Evaluation of a DNA Aβ 42 Vaccine in Aged NZW Rabbits: Antibody Kinetics and Immune Profile after Intradermal Immunization with Full-Length DNA Aβ 42 Trimer

Animals and immunizations

Sixteen four- to five-year-old NZW rabbits (ten females, six males, 3.5 to 5 kg) were purchased from Harlan (Indianapolis, IN). The rabbits were randomly separated in two groups, in which one group received a high dose immunization regimen receiving 16 μg DNA per immunization time point and the other group received a low dose regimen (8 μg DNA/immunization). The intradermal DNA immunizations with the two plasmid DNAs were performed into skin of the outer ear using the Helios gene gun (Bio-Rad, Hercules, CA). In brief, DNA coated gold particles were injected onto the skin of the shaved ears with a helium pressure of 400 psi for a total of five immunizations. The first three immunizations were done in biweekly intervals, followed by two vaccinations in monthly intervals. Two month following these five immunizations, nine of the rabbits were euthanized for pathological examination and cellular immune responses. One animal had died after the fourth immunization due to bacteremia secondary to a tooth root abscess. Immunization was continued in six of the rabbits for five additional time points (total of ten immunizations). Animal experimentation was performed in accord to the guide of the ARC at UT Southwestern Medical Center. Animal use for this study was approved by the UT Southwestern Medical Center IACUC (Institutional Animal Care and Use Committee).

The plasmid system used for DNA Aβ₄₂ trimer immunization is illustrated in Fig. 1. A dual plasmid system, activator and responder plasmid is used for expression of the Aβ_1-42 trimer (3 copies of Aβ_1-42 in a row): The activator plasmid codes for the transcription factor Gal4, the responder plasmid codes for Aβ_1-42 trimer. Gal4 transcription factor transcription is driven by a CMV promoter. The Gal4 protein binds as a homodimer to UAS sites present upstream of the promoter on the responder plasmid. Gal4 binding drives transcription of the Aβ_1-42 trimer sequence containing also an Adenovirus E3 leader sequence and an endosomal targeting sequence derived from mouse MHC class II (H2-DM) (Fig. 1A). Expression of Aβ_1-42 trimer from the plasmid was verified by analysis of mouse skin which was injected with the plasmids via gene gun. Protein lysates from mouse ear (24 h following DNA immunizations) were run on a 4–20% SDS PAGE and Aβ protein expression was detected with an anti-human Aβ₄₂ antibody (Fig. 1B). UAS-monomer transfection (Mon, 1 copy of Aβ_1-42) in mouse ear resulted in a unique band at about 10 kDa, UAS-trimer (Tri) transfection resulted in double fragments at 19 kDa, and dimerized bands of these doublets at about 40 kDa, respectively. A 19 kDa protein contains the leader sequence, three copies of Aβ_1-42, and the endosomal targeting sequence (see Fig. 1A). The earlysate transfected with control DNA (Gal4/UAS-Luc) showed no protein bands detectable with the Aβ₄₂ antibody (Luc).

Antibodies and peptides

As a standard antibody to determine the anti-Aβ IgG immune response a C-terminal Aβ specific rabbit monoclonal antibody recognizing Aβ peptides ending amino acid 42 was used (clone 1-11-3, Biolegend). Other anti-Aβ antibodies used in this study were the mouse moab 6E10 (Biolegend) and rabbit A11 (Life Technologies). As comparison to antisera generated in peptide immunized rabbits we purchased polyclonal rabbit antisera detecting Aβ_1 - 15 or Aβ₄₂ from GeneTex (Irvine, CA). A panel of secondary anti-rabbit IgG antibodies was used: anti-rabbit Ig, anti-rabbit IgG (Southern Biotech, Santa Cruz), anti-rabbit IgM (Southern Biotech), anti-rabbit IgA (Pierce), and three anti-rabbit IgG moabs (Southern Biotech, SIGMA). Aβ peptides used in this study had been purchased from rPeptide, AnaSpec, New England Peptide, Bachem, and American Peptide Company. Full-length Aβ_1-42 peptides (rPeptide) were pretreated and fibrillized as described in Stine et al. [18].

