Characterization and preclinical evaluation of the cGMP grade DNA based vaccine, AV-1959D to enter the first-in-human clinical trials

Abstract

The DNA vaccine, AV-1959D, targeting N-terminal epitope of Aβ peptide, has been proven immunogenic in mice, rabbits, and non-human primates, while its therapeutic efficacy has been shown in mouse models of Alzheimer's disease (AD). Here we report for the first time on IND-enabling biodistribution and safety/toxicology studies of cGMP-grade AV-1959D vaccine in the Tg2576 mouse model of AD. We also tested acute neuropathology safety profiles of AV-1959D in another AD disease model, Tg-SwDI mice with established vascular and parenchymal Aβ pathology in a pre-clinical translational study. Biodistribution studies two days after the injection demonstrated high copy numbers of AV-1959D plasmid after single immunization of Tg2576 mice at the injection sites but not in the tissues of distant organs. Plasmids persisted at the injection sites of some mice 60 days after vaccination. In Tg2576 mice with established amyloid pathology, we did not observe short- or long-term toxicities after multiple immunizations with three doses of AV-1959D. Assessment of the repeated dose acute safety of AV-1959D in cerebral amyloid angiopathy (CAA) prone Tg-SwDI mice did not reveal any immunotherapy-induced vasogenic edema detected by magnetic resonance imaging (MRI) or increased microhemorrhages. Multiple immunizations of Tg-SwDI mice with AV-1959D did not induce T and B cell infiltration, glial activation, vascular deposition of Aβ, or neuronal degeneration (necrosis and apoptosis) greater than that in the control group determined by immunohistochemistry of brain tissues. Taken together, the safety data from two different mouse models of AD substantiate a favorable safety profile of the cGMP grade AV-1959D vaccine supporting its progression to first-in-human clinical trials.

Keywords: Alzheimer's disease (AD); Biodistribution; DNA Aβ-vaccine; Immunohistochemistry (IHC); Magnetic resonance imaging (MRI); Mouse models of AD; Safety toxicology.

Fig. 1.

Biodistribution of AV-1959D followed by EP-mediated single administration in male (A) and female (B) Tg2576 mice. Tissue samples were taken and processed 2 days after the single administration. Plasmid copy numbers were determined by qPCR. Primers and probe were designed to detect 76 bp sequence present in AV-1959D plasmid in the region encoding MultiTEP. Individual animal data and geometric mean values are provided in a log scale. The black dotted line indicates the limit of detection, LOD. The blue dotted line indicates the limit of quantification LOQ of qPCR assay performed in this study. The red dotted line indicates the LOQ required by FDA (FDA, 2007) and the upper limit, which does not require testing of the plasmid persistence at the later point.

Fig. 2.

Dynamics of humoral immune responses in Tg2576 mice vaccinated with AV-1959D/EP at doses of 5 μg/mouse (n = 10F/10M), 25 μg/mouse (n = 10F/10M) and 50 μg/mouse (n = 10F/10M) or injected with PBS (n = 10F/10M) for our acute and long-term safety/toxicology study. Sera were collected 14 days after the third (on Day 56) and the fourth (on Day 86) immunizations, as well as at sacrificial end-points (Days 76, 102 and 158). Concentrations of anti-Aβ antibodies were determined by ELISA as described in Materials and Methods and calculated using a calibration curve generated with 6E10 monoclonal antibody. Each point represents the mean value of antibody concentration (average ± SEM) in a group (n = 10).

Fig. 3.

AV-1959D vaccinations generated humoral immune responses in all Tg-SwD/I mice. Concentrations of anti-Aβ antibodies in vaccinated male and female mice on Day 2 (n = 8F/8M) and Day 14 (n = 8F/7M) after the last immunization were determined by ELISA as described in Materials and Methods and calculated using a calibration curve generated with 6E10 monoclonal antibody. The lines represent mean values of antibody concentrations.

Fig. 4.

No vasogenic edema was detected by magnetic resonance imaging (MRI) in control and vaccinated Tg-SwD/I mice. (A) Representative T2WI and T2 Map images from males/females and control/immunized Tg-SwDI mice (n = 8/group) at day 2 immunization. No overt increases in edema were observed visually. (B) No significant changes in edema (T2 values) were observed among experimental groups at day 2 immunization. (C) At day 14 post immunotherapy, there were no significant differences between control and immunized males. (D) Whole-brain volumes were also assessed, and while no differences in brain volume were found between control and immunized groups (either males or females), control males had significantly smaller brain volumes than females (control and immunized) (two-way ANOVA, Tukey's multiple comparisons, *p < .05). (E) Similarly, ventricular volumes (3rd, 4th ventricles) were also quantified, but no significant differences between male and female, nor control vs. immunized mice were observed (two-way ANOVA, Tukey's multiple comparisons).

Fig. 5.

SWI hypointensities. (A) Representative SWI images at days 2 and 14 posttreatment in male/female and control/immunized Tg-SwDI mice (n = 8/group). No differences were apparent visually. (B) Anterior-posterior quantification of the SWI hypo-intensities at day 2 post-immunization revealed no significant differences between male and female groups (two-way ANOVA, Tukey's multiple comparisons). C) The average number of hypo-intensities over the whole brain was assessed at day 2 within the brains of treated Tg-SwDI mice and did not differ among groups. (D) There were no significant differences in SWI hypo-intensities at day 14 post-immunization in males and females. However, both control and immunized males at the level of the dorsal hippocampus had significantly increased hypo-intensities (slice 5) compared to control and immunized females (two-way ANOVA, Tukey's multiple comparisons, ***p < .001). E) When the average numbers of hypo-intensities were evaluated at day 14 after treatment across all MRI slices, no significant differences were observed, n = 8/group.

