Pathogenicity, Stability, and Immunogenicity of a Knobless Clone of

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1 INFECTION AND IMMUNITY, Mar. 1985, P /85/ $02.00/0 Copyright 1985, American Society for Microbiology Vol. 47, No. 3 Pathogenicity, Stability, and Immunogenicity of a Knobless Clone of Plasmodium falciparum in Colombian Owl Monkeys SUSAN G. LANGRETH* AND ELIZABETH PETERSON Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland Received 22 October 1984/Accepted 17 December 1984 The pathogenicity, immunogenicity, and morphological stability of a knobless clone of strain FCR-3 of the human malaria parasite Plasmodium falkiparum was investigated in Aotus monkeys. An early knob-bearing (K+), wild-type isolate of strain FCR-3 and the D3 knobless (K-) clone were adapted to Aotus monkey erythrocytes in continuous culture, establishing the parasites in Aotus cells without exposure to in vivo cellular or humoral immune responses. All monkeys, intact or splenectomized, which were infected with wild-type FCR-3 adapted to Aotus cells in vitro, developed virulent infections and had to be drug treated. The intact nonsplenectomized animals which received knobless D3 cloned parasites did not develop virulent infections even after multiple infections. The splenectomized monkeys which received the K- D3 clone had virulent infections. Late-stage wild-type K+ parasites sequestered in both intact and splenectomized monkeys, whereas late-stage D3 K- parasites did not sequester in the splenectomized animals. These results suggest that two elements affected the pathogenicity of the malaria parasites in these experiments. Knobs on K+-infected erythrocytes enabled these parasites to sequester, presumably by attachment to capillary endothelium. When present, the spleen eliminated circulating K- late-stage erythrocytes, presumably by selection on the basis of their nondeformability. Although clone D3 K- parasites are nonvirulent in intact monkeys, they induced some immunological protection against challenge with wild-type K+ parasites. The surface morphology of K-- infected erythrocytes remains unaltered throughout these experiments, suggesting that loss of knobs is a stable condition. Infection with the human malaria parasite Plasmodium falciparum results in visible alterations at the surface of the host erythrocyte. These alterations, called knobs, are 70- to 100-nm electron-dense cuplike protrusions beneath the erythrocyte unit membrane. They have been observed in infected erythrocytes (IRBCs) of both humans and Aotus monkeys in vivo and in vitro (1, 15, 21, 30). They are reported to contain histidine-rich protein of parasite origin (8, 12, 13, 18) and are antigenically distinct from the rest of the erythrocyte surface (14). Individuals with immunity to P. falciparum have antibodies in their sera which bind to the knobs (16). The knobs appear on the red cell surface at the stage when rings mature into trophozoites. This timing correlates with the sequestration of late stages from the peripheral circulation. The knobs are believed to be the sites of attachment of IRBCs to the capillary endothelium in the deep vasculature, thereby obstructing blood circulation and avoiding passage through the spleen (22). With the development of methods to maintain erythrocytic stages of P. falciparum in continuous culture (28), parasite variants which did not produce knobs soon were detected in isolates from several geographical areas (17). Cultures have been enriched for knobless variants by plasma expander selection techniques (25). Knobless (K-) singlecell isolates then have been cloned from such cultures (31). Merozoites harvested from such a knobless clone have been used to immunize Aotus monkeys, with muramyl dipeptide as the adjuvant (29). The stability and pathogenicity of viable knobless parasites of P. falciparum have not been vigorously examined in vivo. It is unknown whether knobless parasites are more or less virulent than wild type or whether loss of knobs is a stable condition. It also is unknown whether knobless (K-) * Corresponding author. 