Building a fast selection method that is feasible in a clinical setting

Based on discussion with Dr. med Andreas Kronenberg

Input:
Dr. med. Kronenberg pointed out the necessity of developing of a quick selection method for specific phages within our library. This method should be feasible in a clinical setting and would give our project clinical relevance.

Adjustment:
We have designed and built a bioreactor, that can mix the phage library with the novel host and measure the optical density continuously. To learn more about our bioreactor, visit the <a class="a-link-neg-margin" href="https://2019.igem.org/Team:ETH_Zurich/Hardware">hardware page.</a>

Reducing biosafety risk

Based on discussion with Dr. Ursula Jenal

Input:
Dr. Jenal suggested we do a risk assessment to evaluate the potential impact of our phage libraries on the environment.

Adjustment:
You can find our risk assessment <a class="a-link" href="https://static.igem.org/mediawiki/2019/5/58/T--ETH_Zurich--RiskAssessment_pdf.pdf">here.</a> We concluded that the risk to impact the ecosystem by generating phages with a broad-host range is low. However, as this risk cannot be neglected, we implemented additional safety measures in the lab once we were working with phage libraries as indicated in the report.

Improving infectious potential through phage training

Based on discussion with Dr. Grégory Resch and Dr. Jean-Paul Pirnay

Input:
Dr. Resch and Dr. Pirnay highlighted that even though the tail fiber protein is very important for host specificity, the infectious potential (the ability of the phage to kill its host) is influenced by the whole genome. Achieving binding but not infection would not be of interest in clinical applications where the main goal is lysis of pathogenic bacteria.

Adjustment:
To address this problem, we adapted our selection process to include iterations of phage training. Phage training consists in performing the phage selection procedure multiple times. In practice this is accomplished by isolating the amplified phages and infecting the target host strain again. During each iteration mutations are accumulated across the whole phage genome. In this manner the selected phage has an increased infectivity with respect to the phage isolated during the first selection step.

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Input

Prof. Dr. med. Jürg Hafner

Department of Dermatology, University Hospital Zurich
Has experience with resistant bacteria through his work with patients who are having chronic wounds.

• Chronic wounds are very common: 1% of the population is expected to deal with a chronic wound in his or her lifetime. They are mainly caused by diabetes and insufficient blood flow in arteries and veins. Roughly 80% of these wounds are populated by bacteria, and in a third of these cases pathogens like Methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes and Pseudomonas aeruginosa are found, which are not part of the natural microbiome.
• From literature it is hard to verify the influence of bacteria on wound healing as no randomized clinical trials have been carried out to study this phenomenon yet. However, empirical data suggests, that bacteria can form biofilms on wounds, which presumably influence wound healing in a negative way. Thus, it is likely that bacterial infections are relevant for wound healing and for this reason should be studied more extensively.
• The current treatment of chronic wounds includes periodical debridement of the biofilm and topical disinfection using octenilin for example. Additionally, treatment to remove the cause of the disease is prescribed including the decrease of venous congestion, the release of water from the leg and the use of catheters to improve blood flow. In case of non-healing wounds, surgical procedures are necessary. The application of antibiotics is not desirable as it poses the risk to select for antibiotic resistant bacteria.
• Currently phages are not being used to treat chronic wounds. However, phage therapy would be a promising strategy to fight bacterial infections as it would remove the need for antibiotics. For various other conditions the systemic administration of phage therapy could be used.

Désirée Schwendimann

Assistant doctor in anesthesia, Inselspital Bern
Has come in contact with patients that are infected by multi-resistant bacteria.

• Ms. Schwendimann gave us an insight into how antibiotic resistance affects her work as an anesthetist.
• Thorough cleaning is necessary whenever a hospital has a patient with resistant bacteria and various safety measures have to be followed.
• In case of a surgery, the whole operation room has to be deep cleaned, before it can be used again.
• She mentions that hospitals in the Netherlands have a system that limits the occurrence of antibiotic resistances. Nurses have less patients, which leads to fewer mistakes with cleaning hands between patients, surgeries are performed less frequently on elderly people and they always have immunologists in their teams that check for the presence of bacteria.

[1] Clinical Indications and Compassionate Use of Phage Therapy: Personal Experience and Literature Review with a Focus on Osteoarticular Infections. Patey O. McCallin S. Mazure H. Liddle M. Smithyman A. Dublanchet A. Viruses, Vol. 11, No. 18, 2019

Input

Dr. Grégory Resch

Department of Fundamental Microbiology (DMF), University of Lausanne
Expert in the field of phage research

• No major side effects from phage therapy were observed in clinical trials to date and endotoxin shock from the lysis of the infectious bacteria has never been reported.
• Treatment of a patient in France with an intravenously applied phage cocktail for over six weeks did not show any adverse effects.
  
