Viruses and cells
Mopeia/Lassa reassortant virus (clone ML29) was previously tested as a vaccine against Lassa fever
[10, 26, 27]. Vero E6 cells (#CRL-1586, ATCC) were cultured in Dulbeccos’s modified Eagle’s medium (DMEM, GIBCO-BRL) supplemented with 10% fetal bovine serum (FBS, GIBCO-BLR), 1% penicillin-streptomycin, and 2 mM L-glutamine. To produce the ML29 inoculum, Vero cell monolayers were infected with ML29 master stock at a multiplicity of infection (MOI) of 0.01 and incubated 1 hour at 37°C in 5% CO2, then washed with PBS and covered in DMEM 2% FBS. Supernatants were collected at 48 and 72 hours after infection, stored at -70°C, and titrated on Vero cells. Supernatants usually had about 107 plaque forming units (pfu)/ml. Stocks of lymphocytic choriomeningitis virus-Armstrong (LCMV-ARM) and LCMV-WE (described extensively in
) were similarly produced and titrated on Vero E6 cells and stored at -70°C.
SIVmac251 (originally supplied by Ron Desrosiers) was amplified on rhesus macaque PBMC and given a final passage on CEMX174 cells as described
. Monkeys were infected by inoculating 10 TCID50 per animal by the saphenous vein.
Whole blood collected in heparin was used to stain for cell surface and intracellular markers (especially for cell-mediated immunity assays). Fresh monkey PBMC for transcriptome analyses were obtained from 10 ml of heparin blood by Ficoll-hypaque isolation
 and resuspended in RPMI medium 1640 with 10% FBS (Sigma) for RNA extraction as described below
. Residual PBMC were frozen at -140°C at a concentration of 107 cells/ml. Upon necropsy, splenocytes were obtained for cell-mediated immunity assays: fresh tissue pieces were pressed through mesh, exposed to red cell lysis buffer 5 minutes, then suspended in freezing media and stored at −140°C at a concentration of 107 cells/ml. Serum samples and plasma samples were stored at −140°C before being analyzed for virus or antibody content.
Animals and immunization
Rhesus macaques (Rh) were purchased from the Caribbean Primate Colony. Experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Maryland School of Medicine. Of 16 monkeys inoculated with SIVmac251 as previously described
, 5 were dying with symptoms of SIV disease and had to be euthanized almost a year after SIV infection and before the onset of the ML29 vaccinations. Of the remaining 11 animals, 8 were inoculated with the ML29 vaccine candidate and 3 were inoculated with LCMV-Armstrong, known to be benign in AIDS monkeys (Salvato MS and Zapata JC, not shown). Of the 8 ML29-vaccinees, 5 were inoculated s.c. with 0.5 ml 103 pfu ML29 and 3 were inoculated i..g. with 2 ml 106 pfu ML29. In addition, 5 healthy control animals were vaccinated s.c. with 0.5 ml of ML29 containing 103 pfu. Blood was collected every week for flow cytometry, viral loads, antibody detection, clinical chemistry, hematology, and transcriptome profiles as described
. Six weeks after their first vaccination, 6 macaques (ML-1, SIV/ML-1, SIV/ML-2, SIV/ML-6, SIV/ML-7, SIV/ARM-1) were boosted s.c. with 103 pfu ML29. Animals were monitored daily for weight loss, rashes and clinical signs of SIV or arenavirus disease.
For weight loss over 10% of body weight and in consultation with the veterinary staff, monkeys were euthanized and total blood and tissues [lung, spleen, mesenteric lymph nodes (LN), liver, stomach, ileum, kidney, heart, cerebrum, and cerebellum] were collected. A portion of each tissue was submerged in MEM with 10% FCS for virus and RNA isolation. The remaining tissue portions were flash frozen in liquid nitrogen to be used in virus isolation or fixed in 10% neutral formalin for the preparation of histological sections. Also, at the end of the study surviving healthy animals were euthanized and blood and tissues collected.
Detection of SIV and arenavirus in monkey tissues
SIV viral loads were evaluated from plasma samples using real-time NASBA to determine the number of RNA copies per milliliter (Advanced Biosciences Laboratory, Kensington, MD). This technique can detect 100 SIV RNA copies per ml plasma. Based on SIV-infected animal set points
[48, 49], monkeys were considered slow AIDS progressors (<104 RNA molecules/ml), median progressors (104 to 106 RNA molecules/ml), and rapid progressors (>106 RNA molecules/ml). Of the eight SIV-infected animals inoculated with ML29, two were slow progressors (SIV/ML-2, SIV/ML-5), 5 were median progressors (SIV/ML-1, SIV/ML-4, SIV/ML-6, SIV/ML7, and SIV/ML-8) and one was a rapid progressor (SIV/ML-3). Of the three monkeys inoculated with the mild LCMV-ARM, one was a slow progressor (SIV/ARM −2) and two were rapid progressors (SIV/ARM −1 and SIV/ARM −3).
