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(12) United States Patent (10) Patent No.: US 8,883,481 B2 Dormitzer Et Al

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(12) United States Patent (10) Patent No.: US 8,883,481 B2 Dormitzer Et Al USOO8883481 B2 (12) United States Patent (10) Patent No.: US 8,883,481 B2 Dormitzer et al. (45) Date of Patent: Nov. 11, 2014 (54) REVERSE GENETICS METHODS FOR VIRUS (56) References Cited RESCUE U.S. PATENT DOCUMENTS (75) Inventors: Philipso Dormitzer, Weston, MA (US); 6,455,298 B1* 9/2002 Groner et al. .............. 435/235.1 Björn Keiner, Marburg (DE); Pirada 2008/O124803 A1 5/2008 Billeter et al. Suphaphiphat, Brookline, MA (US); Michael Franti, Quebec (CA); Peter FOREIGN PATENT DOCUMENTS Mason, Sommerville, MA (US); WO WO-2003/091401 4/2003 ennifer Uhlendorff, Neuss (DE): WO WO-2005/062820 7/2005 Mikhail Matrosovich, Marburg (DE) WO WO-2007/047459 7/2005 WO WO-2010/046335 4/2010 (73) Assignee: Novartis AG, Basel (CH) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer the term of this Wang et al. Cloning of the canine RNA polymerase I promoter and patent is extended or adjusted under 35 establishment of reverse genetics for influenza A and B in MDCK U.S.C. 154(b) by 17 days. cells. Virology Journal 2007, vol. 4, p. 102-113.* Nicolson et al. Generation of influenza vaccine viruses on Vero cells (21) Appl. No.: 13/503,353 by reverse genetics: an H5N1 candidate vaccine strain produced under a quality system. Vaccine 2005, vol. 23, pp. 2943-2952.* Koudstaal et al. (Apr. 28, 2009). "Suitability of PerC6 cells to gen (22) PCT Filed: Oct. 20, 2010 erate epidemic and pandemic influenza vaccine strains by reverse genetics.” Vaccine 27(19):2588-93. (86). PCT No.: PCT/B2010/054752 Neumann (2003). “Reverse genetics for the control of avian influ enza.” Avian Dis. 47(3 Suppl):882-7. S371 (c)(1), Suphaphiphatet al. (Apr. 2010). “Human RNA Polymerase I-Driven (2), (4) Date: Oct. 11, 2012 Reverse Genetics for Influenza A Virus in Canine Cells.' J. Virol. vol. 84 No. 73721-3725. (87) PCT Pub. No.: WO2011/048560 Whiteleyetal. (Jul. 2007). "Generation of candidate human influenza vaccine strains in cell culture—rehearsing the European response to PCT Pub. Date: Apr. 28, 2011 an H7N1 pandemic threat.” Influenza. Other RespiViruses. 1(4): 157 66. (65) Prior Publication Data Zhang et al. (Sep. 2009). "A one-plasmid system to generate influ enza virus in cultured chicken cells for potential use in influenza US 2013/0034582 A1 Feb. 7, 2013 vaccine.” JVirol. 83(18):9296-303. International Search Report mailed on Feb. 21, 2012, for Interna tional Patent Application No. PCT/IB2010/054752, filed on Oct. 20, Related U.S. ApplicationO O Data International2010. Preliminary Report on Patentability mailed on Jul. 27. (60) Provisional application No. 61/279.487, filed on Oct. 2012, for International Patent Application No. PCT/IB2010/054752, 20, 2009. filed on Oct. 20, 2010. s Xuan, Bai (2008). “The Construction of Rotavirus Group a Reassort ment strain by Reverse Genetics.” China Master's Thesis Full Text (51) Int. Cl. Database, No. 12. Available online at <http://www.dissertationtopic. CI2N 7/00 (2006.01) net? doc/1470 105>. CI2N 7/02 (2006.01) A6 IK39/45 (2006.01) * cited by examiner (52) U.S. Cl. CPC ........ CI2N 7/00 (2013.01); C12N 2760/16151 Primary Examiner — Louise Humphrey (2013.01) (74) Attorney, Agent, or Firm — Atsuko Polzin; Otis USPC ...................... 435/239; 435/235.1; 424/206.1 Littlefield (58) Field of Classification Search CPC ........... C12N 7/00; C12N 2760/16151; C12N (57) ABSTRACT 2760/16251; C12N 2760/16152; C12N A method for rescuing a virus by reverse genetics is provided 2760/16252; C12N 2760/16134; C12N in which cells are added after transfection. 2760/16234: A61K 39/145 See application file for complete search history. 8 Claims, 4 Drawing Sheets U.S. Patent Nov. 11, 2014 Sheet 1 of 4 US 8,883,481 B2 FGURE O ?ae?***…×××××××××××××××, %. U.S. Patent Nov. 11, 2014 Sheet 2 of 4 US 8,883,481 B2 FGURE 2 ---------------------------|90+=00|| –––.Z0+='00"| U.S. Patent Nov. 11, 2014 Sheet 3 of 4 US 8,883,481 B2 FIGURE 3 U.S. Patent Nov. 11, 2014 Sheet 4 of 4 US 8,883,481 B2 FGURE 4 ------------'-- - - - - - - - - - - - --------------------------------------------------.l . YY YYYYYYYYYYYYYYYYYY YYYYYY 33YYYX Y33. US 8,883,481 B2 1. 