• Non-purified phages, which contain high levels of endotoxins from phage amplification in the bacterial host, may lead to inflammation. Therefore, purification is necessary and can only be done by specifically equipped facilities. Currently theses are the Queen Astrid Military Hospital in Belgium, Pherecydes Pharma in France and USC San Diego and Adaptive Phage Therapeutics in the USA.
• Concerning interactions with the microbiome in case of oral application, a study from Nestlé targeting patients with diarrhea found that phages might not be as aggressive against commensal bacteria as antibiotics. However, results were influenced by the fact that many phages were likely already killed by the stomach acid and didn't reach the intestine.
• A prophage is a DNA sequence of phage origin that got incorporated into the genome of bacteria. Temperate phages will be silent in the host, but can be reactivated and be an indirect trigger for various human diseases. The software PHASTER can be used to check whether a bacterium has a phage sequence in its genome.

Dr. Jean-Paul Pirnay

Laboratory for Molecular and Cellular Technology, Queen Astrid Military Hospital Brussels
Expert in the field of phage research

• Purification of the phage solution is necessary before application in patients, otherwise the endotoxin produced by bacteria during phage amplification can cause severe complications, especially in intravenous applications. However, purification will always lead to the reduction of variability, as some phages stick to the filtration column and will be removed.
• The phage genome has to be sequenced to ensure the absence of toxic genes.
• To improve patient safety and efficacy of the treatment, researchers should aim to better understand the role of the immune system on the one hand and the synergy or disynergy between phages and antibiotics or between multiple phages on the other hand. Furthermore, phage resistance needs to be studied as well.

[1] A Systemic and Critical Review of Bacteriophage Therapy Against Multidrug-resistnt ESKAPE Organisms in Humans. Haddad LE. Harb MA. Stibich MA. Chemaly RF. Clinical Infectious Diseases, Vol. 69, pp. 167 – 178, 2019

Input

Dr. Ursula Jenal

Jenal & Partners Biosafety Consulting

• T7 phage is categorized as BSL-1. Its narrow host range among bacterial strains, incapability to infect eukaryotic cells and decreased efficiency of DNA packaging under UV light reduces its capacity to survive in nature and disbalance the ecosystem in case of an accidental release from the laboratory.
• If we randomize the tail fiber protein of T7 however, the situation has to be analyzed anew, as no laws are in place that regulate this case. Therefore, a risk assessment is necessary. We conducted one that you can find <a class="a-link" href="https://static.igem.org/mediawiki/2019/5/58/T--ETH_Zurich--RiskAssessment_pdf.pdf">here.</a>

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Dr. Grégory Resch

Department of Fundamental Microbiology (DMF), University of Lausanne
Expert in the field of phage research

• Infectious phages from our library most probably already exist in nature, but haven't been found and characterized yet. Therefore, they should not be considered to be GMO and most likely do not affect the environment.
• If a phage is created, that can infect and lyse many bacterial strains, it can only be used in a highly controlled environment to prevent destabilization of the ecosystem.

[1] Contained Use of Bacteriophages: Risk Assessment and Biosafety Recommendations. Verheust C. Pauwels K. Mahillon J. Helsinki DR. Herman P. Applied Biosafety, Vol. 15, No. 1, pp. 32 – 44, 2010

Input

Pia Stadelmann

Swissmedic

• Swissmedic explained us the legal framework for the use of bacteriophages.
• Phages are considered to be medicinal products and therefore the provisions of the Therapeutic Product Act (HMG) apply.
• They must have a production authorization granted by Swissmedic and comply with Good Manufacturing Practice (GMP).
• For clinical trials, a license from the ethics commission and Swissmedic is required.
• The use of the product under a temporary license (“compassionate use”) is only possible if the product is already in clinical trials.

