Team:William and Mary/Implementation
Proposed Implementation
While the focus of our project this year is design and modeling, we want our project to be based on a probiotic that could be realistically implemented as a broad-spectrum antiviral therapeutic. For implementation of our probiotic, we have described a series of safety tests along with clinical trials that will be necessary to determine if our probiotic is safe to be implemented as a broad-spectrum antiviral therapeutic. We have also proposed a method of administration for the probiotic along with safety measures required for its implementation.
.
Preclinical Safety Testing
Feedback from Experts
For our integrated human practices, we consulted 10 experts including microbiologist Dr. Lappan, E.N.T. Dr. Cervin, allergist and immunologist Dr. Mikita, microbiology expert Ms. Mapstone, E.N.T. Dr. Shikani, pediatrician Dr. Turner, allergist and immunologist Dr. Kramer, pulmonologist and lipid mediator expert Dr. Shelhamer, drug developer Dr. Xiaokun and vaccine manufacturing expert Ms. Gale.
Dr. Lappan supported the development of new bacterial chassis for use as probiotics such as Corynebacterium, and stated along with several experts that although there is a lack of gram-negative bacterial strains that are used as nasal probiotics, they can be used as probiotics as long as there are steps taken to ensure their safety. To do this, Dr. Cervin recommended analysis of whole genome sequencing data, particularly the analysis of genes encoding toxins and antibiotic resistance, and suggested against using species that colonize the nasal cavity. Dr. Mikita stated that she would need to be provided with data from clinical trials showing that our probiotic would be safe, including that it decreases sinus infections and improves ciliary clearance. Along those terms, Ms. Mapstone suggested running certain safety tests such as growing the probiotic on agar plates that mimic the environment of the nasal microbiome to test the survival of the probiotic within the human body. Finally, Ms. Mapstone mentioned that our probiotic would need to go through official safety verification testing including cytotoxicity assays and co-culture. Below we have summarized the process by which the probiotic would be tested if it were to be implemented in the real world, which incorporates the feedback we received from experts (3):
.
Procedure for Preclinical Safety Testing
Identification and Screening of Probiotic In Vitro
Nucleotide Sequencing of Bacterial Genomes: Isolates of both engineered Neisseria cinerea strains would be identified using 16S rRNA gene sequencing (Yasmin et al., 2020, Martinez et al., 2015). The phenol-chloroform extraction DNA method would be followed by PCR, gel electrophoresis, purification, and NanoDrop quantification. Gene sequencing of our Neisseria cinerea strains would be performed via Illumina sequencing and the sequences would be analyzed through the use of the NCBI GenBank BLAST function to survey for genes encoding toxins or biogenic amine as well as genes associated with antibiotic or multidrug resistance.
Testing of Probiotic Strains Under Conditions in Nasal Cavity: Isolates of each probiotic strain would be tested for their ability to grow in conditions found in the nasal cavity. Isolates would be grown in various conditions, including a range of pHs (adjusted through use of HCl) and a range of bile salt and phenol concentrations (Yasmin et al., 2020). The amount of colonies would be determined per mL through plating (Yasmin et al., 2020).
Testing for Production of Exopolysaccharides: To test for the production of exopolysaccharides, the Neisseria cinerea cultures would be incubated and centrifuged, followed by the addition of trichloroacetic acid and another round of centrifugation for the removal of precipitated proteins (Liu et al., 2011). Ethanol would be added to the EPS, then centrifuged and kept at 4 degrees Celsius. After 24 hours, the EPS would be dialyzed and freeze-dried, then quantified as described by Nikolic et al. (Nikolic et al., 2012).
