Cloned Plasmodium knowlesi Malaria

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1 INFECTION AND IMMUNITY, June 1983, p /83/ $02.00/0 Copyright C 1983, American Society for Microbiology Vol. 40, No. 3 Splenic Requirement for Antigenic Variation and Expression of the Variant Antigen on the Erythrocyte Membrane in Cloned Plasmodium knowlesi Malaria JOHN W. BARNWELL,'* RUSSELL J. HOWARD,1 HAYDEN G. COON,2 AND LOUIS H. MILLER1 Laboratory ofparasitic Diseases, National Institutes ofallergy and Infectious Diseases,1 and the Laboratory of Cell Biology, National Cancer Institute,2 National Institutes of Health, Bethesda, Maryland Received 29 November 1982/Accepted 2 March 1983 Variant antigens appear on the surface of Plasmodium knowlesi-infected erythrocytes as the asexual parasite matures and are detected by antibodymediated schizont-infected cell agglutination (SICA). We now show that cloned parasites can undergo antigenic variation in nonsplenectomized monkeys. In addition, we previously described a new P. knowlesi phenotype in which uncloned parasites passaged in splenectomized monkeys were no longer agglutinable by immune sera. We-have designated this new phenotype SICA[-] and the one expressing the variant antigen SICA[+]. Cloned parasites can also switch from SICA[+] to SICA[-] in splenectomized monkeys. The switch from SICA[+] to SICA[-] is a gradual process that requires sequential subpassage in several monkeys. After passage in one monkey, the agglutination titer decreased 4- to 16- fold. Decreased agglutination was associated with decreased antibody binding on all infected erythrocytes as measured by fluorescein-conjugated anti-rhesus monkey immunoglobulin. The asexual malaria parasite can therefore alter its expression of variant antigen in response to the host environment (antivariant antibody or splenectomy). When cloned SICA[-] parasites were inoculated into intact monkeys, two courses of parasitemia were observed: fulminant parasitemia (>20%) and parasitemia that was controlled. Fulminant infections were associated with conversion of the parasite from SICA[-] to SICA[+], i.e., from nonexpression to expression of the variant antigen on the erythrocyte surface. Parasitized erythrocytes remained SICA[-] in those infections that were controlled. It appears, therefore, that the expression of the variant antigen on the erythrocyte surface may influence parasite virulence. The chronic persistence of parasites in a host despite the concurrent presence of potentially parasiticidal immune responses is characteristic of infections with several parasitic protozoa. Some of these protozoan parasites have the ability to antigenically vary the molecules that are targets of antiparasitic immunity and thus escape complete elimination from an immunocompetent host. For example, African trypanosomes undergo antigenic variation during the course of an infection by repeatedly expressing different surface coat glycoproteins (24). The asexual intraerythrocytic parasites of malaria also often cause chronic infections that persist for long periods. The most direct evidence that the chronicity of malaria infections may be due in part to antigenic variation of asexual parasites comes from studies on the primate malaria parasite, Plasmodium knowlesi, in rhesus monkeys. In 1938, Eaton (12) found that when the asexual intraerythrocytic parasites of P. knowlesi had matured to the schizont stage the infected erythrocytes were agglutinated by sera from rhesus monkeys immune to P. knowlesi, but not by sera from normal rhesus monkeys. Uninfected rhesus monkey erythrocytes or erythrocytes containing immature asexual parasites were not agglutinated by the immune sera. This indicated that a new antigen must have appeared on the surface of schizont-infected erythrocytes. Twenty-seven years later, Brown and Brown (5) demonstrated by the antibody-mediated agglutination of schizont-infected erythrocytes that the new parasite-dependent antigen expressed on the surface of P. knowlesi-infected erythrocytes was antigenically variable. If a monkey was infected with a particular P. knowlesi population, serum collected 2 or more weeks after drug cure of this infection would agglutinate the erythrocytes infected with that parasite population. This variant phenotype did not change if passaged in naive, nonimmune monkeys. However, reinoculation of this variant population into a monkey with antibodies specific for this 985

2 986 BARNWELL ET AL. variant type resulted in the appearance of a parasite population that expressed a new variant antigen on the surface of the schizont-infected erythrocytes which was not recognized by antibodies to the first variant population. Suppression of the initially high parasitemias of P. knowlesi in rhesus monkeys with antimalarial drugs resulted in chronic infection with repeated waves of parasitemia. During chronic infection each recurrent wave of parasitemia consisted of a parasite population that was of a new variant type (3, 5). Since the agglutination assay that defined the variant types was termed the schizont-infected cell agglutination (SICA) test, the variant antigen expressed on the surface of P. knowlesi-infected erythrocytes was referred to as the SICA antigen. These studies led to the suggestion that chronicity in