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Protection against measles virus encephalitis by monoclonal antibodies binding to a cystine loop domain of the H protein mimicked by peptides which are not recognized by maternal antibodies

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Protection against measles virus encephalitis by monoclonal

antibodies binding to a cystine loop domain of the H protein

mimicked by peptides which are not recognized by maternal

antibodies

Diana Ziegler, ~,2 Phillippe Fournier, 1 Guy A. H. Berbers, 4 Heiko Steuer, ~ Karl-Heinz WiesmCiller, s

Burkhard Fleckenstein, S Francois Schneider, 1 GOnther Jung, 3 Chwan-Chuen King 6

and Claude P. Muller 1,2

1 Laboratoire National de Sante, PO Box 1102, L-1011 Luxembourg, Luxembourg

z.3 Medizinische Fakult~it 2 and Institut fCir Organische Chemie 3, Universitat TCibingen, D-72076 T~bingen, Germany 4 Rijksinstituut voor Volksgezondheid en Milieu, NL-3720 Bilthoven, Netherlands

s Naturwissenschaftliches und l'4edizinisches Institut, D-72762 Reutlingen, Germany

6Graduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Republic of China

After immunization with measles virus (MV) several monoclonal antibodies (HAbs) were obtained, which reacted with peptides corresponding to the amino acids 3 6 1 - 4 1 0 of the haemagglutinin pro- tein (HV-H). Three of these HAbs (BH6, BH21 and BH216) inhibited haemagglutination, neutralized HV in vitro and protected animals from a lethal challenge of rodent-adapted neurotropic HV. These HAbs reacted with the 15-met peptides H381 and H 3 8 6 defining their overlapping region 3 8 6 - 3 9 5 as a sequential neutralizing and protective epitope, which can be imitated by a short peptide. H381 and H 3 8 6 share two Cys residues (C3s~KGKIOJ~LCss4ENPEWA) and for optimal

MAb

binding of peptide (or HV) disulphide bonds were required in addition to a linear C-terminal extension.

Other NAbs bound to peptides C- (BH147, BH195) and N-terminally (BH 168, BH 171) adjacent to the loop but did not neutralize or protect. When sera from measles patients or from women of child- bearing age were tested with the peptides cor- responding to this haemagglutinating and neutral- izing epitope (HNE), none of the sera recognized the 15-met peptides of this region, while some reactivity was found to 30-mers homologous to different wild-type mutants. Its lack of recognition

by

maternal antibodies and its high degree of conservation would make the HNE loop an attractive candidate to include into a subunit vaccine, which could be administered during early childhood, in- dependent of immune status.

Introduction

Current live attenuated measles virus (MV) vaccines have

effectively reduced the morbidity and mortality of measles

world-wide. However, while the incidence of measles has been

reduced to sporadic cases in the northern hemisphere, the lack

of resistance to passively transferred maternal antibodies and

the thermal lability of the live measles vaccines are major

impediments to their successful application in developing

countries and to the global eradication of measles (Weiss,

1992). Although some progress has been made towards

Author for correspondence: C. P. Muller. Fax + 352 490686. e-mail claude.muller@santel.lu

improved heat stability of live attenuated measles vaccines,

subunit vaccines could potentially be distributed without the

need for a cold chain. In addition, peptide-based vaccines could

potentially be resistant to maternal antibodies, thus allowing

early vaccination independent of immune status.

Maternal antibodies (Albrecht

et al.,

1977) and hyper-

immune globulin (Janeway, 1949) demonstrate the importance

of antibodies in protecting exposed subjects from measles. MV

haemagglutinin (MV-H) is known to be the prime target for

neutralizing and protective antibodies (Giraudon & Wild,

1985; Varsanyi

et al.,

1987).

Competitive binding assays with monoclonal antibodies

(MAbs) have been used to identify distinct antigenic sites of

the H protein (Sheshberadaran & Norrby, 1986; Carter et

al.,

'.47! 0001-3983 © 1996 SGM

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1982; Giraudon & Wild, 1985; ter Meulen et al., 1981). Hu et

al. (1993) and Liebert et al. (1994) have m a p p e d neutralizing epitopes of the M V - H b y sequencing escape mutants resistant to neutralizing antibodies. Thus, amino acids were identified that are important for the integrity of the M A b binding sites. Neutralizing antibodies are m o s t l y directed against con- formational epitopes. However, linear epitopes have also been found to be neutralizing and sometimes protective e.g. in foot- and-mouth disease virus (Francis et al., 1991), influenza virus (Shapira et al., 1984), polio virus (Emini et at., 1983) and human immunodeficiency virus (Wang et al., 1991). In morbilliviruses, the potential of peptides for immunization has been demon- strated with a neutralizing and protective epitope of the M V fusion protein (Obeid et al., 1995; Steward et al., 1995).

