Team:William and Mary/Human Practices
Integrated Human Practices
Overview
Our project aims to determine the feasibility of a PUFA-secreting nasal probiotic by reviewing literature, mathematically modeling proposed systems, and consulting experts. Throughout the engineering cycle of our project, we consulted medical professionals with expertise in PUFAs, rhinology, and allergy & immunology. Additionally, we interviewed experts in probiotic and drug development and manufacturing. Their invaluable expertise guided us as we designed our probiotic to be as safe, effective, and appealing-to-use as possible, helping us to overcome many obstacles we faced. Furthermore, through our human practices, we addressed major questions about probiotic implementation and accessibility.
Accessibility stands as a key theme of our project. We have watched the pandemic deplete hospitals of essential equipment and increase demand for potentially life-saving drugs, exacerbating global inequalities. In our own country, we have watched these inequities grow. Though a select few can fly to world-class hospitals for treatment with expensive, experimental drugs, their experiences starkly contrast that of the average American. As the American death toll climbs past 310,000, and global deaths surpass 1.6 million, the need for therapies accessible to all people becomes as urgent as ever.
An overview of our human practices, and how they shaped our project, is described below.
Integrating Feedback into Design
Integrating Feedback into Safety Considerations and Proposed Implementation
Integrating Feedback into Accessibility Considerations
Medical Professionals
The medical professionals among our experts helped us to understand and address the needs of our future stakeholders. Medical doctors would oversee the prescription and administration of our antiviral probiotic, so we consulted medical doctors to inform our vision for the implementation of our project. Please visit our implementation page to learn more.
Dr. James Shelhammer
Pulmonologist and lipid mediator expert Dr. James Shelhamer guided our circuit design. Prior to meeting Dr. Shelhamer, we had considered the use of DHA to inhibit phospholipase cPLA2, which positive strand RNA viruses may manipulate to enhance their own replication. Dr. Shelhamer explained to us that the effect of DHA on cPLA2 varies based on factors such as cell type, and that DHA may activate cPLA2 activity. Realizing that DHA may not be a reliable inhibitor of cPLA2, we investigated other antiviral properties of PUFAs. Dr. Shelhamer guided us throughout our research, cautioning us on the use of anti-inflammatory bioactive lipids such as DHA and leading us to design a “smart” circuit. Additionally, Dr. Shelhamer provided invaluable guidance on TheraPUFA’s implementation, including environmental concerns associated with a genetically modified probiotic.
Dr. James Shelhamer worked at the NIH Clinical Center's Critical Care Medicine Department for 33 years, 24 of which he served as deputy chief. As an expert in pulmonary medicine, he has researched lung cell and molecular biology, including human phospholipase cPLA2, macrophages, and lung epithelial cells. He has also investigated receptors for lipid mediators, regulatory mechanisms, lipidomics, and PUFAs.
July 3, 2020
We initially contacted Dr. Shelhamer during an early phase of our research, when we were considering DHA as a potential inhibitor of cPLA2. Since cPLA2 is implicated in membrane rearrangement, a common strategy of positive strand RNA viruses, we hoped DHA could suppress membrane rearrangement through inhibition of cPLA2. However, despite research that had observed an inhibitory effect of DHA on cPLA2, Dr. Shelhamer had conducted research suggesting that DHA in fact activated cPLA2! After reading his paper, we contacted him for an interview.
Dr. Shelhamer provided much clarification during this early stage of research. He addressed the complexity of lipid mediators, explaining that their effect depends on various factors, such as cell type and phase of inflammation. He also distinguished between two major ways in which PUFAs can affect human cells: 1) receptor-mediated reactions and 2) accumulation within cell membranes, which influences downstream metabolites. Dr. Shelhamer’s comments enlightened us to the complexity of the immune response--for example, metabolites such as prostaglandin E2 (PGE2) can be both pro- and anti-inflammatory.
Additionally, Dr. Shelhamer’s feedback led us to consider that DHA may not be a reliable inhibitor of cPLA2. We began to investigate the broader antiviral effects of omega-3 PUFAs beyond inhibiting cPLA2 (although that is still a potential mechanism of action). We also began research on potential side-effects of omega-3 PUFAs following our interview with Dr. Shelhamer. Some degree of inflammation is necessary to efficiently clear viruses from the body, and a probiotic that secretes only anti-inflammatory PUFAs like DHA or EPA may prevent this necessary inflammation. Dr. Shelhamer pointed us to studies where inhibition of cPLA2 decreased the inflammatory response to bacterial pneumonia, and inhibition of a downstream enzyme, 5-Lipoxygenase, which metabolizes AA to leukotrienes, increased the morbidity of pneumonia in one model.
Dr. Shelhamer’s comments led us to consider the therapeutic potential of AA, and inspired us to design a system where PUFAs with pro-inflammatory metabolites like AA are balanced with PUFAs with anti-inflammatory metabolites.
