Method for Rapid High Volume Production of Synthetic Vaccines

Abstract

The present invention features an on demand programmable machine and process for producing synthetic vaccine or other protein product(s). This system includes modular machine components with programmable inputs to responsively and rapidly synthesize selected protein product(s) in desired amounts. When the device is programmed to produce multiple proteins in a single run, the amount of each protein produced is independently controlled. The programmability and modularity is scalable permitting e.g., low volume output to manage an incipient local outbreak, and in cases where higher production volumes are necessary to prevent epidemic or pandemic events, high levels of production can be planned at the outset or scaled up as greater need is recognized.

Claims

1. An improved machine for bio-synthesizing polypeptides in a cell-free apparatus wherein at least one improvement is selected from the group consisting of: eliminating transcription as a step in protein production; avoiding addition of RNA polymerase; providing high energy phosphates in controlled feed amounts from an auxiliary production source; attaching ribosomes to a solid support; with a result that a flow through continuous peptide/protein production is possible; optimizing production through use of a restricted set of codons and associated tRNAs; selecting ribosomes from organelles or organisms to allow rapid synthetic processing and to lessen pathogenic danger.

2. The machine of claim 1 comprising a bioactive element affixed to a solid support.

3. The machine of claim 2 wherein said solid support is configured in a format selected from the group consisting of: particles, strands, precipitates, gels, sheets, tubings, spheres, membranes, vesicles, containers, capillaries, pads, slices, films, plates and slides.

4. The machine of claim 2 wherein said solid support comprises a composition selected from the group consisting of: silicon, glass, polycarbonate, (poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene, a charged hydrophobic membrane, nylon, a natural fiber, a silkworm silk, a protein, insect, silk and arachnid silk.

5. The machine of claim 1 configured to incorporate at least one changeable module.

6. The machine of claim 5 comprising at least one module comprising suspended particles.

7. The machine of claim 6 wherein said suspended particles are dispersed within a gaseous phase.

8. The machine of claim 1 wherein mRNA in said machine encodes at least one antigenic and immunogenic peptide.

9. The machine of claim 1 comprising mRNA designed in accordance with an artificial intelligence induced algorithm.

10. The machine of claim 1 comprising at least one chaperone protein.

11. The machine of claim 10 wherein said at least one chaperone protein is selected from the group consisting of: DnaK/DnaJ/GrpE, GroEL, GroES, Hsp60s, Hsp10, Hsp40, Cpn60, mt-Cpn60, Cpn10, mt-Cpn10, ch-Cpn20, ch-Cpn60, HSP65 and Clp.

12. The machine of claim 11 comprising at least one Hsp60 selected from the group consisting of: Hsp60A, Hsp60B, Hsp60C and Hsp60D.

13. The machine of claim 10 wherein said at least one chaperone protein is a gene expression product selected from the group consisting of: BBS10, BBS12, TCP1, CCT2, CCT3, CCT4, CCT5, CCT6a, CCT6b, CCT7, CCT8, CLP8, HSPD1, HSPE1, CD74, calnexin, calreticulin, and MKKS.

14. The machine of claim 1, further comprising optimizing content of an ion comprising an element selected from the group consisting of: Mg, Ca, Se, Fe, Cu and S.

15. The machine of claim 1, wherein an ATP/GTP generator operates with a temperature difference of at least 2 C. compared to a portion of said machine with said solid support attached ribosomes.

16. The machine of claim 15 wherein said temperature difference is selected from the group consisting of at least: 2 C., 3 C., 4 C., 5 C., 7 C., 10 and 12 C.

17. The machine of claim 1 comprising at least one: ribosome, tRNA, recognition factor, initiation factor, elongation factor, chemical energy source, RRF, aminoacyl tRNA synthetase, methionyl tRNA formyltransferase, pyrophosphatase, amino acid, ATP-generating catalytic module and RNA polymerase.

18. The machine of claim 17 wherein the at least one chemical energy source is selected from the group consisting of: phosphoenol pyruvate, acetyl phosphate, creatine phosphate, 3-phospho-glycerate, inorganic phosphate, and nucleoside triphosphate.