malaria was partly due to the variation of the SICA antigen on the surface of infected erythrocytes (3, 5, 8, 25). Recently, we reported the existence of a new phenotype of P. knowlesi that was revealed by the passage of uncloned parasite lines in splenectomized monkeys (1). Previous studies on antigenic variation in P. knowlesi have always been performed in monkeys with spleens, and the schizont-infected erythrocytes were always agglutinated by the appropriate antisera. P. knowlesi parasites that were passaged in intact (nonsplenectomized) rhesus monkeys and that were agglutinable by immune sera were designated as the SICA[+] phenotype. After passage of the SICA[+] parasites in splenectomized rhesus monkeys, the infected erythrocytes could not be agglutinated by variant-specific antisera, by cross-reactive sera from chronically infected monkeys, or by antisera raised in intact monkeys against the parasites from splenectomized monkeys. The production of these nonagglutinable parasitized erythrocytes was designated as the SICA[-] phenotype of P. knowlesi. Thus, we found that with uncloned parasites an alteration in or loss of variant SICA antigen expression had occurred in the absence of the spleen. These experiments, however, did not answer the question of whether the loss of agglutinability results from the selection of a genetically distinct SICA[-] subpopulation in the original SICA[+] parasite population, or whether the original SICA[+] parasites had the ability to modulate the expression of the variant SICA antigen on the surface of infected erythrocytes and whether this expression was influenced by the spleen. To address this question and to define the role INFECT. IMMUN. of the spleen in SICA antigen expression and variation we cloned SICA[+] P. knowlesi parasites and now present evidence that (i) cloned parasites are able to vary their variant SICA antigen type in intact, but not spleenless, monkeys; (ii) the switch of cloned SICA[+] parasites to SICA[-] observed in the absence of a spleen results from a reduction in variant SICA antigen expressed on all infected erythrocytes; and (iii) SICA[-] parasites are less virulent than SICA[+] parasites in intact rhesus monkeys. MATERIALS AND METHODS Monkeys and malaria parasites. Rhesus monkeys (Macaca mulatta) of either sex weighing between 4 and 10 kg were used in the experiments described in this paper. Monkeys were inoculated intravenously with either fresh or cryopreserved parasitized erythrocytes containing ring stage parasites of the Malaysian H strain of P. knowlesi (9) to initiate infections. Tissue culture of malaria-infected erythrocytes. Parasitized erythrocytes containing ring stage parasites were cultured in polystyrene flasks at 2 x 107 to 3 x 107 erythrocytes per ml of medium containing RPMI 1640 supplemented with 30 mm HEPES (N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 28.5 mm NaHCO3, 2 g of glucose per liter, 10 mg of hypoxanthine per liter, 25 mg of gentamicin per liter, and 15% serum (human O+, normal rhesus monkey, or horse). The flasks were gassed with a mixture of 6% C02-3% 02-91% N2, sealed, and incubated for 20 to 24 h until parasites were at the two- to four-nucleus stage of schizont maturation. Cloning procedure. P. knowlesi parasites were cloned by micromanipulation as follows. An intact (nonsplenectomized) rhesus monkey was infected with SICA[+] P. knowlesi that had been passaged only in intact animals. When the parasitemia was above 2% and only schizont stage parasites were in the circulation, blood was drawn from the donor monkey in heparinized syringes, washed once in RPMI mm HEPES (ph 7.2) (no NaHCO3), and diluted to 4 x 106 erythrocytes per ml in RPMI mm HEPES with 15% normal monkey serum (ph 7.2), but lacking NaHCO3 supplementation. The diluted cells were placed in a 10- by 8-mm circular chamber sealed to a large cover slip (with HiVac grease) and overlayered with methylsilicone (a nontoxic oil). With the aid of an inverted microscope (at -900x magnification) a single schizont-infected erythrocyte was selected and aspirated into a siliconized glass micropipette (-10-,um inner diameter). The pipette containing the parasitized erythrocyte was withdrawn through the methylsilicone layer, and a 96- well flat-bottom culture plate was positioned on the stage. The pipette was lowered to the bottom of the well, and the parasitized erythrocyte was expelled under microscopic observation (-225 x) into 100,ul of HEPES-buffered RPMI 1640 with 15% serum from the monkey chosen as recipients of the infected erythrocyte. These procedures assured that only one cell was placed in the well. Then 100,ul of RPMI 1640 (with 57 mm NaHCO3) containing serum (15%) and erythrocytes (10% packed cell volume) from the recipient monkey was placed in the well. The plate was immediately placed in a gas-tight box in an atmosphere of 6% COz-3% 02-91% N2 at 37 C and incubated for 8 to 10 h. This culture period allowed the parasite to mature and to reinvade the recipient monkey's erythrocytes. At the end of the incubation period, the contents of a