July 27, 2020
We contacted Dr. Shelhamer for a second interview after completing more work on our probiotic design. To determine when our probiotic should switch from pro-inflammatory to anti-inflammatory PUFAs, we asked what naturally triggers the switch from pro- to anti-inflammatory processes in the human body. Dr. Shelhamer explained that a variety of factors can encourage this switch, but timing may be the driving force. Timing may be related to the half-life of neutrophils and the macrophages that subsequently consume them, or to the decline of pro-inflammatory cytokines. He confirmed that interleukin 10 (IL-10) would be a potential marker that our probiotic could sense to determine if the immune response has “switched” to anti-inflammatory.
We then discussed implementation of the probiotic, including safety features. While we want our probiotic to be as accessible as possible, immunocompromised patients would be ineligible for the treatment. Even commensal, nonpathogenic strains (BSL1) of bacteria can cause disease in immunocompromised patients. Dr. Shelhamer stated that, for patients to be open to a probiotic like ours, we would require extremely good safety and effectiveness data.
When I asked if a kill-switch could ensure the safety of the probiotic, Dr. Shelhamer suggested additional safety measures. Not only do we have to control the probiotic within a patient’s body, but also within the environment when the probiotic inevitably “gets out.” The probiotic could leave the nasopharyngeal cavity through mucus and saliva, or even through feces. Dr. Shelhamer pointed to a University of Arizona study where coronavirus was detected in sewer samples, even though coronavirus primarily targets cells within the respiratory tract.
For administration of the probiotic, Dr. Shelhamer suggested the use of a swab rather than a nasal spray. A spray may place the probiotic too deep within the respiratory tract too quickly, although “although it would probably get there, anyway.” Furthermore, Dr. Shelhamer agreed that prophylactic administration of the probiotic would be appropriate.
Dr. Anders Cervin
E.N.T. Dr. Anders Cervin provided his expertise on the nasopharyngeal microbiome and on probiotics. He guided us on probiotic implementation, suggesting a once-daily administration via nasal irrigation or spray. He also identified safety concerns and helped us to determine what groups would be eligible for the probiotic.
Dr. Anders Cervin is the Garnett Passe and Rodney Williams Foundation Chair in Otolaryngology at The University of Queensland in Australia, as well as the author of over fifty peer-reviewed articles. He has conducted research on chronic sinusitis and otitis media, and has penned articles on the use of probiotics to treat both conditions. We contacted Dr. Cervin for his expertise on the nasopharyngeal microbiome and on probiotics.
July 28, 2020
Dr. Cervin elucidated the role of the nasopharyngeal microbiome, stating that a balanced microbiome is thought to correlate with health benefits. However, researchers have not defined a “balanced” microbiome; there is no consensus on a common healthy profile for the nasopharyngeal microbiome. Dr. Cervin elaborated that, while moderate levels of S. aureus and Corynebacteria are considered normal/healthy, clinical studies focus on pathogenic bacteria rather than profiling healthy bacteria. Pathogenic bacteria include gram negative P. aeruginosa, which is considered a “red flag.”
While gram negative bacteria like P. aeruginosa can cause considerable damage within the respiratory tract, other gram negative strains colonize the nasopharyngeal microbiome without causing disease in healthy humans. These strains have not been used for nasal probiotic studies, despite being native to the upper respiratory tract and BSL1. We asked Dr. Cervin how we could ensure the safety of gram negative bacteria, so we could use BSL1 strains within probiotics. He suggested that we consult whole genome sequencing data, searching for genes encoding toxins and antibiotic resistance. As well, for genetically-modified bacteria, he suggested using species that do not colonize, though this would require repeated re-administration (similar to a standard, daily intranasal corticoid spray for allergic rhinitis).
Dr. Cervin stated that he would be open to prescribing a nasal probiotic, though the probiotic should exclude immunocompromised individuals. He also stated that he believes patients would be open to a nasal probiotic. He suggested that the probiotic be administered via nasal irrigation or a spray.
Dr. Kramer
We interviewed Dr. Kramer for his expertise on probiotics, allergy, and immunology. He explained that a healthy microbiome is associated with health and immune benefits. He stressed that scientific data demonstrating the effectiveness, safety, and quality of the probiotic along with proper approval or registration will be crucial in obtaining the approval of doctors and the public. He emphasized the importance of having extensive safety data when implementing genetically engineered organisms within the probiotic, especially if using gram negative strains which are less well-characterized for use in nasal probiotics. He also provided guidance on the administration of the probiotic and suggested that a nasal spray might be effective. Certain populations, such as the elderly and the immunocompromised, could be left out of the treatment depending on the dose and bacterial strains implemented.
