Bioconjugate Chem. 1990, 1, 375-375
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Synthesis and Characterization of Gentiobiose Heptaacetate Conjugate Vaccines That Produce Endotoxin-Neutralizing Antibodies1p2 Apurba K. Bhattacharjee,'J Jerald C. Sadoff,t Hugh Collins,t Alan S. Cross,t Ali H. Khalil,' Marcia M. Bieber,t Craig Wright,$ and Nelson N. H. Tengt Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, DC 20307-5100, Cancer Biology Research Laboratory, Stanford University School of Medicine, Stanford, California 94305, and Univax Biologics, Inc., Rockville, Maryland 20852. Received July 23, 1990
We have prepared aminoethyl (AE), aminopropyl (AP), and aminopentyl (APT) derivatives of gentiobiose heptaacetate (GH). These spacer compounds (AEGH, APGH, APTGH) have been coupled to succinylated diphtheria toxoid (SuceDT) to produce conjugate vaccines. These conjugates all bind to the anti-lipid A human monoclonal antibody A6(H4C5)in an ELISA binding assay. Rabbits immunized with the APGH conjugate vaccine in either Freund's complete adjuvant or aluminum hydroxide gel produced antibody levels of 5120 and 3600 ELISA units, respectively, campared to an antibody level of less than 20 ELISA units for the prebleed sera. Sera from mice immunized with either the aminopropyl or the aminopentyl conjugate had antibody levels of 5120 and 2560 ELISA antibody units, respectively. These antibodies neutralized endotoxin in a Limulus lysate neutralization assay. Protection against the local Shwartzman reaction was demonstrated (p 0.05) in eight out of nine rabbits immunized with the SuoDT-APGH conjugate vaccine compared to three out of 10 rabbits immunized with the carrier protein SuoDT. Passive transfer experiments demonstrated that four out of five rabbits receiving immune serum were protected from Shwartzman reaction compared to one out of five rabbits receiving normal serum (p < 0.1). These results indicated that epitopes contained in gentiobiose heptaacetate when properly presented as conjugate vaccines were capable of inducing neutralizing antibodies against endotoxin.
Endotoxin is the causative agent for shock in Gramnegative bacteremia. It has been estimated that septic shock is responsible for about 30 000 deaths in the United States every year ( 1 , 2). The problem of treatment is compounded by the increase in antibiotic resistance in these organisms ( 3 , 4 ) . Attempts have been made to develop vaccines to protect against endotoxic shock. Immunization of animals with Gram-negative bacilli protects them against infection with homologous organisms (5-7). Rough mutants of Gramnegative bacilli contain LPS determinants in their core portion that are shared by most Gram-negative bacteria (8, 9). Immunization with LPS of some rough mutants such as the Re mutant of Salmonella and the 55 (Rc) mutant of Escherichia coli have been shown to provide cross-protection against challenge with heterologous organisms (10-14). Ziegler et al. (15)have shown that antisera obtained from healthy human volunteers immunized with the 55 mutant lowered mortality of patients from Gram-negative bacteremia and endotoxic shock. Teng et al. (16)have developed a human IgM monoclonal antibody, A6(H4C5), against a 55 LPS epitope. This antibody was shown t o react t o a wide variety of Gram-negative
* To whom correspondence should be addressed. t
Walter Reed Institute of Research.
* Stanford University School of Medicine.
Univax Biologics, Inc. The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense. In conducting the research described in this paper, the investigators adhered to the G u i d e f o r Laboratory A n i m a l Facilities and Care as promulgated by the committee of the guide for laboratory animal facilities and care of the institute of 8
laboratory animal resources, National Research Council.
