c mice

EXPERIMENTAL

PARASITOLOGY

52,

Plasmodium

yoelii: Antibody and the Maintenance Immunity in BALB/c Mice

ROBERT The John

Curtin

School

18-24 (1981)

of

R. FREEMAN’ Medicul

Reseurc~h,

(Accepted

AND

R. PARISH

CHRISTOPHER

Au.struliurz

for publication

Notionul

of

Univer-sity,

Cutthrrru.

Australia

23 June 1980)

FREEMAN, R. R., AND PARISH, C. R. 1981. P/u.~rnodiurrt yorlii: Antibody and the maintenance of immunity in BALBic mice. Exprimentul Purusitology 52, 18824. Subpatent persistence of parasitemia was detected for up to 7 weeks after infection of BALBic mice with Plusmodium yoelii. Serum taken from recovered mice maintained parasitemias in recipient mice at a subpatent level when transferred repeatedly at 2-day intervals. Single doses of serum from convalescent donors delayed the course of infection in recipients. Small doses of transferred hyperimmune serum had the same effect, whereas large doses (~0.5 ml) totally suppressed parasitemia. Only a single secondary challenge of recovered mice was required in order to produce a maximally protective hyperimmune serum. Mice completely protected from a primary challenge with P. yoelii by transfer of hyperimmune serum were not at all resistant to a second challenge given some weeks later. After transfer of hyperimmune serum into mice with established P. yorlii infection, parasitemia fell to subpatent levels within 48 hr. During the first 21 hr after serum transfer, a progressive reduction in the proportion of ring forms present in blood smears was observed. INDEX DESCRIPIORS: P/o.smodium yoelii; Protozoa, parasitic: Malaria, rodent: Immunity, to malaria: Serum transfer; Mouse, BALBic

nisms may play a role in the acquisition and maintenance of immunity to P. yoelii (Finerty and Krehl 1977; Playfair et al. 1979; Roberts and Weidanz 1979). In the present study the role of antibody in the maintenance of immunity to P. yoelii has been investigated. It is demonstrated that serum transferred from recovered mice harboring subpatent P. yoelii infections can maintain challenge infections at the subpatent level in recipients, and that the antibody titer of immune serum, as well as its protective potential on passive transfer, is substantially boosted when recovered mice are rechallenged with P. yoelii. Furthermore, the results indicate that protective antibody acts in vivo against the schizont and/or merozoite stages of this parasite.

INTRODUCTION

The rodent malaria parasite Plasmodium yoelii (Landau and Killick-Kendrick 1966) produces generally self-limiting infections in laboratory mice (Topley et al. 1970), and this experimental model has been used in a number of recent studies of acquired immunity to malaria. The development of immunity to P. yoefii has been shown to be T cell dependent (Clark and Allison 1974; Weinbaum et al. 1976; Jayawardena et al. 1977) and B cell dependent (Weinbaum et al. 1976). Passive transfer experiments have shown that a protective humoral factor, presumably antibody, is present in the serum of recovered mice (Jayawardena et al. 1978; Murphy and Lefford 1979) while other work has indicated that antibodyindependent cell-mediated immune mecha-

MATERIALS

18 Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.

METHODS

Mice. Female BALB/c mice bred under specific pathogen-free conditions were used from 8 weeks of age. Parasite. Strain 17X of Plusmodium

’ Present address for all correspondence: Department of Immunochemistry, The Wellcome Research Laboratories, Langley Court, Beckenham BR3 3BS, Kent, England, U.K.

0014-4894/81/040018-07$02.00/O

AND

Plasmodium yoelii: MOUSE IMMUNITY yoelii was derived from a cloned line provided by Dr. D. Walliker, Institute of Animal Genetics, Edinburgh, Scotland, U.K. The parasite stock was stored at -70 C as a stabilate of infected mouse blood in the presence of 15 units/ml of heparin and 8% (v/v) glycerol. Infections and scoring of parasitemia. Infections were induced by intravenous (iv) or intraperitoneal (ip) injection of washed, parasitized red cells, at various doses as specified in the text. To score parasitemia, thin smears of tail blood were fixed in methanol, stained with Giemsa stain, and examined at x 1250 magnification. Blood subinoculation tests for subpatent infection. A group of 20 mice recovered from P. yoefii was used to investigate persistence of subpatent infection. On the days indicated in the text, lo-p1 samples of tail blood were taken from 3 randomly selected donors. These were pooled in 0.5 ml Alsever’s solution, and this was injected ip into recipient mice. Recipients were regularly checked for patent parasitemia, but if no parasites were observed by 30 days after subinoculation, the result was scored as negative. After a negative result had been obtained, total blood subinoculations from 3 donors into 3 recipients was performed. Failure to detect parasites in smears taken from recipients up to 30 days later confirmed the blood negative status of the donors. Indirect fluorescent antibody (ZFA) assay. The IFA assay was performed according to the method of Voller and O’Neill (1971), with modifications. Serum dilutions were made in phosphate-buffered saline supplemented with 10% (v/v) fetal calf serum. The second antibody was a rabbit anti-mouse immunoglobulin reagent, labeled with fluorescein isothiocyanate and immunoabsorbant-purified. Counterstaining was with 0.01% (w/v) Evans blue in PBS. Serum transfers. Serum from recovered mice, hyperimmune serum, and normal mouse serum (NMS) was stored at -20 C.