October 30, 2020
We interviewed Dr. Kramer for his expertise on probiotics, allergy, and immunology. For background, we asked him whether a balanced nasopharyngeal microbiome was associated with health and immune benefits, including resistance to opportunistic pathogens and to noninfectious conditions, such as allergies and rhinitis. Dr. Kramer responded that, intuitively, a healthy nasopharyngeal microbiome is associated with health benefits. However, he noted that this concept is just beginning to be acknowledged within the scientific community. As well, while commensal bacteria may be able to reign in opportunistic pathogens, such pathogens may contribute to self-sustaining inflammation. Therefore, simply targeting pathogens may be insufficient to resolve the chronic conditions they cause. Scientific data must demonstrate the effectiveness of the probiotic, in addition to its safety and quality. Scientific data and proper registration or approval will be crucial in obtaining the approval of doctors and the public.
Dr. Kramer carefully considered the different routes for probiotic approval. A nasal probiotic could not qualify as a food supplement, and attempting to pass the probiotic through food/drug safety regulations as a medical device could be challenging if not impossible. While the nasal probiotic could be approved as a drug, our interviewee warned that the drug approval route is expensive.
Regardless of classification, to utilize genetically engineered organisms within the probiotic, researchers would require “water proof” safety data. Researchers would also need strong safety data to support the usage of a gram-negative strain, since gram-negative strains have not been as well-characterized as gram-positive strains for use in nasal probiotics.
To administer the nasal probiotic, Dr. Kramer suggested that a nasal spray could be effective, although most of the product would be swallowed. Depending on the dosage of the spray and the strains used with it, some groups of patients, including elderly and immunocompromised patients, may be excluded from treatments. Given scientific data on safety and effectiveness, Dr. Kramer suggested that eligible patients would be open to a nasal probiotic. However, patients and researchers alike may be biased to prefer a gut probiotic over a nasal probiotic, possibly due to a bias from the availability of probiotic food supplements.
Dr. Cecilia Mikita
We consulted Dr. Cecilia Mikita, an allergist and immunologist, for her expertise on the nasopharyngeal microbiome and her feedback on a nasal probiotic. She explained that non-pathogenic bacteria within the nasopharyngeal microbiome may have health benefits and may also protect against infectious diseases and non-infectious conditions. Dr. Mikita said for her to consider prescribing a nasal probiotic, she would look for data that demonstrates decreased sinus infections, improved ciliary clearance, and decreased SNOT score following probiotic administration. She said she was not opposed to genetically engineered probiotics if FDA approved, and believes her patients would similarly be open to such probiotics. Immunocompromised and elderly patients as well as those recovering from a previous sinus surgery would be ineligible to receive the probiotic. Additionally, she recommends the administration of the probiotic in the form of a spray.
August 10, 2020
Dr. Mikita considers the nasopharyngeal microbiome when evaluating patients with chronic rhinosinusitis and possible immunodeficiency disorders. In addition to testing for influenza and respiratory syncytial virus (RSV), the Biofire Respiratory Panel is a diagnostic assay using a nasopharyngeal sample that tests for 17 viral and 3 bacterial pathogens which commonly cause respiratory illnesses. While she has not observed specific profiles for healthy patients versus patients with chronic or acute illness, she has observed that children often have one or two specific respiratory pathogens. Using other laboratory techniques, nasopharyngeal samples may identify “mixed respiratory flora” when normal flora is identified. The lab results do not identify specific species, but the microbes are common and non-pathogenic.
Non-pathogenic bacteria within the nasopharyngeal microbiome may contribute to health benefits. It is possible that they may protect against noninfectious conditions, such as allergies, as well as infectious diseases, such as influenza. To keep the natural microbiome in check, "Judicious use of antibiotics is of utmost importance," Dr. Mikita explained. In addition to a balanced nasopharyngeal microbiome, Dr. Mikita emphasized the importance of normal respiratory anatomy and function. This includes mucociliary clearance, the lungs' way of removing inhaled particles, including pathogens, from the airways.
While Dr. Mikita has not herself prescribed a nasal probiotic, she has heard of nasal probiotics and has prescribed oral probiotics. For her to consider a nasal probiotic, she would have to review the research literature on specific products, evaluating safety, efficacy, and clinical outcomes including decreased infections, improved ciliary clearance, and decreased SNOT score (validated questionnaire). She is not opposed to genetically-engineered probiotics, if there was an FDA-approved genetically-modified probiotic available. She believes her patients would be open to a nasal probiotic, as well: "They are always looking for therapeutic options," Dr. Mikita said. Unfortunately, immunocompromised and elderly patients, as well as patients who are recovering from previous sinus surgeries, would not be eligible for the probiotic.
To administer the potential probiotic, Dr. Mikita recommends a nasal spray, as a swab would be "patient dependent and likely not administered appropriately."