organisms in ELISA and with a wide variety of isolated LPS, including a strong reaction with lipid A. In the mouse mucin model of lethal bacteremia this antibody showed significant protection against lethal challenge with Pseudomonas aeruginosa, E. coli, and Klebsiella pneumonia, but not against the Gram-positive organism Streptococcus pneumonia (16). Protection against the local Shwartzman reaction induced by E. coli and Klebsiella endotoxin was also demonstrated with this monoclonal antibody. In a recent report DeMaria et al. (17) showed that immunization of human volunteers with vaccines prepared from unheated and boiled Salmonella minnesata R595 (Re chemotype) produced antibodies that showed enhanced protective activity in mice against challenge with viable Gram-negative bacilli and endotoxin. Bieber et al. (Bieber, M. M., Ozguc, M., and Teng, N. N. H., unpublished results) have shown that the binding between human anti-J5 monoclonal A6(H4C5) and lipid A could be inhibited (98%) with gentiobiose octaacetate (a structure similar to the core disaccharide present in lipid A), compared to 80% inhibition with @-glucosepentaacetate and no inhibition with galactose pentaacetate. These results indicated that a 1,6-linked @-glucosedisaccharide was required for the antibody binding and that acetate groups fulfilled the requirements for acyl substitution a t C2 and C3. We decided to prepare a synthetic vaccine using gentiobiose heptaacetate as the epitope and linking it to carrier proteins through spacer arms of different lengths. In this report we describe the preparation of such conjugate vaccines and the immunogenicity of these vaccines in rabbits and mice. Antisera raised in rabbits were tested for endotoxin-neutralizing activity
1043-1002/90/2901-0375$02.50/0 0 1990 American Chemical Society
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Bioconjugate Chem., Vol. 1, No. 6, 1990
with 55 LPS and for protective activity against local Shwartzman reaction with E.coli 0 6 LPS and E.coli 0111: B4 LPS. EXPERIMENTAL PROCEDURES
2-Aminoethanol, 3-aminopropanol, 5-aminopentanol, gentiobiose octaacetate, silica gel, TLC plates, and 1,4dioxane were purchased from Sigma Chemical Co. (St. Louis, MO). Diphtheria toxoid and tetanus toxoid were obtained from Massachusetts Biologic Laboratories (Boston, MA). Chloroform, dichloromethane, and ethyl acetate were purchased from Aldrich Chemical Co. (Milwaukee,WI). Phosphatase-labeled anti-human, antirabbit, and anti-mouse immunoglobulins were purchased from Kirkegaard and Perry Laboratories (Gaithersburg, MD). HBr (33%)in glacial acetic acid was purchased from Fluka Chemical Corp. (Hauppage, NY). Succinic anhydride and l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) were purchased from Pierce Chemical Co. (Rockford, IL). Melting points were recorded with a Fisher Johns melting point apparatus and are corrected. Preparation of Acetobromogentiobiose. Gentiobiose octaacetate (I; 1 g) was dissolved in dichloromethane (2.5 mL) and 33% HBr in glacial acetic acid (5 mL) was added to this solution. After 45 min at 5 "C, dichloromethane (30 mL) and ice water (30 mL) were added. The organic phase was separated, successively washed with aqueous sodium hydrogen carbonate solution and water, dried (sodium sulfate), and evaporated to dryness. AcetobromogentiobioseI1 was crystallized from diethyl ether. The crystalline compound gave a single spot on TLC in ethyl acetateln-hexane (2:l) moving slightly faster than gentiobiose octaacetate. Preparation of 2-Aminoethyl2,3,4,2',3',4',6'-Hepta0-acetylgentiobioside (AEGH). The acetobromogentiobiose (284 mg) was dissolved in dry chloroform (5 mL), and Drierite (0.5 g) was added followed by 2-aminoethanol (0.2 mL in 0.8 mL of chloroform). The reaction mixture was stirred in the dark at room temperature for 16 h and filtered and the filtrate was washed with ice water (2 X 10 mL), dried (sodium sulfate), and evaporated to dryness. The solid was dissolved into dichloromethane (0.9 mL) and AEGH was purified by chromatography on silica gel using ethyl acetate/ethanol(19:1) as the eluant. The purified AEGH was crystallized from hot ethanol (yield 41 '4 ), mp 176-177 "c. Anal. Calcd for C28H40018N:c , 49.56; H, 5.94; N, 2.06. Found: C, 49.44; H, 6.05; N. 2.10. 