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IgA-based oral therapy for protection of piglets against infection with enterotoxigenic E. coli.

Periodic Reporting for period 1 - ImmunoFarm (IgA-based oral therapy for protection of piglets against infection with enterotoxigenic E. coli.)

Reporting period: 2015-05-01 to 2017-04-30

After weaning, the immune protection acquired during the lacteal period is lost, exposing animals to a number of infections such as Postweaning diarrhoea (PWD) in farm piglets. PWD is a common disease that generates significant economic losses to pig farms and it is caused by enterotoxigenic E. coli (ETEC) bacteria (mainly F4 and F18 strains) that infect the piglets after they are weaned. The oral administration of antibodies as passive immunotherapy agents is a very promising solution which prolongs maternal lactogenic immunity against post-weaning infections. For this, recombinant antibodies, in particular light chain-devoid IgAs, were produced in the seeds of Arabidopsis thaliana and Glycine max (soya) to be delivered to the animals as non-purified formulations. The aim of this project has been to generate a cost-effective prophylactic product intended to prevent PWD in piglets.
To achieve the main goals of this project, all the sections described in the project proposal were covered:
1: Cloning.
• Four different anti ETEC-F18 antibodies in an IgA-like format were cloned by means of Gateway (Fig. 1)
• All four constructs were transformed directly to both A. thaliana and G. max (soya). The project described an intermediate step of transient expression in N. benthamiana, but because we already had selected the 4 best VHH candidates, this step was not necessary.

2: In vitro testing.
• Competitive ELISA: In order to determine if the different antibody variants behaved in a competitive manner, a competitive ELISA setting was designed where all the different antibody versions were tested against each other. The outcome of the competitive ELISA setting was that every combination presented competition.
• ETEC bacteria – gut villus adhesion assays: In order to test the inhibitory function of the antibodies on ETEC adhesion, in vitro binding inhibition assays were performed. The results showed that the samples of soybean seed extract containing anti-FedF antibodies were able to prevent the attachment of E.coli to the piglet gut villae, whereas negative controls consisting of wild type soybean seed extracts and PBS were not able to prevent the binding (Fig. 3).
• Integrity testing: Simplified IgA antibodies are cleaved in a way that seems dependent on the VHH (Fig. 2), as the same format suffers more or less cleavage depending on which VHH is fused. The sequences of these antibody fusions were therefore sent for modelling. The modelling results (Fig. 4) conclude that probably a cleavage point in the linker between CH2 and CH3, KPKVNTFR is the coausant of the cleavage of our IgA antibodies.
• Quantification of antibodies present in the seed extracts: The concentration of functional antibodies present in the seed extract was calculated by means of ELISA tests. Results of the quantification are shown in Table 1.

3: In vivo testing.
Four different experimental groups consisting of 5 F18-seronegative piglets each were prepared as described in Table 2. As defined in Fig. 5 the experimental groups were provided with pig feed complemented with the elite IgA crushed seeds for 10 days. Blood and faeces samples were taken throughout the experiment as well as piglet weight data, feed intake and vitality observations. Piglets were challenged with O138 ETEC strain on third and fourth days after in-feed prophylaxis. The prophylactic capacity of seed cocktails was evaluated following four approaches: (i) bacterial excretion in faeces, traced during 2 weeks after the bacterial challenge, (ii) evolution of the Ig seroconversion rate by quantification of anti F18 fimbria-specific antibodies in blood samples, which evidences the exposure to ETEC pathogen, (iii) weight gain of challenged piglets and (iv) feed intake of challenged piglets. During the course of the experiment, two of the piglets of the control group deceased, however, after post-mortem physiological and microbiological examination it could not be concluded that the cause of the death was the ETEC infection. As shown in figure 6a and 6b, no differences could be observed in the parameters of F18-ETEC shedding and weight gain between the experimental and control groups, whereas Fig. 6c shows only slight differences in feed intake between the experimental and the control groups. however data is not conclusive and further challenges should be made with previous adjustment of the ETEC-challenging dose.
It has been shown that the best suited antibody isotype for passive immunotherapy is IgA, the most abundant isotype in the mucosal immune system. Therefore, the design and production of the antibodies against ETEC-F18 in this project has been focused on simplified IgA versions. Plant-expression systems are an attractive platform for production of IgA antibody cocktails. They are cost-effective, highly scalable and have a low risk of contamination with mammalian pathogens.
The ideal platform for production of antibodies aimed at oral passive immunization for veterinary, and even medical uses is such that can produce bulk quantities in short time, does not need expensive purification or formulation and can be stock-piled without the need of refrigeration. Thus, commercial seed crops are ideal cost-effective production platforms and offer the added benefits of being a natural source of protein and providing a stable environment for storage and maintenance. Moreover, this proof of concept might as well be very valuable for public health emergencies such as viral outbreaks, because large quantities of antibodies could be stock-piled in a ready-to-use formulation and could be quickly delivered to the population if required, without the need of syringes or medical instrumental, just as a food additive. Soybean being a commercial crop can be scaled up at a cost effective scale and is an important source of protein used in animal feed, demonstrating its convenience to be used as a feed additive.
With our ImmunoFarm project, and together with a partner project in the laboratory, not only we have generated a feed complement that is currently being evaluated by a company for its future commercialization, but also, we have paved the way to a great variety of uses of in-seed production of antibodies aimed for oral passive immunization, which will be of great socio-economic impact.
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Table 2
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Table 1
Figure 2