Vol. 56, No. 11

Protection against Salmonella typhi Infection in Mice afterImmunization with Outer Membrane Proteins Isolated

from Salmonella typhi 9,12,d,ViARMANDO ISIBASI,l* VIANNEY ORTIZ,2 MARTIN VARGAS,' JORGE PANIAGUA,1 CE'SAR GONZALEZ,'

JOSE MORENO,' AND JESUS KUMATE3Laboratory of Immunochemistry, Unidad de Investigaci6n Biomedica, Instituto Mexicano del Seguro Social,

P.O. Box 73-032,1 Laboratory of Experimental Amebiasis, Instituto Nacional de Higiene,and Secretaria de Salud,3 Mexico City, Mexico

Received 25 March 1988/Accepted 21 July 1988

The current studies were undertaken to assess the ability of the outer membrane proteins (OMPs) ofSalmonella typhi to induce protection against challenge with the bacteria in mucin. OMPs were isolated as

described by Schnaitman (J. Bacteriol. 108:553-556, 1971) and were found to be contaminated withapproximately 4% lipopolysaccharide (LPS). Immunization with as little as 30 ,ug of OMPs conferred 100%protection to mice challenged with up to 1,000 50% lethal doses (LD50) of two strains of S. typhi (9,12,d,Vi andTy2). In addition, 30% protection against challenge with up to 500 LD50 of Salmonella typhimurium was

achieved. Immunization with LPS at doses equivalent to those found in the OMPs was considerably inferior tothe OMPs in the induction of an immune status. Moreover, LPS was effective only when the challenge was

performed with S. typhi 9,12,d,Vi (40% protection to 100 LDso). An antiserum raised in rabbits reacted mainlyagainst the bands of the molecular weights corresponding to the so-called porins contained in the OMPpreparation as shown by Western blotting (immunoblotting). This rabbit antiserum protected 100% of miceagainst challenge with 100 LDSO of either strain of S. typhi and 80% of mice against challenge with the same

LD50 of S. typhimurium. These results indicate the usefulness of OMPs in the induction of active immunityagainst S. typhi in mice.

Typhoid fever remains a major public health problem indeveloping countries. As yet, little is known about themicrobial factors that determine pathogenicity and those thatelicit a protective immune response in humans with typhoidinfection. Currently available vaccines containing eitheracetone- or heat-killed Salmonella typhi are of limited use-fulness because of short-lived protection and development ofunacceptable side effects resulting mainly from the presenceof endotoxin (16). Some attempts to develop a better vaccinehave yielded the orally administered strain S. typhi Ty2la,developed by Germanier and Furer (11), which is deficient inthe enzyme UDP-galactose-4-epimerase. This vaccine isdevoid of side effects and proved to be very effective in afield trial in Egypt, showing protection of 92% (36). In laterstudies performed in Chile, however, the results were not sostraightforward, and protection only ranged from 19 to 67%(10). Another orally administered strain which is streptomy-cin dependent has been shown to be ineffective (21). Themain problem with these oral typhoid vaccinations is theirhigh cost.There has been some controversy through the years

regarding the nature of the protective immunogens amongthe components of members of the family Enterobac-teriaceae. Antibodies against the somatic antigen lipopoly-saccharide (LPS) 0 correlate well with previous infectionbut not with protection against salmonellae both in humanswith typhoid (14) and in mice infected with Salmonellatyphimurium (31). Similarly, antibodies against the flagellarantigen H have not been found to be protective (14). More-over, despite some studies indicating the ability of thecapsular polysaccharidic antigen Vi to induce high titers of

* Corresponding author.