Necropsy reports and histology

Nine of the rabbits underwent necropsy and histological evaluation of target tissues by a veterinarian pathologist.

Plasma and splenocyte collections

Blood was collected prior to the first, after the second, third, and fourth, and two, eight, and sixteen weeks past the fifth immunization. Antibody levels were determined from all blood samples. Blood chemistry and complete blood counts (CBC) were determined from samples prior to the first, after the fourth immunization, and from blood samples drawn eight weeks past the fifth immunization. Ten days following the final blood draw the rabbits were euthanized. Lymphocytes from blood were isolated by density separation centrifugation using Lympholyte^® Cell Separation Media (Cedarlane Labs, Burlington, ON). Spleens and bone marrow were aseptically removed and processed for tissue culture. Tissue culture was performed as previouslydescribed [10].

Antibody ELISA

Aggregated Aβ_1-42 and α-synuclein peptides were prepared as described previously [9]. Briefly, the peptide solution was prepared by adding 250 μl phosphate buffered saline (PBS pH 7.4) to 1 mg of lyophilized Aβ_1-42 (counterions TFA, NaOH, or HCl) or α-synuclein, followed by an overnight incubation at 37°C.

Anti-Aβ antibodies in rabbit plasma are measured according to standard procedures. High binding 96-well plates were coated with human Aβ_1-42 peptide (2 μg/ml) in 50 mM carbonate buffer pH 9.6 overnight at 4°C. Standard curves were included by adding serial dilutions of the rabbit anti-Aβ monoclonal antibody 1-11-3 in PBS/1.5% BSA. Plasma samples were diluted and added at concentrations ranging from 1:200 to 1:5000 to measure the antigen specific antibody concentrations. ELISAs were repeated three to four times and showed an inter-assay variability of 5–8%. Data from one ELISA werepresented.

Dot blot assays

2 μl of Aβ peptide solutions (0.5 μg/μl) were spotted in duplicates onto nitrocellulose filters and air dried. After blocking of the membranes in TBS-T containing 10% milk, further incubation with the respective antibodies (mouse- anti-human Aβ_1 - 6 (6E10) and rabbit- anti-human Aβ_x - 42 (1-11-3) both 1:10,000, or oligomeric Aβ (A11, 1:2,000) was performed overnight at 4°C. Rabbit plasma was used at a dilution of 1:200. HRP labeled secondary antibodies (anti-mouse IgG and anti-rabbit IgG) were used in dilutions of 1:50,000. Antibody binding intensities were measured with an Opti4CN detection kit as recommended by the manufacturer(BioRad).

ELISA and ELISPOT assays

ELISA assays for antibody levels and titers in rabbit plasma were performed according to standard procedures. IgG, IgA, and IgM antibody isotypes were analyzed. Secondary isotype antibodies used had been cross adsorbed with rabbit IgG or IgM, respectively (Southern Biotech). The used polyclonal anti-rabbit IgA antibody was noted to show no cross-reactivity with other rabbit Ig isotypes (Pierce). As standard a monoclonal rabbit-anti-human Aβ₄₂ IgG antibody (clone 1-11-3, Biolegend) was used. For testing of antibody avidity, a 90-min incubation step with the chaotropic salt NaCN in different molarities (5 M, 2 M, 1 M, 0.5 M, 0.25 M, 0.125 M, 0.006  M) was inserted in the ELISA protocol between the primary antibody binding and the detection step (NaCN ELISA). ELISPOT assays to determine frequencies of cytokine secreting cells were performed according to standard procedures (48 h), and as previously described using commercial available antibody sets for rabbit IFNγ, IL-17, and IL-4 (Kingfisher Biotech, Saint Paul, MN) [13, 16]. For maximal T cell stimulation, Concanavalin A (Con A) was used at a concentration of 2.5 μg/ml.

