- Project summary. 1.Project Title
Chemically Programmed Immunity and Adaptive Vaccinology
This One Health proposal is based on an invention by the Nobel Laureate Dr Kary Mullis (December 28, 1944-August 7, 2019). The platform consists of nucleic acids (RNA, DNA, or single-stranded or double- stranded of either or both) chemically modified to provide a linker through a nanoparticle or nanofiber with nuclease resistance; the multivalent aptamers bind pre-existing immune cell receptors or natural or induced antibodies through the nanostructures to other aptamers pre-selected in vitro to bind to a moiety on the pathogen of interest. The pre-existing, anti-ligand activity is intended to re-direct an effective immediate immune response (including pre-existing successful vaccination technology) against the pathogen targeted by the in vitro selected nucleic acid binding sequence. This linker construct is in some instances intended to trigger further immune memory for future antibody, cell immune, toll-like receptor, or other adaptive and innate immune responses through a natural cascade, providing for autogenous vaccine development. The second part of this proposed platform consists of a vector delivery system consisting of the linker synthetic nanoparticles or nanofibers and the synthetic nucleic acid aptamers linked in such a way to retain the above activity upon release of the aptamer that will facilitate dermal, oral, nasal, respiratory, or parenteral delivery of the effective nucleic acid. In the best example of this complete platform, the composite will transform or transfect a target cell for the mass production of a biosynthetic counterpart of the fully synthetic composite, which performs the same anti-pathogen, anti-parasite function, allowing for scale-up by fermentation for manufacture or in vivo amplification. Parasite pathogens have been selected for the targets under this proposal because of the difficulty and underserved need for developing vaccines against them and because animal models, and in some cases side-by-side in field zoonotic disease, allow for animal field testing under conditions which are most appropriate for predicting a human response from animal data. This composite structure also allows for tracer technology to be incorporated while maintaining function in order to determine the biological fate of such composites in vivo and in situ.
2. Platform Description and Rationale:
The platform has been described in detail in enabling US and international patents and peer reviewed scientific papers. The platform and examples will be briefly described here. The details will be given by reference to save space in this expression of interest. The technology is presented in two complementary parts: (1) the nucleic acid aptamer with a chemically conjugated ligand to recruit an existing immune response to natural or vaccine-derived immunogen attached to an aptamer which links the sequences previously selected for DNA binding in vitro to the target pathogen or parasite; and (2) the synthetic nanoparticle/nanofiber vector to carry the aptamers into the host and target tissues by a convenient port of entry, releasing them at the nidus of infection or infestation. Also, a selection process and ruggedized prototype device for use under field conditions to collect target pathogens and isolate an appropriate binding aptamer for amplification and therapeutic use has been designed and undergone preliminary tests. A nanosprayer for topical and respiratory/mucous membrane delivery has also been developed; however, several types of nanospray delivery systems are now commercially available (for cosmetics). The same aptamers and vectors designed for therapeutics have been initially designed for diagnostics using fluorescence dequenching, thermochemiluminescence, magnetic capture, electron microscopy contrast media, and visible light microscopy staining and marking and diagnostically demonstrated against spotted fever type rickettsia, Bacillus anthracis, botulinum toxin, Francisella tularensis (in the field to confirm collection and isolation), and Shiga toxin. Furthermore, the synthetic vectors have been made which transfect and transform bacterial, animal and human cells, traceable by PCR, gene expression, fluorescence, and staining. The bacterial hosts have shown not only the physical and biological evidence of genetic transfer, but also subsequent production of transferable genetic material and vector biosynthetic polymer and transfer of the genetic material to naïve bacterial hosts and their transfer in the same way for three iterations. This biosynthetic form was also transferable to human and animal cells and detected by PCR. The transfer polymer made synthetically or biosynthetically was diazoluminomelanin, a poly (hydroxy) ortho phenylene, made from 3-amino-tyrosine or tyrosine by nitration, reduction and diazotization. This polymer when activated absorbs a wide range of electromagnetic radiation from UV to visible light to microwave and radiofrequency radiation. The polymer has a high affinity for DNA, protects it from nucleases and radiation damage until saturated (high energy pulsed radiation) then can cut or destructively damage DNA or RNA by free radical generation. The microwave killing mechanism has been demonstrated against anthrax and other bacilli spores. Biosynthetic polymer containing the transferable genetic material is readily transferred to bacteria, animal and human cells.