Linear binding sites of MV-H-specific antibodies have been m a p p e d with sera from late convalescent donors b y peptide ELISA using a full set of overlapping peptides of the M V - H protein (Muller et al., 1993). Using peptide 185-195 of the MV-H, a rabbit polyclonal serum was obtained which neutralized in vitro (M~ikel/i et al., 1989).

In the present study, we have d e v e l o p e d M A b s that defined a cystine loop protruding at the M V - H surface that represents a neutralizing and protective epitope that can be mimicked b y a short linear peptide and that is not recognized b y human maternal antibodies.

Methods

• Reagents. The MV Edmonston strain (ATCC VR-24) was grown in Vero cell cultures and its early passage supematants were used for neutralization assays. Haemagglutination-inhibition (HAl) assays and MV ELISA were performed with MV concentrated by ultrafiltration. For Westem blot and for one immunization (BH216) the virus was further

concentrated and purified by sucrose-gradient centrifugation (Muller et

al., 1995). Virus used for all other immunizations was purified by affinity chromatography and inactivated with fl-propiolactone. Ltk cells trans- fected with H (Ltk H; kindly provided by F. Wild and P. Beauverger; Beauverger et al., I993, 1994) and a persistently infected human Epstein- Barr virus (EBV) -transformed B cell line (WMPTT; gift from B. M. Chain, University College Medical School, London, UK) were used to test the reactivity of the MAbs with MV or MV-H protein by flow cytometry as described (Muller et al., 1995).

Production of MAbs.

8-10-week-old BALB/c mice were immu- nized by intraperitoneal or multifocal subcutaneous injections with purified native or denatured (5 min boiling in I mg/ml SDS under non- reducing conditions) MV emulsified in incomplete Freund's adjuvant (Sigma). The animals were boosted with the same antigen preparation on day 21 and 38 (denatured MV) or day 49 (native MV). On day 4I or 52, spleen cells were explanted and fused with Sp2/0 myeloma cells to generate MAbs. Hybridomas specific for MV by ELISA or by Western blot were cloned by limiting dilution in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 mM-hypoxanthine, I mM- glutamine, 1 raM-sodium pyruvate, 100 IU/ml penicillin, 100 ,g/ml streptomycin and 10% fetal bovine serum (FBS) (all from Gibco). Antibodies were purified from the same but FBS-free medium or from ascites and purified by affinity chromatography on a protein G column (Pharmacia). Concentrations were estimated by A280 determination. Ig

subclasses and isotypes were determined with the mouse MAb isotyping kit (Hycult Biotechnology)

P e p t i d e s . 121 15-mers overlapping by 10 amino acids and covering

the whole sequence of the MV-H protein (Edmonston strain; Alkhatib & Briedis, 1986) and truncation, elongation and substitution analogues of peptide H381 and H386 were synthesized by simultaneous multiple peptide synthesis (Wiesmtiller eta]., 1992). The peptides were modified at the C terminus by two e-amino caproic acid residues and one lysine residue which separate the peptide from the biotin residue which was coupled to the Ne-amino group of Iysine amide. In Fig. 4 peptides were biotinylated at their N terminus using a similar spacer, All peptides are designated by the position of their N-terminal amino acid. Peptides were reduced with I40 mM-2-mercaptoethanol. Even much higher concen-

trations did not directly interfere with the ELISA assay, as was measured

with an antibody specific for a peptide containing a single Cys. During the different ELISA steps, reduced peptides were protected from oxidation with 0"0I % vitamin C. Peptides were oxidized by bubbling air through a 5 ,g/ml solution.

• FLISA. The ELISA based on the biotinylated peptides was described previously (Foumier et al., I996). In brief, microtitre plates (Maxisorp; NUNC) were coated with 20 ~g/ml highly purified streptavidin (a gift from L. Seik, Mediagnost, T/ibingen, Germany). Biotinylated peptides (5 p.g/ml) were immobilized by incubation for at least I h at room temperature. Binding of the biotinylated peptides to the plate was verified by competition with biotinylated alkaline phosphatase (Vector Laboratories). Free binding sites were saturated with 1% BSA (Sigma) in TBS. In some assays with MAbs the blocking buffer contained I0 % BSA, 10 % saccharose and 2 % v/v normal goat serum ([CN) in PBS. The plates were either incubated for 1'5 h at 37 °C or overnight at 4 °C with mouse sera or hybridoma supematants diluted 1 : 200- or 1 : 20-fold, respectively, in dilution buffer (10 mM-TBS, 1% BSA, 0"1% Tween, pH 7'4).

The ELISAs were developed with alkaline phosphatase-conjugated goat immunoglobulin (whole molecule) specific for the v-chain of mouse or human IgG (Sigma) at a dilution of I:500 in dilution buffer using p-nitrophenylphosphate (Sigma) as a substrate. Absorbance was measured at 405 nm.

