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' " -'I<5'-:""7"'17 a.” - .. :4- 'y‘“ 15 I 1”?"5’3'I’9MIII/ I7I , _ .5! ,/ 555 -J( J . vrf’i‘hu/ [‘4‘ 1’9? Ely/1’31 I. u. , J,. J u; 5’. m, ”a; 1:54:2‘5555 v.1"; 5 55,551,; Ar',’ If?“ ’36.; 1‘ 4 v' V r?) F - ' as»: face-u haiku 472%"— "v n—ah' r j .. v- 4’, », 1,’,'/‘§'f: ) / ., I." .3121. A! 70'" lief: 1?” rfz/ '1: ‘2‘! .- , "gm. -. . 'r' ’gfii'"; [.,’/;;S' I: f u . I W! ' 11?}!!‘v-MJVJ _,'-'- ..‘ ‘I-u-E :- . ‘ "r. .. , '6:1{("’{/‘5.'c‘7’"‘1, 'J‘ {a ., -'. ‘1‘)?“ ”fl... _ I . - JIé. “1.1.11.1; 41,} 3f?” v 7""5"! 5 . H 1”” 1" a 3’ '/ H ' A .7" Jpn. ': :JIl/I A? It,“ "I - ' If) ‘.‘,r;.Vc- - 'J '-_,'3 3"}; yy' 1? '4 $3.71,” “,"'J.' 21:15.; J ) ' ' ..‘ I' 7 , l‘- , . '.’ . . 4,1, . 1/ g, I . 7r ’5’; f'iéég {’1' ' . .1" " u— I ' {yl‘gn W“ j LIBRARY Michigan State University ‘ - This is to certify that the dissertation entitled Immune Recognition of Parasite - Dependent Antigens Associated With Plasmodium Falciparum - Infected Erythrocytes presented by Hassan M. EISaid has been accepted towards fulfillment of the requirements for Doctoral degree in Philosophy Date M311 12, 1988 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 1? MSU 5 RETURNING MATERIALS: Piace in book drop to LIBRARIES remove this checkout from -—. your record. FINES wiH be charged if book is returned after the date stamped below. IMMUNE RECOGNITION OF PARASITE-DEPENDENT ANTIGENS ASSOCIATED WITH BLAfiMQDIflM.EALQIRABHH'INFECTED ERYTHROCYTES By Hassan Mohamed Elsaid A DISSERTATION Submitted to Michigan State University' in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1988 ABSTRACT IMMUNE RECOGNITION OF PARASITE-DEPENDENT ANTIGENS ASSOCIATED WITH ELfififlQDIfln,EALQIEABgfl-INFECTED ERYTHROCYTES By Hassan Mohamed Elsaid The intraerythrocytic infection of Elgfimgginm fialgipgznm constitutes the primary mechanism by which the parasite evades recognition by human immune mechanisms. In this study we investigated the ability of human immune sera and peripheral blood monocytes to recognize erythro- cytes infected with this parasite. Sera from adults living in malaria endemic areas of Sudan, Nigeria and Irian Jaya contained immunoglobulins specific for parasite-dependent antigens expressed on infected, but not non-infected, erythrocytes., Six in 21:19- adapted parasite strains of different phenotypes, regarding expression of knob structures and cytoadherence affinity to human endothelium/C32 melanoma cells, showed diverse antigenic repertoires on infected erythrocytes. The differences in levels of recognition of individual strains correlated with the drift from knobby and endothelium-binder to knobless and endothelium-non-binder phenotypes. The expression of knob structures alone on erythrocyte surface was not sufficient for conferring antigenicity or cytoad- herence ability to infected_erythrocytes. Two strains failed to react with any of the sera employed in the study. These parasites may display a rare antigenic repertoire or completely lost expression of erythrocyte surface antigens. Sera that opsonized infected erythrocyte surface could also inhibit their cytoadherence to human C32 melanoma cells. Endothelium-binder parasites may express deter- minants common to the different isolates of the parasite, or immune sera may contain a mixture of immunoglobulin specifi- cities directed at distinct epitopes expressed on antigeni- cally diverse isolates, however, associated with the cytoad- herence molecules on infected erythrocytes. Human peripheral blood monocytes were unable to recognize infected erythrocytes, as indicated by their inability to phagocytose the infected targets. In the presence of immunoglobulin molecules specific for erythro- cyte surface antigens, monocytes could recognize, bind and phagocytose infected erythrocytes. Primary infection sera were devoid of reactivity to infected erythrocyte surface, although specificity for parasite antigens was evident in both 196 and IgM immunoglobulin classes. Although parasite- dependent antigens on surface of infected erythrocytes may be variant, they apparently elicit specific antibodies, and for infection with a given parasite, probably play a major role in controlling rising parasitemia. ACKNOWLEDGMENTS I would like to thank Dr. James B. Jensen for serving as my research adviser and for support throughout this study. Special thanks are extended to my committee members for their guidance and support: Drs. H. Hasouna, T. W. Schillhorn Van-veen, R. K. Mass and W. J. Esselman. Special thanks to Dr. J. F. Williams for his encouragement and support. Above all others I thank my parents for their patie- nce, support, continuous encouragement and love they have provided through the years. ii TABLE OF CONTENTS LIST OF TABLES...........................................v LIST OF FIGURES..........................................vi INTRODUCTION..............................................l REVIEW OF LITERATURE......................................3 Acquired resistance to natural Plasomdium infection..3 Falciparum-dependent antigens induced on infected erythrocytes...................................6 Immune responses against bloodstage infection.......lo Antibody-mediated immunity....................10 Cell-mediated immunity........................12 Activation of monocytes/macrophages during malaria.......................................14 Bibliography........................................26 CHAPTER 1. DIVERSITY OF ANTIGENS INDUCED ON ERYTHROCYTES BY CULTURE-ADAPTED ELASMQDIQM EALQIEABEM DETECTED BY HUMAN IMMUNE SERA..................................?.....37 Abstract............................................38 Introduction........................................39 Materials and methods...............................41 Parasites.....................................41 ParaSite cultureSOOOOOOOOOOOOOOOOOOOOOOOOO0.0.41 iii Synchronized parasite cultures................42 Human sera....................................42 Indirect surface immunofluorescence assay.....43 Trypsin treatment of infected erythrocytes....43 Results.............................................44 Discussion..........................................57 References..........................................62 CHAPTER 2. FUNCTIONAL IMPLICATIONS OF IMMUNOGLOBULIN- MEDIATED RECOGNITION OF 2LA§MQDIQM EALQIEARQn-INFECTED ERYTHROCYTE ANTIGENS.....................................67 Abstract............................................68 Introduction........................................69 Materials and methods...............................72 Parasites.....................................72 Human sera....................................72 Indirect immunofluorescence antibody assay....73 Indirect surface immunofluorescence assay.....73 Cell lines....................................74 In 213:9 cytoadherence-inhibition assay.......74 Human peripheral blood mononuclear cells......75 Phagocytosis-promoting activity of human sera.75 Statistical analysis..........................75 Results...............;.............................76 Discussion..........................................84 ReferenceSOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. ......... 0.091 iv LIST OF TABLES CHAPTER 1 Table l Titers of surface immunofluorescence (SIFA) of 40 adult malaria sera tested with six strains of £0 WOOOOOOOOOOOOOOOOOOOOOOOO0.000......000051 Table 2 Distribution of 40 adult malaria sera according to their titers of surface immunofluorescence (SIFA) with different parasite phenotypes..................54 Table 3 Distribution of 40 adult malaria sera according to the geographical origin of tested individuals and paraSite strains...OOOOOOOOOOOOOOOOOOOOO0.00.00.00.054 CHAPTER 2 Table 1 Results of indirect immunofluorescence (IPA), surface immunofluorescence (SIFA), phagocytosis.and cyto- adherence-inhibition of ItGC32-IRBCs for 40 adult human sera...................;......................78 Table 2 Correlation between titers of indirect immuno- fluorescence (IFA), surface immunofluorescence (SIFA), and levels of phagocytosis and cytoadherence- inhibition of ItGCBZ-IRBCs for 40 adult human sera..79 LIST OF FIGURES CHAPTER 1 Figure 1 Surface immunofluorescence of 2. falgipazgm developmental stages. Synchronized parasite cultures were tested for SIFA at different periods during one cycle of development. The stages of parasite development: ring, time 0; trophozoite, 34 h; schizont, 40 h; segmenter, 44 hOOOOOOOOOOOOOOO0.0.00.0000...0.0.0.0000...0.0.0.47 Figure 2 Effect of trypsin treatment of infected erythrocytes on surface immunofluorescence. Infected erythrocytes of strain FCR-3-TC were in- cubated for 10 min with the indicated concentrations of trypsin and assayed for SIFA using a pool of human immune sera.........................................49 Figure 3 Surface immunofluorescence of infected erythrocytes enriched for knobby parasites. Parasites of strains FCR-3-TC and 6-73 were enriched for knobby parasites by repeated gelatin separations (Gel. 1-7). Parasites were tested for SIFA after enrichments 3, 5 and 7. Original preparations were tested simultaneously...............................52 Figure 4 Distribution of 35 adult serum samples according to their titers of surface immunofluorescence. Open circle, sera recognized > two parasite strains including ItGC32; filled circle, sera recognized one strain only; open square, sera recognized > two strains not including ItGC32........................55 vi CHAPTER 2 Figure 1 Correlation between levels of phagocytosis and cytoadherence-inhibition of infected erythrocytes mediated by 40 adult serum samples. Opsonized infected erythrocytes (strain ItGC32) were tested for immunophagocytosis by human peripheral blood monocytes and cytoadherence-inhibition to C32 melanoma cells. r - 0.66, r is significant at 1% level.............OOOOOOOOOOOOOOOOOCOOOOIO0.0.00.0..80 Figure 2 Opsonizing effect of adult sera from malaria endemic areas. Infected (IRBCS) and non-infected (NRBCS) erythrocytes were opsonized with pooled adult sera (HIS) from Irian Jaya (7 samples) and pooled normal human sera (NHS) ‘ and tested for immunophagocytosis by human peripheral blood monocytes. Non-Opsonized (NOS) erythrocytes were tested simultaneously..........................82 vii INTRODUCTION Malaria is caused by protozoan parasites of genus Plasmodium. More than 100 plasmodium species have been described that affect a wide range of vertebrates with relative host specificity. Malaria affects humans, primates, rodents, birds and reptiles. The infection is initiated through the bite of the mosquito vector during which sporozoites are inoculated into the blood stream of the host. In the liver, the sporozoites undergo exo- erythrocytic schizogony. Infected hepatocytes rupture releasing several thousand merozoites which invade red blood cells (RBCs). In RBCs, the parasites undergo erythrocytic asexual schizogony. Most of the clinical manifestations of the disease are associated with this asexual cycle of replication. Under certain conditions, a proportion of newly-invading merozoites differentiate, without cell division, into male and female gametocytes (sexual erythrocytic stage). Sexual reproduction occurs in the gut of the arthropod vector after ingestion of gametocytes. 2 The disease in humans is due to four species, 2. film. 2- sixes. 