Project Description

Describe how and why you chose your iGEM project.


PROBLEM

COVID-19 pandemic began in December 2019 and novel effective vaccines have been developed at a speed faster than ever. Most successful vaccines and the emergency use authorization (EUA) to COVID-19 vaccines were issued for Pfizer-BioNTech mRNA vaccine in December 2020 and Oxford-AstraZeneca adenovirus vaccine in January 2021.

Current vaccine explained

The below short video we made explains the principles of mRNA and adenovirus vaccines compared to the traditional protein vaccine.

The safety issues

The mRNA and adenovirus vaccines have features of delivering antigen genes to host cells where, in the case of SARS-CoV-2, the Spike protein will be produced and trigger the immune response. However, the safety of the Spike proteins was concerned1 with toxicities including disrupting lipid metabolism in liver, heart, kidney damages2, and even crossing the blood-brain barrier in mice3.

In our project, we’d like to seek a safer vector which can transfer and express antigen gene in the non-human cells in the human body. Commensal bacteria such as E. coli living in human intestine may be a good choice to express antigen proteins and induce intestine immune response.


INSPIRATION

iGEM 2021 project - Mingdao

The bacteriophages are viruses infecting and killing bacteria. The previous iGEM project of Mingdao in 2021 utilized the engineered reporter phage to detect Salmonella bacteria in food poisoning. Based on the skills built up, we, as Mingdao iGEM team in 2022, were inspired to extend the technique of phage engineering to achieve our goal.

iGEM 2020 project - TUDelft

In the previous iGEM project of TUDelft in 2020, they aimed to kill locusts by the toxic proteins produced by the gut bacteria which were infected with the engineered phages carrying toxin gene (Cry7Ca1, a gene from Bacillus thuringiensis) through the spray. Their mode of phage - gut commensal bacteria - protein production encouraged us to design in a similar way to create phage vaccine in a relationship of phage, intestine commensal E. coli and antigen protein production.

Clinical trial - BiomX, Inc

For clinical application, an Israel biotech company, BiomX, Inc., has completed a preclinical study demonstrating that a feasible approach to cure colorectal cancer in mice by cytokines production in bacteria surrounding the tumor through the infection of the engineered phages carrying genes of GM-CSF, cytosine deaminase, IL-15 in a similar mode of phage - tumor co-existing bacteria - cytokine protein production. The success of the trials gives us hope to believe that a potential phage vaccine can be created in such a way with synthetic biology.


GOAL & AIMS

Build an antigen model to study vaccine candidate

The chicken ovalbumin (OVA) is a glycoprotein, which is a major component of chicken egg whites, and harbors immunogenic properties in vaccination experiments4. The recombinant OVA protein is readily purified in E. coli BL21 system driven by T7 promoter and triggered by IPTG induction. Therefore, it’s considered as one of the well-known model antigens used to study vaccine effectiveness and immune responses in animal models either using the purified proteins with adjuvants5 or using the antigen-encoded gene carried by vectors6 (e.g., viruses, liposomes, nanoparticles., etc.).

To demonstrate the vaccine effectiveness, our goal is to build up the OVA antigen expression system driven under T7 promoter. The following are our specific aims:

  1. Construct the BioBrick basic part of OVA gene which codon is optimized for E. coli expression.
  2. Improve the expression system by introducing a leader sequence of T7 phage gene 10 (g10) in front of the canonical RBS
  3. Build up the BioBrick composite part of T7 promoter-g10.RBS-OVA-T7 terminator
  4. Test the functionality of the above parts

Engineer phages carrying antigen gene to express proteins in targeted bacteria

T7 bacteriophages are one of the model phages in the lab. T7 promoter regulation is well studied and readily controlled in E. coli system. The genome of the phages can be edited in many ways including a commercially available kit. In addition, and importantly, T7 phages are able to infect E. coli and have potential to infect the intestinal commensal E. coli.

To create T7 phage vaccines, the aims are listed below.