In vivo

challenge/protection experiments,

13-16-day-old,

specific-pathogen-free (SPF) BALB/c mice were treated with a single intraperitoneal injection of purified MAbs (56-364 p,g). I day later they were challenged by intracerebral injection of 12500 TCID60 of BALB/c- adapted CAM MV strain derived from the CAM/RB strain (a gift from U. G. Liebert, Wiirzburg, Germany; Liebert & ter Meulen, 1987).

Human sera. Blood was drawn after informed consent from 320

consecutive out-patients visiting the Laboratoire National de Sant6 (Luxembourg) for a diagnostic venous puncture. Neutralization (NT) and HAI titres were performed as described earlier (Norrby & Gollmar, 1972) with some minor modifications (Muller et al., 1995). Complement-fixing (CF) titres of human sera were determined with a commercial kit (Virion, CH-6330 Cham). Mean log 2 titres were 7'7 q- I2, 7"2 -}- 1'8, 4'4 -f- I"9 for NT, HAI and CF, respectively. Twenty sera from women of child-bearing age with high NT and HA[ titres were tested (Table 2) with different peptides corresponding to the epitope described here. Donors were selected irrespective of their vaccination status. Five additional sera with elevated MV titres were obtained from a recent measles outbreak in Taiwan (Lee et aI., 1992, 1995).

Results

Epitope mapping with MV-H peptides

A panel of 60 H-specific M A b clones was obtained after

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BH171

1500

1000

500

H 361 •366

0 . . .

BH

1500

1000 500

147

H386/394/396

X v

B H 6

1500

H3811H386

1000 500

BH 216

H381/H386

1500 1000 500

o

J'-J

, , ~ i , r H Peptides

BH 168

H3611366

iiiiiiiiiiiiiiiiii

iiii i ii!iiiiii!iiiiiiiiii iiiiiiiiiiii

ddu,N,mad,! ,hm,,HI, ,m!d,,,!d0Ul,ao,=h,!,ml

BH 195

I iidmibnu,,,unlm,,dld~nml~dlHi

3u.111~8

n 6m

I/m39,h,a,

11

i 3g9,

6Billuh,,=~a,=,,m,un=,

BH 21

H38' H386

n'ltm, lumunuu, twnutRd!mm~ummu, at mnlgllmllhmnu,

vtlllPm!

H Peptides

Fig, 1. Binding of selected MAbs (supernatant dilution 1:20) with 121 overlapping biotinylated 15-mer peptides covering the whole sequence of the MV- H protein. The peptides were immobilized by their C-terminal biotin on streptavidin- coated microtitre plates. The absorbance is expressed as .A4o s ( X 1 03). Numbers along the x-axis indicate the position in the MV-H sequence of the N-terminal amino acid of the peptides.

immunization with native or denatured MV. Linear epitopes of these clones were mapped using an ELISA based on bio- tinylated peptides, Seven MAbs were found to react with

peptides corresponding to the amino acids 3 6 1 - 4 1 0 (Fig. 1). MAbs BH168 and BH171 reacted with peptides H361/H366; BH6, BH21 and BH216 with H381/H386; and BH147 and

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Table 1. Characteristics of purified MAbs (1 mg/ml) mapping to H361-410, obtained after immunization with native MV or denatured non-reduced MV

FAGS WB*

M A b s

(red/non-red) ELISA MVI" NT titre HAl titre EBV-MV§ Ltk-H ELISA

peptidest

BH17111 + / + 200 < 2 < 20 -- -- H361/H366 BHI68]] + / + 200 < 2 < 20 -- -- H361/H366 BH6¶ - - / + 200000 100 20480 + + + + + + H38I/H386 BH21~ - - / + 200000 100 20480 + + + + + + H38I/H386 BH216~ - - / + 200000 i00 20480 + + + + + + H381/H386 BHI4711 + / + 100 < 2 < 20 -- -- H386/H391/H396 BH195

II

+ / + 100 < 2 < 20 -- -- H386/H391/H396

Western blot (WB) with reduced (red) or non-reduced (non-red) MV. 4- Reactivity with either MV-infected diploid human cells or purified MV.

See Fig. 1.

§ Persistently MV-infected EBV-transformed cells. IJ Generated against non-reduced denatured MV. ,J Generated against native MV.

BHI95 with H386/H391/H396. The specificity of the binding has been confirmed by competition with free amide peptides (data not shown). The MAbs were of the IgGI~c isotype subclass except for BH216 which was an IgG2b~c.