2- salaries and 2- omele- flasmgsim falciparum is known to be the most virulent of the four species of malarial parasites that affect humans. A unique feature of falciparum malaria (malignant tertian malaria) is that only erythrocytes infected with young forms of the parasite (rings) are found in the peripheral blood of infected subjects. Erythrocytes infected with more mature asexual stages (trophozoites and schizonts) are sequestered in the postcapillary venules of various organs where they adhere to endothelial cells (Bignami and Bastianelli, 1889). This phenomenon is of major importance for the establishment of falciparum infection and in the develop- ment of the pathology associated with the disease. A host immune response that can inhibit peripheral sequestration is expected to influence the outcome of infection. REVIEW OF LITERATURE Acquired resistance to natural plasmodium infection The capacity of a host to combat natural plasmodium infection is largely dependent on the innate resistance of that host and on its immune effector mechanisms, both of which are genetically determined. Generally, the immune response of the mammalian host to malaria is markedly complex. It involves the elaboration of specific and non- specific antibody- and cell-mediated responses accompanied by immunopathological alterations. Deviations in immune reactivity of the host during malaria are mostly parasite- dependent, however, the genetic constitution of the host through its innate resistance tends to modify the outcome of the infection process. Immunosuppression, polyclonal lymphocyte activation, immune complex disease and auto- sensitization all are deviations of immune responsiveness associated with plasmodium infections in humans and animals (Voller, 1974; Warren and Weidanz, 1976; Wyler, 1976: Weidanz, 1982). Acute plasmodium infections, in non-immune hosts, are characterized by two major host responses. h. 4 First, polyclonal activation of T and B lymphocytes, with enhanced synthesis of polyclonal IgM and IgG (Cohen and Butcher, 1971). A minority of the developing antibodies show parasite specificity (Cohen et 51, 1961). Immuno- suppression of the host responses may be associated with this phenomenon (Jayawardena, 1981). Secondly, activation of the reticuloendothelial (RE) system occurs along with the stimulation of myelopoiesis in the spleen and bone marrow with the development of splenomegaly due to splenic myeloid hypertrophy (wyler and Gallin, 1977). In humans, falciparum malaria occurs as an acute disease in infants and children. The period of greatest risk is from six months to five years of age. Infants born to immune mothers are relatively resistant during the first 3 months of life and, thereafter, all children in endemic areas suffer severe and recurrent attacks of malaria frequently with a fatal course (Playfair, 1982). The acquisition of protective immunity to falciparum malaria is characterized by its slow development. The improvement proceeds in stages; the disease becomes infrequent in later childhood; the attacks become less severe, then later the levels of parasitemia is reduced. So by adult life, residents of endemic areas of malarial transmission rarely show the acute form of the disease despite continued exposure. The disease usually occurs as a chronic blood- stage infection due to repeated exposures to the parasite, S with intermittent, low-grade parasitemia (McGregor, 1960). However, after as little as six months abroad, or after elimination of the parasite by chemotherapy, clinically resistant patients may again become susceptible to serious malaria (Maegraith, 1974).1 Sterile immunity against falciparum malaria never develops in the human host and such a state of acquired resistance is an actual premunition (Sargent, 1963). Sterile immunity to plasmodium infections is, however, often seen in animal models. 2. pgxgnei in rats and 2. chabgugi in mice induce complete resistance to reinfection after a brief primary malady (Cox and Voller, 1966). The development of these two extreme types of responses, premunition or sterile immunity, is largely dependent on the experimental model, where a plasmodium species is used to infect a "permissive” rather than a "natural" host. However, in most natural infections, including human malaria, the immune response is characterized by the acquisition of partial resistance which allows the ”immune" host to control, but not to eliminate infection (Jayawar- dena e; 31, 1982). It is noteworthy that acquired immunity to plasmodium infection is usually stage-specific. Rats immune to 2. bgzgnei infection induced by sporozoites are resistant to sporozoite challenge. Sporozoite-vaccinated mice are 6 resistant to sporozoite infection only and not to blood- stage challenge (Nussenzweig et a1, 1969). Falciparumrdependent antigens induced on infected erythrocytes The intra-erythrocytic infection of plasmodia con- stitute the primary mechanism by which the parasite evades recognition by the host defensive mechanisms (Cohen and Lambert, 1982). Recognition of host red blood cells infected with the parasite is expected to be a fundamental step for the mounting as well as for the execution of a competent immune response against bloodstage infection (Rommel, 1985). The asexual erythrocytic cycle of falciparum parasite takes about 48 hours to complete. With the progress of development of the intracellular parasite, from ring form to mature schizont, the infected erythrocyte tolerate profound morphological and structural alterations (Howard, 1982; Hommel, 1985). Red blood cells infected with mature parasite stages are no longer biconcave disks, but acquire a spherical morphology. Alterations in surface membrane involve the insertion of new anion channels, acquisition of new antigenic determinants, expression of binding sites to host cells and the expression of electron dense protrusions termed "knobs" (Trager gt a1, 1966; Miller, 1969; Aikawa gt 7 31, 1975: Kilejian gt :1, 1977: Sherman, 1979; Kilejian, 1979, 1980, 1981; Howard, 1982; Hommel gt 31, 1983; Hommel, 1985). Another class of alterations involves the parasite- dependent modification of membrane structures of host origin. Some of these changes in host components could expose neoantigens, which were formerly cryptic, or create new antigens on formerly nonimmunogenic self components (Howard, 1982). The onset of erythrocyte surface alterations, espe- cially knob expression, coincides with the disappearance of infected RBCs form the peripheral circulation (Miller, 1969). The diseased erythrocytes sequester in the vascula- ture visceral organs such as heart, placenta and in the brain (Miller, 1969). Although the mechanisms of parasite sequestration are not fully elucidated, ultrastructural studies have suggested that endothelial-erythrocyte adhesions occur at the knob sites (Luse and Miller, 1971; Aikawa gt Q1, 1972). This phenomenon is of major impor- tance for the establishment of falciparum infection and in the development of the pathology associated with the disease. Peripheral sequestration is essential for the parasite survival by eluding the filtering action of the spleen. Also, the low oxygen tension in tissue micro- environment favors parasite development and the reinvasion of new red blood cells (Howard and Barnwell, 1984; Hommel, 1985). 8 Of special immunological interest is the expression of parasite-dependent antigens on infected erythrocytes. The existence of parasite-dependent antigens on falciparum- infected erythrocytes was demonstrated through immuno- electronmicroscopy of infected RBCs Opsonized with monkey sera (Kilejian gt :1, 1977: Langreth and Reese, 1979). The antigenic variation of surface determinants was revealed in studies employing surface immunofluorescence techniques using immune monkey sera (Hommel gt :1, 1983). A subse- quent study, using sera from Gambian human subjects, confirmed the presence of neoantigens on in yitzg-cultured falciparum parasites (Mendis gt :1, 1983). In a major step toward the elucidation of host reaction to such deter- minants, Marsh and Howard (1986) reported the presence of antibodies specific to erythrocyte surface antigens in sera of Gambians naturally infected with E. tg1gipgggm. This study confirmed the antigenic diversity of surface antigens on natural parasite isolates and indicated the polyspecific nature of adult sera in comparison to the limited specifi- city of children sera. Gambian children were found to develop isolate-specific humoral response to surface antigens of their own infected red blood cells, but did not cross-react with parasites derived from other infections, while adult sera could recognize multiple parasite isolates (Marsh and Howard, 1986). 9 The use of biochemical and molecular techniques has been instrumental in identifying these determinants and in defining the observed antigenic diversity (McBride gt Q1, 1982). Hall gt 31, (1984) have showed that the 195 KB major glycoprotein of falciparum schizonts, contain both constant and strain-specific domains. Several subsequent studies have confirmed the existence of variable surface determinants on infected erythrocytes and merozoites (Aley gt Q1, 1986; Coppel gt 31, 1986; Scaife gt 31, 1986: Lyon gt 31, 1987: McBride and Heidrich, 1987). However, other studies have also detected common antigens on different geographical parasite isolates (Cheung gt 31, 1986; Jendoubi and da Silva, 1987). Immune responses against bloodstage infection The acquired immunity to falciparum malaria is pre- dominately strain- and stage-specific (Jefferey, 1966: Garnham, 1970: Mitchell gt g1, 1977). This implies that acquired immunity can only be mediated by mechanisms which involve a specific effector process, either a specific antibody that acts alone or in collaboration with comple- ment and/or effector cells such as macrophages, direct T- cell cytotoxicity, or production of cytotoxic mediators following specific interaction of T-cells with malaria antigens (Cohen and Lambert, 1982). It is noteworthy that 10 most of the following evidence, regarding host immune response to plasmodium infection, has been accumulated as results of studies done in animal models. Caution is advised when extrapolating these results to the human situation. Antibodybmediated immunity Malaria infection provides a potent stimulus for the synthesis of immunoglobulins, especially IgG and IgM (Cohen and Butcher, 1969). After sporozoite-induced infections in human volunteers, immunoglobulin (19) levels remain un- changed until the onset of parasitemia, indicating that neither sporozoite invasion nor pre-erythrocytic plasmodium development stimulates Ig synthesis. Soon after the appearance of the parasitemia levels of IgM, IgG and IgA increase, while 190 shows little change. Repeated infec- tions were found to be associated with considerable eleva- tion of IgG and IgM (Tobie gt Q1, 1966). In clinically immune West African adults, IgG production was found to be about seven times greater than that of normal non-infected europeans. Upon prophylactic therapy, IgG production was reduced. This reduction indicates that about one third of the total IgG produced by immune subjects is malaria- induced (Cohen gt Q1, 1961). However, much of this antibody response is non-specific, since only about 5% of 11 the total IgG reacts with plasmodium antigens (Cohen gt g1, 1961; Cohen, 1980). Polyclonal B lymphocyte activation, due to unidenti- fied 2. fig1g1pgtgm products, has been associated with acute plasmodium infections (Greenwood, 1974). This phenomenon has been detected in humans suffering falciparum malaria (Rosenberg, 1978). Supernatants from in XIIIQ cultures of PF induce activation of human T and B lympho- cytes from both malaria-immune and non-immune donors (Greenwood gt g1, 1979). McGregor and Barr, (1972) have shown that specific immune responses following immunization of Gambian children with tetanus toxoid is greatly reduced while accompanied by non-specific polyclonal activation. Circulating immune complexes and autosensitization were found to be associated with polyclonal activation of B lymphocytes during acute falciparum malaria (Houba, 1979). Apart form polyclonal activation, there is evidence that specific antibodies play a major role in controlling the asexual erythrocytic development of plasmodium. Specific antimalarial antibody titers rise with repeated 2. fig1gipgtnm bloodstage infection (McGregor and Williams, 1978). Passive transfer of purified serum IgG from clini- cally immune inhabitants of holoendemic areas has been shown to cure children with acute malaria due to 2. fg1gipgttm or B. mg1gtigg and resulted in dramatic decrease in the levels of parasitemia (Cohen gt g1, 1961). 