Dr. Turner
Dr. Turner is a pediatrician and expert in infectious disease. He provided guidance related to safety considerations, including guidance on safe bacterial strains for a nasal probiotic. Dr. Turner recommended the usage of Lactobacillus, which are widely implemented in probiotics, over BSL-1 Corynebacterium and Neisseria due to their association with disease. He explained the rigorous regulations associated with nasal probiotics, which cannot be regulated as food supplements. He discussed daily administration of a nasal product, but cautioned that, upon administration by spray or a swab, the bacteria would be carried to the posterior nasopharynx by ciliated cells within minutes. His feedback led us to consider strategies to extend the retention time of the probiotic.
August 11, 2020
Dr. Turner emphasized the need for safety regulations to be incorporated within the design of the probiotic, as there are many more regulatory barriers for nasal probiotics than oral ones, which are considered food supplements by the FDA. He stated that he would consider prescribing an engineered nasal probiotic to his patients if it was proven to be safe and effective. He felt that his patients would be open to using it, although the number of interested patients would likely decrease if the probiotic was found to be only effective with certain groups of patients or found to be only moderately effective against viral infection rather than highly effective.
Dr. Turner also mentioned that he recommends the use of Lactobacillus for a nasal probiotic, as it is widely used. He felt that BSL-1 Corynebacterium and Neisseria species are not ideal choices due to their association with disease, particularly in immunocompromised patients, and their lack of use in the development of nasal probiotics.
When asked about the ideal way to administer a nasal probiotic, Dr. Turner stated that he was unsure, but stated that regardless of the method of administration, the bacteria would be carried to the posterior nasopharynx by ciliated cells in a manner of minutes. Although he was unsure about how often a nasal probiotic should be administered, he mentioned that PrEP Biopharm, a company at which he previously worked, recommended the daily administration of their antiviral nasal powder, which was designed to be administered prophylactically in the form of a spray.
Dr. Shikani
Dr. Shikani provided our team with critical information for potential delivery methods of our drug. Having designed a nasal steroid-antibiotic himself, Dr. Shikani recommended that our team use a mucoadhesive poloxamer gel to maintain a high dosage, combating mucociliary clearance. Dr. Shikani’s own research experience led him to recommend a nasal spray as a delivery method, since many of his patients were willing to use nasal sprays. However, he outlined the many regulation obstacles we would face if we were to design this probiotic.
October 18, 2020
Dr. Alan Shikani serves as the chief of Otolaryngology-Head and Neck Surgery at Union Memorial, Good Samaritan Hospital and Sinai Hospital in Baltimore. He founded and currently directs the Maryland Ear, Nose and Throat Center. In addition to treating patients, Dr. Shikani conducts extensive research in otolaryngology. He has published numerous articles in peer-reviewed medical journals and has created ENT inventions currently on the market. We sought Dr. Shikani’s expertise on the nasal microbiome and on nasally-delivered therapies.
Dr. Shikani has researched the use of combinations of steroids and antibiotics to treat refractory sinusitis, or chronic, antibiotic-resistant sinusitis. Patients self-administered the steroid-antibiotic therapeutic twice a day using a nebulizer, then returned to the office once a week for deep administration of the therapeutic in a topical gel. When I asked how he maintained high therapeutic dosage despite mucociliary clearance, Dr. Shikani explained that he worked with a polymer chemist to select a mucoadhesive poloxamer gel. The thermally-reversible gel was a liquid when refrigerated, but solidified at body temperature. He coined a name for this protocol: “Rhinotopic therapy” and has used it for treating different types of refractory sinusitis. He also is working on nebulizable microspheres that could withstand the mucociliary conditions within the sinus cavities.
When asked whether he had ever administered a nasal probiotic, Dr. Shikani responded that he had administered a nasal spray containing Lactobacillus for the treatment of sinusitis. Patients were open to such treatment, though some had concerns regarding the probiotic and their sense of smell. Unfortunately, there is little objective data available in the literature and Dr. Shikani found the probiotic difficult to assess on anecdotal evidence.
Dr. Shikani recommended a spray for the delivery of our potential probiotic, then described the regulation obstacles we would face if we were to ever implement our designs. “The sinuses are very complicated,” he elaborated, mentioning how ostiomeatal obstructions, the immune systems of patients, and mucociliary factors could complicate our work.
Drug Developers, Probiotic & Microbiome Experts
Dr. Xiaokun
Dr. Xiaokun is the founder of Convalife, a Shanghai-based drug development company. Given that Convalife is also working on developing a SARS-CoV-2 drug, Dr. Xiaokun was able to offer us valuable information on testing and developing a similar probiotic. He recommended that our team adopt a “cocktail therapy” approach, where multiple aspects of the virus are targeted instead of just a single portion. This method allows increased effectiveness and directedness of the virus. Further, he recommended that we know all possible toxicological and metabolic effects of our probiotic. Dr. Xiaokun mentioned that this knowledge, in addition to standardized procedures before clinical testing, should ensure the safety of our probiotic.