Functional activity of MV-H peptide-specific HAbs

The above MAbs recognized the MV both in a certified diagnostic ELISA (Enzygnost; Behringwerke) or when purified MV was coated onto the plates (Table 1). BH6, BH21 and BH216 (obtained after immunization with native MV) reacted about 500-fold more strongly than BH171, 168 or 195, which were derived from animals immunized with (non-reduced) denatured virus (data not shown). Under non-reducing con- ditions they all identified a 160 kDa protein in the Western blot, corresponding to the dimeric form of the H protein (Gerlier et al., 1988; Rima, 1983). This was confirmed both with purified MV and recombinant H protein. Under reducing conditions, BH168, BH171, BH147 and BH195 reacted with an 80 kDa protein, while the reactivity of BH6, BH21 and BH216 with the MV was lost. MAbs BH6, BH21 and BH216 recognized H on the surface of the MV-infected EBV- transformed cell line and the H-transfected Ltk- cells. BH171, BH168, BH147 and BH195 were essentially negative on these cells. (Fig. 2). Recombinant H protein inhibited binding of BH21, BH6 and BH216 to peptide H386, demonstrating that the same antibodies react with both antigens. Only BH6, BH21 and BH216 neutralized MV in vitro and inhibited haemag- glutination of monkey erythrocytes (Table 1). Although the binding sites of these peptides partially overlapped (amino acids 386-400) with those of BH147 and BH195, the latter did not display any functional activities.

Challenge/protection experiments with MAbs

T w o - w e e k - o l d B A L B / c mice received between 65 and 344 mg MAb i day before and 4 days after the intracranial injection of 1"25 x 104 TCIDs0 of CAM/BALB brain homo- genate. Fig. 3 shows that 62-{-6 I~g BH6, BH21 or BH216 was sufficient to protect animals from lethal challenge. In contrast, a 5-6-fold higher dose of MAb BH147 and BH195 (which recognize an overlapping region) or BH168 and BH171 did not protect in vivo.

Role of the disulphide bond

BH6, BH2I and BH216 recognized MV only under non- reducing conditions, suggesting that a disulphide bridge is required for binding. The iS-mer peptide H38I has a Cys in position 381 and shares two additional Cys residues at positions 386 and 394 with peptide H386. A 20-mer peptide (381-400), corresponding to the sequence of peptide H381 and H386, inhibited binding of MAbs BH6, BH2I and BH216 to these 15-mers, but inhibition was lost when the three Cys residues were replaced by amino butyric acid (Abu; data not shown).

Fig. 4(a) shows that BH6 and BH2I reacted about fourfold more strongly with peptide H381 than with H386, while BH216 reacts equally well with H38I and H386. The three MAbs recognized the oxidized peptides 4---64-fold more efficiently than the reduced species. MAb BH195 reacts with peptide H386 and H39I irrespective of the disulphide bond.

Fig. 4(b) shows that the substitution of Cys-386 by Abu decreases binding of MAbs BH6 and BH2I to oxidized H386 almost I00-fold. Substitution of Cys-394, either alone or together with Cys-386 completely blocks binding of these MAbs. Binding of BH216 was considerably less sensitive to

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WMPTT MV-infected Ltk-H ~ BH171 ~ BH171 " ~ . . . . BH168 BH168

a BHI47

B 47

BH195 BH195

dlL

, i t . _

0

10 100 1000 10 100 10'00

Arbitrary fluorescence units

Fi N. ;L Binding of MAbs to IvlV-infected EBV-transformed human B cells (WMPTT; left panels) and ktk-H cells (right panels) measured by flow cytometry. Background binding corresponds to the FITC-conjugate alone on EBV-MV cells (GaM) or Ltk-H cells. The difference in background between the WMPT and the Ltk-H cells is mainly due to differences in autofluorescence and to a lesser extend to higher background binding of the FITC-conjugate. Background binding of MAbs to the uninfected EBV cells and to Ltk- wild-type cells was similar to the background of GaM on the corresponding H expressing cells (data not shown).

reduction and to Abu substitution. Virtually no difference in binding was found between oxidized and reduced Abu- derivatives. The reaction of BH195 is independent of Abu substitution or the oxidation state. The results showed that the cystine loop is required for optimal binding of the neutralizing antibodies BH6, BH21 and to a lesser extent BH216. However, it also shows that some of the reactivity is to a linear part of the peptide. Therefore, peptide H386 was truncated from the C terminus.

C-terminal truncation of peptide H386 showed that both in the reduced or oxidized state the 14-mer is required for

iiiiiiiiiiliiiiiiiiii ili iii

iiiii iii iiiiiiiiiliiiiiiiiiiiiiiiiiiiiiiiiiiiiii!iiiiiiil

100 80

|

-l-BH171 ~ 60

~

-.~- BH195 I L ~ 4O 20 ~ <" ~ ) < ' ~ > <- ~ X - - X - - > <" ~ X - - X - - "~<--"

&0

10 20 "I 100~ _ _ _ = = _ - = = = = - = = = = = = = = - - ~= 80 (b) ¢/b 6O 40 20 x - - ~x- - - x - - x - - x - - x - - x - - - x - - x - - x - - x - - x - - x - - ! 10 210 Time (days)

Fig. 3. Challenge/protection experiment with mice passively immunized on day - 1 with 315 pg BH6, 344 pg BH171,330 I~g BH195 (a) or 62 _+ 6 pg BH6, BH21 or BH216 (b), On day O, mice were challenged with an intracranial injection of CAM mouse-adapted Iv]V. Naive mice were challenged without prior antibody transfer. Each group represents 6-8 mice from two independent experiments.

maximal binding of MAbs BH6, BH21 and BH216 (Fig. 4c). The antibodies did not react with the 10-mer or shorter peptides even at the highest concentrations used (not shown). C-terminal elongation did not further enhance binding. Thus, in addition to the S--S loop, the C-terminal linear tail contributes to the binding affinity. It is therefore not surprising that peptide H383-397, which lacked both, did not react with anti- MV-serum (Obeid et al., 1994).

