www.elsevier.com/locate/jneuroim

Journal of Neuroimmunolo

Mimotopes of conformational epitopes in fibrillar h-amyloid

Goar Gevorkiana,1, Irina Petrushinab,1, Karen Manoutchariana, Anahit Ghochikyanc,

Gonzalo Aceroa, Vitaly Vasilevkob, David H. Cribbsb,2, Michael G. Agadjanyanc,*,2

aInstituto de investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, MexicobThe Institute for Brain Aging and Dementia, University of California Irvine, Irvine, CA 92697, United States

cDepartment of Immunology, The Institute for Molecular Medicine, 16371 Gothard Street, Huntington Beach, CA 92649, United States

Received 21 April 2004; received in revised form 7 June 2004; accepted 7 June 2004

Abstract

In Alzheimer’s disease (AD) h-amyloid peptide accumulates in the brain in different forms including fibrils. Amyloid fibrils could be

recognized as foreign by the mature immune system since they are not present during its development. Thus, using mouse antisera raised

against the fibrillar form of Ah42, we have screened two phage peptide libraries for the presence of foreign conformational mimotopes of Ah.Antisera from wild type animals recognized predominately peptides with the EFRH motif from Ah42 sequence, whereas amyloid precursor

protein (APP) transgenic mice recognized mainly phage clones that mimic epitopes (mimotopes) within the fibrillar Ah42 but lack sequence

homology with this peptide.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Antibodies; Transgenic mice; Antigens; Epitopes; Mimotopes; Immunotherapy

1. Introduction

Alzheimer’s disease (AD) is the most common form

of0 dementia in the elderly and is characterized by two

distinct neuropathological lesions in the brain: extracel-

lular senile plaques and intracellular neurofibrillary tangles

(NFT). The plaques are composed primarily of the h-amyloid peptide (Ah), whereas the NFT are composed of

hyperphosphorylated tau protein (Selkoe, 2001). The Ahpeptide is the product of the proteolytic processing of the

amyloid precursor protein (APP), and there is now a

general consensus that this highly amyloidogenic peptide

is a critical element in the onset and progression of AD

(Hardy and Selkoe, 2002). Thus, reducing the production

or enhancing the degradation and clearance of this peptide

has been a major focus of many laboratories. However,

0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jneuroim.2004.06.004

* Corresponding author. Tel.: +1 714 596 7821; fax: +1 714 596 3791

E-mail address: magadjanyan@immed.org (M.G. Agadjanyan).1 Both authors contributed equally to this work.2 These senior authors contributed equally to this work.

.

key tenets of the bamyloid hypothesisQ of AD have

evolved over the last 12 years and important tests of the

hypothesis are now underway in animal models and

humans (Hardy, 2002; Hardy and Selkoe, 2002). In

particular, Ah-immunotherapy has received considerable

attention as one possible strategy for reducing Ah levels

in the brain (Monsonego and Weiner, 2003; Schenk,

2002; Weiner and Selkoe, 2002). The seminal report by

Schenk et al., 1999, showing that active immunization of

APP transgenic (APP/Tg) mice with fibrillar Ah peptide

in combination with a powerful adjuvant induced anti-

bodies specific for Ah that promoted the clearance of Ahfrom the mouse brain, initiated a new field of study in

AD.

Subsequent studies using passive transfer of anti-Ahantibodies also reduced Ah plaque deposition, neuritic

dystrophy, astrogliosis in the brains of APP/Tg mice, and

diminished learning deficits in mice confirmed the role of

the anti-Ah antibodies in the clearance of Ah (Bard et al.,

2000; DeMattos et al., 2001; Janus et al., 2000; Morgan

et al., 2000; Schenk, 2002). A number of groups have

demonstrated that active immunization induces antibodies

gy 156 (2004) 10–20

G. Gevorkian et al. / Journal of Neuroimmunology 156 (2004) 10–20 11

that preferentially recognize a linear epitope in the N-

terminal region of Ah42 (Cribbs et al., 2003; Dickey et

al., 2001; Lemere et al., 2000, 2001; Town et al., 2001).

These results have been confirmed using proteolytic

cleavage of intact, immobilized immune complexes

consisting of polyclonal antibodies from APP/Tg mice

bound to Ah42, followed by high-resolution mass-spec-

trometry. In this report, the linear Ah42 epitope spanning

residues 4–10 was identified as the sequence binding to

the antibodies (McLaurin et al., 2002). Previously,

monoclonal antibodies to residues 3EFRH6 within the

Ah42 sequence were shown to be effective in disaggre-

gation of fibrillar Ah42 in vitro (Solomon et al., 1996,

1997). Thus, the major linear B cell antigenic epitope in

Ah42 is within the sequence containing residues 1–10.

