Immunology 1990 70 315-320

Malaria exoantigens induce T-independent antibody thatblocks their ability to induce TNF

C. A. W. BATE, J. TAVERNE, A. DAVE & J. H. L. PLAYFAIR Department of Immunology, University College &Middlesex School of Medicine, London

Acceptedfor publication 13 March 1990

SUMMARY

Much of the pathology of malaria may be due to the interactions of cytokines, especially tumournecrosis factor (TNF), with various cell types, including endothelial cells, with consequentwidespread systemic effects. It has been shown previously that heat-stable exoantigens in thesupernatants of blood-stage parasite cultures induced the release of TNF in vitro from activatedmacrophages and behaved like toxins in vivo, that mice immunized with the antigens are protectedfrom the toxic effect and that their serum specifically blocks the ability of the antigens to stimulate the

production ofTNF. It is reported here that the inhibitory antibody is mainly IgM and that it appearsto be T-independent, as the titres of antisera from T-deficient and immunologically intact mice were

similar. Antisera raised against exoantigens from two species of rodent parasite inhibited TNFproduction by those ofthe human parasite Plasmodiumfalciparum, and vice versa, indicating that theTNF-inducing moieties of the exoantigens cross-react and therefore presumably contain commonepitopes. Thus vaccination with these exoantigens might provide a means of protection against theclinical effects of malaria and of generating anti-disease immunity by reducing cytokine production.However, these findings imply that it will be necessary to confer on these antigens the ability to

stimulate T cells and generate memory before they can provide a useful basis for an anti-diseasevaccine. The results obtained are discussed in relation to some of the epidemiological features of

malaria.

INTRODUCTION

It has been known for some time that individuals who live inareas where malaria is endemic normally acquire immunity tothe symptoms of the disease while their parasitaemia is still high(McGregor et al., 1956). The basis of this anti-disease immunityis not yet understood, but it has been argued that it may be dueto the development of antibody against toxic malarial antigensthat induce pathology indirectly via a cytokine network (Play-fair et al., 1990). There is considerable evidence to support theview that many of the clinical symptoms may be associated inparticular with the release of tumour necrosis factor (TNF) byactivated macrophages of the infected host (Clark, 1987), and ithas been shown that TNF is released into the circulation duringacute infection. Thus, for example, the amount of TNF in theserum of patients infected with Plasmodiumfalciparum is foundto correlate with the severity of the disease (Kern et al., 1989)and TNF appears in the serum ofmice infected with P. vinckei inparallel to the development of the parasitaemia (Clark &Chaudhri, 1988).

Furthermore, mononuclear cells from patients in the acutephase of the disease have an enhanced capacity to secrete TNF

Correspondence: Dr J. Taverne, Dept. of Immunology, ArthurStanley House, 40-50 Tottenham St, London WIP 9PG, U.K.

(Kwiatkowski et al., 1989), as do macrophages in the spleensand livers of mice infected with rodent malarial parasites(Taverne, Treagust & Playfair, 1986; Taverne et al., 1987).Neutralizing antibody against TNF has been shown to protectmice from developing cerebral malaria (Grau et al., 1988);although this syndrome differs in many respects from cerebralmalaria in man, TNF enhances the expression ofan intracellularadhesion molecule on endothelial cells to which schizonts of P.falciparum bind (Berendt et al., 1989). It may thus alsocontribute to the development of cerebral malaria in man byenhancing the attachment ofparasites to capillaries in the brain.

It has been shown previously that soluble parasite antigensfrom the blood-stage rodent and human parasites stimulateactivated macrophages to release TNF in vitro (Bate, Taverne &Playfair, 1988; Taverne et al., 1990) and that those of the rodentparasites can also do so in vivo (Bate, Taverne & Playfair, 1989).Furthermore, Kwiatkowski et al. (1989) reported that whenhuman monocytes were cultured with P. falciparum more TNFwas detected in the medium at the time of schizont rupture,which is also the time in the growth cycle of the parasite whenexoantigens are released and fever becomes manifest. It issuggested, therefore, that the liberation of such exoantigensduring infection may cause the production of cytokines that areassociated with some of the pathological changes of malaria.