3 VOL. 40, 1983 well were drawn into a syringe and injected intravenously into the recipient monkey. This cloning procedure has led to infection in six of seven animals inoculated. Cryopreservation and reconstitution to isotonicity of parasitized erythrocytes. Cryogenic preservation of P. knowlesi was based on procedures developed for the low-temperature storage of human erythrocytes for transfusion purposes (19). When the majority of parasites were at the ring stage, blood was collected from P. knowlesi-infected monkeys in heparinized syringes and centrifuged, and the plasma was discarded. After the addition of cryoprotectant, the blood was distributed in 1.0- to 1.5-mi volumes in NUNC (InterMed, Denmark) plastic freezing vials and placed at -65 to -70 C for 18 h. The vials were then transferred to liquid nitrogen for long-term storage. Thawed cryopreserved, infected blood was reconstituted to isotonicity with graded salt concentrations (19) and cultured as described above. Parasite survival ranged from 70 to 85%. SICA. SICA assays were performed in U-bottom microtiter trays as previously described (1). Parasitized erythrocytes for use in agglutination tests were obtained either directly from rhesus monkeys or from cryogenically preserved infected blood. The infected blood contained (fresh or cryopreserved) ring stage parasites that were matured to the schizont stage in vitro as described above. The erythrocytes containing mature parasites were separated from uninfected erythrocytes and erythrocytes containing immature parasites (rings and young trophozoites) by density centrifugation over Percoll (Pharmacia, Upsala, Sweden) as previously described (1). Surface-specific immunofluorescence of schizont-infected erythrocytes. Cryopreserved erythrocytes infected with ring stage parasites were thawed and cultured in vitro to the schizont stage. The erythrocytes were washed twice in phosphate-buffered saline or Hanks balanced salt solution (ph 7.2) and adjusted to 2.5 x 108/ml. A sample (180,ul) of the cell suspension (3 to 5% parasitized erythrocytes) was added to 20,ul of normal rhesus serum or immune rhesus serum and incubated for 30 to 40 min at 25 C in 12- by 75-mm glass test tubes. The cells were washed twice in Hanks balanced salt solution. A 100-pAl sample of F(ab')2 fragments of fluorescein isothiocyanate-conjugated goat anti-monkey immunoglobulin G (Cappel Laboratories, Cochranville, Pa.) diluted 1:10 in phosphatebuffered saline was added to the pellet of erythrocytes. After incubation for 30 min at 25 C, two washes in Hanks balanced salt solution (1,000 x g for 1 min at 25 C), and suspension in Hanks balanced salt solution containing 0.5% Formalin, surface fluorescence was examined under UV illumination. The percentage of infected erythrocytes that showed surface immunofluorescence was determined by first counting under phase light the number of erythrocytes in a field (400x magnification) that contained refractive pigment granules and then, after switching to UV illumination, counting the number of fluorescence-positive cells in the same field. Antisera. Sera specific for the variant SICA antigen type of each clone were obtained either from rhesus monkeys in which the original cloning was done or from monkeys given a large inoculum (2.4 x 108 parasites) of a cloned line. After parasitemias reached SPLEEN AND ANTIGEN EXPRESSION IN MALARIA 987 5% or greater and parasitized erythrocytes were collected for cryopreservation and SICA testing, the infections were rapidly cured by five daily injections of chloroquine (10 mg of base per kg of body weight). Serum collected 2 or 3 weeks after drug care is specific for the variant type of the parasites of the infection. In some instances 3 to 4 weeks after drug cure 4 x 10' freeze-thawed (three times), schizont-infected erythrocytes in Freund incomplete adjuvant were injected intramuscularly to boost the variant-specific titers without increasing cross-reactivity to other variant types (see below). Antisera specific for five SICA[+] clones, Pkl(A+), Pk2+, Pk3+, Pk4+, and Pkl(B+)1+, were made in the manner described above (see below). Antisera for Pkl(A+) and Pkl(B+)1 + were made by infection and cure followed by one injection of homologous killed parasites in Freund's incomplete adjuvant (antisera 398D [9 July 1981] and 529R [10 September 1981], respectively.) Hyperimmune sera from chronically infected nonsplenectomized monkeys that are cross-reactive to many different variants of the Malaysian H strain of P. knowlesi were obtained as previously described (1). RESULTS SICA[+] clones of P. knowlesi. Four different clones of P. knowlesi were derived from a monkey infected with an uncloned variant population of the Malaysian H strain. These four clones were designated Pkl(A+), Pk2+, Pk3+, and Pk4+. The positive sign after each clone numeral indicates the schizont-infected erythrocytes could be agglutinated by cross-reacting sera from chronically infected monkeys and therefore were SICA[+]. A fifth SICA[+] clone was derived by reinfecting a monkey with the clone Pkl(A+) 6 weeks after drug cure of a previous infection with the same clone. The parasite population that resulted was a new variant, Pkl(B+), as defined by the failure of Pkl(B+)- infected erythrocytes to be agglutinated by anti- Pkl(A+)-specific sera (see below). The new variant, Pkl(B+), was cloned into another monkey and designated Pkl(B+)1 +; the second arabic numeral indicates a recloning of this cloned line. Antisera raised against the clones Pkl(A+), Pk2+, Pk3+, and Pk4+ were variant specific in that they did not cross-react with each other. Pkl(B+)1+ did not cross-react with Pkl(A+), but reacted with antisera against Pk2+ and Pk3 +. P. knowlesi clones grown continuously in vitro become SICA[-] after 3 to 4 weeks in culture. However, SICA[+] clone populations can be maintained and expanded in vivo in naive, nonimmune rhesus monkeys. Therefore, parasitized erythrocytes for study in the SICA test or by immunofluorescence of the infected erythrocyte surface must be grown in monkeys. Conversion of cloned SICA[+] P. knowlesi to SICA[-] after passage in splenectomized rhesus monkeys. After passage of the uncloned SICA[+] parasites in splenectomized rhesus

