Jul 9, 1981 - course of an infection by repeatedly expressing ... the passage of uncloned parasite lines in sple- ..... Early in infection after becoming patent, the parasit- emias in these monkeys ... afternoon Giesma-stained blood film. VOL.
INFECTION
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IMMUNITY, June 1983, p. 985-994
Vol. 40, No. 3
0019-9567/83/060985-10$02.00/0 Copyright C 1983, American Society for Microbiology
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 of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases,1 and the Laboratory of Cell Biology, National Cancer Institute,2 National Institutes of Health, Bethesda, Maryland 20205 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 16fold. 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 rocytes were agglutinated by sera from rhesus despite the concurrent presence of potentially monkeys immune to P. knowlesi, but not by sera parasiticidal immune responses is characteristic from normal rhesus monkeys. Uninfected rheof infections with several parasitic protozoa. sus monkey erythrocytes or erythrocytes conSome of these protozoan parasites have the taining immature asexual parasites were not ability to antigenically vary the molecules that agglutinated by the immune sera. This indicated are targets of antiparasitic immunity and thus that a new antigen must have appeared on the escape complete elimination from an immuno- surface of schizont-infected erythrocytes. competent host. For example, African trypano- Twenty-seven years later, Brown and Brown (5) somes undergo antigenic variation during the demonstrated by the antibody-mediated aggluticourse of an infection by repeatedly expressing nation of schizont-infected erythrocytes that the different surface coat glycoproteins (24). The new parasite-dependent antigen expressed on asexual intraerythrocytic parasites of malaria the surface of P. knowlesi-infected erythrocytes also often cause chronic infections that persist was antigenically variable. If a monkey was for long periods. The most direct evidence that infected with a particular P. knowlesi populathe chronicity of malaria infections may be due tion, serum collected 2 or more weeks after drug in part to antigenic variation of asexual parasites cure of this infection would agglutinate the comes from studies on the primate malaria para- erythrocytes infected with that parasite populasite, Plasmodium knowlesi, in rhesus monkeys. tion. This variant phenotype did not change if In 1938, Eaton (12) found that when the asexu- passaged in naive, nonimmune monkeys. Howal intraerythrocytic parasites of P. knowlesi had ever, reinoculation of this variant population matured to the schizont stage the infected eryth- into a monkey with antibodies specific for this 985
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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 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, mon-
keys; (ii) the switch of cloned SICA[+] parasites 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.
to
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 1640-30 mM HEPES (pH 7.2) (no NaHCO3), and diluted to 4 x 106 erythrocytes per ml in RPMI 1640-30 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 96well 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
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SPLEEN AND ANTIGEN EXPRESSION IN MALARIA
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
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 antiPkl(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
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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
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 infected with clone:
Seraa
Reactivity of serum Intact rhesus
immunofluorescence)b Passage in splenectomized rhesus monkeysc First
Pkl(A+)
398D, 9 July 1981 Variant-specific 20,480 640 for Pkl(A+) (99.6 ± 0.1) (99.2 ± 0.4) 331, 15 July 1974 Cross-reactive 10,240 320 (chronic) (99.5 ± 0.1) (99.1 ± 0.4) 529R, 10 Septem- Variant-specific NEG (0) NEG (0) ber 1981 for Pkl(B +)1 +
Second
Third
Fourth
NEG (0)
NEG (0) NEG (0)
NEG (0)
NEG (0) NEG (0)
NEG (0)
NEG (0) NEG (0)
Pkl(B+)1+ 529R, 10 Septem- Variant-specific 81,920 20,480 1,280 20 (0) ber 1981 for Pkl(B+)1+ (99.4 ± 0.3) (99.3 ± 0.4) (98.9 ± 0.4)
NEG (0)
331, 15 July 1974 Cross-reactive 10,240 1,280 160 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) _____ 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+.
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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
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