Fig. 6.

Prussian blue comparisons to SWI hypo-intensities. (A) Representative SWI and Prussian Blue images at day 14. Increased magnification of the original SWI and Prussian Blue images (1.25, 10 and 20 ×) illustrate the appearance of numerous hypo-intensities and Prussian Blue puncta and aggregates. The signal loss (dark gray, black pixels) in the SWI MRI images as a result of iron deposition was confirmed by the high-magnification Prussian Blue histology. The locations of the SWI and the positive Prussian Blue profiles overlapped reasonably well. Histological sections have considerably higher densities of Prussian Blue compared to SWI due to micron vs. mm resolution. As in Fig. 5, males at the level of dorsal hippocampus had increased hypo-intensities and the appearance of increased numbers and larger size of puncta and aggregates, and Prussian Blue staining confirmed these findings. Females tended to have fewer and smaller hypo-intensities and Prussian Blue staining puncta. Data shown are from SWI minimum intensity projections. Male and female mice shown are from control Tg-SwDI mice, and immunized mice showed very similar patterns. (B) Summation of all Prussian Blue puncta observed on 3 histological sections spanning the MRI slice confirmed no significant differences between any of the experimental groups at day 14 post-immunization.

Fig. 7.

Number of microhemorrhages in the brains of Tg-SwDI mice vaccinated with AV-1959D. (A) No differences in the number of microhemorrhage profiles in mice were ben detected between control (n = 7/gender/time point) and vaccinated animals (n = 8/gender/time point) terminated at days 2 and 14 after the last immunization. Bars represent average ± SEM. (B) Photomicrographs show representative images of the coronal brain sections of immunized and control mice stained with Prussian Blue. Encircled areas indicate Prussian Blue profiles in CA1 (field CA1 of hippocampus), cc (corpus callosum), Cx (cortex), Th (thalamus). Scale bars: low magnification =2.5 mm, enlarged images =100 μm

Fig. 8.

No increase in vascular deposition of Aβ was observed in AV-1959D/EP vaccinated Tg-SwDI mice of H2^b haplotype compared with control animals 2 and 14 days after the last immunization. (A) 10–11 mo old Tg-SwDI mice were vaccinated with 50 μg/mouse of AV-1959D at days 0, 14, 44, 74 and mice were terminated at days 2 and 14 after the last immunization (n = 8/gender/time point for experimental and 7/gender/time point for control groups). Brain sections were stained with ThS followed by the quantitative data analyses of the punctate ThS-stained blood vessel profiles in the CAA-prone hippocampal and thalamic regions of mouse brains. Bars represent average ± SEM. (B) Photomicrographs depict representative half brain slices showing hippocampal and thalamic areas on the same plane for every analyzed group. Boxed areas depict high-power views of CAA profiles in subiculum and thalamus. Scale bars: low magnification = 1 mm, enlarged images = 50 μm.

Fig. 9.

No increase in microglia activation was observed in AV-1959D/EP vaccinated Tg-SwDI mice compared with control animals 2 and 14 days after the last immunization. (A) Bars represent the average percent of Iba-1 positive area ± SEM. (B) Photomicrographs of representative images show reactive microglia in the hilar region of dentate gyrus vs. hyper-ramified microglial profiles in the adjacent layers in the brains of immunized (n = 8/gender/time point) and control (n = 7/gender/time point) female (F) and male (M) mice. Scale bars = 100 μm.

Fig. 10.

No increase in astrocyte activation was observed in AV-1959D/EP vaccinated mice compared to control animals 2 and 14 days after the last immunization. (A) Bars represent the average density of GFAP positive cells ± SEM. (B) Photomicrographs of diffuse reactive astrogliosis in the hilar region of dentate gyrus with pronounced upregulation of GFAP expression and overlap of the astrocyte processes vs. mild to moderate reactive astrogliosis in adjacent layers in the brains of immunized (n = 8/gender/time point) and control (n = 7/gender/timepoint) female (F) and male (M) mice. Scale bars = 100 μm.

Fig. 11.

No increased infiltration of T and B cells was observed in AV-1959D/EP vaccinated mice compared with control animals 2 and 14 days after the last immunization. Table represents average number ± SEM of the infiltrating lymphocytes per brain section. Photomicrographs of the brain sections from immunized (n = 8/gender/time point) and control (n = 7/gender/timepoint) mice stained with anti-CD3 and anti-B220. High-power views are taken from the circled areas: S1 (primary somatosensory cortex), RS (retrosplenial cortex), Th (thalamic nucleus), DG (dentate gyrus), Hb (habenular nucleus). Scale bars: low magnification = 2.5 mm, enlarged images =100 μm.

Fig. 12.

No increase in the number of necrotic/apoptotic cells was observed in AV-1959D/EP vaccinated mice compared with control animals 2 and 14 days after the last immunization. The Table summarizes the average number ± SEM of TUNEL-positive cells and anti-caspase-3-positive area per brain section. Photomicrographs of brain sections from immunized (n = 8/gender/time point) and control (n = 7/gender/timepoint) mice analyzed via TUNEL for nuclear fragmentation and formation of apoptotic bodies, and anti-caspase-3 immunostaining for apoptosis. High-power images taken from circled areas: DLG (dorsal lateral geniculate nucleus of thalamus), PtA (parietal association cortex), CA1 (field CA1 of hippocampus), S1 (primary somatosensory cortex), cc (corpus callosum), DG (dentate gyrus). Scale bars: low magnification =2.5 mm, enlarged images =100 μm.