2 REVERSE GENETCS METHODS FOR VIRUS mixture of cells; (iii) culturing the mixture of cells in order to RESCUE produce virus; (iv) purifying the virus obtained in step (iii) and optionally (V) formulating the virus into a vaccine. CROSS REFERENCE TO RELATED Reverse Genetics APPLICATIONS Reverse genetics can be used for the production of a wide variety of RNA viruses, including positive-strand RNA This application is a U.S. National Phase patent application viruses 1.2, negative-strand RNA viruses 3,4 and double of PCT/IB2010/054752, filed Oct. 20, 2010, which claims stranded RNA viruses 5. Thus, the present invention pro priority to U.S. provisional patent application Ser. No. vides a method for producing a recombinant virus wherein 10 the virus is obtained using the reverse genetics method of the 61/279,487 filed Oct. 20, 2009, all of which are hereby incor present invention. porated by reference in the present disclosure in their entirety. Known reverse genetics systems involve expressing DNA SUBMISSION OF SEQUENCE LISTING AS molecules which encode desired viral. RNA (VRNA) mol ASCII TEXT FILE ecules from pol I promoters, bacterial RNA polymerase pro 15 moters, bacteriophage polymerase promoters, etc. Further The content of the following submission on ASCII text file more, where a virus requires certain proteins to form an is incorporated herein by reference in its entirety: a computer infectious virus, the systems also provide these proteins e.g. readable form (CRF) of the Sequence Listing (file name: the system further comprises DNA molecules that encode 223.002124600SeqList.txt, date recorded: Apr. 13, 2012, size: viral proteins such that expression of both types of DNA leads 18 KB). to assembly of a complete infectious virus. Where reverse genetics is used for the expression of vRNA, TECHNICAL FIELD it will be evident to the person skilled in the art that precise spacing of the sequence elements with reference to each other This invention is in the field of reverse genetics. Further is pivotal for the polymerase to initiate replication. It is there more, it relates to manufacturing vaccines for protecting 25 fore important that the DNA molecule encoding the viral against various viruses. RNA is positioned correctly between the pol I promoter and the termination sequence, but this positioning is well within BACKGROUND ART the capabilities of those who work with reverse genetics sys temS. Reverse genetics permits the recombinant expression and 30 Generally, reverse genetics is suitable for expression of any manipulation of RNA viruses in cell culture. It is a powerful viruses which are known to require production of genomic tool in Virology and vaccine manufacture because it allows RNA during their life-cycle. Such viruses include, but are not rapid production of recombinant viruses (including reassor limited to, positive-strand and negative-strand RNA viruses, tants) and/or their mutation. The method involves transfect such as those described below. Preferably, the virus is an ing host cells with one or more expression constructs that 35 orthomyxovirus, e.g., an influenza virus. The methods of the encode the viral genome and isolating the virus from the cells. invention are further suitable for non-segmented as well as One drawback with virus rescue by reverse genetics is that segmented viruses. the process is inefficient and frequently results in an unsatis Where the virus is a positive-strand RNA virus it is often factory virus yield. Thus there remains a need in the art to Sufficient to transfect a cell with an expression construct provide alternative and more efficient reverse genetics meth 40 comprising the viral genome. For example, the transfection of ods. plasmids containing the poliovirus genome resulted in the recovery of infectious poliovirus 1.2. Reverse genetics for SUMMARY OF PREFERREDEMBODIMENTS negative-strand RNA viruses has presented more challenges as the antisense viral RNA is usually non-infective and The inventors have now surprisingly discovered that the 45 requires an RNA polymerase to complete the life cycle. Thus, poor efficiency of reverse genetics methods can be improved the viral polymerase must be supplied, either as protein or as significantly by transfecting cells with the reverse genetics a gene for in situ protein expression. construct(s) and Subsequently adding more cells to the trans Where the virus requires a protein for infectivity, it is fected cells. The inventors surmise that the observed increase generally preferred to use bi-directional expression con in efficiency is due to the fact that transfection is harmful to 50 structs as this reduces the total number of expression con the cells. Thus, any viruses which are rescued need to expand structs required by the host cell. Thus, the method of the in sick cells or wait until the virus is transferred to healthy invention may utilise at least one bi-directional expression cells. This results in a loss of viable virus. The addition of construct wherein a gene or cDNA is located between an cells to the transfected host cells allows the rescued virus to upstreampol II promoter and a downstream non-endogenous multiply on a healthy cell substrate which increases the virus 55 pol I promoter. Transcription of the gene or cDNA from the yield significantly. pol II promoter produces capped positive-sense viral mRNA In one embodiment, the invention provides a method for which can be translated into a protein, while transcription preparing a virus comprising the steps of (i) transfecting a from the non-endogenous pol I promoter produces negative culture of host cells with at least one expression construct sense VRNA.
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