High-resolution mapping of B-cell epitopes within an antigenic sequence from Eimeria tenella.

Overlapping hexapeptides representing part of an Eimeria tenella antigenic sequence, shown to induce partial immunity to homologous challenge in chickens, were synthesized on polypropylene pins (Pepskan technique; Cambridge Research Biochemicals, Cambridge, United Kingdom). The binding to these hexapeptides of antibodies from chickens infected and rabbits immunized with five species of Eimeria was studied, using the coated pins as the solid phase of an enzyme-linked immunoassay. Antibody binding to most regions of the sequence was demonstrated, with peak areas of antigenicity correlating with the most hydrophilic regions. A particularly hydrophilic and antigenic area towards the N terminus of the sequence consists of a peptide motif repeated five times in the native antigen. Homologous antisera (chicken and rabbit anti-E. tenella antisera) differed in their pattern of reactivity from heterologous sera raised against other Eimeria species. While the former bound to fewer of the hexapeptides than the latter, they did so very strongly, indicating affinity maturation of the antibody response to E. tenella-specific sequences. No antibody reactivity to two regions of the sequence was detected. These regions occur in relatively hydrophilic areas and so are unlikely to be situated in transmembrane domains or in the interior of globular proteins. Synthetic peptides, as used in these experiments, make possible analysis of the fine specificity of immune responses and thus have a role to play in the development of novel vaccines for the control of coccidiosis.     

Reversal of HLA restriction by a point mutation in an antigenic peptide

The HLA restriction of a human T cell clone specific for residues 307-319 of influenza haemagglutinin was changed by the introduction of a point substitution in the peptide. Cells expressing HLA Dw1, DR4 Dw4, Dw13, Dw14, or Dw15 could present the natural haemagglutinin peptide to varying extents to the clone, but DR4 Dw10 could not. Replacement of glutamine-312 of the peptide with arginine produced an analogue that was recognized by the T cell clone only in the context of DR4 Dw10; neither DR1 nor any of the other DR4 alleles could present this peptide to the clone. The inability to bind either the natural or modified peptide was shown not to be the cause of the non-responsiveness. Rather, the differential stimulation of the clone appeared to arise from sequence variations between the DR alleles in residues comprising the beta-chain helix, which either affected recognition by directly contacting the T cell receptor or modified the conformation of the bound peptide. The latter effects were sufficiently subtle to modulate recognition by changing the fine details of the bound peptide but not to eliminate the capacity of the restriction element to bind the peptide.     

Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates

Hepcidin is an antimicrobial peptide and iron-regulatory molecule that is conserved among vertebrates. Mutations or over-expression of the human hepcidin gene have been found in patients with hemochromatosis and refractory anemia. To further understand the function and regulation of hepcidin, animal models are needed. We sequenced cDNA, genes and upstream regions of zebrafish hepcidin and analyzed gene expression by kinetic PCR. Zebrafish hepcidin genes consist of two introns and three exons that encode a prepropeptide (91 amino acids). The amino acid sequences and gene organization were remarkably conserved between zebrafish and other species. Elevated gene expression was observed in abdominal organs, skin, and heart in fish that developed signs of infection following bacterial injection. Zebrafish may be a suitable model organism for further study of hepcidin gene regulation.     

Recruitment of alloreactive cytotoxic T lymphocytes by an antigenic peptide

To investigate the requirements for induction of cytotoxic T lymphocytes (CTL) by peptides we chose the 16-residue nucleoprotein peptide (NPP; 365-380) from the influenza virus A/NT/60/68 as model substrate that is recognized in conjunction with major histocompatibility complex H-2d. Here we present that CTL can be raised from naive animals by repeated in vitro stimulation with high concentrations of peptide. The frequency of this response can be boosted by immunization of the animals with NPP-conjugated to ovalbumin as a carrier. However, in contrast to NPP-specific CTL lines raised from virus-primed animals none of the peptide-induced CTL lines were able to lyse virus-infected targets. Although they did not show an apparent difference in fine specificity of the peptide recognized, their affinity to the target cells was 100-fold lower than that of CTL from virus-primed animals as estimated from the peptide concentration needed to achieve significant lysis. In addition, the activity of peptide-induced CTL was very sensitive to blocking by anti-CD8 antibodies as compared to virus-specific CTL. Furthermore, all peptide-induced CTL showed a high second reactivity for allogeneic H-2k targets. Therefore, it is argued that high epitope density achieved by high peptide concentrations can in vitro recruit lymphocytes of another specificity. For the tested peptide the reactive T lymphocytes showed high alloreactivity.     