4 988 BARNWELL ET AL. monkeys, we observed that the infected erythrocytes were not agglutinated by immune sera (1). We designated this new phenotype as SICA[-]. In the present study, two clones of P. knowlesi, Pkl(A+) and Pkl(B+)1+, were serially passaged in splenectomized monkeys to test whether cloned parasites would show the same phenotypic change (Table 1). With each passage in these splenectomized animals, agglutination titers in the SICA test decreased 4- to 16-fold. In the case of clone Pkl(A+), the schizont-infected erythrocytes by the second passage in a spleenless monkey (day 13) were nonagglutinable with either serum specific for the SICA type of the parasite inoculated or with chronic immune serum. Clone Pkl(B+)1 + passaged at shorter intervals was almost nonagglutinable by the third passage in splenectomized monkeys (day 11). Although passage of two P. knowlesi SICA[+] clones in splenectomized monkeys rendered the parasitized erythrocytes nonagglutinable (SICA[-]), it was still not clear whether some parasitized erythrocytes were becoming SICA[-] (had lost surface variant antigens) while others remained SICA[+]. Cryopreserved, infected erythrocytes taken from intact monkeys or after each subpassage in splenectomized monkeys were thawed and cultured in vitro to the schizont stage (-20 h in culture) and then tested for surface immunofluorescence. Immunofluorescence of parasitized erythrocytes from intact monkeys for either clone Pkl(A+) or INFECT.1IMMUN. Pkl(B+)1+ showed that almost 100% of the infected erythrocytes gave surface-specific immunofluorescence (Table 1). After the first and second passages in splenectomized monkeys the SICA titers had decreased by 4- to 16-fold. Even though the SICA titers had decreased, almost 100% of the parasitized erythrocytes still showed surface fluorescence (Table 1). The intensity of fluorescence was reduced, however. After two to four passages in splenectomized animals, when the infected erythrocytes were no longer agglutinated by variant-specific or cross-reactive sera, the immunofluorescence with these sera was negative. Conversion of SICA[-J clones to the SICA[+] phenotype and relationship with virulence. Since parasites of the SICA[+] phenotype can alter their phenotype to SICA[-] in splenectomized monkeys, we next examined whether the reverse would occur in monkeys with spleens, that is, conversion from SICA[-] to SICA[+]. The origin and cloning of the SICA[-] clone, SC, has been described previously (1). Clone Pkl(A-)1- was derived by serial passage of clone Pkl(A+) in three splenectomized monkeys. The nonagglutinable (SICA[-]) parasite population, Pkl(A-), was then recloned in a splenectomized monkey, and the parasites of the resulting infection were designated as Pkl(A-)1-. In three intact naive, nonimmune monkeys infected with clone SC, the parasites of the TABLE 1. Effect of passage of cloned SICA[+] parasites in splenectomized monkeys on expression of variant SICA antigen as measured by agglutination and surface immunofluorescence Agglutination titers (% infected erythrocytes positive by surface Erythrocytes immunofluorescence)b infected with Seraa Reactivity of serum Passage in splenectomized rhesus monkeysc clone: Intact rhesus First Second Third Fourth Pkl(A+) 398D, 9 July 1981 Variant-specific 20, NEG (0) NEG (0) NEG (0) for Pkl(A+) (99.6 ± 0.1) (99.2 ± 0.4) 331, 15 July 1974 Cross-reactive 10, NEG (0) NEG (0) NEG (0) (chronic) (99.5 ± 0.1) (99.1 ± 0.4) 529R, 10 Septem- Variant-specific NEG (0) NEG (0) NEG (0) NEG (0) NEG (0) ber 1981 for Pkl(B +)1 + Pkl(B+)1+ 529R, 10 Septem- Variant-specific 81,920 20,480 1, (0) NEG (0) ber 1981 for Pkl(B+)1+ (99.4 ± 0.3) (99.3 ± 0.4) (98.9 ± 0.4) 331, 15 July 1974 Cross-reactive 10,240 1, NEG (0) NEG (0) (chronic) (99.6 ± 0.2) (99.0 ± 0.2) (90.5 ± 2.8) 398D, 9 July 1981 Variant-specific NEG (0) NEG (0) NEG (0) * NEG (0) MEG (0) Ifor Pkl(A+) a Monkey number and date of serum collection. b Agglutination titers are written as reciprocals. Negative (NEG) indicates no agglutination at a 1:10 dilution. The numbers in parentheses are the percentage of infected erythrocytes that reacted by indirect immunofluorescence and are the mean values (+ standard deviations) obtained in four separate experiments in which 350 to 600 infected erythrocytes were scored each time. Erythrocytes infected with mature parasites were identified under phase-contrast microscopy by observing the refractile hemozoin pigment granules in erythrocytes. c Parasites were subpassaged every 6 to 7 days for Pkl(A+) and every 3 to 4 days for Pkl(B+)1+.

