Alzheimer’s Disease Peptide Epitope Vaccine Reduces Insoluble But Not soluble-Oligomeric

Neurobiology of Disease

Alzheimer’s Disease Peptide Epitope Vaccine Reduces Insoluble But Not Soluble/Oligomeric A?Species in Amyloid Precursor Protein Transgenic Mice

Irina Petrushina,1*Anahit Ghochikyan,3*Mikayel Mktrichyan,3Gregory Mamikonyan,3Nina Movsesyan,1

Hayk Davtyan,3Archita Patel,1Elizabeth Head,1,2David H.Cribbs,1,2?and Michael G.Agadjanyan1,3?

1The Institute for Brain Aging and Dementia and2Department of Neurology,University of California,Irvine,Irvine,California92697-4540,and3The Institute for Molecular Medicine,Department of Immunology,Huntington Beach,California92647

Active vaccination of elderly Alzheimer’s disease(AD)patients with fibrillar amyloid-?peptide(A?42),even in the presence of a potent Th1adjuvant,induced generally low titers of antibodies in a small fraction(?20%responders)of those that received the AN-1792 vaccine.To improve the immunogenicity and reduce the likelihood of inducing adverse autoreactive T-cells specific for A?42,we previously tested in wild-type mice an alternative approach for active immunization:an epitope vaccine that selectively initiate B cell responses toward an immunogenic self-epitope of A?in the absence of anti-A?T cell responses.Here,we describe a second generation epitope vaccine composed of two copies of A?1–11fused with the promiscuous nonself T cell epitope,PADRE(pan human leukocyte antigen DR-binding peptide)that completely eliminates the autoreactive T cell responses and induces humoral immune responses in amyloid precursor protein transgenic2576mice with pre-existing AD-like pathology.Based on the titers of anti-A?1–11antibody exper-imental mice were divided into low,moderate and high responders,and for the first time we report a positive correlation between the concentration of anti-A?1–11antibody and a reduction of insoluble,cerebral A?plaques.The reduction of insoluble A?deposition was not associated with adverse events,such as CNS T cell or macrophage infiltration or microhemorrhages.Surprisingly,vaccination did not alter the levels of soluble A?.Alternatively,early protective immunization before substantial neuropathology,neuronal loss and cogni-tive deficits have become firmly established may be more beneficial and safer for potential patients,especially if they can be identified in a preclinical stage by the development of antecedent biomarkers of AD.

Key words:immunotherapy;Alzheimer’s disease;epitope vaccine;antibody;PADRE;?-amyloid

Introduction

The age-related accumulation of amyloid-?(A?)in the CNS has been hypothesized to play a central role in a cascade of events that eventually induces neuronal loss in affected brain regions in Alz-heimer’s disease(AD)(Selkoe,1991,1994;Hardy and Higgins, 1992;Price and Sisodia,1994;Esler and Wolfe,2001;Hardy and Selkoe,2002).Previously,major support for the amyloid cascade hypothesis emerged from A?-immunotherapy studies demon-strated that anti-A?antibodies were capable of reducing AD-like pathology(Schenk et al.,1999)and improving behavior in amy-loid precursor protein(APP)transgenic(Tg)mice(Chen et al., 2000;Janus et al.,2000;Morgan et al.,2000).First,immunother-apy clinical trial in AD patients using the AN-1792vaccine con-taining B and T cell“self epitopes”of A?42was halted during Phase IIa,when?6%of the participants developed aseptic me-ningoencephalitis(Schenk,2002;Weiner and Selkoe,2002;Hock et al.,2003;Nicoll et al.,2003;Orgogozo et al.,2003;Ferrer et al., 2004;Bayer et al.,2005;Fox et al.,2005;Gilman et al.,2005; Masliah et al.,2005;Patton et al.,2006).Importantly,only vacci-nated participants(n?18)developed meningoencephalitis, whereas none of the control patients(n?72)injected with pla-cebo developed adverse events(Orgogozo et al.,2003).Data from these trials suggest that the aseptic meningoencephalitis may have been caused by a T cell-mediated autoimmune response(Nicoll et al.,2003;Ferrer et al.,2004),and that anti-A?antibodies were not responsible for the observed adverse effects after active vac-cination.In fact,low/moderate titers of anti-A?antibodies gen-erated in a small subset of immunized patients(19.7%)were capable of reducing parenchymal amyloid pathology(Nicoll et al.,2003,2006;Ferrer et al.,2004;Masliah et al.,2005;Patton et al.,2006;Boche et al.,2007;Nitsch and Hock,2007)and dimin-ishing progressive cognitive decline associated with the disease (Hock et al.,2003;Gilman et al.,2005).However,?80%of the immunized subjects failed to develop anti-A?antibody titers (“nonresponders”),indicating that the A?self-antigen in the

Received July13,2007;revised Oct.1,2007;accepted Oct.7,2007.

This work was supported by National Institutes of Health Grants AG20241and NS50895(D.H.C.)and Alzheimer’s

Association Grant IIRG-03-6279(M.G.A.).Additional support for mice and the production of peptides was provided

by University of California,Irvive Alzheimer’s Disease Research Center Grant P50AG16573.Dr.Nina Movsesyan was

supported by National Institute on Aging training Grant AG00096.We thank Adrine Karapetyan for technical help

and valuable comments.

*I.P.and A.G.contributed equally to this work.

?D.H.C.and M.G.A.contributed equally to this work as senior authors.

Correspondence should be addressed to Dr.Michael G.Agadjanyan,The Institute for Molecular Medicine,16371

Gothard Street,H,Huntington Beach,CA92647-3652.E-mail:magadjanyan@https://www.wendangku.net/doc/e511097271.html,.

DOI:10.1523/JNEUROSCI.3201-07.2007

Copyright?2007Society for Neuroscience0270-6474/07/2712721-11$15.00/0

The Journal of Neuroscience,November14,2007?27(46):12721–12731?12721

AN-1792vaccine was not a strong immunogen,thus suggesting that alternative immunotherapeutic strategies should be pursued.

Based on the results generated in mouse models of AD(Bard et al.,2000;DeMattos et al.,2001;Dodart et al.,2002),a new clinical trial,AAB-001(Elan and Wyeth Pharmaceuticals,http:// https://www.wendangku.net/doc/e511097271.html,/investorrelations/events/elanwyethsymposium_adpd. asp),has been initiated by using passive transfer of a humanized monoclonal anti-A?antibody(bapineuzumab)in an attempt to avoid the problems associated with active immunization of el-derly AD patients.However,the design of this new trial is associ-ated with additional challenges such as multiple injections of high concentrations of anti-A?antibody every13weeks,the high cost of this monoclonal humanized antibody as well as possible side effects of passive vaccination,including microhemorrhages ob-served in passively immunized very old APP Tg mice(Pfeifer et al.,2002;Wilcock et al.,2004;Racke et al.,2005).This suggests that development of safe active immunotherapeutic strategies may still be desirable.

Previously,we engineered and tested a first generation epitope vaccine in wild-type mice(Agadjanyan et al.,2005),and here we report the development and testing the safety and efficacy of therapeutic vaccination of APP Tg2576mice with pre-existing AD-like pathology with a second generation epitope vaccine composed of two copies of the B cell epitope,A?1–11in tandem with pan human leukocyte antigen DR-binding peptide(PA-DRE),a synthetic,foreign promiscuous T cell epitope[pre-existing AD-like pathology implies the accumulation of soluble oligomeric forms of amyloid-beta peptide leading to the impair-ment of cognitive functions in?6-month-old APP Tg2576mice (Lesne et al.,2006)].