B cell ELISPOT

MultiScreen-IP plates (Millipore, Billerica, MA) were washed with 70% ethanol, rinsed three times with phosphate-buffered saline (PBS), and then coated with 100 μl of Aβ₄₂ (4 μg/ml) in PBS at 4°C overnight. Plates were washed six times with PBS and blocked with RPMI with 2% BSA for 2 h at room temperature. Rested splenocytes and bone marrow cells were plated in 200 μl of medium and incubated 48 h at 37°C. Plates were washed six times in PBS with 0.25% Tween 20 (Sigma) and incubated with 100 μl of 1:250 HRP conjugated goat-anti-rabbit IgG or goat-anti-rabbit IgA for 1 h at room temperature. The plates were again washed with PBS-T and a final wash in PBS, and developed with 100 μl of AEC substrate for 20 to 30 min at room temperature, and the reaction was stopped with deionized water. Spots were air dried and automatically counted with an ELISPOT reader.

Analysis of cell proliferation by CFSE dilution

Cells for labeling were re-suspended in PBS and immediately mixed with an equal volume of CFSE diluted in PBS (1:1). The cells were incubated for 5 min at room temperature with repeated mixing to obtain an even CFSE labeling of all cells. The labeling was stopped by adding of an equal volume of complete RPMI medium and removal of the solution from the cells by centrifugation. The cells were plated in round-bottom 96-well cell culture plates at concentrations of 1×10⁶ cells per well and re-stimulated with Aβ₄₂ peptide or ConA for six days. After harvesting cells were re-suspended in FACS buffer (PBS/1% BSA/ 0.1% NaN₃) and stained with a mouse-anti-rabbit CD4 antibody (clone clone RTH1A, Kingfisher) followed by an APC conjugated rat anti-mouse IgG1 antibody (eBioscience). Fluorescence of the cells was measured using an Accuri C6 Flow Cytometer (Ann Arbor, MI) and analyzed with CFlow Plus and FCS Express Version 3 software programs.

AD mouse model brain immunohistochemistry

For plaque staining formalin fixed brain sections were treated with 70% formic acid for antigen retrieval. Non-specific staining was blocked by a 1-h incubation in 2% BSA/2% goat normal goat serum/0.3% Triton X100 in PBS at room temperature. Tissues were then incubated with primary antibodies (rabbit plasma, dilution 1:250) or control antibody (McSA1(Medimabs, Canada), 1:500) overnight at 4°C. Secondary antibody binding (goat-anti-rabbit IgG HRP, goat-anti-mouse IgG HRP) was detected with a 3,3’-diaminobenzidine (DAB) substrate kit. Sections were scanned using a NanoZoomer digital pathology system and analyzed with NDP viewer software (Hamamatzu, Japan).

Statistics

For statistics (unpaired t test with two tailed p values, Mann-Whitney t test, column statistics), we used GraphPad Prism version 6 for Windows (San Diego, CA, http://www.graphpad.com). p-values of≤0.05 were considered significant.

Blood work and general histology

Blood from the sixteen rabbits used in this study was tested before the vaccination and following the 4th vaccination for signs of inflammation (CBC), or signs of metabolic changes (blood chemistry). No changes were observed (data not shown). All nine rabbits undergoing necropsy were in good body condition with adequate body fat stores and no appreciable postmortem autolysis. No gross abnormalities were found. Two female rabbits had small cysts in their kidneys; these cysts in the renal cortex are not uncommon in aged rabbits. Two other female rabbits had developed uterine leiomyoma, which again is likely due to the age of these rabbits, and the fact that they were retired breeders. Three different HE stained brain regions were examined and showed no sign of lymphocyte infiltration or inflammation (Fig. 2). One female rabbit of the six remaining animals that received continued immunizations (10x) had a mammary adenocarcinoma, which was surgically removed. This animal was euthanized later due to the development of a tooth root abscess of the premolars. The pathology report for this rabbit showed metastasis of the adenocarcinoma to the uterus. The brain showed no sign of inflammation. Two male rabbits (both seven years old) died after this study was completed. One of them died 3 months after the last immunization. Necropsy and histological examinations showed no cause of death (undetermined). The brain of this rabbit showed no signs of inflammation. The second male rabbit was euthanized 4 months after the 10th immunization due to a soft tissue tumor (basal cell carcinoma) on one of his hind legs.

