immune responses mediating survival of naive balb/c mice experimentally infected with lethal rodent...

Immune responses mediating survival ofnaive BALB/c mice experimentally infected with

lethal rodent malaria parasite, Plasmodium yoeliinigeriensis

Balwan Singh, Bishnu P. Nayak, Kanury V. S. Rao, Pawan Sharma*

Immunology Group, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi, India

(Received 27 September 1999; accepted 7 January 2000)

ABSTRACT – The rodent malaria parasite, Plasmodium yoelii nigeriensis is known to cause fatal malariainfections in BALB/c mice. However, we found that nearly 5% of inbred BALB/c mice could overcomeprimary infections initiated with lethal inoculum of P. y. nigeriensis asexual blood-stages, without anyexperimental intervention. These ’survivor’ mice developed peak parasitemia levels of about 5% andsuccessfully resolved their infections in about two weeks time; infected blood collected during thedescending phase of infection in these mice and subinoculated in naive recipients resulted in a normallethal course of infection. Typically, the parasites in survivor mice looked ’sick’ compared to those inthe susceptible mice. In experiments to define temporal basis of this protection, we found that purifiedsplenic B cells isolated from such a survivor mouse, plus T cells from an infected or naive mouse, couldadoptively transfer this protection to an X-irradiated, naive mouse against a lethal parasite challenge.Purified T cells or B cells alone from the survivor mouse donor provided no protection to the X-irradiated, naive recipient. Passive transfer of sera collected from survivor mice animals a week afterrecovery from infection was also able to substantially alter the course of preestablished P. y. nigeriensisinfection. These findings are discussed in the light of recent reports on the genetic control of bloodparasitemia in mouse malaria models. In the generally lethal malaria infections such as those caused byP. y. nigeriensis in mice and by Plasmodium falciparum in naive children, it is not clear what constitutes aprotective immune response in cases which survive primary infections without any experimental ortherapeutic intervention. An understanding of these mechanisms and their regulation would helpdesign better vaccination strategies. © 2000 Éditions scientifiques et médicales Elsevier SAS

Plasmodium yoelii / rodent malaria parasite / protective immunity / B cells

1. Introduction

The natural history of primary malaria infections innaive humans with no apparent erythrocyte-relateddisorder/dysfunction, seems to indicate that the innateimmune mechanisms play an important role in protectionagainst malaria. It has been argued that such mechanismsmay be working by minimizing morbidity of infection in anaive host on the one hand, and providing a chance for the

acquired immune responses to develop on the other [1, 2].Out of nearly 300 to 500 million people infected everyyear with malaria, about 1.5 to 2.7 million succumb toinfection, giving malaria a mortality rate of less than 1%,although among the untreated Plasmodium falciparuminfections in nonimmune humans, mortality rates could goas high as 10% [3]. It appears, nevertheless, that in manycases, malaria parasites including P. falciparum are, infact, tolerated fairly well. For example, according to atheoretical multiplication rate of 16 times per 48 h for theasexual erythrocytic cycle of the parasite, a P. falciparuminfection initiated with as few as 20 sporozoites in a naivesubject should result, within a fortnight after rupture of the

* Correspondence and reprints.a Present address: Emory Vaccine Center, Department of Microbiology and Immu-nology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

Microbes and Infection, 2, 2000, 473−480© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