19

For serum from recovered mice, the time interval between infection and serum collection is specified in the text. Some hyperimmune donors had been rechallenged with P. yoelii four to six times at monthly intervals, serum collection being at 7 days after the final challenge. Other hyperimmune serum donors were only re, challenged once, 4 weeks after the initial infection, and serum was collected 7 days later. In the context of this paper the term “hyperimmune serum” indicates that the donors had been rechallenged with P. yoefii at least once. Serum transfer was always by the ip route, and various doses were used as specified. Various challenge doses of parasitized red cells were used, and the timing of challenge with respect to serum transfer was variable, as specified in the text. RESULTS

Course of primary infection. A maximum parasitemia of about 10% was reached 12 days after ip inoculation of 5 x lo4 Plasmodium yoefii-parasitized red cells from a donor mouse which had been infected from the frozen stabilate (Fig. 1). Thereafter parasitemia declined, becoming subpatent by Day 16. The infection was confined to basophilic erythrocytes, their availability limiting parasitemia during the ascending phase of infection. Repeated passage of the infection resulted in higher maximum parasitemia and longer duration of patent infection. In most of the experiments described, infections were initiated with parasites which had undergone repeated passage, and the control groups showed higher peak parasitemias than were observed in this initial experiment. Persistence of subpatent infection. By subinoculation of blood from recovered mice, it was found that subpatent P. yoelii infection persisted for about 3 weeks after the disappearance of parasites from blood smears (Fig. 1). By Day 60 postinfection,

20

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FIG. I. Course of Phsmodiutn yorlii parasitemia in a group of five BALBlc with 5 x IO4 parasitized red cells, given ip. The result of secondary challenge dotted curve indicates the infectivity of the secondary challenge inoculum previously infected with P. yoclii. The results of blood subinoculation assays are shown below the abscissa.

mice infected on Day 0 (2”) is also shown. The in a group of mice not for subpatent infections

whole blood subinoculations did not give A delay of 4 days was observed in the rise to patent infections, indicating that groups treated with serum from 3- or 6total parasite clearance from the blood had week recovered donors, the 1% parasitemia been achieved. level being reached by Day 10. In the group Resistance to reinfection. Recovered receiving serum from 9-week recovered mice were resistant to superinfection during the subpatent period. To determine the immune status of mice following blood clearance, a group of five mice was challenged iv with lo5 P. yoelii-parasitized red cells 93 days after primary P. yoelii infection. These mice were from the same group shown to be subinoculation negative on Days 44 and 60 after primary infection. Patent parasitemias did not develop following the secondary challenge (Fig. 1). Passive serum transfers from recovered mice. Groups of four mice were injected ip

with 0.5 ml of serum from syngeneic donors which had recovered from P. yoelii infection given 3, 6, or 9 weeks prior to serum collection. A control group received NMS. Two hours later the serum recipients were challenged with lo4 P. yoelii-parasitized red cells, given intravenously. In the controls, parasitemia reached 1% by Day 6 (Fig. 2).

FIG. 2. Course of Plasmodium yoelii parasitemia during the ascending phase in groups of four BALBic mice injected ip with 0.5 ml of NMS (0) or 0.5 ml of serum from donors recovered from P. yoelii infection given 3 weeks (*), 6 weeks(D), or 9 weeks (A) prior to serum collection. Serum recipients were challenged iv with lo4 P. yoelii-parasitized red cells 2 hr after serum transfer.

