IMMUNOLOGICAL INVESTIGATIONS,21(5), 423453 (1992)
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
ENVIRONMENTAL, GENETIC AND IMMUNOLOGICAL FACTORS IN HUMAN RESISTANCE TO SCHISTOSOMA MANSONI Alain J. Dessein, Patricia Couissinier, Christian Demeure, Pascal Rihet, Sibylle Kohlstaedt, Denise Carneiro-Carvalho,Monique Ouattara, Vkronica Goudot-Crozel, HClia Dessein, and Main Bourgois Centre d’Immunologie de Marseille-Luminy Marseille, France Laurent Abel DBpartement de biomathkmatiques Hospital PitiB-SalpBtriBre, Paris, France Edgar M. Carvallo Laboratorio de Immunologia Hospital E. Santos Salvador AIuizio Prata Faculdade de Medicina do Triangulo Mineiro Uberaba, Brazil
INTRODUCTION In hyperendemic areas of Schistosomiasis due to S. mansoni, 3 to 10% of infected subjects developsevere clinical diseasecharacterizedby hepatosplenomegaly, protal blood hypertension and, in some cases, abdominal ascites (reviewed in Ref. 1). Individuals with decompensated hepatosplenomegaly have a poor prognosis, since they require health care that is seldom available in the rural areas of developing countries. Until recently a sizeable fraction of the subjects with hepatosplenomegaly would have evolved toward decompensated forms; fortunately, these individuals benefit greatly from treatment with Oxamniquine or Praziquantel that stops the evolution of their disease and, for some of them, leads to the regression of fibrosis and the reduction of portal hypertension (2-6). However, chemotherapy for the control of morbidity has several limitations: 1. It has little effect on hepatosplenomegaly in whites (7), who represent a sizeable 423
Copyright 8 1992 by Marcel Dekker, Inc.
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
424
DESSEIN ET AL.
fraction of the population in the endemic areas of Latin America and who run a much higher risk of developing hepatosplenomegaly than blacks (8-9). 2. Therapy must be repeated at regular time intervals, since reinfection occurs within a year or two after treatment in hyperendemic area (1&15); reinfection intensities may be as high as preinfection levels in the most susceptible individuals. 3. Drug resistant strains may be selected especially if repeated mass chemotherapy has to be used (16-19). 4. Therapy is costly and out of reach of most families (20); the family income for one month is needed to treat a family with two children, in our study area. 5. A large fraction of the population of certain endemic area is not reached by chemotherapy programs, and this situation becomes worse if chemotherapy has to be repeated at regular time intervals. Thus, additional control methods that could be combined with chemotherapy are sought for the control of schistosomiasis. Vector control by mollusciciding has limited effects (21-22); health education, sanitation (including toilet construction), decontamination of the water for domestic use, and construction of water pipe systems are probably the most effective control measures. The implementation of such expensive programs suffers from the lack of adequate resources and from an insufficient long term government commitment (21-23). For these reasons, efforts are made to develop control methods that would be less expensive, easy to carry out, and that would not require a major financial effort in developing countries where schistosomiasis is only one of several important health problems. The control of schistosomiasis by vaccination has several advantages over other control methods: its cost could be low especially if engineered vaccines are used, it would have long lasting effects, and it would be effective against variants of the parasite, if it includes a variety of protective Ag. Finally, an antischistosome vaccine could be part of a multivalent vaccine and distributed within existing vaccination programs. The first vaccines to be developed were made of either inactivated organisms that had lost their pathogenicity or of relatively safe viruses that were immunologically cross-reactive with human pathogens. Later on, subunit vaccines were developed with semipurified toxins, These vaccines were, in general, potent and safe with few exceptions (reviewed in Ref. 24). It was hoped that the original approaches used for developing vaccines against bacteria and certain viruses could be extended to vaccines against eucaryote parasites such as schistosomes. Unfortunately, the adaptation of eucaryote parasites to their human host and their antigenic complexity make this task more difficult than was originally expected. Successful human vaccines protect against bacterial or viral infections that induce a strong immunity: individuals recovering naturally or after chemotherapy from infection by these pathogens are, in general, protected against reinfection by the same infectious agent. This is a major difference with infections by a number of eucaryote parasites, including helminths, that do not induce high and long-lasting protective immunity in a large fraction of human populations. Moreover, the immune responses induced by eucaryote parasites often have profound deleterious effects on host tissues, as in
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
HUMAN RESISTANCE TO S. MANSONZ
425
severe schistosomiasis (25-27), and great care must be taken that vaccines do not exacerbate immunopathological reactions. Finally, antigen variation (28-32), antigen modulation (33-34), host antigen acquisition (35-37), and possibly mimicry (38-41) are used by parasites to escape host immunity and, thus, vaccine induced immunity. After a period of high optimism, it was realized that our knowledge of the biology of parasitic infections may not be sufficient to allow a rational approach to the problem of vaccination. Among the important questions that had to be reevaluated were the mechanisms of adaptation of parasites to their hosts, including the immunogenicity, variability and pathogenicity of parasite Ag, and the effects of environmental and host genetic factors on human capacities to resist infection and disease. Our laboratory, together with those of our Brazilian colleagues, have addressed these questions in schistosome infections.