Phenotypic consequences of nanoparticle composite transformation or transfection and fail-safe system for its destruction have been demonstrated.
Data for treatment of anthrax in mice using Sterne and Ames strain Bacillus anthracis has been collected. The data for immediate recruitment of the immune system in mice were very robust because although survival of the more pathogenic strain was limited, one must keep in mind that the anthrax was delivered by inhalation at very high multiple lethal doses and the aptamer was delivered most effectively after a 3 hour delay by inhalation and was only against the protective antigen of the anthrax toxin and had to mobilize the immune system within 3 days with total lethality at 4 days. Alpha 1,3 galactose aptamer was the immune linker, but the mice do not make antibody against this epitope and human serum, containing natural anti-gal antibody had to be used to direct the mouse immune system after immunization of the mice with human serum. Independent experiments at the US Army Medical Institute of Infection Disease showed that lung clearance of spores was superior in the presence of aptamer: approximately 67 CFU (colony forming units) of bacilli per gram of lung of a treated mouse vs an estimated 44,600 CFU bacilli per gram of lung of an untreated mouse, a 665-benefit ratio.
No human in vivo toxicity data are available, only in vitro data with human cells. However, experiments were also performed using the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure, which showed DALM to be non-toxic. Clinical trials with polymeric nanoparticles and DNA vaccines which are very similar to these agents have been performed with favourable results and some are still underway (see references). DNA vaccines have been shown to be well tolerated and safe.
3. Research and Technical Objectives – Statement of work
Objectives
The objectives of this proposal are to demonstrate the effectiveness of the platform technology and delivery systems described in this proposal in appropriate in vitro and in vivo models, safety and efficacy of such a demonstrably effective form against a clinically significant parasitic disease with both natural animal and human correlates, first in clinical animal models then in human clinically derived samples, and finally in human subjects.
4. Endpoints
The endpoints are to produce a highly reproducible and clinically relevant, for safety and efficacy, composite nanoparticle aptamer platform which can be readily re-directed toward many infectious and parasitic targets by mere substitution of components without extensive re-testing and field trials.
5. Target pathogens
Pathogen 1: Malaria (Plasmodium falciparum and others): There is no current effective vaccine for this disease and resistance to the most effective treatment with artemisinin is spreading. However, natural resistance has been demonstrated in endemic areas in children, but the effective response requires IgM and not IgG and the target antigen galactose-alpha-1,3-galactose. Vaccines that lead to IgG response development will not be as effective as those that direct the immune system toward an IgM response to alpha gal.
Pathogen 2: Heartworms (Dirofilaria immitis): this disease is not only pervasive in canines across the world, but the current treatment uses the same macrocyclic lactones, to which resistance is developing and spreading, used in human filariasis; macrocyclic lactones are the only class of drugs currently available to treat these filarial diseases, which means the development of resistance would eliminate any effective treatment, and they involve altering or blocking the release of an antigen which interferes with the immune response to these filaria and which is effectively activated upon treatment to eliminate microfilaria. There is a robust antibody response which is interfered with by soluble antigen/antibody complex formation which could be overcome by the appropriate redirection or facilitation of immune targeting.
Pathogen 3: Dracunculus medinensis, Guinea worm; the hope to eradicate this parasite, now only in Africa, has recently shown signs of failure after finding that the once thought to be rare infection in dogs (over a thousand infected dogs discovered) is now commonly present in them in Chad. This infection of dogs in areas of re-emerging human infection allows for studies of the platform response in dogs under field conditions which would be most relevant to concurrent human disease.