Materials and Methods

Mice,epitope vaccine,peptide immunogens,and experimental protocol. Aged(?9.4months old)female APP Tg2576mice were bred and pro-vided by the animal facility associated with the University of California at Irvine(UCI)Alzheimer’s Disease Research Center.All animals were housed in a temperature and light-cycle controlled facility,and their care was under the guidelines of the National Institutes of Health and an approved Institutional Animal Care and Use Committee protocol at University of California at Irvine.

To engineer an epitope AD vaccine,we synthesized the N terminus of an immunodominant B cell epitope of A?1–11(McLaurin et al.,2002; Bard et al.,2003;Cribbs et al.,2003)in tandem with a promiscuous foreign T cell epitope,so called pan-DR epitope,PADRE(Alexander et al.,1994).The peptide2A?1–11-PADRE was synthesized as a multiple antigenic peptide(MAP),containing a core matrix of4branching lysines (Tam,1988;Chai et al.,1992)to generate2A?1–11-PADRE-MAP(In-vitrogen,Carlsbad,CA).A?42peptide was synthesized at the Peptide Core Facility at the Institute for Brain Aging and Dementia at UCI by solid-phase Fmoc amino acid substitution and purified by reverse-phase high-pressure liquid chromatography.

Mice were immunized with2A?1–11-PADRE-MAP or fA?42as de-scribed previously(Cribbs et al.,2003;Petrushina et al.,2003;Agadjan-yan et al.,2005).Briefly,2A?1–11-PADRE-MAP(500?g/ml)or the fibril-lar(Schenk et al.,1999;Cribbs et al.,2003)A?42peptide(500?g/ml) were mixed with Quil A,a Th1-type conventional adjuvant,and mice were injected with50?g of antigen subcutaneously.Experimental mice at age9.4?0.9months old were immunized with2A?1–11-PADRE-MAP(n?21)or fA?42(n?12),whereas the control group of APP Tg 2576mice(n?11)was injected with an adjuvant only.All mice were boosted at monthly intervals.Cellular immune responses were analyzed in five mice from each group killed at9d after the fourth immunization. We continued to boost the remaining mice at monthly intervals and sera was collected at8–10d after the third,fourth,sixth and10th immuniza-tions and used for detection of anti-A?antibodies.At the end of the study,after10vaccinations when animals were19.4?0.9months old, neuropathological changes were compared across groups in response to treatment.In these animals,cellular immune responses also were analyzed.

T cell proliferation.The analysis of T cell proliferation was performed in splenocyte cultures from individual animals,as described previously (Cribbs et al.,2003;Agadjanyan et al.,2005).In addition,CD4?T cell proliferation was assessed using FACS assay according to the manufac-turer’s instructions(BD Biosciences,San Jose,CA).Briefly,to detect antigen-specific proliferation of CD4?T cells,we stained splenocyte cul-tures by1?M succinimidyl ester of carboxyfluorescein diacetate(CFSE; Invitrogen)for10min at37°C.After washing,the cells were incubated for3d in culture media alone or with PADRE(5?M).After incubation, the cultures were stained with phycoerythrin(PE)-labeled rat anti-mouse CD4monoclonal antibodies(BD Biosciences).Because dead cells might fluoresce nonspecifically,these cells were excluded from the assay using a nucleic acid dye(7-amino actinomycin D from BD PharMingen, San Diego,CA),and proliferation of viable cells was analyzed by FACS-can flow cytometer(BD Biosciences)as described by the manufacturer. CD4?population was separately analyzed using CellQuest software(BD Biosciences).

Production of cytokines by immune splenocytes.The same splenocytes used to assess T cell proliferation were used for detection of Th1(IFN?) or Th2(IL4)lymphokines by ELISPOT(BD PharMingen)as described previously(Cribbs et al.,2003;Agadjanyan et al.,2005).In addition,the FACS method was used for detection of specific cytokines by CD4?T cells(Pala et al.,2000).Briefly,the cultures of splenocytes from experi-mental and control animals were restimulated for3–4d with PADRE peptide(5?M),and then for4–6h with PMA(phorbol12-myristate 12-acetate)and ionophore(ionomycin)(both from Sigma,St.Louis, MO).In addition,we used Brefeldin A(BD Biosciences)to block cyto-kine secretion,which increases intracellular accumulation.Surface stain-ing was performed using FITC-labeled anti-mouse CD4monoclonal an-tibodies(MoAb;BD PharMingen).Cells were washed,fixed, permeabilized,and CD4?T cell subset producing IL-4or IFN?was de-tected using the appropriate PE-labeled anti-mouse cytokine antibodies (BD PharMingen).

Detection of anti-A?antibodies by ELISA.Total anti-A?42antibodies were detected as described previously(Cribbs et al.,2003;Petrushina et al.,2003)with small modifications:to develop the color reaction we used the3,3?,5,5?tetramethylbenzidine(TMB)peroxidase substrate(Pierce, Rockford,IL),and plates were analyzed spectrophotometrically at450 nm.The standard curve for determination of anti-A?antibody concen-trations in the sera was based on different known concentrations of monoclonal antibody20.1kindly provided by Dr.Van Nostrand(Stony Brook University,Stony Brook,NY).The concentrations of antibody were recorded in micrograms per milliliter.To determine the specific isotypes,pooled sera from mice were diluted1:2500and tested in dupli-cate.As we reported previously,mice of H2bxs immune haplotype(APP Tg2576)do not express IgG2a,producing IgG2c anti-A?antibodies instead(Petrushina et al.,2003).Therefore,in our experiments we used anti-IgG2a b-specific antibodies(BD PharMingen)along with anti-IgG1-,IgG2b-and IgM-specific antibodies(Zymed,San Francisco,CA). Detection of A?plaques in human brain tissues.Sera from immunized mice were also screened for the ability to bind to A?plaques in the human brain as we described previously,using immunohistochemistry (Ghochikyan et al.,2003;Agadjanyan et al.,2005).A digital camera (Olympus,Tokyo,Japan)was used to capture images of the plaques at 20?magnification.The binding of antisera(dilution1:1000)to the ?-amyloid plaques was blocked by preabsorption of the sera with5?M A?1–15peptide(1h,37°C).

Immunohistochemistry.To analyze the effect of active immunization with the epitope vaccine on neuropathological changes in APP Tg2576 mice(19.4?0.9months old),the brains were processed for immuno-histochemistry and histochemistry by previously published methods (Ghochikyan et al.,2003;Agadjanyan et al.,2005).Animals were killed under deep Nembutal sodium solution(150mg/kg,i.p.)anesthesia.To ensure proper fixation and immunostaining of brain tissues,mice were exsanguinated by transcardial perfusion with normal saline.Then brains

12722?J.Neurosci.,November14,2007?27(46):12721–12731Petrushina et al.?Testing the Epitope Vaccine in APP Tg2576Mice

were removed and bisected in midsagittal plane.The right hemisphere

was snap frozen for biochemical analysis,whereas the left hemisphere

was fixed in4%paraformaldehyde for immunohistochemical analysis.

Forty-micrometer-thick free-floating coronal sections of fixed hemi-

brains were collected using a vibratome.To assess the extent of neuropa-

thology and neuroinflammation that occurs in the brains of mice,the

following primary antibodies were used.A?deposits were detected with anti-A?42(dilution,1:2000;Invitrogen)and anti-A?40(1:10000;Invitro-gen).Activated microglia were detected with the anti-I-A/I-E[marker of

major histocompatibility complex(MHC)II alloantigens;1:200;BD

PharMingen]and anti-CD45(1:300;Serotec,Raleigh,NC)antibodies.

