The codon-optimized HIV-1 HXB2 env
and the OVA genes were cloned separately into the VRC vector (DNA-gp120 and DNA-OVA, respectively). The empty VRC vector was provided by Dr G. Nabel (Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health). Plasmids were prepared using the Endo-free plasmid Giga kit (QIAGEN, Valencia, CA) and had endotoxin levels <0.1
Preparation of PEI-DNA complexes.
Plasmid DNA was complexed with in vivo
-jetPEI (Polyplus transfection, Illkirch, France) according to the manufacturer's guidelines. Specifically, 0.7
plasmid DNA solution in 5% glucose was mixed with jetPEI solution to achieve a final DNA concentration of 0.4
and an N/P ratio of 7.5. The mixture was incubated for at least 30
min at room temperature in order for the complexes to form before being injected into the mice.
Mice and immunizations.
Balb/c female mice, 8–12-weeks old, were purchased from Charles River Laboratories (Wilmington, MA). C57BL/6 (B6) mice, B6.PL mice carrying the Thy1.1 allele, and transgenic OT-I mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions. Research on mice was approved by the Beth Israel Deaconess Institutional Animal Care and Use Committee. Mice were immunized IM with 40
μg of DNA in solution in a 100-μl total injection volume (50
μl delivered into each quadriceps muscle). Non-invasive aerosol applications were performed using the technique described by Bivas-Benita et al.30
In short, 50
μl formulation consisting of 40
μg DNA complexed to jetPEI was sprayed directly into the airways of mice with the Penn-Century MicroSprayer (Penn-Century, Wyndmoor, PA). Identical procedures were used for the gp120 and OVA-expressing plasmids.
Lymphocyte isolation for tetramer and phenotypic analysis of T cells.
Blood samples from individual mice were collected in RPMI 1640 supplemented with 40
heparin and peripheral blood mononuclear cells were isolated by density gradient centrifugation using Lympholyte-M (Cedarlane, Burlington, NC). Cells collected from the gradient interface were washed with phosphate-buffered saline (PBS) containing 2% fetal bovine serum and stained at room temperature for 20
min with phycoerythrin-conjugated H-2Dd
/p18 or SIINFEKL tetramer. Cells were then stained with anti-CD3e-APC-Cy7 (145-2C11) and anti-CD8a-PerCP-Cy5.5 (53-6.7) from BD Biosciences (San Jose, CA) at room temperature for an additional 15
min. Following staining, cells were washed once and fixed with PBS containing 2% paraformaldehyde.
Lungs and MLN were removed aseptically and the airway luminal cells were removed from the lungs by four consecutive BALs through a cannula in a volume of 2
ml PBS to ensure efficient recovery. Following the lavage, lungs and MLN were cut into small pieces using straight scissors and incubated with 1.33
collagenase-D and 0.2
DNase (Roche, Indianapolis, IN) in HBSS for 1 hour and 30
min, respectively, at 37
°C with vigorous shaking. Tissue fragments were then crushed through a 70-μm pore size filter and cells were washed once. Lung cells were resuspended in 40% Percoll (Sigma-Aldrich, St Louis, MO), layered over 67% Percoll, and centrifuged at 1,900
r.p.m. for 25
min. The lymphocytes were present in the interface between the two Percoll layers. For tissue tetramer staining, lymphocytes were collected from the gradient interface, washed, and stained as described above for blood samples. MLN samples were washed twice after the filtration step and similarly stained. Samples were analyzed using an LSR II flow cytometer (BD Biosciences, San Jose, CA) and FlowJo software (Tree Star, Ashland, OR).
PCR analysis of plasmid DNA.
B6 mice were pulmonary immunized with PEI-DNA-OVA and DNA was isolated from their lungs using the Qiagen DNeasy kit (Qiagen, Valencia, CA). For the PCR reaction, the forward primer (5′-CTCAAAAGACAGCGGCCGCGCC
ACCATGGGCTCCATCGGCGCAGCAAGCA) and the reverse primer (5′-AGCTTTCGGATCC
TTAAGGGGAAACACATCTGCCAAAGAAGAGAACGGCG) were used with the Platinum PCR SuperMix High Fidelity for PCR amplification (Invitrogen, Grand Island, NY). The plasmid DNA-OVA used for immunization served as positive control for the reaction. Each reaction consisted of 45
μl Platinum PCR SuperMix High Fidelity, 200
final concentration of each primer solution, and 200
ng genomic DNA template. PCR products were then subjected to agarose gel electrophoresis and 2-log DNA ladder was used to determine the amplicon's mass (New England BioLabs, Ipswich, MA).
In vivo antigen presentation assays.