5 VOL. 40, 1983 resulting infection did not convert to the SICA[+] phenotype, but remained SICA[-], since a battery of sera from chronically infected hyperimmune monkeys did not agglutinate the infected erythrocytes (Table 2, Fig. 1A). Early in infection after becoming patent, the parasitemias in these monkeys increased 8- to 10-fold daily. However, after reaching peak parasitemias of 1 to 6% the parasitemias began to decline. A fourth monkey infected with clone sc followed the same course of parasitemia, but the SICA type of the resulting infection was not determined. Two intact rhesus monkeys that had previously experienced a single SICA[+] P. knowlesi infection were also inoculated with cloned sc parasites. In these two monkeys the schizontinfected erythrocytes from the resulting infection were agglutinated by the sera of chronically infected monkeys and had therefore converted to the SICA[+] phenotype (Table 3, Fig. 1B). The schizont-infected erythrocytes of the original challenge inocula were not agglutinated by chronic sera. Parasitemias in these two monkeys also increased 8- to 10-fold each day, but did not decline, and reached parasitemias of 60 and 25%. Two other intact rhesus monkeys were inoculated with the SICA[-] clone, Pkl(A-)1-. One monkey had not experienced a previous P. knowlesi infection, whereas the second intact animal had experienced a single SICA[+] infection 9 months before reinfection with Pkl(A-)1- parasites. The parasite populations in both monkeys reconverted from SICA[-] to SICA[+] as the schizont-infected erythrocytes from the infections were agglutinated by the TABLE 2. SPLEEN AND ANTIGEN EXPRESSION IN MALARIA cn 10 ui 1 X- 0.1 X t 0.0 0o.01S ;--i It / - I2 <0.01 I DAYS AFTER INFECTION FIG. 1. Mean of parasitemias in nonsplenectomized rhesus monkeys inoculated with cloned SICA[-] P. knowlesi. (A) Four monkeys in which parasitemias were controlled. In three monkeys the parasites remained SICA[-] during the infection. The parasites in the fourth monkey were not SICA typed. (B) Four monkeys in which parasites reconverted to SICA[+] at some point during the infection and the parasitemias were not controlled. The mean prepatent period was days for the monkeys in group A and days for the monkeys in group B. chronic sera (Table 3, Fig. 1B). Again, parasitemias rapidly rose to dangerously high levels (21 and 27%) in these intact animals; one animal succumbed to the infection before antimalarial drugs were given. Agglutination titers and courses of parasitemia of cloned SICA[-] parasites passaged into rhesus monkeys with spleensa Monkey Previous SICA[-1 Agglutination Peak no. no.infection inoculum c ~~titers of paaieib Outcome of infection chronic.oi sera paiasitemia 705D No SC Not done 4.8 Controlled infection 780F No sc NEG 6.0 Controlled infection 616G No Sc NEG 1.2 Controlled infection 335A No sc NEG 5.4 Controlled infection 553G No PK1(A-)1-1,280-5, Died W962 Yes sc Died 259P Yes Sc , Required chloroquine 612G Yes Pkl(A-) , Required chloroquine a Nonsplenectomized rhesus monkeys were inoculated with cryopreserved samples of cloned SICA[-] parasites. Agglutination tests with six to eight different antisera from chronically infected, hyperimmune monkeys were performed on the parasitized erythrocytes of the resulting infections at the parasitemias noted in the table. NEG indicates no agglutination at 1:10 dilution of antisera, and titers of positive assays are given as a range. After it was noted that the rising parasitemias in monkeys 553G and W962 caused death, monkeys were treated with intramuscular chloroquine when the parasitemia rose above 20%. b Peak parasitemias are recorded as the percentage of erythrocytes infected with parasites (ring stage) in an afternoon Giesma-stained blood film.

6 990 BARNWELL ET AL. INFECT. IMMUN. TABLE 3. Dependence of antigenic variation of cloned P. knowlesi in intact rhesus monkeys on the presence of agglutinating antibodies specific for the infecting variant type Schizont-infected erythrocyte agglutination titersa Antisera specific for parasites Monkey Previous Challenge infection Antibodies to parasites of challenge inoculum Antigenic no. infection of challenge inoculum geac (titer) Parasites of Parasites from variation challenge resulting infection 398D Pkl(A+) Pkl(A+) homologous Yes (20,480) 20,480 NEGb Yes 398H Pk2+ Pk2+ homologous Yes (1,280) 1,280 NEGb Yes 529R Pkl(B+)1 + Pkl(B+)1+ homologous Yes (81,920) 81,920 NEGb Yes 506H Pk3+ Pkl(A+) heterologous No (NEG)C 20,480 20,480 No 921H Pk4+ Pk2+ heterologous No (NEG)C 1,280 1,280 No a Agglutination titers are written as reciprocals. NEG indicates no agglutination at a 1:10 dilution of antisera. Monkeys were challenged with 106 parasites. Reinfection parasitemias were evident by day 3 or 4 in all cases and rose 8- to 10-fold daily thereafter in homologously or heterologously challenged monkeys. b Parasitized erythrocytes from the infections resulting from challenge, although not agglutinated by prechallenge sera or sera specific for the parasite clones in the challenge inocula, were agglutinated by sera from chronically infected monkeys. This indicated that the parasitized erythrocytes remained SICA[+], but of a new variant type but recognized by antibodies in the prechallenge sera. Titers with chronic immune sera range from 1:2,560 to 1:10,240. c Prechallenge sera from monkeys 506H and 921H agglutinated erythrocytes infected with clones Pk3+ and Pk4+, respectively, at titers of 1:2,560 and 1:640, but as indicated were negative for agglutination against erythrocytes infected with clones Pkl(A+) or Pk2+. Antigenic variation of cloned SICA[+] P. knowlesi. To test the capacity