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Keywords:

  • malaria;
  • Plasmodium falciparum;
  • circumsporozoite protein;
  • T-helper cell epitope;
  • polymorphism
  • malaria;
  • Plasmodium falciparum;
  • protéine circumsporozoïte;
  • épitope de cellules T-helper;
  • polymorphisme
  • Malaria;
  • Plasmodium falciparum;
  • proteína del circumesporozoito;
  • epítope de CD4 + ;
  • Polimorfismo

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective  To investigate the extent of genetic variations in T-helper-cell epitopic regions of circumsporozoite (CS) protein in Plasmodium falciparum field isolates collected from different regions of India at different phases of malaria transmission.

Methods  Genomic DNA was isolated from 507 P. falciparum wild-parasite isolates obtained from six geographical locations of India at three time points coinciding with malaria transmissions. The T-helper-cell epitopic regions were polymerase chain reaction (PCR)-amplified and the products were purified and then sequenced.

Results  Based on sequences, nine variants were found among isolates and they were categorized into nine groups (V-1 to V-9), where V-1 and V-2 were observed in all three time points (TP). The variants V-1 to V-4 in TP-1; V-1, V-2, V-5 to V-8 in TP-2; and V-1, V-2, V-5 and V-9 in TP-3 were present and they showed restricted heterogeneity. During peak transmission (TP-2), parasite populations were more diverse and heterogeneous and the variants regionally unbiased and restricted. However, the alleles of V-6 and V-9 in both Th2R and Th3R showed identical sequence variation with those observed in other geographical regions of the world. The remaining seven groups did not show such similarity.

Conclusion  The Th2R and Th3R epitopes are implicated in host immune response to P. falciparum. The polymorphism in these epitopic regions indicates antigenic diversity, which may cause adverse outcome of a subunit vaccine including the CS prototype variant. Therefore, the formulation of a vaccine considering the restricted local repertoire parasite populations may be helpful.

Protéine circumsporozoïte de Plasmodium falciparum: les variations épidémiologiques entre les isolats répandus en Inde

Objectif:  Investiguer l’ampleur des variations génétiques dans les régions épitopiques de la protéine circumsporozoïte (CS) des cellules T-helper dans des isolats de Plasmodium falciparum provenant de différentes régions de l’Inde à différentes phases de la transmission de la malaria.

Méthodes:  De l’ADN génomique a été isoléà partir de cinq cent sept (507) parasites sauvages de P. falciparum obtenus dans six zones géographiques de l’Inde à trois périodes de temps coïncidant avec la transmission de la malaria. Les régions épitopiques des cellules T-helper ont été amplifiées par PCR et les produits ont été purifiés puis séquencés.

Résultats:  Sur la base de séquences, neuf variantes ont été trouvées parmi les isolats et ont été classifiées en neuf groupes (V-1 à V-9), où V-1 et V-2 ont été observées dans les trois périodes de temps (TP). Les variantes V-1 à V-4 étaient présentes dans le TP-1; V-1, V-2 et V-5 à V-8 dans le TP-2; V-1, V-2, V-5 et V-9 dans le TP-3, et elles démontraient une hétérogénéité restreinte. Durant le pic de transmission (TP-2), les populations de parasites étaient plus variées et hétérogènes et les variantes étaient non biaisées et restreintes régionalement. Cependant, les allèles de V-6 et V-9 à la fois dans les épitopes Th2R et Th3R montraient la même variation de séquence identique à celles observée dans d’autres régions géographiques du monde. Les sept autres groupes n’ont pas révélé cette similarité.

Conclusion:  Les épitopes Th2R et Th3R sont impliqués dans la réponse immunitaire de l’hôte àP. falciparum. Le polymorphisme dans ces régions épitopiques indique une diversité antigénique, qui pourrait mener à des résultats défavorables pour un vaccin à sous-unités comprenant une variante prototype CS. Par conséquent, la formulation d’un vaccin en tenant compte du répertoire local limité des populations de parasites pourrait être utile.

Proteína de circumsporozoito de Plasmodium falciparum: variaciones epidemiológicas entre aislados de campo prevalentes en India

Objetivos:  Investigar la extensión de la variación genética en regiones epitópicas de CD4 +  de la proteína del circumesporozoito (CS) en aislados de Plasmodium falciparum recolectados de diferentes regiones de la India en diferentes fases de transmisión de malaria.

