rologie i - European Congress of Virology

5 th European Congress of VirologyREF 056Evaluation of “In Line” tools for viruses characterization during Vaccineproduction processMarc FIORUCCI 1 , Alexandra BEAUFRÈRE 1 , Cyril PAYA 1 , SteveCRUSSARD 1 , Blandine DE SAINT VIS 1 , Jean REYES 1 , ChristopheGAILLARD 1Merial, Lyon, FRANCEMonitoring the process and optimizing production yields of viruses isa key step for vaccine industry. As a consequence a characterization ofviruses in a real time manner would be an advantage for manufacturers.In this study, three different technics have been evaluated allowing a rapidcharacterization of viruses: 2 virus counters (Flow cytometry and qViro)and a molecular amplification test (qPCR). This study shows that flowcytometry (FCM), with a specific staining, is able to qualitatively characterizeviral particles but relation between FCM titers and infectious titeris not obvious. qViro, is a fast and nonspecific technic based on coulterprinciple allowing a direct counting of viral particle. Although, qVirowas easy to setup and handle, relation between qViro titer and infectioustiter has not been yet demonstrated probably due to the complexity of thesample. Finally, qPCR has been evaluated and a strong relation is shownwith infectious titer. Interestingly qPCR titers were not impacted by thenature of the sample allowing the use of this test throughout the productionprocess.In conclusion, this study describes the ability of new tools to characterizeviruses particles in a real time manner. A bioequivalence has been shownbetween qPCR titre and infectious titer whereas viral counters are moreadapted for qualitative characterization of virus particles.REF 057Obtaining recombinant properly folded neuraminidase NA ofInfluenza Virus using a novel system for stable, high level expressionfrom the T7 promoter in Escherichia coli cellsAgnieszka ROMANIK 1 , Malgorzata KESIK BRODACKA 1 , ViolettaSACZYNSKA 1 , Violetta CECUDA ADAMCZEWSKA 1 , KatarzynaFLORYS 1 , Boguslaw SZEWCZYK 2 , Grazyna PLUCIENNICZAK 1 ,Andrzej PLUCIENNICZAK 11 The Institute of Biotechnology and Antibiotics, Warsaw, POLAND;2 University of Gdansk, Gdansk, POLANDNeuraminidase NA is a second immunodominant glikoprotein of InfluenzaVirus. NA is an enzyme that removes sialic acids from the surface of thecells, hence newly formed virions can be released from infected cells.Production of the recombinant NA (rNA) protein is crucial in developingnew influenza subunit vaccines or as an antigen in NA antibody testsaccording to DIVA (Differentiating Infected from Aaccinated Animals)strategy. Recombinant NA is produced mainly in insect or mammaliancells. Using bacterial expression system to produce this protein is not incommon usage. Main problem in using the prokaryotic expression systemis obtaining of stable recombinant protein with high level of expression.Here we report obtaining of the recombinant NA of avian H5N1Influenza Virus in Escherichia coli cells using a new system for stableexpression of protein from T7 promoter. In order to provide expressionin bacterial cells we deleted first 33 amino acid of NA protein (wholetransmembrane domain) and added six histidine to N terminus of theprotein. The recombinant protein was expressed in bacterial cells as inclusionbodies. We purified and renaturated protein on column packed withNiNTA resin. We received pure, properly folded rNA, which was confirmedby SDS PAGE and ELISA test. rNA protein obtained with