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31st Clinical Virology Symposium USE OF RECOMBINANT VIRUS TECHNOLOGY TO PRODUCE A NON-INFECTIOUS, WHOLE PROCESS EBOLA CONTROL Daytona Beach, FL April 26-29, 2015 Catherine E. Huang, Rajeswari Vemula, Cristine Chisholm and Bharathi Anekella SeraCare Life Sciences, Gaithersburg, MD INTRODUCTION RESULTS AND DISCUSSION Stability Studies Performance of AccuPlex rEbola GP/NP Reference Material Cepheid Xpert Ebola Assay BM-20 ˚C 1.00E+05 1.00E+04 BM 4 ˚C Figure 6: Real Time Stability of recombinant Sindbis virus at RT, 4 ˚C and -20 ˚C. 1.00E+03 BM RT (25 ˚C) 1.00E+02 1.00E+01 0 2 4 6 8 10 40 40 40 35 35 CONCLUSIONS 35 NP Lot 1 GP Lot 1 GP Ct 25 NP Ct 30 GP Lot 2 25 20 Ct Value Ct Value 30 30 NP Lot 2 25 NP Lot 3 GP Lot 3 20 20 15 15 15 2 3 4 5 6 7 8 9 10 Run2 Run3 Run4 Run5 Run6 Run1 Run7 Run2 Run3 Run4 Run5 Run6 Run7 Performance runs Performance runs PCR Assays: Ebola reference material was tested using a lab developed TaqMan assay targeting either GP/NP or common viral vector sequence. Testing was also performed using assay developed by Cepheid and RUO assay from PrimerDesign 1.00E+06 Performance on NP TaqMan Assay Performance on GP TaqMan Assay Run1 Figure 4a: Testing of AccuPlex rEbola GP/NP Reference Material on Cepheid Xpert Ebola Assay Ten replicates were tested and the assay targets GP and NP regions. All ten replicates were tested positive and the Ct values were between 28-30. Figure 4b: Inter-assay performance of AccuPlex rEbola GP/NP Reference Material Three validation lots were tested on 7 different runs in triplicate on both GP and NP real time TaqMan assay. Figure 4c: Testing of AccuPlex rEbola GP/NP Reference Material on PrimerDesign assay Ten replicates were tested and the assay targets VP24 (blue lines) and NP (green lines). All ten replicates were tested positive and the Ct values were between 24-25. Positive control Positive control Construct Construct Stress stability was performed on AccuPlex rEbola GP/NP Reference Material at 37 ˚C and multiple freeze thaws to determine a preliminary expiration date. Based on the data, the reference material currently has an expiration of 24 months at 2-8 ˚C (Figure 5a and 5b). Real time stability studies are ongoing. Stability Studies Stability at 37 ˚C Stability - Freeze/Thaw 1.00E+07 1.00E+06 1. C Xiong, R Levis, P Shen, S Schlesinger, CM Rice, and HV Huang. Science 3 March 1989: 243# (4895), 1188-1191. 2. S Altman-Hamamdzic1, C Groseclose1, J-X Ma2, D Hamamdzic3, NS Vrindavanam1, LD Middaugh1, NP Parratto1 and FR Sallee. Gene Therapy (1997) 4, 815–822. 3. Schlesinger S. 2000. Alphavirus expression vectors. Adv Virus Res.;55:565-77. 4. Sofer G. 2003. Virus Inactivation in the 1990s —and into the 21st Century Part 4, Culture Media, Biotechnology Products, and Vaccines. BioPharm International. January: pp50-57. 1.00E+06 Copies/mL Additional real time studies were performed on a different recombinant virus at room temperature, 4 ˚C and -20 ˚C (Figure 6). No downward trends were detected across the nine (9) months of storage, even for samples stored at ambient temperatures. This demonstrates that the viral coat proteins form a stable protective barrier that prevents nucleases in complex clinical matrices such as plasma from degrading the target RNA sequence. 1.00E+05 1.00E+05 1.00E+04 1.00E+04 NS 1D 3D 5D 11D 22D SeraCare developed stable, well-characterized Ebola positive reference material using recombinant technology The recombinant genome size is comparable to many human pathogenic RNA viruses so offer a similar secondary structural complexity to what is found in the patient specimens. AccuPlex recombinant Ebola GP/NP reference material is replication defective and non-infectious to humans to ensure added safety for laboratory staff. AccuPlex rEbola GP/NP Reference Material is a multiplex control and designed to contain one recombinant virus bearing a portion of the GP gene and a second recombinant virus bearing a portion of the NP and VP24 genes. The virus is enveloped and must go through an extraction process prior to RNA detection. This makes the reference material a more efficient whole process control than plasmid or other biosynthetic material because it better mimics patient samples. This reference material