Astrocytes were labeled with anti-glial fibrillary acidic protein(GFAP;

1:3000)antibodies(Eng et al.,2000).Infiltration of T cells and macro-

phages were analyzed using anti-CD3-?(1:50;Santa Cruz Biotechnol-

ogy,Santa Cruz,CA),anti-CD4,anti-CD8(1:250;Novocastra Laborato-

ries,Newcastle,UK),and anti-F4/80(1:50;Serotec)antibodies,

respectively.The tissues from all animals within a given experimental

group were processed in parallel.Sections to be immunostained with

anti-A?antibodies were pretreated in90%formic acid for4min to enhance A?staining(Kitamoto et al.,1987).Sections for the staining with anti-CD45and anti-I-A/I-E(anti-MHC II)were pretreated with

proteinase K(0.03mg/ml)for5–7min at room temperature.Hydrogen

peroxide-quenched and blocked sections were incubated with primary

antibody overnight at4°C.Sections were then washed and incubated

with appropriate biotinylated secondary antibodies(1h at room temper-

ature).After multiple washes,the tissues were incubated in ABC for1h,

and color development was performed using DAB(3,3?-

diaminobenzidine)substrate kit(Vector Laboratories,Burlingame,CA).

Sections were mounted on Vectabond-coated slides(Vector Laborato-

ries),dehydrated,and covered using DPX(BDH Laboratory Supplies,

Poole,UK).Fibrillar A?deposits were visualized using Thioflavin S (ThS)as described by(Schmidt et al.,1995).Briefly,mouse brain sections

were washed with Tris buffer and stained for10min with a solution of

0.5%ThS in50%ethanol.Finally,sections were washed in50%ethanol

and Tris buffer,then dried and covered using Vectashield(Vector

Laboratories).

Quantitative image analysis.NIH imaging was used to analyze the area

occupied by?-amyloid and glial reactivity as described previously(Head et al.,2001).Immunostaining was captured using a Sony(Tokyo,Japan)

high-resolution CCD video camera(XC-77)and NIH image1.59b5soft-

ware.For every animal,12images(525?410?m each)of the frontal parietal region in the cortex at approximately the same plane(0.74to ?2.9mm with respect to bregma)of two adjacent sections were captured with a20?or40?objective.The samples included six images from the

superficial layer and the remaining six from the deep layer.NIH imaging

was used to analyze the area occupied by?-amyloid(A?load)relative to the background,and expressed as the percentage of area occupied.The threshold for detection of immunoreactivity was established and then held constant throughout the image analysis.ThS-positive plaques were counted by visual inspection of cortical region of all stained sections although blind with respect to treatment condition;a mean semiquanti-tative score was independently determined for each slide by two observers.

Prussian blue staining for microhemorrhage.Staining for hemosiderin

deposits was performed on duplicate adjacent coronal sections of the

mice brains containing similar regions located at approximately?1.5

bregma point,50?m thick,collected from immunized and naive,age-matched APP Tg2576mice.The sections were stained with Prussian blue working solution(equal parts of freshly made5%potassium ferrocianide and5%hydrochloric acid)for30min at room temperature,washed in deionized water,and counterstained with Nuclear fast red.Possible hem-orrhage events in the form of the number of Prussian blue-positive pro-files were counted in the brains of each mouse on all sections by two independent observers,and the average number of hemosiderin deposits was calculated per each brain hemisphere.

Biochemical analysis.Biochemical analysis of the brain tissue was pro-

cessed as described previously(Kawarabayashi et al.,2001),except that

cortices of right hemispheres of brains were used.Briefly,frozen cortices

were thawed,minced and then homogenized in50m M Tris-HCl buffer containing2%SDS,pH8.0,and a mixture of protease inhibitors(MP Biomedicals,Solon,OH).Homogenates were centrifuged(100,000?g, 1h,4°C)and supernatants were stored at?70°C for additional analysis of soluble?-amyloid.Seventy percent formic acid was added to the pel-lets for extraction of SDS-insoluble?-amyloid.After sonication samples were centrifuged(100,000?g,1h,4°C)and supernatants were stored for analysis of insoluble?-amyloid.After neutralization of formic acid with 1.0M Tris-base/0.5M NaH2P04,concentrations of insoluble and soluble A?40and A?42were analyzed using?-amyloid ELISA kits(Invitrogen) according to the manufacturer’s recommendations.Plates were analyzed spectrophotometrically at450nm via a microplate reader,and the con-centrations of A?40and A?42were calculated using standard curves for A?40and A?42peptide by comparing the sample’s absorbance with the absorbance of known concentrations of a https://www.wendangku.net/doc/e511097271.html,ing the wet weight of cortex region in the original homogenate,the final values of A?were expressed as micrograms per gram wet weight of cortex.

Dot blot assay and combination of IP and WB.Soluble fractions from cortical homogenates of experimental and control mice used in ELISA were also used for dot blot assay and for both immunoprecipitation(IP) and Western blot(WB).Total protein concentration in the homogenates was determined using bicinchoninic acid assay(Pierce)and adjusted to4 mg/ml with PBS.

Dot blot.Two microliters of sequential dilutions of homogenates were applied to nitrocellulose membrane(GE Healthcare,Piscataway,NJ), air-dried,and blocked with5%fat-free dry milk in TBST(10m M Tris-HCl,pH8.0,150m M NaCl,0.05%Tween20).Oligomers were detected by using HRP-conjugated A11polyclonal antibody(kindly provided by Dr.C.Glabe,University of California,Irvine,Irvine,CA).Blots were developed with ECL detection system(Santa Cruz Biotechnology).Au-toradiograms were scanned,and densitometry of A?oligomer spots was performed with NIH Image J software,version1.36b.The relative optical density was calculated and presented as average value?SD for each group.

IP and WB.Aliquots of homogenates were pooled into four groups (based on low,moderate,high anti-A?antibody responders and control nonimmunized mice,200?g of total protein in each pooled aliquot), diluted to500?l with PBS and incubated(overnight at4°C)with anti-A?20.1monoclonal antibody immobilized on protein G-sepharose.The beads were washed three times in PBS,and proteins were eluted in20?l of SDS-PAGE loading buffer by boiling.Samples were subjected to elec-trophoresis on12.5%SDS-Tris polyacrylamide gel and proteins were electrotransferred to polyvinylidene difluoride membrane(GE Health-care).The membrane was boiled for2min and blocked with5%fat-free dry milk followed by detection of A?oligomers using anti-A?biotinyl-ated20.1monoclonal antibody.Three experiments with the same ho-mogenates were performed and Western blots were scanned and con-verted into digital files.Densitometry of A?oligomer bands were performed using NIH Image J software,version1.36b and data(aver-age?SD)were presented from three experiments.

Statistical analysis.All statistical parameters(mean,SD,significant difference,etc.)used in experiments were calculated using Prism3.03 software(GraphPad Software,San Diego,CA).Statistically significant differences were examined using an ANOVA and post hoc comparisons were done using Tukey’s test(p?0.05was considered as statistically different).

Results

The second generation epitope vaccine stimulates

CD4?IFN??T cells specific to PADRE without activation of autoreactive anti-A?T helper cells

To circumvent the side effects of the AN-1792vaccine against AD,we engineered a vaccine in which a foreign T helper cell epitope was incorporated with two copies of the B cell epitope of A?42.APP Tg2576mice vaccinated with2A?1–11-PADRE-MAP induced T cell responses directed against the foreign antigenic determinant,PADRE,but not against self A?antigen.More spe-cifically,splenocytes isolated from vaccinated animals prolifer-ated after re-stimulation with PADRE,but not A?40(Fig.1A).In

Petrushina et al.?Testing the Epitope Vaccine in APP Tg2576Mice J.Neurosci.,November14,2007?27(46):12721–12731?12723

contrast,control APP Tg2576mice vacci-nated with fibrillar A?42antigen(fA?42) that has self B and T cell antigenic deter-minants induced only autoreactive T cells activated after restimulation with A?40 (Fig.1A).

