met het GGO bij contact met de intacte huid en dat dit tot een locale reactie zal leiden (A4.3).

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4 met het GGO bij contact met de intacte huid en dat dit tot een locale reactie zal leiden (A4.3). - Onder A4.14 geeft u dat verbanden die 24 uur na vaccinatie door de proefpersoon zelf worden verwijderd buiten het ziekenhuis worden gedesinfecteerd met alcohol en in een plastic zak worden gedaan. Het is onduidelijk of dit afval vervolgens door het ziekenhuis (als ML-I afval) wordt afgevoerd of door de proefpersonen zelf wordt afgevoerd. In het tweede geval dient de methode van desinfectie nader te worden beschreven en de effectiviteit van de desinfectie nader te worden onderbouwd. - U geeft in appendix 1, onder punt 3 dat MVA niet in het genoom van gastheercellen kan integreren. U dient dit kort te onderbouwen op basis van relevante literatuur. Ik stel u in de gelegenheid om de aanvraag tot aan te vullen met de thans ontbrekende gegevens. Indien u de gevraagde gegevens niet voor deze datum kunt aanvullen, dient u dit schriftelijk met redenen omkleed binnen 14 dagen mede te delen. Hierbij kunt u een voorstel doen voor een andere datum. In verband met het ontbreken van de benodigde gegevens wordt op grond van artikel 4:15 van de Algemene wet bestuursrecht de wettelijke beslistermijn opgeschort met ingang van de dag na die waarop deze brief is verzonden. De beslistermijn wordt opgeschort tot de dag waarop de aanvraag is aangevuld. Ik maak u erop attent dat op grond van artikel 4:5 van de Algemene wet bestuursrecht uw aanvraag buiten behandeling kan worden gelaten, indien de gevraagde gegevens niet voor de hierboven vermelde datum zijn ontvangen. Hoogachtend, dr. H.C.M. van den Akker, Bureau GGO Ministerie van IenM PorM/RB IM /00 Pagina 3 / 3

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20 13 competent in mast avian and mammalian cells.[3, 20] Compared to CVA, MVA has lost 3lkb 6 passage 583 (kindly provided by Prof. G. Sutter, LMU Munich) under serum-free conditions. 7 The MVA-F6 itself was clonally isolated in CEF cells (at the 58O passage) from MVA material to the Bavarian State Vaccine Institute in MVA itself was originally derived by 12 vaccinia virus Ankara (MVA) at passage 516.[18-19] In contrast to MVA, CVA is replication 11 Ankara) in CEF until the virus developed an attenuated phenol:ype and was named modified 10 long term serial culture of conventional vaccinia virus CVA (Chorioallantoic vaccinia virus 8 passage on CEF ceils, a virus preparation which has been provided as standard seeding 4 3 A2.7. Describe the parental vector. 5 MVA-F6-sfMR was obtained by serial passaging of the original MVA-F6 clonal isolate, CEF 11 L 34 pathogenicity category of the parental vector been changed in respect 35 of the pathogenicity category of the parental virus? 38 mammals Between the deletion III flanking region 1 and deletion III flanking region 2 the following RNA polymerase complex.{22] It is thus only expressed in poxvirus-infected cells under 50 nature and t is not recognized by mammalian RNA pol II (RNA polymerase II). 40 A2.9. Describe the structural genes and regulatory sequences present in the regulatory sequences and structural genes are present: 46 Promoter: psynhi (synthetic promoter II), a short synthetic promoter that was specially 47 designed for early-late expression of genes under the transcriptional control of the pox-viral 49 control of poxvirus-derived polymerases. The promoter thus does not occur in poxviruses in 41 vector and in the DNA inserted in the vector No, the virus was and is still replication deficient and not pathogenic in humans and,-. 33 A2.8. Have any characteristics of the parental vector that are critical for the VI: Deletion sites; Flank.1: deletion III flanking region 1; Flank 2: deletion III flanking Figure 2 Genome of MVA-F6-sfMR characterized deletion sites.{3, 21] These genetic alterations have rendered the virus 18 be used as a control vaccine in the MVA-HA clinical trials. 31 region 2 REGION REGION TERMINAL CENTRAL CONSERVED REGION TERMINAL LEFT RIGHT H I VII VI III 14 ( 45%) of its genetic material, divided over six large and multiple small but fully 16 replication deficient and because of the nature and size of the exerts, the virus cannot 17 revert into the old replication competent phenotype. This parental vector MVA-F6-sfMR will 21 Flank 1 Flank The genetically modified viral vector

