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Is Immunity to malaria really short-lived?
Parasitolo~ Today, vol. 8, no. I I, 1992
375
Is Immunity to Malaria Really Short-lived? P. Deloron and C. Chougnet Protection against Plasmodium falciparum malaria is usually considered to be the cumulative product of repeated exposure to parasites, and thus a function of age, in endemic areas. The recent outbreak of malaria in the central highlands of Madagascar gave Philippe. Deloron and Claire Chougnet the opportunity to compare the incidence of malaria in children and young adults exposed to malaria for the first time, with that in older adults who spent their childhood in the study a r e a before malaria control was introduced Protection, as well as immune responses to two major P. falciparum antigens, was not related to age. Individuals older than 40 years were more protected than were younger adults. This increased protection was probably due to immunological memory.
Malaria remains one of the most important diseases in tropical areas. Children, especially those under five years of age, are usually the main target of morbidity and mortality. In endemic areas, repeated infections result in immune responses that do not eliminate parasites but that decrease the clinical impact of the disease. Numerous epidemiological studies conducted in areas of stable malaria have demonstrated an age-related increase in malaria-specific immune responses, as well as an agerelated decrease in malaria-related morbidity. It is generally held that protection against P. falciparum malaria is the result of repeated exposures to parasites over a period of many years and that its persistence requires regular contact with the parasite. However, this model has been documented poorly. Where malaria transmission is unstable, and especially during epidemics, a different situation may occur. Such a situation developed recently in the central highlands of Madagascar, where an epidemic of falciparum malaria started in the mid-eighties in an area where falciparum malaria had been absent for almost three decades. Thus, in this community, children and young adults, who were all naive, were exposed suddenly to malaria-infected mosquitoes. Adults older ~:han40 years, who had spent their chilclhood in this area before malaria control, had probably had many previous infections and might have retained some degree of ~) 1992, ElsevierSciencePublishers Ltd, (UK)
immunological memory. The aim of this paper is to relate the immunological alterations that occurred in the population of this area after the 1987 outbreak with the development of clinical protection against the disease. We also highlight the differences between the immune response of such a population and that of the inhabitants of areas of highly endemic and stable malaria. Before 1950, the central highland plateau of Madagascar was a malariahyperendemic area (Box I). As a result of control activities during the 1950s and 1960s, malaria transmission decreased dramatically and the highland plateau was declared a 'surveillance area' in 1960. Unfortunately, control activities decreased subsequently. The time of the reappearance of falciparum malaria in the central highlands of Madagascar is not known accurately but was dramatized by a major outbreak in late 1987 (Ref. I). However, chart reviews showed an increase of malaria cases in the paediatric wards of Antananarivo from 1985 (Ref. 2). Beginning in October 1987, we have conducted a series of investigations in the population of Manarintsoa, a village of 1400 inhabitants, located 20 km west of Antananarivo, the capital city of Madagascar. Three surveys were conducted during the period 1987-1989 during which malaria morbidity was followed among this population. The first study was conducted for six months during the 1988 transmission season. It consisted of passive case detection of malaria attacks among the outpatients presenting to the Manarintsoa health centre 3. Subsequently, 76 individuals were followed weekly from January to June 1988 (Refs 4-6). Finally, a second cohort study which involved 27I inhabitants, who were followed monthly during the next transmission season, was undertaken from December 1988 to June 1989 (P. Deloron and C. Chougnet, unpublished). During these two studies, subjects who presented with one or more malaria attacks during the follow-up were considered as non-protected; the others were considered as protected. Malaria attacks were defined either as the presence of parasites in blood, with associated fever (axillary temperature
~37.5°C), abdominal pain, diarrhoea, vomiting or headache, or as high parasite density (~5000 per I~1 of blood in the first study; ~1500 per I~1 in the second). Data from these three studies were used to assess the relationship between age and morbidity and between age and the evolution of immune responses to defined P. falciparum antigens. Protection in the O v e r 40s
In the survey of 2776 outpatients of the Manarintsoa health centre, the parasite rate averaged 63.2% and did not vary with age (between 51.0% and 68.8% in all age groups). However, among patients infected with P. falciparum, those between two and 39 years old were more likely to have fever (indicating an acute malaria attack) than those older than 40 (52.6% versus 29.5%; P ~104). Similarly, the lowest spleen rate was observed in this age group (3.9% versus 51.1%; P ,~104 ) (Ref. 3). These data were derived from passive case detection, thus parasite rates may not relate accurately to the prevalence in the population as a whole because the proportion of symptomatic individuals who report to the clinic may vary with age. However, this is unlikely to interfere with the fever rate in patients of the health centre who are infected with P. falciparum. These findings are in contrast with the fact that individuals of all ages were similarly exposed to mosquito bites, as described later in this paper. The fact that individuals older than 40 years responded differently than those in other age groups is supported by the two cohort studies. In the 1988 study, a total of 148 malaria attacks were identified in 61 of the 76 individuals of the cohort; 85% of these malaria attacks had symptomatic parasitaemia. Villagers older than 40 years were more likely to be protected against falciparum malaria attacks than were younger individuals (42.9% versus 14.5%, P ~0.005) (Ref. 4). Similar results were obtained in the 1989 study dealing with a greater number (271) of inhabitants of Manarintsoa, 65.3% being considered as non-protected and 34.7% as protected. The proportion of protected
