Measles Virus Peptides Research Articles

Immune alterations in subacute sclerosing panencephalitis reflect an incompetent response to eliminate the measles virus.

In subacute sclerosing panencephalitis (SSPE) the persistence of measles virus (MeV) may be related to the altered immune response. In this study, cytokine responses of lymphocytes and monocytes were evaluated in SSPE compared to controls with non-inflammatory (NICON) and inflammatory (ICON) diseases. Patients with SSPE (n = 120), 78 patients with ICON and 63 patients with NICON were included in this study. Phenotypes of peripheral blood mononuclear cells (PBMC) have been analyzed by flow cytometry. CD3 and CD28, and S. aureus Cowan strain I (SAC) stimulated and unstimulated cells were cultured and IL-2, IL-10, IFN-γ, IL-12p40, IL-12p70 and IL-23 were detected in supernatants by ELISA. MeV peptides were used for MeV-specific stimulation and IFN-γ secretion of PBMC was measured by ELISPOT. Spontaneous and stimulated secretions of IL-10 were lower in SSPE compared to both control groups. T cell stimulation induced lower IFN-γ production than ICON group, but higher IL-2 than NICON group in SSPE. Stimulated PBMC produced lower IL-12p70 in SSPE and had decreased CD46 on the cell surface, suggesting the interaction with the virus. IFN-γ responses against MeV peptides were not prominent and similar to NICON patients. The immune response did not reveal an inflammatory activity to eliminate the virus in SSPE patients. Even IL-10 production was diminished implicating that the response is self-limited in controlling the disease.

Role of the Th1 and Th17 Pathway in Subacute Sclerosing Panencephalitis

Subacute sclerosing panencephalitis (SSPE) is a progressive and fatal disease caused by reactivation of a mutated measles virus in brain tissue. The process of reactivation is yet to be elucidated. In this study, the possible roles of the Th1 (interleukin [IL]-12, interferon [IFN]-γ) and the Th17 axis (IL-23, IL-17, IL-22), particularly of IL-17, in the pathogenesis of SSPE were investigated. Briefly, mononuclear cells from SSPE patients were stimulated using measles virus peptide, and the release of IL-12, IL-23, IL-22, IFN-γ, and IL-17 cytokines was measured using enzyme-linked immunosorbent assay and/or enzyme-linked immunosorbent spot assay (ELISpot). We found that in comparison to the mononuclear cells obtained from healthy donors, cells from SSPE patients exhibited increased levels of IL-12, IL-23, IL-17, IL-22, and IFN-γ cytokines in response to measles virus stimulation. However, the same result was not obtained with cytomegalovirus and phytohemagglutinin. Using flow cytometry, mononuclear cells obtained from SSPE patients and healthy controls were also analyzed for the presence of intracellular IL-17 in response to measles virus stimulation. On stimulation, the number of IL-17-positive cells were found to be higher among mononuclear cells obtained from the patients. In addition, the numbers of IL-17- and IFN-γ-positive cells were significantly increased in SSPE patients. In conclusion, this study demonstrates that both the IL-12/IFN-γ and the IL-23/IL-17/IL-22 pathways are functionally abnormal in SSPE pathogenesis. Targeting these pathways and their specific pro-inflammatory mediator production may provide a new strategy to suppress SSPE development.

IFN-gamma response against measles virus peptides in subacute sclerosing panencephalitis patients

IFN-gamma response against measles virus peptides in subacute sclerosing panencephalitis patients

Importance of HLA-DQ and HLA-DP polymorphisms in cytokine responses to naturally processed HLA-DR-derived measles virus peptides

We studied the association between class II human leukocyte antigen (HLA)-DRB1*0301 presented measles virus (MV) peptide-specific cytokine responses and DQB1 and DPB1 alleles among 313 individuals who received two doses of measles–mumps–rubella-II vaccine. The overall median IFN-γ secretion levels (first and third quartiles) for the 19-amino acid MV phosphoprotein (MV-P)- and 14-amino acid MV nucleoprotein (MV-N)-derived peptides were 27.7 pg/ml (1.8, 109.4) and 1.9 pg/ml (−6.2, 13.0), respectively; median IL-4 secretion levels were −0.6 pg/ml (−7.1, 6.2) and 2.4 pg/ml (−3.2, 9.3), respectively. Primary statistical analyses were adjusted for previously identified DRB1 associations. A marginally significant increase in the frequency of the DQB1*0604 ( p = 0.02) allele was found among subjects who demonstrated detectable IL-4 levels to the MV-P peptide. Further, DPB1*0201 ( p = 0.02) and DPB1*1301 ( p = 0.09) alleles provided suggestive evidence of an association with MV-P-induced IL-4 secretion. Examination of IFN-γ responses to MV-P and MV-N indicated that none of the individual alleles of the DQB1 and DPB1 loci were associated with peptide-induced T cell response. An increase in the frequency of DPB1*0501 ( p = 0.01) was found among subjects who failed to produce MV-N peptide-specific IL-4 responses. These data further confirm that HLA-DRB1 alleles are the major restriction molecules for MV-P and MV-N measles virus antigen presentation to T cells. We speculate that MV-P and MV-N peptides derived from DRB1*0301 could potentially be recognized in association with different HLA molecules, including DQB1 and DPB1; however, statistical adjustments for the effect of HLA-DR locus could potentially alter these genetic relationships. This concept provides important information supporting the use of promiscuous peptides in a peptide-based vaccine approach.