However, there is considerable evidence that fibrillar

Ah42 possesses conformational epitopes that can induce a

humoral response in humans as wells as mice. Gaskin et

al. reported that four human monoclonal IgM antibodies

derived from one AD patient recognized conformational

antigenic determinants in fibrillar Ah42. This antigenic

epitope/s was conserved in amyloid plaques in situ, which

indicates that humans may recognize this unique con-

formational antigenic determinant/s in fibrillar Ah42 as

non-self (Gaskin et al., 1993b). Recently, two conforma-

tion-specific mouse monoclonal IgM antibodies against

different disease-related amyloid fibrils including Ah40

fibrils were characterized (O’Nuallain and Wetzel, 2002),

although binding of these antibodies to amyloid plaques

in brain tissue from AD patients has not yet been

demonstrated. More recently, polyclonal rabbit antibodies

to the fibrillar Ah42 have been generated, and these

antibodies bound to the fibrillar full-length Ah. Although

the same antisera were capable of binding to linear Ah1–8

sequence, the affinity of binding to fibrillar Ah42 was

almost 1000 times higher. These authors suggested that

the anti-fibrillar antibodies might preferentially bind to the

conformation of Ah1–8 that is stabilized in fibrillar form

of Ah42 (Miller et al., 2003). These results collectively

suggest that the full-length fibrillar Ah42 contains

conformational antigenic determinants that are not formed

by the shorter linear peptides.

In the current study we assayed for conformational B

cell epitopes within the fibrillar form of Ah42 using anti-

Ah42 antisera raised in APP/Tg or wild type mice with

the help of two phage display peptide libraries. We report

that in APP/Tg mice immunization with fibrillar Ah42

generates antibodies that recognize not only the linear

EFRH epitope of this antigen but also a number of

mimotopes expressed by phage clones. One such linear

mimotope expressed by several phage clones was

synthesized, and binding to anti-Ah42 antibodies was

confirmed. Interestingly, anti-Ah42 antisera generated in

immunized wild type mice recognized predominately

linear peptides with the EFRH motif from the Ah42

sequence.

2. Materials and methods

2.1. Preparation of peptides

All peptides were synthesized at the Peptide Core

Facility at the Institute for Brain Aging and Dementia at

UCI. We routinely obtain very high quality Ah synthetic

peptides (N99% purity). Peptides were synthesized by the

solid-phase Fmoc method, purified by reverse-phase

HPLC, as previously described (Cribbs et al., 1997,

2003). h-Amyloid peptides 1–42 (Ah42), 1–40 (Ah1–40),

1–28 (Ah1–28), and 25–35 (Ah25–35), as well as 15- and 17-

mer overlapping small peptides spanning aa 1–15 (Ah1–15),

6–20 (Ah6–20), 11–25 (Ah11–25), 16–30 (Ah16–30), 21–35

(Ah21–35), and 26–42 (Ah26–42) of Ah were prepared. In

addition, we synthesized (Multiple Peptide Systems, San

Diego, CA) high quality (N99% purity) 11- and 16-mer

synthetic mimotopic antigens (MAG) GDLKRTTGGGC

(MAG11) and LDLKRTTTTHTSGGGC (MAG16). Both

MAG peptides were based on data generated from screen-

ing of phage display libraries against anti-Ah42 sera

produced in APP/Tg mice (see Results).

In addition, we conjugated MAG11 to Imject Malei-

mide-Activated Ovalbumin (OVA) to obtain MAG11–

OVA. Two milligrams of peptide MAG11 was conjugated

with powdered Imject Maleimide-Activated Ovalbumin,

and the conjugate was purified by gel filtration according

to the manufacturer’s protocol (Pierce, Rockford, IL).

Briefly, 10 fractions of eluate were collected and the

amount of protein in each fraction was tested by Bio-Rad

protein assay (Bio-Rad, Hercules, CA). Two fractions of

the eluate showing the highest presence of protein were

combined and stored at 4 8C.

2.2. Preparation of antigen and immunization of mice

Six- to eight-week-old APP/Tg 2576 {(H2bxs) UCI

Transgenic Mouse Facility)}, BALB/c (H2d), C57Bl/6

(H2b), C3H (H-2k), and F1B6SJL (H2bxs) mice (Jackson

Laboratories, Bar Harbor, ME) were housed in the animal

facility at UCI in a temperature and light-cycle controlled

facility, and their care was under the guidelines of the NIH

and UCI. Each mouse was injected subcutaneously (s.c.)

with 100 Ag of fibrillar Ah42 formulated in Th2-type

adjuvant Alum (Cribbs et al., 2003). The protocol for

preparation of fibrillar Ah42 was adapted from Kayed et al.

(2003). Briefly, we dissolved lyophilized Ah42 peptide in

1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma) and incu-

bated it at room temperature, 15 min. The seedless Ahsolution was added to ddH2O (V/V=1:10) and after addi-

tional 15 min incubation at room temperature the samples

were centrifuged (14,000�g, 15 min). The supernatant

fraction was transferred to a siliconized tube and subjected

to a gentle stream of nitrogen to evaporate the HFIP (10

min). The samples were then stirred at 500 rpm using a

Teflon coated micro stir bar for 9 days. Fibril formation was

G. Gevorkian et al. / Journal of Neuroimmunology 156 (2004) 10–2012

monitored by thioflavin T fluorescence and UV light

scattering. The solutions were centrifuged at 14,000�g for

20 min, the fibril pellet was washed 3� with the ddH20, and

then resuspended in the desired buffer. The morphology was

verified by AFM or negative stain EM. After the first

immunization, two boosts were performed at 2-week

intervals with the same peptide. Mice from each exper-

imental group (six mice of APP/Tg, six C57BL/6, six C3H,

eight F1B6SJL and twelve BALB/c) were bled 8 days after

last boost, and individual sera were used to detect the

presence of specific anti-Ah42 antibodies by ELISA and/or

for screening of the phage display peptide libraries.