19. The machine of claim 18 wherein a nucleoside triphosphate is selected from the group consisting of: ATP and GTP.

20. The machine of claim 17 wherein the at least one initiation factor is selected from the group consisting of: IF1, IF2 and IF3.

21. The machine of claim 17 wherein the at least one elongation factor is selected from the group consisting of: EF-Tu, EF-Ts, EF4 and EF-G.

22. The machine of claim 17 wherein the at least one release factor is selected from the group consisting of: RF1, RF2 and RF3.

23. The machine of claim 17 wherein the at least one RNA polymerase comprisesT7 RNA polymerase.

24. The machine of claim 17 comprising at least one anti-oxidant.

25. The machine of claim 24 wherein said at least one antioxidant is selected from the group consisting of: superoxide dismutase, melatonin, ascorbic acid, glutathione, lipoic acid, uric acid, carotene, tocopherol, ubiquinol, DnaK/DnaJ/GrpE, GroEL/GroES and n-acetyl cysteine.

26. The machine of claim 17 comprising at least one component selected from the group consisting of: an ion or complex comprising an element selected from the group consisting of: Mg, Ca, Se, Fe, Cu, S and Fe; Nm23-H6, nucleoside diphosphate kinase, chaperone, spermine synthase, spermine oxidase, 3-aminopropanal, N1-acetylspermine, N-acetyl-3-aminopropanal and polyamine.

27. The machine of claim 26 wherein said chaperone is selected from the group consisting of: a gene expression product selected from the group consisting of: BBS10, BBS12, TCP1, CCT2, CCT3, CCT4, CCT5, CCT6a, CCT6b, CCT7, CCT8, CLP8, HSPD1, HSPE1, CD74, calnexin, calreticulin, and MKKS.

28. The machine of claim 26 wherein said polyamine is selected from the group consisting of: including, but not limited to: putrescine (PUT), NH.sub.2(CH.sub.2).sub.4NH.sub.2; spermidine (SPD), NH.sub.2(CH.sub.2).sub.3NH(CH.sub.2).sub.4NH.sub.2; spermine (SPM) and NH.sub.2(CH.sub.2).sub.3NH(CH.sub.2).sub.4NH(CH.sub.2).sub.3NH.sub.2.

29. A method for in vitro production of protein product, said method comprising operating a ribosomal based peptide synthesizer, said synthesizer comprising one or more ribosomes comprising rRNA and protein; initiation factors; elongation factors; release factors; ribosome recycling factors; a sufficient number and variety of aminoacyl tRNA synthetases dependent on the amino acids actually used; a sufficient number and variety of amino acids dependent on the amino acids actually used; at least one mRNA; methionyl tRNA formyltransferase; an ATP/GTP generator; nucleoside diphosphate kinase; and pyrophosphatase.

30. The method of claim 29 wherein the tRNAs are minimized to correspond to mRNA sequences to be translated.

31. The method of claim 29 wherein the aminoacyl tRNA synthetases are minimized to correspond to mRNA sequences to be translated.

32. The method of claim 29 wherein ribosomes are immobilized on a solid or semisolid substrate and a reaction mixture is flowed past the immobilized ribosomes.

33. The method of claim 29 wherein the protein product is an intermediate for vaccine.

34. The method of claim 29 wherein the synthesizer comprises multiple mRNA templates of differing sequences.

35. The method of claim 29 wherein the mRNA is assessed to eliminate sequences endogenous to the intended recipient.

36. The method of claim 29 wherein said synthesizer comprises at least one polyamine.

37. The method of claim 36 wherein said at least one polyamine is selected from the group consisting of: putricine, spermine and spermididine.

38. The method of claim 37 wherein a spermidine/ribosome ratio is selected from the group consisting of about: 3:1, 5:1, 7:1, 10:1, 12:1, 15:1, 20:1 and 25:1.

39. The method of claim 26 wherein a number of multiple mRNA templates is selected from the group consisting of at least: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20 and 25.

40. The method of claim 29 wherein the algorithm seeks to optimize one or more factors selected from the group consisting of: immunogenic potential, cost of production, shelf life, biological half-life, solubility, viscosity effect, potential for induction of auto-immune response, protein-protein interactions, three-dimensional stability, cross reactivity and synthetic rates and accuracy.

