Immunology and Immunochemistry of Synthetic and Semisynthetic

5 Immunology and Immunochemistry of Synthetic and Semisynthetic Salmonella O-Antigen-Specific Glycoconjugates ALF A. LINDBERG, RALFH WOLLIN, GERT BRUSE, ERIK EKWALL, and STEFAN B. SVENSON

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: September 29, 1983 | doi: 10.1021/bk-1983-0231.ch005

National Bacteriological Laboratory, Department of Bacteriology, S-105 21 Stockholm, Sweden

The lipopolysaccharide (LPS) molecule is an interesting component of the envelope of all bacteria belonging to the Enterobacteriaceae. The polysaccharide part constitutes a sub stantial portion of the cell envelope. The recognition of an antigenic specificity in the polysaccharides, the O-antigens, has led to the common use of antibodies for identification of these bacteria through serotyping. The O-polysaccharide is covalently linked to lipid A where the well-known noxious endotoxic activity of all Enterobacteriaceae resides (1). The toxic activity of lipid A has often been considered to be the greatest biological significance of the LPS, and the importance of the O-polysaccha ride has been grossly overlooked. In this communication we will review our research, which has focused on the O-polysaccharide chains with three specific goals in mind: (i)

The use of small synthetic saccharide haptens identical to regions of various O-polysaccharide chains for the production of specific diagnostic antisera,

(ii)

the use of bacteriophage-associated endoglycosidases for the production of larger saccharides from O-poly saccharide chains, and

(iii)

the production and use of Salmonella O-antigen-specific saccharide glycoconjugates (glycoproteins and glycolipids) for studies of humoral and cell-mediated immune reactivity after salmonellosis, and for studies of their use as immunogens, e.g., vaccines.

Salmonella Oligosaccharide-Protein of O-Antigen-specific Antibodies

Conjugates for the Production

The serogroup classification of Salmonella bacteria, accord ing to the Kauffmann-White scheme (2), is based on the antigenic specificities which reside in the polysaccharide chain (the 0097-6156/83/0231-0083$10.00/0 © 1983 American Chemical Society

Anderson and Unger; Bacterial Lipopolysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

BACTERIAL LIPOPOLYSACCHARIDES


84

0-antigen) of the l i p o p o l y s a c c h a r i d e (LPS) of the b a c t e r i a l c e l l envelope. The polysaccharide chain, which as f a r as we know i s composed of polymerized repeating u n i t s comprising from three to f i v e monosaccharides (3), contains s e v e r a l a n t i g e n i c determinants: one s p e c i f i c f o r the serogroup and one or more i n common with other serogroups (2). Conventional procedures f o r the p r e p a r a t i o n of a n t i s e r a against Salmonella O-antigens i n v o l v e immunization of r a b b i t s with whole heat- or f o r m a l i n - k i l l e d b a c t e r i a . However, t h i s procedure r e s u l t s i n the production of a n t i b o d i e s of s e v e r a l d i f f e r e n t s p e c i f i c i t i e s , i n c l u d i n g a n t i b o d i e s with s p e c i f i c i t y f o r surface e n t i t i e s other than the 0-antigen. To render these a n t i s e r a O - a n t i g e n - s p e c i f i c , absorptions with other b a c t e r i a are done i n order to remove a n t i b o d i e s with undesired s p e c i f i c i t i e s . Absorptions are, however, o f t e n incomplete and the t i t e r of the d e s i r e d a n t i b o d i e s may decrease as w e l l . We assumed that i f saccharides i d e n t i c a l to known a n t i g e n i c determinants of Salmonella O-antigens could be synthesized or obtained by p a r t i a l h y d r o l y s i s of O-polysaccharide chains, and coupled to immunogenic c a r r i e r molecules, the r e s u l t i n g glycocon jugates would be s u i t a b l e as antigens f o r e l i c i t i n g i n r a b b i t s a n t i b o d i e s with more defined O-antigenic s p e c i f i c i t y . We have used the O-antigens of Salmonella serogroups A-E as a model system i n these s t u d i e s s i n c e the s t r u c t u r e s of the O-chains are w e l l e s t a b l i s h e d (4). Synthetic Disaccharide