6. Pre-Clinical Approach:
The worst case non-clinical/pre-clinical development approach has already been established by previous Department of Defence research on anthrax spore protection using a mouse/Sterne/Ames anthrax spore inhalation and immediate mobilization of the immune system by inhalation aptamer composites and looking for prolonged survival and clearing of inhaled anthrax spores. The dose ranges have been pre-determined by in vitro neutralization molar ratio data (for anthrax toxins but of course not the proposed targets). The general toxicity issues are the activation of toll-like receptor innate immunity and direction toward Th1 vs Th2 immune pathways (i.e., avoiding alpha gal meat allergy-like responses by appropriate cellular immune cell targeting along with parasite targeting) which can be adjusted by making alterations in the nucleic acid sequences of the aptamers. Since the immune response can be recruited during concomitant infections, the reduction in microfilaria or other circulating parasitic forms (i.e., in malaria), we can directly assess the response in an individual human or animal as its own control.
- 7. Clinical development plan and regulatory strategy:
The strategy for clinical development through Phase I of the proposed pathogens uses constructs which are essentially variations of the same form and these lead to certain derived, in-depth analyses of immunological mechanisms of action of the platform and toxicity. Each pathogen/parasite addressed will take a similar approach by substitution: the anti-malarial aptamer composite will contain a bifunctional nanoparticle coated with a nucleic acid with galactose-alpha-1,3-galactose moiety at the exposed end to engage natural human antibody against the alpha gal and an anti-B1 lymphocyte receptor(s) aptamer, and/or including anti-IgM aptamers, on the same nanoparticle surface to directly stimulate IgM production for the removal of the malarial parasites. These composites can be tested against peripheral human monocytic/lymphocytic cells first (binding and stimulation assays) and antibody in human serum for binding. The Phase I test would look at the toxicity of the alpha gal/anti-B1 lymphocyte aptamer nanoparticle composite. Initially, a model form will be tested (for toxicity and efficacy) in the avian model of malaria infection by using poultry immunized against alpha gal and a nanoparticle with an alpha gal nucleic acid moiety and an anti-avian IgY aptamer. The poultry will be infected with avian malaria and the various component controls and complete platform. The anti-heartworm platform, which can be extended to human filarial parasites by substitution of appropriate aptamer specific parasite or general cross-reacting filarial antigen moieties, will consist of a bifunctional nanoparticle coated with an aptamer selected against the endosymbiont Wolbachia and another aptamer against one or more excretory/secretory antigens (currently used as diagnostic antigens in dogs). Domestic (pet) dogs with and without concurrent heartworm infections from endemic heartworm areas will be recruited for treatment/vaccination with the preparation following in laboratory testing of efficacy and toxicity in dogs and monitored for microfilaria and immunological responses indicating adult female heartworm presence. The anti-dracunculid composite will be composed of the same nanoparticle types as tested above, species specific anti-excretory/secretory antigens aptamer(s) for the parasite (many have already been cloned) and an anti-measles/distemper virus or anti-measles/distemper antibody epitope aptamer. Cross reacting anti-measles/distemper aptamers will allow the same composite prep to be used in both dogs and humans, who have been immunized against these viruses and will either be challenged with the currently available vaccines at time of treatment or those demonstrating high antibody titers against these viruses at the time. The planned licensure pathway for approval of a phase 1 clinical trial will be through a San Antonio clinical trial firm to be hired.
