Onconetix, Inc. 10-K

Item 1. Business Overview We are a biotechnology company focused on the research and development of transformational vaccines to prevent infectious diseases worldwide. We hold exclusive, global rights to novel technology licensed from renowned research institutions around the world, including St. Jude Children’s Research Hospital, the University of Oxford, Cincinnati Children’s Hospital Medical Center, and the University of Texas Health at San Antonio. We believe that our pipeline and vaccine platform are synergistic for developing next generation preventive vaccines to improve both health outcomes and quality of life globally. We seek to develop vaccines that provide long-lasting immunity to harmful viral and bacterial pathogens that cause infections in patient populations with high unmet needs. Our most advanced vaccine candidate is a live-attenuated, intranasally delivered, serotype independent Streptococcus pneumoniae vaccine to prevent middle ear infections, also known as acute otitis media (AOM), and pneumococcal pneumonia. AOM is a significant burden globally, particularly in young children, and pneumococcal pneumonia primarily impacts the elderly population. Additionally, we believe that this attenuated bacterium can serve as a platform to protect against other infectious agents that cause acute otitis media, such as non-typeable Haemophilus influenzae and Moraxella catarrhalis , by anchoring antigens from these pathogens on the surface of BWV-201, our attenuated Streptococcus pneumoniae bacterial vaccine. We hold a global, exclusive license to this technology, which was generated from the laboratory of Jason Rosch, Ph.D., of St. Jude Children’s Research Hospital. Our influenza programs are based on technology developed by Sunetra Gupta, Ph.D. at the University of Oxford, for which we hold a global, exclusive license for use of epitopes of limited variability, ELVs, to develop novel influenza vaccine candidates. Identified through a proprietary computational research and discovery process, we believe a vaccine formulated with these epitopes from different influenza strains will produce a viable universal influenza vaccine candidate. We are exploring the development of these influenza ELVs utilizing our norovirus shell and protrusion (S&P) nanoparticle vaccine platform, licensed from Cincinnati Children’s Hospital Medical Center, or CHMC. We are also utilizing this platform to develop a vaccine for the prevention of gastroenteritis caused by norovirus or rotavirus, as well as novel vaccines for malaria and monkeypox. The final candidate in our vaccine pipeline is a live-attenuated, orally delivered vaccine to prevent Chlamydia, for which we have a global, exclusive license to this technology originated from the University of Texas Health at San Antonio. We leverage the expertise of each of our collaborators to pursue the discovery and development of vaccines for these diseases, each of which represent high unmet needs globally. In addition, we have expertise in identifying business development opportunities for our platform vaccines technologies and portfolio. This allows for both internal pipeline expansion and the ability to generate non-dilutive revenue from potential licensing partners to utilize our discovery engine vaccine platform. There is potential for adjunctive or next generation therapeutic exploration to enhance current standard of care options. Vaccination has been used as an effective method of protecting individuals against harmful diseases by utilizing the body’s natural defense system to develop resistance or immunity to infections (World Health Organization, https://www.who.int/news-room/q-a-detail/herd-immunity-lockdowns-and-covid-19 ). The body’s immune system naturally creates antibodies and cell — mediated immunity to defend against foreign pathogens. Vaccines introduce or present these foreign pathogens, prompting the body’s immune system produce a response protective against the pathogen without exposing the body to the relevant lethal or harmful infection (World Health Organization, https://www.who.int/news-room/q-a-detail/herd-immunity-lockdowns-and-covid-19 ). While vaccines are generally able to provide resistance against disease, many infectious diseases can evolve or mutate leading to shortcomings of traditional vaccines, such as yearly reformulations. We believe our vaccine candidates can provide an alternative to the current standards of care by harnessing durable and long-lived immune response to specific or multiple antigens. The global vaccine market has recently experienced significant growth caused by rising awareness of the importance of immunization and vaccination benefits in emerging markets as well as by projects to fuel further global market expansion. For instance, The World Health Organization (WHO) has undertaken initiatives to increase immunization awareness through its Global Vaccine Action Plan and Global Immunization Vision and Strategy. As such, market research professionals project the global vaccine market size to reach $73.78 billion by 2028, representing a compounded annual growth rate (CAGR) of 7.3% over the forecast period, driven by rising prevalence of infectious diseases, increasing government funding for vaccine production and growing emphasis on becoming immunized. 1 This market acceleration has been coupled with various strategic transactions in the sector, including consolidations and mergers and acquisitions in recent years. Major market participants have strategically acquired start-ups and mid-sized companies to broaden their products portfolios and service offerings. For instance, in February 2019, Bharat Biotech acquired Chiron Behring Vaccines, one of the leading manufacturers of rabies vaccines across the globe. Additionally, in October 2018, Emergent BioSolutions, a multinational specialty biopharmaceutical company, acquired PaxVax for $270 million, and in July 2017 Sanofi acquired Protein Sciences for $650 million. In the pneumococcal disease market specifically, for which we are targeting for our Streptococcus pneumoniae vaccine candidate, GlaxoSmithKline acquired Affinivax for up to $3.3 billion in May 2022. The appetite of these companies to buttress their vaccine programs and pipelines reflects the increasing importance of vaccines in the healthcare sector, both nationally and worldwide. The U.S. Centers for Disease Control, or CDC, its Advisory Committee on Immunization Practices, or ACIP, and similar international advisory bodies develop vaccine recommendations for both children and adults. New pediatric vaccines that receive ACIP preferred recommendations are almost universally adopted, and adult vaccines that receive a preferred recommendation are widely adopted. We believe that our vaccine candidates will be well-positioned to obtain these preferred recommendations, by virtue of their longer and more durable immunity, which could drive rapid and significant market adoption. PIPELINE Our vaccine candidates are being developed in a manner that is scalable, designed to be cost-effective and provide long term benefit to patients from infectious agents. The FDA regulatory approval process is lengthy and time -consuming , and we may experience significant delays in the clinical development and regulatory approval of our vaccine candidates. Our vaccine candidates are in early stages of development and may fail in development or suffer delays that materially and adversely affect their commercial viability. We may be unable to complete development of or commercialize our vaccine candidates or experience significant delays in doing so due to regulatory or other uncertainties. Strategy We aim to identify, discover and develop novel preventive vaccines for infectious diseases. Key elements of our strategy include: ● Investment in advancing the development of our novel vaccine pipeline programs through IND-enabling activities and Phase I clinical studies. ● We plan to advance our main vaccine programs: S. pneumoniae induced AOM and pneumococcal pneumonia, influenza, norovirus-rotavirus, malaria, and Chlamydia. ● Our in-licensed vaccine candidates are carefully selected based on the following criteria: area of significant unmet medical need for preventive long-term vaccine; strong scientific rationale and established clinical and regulatory pathways; defined competitive landscape and potential future commercial opportunity; and license exclusivity. ● Prioritizing the research and development for our lead vaccine candidate, BWV-201, through Phase I. ● We plan to develop an intranasally delivered, serotype independent Streptococcus pneumoniae vaccine, capable of protecting young children against acute otitis media, also known as middle ear infections, and the elderly against pneumococcal pneumonia. In collaboration with St. Jude Children’s Research Hospital, we are exploring the potential to anchor antigens from additional otopathogens to the surface of this vaccine, including non-typeable Haemophilus influenzae and Moraxella catarrhalis. ● Maximize and utilize the value of our collaborators and third-party vendors. ● We will combine disciplined business strategies to further expand the potential synergies with current collaborators. 2 ● Deploy and expand our proprietary norovirus S&P nanoparticle platform. ● Our immunogenic multi-purpose vaccine platform technologies can be utilized with an array of infectious disease agents to access multiple development pathways and allow for potential next-generation life cycle management to expand our pipeline and pursue business development opportunities. There is potential for the platform to pursue adjunctive therapies to currently available drugs, and for current therapies to be re-optimized and formulated to protect against multiple antigens. We plan to utilize this platform to explore the potential to formulate our influenza vaccine candidates by presenting patented epitopes of limited variability within the platform. Management and History Blue Water Vaccines Inc. was founded in October 2018 by our Chief Executive Officer, or CEO, Joseph Hernandez, with the initial goal of developing a transformational universal flu vaccine to treat and prevent infections in patients globally. Our initial technology, licensed from the University of Oxford, provides a novel approach to developing a universal influenza vaccine. Subsequently, our team has identified other program candidates and technologies to broaden and diversify our vaccine pipeline. Mr. Hernandez, our Chairman and CEO, is a veteran entrepreneur, philanthropist, and operator with a broad skillset of founding, building, and selling companies, as well as executing business development transactions and securing private and public capital, including Digene, Noachis Terra and Blue Water Acquisition Corp. Mr. Hernandez was responsible for our initial $7 million seed funding round from investors including CincyTech. In addition to his position as our Chairman and CEO, Mr. Hernandez also served on the board of directors for Clarus Therapeutics, Inc. (OTCpink: CRXTQ) until August 2022, and serves on the board of certain private companies. Subsequently, a team of veteran industry executives and advisors were assembled, bringing valuable expertise to our growing infectious disease company. Jon Garfield, our Chief Financial Officer, has over 20 years of financial leadership experience, including with healthcare companies. Mr. Garfield regularly provides consulting services to private equity funds and privately held companies and served as the CEO of Unity MSK from February 2021 to January 2023, and served as interim Chief Financial Officer of Blue Water Vaccines Inc. from September 2021 until the consummation of our initial public offering in February 2022, upon which he became our full-time Chief Financial Officer. Erin Henderson, who serves as our Chief Business Officer and Corporate Secretary, has over 20 years of leading strategic transactions, governmental and stakeholder relations and corporate expansion. Previously, since 2010, she was the Managing Principal at The Aetows Group, a management consulting firm serving both the public and private sectors. Andrew Skibo is our Head of Biologic Operations and was recently Head of Global Biologics Operations at MedImmune/AstraZeneca and previously worked for Amgen and Genentech (now Roche), where he was responsible for operations, engineering, construction, and validation for large-scale capital projects related to bio-pharmaceutical manufacturing. Ali Fattom, Ph.D. is our Head of Science and Discovery and was recently Chief Scientific Officer at BlueWillow Biologics, where he led their efforts to develop viral vaccines for various infectious diseases, including HSV, RSV, and influenza, and previously worked for Nabi Biopharmaceutical and The National Institutes of Health. Dr. Fattom is also an Adjunct Professor at the University of Michigan. Additionally, members of our Board of Directors have extensive expertise in the fields of life sciences, business, and finance. In addition to Mr. Hernandez, our directors include Vuk Jeremić, previous Chair of the Council of Europe’s Committee of Ministers and previous President of the United Nations General Assembly, Simon Tarsh, a retired Deloitte Consulting Managing Director with experience in Life Sciences, Timothy Ramdeen, who has nearly a decade of experience in private equity and hedge fund investing, capital markets, and company formation, and James Sapirstein, R.Ph., M.B.A, President, CEO and Chairman of First Wave BioPharma, Inc. (Nasdaq:FWBI). Our Scientific Advisory Board includes Sunetra Gupta, Ph.D. Professor of Theoretical Epidemiology at The University of Oxford, a leading voice in infectious disease globally; and John Rice, Ph.D., Managing Director at CincyTech, with more than 30 years of biotechnology advising experience. 3 Subject to certain non-compete restrictions, our chief executive officer, Joseph Hernandez, and other key personnel may pursue other business or investment ventures while employed with us. Accordingly, they may have conflicts of interest in allocating time among various business activities and potentially competitive fiduciary and pecuniary interests that conflict with our interests. See “Risk Factors — Our Chief Executive Officer, Joseph Hernandez and our Chief Financial Officer, Jon Garfield, hold certain management positions and directorships of other companies and may allocate their time to such other businesses, which may cause conflicts of interest in their determination as to how much time to devote to our affairs and potentially competitive fiduciary and pecuniary interests that conflict with our interests.” For a complete discussion of the business affairs of our officers, directors and other personnel, please see “Management — Executive Officers and Directors.” Any such additional business activities or ventures may present conflicts to our interests. We do not believe that any such potential conflicts would materially affect our ability to conduct our operations. Our Infectious Disease Vaccine Candidates Infectious diseases are one of the leading causes of death worldwide. Infectious disease is caused by microorganisms or pathogens, including viruses, bacteria, fungi, and parasites that infect an individual and cause disease. Diseases often cause high fever, inflammation, or other symptoms. While some diseases can be treated with drugs or therapeutics, some infectious agents evolve to become resistant to commonly used drugs, such as antibiotics, and can become difficult to control. Infectious diseases can be passed from person to person or transmitted by insects or other animals. In many cases, vaccines are used to elicit a protective immune response in the absence of an infection to render an individual immune to a particular infectious disease. BWV-201: Streptococcus pneumoniae (S. pneumoniae) Vaccine Our BWV-201 vaccine candidate is an intranasally delivered, live-attenuated, serotype-independent vaccine, for which early data supports further investigation to pursue a long-term preventive intranasal vaccine for S. pneumoniae induced acute otitis media, or AOM, and pneumococcal pneumonia. We in-licensed the novel live-attenuated S. pneumoniae strain from St. Jude Children’s Research Hospital, or St. Jude, as a potential serotype independent vaccine. Researchers from St. Jude developed a strain of S. pneumoniae that contains greatly reduced virulence, yet can transiently colonize the nasopharyngeal cavity, inducing immune responses to significantly decrease the incidence of AOM and sinusitis as demonstrated in animal models. Our vaccine production is a straightforward process, utilizing the entire attenuated bacterium with purification and concentration steps only in the downstream process, thereby reducing the time and cost of production significantly compared to commonly used polysaccharide or conjugate vaccines. There is potential for this vaccine to provide a long-term, leading alternative treatment for AOM and pneumococcal pneumonia and subsequent introduction of a novel preventative standard of care. The development of a novel vaccine could eradicate potential short-term pain and/or long-term harmful side effects from contracting the bacteria, as well as eliminate or decrease the need for antibiotic treatment. Complications from AOM include sensorineural hearing loss, or SNHL, in adults but are more relevant for the endangerment of children, while pneumococcal pneumonia primarily impacts elderly adults and can lead to hospitalization and other subsequent infections. 4 Based on information from the American Academy of Pediatrics, over 5 million cases of AOM are reported annually in the U.S., resulting in approximately 30 million medical care visits and over 10 million antibiotic prescriptions, representing approximately $4.3 billion spent on treatment in the U.S. alone. AOM is the most common condition treated with antibiotics in the United States and increasing antibiotic resistance among the organisms responsible for AOM is of concern to researchers and public health officials globally. Additional statistics supporting the need for a novel preventive vaccine: ● The global AOM rate is 10.85%, or 709 million cases per year, with 51% occurring in children under 5 years old (Tong et al. BMC Health Serv Res. 2018; 18: 318). ● By 3 years of age, 80% of children globally are expected to have at least one episode of AOM. (Vergison A, Lancet Infect Dis. 2010 Mar;10(3):195-203. Doi: 10.1016/S1473-3099(10)70012-8. PMID: 20185098.). ● Current treatment for AOM is by antibiotic prescription, with more than 80% of all consultations resulting in a prescription. (Haggard, M. Eur J Pediatr 170, 323 – 332 (2011). https://doi.org/10.1007/s00431-010-1286-4 ). ● Even with the introduction of the pneumococcal conjugate vaccine (PCV13) in 2010, 26-36% of cases of AOM in U.S. were caused by S. pneumoniae. (Casey JR, Kaur R, Friedel VC, Pichichero ME. Acute otitis media otopathogens during 2008 to 2010 in Rochester, New York. Pediatr Infect Dis J . 2013;32(8):805-809. Doi:10.1097/INF.0b013e31828d9acc). ● Worldwide cases of AOM due to S. pneumoniae is estimated to be 30-50%. (Bergenfelz C, Hakansson AP. Curr Otorhinolaryngol Rep. 2017;5(2):115-124. Doi: 10.1007/s40136-017-0152-6. Epub 2017 May 20. PMID: 28616365; PMCID: PMC5446555.). ● An estimated $4.3 billion USD is spent on AOM treatment each year in the U.S. alone. (Tong S, BMC Health Serv Res. 2018 May 2;18(1):318. Doi: 10.1186/s12913-018-3139-1. PMID: 29720156; PMCID: PMC5932897.). The current standard of care treatment for AOM in children is reliant on antibiotics. The resolution rate of AOM in children is 81% without antibiotic treatment vs. 93% with antibiotic treatment. Antibiotic treatment of AOM in children has limitations, including recurrence within 30 days. Pneumococcal pneumonia, caused by colonization of S. pneumoniae in the lungs, primarily impacts elderly adults and, according to the CDC, results in approximately 150,000 hospitalizations in the United States alone each year. In addition to the disease burden, pneumococcal pneumonia accounts for approximately $1.3 billion in direct medical costs annually plus costs associated with lost productivity (O’Brien K, Pneumococcus, Pneumococcal Disease and Prevention, The Vaccine Book (Second Edition), Academic Press, 2016, Pages 225-243, ISBN 9780128021743). While there are currently available pneumococcal vaccines, outlined below, these vaccines provide limited levels of protection against pneumonia, as they are administered intramuscularly and do not elicit strong mucosal immunity (Berild JD, 2020. Pathogens, 9(4), 259. DOI: 10.3390/pathogens9040259). This technology from St. Jude is delivered intranasally, which is hypothesized to provide adequate levels of mucosal immunity to prevent non-invasive pneumococcal disease, including pneumonia and AOM. 5 The CDC recommends broad pneumococcal vaccines for children younger than 2 and for adults over 65 years of age (CDC). The CDC also recommends vaccinations for children and adults aged 2 through 64 either previously unvaccinated or partially vaccinated. Three vaccines are currently approved in the U.S. and other countries: (i) Prevnar13 or PCV13 (under 18), (ii) Prevnar20 or PCV20 (Pfizer) and (iii) Pneumovax or PPSV23 (Merck). An additional vaccine, Synflorix, is approved for use outside of the U.S. for the prevention of pneumococcal disease and S. pneumoniae induced AOM for the 10 serotypes included in the vaccine. Therefore, an effective serotype independent S. pneumoniae AOM vaccine could significantly impact pediatric healthcare demand and may reduce hospitalizations for pneumococcal pneumonia in older adults. As a preventative treatment, the vaccine’s advantages include reduction of near-term pain; reduction of recurrent AOM that may result in the need for tympanostomy tube placement; lessening of antibiotic usage, which would decrease the number of antibiotic resistant organisms in the environment; avoiding potential long-term hearing loss; and prevention of hospitalizations and deaths caused by pneumococcal pneumonia. Previous live, attenuated strains of S. pneumoniae were generated by deleting several highly immunogenic virulent genes and therefore may not be optimal vaccine candidates. Some of these deletions include antigens that induce antibody responses following pneumococcal carriage and otitis media in young children and therefore may not be optimal vaccine candidates. Our technology in-licensed from St. Jude focuses on candidate genes essential for microbial adaptation to the host environment while maintaining virulence determinants. The St. Jude researchers developed a S. pneumoniae strain with a deletion in ftsY , a central component of the signal recognition pathway (SRP). SRP mutants have greatly reduced virulence, although virulence factors are still produced. The S. pneumoniae ftsY deletion strain may potentially make an ideal live-attenuated vaccine, as it can transiently colonize the nasopharyngeal cavity without inducing immune responses to virulence protein antigens but does not cause invasive disease. Our candidate vaccine is a live-attenuated serotype-independent vaccine, that early data supports further development to pursue a potential long-term preventive intranasal treatment. BWV-201 will likely require two doses to provide life-long protection. BWV-201 has the ability to transiently colonize the nasopharyngeal cavity and significantly decrease the incidence of AOM and sinusitis in animal models. The vaccine candidate is derived from the noninvasive serotype 19F strain BHN97, which normally causes sinusitis/purulent rhinitis and AOM. As previously noted, the ftsY gene was deleted by St. Jude researchers, and is designated BHN97∆ftsY (Rosch, Jason W et al. EMBO molecular medicine vol. 6,1 (2014): 141-54. Doi:10.1002/emmm.201202150). We are also exploring the potential for BWV-201 to present antigens from additional AOM-causing pathogens, such as non-typeable Haemophilus influenzae and Moraxella catarrhalis . Based on preliminary data from St. Jude, we are able to present additional antigens and following intranasal vaccination with the new construct, vaccinated mice generate antibodies to both non-typeable Haemophilus influenzae and Moraxella catarrhalis , in addition to generating antibodies from various strains of S. pneumoniae. Our vaccine production is a straightforward approach, utilizing the entire bacterium with purification and concentration steps only in the downstream process thereby significantly reducing the time and cost of production compared to polysaccharide or conjugate vaccines. Preclinical data colonization and invasiveness and Otitis Media/Sinusitis Efficacy Our pre-clinical data has shown encouraging results from the research and development of BWV-201 as a potential intranasally delivered vaccine candidate. Multiple animal models have demonstrated protection from AOM. To demonstrate vaccine efficacy against AOM and sinusitis, mice were immunized (prime and two boosts) with Prevnar 7 (PCV7), Prevnar 13 (PCV13), Pneumovax (PCV23), D39x and BHN197 caxP and ftsY deletion mutants. Deletion of ftsY, a central component of the signal recognition particle (SRP) pathway show heightened sensitivity to environmental stress and have greatly diminished virulence. Deletion of caxP, a calcium/magnesium transporter, renders host physiological conditions in blood and mucosa toxic to the bacterium. BHN97ftsY serotype 19F is also characterized in PCV7, PCV13, and PCV23 (Rosch, Jason W et al. EMBO molecular medicine vol. 6,1 (2014): 141-54. Doi:10.1002/emmm.201202150). 6 In this head-to-head preclinical study, mice (n=25-31) were either mock-vaccinated (PBS) or live-attenuated vaccinated (with deletions of either type 2 or 19F backgrounds). PPV23 was used as a negative control. Two weeks following the second boost, the bioluminescent strain BNH97x (type 19F), a serotype included in Prevnar 7, Pneumovax and BHN97ftsY (referred to as homologous challenge) were introduced to the mice and imaged twice daily for development of AOM and sinusitis. Only BHN97∆ftsY (BWV-201), and to a lesser extent Prevnar 7, showed significant reduction in AOM and only BHN97∆ftsY demonstrated significantly reduced sinusitis compared to mock infected animals. The incidence of AOM was significantly ( p <0.05 compared to mock) lower in BHN97∆ftsY — vaccinated mice (Figure A-below). Only BHN97∆ftsY vaccine significantly decreased the incidence of sinusitis ( p < 0.05). Measurement of luminescence at 24 and 72 h confirmed protection engendered by BHN97∆ftsY. Figure 7. Vaccine protection against otitis media and sinusitis. Mice (n=25 – 31 per group, performed at least twice for each group) were mock-vaccinated with PBS (Mock) or vaccinated with live-attenuated vaccines deleted for caxP or ftsY on either a type2 (D39∆caxP, D39∆ftsY) or type19F (BNH97∆caxP, BNH97∆ftsY) background. Mice were challenged with a bioluminescent S. pneumoniae strain BNH97X (type19F) and imaged twice daily for development of AOM or sinusitis. A. The proportion of mice developing an infection of the ear or sinus by Xenogen imaging. * =p<0.05 by Chi-squared test compared to the mock vaccinated group. PPV23 was used as a negative control (60% otitis and 80% sinusitis). Errors bars represent standard error of the mean. PCV7 is Prevnar 7, PPV23 is Pneumovax and BHN97∆ftsY is BWV-201. To determine if BHN97∆ftsY, or BWV-201, (serotype 19F) can induce heterotypic AOM protection (AOM caused by a S. pneumoniae serotype not contained in the vaccine), mice (n=20) were immunized as detailed above and challenged with BHN54 (serotype 7), which causes otitis media in about 50% of challenged animals. The control vaccine Prevnar 13 contains serotype 7; therefore, this study compares heterotypic (BHN97∆ftsY) versus homotypic (Prevnar 13) vaccine protection. BHN97∆ftsY had a 10-fold lower incidence of AOM, (*p < 0.05) when compared to mock immunized animals, demonstrating that the attenuated vaccine does induce heterotypic protection. Bioluminescent signaling as well as, reduction in weight loss also demonstrated secondary analysis supporting vaccine protection. 7 BHN97∆ftsY induced protection from AOM was additionally confirmed in a chinchilla (n=20) animal model. The animals were immunized (prime and two boosts) and then challenged with BHN97 two weeks after the final boost. Vaccinated animals had a decreased incidence of culture-positive ears and had a significantly decreased number of recoverable bacteria from the middle ear (A). Following vaccination, a reduction in the number of culture positive ears in vaccinated group compared to the mock animals was observed (B) as well as significant reduction in recoverable CFUs from middle ear 7 days post challenge (C) * = p < 0.05 by Mann — Whitney. Figure 8. Vaccine protection in a chinchilla model of otitis media. The BHN97strain is capable of causing otitis media in chinchillas via intranasal administration as observed by recoverable bacterial colony forming units (CFUs) from the middle ear (A) following challenge. B, C Following vaccination with BHN97 ∆ftsY (BWV-201), a reduction in the number of culture positive ears in the vaccinated group compared to the mock animals was observed (B) as well as a significant reduction in recoverable CFUs from the middle ear at 7days post challenge (C). * =p<0.05by Mann — Whitney. Vaccine is BHN97 ∆ftsY (BWV-201). 