Métodos:  Se aisló ADN genómico de quinientos siete (507) aislados de P. falciparum obtenidos de seis lugares geográficos de la India en tres momentos diferentes, coincidiendo con la transmisión de malaria. Las regiones epitópicas de CD4 +  fueron amplificadas mediante PCR y los productos purificados y secuenciados.

Resultados:  Basándose en la secuencia, se encontraron nueve variantes entre los aislados y fueron categorizados en nueve grupos (V-1 a V-9), donde V-1 y V-2 fueron observados en los tres puntos de tiempo (PT). Las variantes V-1 a V-4 en PT-1; V-1, V-2, V-5 a V-8 en PT-2; y V-1, V-2, V-5 y V-9 en PT-3 estaban presentes y mostraban un heterogeneidad restringida. Durante el pico de transmisión (PT-2), las poblaciones parasitarias eran más diversas y heterogéneas y las variantes regionales sin sesgo y restringidas. Sin embargo, los alelos de V-6 y V-9 tanto en Th2R como Th3R mostraron una variación de secuencia idéntica con aquellos observados en otras regiones geográficas del mundo. Los siete grupos restantes no mostraron dicha similitud.

Conclusión:  Los epítopes Th2R y Th3R estaban implicados en la respuesta inmune del hospedero P. falciparum. El polimorfismo en estas regiones epitópicas indican diversidad antigénica, lo cual puede causar resultados adversos de una subunidad de vacuna, incluyendo el prototipo variante CS. Por lo tanto, la formulación de una vacuna teniendo en cuenta el repertorio local restringido de la población parasitaria podría ser útil.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Malaria caused by Plasmodium falciparum remains a serious disease in the tropical countries, endangering the life and development of infants and young children (Nwagwu et al. 1998). The control of malaria is mainly based on prompt treatment of febrile patients and vector control using insecticides. However, these control measures cannot be sustained because of the development of parasite resistance to commonly used antimalarials and development of insecticide resistance in anopheline vectors (Marsh 1992). Efforts are being made to develop suitable, effective subunit vaccines against malaria and some of them have undergone human trials also (Ballou & Cahill 2007). So far not a single vaccine formulation has been found to be fully effective to combat natural or challenge infection against overall P. falciparum populations.

Among all stage-specific antigens that have been identified for vaccine formulations, the circumsporozoite (CS) protein is the leading candidate. The CS protein is the predominant protein found on the surface of sporozoite; it has approximately 420 amino acids with a molecular weight of 58 kDa (Escalante et al. 2002). Sequence analysis of P. falciparum CS gene showed two non-repetitive regions at 5′ and 3′ ends and a variable central region containing immunodominant B-cell epitopes with multiple repeats of four amino acid motifs (McCutchan et al. 1989). The C-terminal part of the sequence contains two polymorphic T-cell epitopic regions, Th2R and Th3R, flanking the highly conserved RII region (Figure 1) and spanning amino acid residues from 326 to 343 (Th2R) and 361 to 380 (Th3R), respectively (Good et al. 1987; De la Cruz et al. 1988; Jalloh et al. 2006).

image

Figure 1.  Schematic diagram of the P. falciparum circumsporozoite protein (CSP) and sequence of the oligonucleotide primers, P1 and P2 for amplification of the T-cell epitopes Th2R and Th3R. The sequences of RI and RII regions are highly conserved among the CSP of different Plasmodia. Two flanking non-repeat regions contain polymorphic T-cell determinants (Dame et al. 1984).

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Extensive polymorphism found in the T-helper-cell epitope raised doubts about the feasibility of a T-helper-cell epitope-based vaccine. The variants can be categorized into groups, which renewed hopes about the possibility of a T-cell epitope-based subunit polyvalent vaccine (Doolan et al. 1992). However, if the variations are restricted and are regionally unbiased, then the prototype variants could be included into a subunit polyvalent vaccine against P. falciparum. A recent study on vaccine formulation RTS,S based on a section of the CSP including the T-cell epitope has claimed protection against sporozoite challenge (Ballou et al. 1987; Allouche et al. 2000). Although the data of variations in T-helper-cell epitopic regions are available from other geographic regions of the world, the data from India, where malaria situations are very critical, are scanty. India is geographically diverse in terms of topography, climate, vector availability and malaria endemicity. Hence we aimed at assessing the extent of variations in CSP-Th2R and Th3R of P. falciparum isolates prevalent at different geographic locations and at different transmission periods.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study site