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liver schizonts, in 100% of the host RBCs invaded by theparasite. However, in systematic studies on patients suffer-ing from primary attacks of P. falciparum, Pierre Druilheand colleagues [1, 2, 4] found that the actual averageparasitemia hovered around 0.1% not only at ten days butfor weeks thereafter. Moreover, it is a commonly observedbut rarely documented fact that a small proportion ofpeople in the endemic areas never come down withsymptomatic malaria despite rampant symptomaticmalaria infections in their siblings and other familial con-tacts. Nonspecific mechanisms such as those implicatingnatural killer (NK) cells or certain cytokines have beenproposed to mediate some degree of protection against theasexual blood stages of the parasite [5, 6] although theprecise mechanism of such protection remains controver-sial [7, 8]. In the generally lethal malaria infections such asthose caused by Plasmodium yoelii nigeriensis in miceand by P. falciparum in naive children, it is not clear whatconstitutes a protective immune response in patients whosurvive primary infections without any experimental ortherapeutic intervention. While a large body of literatureexists on the role of acquired immune responses in immu-nity against malaria (reviewed in [9, 10]), relatively little isknown about the native or early immune mechanismseffective against a potentially lethal primary malaria infec-tion. We believe that an understanding of these mecha-nisms and their regulation would help in the design ofbetter vaccination strategies. However, operational andethical constraints would make it difficult to carry out suchstudies on untreated primary malaria infections in humans;therefore, suitable animal models will have to be used.Although correlation between animal models and humansis far from perfect, studies in animal models have provideduseful insight into the mechanism of protective immunity[9, 10]. Moreover, lately murine models have also beensuccessfully used to identify the host’s natural resistancegenes against at least three different infectious agents, viz.,Leishmania donovani, Mycobacterium species and Sal-monella typhimurium, and the knowledge gained wasutilized to identify, by comparative genomics, the homolo-gous genes for natural resistance in humans [11]. Signifi-cantly, two recent studies on the murine malaria parasite,P. chabaudi, have provided evidence for natural resistanceto severe malaria in susceptible and resistant strains ofmice [12, 13]; these studies demonstrate that resistanceloci named char1 and char2 map to mouse chromosomes9 and 8, respectively [12]. However, these studies containno data regarding possible immune mechanisms operat-ing in natural resistance against malaria in mice.

The present study stemmed from a serendipitous obser-vation that not all BALB/c mice succumbed to infectionwith P. yoelii nigeriensis, a rodent malaria parasitedescribed to cause 100% mortality in mice [14–16]. Typi-cally, a naive BALB/c mouse, injected intraperitoneally(i.p.) with 1 × 104 parasitized erythrocytes, would developprogressively high levels of peripheral blood parasitemia,reaching > 50% parasitemia in 8–10 days, and die [14].However, we observed that some 5% of mice were able tosurvive this potentially lethal infection without any experi-mental intervention. A closer examination revealed thatalthough these mice took up the infection, they were,

nevertheless, able to resolve it, presumably through theirnative or early immune mechanisms. Subsequent experi-ments showed that the protection mechanism involved aT-cell-dependent B-cell response. Surprisingly, theresponse did not share the classical characteristic ofacquired immunity in that there was no development ofmemory. However, this immunity was stable, surviving atleast four lethal challenges with the asexual blood stagesof the parasite. Finally, serum IgG fractions obtained frommice which had survived infection were capable of inhib-iting in vitro growth of the human malaria parasite, P.falciparum.

2. Materials and methods2.1. Mice and parasites

Six- to eight-week-old, female BALB/c (H-2d) mice wereobtained from the National Institute of Nutrition, Hydera-bad (India). The animals were housed in our institutionalanimal care facility under specific pathogen-free condi-tions; they were looked after and used in the experimentsfollowing guidelines set forth in the National Institutes ofHealth manual [17].

The rodent malaria parasite, P. y. nigeriensis, was origi-nally obtained from Dr S. K. Puri of the Central DrugResearch Institute, Lucknow (India) and maintained inBALB/c mice at our animal care facility by serial, i.p.inoculation of the asexual blood-stages of the parasite. Wehad earlier found that animals infected i.p. with inocularanging from 1 × 104 to 106 parasitized erythrocytes (PE)developed almost similar patterns of parasitemia after thepatent period. We now routinely initiate all blood-stageinfections in mice with an i.p. inoculum of 1× 104 PE, andhave used this model in our studies on the evaluation ofsynthetic peptide immunogens based on conserved aminoacid sequences in the malaria antigens [18]. In the presentstudy also, all P. y. nigeriensis infections were initiatedwith i.p. inoculation of 1 × 104 PE per animal; the para-sitemia was monitored by examination of Giemsa-stainedthick and/or thin smears of tail blood under an oil immer-sion lens. In some groups of mice, experimental P. y.nigeriensis infections following a normal course wereterminated by treatment with pyrimethamine (25 mg/kgdose) plus sulphadoxin (1.25 mg/kg dose); one week afterthe termination of infections, serum samples were col-lected from these mice as described below.