8 A potential advantage of an attenuated S. pneumoniae vaccine such as BHN97∆ftsY is that immune responses are directed to bacterial proteins rather than just polysaccharides and should not be limited to serotype specific protection. Purified polysaccharide (PPV) vaccines such as Pneumovax (produced by Merck &Co.) and pneumococcal conjugate vaccines such as Prevnar 7/13/20 (produced by Wyeth/Pfizer) or Synflorix (produced by GlaxoSmithKline plc) are generally considered serotype specific, inducing protection to disease caused only by pneumococcal strains contained in the vaccines. Utilizing BWV-201 as a platform to protect against other pathogens Although S. pneumoniae remains the leading cause of acute otitis media, other otopathogens are known to cause the disease, including non-typeable Haemophilus influenzae and Moraxella catarrhalis . To holistically address acute otitis media, we plan to evaluate the possibility of adding antigens from non-typeable Haemophilus influenzae and Moraxella catarrhalis to the surface of the S. pneumoniae bacteria that make up BWV-201. To date, Dr. Jason Rosch at St. Jude Children’s Research Hospital has successfully anchored proteins from both additional pathogens to BWV-201 and has performed ELISAs to ensure antibodies were generated against each pathogen. Newly generated data, not yet published. Dr. Rosch engineered the live vaccine to express protective epitopes of non-typeable Haemophilus influenzae and Moraxella catarrhalis on the cell surface of BWV-201. Shown in the figure above, the novel vaccine construct raised antibodies against all three pathogens following intranasal vaccination by ELISA. Future development of this vaccine construct will include challenge studies in mice to determine efficacy of this vaccine construct in preventing disease caused by each pathogen. Mice will be vaccinated with the new construct, and will subsequently be exposed to S. pneumonaie , both heterologous and homologous strains, as well as non-typeable Haemophilus influenzae and Moraxella catarrhalis. Upon completion of this study and results showing decreased incidence of AOM in mice, we plan to pursue development of this vaccine construct and transfer to a partner CMO for manufacturing optimization. 9 BWV-101: UNIVERSAL INFLUENZA & BWV-102: H1 INFLUENZA The company’s influenza vaccine programs are focused on developing transformational and novel influenza vaccines: BWV-101 for an influenza vaccine to provide protection against H1, H3 and Flu B infections; and BWV-102 for a H1 only vaccine. This program is licensed from the University of Oxford in which all relevant studies were performed to support our hypothesis. Our goal is to develop a vaccine that protects against all influenza strains that commonly infect humans by targeting specific parts of the influenza viruses, which are of limited variability across flu strains and induce a strong protective immune response. This proof of concept will be leveraged to develop BWV-101 by studying the cross-reactivity of different flu strains, H1, H3 and influenza B. The BWV-101 vaccine candidate may potentially provide a therapeutic benefit that negates the need for annual vaccination, vaccine reformulation, and provide long-lasting broad protection against the flu to millions globally (Thompson et al. Nature Communications. 2018. 9:385). Influenza Influenza is a viral infection of the respiratory system, causing an infected person to suffer from certain symptoms, including fever, muscle aches, runny nose, cough, congestion, headaches, and fatigue. The four types of influenza viruses include type A, B, C, and D. The type A and B influenza viruses are referred to as human influenza viruses that are primarily responsible for seasonal flu epidemics each year. Type A flu viruses are further divided into two subtypes, named based on differences in two viral surface proteins called hemagglutinin (H) and neuraminidase (N). Influenza types C and D present a lower priority for vaccination, as Type C viruses cause a mild respiratory illness in humans and has not been associated with human epidemics, and Type D viruses primarily affect cattle and are not known to cause illness in humans ( https://www.cdc.gov/flu/about/viruses/types.htm ). Figure 3. This graphic shows influenza virus types including the two types of influenza viruses (A,B) that cause most human illness and that are responsible for the flu season each year. Influenza A viruses are further classified into subtypes, while influenza B viruses are further classified into two lineages: B/Yamagata and B/Victoria. There is a major unmet need for the development of a novel universal flu vaccine as a prophylactic therapy. Influenza is a major respiratory pathogen. The WHO estimates there are an estimated 1 billion cases of influenza infection with 3-5 million severe cases and 290,000-650,000 related respiratory human deaths worldwide every year. The estimate does not take into account deaths from other diseases such as cardiovascular disease, which can be influenza related. The next influenza pandemic is believed by many experts to be a potentially devastating global health threat. Influenza mortality rates are highest for the very young and elderly. 10 The global influenza vaccine market was valued at $3.96 billion in 2018, and is projected to reach $6.20 billion by 2026, representing a CAGR of 5.9% from 2019 to 2026. Currently, the standard of care and most effective protection against flu is through annual vaccination. The WHO estimates that worldwide, approximately $4 billion is spent on influenza vaccines annually. However, the flu also a major cause of work absenteeism, leading to an estimated annual productivity loss in the U.S. of $87 billion. Flu vaccination consists of a yearly injection of attenuated or inactivated (dead) influenza viruses to induce humoral immunity in the form of the antibodies against the current circulating or anticipated seasonal influenza strains. The induction of antibody-producing B-cells through vaccination allows the immune system to defend the body against the influenza virus circulating during the winter months. An annual seasonal flu vaccine is the best way to help protect against flu. Vaccination has been shown to have many benefits including reducing the risk of flu illnesses, hospitalizations and even the risk of flu-related death in children. The CDC recommends use of any licensed, age-appropriate influenza vaccine during the 2020-2021 influenza season, including inactivated influenza vaccine (IIV), recombinant influenza vaccine (RIV), or live-attenuated influenza vaccine (LAIV). No preference is expressed for any influenza vaccine over another. Both trivalent and quadrivalent influenza vaccines will be available. The trivalent vaccines formulation will include A(H1N1) pdm09, A(H3N2) and B/Victoria. The quadrivalent vaccine formulations will include A(H1N1) pdm09, A(H3N2) and B/Victoria, plus B/Yamagata ( https://www.cdc.gov/flu/about/viruses/types.htm ). The current influenza vaccines induce antibodies that target regions of the virus that are highly variable and have serious shortcomings, as they: (i) must be administered annually, (ii) typically provide protection to only 50% of the individuals who receive it; and (iii) need to be updated annually and reformulated 6 months prior to influenza season, such that strains that are subsequently prevalent during the applicable “flu season” are not protected against by the vaccine. Our Proprietary Epitope Discovery Using the technology that we have exclusively licensed from the University of Oxford, we are developing a universal influenza vaccine. Our exclusive license agreements include patented influenza epitopes of limited variability, or ELV, identified through a proprietary computational research and discovery process, discovered by Dr. Sunetra Gupta and her team at the University of Oxford. We have acquired intellectual property for cross-protective epitopes to be used for our vaccine candidates that were developed and identified through a unique computational discovery process at Oxford University. The data produced through computational analysis at Oxford has shown that antigen evolution in influenza is limited to certain regions of the virus that facilitate binding and entry to host cells and these regions of limited antigenic variability are naturally immunogenic and therefore may be used to develop universal immunity to influenza viruses. We have identified epitopes of limited variability in H1 influenza that have circulated throughout history (since 1918) and make ideal vaccine targets and have completed similar analysis of H3 and Flu B strains for similar epitopes which will be used to produce our lead vaccine candidate BWV-101 as a universal vaccine for influenza infection. Due to the cross-reactive nature of the H1 epitopes in pre-pandemic H1 influenza A, we are also pursuing the development of a stand-alone H1 vaccine (BWV-102). These epitopes are able to be formulated into a vaccine candidate using our virus-like particle (VLP) platform technologies and may be evaluated using other vaccine technologies through partnerships in order to accelerate development of potential vaccines or to explore adjunct therapies (Thompson et al. Nature Communications. 2018. 9:385). Figure 4. Current influenza vaccine targets. Antigenic Drift (Thompson et al. Nature Communications. 2018. 9:385) A single conformational epitope is typically 8 to 15 amino acids in length and in an extreme circumstance (where every change creates an escape mutant), a single epitope could theoretically vary from 208 to 2015 different ways. Therefore, a highly variable virus like influenza should be able to mutate in countless ways during each subsequent season. This would inevitability lead to an explosion of genetic diversity and numerous circulating strains. However, it seems that there is a constraint limiting how influenza evolves, leading to a single or limited number of strains dominating each season. In 2007, Sunetra Gupta led a group of researchers at the University of Oxford who published a proprietary mathematical model proposing that the single strain dominance, typically seen worldwide annually, could be explained by hypothesizing that epitopes of ‘limited variability’ exist (Antigenic Drift Hypothesis). The model hypothesizes that while there is a significant amount of mutation of influenza strains, this variability occurs in a specific portion of the virus, while certain epitopes are required to remain relatively constant and are more limited in their variability in order for the virus to infect individuals, thus clarifying how influenza is not as variable as commonly thought. 11 Antigenic Drift Hypothesis Illustration Figure 5. Identification of a site of limited variability in the head domain of the H1 HA. b,c Location of ABS of lowest variability containing position 147 with position 147 shown in yellow and the rest of the site colored in red. d Phylogenetic trees of pre-pandemic and post-pandemic highlighted rectangle H1N1with tips colored according to the conformation of the epitope of limited variability (hereafter called OREO). Please note the re-introduction of H1N1influenza in 1977 involved a strain which previously circulated in 1949/50. 12 The Antigenic Drift Hypothesis suggests the existence of epitopes of limited variability mediate a population’s immunity to influenza strains. As a particular influenza strain circulates in the population, immunity to a specific pattern of epitopes is induced. This leads the virus to change its antigenic configuration and cycle through its limited repertoire of antigenic conformations. However, population immunity also changes due to birth and death within the population (i.e. individuals in the population who had experienced and developed immunity to certain conformations die). This allows prior epitope conformations to reappear. The loss of herd immunity to these epitope of limited variability causes the emergence of epidemics ( Thompson et al. Nature Communications. 2018. 9:385 ). Oxford scientists have identified the naturally antigenic regions that drive immunity to influenza by evaluating serum from these from various age groups of humans using assays and ELISAs reveal periodic cross-reactivity to ELV. Pseudotype microneutralisation data reveals a cyclical pattern of epitope recognition. The studies of children’s sera were used to detect antibodies and demonstrated that young children ages 6 to 12 had immunity to historical influenza strains that circulated many years prior to when they were born and they could never have possibly been exposed to, one of which that last circulated in 1934. Mutagenesis of the identified regions of limited variability in various historical viruses removed the protective immunity. Furthermore, vaccination of mice, as shown below, with these regions of the influenza virus produced an identical immune response that was observed in the children. For example, the mice vaccinated with either the region from the influenza virus circulating in 2006 or 1977 were protected against infection with an influenza with a virus that last circulated in 1934, replicating the immunity seen in children ages 6 to 12. (Thompson et al. Nature Communications. 2018. 9:385) 13 Figure 6. Sequential vaccination using chimeric HA constructs. Five groups of mice were sequentially vaccinated with 2009-like (blue), 2006-like (red),1995-like (orange), 1977-like (green) and 1940-like (pink) epitope sequences substituted into H6, H5 and H11 Has. Two further control groups were sequentially vaccinated with H6, H5 and H11 constructs without any sequence substituted into the Has (vaccinated controls). Further two groups were mock vaccinated (unvaccinated controls). c,d,f,g Pseudotype microneutralisation assays using 0.5μl of sera from the bleed at 21 weeks. Error bars are mean ± s.e.m.n=6 for experimental groups and control groups. The values provided are an average of two replicates. 14 This work demonstrated that vaccination with just four variants of one region of limited variability in H1 influenza was able to elicit immunity to all historical H1 influenza strains. As these regions periodically reappear and disappear over time, vaccination with all of the possible variants would be expected to provide protection against future influenza strains as well. The identified epitopes are restricted in their variability due to presence of a receptor-binding site and small alpha helix structure between disulphide bonds. The following research findings form the basis for our influenza vaccine candidates: 1. Epitopes of limited variability which are under strong immune selection exist within influenza. 2. These epitopes drive the antigenic evolution of influenza. 3. These epitopes cycle between a limited number of different conformations. 4. Epitopes of limited variability would make ideal vaccine targets. BWV-101: Universal Influenza Vaccine Our approach to developing a novel, universal flu vaccine for the prevention and protection against human influenza strains and potential pandemic strains by targeting specific limited variability epitopes includes the following steps and processes. We are exploring development of an influenza vaccine utilizing both the S & P nanoparticles to determine the most effective and efficient presentation of our ELVs and the versatile S&P nanoparticle vaccine platform from CHMC with the H1 influenza antigens. Data in preclinical mice (Rotavirus-specific-antibody-free BALB/c mice, n=5-7) challenge studies inserted M2e, a spike protein of influenza, into a P-particle loop; showed mice that were vaccinated had 100% protection when injected with lethal doses of influenza (Tan et al. JOURNAL OF VIROLOGY, Jan. 2011, p. 753 – 764). This dual approach will allow us to gain valuable information as we further the development and manufacturing of the BWV-102 program and utilize it for the development of BWV-101. We are currently assessing the ELVs to determine the most effective and efficient route of antigen presentation. Additionally, we are currently optimizing antigens for H3 and Flu B to be included with the identified H1 antigens to finalize our universal influenza vaccine formulation. We are using established manufacturing methods, including E.coli fermentation to produce our chimeric proteins, to reduce the cost and increase the efficiency and scalability of our manufacturing process for the vaccine. The antigens will be displayed by a proprietary VLP that can be produced in E. coli (Pharmaceutics 2019, 11, 472; doi:10.3390/pharmaceutics11090472). Our research and discovery model uses bioinformatics and phylogenetic analysis to identify possible sites of epitopes of limited variability before confirming their existence experimentally. To date, we have identified naturally immunogenic epitopes for H1, H3 and influenza B. Bioinformatics studies and wet lab studies suggest that these epitopes, especially H1N1, and the chimeric scaffold configuration of our vaccine induce immunity due to induction of broad cross-reactive antibodies in other strains such as H10N3 (bird flu), and pandemic strains including H5NX, H7NX, and H9NX. H9NX (Thompson et al. Nature Communications. 2018. 9:385). Therefore, we foresee the development of H1N1 vaccine as a priority due to its high cross-reactive priorities. 15 BWV-102 Stand-Alone H1 Vaccine We are developing our H1 stand-alone influenza prophylactic product, BWV-102, to address potential pandemic zoonotic H1 strains, specifically the G4 EA H1N1 identified by scientists and reported in June 2020, as a potential next pandemic strain. BWV-102 is being developed using the H1 ELVs identified by the team at the University of Oxford. While the product is designed to protect against infection from any H1 strain, there is potential for cross protection from H5 and H10 strain infections as well. Preclinical studies were conducted in Balb C mice (n=6) using a prime-boost-boost protocol (Thompson et al. Nature Communications. 2018. 9:385). The proposed Phase I clinical study will employ this prime — boost protocol; however, it is possible that a single dose of the vaccine candidate will confer protection against current and historical H1 strains with a prime-boost dose or a single dose. As reported in 2020, the G4 EA H1N1 strain is the most prevalent influenza strain circulating among swine populations in China. The strain was first identified in 2016 and has been monitored by scientists in China through their swine surveillance program. The strain has genes from a mix of pig, avian and human viruses, including genes from the 2009 H1N1 flu pandemic virus. Currently, the G4 EA H1N1 strain is not transmissible human to human, however, scientists hypothesize that there is a high likelihood of strain reassortment occurring that could make human to human transmissibility possible. The current H1N1 influenza strain circulating may provide some protection against disease induced by G4 EA H1N1 infection. The ability of the BWV-102 ELVs to induce an immune response and protection against heterologous challenge with historical strains was assessed in Balb-C mice (n=6) ( Thompson et al. Nature Communications. 2018. 9:385 ). We are currently assessing the ELVs in combination with the S 60 particle, P 24 particle and a proprietary VLP, currently in development, to determine the most effective and efficient route of antigen presentation. Manufacturing of the product is expected to occur in E. coli (Pharmaceutics 2019, 11, 472; doi:10.3390/pharmaceutics11090472) . We anticipate results of the VLP presentation assessments in the first half of 2022. BWV Norovirus (NoV) S&P Nanoparticle Versatile Vaccine Platform Bioengineering the shell (S) and protruding (P) domains of the norovirus capsid protein, polyvalent nanoparticles and polymers/oligomers provide a versatile vaccine platform with wide applications Our Approach to Stimulating the Immune System for Infectious Disease Protection Our S&P platform was co-invented by two researchers, Xi Jason Jiang, Ph.D., and Ming Tan, Ph.D., of the Division of Infectious Disease at the Cincinnati Children’s Hospital Medical Center. The pre-clinical research conducted at CHMC provided encouraging data that supports further investigation and development of the platform for our vaccine candidates. The S&P platform combines two or more immunogenic components, a norovirus antigen plus at least one additional antigen, together creating novel constructs. The norovirus nanoparticle enhances immunogenicity of the inserted antigen. The S & P particles themselves also act as antigens, and are large enough to trigger an immune response to a foreign substance. By combining the norovirus nanoparticle with one or more antigens from other infectious disease(s), the immune system is stimulated to create antibodies to both the norovirus and the additional antigen(s). Key Elements of our Platform We are leveraging our disruptive norovirus nanoparticle platform to develop novel, broad-spectrum vaccines for adult and child infectious disease prevention by taking advantage of: ● Flexible and Scalable discovery platform engine. We believe we are able to design and create novel vaccines that are stable and scalable for broad spectrum prophylactics. Through this platform’s adaptability, we may opportunistically expand our pipeline and potentially collaborate with third parties for additional vaccines, as well as therapeutics. ● Cost-effective and Rapid Production of Novel Vaccines. We are potentially able to reduce the cost and time to manufacture a vaccine candidate by utilizing an E.coli expression platform, compared to traditional vaccine production which uses other, longer production-time platforms, such as Chinese Hamster Ovary (CHO) cells. We have bioengineered these nanoparticles to be stable and effective, as determined through animal immunogenicity studies, using E.coli expression which may provide cost savings and efficiency compared to other VLPs needing a eukaryotic expression system. (Pharmaceutics 2019, 11, 472; doi:10.3390/pharmaceutics11090472). ● Multi-antigen and Pathogen Capabilities. One of the key features of our platform is its ability to carry multiple antigens at a time, thereby creating a multi-targeted vaccine. It also provides the opportunity to develop vaccines for protection against not only viral pathogens, but also bacterial and potentially parasitic and fungal pathogens. ● Therapeutic potential. We believe our platform may offer opportunities to develop non-infectious disease therapeutic products, for example being used as a carrier or vehicle to transport drugs to specific target locations. 16 Viral capsid proteins are responsible for many basic functions necessary for viral life cycles, such as viral attachment and entry, and thus can elicit neutralizing antibodies against viral infection after immunization to humans and animals. Consequently, viral capsid proteins are promising vaccine targets against viral infection. Indeed, various capsid protein nanoparticles and complexes have been developed and used as nonreplicating subunit vaccines to combat various infectious diseases. Unlike traditional live-attenuated and inactivated virus vaccines that need cultivation of infectious virions and are associated with certain safety concerns, the nonreplicating VLP vaccines derived from bioengineered viral capsid proteins do not involve an infectious agent and, therefore, may be safer and have lower manufacturing costs than traditional vaccines. Thus, VLP vaccines represent a next generation of innovative vaccine strategy. Structure ● The NoV (VP1) capsid structure consists of two major domains: (i) a N-terminal shell (S) domain and (ii) a C-terminal protruding (P) domain. The S domain builds the interior shell of the capsid and the P domain forms the dimeric protrusions of the capsid. ● The protrusions (P) of norovirus capsid interact with viral glycan receptors for attachment to host cells to initiate an infection. ● The S domain interacts homotypically and drives self-formation of an approximately 60 nm VLP. ● The P domain exhibits homotypic interactions, forming a 24 nm VLP with dimeric protrusions for stabilization of the viral capsid. Additionally, it can also form oligomers or polymers. Figure 1. Lineage structures of norovirus capsid protein or viral protein 1 (VP1) and various nanoparticles derived from full-length or truncated VP1. The N-terminal shell (S) (green) and the C-terminal protruding (P) (dark blue) domains with a short flexible hinge (light blue) in between (with amino acid numbers based on GI.1 Norwalk virus VP1) are shown. (A) Production of full-length norovirus VP1s via a eukaryotic expression system self-assembles into virus-like particles (VLPs). (B) Production of the S or P domain via the Escherichia coli expression system self-assembles into S or P nanoparticles. 17 Due to the homotypic interaction attributed to the norovirus capsid domains, researchers at CHMC, through bioengineering, designed and generated two subviral nanoparticles, the 24-valent P 24 and the 60-valent S 60 nanoparticles, and P-derived polymers to serve as a multifunctional vaccine platform against different pathogens and illnesses. ● These nanoparticles and polymers are easily produced, highly stable, and extremely immunogenic which we believe makes them compelling platforms to serve to display foreign antigens, self-assembling into chimeric nanoparticles or polymers as vaccine candidates. ● There are several preclinical studies that showed P 24 /S 60 chimeric vaccine candidates that can display different foreign antigens and epitopes, as set forth below in Tables 1 and 2. Therefore, there may be additional candidates to further explore as human vaccines. (Xia et al. ACS Nano 2018, 12, 10665−10682) . ● Such VLPs and capsid-like nanoparticles may be excellent vaccine candidates against corresponding viral pathogens because they can retain arrays of antigenic epitopes that faithfully mimic those of the native virions, and these repeated viral antigens and epitopes stimulate strong immune responses in their animal and human hosts. In addition, such highly immunogenic subviral nanoparticles may also serve as versatile platforms that are able to display foreign antigens for improved immune responses to facilitate development of novel vaccines against various pathogens and diseases. ● The fact that the P 24 VLP nanoparticles and polymers are composed of authentic norovirus antigens and retain norovirus-specific molecular patterns make it an excellent vaccine candidate against the norovirus. ● In addition, the natures of self-formation, high stability, polyvalence, and high immunogenicity, as evidenced by animal studies conducted in gnotobiotic pig models and mouse models, results included herein, of the nanoparticles and polymers make them strong vaccine candidate platforms to display foreign antigens, resulting in chimeric nanoparticles as vaccine candidates against further pathogens and diseases. Our multifunctional vaccine platform is a robust discovery engine and has broad application using both S 60 and P 24 nanoparticles to target multiple pathogens and illnesses. The P 24 nanoparticle has also been used to display multiple viral epitopes for enhanced immunogenicity for novel subunit vaccine development, see Table 1 below. These include the M2e epitope of the matrix 2 (M2) protein and the HA2 protein B cell epitope of influenza viruses, the B cell epitope of VP3 of enterovirus 71 (EV71), the 4E10 and 10E8 epitopes of human immunodeficiency virus type 1 (HIV-1), among others. Table 1. Summary of norovirus nanoparticles and polymers as vaccine candidates and platforms to display foreign antigens and epitopes. Nanoparticle/ Polymer Antigen/Epitope to be Displayed (Pathogen) Chimeric Products as Vaccine Candidate Immunity against Pathogens or Diseases S 60 VP8* (rotavirus) S 60 – VP8* Rotavirus P 24 P domain (norovirus) P 24 Norovirus P 24 VP8* (rotavirus) P 24 – VP8* Rotavirus and norovirus P 24 M2e (influenza virus) P 24 – M2e Influenza virus P 24 HA2 B cell epitope (influenza virus) Trivalent HA2-PP (P 24 -HA2:90-105) Influenza A virus and influenza B virus P 24 VP3 B cell epitope (EV71) PP-71-6 (P 24 -71-6) EV71 P 24 4E10/10E8 epitopes (HIV-1) 4E10-PP/10E8-PP HIV-1 P 24 Amyloid-beta, Aβ PP-3copy-Aβ1-6 Alzheimer’s disease P polymer P domains (noroviruses) NoV P GI -NoV P GII GST NoV P + Different noroviruses P polymer P domain (HEV) NoV P-HEV P Norovirus and HEV P polymer P domain (astrovirus) P domain (HEV) Ast P-HEV P-NoV P Norovirus, astrovirus, and HEV P polymer P domain (astrovirus) P domain (HEV) VP8* (rotavirus) Ast P-HEV P-VP8* Rotavirus, astrovirus, and HEV Note: EV71, enterovirus 71; HIV-1, human immunodeficiency virus type 1; HEV, hepatitis E virus; Ast, astrovirus, NoV, norovirus, P, protruding domain; P+, the P domain with an end-linked cysteine-containing peptide that can self-assemble into oligomers; PP, P particle; GI, norovirus genogroup I; GII, norovirus genogroup II. Please see the main text for details. 