Parasitology Today, vol. 8, no. I I, 1992
376
Box I. Historical Aspects of M a l a r i a in the C e n t r a l Highlands of Madagascar Since the beginning of the century, Madagascar has been known to be hyperendemic for falciparum malaria. Antimalarial control activities were initiated in 1949. In the central highlands, with a tropical mountain climate, insecticide spraying was highly effective, leading to the disappearance of one of the two malaria vectors (Anophelesfunestus)and to a dramatic reduction in the population of the ocher (An. gambiae). Malaria decreased in parallel and was hypoendemic ten years later. To consolidate malaria eradication, mass chloroquine administration was initiated in 1959. A further reduction of malaria transmission was obtained, but a few loci remained. In the mid 1960s, control activities gradually decreased due to financial and administrative difficulties. In the period 1970 to 1985, entomological and parasitological data are almost lacking, but malaria was not reported to be actively transmitted. Following the 1987 outbreak, falciparum malaria was hyperendemic, although the level of transmission was very low with fewer than two infective bites per person per year. After chloroquine administration and insecticide spraying, transmission decreased and no mosquitoes were found to be infected in 1991; the area is now hypoendemic for malaria.
Mass Drug Administration 1987 Outbreak
Vector Control
Hyperendemy
1950
1960
1970
1980
1990
I
I
I
I
I
Mesoendemy
50-75%
8%
Preeradication
Hyper endemy
0.9%
33% 80% Spleen rates
individuals differed significantly between age groups, reaching 61.1% in individuals older than 40 years (Fig. I ). This difference in proportion remained significant when the group of individuals less than 40 years was split in two, three or six age groups (all P <0.01). However, when individuals older than 40 years were omitted from the calculations, no differences between age groups were detected (all P >0.05). Antimalarial drug consumption (as studied by urine tests) and mosquito exposure did not appear to vary with either age or location of the house inside the village. Entomological surveys failed to identify geographical variations of Anopheles sporozoite rates inside the village. In addition, biting peak by both An. gambiae and An. funestus occurred between 22.00 and 3.00h, suggesting that no environmental factor modified exposure to mosquitoes s. Humoral
and Cellular Responses
Humoral and cellular responses of the subjects enrolled in the 1988 cohort to synthetic peptides reproducing dominant epitopes of two major P. falciparum antigens [Pf155/RESA and
circumsporozoite (CS) protein] were investigated at the time of enrolment, at the beginning of the rainy season6'7. Antibodies to the CS protein repeat were found in 23% of individuals. Titres of these antibodies were rather low. The mean antibody titre was higher in the oldest (>40 years) individuals (P <0.02) but did not differ significantly among other age groups (OD = 0.17, 0. I 8, 0.09 and 0.32 in the 9-19, 20-29, 30-39 and I>40 years age groups, respectively), demonstrating that all age groups were exposed to infected mosquito bites. The higher anti-CS protein antibody titre in subjects older than 40 years may reflect a secondary response in those with immunological memory. The overall antibody prevalence rate was lower than that in adult populations living in areas of stable and intense malaria transmission, such as much of tropical Africa 8-1J , where such antibodies are found in more than 70% of the adult population. However, the antibody prevalence rate in our study subjects is similar to the rate observed in American citizens (27%) who have stayed for one to two years in tropical Africa 12. Antibodies to Pf155/RESA were observed in 35%-64% of our subjects (according to the peptide), a
prevalence rate that is similar to those observed in other African populations ~°'t~. However, the antibody titre did not vary with age, contrasting with the results of numerous studies conducted in areas of stable malaria. In such areas an age-related increase of the humoral response to both Pf155/ RESA ~°'~ and CS protein 8'9, as well as to several other P. falciparum antigens ~3 has been reported consistently. Cellular responses were assessed by lymphocyte proliferation. The percentage of cellular responders to CS protein and to Pf155/RESA peptides ranged from 29-35%. Mean stimulation indexes were low (mean stimulation indexes between three and six with any peptide) 6'7. The percentage of cellular responders to Pf155/RESA peptides was not lower in this area of Madagascar than in areas of high and stable malaria transmission, such as Liberia j4 or the Gambia Is'l 6. Levels of responses were also similar in the three areas. The levels of proliferative responses to the central repeat of Pf155/RESA varied among age groups, being highest in the oldest. However, no trend of an agerelated variation was observed, and these levels did not significantly differ before 40 years of age. Similarly, no
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Parasitology Today, vol. 8, no. I I, 1992
age-related increase of cellular responses to Pf155/RESA was reported in the studies from Liberia and the Gambia.
a 60
Does Protection Reflect C u m u l a t i v e Exposure to the Parasite?