This study was conducted in six locations of India (Figure 2), two northern (New Delhi/Delhi and Ghaziabad/Uttar Pradesh), one north-eastern (Guwahati/Assam), two eastern (Kolkata/West Bengal and Sundargarh/Orissa), and one central (Jabalpur/Madhya Pradesh) areas. Although these six areas are endemic for malaria, they are different as per natural climate, rainfall, vegetation, etc.

image

Figure 2.  Map of India showing locations of six study areas. •, Delhi; bsl00066, Uttar Pradesh; inline image, Madhya Pradesh; ◆, Orissa; bsl00001, Assam; inline image, West Bengal.

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The principal vector of urban malaria in both Delhi and Kolkata is thought to be Anopheles stephensi. Early and prolonged monsoons are responsible for transmission of both Plasmodium vivax and P. falciparum (Hati et al. 2000; Biswas et al. 2001).

The Ghaziabad district of Uttar Pradesh in northern India is endemic for malaria, having seasonal transmission of both P. vivax and P. falciparum. This area also shows intensive transmission of both the species during early or late rainy seasons. In this area, among the anopheline population, An. culicifacies is the most abundant vector followed by An. annularis and An. subpictus (Biswas 2000).

The north-eastern area is highly endemic for malaria and is known for its high rainfall and persistent malaria transmission. More than 70% of malaria infections are due to P. falciparum and this species is responsible for enhanced morbidity and mortality. An. minimus is the major vector species, which is highly anthropophilic thus responsible for enhanced transmission. In this area, P. falciparum transmission is perennial (Dev et al. 2004).

The eastern area (Sundargarh/Orissa) is characterized by a tropical climate and receives high rainfall from the south-east monsoon and the retreating north-east monsoon. About 34% of the areas are covered with forest. P. falciparum causes more than 90% of the total malaria cases and number of deaths is also high. The whole district is under the influence of two malaria vectors, An. culicifacies and An. fluviatilis. The peak prevalence period of An. culicifacies is between April and September, whereas for An. fluviatilis, it is during October to January (Sharma et al. 2006; Biswas et al. 2008).

In central India, P. vivax and P. falciparum are prevalent in people of all age groups, especially in older children (Singh et al. 2000). In this area, malaria is highly seasonal; P. vivax cases are common during the dry season (February to June), whereas P. falciparum cases are available during the post-monsoon period and autumn season (July to January).

Sample collection

National Institute of Malaria Research (NIMR), the Institutional Ethics Committee approved this study. The study began in June 2005 and continued up to January 2008. In order to reduce the patient recruitment time, health centres/dispensaries close to well-defined communities were identified to conduct the activities during the malaria season; mobile clinics were also organized, which received at least 15–20 febrile patients per day. Patients with a history of fever attended malaria and outdoor clinics and were examined for malaria by microscopy. Patients diagnosed with malaria were treated with recommended antimalarials as per National Drug Policy. Patients with P. falciparum infection were enrolled in the study after obtaining their informed written consent. Finger-prick blood samples from the study patients, belonging to all age groups, were collected in Eppendorf tubes with anticoagulant and were stored at −20 °C until use. Blood samples were collected at three consecutive time points based on the intensity of transmission, i.e. Time point-1 (TP-1) from June to August; Time point-2 (TP-2) from September to November; Time point-3 (TP-3) in December and January.

Preparation of genomic DNA

Genomic DNA was prepared from parasitized blood samples by a modification of the method described previously (Foley et al. 1992). Essentially, 50 μl of blood samples were washed three times, each time by vortexing in 1 ml of ice-cold 5 mm di-sodium phosphate buffer (pH 8.0) and then centrifuging (10 000 g, for 10 min at 4 °C). The final pellet was suspended in 50 μl of sterile distilled water, heated in a boiling water bath for 20 min, cooled slowly at room temperature and centrifuged as before. Forty microlitres of supernatant was taken and 5 μl of this was used in a 20-μl polymerase chain reaction (PCR) mixture.