Animals were bled from the retroorbital plexus andsmall volumes of serum samples were collected. Bothpreinfection (day 0) and test (day 14 after complete reso-lution of infection) mouse sera were heat-inactivated,adsorbed with fresh, washed, normal human erythrocyte’ghosts’ to remove the heterophile antibodies before beingused in various assays as described below. For someexperiments, purified immunoglobulin G (IgG) fractionswere obtained from the preinfection and test mouse seraby ammonium sulfate precipitation of the sera to get thegamma globulin fraction, followed by ion-exchange chro-matography on an Econo-Pac(R) IgG purification column(Bio-Rad Laboratories, Richmond, CA, USA) as describedpreviously [19]. After their purity was ascertained by SDS-

Original article Singh et al.

474 Microbes and Infection2000, 473-480

PAGE and immunoblotting, the purified IgG fractions weredialyzed against plain RPMI-1640 medium, i.e., themedium supplemented with 25 mM of HEPES and 0.2%sodium bicarbonate but without serum, passed throughsterile 0.22-µm membrane filters and used in the sero-therapy experiments and in the parasite growth inhibitionassays as described below.

The human malaria parasite, P. falciparum (strain FID-3)was maintained in continuous culture using the candle jarmethod of Trager and Jensen as described elsewhere [20].The detergent-soluble extracts of the parasite proteinswere prepared and used as antigens in an enzyme-linkedimmunosorbent assay (ELISA) or the immunoblotting assayas described in our previous papers [18–20].

2.2. Enrichment of cell populations and adoptive transferexperiments

Splenic cell suspensions enriched for B or T cells wereprepared essentially as described [21]. Briefly, the spleenwas removed aseptically from a mouse under anesthesia(ether) and disrupted to obtain a single-cell suspension.The erythrocytes in the cell suspension were lysed withTris-ammonium chloride, pH 7.4, and the adherent cellswere removed by panning on plastic tissue culture platesat 37 °C for 90 min in a CO2 incubator. Nonadherent cellswere collected and resuspended to a concentration of 5 ×107 cells/mL. For enrichment of B cells, we removed Tcells by incubating the nonadherent, mononuclear cellswith magnetic beads (Dynabeads from Dynal, Oslo, Nor-way, 4 × 108 beads/mL) coated with monoclonal antibodyspecific for mouse Thy1.2, followed by their depletion ona magnet, as recommended by the manufacturer. Afterreadjusting the cell suspension to the original concentra-tion, the panning procedure using Dynabeads wasrepeated. The resultant enriched B cells were then washedand tested for viability by trypan blue dye exclusion, and aviable cell count was obtained by using a hemocytometer.

For preparing T-cell-enriched suspensions, nonadher-ent splenic lymphocytes (5 × 107 cells/mL) obtained asdescribed above were subjected to two rounds of panningon Dynabeads coated with mouse anti-B220 monoclonalantibody (Dynal, 4 × 108 beads/mL) following a protocolrecommended by the manufacturer. The resultant T-cell-enriched suspension was washed, and counted for viablecells.

Splenic cell suspensions so prepared were used in theadoptive transfer experiments which involved intravenousinoculation of 1 × 106 cells per animal; in general, therecipients were X-irradiated (550 rads/mouse) about 18 hprior to the scheduled cell transfer.