of our cloned SICA[+] P. knowlesi parasites to vary the SICA antigen, three intact monkeys (398D, 398H, and 529R) were reinfected with the same parasite clones with which the monkeys had been initially infected (homologous variant challenge; Table 3). In all three monkeys, the parasites changed their variant type. Sera obtained from each monkey before reinfection agglutinated the parasitized erythrocytes of the challenge inoculum, but not the parasitized erythrocytes of the resulting infection. The parasites were not SICA[-], but a new variant phenotype, since cross-reactive TABLE 4. sera from chronically infected animals agglutinated these parasitized erythrocytes as well as the schizont-infected erythrocytes of the challenge inoculum (Table 3). It has been suggested that antigenic variation in uncloned P. knowlesi malaria is induced by and dependent upon the presence of antibody of the appropriate variant specificity (4, 7). The role of antibody to the SICA antigen in antigenic variation was reexamined with cloned P. knowlesi variant types by heterologous variant challenge (Table 4). Two monkeys (506H and 921H) were first infected with SICA[+] clones Pk3+ and Pk4+, respectively. Sera obtained from Lack of antigenic variation of cloned P. knowlesi in splenectomized rhesus monkeys with circulating antibodies specific for the infecting variant type Schizont-infected erythrocyte agglutination titersb Antisera specific for parasites of Previous Challenge ib di i infection infection of challenge inoculum Monkey Monkey no.' infevtious Chalengeo no.a Antibodies to parasites challenge inoculum Antigenic (titer) Parasites of Parasites from variation challenge resulting infection 608H Uncloned para- Pkl(B+)1+ Yes (5,120) 81,920 5,120 No sites 398H Pk2+ (x3) Pkl(B+)1+ Yes (20,480) 81,920 5,120 No 184B Pkl(A+) Pkl(A+) Yes (5,120) 40,960 2,560 No 195E Pkl(B+)1+ Pkl(B+)1+ Yes (20,480) 81, No a Monkey 608H was infected with an uncloned variant population, drug cured, and immunized twice with killed (freezed-thawed three times) parasitized erythrocytes (4 x 108) in Freund incomplete adjuvant. Monkeys 184B and 195E were infected once with the indicated clone, drug cured, and immunized once with parasites in Freund incomplete adjuvant. Monkey 398H was infected three times and drug cured after each infection. Monkeys were challenged with approximately 1 x 108 to 5 x 108 parasites 3 to 6 weeks after splenectomy. b Agglutination titers are written as reciprocals. Prechallenge sera was taken after splenectomy just before challenge with P. knowlesi clones.

7 VOL. 40, 1983 these two intact monkeys just before reinfection agglutinated erythrocytes infected with clones Pk3+ or Pk4+, but did not agglutinate erythrocytes infected with Pkl(A+) or Pk2+ parasites, respectively, the cloned parasites used to reinfect these two monkeys. In these two cases of heterologous variant challenge, the parasite populations did not change their variant type. The course of the parasitemia in monkeys challenged with the homologous variant was identical to that of monkeys challenged with the heterologous variant in that the mean prepatent periods did not differ significantly, 3.3 ± 0.6 versus 3.5 ± 0.7 days, respectively. Effect of splenectomy on spleen in induction of antigenic variation. As described above, cloned parasites will undergo antigenic variation when inoculated into a monkey that has antibodies to the variant antigen of the inoculum. Since expression of SICA antigen was spleen dependent (see above), we determined whether the spleen was required, in addition to antibody, to induce antigenic variation. In four splenectomized monkeys the prechallenge sera and variant-specific antisera agglutinated the schizont-infected erythrocytes of the challenge inocula and schizont-infected erythrocytes of the parasite populations that resulted from the challenges (Table 4). Therefore, there was no change in variant type, although the monkeys had circulating antibody. As expected from our previous results on the passage of SICA[+] parasites in splenectomized monkeys, the SICA titers decreased. DISCUSSION Antigenic variation of cloned parasites. Brown and Brown (5) were the first to show by antibody-mediated agglutination that a parasite-dependent antigen expressed on the surface of P. knowlesi-infected erythrocytes, the SICA antigen, was variable in antigenic specificity. Thus, during a single chronic infection with an uncloned parasite population it was found that each recurrent wave of parasitemia contained a parasite population of a different SICA antigen phenotype (3). Furthermore, Brown et al. (7) demonstrated that antigenic variation would occur if parasites of a particular variant type were inoculated into a monkey that had circulating agglutinating antibodies to the parasitized erythrocytes. If the antibodies were specific for some other variant type, antigenic variation did not occur. Since these studies were performed with uncloned parasites, antigenic variation could have resulted either from selection of mutants or from the expression of genes which code for different SICA antigens. We have now shown that cloned parasites can undergo antigenic variation when inoculated SPLEEN AND ANTIGEN EXPRESSION IN MALARIA 991 into monkeys that have antibodies to that variant type (Table 3). Thus, cloned malaria parasites, like cloned African trypanosomes (14), can undergo antigenic variation. Certain differences are evident, however. First, African trypanosomes express the variant antigen on the parasite surface; the variant antigen in P. knowlesi is expressed on the surface of the host erythrocyte. Whether the variant SICA antigen is