Polymerase chain reaction

The oligonucleotide primers, P1 and P2 (Figure 1), used for PCR to amplify the T-cell epitopic region correspond to nucleotide 1008–1028 and 1323–1347 in the sequence of 7G8 clone, respectively (Lockyer et al.1989). In addition to the genomic DNA preparation, each reaction mixture contained 50 pm each of the forward and reverse primers, 10 mm each of deoxynucleotide triphosphates and 3 U Taq polymerase (Bangalore Genei, Bangalore, India). Amplification consisted of denaturation at 95 °C for 2 min, annealing at 55 °C for 1 min, amplification at 72 °C for 3 min for 30 cycles and final extension at 72 °C for 10 min. The PCR-amplified products were purified using a commercial QIA quick® PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.

DNA sequencing

Purified PCR-amplified products of T-helper-cell epitopic regions were sequenced (using the same primers used to amplify the T-helper-cell epitopic regions) in an automated sequencer (Genetic Analyzer 310, ABI-PRISM 377; Applied Biosystems, Foster City, CA, USA) following manufacturer’s protocol.

Data analysis

The nucleotide sequences were aligned and the deduced amino acid residues numbered, taking 7G8 sequences as the standard (Dame et al.1984). The nucleotide sequences obtained from six geographical regions at the three time points were analysed to compare and estimate the levels of significance for geographic and period variations using statistical tools available at http://www.physics.csbsju.edu. The significance of group differences was calculated by using the one-way anova by testing the null hypothesis among the nucleotide sequences of no genetic differentiation among the parasite populations in a given time point irrespective of differences in geographic locations. For all sets of comparisons, P-values <0.05 were considered significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Parasitized blood samples obtained from symptomatic P. falciparum malaria patients were used for PCR amplification of the T-helper-cell epitope of CSP gene directly without in vitro growth to avoid possible selection of some of the clones during culture. Parasitaemia in these samples varied from 1000 to 100 000 parasites/μl of blood. Altogether, 507 P. falciparum isolates from different geographical regions of India, viz. Delhi (DL), Orissa (OR), Uttar Pradesh (UP), Assam (AS), Madhya Pradesh (MP) and West Bengal (WB) were studied at three time points of transmission period. Of the 507 P. falciparum isolates, 157 were collected during the first time point (June–August), 213 during the second time point (September–November) and 137 during the third time point (December–January). The DNA and deduced amino acid sequences of Th2R and Th3R of individual isolates were compared with 7G8, LE5 and 3D7 sequences (Tables 1–3). We observed that polymorphisms resulted mainly from nucleotide substitution in the first codon and less frequently in the second position. Nearly all the point mutations were found to be non-synonymous and confined to both Th2R and Th3R. It is interesting to note that all the isolates in the present study that share identical Th2R sequences also share the identical Th3R sequence. The sequence variations in Th2R and Th3R are linked in each isolate, thus allelic linkage has been observed. Among the 507 isolates collected over a span of 3 years at three time points, nine Th2R and eight Th3R allelic forms were found. The variants were found to be restricted into four groups in TP-1 (V-1, V-2, V-3 and V-4) and TP-3 (V-1, V-2, V-5 and V-9) and six groups in TP-2 (V-1, V-2, V-5, V-6, V-7 and V-8).

Table 1.   Nucleotide sequence variation in the Th2R and Th3R epitopic regions of circumsporozoite gene and categorization of sequences into groups (Time point 1)
 326Th2R342361Th3R380
  1. V, variant.

  2. V-1 (n = 47): DL (7), UP (7), MP (6), OR (10), AS (9), WB (8); V-2 (n = 49): DL (8), UP (9), MP (5), OR (12), AS (10), WB (5); V-3 (n = 35): DL (5), UP (4), MP (5), OR (9), AS (6), WB (6); V-4 (n = 26): DL (3), UP (4), MP (3), OR (7), AS (6), WB (3).