760 trophozoites and schizonts will sequester from the peripheral circulation of an infected animal. If they do not sequester, it is uncertain whether they will be recognized or destroyed by the spleen. To investigate these parameters, a knobless clone and its parent wild-type (K+) strain of P. falciparum were adapted to owl monkey erythrocytes. Human malaria parasites are usually adapted to monkey cells by serial passage through several splenectomized animals. Because of reports by J. Barnwell et al. (3, 4), M. Hommel et al. (9, 10), and P. David et al. (7) of changes in antigenicity upon passage of malaria parasites through splenectomized primates, we did not adapt the K- clone and K+ wild-type parasites in this way. Instead, the parasites were adapted in vitro by continuous culture in owl monkey erythrocytes by recently developed modifications of human cell culture methods (23). In this report we present evidence that a knobless clone of P. falciparum (FCR-3 clone D3) is not pathogenic but is immunogenic in intact Colombian owl monkeys. Late-stage parasites of the K- clone, which lack knobs to bind to capillary endothelium, do not sequester and therefore are subject to destruction by the spleen. In addition, the surface morphology of Aotus erythrocytes infected with the K- clone remains knobless in cells isolated from infected intact or splenectomized owl monkeys and after long-term culture. MATERIALS AND METHODS Monkeys. Adult, healthy Colombian owl monkeys (Aotus trivirgatus griseimembra) were obtained from South American Primates, Miami, Fla.; Walter Reed Army Institute of Research, Washington, D.C.; Litton Bionetics, Rockville, Md.; and the National Institutes of Health, Poolesville, Md. All animals were karyotyped and determined to be type II, III, or IV (Table 1).

2 VOL. 47, 1985 TABLE 1. Comparison of parasitemias in intact and splenectomized monkeys infected with FCR-3 wild type or FCR-3 clone D3 (K-)a Monkeysb parasitemia (%) Drug treated FCR-3 wild type, intact 115 (M, II) 4.1 Yes 5335 (F, II) 6.5 Yes 5336 (F, III) 5.5 Yes 7921 (F, II) 0.8 Yesc 8443 (M, II) 2.4 Yesc FCR-3 wild type, splenectomized 217J (F, II) 4.4 Yes Clone D3, intact 6902 (M, II) 0 No 8490 (M, II) 0 No WR 159 (M, II) 0 No 5342 (F, II) 0.02 No 114 (F, IV) 0 No 216J (F, III) No WR 157 (F, IV) 0 No Clone D3, splenectomized 209J (M, III) 7.6 Yes WR 135 (M, IV) 2.9 No 5341 (F, III) 7.6 Yes a Peak parasitemias for each group cannot be averaged because of the intervention by drug treatment. Criteria for drug treatment are detailed in the text. bsex (F, female; M, male) and karyotype are within parentheses. c Monkeys were drug treated when patent, with reticulocytes > 20%, and hematocrit < 20. Animals were anesthesized with 0.1 to 0.15 ml of ketamine (100 mg/ml) for venipuncture and for infection with parasitized erythrocytes via the femoral vein. Blood was drawn without anticoagulant for serum samples at 7- to 10-day intervals. The last preimmune blood draw was 7 days before infection. Blood was taken from ear pricks for daily monitoring of parasitemias on blood films and for samples for electron microscopy. At peak parasitemias, blood with 10% anticoagulant (heparin or citrate phosphate dextrose) was drawn for recovery of parasitized erythrocytes for in vitro culture, cryopreservation, and preparation of antigen slides for immunofluorescence assays (IFAs). Parasites. A stabilate of P. falciparum FCR-3 (FMG) from the Gambia (11), cryopreserved within 2 months of its original in vitro adaptation, was used throughout these experiments. This parental wild-type strain FCR-3 is morphologically and antigenically heterogeneous (15-17). The D3 knobless clone of strain FCR-3 was obtained by microscopic selection by Trager et al. (31). Both wild-type FCR-3 and the D3 clone were kindly provided by William Trager. Parasite cultivation. Parasites (FCR-3 wild type and clone D3) grown in human 0+ erythrocytes with A' serum by the Trager-Jensen (28) method were adapted to culture in Aotus monkey erythrocytes (23). The adapted parasites were maintained in vitro in