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Hyperimmune serum (Bate et al., 1988) and primary antiseramade against the exoantigens (Bate et al., 1989; Taverne, Bate &Playfair, 1989; Taverne et al., 1990) specifically inhibit theinduction of TNF by these exoantigens, but not by bacteriallipopolysaccharide (LPS). Again, when mice sensitized to TNFby D-galactosamine treatment were immunized with the anti-gens they were specifically protected against death caused byinjection of the antigens (Bate et al., 1989). In the present studyevidence is described that shows that the blocking antibodygenerated by injection of malarial exoantigens is predominantlyT-independent, an observation that may explain some of thefeatures of anti-disease immunity and which has implicationsfor vaccine design.

MATERIALS AND METHODS

MiceOutbred females, at least 6 weeks old, were used (Tuck No 1, A.Tuck & Sons, Battlesbridge, Essex). Female BALB/c nude micewere obtained from the National Institute of Medical Research,Mill Hill, London.

Rodent parasitesPlasmodium yoelii 17X, its lethal variant (Freeman & Holder,1983), and P. berghei ANKA were used (both from Dr N.Wedderburn).

Parasite exoantigensSoluble antigens from rodent parasites were obtained aspreviously described (Bate et al., 1989) except that 108/mlparasitized erythrocytes were incubated overnight in phos-phate-buffered saline (PBS) at 370 upon rollers. The super-natants were collected, boiled for 5 min, centrifuged, filtered andstored at 4°. The medium of a continuous flow culture systemwas used as a source of exoantigens of P. falciparum Nf 54(Taverne et al., 1990). It was boiled, centrifuged, concentrated10 times, filtered and stored at 4'.

AntiseraMice were injected i.p. with 0-2 ml of boiled supernatants andthey were generally bled 10-12 days later. Sera were pooled fromthree or more mice and then heat-inactivated at 560 for 30 min.These primary antisera were titrated for their ability to block theinduction of TNF by parasite exoantigens by mixing equalvolumes of serial dilutions with a constant dilution of anexoantigen preparation before addition to peritoneal macro-phages. The inhibitory titre is defined as the reciprocal of thedilution that blocked 50% ofTNF release. Since this varies withthe concentration of antigen, the figures quoted are alwayscomparative titres, determined against the same preparation ofantigen in the same test. Antisera were depleted of IgG and IgMon isotype-specific agarose columns (Immobilized anti-mouseimmunoglobulins, Calbiochem, La Jolla, CA). The affinity-purified IgG and IgM fractions were then eluted with 0 1 Mglycine-HCl buffer at pH 2 5 into 1 0 M Tris buffer at pH 8 anddialysed against PBS.

Hyperimmune sera against lethal P. yoetii were kindlyprovided by Mr J. B. de Souza of this department. They wereobtained from mice that had been vaccinated with Triton X- 100lysates of parasitized erythrocytes, injected with saponin asadjuvant, then infected and bled 5-10 days after recovery(Playfair & De Souza, 1986).

Antisera from mice depleted of CD4+ T cellsA group of mice was treated for three consecutive days byinjection, first i.v. then twice i.p., of 400 ,ug/mouse of a ratmonoclonal antibody (YTS 191 1; from Dr N. Wedderburn)against the mouse L3/T4 epitope P (Cobbold et al., 1984) beforeinjection of parasite exoantigens. This treatment consistentlydecreased the number of spleen cells staining by FACS analysisfrom about 40% to about 0-1% (Hutchings & Cooke, 1990).

Peritoneal cellsCells were obtained as described previously (Bate et al., 1989).Briefly, they were collected from mice given I ml of 4%thioglycollate (Difco, Detroit, MI) i.p. 3-5 days previously,using Hanks' balanced salt solution (HBSS) containing 1 U/mlof heparin and S yig/ml polymyxin B (Sigma, St Louis, MO).Washed cells were suspended in RPMI-1640, 5% fetal calfserum (FCS) and polymyxin B, counted, and dispensed into 96-well microtitre plates (Nunc, Roskilde, Denmark) in 100 ,lvolumes at 5 x 105 cells/well. Cells were incubated for 2-3 hr at37c in an atmosphere of 5% CO2 in air to allow macrophages toadhere and a further 30 min with an added 100 ,l ofindomethacin (Sigma) at 2 yg/ml. Non-adherent cells were thenremoved and the medium was replaced with 200 pl of RPMI-1640 containing the test stimulants. Next day, the supernatantswere collected and assayed for TNF. A 1/10 dilution was madein RPMI containing 5% FCS and 1 pg/ml emetine (Sigma) andstored at -200, in case retitration was needed. Macrophagesincubated in serial concentrations of LPS or in medium alonewere always included as positive and negative controls on theresponsiveness of the cells.