Haptens

In serogroups A, B and D the t e t r a s a c c h a r i d e repeating u n i t s are i d e n t i c a l except f o r the nature of the dideoxyhexose l i n k e d to the D-mannose residue (Figure 1). Even before the s t r u c t u r e of the O-chain was e l u c i d a t e d i t had been c o n v i n c i n g l y shown that paratose f o r serogroup A, abequose f o r serogroup B, and t y v e l o s e f o r serogroup D were immunodominant sugars i n the serogroup s p e c i f i c i t y (5). Dideoxyhexoses, c o v a l e n t l y l i n k e d to ovalbumin or bovine serum albumin (BSA) as the c a r r i e r p r o t e i n gave r i s e to a n t i b o d i e s i n r a b b i t s . The a n t i b o d i e s could p r e c i p i t a t e the s y n t h e t i c antigen, but d i d not a g g l u t i n a t e the b a c t e r i a (5). T h i s was i n agreement with the r e s u l t s of Goebel (6, 7) and McCarthy (8) who d i d not f i n d any a n t i b a c t e r i a l a n t i b o d i e s i n r a b b i t s immunized with a r t i f i c i a l antigens c o n t a i n i n g only the terminal sugar of an immunologically a c t i v e o l i g o s a c c h a r i d e u n i t . Therefore the f i r s t s y n t h e t i c work was d i r e c t e d to produce d i s a c c h a r i d e s with the dideoxyhexose i n the non-reducing end position. The synthesis of the d i s a c c h a r i d e s e n t a i l e d the p r e p a r a t i o n of 3,6-dideoxyhexosyl ( p a r a t o s y l , abequosyl and t y v e l o s y l ) precur sors s u i t a b l e f o r j o i n i n g to an a p p r o p r i a t e l y d e r i v a t i z e d D-mannose residue (9, 10). In order to couple the synthesized d i s a c c h a r i d e s to c a r r i e r p r o t e i n s i t was necessary to have an aglycone c o n t a i n i n g a s u i t a b l e r e a c t i v e group such as a j>-isothiocyanatophenyl group


5.

LINDBERG ET AL.

Salmonella O-Antigen-Specific Glycoconjugates

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LIPID A

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POSITIVE IN I F L

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SALMONELLA SEROGROUP A

I

OH

B

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0/62

c" ^ ^ « ° " 0 "

NHCNHCH -" 2

85

63/63

| OTHER SALMONELLA

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AND ENTEROBACTERIA

SEROGROUP B

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BACTERIA

(04 l i n k e d to the D - g a l a c t o s y l group of the repeating u n i t (Figure 7, I I ) , as i n the a n t i g e n i c determinant 012 , i s compatible with h y d r o l y s i s by phages P22 and P27 (37). The data even suggested that these phages p r e f e r e n t i a l l y hydrolysed the L-Rhaj>(al-*3)D-Gal l i n k a g e where the D-galactose r e s i d u e was g l u c o s y l a t e d (37). However, the a, 1*4 l i n k e d D-glucose residue made the s u b s t r a t e r e s i s t a n t to the phage 9NA and KB1 endo-a-rhamnosidases. ( i i ) The presence of a D-glucose residue a,l->-6 l i n k e d to the D-galactose of the repeating u n i t (Figure 7, I I I ) , a consequence of l y s o g e n i c conversion with phage P22, made i t impossible f o r the phage P22 and P27 enzymes to hydrolyse the 0-chain (37). However, the a,1+6 l i n k e d D-glucose, the a n t i g e n i c determinant 01, permitted h y d r o l y s i s of the 0-chain by the phage 9NA and KB1 enzymes (37). For these enzymes the data suggested that h y d r o l y 2




ABE

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- d i s a c c h a r i d e , which i s the determinant of 0 - a n t i gen 4 (Figure 1). The median IgG t i t e r s i n convalescent sera ( c o l l e c t e d 2-6 weeks a f t e r onset of Salmonella serogroup B i n f e c t i o n ) from a l l 10 p a t i e n t s showed the same tendency: t i t e r s were 1380, 1510 and 950 against the Salmonella BO-modified LPS, AMRGITC-BSA and AM-(CH^^-BSA antigens, r e s p e c t i v e l y . The correspond i n g median t i t e r s i n sera from 10 Salmonella DO-infected p a t i e n t s were 380, 135, and 60, r e s p e c t i v e l y . These r e s u l t s demonstrate the e x c e l l e n t s p e c i f i c i t y of the AMRG- and AM-glycoconjugates. Cell-mediated Immune Reactions and O-Polysaccharides