also found on the parasite or merozoite surface is unknown at this time. Second, antibody to the trypanosome variant antigen in the presence of complement causes lysis of the parasite (17). Antibody to the malaria variant antigen plus complement causes no lysis of the infected erythrocyte (7). Third, the function of the variant antigen probably differs for the two parasites. It is believed that the variant antigen in African trypanosomes covers the parasite surface so that antibody cannot bind to nonvariant determinants on the trypanosome membrane (10). We present evidence that the expression of malarial variant antigen in the erythrocyte membrane is associated with increased parasite virulence. Fourth, it is currently believed that in African trypanosomes variant-specific antibody selects for trypanosomes that have expressed alternate variant antigen genes (23). What environmental factors, if any, control the rearrangement of the genomic DNA in the trypanosome variant antigen gene family is not known. In P. knowlesi malaria, variant-specific antibody does not appear to be selective, either with uncloned parasite populations (4) or with cloned parasite populations (Table 4, results of homologous and heterologous variant challenge). Rather, in the case of asexual malaria parasites, variant-specific antibody may be inductive for antigenic variation (4). Although it was shown that the surface glycoprotein of African trypanosomes varied in cloned parasites, it was argued that rapid mutation and selection could explain the variation (14, 20). From the subsequent N-terminal amino acid sequencing of four variant trypanosome glycoproteins (2) and genomic DNA studies (15, 25), it is now known that the genes for many variants are within the genome of a single trypanosome. Similar data should now be developed for malaria parasites. Failure to express the variant antigen on the erythrocyte surface in splenectomized monkeys. We previously described that passage of uncloned P. knowlesi in splenectomized monkeys resulted in a new parasite phenotype (1) that is characterized by the inability of immune sera from chronically infected monkeys to agglutinate erythrocytes infected with this phenotype of P. knowlesi. We designated the usual phenotype that was able to be agglutinated as SICA[+]

8 992 BARNWELL ET AL. and the new nonagglutinable phenotype that was derived by passage of SICA[+J parasites in splenectomized monkeys as SICA[-]. We have now shown that the loss of agglutinability of P. knowlesi-infected erythrocytes after passage in splenectomized monkeys also occurs with cloned SICA[+] parasites. The loss, however, was not immediate. Passage of a SICA[+] clone in a splenectomized monkey resulted in a decrease in the agglutinability of the schizont-infected cells. With each subsequent passage in splenectomized monkeys the agglutination titer decreased so that after two to three passages the parasitized erythrocytes were not agglutinated by high-titered variant-specific or cross-reactive immune sera from chronically infected monkeys. This decrease in agglutinability appears to result from a decrease in the quantity of antigen expressed on the infected erythrocyte surface rather than a decrease in the number of positive cells as indicated by the surface immunofluorescence study (Table 1). The percentage of fluorescent-reactive, infected erythrocytes did not differ significantly between parasitized erythrocytes from the intact monkey or from parasites passaged once or twice in splenectomized monkeys, even though agglutination titers were reduced 16-fold or more. However, in splenectomized animals the intensity of the fluorescence of individual infected erythrocytes appeared to be decreased compared with the parasitized erythrocytes from an intact monkey. After three to four passages in splenectomized monkeys when the parasitized erythrocytes were nonagglutinable, the parasitized erythrocytes were also not positive for immunofluorescence, even with antisera from chronically infected, hyperimmune monkeys. These results exclude the possibility that passage of SICA[+] parasites in splenectomized monkeys resulted in the selection of a genotypically distinct SICA[-] subpopulation present in the original SICA[+] parasite population. Rather, our results show that SICA[+] parasites were able to alter the phenotype of the parasitized erythrocyte to SICA[-]. During the transition from SICA[+] to SICA[-J, there appeared to be a continual decrease in the quantity of SICA antigen expressed on the surface of all infected erythrocytes. However, at present, we do not know whether the SICA[-] parasite continues to produce SICA antigen, but does not insert it into the erythrocyte membrane, or whether synthesis of the SICA antigen has stopped altogether. SICA[-] cloned parasites were also capable of reconverting to SICA[+J on a single passage in monkeys with a spleen. Although this reconversion was generally associated with the intact monkeys having experienced a previous P. knowlesi infection, the exact conditions that allowed this to occur were not defined. The conversion of the SICA[-] parasites to SICA[+] could have resulted from the selection of a minor SICA[+] population in the overall cloned SICA[-] population. This possibility is unlikely, however, since surface immunofluorescence of the cloned SICA[-] populations did not reveal any SICA[+] cells in 2 x 104 infected erythrocytes examined. Additionally, the prepatent periods in monkeys in which parasites converted to SICA[+] were not different from the prepatent periods in monkeys in which parasites did not convert to SICA[+] (Fig. 1). Although the presence of the