Passive protection of diabetic rats with antisera specific for the polysaccharide portion of the lipopolysaccharide isolated from Pseudomonas pseudomallei

LE BRYAN, S WONG, DE WOODS, DAB DANCE, W CHAOWAGUL. Passive protection of diabetic rats with antisera specific for the polysaccharide portion of the lipopolysaccharide isolated from Pseudomonas pseudomallei. Can J Infect Dis 1994;5(4) : 17 0 -1 7 8 . Polyclonal and monoclonal antisera raised to tetanus toxoid-conjugated polysaccharide of lipopolysaccharide (LPS) and purified LPS of Pseudomonas pseudomallei that reacted with a collection of 41 strains of this bacterium from 23 patients are described. The common antigen recognized by these sera was within U1e polysaccharide component of the LPS of the cells. The sera were specific for P pseudomallei in U1at none of 37 strains of other bacteria. including 20 Gram-negative and three Gram-positive species. were recognized . although cross-reaction occurred using the anticonjugate serum with some strains of Pseudomonas cepacia serotype A. a closely related bacterium. Passive protection studies using a diabetic rat model of P pseudomallei infection showed that partially purified rabbit polyclonal and mouse monoclonal antisera were protective when the median lethal dose was raised by four to five orders of magnitude. The wide distribution of the polysaccharide antigen among isolates of P pseudomallei used in this study and the protective role of antibody to U1e conjugated polysaccharide antigen suggest potential as a vaccine .

P SEUDOMONAS PSEUDOI\IIALLEI IS T ilE CAUSATIVE ORGAN - ism of melioidosis, a disease of both humans and animals.In recent years the incidence of melioidosis.which is most commonly found in SouU1easl Asia and northern Australia.has been found lo be higher than once considered (l ,2).The fu lminating septicemic form is probably only the most obvious manifestation of a disease spectrum varying from this e>..' 1rcme lo mild or subclinical forms of the disease.
Investigations in Thailand have shown that clinically apparent severe melioidosis is an important cause of morbidity and mortality in lhal country and is more widespread than appreciated until recenlly.11. seems likely that a majority of patients is asymptomatic after infection.and some may harbour the organism for many years.Clinically apparent infection may present as localized acute suppurative or chronic granu lomatous illness or septicemic disease from either a demonstrable or a nondemonslrable primary site.Pneumonic manifestations are common in severe disease.Severe disease is worse with certain risk factors .especially diabetes mellitus.bul oilier underlying diseases have also been detected (1.2).Recenlly neurological melioidosis has been described probably due lo exotoxinmediated pailiology in iliat iliere was absence of direct infection of the central nervous system (3).
The pattern of antimicrobial susceptibility of P pseudomalleihas been well defined.Chloramphenicol, doxycycline.tetracycline .kanamycin and lrimelhoprim-sulfamelhoxazole have been used for treatment.More recenlly ceflazidime.piperacillin.amoxicill in /clavulanic acid or imipenem-cilaslatin have been used.In vitro susceptibili ty studies show that imipenem is lhe most active of available drugs, with piperacillin, doxycycline.amoxycillin/clavulanic acid, cefixime.cefelamel.azlocillin and ceftazidime a lso being very active .
Untreated disseminated melioidosis has a mortality rate of close lo 90%.Antimicrobial therapy reduces mortality and improves outcome bulilierapeulic failure is still common (2,4).
No effective meiliod of prevention of melioidosis exists.We believe il is reasonable lo conclude U1al blood borne antibody would be potentially helpful because of ilie septicemic nature of severe disease.We therefore undertook to develop antisera to an antigenic preparation of P pseudomallei ilial wou ld react widely with strains a ti s ing from different palicnts and that wou ld prevent or reduce the severity of disease in infected animals.We report here ilic results of our studies lo provide such a preventive approach lo melioidosis.