12 The decreased parasitemia following the passive transfer of immunoglobulins, and the observation that protective anti- body does not damage intracellular parasites (Cohen and McGregor, 1963) indicates that either mature schizonts or merozoites, or both are likely the targets of the protec- tive IgG response (Perrin and Dayal, 1982). The precise mechanism by which humoral antibodies confer protection has not been clearly established (Cohen gt g1, 1969). However, specific antibody as an effector recognition molecule is extremely important for Ig- dependent cell-mediated mechanisms, immunophagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC), all . are powerful mechanisms by which asexual blood stages of the parasite may be controlled. Cell-mediated.imnunity Although most investigators agree that both cellular and humoral factors are involved in the slow development of immunity to malaria, the precise role of cell-mediated effector mechanisms is poorly understood, especially in falciparum malaria. The contribution of cell-mediated responses to protection has been explored mainly in rodent models. The evidence that cell-mediated mechanisms may be important for the development of immunity is based on the following observations. l3 l-Animals deficient in T lymphocytes are unable to respond efficiently to malaria. Normal mice are able to control B. ygg111 infections (Roberts gt g1, 1978). In contrast, congenitally athymic or T-cell-deprived mice are unable to mount effective resistance and infections are fatal in 30-35 days. Even if infection in T-cell-deficient animals is controlled temporarily by chemotherapy, resistance fails to develop (Roberts and Weidanz, 1978). This indicates the need for T-cells in the induction of immunity to this parasite (Clarke and Allison, 1974). 2-Adoptive transfer of T-cells from animals immune to malaria can transfer such resistance to non-immune recipients. Splenic T-cells from mice immune to 2. bgtgng; or to 2. ygg111 were found to confer partial immunity to non-immune recipients (Phillips, 1970). However, better protection resulted from transfer of both T-cells and B- cells (Jayawardena, 1978) or whole spleen cell preparations (Grvely and Kreier, 1976). 3-Cells of the reticuloendothelial system play an important role in the control and immunopathology of plasmodium infections, both as regulatory and as effector cells. Most of their functions were found to be under the positive control of T lymphocytes (Jayawardena, 1981). Although the function of T-cells is pivotal for efficient immunity to malaria, the precise effector mechanism(s) through which T-cells confer immunity have not been l4 completely clarified (Playfair, 1982). Attempts to demo- nstrate a cytolytic function of T lymphocytes in yittg have been unsuccessful (Phillips gt g1, 1970). This observation is not surprising in view of the fact that T-cell recogni- tion is restricted by MHC expression. So, although para- site antigens are expressed on the infected RBCs (Hommel, 1985), the lack of MHC products on mammalian non-nucleated cells may lead to the non-recognition by cytotoxic T-cells (Cohen and Butcher 1971). Since protection from lethal malaria infection is conferred by passive transfer of immune sera, it has been suggested that the protective_ activity mediated by T-cells is related to their helper function for protective antibody synthesis (Perrin and Dayal 1982). Activation of monocytes/macrophages (Mn/Hf) during acute ' malaria Although there is substantial evidence for activation of the monocyte/macrophage system during plasmodium infections, little is known about its actual role as an effector system against bloodstage infection. The follow- ing are the manifestations of involvement of Mn /Mf during malaria. Activation of Mn/Mf cells is local in nature and usually occurs in the spleen and liver during acute 15 infection. Shear gt 31, (1979) found that splenic macro- phages of B. bgtgngi-infected mice are efficient in phagocytosis of infected reticulocytes, however, peritoneal macrophages of the same animals were not. Upon thioglyco- late induction this later population attained active phagocytic function. Although the activation of Mn/Mf is local in nature, systemic inflammatory mediators, products of activated Mf, can be detected in the circulation of acutely infected subjects. Wigzell and his co-workers (Ojo-Amaize gt Q1, 1981) found a positive correlation between the degree of falciparum parasitemia, NK activity in_21ttg and interferon titers (a and p interferons) in sera of acutely infected West African children. Serum levels of endogenous tumor necrosis factor (TNF), a potent effector and regulatory Mf product, were found elevated in Thai patients acutely infected with 2. tg1gipgtgm and 2. 21233 (Scuderi gt Q1, 1986). Although r-TNF has no effect on PF in yittg, administration of mouse r-TNF into mice infected with a lethal variant of 2. ygg111 significantly reduced parasit- emia and prolonged the survival of infected mice (Taverne gt Q1, 1987). Although TNF may be unable to inhibit the proliferation of the parasite, exogenous r-TNF is expected to activate the RE system in 2129 through an amplification mechanism mediated by its autocrine ability to activate Mn/Mf, stimulating the release of interleukin-1 (IL-1), 16 interferon and endogenous TNF, which in turn can activate Mn/Mf-, NK-, or T cell-dependent effector mechanisms (Kornbluth and Edington, 1986; Dinarello gt g1, 1986). Locally produced inflammatory mediators are expected to exert systemic influence on cells of other tissues and may predispose the pathology in other organs. TNF is known to exert a profound systemic procoagulant activity on endothelial cells with the production of IL-1, which potentiates this effect (Nawroth gt g1, 1986). Procoagu- lant activity is likely to predispose and enhance sequest- ration of erythrocytes infected with mature stages of the parasite in peripheral capillaries. This may lead to serious pathological complications such as cerebral malaria, a pathognomonic feature of falciparum infection (Daroff gt Q1, 1967). It is noteworthy that peripheral blood lymphocytes from clinically "immune" patients suffering chronic bloodstage infection can respond specifically to stimula- tion with PF erythrocyte antigens by release of IL-2 and gamma-interferon 1n 21ttg (Troye-Blomberg gt Q1, 1984). .However, patients with acute falciparum malaria only exhibit a weak and short-lived 1n yittg lymphoproliferative response to the same antigens. Although the antigen- induced production of IL-2 in yittg was low and of short duration, antigen-specific production of gamma-interferon was not impaired (Troye-Blomberg gt g1, 1985). 17 This indicates the presence of antigen-specific T cell- suppression during acute falciparum infection, however, T cell-dependent Mf activation mediated by gamma-interferon may not be impaired. Monocytosis is a classical feature of acute plasmodial infections. Infection with P. ygg111 in mice induces massive blood monocyte response. It is interesting to note that this response is impaired in T-cell-deprived mice. This may indicate that Mn/Mf activation is a T-cell- dependent phenomenon (Jayawardena gt :1, 1977). Mono- cytosis was also observed in Gambian children with acute falciparum malaria. Transient monocytosis (15%) was accompanied by anemia, reticulocytosis and erythrophago- cytosis of infected and non-infected RBCs (Facer and Brown, 1981). Cell-traffic studies, using radio labelled effector cells, suggested that both lymphoid and myeloid cells are specifically attracted to the spleen and later to the liver during acute infection. In adoptive transfer models using P. ygg111, these changes in cell traffic were shown to be T-cell-dependent, that is analogous to the traffic of cells to the injection site of the delayed type hypersensitivity test (Playfair gt Q1, 1979). Fewer macrophages accumulate in the spleens of nude mice infected with 2. bgtghgi than in normal mice. Although 2. ygg111-infected normal mice show enhanced phagocytosis, as judged by accelerated carbon 18 clearance, this is not observed in athymic mice at a similar stage of infection (Roberts and Weidanz, 1978). An endogenous spleen-derived chemotactic factor of lymphocytic origin was detected in extracts of spleens of plasmodium- infected mice and monkeys, but not from control animals, that promoted the attraction and proliferation of peri- pheral blood monocytes in the spleens of acutely infected animals. While parasite extracts were found to be completely deficient as chemoattractants for the same cells (Wyler and Gallin, 1977). This indicates that both the accumulation and the functional activity of splenic macrophages are regulated by T lymphocytes. Most of the pathology observed in acute malaria is associated with activation of splenic Mn/Mf cells. The spleen is the organ which shows the earliest changes in animals infected with malaria. Splenomegaly is a hallmark of plasmodial infections and its rate of increase in human populations was used for the evaluation of malaria prevale- nce within a population (Boyd, 1949). Spleen enlargement is usually associated with acute infections, and it is likely to remain enlarged during chronic and repeated infections. However, if the host could control the infec- tion or the infection is terminated by chemotherapy, there is a fairly rapid return to normal spleen size (Voller, 1974). In areas where there is massive and sustained malaria exposure, most of the children will show enlarged 19 spleens. However, the spleen enlargement declines with increasing age, and this correlates with the increasing degree of effective immunity. Thus, in endemic areas spleen rates are high in children, but low in adults (Crane, 1972). Both the accumulation of peripheral blood monocytes and in gitn proliferation of splenic myeloid precursors are thought to be contributing to the splenic myeloid hyperplasia, which is the main cause of the hyper- splenism (Wyler and Gallin, 1977). Expansion of the splenic macrophage