All cells were incubated at 37°C in 5% CO2
under humidified conditions. Culture conditions were as follows: 293 cells were maintained in Dulbecco modified Eagle medium (DMEM; Gibco-BRL, Bethesda, Md.) supplemented with 10% fetal calf serum (FCS; Sigma, St. Louis, Mo.), penicillin, streptomycin, and l-
glutamine (Gibco-BRL). P815 (H-2d
mastocytoma) cells and EL4 (H-2b
thymoma) cells were maintained in RPMI 1640 medium (Gibco-BRL) supplemented with 10% FCS, penicillin, streptomycin, and l-
glutamine (Gibco-BRL). Primary splenocytes were prepared and cultured in RPMI 1640 medium supplemented with 10% FCS (HyClone, Logan, Utah), 50 μM β-mercaptoethanol (Gibco-BRL), penicillin, streptomycin, and l-
glutamine (Gibco-BRL); this is referred to as CR10-β medium. Baby hamster kidney (BHK) cells were maintained as described previously (27
). The NIH 3T3 mouse fibroblast cell line was originally obtained from the American Type Culture Collection and was maintained in DMEM supplemented with 10% FCS, penicillin, and streptomycin.
Amplicon vector construction.
A codon-optimized version of HIV-1 gp120 DNA was kindly provided by Jürgen Haas (Max-von-Pettenkofer Institut, Munich, Germany). This gp120 expression vector was derived from HIV-1 MN, except for the V3 loop, which was derived from HIV-1 LAI and contains an epitope recognized by BALB/c mice in the context of H-2Dd
). The codon-optimized version of gp120 was selected to allow high-level, Rev-independent expression as described previously (4
). The HSV-1 amplicon vector pHSVPrPUC (13
) contains the ColE1 plasmid origin, an HSV-1 packaging signal and origin, and a multiple cloning site situated downstream from the HSV-1 immediate-early 4/5 promoter (IE4/5) and upstream from the simian virus 40 polyadenylation signal. A Not
I 1.6-kb fragment encoding the synthetic gp120 sequence was removed from the pSyngp120v3LAI plasmid and cloned directionally into a Sal
HI-cut (blunted) pHSVPrPUC vector.
Helper virus-free HSV amplicon packaging.
Packaging of virus vectors was performed as described previously (9
). Briefly, on the day prior to transfection, 2 × 107
BHK cells were seeded in a T-150 flask and incubated overnight at 37°C in 5% CO2
. On the day of transfection, 1.8 ml of OptiMEM (Gibco-BRL), 25 μg of pBAC-V2 DNA (53
), 7 μg of pBSKS (vhs
), and 7 μg of amplicon vector DNA were combined in a sterile polypropylene tube. Then, 70 μl of Lipofectamine Plus reagent (Gibco-BRL) was added over a period of 30 s to the DNA mix, and the mixture was incubated at 22°C for 20 min. In a separate tube, 100 μl of Lipofectamine (Gibco-BRL) was mixed with 1.8 ml of OptiMEM, and this mixture was also incubated at 22°C for 20 min. After the incubations, the contents of the two tubes were combined over a period of 30 s and incubated for an additional 20 min at 22°C. During this second incubation, the medium in the seeded T-150 flask was removed and replaced with 14 ml of OptiMEM. The transfection mix was added to the flask and allowed to incubate at 37°C for 5 h. The transfection mix was diluted with an equal volume of DMEM plus 20% FBS, 2% penicillin-streptomycin, and 2 mM hexamethylene bisacetamide (HMBA) and then incubated overnight at 34°C. The following day the medium was removed and replaced with DMEM plus 10% FBS, 1% penicillin-streptomycin, and 2 mM HMBA. The packaging flask was incubated an additional 3 days before the virus was harvested and stored at −80°C until purification. Viral preparations were subsequently thawed, sonicated, clarified by centrifugation, and concentrated by ultracentrifugation through a 30% sucrose cushion. Viral pellets were resuspended in 100 μl of phosphate-buffered saline (PBS) and stored at −80°C until use. Titers of vectors were determined as described previously by using expression and transduction titering methods (10
). Virus was diluted in sterile Dulbecco PBS (D-PBS; Gibco-BRL) to the appropriate concentration prior to injection. In addition, two separate irrelevant amplicons were prepared as controls by using the same vector system described above; one contained the gene for Escherichia coli
β-galactosidase (LacZ), and one contained the gene for murine intestinal alkaline phosphatase (MIAP).