Conjugation of PS to tetanus toxoid:
The method was ada pled from described methods (8-1 0).A solution of rs (5 mg/mL) equ ilibrated at 4 °C was rapidly brought to pH 10.5 with 0 .1 N sodium hydroxide.and 100 mg/mL cyanogen bromide was added to a final concentration of 0.4 mg/mL rs .The pH was maintained al 10.5 for 6 mins.after which the pH was brought to 8.5 with 0.5 M sodium bicarbonate.Adipic acid dihydrazide (20 mg) was added next and ilie mixture incubated at 4°C for 16 h.The mixture was dialyzed against 500 volumes of water for 48 h, after which the pH was adjusted with hydrochloric acid to 4 .7. Tetanus toxoid (10 mg) (Insliiul Armand Frappier) and l-elhyl-3-(3-dimethylaminopropyl) carbodiimide (100 mg) were added and lhe reaction mixture stirred for 4 h at 25°C.This conjugation mixture was thereafter dialyzed against 500 volumes of phosphate buffered saline (PBS) (pH 7.4) ovemight at 4 °C and lyophilized.The conjugated PStetanus toxoid was purified from LPS, PS and tetanus toxoid by Sephacryl S-500 column chromatograph (Pharmacia LKB Biotechnology).A volume of 0.55 mL conjugate mixture was loaded on a Sephacryl S-500 column with 28 mL bed volume at a f1ow rate of 0.5 mL 0.05 M phosphate buffer (pH 7.0)/min and 0.8 mL fractions collected.From the elution profile determined by protein assay (BCA Micro-protein Assay, Illinois) and carbohydrate test (11).a peak containing both protein and carbohydrate was collected.The pooled fractions were lyophilized and reconstituted with 1 mL sterile water for injection.Production of anticonjugate antibodies: A New Zealand White female rabbit was immunized by intravenous injection of 1 mL of 50 !lg/mLconjugate solution on days 1, 10 and 17.Blood was taken on day 24 and the serum was collected and tested using enzyme-linked immunosorbent assay (ELISA) with LPS.PS and tetanus toxoid as antigens.The rabbit was terminally bled on day 32, serum was collected and frozen in aliquots at -70°C.A 1. 5°C maximal temperature response occurred with the initial injection, suggesting, despite purification, some minor LPS contamination of this preparation.ELISA procedures: Round bottomed microtitre plates (Dynatech Laboratories Inc, Virginia) were coated with 5 11g bovine serum albumin, Pseudomonas aeruginosa 503 LPS.P aeruginosa serotype A and serotype E LPS.P pseudomallei LPS, P pseudomallei PS.PS-tetanus toxoid conjugate or tetanus toxoid in 100 11L of 0 .05M carbonate buffer, pH 9.6.Plates were incubated at 4°C overnight or 37°C for 1 h.The plates were washed three times in PBS containing 0.05% Tween-20.A blocking step was performed with 1% skim milk in PBS.One hundred microlitres of each of the serum dilutions were prepared in PBS with 1% skim milk and incubated in appropriate wells for 1 hat 37°C.After washing, 100 !lL of horseradish peroxidase conjugate antirabbit immunoglobulin (Ig) G was added and incubated for 1 hat 37°C.After washing, 100 11L per well of substrate ( 2-2' -azinodi[3-ethyl-benzthiazoline sulphonate]) (KPL Inc , Maryland) was added and colour allowed to develop for at least 10 mins before the absorbance at 405 nm was measured using a Bio-Tek Instrument Inc EIA au to reader model EL 310 (Mandell Scientific Co Ltd).Specificity ofthe antipolysaccharide reaction: Reaction of the antibody with a range of bacteria and LPS.PS and tetanus toxoid preparations was determined by ELISA and immunoblot assays.Bacterial cells were grown ovemight either in brain heart infusion broth or tryptic soya agar.The cells were washed and adjusted to 0.6 optical density 600 nm in PBS.One millilitre of cell suspension was collected in a microfuge tube.After the pellet was resuspended in 50 !lLPBS, the suspension was boiled for 10 mins at l00°C, and 25 11g of proteinase Kin a 10 !lL volume was added and incubated for 1 h at 60°C to ensure no residual contaminating P pseudomallei protein remained.The final volume was made up to 500 !lL with 0.5 M carbonate buffer (pH 9.6).One hundred microlitres of each antigen preparation was added per well of the ELISA plate and incubated at 4°C ovemighl.After washing and blocking as described above, 100 !lL of a 1:1000 dilution of antiserum was added per well and plates were incubated at 37°C for 1 h.Absorbance readings at 405 nm were determined after addition of peroxidase-anti-IgG conjugate and substrate as described above.The control was an identical dilution of normal rabbit serum collected from the same rabbit before immunization.