2.3. ELISA

Serum samples collected from mice at different timepoints during infection were tested for the presence ofantiparasite antibodies in an ELISA using parasite lysate asthe capture antigen. Procedures employed for the prepa-ration of the parasite lysate (FID-3 isolate of P. falciparumor P. y. nigeriensis) and for performing the assay wereessentially as described previously [18–20]. Briefly, thewells of flat-bottomed Immulon-2(R) plates (DynatechLaboratories Inc., Chantilly, VA, USA) were coated with

the previously determined optimal concentration of cap-ture antigen; the uncovered reactive sites were blockedwith 5% milk powder solution in PBS. The antigen-coatedwells were then sequentially incubated with appropriatedilutions of the first antibody followed by optimally diluted,enzyme-labeled secondary antibody (horseradishperoxidase-labeled anti-mouse IgG), with thorough wash-ing of plates in between the incubations. The enzymereaction was developed using o-phenylenediamine dihy-drochloride as the chromogen and hydrogen peroxide asthe substrate. After stopping the reaction with sulfuricacid, OD490 of the reaction product in the wells wasrecorded using a microplate reader (Molecular Devices,Palo Alto, CA, USA). In the end-point titrations, the lastdilution of a test serum yielding an OD490 value twice ormore of that obtained with the respective preimmuneserum (diluted 1/100) was taken as the end-point titer.

2.4. Immunoblotting

The reactivity of the survivor mice sera with the parasiteprotein(s) was further ascertained by immunoblotting. Thewhole parasite (P. falciparum as well as P. y. nigeriensis)lysate was fractionated on a 10% gel by SDS-PAGE underreducing conditions and transferred onto nitrocellulosemembrane following standard procedures. After the uncov-ered reactive sites of the nitrocellulose membrane wereblocked by saturating it with 5% nonfat milk powdersolution in PBS overnight, the membrane was probed withvarious preimmune and test mouse sera. The parasiteproteins separated by SDS-PAGE and transferred ontonitrocellulose paper, were incubated first with mouse seraand then with the horseradish peroxidase-labeled anti-mouse IgG antibodies. The final enzyme reaction wasdeveloped using H2O2 as the substrate and 4-chloro-1-naphthol as the chromogen.

2.5. Transfer of test IgG to mice with high parasitemia

Two infected BALB/c mice showing high parasitemia (>10%) were injected, i.v., with the purified IgG from survi-vor mouse (500 µg/ animal) three times at an interval ofthree days each. Two control mice, similarly infected,received the same quantity of normal (preinfection) IgG ina similar fashion. The course of parasitemia in both thegroups was followed by microscopy.

2.6. Parasite (P. falciparum) growth inhibition assay

The in vitro cultures of the FID-3 strain of P. falciparumwere synchronized at the ring stage by two treatments with5% sorbitol solution [22] and incubated further for about30 h, so that at the time of setting up of the assay, nearly90% of the parasites were > 4N segmenters.

Initially, preinfection and test sera were investigated forantiparasite activity in the synchronized cultures grown inthe RPMI-1640 medium supplemented with 10% humanserum plus 5% of appropriate mouse serum. The cultureswere incubated in a candle-jar at 37 °C for about 40 hwith a change of culture media containing appropriatesera at 24 h. Additional controls included culture wellswith no mouse serum and wells with known negative(noninhibitory) and inhibitory sera as described [19].

Subsequently, various concentrations of purified IgGfractions obtained from the preinfection and test mouse

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sera were incorporated in this assay system to study theirpossible effect on parasite growth. Each concentration wastested in triplicate as described [19, 20]. Purified IgGfractions obtained from serum samples from a rabbit immu-nized with a 12-residue peptide sequence in the acidic-basic repeat antigen of P. falciparum, and previously shownto exert a dose-dependent inhibitory effect on the P. falci-parum merozoite re-invasion of human erythrocytes [19],were included as positive controls. Serum samples fromanother rabbit immunized with ’P-8’, a 21mer syntheticpeptide construct based on the P. falciparum merozoitesurface protein-1 [20], were used as negative control. Aparallel set of culture wells with appropriate mouse serawas monitored every 6 h by microscopy for any possibletoxic effect of mouse sera on the parasite or parasitisederythrocytes.

At the end of the assay, smears were drawn from ali-quots taken from each well, stained with Giemsa andexamined under a microscope by two research workers.Percent parasitemia was calculated as number of PE in atotal of 100 erythrocytes; at least 10 000 cells were countedto determine parasitemia in each smear.