Four out of 12 analyzed rabbit brains showed positive staining with Prussian Blue (one to three cortical lesions, data not shown).

Antibody immune response in DNA Aβ₄₂ immunized rabbits

Antibody levels and total antibody amounts were measured for anti-Aβ₄₂ antibodies of the IgG, IgA, and IgM isotypes. In blood samples taken prior to the first immunizations, baseline levels of anti-Aβ₄₂ antibody levels were found (< 20 μg/ml plasma). An analysis of the antibody titers found in the rabbits after three immunizations demonstrated the development of an anti-Aβ₄₂ IgG antibody response. In Fig. 3, the representative titers for two rabbits which had received high dose DNA Aβ₄₂ immunizations, and two rabbits which had received the low dose immunization are shown (Fig. 3A). Half maximal binding was determined for rabbit 828D as 1:25,000, 1:3,200 for rabbit 831D, 1:12,800 for rabbit 833D, and 1:1,600 and 1:5,000 for rabbit 858D. This second increase of antibody binding to Aβ_1-42 at higher dilutions of the plasma samples is indicative of a second antibody epitope, which becomes more accessible at higher antibody dilutions due to antibody hinderance and steric competition at lower plasma dilutions. Similar findings were made in all of the sixteen rabbits. Further analyses to the epitope binding of the anti-Aβ₄₂ antibodies produced in these four rabbits showed a strong binding to full-length Aβ_1-42 peptide, and much lesser binding to a panel of shorter Aβ peptides (linear epitopes) spanning this peptide at this early time point (Fig. 3B). Shown are the binding pattern for the antibodies from the same rabbits for which the antibody titer analysis is depicted, but similar results were obtained for all sixteen animals. The binding to the linear epitopes at this early time point (3 immunizations) is low in rabbits 828D and 831D, much higher in rabbit 833D, and antibodies produced in rabbit 858D bound stronger to a mid-region Aβ peptide, Aβ_10 - 26 and Aβ_17 - 31, than to the typical B cell epitope of Aβ₄₂, which is Aβ_1 - 16 (Fig. 3B).

Antibody levels increased to mean antibody levels of 134.4±47.14 μg/ml plasma after 3 immunizations, and to 277.1±118.4 μg/ml plasma after 5 immunizations. No significant differences were found between the low dose (8 μg DNA/immunization) and high dose group (16 μg DNA/immunization) (Fig. 4A). Antibody levels declined slightly to 246.6±109.5 μg/ml (n = 15, p = 0.461) after a resting period of two month following the 5th immunization. The analyses of antibody producing plasma cells in spleen from the immunized rabbits confirmed that a good humoral immune response had been elicited in the rabbit model with the DNA vaccine. Anti-Aβ_1-42 IgG antibody producing plasma cells were found in these cultures with mean values of 103.7 anti-Aβ₄₂ IgG antibody forming units in splenocytes from nine rabbit analyzed (p > 0.0001 compared to controls with 14.4 antibody-forming cells AFC/10⁶ cells (Fig. 4B). Splenocyte cell culture supernatant (48 h) contained an average of 0.2 μg anti-Aβ₄₂ IgG antibody/ml medium confirming the presence of Aβ₄₂ specific plasma cells in spleens of DNA Aβ₄₂ immunized rabbits(Fig. 4C).

Antibody epitopes were determined for all sixteen rabbits following the 4th immunization. Anti-Aβ antibodies of the IgG (Fig. 4D), IgA (Fig. 4E), and IgM (Fig. 4F) isotypes bound to a wide panel of small Aβ peptides including N-truncated (Aβ_3 - 42, Aβ_4 - 17, Aβ_5 - 17, Aβ_6 - 17) and pyroglutamate modified Aβ peptides (³Pyr-Aβ_3 - 42 and ¹¹Pyr-Aβ_11 - 42).

In the six rabbits, which received continued immunizations in monthly intervals, antibody levels did not increase further (Fig. 5). These six rabbits had anti-Aβ₄₂ IgG antibody levels of 247.9±57.63 μg/ml plasma after five DNA immunizations, and an antibody level of 246.4±50.29 μg/ml after ten DNA Aβ₄₂ immunizations. Following the stop of further vaccinations, the antibody levels declined to 162.7±32.5 μg/ml after a two-month period, and to 105±37.58 μg/ml after a four-month period (Fig. 5A). From these data, the antibody half-life was calculated as three months (Fig. 5B).