Immunoblots were performed in the following manner.Bacterial cells were adjusted to 0.6 optical density 600 nm in saline and pelleted in microfuge tubes.The ce lls were washed once in saline and resuspended in 50 !lLsample buffer (4% sodium dodecyl sulphate [sos].10% glycerol, 5% 2-mercaptoethanol and trace of bromophenol blue in Tris buffer, pH 6.7).The samples were boiled for 10 mins at l00°C followed by addition of proteinase K (25 11g dissolved in 10 11L sample buffer) and DNAase ( 10 11g dissolved in 10 !lL sample buffer) to each san1ple.The samples were incubated for 60 mins at 60°C.Tetanus toxoid (40 11g protein per well), LPS and PS (40 !lg/well) and the whole cell LPS preparations were loaded onto a 15% sos-urea polyacrylamide gel.and electrophoresis was carried out (12).Gels were thereafter blotted on Immobilon transfer membrane 'Millipore PVDF' (Millipore Canada Ltd).Immunoassay was perfom1ed by initial blocking of membranes using 1% skim milk in Tris buffered saline (pH 7.4).followed by washing with Tris buffered saline.The antitetanus toxoid/PS antibody was used at.a 1:1000 dilution, incubated for 1 h at 3rC, washed with Tris buffered saline, treated with horseradish peroxidase conjugated antirabbit IgG (ICN lmmunoBiologicals, Illinois) for 1 h at 37°C and the substrate 4-chloro-1-naphthol (Bio Rad) added to allow colour development.

Absorption of the antitetanus toxoid/PS antiserum:
Purified LPS ( 1 !lgf!lL ) from P pseudomallei 304b was digested with proteinase Kat a concentration of 2 pg/ ~Lg of LPS.One microgram of the digested LPS was added to 20 pL of 1:2 and 1:20 diluted serum and incubated overnight at 4°C.The precipitate was removed by centrifugation using a microfuge at 12,718 g for 15 mins.Thereafter the ELISA procedure was used to detennine the reduction in titre using sodium hydroxide-lysed whole cell antigen preparations from P pseudomallei strain 304b and a final dilution of PS antiserum of 1:2000.Monoclonal antibody production: Polysaccharide antigens were from sos PAGE gels in an electroelutor (Tyler Research Instruments).The antigens were eJ\.'tensively dialyzed against distilled water to remove sos and sub- sequenlly lyophilized to concentrate the preparation.A BALB/C mouse (Simonsen Labs Inc, California) was immunized by initial intraperitoneal injection of 100 )lg of electroeluted antigen preparation in a 50 JlL volume and 50 )lg of N-acetylmuramyl-L-alanyl-D-isoglutamine (Sigma).The same quantity of antigen was injected intravenously once a week for three more weeks.Subsequently the mouse was sacrificed.the spleen removed and fusion performed using the NS-1 myeloma cell line (12).Monoclonal antibody MCA PS-Pp-W was identified by reaction with partially purified LPS of P pseudomallei on Western blot as well as by ELISA.The specificity of the monoclonal antibody for other bacteria was determined by reaction witl1 a variety of other bacteria.Agglutination reactio n: Bacterial cells (50 )lL of 2 .0A6oo suspension /mL in PBS) were suspended on a glass slide and an equal volume of undiluted serum was mixed with the cells.A positive agglutination result could be visualized within 1 min.A control was performed with normal rabbit serum.