Plasmodium

yoelii:

donors, the delay in the onset of patent parasitemia was reduced by 2 days, and the 1% level was reached by Day 8. In the next experiment, serum from donors recovered from P. yoetii infection given 3 weeks previously was used. One group received a single dose of 0.5 ml, 1 hr before challenge with 2 x lo6 P. yoeliiparasitized red cells. Parasitemia in this group was delayed, compared with controls, by 3-4 days as expected (Fig. 3). A second group received two OS-ml doses of immune serum on Days 0 and 4, and parasitemia was delayed by a further 2 days. A third group received multiple doses of immune serum, on Days 0,2,4,6, and 8. Patent parasitemia was suppressed for as long as the serum transfers continued, and no parasites were seen on or before Day 14 after challenge. However, patent infections were observed in three of the five mice in this group between Days 20 and 26. Hyperimmune serum transfer. Groups of four mice were injected ip with 0.5, 0.3, or 0.1 ml of serum from donors hyperimmunized against P. yoelii, and a control group received 0.7 ml of NMS. One hour later, all mice were challenged iv with lo4

days after

infection

FIG. 3. Mean parasitemias in groups of five BALB/c mice infected iv on day 0 with 2 x IO* Plasmodium yoelii-parasitized red cells. Serum transfers (ip). were as follows: 0.5 ml NMS on Day 0 (0); 0.5 ml 3-week convalescent serum on Day 0 (0); 0.5 ml 3-week convalescent serum on Days 0 and 4 (A); 0.5 ml 3-week convalescent serum on Days 0, 2, 4, 6, and 8 (W).

21

MOUSE IMMUNITY

P. yoelii-parasitized red cells. Mice receiv-

ing 0.5 ml of hyperimmune serum did not develop patent infections during an observation period of 30 days (Fig. 4). Doses of 0.3 or 0.1 ml were subprotective, delaying the onset of patent parasitemia by 8 and 2 days, respectively. Multiple immunization was found to be unnecessary for the production of a protective hyperimmune serum. As shown in Fig. 5, 0.5 ml of serum taken 7 days after a secondary P. yoelii challenge completely suppressed patent infection on passive transfer. The same protected recipients and the group which had initially received NMS were rechallenged with P. yoefii 49 days after the primary challenge. The group originally unprotected after NMS treatment was solidly immune to reinfection as expected, whereas the mice originally protected by transfer of hyperimmune serum were fully susceptible to the secondary challenge (Fig. 5). Effect of hyperimmune into previously infected

serum transferred mice. Eight mice were infected with lo5 P. yoelii-infected

red cells. Five days later, when mean parasitemia was 7.7%, four mice were injected ip with 1.0 ml of hyperimmune serum, and four mice were injected with 1.O

0.1 0

2

4

6

FIG. 4. Mean

6

IO I2 I4 16 I6 20 22 24 26 days alter infection

parasitemias

in groups

28 30

of four

BALB/c mice infected iv with IO4 Plasmodium .voe/iiparasitized red cells on Day 0, 1 hr after ip transfer of 0.7 ml NMS (O), or 0.5 ml (O), 0.3 ml (A), or 0.1 ml (W) of hyperimmune serum.

22

FREEMAN

FIG. 5. Mean parasitemias in groups of five BALBic mice following primary and secondary (2”) iv challenges with I@ Plumodium yoelii-parasitized red cells. Groups were injected ip with 0.5 ml NMS (0) or 0.5 ml hyperimmune serum (0) I hr before the primary challenge.

AND

PARISH

contrast, in the group injected with hyperimmune serum, the proportion of rings fell to 2% after 21 hr, while the proportion of schizonts increased to 47% of asexual forms. Antibody titers. The IFA assay revealed that the serum titer of antibody against the parasite was reduced in mice recovered 11 weeks after P. yoelii infection, compared with mice immediately after recovery (Table II). However, 1 week after a secondary challenge with P. yoefii, the serum titer was substantially boosted. DISCUSSION