Auto-Aggregation Assay: Cultures of Neisseria cinerea isolates from both of our probiotic strains would be centrifuged and grown overnight (Yasmin et al., 2020, Xu et al., 2009). Cells would be washed with PBS and suspended in PBS until an absorbance of about 0.5 is reached at 600 nm (Yasmin et al., 2020, Xu et al., 2009). 1 mL of each suspension would be vortexed and incubated for varying amounts of time. Absorbance of supernatants of suspensions grown at different time intervals would be measured (Yasmin et al., 2020, Xu et al., 2009). The percentage of auto-aggregation would be calculated through equation 1 provided by Yasmin et al. (Yasmin et al., 2020).
Cell Surface Hydrophobicity Test: The ability of Neisseria cinerea isolates from both of our probiotic strains to bind with hydrocarbons would be tested by growing cells overnight, centrifuging cells, washing them with PBS and suspending them in PBS until an absorbance of about 0.5 is reached at 600 nm (Yasmin et al., 2020, Kotzamanidis et al., 2010). Hydrocarbon would be added to suspensions, which would be briefly incubated, vortexed and kept for 20 minutes for phase separation to occur. Absorbance would be measured for the aqueous phase at 600 nm (Yasmin et al., 2020, Kotzamanidis et al., 2010). Equation 2 provided by Yasmin et al. would be used to calculate the percentage of hydrophobicity (Yasmin et al., 2020).
Free Radical Activity Test: DPPH would be added to an aliquot of Neisseria cinerea cells from both of our probiotic strains; they would be mixed and kept in an area with low levels of light and absorbance would be measured after 30 minutes (Yasmin et al., 2020, Chen et al., 2006). Free radical activity would be calculated using equation 3 provided by Yasmin et al. (Yasmin et al., 2020).
Hydrogen Peroxide Resistance Test: Aliquots of Neisseria cinerea culture of both probiotic strains would be mixed with NaCl and a range of H2O2 concentrations (Yasmin et al., 2020, Oberg et al., 2011). Viability would be analyzed after an hour of incubation (Yasmin et al., 2020, Oberg et al., 2011).
Sodium Nitrite Depletion Test: Sodium nitrate solution would be added to Luria broth for anaerobic inoculation and incubation of Neisseria cinerea cultures of both our probiotic strains (Yasmin et al., 2020, Wu et al., 2012). Initial and final absorbance would be calculated using the colorimetric nitrite method (Yasmin et al., 2020, Yan et al., 2008). Percentage of nitrite depletion would be calculated using equation 4 provided by Yasmin et al. (Yasmin et al., 2020).
Antibacterial Activity Test: Through use of the ager well diffusion method (Ren et al., 2014), isolates of our engineered Neisseria cinerea strains would be tested against bacterial pathogens of humans such as Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus. Antibacterial activity would be quantified through the measurement of the clear zone diameter of isolates tested against the different pathogens (Yasmin et al., 2020, Ren et al., 2014).
Cholesterol Reduction Assay: Cholesterol stock solution and bile salt would be added to Luria broth, which would be inoculated with Neisseria cinerea cultures of both our probiotic strains, then incubate and withdraw for specified time intervals for centrifugation (Yasmin et al., 2020). Cholesterol amount would be quantified through use of the modified colorimetric method (Yasmin et al., 2020, Rudel et al., 1973, Liong et al., 2005). This method involves adding KOH and ethanol to bacterial culture, vortexing the mixture, a 30-minute incubation, and a cooling period. Next, it involves adding hexane and Milli-Q water to the mixture and vortexing for a second time, then evaporating the upper (hexane) layer. Finally, the method requires addition of o-phthalaldehyde reagent and H2SO4, each followed by a vortexing step, and measurement of absorbance at 550 nm (Yasmin et al., 2020, Rudel et al., 1973, Liong et al., 2005). Absorbance would be calculated using equation 5 provided by Yasmin et al. (Yasmin et al., 2020).
