first ;H ' lil‘llllllllllllll Hill! V , l 3 1293 01090 7750 K‘Memm’ ‘5 ‘Ii "cl-"4.0.1 hfi pg mm ‘- This is to certify that the thesis entitled LONGITUDINAL STUDIES ON THE IMMUNE RESPONSE TO PLASMODIUM FALCIPARUM IN SUDAN MEASURED I_N VITRO presented by John Alan Vande Waa has been accepted towards fulfillment of the requirements for Asa—degree in Microbiology fl W Major fiessor Date\77T/47 /Z/7i/ 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution Vu£&$-; 3,.“ ins-.316 Umxwfii‘lfiy ,, M ,h _. ‘P a, -. "I '- I X 1 8 .‘ i - I r‘ -V ‘ MSU LlBRARlES ”- RETURNING MATERIALS: Place in book drop to remove this checkout from your record. [lfl§§_will be charged if book is returned after the date stamped below. LONGITUDINAL STUDIES ON THE IMMUNE RESPONSE TO PLASMODIUM FALCIPARUM IN SUDAN MEASURED IN_VITRO By John A. Vande Haa A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1984 ABSTRACT LONGITUDINAL STUDIES ON THE IMMUNE RESPONSE TO PLASMDDIUM FALCIPARUM IN SUDAN MEASURED IN_VITRO By John A. Vande Haa Inhibition of Plasmodium falciparum i vitro by human immune serum provides needed information in understanding antimalarial immune mechanisms. Longitudinal, dry season to wet season, changes in antimalarial activities were studied in sera isolated from 62 individuals living in an area of hyperendemic but unstable malaria. Highly synchronous cultures of E; falciparum were used to distinguish and quantitate two antimalarial activities, merozoite invasion inhibition and intraerythrocytic parasite retardation. In 54% of the individuals, intraerythrocytic parasite retardation activity increased significantly, nearly 3-fold, in wet season sera as compared to dry season sera. Merozoite invasion inhibition activity was moderate and did not change seasonally. Merozoite invasion inhibition was, however, correlated to parasite-specific IgG titers, and total serum IgG concentration. These results confirm earlier studies which demonstrate two antimalarial activities in Sudanese sera and provides evidence that intraerythrocytic parasite retardation activity plays a role in antimalarial immunity. LIST OF TABLES. . . LIST OF FIGURES . . LITERATURE REVIEW . Introduction . . . Innate resistance. Introduction. . Sickle cell anemia. Glucose-G-phosphate Clinical-epidemiological observations Premunition immunity. . . . . . . . . Characterization of the immune response . de TABLE hydrogenas Thalassemias. . . . . . . . . . . Duffy blood group . . . . . . . . Acquired immunity . . . . . . . . . . . Introduction. . . . . . . . . . . . . Introduction. . . . . . . . . . Passive transfer. . . . . . . . Non-specific response. . . Parasite-specific response In Vitro inhibition . . . . . Immunoprecipitation . . . . . : CEll-mediated immune response ARTICLE: PLASMODIUM FALCIPARUM AbStraCto O O O I I O I I O 0 Introduction. . . . . Materials and Results . . . Discussion. . References. . BIBLIOGRAPHY. . Methods LONGITUDINAL STUDIES ON THE ii OF CONTENTS e O O O O O IMMUNE RESPONSE TO IN SUDAN MEASURED yivrrao. . . Page iii iv DmmflmbébhwwNNt-fi Table LIST OF TABLES Page Immunoglobulin concentrations and antimalarial activites in serum samples collected from the same individuals during dry and wet seasons, 1982. .. . .. . .. . .. . 28 Correlation coefficients between parasite-specific immunoglobulin classes, total serum immunoglobulin concentrations by classes, merozoite invasion inhibition, and inhibition of intraerythrocytic parasite development (crisis-form activity).. . .. . .. . .. . .. . .. . 29 iii Figure LIST OF FIGURES Page Antimalarial activities of paired serum samples collected in dry and wet seasons on Plasmodium falciparum in vitro from 62 individuals. (A) Inhibition of intraeryfhrocytic parasite deveTOpment. crisis-form activity) as determined by the reduction of I thypoxanthine incorporation into synchronized parasites grown in the presence of 25% immune serum as compared to parasites grown in 25% nonimmune sera. (B) Inhibition of merozoite invasion as determined by the reduction of newly formed ring stages from segmenting schizont culture incubated in 25% nonimmune serum. .All data points from each serum sample epresent mean inhibition values from 3 experiments (SJL - 10% inhibition) .. . .. .. .. .. .. .. .. .. . 23 Inhibition of intraerythrocytic parasite develOpment of serum samples longitudinally collected from 19 individuals during the dry season, 1982, wet season, 1982, and dry season, 1983. .All data points from each serum represent mean inhibition values from 3 experiments (5.0.t10%inhibition)................. 26 Distribution pattern of in vitro merozoite invasion inhibition as related to falciparum parasite-specific IgG IJKA. titer. Although correlation coefficient (Table 2; P < 0.001) between 196 I.F.A. titers and antimerozoite activity was positive, the distribution demonstrated that one could not accurately predict the degree of merozoite invasion inhibition as a function of IgG IJKA. titer .. 31 iv LITERATURE REVI EN The Imnune Response to Plasmodium falciparum Introduction Immunity to malaria, which develOps among most individuals living in malarious regions, is a well documented phenomenon. In areas of high or consistent malaria endemicity, where there is little, if any, implemen- tation of control measures, such immunity provides considerable protection from serious malarial disease. Although well documented, the majority of evidence which supports the occurrence and efficaty of this immunity, has been obtained from clinical and epidemiological observa- tions. The Use of clinical parameters as indicators of immunity are inherently limited in specificity and sensitivity, especially'under field conditions, but have been the only available indicators of protection. Characterization of the immune mechanisms responsible for the observed protection has been attempted using a variety of techniques and immunolo- gical markers. Interpretation of experimental findings has been severely complicated due to the nature of investigations involving human subjects, inaccessibility to modern laboratory facilities, and the consequent in- ability to adequately correlate lg 1112 and in vitro observations. Although the mechanisms of protective immunity are not well understood, a great deal is known about the immunological components and parameters associated with extensive exposure to malaria infections. It is the aim of this paper to investigate some aspects of acquired immunity to malaria in individuals indigenous to malarious regions. Natural, or innate immunity, among indigenous of the trapics, although not actively acquired, does exist due to selective pressure exerted by the disease. It is important to emphasize these factors since they require consideration when interpreting epidemiological and inmunol- ogical results. Immune protection acquired in malarious areas is characterized by both age and exposure dependence. It is these depend- encies to which immunological parameters need to be compared, since they are a principle measurement of protection. Parasite-specific and non- specific serum components have been extensively examined and correlated with acquired inmunity. More recently, parasite-specific inhibition by immune serum has been analyzed _i_n_ vitro, has provided much needed in- formation about mechanisms and target antigens. Lastly, cel l—mediated