INFECTION AND IMMUNITY, Sept. 1984, p. 537-5430019-9567/84/090537-07$02.00/0Copyright © 1984, American Society for Microbiology

Protection of Mice Against Vibrio vulnificus Disease by Vaccinationwith Surface Antigen Preparations and Anti-Surface Antigen

AntiseraARNOLD S. KREGER,* LARRY D. GRAY, AND JACQUELINE TESTAt

Department of Microbiology and Immunology, The Bowman Gray School of Medicine of Wake Forest University,Winston-Salem, North Carolina 27103

Received 21 March 1984/Accepted 21 May 1984

Vaccination of mice with either Formalin-killed cells or cell extracts of a virulent strain and a weakly virulentstrain of Vibrio vulnificus or with rabbit antisera (AS) against the Formalin-killed cells and cell extractsprotected against the virulent strain of V. vulnificus. However, the virulent strain vaccines and AS elicited a

significantly stronger immune response than the weakly virulent strain vaccines and AS. Adsorption of the ASwith either the homologous or heterologous Formalin-killed cells significantly reduced the ability of the AS toprotect mice. The major protective antigen(s) in the cell extracts migrated in the void volume of Sephacryl S-400 superfine, was not effectively sedimented by centrifugation at 100,000 x g for 2 h, had an isoelectric pointof 3.8 to 4.2, and was sensitive to boiling or autoclaving for 15 min, periodate oxidation, and exposure to pH 12but was resistant to 56°C, trypsin, pronase, RNase, neuraminidase, and pH 4.5. Electron microscopy revealedthat the virluent strain possessed a more dense ruthenium red-staining layer on its outer membrane and had a

much smoother surface than the weakly virulent strain. Our results provide evidence that a major protectiveantigen and virulence factor of V. vulnificus is a heat-labile, acidic polysaccharide located on the bacterialsurface.

The halophilic bacterium Vibrio vulnificus, previouslycalled "lactose-positive (L+) Vibrio" and Beneckea villni-fica (1, 9, 13), causes acute, fulminating wound infectionsand septicemia in humans (2-4, 7, 8, 12-14, 16, 21, 22, 25, 31,33). In addition, mice experimentally infected by subcutane-ous injection with V. vulnificus exhibit extensive edema andtissue necrosis at the initial site of infection and a rapidlydeveloping, fatal septicemia (5, 26) similar to that observedduring human disease. Putative virulence factors producedby V. vulnificlus include extracellular cytolysin, proteases,and siderophores (18, 28, 29; M. M. Carruthers and W. J.Kabat, Abstr. Annu. Meet. Am. Soc. Microbiol. 1981, B59,p. 24; M. D. Poole, J. H. Bowdre, and D. Klapper, Abstr.Annu. Meet. Am. Soc. Microbiol. 1982, B155, p. 43). Inaddition, Kreger et al. (17) proposed that virulence is depen-dent, at least in part, on antiphagocytic surface antigen(s)and, therefore, that surface antigen preparations be evaluat-ed for immunogenic activity against V. vulnificus disease. Inthis report, we describe the ability of various surface antigenpreparations and anti-surface antigen antisera (AS) to immu-nize mice against fatal experimental disease caused by V.vulnificus.

MATERIALS AND METHODSBacteria and growth conditions. A virulent strain (E4125)

and a weakly virulent strain (A1402) of V. vulnificus werekindly supplied by R. E. Weaver and D. G. Hollis of theCenters for Disease Control (Atlanta, Ga.). Strain E4125 hada 50% lethal dose (LD50) value for 6- to 8-week-old (ca. 25- to30-g), Dub:(ICR) strain, randomly bred albino mice (Domin-ion Laboratories, Inc., Dublin, Va.) of ca. 5 x 106 CFUinjected subcutaneously (s.c.). Strain A1402 had an LD50value for mice of >2 x 109 CFU. Frozen specimens of V.

* Corresponding author.t Present address: Swiss Institute for Experimental Cancer Re-

search, 1066 Epalinges, Switzerland.

vulnificus and seed culture suspensions were prepared as

previously described (18), except the seed cultures weregrown on Columbia agar (BBL Microbiology Systems,Cockeysville, Md.) rather than on Columbia agar containing5% sheep blood. Flask cultures were prepared by inoculating2-liter flasks containing 200 ml of heart infusion broth (DifcoLaboratories, Detroit, Mich.) with 1 ml of seed culturesuspension (ca. 1010 CFU) and incubating the inoculatedmedium for 7 to 8 h at 30°C on a gyratory shaker operating at220 cycles per min.

