Localization of a T-cell epitope within the nucleocapsid protein of avian coronavirus.

In a previous study, two murine T-cell hybridomas generated after immunization with infectious bronchitis virus (IBV) were shown to be responsive to the internally localized viral nucleocapsid protein. In the present study, the antigenic determinants were mapped using recombinant expression products and synthetic peptides. Both hybridomas recognized the region spanning amino acid residues 71 to 78 of the nucleocapsid protein. The experimentally determined epitope corresponded with predicted motifs. Both an I-Ed binding motif and a predicted cleavage site for the aspartyl protease cathepsin D were contained within the sequence. The epitope was shown to prime cellular immune responses to IBV in the chicken.     

Sequence of the membrane protein gene from avian coronavirus IBV

cDNA clones prepared from genomic RNA of coronavirus IBV have been sequenced. The nucleotide sequence for the complete 5' region of mRNA C, which is not present in mRNAs A and B, has been determined. A sequence of 1224 bases is presented which contains a long open reading frame predicting a polypeptide of molecular weight 25 443. This is in agreement with the molecular weight of 23 000 reported for the unglycosylated form of the membrane polypeptide.     

Class II-restricted bifunctional T-cell hybridomas reactive to self- and foreign myelin basic protein.

To study the cross-reactivity and functional properties oF murine T cells specific for myelin basic protein (MBP), a panel of 15 interleukin-2(IL-2)-releasing T-cell hybridomas was produced from SJL/J mice immunized either with human MBP or alternatively with a peptide corresponding to the known encephalitogenic sequence for SJL/J mice at positions 89-106. Hybridomas were I-As-restricted and activated by an MBP challenge as low as 20 nM. Cross-reactivity to other MBP indicated at least three immunodominant specificities for xenogeneic determinants, which could be further subdivided on the basis of antigen-independent reactivity to allogeneic stimulator cells. In addition, two self-specificities were demonstrated, one of which was to a determinant outside the 89-106 region. Irrespective of specificity pattern (self or foreign), all hybridomas effected antigen-dependent cytotoxicity of an antigen-presenting B-cell hybridoma (LS-102.9), which was mediated by cell contact or at close range. These findings suggest an approach to identifying new autoantigenic epitopes on MBP, and to studying T-cell-mediated effector pathways in myelin autoimmunity.     

Phylogeny of antigenic variants of avian coronavirus IBV

The sequences of the peplomeric S1 protein of four serologically distinct strains of the infectious bronchitis virus (IBV), an avian coronavirus, have been determined. The S1 protein is thought to contain the serotype-specific neutralization epitopes and to be the main target of antigenic variation. An alignment with sequences of three strains published previously showed that from the 545 amino acid residues only 243 have been conserved. Clustering of substitutions suggests that most serotype determinants are located within the first 300 amino acid residues of S1. A phylogenetic tree of the S1 sequences showed very variable rates of divergence. Differences in topology with a tree based on RNAse-T1 fingerprint data indicate that some of the IBV strains have arisen by genetic recombination.     

Evidence of genetic diversity generated by recombination among avian coronavirus IBV

Summary.  Previously, we demonstrated that the DE072 strain of IBV is a recombinant which has an IBV strain D1466-like sequence in the S gene. Herein, we analyzed the remaining 3.8 kb 3′ end of the genome, which includes Gene 3, Gene 4, Gene 5, Gene 6, and the 3′ non-coding region of the DE072 and D1466 strains. Those two viruses had high nucleotide similarity in Gene 4. However, the other individual genes had a much different level of sequence similarity with the same gene of the other IBV strains. The genome of five IBV strains, of which the complete sequence of the 3′ end of the genome has been determined, were divided at an intergenic (IG) consensus sequence (CTGAACAA or CTTAACAA) and compared phylogenetically. Phylogenetic trees of different topology indicated that the consensus IG sequences and the highly conserved sequence around this regions may serve as recombination ‘hot spots’. Phylogenetic analysis of selected regions of the genome of the DE072 serotype field isolates further support those results and indicate that isolates within the same serotype may have different amounts of nucleotide sequence similarity with each other in individual genes other than the S gene. Presumably this occurs because the consensus IG sequence serves as the template switching site for the viral encoded polymerase. Received December 1, 1999 Accepted March 24, 2000     

Chemical and functional analysis of MHC class II-restricted T cell epitopes.

Various aspects of antigen degradation and presentation are reviewed, in particular with respect to fragmentation of native vs. denatured proteins, different enzymatic machinery present in different cells and individuals, characterization of epitopes and their persistence on antigen-presenting cells as well as their capacity to interact with different MHC class II molecules. Finally, the structure of antigenic peptides is discussed.     