Arenaviruses were detected by three different methods: 1) conventional plaque assays that are sensitive to 20 infectious particles in 1 ml solution and were used for assessments of plasma or tissue viremia for arenaviruses ML29, LCMV-Armstrong, and LCMV-WE
; 2) RT-PCR that detects 10 viral genomes in 200 μl serum and was used in all tissues samples (as described in
; and 3) a sensitive 2-step amplification assay in which infected plasma or cells are co-cultivated with Vero cells one week, then the media is subjected to plaque assay. The 2-step assay increases the plaque detection up to 1 infectious particle per ml
Flow cytometry to characterize PBMC
Ten ml of whole blood was collected in heparin tubes, and 100 μl was mixed with each antibody panel (mixtures of fluorochrome-conjugated antibodies supplied by BD Bioscience San Jose, CA). Panel 1 included mouse anti-human CD20-FITC (clone 2H7), CD3-PE (clone SP34-2), CD4-APC (clone L200), and CD8-PerCP (clone SK1) to detect B and T lymphocyte populations. Panel 2 included mouse anti-human CD16-FITC (clone 3 G8), CD56-PE (clone NCAM16.2), CD4-APC (clone L200), and CD8-PerCP (clone SK1) to detect NK, gamma-delta T cell (γδT cell), and NKT cell populations. Panel 3 included CD14-FITC (clone M5E2) and CD4-APC (clone L200), to detect monocyte and T cell populations. Panel 4 included CD3-PE, and TCR Vgamma9-FITC (clone IMMU 360, Beckman Coulter). After 30 min, FACS Lysing Solution (BD) was added for red cell lysis and incubated 20 min at room temperature. Cells were washed twice with PBS and fixed in 2% formaldehyde, then collected in a FACScalibur (BD) and data were analyzed with FlowJo Software (Tree Star, San Carlos, CA).
Intracellular staining for virus-specific IFN γ responses
Whole heparinized blood (0.5 ml) was stimulated with <50 μl of ML29 (5 × 105 pfu) or MEM (control) at 37°C, 5% CO2 overnight. 25 μl of 1:10 dilution of monensin (Golgiplug, BD) was added to each sample 4 hours prior to staining. Then cells were stained for surface antigens with anti-CD4-APC (clone L200) and CD8 (clone SK1) and stained for intracellular IFNγ using monoclonal antibody 4S.B3 and the Cytofix Cytoperm kit (BD). Samples were analyzed on a FACSCalibur instrument with 50,000 events in the lymphocyte gate and results were analyzed using FlowJo software (TreeStar).
PBMC from immunized animals were used in IFNγ ELISPOT (U-CyTech B.V., Ultrecht, The Netherlands) according to the manufacturer's recommendations with slight modifications
. Briefly, 2 × 106 cells in 0.5 ml of RPMI-1640 (Invitrogen) with 5% FBS, 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 25 mM Hepes buffer were stimulated by co-incubation overnight at 37°C with 2 × 106 pfu of ML29. After stimulation, the cells were washed, resuspended in the same medium, and 0.3–0.4 × 106 cells/well were added to ELISPOT 96-well plates pre-coated with mouse anti-monkey IFNγ. The plates were incubated at 37°C for 5 h, washed, and incubated with gold-conjugated anti-biotin. The spot-forming cells (SFC) secreting IFNγ were developed with activator solution and counted (Immunospot 3.2 Analyzer, C.T.L. Cellular Tech., Ltd.)
Anti-LAS-GPC antibodies in serum samples were measured by IgG ELISA as previously described
. Supernatants of ML29-infected Vero E6 cells were concentrated in 15 ml Amicon tubes then sonicated using a Misonix-S4000 sonicator (MISONIX, Newtown, CT). This concentrated-antigen was suspended in carbonate-bicarbonate buffer (pH 9.6), and 100 μl of antigen was adsorbed to the wells of microtitration plates overnight at 4°C. After washing the 96-well plates 6 times with PBS-Tween (0.05%), two-fold dilutions (1/50 to 1/400) of plasma were added and incubated for an hour at 37°C. Wells were washed 5 times, and 100 μl of 5,000-times diluted peroxidase-conjugated goat anti-monkey IgG (A-2054, Sigma) was added and incubated one hr, then washed 6 times, then substrates were added, incubated for 30 min in the dark, then stopped with 100 μl/well of 1 M phosphoric acid and read at OD450 on a Wallac 1420 plate-reading spectrophotometer.
Gene expression from monkey PBMC cDNA
Total RNA was isolated from fresh PMBC samples, using the Trizol method (Invitrogen, Carlsbad, CA) followed by a cleaning step with RNeasy mini kit (Qiagen, Valencia, CA). Quality and quantity of all RNA samples were evaluated on an Agilent 2100 BioAnalyzer 116 (Agilent Technologies, Palo Alto, CA) by looking at 18 and 28 s rRNA peaks and by the RIN (RNA integrity number). High quality RNA was labeled and hybridized according to Affymetrix protocols using the human GeneChip U133 Plus 2.0 array (Affymetrix, Santa Clara, CA) as described previously
. This chip covers the whole human genome using 54,000 probesets representing approximately 22,000 genes and has been validated for use with non-human primates
[50–52]. Although many PBMC-RNA samples were analyzed, the only ones from sufficiently large groups to yield statistically significant data were the SIV-infected samples (n = 8), the SIV/ML29 s.c. week 1 samples (n = 5) and the SIV/ML29 s.c. week 2 samples (n = 5). Smaller groups included SIV/ML29 i.g. weeks 1 and 2, SIV/LCM s.c. weeks 1 and 2, ML29 i.v. weeks 1 and 2 and the uninfected samples.