Aotus erythrocytes for 1 month before monkeys were infected. The erythrocytes of each monkey were tested for their ability to support the growth of both the D3 clone and the wild-type strain in vitro before in vivo infections. Erythrocytes from infected monkeys exhibiting parasitemias of to 7.0% were placed into culture to expand parasite populations for cryopreservation and electron microscopy. P. FALCIPARUM IN COLOMBIAN OWL MONKEYS 761 Infection of monkeys from cultured parasites. Inocula were prepared from cultures at parasitemias of approximately 2%, with ring stages predominant. The parasitized erythrocytes were washed in RPMI 1640 medium to remove the AB serum and were diluted in RPMI 1640 medium without serum to give an inoculum of 0.5 ml containing a specific number of parasitized erythrocytes, usually 5 x 105. After infection of the animals, the extra syringes which had been prepared with infecting doses were returned to the laboratory, and the parasitized erythrocytes were returned to culture, passing the cells through a 23-gauge needle, to simulate conditions of animal infection. These inoculum controls were cultured with 10% human AB serum at 2% hematocrit for 4 to 7 days to ascertain viability of the infecting parasites. Approximately 3 months before infection, several monkeys were splenectomized (217J, 209J, WR 135, and 5341). Monkeys 115, 7921, 8443, and 217J (splenectomized) received doses of 5 x 105 FCR-3 wild-type parasites. Monkeys 6902, 8490, WR 159, 5342, 209J (splenectomized), WR 135 (splenectomized), and 5341 (splenectomized) received initial doses of 5 x 105 clone D3 parasites. The intact monkeys in this group subsequently received increasingly higher doses of clone D3, up to 1 x 107 parasites, at approximately monthly intervals. Monkeys 114, 216J, and WR 157 initially received 5 x 106 clone D3 parasites, followed by two doses of 1 x 107 clone D3 parasites at approximately monthly intervals. Monkeys 6902 and 8490 were challenged with 5 x 105 FCR-3 wild-type parasites 1 month after their second infection with clone D3. Monkeys 114 and 216J were challenged with S x 105 FCR-3 wild-type parasites after receiving three doses of clone D3. Monkeys 5335 and 5336, never before exposed to P. falciparum, also were given 5 x 105 wild-type parasites at the time of these challenge infections. Parasitemias in monkeys were monitored by Giemsastained blood films. Counts were based on a minimum of 20,000 erythrocytes. The following criteria were used for determining when to drug treat experimental animals: parasitemia above 5% (if predominantly ring stages) or above 4% (if late stages prominent); reticulocytes above 20% of the total erythrocytes; hematocrit below 20; anorexia exceeding 24 h; or moribund animal. Monkeys were treated with a total of 55 to 65 mg of amodiaquine (no. BG58821/2977 AK; Walter Reed Army Institute of Research) given intramuscularly in water (50 mg/ml) over 3 to 4 days. Recrudescent monkeys were treated when their parasitemias exceeded 3.5%. Microscopy. IFAs were performed as described previously (33), with antigen slides prepared with FCR-3 wild-type cultured parasites. Dilutions of heat-inactivated sera from 1:20 to 1:10,000 were tested, and titers reported are the reciprocal of the last dilution giving positive fluorescence of merozoites within schizonts. For all assays, the control normal serum was a pool prepared from 10 naive Aotus monkeys, screened for negative response to P. falciparum by IFA and to human erythrocyte surface proteins in a hemagglutination assay. Slides were examined by epifluorescence microscopy. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were performed on both the wild-type FCR-3 and the D3 clone parasites from cultures in human O+ erythrocytes, from cultures in Aotus erythrocytes before infection of animals, from isolates from all monkeys exhibiting parasitemias, and from isolates from infected monkeys which were placed back into culture in Aotus erythrocytes for 1 to 14 days. Methods for fixation and processing for TEM were as described previously (15). TEM

