F4-related mutation and expression analysis of the aminopeptidase N gene in pigs.

Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3' untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT-) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R-) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R- pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R- pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.

Differentiation of F4 receptor profiles in pigs based on their mucin 4 polymorphism, responsiveness to oral F4 immunization and in vitro binding of F4 to villi.

F4(+) enterotoxigenic Escherichia coli (F4(+) ETEC) are an important cause of diarrhoea and mortality in piglets. F4(+) ETEC use their F4 fimbriae to adhere to specific receptors (F4Rs) on small intestinal brush borders, resulting in colonization of the small intestine. To prevent pigs from post-weaning diarrhoea, pigs should be vaccinated during the suckling period. Previously, we demonstrated that F4acR(+), but not F4acR(-) piglets could be orally immunized with purified F4 fimbriae resulting in a protective immunity against F4(+) ETEC infections, indicating that this immune response was F4R dependent. Recently, aminopeptidase N has been identified as a glycoprotein receptor important for this oral immune response. However, in some oral immunization experiments, a few F4acR(+) piglets did not show an antibody response upon oral immunization, suggesting additional receptors. Therefore, the binding profile of F4 to brush border membrane (glyco)proteins was determined for pigs differing in F4-specific antibody response upon oral immunization, in in vitro adhesion of F4(+)E. coli to small intestinal villi, and in Muc4 genotype. Six groups of pigs could be identified. Only two groups positive in all three assays showed two high molecular weight (MW) glycoprotein bands (>250kDa) suggesting that these high MW bands are linked to the MUC4 susceptible genotype. The fact that these bands were absent in the MUC4 resistant group which showed a positive immune response against F4 and was positive in the adhesion test confirm that at least one or perhaps more other F4Rs exist. Interestingly, two pigs that were positive in the villous adhesion assay did not show an immune response against F4 fimbriae. This suggests that a third receptor category might exist which allows the bacteria to adhere but does not allow effective immunization with soluble F4 fimbriae. Future research will be necessary to confirm or reveal the identity of these receptors.

Maltose-binding protein is a potential carrier for oral immunizations.

Maltose binding protein (MBP) is often fused to a relevant protein to improve its yield and facilitate its purification, but MBP can also enhance the immunogenicity of the fused proteins. Recent data suggest that MBP may potentiate antigen-presenting functions in immunized animals by providing intrinsic maturation stimuli to dendritic cells through TLR4. The aim of this study was to examine if an MBP-specific immune response can be elicited by oral administration of MBP. Therefore, in a first experiment the MBP specific immune response was analyzed after oral immunization with MBP or MBP+CT to piglets and both the systemic and mucosal immune responses were examined Although no high systemic response was observed in the MBP-group, a local mucosal IgM MBP-specific response in the jejunal Peyer's patches was observed. In the second experiment MBPFedF was orally administered to piglets. A significant systemic response against MBP and a weak response against FedF were found after oral administration of MBPFedF+CT. Also the presence of MBP-specific IgA ASC in the lamina propria indicates that a local intestinal immune response against MBP was induced. Our data suggests that MBP can cross the epithelial barrier reaching the gut-associated lymphoid tissue after oral administration to pigs, which implicates that MBP could act as a carrier and delivery system for fused proteins to target the vaccine antigens to intestinal immune cells.

Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea in human and animal. In piglets, ETEC having F4 fimbriae (F4(+) ETEC) induce severe diarrhea, dependent on the presence of receptors for F4 (F4R). In this study, porcine aminopeptidase N (pAPN) was identified as an F4R by comparative proteomic analysis of brush border proteins of F4R(+) and F4R(-) pigs and by adherence/internalization experiments on pAPN-transfected cells. Binding of F4 fimbriae to pAPN depended on sialic acid containing carbohydrate moieties, and resulted in clathrin-mediated endocytosis of the fimbriae. Endocytosis via pAPN was not restricted to F4 fimbriae, but was also observed for anti-pAPN antibodies. Both F4 fimbriae- and pAPN-specific antibodies were taken up in vivo by porcine enterocytes and induced subsequently a rapid immunoglobulin A and G response. In conclusion, we identified pAPN as an endocytotic receptor for F4 fimbriae and highlight the opportunity to target vaccine antigens to this epithelial receptor.