4O ,,i,,a
All the studies we conducted in the central highlands of Madagascar showed that individuals older than 40 years were more protectecl against clinical falciparum malaria, but not against infection, than were those younger, despite being submitted to similar exposure to infective mosquito bites. In addition, they had higher humoral and cellular responses to some major epitopes of two parasite antigens. Individuals over 40 years spent their childhood in a malaria hyperendemic area and probably experienced numerous infections over a period of several years. Conversely, younger subjects were almost, if not totally, naive before the outbreak. Although an agedependent immunity not related to parasite exposure might eventually occur 17, the difference in response between subjects older and younger than 40 is likely to be due to a difference in past exposui-e to malaria parasites. Conclusive evidence would have required the study of a control group covering the same age range, exposed to the same present malaria epidemic but without having been exposed to the previous period of transmission. Such a control group did not exist in Madagascar. The persistence of some level of protection after almost 30 years is surprising as malaria immunity is thought to wane within one or two years after the end of exposure to the parasite. Although the lack of immunological memory has been demonstrated for transmission-blocking immunity '8, it has been only poorly documented in the case of clinical protection. West African immigrants to France are susceptible to clinical malaria when they return to their country after several years. However, the case fatality rate has never been estimated and they rarely have lethal cerebral malaria. P. falciparum malaria attack is less severe in such individuals than in naive individuals. Similarly, fever and parasite clearance times are shorter than in non-immune individuals ~9. These migrants, in common with the inhabitants of Madagascar, may still harbour immune memory cells that were primed many years before. Moreover, it is conceivable that anti-malarial
0
20
0 9-19
b
20-29
30-39
40+
8O
60
40
20
0 1-10
11-19
20-29
30-39
40+
Age (years) Fig. I. The age distribution of protection against Plasmodium falciparum malaria in two cohorts of inhabitants of Manarintsoa, in the central highlands of Madagascar, followed during the 1988 (a) (n = 76), and 1989 (b) (n = 271) malaria transmission seasons. Followup was conducted weekly in 1988 and monthly in 1989. Protection was defined by the absence of any episode of malaria attack during the follow-up. The number of individuals followed in each age group is indicated above the bars.
immunity was sustained by infection with organisms possessing antigens crossreacting with malaria antigens2°. A new parasite exposure will allow these cells to expand and initiate a secondary immune response. In this respect, antibodies to Pf155/RESA are able to persist for more than six years after the cessation of antigen exposure (A.J. Sulzer, P. Deloron and P. Millet, unpublished). Similarly, humoral and cellular responses to both CS protein and Pf155/RESA may persist in migrants from West Africa after they have spent up to 13 years in France without returning to their native area2~. A number of epidemiological studies in endemic areas have shown protection to be a function of age. This fits with the model usually adopted for the acquisition of protection, which is believed to result from the cumulative
effects of repeated exposure to parasites. The effects of age may occur independently of exposure, but age and exposure are synonymous in populations spending their entire life in a malaria-endemic area. Only very peculiar epidemiological situations, such as the one that had happened in the central highland of Madagascar, allow the effect of age to be studied independently of exposure as well as duration of memory mechanism of malaria immunity. The recent disappearance of falciparum malaria transmission in the central plateau of Madagascar since 1991 provides a unique opportunity for such studies. Acknowledgements
We thank the population of Manarintsoa for participating in these successivestudies. We also thank all the people who took part
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Parasitology Today, vol. 8, no. I I, 1992
in the studies reported here, in particular Marie Danielle Andriamangatiana-Rason, Pierre Coulanges, Didier Fontenille, Jean Paul Lepers and Jean Aime Ramanamirija from the Madagascar Pasteur Institute, and Pascal Astagneau, B6atrice Dubois and Florence Migot from INSERM U I3. This work was supported in part by grants from AUPELFAJREF and from the Tropical Pathology Society. References
I Lepers, J.P. et al. (1988) Lancet i, 586 2 Razanamparany, M. et al. (1989) Arch. Inst. Pasteur Madagascar 56, 91-96 3 Lepers, J.P. et al. (1990) Bull. WHO 68, 217-222 4 Astagneau, P. et al. ( 1991 ) Am, J. E_pidemiol, 133, 177-184 5 Fontenille, D, et al, (1990) Am. J. Trap. Med.