3 762 LANGRETH AND PETERSON samples were examined in a JEOL 100CX or Philips 400 transmission electron microscope, operating at 60 kv. For SEM, cultured cells were routinely fixed by a modification of the procedure of Arnold et al. (2). Cells were washed with culture medium (RPMI 1640 medium) without serum, prefixed with 0.1% glutaraldehyde in RPMI 1640 medium (ph 7.0) for 15 min at room temperature, and fixed with 2.0% glutaraldehyde in phosphate-buffered saline (ph 7.3) for 45 min at room temperature. Cells were resuspended in RPMI 1640 medium and allowed to settle for 30 min onto cover slips which had been pretreated with 0.1% poly-l-lysine (ph 7.3) for 30 to 60 min. Unattached cells were washed off with RPMI 1640 medium, and the cover slips were rinsed with distilled water, dehydrated with increasing concentrations of ethanol, and transferred to Freon 113. Samples were then critical-point dried from CO2 with a Polaron criticalpoint drying apparatus or air dried from Freon 113 in a vacuum dessicator at room temperature (20). After the samples were coated with gold-palladium, they were viewed with a JEOL JSM-35 scanning electron microscope at an accelerating voltage of 25 kv and recorded with Polaroid P/N 55 film. Samples of blood from infected animals were collected in heparinized blood capillary tubes and processed for SEM by the protocol described for culture cells except that samples were usually held overnight at 4 C in fixative. For samples which contained no late-stage parasites, cells were cultured overnight to allow parasite maturation and were then processed for SEM. To identify parasitized cells for SEM analysis, a light microscopy step was included in sample preparation. After fixation and attachment to glass cover slips, samples were rinsed with RPMI 1640 medium and stained with Giemsa stock solution diluted 1:5 with phosphate buffer (ph 6.8) for 10 min at room temperature, in a modification of the procedure of Wetzel et al. (32). After the cover slips were rinsed with RPMI 1640 medium, they were inverted over a bufferfilled well on a microscope slide, and cells with Giemsastained parasites in areas near an orientation pattern were photographed for later comparison with scanning electron micrographs of the same cells. Routine processing for SEM then was continued. RESULTS Pathogenicity. Both the wild-type strain FCR-3 and the D3 clone parasites were cultured continously in Aotus erythrocytes for 1 month before infection of monkeys. The erythrocytes of all monkeys in these experiments supported the growth in vitro of both wild-type and D3 parasites under standard culture conditions (23). Course of infection in vivo. Data comparing the wild-type (K+) and clone D3 (K-) infections in intact and splenectomized monkeys are presented in Table 1. All monkeys which received the wild-type FCR-3 parasites developed virulent infections (high parasitemias or reticulocytes > 20% total red cells or both). Throughout their infections ring-stage parasites were the predominant form in the peripheral circulation. Monkeys 8443 and 7921 went into reticulocyte crisis (reticulocytes > 20%, 5% normoblasts, hematocrit < 20) when their parasitemias were 2.4 and 0.8%, respectively. The monkeys infected with wild-type FCR-3 required drug treatment according to the preestablished criteria (see above). Two of the animals which received wild-type FCR-3 subsequently recrudesced (217J splenectomized and 115), exhibiting low transient parasitemias. After receiving a single low dose of the K- D3 clone, the splenectomized monkey 209J developed a virulent infection INFECT. IMMUN. and was drug treated. This animal recrudesced three times and also was drug treated at the third recrudescence. One year after recovery from the third recrudescence, monkey 209J again was infected with a low dose (5 x 105) of the K- D3 clone. The monkey became patent 12 days later. The parasitemia peaked at 0.17% on day 17 postinfection and was negative by day 27. One month later the monkey again was challenged with a higher dose (1 x 106) of D3 but did not become patent. Two