Immunogenicity of two FMDV nonameric peptides encapsulated in liposomes in mice and the protective efficacy in guinea pigs.

It has been predicted that nonameric peptides I (VP1(26-34), RRQHTDVSF), II (VP1(157-165), RTLPTSFNY) and III (VP1(45-53), KEQVNVLDL) from the VP1 capsid protein of the foot-and-mouth disease virus (FMDV) are T cell epitopes. To investigate whether these peptides have immunological activity, BALB/c mice were immunized with peptide I, II or III conjugated with immunostimulating complexes (ISCOMs). A cytotoxic T lymphocyte assay was used to evaluate the cytotoxic activity induced by peptides along with by measuring peptide-specific T-cell proliferation and CD8(+) T lymphocyte numbers in whole blood and interferon (IFN)-γ production in peripheral blood mononuclear cells induced by peptides. To further identify the protective efficacy of peptides, an FMDV challenge assay was done in guinea pigs. Peptides I and II stimulated significant increases in T-cell proliferation, CD8(+) T lymphocytes, and IFN-γ secretion and cytotoxic activity compared to controls. The FMDV challenge assay indicated peptides I and II can protect over 60% of animals from virus attack. The results demonstrate that peptides I and II encapsulated in liposomes should be CTL epitopes of FMDV and can protect animals from virus attack to some extent.

Reconstruction of a chicken BF2 protein complex and identification of binding nonamer peptides derived from avian influenza virus hemagglutinin.

In order to reconstruct the system for identification of short antigenic peptides, the chicken BF2 gene of Chinese Sanhuang (SH) chicken line was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich peptide, by splicing overlap extension PCR (SOE-PCR). The MBP-BF2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/E. coli TB1 system. The purified MBP-BF2-(G4S)3-beta(2)m protein was cleaved by Factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the BF2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD). In addition, the refolded BF2-(G4S)3-beta2m protein was used to bind three predicted nonameric peptides derived from the hemagglutinins of the avian influenza virus (AIV) H5N1 and H9N2 subtypes. The BF2-(G4S)3-beta2m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and tandem mass spectrometry analysis, respectively. The results indicate that the secondary structures and predicted three-dimensional crystal structure of BF2-(G4S)3-beta(2)m are similar to those of the monomers of chicken BF2 and beta(2)m. The BF2-(G4S)3-beta(2)m protein could bind two of the three predicted nonamer peptides derived from AIV hemagglutinin. The experimental system demonstrated that the reconstructed BF2-(G4S)3-beta(2)m protein complex can be used to identify nonamer peptides, including T-cell epitopes in chicken.

Extensive analysis of different allelelic structures of the chicken BF2 and beta2m proteins.

No information is available to date on the different allelelic structures of the chicken MHC class I (BF2) and beta2m proteins. To elucidate the structure, new allelic beta2m and five different BF2 genes were expressed solubly and purified in a pMAL-p2X/E. coli TB1 system. The 2D structure was detected by circular dichroism (CD) spectroscopy, and the 3D structures of their peptide-binding domain (PBD) were analyzed by homology modeling. The sequence lengths of the alpha-helix, beta-sheet, turn, and random coil in the five BF2 proteins were 69-73 aa, 67-72 aa, 35-37 aa, and 94-98 aa, respectively. The new beta2m protein displayed a typical beta-sheet. Homology modeling of the different BF2 and beta2m proteins demonstrated similarities to the structure of human and rat MHC class I proteins. The 3D structure, however, revealed that the BF2 and beta2m structures were unique. The correct refolding of recombinant BF2 and beta2m proteins might be a powerful tool to further detect antigenic peptides.

Structures and homology modeling of chicken major histocompatibility complex protein class I (BF2 and beta2m).

In order to elucidate the two-dimensional (2D) and three-dimensional (3D) structures of chicken major histocompatibility complex (MHC) class I protein (BF2 and beta2m) and further reconstruct their complex identifying the virus-derived antigenic peptides, the mature protein of BF2 and beta2m genes were expressed solubility in pMAL-p2X/Escherichia coli. TB1 system. The expressed MBP-BF2- and MBP-beta2m-fusion proteins were purified, and cleaved by the factor Xa protease. Subsequently, the monomers were further separated, and the purified MBP-BF2, -beta2m, and MBP were analyzed by circular dichroism (CD) spectrum. The contents of alpha-helix, beta-sheet, turn, and random coil in BF2 protein were 72, 102, 70, and 90 amino acids (aa), respectively. The beta2m proteins displayed a typical beta-sheet and the contents of alpha-helix, beta-sheet, turn, and random coil were 0, 46, 30, and 22 aa, respectively. Homology modeling of BF2 and beta2m proteins were similar as the 3D structure of human MHC class I (HLA-A2). The results showed that pMAL-p2X expression and purification system could be used to obtain the right conformational BF2 and beta2m proteins, and the 2D and 3D structures of BF2 and beta2m were revealed to be similar to human's. The recombinant BF2 and beta2m-based proteins might be a powerful tool for further detecting antigenic peptides.

Characterization of BF2 and beta2m in three Chinese chicken lines.

Twenty-four BF2 genes and 10 beta(2)m genes from Chinese Sanhuang (SH), Wuji (WJ), and Zhenzhu (ZZ) chicken lines were cloned, and the amino acid replacement rates of the BF2 polypeptide binding domain were investigated. For this purpose, 13 BF2 genes from the SH-chicken line (BF2*01sh-BF2*13sh), six BF2 genes from the WJ-chicken line (BF2*01wj-BF2*06wj), and five BF2 genes from the ZZ-chicken line (BF2*01zz-BF2*05zz) were analyzed. The overall conservation of BF2 alleles could be observed within the sequences, and relative conservation was also displayed in the peptide-binding domain, CD8(+) interaction sites, and beta(2)m contact sites. Based on the amino acid similarity, BF2 from the three chicken lines could be divided into eleven gene groups, and five novel gene groups were observed. Although the amino acid similarity among the different alleles was 75.7-99.2%, within an allelic group the members shared >91% amino acid identity with each other. In addition, beta(2)m genes from the three Chinese chicken lines were also clustered into two gene groups: I and II. Between groups I and II, the amino acid identical ratio was much lower (81.9-84.0%). Group I is close to that of the reported chicken beta(2)m, whereas group II represents a new allelic group. The results suggest that five new BF2 groups and a new beta(2)m group exist in the three Chinese chicken lines.