Direct measurement of activation of PADRE-specific CD4?T helper cells in vaccinated APP Tg2576mice by a FACS assay confirmed these results.CD4?T cells from mice vaccinated with the pep-tide epitope vaccine induced strong prolif-eration of this subset of T cells after re-stimulation with nonself PADRE peptide, but not A?40(Fig.1B).In contrast,vacci-nation with fA?42formulated in QuilA ad-juvant induced proliferation of CD4?T cells activated after re-stimulation with self-antigen A?40,but not with nonself T cell epitope PADRE.Of note,CD4?T cells from mice injected with adjuvant did not proliferate after restimulation either with PADRE or A?40peptides(Fig.1B).

To further investigate T cell responses to vaccination,we analyzed Th1(IFN?) and Th2(IL-4)cytokine production by splenocytes and CD4?T cells from im-munized and control APP Tg2576mice

using ELISPOT and FACS assays,respec-

tively(Fig.1C,D).In these experiments,splenocytes from mice vaccinated with epitope vaccine were restimulated with PADRE, whereas splenocytes isolated from animals immunized with fA?42were restimulated with A?40.Groups of APP Tg2576mice injected with the epitope vaccine induced a strong IFN?(Th1) response based on the number of splenocytes producing this lym-phokine,whereas mice immunized with fA?42had low re-sponses.Because we used a Th1adjuvant in both the epitope and fA?42vaccinations,it was not surprising that both groups of mice generated less IL-4(Th2)than IFN?(Th1),whereas splenocytes from naive mice did not generate either IL4or IFN?cytokines (Fig.1C).We confirmed these results by measuring the produc-tion of IL-4or IFN?cytokines in CD4?T helper cells.We found that the number of PADRE-specific CD4?IFN??T helper cells in mice immunized with the epitope vaccine was higher than the number of anti-A?CD4?IFN??T cells in mice immunized with fA?42(Fig.1D).The same was true for IL-4-producing anti-PADRE and anti-A?CD4?T cells,although both groups had significantly lower number of CD4?T cells producing IL-4than IFN?(Fig.1D).Thus,CD4?T cells and splenocytes isolated from both vaccinated groups predominantly produced a Th1-type cytokine,IFN?,consistent with antigens being formulated in a Th1-type adjuvant,Quil A.Cellular immune responses ana-lyzed after4(Fig.1)and10(data not shown)immunizations showed the same specificity and profile.Collectively,the analyses of T cells demonstrated that the epitope vaccine did not activate autoreactive T cells,but stimulated nonself PADRE-specific CD4?IFN??T lymphocytes.

Anti-PADRE-specific CD4?IFN??T lymphocytes help B cells to produce therapeutically potent anti-A?1–11specific antibody in vaccinated APP Tg2576

To determine the ability of activated PADRE-specific T cells to stimulate anti-A?B cells,we measured antibody concentrations in the sera pooled from each group of animals after three,four, six,and10immunizations.After three injections of the epitope vaccine the concentrations of anti-A?antibodies in the sera were higher than that in mice immunized with fA?42.However,after the sixth immunization this difference in anti-A?antibody titers steadily diminished,becoming equal after the last two boosts in both groups(Fig.2A).Of note,mice immunized with the Quil A adjuvant alone did not induce anti-A?antibodies(data not shown).

As mentioned above,these data were generated with sera pooled from each of the group;however,in individual animals we found significant variability in anti-A?antibody responses(Fig. 2B).Concentrations of anti-A?1–11antibodies after10immuni-zations were high in four mice immunized with the epitope vac-cine(176.8?163.56?g/ml),whereas an additional eight and four vaccinated animals induced moderate(22.2?14.16?g/ml) and low levels(1.2?1.5?g/ml)of anti-A?1–11antibodies,re-spectively(Fig.2C–E).This individual variability in humoral im-mune responses was also detected in the group of mice immu-nized with fA?42antigen(Fig.2C–E):four mice generated high (82.65?22.68?g/ml),one mouse had moderate(23.3?g/ml), and two animals had low concentrations(0.9?0.7?g/ml)of anti-A?42antibodies.Thus,the epitope vaccine was at least as effective as fA?42in induction of antibodies after10immuniza-tions,but was significantly more effective at initiating the anti-body immune responses(Fig.2A).

To characterize the types of humoral immune responses from each vaccination group,we measured the production of IgG1, IgG2a b,IgG2b,and IgM anti-A?antibodies in the sera collected from individual animals after a total of10injections of the epitope vaccine or fA?42.All mice vaccinated with the epitope vaccine generated comparable amounts of IgG1,IgG2a b,and as a result,the average of IgG1/IgG2a b ratio was close to1(Fig.2F). This ratio implied that the epitope vaccine formulated in Th1

Figure1.The second generation peptide epitope vaccine induces T cell response specific to promiscuous and nonself epitope, PADRE.Splenocyteswereisolatedfromindividualmice(n?5)afterfourthimmunizationandrestimulated invitro withPADREor

A?

40peptides.A,Proliferationofsplenocyteswasdetectedby3[H]thymidineincorporation.B,D,ProliferationofCD4?Tcells(B)

and production of cytokines by this T cell subset(D)were detected by flow cytometry in pooled splenocyte cultures.Production of

IFN?and IL4cytokines by pooled immune splenocytes was detected by ELISPOT assay(C).The experiment was repeated after the

10th immunization with similar results.***p?0.001.

12724?J.Neurosci.,November14,2007?27(46):12721–12731Petrushina et al.?Testing the Epitope Vaccine in APP Tg2576Mice

adjuvant induced a Th1type humoral response.Similar results

were observed in mice immunized with fA ?42;immunization in

these animals induced anti-A ?42antibodies of all IgG isotypes

tested,and IgG1/IgG2a b ratio was ?1(Fig.2F ).Antibodies gen-erated by the epitope vaccine recognized human A ?deposits

when used in immunohistochemical experiments in an AD case,

and this binding was blocked by preabsorption of sera with A ?42,

A ?1–11,or 2A ?1–11peptides equally well (data not shown).Thus,

as was expected,the epitope vaccine induced antibodies specific

to the A ?1–11sequence of A ?42.

The link between anti-A ?1–11antibody concentrations and

clearance/reduction of AD-like neuropathology in the brains

of APP Tg 2576mouse model of AD

To demonstrate the therapeutic efficacy of anti-A ?antibodies

generated in response to the peptide epitope vaccine,we analyzed

neuropathological changes in aged (19.4?0.9months old)ex-perimental and control APP Tg 2576mice (after 10injections).

Figure 3shows a significant decrease in cortical plaque burden in

APP Tg 2576mice immunized with both the epitope vaccine and

the fA ?42antigen,compared with the control adjuvant-only in-jected group.More specifically,in brains of mice immunized

with the epitope vaccine or fA ?42,we de-tected significantly less A ?42or A ?40load (diffuse and cored plaques)than in brains of control animals (Fig.3A ,B ).Addition-ally,we demonstrated significant reduc-tion of ThS-positive A ?deposits (cored plaques)in the brains of experimental mice versus controls (Fig.3C ).Collec-tively,these results demonstrate that im-munization with epitope vaccine is effec-tive in reducing cerebral A ?burden in APP Tg 2576mice,which were ?9months old at the start of the study.

