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Isolation of an (O,H,Vi)-Free Immunoprotective Antigenic Fraction with Mannose Receptor-Like Activity from Salmonella typhiAuthor(s): Hlne Jouin, Anne-Marie Staub and Joseph E. AloufSource: The Journal of Infectious Diseases, Vol. 143, No. 1 (Jan., 1981), pp. 106-113Published by: Oxford University Press

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THE JOURNAL OF INFECTIOUS DISEASES * VOL. 143, NO. 1 * JANUARY 1981c 1981 by The University of Chicago. 0022-1899/81/4301-0018$00.81

Isolation of an (O,H,Vi)-Free ImmunoprotectiveAntigenic Fraction withMannose Receptor-LikeActivity from Salmonella typhiH61bneJouin, Anne-Marie Staub, andJoseph E. Alouf

From the UnitddesAntighnesBactdriens,nstitutPasteur,and the CentreNationalde la Recherche cientifique,Paris,FranceAn antigenic ractionof SalmonellayphistrainTY6S,obtained roma bacterialpelletby washingswith 1.5 MNaCl andultracentrifugationna 5% -20% sucrosedensitygra-dient, inducedserumantibodies n rabbits hat protectedchickenembryoschallengedwith lethal doses of the virulentmicroorganism. heantigenicpreparationwas free ofVi, O, andH antigens. t containedabout 85%proteinandRd1P' ipopolysaccharide.Theproteinmoietycomprisedhreepolypeptides f 45,000,32,000,and20,000daltons,respectively.naddition, hepurifiedpreparationxhibited heproperties f a bacterialadhesinwithreceptor-like ctivity or mannosyl esidues,as shownby agglutination ftargetcellscontaining hese residuesat their surfaces.

Salmonella typhi is the causative agent of typhoidin humans. Evaluation of the effectiveness of ty-phoid vaccines or of the immunoprotective effectsof antisera from convalescent patients or im-munized humans or animals cannot be undertakenon mice or other laboratory animals because theyare not susceptible to infection by S. typhi. In con-trast, chicken embryos are susceptible to infectionwith human strains of this species. Thus, they area useful biological tool for the study of the protec-tive properties of immune sera to S. typhi [1, 2].The protective effect of these sera was not relatedto their content of antibodies (agglutinins, hemag-glutinins, or precipitins) to O, H, and Vi antigens[1]. This finding is in agreement with studies thathave reported that antibodies to O, H, and Vi an-tigens are of little or questionable importance inreflecting the state of protective immunity to ty-phoid fever [3].We postulated in a previous communication [2]that the immunoprotective properties of the anti-sera could be due to a still unidentified antigen(s)present on the surface of S. typhi organisms. Wereport here the isolation and some properties of aVi-free immunoprotective preparation from S.typhi strain TY6S (O-H+Vi+),which lacks O anti-gen. The chicken embryo challenge was under-taken with strain TY2 (O*H+Vi+),solated from atyphic patient.

Received or publicationMay29, 1980,andin revised ormAugust15, 1980.Please address equestsor reprintso Dr. JosephE. Alouf,Unite des AntighnesBacttriens,InstitutPasteur, 28, rue duDocteur-Roux, 5724 Paris Cedex15,France.

We also describe receptor-like properties of theisolated fraction related to adherence of S. typhito mannose residues on yeast cells or on erythro-cytes coated with mannans.

Materials nd MethodsBacterial strains. The virulent TY2 strain of S.