Sequence alignment

The sequence H381-405 of different MV isolates is fully conserved in all vaccine strains (Fig. 5). Limited mutations have been found in recent wild-type isolates. Most mutations were found in the IP3 strain, a subacute-sclerosing panencephalitis (SSPE) isolate from the early 1970s, which was shown to have a markedly reduced haemadsorption activity (Cattaneo el al., 1989; Burnstein et aL, 1974). Escape mutants have been described in this region by Hu et al. (1993) and Liebert et al. (1994). The H protein of the MV shares about 60% identity to rinderpest virus H (Tsukiyama et al., 1987; Yamanaka et al., I988) and about 34 % with canine distemper virus H (Curran et al., 1991). The amino acid sequence 381-400 of the H protein of different morbilliviruses (Fig. 5) shows very little identity

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BH6 BH21

Y

J

T I T BH216

J

BH195

/-

/-

CFQQACKGIQALCE CKGKIQALCENPEWA QALCENPEWAPLKDN

I<S J_J

7

_ /

f

CKGKIQALCENPEWA

f

.j_7-

..S

S

J

Z

..J....ll ..J.... I .F

t ~ Dilution

Fig. 4. For legend see opposite.

CKGKIQALCENP CKGKIQALCENP~ CKGKIQALCENPEW CKGKIQALCENPEWA CKGKIQALCENPEWAPL !48,

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N A M E S S E Q U E N C E S R E F E R E N C E S < . . . H 3 8 1 . . . > < . . . H 3 8 6 . . . . > < . . . H 3 9 1 . . . . > E D W T * C F Q Q A C K G K I Q A L C E N P E W A P L K D N A l k h a t i b a n d B r i e d l s , 1 9 8 6 P A M M E A H A * . . . C V . . . G e r a l d e t a t . , 1986 M E A H A A * . . . N . . . R o t a e t a l . , 1 9 9 2 M E A H A A A * . . . R . . . R o t a e t a l . , 1 9 9 2 M E A H A A D * . . . N . . . R o t a e t a l . , 1 9 9 2 M V H G R N A A I . . . S H u e t a l . , 1 9 9 3 M V 0 8 4 1 1 " - - - H . . . H u m m e l e t a l . , 1 9 9 4 M V 0 8 4 1 4 " - - - H . . . H u m m e l e t a t . , 1 9 9 4 $ 7 3 8 6 9 " . . . N . . . S a i t o e t a l . , 1 9 9 4 I P - 3 - C a . . . R - E V . . . D . . . G C a t t a n e o e t a l . , 1 9 8 9 A . . . C a t t a n e o e t a l . , 1 9 8 9 B . . . N . . . H C a t t a n e o e t a l . , 1 9 8 9 M I B E . . . G C a t t a n e o e t a t . , 1 9 8 9 S H . . . S c h u l z e t a l . , 1 9 9 2 T T . . . S c h u l z e t a l . , 1 9 9 2 C L . . . S c h u l z e t a l . , 1 9 9 2 S E . . . S c h u l z e t a l . , 1 9 9 2 A I K - c . . . M o r i e t a l . , 1 9 9 3 C h i - 1 . . . N . . . R o t a e t a l . , 1 9 9 4 S a n D . . . N . . . R o t a e t a l . , 1 9 9 4 C h i - 2 . . . R . . . R o t a e t a l . , 1 9 9 4 M c l . . . R o t a e t a l . , 1 9 9 4 J M . . . R o t a e t a t . , 1 9 9 4 E ~ ... R o t a e t a l . , 1 9 9 4 C A M . . . R o t a e t a t . , 1 9 9 4 M O E . . . R o t a e t a l . , 1 9 9 4 Z A G . . . R o t a e t a t . , 1 9 9 4 Y A ... K o m a s e e t a l . , 1 9 9 0 L e c - W I . . . D . . . H u e t a l . , 1 9 9 3 C A M / N C 3 2 . . . S . . . L i e b e r t e t a l . , 1 9 9 4 C A M / K 7 1 . . . K . . . L i e b e r t e t a l . , 1 9 9 4 R I N D L - R R E - - R E - P P P F - N S T D - E - - E A G T s u k i y a m a e t a l . , 1 9 8 7 R I N D K - R L E - - R F R P P P F - N S T D - E - - E A G Y a m a n a k a e t a t . , 1 9 8 8 C D V - L E S - - Q R - T Y P M - N Q A S - E - F G G R C u r r a n e t a l . , 1 9 9 1 *: E M B L r e f e r e n c e s