The Yersinia enterocolitica invasin protein promotes major histocompatibility complex class I- and class II-restricted T-cell responses

Yersinia enterocolitica invasin (Inv) protein confers internalization into and expression of proinflammatory cytokines by host cells. Both events require binding of Inv to beta1 integrins, which initiates signaling cascades including activation of focal adhesion complexes, Rac1, mitogen-activated protein kinase, and NF-kappaB. Here we tested whether Inv might be suitable as a delivery molecule and adjuvant if used as a component of a vaccine. For this purpose, hybrid proteins composed of Inv and ovalbumin (OVA) were prepared, applied as a coating to microparticles, and used for vaccination. Fusion of OVA to Inv did not significantly disturb the ability of Inv to promote host cell binding, internalization, and interleukin-8 (IL-8) secretion when applied as a coating to microparticles. The microparticles were used for vaccination of mice adoptively transferred with OVA-specific T cells from OT-1 or DO11.10 mice. Administration of OVA-Inv-coated microparticles induced OVA-specific T-cell responses. OVA-specific CD4 T cells produced both gamma interferon (IFN-gamma) and IL-4 as determined by enzyme-linked immunosorbent assay. Likewise, pronounced OVA-specific CD8 T-cell responses associated with IFN-gamma production were observed. Together, these results suggest that Inv might be an attractive tool in vaccination as it confers both host cell uptake and adjuvant activity by engagement of beta1 integrins of host cells, which leads to CD4 as well as CD8 T-cell responses.     

Synthesis and subcellular localization of the murine coronavirus nucleocapsid protein

The synthesis and processing of the nucleocapsid protein (pp60) of the JHM strain of murine coronaviruses were examined. Pulse-chase experiments showed that pp60 was synthesized initially as a protein of approximately 57,000 in molecular weight (p57). Immunoprecipitation using mouse anti-JHMV antiserum indicated that p57 was virus specific. Immunoprecipitation with monoclonal antibodies specific for pp60 showed that p57 was antigenically related to pp60 and was not phosphorylated, while the intracellular protein that comigrated with the virion nucleocapsid protein, pp60, was phosphorylated. The p57 was found exclusively in the cytosol while the majority of pp60 was associated with the membrane fraction but pp60 was not an integral membrane protein.     

Major histocompatibility complex (MHC) restricted antigen recognition: high frequency of human T-cell clones recognizing novel MHC class II determinants

We have used antigen-specific human T-cell clones to study the relationship between MHC and antigen recognition specificities expressed by T cells. Tetanus toxoid (TT)-specific T-lymphocyte clones were derived from a immunized HLA-DR2,7 heterozygous donor by limiting dilution from peripheral blood mononuclear cells (PBM) restimulated with TT in vitro. Clones were screened for MHC-restricted antigen recognition against antigen-presenting cells (APC) from a panel of HLA-typed donors, using an in vitro T-cell proliferation assay. Several distinct patterns of antigen recognition were identified. In addition to T cells that recognized TT in association with donor class II MHC antigens, we found clones that simultaneously expressed self-restricted antigen recognition and alloreactivity, and clones with specificity for antigen in the context of MHC antigens not expressed by the T-cell donor. This was confirmed in inhibition studies using well-characterized monoclonal antibodies against class II MHC antigens to block specific proliferative responses. We propose a possible structure for the determinant recognized by two of the clones. These results suggest that the T-cell antigen receptor undergoes random or antigen-dependent changes in vitro, and that this may be a mechanism for somatic diversification of the T-cell repertoire.     

Association of the chicken MHC B haplotypes with resistance to avian coronavirus

Clinical respiratory illness was compared in five homozygous chicken lines, originating from homozygous B2, B8, B12 and B19, and heterozygous B2/B12 birds after infection with either of two strains of the infectious bronchitis virus (IBV). All chickens used in these studies originated from White Leghorn and Ancona linages. IBV Gray strain infection of MHC homozygous B12 and B19 haplotype chicks resulted in severe respiratory disease compared to chicks with B2/B2 and B5/B5 haplotypes. Demonstrating a dominant B2 phenotype, B2/B12 birds were also more resistant to IBV. Respiratory clinical illness in B8/B8 chicks was severe early after infection, while illness resolved similar to the B5 and B2 homozygous birds. Following M41 strain infection, birds with B2/B2 and B8/B8 haplotypes were again more resistant to clinical illness than B19/B19 birds. Real time RT-PCR indicated that infection was cleared more efficiently in trachea, lungs and kidneys of B2/B2 and B8/B8 birds compared with B19/B19 birds. Furthermore, M41 infected B2/B2 and B8/B8 chicks performed better in terms of body weight gain than B19/B19 chicks. These studies suggest that genetics of B defined haplotypes might be exploited to produce chicks resistant to respiratory pathogens or with more effective immune responses.     

