining diseases of CNS and usefulness for diagnosis of particular methods as well as analysis of results obtained with single sample of serum or CNS. An advantage of ELISA for diagnostic purposes and CSF samples were confirmed.     

Screening random peptide libraries with subacute sclerosing panencephalitis brain-derived recombinant antibodies identifies multiple epitopes in the C-terminal region of the measles virus nucleocapsid protein

Infectious and inflammatory diseases of the CNS are often characterized by a robust B-cell response that manifests as increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands. We previously used laser capture microdissection and single-cell PCR to analyze the IgG variable regions of plasma cells from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Five of eight human IgG1 recombinant antibodies (rAbs) derived from SSPE brain plasma cell clones recognized the measles virus (MV) nucleocapsid protein, confirming that the antibody response in SSPE targets primarily the agent causing disease. In this study, as part of our work on antigen identification, we used four rAbs to probe a random phage-displayed peptide library to determine if epitopes within the MV nucleocapsid protein could be identified with SSPE brain rAbs. All four of the SSPE rAbs enriched phage-displayed peptide sequences that reacted specifically to their panning rAb by enzyme-linked immunosorbent assay. BLASTP searches of the NCBI protein database revealed clear homologies in three peptides and different amino acid stretches within the 65 C-terminal amino acids of the MV nucleocapsid protein. The specificities of SSPE rAbs to these regions of the MV nucleocapsid protein were confirmed by binding to synthetic peptides or to short cDNA expression products. These results indicate the feasibility of using peptide screening for antigen discovery in central nervous system inflammatory diseases of unknown etiology, such as multiple sclerosis, neurosarcoidosis, or Behcet's syndrome.     

Subacute sclerosing panencephalitis and multiple sclerosis: in vitro measles immunity and sensitization to myelin basic protein.

Three children with subacute sclerosing panencephalitis (SSPE), 12 patients with multiple sclerosis (MS) and 12 healthy persons were studied by the macrophage migration inhibition factor (MIF) assay with measles and rubella antigens and with myelin basic protein. For the SSPE patients the mean migration indexes +/- standard deviation were 44.1 +/- 10.9 for measles antigen, 38.7 +/- 12.3 for rubella antigen and 49.8 +/- 25.7 for myelin basic protein; for the MS patients the indexes were 103.0 +/- 10.6, 93.8 +/- 15.0 and 89.3 +/- 19.9; and for the healthy subjects the indexes were 68.8 +/- 22.6, 77.7 +/- 31.3 and 100.1 +/- 6.5. The results of this study showed increased cellular immunity to measles and rubella in SSPE patients as compared with healthy persons, and absence of immunity to measles in MS patients. Patients with MS showed hypersensitivity to myelin basic protein during clinical exacerbations that was not associated with changes in immunity to measles, whereas all SSPE patients showed a significant response regardless to stage of illness.     

Detection of IgG antibody to measles virus by radioimmunoassay technique

A highly sensitive procedure of solid-phase radioimmunoassay (RIA) was developed for the detection of measles IgG antibody. HeLa cells persistently infected with measles virus were used as a solid-phase antigen. This technique was applied to the detection of measles IgG antibody in patients with subacute sclerosing panencephalitis (SSPE) and multiple sclerosis. Normal subjects having experienced natural measles or measles vaccination and patients with various neurological diseases of non-virus nature were also examined as control groups. Measles antibody was detected at high titers in both the sera and cerebrospinal fluid of SSPE patients. Moreover, RIA/HI ratios of SSPE patients were significantly higher than those of normal subjects, suggesting the presence in the formers of antibodies to nucleocapsids at high titers as well as to viral envelopes. On the other hand, no significant difference was found in both RIA and HI titers between the sera of multiple sclerosis and those of various neurological diseases.     

Immune response in subacute sclerosing panencephalitis: reduced antibody response to the matrix protein of measles virus.

Immune precipitation was used to study the humoral immune response of patients with subacute sclerosing panencephalitis (SSPE). Patients with SSPE have a progressive infection of the CNS by measles or a measles variant despite high serum antibody levels to measles virus as measured by standard serologic techniques. However, when the antibody response to individual measles virus proteins was measured, we found a striking reduction in the ability of sera from patients with SSPE to precipitate the matrix (M) protein as compared to the precipitation of the M protein by sera from normal adults who had natural measles infection in childhood, or by convalescent sera obtained 3 to 5 weeks after a naturally occurring measles infection. The decreased antibody response to the M protein in sera from patients with SSPE occurred despite a vigorous antibody response to the other viral proteins, suggesting a selective defect in the production of antibody to a single viral protein. The reduced anti-M antibody in sera from patients with SSPE was demonstrated whether immune precipitation was performed with wild-type measles virus or SSPE virus proteins. These results suggest that in SSPE only small amounts of the M protein are produced. This result may help explain how measles virus persists in the central nervous system of patients with SSPE.     

