Shigella flexneri O-antigen specific enzyme immunoassay: lipopolysaccharides and synthetic glycoconjugates as antigens

Serodiagnosis

and Immunotherapy

(1988) 2,63%78

Shigelfu flexneri O-antigen specific enzyme immunoassay: lipopolysaccharides and synthetic glycoconjugates as antigens

Alf A. Lindberg*, Erik Ekwall* and Nils I. A. Carlint

+ Karolinska Institute, Department of Clinical Bacteriology, Huddinge Hospital. S-14186 Huddinge, Sweden; and Department of Vaccine Production, National Bacteriological Laboratory, S-10521 Stockholm. Sweden Sera were collected from either mice or rabbits immunized with heat-inactivated ShigellaJiexneri serotype 1b or 2a bacteria or from humans with bacillary dysentery caused by serotype 1b or 2a. The sera were assayed for their antibody response by an enzyme-immunoassay (EIA). The sensitivity and specificity of the assay was examined by a panel of phenol-water extracted lipopolysaccharides (LPS) from all S. flexneri serotypes, including a complete core rough mutant, and artificial glycoconjugates synthetic or chemically derived di-, tri-, tetra- and octasaccharides, which are elements of the 0-antigenic S. jexneri polysaccharide chain, covalently linked to bovine serum albumin (BSA). In terms of sensitivity the LPS antigens were superior to the artificial glycoconjugate antigens. The chemically and immunochemically defined LPS antigens did not, however, permit a serotype-specific immunodiagnosis. The results indicated that besides antibodies against serotype-specific epitopes, antibodies also appeared to be formed against epitopes common to all serotypes. The artificial SaccharideeBSA glycoconjugates were also unable to allow a serotypespecific diagnosis with one possible exception, the group-antigen 6BSA glycoconjugate. The results suggest that S.Jexneri species-specific serodiagnosis can be obtained using characterized LPS antigens. Keywords:

Shigella.flexneri,

enzyme immunoassay,

lipopolysaccharides.

glycoconju-

gates Introduction Bacillary dysentery caused by ShigelfaJIexneri is one of the major causes of gastrointestinal illness in developing countries and often diagnosed on the clinical picture’. The

patient suffers from painful cramps and a mild to moderate fever’s3.Untreated the illness in mal- or under-nourished individuals has been reported to have a mortality up to 1.3%4. The microbiological diagnosis is based on the isolation of S. jkmeri bacteria from

faecal

specimens,

and

their

subsequent

identification

by biochemical

tests and

serology’. Unfortunately the isolation procedure suffers important drawbacks. Firstly, Shigella are sensitive and often die during transport to the laboratory. Secondly. there are no good enrichment procedures and no particular selective solid media favouring the growth of the Shigella. Hence the Shigella are not infrequently overlooked in the

diagnostic laboratory. 63 OSR-4786/88/010063+

16 $03.00/O

:c; 1988 Academic

Press Limited

64

A. A. Lindberg

et al.

An alternative to isolation and identification of the causative micro-organism is the demonstration of an antibody response towards epitopes specific to the organism. The serotype designations of S. jkwrri (serotypes la, lb, 2a etc.) are based on the immunochemical specificities found in the lipopolysaccharide (LPS) of the cell envelope of the bacterium. All S. ,flrxnevi of serotypes l-5 have an 0-antigenic polysaccharide chain built of repeating tetrasaccharide units: -at-Rhap-( l--2) -aL-Rhap-(I --3)-at-Rhap-( I--,3)-D-GlcpNAc. (J) (JJ) (JJJ) The repeating units are joined by 81-2 linkages6.7.Serotypes la and 1b have in common a type-specific epitope 1 found only in these two serotypes; D-glucose al-4 linked to the N-acetyl-o-glucosamine residue6(Figure I). The difference between serotypes la and I b is that in 1b the L-rhamnose III residue has an 0-acetyl group linked to C-26. This results in group-antigen 6 specificity which is found also in serotypes 3a, 3b, 4b (Figure 1). In serotypes 2a and 2b, D-glucose linked 14 to L-rhamnose III is the common type I1 epitope. Serotype 2b differs from 2a by having also a D-glucose linked al-3 to Lrhamnose I.

Basic

structure

Substitutents

(I 11:6.7,8)

(IV:3.4)

(-:7,8)

Figure

I

Shigel/u

Jexneri

BGt

0

- acetyl

0

=Glucosyl

group residue

w

T

Q-yg

yqf$(w:6J

F

T

O-antigen

structures, Abbreviations: glucosamine.

@I-4)

(-13-4)

Rha,

rhamnose;

