Corresponding Author Chae Seung Lim,Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Guro 2 Dong, Guro Gu, Seoul 152-703 Korea. Tel.: +82 2 26263245; Fax: +82 2 26261465; E-mail: email@example.com
The Ookinete surface proteins of Plasmodium vivax (P. vivax), Pvs25 and Pvs28, were candidates for the transmission blocking vaccine (TBV), which exhibited great antigenic diversities among various isolates. Polymorphisms of these genes in the isolates from Republic of Korea (ROK) were analysed, which provided valuable baseline data for the field trials of TBV-based vaccines. A total of 98 isolates were collected over 11 years from 1996 to 2007. pvs25 and pvs28 genes from the above isolates were amplified, sequenced and compared against Sal-1 strain. Sequencing analysis of PCR products from P. vivax pvs25 revealed two allelic types, Q97T130 and E97/T130 alleles with the frequencies of 54.5% and 45.5%, respectively, in comparison with Sal I type sequence (E97/I130). From pvs28 gene, polymorphisms at M52L and T140S in the first and third EGF-like domains in comparison to Sal-1 strain were detected, respectively. Six GSGGE tandem repeats followed by GSGGDT or SSGGDT were identified at the end of the fourth EGF-like domain in all Korean isolates. Interestingly, different tandem repeats of amino acid substitutions were observed from isolates collected after 2006 in comparison with preceding years. The ROK isolates revealed limited sequence polymorphisms in pvs25 and tandem repeats in pvs28 in comparison with reported isolates from other nations. Current observations suggested the rapid progresses of genetic changes among Korean isolates.
Polymorphismes des séquences de protéines de surface Ookinete (Pvs25 et Pvs28) de Plasmodium vivax provenant d’isolats cliniques de Corée
Les protéines de surface Ookinete de Plasmodium vivax (P. vivax), Pvs25 et Pvs28, étaient des candidats pour le Vaccin Bloquant la Transmission (TBV), qui a démontré une grande diversité antigénique pour les différents isolats. Les polymorphismes de ces gènes dans les isolats provenant de la République de Corée ont été analysés, ce qui a fourni des données de référence précieuses pour les essais sur le terrain de vaccins basés sur le TBV. 98 isolats ont été collectés sur 11 ans, de 1996 à 2007. Les gènes Pvs25 et pvs28 de ces isolats ont été amplifiés, séquencés et comparés à la souche Sal-1. L’analyse du séquençage des produits PCR de P. vivax pvs25 a révélé deux types alléliques, les allèles Q97T130 et E97/T130 avec des fréquences de 54,5% et 45,5%, respectivement, en comparaison avec le type de séquence Sal-1 (E97/I130). Pour le gène pvs28, des polymorphismes au niveau de M52L et T140S dans les 1er et 3ème domaines semblables à EGF en comparaison à la souche Sal-1 ont été détectés, respectivement. Six séquences GSGGE répétitives en tandem suivies par GSGGDT ou SSGGDT ont été identifiées à la fin du 4ème domaine semblable à EGF dans tous les isolats coréens. De façon intéressante, différentes substitutions répétitives d’acides aminés en tandem ont été observées dans des isolats collectés après 2006 en comparaison avec les années précédentes. Les isolats de la République de Corée ont révélé des polymorphismes de séquences limités dans pvs25 et des répétitions en tandem dans pvs28 en comparaison avec des isolats rapportés dans d’autres nations. Les observations actuelles suggèrent la rapide progression de modifications génétiques dans les isolats coréens.
Polimorfismo en la secuencia de proteínas de superficie de oocinetos de Plasmodium vivax (Pvs25 and Pvs28) provenientes de aislados clínicos en Korea
Las proteínas de superficie de oocinetos de Plasmodium vivax (P.vivax), Pvs25 y Pvs28, eran candidatas para la vacuna de bloqueo de la transmisión (VBT) la cual presentaba una gran diversidad antigénica entre aislados. Se han analizado los polimorfismos de estos genes en aislados de la República de Korea (RDK), arrojando datos de base valiosos para ensayos de campo de vacunas derivadas de VBT. Se recolectaron 98 aislados a lo largo de 11 años, entre 1996 y 2007. Se amplificaron los genes pvs25 y pvs28, se secuenciaron y compararon con los de la cepa Sal-1. El análisis de la secuencia de productos de PCR de P.vivax pvs25 reveló dos tipos alélicos, Q97T130 y E97/T130 con unas frecuencias de 54.5% y 45.5%, respectivamente, en comparación con la secuencia de tipo Sal I (E97/I130). En el gen pvs28, se detectaron polimorfismos en M52L y T140S en el primer y tercer dominio del tipo EGF respectivamente, en comparación con la cepa Sal-1. Se identificaron seis repeticiones en tandem GSGGE seguidas por GSGGDT o SSGGDT al final del cuarto dominio tipo EGF en todos los aislados Koreanos. Curiosamente, se observaron diferentes repeticiones en tandem de sustituciones de amino ácidos en aislados recogidos después del 2006, en comparación con aquellos procedentes de años anteriores. Los aislados de RDK revelaron un polimorfismo limitado en la secuencia de pvs25 y las repecticiones en tandem de pvs28 en comparación con los aislados reportados en otros países. Las observaciones actuales sugieren un rápido progreso en cambios genéticos de los aislados koreanos.
