zyxwvutsrqp zyxwvutsrqpo zyxwvutsrq zyxwvutsrqp Eur. J. Biochem. 179,651 -657 (1989) FEBS 1989 Synthesis of glycoconjugates derived from various lipopolysaccharides of the Vibrionaceae family Joseph H. BANOUB, Derek 11. SHAW, N. Anthony NAKHLA and Howard J. HODDER Department of Fisheries and Oceans, Experimental Sciences Division, St. John's, Newfoundland (Received April b/October 5, 1988) - EJB 88 0401 zyxwvut zyxwvu Conjugation of simple ketoses (such as 3-deoxy-~-manno-2-octu~osonic acid and N-acetylneuraminic acid) and of various 0-specific polysaccharides (from Aerornonas hydrophila and Aeromonas salmonicida) to the bifunctional spacer 2,6-hexanediamine, was achieved by reductive amination. The saccharide - 1-(ti-amino)-hexanealkyamines obtained were converted into the corresponding isothiocyanate derivatives and coupled to the free c-amino group of lysine residues of the protein carrier bovine serum albumin. In similar manner, the aldehyde group introduced by selective periodate oxidation into the partially 0-deacylated lipopolysaccharide of Vibrio anguillarum was conjugated to 1,6-hexanediamine, converted into the corresponding isothiocyanate and covalently attached to bovine serum albumin. During the past decade, artificial glycoconjugates or This paper presents a new method for coupling different neoglycoproteins, obtaincd by covalent attachment of poly- 0-specific polysaccharides and a partially 0-deacylated saccharide to protein, have been extensively used for anti- oxidized lipopolysaccharide of representative species of the body -polysaccharide interactions [l] and found to be of Vibrionaceae family to the lysine residues of the carrier progreat utility as vaccines against encapsulated bacteria [2]. tein bovine serum albumin. The method presented here is Numerous methods, each with their own merits and pur- based on the attachment of the new spacer (or bridging arm) poses, have been developed for the synthesis of glycocon- 1,6-hexanediamine, by reductive amination, to the ketonic jugates [3]. Most of these studies have generally resorted to carbonyl group of the only dOclA residue of the corethe use of naturally occurring bacterial oligo- and polysac- oligosaccharide portion [lo, 111 of the native 0-specific charides or chemically synthesized carbohydrate haptens polysaccharides of Aeromonas hydrophila and Aeromonas which mimic the natural saccharide sequences of the carbo- salmonicida and to the core heptose aldehyde group of the hydrate portion of the conjugate. One method that is both partially deacylated oxidized lipopolysaccharide of Vihrio simple and effective is the direct covalent attachment of reduc- mguillarum [ 121. The polysaccharide - 1-alkylamine derivaing carbohydrates to the amino groups of proteins by re- tives obtained are purified and converted into the correspondductive amination using sodium cyanoborohydride [4, 51. A ing (6-isothiocyanato)-hexane alkylamine derivatives and major disadvantage of this method is the opening of the ring then coupled to the lysine residues of bovine serum albumin structure of the terminal reducing sugar to generate an acyclic by the procedure of McBroom et al. [13]. amine which, in certain cases, could be detrimental to the biological specificities of the glycoconjugate [6].However, this disadvantage is of no importance if the haptenic saccharide is MATERIALS AND METHODS large, as in the case of bacterial polysaccharide [7]. Another Bacterial cultures drawback to this method is that it is not possible to conjugate oligosaccharides having reducing ketose residues, such as the A virulent strain of Aeromonas hydrophila (strain SJ-44) terminal 3-deoxy-~-manno-2-octulosonk acid (dOclA), to was obtained from Dr R. Lallier (Faculty of Veterinary Mediproteins [8]. This is consistent with the fact that a successful cine, University of Montreal, St Hyacinthe, Quebec). The reductive amination attachment of oligosaccharides contain- virulent strains of Aeromonas sulmonicida (strain SJ-15) and ing terminal dOclA residues to proteins can be achieved only Vihrio anguillarum (strain SJ40T) were kindly supplied by Dr if a functionalized spacer molecule is introduced at the ketonic T. P. T. Evelyn (Pacific Biological Station, Nanaimo, BC). carbonyl group of dOclA [8, 91. Correspondence to J. H. Bdnoub, Department of Fisheries and Oceans, Experimental Sciences Division, Science Branch, P. 0. Box 5667, A1C 5x1 St. John's, Newfoundland, Canada Abbreviation$. Albumin, bovine serum albumin; 0-specific polysaccharide is defined as the combined 0-chain polysaccharide/ core oligosaccharide portion of the lipopolysaccharide; LD-Hep, L-g!ycero-D-manno-heptose; L-Rha, 1.