18 The S 60 Nanoparticle as a Multifunctional vaccine platform Recent technology has generated S nanoparticles using an E. coli system with stabilized expression and self-assembly. The S nanoparticles feature exposed C-terminal flexible hinge sites that offer ideal fusion sites for displaying foreign antigens. Researchers at CHMC have developed a technology to produce uniform 60-valent NoV S 60 nanoparticles with high efficiency using a simple bacterial expression system. This was achieved by taking advantage of the homotypic interactions of the NoVVP1 S domain that naturally builds the interior shells of NoV capsids, as well as several modifications to stabilize the S domain proteins and enhance the inter-S domain interactions, respectively. Specifically, we introduced an R69A mutation to destruct the exposed protease cleavage sites on the surface of the native shell that otherwise leads to easy degradation of the S proteins. In addition, we introduced triple (V57C/Q58C/S136’C) cysteine mutations to establish inter-S domain disulfide bonds between two pairs of sterically close residues that belong to two neighboring S domains. This led to significantly enhanced stability and yields of the self-assembled S 60 nanoparticles produced by the simple E. coli system. The below bullets are supported by published data by Ming Tan, the co-inventor of the S&P platform, and his research team at CHMC. ● An important feature of our technology was to rationally introduce intermolecular disulfide bonds to stabilize the S 60 nanoparticles. This approach could also be used to stabilize other viral protein particles or complexes. ● The 60 freely exposed C-termini are a key feature facilitating the S 60 nanoparticle to be a useful vaccine platform. Foreign antigens or epitopes can simply be fused to the end of the S domain via flexible linker through recombinant DNA technology. ● Uniform 60-valent NoV VLPs or S particles produced in a bacterial expression system have not been produced before. ● Importantly, our S 60 nanoparticles maintained the native conformation with authentic antigenicity; thus, our NoV S 60 nanoparticle technology represents a significant bioengineering advancement as uniform 60-valent NoV VLP or S particle via an expression system have never been produced before (Xia et al. ACS Nano 2018, 12, 10665−10682). ● Uniform complexity and size of vaccine particles are important factors in quality control of vaccine products, as variations in complexity and size will result in variations in immunization outcomes of the vaccines. Broad application to fuse several antigens to the S 60 nanoparticle based on multiple studies shown below conducted by CHMC (Xia et al. ACS Nano 2018, 12, 10665−10682) CHMC has been able to fuse several antigens to the S 60 nanoparticle to the same exposed S domain C-terminus via the same linker. These included (1) the rotavirus (RV) surface spike protein VP8*; (2) the HA1 antigen or receptor-binding domain (RBD) (223 amino acids) of the hemagglutinin (HA) of anH7N9 influenza A virus; (2) the TSR antigen (67 amino acids) of the circumsporozoite surface protein (CSP) of the malaria parasite Plasmodium falciparum; (3) the protruding domain antigen (187 amino acids) of a hepatitis E virus; (4) a longer version of the RV VP8*antigen (231 amino acids); and (5) the VP8*antigen (159 amino acids) of the murine RV (mRV) EDIM strain (Table 1). Particle formations of these fusion proteins have been shown by gel-filtration and/or EM (Table1). In addition, they have shown that the S 60 nanoparticle-displayed HA1 and mRV VP8*antigens elicited significantly higher HA1- and mRV VP8*-specific antibody titers, respectively, than those elicited by the free HA1 or mRV VP8*antigens (Table 2). 19 Table 2. List of Antigens That Have Been Displayed by the S 60 Nanoparticles epitope/antigen size (residue) yield (mg/L bacteria culture) S 60 – antigen particle formation significant immune enhancement in mice f RV VP8* antigen 159 ~40 yes yes HA1 antigen a 223 ~10 yes yes TSR/CSP antigen b 67 ~10 yes ND full RV VP8* antigen c 231 ~20 yes ND murine RV VP8* antigen d 159 ~5 yes yes HEV protruding domain antigen e 187 ~10 yes ND a HA1 antigen containing the receptor binding site is the head portion of the hemagglutinin (HA) of H7N9 influenza A virus. b TSR/CSP antigen is the C-terminal portion of the major surface protein of acircumsporozoite (CSP) that plays a key role in host cell invasion of the malaria parasite Plasmodium falciparum. c Full RV VP8*antigen is the full-length VP8*domain of the spike protein of a human P[8] rotavirus. d Murine RV VP8*antigen is the core portion of the VP8*protein constituting the head of the spike protein of a murine rotavirus EDIM strain. e HEV protruding domain antigen is part of the protruding domain of a hepatitis E virus capsid. f Immune enhancements of the S 60 nanoparticle-displayed antigens were measured in mice using free monomeric antigens as control for comparisons. “ND” = not determined. S 60 nanoparticles may serve as a polyvalent vaccine platform (Xia et al. ACS Nano 2018, 12, 10665−10682) ● We believe the self-assembled, polyvalent S 60 nanoparticle with 60 flexibly exposed S domain C-termini is an ideal vaccine platform for antigen presentation and immunogenicity enhancement. ● This has been supported by studies showing that when Hisx6 tag was fused to the hinge of the S domain via a linker, fusion proteins self-formed into the S 60 nanoparticles. ● This has also been demonstrated by constructing a chimeric, and reconfirmed by cyroEM density map, S 60 nanoparticle displaying 60 RV (rotavirus) VP8* proteins, the major rotavirus neutralizing antigen. The S 60 -VP8*particles can be easily produced with high stability. The chimeric nanoparticle induced higher immunoglobulin, or IgG, response in mice (n=6) toward the displayed VP8*antigen than soluble VP8* antigen. Mouse sera experiments were completed analyzing vaccinated versus the control group to show neutralizing activity against RV infection. The statistical differences between the groups are (*P < 0.05, **P < 0.01, ***P < 0.001) as shown below (Figure 2) (Xia et al. ACS Nano 2018, 12, 10665−10682). ● The RV surface spike protein, VP8* was tested for feasibility of the S 60 nanoparticle by the analysis using EM micrograph examination and ESI-MS analysis. S 60 -VP8*particles exhibited stronger blockade in mice (n=6) sera after vaccination (P=0.0003) (Xia et al. ACS Nano 2018, 12, 10665−10682). ● The polyvalent B- and T-cell epitopes of the antigens on the polyvalent VLP platform led to induction of stronger humoral and cellular immune responses, respectively, in animals and humans compared with those elicited by the monovalent epitopes of the free antigen. Thus, the polyvalent VLP platform is likely to increase the immunogenicity of the displayed antigens. Mouse sera experiments were completed analyzing vaccinated versus the control group to show neutralizing activity against RV infection. The statistical differences between the groups are (*P < 0.05, **P < 0.01, ***P < 0.001) as shown below. (Xia et al. ACS Nano 2018, 12, 10665−10682). 20 Figure 2. S 60 -VP8*particles enhanced immunogenicity toward the displayed RV VP8*antigens. The same dose/dosage of the S 60 -VP8*particles, free VP8*antigens, and S 60 nanoparticles without VP8*was given to mice (N=6), respectively, followed by measurements of theVP8*-specific IgG responses (A), 50% blocking titers (BT50) against RV VP8*-glycan ligand interaction (B), and neutralization activity against RV infection/replication in culture cells (C) of the resulting mouse antisera. (A) VP8*-specific IgG responses/titers elicited by theS60-VP8*particles, free VP8*antigens, and the S60nanoparticles, respectively. (B) BT50against RV VP8*−ligand interactions by the mouse sera after vaccination with the same three immunogens, respectively. (C) Neutralizing activity against RV infection/replication in culture cells by mouse sera after immunization with the same three immunogens, respectively. In all these experiments mouse sera after immunization with diluent (PBS) are used as negative controls. 21 The P 24 Nanoparticle as a versatile platform (Tan et al. Nanomedicine, 2012. 7.6,1-9) The crystal structure of norovirus VLPs indicates that P domain is involved in strong dimeric interactions forming dimeric protrusions on the viral surface. The oligomeric interactions of the P domains are also observed at the five-fold axes to further stabilize the capsid structure. When the P domain protein was expressed using the E. coli system, it self-assembled into P dimers, as well as 24 valent P nanoparticles, P 24 . P dimers and P 24 nanoparticles can exchange dynamically, depending on concentration of the P domain protein, indication that the assembled P 24 particles at this stage were unstable and easy to disassemble back into P dimers. To facilitate P 24 nanoparticle formation, inter-P domain disulfide bonds were introduced through fusion of a cysteine-containing peptide to the end of the P domain. During the P 24 nanoparticle assembly, the cysteine patches were brought to the center of the P 24 nanoparticles, resulting in sterically close contact and thus forming inter-P domain disulfide bonds that significantly stabilized the P 24 nanoparticles, which could no longer disassemble back into the P dimers. ● P 24 nanoparticles can be produced using an E. coli expression system faster and a lower cost than VLPs. ● Both VLP and P 24 nanoparticles without adjuvant produce innate, humoral, and cellular immunity. ● The platform can be used to display foreign antigens, epitopes and viral pathogens and non-infectious disease. ● Studies have demonstrated immune response against flu, rotavirus, and norovirus using bi- or trivalent vaccine candidates developed using this approach, noting the potential for the development of a universal flu vaccine. Pre-clinical studies in influenza and rotavirus are provided below supporting our vaccine candidate programs. See — Our Infectious Disease Vaccine Candidates . BWV-301 Norovirus-Rotavirus Vaccine Program We are developing BWV-301 to prevent acute gastroenteritis, or AGE, caused by norovirus and rotavirus, utilizing the P 24 nanoparticle of our vaccine platform. The vaccine is based on one or two doses of the norovirus P 24 nanoparticle presenting 24 rotavirus VP8* antigens. Most cases of gastroenteritis are caused by viruses. The CDC reports that viral gastroenteritis infections cause 200,000 deaths in children worldwide each year. Common symptoms of viral gastroenteritis causes nausea, vomiting, diarrhea, anorexia, weight loss, and dehydration. Gastroenteritis Gastroenteritis, often called stomach flu, is inflammation of the gastrointestinal tract — the stomach and intestine. Symptoms may include diarrhea, vomiting and abdominal pain. Fever, lack of energy and dehydration may also occur. While gastroenteritis is usually caused by viruses, bacteria, parasites, and fungus can also cause gastroenteritis. Eating improperly prepared food, drinking contaminated water or close contact with a person who is infected can spread the disease. Norovirus and rotavirus are two viruses that cause gastroenteritis in adults and children. In 2015, there were two billion cases of gastroenteritis, resulting in 1.3 million deaths globally. Children and those in the developing world are affected the most. In 2011, there were about 1.7 billion cases, resulting in about 700,000 deaths of children under the age of five. In the developing world, children less than two years of age frequently get six or more infections a year. It is less common in adults, partly due to the development of immunity. In adults, norovirus is the most common cause of severe disease. Rotavirus, however, is the common cause of AGE in children. Norovirus Norovirus causes significant debilitating AGE, with a reported 700 million infections and 20% of all diarrheal cases reported annually worldwide, according to the CDC. About 200 million cases are seen among children under 5 years old, leading to an estimated 50,000 child deaths every year. Norovirus is the cause of approximately 20% of all AGE cases worldwide each year. It is estimated that 68.9 cases of norovirus infection occur in every 1000 people. In North America, norovirus induced AGE tends to be seasonal, occurring in cooler, rainy months and particularly impacts groups in close proximity, such as in schools, dormitories, medical facilities, and cruise ships. Norovirus costs $60.3 billion worldwide each year (CDC). Globally, norovirus resulted in a total of approximately $4.2 billion in direct health system costs and approximately $60.3 billion in societal costs per year. Disease among children younger than 5 years cost society $39.8 billion, compared to $20.4 billion for all other age groups combined. Costs per norovirus illness varied by both region and age and was highest among adults ages 55 years and older. Productivity losses represented 84-99% of total costs varying by region. While low and middle income countries and high income countries had similar disease incidence (10,148 vs. 9,935 illness per 100,000 persons), high income countries generated 62% of global health system costs (Bartsch et al. PloS One 2016; 11:e0151219). 22 In North America, the median yearly cost of outbreaks was $7.6 million in direct medical costs, and $165.3 million in productivity losses. An average of approximately 113,000 hospitalizations, 8.2-122.9 million missed school/work days, $0.2-$2.3 billion in direct medical costs, and $1.4-$20.7 billion in productivity losses was due to sporadic illness. The total economic impact of norovirus infection was $10.6 billion based on the current incidence estimate 68.9 cases per 1000 population, or approximately $0.15 million per person infected. The total economic burden is greatest in young children but the highest cost per illness is among older age groups in some regions. These large costs overwhelmingly are from productivity losses resulting from acute illness. Low, middle, and high income countries all have a considerable economic burden, suggesting that norovirus gastroenteritis is a truly global economic problem. There is not a norovirus vaccine on the market presently. There are, however, a number of rotavirus vaccines currently marketed around the world. RotaTeq, owned by Merck, a live, oral pentavalent vaccine and Rotarix, owned by GSK, a monovalent, human, live-attenuated vaccine are recommended by the WHO for global use in children and approved for use in the U.S., Canada and Europe. Other monovalent vaccines are available but only approved for use in one country, either China, Vietnam or India. Development P 24 VLPs produced in E. coli and norovirus VP1 VLPs produced in a baculovirus expression system were both demonstrated to elicit innate, humoral and cellular immunity in a mouse model, indicating that both constructs have potential as norovirus virus candidates. In addition, when delivered intranasally both constructs were able to induce partial cross-variant protection against diarrhea in a gnotobiotic pig model. Ramesh et al. Vaccines 2019, 7, 777. Rotavirus Rotavirus is the most common cause of diarrheal disease among infants and young children, causing an estimated 111 million episodes of diarrhea annually, 2 million hospitalizations and 352,000-592,000 deaths annually, according to the CDC. After the introduction of live-attenuated oral vaccines the incidence of rotaviral hospitalizations and deaths have significantly declined. However, there is still a need for efficacious, cost-effective rotavirus vaccines. The rotavirus vaccine is recommended by the CDC and ACIP as a prevention for children. However, managing the symptoms is the only way to help adults and children infected with either of the viruses. Due to the potential of death, most treatments are focused on dehydration prevention and management. Treatment involves getting enough fluids. For mild or moderate cases, this can typically be achieved by drinking oral rehydration solution (a combination of water, salts and sugar). In those who are breastfed, continued breastfeeding is recommended. For more severe cases, intravenous fluids may be needed and care provided in the hospital. Fluids may also be given by a nasogastric tube. Zinc supplementation is recommended in children. Antibiotics are generally not needed. However, antibiotics are recommended for young children with a fever and bloody diarrhea. To determine the potential of the P 24 VLP to serve as a rotavirus vaccine candidate, the 159 amino acid VP8* protein was inserted into a P 24 domain surface loop. The fusion proteins self-assembled into P 24 VLPs, and the 24 rotavirus VP8* antigens were demonstrated by cryo-EM to be displayed on the outermost surface of the chimeric P 24 VLP. Mice (n-5-7) immunized intranasally with the P 24 -VP8* or intramuscularly with Freund’s adjuvant elicited significantly higher rotavirus neutralizing antibodies than the free VP8* immunized under the same conditions (IN or IM). (P >0.05), (Tan et al. J. Virol. 85(2):753-764. 2011. P 24 -VP8* VLPs were further characterized as a potential rotavirus vaccine in mouse and gnotobiotic pig challenge studies. A construct consisting of P 24 and the VP8* antigen from the murine rotavirus EDIM strain was constructed and tested using a murine rotavirus challenge model. Mice (n=5-7) were immunized with P 24 -mouseVP8*, mouseVP8* alone or P 24 -human VP8* 3 times intranasally without adjuvant. Rotavirus shedding was significantly lower in animals immunized with P 24 -mouseVP8* than mock vaccinated or animals that received mouseVP8* only or P 24 -humanVP8* * (P >0.05) (Tan et al. J. Virol. 85(2):753-764. 2011). 23 Additionally, an immunogenicity study was conducted in gnotobiotic pigs (n=25). A construct of P 24 and the VP8* antigen corresponding to human rotavirus Wa strain was tested in a gnotobiotic pig challenge model. Animals were immunized intramuscularly (IM) three times with either P 24 -WuVP8* with luminium hydroxide adjuvant or luminium hydroxide alone and were challenged with human Wa rotavirus 7 days post dose three. Animals immunized with P 24 -WuVP8* showed a significant reduction in the mean duration of diarrhea, virus shedding and significantly lower fecal cumulative consistency scores compared to adjuvant only control group (*, p < 0.05; **, p < 0.01). (Ramesh et al. Vaccines 7: 177 2019; doi:10.3390/vaccines7040177). Figure 9. .P24-VP8* vaccine protected against VirHRV diarrhea and reduced overall virus shed among vaccinated pigs. Fecal consistency (A) and virus shedding (B) were monitored daily from post challenge day (PCD) 1 to PCD 7 after the challenge with VirHRV. Fecal consistency scores≥2 were considered to be diarrheic (dashed line indicates the threshold of diarrhea). Statistical significance between vaccinated and control groups, determined by multiple t tests, are indicated by asterisks (*,p<0.05; **,p<0.01). Additionally, serum samples were collected from the pigs at the times of P 24 -VP8* vaccine administration (PID 0, PID 10, PID21 and PID 21) and VirHRV challenge (PID 27) and upon euthanasia (PCD 7). The P 24 -VP8* vaccine was highly immunogenic in Gn pigs. It induced strong VP8*-specific serum IgG and Wa-specific virus-neutralizing antibody responses from post-inoculation day 21 to PCD 7. Comparisons between groups at the same time points were carried out using Student’s t-test and significant differences are identified by *** (n = 10 – 15; p < 0.001). Tukey-Kramer HSD was used for the comparison of different time points within the same group, where different capital letters (A, B, C,D) indicate a significant difference, p < 0.01, and shared letters indicate no significant difference. These findings support further investigation of the noro-rotavirus dual nanoparticle vaccine. (Ramesh et al. Vaccines 7: 177 2019; doi:10.3390/vaccines7040177) 24 Figure 10. Geometric mean VP8*-specific IgG (A) and IgA (B) and Wa-HRV neutralizing (C) antibody titers in serum collected from Gn pigs at PID 0, 10, 21, 28, and PCD 7. Pigs were vaccinated with P24-VP8* vaccine or Al(OH)3 adjuvant only. Each serum specimen was tested at an initial dilution of 1:4. Negative samples were assigned an arbitrary value of 2 for calculation and graphical illustration purposes. Comparisons between groups at the same time points were carried out using Student’s t-test and significant differences are identified by *** (n = 10 – 15; p < 0.001). Tukey-Kramer HSD was used for the comparison of different time points within the same group, where different capital letters (A, B, C, D) indicate a significant difference, p < 0.01, and shared letters indicate no significant difference. An effective norovirus culture-based neutralization assay is not available, due to the lack of an efficient cell culture system to produce human norovirus. Therefore, a surrogate neutralization assay has been developed in the field, measuring the ability of antisera to block norovirus VLP binding to host receptors. In addition to generating rotavirus neutralizing antibody, Tan et al (J. Virol. 86:753-764. 2011) demonstrated that anti- P 24 -VP8* mouse sera blocked norovirus VLP binding, indicating that the insertion of the VP8* fragment did not inhibit induction of norovirus VLP binding antibodies and suggesting the P 24 -VP8 construct could potentially serve as a single vaccine against both rotavirus and norovirus disease (P >0.05). 25 Our Vaccine We hold the exclusive global license for the novel norovirus-rotavirus combination vaccine (except in China and Hong Kong) from Cincinnati Children’s Hospital Medical Center, or CHMC, CHMC researchers engineered the norovirus major structural protein VP1 such that the N-terminal shell (S) and C-terminal protruding (P) domains of VPI could be expressed as separate S 60 and P 24 VLPs. Unlike norovirus VLPs composed of the intact VP1 protein or the unmodified S 60 fragment, our S 60 and P 24 VLPs can be expressed in E. coli . The researchers demonstrated that S 60 VLPs could be used to present foreign antigens on the surface of the S 60 VLP. Further, it has also demonstrated that foreign antigens could also be expressed on the surface of the P 24 VLP. The proposed norovirus-rotavirus vaccine is based on the P 24 VLP technology. Our vaccine production is based on an E.coli expression platform. Development Following IND submission, if accepted, we intend to initiate our Phase I clinical trial in healthy adults ages 18 to 54. If approved, we believe our vaccine is well positioned to receive a recommendation from the CDC, ACIP, and similar international advisory groups for inclusion in vaccine programs. BWV-302: Norovirus-malaria vaccine program Additionally, we are currently investigating a malaria vaccine, BWV-302, utilizing our norovirus platform. The vaccine is designed to offer protection from both norovirus and malaria, infectious diseases that occur frequently together in geographic regions. The vaccine utilizes a protein identified on the surface of the plasmodium parasite being presented on the surface of the norovirus nanoparticle. Malaria Malaria can be a deadly disease caused by protozoan parasites from the Plasmodium family, primarily spread by mosquitos (CDC, https://wwwnc.cdc.gov/travel/diseases/malaria) . Malaria may also, at times, be transmitted through blood transfusion, organ transplantation and from mother to fetus. (CDC, https://wwwnc.cdc.gov/travel/yellowbook/2020/travel-related-infectious-diseases/malaria ). While transmission through blood transfusion is rare in the U.S., there are no approved blood tests currently available to screen blood donation for malaria. There were approximately 219 million cases of malaria reported in 2019 globally, resulting in approximately 409,000 deaths, of which approximately 67% were children. (WHO, https://www.who.int/news-room/fact-sheets/detail/malaria ). Symptoms of malaria normally manifest themselves within 7 to 10 days of exposure, and can at times, be mistaken for other illnesses, including influenza. Severe malaria is life-threatening and can cause multi-organ failure in adults and severe anemia, metabolic acidosis and cerebral malaria in children. The World Health Organization estimates that almost half of the global population is at risk of contracting malaria. Infants, children under 5 years of age, pregnant women and immune compromised individuals are highest risk of developing the disease. Additionally, non-immune migrants, mobile populations and travelers are at risk of developing severe disease. Neurological issues in children may continue to persist after cerebral malaria, including ataxia, palsy, speech impairment, deafness and blindness. 26 More than 100 species of Plasmodium have been identified. Four of the species have been recognized as naturally infecting humans, while one that infects macaques and has been identified as a cause of zoonotic malaria. In rare cases, additional species may infect humans. The primary four parasites that cause human infection are P. falciparum, P. vivax, P. ovale and ( https://www.cdc.gov/malaria/about/biology/index.html ). P. knowlesi is naturally occurring in macaques in Southeast Asia and has recently been reported as the cause zoonotic malaria, especially in Malaysia. P. falciparum is found world-wide, can cause severe malaria and is the predominate human malaria causing species around the world. There is currently one vaccine for malaria, RTS,S/AS01 (MVI-GSK) targeting the falciparum CS protein, which received a positive opinion from the European Medicines Agency (EMA) for use outside of the European Union in infants 6 weeks of age and older. ( https://www.ema.europa.eu/en/news/first-malaria-vaccine-receives-positive-scientific-opinion-ema ) According to the EMA, the World Health Organization and the relevant regulatory agencies for countries outside of the European Union can authorize its use. The vaccine is currently being administered to infants and children in parts of Africa within high transmission regions. The vaccine’s efficacy appears to wane after five years (Laurens MB. RTS,S/AS01 vaccine (Mosquirix™): an overview. Hum Vaccin Immunother. 2020;16(3):480-489. Doi:10.1080/21645515.2019.1669415). The recommended course of action for preventing malaria is prevention of mosquito bites, and for those most vulnerable, a preventative treatment with sulfadoxine-pyrimethamine, especially in high transmission areas (WHO). In certain regions, the WHO has recommended the addition of amodiaquine to children under 5 years of age monthly during the high transmission season, along with sulfadoxine-pyrimethamine. Many regions employ mosquito control measures to reduce mosquito populations, however, 73 countries have reported mosquito resistance to at least 1 of the 4 most commonly used insecticides, while 23 countries have reported mosquito resistance to all of the commonly used insecticides. Once malaria is diagnosed, the two most common treatments are Chloroquine phosphate and Artemisinin-based combination (ACT) therapies. Chloroquine is the preferred treatment, however, some malaria parasites have become resistant to chloroquine and it may not be an effective treatment. ACT is a combination of two or more drugs that work against the malaria parasite in different ways. This is usually the preferred treatment for chloroquine-resistant malaria. However, as recently reported in Nature Medicine, there is growing concern about Artemisinin — derivative resistant P.falciparum in the Greater Mekong subregion (Cambodia, Thailand, Vietnam, Myanmar and Laos) ( https://www.nature.com/articles/s41591-020-1005-2.pdf ). Previous occurrences of resistant strains also first appeared in the Greater Mekong subregion and then spread to other parts of the world. ( https://www.nature.com/articles/s41591-020-1005-2.pdf ). Our Vaccine We hold the exclusive global license for the novel norovirus-malaria combination vaccine from Cincinnati Children’s Hospital Medical Center, or CHMC, CHMC researchers engineered the norovirus major structural protein VP1 such that the N-terminal shell (S) and C-terminal protruding (P) domains of VPI could be expressed as separate S 60 and P 24 VLPs. Unlike norovirus VLPs composed of the intact VP1 protein or the unmodified S 60 fragment, our S 60 and P 24 VLPs can be expressed in E. coli . The researchers, Xi Jason Jiang, Ph.D., and Ming Tan, Ph.D., demonstrated that S 60 VLPs could be used to present foreign antigens on the surface of the S 60 VLP. Further, it has also demonstrated that foreign antigens could also be expressed on the surface of the P 24 VLP. (see BWV Norovirus (NoV) S&P Nanoparticle Versatile Vaccine Platform ). The proposed norovirus-malaria vaccine, P-CS)TSR is based on the P 24 VLP technology. Our vaccine production is based on an E.coli expression platform. 27 The circumsporozoite (CS) protein is the major surface component of P. falciparum sporozoites and is essential for host cell invasion. Our vaccine, developed by Jiang and Ming from CHMC, combines a small domain of the CS protein with the norovirus P 24 particle creating a chimeric nanoparticle capable of eliciting an immune response. A mouse immunization study was conducted using the P 24 particle presenting the small domain of the CS protein. Mice (n=16) were immunized three times with the chimeric nanoparticle using aluminum hydroxide as an adjuvant, 3D7-His, 3D7-GST and PBS. Sera was collected and evaluated. High antibody titers, as determined by ELISA, were observed after the second immunization and higher titers were observed after the third immunization. The antibodies were also shown to recognize the plasmodium falciparum 3D7 strain using immunofluorescence assays. These data demonstrate the potential of our vaccine candidate against malaria. We expect to conduct an animal challenge study to further analyze the protective nature of BWV-302 and support an IND application. Table 3. Mouse malaria antibody titer post-immunization Antibody titer after 2 nd immunization Antibody titer after 3 rd immunization Figure 11. IFA of plasmodium sporozoites (3D7) stained with anti-P 24 particle presenting the small domain of the CS protein mouse sera Development We anticipate conducting an animal challenge study for BWV-302 in the second half of 2023. Upon completion, the technology will be transferred to a partner contract manufacturing organization (CMO) for process optimization, GMP production and toxicology studies, as well as other studies required by the FDA for IND submission, currently anticipated for the second half of 2022. Following IND submission immediately upon completion of the toxicology study, if successful, we intend to initiate our Phase I clinical trial in healthy adults ages 18 to 54 upon acceptance by the FDA. Exploration of a Novel Monkeypox Vaccine Using BWV VLP Platform In addition to norovirus, rotavirus, and malaria, we are exploring the potential to utilize the norovirus S&P platform to create a novel monkeypox vaccine. Research into the viability of this vaccine candidate is ongoing and includes insertion of selected monkeypox antigens into the S&P particles and sequence optimization, establishing the optimal expression system to enhance future manufacturing of the vaccine product, as well as immunogenicity and efficacy studies at various stages of development. To date, antigens of interest have been identified and the vaccine construct has been generated in small amounts using our VLP platform licensed from CHMC. Immunogenicity studies in mice are ongoing and results will inform our decision to move forward with a challenge study, which will evaluate the in vivo efficacy of this vaccine in the mouse model. Given this vaccine is in early stages of development and optimization, study designs and development paths are flexible. Upon completion of immunogenicity and efficacy studies with promising results, this technology may be transferred to a partner CMO for process optimization, GMP production and toxicology studies, as well as other studies required by the FDA for IND submission. 