additional splenectomized animals, naive to malaria, received a single low dose (5 x 105) of the K- D3 clone and developed virulent infections. One animal (5341) had a peak parasitemia of 7.6% and was drug treated. The second animal (WR 135) had parasitemias in excess of 1% for 10 days but did not exceed 3% and was not drug treated. Reticulocytes exceeded 20% in this animal immediately after clearance of the parasites, and the animal subsequently became chronic at low parasitemia (<0.05%). During the first experiment, four intact monkeys received multiple doses of the K- D3 clone. Three animals (WR 159, 6902, and 8490) never became patent with D3, and the fourth (5342) showed transient parasitemias never exceeding 0.02%. After receiving two doses of D3 parasites, monkeys 8490 and 6902 were challenged with the wild-type FCR-3. Although they developed parasitemias of 1.2 and 3.6%, respectively, the animals controlled their infections without drug treatment. Their hematocrits and reticulocyte percentages remained within acceptable ranges throughout their infections. In contrast, the naive monkey 5335 had to be drug treated when its parasitemia reached 6.5%. In a second experiment, three intact monkeys (114, WR 157, and 216J) received multiple high doses of clone D3. Two animals (114 and WR 157) never became patent, whereas monkey 216J exhibited a parasitemia (0.005%) on only 3 days. Monkeys 114 and 216J and a naive animal (5336) were subsequently challenged with the wild-type strain. All three became patent 9 to 10 days later and were drug treated 16 to 17 days postchallenge when their parasitemias were 5.9, 5.9, and 5.8%, respectively. The naive animal (5336) recrudesced and was again drug treated (peak parasitemia, 3.7%). Parasitemia did not recur in monkey 114. The recrudescent parasitemia of monkey 216J did not exceed 0.20%. An analysis was made of the proportion of late stages present in the peripheral circulation of every animal in these experiments on all days when their parasitemia exceeded 1%. These data are summarized in the form of a histogram (Fig. 1) and include values for eight FCR-3-infected animals (seven intact and one splenectomized) and three splenectomized D3-infected animals. None of the seven intact D3- infected animals ever had parasitemias exceeding 0.02%, and therefore do not provide data to be included in this summary. Ring forms were predominant in FCR-3-infected animals, with >87% of the data points showing 20% or fewer late stages (Fig. 1). In no instance did late stages exceed 50% in these animals. In contrast, the proportion of late stages in the peripheral circulation of the D3-infected, splenectomized animals was frequently high, with approximately 45% of the data points showing >50% late stages. Antibody response. Before infection, the serum antibody titers against P. falciparum FCR-3 were negative for all monkeys as determined by IFA. Monkeys infected with P. falciparum FCR-3 wild-type strain in Aotus cells from culture developed humoral immune responses as detected by IFA (Table 2). Monkeys infected with clone D3 also developed antibody titers (Tables 2 and 3). The response to D3 was dose dependent in that the monkeys receiving higher amounts of D3 (5 x 106) rapidly produced higher antibody

4 VOL. 47, c z 0' U t 0 50 LATE STAGES (%) FIG. 1. Frequency distribution of percentage of total number of days when infected animals had late-stage parasites (trophozoites and schizonts) as the indicated percentage of their total IRBCs. Solid bars are total percentages for eight animals infected with FCR-3 wild type, seven intact and one splenectomized. Patterned bars are total percentages for three splenectomized animals infected with D3 K-. No intact animals infected with D3 developed significant parasitemias. titers (114, 216J, and WR 157). Monkeys 8490 and 6902, which were infected twice with D3, followed by a wild-type challenge, produced high IFA titers 1 month after challenge (Table 3). Monkeys 114 and 216J, which received three large doses of D3 developed high IFA titers before challenge with wild-type