As described previously,data from the first human clinical trial indicated that AD patients responding to the AN-1792vac-cine and producing anti-A ?antibody titers over 1:2200showed some slowing of cognitive decline and localized reduction of plaques (Hock et al.,2003;Gilman et al.,2005;Masliah et al.,2005;Nicoll et al.,2006).To address this question in our preclin-ical trials,we grouped all mice vaccinated with the epitope vac-cine based on the concentrations of generated anti-A ?antibodies (Fig.2C–E ),and analyzed the levels of soluble and insoluble A ?in the brains of experimental and control animals.The rationale for using A ?as an outcome measure reflecting possible func-tional improvements was based on previous work showing that A ?is correlated with behavior in APP Tg 2576mice (Hsiao et al.,1996;Westerman et al.,2002)and that vaccination of APP Tg mice with fA ?42leads to behavioral improvements and reduced A ?(Chen et al.,2000;Janus et al.,2000;Morgan et al.,2000;Das et al.,2001,2003).We observed a positive correlation between the concentration of anti-A ?1–11antibodies and reduction of cortical A ?42load in the brains of vaccinated APP Tg 2576mice (coeffi-cient of correlation is ?0.83,p ?0.05).As shown in Figure 4A ,there are no differences in A ?42load between the group of mice with low concentrations of anti-A ?1–11antibodies and control mice.Although A ?42load is noticeably reduced in the brains

of Figure 2.The second generation epitope vaccine selectively initiates B cell responses toward an immunogenic epitope of A ?1–11,whereas T cell help is provided by a genetically linked nonself T cell epitope,PADRE.A ,B ,Concentration of anti-A ?antibody detected in the pooled sera after three,four,six,and 10immunizations (A )or in individual animals after 10immunizations (B ).C–E ,APP Tg 2576mice immunized with epitope or fA ?42vaccines generated low (C ),moderate (D ),or high (E )concentrations of anti-A ?antibody.F ,Detection of IgG1,IgG2a b ,IgG2b,and IgM isotypes of anti-A ?antibody.

Figure 3.A–C ,The second generation epitope vaccine inhibits A ?deposition in the brains of 19.4?0.9-month-old APP Tg 2576mice.Image analysis of A ?42(A ),A ?40(B ),and Thioflavin S (C )staining in cortex of mice vaccinated with epitope vaccine,fA ?42,or control mice.Vaccinated mice showed a significant reduction in A ?42/40load (cored and diffuse)and in the number of ThS-positive (cored)plaques (*p ?0.05)compared with the control animals.Error bars represent the average ?SE for n ?6in the group of control mice,and n ?16and n ?7in the groups of mice vaccinated with epitope and fA ?42vaccines,respectively.Petrushina et al.?Testing the Epitope Vaccine in APP Tg 2576Mice J.Neurosci.,November 14,2007?27(46):12721–12731?12725

mice with moderate concentration,this reduction was not signif-icant,whereas in the mice with a high concentration of serum anti-A ?1–11antibodies A ?42load was reduced significantly (p ?0.018).Immunostaining of adjacent brain sections with anti-A ?40-specific antibodies revealed a marked reduction of A ?40load in all groups of experimental mice versus the controls,inde-pendent of serum antibody concentrations (Fig.4B ).However,ThS staining also showed positive correlation between the con-centrations of anti-A ?1–11antibodies and reduction of cortical cored plaques (Fig.4C ),which in APP Tg 2576mice contain both A ?40/42peptides (Kawarabayashi et al.,2001).Similar effects were observed in the hippocampal regions of vaccinated APP Tg 2576mice stained with anti-A ?42.Representative images of hip-pocampal regions stained with anti A ?42-specific monoclonal antibodies (D-G)or ThS (H-K)are shown in Figure 4D–K .To further confirm an association between serum antibody concentrations and differential effects on reducing A ?40or A ?42,we measured insoluble A ?40and A ?42in cortical homogenates of experimental and control mice by sandwich ELISA.Insoluble A ?40and A ?42concentrations were significantly lower in the cortex of mice vaccinated with the epitope vaccine or fA ?42,com-pared with control animals (Fig.5A ,B )(p ?0.001and p ?0.05,respectively).However,when we compared A ?levels in groups with low,moderate,and high titers of antibodies,we found a significant reduction of A ?40and A ?42only in mice with anti-A ?1–11antibody levels ?50?g/ml (Fig.5C ,D )(p ?0.05and p ?0.001,respectively).Collectively,these results demonstrate that high concentrations of anti-A ?antibody are critical for effective reduction of insoluble A ?in APP Tg 2576mice with pre-existing AD-like pathology.

A potential problem of immunotherapy is that reduction of insoluble A ?may lead to increased levels of soluble forms of this peptide (Patton et al.,2006),possibly oligomers,which are known to be more neurotoxic and can impair cognitive function (Klein et al.,2001;Gong et al.,2003;Cleary et al.,2005;Klyubin et al.,2005;Lesne et al.,2006).Accordingly,we measured soluble A ?40and A ?42levels in detergent-extracted samples of cortical homogenates of control mice and animals vaccinated with epitope vaccine or fA ?42by capture ELISA (Fig.5A ,

B ).There were no statistically significant differences between experimental and control mice.In addition,we evaluated the levels of A ?oli-gomers in the cortical homogenate by dot blot using A11anti-oligomeric antibodies (Kayed et al.,2003).All available homog-enates from low (n ?3),moderate (n ?7),and high (n ?3)responders as well as control (n ?5)mice were used in these experiments.No differences were found between all three groups of immunized mice as well as between vaccinated and control animals (Fig.5E ,F ).Of note,no oligomers were detected in brain homogenates from wild-type mice (data not shown).To confirm these results and to measure the relative levels of high molecular weight (?5-to 6-mers)oligomers,the cortical homogenates were subjected to IP followed by WB.The levels of A ?5-mers,9-mer,and 12-mer oligomers in detergent-extracted samples of cortical homogenates obtained from experimental and control mice did not significantly differ,and wild-type mice did not de-posit A ?-reactive oligomers (Fig.5G ,H ).Therefore,the level

of

Figure 4.A–C ,The anti-A ?antibody concentration positively correlates with the reduction of A ?42and Thioflavin S,but not A ?40load in the cortex areas of APP Tg 2576mice immunized with epitopevaccine.Errorbarsrepresenttheaverage ?SEforcontrolmice(n ?6)andmicewithlow(n ?4),moderate(n ?8),andhigh(n ?4)anti-A ?antibodyconcentrations.*p ?0.05;**p ?0.01).D–K ,Thesameresultswereobtainedafterstainingofhippocampalregionswithanti-A ?42-specificmonoclonalantibody(D–G )orThioflavinS(H–K ).Representativeimagesofhippocampal regions of control mice (D ,H )and mice with low (E ,I ),moderate (F ,J ),and high (G ,K )anti-A ?antibody concentrations.Original magnifications,4?.Scale bar,500?m.

12726?J.Neurosci.,November 14,2007?27(46):12721–12731Petrushina et al.?Testing the Epitope Vaccine in APP Tg 2576Mice

oligomeric,toxic forms of A?is not reduced in the brain of APP Tg2576mice at least when vaccination was initiated in mice with pre-existing AD-like pathology.These differences between re-ducing insoluble and soluble A?42in vivo are not connected to the differences in binding affinity of anti-A?1–11antibodies to these forms of?-amyloid as we previously demonstrated (Mamikonyan et al.,2007).

The effect of vaccination on microglial activation,lymphocyte infiltration,and microhemorrhages

To detect inflammation-related pathology in the brains of ani-mals immunized with the epitope vaccine,the same brain regions

used for A?burden studies were evaluated

for microglial activation(MHC class II,

CD45),astrocyte hypertrophy(GFAP),

and microhemorrhages in blood vessels

(Prussian blue).Quantitative image anal-

ysis of MHC class II(I-A b/I-E b)positive

cells demonstrated significantly decreased

microglial activation in APP Tg2576mice

vaccinated with the epitope vaccine,and

this decrease was comparable with that

seen in the cerebral cortex of mice immu-

nized with fA?42(Fig.6A).Representative

MHC class II immunoreactivity is shown

for mice vaccinated with the epitope vac-

cine for comparison with control animals

(Fig.6B–E).These data are similar to the

results obtained after immunostaining of

the brain tissues with anti-CD45antibod-

ies(Fig.6F–I).Thus,immunization with

the epitope vaccine decreased microglial

activation in the brains of aged APP Tg

2576mice.