GlcNAc,

N-Acetyl-D-

Shigella

jlexneri

enzyme immunoessay

65

The fact that all S. @xneri of serotypes I-5 have in common an 0-antigenic polysaccharide chain in their LPS built of the samebasic tetrasaccharide repeating unit raises the possibility that a common epitope(s) in the chain may represent all these serotypes. In addition the presence of the various combinations of type- and groupantigen specific epitopes raises the possibility of establishing a S. .flr.uneri serotypespecific immunoassay. In this paper we report the testing of thesehypotheses by enzymeimmunoassays(EIA) based upon chemically defined LPSs and synthetic glyco-conjugates as antigens. Materials and methods Bacterial strains Shigella~flexneri strains of serotypes la, lb, 2a, 2b, 3a, 3b, 4a. 4b, 5a, 5b, X. Y. 6 were from the strain collection of the National Bacteriological Laboratory, Stockholm. S. ,flexneri 4bR, a rough mutant devoid of O-antigen, was available from earlier work’. Preparation of lipopolysaccharide and their chemical and immunochemical characterization Individual colonies of bacterial strains selectedfor LPS production were tested for their antigenic specificity by agglutination with monoclonal anti S.$exneri antibodies- I”. The bacteria were then grown in submergedculture and LPS extracted by hot phenol-water” for smooth bacteria and by phenol-chloroform-light petroleum” for rough bacteria. The LPS preparations were subjected to sugar analysis’“, and protein” and RNA (A,,,,) determination. Generally protein content was lessthan 2% and RNA lessthan 10%. For nuclear magnetic resonance (NMR) spectroscopy, LPS was subjected to hydrolysis by acetic acidIs. The resulting 0-antigenic polysaccharide was lyophilized and analysed by ‘H- and ‘C-NMR spectroscopy using a JEOL GX 270 instrument. Spectra were recorded at 70” in D,O with tetramethylsilane as external standard. Only LPSs that showed the expected6 signals in NMR were used. The LPSs were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis. as described elsewhere9.1h, to check for presenceof O-antigen. lmmunochemically, LPS was assayed in EIA for antigenicityi7. Briefly. LPS was coated to polystyrene microtiter plates (A/S NUNC, Roskilde. Denmark) in different concentrations (50 to 0.01 ug) in coating-buffer (0.05 M carbonate buffer. pH 9.6). Residual binding sites were blocked by addition of a 1% solution of bovine serum albumin (BSA) in the same buffer. Plates were washed and incubated with a single concentration of monoclonal antibody or rabbit antisera. Plates were incubated, washed and an alkaline phosphatase rabbit anti-mouse immunoglobulin was added (diluted 1! 4000). Alternatively an alkaline phosphatase goat anti-rat immunoglobulin (diluted I:i 8000) (Sigma Chemical Co., St. Louis, MO, U.S.A.) or an alkaline phosphatase goat anti-rabbit IgG conjugate (diluted l/8000) (Sigma) was used. After incubation plates were developed as described”. The antigen concentration giving an A,,, = 0.1 at 100min was calculated. Preparution

qf synthetic and semi.qnthetic g1Jvoconjugate.p

The procedures used for N-de-acetylation and de-amination of 0-antigenic polysacchar-

66

A. A. Lindberg

et al.

ides have beendescribed previously”, as have the conditions for coupling of the resulting oligosaccharides to protein carriers2’. The octasaccharide-BSA conjugate used in this study was obtained by phage Sf6 endo-rhamnosidase cleavage of the 0 side chain as described’. The resulting octasaccharide was derivatized and coupled to BSA as described”. The synthesis and coupling of the glycoconjugates a-D-Glcp-( 1-4)-a-r>GlcpNAc- 1-O-BSA and-a-L-Rhap-( I-3)-a-L-Rhap(-20Ac)- I-O-BSA has been described earlier2’,22.All glycoconjugates had a carbohydrate/protein ratio in the range of 15525mol of carbohydrate/m01 of protein. Monoclonal antibodies The generation and immunochemical characterization of the different mouse and rat monoclonal antibodies was carried out as described previously8-‘0. The designations used for these antibodies attempt to correlate to the type and group antigen designation used in Shigella serology‘: Monoclonal Antibody to Shigella flexneri, i.e. MASF, followed by either a Roman numeral (I, II, etc.) indicates a type antigen, e.g. MASF I, and Arabic numeral(s) indicates a group antigen, e.g. MASF 3,4. The Arabic numeral after the antigen designation indicates our numbering of the specific hybridoma. Polyvalent mouseand rabbit antisera Sera were obtained from Balb/c mice and New Zealand white rabbits immunized with whole heat-killed S. flexneri bacteria according to standard manuals5. Patients Convalescent sera (collected 2-4 weeks after the onset of disease)from five patients with bacteriologically verified Shigella JEexneri infections of either serotype lb or 2a were available from other serological studies23,24 . One 23-year-old male Swedish (RS 380) and two, 8- (SP 40) and 17- (SP 17) month-old, Vietnamese patients suffered from serotype 1b infections whereas one 34-year-old female Swedish (RS 203) and one 16-month-old (SP 15) Vietnamese patient had serotype 2a infections. All sera were kept frozen until analysed. Enzyme immunoassay (EIA) for titre determination The EIA was performed in accordance with Engvall & Perlmann17 modified for polystyrene microtitre plates (A/S NUNC, Roskilde, Denmark) as previously described”. The wells were coated with 100ul of LPS antigen (10 pgmll’ in 0.05 M carbonate buffer, pH 9.6) at 2&25”C for 18h. Control wells were treated with coating buffer only. Before use the plates were washed three times with washing buffer, 0.15 M NaCl containing 0.05% (v/v) of Tween 20. The sera to be assayed were diluted in incubation buffer, phosphate-buffered saline, pH 7.4, containing 0.05% (v/v) of Tween 20 (PBS-T). Aliquot (100 ~1) of diluted sera to be tested, one positive and one negative control serum and incubation buffer only, all in duplicates, were added to the microtitre plates. They were incubated at 22°C for 2 h and washed as before. Alkaline phosphate conjugated antibodies (100 ul), diluted in incubation buffer, were then added to the wells and the microtitre plates incubated at 22°C for 18 h. The conjugates used were swine anti-human IgG (diluted 1:350) (Orion Diagnostica Oy, Helsinki, Finland), goat anti-