Killed cell vaccines. The bacteria obtained from one flaskculture, or from several Columbia agar cultures grown asdescribed for electron microscopy studies, were collected bycentrifugation (10,000 x g, 15 min, 4C), washed twice withsterile phosphate-buffered saline (PBS; 0.02 M Na2HPO4-HCl-0.15 M NaCl [pH 7.4]), and suspended in 50 ml of PBS.The number of CFU per ml of suspension (usually ca. 5 x1010 CFU/ml) was determined by plate counts, and thebacteria in 10-ml portions of the suspension were killed byexposure to (i) 0.8% (vol/vol) Formalin for 1 day at ca. 25°C,(ii) 56°C for 15 min, (iii) 100°C for 15 min, and (iv) autoclav-ing (121°C) for 15 min. The heat-treated suspensions were

cooled to room temperature, adjusted to contain 0.8% For-malin, and kept for 1 day at ca. 25°C. The vaccines were

stored at 4°C, and 1-ml portions were diluted with sterile PBS,before being used in the active immunization studies, tocontain the number of bacteria equivalent to ca. 5 x 109CFU/ml.

Cell extracts. The bacteria obtained from four flask cul-tures were washed twice with sterile PBS and suspended inPBS to a final volume of 80 ml. The suspension was mixedfor 20 s in a Waring commercial blender (model 7011S;Waring Products Div., Dynamics Corp. of America, NewHartford. Conn.) adjusted to the low setting, and the super-natant fluids were obtained by centrifugation (24,000 x g, 20min, 4C), followed by membrane filtration (pore size, 0.2,um). The results of plate counts performed before and after

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mixing the washed bacteria in the blender indicated that thetreatment did not cause detectable loss of bacterial viability.The virulent strain cell extract (CE) was further fractionated,in some experiments, into an insoluble or sedimentablefraction and a soluble or nonsedimentable fraction by cen-trifugation at 100,000 x g for 2 h.

Antisera. Rabbit antisera (AS) against Formalin-killedcells (FKC) of V. vulnificus E4125 and A1402 were obtainedas previously described (17). AS had agglutination titers of500 to 1,000 when tested against the homologous and heter-ologous FKC by a tube agglutination procedure (11). ASagainst the CEs of the two strains were obtained by vaccinat-ing New Zealand white rabbits s.c. on day 0 with 1.2 ml of anemulsion composed of equal parts of CE and Freund com-plete adjuvant and on day 28 with 1.2 ml of an emulsioncomposed of CE and Freund incomplete adjuvant. Therabbits were exsanguinated 2 weeks after the second vacci-nation, and AS were lyophilized. AS had agglutination titersof 1,000 to 2,000 against the homologous and heterologousFKC. In some experiments, 5-ml samples of heat-inactivatedAS were adsorbed twice with the homologous or heterolo-gous FKC as previously described (17).

Assays. The assay for protective antigen (PA) activity wasperformed by determining the ability of the preparations toactively immunize 6- to 8-week-old (ca. 25- to 30-g),Dub:(ICR) strain, randomly bred albino mice against fatal V.vulnificus disease. Groups of mice (10 mice per group) wereinjected intraperitoneally on days 0, 3, 6, and 9 with 0.2-mlportions of the preparations being examined, and the micewere challenged s.c. on day 16 with 100 or 10 LD50s of V.vulnificus E4125. The percent survival values were deter-mined after observing the animals for 3 days postinfection.The ability of the rabbit AS to passively immunize miceagainst fatal V. vulnificus disease was determined by intra-peritoneal injection of heat-inactivated sera (0.5 ml) intogroups of mice (10 mice per group) 3 to 4 h before challengewith 100 or 10 LD50s of V. vulnificus E4125. The bacterialsuspensions used to challenge the mice in the active andpassive immunization studies were prepared by growing thebacteria in heart infusion broth (as described for the prepara-tion of the vaccines), washing the bacteria with PBS, andsuspending the bacteria to yield the required LD50s in 0.1 mlof PBS.The CEs were assayed for protein (crystalline bovine

albumin standard) by the method of Lowry et al. modifiedfor samples containing insoluble proteins (19), for neutralcarbohydrate (glucose standard) by the phenol-sulfuric acidprocedure modified by Nowotny (23), and for lipopolysac-charide endotoxin (Escherichia coli endotoxin standard) bythe Limulus amoebocyte lysate method with commerciallyavailable lysate (Pyrostat; Worthington Diagnostics, Free-hold, N.J.) as recommended by the manufacturer.Gel filtration. A 5-ml sample of CE from V. vulnificus