Stable Isotope Tagging of Epitopes

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.

Identification of Class II HLA-DRB1*03-Bound Measles Virus Peptides by 2D-Liquid Chromatography Tandem Mass Spectrometry

Two-dimensional liquid chromatography (2D-LC), combined with gas phase fractionation tandem mass spectrometry, was used to identify 13 naturally processed peptides originating from measles virus that were presented by HLA-DRB1*03 class II molecules. The peptides are from three of the six measles structural proteins: phosphoprotein, nucleocapsid, and hemagglutinin. These peptides provide an important first step toward understanding the mechanism of immune response to measles virus and development of a new generation of peptide-based vaccines.

Naturally processed measles virus peptide eluted from class II HLA-DRB1*03 recognized by T lymphocytes from human blood

This is the first report of the direct identification of a HLA-DRB1*03 measles-derived peptide from measles virus infected EBV-transformed B cells. We purified HLA-DR3-peptide complexes from EBV-B cells infected with measles virus (Edmonston strain) and sequenced the HLA-DR3-peptides by mass spectrometry. A class II peptide, derived from a measles phosphoprotein, ASDVETAEGGEIHELLRLQ (P1, residues 179–197), exhibited the capacity to stimulate peripheral blood mononuclear cells to proliferate. Our data provides direct evidence that the antigenic peptide of measles virus was processed by antigen-presenting cells, presented in the context of HLA class II molecules, and was recognized by peripheral blood T cells from healthy individuals previously immunized with measles vaccine. The approach described herein provides a useful methodology for the future identification of HLA-presented pathogen-derived epitopes using mass spectrometry. The study of cell-mediated immune responses to the measles-derived peptide in immune persons should provide significant insight into the design and development of new vaccines.

A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.

We studied the natural MHC class I display of measles virus (MV) epitopes. Peptide ligands associated with HLA-A*0201 were purified from a B lymphoblastoid cell line prior to and after infection with MV. Infection-induced peptides were revealed using microcapillary reversed phase high performance liquid chromatography electrospray ionization/mass spectrometry (microLC-ESI/MS) by subtraction of the "infected" and "uninfected" ion traces. Three naturally processed viral epitopes derived from different MV proteins were identified through tandem MS sequencing. These peptides were expressed at widely divergent levels of HLA-peptide complexes, but had similar binding capacities to HLA-A*0201. The most abundant viral peptide species, identified as residues 84-92 (KLWESPQEI) of the MV nonstructural C protein, was expressed at an unprecedented high density (> 10(5) copies per cell) and was immunogenic in HLA-A2/Kb-transgenic mice. Furthermore, natural mutants of this epitope, occurring in persistent lethal MV strains, were shown to have lost their HLA-A*0201 binding capacity. Thus, here we report for the first time the direct discovery through microLC-ESI/MS of a uniquely dominant viral HLA class I ligand, KLWESPQEI, with features eligible for immune selection pressure.

Neonatal and early life immune responses to various forms of vaccine antigens qualitatively differ from adult responses: predominance of a Th2-biased pattern which persists after adult boosting.

Induction of neonatal immune responses to vaccine antigens is believed to be of limited efficacy because of immune immaturity and particular susceptibility to tolerogenic signals during this period of life. To characterize particular features of neonatal immune responses to vaccine antigens, we assessed the capacity of BALB/c mice at different stages of immunological maturation to respond to a selection of vaccine antigens and presentation systems. Significant B and T cell responses to vaccine antigens (tetanus and measles virus peptides, tetanus toxoid, live viral attenuated measles virus, canarypox recombinant measles vector or bacillus Calmette-Guérin) were obtained as early as the first week of life. However, these neonatal responses differed qualitatively from adult responses by a decreased IgG2a/IgG1 ratio of vaccine-specific antibodies, the secretion of significantly higher interleukin-5 and lower interferon-gamma levels by vaccine-specific T cells and an impaired induction of cytotoxic T cell precursors. This pattern of biased Th2 versus Th1 responses induced upon early exposure to vaccines was not reversed by decreasing the doses of vaccine antigens. It did not disappear with aging and was still reflected in adult responses to booster immunization with the corresponding antigen. Thus, neonatal immunization can induce significant vaccine specific responses with a predominance of a Th2 pattern which can persist in boosted adult mice.