IFN-γ expression and infectivity of Toxoplasma infected tissues are associated with an antibody response against GRA7 in experimentally infected pigs.

Toxoplasma gondii, an obligate intracellular parasite, can be transmitted to humans via the consumption of infected meat. However, there are currently no veterinary diagnostic tests available for the screening of animals at slaughter. In the current work, we investigated whether cytokine responses in the blood, and antibody responses against recombinant T. gondii GRA1, GRA7, MIC3 proteins and a chimeric antigen EC2 encoding MIC2-MIC3-SAG1, are associated with the infectivity of porcine tissues after experimental infection with T. gondii. Two weeks after experimental infection of conventional 5-week-old seronegative pigs, an IFN-γ response was detected in the blood, with a kinetic profile that followed the magnitude of the GRA7 antibody response. Antibody responses to GRA1, MIC3 and EC2 were very weak or absent up to 6 weeks post infection. Antibodies against GRA7 occurred in all infected animals and were associated with the presence of the parasite in tissues at euthanasia a few months later, as demonstrated by quantitative real-time PCR and isolation by bio-assay. Remarkably, although brain and heart tissue remained infectious, musculus gastrocnemius and musculus longissimus dorsi were found clear of infectious parasites 6 months after experimental infection. Seropositive response in a GRA7 ELISA indicates a Toxoplasma infection in pigs and is predictive of the presence of infectious cysts in pig heart and brain. This new ELISA is a promising tool to study the prevalence of Toxoplasma infection in pigs. Clearance of the infection in certain pig tissues suggests that the risk assessment of pig meat for human health needs further evaluation.

Effect of plasmid DNA encoding the porcine granulocyte-macrophage colony-stimulating factor on antigen-presenting cells in pigs.

We previously demonstrated that intradermal (ID) delivery of plasmid DNA encoding the porcine granulocyte-macrophage colony-stimulating factor (GM-CSF) 7 days before DNA vaccination enhances both cellular and humoral responses in pigs. In the present work, we studied the effect of the GM-CSF gene on antigen-presenting cells (APC) in pigs. We demonstrated that ID delivery of this gene significantly increased the number of epidermal CD1(+) cells (Langerhans' cells, skin dendritic cells) at the injection site at day 7. This was accompanied by an enhanced percentage of APC at the immune induction site following DNA vaccination, whereas a positive effect on APC maturation could not be demonstrated. Taken together, our data suggest that both DC recruitment to the immunization site and expansion of APC in the draining LN following DNA vaccination might contribute to the immune enhancing effect of plasmid encoded GM-CSF in pigs.

An enhanced GRA1-GRA7 cocktail DNA vaccine primes anti-Toxoplasma immune responses in pigs.

The protozoan parasite Toxoplasma gondii is the causative agent of a worldwide zoonosis and high prevalencies can be found both in animals and humans. An important source of human contamination with T. gondii is the consumption of raw or undercooked meat products. In this study, we evaluated whether DNA vaccination against T. gondii in pigs is able to generate immune responses known to be protective against tissue cyst formation. A GRA1-GRA7 DNA vaccine cocktail was enhanced by codon optimization of the encoding antigens and addition of heat labile enterotoxin expressing vectors as genetic adjuvant. Pigs vaccinated intradermally with this enhanced GRA1-GRA7 DNA vaccine cocktail developed high antibody levels against GRA1, GRA7 and a T. gondii lysate, and lymphocyte proliferation and production of IFN-gamma could be detected in these animals after challenge with the parasite. These results indicate that pigs can be efficiently primed against T. gondii infection by means of a DNA vaccine.

Comparison of immune responses in parenteral FaeG DNA primed pigs boosted orally with F4 protein or reimmunized with the DNA vaccine.