immune responses are reviewed in regard to their correlation with acquired imnunity in light of the recent advances in _i_n vitro culture. Innate resistance Introduction. Under the selective pressure of malarial disease, especially falciparum malaria, some individuals from the tr0pics have devel0ped a natural resistance to severe infection. This natural, or innate, inmunity generally interferes physiologically with parasite de- veTOpment, or host-cell invasion. As obligate intracellular parasites, Plasmdiumgphare dependent upon erythrocytes for continuous asexual reproduction. Accordingly, three abnormal blood diseases have evolved in man under selective pressure from malaria that, while pathogenic to man are even more so to the parasite. These blood diseases are sickle cell anemia, the thalassemias and glucose-6-phosphate dehydrogenase deficiency. Sickle cell anemia. The most wel l-known of these conditions is sickle cell anemia. Sickle cell hemoglobin (Hb) results from the pre- sence of the sickle cell gene S and may be expressed as HbSS (homozygous), which is lethal, or HbAS (heterozygous), which is non- lethal. Heterozygous carriers of the sickle gene are less susceptible to Plasmodium falciparum than individuals without this gene. The correla- tion between HbS and resistance to malaria was first suggested by Allison (I). Mechanisms through which HbS confers resistance were studied by Luzzato _e_t 1L. (45) and finally demonstrated in m by Friedman _e_t _a_l_. (30,31) and Pasvol 3&(68). Since sickling occurs more readily in low oxygen concentrations and when the red cell cytosol becomes acidi- fied, and since the presence of the parasite induces both of these conditions, erythrocytes containing HbS undergo sickling when parasitized killing the parasite, thus conferring a certain innate resistance to sickle-cell carriers (45). Thus, the selective pressure by the parasite on the heterozygote has maintained this gene in the p0pulation deSpite the lethality of homozygosity. Glucose-G-phosphate dehydrgenase. Glucose-G-phosphate dehydro- genase deficiency in erythrocytes is a genetic trait found with relati ve- ly high frequencies in malarious p0pulations. Allison (2) suggested that the Gd" gene was selected through the generations by infections of _P_. falciparum. The most protected by this deficiency are females hetero- zygous for the gene, GdT/Gd', whereas males are either Gd“ or Gd" since the gene is sex-linked (43,46). The mechanism of protection against malaria in the heterozygote is not understood. According to Luzzato _e__t_: 31; (47), parasites which invade deficient erythrocytes _i_n_ vitro are developmentally inhibited and only a fraction are able to complete the asexual cycle. Thalassemias. Similarly, thalassemia syndromes also have inhibitory effects on the intracellular growth of the parasite, presumably due to the retention of fetal hemoglobin or reduced iron, two conditions that do not favor parasite develOpment (67,65). Duffy bloodgroup. In addition to the genetic red cell abnormal- ities described which interfere with parasite devel0pment, a red cell surface component associated with Duffy blood group antigen, apparently determines erythrocytic susceptibility to P_._ M invasion. This was first observed by Boyd 3.12 _a_]_:_ (4) who noted that American blacks were often refractory to P_.flm infections. Further studies demonstrated that Duffy positive individuals were susceptible whereas Duffy-negative individuals were resistant to vivax infections (62). It is not clear how Duffy blood group antigens interact with the parasite; _i_g_ 11331:. merozoites can attach to Duffy-negative cells but cannot subsequently complete the invasion process (61). Such natural resistance to malaria infections needs to be differentiated from acquired imunity during investigations of inmune mechanisms. Despite the potential interference that innate factors could cause in the interpretation of inmune responses, these factors are seldom considered. Acquired inmunity Introduction. Some of the earliest reports of an acquired immunity to malaria were made from malarious regions among indigenous papulations. Daniels (24) observed that native children suffered more acutely from malaria, as measured by fevers, than did native adults. Spleen rates among children were also observed to be much higher than rates among adults (24,38). Further observations on indigenous populations found an age dependent expression of this immunity, where if the children survived they appeared to produce a tolerance to infection despite detectable parasitemia (38). Spleen rates and rates of infection were also found to decrease with age (71,12,48,14) but a predictive pattern of acquired immunity was not observed due to differences in geographic location, parasite species and endemicity (14). Since these early observations, investigators have worked under relatively consistent conditions by taking advantage of the stable malaria, predominately P_._ falciparum, in many regions of Africa. Chloro- quine also was available, as were effective insecticides, which provided a certain amount of control over eXposure and, consequently, unexposed groups from one village could be compared to villages under high rates of exposure. These relatively controlled conditions plus more consistent diagnosis, clinical description and quantitation of parasitemia provided consistency among investigators which lead to the identification of a predictive pattern of acquired immunity. Clinical-epidemiolgical observations. McGregor (51), consolidating the research done in the past with that of his own experience in the Gambia, prOposed that acquired immunity could be divided into successive stages, depending upon age and exposure. Since this work, many investi- gators have confirmed and expanded McGregor's observations. The first stage of immunity is the protection from malarial infections which the mother presumably provides for the develOping fetus. The mechanisms through which the mother confers this protection are not known but the placenta is thought to be a major factor (60). The placenta sequesters a tremendous quantity of parasites (9,32). However, in holoendemic areas transplacental transmission of infection is rare, but becomes more frequent in areas of lower malaria endemicity 922) which suggests an immune response. The second stage of immunity is considered to be the passive transfer of protection from mother to newborn. The evidence for this protection is derived from the many observations that infants, within the first 2-3 months, suffer malaria much less frequently than do the other age groups of children, and when infections do occur parasite- mias are low and symptoms mild (3,9,51,57,37). The nature of such protection is not known, but the period of the protection roughly corre- lates to the duration of maternal antibodies in the infantfs serum (53,10). However, the presence of fetal hemoglobin, inhibitory to para- site deveTOpment, is also considered to play a protective role (67). As the passive immunity declines, susceptibility increases, signaling the 3rd stage of immunity. Beginning at approximately 6 months and lasting through 2 years of age children suffer through the most dangerous period of exposure to malaria infection. Frequency of infection progressively increases, parasitemias rapidly