Verification of gp120 expression.
To verify gp120 expression, 293 cells were first seeded in 12-well plates and allowed to adhere overnight at 37°C in 5% CO2. Cells were then either infected with HSV:gp120 amplicon particles or with HSV:MIAP amplicon particles as a negative control. On the day of infection, medium was removed, and cells were infected at a multiplicity of infection (MOI) of 0.5 in a volume of 0.5 ml at 37°C in 5% CO2. After 1 h, 1.5 ml of supplemented DMEM was added to the wells, and the cells were incubated for 48 h. Medium was collected from the wells and concentrated by using the Microcon microconcentrator (M-50; Millipore, Bedford, Mass.). Concentrated supernatants were combined with an equal volume of Laemmli buffer and were separated on 4 to 15% gradient sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad, Hercules, Calif.). Full-range rainbow molecular weight markers (Amersham/Pharmacia, Piscataway, N.J.) were added as a standard. The gels were transferred to nitrocellulose and were blocked in PBST buffer (1× PBS, 0.1% Tween 20) containing 5% nonfat milk. The blots were incubated with the AD3 anti-gp120 monoclonal antibody (AIDS Repository, Rockville, Md.) for 1 h at room temperature. Blots were extensively washed with PBST. The blots were then incubated with sheep anti-mouse horseradish peroxidase secondary antibody (Amersham). Blots were again washed extensively with PBST. Enhanced chemiluminescence (Amersham) was performed for 1 min, and the blots were exposed to X-ray film (Kodak, Rochester, N.Y.).
The H-2Dd-restricted 10-mer epitope peptide from the V3 loop (RGPGRAFVTI) was synthesized by Alpha Diagnostics (San Antonio, Tex.), purified by high-pressure liquid chromatography and analyzed by mass spectroscopy.
Female BALB/c mice aged 5 to 6 weeks were obtained from Taconic Laboratories (Germantown, N.Y.) and maintained according to University of Rochester guidelines. Prior to all injections, mice were bled orbitally, and sera were stored at −80°C until testing. For allogeneic CTL cultures, female C57BL/6 mice, aged 5 to 6 weeks, were utilized and handled as indicated above. Mice were injected by using a 28G 0.5-in. 1-ml insulin syringe (Becton Dickinson, Franklin Lakes, N.J.). Injections were given intramuscularly (i.m.) in the right hind hamstring muscle, i.d. at the base of the tail, or intraperitoneally (i.p.) in the body cavity. After inoculation, mice were incubated for 21 to 22 days prior to sacrifice and analysis.
At euthanization, sera were obtained by cardiac puncture, and spleens were removed by using sterile technique with sterile forceps and tweezers and transferred to 60-mm tissue culture dishes containing 5 ml of RPMI 1640 medium (Gibco-BRL). The spleens were aseptically transferred to 100-mm tissue culture dishes containing 10 ml of CR10-β. Autoclaved frosted glass slides (VWR, Media, Pa.) were used to gently grind the spleens to obtain a single cell suspension. Splenocytes were centrifuged, and the cell pellets were resuspended in 10 ml of CR10-β, counted, and diluted to a concentration of 4 × 107 cells/ml.
Two to three days before the CTL cultures were prepared, lipopolysaccharide (LPS) blasts were prepared as stimulator cells for the CTL cultures. Syngeneic BALB/c mice were euthanized, and their spleens were processed as described above. Naive splenocytes were cultured in T-75 flasks at a concentration of 4 × 107 cells/flask in 30 ml of CR10-β supplemented with 150 μl of LPS (1 mg/ml in sterile H2O, from E. coli serotype O55:B5) and 30 μl of dextran sulfate (7 mg/ml in sterile PBS). Flasks were incubated at 37°C in 5% CO2. After the incubation period, flasks were examined to look for blast-like cells. LPS blasts were washed extensively with RPMI 1640 medium and then pulsed with the RGPGRAFVTI peptide for 60 min in a 37°C water bath. Blasts were irradiated in a cesium-137 source for 10 min (ca. 3,000 rads) and then diluted to a concentration of 107 cells/ml prior to use.