Bactericidal activity o f the immune serum:
The method was adapted from previously desc1ibed methods (13,14).Anti-PS rabbit serum used in this assay was stored at -70°C in aliquots.and the serum was never refrozen.Bacteria were grown overnight or until midlogarithmic growth phase in brain heart infusion broili.then washed and resuspended in saline to a concentration of 10 7 colony-forming units (CFU)/mL.The saline suspension was diluted 10-fold in Hank's buffered saline solution (Gibco) containing 0.1 % (W /V) gelatin (Sigma).At time zero.ilie bacterial suspension (10 6 CFU/ mL) was diluted 10-fold by adding il to a tube Figure 2) Elution profile from a Sephacryl S -500 coLumn of carbohydrate readiue material from purified and hydrolyzed lipopolysaccharide coryugated to tetanus toxoid.A562 Reading in protein assay containing Hank's buffered saline solution with 0 .1% gelatin and 10% (V /V) anti-PS rabbit serum and 10% (V /V) guinea pig complement (ICN ImmunoBiologicals).Control tubes contained the same reaction miA1:ure, but with normal rabbit serum and without serum.Tubes were incubated at 37°C, and at various intervals samples were taken, serially 10-fold diluted in saline, and 0.1 mL aliquots were plated in duplicate onto tJypticase soya agar plates for viable counts.Animal pro tection s tudies: Three separate experiments were perfom1ed using an animal model of septicemic P pseudomallei infection in diabetic rats (15).Forty-five male Sprague-Dawley rats with an average weight of 50 g were used for each experiment.Animals were given intraperitoneal injections of either PBS or streptozotocin (Sigma, 80 mg/kg body weight for each of two consecutive days) to induce diabetes.Urine glucose levels were monitored (Diastix, Miles Canada, Inc) to assess ilie onset of diabetes.Diabetic animals were given 10 mU/g body weight NPI-1 insu lin (Connaught Novo) intraperitoneally when urine glucose levels reached 160 mmol/L.Three days post insulin injection , groups of 15 animals (five nondiabetic-nonimmunized, five diabetic-nonimmunized and five diabetic-immunized) were inoculated intrapeiitoneally with 10 4 , 10 5 or 10 6 P pseudomallei strain 316c organisms.Passive immunization was achieved by intraperitoneal injection of sterile IgG (anti-PS serum) or IgM (monoclonal antibody) partially purified by ammonium su lphate precipitation ( 1.5 mg in 0.1 mL PBS) at the time of inoculation (16).Nonimmunized animals received intraperitoneal injections of 0.1 mL PBS.The experimental end-point was death and median lethal dose (LD5o) values were determined using th e method of Reed and Muench (17).

LPS patterns of P p s e udomallei strains:
F igure 1 shows the LPS staining patterns of 12 strains of proteinase K-digested whole cells of P pseudomallei using the method of Hitchcock and Brown (18).This set consists of strains isolated from six different patients (365 , 392.316.305.415 and 443) w1th mu ltiple isolations from each patient taken early and late in the course of antimicrobial therapy (signified by the lower case letters) (Figure 1) .The pattern in all cases was that of a smooth LPS , w1th most higher molecular weight bands clustered in a group in the upper gel consistent w1th long chain LPS.The results of these gels are also consistent w1th a core and lipid A region to the LPS.

Preparatio n of P p s e udomallei PS c o njugat e d to t e t anus tox oid and an t iserum:
LPS was purified from P pseudomaLLei strain 304b, the PS removed following acid hydrolysis and the PS conjugated to tetanus toxoid as described above.Figure 2 s h ows the elution profil e from a Sephacryl S-500 column of chromatographed material previously subjected to the conjugation procedure as described.A fraction representing larger molecular weight mater ial in a small protein peak immediately preceding the main protein peak of tetanus toxoid and which contained carbohydrate (fractions 13 to 1 7) was selected for inj ection into the rabbit.No other PS was detected.indicating that all PS was conjugated due to the gross excess of tetan u s toxoid.The serum obtained after three injections (32 days after the initial injection) was termed anti-PS serum and used for a ll subsequent assays and tests other than those using monoclonal antibody.Specificity of anti-PS serum: Anti-PS serum at two different dilutions was absorbed w1th proteinase Ktreated LPS of P pseudomalle i 304b, as described above , to confirm that specificity was not directed to any protein impurity in the antigen preparation.Reactivity w1th 304b antigen was then detem1ined relative to untreated serum and after absorption w1th proteinase K-treated LPS.A control containing only proteinase K was used in the event this treabnent in itself lowered the titre through proteolysis of antibody.Results of absorption studies (Table 1) showed a dose response in that a substantially greater reduction in ELISA activity resulted after LPS absorption of the more dilute than of the less dilute a ntiserum preparation .The anti-PS serum reacted w1th a diffuse region in the higher molecular band region of either LPS extracted from P pseudomallei or of proteinase K-digested whole cells on immunoblots, also consistent w1th a reaction to LPS (Figure 3. left).No immunoblot reaction was detected in the region of LPS gels where lipid A was eA.rpected to migrate.Further, a monoclonal antibody reacting w1th proteinase K-treated whole cells and LPS of strain 304b gave a very similar b lot reaction w1th P pseudomatlei strains (Figure 3, right) .The anti-PS serum gave a strong reaction w1th tetanus toxoid.tetanus toxoid -Ps conjugate, purified preparations of the hydrolyzed PS and LPS from P pseudomatlei 304b in ELISA reactions.No reactivity was found w1th LPS from two serotypes of P aeruginosa (Table 2).
