Protective cellular immune responses depend on MHC presentation of pathogen-derived Ag fragments. MHC diversity renders this process sensitive to point mutations coding for altered amino acid sequence of the short target Ag-derived peptides epitopes. Thus, in a given host, a pathogen with an altered epitope sequence will be more likely to escape detection and elimination by the immune system. At a population level, selection by immune pressure will increase the likelihood of polymorphism in important pathogen antigenic epitopes. This mechanism of immune evasion is found in viruses and other pathogens. The detection of polymorphic hot spots in an Ag is often taken as a strong indication of its role in protective immunity. We provide evidence that polymorphisms in the T cell epitopes of a malaria vaccine candidate are unlikely to have been selected by immune pressure in the human host.

The circumsporozoite protein (CS)4, is a major component of the surface of the sporozoite. This is the form of a malaria parasite injected by the mosquito into the vertebrate host to initiate the malaria infection. CS has long been considered an Ag crucially implicated in protective immunity against the pre-erythrocytic (PE) stages of the Plasmodium infection (1). The gene coding for this Ag was the first Plasmodium gene to be cloned (2), and the first Plasmodium falciparum subunit vaccine tested in human volunteers was based on CS (3). CS-based vaccines are still at the forefront of malaria vaccine development (4, 5).

Immunity to the PE stages of malaria depends to a large extent on cellular responses (6). Sequence analysis of the pfcs gene from geographically diverse isolates of P. falciparum revealed two polymorphic regions in the otherwise highly conserved 3′-end region of the gene where nearly all the mutations observed were found to be nonsynonymous, prompting the hypothesis that these were selected by cellular immune pressure (7). Indeed, the variable residues mapped to two previously identified immunodominant human T cell epitope regions (8). Although relatively few surveys of pfcs polymorphism were conducted, >20 variants have been described so far (9, 10) for each of these CD4+ T cell epitopes, Th2R and Th3R. Such a high degree of immune-driven polymorphism poses a serious challenge to the use of CS as a basis for a vaccine. However, although these observations, associated with vaccination studies and epidemiological investigations, provided compelling support for a critical role of these epitopes in naturally acquired sterilizing immunity against P. falciparum PE stages, formal evidence for such a role is as yet unavailable.

We reasoned that the extensive Th2R/Th3R polymorphism observed in the natural parasite population could be exploited to address this issue. If the immune responses dependent on these epitopes were to play a determinant role in the success of sporozoite inoculations, then the extent of Th2R and Th3R epitope diversity in the parasite populations circulating in the blood of residents in endemic areas should vary with their immune status. Diversity should be high in relatively nonimmune children and low in adults. Therefore, we conducted a large scale survey of pfcs Th2R and Th3R diversity in 283 admission blood samples collected from patients presenting with malaria in three distinct endemic regions on the Thai-Burmese border. Although these samples were taken from patients living in areas of low unstable transmission (∼0.1–10 infective bites/person/year for P. falciparum), there is abundant evidence that significant immunity is gained by this exposure as evidenced by the increased risks of severe malaria in children and the marked differences in therapeutic responses to antimalarial drug treatments with increasing age.

Blood samples were collected on admission from consenting symptomatic patients after a diagnosis of P. falciparum malaria was established by microscopic examination of thin and tick blood smears stained with Giemsa. One hundred patients from the Mae La clinic were sampled: n = 35 during 1996, n = 45 during 1999, and n = 20 during 2001. One hundred eighty-three samples were obtained from patients admitted to the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University (Bangkok, Thailand) from June to September 2001 with infections acquired in Ratchaburi, Thailand (n = 140), and in Kanchanaburi, Thailand (n = 43). In all cases, admission blood samples were collected in EDTA tubes. Two hundred microliters of blood were centrifuged, and the RBC pellets were stored at −20°C.

Amplification of the pfcs 3′-end region by the PCR and genotyping by sequence-specific oligonucleotide probes (SSOPs) were conducted as previously described (9) with minor modifications. Briefly, 5 μl of the lysed freeze-thawed infected RBC pellet were mixed with 150 μl of PBS solution, and the parasites were pelleted by centrifugation for 5 min at 5000 × g. Fifty microliters of amplification reaction mixture, containing 1× PCR buffer, 200 nM of each primer, 250 μM dNTPs, 2.0 mM MgCl2, and 2.5 U of TaqDNA polymerase (Applied Biosystems), were added directly to the pellet before initiating the amplification reaction using a using a PTC 200 (MJ Research).