Safety Evaluation Testing of Probiotic In Vitro
Antibiotic Susceptibility Test: Neisseria cinerea isolates from both of our probiotic strains would be tested for their sensitivity to a wide range of antibiotics (Yasmin et al., 2020). Specifically, this would be done using the antibiotic disc diffusion method (Vijayakumar et al., 2015), which involves growing tested isolates on agar plates and placing antibiotic disks on the plates for 30 minutes before incubating for 2 days. This would be followed by measuring the zone of inhibition for each disc, with specified ranges indicating different levels of susceptibility (as stated by Yasmin et al.) (Yasmin et al., 2020).
Hemolytic Activity: Neisseria cinerea isolates from both of our probiotic strains would be grown on Columbia agar plates and incubated for 2 days, followed by an analysis of the lysis of blood cells present around the bacterial colonies (Yasmin et al., 2020, 17). This would be classified as either green zones (α-hemolysis), clear zones (β-hemolysis), or no zones (γ-hemolysis) present, with only those with no zones considered safe for use (Yasmin et al., 2020, Mangia et al., 2019).
Cytotoxicity Activity Test: The cytotoxic activity of the Neisseria cinerea isolates from both of our probiotic strains would be measured using a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Yasmin et al., 2020, Mohanty et al., 2019). This assay involves adding tested bacterial isolates cell-free filtrate to Caco-2 cell culture medium and adding MTT solution (after incubation) and incubating for another 2 hours (Yasmin et al., 2020, Mohanty et al., 2019). Finally, dimethyl sulfoxide would be added to the cell culture medium followed by an absorbance and optical density measurement (Yasmin et al., 2020, Mohanty et al., 2019).
.
Form of Administration
After extensive safety testing and clinical trials (listed above), if our probiotic is found to be safe based on the results found, we would then develop a form of administration for patients whom the probiotic could benefit. Based on the feedback we have received from experts, we envision our probiotic to be administered at least once per day in the form of a nasal spray. Dr. Cervin, Dr. Mikita, Dr. Lappan and Dr. Shikani recommended using a nasal spray rather than a swab to ensure that the probiotic will be administered correctly [see our HP page for more details on interviews]. According to several of the experts we interviewed, many patients are open to using engineered probiotics in the form of a spray, as they are already being produced by biotechnology companies. For example, Dr. Turner informed us that the company PrEPBiopharm produces a prophylactic antiviral nasal spray that is designed to be administered at least once daily. Along with the nasal probiotic created by PrEPBiopharm, Ms. Mapstone, who is working on the development of a breast milk probiotic for preterm babies, also designed her probiotic to be administered at least once per day. Additionally, Dr. Shikani informed us that he had previously used a Lactobacillus nasal spray for patients with sinusitis, and that such patients were open to the treatment. Dr. Shelhamer shared our vision of administering the probiotic prophylactically, as this would allow for a greater antiviral effect (Braz-De-Melo et al., 2019). However, as several experts noted, our probiotic would not be eligible for immunocompromised and elderly patients, as although we plan to use BSL-1 bacterial strains, which do not cause disease in healthy humans, these strains have been found to cause disease in the immunocompromised and the elderly.
.
Clinical Trials
Animal Testing
Before our probiotic would be tested in human clinical trials, it would be tested on mice under conditions approved by the Institutional Animal Care And Use Committee. The nasal administration of our engineered probiotic strains would occur daily with the amount of bacterial cells administered per day (for three days) totalling 20 uL of bacterial solution at varying concentrations of 0, 1, or 10 mg/ml for each mouse (0 mg/ml for control) (Hori et al., 2001). Mice would be 10 and 11 weeks old (Hori et al., 2001). The mice would be infected with SARS-CoV-2 after being anesthetized in the method used by Hori et al. (Hori et al., 2001) and viral titers would be quantified in nasal washes after three days through the method used by Tamura et al. (Tamura et al., 1996) followed by the method used by Hori et al. (Hori et al., 2001). Survival rate of mice infected with SARS-CoV-2 would be measured based on survival of mice who have received administration of PBS after viral infection for a two-week period (Hori et al., 2001). Cytokine concentration would also be measured in the supernatant of mediastinal lymph node cell culture through the use of a sandwich enzyme-linked immunosorbent assay (ELISA) (Hori et al., 2001).