Shigella flexneri

enzyme immunoessay

67

mouse Ig (diluted 1:lOOO) and goat anti-rabbit IgG (diluted I :4000) (Sigma). After having been washed again the wells were filled with 100ul enzyme substrate solution (paranitrophenyl phosphate I mg ml-‘) in 1.0 M diethanolamine-HCI buffer, pH 9.8, containing 0.5 mM HgCl,, and incubated. The colour was determined in a Titertek Multiscan photometer (Flow Laboratories Ltd, Irvine, Scotland) at 405 nm after 25, 50 and lOOmin. The absorbance seen in control wells was uniformly less than 0.100 at 405 nm per 100 min and was not accounted for. The intra-assay variation was lessthan f 5% and the inter-assay variation lessthan f 15%, as estimated with the positive and negative control sera. No adjustment of the titres in individual sera was therefore considered necessary. The EIA titres in this study are expressed as end-point titres defined as the reciprocal serumdilutions giving an A,, at 100min of 0.1. For end-point titrations, IO-fold dilution steps from 10 -’ to IO- ’ were tested. For calculation of the end-point titre the mean of the absorbance values at each dilution step was used.

Results

Immunochemical characterization of S.JIexneri LPSs used in this study The LPSs used were tested in EIA antigen saturation experiments with mouse monoclonal anti-S.Jlexneri antibodies (MASF I- 1, II- 1, IV-l, V- 1, VI- 1,7,8- 1, MASF 6-l and MASF R3 core-l) or rat monoclonal antibodies (MASF IV-2 and Y-5) (Table 1). The LPSs tested represented all the establishedserotypes of S.Jtexneri and a rough mutant of S..fEexneri, 4 bR, which has the complete core=coli R3 core25,2h. As expected the LPSs tested reacted with the monoclonal antibodies specific for their type-antigen determinant (I, II, etc.). They also showed binding for monoclonal antibodies specific for their respective group antigen determinants (3,4; 6; 7,8). In addition, seven of the LPSs from smooth strains also reacted with the core-specific MASF R3 antibody. These results show that the S.Jexneri LPSs used have (i) the expected type-antigen specific epitopes, (ii) the expected group-antigen specific epitopes and (iii) core structures exposed since several react with the core-specific monoclonal antibody. SDS pal-vacrylamide gelelectrophoresisof LPS preparations used as antigens in EIA The different LPSs used were subjected to SDS-PAGE (Figure 2). A heterogeneity in respect to amount and average chain length of O-antigen between the different preparations was apparent. Several LPSs had the majority of staining material in the bottom of the gel, i.e. LPSs of serotype 1b, 3a and X, indicating an abundance of LPSs molecules without 0 repeating units, or with only a few. This is in agreement with the antigen saturation experiments where these three preparations were shown to have reactivity with the MASF R3 core-l antibody in the samerange as the rough 4bR LPS (Table 1). Specifcitl, of’ the lipopolysaccharides when usedas antigens in enzyme immunoassays Sera were collected from mice and rabbits which had been immunized with heatinactivated bacteria, either S.JIexneri serotype 1b or serotype 2a. Samples taken before immunization showed EIA IgG end-point titres < 0.2 against all the LPS antigens used in the panel. Serum samples were also collected from individuals who had bacillary

0.80

0.01

MASF

Entries left blank mean that antigen

k (-:7,8) Y (-:3,4) 4bR

LPS serotype MASF I-l _____ ~~~ la (1:3,4) I.00 0.40 lb (1:6) 2a (11:3,4) 2b (11:7,8) 3a (111:6,7,8) 3b (111:3,4,6) 4a (IV:3,4) 4b (IV:6) Sa (V:3,4) 5b (V:7.8)

0.50

MASF

concentrations

II-I

0.60 0.90

MASFIV-2

> 50 pg were needed

IV-I

V-l

0.04

of 0.1.

MASFVI-I

for an absorbance

0.03 0.09

MASF

0.50

0.50

0.05 0.05

MASF

7,8-l

0.04

0.30

0.01

0.01

MASFY-5

0.05

0.35

0.02

0.20 0.20 I.50 0.85 7.0

0.20 I.30

0.20 0.50 0.02

MASF

MASF6-1

R3 core-l

Table 1. Antigen saturation experiments by enzyme-immunoassay using LPS from all ShigellaJlrsneri serotypes and monoclonal antibodies specific for type- and group-antigens of S.Jie.xneri (data are micrograms of antigen required for an absorbance value at 405 nm, 100 min, of 0.1)

I

?

?