Humoral and cellular immune responses to matrix protein of measles virus in subacute sclerosing panencephalitis.

The immune response to matrix (M) protein of measles virus was examined in patients with subacute sclerosing panencephalitis (SSPE) and controls. Antibodies specific for M and nucleocapsid (NC) proteins in 11 serum and 8 cerebrospinal fluid (CSF) samples from patients with SSPE were quantitated by enzyme-linked immunosorbent assay by using affinity-purified measles virus proteins. Geometric mean anti-NC antibody titers were higher in the serum (6.58 +/- 0.98 [mean +/- standard deviation]) and CSF (4.38 +/- 0.74) of SSPE patients compared with controls. Anti-M antibodies were present in the serum and CSF of all SSPE samples tested but in titers lower than those of anti-NC antibodies. Geometric mean anti-M antibody titer was 3.35 +/- 0.53 in sera from patients with SSPE compared with 3.05 +/- 0.66 in sera from patients with other neurological diseases and 3.12 +/- 0.74 in sera from healthy individuals. Geometric mean anti-M antibody titer was 2.59 +/- 0.86 in the CSF of eight patients with SSPE compared with a mean less than 1.00 for patients with other neurological disease (controls). Intrathecal synthesis of anti-M or anti-NC antibodies was established in four patients with SSPE. The cellular immune responses to M, F, HA, and NC proteins were examined in four of the patients with SSPE by lymphoproliferation and were not significantly different from those in five healthy controls. The results demonstrate humoral and cellular immune responses to M protein in patients with SSPE and indicate that it is unlikely that a defect in the immune response to this virus component accounts for the disease process in the patients studied.     

Elevated interleukin-12 and CXCL10 in subacute sclerosing panencephalitis

Immunosuppression associated with measles virus (MV) can be demonstrated by cytokine production failure in subacute sclerosing panencephalitis (SSPE) and may have implications on the pathogenesis of the disease. Cytokines (IL-12, IL-10, IL-4, IL-17, IL-18, IFN-alpha, IFN-gamma) and chemokines (CXCL8, CXCL10, CCL2 and CCL5) were measured in the cerebrospinal fluid (CSF) and serum samples from 60 patients with SSPE, 36 patients with infectious and/or inflammatory (IN) and 28 with other non-inflammatory (NIN) neurological diseases by ELISA. IL-12 p70+p40 was elevated in CSF and sera of SSPE when compared to the NIN group. However, the CSF levels of IL-12 p70 alone were not increased, indicating an increase of p40. The CSF of SSPE patients also showed relatively higher levels of IL-10 than that of the NIN group. CXCL10 levels in CSF were significantly higher in SSPE, whereas CXCL8 was increased in sera compared to NIN. No difference was detected in IFN-gamma, IFN-alpha, IL-17, IL-18, IL-4 or CCL2 and CCL5 levels. These results demonstrate that immune response against MV in SSPE may be impaired, although some T cell/Th1 inducing stimulations are present.     

Antigen discovery in chronic human inflammatory central nervous system disease: panning phage-displayed antigen libraries identifies the targets of central nervous system-derived IgG in subacute sclerosing panencephalitis.

The presence of increased IgG in the brains of humans with infectious and inflammatory CNS diseases of unknown etiology such as multiple sclerosis may be a clue to the cause of disease. For example, the intrathecally synthesized oligoclonal bands in diseases such as subacute sclerosing panencephalitis (SSPE) or cryptococcal meningitis have been shown to represent Ab directed against the causative agents, measles virus (MV), or Cryptococcus neoformans, respectively. Using SSPE as a model system, we developed a strategy to identify the antigenic targets of the intrathecal disease-relevant IgG in chronic human inflammatory and demyelinating diseases of the CNS. Libraries of cDNA Ags were displayed on the surface of T7Select bacteriophage and biopanned on IgG extracted from the brain of an SSPE patient, or on a monospecific recombinant Fab identified from SSPE brain. After three or six rounds of biopanning on either Ab, positive phage-displayed Ags reacting with IgG were enriched to 35-77% of all panned clones. Sequence analysis of the positive clones identified fragments of the nucleocapsid protein of MV, the cause of SSPE. The sensitivity of the system was determined by diluting the positive clones from this SSPE phage-displayed library at a ratio of 10(-6) into another phage-displayed library that did not contain any detectable MV Ags; after six rounds of panning, the positive clones comprised 34% of all phage and were also shown to be MV nucleocapsid specific. This strategy will be useful to identify potentially rare Ags in diseases of unknown cause.     