A template for PCR amplification was obtained from a subset of 50 samples collected in Mae La and 50 parasite isolates originating from Ratchaburi and Kanchanburi, as described above. The polymorphic regions present in three unlinked single-copy P. falciparum genes were as follows: block 2 of the merozoite surface protein 1 (msp1), block 3 of the merozoite surface protein 2 (msp2), and the RII region of the glutamate rich protein (glurp) were amplified according to a previously published nested PCR protocol (11). The products were electrophoresed on 3% MetaPhor gels in Tris-borate-EDTA buffer, and the DNA was visualized on an UV transilluminator following ethidium bromide staining. Fragment sizes were assigned to different bin sizes visually; these were labeled starting with A for the largest size, then B for the next size, and so forth. Multiplicity of infection of a sample represented the highest number of distinguishable allelic variants (i.e., resolvable bands of amplified product) observed for a given genetic marker.

In an initial study, fragments corresponding to the 3′-end region of pfcs, obtained by PCR amplification from 95 randomly selected samples, were cloned and sequenced. This served to assess the frequency of the Th2R and Th3R allelic variants found in parasites from these sites and to validate the use of a high-throughput method based on hybridization with type-specific oligonucleotides (PCR-SSOP), previously developed for the analysis of West African parasites (9), for the assessment of the pfcs diversity in Thai parasites.

Nearly all the point mutations observed were nonsynonymous and confined to the two epitope regions (12). However, although numerous variants were detected, 11 Th2R and 9 Th3R allelic forms, the Th2R*05 and the Th3R*01 variants, as previously classified (9), were present at very high frequencies (84 and 90%, respectively) and the combination Th2R*05 + Th3R*01 (henceforth referred to a *5/*1) accounted for >70% of the fragments sequenced (12). The predominance of the pfcs *5/*1 type was confirmed by PCR-SSOP analysis of all 283 isolates collected, as P. falciparum parasites harboring this specific variant were detected in 81% of the samples. Frequency of the pfcs *5/*1 allele did not vary significantly between the three sites (82, 79, and 77% for the samples collected at Mae La, Ratchaburi, and Kanchanburi, respectively), nor between adults and children or between patients experiencing their first attack and those previously infected one or more times (Table I). Moreover, the use of samples collected from Mae La between 1996 and 2001 provided a clear demonstration of the temporal stability of this high frequency of the pfcs *5/*1 allele (Table I).

Table I.

Frequency of the pfcs gene bearing Th2R*05 and Th3R*01 allelic types

SiteGroupnTh2R*05aTh3R*1b*5/*1
n%n%n%
Ratchaburi Total 140 113 80.7 114 81.4 111 79.3 
 1 episode 44 36 81.2 37 84.1 36 81.2 
 >1 episode 95 76 80.0 78 82.1 74 77.9 
 <18 yearsc 44 40 90.1 39 88.6 39 88.6 
 >19 years 92 69 75.0 73 79.3 68 73.9 
Kanchanaburi Total 43 34 79.1 38 88.4 33 76.7 
 1 episode 27 21 77.8 23 85.2 21 77.8 
 >1 episode 13 10 76.9 11 84.6 10 76.9 
 <18 yearsc 100.0 100.0 100.0 
 >19 years 35 26 74.3 30 85.7 25 71.4 
Mae La Total 100 82 82.0 84 84.0 82 82.0 
 1996 35 26 74.3 28 80.0 26 74.3 
 1999 45 42 93.3 42 93.3 42 93.3 
 2001 20 14 70.0 14 70.0 14 70.0 
 <10 years 53 41 77.4 41 77.4 41 77.4 
 >40 years 47 41 87.2 41 87.2 41 87.2 
SiteGroupnTh2R*05aTh3R*1b*5/*1
n%n%n%
Ratchaburi Total 140 113 80.7 114 81.4 111 79.3 
 1 episode 44 36 81.2 37 84.1 36 81.2 
 >1 episode 95 76 80.0 78 82.1 74 77.9 
 <18 yearsc 44 40 90.1 39 88.6 39 88.6 
 >19 years 92 69 75.0 73 79.3 68 73.9 
Kanchanaburi Total 43 34 79.1 38 88.4 33 76.7 
 1 episode 27 21 77.8 23 85.2 21 77.8 
 >1 episode 13 10 76.9 11 84.6 10 76.9 
 <18 yearsc 100.0 100.0 100.0 
 >19 years 35 26 74.3 30 85.7 25 71.4 
Mae La Total 100 82 82.0 84 84.0 82 82.0 
 1996 35 26 74.3 28 80.0 26 74.3 
 1999 45 42 93.3 42 93.3 42 93.3 
 2001 20 14 70.0 14 70.0 14 70.0 
 <10 years 53 41 77.4 41 77.4 41 77.4 
 >40 years 47 41 87.2 41 87.2 41 87.2 
a