TNF assayTNF was assayed colorimetrically by its cytotoxicity for L929cells (from the European Collection of Animal Cell Cultures,Salisbury, Wilts), seeded at 2 5 x 104 cells/well 24 hr earlier asdescribed previously (Bate et al., 1989). One unit is defined as thequantity of TNF required to kill 50% of the tumour cells.

ELISA assaysTests were performed by standard procedures in 96-wellmicrotitre plates (Sterilin, Teddington, Middlesex) coated withvarying concentrations of boiled samples of parasite exoanti-gens diluted in PBS. Wells were blocked with 2% bovine serumalbumin and washed with PBS containing 5% Tween 20. Serialdilutions of antisera to be tested were made in the washingbuffer and added, followed by biotin-conjugated goat anti-mouse IgM, IgG (Sigma) or sheep anti-mouse IgG3 (TheBinding Site Ltd, Edgbaston, Birmingham) and then streptavi-din peroxidase (Sera-Lab Ltd, Crawley Down, Sussex), alldiluted according to the manufacturer's instructions. Wells werewashed with washing buffer between each step. Finally, colourwas developed with 0-phenylene diamine (1 mg/ml; Sigma) in0 01 M citrate buffer, pH 6, containing 1/3000 H202 and read atOD 450 nm.

Other reagentsLipopolysaccharide (phenol extract of Escherichia coli 055: B5;LPS) was obtained from Sigma.

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Malaria exoantigens

2-100 -go

0 80 *Q. 70-LL 60 -I 50

c 40 _- >2o 30-D 20

10

8_0 40 160 640 2560 10,240Reciprocal of dilution of antiserum

Figure 1. The inhibitory effect of antiserum on TNF production inresponse to exoantigens of lethal P. yoelii. Antiserum dilutions were

mixed with a constant amount of a boiled P. yoelii supernatant andincubated overnight with mouse peritoneal macrophages; the figures are

means of duplicates. Secreted TNF in the absence of antiserum was

104,500 U/ml. TNF production by 0-1 ,Ig/ml LPS was not decreased by1/40 dilution of the antisera. The results shown are of samplesrepresentative of nine separate pools of the primary antiserum and fourofhyperimmune serum. Primary antiserum against P. yoelii exoantigens(0); hyperimmune serum (0); antiserum made against normal erythro-cyte supernatant (0).

RESULTS

Inhibition of TNF production by antiserum

Previously it has been reported that antiserum from hyperim-mune mice inhibited TNF induction by exoantigens from thehomologous parasite in vitro (Bate et al., 1988) and that antiseramade by direct injection of boiled supernatants from parasitecultures into mice specifically inhibited induction of TNF bythese antigens in vitro (Bate et al., 1989; Taverne et al., 1990) andin vivo (Bate et al., 1989). Comparative titrations of the primaryantisera made against the exoantigens showed that they alwayspossessed greater blocking activity (average titre about 10,000)than hyperimmune serum (highest titre 800). Control sera raisedagainst boiled supernatants of normal red blood cells andnormal mouse serum sometimes showed some inhibition at a

1/40 dilution but not at higher dilutions.Figure 1 illustrates the results characteristic of several

experiments. The blocking activity of a primary antiserumagainst the exoantigens of lethal P. yoelii was compared with a

hyperimmune serum by titration against a concentration ofhomologous parasite exoantigens that induced 104,500 U/ml ofTNF from peritoneal macrophages in the presence of 5 yg/mlpolymyxin B; it shows that the antisera blocked TNF produc-tion in a dose-dependent manner. The presence of blockingactivity in hyperimmune serum obtained from mice that hadbeen vaccinated and had then recovered from a challengeinfection confirms that triggering exoantigens are presentduring the course of infection. Preincubation of macrophageswith antiserum and then its removal did not affect subsequenttriggering by parasite exoantigens, indicating that the inhibitionwas not mediated by an effect on the cells themselves.