  3. DL, Delhi; UP, Uttar Pradesh; MP, Madhya Pradesh; OR, Orissa; AS, Assam; WB, West Bengal.

  4. Numbers in parentheses against each state indicate the number of isolates from that region.

  5. Dashes indicate identical amino acid residues.

7G8PSDKHIEQYLKKIKNSISIKPGSANKPKDELDYENDIE
LE5QKTQLQA
3D7KENQLA
V-1QQKKKNTQLDQQD
V-2QQKKKENIQLDQRAD
V-3QENTQLQEENTQD
V-4QENTQLQDAD
Table 2.   Nucleotide sequence variation in the Th2R and Th3R epitopic regions of circumsporozoite gene and categorization of sequences into groups (Time point 2)
 326Th2R342361Th3R380
  1. V, variant.

  2. V-1 (n = 40): DL (4), UP (5), MP (6), OR (8), AS (9), WB (8); V-2 (n = 29): DL (4), UP (4), MP (5), OR (6), AS (6), WB (4);V-5 (n = 23): DL (4), UP (3), MP (3), OR (7), AS (4), WB (2); V-6 (n = 42): DL (8), UP (5), MP (5), OR (9), AS (9), WB (7); V-7 (n = 37): DL (5), UP (4), MP (4), OR (8), AS (10), WB (6); V-8 (n = 42): DL (6), UP (7), MP (5), OR (10), AS (7), WB (7).

  3. DL, Delhi; UP, Uttar Pradesh; MP, Madhya Pradesh; OR, Orissa; AS, Assam; WB, West Bengal.

  4. Numbers in parentheses against each state indicate the number of isolates from that region.

  5. Dashes indicate identical amino acid residues.

7G8PSDKHIEQYLKKIKNSISIKPGSANKPKDELDYENDIE
LE5QKTQLQA
3D7KENQLA
V-1QQKKKNTQLDQQD
V-2QQKKKENIQLDQRAD
V-5QKRQLA
V-6QLA
V-7QKNTQLDND
V-8QQLDQA
Table 3.   Nucleotide sequence variation in the Th2R & Th3R epitopic regions of circumsporozoite gene and categorization of sequences into groups (Time point 3)
 326Th2R342361Th3R380
  1. V, variant.

  2. V-1 (n = 39): DL (5), UP (4), MP (6), OR (9), AS (8), WB (7); V-2 (n = 30): DL (4), UP (4), MP (5), OR (6), AS (6), WB (5); V-5 (n = 25): DL (2), UP (3), MP (3), OR (4), AS (7), WB (6); V-9 (n = 43): DL (8), UP (7), MP (5), OR (9), AS (9), WB (5).

  3. DL, Delhi; UP, Uttar Pradesh; MP, Madhya Pradesh; OR, Orissa; AS, Assam; WB, West Bengal.

  4. Numbers in parentheses against each state indicate the number of isolates from that region.

  5. Dashes indicate identical amino acid residues.

7G8PSDKHIEQYLKKIKNSISIKPGSANKPKDELDYENDIE
LE5QKTQLQA
3D7KENQLA
V-1QQKKKNTQLDQQD
V-2QQKKKENIQLDQRAD
V-5QKRQLA
V-9QKQLN

All nine allelic variants appeared to be regionally unbiased in the sense that a similar type of variants was found in different geographical regions of India (Figure 3). Of the 157 isolates studied during TP-1, 47 (29.9%), 49 (31.2%), 35 (22.3%) and 26 (16.6%) belonged to group V-1, V-2, V-3 and V-4, respectively. Of the 213 isolates studied during TP-2, 40 (18.8%), 29 (13.6%), 23 (10.8%), 42 (19.7%), 37 (17.4%) and 42 (19.7%) belonged to group V-1, V-2, V-5, V-6, V-7 and V-8, respectively. Of the 137 isolates studied during TP-3, 39 (28.5%), 30 (21.9%), 25 (18.2%) and 43 (31.4%) belonged to V-1, V-2, V-5 and V-9, respectively. Their frequency distribution in different regions at three time points is shown in Figure 4. The differences in distribution patterns of different alleles among the parasite populations of six areas in a given time point were significant. The allelic forms of groups V-1 and V-2 were prevalent throughout the season in six areas; only frequency distribution was variable at three time points. During TP-1, of the four variant forms, V-1 and V-2 were predominant; also V-3 was found in more isolates compared with V-4 (P = 0.011). Six variants were available in TP-2, where V-1 and V-2 were common.

image

Figure 3.  Distribution of P. falciparum circumsporozoite protein T-helper-cell epitopic alleles at three different time points. V, variant; TP, time point. Numbers in parentheses indicate sample size in each time point.