- 8. Mechanism of action:
Because the mechanism involves re-directing an immune response from an ineffective but pre-existing abundant one, the reduction in numbers of immature circulating forms of the parasites or adult parasite fertility and viability will be used to assess the effectiveness of the mechanism of action. Furthermore, levels of isotypes and idiotypes of antibodies and specific committed cellular immunity will be assessed by quantity and specificity of response to the effective antigens/immunogens. The reappearance of disease in an endemic area in individuals (animals or humans) treated and cleared in this way, by adaptive vaccinology, will be assessed for reappearance of infection over time and the extent of signs and symptomology of apparent disease. The effectiveness of re-treatment will be evaluated and the necessity to further re-direct the response.
- 9. Chemistry, Manufacturing & Control (CMC) Development
The large-scale manufacture of the platform involves methods already being used in commercial production of aptamers (SELEX) and newer methods described in the publications and patents referenced, including an automated method, and a field diagnostic system for finding binding sequences and cloning them for fermentation production.
- 10. Timeline and Key milestones:
Timeline – Decision/key milestones
From initiation of project: 3-6 months to select aptamers for potential molecular targets; starting after selection/conjugation of aptamers to nanoparticles and testing of binding activity, determining quality and quantity reliability (first milestone that must be passed): 6 months; testing in in vitro cellular models, animal and human, and laboratory models if necessary (mice and chickens or other poultry for malarial responses; dogs for microfilaria and adult heartworm responses; dracunculid responses will have to be done in the field in Chad in dogs); at least one, and preferably, the first two laboratory animal models must be passed without overt toxicity to proceed. By 2.5 years from the initiation, at least phase 1 trials should begin for one of the specific anti-parasite composites. Phase 2 and 3 will most likely exceed the 3-year limit of this funding proposal.
- 11. Vaccine Development Timeline: Because changing the target for vaccination only requires a substitution into the platform, human trials could occur within 3 months of identifying a target. The approval of prior tests of the DNA/nucleic acid and nanoparticle platforms for use in humans as safe and the concurrent use of similar preparations in domestic animals (under USDA/APHIS biologicals rules) or in-country animals would greatly accelerate the process.
The aptamer platform composite approach does not require the prior development of immunity against the target organism but employs pre-existing or concomitant immunity to provide protection. In anthrax trials with naïve mice at multiple lethal doses of anthrax spores, immune protection against lethal anthrax toxin was initiated as early as 2 hours after exposure and maintained in an advancing infection at periods of 24 hours until 14 days when primary immunity took over. This worst case would not be expected with the parasites described since they are not uniformly fatal at 4 days as the overwhelming anthrax spore challenge would be. Also, the parasites themselves, when present as initial larval forms or subsequent adults maintain an active but ineffectual immune response, which the aptamer composite platform would re-direct for the elimination of the parasites. The presence of the nanoparticle-based aptamer composite platform is anticipated to continue for at least a year and it remains to be seen if the re-directed immune pathway will persist after the composite platform has broken down, but this is possible if not probable and will be explored as part of the experimental phase with subsequent re-challenges with parasites. Potentially no boosting will be required.
- 12. Scale up to target doses
The estimated time of release, if anti-ligand targets are already available for aptamer binding selection, and if no clinical trials are required before such an emergency response (except for safety), then the best time would be 3 months and worst 6 months, starting from the beginning of the process. If the aptamer sequence is already known, production and delivery could occur in 2-4 weeks. The platform is designed for single dose use at best, but can be used, as it was with anthrax, in multiple serial doses for severe overwhelming potential lethal disease. The questions about manufacture process have been addressed elsewhere in this pre-proposal.
- 13. Budget
Total budget estimate
The initial pre-clinical development approximate cost will be at least $1.8M per year, which includes all the manufacturing technology development for scale, initial in vitro cell culture and animal testing. It will also include testing for toxicity of the nanoparticle and nucleic acid constructs unless data can be accepted on similar DNA and nanoparticle constructs already being used in human and animal clinical trials corresponding to this purpose (if not this may raise the cost to $3M yearly). The production of the scaled-up end-product for each parasite for use in testing, clinical trials and/or emergency response would be at least $550,000 for the custom made (not final manufacturing cost) for 100,000 doses. The cost objective for the final product for distribution for medical and veterinary use is 16 to 25 cents a dose at today’s costs of intermediate scale up manufacturing of nucleic acids (of bifunctional target length aptamers) and nanoparticles, or as close to this cost as possible. The major pass through costs would be for clinical trials, if they are to be funded all or in part, about $55M. Otherwise work up to that time (over a minimum of 3 years) would be $5.9M to $6.55M.