Human Safety Testing
Subjects: Only healthy patients from ages 18 to 50 would be permitted to participate in this study. Elderly and immunocompromised patients would be excluded from this study. 100 subjects would take part in the study and would be divided into two groups in which one half of the subjects would be treated with the probiotic and the other half would be the control group which would not receive probiotic treatment. Use of any other nasal or respiratory probiotics would be prohibited 1 month prior to the study and throughout its duration.
Experimental Design: The study will be conducted over the course of 16 weeks with the probiotic being administered 2 times daily in doses of 10^8 colony-forming units/day (De Boeck et al., 2020) in the form of a nasal spray for those in the probiotic group. Those in the control group would be given a saline solution in place of the probiotic treatment.
Safety Evaluation: Throughout the trials, an analysis of adverse events, clinical chemistry, hematology and hemodynamic parameters would be conducted as described by Lefevre et al. (Lefevre et al., 2017).
.
Safety Elements of Probiotic
Environmental Kill Switch
To ensure the containment of our probiotic both outside of the body and inside of it, we have selected two kill switches for our engineered strains of Neisseria cinerea using parts that are native to Neisseria species. For our first kill switch, which is temperature-sensitive to prevent escape of the probiotic into the environment, combines the use of the FitAB toxin-antitoxin system native to Neisseria gonorrhoeae (Mattison et al., 2006) and CssA, an RNA thermometer native to Neisseria meningitidis (Barnwal et al., 2016). FitA is an antitoxin that binds toxin FitB, inhibiting its toxic effects (Lobato-Márquez et al., 2016). CssA prevents protein translation at temperatures below 37 degrees Celsius through the formation of a hairpin structure. At temperatures of 37 degrees Celsius, it is able to unfold to allow protein translation to occur (Barnwal et al., 2016). For this kill switch, we have selected the native FitAB promoter (Wilbur et al., 2005), CssA RBS sequence for FitB, and complete CssA sequence for FitA, along with the ThiC terminator region from Neisseria meningitidis (Righetti et al., 2005).
Inside the Body Kill Switch
To ensure that our probiotic is safe to use in the human body, we have selected another chemically-inducible kill switch to allow for the removal of the probiotic without the use of antibiotics. We have selected the aspirin-inducible kill switch described by Chen et al., 2019. This kill switch would consist of aspirin-inducible promoter, Psal, native to Acinetobacter baylyi ADP1 (Chen et al., 2019). The transcriptional effector of this promoter, SalR, has two forms, SalRr, a transcriptional repressor, and SalRa, a transcriptional activator (Chen et al., 2019). In the presence of aspirin, it changes to its activator form (Chen et al., 2019). We have selected constitutive promoter PopaB from Neisseria gonorrhoeae to express SalR (Ramsey et al., 2012). After looking into toxin-antitoxin systems that are native to Neisseria, we selected Ngζ_1, a zeta toxin found in the Neisseria gonorrhoea ngε_1 / ngζ_1 toxin-antitoxin system that causes cell lysis (Rocker et al., 2018). In this kill switch, the zeta toxin will only be expressed in the presence of aspirin (Rocker et al., 2018). For this kill switch, we would place expression of Ngζ_1 under the control of the Psal promoter, and would use the CssA RBS and ThiC terminator.
.
References
Chen F.A., Wu A.B., Shieh P., Kuo D.H., Hsieh C.Y. Evaluation of the antioxidant activity of Ruellia tuberosa. Food Chem. 2006;94:14–18. doi: 10.1016/j.foodchem.2004.09.046.
Chen, J. X., Steel, H., Wu, Y. H., Wang, Y., Xu, J., Rampley, C. P., ... & Huang, W. E. (2019). Development of aspirin-inducible biosensors in Escherichia coli and SimCells. Applied and environmental microbiology, 85(6).