28 Monkeypox is a viral zoonosis, or a virus transmitted from humans to animals, and is a member of the same genus as the smallpox virus, Orthopoxvirus . While clinical symptoms of monkeypox are less severe than those of smallpox, several recent outbreaks and the eradication of smallpox in 1980 have brought global attention to the prevention of monkeypox spread. Monkeypox primarily occurs in central and west Africa, often in proximity to tropical rainforests, but has been increasing in urban areas, particularly with a recent outbreak in 2022 that spread to 110 countries and caused approximately 85,000 cases as of January 2023. There are currently two approved vaccines for the prevention of monkeypox infection in the United States: JYNNEOS and ACAM2000. Both vaccines were originally approved to prevent smallpox infection but have been approved for use in monkeypox. JYNNEOS is a 2-dose vaccine, with doses given 4 weeks apart and is a live-attenuated, non-replicating vaccine while ACAM2000 is a live, replication-competent vaccinia virus given via bifurcated needle in a single dose. Both are designed to elicit an immune response to prevent monkeypox and smallpox infection without causing disease. While vaccines have shown efficacious in preventing disease, there remains a need for additional vaccination options, particularly those that are not comprised of live virus. BWV-401: Chlamydia Vaccine Chlamydia Background Chlamydia is a sexually transmitted infection caused by the bacterium Chlamydia trachomatis and can impact both men and women. According to the Centers for Disease Control and Prevention, there were about 1.6 million new cases of chlamydia reported in 2020 in the United States and globally, the World Health Organization estimates about 129 million new cases each year. Additionally, given high estimations of asymptomatic cases and low availability of diagnostic testing in low- and middle-income countries, these annual estimates may be an underrepresentation. Currently, there is no vaccine available to prevent chlamydia infection, and the main treatment is through antibiotic regimens with the possibility of reinfection after antibiotics have treated the disease. If left undetected or untreated, Chlamydia represents a major cause of pelvic inflammatory disease and infertility in women. It is estimated that about 10 – 15% of women that experience untreaded chlamydia develop pelvic inflammatory disease and face chronic pain or fertility problems later in life. Additionally, should women contract chlamydia during pregnancy or give birth with an active infection, newborns may develop eye infections or pneumonia resulting from the disease. 29 BWV-401 Approach BWV-401 is an orally delivered, live-attenuated chlamydia vaccine derived from a murine strain, Chlamydia muridarum, developed in the laboratory of Guangming Zhong, M.D., Ph.D. at the University of Texas Health at San Antonio. By administering this vaccine orally, BWV-401 may elicit transmucosal immunity and provide protection against chlamydia in the genital tract post-vaccination without altering the gut microbiota or the development of gut mucosal resident memory T cell responses to non-chlamydial infection. In the initial publication establishing this approach as a viable vaccine development pathway, mice were intragastrically inoculated with C. muridarum to mimic oral immunization. Following each inoculation, both vaginal and rectal swabs were periodically taken to monitor viable C. muridarum colonization or organs/tissues were harvested to titrate viable organisms. Through this study, researchers identified the following key findings supporting further development of this vaccine candidate. ● GI tract C. muridarum induces transmucosal protection against genital tract infection. C. muridarum colonization in the gastrointestinal tract correlated with reduced C. muridarum infection in the genital tract of the same mice. First, the extent to which C. muridarum organisms spread from the genital tract into the GI tract inversely correlated with their course of shedding in the genital tract. Second, the coinoculation of C. muridarum organisms into the GI tracts of mice infected vaginally with plasmid-deficient C. muridarum significantly shortened the course of vaginal infection. Finally, the reduced spreading of plasmid-free C. muridarum into the GI tract also minimized immunity against reinfection in the genital tract (Fig. 1). Figure 1. Effect of intragastric inoculation as an oral vaccination on genital tract susceptibility to C. muridarum challenge infection. C57BL/6J mice intragastrically inoculated with buffer only (control group, n 8) (a and a1) or 2x10E5 IFU of wild-type C. muridarum (clone CM-mCherry, immunization group, n 8) (b and b1) were challenged intravaginally on day 56 with 2x10E5 IFU of wild-type C. muridarum clone G13.32.1. (A) Mice were monitored for live organism shedding by the collection of both vaginal (a and b) and rectal (a1 and b1) swab specimens over the time course displayed along the x axis. The results are expressed as the log10 number of IFU per swab specimen along the y axis. Black bars, titers of G13.32.1; red bars, titers of CM-mCherry; dark red bars, titers of both G13.32.1 and CM-mCherry. Note that on days 3, 7, and 14 after intravaginal challenge (designated in parentheses as 3=, 7=, and 14=, respectively) after intragastric immunization, immunized mice displayed a >1,000-fold decrease in the number of IFU by evaluation of vaginal swab specimens at each time point (*, P< 0.05, Wilcoxon rank-sum test). The overall shedding course was also significantly reduced (*, P<0.05, Wilcoxon rank-sum test, AUC, for panel b versus panel a). (B) All mice were sacrificed on day 128 after intragastric immunization (or day 63= after challenge) for evaluation of the upper genital tract pathology both macroscopically (a and b) and microscopically (d and e). (a and b) Representative macroscopic images of one entire genital tract from the control (a) and immunization (b) groups are shown. White arrows, oviducts positive for hydrosalpinges. Magnified images of oviduct/ovary regions are shown on the right of the overall genital tract images, with the white numbers indicating the hydrosalpinx scores. (c) Both the incidence and the severity of hydrosalpinx were quantitated. The group immunized in the GI tract developed a significantly lower incidence ($, P<0.05, Fisher’s exact test) and a reduced score (*, P<0.05, Wilcoxon rank-sum test) compared with those for the control mice. (d and e) Microscopically, severely dilated oviducts (marked with a white line with arrows at both ends) were easily identified from control mice, as shown in the representative image (d), while the immunized mice mostly displayed normal oviduct cross sections ©. (d1 and e1) The inflammatory cells were identified using a 100x objective lens, as shown in the representative images from the control (d1) and immunized (e1) mice. The areas observed with a 100x objective lens are marked with white squares in the 10x images. (f) The severity of the inflammatory infiltration was semiquantitated using the criteria described in the Materials and Methods section. Note that the immunized mice developed scores significantly decreased (*, P<0.05, Wilcoxon rank-sum test) compared with those for the control mice. 30 ● Transmucosal protection is rapidly induced, durable, and independent of sustained C. muridarum colonization in the gastrointestinal tract. Both the time required for GI tract C. muridarum induction of transmucosal protection and the duration of protection were determined (Fig. 2). One week after intragastric inoculation with CM-mCherry, mice gained significant resistance to intravaginal challenge infection with G13.32.1, with G13.32.1 shedding being reduced by >100-fold on day 3 and the course of infection being shortened by ~1 week, leading to a significant reduction in both the overall infection course and the upper genital tract pathology. The protection was enhanced over time (Fig. 1) and lasted 20 weeks. Whether the transmucosal protection was dependent on ongoing CM-mCherry colonization in the GI tract was further determined (Fig. 3). Mice with or without CM-mCherry in the GI tract for 28 days were either left untreated or treated with doxycycline daily for 2 weeks. After resting for another 2 weeks, the mice were vaginally challenged with G13.32.1. Mice colonized with CM-mCherry in the GI tract for 56 days became highly resistant to intravaginal challenge infection and hydrosalpinx induction, as described above. Importantly, after the immunized mice received daily doxycycline treatment between days 28 and 42, which completely cured the GI tract CM-mCherry infection, the mice still maintained a robust resistance to intravaginal challenge infection and hydrosalpinx development. Thus, within 4 weeks, intragastrically inoculated C. muridarum induced a robust memory response that was protective. Mock-immunized mice similarly treated with doxycycline developed severe hydrosalpinx after the same intravaginal challenge, suggesting that the doxycycline treatment protocol did not affect chlamydial pathogenicity in the upper genital tract. It is worth noting that although the immunized mice were resistant to challenge infection with C. muridarum in the genital tract, the GI tract remained susceptible to colonization by the C. muridarum organisms. Figure 2. Intragastric immunization elicits rapid and durable protective immunity to genital tract challenge. C57BL/6J mice with (n 5) or without (n 5) prior intragastric immunization with 2x10E5 IFU of CM-mCherry for 1 week (1W) (a) or 20 weeks (20W) (b) were challenged vaginally with clone G13.32.1. The mice were monitored for C. muridarum shedding by evaluation of both vaginal and rectal (not shown) swab specimens on days 3 and 7 postinfection (3= and 7=, respectively) and weekly thereafter. The results are expressed as the log10 number of IFU per swab specimen. Mice were significantly resistant to a genital tract challenge only 1 week after immunization in the GI tract (*, P<0.05, Wilcoxon rank-sum test, AUC), and the resistance increased and lasted for up to 20 weeks (**, P< 0.01, Wilcoxon rank-sum test, AUC). All mice were sacrificed on day 56 after the challenge infection, and the upper genital tract was evaluated for the incidence (in percent) of hydrosalpinx and the severity score (mean + standard deviation). Immunization via the GI tract resulted in significant protection against hydrosalpinx induced by the vaginal infection (#, P<0.05, Fisher’s exact test; *, P<0.05, Wilcoxon rank-sum test; **, P<0.01, Wilcoxon rank-sum test). 31 Figure 3. The durable transmucosal protection induced by intragastric immunization is not dependent on long-term gastrointestinal infection. Groups of C57BL/6J mice immunized intragastrically with 2x10E5 IFU of CM-mCherry (n 5 for panel a and n 7 for panel b) or not immunized (n 6) © were treated on day 28 with doxycycline (20 ug/kg of body weight intragastrically once daily) for 2 weeks (days 28 to 42) (b and c) or were not treated with doxycycline (a). The doxycycline-treated mice were then rested for 2 weeks (days 43 to 56). On day 56 after immunization in the GI tract, all mice were intravaginally challenged with 2x10E5 IFU of clone G13.32.1. (A) Mice were monitored for the shedding of chlamydiae by evaluation of both vaginal (a to c) and rectal (a1 to c1) swab specimens over the course of infection (the days after challenge infection are designated 3= to 56= in parentheses). Results are expressed as the log10 number of IFU per swab specimen. Mice in the immunization plus doxycycline treatment group displayed no IFU in the rectal swab specimens prior to the intravaginal challenge (days 31 to 56) (b) but maintained transmucosal protection against chlamydial infection in the genital tract (*, P<0.05, Wilcoxon rank-sum test, for panel b1 versus panel c1), equivalent to the findings for immunized mice not treated with doxycycline (*, P<0.05, Wilcoxon rank-sum test, for panel a1 versus panel c1). These two groups maintained similar levels of protection (*, P<0.05, Wilcoxon rank-sum test, for panel b1 versus panel a1). (b and c) The genital tract G13.32.1 organisms spread to the GI tracts. (a and a1) Black bars, G13.32.1 alone; dark red bars; both CM-mCherry and G13.32.1. (B) On day 114 after intragastric immunization, all mice were sacrificed to evaluate the upper genital tract pathology macroscopically. Representative images of the entire genital tracts from the groups receiving immunization without doxycycline treatment (a2), immunization plus doxycycline treatment (b2), or doxycycline treatment without immunization (c2) are shown. White arrows, oviducts positive for hydrosalpinges. Magnified images of oviduct/ovary regions are shown on the right of the overall genital tract images, with the white numbers indicating the hydrosalpinx scores. Both the incidence of hydrosalpinx and the hydrosalpinx severity score (mean standard deviation) are listed above the corresponding images. Regardless of doxycycline treatment, immunized mice were significantly protected from the development of hydrosalpinx (*, P<0.05, Wilcoxon rank-sum test, for the immunization alone group in panel a2 versus panel c2 and for the immunization plus doxycycline treatment group in panel b2 versus panel c2). 32 ● Gastrointestinal tract Chlamydia muridarum is nonpathogenic. Having demonstrated the strong transmucosal protective immunity induced by GI tract C. muridarum, researchers next evaluated whether C. muridarum colonization in the GI tract is pathogenic. Since long-lasting C. muridarum colonization is restricted to the cecum, colon, and rectum, researchers carefully examined the mouse colons. There was no significant difference in the gross appearance or length of the cecum, colon, and rectum between mice with C. muridarum colonization and mice without C. muridarum colonization for 7, 28, or 56 days, suggesting that C. muridarum did not cause colitis. C. muridarum inclusions were microscopically localized in the colon mucosal epithelial cells. Despite the presence of clusters of C. muridarum-infected epithelial cells, the epithelial tissue architecture remained intact when the adjacent sections were examined following hematoxylin-eosin (H&E) staining. Furthermore, there was a general lack of significant inflammatory infiltration, although scattered inflammatory cells were always detectable. Compared to control colonic tissue, no significant difference was found between infected and noninfected mice (data not shown). We believe that this data, presented by Zhong et al., is sufficient to pursue development of this vaccine candidate. Given the high numbers of Chlamydia cases both in the United States and around the globe each year, as well as the lack of an available Chlamydia vaccine, we believe this vaccine will serve a high unmet need. We hold a global, exclusive right to develop a novel Chlamydia vaccine from this technology at the University of Texas Health at San Antonio. BWV-401 Development As the approach to utilize an attenuated murine strain of Chlamydia is novel, we plan to establish the infectivity of C. muridarum in a non-human primate model. This will provide robust data supporting potential efficacy of this vaccine candidate in humans, once we reach clinical trials. In collaboration with Dr. Zhong and the University of Texas Health at San Antonio, we will develop a protocol to test both wild-type C. muridarum and our attenuated strain in non-human primates and complete necessary endpoints for this study. Proper endpoints will allow us to determine the ability of murine strain C. muridarum to infect non-human primates and the efficacy of the attenuated strain, which will represent our vaccine candidate, following challenge of the non-human primates with human Chlamydia strain, Chlamydia trachomatis. Following completion of the non-human primate study, we plan to transfer this technology to a partner CMO for process optimization, GMP production and toxicology studies, as well as other studies required by the FDA for IND submission. Government Regulation and Product Approval The FDA and other regulatory authorities at federal, state and local levels, as well as in foreign countries, extensively regulate, among other things, the research, development, testing, manufacture, quality control, import, export, safety, effectiveness, labeling, packaging, storage, distribution, record keeping, approval, advertising, promotion, marketing, post-approval monitoring and post-approval reporting of drugs and biologics such as those we are developing. Small molecule drugs are subject to regulation under the Food, Drug, and Cosmetic Act, or FDCA, and biological products are additionally subject to regulation under the Public Health Service Act, or PHSA, and both are subject to additional federal, state, local and foreign statutes and regulations. We, along with third-party contractors, will be required to navigate the various preclinical, clinical and commercial approval requirements of the governing regulatory agencies of the countries in which we wish to conduct studies or seek approval or licensure of our product candidates. 33 United States U. S. Biopharmaceuticals Regulation The process required by the FDA before drug and biologic product candidates may be marketed in the United States generally involves the following: ● completion of extensive preclinical laboratory tests and animal studies performed in accordance with applicable regulations, including the FDA’s Good Laboratory Practice, or GLP, regulations; ● submission to the FDA of an investigational new drug application, IND, which must become effective before clinical trials may begin; ● approval by an independent institutional review board or ethics committee at each clinical site before the trial is commenced; ● performance of adequate and well-controlled human clinical trials in accordance with FDA’s Good Clinical Practice, or GCP, regulations to establish the safety and efficacy of a drug candidate and safety, purity and potency of a proposed biologic product candidate for its intended purpose; ● preparation of and submission to the FDA of a new drug application, or NDA, or biologics license application, or BLA, as applicable, after completion of all pivotal clinical trials; ● satisfactory completion of an FDA Advisory Committee review, if applicable; ● a determination by the FDA within 60 days of its receipt of an NDA or BLA to file the application for review; ● satisfactory completion of an FDA pre-approval inspection of the manufacturing facility or facilities at which the proposed product is produced to assess compliance with current Good Manufacturing Practice requirements, or cGMPs, and of selected clinical investigation sites to assess compliance with GCPs; and ● FDA review and approval of an NDA, or licensure of a BLA, to permit commercial marketing of the product for particular indications for use in the United States. Preclinical and Clinical Development Prior to beginning the first clinical trial with a product candidate, we must submit an IND to the FDA. An IND is a request for authorization from the FDA to administer an investigational new drug product to humans. The central focus of an IND submission is on the general investigational plan and the protocol or protocols for preclinical studies and clinical trials. The IND also includes results of animal and in vitro studies assessing the toxicology, pharmacokinetics, pharmacology and pharmacodynamics characteristics of the product, chemistry, manufacturing and controls information, and any available human data or literature to support the use of the investigational product. An IND must become effective before human clinical trials may begin. The IND automatically becomes effective 30 days after receipt by the FDA, unless the FDA, within the 30-day period, raises safety concerns or questions about the proposed clinical trial. In such a case, the IND may be placed on clinical hold and the IND sponsor and the FDA must resolve any outstanding concerns or questions before the clinical trial can begin. Submission of an IND therefore may or may not result in FDA authorization to begin a clinical trial. 34 Clinical trials involve the administration of the investigational product to human subjects under the supervision of qualified investigators in accordance with GCPs, which include the requirement that all research subjects provide their informed consent for their participation in any clinical study. Clinical trials are conducted under protocols detailing, among other things, the objectives of the study, the parameters to be used in monitoring safety and the effectiveness criteria to be evaluated. A separate submission to the existing IND must be made for each successive clinical trial conducted during product development and for any subsequent protocol amendments. Furthermore, an independent institutional review board for each site proposing to conduct the clinical trial must review and approve the plan for any clinical trial and its informed consent form before the clinical trial begins at that site, and must monitor the study until completed. Regulatory authorities, the institutional review board or the sponsor may suspend a clinical trial at any time on various grounds, including a finding that the subjects are being exposed to an unacceptable health risk or that the trial is unlikely to meet its stated objectives. Some studies also include oversight by an independent group of qualified experts organized by the clinical study sponsor, known as a data safety monitoring board, which provides authorization for whether or not a study may move forward at designated check points based on access to certain data from the study and may halt the clinical trial if it determines that there is an unacceptable safety risk for subjects or other grounds, such as no demonstration of efficacy. For purposes of biopharmaceutical development, human clinical trials are typically conducted in three sequential phases that may overlap or be combined; ● Phase 1. The investigational product is initially introduced into patients with the target disease or condition. These studies are designed to test the safety, dosage tolerance, absorption, metabolism and distribution of the investigational product in humans, the side effects associated with increasing doses, and, if possible, to gain early evidence on effectiveness. ● Phase 2. The investigational product is administered to a limited patient population to evaluate the preliminary efficacy, optimal dosages and dosing schedule and to identify possible adverse side effects and safety risks. ● Phase 3. The investigational product is administered to an expanded patient population to further evaluate dosage, to provide statistically significant evidence of clinical efficacy and to further test for safety, generally at multiple geographically dispersed clinical trial sites. These clinical trials are intended to establish the overall risk/benefit ratio of the investigational product and to provide an adequate basis for product approval. In some cases, the FDA may require, or companies may voluntarily pursue, additional clinical trials after a product is approved to gain more information about the product. These so-called Phase 4 studies may be made a condition to approval of the application. Concurrent with clinical trials, companies may complete additional animal studies and develop additional information about the characteristics of the product candidate and must finalize a process for manufacturing the product in commercial quantities in accordance with cGMP requirements. The manufacturing process must be capable of consistently producing quality batches of the product candidate and, among other things, must develop methods for testing the identity, strength, quality and purity of the final product, or for biologics, the safety, purity and potency. Additionally, appropriate packaging must be selected and tested and stability studies must be conducted to demonstrate that the product candidate does not undergo unacceptable deterioration over its shelf life. During all phases of clinical development, regulatory agencies require extensive monitoring and auditing of all clinical activities, clinical data, and clinical study investigators. The FDA or the sponsor or its data safety monitoring board may suspend a clinical study at any time on various grounds, including a finding that the research patients or patients are being exposed to an unacceptable health risk. Similarly, an institutional review board can suspend or terminate approval of a clinical study at its institution if the clinical study is not being conducted in accordance with the institutional review board’s requirements or if the biological product candidate has been associated with unexpected serious harm to patients. There are also requirements governing the reporting of ongoing clinical trials and completed clinical trial results to public registries. Sponsors of clinical trials of FDA-regulated products are required to register and disclose certain clinical trial information, which is publicly available at www.clinicaltrials.gov . 35 NDA/BLA Submission and Review Assuming successful completion of all required testing in accordance with all applicable regulatory requirements, the results of product development, nonclinical studies and clinical trials are submitted to the FDA as part of an NDA or BLA, as applicable, requesting approval to market the product for one or more indications. The application must include all relevant data available from pertinent preclinical studies and clinical trials, including negative or ambiguous results as well as positive findings, together with detailed information relating to the product’s chemistry, manufacturing, controls, and proposed labeling, among other things. The submission of an application requires payment of a substantial application user fee to the FDA, unless a waiver or exemption applies. The FDA has sixty days from the applicant’s submission to either issue a refusal to file letter or accept the application for filing, indicating that it is sufficiently complete to permit substantive review. Once an NDA or BLA has been accepted for filing, the FDA’s goal is to review standard applications within 10 months after it accepts the application for filing, or, if the application qualifies for priority review, six months after the FDA accepts the application for filing. In both standard and priority reviews, the review process is often significantly extended by FDA requests for additional information or clarification. The FDA reviews an NDA to determine whether a drug is safe and effective for its intended use and a BLA to determine whether a biologic is safe, pure and potent. FDA also reviews whether the facility in which the product is manufactured, processed, packed or held meets standards designed to assure and preserve the product’s identity, safety, strength, quality, potency and purity. The FDA may convene an advisory committee to provide clinical insight on application review questions. Before approving an NDA or BLA, the FDA will typically inspect the facility or facilities where the product is manufactured. The FDA will not approve an application unless it determines that the manufacturing processes and facilities are in compliance with cGMP requirements and adequate to assure consistent production of the product within required specifications. Additionally, before approving an application, the FDA will typically inspect one or more clinical sites to assure compliance with GCPs. If the FDA determines that the application, manufacturing process or manufacturing facilities are not acceptable, it will outline the deficiencies in the submission and often will request additional testing or information. Notwithstanding the submission of any requested additional information, the FDA ultimately may decide that the application does not satisfy the regulatory criteria for approval. After the FDA evaluates an application and conducts inspections of manufacturing facilities where the investigational product and/or its drug substance will be manufactured, the FDA may issue an approval letter or a Complete Response letter. An approval letter authorizes commercial marketing of the product with specific prescribing information for specific indications. A Complete Response letter will describe all of the deficiencies that the FDA has identified in the application, except that where the FDA determines that the data supporting the application are inadequate to support approval, the FDA may issue the Complete Response letter without first conducting required inspections, testing submitted product lots and/or reviewing proposed labeling. In issuing the Complete Response letter, the FDA may recommend actions that the applicant might take to place the application in condition for approval, including requests for additional information or clarification, which may include the potential requirement for additional clinical studies. The FDA may delay or refuse approval of an application if applicable regulatory criteria are not satisfied, require additional testing or information and/or require post-marketing testing and surveillance to monitor safety or efficacy of a product. If regulatory approval of a product is granted, such approval will be granted for particular indications and may entail limitations on the indicated uses for which such product may be marketed. For example, the FDA may approve the application with a risk evaluation and mitigation strategy, or REMS, to ensure the benefits of the product outweigh its risks. A REMS is a safety strategy to manage a known or potential serious risk associated with a product and to enable patients to have continued access to such medicines by managing their safe use, and could include medication guides, physician communication plans, or elements to assure safe use, such as restricted distribution methods, patient registries and other risk minimization tools. The FDA also may condition approval on, among other things, changes to proposed labeling or the development of adequate controls and specifications. Once approved, the FDA may withdraw the product approval if compliance with pre- and post-marketing requirements is not maintained or if problems occur after the product reaches the marketplace. The FDA may require one or more Phase 4 post-market studies and surveillance to further assess and monitor the product’s safety and effectiveness after commercialization, and may limit further marketing of the product based on the results of these post-marketing studies. 