E4125 was applied to a water-cooled (5°C) column (2.6 by 66cm) of Sephacryl S-400 superfine (Pharmacia Fine Chemi-cals, Piscataway, N.J.) equilibrated with PBS and was elutedin the downward flow mode at a flow rate of ca. 16 ml/h withthe equilibrating buffer. Fractions (4 ml) collected at 5°Cwere pooled (4 fractions per pool), and the pools were

assayed for PA activity against 10 LD50s of V. vulnificusE4125. Blue dextran was used to estimate the column voidvolume.

Isoelectric focusing. A 3-mi sample of the soluble fractionof a CE obtained from V. vulnificus E4125 was dialyzed for18 h at 4°C against 8 liters of 0.02 M Na2HPO4-HCl (pH 7),and the solution was fractionated by high-speed electrofo-

cusing (20, 34) in a pH 3.5 to 10 glycerol density gradientformed at 15 W for 17 h with an LKB 8100-1 column (LKBInstruments Inc., Gaithersburg, Md.). The pH of each 4-mlfraction was determined at 4°C, the pHs of the fractions wereadjusted to pH 6 to 8, and the fractions were diluted with anequal volume of PBS (0.067 M Na2HPO4-0.077 M NaCl [pH7]) before being assayed for PA activity against 10 LD50s ofV. vulnificus E4125.Electron microscopy. Columbia agar in petri dishes was

streaked for confluent growth with rapidly thawed speci-mens of bacteria, the inoculated medium was incubated forca. 12 h at 30°C, and bacteria to be examined by transmissionelectron microscopy were washed from the plates and fixed(2 to 4 h, 4°C) with a 5- to 10-ml solution of 1.3% glutaralde-hyde (wt/vol) and 0.17% ruthenium red (wt/vol) in 0.07 Msodium cacodylate buffer (pH 7.3). The cells were washedwith cacodylate buffer and postfixed (2 h, 25°C) with 1.3%osmium tetroxide (wt/vol)-ruthenium red in cacodylate buff-er. The fixed cells were washed, dehydrated by exposure toincreasing concentrations of ethanol (the 25% ethanol solu-tion was saturated with uranyl acetate), embedded in Epon,thin sectioned, stained sequentially with uranyl acetate andlead citrate, and examined with a Phillips EM-400 electronmicroscope at 80 kV.The surface structure of bacteria examined by scanning

electron microscopy was stabilized by incubating (1 h, 25°C)cubes of Columbia agar cultures of V. vulnificus with heat-inactivated (56°C, 30 min) antiserum (diluted 1:2 with PBS)raised against the CE of V. vulnificus. The cells were washedwith cacodylate buffer, fixed (1 h, 25°C) with glutaraldehydein cacodylate buffer, washed, and postfixed (2 h, 25°C) withosmium tetroxide. The fixed cells were washed with cacody-late buffer, dehydrated in ethanol, critical-point dried in

TABLE 1. Active immunization of mice against disease causedby V. vulnificus E4125 by vaccination with killed cells of V.

vulnificus E4125 and A1402% Survival after

Vaccine strain Treatment" challenge with:100 LD5Osb 10 LD5osb

E4125 Formalin 90 (0.0001) 100 (<0.0001)56°C + Formalin 80 (1) 100 (1)100°C + Formalin 10 (0.001) 90 (1)121°C + Formalin 0 (0.0001) 50 (0.03)

A1402 Formalin 20 (0.47) 50 (0.03)56°C + Formalin 10 (1) 40 (1)100°C + Formalin 0 (0.47) 30 (0.65)121°C + Formalin 0 (0.47) 10 (0.14)

PBS-F (control)' 0 0

" The bacteria were killed by exposure to Formalin (final concentration.0.8% [vol/vol]) or by heating the bacterial suspension for 15 minm followed byFormalin exposure, as described in the text.

b The P values for the comparison of the two Formalin-killed vaccines withthe PBS-F (control) vaccine and for the comparison of the three heat-treatedvaccines with the Formalin-killed vaccine are within parentheses. Because ofmultiple-comparison corrections, the 95% significance levels are P = 0.025 forthe Formalin-killed vaccines compared with the PBS-F vaccine and P = 0.017for the heat-treated vaccines compared with the Formalin-killed vaccine. Inaddition to estimation of P values by the Fisher exact test, logistic regressionanalysis of the data indicated that the virulent strain vaccine (E4125) elicited asignificantly stronger immune response than the weakly virulent strainvaccine (A1402) and that the immunogenic activity of both vaccines wassignificantly reduced by heating.