serum antibodies, there is some controversy about its role inthe induction of protective immunity against S. typhi infec-tion (28). However, one such study (1) with a 17-monthfollow-up suggested that immunization with Vi confers a

protective status against typhoid in human volunteers. Re-cently, attention has been addressed to the role of the outermembrane proteins (OMPs) of gram-negative bacteria in theinduction of specific immunity. Among these are the so-

called major proteins which include porins (OmpC, OmpF,OmpD, PhoE, etc.) and the heat-modifiable protein (OmpA)(26). Studies in mice have shown that immunization withOMPs from Neisseria gonorrhoeae and Neisseria meningiti-dis results in protection against the infections caused bythese bacteria (3, 37). In subsequent studies, it was shownthat immunization with OMPs from other gram-negativebacteria such as Haemophilus influenzae (13), Shigella flex-neri (2), and Pseudomonas aeruginosa (12) also induces a

protective status in experimental animals.Recent studies have demonstrated that OMP preparations

from a rough strain of S. typhimurium protect mice againstinfection with the bacteria as long as these OMPs are linkedto a polysaccharide which functions as an adjuvant (18, 32).Similarly, rabbit anti-OMP serum protects against S. typhi-murium infection in mice (19). Protein antigens may haveadvantages over polysaccharidic antigens because the for-mer have the capacity to induce antibodies of higher affinity(6) and also to elicit cell-mediated immunity (35), whichmight result in longer-lived protection against the bacteria.One major problem in the study of typhoid fever is the fact

that its causative agent, S. typhi, is virulent only for humansand chimpanzees (9), thereby precluding easy and extensiveexperimental murine manipulations. Murine infection withthe natural mouse pathogen S. typhimurium is the most

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INFECTION AND IMMUNITY, Nov. 1988, p. 2953-29590019-9567/88/112953-07$02.00/0Copyright © 1988, American Society for Microbiology

2954 ISIBASI ET AL.

widely accepted model for the study of immunity in typhoidfever (25). This model, however, has the disadvantage ofusing a different Salmonella strain, which brings about theneed to test the validity of the observations obtained withsuch a model in animals, and eventually humans, infectedwith S. typhi. To solve this problem, some investigatorshave used mice injected intraperitoneally (i.p.) with S. typhiin mucin (30). Although imperfect, such a model has becomewidely accepted as a test for the usefulness of the differenttyphoid vaccines available. The current studies were done toexamine the role of the OMPs of S. typhi in the induction ofprotection against an i.p. challenge with S. typhi in mucin inmice. In addition, the role of rabbit anti-OMP antiserum inthe passive transfer of immunity was also assessed. Theresults suggested that OMPs are valuable immunogens in theprotection against Salmonella infection in mice and thatpartial cross protection against at least two Salmonellaspecies can be obtained.

MATERIALS AND METHODS

Bacterial strains. The virulent strain S. typhi 9,12,d,Vi wasoriginally isolated from a patient with typhoid fever and hasbeen maintained in culture in our laboratory since 1979. S.typhi Ty2 (the strain used in the elaboration of the parenteraltyphoid vaccine) and S. typhimurium 1,4,5,12 were kindlydonated by the Instituto Nacional de Higiene in MexicoCity, Mexico.

Mice. Outbred NIH strain mice were initially obtainedfrom the National Institutes of Health, and BALB/c micewere from Jackson Laboratory (Bar Harbor, Maine). Bothstrains have been bred and kept in our animal facilities.

Isolation of OMPs. OMPs were obtained as described bySchnaitman (29). Briefly, cultures of S. typhi 9,12,d,Vi, S.typhi Ty2, and S. typhimurium 1,4,5,12 were grown inminimum salts medium (medium A) containing 5% yeastextract and 12.5% glucose and incubated in a shaker at 37°Cfor 18 h until the mid-exponential-growth phase wasreached. Cultures were harvested and suspended in 0.01 MHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid) buffer. Bacteria were treated in an OmniMixer (IvanSorvall, Inc., Norwalk, Conn.) to remove flagella and thenwere disrupted by sonication with an Ultratip Lab-sonicsystem (Lab-Line, Melrose Park, Ill.) and centrifuged at7,000 x g for 10 min to remove intact cells. The supernatantfluid was centrifuged at 200,000 x g for 45 min to obtain cellenvelopes. The cytoplasmic membrane was removed with0.01 M HEPES containing 2% Triton X-100, followed bycentrifugation at 200,000 x g for 45 min. To solubilize theOMP, the Triton X-100-insoluble fraction (outer membraneand peptidoglycan) was suspended in 0.05 M Tris containing5 mM EDTA and 2% Triton X-100 (pH 7.4) and was allowedto stand for 10 min at 23°C. Finally, the suspension wascentrifuged as above, and the OMPs were recovered in thesupernatant fraction. Protein concentration was determinedby the method of Lowry et al. (22). LPS contamination of theprotein samples was determined indirectly by measuring theconcentration of 2-keto-3-deoxyoctulosonic acid (17), usingas a standard 2-keto-3-deoxyoctulosonic acid obtained fromSigma Chemical Co. (St. Louis, Mo.).Anti-OMP antisera. Anti-OMP antisera were raised in