typhi possessing O, H, and Vi antigens and thenonvirulent O- strain TY6S possessing H and Viantigens were used (provided by Dr. L. Le Minor,the World Health Organization International Sal-monella Center, Institut Pasteur, Paris). Thestrains were maintained lyophilized.Preparation of the Vi-free fraction. Fraction-ation of S. typhi strain TY6S was carried out bythe method of Forsberg et al. [4] for Pseudomonasaeruginosa. Bacterial cells in the early logarithmicphase of growth were harvested from the mediumby centrifugation at 16,300 g for 20 min at 4 C andwere suspended in 0.5 MNaCl to a concentrationof about 5.0 x 1010cells/ml. All subsequent oper-ations were carried out at 4 C (figure 1). The cellsuspension was centrifuged immediately at 16,300g for 30 min, and the opalescent supernatant wasremoved by aspiration and retained. The cellswere suspended again in the initial volume of 0.5 MNaC1 and centrifuged, and the supernatant wasseparated and retained. This procedure was re-peated once. The three supernatants were pooled,centrifuged at 35,000 g for 30 min to eliminate anyremaining cells, and filtered (pore size, 0.22 tm;Millipore Corp., Bedford, Mass.).The filtrate (fraction I) contained large amounts

106

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Immunoprotective S. typhiFraction 107

TY6S strain cells (logarithmic phase)centrifugation 16,300xg, 20 min

Cells Supernate (discarded)3 times + 0.5 M NaC1centrifugation 16,300xg, 30 min

Cells(discarded) Pooled supernates

centrifugation 35,000xg, 20 min

Pellet Supernate filtered(discarded) through 0.22 p Millipore(Fraction I)

ultracentrifugation105,000xg, 2 h

Pellet Supernate(discarded)

0.5 M NaC1 washing (twice), IPellet Supernates

(Fraction II) (discarded)ultracentrifugation in 5-20 lsucrose gradient (250,000xg, 18 h)

Bottom Top(Fraction P) (Fraction Vi)

Figure1. Flow diagramof the purificationof a cell-surfaceantigenicextract from Salmonella yphi strainTY6S. Millipore = filter from MilliporeCorp., Bed-ford, Mass.of proteins and Vi antigen in addition to lipids andcarbohydrates. This material was ultracentrifugedat 105,000 g for 2 hr. The pellet was washed twicewith 0.5 M NaC1, and the supernatant was dis-carded. The pellet (fraction II), dissolved in 0.5 MNaC1, was applied to a 5%o-20%oucrose densitygradient and centrifuged at 4 C at 250,000 g for 18hr (figure 2). A protein-rich fraction completelyfree from Vi antigen (fraction P) was found in thebottom of the gradient; Vi antigen was in the top.Immunization. Male and female outbred Bous-cat white rabbits, weighing 2-3 kg, were obtainedfrom an animal colony at the Institut Pasteur,Garches, France. Rabbits having natural anti-bodies to Vi antigen were not used.Groups of six (or 12) rabbits were immunized byfour injections each containing about 0.1 mg ofantigen. On day 0, the first injection, consisting of1 ml of antigen and 2 ml of complete Freund's ad-juvant (Difco Laboratories, Detroit, Mich.), wasgiven sc in the back. On day 22 an injection was

mg1.1.1.21.1

oc. o---o proteins,c ... Vio.0.7O*o.

0. A-------4Q3\42.1o3 /" "

o t mu1 4 rbnboto tbenubr to

Figure2. Ultracentrifugation attern of fraction II(see figure1)of SalmonellayphistrainTY6S.Thesam-ple (100Ml)was layeredon a 5%-20% sucrosedensitygradientand centrifuged t 250,000g for 18 hr at 4 C.The total volumeof the fractionscollectedwas 500 l.Vi = Vi antigen.given im in the leg. The remaining injections weregiven on days 24 and 27, iv in the marginal earvein. The rabbits were bled seven days after thelast injection, and the sera were stored at - 180 C.No antibodies to O or H antigens were detectablein the sera before or at the end of immunization.