' PBS (0.02 M Na,HPO4-HCI-0.15 M NaCI [pH 7.4]) supplemented with0.8% Formalin.

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IMMUNIZATION AGAINST VULNIFICUS DISEASE 539

> 60 100 LD50. after Vaccinationwith E4125 CE

$ 40 \10 LD505 afterVaccination

with Al 402 CE20

_ 100 LD50s after Vaccinationwith A1402 CE

1 2 4 8 16 32 64 128Reciprocal of Vaccine Dilution

FIG. 1. Active immunization of mice against disease caused byV. v,iulnificus E4125 by vaccination with CEs of V. vitinificus E4125and A1402. Mice were vaccinated, as described in the text, withtwofold serial dilutions of the CEs before s.c. challenge with 100 and10 LD0s, of V. vidinificus E4125.

carbon dioxide, coated with gold-palladium, and examinedwith a Phillips EM-501 electron microscope at 30 kV.

Statistics. The Fisher exact test (35) was used to calculatetwo-tailed P values. A multiple-comparison correction was

used, when necessary, to correct the P values (<0.05)considered statistically significant. The data were also exam-ined by logistic regression analysis.

RESULTS AND DISCUSSION

Active immunization against experimental V. vulnificusdisease. Two observations were made during the activeimmunization studies with FKC and CEs of V. vilnificuisE4125 (virulent strain) and A1402 (weakly virulent strain).First, vaccination either with FKC obtained from heartinfusion broth cultures (Table 1) or Columbia agar cultures(data not shown) or with the CEs (Fig. 1) actively immunizedmice against fatal disease caused by s.c. challenge with V.lvulnificus E4125. However, the virulent strain vaccineselicited a significantly stronger immune response than didthe weakly virulent strain vaccines (Table 1 and Fig. 1). Thisobservation suggests either a quantitative or a qualitativedifference in the PA(s) possessed by the two bacterialstrains. For example, the weakly virulent strain may havereduced amounts of the same PA possessed by the virulentstrain, or the weakly virulent strain may possess a structural-ly altered but cross-reacting PA which elicits a weakerimmune response than does the virulent strain PA.

Second, the immunogenic activity of the killed cell vac-

cines of both strains was significantly reduced when thebacteria were killed by boiling or autoclaving rather than byFormalin treatment (Table 1). This observation indicatesthat the major PA in the vaccines is not the heat-stablelipopolysaccharide endotoxin (LPS) of the bacteria. Howev-er, part or all of the residual immunogenic activity in theboiled and autoclaved vaccines may be a function of theheat-stable bacterial LPS. Alternatively, the residual activitymay indicate incomplete inactivation of the heat-labile PA.

Passive immunization against V. vulnificus disease. Threeobservations were made during the passive immunizationstudies with rabbit AS raised against V. vulnJifi(s FKC(Table 2) and CEs (data not shown). First, the AS passivelyprotected mice against fatal disease caused by s.c. challengewith V. vulnifiuius; however, the AS raised against thevirulent strain vaccines protected significantly better thandid the AS raised against the weakly virulent strain vaccines.This observation supports the idea that the immunity elicitedby the vaccines is humoral or antibody mediated rather thancell mediated and confirms the results obtained during theactive immunization experiments, which indicated a quanti-tative or qualitative difference in the PAs possessed by thevirulent and weakly virulent bacteria.

Second, the observation that adsorption of the AS with thehomologous FKC markedly reduced the ability of the AS toprotect mice confirmed our suspicion that the PA is locatedon the bacterial surface. We suspected, before performingthe adsorption studies, that the PA was located on or looselyattached to the bacterial surface because of our previousobservation that CEs containing the antigen could be ob-tained from V. l'ulnificuis by a procedure which did not causedetectable cell lysis or loss of viability.