  1. Test the T7 phage susceptibility of the isolated intestinal E. coli
  2. Engineer T7 phage genome to carry GFP or OVA genes by the T7Select® 415-1 Cloning Kit (Merck Ltd.)
  3. Measure the GFP or OVA protein expression in the engineered phage-infected E. coli.

FLOWCHART & EXPECTED RESULTS

Below is a short animation that describes our experimental flowchart and the expected results.

Intestine immune responses

Currently, COVID-19 vaccines and most of others were administered through intramuscular injection. To evaluate the potential effectiveness of an oral vaccine, we researched scientific papers working in field of intestine immune responses. The human small intestine with well-structured gut-associated lymphoid tissue (GALT) and gut regional lymph nodes can trigger immune responses including B-cell antibody producing7. However, in addition to pathogens, the food antigens and commensal microbial antigens increase the complexity of the tolerance of the small intestinal immune system. Our target of E. coli predominantly lives in the large intestine of humans8,9, where, regardless of fewer studies, the leukocyte populations and well-developed lymphoid tissues have been identified and are able to induce varies immune responses predominantly against microbial antigen7,9. Therefore, in our design, the T7 phage and the bacterial target (e.g., E. coli) and the localization of the immune system may be ideal to induce immune responses against foreign pathogenic antigen in the large intestine.

Adjuvant-like properties of phage and bacterial debris

Adjuvants play an important role in vaccines for stimulating optimal immune responses to antigens such as aluminum-based and saponin-based materials, as well as water-in oil emulsion or Toll-like receptors (TLRs) ligand-based adjuvants10. In addition, the bacterial envelopes including E. coli have potential to enhance and improve the protective immunity11. The immunostimulatory properties are named bacterial ghosts (BG) to define this adjuvant-like features. Furthermore, bacteriophage-based vaccines were proved self-adjuvanted nature, in which adding traditional adjuvants such as aluminum or liposomes did not further increase the intensities of immune responses12. In sum, we believe that the antigen produced by E. coli infected and killed by phage carrying antigen-encoded gene will evoke desirable immune responses to generate specific antibodies.


APPLICATION IN THE REAL WORLD

For safety concerns, the use of phage therapy was considered in clinical practices13. In addition to BiomX, using the engineered phages to treat a patient with a drug-resistant Mycobacterium abscessus has been reported in a clinical trial in UK in 201914, which was the first case of GM phage treatment in the world.

To apply our engineered phage vaccine in the real world, Prof. Ming-Shiou Jan of Chung Shan Medical University helped us apply the animal use form reviewed by Institutional Animal Care and Use Committee (IACUC). The evaluation is already underway. Nevertheless, we aimed and tried to make a proof of concept of a mode of the engineered phage - intestinal E. coli - antigen production by building up the composite BioBrick parts containing antigen gene, engineering phages, and demonstrating the antigen production in the engineered phage-infected intestinal E. coli.