Fi 9. 5. Alignment of the haemagglutinin-neutralizing epitope (HNE) region (amino acids 3 8 1 - 4 0 5 of MV-H) of different MV isolates (EDVVq- to YA), of in vitro induced antibody escape mutants (Lec-Wi to CAM/K71 ) and of other morbilliviruses (rinderpest virus L to canine distemper virus). Only amino acids differing from the Edmonston wild-type strain (EDWT; Alkhatib & Briedis, 1986) are shown. The peptides used in this study are indicated above the alignments. We thank B. Rima (Belfast, UK) for making available pa~s of his data bank.

but the Cys residues are fully conserved, indicating that the local loop formed by disulphide bound is functionally critical.

Reactivity of

human sera

with HNI=

peptides

To study whether maternal antibodies could react with this neutralizing epitope, a panel of sera obtained from women of child-bearing age (Table 2) was tested against peptides H381 and H386 and against 30-mer peptides corresponding to most of the mutations shown in Fig. 6. The sera were selected solely

on the basis of high NT and HAl titres (see Methods). Essentially, three types of reactions were found (Fig. 6): three sera reacted strongly with the same 9 of the 11 30-mers, 10 gave a weak reaction and 7 sera did not react with any peptide. One of the two peptides that were not recognized by any serum contained an additional Cys in position 40I. No reactivity was found against the 15-mers H381, H386 or H391. We also tested sera collected at a recent MV outbreak in Taiwan ($13, $22, $39, $41, $42; Lee et al., 1992; Table 2). One serum reacted with a single 30-mer and not with any of the 15- mer peptides of the neutralizing epitope described here (Fig. 6).

Discussion

After immunization with native or denatured MV, several MAbs were obtained that reacted with peptides corresponding to the sequence H361-410 (Table 1). Three MAbs (BH6, BH21 and BH216) inhibited virus-induced haemagglutination of monkey erythrocytes, neutralized the virus in vitro, and protected animals from a lethal challenge of rodent-adapted neurotropic MV. These three MAbs reacted with the 15-mer peptides H381 and H386 defining H381-400 as a linear neutralizing and protective epitope. These peptides correspond to a small cysteine cluster region (Cys-381, Cys-386 and Cys- 394). In Western blots, BH6, BH21 and BH216 reacted with MV only under non-reducing conditions. For optimal binding, peptide H381 and H386 had to be oxidized, suggesting that a disulphide bond is required, which is formed by both peptides. Since these peptides share Cys-386 and Cys-394, but not Cys- 381, a loop formed by the former two residues is the likely target structure for the neutralizing antibodies. This interpret- ation is also supported by binding studies with substitution analogues in which the -SH group of the Cys-386 and/or Cys- 394 is replaced by -OH. It is therefore somewhat surprising that H381 binds MAbs BH6 and BH21 fourfold better than H386. Data from site-directed mutagenesis of individual Cys residues are more compatible with a loop structure formed by Cys-381 and Cys-394 (Hu & Norrby, 1994). It is therefore possible that differences in binding affinity to the overlapping region 386--395 of peptides H381 and H386 only reflect the more or less tight curvature of the Cys loop in these two peptides.

Studies with the Abu analogues and the C-terminal truncations showed that there was also a low-affinity binding component to the linear C-terminal tail of H386. Recognition of amino acids of the C-terminal tail [mainly (E)WA-COOH] by BH6, BH21 and BH216 does not require oxidation.

Fig. 4. Titration of MAb BH6, BH21, BH216 and BH195 on biotinylated peptide analogues of H386 immobilized to streptavidin-coated plates. The concentrations of MAbs corresponding to the I :2 dilutions were: I 0 l~glml BH6; 3"5 p.g/ml BH21 ; 11.2 l~g/ml BH216; 2.6 p.glml BH195. (a) Reactivity of these MAbs with peptide 1-1386 (I 5-mer) and adjacent 15- mers H381 and H391 ; (b) shows the reaction with amino-butyric acid (X) single and double substitution analogues of H386: H386(Cys-3861394 ~ Abu), H386(Cys-386 ~ Abu), H386(Cys-394 ~ Abu) ; (c) displays binding to C-terminal truncation and elongation analogues of H386. A , oxidized peptides; 0 , reduced peptides. The data is shown as A4o 5 on the y-axis, the scale ranging for all panels from 0 to 2.0. The X-axis shows the dilutions ranging in fourfold dilution steps from undiluted to 1 : 16 384 of the above stock solutions.