Plasmodium vivax (P. vivax) accounts for over half of all malarial cases in the world, as many as 400 million cases per year (Price et al. 2007). During the transmissions of P. vivax between human host and the mosquito vector, antibodies against Pvs25 and Pvs 28 block the development of malaria parasites in the mosquito host (Kaslow 1992). Hence, Pvs25 and Pvs 28 were accepted as leading candidates and targets in the Transmission Blocking Vaccine (TBV) Program (Sattabongkot et al. 2003; Malkin et al. 2005). These proteins contained conserved structures, four epidermal growth factor (EGF)-like domains (Tsuboi et al. 1998), and various polymorphisms in pvs25 and pvs28 genes from isolates from different endemic areas were reported. The antigenic variations observed in target molecules could present difficulties in developing effective vaccines (Tsuboi et al. 1998). Thus, we analysed nucleotide polymorphisms of pvs25 and pvs28 genes in clinical isolates from the Republic of Korea (ROK). The results from these Korean isolates strongly suggest potential use of these proteins as valuable baseline data for future field trials of TBV-based vaccines.
Materials and methods
Between 1996 and 2007, blood samples were collected with EDTA blood drawing tubes from 98 patients diagnosed with P. vivax by microscopic Giemsa stain. All cases received supervised treatments of 2000 mg hydroxychloroquine sulphate, given orally (800 mg initial dose, followed by 400 mg at 6, 24 and 48 h). After hydroxychloroquine therapy, outpatients received oral primaquine (15 mg/day for 14 days). Whole blood from each patient was collected and stored at −80 °C. The DNA was extracted from frozen pellets using AquaPure genomic DNA kits (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturer’s instructions. All patients provided informed consent. This study was approved by the Ethical Review Committee of Research in Korea University Ansan Hospital.
Plasmodium vivax DNA was extracted from collected whole blood by a commercial InstaGene matrix kit or AquaPure genomic DNA kit (Bio-Rad Laboratories, Hercules, CA). The following primers (5′–3′) were used for PCR: pv25F2 5′-CACCGACCACAAAAACTTAT-3′ (AF083502, 158–177) and pv25R2 5′-AACGTAAAGCCTTCCATACA-3′ (AF083502, 814–795) for the P. vivax pvs25 gene; Pvs28F3 5′-CTTTTGCGCCTCCCCCTTGTTCA-3′ (AF083503, 39–61) and Pvs28R3 5′-GCTTCTCCCCTTGTGTATGTTTGT-3′ (AF083503, 793–816) for the P. vivax pvs28 gene. The primers were used at a final concentration of 0.1 μm in 30 μl of reaction mixture (10 mm Tris–HCl, pH 8.3, 50 mm KCl, 1.5 mm MgCl2, 0.2 mm each dNTP) with 2 μl DNA and 2.5 units of ExTaq polymerase (Takara Bio Inc, Shiga, Japan). Reaction mixtures were cycled 30 times in a DNA thermal cycler (Eppendorf Scientific Inc, Hamburg, Germany). Each cycle included denaturation at 95 °C for 1 min, annealing at 56 °C for 1 min and extension at 72 °C for 3 min. Amplified products were size-fractionated by electrophoresis in 1.5% agarose containing 0.5 mg ethidium bromide/ml. PCR products were gel-purified using QIAGEN DNA purification kit (Qiagen, Germany) by following the manufacturer’s instructions. DNA fragments were sequenced in both directions for each PCR product using an ABI-3100 DNA sequencer.
Sets of pvs25 and pvs28 gene sequence alignments were performed manually. Both coding and non-coding sequences were analysed. We specified the nucleotide and amino acid positions against the reference strain; Sal-I pvs25 (AF083502) and pvs28 (AF083503). Nucleotide sequence data of different haplotypes in this study were submitted to GenBank under Accession numbers GU971416 to GU971611.
Amplifications of pvs25 and pvs28 were successful for 98 P. vivax isolates using specific primers, resulting in amplification of 657 bp and 793 bp DNA fragments for pvs25 and pvs28 genes, respectively.
From 98 PCR-amplified products of pvs25 genes in Korean isolates, two distinct allelic types were identified. Compared to Sal-1, five synonymous and 20 non-synonymous nucleotide substitutions from 557 bp coding sequences were also detected in all sequenced samples. Two amino acid substitutions, E97Q and I130T, were found in comparison with Sal-1 strain. Two haplotypes, Q97T130 and E97/T130, were identified with frequencies of 68.3% and 31.7%, respectively, which were distinct from Sal I type sequence (E97/I130) (Figure 1). Until in 2000, Q97T130 was the only Pvs 25 allele type, however, since 2006, frequency of E97/T130 allele type in our sample collections had increased up to 80%. The substantial frequencies are listed in Figure 1, according to the year of collection.