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Dr. Jean-Paul Pirnay

Laboratory for Molecular and Cellular Technology, Queen Astrid Military Hospital Brussels
Expert in the field of phage research

• Regulatory issues include the declaration of phages as medicinal products, which require Good Manufacturing Practice, preclinical studies, phase I, II and III clinical trials and centralized marketing authorization. This is too expensive as phages are biological entities that cannot be patented. Moreover, this process is also not feasible for an evolving therapy.
• In Belgium phages were used earlier under the WMA declaration of Helsinki, Article 37, which enables their use for desperate cases without the need to perform clinical trials and work under GMP conditions. While demonstration of the efficiency of phage therapy is not feasible from compassionate use cases only, the generated data suggests that phage therapy is safe, as no adverse effects were detected. The main problem with this approach is however, that too much time is lost in case of critically ill patients.
• The Dedicated PT framework is a new concept elaborated by scientists in cooperation with Belgian Authorities. It includes the characterization of phages, their storage in a banking system, the production of a single phage (active pharmaceutical ingredient) using a suitable host according to a monograph and external quality testing by a "Belgian Approved Laboratory". Then approved phages can be bought in a pharmacy upon prescription as magistral preparations.

Dr. Grégory Resch

Department of Fundamental Microbiology (DMF), University of Lausanne
Expert in the field of phage research

• The phage from which the library is derived needs to be well characterized and has to undergo a clinical trial.
• The evolved phages don't have to undergo this procedure again, as they underwent only an accelerated natural selection process. Instead they only have to be sequenced to exclude the existence of dangerous sequences.
• A physician with no other treatment option can contact Swissmedic via a special demand form. Phages that were authorized in another country with similar medical standards can be used in this case.

[1] Clinical Indications and Compassionate Use of Phage Therapy: Personal Experience and Literature Review with a Focus on Osteoarticular Infections. Patey O. McCallin S. Mazure H. Liddle M. Smithyman A. Dublanchet A. Viruses, Vol. 11, No. 18, 2019

Input

Dr. med. Andreas Kronenberg

Member of scientific board at the Swiss Center for Antibiotics Resistance
Group Leader at Institute for Infectious Diseases, University of Bern

• Andreas Kronenberg recognizes the importance of our project.
• Identification of infecting phages needs to be more rapid than the current method which takes 24 – 48 h.
• Each bacterial infection case is different, and a therapy that takes this into account and that can be deployed in all cases is desirable.

Dr. Jean-Paul Pirnay

Laboratory for Molecular and Cellular Technology, Queen Astrid Military Hospital Brussels
Expert in the field of phage research

• Efficacy of infection depends on many parameters including but not limited to the tail fiber protein. Many parameters play an important role: attachment, high replication number, rapid infection, induction of resistance response and optimal working temperature (37 °C).
• Mutations in the whole genome would be necessary for maximal efficacy in library generation.
• For clinical applications a concentration of 10^7 PFU/ml is necessary.
• The entire phage genome needs to be sequenced to ensure the absence of toxic genes.
• The stability of phages has to be tested after their amplification.

Prof. Dr. med. Jürg Hafner

Department of Dermatology, University Hospital Zurich
Has experience with resistant bacteria through his work with patients who are having chronic wounds.

• A biological tool to kill bacteria would be very useful because the use of antibiotics should be limited to avoid the generation of resistant bacteria.
• A first application for our product, which could be easily tested, are chronic dermal wounds. However, systemic intra-venous treatment for various conditions could be considered at a second stage.

Dr. Grégory Resch

Department of Fundamental Microbiology (DMF), University of Lausanne
Expert in the field of phage research

• He appreciates that our method allows for adaptation in the course of treatment and that new phages can be identified to account for the potential emergence of resistance.
• Suggests using T3 sequence as a positive control to verify whether our system is working.
• Compare the results to classical phage training.
• Changing the tail fiber sequence alone might not lead to the maximum efficacy of infection.
• Phages might aggregate before application. This can be avoided by leaving the phages on different plates and only mixing shortly prior to application.
• The stability of phages can be evaluated via titer measurement.

Scientifica

Scientifica is a fair that aims to educate the broad public about the most recent scientific achievements. It includes special activities for children as well as a specific program for politicians and economists. Scientists have the opportunity to explain their projects, raise enthusiasm for science and get valuable feedback from a very diverse audience. We shared the iGEM booth with last year’s team, AROMA, and had the opportunity to present our project and explain the principles of synthetic biology in general.

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• The project was received well by the public and visitors were open to the idea of using phages to treat bacterial infections.
• The concerns that were raised more often were the price of phage therapy, a comparison of modified phages to naturally occurring phages, biosafety issues and the efficacy of treatment.

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Presentation at the Intra-Departmental Seminar

The Seminar is a series of talks that takes place during the semester at the Department of Biosystems Science and Engineering (D-BSSE) of ETH. The talks are great for interaction and the sharing of expertise with scientists from different backgrounds. We were given the opportunity to present our project during the seminar on the 4th of October. During the discussion some good questions were raised that helped to direct our work in the last weeks before the Jamboree.

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