rise and the infections become increas- ingly virulent, peaking at 2 years. Anemia, fever and splenomegaly, all severe and often fatal, are characteristic symptoms of malaria during this period (3,9,54,18). Following the period of greatest morbidity and mortality, by 3 years of age, children begin to demonstrate some degree of resistance. This stage of immunity is characterized by a tolerance to infection, with a reduction in clinical symptoms despite marked parasite- mias. Beginning at 3 years, children usually have the frequency of infection and high parasitemias characteristic of 2 year olds, but ane- mia, fever and mortality are significantly reduced. ‘The protection gradually increases with age and in addition to the maintenance of tolerance, there is a steady decrease in parasitemias, frequency of infection and splenomegaly through ages 5-10 (3,54,51,58,37). Finally, a stable immunity is reached sometime between 15 to 30 years 3,14,54,51,58). By this time, infections when they do occur are usually mild, with low parasitemias, are often subclinical, and self-limiting. Investigations of the parameters of this stable immunity in adults have become the principle means of elucidating the inmune mechanism(s) Operating against the disease. Premunition inmunity. The increasing protection with age and exposure suggests a slowly acquired inmunity. When a stable inmunity is reached in adults the protection is not absolute. Adults infrequently suffer from malaria although the infections are usually mild. Because of the limitation in diagnosis, adults may carry subclinical , or chronic infections, which go undetected. Consequently, the degree of inmunity is difficult to ascertain. Sergent _e_t__a__l_._ (72) were the first to pr0pose that maintenance of imunity to Sporozoa was an imunity to superin- fection, which he termed "premunition" imunity. Epidemiological studies on the duration of this inmunity have provided evidence to support this suggestion. In areas where malaria incidence had been dramatically reduced or virtually eliminated by chemOprOphylaxis and insecticides, followed by a resurgence of disease when control were measures were terminated, malaria returns but at a lower incidence than before control even though infected mosquito inoculation rates return to precontrol values. Based on this criteria, the duration of acquired immunity decreases steadily but has been shown to prevail in the absence of infection possibly for up to 11 years (21,27). However, as it is difficult to determine the degree of production in a papulation, much less an individual, the duration of protection using these criteria is not easily assessed. More predictable observations have been made from immune individuals who have left malarious regions, were radically cured of the disease, and were free of possible reinfection. After only six months abroad, when such individuals return to malarious regions they again become susceptible to malaria infections, which may be severe (49). Because so little data is available on humans, it is not known whether premunition immunity is the result of maintenance of a subclinical infection or repeated exposure and self-cure. Due to the limitations in determining subclinical infections, sterilizing immunity in humans has yet to be determined. Characterization of the inmune response Introduction. The immune mechanisms responsible for protection in humans are not well understood, due, in part, to difficulties in determining and defining protection, and then differentiating the protective from nonprotective immune responses. The determination of protection ultimately requires verification fl 1119 by controlled parasite challenge. Obviously, work of this nature cannot be accomplished routinely, and consequently, protection can only be defined by clinical and epidemiological correlations. In general, the assumption is made by most investigators that every adult from a malarious region is imune. Adults who have recently experienced a malaria episode are often considered to be hyperimmune. Immune responses of these ”protected" individuals were first studied by passive transfer experiments, but most work has been focused on correlating immunological and serological parameters to clinical and epidemiological observations. The emphasis of most of this work has been on humoral immune responses. Evidence that cell-mediated immune responses also participate has not been conclusive due to the difficulty of conducting apprOpriate studies using infected humans. Recently, _i_n_ vitro inhibition techniques have provided a great deal of information concerning antiparasite inmune responses, both humoral and cell-mediated. Passive transfer. The first passive transfer experiments were those of Sotiriades (74), who injected blood from adults who had recently recovered from a malaria episode into an individual with an acute infection. One to two days later the recipient showed signs of recovery, demonstrating reduction in both parasitemia and fever. In later experiments, Lorando and Sotiriadhs (44) controlled for possible cross-infectivity from the blood by adding quinine to the sample prior to injection. Although the quinine concentration received by the injection was not therapeutic, the immune response may have been altered. To investigate which serum component(s) were responsible for passive protection, Cohen _e_t g_1_._ (15) obtained pooled serum from adult Gambians, purified the IgG fraction, and gave several injections of this 196 to children with acute falciparum infections. Parasitemias were reduced significantly, usually within 48 hrs, and some, but not all, recovered without the aid of chemotherapy. Following a similar procedure in Nigeria, Edozian e_t_a_l_._(29) isolated the IgG fraction from umbilical cord blood and adult serum which separately could reduce parasitemia and symptoms in children with acute infections. Using 196 from adults, cross-protection was observed from East to West Africa (55,82) but serum IgG from Malaysia failed to protect Nest Africans (19). Human immune IgG was found to partially protect Agtgs monkeys from acute falciparum infections (26). From these studies it is apparent that 190 can have significant influence upon the course of infection. Since parasitemias decrease the IgG appears to be affecting the erythrocytic cycle (15,19). Further characterization of the inmune mechanisms could not be done using 10 these methods. It was also difficult to assess the degree of protection provided by the 190 since the recipients were at an age where they could already express a certain degree of tolerance. Thus, the partial protection observed was probably the result of both the donated IgG and the children's own immune response. Serology. The evidence that serum, as 196, could provide partial protection by passive transfer led to many investigations involving analysis of serum commnents found among individuals exposed to malaria. Several serological studies have measured both non-parasite-Specific imune components, such as total serum immunoglobulin concentrates, as well as parasite-specific inmune components, including inmunofluorescence titers and imunoprecipitation to parasite antigens. Non-specific response. The non-specific serological studies have demonstrated that total immunoglobulin concentrations among malarious papulations are significantly elevated. Children who have been exposed to malaria have greater concentrations of 196 than children under chemOprophylaxis (54). Among adults it was found that chemoprOphylaxis reduced the elevated IgG concentration (34). Cohen and McGregor (16) found that among African adults in malarious areas 196 synthesis and catabolism were nearly seven times those of European controls. This hypergammaglobulinemia was found in all age groups with detectable parasitemias and 19 concentrations increased directly with spleen size through 15 years of age (54,58). However, only during the first two years were IgM concentrations higher among parasitized children (58). No differences were observed between parasitized and nonparasitized adults (50,58). No consistent relationships could be demonstrated for IgA, IgD or IgE concentrations among any age group (52). 