S p e c i f i c i t y f o r Salmonella

In Vivo T e s t i n g . In demonstrations of cell-mediated immune responses such as delayed h y p e r s e n s i t i v i t y r e a c t i o n s i n s k i n , most i n v e s t i g a t o r s have used crude e x t r a c t s of whole Salmonella b a c t e r i a (54, 55). This had made i t d i f f i c u l t to evaluate which component(s) had e l i c i t e d the cell-mediated immunity. In a study of the s p e c i f i c i t y of delayed h y p e r s e n s i t i v i t y r e a c t i o n s i n Salmonella i n f e c t e d c a t t l e to intracutaneous i n j e c t i o n of t e s t antigen s o l u t i o n s , i t was found that the animals reacted mainly against e x t r a c t s from O - a n t i g e n i c a l l y homologous b a c t e r i a (56). T h i s s t r o n g l y suggested that the O-polysaccharides were important i n t h i s context. In a subsequent study calves were experimentally i n f e c t e d o r a l l y with a s u b l e t h a l dose of the c a l f v i r u l e n t s t r a i n S^. typhimurium SVA44 and s k i n t e s t e d 3 weeks l a t e r (Table I) (57). The LPS from S. typhimurium SH4809 (04,5,12) i n a dose of 50 yg e l i c i t e d w i t h i n 48 h i n i n f e c t e d calves an i n c r e a s e i n double s k i n f o l d t h i c k n e s s , with a mean of 4.5 mm. The corresponding f i g u r e i n the u n i n f e c t e d calves was 1.3 mm, a h i g h l y s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e . I n j e c t i o n of an O - a n t i g e n i c a l l y unrelated LPS (S. thompson), or of p o l y - or o l i g o s a c c h a r i d e s from the S. typhimurium O-polysaccharide, f a i l e d to e l i c i t a s k i n r e a c t i o n i n both groups of c a l v e s . Furthermore the [GM(A)R] -ITC-BSA g l y c o conjugate f a i l e d to give a p o s i t i v e r e a c t i o n (data not shown). However, the covalent l i n k a g e of the same octasaccharide s t r u c t u r e v i a the i s o t h i o c y a n a t e route to dodecylamine i n s t e a d of BSA y i e l d e d a g l y c o l i p i d which upon i n j e c t i o n i n i n f e c t e d c a l v e s gave r i s e to a h i g h l y s i g n i f i c a n t l y increased s k i n s w e l l i n g as compared to the s w e l l i n g seen when the same semisynthetic g l y c o l i p i d was 2


103



104

3000 -

MONTHS AFTER

ONSET

OF

DISEASE

Figure 10. IgG antibody titers against three Salmonella O-antigen 4-containing antigens as estimated by ELISA in serum samples from a patient with S. typhimurium infection. Key: A, Salmonella BO LPS IO~ OX;; AMRG-BSA; and o, AM-(CH \-BSA.



Significance infected vs. controls

controls

P. typhimurium

(O.8)

8

No.

O.25. typhimurium SH4809 (04,5,12) LPS

585

[GM(A)R] -ITC-BSA

-3 ELISA end-point t i t e r x 10

AMRG-ITC-BSA (04,12)

AM-ITC-BSA (04)

Immunogen and 0-antigen s p e c i f i c i t y

TABLE I I . Antibody Response i n Rabbits Immunized with Various JS. typhimurium Saccharide-BSA Conjugates*.


110

BACTERIAL LIPOPOLYSACCHARIDES (v)

The a n t i - s a c c h a r i d e antibody response appears to be optimal when the molar r a t i o of saccharide to BSA i s around 20. T h e o r e t i c a l l y there are 57 s i t e s i n the BSA molecule to which the hapten can be l i n k e d .

(vi)

A n t i b o d i e s with s p e c i f i c i t y f o r the l i n k a g e arm between the hapten and c a r r i e r are formed as demonstrated f o r the d i s a c c h a r i d e haptens. The l a r g e r the saccharide the l e s s pronounced i s t h i s tendency.


The c h a r a c t e r i s t i c s of the antibody response discussed above d i d not r e l a t e to t h e i r biological activity. Therefore a s e r i e s of experiments was done i n order to i n v e s t i g a t e these aspects. (i)

The b a c t e r i c i d a l a c t i v i t y , e.g. the a b i l i t y of e l i c i t e d r a b b i t a n t i b o d i e s p l u s guinea p i g complement to k i l l b a c t e r i a when mixed and incubated at +37°, was assayed f o r the anti-AM-ITC-BSA (04) and TM-ITC-BSA (09) a n t i s e r a (12, 15). These a n t i s e r a were as e f f e c t i v e i n t h i s respect as were a n t i s e r a e l i c i t e d by whole h e a t - k i l l e d b a c t e r i a . Furthermore they were, as expected, a b s o l u t e l y s p e c i f i c , e.g. a b a c t e r i c i d a l e f f e c t was seen only on b a c t e r i a with an 0-antigen i d e n t i c a l to the antibodye l i c i t i n g haptenic s t r u c t u r e .