2.3. Detection of the titer of anti-Ab42 antibodies and

peptide competition assay

The same fibrillar Ah42 used for immunization was used

for ELISA. Binding of polyclonal sera to fibrillar Ah42 was

detected as previously described (Kayed et al., 2003; Cribbs

et al., 2003). Briefly, fibrillar Ah42 peptide was diluted in

coating buffer (0.1 M sodium bicarbonate, pH 9.6) to 2.5

AM concentration, and 96 wells plates were coated at 37 8Cwithin only 2 h (the fibril formation was checked by

thioflavin T fluorescence). Plates were then washed,

blocked, and primary sera from experimental and control

mice were added in duplicate at an initial dilution of 1:1000,

or as indicated, and diluted serially in Tween-20 Tris Buffer

Solution (TTBS) to 1:64,000. After incubation and washing,

anti-mouse IgG (whole molecule) conjugated with horse-

radish peroxidase (HRP) was added as recommended by

manufacturer (Jackson Laboratories). Plates were incubated,

washed, and OPD substrate solution (o-phenylenediamine

in 0.05 M phosphate–citrate buffer, pH 5.0, Sigma) was

added to develop the reaction. All plates were analyzed

spectrophotometrically at 405 nm.

Using anti-Ah42 antisera generated in APP/Tg animals

and mice of different haplotypes, we also analyzed binding

of antibodies to fibrillar Ah42 in the presence of small

overlapping peptides Ah1–15, Ah6–20, Ah11–25, Ah16–30,

Ah21–35, and Ah26–42 of Ah42. In this competition ELISA

we also used peptides spanning Ah1–28, Ah25–35 and

fibrillar Ah42. Briefly, plates coated with fibrillar Ah42

were washed and blocked, as described above. Antisera

from immunized and control mice, at approximately the

equal concentrations, were mixed with small overlapping

peptides, Ah1–28, Ah25–35 or with control fibrillar Ah42

peptide (all peptides at final concentration from 0.019 to 5

AM as indicated in Figs. 2 and 3 legends). After incubation

(1 h, 37 8C) the peptide-treated antisera were added in

duplicate to Ah42-coated plates. Using secondary anti-

mouse IgG-HRP antibodies and OPD substrate the reaction

was developed, the plates were analyzed for binding as

described above, and the percent of inhibition by small

peptides, as well as by control peptides were calculated

using binding of sera without competing peptides to fibrillar

Ah42 as 100% (Cribbs et al., 2003).

2.4. Binding of anti-Ab42 antibodies to mimotopes MAG11

and MAG16

In order to correctly compare the levels of binding of

anti-Ah42 antisera from APP/Tg and wild type mice to

MAG11 and MAG16 peptides, we first detected the concen-

tration of anti-Ah42 antibodies in the experimental sera.

Using 6E10 monoclonal antibody that recognizes 1–17aa of

Ah42 (Signet Labs, Dedham, MA), we generated a standard

curve and determined the anti-Ah42 antibody concentration

in the sera of immunized mice. Briefly, individual mouse

serum and the 6E10 antibody were incubated in the wells

coated with fibrillar Ah42. The plates were washed, and

secondary anti-mouse IgG-HRP antibodies were added

followed by OPD substrate solution. All plates were

analyzed spectrophotometrically, average dataFS.D. were

calculated for each group of mice, and results were recorded

in Ag per ml. Equal concentrations of antisera from wild

type and APP/Tg mice were used to detect binding of anti-

Ah42 antibodies to mimotopes. The same ELISA procedure

outlined above was used in these experiments, except

MAG11, MAG16, and MAG11–OVA were used for coating

the 96 wells plates (2.5AM peptide in bicarbonate coating

buffer, pH 9.7).

2.5. Screening of phage display peptide library with

polyclonal anti-Ab42 antibodies

Selection of phage by biopanning was performed

essentially as previously described (Gevorkian et al.,

2000; Gomez-Roman et al., 2002). Briefly, Maxi Sorp

microtiter plates (Nunc, Roskilde, Denmark) were coated

with goat anti-mouse IgG (Zymed, CA, USA) at a

concentration of 5 Ag/ml and blocked with PBS supple-

mented with 2% BSA (bovine serum albumin). After

washing, polyclonal anti-Ah42 antisera (1:200 dilution)

