- Open Access
Genetic polymorphism of merozoite surface protein-1 and merozoite surface protein-2 in Plasmodium falciparum field isolates from Myanmar
© Kang et al; licensee BioMed Central Ltd. 2010
- Received: 30 December 2009
- Accepted: 17 May 2010
- Published: 17 May 2010
Merozoite surface protein-1 (MSP-1) and MSP-2 of Plasmodium falciparum are potential vaccine candidate antigens for malaria vaccine development. However, extensive genetic polymorphism of the antigens in field isolates of P. falciparum represents a major obstacle for the development of an effective vaccine. In this study, genetic polymorphism of MSP-1 and MSP-2 among P. falciparum field isolates from Myanmar was analysed.
A total of 63 P. falciparum infected blood samples, which were collected from patients attending a regional hospital in Mandalay Division, Myanmar, were used in this study. The regions flanking the highly polymorphic characters, block 2 for MSP-1 and block 3 for MSP-2, were genotyped by allele-specific nested-PCR to analyse the population diversity of the parasite. Sequence analysis of the polymorphic regions of MSP-1 and MSP-2 was also conducted to identify allelic diversity in the parasite population.
Diverse allelic polymorphism of MSP-1 and MSP-2 was identified in P. falciparum isolates from Myanmar and most of the infections were determined to be mixed infections. Sequence analysis of MSP-1 block 2 revealed that 14 different alleles for MSP-1 (5 for K1 type and 9 for MAD20 type) were identified. For MSP-2 block 3, a total of 22 alleles (7 for FC27 type and 15 for 3D7 type) were identified.
Extensive genetic polymorphism with diverse allele types was identified in MSP-1 and MSP-2 in P. falciparum field isolates from Myanmar. A high level of mixed infections was also observed, as was a high degree of multiplicity of infection.
- Falciparum Isolate
- Variable Central Region
- Similar Frequency Pattern
- Effective Malaria Vaccine
Malaria is a major human health-threatening disease, which resulting in approximately 200-300 million clinical cases and 1-3 million deaths each year worldwide. Plasmodium falciparum causes the most severe form of the disease and is responsible for most malaria morbidity and almost all malaria mortality. Despite enormous efforts for malaria control and prevention, multiple factors, including insecticide resistance in the mosquito vectors, the lack of effective vaccines, and the emergence and rapid spread of drug-resistant strains, are contributing to the global worsening of the malaria situation. Therefore, there is an urgent need for the development of effective malaria vaccine. However, extensive genetic diversity in natural parasite populations is a major obstacle for the development of an effective vaccine against the human malaria parasite, since antigenic diversity limits the efficacy of acquired protective immunity to malaria [1–3]. Therefore, it is important to investigate the genetic diversity of malaria parasites, particularly the genetic diversity of vaccine candidate antigens, in different geographic regions to develop effective malaria vaccines.
Merozoite surface protein-1 (MSP-1) of P. falciparum is a major surface protein, with an approximate molecular size of 190 kDa, that plays an important role in erythrocyte invasion by the merozoite . The protein is a principal target of human immune responses [5–7] and is a promising candidate for a blood stage subunit vaccine [4, 8]. The MSP-1 gene has 7 variable blocks that are separated either by conserved or semi-conserved regions. Block 2, a region near the N-terminal of the MSP-1 gene, is the most polymorphic part of the antigen and appears to be under the strongest diversifying selection within natural populations . Up to now, four different allelic types of block 2 have been identified: MAD20, K1, RO33, and MR [9–12].
MSP-2 of P. falciparum is another leading candidate antigen for subunit malaria vaccine . It comprises highly polymorphic central repeats flanked by unique variable domains and conserved N- and C-terminal domains [1, 14]. The MSP-2 alleles generally fall into two allelic types, FC27 and 3D7, which differ considerably in the dimorphic structure of the variable central region, block 3. Due to their polymorphic features, the MSP-1 and MSP-2 genes have been employed as polymorphic markers in studies of malaria transmission dynamics in natural isolates of P. falciparum.
The morbidity and mortality rates due to malaria have been declining gradually in recent years in Myanmar, but malaria still remains one of the most serious problems threatening human health in the country, resulting in approximately 60% of malaria deaths in the South-East Asia region . Information on the nature and extent of population diversity within malaria parasites circulating in the country is essential not only for understanding the mechanism underlying the pathology of malaria but also for establishing a proper control strategy. However, only limited data are available on the genetic diversity of P. falciparum populations of the country. This study was designed to analyse the genetic diversity of MSP-1 and MSP-2 in field isolates of P. falciparum collected in a rural area outside of Mandalay, Myanmar.