Culture conditions. S. typhi strain TY6S wasincubated at 37 C for 12 hr in the following me-dium, containing, per liter: KH2PO4, 13.60 g;KC1, 0.50 g; (NH4)2SO4, 0.75 g; MgSO4, 0.05 g;nicotinic acid, 0.01 g; ammonium ferric citrate,0.02 g; CaC12,0.02 g; glucose, 3.00 g; tryptophan,1 mM;and the other 19 L-aminoacids, 50 1M. ThepH was adjusted to 7.5. Cells cultured under theseconditions did not contain H antigen detectable byagglutination with standard antisera to H antigen(Institut Pasteur Production, Paris, France).S. typhi strainTY2 was first incubatedin the fol-lowing liquid medium, containing, per liter:Liebig meat extract (Brooke Bond Co., Agnitres,France), 5 g; peptone (Difco), 10 g; and NaCl, 2.5g. The pH was adjusted to 7.5. When the culturewas developed, it was transferred into the samemedium containing 13 g of agar/liter.Chicken embryo test. Fertile eggs from whiteLeghorn chickens (France-Ponte, Arpajon, France)were incubated with automatic turning, controlledtemperature (37.3-37.8 C), controlled humidity(50%-55%0), and air circulation. Eleven-day-old



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108 Jouin, Staub, andAlouf

living chicken embryos were selected for inocula-tion. The undiluted rabbit sera (10 Al)were addedto 190 al of 0.15 M NaCl containing 20 to 2.0 x104 living S. typhi TY2 organisms. Small windowswere cut in the shell with a drill. The serum-bacter-ia mixtures (10 eggs per mixture) were injectedwith a tuberculin syringe onto the chorioallantoicmembrane, and the windows were sealed with ad-hesive tape. The eggs were then placed horizontal-ly in the incubator with the window up. Viabilitywas assessed by candling after a three-day incuba-tion. The surviving embryos were counted. Con-trol procedures consisted of the injection of 200 Clof 0.15 MNaCI. The LD50 of S. typhi was checkedby injection of the serum-free, diluted bacterialsuspensions. The LDso always corresponded to theinoculation of one bacterium. The results were ex-pressed as the number of LDsoneutralized by 10 jlof antiserum. Normal rabbit sera never protectedagainst more than 20 LD5s0. This value was thelower limit for the assessment of the protectivetiter of immune sera.

Assay of Vi antigen. This homopolysaccha-ride was assayed as described elsewhere [5] by

HAI of sheep erythrocytes sensitized with purifiedantigen by antiserum to Vi antigen.Chemical assays. Concentrations of proteinswere determined by the method of Lowry et al. [6]with use of bovine serum albumin as the standard.Concentrations of sugars were determined by thephenol-H2SO4 method with use of glucose as astandard [7]. Lipids were extracted by the methan-ol-chloroform method [8]. Heptose concentra-tions were determined as described by Osborn [9].Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). The gels wereelectrophoresed according to the method of Laem-mli [10]. The molecular weights were determinedby the method of Weber and Osborn [11].Yeast cell aggregate test. Saccharomyces cer-evisiae cells harvested from a standard mediumwere suspended in phosphate-buffered saline to aconcentration of 107cells/ml as describedby Ofeket al. [12]. S. typhi strain TY6S cells (2.5 x109/ml) were incubated with the yeast cell suspen-sion at room temperature(about 24 C) for 30 min.Aggregation was then observed microscopically.Mannose-sensitized erythrocyte agglutination

20000

(UU,

( 200

oN

0c 2C ........

1 11LA-0

RabbinumberI I12c 4

Rabbit number

P ViO00(ULA.,r

20(3

Noc

0 2

Rabbit numberFigure3. Serumantibody itersbefore( - - ) and after(-) immunization f rabbitswithfractionsI (left), P(middle),and Vi (right)of Salmonella yphistrainTY6S(see figure1).The resultsaresignificant or titersexceedingneutralization f 20 LDsof S. typhi n chickenembryos.



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Immunoprotective. typhiFraction 109

Table 1. Chemical ompositionof fractionP of Sal-monellatyphistrainTY6S.Component Weight mg) Percentageweight*Dry weight,total 7.35 100Proteins 6.40 87Lipids 0.60 8Neutral ugarst 0.80 10Phosphorus 0.17 2.3

NOTE. Forprocedures,eefigure1.* Becauseof thelimitations f mostanalyticalmethods, hecalculatedotalwithrespecto dryweightdoes notequal100%.t The amountof heptosepresent n the neutral ugarswasabout 0.3 mg.test. Sheep red blood cells (2.5%0suspension inphosphate-buffered saline) were sensitized withyeast mannans by the benzoquinone method ofTernynck and Avrameas [13]. The agglutinationof these cells by S. typhi cells was carriedout simi-larly to that of yeast cells.Results

