INFECTION AND IMMUNITY, Feb. 1990, p. 373-377 Vol. 58, No. 20019-9567/90/020373-05$02.00/0Copyright © 1990, American Society for Microbiology

Synthesis and Characterization of Escherichia coli 0180-Polysaccharide Conjugate Vaccines

S. J. CRYZ, JR.,'* A. S. CROSS,2 J. C. SADOFF,2 AND E. FURER'Swiss Serum and Vaccine Institute, CH-3001 Bern, Switzerland,' and Walter Reed Army

Institute of Research, Washington, D.C. 20307-51002

Received 28 July 1989/Accepted 30 October 1989

Nontoxic, serologicaily reactive 0 polysaccharide was derived from Escherichia coli 018 lipopolysaccharideby acid hydrolysis, extraction with organic solvents, and gel filtration chromatography. Oxidized 0polysaccharide was covalently coupled to either Pseudomonas aeruginosa toxin A or cholera toxin by usingadipic acid dihydrazide as a spacer molecule in the presence of carbodiimide. The resulting conjugates werecomposed of approximately equal amounts of 0 polysaccharide and protein and were nontoxic andnonpyrogenic. Both conjugates engendered an immunoglobulin G antibody response in rabbits that recognizednative 018 lipopolysaccharide. Such antibody was able to promote the uptake and killing of an E. coli 018strain bearing the Kl capsule by human polymorphonuclear leukocytes. Immunoglobulin G isolated from thesera of rabbits immunized with either conjugate afforded protection against an E. coli 018 challenge whenpassively transferred to mice.

Escherichia coli is the leading cause of bacterial noso-comial infections (16, 35, 42). The introduction of broad-spectrum antibiotics with in vitro activity against E. coli hashad a negligible impact upon mortality rates, evidenced bythe fact that E. coli bacteremia carries an attendant mortalityrate ranging from 10 to 30% (2, 22, 37, 39, 42). In addition,the incidence of E. coli nosocomial infections does notappear to be abating in the face of infection control mea-sures.Both capsular (K) and lipopolysaccharide (LPS) (0) anti-

gens are important E. coli virulence factors (4, 5, 20, 28).Both structures can confer serum resistance, a characteristicwhich potentiates the ability of E. coli to invade and persistin the bloodstream. Several investigators have shown thatanti-capsular and anti-LPS antibodies afford protectionagainst experimental E. coli infections (6, 17, 18, 27). Alimited number of both 0 and K types causes the majority ofE. coli septicemias, making both antigens potential vaccinecandidates (4, 20, 24, 25). However, there are two majordrawbacks to the use of E. coli capsular antigens as humanvaccine candidates: (i) approximately 40% of bacteremicisolates cannot be capsule typed; and (ii) the Ki and K5antigens, which account for more than 20% of K-typableblood isolates, are poorly immunogenic due to antigenicrelatedness to mammalian glycosaminoglycans.We have shown that 0 polysaccharide (0-PS) derived

from Pseudomonas aeruginosa LPS, when covalently cou-pled to either tetanus toxoid or toxin A (ToxA), yieldsconjugates that are safe and immunogenic in humans (8, 10,11). Cholera toxin (CT) has also been found to serve well asa carrier for small-molecular-weight, nonimmunogenic mol-ecules (29, 32). In the present study, we describe thesynthesis and characteristics of E. coli 018 O-PS-ToxA andO-PS-CT conjugate vaccines. 018 LPS was chosen to serveas a prototype conjugate after which conjugates of otherserotypes could be modeled, because it is frequently ex-pressed by E. coli blood isolates. Conjugates produced witheither of these proteins were found to be nontoxic for

* Corresponding author.

animals, immunogenic, and capable of eliciting a protectiveantibody response.

MATERIALS AND METHODSBacterial strains and growth conditions. E. coli 205 (O

serotype 018:K nontypable) and strain 133 (06;K53) arehuman blood isolates provided by A. Brauner, KarolinskaHospital, Stockholm, Sweden. Strain 205 produces a cap-sule, as evidenced by the presence of acidic polysaccharidereleased into the growth medium. E. coli BORT is an018:K1 bacteremic isolate from the Walter Reed ArmyInstitute of Research culture collection. The strains werestored at -70°C in a 10% solution of skim milk. Cultureswere not passaged more than four times, because this resultsin a loss of 0 antigen. Cultures were grown on Trypticasesoy broth (BBL Microbiology Systems, Cockeysville, Md.)supplemented with 1% (vol/vol) glucose.