3-Aminopropyl2,3,4,2',3',4',6'-hepta-O-acetylgentiobioside (APGH) was synthesized the same way as described above except that 3-aminopropanol was used in place of 2-aminoethanol (yield 62 % ). The crystalline APGH had mp 166-167 "C. Anal. Calcd for C29H42018N: C, 50.25; H, 6.06; N, 2.02. Found: C, 50.31; H, 6.21; N, 1.90. 5-Aminopentyl2,3,4,2',3',4',6'-hepta-O-acetylgentiobioside (APTGH) was synthesized the same way with 5-aminopentanol as the aglycon (yield 40%). The crystalline APTGH had mp 145-146 "C. Anal. Calcd for C31I146Old: C, 51.67; H, 6.43; N, 1.94. Found: C, 51.57; H, 6.49; N, 1.93. Succinylation of Diphtheria Toxoid. Diphtheria toxoid (DT) solution (10 mL, 8.0 mg of protein/mL) was dialyzed against 1L of 0.05 M NaCl at 5 "C for 16 h. Succinic anhydride (60 mg) was added to the dialyzed DT slowly with stirring during 2 h while the pH was maintained between 8 and 8.5 by adding small amounts of 3 M NaOH solution. The reaction mixture was dialyzed against three changes of water (3 X 1 L) in 24 h and then lyophilized to yield 70 mg (88%). Estimation by the TNBS reagent (18) showed the presence of less than 1%primary amine.
Bhattacharjee et al.
Preparation of Conjugate Vaccine. Succinylated diphtheria toxoid (SuoDT, 50 mg) was dissolved in 20% dioxane (10 mL) and EDC (200 mg) was added to it followed by a solution (8 mL) of APGH (25 mg) in 20% dioxane. The pH of this reaction mixture was maintained between 5.5 and 5.9 by intermittent addition of 0.1 N HC1 for 1 h at room temperature. A second 200 mg of EDC was added and the reaction continued for one more hour. The reaction mixture was dialyzed extensively against several changes of distilled water for 2 days at 5 "C and was then freeze-dried (yield 32 mg, 61 % ). The conjugate SueDT-APGH was found to contain 5.25% APGH by the phenol sulfuric acid method (19). Several batches of conjugate vaccines were prepared the same way as described above. SueDT-APTGH was prepared the same way as described above. The reaction sequence is shown in Scheme I. Tetanus toxoid (TT)was coupled to APTGH by the same procedure to give the conjugate TT-APTGH. Enzyme Linked Immunosorbent Assay (ELISA). The ELISA was performed in 96-well flat-bottom polystyrene microtiter plates (Costar, Cambridge, MA) essentially by the method of Engvall and Perlmann (20). The wells were coated with the conjugate TT-APTGH at a concentration of 10 pg/mL in PBS (0.01 M NaH2P04, 0.14 M NaC1,0.02% NaN3, pH 7.4) at 37 "C for 3 h. Excess reactive groups were blocked with 1%casein (Fisher Scientific, Columbia, MD) in PBS at 37 "C for 1 h. The wells were washed with PBS between steps to remove unbound material. The antigen-coated plates were incubated with serial 2-fold dilutions of antibodies for 16 h at room temperature (25 "C). Incubation with the second antibody was done for 16 h at room temperature. Disodium p-nitrophenyl phosphate (Sigma Chemical Co.) at a concentration of 1 mg/mL in 1.0 M diethanolamine buffer (with 1 mM MgClz), pH 9.8, was used as substrate. Absorbance was read on a Dynatech plate reader (Dynatech Laboratories, Alexandria, VA) at 410 nm. Antibody units were calculated as the mean of the product of the optical density (in the range of 0.3-1.0) and the reciprocal serum dilution at three different 2-fold dilutions. SDS-PAGE and Western Transfer. Sodium dodecy1 sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli (21) as described previously (22). Western transfer of electrophoretically separated glycoproteins to nitrocellulose membrane (Bio-Rad Laboratories, Richmond, CA) was performed in a transblot apparatus (Bio-Rad)as described before (22). Immunization of Rabbits. New Zealand white rabbits, obtained from Hazelton Research Products (Denver, PA) (2-2.5 kg), were injected intramuscularly with SucvDTAPGH (lot no. IIIA-103, 100 pg) in Freund's complete adjuvant. Booster injections were given with the conjugate (100 pg) in saline. Four New Zealand white rabbits (22.5 kg) were immunized with SuoDT-APGH (lot no. IIIA171,100 pg) mixed with aluminum hydroxide gel with a gel to protein ratio of 20:l (w/w). The first booster injection was given with the same adjuvant. The subsequent injections were give with SuoDT-APGH (50 pg) in saline. Immunization of Mice. One group of C57 black mice (5-6 weeks old), obtained from Charles River Laboratories (Wilmington,MA), were immunized intraperitoneally with Suc-DT-APTGH (lot no. V-13, 10 pg) mixed with aluminum hydroxide gel with a gel to protein ratio of 20:l (w/w). A second group of C57 black mice (five mice in a group) were immunized the same way with Suc-DTAPGH (lot no. IIIA-171). The first booster injections were
Bioconjupte Chem., Vol. 1, No. 6, 1990 377
Gentiobiose Heptaacetate Conjugate Vaccines Scheme I.' Synthesis of hepta-0-acetyl-gentiobiose conjugate vaccines
Ro*::w CHZOR
ACOH nor
OR
I1
I
Bf
1 I
2
HO-CH2-(CH2)n-CH2
-NH2
CHCI3
Ro+$L&% CH2
O-CH2-(CH2)n-CH2
/
CHZOR
'
O
W
$
-NHp
RO
I
2
3
20%
DIOXANE
I11
EDC 0 P-t-OH
% O-CHZ-(CHZ)lr-CHZ-NH-C
Iv
0 I -P
-L3
R n
=
0, lor 1
P
1
CARRIER PROTEIN
a I, Gentiobiose octaacetate; 11, acetobromogentiobiose;111, aminoalkylhepta-0-acetylgentiobioside; IV, carrier protein conjugate of (aminoalky1)hepta-0-acetylgentiobiose.
given the same way as the primary immunization. The subsequent injections were given with conjugate (10 pg) in saline. Production of the Local Shwartzman Reaction. The skin sites in 1.5-kg New Zealand white rabbits were prepared by intracutaneous injection of 0.1 mL of endotoxin (E.coli 0 6 LPS, or E. coli Olll:B4 LPS prepared by the hot phenol extraction method) in saline containing 0.1 mg of LPS. The reaction was provoked 23 h later by injection of 0.1 mL of endotoxin (0.025 mg) solution into the marginal ear vein. For the active immunization experiment, nine rabbits were immunized with Suc-DTAPGH vaccine as described in the methods, and 10 rabbits were immunized with the carrier protein SueDT the same way to be used as the control. For the passive protection experiment, 15 mL of immune serum (2500 ELISA units/mL) was given intravenously to each of the test rabbits and 15 mL of normal rabbit serum was given intravenously to each of the control rabbits 2 h before the provocative dose. Any hemorrhage or necrosis of the skin occurring after the provocative dose was recorded as a positive reaction. Preparation of APGH-CoupledAffinity Adsorbent. Affigel-15 (Bio-Rad Laboratories, Richmond, CA) was washed three times with ethanol to remove additives. APGH (10 mg) was taken into ethanol (2 mL) when it gave a suspension. 1,4-Dioxane was added to a final concentration of 20% when the solution became clear. This solution was added to 2 mL of washed Affigel-15 and the mixture was shaken for 4 h at room temperature. The gel was filtered, washed once with ethanol, suspended into 0.1 M tris-C1 buffer, pH 7.4, and shaken for another 4 h to block excess active groups. The gel suspensionwas filtered, washed with PBS, pH 7.4, and packed into a column. Affinity Chromatography. The Affigel-15-APGH
column (2 mL) was washed with 0.05 M NaCl (10 mL). Immune rabbit serum (1.0 mL) was cycled (3X) through the column. The column was then washed with PBS until the OD of the effluent was