Hyg. 43, 107-115 6 Chougnet, C. et aL (1990) Immunol. Lett. 25, 231-235 7 Chougnet, C. et aL (1990) Am, J. Trap. Med, Hyg. 43, 234-242 8 Del Giudice, G, et al. (1987) Am, J. Trap. Med. Hyg, 36, 203-212 9 Esposito, F. et al. (1988) Trans. R, Sac, Trap. Med. Hyg. 82, 827-832 I 0 Deloron, P, et al. (1989) Am. J. Trap. Med. Hyg. 41,395-399 II Deloron, P, and Cot, M, (1989) Trans. R. Sac. Trap, Meal Hyg. 84, 191-195 12 Miller, K.D, et al. (1988) J. Infect, Dis. 158, 868-87 I 13 Marsh, K. et al. (1989) Trans. R. S0c. Trap. Med. Hyg. 83, 293-303 14 Petersen, E. et oL (1989) Am. J. Trap. Med, Hya~. 4 I, 386-394 15 Troye-Blomberg, M, et al. (1989)J. ImmunoL 143, 3043-3048
16 Riley, E.M. et al. (1991) Trans, R. Sac, Trap. Med. Hyg. 85, 436-443 17 Baird, J.K. et al. (1991 ) Am, J, Trap. Med. Hyg. 45, 65-76 18 Ranawaka, M.B.R, et al. (1988)Infect. Immun. 56, 1820-1824 19 Charmot, G, et al. (1990) in Chimioth~rapie des parasitoses sev~res (Vachon, F. et al., eds), pp 29-38, Arnette 20 McLaughlin, G.L., Benedik, M.J. and Campbell, G.H. (1987) Am. J. Trap. Med. Hya~. 37, 258-262 21 Chougnet, C., Deloron, P. and Save[, J, (I 991 ) Ann. Trap. Med. Parasitol, 85, 357-363
Philippe Deloron and Claire Chougnet are at INSERM UI3, Institut de M6decine et d'Epid~miologie Africaines, 75944 Paris Cedex 19, France.
A Superfamily of Trypanosomacruz/ Surface Antigens O. Campetella, D. S nchez, J.J.Cazzulo and A.C.C. Frasch Several genes of Trypanosoma cruzi encode surface antigens that include a n amino acid motif that is conserved among bacterial neuraminidases. Oscar Campetella, Daniel Sdnchez, Juan Jos~ Cazzulo and Alberto Carlos Frasch here suggest grouping these gene families in a superfamily. Recent results from several laboratories have started to clarify the relationship between the surface antigens of Trypanosoma cruzi, the agent of American trypanosomiasis, Chagas disease. The information obtained from the cloning and sequencing of genes for T. cruzi antigens has shown that they share one major characteristic: the presence of tandemly repeated amino acid motifs, that are the targets of the immune response during natural and experimental infections (see Ref. I for review). Data from the sequencing of the region 5' to the repeated motifs of some of these genes, as well as other related genes lacking repeated units, have added important information. Several genes encoding surface antigens were found to have significant sequence homologies, including up to four copies of partially or completely conserved sequences encoding the motif Ser-XAsp-X-Gly-X-Thr-Trp. This motif is conserved among bacterial neuraminidases2 and its presence in members of a superfamily of surface antigens might suggest that they originated in an ancestral (neuraminidase?) gene.
Families and Superfamilies
The first gene encoding a T. cruzi antigen, named TSA-I, was cloned in 1986 (Ref. 3). Its partial sequence showed a repeated nonapeptide sequence motif. Some years later, the whole sequence of this gene was obtained 4. The deduced amino acid sequence on the amino side of the repeat turned out to be highly homologous, although not identical, to that of two other sequences obtained independently by Kahn and co-workers (SA85-1, Ref. 5) and Takle and Cross (Tt34cl, Ref. 6). Unlike with TSA-I, these two genes lack a repetitive amino acid motif. All three have copies of the bacterial neuraminidase motif 2. One of these proteins, SA85-1, has been considered to be the neuraminidase of T. cruzi s, but this seems now not to be the case (see below). All these genes are highly homologous, and thus comprise a gene family (the gp85 family, Refs 5,6) expressed in the trypomastigote stage. The estimated molecular weight of these proteins is around 85kDa and they have, at least before processing, carboxy terminal extensions that are compatible with the post-translational addition of glycosylphosphatidilinositol (GPI) anchors6. Other genes encoding proteins of 120-200kDa that also contain the conserved neuraminidase motifs have been identified 7-9, These proteins are
also expressed in the trypomastigote stage, and are anchored on the parasite surface by GPI, allowing their easy shedding into the medium 7'r°. One of these genes has been isolated and sequenced by our group while characterizing a major shed acute phase antigen (SAPA) 7.1°. Pereira and coworkers have cloned and sequenced the gene encoding the enzyme neuraminidase8 and have found that it contains the same amino acid repeats as are present in SAPA ~° and the conserved motifs that are present in bacterial neuraminidases2'8. In addition, Pereira's neuraminidase and SAPA are more than 80% identical in the non-repeated sequences, thus showing that they belong to the same gene family. Members of the SAPA protein family have been shown to have not only most of the neuraminidase activity of the parasite, but also most of the trans-sialidase activity of the parasite 9. Trans-sialidase is an enzyme unique to T. cruzi. At variance with all other sialyl-transferases described in eukaryotes that use cytidine monophosphate-sialic acid as donor, trans-sialidase uses sialylated substrates other than the nucleotide derivative, like fetuin, as donor molecules 9'11-j3 Recently, Nussenzweig and co-workers have demonstrated that neuraminidase and trans-sialidase activities are in the same molecule 14. Members of this gene family are characterized by a 5' domain probably encoding the region of the protein with enzymatic activity and a (~ 1992,ElsevierSciencePublishersLtd,(UK)
375
Is Immunity to Malaria Really Short-lived? P. Deloron and C. Chougnet Protection against Plasmodium falciparum malaria is usually considered to be the cumulative product of repeated exposure to parasites, and thus a function of age, in endemic areas. The recent outbreak of malaria in the central highlands of Madagascar gave Philippe. Deloron and Claire Chougnet the opportunity to compare the incidence of malaria in children and young adults exposed to malaria for the first time, with that in older adults who spent their childhood in the study a r e a before malaria control was introduced Protection, as well as immune responses to two major P. falciparum antigens, was not related to age. Individuals older than 40 years were more protected than were younger adults. This increased protection was probably due to immunological memory.
