1270 NATURE MEDICINE • VOLUME 8 • NUMBER 11 • NOVEMBER 2002 ARTICLES The β-amyloid plaques in the brain are a principal target in the de- velopment of strategies to treat Alzheimer disease (AD) 1,2 , because the β-amyloid protein is associated with impaired long-term po- tentiation 3 , age-related memory impairment 4 , neurotoxicity 5 and formation of neurofibrillary tangles 6 . Active or passive vaccination against β-amyloid reduces the extent of amyloid plaques in the brains of transgenic mice expressing AD-causing mutations 7–9 . This reduced β-amyloid burden is associated with restored cogni- tive function 10–12 . The biological mechanisms of β-amyloid re- moval from brain tissue are unknown, but may involve a combination of microglial F c -receptor mediated phagocytosis 8 , se- questration of plasma amyloid β-peptide (Aβ) followed by drainage of soluble cerebrospinal fluid (CSF) Aβ into plasma 13,14 and antibody-mediated disaggregation of amyloid fibrils 15 . Antibody-mediated removal of β-amyloid from brain parenchyma can occur within several days in vivo 16 . Active immunization with a pre-aggregated synthetic Aβ 42 preparation, was therefore tested as part of the AN1792 multicenter trial for mild to moderate AD. Here we report the results of immunohistochemical investigations with sera and CSF obtained as part of an additional adjunct study involving the Zurich cohort of 30 patients. In this double-blind randomized study, 24 patients received a primary and a booster intramuscular injection of the active vaccine, with an intermittent four-week time period, and 6 received placebo 17 . Presence of antibodies against β-amyloid in human immune sera On immunohistochemical analysis, 24 human immune sera ob- tained 55.4 ± 5.9 days (mean ± s.d.) after the booster injection recognized both β-amyloid plaques and vascular amyloid on brain sections of doubly transgenic APP Sw ×PS1 M146L mice 18–20 , as indicated by staining, whereas most of the corresponding pre- immune sera did not stain any specific structures in adjacent brain sections (Fig. 1a and b). Nine pre-immune sera produced weak staining of β-amyloid plaques; in four of these cases, vacci- nation resulted in greater staining intensities. Antibodies against β-amyloid also stained diffuse Aβ deposits, but the reactions with β-amyloid plaques generally gave higher-intensity signals (Fig. 1c and d). In addition, the antibodies stained vascular amy- loid in subarachnoidal (Fig. 1e) and perforating blood vessels (Fig. 1f). The presence of vascular amyloid in transgenic mice ex- pressing amyloid precursor protein (APP) under the control of neuronal promoters is well established 21,22 . Staining of adjacent sections with either thioflavin S (Fig. 1g) or immune serum (Fig. 1h) produced similar staining patterns, indicating that the anti- bodies against amyloid reacted with structures within the β- sheet conformations detected by thioflavin S. Pre-absorption of the immune sera with aggregated synthetic Aβ 42 removed all im- munoreactivity, indicating specific interaction of the antisera with the Aβ-containing components of plaque and vascular amyloid (Fig. 1i and j). We were unable to use soluble Aβ 42 for pre-absorption experiments, because Aβ 42 aggregates rapidly in vitro under the conditions used for pre-absorption. Absence of cross-reactivity with endogenous APP or its derivatives In contrast to the consistent staining of pathological structures, the immune sera did not stain neurons or any cellular compo- nents—despite the fact that transgenic human APP is predomi- nantly expressed by neurons and, to a lesser extent, glial cells 23 . This finding indicated that the antibodies generated in AD pa- tients in response to vaccination did not cross-react with full- length human APP, intracellular Aβ or other physiologic APP derivatives. This hypothesis was supported by western-blot analyses that showed no cross-reactivity of the human immune sera with full-length APP, C-terminal derivatives or Aβ in protein Generation of antibodies specific for β-amyloid by vaccination of patients with Alzheimer disease CHRISTOPH HOCK, UWE KONIETZKO, ANDREAS PAPASSOTIROPOULOS, AXEL WOLLMER, JOHANNES STREFFER, RUTH C. VON ROTZ, GABRIELA DAVEY, EVA MORITZ & ROGER M. NITSCH Division of Psychiatry Research, University of Zurich, Zurich, Switzerland C.H. and U.K. contributed equally to this study. Correspondence should be addressed to R.M.N.; email: nitsch@bli.unizh.ch Published online 15 October 2002; doi:10.1038/nm783 To characterize antibodies produced in humans in response to Aβ 42 vaccination, we carried out immunohistochemical examinations of the brains of both transgenic mice and human patients with β-amyloid pathology. We collected sera from patients with Alzheimer disease who received a primary injection of pre-aggregated Aβ 42 followed by one booster injection in a placebo-con- trolled study. Antibodies in immune sera recognized β-amyloid plaques, diffuse Aβ deposits and vascular β-amyloid in brain blood vessels. The antibodies did not cross-react with native full- length β-amyloid precursor protein or its physiological derivatives, including soluble Aβ 42 . These findings indicate that vaccination of AD patients with Aβ 42 induces antibodies that have a high degree of selectivity for the pathogenic target structures. Whether vaccination to produce anti- bodies against β-amyloid will halt the cognitive decline in AD will depend upon clinical assess- ments over time. © 2002 Nature Publishing Group http://www.nature.com/naturemedicine