Additional funding not applicable.
- 14. Application organization/consortium
Experience and track record of applicant organization and cooperating partner(s)
John G. Bruno: B.S., Microbiology with Chemistry Minor, Univ. of Arizona (1985) , Ph.D., Microbiology with emphasis on Immunology, Univ. of Arizona, Tucson (1991), Jan 2003-August 2018 Senior Scientist, Vice President and Chief Science Officer- Operational Technologies Corporation (OpTech), OTC Biotechnologies, LLC, and Pronucleotein Biotechnologies, Inc., San Antonio, TX., September 2018 – Present, Principal Scientist and Director of Biotechnology – Nanohmics, Inc., Austin, TX., 68 Grants/Contracts; Funding > $16M; 104 Peer-Reviewed Journal Articles; 16 Total U.S. and WIPO/PCT/EU Patents .
Ronald M. Cook: UC Berkeley College of Chemistry (BS 1969); University of Washington, Seattle, Department of Chemistry, Ph.D. 1974; postdoctoral, University of California, San Francisco, (Dept of Microbiology and Immunology), 1974-76; founded Biosearch, Inc in 1977 to supply synthetic peptides (enkephalins) and protein bioconjugates for research; 1980, synthesis of nucleic acids, Biosearch became leading innovator in synthetic DNA oligonucleotide chemistry; 1982, the company developed first commercially viable Oligo synthesizer (SAM I); one of the first SAM units was placed with Kary Mullis, at Cetus, enabling the invention of PCR in 1983; 1990-1993, Principal Consultant to Beckman Instruments; 1993- 2015, Biosearch Technologies, Inc.: President/CEO, Chief Technical Officer; Chairman, Light Speed Genomics, a Santa Clara company; Chairman, DNA Technology, a Danish oligo-manufacturing company; Chairman, Vitra Bio, a German manufacturer of specialty glass for solid phase chemistry; consultant and/or served on Scientific Advisory Boards of Beckman Instruments, Solexa (Cambridge, England, RMC was a co-founder), developing next generation DNA sequencing technology; and Mosaic (Cambridge, Ma), developing solid phase PCR and contingent diagnostic applications; 2015-2019. LGC-Genomics, Chief Scientific Officer, Chairman, Co-Founder Optical Biosystems.
George W. Irving III; DVM (1965), MS (1970), Diplomate American College of Laboratory Animal Medicine (1972), and American College of Veterinary Preventive Medicine (1977); served 30 years as USAF Military Officer, and 23 Years as VP at Conceptual MindWorks, Inc., directing research in Chemical & Bio Warfare Agent defense, Non-Lethal Weapons Bio Effects, and Pharmaceutical/Vaccine Development.
Johnathan L. Kiel: DVM (1974, Texas A&M); PhD (Microbiology and Biochemistry, 1981, Texas Tech); Diplomate American College of Veterinary Microbiologists (1984), Charter Diplomate in Veterinary Parasitology (2011); served 38 years total, as a military officer, as a Civil Servant (GS14-15) research veterinarian, and Senior Scientist (Brigadier General Equivalent) in the Senior Executive Service in electromagnetic bioeffects and biosurveillance and counterproliferation of biological warfare and other infectious agents; 106 peer-reviewed articles; 30 patents. The fundamental form of this technology was invented by Dr Kary Mullis (deceased 7 August 2019), the recipient of the Nobel Prize, for inventing, PCR in 1993; with his collaboration, the platform was extended to other applications and delivery systems by the researchers of the United States Air Force at the Air Force Research Laboratory.
