Doubly Phosphorylated Peptide Vaccines to Protect Transgenic P301S Mice against Alzheimer s Disease Like Tau Aggregation
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1 , 1-11 manuscripts; doi: /vaccines20x000x Article OPEN ACCESS vaccines ISSN X Doubly Phosphorylated Peptide Vaccines to Protect Transgenic P301S Mice against Alzheimer s Disease Like Tau Aggregation Monique Richter 1,2, Agneta Mewes 1,2, Manuela Fritsch 1,2, Ute Krügel 3, Ralf Hoffmann 1,2 and David Singer 1,2, * Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Leipzig 04103, Germany Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig 04103, Germany Rudolf Boehm Institute for Pharmacology and Toxicology, Universität Leipzig, Leipzig 04107, Germany Supplementary Files Content Figure S1. Illustration of hippocampal regions quantified after immunohistochemistry using phosphorylation-dependent antibodies. Figure S2. Representative immunoblots of brain homogenates of P301S mice for analysis of total tau and phospho-tau. Total tau was stained by mab Tau5 and phospho-tau with mabs AT8, AT100 and AT180. Table S1. IgG titers of mice immunized with Tau [ps202/pt205], (BT = behavioral test, x =no blood sample available, IR = immune response). Table S2. IgG titers of mice immunized with Tau [pt212/ps214], (BT = behavioral test, x =no blood sample available, IR = immune response). Table S3. IgG titers of mice immunized with Tau [pt231/ps235], (BT = behavioral test, x =no blood sample available, IR = immune response). Table S4. IgG 1/2a/2b/2c titers of mice immunized with Tau [ps202/pt205]. Table S5. IgG 1/2a/2b/2c titers of mice immunized with Tau [pt212/ps214]. Table S6. IgG 1/2a/2b/2c titers of mice immunized with Tau [pt231/ps235].
2 S2 Figure S1. Illustration of hippocampal regions quantified after immunohistochemistry using phosphorylation-dependent antibodies. Exemplarily shown is the hippocampal formation of an untreated P301S mouse (48 weeks of age) using mab AT8 (Tau[pS202/pT205]). Within the hippocampal formation, the pyramidal cell layer of region CA1 (blue), CA2/3/4 (black) and the granular cell layer of the dentate gyrus (red, DG) are marked (left). In the indicated areas cells positively stained for phospho-tau were counted as exemplified in the magnified picture (right). Figure S2. Representative immunoblots of brain homogenates of P301S mice for analysis of total tau and phospho-tau. Total tau was stained by mab Tau5 and phospho-tau with mabs AT8, AT100 and AT180. Samples S1 S3: mice immunized with Tau [pt212/ps214], S4 S10: mice immunized with Tau [pt231/ps235] and S11 S13: placebo-treated P301S mice. As reference (Ref) an untreated P301S mouse was included. Soybean trypsin inhibitor (SBTI) was used as external standard.
3 S3 Table S1. IgG titers of mice immunized with Tau [ps202/pt205], (BT = behavioral test, x = no blood sample available, IR = immune response). Mouse number PreImmune Priming 1. Boost 2. Boost 1 month 2 month 3 month 4/5 month 6/7 month final Weeks after immunization /27 31/35 38 Age of mice (weeks) /37 41/ , , ,000 10,000 10, , , ,000 10, , , ,000 10,000 10, , ,000 10, , , , ,000 10,000 10, , ,000 BT 100,000 10,000 10,000 10,000 10, ,000 10,000 BT 10, x x x , ,000 10,000 BT , ,000 BT 100,000 10,000 10,000 10, x , ,000 10,000 10, , ,000 10,000 10,000 10,000 10, , , , , ,000 10,000 10,000 10, x ,000 10, , , , ,000 10,000 10,000 10, , , , ,000 10,000 10, ,000 10, ,000 10,000 10,000 10, ,
4 S4 Table S1. Cont. Mouse Number PreImmune Priming 1. Boost 2. Boost 1 month 2 month 3 month 4/5 month 6/7 month Final Weeks after Immunization /27 31/35 38 Age of Mice (Weeks) /37 41/ ,000 10, ,000 10,000 10,000 10, ,000 10, ,000 10,000 10, , ,000 10,000 10,000 10, ,000 10,000 10,000 10, Total number of mice n = 39 n = 40 n = 40 n = 40 n = 36 n = 40 n = 40 n = 28 n = 17 n = 19 Total number of mice with IR n = 0 n = 15 n = 25 n = 26 n = 24 n = 27 n = 26 n = 19 n = 13 n = 14 [%] of mice with IR 0% 37.5% 62.5% 65% 66.7% 67.5% 65% 67.8% 76.5% 73.7%
