MVA Flu. Figuur 1: A: Middels homologe recombinatie (op basis van de homologie van de Del III flanks in het MVA

Transcriptie

1

2

3

4 Figuur 1: A: Middels homologe recombinatie (op basis van de homologie van de Del III flanks in het MVA genoom en het plasmide) in CEF cellen wordt in de geïnfecteerde+getransfecteerde cel de regio tussen de Del III flank 2 en Del III flank 1 ingebouwd in het genoom van nieuwe MVA virions die door de cel worden gemaakt. Dit resulteert in een gemixte virus populatie van normaal MVA virus en recombinant MVA virus. Vervolgens wordt deze mix specifiek gezuiverd voor het recombinant MVA virus op basis van de marker (positieve selectie). Dit resulteert in een zuivere populatie van recombinant virus. MVA Flu Figuur 2: Omdat in het uiteindelijke vaccin virus de marker niet nodig is wordt vervolgens het recombinant virus een aantal maal gepasseerd met selectie voor virus dat de marker niet meer heeft (negatieve selectie op basis van cytopathologisch effect zonder marker expressie. Dit is mogelijk omdat door de repeat van een deel van Del III flank 1, de tussenliggende regio middels homologe recombinatie uit het genoom wordt gerecombineerd (2), wat resulteert recombinant MVA virus zonder marker. Dit is het uiteindelijke MVA flu (3), het restmateriaal wordt afgebroken.

5 Hieronder de uitgebreide beschrijving zoals deze is terug te vinden in een voorbeeld IMPD van een MVA flu vaccin. 2.1.S.2.2 Description of Manufacturing Process and Process Controls The HA gene from influenza virus A was ordered as a synthetic gene from Geneart (Regensburg, Germany). In the synthetic sequence the gene was preceded by a sequence encoding for synthetic promotor II (psynii) and flanked by BamHI and NotI restriction sites. By restriction digest with BamHI and NotI the synthesized insert (psynii + HA) was cut out of the manufacturer plasmid. The pmkiii MVA shuttle vector (encoding an antibiotic resistance gene (for growth in bacterial cells) and mcherry (under control of a vaccinia promotor thus usable as a selection marker for recombinant MVA)) was also digested with BamHI and NotI. The insert was ligated into the vector using T4 ligase and subsequently the plasmid DNA was transformed into competent cells. Single colonies from the transformation were grown in 3ml cultures and DNA was isolated to perform sequence analysis and restriction analysis. The presence of the insert in the shuttle vector was confirmed by PCR, restriction analysis, sequence identity was verified and the DNA was amplified to obtain a master stock (pmkiii psynii HA) to be used for transfection of MVAinfected cells. To obtain a serum free batch of the MVA vector backbone the MVA F6 isolate was passaged 6 times on Chicken Embryo Fibroblasts (CEF) under serum free conditions using nothing but Virus production medium (VP SFM, ref 11681, GIBCO, Life Technologies), resulting in the seed stock of the empty vector: MVA F6 sfmr. In order to produce the MVA HA sfmr virus, CEF cells were infected with the MVA F6 sfmr virus and subsequently transfected with the pmkiii psynii HA DNA. Because of the flanking regions present in the shuttle vector that overlap with the deletion site III flanking regions in the MVA genome, the psynii HA and vaccinia promotor mcherry are incoorperated in the MVA genome through homologous recombination. Subsequently the transfection/infection mix was harvested after an incubation period of two days and titrated on CEF cells. Individual red fluorescent virus plaques were picked and passaged on CEF cells via limiting dilution. This was repeated until the virus preparation was pure recombinant MVA HA and was free of empty vector (checked by PCR with primers that are specific for the HA gene and the deletion III flanking regions). After this positive selection based on mcherry expression, serial passaging of this recombinant virus was performed to lose the mcherry gene (also by homologous recombination due to a small repeat of the deletion III flanking region 1 in front of the mcherry gene). After this negative selection in which non fluorescent virus plaques were picked, the endproduct was a pure MVA HA preparation. This virus preparation was tested with the deletion III and HA specific PCR and MVA and HA expression were confirmed with monoclonal antibodies against Vaccinia and HA by immunocytochemistry and in cell lysates of CEF cells infected with the respective virus. Once the identity of the virus was confirmed the seed stock was amplified to obtain the master seed stock (MVA HA sfmr) of which again the identity was confirmed. Furthermore general sterility (blood agar smear) testing and Mycoplasma PCR were performed on the virus stock and these tests were all negative. Potency was tested by titration of the virus on CEF cells and a growth curve was performed that confirmed similar growth of the MVA HA sfmr virus compared to the MVA F6 sfmr virus. Genetic stability of the virus was tested by 5 low MOI (multiplicity of infection) passages of the virus on CEF cells. The virus from the final passage was characterized by an identity PCR based on primer sites, specifically designed for the six deletion sites. Purity of the virus preparation (>95% recombinant virus) was tested by double staining of MVA HA sfmr infected CEF cells for vaccinia and HA protein expression with specific monoclonal antibodies.

6 This master seed virus: MVA HA sfmr is used as seed virus by IDT to produce the actual clinical batch of the vaccine. This will be done via a multi step growth process on chicken embryo fibroblasts that have been isolated from SPF eggs, resulting in an endvolume of 100 liter. Samples are taken from the master seed virus and working seed virus to determine genetic identitiy and potency. For the final product genetic identity, potency, purity, stability and sterility will be tested by IDT based on the quality requirements that will be described in the quality agreement with IDT. Tabel 1

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44