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image

Figure 4.  Frequency distribution of different T-helper-cell epitope haplotypes (V-1 to V-9) in six regions of India. V, variant; TP, time point; DL, Delhi; UP, Uttar Pradesh; MP, Madhya Pradesh; OR, Orissa; AS, Assam; WB, West Bengal.

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We observed four new variant forms (V-5 to V-8) among the isolates studied in this period. Among the six forms V-1, V-6, V-7 and V-8 alleles were found in more number of isolates in all the areas during TP-2 (P = 0.019). During TP-3, variant forms V-1, V-2 and V-5 were present; we also observed a new allelic form V-9, which was present in most isolates. The overall distribution of these four variants in six areas during TP-3 was significant (P = 0.023). Among the nine allelic forms, some appeared to be new on comparison of T-helper-cell epitopic regions of Pf-CSP gene among global parasite lines and isolates (Tables 4 and 5). As reported earlier (Allouche et al. 2000), several amino acid substitutions were observed at the 3′ end of the T-cell epitope and on alignment of the sequences; the most abundant Th2R allele was PSDKHIEQ/KYLKKIQ/KNS. Similarly in Th3R, the most abundant allele was GSAN/DKPKDE/QLDYEN/D. These alleles were observed in Africa, South America, Southeast Asia and Middle-east Asia. The Th2R sequences of the Indian isolates were more similar to LE5, whereas Th3R sequences were more similar to 7G8, LE5 and 3D7.

Table 4.   Geographical distribution of point mutations in Th2R of CSP among P. falciparum isolates and laboratory lines
328            341ParasiteReference
DKHIEQYLKKIKNS7G8Allouche et al. 2000
QKTQLE5Allouche et al. 2000
KENQ3D7Allouche et al. 2000
TT9-98Lockyer et al. 1989
QKTQ3661:3991-10Allouche et al. 2000; Lockyer et al. 1989
QWel; T9-101; T4R; V-6Lockyer et al. 1989; Doolan et al. 1992; Allouche et al. 2000; Present study
KIt G2 G1Lockyer et al. 1989; Doolan et al. 1992
QKQQ3668,10Lockyer et al. 1989; Allouche et al. 2000
QKQ40610: 4191-9; V-9Lockyer et al. 1989; Allouche et al. 2000; Present study
KENQNF54: 4271-4,6-10Lockyer et al. 1989
NT4275Lockyer et al. 1989
QKRQMS 2Doolan et al. 1992
QKRQBRA 2Allouche et al. 2000
QKQR3662-7Allouche et al. 2000
QKQYThai isolateAllouche et al. 2000
TEQThai II 807,836,837Allouche et al. 2000
QYThai IV 827,835A,842Allouche et al. 2000
KETQThai VII835bAllouche et al. 2000
EQThai V828Allouche et al. 2000
QQKKNTGroup IBhattacharya et al. 2006
QKKENIQGroupIIBhattacharya et al. 2006
QENTQGroup III; V-3Bhattacharya et al. 2006; Present study
QENTQGroup III; V-4Bhatia & Bhattacharya 2006; Present study
QKRQGroup IV; V-5Bhatia & Bhattacharya 2006; Present study
QQKKKNTQV-1Present study
QQKKKENIQV-2Present study
QKNTQV-7Present study
QQV-8Present study
Table 5.   Geographical distribution of point mutations in Th3R of CSP among P. falciparum isolates and laboratory lines
364             378ParasiteReference
GSANKPKDELDYEND7G8Allouche et al. 2000
QALE5, 3661,5,8-10; 3991-10Doolan et al. 1992; Lockyer et al. 1989
A3D7Allouche et al. 2000
AT9-98: NF54:4061,7,8 4271-10:41910; V-5 & V-6Lockyer et al. 1989; Present study
DIt G2 G1:WelLockyer et al. 1989
N3662-4,6,7 40610:4191-9; V-9Lockyer et al. 1989; Present study
R4062Lockyer et al. 1989
D4066,9Lockyer et al. 1989
DQWell, FCR3Allouche et al. 2000; Lockyer et al. 1989
QNGam336b,406b,419Allouche et al. 2000
RAGam 406cAllouche et al. 2000
ADGam 406dAllouche et al. 2000
QAE7,PNG2, Thai VIIIDoolan et al. 1992; Allouche et al. 2000
GSHB3, D10, X5,BRA1Doolan et al. 1992; Allouche et al. 2000
NBra S34, Thai VAllouche et al. 2000
DQCSPNG3Allouche et al. 2000
DThai VIAllouche et al. 2000
GEThai VIIAllouche et al. 2000
DQQDGroup I; V-1Bhattacharya et al. 2006; Present study
DQRADGroup II; V-2Bhattacharya et al. 2006; Present study
QEENTQDGroup IIIBhattacharya et al. 2006
DADGroup IVBhattacharya et al. 2006
QEENTQDV-3Present Study
QDADV-4Present Study
DNDV-7Present Study
DQADV-8Present Study