Third, despite the previous observation that weakly viru-lent strain FKC elicited a significantly weaker immuneresponse than did virulent strain FKC (Table 1), adsorptionof the virulent strain AS with the weakly virulent strain FKCwas as effective in reducing the protective activity of the ASas was adsorption of the AS with the virulent strain FKC.Incubation of the weakly virulent strain FKC with normalrabbit serum before adsorbing the AS with the FKC did notreduce the protective antibody-adsorbing ability of the cells(Table 2). Thus, the ability of the FKC to adsorb theprotective antibodies does not appear to be caused bynonspecific binding of immunoglobulins by the FKC. If thereis a quantitative difference in the PAs possessed by the twobacterial strains (i.e., more antigen on the virulent bacteria

TABLE 2. Passive immunization of mice against disease causedby V. 'ilni (fi-is E4125 by vaccination with AS raised against FKC

of V. v'ulnificius E4125 and A140)2% Survival after

Serum preparation Adsorbed with challenge with:FKC of:100 LD5,s" 10 LDs,,s'

Anti-killed E4125 80 (0.001) 100 (<0.0001)E4125 0 (0.001) 20 (0.001)A1402 0 (0.001) 20 (0.001)A1402 (after 0 (0.001) 30 (0.003)

incubationof FKC withN RS")

Anti-killed A1402 0 (1) 60 (0.011)A140' ()((1) 0 (0.011)E4125 0 (1) 0 (0.011)

NRS (control) 0 0

" The P values for the comparison of the two unadsorbed AS with thenormal rabbit serum (control) and for the comparison of the adsorbed AS withthe unadsorbed AS are within parentheses. Because of multiple-comparisoncorrections, the 95% significance levels are (i) P = 0.025 for the unadsorbedAS compared with the normal rabbit serum and for the adsorbed anti-A1402AS compared with the unadsorbed anti-A1402 antiserum and (ii) P = 0.017 forthe adsorbed anti-E4125 AS compared with the unadsorbed anti-E4125antiserum.

' Samples (5 ml) of heat-inactivated normal rabbit serum (NRS) wereadsorbed with the FKC as previously described for the adsorption of AS (17).and the cells then were used to adsorb the antiserum.

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540 KREGER, GRAY, AND TESTA

than on the weakly virulent bacteria), our results may beexplained by the idea that the reduced amount of antigen inthe vaccines of the weakly virulent strain results in theireliciting a weaker immune response than the virulent strainvaccines; however, the large numbers of weakly virulentstrain bacteria used in the adsorption procedure possesssufficient antigen to effectively adsorb and remove theprotective antibodies in the AS. If there is a qualitativedifference in the PAs possessed by the two bacterial strains(i.e., the weakly virulent strain PA is a structurally modifiedbut cross-reacting form of the virulent strain PA), our resultsmay be explained by the idea that the physicochemicalmodification or difference in the weakly virulent strain PAreduces its immunogenic activity but does not prevent itsrecognition and binding of antibodies raised against thevirulent strain PA. The results of additional studies tocharacterize the physicochemical and immunochemicalproperties of highly purified PA preparations should indicatewhether our observations result from a quantitative orqualitative difference in the PAs possessed by the two V.vulnificus strains.Experiments were not performed to determine the mecha-

nism(s) for the strong passive immunity conferred by theantibodies raised against the virulent V. wulnificius strain.One possible mechanism is that the antibodies enhanced invivo phagocytosis and clearance of V. vu/nificus. The viru-lent V. vulnificus strain used in our studies is resistant to invitro phagocytosis, whereas the weakly virulent strain isreadily phagocytized in vitro by human polymorphonuclearleukocytes (17). In addition, AS raised against FKC of thevirulent strain significantly enhances both the amount andrate of phagocytosis of V. vulnificus (17). Furthermore, micevaccinated with various killed V. vulnificuts isolates andintravenously challenged with the viable bacteria have beenreported to clear the bacteria from the bloodstream signifi-

14

12-

10

4 - F

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Fraction

FIG. 2. Isoelectric focusing of PA preparation obtained by high-speed centrifugation of V. vulnificus E4125 CE. The pH of each 4-mlfraction was determined at 4°C (0), the pHs of the fractions were

adjusted to pH 6 to 8, and samples (0.2 ml of a 1:2 dilution) of thepH-adjusted fractions were examined for PA activity as described inthe text. The fractions containing the most PA activity (elicited100% survival of vaccinated mice challenged with 10 LD5os of V.

tlulnificus E4125) are indicated with asterisks. Fractions 3 and 6 alsocontained some PA activity (elicited 80 and 60% survival, respec-

tively).

