Profiles of Intestinal Membrane Vesicles and Viruses Associated to IBD – PRIMAVERA

To validate the commercial ILM device, the Videodrop, purified bacteriophage and membrane vesicle preparations from stool were obtained. The concentrations of the phage preparations were determined by different techniques: phage enumeration by lysis plates, quantitative PCR (qPCR), epifluorescence microscopy, ILM and NTA.
We enumerated viruses and vesicles in the stools of 40 individuals: 20 healthy and 20 patients with Crohn's disease, 10 of whom were in inflammatory flare at the time of collection, and 10 in remission.
Finally, we performed viral analysis of stool and blood samples from a cohort of five healthy volunteers and five IBD patients (participating in the Suivitech study). Blood virome analysis was added to the project to complement our data on microbiota dysregulation in IBD, correlating with altered intestinal permeability that could lead to translocation of intestinal viruses into the bloodstream. The viral DNA extraction and amplification protocol was developed in order to obtain a quantitative representation of the viruses.
Finally, we have developed a set-up allowing the simultaneous acquisition of interferometric and fluorescence signals. This simultaneous acquisition provides an additional parameter in the discrimination of signals from complex mixtures of nanoparticles.

We have shown that the Videodrop does not detect objects smaller than 90nm in diameter. As the majority of phages are smaller in diameter, pending the development of more sensitive devices by our partners, we used another technique for virus enumeration. However, as the majority of intestinal EVs are larger than 90 nm, we used the Videodrop to quantify them. We did not observe any difference in the amount of EVs between patients and controls, but we did observe more viruses in relapsing patients compared to healthy patients.

We observed that the blood plasma virome is dominated by bacteriophages infecting Proteobacteria, Bacteriodetes and Firmicutes in both cohorts. Although we did not observe quantitative differences in the viral load of IBD patients and healthy individuals, we found an increase in viral DNA overlap in blood and stool of IBD patients compared to healthy volunteers, consistent with the observed increase in intestinal permeability in IBD. Furthermore, in both blood and stool samples, an increase in the prevalence of temperate phages was also observed.

Fecal and blood viruses from 10 patients and 10 additional healthy subjects are being analysed. We will seek to identify the primary hosts of intestinal phages that are more abundant in Crohn's disease patients than in healthy individuals. This will allow us to understand whether their increased numbers in Crohn's disease play an active role in the disease, for example if phages in Crohn's patients are found to target a key species for symbiosis. The second objective of this project is to further characterise the phages found in the blood, and their similarity to intestinal viruses, in order to understand whether the altered membrane permeability in patients allows increased transport of intestinal phages across the intestinal epithelium, which could participate in the systemic inflammation observed in these patients. Finally, we will develop and use techniques for the preparation of phage DNA prior to sequencing that allow the identification of both single and double-stranded DNA phages, which is not possible with currently available methods.

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The enteric virome is an enormous and very diverse component of our intestinal microbiota whose effects are only now coming to light. It includes viruses that infect eukaryotic cells, but mostly phages that infect bacteria. Recent studies indicate that differences in enteric virome composition are markers of inflammatory bowel diseases, and in particular Crohn’s disease (CD). In addition, there are more and more reports of membrane vesicles secreted by the gut microbiota and by human cells, and their role is just starting to be revealed. Overall, there are good indications that the type and number of viruses and vesicles present in the gastrointestinal tract are indicators of the physiological state of individuals.
Despite these clues, the study of the enteric virome in health and disease is still in its infancy. This is even truer concerning vesicles. The paucity of studies is related to the technical difficulties of describing these bio-nanoparticles. For viruses, metagenomics is the reference method. However, it generally cannot give good estimates of viral abundance nor predict the host of viruses. Such titers are estimated by techniques based on epifluorescence microsocopy, but the method cannot accurately discriminate viral particles from vesicles, and is not well adapted to detect particles with few nucleic acids. Transmission electron microscopy, although valuable to distinguish viruses from vesicles, is not quantitative, very time consuming and necessitates expensive equipment. A recent method based on the Brownian motion of nanoparticles has been developed (Nanotracking analysis) to analyze vesicles and viruses, but this method fails to discriminate viruses from vesicles but also to detect small viruses.
A new imaging method, which we designate as interferometric microscopy, developed by the partner 2 of this proposal, has the potential to revolutionize the diagnostic of Crohn’s disease, based on stool bio-nanoparticles profiles. Indeed, interferometry enables to obtain information on the number and types of nanoparticles in a liquid suspension without purification nor expensive consumables. It takes advantage of interference phenomena between two signals: the first one being the direct signal from the light source, and the second one being the scattering signal from the particle. This project will allow to assess the market potential of the interferometer equipment and, in a second step, launch its commercialization.

We want to treat in parallel the same intestinal samples of a cohort of healthy people and CD patients from Saint-Antoine Hospital (partner 3) with interferometry and metagenomics approaches. This will serve first to validate by metagenomics that CD patients virus richness is far greater than that of healthy subjects (Norman et al. 2015). Second, we will determine whether this increased richness correlates with high virus titers by interferometric profile analyses. We expect that the differences in gut bio-nanoparticles composition will be captured by interferometry as efficiently as by the metagenomics approach, opening the way to an almost immediate and cheap test to orient CD diagnostic.