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The CS protein of P. falciparum is one of the most highly characterized malaria vaccine candidate antigens, whose components have undergone various clinical trials (Nussenzweig & Nussenzweig 1985, 1989; Bojang et al. 2001; Alonso et al. 2004). For any vaccine candidate component, baseline information on naturally acquired immune responses and genetic diversity in the candidate molecule from various geographically diversified areas are required for its inclusion in a successful trial. Although a lot of data have been generated from areas of high malaria transmission, these types of information from low or moderate malaria transmission areas are lacking. From epidemiologic and immunologic standpoints, the development of natural immunity is affected by the complexity/multiplicity of infection that correlates with the genetic diversity of parasites in a given situation of malaria transmission. Keeping this in view, we investigated the extent of genetic variations in T-helper-cell epitopic regions of CS protein in P. falciparum isolates prevalent in India. The study was undertaken based on earlier observations that P. falciparum parasites display genetic diversity in regions where malaria transmission is low or moderate (Bhattacharya et al. 2006; Anitha et al. 2007; Zakeri et al. 2007).

Malaria prevalence correlates with transmission intensity. Therefore, increased transmission enhances the parasite reproduction rate. In areas with high malaria transmission, the parasite populations remain diversified, to avoid selection pressure, by exhibiting polymorphisms. The P. falciparum parasite populations of Africa exhibit a high degree of genetic diversity compared with other parts. This could explain why in Africa malaria transmission intensity is very high compared with that in South America, Southeast Asia and Middle-east Asia (Jongwuitiwes et al. 1994; Escalante et al. 2002; Zakeri et al. 2007). High genetic diversity in CS protein may be due to intragenic recombination and causes generation of new alleles on the T-cell epitopes to make the parasites more adjustable during high transmission (Lockyer 1991). In regions of low-to-moderate malaria endemicity, a representative population of major allelic forms along with minor variants in different parasite populations may exist (Kumkhaek et al. 2005).

It seems that CS protein is under positive natural selection; therefore it is possible that convergent substitutions of amino acids may happen (Escalante et al.1998). In the CSP gene, most of the sequence variations lie within the B- and T-cell epitopic regions of which the 3′ position is highly polymorphic (Escalante et al. 2002). The epitope-specific T cells may modulate their effector functions by altered peptide ligand (APL) antagonism (Plebanski et al. 1997). The coexistence of closely related variants may provide an altered activation signal to the T cells causing inactivation of some of its effector functions. All changes were non-synonymous mutations resulting in changes in amino acids and there was total absence of synonymous mutations as reported earlier (Rich et al. 1997). Within Th2R and Th3R epitopes, mutations appeared in certain positions of amino acids by both hydrophilic and hydrophobic replacements. The presence of non-synonymous mutations in the immunodominant T-cell epitopic region of Pf-CSP gene suggests that the mutations were a result of selection pressure exerted by the T cells (De la Cruz et al. 1987). This is applicable for four new variants (V-5 to V-8) that appeared during peak transmission period.

However, the prevalence of non-synonymous mutations V-1 and V-2 during all three time points in six locations seems to be due to the propagation of these forms in population rather than phenotypic selection (Arnot 1989; Caspers et al. 1989). Previous studies demonstrated the role of T- and B-cell-mediated immunity in malaria and how the non-synonymous mutations resulting in amino acid changes may affect the epitopic conformation and the consequences of immune responses in individuals residing in malaria-endemic areas (Kumar et al. 2006). Sequence analysis of natural parasite populations in a given area may predict the outcome of vaccination (Weedall et al. 2006).