Linear anti-Aβ B-cell epitopes are distributed over the entire amino acid sequence Aβ_1 - 40

For characterization of the linear anti-Aβ specific B-cell epitopes, we used a panel of shorter Aβ peptides spanning the Aβ_1 - 40 sequence, Aβ_1 - 16, Aβ_6 - 20, Aβ_10 - 26, Aβ_17 - 31, Aβ_22 - 35, and Aβ_29 - 40, in a direct ELISA assay. Antibody epitopes were determined from antibodies of 3x, 5x, and 10x immunized rabbits, as well as from the antibodies following the two or four-month interval (plasma dilutions 1:400 (3x), Fig. 3B; plasma dilutions 1:1000 (5x, 10x), Fig. 5C-F). These data show a wide epitope binding, detecting epitopes throughout the smaller 11–16 amino acid long peptides spanning the entire Aβ_1 - 40 sequence.

Antibody isotypes

The rabbit humoral immune response consists of four antibody isotypes: IgM, IgG, IgE, and IgA. While humans and mice have four or five IgG subclasses respectively, there is only one IgG subclass in the rabbit. We analyzed the binding of antibody isotypes, IgM, IgG, and IgA to full-length Aβ_1-42, N-truncated (Aβ_4 - 42, Aβ_5 - 42) and Pyroglutamated Aβ peptides (³PyrAβ_3 - 42 and ¹¹PyrAβ_11 - 42) in DNA Aβ₄₂ immunized rabbits (Fig. 6A). Commercially available rabbit antisera against Aβ_1 - 15 and Aβ_1-42 showed similar binding of antibody isotypes indicative that the rabbit produces all three isotypes in an immune response (data not shown). The binding to Aβ_4 - 42 or Aβ_5 - 42 and ³PyrAβ_3 - 42 contained mainly IgG and IgM antibodies and significantly less IgA antibodies (p values of 0.0317, 0.022, 0.022, Mann-Whitney). In Fig. 6B, the results for IgG antibody binding to Aβ_1-42, Aβ_4 - 42, Aβ_5 - 42, and ³PyrAβ_3 - 42 from six rabbits are shown. When the rabbit sera were tested against a different brain antigen, α-synuclein, they displayed the same binding pattern: strong binding of IgG and IgM antibody isotypes and significant less binding of antibodies with the IgA isotype. From the two commercially available antisera (anti-human Aβ1–15 and anti-human Aβ_1-42), only the serum with Aβ_1-42 specificity contained some IgG antibodies binding to Aβ_4 - 42, Aβ_5 - 42, ³PyrAβ_3 - 42, and α-synuclein (Fig. 6B).

Since there is only one IgG antibody isotype in the rabbit and a lack of information about additional biological features of the antibodies (differences in complement binding or Fc receptor binding), it is not possible to classify the immune response based on isotyping data in this species.

Dot blot assay and avidity ELISA

For further specification of the antibody binding to Aβ, a dot blot assay was performed. Peptides were spotted on nitrocellulose membranes and probed with the commercially available antibodies 6E10, 1-11-3, and A11, or the different sera from rabbits immunized in this study. As shown in Fig. 7A, the 6E10 antibody (Aβ_1 - 16) bound well to Aβ_1-42 and Aβ_1 - 40, while 1-11-3 bound to Aβ_1-42, and not to Aβ_1 - 40 consistent with the epitope specificity of this antibody in the C-terminal end of the Aβ_1-42 peptide. A11 bound weakly to Aβ_1-42/HFIP and Aβ_1 - 40 indicative of some degree of oligomeric Aβ conformers in these preparations. Antibodies in the six rabbit plasma samples bound well to Aβ_1-42 and Aβ_1 - 40. Also, all six rabbit plasma samples bound to α-synuclein with the highest intensity found for rabbit 858D, and did not bind to amylin, another amyloid peptide.