New Zealand rabbits (2.5 to 3 kg) intradermally injected withdifferent concentrations of OMP. The first two injections of1 mg/ml were given in Freund complete adjuvant on days 0and 7. After day 15, the rabbits received three furtherinjections of 0.15, 0.5, and 1.0 mg/ml given every other day

without Freund complete adjuvant. Rabbits were bled 10days after the last injection.SDS-PAGE and Western blots (immunoblots). Sodium do-

decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing conditions with thediscontinuous buffer systems of Laemmli (20) in a verticalslab-gel electrophoresis unit (LKB Instruments, Inc., Rock-ville, Md.). The separating gel contained 11.2% acrylamide,2.5% bisacrylamide, and 0.19% SDS in 0.35 M Tris hydro-chloride buffer (pH 8.8). The stacking gel contained 5%bisacrylamide and 0.1% SDS in 0.12 M Tris hydrochloridebuffer (pH 6.8). The running buffer was 0.025 M Trishydrochloride-0.192 M glycine (pH 8.3)-0.1% SDS. Electro-phoresis was performed at 30 mA per plate.

Electrophoretic transfer of OMPs from polyacrylamidegels to nitrocellulose paper (NCP) was done in an LKBTransphor electroblotting unit with the transfer buffer de-scribed by Towbin et al. (33). The proteins were electropho-resed at 100 mA for 18 h. NCPs were immersed for 1 h at37°C in 0.15 M NaCl-0.01 M phosphate buffer (pH 7.2) (PBS)containing 6 mM EDTA, 0.25% gelatin, and 0.1% Tween 20.After being washed in PBS plus 0.1% Tween 20 (PBS-T), theNCP was incubated for 3 h at 25C in a 1:100 dilution of thetest serum samples in 0.25% gelatin-PBS. After beingwashed in PBS-T, the NCP was incubated for 1.5 h at 25°Cwith optimal concentrations of horseradish peroxidase-con-jugated goat anti-rabbit immunoglobulin G (Organon Tekni-ka, Malvern, Pa.). After the NCP was washed twice more inPBS-T and PBS, respectively, it was soaked in a solutioncontaining PBS with 2 mM 4-chloro-2-naphthol and 0.08%H202. After a wash in tap water, the NCP was photographedand air dried in the dark.

Evaluation of active and passive protection. For the activeprotection assays, groups of 10 NIH mice, each weighing 18to 20 g, were twice immunized i.p., with a 2-week intervalbetween immunizations, with various concentrations ofOMPs from S. typhi 9,12,d,Vi dissolved in a 50 mM Tris-5mM EDTA buffer (pH 7.8). Preliminary studies showed that30 jig of OMP was the optimal concentration to elicitprotective immunity in NIH mice. Ten days after the lastinjection, mice were challenged i.p. with 20 to 1,000 50%lethal doses (LD50) of one of the three bacterial strainssuspended in 5% mucin. Control groups consisted of 10 micein which 200 ,ul of Tris-EDTA buffer with 5% mucin wasadministered i.p. For the passive protection studies, groupsof 10 mice each were given 200 ,ul of either heat-inactivated(56°C for 30 min) rabbit OMP immune serum or normalrabbit serum (control) intravenously and challenged i.p. 1 hlater with 50 and 100 LD50 of one of the Salmonella strainsin mucin. Protection was evaluated as the percent survivalduring the 10 days following the challenge.To determine the role of the LPS contamination of the

OMP preparations in the induction of immunity, groups of 10mice were immunized i.p. with a concentration of LPS (1.2,ug) equivalent to that contained in 30 ,g of OMP andchallenged as described above for the active immunizationstudies.