from wildtype or APP/Tg mice were added, and plates

were incubated for 1 h at 37 8C. Two phage display

peptide libraries (New England Biolabs, Beverly, MA,

USA) of linear heptapeptides and dodecapeptides were

screened against polyclonal anti-Ah42 sera raised in APP/

Tg mice, whereas sera generated in C57BL/6 mice were

used only with the heptapeptides library. In both libraries

random peptides were expressed at the N-terminal region

of the minor coat protein pIII of the M13 phage. After

washing, 1011 plaque-forming units (PFU) from each

library were added to the plates and incubated for 3 h

at 4 8C, followed by 1 h at room temperature. Non-

specific phages were washed off, and affinity-selected

phages were eluted by glycine–HCl (pH 2.2) and

neutralized by Tris–base. A total of three rounds of

biopanning were performed, and the eluate from each

round was amplified and used for each subsequent round

(second and third) or for characterization of clones (third

round). Individual phage clones were picked at random

from the plated eluates, and used in ELISA to detect the

Fig. 1. Titer of anti-Ah42 antisera generated in APP/Tg and wildtype mice

of four different MHC haplotypes. Total Ig was detected in serum from

individual mice (n=number of animals) after three immunizations with

fibrillar Ah42 formulated in Alum, a Th2-type adjuvant. Data represent the

average titerFS.D. Data was analyzed by one-way ANOVA (see Section 2)

to show with statistical significance that APP/Tg (in all dilutions P at least

b0.001) and C57BL/6 (in all dilutions P at least b0.01) mice antibody

responses were lower than that of mice of other haplotypes. The

experiments with APP/Tg, BALB/c, and C57BL/6 mice were repeated

twice with similar results.

G. Gevorkian et al. / Journal of Neuroimmunology 156 (2004) 10–20 13

binding to anti-Ah42 antisera and sequencing of phage-

bearing peptides.

2.6. Detection of binding by anti-Ab42 antisera to selected

phage clones by ELISA and DNA sequencing

Plates were coated with goat anti-mouse IgG as described

above. After washing, polyclonal anti-Ah42 antibodies were

added, and plates were incubated for 1 h at 37 8C. Afterwashing, 1010 PFU/ml of each individual clone were added

to plates and incubated for 3 h at 4 8C, followed by 1 h at

room temperature. After washing, a rabbit anti-M13

polyclonal antibody prepared in our laboratory was added

and incubated for 1 h at 37 8C. An anti-rabbit IgG (Zymed)

conjugated with peroxidase was then incubated for an

additional hour at 37 8C followed by a wash step, and an

ABTS single solution (Zymed) was added. The absorbance

values were measured at 405 nm. All experiments were

performed three times in duplicate. The single-stranded

DNA was isolated from each phage clone and individual

peptide sequence was determined or deduced as we

described previously (Gevorkian et al., 2000; Gomez-

Roman et al., 2002).

2.7. Statistical analysis

Data was analyzed by one-way ANOVA test, using

Tukey’s Multiple Comparison Post-Test, of Graph Pad

Prism 3.03 Software.

3. Results

3.1. The presence of conformational antigenic determinants

in Ab42 peptide

Our previous results suggested that conformation-

specific polyclonal antibodies may be induced following

immunization of BALB/c mice with fibrillar human Ah42

(Cribbs et al., 2003). Now we have investigated anti-Ah42

antisera in wildtype mice of four different MHC hap-

lotypes and, more importantly, in APP/Tg 2576 mice for

the generation of conformation-specific antibodies in

fibrillar Ah42 immunized mice. Alum, a Th2 type

adjuvant, was used to boost the humoral immune

response. Immunization of APP/Tg 2676 mice of H2b x s

haplotypes induced the lowest, whereas immunization of

MHC-matched non-transgenic F1B6SJL (H2b x s) animals

induced the highest titer of anti-Ah42 antibodies (Fig. 1).

BALB/c (H2d) and C3H (H-2k) mice, which are not

MHC-matched with APP/Tg 2576, generated anti-Ah42

antibody response levels similar to that of F1B6SJL. At

the same time, humoral immune response was signifi-

cantly impaired not only in APP/Tg 2576 animals, but

also in the background wild type strain—C57Bl/6 (H2b)

mice. Notably, the level of anti-Ah42 antibody in another

parental wild type strain, SJL (H2s), was not impaired

(data not shown and Petrushina et al., 2003).

Our initial goal was to determine whether antibodies

generated in these animals bind to the fibrillar Ah42 better

than to the short linear peptides. Thus, we analyzed the

mouse sera for the presence of conformational antigenic

determinant/s on fibrillar Ah42 by using a competition

assay described earlier (Cribbs et al., 2003). In order to

standardize anti-Ah42 antibody levels, we have used

different dilutions of antisera generated in wild type or

APP/Tg mice. Pre-incubation of antisera from both wild

type and APP/Tg mice with Ah42 resulted in almost

complete loss of antibody binding to the bound fibrillar

Ah42. However, binding of antisera from wild type mice

was inhibited between 50% and 80% by peptides Ah1–15

and Ah1–28. Conversely, binding of antisera from APP/Tg

2576 mice was only inhibited to approximately 27% by

peptides Ah1–15 or Ah1–28 (Fig. 2). To further confirm the

data from APP/Tg mice, we performed additional

inhibition studies using individual antisera from these

animals and different concentrations of Ah1–15, Ah6–20,

Ah42, as well as a mixture of Ah11–25 Ah16–30, Ah21–35,

and Ah26–42 (Fig. 3). Peptide Ah1–15 even at a

concentration of 5 AM only blocked binding of serum

antibody to Ah42 antigen between 53% and 67%, and

this was significantly lower than inhibition with Ah42

(Pb0.001). As expected, the peptides Ah6–20 and mixture

of Ah11–25+6–30+21–35+26–42 even at 5 AM were ineffective.