In an ELISA protocol using different molarities of the chaotropic salt NaCN to test for antibody binding avidities, the antigen binding specificities seen in the dot blot were confirmed (plasma dilutions 1:500, Fig. 7B). All rabbit plasma samples bound with high avidity to Aβ_1-42 and the deletion mutant of Aβ_1-42 lacking residue 22 (Δ22 Aβ_1-42, not shown in the dot blot). Avidity of binding for the rabbit antibodies was compared to the control antibodies 6E10 and 1-11-3. High avidity to Δ22 Aβ_1-42 was found for moab 11-3-1, the rabbit plasma antibodies bound to Δ22 Aβ_1-42 with similar strength as moab 6E10. Highest Aβ_1 - 40 binding was detected for the moab 6E10 with a substantial decrease of binding at NaCN concentration of 0.5 M. No binding was found for moab 1-11-3 consistent with the specificity of the antibody for Aβ peptides ending in residue Aβ₄₂. The six rabbit plasma samples showed an intermediate avidity to Aβ_1 - 40 with loss of binding at NaCN concentrations at 0.5 M, except plasma antibodies from 828D which showed loss of binding at 1 M NaCN. No binding to α-synuclein was found for moabs 6E10 and 1-11-3. The six rabbit plasma samples tested in this assay showed similar avidities in binding to α-synuclein with loss of binding at salt concentrations of 0.5 M NaCN. Plasma antibodies of 858D, which showed a strong signal in the dot blot assay (Fig. 6A), also showed the strongest binding to α-synuclein in the NaCN ELISA; binding was clearly detectable at concentrations up to 1 M NaCN (Fig. 7B).

Cross-reactivity of rabbit plasma samples with α-synuclein

The α-synuclein binding was further evaluated in a series of ELISA assays. Plasma samples from 15 DNA Aβ₄₂ immunized rabbits were tested after the 5th immunization time point for binding to Aβ_1-42, full-length α-synuclein (1–140), a deletion mutant α-synuclein/Δ61–95, α-synuclein 1–60, amylin, which is a 37 aa peptide, IAPP (Islet Amyloid Peptide), which is a 5 aa peptide, and SAP (Serum Amyloid P component), which is a 6 aa peptide, and a mutant Aβ_1-42 peptide which lacks residue 22 (Δ22Aβ_1-42), which has been described as highly oligomeric [19] (plasma dilution 1:500, Fig. 8A), High binding was found for Aβ_1-42, Δ22Aβ_1-42, and all three α-synucleins, low binding was found for amylin, SAP, and IAAP (p values (Mann-Whitney test) for the comparison of binding to Aβ_1-42 and the latter three peptides was p < 0.0001 and 0.0007, respectively). From these 15 samples the data of the six rabbits, which received continued immunizations (ten times) were extracted and shown in Fig. 8B to allow a better comparison with the data showing the cross-reactivity following the 10th immunization (Fig. 8C).

Analyses of T cell responses in the immunized rabbits

IFNγ secretion was determined by an ELISPOT assay for all of the rabbits (n = 15) one week following the 5th immunization from peripheral blood lymphocytes. No IFNγ secreting cells were detected after Aβ₄₂ peptide re-stimulation while high numbers of IFNγ spots were found in wells which had been stimulated with ConA (data not shown). Further T cell responses were determined from splenocytes in eight of the rabbits four months following the fifth DNA Aβ_1-42 immunization by ELISPOT assays for IFNγ, IL-17, and IL-4 (Fig. 9) and a CFSE proliferation assay (Figs. 10 and 11).

The analyzed rabbits showed high variability in the cellular assays between the individual animals, which was expected since the cohort consisted of aged non-inbred breeders. The ELISPOT assays showed low numbers of IFNγ secreting cells but with significant differences in response to Aβ₄₂ peptide re-stimulation in three of the eight rabbits (831D, 835D, and 855D). The IL-17 ELISPOT showed increased numbers of IL-17 secreting cells in all eight rabbits analyzed, and again for three of the rabbits significantly increased numbers of IL-17 secreting cells were present upon peptide re-stimulation (830D, 833D, and 836D). IFNγ and IL-17 producing cells were not overlapping as they were found in different animals. No significant differences between medium controls and Aβ₄₂ peptide re-stimulated splenocyte cultures were found in regard to IL-4 secreting cells.