-rhamnose; L-Qui3NAc, 3-acelamido-3,6-dideoxy-~-glucosc ; D-QuI~NAc, 2-acetamido-2,6-dideoxyD-glucose: dOclA, 3-deoxy-~-rnanno-2-octulosonic acid. zyxw Purification and hydrolysis of the lipopolysaccharides Lipopolysaccharides were extracted from lyophilized bacterial cells by the aqueous phenol method and were recovered as previously described [12, 14, 151. Production of the 0-specific polysaccharides was achieved by hydrolysis of the lipopolysaccharide in 1 %, aqueous acetic acid for 90 min at 100"C, followed by gel chromatography on Sephadex G-50 as previously described [12, 14, 151. 652 zyxwvutsrqpo Chemicals Crystalline bovine serum albumin (referred to as ‘albumin’ here) was obtained from Schwarz-Mann (Orangeburg, NY) and 1,6-hexanediamine hydrochloride from Pfaltz and Bauer (Waterbury, CT); N-acetylneuraminic acid, the crystalline ammonium salt of dOclA and sodium cyanoborohydride were obtained from Sigma Chemical Co. (St Louis, MO). Ana(yticu1procedures Protein was measured by the method of Lowry et al. [16] and sugars were measured by the procedure of Dubois et al. [17]. Unconjugated lysine was determined with an amino acid analyzer using albumin as a standard. The molecular masses of the glycoconjugates were determined by gel filtration chromatography as described by Hopwood and Robinson [18]. Thin-layer chromatography was used to differentiate between dOclA or N-acetylneuraminic acid and their alkylamine derivatives. TLC analysis was carried out on 0.25-mm precoated plates of silica gel 60F254 (E. Merck, Darmstadt, FRG) developed with chloroform/methanol/ water (10: 3: 1, by vol.). 13C-NMRspectra of the 1-(6-amino)hexane alkylamine derivatives of dOclA and N-acetylneuraminic acid were measured in DzO, with a Varian CFT20 spectrometer operating at 20 MHz in the pulse Fouriertransform mode with complete proton decoupling. Chemical shifts are reported downfield from external tetramethylsilane. into their corresponding 1-(6-isothiocyanato)-hexane alkylamine derivatives by the method of McBroom et al. [13]. Preparution of the 0-specifi:c-polysuccharideI - (6-isothiocyanato)-hexaneulkylamine derivatives The O-specific polysaccharide (30 pmol: A . hydrophila, 159 mg; salmonicidu, 313.5 mg) was dissolved in water ( 5 ml). This solution was added to a rapidly stirred mixture, adjusted to pH 8.0, of sodium cyanoborohydride (0.3 mmol) and 1,6hexanediamine hydrochloride (3 mmol) in water ( 2 ml). The reaction was allowed to proceed for 48 h while maintaining constant pH by titration with 0.1 M sodium hydroxide. The reaction mixture was then applied to a column of Bio-Gel P-2 (2.5 x 90 cm) and eluted with 0.1 M acetate pH 5.0. Fractions were collected and assayed for carbohydrate and checked by 13C-NMR for the incorporation of the alkylamine spacer. The O-specific-polysaccharide- 1-(6-amino)-hexane alkylamine derivatives were converted into the corresponding 1-(6-isothiocyanato)-hexane derivatives as follows. To 24 pmol of the O-specific-polysaccharide - 1 -(6-amino)-hexane alkylamine derivative dissolved in 80% aqueous ethanol (2 ml), thiophosgene (1.3 mol) was added dropwise, while a constant pH of 7.0 was maintained by titration with 1 M sodium hydroxide in 80% aqueous ethanol. When consumption of sodium hydroxide had ceased, the reaction mixture was freed of excess thiophosgene by repeated evaporation with methanol; the yellow product was dried under vacuum. z zyxwvutsrq zyxwvutsrqp zyxwvuts General conditions ,for coupling dOclA and N-acetylneuraminic acid to the spacer I ,&hexanediamino: preparation of the 1-(6-amino)-hexane alkylamine derivatives Method A . The conjugation of dOclA to 1,6-hexanediamine hydrochloride was carried out using a modification of the Svenson and Lindberg method [19]. dOclA (23.8 mg, 0.1 mmol) was dissolved in water (15 ml) and adjusted to pH 10 with 0.1 M sodium hydroxide. This was then added to a rapidly stirred mixture of 1,6-hexanediamine hydrochloride (10 mmol) and sodium cyanoborohydride 0.06 g (1 mmol) pre-adjusted to pH 8.0. The reaction was allowed to proceed at room temperature for 48 h with the pH controlled at 8.0 by addition of 0.1 M sodium hydroxide. The reaction mixture was then applied to a column of Bio-Gel P-2 (2.5 x 90 cm) in distilled water adjusted to pH 6.0 with dilute acetic acid. Fractions were assayed for carbohydrate and the purity of the dOclA - 1-(6-amino)-hexane alkylamine