69

Shigella J¶exneri enzyme immunoessay

l

5

10

15

Figure 2. SDS- PAGE of Shigella Jexneri lipopolysaccharides. Lanes I to IS show lipopolysaccharidcs of serotypes la, I b. 2a. 2b. 3a. 3b. 4a, 4b, Sa, 5b, X. Y and 6. S. sonnei (P. .shige//oides) and S @sneri 4bR

dysentery caused either by S.Jexneri serotype 1a or serotype 2b and where the bacteria had been isolated from faecal specimens. S..flexneri serotype lb. Serum samplescollected from one mouse, one rabbit and three patients were tested against the panel of 14 LPSs (Figure 3). The mouse serum showed high titres > 5 x lo3 against 11 of the 14 LPS antigens [Figure 3(a)]. The highest titre was seenagainst the S.flexneri 1b LPS. From the high titre against the serotype 1a LPS we surmisethat antibodies specific for the type-antigen I epitope had been formed. The high titres against the 3a, 3b and 4b LPSs indicate that antibodies against the group-antigen 6 epitope had been elicited. The equally high titres against severa) of the other LPS antigens from serotypes 2a, 5a and the “basic” serotype Y, show that antibodies also had been formed against group-antigen 3,4 (seen with the Y, 2a and 4a LPSs) and probably against common hitherto undefined epitopes. A high titre was also seenagainst the rough core LPS. The rabbit serum yielded titres very similar to those seen in the mouse serum [Figure 3(b)]. The end-point titres were about IO-fold higher which most likely was a consequenceof the immunoglobulin enzyme conjugate used for detection of the antibody responses.The fact that titres > 5 x lo4 were seenagainst S..fle.xneri LPSs from serotypes 1b, 2a, 3a, 3b, 4b and Y points to the formation in the rabbit of antibodies recognizing group-antigens 3,4 and 6 and perhaps other common unidentified epitopes. The titre seen against the serotype la LPS can be a consequence of antibodies against the type-antigen I and the group-antigen 3,4 epitopes both or either of them. It is notable that the rabbit responded with antibody formation against the serotype X but not the rough core LPS, whereasin the mouseserum the reverse situation was seen.

70

A. A. Lindberg

et al.

(b) 50 30 20 IO 753 2 I0.7o-5 - i200 -

(d)

%850 5 305 20: .= IO E 7.-z 56I; 32/ 0.7 0.5 100 80 50 30 20

-

IO 7 5 3 2 0.7 0.51l.L

(e:

la bZab3ab4ab5ab

x y R”6”

r

D-A

l--l I

la b 20 b 3a b

4a

b 5a b x y R “6”

Figure 3. Enzyme immunoassay end-point titres against 14 different Shigellaflexneri lipopolysaccharides in sera from S. jkxneri serotype 1b immunized animals or infected patients. Sera analysed: (a) mouse NC8; (b) rabbit; (c) Swedish patient R 380, (d) Vietnamese patient SP 17; (e) Vietnamese patient SP 40. Antigens used: la-Y, Shigellaffexneri serotypes la-Y; R, S. jlexneri 4bR; ‘W’, Shigellaflexneri serotype 6. In (b) end-point titre x IO’, not x 10’.

The antibody responseseenin the Swedish patient (RS 380,23 years) follows a pattern very similar to what was seenin the experimental animals [Figure 3 (c)]. Thus high titres > 10 x lo3 were seenagainst eight of the 14 LPS antigens. From the equally high titres against the I a, 1b, 3a, 3b and 4b LPSs, we surmise that type-antigen I as well as groupantigen 6 specific antibodies had been formed. The high titres against the 3a, 3b and 4b LPSs confirm the formation of antibodies with group-antigen 6 specificity. Again the high titres against the S.JIexneri Y, 4a and 5a LPSs suggestthe formation of antibodies

Shigellajlexneri

enzyme immunoessay

71

against the group-antigen 3,4 epitopes, and most likely against hitherto undefined S. flexneri epitopes. A lower, but significant, titre was seenagainst the rough core LPS. Sera from two Vietnamese patients were titred (SP 17. 17 months old, and SP40, 8 months old) [Figures 3(d) and (e)]. The antibody responses in these two patients appeared restricted lvhen compared to that seen in the Swedish patient who was much older. The highest titres were seen against the S. flexneri serotype la and lb LPS antigens. This is indicative of antibody formation against type-antigen I. The high titres against the 3a, 3b and 4b LPSs suggestedformation also against the groupantigen 6 epitope. The relatively high titres seenagainst the Y LPS suggestthat group-antigen 3.4 recognizing antibodies also had been elicited. The overall pattern seenin the two experimental animals and the three humans points in the direction of a polyclonal responseagainst the S.,flexneri serotype I b polysaccharide with antibodies being formed against (i) the type-antigen I epitope, (i) group-antigen 6 epitope, (iii) group-antigen 3,4 epitopes, and (iv) presumably against epitopes which so far have not been identified. It is noteworthy that low titres, i.e. < 2 x 103,were seldom observed. Only against S.Jlexneri serotype 6 LPS, with a structure different from LPSs from serotypes Y, X and l-5, were low titres uniformly seen. S.jlexneri serotype 2~. Serum samplesobtained from one mouse, one rabbit and two patients either immunized or infected with S.flexneri serotype 2a bacteria were assayed against the same panel with 14 LPSs [Figure 4(a) and (b)]. The S.Jiexneri serotype 2a LPS has two described antigenic epitopes: the type-antigen 11and the group-antigen 3,4 epitopes (Figure 1). The type II epitope is found only in S.flexneri serotype 2a and 2b LPS. The group-antigen 3,4 epitope(s) are found in serotype la, 2a. 3b, 4a, 5a and Y LPSS. The mouseserum (NC1 2) had end-point titres > 5 x 1O3against eight of the 14 LPSs in the test panel [Figure 4(a)]. The highest titre was seenagainst the 2a LPS. A low titre was observed against the type 2b LPS, which could indicate that the antibodies have a low affinity against the type-antigen II epitope, as it is present in this LPS. The high titres against the group-antigen 3,4 epitope containing LPS demonstrate that a significant portion of the antibodies formed are directed against these epitopes. It is noteworthy that no detectable antibody titres were elicited against the rough core LPS by immunization with S. ,j!exneri serotype 2a bacteria. The rabbit serum contained antibody titres which for 7/14 LPSs gave end-point titres > 5 x IO3[Figure 4(b)]. The titres were equally high against six of these LPSs: 1b, 2a. 3a. 3b. 4b and Y. This shows that antibodies against the group-antigen 3.4 and the typeantigen 11(as present in the 2a but not 2b polysaccharide) had been elicited. The high titre against the S.,flexneri serotype 1b, 3a and 4b LPSs. but non-measurable against the rough core LPS, demonstrates that antibodies had been formed against O-polysaccharide chain epitopes which so far have not been defined. The two patients (Swede RS203, 34 years old, and Vietnamese SPl5, 16 months old) responded with titres which were > 5 x lo3 against seven and nine of the 14 LPSs, respectively [Figures 4(c) and (d)]. The three highest titres were seenagainst the 2a, 1b and 1a LPSs in the RS203 serum [Figure 4(c)]. In the SPl5 serum the titres were highest against the Y, 2a and 1b LPSs, in that order [Figure 4 (d)]. Again all the titre estimates point towards a polyclonal response with specificities against the type-antigen II, the group-antigen 3,4 and unknown 0-polysaccharide chain epitopes. Also here the titres against the core LPS and the S. fkxneri serotype 6 LPS were either very low or nonmeasurable.