Interestingly, fibrillar Ah42 was still a very potent inhibitor

at concentrations as low as 0.313 AM (about 60%

Fig. 3. Conformation-specific polyclonal anti-Ah42 antibodies in the

immune sera from individual APP/Tg mice. Inhibition studies were

performed with indicated concentrations of Ah1–15, Ah6–20,

Ah11–25+16–30+21–35+26–42, and Ah42 peptides and anti-Ah42 sera.

Averaged data with antisera at dilution of 1:4000FS.D. are presented

(n=6). The ANOVA analysis (see Section 2) showed with statistical

significance that linear Ah1–15 peptide inhibited significantly weaker than

fibrillar Ah42 peptide ( Pb0.001 at concentrations 5–0.313 AM; Pb0.05 at

concentrations 0.078 AM; and Pb0.01 at concentrations 0.019 AM).

Fig. 2. The presence of conformation-specific polyclonal anti-Ah42 antibodies in the sera of Ah42 immunized APP/Tg 2576 mice and wild type animals of

different haplotypes. An ELISAwas performed to measure the ability of six small overlapping peptides, as well as Ah1–28, Ah25–35, and Ah42 peptides (all at

final concentration 2.5 AM) to inhibit the binding of polyclonal anti-Ah42 antibodies to Ah42 (concentrations of pooled sera are indicated in parenthesis). The

inhibitory potency of both Ah1–28 and Ah1–15 were similar, whereas the inhibition with Ah42 was significantly higher, especially with sera from APP/Tg mice.

These experiments were repeated twice with similar results.

G. Gevorkian et al. / Journal of Neuroimmunology 156 (2004) 10–2014

inhibition), while Ah1–15 was virtually inactive at this

concentration (Pb0.001). The results from competition

assays argue for the presence of conformation-specific

polyclonal anti-Ah42 antibodies generated in wild type

mice of different haplotypes and at much higher levels in

APP/Tg 2576 animals.

3.2. Screening of phage display libraries against polyclonal

anti-Ab42 antibodies raised in wild type mice

In order to identify the linear peptides that mimic

conformational epitope/s possessed in fibrillar Ah42 peptide,

we used the well-characterized phage display peptide library

technique. Phage display peptide libraries provide an

excellent source of a large number of epitopes and have

been widely used in studies on antigen–antibody binding

(Manoutcharian et al., 2001; Mertens et al., 2001; Scott and

Smith, 1990). Accordingly, a phage display library of linear

heptapeptides was screened against polyclonal anti-Ah42

sera raised in C57BL/6 mice, the weakest responder among

wild type lines. Three rounds of biopanning were per-

formed, and enrichment of affinity-selected clones was

selected in each round. After the third round of biopanning,

22 plaques were randomly picked and amplified. DNA

sequences of the heptapeptide inserts, and the deduced

Fig. 4. (a, b, c) Binding of anti-Ah42 antibodies generated in C57BL/6 (a) and APP/Tg (b, c) mice to phage clones, isolated after screening of 7-mer (a, b) and

12-mer (c) phage display libraries (see details in Section 2). The experiments with isolated phage clones were repeated three times with the similar results.

DOD=experimental OD (binding to a phage clone displaying a peptide) minus background (binding to a control phage clone with irrelevant peptide). Number

of positive phage clones presented in parentheses.

G. Gevorkian et al. / Journal of Neuroimmunology 156 (2004) 10–20 15

G. Gevorkian et al. / Journal of Neuroimmunology 156 (2004) 10–2016

amino acid sequences were determined. All selected phage

clones were amplified and used in ELISA to confirm

binding of selected phage clones with appropriate anti-Ah42

and control sera. Peptide inserts of 17 positive clones (from

22 selected) were identified. Sixteen clones have peptide

inserts comprised of the linear 3EFRH6 immunodominant

region of Ah42: seven clones had EFRH, five clones XFRH,

two clones EXRH, one clone XXRH, one clone EFRX (Fig.

4a). One of 17 positive clones had peptide insert YELPRLT,

non-homologous to immunodominant linear 3EFRH6 epit-

ope. However, this phage clone binds to Ah42 antibodies

very weakly (Fig. 4a). None of the negative phage clones

had homology with this immunodominant region (data not

shown). Similar results were generated with anti-Ah42 sera

isolated from high responder mice of H2k haplotype (data

not shown). Therefore, wild type mice of two haplotypes

predominately recognized the linear immunodominant

epitope EFRH spanning N-terminal amino acids 3–6.