36 Expedited Development and Review Programs The FDA offers a number of expedited development and review programs for qualifying product candidates. The fast track program is intended to expedite or facilitate the process for reviewing new products that meet certain criteria. Specifically, new products are eligible for fast track designation if they are intended to treat a serious or life-threatening disease or condition and demonstrate the potential to address unmet medical needs for the disease or condition. Fast track designation applies to the combination of the product and the specific indication for which it is being studied. The sponsor of a fast track product has opportunities for frequent interactions with the review team during product development and, once an NDA or BLA is submitted, the product may be eligible for priority review. A fast track product may also be eligible for rolling review, where the FDA may consider for review sections of the NDA or BLA on a rolling basis before the complete application is submitted, if the sponsor provides a schedule for the submission of the sections of the application, the FDA agrees to accept sections of the application and determines that the schedule is acceptable, and the sponsor pays any required user fees upon submission of the first section of the application. A product intended to treat a serious or life-threatening disease or condition may also be eligible for breakthrough therapy designation to expedite its development and review. A product can receive breakthrough therapy designation if preliminary clinical evidence indicates that the product, alone or in combination with one or more other drugs or biologics, may demonstrate substantial improvement over existing therapies on one or more clinically significant endpoints, such as substantial treatment effects observed early in clinical development. The designation includes all of the fast track program features, as well as more intensive FDA interaction and guidance beginning as early as Phase 1 and an organizational commitment to expedite the development and review of the product, including involvement of senior managers. Any marketing application for a drug or biologic submitted to the FDA for approval, including a product with a fast track designation and/or breakthrough therapy designation, may be eligible for other types of FDA programs intended to expedite the FDA review and approval process, such as priority review and accelerated approval. A product is eligible for priority review if it has the potential to provide a significant improvement in the treatment, diagnosis or prevention of a serious disease or condition. Priority review designation means the FDA’s goal is to take action on the marketing application within six months of the 60-day filing date. Additionally, products studied for their safety and effectiveness in treating serious or life-threatening diseases or conditions may receive accelerated approval upon a determination that the product has an effect on a surrogate endpoint that is reasonably likely to predict clinical benefit, or on a clinical endpoint that can be measured earlier than irreversible morbidity or mortality, that is reasonably likely to predict an effect on irreversible morbidity or mortality or other clinical benefit, taking into account the severity, rarity, or prevalence of the condition and the availability or lack of alternative treatments. As a condition of accelerated approval, the FDA will generally require the sponsor to perform adequate and well-controlled post-marketing clinical studies to verify and describe the anticipated effect on irreversible morbidity or mortality or other clinical benefit. In addition, the FDA currently requires as a condition for accelerated approval pre-approval of promotional materials, which could adversely impact the timing of the commercial launch of the product. Fast track designation, breakthrough therapy designation and priority review do not change the standards for approval but may expedite the development or approval process. Even if a product qualifies for one or more of these programs, the FDA may later decide that the product no longer meets the conditions for qualification or decide that the time period for FDA review or approval will not be shortened. Orphan Drug Designation Under the Orphan Drug Act, the FDA may grant orphan designation to a drug or biologic intended to treat a rare disease or condition, which is a disease or condition that affects fewer than 200,000 individuals in the United States, or more than 200,000 individuals in the United States for which there is no reasonable expectation that the cost of developing and making available in the United States a drug or biologic for this type of disease or condition will be recovered from sales in the United States for that drug or biologic. Orphan drug designation must be requested before submitting an NDA or BLA. After the FDA grants orphan drug designation, the generic identity of the therapeutic agent and its potential orphan use are disclosed publicly by the FDA. The orphan drug designation does not convey any advantage in, or shorten the duration of, the regulatory review or approval process. 37 If a product that has orphan drug designation subsequently receives the first FDA approval for the disease for which it has such designation, the product is entitled to orphan drug exclusive approval (or exclusivity), which means that the FDA may not approve any other applications, including a full NDA or BLA, to market the same drug or biologic for the same indication for seven years, except in limited circumstances, such as a showing of clinical superiority to the product with orphan drug exclusivity or if the FDA finds that the holder of the orphan drug exclusivity has not shown that it can assure the availability of sufficient quantities of the orphan drug to meet the needs of patients with the disease or condition for which the drug was designated. Orphan drug exclusivity does not prevent the FDA from approving a different drug or biologic for the same disease or condition, or the same drug or biologic for a different disease or condition. Among the other benefits of orphan drug designation are tax credits for certain research and a waiver of the NDA or BLA application fee. A designated orphan drug may not receive orphan drug exclusivity if it is approved for a use that is broader than the indication for which it received orphan designation. In addition, exclusive marketing rights in the United States may be lost if the FDA later determines that the request for designation was materially defective. Post-Approval Requirements Any products manufactured or distributed by us pursuant to FDA approvals are subject to pervasive and continuing regulation by the FDA, including, among other things, requirements relating to record-keeping, reporting of adverse experiences, periodic reporting, product sampling and distribution, and advertising and promotion of the product. After approval, most changes to the approved product, such as adding new indications or other labeling claims, are subject to prior FDA review and approval. There also are continuing user fee requirements, under which the FDA assesses an annual program fee for each product identified in an approved NDA or BLA. Biopharmaceutical manufacturers and their subcontractors are required to register their establishments with the FDA and certain state agencies, and are subject to periodic unannounced inspections by the FDA and certain state agencies for compliance with cGMPs, which impose certain procedural and documentation requirements upon us and our third-party manufacturers. Changes to the manufacturing process are strictly regulated, and, depending on the significance of the change, may require prior FDA approval before being implemented. FDA regulations also require investigation and correction of any deviations from cGMPs and impose reporting requirements upon us and any third-party manufacturers that we may decide to use. Accordingly, manufacturers must continue to expend time, money and effort in the area of production and quality control to maintain compliance with cGMPs and other aspects of regulatory compliance. The FDA may withdraw approval if compliance with regulatory requirements and standards is not maintained or if problems occur after the product reaches the market. Later discovery of previously unknown problems with a product, including adverse events of unanticipated severity or frequency, or with manufacturing processes, or failure to comply with regulatory requirements, may result in revisions to the approved labeling to add new safety information; imposition of post-market studies or clinical studies to assess new safety risks; or imposition of distribution restrictions or other restrictions under a REMS program. Other potential consequences include, among other things: ● restrictions on the marketing or manufacturing of a product, complete withdrawal of the product from the market or product recalls; ● fines, warning or untitled letters or holds on post-approval clinical studies; ● refusal of the FDA to approve pending applications or supplements to approved applications, or suspension or revocation of existing product approvals; ● product seizure or detention, or refusal of the FDA to permit the import or export of products; ● consent decrees, corporate integrity agreements, debarment or exclusion from federal healthcare programs; ● mandated modification of promotional materials and labeling and the issuance of corrective information; ● the issuance of safety alerts, Dear Healthcare Provider letters, press releases and other communications containing warnings or other safety information about the product; or ● injunctions or the imposition of civil or criminal penalties. 38 The FDA closely regulates the marketing, labelling, advertising and promotion of biopharmaceutical products. A company can make only those claims relating to safety and efficacy, purity and potency that are approved by the FDA and in accordance with the provisions of the approved label. However, companies may share truthful and not misleading information that is otherwise consistent with a product’s FDA approved labelling. The FDA and other agencies actively enforce the laws and regulations prohibiting the promotion of off-label uses. Failure to comply with these requirements can result in, among other things, adverse publicity, warning letters, corrective advertising and potential civil and criminal penalties. Physicians may prescribe legally available products for uses that are not described in the product’s labelling and that differ from those tested by us and approved by the FDA. Such off-label uses are common across medical specialties. Physicians may believe that such off-label uses are the best treatment for many patients in varied circumstances. The FDA does not regulate the behavior of physicians in their choice of treatments. The FDA does, however, restrict manufacturer’s communications on the subject of off-label use of their products. U.S. Market Exclusivity A biological product can obtain pediatric market exclusivity in the U.S., which, if granted, adds six months to existing exclusivity periods, including some regulatory exclusivity periods tied to patent terms. This six-month exclusivity, which runs from the end of other exclusivity protection or patent term, may be granted based on the voluntary completion of a pediatric study in accordance with an FDA-issued “Written Request” for such a study. The Biologics Price Competition and Innovation Act of 2009, or BPCIA, created an abbreviated approval pathway for biological products shown to be biosimilar to, or interchangeable with, an FDA-licensed reference biological product. This amendment to the PHSA attempts to minimize duplicative testing. Biosimilarity, which requires that there be no clinically meaningful differences between the biological product and the reference product in terms of safety, purity, and potency, can be shown through analytical studies, animal studies, and a clinical trial or trials. Interchangeability requires that a product is biosimilar to the reference product and the product must demonstrate that it can be expected to produce the same clinical results as the reference product and, for products administered multiple times, the biologic and the reference biologic may be interchanged after one has been previously administered without increasing safety risks or risks of diminished efficacy relative to exclusive use of the reference biologic. However, complexities associated with the larger, and often more complex, structure of biological products, as well as the process by which such products are manufactured, pose significant hurdles to implementation that are still being worked out by the FDA. The FDA will not accept an application for a biosimilar or interchangeable product based on the reference biological product until four years after the date of first licensure of the reference product, and the FDA will not approve an application for a biosimilar or interchangeable product based on the reference biological product until 12 years after the date of first licensure of the reference product. “First licensure” typically means the initial date the particular product at issue was licensed in the U.S. Date of first licensure does not include the date of licensure of (and a new period of exclusivity is not available for) a biological product if the licensure is for a supplement for the biological product or for a subsequent application by the same sponsor or manufacturer of the biological product (or licensor, predecessor in interest, or other related entity) for a change (not including a modification to the structure of the biological product) that results in a new indication, route of administration, dosing schedule, dosage form, delivery system, delivery device or strength, or for a modification to the structure of the biological product that does not result in a change in safety, purity, or potency. The BPCIA is complex and continues to be interpreted and implemented by the FDA. In addition, government proposals have sought to reduce the 12-year reference product exclusivity period. Other aspects of the BPCIA, some of which may impact the BPCIA exclusivity provisions, have also been the subject of recent litigation. As a result, the ultimate implementation and impact of the BPCIA is subject to significant uncertainty. 39 Pediatric Study Plan and Pediatric Exclusivity Under the Pediatric Research Equity Act, as amended, or the PREA, certain NDAs and certain NDA supplements must contain data that can be used to assess the safety and efficacy of the product candidate for the claimed indications in all relevant pediatric subpopulations and to support dosing and administration for each pediatric subpopulation for which the product is safe and effective. The FDA may grant deferrals for submission of pediatric data or full or partial waivers. The PREA requires that a sponsor who is planning to submit a marketing application for a product candidate that includes a new active ingredient, new indication, new dosage form, new dosing regimen or new route of administration submit an initial Pediatric Study Plan, or the PSP, within 60 days of an end-of-phase 2 meeting or, if there is no such meeting, as early as practicable before the initiation of the phase 3 or phase 2/3 study. The initial PSP must include an outline of the pediatric study or studies that the sponsor plans to conduct, including study objectives and design, age groups, relevant endpoints and statistical approach, or a justification for not including such detailed information, and any request for a deferral of pediatric assessments or a full or partial waiver of the requirement to provide data from pediatric studies along with supporting information. The FDA and the sponsor must reach an agreement on the PSP. A sponsor can submit amendments to an agreed-upon initial PSP at any time if changes to the pediatric plan need to be considered based on data collected from preclinical studies, early phase clinical trials and/or other clinical development programs. Unless otherwise required by regulation, the PREA does not apply to a drug for an indication for which orphan designation has been granted, except that the PREA will apply to an original NDA for a new active ingredient that is orphan-designated if the drug is a molecularly targeted cancer product intended for the treatment of an adult cancer and is directed at a molecular target that the FDA determines to be substantially relevant to the growth or progression of a pediatric cancer. A drug can also obtain pediatric market exclusivity in the United States. Pediatric exclusivity, if granted, adds six months to existing exclusivity periods and patent terms. This six-month exclusivity, which runs from the end of other exclusivity protection or patent term, may be granted based on the voluntary completion of a pediatric study in accordance with an FDA-issued “Written Request” for such a study. Patent Term Restoration and Extension Depending upon the timing, duration and specifics of the FDA approval of our product candidates, some of our U.S. patents may be eligible for limited patent term extension. The provisions of the Drug Price Competition and Patent Term Restoration Act, informally known as the Hatch-Waxman Act, permit a patent restoration term of up to five years as compensation for patent term lost during product development and the FDA regulatory review process. However, patent term restoration cannot extend the remaining term of a patent beyond a total of 14 years from the product’s approval date. The patent term restoration period is generally one-half the time between the effective date of an IND and the submission date of a BLA plus the time between the submission date of a BLA and the approval of that application. Only one patent applicable to an approved product is eligible for the extension and the application for the extension must be submitted prior to the expiration of the patent. The USPTO, in consultation with the FDA, reviews and approves the application for any patent term extension or restoration. In the future, we may apply for restoration of patent term for one of our currently owned or licensed patents to add patent life beyond its current expiration date, depending on the expected length of the clinical trials and other factors involved in the filing of the relevant BLA. Many other countries also provide for patent term extensions or similar extensions of patent protection for biologic products. For example, in Japan, it may be possible to extend the patent term for up to five years and in Europe, it may be possible to obtain a supplementary patent certificate that would effectively extend patent protection for up to five years. Federal and State Fraud and Abuse, Data Privacy and Security, and Transparency Laws and Regulations In addition to FDA restrictions on marketing of pharmaceutical products, federal and state healthcare laws and regulations restrict business practices in the biopharmaceutical industry. These laws may impact, among other things, our current and future business operations, including our clinical research activities, and proposed sales, marketing and education programs and constrain the business or financial arrangements and relationships with healthcare providers and other parties through which we market, sell and distribute our products for which we obtain marketing approval. These laws include anti-kickback and false claims laws and regulations, data privacy and security, and transparency laws and regulations, including, without limitation, those laws described below. 40 The U.S. federal Anti-Kickback Statute prohibits any person or entity from, among other things, knowingly and willfully offering, paying, soliciting or receiving remuneration to induce or in return for purchasing, leasing, ordering or arranging for or recommending the purchase, lease or order of any item or service reimbursable under Medicare, Medicaid or other federal healthcare programs. The term “remuneration” has been broadly interpreted to include anything of value. The U.S. federal Anti-Kickback Statute has been interpreted to apply to arrangements between pharmaceutical manufacturers on the one hand and prescribers, purchasers and formulary managers on the other. Although there are a number of statutory exceptions and regulatory safe harbors protecting some common activities from prosecution, the exceptions and safe harbors are drawn narrowly. Practices that involve remuneration that may be alleged to be intended to induce prescribing, purchases or recommendations may be subject to scrutiny if they do not qualify for an exception or safe harbor. Several courts have interpreted the statute’s intent requirement to mean that if any one purpose of an arrangement involving remuneration is to induce referrals of federal healthcare covered business, the statute has been violated. A person or entity does not need to have actual knowledge of this statute or specific intent to violate it in order to have committed a violation. In addition, the government may assert that a claim including items or services resulting from a violation of the U.S. federal Anti-Kickback Statute constitutes a false or fraudulent claim for purposes of the federal civil False Claims Act or the civil monetary penalties laws. Federal civil and criminal false claims laws and civil monetary penalties laws, including the federal civil False Claims Act, which can be enforced by individuals through civil whistleblower and qui tam actions, prohibit any person or entity from, among other things, knowingly presenting, or causing to be presented, a false claim for payment to the federal government or knowingly making, using or causing to be made or used a false record or statement material to a false or fraudulent claim to the federal government. A claim includes “any request or demand” for money or property presented to the U.S. government. Several pharmaceutical and other healthcare companies have been prosecuted under these laws for allegedly providing free product to customers with the expectation that the customers would bill federal programs for the product. Other companies have been prosecuted for causing false claims to be submitted because of the companies’ marketing of products for unapproved, and thus non-reimbursable, uses. The federal Health Insurance Portability and Accountability Act of 1996, or HIPAA, created additional federal criminal statutes that prohibit, among other things, knowingly and willfully executing a scheme to defraud any healthcare benefit program, including private third-party payors and knowingly and willfully falsifying, concealing or covering up a material fact or making any materially false, fictitious or fraudulent statement in connection with the delivery of or payment for healthcare benefits, items or services. Also, many states have similar fraud and abuse statutes or regulations that apply to items and services reimbursed under Medicaid and other state programs, or, in several states, apply regardless of the payor. 41 In addition, we may be subject to data privacy and security regulation by both the federal government and the states in which we conduct our business. HIPAA, as amended by the Health Information Technology for Economic and Clinical Health Act, or HITECH, and their respective implementing regulations, impose specified requirements on certain types of individuals and entities relating to the privacy, security and transmission of individually identifiable health information. Among other things, HITECH makes HIPAA’s security standards directly applicable to “business associates,” defined as independent contractors or agents of covered entities, which include certain healthcare providers, healthcare clearinghouses and health plans, that create, receive, maintain or transmit individually identifiable health information in connection with providing a service for or on behalf of a covered entity. HITECH also increased the civil and criminal penalties that may be imposed against covered entities, business associates and possibly other persons, and gave state attorneys general new authority to file civil actions for damages or injunctions in federal courts to enforce HIPAA and seek attorney’s fees and costs associated with pursuing federal civil actions. In addition, state laws govern the privacy and security of health information in certain circumstances, many of which are not pre-empted by HIPAA, differ from each other in significant ways and may not have the same effect, thus complicating compliance efforts. The federal Physician Payments Sunshine Act requires certain manufacturers of drugs, devices, biologics and medical supplies for which payment is available under Medicare, Medicaid or the Children’s Health Insurance Program, with specific exceptions, to report annually to the Centers for Medicare & Medicaid Services, or CMS, information related to payments or other transfers of value made to physicians and teaching hospitals, and applicable manufacturers and applicable group purchasing organizations to report annually to CMS ownership and investment interests held by physicians and their immediate family members. We may also be subject to state laws that require pharmaceutical companies to comply with the pharmaceutical industry’s voluntary compliance guidelines and the relevant compliance guidance promulgated by the federal government, state laws that require drug manufacturers to report information related to payments and other transfers of value to physicians and other healthcare providers, marketing expenditures or drug pricing, and state and local laws that require the registration of pharmaceutical sales representatives. Because of the breadth of these laws and the narrowness of available statutory exceptions and regulatory safe harbors, it is possible that some of our business activities could be subject to challenge under one or more of such laws. If our operations are found to be in violation of any of the federal and state laws described above or any other governmental regulations that apply to us, we may be subject to significant criminal, civil and administrative penalties including damages, fines, imprisonment, disgorgement, additional reporting requirements and oversight if we become subject to a corporate integrity agreement or similar agreement to resolve allegations of non-compliance with these laws, contractual damages, reputational harm, diminished profits and future earnings, disgorgement, exclusion from participation in government healthcare programs and the curtailment or restructuring of our operations, any of which could adversely affect our ability to operate our business and our results of operations. To the extent that any of our products are sold in a foreign country, we may be subject to similar foreign laws and regulations, which may include, for instance, applicable post-marketing requirements, including safety surveillance, anti-fraud and abuse laws, implementation of corporate compliance programs, reporting of payments or transfers of value to healthcare professionals, and additional data privacy and security requirements. Healthcare Reform Coverage and Reimbursement The future commercial success of our product candidates, if approved, will depend in part on the extent to which third-party payors, such as governmental payor programs at the federal and state levels, including Medicare and Medicaid, private health insurers and other third-party payors, provide coverage of and establish adequate reimbursement levels for our product candidates. Third-party payors generally decide which products they will pay for and establish reimbursement levels for those products. In particular, in the United States, no uniform policy for coverage and reimbursement exists. Private health insurers and other third-party payors often provide coverage and reimbursement for products based on the level at which the government, through the Medicare program, provides coverage and reimbursement for such products, but also on their own methods and approval process apart from Medicare determinations. Therefore, coverage and reimbursement can differ significantly from payor to payor. 