Localization of measles virus antigens in subacute sclerosing panencephalitis in ferrets

Young adult male ferrets were inoculated intracerebrally (i.c.) with a cell-associated encephalitogenic subacute sclerosing panencephalitis (SSPE) virus strain to study the pathogenesis of the disease at the ultrastructural level. Most became acutely ill in 8-13 days. Areas of the brain were examined with indirect immunoperoxidase labeling techniques to detect measles antigen. None of these animals showed the characteristic viral nucleocapsids or marked inflammatory response associated with SSPE. However, all had positive immunolabeling of unstructured virus antigen, especially in post-synaptic regions in all areas of the brain that were examined. One ferret, immunized with measles vaccine 40 days prior to challenge with SSPE, became ill 18 days post inoculation (p.i.). Perivascular cuffings of inflammatory cells and large cytoplasmic inclusions of fuzzy nucleocapsids were found in the brain and spinal cord. The study indicates that ferrets which become acutely ill after inoculation with cell-associated SSPE virus do so before there is a marked cellular immune response or formation of virus nucleocapsids.     

Protective effect of measles virus inoculation on subacute sclerosing panencephalitis virus-infected mice

The Biken strain of subacute sclerosing panencephalitis (SSPE) virus caused a fatal neurologic disease in adult mice after intracerebral inoculation. However, the mice were completely protected from the disease when a high dose of measles virus was given intracerebrally after the SSPE virus infection. The measles virus inoculation induced interferon production and immune responses. An experiment with athymic nude mice showed that interferon and anti-measles antibody were able to prolong the incubation period of the disease but not to protect the SSPE virus-infected nude mice from death. For complete protection, T lymphocytes appeared to be essential. The present study suggested that the protective effect of measles virus inoculation is basically due to the induction of immune responses and that SSPE virus infection in mice is susceptible to immune reactions.     

Cloning the antibody response in humans with inflammatory central nervous system disease: analysis of the expressed IgG repertoire in subacute sclerosing panencephalitis brain reveals disease-relevant antibodies that recognize specific measles virus antigens.

The presence of increased IgG in the brains of humans with infectious and inflammatory CNS diseases of unknown etiology such as multiple sclerosis may be a clue to the cause of disease. For example, the intrathecally synthesized oligoclonal bands (OGBs) in diseases such as subacute sclerosing panencephalitis (SSPE) or cryptococcal meningitis have been shown to represent Ab directed against the causative agents, measles virus (MV) or Cryptococcus neoformans, respectively. Using SSPE as a model system, we have developed a PCR-based strategy to analyze the repertoire of IgG V region sequences expressed in SSPE brain. We observed abnormal expression of germline V segments, overrepresentation of particular sequences that correspond to the oligoclonal bands, and substantial somatic mutation of most clones from the germline, which, taken together, constitute features of Ag-driven selection in the IgG response. Using the most abundant or most highly mutated gamma H chain and kappa or lambda L chain sequences in various combinations, we constructed functional Abs in IgG mammalian expression vectors. Three Abs specifically stained MV-infected cells. One Ab also stained cells transfected with the MV nucleoprotein, and a second Ab stained cells transfected with the MV-fusion protein. This technique demonstrates that functional Abs produced from putative disease-relevant IgG sequences can be used to recognize their corresponding Ags.     

Measles virus encephalitis in ferrets as a model for subacute sclerosing panencephalitis

Young adult ferrets were used as experimental animals to study subacute sclerosing panencephalitis (SSPE). When cells infected with cell-associated measles virus strains isolated from SSPE patients were inoculated intracerebrally (i.c.) into ferrets, they developed an acute encephalitis and died within 1 to 3 weeks without detectable antibody formation. Immunization with live measles vaccine 5 weeks before i.c. inoculation changed the course of the infection in about 50% of the ferrets. These animals developed a subacute encephalitis within weeks or months after inoculation. Cell-associated measles virus was isolated from their brains and high measles antibody titers were found in their sera, comparable to those in sera of SSPE patients. Measles virus specific immunoglobulins (IgG) were present in their brains and determination of IgG/albumin ratios indicated that antibodies were synthesized in the brain in response to the persistent measles virus infection. Measles specific oligoclonal IgG bands were found in the sera and spinal fluids of these animals. Therefore, subacute ferret encephalitis has virological and immunological characteristics in common with SSPE, indicating that it may serve as a model for the human disease. Other animal models of SSPE are described briefly.     

