Zbl. Bakt. Hyg. A 259, 367-377 (1985)

Proteins from Salmonella R-Mutants Mediating ProtectionAgainst Salmonella typhimurium Infection in MiceII. Protection Tests Performed with Proteins Freefrom Lipopolysaccharide

S. SCHLECHT and NIRUPAMA BHATNAGAR 1

Max-Planck-Institut fur Immunbiologie, Freiburg i. Br.

With 2 Figures ' Received August 14, 1984 . Accepted October 18, 1984

Abstract

Lipopolysaccharide-free prote ins obtained from R-mutants of Salmonella typh imurium,S. minnesota and S. dublin as well as those from an S-form of S. typhimurium mediatedprotection in mice against experimental infection with S. typbimurium. The protection wasmeasured by LD50 and is statistically significant. The level of protection of all the prepara­tions was similar. Non-bacterial proteins had no effect under similar experimental condi ­tions. Protection afforded by the purif ied proteins was lower than that of the correspondingcomplex crude extract; supplementation of proteins with lipopoly saccharide and phospholi­pids in model membrane vesicles enhanced their potency . However, lipopolysaccharides orphospholipids alone were not able to increase the efficacy of purified proteins. Some otherfractions obtained by gel filtration of the crude extract also afford protection.

Zusammenfassung

Aktive Immun isierung mit isolierten, lipopolysaccharidfreien Proteinen aus R-Mutantenvon S. typbimurium, S. minnesota und S. dublin sowie aus einer S-Form von S. typhimuriumvermittelt im Mausversuch einen sratistisch signifikanten Schutz gegen experimentelle lnfek­tion mit S. typh imurium, gemessen anhand der LD5o• Die Hohe des Schutzes liegt fur ailePraparate auf gleichem Niveau. Proteine nicht-bakterieller Herkunft weiscn dagegen keinesignifikante Schutzwirkung auf. Der Schutz durch die gereinigten Salmonella-Proteine istgeringer als der durch die zugehorigen, komplex zusammengesetzten Rohextrakte hervorge­rufene . Supplementierung der Proteine mit Lipopolysaccharid in Verbindung mit Phospholi­pid im Vesikel-Modell fiihrt in einigen Fallen wieder zu einer Verstarkung des Schutzes; eineRekonstituierung der Komponenten zu ihrer natiirlichen Anordnung ist aber offenbar vonnoch unbekannten Parametern abhangig, Lipopolysaccharid oder Phospholipid allein warennicht in der Lage, die Prote inwirkung zu verstarken. Neben den lipopolysaccharidfreienProtein-Praparaten rufen auch andere Fraktionen, die bei der Gelfiltration der Rohextrakteerhaltcn werden, Infektionsschutz hervor.

I Present address: Dept . of Experimental Medicine, P. G. I, Chandigarh-160012, India

368 S.Schlecht and N. Bhatnagar

Introduction

Surface proteins of various bacteria have been implicated to afford protectionagainst infections caused by these organisms, e.g. Brucella, Pasteurella, Pseudomonas,Escherichia or Neisseria (1, 5,6, 15,23,27,33). Attempts have also been made todevelop a vaccine based on Salmonella proteins (3, 13, 17, 24, 26, 28). However, allpreparations obtained from Salmonella were contaminated with other subcellular com­ponents. Under such circumstances the role of various contaminants as possible immu­nogens or at least in enhancing the efficacy of the proteins can not be ruled out. In thisrespect Kuusi and co-workers (14) showed tli.at porin preparations from S. typhimu­rium, when freed from minimal amounts of contaminating lipopolysaccharide (LPS),lost their protective power. Thus, in order to evaluate the efficacy of Salmonellaproteins for protection, it is essential to free them from contaminants which maypossess immunological activity.

In preceeding papers (4, 20, 31), methods have been described for extraction andpurification of proteins from smooth strains as well as from rough mutants of Salmo­nella. The proteins could be obtained free from LPS, the main antigen of the bacterialsurface. They still contain some, if only minimal amounts of phospholipids (PL) andnucleic acids (NA). This paper demonstrates that active immunization of mice withsuch purified proteins affords significant protection against experimental infectionwith S. typhimurium.