population associated with an increase in macrophage colony forming cells in the spleen and bone marrow, as well as the presence of chemotactic factors and splenic macrophage migration inhibition activity, are all augmented after primary plasmodial infection in rodents (Coleman gt Q1, 1976). There is increasing evidence that local activation of splenic macrophages, which may be associated with splenic myelopoiesis, induces the appearance of an immunosuppres- sive, Ia’, macrophage-like population. It was shown that treatment of mice with i-carrageenan, a macrophage suppres- sive agent, induces splenic, Mf-like cells capable of inhibiting the lytic function of specific cytotoxic T lymphocytes (Yang and Cudkowicz, 1978). Similar popula- tions were also induced by hydrocortisone acetate, silica and Q. pgtygm (Hochman and Cudkowicz, 1977; Savary and Latzova, 1978; Ojo gt 31, 1978). Local irradiation of bone 20 marrow by the bone-seeking isotope Sr89 induced massive destruction of bone marrow associated with splenic myelo- poiesis along with the appearance of adherent, suppressive Mf-like cells capable of inhibiting T-cell and NK cell functions (Cudkowicz and Hochman, 1979; Haller and Wigzell, 1977). It is interesting to find that a similar population of adherent, suppressive, Mf-like cells are induced in the spleens of mice acutely infected with 2. gngtgugi. Both T- cell and B-cell functions were impaired, as judged by the mitogenic response to Con A and LPS and anti-sheep RBCs plaque formation in spleens of infected animals. However, no suppressive activity was detected in other organs. When mixed with spleen cells from non-infected animals, spleen adherent cells of infected mice also suppressed anti-SRBCs and T and B cell mitogenic responses (Correa gt g1, 1980). Although simple malarious splenomegaly is usually associated with acute infections of children or non-immune adults, massive and persistent splenomegaly can occur as a pathological condition in adults in association with atypical response to chronic infection (Crane, 1972). Tropical Splenomegaly Syndrome (TSS) has been observed in adults living in endemic areas of West Africa (Lowenthal and Jones, 1972), Uganda (Ziegler, 1973), India (Cottoir and Marell, 1950) and New Guinea (Crane and Pryor, 1971). T88 is characterized by persistent splenomegaly, anemia, hepatic sinusoidal lymphocytosis, macroglobulinemia and 21 exceptionally high levels of antimalarial antibodies (Bryceson gt 31, 1976) Other serological features of TSS include high levels of cold agglutinins, antiglobulins such as rheumatoid factors (Ziegler gt g1, 1969) and the occasional appearance of monomeric (7S) IgM in serum (Fauknle and Greenwood, 1977). High levels of polyclonal cryoglobulins containing IgG, IgM, IgA and C3, but apparently no malarial antigens, have been also reported (Ziegler, 1973; Wells, 1970). Circulating immune complexes have been demonstrated in patients with TSS, however, malarial antigens were not detected in these complexes.(Ziegler, 1973). This suggests that these complexes mostly result from interactions between immunoglobulin molecules, and include rheumatoid factors (19S and 7S)-IgG complexes and idiotype anti- idiotype complexes (Cohen and Lambert, 1982). The finding- of low levels of C3 and the anticomplementary activity of TSS sera suggested in 2129 fixation of complement and supported the hypothesis that T88 is an immune complex disease (Ziegler and Stuiver, 1972). Although the patho- genesis of T88 is not fully understood, it has been suggested that a basic defect in suppressor T cell activity may favor the expression of polyclonal B cell activation (Fankule and Greenwood, 1976). It is noteworthy that no evidence of impairment in cellular or humoral immunity have been demonstrated in patients with TSS, as indicated by 22 normal cutaneous skin reactivity to mumps antigen, antibody response to E. gg11 Vi antigen and normal lymphoprolifera- tive response to T cell mitogens in yitrg. Thus, gross immunological deficiency cannot be associated with the , intense lymphoreticular proliferation observed in this syndrome (Ziegler gt Q1, 1969; Crane, 1977). Although phagocytosis constitutes a central effector mechanism in controlling infection, the role played by phagocytic cells in general and Mn/Mf in particular in combating plasmodial infections is not fully elucidated. However, in 2129 studies have demonstrated that phago- cytosis of infected RBCs occurs in RE tissues during acute plasmodial infections. Taliaferro and Cannon (1936) were the first to observe that RBCs infected with 2. btgg11; igngm, as well as non-infected RBCs, are found in the phagocytic vacuoles of spleen macrophages of Panamanian monkeys. Also, they were able to correlate between the phagocytosis of parasitized RBCs and the hyperplasia of the spleen tissue as manifested by splenomegaly. Zuckerman (I945) found that chicken peripheral blood monocytes are able to phagocytose 2. gg111nggggm and 2. 1gphutgg infected RBCs only in the presence of opsoniz- ing antibodies of immune sera from infected chicken. Russel gt Q1, (1963) reported that the splenomegaly and hepatomegaly associated with human plasmodial infections are due to hyperplasia of the spleen phagocytic cells upon 23 ingestion of parasitized RBCs. Also, they demonstrated that at the time of crisis in infection, as the parasite count starts to decline, phagocytes have been observed to avidly engulf parasitized RBCs and even non-infected RBCs. Sheagren gt g1, (1970) found that the phagocytic function of mononuclear phagocytes is increased during natural human malaria infections, as shown by the increase in the rate of clearance of colloidal carbon from the blood stream of naturally infected patients. Verns, (1980) described phagocytosis of 2. fg1g1pgzum infected RBCs by peripheral blood monocytes in blood films of patients with acute falciparum malaria. This observa— tion was later confirmed by Facer and Brown (1981) who found peripheral monocyte erythrophagocytosis of PF- infected RBCs and normal RBCs in Gambian children with acute falciparum malaria. The affected children showed anemia, reticulocytosis and circulating normoblasts. Erythrophagocytosis correlated with RBC-bound IgG and complement components C3b and C4b. 1n yitzg studies have also contributed to the under- standing of the requirements for Mn/Mf-mediated endo- cytosis. However, controversial observations have been encountered concerning the ability of human peripheral blood (PB) monocytes to internalize erythrocytes infected with in gitzg adapted strains of B. 131gipgtgm (Khusmith and Druilhe, 1983). It is important to consider that the 24 in yitzg cultured parasite may be phenotypically distinct from the natural pathogen. 1n yittg adapted strains can lose their natural affinity to bind human endothelium and Mn/Mf (8'). Also, long term culture of the parasite can lead to the permanent loss of the knob structures on infected RBCs (Hommel, 1983: David gt 31, 1983). The lack of standardized methodology for the assay of endocytosis has resulted in additional controversy. Trubowitz and Masek, (1968) have observed ingestion of PF merozoites by neutrophils, but failed to detect any ingestion of SIRBCs by peripheral blood monocytes in the absence of antisera. Cohen gt 31, (1961), Brown (1969) and Phillips gt g1, (1970) found that splenic Mf from 2. Kngx1gg1-infected rhesus monkeys were able to phagocytose parasitized RBCs. Criswell gt Q1, (1971) and Tosta and Wedderburn (1980) reported that spleen Mf from 2. ngtgngi-infected rodents were able to endocytose infected RBCs only in the presence of immune serum. Shear gt Q1, (1979) demonstrated that splenic Mf from 2. bgtgngi-infected mice are more efficient .in phagocytosis of parasitized reticulocytes than Mf from non-infected animals. The recognition and ingestion of infected reticulocytes were found to be mediated by opsonic IgG molecules found in sera of infected animals. Khusmith gt Q1, (1982) found that human monocytes from patients with falciparum malaria as well as non-infected subjects do ingest merozoites of PF in the absence of human 25 immune sera, but rarely phagocytose parasitized or non- infected RBCs in the absence of immune sera. Khusmith and Druilhe, (1983) demonstrated that cytophilic binding of human immune serum or its purified IgG fraction to human monocytes stimulated endocytosis of PF merozoites. Cyto- philic binding of the same fractions did not mediate endocytosis of SIRBCs. Celada gt g1, (1982) found that PB monocytes from normal blood donors ingest PF-SIRBCs opsonized by sera from patients living in areas with endemic malaria. In contrast, sera obtained from patients recovering from a first infection or normal pooled sera do not promote phagocytosis of infected RBCs. Immune sera did not support the endocytosis through cytophilic binding to effector cells. The activity of immune sera were found associated with its IgG fraction. Infected RBCs containing schizonts and trophozoites were preferentially ingested as compared to ring forms. Celada gt g1, (1984) reported that both human neutrophils and monocytes are able to endocytose preferentially PF-infected RBCs in the presence of immune sera. 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Immune phagocytosis in murine malaria. 11_Expt_ngg1 149:1288. 97. Sherman, I. 1979. Biochemistry of Plasmodium (Malaria parasites). n1gzgt1g12_3g21 43:453 98. Tait, A. 1981. Analysis of protein variation in 2. 1g1g1pgtgm by two-dimensional gel electrophoresis. M01. Biochem1_£aras11211 2:205. 99. Taliaferro, W. H. and Cannon, P. 1936. The cellular reactions during primary infections and superinfection of R. btgg111gngm in Panamanian monkeys. 11_13t2_n1g1 59:72. 100. Taverns, J., Tavernier, J., Fiers, W. and Playfair, J. H. L. 1987. Recombinant tumor necrosis factor inhibits malaria parasites 1n 2129 but not 13 21tzg. Q11nt_gxp1 immunell 67:1- 101. Trager, W. , Rudzinska, M. A. and Bradbury, P. C. 1966. The fine structure of £._£g1g1pgzgm and its host erythrocyte in natural malaria in man. flu11, E39: 35:883. 102. Tobie, E., Abele, C., Wolfe, S., Contacos, P. and Evans, C. 1966. Serum immunoglobulin levels in human malaria and their relationship to antibody production. gt Immuneli 973493- 35 103. Tosta, C., and Wedderburn, C. 1968. Immune phago- cytosis of E. 2gg111-infected erythrocytes by macrophages and eosinophiles. Q11n2_gxpt_lmmgng11 42:114. 104. Troye-Blomberg, M., Romero, P., Bjorkman, A., Patar- royo, M. and Perlmann, P. 1984. Regulation of the immune response in 2. tg1g1pgzgm malaria III. Proliferative response to antigen 1n 21ttg and subset composition of T cells from patients with acute infection or from immune donors. slin1_exnl_1mmuncli 58:380. 105. Troye-Blomberg, M., Andersson, G., Stoczkowska, M., Shabo, R., Romero, P., Patarroyo, M., Wigzell, H. and Perlmann, P. 1985. Production of IL-2 and INF-g by T cells from malaria patients in response to g. fg1g1pgttm or erythrocyte antigens 13 21tz_. 11_1mmgng1t 135:3498. 106. Trubowitz, S. and Masek, B. 1968. 3. fig1g1pgttm phagocytosis by polymorphonuclear leukocytes. §g1gngg 162:273. 107. Udeinya, I. J., Miller, L. H., McGregor, I. A. and Jensen, B. J. 1983. 2. tg1g1pgzgm strain-specific antibody blocks binding of infected erythrocytes to amelanotic melanoma cells. Ngtntg 303:429. 