Statistical significance of the results of this assay wasdetermined using Student’s t test.

3. Results and discussion3.1. Some naive mice protected themselves againstpotentianlly fatal murine malaria

A large majority of the mice in groups of 5 to 20, onbeing experimentally infected with P. y. nigeriensis, devel-oped progressively high parasitemia, finally succumbingto infection around day 9 or 10 postinoculation; the courseof parasitemia we observed in three groups of such mice isshown in figure 1 (groups I, II, & III). The infection in thesemice became patent on day 3 postinoculation; the averagepercent parasitemia observed on day 5 was 1.18 ± 0.78(range 0.95–1.92), which progressively increased to18.23 ± 1.26 (range 16.7–20.0) on day 6, 24.86 ± 1.93(range 22.6–27.3) on day 7 and 58.94 ± 1.86 (range56.4–60.7) on day 9, finally leading to the death of theseanimals on day 10. These observations are in generalagreement with those made by others indicating lethalvirulence of P. y. nigeriensis in mice [14–16]. Occasion-ally, however, we would come across a mouse in whichpotentially lethal infection got controlled and finallyresolved without any experimental intervention; in fig-ure 1, group IV (curves 1 to 4 representing individualmice) comprises animals that experienced peak para-sitemia of only around 5%, followed by complete recov-ery, as evident from the results of blood slide examinationand blood transfusion in naive recipients. These four ’sur-vivor’ mice came from different batches of infected ani-mals but followed a similar course of parasitemia. Thecourse of parasitemia in the ’survivor’ mice was dramati-cally different from that observed in the susceptible groupsof mice from day 5 through day 9 postinoculation (fig-ure 1, group IV/curves 1–4); the survivor mice developedthe average percent parasitemia of 2.86 ± 1.36 (range1.5–5.0) on day 5, which peaked at 4.07 ± 1.08 (range

3.0–5.3) on day 6 and then declined to 2.61 ± 1.37 (range0.4–4.05) the next day and finally to undetectable levelsfrom day 10 onwards; all these mice survived till they weresacrificed for the adoptive transfer experiments. Moresignificantly, once these mice had cleared their primaryparasitemia, they remained negative for the parasite, asevident from the results of regular slide examination andsubinoculation of their blood sample in the naive recipi-ents. In view of the established lethality of P. y. nigeriensis[14–16], this was an unusual observation and it providedus an opportunity to look at the native or very earlyimmune mechanisms in this case.

A careful examination of the blood smears revealed thata proportion of parasites in a self-limiting infection in thesurvivor mice appeared to be damaged or in ’crisis’ justbefore the infection started getting controlled on day 6postinoculation, in contrast to the healthy appearance andnormal morphology of nearly all parasites in those micewhich finally succumbed to the infection (figure 2). Thereis some sort of vacuolization of the cytoplasm in the crisisforms of the parasite, accompanied by appearance ofcharacteristic dot-like structures in the infected RBC cyto-plasm. These dot-like structures could be cytoplasmicaggregates or remnants of degenerating parasites, althoughthe precise biochemical nature and/or mechanisms respon-sible for their formation remain to be established. More-over, the dot-like structures were so typical that it was,indeed, possible to predict a self-limiting course of infec-tion as and when these were detected. In contrast, we didnot come across such structures in infections treated withantimalarials or in infections which proceeded normally,with lethal outcome. In fact, infections treated with anti-malarials (pyrimethamine + sulfadoxin) presented a pic-

Figure 1. The course of P. y. nigeriensis parasitemia in naiveBALB/c, susceptible (groups I–III) and survivor mice (group IV,curves 1–4). Infections in each mouse were initiated with i.p.inoculation of 1 × 104 PE. Each data point in curves for groups Ito III represent an average value obtained from at least six mice;vertical bars represent standard deviation of the mean. In groupIV, each of the four curves represents values obtained in anindividual survivor mouse.



ture wherein almost all parasites appeared damaged orinhibited in growth as seen in Giemsa-stained bloodsmears. Survivor mice, on the other hand, presented witha heterogenous population of parasites comprised of partlyor fully damaged as well as apparently healthy parasites,as seen in blood smears on day 6 to 8 of infection. Thus,the blood smears of survivor and susceptible mice couldprovide reasonable prognosis of the outcome of infection.