A.

72

A. Lindberg

et

al.

I 200

lb)

-

1

‘% 50 30 20 IO 75-6‘0 2 z c

3 2-

0.7

i & &

?%-m50 -

I -

(c)

I

I

(d)

30 20 IO 7532-

0.7 0.5

I -

I ) 3(

30 b 40 b 50 b x y R “6”

Figure 4. Enzyme immunoassay end-point titres against 14 different ShigelluJIrxneri lipopolysaccharides in sera from S.Jiexneri serotype 2a immunized animals or infected patients. Sera analysed: (a) mouse; (b) rabbit NC9; (c) Swedish patient RS 203; (d) Vietnamese patient SP 15. Antigens used: la-Y Shigellaflexneri serotypes la-Y; R S. jfexneri 4bR; “6” S. Jlexneri serotype 6.

The conclusions which can be drawn from the titrations of sera from two mice, two rabbits and five patients are: (i) a polyclonal response against a number of S.Jiexneri polysaccharide epitopes is elicited; (ii) the response does not allow a type-specific diagnosis to be established, since the titres against the type-antigen epitope-containing LPSs are not necessarily the highest; and (iii) the antibody responses seen may permit an immunological diagnosis of a S. Jexneri infection. Sensitivity and specljicity of S. Jexneri enzyme immunoassays

gl-vcoconjugates

when used as antigens in

The inability of the S.Jiexneri LPS antigens to permit a serotype-specific immunoassay prompted us to investigate whether defined saccharides of the 0-antigenic polysaccharide chain, covalently linked as haptens to BSA, could result in improved specificity. Endpoint titrations were therefore performed on seven of the nine sera studied above using five different S. Jexneri glycoconjugates (Figure 5). Four of the sera from S. JIexneri serotype 1b immunized or infected animals/ individuals were tested. The sensitivity and specificity of two disaccharide conjugates with serotype 1b epitopes (l-BSA and &BSA) were compared with that of the native lb

Shigellaflexneri

enzyme

73

immunoesay

LPS (Table 2). The lb LPS was from two- to 17-fold more sensitive than the 6-epitope glyconconjugate, and from IO- to > 50-fold more sensitive than the I-epitope glycoconjugate. The S.Jle.meri serotype lb LPS does not-according to traditional nomenclature based on the use of absorbed rabbit antisera-express group-antigen 3,4 specificity (Figure I ). However, both the Y 1 and II glycoconjugates gave high titres. High titres were also seenagainst the Y2 conjugate. In sera collected from the rabbit or the two patients immunized/infected with S. ,jle.weri serotype 2a bacteria. the II-BSA glycoconjugate as an antigen was compared with the native 2a LPS antigen (Table 2). The titres seenwith the II-- BSA conjugate were from two- to four-fold lower than with the LPS antigen. The Y l- and Y2-BSA conjugates gave higher titres than the II&BSA antigen. This suggeststhat a significant portion of the antibodies formed in the rabbit or the two patients were directed against intra-chain determinants like the a-L-Rhap-( l-3)-r.-Rhap and u-r-Rhap-( 1 3)-i,GlcpNAc disaccharides, or parts thereof. In terms of sensitivity the lb and 2a LPSs and the Y IlBSA and Y2 BSA conjugates were superior to the “epitope-specific” I-, II- and 6BSA conjugates. In terms of specificity neither of the I--, II-- and 6BSA conjugates permitted an epitope-specific diagnosis, with the possible exception of the 6BSA conjugate. A study on a larger

Epltope

a-D-Glc~-(l-4)-~-lJ-Glc~NAc -

I

a-&-Rhap-(l-3)-a-&-Rhap

6

2 I OAc

a-L-Rhap-(l-2)-a-&-Rhae-(l-3)-a-L-Rhae

II

a-D-G1 -

1: CE

a-L-Rhap-(l-3)-6-D-GlcpNAc-[l-2)-~-~-Rhap-~l-2~a-L-Rhap-(l-3)-a-&-Rhae-o)-B-D-GlcENAc-(l-2)-

Yl

a-&-Rhap-(l-2)-a-&-Rhap

a-&-Rhap-(l-2)-a-&-Rhae-(l-3)-a-L-Rhap Figure

5. Structure

of saccharides

Y2 linking

the reducing

end to bovine

serum

albumin

74

A. A. Lindberg

et al.