Immunoglobulin isotypes responsible for inhibitory activity

To determine the Ig isotypes responsible for the blockingactivity, a pool of antiserum made against the exoantigens oflethal P. yoelii was depleted of IgM or IgG by chromatographyon anti-mouse IgM or IgG agarose, and to confirm that all theactivity was indeed due to immunoglobulin and not to any other

o 100 -

3 90

-D 8070

U-z 60-'- 50co 40-t30-20-

c0 080 400 2000 10,000 50,000

Reciprocal of dilution of ontiserum

Figure 2. Inhibition by affinity-purified IgM and IgG from primaryantiserum of TNF production in response to exoantigens of lethal P.yoelii. Aliquots of an antiserum eluted from anti-mouse IgM or anti-mouse IgG agarose were titrated against a concentration of exoantigensthat induced 22,190 U/ml of TNF. Similar results were obtained in twoother experiments. Original antiserum (0); IgM (0); IgG (El).

inhibitors that might have been present in the serum, samples ofantiserum were depleted sequentially of IgM, then of IgG.Antiserum depleted of IgG consistently lost less blockingactivity than that depleted of IgM, while serum depleted of bothisotypes lost all activity. For example, when serum with a titre of10,240 was depleted ofIgG its titre was reduced to 7240, whereasit was reduced to 640 by depletion of IgM. Conversely, eluatescontaining antibody recovered from the anti-IgM column hadhigher inhibitory titres than those from the anti-IgG column(4472 and 165, respectively) (Fig. 2).

ELISA assays with antiserum against exoantigens

In preliminary experiments in which hyperimmune sera weretitrated by ELISA against a constant amount of a boiled P.yoelii supernatant adsorbed to the plate, antibody binding toparasite exoantigens was detected when antibody against mouseIgM was used in the test, but none with anti-mouse IgG. Infurther experiments with primary antiserum against the exoanti-gens, specific dose-related binding of IgM was again detectedand binding of IgG was also seen, provided antibody againstmouse IgG3 was employed. Some non-specific binding ofantibody to a control normal erythrocyte supernatant alsooccurred, but to a lesser degree. The results of representativeexperiments are shown (Fig. 3). Although the antibody thatbinds to exoantigens as detected by ELISA is not necessarily thesame as that responsible for blocking their TNF-inducingability, it is noteworthy that the antibody detected in theprimary antisera was again predominantly of the T-independentisotypes.

Time-course of the development of inhibitory antibody

The observation that the inhibitory antibody in antiserumagainst exoantigens was predominantly IgM and that the IgGbinding activity was mainly in the IgG3 fraction suggested thatthe exoantigens might be T cell-independent. The time-courseof appearance in the serum of blocking antibody was thereforedetermined to see if it was characteristic of such responses.Pooled sera, from groups of mice bled at intervals afterimmunization with parasite exoantigens of lethal P. Yoelii, weretested against one concentration of the antigen preparation andtheir titres determined and plotted against time after immuniza-tion (Fig. 4). Serum taken at Day 3 did not inhibit TNFproduction but some inhibition was detectable at Day 4 and

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(a ) ( b )

40 160 640 2560 40 640 10,240Reciprocal of antiserum dilution

10 20 40 80 160 320 640Reciprocal of antiserum dilution

Figure 3. Absorption to P. yoelii exoantigens of IgM and IgG3 fromprimary antiserum as shown by ELISA. Plates were coated with a boiledP. yoelii (non-lethal) supernatant diluted 1/4 or a control supernatant,followed by doubling dilutions of the primary homologous antiserumand then (a) goat anti-mouse IgM or (b) sheep anti-mouse IgG3. Thefigures are means of duplicates. Similar results were obtained with threeother samples of primary antiserum. Parasite exoantigens (0); normalerythrocyte supernatant (0).

10,000

1000 -

C- 100 -I

* i I

0 10 20 30 40 50 60Day after immunization

Figure 4. Time-course of development of inhibitory antibody. Serumfrom groups of three mice collected at various times after injection ofexoantigens of lethal P. yoelii was pooled and titrated against a

concentration of exoantigens that induced 67,500 U/ml of TNF.