De Boeck, I., van den Broek, M., Allonsius, C. N., Spacova, I., Wittouck, S., Martens, K., Wuyts, S., Cauwenberghs, E., Jokicevic, K., Vandenheuvel, D., Eilers, T., Lemarcq, M., De Rudder, C., Thys, S., Timmermans, J. P., Vroegop, A. V., Verplaetse, A., Van de Wiele, T., Kiekens, F., Hellings, P. W., … Lebeer, S. (2020). Lactobacilli Have a Niche in the Human Nose. Cell reports, 31(8), 107674. https://doi.org/10.1016/j.celrep.2020.107674
Hori, T., Kiyoshima, J., Shida, K., & Yasui, H. (2001). Effect of intranasal administration of Lactobacillus casei Shirota on influenza virus infection of upper respiratory tract in mice. Clinical and diagnostic laboratory immunology, 8(3), 593–597. https://doi.org/10.1128/CDLI.8.3.593-597.2001
Kotzamanidis C., Kourelis A., Litopoulou-Tzanetaki E., Tzanetakis N., Yiangou M. Evaluation of adhesion capacity, cell surface traits and immunomodulatory activity of presumptive probiotic Lactobacillus strains. Inter. J. Food Microbiol. 2010;140:154–163. doi: 10.1016/j.ijfoodmicro.2010.04.004.
Lefevre, M., Racedo, S. M., Denayrolles, M., Ripert, G., Desfougères, T., Lobach, A. R., Simon, R., Pélerin, F., Jüsten, P., & Urdaci, M. C. (2017). Safety assessment of Bacillus subtilis CU1 for use as a probiotic in humans. Regulatory toxicology and pharmacology : RTP, 83, 54–65. https://doi.org/10.1016/j.yrtph.2016.11.010
Liu C.F., Tseng K.C., Chiang S.S., Lee B.H., Hsu W.H., Pan T.M. Immunomodulatory and antioxidant potential of Lactobacillus exopolysaccharides. J. Sci. Food Agric. 2011;91:2284–2291. doi: 10.1002/jsfa.4456.
Liong M., Shah N. Bile salt deconjugation ability, bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains. Int. Dairy J. 2005;15:391–398. doi: 10.1016/j.idairyj.2004.08.007.
Lobato-Márquez, D., Díaz-Orejas, R., & García-del Portillo, F. (2016). Toxin-antitoxins and bacterial virulence. FEMS microbiology reviews, 40(5), 592-609.
Mangia N.P., Saliba L., Deiana P. Functional and safety characterization of autochthonous Lactobacillus paracasei FS103 isolated from sheep cheese and its survival in sheep and cow fermented milks during cold storage. Ann. Microbiol. 2019;69:161–170. doi: 10.1007/s13213-018-1416-1.
Martinez I., Stegen J.C., Maldonado-Gomez M.X., Eren A.M., Siba P.M., Greenhill A.R., Walter J. The gut microbiota of rural papua new guineans: Composition, diversity patterns, and ecological processes. Cell Rep. 2015;11:527–538. doi: 10.1016/j.celrep.2015.03.049.
Mattison, K., Wilbur, J. S., So, M., & Brennan, R. G. (2006). Structure of FitAB from Neisseria gonorrhoeae bound to DNA reveals a tetramer of toxin-antitoxin heterodimers containing pin domains and ribbon-helix-helix motifs. Journal of Biological Chemistry, 281(49), 37942-37951.
Mohanty D., Panda S., Kumar S., Ray P. In vitro evaluation of adherence and anti-infective property of probiotic Lactobacillus plantarum DM 69 against Salmonella enterica. Microbial. Pathog. 2019;126:212–217. doi: 10.1016/j.micpath.2018.11.014.