Significantly, we found that parasites collected duringthe descending phase of the self-limiting P. y. nigeriensisinfections retained their usual virulence, as evident fromthe course of parasitemia they induced following theirsubinoculation in naive mice (data not presented).

3.2. Native protection resided with the B cells

As a first step in our effort to understand the basis of thisnative protective response, we adoptively transferred totalsplenocytes from a survivor mouse to an X-irradiated naivemouse. The recipient, challenged with a lethal inoculumof the asexual blood-stage parasites the next day, becamepatent on day 3, but successfully controlled the progres-sion of parasitemia, not letting it exceed 6%, and survived.In fact, we could carry out three successive transfers ofnonadherent, mononuclear splenocytes as every time, thenaive, X-irradiated recipient survived the lethal P. y.nigeriensis challenge (data not presented). Subsequently,we found that purified B cells from the spleen of an intactsurvivor mouse obtained after 14 days of clearing itsprimary infection, were capable of adoptively imparting toan X-irradiated, naive mouse, a similar level of protectionagainst a lethal challenge with the parasite (figure 3).Furthermore, putatively parasite-specific B cells couldimpart this protection only in association with T cells(figure 3, groups A & B), although it did not matter if the Tcells were specifically primed, i.e., obtained from aninfected (∼ 15% parasitemia) intact mouse, or not (i.e.,

from a naive mouse). The X-irradiated mice which receivedB cells alone (figure 3, group C) from the survivor mouse,experienced a longer survival period but eventually failedto control challenge infections and died on day 13 posti-noculation; the adoptive transfer of T cells alone from asurvivor mouse also failed to impart any protection to therecipients (figure 3, group D); and, of the X-irradiatedanimals given B and T cells from a normal mouse, asexpected, none survived the challenge infection (figure 3,group E). Thus, these experiments indicated that the nativeability of the survivor mice to eliminate potentially lethalprimary malaria infections resided with B cells in thespleen, although, it may be emphasized, the cooperationfrom T cells was an essential requirement for inducing thisprotective response. However, we found that the micewhich had survived the untreated primary infections withlethal P. y. nigeriensis remained fully susceptible tore-challenge infections given a month later, and the courseof parasitemia upon reinfection also remained somewhatsimilar to the one observed during the primary self-limitinginfection (unpublished data).

B cells are known to exert their biological effect onpathogens through the antibodies they produce. So thesera were collected from survivor mice as and whenavailable in different groups and analyzed for the presenceof parasite-specific antibodies in assays like ELISA andWestern blotting using parasite protein extract as antigens.The results of an antibody binding ELISA using the P. y.nigeriensis asexual blood-stage parasites lysate as captureantigen indicated that the survivor mice made high levels

Figure 2. Giemsa-stained, thin blood-smear from A. Normalsusceptible mouse infected with P. y. nigeriensis showing healthyparasites (arrow); this animal developed a peak parasitemia ofabout 50% before succumbing to infection on day 9; and, B.’Survivor’ mouse, similarly infected with P. y. nigeriensis, showingunhealthy parasites, with marked pyknosis in the parasite’s cyto-plasm and perforations or ’pits’ in erythrocyte membrane; thismouse successfully resolved its infection (peak parasitemia of onlyabout 5%) and the adoptive transfer of its splenic B cells succes-sfully imparted protection to a naive, X-irradiated mouse.

Figure 3. The course of parasitemia in the X-irradiated BALB/cmice reconstituted with purified splenic B cells from a survivormouse plus primed T cells from an infected mouse (group A) ornaive mouse (group B); B cells alone from a survivor mouse(group C); T cells alone from a survivor mouse (group D); and, Bcells plus T cells from a naive mouse (group E). Each data pointrepresents a mean of values obtained in three mice, and thevertical bars represent standard deviation of the mean.