August 2, 2020
Infection by SARS CoV-2 can cause overactivation of the immune system, resulting in excessive quantities of proinflammatory cytokines. According to a recent paper, PARP-1 inhibitors can inhibit the production of these cytokines, including IL-6, IL-1 and TNF-α (Curtin, 2020, Br. J. Pharmacol.). CVL218 is a PARP-1 inhibitor developed by Chinese Academy of Sciences Shanghai Institute of Materia Medica. Data-driven research has shown the ability of CVL218 to inhibit SARS CoV-2 (Ge et al., 2020, medRxiv preprint).
In addition to CVL218’s ability to inhibit proinflammatory cytokines, CVL218 can target SARS CoV-2 through two other mechanisms. First, molecular simulation has shown that CVL218 can lead to ADP-ribosylation of NHP3. NHP3 is an evolutionary product created by the virus to combat IFN-γ, one of the most important cytokines in the process of SARS-CoV-2 infection. Second, CVL218 can bind to the N-terminal region of the nucleocapsid protein of SARS-CoV-2. By binding to this protein, CVL218 disrupts the highly-ordered RNA conformation necessary for the virus to replicate and transcribe its genome. Currently, Convalife is working on the development of this drug.
Convalife’s in vitro experiments show that CVL218 is able to inhibit both the invasion and replication of SARS-CoV-2, whereas Remdesivir can only inhibit the invasion. EC50 is around 5 micro-mol (for reference, Remdesivir is around 1.5 micro-mol). Through a combination of Remdesivir and CVL218, EC50 is able to decrease to 0.7 micro-mol. Convalife has already finished phase I and II of clinical trials, which have shown the safety of CVL218 and produced promising results. Furthermore, Convalife has received positive responses from the Pre-IND Consultation Program of the U.S. FDA.
Convalife plans to focus their distribution efforts on foreign countries like the U.S., since the number of COVID patients within China is close to zero. Though the government of each recipient country will determine its price, Dr. Shen hopes Convalife’s drug will be accessible to all populations, regardless of income. The relatively simple production procedures for CVL218, along with its small molecular weight, reduces production costs. Further supporting international relationships, Dr. Shen has expressed interest in collaborating with any research institute or drug company world-wide that shows interest in their drug. Convalife has reached out to doctors in the U.S. to ask if they would like to work together.
When asked about major safety concerns or risks that could allow the drug to be misused, Dr. Shen stated that Convalife follows strict, standardized and specific procedures to guarantee the safety and effectiveness of their drug prior to clinical trials. These procedures ensure that Convalife maintains CFC at an appropriate level, and understands the toxicology and metabolism of their drug to a fine-grained level. To further demonstrate the safety of the drug, Dr. Shen cited the success of Convalife’s phase I and II trials. He stated that no level III adverse effects were observed--in fact, nausea and vomiting were the only adverse effects recorded.
In addition to the drug’s safety, Dr. Shen listed three other benefits of Convalife’s drug. First, it targets multiple aspects of the virus, whereas other drugs may only target one aspect. While some single-target drugs may be effective against their respective targets, “most ideal antiviral-drugs, such as an AIDS cocktail, should work on multiple aspects.” Second, Convalife’s drug can be orally administered, whereas Remdesivir must be administered via injection. Third, Convalife’s drug has been shown to be more effective than Remdesivir, and can improve the effectiveness of Remdesivir when administered simultaneously with it.
Though Convalife does not utilize a synthetic biology approach in the development of their drug, Dr. Shen generously offered us feedback on our project. He advised us to ensure its effectiveness and directness, and to consider a “cocktail therapy” that targets multiple aspects of the virus.
Dr. Lappan
Microbiologist Dr. Lappan suggested using Corynebacterium as our probiotic chassis instead of Lactobacillus . She explained that Lactobacillus is typically utilized for gut-related probiotics, and Corynebacterium exists in great abundance in the nasopharyngeal microbiome of healthy children. She explained that, since pathogens in the nasopharynx are typically gram negative, it is difficult to develop gram-negative nasopharyngeal probiotics. Despite the obstacle, she promoted the use of commensal gram-negative bacterial strains, suggesting Corynebacterium pseudodiphtheriticum, Dolosigranulum pigrum , and some Moraxella species.
August 19, 2020
Dr. Lappan promoted the use of Corynebacterium as a nasal probiotic due to its presence in high amounts in the nasopharyngeal microbiome of healthy children, and explained that since Lactobacillus is often used for gut-related probiotics, it is not as promising for use in the nasopharynx. She also explained that there is a lack of gram-negative bacterial species used as nasopharyngeal probiotics, likely due to the fact that more pathogens tend to be found within gram-negative species. However, she mentioned that commensal gram-negative bacterial species exist and can be potentially used as probiotics if tested in the same way as existing probiotic species. She also listed certain species that were found to be part of a healthy nasopharyngeal microbiome: Corynebacterium (“specifically Pseudodiphtheriticum , sometimes accolens”), Dolosigranulum pigrum, and some Moraxella species (however, M. catarrhalis causes disease).