108. Udeinya, I. J., Leech, J. H., Aikawa, M. and Miller, L. H. 1985. An 1n v1tzo assay for sequestration : Binding of B. tg1g1pgtgm-infected erythrocytes to formalin-fixed endothelial cells and amelanotic melanoma cells. J, Ezgto- 23911 32:88. 109. verns, A. 1980. Phagocytosis of 2. tg1g1pgttm parasitized erythrocytes by peripheral blood monocytes. Lgnggt ii:1297. 110. Voller, A. 1974. Immunopathology of malaria. 32112 £392 50:177. 111. Warren, H. S. and Weidanz, W. P. 1976. Malarial immunodepression 1n 21ttg : adherent spleen cells are func- tionally defective as accessory cells in the response to horse erythrocytes. Egtt_11_lmmpng1t 6:816. 112. Weidanz, W. P. 1982. Malaria and alterations in immune reactivity. fit1t1gn__ugg2_fig11t 38:167. 113. Wells, V. J. 1970. Immunological studies in tropical splenomegaly syndrome- Transl_31_§csl_lrspi_ued1 fiygt 64:531. 36 114. Wilson, R. J. M. 1980. Serotyping 2. tg1g1pgtgm malaria with S-antigens. Egtgtg 284:451. 115. wyler, D. J. 1976. Peripheral lymphocyte subpopula- tions in human falciparum malaria. Q11n2_gxp1_1mmgng12 23:471. 116. WYler, D. J. and Gallin, J. 1977. Spleen-derived mononuclear cell chemotactic factor in malaria infections : A possible mechanism for splenic macrophage accumulation. £1.1mmunell 118:478. 117. Yang, P., and Cudkowicz, G. 1978. Depressive effects cf carrageenan on cell-mediated lympholysis induced 1n 21ttg : Antimacrophage and antilymphocyte activities. 11.8eticulcendethell_§221 24:461- 118. Ziegler, J. L. 1973. Cryoglobulinemia in tropical splenomegaly syndrome. slin1_sxnl_1mmuncll 15:65. 119. Ziegler, J. L., and Stuiver, P. C. 1972. Tropical splenomegaly in Rawandan Kindred in Uganda. fi:1_nggtglt 3:79. 120. Ziegler, J. L., Cohen, M. H., and Hutt, M. S. R. 1969. Immunological studies in tropical splenomegaly syndrome in Uganda. fi;1_ng§1211 3:79. 121. Zucherman, A. 1945. In 21ttg opsonic tests with 2. sallinassum and B. lsnhsras. .11_Inf1_nis1 77:28. CHAPTER ONE ' DIVERSITY OF ANTIGENS INDUCED ON ERYTHROCYTES BY CULTURE-ADAPTED W W DETECTED BY HUMAN IMMUNE SERA. Hassan M. Elsaid and James B. Jensen 37 38 ABSTRACT The antigenic properties of erythrocytes infected with six cultured strains of 2. tg1g1pgttm have been investigated using an indirect surface immunofluorescence assay (SIFA). The parasite strains employed display different phenotypes of knob structure expression and cytoadherence properties. Sera of adults living in malaria endemic areas of Sudan, Nigeria and Irian Jaya contained immunoglobulins specific for parasite-dependent antigens expressed on infected erythrocytes. The sera preferentially recognized endotheli- um-binder parasites of strain ItGC32. However, sera of patients with primary falciparum infection failed to react with any of the strains tested. The differences in levels of recognition of individual strains correlated with the drift from knobby and endothelium-binder to knobless and non-binder phenotypes. The expression of knob structures alone on infected erythrocyte surface was not sufficient for conferring antigenicity to infected erythrocytes. Two parasite strains failed to react with any of the serum samples employed in this study. These parasites may display a rare antigenic repertoire or completely lost expression of erythrocyte surface antigens. 39 INTRODUCTION Blasmedism falciparum. the causative agent of malig- nant tertian malaria in humans, represents a species with considerable phenotypic heterogeneity. Isolates of the parasite from individual patients have been shown to diverge regarding their sensitivity to antimalarial drugs, antigenic makup, isoenzyme typing, two-dimensional electrophoretic pattern of their proteins and expression of knobs on infected erythrocytes (Carter and McGregor, 1973: Kilejian, 1980: Wilson, 1980: Tait, 1981: Rosario, 1981: MCBride gt g1, 1982, 1987: Thaithong gt 31, 1984). The major glyco- protein of 2. tg1g1pgtnn associated with schizont and merozoite surfaces, was shown to include both conserved and strain-specific domains (Hall gt 31, 1984: Scaife gt 31, 1986: Cheung gt g1, 1986: Lyon gt g1, 1987). Further, distinct antigenic diversity have been detected among para- site-dependent antigens associated with infected erythro- cytes (Hommel gt Q1, 1983: Mendis gt Q1, 1983: Udeinya gt Q1, 1983 a: Aley gt g1, 1986: Coppel gt g1, 1986). Gambian children naturally infected with 2. tg1g1pgzgm were found to develop isolate-specific humoral response to surface anti- gens on their own infected red blood cells, but their sera did not cross-react with parasites derived from other infections (Marsh and Howard, 1986). 40 The role of merozoite surface antigens as inducers of protective immune responses has been the subject of much research and discussion (Cohen and Lambert, 1982: Perlmann gt Q1, 1984: Hommel, 1985: Wahlgren gt g1, 1986). However, because the parasite antigens associated with the surface of infected erythrocytes have been shown to be diversified (Marsh and Howard, 1986: Coppel gt g1, 1986) their role in protection is less clear. Pooled IgG fractions of sera from adults showing clinical resistance to malaria could effec- tively reduce falciparum parasitemia when passively trans- ferred to children with acute infections (Cohen gt 31, 1969: Cohen and Butcher, 1970). No damage was observed to the intracellular parasite. This may indicate that immune IgG recognized antigens on surfaces of parasitized RBCs and/or merozoites. This observation suggests the importance of parasite neoantigens on infected erythrocytes as targets for host humoral response. In this study we investigate the antigenic properties of erythrocytes infected with culture-adapted strains of 2. £g1g1pgttm displaying different phenotypes as defined by knob structure expression and endothelium-cytoadherence properties. 41 MATERIALS AND METHODS Parasites. Phenotypically, 1n,21tzg-adapted falciparum parasites have been characterized as having knobs and the capacity to bind human endothelium or C32 melanoma cells (K+ 8*): having knobs but no cytoadherence properties (K+ B‘): and having no knobs (K‘). The strains of B. £g1g1pgtgm used in this study include FCR-3-TC, a Gambian isolate from patient TC after accidental infection with strain FCR-3 (West Africa) (Jensen and Trager, 1978: Jensen gt g1, 1981), I-2 (Sudan), G-73 (Irian Jaya) and KAS-l (Kenya). Strain ItGC32, a clone of strain ItG (Brazil) was kindly provided by Dr. J. Leech. This clone is knobby and binds human endothelium/C32 cells (K+B+). Strains FCR-3-K+c3 (K+B') and FCR-3-K'c5 (K'B') are clones of the FCR-3, were kindly provided by Dr. T. Green. Parasite cultures. Parasites were cultured in medium RPMI- 1640 supplemented with 5% human A+ serum and 25 mM HEPES buffer in O+ erythrocytes according to the method of Jensen and Trager, (1977), except for ItGC32 which was incubated in modular incubation chambers (Flow Laboratories) in a gas mixture of 1% 02, 3% C02, 96% N2. 42 Synchronized parasite cultures. Parasite cultures were synchronized by sequential treatment with sorbitol and difluoromethylornithine (DFMO), a gift from Dr. P. McCann, Merrill-Dow (Cincinnati). Briefly, washed cultures were incubated in 5% sorbitol for 15 min at 37°C to eliminate all the mature forms of the parasite (Lambros and Vanderberg, 1979). The ring-stage parasites were allowed to grow for 20-24 h in complete medium supplemented with 5 mM DFMO, which inhibits DNA synthesis resulting in highly synchronous trophozoite-stage organisms. Washed cultures were incubated in complete medium supplemented with 250 uM putrescine, which stimulates the resumption of DNA synthesis and allows the parasite to develop synchronously through schizogony. Parasites could be harvested at any time after sorbitol or DFMO synchronization to isolate organisms at a specific developmental stages. Knobby parasites were enriched by the gelatin flotation technique according to the method of Jensen (1978). Human sera. Thirty five serum samples representing adults from areas having distinct malaria endemicity. Malaria transmission in Irian Jaya (25 samples) and Nigeria (5 samples) is holoendemic, while in Damazin Sudan (5 samples) it is hyperendemic. Sudanese samples were collected immediately following the rainy season when malaria trans- mission was high. Five serum samples from American patients 43 suffering primary malaria infection were also studied. One case (ML) was diagnosed as being infected with B. 21232. All American sera were collected during the acute disease, except (PR), whose sample was collected 2 years after infection. Surface immunofluorescence assay (SIFA). Sera were titrated for the specific recognition of parasite-dependent antigens on infected red blood cells by an indirect immunofluores- cence assay according to the method of Mendis gt g1, 1983. Briefly, washed infected erythrocyte preparations were opsonized with 100 ul test serum at appropriate dilutions for 30 min at room temperature. Cells were washed twice in medium RPMI 1640 then incubated for 20 min at room tempera- ture with appropriate dilution of fluorescein-conjugated goat-antihuman IgG (Cappel Laboratories). After additional washing, wet preparations of infected cells were examined under a glass cover slip for surface fluorescence by ultra- violet microscopy at 100x. Sham cultures of non-infected human erythrocytes, cultured for the same period as parasite cultures were tested simultaneously. Trypsin treatment of infected erythrocytes. Infected erythrocytes were washed twice in Tris-saline buffer containing 2 mM CaClz, pH 7.2. Cells were resuspended in the same buffer containing trypsin (GIBCO Laboratories) at 44 appropriate concentrations. Cells were incubated for 10 min at room temperature, then washed once and resuspended in 0.01 M phosphate-buffered saline (PBS) containing 3 mg/ml soybean trypsin inhibitor (Sigma) and 200 ug/ml pepstatin (Sigma). .Incubation continued for 10 min at room tempera- ture. Infected RBCs were washed twice in PBS and tested for SIFA. RESULTS In order to determine the stage specificity of in- fected-erythrocyte surface antigens, DFMO-synchronized cultures of strain FCR-3-TC were examined for SIFA using a pool of malaria hyperimmune sera at intervals during a cycle of parasite growth. By the late trophozoite-early schizont stage, about 68% of infected RBCs opsonized with immune sera showed specific surface fluorescence of uniform intensity (figure 1). With the progress of development, the percent- age of positive cells did not significantly increase. Non- infected erythrocytes of sham cultures failed to show any fluorescence. Surface fluorescence was abolished by trypsinization of parasite-infected erythrocytes (figure 2). It has been shown that gelatin concentration techni- ques are associated with the presence of knobs (Langreth and Reese, 1979: Jensen, 1978). Thus strain FCR-3-TC and G-73 45 were enriched for knob-bearing parasites by repeated gelatin flotation. When these highly enriched preparations were examined for SIFA using the pool of hyperimmune sera, only FCR-3-TC showed surface fluorescence (figure 3). Electron microscopy of erythrocytes infected with strain G-73 con- firmed the expression of knob structures (data not shown), though this parasite showed no SIFA reactivity with any of the tested sera. Strain KAS-l which could not be enriched using gelatin techniques, and thus presumably K', likewise showed no surface reaction with any of the immune sera. Tables 1, 2 and figure 4 show the pattern of specific recognition of erythrocyte-associated antigens of 6 strains of diverse K and B phenotypes. The majority of sera could opsonize schizonts of the K+ B+ strain ItGC32. Fewer sera showed reactivity with the K+ B‘ strains FCR-3-TC and I-2. None of the sera tested recognize surface determinants on strains G-73 or KAS-l. None of the sera from primary malaria patients showed surface immunofluorescence with any of the strains tested. The SIFA test allows examination of the diversity of infected-erythrocyte surface antigens and to some degree the ' spectrum of antibodies found in malaria immune sera from geographically and endemicaly distinct areas. From the parasite antigen perspective, the ItGC32 strain reacted with significantly more sera than any other strain. 