Miscellaneous. S. typhi 0901 LPS was extracted by thephenol-water method (38). This LPS was electrophoresed inpolyacrylamide gels and stained by the technique describedby Tsai and Frasch (34). Flagellar antigen was obtained asdescribed by Ibrahim et al. (15). Rabbit antibody titersagainst S. typhi OMPs and flagella were determined by anenzyme-linked immunosorbent assay technique (7). TheLD50 for each of the Salmonella strains used in the current

INFECT. IMMUN.

ROLE OF PORINS IN PROTECTION AGAINST S. TYPHI 2955

1 2 3 4 5

97 Kd

66 Kd

42 Kd

31 Kd

21 Kd

14 Kd

FIG. 1. SDS-PAGE of Salmonella OMPs. Lanes: 1 and 5, mo-lecular size markers (all from Bio-Rad Laboratories, Richmond,Calif.): phosphorylase b (95 kDa), bovine serum albumin (66 kDa),chick ovalbumin (42 kDa), carbonic anhydrase (31 kDa), soybeantrypsin inhibitor (21 kDa), and hen egg lysozyme (14 kDa); 2, S.typhi 9,12,d,Vi; 3, S. typhi Ty2; 4, S. typhimurium.

studies was calculated as described by Reed and Muench(27).

RESULTS

OMP preparation. S. typhi and S. typhimurium OMPsisolated as described above had a 4% contamination withLPS. Figure 1 shows the SDS-PAGE pattern of the OMPs ofS. typhi 9,12,d,Vi, S. typhimurium, and S. typhi Ty2. Thereare two major groups of proteins. One is between 36 and 41kilodaltons (kDa), which corresponds to the molecular sizeranges of the known gram-negative porins and OmpA. Theother group of proteins is located between 23 and 28 kDa.These proteins do not correspond to any of those classifiedto date. In addition, a band of approximately 17 kDa and anumber of minor proteins with molecular sizes ranging from17 to 70 kDa can be observed. Figure 2 shows the SDS-PAGE pattern of flagellar antigen of S. typhi 9,12,d,Vi. Twomajor bands of about 58 kDa are observed; similar bands arenot present in OMPs.

Figure 3 shows the Western blot analysis of the anti-OMPimmune rabbit serum. It appears to be reacting with greaterintensity with the protein bands located between 36 and 41kDa which correspond to the so-called porins and OmpA; noreaction against the two similar major bands of the flagellarantigen was observed. Immune serum raised against S. typhi9,12,d,Vi also reacted against the other two strains, which isnot surprising because there is extensive homology amongthe porins of different bacteria belonging to the familyEnterobacteriaceae (8, 23).

Active protection studies. The three Salmonella strainsused in this study had different LD50. Thus, the LD50 for S.typhi 9,12,d,Vi was 1,500 x 102 bacteria, for S. typhi Ty2 itwas 400 x 102, and for S. typhimurium it was 1.6 x 102. Thelatter value is higher than that reported in the literature.However, in these studies S. typhimurium was injected inmucin, which renders the mice more sensitive to the chal-lenge, whereas in most other studies, the bacteria wereinjected in saline. Also, the results were different whenmales or females were used (males being more sensitive).

97 Kd .

66 Kd * 4

43 Kd

31 Kd

21 Kd

14 Kd

FIG. 2. SDS-PAGE of S. typhi 9,12,d,Vi flagellar antigen.Lanes: 1, molecular size markers as described in the legend to Fig.1; 2, flagellar antigen.

Therefore, in each experiment performed in these studies,female mice were always used. Finally, since the resultsobtained with BALB/c mice (data not shown) were verysimilar to those obtained with the NIH strain, all the resultsgiven are from experiments performed with NIH mice.The results of active protection induced by immunization

with doses of OMPs from S. typhi 9,12,d,Vi ranging from1.25 to 30 ,ug are depicted in Fig. 4. Immunization with aslittle as 10 ,ug of OMPs gave 100% protection against achallenge with 20 to 500 LD50 of both strains of S. typhi (Fig.4A and B). Even with doses as high as 1,000 LD50 of S. typhiTy2 (which was almost four times as virulent as the 9,12,d,Vistrain), the degree of protection was 100%. When the mice

45 KD

29 KD-

1 23 4

FIG. 3. Western blot analysis of rabbit anti-OMP antiserum.Lane 1, Molecular size markers: chick ovalbumin (45 kDa) andcarbonic anhydrase (29 kDa). NCP was stained with India ink. Lane2, 3, and 4, S. typhi 9,12,d,Vi, S. typhi Ty2, and S. typhimuriumOMPs, respectively, reacted with rabbit anti-OMP serum and de-veloped with peroxidase-labeled goat antirabbit antiserum.