Malaria remains one of the most important diseases in tropical areas. Children, especially those under five years of age, are usually the main target of morbidity and mortality. In endemic areas, repeated infections result in immune responses that do not eliminate parasites but that decrease the clinical impact of the disease. Numerous epidemiological studies conducted in areas of stable malaria have demonstrated an age-related increase in malaria-specific immune responses, as well as an agerelated decrease in malaria-related morbidity. It is generally held that protection against P. falciparum malaria is the result of repeated exposures to parasites over a period of many years and that its persistence requires regular contact with the parasite. However, this model has been documented poorly. Where malaria transmission is unstable, and especially during epidemics, a different situation may occur. Such a situation developed recently in the central highlands of Madagascar, where an epidemic of falciparum malaria started in the mid-eighties in an area where falciparum malaria had been absent for almost three decades. Thus, in this community, children and young adults, who were all naive, were exposed suddenly to malaria-infected mosquitoes. Adults older ~:han40 years, who had spent their chilclhood in this area before malaria control, had probably had many previous infections and might have retained some degree of ~) 1992, ElsevierSciencePublishers Ltd, (UK)
immunological memory. The aim of this paper is to relate the immunological alterations that occurred in the population of this area after the 1987 outbreak with the development of clinical protection against the disease. We also highlight the differences between the immune response of such a population and that of the inhabitants of areas of highly endemic and stable malaria. Before 1950, the central highland plateau of Madagascar was a malariahyperendemic area (Box I). As a result of control activities during the 1950s and 1960s, malaria transmission decreased dramatically and the highland plateau was declared a 'surveillance area' in 1960. Unfortunately, control activities decreased subsequently. The time of the reappearance of falciparum malaria in the central highlands of Madagascar is not known accurately but was dramatized by a major outbreak in late 1987 (Ref. I). However, chart reviews showed an increase of malaria cases in the paediatric wards of Antananarivo from 1985 (Ref. 2). Beginning in October 1987, we have conducted a series of investigations in the population of Manarintsoa, a village of 1400 inhabitants, located 20 km west of Antananarivo, the capital city of Madagascar. Three surveys were conducted during the period 1987-1989 during which malaria morbidity was followed among this population. The first study was conducted for six months during the 1988 transmission season. It consisted of passive case detection of malaria attacks among the outpatients presenting to the Manarintsoa health centre 3. Subsequently, 76 individuals were followed weekly from January to June 1988 (Refs 4-6). Finally, a second cohort study which involved 27I inhabitants, who were followed monthly during the next transmission season, was undertaken from December 1988 to June 1989 (P. Deloron and C. Chougnet, unpublished). During these two studies, subjects who presented with one or more malaria attacks during the follow-up were considered as non-protected; the others were considered as protected. Malaria attacks were defined either as the presence of parasites in blood, with associated fever (axillary temperature
~37.5°C), abdominal pain, diarrhoea, vomiting or headache, or as high parasite density (~5000 per I~1 of blood in the first study; ~1500 per I~1 in the second). Data from these three studies were used to assess the relationship between age and morbidity and between age and the evolution of immune responses to defined P. falciparum antigens. Protection in the O v e r 40s
In the survey of 2776 outpatients of the Manarintsoa health centre, the parasite rate averaged 63.2% and did not vary with age (between 51.0% and 68.8% in all age groups). However, among patients infected with P. falciparum, those between two and 39 years old were more likely to have fever (indicating an acute malaria attack) than those older than 40 (52.6% versus 29.5%; P ~104). Similarly, the lowest spleen rate was observed in this age group (3.9% versus 51.1%; P ,~104 ) (Ref. 3). These data were derived from passive case detection, thus parasite rates may not relate accurately to the prevalence in the population as a whole because the proportion of symptomatic individuals who report to the clinic may vary with age. However, this is unlikely to interfere with the fever rate in patients of the health centre who are infected with P. falciparum. These findings are in contrast with the fact that individuals of all ages were similarly exposed to mosquito bites, as described later in this paper. The fact that individuals older than 40 years responded differently than those in other age groups is supported by the two cohort studies. In the 1988 study, a total of 148 malaria attacks were identified in 61 of the 76 individuals of the cohort; 85% of these malaria attacks had symptomatic parasitaemia. Villagers older than 40 years were more likely to be protected against falciparum malaria attacks than were younger individuals (42.9% versus 14.5%, P ~0.005) (Ref. 4). Similar results were obtained in the 1989 study dealing with a greater number (271) of inhabitants of Manarintsoa, 65.3% being considered as non-protected and 34.7% as protected. The proportion of protected