Identification of interferon-resistant subpopulations in several strains of measles virus: positive selection by growth of the virus in brain tissue.

Subacute sclerosing panencephalitis (SSPE) is a chronic and usually fatal central nervous system disease caused by a persistent infection with measles virus. The pathogenic mechanisms of the disease are poorly understood, but restricted expression of viral antigens within the infected tissue appears to be involved. We have previously proposed that interferon (IFN) plays a role in the pathogenesis of SSPE by interacting with viral subpopulations that are relatively resistant to IFN-mediated inhibition. Such IFN-resistant viral subpopulations have now been identified in six independent strains of measles virus, two derived from patients with measles and four derived from patients with SSPE. By means of a replicative-plating procedure, these IFN-resistant viruses were found to be heterogeneous with respect to their growth in the presence of high levels of IFN. One viral form replicates fully, with complete destruction of the infected-cell culture, whereas the other form induces a restricted, self-limited form of cytopathic effect, similar to that seen with cell-associated strains of measles virus isolated from SSPE patients. Passage of a virus stock containing both of these viral forms through the central nervous system tissue of newborn hamsters strongly selects for the viral form associated with the self-limiting type of cytopathic effect. The presence of this form of IFN-resistant virus coupled with chronic production of IFN within the central nervous system may account for viral persistence in SSPE patients.     

Epidemiology of Subacute Sclerosing Panencephalitis (SSPE) in Germany from 2003 to 2009: A Risk Estimation

Subacute sclerosing panencephalitis (SSPE) is a fatal long-term complication of measles infection. We performed an estimation of the total number of SSPE cases in Germany for the period 2003 to 2009 and calculated the risk of SSPE after an acute measles infection. SSPE cases were collected from the Surveillance Unit for Rare Paediatric Diseases in Germany and the Institute of Virology and Immunobiology at the University of Würzburg. The total number of SSPE cases was estimated by capture-recapture analysis. For the period 2003 to 2009, 31 children with SSPE who were treated at German hospitals were identified. The capture-recapture estimate was 39 cases (95% confidence interval: 29.2–48.0). The risk of developing SSPE for children contracting measles infection below 5 years of age was calculated as 1∶1700 to 1∶3300. This risk is in the same order of magnitude as the risk of a fatal acute measles infection.     

Selective killing of subacute sclerosing panencephalitis virus-infected cells by liposomes containing fragment A of diphtheria toxin

A more than 99% reduction in infectivity of cultures infected with subacute sclerosing panencephalitis (SSPE) virus was obtained by treating the cultures with liposomes containing fragment A of diphtheria toxin. In the mixed cultures of human embryonic lung (HEL) cells and SSPE virus-infected HEL cells (SSPE cells), SSPE virus in SSPE cells invaded the surrounding HEL cells successively, forming syncytial giant cells to destroy the whole cultures. By adding liposomes containing fragment A of diphtheria toxin to the mixed cultures, we were successful in killing the SSPE cells selectively and as the result of elimination of SSPE cells from the cultures, the cultures appeared to be cured by uninfected HEL cell growth.     

Multiple enhancers located in a 1-Mb region upstream of POU3F4 promote expression during inner ear development and may be required for hearing

Although the exact pathogenesis of subacute sclerosing panencephalitis (SSPE) remains to be determined, our previous data suggested a genetic contribution to the host susceptibility to SSPE. During chronic viral infection, virus-specific cytotoxic T lymphocytes display poor effector functions. Since co-inhibitory molecules are involved in the suppression of T lymphocytes, we investigated whether single nucleotide polymorphisms (SNPs) of genes encoding co-inhibitory molecules contributed to a susceptibility to SSPE. Association studies on a total of 20 SNPs in 8 genes (CTLA4, CD80, CD86, PD1, PDL1, PDL2, BTLA and HVEM) and subsequent haplotype analysis of 4 SNPs in the PD1 genes were performed in Japanese and Filipino SSPE patients and controls. Then, we investigated a functional difference in promoter activity of two haplotypes and compared the expression levels of PD1 between SSPE and controls. The frequency of GCG(C) haplotype of PD1 containing ?606G allele was significantly higher in SSPE patients than in controls both in Japanese and in Filipinos. The promoter activity was significantly higher in the construct with ?606G allele than in that with ?606A allele. The expression levels of PD1 were significantly higher in SSPE patients than in the controls. Our results suggested that the PD1 gene contributed to a genetic susceptibility to SSPE.     

Functional and nonfunctional measles virus matrix genes from lethal human brain infections.