The preceding results argue strongly that the major reaction of the anti-PS serum was w1th PS components of LPS conjugated to tetanus toxoid.

Bacteria recognized by anti-PS s e rum:
A large series of proteinase K-treated whole cells of bacteria was examined for reactivity w1th anti-PS serum by the ELISA procedure.Results consistently showed that the only bacterial species recognized, with one exception in tl1e group tested, wasP pseudomatlei (Table 2).The exception was a group of several strains belonging to serotype A of P cepacia, a bacterium known to be closely related to P pseudomallei (19).Other serotypes of P cepacia did not react (Table 2 , Figure 3, left).Although only one example of serotypes other tl1an type A is shown in Table 2 , several isolates of each were tested.P cepacia serotype A strains consistently reacted by ELISA and in immunoblots (Figure 3 , left) with the anti-PS serum, but not with a monoclonal antibody (IgM) raised against P pseudomatlei LPS (Table 2, Figure 3, right).Jmmunoblot results for the two antisera against whole cell digests of P pseudomatleistrains 415b, 305a, 304b and 199a as well as isolates of P cepacia.Aeromonas hydro-phiLia and Yersinia enterocoLitica are shown in Figure 3. Whole cell digests of P aeruginosa serotype A, P aerugi- Salmonella typhimurium were a lso examined using both anti-rs serum and the monoclonal antibody.The results of both ELISA (Table 2) and immunoblot reaction (data not shown) were negative.
Interestingly every strain of P pseudomallei examined reacted with the anti-PS serum by the ELISA as well as the immunoblot method (Ta ble 2, Figure 3, left).Many of these strains were also examined with the monoclonal antibody by the ELISA and immunoblot reaction and all were positive (Table 2, Figure 3 .right).P pseudomallei NCTC 8708.230. 3 04b, 392a and 392f.and LPS extracted from P pseudomallei 304b were also positive by immunoblot assay using both antisera (data not shown).as well as by ELISA (Tabl e 2).In a ll.str ains from 23 different patients reacted positively by ELISA as did all of the multiple isolates from the same patient (Table 2).Animal protection studies: Streptozotocin-induced diabetic rats were found to be susceptible to P pseudomallei 316c.a different strain from that used to isolate the LPS for antibody.LD5o values for P pseudomallei in streptozotocin-treated rats were markedly reduced compared with control animals (Table 3) .In three separate experiments the LD5o was raised to greater than 1x10 8 organisms by injection of the anti-rs serum and to greater than lxl0 8 organisms in two e:>..rperiments by injection of the monoclonal antibody.These studies clearly showed that.und er these test condit ions, pass ive administration of anti-PS or monoclonal antibody directed to an LPS antigen of strain 304b was protective for another P pseudomallei strain .
Bactericidal and agglutinating activity: It was not possible to show any bactericidal activity of the an U-Ps serum or monoclonal antibody against the homologous strain 304b .Anti-PS serum agglutinated several strains of P pseudomallei but the monoclonal antibody did not.

DISCUSSION
Several important findings emerge from this series of investigations.There was apparently only a single serotype of LPS recognized by the anti-PS serum or the monoclonal serum raised against a prototype strain of P pseudomallei.Strains originated from 23 different patients indicating that a single serotype is widespread in this group.There could be several reasons for this .The patients cou ld represent a cluster.There is no evidence to support the view that a single strain spread among the different patients.Strains arose from different individuals and locations a t different times with no communality among the patients .Preliminary ribotyping studies a lso indicate that these stra ins do not represent a single clone (unpublished data).However. it is possible tl1at a single serotype exists in the Thailand region from which these isolates originated, and that other types exist elsewh ere or in lower numbers and were not detected in this series.
