A VLP vaccine for epidemic Chikungunya virus protects non-human primates against infection

Transcription

1 A VLP vaccine for epidemic Chikungunya virus protects non-human primates against infection Wataru Akahata, Zhi-yong Yang, Hanne Andersen, Siyang Sun, Heather A. Holdaway, Wing-Pui Kong, Mark G. Lewis, Stephen Higgs, Michael G. Rossmann, Srinivas Rao and Gary J. Nabel SUPPLEMENTARY METHODS SUPPLEMENTARY FIGURES

2 SUPPLEMENTARY METHODS Transfection of CHIKV C-E and E vector. To confirm expression of CHIKV C-E and E proteins, we transfected 293T cells using a FuGENE TM 6 Transfection Reagent kit (Roche Diagnostics GmbH) with 3 µg of the plasmid DNAs, following the manufacturer s recommendations. Cell culture. We cultured 293T and 293A (human embryonic kidney cells), Vero (African green monkey kidney epithelial cells), HeLa (human cervical adenocarcinoma) and BHK (baby hamster kidney cells) in Dulbecco s modified Eagle s medium (DMEM; GIBCO BRL) containing 10% heat-inactivated fetal bovine serum (FBS) (GIBCO BRL). We grew the 293-derived suspension cell line 293F (Invitrogen) in FreeStyle TM 293 Expression medium (Invitrogen). Production of pseudotyped lentiviral vectors. 2 µg of vesicular stomatitis virus glycoprotein (VSV-G), 2 µg of pngvl-4070a amphotropic MuLV gp70 expression vector or 500 ng of empty vector served as positive and negative controls for these pseudotyped reporters respectively. After calcium phosphate transfection (Invitrogen) overnight, we replenished the culture media. 48 hours later, we harvested supernatants, filtered them through a 0.45 µm syringe filter, stored them in aliquots, and froze them at 80ºC. We standardized the viruses by the amount of HIV-1 Gag p h after transfection, we harvested CHIKV pseudotyped lentiviral vectors and normalized them according to HIV-1 Gag p24 levels before infection, as previously described 1. Virus preparation. We prepared CHIKV (strain LR2006 OPY-1) and determined the virus titers as previously described 2. Briefly, we transfected viral RNA transcribed from plasmid

3 CHIK-LR ic into BHK-21 cells by electroporation. We aliqotted the supernatants from the transfected cells, titrated the stock virus, and determined tissue culture infectious dose 50% (TCID 50 ) endpoint titers using Vero cells. To produce virus for vertebrate challenge, we infected C6/36 (Aedes albopictus) cells grown to confluence in T150 flasks with stock virus at a multiplicity of infection of Supernatants were collected 48 hrs after infection, aliquotted, and titrated to determine TCID 50 endpoint titers on Vero cells. Buoyant density gradient sedimentation analysis and purification of VLPs. We harvested the supernatants 72 h after transfection of C-E plasmid and filtered them through a 0.45 μm pore size filter, then layered them onto a 60% Optiprep (Iodixanol) medium (Invitrogen) and centrifuged them at 50,000 x g for 1.5 h with a Surespin 630 rotor (Sorvall). We removed the supernatants to leave 4 ml above the virus band and mixed them to a 20% final concentration of OptiPrep. Next, we centrifuged them at 360,000 x g for 3.5 hr with an NVT100 rotor (Beckman) to form a density gradient. After the centrifuge, we collected, weighed, and plotted the densities of 500 µl of each fraction. Total VLP concentration was measured with the Bradford method (Bio-Rad) following the manufacturer s recommendations. We separated 20 µl of each fraction on a 4% 15% SDS-PAGE gel, transferred them onto an Immobilon-P membrane, and blotted them with sera from mice injected with a readily available, established CHIKV strain S-27 (ATCC: VR-1241AF TM ) and goat anti-mouse immunoglobulins linked to horseradish peroxidase (Santa Cruz Biotechnology). Neutralization of CHIKV E pseudotyped lentiviral vectors by mouse and monkey antisera. We performed the neutralization assay as described previously 1.One day prior to infection, we plated a total of A cells into each well of a 96-well dish. We first titrated CHIKV E-pseudotyped lentiviral vectors encoding luciferase by 4-fold serial dilution starting at