Nikolic M., López P., Strahinic I., Suárez A., Kojic M., Fernández-García M., Topisirovic L., Golic N., Ruas-Madiedo P. Characterisation of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11 and its non-EPS producing derivative strains as potential probiotics. Inter. J. Food Microbiol. 2012;158:155–162. doi: 10.1016/j.ijfoodmicro.2012.07.015.
Oberg T., Steele J., Ingham S., Smeianov V., Briczinski E., Abdalla A., Broadbent J.R. Intrinsic and inducible resistance to hydrogen peroxide in Bifidobacterium species. J. Indus. Microbiol. Biotechnol. 2011;38:1947–1953. doi: 10.1007/s10295-011-0983-y.
Ren D., Li C., Qin Y., Yin R., Du S., Ye F., Liu C., Liu H., Wang M., Li Y., et al. In vitro evaluation of the probiotic and functional potential of Lactobacillus strains isolated from fermented food and human intestine. Anaerobe. 2014;30:1–10. doi: 10.1016/j.anaerobe.2014.07.004.
Righetti, F., Materne, S. L., Boss, J., Eichner, H., Charpentier, E., & Loh, E. (2020). Characterization of a transcriptional TPP riboswitch in the human pathogen Neisseria meningitidis. RNA biology, 17(5), 718-730.
Rocker, A., Peschke, M., Kittilä, T., Sakson, R., Brieke, C., & Meinhart, A. (2018). The ng_ζ1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis. Nature communications, 9(1), 1-11.
Rudel L.L., Morris M. Determination of cholesterol using o-phthalaldehyde. J. Lipid Res. 1973;14:364–366.
Tamura S, Miyata K, Matsuo K, Asanuma H, Takahashi H, Nakajima K, Suzuki Y, Aizawa C, Kurata T. Acceleration of influenza virus clearance by Th1 cells in the nasal site of mice immunized intranasally with adjuvant-combined recombinant nucleoprotein. J Immunol. 1996;156:3892–3900.
Vijayakumar M., Ilavenil S., Kim D.H., Arasu M.V., Priya K., Choi K.C. In-vitro assessment of the probiotic potential of Lactobacillus plantarum KCC-24 isolated from Italian rye-grass (Lolium multiflorum) forage. Anaerobe. 2015;32:90–97. doi: 10.1016/j.anaerobe.2015.01.003.
Wilbur, J. S., Chivers, P. T., Mattison, K., Potter, L., Brennan, R. G., & So, M. (2005). Neisseria gonorrhoeae FitA interacts with FitB to bind DNA through its ribbon− helix− helix motif. Biochemistry, 44(37), 12515-12524.
Wu Y.Y., Liu F.J., Li L.H., Yang X.Q., Deng J.C., Chen S.J. Isolation and identification of nitrite-degrading lactic acid bacteria from salted fish. Adv. Mater. Res. 2012;393:828–834. doi: 10.4028/www.scientific.net/AMR.393-395.828.
Xu H., Jeong H., Lee H., Ahn J. Assessment of cell surface properties and adhesion potential of selected probiotic strains. Lett. App. Microbiol. 2009;49:434–442. doi: 10.1111/j.1472-765X.2009.02684.x.
Yan P.M., Xue W.T., Tan S.S., Zhang H., Chang X.H. Effect of inoculating lactic acid bacteria starter cultures on the nitrite concentration of fermenting Chinese paocai. Food Control. 2008;19:50–55. doi: 10.1016/j.foodcont.2007.02.008.
Yasmin, I., Saeed, M., Khan, W. A., Khaliq, A., Chughtai, M., Iqbal, R., Tehseen, S., Naz, S., Liaqat, A., Mehmood, T., Ahsan, S., & Tanweer, S. (2020). In vitro Probiotic Potential and Safety Evaluation (Hemolytic, Cytotoxic Activity) of Bifidobacterium Strains Isolated from Raw Camel Milk. Microorganisms, 8(3), 354. https://doi.org/10.3390/microorganisms8030354