Isolation of LPS and O-PS. Cultures for LPS isolation weregrown in an 80-liter fermentor (Giovanola AG, Monthey,Switzerland) with high aeration and agitation. Stationary-phase cultures were harvested by centrifugation and washedin Tris-NaCl buffer (0.05 M Tris-0.3 M NaCl [pH 9]). LPSwas extracted by the hot phenol-water method of Westphalet al. (40). The extracted crude LPS was dialyzed exten-sively against distilled water. LPS was pelleted by centrifu-gation at 100,000 x g for 3 h and suspended in Tris-NaClbuffer containing 0.1 M MgSO4 (pH 7.5). RNase and DNase(Boehringer Mannheim AG, Mannheim, Federal Republic ofGermany) were added to a final concentration of 20 ,ug/ml.After incubation for 3 h at 37°C with gentle shaking, 200 jxgof pronase (Boehringer) per ml was added. This solution wasstirred for 16 to 18 h at 4°C. The LPS was pelleted bycentrifugation at 100,000 x g and suspended in distilledwater. This procedure was repeated twice, and the final LPSpellet was suspended in distilled water and lyophilized. LPSpurified in this manner contained less than 2% (wt/wt)protein or nucleic acids.O-PS was obtained from LPS by hydrolysis in dilute acetic

acid. LPS (approximately 1 g) was suspended in 220 ml of a1% (vol/vol) acetic acid solution. The solution was placed ina glass vial with a sintered glass top and heated for 150 min

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374 CRYZ ET AL.

at 100°C. After cooling, the material was centrifuged, and thelipid-A-containing pellet was discarded. The supernatantwas then extracted three times with an equal volume ofchloroform-methanol (3:1, vol/vol). The O-PS-containingaqueous phase was concentrated by rotary evaporationunder reduced pressure. The concentrate was passedthrough a 5- by 40-cm AcA 34 column (LBK Produkter,Bromma, Sweden) equilibrated in distilled water. The pyro-gen-containing material, which eluted in the void volume,was discarded, and fractions with a molecular weight ofc50,000 were pooled, concentrated, sterilized by passagethrough a 0.22-,um-pore-size filter, and lyophilized. O-PSprepared in such a manner was nonpyrogenic (see below)when administered intravenously to rabbits at a dose of 5,ug/kg of body weight.

Purification of CT and ToxA. CT was purified from theculture supernatants of Vibrio cholerae 569B as previouslydescribed (14). ToxA was purified as described by Cryz et al.(9).

Biological assay for CT, ToxA, and their respective conju-gates. The biological activity of CT and of the O-PS-CTconjugate was determined in a Y-1 adrenal cell assay (13).Endpoints are expressed as the smallest quantity of materialnecessary to result in rounding of250% of cells. The activityof ToxA and of the O-PS-ToxA conjugate was tested in aCHO cell assay (8).

Synthesis of conjugates. Lyophilized O-PS (60 mg) wasreconstituted in 12 ml of distilled water. Solid NaIO4 (258mg) was added, and the reaction was allowed to proceed at22°C for between 2 and 20 min. The oxidation reaction wasstopped by the addition of ethylene glycol. The mixture wasconcentrated by rotary evaporation under reduced pressure,and the oxidized O-PS was separated from other reactantsby filtration over Sephadex G-25 (Pharmacia, Uppsala, Swe-den). The degree of oxidation was determined by quantitat-ing reducing sugars by the ferric ferricyanide method of Parkand Johnson (26) with glucose as a standard. The percentageof sugars oxidized for 018 O-PS after 2, 5, 10, and 20 minwas 65, 78, 83, and 100%, respectively.