3.3. Screening of phage display libraries against polyclonal

anti-Ab42 antibodies raised in APP/Tg 2576 mice

Next, we screened anti-Ah42 antisera from APP/Tg2576

mice against the same phage display peptide library

(heptapeptides) that had been used against antisera from

Fig. 5. (a, b, c, d) Binding of anti-Ah42 antisera (equivalent to 0.4 Ag/ml antibod

haplotypes (n=4) to MAG11 and MAG16 mimotopic peptides (mimotopes). Dilu

generated with monoclonal antibody 6E10.

wild type mice. Sixteen positive clones were selected from

biopanning and DNA (data not shown), and amino acid

sequences of their inserts were determined (Fig. 4b). Only 4

out of 16 ELISA-positive clones had the sequence EFRH,

and one clone had EFRH motif (XFXH). The inserts of the

other positive clones had sequences that did not show

homology with Ah42, suggesting that they may represent

peptides that mimic Ah42 epitopes. From these phage

clones, five had similar inserts with the GDXKRTT motif:

four clones with GDLKRTT sequence and one clone with

GDFKRTT sequence. To confirm these results, we screened

anti-Ah42 antisera from APP/Tg mice against a phage

display peptide library of linear dodecapeptides. Thirteen

ELISA-positive clones were generated, and only two of

them had the EFRH sequence motif, whereas all the other

clones had inserts that lacked homology with the Ah42

peptide. Interestingly, two clones had the sequence LDLK-

RTTTTHTS (two clones). Thus, DXKRTT motif was

encountered in seven phage clones isolated collectively

from two different libraries: GDFKRTT (one clone),

GDLKRTT (four clones), and LDLKRTTTTHTS (Fig.

4b,c). Thus, anti-Ah42 sera from APP/Tg mice recognized

predominantly phage clones carrying non-self peptides

(mimotopes) and very few clones containing the linear

epitope EFRH of Ah42.

ies) generated in individual APP/Tg (n=6) and wild type mice of different

tions of antisera in ELISA were adjusted according to the standard curve

G. Gevorkian et al. / Journal of Neuroimmunology 156 (2004) 10–20 17

3.4. Binding of mimotopic peptide MAG11 or MAG16 to anti-

Ab42 sera isolated from mice of different haplotypes and

APP/Tg animals

Two of the mimotopes with DLKRTT motif identified in

screening of phage display libraries against anti-Ah42 sera

from APP/Tg 2576 mice were synthesized as following: the

7-mer (GDLKRTT) and 12-mer (LDLKRTTTTHTS) pep-

tides cores plus three amino acids from the N-terminal

region of the minor coat protein of the M13 phage (GGG)

and Cys residue (MAG11 and MAG16). Along with these

peptides, we generated MAG11 and MAG16 peptides

conjugated to OVA and all four peptides were used for

detection of binding to anti-Ah42 antibodies from wild type

and APP/Tg 2576 immunized mice. Because titers of anti-

human Ah42 antibodies in the sera of APP/Tg and wild type

mice were very different (see Fig. 1), we first determined the

concentrations of these antibodies in the antisera of

individual BALB/c, C57BL6, F1B6SJL, and APP/Tg mice

using a standard curve generated with monoclonal antibody

6E10 (data not shown). Immunization of C57BL6 and APP/

Tg 2576 mice with fibrillar Ah42 induced approximately six

times less anti-Ah42 antibodies than in BALB/c and

F1B6SJL animals (41.6F9.01 vs. 250.2F31.77 Ag/ml),

and these data correlated with results previously reported

with immunization of C57BL6 (H2b) and B6D2F1 (H2bxd)

mice by Ah immunogen (Spooner et al., 2002). Accord-

ingly, to compare binding of anti-Ah42 antibodies to MAG11

and MAG16 peptides, we used approximately 0.4 Ag/ml

anti-Ah42 antibodies in each ELISA. We demonstrated that

anti-Ah42 antibodies from two APP/Tg mice bound to

MAG11 and MAG16 peptides strongly. Antisera from two

other mice bound these peptides weakly, whereas the sera

from the remaining two mice did not show significant

binding (Fig. 5a). Anti-Ah42 sera from F1B6SJL mice also

Fig. 6. Conjugation of MAG11 and MAG16 to the carrier protein (OVA) had

masked some antigenic determinants of mimotopes. Binding of anti-Ah42

antisera (equivalent to 0.4 Ag/ml of antibodies) generated in APP/Tg (n=4,

responders only) mice to MAG11, and to OVA–MAG11 or OVA–MAG16,

was measured by ELISA three times with similar results. The data analysis

(see Section 2) showed with statistical significance ( Pb0.05) that

conjugation with OVA inhibited binding of the antisera to MAG peptides.

reacted to MAG11 and MAG16 peptides. One animal

responded significantly, whereas the other three mice had

a very weak response (Fig. 5b). All four BALB/c and the

four C57BL6 animals immunized with fibrillar Ah42

peptide did not recognize MAG11 and MAG16 mimotope

peptides (Fig. 5c,d). Next, we compared binding of four

positive antisera from APP/Tg mice (two high and two

intermediate responders) to MAG11 directly or to MAG11

and MAG16 conjugated with OVA. It appears that anti-Ah42

antibodies bound to MAG11 peptide significantly (pb0.05)

better than to MAG11–OVA or MAG16–OVA coated plates

(Fig. 6).