Material and Methods

Vaccine

a) Urea extracts were obtained from S-form and R-mutants of S. typhimurium (S. tm), S.minnesota (S. m), and S. dublin (S. du) as described previously (20, 31). Preparationscontaining 40 or 20 ug of protein were used as single dose.

b) Different fractions of urea extracts, obtained by gel filtration and/or ion exchangechromatography (4). Mostly LPS-free protein preparations were used, the single dose con­taining in general 40 I-tg and in exceptional cases 20 or 10 ug protein (see Text). With theother fractions the dry weight equivalent to the corresponding protein fraction was given. Ifnecessary, suspensions underwent a short treatment in an ultrasonic bath for better distribu­tion of material.

c) LPS-free proteins supplemented either with LPS from S. tm 1135 (S), S. tm his 386(Ra), S. tm SL 1032 (Rdj ) , S. m R 595 (Re) or with PL from S. trn TV 227 (Ra), S. tm SL1032 (Rd1) or a combination of both substances. To form membrane vesicles the suspen­sions were sonicated as described in (12) for 4 min in a Branson Sonifier Cell Disrupter B15with microtip. The vesicles were used for immunization within 2 h.

d) Non-bacterial proteins. Bovineserum albumin (BSA), purified and crystallized (Serva,Germany) and ovalbumin Lot 47C-3928 (Sigma) were used. The single dose contained 40ug of protein.

Each vaccine preparation was suspended in 0.9 % saline containing 0.001 % Hibitan,Final volume of each dose was 0.2 ml.

Animals

Female NMRI mice (WIGA, Sulzfeld, Germany) weighing 18-20 g were used. The ani­mals were randomized in groups of 8 or 10. They were fed on standard feed (Herilan MR 5)and water ad libitum and kept at 22-23 "C.

Proteins from Salmonella R-Mutants 369

Immunization and infection

The mice were immun ized intraperitoneall y (i. p.) twice at intervals of 14 days as descri­bed earlier (20). Ten days after the second immunization the animals were infected with ten­fold dilutions of S. typhimurium C5 through the i. p. route and were monitored for aminimum of 15 days, in some cases for a prolonged period of 35 days. The LDso (25) wascalculated on the basis of mortality after 15 days.

Results

1. Protection by LPS-free proteins

LPS-free protein preparations obtained from bo th S-form and R-mutant s of Salmo ­nella were found to be protective against S. typhimurium infection. The efficacy is

Table 1. Protection mediated by LPS-free proteins of Salmonella against experimental infec­tion with S. typhimurium in NMRI mice

Mate rial used for Prepa- Single Contamination LDso in Mice afterImmunization ration Dose with LPS Challenge with

Protein S. typhimurium C5f.lg

Non immunized anima ls 6.3 x 101 - L2 X 103

0.9 % NaCl solution L2 x 102 n.s,Bovine Serum Albumin (Serva) 40 LO x 103 n.s.Ovalbumin (Sigma) 40 4.1 x 103 n.s,

S. typhimurium 1135 (S) UE 1 20 + 1.4 X 107**Protein 10 3.3 X 104**

S. typhimurium his 386 (Ra) UE 1 40 + L7 X 106**Protein 40 3.0 x 104**

S. typhimurium TV 166 (Rb2) UE 3 40 + 4.6 X 105**Protein 20 2.3 x 103 n.s.

S. typhimurium SL 1032 (Rd1) UE 1 40 + 6.5 X 105..Protein 40 3.5 x 104*

S. typhimur ium SL 1102 (Re) UE 1 40 + 4.0 X 104••

Protein 40 L2 x 104•

S. minnesota R 345 (Rb2) UE3 40 + 2.1 X 104*Protein 40 L1 x 104..

S. dublin R1 UE 40 + 9.2 X 104..

Prote in 40 3.1 X 104..

Mice were immunized i.p. twice at an interval of 14 days and infected i. p. 10 days after thesecond immunization by graded amounts of S. typhimurium C5. The LDso (25) presents thefinal mortality at 15 days p.i,UE: Urea extract, dialyzed and lyophilized (20).Protein : LPS-free pro tein of the corresponding urea extract, obtained after gel filtration or

ion exchange chromatography (4).+: LPS present

no LPS present; ~-hydroxymyristic acid content < 0.001 % according to (4).*, **: significant (P <: 0.01) or highly significant (P <: 0.001 ) difference in mortality as

compared to corresponding non immunized anima ls (34).n.s.: difference in mortality statistically not significant.