derivative was checked by I3C-NMR. Method B. The conjugation of N-acetylneuraminic acid to 1,6-hexanediamine hydrochloride was carried out under similar conditions to the procedure of Roy et al. [8]. NAcetylneuraminic acid (100 mg, 0.32 mmol) was dissolved in 0.2 M phosphate buffer ( 5 ml) at pH 6.0. To this solution sodium cyanoborohydride (201 mg, 3.2 mmol) and 1,6-hexanediamine (3.71 g, 32 mmol) were added. The reaction was allowed to proceed for 24 h at pH 6.0. The reaction mixture was then applied to a Sephadex G-I 5 column (1.5 x 90 cm) made up in distilled water adjusted to pH 6.0 with acetic acid. Fractions were assayed for carbohydrate and the purity of the N-acetylneuraminic-acid - 1-(6-amino)-hexane alkylamine derivative was checked by 13C-NMR. Formation of the saccharide-albumin glycoconjugates The 1-(6-isothiocyanato)-hexane alkylamine derivatives of dOclA and N-acetylneuraminic acid and the corresponding O-specific polysaccharides were covalently attached to the free &-aminogroup of the lysine residues of the protein carrier bovine serum albumin. The saccharide-isothiocyanate derivatives (177 pmol) dissolved in phosphate buffer at pH 9.0 (2 ml), were each added to a rapidly stirred solution of albumin (I pmol) dissolved in the same buffer. The reactions were allowed to proceed for periods varying over 48 - 96 h at room temperature, while the pH was maintained constant by titration with 10mM sodium hydroxide. The coupling was assumed to be complete when consumption of sodium hydroxide had ceased; the resulting glycoconjugates were extensively dialyzed against distilled water and freeze-dried. The glycoconjugates were purified by gel chromatography on Bio-Gel P-150 (2.50 x 90 cm) eluted with 0.1 M phosphate pH 9.0. Fractions were assayed for carbohydrate and protein. zyxwvutsrqpo Preparation of the partially O-deacylated oxidized lipopolysaccharide - I - (6-isothiocyanato)-hexane alkylamine derivative and its ulbumin glycoconjugate V . anguillarum lipopolysaccharide (100 mg, 9.9 pmol) was dissolved in 0.25 M sodium hydroxide (5 ml) and heated at 56 T in a water bath for 60 min. The solution was cooled to room temperature and centrifuged at 2000 rpm for 15 min at 4°C. The pellet was discarded and the supernatant neutralized with acetic acid, dialyzed against distilled water and lyophilized. The partially O-deacylated lipopolysaccharide obtained was dissolved in 0.1 M sodium metaperiodate (10 ml) and placed in the dark for 15 min at room temperature; ethylene glycol (2 ml) was then added to the reaction mixture. The solution was extensively dialyzed and subsequently lyophilized. zyxwvutsr Preparation of the I- (6-isothiocyanato)-hexanealkylamine derivatives o f dOclA and N-acetylneuraminic acid The 1-(6-amino)-hexdne alkylamine derivatives of N-acetylneuraminic acid or dOclA (0.3 mmol) were transformed z zyxwvuts 653 The partially 0-deacylated oxidized (i.e. modified) lipopolysaccharide (89 mg z 8.8 pmol) was dissolved in water and adjusted to pH 10. This solution was added to a rapidly stirred mixture (adjusted to pH 8.0) of sodium cyanoborohydride (0.9 mmol) and 1,6-hexanediamine hydrochloride (9 mmol) in water (2.0 ml). The reaction was allowed to proceed for 48 h while maintaining constant pH. The reaction mixture was then processed as described earlier and converted into the 1-(6-isothiocyanato)-hexane alkylamine derivative by the procedure of McBroom et al. [13]. The modified-lipopolysaccharide - 1-(6-isothiocyanato)hexane alkylamine derivative (8 pmol) dissolved in phosphate buffer pH 9.0 (2 ml) was added to a rapidly stirred solution of albumin (0.08 pmol) dissolved in the same buffer. The reaction was allowed to proceed for 48 h at room temperature and processed as previously described. The glycoconjugate was purified by gel chromatography on Bio-Gel P-150eluted with 0.1 M phosphate pH 9.0. buffer ions [8]. By varying these parameters, successful covalent attachment of oligosaccharides having aldose end groups to protein carriers was achieved [22, 231, whereas no direct coupling of ketoses to proteins has been observed [8, 241. Roy et al. have indicated that the unsuccessful coupling of ketoses to proteins was due to the lower reactivity of the ketonic carbonyl group of the ketose residue as compared to the aldehydic group, in addition to steric effects introduced by using a large macromolecular protein carrier as a receptor molecule [8]. They concluded that the conjugation of terminal ketose residues of oligosaccharides to proteins, by