42 In the United States, the European Union, or EU, and other potentially significant markets for our product candidates, government authorities and third-party payors are increasingly attempting to limit or regulate the price of products, particularly for new and innovative products, which often has resulted in average selling prices lower than they would otherwise be. Further, the increased emphasis on managed healthcare in the United States and on country and regional pricing and reimbursement controls in the EU will put additional pressure on product pricing, reimbursement and usage. These pressures can arise from rules and practices of managed care groups, judicial decisions and laws and regulations related to Medicare, Medicaid and healthcare reform, pharmaceutical coverage and reimbursement policies and pricing in general. Third-party payors are increasingly imposing additional requirements and restrictions on coverage and limiting reimbursement levels for products. For example, federal and state governments reimburse products at varying rates generally below average wholesale price. These restrictions and limitations influence the purchase of products. Third-party payors may limit coverage to specific products on an approved list, or formulary, which might not include all of the FDA-approved products for a particular indication. Similarly, because certain of our product candidates are physician-administered, separate reimbursement for the product itself may or may not be available. Instead, the administering physician may only be reimbursed for providing the treatment or procedure in which our product is used. Third-party payors are increasingly challenging the price and examining the medical necessity and cost-effectiveness of products, in addition to their safety and efficacy. We may need to conduct expensive pharmacoeconomic studies in order to demonstrate the medical necessity and cost-effectiveness of our product candidates, in addition to the costs required to obtain the FDA approvals. Our product candidates may not be considered medically necessary or cost-effective. A payor’s decision to provide coverage for a product does not imply that an adequate reimbursement rate will be approved. Adequate third-party payor reimbursement may not be available to enable us to realize an appropriate return on our investment in product development. Legislative proposals to reform healthcare or reduce costs under government insurance programs may result in lower reimbursement for our product candidates, if approved, or exclusion of our product candidates from coverage and reimbursement. The cost containment measures that third-party payors and providers are instituting and any healthcare reform could significantly reduce our revenue from the sale of any approved product candidates. The United States and some foreign jurisdictions are considering enacting or have enacted a number of additional legislative and regulatory proposals to change the healthcare system in ways that could affect our ability to sell our product candidates profitably, if approved. Among policy makers and payors in the United States and elsewhere, there is significant interest in promoting changes in healthcare systems with the stated goals of containing healthcare costs, improving quality and expanding access. In the United States, the pharmaceutical industry has been a particular focus of these efforts, which include major legislative initiatives to reduce the cost of care through changes in the healthcare system, including limits on the pricing, coverage, and reimbursement of pharmaceutical and biopharmaceutical products, especially under government-funded healthcare programs, and increased governmental control of drug pricing. There have been several U.S. government initiatives over the past few years to fund and incentivize certain comparative effectiveness research, including creation of the Patient-Centered Outcomes Research Institute under the ACA. It is also possible that comparative effectiveness research demonstrating benefits in a competitor’s product could adversely affect the sales of our product candidates. The ACA became law in March 2010 and substantially changed the way healthcare is financed by third-party payors, and significantly impacts the U.S. pharmaceutical industry. Among other measures that may have an impact on our business, the ACA established an annual, nondeductible fee on any entity that manufactures or imports specified branded prescription drugs and biologic agents; a new Medicare Part D coverage gap discount program; and a new formula that increased the rebates a manufacturer must pay under the Medicaid Drug Rebate Program. Additionally, the ACA extended manufacturers’ Medicaid rebate liability, expands eligibility criteria for Medicaid programs, and expanded entities eligible for discounts under the Public Health Service Act. At this time, we are unsure of the full impact that the ACA will have on our business. 43 Since its enactment, there have been judicial and Congressional challenges to certain aspects of the ACA, as well as recent efforts by the Trump administration to repeal or replace certain aspects of the ACA, and we expect such challenges and amendments to continue. Since January 2017, President Trump has signed two Executive Orders and other directives designed to delay the implementation of certain ACA provisions or otherwise circumvent requirements for health insurance mandated by the ACA. Concurrently, Congress has considered legislation that would repeal or repeal and replace all or part of the ACA. While Congress has not passed comprehensive repeal legislation, two bills affecting the implementation of certain taxes under the ACA have been signed into law. The Tax Cuts and Jobs Act of 2017, or Tax Act, includes a provision that repealed, effective January 1, 2019, the tax-based shared responsibility payment imposed by the ACA on certain individuals who fail to maintain qualifying health coverage for all or part of a year that is commonly referred to as the “individual mandate.” On January 22, 2018, President Trump signed a continuing resolution on appropriations for fiscal year 2018 that delayed the implementation of certain ACA-mandated fees, including the so-called “Cadillac” tax on certain high cost employer-sponsored insurance plans, the annual fee imposed on certain health insurance providers based on market share, and the medical device excise tax on nonexempt medical devices. The Bipartisan Budget Act of 2018, or the BBA, among other things, amended the ACA, effective January 1, 2019, to increase from 50% to 70% the point-of-sale discount that is owed by pharmaceutical manufacturers who participate in Medicare Part D and to close the coverage gap in most Medicare drug plans, commonly referred to as the “donut hole.” In July 2018, CMS published a final rule permitting further collections and payments to and from certain ACA qualified health plans and health insurance issuers under the ACA adjustment program in response to the outcome of federal district court litigation regarding the method CMS uses to determine this risk adjustment. In December 2018, a U.S. District Court Judge in the Northern District of Texas, or Texas District Court Judge, ruled that the individual mandate is a critical and inseverable feature of the ACA, and therefore, because it was repealed as part of the Tax Act, the remaining provisions of the ACA are invalid as well. While the Texas District Court Judge, as well as the Trump administration and CMS, have stated that the ruling will have no immediate effect, it is unclear how this decision, subsequent appeals, and other efforts to repeal and replace the ACA will impact the ACA. In addition, other legislative changes have been proposed and adopted since the ACA was enacted. In August 2011, the President signed into law the Budget Control Act of 2011, as amended, which, among other things, included aggregate reductions to Medicare payments to providers of 2% per fiscal year, which began in 2013 and, following passage of subsequent legislation, including the BBA, will continue through 2027 unless additional Congressional action is taken. In January 2013, the American Taxpayer Relief Act of 2012 was enacted which, among other things, reduced Medicare payments to several types of providers and increased the statute of limitations period for the government to recover overpayments to providers from three to five years. Further, there has been increasing legislative and enforcement interest in the United States with respect to drug pricing practices. Specifically, there have been several recent U.S. Congressional inquiries and proposed and enacted federal and state legislation designed to, among other things, bring more transparency to drug pricing, review the relationship between pricing and manufacturer patient programs, and reform government program reimbursement methodologies for drugs. At the federal level, the Trump administration’s budget proposal for fiscal year 2019 contains further drug price control measures that could be enacted during the 2019 budget process or in other future legislation. Additionally, the Trump administration released a “Blueprint” to lower drug prices and reduce out of pocket costs of drugs that contains additional proposals to increase manufacturer competition, increase the negotiating power of certain federal healthcare programs, incentivize manufacturers to lower the list price of their products and reduce the out of pocket costs of drug products paid by consumers. In August 2022, Congress passed the Inflation Reduction Act of 2022, which included a provision allowing Medicare to negotiate drug prices directly with pharmaceutical manufacturers. This provision may impact pricing strategies and determinations in the future. The U.S. Department of Health and Human Services, or HHS, has already started the process of soliciting feedback on some of these measures and is implementing others under its existing authority. For example, in September 2018, CMS announced that it will allow Medicare Advantage plans the option to use step therapy for Part B drugs beginning January 1, 2019. On January 31, 2019, the HHS Office of Inspector General proposed modifications to U.S. federal Anti-Kickback Statute safe harbors which, among other things, may affect rebates paid by manufacturers to Medicare Part D plans, the purpose of which is to further reduce the cost of drug products to consumers. In addition, CMS issued a final rule, effective on July 9, 2019, that requires direct-to-consumer television advertisements of prescription drugs and biological products, for which payment is available through or under Medicare or Medicaid, to include in the advertisement the Wholesale Acquisition Cost, or list price, of that drug or biological product if it is equal to or greater than $35 for a monthly supply or usual course of treatment. Prescription drugs and biological products that are in violation of these requirements will be included on a public list. Congress and the Trump administration have each indicated that it will continue to seek new legislative and/or administrative measures to control drug costs. At the state level, legislatures have increasingly passed legislation and implemented regulations designed to control pharmaceutical and biological product pricing, including price or patient reimbursement constraints, discounts, restrictions on certain product access and marketing cost disclosure and transparency measures, and, in some cases, designed to encourage importation from other countries and bulk purchasing. In addition, regional healthcare authorities and individual hospitals are increasingly using bidding procedures to determine which drugs and suppliers will be included in their healthcare programs. Furthermore, there has been increased interest by third party payors and governmental authorities in reference pricing systems and publication of discounts and list prices. These measures could reduce future demand for our products or put pressure on our pricing. 44 Additionally, in May 2018, the Trickett Wendler, Frank Mongiello, Jordan McLinn, and Matthew Bellina Right to Try Act of 2017, or the Right to Try Act, was signed into law. The law, among other things, provides a federal framework for certain patients to access certain investigational new drug products that have completed a Phase 1 clinical trial and that are undergoing investigation for FDA approval. Under certain circumstances, eligible patients can seek treatment without enrolling in clinical trials and without obtaining FDA permission under the FDA expanded access program. There is no obligation for a drug manufacturer to make its drug products available to eligible patients as a result of the Right to Try Act. Foreign Regulation In order to market any product outside of the United States, we would need to comply with numerous and varying regulatory requirements of other countries regarding safety and efficacy and governing, among other things, clinical trials, marketing authorization, commercial sales and distribution of our product candidates. For example, in the EU, we must obtain authorization of a clinical trial application, or CTA, in each member state in which we intend to conduct a clinical trial. Whether or not we obtain FDA approval for a drug, we would need to obtain the necessary approvals by the comparable regulatory authorities of foreign countries before we can commence clinical trials or marketing of the drug in those countries. The approval process varies from country to country and can involve additional product testing and additional administrative review periods. The time required to obtain approval in other countries might differ from and be longer than that required to obtain FDA approval. Regulatory approval in one country does not ensure regulatory approval in another, but a failure or delay in obtaining regulatory approval in one country may negatively impact the regulatory process in others. Further, some countries outside of the United States, including the EU member states, Switzerland and the United Kingdom, have also adopted data protection laws and regulations, which impose significant compliance obligations. In the EU, the collection and use of personal health data is governed by the provisions of the General Data Protection Regulation, or GDPR. The GDPR became effective on May 25, 2018, repealing its predecessor directive and increasing responsibility and liability of pharmaceutical companies in relation to the processing of personal data of EU subjects. The GDPR, together with the national legislation of the EU member states governing the processing of personal data, impose strict obligations and restrictions on the ability to process personal data, including health data from clinical trials and adverse event reporting. In particular, these obligations and restrictions concern potentially burdensome documentation requirements, granting certain rights to individuals to control how we collect, use, disclose, retain and process information about them, the information provided to the individuals, the transfer of personal data out of the EU, security breach notifications, and security and confidentiality of the personal data. The processing of sensitive personal data, such as physical health condition, may impose heightened compliance burdens under the GDPR and is a topic of active interest among foreign regulators. In addition, the GDPR provides for more robust regulatory enforcement and fines of up to €20 million or 4% of the annual global revenue of the noncompliant company, whichever is greater. Data protection authorities from the different EU member states may interpret the GDPR and national laws differently and impose additional requirements, which add to the complexity of processing personal data in the EU. Guidance on implementation and compliance practices are often updated or otherwise revised. European Union European Union Coverage Reimbursement and Pricing In the European Union, pricing and reimbursement schemes vary widely from country to country. Some countries provide that drug products may be marketed only after a reimbursement price has been agreed. Some countries may require the completion of additional studies that compare the cost-effectiveness of a particular drug candidate to currently available therapies, or so called health technology assessments, in order to obtain reimbursement or pricing approval. For example, the European Union provides options for its member states to restrict the range of drug products for which their national health insurance systems provide reimbursement and to control the prices of medicinal products for human use. European Union member states may approve a specific price for a drug product or may instead adopt a system of direct or indirect controls on the profitability of the company. 45 EU Drug regulation In order to market any product outside of the United States, we would need to comply with numerous and varying regulatory requirements of other countries and jurisdictions regarding quality, safety and efficacy and governing, among other things, clinical trials, marketing authorization, commercial sales and distribution of our products. Whether or not we obtain FDA approval for a product, we would need to obtain the necessary approvals by the comparable foreign regulatory authorities before we can commence clinical trials or marketing of the product in foreign countries and jurisdictions such as in China and Japan. Although many of the issues discussed above with respect to the United States apply similarly in the context of the EU, the approval process varies between countries and jurisdictions and can involve additional product testing and additional administrative review periods. The time required to obtain approval in other countries and jurisdictions might differ from and be longer than that required to obtain FDA approval. Regulatory approval in one country or jurisdiction does not ensure regulatory approval in another, but a failure or delay in obtaining regulatory approval in one country or jurisdiction may negatively impact the regulatory process in others. Failure to comply with applicable foreign regulatory requirements, may be subject to, among other things, fines, suspension or withdrawal of regulatory approvals, product recalls, seizure of products, operating restrictions and criminal prosecution. Non-clinical studies and clinical trials Similarly to the United States, the various phases of non-clinical and clinical research in the EU are subject to significant regulatory controls. Non-clinical studies are performed to demonstrate the health or environmental safety of new chemical or biological substances. Non-clinical studies must be conducted in compliance with the principles of good laboratory practice (GLP) as set forth in EU Directive 2004/10/EC. In particular, non-clinical studies, both in vitro and in vivo, must be planned, performed, monitored, recorded, reported and archived in accordance with the GLP principles, which define a set of rules and criteria for a quality system for the organizational process and the conditions for non-clinical studies. These GLP standards reflect the Organization for Economic Co-operation and Development requirements. Clinical trials of medicinal products in the EU must be conducted in accordance with EU and national regulations and the International Conference on Harmonization (ICH) guidelines on good clinical practices (GCP) as well as the applicable regulatory requirements and the ethical principles that have their origin in the Declaration of Helsinki. Additional GCP guidelines from the European Commission, focusing in particular on traceability, apply to clinical trials of advanced therapy medicinal products. If the sponsor of the clinical trial is not established within the EU, it must appoint an entity within the EU to act as its legal representative. The sponsor must take out a clinical trial insurance policy, and in most EU member states, the sponsor is liable to provide ‘no fault’ compensation to any study subject injured in the clinical trial. Certain countries outside of the United States, including the EU, have a similar process that requires the submission of a clinical study application (CTA) much like the IND prior to the commencement of human clinical studies. A CTA must be submitted to each country’s national health authority and an independent ethics committee, much like the FDA and the IRB, respectively. Once the CTA is approved by the national health authority and the ethics committee has granted a positive opinion in relation to the conduct of the trial in the relevant member state(s), in accordance with a country’s requirements, clinical study development may proceed. The CTA must include, among other things, a copy of the trial protocol and an investigational medicinal product dossier containing information about the manufacture and quality of the medicinal product under investigation. Currently, CTAs must be submitted to the competent authority in each EU member state in which the trial will be conducted. Under the new Regulation on Clinical Trials, which is currently expected to become applicable by early 2022, there will be a centralized application procedure where one national authority takes the lead in reviewing the application and the other national authorities have only a limited involvement. Any substantial changes to the trial protocol or other information submitted with the CTA must be notified to or approved by the relevant competent authorities and ethics committees. Medicines used in clinical trials must be manufactured in accordance with good manufacturing practice (GMP). Other national and EU-wide regulatory requirements also apply. 46 Marketing Authorizations To market a medicinal product in the EU and in many other foreign jurisdictions, we must obtain separate regulatory approvals. More concretely, in the EU, medicinal product candidates can only be commercialized after obtaining a Marketing Authorization (MA). To obtain regulatory approval of an investigational medicinal product under EU regulatory systems, we must submit a marketing authorization application (MAA.) The process for doing this depends, among other things, on the nature of the medicinal product. There are two types of Mas: ● the “Union MA”, which is issued by the European Commission through the Centralized Procedure, based on the opinion of the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) and which is valid throughout the entire territory of the EU. The Centralized Procedure is mandatory for certain types of products, such as (i) medicinal products derived from biotechnology medicinal products, (ii) designated orphan medicinal products, (iii) advanced therapy products (such as gene therapy, somatic cell therapy or tissue-engineered medicines), and (iv) medicinal products containing a new active substance indicated for the treatment certain diseases, such as HIV/AIDS, cancer, neurodegenerative diseases, diabetes, other auto-immune and viral diseases. The Centralized Procedure is optional for products containing a new active substance not yet authorized in the EU, or for products that constitute a significant therapeutic, scientific or technical innovation or that the granting of authorization would be in the interest of public health in the EU; and ● “National Mas”, which are issued by the competent authorities of the EU member states and only cover their respective territory, are available for products not falling within the mandatory scope of the Centralized Procedure. Where a product has already been authorized for marketing in an EU member state, this National MA can be recognized in another member state through the Mutual Recognition Procedure. If the product has not received a National MA in any member state at the time of application, it can be approved simultaneously in various member states through the Decentralized Procedure. Under the Decentralized Procedure an identical dossier is submitted to the competent authorities of each of the member states in which the MA is sought, one of which is selected by the applicant as the Reference member state. Under the above-described procedures, in order to grant the MA, the EMA or the competent authorities of the EU member states make an assessment of the risk-benefit balance of the product on the basis of scientific criteria concerning its quality, safety and efficacy. Under the Centralized Procedure, the maximum timeframe for the evaluation of a MAA by the EMA is 210 days. Where there is a major public health interest and an unmet medical need for a product, the CHMP may perform an accelerated review of a MA in no more than 150 days (not including clock stops). Innovative products that target an unmet medical need and are expected to be of major public health interest may be eligible for a number of expedited development and review programs, such as the PRIME scheme, which provides incentives similar to the breakthrough therapy designation in the US PRIME is a voluntary scheme aimed at enhancing the EMA’s support for the development of medicines that target unmet medical needs. It is based on increased interaction and early dialogue with companies developing promising medicines, to optimize their product development plans and speed up their evaluation to help them reach patients earlier. Product developers that benefit from PRIME designation can expect to be eligible for accelerated assessment but this is not guaranteed. The benefits of a PRIME designation include the appointment of a CHMP rapporteur before submission of a MAA, early dialogue and scientific advice at key development milestones, and the potential to qualify products for accelerated review earlier in the application process. Mas have an initial duration of five years. After these five years, the authorization may be renewed for an unlimited period on the basis of a reevaluation of the risk-benefit balance, unless the EMA decides, on justified grounds relating to pharmacovigilance, to mandate one additional five-year renewal period. Data and marketing exclusivity The EU also provides opportunities for market exclusivity. Upon receiving MA, new chemical entity, or reference product candidates, generally receive eight years of data exclusivity and an additional two years of market exclusivity. If granted, the data exclusivity period prevents generic or biosimilar applicants from relying on the pre-clinical and clinical trial data contained in the dossier of the reference product when applying for a generic or biosimilar MA in the EU during a period of eight years from the date on which the reference product was first authorized in the EU. The market exclusivity period prevents a successful generic or biosimilar applicant from commercializing its product in the EU until 10 years have elapsed from the initial authorization of the reference product in the EU. The overall 10-year market exclusivity period can be extended to a maximum of eleven years if, during the first eight years of those 10 years, the MA holder obtains an authorization for one or more new therapeutic indications which, during the scientific evaluation prior to their authorization, are held to bring a significant clinical benefit in comparison with existing therapies. However, there is no guarantee that a product will be considered by the EU’s regulatory authorities to be a new chemical entity, and products may not qualify for data exclusivity. 