describedmethod can be directly used in immunology test and vaccines for influenzavirus.REF 058AIDS vaccine Thai RV 144 correlate of protection: Envelope gp120V2 loop, which induces protective neutralizing IgG antibodies, is amarine Conus mu conotoxin binding to the voltage gated Na+ sodiumchannelGuy Mong Ky TRAN 1,4 , Adrien CAPRANI 2,31 Retired, ex Public Health resident, ex European AIDS ClinicalSociety (EACS) Scientific Council member, Chatenay Malabry, FRANCE;2 Association Positifs, Paris, FRANCE; 3 CNRS Jussieu, Paris, FRANCE;4 Association Positifs.org, Scientific Advisor, Paris, FRANCEThai RV 144 vaccine efficacy is 31,2%; protective IgG target the gp120V1 V2 loops. We analyse the V2 loop (Zolla Pazner S, 2013) by aminoacid (AA) sequences comparison by BLASTP with visual search and threedimensional (3D) structure (Xue T, 2003; Pallaghy PK, 1997). The 2 Thaivaccine strains V2 loops were screened on toxins binding to the voltagegated Na+channel (NaCh). Result: 1) 3 mu conotoxin active site AAs(K13, Q14, K16) (Conus Geographicus, Kinoshitai, Striatus, Betulinuschimera) (Ekberg J, 2008) are found in the Thai V2 loop (V172 crucial):Thai V2 166 RDKKQ KVHALFY R 178conotoxin 10 RDKKQCKVHALCCGR 242) The vaccine MN strain V2 loop mimics the scorpion toxin N terminusactive site (Kharrat R, 1989); its deletion abolishes the toxicity (El AyebM, 1986). Interestingly antibodies against N terminus induce broad crossreactive protection (Devaux C, 1999). The toxin precursor (Cn7, AaH, BotIX chimera) (Possani LD, 2000) is included.MN strain 157 CSFQMTGLEDKVKKEYALLYK 178Scorpion 13 CLFMTGVEAEIKVKKEGYALQYK 103) V2/V3 loops of HIV 2/SIV PBJ14 (fatal AIDS) were 3D superimposedon spider atratoxin (Atx)/versustoxin, 2 NaCh ligands. V2YxxxWYxxDxxC is conserved in HIV 2. V3 is SGLVFH: Atx 4 KR MKY AWYNQQ C TGLFKKC 42HIV 2 KK MK Y AWY QD C SGLVFHThe scorpion venom concept of AIDS (Tran GMK,1989) is confirmed bythe homology between the Thai V2 loop and mu conotoxin, a NaCh ligand.Omega 3, a NaCh modifier, is efficient in AIDS (Caprani A, 2012). AIDSvaccine should target V2/V3, avoid mitigating IgA (Haynes BF, 2012) andadd Mannose vaccine. ACK: No.REF 059Thai AIDS vaccine RV 144: Molecular homology between HIV 1 gp120 envelope first conserved region C1, which induces IgA antibodiesincreasing AIDS risk, and the complement receptor CR1Guy Mong Ky TRAN 1,4 , Adrien CAPRANI 2,31 Retired, ex Public Health resident, ex European AIDS Clinical Society(EACS) Scientific Council, 31 Av du Bois, 92290 Chatenay Malabry,FRANCE; 2 CNRS Jussieu, Paris, FRANCE; 3 Association Positifs, Paris,FRANCE; 4 Association Positifs, Scientific Advisor, Paris, FRANCEThai RV 144 vaccine, despite gp120 V1/V2 loops neutralizing IgG, has alow 31% efficacy, mitigated by IgA antibodies against the 1st conservedregion C1 (odds ratio 3.15) (Haynes BF, 2012). We analyse the biologicalsignificance of C1 by BLASTP and visual amino acid (AA) sequencescomparison: We screened C1 of the vaccine 2 Thai strains and all HIV1 strains (Los Alamos, Kuiken C, 2012) on Homo Sapiens and founda molecular homology between C1 (112 122) and complement receptorCR1 (2089 2099):C1 WDQSLKPCVKL, C1 WGPKLKPCVKL (strain37 cpx) (Powell RL, 2007), CR1 WGPKL(H,P)CSRV.The tip of the CR1 loop (Proline P2095) is in retro inverso: 2096 (H,P)2095, instead of 2095 PH 2096. Such tip inversion exists: EpidermalGrowth Factor (EGF) and Transforming Growth Factor alpha (TGF a) bindS134 Virologie, Vol 17, supplément 2, septembre 2013