spleen is required for the maintenance of the SICA[+] phenotype, how the spleen or a dent spleen-depen- mechanism acts as a positive modulator for the expression of the SICA antigen on the surface of infected erythrocytes is unknown at present. It appears not to be due to some change in the properties of erythrocytes from splenectomized monkeys or merely a requirement for infected erythrocytes to pass through the spleen to unmask parasite-dependent antigens on the erythrocyte surface. SICA antigen, albeit reduced, is still expressed on infected erythrocytes when initially passaged in splenectomized monkeys. Also, SICA antigen is not always reexpressed when SICA[-] parasites are initially passaged into monkeys with spleens. We currently favor the possibility that intraerythrocytic malaria parasites are capable of responding to stimuli of splenic origin. It is unclear what biological advantage or parasite function causes expression of SICA antigen on the erythrocyte surface to be inducable rather than constitutive. Function of the variant antigen. Two facts suggest that the SICA molecule or some portion of it may be important for parasite survival. First, this molecule, recently identified as a parasite protein (R. J. Howard, J. W. Bamwell, and V. Kao, Proc. Natl. Acad. Sci. U.S.A., in press), is inserted into the outer surface membrane of infected erythrocytes, where it is accessible to immune recognition. Second, the fact that the parasite varies the antigenic determinants of this molecule in response to specific antibody makes it apparent that the SICA molecule or some portion of it may be important for parasite survival. This does not indicate what function this parasite molecule may serve for the parasite. In this regard, the SICA[-] of phenotype P. knowlesi provides a tool in ascertaining the role of the variant SICA molecule. Since INFECT. IMMUN. both SICA[+] and SICA[-] parasites rapidly multiply in and kill splenectomized monkeys, it is not likely that the exposed variant of the part SICA molecule serves some metabolic function such as membrane transport for the

9 VOL. 40, 1983 parasite. Why, then, would a parasite insert an immunogenic component into the erythrocyte membrane, where it is exposed to the host immune system? The parasite would then have to expend additional energy to vary this component to escape the antibody. Our results on infecting intact rhesus monkeys with SICA[-] parasites (Table 3, Fig. 1) may help in identifying a function of the variant molecule. P. knowlesi is normally a virulent malaria parasite in intact rhesus monkeys. The primary infection with SICA[+] parasites is usually fulminating with parasitemias, increasing 8- to 10-fold or more daily until high parasitemic levels are reached and the monkeys die (6, 13, 24). We have observed that infection of intact rhesus monkeys with SICA[-] cloned parasites also resulted in 8- to 10-fold daily increases of parasitemia at low parasitemic levels (<1%). However, unlike SICA[+] infections in intact monkeys, if the SICA[-] parasites remained SICA[-] during the infection, the monkeys were able to control their infection and survive. In contrast, if the SICA[-] parasites reconverted to SICA[+] (i.e., expressed variant SICA antigen again) at some point during the acutely rising infection, the monkeys did not control the infection, and parasitemias continued to rise to high parasitemic levels (>20%o parasitemia) 6 to 8 days postinfection. These data demonstrate a positive correlation between the expression of SICA variant antigen (SICA[+] phenotype) and increased virulence of P. knowlesi in the rhesus monkey. This appears paradoxical since one would have thought that a parasite that did not expose an antigen on the erythrocyte surface would be more virulent. If the spleen was removed, the difference in virulence between SICAH-] and SICA[+] parasites disappeared, and both phenotypes rapidly multiplied and would have killed the monkeys. The difference in virulence between SICA[+] and SICA[-] parasites was seen during a primary infection. During a primary infection when parasitemias rise rapidly to produce a fatal infection, the variant SICA type of SICA[+] parasites does not change; therefore, the virulence of the SICA[+] parasite is unrelated to antigenic variation per se. If the failure to express SICA antigen was the only major change in the switch from the SICA[+] to the SICA[-] phenotype, then our results and the considerations discussed above lead us to suggest that the variant SICA antigen may function to help the parasite escape a spleen-dependent immune response. The spleen is the major organ of host defense in malaria (21, 27). In addition to its role in defense, we now know that the spleen induces the expression of a P. knowlesi molecule, the variant SICA antigen, which may then permit SPLEEN AND ANTIGEN EXPRESSION IN MALARIA 993 the parasite to escape a spleen-dependent immune response as well as variant-specific antibody responses. A parallel mechanism may exist in Plasmodium falciparum. P. falciparum-infected erythrocytes escape the spleen by binding to venular endothelium (18, 22), and the membrane ligand for endothelial binding may be of parasite origin (16). As with variant SICA antigen in P. knowlesi, the spleen influences the expression of the P. falciparum-binding ligand in squirrel monkeys (11). We therefore believe that the interaction between the host's spleen and the asexual malaria parasite described for P. knowlesi in this paper is applicable to the immunobiology of other simian (26) and human malarias. ACKNOWLEDGMENTS We thank D. Hudson and D. 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