4 1:50 serum dilution. We then incubated similar amounts of pseudotyped lentiviral vectors (with p24 levels of approximately 50 ng ml 1 ) with the indicated dilutions of mouse antisera for 60 minutes at room temperature prior to adding the virus:sera solution to 293A cells (10 4 cells per well in a 96-well dish, 50 µl per well, in triplicate). Sera from non-immune mice or monkeys served as a negative control. After a 24 hour incubation, we lysed cells using cell lysis buffer (Cell Signal) and measured luciferase activity using Microbeta JET (PerkinElmer) following incubation with Luciferase assay reagent (Promega), according to the manufacturer s protocol. All the experiments were performed in triplicate. We independently repeated the assays for each figure at least three times. We calculated inhibition values as follows: inhibition (%) = [1 (luciferase activity (cps) in pseudotyped lentiviral vector infected cells incubated with the indicated dilutions of mouse antisera) / (luciferase activity (cps) in pseudotyped lentiviral vector infected cells incubated with the same dilutions of non-immune mouse serum)] 100. We calculated the IC 50 with Prism 5 software. Electron microscopy. The Image Analysis Laboratory at the National Cancer Institute examined the morphology of the VLPs. We purified VLPs by Optiprep density centrifugation and then fixed them in 4% formaldehyde in PBS. Negative-stain electron microscopy for viral diagnosis has been described previously 3. Briefly, we placed 1.0 µl of the sample onto a carboncoated Formvar-filmed copper grid (Tousimis Research Corp.) and allowed the VLPs to attach. We then added 2 µl of 1% PTA solution (phosphotungstic acid, ph 7.0) (Fisher Scientific) to negatively stain the VLPs, and examined the grid by electron microscope (Hitachi H7000) operated at 75 kv. We captured digital images with a CCD camera (AMT). Plaque assays. We tested serum samples for CHIKV neutralizing antibody using a standard plaque reduction neutralization test (PRNT). Briefly, we heat-inactivated monkey sera

5 at 56 C for 30 minutes and diluted them in virus diluent (PBS/5% BSA). The serum dilutions were 1:100, 1:1000, 1:10,000 and 1:100,000, since at higher concentrations we observed cell toxicity. We mixed diluted serum samples with an equal volume of 40 PFU CHIKV (strain LR2006 OPY-1) and incubated them for 1 hr at 37 C. We inoculated six-well plates of confluent Vero cells with 200 µl of the serum-virus mixtures in duplicate and incubated them at 37 C for 1 hr. Plates were overlaid with 3 ml of medium containing 0.9% agarose (Lonza Rockland, Rockland, ME) and incubated them at 37 C in a 5% CO 2 incubator for 2 days. We then added a second overlay medium containing neutral red and 1% agarose and incubated the plates overnight before visualizing and counting plaques. We used a plaque assay to measure the plasma viremia in the monkeys after challenge. For this purpose, we inoculated six-well plates of confluent Vero cells with 200 µl of plasma- PBS mixtures in duplicate and carried out the assay as described previously. The plasma dilutions were 1:200, 1:400, 1:800, 1:1000, 1:10,000 and 1:100,000. Animal experiments and blood collection of monkeys. In the monkey pilot experiments, we injected two rhesus macaques (Macaca mulatta) (ZE58, weight 5.2 kg, 3 years old; ZE79, weight 4.4 kg, 3 years old) intravenously with PFU of CHIKV (strain LR2006 OPY-1), and collected blood on hours 0, 6, 24, 48, 72, 96, 120, 144 and sacrificed the monkeys 144 h after challenge. We used gauge needles and either syringes or vacuum tubes to collect blood, with EDTA as an anticoagulant. The maximum blood volume removed did not exceed 20% (12 ml kg 1 ) per month, with no more than 15% (9 ml kg 1 ) removed during any single draw. We measured whole blood cells using a hematology analyzer (IDEXX Laboratories, Inc.).

6 The Animal Care and Use Committee, Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases ( reviewed and approved all animal experiments. We performed all experiments in accordance with relevant federal and National Institutes of Health guidelines and regulations. Detection of CHIKV RNA by quantitative RT-PCR. To isolate RNA, we centrifuged serum samples at 10,000 x g for 1 hr, poured off the liquid and added 1 ml of RNA-STAT 60 (Isotex Diagnostics). We then incubated the samples at RT for 5 min and resuspended them in 250 µl of chloroform by vortexing. We again centrifuged the samples at 10,000 x g for 1 hr, removed the aqueous top layer, added 0.5 ml isopropanol and 10 µl trna (10 µg ml 1 ), and allowed the samples to precipitate overnight at 20 C. Then we centrifuged the samples for 1 hr, washed them with cold 75% ethanol, and centrifuged them for another hour. We resuspended the RNA in 30 µl RNAse-free water. For RT-PCR, we added 10% RNA to TaqMan reagents (Applied Biosystems) along with primers and probe (listed below) and amplified the samples in a 7700 Sequence Detection System (Applied Biosystems). Briefly, we reverse-transcribed the samples at 48 C for 30 min, held them at 95 C for 10 min, then ran them for 40 cycles of 95 ºC for 30 s and 60 C for 1 min. We compared the signal to a standard curve of known concentrations of plasmid containing the LR2006 OPY-1 sequence starting at 10 7 down to 1 copy per ml and multiplied by 10, giving a detection range from copies per ml. We performed all experiments in triplicate. We designed the primers and probe to bind to a highly conserved region on the E1 structural protein gene. Primer sequences: CHIK-F 5'- AAGCTCCGCGTCCTTTACCAAG-3' and CHIK-R 5'-CCAAATTGTCCTGGTCTTCCT-3'. Probe sequence: CHICK-P FAM-CCAATGTCTTCAGCCTGGACACCTTT-TAMRA as described previously 4

