Four to 7-wk-old female BALB/c mice were purchased from the The Jackson Laboratory (Bar Harbor, Maine) and housed in a temperature and light-cycle controlled facility. APPswe/PS1deltaE9 transgenic mice (stock number 004462) were also purchased from The Jackson Laboratory. Animal use protocols were approved by the UT Southwestern Medical Center Animal Care and Use Committee (Dallas, TX).
2.2. Construction of plasmids
The Aβ42 monomer and trimer genes were chemically synthesized and cloned into the immunization vector system [14
]. A set of complementary oligonucleotides of the Aβ42 DNA sequence were designed using the DNA builder program and custom synthesized (Sigma, St. Louis, MO). These oligonucleotides were designed after the respective Aβ42 amino acid sequence using multiple codons for a particular amino acid allowing a more flexible design of the nucleotide sequence to avoid hairpins, primer dimer structures and other inappropriate matches among the sequences which can hinder gene synthesis by Polymerase chain reaction (PCR). A total of 32 oligonucleotides (end concentration 250 μM) were mixed for the first PCR reaction to assemble them and built the designed gene sequence (30 cycles: 94°C for 15 s, 55°C for 30 s and 72°C for 45 s; Platinum®Taq DNA Polymerase, Invitrogen, Carlsbad, CA). A second PCR was used to amplify the full-length product using a forward and a reverse primer (30 cycles: 94°C for 15 s, 55°C for 30 s and 72°C for 45 s). PCR products from this second run were purified by gel electrophoresis, digested with restriction enzymes (Promega, Madison, WI ) and cloned into the polycloning site of the plasmid vector (EcoRI/XbaI digestion). Bacteria were transformed with the ligated plasmids and clones were identified by sequence analysis (Applied Biosystem, CA, Sequencing core of UTSW). An adenovirus E3 gene leader sequence [19
] and an endosomal targeting sequence [20
] were cloned up and down stream of the Aβ42 gene, respectively. For the control immunizations corresponding plasmids were constructed. Plasmid pGal4/UAS-Luc consists of the same binary plasmid system as pGal4/UAS-Aβ42 trimer or monomer but without the E3 leader and endosomal targeting sequence, in which the transcription of the Luc gene is driven by binding of the Gal4 transcription factor. In pCMV-Luc, transcription is driven by a CMV promoter [21
2.3. Transfection of cells, ear skin and detection of expressed proteins by Western blot and ELISA
HEK293 cells (8 × 105) were transiently transfected with 2 μg plasmid DNA by Fugene 6 Reagent (Roche, Indianapolis, IN) or by gene gun bombardment at 200 psi (3 shots with 1 μg plasmid). After 24 h of growth (37 °C, 10% CO2) in Dulbecco’s Modified Eagles Medium supplemented with 10% heat inactivated Fetal Bovine Serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine (Invitrogen), the cells were washed with PBS, proteins were extracted with reporter lysis buffer (Promega), and protein concentrations were determined using a NanoDrop® Spectrophotometer. Cell lysates were resolved in 4–20% SDS-PAGE followed by Western Blot analysis to detect expressed Aβ trimer or monomer. An anti-Aβ1–16 monoclonal antibody (6E10, Signet, MA) was used for detection of the expression of Aβ42 trimer or Aβ42 monomer, and an anti-Gal4 polyclonal antibody (SC-729, Santa Cruz, CA) was used for the detection of Gal4 expression. Cell lysates transfected with control vector pUAS-Luc or CMV-Luc were used as the negative controls. For in-vivo expression of Aβ42, the mouse ear was transfected with Luc or Aβ42 constructs by gene gun (300 psi, 1 μg in each side of the ear). Ears were collected 24 hours after transfection and homogenized with a polytron device in 500 μl reporter lysis buffer (Promega). After measurement of the protein concentrations, extracted proteins were loaded on 4–20% SDS-PAGE followed by Western Blot analysis to detect expressed Aβ42. Here we used a polyclonal antibody Aβ42 antibody, SC5399 (Santa Cruz Biotechnology, Santa Cruz, CA), because the secondary anti-rabbit IgG antibody did not show cross-reactivity with other mouse proteins in these particular lysates. An ear lysate transfected with control vector pUAS-Luc was used as the negative control. Antibody binding was visualized with enhanced chemiluminescence detection using Luminol reagent as recommended by manufacturer (Thermo Scientific, Waltham, MA). Pixel densities of the bands were analyzed using ImageJ software (rsb.info.nih.gov/ij) and normalized against APP expression level in the same lane. In-vivo expression of Aβ42 in ear skin in the same lysates was also confirmed by ELISA. The ELISA plates were coated with the ear lysates (1 mg protein/ml coating buffer) and incubated overnight at 4°C. The plates were blocked with blocking buffer (PBS/1.5% BSA) for 30 min and incubated with anti-Aβ monoclonal antibodies, 6E10 and 4G8 (1ng/ml, Covance, Madison, WI), for 1 hour at room temperature. After washing the plates were incubated with a goat-anti-mouse IgG/HRP labeled secondary antibody for another hour. Antibody binding was detected with ABTS substrate and the plates were read at 405 nm. The concentration of Aβ42 was calculated with a standard curve for Aβ42 peptide and non-specific binding was subtracted from second antibody control and pUAS-Luc transfected lysate control.
