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Using a protein approach with Waddlia chondrophila and zebrafish as model organisms to conquer the female tubal factor subfertility market

Periodic Reporting for period 1 - PROZE (Using a protein approach with Waddlia chondrophila and zebrafish as model organisms to conquer the female tubal factor subfertility market)

Reporting period: 2017-09-01 to 2018-08-31

Worldwide 10-15% of couples are struggling to conceive, and approximately 25% of women will experience at least one miscarriage during their life. Early pregnancy loss and infertility are of a large emotional burden for the women and their partners, and diagnostic work-up and treatment requires hospital facilities and financial resources from the society.

Intracellular bacteria, such as Chlamydia trachomatis, may cause infection of the upper genital tract, leading to tubal pathology and infertility. Recently, several studies identified Waddlia chondrophila, an zoonotic emerging intracellular bacterium belonging to the order of Chlamydiales, to be associated with adverse pregnancy outcome and infertility problems such as tubal factor infertility.

At the moment, the diagnostics of W. chondrophila is performed by PCR, microimmunofluorescence (MIF) and an ELISA based on Waddlia whole outer membrane proteins. The PCR only detects an ongoing active infection as bacterial DNA still needs to be present in the samples. The MIF is time consuming and requires assessment of 2 independent reviewers. The whole outer membrane protein ELISA needs calibration prior each run and preparation of the outer membrane proteins is very time consuming and leads to differences in batch production. In conclusion, there is a need for an easy-to-use diagnostic serology test for W. chondrophila.

The aim of TubaScan is to develop an easy-to-use diagnostic test to measure antibodies against W. chondrophila in human serum. The ELISA will be based on W. chondrophila specific peptides, which makes the production easier and results in less cross reaction with other Chlamydiales.
The one-year PROZE project consisted of four different aims, which are described below.

1. Waddlia chondrophila culture and protein techniques.
At the start of the project the protocols and techniques to culture W. chondrophila were not present at TubaScan. During the PROZE project, we successfully set-up the W. chondrophila culture, this includes the culturing of Acanthamoeba castellanii in which W. chondrophila grows. Stocks of amoeba and W. chondrophila are stored for future usage and protocols are available within TubaScan.

2. Peptide selection
The selection of the peptides started with the usage of a database from our collaborator in Lausanne (Switzerland). This database contains the genomes of 77 Chlamydiales and it is possible to compare these genes and find similarities and differences. In addition, the results from a paper by dr. Julia Lienard were used as well, as she determined among others which outer membrane protein were able to react with human sera (including W. chondrophila positive serum). The combination of these two sources led to the selection of six promising proteins.

Those six proteins were ran through BLASTp. BLAST is an online program that compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. After this step, two proteins were excluded because they showed more homology with related Chlamydiales. At TubaScan, we do not have the expertise to identify the peptides which are most immunogenic, therefore we send the sequence of the four proteins of interest to GenScript who provided us with the immunogenic regions of each protein. Again, we used Blastp to select the most promising peptide regions, which led to the final selection of 12 peptides. These 12 peptides were produced by GenScript with a purity of >95%.

3. Sensitivity and specificity of the outer membrane ELISA versus the peptide ELISA
After the selection and production of the peptides, we used them to develop the peptide ELISA. The peptides were used in different combinations to coat the ELISA plates. Based on the results obtained from several experiments we concluded that the peptide combination of these four peptides do not influence the results. We did found out that a sample dilution of 1:50 and detection antibody concentration of 1:5000 were optimal.

If we make combinations (max 8 peptides per coating) of all peptides, we can see differences in OD value (the final outcome of the ELISA) between Waddlia negative/ doubtful samples and Waddlia positive serum samples. However, we only observe these differences when we preselect samples with a well-defined status (negative, doubtful or positive). Once we measure a larger batch of samples we do not observe these differences anymore, and we cannot clearly distinguish between the three different groups.

We came across several challenges for the WaddliaSCAN:

a. Better distinguish between negative, doubtful and positive samples
b. Determine cross-reaction with relevant Chlamydiales
c. Validation of peptide ELISA with a second sample cohort
d. Need for a positive control

4. Zebrafish model and CRISPR/Cas9
The associate was trained by the zebrafish expert from the MMI at the VUmc in a 2-week zebrafish course. In addition, there is a postdoc in TubaScan with extensive zebrafish experience. She has learned both the Innovative Associate and a junior researcher how to perform infections. All protocols needed to perform zebrafish injections (medium preparation, W. chondrophila preparation, and injections) are prepared and stored in TubaScan’s protocol database.

We decided to use the CRISPR/Cas9 system to make a Toll-like-receptor 2 (TLR2) knock-out. TLR2 is one of the important immune receptors which recognize bacteria. All needed primers, guide RNA and Cas9 protein are ordered and tested using a housekeeping gene. All materials to make the TLR2 knock out zebrafish are present and injections has started in September.
Based on the limited number of people working on W. chondrophila, of which almost all in an academic research only setting, and the questionable clinical relevance and pathogenicity of W. chondrophila we decided that the development of a commercial WaddliaSCAN is not realistic. The costs to develop an official CE-IVD approved diagnostic test, which can be used in both research and diagnostics, are too high in relation to the demand of the market.

Together with our collaborator in Lausanne (Switzerland), we aim to continue to work on the Waddlia peptide ELISA. However, the focus will be to develop the assay for research only. We do feel, that there is a clinical relevance for W. chondrophila, and the possibility to perform more and better research with an new available peptide ELISA would be of great interest.

The W. chondrophila expertise of the associate have led to many new collaborations, and the start and continuation of projects. So far, the PROZE project has led to:
- Improved and stronger collaborations in both the diagnostic, human and veterinary field
- More awareness and knowledge creation on W. chondrophila through presentations at two conferences
- Start of new projects which focus on OneHealth

All of these above points will increase the visibility of W. chondrophila, and it will lead to more research. The results of these new and ongoing projects will lead to more societal implications. Questions can be answered in relation of W. chondrophila and female reproductive health. In addition, our intended research will also provide information for individuals who work in close contact with farm animals and the association with W. chondrophila infection. Lastly, we aim to investigate W. chondrophila in the environment, such as in drinking and well water.