The results of this study establish that the initial phase of patent Plasmodium yoelii parasitemia in BALB/c mice is followed by ml of NMS. Transfer of hyperimmune serum caused rapid clearance of patent a phase of subpatent parasitemia, when parasitemia, to below 0.1% after 24 hr (Fig. persisting parasites are detectable only by 6). Parasitemia remained subpatent for 10 blood subinoculation. In this way P. yoelii days, then reemerged to reach about 1% is a typical malaria parasite. Patent recrudescences were not observed, but this before clearance. Smears taken at 5, 13, and 2 1 hr after does not detract from the potential relevance serum transfer were scored for proportions of the model to human malaria, since only of rings, trophozoites, and schizonts (Table a single clinical episode is usually observed falciparum I). During the first 21 hr after NMS transfer, in infections with Plasmodium the proportion of rings remained at 50-60% (Kitchen 1949). Convalescent mice were resistant to and schizonts remained at about 10%. In superinfection during the subpatent phase, and remained resistant to challenge after IOOr complete clearance of primary blood parasitemia. However, BALB/c mice recovered from P. yoelii infection have been shown to be susceptible to challenge with a heterologous malaria parasite, Plasmodium vinckei petteri (Freeman 1978), demonstrating that acquired immunity to P. yoefii is specific. A single dose of serum taken during the phase of subpatent infection (i.e., 3 to 6 weeks after infection) delayed the course of primary P. yoelii parasitemia in recipients by 4 days. Repeated doses extended the delay in the onset of patent parasitemia, t,me oher serum transfer (hours) and multiple doses held parasitemia at the FIG. 6. Mean parasitemias in groups of four BALBic subpatent level for as long as the serum mice infected iv on Day -5 with IO’ Plasmodium transfers continued. It seems reasonable to yoelii-parasitized red cells, and injected ip with I .O ml NMS (0) or hyperimmune serum (0) at t = 0 hr. conclude that the suppression of persisting

Plasmodium

yoelii:

23

MOUSE IMMUNITY

TABLE I Disappearance of Plasmodium yoelii Ring Forms following Hyperimmune Serum Transfer into Mice with Established Parasitemia Serum transferred

Parasite morphology”

Hours after serum transfer

Percentage parasitemia”

Rings’

Trophozoites

Schizonts

NMS

5 13 21

10.6 8.8 10.5

57 + 7 55 k 5 62+ 13

40 k 7 38 2 6 25 + 6

724 723 13 k 7

Hyperimmune

5 13 21

5.9 3.8 0.9

42 k 5 17 t 5 2+2

53 k 5 71 +6 51 +3

523 12 k 7 47 k 4

’ Mean in groups of four mice. Mean parasitemia at I = 0 hr was 7.7%. Ir Expressed per 100 parasitized red cells i SD. ’ P. yoelii ring forms are not well defined in Giemsa-stained smears, and the count included small trophozoites.

infection in the subpatent state may be an antibody-mediated function. Serum from mice recovered 9 weeks after P. yoefii infection was less protective, in that challenge infections in recipients were delayed by only 2 days. It was likely that the donors had eliminated blood parasitemia by this stage, and that the titer of serum antibody had declined (Table II). However, recovered mice retained the capacity to mount a strong secondary antibody response, and the IFA serum titer was substantially boosted within a week of secondary challenge. The higher IFA titers in the serum of rechallenged mice correlated with the increased protective potential of such serum on passive transfer. By dose-response testing, it was found that 0.1 ml of hyperimmune serum produced a delay in parasitemia similar to that observed after

transfer of 0.5 ml of convalescent serum. The simplest interpretation is that hyperimmune serum contains an increased concentration of protective antibody. The serum IFA titers observed in this study are consistent with this interpretation. It was found that multiple rechallenges were not necessary in order to produce a hyperimmune serum of maximal protective potential, and that a single secondary challenge was sufficient. Thus, it seems likely that the difference between serum from convalescent donors and serum from hyperimmune donors is merely quantitative. Jayawardena et al. (1978) reported that a dose of hyperimmune serum which completely protected intact recipients from challenge with P. yoelii was ineffective in T-cell-deprived recipients (although a long delay in the onset of patent parasitemia was observed in the deprived recipients). These workers suggested that, in intact mice, transferred hyperimmune serum inhibits TABLE II parasitemia until a T-cell-dependent proAnti-Plusmodium yoelii IFA Titers of Normal tective response is mounted by the recipiand Immune BALB/c Sera ents, the result being that no parasitemia is IFA titer Serum detected. It might therefore be expected that mice protected from P. yoelii challenge NMS ‘Cl:20 by transfer of hyperimmune serum should 3-Week convalescent I :2,560 I I-Week convalescent 1:640 be resistant to secondary challenge. How1:20,480 P. yoelii hyperimmune” ever, in the present study it was found that protected by transfer of I’ Serum from mice taken 7 days after a secondary mice initially hyperimmune serum were fully susceptible challenge with P. yoelii.