4. Discussion

Conformation-specific monoclonal anti-Ah42 antibodies

generated in APP/Tg mice and AD patient have been

previously described (Gaskin et al., 1993a; O’Nuallain and

Wetzel, 2002). Data recently generated by our group also

suggests that active immunization of wild type mice with

fibrillar human Ah42 induces antibodies that may recognize

conformational antigenic determinants on this peptide

(Cribbs et al., 2003). More recently, it was demonstrated

that rabbits injected with fibrillar self-Ah42 peptide induced

antibodies that bind to fibrillar form of Ah42 about 1000-

fold stronger than to linear peptide Ah1–8 (Miller et al.,

2003). These results collectively suggest that the full-length

fibrillar Ah42 contains some conformational antigenic

determinant/s that is not present in the shorter linear

peptides. In this study we directly investigated the presence

of such conformational epitope/s (mimotope/s) on fibrillar

Ah42 peptide using mouse polyclonal anti-Ah42 sera and

phage display library technique.

First, we raised polyclonal anti-fibrillar Ah42 antibodies

in wild type mice of four different haplotypes and APP/Tg

2576 animals, and detected the titer of these antibodies

(Fig. 1). Next, we analyzed the binding of antibodies to

different short linear peptides or fibrillar Ah42. Because

adsorption of a peptide to an ELISA plate may mask some

of the peptide’s epitopes, we decided to detect the presence

of conformational antigenic determinants in fibrillar Ah42

by using a competition assay where the small peptides are

in solution and the fibrillar Ah42 is bound to the ELISA

plate. As we expected from our previous data (Cribbs et al.,

2003), fibrillar Ah42 peptide almost completely inhibited

binding of antisera from all wild type and APP/Tg mice to

the bound Ah42. Only two short peptides, Ah1–15 and Ah1–

28, also had substantial inhibitory activity, although it was

less than that for fibrillar Ah42, specifically in case of APP/

Tg mice (Fig. 2). These data were confirmed with antisera

from APP/Tg mice when we used different concentrations

of Ah1–15, Ah6–20, Ah11–25+ 16–30+21–35+26–42, and fibrillar

Ah42 peptides (Fig. 3). Thus, the results from competition

assays argue for the presence of conformation-specific

polyclonal anti-Ah42 antibodies generated in wild type

G. Gevorkian et al. / Journal of Neuroimmunology 156 (2004) 10–2018

mice of different haplotypes, and especially in APP/Tg

2576 mice.

To identify linear peptides that mimic conformational

epitope/s within fibrillar Ah42 we utilized the phage display

technique which has proven useful in identifying mimotopes

of conformational epitopes. Antisera from C57BL6 wild

type mice frequently recognized the linear immunodominant

epitope EFRH spanning the N-terminal amino acids 3–6 of

Ah42. In total, 16 positive phage clones were identified, and

from all of these clones seven had the EFRH motif (Fig. 4a).

Previously, phage clones carrying the 3EFRH6 epitope of

Ah42 were described by binding to two different mono-

clonal antibodies (Frenkel et al., 1998). In our report, we

demonstrated that polyclonal anti-fibrillar Ah42 serum from

wild type mice of two different haplotypes (low and high

responders) also recognized this linear antigenic determi-

nant of Ah42 (Fig. 4a and data not shown, respectively). In

this mouse model, human fibrillar Ah42 is not a self-antigen,

so it was not surprising that the majority of antibodies

recognize the N-terminal region of Ah42 that differs from

the rodent sequence by three aa at positions 5, 10, and 13

(Hilbich et al., 1991). Thus, at least some of the immune

responses were likely due to recognition of the non-self

human residues in the human peptide used for immuniza-

tion. Next, we decided to screen two different phage

libraries against anti-fibrillar Ah42 serum from APP/Tg

2576 mice. In two separate experiments we detected only 6

out of 29 ELISA positive clones that had a sequence EFRH,

and one clone that had a EFRH motif (XFXH). These results

were similar to those generated with M13 phage display

system and polyclonal antibodies from rabbits immunized

with fibrillar Ah42 (Miller et al., 2003) (rabbit and human

Ah42 are identical). These authors identified five phage

clones that expressed peptides with a 2AEFRH6 motif.

However, we also detected 23 positive clones that did not

have any homology with the Ah42 immunogen. Whether or

not these data represent differences in animal models

remains to be determined, however in our experiments the

DXKRTT motif was encountered in seven phage clones

isolated from two different libraries. Thus, antisera from

wild type animals recognized primarily the EFRH epitope

(Fig. 4a), whereas anti-Ah42 sera from APP/Tg mice

recognized very few clones carrying the linear epitope

EFRH (Fig. 4b). On the contrary, the sera from immunized

APP/Tg mice identified predominantly phage clones carry-

ing mimotopes of fibrillar Ah42 (Fig. 4b,c), which suggests

that different antibody repertoires were induced following

Ah-immunization of transgenic and wild type mice.