Day 5, and the titres increased to reach a peak at about Day 12and then declined. The rapid and transient nature of thisresponse is that predicted for a T-independent antigen. Further-more, titres at 10-12 days were not enhanced by injection of theparasite exoantigens with 25 jug of saponin, as an adjuvant, nor

were they boosted by two or three further injections of theantigens (data not shown).

Antisera from T-cell deficient mice

To confirm that the antigens were indeed T-independent,antisera were made against exoantigens of lethal P. yoelii in twogroups of mice that were depleted of T-helper cells by treatmentwith antibody against CD4 and in two groups of nude mice. Theresults of typical titrations show that there was no significantdifference between the blocking activity of these antisera and

Figure 5. Inhibition ofTNF production by exoantigens of lethal P. yoeliiby antiserum from T-deficient mice. Antisera from (a) mice depleted ofCD4+ T cells and (b) nude mice were titrated against a concentration ofexoantigens that induced 102,400 U/ml of TNF in the presence ofpolymyxin and compared with control antisera made at the same timeagainst the same boiled supernatants. The figures are means ofduplicates. In each case two groups of mice were immunized, on

different occasions, and gave similar results; titrations of one ofeach are

shown. Control antisera (0); antisera from T-deficient mice (0).

that of control mice indicating that the exoantigens can induceinhibitory antibody in the absence of functional T cells (Fig. 5).

Cross-reactivity of antisera against exoantigens of differentPlasmodium species

Previously it has been observed that hyperimmune serum

against lethal P. yoelii does not inhibit the induction ofTNF byexoantigens of P. berghei (Bate et al., 1988) but, by contrast,primary antiserum made against exoantigens of P. yoelii blocksthe activity of boiled culture medium from P. falciparum(Taverne et al., 1990). As antisera made against boiled super-

natants are now seen to be more potent inhibitors of TNFinduction by the exoantigens than hyperimmune sera, the abilityof those made against exoantigens of different Plasmodiumspecies to cross-react with their various exoantigens was

investigated; typical results are shown (Table 1). Inhibitorytitres cannot be compared as the amount ofimmunizing antigenused in each case is difficult to quantify and small differencesbetween the times when mice are bled can make a big differenceto the titre (as shown above in Fig. 4). It can be seen, however,that the TNF-inducing ability of exoantigens from two speciesof rodent parasite (P. yoelii and P. berghei) was inhibited byantisera against either, and that cross-reactions were observedwith antisera made against the exoantigens of the rodentparasite P. yoelii and the human parasite P.falciparum. None ofthese antisera, even at a 1/40 dilution inhibited TNF release by01 jpg/ml LPS.

DISCUSSION

These results show clearly that exoantigens of rodent andhuman malarial parasites injected into mice act as T-indepen-dent antigens. Since protection, in terms of anti-parasiteimmunity, has been shown to be T cell-dependent in everysystem tested, not much attention in the past has been paid to T-independent antibodies. Indeed, Taylor et al. (1982) reportedthat P. yoelii lacks both types 1 and 2 T-independent antigens.They did not find antibody against the parasite in sera frominfected nude mice, using both immunofluorescence tests withparasitized erythrocytes and radioimmunoassay with a parasiteextract (which itself failed to cause polyclonal activation of B

318 C. A. W. Bate et al.

044

0-3

0-2

0*1

0.0

E

0

0

0

t 100

° 80CL-

z 60

C 40

D 20

0

Malaria exoantigens

Table 1. Cross-reactions of antisera made against exoantigens of different Plasmodium species

TNF U/mlStimulating 0%antigen Antiserum against Dilution - Antibody + Antibody inhibition

P. yoelii (lethal) P. yoelii (lethal) 1/2560 63,340 5220 91 8P. yoelii (non-lethal) 1/2560 63,340 10,450 83 5P. berghei 1/320 6400 1600 75P. falciparum 1/160 76,800 9953 87

P. berghei P. berghei 1/320 7350 1050 86P. yoelii (lethal) 1/1280 7350 800 89

P. falciparum P. falciparum 1/400 51,200 8450 84P. yoelii (lethal) 1/400 51,200 8450 84

Dilutions of these primary antisera were incubated with concentrations of triggering exoantigensthat induced similar amounts of TNF from mouse macrophages; the results shown are the highestdilutions giving more than 75% inhibition. TNF release by 01I Mg/ml ofLPS was not inhibited by anyof the antisera at a 1/40 dilution.