Another interpretation is that there is only one serotype of P pseudomallei.Cu rrent serologica l procedures do not discriminate serotypes (2,20).Two serotypes reported earlier (21) apparently reflect smooth and rough LPS variation and not two serotypes (unpublished data).The indirect h em agglutination (IHA) test a pparently recognizes a rs antigen that is likely to be th e LPS .Using this test.Atthasampunna et a! (22) found that 15% of Thais had a ntibody toP pseudomallei.Embi et   A second finding is that the antigen recognized by the monoclonal serum is also recognized by the anti-PS serum.All of our results are consistent with this being a repealing side chain ofthe LPS structure.Further.this antigen is widely distributed in P pseudomallei strains but is not found in a wide variety of other Gram -negative and some Gram-positive bacteria.The only exception to this is the occurrence of this antigen in most of the serotype A isolates of P cepacia examined .The last observation is highly consistent with the close relationship be tween these two bacteria by a variety of methods (5).Furthermore.some simila rity in structure of LPS from these two bacteria in the inner core region was recently reported (25).Monoclonal antibody did not react with P cepacia isolates.suggesting that there is more than one epitope included in the LPS structure and recognized by the anli-PS serum .It re mains an unlikely possibi lity that the latter serum recognizes a minor non-LPS epitopc found in both P cepacia and P pseudomallei.a lthough our results support the view that most activity is to an LPS antigen .
A third and very important outcome is that a vaccine directed towards the PS portion of the LPS could pote ntially be important in control of melioidosis.This view is supported by the widespread distribution of the protective e pitope or epilopes in P pseudomallei strains.the capability to conj ugate the PS to a protein carrier.and eviden ce for protection in an an imal model using both polyclonal and monoclonal sera.Results from IliA testing suggest that rs is recognized in natural infections and an tibodies arc produced against it.It is conceivable that such antibody is a major factor in controlling the frequency and severity of melioidosis.There is no definite evidence to confirm eith er of th ese specu la tions.but current findings are cons istent with such an interpretation.The animal model used in our stud ies may not be an idea l model for melio idosis.but no generally accepted an imal model exists.At least two aspects of the strcptozotocin-induccd diabetic rat mode l are consistent with human disease .The disease is more severe in compromised patients including those with diabetes.leukemia.other malignancies.chronic rena l and liver failure.alcoholism.malnutrition and other similar examples (2).Secondly.severe human disease has a bacteremic component to it.as does the diabetic rat model.In these studies antibody was given simultaneously with the infecting dose of bacteria and was highly protective.Similarly.in actively immunized humans.a ntibody is expected to present at the time of exposure to the bacterium .Therefore.while not absolutely predictive.our protection studies argue strongly th at antibodies to the same e pitope or epitopes in huma ns a re likely protective .At this lime we do not have any inform a tion on the possible mechanism of protection.except that we have been unable to detect bactericidal activity.Clearly the passively supplied antibody could be e nha ncing phagocytosis.We are pursing more complete mechanistic understanding using human studies where we feel U1ese fmclings will ha ve more clefmite relevance.
A potential problem with a vaccine developed from LPS is toxicity.We have not examined this aspect of the conjugated an tigen in d e tail.However.past experience indicates that toxicity is expected to be minor.As noted above .we did observe a temperatu re rise in a rabbit injected with the conjugate.Th e major probl em in e limina ting some degree of pyrogenicity would be in purifying the vaccine adequately to ensure that contamination with LPS is below biologically s ignificant levels.Toxicity assoc iated with the PS and the carrier tetanus toxoid is expected to be small based on results with other conjugated protein -Ps vaccines (10).A major attraction of this putative vaccine antigen is its prese nce in all strains of P pseudomalle i examined to elate .an uncommon finding for LPS antigens of Gram-negative bacteria.The cross-reaction with P cepacia could be an added advantage because this organism is an opportunistic pathogen .Both organisms have produced disease in cystic fibrosis patients (26.27).and the a ntigen may be a potential vaccine cand idate for that group of patients .
CAN J INFECT Dis VOL 5 No 4 JULY 1 AuGUST 1994

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control normal rabbit serum; all examinations were the overage of three or more experiments.0 indicates ~0.05.ELISA Enzyme-linked immunosorbent assay; FH Foothills Hospital strain collection; LPS Lipopolysaccharide; NO Not done CAN J INFECT D1s VOL 5 No 4 JuLv 1 AuGusT 1994