When asked about whether a nasal probiotic should be administered using a spray or a swab, she stated that it would depend on the probiotic species and the time for their retention in the nasopharynx. However, she did mention that administering the probiotic using a spray would be much more effective than administering the probiotic using a swab, and felt that it would be easier to quantify the amount of bacteria delivered through the use of spray compared to a swab. As for nutrient sources for bacteria in the nasopharynx, Dr. Lappan stated that while this is an overlooked area in nasopharyngeal microbiome research, she felt that it was plausible to think that commensal bacteria in the nasopharynx could be providing protection against pathogenic species by consuming the existing nutrient sources.
Dr. Mapstone
Lydia Mapstone is the co-founder and CEO of Boobybiome, a company developing a breast milk probiotic. She suggested the use of multi-species probiotic, explaining that the interactions between multiple probiotic strains and the microbiome can make the probiotic more effective. Ms. Mapstone also provided guidance on probiotic testing, safety, implementation, and accessibility. Finally, she discussed public opinion on probiotics.
Lydia Mapstone is a PhD student at the University College London, as well as the co-founder and CEO of Boobybiome, a company that aims to provide a breast milk probiotic for premature infants. Ms. Mapstone was also a member of University of Edinburgh's OG team for the 2017 iGEM season. We consulted Ms. Mapstone for her expertise on probiotics.
September 14, 2020
First, we asked Ms. Mapstone how she went about selecting strains for her probiotic; specifically, why she chose to pursue a multi-species probiotic over a single-strain probiotic. She explained that the human microbiome is a complex community, then provided an analogy she learned from an expert in infant microbiology. If you are at a party and two people enter, no one will notice. However, if ten people who know each other and the people at the party enter, then they will change the dynamics of the party. Similarly, a multi-species probiotic formulated with the relationships between strains in mind may be more effective than a single-species probiotic.
When choosing which strains to include, Ms. Mapstone stressed the importance of shaping the developing infant gut. The infant gut differs from a mature gut in both pH and oxygen; it is not a completely anaerobic environment. Therefore, infants require bacteria that make the gut into a suitable environment for obligate anaerobes. If researchers simply introduced bifidobacteria, they may not even survive.
To investigate whether the selected strains interact well with the immune system, Ms. Mapstone suggests introducing the probiotic to a mammalian cell line and measuring IL-10, IL-10, TNF-α. Additionally, to investigate whether the strains can survive in the human body, Ms. Mapstone suggests growing the bacteria on agar plates that mimic the environment of the microbiome in terms of bile salts and pH. Finally, Ms. Mapstone plans to outsource the safety verification of the probiotic, which includes cytotoxicity assays and co-culture. The eventual product will be regulated as a food supplement, rather than a drug (developing a drug is much more expensive).
After discussing the safety of the probiotic in the infant, I asked how researchers could ensure the safety of the probiotic if it were to escape into the environment. Ms. Mapstone explained that the bacterial strains she intends to use are already present in breastmilk, meaning they are already in the environment. In addition to selecting native, non-engineered strains, Ms. Mapstone suggested strategically selecting obligate anaerobic bacteria for probiotics, since these bacteria cannot survive outside the body, anyway.
Finally, for the format of the probiotic, Ms. Mapstone stated that she originally considered a freeze-dried probiotic. However, recent literature suggests that a liquid probiotic may be more likely to survive in the body, though this format may be more expensive and reduce shelf life. The liquid probiotic would be administered about once a day.
When I asked about public perception of a breast milk probiotic, Ms. Mapstone stated that she anticipated a positive reaction. Due to the rise in popularity of live cultures in yogurts and kombucha, Ms. Mapstone feels as though the public is already familiar with probiotics. Additionally, the public is generally familiar with the importance of breast milk over formula. Ms. Mapstone hopes that consumers could be open to a breast milk probiotic as a means to “bridge the gap” between milk and formula. A breast milk probiotic may be even more attractive to consumers if it is tailored to individual infants. Ms. Mapstone hopes to create a database of breast milk donors to better tailor the probiotic based on diet and genetic factors.