REFERENCE

  1. Theoharides TC, Conti P. Be aware of SARS-CoV-2 spike protein: There is more than meets the eye. J Biol Regul Homeost Agents. 2021 May-Jun;35(3):833-838. doi: 10.23812/THEO_EDIT_3_21. PMID: 34100279.
  2. Nguyen V, Zhang Y, Gao C, Cao X, Tian Y, Carver W, Kiaris H, Cui T, Tan W. The Spike Protein of SARS-CoV-2 Impairs Lipid Metabolism and Increases Susceptibility to Lipotoxicity: Implication for a Role of Nrf2. Cells. 2022 Jun 14;11(12):1916. doi: 10.3390/cells11121916. PMID: 35741045; PMCID: PMC9221434.
  3. Rhea EM, Logsdon AF, Hansen KM, Williams LM, Reed MJ, Baumann KK, Holden SJ, Raber J, Banks WA, Erickson MA. The S1 protein of SARS-CoV-2 crosses the blood-brain barrier in mice. Nat Neurosci. 2021 Mar;24(3):368-378. doi: 10.1038/s41593-020-00771-8. Epub 2020 Dec 16. PMID: 33328624; PMCID: PMC8793077.
  4. Geary TW, Reeves JJ. Production of a genetically engineered inhibin vaccine. Vaccine. 1996 Sep;14(13):1273-9. doi: 10.1016/s0264-410x(96)00014-x. PMID: 8961517.
  5. Hafner AM, Corthésy B, Merkle HP. Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant. Adv Drug Deliv Rev. 2013 Oct;65(10):1386-99. doi: 10.1016/j.addr.2013.05.013. Epub 2013 Jun 7. PMID: 23751781.
  6. Gregoriadis G, Bacon A, Caparros-Wanderley W, McCormack B. A role for liposomes in genetic vaccination. Vaccine. 2002 Dec 20;20 Suppl 5:B1-9. doi: 10.1016/s0264-410x(02)00514-5. PMID: 12477411.
  7. Spencer J, Sollid LM. The human intestinal B-cell response. Mucosal Immunol. 2016 Sep;9(5):1113-24. doi: 10.1038/mi.2016.59. Epub 2016 Jul 27. PMID: 27461177.
  8. Katouli M. Population structure of gut Escherichia coli and its role in development of extra-intestinal infections. Iran J Microbiol. 2010 Jun;2(2):59-72. PMID: 22347551; PMCID: PMC3279776.
  9. Mowat AM, Agace WW. Regional specialization within the intestinal immune system. Nat Rev Immunol. 2014 Oct;14(10):667-85. doi: 10.1038/nri3738. Epub 2014 Sep 19. PMID: 25234148.
  10. Lee W, Suresh M. Vaccine adjuvants to engage the cross-presentation pathway. Front Immunol. 2022 Aug 1;13:940047. doi: 10.3389/fimmu.2022.940047. PMID: 35979365; PMCID: PMC9376467.
  11. Lim J, Koh VHQ, Cho SSL, Periaswamy B, Choi DPS, Vacca M, De Sessions PF, Kudela P, Lubitz W, Pastorin G, Alonso S. Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity. Front Immunol. 2019 Nov 22;10:2737. doi: 10.3389/fimmu.2019.02737. PMID: 31824511; PMCID: PMC6883722.
  12. Zhu J, Jain S, Sha J, Batra H, Ananthaswamy N, Kilgore PB, Hendrix EK, Hosakote YM, Wu X, Olano JP, Kayode A, Galindo CL, Banga S, Drelich A, Tat V, Tseng CK, Chopra AK, Rao VB. A Bacteriophage-Based, Highly Efficacious, Needle- and Adjuvant-Free, Mucosal COVID-19 Vaccine. mBio. 2022 Aug 30;13(4):e0182222. doi: 10.1128/mbio.01822-22. Epub 2022 Jul 28. PMID: 35900097; PMCID: PMC9426593.
  13. Suh GA, Lodise TP, Tamma PD, Knisely JM, Alexander J, Aslam S, Barton KD, Bizzell E, Totten KMC, Campbell JL, Chan BK, Cunningham SA, Goodman KE, Greenwood-Quaintance KE, Harris AD, Hesse S, Maresso A, Nussenblatt V, Pride D, Rybak MJ, Sund Z, van Duin D, Van Tyne D, Patel R; Antibacterial Resistance Leadership Group. Considerations for the Use of Phage Therapy in Clinical Practice. Antimicrob Agents Chemother. 2022 Mar 15;66(3):e0207121. doi: 10.1128/AAC.02071-21. Epub 2022 Jan 18. PMID: 35041506; PMCID: PMC8923208.
  14. Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, Gilmour KC, Soothill J, Jacobs-Sera D, Schooley RT, Hatfull GF, Spencer H. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. 2019 May;25(5):730-733. doi: 10.1038/s41591-019-0437-z. Epub 2019 May 8. PMID: 31068712; PMCID: PMC6557439.