Expression and refolding of OLA Ⅰ protein with peptides derived from sheeppox virus / 生物工程学报

The aim of this study was to refold the OvisAries leukocyte antigen (OLA) class Ⅰ protein with peptides derived from sheeppox virus (SPPV) to identify SPPV T cell epitopes. Two pairs of primers were designed based on the published sequence of a sheep major histocompatibility complex Ⅰ to amplify the heavy chain gene of OLA Ⅰ α-BSP and the light chain gene of OLA Ⅰ-β2m. Both genes were cloned into a pET-28a(+) expression vector, respectively, and induced with ITPG for protein expression. After purification, the heavy chain and light chain proteins as well as peptides derived from SPPV were refolded at a ratio of 1:1:1 using a gradual dilution method. Molecular exclusion chromatography was used to test whether these peptides bind to the OLA Ⅰ complex. T-cell responses were assessed using freshly isolated PBMCs from immunized sheep through IFN-γ ELISPOT with peptides derived from SPPV protein. The results showed that the cloned heavy chain and light chain expressed sufficiently, with a molecular weight of 36.3 kDa and 16.7 kDa, respectively. The protein separated via a SuperdexTM 200 increase 10/300 GL column was collected and verified by SDS-PAGE after refolding. One SPPV CTL epitope was identified after combined refolding and functional studies based on T-cell epitopes derived from SPPV. An OLA Ⅰ/peptide complex was refolded correctly, which is necessary for the structural characterization. This study may contribute to the development of sheep vaccine based on peptides.

Homology modeling and epitope prediction of Der f 33

Dermatophagoides farinae (Der f), one of the main species of house dust mites, produces more than 30 allergens. A recently identified allergen belonging to the alpha-tubulin protein family, Der f 33, has not been characterized in detail. In this study, we used bioinformatics tools to construct the secondary and tertiary structures and predict the B and T cell epitopes of Der f 33. First, protein attribution, protein patterns, and physicochemical properties were predicted. Then, a reasonable tertiary structure was constructed by homology modeling. In addition, six B cell epitopes (amino acid positions 34-45, 63-67, 103-108, 224-230, 308-316, and 365-377) and four T cell epitopes (positions 178-186, 241-249, 335-343, and 402-410) were predicted. These results established a theoretical basis for further studies and eventual epitope-based vaccine design against Der f 33.

A novel M2e-multiple antigenic peptide providing heterologous protection in mice

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F arrow right I, 21/21) and 212 (G arrow right S, 19/21) or (G arrow right N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G arrow right D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L arrow right F) and 129 (M arrow right I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W arrow right C), and 5/21 specimens showed another novel change at codon 115 (G arrow right A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.

A hybrid approach for predicting promiscuous MHC class I restricted T cell epitopes.

In the present study, a systematic attempt has been made to develop an accurate method for predicting MHC class I restricted T cell epitopes for a large number of MHC class I alleles. Initially, a quantitative matrix (QM)-based method was developed for 47 MHC class I alleles having at least 15 binders. A secondary artificial neural network (ANN)-based method was developed for 30 out of 47 MHC alleles having a minimum of 40 binders. Combination of these ANN-and QM-based prediction methods for 30 alleles improved the accuracy of prediction by 6% compared to each individual method. Average accuracy of hybrid method for 30 MHC alleles is 92.8%. This method also allows prediction of binders for 20 additional alleles using QM that has been reported in the literature, thus allowing prediction for 67 MHC class I alleles. The performance of the method was evaluated using jack-knife validation test. The performance of the methods was also evaluated on blind or independent data. Comparison of our method with existing MHC binder prediction methods for alleles studied by both methods shows that our method is superior to other existing methods. This method also identifies proteasomal cleavage sites in antigen sequences by implementing the matrices described earlier. Thus, the method that we discover allows the identification of MHC class I binders (peptides binding with many MHC alleles) having proteasomal cleavage site at C-terminus. The user-friendly result display format (HTML-II) can assist in locating the promiscuous MHC binding regions from antigen sequence. The method is available on the web at www.imtech.res.in/raghava/nhlapred and its mirror site is available at http://bioinformatics.uams.edu/mirror/nhlapred/.

A survey of the Th2R and Th3R allelic variants in the circumsporozoite protein gene of P. falciparum parasites from western Thailand.

Allelic variation in the Plasmodium falciparum circumsporozoite protein (CS) gene has been determined by sequencing the immunodominant T-cell epitopes, Th2R and Th3R, from 95 isolates from two malaria-endemic areas in the west of Thailand. Comparison with a reference sequence revealed only non-synonymous point mutations in the two epitope regions. Point mutations were found outside these epitopes in a minority of samples, and all but four were also non-synonymous. A relatively high number of variants, 11 Th2R and 9 Th3R, were detected and comprised some that had not been previously observed. However, the Th2R*05 and the Th3R*01 allelic variants predominated, as they were found in more than 70% of the 101 sequences obtained.