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.     

First signature of islet beta-cell-derived naturally processed peptides selected by diabetogenic class II MHC molecules

The diversity of Ags targeted by T cells in autoimmune diabetes is unknown. In this study, we identify and characterize a limited number of naturally processed peptides from pancreatic islet beta-cells selected by diabetogenic I-A(g7) molecules of NOD mice. We used insulinomas transfected with the CIITA transactivator, which resulted in their expression of class II histocompatibility molecules and activation of diabetogenic CD4 T cells. Peptides bound to I-A(g7) were isolated and examined by mass spectrometry: some peptides derived from proteins present in secretory granules of endocrine cells, and a number were shared with cells of neuronal lineage. All proteins to which peptides were identified were expressed in beta cells from normal islets. Peptides bound to I-A(g7) molecules contained the favorable binding motif characterized by acidic amino acids at the P9 position. The draining pancreatic lymph nodes of prediabetic NOD mice contained CD4 T cells that recognized three different natural peptides. Furthermore, four different peptides elicited CD4 T cells, substantiating the presence of such self-reactive T cells. The overall strategy of identifying natural peptides from islet beta-cells opens up new avenues to evaluate the repertoire of self-reactive T cells and its role in onset of diabetes.     

Characterization of naturally processed and presented peptides associated with bovine major histocompatibility complex (BoLA) class II DR molecules

Differential regulation of genetic resistance to infectious disease may partially be explained by variation in the binding affinity and the repertoire of pathogen-derived antigenic peptides associated with major histocompatibility complex (MHC) molecules. In this study, we investigated characteristics of peptides that bind to the bovine MHC allele BoLA-DRB3*2703, which is associated with occurrence of clinical mastitis in Holstein dairy cattle, and assigned a putative peptide-binding motif to this allele. This was achieved by in vitro expression of allele *2703 as well as a control allele, BoLA-DRB3*1201 which is present at high frequency in Holsteins. Transfected cell lines alone (for allele *1201) or in combination with blood mononuclear cells from an animal homozygous for allele *2703 were used as the source of naturally processed and presented peptides. Subsequent to elution of peptides from BoLA-DR+ cells, their sequences were determined by electrospray ionization mass spectrometry. Eluted peptides were between 13 and 20 amino acids long and the majority were in sets of overlapping sequences. These peptides were derived from intra- and extracellular proteins, as well as foreign proteins present in the culture medium. Some peptides had originated from molecular chaperones present in the endoplasmic reticulum, such as ER-60 and GRP78, pointing to some degree of overlap and cross-sampling between MHC class I and class II antigen presentation pathways. Consistent with reports of human and mouse MHC class II-associated peptides, putative peptide-binding motifs could be assigned to alleles *2703 and *1201, comprising a hydrophobic or an aromatic residue at relative position 1, a hydrophobic residue at position 4 and a small residue at position 6 of the eluted peptides. These findings provide the foundation for future studies of molecular mechanisms of MHC-disease associations of cattle.     

Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS3_1406–1415 and NS5B_2594–2602). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.     

Identification,localization and receptor characterization of novel mammalian substance P-like peptides

Hemokinin-1 (HK-1) is a novel substance P (SP)-like peptide that is encoded by the preprotachykinin C (PPT-C) gene recently identified in mouse B cells and shown to be a potentially important regulator of B cell development (Nat. Immunol. 1 (2000) 392). We have now isolated and characterized the human and rat orthologs of PPT-C and examined activities of human and mouse HK-1 on the three tachykinin receptors, neurokinin-1-3 (NK1-3). The rat PPT-C polypeptide is highly homologous to mouse PPT-C and contains the same processing sites to generate predicted HK-1. The human PPT-C polypeptide is also homologous to mouse PPT-C, however, it contains two potential monobasic cleavage sites rather than a single dibasic cleavage site at the amino-terminal end of the predicted HK-1 peptide. Thus, human PPT-C has the potential to generate full length predicted HK-1 as well as a truncated version (HK-1(4-11)). Polymerase chain reaction analysis revealed that both human and mouse PPT-C were expressed in a variety of tissues with strong signals detected in the skin of both species and in the mouse brain. Binding and functional analysis indicated that human and mouse HK-1 peptides were nearly identical to SP in their overall activity profile on the three NK receptors with the most potent affinity for the NK1 receptor. The results indicate that PPT-C encodes another high affinity ligand of the NK1 receptor which may play an important role in mediating some of the physiological roles previously assigned to the NK1 receptor.     