11 Parasite-specific response. Parasite-specific serological studies have shown greater correlations with acquired immunity among p0pulations. Parasite—specific immunoglobulins could be detected by inmunofluorescence in the cord blood and serum of the neonate and were determined to be of maternal origin (77,56). From 2 months to about 1 year, immunofluorescent antibody titers (IJfiAm) decreased, but after this period titers progressively increased until they stabilized among the 20- 30 year olds (28). These changes in IJRA. titers correlated quite closely with the clinical-epidemiological observations of the develOpment of acquired immunity. In young children the I.F.A. titers correlated well to Spleen size and, although uncommon among African adults, Splenomegaly was also correlated to IJZA. titers (33). Little is known about IgM IJfiA. titers among malarious p0pulations but.much like IgM serum concentrations and primary infections, IgM titers are the first to appear and gradually diminish while 190 titers rise*(20). Sustained IgM I.F.A. titers were found to be a distinctive feature of trapical Splenomegaly syndrome (23). Although I.F.A. titers are relatively stable in adults, over several years of malaria control measures 19 titers in .all age groups significantly decreased (21). Other methods for measuring parasite-specific antibody titers, such as the passive agglutination methods using parasite antigens coated on red cells, can be sensitive but have not been useful for epidemiological work (80). Some of the more recent enzyme linked immunosorbent assays (ELISA) have been used successfully and have given similar results to IJKA. titers (78). However, with low antibody titers this method does not appear to be as sensitive as I.F.A. (79). Despite the association between I.F.A. titers and acquired imunity, the relationship is far from absolute and does not 12 predict the degree of protective immunity of an individual, but simply represents the degree of exposure received by a papulation (79). Inmunoprecipitation. Antigens used for I.F.A. determinations are usually infected red cells and as such have limited specificity with regard to protective antigens. A more specific analysis of the antibody response in acquired immunity is achieved by immunOprecipitation of plasmodial antigens. The first such studies were those of McGregor and Wilson (59). Using gel diffusion techniques they demonstrated that, much like I.F.A. titers, the number of precipitated antigens increased with age. When parasite fractions were used they found a heat labile fraction that responded more specifically and was correlated with the epidemiology of acquired immunity. Both the number of individuals with precipitating antibodies and the number of heat labile antigens precipitating increased with age. These antibodies were present in the sera of newborns, their titers decreased during the first year and then increased until approximately 10 years of age when they became stable (59). It was also found that both the number of individuals with precipitating antibodies and the number of precipitated antigens decreased after malaria control measures were introduced (21). A heat stable parasite fraction could be found circulating in the serum of infected individuals which demonstrated a considerable degree of serological diversity, and has recently been used for serotyping Plasmodium isolates (84,83). The importance of this circulating antigen to the maintenance of protection has yet to be confirmed. In vitro inhibition. The serological information, most notably IJBA. titers and immunOprecipitation, are reasonably associated with clinical-epidemiological observations of acquired inmunity. However, these associations are derived from p0pulation studies, and when applied 13 to individuals such associations and their predictive values for protection become diminished (63). In order to analyze the immune response of an individual it is obvious that a great deal of clinical information concerning malaria must be obtained. Even with extensive clinical information there is still uncertainty about how protected the individual may be. Since protection for each individual cannot be realistically determined by challenge _i_n_ _v__i_y_o, _i_n y_i_t_r_g assays for antiparasitic activities have been utilized. Phillips gt 3L (69) first used human immune serum from adult Gambians on cultures of P_._falciparum. Of the 15 sera tested, only two significantly inhibited parasite multiplication. Similar results were later reported by Wilson and Phillips (85). These early studies were conducted before continuous _i_fl vitro cultures of 3:. falciparum were achieved, and thus, the degree of serological induced inhibition could not be reliably assessed. The develOpment of successful culture techniques for continuous in vitro pr0pagation of .P_. falciparum, develOped by Trager and Jensen (76), has provided a much needed tool for analyzing antiparasitic mechanism found in human immune serum. Since then many investigators have used _i_n vitro inhibition of P_._ falciparum to study inmune mechanisms and have attempted to associate this inhibition with acquired immunity. Inhibition of parasite multiplication has been observed by many investigators using adult immune serum obtained from endemic areas. However, not all sera from endemic areas are inhibitory to cultured parasites, and since many studies of inmune mechanisms have been conducted using pooled serum samples, the results may be of questionable value. For example, a positive correlation between 190 I.F.A. titers and in vitro parasite inhibition has been reported by some, 14 but not all investigators (70,35). Brown _e_t _a_l_._ (6) found inhibition of cultures by 190 which also precipitated schizont antigens. Attempts to determine the protective antigens and the parasite strain specificity of those antigens by the inhibitory 196 have not been conclusive (7L Some sera which had no antiparasitic activity in litrg_immun0precipitated many of the same, and in some cases, exactly the same antigens as the inhibitory IgG (8). The mechanism through which serum, or 196, is acting on the parasite appeared to be inhibition of merozoite invasion, but this was not conclusive» Much of the observed variations between 190, in vitro inhibition and immunoprecipitation may be the result of methodology, but this remains to be determined. Jensen gt gj;_(39) used highly synchronous cultures of E; falciparum and demonstrated two different antiparasitic activities in most adult immune serum from Sudan--antimerozoite and intracelltflar growth inhibition. Serum obtained from Indonesia analyzed similarly demonstrated only inhibition of merozoite invasion, but these sera had no intracelllrlar growth inhibition activity (41). Further characterization of these two antiparasitic activities are needed, but the intracellular growth inhibition found in the serum dose not appear to be antibody, yet in the Sudanese sera appears to be associated with clinical immunity (40). 