Immunoprotective properties of fraction P.Rabbits were immunized with fractions I, P, andVi. The first two preparations (figure 3) inducedthe appearance of a protective activity of varioustiters in immune sera, depending on the individualanimal. In contrast, fraction Vi did not elicit such

an activity. Chicken embryos were not protectedwhen they were challenged with Salmonellatyphimurium instead of S. typhi, a difference in-dicating the specificity for this species. Fraction Padsorbed on aluminum hydroxide as adjuvantelicited protective activity measured in rabbit sera.However, the titer was twofold lower than thatobtained with complete Freund's adjuvant.Physical and chemical characteristics of frac-tion P. The chemical composition of purifiedfraction P is shown in table 1. A high protein con-tent (85%) was found, with low amounts of lipids(8%) and neutral carbohydrates (10%). Heptosesrepresented about 40% of the total neutral carbo-hydrates. Ribose, deoxyribose, and Vi antigen(D-N-acetylgalactosaminuronic acid, partly O-ace-tylated) were not detected.

SDS-PAGE of fraction P (figure 4) disclosed amajor protein band with two faint components oflower molecular weights. The migration positionof the three bands was compared with those of thereference proteins of different molecular weights;the values were 45,000, 32,000, and 20,000, re-spectively. Whether the two faint bands corre-spond to degradation products of the major com-ponent is not clear. This possibility is suggested bythe observation of a single band exhibited by somepreparations in SDS-PAGE.

7~472~

o 67.0o 1x450- 2 8

32.0250 3

S2o0.0_0 178 4

0 6 0.7 0.8 0.9 1.0 1.1Mobility (cm)

Figure4. Results of sodiumdodecyl sulfate-polyacrylamide eldiscelectrophoresisf fractionP (seefigure 1) of Salmonella yphi strainTY6S: (left) fractions and (right)relativemobility, as distancein cmfromthe cathode.The standardmolwt markerproteinsare: (1) bovineserum albumin, 67,000; (2) oval-bumin, 45,000; (3) chymotrypsino-gen, 25,000; and (4) myoglobin,17,800.Threecomponents a, b, andc) of fractionP areseen.



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110 Jouin, Staub,andAlouf

Figure5. Resultsof geldiffusion mmunochemicalnalysisof fractionP(see figure1) of Salmonella yphistrainTY6S.Left: Theupperwellcon-tains rabbitantiserum o fractionP, and thebottom well contains ractionP. Right:Thecontentsof the wells are the same,but thegelcontainshep-tose (100 mg/ml).

Immunochemical analysis of fraction P. Gel-immunodiffusion of fraction P against homolo-gous rabbit antisera disclosed a main precipitationzone and another faint band (figure 5, left).The same immunochemical analysis was under-taken in gels in which glucose, galactose, N-acetyl-glucosamine, and heptose, which are componentsof the core moiety of S. typhi lipopolysaccharides,were incorporated. Immunoprecipitation was in-hibited in the presence of heptose (figure 5, right),an inhibition suggesting that fraction P containslipopolysaccharide with heptose as the immuno-dominant group. As shown in table 1 this sugarwas in fraction P. This finding was confirmed bytesting the antisera with the three different lipo-polysaccharides from Salmonella R forms withterminal heptose (figure 6) [14], provided by Dr.O. Liideritz, Max-Planck Institut fir Immunbio-logie, Freiburg, Federal Republic of Germany.Only Rd1P*polysaccharide was shown to react (inthe ring test) with the rabbit antiserum to fractionP. This fraction very likely contained lipopolysac-charide of Rd1P' type.Rabbits were then immunized with a rough mu-