46 Reactivity generally followed a pattern in which K+ B+ > K+ B' > K’ strains. Although there were some exceptions. Four sera recognized K+ B' strains but did not react with ItGC32, whereas five sera recognized the K+ and K‘ clones of FCR-3, but had higher titers to the K+ clone. One serum sample form Irian Jaya reacted with the K’ clone but not with the K+ clone. As mentioned above the G-73 and KAS-l strains did not react with any sera (table 1). An examination of the spectrum of antibodies to SIFA demonStrated that sera from holoendemic Irian Jaya and Nigeria reacted with more strains and phenotypes than sera from hyperendemic Sudan. Moreover, Sudanese sera reacted more strongly with the local I-2 strain than with FCR-3 K+ clone, but were nonreactive to the K' clone (table 3). 47 Figure 1. Surface immunofluorescence of R. fig1g1pgrnm developmental stages. Synchronized parasite cultures were tested for SIFA at different periods during one cycle of development. The stages of parasite development: ring, time 0: trophozoite, 34 h: schizont, 40 h: segmenter, 44 h. 48 .00.. c a .m _ .w mo. P m S an mm 1 O . are «averse—n6 waives» mom—3638.. moan”? 926.8338. mesons 16:3 ._ 49 Figure 2. Effect of trypsin treatment of infected erythrocytes on surface immunofluorescence. Infected erythrocytes of strain FCR-3-TC were incubated for 10 min with the indicated concentrations of trypsin and assayed for SIFA using a pool of human immune sera. 50 docs m um. m e .w .w mo. p m m x mm. o IT . do Loo .362: co\3_ locum N . _t_flwu|. 51 Table 1. Titers of surface immunofluorescence (SIFA) of 40 serum samples tested with six strains of P. falciparum. Strains Sera Source ItGC32 FCR3-K+ 1-2 G-73 FCR3-K— KAS-l TC Pr.Inf. * O O 0 0 0 0 JH , 0 0 0 0 0 0 LM , 0 0 0 0 0 0 ** MZ , O 0 0 0 O 0 PR , 0 O 0 0 O 0 1 Sudan 5 0 0 0 0 0 2 , 5 O O -0 O 0 3 , 80 0 10 0 0 0 4 , 640- O 10 O 0 0 5 , 640 20 80 O O 0 6 Nigeria 0 0 5 0 0 0 7 , 40 40 10 O 0 0 8 , 40 20 10 O 0 O 9 , 40 10 10 O 5 0 10 , 80 80 0 O 10 0 11 I.Jaya 640 0 5 0 0 0 12 , O 80 40 O 10 0 l3 , O 5 0 0 ND 0 l4 , 5 0 O 0 O 0 15 , 5 0 0 O O 0 16 , 4O 0 O 0 0 0 l7 , 80 4O 0 0 0 O 18 , 80 10 0 0 4O 0 l9 , 160 O O O 10 0 20 , 160 80 0 0 0 0 21 , 640 5 40 0 0 0 22 , 640 5 O O O 0 23 , 640 0 5 0 0 0 24 , 0 ND 0 0 ND 0 25 , 0 0 0 0 O 0 26 , 0 160 0 O O 0 27 , 0 0 O O O 0 28 , O 0 O 0 0 0 29 , 5 0 0 0 0 0 30 , 40 40 0 O 40 0 31 , 40 0 0 0 0 0 32 , 40 O O 0 0 0 33 , 160 80 0 O 0 0 34 , 640 80 0 O 0 0 35 , 640 10 0 0 0 0 * 0 indicates titer < 5. ** Acute infection with P.vivax. Pr. Inf.: Sera of primary infection patients. ND: not done 52 Figure 3. Surface immunofluorescence of infected erythrocytes enriched for knobby parasites. Parasites of strains FCR-3-TC and G-73 were enriched for knobby parasites by repeated gelatin separations (Gel. 1-7). Parasites were tested for SIFA after enrichments 3, 5 and 7. Original preparations were tested simultaneously. Q aanL-J 190 oN 2 199 9 199 L 199 53 X SIFA positive IRBC N 0| \1 0| 0 0| “001 W \\\\\\\\\\\\\\\\\\\\\\V i \\\\\\\\\\\\\\\\\\\\\\\\V 1 a\\\\\\\\\\\\\\\\\\\\\‘ GIL-9 E Ol-QHOJ m 54 Table 2. Distribution pattern of 40 malaria sera tested for recognition (SIFA) of six different parasite phenotypes. Parasites* Sera Number K+B+ K+B- , K-B- ItGC32 FCR3-K+ I-2 G-73 FCR3-K- KAS-l Immune 35 27 17 10 O 6 0 sera Primary 5 0 0 0 0 0 0 infection * Parasite phenotypes: K+, knobby: K-, knobless: B+, binder to human endothelium: B-, non-binder. Table 3. Distribution pattern of 40 malaria sera according to the geographical origin of sera and parasites. Sera* Strains Source Number ItGC32 I-2 FCR3-K+ FCR3-K- G-73 KAS-l (Brazil)(Sudan)(West Africa) (I.Jaya)(Kenya) Sudan 5 5 3 1 0 0 0 Nigeria 5 4 4 4 2 0 0 I. Jaya 25 18 4 12 4 o 0 Primary 5 0 0 0 0 0 0 infection (USA) * Sera were tested for surface immunofluorescence (SIFA) using the indicated parasite strains, see materials and methods. 55 Figure 4. Distribution pattern of 35 serum samples according to their surface immunofluorescence titers. Open circle, sera recognized > two parasite strains including ItGC32: filled circle, sera recognized one strain only: open square, sera recognized > two strains not including ItGC32. 56 SIFA TITER. who . 8188 30 . 95 O 88 0 BE .3 - mg 8 so 8 8 3 . 088 so 8m i. 8 as 0 zoom» Tn mmoux+ momuxl 93:6 04 p. 3.2333 305.6 a. 57 DISCUSSION The expression of parasite-dependent antigens on erythrocytes infected with 2. tg1g1pgxnm correlated with the maturation of the intraerythrocytic parasite 1n 21trg. A majority of erythrocytes infected with late trophozoite and early schizont stages, showed specific surface fluorescence when opsonized with pooled immune sera. However, specific opsonization of 100% of cells containing mature parasites was never achieved. This observation may imply the anti- genic heterogeneity of IRBCs in culture. Ring-infected and non-infected RBCs failed to show any specific fluorescence. Trypsinization of infected erythrocytes abolished surface fluorescence. This indicates the protein nature of erythro- cyte neoantigens. Our results confirm the similar findings of Mendis gt 31, (1983). The nonreactivity of strains G-73 and KAS-l also supports the apparently contradictory observations of Marsh gt g1, (1986) in which Gambian immune sera failed to recognize culture-adapted isolates from the same locality. Obviously, strains G-73 and KAS-l express anomalous, or no parasite antigens on their host erythrocytes. Previous studies (Kilejian gt g1, 1977: Langreth and Reese, 1979) have suggested that erythrocyte surface antigens recognized by monkey immune sera were associated 58 with the knob structures. As we indicated above, IRBCs showed antigenicity by the trophozoite stage of development. Expression of knobs and the acquisition of cytoadherence capability by IRBCs occur concurrently during the same phase of intraerythrocytic development (Trager gt g1, 1966: Luse and Miller, 1971). Our results show that human immune sera recognized surface antigens on infected RBCs regardless of the K and B phenotypes of parasites. Sera detected deter- minants on RBCs infected with FCR-3 K' parasites but failed to react with erythrocytes infected with the K+ parasites of strain G-73. It is plausible to conclude that neither the presence of knobs on G-73 nor their absence on FCR-3-K’ clone influenced the qualitative recognition by sera. The expression of surface antigens on K' B’ parasites is in- dependent of the co-expression of knob structures and cyto- adherence molecules. The polymorphism, or complete loss of this class of surface antigens, may be also independent of the K and B phenotype of the parasite, as indicated by the reaction of strains G-73 and KASI with immune sera. On the other hand, the expression of cytoadherence molecules seems to be associated with co-expression of knobs since the K' B+ phenotype has never been reported. It is noteworthy that knob structures are located under rather than on the erythrocyte plasma membrane (Trager gt g1, 1966: Leech gt g1, 1984: Pologe gt g1, 1987), which explains why monoclonal antibodies directed to the histidine-rich protein 59 of knobs could not recognize the target antigen on surfaces of unfixed IRBCs and had no effect on 1n 21tzg-cytoadherence (Taylor gt Q1, 1987). Nonetheless, cytoadherence was shown to be associated with erythrocyte membrane over knob sites (Luse and Miller, 1971). Thus, it is probable that the antigenic cytoadherence molecules are expressed on the erythrocyte membrane over knob sites. Polymorphism or loss of the binding molecules can occur without loss of knobs as indicated by the K+ B' phenotype of most of 13 21ttg-adapted parasite strains (Udeinya gt Q1, 1983 b). This observation explains why the physical presence of knobs alone is not sufficient for conferring antigenicity (strain G-73), or for cytoadherence (K+ 8' strains) of infected RBCs. Specific antibodies and spleen have been suggested as selective immune pressures that control parasite antigenic drift 13 2129 (David gt g1, 1983: Hommel gt 31, 1983). Also, it has been demonstrated that 1n 21ttg, newly isolated parasites (assumed to be K+ B+) rapidly loose their cytoad- herence properties, changing to K+ B' (Udeinya gt g1, 1983 b), and eventually become knobless (K‘ B’). This phenotypic drift is probably due to the loss of immunologic selective pressures under 1n 21trg culture conditions. The observed differences in the levels of recognition of individual strains correlate with the drift from the K+ B+ to K‘ B' phenotypes. The majority of sera, that represent different geographical backgrounds, recognized the endothelium-binding 6O parasites with preferential specificity and significantly higher titers than any other strain. It is likely that strain ItGC32, which is K+ 8*, is phenotypically similar to fresh parasite isolates. This strain may display a distinct ' class of surface antigens associated with the B+ phenotype. It is probable that more immunogenic determinants or common antigens are associated with the cytoadherence molecules. The differences in expression of this class of antigens on K+ B' as well as K' B' IRBCs may explain their failure to bind endothelium/C32 cells. Although neoantigens on IRBCs may show antigenic diversity, common epitopes may still exist. It has been shown that the host receptors involved in adherence of IRBCs to human endothelium, melanoma cells and monocytes are iden- tical. The monoclonal antibody OKMS can recognize this activity and can block cytoadherence to such target cells (Barnwell gt Q1, 1985). This observation implies that cytoadherence molecu1e(s) should retain structurally and antigenically conserved epitopes that would allow the antigenically diverse parasites to bind host ligands of limited, or no variation. The observation that sera from endemic areas were able to recognize infected RBCs while sera from acutely infected patients could not, suggests the importance of multiple exposures to malaria as a necessity to develop and maintain serum reactivity. Also, it may signify that erythrocyte 61 surface antigens are of limited immunogenicity. These assumptions are supported by the observation that poly- specific sera showed significantly higher titers to ItGC32 than monospecific sera. Multiple exposures to parasites of diverse antigenic makeup are expected to increase the spectrum of recognition of variable antigens and to poten- tiate immune response to conserved determinants of low immunogenicity. This notion can partially explain both the slow development of acquired immunity to falciparum malaria and the polyspecificity of adult sera in comparison to mono- specific pediatric sera (Marsh and Howard, 1986). 