'These authors have suggested that the nonantibody, antiparasitic factor associatedlwith intraerythrocytic parasite inhibition may be a product of cell—mediated immunity (40). Cell-mediated imune response. The studies of cell-mediated iumune responses in man have been severely limited because of the inability to control and manipulate cellular factors i vivo in man. Consequently, most work in this area has not been conclusive when applied to acquired immunity in malaria. There are measurable changes in lymphocytes during 15 malarial infections, but no consistent patterns have been recognized except a general reduction in peripheral T-cells. Changes in B-cells, null cells and K-cells have been noted, but such changes vary unpredictably (86,36,81). Ojo-amaise 3 EL (66) found a positive correlation between parasitemia and both gamma-interferon and natural killer cell activity in malarious children. _I_n .‘LLPLQ inhibition of P_. falciparum has also been used to study peripheral effector cells. Peripheral lymphocytes from inmune individuals significantly inhibited the multiplication of g._ falciparum but only in the presence of malaria antibody, which represents an antibody-dependent cellular cytotoxicity response to malaria (36,5). Polymorphonuclear leukocytes (neutrOphils) and monocytes from noninmunes were found to ingest infected red cells in culture in the present of IgG from individuals living in endemic areas (11). Similar opsonization activity was found by Khusmith and Druilhe (42) using nonimmune monocytes and immune IgG upon the ingestion of merozoites, but not infected cells. The many studies of antiparasitic activities associated with specific antibodies and those using various cellular components of the inmune system suggest that acquired imnunity to malaria is complex, involving both humoral and cellular immune mechanisms. However, the interactions of these various components and their relationship to clinical inmunity is still obscure. LONGITUDINAL STUDIES ON THE IMMUNE RESPONSE TO PLASMODIUM FALCIPARUM IN SUDAN MEASURED I!_VITRO John A. Vande Haa and James B. Jensen Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824-1101 16 17 ABSTRACT Inhibition of Plasmodium falciparum i vitro by human immune serum provides needed information in understanding antimalarial immune mechanisms. Longitudinal, dry season to wet season, changes in antimalarial activities were studied in sera isolated from 62 individuals living in an area of hyperendemic but unstable malaria. Highly synchronous cultures of g; falciparum were used to distinguish and quantitate two antimalarial activities, merozoite invasion inhibition and intraerythrocytic parasite retardation. In 54% of the individuals, intraerythrocytic parasite retardation activity increased significantly, nearly 3-fold, in wet season sera as compared to dry season sera. Merozoite invasion inhibition activity was moderate and did not change seasonally. Merozoite invasion inhibition was, however, correlated to parasite-specific IgG titers, and total serum IgG concentration. These results confirm earlier studies which demonstrate two antimalarial activities in Sudanese sera and provides evidence that intraerythrocytic parasite retardation activity plays a role in antimalarial immunity. 18 INTRODUCTION Acquired immunity to malaria in the human host is well documented but not well understood (20,22). Evidence is available suggesting that both humoral (4,6) and cell-mediated (1,8) inmune responses are essential components of this immunity. The _i_n_ vitro cultivation of Plasmodium falciparum, as described by Trager & Jensen (23) provides a means of analyzing serum antimalarial components and mechanisms of this immunity. Data from i_n_ m inhibition assays substantially improves our understanding of malaria immunology, heretofore based on clinical observations (18). It was previously shown that both purified IgG and a non- imunoglobulin'serum component, found in persons living in malarious endemic areas of Sudan, inhibited P_._ falciparum _i_n_ vitro (13). The latter component has been termed crisis-form factor (C.F.F.), since it retards i ntraerythrocytic parasite development generating typical crisis- forms _i_g vitro (11). Thus, sera from malarious regions in the Sudan demonstrate two distinct antimalarial activities, merozoite invasion inhibition, which has been demonstrated to be antibody dependent (5), and intraerythrocytic parasite retardation, an activity suspected to be mediated through non-antibody mechanisms or components (13). In the present experiments using highly synchronous cultures of £1 falciparum, we have been able to distinguish and specifically quantitate these two antimalarial activities in individual sera. Re have determined the longitudinal changes in these two activities in paired sera obtained during the low transmission dry season and malarious wet season, respectively, from subjects living in an area of unstable hyperendemic 19 falciparum malaria. We have found profound seasonal changes in crisis- form activity but could not demonstrate any changes in merozoite invasion inhibition. MATERIALS AND METHODS Subjects . All 62 individuals studied were adult male volunteers, ages 20-66 years old, residents of the Blue Nile Province, Sudan. In this area, malaria is hyperendemic but unstable. Malaria incidence during the dry season, particularly'in early June, is almost undetectable except in young children where the incidence is approximately 1-5%, as determined by thick films stained with Giemsa. However, the incidence of malaria for the entire p0pulation profoundly increases after the rains begin in June and July, reaching a peak during October and November of over 50% (unpublished observations). The p0pulation in this area is exposed primarily to P; falciparum, with rare infections of P; vivax and E; malariae. Samples were collected from Sennar army camp and the local villages of Sheihk Talha, Um Shoka, Had Hashim and Ismail, all within 80 kilometers of Sennar and situated along the Blue Nile River. £23!!! Paired serum samples were obtained from each subject, the first sample during the end of the dry season, June, 1982, and the second sample following the peak of transmission in october or November, 1982. A third sample was obtained from nineteen subjects during the following dry season, June, 1983. Blood samples were taken by venipuncture in siliconized vacutainers (Becton-Dickenson), allowed to clot, refrigerated overnight at 4°C and serum separated from formed elements by 20 centrifugation. Once separated, the sera were immediately frozen and kept at -20°C until transported to our laboratory at Michigan State University, where they arrived still frozen as described previously (12), and stored at -20°C until analyzed. All