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of parasite-specific IgG antibodies; a pool of test seraobtained from four survivor mice yielded an ELISA titer of1/80 000 for parasite-specific IgG antibodies. In an immu-noblot, this sera pool recognized several protein bands inboth P. y. nigeriensis and P. falciparum parasite lysates(figure 4); significantly, the pattern of proteins recognizedby the survivor mouse sera was different from that obtainedwith sera from mice in which P. y. nigeriensis infectionshad been drug-terminated at parasitemia levels of about15%. The survivor mouse sera strongly reacted with pro-tein bands at relative molecular masses of approximately28, 40, and 43 kDa. The sera pool from the drug-curedmice reacted with fewer protein bands and its reactivitywith these three bands was rather weak. The cross-reactivity of anti-P. y. nigeriensis sera with the humanmalaria parasite is well established, and the results pre-sented here once again highlight the presence of pan-specific antigenic proteins, some of which may, indeed, beimportant in inducing protection against malaria.

3.3. Parasite-inhibitory potential of IgG purified from thesurvivor mouse sera

Purified IgG obtained from the high-titre sera of survi-vor mice, i.e., sera collected about 14 days after theycleared the primary infection, was tested for its effect on apatent P. y. nigeriensis infection in mice (figure 5). In thisexperiment, two control mice having parasitemia of about15% when they received the sterile normal mouse IgG(500 µg/animal), i.v., continued to go through progressiveparasitemia and succumbed to infection two days laterwith peak parasitemia of 44.9% and 64.2%, respectively(figure 5, group I). Two experimental mice showing para-sitemia of 11 and 17%, respectively, (figure 5, mouse 1and 2) were injected, i.v., with the purified survivor mouseIgG (500 µg/ animal) three times at an interval of threedays each, and the course of parasitemia was followed bymicroscopy. In one of these animals (figure 5, mouse 1,with 17% parasitemia on the first day of the passivetransfer), the parasitemia declined to subpatent level afterthe second dose of the survivor mouse IgG but thenfluctuated between ∼ 20 to 40% for several days, eventu-ally leading to death of the animal on day 18 of the firstdose of passive transfer. The other experimental animal(figure 5, mouse 2 with 11% parasitemia on the day of firstinjection of IgG) controlled the progressive increase inparasitemia for some days and extended its survival timeby seven days compared to the control mice, and died onday 9 after the first dose of passive transfer, with peakparasitemia of 54.13%. Thus, the survivor mouse IgGprovided some level of protection even against preestab-lished high-parasitemia malaria infections in terms ofextended survival time of the experimental animals, eventhough these animals eventually succumbed to infection.Additional control groups in this experiment includedanimals showing parasitemia ranging from about 7 to20%, divided into five groups of three animals each andgiven, respectively, purified B cells alone from the survivormice, the infected/drug-cured animals or normal mice(figure 5, groups II, III, and IV, respectively), or serum fromthe infected/drug-cured animals or normal mice (figure 5,groups V and VI, respectively). Mice in none of these

control groups survived beyond two days of B-cell/serumtransfer and died with parasitemia ranging from 50 to67%.

More significantly, sera from native immune animalscollected within two weeks of clearing their primary infec-

Figure 4. Immunoblot of parasite proteins probed with serapool from survivor mice (top panel) or drug-cured mice (bottompanel). Lane 1, proteins from normal human erythrocyte mem-branes, lane 2, whole parasite lysate of P. y. nigeriensis, and lane 3,whole parasite lysate of P. falciparum.