7 REFERENCES 1. Yang, Z.-Y. et al. Immunization by avian H5 influenza hemagglutinin mutants with altered receptor binding specificity. Science 317, (2007). 2. Tsetsarkin, K.A., Vanlandingham, D.L., McGee, C.E. & Higgs, S. A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS. Pathog. 3, e201 (2007). 3. Palmer,E.L. & Martin,M.L. Electron Microscopy in Viral Diagnosis. CRC Press, Boca Raton, FL (1988). 4. Pastorino, B. et al. Development of a TaqMan RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses. J Virol. Methods 124, (2005). 5. Naldini, L., Blomer, U., Gage, F.H., Trono, D. & Verma, I.M. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc. Natl. Acad. Sci. USA 93, (1996). 6. Huang, Y., Yang, Z.-Y., Kong, W.-P. & Nabel, G.J. Generation of synthetic Severe Acute Respiratory Syndrome coronavirus pseudoparticles: implications for assembly and vaccine production. J. Virol. 78, (2004).

8 Supplementary Figure 1. Schematic representation of pseudotyped lentiviral vectors and characterization of CHIKV E pseudotyped lentiviral vectors by buoyant density sedimentation and Western blot analysis. (a) Plasmids encoding the indicated CHIKV Env strains (E or E OPY-1 ) were cotransfected with a transducing vector encoding a luciferase reporter gene (phr CMV-luciferase plasmid) and a packaging plasmid expressing human immunodeficiency virus type 1 (HIV-1) structural proteins (pcmvδr8.2) into 293T cells. phr CMV-luciferase and pcmvδr8.2 plasmid were previously described (figures modified from ref. 5 ). In brief, the phr CMV-luciferase plasmid comprises HIV-1 long terminal repeats

9 (LTRs), splice donor (SD), the packaging signals (ψ), Rev responsive element (RRE) and a luciferase reporter gene under the control of a human CMV promoter. pcmvδr8.2 provides all HIV-1 structural proteins except HIV-1 envelope under the control of a CMV promoter and ψ sequences were deleted. 48 h after transfection, supernatants were harvested and run on sedimentation gradients as described previously 6. (b) Quantification of gradient fractions is shown with the indicated strains, showing colocalization of Env with the Gag fraction of the expected buoyant density for lentiviral particles ( g ml 1 ) (upper panel). Western blot analysis of gradient fractions for CHIKV E1/E2 and Gag are shown (lower panel).

10 Supplementary Figure 2. Cryo-EM reconstruction of Sindbis virus. Comparison of the cryo-em reconstruction of CHIKV VLP (Fig. 2c) with Sindbis shows that CHIKV VLP is structurally similar to alphaviruses. Shaded-surface representation of the 3D density map of Sindbis virus is viewed along an icosahedral 2-fold axis. The white triangle marks the boundary of an icosahedral asymmetric unit. The numbers show the positions of the icosahedral 2-, 3-, and 5-fold axes limiting an asymmetric unit. The central cross-section through the cryo-em maps of CHIKV VLP (Fig. 2c) and Sindbis virus (left) reveals similar orientations of the icosahedral (2-, 3-, and 5-fold) axes as well as the quasi-threefold (q3) axis as shown with white lines. The map is calculated to 18 Å resolution.

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12 Supplementary Figure 3. Viremia and hematologic changes after CHIKV LR2006 OPY-1 challenge in monkeys. (a) Two naïve rhesus monkeys (diamond: ZE58 and circle: ZE79) were challenged intravenously with PFU of the CHIKV strain LR2006 OPY-1. The viremia in the monkeys after challenge was measured by quantitative RT-PCR (limit of detection = 40 RNA copies ml 1 ). Blood was collected at the indicated times after injection. (b) Monkeys injected with PBS (Control) or immunized with VLPs were challenged intravenously with PFU of the CHIKV strain LR2006 OPY-1 15 weeks after the final boost. The percentage of indicated cells in the monkeys white blood cells and total white blood cells were measured using a hematology analyzer after challenge with CHIKV. Error bars represent the standard error of the mean. An unpaired two-tailed t test was used for statistical analysis (lymphocytes, Control vs. VLPs at 1 day, P = ; neutrophils, Control vs. VLPs at 1 day, P = ).