- 15. Dissemination plan of study results and data sharing:
Peer-reviewed journal papers will be used when applicable to share data. Clinical trial registration and studies will be managed by a San Antonio clinical trials firm (such as Clinical Trials of Texas, Inc.). Because the targets for this project involve tropical diseases as well, we will reach out to public health and medical authorities of potential target countries such as Chad and other African countries through the World Health Organization and the OIE, World Organisation for Animal Health. All patient data will be protected in accord with US and international laws. Patent applications, both US and international, will be used to disclose the processes, but non-exclusive licensing will be used as much as possible to make the technology globally available. Procedures in accord with the USDA/APHIS will be followed to license the resulting animal biologicals produced from the project platform.
- 16. References.
- 1. Mullis, K.B., Vivekananda. J., Kiel, J.L., Cook, R.M. Chemically Programmable Immunity, US Patent 8,604,184 B2, December 10, 2013.
- 2. Kiel, J.L., Tijerina, A., Holwitt, E.A., Sloan, M.A., Woitaske, M., and Fan, M. Compositions, Methods and Uses for Biosynthetic Plasmid Integrated Capture Elements, US Patent 8,628,955 B2, Jan. 14, 2014.
- 3. Kiel, Johnathan L., Holwitt, Eric A., Fan, Michael (Maomian), Roper, Shelly D., Methods and Compositions for Rapid Selection and Production of Nucleic Acid Aptamers, US Patent 9,273,345 B2, March 1, 2016.
- 4.Vivekananda, J., and Kiel, J. L. Anti-Francisella tularensis DNA aptamers detect tularemia antigen from different subspecies by Aptamer-Linked Immobilized Sorbent Assay, Laboratory Investigation 86: 610-618, 2006.
- 5. Fan, M., McBurnett, S. R., Andrews, C. J., Allman, A. M., Bruno, J. G., and Kiel, J. L. Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers. J Biomol Tech 19(5), 311–319 (December 2008).
- 6. Bruno, J G. Aptamer–biotin–streptavidin–C1q complexes can trigger the classical complement pathway to kill cancer cells In Vitro Cell.Dev.Biol.—Animal (2010) 46:107–113.
- 7. Mutapi, F., Billingsley, P.F., and Secor, W.E. Infection and treatment immunizations for successful parasite vaccines. Trends in Parasitology March 2013, Vol. 29, No. 3, 135-141.
- 8. Bruno, J.B., Richarte, A.M., Savage, A.A., and Sivils, J.C. Development and Characterization of DNA Aptamers Which Bind Kinesins from Leishmania Promastigotes. J. Bionanosci. 8, 1–12, 2014.
- 9. Aguilar, R., et al. Antibody responses to α-Gal in African children vary with age and site and are associated with malaria protection. Scientific Reports. 3 July 2018, DOI:10.1038/s41598-018-28325-w.
- 10. Kristian, S.A., Hwang, J.H., Hall, B., Leire, E., Iacomini, J., Old, R., Galili, U., Roberts, C., Mullis, K.B., Westby, M., and Nizet, V. Retargeting pre-existing human antibodies to a bacterial pathogen with an alpha-Gal conjugated aptamer. J Mol Med (Berl). 2015 June; 93(6): 619–631.
- 11.Slatko, B.E.,Taylor, M.J., and Foster, J.M. The Wolbachia endosymbiont as an anti-filarial nematode target. Symbiosis (2010) 51:55–65.
- 12. Pati, R., Shevtsov, M., and Sonawane, A. Nanoparticle Vaccines Against Infectious Diseases. Frontiers in Immunology (9), October 2018, Article 2224.
- Don’t lose sight of the versatility of this approach (just because the original proposal involves its use against parasites),especially for COVID-19, where the path the immune response takes is the difference between recovery or death or long term chronic disease.
Global Biosurveillance 