paired serum samples were dialyzed concurrently to remove any chloroquine or other drugs and to equilibrate them with the parasite culture medium. Dialysis was conducted at 4°C in 10,000-12,000 MH cutoff membranes (SpectrOpor), first in 0.015 M phosphate-buffered saline, pH 7.2, for two 1:100 dilutions and finally l:IOO in RPMIs 1640 supplemented with 25 mM HEPES and 0.21% sodium bicarbonate, dialysate was changed at 24 h intervals. After dialysis, the paired sera were filter sterilized through 0.45 m pore membranes (Schleicher & Schull) and heat inactivated at 56°C for 30 min before testing for antiparasitic activity. Parasite strain The paired sera were all tested for antiparasitic activity against the FCMSU-l/Sudan strain, isolated in the area of study and transported to Michigan State University (Divo, Vande Naa & Jensen, manuscript in preparation). Stock cultures were maintained in 0+ erythrocytes using the candle jar method (9). Merozoite reinvasion inhibition Parasite cultures, grown to 10-15% parasitemia, were synchronized to 6-8 h age differential using a combination of the sorbitol lysis (15) and gelatin flotation methods (10) as previously described (12). At the time of segmentation and merozoite release, the gelatin-concentrated schizont- infected cells were diluted to 25% parasitemia using freshly washed 0+ erythrocytes and inmediately dispensed into 96-well microtiter plates (Linbro). Each well contained 1.5 pl cells and 100 pl of RPMI 1640 21 containing 10% v/v pooled nonimmune human serum (RP-10) and 25% v/v dialysed test serum. 'The plates were incubated in a candle jar at 37°C for 4 h to allow merozoites to invade the fresh cells. After 4 h, thin films from each well were made and stained with Giemsa to determine the degree of merozoite invasion inhibition by comparing the number of newly invaded ring-stage parasites in the test serum to the number observed in identical cultures eXposed to an equal concentration of dialyzed nonimmune serum. Intraerythrocytic retardation Parasites were presynchronized as described above. Merozoite invasion was allowed to occur for 4 h, and was terminated by lysing all remaining schizonts with 5% aqueous sorbitol , producing a culture of young ring-stage parasites with a 4 h age differential. These ring-stage parasites were diluted to 2-3% parasitemia with fresh 0+ erythrocytes and dispensed into 96-well microtiter plates. Each well contained 2 ul cells, 200 (n 25% immune sera in RP-lO and 2 uCi of [3H]hypoxanthine~(10 Ci/mmole, New England Nucleari. These plates were incubated in a candle jar at 37°C for 40 h, and then harvested onto glass-fiber filters using a Bellco Microharvester. The incorporation of [3H1hypoxanthine into parasite nucleic acids was measured by liquid scintillation spectrometry as described (12). Thin films were also made from wells in which [3H1hypoxanthine was omitted and stained with Giemsa. Parasite retardation was morphologically determined by comparing the extent of parasite development in test serum to develOpment in dialyzed nonimmune serum (11). 22 Serum immunogl obul ins Indirect fluorescent antibody (I.F.A.) titers were determined using traphozoite and schizont stage parasites of the FCMSU-l/Sudan strain according to the methods of Hall et al. (7L. Class-specific, fluorescein conjugated anti-human antibodies (Cappel laboratories) were used to determine specifically the IgG, IgM and IgA titers for each serum. Total serum concentrations of IgG, IgM and IgA were determined by single radial immunodiffusion (R.I.D.) using EndOplate Immunoglobul in Test.Kits (Kallestad LabsL. Ring diameters were measured using the endpoint method described by Mancini, Carbonara & Heremans (1965). RESULTS Intracellular parasite retardation, merozoite invasion inhibition, and parasite-specific and non-specific antibody responses were commred for paired dry season (DS and wet season (NS) serum samples from 62 individuals. Data describing the degree of intracellular parasite retardation and merozoite invasion inhibition in individual inmune sera are presented in Figure 1. The mean difference in parasite develOpmental retardation from dry to wet season, 39% in 05 vs 66.5% NS, was highly significant (P < 0.001). Intracellular retardation profoundly increased from the DS to the HS in nearly 55% of the subjects. In this high responding group, the mean intracellular parasite retardation activity increased nearly 3-fold, from 24.5% to 69.0% inhibition. In 35% of the subjects, intracellular parasite retardation activity did not significantly change (two standard deviations above or below the mean, 5.0. _+_10% inhibition); most of these had elevated inhibitory serum at both sample times. In less than 10% of 23 Fig. 1. Antimalarial activities of paired serum samples collected in dry and wet seasons on Plasmodium falciparum 31 m from 62 individuals. (A) Inhibition of intraerythrocytic parasite development (crisis-form activity) as determined by the reduction of [3H1hypoxanthine incorporation into synchronized parasites grown in the presence of 25% immune serum as compared to parasites grown in 25% nonimmune sera. (8) Inhibition of merozoite .invasion as determined by the reduction of newly formed ring stages from segmenting schizont culture incubated in 25% nonimmune serum. All data points from each serum sample represent mean inhibition values from 3 experiments (5.0. i 10% inhibition). ,. a 3. ye. ,3...A . fl 7,1“? 9} . e... , _,_ B . 25.1.1.2? o 1.1 ..§; .1. \\ DRY SEASON WET DRY moi «w «F 70. so- 0 o 30- 20- w- o- -m- zoEmiz. x -20- 25 the subjects parasite deveTOpment retardation activity decreased from OS to NS. The data shown were obtained in experiments using [3H]hypoxanthine incorporation as a measure of parasite maturation, the retardation of growth by was confirmed morphologically in Giemsa-stained thin films. In contrast to the significant increase in intracellular retardation activity seen in the NS sera, the degree of merozoite invasion inhibition did not change from OS (15.7%) to NS (15.4%). Most sera collected had low levels of merozite invasion inhibition. A third serum sample was obtained from 19 of the same individuals during the following 05, June, 1983. Antiparasite activities of these DH-HS-DS samples were tested together in the same microtiter plate to minimize inter-experiment variability. These data, shown in Figure 2, demonstrated again a highly significant increase in parasite retardation activity that returned to approximately the same degree of inhibition measured in the sera from the previous dry season (21.4% to 66.5% to 23.8%, DS-HS-DS, respectively). A comparison of total mean DS and HS parasite inhibition and antibody concentrations measured in these sera is shown in Table 1. Only intracellular parasite retardation changed significantly from the DS to NS (P < 0.001). To investigate the relationships between merozoite invasion inhibition, intracellular parasite retardation and humoral inmune factors, correlation coefficients were determined comparing each parameter among individuals. These data are summarized in Table 2. Intracellular parasite retardation was not significantly correlated with merozoite invasion inhibition or humoral immune factors, with the exception of a weak correlation between intracellular parasite retardation and I.F.A. titers. As expected, there was a significant (P < 26 Fig. 2. Inhibition of intraerythrocytic parasite development of serum samples longitudinally collected from 19 individuals during the dry season, 1982, wet season, 1982, and dry season, 1983. All data points from each serum represent mean inhibition values from 3 experiments (S.D. 1 10% inhibition). 