Fine mapping of an apparently targeted latent human herpesvirus type 6 integration site in chromosome band 17p13.3

An unusually high level of latent HHV-6 infection has been documented in the peripheral blood and/or bone marrow cells of a small group of patients with predominantly malignant lymphoid disorders, and in at least one healthy individual. We have shown previously in peripheral blood mononuclear cells (PBMCs) of three patients, two with a history of lymphoma and one with multiple sclerosis, a specific target site for latent integration of the full-length HHV-6 viral genome on the distal short arm of chromosome 17, in band p13.3. Fluorescence in situ hybridization (FISH) procedures were used to map more precisely the location of the viral integration site in one of those patients, relative to two known oncogenes mapped previously, namely CRK, and the more telomeric ABR oncogene. It is shown that the HHV-6 integration site is located at least 1,000 kb telomeric of ABR, and is very likely to map close to or within the telomeric sequences of 17p. This finding is significant given that human telomeric-like repeats flank the terminal ends of the HHV-6 genome. Cytogenetic studies showed evidence of karyotype instability in the peripheral blood cells infected latently.     

Antibodies to highly conserved peptide sequence of staphylococcal and streptococcal superantigens in Kawasaki disease

Superantigen-mediated disease such as toxic shock syndrome is seen in patients who have a weak antibody response to the antigen toxic shock syndrome toxin 1 (TSST-1). We hypothesized that there may be deficiency in antibody production to staphylococcal and streptococcal toxins in Kawasaki disease (KD) children. A peptide was constructed from the homologous portion of the staphylococcal enterotoxins (SE) and streptococcal pyrogenic enterotoxins (SPE), and antibodies to the peptide were made. The anti-peptide antibody immunoblotted several of the SE toxins and SPE toxins. Presence of the peptide antibodies was investigated via ELISA in the sera of acute KD (n = 30), convalescent KD (n = 12), control adults (n = 10), and children (n = 19). The mean anti-peptide antibodies were indistinguishable between control children and KD before treatment with immunoglobulin (P = 0.7) but rose significantly after therapy (P < 0.01). The adults had significantly higher antibodies than the KD, both acute and late (P < 0.0001) and the control children (P < 0.0001). Thus, KD patients do not have a defective serological response against toxins such as SPE/SE/TSST-1. Normal children have significantly lower antitoxin antibody levels to the toxins compared to the adults.     

Evidence of an antigenic shift among Palyam serogroup orbiviruses

The Japanese isolates of Palyam serogroup viruses isolated from 1985 to 2001 were investigated for the genome sequence of segments 2 and 7 and were phylogenetically analyzed in comparison with Australian and African isolates of the same serogroup. The nucleotide sequences of segment 7 were highly conserved within Japanese isolates (95.1 to 100%) and between Japanese and Taiwanese isolates (96.0 to 100%), whereas the identities between Japanese and Taiwanese isolates and Australian and African isolates were fairly conserved (84.2 to 92.0%). Phylogenetic analysis based on segment 7 revealed three clusters according to geographical origin. As a result of the nucleotide sequence analysis of segment 2, which encodes a serotype-specific antigen, Japanese isolates were classified into two groups by genome length and nucleotide identities. Four of the nine Japanese isolates were categorized into the same group as prototype strain K-47 of the Chuzan virus, and the remaining isolates were categorized into the same group as the D'Aguilar virus and Nyabira virus. Phylogenetic analysis based on segment 2 revealed two clusters, the cluster containing Chuzan virus and the cluster containing the D'Aguilar and Nyabira viruses. To examine the antigenic relationship among viruses categorized in different clusters, we conducted a cross-neutralization test. KSB-29/E/01, isolated in 2001 in Japan, was neutralized by antiserum not only to strain B8112 of D'Aguilar virus but also to Chuzan virus. These results indicated that genetically and antigenically unique characteristics of KSB-29/E/01 were attributed to genetic reassortment of segment 2 between Chuzan virus and D'Aguilar virus.     

Fine characterization of the antigenic site within the flap region in the protease protein of HIV-1

Summary.  The aspartate protease encoded by human immunodeficiency virus type 1 is essential for cleavage of the gag and gag-pol precursor proteins. The majority of HIV-1-antibody-positive sera react with the protease. In this study we used a substitution set of peptides for detailed characterization of the earlierdefined antigenic site (aa 44–58) within the central “flap”region, also important in the context of conformationalflexibility during protease inhibitor binding. We found thatisoleucine at position 54 was important for creating anantigenic site required for binding of anti-HIV-1 sera. The identification of structurally essential amino acids in theflap region of HIV-1 PR may have important implications infuture development of antiviral drugs. Accepted September 9, 1999/Received June 1, 1999     

Antibody reactivity of conformational peptide mimics of a conserved H5N1 neutralization site in different fusion proteins

Several peptide mimics of a conserved H5N1 avian influenza virus neutralization site recognized by 8H5 mAb have been reported previously. In this study, the secondary and possibly higher structural orders of the peptide mimics 122 and 125 were investigated and found to be closely related to the specific binding with 8H5 mAb. These two peptide mimics were fused to three different carrier proteins, and the antibody binding activities were recovered in 4 of the 11 fusion proteins. HEV structural protein p239 and HBc were more suitable than the outer membrane protein T47 of the Treponema pallidum particle for the recovery of reactivity. The increase in the copy number of peptide mimics was important for the recovery of antibody-binding activity and the interaction between peptide and carrier protein may affect the spatial structure of both the peptide and the carrier protein. These results are likely to be of relevance for conformational peptide mimics in diagnostic tests, vaccine and inhibitors.     