tions, were found to exert a growth inhibitory effect on thehuman malaria parasite, P. falciparum as well (table I).Our initial studies with various mouse sera showed thatincorporation of as little as 5% test sera (collected about14 days after clearing the primary infection) from thesurvivor mice caused nearly 40% inhibition of parasitegrowth in this assay. In order to establish that this inhibi-tory effect was indeed due to antibodies and not someother factor in the serum, we repeated this assay withpurified IgG fractions from various sera. Both known posi-tive and negative rabbit sera controls gave the expectedresults: the positive control, viz., rabbit anti-AB-1 IgGfraction causing about 50% growth inhibition at 500 µg/mL concentration and the negative control, rabbit anti-MSP-1 peptide causing only about 10% inhibition of the

parasite at 2.0 mg/mL. These results are in agreement withour observations reported earlier [19, 20]. As evident fromthe data presented in table I, the survivor mouse test IgGisolated from the same pool that gave high ELISA titers andreacted with the parasite-lysate in an immunoblot (fig-ure 4), caused nearly 70% inhibition of the P. falciparumasexual blood-stage parasites’ growth when used at aconcentration of 300 µg/mL (table I); the degree of growthinhibition observed was highly significant (P < 0.01).

Our finding that B cells or antibodies, in associationwith T cells, provided protection against asexual blood-stages of malaria parasite is in line with what several othershave demonstrated since 1961 [23–27]. The present work,however, attempts to document in a preliminary way,some basis of protective immunity seen in the untreatedprimary malaria infections with a potentially lethal strainof the parasite. The data presented in this communicationshow that although the native protective immunity wasmediated by a T-cell-dependent B-cell response, it did notseem to generate any memory, which was unlike a classi-cal humoral immune response. The test serum from survi-vor mice also recognized some specific parasite proteinsin the immunoblots (28-, 40-, and 43-kDa bands); we donot know as yet about the identity of these proteins.Hopefully, further characterization of these proteins bygene cloning and epitope mapping may reveal sequencesputatively responsible for inducing protective immunity inprimary malaria infections. On the other hand, the hostsusceptibility/resistance trait has been mapped to a locuson chromosome 8 of mouse which contains genes forseveral interesting host proteins such as the scavengerreceptor protein expressed by professional phagocytes,and the cytokine interleukin-15 [13]. Further studies areneeded to explore the role of these proteins in the naturalresistance of the host to malaria.

References

[1] Druilhe P., Bouharoun-Tayoun H., Natural immunities,Res. Immunol. 142 (1991) 637–643.

[2] Druilhe P., Perignon J.L., Mechanisms of defense against P.falciparum asexual blood stages in humans, Immunol. Lett.41 (1994) 115–120.

[3] World Health Organization Malaria, WHO Fact Sheet(revised) 94 (1996) 1–3.

Figure 5. Effect of the survivor mouse IgG on the course ofestablished, patent infection. Course of parasitemia in groups ofexperimentally infected mice showing high parasitemia and trea-ted with the purified ’survivor mouse’ IgG (mice numbers 1 and2), normal mouse IgG (group I), or purified splenic B cells fromthe survivor mice (group II), infected/drug-cured mice (groupIII), naive mice (group IV), or serum from the infected/drug-cured animals (group V), and naive mice (group VI). Each datapoint represents an overall mean of quadriplicate values obtainedin an individual mouse (mice 1 and 2), two animals (group I) orthree mice (groups II–VI).

Table I. In vitro growth of P. falciparum in the presence of IgG purified from mouse sera collected on day 0 (naive IgG)and 14 days after recovery (test IgG) of mice infected with P. y. nigeriensis*.

IgG conc. (µg/mL)Percent parasitemiaa

Percent inhibition Student’s t test (P values)Naive IgG Test IgG

300 0.73 ± 0.08 0.23 + 0.07 68.44 < 0.01150 0.75 ± 0.11 0.43 + 0.09 42.66 < 0.0575 0.74 ± 0.09 0.63 + 0.07 14.86 < 0.2037.5 n.d. 0.67 ± 0.08 09.46 n.d.

* Parasitemia at 48 h is presented; initial parasitemia (0 h) was 0.21%. a Data represent mean ± standard deviation of values obtained in triplicate wellsfor each category of serum concentration. Results obtained with the positive and negative control IgG were within expected range of values and arepresented in the text. n.d., not done.



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