Adipic acid dihydrazide (ADH) was coupled to ToxA orCT as previously described (8). The degree to which ToxAwas substituted with ADH on a molar ratio was 12:1,whereas that for CT was 8:1. Carrier protein-ADH wasdissolved in 50 mM phosphate buffer (pH 7) at a finalconcentration of 2 mg/ml. An equal amount of oxidized O-PS(2 mg/ml) was added and incubated at 22°C for 1 h.NaCNBH3 was added to a final concentration of 10 ,umol andwas incubated at 22°C until it became opalescent. Themixture was extensively dialyzed against phosphate-buffered saline (PBS; pH 7.4) and applied to a SephadexG-100 column. Void volume fractions, which contained theconjugates, were collected.ELISA. A stock solution of LPS was prepared by dissolv-

ing 5 mg of LPS in 1 ml of 36 mM triethylamine in distilledwater. This solution was stored at 4°C. Microdilution plates(Immunolon; Dynatech, Buchs, Switzerland) were coated byplacing 150 ,ul of a 10-,ug/ml LPS solution (diluted from thestock solution) in PBS (pH 7.2), followed by incubation at37°C for 2 h. The plates were stored at 4°C and washed threetimes with PBS-T (PBS containing 0.02% [vol/vol] Tween20) just before use. Sera to be tested were serially diluted(starting at a 1:10 dilution) in PBS-T, and 150 ,ul was addedto each well. The plates were incubated at 37°C for 1 h andwashed three times with PBS-T. A 1:5,000 dilution ofperoxidase-labeled goat anti-rabbit immunoglobulin G (IgG;Nordic Immunological Laboratories, Tilburg, The Nether-

lands) in PBS-T was added (150 [lI per well). The plates werethen incubated for 90 min at 37°C and washed three times inPBS-T, and 150 ,ul of substrate solution (13) was added perwell. The plates were incubated for 10 to 30 min at roomtemperature, A405 was measured with a Titertek Multiscan(Flow Laboratories, Inc., Hamden, Conn.). Titers are ex-pressed as enzyme-linked immunosorbent assay (ELISA)units, which were calculated by multiplying the reciprocal ofa serum dilution within the linear portion of the dilutioncurve (A405, 0.4 to 1.0) by the corresponding A405 value.

Immunogenicity studies. New Zealand White rabbits (2 to2.5 kg) were immunized intramuscularly with 50 pug of O-PS(as a conjugate) in 0.5 ml of PBS on days 0 and 14. Serumsamples were obtained on days 0, 14, and 28.

Safety and pyrogenicity studies. The toxicity of the conju-gate vaccine was evaluated by the intraperitoneal adminis-tration of 50 and 500 ,ug per mouse and guinea pig, respec-tively, as described under article V.2.1.5 of the EuropeanPharmacopoeia (12). For pyrogenicity studies, rabbits (NewZealand White, 2 to 2.5 kg) were administered graded dosesof antigen intravenously. Temperatures were recorded at30-min intervals for 4 h postinfusion. A negative responsewas less than a 0.3°C rise in temperature at any given timepoint.

Quantitative analysis. The protein content of the conjugatevaccine was determined by the method of Lowry et al. (19).The carbohydrate content was measured by the tryptophanereaction (41) with purified E. coli 018 O-PS as a standard.

Isolation of IgG. IgG was purified from pooled rabbitserum samples as described previously (23). The IgG wasconcentrated approximately threefold by suspending thefinal precipitate in PBS equal to 33% of the original volumeof serum. This concentrate was filter sterilized and stored at-200C.Opsonophagocytosis assay. Opsonophagocytic assays were

preformed in microdilution plates as previously described(31). The BORT strain of E. coli (018;K1) was used. The testsera had to be stored frozen, so fresh human serum was usedas a complement source. The assay mixture, which con-tained 106 E. coli BORT cells and 106 human polymorpho-nuclear leukocytes per ml and a 10% final concentration oftest serum, was incubated for 120 min at 37°C with gentleshaking.

Protection studies. For passive protection studies, NMRImice (female, 18 to 20 g, in groups of 8 to 10) each received0.2 ml of the rabbit anti-018 conjugate IgG preparation(equal to 3.8 mg of total protein) intravenously 8 to 20 hbefore challenge. Control mice received a similar amount ofpreimmune rabbit IgG. Two different challenge models wereused. In one model, graded doses of E. coli 205 weresuspended in 3.5% (wt/vol) hog gastric mucin (Sigma Chem-ical Co., St. Louis, Mo.) and injected intraperitoneally in 0.5ml. In the second model system, the gastric mucin wasomitted. Mortality rates were recorded for 5 days postchal-lenge. All deaths occurred within 72 h of challenge. The 50%lethal dose was determined by the method of Reed andMuench (30).