reductive amination, would require the introduction of a small functionalized spacer molecule at the ketonic carbonyl group [8]. This conclusion was based on the fact that a successful conjugation of dOclA by reductive amination was achieved using an excess of the small receptor glycine molecule [8]. We therefore chose to use 1,6-hexanediamine as a novel bifunctional spacer (during the conjugation of dOclA by reductive amination) as it leaves the unconjugated functional 6-amino group free for further chemical manipulation aimed at covalent attachment to carrier proteins. Hence 3-deoxy-~-manno-2-octu~osonic acid (0.1 mmol) was reacted with an excess of the novel spacer 1,6-hexanediamine (10 mmol) in the presence of sodium cyanoborohydride. At pH 8.0, the imine derivative (Fig. 1) formed between the ketonic carbonyl group and the primary amino group of the spacer, was selectively reduced to the secondary amine to form the dOclA - 1-(6-amino)-hexane alkylamine derivative 2,1 3 C - N M R (DzO); G/ppm = 174.10 (C-1, carboxylic acid), 78.82 (C-4), 75.76 (C-5), 74.54 (C-6), 72.03 (C-7), 64.20 (C-8), 47.84 (C-2), 40.40 (C-3 and a-CH2, spacer), 27.54 (B-CH,, spacer) and 26.13 (7-CH2, spacer). This latter compound was subsequently transformed into the dOclA - 1-(6-isothiocyanato)-hexane alkylamine derivative 3 . The conversion of the isothiocyanate derivative from the compound 2 was almost quantitative. Similarly, N-acetylneuraminic acid was conjugated to the 1,6-hexanedkamine spacer using the molar ratio of sugar/reducing agent/spacer of 1 : 10: 100; these are similar proportions of the dOclA conjugation. The N-acetylneuraminicacid - 1-(6-amino)-hexane alkylamine derivative showed the following 13C-NMR(D20):G/ppm = 175.60 (C-I, carboxylic acid), 174.88 (CO, NHAc), 71.83 (C-8), 70.42 (C-6), 68.70 (C-7), 68.07 (C-4), 64.35 (C-9), 55.07 (C-2), 47.91 (C-5), 40.45 (a-CH2, spacer), 35.19 (C-3), 27.54 (8-CH2, spacer), 26.22 (y-CH2, spacer) and 23.03 (CH3, NHAc). This latter compound was subsequently transformed into the corresponding isothiocyanate derivative. zyxwvuts zyxwvu zyxw zyxwvutsrqp Immunization procedures New Zealand white rabbits (approximate mass 2 - 3 kg) were immunized intravenously [20] on five successive days with increasing amounts (0.66 - 3.4 kg) of the various conjugates. A booster dose of 3.4 pg was administered 15 days after the last injection and the rabbits were bled 10 days later. Immunodiffusion Double radial immunodiffusion was performed in 0.9% agarose gels in phosphate-buffered saline containing 2% poly(ethylene glycol) ( M , 4000) [21]. Passive hemagglutination Passive hemagglutination tests were carried out in microtiter plates (Linbro Division, Flow Laboratories, Inc., Hamden, CN). Human type 0 erythrocytes (1 YOsuspension in phosphate-buffered saline) were incubated with alkali-treated lipopolysaccharide preparations (0.25 M NaOH at 56 "C for 60 min) at a concentration of 30 pg/ml for 90 min, at 37°C. The coated erythrocytes were then washed five times with phosphate-buffered saline. For titration of antisera, 25 p1 0.5% erythrocyte suspension was mixed with 25 pl serum (twofold dilutions) and left at room temperature for 2 h. The serum titre was the highest dilution of the serum giving maximal strong hemagglutination. Previously prepared rabbit antisera to the lipopolysaccharides of A . salmonicida and V. anguillarum and also to whole cells of V . anguillarum were also used as reference antisera. RESULTS N zyxwvutsrqp zyxwvutsrqp zyxwvuts Preparution of the I - (6-isothiocyanato)-hexane alkylamine derivutives o j 3-deoxy-o-manno-2-octulosonic acidand N-aretylneuraminic acid It has been shown that the amount of the cyclic form of a reducing sugar is the rate-limiting step in the reductive amination reaction using sodium cyanoborohydride, and also that it is dependent on temperature, pH and the presence of Preparation of the 0-specific-polysuccharide I - (6-isothiocyanuto)-hexane alkylarnine derivatives Conjugation of' the 0-spectfic polysaccharide of A. hydrophila to the 1,6-hexanediamine spacer and formation of the corresponding isothiocyanate derivative. A . hydrophila is a gram-negative bacterium associated with both aquatic and terrestrial environments. Classically, the species has been regarded as an opportunistic pathogen of freshwater fish. The chemical structure of the repeating unit [14] of the native 0-specific polysaccharide (of strain SJ-44) is indicated in the following structure. OAC (21 %) I 2 [4)-a-~-Rhap-( 1+3)-jl-~-GlcpNAc-(l] 4core oligosaccharide 1+ dOclA 654 zyxwvutsrqp cot -No+ I I CHI I nocn I nocn I CHOH I C=NICH21,NHz I I CHOH F""" CHOH zyxwvutsrqpo zyxwvutsrqponmlkjih zyxwvutsr zyxwvutsrqp zyxwvutsrqp zyxwvutsrqponmlk zyxwvutsr zyxwvut I I!HzOH CH2OH I ~. ........... kH C02-Na' I S I " I c=o I F CHNHICHZIINH C NHICHZI, H NaCNBH, I I cwz I HOCH I HOCH I cnon I C HOH I CHNHICHZII NH FHZ I c=o HO&i . .......