Parasitology Today, vol. 8, no. I I, 1992
376
Box I. Historical Aspects of M a l a r i a in the C e n t r a l Highlands of Madagascar Since the beginning of the century, Madagascar has been known to be hyperendemic for falciparum malaria. Antimalarial control activities were initiated in 1949. In the central highlands, with a tropical mountain climate, insecticide spraying was highly effective, leading to the disappearance of one of the two malaria vectors (Anophelesfunestus)and to a dramatic reduction in the population of the ocher (An. gambiae). Malaria decreased in parallel and was hypoendemic ten years later. To consolidate malaria eradication, mass chloroquine administration was initiated in 1959. A further reduction of malaria transmission was obtained, but a few loci remained. In the mid 1960s, control activities gradually decreased due to financial and administrative difficulties. In the period 1970 to 1985, entomological and parasitological data are almost lacking, but malaria was not reported to be actively transmitted. Following the 1987 outbreak, falciparum malaria was hyperendemic, although the level of transmission was very low with fewer than two infective bites per person per year. After chloroquine administration and insecticide spraying, transmission decreased and no mosquitoes were found to be infected in 1991; the area is now hypoendemic for malaria.
Mass Drug Administration 1987 Outbreak
Vector Control
Hyperendemy
1950
1960
1970
1980
1990
I
I
I
I
I
Mesoendemy
50-75%
8%
Preeradication
Hyper endemy
0.9%
33% 80% Spleen rates
individuals differed significantly between age groups, reaching 61.1% in individuals older than 40 years (Fig. I ). This difference in proportion remained significant when the group of individuals less than 40 years was split in two, three or six age groups (all P <0.01). However, when individuals older than 40 years were omitted from the calculations, no differences between age groups were detected (all P >0.05). Antimalarial drug consumption (as studied by urine tests) and mosquito exposure did not appear to vary with either age or location of the house inside the village. Entomological surveys failed to identify geographical variations of Anopheles sporozoite rates inside the village. In addition, biting peak by both An. gambiae and An. funestus occurred between 22.00 and 3.00h, suggesting that no environmental factor modified exposure to mosquitoes s. Humoral
and Cellular Responses
Humoral and cellular responses of the subjects enrolled in the 1988 cohort to synthetic peptides reproducing dominant epitopes of two major P. falciparum antigens [Pf155/RESA and
circumsporozoite (CS) protein] were investigated at the time of enrolment, at the beginning of the rainy season6'7. Antibodies to the CS protein repeat were found in 23% of individuals. Titres of these antibodies were rather low. The mean antibody titre was higher in the oldest (>40 years) individuals (P <0.02) but did not differ significantly among other age groups (OD = 0.17, 0. I 8, 0.09 and 0.32 in the 9-19, 20-29, 30-39 and I>40 years age groups, respectively), demonstrating that all age groups were exposed to infected mosquito bites. The higher anti-CS protein antibody titre in subjects older than 40 years may reflect a secondary response in those with immunological memory. The overall antibody prevalence rate was lower than that in adult populations living in areas of stable and intense malaria transmission, such as much of tropical Africa 8-1J , where such antibodies are found in more than 70% of the adult population. However, the antibody prevalence rate in our study subjects is similar to the rate observed in American citizens (27%) who have stayed for one to two years in tropical Africa 12. Antibodies to Pf155/RESA were observed in 35%-64% of our subjects (according to the peptide), a
prevalence rate that is similar to those observed in other African populations ~°'t~. However, the antibody titre did not vary with age, contrasting with the results of numerous studies conducted in areas of stable malaria. In such areas an age-related increase of the humoral response to both Pf155/ RESA ~°'~ and CS protein 8'9, as well as to several other P. falciparum antigens ~3 has been reported consistently. Cellular responses were assessed by lymphocyte proliferation. The percentage of cellular responders to CS protein and to Pf155/RESA peptides ranged from 29-35%. Mean stimulation indexes were low (mean stimulation indexes between three and six with any peptide) 6'7. The percentage of cellular responders to Pf155/RESA peptides was not lower in this area of Madagascar than in areas of high and stable malaria transmission, such as Liberia j4 or the Gambia Is'l 6. Levels of responses were also similar in the three areas. The levels of proliferative responses to the central repeat of Pf155/RESA varied among age groups, being highest in the oldest. However, no trend of an agerelated variation was observed, and these levels did not significantly differ before 40 years of age. Similarly, no
377
Parasitology Today, vol. 8, no. I I, 1992
age-related increase of cellular responses to Pf155/RESA was reported in the studies from Liberia and the Gambia.
a 60
Does Protection Reflect C u m u l a t i v e Exposure to the Parasite?
