Jussi J. Joensuu

Glycosylated F4 (K88) fimbrial adhesin FaeG expressed in barley endosperm induces ETEC-neutralizing antibodies in mice

The F4-positive enterotoxigenic Escherichia coli (ETEC) strains are a frequent cause of porcine post-weaning diarrhea. Orally administered F4 fimbriae or FaeG, the major subunit and adhesin of F4, induce a protective mucosal immune response in F4 receptor-positive piglets. Feed plants carrying immunogenic subunit proteins can offer great advantages for oral vaccination of domestic animals. Here, we describe high-level endosperm-specific production (1% of total soluble proteins) of FaeG in the crop plant barley. The endoplasmic reticulum-targeted recombinant endospermic FaeG (erFaeG) was shown to be heterogeneously glycosylated. The erFaeG showed resistance at digestive conditions simulating piglet gastric fluid. Glycosylation did not abolish the immunogenic character of the FaeG protein, since erFaeG was able to induce F4 fimbria-specific antibodies in mice. Biological activity of these anti-F4 antibodies was demonstrated in vitro by blocking the attachment of the F4+ ETEC to the F4 receptors present on porcine intestinal enterocytes.

Fimbrial Subunit Protein FaeG Expressed in Transgenic Tobacco Inhibits the Binding of F4ac Enterotoxigenic Escherichia coli to Porcine Enterocytes

Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed. Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants. In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein. Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment. For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast. The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein. The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions. Plant-produced FaeG proved to be stable up to 2 h under these conditions. The binding and inhibition properties were tested with isolated piglet villi. These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner. Thus, the first two prerequisites for the development of an oral vaccine have been met.

Transgenic plants for animal health: plant-made vaccine antigens for animal infectious disease control

A variety of plant species have been genetically modified to accumulate vaccine antigens for human and animal health and the first vaccine candidates are approaching the market. The regulatory burden for animal vaccines is less than that for human use and this has attracted the attention of researchers and companies, and investment in plant-made vaccines for animal infectious disease control is increasing. The dosage cost of vaccines for animal infectious diseases must be kept to a minimum, especially for non-lethal diseases that diminish animal welfare and growth, so efficient and economic production, storage and delivery are critical for commercialization. It has become clear that transgenic plants are an economic and efficient alternative to fermentation for large-scale production of vaccine antigens. The oral delivery of plant-made vaccines is particularly attractive since the expensive purification step can be avoided further reducing the cost per dose. This review covers the current status of plant-produced vaccines for the prevention of disease in animals and focuses on barriers to the development of such products and methods to overcome them.

The polymeric stability of the Escherichia coli F4 (K88) fimbriae enhances its mucosal immunogenicity following oral immunization

Only a few vaccines are commercially available against intestinal infections since the induction of a protective intestinal immune response is difficult to achieve. For instance, oral administration of most proteins results in oral tolerance instead of an antigen-specific immune response. We have shown before that as a result of oral immunization of piglets with F4 fimbriae purified from pathogenic enterotoxigenic Escherichia coli (ETEC), the fimbriae bind to the F4 receptor (F4R) in the intestine and induce a protective F4-specific immune response. F4 fimbriae are very stable polymeric structures composed of some minor subunits and a major subunit FaeG that is also the fimbrial adhesin. In the present study, the mutagenesis experiments identified FaeG amino acids 97 (N to K) and 201 (I to V) as determinants for F4 polymeric stability. The interaction between the FaeG subunits in mutant F4 fimbriae is reduced but both mutant and wild type fimbriae behaved identically in F4R binding and showed equal stability in the gastro-intestinal lumen. Oral immunization experiments indicated that a higher degree of polymerisation of the fimbriae in the intestine was correlated with a better F4-specific mucosal immunogenicity. These data suggest that the mucosal immunogenicity of soluble virulence factors can be increased by the construction of stable polymeric structures and therefore help in the development of effective mucosal vaccines.

Plant biotechnology for deeper understanding, wider use and further development of agricultural and horticultural crops

Yhteyttavat kasvit ovat ravinnon ja bioenergian ensisijainenlahde maapallolla. Viljelykasvien ominaisuuksia muokkaamalla seka kasvien biosynteesikoneistoa eri tavoin hyodyntamalla pyritaan vastaamaan haasteisiin, joita vaestonkasvu ja sen myota lisaantyva energian seka ravinnon tarve tuovat ihmiskunnalle. Lisaksi on pyrittava hillitsemaan ilmastonmuutosta. Taman katsauksen tarkoituksena on osoittaa, kuinka kasvibioteknologia auttaa vastaamaan naihin haasteisiin. Esimerkkeina kaytetaan mm. perunan jalostukseen, viruskestavyyteen ja kylmyydensietoon seka sadelatvan (gerberan) kukankehitykseen ja kukintaan kohdistuvaa bioteknologista tutkimusta. Se on tuottanut tietoa tarkeiden kasvigeenien sijainnista ja rakenteesta kasvigenomeissa, geenien saatelysta kehityksen aikana seka geenien reagoinnista ympariston muutoksiin ja taudinaiheuttajien tartuntaan. Naita tietoja tarvitaan, jotta kasvien tuottavuutta ja kasvituotteiden laatua voidaan entisestaan parantaa mm. biotekniikan keinoin. Molekyyligeneettiset tutkimukset tuottavat myos geenimerkkeja, joilla voidaan tehostaa perinteista risteytysjalostusta. Geenitekniikan avulla on voitu kehittaa uusia viruskestavyyden mekanismeja kasveihin. Biotekniikan menetelmia voidaan hyodyntaa myos syotavien rokotteiden tuottamiseen seka farmasianteollisuuden tarvitsemien proteiinien tuotantoon kasveissa. Toisaalta biotekniikan menetelmia tarvitaan geenivarojen kartoittamiseen seka geneettisen monimuotoisuuden ja geenivirtojen selvittamiseen kasvipopulaatioissa. Nailla tutkimuksilla on merkitysta niin kasvien geenivarojen hyodyntamiselle kuin riskien tunnistamiselle seka sellaisten viljelykaytantojen kehittamiselle, joilla voidaan ehkaista maa- ja puutarhatalouden haittavaikutuksia viljely- ympariston ulkopuolella. Tulevaisuudessa kasvibiotekniikalla nahdaan entista suurempi merkitys haettaessa ratkaisuja suuriin haasteisiin, jotka liittyvat ihmiskunnan selviytymiseen. Tahan tarvitaan kasvibiotekniikan osaamisen kartuttamista myos kehitysmaissa.