The wild isolates collected directly from patients contain a heterogeneous population of parasites; a mixture of several genotypes, which is a common feature of endemic areas (Plebanski et al. 1997; Allouche et al. 2000). The degree of heterogeneity in Th2R and Th3R in P. falciparum parasites of an endemic region in a given locality and season is considerably higher. The Pf-CSP gene displayed restricted diversity at Th2R and Th3R epitopes. An earlier study from India reported only four haplotypes of Th2R and Th3R (Bhattacharya et al. 2006). In our study, nine Th2R and eight Th3R haplotypes were observed in clinical isolates collected at three time points from six areas. P. falciparum isolates collected from different parts of Africa showed high polymorphism in CSP-Th2R and Th3R (Escalante et al. 2002). Studies from other parts of the globe, such as Brazil, Papua New Guinea, Vanuatu, Thailand and Myanmar demonstrated that Th2R and Th3R polymorphisms in P. falciparum isolates of these areas are relatively less frequent than in Africa (Escalante et al. 2002; Jalloh et al. 2006). We observed that T-cell epitopic diversity is not very high, taking into consideration geographical and temporal variations. The dominant Th2R and Th3R haplotypes V-1 and V-2 are present in the majority of the isolates, as reported in earlier studies (Yoshida et al. 1990; Shi et al. 1992). Prevalence of these types of similar haplotypes in parasite populations may be due to the transmission through anopheline vectors locally. The availability of four new forms (V-5 to V-8) in TP-2 coincides with peak P. falciparum transmission. These CSP variants in TP-2 may not be the target of the immune system but can be propagated in the population through the local anopheline vectors responsible for malaria transmission (Kumkhaek et al. 2005).

Our results suggest that variations in CSP-Th2R and Th3R epitopes may not be due to frequency-dependent selection, as the frequency of predominant alleles was more or less stable over the study period. Similar observations have been made from Vietnam and The Gambia for the P. falciparum erythrocytic-stage merozoite surface protein (Conway 1997; Ferreira et al. 1998). Moreover, our findings demonstrate the temporal patterns of CSP-T epitope diversity in Indian isolates prevalent in areas with different malariogenic conditions. The observed polymorphisms in CSP gene could be due to the selective pressure of the host immune system, thus causing constraints on parasites for evolving immune evasion (Plebanski et al. 1997). It is more likely that parasites adapt to meet their biological and survival requirements (Jalloh et al. 2006).

Previous findings and the present study suggest that polymorphisms in CSP-T epitopes may be driven and balanced by some forces resulting from host–parasite interaction associated with environmental factors. On comparison of sequence diversity in Th2R and Th3R epitopes of Indian isolates with those of other geographic areas, the alleles in two groups (V-6 and V-9) in both Th2R and Th3R showed identical sequence patterns as those observed in other geographical regions of the world (Lockyer et al.1989; Doolan et al. 1992). However, three haplotypes from Th2R (V-3, V-4 and V-5) and Th3R (V-1, V-2, V-3), showed sequence patterns identical with previous isolates from India (Bhatia & Bhattacharya 2006; Bhattacharya et al. 2006). On the other hand, the remaining four haplotypes observed as new variants among Indian isolates and the variations in Th2R and Th3R are regionally unbiased, and variants detected in overall isolates can be put in nine groups. Moreover, allelic linkage of sequence variations in Th2R and Th3R epitopic regions was also seen in most of the variants.

Thus we may conclude from this study that the sequence diversity at T epitopic regions of P. falciparum CSP gene among isolates appears to be regionally unbiased and restricted in Indian settings. In the isolates from six areas, mutation patterns remained unique. The number of allelic variants increased in the peak transmission phase (TP-2) of P. falciparum. The host factors and environmental factors may play a crucial role in development of these variations for parasite’s adaptability. Therefore, in the development of a malaria vaccine strategy and for future vaccine trials, local parasite populations need to be considered.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study received financial support from the intramural grant of the National Institute of Malaria Research (Indian Council of Medical Research), Delhi, India. We are grateful to Dr C.R. Pillai, NIMR, Delhi for providing some of the parasite isolates and Dr R.H. Das, Institute of Genomics and Integrative Biology, Delhi, for providing sequencing facilities.

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  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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