4O ,,i,,a
All the studies we conducted in the central highlands of Madagascar showed that individuals older than 40 years were more protectecl against clinical falciparum malaria, but not against infection, than were those younger, despite being submitted to similar exposure to infective mosquito bites. In addition, they had higher humoral and cellular responses to some major epitopes of two parasite antigens. Individuals over 40 years spent their childhood in a malaria hyperendemic area and probably experienced numerous infections over a period of several years. Conversely, younger subjects were almost, if not totally, naive before the outbreak. Although an agedependent immunity not related to parasite exposure might eventually occur 17, the difference in response between subjects older and younger than 40 is likely to be due to a difference in past exposui-e to malaria parasites. Conclusive evidence would have required the study of a control group covering the same age range, exposed to the same present malaria epidemic but without having been exposed to the previous period of transmission. Such a control group did not exist in Madagascar. The persistence of some level of protection after almost 30 years is surprising as malaria immunity is thought to wane within one or two years after the end of exposure to the parasite. Although the lack of immunological memory has been demonstrated for transmission-blocking immunity '8, it has been only poorly documented in the case of clinical protection. West African immigrants to France are susceptible to clinical malaria when they return to their country after several years. However, the case fatality rate has never been estimated and they rarely have lethal cerebral malaria. P. falciparum malaria attack is less severe in such individuals than in naive individuals. Similarly, fever and parasite clearance times are shorter than in non-immune individuals ~9. These migrants, in common with the inhabitants of Madagascar, may still harbour immune memory cells that were primed many years before. Moreover, it is conceivable that anti-malarial
0
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20-29
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8O
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Age (years) Fig. I. The age distribution of protection against Plasmodium falciparum malaria in two cohorts of inhabitants of Manarintsoa, in the central highlands of Madagascar, followed during the 1988 (a) (n = 76), and 1989 (b) (n = 271) malaria transmission seasons. Followup was conducted weekly in 1988 and monthly in 1989. Protection was defined by the absence of any episode of malaria attack during the follow-up. The number of individuals followed in each age group is indicated above the bars.
immunity was sustained by infection with organisms possessing antigens crossreacting with malaria antigens2°. A new parasite exposure will allow these cells to expand and initiate a secondary immune response. In this respect, antibodies to Pf155/RESA are able to persist for more than six years after the cessation of antigen exposure (A.J. Sulzer, P. Deloron and P. Millet, unpublished). Similarly, humoral and cellular responses to both CS protein and Pf155/RESA may persist in migrants from West Africa after they have spent up to 13 years in France without returning to their native area2~. A number of epidemiological studies in endemic areas have shown protection to be a function of age. This fits with the model usually adopted for the acquisition of protection, which is believed to result from the cumulative
effects of repeated exposure to parasites. The effects of age may occur independently of exposure, but age and exposure are synonymous in populations spending their entire life in a malaria-endemic area. Only very peculiar epidemiological situations, such as the one that had happened in the central highland of Madagascar, allow the effect of age to be studied independently of exposure as well as duration of memory mechanism of malaria immunity. The recent disappearance of falciparum malaria transmission in the central plateau of Madagascar since 1991 provides a unique opportunity for such studies. Acknowledgements
We thank the population of Manarintsoa for participating in these successivestudies. We also thank all the people who took part
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in the studies reported here, in particular Marie Danielle Andriamangatiana-Rason, Pierre Coulanges, Didier Fontenille, Jean Paul Lepers and Jean Aime Ramanamirija from the Madagascar Pasteur Institute, and Pascal Astagneau, B6atrice Dubois and Florence Migot from INSERM U I3. This work was supported in part by grants from AUPELFAJREF and from the Tropical Pathology Society. References
I Lepers, J.P. et al. (1988) Lancet i, 586 2 Razanamparany, M. et al. (1989) Arch. Inst. Pasteur Madagascar 56, 91-96 3 Lepers, J.P. et al. (1990) Bull. WHO 68, 217-222 4 Astagneau, P. et al. ( 1991 ) Am, J. E_pidemiol, 133, 177-184 5 Fontenille, D, et al, (1990) Am. J. Trap. Med.