We previously showed that an intradermal (i.d.) FaeG DNA prime (2x)-oral F4 protein boost immunization induces a systemic response and weakly primes a mucosal IgG response in pigs, especially when plasmid vectors encoding the A and B subunit of the E. coli thermo-labile enterotoxin (LT) are added to the DNA vaccine. In the present study, we evaluated whether addition of 1alpha,25-dihydroxyvitamin D(3) (vitD(3)) to the DNA vaccine could further enhance this mucosal priming and/or modulate the antibody response towards IgA. To further clarify priming of systemic and mucosal responses by the i.d. DNA vaccination, we firstly compared the localization of the F4-specific antibody response in pigs that were orally boosted with F4 to that in pigs that received a third i.d. DNA immunization and secondly evaluated cytokine mRNA expression profiles after i.d. DNA vaccination. The i.d. DNA prime (2x)-oral F4 boost immunization as well as the 3 i.d. DNA vaccinations induced mainly a systemic response, with a higher response observed following the heterologous protocol. Co-administration of vitD(3), and especially of the LT vectors, enhanced this response. Furthermore, only the heterologous immunization resulted in a weak mucosal priming, which appeared to require the presence of the LT vectors or vitD(3) as adjuvants. In addition, the LT vectors strongly enhanced the FaeG-specific lymphocyte proliferation and this was accompanied by the absence of a clear IL-10 response. However, despite two DNA immunizations in the presence of these adjuvants and an oral F4 boost, we failed to demonstrate the secretory IgA response needed to be protective against enterotoxigenic E. coli.

Optimized FaeG expression and a thermolabile enterotoxin DNA adjuvant enhance priming of an intestinal immune response by an FaeG DNA vaccine in pigs.

One of the problems hindering the development of DNA vaccines is the relatively low immunogenicity often seen in humans and large animals compared to that in mice. In the present study, we tried to enhance the immunogenicity of a pcDNA1/faeG19 DNA vaccine in pigs by optimizing the FaeG expression plasmid and by coadministration of the plasmid vectors encoding the A and B subunits of the Escherichia coli thermolabile enterotoxin (LT). The insertion of a Kozak sequence and optimization of vector (cellular localization and expression) and both vector and codon usage were all shown to enhance in vitro FaeG expression compared to that of pcDNA1/faeG19. Subsequently, pcDNA1/faeG19 and the vector-optimized and the vector-codon-optimized construct were tested for their immunogenicity in pigs. In line with the in vitro results, antibody responses were better induced with increasing expression. The LT vectors additionally enhanced the antibody response, although not significantly, and were necessary to induce an F4-specific cellular response. These vectors were also added because LT has been described to direct the systemic response towards a mucosal immunoglobulin A (IgA) response in mice. Here, however, the intradermal FaeG DNA prime-oral F4 boost immunization resulted in a mainly systemic IgG response, with only a marginal but significant reduction in F4+ E. coli fecal excretion when the piglets were primed with pWRGFaeGopt and pWRGFaeGopt with the LT vectors.

Plasmid-encoded GM-CSF induces priming of the F4(K88)-specific serum IgA response by FaeG DNA vaccination in pigs.

We have used FaeG DNA to immunise piglets by the intradermal (ID) and the intramuscular (IM) route in a heterologous prime/boost model. ID immunisation with DNA resulted in a better induction of cellular immunity, whereas only the IM immunisation could prime an F4(K88)-specific serum IgA response. However, ID administration of plasmid-encoded GM-CSF 1 week before the ID immunisation enhanced the F4-specific humoral and cellular immune response and even primed the F4-specific IgA response more efficiently than the IM immunisation did.

Priming of piglets against enterotoxigenic E. coli F4 fimbriae by immunisation with FAEG DNA.

Early vaccination is necessary to protect pigs against postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). However, at present no commercial vaccine allows successful vaccination. This is partly due to the presence of maternally derived antibodies. Since DNA vaccines are suggested to be superior to protein vaccines in young animals with maternal antibodies, we determined whether the fimbrial adhesin (FaeG) of F4ac(+) ETEC could be used as a plasmid DNA vaccine to prime piglets in a heterologous prime-boost approach. Hereto, pcDNA1/faeG19 was constructed and expression of rFaeG in Cos-7 cells was demonstrated. Thereafter, pigs were immunised (days 0, 21 and 42) intramuscularly by injection or intradermally by gene gun and humoral and cellular immune responses were analysed. Even though responses were low, results demonstrated that intramuscular injection was superior to gene gun delivery for priming the humoral immune response since higher antibody titres were raised, whereas gene gun delivery better induced a cellular response, evaluated by a lymphocyte proliferation assay. Effective priming of the humoral immune response was evidenced by high IgG titres 1 week after a protein boost with purified F4. The low responses to the pcDNA1/faeG19 DNA vaccination suggest that delivery of the DNA and/or the expression of the faeG gene should be improved.