P. yoefii

24

FREEMAN

to a second P. yoelii challenge given some weeks after the first. The results indicate that the complete elimination of P. yoelii parasitemia, mediated by transferred hyperimmune serum, can be achieved in the absence of a specific immune response on the part of the recipient. The protective effect of hyperimmune serum was most clearly seen after transfer into mice with overt, established P. yoelii infections. Parasitemia declined dramatically within 24 hr and was undetectable in blood smears by 48 hr after transfer. As parasitemia declined, it was clear from examination of blood smears that reinvasion of new host red cells was impaired, although the proportion of trophozoites remained high. These observations suggest that protective antibody acts against schizonts and/or merozoites of P. yoelii. The results of this study show that antibody-mediated mechanisms are of central importance in the acquisition and maintenance of immunity to P. yoefii. The conclusions are consistent with the findings of Murphy and Lefford (1979), who found no evidence of protective cell-mediated immunity operating against P. yoelii. Cell-mediated mechanisms have been found only in mice vaccinated with killed P. yoelii preparations (Finerty and Krehl 1977; Playfair et ul. 1979) or BCG (Clark et al. 1976), or after cure of acute infection in immunodeficient mice by chemotherapy (Roberts and Weidanz 1979). Such manipulations of the immune system may induce immune responses quite different from those expressed by untreated mice in response to self-limiting Plasmodium yoelii infection. It is clear that the response of untreated mice is protective, provides a long-lasting immunity to reinfection, and is essentially antibody mediated. REFERENCES CLARK,

I. A., AND ALLISON. A. C. 1974. Bahesirr and PIasmodium herghei yoelii infections nude mice. Notur-r (London) 252, 328-329.

croti

miin

AND

PARISH

CLARK, 1. A., ALLISON. A. C., AND Cox, F. E.G. 1976. Protection of mice against Buhesiu and Plusmedium with BCG. Nurure (London) 259, 309-311. FINERTY, J. F., AND KREHL, E. P. 1977. Delayed immune reactions in mice immunized with malarial antigen. American Journal of Tropkul Medicine und

Hygiene

26, 377-381.

FREEMAN, R. R. 1978. “Malarial Infections in Laboratory Mice.” Ph.D. thesis. Australian National University. Canberra. JAYAWARDENA, A. N.. TARCETT, G. A. T., CARTER. R. L., LEUCHAHS, E.. AND DAVIES, A. J. S. 1977. The immunological response of CBA mice to P. you/ii. I. General characteristics, the effects of T-cell deprivation and reconstitution with thymus grafts.

1mmunolog.v 32, 849-859. JAYAWARDENA, A. N..TARGETT,G. A. T., LEUCHARS, E.. AND DAVIES. A. J. S. 1978. The immunological II. The passive response of CBA mice to P. y&ii. transfer of immunity with serum and cells. Immunology 34, 157~ 165. KITCHEN, S. F. 1949. Fulcipurum malaria. In “Malariology” (M. F. Boyd, ed.), Vol. 2, pp. 995p 1016. Saunders, Philadelphia. LANDAU. 1.. AND KILLICK-KENDRICK, R. 1966. Rodent plasmodia of the Republique Centrafricaine: The sporogony and tissue stages of Plusmodium c.huhuudi and P. herghei yoelii. TrunsuctionJ of the

Ro.vul Society

of Tropical Medicine and Hygiene 60,

633-649. MURPHY, J. R., AND LEFFORD, M. J. 1979. Host defenses in murine malaria: Evaluation of the mechanisms of immunity to Plusmodium yoelii infection. Infecfion und Immunity 23, 384-391. PL.AYFAIR. J. H. L., DESOUZA. J. B., DOCKRELL. H. M., AGOMO, P. U., AND TAVERNE. J. 1979. Cell-mediated immunity in the liver of mice vaccinated against malaria. Nurure (London) 282, 731-734. ROBERTS. D. W., AND WEIDANZ. W. P. 1979. T-Cell immunity to malaria in the B-cell deficient mouse.

Americun Journul Hygiene 28, 1-3.

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und

TOPLEY. E., BRUCE-CHWATT, L. J.. AND DORHELL., J. 1970. Haematological study of a rodent malaria model. Journul of Tropkul Medicine und Hygiene 73, l-8. VOLLER, A., AND O’NEILL, P. 1971. Immunofluorescence method suitable for large scale application to malaria. Bulletin of the World Heulth Orgunizution 45, 524-529. WEINBAUM. F. I., EVANS, C. B., AND TIGELAAR, R. E. 1976. Immunity to PluJmodium hrrghei voelii in mice. I. The course of infection in T-cell and Bcell deticient mice. Journul of Immunology 117, 1999 - 2005.