Ms. Mapstone’s probiotic, especially if tailored to individual infants, could be of great benefit to premature infants who would otherwise drink sterilized breast milk. However, the price of this future product could act as a barrier to mothers and infants who need it. When asked how she could make the probiotic as accessible as possible, Ms. Mapstone stated that she would work with the National Health Services to ensure a low price. Ms. Mapstone explained that, in countries without such a contract between producers and the government, such as in the United States, the probiotic could sell for 10x as much. Additionally, Ms. Mapstone aims to reduce manufacturing costs in order to lower the price of the end product. Despite the high cost of production from ELISA kits and testing, Ms. Mapstone is pursuing other ways to streamline the production process. For example, if she can identify effective bacterial strains, she could reduce the number of bacterial species necessary for the probiotic (if 3 strains could do the work of 10, for example).
Ms. Karolyn Gale
Ms. Karolyn Gale is the Senior Director of regulatory affairs at biotechnology company Emergent BioSolutions, which has partnered with third party pharmaceutical and biotechnology innovators to manufacture potential COVID-19 vaccines. She discussed vaccine accessibility and regulation, addressing concerns related to the speed of vaccine development related to Operation Warp Speed.
September 29, 2020
Ms. Karolyn Gale is the Senior Director of regulatory affairs at biotechnology company Emergent BioSolutions, responsible for reviewing documents and launching products. Emergent BioSolutions develops and manufactures medical countermeasures against public health threats such as botulism, chikungunya, and anthrax. Such countermeasures include both human and equine hyperimmune plasma, as well as NARCAN® Nasal Spray to address the opioid crisis.
Due to the pandemic, Emergent BioSolutions is developing human and equine plasma derived hyperimmune treatments, and partnering with third-party pharmaceutical and biotechnology innovators to manufacture potential COVID vaccines. These projects are supported by Operation Warp Speed, which provides funding and support for public-private partnerships to develop a vaccine against SARS-CoV-2.
When asked if drug regulations could or even should be improved, Ms. Gale affirmed her confidence in the current regulations. Though the public may perceive vaccine progress as “too fast” under Operation Warp Speed, Ms. Gale explained that the concerted effort of public and private researchers is to thank for rapid vaccine development, rather than major protocol changes. “There is no exception to good manufacturing practices,” she added, emphasizing that “no shortcuts” have been taken throughout the development process. She went on to add that vaccine developers have become increasingly transparent throughout the pandemic, openly sharing information such as clinical trial protocols on the internet.
I next asked how vaccine distribution could be managed with accessibility and fairness in mind, once a vaccine is successfully developed. Ms. Gale responded that distributors must consider factors such as risk to exposure and risk of severe infection. While front-line medical professionals are clearly at high risk of exposure, it may be more difficult to determine who will become extremely sick, versus who would experience mild symptoms or even remain asymptomatic. While Ms. Gale does not work with product pricing in regulatory affairs, she suggests that manufacturers engage in cross-functional discussions with key opinion leaders and government agencies to determine the best distribution strategy.
Opinions shared in this interview are specific to the individual, and not necessarily that of Emergent BioSolutions.
Protection of Human Subjects Protocol
To ensure that our interviews with medical doctors were conducted in a professional manner, especially with respect to informed consent, we submitted a protocol for the protection of human subjects to our university. Prior to the submission of the protocol, our entire team underwent on-line CITI training in both protection of human subjects and research ethics. The Protection of Human Subjects Committee (PHSC) at our university approved our protocol, which outlined procedures for obtaining informed consent.
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Integrating Feedback and Guidance
Interviews with stakeholders and experts in probiotics and drug development shaped our project in multiple ways.
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Design
Dr. Shelhamer helped us to carefully consider the interactions between our probiotic and the immune system. Specifically, he outlined potential effects of anti-inflammatory PUFAs such as DHA, and how these PUFAs could result in immunosuppression if administered prior to infection or in excess. His feedback led us to investigate the use of AA in addition to DHA, and to design a “smart” circuit that could determine when to secrete DHA over AA. Dr. Shen Xiaokun’s drug development expertise encouraged us to target multiple aspects of the viral life cycle with our probiotic. Similarly, an M.D. suggested that simply targeting pathogens may be insufficient to resolve the chronic conditions, such as self-sustaining inflammation, that they cause. PUFAs address this concern by influencing inflammation and immune function in addition to suppressing viruses. Furthermore, the ability of PUFAs to lyse viral envelopes, disrupt membrane replication by positive-strand RNA viruses, and regulate inflammation allows PUFAs to combat viruses in multiple ways, echoing Dr. Xiaokun’s suggestion.
Please see our Design page for more information about our project design.
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Safety and Implementation
Many of our contacts provided invaluable feedback on the implementation and safety considerations of our project. This feedback led us to design the probiotic as a prophylactic treatment, administered once per day via nasal spray. Though Dr. Shelhamer voiced concern over a spray, which may deposit bacteria deep within the lungs very quickly, we have investigated additional safety precautions to address this concern. To ensure that the probiotic will survive within the nasal cavity long enough to have an effect, despite mucociliary clearance, we investigated the poloxamer gel suggested by Dr. Shikani.