provides means for monitoring all steps of PCR assays and are useful for assay validation and as run controls. The SeraCare AccuPlex rEbola GP/NP Reference Material shows consistent performance on all tested platforms. AccuPlex rEbola GP/NP Reference Material is formulated in defibrinated plasma allowing at least 9 months of stability at room temperature. This corresponds to more than 2 years of stability at 2-8 ºC when using conservative models. REFERENCES 1.00E+07 Copies/mL Stability Studies: Performance studies were conducted on 3 Ebola validation lots over multiple days using TaqMan assay targeted to GP and NP region. Stress stability studies were conducted on one lot of Ebola product by subjecting it to freeze thaw cycles and holding the samples at 37 ˚C for extended periods to simulate adverse shipment and storage conditions. Real Time Stability studies on Ebola reference material is ongoing. Here we present real time stability data for another recombinant Sindbis virus that has been formulated in a similar matrix and stored at ambient room temperature (approximately 25 ˚C). Vials were tested periodically over 9 months using a laboratory developed TaqMan real time PCR test. 1.00E+07 Storage Duration In Months Number of datapoints Design and Development of Recombinant Ebola Virus: synthetic constructs were designed targeting glycoprotein gene (GP), nucleoprotein gene (NP) and VP24 of Ebola Zaire isolate H. sapienswt/GIN/2014/Gueckedou-C05; GenBank accession number KJ660348.2. The capped RNA are introduced into baby hamster kidney cells. The recombinant virus was cultured and purified from the cell debris. The purified virus was heat inactivated as a further safety precaution4. After titering the viruses using a laboratory developed TaqMan PCR assay, the viruses were combined and diluted into defibrinated human plasma and 0.09% sodium azide as a preservative. Viral Load (copies/mL) SeraCare developed multiplex recombinant reference material, AccuPlex rEbola GP/NP Reference Material, that targets the GP/NP regions. The reference material is formulated in defibrinated plasma with a fill volume of 250µL and can be stored at 2-8 ˚C. The Limit of Detection (LOD) was estimated for 3 PCR assays, Cepheid Xpert® Ebola assay, PrimerDesign assay and in-house developed TaqMan assay. Based on the LOD a low positive process control was manufactured (Figure 4). Performance studies were conducted over multiple days using multiple operators. The %CV for AccuPlex rEbola GP/NP Reference Material on all 3 assays was <2% 1 MATERIALS AND METHODS Real Time Stability 1.00E+08 Ct Value Outbreaks of new viral communicable disease and emergence of new viral strains, such as the recent outbreak of Ebola 2014 strain in Guinea, can represent public health emergencies. This outbreak mobilized the IVD laboratories to develop rapid and sensitive PCR assays to help diagnose and treat those infected while helping contain the rapid spread. As the test developers design, manufacture and validate diagnostic assays to prepare for these threats, positive reference materials are needed. SeraCare has developed the AccuPlex™ rEbola GP/NP Reference Material using recombinant virus technology. The use of recombinant virus, containing target sequences from Ebola virus has many advantages as a NAT quality control material. First, it mimics clinical samples because it undergoes the entire extraction procedure. Second, the product is non-infectious and ensures biological safety for lab personnel. Recombinant virus vector has deletion of about 1/3 of genome, which makes it incompetent to replicate in human or animal host. SeraCare’s AccuPlex rEbola GP/NP Reference Material was made by replacing structural proteins required for replication with selected Ebola target sequences. Finally, recombinant viruses have an extended stability at 2-8 ˚C; control materials do not require freezer storage. RESULTS AND DISCUSSION NS 1 F/T 2 F/T 3 F/T ACKNOWLEDGMENTS Figure 5a: Stability of AccuPlex rEbola GP/NP Reference Material across multiple freeze thaw cycles Figure 5b: Stability of AccuPlex rEbola GP/NP Reference at 37 ˚C The authors are most grateful to Dr. Powell at PrimerDesign for testing the samples and to Medha Kulkarni at Cepheid for testing the samples on Xpert Ebola Assay.