~~ - rlOFH COz -No' COz -NO' B S A ,,,' I CHOH I CHOH I +. I CSCI, ~HOCH I Hocn I I CHOH I CHOH CHIOH 2 4, I CHNHICHzI( NHz y 2 CHpOH CH~OH N=C= 5 2 Fig. 1. Covalent atfachmenfofthe I-(6-isothiocyanato)-hexanealkylamine derivative of dOclA to the €-aminogroups of albumin Iysine residues. BSA = bovine serum albumin The dOclA reducing end of the core-oligosaccharide portion of the 0-chain polysaccharide was reacted with an excess of the spacer 1,6-hexanediamine in the presence of sodium cyanoborohydride at pH 8.0. The resulting O-specificpolysaccharide - 1-(6-amino)-hexane alkylamine derivative was purified by gel chromatography and then transformed into the corresponding (6-isothiocyanato)-hexane alkylamine derivative. Conjugation of the 0-specific polysaccharide of A. salmonicida to the I,6-hexanediamine spacer and Jormation of the corresponding isothiocyanate derivative. The bacterium A . salmonicida is the causative agent of furunculosis in salmonid fishes and is an important pathogen causing high mortalities is aquaculture and fish hatchery operations. The chemical structure of the repeating unit of the native 0-specific polysaccharide [15] is indicated in the following structure. and the corresponding 0-specific polysaccharides to the free &-aminogroup of the lysine residues of albumin was achieved by reacting an excess of the saccharide isothiocyanate derivative (177 pmol) dissolved in the same buffer at pH 9.0 to 1 pmol albumin dissolved in the same buffer. The relative amounts used for the formation of the saccharide-albumin glycoconjugates were optimized, based on the premise that the highest degree of substitution of the lysyl residues in albumin by the dOclA-l-(6-isothiocyanato)hexane alkylamine derivative was achieved at a molar ratio of ligand/individual lysyl group of about 3 : 1 [8]. Using this ratio, the dOclA-albumin and N-acetylneuraminic-acid albumin glycoconjugates were obtained; virtually all of the 59 available lysine residues were derivatized. For the 0-specific-polysaccharide - albumin glycoconjugates of A . hydrophila and A . salmonicida approximately zyxwvutsrqpo a-D-Glcp (35%) 1 1 4 X-D-GIC~ 1 1 3 OAc (75%) 1 4 [+4)-a-Rhap-(l-t3)-P-~-ManpNAc-(l]~~ -core oligosaccharide 1+dOclA The native 0-specific polysaccharide of A . salmonicida has a core oligosaccharide containing a terminal dOclA reducing residue. The conjugation of t h s 0-specific polysaccharide was performed essentially under the conditions used for A . hydrophila (see above). 10 mol polysaccharide/mol albumin was introduced, using the same conditions. The chemical composition of the glycoconjugates was determined by measurement of protein and carbohydrate content, and molecular mass, as described in Materials and Methods. The degree of conjugate substitution was also confirmed by the amount of unconiugated lysine residues, as indicated by amino acid analysis. This deFormation qf the saccharide - albumin glyconjugates gree of substitution was not immoved when the ratio of the Covalent attachment of the 1-(6-isothiocyanato)-hexane igand to individual lysyl groupwere changed to 1 : 1, 2: 1, or alkyldmine derivatives of dOclA and N-acetylneuraminic acid 5 :1 . z zyxwvu 655 0) 0 - SPECIFIC POCYSACCHARIM b) Q- SPECIFIC POLYSACCHAROE LIPID A CORE OLIWSACCHARIDE DEACYLATED LIPID A I1 NH,ICH,l,NH2,NaBH,CN zyxwvutsrqpo zyxwvut zyxwvutsrqponmlk zyxwvutsrq 21 csc12 CHNH (CH,I, N = C= S C) ~I ~ C I F POLYSACCHARN I C OL IGOSACCHARIDE I zyxwvutsrqpo zyxwvuts zyxwvutsrq d) Q - SPECIFIC POLYSACCHARIM CORE DEACYLATED LIPID A OL IGOSACCHARIDE 0 Fig. 2 . Formation of modifird-lipopolysaccharide -albumin conjugate. ( a ) Intact lipopolysaccharide; (b) partially deacylatcd and selcctively oxidized lipopolysaccharide (i.e. modified lipopolysaccharide); (c) 1-(6-isothiocyanatoz)-hexane alkylamine dcrivative of modified lipopolysaccharide; (d) modified-lipopolysaccharide-albumin conjugate. BSA = bovine serum albumin Preparation of the 1- (6-isothiocyanato)-hexaneulkylumine derivative of purtially 0-deacylated oxidized V. anguillarum lipopolysaccharide and its albumin glycoconjugute V. anguillarum, a gram-negative bacterium