An HLA-DRB1-derived peptide associated with protection against rheumatoid arthritis is naturally processed by human APCs

Predisposition to rheumatoid arthritis (RA) is thought to be associated with HLA-DR1, -DR4, and -DR10. However, many epidemiological observations are better explained by a model in which the DQ alleles that are linked to these DR alleles, i.e., DQ5, DQ7, and DQ8, predispose to RA, while certain DR alleles have a dominant protective effect. All protective DRB1 alleles, e.g., *0402, *1301, and *1302, encode a unique motif, (70)DERAA(74). The protection may be explained by the presentation of DRB1-derived peptides by DQ to immunoregulatory T cells, because it was demonstrated in various autoimmune disease models that T cell responses to certain self-Ags can be involved in disease suppression. The aim of this study was to analyze whether peptides carrying the DERAA motif are naturally processed by human APC and presented in the context of the RA-predisposing DQ. Using a synthetic peptide carrying the DRB1*0402-derived sequence (65)KDILEDERAAVDTYC(79), we generated DERAA peptide-specific DQ-restricted T cell clones (TCC) from a DQ8 homozygous individual carrying DERAA-negative DR4 alleles. By analyzing the proliferation of these TCC, we demonstrated natural processing and presentation of the DERAA sequence by the APC of all the individuals (n = 12) carrying a DERAA-positive DRB1 allele and either DQ8 or the DQ8-related DQ7. Using a panel of truncated synthetic peptides, we identified the sequence (67)(I)LEDERAAVD(TY)(78) as the minimal determinant for binding to DQ8 and for recognition by the TCC. These findings support a model in which self-MHC-derived peptide can modulate predisposition to autoimmune disease in humans.     

Mass tag-assisted identification of naturally processed HLA class II-presented meningococcal peptides recognized by CD4+ T lymphocytes

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.     

Identification of immunogenic MHC class II Tyrosinase-derived peptides using HLA-DR1 and HLA-DR4 transgenic mice

The immunogenicity of "novel" MART-1 and Tyrosinase class-II peptides was assessed in transgenic mice. Tyrosinase(141-161) peptide was found to be immunogenic and endogenously processed in the HLA-DRbeta1*0101 and HLA-DRbeta1*0401 transgenic mice with peptide specific production of IFNgamma or IL-5 respectively. The MART-1(29-43) peptide was only found immunogenic in HLA-DRbeta1*0101 mice.     

HLA class II diversity in Australian Aborigines: unusual HLA-DRB1 alleles

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M77670 (DRB1 ^* AB3), M77671 (DRB1 ^* AB4), MM77672 (DRB1 ^* AB2), MM77673 (DRB1*A01), M81670 (DRB1*0410), and M81700 (DRB1 ^* 0411).     

Identification and characterization of novel adeno-associated virus isolates in ATCC virus stocks

Adeno-associated viruses (AAVs) depend on a helper virus for efficient replication. To identify novel AAV isolates, we screened a diverse set of virus isolates for the presence of AAV DNA. AAVs found in 10 simian adenovirus isolates showed greater than 96% homology to AAV1 and AAV6 but had distinct biological properties. Two representatives of this group, AAV(VR-195) and AAV(VR-355), were studied in more detail. While the novel AAVs had high sequence homologies and required sialic acid for cell binding and transduction, differences were observed in lectin competition, resulting in distinct tropisms in human cancer cell lines.     