As an allogeneic control for CTL expansion and killing assessment, one C57BL/6 mouse was euthanized per experiment (as described above), and its spleen processed as described above. The splenocytes were diluted to a concentration of 2 × 107 cells/ml and irradiated along with the LPS blasts.
The rat anti-mouse gamma interferon monoclonal antibody AN18 was produced from the corresponding hybridoma, which was kindly provided by Edith Lord (University of Rochester Medical Center). Hybridoma supernatants were concentrated by using the Cellmax system (Spectrum Laboratories, Laguna Hills, Calif.) according to the manufacturer's instructions. Ninety six-well membrane plates (Millipore) were coated with the AN18 monoclonal antibody diluted in 1× D-PBS (Gibco-BRL), and plates were washed with RPMI 1640 medium (Gibco) and blocked for at least 1 h at room temperature with CR10-β. Splenocytes from immunized animals were diluted in a background of syngeneic splenocytes to allow each enzyme-linked immunospot (ELISPOT) well to contain a fixed cell concentration of 106 cells per well. Splenocytes were cultured either in 2 μM (final concentration) of the V3 peptide diluted in CR10-β or in CR10-β alone for 18 h at 37°C in 5% CO2. Plates were washed extensively with PBST. Biotinylated rat anti-mouse gamma interferon was added to the wells (clone XMG1.2; PharMingen, San Diego, Calif.) and incubated for 2 h at room temperature. Streptavidin-alkaline phosphatase was added (Jackson Immunoresearch, West Grove, Pa.) to the well, and the mixture was allowed to incubate 1 h at room temperature. Plates were washed as described above. Vector Blue substrate (Vector Laboratories, Burlingame, Calif.) was prepared according to the manufacturer's instructions, added to the plates, and incubated for 10 min at room temperature in the dark. Plates were then washed extensively with distilled H2O, allowed to dry overnight, and then counted and analyzed by Zellnet Consulting, Inc. (New York, N.Y.).
To expand the CTL population from the inoculated animals to a level that could be observed by CTL assay, CTL cultures were prepared. Multiple wells were set up per animal to be tested. A 1-ml portion of CR10-β was combined with 0.1 ml of experimental splenocytes (4 × 106 cells) and either 0.1 ml of irradiated, V3-peptide-pulsed LPS blasts (106 cells) to determine the peptide-specific response or 0.1 ml of irradiated C57BL/6 cells (2 × 106 cells) to test for the allogeneic response. Cultures were incubated for 5 days at 37°C in 5% CO2.
The cytotoxic activity of gp120-specific CTLs was determined by JAM assay (29
). Briefly, P815 and EL4 cells were seeded at 2 × 105
cells/ml on the day prior to the assay. On the day of the assay, the cells were centrifuged and resuspended in RPMI 1640 medium supplemented with 10% FCS. The cells were labeled with [3
H]thymidine (5 μCi/ml; New England Nuclear, Boston, Mass.) for 3 h at 37°C in 5% CO2
, washed with RPMI 1640 medium, and diluted to a concentration of 105
cells/ml in CR10-β. CTL effectors were washed extensively with RPMI 1640 medium and resuspended in a fixed volume of CR10-β. The CTLs were added to 96-well U-bottom plates (Falcon) in triplicate, to give responder/target ratios from 100:1 to 1.6:1. Ten thousand radiolabeled targets were then added either in the presence or in the absence of the V3 peptide. Plates were centrifuged briefly to pellet CTL effectors and targets and then incubated at 37°C in 5% CO2
. After 3 h, plates were harvested onto glass filters (Perkin-Elmer Wallac, Gaithersburg, Md.) by using a 96-well automated harvester (Tomtec, Hamden, Conn.). After they were dried, the filters were subjected to scintillation counting analysis by using the Microbeta 1450 Trilux counter (Perkin-Elmer Wallac). Cytotoxicity was determined according to the following formula: percent cytotoxicity = [(S
) ÷ S
] × 100, where E
is the experimentally retained DNA in the presence of killers (in counts per minute) and S
is the retained DNA in the absence of killers (spontaneous) as described previously (29
In some experiments, CD8+-cell depletion was accomplished with the use of anti-CD8a Ly-2 magnetic beads (Miltenyi-Biotec), according to the manufacturer's instructions. To verify successful elimination of the CD8+ cells, splenocytes were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD8 (Ly-2; BD PharMingen) and were examined by using a FACScan cytometer (Becton Dickinson).