Blood samples and genomic DNA extraction
A total of 63 P. falciparum infected blood samples used in this study were collected from patients attending the Wet-Won station hospital, Pyin Oo Lwin Township, Mandalay Division, Myanmar during 2004-2006 . All blood samples were collected after informed consent and the use of the samples for this study was approved by the Department of Health, The Union of Myanmar, and the Ethic Committee of the Centers for Disease Control and Prevention, Korea. Genomic DNA was extracted from 100 μl of whole blood sample by using a QIAamp Blood Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instruction.
Allelic typing of MSP-1 and MSP-2
The oligonucleotide primers specific for the polymorphic regions (block 2 of MSP-1 and block 3 of MSP-2) were designed as described previously . The two genes were amplified by nested PCR. An initial amplification of the outer regions of the two genes was followed by a nested PCR with family-specific primer pairs. All reactions were carried out in a final volume of 40 μl containing 0.2 mM dNTP, 1 μM of each primer, and 2.5 U of Ex Taq DNA polymerase (Takara, Otsu, Japan). In the first round reaction, 4 μl of genomic DNA was added as a template. In the nested reaction, 1 μl of the first PCR product was added. Each amplification profile consisted of initial denaturation at 94°C for 5 min, followed immediately by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min. The final cycle had a prolonged extension at 72°C for 10 min. Each PCR product was electrophoresed on 1.5% agarose gels and the DNA was visualized by ultraviolet transillumination after staining with ethidium bromide. The number and size of the resulting amplified products were analysed.
Allelic distribution and multiplicity of infection
The prevalence of each allelic type was determined as the presence of PCR products for the type in the total number of amplified bands for the corresponding locus. The multiplicity of infection (MOI), or complexity of infection, was estimated by the average number of PCR fragments per infected individual, as described previously [18, 19].
Sequencing analysis of MSP-1 and MSP-2
For sequence analysis of MSP-1 and MSP-2, all PCR products (128 for MSP-1 and 148 for MSP-2) obtained by allelic typing PCR were purified from the gel and cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). Each ligation mixture was transformed into E. coli DH5α competent cells and positive clones were screened for the presence of plasmid with the appropriate insert. Sequencing reactions were performed using the BigDye Terminator Cycle Sequencing Ready Reaction Kit in an ABI 377 automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA). To verify the sequences, sequence analysis was performed by analysing at least two plasmid clones containing each gene insert. Analysis of the primary structures of the deduced amino acid sequences was done with DNASTAR (DNASTAR, Madison, WI, USA). Nucleotide sequences reported in this paper are available in the GenBank database under accession numbers EU445555-EU445557, EU445559-EU445566, GQ861442-GQ861443, and GQ861445 for MSP-1, and numbers EU647447-EU647468 for MSP-2.
Allelic polymorphism of MSP-1 and MSP-2
Allele typing and diversity profiles of P. falciparum isolates from Myanmar based on genetic diversity of MSP-1 and MSP-2.
No. of samples
PCR product size (bp)
Multiplicity of infection (MOI)
K1 + MAD20
FC27 + 3D7
Sequence analysis of MSP-1
Sequence analysis of MSP-2
The genetic structure of P. falciparum populations plays a highly important role in the natural acquisition of immunity in malarial infections [2, 20]. Therefore, knowledge of the genetic structure of these populations is necessary to develop strategies to control the disease, including the design of effective vaccines against P. falciparum. In this study, genetic polymorphism of two merozoite surface proteins, MSP-1 and MSP-2, of 63 P. falciparum isolates collected in Myanmar, where malaria is endemic or hypoendemic, was analysed . To our knowledge, no such study has been done in Myanmar to date, and therefore this study provides the first estimate of the genetic diversity of P. falciparum wild-type isolates circulating in Myanmar.
Allele-specific PCR typing of MSP-1 (block 2) and MSP-2 (block 3) showed that P. falciparum populations in Myanmar have a highly complex genetic diversity. For MSP-1, both types of K1 and MAD20 with different length of amplified products (120-210 bp for K1 and 140-250 bp for MAD20) were identified. Most of the isolates (63.5%) were mixed infections which harbored both allele types. Several similar studies in different geographic areas which used block 2 of MSP-1 as a polymorphic marker reported important variations in the frequency of the genotypes. MAD20 (57/63, 90.5%) was the predominant allele in the P. falciparum population in Myanmar, which is consistent with the situations in Thailand, Iran, Pakistan and Colombia [21–24]. On the other hand, in studies in French Guiana, Kenya and Peru, MAD20 is the less frequent type and K1 is the most frequent [11, 25, 26]. Allele typing for MSP-2 also showed that both FC27 and 3D7 allele types were identified among the isolates. The frequency of FC27 and 3D7 allele type was 80.9% (51/63) and 87.3% (55/63), respectively, but a high proportion of the isolates (68.3%, 43/63) contained both allele types. Similar frequency patterns are observed in Thailand, Iran, Pakistan and Cameroon [21, 23, 24, 27], but not in Brazil, where FC27 type is more prevalent . These results collectively suggest that diverse allelic variations of MSP-1 and MSP-2 exist in P. falciparum Myanmar isolates and that most of the infections were mixed. This is similar to observations made in other endemic areas [11, 17, 21, 22, 27–29].