TABLE 3. Stability of PA in CE of V. vulnificuis E4125

% Survival after challenge with:Treatment of CE"

100 LD5,s" 10 LD,s",s

4°C, 14 days (pH 7.4) (control) 90 10056°C. 15 min 100 (1) 100 (1)100°C, 15 min 10 (0.001) 70 (0.21)121°C, 15 min 0 (0.0001) 20 (0.001)pH 12, 4°C, 24 h 10 (0.001) 90 (1)37°C, 2 h (control) 100 100Trypsin 100 (1) 100 (1)Pronase 100 (1) 100 (1)RNase A 100 (1) 100 (1)Neuraminidase 100 (1) 100 (1)pH 4.5, 4°C, 24 h (control) 100 100Periodate oxidation (pH 4.5, 4°C, 10 (0.0001) 90 (1)

24 h)'pH 7.4, 25°C, 16 h (control) 90 100Periodate oxidation (pH 7.4, 25°C, 0 (0.0001) 10 (0.0001)

16 h)d" The heat and enzyme treatments were done in PBS (0.02 M Na2HPO4-

HCI-0.15 M NaCI [pH 7.41). The enzyme treatments (final concentration. 100,ug/ml) were done at 37'C for 2 h. The pH 12 and 4.5 preparations wereadjusted to pH 6 to 8 before injection into mice. All of the treated preparationswere diluted with an equal volume of PBS before being assayed for PAactivity.

h The P values for the comparison of the treated CEs with the control CEsare within parentheses. The 95% significance level for the CEs subjected toperiodate oxidation is P = 0.05. Because of multiple-comparison corrections.the 95% significance level for the other treated CEs is P = 0.0125.

As described by Spiro (30)."As described by Flesher et al. (10).

cantly faster than nonvaccinated mice (M. L. Tamplin, J. B.Cornette, G. E. Rodrick, H. Friedman, and N. J. Blake,Abstr. Annu. Meet. Am. Soc. Microbiol. 1982, B21. p. 21).Another possible explanation for the observed passive im-munity is that the antibodies enhanced complement-mediat-ed lysis of V. vulnificus. Various isolates of V. vuinificushave been reported to be more resistant to human comple-ment-mediated lysis than are various isolates of Vibrioparahaemolyticus, a relatively noninvasive vibrio (6). Inaddition, Tamplin et al. (32) found that various isolates of V.vulnificus were significantly weaker activators of the com-plement cascade than were various isolates of V. parcahae-molyticus and Vibrio cholerae.

Properties of the PA. The protein, neutral carbohydrate,and LPS contents in the virulent and weakly virulent strainCEs were similar and were ca. (per ml): 550 ,ug of protein, 16pLg of carbohydrate, and 0.5 ng of LPS, respectively. The PAin the virulent strain CE migrated in the void volume ofSephacryl S-400 superfine (data not shown) and, therefore,appeared to have an apparent molecular weight of .2 x 106to 8 x 106. PA assays with twofold serial dilutions of thenonsedimentable and sedimentable fractions of the CErevealed that a 64-fold dilution of the nonsedimentablefraction elicited significant active immunity; however, onlythe undiluted sedimentable fraction elicited significant im-munity (data not shown). The observation that most of thePA activity was nonsedimentable by centrifugation at100,000 x g for 2 h supports the idea that most of the PA inthe CE exists as a soluble polymer rather than in associa-tion with insoluble outer membrane fragments. The smallamount of sedimentable PA activity may have been causedby aggregation of the PA or by binding of the PA tosedimentable outer membrane fragments. Alternatively, thesmall amount of sedimentable PA activity may be a functionof a membrane-bound PA which is different than the major