The Th2R*5 epitope has the following amino acid sequence: PSDKHIEQYLKKIQNSL.

b

The Th3R*1 epitope has the following amino acid sequence: NKPKDELDYEND.

c

The age distribution of the younger patients recruited in Ratchaburi was as follows: 8–10 years old, n = 8; 11–15 years old, n = 20; and 16–18 years old, n = 16. Six of the seven younger patients recruited in Kanchanaburi were 17–18 years old, and the last one was 13 years of age.

The marked geographical and temporal stability of the predominance of a single allelic pfcs type in this large number of samples was unexpected, especially when it was maintained across all age groups. Indications of restricted diversity were obtained in some of the previous surveys of pfcs polymorphism, although the number of isolates analyzed were low. Nineteen of the 23 parasite isolates collected from diverse areas in Brazil were found to be of the *01/*01 type, the remainder being *04/*01 (13). P. falciparum parasites in 22 isolates collected from widely separated areas in Papua New Guinea were all found to be of the pfcs *5/*4 Th2R/Th3R type (14). The single survey that had been conducted in Thailand previously, with samples collected in 1988–1989 from the same district as that of Mae La, was of particular interest because 12 of the 19 (63%) P. falciparum isolates were also found to be of the pfcs *5/*1 allelic type (15). These results contrast with the unrestricted pfcs diversity noted for African parasites. In the initial Gambian study, seven Th2R and six Th3R allelic types were detected in only five clinical isolates (16). Multiple types were also found in isolates collected from 93 children in another Gambian village, and most parasites were randomly distributed among 8 nearly equally prevalent pfcs Th2R or Th3R variants (9). Because the potential for sexual recombination is expected to decrease with infrequent passage through the mosquito vector (17), these two patterns of diversity were interpreted as resulting from variations in the overall genetic diversity of P. falciparum populations as function of the transmission intensities in these sites. Our observations would be consistent with this interpretation because relatively low numbers of infective bites per person per year (0.1–10) are calculated for the Thai sites. The overall genetic diversity had not been determined for the parasites analyzed in the pfcs surveys conducted in Brazil and Papua New Guinea; however, there were indications from the initial study in Thailand that the parasites bearing the same pfcs type were genetically heterogeneous (15).

Therefore, we selected 55 samples collected from Mae La, 41 of which were classed as harboring *5/*1 parasites, and conducted genotyping based on three standard P. falciparum polymorphic molecular markers (11). The extent of parasite genetic diversity was high because numerous distinct allelic variants were observed for each of the markers (12, 13, and 7 for msp1, msp2, and glurp, respectively). Remarkably, each of the samples analyzed exhibited a unique genotyping pattern (Table II), and in 32 of the 55 samples, the parasite population was probably clonal (with a single allelic variant found for each of the genetic markers). The proportion of mixed genotypes and the frequency distribution of the distinct allelic forms did not differ between the *5/*1 parasites and those bearing other pfcs types. A similar finding was obtained for a subset of 50 samples collected from Ratchaburi (data not shown).

Table II.