To further investigate the allelic diversity of MSP-1 and MSP-2 in P. falciparum isolates from Myanmar, sequence analysis of MSP-1 and MSP-2 was performed. Sequence analysis of MSP-1 block 2 showed that a total of 14 alleles of MSP-1, 5 for K1 type and 9 for MAD20 type, were identified. Allelic diversity of MSP-1 block 2 in P. falciparum Myanmar isolates was due to different numbers of unique tripeptide repeats, which is similar to previous studies [14, 30]. Sequence analysis of MSP-2 block 3 also showed high allelic diversity, with seven alleles for FC27 and 15 alleles for 3D7. As reported in previous studies on parasites from different geographic origins [14, 28, 30], the sequences belonging to the FC27 family of P. falciparum isolates from Myanmar were generally conserved but varied in the number of repeats. The 3D7 displayed more extensive sequence diversity. Besides the major polymorphic characters in the R1 and R2 regions, several non-synonymous amino acid substitutions were identified in family specific regions (E1, E2, and E3) of 3D7 type alleles and the variations make the genetic diversity of 3D7 allele type much greater than FC27 type alleles. Interestingly, duplication of PT motif at the 3' end of block 2 was identified in two 3D7 type alleles (alleles 10 and 11). This proliferation of the PT motif had been identified in non-Asian parasites previously , but it is the first description of PT duplication in Asian parasite. MSP-2 was more polymorphic than the MSP-1 in P. falciparum isolates from Myanmar, which is also consistent with previous studies [17, 21, 27, 31, 32].
Although it seems likely that nonreciprocal recombination events, such as replication slippage and gene conversion, during the mitotic (asexual) replication of the parasite also play a plausible role in creating allele variation [6, 33], allelic diversity of P. falciparum MSP-1 and MSP-2 is mainly generated by meiotic recombination events involving genetically distinct parasite clones that infect the same mosquito vector [34, 35]. Therefore, the proportion of mixed infections and the number of clones per individual is one of the pre-requisites to generate new genotypes and to increase the diversity of the parasitic population . Multiple clonal infections with different genotypes of P. falciparum were identified among Myanmar P. falciparum isolates in a high proportion (79.4% for MSP-1 and 87.3% for MSP-2). And a high level of MOI (2.03 for MSP-1 and 2.35 for MSP-2) was also found. Sequence analysis of MSP-1 and MSP-2 also showed that diverse alleles (14 for MSP-1 and 22 for MSP-2) were identified among the isolates. Although direct comparison could be impossible due to the different size of blood samples used in each study, this is less than holoendemic areas such as Senegal (33 for MSP-1 and 17 for MSP-2) , Uganda  and Gabon (25 for MSP-1 and 19 for MSP-2)  but more than low endemic Asian countries including Thailand (10 for MSP-1 and 17 for MSP-2)  and Iran (9 for MSP-1 and 11 for MSP-2) . These extensive allelic variations were also identified in circumsporozoite protein (CSP), MSP-1, and MSP-3α  and apical membrane antigen-1 (AMA-1)  of P. vivax isolates from Myanmar. The unique geographic location of Myanmar, which is surrounded by five neighbouring malaria endemic countries, appears to contribute to the large diversity of parasite genotypes in this country. Migration of people within the country and between neighbouring countries may also introduce a P. falciparum population with different alleles to the country, resulting in an extensive sequence variation in the parasite. Further studies associated with antibody responses against MSP-1 and MSP-2 in Myanmar patients are needed to evaluate the impact of this polymorphism on the immune response to the antigens, since the genetic diversity would not necessarily reflect selection acting at protein level. Studies using a larger number of blood samples collected from different geographic areas in Myanmar are also required not only to determine the nationwide parasite heterogeneity and detailed malaria epidemiology but also to implement malarial control programmes in the country.
A major finding of this study was that P. falciparum field isolates in Myanmar exhibited a high degree of genetic polymorphism in MSP-1 and MSP-2. Moreover, most of the infections were mixed with a high level of MOI. These results collectively suggested the highly complex population structure of the parasite in Myanmar.
This study was partially supported by a grant of Korea Centers for Disease Control and Prevention (2008-E00151-00).
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