High-pressure liquid chromatography. A SI 300 Polyolcolumn (Serra Fine Biochemicals, Heidelberg, Federal Re-public of Germany) was used to determine the molecularweight of O-PS, CT, ToxA, and the conjugates (resolutionrange, 6 x 103 to 6 x 105 daltons). The elution profile wasmeasured at 280 and 220 nm with a Uvikon 735LC detector(Kontron Instruments, Zurich, Switzerland) coupled to aSpectra-Physics SP 4290 integrator (Spectra-Physics, SanJose, Calif.).

INFECT. IMMUN.

E. COLI 018 O-PS CONJUGATE VACCINES 375

TABLE 1. Characteristics of E. coli 018 0-PS conjugates

Composition(% dry Molecular Biological Pyro-dConjugate weight) wta Toxicityb activityc genicityd

(%) (pLg/kg)O-PS Protein

0-PS-CT 43.4 56.6 >600,000 Nontoxic <0.01 >50-PS-ToxA 50 50 >600,000 Nontoxic <0.01 >5

a Determined by high-pressure liquid chromatography.b Mice and guinea pigs each received 50 or 500 p.g, respectively, of vaccine

intraperitoneally. No overt signs of toxicity were observed.c Expressed as percent activity of native CT or ToxA as determined in the

Y-1 adrenal and CHO cell assay systems, respectively.d The administration of 5 p.g of conjugate per kg of rabbit body weight

evoked less than a 0.3°C rise in temperature.

Statistics. Significance was determined by chi-square anal-ysis.

RESULTSLipid A and trace quantities of native LPS were removed

from acetic acid-treated LPS by extraction in organic sol-vents and gel filtration chromatography. The final 0-PSpreparation was nonpyrogenic when intravenously adminis-tered to rabbits at a dose of 5 jig/kg of body weight. Althoughserologically reactive, 0-PS failed to elicit an anti-018 LPSimmune response in rabbits when injected alone (100 jig perdose) or together with toxin A in an uncoupled state (datanot shown). This lack of immunogenicity is most likelyattributable to its low molecular weight (c50,000).The characteristics of two conjugates are shown in Table

1. Conjugates were composed of approximately equalamounts of 0-PS and carrier protein and possessed a molec-ular weight of >600,000, greater than that of CT (-94,000),ToxA (66,000), or 0-PS (.50,000) (Fig. 1). The conjugateswere nontoxic for mice and guinea pigs and displayed nodetectable biological activity for cultured cells. The conju-gates showed no increase in either toxicity or biologicalactivity after incubation at 37°C for 4 weeks. They were alsononpyrogenic for rabbits at a dose of 5 ,ug/kg of body weightwhen administered intravenously. In contrast, native E. coliLPS evoked an elevation in temperature (>0.7°C) whengiven at doses as low as 0.05 .i.g/kg.The ability of these two conjugates to elicit an anti-018

LPS antibody response was determined in rabbits (Table 2).Both conjugates engendered anti-018 LPS antibody afteradministration of a single dose of vaccine. A second immu-nization with the ToxA conjugate evoked a modest (twofold)rise in titer, whereas the response to the 0-PS-CT vaccinewas more pronounced (sevenfold rise in titer). Final anti-LPS antibody titers after two immunizations were compara-ble for both conjugates. Immunization also engendered a risein anti-carrier protein antibody (data not shown).The ability of rabbit anti-conjugate antibody to promote

the uptake and killing of an E. coli 018:K1 strain by humanpolymorphonuclear leukocytes was determined. Preimmunesera did not possess any detectable opsonic antibody. Themean percent kill was 93% (range, 85 to 98%) after immuni-zation with the 0-PS-CT conjugate and 83% (range, 81 to88%) for the 0-PS-ToxA conjugate. In most animals, peaklevels of opsonic antibodies were seen on day 28, whengreater than 80% of the initial bacterial inoculum was killed.Phagocytosis was complement dependent. No killing wasobserved when postimmunization sera were incubated withfresh serum serving as a complement source but in theabsence of human polymorphonuclear leukocytes.