H-2Dd streptavidin-allophycocyanin tetramers containing the V3 peptide were prepared by the NIH Tetramer Facility (Emory University, Atlanta, Ga.). Tetramer staining proceeded as follows: 1 × 106 to 2 × 106 freshly isolated splenocytes were washed once and resuspended in 50 μl of ice-cold fluorescence-activated cell sorting (FACS) buffer (1× PBS [pH 7.2] containing 0.09% sodium azide and 2% fetal bovine serum). For staining, 50 μl of FACS buffer containing 0.1 μg of FITC-conjugated anti-mouse CD8 (Ly-2) and 0.5 μg of tetramer were added to the cells, mixed well, and kept on ice for 1 h in the dark. The cells were subsequently washed with FACS buffer twice, resuspended in 400 μl of FACS buffer, and analyzed on a FACSCalibur (BD PharMingen) by using CellQuest software. A total of 100,000 events were collected, and the histograms shown represent cells gated initially by forward scatter and side scatter.
ELISA (enzyme-linked immunosorbent assay) plates were coated overnight at 4°C with recombinant soluble HIV-1 MN gp160 (Protein Sciences, Meridien, Conn.), diluted in coating buffer (0.1 M NaHCO3 [pH 8.6]). Plates were washed with PBS-T buffer and then blocked for 2 h at 37°C by using PBS-T containing 3% bovine serum albumin (Sigma). Plates were washed again in PBS-T, and sera were diluted, added to the ELISA plates in duplicate, and incubated for 1 h at 37°C. Plates were washed as described above. Anti-mouse immunoglobulin G (IgG) Fc-specific secondary antibody (Sigma) was added to the plates, and they were incubated for 1 h at room temperature. After washing, ABTS [2,2′azinobis(3-ethylbenzthiazolinesulfonic acid)] substrate (Sigma) was added to the plates and, after enough time for color development, the absorbance was measured at 405 nm. Endpoint titers were ascertained by using a scatter plot with optical density (OD) values on the y axis and dilution−1 on the x axis, for which the x-axis scale was logarithmic. After the data were plotted, a logarithmic curve fit was applied to each individual dilution series, and the point where the curve fit intersects the positive-negative cutoff value was determined. This cutoff value was calculated as the mean OD of all preimmune sera plus four standard deviations. For the experiment involving the preinfection of mice with wild-type HSV-1, the cutoff value was calculated slightly differently due to the fact that the HSV-1-preinfected mice exhibited an elevation in their background level of serologic reactivity to HIV-1 gp120. The cutoff value was calculated as the mean OD plus four standard deviations of all sera collected from either preinfected or naive mice.
Sera were tested for anti-HSV IgG antibodies by ELISA as described previously (20
). Briefly, ELISA plates were coated with UV-inactivated HSV overnight at 4°C. Plates were washed with PBST and blocked with PBS-0.1% bovine serum albumin for 1 h at 37°C. Plates were again washed with PBST. Sera were diluted and incubated in duplicate on the HSV-coated plates overnight at 4°C. Plates were washed with PBST and treated with alkaline phosphatase-conjugated goat anti-IgG (Cappel, Worthington, Pa.) for 3 h at 37°C. Plates were washed with PBST and incubated with p
-nitrophenyl phosphate substrate (Sigma), and the absorbance was read at 405 nm.
Virus neutralization assays.
Sera from mice subjected to preinfection with HSV-1 (Patton) strain were analyzed for the presence of virus-neutralizing antibodies as follows. Briefly, sera obtained either prior to or 5 weeks after HSV-1 (Patton) infection were diluted 1:20 and 1:100 in PBS and then combined with an equal volume (1,000 infectious units [i.u.], 100 μl) of HSV:lacZ amplicon particles. After incubation at 37°C for 135 min, this preparation was then added to Vero cells in 24-well tissue culture plates for 60 min at 37°C. DMEM medium containing 5% FCS was then added to each well (1.0 ml), and cells were allowed to incubate at 37°C in 5% CO2 for 48 h. At the end of the given time period, the cells were fixed with 1% glutaraldehyde, washed with 1× PBS, and analyzed for β-galactosidase activity by a histochemical staining method with X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) as the substrate. For each serum and dilution tested, the percent reduction in LacZ-positive cells was calculated relative to cultures that were exposed to untreated HSV:lacZ amplicon particles.