Astrocytes in both experimental and

control mice were detected using the

astrocyte-specific marker(GFAP)and a

quantitative image analysis indicated that

vaccinated mice had a lesser degree of as-

trocytosis compared with the control

group.However,immunization with

epitope vaccine led to a14%reduction,

whereas immunization with fA?42re-

sulted in?53%reduction in GFAP im-

munoreactivity(Fig.6J).Importantly,im-

munization with the epitope vaccine

reduced astrocytosis to?50%of the mice

with high sera concentrations of antibod-

ies and this decrease is statistically signifi-

cant(Fig.6K).

Previously,it was reported that very

old APP Tg mice receiving passive transfer

of anti-A?monoclonal antibodies devel-

oped cerebral microhemorrhages(Pfeifer

et al.,2002;Wilcock et al.,2004).More

recently,it was shown that cerebral amy-

loid angiopathy-associated microhemor-

rhages are increased in?20-month-old

APP?presenilin1(PS1)transgenic mice

immunized eight times with fA?42formu-

lated in complete Freunds adjuvant

(CFA)/incomplete Freunds adjuvant

(IFA)(Wilcock et al.,2007).To assess

this potential adverse side effect of epitope vaccine,we selected adjacent coronal sections of mouse brains located at?1.5bregma and visualized microhe-morrhages by detecting hemosiderin(byproduct of degrada-tion of hemoglobin,occurring at the sites of previous micro-hemorrhages)deposits using Prussian blue staining. Characteristic blue hemosiderin-positive profiles were ob-served primarily in the neocortical,leptomeningeal,hip-pocampal and thalamic areas of the brain.These microhem-orrhages were detected at a rate of2.8?0.8per hemibrain in control nonimmunized mice and2.6?1.33in animals immu-nized with the epitope vaccine.However,vaccination of a

Figure5.The reduction of insoluble,but not soluble A?

40and A?

42

levels are positively correlated with the concentrations of

anti-A?

1–11antibody.A,B,A significant decrease in the total(parenchymal and vascular)levels of insoluble A?

40

and A?

42

in

cortical homogenates was observed after epitope vaccine and fA?

42immunization.C,D,The significant reductions in insoluble

A?

40and A?

42

levels were observed only in groups with high titers of antibodies.*p?0.05and***p?0.001.However,the

levels of soluble A?

40and A?

42

in cortical homogenates did not differ between experimental and control groups(A,B).E–H,The

level of oligomeric forms of A?detected in cortical homogenates using dot blot(E,F)or by combination of IP with WB(G,H)also

was not changed after vaccination.In dot blot assay,homogenates from individual animals were diluted and analyzed using A11

oligomer-specific antibody(E),and relative optical density(F)was presented as the average?SD.Pooled homogenates of each

group of mice were analyzed by IP/WB(G),and no significant difference between the density and thickness of oligomeric bands

(H)was detected(with the average?SD calculated from three independent experiments).Lanes in WB:a,wild-type;b,control;

c,low,?5?g/ml;d,moderate,5–50?g/ml;e,high,?50?g/ml.

Petrushina et al.?Testing the Epitope Vaccine in APP Tg2576Mice J.Neurosci.,November14,2007?27(46):12721–12731?12727

group of APP Tg 2576mice with fA ?42had resulted in 3.97?1.24hemosiderin-positive profiles.Although not statistically significant,there was nonetheless approximately a 41%in-crease compared with the control group,and these data re-semble the results generated by (Wilcock et al.,2007)with APP ?PS1aged mice.Thus,immunization with the second generation epitope vaccine did not increase the incidence of cerebral microhemorrhages in ?19-month-old APP Tg 2576mice (Fig.6L ).Although there is only one unconfirmed report about lymphocyte infiltration detected in the brains of wild-type mice immunized with A ?42formulated in CFA/IFA fol-lowed by injection with pertussis toxin (Furlan et al.,2003),we also investigated whether immunization with the epitope vac-cine formulated in Th1adjuvant might also induce T cell in-filtration into the brain.Our standard methodology for de-tecting T cells in brain vessels of mice (Ghochikyan et al.,2003)did not identify CD3-,CD4-,and CD8-positive T cells or F4/80-positive macrophages in the brains of mice immu-nized with our second generation epitope vaccine (data not shown).Thus,reduction of insoluble A ?deposition in immu-nized APP Tg mice was not associated with deleterious side effects,including cerebral inflammation and microhemorrhages.

Discussion

Published results from the recently halted AN-1792clinical trial in patients with AD suggested that aseptic meningoencephalitis was linked to an adverse T cell-mediated autoimmune response (Nicoll et al.,2003;Ferrer et al.,2004).Presence of anti-A ?anti-bodies correlated with effective reduction of A ?pathology in patients that came to autopsy (Nicoll et al.,2003;Ferrer et al.,

2004;Masliah et al.,2005),suggesting a possible therapeutic ben-efit of vaccination (Bayer et al.,2005;Fox et al.,2005;Gilman et al.,2005).We analyzed the effect of different concentrations of anti-A ?antibodies in serum on neuropathological changes in the brains of actively immunized APP Tg 2576mice in the absence of an autoreactive anti-A ?T cell response.We have obtained results somewhat similar to data described in the AN-1792clinical trial:(1)the higher the concentrations of anti-A ?antibody specific to the N terminus of A ?42,the larger the therapeutic effect.The NTB composite z -scores were regressed on the geometric mean anti-body titers,although this correlation was not significant (Gilman et al.,2005).(2)AN-1792vaccine reduced the number of A ?plaques,but not the level of soluble A ?in the brains (Patton et al.,2006).To circumvent the side effects of the AN-1792vaccine and to reduce the potential for cell-mediated autoimmune toxicity in AD patients,we engineered the first (Agadjanyan et al.,2005)generation of epitope vaccine and tested it in wild-type mice.Here,we report on design and testing of the second generation peptide epitope vaccine in APP Tg 2576mouse model (Hsiao et al.,1996;Kawarabayashi et al.,2001;Westerman et al.,2002),and demonstrated that this prototype AD vaccine induced strong anti-PADRE cellular and anti-A ?humoral responses (Figs.1,2).If this proves to be predictive for human trials and vaccinated individuals induce strong anti-A ?antibody responses without generating potentially harmful autoreactive T helper cells,our epitope vaccine approach may represent a reasonable alternative to a passive vaccination strategy with humanized anti-A ?antibody.

The AN-1792clinical trial data demonstrated significant in-dividual variability in anti-A ?antibody responses in

vaccinated

Figure 6.The second generation epitope vaccine reduces glial activation without increasing brain microhemorrhages.A ,Image analysis of cortex areas from vaccinated or control mice was performed after staining with anti-I-A/I-E antibody.Vaccination either with the epitope vaccine,or fA ?42decreased microglial activation in cortical region of immune mice (*p ?0.05)compared with that in control mice.B–I ,Representative pictures of brain sections stained with anti-I-A/I-E (B–E )and anti-CD45(F–I )antibodies showing a decreased immunoreactivity also in the hippocampal brain regions in mice immunized with the epitope vaccine (C ,E ,G ,I )compared with control animals (B ,D ,F ,H ).Original magnifications:B ,C ,F ,G ,5?;D ,E ,H ,I ,40?.J ,K ,Image analysis of cortical regions from vaccinated or control mice was performed after staining with anti-GFAP antibody.Astrocytosis was significantly (*p ?0.05)decreased in mice immunized with fA ?42.A reduction in GFAP load was slightly reduced in all mice vaccinated with the epitope vaccine,but it was significant only in mice with high concentration anti-A ?antibody.L ,The number of Prussian blue-positive profiles in the brain hemispheres of experimental and control mice did not differ significantly.