370 S.Schlecht and N. Bhatnagar

5,typhimurium (5 )

5,typhimur ium his 386 (Ra)

5.typhim ur ium 5L 1032 (Rd, )

5.dub lin Rl (Ra)

100

80

60

>­oc~ .£ 40o- rn<1lt::~ au.~<1l

.~ '0~ 20:JE:JU

/1,:;;'~ \Z"~"!./~_' ~ ':.."".! r:." ~":':~':..'7"':':, ,!:,'::'~~1"! ''':''':'-' .....:.',; •• :..' . • .

I ,

f -'i/ i

/ :.....;

5 ::! j" .';,.

I'!"",1' ;s :

'I":!, f

"",!n,."~r.:..:t ,t;

l:r.~.

tI~

"d~.

5 10 15 20 25 30

Days p. i.35

Fig. 1. Relative cumulative frequency of mortality in NMRI mice immunized with LPS-freeproteins from different Salmonellastrains and challenged with S. typhimurium C5. Immuni­zation and challenge procedures were the same as given in Table 1. The single dose ofprotein was 10 ug for S. typhimurium 1135 (S) and 40 ug for the other strains. Challengewas performed in the range of 102 to 107 infecting bacteria in animals vaccinated with S.dublin Rl and of 104 to 107 bacteria with the other vaccinated groups.

represented as LDso (Table 1). The mortality in immunized animals is remarkablylower than in the nonimmunized group or in animals treated with proteins of non­bacterial origin. Furthermore, it also differs in most cases statistically (34) from that ofthe infection controls to a significant or even highly significant degree. Amongst theproteins of the seven mutants used, only one preparation, obtained from S. typhimu­rium TV 166, was ineffective. Protection elicited by the crude urea extract in mostcases was better than that by the corresponding purified proteins.

Fig. 1 shows some typical examples of the time course of mortality in animals. Thesurvival rate can be considered as stabilized 15 days after challenge. Death after thisperiod occurs only in exceptional cases. Thus, evaluation of protection on the basis ofLDso after 15 days can be considered as adequate. Table 2 shows the relation betweenthe dose of the vaccine and protection. At lower levels of the protein, variation in theLDso values is not very significant.

2. Other cellular components mediating protection

Other fractions obtained by column chromatography of the urea extracts (4) containvarying amounts of LPS, PL and NA in addition to smaller amounts of differentpolypeptide species. Table 3 demonstrates that peak 1 which is eluted with the void

Proteins from Salmonella R-Mutants 371

Table 2. Dose dependence of protection mediated by LPS-free protein against S. typhimu­rium infection in NMRI mice

Bacterial Strain

S. typhimurium 1135 (S)

S. typhimurium SL 1032 (Rdt )

Single Dose LDsoProtein Control ImmunizedI!g Animals Animals

1 1.2 X 103 7.0 X 103

10 3.3 X 104

4 1.2 X 102 1.4 X 104

40 3.5 X 104

200 6.9 X 101

Immunization and challenge procedures as described in Table 1.

volume is as effective as the main pro tein peak in providing protection . Material fromthis peak contains the highest amount of LPS. Other fractions also proved to beeffective, and the same was observed consistently with other Salmonella mutants.However, no general correlation appears to exist between chemical composition andprotective efficacy of the materials.

3. Protection by LPS-free prote ins supplemented with LPS and/or PL

Various LPS-free proteins were supplemented with different amounts of LPS fromthe S. typhimurium S-form or from Salmonella R-mutants as well as with PL fromSalmonella R-mutants. When mice were immunized with mixtures of either protein +LPS or protein + PL (12) no adjuvant activity could be observed at all.

Supplementation of pro tein with S-form LPS from S. typhimurium together with PLof the R-mutant TV 227, however, brought abou t a remarkable enhancement of theprotective activity of Salmonella protein (Table 4). Combinations of the same LPSwithphospholipid from other Salmonella R-mutants or combinations of PL from mutantTV 227 with another R-LPS, his 386, were either less or not at all effective.