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2956 ISIBASI ET AL.

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with S. typhi Ty2; (C) S. typhimurium challenge. Data are expressed as percent survival after a 10-day observation period. Doses used forimmunization were 1.25 ,ug (0), 10 ,ug (l), and 30 ,ug (0). Some mice were not immunized (A).

were challenged with S. typhimurium, however, the resultswere different, since immunization with 30 ,ug (the highestdose used) gave a protection of about 45% against 1,000LD50 (Fig. 4C). Nevertheless, the results obtained for im-munized mice challenged with S. typhimurium were some-what variable and not as reproducible as those obtained formice challenged with both strains of S. typhi. Controlsinjected i.p. with 5% mucin as well as uninjected mice (datanot shown) all died with as little as 50 LD50 of S. typhi9,12,d,Vi and 20 LD50 of S. typhi Ty2 and S. typhimurium.In Fig. 4 it can also be seen that there is a linear correlationbetween the virulence of the bacterial strain used and thedose of OMP required to obtain protective immunity. Theseresults suggest that vaccination of mice with OMP confersprotective immunity against the live bacteria.

Figure 5 shows the 10-day-survival kinetics of a group ofmice immunized twice with 30 ,ug of S. typhi OMPs. As seen,this immunization schedule gave 100% protection against500 LD50 of both S. typhi strains, whereas only 30% protec-tion against S. typhimurium was seen, which raised to 60%when only 20 LD50 were used for the challenge. Theseresults indicate that these OMPs contain an immunogencapable of inducing active protection against a challengewith live bacteria. In addition, some degree of cross protec-tion between S. typhi and S. typhimurium was observed.To assess the role of the LPS contained in the OMP

preparation in the induction of active immunity against S.

typhi, we immunized mice with purified LPS obtained fromS. typhi 0901. Figure 6 shows the results of immunization ofmice with 1.2 ,ug of LPS compared with immunization with30 jig of OMPs, which contains approximately 4% LPS (or1.2 ,ug) as determined by the 2-keto-3-deoxyoctulosonic acidcontent in the samples. This concentration of LPS gave aprotection of only 40% against a challenge with 100 LD50 ofS. typhi 9,12,d,Vi, whereas no protection at all was elicitedagainst the other two strains, despite the fact that theantigenic serotypes of the polysaccharides in both strains ofS. typhi are 9,12. Thus, the degree of protection induced byLPS is considerably lower than that induced by the wholeOMPs, suggesting a role for the proteins in the induction ofprotective immunity against infection with S. typhi.

Passive transfer of protection with rabbit anti-OMP serum.Finally, to determine the role of humoral immunity in theprotection of mice against S. typhi by OMPs, we performedpassive immunization studies by injecting rabbit anti-OMPantiserum into mice 1 h before a challenge with the livebacteria in mucin. The results shown in Fig. 7 indicate thatOMP-immune rabbit serum was able to confer 100% protec-tion against a challenge with 100 LD50 of both strains of S.typhi and 80% protection against S. typhimurium, although itwas not as effective as the active immunization, which wasable to protect against up to 500 LD50. Controls injected withrabbit anti-OMP serum absorbed with OMPs from S. typhi9,12,d,Vi showed no protection at all (data not shown).

I I

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so

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50-

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D AY S PO ST - CH ALL ENSEFIG. 5. Kinetics of survival of a group of mice immunized with 30 jig of S. typhi 9,12,d,Vi OMPs during the 10-day observation period.

, Challenge with 20 LD50; -----, challenge with 500 LD50 of the indicated bacterial strain.