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Table 2. Characteristics of sera from women of child-bearin 9 age (samples HS1005- HS1163) and from measles patients from an outbreak in Taiwan (samples $13-$51)

Sample Age (years) MV exposure* HAlt NTI CFI" HNE-ELISA:I:

HS1005 39 ? 8'0 9"0 7 + HS1006 31 ? 8'5 8'5 5 + HS1010 38 D (7) 10'0 11"0 6 + HSI013 29 D (10) 10'0 II'0 6 + + + HS1018 27 ? 9'5 8"5 6 + HS1024 27 ? 8'0 8'5 5 + HS1031 23 ? 8'5 6"0 3 + HS1036 34 7 8'0 7"5 4 -- HS1039 31 D (3-8) 10"0 10"0 8 + + + HSl118 25 D (5-10) 8'0 8"5 7 -- HSl122 36 D (9) 9'0 9-0 7 -- HSl135 33 D (11) 8"5 8"5 6 -- HS1144 46 2 9"0 9'0 NT _4_ HSl151 31 D (8-9) 9'0 9"0 7 + HSl180 26 D (6--7) 8"5 7"5 6 -- HSl196 29 D (12) 8"5 8"0 5 -- HSl199 25 ? 8"0 8"0 4 -- HS1204 26 ? 8"0 8"5 6 + HS1245 32 D (3-5) 8'5 NT 5 + HSl163 45 ? 10"5 8"5 7 + + + Mean _SD 8"3+2"0 8"6___1"2 5"7-}-1"3 $39 8 D[I2] 8'6 0 6 $41 10 D[8] 9"2 7"3 6 $42 2 D[22] 7"3 6"9 I0 $45§ 42 D[I] 8"6 6"3 3 S51§ 42 D[3] 8'6 6"9 3

* D( ) = disease at the age of (); D[] = disease, serum drawn [days after rash]. Jr Log 2 titre.

+ + +, strong; +, weak; --, negative reaction with 30-mer peptides as shown in Fig. 6. § Serum of the same donor.

T w o additional MAbs, BH147 and BH195, mapped to the sequence H 3 8 6 - 4 1 0 partially overlapping with the peptides recognized b y the above neutralizing MAbs. In contrast to the neutralizing antibodies, BH195 was insensitive to Abu sub- stitution and to reduction, demonstrating that these M A b s did not require the loop structure for binding. C-terminal trunc- ation analogues demonstrated that Trp-399 was critical for binding of BH195. The comparison of BH195/BH147 which did not neutralize, with B H 6 / B H 2 1 / B H 2 1 6 highlights the importance of the Cys loop for neutralization.

Two MAbs (BH171 and BHI68) reacted with a region located N-terminal of the loop. This region corresponds to the most hydrophilic region of the protein and neutralizing M A b s have induced escape mutants with mutations in this region (377R --* Q, 378M ~ K; Liebert et al., 1994). O u r antibodies, however, did not neutralize the virus, inhibit haemagglu- tination or protect in vivo. M/ikel/i et al. (1989) have also found reactivity to this region in an anti-MV polyclonal serum, but anti-peptide H 3 6 8 - 3 7 7 antibodies did not display any func-

tional activity, although they reacted with MV. These results suggest that this region m a y contain a neutralizing epitope that is also not readily mimicked b y the corresponding linear peptide.

O u r antibodies against the H 3 8 1 - 4 0 0 region define a local loop protruding from the surface of the MV-H that contains an epitope involved in haemagglutination and neutralization ('HNE loop') and which can be imitated b y short peptide loops. Such peptides are of potential interest for incorporation into a subunit vaccine. Proteins and peptides usually induce antibodies of the IgG1 subtype, which lacks efficiency for complement fixation and FcyRI binding of the IgG2a isotype (Neuberger & Rajewsky, 1981; Ravetch & Kinet, 1991), the most important effector antibody in viral infections (Ishizaka et al., 1995). O u r in vivo challenge experiments demonstrated that protection via MAbs targeted to the HNE loop is independent of these effector functions, since both IgG1 (BH6 and BH21) and IgG2b (BH216) isotypes were protective.

In human immunodeficiency virus (HIV), such a loop (V3)

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~ F Q Q A C K G K [ Q A L C E N P E W A P L K D N R I P S . . . ~ V . . . . . . N . . . . . . . R . . . . .. .. .. R - l g E . . . . D . . . ~ .... . . . .. . . N . . . l-I .... . . . I I .... . . . ( 3 .... . . . . .. . D . . . . . . . K . . . . C F Q Q A C K G K 1 Q A L C E N P E W A P L K D N R I P S . . . ~ _ V . . . . . . R . . . . . .. .. . R - ] ~ Y ... l ~ . . . O .... . . . .. . . i N . . . H .... . . . H .... .. . . . .. E l . . . . .. . . . .. ~ . . . . . . . K . . . . 150 650 I / i l I I | I I Mean A4o 5

(a)

115C 200

k

d

700 1 2 0 0 1 5 0 650 1150 I I I I (d) (e) 600 100 660 [00

(b)