STUDY AREA AND EFFECT OF ENVIRONMENTAL FACTORS ON INFECTION INTENSITIES
Our study was carried out in Caatinga do Moura, a village in the state of Bahia, Brazil. The village is free of human parasitic diseases such as leishmaniasis, malaria, or Chagas disease. A short and narrow river runs through Caatinga, and this is the principal source of water for agricultural and domestic use. Since the village is in a semi-arid area, irrigation is an absolute requirement for agriculture; the various irrigation canals represent ideal breeding sites for B. glabratu snails and are, during the dry season, covered by hundreds of snails, some of which are infected by S. munsoni. In the river, however, snails are scarce, and infective cercariae are shed mainly in irrigation canals and then carried by the water flow to the river. Since the river is narrow, only a few sites (four in this study) are suitable for bathing and washing; these places are located 20 to 50 meters apart on the river and receive water from the same irrigation canals. Thus, water infectivity was relatively uniform for all subjects frequenting these sites, as confirmed by sentinel mice immersed in the river at the different sites. Under these conditions, exposure to the parasite was mostly dependent on the frequency, the duration, and the nature of the water contacts of study subjects. These variables were evaluated seven days a week, every other week, by observers who visited river sites every hour from sunrise to sunset. Daily observations were recorded and individual data transferred to personnel records at the end of the week. Examples of a daily record and of an individual record are shown in Fig. 1. These observations were further validated by cross-interviews with study subjects and with their relatives. Based on these results, subjects were classified into four different water groups (see legend for Fig. 2). Infection intensities were quantified by individual fecal egg counts that were the arithmetic mean of at least five determinations with the Kato-Katz method (42). The effect of water contact levels, age, and sex on the raw egg count values are presented in Fig, 2 a, b. There was a strong effect of water contact on infection
DESSEIN ET AL.
426
DATA
AVALI&O
DO GRAU DE CONTACTO CI~GUADO RIO
1
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
I
FIGURE 1 Water contact evaluation: daily records and bi-annual individual records. Water contacts were evaluated by observers who visited river sites (D.A), (I), (PO), and (G), every hour from 8 a.m. to 6 p.m.. The name (nome), the origin (casa) of the subjects present in the water, their water re-
HUMAN RESISTANCE TO S. MANSONZ
427
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
s YESES. I V A U I ~ X O DO GRAU DE CONTICTO
J U L
A G 0
S E T
0 U T
N
0 V
r
I
D E 2
I
1
1
1
1
lated activities (washing clothes (R), dishes (P), hair (C), and bathing (B) were recorded at each site (Fig. 1, p. 424). At the end of the week, data were transfered to bi-annual individual records (Fig. 1, p. 425).
DESSEIN ET AL.
428
b)
a’
lwJk
-High water contact .- .- -
(w = 3 + 4 ) Low water Contact
(w=l)
P
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
d
I
2
3
4
WATER CONTACT CLASSES ( W )
0 u1
200
c
0
3
a
100
4
.3
+*
0
.