41. The method of claim 29 wherein said one or more ribosomes are of human origin.

42. The method of claim 29 wherein said one or more ribosomes are of mitochondrial origin.

43. The method of claim 29 wherein said one or more ribosomes are of human microbiome origin.

44. The method of claim 29 wherein said one or more ribosomes are of E. coli origin.

Description

EXAMPLES

Example 1Oil Mite

[0107] Oil tar sands workers near Vancouver develop hives, then fever. About one third of the fevers progress to rigor mortis like conditions with tetanic cramps throughout skeletal muscle. Several workers are placed on ventilators. An unidentified toxin is suspected. A criminal investigation rules out illegal drug use as the cause of this new malady. A toxin related to tar sands extraction methods is suspected. Comparison to industrial samples obtained at various stages in drilling/production from other extraction fields does not reveal a difference that might be responsible for the incapacity of these workers. Soil samples are taken to assess for possible causes. Although the sampling technicians are provided and diligently use sub HEPA size protective gear and positive ventilation garb, four days later two technicians who carpool together to the site sport the telltale rash and are prophylactically hospitalized. Three days later one technician ceases eating and becomes feverish. Her nurse develops the telltale rash. The nurse never had contact with the site, the protective gear or clothing from the technicians. The only possible exposure seemed to be contagion from airborne or body contact. It was discounted that this nurse had contact with bodily fluids. The aide most likely to have contacted fluids developed no symptoms. Then the other technician's girlfriend who had driven the truck back to base is staying in hospital. As the female technician approaches rigor, the girlfriend sports the rash, though both technicians had been segregated in isolation. A forensic disease technician samples fabric, mats and air from the truck and several other vehicles on site. No toxins are noted, but a common mite is found. Several vehicles and the portable breakrooms and toilet facilities (RV style) are vacuumed to collect live arthropods and their remains. These are worked up on the theory that a zoonotic disease might be operational.

[0108] A DNA virus is identified; and both technicians, but not the girlfriend test positive for the viral DNA in their blood and cutaneous biopsies. The viral genome is sequenced and all possible reading frames are processes as messenger RNA. The diseasebeing probably zoonotic and involving distant species, suggests a fungal mitochondrion based protein synthesis system to be used as the basis for synthetic machinery. Since arthropods and mammals are widely separated on the taxonomic charts, a closest similarity of mitochondrial ribosome and related tRNAs, factors and controls are made available for synthesizing 35 encoded protein sequences in all reading frames. Concurrently the sequences of these proteins are compared using international databases to human proteins and all nucleic acids. All sites in the area are treated with insecticides to eliminate the infected arthropods. Several available algorithms are used to select polypeptides with likely excellent immunogenicity in humans. A composite sampling of blood from twenty-seven of the thirty one workers known to have presented symptoms is reactive to seventeen of the hundred or so proteins presented. Six are highly reactive to these antibodies. These sequences are compared with the available human genomes for potential auto-recognition. As a back-up the other eleven are similarly assessed. Likely mimicry having been discounted, the mitochondrial RNA and ribosomal systems are commandeered to batch produce all six polypeptides. Three of the longer proteins with in excess of 180 amino acid residues are synthesized as intact molecules but also produced in 60 amino acid length fragments walked at 15 residue steps.

[0109] These polypeptides are injected into 4 strains of mouse and one canine with no apparent ill effect. Three volunteers from the region, two tar sands workers, one of whom was merely clerical with scant physical presence at the site, and a pipeline worker, act as phase 1 safety screeners, each receiving 35 g protein in a single intra-muscular injection. With no deleterious results apparent, a small dose is injected IM into one suffering worker. No allergic reaction was noted. Others then are vaccinated. Three weeks to three months later samples obtained from those vaccinated reveal that seventy-three percent have detectable antibody following only one dose. All recipients are offered a second vaccination at their discretion. Vials with material sufficient to produce 2500 vaccine doses are stored in 180 freezers. A panel is studying recommendations for possible vaccination of all area workers and to plan responses for any similar recurrence.