5 S5 Table S2. IgG titers of mice immunized with Tau [pt212/ps214], (BT = behavioral test, x = no blood sample available, IR = immune response). Mouse Number PreImmune Priming 1. Boost 2. Boost 1 month 2 month 3 month 4/5 month 6/7 month Final Weeks after Immunization /27 31/35 38 Age of Mice (Weeks) /37 41/ , ,000 10,000 10, , , , ,000 10,000 10,000 10, ,000 10, x x x x x , , ,000 10,000 10,000 x x ,000 BT ,000 BT 10, , ,000 10,000 10, x , ,000 10,000 10,000 10, , , , , ,00 10,000 10,000 x , , ,000 10,000 10,000 10, , ,000 10,000 10,000 10,000 10,000 10, x x 10, x , , , ,000 10,000 10, ,000 10, , ,
6 S6 Table S2. Cont. Mouse Number PreImmune Priming 1. Boost 2. Boost 1 month 2 month 3 month 4/5 month 6/7 month Final Weeks after Immunization /27 31/35 38 Age of Mice (Weeks) /37 41/ ,000 10, x , , ,000 10,000 10, ,000 10, , ,000 10,000 10, , ,000 10, , ,000 10, , , ,000 10,000 10,000 10, x ,000 10, ,000 10,000 10,000 10, ,000 10,000 10,000 10,000 10,000 Total number of mice n = 40 n = 40 n = 40 n = 40 n = 35 n = 39 n = 38 n = 27 n = 16 n = 19 Total number of mice with IR n = 2 n = 9 n = 35 n = 40 n = 34 n = 38 n = 37 n = 24 n = 14 n = 17 [%] of mice with IR 5% 22.5% 77.8% 100% 97.1% 97.4% 97.4% 88.9% 87.5% 89.5%
7 S7 Table S3. IgG titers of mice immunized with Tau [pt231/ps235], (BT = behavioral test, x =no blood sample available, IR = immune response). Mouse Number PreImmune Priming 1. Boost 2. Boost 1 month 2 month 3 month 4/5 month 6/7 month Final Weeks after Immunization /27 31/35 38 Age of Mice (Weeks) /37 41/ , , ,000 10,000 10, , , , ,000 10,000 10, ,000 10,000 10, , , ,000 10,000 x x x 10, , ,000 BT 10, ,000 BT , , ,000 BT 100, , ,000 10, ,000 10,000 BT 10,000 10,000 10,000 10, , ,000 BT , , , ,000 10,000 10,000 10,000 10,000 10, , , ,000 10,000 10,000 10,000 10,000 10, , ,000 10,000 10,000 10, , ,000 10, , , , ,000 10,000 10,000 10,000 10, ,000 10, , , , , ,000 10,000 10,000 10, , , , ,000 10,000 x x x 0 10,000 10,000 10,000 10, x ,000 10,000 10, ,000 10,000 10, x , ,000 10,000 10, ,000 x x x x x
8 S8 Table S3. Cont. Mouse Number PreImmune Priming 1. Boost 2. Boost 1 month 2 month 3 month 4/5 month 6/7 month Final Weeks after Immunization /27 31/35 38 Age of Mice (Weeks) /37 41/ , , ,000 10,000 10, , , , ,000 10,000 10, , , ,000 10,000 10, ,000 10, ,000 10,000 10, ,000 10, ,000 10,000 10, ,000 10,000 10,000 10, , , ,000 10,000 10, ,000 10,000 10, ,000 10,000 10,000 10, x x x x x x x x x x x Total number of mice n = 39 n = 39 n = 39 n = 37 n = 32 n = 37 n = 36 n = 26 n = 17 n = 20 Total number of mice with IR n = 5 n = 17 n = 39 n = 37 n = 32 n = 37 n = 35 n = 26 n = 16 n = 20 [%] of mice with IR 12.8% 43.6% 100% 100% 100% 100% 97.2% 100% 94.1% 100%
9 S9 Table S4. IgG 1/2a/2b/2c titers of mice immunized with Tau [ps202/pt205], mo = months after the 2. boost. IgG1 IgG2a IgG2b IgG2c Tau [ps202/pt205] Mouse Number 2. boost 2 mo 5 mo final 2. boost 2 mo 5 mo final 2. boost 2 mo 5 mo final 2. boost 2 mo 5 mo final ,000 10, ,000 10, , ,000 10, , , , ,000 10, , ,000 10,000 10,000 10,000 10, , ,000 10, , , ,000 10, mean value
10 S10 Table S5. IgG 1/2a/2b/2c titers of mice immunized with Tau [pt212/ps214], mo = months after the 2. boost. IgG1 IgG2a IgG2b IgG2c Tau [pt212/ps214] Mouse Number 2. boost 2 mo 5 mo final 2. boost 2 mo 5 mo final 2. boost 2 mo 5 mo final 2. boost 2 mo 5 mo Final #289 10, #291 10, # ,000 10, #390 10, # #392 10,000 10, # # ,000 10, ,000 10, #499 10, , , # ,000 10, # ,000 10,000 10, #547 10,000 10, mean value
11 S11 Table S6. IgG 1/2a/2b/2c titers of mice immunized with Tau [pt231/ps235], mo = months after the 2. boost. IgG1 IgG2a IgG2b IgG2c Tau [pt231/ps235] Mouse Number 2. boost 2 mo 5 mo final 2. boost 2 mo 5 mo final 2. boost 2 mo 5 mo final 2. boost 2 mo 5 mo Final #275 10,000 10, #282 10, # ,000 10, #383 10, # , ,000 10, , , #451 10,000 10, # , , # , , # , ,000 10,000 10, ,000 10,000 10, #481 10,000 10, , #506 10, # , , , mean value
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