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W., Howard, R. J. and Miller L. H. 1982. Altered expression of B. 3ng21gg1 variant antigen on the erythrocyte plasma membrane in splenectomized rhesus monkeys. g2_1mn2ng11 128:224-226. 3. Barnwell, J. W., Howard, R. J., Coon, H. and Miller L. H. 1983. Splenic requirement for antigenic variation and expression of the variant antigen on the erythrocyte membrane in cloned Plasmodium knowlesi malaria. Intggtt INNER; 40:985-994. 4. Barnwell, J. W., Ockenhouse, C. F. and Knowles, D. M. 1985. Monoclonal antibody OKMS inhibits the 13 21tzg binding of B. tg1g1pgztm-infected erythrocytes to monocytes, endothelial and C32 melanoma cells. 12_1mm2ng11 135:3494. 5. Carter, R. and McGregor, I. A. 1973. Enzyme-variation in B. falsinanm in The Gambia. WM... §2g1 67: 830-837. 6. Cheung, A., Leban, J., Shaw, A. R., Merkli, B., Stocker, J., Chizzolini, C., Sander, C. and Perrin, L. H. 1986. Immunization with synthetic peptides of a 21ggmg§1gm surface antigen induces anti-merozoite an- falsieam tibedieS- W 83: 8328-8332 7. Cohen, S., McGregor, I. A., and Carrington, S. 1961. Gamma-globulin and acquired immunity to human malaria. Hgtuzg 192:733. 8. Cohen, S. and McGregor, I. A. 1963. Gammaglobulin and acquired immunity to malaria. In: Granham, P., Pierce, A., and Roitt, I. Immunity to Protozoa pp. 123. Blackwell Scientific Publications, Oxford. 9. Cohen, S. and Lambert, P. 1982. In: Immunology of parasitic infections. ed. Cohen, S. and Warren, K. Black- well Publications, Oxford. 63 10. Coppel, R. L., Culvenor, J. G., Bianco, A. E., Crew- ther, P. , Stahl, H. , Brown, G. , Anders, R. F. and Kemp, D. J. 1986. Variable antigen associated with the surface of erythrocytes infected with mature stages of 21ggmgg1gm falsinsznm . u911_Biccnem1_£arasitcll 20:265-77- 11. David, P. H., Hommel, M. , Miller, L. H., Udeinya. I. J. and Oligino, L. D. 1983. Parasite sequestration in 2. tg1g1pgztm malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes. Ezgg1_Ngt12_Aggg1 £911_HSAI 8835075 12. Hall, R. , Osland, A. , Hyde, J. , Simmons, D. , Hope, I. and Scaife, J. 1984. Processing, polymorphism, and biochemical significance of P190, an major surface antigen of the erythrocytic forms of Plasmodium falciparum. Hell Bicshsm1_zaras11211 11:61-80. 13. Hommel, M., David, P. H., Oligino, L. D. and David, J. 1982. Expression of strain specific surface antigens on B. falsinarun-infected erythrocytes. Parasite_1mmun911 4:405- 419. 14. Hommel, M. 1985. Antigenic variation in malaria parasites. Imnnng1gg2_19§g2 6:28-33. 15. Hommel, M. , David, P. H. and Oligino, L. D. 1983. Surface alterations of erythrocytes in £1ggmgg1gm 1g1g1pgttm malaria antigenic variation, antigenic diversity, and the role of the spleen. l1_fixpt_ngg1 157:1137-1148. 16. Jensen, J. B. and Trager, W. 1977. £1ggmgg1gn 1g1g1pgttn in culture: use of outdated erythrocytes and description of the candle jar method. 11_£gtgg1tg12 63: 883-886. 17. Jensen, J. B. 1978. Concentration from continuous culture of erythrocytes infected with trophozoites and schizonts of Plasmodium falsinarum Am1_11_Tr921_Medl_nxsi 27: 1274-1276 18. Jensen, J. B. and Trager, W. 1978. £1ggmgd1um fg1g1pgxgm in culture: establishment of new strains. Am, J. IIQRI_M§§1_HYQI 273743 19. Jensen, J. B., Capps, T. and Carlin, J. 1981. Clinical drug-resistant falciparum malaria acquired from cultured parasites. Am1_11_mtgpt_ng§1_fl2g1 30:523-525. 64 20. Kilejian, A. , Abati, A. , and Trager, W. 1977. 2. 131g1pgxgm and 2. gggtng21: Immunogenicity of "knob-like protrusions "on infected erythrocyte membranes. £291 Bgtgg1tg11 42:157-164. 21. Kilejian, A. 1980. Stage-specific proteins and glycoproteins of £1g§mgg1gm fg1g1pgtnm: Identification of antigens unique to schizonts and merozoites. EIQ£1_NQE11 A§e§1_§§11_flfibi_ 77:3695‘3599- 22. Lambros, C. and vanderberg, J. 1979. Synchronization of Plaemccinn falciparum erythrocytic stages in culture- 11 Egtgg1t91hIL 65:418-420. 23. Langreth, S. G. and Reese, R. T. 1979. Antigenicity of infected-erythrocyte and merozoite surfaces in falciparum malaria. l1_Exnt_Hgg1 150:1241-1254. 24. Leech, J. H., Barnwell, J. W., Aikawa, M. , Miller, L. H. and Howard, R. J. 1984. £1ggmgd1gm 111g1pgtgm malaria: association of knobs on the surface of infected erythrocytes with a histidine-rich protein and the erythrocyte skeleton. £1.9ell_nicli. 98:1256-64. 25. Luse, s. A. and Miller, L. H. 1971. E. 1g1g1pgtnn ultrastructure of parasitized erythrocytes in cardiac vessels. Am1_11_Trcnl_Med1_fixsl 20:655- 26. Lyon, J. A., Haynes, J. D., Diggs, C. L., Chulay, J. D., Haidaris, C. G. and Pratt-Rossiter, J. 1987. Mono- clonal antibody characterization of the 195-kd major surface glycoprotein of £1ggmgg1gm tg1g1pgzgm malaria schizonts and merozoites: identification of additional processed products and a serotype-restricted repetitive epitope. ;2_Immgng11 138:895-901 27. Marsh, K. , Howard, R. J. 1986. Antigens induced on erythrocytes by E. 131g1pgtgn: expression of diverse and conserved determinants. §g1gngg 231:150-3 28. Marsh, K. , Sherwood, J. A. and Howard, R. J. 1986. Parasite-infected-cell-agglutination and indirect immuno- fluorescence assays for detection of human serum antibodies bound to antigens on £1ggmgg1tm fg1g1pgzgm-infected erythro- cytes. £1.1mmuncll_nethcde 913107-15 29. McBride, J., Walliker, D. and Morgan, G. 1982. Antigenic diversity in the human malaria parasite B. 131; ciparum. Science 217:254-257- 65 30. McBride, J. and Heidrich, H. 1987. Fragments of the polymorphic Mr 185,000 glycoprotein from the surface of isolated 21ggng§1nmpfig1g1pgztm merozoites form an antigenic complex. Mell_nicchem1_2eraeitcll 23:71-84 31. Mendis, K., David, P. H., Hommel, M., Carter, R. and Miller, L. H. 1983. Immunity to malarial antigens on the surface of 2. 131g1pgznn-infected erythrocytes. An1_11 Trcni_flec1_flxsi 32:926- 32. Perlmann, H., Berzins, K., Wahlgren, M., Carlsson, J., Bjorkman, A., Patarroyo, M. and Perlmann, P. 1984. Antibodies in malarial sera to para- site antigens in the membrane of erythrocytes infected with early asexual stages 01.2. falciparum. 11.nxnl_nec1 159:1686-1704- 33. Pologe, L. G., Pavlovec, A., Shio, H. and Ravetch, J. V. 1987. Primary structure and subcellular localization of the knob-associated histidine-rich protein of E1ggmgd1gn falciparum” EIQ§1_Hc§11_bsi§1_§911_9551 34:7139‘7143 34. Rosario, V. 1981. Cloning of naturally occurring mixed infections of malaria parasites. 591gngg 212:1037-1038. 35. Scaife, J., Bone, N., Goman, M., Hall, R., Hope, I. A., Hyde, J. E., Langsley, G., Mackay, M., Oquendo, P. and Simmons, D. 1986. Antigens of P1ggmgg1gn tg1g1pgtnn blood stages with clinical interest cloned and expressed in E. .ccli- Bareeitclccx 92 Sappl= 8119-37 36. Sherwood, J. A., Roberts, D. D., Spitalnik, S. L., Marsh, K., Harvey, E. B., Miller, L. H. and Howard, R. J. 1986. Parasitized erythrocyte antigens and thrombospondin adhesion in the immunology and pathogenesis of falciparum malaria. Trane1_Aeecc1_Am1_£hxeiciane 99:206-13. 37. Tait, A. 1981. Analysis of protein variation in 2. 131g1pgtnn by two-dimensional gel electrophoresis. ng11 nicchem1_zerae11211 2:205. 38. Taylor, D. W., Parra, M., Chapman, G. B., Stearns, M. E., Rener, J., Aikawa, M., Uni, S., Aley, S. B., Panton, L. and Howard, R. J. 1987. Localization of £1ggmgg1nm fg1_ g1pgtun histidine-rich protein 1 in the erythrocyte skeleton under knobs. Hell_nicchem1_£ereeitcli 25:165-74 39. Thaithong, S., Beale, G. H., Fenton, B., McBride, J., Rosario, V., Walker, A. and Walliker, D. 1984. Clonal diversity in a single isolate of the malaria parasite B._falcicerum. Trane1_Bi_§cc1_Trcnl_uec1_H¥c1 78:242-245- 66 40. Trager, W., Rudzinska, M. A. and Bradbury, P. C. 1966. The fine structure of £,_tg1g1pg;nm and its host erythrocyte in natural malaria in man. £2111_flHQ1 35:883. 41. Trager, W. and Jensen, J. B. 1976. Human malaria parasites in continues culture. §g1gngg 193:673-675. 42.a Udeinya, I. J., Miller, L. H., McGregor, I. A. and Jensen, J. B. 1983.,{£. tg1g1pgznn strain-specific antibody blocks binding of infected erythrocytes to amelanotic melanoma cells. Ngtnrg 303:429. 43.b Udeinya, I. J., Graves, P., Carter, R., Aikawa, M. and Miller, L. H. 1983. 2. 1g1g1pgznm: effect of time in continuous culture on binding to human endothelial cells and amelanotic melanoma cells. Expt_£g:gg1tg11 56:207-214. 44. Wahlgren, M., Bj:orkman, A., Perlmann, H., Berzins, K. and Perlmann, P. 1985. Anti-£1ggmgg1gm fg1g1pgtgn anti- bodies acquired by residents in a holoendemic area of Liberia during development of clinical immunity. Am1_11 W 35322-9 45. Wilson, R. J. M. 1980. Serotyping 2. fg1g1ngxnn malaria with S-antigens. ngtnzg 284:451. CHAPTER TWO FUNCTIONAL IMPLICATIONS OF IMMUNOGLOBULIN-MEDIATED RECOGNITION 01" W W'INFECTED ERYTHROCYTE ANTIGENS. Hassan M. Elsaid and James B. Jensen 67 68 ABSTRACT The functional significance of immunoglobulin-mediated recognition of £1ggmgg1nm £g1g1pgxnn-infected erythrocytes has been examined. Sera of adults from malaria endemic areas have been tested for the specific recognition of ' .erythrocytes infected with the endothelium-binding parasites of strain ItGC32 using an indirect surface immunofluores- cence assay (SIFA). Also, the sera were assayed for 1n 21ttg cytoadherence-inhibition of infected erythrocytes to human C32 melanoma cells, and for promotion of immuno- phagocytosis of infected erythrocytes by human peripheral blood monocytes. Sera that recognized infected erythrocytes could promote cytoadherence-inhibition and endocytosis by monocytes. No correlation could be detected between levels of recognition of erythrocyte surface antigens (SIFA) and titers of fluorescent antimalarial antibodies detected by conventional indirect fluorescent antibody assay (IFA). 