tant of Salmonella minnesota, which has theRd1P' lipopolysaccharide. The antisera obtaineddid not protect chicken embryos from challengewith S. typhi strain TY2. This result indicated thatthe lipopolysaccharide present in fraction P wasnot a significant immunoprotective moiety of thisfraction. Fraction P was free of H antigen as dem-onstrated by immunologic tests.Toxicity of fraction P. Groups of five Swissmice (weighing 20 g) were inoculated with fivefolddilutions of fraction P in pyrogen-free saline.Each animal received 0.5 ml ip. Mice inoculatedwith pyrogen-free saline served as controls. Themice were observed for five days after inoculation.The LDso was calculated by the method of Reedand Muench [15] as 2.5 yg. This toxicity was verylikely due to the lipopolysaccharide present infraction P (about 20%7on the basis of heptosecontent).Aggregation of yeast cells by fraction P.Yeast cells are aggregated by S. typhi cells (figure7, left) under the conditions described in Materialsand Methods. A similar aggregation as shown bythe formation of large clumps was observed by in-

R 01igosaccharidechemotypeGIcNAc Gal Hep KDO- P- EtN

P P-P-EtNRa GIc - Gal -- Ge --- Hep -- Hep --4 KDO - KDO [LIPID A)

KDO-P - E NP - P--EtN

Rd,P+ Hep -4 Hep -* KDO -- KDO -4 [LIPID A]KDO - P - EtN

RdP- Hep -- Hep DO--0 , KOO -4 [LIPID A]KDO - P - FtN

Rd Hep -41 ( cDO - 0KDO -- LIPID A]

Figure6. Structures of lipopoly-saccharides f four core-defectiveRmutantsof Salmonella 14].



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Immunoprotective. typhiFraction 111

c 4

Figure7. Aggregation f yeastcells: eft, clumpingn thepresence f SalmonellayphistrainTY6S;middle,clump-ingin thepresence f 5mgof fractionP/ml (see figure1);andright, nhibition f clumpingn thepresence f 5 mgoffractionP/ml with mannose 5 mg/ml) (x 400).cubation of fraction P at a concentration of 5mg/ml with yeast cells (107/ml) (figure 7, middle).Clumping by both S. typhi cells and fraction Pwas inhibited in the presence of D-mannose, man-nans, or methyl a-D-mannopyranoside (5 mg/ml)in the incubation medium (figure 7, right). In con-trast, no inhibition was found with galactose, glu-cose, N-acetylgalactosamine, or N-acetylglucos-amine at the same concentration.A similar agglutination of D-mannose-sensi-tized erythrocytes by fraction P and S. typhi cellswas also observed. D-mannose inhibited agglutina-tion.These findings indicate that fraction P possessesa specific receptor-like activity for mannose re-sidue.Serum absorption by erythrocytes. Absorp-tion was performed with 50 plof serum incubatedat 4 C for 10 min with 0.5 ml of erythrocyte sus-pension.The protective properties of rabbit sera im-munized with fraction P were suppressed after ab-sorption with mannose-sensitized erythrocytes aswell as with unsensitized erythrocytes.DiscussionThe immunity of humans against typhoid fever re-

sulting from vaccines preparedfrom bacterial cellsof S. typhi appears to be humoral as well as cell-mediated [3, 16, 17]. However, although S. typhiwas discovered 100 years ago and immunizationagainst the disease has been performed for over 65years, our knowledge of the antigen(s) involved inimmunoprotection is still very limited, as moststudies on salmonella antigens have been predom-inantly directed toward serologic classification[14, 18].Several lines of evidence suggest that the classi-cal and widely studied cell surface antigens O, H,and Vi are of little importance in reflecting thestate of immunity to the disease [1, 3]. This find-ing led us to postulate that another cell-surface an-tigen(s) still to be identified must be involved inimmunoprotection [2]. The chicken embryo, whichis known as an excellent model for the study of in-fection and immunity to microorganisms patho-genic for humans [19-21], proved a valuable ex-perimental tool for the assessment of the immuno-protective properties of antisera to S. typhi [5].This model was used in the present study.We report here the isolation and purification ofa protein-rich, immunogenic preparation (fractionP) distinct from the O, H, and Vi antigens of S.typhi. Sera from rabbits immunized with this pre-paration, which was fractionated from the super-