Next we demonstrated binding of anti-fibrillar Ah42 sera

from APP/Tg mice to the mimotopes GDLKRTT and

LDLKRTTTTHTS, frequently identified in screening of

two phage display libraries with anti-Ah42 sera from APP/

Tg mice. In an attempt to conserve the native structure of

these peptides, we added three amino acids from N-terminal

region of the minor coat protein of the M13 phage (GGG) to

the C-terminus. In addition, we added a cysteine residue to

facilitate conjugation to the carrier protein. Therefore, we

generated MAG11 and MAG16 peptides along with MAG11–

OVA and MAG16–OVA conjugates, which were used for

detection of binding by anti-Ah42 antisera. To compare

binding of anti-Ah42 antibodies from each mouse to MAG11

and MAG16 peptides in ELISA, we used anti-Ah42 sera

equivalent to approximately 0.4 Ag/ml of antibody. Sera

from at least two APP/Tg mice recognized MAG11 and

MAG16 strongly, whereas sera from two other animals

bound these peptides weakly, and the remaining two sera

did not show appreciable binding (Fig. 5a). Surprisingly,

one out of four B6SJLF1 mice, that shared the same MHC

haplotype with the APP/Tg mice, also had antibodies

binding to MAG11 and MAG16 peptides. In contrast, anti-

Ah42 sera from two other mice strains (BALB/c or C57BL6)

with MHC haplotypes different from APP/Tg animals did

not recognize either MAG11 or MAG16 (Fig. 5c,d).

These results identified for the first time the linear

foreign peptides that mimic fibrillar Ah42 without having

any homology. Molecular mimicry is defined as similar

structures within very different macromolecules. The con-

cept of molecular mimicry is taken primarily from the

classical biochemistry of receptor–ligand or antibody–

antigen interactions. Previously, we reported that immuni-

zation with a peptide that mimics a carbohydrate molecule

induced antibodies against a viral glycoprotein (Agadjanyan

et al., 1997). In the current report we demonstrate that

conformation-specific polyclonal anti-Ah42 antibodies can

bind to non-homologous linear peptides, which mimic

conformational epitopes of fibrillar Ah42. Thus, we have

identified antigenic mimotopes of fibrillar Ah42. We realize

that antigenic and immunogenic properties of epitope/

mimotopes may not correlate directly (Deroo and Muller,

2001; Van Regenmortel, 2001), and in order to demonstrate

the immunogenicity of mimotopic peptides we need to

determine whether anti-Ah42 antibodies are induced after

immunization of mice with these peptides. However, first

we analyzed whether conjugation with the carrier protein

influences binding of anti-Ah42 antisera from APP/Tg mice

to the mimotopic peptides MAG11 or MAG16. Anti-Ah42

antibodies bound to non-conjugated MAG peptide signifi-

cantly better than to conjugated Mag11 and MAG16 (Fig. 6).

Therefore, conjugation of these peptides with OVA appears

to mask some antigenic determinants of the mimotopes.

While other carrier proteins and/or methods of conjuga-

tion may be required for preparation of immunogenic

versions of mimotopes and testing for their antigenic

properties, we believe that it will be extremely important

to detect both antigenic and immunogenic mimotopes in

fibrillar Ah42 in humans. It is likely that different organisms

may see the mimotopic structures of the same antigen quite

differently. The characterization of antibodies generated

after vaccination of AD patients and immunization of APP/

Tg mice supports this hypothesis. Although the Ah42

epitope recognized by human antibodies was not determined

directly, it was shown that, while sera from APP/Tg mice

G. Gevorkian et al. / Journal of Neuroimmunology 156 (2004) 10–20 19

recognized Ah42 monomers, tetramers, hexamers and large

oligomers, the human antibodies appear to bind only to the

aggregated form of Ah42 (Hock et al., 2002; McLaurin et

al., 2002; Spooner et al., 2002; Dodart et al., 2003).

Accordingly, we are planning to screen the same phage

display libraries against human anti-Ah42 sera in order to

detect human mimotopes of conformational epitopes in h-amyloid fibrils. Identification of linear peptides that mimic

human Ah42 structures may provide significant advantages

over immunization with fibrillar Ah42. First, the mimotope

will be non-toxic. Second, the immunogenicity may be

significantly enhanced in humans, because it will be a non-

self antigen. Third, the mimotopic short peptide (non-self B

cell epitope) can be attached to a foreign T cell epitope to

amplify the humoral immune response to the antigenic

mimotopes. Thus, the identification of human mimotopes of

fibrillar or possibly oligomeric forms of Ah42 may provide

novel molecular targets for generating a safe and effective

epitope vaccine against AD.

Acknowledgements

This work was supported by R01 grants from NIH

AG20241 for D.H. Cribbs and M.G. Agadjanyan; AI

44809 for M.G. Agadjanyan, and IIRG grant by Alzheimer’s

Association (IIRG-03-6279) for M.G. Agadjanyan and D.H.

Cribbs.

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