(f) 600 100 (c) I I I l i I 600 LO0 I I I (h) 60o Fig. 6. Reactivity of human sera (Table 2) with the 15-mers H381, H386 and H391 and with different 30-mer peptides of the H 3 8 1 - 4 1 0 region homologous with virus isolates of Fig. 5. The upper panels show three examples of sera from women of child-bearing age with strong (HSI 155), weak (HSI 144) and negative (HSI 122) reaction with the 30-mers. The strong ( + + + ) , weak ( + ) and negative (--) reaction of the other women's sera are shown in Table 2. The lower panel shows five sera from a measles outbreak in Taiwan (same order as in Table 2). Data represent mean A4o 5 ( x I 03) of a biotinylated peptide ELISA. Backgrounds [I 0 0 - 2 0 0 x (A4o 5 x 103)] defined as the reactivity of non-reacting peptides were truncated.

has emerged as the principal neutralizing linear determinant in the envelope protein (Wang et aL, 1991; Ahlers et al., 1993). Peptides mimicking this structure have generated neutralizing and protective antibodies in chimpanzees (Fultz et al., 1992; Goudsmit et al.,. 1988). Clinical studies are underway to investigate the potential of the V3 loop as a human vaccine (Walker & Fast, 1994). However, the V3 loop as a target for protective antibodies suffers from its extensive sequence variability (Javaherian el al., 1989; Goudsmit el al., 199I). In contrast, only limited numbers of mutations of the HNE loop of MV have been reported (Fig. 5) suggesting that extensive variability in this region cannot be tolerated. Although the region bears little identity to other morbilliviruses such as rinderpest virus and canine distemper virus, the conservation of the cysteines suggests that the HNE loop is essential and will not easily disappear under the selective pressure of antibodies.

In this context, observations in the rodent-adapted CAM strain which have associated this region with neurovirulence are of interest (Liebert el al., 1994). While an E-395--* K mutation removed neurovirulence, the G-388--* S mutation lowered neuropathology (Liebert et al., 1994). The IP3 wild- type isolate derived from an SSPE patient also showed

mutations in this region (Fig. 5; Cattaneo et al., 1989; Burnstein et aL, 1974). Further studies with mutants will be required to test whether antibodies to this region may also potentiate disease.

One of the benefits of peptide-based MV vaccines is their potential to escape maternal antibodies transferred from mother to the child. Such a subunit vaccine could conceivably be used to induce neutralizing antibodies in the presence of antibodies from vaccinated or wild4ype MV-infected mothers. We showed (Muller et al., 1993) that sera from late con- valescent donors did not react with 15-mers corresponding to the HNE loop of the Edmonston strain. It is conceivable that antibodies were missed which were induced by wild-type MV with non-cross-reactive mutations in the HNE loop. To minimize this possibility in the present study, high titre sera from women of child-bearing age and from patients recovering from measles (Lee et a]., 1992, 1995) were reacted here with a set of 30-mer peptides corresponding to most known muta- tions in this region. Few sera reacted strongly with the longer peptides but none reacted with the 15-mers which fully represented the neutralizing and protective epitope. A number of isolates from a single outbreak showed no mutations in this region, confirming that the HNE loop is not under a strong

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selective pressure from neutralizing antibodies (Outlaw & Pringle, 1995). This suggests that HNE l o o p peptides could potentially be suitable for immunization in the presence of neutralizing M V antibodies. Further studies will be required, however, to investigate cross-reactivity of HNE loop-specific antibodies of isolates obtained from different geographical areas.

Note added in proof. Cystine loops have recently been described

in a number of proteins as a 'noose' motif which provides high surface accessibility to the amino acids contained in the loop (Lapthom et at., Nature Structural Biology 2, 266--268). The HNE loop described here would therefore represent a Haemagglutinin Noose Epitope.

We thank Wim Ammerlaan for flow cytometry measurements and Dr Giesendorf for the generous gift of Enzygnost plates. We are grateful to Dr B. Rima (Belfast, UK) for permitting us to use data from his partially unpublished sequence data bank. Parts of this work were done by D.Z. and P. F. in partial fulfilment of their doctoral theses. The financial support of the Centre de Recherche Public-Sant6, Luxembourg (CRP91/06) and of the EC-Biotechnology Programme (project PL920131) are gratefully acknowledged.

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Received 4 March 1996; Accepted 7 June 1996

數據

Fig,  1.  Binding of selected MAbs  (supernatant dilution  1:20)  with  121  overlapping biotinylated 15-mer peptides  covering the whole sequence of the  MV-  H  protein
Table  1. Characteristics  of  purified  MAbs (1  mg/ml)  mapping  to  H361-410,  obtained  after  immunization with  native  MV or denatured  non-reduced  MV
Fig.  3. Challenge/protection  experiment with  mice  passively immunized  on  day  -  1 with  315  pg BH6,  344  pg  BH171,330  I~g BH195  (a)  or  62 _+ 6  pg  BH6,  BH21  or BH216  (b),  On day O, mice were challenged  with  an intracranial  injection
Fig.  4. Titration of MAb  BH6,  BH21,  BH216  and BH195  on biotinylated peptide analogues of  H386  immobilized to  streptavidin-coated plates
+3

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