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112 Jouin,Staub,andAlouf

natant of O-H strains of S. typhi obtained bycell washing with 0.5 M NaCl (figure 1), wereshown specifically to protect chicken embryoschallenged with a virulent (Oi') human strainof S. typhi. In contrast, no protection was af-forded by the same sera upon infection of the em-bryo with S. typhimurium under the same condi-tions.Fraction P mainly comprised proteins (about85%7)with smaller amounts of lipids and carbohy-drates, including Rd1P' type lipopolysaccharide.The protein moiety disclosed slight heterogeneityby SDS-PAGE (apparent mol wt, 20,000-45,000).The protective properties of the rabbit antisera tofraction P did not appear to involve any contribu-tion of the antibodies to the lipopolysaccharidepresent in this fraction.

Like intact S. typhi cells as shown here, and likeseveral human strains of Escherichia coli [22],fraction P aggregated yeast cells, the surfaces ofwhich are known to be covered with mannans[23]. It also agglutinated erythrocytes coated withmannans. Aggregation or agglutination was pre-vented by prior incubation of either fraction P orS. typhi cells with D-mannose or methyl a-D-man-nopyranoside but not with other sugars tested atthe same concentration.It has been reported that certain strains of E.coli and S. typhi produce adherence factors (adhe-sins) at their surface that mediate specific attach-ment of the organisms to mannose or mannose-like receptors on the surface of human epithelialcells, macrophages, and polymorphonuclear cells[12, 22, 24-26] as well as of various animal cells[27, 28]. The binding of the bacterial strains ontarget cells is specifically inhibited by D-mannoseand methyl a-D-mannopyranoside [24, 28]. Amannose-sensitive adhesin of E. coli has beenshown to be associated with type 1 pili [29]. Thisadhesin is protein in nature. Recently, Eshdat etal. [30] isolated from E. coli extracts, by differen-tial centrifugation and gel filtration, a protein thatappears to be involved in the mannose-specificadherence of the organism to eukaryotic cells.This protein (mol wt, 36,000) was different fromthat of type 1 pili and appeared to be a surfacecomponent of the cell.The mannose-specific lectin-like properties offraction P reported in the present study indicatethat this purified preparation contains S. typhi ad-hesin. It is likely but not certain that this cell-bind-

ing factor is a component of the protein moiety offraction P (relative mol wt, 20,000-45,000). Themild treatment of S. typhi cells used to obtainfraction P and the presence of some lipopolysac-charide in this fraction suggest that the protein(s)present in fraction P originates from the outermembrane. Several outer membrane proteins havebeen shown to be virulence factors in the mouseinfected with S. typhimurium [31]. As adherenceto endothelial and mucosal surface is considered aprerequisite for successful invasion of host tissuesby many bacterial pathogens [32], S. typhi adhesinmay be an important virulence factor. The im-munoprotective properties of the immune rabbitsera to fraction P might be due to the neutraliza-tion of adhesins at the surface of virulent S. typhicells, which would prevent adherence onto chickenembryo cells.Thus, it was of interest to determine whether theprotective antibodies in antisera to fraction P wereto the mannose-like receptor. We did indeed finddisappearance of the immunoprotective propertiesafter serum absorption with mannose-sensitizederythrocytes. However, this finding was not con-clusive because a similar result was obtained withunsensitized erythrocytes. This result may be dueto either nonspecific adsorption of the antibodiesor combination with mannose residues of mem-brane glycoproteins of control erythrocytes [33].This problem must be investigated further beforethe answer is known.