parasites (Table 3). Stability of phenotype. The surface morphologies of erythrocytes infected with wild-type and D3 parasites were studied by TEM and SEM. These surface morphologies appeared to be the same, whether the parasites were cultured in human or Aotus erythrocytes. In TEM, most trophozoiteand schizont-irbcs in wild-type FCR-3 cultures had distorted surfaces and knob structures. All trophozoite- and schizont-irbcs observed in D3 clone cultures had relatively smooth surfaces and lacked knobs as determined by TEM. A few (<10%) late-stage IRBCs from FCR-3 wild-type cultures TABLE IFA titers on monkeys with virulent infections' Titer Monkey Infecting Preinfec- At drug treat- At 1 mo Parasites Ptionfec- ntdu treat- posttreatment 115b Wild type (K+) <20 20 (21) Jb.C Wild type (K+) <20 <20 (21) Wild type (K+) <20 80 (25) b Wild type (K+) <20 40 (17) Wild type (K+) < (31) Wild type (K+) < (30) Jb.C D3 clone (K-) < (33) 640 a All animals were drug treated at peak parasitemia except monkeys 7921 and 8443, who were treated when reticulocytes > 20%o and hematocrits < 20. b Had recrudescences. c Monkeys 217J and 209J were splenectomized. P. FALCIPARUM IN COLOMBIAN OWL MONKEYS 763 had the D3 surface morphology as determined by TEM as expected, since the D3 clone was derived from the FCR-3 wild-type strain. The IRBC surface morphology of the parasites isolated from each monkey at peak parasitemias retained the same morphology as that of the parasites in the infecting dose, as determined by TEM. The presence or absence of a spleen had no observed effect on IRBC surface morphology. Clone D3-parasitized erythrocytes isolated from monkeys 5342 (intact) and 209J (splenectomized) remained knobless, smooth, and undistorted (Fig. 2A). Isolates from the various recrudescences of monkey 209J also retained typical D3 morphology. Parasitized erythrocytes isolated from monkeys infected with FCR-3 wild type (intact 115, 5335, 7921, and 8443 and splenectomized 217J) continued to exhibit the heterogeneous surface morphologies characteristic of this strain. Most late-stage IRBCs showed knob-bearing, distorted surfaces (Fig. 2D). Isolates from monkeys 6902 and 8490 after challenge with FCR-3 wild type were of K+ morphology. SEM, in combination with Giemsa-stained light microscopy, also was used to assess the surface morphology of cells infected with late-stage FCR-3 wild-type and D3 parasites. Knobs were observed on the surface of most late-stage FCR-3 wild-type infected cells when the parasites were cultured in either human or Aotus erythrocytes, and the relative shape and frequency of the knobs appear similar in both host cell types. Samples isolated from each FCR-3-wildtype-infected animal at the peak parasitemia, before drug treatment, showed the same predominantly knobby morphology as that seen in the culture material. A human O cell infected with a trophozoite of the FCR-3 wild-type strain is shown in Fig. 2E and is a typical example of the K+ morphology of late-stage infected cells. Figure 2F shows a trophozoite-infected Aotus erythrocyte isolated from an animal after infection with FCR-3 wild type. Erythrocytes infected with late stages of clone D3 parasites were uniformly knobless and relatively undistorted. Many trophozoite-infected cells appear to have a single, large depressed area on the surface. This morphology is similar in cultures of human and Aotus erythrocytes and in IRBCs isolated from splenectomized animals. The knobless morphology typical of this clone is seen in Fig. 2B and C. Figure 2B shows a D3 trophozoite-infected human O+ erythrocyte in culture, and Fig. 2C shows a trophozoite-infected Aotus erythrocyte isolated from an animal after infection with clone D3. TABLE 3. IFA titers on FCR-3 clone D3-infected intact monkeys Titer Monkey Preinfec- At 1 mo At 1 mo At 1 mo tion postinfec- postinfec- postinfection no. 1 tion no. 2 tion no. 3 Low dose of D3 WR159 < a < < ,120b 6902 < " High dose of D3 114 < ,280 2, Ja <20 2,560 1,280 2,560 WR157 <20 1,280 2,560 2,560 a Transient patency. b Infection number 3 was with wild-type parasites.