E

C4

to0

aI

.0

0..0.4

A

B

0 10 20 30 40Retention Time (min)

FIG. 1. High-pressure liquid chromatography of ToxA, CT, 0-PS, and 0-PS-ToxA conjugates. (A) Elution profiles of ToxA (peak1), CT (peak 2), and oxidized 0-PS (peak 3). (B) Elution profile ofthe 0-PS-ToxA conjugate.

Next, the ability of anti-conjugate antibody to affordprotection against a lethal E. coli challenge was determined.IgG preparations were prepared from normal rabbits orthose immunized with either an 018 0-PS-ToxA, 0180-PS-CT, or 06 0-PS-ToxA conjugate. The passive trans-fer of IgG containing a high anti-018 LPS titer conferredsignificant protection (P < 0.05) against challenge with anencapsulated 018 strain of E. coli (Table 3). In contrast, ananti-06 IgG preparation was ineffective, demonstrating theserospecificity of protection in this model. This protectiveeffect was also evident when the challenge organism wasdelivered in mucin, where the mean lethal dose for micereceiving normal rabbit IgG was <2.5 x 102 E. coli 205bacteria, whereas for those receiving anti-018 0-PS-ToxAIgG it was 1.9 x 104 bacteria.Given the known importance of the Kl capsule in extra-

intestinal E. coli infections (4-6, 28), we next evaluated theability of anti-conjugate antibody to afford protection against

TABLE 2. Anti-E. coli 018 LPS IgG ELISA antibody responseafter immunization with conjugate vaccines

Geometric mean IgG ELISA units (range)Immunogen'

Preimmunization Day 14 Day 28

O-PS-ToxA <10 608 (228-1,570) 1,276 (918-1,987)O-PS-CT <10 110 (10-450) 710 (180-1,440)

a Three rabbits were immunized on days 0 and 14 with an amount ofconjugate equal to 50 p.g of O-PS.

L

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376 CRYZ ET AL.

TABLE 3. Protection afforded against E. coli 018 infection bypassive transfer of anti-conjugate IgG

IgG transferreda IgG ELISA titer Mortality pb018 LPS 06 LPS (

Normal rabbit <10 <10 90Anti-018 0-PS-ToxA 940 NDc 10 <0.01Anti-018 0-PS-CT 448 <10 20 <0.01Anti-06 0-PS-ToxA 66 669 80 >0.05

a Groups of 10 mice were used. Each mouse received 3.8 mg of rabbit IgGintravenously in 0.2 ml 20 h before challenge with 107 E. coli 205 (018:Knontypable) cells.

b p was determined by chi-square analysis by comparison of the mortalityrate for mice that received normal rabbit IgG with that in mice that receivedanti-conjugate IgG.

c ND, Not done.

an E. coli 018 strain bearing the Kl capsule (Table 4). Themortality rate for mice that received normal rabbit IgG was100%. In contrast, the passive transfer of anti-conjugateantibody reduced the mortality rate to 25% (P < 0.05).

DISCUSSION

Previous studies have demonstrated the ability of poly-clonal or monoclonal anti-LPS antibody to protect againstexperimental E. coli infections (17, 18, 27). The beneficialeffect of anti-LPS antibody is most likely mediated by itsopsonic potential, which acts to eradicate the invadingbacteria by phagocytic cells. Although most individualspossess naturally acquired antibody to several E. coli 0types, these levels do not appear to be protective (21). Onepossible means to bolster antibody titers is by the passivetransfer of intravenous immune globulins. Studies to deter-mine the levels of anti-E. coli opsonic antibody in severalnormal intravenous immune globulins indicate that mostpreparations would not be expected to routinely containsubstantial amounts of functional antibody to many E. coli 0types (1, 31, 38). Although it is effective against otherbacterial pathogens, the failure of a normal intravenousimmune globulin to protect against E. coli septicemia inpatients with chronic lymphocytic leukemia (3) indicates theneed for evaluating an intravenous immune globulin withdocumented increased levels of anti-LPS antibody.