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IMMUNIZATION AGAINST V. V'ULNIFICUS DISEASE 541

PA in the CE. The PA in the virulent strain CE had anisoelectric point of 3.8 to 4.2 (Fig. 2) and was sensitive toboiling or autoclaving for 15 min, periodate oxidation, andexposure to pH 12 but was resistant to 56°C, trypsin,pronase, RNase, neuraminidase, and pH 4.5 (Table 3). Partor all of the residual immunogenic activity in the boiled andautoclaved CEs may be a function of the small amount ofheat-stable LPS in the CEs. Alternatively, the residualactivity may indicate incomplete inactivation of the heat-labile PA in the CEs. Both the PA obtained by sequentialhigh-speed centrifugation and isoelectric focusing of thevirulent strain CE and the PA in the weakly virulent strainCE had the same sensitivity and resistance to inactivation asdid the PA in the virulent strain CE (data not shown). Thus,the data support the idea that the major PA in the CEs fromboth strains is associated with a heat-labile, acidic polysac-charide. In this regard, the K antigen of V. parahaemolyticusis an acidic polysaccharide located on the bacterial surfaceand is a PA against fatal disease in mice intraperitoneallychallenged with the homologous bacterial strain (24). How-

ever, the K antigen of V. parahaemolvticus is significantlymore heat stable (100°C for 2 h) than is the PA of V.vulnificus (24).Electron microscopic examination of virulent and weakly

virulent strains of V. vulnificus. Before performing the immu-nization experiments described in this paper, we observedthat (i) virulent strain colonies on Columbia agar had twicethe diameter of weakly virulent strain colonies (unpublisheddata), although no significant difference in growth rate inbroth was noted for the two strains (18), (ii) virulent strainbacteria grown in broth and sedimented by centrifugation(5,100 x g, 10 min) or virulent strain FKC allowed tosediment spontaneously while stored at 4°C for several dayswere more easily dispersed than were similar preparations ofthe weakly virulent strain bacteria (unpublished data), and(iii) virulent strain bacteria were significantly more resistantto in vitro phagocytosis by human polymorphonuclear leu-kocytes than were weakly virulent strain bacteria (17). Thesethree observations together with the results of our immuni-zation studies strongly suggest differences in the surface

A

FIG. 3. Electron microscopy of virulent strain E4125 and weakly virulent strain A1402 of V. i'ulnificus. (A) and (B) are transmissionelectron micrographs of strains E4125 and A1402, respectively, after the staining of the cell surface with ruthenium red. Line markers = 0.5p.m. (C) and (D) are scanning electron micrographs of strains E4125 and A1402. respectively. Line markers = 1 p.m.

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structures of the virulent and weakly virulent V. vulnificusstrains. Examination of the surfaces of the two strains bytransmission and scanning electron microscopy further sup-ports that idea. The virulent V. vulnificeus strain possessed amore dense ruthenium red-staining layer on its outer mem-brane and had a much smoother surface than the weaklyvirulent V. vulnificus strain (Fig. 3). Our physicochemicaldata support the idea that the PA is an acidic polysaccharide,and ruthenium red is thought to be a specific stain for acidicpolysaccharides; however, further studies are needed todetermine whether the ruthenium red-staining substance(s)on the bacterial surface is the PA described in our communi-cation. Recently, in vitro passage of Baciteroides fragilis23745 has been reported to select for a small colony typevariant which has reduced virulence and significantly lessruthenium red-staining capsular polysaccharide and is signif-icantly more sensitive to complement-dependent phagocyto-sis than are the parental, large colony type cells (15, 27).

In conclusion, our current hypothesis to explain the datain this communication, the results obtained during ourprevious phagocytosis studies (17), and the data of Car-ruthers and Kabat (6) and Tamplin et al. (32; M. L. Tamplin,J. B. Cornette, G. E. Rodrick, H. Friedman, and N. J.Blake, Abstr. Annu. Meet. Am. Soc. Microbiol. 1982, B21,p. 21) is that V. vulnificus virulence is associated with thepossession of a heat-labile, acidic polysaccharide surfacecomponent that has PA activity and aids the bacterium inresisting in vivo phagocytosis or complement-mediated lysisor both. Studies are in progress to obtain highly purifiedpreparations of the PA so that we can subject our hypothesisto further experimental evaluation.

ACKNOWLEDGMENTSWe thank R. E. Weaver and D. G. Hollis (Centers for Disease

Control) for supplying the bacterial strains used in this study.This investigation was supported by Public Health Service grants

Al-18184 and HL-16769 from the National Institute of Allergy andInfectious Diseases and the National Heart. Lung, and BloodInstitute, respectively.

LITERATURE CITED1. Baumann, P., L. Baumann, S. S. Bang, and M. J. Woolkalis.

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