5 764 LANGRETH AND PETERSON INFECT. IMMUN. FIG. 2. Transmission and scanning electron micrographs of cells containing D3 K- (A, B, and C) and FCR-3 wild-type K+ (D, E, and F) late-stage parasites. (A) K- schizont-irbc from the third recrudescence of the splenectomized monkey which received clone D3. (B) Human O+ erythrocyte in culture containing a D3 K- trophozoite. (C) D3 trophozoite-infected Aotus erythrocyte from an infected monkey. (D) A K+ schizont-irbc from an intact Aotus monkey infected with FCR-3 wild type; the isolate was cultured for 1 day to mature the parasites. (E) Human O+ erythrocyte in culture containing an FCR-3 wild-type trophozoite. (F) Aotus erythrocyte taken from an infected monkey containing an FCR-3 wild-type trophozoite. Arrowheads indicate knobs on the cell surface. Bars represent 1,um. DISCUSSION These studies in Colombian Aotus monkeys directly compare the pathogenicity of a heterogeneous wild-type K+ strain of P. falciparum, FCR-3, and the cloned D3 K- line derived from FCR-3. This cloned line lacks the surface distortions and knobs of erythrocytes infected with the parental strain. The wild-type K+ strain caused virulent infections in both intact and splenectomized monkeys, and late-stage IRBCs were sequestered from the peripheral circulation regardless of whether the host monkey was splenectomized. The cloned K- line was virulent in splenectomized animals, and late-stage IRBCs did not sequester in these monkeys. In intact animals, the K- cloned line was essentially avirulent; parasitized cells were seen only on rare occasions, and these few were ring forms. Two major factors appear to play crucial roles in determining virulence in these experiments: the surface of latestage IRBCs is K+ or K-, and the animal is splenectomized or intact. Our results suggest the following: (i) the presence or absence of knobs determines whether late-stage parasites will sequester or circulate, and (ii) if late stages do circulate, the presence or absence of a spleen determines whether these circulating late-stage parasites will be eliminated or contribute to a virulent infection. The mechanism of this very rapid splenic destruction of K- IRBCs is probably not antibody mediated but is based on recognition of nondeformability such as occurs with senescent erythrocytes. Cranston et al. (6) reported that erythrocytes infected with late-stage P. falciparum parasites (trophozoites and schizonts) lose the ability to deform, as measured by sheer stress in vitro. They compared the cloned D4 line of FCR-3 (31), which is K-, with two K+ strains. They found that loss of deformability is related to parasite maturation in both K+ and K- strains. Since the spleen is known to recognize and destroy nondeformable erythrocytes, these authors suggested that the loss of deformability is the determining factor in splenic elimination of late-stage P. falciparum IRBCs. The roles of the spleen in malaria are varied and complex. It is a major site of parasite and erythrocyte destruction (26, 34), and both specific and nonspecific immune mechanisms have been demonstrated (24, 27). In addition, David et al. (7) and Hommel et al. (9, 10) have suggested that the spleen may modulate antigenic expression, sequestration, and in vitro cytoadherence properties of erythrocytes infected with latestage P. falciparum. These studies compared two lines of K+ parasites which were sequentially passaged in several squirrel monkeys. The surface and knob morphologies of the wild-type- and D3 K--IRBCs did not change throughout the course of our experiments. Thus, these major morphological structures appear to be stable in vivo as well as in vitro. Variation of

