Proof of Concept

Figure 1. Visual representation of our four pillars


The proof of concept of our project relies on four main pillars as illustrated in figure 1. Each of the pillars was experimentally investigated as can be found in the results section. Here we describe how each pillar touches upon our project and how our results support our proof of concept.

Delivery Platform

We opted for an approach where we target the avian influenza virus at the most likely site of infection. Since avian influenza is an airborne virus, this will most likely be the lungs of poultry. According to our literature review we found Limosilactobacillus reuteri to be present within the lung microbiomes of chickens, hence we chose L. reuteri as the delivery platform of our nanobodies. Since the presence of L. reuteri is already proven by literature we did not require an extensive experimental investigation for our proof of concept. Nonetheless, we were still interested in confirming if Dutch poultry contain L. reuteri in measurable amounts. For this we did a microbiome analysis, for which we are still awaiting the sequence results.

Nanobody Secretion

Being able to insert a plasmid into L. reuteri is a foundational step in our proof of concept regarding the nanobody secretion pillar. If we are able to transform L. reuteri DSM 20016 with our electroporation protocol it would enable us to use this protocol to also transform our nanobody of interest into L. reuteri. Our results indicate that while L. reuteri DSM 20016 appears to have a low transformation efficiency, the transformation by electroporation is possible. We observed a single band at the expected size when transforming L. reuteri with the pYTK001 GFP vector. This band is a strong indication at least one transformation was successful, which would open up the possibility to, given more time, further optimize the protocol and eventually transform our pTRKH3 expression vector with our nanobody into L. reuteri DSM 20016. All-in-all, it seems our transformation efficiency is a technical problem and not a biological one, meaning it is solvable by optimization.

Nanobody Efficacy

The third foundational aspect of our project revolves around the ability to produce, purify, and experiment with the anti-avian influenza nanobodies. In order to develop and evolve our project the nanobody efficacy pillar is essential. With our Western blot results we have shown clear evidence we are able to produce and purify several nanobodies, either monovalent, as well as bivalent nanobodies. We even went beyond our proof of concept by producing and purifying an unpublished bivalent nanobody, namely the R1A-B6 linked to R1A-A5 by a (gly-gly-gly-gly-ser)6 linker.

The produced nanobodies were also used for ELISA experiments to assess the binding antigen-specific reactivity of our anti-avian influenza nanobodies to hemagglutinin. Due to time constraints we have attempted this experiment only once, which resulted in no measurable binding for any hemagglutinin. However, since even our positive result showed low absorbance, it can be inferred that we ran into a technical problem. By optimizing the ELISA protocol we should be able to measure binding affinities of our nanobodies, especially for the nanobodies which already showed binding affinity to several of the hemagglutinins. Since our range of avian influenza subtypes is much broader than literature, we should be able to expand further than what is currently known.

Project Safety

The fourth pillar of our project is the biocontainment of our therapeutic organism. Since we intend to apply our bacterium in-situ in the lungs of chickens where the expressed and secreted nanobodies can be diffused into the lung mucus. Such an application, though theoretically safe, will require a robust biocontainment strategy to prevent the bacterium from escaping the chicken lungs. For this purpose we designed a series of TlpA-based temperature kill-switches and a CarH-based light kill-switch. These cause the expression of lethal CRISPR-Cas9 complexes targeting 16s rRNA when the temperature drops below 36℃, 39℃ and 41℃, or when exposed to light respectively.

Though the assembly of these final constructs was not managed in time, we did construct a series of temperature sensor test-constructs, where the 6 temperature sensor variants control mRFP1 expression. These were all transformed into DH5α, and some in E. coli BL21, and the 36℃ constructs were tested at 37℃ for activity. This showed that the cells became fluorescent at this temperature. We also showed that one of the 4 constitutive promoter components was functional, and is affected by temperature changes as well.

The next steps would have been further transformation into the E. coli BL21 strain, which had shown better expression, and then electroporation transformation into L. reuteri DSM 20016. The microtiter plate fluorescence experiment has been mostly optimized, so we could then compare fluorescence at different temperatures in the 3 bacterial strains.

We had some trouble synthesizing the light sensor, and would need to get into contact with the original creators to acquire the template. We also need the CRISPR-Cas9 expression cassettes from the original creators, as we could not get them in time.

Finally we could then assemble all these components into the final kill-switches and test their effects on growth-rate at different temperatures and light-exposure. Additionally, the kill-switches could be incorporated into L. reuteri DSM 20016 genome to create the final OR-gated containment strain.