28 .o.~.¢ an umcvscouuo me meo'uocucoucou :.p=aopuo==sl. sacom mcmu_a ucponvcm peooca.uo¢o mo.o x a .beeo_c.ea_a be: u .m.z mucou_~pcm—m mo upo>op o=_ELouou a» vow: anon-» m.ucou=umw a ”Hoo.o v my o.mm i “.ma m.mo No c.5m . H.¢~i o.mm we .a. covu'a.gc. oceanopo>oc ou—mucen upaxuocgaxyoocucu mz ~.~m i m.~Hi «.ma No o.Hm t H.oi s.m~ me an. cowapnpgcp covme>=_ oppouocoz ma mom i mm mm~ No man i an “mo we aapv\ue. ¢o .Nmmfi .mcomoom no: one age mcwcav m—o=c¢>pv=_ mama osu seem touuoppou morasem Eacom :_ mo—up>vpom pewcmposruce can mcovuocucoucou cvpzncpaocassu .~ upon» 29 .bmoe-e a no.9 v no acovsumv doo.o v a: .»u*>_uoe canon» scam mwmvcu. :o—urawgc— acosaopo>mu oupmocom uwuauocguxgoocucmn au—>wuua =o.».a_;=_ ccwmo>=. ou.o~cgo:n eo.c - - - - - - - ee_b.e.ee. nou_c~ocoz so.o e3.o - - - - - - ..o\ee. (a3 mo.e ae.e ec.o- - - - - - .Pe\ae. zen mo.o e3~.o emm.o eo.o- - - - - ..e\ae. we“ mo.o ~o.o mo.e- o~.o- ee.c - - - .<.a.~ «no o3.o- m~.o m3.o «me.o e~e.o No.o - - .<.a._ an“ nm~.o «me.o so.o o~.o «am.o mo.o eme.o - .<.a.~ woo sa.>_boe eo.»_e.ee_ pose, peso, peso. .<.e._ .<.a.~ .<.e.~ e293 corset: <3 :3 e3 .3 :3 S3 .eau*>—uuo scomtm—m—cu; peasaopo>ou mapmocon u.uxuocguxuoecucv mo copu_n_=:. can .:e_a_avgc_ ccwme>cp oupouocoe .mommopu.aa.mcowuecucoucou cwpancpuocanep sacom pope» .mommepu c—panopuo==EI* uvmvuoamiou_mocen coozuon mu=o_u_wmoou co.uepogcou .~ opaoh 30 0.001) correlation between serum IgG I.F.A. titers and merozoite invasion inhibition. However, when these data are presented graphically (Figure 3), it is clear that the large individual variation prevents the use of IgG I.F.A. titers against falciparum schizonts as a reliable index of merozoite inhibition. Merozoite invasion inhibition was also correlated with total serum 190 levels, though not as strongly. Statistically significant relationships also existed between 196 I.F.A. titers and IgM I.F.A. titers (0.43), 190 I.F.A. titers and total serum IgG concentration (0.31), and between total serum IgG concentration and total serum IgA concentration (0.35). In addition, significant correlations existed, between IgM I.F.A. titers and total serum IgG concentration (0.62), and between IgM I.F.A. titers and total serimi IgM concentration (0.40). To assess our papulations exposure to other parasitoses we surveyed the villages for urinary and intestinal protozoa and helminths. The actual incidence of these parasitoses were not determined at the time of our survey, however, the relative frequencies of each parasite could be determined, primarily among the children. The commrative frequencies of the intestinal protozoa were Entamoeba coli > Giardia lamblia > Entamoeba histolytica > Chilomastix mesnili > Endolimax nana. The comparative frequencies of urinary or intestinal helminths found were Schistosoma haematobium > Enterobius vermicularis > Hymenolepis nana > Taenia saginata > _S_._ mansoniirare). The frequencies of all these parasites varied extensively from village to village, especially S_. haematobium where in one village over 85% of the children were passing S_. haematobium eggs while in another only 5% of 200 children were infected. No association could be made between these parasitoses and the seasonal antimalarial immune responses of the individuals in our study. Furthermore the soldiers who contributed more than 25% of the paired 31 Fig. 3. Distribution pattern of in M merozoite invasion inhibition as related to falciparum parasite-specific IgG IJfiA. titer. Although correlation coefficient (Table 2; P < 0.001) between IgG I.F.A. titers and antimerozoite activity was positive, the distribution demonstrated that one could not accurately predict the degree of merozoite invasion inhibition as a function of IgG I.F.A. titer. 000000 000 @000 l 5' 20 T T (280 2560 000 o a» oomoooo o —8 to O o a comp mo 0 —% 0 cm ooooocn o (I) 0 m3 000000 @000 o #8 o o oo o o “'8 o oo o #8 T I l T I I T O O O O O 0 O n V n N - T NOLUBIHNI % (FA TITER 33 serum samples examined were essentially free of parasites, except for occasional g; lamblia. Nonetheless, these individuals had as much malaria as did the villagers and their sera varied in antiplasmodium activity from OS to NS to the same extent as sera from villagers with significant prevalent parasitoses. DISCUSSION Re have found that sera from Sudanese adults contained both merozoite invasion inhibition and intracellular parasite retardation activities. From dry season to wet season, intracellular parasite retardation activity increased significantly (P < 0.001), whereas merozoite invasion inhibition did not change. During the following dry season, 1983, intracellular parasite retardation activity, hereafter called crisis-form activity, returned to levels corresponding to the previous dry season, 1982. This seasonal rise and fall in serum concentration of crisis-form factor was apparently due to exposure to falciparum malaria, because in our parasitological surveys of this population, we did not find any other parasite or infectious agent the transmission of which fluctuated with changes in rainfall. Although there were differences in the relative frequencies of other parasite infections from village to village, there were no differences in antimalarial activity between villages. Furthermore, preceding the rainy season, 1983, the study area was extensively sprayed to control mosquito populations and the crisis-form activity in sera collected during October and November 1983, remained uniformly low (unpublished observations). Some of the sera did not change seasonally, but maintained reasonably high concentrations of crisis-form factor in dry and wet 34 seasons. He do not yet know the actual kinetics or serum half-l ife of this activity, so we can only speculate as to how long C.F.F. remains active in the serum. These individuals could possibly have been exposed to malaria during the dry season or alternatively could harbor subclinical malaria infections, but the latter possibility is less likely in this population since splenomegaly is rare and IgG I.F.A. titers uniformly low (14). Sera from a few individuals decreased in crisis-form activity during the wet season, which could be explained by a malaria infection that occurred during the dry season but not in the wet season. In this p0pulation crisis-form activity was correlated with 190 I.F.A. titers. However, since schizont-specific I.F.A. titers are more of an indicator of the degree of exposure, or recent exposure to falciparum infections (17) rather than a reliable index of protection (13), we postulate that the correlation between 190 I.F.A. titers and crisis-form activity results from parasite exposure which induced the production of crisis-form factor. The seasonal, and apparently parasite-specific, induction of crisis- form activity indicates that C.F.F. may be an integral part of the inmune response to malaria in the individuals studied. The ability to produce or maintain high concentrations of C.F.F. and the resultant suppression of malaria infections requires further