21 6 are fillers) 7-9 hemaggiutinin protein of one of the following subtypes: Hi, H2, H3, H4, H5, H6, H7, H8, 13 gene sequence deletion 3.pdf). 12 promoter and location of the recombinant gene is provided as a separate file (see MVA-HA 11 recombinant MVA-HA virus. The sequence of the deletion III site including the stuffers, 10 H9, HiO, Hil, H12, H13, H14, H15, H16. Only one HA gene will be incorporated per 4 Stuffers (non-structural sites (not expressed in infected cefls)): 5-8 Structural gene: the coding region of the HA gene coding for influenza A virus MCC Restriction sites: NoU and HpaI (A + KOZAK sequence which is beneficial for translation whereas the two C s C) the ceils may lead to complementation or recombination The viral parental vector is cultivated in chicken embryo fibroblasts (CEF cells) that are 30 isolated from embryonated eggs of selected SPF chicken flocks (such eggs are used also for 32 before they are used to cultivate the virus. The genetic material that is present in these modified organism, also stating any helper sequences that are present Figure 2 Genome of MVA-based influenza vaccine vector VI: Deletion sites; Flank 1: deletion III flanking region 1; gene: influenza virus 49 hemagglutinin gene; promoter; psynhipromoter; Flank 2: deletion III flanking region 2; REGION 23 the recombinant MVA-HA virus. 25 A2.1O. Describe the origin of the celisiceli lines in which the viral parental 16 virus. DNA coding for an HA subtype can be used as a vaccine. This form of vaccination 18 studies with such vaccines have demonstrated that DNA encoding hemagglutinin is safe, 19 weli-tolerated and immunogenic, irrespective of the HA subtype that the DNA encodes.[ ] 22 Conclusively, selection of the different HA subtypes does not alter the risk-assessment of vector is cultivated, indicating which genetic components present in REGION TERMINAL LEFT 1 1 V II A2.11. Summarise the information in a diagram (map) of the genetically genes and the HA proteins they encode are harmless outside the context of an influenza 31 the production of most licensed seasonal influenza virus vaccines). CEF are passaged once 14 The different HA subtypes are all found in viruses that originate from water fowl. The HA 17 demonstrates the effect of HA expression outside the context of an influenza virus. Clinical 33 celis does not lead to interaction with the parental vector. CENTRAL CONSERVED REGION TERMINAL RIGHT rj [ III Iv Flank 1 stuffer N ioter 3 1 There is no sequence homology of the psynli promoter with poxvirus sequences or other 2 known viral sequences.

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31 MVA-F6-sfMR Visit MVA-F6-sfMR Visit 2 V V 1 MVA-HA-sfMR / Primary MVA-HA-sfMR / Primary months vaccination wks wks wks wks wks Strategy Vaccination Prior to lst wks 6-12 wks or Time points.[8] Furthermore the MVA infection is self-limiting, after the virus enters the celi, viral 35 against influenza and MVA virus and isolation of PBMCs to test T cell immunity against 38 4 Wks= weeks 25 The data obtained in macaques are confirmed by data from a clinical trial described by 28 Rochlitz et al found that after intramuscular immunization no viral DNA and no MVA virus 32 immunization contains the GMO. 34 The samples will be used to perform Clinical Chemistry analysis, serology for antibodies 36 influenza and MVA virus antigens. Since it is highly unlikely that these samples contain the 37 GMO they can be handled without IG-restrictions. 10 syringe is filled with the MVA-based vaccine from the vial and subsequently it is 12 immunization the injection site will be cleaned with 70% ethanol and covered with a 9 well. The infection site will be disinfected with 70% ethanol two times. Subsequently, a 14 to stay on for at least 24 hours subjects. Conclusively, it highly unlikely that the blood from the test subject 3-4 weeks after 22 detected only up to day 4 after immunization and no live virus was found at any of the time 24 proteins are produced but no new virus particles are formed and thus also not excreted. 26 Rochlitz et al, in which humans were immunized with recombinant MVA with dosages 29 was detected in the blood of immunized subjects on 1 hour after immunization and also not 30 at 8 days after immunization.[32] Virus was also not detected in the urine of the test- 20 weeks after administration of the MVA-based vaccine. It is highly unlikely that these 27 comparable to those that are to be used in the trials described in the current application. 16 A3.8. Are samples taken from the test subjects that do or may contain GMOs, 17 and which tests are carried out with these samples? 19 The only samples that will be taken from the test subjects are blooci samples, earliest samples contain the GMO. In macaques that were immuriized with MVA, only viral DNA was 5 =Blood withdrawal, in the period of weeks after the first vaccination there can be several moments 8 surgery sheets and the area around the injection site will be covered with surgery sheets as 13 waterproof band aid in order to prevent leakage of vaccine preparation. This band aid has week periods. to withdraw blood 6 A3.7. How will the GMO preparation be administered to the test subject? 7 The test subject will be seated in a chair and with one arm free. The arm will rest on Only one vaccination, blood withdrawal and physical examination will occur in each of the 3-4 and Primairy visit: informed consent, blood withdrawal 11 administered intramuscularly via bolus injection in the deltoid region of the upper arm. After V ccination, Blood withdrawal and physical examination (mcl 7day daily diary card) 3 V V MVA-F6-sfMR MVA-HA-sfMR / Primary 2 1 Immunization strategies

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