cells). Their results are not surprising, however, in view of thenature of the antigens they used and the sensitivity of those testscompared with the blocking assay used here. Both parasitizederythrocytes and the extract would have been relatively free ofparasite exoantigens, while it has been found that hyperimmunesera, which contain significantly more total antibody thanserum taken during infection (Playfair & De Souza, 1979),nevertheless contain less blocking antibody than primaryantisera made solely against the boiled exoantigens. Antibodytitres measured in terms of the ability of an antiserum to blockTNF induction by the antigens are considerably greater thanthose determined by ELISA, perhaps because the part of themolecule that interacts with a receptor on the surface of themacrophage is too small a part of the whole to be detectable by abinding assay done with the whole antigen. Nonetheless it isprobably significant that the blocking antibody was mostly IgMand that the only IgG antibody detectable by ELISA with boiledexoantigens was IgG3, an isotype also known to be induced byT-independent antigens in mice, and to share some propertieswith IgG2 in man in that these isotypes are commonly producedin response to polysaccharide antigens (Hammarstr6m & Smith,1986). There is preliminary evidence to suggest that parasiteexoantigens are not proteins, but may be glycolipids: theirability to stimulate macrophages to produce TNF is notdestroyed by treatment with various proteases, but appears tobe affected by some glycosidases and lipases, and they bind somelectins (C. Moreno unpublished data).

If experiments with mice are any guide, it might be predictedthat in man IgM and IgG2 would be the major antibody isotypesthat react with cytokine-inducing parasite exoantigens, and thatthe best way to detect or isolate the antigens would be to useserum taken soon after a primary infection. However, somesoluble antigens of P.falciparum which induce the production ofTNF by mouse macrophages and human monocytes (P. H.Jakobsen and D. Kwiatkowski, unpublished data) were purifiedby affinity chromatography using IgG from pooled serum froman endemic area (Jepsen & Andersen, 1981), perhaps becauseIgG2 comprises about 23% of the total human IgG whereas inthe mouse IgG3 accounts for less than 2%. These antigens sharesome properties with bacterial lipopolysaccharide (Jakobsen,

Baek & Jepsen, 1988), at least one contains carbohydratemoieties (Jakobsen et al., 1987), and at least one (antigen 7)appears to be serologically related to the exoantigens used in thisstudy. Indeed, the results of the cross-inhibition experiments(Table 1) with primary antisera against the exoantigens ofhuman and rodent parasites suggest that the cytokine-inducingcomponent has been conserved during evolution: it has beenshown previously that the same seems to be true for the cellularreceptors concerned, in that exoantigens derived from bothhuman and rodent parasites induce both human monocytes andmouse macrophages to secrete TNF (Taverne et al., 1990).

At least seven exoantigens of P. falciparum have beenidentified and it may be significant that most children in theGambia do not develop precipitating antibody against one ofthem, antigen 7, until they are about 4 years old (P.H. Jakobsen,personal communication). This is about the time that theydevelop some immunity to the clinical manifestations of malaria(McGregor et al., 1956). There is also evidence that individualswho return to endemic areas after a period of absence developsymptoms of disease at lower parasite densities than those whohave never left the area. If, unlike anti-parasite immunity, thiskind of anti-disease immunity depends upon antibody producedin response to T-independent antigens, and its maintenancetherefore demands continuous antigenic stimulation, for use in avaccine it would need to be ensured that the parasite exoantigenscan interact with T cells and provoke memory (Playfair et al.,1990). Finally, it is worth recalling that the incidence of severemalaria in Africa, unlike that of tuberculosis, has not shown theexpected increase following the emergence of AIDS (Morrow,Colebunders & Chin, 1989). Perhaps this too points to the valueofT-independent immune mechanisms in protection against theclinical complications of malaria.

ACKNOWLEDGMENTSThis work was supported by a grant from the Medical ResearchCouncil. We wish to thank Dr Carlos Moreno (Hammersmith Hospital)for his helpful advice and we are grateful to Dr. K. Rockett (of thisdepartment) and Professor G. A. T. Targett (London School of Hygieneand Tropical Medicine) for supplying us with spent medium fromcultures of P. falciparum.

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