characterization. However, Jensen et al. 913) have recently demonstrated an association between serum crisis-form activity and clinical inmunity. Although our data suggest a parasite-specific dependence of the production of C.F.F., the triggering or induction mechanisms are not known. Nonetheless, the degree of malaria exposure that these pe0ple experience, primarily during the wet season, is sufficient to induce highly inhibitory concentration of C.F.F. when necessary. Since crisis-form activity was correlated to 190 I.F.A. 35 titers, which in turn reflects the degree of exposure to malaria infections, crisis-form activity would predictably be greater in holoendemic areas of Africa. The source of crisis-form factor is not know. However, some investigators working with animal models have suggested that crisis-forms result from the action of mononuclear cell secretions, i.e. monokines, lymphokines, lymphotoxins or tumor necrosis factor (2,3,21,24), and that these factors may be T-cell regulated (1,8). In this p0pulation most of the sera only moderately inhibited merozoite invasion, with few individuals inhibiting invasion 40% or more. These results are similar to those reported previously where only 2 of 12 sera collected from holoendemic southern Sudan had appreciable antimerozoite activity (13). Furthermore, they support the observations of Philips et al. (19) who found only 2 of 15 Gambian sera inhibitory to short-term cultures of _P; falciparum. However, it was the impression of these researchers that the inhibition may have been directed against the late deveTOpmental stages of the parasite. They reported that in the presence of the inhibitory sera, the segmenters appeared abnormal, some were lysed, and there was a measurable reduction in the incorporation of radiolabeled precursors into parasite macromolecules. Although these observations were made before the deveTOpment of techniques for continuous cultivation of P_._ falciparum, they parallel our own experience with sera collected in different regions of Sudan. In contrast to the situation in Sudan, many sera obtained from an Indonesian papulation inhibited merozoite invasion over 80%, but these sera had 8 to 15 times more parasite-specific antibody and no CJRF. activity (14). Our assay for antimerozoite activity is based on assessing new ring formation and does not determine if merozoites allowed 36 to invade in the presence of antibody are subsequently damaged. If damaged, the inhibition of the parasite by this mechanism would obviously be enhanced. Furthermore, this p0pulation generally experiences P; falciparum infections only during the wet season and the degree of such exposure is reflected in the relatively low 190 IJKA. titers and, subsequently, low merozoite invasion inhibition activity. Presumably, greater antigenic stimulation in areas of higher malaria incidence would produce higher parasite specific IgG IJRA. titers and merozoite invasion inhibition. Our finding that merozoite invasion inhibition did not change with seasonal parasite exposure was somewhat unexpected, especially since it was measured against a parasite strain isolated from one of the villages included in our study. As a primary antiparasitic defense, antimerozoite antibody titers would be expected to rise anamnestically to the exposure from an infection during the wet season, but such was not observed. The triggering of antibody production by antigenic stimulation, in this case antibodies responsible for merozoite invasion inhibition, is well understood. However, there are many factors which can suppress an antibody response during a malaria infection (22). The rapid rise in crisis-form activity may be a key factor in suppressing the increase of antimerozoite antibody production by rapidly clearing the infection and reducing antigenic stimulation. Merozoite invasion inhibition was correlated with 190 IJKA. titers, as would be expected from an antibody-mediated activity. However, the use of whole traphozoites and schizonts as antigens in the I.F.A. assay limits the specificity of this test which does not distinguish inhibitory from noninhibitory antibodies. Such lack of specificity was demonstrated by several sera with high 196 IJhA. titers that did not 37 appreciably inhibit merozoite invasion. Clearly, a purer antigen preparation would be required to improve the predictive value of IgG IJZA. titers for merozoite invasion inhibition. It is also important to note that there was no correlation between merozoite inhibition and CJLF. activity. This lack of correlation supports the validity of our assay system using whole sera, in that a 4 h incubation period apparently is sufficient to allow for reinvasion inhibition without interference with intraerythrocytic parasite develOpment. There was a significant correlation between merozoite invasion inhibition and total serum IgG concentration. Hypergammaglobulinemia, to varying degrees, is a common characteristic of peOple living in malarious regions (18) and the papulation examined in this study was no exception. These moderately elevated serum IgG concentrations were also correlated with parasite-specific IgG I.F.A. titers and this in turn was probably responsible for the merozoite invasion inhibition observed. No correlation was found between parasite-specific IgM and IgA IJRA. titers and merozoite invasion inhibition. The reliability of this data is, however, limited since there were so few individuals who demonstrated 19M or IgA I.F.A. titers, all of which were comparatively low. A larger, or younger, p0pulation with a greater range of 19M or IgA IJLA. titers may have provided more definitive results. Interestingly, the one individual who had the highest antimerizoite activity not only had a high 190 IJRA. titer, as expected, but also had a uniquely high IgM I.F.A. titer. Whether this was due to both immunoglobul in classes remains to be determined. Other statistically significant correlations existed between the class-specific I.F.A. titers and total serum immunoglobulin concentrations, but their relationship, if any, to parasite inhibitory activities are unclear. 38 In summary, the combined action of two antimalarial activities, merozoite invasion inhibition and crisis-form activity, could provide a substantial inhibitory effect on parasite growth and multiplication. Our data strongly support the hypothesis that production of crisis-form factor is positively correlated with exposure to falciparum malaria and thus, may play a significant role in the acquired immunity to malaria in Sudan. 39 ACKNOWLEDGEMENTS The authors gratefully acknowledge Alan A. Divo for his assistance in the field and the staff of The Malaria Training Center for their assistance and logistical support. This research was sponsored by NIH, grant no. AI -16312, under the au5pices of Michigan State University - Ministry of Health "Collaborative Research on Parasitic Diseases in Sudan" project and contract AID/DSPE-C-OO67 of the U.S. Agency for International Development. This paper is listed as article #11165 of the Mighican Agricultural Experiment Station. 10. 40 REFERENCES Allison A.C. 8 Eugui E.M. 1983. The role of cel l-mediated immune responses in resistance to malaria, with special reference to oxidant stress. Ann. Rev. of Inmunal. 1:361-392. 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