6 VOL. 47, 1985 surface IRBC antigens, such as occurs in the Plasmodium knowlesi-rhesus model, may be a cause of recrudescences in primate malarias (5). Antigenic variation of IRBC surface components has recently been reported in P. falciparum infections in squirrel monkeys in a cloned parasite line and in recrudescent populations of the Indochina I strain (9). Strainspecific high-molecular-weight P. falciparum IRBC surface antigens which are related to in vitro cytoadherence also have been reported (19). The relationship of these antigens to the IRBC surface antigens in D3 K- cells and to loss of knobs is not known. Analysis of surface antigenic determinants in original and recrudescent animal isolates of the D3 K- line is in progress. Although parasites of the cloned D3 knobless line have reduced pathogenicity in intact monkeys, they induced a humoral immune response, as measured by IFA titers, and this response was dose dependent (Table 3). To determine whether the immune response induced by D3 was protective, monkeys were subsequently challenged with homologous D3 or heterologous FCR-3 wild-type parasites. Prior exposure to the D3 clone provided solid protection for the splenectomized monkey 209J when later challenged with homologous D3 parasites. Two experiments demonstrated partial protection after heterologous FCR-3 wild-type challenge. In experiment 1, two D3 monkeys eliminated their FCR-3 wild-type infections without drug treatment. In experiment 2, the D3 monkeys received higher doses of D3 parasites and developed higher antibody titers as measured by IFA but were less protected than the monkeys in experiment 1 when challenged with wild-type FCR-3. The monkeys had to be drug treated to control their initial parasitemias from wild-type infection, but only the control naive animal (5336) had to be drug treated a second time because of a high recrudescent parasitemia. Due to the extremely limited supply of Colombian owl monkeys, animals in experiments 1 and 2 differed by sex and karyotype (Table 1), and these variables may have contributed to the observed differences in protection. The genetics of the immune response in Aotus monkeys have not yet been well studied. Partial rather than complete protection is consistent with the suggestion that the wild-type strain is both antigenically as well as morphologically more heterogeneous than the D3 cloned line. The adherence of late-stage K+ parasitized erythrocytes to capillary endothelium via knobs contributes to the pathogenesis of falciparum malaria. Sequestered parasites obstruct local blood circulation, leading to local anoxia and ischemia. In addition, these sequestered parasites avoid attack by both specific and nonspecific immunological mechanisms, including splenic destruction. The use of knob-specific antisera to neutralize knob function may be an additional approach to controlling the pathogenicity of this disease. Parasite sequestration has been reversed in vivo and in K+ IRBC cytoadherence in vitro (7) with squirrel monkey immune serum. Antibodies to knobs have been identified in the sera of immune owl monkeys (16). Thus, although most attempts to develop immunity to falciparum malaria use merozoite antigens, knob-endothelium binding components also are possible candidate antigens for a multivalent vaccine. ACKNOWLEDGMENTS We thank William Trager for providing us with an early isolate of wild-type FCR-3 and the D3 cloned line and William Ellis for antimalarial drugs. We are indebted to Donald Krogsted and William Collins for helpful discussions and advice and to Robert Reese for P. FALCIPARUM IN COLOMBIAN OWL MONKEYS 765 karyotyping several of the monkeys. The technical assistance of G. Patrick Riordan, Susan Kenney, and Ina Ifrim is gratefully acknowledged. This work was supported by funds from the U.S. Agency for International Development (PASA no. DZ/DSB ) and the United Nations Development Program/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases (Project no ). LITERATURE CITED 1. Aikawa, M., J. R. Rabbege, and B. T. WeUde Junctional apparatus in erythrocytes infected with malarial parasites. Z. Zellforsch. U. Mikroskop. Anat. 124: Arnold, J. D., A. E. Berger, and 0. L. Allison Some problems of fixation of selected biological samples for SEM examination. Scanning Electron Microscopy/1971: BarnweUl, J. W., R. J. Howard, H. G. Coon, and L. H. Miller Splenic requirement for antigenic variation and expression of the variant antigen on the erythrocyte membrane in cloned Plasmodium knowlesi malaria. Infect. Immun. 40: Barnwell, J. W., R. J. Howard, and L. H. 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