The Escherichia coli F18 receptor locus (ECF18R) has been genetically mapped to the halothane linkage group on porcine Chromosome (Chr) 6. In an attempt to obtain candidate genes for this locus, we isolated 5 cosmids containing the alpha (1,2)fucosyltransferase genes FUT1, FUT2, and the pseudogene FUT2P from a porcine genomic library. Mapping by fluorescence in situ hybridization placed all these clones in band q11 of porcine Chr 6 (SSC6q11). Sequence analysis of the cosmids resulted in the characterization of an open reading frame (ORF), 1098 bp in length, that is 82.3% identical to the human FUT1 sequence; a second ORF, 1023 bp in length, 85% identical to the human FUT2 sequence; and a third FUT-like sequence thought to be a pseudogene. The FUT1 and FUT2 loci therefore seem to be the porcine equivalents of the human blood group H and Secretor loci. Direct sequencing of the two ORFs in swine being either susceptible or resistant to adhesion and colonization by F18 fimbriated Escherichia coli (ECF18) revealed two polymorphisms at bp 307 (M307) and bp 857 (M857) of the FUT1 ORF. Analysis of these mutations in 34 Swiss Landrace families with 221 progeny showed close linkage with the locus controlling resistance and susceptibility to E. coli F18 adhesion and colonization in the small intestine (ECF18R), and with the locus of the blood group inhibitor S. A high linkage disequilibrium of M307-ECF18R in Large White pigs makes the M307 mutation a good marker for marker-assisted selection of E. coli F18 adhesion-resistant animals in this breed. Whether the FUT1 or possibly the FUT2 gene products are involved in the synthesis of carbohydrate structures responsible for bacterial adhesion remains to be determined.

BackgroundRecombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines.Methodology/Principal FindingsPeptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection.Conclusions/SignificanceAP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.

The alpha(1,2)fucosyltransferases (FUT1 and FUT2) contribute to the formation of blood group antigen structures, which are present on cell membranes and in secretions. In the present study we demonstrate that both FUT1 and FUT2 are expressed in the pig small intestine. FUT1 polymorphisms influence adhesion of F18 fimbriated Escherichia coli (ECF18) to intestinal mucosa, and FUT2 is associated with expression of erythrocyte antigen 0. The FUT1 polymorphisms result in amino acid substitutions at positions 103 (Ala-->Thr) and 286 (Arg-->Glu). Tightly controlled expression of the FUT2 gene results in either an abundance or an absence of mRNA in small intestinal mucosa. ECF18-resistant animals were shown to be homozygous for threonine at amino acid 103 of the FUT1 enzyme. Susceptibility to ECF18 adhesion appeared to be solely dependent on the activity of FUT1 in intestinal epithelia. In intestinal mucosae of ECF18-resistant pigs which expressed FUT1 but not FUT2 RNA, the levels of alpha(1,2)fucosyltransferase activity were significantly lower (28- to 45-fold, P<0.001) than in susceptible pigs. Moreover, lysates of CHO cells transfected with FUT1 constructs encoding threonine at amino acid position 103 also showed significantly reduced enzyme activity compared with constructs encoding alanine at this position. Our genetic and enzymatic studies support the hypothesis that the FUT1 enzyme, and particularly the amino acid at position 103, is likely important in the synthesis of a structure that enables adhesion of ECF18 bacteria to small intestinal mucosa.

In mammals, red/yellow and brown/black colorations are determined by the distribution of two pigments, phaeomelanin and eumelanin, respectively, the relative amounts of which are controlled primarily by two loci, extension and agouti. Dominant alleles at the extension locus increase brown/black pigmentation, while recessive alleles block eumelanin synthesis, thereby extending red/ yellow pigmentation within the hair follicle melanocyte. Robbins and associates (1993) have shown that the pigmentation phenotypes in mice controlled by the extension locus result from point mutations altering the function of the melanocyte-stimulating hormone receptor (MSHR). Johannson and colleagues (1994) demonstrated cosegregation of the chestnut (red) coat color in horses and polymorphisms at the MSHR locus. The black and red coat colors, respectively, in cattle are also controlled at the extension locus, the red color being due to a recessive allele (Searle 1968). The Holstein breed is stratified in red and black subpopulations. Gene flow from the black and white to the red and white subpopulation is through rare black carriers of the recessive red allele.To develop a direct test for determining the presence of the recessive allele causing red coat color in black Holstein cattle, we attempted to PCR amplify the bovine MSHR gene, assuming that the molecular basis of the bovine coat color phenotypes determined at the extension locus was similar to the one in horses and mice. The human (Chhajlani and Wikberg 1992) and mouse (Mountjoy et al. 1992) MSHR sequences were aligned to identify highly homologous regions suitable for designing primers based on the human sequences. In a first attempt we tried to amplify bovine DNA corresponding to a 544-bp segment of the human sequence using primer 1 and primer 2 (see Table 1). PCR was carried out in a reaction volume of 25 Ixl containing 100 ng of bovine or human genomic DNA, 10 mM Tris-HC1 pH 8.9, 50 mM KC1, 200 ~M of each deoxynucleotide, 0.4 ~M of each primer, and 2.5 U of Taq DNA polymerase (Boehringer Mannheim, Germany) in a thermal cycles (Hybaid Teddington, UK). MgC12 concentrations ranging from 1.5 to 3.0 mM with 0.5-mM increments were tested to optimize the PCR for specific amplification. After initial denaturation for 5 min at 95~ the PCR profile consisted of a denaturation step at 94~ for 30 s, an annealing step at 62~ for 30 s, and an elongation step at 72~ for 30 s for a total of 30 cycles, followed by a final extension of 7 min at 72~ The PCR product obtained when using bovine template DNA of a black Holstein animal consisted of two major bands between 500 and 600 bp, that is in the range of the human band at 544 bp. The two bovine bands were recovered from the agarose gel according to Heery and coworkers (1990) and subjected individually to another round of *Present address:

Induction of high frequencies of specific T cells by vaccination requires prime-boost regimens. To reach optimal immune responses, it is necessary to use different vectors for priming and boosting as e.g. DNA vaccination followed by boosting with a recombinant viral vector. Here, we show that vaccines based on virus-like particles (VLP) displaying peptide epitopes are equally effective to induce CTL responses if used in a homologous or heterologous prime-boost setting. Strikingly, high frequencies (>20% of CD8(+) cells) of protective CTL could be induced and maintained by weekly injection of VLP. Thus, the use of VLP may avoid the requirement for complicated heterologous prime-boost regimens, facilitating the development of effective T cell-based vaccines.