Subacute sclerosing panencephalitis (SSPE) is a lethal disease induced by the persistence of measles virus in the human brain. In many SSPE cases, the viral matrix (M) protein cannot be detected; in others, M proteins of the expected size are found and sequence analysis of M cDNAs has confirmed that the reading frames are intact, showing only several missense mutations. To determine whether these alterations result in nonfunctional proteins, we have replaced the M gene of an infectious full-length genomic cDNA (from vaccine strain Edmonston) with different M genes derived from four patients with SSPE. One of the SSPE M genes tested proved to be functionally competent, giving rise to a virus yielding titers similar to those of viruses containing the M gene from control lytic strains. The other three SSPE M genes were not functionally competent in the same test. In all three cases, the inactivating changes resided in the carboxyl-terminal half of the M protein, as shown by the exchange of either of the two genes halves. In summary, mutational M gene alterations, which either prevent synthesis of M protein altogether or only allow synthesis of nonfunctional M protein, have been detected by us and by others in 9 of 10 SSPE cases. The one functional M gene appears to be an exception to the rule, indicating that M gene alteration might not be an absolute requirement for disease development.     

Generation of recombinant adenovirus expressing siRNA against the L mRNA of measles virus and subacute sclerosing panencephalitis virus

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by prolonged persistent infection of the central nervous system with a measles virus (MV) mutant called SSPE virus. At present, there is no effective treatment to completely cure SSPE and development of a new therapeutic measure(s) against this fatal slow virus infection is needed. We previously reported that replication of MV and SSPE virus was effectively inhibited by small interfering RNA (siRNA), either chemically synthetic or plasmid-driven ones, that were targeted against different sequences of the mRNA for the L protein of MV. In this study, we have generated recombinant adenovirus expressing the siRNAs (rAd-siRNA-MV-L2, -L4 and -L5) and demonstrated that these rAd-siRNAs efficiently inhibited replication of MV and SSPE virus in a dose-dependent manner. Due to their high capacity for gene delivery to nerve cells and the potential to inhibit SSPE virus replication, the rAd-siRNAs could be a good candidate for a novel therapeutic measure against SSPE.     

Th2-mediated host protective immunity to intestinal nematode infections.

Despite many years of study, relatively little is known about the effector mechanisms that operate against intestine-dwelling nematodes. Most of the current understanding comes from studies of laboratory model systems in rodents. It is clear that when an intestinal helminth infection takes place the immune system generates a strong Th2-mediated response, which regulates a variety of responses characteristic of helminth infections such as eosinophilia, intestinal mastocytosis and elevated IgE production. The ability to modulate the host''s immune response in vivo with cytokine-specific monoclonal antibodies and recombinant cytokines, together with the use of animals with disruption of key genes involved in the immune response, have provided powerful tools with which to dissect the potential effector mechanisms operating. In the absence of a T-cell compartment the host is unable to expel the parasite. If a Th1-dominated response is generated, protective immunity is almost universally compromised. Thus, it it would appear that some aspect of a Th2-mediated response controls effector mechanisms. Although it is clear that for some infections the mast cell appears to be involved in protection, probably through the generation of a non-specific inflammatory response, how these cells become activated remains unclear. Data from infections in transgenic animals suggest that activation is not through the high-affinity receptor for IgE. Such studies also call into doubt the importance of conventional interactions between effector leucocytes and antibody. There is little evidence to support a protective role for eosinophilia in any system. New data also imply that, although interleukin 4 (IL-4) is generally important (and can exert effects independent of an adaptive immune response), it is not always sufficient to mediate protection; other Th2 cytokines (e.g. IL-13) may warrant closer investigation. It is apparent that a number of potential Th2-controlled effector mechanisms (some of which may be particularly important at mucosal surfaces) remain to be explored. Overall, it is likely that worm expulsion is the result of a combination of multiple mechanisms, some of which are more critical to some species of parasite than to others.     

A serum protein affecting the regulation of the immune system

A thermostable alpha₂ globulin inhibiting the immunoglobulin/anti-immunoglobulin reaction was demonstrated working with subacute sclerosing panencephalitis (SSPE) and control serum IgG. This protein was isolated from SSPE and normal human blood, it inhibits the immunoglobulin/anti-immunoglobulin reaction but no other antigen/antibody reactions when applying different immunochemical methods such as nitrocellulose immunofixation, 2 site immunoradiometric assay, solid phase radioimmunoassay in coated cups. This was demonstrated, working on the one hand with measles virus strain Edmonston or SSPE virus strain D.R. and SSPE serum and on the other hand with IgG from SSPE and control serum. This alpha₂ globulin, an inhibiting protein, appears to be related to normal immunosuppressive protein.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.