12728?J.Neurosci.,November 14,2007?27(46):12721–12731Petrushina et al.?Testing the Epitope Vaccine in APP Tg 2576Mice

AD patients(Bayer et al.,2005;Gilman et al.,2005).As men-tioned by Patton et al.(2006),only59individuals in the immu-nized cohort induced desirable antibody titers to immunization with fA?42.Importantly,some of these AD patients showed a trend toward slowing of cognitive decline associated with the disease(Hock et al.,2003),improvement in the memory domain of the NTB and the decreased CSF tau levels(Gilman et al.,2005). Collectively,these data suggested that higher titers of antibodies might be beneficial for AD patients.Because the concentrations of anti-A?antibodies in individual APP Tg2576mice vaccinated with our epitope vaccine were not uniform either(Fig.2B)we were able to analyze the association between the neuropatholog-ical changes in vaccinated APP Tg2576mice with the concentra-tion of anti-A?antibodies in the sera of these animals.We ob-served that the concentration of anti-A?antibodies was associated with a therapeutic effect in APP Tg2576mice(Fig.4). Our data also indicated that high concentrations of anti-A?an-tibodies are critical,selectively for the reduction of A?42deposits, whereas A?40deposits and cored plaques are more responsive to A?immunotherapy overall and can be cleared even with low lev-els of anti-A?antibodies(Fig.4).Previous data with APP Tg mice have demonstrated that although A?plaques were reduced,the total level of soluble and insoluble A?did not differ between immunized and control mice(Janus et al.,2000).Clinical studies demonstrated that immunizations with AN-1792vaccine mobi-lize parenchymal plaques and reduce the level of insoluble A?, but increase the level of vascular and soluble?-amyloid(Masliah et al.,2005;Nicoll et al.,2006;Patton et al.,2006).These data suggest that the increased level of soluble A?may produce con-ditions that favor formation of toxic oligomeric species of A?.To address this issue,we analyzed the levels of soluble and insoluble A?40and A?42in cortex homogenates obtained from immunized and control APP Tg2576mice and demonstrated that vaccina-tion significantly decreased the levels of insoluble A?40and A?42. Importantly,vaccination did not increase the levels of the more toxic soluble A?in mouse model of AD(Fig.5A,B)in contrast to data reported with the AN-1792vaccine(Patton et al.,2006). Additionally,we demonstrated that only the high concentrations of anti-A?antibodies significantly decrease both insoluble A?40 and A?42in cortical tissues of experimental mice(Fig.5C,D). Thus,our vaccine did not affect the levels of soluble A?40and A?42in the brains of mice with pre-existing AD-like pathology. Previously it was demonstrated that A?neurotoxicity requires insoluble fibril formation(Loo et al.,1993;Lorenzo and Yankner, 1994).However,emphasis has shifted to soluble oligomers as pathological species of A?42(Gong et al.,2003;Cleary et al.,2005; Klyubin et al.,2005;Lesne et al.,2006),although aggregation-related toxicity was reported almost a decade earlier(Pike et al., 1991).In APP Tg2576mice,memory deficits are first detected in 6-month-old mice accumulating12-mer oligomers(Lesne et al., 2006)and before overt plaque deposition.Accordingly,we ana-lyzed the levels of oligomers in the brains of APP Tg2576mice using an anti-oligomeric antibody(A11),and have demonstrated that immunization with the epitope vaccine did not induce a reduction in immunized compared with control animals(Fig. 5E,F).We further confirmed these by dot blot experiments using A?fractions immunoprecipitated from cortical homogenates and then detecting oligomers by Western blotting.Of note,in these experiments we used12.5%SDS-Tris polyacrylamide gel that allowed the detection of oligomers?20kDA.The levels of A?oligomers in the brains of experimental and control mice were not significantly different and no oligomers were detected in wild-type mice(Fig.5G,lane a).These results suggest that either higher concentration of anti-A?antibodies are needed to signif-icantly reduce oligomeric A?in brains of immunized mice,or more likely that immunization should be initiated at an earlier age,in mice either without pre-existing A?pathology or with early-stage AD-like pathology.Whereas our ongoing prevention immunization studies with the DNA epitope vaccine may allow us to address these hypotheses,we need to mention that multiple transcutaneous immunizations of young PSAPP mice without pre-existing AD-like pathology with fA?42and cholera toxin gen-erated high titers of anti-A?antibodies(?250?g/ml)that re-duced the levels of both insoluble and soluble cerebral A?de-tected in aged mice(Nikolic et al.,2007).Interestingly,analysis from two cases from the AN-1792clinical trial report also suggest that“anti-amyloid immunization may be most effective not as therapeutic or mitigating measures,but as a prophylactic mea-sure when A?deposition is still minimal”(Patton et al.,2006).

It is difficult to predict the effects of frequent passive delivery of high concentrations of humanized anti-A?antibodies in on-going passive immunotherapy trials with AD patients because this treatment approach may still induce undesirable side effects, for example microhemorrhages.Although these clinical studies are important for the future of A?immunotherapy,an effective active immunization approach is still feasible providing that the AD vaccine is safe,induces an adequate antibody response to important B cell epitope of A?,and is free of harmful autoreactive T cell responses.The second generation epitope vaccine de-scribed in this study induced peripheral anti-PADRE-specific Th1-type proinflammatory responses without infiltration of T cells or macrophages in the brains of vaccinated APP Tg2576 mice.It is known that AN-1792vaccine caused an increase in cerebral vasculature deposition of A?(Masliah et al.,2005;Nicoll et al.,2006;Patton et al.,2006).Previously it was also shown that active A?42vaccination of double transgenic(APP?PS1)mice resulted in significantly increased cerebral amyloid angiopathy and associated microhemorrhages(Wilcock et al.,2007).Al-though we did not directly investigate the effect of epitope vac-cine on A?deposition in cerebral vasculature,we demonstrated that anti-A?1–11antibodies did not increase the incidence of ce-rebral microhemorrhages in the brains of immunized mice(Fig. 6L).Thus,we suggest that an epitope vaccine could be used as a safe and effective measure for the treatment of people with early preclinical stage AD especially if they can be diagnosed by mea-suring Tau/A?and pTau/A?ratio in CSF(de Jong et al.,2006; Fagan et al.,2007a,b)and/or detecting of accumulation of A?in the brains using Pittsburgh Compound-B positron emission to-mography scan(Klunk et al.,2004).