From the amplified products of pvs28 genes, six synonymous and 35 non-synonymous nucleotide substitutions from 765 bp coding sequences were discovered in all sequenced samples. Compared to Sal-1 strain, amino acid substitutions, M52L and T140S, were detected in the first and third EGF-like domains, respectively. At the end of the fourth EGF-like domain, six GSGGE tandem repeats followed by GSGGDT were identified in all Korean isolates. Interestingly, amino acid tandem repeats revealed different patterns from the isolates collected before and after year 2006. In 2006, isolates sequences with GSRGE/GCGGE/SSRGE from 3rd to 5th tandem repeats and SSGGDT at 7th repeat region were found (Figure 2). However, in 2007, isolates with SSRGE sequences at 5th tandem repeat and SSGGDT at 7th repeat region were detected (Figure 2). The substantial frequencies by collected years are listed in Figure 1.
The major Korean alleles of pvs25 (Q97T130) and pvs28 from early remerging periods (1996–1997) seemed to have decreased rapidly in recently collected samples, while new alleles with minor frequencies appeared and increased in frequency over time (Figure 1).
Pvs25 and Pvs28 belong to families of cysteine-rich protein families, P25 and P28; they contain 22 and 20 cysteine residues, respectively. The cysteine residues, comprising four tandem EGF-like motifs were aligned with the consensus amino acid sequences of zygote/Ookinete surface proteins of other malaria parasites (Tsuboi et al. 1998). The antigenic variations observed in target molecules of malaria could hinder the development of the effective vaccine (Sattabongkot et al. 2003; Zakeri et al. 2009). As previously reported, the antigenic variations of Pvs25 seemed to be limited than Pvs28 in Korea (Tsuboi et al. 1998; Zakeri et al. 2009). In our study, pvs25 of P. vivax in Korean isolates was categorized into two main allelic subtypes, whereas pvs28 consisted of one major allelic type of six GSGGE tandem repeats, followed by GSGGDT and numerous minor allelic types including repeat region (Tables 1 and 2). In pvs25, two main point mutations were found in Korean isolates at E97Q and I130T substitutions in comparison with Sal-1 strain. Two main haplotypes, Q97/T130 and E97/T130, in EGF-2 and EGF-3 regions were distinct in Korean isolates from Sal I type sequence (Table 1). Other minor polymorphic types rose from two main haplotypes.
Table 1. Comparison of amino acid polymorphisms in the Pvs25 gene of P. vivax isolates between Korean and world wide isolates
In Korean pvs28 isolates, three single amino acid polymorphisms, M58L (in EGF-1 domain), T140S (in EGF-3 domain) and A219P (in THR domain), were identified. The most striking variations were the six tandem repeats of GSGGE, followed by GSGGDT in all Korean isolates in comparison against Sal-1 (5xGSGGE+ GSGDT). One regular amino acid insertion (GSGGDT) occurred at the 7th repetitive position in all Korean isolates. Among the minor types, amino acid changes were found at 1st and 3rd amino acid positions of the repeats. These sequences were only found in Korean isolates collected after 2006. Interestingly, the rapid genetic changes have been observed frequently in recent years, suggesting the accelerated mutation tendency in pvs25 and pvs28.
Since the first report of autochthonous P. vivax transmission in 1993, transmissions reached 4142 cases in 2000. The rapid increases in recent years could be related to the presence of these new allelic types of P. vivax in Korea. Most of the P. vivax malaria cases occur in summer from June through September, when the major transmission vector in ROK, Anopheles sinensis and An.kleini mosquitoes, are also isolated. Considering the habitual status of P. vivax in Korea, the vector-related changes could have accelerated the current observations; increased frequency of minor allelic types of Korea isolates. The Pvs25 and Pvs28 are the targets of naturally acquired host immunity. In pvs28, the amino acid sequences of the new variants are different from early reemerging strains and worldwide isolates. The recent appearance of polymorphisms in repeat region mutations also might be attributed to selection by vector population changes, and/or the preferential production of specific variants.
Previously, the TBV study in Thailand demonstrated that Sal-I-based vaccines were effective against two allelic types of pvs25 and pvs28 in natural isolates of P. vivax, suggesting the wide range applicability of Sal-I-based vaccines (Sattabongkot et al. 2003). The newly detected amino acid substitutions among Korean isolates revealed wide range of variations in comparison to the isolates from other nations. However, main allelic types coincided with previous reported amino acid substitutions (Tsuboi et al. 1998; Sattabongkot et al. 2003; Zakeri et al. 2009). However, owing to the occurrence of minor allelic types, Pvs25 and Pvs28 Sal-I-based TBVs need to be evaluated in Korea.
Financial support: This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (KRF-2008-314-E00075). Disclosure: The view of the authors (Jetsumon Sattabongkot) did not purport to reflect the position of the US Department of the Army or Department of Defense.