(ii)

In clearance s t u d i e s , where the a b i l i t y of the a n t i bodies to a i d i n the clearance of b a c t e r i a from the blood was followed, we found that p a s s i v e l y administered r a b b i t a n t i b o d i e s e l i c i t e d a g a i n s t the AM-ITC-BSA and [GM(A)R] -ITC-BSA conjugates were as e f f i c i e n t as a n t i - O - a n t i b o d i e s e l i c i t e d by whole h e a t - k i l l e d b a c t e r i a (64). When mice were a c t i v e l y immunized w i t h the glycoconjugates the l a r g e r saccharide haptens e l i c i t e d an antibody response which was a l s o e f f i c i e n t i n pro moting c l e a r a n c e . There was a s t r i c t c o r r e l a t i o n between the antibody t i t e r and the clearance r a t e (64). 2

(iii)

In i n v i t r o phagocytosis experiments where the a b i l i t y of i n d i v i d u a l sera c o l l e c t e d from mice before and a f t e r immunization with the saccharide-ITC-BSA conjugates were t e s t e d f o r t h e i r a b i l i t y to promote the uptake of b a c t e r i a by a c t i v a t e d mouse p e r i t o n e a l exudate c e l l s i n the presence of complement we found that a l s o here the immune s e r a e l i c i t e d by the [GM(A)R] -ITC-BSA conjugates (n = 2 or 3) were e f f i c i e n t (P . typhimurium s t r a i n , but not against challenge with an O - a n t i g e n i c a l l y heterologous J3. e n t e r i t i d i s s t r a i n (the b a c t e r i a were Isogenic with exception of the 0-antigen) (65)• From experiments where s e r i a l d i l u t i o n s of the r a b b i t a n t i s e r a were given p r i o r to challenge with 25 x ^Q doses we could conclude a f t e r p l o t t i n g the a n t i S. typhimurium LPS (04,12) antibody t i t e r against the percentage of s u r v i v ing mice that a l l a n t i s e r a were e q u a l l y e f f i c i e n t i r r e s p e c t i v e of whether they had been e l i c i t e d by a glycoconjugate or h e a t - k i l l e d b a c t e r i a (Figure 12).


ld

(v)

I n j e c t i o n of b a c t e r i a l endotoxins i n t o h e a l t h y man or animals r e s u l t s , among other t h i n g s , i n a f e v e r response (66). I t has long been recognized that repeated i n j e c t i o n s of the endotoxin can l e a d to t o l e r a n c e to the pyrogenic a c t i v i t y . V i r t u a l l y a l l s t u d i e s have been done with p r e p a r a t i o n s c o n t a i n i n g the t o x i c l i p i d A component, e i t h e r i n i t s n a t i v e s t a t e or a f t e r v a r i o u s more or l e s s e f f i c i e n t chemical d e t o x i f i c a t i o n proce dures. Since l i p i d A i s r e q u i r e d f o r the immunogenicity of the endotoxin the degradation procedures have l e f t i n v e s t i g a t o r s e i t h e r with only a p a r t i a l l y d e t o x i f i e d product, or a completely d e t o x i f i e d product which a c c o r d i n g l y has been rendered nonimmunogenic. I t has t h e r e f o r e p r e v i o u s l y been impossible to e s t a b l i s h i f endotoxin t o l e r a n c e can be acquired i n the absence of the l i p i d A component. The problem was addressed using a glycoconjugate c o n s i s t i n g of the S. typhimurium SH4809 dodecasaccharide c o v a l e n t l y l i n k e d to human serum albumin ([GM(A)R]^-ITCBSA) (67). The conjugate, shown to be f r e e of contamin a t i n g p r o t e i n and endotoxic a c t i v i t y , e l i c i t e d i n r a b b i t s immunized e i t h e r i n t r a v e n o u s l y or i n t r a p o p l i t e a l l y a d e f i n i t e t o l e r a n c e (Figure 13). The i n t r a v e n o u s l y immunized group 1 showed, when i n j e c t e d with the parent S. typhimurium SH4809 LPS, a mean f e v e r index s t a t i s t i c a l l y d i f f e r e n t from the c o n t r o l group (P

Immunology and Immunochemistry of Synthetic and Semisynthetic

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