47 Pediatric Development In the EU, MAAs for new medicinal products candidates have to include the results of trials conducted in the pediatric population, in compliance with a pediatric investigation plan (PIP) agreed with the EMA’s Pediatric Committee (PDCO). The PIP sets out the timing and measures proposed to generate data to support a pediatric indication of the drug for which MA is being sought. The PDCO can grant a deferral of the obligation to implement some or all of the measures of the PIP until there are sufficient data to demonstrate the efficacy and safety of the product in adults. Further, the obligation to provide pediatric clinical trial data can be waived by the PDCO when these data is not needed or appropriate because the product is likely to be ineffective or unsafe in children, the disease or condition for which the product is intended occurs only in adult populations, or when the product does not represent a significant therapeutic benefit over existing treatments for pediatric patients. Once the MA is obtained in all EU Member States and study results are included in the product information, even when negative, the product is eligible for six months’ supplementary protection certificate extension (if any is in effect at the time of authorization). Post-Approval Requirements Similar to the United States, both MA holders and manufacturers of medicinal products are subject to comprehensive regulatory oversight by the EMA, the European Commission and/or the competent regulatory authorities of the member states. The holder of a MA must establish and maintain a pharmacovigilance system and appoint an individual qualified person for pharmacovigilance who is responsible for oversight of that system. Key obligations include expedited reporting of suspected serious adverse reactions and submission of periodic safety update reports (PSURs). All new MAA must include a risk management plan (RMP) describing the risk management system that the company will put in place and documenting measures to prevent or minimize the risks associated with the product. The regulatory authorities may also impose specific obligations as a condition of the MA. Such risk-minimization measures or post-authorization obligations may include additional safety monitoring, more frequent submission of PSURs, or the conduct of additional clinical trials or post-authorization safety studies. The advertising and promotion of medicinal products is also subject to laws concerning promotion of medicinal products, interactions with physicians, misleading and comparative advertising and unfair commercial practices. All advertising and promotional activities for the product must be consistent with the approved summary of product characteristics, and therefore all off-label promotion is prohibited. Direct-to-consumer advertising of prescription medicines is also prohibited in the EU. Although general requirements for advertising and promotion of medicinal products are established under EU directives, the details are governed by regulations in each member state and can differ from one country to another. The aforementioned EU rules are generally applicable in the European Economic Area (EEA) which consists of the 27 EU member states plus Norway, Liechtenstein and Iceland. For other countries outside of the EU, such as countries in Latin America or Asia (e.g. China and Japan), the requirements governing the conduct of clinical studies, product licensing, pricing and reimbursement vary from country to country. In all cases, again, the clinical studies are conducted in accordance with GCP and the applicable regulatory requirements and the ethical principles that have their origin in the Declaration of Helsinki. If we fail to comply with applicable foreign regulatory requirements, we may be subject to, among other things, fines, suspension or withdrawal of regulatory approvals, product recalls, seizure of products, operating restrictions and criminal prosecution. Privacy and data protection laws We are also subject to laws and regulations in non-US countries covering data privacy and the protection of health-related and other personal information. For instance, EU member states and other jurisdictions have adopted data protection laws and regulations, which impose significant compliance obligations. Laws and regulations in these jurisdictions apply broadly to the collection, use, storage, disclosure, processing and security of personal information that identifies or may be used to identify an individual, such as names, contact information, and sensitive personal data such as health data. These laws and regulations are subject to frequent revisions and differing interpretations, As of May 2018, the General Data Protection Regulation (GDPR) replaced the Data Protection Directive with respect to the processing of personal data in the European Union. The GDPR imposes many requirements for controllers and processors of personal data, including, for example, higher standards for obtaining consent from individuals to process their personal data, more robust disclosures to individuals and a strengthened individual data rights regime, shortened timelines for data breach notifications, limitations on retention and secondary use of information, increased requirements pertaining to health data and pseudonymised (i.e., key-coded) data and additional obligations when we contract third-party processors in connection with the processing of the personal data. The GDPR allows EU member states to make additional laws and regulations further limiting the processing of genetic, biometric or health data. Failure to comply with the requirements of GDPR and the applicable national data protection laws of the EU member states may result in fines of up to €20,000,000 or up to 4% of the total worldwide annual turnover of the preceding financial year, whichever is higher, and other administrative penalties. 48 Japan Japanese drug regulation Non-clinical studies and clinical trials Being a member of the International Conference on Harmonization (ICH), Japan has pharmaceutical regulations fundamentally similar to those of the United States or EU. Non-clinical studies are performed to demonstrate the health safety of new chemical or biological substances. Non-clinical studies must be conducted in compliance with the principles of Japanese good laboratory practice (GLP) which reflect the Organization for Economic Co-operation and Development requirements. Currently, Japan and EU have a mutual recognition agreement for GLP, and data generated compliant with EU requirements will be accepted by the Japanese authorities. There is no similar agreement with the United States. Clinical trials of medicinal products in Japan must be conducted in accordance with Japanese regulations based on ICH guidelines governing good clinical practices (GCP). They focus on ethics of the clinical trial and protection of the privacy of the trial subjects. If the sponsor of the clinical trial is not established within Japan, it must appoint an entity within the country to act as its caretaker who should be authorized to act on the sponsor’s behalf. The sponsor must take out a clinical trial insurance policy, and, according to the industry agreement, should put in place a common compensation policy for the injuries from the trial. Prior to the commencement of human clinical studies, the sponsor must complete evaluation of the safety of the investigative product, and submit a clinical trial notification and the protocol to the authorities in advance, upon agreement of the IRB of the participating institutions. When the authorities do not comment on the notification, the sponsor may proceed with the clinical trial. Any substantial changes to the trial protocol or other information submitted must be cleared by the IRB and notified to the authorities. Medicines used in clinical trials must be manufactured in accordance with good manufacturing practice (GMP). Product approval To market a medicinal product in Japan, we must obtain regulatory approval. To obtain regulatory approval of an investigational medicinal product, we must submit a new drug application. The process for doing this depends, among other things, on the nature of the medicinal product and there are currently a few different pathways for approval. If the product is designed for treating certain “difficult diseases” or those whose patient size is limited, we may be able to obtain designation as an orphan drug product if it demonstrates unique therapeutic value. Approval application for such designated orphan products will be processed on an expedited basis and the authorities’ requirement for clinical data will be much limited. Separately, the latest amendment to the law introduced separate pathways for (i) truly innovative products with a unique mode of action and (ii) those which will satisfy unmet medical needs. These products will also be processed on an expedited basis. The evaluation of applications will be based on an assessment of the risk-benefit balance of the product on the basis of scientific criteria concerning its quality, safety and efficacy. Once the review organization complete its review task, the matter will be considered by the advisory committee of experts, and the government will grant approval upon positive recommendation from the committee. The volume and quality of the clinical data will be the key determinant of the approval decision. Clinical trial data generated overseas will be accepted as part of the data package consistent with the ICH recommendation. Typically, a limited dose response clinical trial for Japanese subjects is required to ensure that data are extrapolatable for the Japanese population. In a more recent development, the authorities encourage manufacturers to organize an international joint clinical trial with some Japanese participation under a joint protocol, to expedite the clinical trial process. Regulatory approval does not expire. 49 Licensing requirement Separate from the approval requirement, it is also mandatory to possess a distribution license of an appropriate class for the manufacturer to commercially distribute the product in Japan. Non-Japanese companies who possess only the product approval may designate an appropriate license holder in Japan to commercially distribute the product, rather than distributing it on its own. The license is valid for 5 years. Intellectual Property Exclusive License Agreement with Children’s Hospital Medical Center, d/b/a Cincinnati Children’s Hospital Medical Center On June 1, 2021 (the “Effective Date”), the Company entered into a license agreement with Children’s Hospital Medical Center, d/b/a Cincinnati Children’s Hospital Medical Center (“CHMC”) to develop and commercialize certain CHMC patents and related technology directed at a VLP vaccine platform that utilizes nanoparticle delivery technology, which may have potential broad application to develop vaccines for multiple infectious diseases (“the CHMC Agreement”). The license is exclusive, worldwide, and is for all uses (other than the “Excluded Field” of immunization against, and prevention, control, or reduction in severity of gastroenteritis caused by Rotavirus and Norovirus in China and Hong Kong). The license is sublicensable with prior CHMC written approval consistent with the terms of the CHMC Agreement. The CHMC Agreement includes the below patents, which we refer to as the “Licensed Patents”, and any divisionals, continuations and continuations-in-part thereto (solely to the extent that the claims in the continuations-in-part are directed to the subject matter specifically claimed in the Licensed Patents, and they have the same priority date as the Licensed Patents, but do not include any different or additional claims), and any patents resulting therefrom: U.S. Patent Application No. U.S. Patent No. Granted Claim Type U.S. Expiration Foreign Counterparts 12/797,396 8,486,421 Compositions of the vaccine/vaccine platform 1/13/2031 CN107043408B EP2440582B1 JP5894528B2 13/924,906 9,096,644 Method of treatment 9/20/2030 CN107043408B EP2440582B1 JP5894528B2 13/803,057 9,562,077 Compositions of the vaccine platform 11/8/2033 none 16/489,095 pending pending** [3/15/2038]* Pending applications in Canada, China, EU, Hong Kong and Japan 63/149,742 (filed 2/16/2021) pending pending** [February 2042] # TBD 63/162,369 (filed 3/17/2021) pending pending [March 2042] # TBD * Projected expiration if patent issues: 20 years from earliest non-provisional application filing date. # Non-provisional application not yet filed. Expiration projected 21 years from provisional application filing date. Dependent on timely conversion to non-provisional application and issuance of patent. ** This is a pending application. Claim type will be determined after U.S. prosecution is complete. The claim type sought includes compositions of the vaccine and vaccine platform. The CHMC Agreement also grants the Company a non-exclusive limited license to use and copy internally any technical information in existence and known before the Effective Date by CHMC solely as necessary for the use and practice of the Licensed Patents (the “CHMC Technology”). The term of the CHMC Agreement begins on the Effective Date and extends on a jurisdiction by jurisdiction and product by product basis until the later of: (i) the last to expire Licensed Patent; (ii) ten (10) years after the first commercial sale; or, (iii) entrance onto the market of a biosimilar or interchangeable product. CHMC has reserved the right to practice, have practiced, and transfer the Licensed Patents and CHMC Technology for research and development purposes, including education, research, teaching, publication and public service, but not to use or practice the Licensed Patents or CHMC Technology in Field of Use for any commercial or profit purpose. 50 The Licensed Patents granted to the Company under the CHMC Agreement are also subject to any rights of the United States federal, state and/or local Government(s), as well as nonprofit entities, if certain patents or technologies were created in the course of Government-funded or non-profit entity-funded research. The CHMC Agreement also contains compulsory licensing provisions under which CHMC must notify the Company in writing whenever CHMC may become aware of third parties that are interested in obtaining rights to the Licensed Patents or CHMC Technology for purposes that are beyond the scope of the Company’s development and commercialization plan. The Company may elect to pursue the new purposes itself (and negotiate commercially reasonable development targets), or enter into sublicense negotiations with the interested third party. However, if the Company fails to meet its development targets for the new purposes or fails to enter into a sublicense agreement with the interested third party within nine (9) months of the notice from CHMC, then the new purpose will be excluded from the license grant and CHMC will be free to pursue licensing of the Licensed Patents or CHMC Technology within the Excluded Field to an interested third party. Any patented modification, alteration or improvement of any invention claimed in a Licensed Patents or CHMC Technology which is conceived or reduced to practice solely by the Company (“Company Improvement”) is owned by the Company; however, for any such Company Improvement, the Company will automatically grant to CHMC a worldwide, perpetual, sublicensable, nonexclusive, paid-up, royalty-free license to use any Company Improvements solely for clinical or non-clinical, non-commercial research, testing, educational and patient care purposes. The CHMC Agreement also provides the Company with an option to license any CHMC or jointly patented modification, alteration or improvement of any invention claimed in a Licensed Patent (“CHMC Improvement” and “Joint Improvement, respectively”), with option fee for each Improvement that the Company elects to include in the license grant of the CHMC Agreement. The Company is required to pay CHMC an aggregate of up to $59.75 million upon the achievement of specified development milestones, of approximately $0.5 million, regulatory milestones, of approximately $1.25 million and commercial milestones, of approximately $58 million (excluding any royalty arrangements). In the event the Company enters into a sublicense agreement with a third party who is not an affiliate, then the Company is obligated to pay CHMC a percentage of all non-royalty sublicensing revenue. Specifically, the Company must pay twenty-five percent (25%) for revenue received from the sublicensee prior to first net sale of a licensed product, fifteen percent (15%) for revenue received after first net sale of a licensed product or five percent after the first sale of a second licensed product. No annual maintenance fee is required. Pursuant to the CHMC Agreement, the Company paid to CHMC a one-time $25,000 initial license fee; thereafter, in fiscal year ended December 31, 2022, the Company paid $200,000 in deferred license fees. Under the CHMC Agreement, the Company is obligated to use commercially reasonable efforts to bring licensed products to market through diligent research and development, testing, manufacturing and commercialization and to use best efforts to make all necessary regulatory filings and obtain all necessary regulatory approvals, and achieve milestones relating to development and sales, and report to CHMC on progress. The Company will also be obligated to pay the agreed upon development milestone payments to CHMC. Development milestones include: (i) IND filings of each Licensed Product; (ii) BLA or equivalent allowed for Licensed Product in U.S. or E.U.; (iii) first commercial sale of licensed product in the U.S.; (iv) first commercial sale of licensed product in the E.U.; (v) first commercial sale of licensed product in Japan; (vi) first commercial sale in Rest of World (ROW); (vii) conclusion of the first calendar year. Pursuant to the terms of the CHMC Agreement, if the Company fails to achieve milestones or make milestone payments on certain milestones, and cannot mutually agree with CHMC on an amendment to the milestones, then CHMC will have the option of converting any and all of such exclusive licenses to nonexclusive licenses. In addition to the fees discussed above, beginning on the first Net Sale, the Company will pay CHMC running royalties on a quarterly basis as a percentage of Net Sales (as defined in the CHMC Agreement) of the Company, its affiliates and any subsidiaries. Similarly, in the event the Company enters into a sublicense agreement, the Company shall pay CHMC a percentage of all non-royalty sublicensing revenues received from the sublicensee. There is a 5% royalty rate for products and processes for P-Particle VLP Bivalent vaccine for norovirus and rotavirus; a 4% royalty rate for products and processes for Universal Flu Vaccine(s); and a 2% royalty rate for all other products or processes for other indications. To date, no payments have been made related to the milestones or royalties. Before any Valid Claims (as defined in the CHMC Agreement) exist, the running royalty rates are reduced by fifty percent (50%). The CHMC Agreement also contains an anti-stacking provision pursuant to which in the event the Company is legally required to pay royalties to one or more third parties whose patent rights dominate the Licensed Patents, and would therefore be infringed by exercise of the license rights granted in the CHMC Agreement, the Company may reduce running royalty payments by fifty percent (50%). In the event the Company grants sublicenses, the Company is obligated to pay CHMC as follows: (i) specified percentage of revenue received prior to first Net Sale of first Licensed Product; (ii) specified percentage for revenue received after first Net Sales of first Licensed Product but before first Net Sales of second Licensed Product; or (iii) specified percentage for revenues received after first Net Sales of second Licensed Product. 51 CHMC reserved the first and sole right, using in-house or outside legal counsel selected by CHMC, to prepare, file, prosecute, maintain and extend patents and patent applications, and the Company agreed to reimburse CHMC for its legal and administrative costs incurred in the course of doing such. The Company also agreed to reimburse CHMC for incurred legal fees of approximately $177,100 as of the Effective Date. CHMC will provide the Company a reasonable opportunity to comment during prosecution and will consider the Company’s comments, but CHMC retained control over all final decisions. If CHMC elects to not be responsible for the prosecution or maintenance of any such patents, the Company will receive a sixty (60) days’ written notice upon which the Company may elect, at the Company’s expense, to assume the responsibilities and obligations to prosecute and maintain the patents (among other things); thereafter, the Company will use reasonable efforts to give CHMC an opportunity to comment, but the final decision with respect to such matter will remain with the Company. The CHMC Agreement contains no CHMC representations or warranties. The CHMC Agreement also requires the Company to indemnify CHMC and other related parties against all claims, suit, actions, demands, judgments, or investigations arising out of any product the Company produces under the CHMC Agreement, as set forth in the CHMC Agreement, and requires the Company, beginning with the earlier of the first clinical trial or commercial sale or other commercialization to obtain liability insurance. CHMC will have the first and sole right but not the obligation, at its own expense, to initiate an infringement suit or other appropriate actions against third party infringers and receives all therefrom. For joint suits initiated against third party infringers and receives damages or profits recovered therefrom. In the event CHMC does not, within six (6) months after becoming aware of infringement, secure cessation of the infringement, the Company will have the right to initiate suit at its own expense. Any damages or profits that the Company recovers will be treated as Net Sales subject to royalties after the Company has been compensated for its costs in handling such action. In the event of a joint infringement suit, the Company and CHMC will agree in writing who will control the action and how cost and recoveries will be shared. The Company may terminate the CHMC Agreement for convenience, at any time prior to first commercial sale of a product or process by providing one hundred and eighty (180) days’ written notice to CHMC. It may also terminate for a CHMC uncured material breach. CHMC may terminate the CHMC Agreement for an uncured Company material breach or insolvency or bankruptcy. In the event the Company’s material breach is for failure to meet any of the milestone payments, the Company is entitled to a nonexclusive license to continue developing indications that have already entered development at any stage or in which the Company has invested in developing. CHMC may also terminate the CHMC Agreement to the fullest extent permitted by law in the countries of the worldwide territory, in the event the Company or its affiliates challenge or induce others set up challenges to the validity or enforceability of any of the Licensed Patents and the Company will be obligated reimburse CHMC for its costs, including reasonable attorneys’ fees. In addition to the CHMC Agreement, the Company also entered into a sponsored research agreement dated June 30, 2022 with CHMC for research related to the CHMC Agreement (the “CHMC SRA”). Pursuant to this research agreement, the Company is obligated to pay CHMC an aggregate amount not -to-exceed $247,705. Option Agreement between Oxford University Innovation Limited and Blue Water Vaccines Inc. On December 18, 2018, the Company entered into an option agreement with Oxford University Innovation Limited (“OUI”), pursuant to which the Company paid an option fee of between $25,000, to OUI in exchange for a period of exclusivity, in advance of a fundraising of fifteen million dollars ($15,000,000). Under the option agreement, the Company has the right to exercise the option for the grant of the right to the Company to an exclusive, worldwide license to PCT Patent Application number PCT/GB/2017/052510, any patents granted in response to that application, any corresponding foreign patents and applications deriving priority from that application, and any addition, continuation, continuation-in-part, division, reissue, renewal or extension based thereon, and related know-how and confidential information (the “OUI Technology”). Exercise of the option by the Company was conditional upon the Company submitting a business plan for the subsequent two years, including a development plan for the OUI Technology and a financial projection, demonstrating the Company’s ability to develop the OUI Technology and evidence of the Company’s solvency and receipt of fifteen million dollars ($15,000,000) in funds for the development of the OUI Technology. The Company has agreed that, as a condition precedent to the license becoming effective, it must provide funding for three years of salary for Dr. Craig Thompson in Oxford’s Department of Zoology of four hundred and twenty thousand pounds (£420,000). No additional funds are required to fulfill the three-year salary commitment, at this time, and none are anticipated prior to the completion of the three year term. 52 License Agreement between Oxford University Innovation Limited and Blue Water Vaccines Inc. On July 16, 2019, the Company entered into an exclusive, worldwide agreement (“OUI Agreement”) with Oxford University Innovation Limited (“OUI”), pursuant to which the Company obtained an exclusive worldwide license for all fields to PCT Patent Application number PCT/GB/2017/052510, entitled “Immunogenic Composition,” any patents granted in response to that application, any corresponding foreign patents and applications deriving priority from that application, and any addition, continuation, continuation-in-part, division, reissue, renewal or extension based thereon, and a nonexclusive license to related know-how and confidential information, as set forth in the below chart (the “Licensed Technology”): U.S. Patent Application No. U.S. Patent No. Granted Claim Type U.S. Expiration Foreign Counterparts 16/326,749 11,123,422 Compositions and method of treatment 8/25/2037 Pending applications in Australia, Canada, China, EU and Japan 17/458,712 pending pending** [8/25/2037]* * Projected expiration if patent issues: 20 years from earliest non-provisional application filing date. ** This is a pending application. Claim type will be determined after U.S. prosecution is complete. The claim type sought includes compositions of the compositions and method of treatment. The OUI Agreement has a term concluding ten years following the last to expire of all licensed patents and patent applications as defined under the terms of the OUI Agreement. The license was conditional upon the Company entering into a separate agreement with Oxford University to provide funding for three years’ salary for Dr. Craig Thompson in the University’s Department of Zoology, which amounted to four hundred and twenty thousand pounds (£420,000), which was paid by the Company in January 2020. No additional funds are required to fulfill the three-year salary commitment, at this time, and none are anticipated prior to the completion of the three year term. Improvements to the Licensed Technology as defined in the OUI Agreement belong to OUI and are included in the Licensed Technology. All Company Improvements of belong to the Company. The Company granted to OUI, and OUI subsequently granted to Oxford University, a non-transferable, irrevocable, perpetual, royalty-free license to use and publish the Licensed Technology and the Company’s Improvements upon the Licensed Technology for non-commercial use. If a Licensed Product is covered by the Medicines Access Policy of Oxford University to promote, the Company shall adhere to the requirements of the Medicines Access Policy. The Company is required to pay OUI milestone payments of up to an aggregate of $51.25 million upon the achievement of specified development milestones, of approximately $2.25 million, regulatory milestones, of approximately $9.5 million and commercial milestones, of approximately $39.5 million (excluding any royalty arrangements). An annual maintenance fee, or minimum sum, $10,000 to $20,000 will be required beginning in 2023 through launch, increasing to $250,000, which would be the highest “minimum sum” of royalties in any year prior until expiration or revocation of the last valid claim covering a licensed product, in which case the annual maintenance fee will no longer be required and the “step down” royalty rate will apply. The Company did not pay a signing fee to OUI and is obligated to pay a 6% royalty on all net sales of licensed products, as defined in the OUI Agreement, as well as royalties of 25% on any sums received by the Company from any sublicensee (including all up-front, milestone and other one-off payments received by the Company from any sub-licenses or other contracts granted by the Company with respect to the licensed technology). After the expiration or revocation of the last Valid Claim (as defined in the OUI Agreement) covering a Licensed Product, a “step down” royalty rate shall apply to such Licensed Technology and no minimum sum will be payable by the Company. If the Company has to pay royalties to a third party to use a proprietary manufacturing process proprietary adjuvants in order to make or have made a Licensed Product, the Company will be able to deduct from all royalty payments, up to a maximum amount of twenty-five percent (25%) of the royalties due to OUI. The OUI Agreement entitles the Company to supply a commercially reasonable quantity (not exceeding 5% of units sold in any quarter) of licensed products for promotional sampling. 