.l) and the nontransmission hypothesis was rejected (model 2vs 1,x2=11.7,2dfs,p>.01). Both the recessiveand thedominant major genemodel did not fit the data, implying that the major gene was co-dominant. Parameter estimates indicate that about 5% of the study population is homozygous for the deleterious allele and predisposed to high infections, 60% is homozygous resistant, and 35% is heterozygous with an intermediate, although fairly good, level of resistance. The predicted distributions of phenotypes under the co-dominant major gene hypothesis are shown in Fig. 4. The most likely genotypes of family members according to their E2 values are indicated in Fig. 3. To evaluate the fit of the codominant major gene model to the data we computed the expected proportions of individuals in the five genotype classes defined in Fig. 3, that is, aa (E2
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
ENVIRONMENTAL, GENETIC AND IMMUNOLOGICAL FACTORS IN HUMAN RESISTANCE TO SCHISTOSOMA MANSONI Alain J. Dessein, Patricia Couissinier, Christian Demeure, Pascal Rihet, Sibylle Kohlstaedt, Denise Carneiro-Carvalho,Monique Ouattara, Vkronica Goudot-Crozel, HClia Dessein, and Main Bourgois Centre d’Immunologie de Marseille-Luminy Marseille, France Laurent Abel DBpartement de biomathkmatiques Hospital PitiB-SalpBtriBre, Paris, France Edgar M. Carvallo Laboratorio de Immunologia Hospital E. Santos Salvador AIuizio Prata Faculdade de Medicina do Triangulo Mineiro Uberaba, Brazil
INTRODUCTION In hyperendemic areas of Schistosomiasis due to S. mansoni, 3 to 10% of infected subjects developsevere clinical diseasecharacterizedby hepatosplenomegaly, protal blood hypertension and, in some cases, abdominal ascites (reviewed in Ref. 1). Individuals with decompensated hepatosplenomegaly have a poor prognosis, since they require health care that is seldom available in the rural areas of developing countries. Until recently a sizeable fraction of the subjects with hepatosplenomegaly would have evolved toward decompensated forms; fortunately, these individuals benefit greatly from treatment with Oxamniquine or Praziquantel that stops the evolution of their disease and, for some of them, leads to the regression of fibrosis and the reduction of portal hypertension (2-6). However, chemotherapy for the control of morbidity has several limitations: 1. It has little effect on hepatosplenomegaly in whites (7), who represent a sizeable 423
Copyright 8 1992 by Marcel Dekker, Inc.
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
424
DESSEIN ET AL.
fraction of the population in the endemic areas of Latin America and who run a much higher risk of developing hepatosplenomegaly than blacks (8-9). 2. Therapy must be repeated at regular time intervals, since reinfection occurs within a year or two after treatment in hyperendemic area (1&15); reinfection intensities may be as high as preinfection levels in the most susceptible individuals. 3. Drug resistant strains may be selected especially if repeated mass chemotherapy has to be used (16-19). 4. Therapy is costly and out of reach of most families (20); the family income for one month is needed to treat a family with two children, in our study area. 5. A large fraction of the population of certain endemic area is not reached by chemotherapy programs, and this situation becomes worse if chemotherapy has to be repeated at regular time intervals. Thus, additional control methods that could be combined with chemotherapy are sought for the control of schistosomiasis. Vector control by mollusciciding has limited effects (21-22); health education, sanitation (including toilet construction), decontamination of the water for domestic use, and construction of water pipe systems are probably the most effective control measures. The implementation of such expensive programs suffers from the lack of adequate resources and from an insufficient long term government commitment (21-23). For these reasons, efforts are made to develop control methods that would be less expensive, easy to carry out, and that would not require a major financial effort in developing countries where schistosomiasis is only one of several important health problems. The control of schistosomiasis by vaccination has several advantages over other control methods: its cost could be low especially if engineered vaccines are used, it would have long lasting effects, and it would be effective against variants of the parasite, if it includes a variety of protective Ag. Finally, an antischistosome vaccine could be part of a multivalent vaccine and distributed within existing vaccination programs. The first vaccines to be developed were made of either inactivated organisms that had lost their pathogenicity or of relatively safe viruses that were immunologically cross-reactive with human pathogens. Later on, subunit vaccines were developed with semipurified toxins, These vaccines were, in general, potent and safe with few exceptions (reviewed in Ref. 24). It was hoped that the original approaches used for developing vaccines against bacteria and certain viruses could be extended to vaccines against eucaryote parasites such as schistosomes. Unfortunately, the adaptation of eucaryote parasites to their human host and their antigenic complexity make this task more difficult than was originally expected. Successful human vaccines protect against bacterial or viral infections that induce a strong immunity: individuals recovering naturally or after chemotherapy from infection by these pathogens are, in general, protected against reinfection by the same infectious agent. This is a major difference with infections by a number of eucaryote parasites, including helminths, that do not induce high and long-lasting protective immunity in a large fraction of human populations. Moreover, the immune responses induced by eucaryote parasites often have profound deleterious effects on host tissues, as in
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
HUMAN RESISTANCE TO S. MANSONZ
425
severe schistosomiasis (25-27), and great care must be taken that vaccines do not exacerbate immunopathological reactions. Finally, antigen variation (28-32), antigen modulation (33-34), host antigen acquisition (35-37), and possibly mimicry (38-41) are used by parasites to escape host immunity and, thus, vaccine induced immunity. After a period of high optimism, it was realized that our knowledge of the biology of parasitic infections may not be sufficient to allow a rational approach to the problem of vaccination. Among the important questions that had to be reevaluated were the mechanisms of adaptation of parasites to their hosts, including the immunogenicity, variability and pathogenicity of parasite Ag, and the effects of environmental and host genetic factors on human capacities to resist infection and disease. Our laboratory, together with those of our Brazilian colleagues, have addressed these questions in schistosome infections.