belonging to the family Vibrionaceae, is an important pathogen of marine and estuarine fish, causing the hemorrhagic septicemia vibriosis. The chemical structure of the 0-specific polysaccharide of the phenol-soluble cellular lipopolysaccharide is as follows [12], where R is a propionyl group. the core oligosaccharide of A . hydrophila chemotype I [27] directly to albumin by the method of McBroom et al. [13], without the introduction of the spacer molecule. This method proved to be unsuccessful. We have established that only the terminal non-reducing LD-heptosyl residue of the inner region of the core oligosaccharide of V . unguillarum would be susceptible to a selective rapid periodate oxidation yielding a D-mannosyl residue possessing an aldehyde group at C-6 (see Methods). It thus became essential to modify the complete V . anguillurum B-~-Quip3NAc-(l[ -+~)-P-L-Q~~~~NAC-(~+~)-~-L-QU~~~NAC-(~-] 4 2 t OMe 7 1 a-~-Quip2NAc 314 T R The inner region of the core oligosaccharide of V. anguillurum, which is essentially identical to that of the various A. hydrophila chemotypes [25-281, is composed of a doubly branched L-glycero-D-manno-heptose substituted by other sugar residues in thc following manncr. L-a-D-Hepp 1 1 6 -+ 3)-L-a-D-Hepp-(l +dOclA 4 T 1 ~t-~-GlcpN-(l+7)-~-a-~-Hepp Although the inner region of the core oligosaccharide of V . anguillurum contains a 2-amino-2-deoxy-~-ghcoseresidue having a free amino group, which conceivably could react with thiophosgene and then act as a coupling site in formation of the glycoconjugate. We have therefore attempted to couple lipopolysaccharide in order to introduce an aldehyde group at C-6 of the terminal non-reducing Lu-heptosyl residue in the inner-core region. This free aldehyde group could then be conjugated to the spacer 1,6-hexanediamine. It is known that lipopolysaccharides are capable of eliciting pathophysiological toxic effects, known as endotoxic effects [29], which are caused by the lipid A portion and that partially deacylated lipid A and lipopolysaccharide samples are considerably less toxic than the natural preparation by a factor of lo3 [29]. Therefore, it was logical to use a partially deacylated oxidized lipopolysaccharide for conjugation to the spacer 1,6-hexanediamine for potential preparation of an albumin glycoconjugate. Hence, the partially 0-deacylated V. anguillurum lipopolysaccharide was selectively periodateoxidized and the product was reacted with an excess of 2,6hexanediamine in the presence of sodium cyanoborohydride at pH 8.0 (Fig. 2). The resulting modified 1-(6-arnino)-hexane alkylamine lipopolysaccharide derivative was purified and converted into the isothiocyanate derivative as described for the other saccharides. This derivative was then covalently 656 zyxwvutsrqp zyxwvu 6 has been shown that immunization with the purified O-antigen is rarely successful as this fraction is usually nonimmunogenic due to it small molecular size [33]. In order to render the polysaccharide immunogenic, it has to be covalently linked to a suitable larger carrier molecule 1321. We have suggested that in lipopolysaccharides of the Vibrionaceae family there is only one dOclA residue (in contrast to enterobacterial lipopolysaccharides) which connects the native polysaccharide to the lipid A [lo]; it has been established that this residue exists in the furanose form in a mutant of Arromonas salmonicida and is substituted through C-6 [ll]. The novel synthesis of glycoconjugates presented in this paper is tailor-made for the various 0-specific antigens derived from Vibrionaceae lipopolysaccharides. It is based on the introduction of the 1,bhexanediamine to the only terminal reducing dOclA residue of the core oligosaccharide portion of the native 0-specific polysaccharides. The anticipated success of this synthesis of glycoconjugates was based on experimental results of covalent attachment by reductive amination of simple ketose monosaccharides such as dOclA and N-acetylneuraminic acid to the spacer 1,6-hexanediamine. To our knowledge, this spacer has not previously been used for glycoconjugate syntheses. It was originally uscd for the covalent attachment of an active-sitedirected inhibitor of human leukocyte elastase to the microspheres of human albumin as a potential therapeutic agent for emphysema [34]. It has been shown by Lemieux et al. [35, 361 that the introduction of a bridging arm, composed of a polymethylene chain between the haptenic polysaccharide and the protein carrier, produced antisera that mainly recognized