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_ ^̂ ^ the world are approximately 12 million cases per year with3 -u an incidence of 540 new cases per 100,000 general population

in developing countries (5). The major causes of the preva-A A Solmon/llo typhi 9,12, d, Vi lence of this disease are related to inadequate disposal of

_\OSalmonella typhi Ty2 feces and inappropriate water supply. As long as these\ OSolw'onelloa typaimrium problems are not solved, enteral diseases will remain one of_\\, the leading causes of morbidity in third world countries. An

alternative is the development of an effective vaccine againstS. typhi. Currently available vaccines are not satisfactorybecause of undesirable side effects, a lack of sustained

b n effectiveness, or both. Attempts to define the antigens capa-.....Zh I I, i' , ,ble of inducing a protective status against gram-negative

0 100 200 300 400 500 600 bacteria have shown that LPS as well as other surface

NUMBER OF LD 50 antigens such as the flagellar (H) antigen, despite eliciting aNUMBEROF10-50 ~good antibody response, do not induce protection (14).

6. Role of LPS in the induction of active protection. Mice Studies of the capsular Vi polysaccharide, despite showingmunized with either 30 ,ug of S. typhi 9,12,d,Vi OMPs (A,O, that it elicits a good antibody response, have given con-.25 ,ug of LPS from S. typhi 0901 (A, 0, O). Results are flicting results with regard to the protection conferred byed as percent survival during a 10-day observation period. these antibodies. Most studies in humans have not found a

close correlation between the presence of anti-Vi antibodiesresults indicate that humoral immunity plays an and a protective status against typhoid (14), and in studies inant role in the protection against infection with S. which protection was obtained with the Vi antigen, then mice. Moreover, the results suggest that besides follow-up has not been long enough to draw conclusions.antibodies, other mechanisms are implicated in the In the current studies, a preparation containing OMP, 4%ion against S. typhi in mice. LPS, and no detectable flagellar antigen derived from S.

typhi induced protection against a challenge of up to 1,000DISCUSSION LD50 (maximum tested) of the relevant bacteria in NIH

mice. In addition, some degree of cross protection was foundcurrent studies were undertaken to define the role of between S. typhi and the closely related S. typhimurium.[Ps of S. typhi in the induction of protective immunity That the protection seen was due to the OMPs is stronglya challenge with live bacteria. An animal model was suggested by the fact that immunization with LPS at thewhich mice were injected with S. typhi obtained from same concentration as that found in the protein preparationfferent sources or with S. typhimurium suspended in conferred minimal if any protection to similar animals. TheseThe major findings of these studies can be summa- results suggest that OMPs are valuable immunogens for thes follows. OMPs are good immunogens in the induc- prevention of typhoid fever. Moreover, the cross protectionprotective immunity against the relevant as well as seen indicates that the immune response is directed at sharedrelated bacteria of the genus Salmonella. LPS or epitopes between these Salmonella species. This observa-

Lr contamination appears to play a minimal if any role tion could also have valuable applications in the future forinduction of the protective status. Humoral immunity the development of a vaccine against more than one speciesin important role in the protection observed in this of gram-negative pathogens. In agreement with these obser-but it appears that other mechanisms may also be vations is the fact that by gene sequencing it has been found

Zd. that there is extensive homology among the OMPs, specifi-toid fever is still an unsolved health problem in the cally the porins, of different gram-negative bacteria (8, 23),ty of the world. Estimates of the number of cases for despite some polymorphism. The fact that the degree of

cross protection was not absolute indicates that responsesdirected against the private epitopes of S. typhi may alsoplay a role in the protective status.

Studies testing the role of OMPs from other gram-negativebacteria have shown that these proteins are good immuno-gens in the induction of protection against the infection of

AASolmonello typhi 9,12,d,Vi animal models with N. gonorrheae (3), N. meningitidis (37),*ESolmonello typhi Ty2 P. aeruginosa (12), H. influenzae (13), and Shigella sonnei* 0 Salmonella typhimurium and S. flexneri (2). In addition, OMPs from other pathogens

such as Chlamydia trachomatis (4) are good immunogens inthe induction of protection against the relevant bacteria.However, none of those studies examined the induction ofcross protection by OMPs. Moreover, the role of cell-mediated immunity against these immunogens has not been

50 100 150 analyzed. In the studies reported here, rabbit anti-OMP

NUMER OF LD-50 serum was capable of passively transferring a protective7. Passive protection of mice by rabbit anti-S. typhi status against a challenge with S. typhi. It is interesting thatVi antiserum. Mice were given 200 >t1 of heat-inactivated the Western blot analysis of such serum showed that itrabbit serum (A, *, 0) or rabbit anti-OMP serum (A, 0, Q) reacted mainly against the OMPs migrating between 37 andlater were challenged with the indicated LD50 of the bacterial 45 kDa, which corresponds to the porins and OmpA. Thesedepicted. Results are expressed as percent survival during a results suggest that these proteins are the main target of thisobservation period. immune response and indicate that humoral immunity di-

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2958 ISIBASI ET AL.

rected against OMPs (probably the porins) plays an impor-tant role in the immunity against S. typhi, although someother acquired mechanisms appear to be involved.