Identification and characterization of YME1L1, a novel paraplegin-related gene

A gene responsible for an autosomal recessive form of hereditary spastic paraplegia (SPG7) was recently identified. This gene encodes paraplegin, a mitochondrial protein highly homologous to the yeast mitochondrial AAA proteases Afg3p, Rca1p, and Yme1p, which have both proteolytic and chaperone-like activities at the inner mitochondrial membrane. By screening the expressed sequence tag database, we identified and characterized a novel human gene, YME1L1 (YME1L1-like1, HGMW-approved symbol). This gene encodes a predicted protein of 716 amino acids highly similar to all mitochondrial AAA proteases and in particular to yeast Yme1p. Expression and immunofluorescence studies revealed that YME1L1 and paraplegin share a similar expression pattern and the same subcellular localization in the mitochondrial compartment. YME1L1 may represent a candidate gene for other forms of hereditary spastic paraplegia and possibly for other neurodegenerative disorders.     

Identification and characterization of novel mammalian neuropeptide FF-like peptides that attenuate morphine-induced antinociception

The two mammalian neuropeptides NPFF and NPAF have been shown to have important roles in nociception, anxiety, learning and memory, and cardiovascular reflex. Two receptors (FF1 and FF2) have been molecularly identified for NPFF and NPAF. We have now characterized a novel gene designated NPVF that encodes two neuropeptides highly similar to NPFF. NPVF mRNA was detected specifically in a region between the dorsomedial and ventromedial hypothalamic nuclei. NPVF-derived peptides displayed higher affinity for FF1 than NPFF-derived peptides, but showed poor agonist activity for FF2. Following intracerebral ventricular administration, a NPVF-derived peptide blocked morphine-induced analgesia more potently than NPFF in both acute and inflammatory models of pain. In situ hybridization analysis revealed distinct expression patterns of FF1 and FF2 in the rat central nervous system. FF1 was broadly distributed, with the highest levels found in specific regions of the limbic system and the brainstem where NPVF-producing neurons were shown to project. FF2, in contrast, was mostly expressed in the spinal cord and some regions of the thalamus. These results indicate that the endogenous ligands for FF1 and FF2 are NPVF- and NPFF-derived peptides, respectively, and suggest that the NPVF/FF1 system may be an important part of endogenous anti-opioid mechanism.     

Identification and characterization of a novel structural glycoprotein in pseudorabies virus, gL.

Herpesvirus envelope glycoproteins play important roles in the interaction between virions and target cells. In the alphaherpesvirus pseudorabies virus (PrV), seven glycoproteins that all constitute homologs of glycoproteins found in herpes simplex virus type 1 (HSV-1) have been characterized, including a homolog of HSV-1 glycoprotein H (gH). Since HSV-1 gH is found associated with another essential glycoprotein, gL, we analyzed whether PrV also encodes a gL homolog. DNA sequence analysis of a corresponding part of the UL region adjacent to the internal inverted repeat in PrV strains Kaplan and Becker revealed the presence of two open reading frames (ORF). Deduced proteins exhibited homology to uracil-DNA glycosylase encoded by HSV-1 ORF UL2 (54% identity) and gL encoded by HSV-1 ORF UL1 (24% identity), respectively. To identify the PrV UL1 protein, rabbit antisera were prepared against two synthetic oligopeptides that were predicted by computer analysis to encompass antigenic epitopes. Sera against both peptides reacted in Western blots of purified virions with a 20-kDa protein. The specificity of the reaction was demonstrated by peptide competition. Since the PrV UL1 sequence did not reveal the presence of a consensus N-linked glycosylation site, concanavalin A affinity chromatography and enzymatic deglycosylation of virion glycoproteins were used to ascertain that the PrV UL1 product is O glycosylated. Therefore, we designated this protein PrV gL. Analysis of mutant PrV virions lacking gH showed that concomitantly with the absence of gH, gL was also missing in purified virions. In summary, we identified and characterized a novel structural PrV glycoprotein, gL, which represents the eighth PrV glycoprotein described. In addition, we show that virion location of PrV gL is dependent on the presence of PrV gH.     

Identification and characterization of a novel non-structural protein of bluetongue virus

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77-79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.