Shigellu jZexneri enzyme immunoessay

75

collection of human sera showed that the 6BSA conjugate permitted an epitope-specific diagnosis (0.001 < PC 0.01 in a comparison between sera from serotype 1b- and ?ainfected patients-data not shown). Discussion

Our own previous studies of the immunochemical specificity of monoclonal antibodies against the S. Jlexneri 0-antigenic polysaccharide chain showed that antibodies were produced (i) which recognized the terminal non-reducing end of the 0-polysaccharide chain, i.e. the RhaI-+RhaII~RhaIII structure”; (ii) which recognized intra-chain epitopes like RhaII+RhaIII-+GlcNAc in serotype Y”; (iii) which recognized typeantigen epitopes like the type I epitope as present in both serotype la and I b S.,flexneri bacteria”; (iv) which required the simultaneous presenceof two group-antigens like the 6 and 7,8 epitopes in S. flexneri serotype 3a bacteria’*; (v) which defined an epitope, provisionally labelled as group-antigen 1, present in all S. jlexneri of serotypes l-5, but also in serotype 6 and S. ciysenteriueserotype 1 bacteria”‘; and (vi) which recognized the core saccharide, i.e. the structure to which the 0-polysaccharide chain is covalently linked8 (Figure 1). This multitude of epitopes present on the S. ,flrxneri 0-antigenic polysaccharides may present us with insurmountable problems when we try to establish serotype-specific immunoassays. However, it may be possible to develop a speciesspecific immunoassay for the diagnosis of dysentery caused by S. fiexneri. The quality of any immunoassay is dependent on the quality of the antigens used for the detection of the antibody responses.The S.,flexnrri LPSs used in this study have been both chemically and immunochemically characterized* (Table 1). When tested against the panel of S.$exneri specific monoclonal antibodies they all showed unique specificities: they reacted with the expected type- and group-antigen specific monoclonal antibodies and showed no crossreactivities. There were two exceptions, however. Firstly, the MASF Y-5 antibodies did not react with the la and Sa LPSs in the EIA. Since they agglutinated the bacterial strains when tested as a coagglutination reagent”‘, we surmise that the affinity of the MASF Y-5 is too low to allow detection in EIA. It is also a well-known fact that strains of serotypes la and also 4a can be found which fail to react with rabbit group-antigen 3,4 serum>.Secondly, seven of the I2 serotype specific LPSs also bound the rough core specific antibody MASF R3 core-l. This means that a significant portion of the core saccharidesare left uncapped by 0-antigenic polysaccharide chains and hence are accessibleto core reactive antibodies. Since the core is the same in all S. flr.xnrri of serotypes I-5. a possibility of a cross-reactivity basedon the common core structure exists. The saccharidesusedas haptens and covalently linked to BSA to form the glycoconjugates had been obtained through chemical synthesis (ILBSA. 6-BSA)?‘,ZZ, chemical degradation using N-de-acetylation and de-amination (I I-BSA, Y2-BSA)‘7 or through enzymatic hydrolysis using phage Sf6 endo-rhamnosidase (Y 1lBSA)‘. These conjugates readily detect rabbit antibodies elicited by immumzation with heat-killed bacteria-‘. However. glyconjugates with disaccharide haptens have failed to detect some of the mousemonoclonal antibodies formed and tetra- and octasaccharides have been required for detection”. In the studies of anti-dextran antibodies formed in man, Kabat Pt ~1. found that di- and trisaccharide haptens were large enough to allow detection. even ifthe largest affinities between antibody and antigen were found with structures of the size of a hexa- or heptasaccharide”. In our studies of the antibody response against the O-