It seems unlikely that other contaminants such as theflagellar H antigen played a role in the results obtained forseveral reasons. First, the bacteria were treated before lysiswith an OmniMixer to remove the flagella; second, OMPpreparations as seen by gel electrophoretic analysis did notcontain flagellar antigen; therefore, if present, these antigenswere at minimal concentrations, insufficient to induce animmune response against them. This is confirmed becausethe rabbit anti-OMP serum failed to react with purifiedflagella in an enzyme-linked immunosorbent assay (data notshown). Currently, we are analyzing the role of a morepurified OMP preparation obtained as described by Nikaido(24), which besides being almost free of LPS is highlyenriched for the porins. Preliminary results with this anti-genic preparation have confirmed our current observations.In addition, we are studying the role of cell-mediated immu-nity in the protection against S. typhimurium in mice.Preliminary results indicate a high degree of cross-reactivityin T-cell responses against porins obtained from S. typhimu-rium, S. typhi, and Escherichia coli in animals immunizedwith either whole acetone-killed S. typhimurium or purifiedporins (J. Moreno et al., manuscript in preparation).The best animal model with which to study the pathogen-

esis and perhaps the nature of the immune response againstSalmonella antigens is the natural infection of mice with thepathogen S. typhimurium. This is done by challenging micewith the bacteria administered either orally or parenterally(25), both resulting in a similar pathogenic picture. Theadvantage of this model is that it closely resembles thepathogenesis of human typhoid. However, it presents somedisadvantages for our studies. First, there are several factorsof natural as well as induced resistance that play a role in thedevelopment of mouse typhoid, and the human counterpartsto these are unknown. Second, the aim of this study was todefine the antigenicity and protective capacity of an antigendirectly derived from the human pathogen and not a cross-reacting antigen from S. typhimurium. For these reasons, thestudies reported here were performed in a model that makesuse of mice challenged with bacteria in mucin. This is knownto increase susceptibility to S. typhi, but it also increasessusceptibility to less aggressive bacteria such as E. coli,which could be a disadvantage. The major advantage of thismodel is that it analyzes the protection induced with antigensderived from the human pathogen against the same patho-gen.

Since our model is an artificial one, these results on theability of S. typhi OMPs to induce protection against paren-teral challenge by Salmonella species in mucin must beconfirmed in a model with an oral administration of themouse-virulent S. typhimurium. Nevertheless, our resultsagree with those obtained by other researchers of micetyphoid. Thus, Kussi and co-workers in a series of studies(18, 19) have found that porins from S. typhimurium areimmunogenic and play a role in the protection against mousetyphoid. Further studies showed that the induction of im-mune status is optimal when the porins are administeredlinked to polysaccharides, which play the role of adjuvants(32). Moreover, it was shown that the protective status couldbe transferred with antiporin serum. The role of cell-medi-ated immunity against OMP in such a model remains to bestudied. Because proteins are T-cell-dependent antigens, it isto be expected that these proteins will induce cell-mediatedresponses.

In conclusion, we found that OMPs and perhaps porinsmight be valuable immunogens for the induction of a protec-tive status against typhoid fever. Our current studies aretesting the specific role of the porins in the induction of thisimmunity and the roles of cell-mediated response and anti-bodies in the induction of this protective status.

ACKNOWLEDGMENTS

This study was supported by project grant PCSABNA-022764from the Consejo Nacional de Ciencia y Tecnologia, Mexico. V.0.and M.V. were recipients of a scholarship from CONACYT.

LITERATURE CITED1. Acharya, I. L., C. L. Lowe, R. Thapa, V. L. Gurubacharya,

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