Safety was our priority when selecting species. Our contacts guided us in identifying potential species and evaluating their safety within a probiotic. Ms. Mapstone, co-founder and CEO of breastmilk probiotic company Boobybiome, suggested that we consider multiple species to have a larger impact on the microbiome. We decided on two potential chasses to be included within our probiotic, both of which are BSL1 and native to the nasopharyngeal region. We paid special interest to species that had been utilized or investigated previously for probiotic use. Though species like Neisseria cinerea and Corynebacteria fit some or all of these criteria, Dr. Turner warned us of the relations of similar species to disease. While investigating strategies to demonstrate the safety of BSL1 gram negative species, we investigated alternative chasses, such as species of Lactobacillus . Then, we researched ways to adapt circuits designed for gram-negative species for use in gram positive species such as Lactobacillus .
In addition to investigating alternative strains, we identified means to improve the safety of all potential strains, including genome-searches for concerning sequences (and sequential knockout of these sequences), genetic kill switches, and the acquisition of “water-proof” safety data. Contacts advised us regarding surveys and assays to assess the safety of the probiotic, including SNOT surveys and cytotoxicity assays. Strong safety data could allow for the implementation of species previously unused in probiotics, such as Neisseria cinerea .
Our contacts warned that the safety verification of the probiotic would be expensive if the probiotic is regulated as a drug. However, a nasal probiotic cannot be regulated as a food supplement, the typical route for probiotics. Additionally, safety data from rigorous drug testing would be imperative in making the probiotic attractive to potential patients.
Though many of our contacts felt that their patients would be open to a nasal probiotic, even if it were genetically-modified, safety concerns could discourage patients from taking TheraPUFA. When we presented our project to incoming college freshmen, they voiced many concerns that future patients may have: How can we determine that the probiotic is safe? If PUFAs inactivate the virus, will the body still be able to develop immunity to that virus? When would the probiotic be administered? Would it be administered prophylactically? What would occur if the probiotic is administered “too late”? These questions provided us with insight on the concerns of potential stakeholders. To answer the questions, we returned to literature review and interviews with medical experts, many of whom recommended prophylactically once daily. Our interviews also informed us of drug regulations and safety testing procedures described previously. In addition to rigorous human practices, we addressed our “stakeholders” questions through our mathematical model, which informed us of the dosage necessary for the probiotic to be effective, and for the effect of the probiotic at different stages of infection.
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Accessibility
The cost of regulating the probiotic as a drug could increase manufacturing costs and eventual price, a barrier to accessibility. Ms. Mapstone provided guidance on keeping the probiotic accessible despite manufacturing and regulation costs. She described strategies for reducing the cost of her own probiotic, such as negotiating contracts with government agencies and streamlining production by formulating an efficient combination of probiotic species. Similarly, Dr. Xiaokun stated that the simple production procedures for his designed drug, along with its small molecular weight, would reduce its manufacturing costs.
Despite best attempts to reduce manufacturing costs, accessibility issues may still pervade during the pandemic, when there is an urgent, overwhelming need for therapeutics. Ms. Gale suggested that distributors first ensure that priority be placed upon front-line medical workers who are at particular risk of exposure. Additionally, though it is difficult to determine which patients will become the most sick, Ms. Gale suggested that priority be placed upon patients at particularly high-risk. Ms. Gale suggests that manufacturers engage in cross-functional discussions with key opinion leaders and government agencies to determine the best distribution strategy.
Finally, despite our efforts to ensure equal accessibility, some patients would be excluded from our probiotic. From our interviews with medical experts, we determined that elderly patients and immunocompromised patients should be excluded from our probiotic due to safety concerns.
Please see our safety and implementation pages.
References
Ge, Y., Tian, T., Huang, S., Wan, F., Li, J., Li, S., Yang, H., Hong, L., Wu, N., Yuan, E., Cheng, L., Lei, Y., Shu, H., Feng, X., Jiang, Z., Chi, Y., Guo, X., Cui, L., Xiao, L., Li, Z., Yang, C., Miao, Z., Tang, H., Chen, L., Zeng, H., Zhao, D., Zhu, F., Shen, X., & Zeng, J. (2020). A data-driven drug repositioning framework discovered a potential therapeutic agent targeting COVID-19. bioRxiv, 2020.2003.2011.986836. https://doi.org/10.1101/2020.03.11.986836
Liu, Y., Chen, L. Y., Sokolowska, M., Eberlein, M., Alsaaty, S., Martinez-Anton, A., Logun, C., Qi, H. Y., & Shelhamer, J. H. (2014). The fish oil ingredient, docosahexaenoic acid, activates cytosolic phospholipase A₂ via GPR120 receptor to produce prostaglandin E₂ and plays an anti-inflammatory role in macrophages. Immunology, 143(1), 81–95. https://doi.org/10.1111/imm.12296