53 In the event that royalties paid to OUI do not amount to the “minimum sum”, as discussed above, under the OUI Agreement for a particular year, the Company is obligated to make up the difference between the royalties actually paid and such minimum sum. The minimum sums vary over time, and reduces to $0 once the “step down” applies. The minimum sums and milestone fees are indexed to the RPI (Retail Prices index for all items which is published in the United Kingdom by the Office for National Statistics, or any replacement of it) and will be increased or decreased as appropriate as set forth in the OUI Agreement. The Company is obligated to use its best efforts to develop and market Licensed Products in accordance with its development plan report to OUI on progress and achieve the following milestones and must pay OUI nonrefundable milestone fees as follows when it achieves them: initiation of first Phase I study; initiation of first Phase II study; initiation of first Phase III/pivotal registration studies; first submission of application for regulatory approval (BLA/NDA); marketing authorization in the United States; marketing authorization in any EU country; marketing authorization in Japan; first marketing authorization in any other country; first commercial sale in Japan; first commercial sale in any ROW country; first year that annual sales equal or exceed certain thresholds. Upon consultation with the Company and at the Company’s expense, OUI shall prosecute, use all reasonable endeavors to maintain and renew the patents throughout the duration of the OUI Agreement. The Company and OUI agreed to inform each other in writing of any misappropriation or infringement of any rights to the licensed technology; however, the Company has the first right to take legal action at its own cost in relation to any such misappropriation or infringement, but must discuss any proposed legal action with OUI and take into account any legitimate interest of OUI in the legal action that it takes. If the Company notifies OUI that it does not intend to take legal action in such matters, OUI may take any legal action at its own cost. All profits or damages recovered after unrecovered costs and expenses are deducted are treated as net sales for which royalties would be due. OUI makes no warranties at all with regard to the Licensed Technology or whether use of it will infringe third party rights. The Company is required to indemnify OUI and Oxford University from all third party claims, damages, and liabilities asserted by third parties arising directly or indirectly from use of the Licensed Technology; marketing of Licensed Products; or breach of the OUI Agreement. The OUI Agreement is governed by English law and the parties agreed to submit to the exclusive jurisdiction of English Courts for resolution of any disputes arising out of or in connection with the OUI Agreement, with the exception of actions relating to intellectual property disputes or confidential information which may be brought in any court of competent jurisdiction. Either party may terminate the OUI Agreement for an uncured material breach. The Company may terminate the OUI Agreement for any reason at any time upon six months’ written notice expiring after the third anniversary of the OUI Agreement. OUI may terminate immediately if the Company has a petition presented for its winding-up or passes a resolution for winding up other than for a bona fide amalgamation or reconstruction or compounds with its creditors or has a receiver or administrator appointed. OUI may also terminate if the Company opposes or challenges the validity of any of the patents or applications in the Licensed Technology; raises the claim that the know-how of the Licensed Technology is not necessary to develop and market Licensed Products; or in OUI’s reasonable opinion, is taking inadequate or insufficient steps develop or market Licensed Products and does not take any further steps that OUI requests by written notice within a reasonable time. Pursuant to the terms of the OUI Agreement, the Company entered into a sponsored research agreement (the “OUI SRA”), dated December 18, 2019 with Oxford University for research related to the OUI Agreement for a period of three years for a total of £420,000. The Company prepaid the full amount to Oxford of $554,802 for the services in January 2020. Pursuant to an amendment to the SRA (the “OUI SRA Amendment”), dated May 16, 2022, the term of the research under the SRA was extended for an additional 18 months, culminating on June 18, 2024. The OUI SRA Amendment also requires that the Company provide additional funding in connection with the research in the amount of £53,500. 54 Exclusive License Agreement between St. Jude Children’s Research Hospital, Inc. & Blue Water Vaccines Inc. On January 27, 2020 (the “Effective Date”), the Company entered into an exclusive, worldwide license agreement with St. Jude Children’s Research Hospital, Inc. (“St. Jude”), pursuant to which St. Jude granted the Company an exclusive license to develop licensed products and produce vaccines for use in humans (“St. Jude Agreement”) under U.S. Provisional Patent Application No. 61/537,290 (U.S. Patent No. 9,265,819 issued on February 23, 2016), and U.S. Provisional Patent Application No. 62/817,748 (filed March 13, 2019), and any issued patents, divisions, continuations, continuations-in-part, to the extent that the claims are directed to subject matter described in the above-referenced patent applications and are entitled to the priority date of the existing patent rights, re-examinations, substitutions, renewals, restorations, additions, or registrations thereof, as well as non-United States counterparts thereof, and extensions and supplementary protection certificates thereon (“Patent Rights”), all as set forth in the below chart: U.S. Patent Application No. U.S. Patent No. Granted Claim Type U.S. Expiration Foreign Counterparts 14/345,988 9,265,819 Compositions and method of treatment 9/19/2032 none 17/602,414 # pending pending** [3/12/2040]* Pending Applications in: Australia, Brazil, Canada, China, Europe, Hong Kong, Japan and Korea * Projected expiration if patent issues: 20 years from earliest non-provisional application filing date. # U.S. National stage entry of WO 2020/183420 (PCT/IB2020/052250). ** This is a pending application. Claim type will be determined after U.S. prosecution is complete. The claim type sought includes compositions and method of treatment. The license is sublicensable consistent with the terms and conditions of the St. Jude Agreement, provided that the Company remains responsible for the performance by each of its sublicensees. The license is subject to any government rights the United States has reserved, and St. Jude retained the right to make, have made, provide and use for St. Jude’s non-commercial research and clinical purposes, including the right to distribute St. Jude’s biological material disclosed and claimed in the Patent Rights for non-profit academic research use to non-commercial entities as is customary in the scientific community and to sell the biological materials as research reagents for research use only by the scientific community. In the event the Company enters into a sublicense agreement with a third party who is not an affiliate, then the Company is obligated to pay St. Jude fifteen percent of any sublicense consideration, subject to specified exclusions, but including any upfront or milestone fees and including any premium paid by sublicensee over Fair Market Value (as defined in the agreement) for the Company’s stock. In exchange for the licenses, the Company paid St. Jude an initial license fee of $15,000 and is required to pay an annual maintenance fee of $10,000 beginning on the first anniversary of the Effective Date (which is waived if all of the developmental milestones scheduled for completion before such annual fee is due have been achieved), milestone payments, patent reimbursement, and running royalties based on net sales of licensed products under the St. Jude Agreement. Under the St. Jude Agreement, the Company is obligated to use commercially reasonable efforts to develop and commercialize the licensed product(s). If the Company fails to achieve the development milestones contained in the St. Jude Agreement, and if the Company and St. Jude fail to agree upon a mutually satisfactory revised time line, St. Jude will have the right to terminate the St. Jude Agreement. On May 11, 2022, the Company and St. Jude entered into a first amendment to the St. Jude Agreement (the “St. Jude Amendment”). The St. Jude Amendment provides for a revised development milestone timeline and a one-time license fee of $5,000. The St. Jude Amendment also provides for an increase in the aggregate milestone payments that are due upon the achievement of specified developmental milestones, from $1.0 million to $1.9 million; specifically, development milestones of $0.3 million, regulatory milestones of $0.6 million, and commercial milestones of $1.0 million. 55 The milestones include the following events: (i) complete IND enabling study by 2022; (ii) Initiate animal toxicology study by last half of 2022; (iii) file IND by last half of 2023; (iv) complete Phase I Clinical Trial by last half of 2024; (v) commence Phase II Clinical Trial by 2025; (vi) commence Phase III Clinical Trial by 2027; and, (vii) regulatory approval, U.S. or foreign equivalent by 2032. Upon achievement of certain development and commercialization milestones, the Company is required to make milestone payments to St. Jude between the achievement of certain milestones (commencement of a Phase III clinical trial through first commercial sale). As of the date of this Report, none of these milestones have been achieved. Additionally, the Company is obligated to make running 5% royalty payments payable, for each licensed product(s) sold by the Company, its affiliates or sublicensees, based on the net sales for the duration of the St. Jude Agreement. Furthermore, the Company is obligated to pay a percentage between 15% of other consideration received for any sublicenses. The Company is responsible for and shall bear all expenses relating to the filing, prosecution, and maintenance of all patent rights licensed under the St. Jude Agreement. The Company has the first right to enforce any patent against infringement, and shall keep St. Jude informed of the status of such; however, before the Company may commence any action with respect to any such alleged infringement, the Company shall take into consideration the views of St. Jude and the potential effect on the public interest. Prior to initial human testing or first commercial sale of a licensed product, and thereafter so long as the licensed products are being sold in any particular country, the Company (and its sublicenses) is required to obtain and maintain insurance to cover its indemnity obligations, and to obtain and maintain product liability insurance coverage. St. Jude represented and warranted that it has good and marketable title to the Patent Rights, but made no other representations and warranties. The term of the agreement commenced on the Effective Date, and shall continue, in each country, until the date of expiration of the last to expire valid claim included within the Patent Rights in that country. Either party may terminate the St. Jude Agreement in the event the other party (a) files or has filed against it a petition under the Bankruptcy Act (among other things) or (b) fails to perform or otherwise breaches its obligations under the St. Jude Agreement, and has not cured such failure or breach within sixty (60) days. The Company may terminate for any reason on thirty (30) days written notice. In addition to the St. Jude Agreement, the Company also entered into a sponsored research agreement (the “St. Jude SRA”) dated May 3, 2021 with St. Jude for research related to the St. Jude Agreement. Pursuant to the St. Jude SRA, the Company is obligated to pay St. Jude an aggregate amount of $73,073 in two parts, Phase I for $57,624 and Phase II for $15,449. This sponsored research project began during the year ended December 31, 2021. The Company entered into a second sponsored research agreement with St. Jude, dated August 29, 2022, pursuant to which the Company is obligated to pay St. Jude an amount of $75,603 which is due within 30 days of the effective date of the agreement. Exclusive License Agreement between the University of Texas Health Science Center at San Antonio & Blue Water Vaccines Inc. On November 18, 2022, the Company entered into a patent and technology license agreement (the “UT Health Agreement”), with the University of Texas Health Science Center at San Antonio (“UT Health”). Under the terms of the UT Health Agreement, the Company holds an exclusive, worldwide license (other than the excluded field of vectors) to certain specified patent rights relating to the development of a live attenuated, oral Chlamydia vaccine candidate, as set forth in the chart below: U.S. Patent Application No. U.S. Patent No. Granted Claim Type U.S. Expiration Foreign Counterparts 15/551,829 10,596,247 Compositions and method of treatment 3/24/2040 none ‌ 63/424,281 pending pending** ‌[11/2/2042]* none * Projected expiration if patent issues: 20 years from earliest non-provisional application filing date. ** This is a pending application. Claim type will be determined after U.S. prosecution is complete. The claim type sought includes compositions of the compositions and method of treatment. 56 An initial non-refundable license fee of $100,000 was due upon execution of the UT Health Agreement and subsequent annual license fees of $20,000 per year for each of the four years ending on December 31, 2026; $40,000 per year for each of the two years ending on December 31, 2028, and $60,000 for the year ending December 31, 2029 and each year thereafter. See Note 7 to our financial statements included elsewhere in this Report for information on milestone payments as well as royalty obligations required under the UT Health Agreement. The UT Health Agreement will expire upon the expiration of the last date of expiration or termination of the patent rights, unless terminated earlier. The Company may terminate the UT Health Agreement for convenience, by providing 90 days’ written notice to UT Health. UT Health may terminate the UT Health Agreement in the event the Company (a) becomes arrears in payment due and does not make payment within 30 days after notification from UT Health or (b) is in breach of any non-payment provision and does not cure such breach within 60 days after notification from UT Health or (c) UT Health delivers notice to the Company of three or more actual material breaches of the UT Health Agreement in any 12-month period or (d) in the event the Company or its affiliates initiates any proceeding or action to challenge the validity, enforceability, or scope of any of the licensed patents. Pursuant to the UT Health Agreement, as disclosed in Note 7 to our financial statements included elsewhere in this Report, the Company is obligated to pay certain milestone and royalty payments in the future, as the related contingent events occur. Specifically, the Company is obligated to pay UT Health a royalty on net sales, being 5% or 3% depending on whether the product is covered by a valid claim or not, as defined in the agreement. The Company is also obligated to pay a 20% royalty on any sums received by the Company from any sublicensee. In addition, the Company is required to pay UT Health milestone payments of up to an aggregate of approximately $2.2 million; specifically, upon the achievement of specified development milestones of approximately $0.7 million and regulatory milestones of approximately $1.5 million. Manufacturing and Supply We currently do not own or operate any manufacturing facilities, but our strategic partnership with Ology Bioservices, Inc. (which was later acquired by National Resilience, Inc.) (“Ology”) provides us with access to substantial resources to facilitate an independent supply path to the market. Ology is a leading global contract manufacturer with deep domain expertise and experience in large and small-scale production of clinical, as well as commercial-stage products. We have entered into agreements with Ology to secure capacity, technical expertise and resources to support the production of our products and processes that are intended to scale to commercial scale at Ology or other commercial manufacturing sites. In July 2019, we entered into a development and manufacturing master services agreement with Ology, which we refer to, as amended, as the Ology Agreement, pursuant to which Ology is obligated to perform manufacturing process development and clinical manufacture and supply of components. Under the Ology Agreement, we will pay Ology agreed upon fees for Ology’s performance of manufacturing services, and we will reimburse Ology for its out-of-pocket costs associated with purchasing raw materials, plus a customary handling fee. The Company entered into an initial Project Addendum on October 18, 2019 and the Company was required to pay Ology an aggregate of approximately $4 million. Due to unforeseen delays associated with COVID-19, the Company and Ology entered into a letter agreement dated January 9, 2020 to stop work on the project, at which point, the Company had paid Ology $100,000 for services. The second Project Addendum was executed May 21, 2021 and the Company is obligated to pay Ology an aggregate amount of approximately $2.8 million, plus reimbursement for materials and outsourced testing, which will be billed at cost plus 15%. During 2022, the Company entered into three amendments to the Ology Agreement, to adjust the scope of work defined in the second Project Addendum. The amendments resulted in a net increase to the Company’s obligations under the second Project Addendum of $154,000. During the years ended December 31, 2022 and 2021, the Company incurred research and development expenses related to the Ology Agreement of approximately $1,329,000 and $328,000, respectively, and had approximately $476,000 and $669,000 recorded as related accounts payable and accrued expenses, respectively, at December 31, 2022, and approximately $164,000 and $115,000 recorded as related accounts payable and accrued expenses, respectively, at December 31, 2021. Either party may terminate a Project Addendum and/or the Ology Agreement upon the material breach of any provision of this Agreement by the other Party if such breach is not cured by the breaching party within thirty (30) calendar days after receipt by the breaching Party of written notice of such default. The Company may terminate the Ology Agreement or the associated Project Addendum for any or no reason upon sixty (60) days’ prior written notice to Ology. 57 Employees As of March 6, 2023, we had 12 employees. None of our employees are represented by a collective bargaining agreement, and we have never experienced any work stoppage. We believe we have good relations with our employees. Properties and Facilities We are currently leasing an office located at 201 E Fifth Street, Suite 1900, Cincinnati, OH 45202, which is renewed on a monthly basis. We also lease office space located at 150 Worth Avenue, Palm Beach, FL 33480, which lease expires on April 30, 2023. All of our research and development is performed on the premises of our third-party providers. Buyback Program On November 10, 2022, the Company’s Board of Directors approved a share repurchase program to allow for the Company to repurchase up to 5 million shares of common stock, with discretion to management to make purchases subject to market conditions. The maximum purchase price is $2.00 per share and there is no expiration date for this program. Fundraising Activities April Private Placement On April 19, 2022, we consummated the closing of a Private Placement (the “April Private Placement”), in which we received approximately $6.9 million in net cash proceeds, pursuant to the terms and conditions of the Securities Purchase Agreement, dated as of April 13, 2022 (the “April Purchase Agreement”), by and among the Company and certain purchasers named on the signature pages thereto. At the closing of the April Private Placement, the Company issued 590,406 shares of common stock, pre-funded warrants to purchase an aggregate of 590,406 shares of common stock and preferred investment options to purchase up to an aggregate of 1,180,812 shares of common stock. The purchase price of each share and associated preferred investment option was $6.775 and the purchase price of each prefunded warrant and associated preferred investment option was $6.774. The aggregate gross proceeds to the Company from the April Private Placement were approximately $8.0 million, before deducting placement agent fees and other offering expenses. H.C. Wainwright & Co., LLC (“Wainwright”) acted as the exclusive placement agent for the April Private Placement. In connection with the April Private Placement, we entered into a registration rights agreement with the purchasers, dated as of April 13, 2022 (the “April Registration Rights Agreement”), pursuant to which we filed a registration statement covering the resale of registrable securities under the April Registration Rights Agreement, which was declared effective on May 20, 2022. Upon the occurrence of any Event (as defined in the April Registration Rights Agreement), which, among others, includes the purchasers being prohibited from reselling the securities acquired in the April Private Placement for more than ten (10) consecutive calendar days or more than an aggregate of fifteen (15) calendar days during any 12-month period, we are obligated to pay to each purchaser, on each monthly anniversary of each such Event, an amount in cash, as partial liquidated damages and not as a penalty, equal to the product of 2.0% multiplied by the aggregate subscription amount paid by such purchaser pursuant to the April Purchase Agreement. Wainwright served as the exclusive placement agent for the April Private Placement and received a cash fee of 7.5% of the aggregate gross proceeds of the offering and received warrants (the “April Wainwright Warrants”) to purchase up to 70,849 shares of our common stock, which was equivalent to 6.0% of the shares and prefunded warrants sold in the April Private Placement. We also agreed to pay Wainwright a management fee equal to 1.0% of the aggregate gross proceeds from the offering and reimburse certain out-of-pocket expenses up to an aggregate of $85,000. We also agreed, upon any exercise for cash of any preferred investment options, to issue to Wainwright warrants to purchase the number of shares equal to 6.0% of the aggregate number of placement shares underlying the preferred investment options that have been exercised (the “April Contingent Warrants”). The maximum number of April Contingent Warrants issuable under this provision is 70,849. 58 August Private Placement On August 11, 2022, the Company consummated the closing of a private placement (the “August Private Placement”), pursuant to the terms and conditions of a securities purchase agreement, dated as of August 9, 2022. At the closing of the August Private Placement, the Company issued 1,350,000 shares of common stock, pre-funded warrants to purchase an aggregate of 2,333,280 shares of common stock and preferred investment options to purchase up to an aggregate of 4,972,428 shares of common stock. The purchase price of each share of common stock together with the associated preferred investment option was $2.715, and the purchase price of each pre-funded warrant together with the associated preferred investment option was $2.714. The aggregate net cash proceeds to the Company from the August Private Placement were approximately $8.7 million, after deducting placement agent fees and other offering expenses. In addition, the investors in the August Private Placement, who are the same investors from the April Private Placement, agreed to cancel preferred investment options to purchase up to an aggregate of 1,180,812 shares of the Company’s common stock issued in April 2022. The pre-funded warrants have an exercise price of $0.001 per share, are exercisable on or after August 11, 2022, and are exercisable until the pre-funded warrants are exercised in full. On September 20, 2022, 945,000 of the pre-funded warrants were exercised, and as such the Company issued 945,000 shares of common stock on that date. The preferred investment options are exercisable at any time on or after August 11, 2022 through August 12, 2027, at an exercise price of $2.546 per share, subject to certain adjustments as defined in the agreement. Wainwright acted as the exclusive placement agent for the August Private Placement. The Company agreed to pay Wainwright a placement agent fee and management fee equal to 7.5% and 1.0%, respectively, of the aggregate gross proceeds from the August Private Placement and reimburse certain out-of-pocket expenses up to an aggregate of $85,000. In addition, the Company issued warrants to Wainwright (the “August Wainwright Warrants”) to purchase up to 220,997 shares of common stock. The August Wainwright Warrants are in substantially the same form as the preferred investment options, except that the exercise price is $3.3938. The form of the preferred investment options is a warrant, and as such the preferred investment options, the pre-funded warrants, and the August Wainwright Warrants are collectively referred to as the “August Private Placement Warrants”. Further, upon any exercise for cash of any preferred investment options, the Company agreed to issue to Wainwright additional warrants to purchase the number of shares of common stock equal to 6.0% of the aggregate number of shares of common stock underlying the preferred investment options that have been exercised, also with an exercise price of $3.3938 (the “August Contingent Warrants”). The maximum number of August Contingent Warrants issuable under this provision is 298,346, which includes 70,849 of April Contingent Warrants that were modified in connection with the August Private Placement. In connection with the August Private Placement, the Company entered into a Registration Rights Agreement with the purchasers, dated as of August 9, 2022 (the “August Registration Rights Agreement”). The August Registration Rights Agreement provides that the Company shall file a registration statement covering the resale of all of the registrable securities (as defined in the August Registration Rights Agreement) with the SEC no later than the 30th calendar day following the date of the August Registration Rights Agreement and have the registration statement declared effective by the SEC as promptly as possible after the filing thereof, but in any event no later than the 45th calendar day following August 9, 2022 or, in the event of a full review by the SEC, the 80th day following August 9, 2022. The registration statement on Form S-1 required under the Registration Rights Agreement was filed with the SEC on August 29, 2022, and became effective on September 19, 2022. Upon the occurrence of any Event (as defined in the August Registration Rights Agreement), which, among others, prohibits the purchasers from reselling the securities for more than ten consecutive calendar days or more than an aggregate of fifteen calendar days during any 12-month period, and should the registration statement cease to remain continuously effective, the Company is obligated to pay to each purchaser, on each monthly anniversary of each such Event, an amount in cash, as partial liquidated damages and not as a penalty, equal to the product of 2.0% multiplied by the aggregate subscription amount paid by such purchaser in the August Private Placement. Legal Proceedings From time to time we may be involved in various disputes and litigation matters that arise in the ordinary course of business. We are currently not a party to any material legal proceedings. Boustead Settlement On April 15, 2022, the Company received a demand letter (the “Demand Letter”) from Boustead Securities, LLC (“Boustead”). The Demand Letter alleged that the Company breached its underwriting agreement with Boustead, in connection with the Company’s February 2022 initial public offering. The Demand Letter alleged that, by engaging H.C. Wainwright & Co., LLC as placement agent for the April Private Placement, the Company breached Boustead’s right of first refusal (“ROFR”) to act as placement agent granted to Boustead under the underwriting agreement and, as a result of selling securities in the April Private Placement, breached the Company’s obligation under the underwriting agreement not to offer, sell, issue, agree or contract to sell or issue or grant or modify the terms of any option for the sale of, any securities prior to February 17, 2023 (the “Standstill”). 59 On October 9, 2022, the Company and Boustead entered into a Settlement Agreement and Release effective as of September 28, 2022, pursuant to which Boustead agreed to waive the ROFR and the Standstill and to release the Company from certain claims with respect to the April Private Placement, the August Private Placement, and all future private, public equity or debt offerings of the Company. As consideration for such waiver, the Company agreed to pay Boustead a cash fee of $1,000,000 plus $50,000 in legal expenses and release Boustead from all claims, subject to certain exceptions. In addition, the Company agreed to issue to Boustead 93,466 shares of restricted common stock in exchange for the cancellation of 111,111 warrants that were issued to Boustead in connection with the initial public offering. Concurrent with the execution of the Settlement Agreement, the Company and Boustead Capital Markets, LLP (“Boustead Capital”) entered into a three-month Advisory Agreement (the “Advisory Agreement”) for which consideration equal to 200,000 shares of restricted common stock, with no vesting provisions, was issued to Boustead Capital upon execution of the Advisory Agreement. Changes in and Disagreements with Accountants None. Corporation Information We were incorporated in Delaware on October 26, 2018. Our principal executive offices are located at 201 E Fifth Street, Suite 1900, Cincinnati, OH 45202, and our telephone number is (513) 620-4101. Our corporate website address is www.bluewatervaccines.com . The information contained on or accessible through our website is not part of this Annual Report on Form 10-K. Available Information We maintain a website at www.bluewatervaccines.com . You may access our annual reports on Form 10-K, quarterly reports on Form 10-Q, current reports on Form 8-K, and amendments to those reports filed or furnished pursuant to Section 13(a) or 15(d) of the Exchange Act with the SEC free of charge at our website as soon as reasonably practicable after such material is electronically filed with, or furnished to, the SEC. The reference to our website address does not constitute incorporation by reference of the information contained on our website, and you should not consider the contents of our website in making an investment decision with respect to our common stock.