Engineering Conferences International ECI Digital Archives Vaccine Technology IV Proceedings Spring 5-21-2012 Rabies virus-like particles expressed in HEK293 cells Diego Fontana Cell Culture Laboratory Biochemistry and Biological Sciences School Universidad Nacional del Litoral Santa Fe - Argentina Ricardo Kratje Cell Culture Laboratory Biochemistry and Biological Sciences School Universidad Nacional del Litoral Santa Fe - Argentina Marina Etcheverrigaray Cell Culture Laboratory Biochemistry and Biological Sciences School Universidad Nacional del Litoral Santa Fe - Argentina Claudio Prieto Cell Culture Laboratory Biochemistry and Biological Sciences School Universidad Nacional del Litoral Santa Fe - Argentina Follow this and additional works at: http://dc.engconfintl.org/vaccine_iv Part of the Biomedical Engineering and Bioengineering Commons Recommended Citation Diego Fontana, Ricardo Kratje, Marina Etcheverrigaray, and Claudio Prieto, "Rabies virus-like particles expressed in HEK293 cells" in "Vaccine Technology IV", B. Buckland, University College London, UK; J. Aunins, Janis Biologics, LLC; P. Alves, ITQB/IBET; K. Jansen, Wyeth Vaccine Research Eds, ECI Symposium Series, (2013). http://dc.engconfintl.org/vaccine_iv/14 This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Vaccine Technology IV by an authorized administrator of ECI Digital Archives. For more information, please contact franco@bepress.com.
RABIES VIRUS-LIKE PARTICLES EXPRESSED IN HEK293 CELLS Diego Fontana, Ricardo Kratje, Marina Etcheverrigaray and Claudio Prieto Cell Culture Laboratory Biochemistry and Biological Sciences School Universidad Nacional del Litoral Santa Fe - Argentina May 20-25, 2012
RABIES Rabies is a fatal disease and represents a severe public health problem in developing countries. Without treatment, rabies infection of humans is almost invariably fatal. Schnell et al.. (2010) The World Health Organization estimates 40.000-70.000 deaths of rabies every year.
RABIES Prevention of rabies in humans depends on a combination of interventions, including: Pre-exposure immunization of people at frequent risk of exposure Provision of post-exposure prophylaxis to potentially exposed patients There is a need to develop safer, cheaper and efficacious vaccine against rabies...
RABIES VIRUS (RV) Enveloped virus of the Rhabdoviridae family Released from the infected cell by budding Negative-stranded RNA virus Encodes 5 structural proteins of which Matrix protein (M2) Glycoprotein (G) Central role in virus budding during virus infection (Adapted from Schnell et al., 2010)
Glycoprotein (G) It is an integral transmembrane protein Organized as a trimerin the virus surface It is the major viral antigen responsible for the induction of protective immunity Induce T helper cells (Celis et al., 1986) Induce cytotoxic T cells (Mcfarlane et al., 1986) Production virus neutralizing antibodies (Perrin et al., 1985) G protein posseses an autonomous exocytosis activity InfectiousparticlescontainingG proteinwerefoundin thesupernatantsof infected cells (Mebatsion et al., 1996)
Objectives Construct a VLP expressing in HEK293 cells the rabies virus Gprotein Construct a recombinant cell line that expresses the G protein and evaluate the particle production Express the matrix protein together with the glycoprotein to evaluate whether the presence of this protein improves particle budding Immunize mice with the VLPs and evaluate the immune response triggered
Recombinantcell line development HEK293 cells Lentiviral vector-mediated transduction
Lentiviral vector production gag, pol rev HEK293 LV Stock -80 C Transfection Harvest VSV Glyco G plv-plk-goi pmdl, prev, pmd2.g (Dull et al., 1998; Segall et al. 2003) plv-plk (Prieto et al., 2011) G protein (PV strain) M2 protein (PV strain) DMEM 10% FBS, 5% CO 2, 37 C 48 h Lentivirus Titer (Lentivirus-Associated HIV p24) LV-G: 5x10 7 LP/ml LV-M2: 7x10 7 LP/ml
Recombinantcell line development HEK293 cells Lentiviral vector-mediated transduction LV-G G protein expressing cell line (HEK-G) LV-G + LV-M2 G and M2 expressing cell line (HEK-G/M2)
Recombinant cell line development Analysis of the G protein expression HEK-G HEK-G/M2 Flow cytometry mab against G protein Goat anti-mouse AlexaFluor 488 98% 98% fluorescent cells fluorescent cells Fluorescense microscopy mab against G protein Goat anti-mouse AlexaFluor 488
Recombinant cell line development Analysis of the M2 protein expression RT-PCR
VLPs analysis Analysis of the particles in the supernatant Sandwich ELISA Abs492nm 2,5 2,0 1,5 1,0 Standard HEK-G/M2 HEK-G M2 improves the particle budding? 0,5 1E-3 0,01 0,1 1 10 Dilutions HEK-G
VLPs analysis Concentration Purification 1. Supernatant harvest 2. Clarification 3. Filtrate 0.45µm 4. Ultracentrifugation through 30% sucrose cushion Abs492nm 2,2 2,0 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 Standard Concentrated HEK-G HEK-G 75-fold concentration Western blot G 206 kda 127 kda 89 kda Anti-rabies virus serum 1E-4 1E-3 0,01 0,1 1 10 TEM Dilutions 32 kda 17 kda Diameter 20-50nm
RCL assay Testing for Replication Competent Lentivirus (RCL) RCL? HEK-G p24 ELISA to detect HIV-1 capsid protein (GAG)
RCL assay Testing for Replication Competent Lentivirus (RCL) HEK-G p24 ELISA to detect HIV-1 capsid protein (GAG)
Immunization protocol Female Balb/c mice Intramuscular route Freud incomplete adyuvant 2 dosis (0 and 12 day) Four groups: A. Commercial human vaccine- VERORAB (n=4) B. Commercial animal vaccine- BAGOVAC (n=4) C. 0.3 µg of VLPs(n=5) D. 3 µg of VLPs(n=5) Blood samples collected on day 19 Analyzed by flow cytometry
Immunization protocol TARGET CELLS FOR ANTIBODIES DETECTION IN RABIES VACCINE CONTROL Diego Fontana, Claudio Prieto, Ricardo Kratje and Marina Etcheverrigaray Poster Nº18 Vero-G cell line vs G protein Sera dilutions Vero-G cell line Secondary conjugated antibody incubation FC GUAVA EasyCyte Graph % fluorescent cells Dilutions
Immunization protocol Sera analysis 100 90 80 Veterinary Vaccine Human Vaccine 0.3 ug VLPs 3 ug VLPs ls % Fluorescent Cell 70 60 50 40 30 20 10 0 10 100 1000 10000 100000 1000000 1E7 Dilutions
Conclusion We constructed two recombinant cell lines HEK-G HEK-G/M2 We confirmed the expression of RV proteins by flow cytometry, fluorescence microscopy, RT-PCR and western blot. The co-expression of the M2 protein, in this case, does not increase the amount of VLPs in the supernatant VLPs concentrated by ultracentrifugation were analyzed by TEM
Conclusion We immunized Balb/c mice with VLPs preparations and we compared it with commercial vaccines The collected blood samples were analyzed by flow cytometry The results obtained indicate that this VLPs are immunogenic and capable of inducing specific antibody responses With these results, and with further analysis, these virus-like particles could be proposed as a rabies vaccine candidate
Thank you! Obrigado!
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