Discussion

In Salmonealla infection, species-specific protective immunity is considered essen­tially due to the O-polysaccharide part of the lipopolysaccharide (e.g. 8, 9). Further­more, heterologous Salmonella S-forms, which do not share common O-antigen, aswell as Salmonella rough mutants including deletion mutants (21) show cross protec­tion (29, 30 ). From these reports it can be concluded that additional antigens which areresponsible for mediating species-overlapping immunity must be present on the surfaceof Salmon ella cells. This is further supported by immunization experiments carried outwith E. coli hybrids expressing S. typhi or S. typhimurium O-antigens (7, 19). Thehomologous protective capacity of such vaccines is smaller than that of the correspon­ding Salmonella vaccines, indicating that some immunizing component is missing inthe hybrids. In accordance, R-mutant vaccines from E. coli are considerably less pro­tective than those from Salmonella (32).

Tab

le3.

Pro

tect

ion

ofN

MR

Im

ice

agai

nst

infe

ctio

nw

ithS.

typh

imur

ium

byim

mun

izat

ion

with

diff

eren

tfr

acti

ons

obta

ined

from

gelf

iltra

tion

Bac

teri

alSt

rain

Pre

para

tion

Con

tam

inat

ion

(%)

Sing

lew

ithD

ose

LD

50Pr

otei

nC

ontr

olIm

mun

ized

CI4

0H

CI6

NA

(or

Dry

Wei

ght)

Ani

mal

sA

nim

als

f1g

S.ty

phim

uriu

mhi

s38

6(R

a)Pe

ak1*

2.37

1.62

0.32

(175

)1.

2x

102

1.7

X1

05

Prot

ein-

Peak

**0

0.01

40.

1340

3.0

x10

4

Peak

6***

0.00

20.

008

0.27

(175

)2.

1X

103

S.du

blin

Rl

(Ra)

Peak

1*4.

944.

340

(80)

8.2

x10

11.

6X

10

5

Prot

ein-

Peak

**0

0.06

0.29

403.

1x

10

4

Peak

5***

0.04

0.06

1.40

(80)

2.4

x1

05

Imm

uniz

atio

nan

dch

alle

nge

asde

scri

bed

inT

able

1.A

naly

tical

data

acco

rdin

gto

(4).

*V

oid

volu

me

mat

eria

lob

tain

edby

the

firs

tge

lfi

ltatio

n,**

LPS

-fre

epr

otei

nob

tain

edaf

ter

2co

nsec

utiv

eor

recy

cled

gel

filtr

atio

ns.

***

Mat

eria

lfo

llow

ing

the

prot

ein

peak

inpu

rifi

cati

onpr

oced

ure

byth

ese

cond

gel

filtr

atio

n.C

140

H~-Hydroxymyristicac

idC

16Pa

lmiti

cac

idN

AN

ucle

icac

ids

w ;j ~ V'J g. '"g. .... ~ ::l Q. ;Z tl:i

e­ ~ S ~ ()Q ~ 0;

Proteins from Salmonella R-Mutants 373

Table 4. Effect of lipopolysaccharide (LPS) and phospholipid (PL) or lipopolysaccharidealone on the protective capacity of LPS-free proteins from R-mutants

Material used for Immunization LDso in Mice afterChallenge withS. typhimurium C5

Non immunized animals 6.6 x 100-2.8 x 101

S. duo R1 (Ra) protein, 20 ug 1.1 X 104

S. duo R1 (Ra) protein, 20 I!g + 20 ug LPS (1135) 8.3 x 104

S. duo R1 (Ra) protein, 20 I!g + 20 ug LPS (R595) 2.2 x 103

S. para. C E. Afr, R3 (Ra) protein,40 I-lg 3.0 x 103

S. para. C E. Afr. R3 (Ra) protein,40 ug+ 10 ug LPS (1135)+300 ug PL1.8 x 105

S. tm. his 386 (Ra) protein, 20 f.lg 6.0 X 102

S. tm. his 386 (Ra) protein, 20 I!g + 10 ug LPS (1135) +300 ug PL3.4 X 105

Immunization and challenge procedures as described in Table 1.LPS was extracted from S. typhimurium 1135 'IS) (35) or from S. minnesota R595 (Re). PLwas extracted from S. typhimurium TV 227 (Ra) by chloroform methanol treatment (10).The mixtures used for immunization as well as protein alone were subjected to ultrasonictreatment to form vesicles (12). Suspensions were used for immunization within 2 h.

