Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever
1 Tulane University Health Sciences Center, New Orleans, LA, USA
2 Autoimmune Technologies, LLC, New Orleans, LA, USA
3 Corgenix Medical Corporation, Broomfield, CO, USA
4 Tulane University Department of Pathology, New Orleans, LA, USA
5 Vybion, Inc., Ithaca, NY, USA
6 Applied Diagnostics Branch, Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA
7 Viral Therapeutics Branch, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases Diagnostic Systems Division, Fort Detrick, MD, USA
Virology Journal 2010, 7:279 doi:10.1186/1743-422X-7-279Published: 20 October 2010
Additional file 1:
Graphic representations of recombinant constructs, mammalian plasmid vector, and single LASV gene expression. Ai. GPC gene with known domains (SP, signal peptide; GP1, glycoprotein 1; GP2, glycoprotein 2; TM, transmembrane; IC, intracellular; ER, endoplasmic reticulum retention signal). Signal peptidase (SPase) and subtilase SKI-1/S1P cleavage sites are indicated. Seven glycosylation sites on GP1 and 4 on GP2 are indicated by Y. Aii. GPC construct with C-terminal FLAG. Aiii. Nucleoprotein gene displaying putative helicase, RNA binding, WD40, repeated [R] domains, and pre-protein cleavage motif. Aiv. NP with C-terminal 6X-HIS. Av. Z gene displaying myristoylation (myr), cyclin/CDK, nuclear receptor box (NR BOX), RING, and late PTAP and PPPY domains. Avi. Z gene with one glycine-6X-HIS domain inserted at amino acid position +3. Avii. Z gene with C-terminal 6X-HIS. B. Mammalian expression vector pcDNA3.1+_intA was used to generate all expression constructs outlined in these studies. C. LASV NP-3'HIS (lane 1), Z-3'HIS (lane 2), Z-5'glyHIS (lane 3), and GPC (lane 4) gene expression were analyzed by western blot. Ci. Intracellular (C) expression of NP-3'HIS (60 kDa), Z-3'HIS (12 kDa), Z-5'glyHIS (15 kDa), and GPC (72 kDa). In the GPC lane, probed with an α-GP1 mAb, expression of monomeric GP1 was also detected (42 kDa). In culture supernatants (S), NP-3'HIS was not detected (Cii, lane 1). Z-3'HIS was present in supernatants at high levels (Cii, lane 2). Disrupting the myristoylation site on the N-terminus of Z prevented the release of the protein from cells (Cii, lane 3). The soluble GP1 component previously described through expression of GPC [11,12] was detected in supernatants (42 kDa) (Cii, lane 4).
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Additional file 2:
Transfection experiments with combinations of tagged and untagged Z, NP, and GPC constructs. HEK-293T/17 cells were transfected in 6-well plates as outlined in Methods, with combinations of LASV gene constructs. VLP were purified through 20% sucrose cushions and subjected to western blot analysis. Blots were probed with αGP1, αGP2, αFLAG M2, αHIS mAbs, or αNP PAb. Lane designations: 1. Z; 2. Z-3'HIS; 3. Z+GPC+NP; 4. Z+GPC-FLAG+NP; 5. Z-3'HIS+GPC+NP; 6. Z-3'HIS+GPC-FLAG+NP; 7. Z+GPC; 8. Z-3'HIS+GPC; 9. Z+GPC-FLAG; 10. Z-3'HIS+GPC-FLAG; 11. Z+NP; 12. Z-3'HIS+NP. The Z-3'HIS+GPC+NP combination consistently generated the highest VLP yields with corresponding incorporation of all three LASV genes.
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Additional file 3:
DNA fragmentation and MTT cytotoxicity analysis of HEK-293T/17 cells transfected with LASV gene constructs. A. Fragmentation assays were performed by resolving 2 μg of genomic DNA from transfected and untransfected cells on agarose gels. A low molecular weight DNA laddering effect consistent with apoptotic DNA fragmentation was not observed in any of the samples (n = 3). B. MTT cytotoxicity analysis of transfected cells, in 96-well format (n = 3). Vector only (pcDNA3.1+:intA), NP, GPC, and GPC-FLAG transfected cells did not display significant cytotoxicity when compared to untransfected controls (293T/17 cell ctrl) [p > 0.05]. Conversely, inclusion of the Z matrix gene, in native (Z) or 3'HIS-tagged format (Z-3'HIS), alone or in combination with any version of LASV GPC and/or NP resulted in significant reduction in MTT incorporation levels [p < 0.05 to p < 0.001, n = 3]. The numbered gel lanes in A. correspond to the bars in B. The p value for each transfection condition compared to the 293T/17 cell control is shown above the corresponding lane.
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