Lymphocytes from spleens and lymph nodes of OT-I mice were washed with PBS and incubated with an equal volume of 10
eFluor 670 (eBioscience, San Diego, CA) in PBS for 10
min at 37
°C, at a final concentration of 5
. To quench the label, ice-cold RPMI 1640 complete medium was added, incubated on ice for 5
min, and then cells were washed twice with PBS. To evaluate antigen presentation in MLN, 5 × 106
labeled cells in 200
μl were adoptively transferred into B6.PL mice by tail vein injection. For pulmonary evaluation of antigen presentation, cells were resuspended in 50
μl PBS and instilled intratracheally in B6.PL mice. BAL, lungs, and MLN were harvested at different time points and the level of eFluor 670 dilution was determined by flow cytometry using Live/Dead fixable aqua dead-cell stain (Invitrogen), specific phycoerythrin-labeled tetramers, anti-CD3e-APC-Cy7 (145-2C11; BD Biosciences), anti-CD8a-PerCP-Cy5.5 (53-6.7; BD Biosciences), and anti-Thy1.2-eFluor 450 (53-2.1; eBioscience). To assess direct antigen presentation in the lung, lungs were removed and digested as described above. Single-cell suspensions were depleted of T cells by MACS separation using CD4, CD5 and CD8 MicroBeads according to the manufacturer's guidelines (Miltenyi Biotec, Auburn, CA). RF33.70 hybridoma cells (a gift from Dr K. Rock, University of Massachusetts, Worcester, MA), which produce IL-2 upon specific recognition of SIINFEKL presented by APCs, were co-cultured for 24
h with lung APCs in a 96-well plate (5 × 104
cells/well). Supernatants were collected and IL-2 levels were determined by the OptEIA mouse IL-2 ELISA kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions.
In vivo airway lymphocyte proliferation.
For luminal staining of antigen-specific CD8+
T cells, a 25
solution of eFluor 670 was prepared in dimethyl sulfoxide. This stock solution was further diluted with 5% glucose solution to a final concentration of 2
. Mice immunized with PEI-DNA-gp120 by the pulmonary route were administered 50
μl of the 2
eFluor 670 solution using the same non-invasive application method we used for immunization, and eFluor 670 dilution was determined 4 days later. Proliferation was further evaluated using the thymidine analog EdU. Pulmonary immunized animals were administered 250
μg of EdU in PBS intraperitoneally. Mice infected intranasally with replication-competent NYCBH strain vaccinia virus expressing HIV-1 B10 (rVac-gp160) were used as positive control. One day after EdU administration, mice were killed and cells isolated from BAL. Cells were stained with anti-CD8a-PerCP-Cy5.5 (53-6.7; BD Biosciences) and evaluated for incorporated EdU using the Alexa Fluor 647 Click-iT EdU flow cytometry assay kit according to the manufacturer's guidelines (Invitrogen).
In vivo CD8+ T-cell depletion.
T-cell depletion in mice was accomplished with sterile-purified rat IgG2b anti-mouse CD8 monoclonal antibody (YTS 169.4, Bio-X-cell; West Lebanon, NH). In all, 50
μl of a 2
antibody solution were sprayed in the lungs of mice by non-invasive pulmonary administration 6 weeks following their immunization. Two days following this treatment, mice were challenged intranasally with 2 × 106
pfu rVac-gp160 and viral titers were evaluated 5 days post challenge in lungs and ovaries using plaque forming assays on CV-1 cells.
Blockage of peripheral T-cell recruitment.
Immunized mice were injected i.p. with 200
μl of 0.4
FTY720 solution (Cayman Chemical, Ann Arbor, MI) to block T-cell recruitment to the lungs. The efficacy of the treatment was confirmed by measuring CD3+
lymphocytes in the peripheral blood of the mice.
Recombinant vaccinia virus challenge.
Groups of mice were challenged 6 weeks after a plasmid DNA immunization with replication-competent NYCBH strain vaccinia virus expressing HIV-1 B10 (rVac-gp160). Mice were anesthetized by intraperitoneal injection with a ketamine (100
) mixture and 2 × 106
pfu rVac-gp160 (10 × LD50) was instilled intranasally. Infected mice were monitored for survival and viral titers in the lungs and ovaries. For the evaluation of viral titers, mice were sacrificed 5 days following vaccinia virus challenge and their lungs and ovaries were harvested and frozen at −80
°C until analysis. A day before the assay, tissues were thawed, homogenized in 100
Tris buffer, pH=8, and frozen again overnight. On the day of the assay the cells were sonicated for 2
min and viral titers were evaluated by plaque forming assay on CV-1 cells. Each mouse tissue was evaluated in duplo plates to assure reproducibility.
Influenza virus challenge.
T-cell responses induced by ovalbumin plasmid immunization were evaluated using the genetically modified influenza strain A/PR/8/34 (PR8, H1N1) encoding the OVA257–264
-restricted epitope in the neuraminidase protein.31
C57BL6 mice were immunized by the pulmonary route with PEI-DNA-OVA and 6 weeks later challenged intranasally with 105
EID50 of the influenza strain.
Statistical analysis. Immune response data are presented as mean±s.e. for each experimental group. Statistical analyses were performed using the two-sided Student's t-test or one-way analysis of variance, and P<0.05 was considered significant.