In the present study, we describe the synthesis andcharacteristics of E. coli 018 O-PS-protein conjugates. Wechose to evaluate ToxA and CT as carrier proteins due toprior favorable experience (29, 32). Tetanus toxoid was notselected as a candidate carrier protein because (i) polysac-charide-tetanus toxoid conjugates were found to elicit anunacceptably high rate of adverse reactions in adults (33) and

TABLE 4. Protection afforded against E. coli 018;K1 by passivetransfer of anti-conjugate IgG

IgG transferreda Igo ELISA titer Mortality pbto 018 LPS (%

Normal rabbit <10 100Anti-018 0-PS-ToxA 940 25 <0.05Anti-018 0-PS-CT 448 25 <0.05

a Groups of 10 mice were used. Each mouse received 3.8 mg of rabbit IgGintravenously in 0.2 ml 8 h before challenge with 107 E. coli BORT, (018;K1)cells.

b P was determined by chi-square analysis by comparison of the mortalityrate for mice that received normal rabbit IgG with that in mice that receivedanti-conjugate IgG.

because (ii) high levels of preexisting anti-tetanus toxoidantibodies in adults tended to suppress the immune responseto the O-PS moiety of a P. aeruginosa O-PS-tetanus toxoidconjugate (11). The conjugates described in the presentstudy were of a high molecular weight, nontoxic, nonpyro-genic, and stable to toxic reversion at physiologic tempera-tures, traits which are desired in a candidate E. coli vaccine.These findings confirm those of previous studies showingthat ToxA and CT can be irreversibly detoxified by theintroduction of ADH followed by covalent coupling to apolysaccharide (8, 10, 32).

E. coli O-PS conjugates produced with either ToxA or CTwere equally immunogenic in rabbits. Of critical importancewas the finding that anti-conjugate antibody bound to native018 LPS and promoted the phagocytosis and killing of an018:K1 strain of E. coli by human polymorphonuclearleukocytes. Antibody engendered by these conjugates con-ferred excellent protection against fatal E. coli 018 experi-mental infections. It is important to note that the level ofprotection afforded was comparable for both an 018:non-K1and an 018:K1 strain of E. coli. This indicates that theknown protective effect of the Kl capsule can be overcomeby anti-LPS antibody.

Seroepidemiological studies indicate that an E. coli vac-cine based upon serospecific LPS antigens must be polyva-lent (4, 20, 24, 25). The fact that approximately 10 0serotypes account for roughly 70% of E. coli bacteremicisolates and that there does not appear to be significantgeographical variances makes such an approach feasible (4,20, 24, 25). Preliminary studies in our laboratory have shownthat conjugate vaccines can also be synthesized with E. coli04 and 06 O-PSs, which are nontoxic and able to engenderan anti-LPS IgG antibody response when parenterally ad-ministered to rabbits.The majority of nosocomial gram-negative bacillary septi-

cemia and pneumonia is caused by E. coli, Klebsiella spp.,and P. aeruginosa (2, 16, 22, 35, 37, 39, 42). The idealimmunotherapeutic or immunoprophylactic agent shouldoffer protection against all three of the above bacterialpathogens (7, 36). Multivalent Klebsiella spp. and P. aeru-ginosa vaccines are currently undergoing evaluations inhumans for safety and immunogenicity (10, 15). The possi-bility of combining these existing vaccines with an E. colicomponent is particularly attractive. One potential problemwith such an approach is the fact that the above-mentionedP. aeruginosa vaccine consists of O-PS coupled to toxin A.Therefore, the simultaneous administration of multivalent E.coli and P. aeruginosa vaccines, which both use toxin A asa carrier, could lead to an unacceptable high rate of adversereactions due to the large quantity of toxin A. Administeringthe two vaccines at different times to avoid this complicationmay result in a markedly reduced immune response to thesecond vaccine due to epitope-specific suppression mediatedby preexisting antitoxin A antibody (11, 34). The use of CTas the carrier protein for E. coli O-PS in such a situationshould circumvent this problem.

Studies are now in progress to evaluate and compare thesafety and immunogenicity of the E. coli 018 O-PS-ToxAand O-PS-CT conjugates described in this report in humans.

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