2.4. Immunization of the mice using the Gene Gun method
All plasmid DNAs were purified using a commercial plasmid maxi kit (Qiagen, Valencia, CA). The purity and concentration of DNA were measured by optical density reading at 260/280 nm and gel electrophoresis. Immunizations were performed as described before [14
]: DNA coated gold particles were bombarded to both sides of the mouse ears with a helium gas pressure of 300 psi (Helios gene gun, Bio-Rad, Hercules, CA). Each vaccination consisted of 1 μg DNA into the skin of both side of the ears for a total of 4 μg DNA for each vaccination point. Mice were immunized weekly for the first 4 times and then biweekly for a total of 6–11 immunizations. Three independent experiments for each immunization vector (Gal4/UAS-Luc, Gal4/UAS-Aβ42 monomer, Gal4/UAS-Aβ42 trimer, CMV-Luc, CMV-Aβ42 trimer) were performed with 4 mice in each group.
2.5. Use of the vaccine generated immune sera for detection of Aβ42 protein in Western blots
Full length human Aβ42 gene was inserted into pMAL vector downstream of the malE gene (encoding gene of maltose-binding protein, MBP) (New England Biolabs, Ipswich, MA) and MBP-Aβ42 was expressed in E. coli BL21. The bacterial pellet was extracted with 1 x SDS loading buffer and 2 μl of supernatant (10 μg/total protein/lane) were applied to a 4–20% SDS PAGE and transferred to a PVDF membrane (Immulon-P, Millipore, Billerica, MA). The membranes were blocked with blocking buffer (TBST containing 3% milk, 1% BSA, 0.05% tween 20), and probed with 1:2000 diluted sera obtained from mice vaccinated with Aβ42 constructs or Luc control constructs as negative control. Lower dilutions of the antisera resulted in high background and could therefore not be used. HRP-conjugated goat anti-mouse IgG (1:5,000 dilution, Bio-Rad) was used as secondary antibody and antibody binding onto the membranes were detected using an enhanced chemiluminescence assay (PerkinElmer Life Sciences, Waltham, Massachusetts).
2.6. ELISAs for detection of anti-Aβ antibody, isotyping and epitope mapping
Blood was collected from mouse tail vein and the serum was analyzed for Aβ-specific antibodies by ELISA. The assay was performed as described earlier (14). Briefly, the plate was coated with 50 μl Aβ42 peptide (2 ug/ml)(BioBasic Inc., Ontario, Canada) in coating buffer and serum was added into the wells in various concentrations in blocking buffer (1% milk, 1% BSA, 0.05% Tween 20 in PBS) and incubated overnight at 4°C. Antibody binding was detected with a secondary HRP conjugated rabbit anti-mouse IgG antibody. The reaction was visualized by adding 3,3′,5,5′tetramethylbenzidine (TMB) (Pierce, Rockford, IL) substrate solution and was read after 15 min of substrate reaction in a spectrophotometer at 405 nm. Further reaction was stopped at 20 min by adding 50 μl 0.5N HCL and read again at 450 nm. Antibody concentrations were calculated using a calibration curve generated with known concentrations of an anti-Aβ42 monoclonal antibody, 4G8 (Covance). The isotypes of anti-Aβ antibodies were detected using rabbit anti-mouse IgG1, IgG2a, IgG2b and IgM as secondary antibodies (Zymed, San Francisco, CA) and an HRP conjugated anti-rabbit Ig antibody as tertiary antibody as previously described (15). Epitope mapping of the immune sera was performed by analysis of the antibody binding to various Aβ42 peptides including Aβ1-28, 1-15, 1-9, 5-14, 9-18, 16-38, and 31-42 following the ELISA procedure described above.
2.7. Detection of senile plaques with vaccine generated immune sera
Brain sections from APPswe/PS1deltaE9 transgenic mice were used to test whether sera of vaccinated animals contain antibodies that bind to senile plaques. Sera from mice vaccinated with DNA Aβ42 trimer and control plasmid (end concentration 1 μg/ml) were added to 5 μm-thick brain sections of formalin-fixed paraffin embedded APPswe/PS1deltaE9 mice. Sections were deparaffinized and pretreated with 90% formic acid, and exogenous peroxidase was quenched. Controls used were: Staining with an antibody specific for Aβ42 (A1976, Sigma) as a positive control; staining of brain sections of a wild-type mouse (C57BL/6) as negative control (no plaques); staining with pre-absorbed immune serum (1 h at 37 °C with Aβ42 peptide, 1 μM final concentration) as a specificity control. Binding of antibodies to brain sections was detected by HRP labeled secondary antibody followed by 5–10 min development with metal-enhanced diaminobenzidine substrate (Pierce). A digital camera (Olympus, Japan) was used to capture images of the plaques at 10× magnification.
2.8. Statistical analyses
Data are expressed as means and standard deviations and a standard Student T-test was used. P values <0.05 were the criterion for statistical significance.