Hyg. 43, 107-115 6 Chougnet, C. et aL (1990) Immunol. Lett. 25, 231-235 7 Chougnet, C. et aL (1990) Am, J. Trap. Med, Hyg. 43, 234-242 8 Del Giudice, G, et al. (1987) Am, J. Trap. Med. Hyg, 36, 203-212 9 Esposito, F. et al. (1988) Trans. R, Sac, Trap. Med. Hyg. 82, 827-832 I 0 Deloron, P, et al. (1989) Am. J. Trap. Med. Hyg. 41,395-399 II Deloron, P, and Cot, M, (1989) Trans. R. Sac. Trap, Meal Hyg. 84, 191-195 12 Miller, K.D, et al. (1988) J. Infect, Dis. 158, 868-87 I 13 Marsh, K. et al. (1989) Trans. R. S0c. Trap. Med. Hyg. 83, 293-303 14 Petersen, E. et oL (1989) Am. J. Trap. Med, Hya~. 4 I, 386-394 15 Troye-Blomberg, M, et al. (1989)J. ImmunoL 143, 3043-3048
16 Riley, E.M. et al. (1991) Trans, R. Sac, Trap. Med. Hyg. 85, 436-443 17 Baird, J.K. et al. (1991 ) Am, J, Trap. Med. Hyg. 45, 65-76 18 Ranawaka, M.B.R, et al. (1988)Infect. Immun. 56, 1820-1824 19 Charmot, G, et al. (1990) in Chimioth~rapie des parasitoses sev~res (Vachon, F. et al., eds), pp 29-38, Arnette 20 McLaughlin, G.L., Benedik, M.J. and Campbell, G.H. (1987) Am. J. Trap. Med. Hya~. 37, 258-262 21 Chougnet, C., Deloron, P. and Save[, J, (I 991 ) Ann. Trap. Med. Parasitol, 85, 357-363
Philippe Deloron and Claire Chougnet are at INSERM UI3, Institut de M6decine et d'Epid~miologie Africaines, 75944 Paris Cedex 19, France.
A Superfamily of Trypanosomacruz/ Surface Antigens O. Campetella, D. S nchez, J.J.Cazzulo and A.C.C. Frasch Several genes of Trypanosoma cruzi encode surface antigens that include a n amino acid motif that is conserved among bacterial neuraminidases. Oscar Campetella, Daniel Sdnchez, Juan Jos~ Cazzulo and Alberto Carlos Frasch here suggest grouping these gene families in a superfamily. Recent results from several laboratories have started to clarify the relationship between the surface antigens of Trypanosoma cruzi, the agent of American trypanosomiasis, Chagas disease. The information obtained from the cloning and sequencing of genes for T. cruzi antigens has shown that they share one major characteristic: the presence of tandemly repeated amino acid motifs, that are the targets of the immune response during natural and experimental infections (see Ref. I for review). Data from the sequencing of the region 5' to the repeated motifs of some of these genes, as well as other related genes lacking repeated units, have added important information. Several genes encoding surface antigens were found to have significant sequence homologies, including up to four copies of partially or completely conserved sequences encoding the motif Ser-XAsp-X-Gly-X-Thr-Trp. This motif is conserved among bacterial neuraminidases2 and its presence in members of a superfamily of surface antigens might suggest that they originated in an ancestral (neuraminidase?) gene.
Families and Superfamilies
The first gene encoding a T. cruzi antigen, named TSA-I, was cloned in 1986 (Ref. 3). Its partial sequence showed a repeated nonapeptide sequence motif. Some years later, the whole sequence of this gene was obtained 4. The deduced amino acid sequence on the amino side of the repeat turned out to be highly homologous, although not identical, to that of two other sequences obtained independently by Kahn and co-workers (SA85-1, Ref. 5) and Takle and Cross (Tt34cl, Ref. 6). Unlike with TSA-I, these two genes lack a repetitive amino acid motif. All three have copies of the bacterial neuraminidase motif 2. One of these proteins, SA85-1, has been considered to be the neuraminidase of T. cruzi s, but this seems now not to be the case (see below). All these genes are highly homologous, and thus comprise a gene family (the gp85 family, Refs 5,6) expressed in the trypomastigote stage. The estimated molecular weight of these proteins is around 85kDa and they have, at least before processing, carboxy terminal extensions that are compatible with the post-translational addition of glycosylphosphatidilinositol (GPI) anchors6. Other genes encoding proteins of 120-200kDa that also contain the conserved neuraminidase motifs have been identified 7-9, These proteins are
also expressed in the trypomastigote stage, and are anchored on the parasite surface by GPI, allowing their easy shedding into the medium 7'r°. One of these genes has been isolated and sequenced by our group while characterizing a major shed acute phase antigen (SAPA) 7.1°. Pereira and coworkers have cloned and sequenced the gene encoding the enzyme neuraminidase8 and have found that it contains the same amino acid repeats as are present in SAPA ~° and the conserved motifs that are present in bacterial neuraminidases2'8. In addition, Pereira's neuraminidase and SAPA are more than 80% identical in the non-repeated sequences, thus showing that they belong to the same gene family. Members of the SAPA protein family have been shown to have not only most of the neuraminidase activity of the parasite, but also most of the trans-sialidase activity of the parasite 9. Trans-sialidase is an enzyme unique to T. cruzi. At variance with all other sialyl-transferases described in eukaryotes that use cytidine monophosphate-sialic acid as donor, trans-sialidase uses sialylated substrates other than the nucleotide derivative, like fetuin, as donor molecules 9'11-j3 Recently, Nussenzweig and co-workers have demonstrated that neuraminidase and trans-sialidase activities are in the same molecule 14. Members of this gene family are characterized by a 5' domain probably encoding the region of the protein with enzymatic activity and a (~ 1992,ElsevierSciencePublishersLtd,(UK)