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一、学习用品(school[sku:l] things) Pen钢笔[pen] Pencil铅笔['pens?l] pencil-case铅笔盒['pens?l-keis] ruler尺子['ru:l?] book书[buk] bag包[b?ɡ] comicbook漫画书['k?mik-buk]postcard明信片[p?ust-kɑ:d] newspaper报纸['nju:s,peip? 'nju:z-] schoolbag书包['sku:lb?ɡ]eraser橡皮[i 'reiz?] crayon蜡笔['krei?n] sharpener卷笔刀story-book故事书 ['st ?:ri-buk] notebook笔记本['n?utbuk] Chinese book语文书English book英语书math book数学书 [m?θ-buk] magazine杂志[,m?ɡ?'zi:n] dictionary词典 ['dik??n?ri]二、人体（body）['b?di] foot脚[fut] head头[hed] face脸[feis] hair头发[hε?]nose鼻子[n?uz]mouth嘴 [mauθ]eye眼睛[ai] ear耳朵[i?]arm手臂[ɑ:m] hand手[h?nd] finger手指['fi? ɡ?] leg腿[leɡ] tail 尾巴[teil] 三、颜色（colours） red红[red] blue蓝[blu:]yellow黄['jel?u] green绿[ɡri:n] white白[hwait]black黑[bl?k] pink粉红[pi?k] purple紫['p?:pl] orange橙['?rind?, '?:-]brown棕[braun] 四、动物（animals）animal['?niml] cat猫[k?t] dog狗[d?ɡ, d?:ɡ]pig猪[piɡ]duck鸭[d?k] rabbit兔['r?bit] horse马[h?:s]elephant大象 ['elif?nt] ant蚂蚁[?nt]fish鱼[fi?] bird鸟[b?:d]eagle鹰['i:ɡl]beaver海狸['bi:v?]snake蛇[sneik]mouse老鼠[maus, mauz] squirrel松鼠['skw?:r?l, 'skwi-, 'skw?-] kangaroo袋鼠[,k?nɡ?'ru:]monkey猴['m??ki] panda熊猫['p?nd] bear熊[bε?]lion狮子['lai?n]tiger老虎['taiɡ?] fox狐狸[f?ks] zebra斑马['zi:br?] deer鹿[di?] goose鹅[ɡu:s] giraffe长颈鹿[d?i'rɑ:f] hen母鸡[hen]turkey火鸡['t?:ki] lamb小羊[l?m] sheep绵羊[?ip] goat山羊[ɡ?ut] cow奶牛[kau] donkey驴['d??ki] squid鱿鱼[skwid] lobster龙虾['l?bst?] shark鲨鱼[?ɑ:k] seal海豹[si:l] sperm whale抹香鲸[sp?:m-hweil] killer whale虎鲸['kil?- hweil] 五、人物（people）['pi:pl] friend朋友[frend] boy男孩[b?i] girl 女孩[ɡ?:l]mother母亲['m?e?] father父亲['fɑ:e?]sister姐妹['sist?] brother兄弟['br?e?] uncle叔叔；舅['??kl]舅 man男人[m?n] woman女人['wum?n] Miss小姐[mis] Mr先.生 lady女士小；姐['leidi] mom妈妈[m?m] dada爸爸[d?d, 'dɑ:dɑ:]parents父母parent['pε?rnt]son儿子[s?n] grandparents祖父母['ɡr?nd,pε?r?nt] grandma/grandmother（外）祖['ɡ母r?nd,m?e?]aunt姑姑[ɑ:nt, ?nt] cousin堂（表）兄弟；堂（表）姐妹['k?z?n]daughter女儿['d?:t?] baby婴儿['beibi]kid小孩[kid]queen女王[kwi:n] classmate同学['klɑ:smeit]visitor参观者['vizit?] neighbour邻居['neib?] principal校长['prins?p?l] university student大学生[,ju:ni'v?:s?ti-'stju:d?nt]pen pal笔友[pen-p?l]tourist旅行者['tu?rist] people人物['pi:pl]robot机器人['r?ub?t, -b?t, 'r?b?t] grandpa/grandfather（外）祖父['ɡr?ndpɑ:]/ ['ɡr?nd,fɑ:e?]六、职业（jobs）[d??bs] teacher教师['ti:t??] student学生['stju:d?nt, 'stu:-]doctor医生[d?kt?] nurse护士[n?:s]driver司机[n?:s] farmer农民['fɑ:m?]singer歌唱家['si??] writer作家['rait?] actor男演员['?kt?] actress女演员['?ktris] artist画家['ɑ:tist] TV reporter电视台记[ri'p?:t?]者 engineer工程师[,end?i'ni?] accountant 会计[?'kaunt?nt] policeman(男)警察[p?'li:sm?n] salesperson销售员['seilz,p?:s?n] cleaner清洁工 ['kli:n?] police警察[p?'li:s] baseball player棒球运动['beisb?:l员- 'plei?] assistant售货员[?'sist?nt] 七、食品、饮（料food & drink） rice米饭[rais]bread面包[bred]beef牛肉[bi:f]milk牛奶[milk] water水['w?:t?, 'w?-] egg蛋[eɡ]fish 鱼[fi?] tofu豆腐['t?ufu:]cake蛋糕[keik] hot dog热狗[h?t-[d?ɡ]hamburger汉堡包['h?mb:ɡ?]jam果酱[d??m] French fries炸薯条[fraids]cookie曲奇['kuki]biscuit饼干['biskit] noodles面条['nu:dl]meat肉[mi:t] chicken 鸡肉['t?ikin]pork猪肉[p?:k]mutton羊肉['m?t?n] vegetable蔬菜['ved?it?bl]salad沙拉['s?l?d]ice冰[ais] soup汤[su:p]ice-cream冰淇淋['aiskri:m]Coke可乐 juice 果汁[d?u:s] tea茶[ti:] coffee咖啡['k?fi] breakfast 早餐['brekf?st] lunch午餐[l?nt?] dinner/supper晚餐['din?] / ['s?p?] meal一餐[mi:l] 八、水果、蔬(fruit/菜 vegetables)[fru:t]['ved?it?bl]

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单项选择题 1、马克思主义政治经济学的理论基础是（ C ） A、劳动价值论 B、剩余价值论 C、辩证唯物主义和历史唯物主义 D、资产阶级古典政治经济学和空想社会主义 解析：劳动价值论是政治经济学的基础，唯物史观和剩余价值理论是马克思的两个伟大发现，选项D是马克思主义政治经济学的理论来源。只有答案C才是理论基础。 4、在生产资料中属于劳动资料的是（ B ） A、原材料 B、机器设备 C、燃料 D、辅助材料 解析：劳动资料是指人的劳动和劳动对象联系起来的媒介物，A、C、D属于劳动对象范畴，即人把自己的劳动加于其上的东西。 8、价值规律是（ A ） A、只在几个社会形态存在的经济规律 B、各个社会形态都存在的经济规律 C、资本主义社会特有的经济规律 D、社会主义社会特有的经济规律 解析：价值是商品的特有属性，非商品的劳动产品不存在价值，在原始社会和共产主义社会不存在商品，因此，商品只是在奴隶社会、封建社会、资本主义社会和社会主义社会存在。 9、基本经济规律是（ D ） A、只存在于资本主义社会的经济规律 B、在一切社会形态都存在和起作用的经济规律 C、几种社会形态共有的经济规律 D、一种社会形态起主导作用的经济规律 解析：此题考核的是基本经济规律的名词解释。需要注意，基本经济规律是一个社会起主导作用的规律。在中国现在虽然有存在私营企业，存在剥削，存在剩余价值，但因为它不占主导地位，所以不能说剩余价值规律是我国的基本经济规律，剩余价值是资本主义的基本经济规律。目前我国的基本经济规律是公有制占主导的按劳分配规律。 11、价值的实体是（ B ） A、具体劳动 B、抽象劳动 C、私人劳动 D、社会劳动 解析：本题考察价值的内涵，即价值一词的名词解释，抽象劳动是形成价值的唯一源泉，因此答案是B。注意抽象劳动本身并不是价值，抽象劳动的凝结才构成价值。 12、社会经济制度更替的一般规律是（ A ） A、生产关系随生产力的发展而逐渐改变自身的性质 B、生产力不断运动 C、生产关系不断变革 D、生产力和生产关系不断进行矛盾运动 解析：社会经济制度属于生产关系的范畴，生产关系的变化归根到底取决于生产力的变化，所以答案为A。 13、市场经济是指（ B ） A、以市场存在为条件的商品经济 B、市场对资源配置起基础性作用的商品经济 C、市场决定一切经济活动的商品经济