SARS冠状病毒N蛋白的克隆与表达

目的　克隆和表达SARS冠状病毒N蛋白 ,并分析其免疫学活性。方法　采用RT PCR从SARS冠状病毒RNA中扩增出编码N蛋白的基因 ;经克隆和测序分析后 ,亚克隆至表达载体pGEX 4T 2 ,转化大肠杆菌JM10 9,PCR和双酶切鉴定 ;阳性菌株经IPTG诱导 ,SDS PAGE分析 ;大量诱导表达N蛋白 ,亲和层析予以纯化 ;免疫印迹分析纯化蛋白对SARS的诊断效果 ;用纯化的融合蛋白免疫小鼠观察其诱导的抗体应答。结果　RT PCR扩增出N蛋白基因的特异片段 ,获得的阳性克隆序列与Gen Bank中登录的SARS冠状病毒的N蛋白基因序列同源性为 99.8% ;N蛋白基因被亚克隆到表达载体pGEX 4T 2 ,在JM10 9中表达了N蛋白 ,表达的蛋白经亲和层析获得纯化 ;纯化蛋白能被SARS病人血清识别 ;免疫小鼠诱导产生了高滴度的抗体。结论　成功构建了SARS冠状病毒N蛋白的重组表达质粒 ,在大肠杆菌中表达的N蛋白融合蛋白具有良好的免疫学活性。     

Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene

The 3' end of the 20-kb genome of the Purdue strain of porcine transmissible gastroenteritis coronavirus (TGEV) was copied into cDNA after priming with oligo(dT) and the double-stranded product was cloned into the PstI site of the pUC9 vector. One clone of 2.0-kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3' end of the genome. Two potential, nonoverlapping genes were identified within the 3'-terminal 1663-base sequence from an examination of open reading frames. The first gene encodes a 382-amino acid protein of 43,426 mol wt, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid sequence homology with other coronavirus nucleocapsid proteins. It is flanked on its 5' side by at least part of the matrix protein gene. The second encodes a hypothetical 78-amino acid protein of 9101 mol wt that is hydrophobic at both ends. A 3'-proximal noncoding sequence of 276 bases was also determined and a conserved stretch of 9 nucleotides near the poly(A) tail was found to be common among TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.     

Destructive potential of the aspartyl protease cathepsin D in MHC class II-restricted antigen processing

Whether specific proteases influence MHC class II antigen presentation is still not clearly defined. Cathepsin D, one of the most abundant lysosomal proteases, is thought to be dispensable for MHC class II antigen presentation, yet in vitro digestions of antigen substrates with endosomes/lysosomes from antigen-presenting cells sometimes reveal a dominant role for pepstatin-sensitive aspartyl proteases of which cathepsin D is the major representative. We tested whether the aspartyl protease substrate myoglobin requires cathepsin D activity for presentation to T cells. Surprisingly, in dendritic cells (DC) lacking cathepsin D, presentation of two different myoglobin T cell epitopes was enhanced rather than hindered. This paradox is resolved by the finding that pepstatin-sensitive myoglobin processing activity persists in lysosomes from cathepsin D-null DC and that this reduced activity, most likely due to cathepsin E, is closer to the optimum level required for myoglobin antigen presentation. Our results indicate redundancy among lysosomal aspartyl proteases and show that while processing activities can be productive for MHC class II T cell epitope generation at one level, they can become destructive above an optimal level.     

Sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes

The 3' end of the 20-kb genome of the Mebus strain of bovine enteric coronavirus (BCV) was copied into cDNA and cloned into the PstI site of the pUC9 vector. Four clones from the 3' end of the genome were sequenced either completely or in part to determine the sequence of the first 2451 bases. Within this sequence were identified, in order, a 3'-noncoding region of 291 bases, the gene for a 448-amino acid nucleocapsid protein (N) having a molecular weight of 49,379, and the gene for a 230-amino acid matrix protein (M) having a molecular weight of 26,376. A third large open reading frame is contained entirely within the N gene sequence but is positioned in a different reading frame; it potentially encodes a polypeptide of 207 amino acids having a molecular weight of 23,057. A higher degree of amino acid sequence homology was found between the M proteins of BCV and MHV (87%) than between the N proteins (70%). For the M proteins of BCV and MHV, notable differences were found at the amino terminus, the most probable site of O-glycosylation, where the sequence is N-Met-Ser-Ser-Val-Thr-Thr for BCV and N-Met-Ser-Ser-Thr-Thr for MHV. BCV apparently uses two of its six potential O-glycosylation sites.     

Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell clones

The identification of ricin toxin A-chain (RTA) epitopes and the molecular context in which they are recognized will allow strategies to be devised that prevent/suppress an anti-RTA immune response in patients treated with RTA-based immunotoxins. RTA-specific human T-cell lines and T-cell clones were produced by in vitro priming of PBMC. The T-cell clones used a limited set of Vbeta chains (Vbeta1, Vbeta2 and Vbeta8) to recognize RTA epitopes. The use of RTA deletion mutants demonstrated that T-cell lines and T-cell clones from three out of four donors responded to RTA epitopes within the domain D124-Q223, whereas one donor recognized the region I1-D124. The response to RTA peptides of T-cell lines and T-cell clones from two donors allowed the identification of immunogenic segments (D124-G140 and L161-T190) recognized in the context of different HLA-DRB1 alleles (HLA-DRB1*0801, and HLA-DRB1*11011 and B1*03011, respectively). The response to L161-T190 was investigated in greater detail. We found that the HLA-DRB1*03011 allele presents a minimal epitope represented by the sequence I175-Y183 of RTA, whereas the HLA-DRB1*11011 allele presents the minimal epitope M174-I184. RTA peptides and an I175A RTA point mutant allowed us to identify I175 as a crucial residue for the epitope(s) recognized by the two HLA-DRB1 alleles. Failure of T-cell clones to recognize ribosome inactivating proteins (RIPs) showing sequences similar but not identical to RTA further confirmed the role of I175 as a key residue for the epitope recognized in the context of HLA-DRB1*11011/03011 alleles.     

Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus

The human coronavirus, associated with severe acute respiratory syndrome (SARS-CoV), was identified and molecularly characterized in 2003. Sequence analysis of the virus indicates that there is only 20% amino acid (aa) identity with known coronaviruses. Previous studies indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly. Yet, little sequence homology between the newly identified SARS-CoV and those previously studied coronaviruses suggests that determination of protein-protein interaction and identification of amino acid sequences, responsible for such interaction in SARS-CoV, are necessary for the elucidation of the molecular mechanism of SARS-CoV replication and rationalization of anti-SARS therapeutic intervention. In this study, we employed mammalian two-hybrid system to investigate possible interactions between SARS-CoV nucleocapsid (N) and the membrane (M) proteins. We found that interaction of the N and M proteins takes place in vivo and identified that a stretch of amino acids (168-208) in the N protein may be critical for such protein-protein interactions. Importantly, the same region has been found to be required for multimerization of the N protein (He et al., 2004) suggesting this region may be crucial in maintaining correct conformation of the N protein for self-interaction and interaction with the M protein.     

Dendritic cells permit identification of genes encoding MHC class II-restricted epitopes of transplantation antigens

Minor or histocompatibility (H) antigens are recognized by CD4+ and CD8+ T lymphocytes as short polymorphic peptides associated with MHC molecules. They are the targets of graft versus host and graft versus leukemia responses following bone marrow transplantation between HLA-identical siblings. Several genes encoding class I-restricted minor H epitopes have been identified, but approaches used for these have proved difficult to adapt for cloning class II-restricted minor H genes. We have combined the unique antigen-presenting properties of dendritic cells and high levels of episomal expression following transfection of COS cells to identify a Y chromosome gene encoding two HY peptide epitopes, HYAb and HYEk.     

Demonstration of MHC class II-restricted cytotoxic T lymphocytes in mice against herpes simplex virus

In humans CD4+ major histocompatibility complex (MHC) class II-restricted T cells dominate cytotoxic T-lymphocyte (CTL) responses to herpes simplex virus (HSV), whereas in the mouse only CD8+ MHC class I-restricted CTL have been reported. In this study, we demonstrate that a minor fraction (around 30%) of the response in draining lymph nodes of acute local HSV infections is attributable to CD4+ CTL mice. Such CTL were identified on the basis of antiserum inhibition studies, negative depletion approaches, as well as their differing antigen processing requirements to CD8+ MHC class I-restricted CTL. A possible role for CD4+ CTL as immunoregulators in local infections is discussed briefly.