Genotypes of P. falciparum isolates collected in Mae La

YearAge (years)Th2R/Th3Rmsp1msp2glurpMOI
K1MAD20R033FC27IC
1996 05/01    
1996 05/01   
1996 05/01    
1996 05/01    
1996 10 05/01    
1996 10 05/01    
1996 42 05/01   D+K 
1996 44 05/01    
1996 46 05/01    
1996 46 05/01    D+K 
1996 52 05/01   A+B  
1996 56 05/01    
1999 05/01    
1999 05/01   D+G C+D 
1999 05/01    
1999 05/01    
1999 05/01 B+D A+B E+G C+D 
1999 05/01    
1999 05/01 B+C   
1999 05/01 E+F A+B 
1999 05/01  A+B 
1999 41 05/01    
1999 41 05/01    
1999 44 05/01    
1999 45 05/01    
1999 46 05/01  A+C   
1999 50 05/01    
1999 50 05/01    
1999 50 05/01    
2001 05/01    
2001 05/01  E+G   
2001 05/01    
2001 05/01    E+K 
2001 05/01    E+K 
2001 10 05/01    C+K 
2001 42 05/01    
2001 43 05/01  B+D  D+F 
2001 47 05/01    A+B 
2001 48 05/01    
2001 52 05/01 D+E  B+C 
2001 54 05/01    
1996 12/09    
1996 66 12/09    
2001 12/09    
2001 10 12/09    
2001 10 12/17    
1996 10 15/10    
1996 10 15/10  
1996 10 15/10  
2001 42 15/10   A+E+K 
1996 18/01  C+G  A+E 
1996 49 18/01    
2001 44 18/01  D+F  
2001 44 21/01  E+G 
2001 60 22/01    
YearAge (years)Th2R/Th3Rmsp1msp2glurpMOI
K1MAD20R033FC27IC
1996 05/01    
1996 05/01   
1996 05/01    
1996 05/01    
1996 10 05/01    
1996 10 05/01    
1996 42 05/01   D+K 
1996 44 05/01    
1996 46 05/01    
1996 46 05/01    D+K 
1996 52 05/01   A+B  
1996 56 05/01    
1999 05/01    
1999 05/01   D+G C+D 
1999 05/01    
1999 05/01    
1999 05/01 B+D A+B E+G C+D 
1999 05/01    
1999 05/01 B+C   
1999 05/01 E+F A+B 
1999 05/01  A+B 
1999 41 05/01    
1999 41 05/01    
1999 44 05/01    
1999 45 05/01    
1999 46 05/01  A+C   
1999 50 05/01    
1999 50 05/01    
1999 50 05/01    
2001 05/01    
2001 05/01  E+G   
2001 05/01    
2001 05/01    E+K 
2001 05/01    E+K 
2001 10 05/01    C+K 
2001 42 05/01    
2001 43 05/01  B+D  D+F 
2001 47 05/01    A+B 
2001 48 05/01    
2001 52 05/01 D+E  B+C 
2001 54 05/01    
1996 12/09    
1996 66 12/09    
2001 12/09    
2001 10 12/09    
2001 10 12/17    
1996 10 15/10    
1996 10 15/10  
1996 10 15/10  
2001 42 15/10   A+E+K 
1996 18/01  C+G  A+E 
1996 49 18/01    
2001 44 18/01  D+F  
2001 44 21/01  E+G 
2001 60 22/01    

Taken together, the genetic analysis of the samples from Thailand demonstrated a strong and stable fixation of a particular pfcs allelic variant in an otherwise highly genetically diverse population of P. falciparum parasites, a pattern hitherto undetected for other polymorphic Ags. This observation is inconsistent with prevalent concepts of the role played by naturally acquired protective immunity in the selection of polymorphisms in the CS T cell immunodominant epitopes (18). The data presented does not exclude an important role for the CS Ag in natural or induced immune responses against PE parasite forms. Nonetheless, it does suggest that natural protective immune responses that depend on the Th2R and Th3R epitopes, or the memory of these, are likely to be short-lived. This contention is consistent with the low frequency of PE-specific T cells in immune adult Africans (19), and with the results from the first efficacy field trial of the RTS,S vaccine, a formulation based on the repeated central part and the C terminus of the CS Ag. In the latter trial, conducted in Gambian adults, protection against clinical attacks was induced relatively efficiently, although the duration of the protective effect waned 2 mo following the last injection (4). Associated molecular analysis of the pfcs Th epitope frequencies did not reveal any evidence that variants that differ from the type present in the formulation were selected against in the vaccinees (20). In a recent extensive analysis of cellular responses in the protected individuals (21), it was found that protection was correlated with responses stimulated by an invariant T cell epitope but not with those linked to the polymorphic Th epitopes. In light of these observations, our data suggest that immune pressure is unlikely to play any significant role in selecting and maintaining the extensive polymorphisms that occur naturally in the CS Th2R and Th3R epitopes. Consequently, these polymorphisms would no longer have to be considered as a potential serious obstacle to confound the universal efficacy of a CS-based vaccine, and the fear that escape variants insusceptible to a particular vaccine formulation would be minimized. This bodes well for experimental vaccines based on the CS Ag.