References1. Joyeux, Y., Jouin, H., Leluc, B., Staub, A.-M. Re-cherches sur l'antigene X responsable de la productiondes anticorps anti-Salmonella typhi, protecteurs pourl'embryon de poulet. C. R. Acad. Sci. [D] (Paris)279:1507-1510, 1974.2. Joyeux, Y., Jouin, H., Leluc, B., Staub, A.-M. Recherchesur l'antigtne X responsable de la production des an-ticorps anti-Salmonella typhi, protecteurs pour l'em-bryon de poulet. Ann. Immunol. (Paris) 128:221-223,

1977.3. Warren, J. W., Hornick, R. B. Immunization against ty-phoid fever. Annu. Rev. Med. 30:457-472, 1979.4. Forsberg, C. W., Costerton, J. W., MacLeod, R. A. Se-paration and localization of cell wall layers of a gram-negative bacterium. J. Bacteriol. 104:1338-1353, 1970.5. Eiguer, T., Staub, A.-M. Etudes immunologiques surl'antighne Vi. I. Propri6t6s immunologiques d'unepr6paration d'antighne Vi: effet de l'ac6tolyse et dutraitement par le formol. Annales de l'Institut Pasteur110:707-726, 1966.



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ImmunoprotectiveS. typhiFraction 113

6. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall,R. J. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275, 1951.7. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A.,Smith, F. Colorimetric method for determination ofsugars and related substances Anal. Chem. 28:350-356,1956.8. Folch, J., Lees, M., Stanley, G. H. S. A simple method forthe isolation and purification of total lipids from animaltissues. J. Biol. Chem. 226:497-509, 1957.9. Osborn, M. J. Studies on the gram-negative cell wall. I.Evidence for the role of 2-keto-3-deoxyoctonate in thelipopolysaccharide of Salmonella typhimurium. Proc.Natl. Acad. Sci. U.S.A. 50:499-506, 1963.10. Laemmli, U. K. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature227:680-685, 1970.11. Weber, K., Osborn, M. The reliability of molecular weightdeterminations by dodecyl sulfate-polyacrylamide gelelectrophoresis. J. Biol. Chem. 244:4406-4412, 1969.12. Ofek, I., Mirelman, D., Sharon, N. Adherence of Escheri-chia coli to human mucosal cells mediated by mannosereceptors. Nature 265:623-625, 1977.13. Ternynck, T., Avrameas, S. A new method usingp-benzo-quinone for coupling antigens and antibodies to markersubstances. Ann. Immunol. (Paris) 127:197-208, 1976.14. Jann, K., Westphal, O. Microbial polysaccharides. In M.Sela [ed.]. The antigens. Vol. 3. Academic Press, NewYork, 1975, p. 1-125.15. Reed, L. J., Muench, H. A simple method of estimatingfifty per cent endpoints. American Journal of Hygiene27:493-497, 1938.16. Collins, F. M. Vaccines and cell-mediated immunity. Bac-teriol. Rev. 38:371-402, 1974.17. Collins, F. M. Cellular antimicrobial immunity. CRC Crit.Rev. Microbiol. 7:27-78, 1979.18. Roantree, R. J. Salmonella O antigens and viru-lence. Annu. Rev. Microbiol. 21:443-466, 1977.19. Buddingh, G. J. The chick embryo for the study of infec-tion and immunity [editorial]. J. Infect. Dis. 121:660-663, 1970.20. Tieffenberg, J., Vogel, L., Kretschmer, R. R., Padnos,D., Gotoff, S. P. Chicken embryo model for type IIIgroup B beta-hemolytic streptococcal septicemia. Infec.Immun. 19:481-485, 1978.21. Powell, C. J., Jr., Finkelstein, R. A. Virulence of Escheri-chia coli strains for chick embryos. J. Bacteriol.91:1410-1417, 1966.

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24. Bar-Shavit, Z., Ofek, I., Goldman, R., Mirelman, D.,Sharon, N. Mannose residues on phagocytes as recep-tors for the attachment of Escherichia coli andSalmonella typhi. Biochem. Biophys. Res. Commun.78:455-460, 1977.25. Mangan, D. F., Snyder, I. S. Mannose-sensitive interac-tion of Escherichia coli with human peripheralleukocytes in vitro. Infec. Immun. 26:520-527, 1979.26. Thorne, G. M., Deneke, C. F., Gorbach, S. L. Hemag-glutination and adhesiveness of toxigenic Escherichiacoli isolated from humans. Infec. Immun. 23:690-699,1979.

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