In the CFSE proliferation assay, high background levels with unspecific proliferation in medium control wells was found for rabbits 830D, 835D, and 855D. Two rabbits, 833D and 835D, showed significantly increased levels of CD4 T cell proliferation in response to the Aβ₄₂ peptide re-stimulation in vitro, which increased from 17% in medium to 25% in Aβ₄₂ peptide containing medium, or 8% to 22% in the second rabbit, respectively. The remaining three rabbits, 829D, 831D, and 836D showed no significant increase in T cell proliferation following Aβ₄₂ peptide re-stimulation (Fig. 10). Representative histograms for the CFSE proliferation are shown in Fig. 11A-G. Figure 11A shows the gating (gate R1) on proliferating of CD4+ T cells following Aβ₄₂ peptide re-stimulation of splenocytes in culture of rabbit 833D, which possessed 28.7% of proliferating CD4 T cells (Figs. 10, 11A, and 11B). Further analyses of these cells with the FCS Express Version 3 software program (shown in Fig. 11C, D), indicated that the clouds of cells seen in Fig. 10A are likely only noise, which do not form separated peaks of dividing cells, which is shown for the CD4 T cell populations following ConA stimulation in Fig. 11G. Only one clear peak for the undivided cells is seen (grey peak in Fig. 10C, D). In Fig. 11E-G, the same analyses were performed for a different rabbit, 831D, which showed no CD4 proliferation in Fig. 10. Also for this rabbit only one peak for the undivided cells can be found (Fig. 11E medium control, Fig. 11F Aβ₄₂ peptide re-stimulation), but the histogram from the ConA stimulated splenocytes of this rabbit formed five clearly separated peaks of dividing cells (black) and one small peak of undivided cells (grey, Fig. 11G). Similar findings of background noise and no clearly dividing cell populations were made in the detailed proliferation analysis of the other six rabbits (data not shown). All rabbits showed high levels of CD4 T cell proliferation in response to ConA stimulation (positive control).

Anti-Aβ₄₂ antibodies from immunized rabbits stained senile plaques in brain sections of 3xTg-AD mice

The staining of brain sections from a 3xTg-AD mouse (B6;129-Psen1^tm1Mpm Tg(APPSwe,tauP301 L)1Lfa/Mmjax) with plasma from the rabbits, which had received DNA Aβ₄₂ immunizations, showed clear staining in the typical areas in which amyloid plaques are present in this AD mouse model, frontal cortex and subiculum of the hippocampus (hippocampus shown in Fig. 12A). In the comparison to a commercial anti-Aβ₄₂ antibody (McSA1, shown in Fig. 12B), we found a substantial overlap of detected amyloid plaques in the areas stained with both antibodies (indicated with blue and green asterisks). In addition to the overlapping areas, unique staining pattern can be identified for both of the antibodies as well. Brain sections from this 3xTg-AD mouse showed no staining with control plasma from a non-immunized rabbit (Fig. 12C). No staining was found in plaque free areas as shown for the cerebellum (Fig. 12D-F).

Conclusions

Positive results for antibody production after DNA gene gun immunization in large mammals has been missing, and the data presented here show that DNA Aβ₄₂ immunization induced a good humoral immune response in aged NZW rabbits, which possess the same Aβ_1-42 sequence as humans. The rabbit skin at the injection site showed no signs of inflammation or irritation in any of the vaccinated animals, and because the rabbit skin is an optimal target to test chemical agents for irritation, it is highly likely that human skin will show the same results following DNA gene gun vaccinations. Brains from the analyzed rabbits showed no sign of inflammation. The humoral immune response was polyclonal and multivalent; detecting N-terminal truncated Aβ peptides as well as several mid-region Aβ peptides very similar to our findings in the mouse [29]. Full-length DNA Aβ₄₂ vaccination into the skin is likely to induce a good humoral immune response in humans as well.