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antigenic polysaccharide of the LPS of Salmonella bacteria, synthetic disaccharides were found to be well-suited for a specific diagnosis30. In work reported here, we immunized mice and rabbits becausethese animals do not naturally harbour or become infected with S. Jiexneri. which is pathogenic only to primates2,‘. Titres in sera drawn before immunization also showed low values, < 0.2 x 103,against all the LPS antigens of the panel. After immunization, all sera from the mice and rabbits immunized with either S. ,flexneri serotype la or serotype 2a bacteria showed high titres against most of the S.Jlexneri LPS antigens [Figures 3(a) and (b), and 4(a) and (b)]. The fact that the anti-rough core LPS titres were low, with the exception of mouse NC8 which had a significant responseafter being immunized with serotype 1b bacteria [Figure 3(a)], indicates that the high titres seenagainst several of the LPSs were directed against the 0-polysaccharide chain and not the core saccharide. Consequently the high titres should be the result of antibodies directed against epitopes on the polysaccharide, one or several, which are shared between the various serotypes. The conformation of the basic S. jlexneri serotype Y polysaccharide in solution has recently been established3’.In its most ordered state, it forms a helical structure where each tetrasaccharide repeating units turns 180” round the longitudinal axis. Only two antigenic specificities, group antigen 3,4, have been assignedto this structure in studies with rabbit antiseras. We identified four distinctly different patterns in six monoclonals closely studied using LPS and a battery of synthetic glycoconjugates as antigens”. One antibody (MASF-YS) which recognized the intraachain a-L-Rhap-( I-3)-P-D-GlcpNAc(I-2)-a-L-Rhap-( l-2) a-L-Rhap structure had a binding specificity close, or identical to the “classical” group-antigen 3,45,27(Table 1). We concluded that the serotype Y polysaccharide chain harbours several epitopes in addition to the 3,4 epitope(s)27and we now suggest that the high titres seenin this study with the Yl-BSA, Y2-BSA and the various LPSs in the EIAs are the result of antibodies elicited against one or more of these epitopes. In sera collected after S. jlexneri serotype lb immunization/infection the titre was highest against the lb LPS in four of the five sera [Figure 3(a)-(d)]. The titre was, however, not much higher than against other LPSs. We assume that this is because antibodies were not predominantly formed against the type-antigen I epitope, i.e. the aD-Glcp-( I~)-P-D-G~c~NAc structure (Figure 1). This interpretation was also supported by finding low titres using the disaccharide-I-BSA glycoconjugate (Table 2). High titres were seenagainst LPSs containing the group-antigen 6 epitope, i.e. the OAc (1-2)-a-LRhap structure being immunodominant in this epitope (Figure 3). The observation that the titres against the synthetic 6BSA conjugate also were high is in accordance with formation of group-antigen 6 specific antibodies (Table 2). The high titres against the Y22BSA conjugate showed that terminal non-reducing end specific antibodies i.e. against the unsubstituted a-L-Rhap-( I-2)-L-Rhap-, had been formed. However, the high titres seenalso against the Y I-BSA conjugate which lacks the natural non-reducing end of the 0-antigenic polysaccharide (Figure 5), demonstrated that antibodies had been formed against intrachain determinants, possibly the “3,4 antigen”. The S.JIrxneri serotype 2a LPS has an 0-polysaccharide chain where only the RhalII residue has a substitution, the o-glucose a-linked to C4 (Figure 1). This type-antigen II epitope was present in the II-BSA conjugate and the native 2a LPS, and in both of the high titres detected in the rabbit and human sera assayed [Figure 4, Table 21. Since equally high titres were detected with the Y l- and Y22BSA conjugates, it is probable that antibodies against the II epitope did not constitute a dominant portion of

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antibodies formed in theseserum samples.Instead, antibodies which were recognized by the basal repeating unit as present in the Y LPS, were in a majority. Again the interpretation is that terminal end and intrachain epitopes, i.e. the “3,4 antigen” as present in the Y polysaccharide, are also present in the 2a polysaccharide, and antibodies against them are formed as a result both of natural infection and immunizations. These studies have been focused on the S. JIexneri serotype lb and 2a LPSs firstly becausethey are among the two most common serotypes seenin dysentery caused by S. flexneri and secondly because they represent an 0-antigenic polysaccharide where the repeating unit has two and one substitution(s), respectively. The data convincingly show that the antibody responsesmay permit a species-specificimmunological diagnosis of a S. jlexneri infection with LPS or the Yl- and Y2---BSA glycoconjugates. The use of saccharides forming branches on the basis Y structure and surmised to be epitopespecific, i.e. the I-- and II-BSA conjugates, did not allow a serotype-specific diagnosis. The reason(s)for this is unclear. The fact that the bBSA conjugate appeared to permit a serotype-specific diagnosis may indicate that we have not chosen the correct structural domains for the epitope-conjugates. In accompanying papersZ7,14 we report on the experiences of assaying sera from healthy individuals and patients with bacillary dysentery in Vietnam using S. jle.uneri LPS antigens, Acknowledgement This work was supported by the Swedish Medical ResearchCouncil (grant no. 16 x-656) and the Swedish Agency for ResearchCooperation with developing countries (SAREC). The skilful technical assistanceof MS Kerstin Karlsson is gratefully acknowledged. References Stall BJ, Glass, RI, features of patients Bangladesh. J Infect 7 Levine MM. Shigella 1.

; :

4. 5. 6. 7. 8. 9. 10. 1 I.