69 INTRODUCTION The immune response to falciparum malaria is complex. Adults in areas of endemic transmission develop a state of acquired resistance to infection. This state is strain- and stage-specific and is acquired over a long period of time (Jefferey, 1966: Garnham, 1970: Mitchell gt g1, 1977), during which the patient endures repeated attacks of blood- stage infection (McGregor, 1960: Jayawardena gt g1, 1982). Both the humoral and cellular components of immunity are thought to be involved in such acquired resistance (Sergent, 1963: Playfair, 1982). However, the particular mechanism(s) by which an immune adult can control bloodstage infection and the stage at which the parasite is susceptible to such effector mechanism(s) are largely unknown (Cohen gt g1, 1969). The intraerythrocytic infection of plasmodia constit- utes the primary mechanism by which parasites evade reco- gnition by the host immune system (Cohen and Lambert, 1982). Recognition of non-self determinants is a pivotal step for mounting and executing an effective immune response. A cytolytic effector function for cytotoxic T-cells against infected red blood cells (RBCs) is doubtful in view of the fact that antigen recognition by primed T-cells is restric- ted by recognition of self HLA-Dr molecules (Phillips gt g1, 70 1970: Cohen and Butcher 1971). In the absence of T-cell recognition, a potential role for other effector cells that do not require MHC-modified determinants for recognition is likely. Monocytes/macrophage (Mn/Hf), and polymorphonuclear leukocytes (PMNs) are potential candidates. An effector role for Mn/Mf and/or PMNs is expected to be minimal in the absence of parasite chemotaxis (Trubowitz and Masek, 1968: Wyler and Gallin, 1977). It is clear that in the absence of recognition by cellular effector mechanisms, specific immunoglobulin (Ig) molecules are viable alternatives for the specific recogni- tion of parasite-infected erythrocytes. Pooled IgG frac- tions of sera from adults showing clinical resistance to malaria could effectively reduce falciparum parasitemia when passively transferred to children with acute infection (Cohen gt g1, 1961: Cohen and McGregor, 1963). This observation implies that specific Ig molecules may function as recognition elements during falciparum malaria 1n 212g. Opsonizing Ig molecules can trigger several powerful humoral and cell-mediated mechanisms, e.g. complement fixation, antibody-dependent cell-mediated cytotoxicity (ADCC) and ' immunophagocytosis by Mn/Mf and PMNs. In a previous investigation we reported that sera of adults living in malaria-endemic areas of Nigeria, Sudan and Irian Jaya, could specifically recognize neoantigens on erythrocytes infected with endothelium-binding cultured 71 parasites (Elsaid and Jensen, 1988). In this report, we extended the search for the functional implications of the observed immunoglobulin-mediated recognition. The sera were tested for the specific recognition of P. tg1g1pgrnm- infected RBCs, as well as for their ability to inhibit cytoadherence to C32 melanoma cells and to mediate immuno- phagocytosis by human peripheral blood monocytes 1n 21tng. Sera were also tested for fluorescent antimalarial anti- bodies using conventional IFA assay. 72 MATERIALS AND METHODS Parasites. £13gngg1gmyt31g1n3zgm strain ItGC32, a clone of strain ItG (Brazil) was kindly provided by Dr. J. Leech. This clone is knobby and binds human endothelium/C32 cells 13,21ttg, designated K+B+. Parasite cultivation was in human O+ erythrocytes. Parasites were cultured in medium RPMI-1640 supplemented with 5% human A+ serum and 25 mM HEPES buffer. Cultures were incubated in modular incubation chambers (Flow Laboratories) in a gas mixture of 1% 02, 3% coz, 96% N2. Human sera. Two groups of human sera were included in this study (table 1). The first group includes thirty five serum samples representing adults of three malaria-endemic areas. These samples include 25 adults from Irian Jaya, 5 adults from Nigeria and 5 adults from the Damazin area of central Sudan. Malaria transmission in Irian Jaya and Nigeria is holoendemic, while the Damazin area is hyperendemic. Sudan samples were collected immediately following the rainy season when malaria transmission was high. The second group contains five serum samples from American patients suffering primary falciparum malaria. One case was diagnosed as infected with B. 21233. The available samples were collec- ted during acute infection, except one case, the sample was 73 collected 2 years after infection during which time he was never subjected to reinfection. Indirect immunofluorescent antibody assay (IFA). Anti- malarial IFA titers were determined for both serum IgG and IgM immunoglobulin classes as described previously (WHO memorandum, 1974). Antigen slides were prepared with schizont-enriched ItGC32 cultured parasites. A pool of control normal human sera were assayed simultaneously. Titers are reported as the reciprocal of the highest dilution giving positive fluorescence of the intraerythro- cytic parasite. Surface immunofluorescence assay (SIFA). This assay was performed as described previously (Mendis gt 31, 1983). Briefly, washed parasite preparations were opsonized with 100 ul test serum at appropriate dilutions for 30 min at room temperature. Cells were washed twice in medium RPMI 1640 then incubated for 20 min at room temperature with optimum dilution of fluorescein-conjugated goat-antihuman IgG (Cappel Laboratories). Wet preparations of infected cells were examined under a glass cover slip for surface fluorescence by ultraviolet microscopy at 100x. Sham cultures of non-infected human erythrocytes cultured for the same period as parasite cultures were tested simultan- eously. 74 Cell lines. Human amelanotic melanoma cell line C32 (American Type Culture Collection no. CRL 1585) were main- tained in medium RPMI-1640 supplemented with 10% heat- inactivated fetal calf serum, 25 mM HEPES buffer and antibiotics. Cytoadherence-inhibition assay. The assay of binding inhibition was performed according to the method of Udeinya gt 31, (1985). Briefly, equal volumes of serum and packed infected RBCs were mixed and incubated for 30 min at 37'C. Opsonized infected cells were suspended in medium RPMI-1640 buffered with 25 mM HEPES buffer and 25 mM sodium bicarbo- nate to bring the hematocrit to 2-4%. The suspension (0.3 ml) was laid over monolayers of C32 cells (3-5 x 104 /cm2) in 12 well tissue culture plates (Corning). Plates were incubated for 1 hour at 37'C. Gently washed monolayers were fixed and stained with 10% Giemsa. The effect of serum on binding was determined by comparing number of infected RBCs (IRBCs) bound in presence of immune serum to number of IRBCs bound in presence of control normal human serum. The percent inhibition of binding was calculated as follows: IRBCs / cell with control serum - IRBCs /cell with test serum % Inhibition = X 100 IRBCs / cell with control serum 75 Human peripheral blood mononuclear cells. Blood samples were collected from healthy volunteers without any history of malaria infection. Peripheral blood mononuclear cells were isolated by density gradient separation on Ficoll- Hypaque (Pharmacia Fine Chemicals) using standard metho- dology. Phagocytosis-promoting activity of immune sera. Equal volumes of packed infected RBCs and appropriate dilution of serum were mixed and incubated for 30 min at room tempera- ture. Washed IRBCs were suspended at a hematocrit of 2% in medium RPMI-1640 buffered with 25 mM HEPES buffer. The target cell suspension (200 ul) was laid over PB Mn mono- layers (1 x 106 mononuclear cells/ml) in Lab-Tek tissue culture slide chambers (Milles Laboratories). The chambers were incubated at 37'C for 45 min. Repeatedly washed mono- layers were fixed in methanol and stained with 10% Giemsa. Monolayers were counted for at least 500 phagocytic cells (duplicate assay) and the results were expressed as the percentage of phagocytic cells ingested one or more IRBCs. Statistical analysis. The correlation coefficient (r) and significance of 'r' were calculated using Minitab computer program (Minitab inc.). 76 RESULTS Table 1 shows that all sera had significant levels of fluorescent antimalarial IgG. One patient, who had been. exposed to falciparum infection two years earlier showed no antimalarial specificity in both IFA and SIFA assays. All .but four sera showed specific antimalarial activity in the IgM class. Twenty seven sera in the first group, represen- ting the three areas under investigation, specifically recognized parasite neoantigens on infected erythrocyte surface showing titers in the range of 5-640. Although three patients with acute falciparum infection as well as a patient with acute vivax infection showed significant IFA titers in both immunoglobulin classes, all failed to show any surface fluorescence with infected RBCs. Generally, levels of IFA titers, in both IgG and IgM classes, were significantly higher than those of SIFA for individual sera. Table 2 shows that no correlation existed between levels of IgG specificities directed to surface antigens and titers of fluorescent malaria antibodies detected by IFA assay, though significant positive correlation (r = 0.68) existed between levels of IFA in the IgG and IgM classes. Using human C32 melanoma cells as the immobilized cell for cytoadherence, adult immune sera showed a wide range of inhibition to IRBCs binding. None of the sera of primary 77 infection patients showed significant reduction in the ability of IRBCs to bind C32 cells. A significant correla- tion (r - 0.76) was detected between SIFA titers and levels of C32 inhibition for individual sera. However, 5 cases in the first group showed marked levels of C32 inhibition although their SIFA titers and levels of phagocytosis were significantly low (table 1). Figure 2 shows that opsonization of infected RBCs with pooled sera of adults from malaria-endemic areas signific- antly promoted the ingestion of RBCs infected with mature stages of the parasite. Non-opsonized IRBCs or those opsonized with normal human sera showed background levels of phagocytosis. Levels of phagocytosis promoted by sera of primary infection patients were significantly lower than those of immune sera. A significant positive correlation (r 8 0.8) was detected between SIFA titers and levels of endocytosis mediated by individual sera. Similar correlation was also evident between levels of phagocytosis and that of binding- inhibition (r = 0.66) (figure 1 and table 2). The levels of phagocytosis, same as those of SIFA and cytoadherence- inhibition, did not show any correlation with levels of IFA in both IgG and IgM classes (table 2). 78 Table 1. Results of indirect immunofluorescence (IFA), surface immunofluorescence (SIFA), phagocytosis and cytoadherence-inhibition of ItGC32-IRBCs for 40 malaria sera. Sera Source % Phago. % Binding SIFA IgG IgM inhibition IFA IFA .Immune Sudan * 2.1 4 62.9 5 320 80 sera , 3 72.8 5 160 o , 26.1 96.3 640 640 O , 27.5 86.4 640 1280 160 , 32.2 82.7 80 1280 160 Nigeria 1.2 37 0 5120 80 , 4.5 57 40 2560 40 , 12 75.3 40 2560 O , 15 96.3 40 20480 80 , 25 98.8 80 5120 320 I.Jaya 2.1 2.7 o 1280 40 , 4.1 88 o 640 so , 4.2 76.5 5 20480 2560 , 4.6 87.7 5 10240 640 , 11.1 86 640 2560 320 , 11.9 73.3 160 5120 320 , 16.1 78.7 40 10240 80 , 22.5 93.3 640 5120 160 , 24 73 640 10240 320 , 24.5 74.3 80 10240 640 , 25 80 160 5120 160 , 36.4 70 80 5120 160 , -2.9 9 0 10240 1280 , -2 10 0 10240 2560 , -O.6 40 0 10240 2560 , 0.9 O 0 10240 1280 , 2 23 0 20480 1280 , 2.5 28.4 5 10240 2560 , 10 38 40 5120 640 , 10.4 90.1 40 5120 640 , 10.6 76.7 160 20480 1280 , 13.7 34.4 40 5120 640 , 21.3 90.7 640 10240 640 , 21.9 75 640 5120 160 , 18.3 75.3 640 320 320 Primary USA 2.8 0 0 2560 640 infection , 4.8 1.4 0 10240 2560 sera , -l.5 O 0 2560 160 O, 6.3 -3.1 0 1280 1280 , 1.6 O O O O * % Phagocytosis of IRBCs by human PB monocytes. 4 % Inhibition of cytoadherence of IRBCs to C32 cells. V P. vivax infection. 79 Table 2. Correlation between titers of indirect immuno- fluorescence (IFA), surface immunofluorescence (SIFA), levels of phagocytosis and cytoadherence-inhibition of ItGC32-IRBCs for 40 malaria sera. Binding IgG IgM SIFA Phago. Inhibition IFA IFA SIFA l Phagocytosis 0.82 1 Binding inh. 0.76 0.66 1 IgG IFA 0.1 0.1 0.12 1 IgM IFA -O.15 -O.15 -0.2 0.68 l Number of data points is 40 R value > 0.39 is significant at 1% level. 80 Figure 1. Correlation between levels of phagocytosis and cytoadherence-inhibition of IRBCs mediated by 39 serum samples. Opsonized infected erythrocytes (strain ItGC32) were tested for immunophagocytosis by human PB monocytes and cytoadherence-inhibition to C32 melanoma cells. r = 0.66, r is significant at 1% level. 81 XINHENHON doc. o 8 8. O as QC 0 . oo oo o 6% 8. oo 3. a. mo 8 o. 0% :3 m 1... Mo u.o nutImoOQ