Study of an antigenic site of human serum albumin with monoclonal antibodies

Two monoclonal anti-HSA antibodies, HA1 and HA2, have been shown to be specific for a univalent fragment of 6000 mol. wt, F1, located near the C-terminus of HSA (Doven, Pesce & Lapresle, Immunology Letters 3, 365-370, 1981). Both monoclonal antibodies have been shown to react with the same site, which includes the following components: the last small loop of HSA (558-567) the disulfide bridge 514-559 and the residue 570. This site is as available on HSA and F1, but partially masked on the 'Inhibitor' fragment from which F1 derives. Polyclonal anti-F1 antibodies, purified from rabbit sera or mouse ascites by affinity chromatography, react with the same site as HA1 and HA2. However, polyclonal antibodies are heterogenous, most probably because they consist of anti-F1 specific antibodies and of antibodies specific against other parts of the albumin molecule which cross-react with F1. In addition, monoclonal antibodies can recognize the mutation of a single amino acid residue in the albumin molecule.     

Preparation of antibodies to a synthetic C terminus of hirudin and identification of an antigenic site

Hirudin is a 65 amino acid anticoagulant peptide produced in the leech. The single polypeptide is cross-linked by three disulfide linkages in the NH2 terminal half of the molecule. A peptide corresponding to the COOH terminus (residues 45-65) was synthesized utilizing lysine 47 as a specific residue to conjugate to thyroglobulin as a carrier for raising antibodies in mice. Using an enzyme-linked immunosorbent assay (ELISA) technique, it was found that the major antigenic domain(s) was located between residues 52-65. The COOH terminal residues Ile-59, Tyr-63, and Leu-64 are crucial for maintaining the antigenic structure. The NH2 terminal region (residues 45-52) that is proximal to the carrier protein, however, was not immunoreactive. A possible mechanism by which antibodies recognize the COOH terminal region of the synthetic peptide and the strategy for raising such antibodies are discussed.     

Genomic sequence of an antigenic variant Newcastle disease virus isolated in Korea

In Korea, extensive Newcastle disease (ND) vaccine programs have been implemented, but ND outbreaks continue to occur occasionally, even in well-vaccinated farms. KBNP-4152 is a virulent ND virus, which has been isolated from vaccinated chickens in Korea. In this study, we conducted a comparison of the antigenicity of KBNP-4152 with that of a vaccine strain, La Sota, via virus-neutralization (VN) and cross haemagglutination-inhibition (HI) tests, and analyzed the genomic sequences. The antigenicity of KBNP-4152 was distinct from La Sota, and the expected genome size was 15,192 nt, as was the case with other recent virulent ND viruses analyzed. Based on the partial F gene, the strain was phylogenetically classified into the VIId genotype, but was distinct from other VII viruses due to amino acid changes at (E347K) and proximal to (M354K), the major linear epitope of HN, as well as relatively low amino acid similarity of the V protein, and a truncated W protein (203 aa vs. 227 aa). Therefore, KBNP-4152 is unique among genotype VII.     

中国汉族单纯型发作性运动诱发性运动障碍家系的致病基因定位研究

目的 在1个中国汉族单纯型发作性运动诱发性运动障碍(paroxyamal kinesigenic dykinesia,PKD)大家系中定位其致病基因所在区域.方法 用商业化的ABI微卫星试剂盒进行全基因组扫描,用Linkage和Genehunter等软件对基因分型的结果 进行参数和非参数连锁分析,并在最可能的阳性区域内进一步选择微卫星位点进行精细定位和单倍型分析.验证全基因组扫描结果 并缩小单纯型PKD家系的新位点区域.结果 全基因组扫描在D3S1580处得到最大的两点LOD值1.75(θ=0);精细定位在D3S3669处得到最大的LOD值2.82(θ=0),NPL值9.83.单倍型分析将致病基因定位于D3S1314和D3S1265之间约10.2 cM大小的区域.结论 该单纯型PKD家系的致病基因定位于3q28-29的D3S1314和D3S1265之间10.2 cM区域,是一个新的PKD致病基因位点.     

A relevant antigenic site in human growth hormone localized in sequence 98–128

The immunoreactivity of six synthetic peptides covering different lengths of human growth hormone antigenic region 73–128 has been studied using antisera from rabbits and guinea pigs and sera from two familial prenatal growth hormone-deficient patients submitted to replacement therapy. This survey disclosed that peptide 98–128 was responsible for the total antigenicity of human growth hormone, strongly indicating that a relevant epitope(s) is located in this region of the molecule.