the epitopes of the polysaccharide portion of the glycoconjugate. One of the primary amino groups of the 1,6-hexanediamine spacer was introduced in the dOclA terminal-reducing residue of the core oligosaccharide portion of the native 0-chain polysaccharide of A . hydrophila and A . salmonicida, under very mild conditions at pH 8.0 by reductive amination. After subsequent conversion to the corresponding polysaccharide-isothiocyanate derivatives, they were covalently attached to the free ,+amino group of lysine residues of the carrier protein bovine serum albumin. The resulting glycoconjugates were immunogenic and elicited both anti-haptenic and anti-albumin antibodies in rabbits. It has been shown that free aldehydic groups can be used successfully in the conjugation of bacterial capsular polysaccharide to proteins as potential human vaccines [36]. It was therefore reasonable to expect that a similar modification could be introduced in lipopolysaccharide molecule. It has also been known for quite some time that deacylated lipid A and lipopolysaccharide are considerably less toxic than the parent preparation [29] and it is anticipated that conjugation of deacylated lipopolysaccharide to a protein carrier will break down the endotoxic conformation of these molecules [29], rendering them considerably less toxic and making them more amenable for potential vaccine use. The V . anguillarum lipopolysaccharide was partially 0-deacylated with mild alkali and modified by selective oxidation of periodate-sensitive terminal core heptose. Attempts at direct covalent attachment of this modified lipopolysaccharide to albumin by reductive amination were unsuccessful ; however, this can be circumvented by the introduction of the spacer 1,6-hexanediamine to the freed aldehyde group obtained (see Fig. 2). As mentioned earlier, the 0-specific polysaccharide of V. anguillarum is a short heteropolymer of a main chain of zyxwvutsrqpo zyxwvutsrqp zyxwvutsr zyxwvutsrq zyxwvu Fig. 3 . Passive hemag~hrtinatiun ritres of' sera to V. anguillarum lipupol~,.sac.c'liuri~~ and lipopol~saccharide-albumin conjugate. The titres of V. mnguillarurn SJ40T lipopolysaccharide (filled blocks) and its albumin conjugale (shaded blocks) wcre measured against (A) antiserum to V. anguillarum SJ40T lipopolysaccharide and (B) antiserum to whole cells of V. anguillarurn SJ-43 attached to the protein carrier bovine serum albumin (Fig. 2); the molar ratio of ligand/individual lysyl group was 1.7:l. It was calculated that in this modified-lipopolysaccharide albumin glycoconjugate an average of 14 molecules modified lipopolysaccharide/moleculealbumin was introduced. Immunological properties of the saccharide-albumin glycoconjugates The Q-specific-polysaccharide - albumin glycoconjugates of A . h-ydrrophila and A . salmonicida and the modifiedlipopolysaccharide - albumin glycoconjugate of V . anguillarum were used as immunogens in rabbits; the antisera were evaluated by immunodiffusion and passive hemagglutination. The immunodiffusion experiments with the respective saccharide-albumin glycoconjugates indicated that these conjugates produced antibodies that gave precipitin lines against their homologous lipopolysaccharides. Passive hemagglutination experiments were performed with the respective antisera to the various saccharide-albumin glycoconjugates and their alkali-treated homologous lipopolysaccharides. In rabbits immunized with the individual saccharide-albumin glycoconjugates of A . hydrophila and A . salmonicida, reasonable titres of 320 and 1280, respectively, were obtained. In the modified-lipopolysaccharide - albumin glycoconjugate of V. anguillarum, a titre of 640 was obtained. Comparative hemagglutination experiments carried out with antisera to V . anguillarum SJ-40T lipopolysaccharide and the whole cell of a different strain, V . anguillarum SJ-43, showed that the antigens of V. anguillarum SJ40T lipopolysaccharide and its modified albumin glycoconjugate had exactly the same titres (see Fig. 3). zyxwvu DISCUSSION The significance and severity of bacterial fish diseases caused by various species of the Vibrionaceae family have increased with the development and expansion of commercial aquaculture [30]. These ventures are increasingly relying on vaccination for disease control. The crude empirical approach using whole cells of killed bacteria as vaccines has prompted the development of novel methods directed toward usage of the cell-surface lipopolysaccharide antigen as a vaccine. It has been shown that Vihrio anguillarum lipopolysaccharide protects fish against vibriosis infections [31]. The protective value of such vaccines has long been attributed to the anti-0-antigen moiety 1321; it z zyxwvutsrq zyxwvutsr 657 zyx 13. McBroom, C. R., Samanen, C. H. & Goldstein. I. J. (1972) Methods Enzymol. 