The fact that P. falciparum parasites bearing the pfcs *5/*1 allelic variant were specifically maintained at high frequency in a large endemic area across time not only attests their transmission efficacy but also denotes that a strong selective pressure is exerted on these two stretches of the CS Ag. We propose that selection is of a biological nature and is related to a functionally significant role of these regions in sporozoite biology. Elegant investigations of have revealed a functional role for CS. In the mosquito, the presence of CS is needed for oocyst development (22), and it is centrally involved in the gliding motility of the sporozoite (23) necessary for salivary gland invasion. In the mammal, the CS protein interacts with proteoglycans associated to the surface of the hepatocyte (24) and is thus implicated in the invasion of these cells by the parasite. The reduction in the binding to hepatoma cells of a CS protein with a deleted Th3R epitope (25) had already prompted the suggestion that selection is not solely linked to immunity. Nonetheless, evidence suggestive of a strong selective pressure in the vertebrate host is not available. Indeed, major variations in receptivity to sporozoites or indications of insusceptibility of some individuals but not others to the sporozoites of a particular parasite line, both indicative of such selection, were not noted in the carefully observed extensive series of sporozoite-induced experimental infections conducted on malariotherapy patients, prisoner volunteers, and more recently on volunteers in vaccination trials. On the other hand, there are clear examples of parasite strains refractory to transmission by, or poorly infective to, one but not another species of Anopheles (26, 27). Evidence that the CS protein might be directly implicated in the differential susceptibility of anopheline species was provided by experimental and epidemiological observations of a contrasting transmission efficacy of Plasmodium vivax parasites bearing the VK210 or the VK247 repeat types by two distinct Anopheles species in Mexico (28). Therefore, we propose that substantial selective pressure on CS is exerted in the mosquito host. This would predict that different parasite lines would exhibit a particular degree of adaptation to anopheline species with which they are coindigenous. This is consistent with the predominance of different Th2R/Th3R allelic types in Brazil (13), Papua New Guinea (14), and Thailand (12), where different Anopheles species act as major carriers. The less restricted pfcs epitope diversity in the African parasites might result from a relative insensitivity of the dominant vector species, Anopheles gambiae and Anopheles funestus, to the pfcs polymorphisms. This would account for the recognized supreme efficiency of A. gambiae as a vector for P. falciparum. It would be of great interest to conduct an analysis, akin to the one presented here, using samples collected from an African hyperendemic area. However, the blood-stage parasites collected must be derived from recently inoculated sporozoites. Thus, the cohort under study must first be cleared of chronic parasitemia and the individuals closely monitored for the appearance of the first parasitemic episode. Ultimately, formal evidence for the hypothesis that selective pressure is exerted in the mosquito will require comparative experimental infections of different species of Anopheles mosquitoes with P. falciparum parasite strains that differ genetically only with respect to the Th2R and Th3R epitopes.

In conclusion, the data presented here challenges the role of immune pressure as the major selective force leading to the generation and maintenance of polymorphism in an acknowledged immunodominant and established vaccine candidate plasmodial Ag. The proposal that the selection acts at the level of the parasite survival in the mosquito host is consistent with disparate observations of Plasmodium infections. A variety of polymorphisms characterize a large number of other parasite proteins, with incompletely known function, that are leading vaccine candidates. Recent investigations are revealing that many of these Ags, hitherto considered as specific to a single parasite stage, are actually expressed in the blood and the PE stages (29). Given the serious implications of antigenic diversity to the development and deployment of an effective malaria vaccine, the nature of the selective pressures acting to maintain these polymorphisms merits investigation in the context of both the vertebrate and insect hosts of Plasmodium.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by a grant from VIHPAL of the Ministère de la Recherche, France, and in part by the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, and The Wellcome Trust. C.K. was supported by a Basic Research grant from the Royal Golden Jubilee Ph.D. Program of the Thailand Research Fund and a grant from Mahidol University.

4

Abbreviations used in this paper: CS, circumsporozoite protein; PE, pre-erythrocytic; SSOP, sequence-specific oligonucleotide probe.

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