Huq MI, Khan MU, Banu H. Holt J. Epidemiological and clinical infected with Shige[la who attended a diarrhea] disease hospital in Dis 1982; 146: 177-83. and Salmonella diarrhoeal disease. Trop Dot 1979; 9: 4-9. Appendix D-15. The prospectsfor immunizingagainstShigella spp. In: Institute of Medicine. New Vaccine Development: Establishing Priorities. Vol Il. Diseases of Importance in Developing Countries. Washington DC: National Academy Press, 1986: 329-36. Mravunac B, Weber D. Shigellosis in the first two years of life. Br Med J 1956; I: 108c-2. Edwards PR, Ewing WH. Identification of Enterobacteriaceae. Minneapolis: Burgess, 1972. Kenne L, Lindberg B, Petersson K, Katzenellenbogen E, Romanowska E. Structural studies of Shigella,flexneri O-antigens.Eur J Biochem 1978;91: 279-84. Carlin NIA, Lindberg AA. Bock K, Bundle DR. The ShigellaJlevnui 0-antigenic polysaccharide chain. Nature of the biological repeating unit. Eur J Biochem 1984; 139: 189-94. Carlin NIA, Lindberg AA. Monoclonal antibodies specific for Shige/lajexneri lipopolysaccharides: clones binding to type I and type lll:6,7.8 antigens,group 6 antigen and a core epitope. infect lmmun 1986; 53: 103-9. Carlin NIA. Lindberg AA. Monoclonal antibodies specific for O-antigenic polysaccharide of Shigellajlexneri: clones binding to II. ll:3.4 and 7,8 epitopes. J Clin Microbial 1983: 18: 1183-9. Carlin NIA, Lindberg AA. Monoclonal antibodies specific for Shigella fle.vzeri lipopolysaccharides: clones binding to type IV, V and VI antigens, group 3,4 antigen and an epitope common to all S.flexneri and S. cfvsenferiae type 1. Infect lmmun 1987; 55: 1412-20. Liideritz 0. Westphal 0, Staub AM, Nikaido H. Isolation and chemical and immunological characterization of bacterial lipopolysaccharidcs. Microbial Toxins, Vol. 4. New York: Academic Press. 197 I : 145-233.

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12. Galanos B. Liideritz 0, Westphal 0, A new method for the extraction of R lipopolysaccharides. Eur J Biochem 1969; 9: 245-9. 13. Sawardecker JS, Sloneker JH. Jeanes A. Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal Chem 1985; 37: 16024. 14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 26575. 15. Driige W, Liideritz 0, Westphal 0. Biochemical studies on lipopolysaccharides of Salmonella R mutants. Eur J Biochem 1968; 4: 12633. 16. Tsai CM. Frasch CE. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 1982; 119: 115-9. 17. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay. Quantitative assay of immunoglobulin G. Immunochemistry 1971; 8: 8714. 18. Karlsson K, Granstrom M, Lindberg AA. Salmonella sp. antibodies. XI. Antigens and Antibodies 2. In: Bergmeyer HQ, ed. Methods of enzymatic analysis. Weinheim, FRG: VCH Verlegsgesellschaft, 1986: 85598. 19. Erbing C, Granath K, Kenne L, Lindberg B. A new method for the N-deacetylation of carbohydrates. Carbohydr Res 1976; 47: C5. 20. Svensson SB, Lindberg AA. Coupling of acid-labile Salmonella specific oligosaccharides to macromolecular carriers. J lmmunol 1978; 25: 323335. 21. Garegg PJ. Norberg T, Konradsson P, Svensson SCT. Synthesis of p-aminophenyl-2-Oacetyl-3-O-a-L-rhamnopyranosyl-a-L-rhamnopyranoside. Carbohydr Res 1983; 116: 308-l 1. 22. Forsgren M, Norberg T. Syntheses of the p-trifluoro-acetamidophenyl glycosides of 2acetamido-2-deoxy-4-O-o-glucopyranosyl-PD-glucopyranose and 2-acetamido-2-deoxy-6-0a-o-glucopyranosyl-b-u-glucopyranose. Carbohydr Res 1983; 116: 3947. 23. Ekwall E, Dac Cam P, Due Trach D, Taube A, Lindberg AA. Shigelfujexneri O-antigen specific enzyme immunoassay: class-specific antibody titres against lipopolysaccharide antigens in healthy Vietnamese and Swedish populations. Serodiag Immunother Infect Dis 1988; 2: 47-61. 24. Ekwall E, Dac Cam P, Van Chan N, Due Trach D, Lindberg AA. ShigeZluJIexneri O-antigen specific enzyme immunoassay: a prospective study of class-specific antibody titres against lipopolysaccharide antigens in Vietnamese children and adults with sero-type 1b and 2a dysentery. Serodiag Immunother Infect Dis, in press. 25. Johnston JH, Johnston RJ, Simmons DAR. The immunochemistry of Shigellufiexneri Oantigens. The biochemical basis of smooth to rough mutation. Biochem J 1967; 105: 79-81. 26. Jansson PE, Lindberg AA, Lindberg B, Wollin R. Structural studies of the hexose region of the core in lipopolysaccharides from Enterobacteriuceae. Eur J Biochem 1981; 115: 571-7. 27. Carlin NIA, Bundle DR, Lindberg AA. Characterization of five Shigellaflexneri variant Yspecific monoclonal antibodies using defined saccharides and glycoconjugate antigens. J Immunol 1987; 138: 4419-27. 28. Carlin NIA, Wehler T, Lindberg AA. Shigefla jlexneri O-antigen epitopes: Chemical and immunochemical analyses reveal that type III and group antigen 6 epitopes are identical. Infect Immun 1986; 53: 110-5. 29. Kabat EA. Mayer MM. Experimental immunochemistry. Springfield: Charles C. Thomas Publisher. 1961. 30. Ekwall E, Svensson SB, Norberg T, Svenungsson B, Lindberg AA. Defined saccharide antigens for sensitive and specific detection of the 0-antigenic antibody response after Salmonella infection. Serodiag Immunother Infect Dis 1987; 1: 57767. 3 I. Bock K, Josephson S, Bundle DR. Lipopolysaccharide solution conformation: antigen shape inferred from high resolution ‘H and ‘C nuclear magnetic resonance spectroscopy and hardsphere calculations. J Chem Sot Perkin Trans 1982; II: 59-70. (Manuscrip

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December 1987)