In order to identify components responsible for species-overlapping protection, prot­eins have been extracted from different Salmonella strains and have been freed fromLPS (4). This paper shows that such protein preparations evoke a certain statisticallysignificant, basal level of stable protection against infection with S. typhimurium. Inaddition to proteins isolated from a S. typhimurium S-form and several S. typhimuriumR-mutants, those from heterologous species such as S. dublin and S. minnesota are alsoequally effective. Protection by all these vaccines is practically on the same level (Table1 and Fig. 2). These results indicate that certain Salmonella proteins probably arecommon antigens shared by the different species. Recently it has been shown (30a) thatimmunoprotection by Salmonella R-mutants is mediated through immunogens and notmerely due to non-specific stimulation of the immune system. Proteins of non-bacterialorigin do not evoke protection.

The lower efficacy of the purified proteins compared to that of whole cells or to thatof the urea extracts using single doses with identical protein contents (Fig. 2) indicatesthat either partial denaturation of the proteins or removal of some other importantcomponent(s) by the process of purification may have occurred, influencing the overallimmunoprophylactic property of the vaccine. With acetone-killed bacteria protectiveactivity was found to be enhanced considerably by adding LPS (30a). However, supple­mentation of LPS-free proteins with either LPS or PL from different Salmonella strainsis without influence on protection.

Different investigators (11, 12, 14) have emphasized the immunopotentiating effectof LPS on antibody formation against enterobacterial and other proteins. In respect toprotection mediated by active immunization, however, our findings demonstrate thatin addition to LPS, presence of PL is necessary to enhance the efficacy of purifiedproteins. Furthermore the origin of PL appears to be important, although the chemicalbackground of this behaviour is unknown. Amongst the LPS's tested, G-LPS from S.typhimurium seems to be more effective than R-LPS's. Thus despite the fact that

374 S.Schlecht and N.Bhatnagar

LD50 ABC108

104

S.tm.1135

S.tm.his386

S.tm.TV166

S.tm.SL 1032

S.tm.SL1102

S.m.R345

S.du.Rl

Fig. 2. Comparison of protection afforded by acetone killed bacteria (A), urea extract (B)and LPS-free proteins (C) in NMRI mice.Conditions of immunization and challengeare thesame as given in Table 1. Single dose with acetone killed bacteria was 1 x 108

. With ureaextracts amounts of material containing 40 lAg protein were used, and with LPS-free proteinsamounts were generally 40 Ilg (* 20 ug, H 10 ug),

proteins, LPS and PL when reconstituted into membrane vesicles function in a physico­chemical way (22), their arrangement according exactly to the natural membraneorganization is apparently difficult and depends on still unknown factors (see also 16).The immunological efficacy of the urea extracts might be considered as an indicationthat original molecular interrelationships between some components still exist to acertain degree in this case.

Other fractions obtained from urea extract by gel filtration were also found to beprotective, especially the material eluted with the void volume containing highamounts of LPS and a few polypeptides. LPS is known to be ineffective in giving anyprotective response in the test system used here (30a). Thus, whether the natural LPS­protein-complex or its protein part alone (28) is responsible for protection, will have to

be ascertained.In conclusion, the results of this study clearly indicate that Salmonella proteins must

be considered as an important component in vaccines of these organisms. They areprobably responsible for interspecies cross-protection. The protein preparations usedhere are mixtures of different polypeptides (4) and identification of those which areresponsible for mediating protection remains an urgent problem. As protein patterns ofbacteria depend on cultivation conditions (2), biosynthesis of the immunological rele-

Proteins from Salmonella R-Mutants 375

vant proteins might be favoured by optimizing this process, resulting in more potentand more consistent vaccines.

An anti-Salmonella vaccine based on protein would have the advantage of being freefrom the toxic side effects of LPS. In preliminary experiments, single doses up to 1 mgof LPS-free protein have been tolerated without any sign of toxicity. Thus, a properdose given together with an appropriate non-LPS adjuvant might enhance the protecti­ve capacity to a level of practical interest.

We wish to thank Mrs. T. Franz for skilful technical assistance. - The project wassupported by the Deutsche Forschungsgemeinschaft.

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Dr. S. Schlecht, Max-Planck-Institut fur Immunbiologie, Postfach 1169, D-7800 Frei­burg i. Br.