STUDY AREA AND EFFECT OF ENVIRONMENTAL FACTORS ON INFECTION INTENSITIES
Our study was carried out in Caatinga do Moura, a village in the state of Bahia, Brazil. The village is free of human parasitic diseases such as leishmaniasis, malaria, or Chagas disease. A short and narrow river runs through Caatinga, and this is the principal source of water for agricultural and domestic use. Since the village is in a semi-arid area, irrigation is an absolute requirement for agriculture; the various irrigation canals represent ideal breeding sites for B. glabratu snails and are, during the dry season, covered by hundreds of snails, some of which are infected by S. munsoni. In the river, however, snails are scarce, and infective cercariae are shed mainly in irrigation canals and then carried by the water flow to the river. Since the river is narrow, only a few sites (four in this study) are suitable for bathing and washing; these places are located 20 to 50 meters apart on the river and receive water from the same irrigation canals. Thus, water infectivity was relatively uniform for all subjects frequenting these sites, as confirmed by sentinel mice immersed in the river at the different sites. Under these conditions, exposure to the parasite was mostly dependent on the frequency, the duration, and the nature of the water contacts of study subjects. These variables were evaluated seven days a week, every other week, by observers who visited river sites every hour from sunrise to sunset. Daily observations were recorded and individual data transferred to personnel records at the end of the week. Examples of a daily record and of an individual record are shown in Fig. 1. These observations were further validated by cross-interviews with study subjects and with their relatives. Based on these results, subjects were classified into four different water groups (see legend for Fig. 2). Infection intensities were quantified by individual fecal egg counts that were the arithmetic mean of at least five determinations with the Kato-Katz method (42). The effect of water contact levels, age, and sex on the raw egg count values are presented in Fig, 2 a, b. There was a strong effect of water contact on infection
DESSEIN ET AL.
426
DATA
AVALI&O
DO GRAU DE CONTACTO CI~GUADO RIO
1
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
I
FIGURE 1 Water contact evaluation: daily records and bi-annual individual records. Water contacts were evaluated by observers who visited river sites (D.A), (I), (PO), and (G), every hour from 8 a.m. to 6 p.m.. The name (nome), the origin (casa) of the subjects present in the water, their water re-
HUMAN RESISTANCE TO S. MANSONZ
427
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
s YESES. I V A U I ~ X O DO GRAU DE CONTICTO
J U L
A G 0
S E T
0 U T
N
0 V
r
I
D E 2
I
1
1
1
1
lated activities (washing clothes (R), dishes (P), hair (C), and bathing (B) were recorded at each site (Fig. 1, p. 424). At the end of the week, data were transfered to bi-annual individual records (Fig. 1, p. 425).
DESSEIN ET AL.
428
b)
a’
lwJk
-High water contact .- .- -
(w = 3 + 4 ) Low water Contact
(w=l)
P
Immunol Invest Downloaded from informahealthcare.com by Chulalongkorn University on 12/29/14 For personal use only.
d
I
2
3
4
WATER CONTACT CLASSES ( W )
0 u1
200
c
0
3
a
100
4
.3
+*
0
.
.l) and the nontransmission hypothesis was rejected (model 2vs 1,x2=11.7,2dfs,p>.01). Both the recessiveand thedominant major genemodel did not fit the data, implying that the major gene was co-dominant. Parameter estimates indicate that about 5% of the study population is homozygous for the deleterious allele and predisposed to high infections, 60% is homozygous resistant, and 35% is heterozygous with an intermediate, although fairly good, level of resistance. The predicted distributions of phenotypes under the co-dominant major gene hypothesis are shown in Fig. 4. The most likely genotypes of family members according to their E2 values are indicated in Fig. 3. To evaluate the fit of the codominant major gene model to the data we computed the expected proportions of individuals in the five genotype classes defined in Fig. 3, that is, aa (E2