2NB, 212-219. 14. Shaw, D. H. & Squires, M. J. (1984) FEMS Microhiol. Lett. 24. 277 - 280. 15. Shaw, 1).H., Lee, Y.-Z., Squires, M. J. & Luderitz, 0. (1983) Eur. J. Biochem. 131, 633 -638. 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1 951) J . B i d . Chcvn. 193, 265 - 275. 17. Dubois, M., Gilles, K. A,, Hamilton, J . K . , Rebers, P. A. & Smith, F. (1956) Anal. Chem. 28, 350-356. 18. Hopwood, J. J. & Robinson, H. C . (1973) Biochem. J. 135,631 637. 19. Svenson, S. B. & Lindberg, A. A. (1979) J. Immunol. Methods 25, 323 - 335. 20. Neter, E . (1956) Bactrriol. Rev. 20, 166-188. 21. Ouchterlony, 0. (1963) Prog. Allergy6, 30-154. 22. Danielson. S. J. & Gray, G. R. (1986) Glycoconjugate J . 3, 363377. 23. Makhlouf, S. E., Davis, L. E., Deepika, P. &Anderson, B. (1986) Glycoconjugate J . 3,351 - 362. 24. Lee, H. S., Sen, L. C., Clifford, A. J., Whitaker, J. R. & Feeney, R. E. (1979) J . Agric. Food Chem. 27, 1094- 1098. 25. Banoub, J. H., Michon, F., Shaw, D. H. & Roy, R. (1984) REFERENCES Carhohydr. Rus. 128,203- 21 6. 26. Banoub, J. H., Choy, Y.-M., Michon, F. & Shaw, D. H. (1983) 1. Stowell. C. P. & Lee. Y. C. (1980) , , Adv. Curhohvdr. Chem. BioCarhohydr. Res. 114, 267 - 276. chem. 37, 225 -281. 27. Michon, F., Shaw, D. H. & Banoub, J. H. (1984) Eur. J . Biochem. 2. Lee, C.-J. (1987) Mol. Immunol. 24, 1005-1019. 145, 107-114. 3. Aplin, J. D. & Wriston, J. C., Jr (1981) C R C Crit. Rev. Biochem. 28. BdnOUb, J. H. & Shaw, D. H. (1981) Ccirhohydr. Res. Y8, 9310,1259 - 1306. 103. 4. Gray, G. R. (1974) Arch. Biochem. Biophys. 163,426-428. 5. Schwartz, B. A. & Gray, G. R. (1977) Arch. Biochem. Biophys. 29. Galanos, C., Luderitz, O., Rietschel, E. T. & Westphal, 0. (1977) Int. Rev. Biochem. 14,239-335. 181, 542-549. 6. Kamicker, B. J., Schwartz, B. A., Olson, R. M., Drinkwitz, D. 30. Akazawa, H. (1968) Bull. Jpn Soc. Sci.Fish. 34, 240-246. 31. Abe, P. M. (1973) M. Sc. Thesis, Oregon State University. C. & Gray, G. R. (1977) Arch. Biochem. Biophys. 183, 39332. Miller, J . M., Spilsbury, J. F., Jones, R. J.. Roe, E. A. & Lowbury. 398. 7. Kleinhammer, G., Himmelspach, K . & Westphal, 0. (1973) Eur. E. J. L. (1977) J. Med. Microhiol. 10, 19-27. 33. Vande Wiel, P. A,, Witvlict, M. H., Evenberg, D., Derks, H. J. J . Immunol. 3, 834-838. G. M. & Coen Beuvcry, E. (1987) Vaccine 5 , 33-37. 8. Roy, R., Katzenellenbogen, E. & Jennings, H. J. (1984) Can. J . 34. Martodam, R. R., Twumasi, D. Y., Liener, I. E., Powers, J. C., Biochem. Cell B i d . 62, 270 - 215. Nishimo, N. & Krejcarek (1979) Proc. Natl Acad. Sci. USA 76, 9. Banoub, J. H. & Hodder, H. J. (1985) Cun. J . Biochem. CeN B i d . 63, 1199- 1205. 2128 -2132. 35. Lemieux, R. U., Baker, D. A. & Bundle, D. R. (1975) J . Am. 10. Banoub, J. H., Shaw, D. H. & Michon, F. (1983) Carhohydr. R e x 123, 117-122. Chem. Soc. 97,4076-4083. 11. Shaw, D . H., Squircs, M. J., Ishiguro, E. E. & Trust, T. J. (1986) 36. Lemieux, R. U.. Raker, D. A. & Bundle, D. R. (1977) Cun. J . Eur. J. Biochem. 161, 309-313. Biochem. 55, 507-512. 12. Banoub, J. H., Michon, F. & Hodder, H. J. (1987) Biochem. Cell 37. Jennings, H . J. & Lugowski, C. (1981) J . Immunol 127, 1011B i d . 6.5,19- 26. 1018. (1 + 4)-linked 3-acetamido-3,6-dideoxy-~-~-glucose alternately substituted through 0 - 2 with side-chain residues of 2-acetamido-2,6-dideoxy-~-glucose, which are extremely acidlabile. The mild conditions used in the present coupling procedure allowed the conjugation of this acid-sensitive modified lipopolysaccharide to the macromolecular protein carrier. In addition, this method does not grossly affect the immunogenic specificities of the carrier protein as the resulting glycoconjugates eluted as a single peak in gel chromatography, indicating that no cross-linking or major alterations of the carrier protein had occurred [19]. To our knowledge, this is the first instance where a lipopolysaccharide of any sort has been covalently conjugated to a carrier protein. It is anticipated that the 0-specific-polysaccharide- albumin and modified-lipopolysaccharide - albumin glycoconjugates may be used as potential fish vaccines and that the antisera produced will possess good bacteriostatic activity against the homologous organisms. zyxwvutsrqp zyxwvutsrqp zyxwvutsr zy zyxwvutsrqp