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Allelic variation of polymorphic vaccine candidates merozoite surface protein-2 in Plasmodium falciparum isolates from South-East of Iran.

INTRODUCTION

Malaria is still one of the most life-threatening parasitic diseases worldwide [1]. Malaria results in approximately 300-500 million clinical cases and 1-3 million deaths each year worldwide mainly of young children [2]. Amongst the four species of Plasmodium that transmit human malaria, Plasmodium falciparum is responsible forthe most severe clinical manifestations of disease and causes most malaria morbidity and almost all malaria mortality [3]. Annually, a big proportion of funding and staffing in the world is devoted to malaria problem [4]. However, a variety of factors, including insecticide resistance in vectors, the lack of efficient vaccine, and the emergence and rapid spread of drug-resistant strains are contributing to the deterioration of global malaria situation [5]. Therefore, there is an urgent need to develop effective malaria vaccines [6].

However, extensive genetic diversity in natural parasite populations is the major barrier for the development of an effective vaccine against human malaria parasites [7], since antigenic diversity limits the efficacy of acquired protective immunity to malaria [8]. Such extreme antigenic diversity increases the ability of the parasite to evade the host immune responses [9]. A true understanding about frequency of vaccine candidate antigens and changes in natural parasite populationsis important to design a successful and effective malaria vaccine and also provides useful facts to interpret immunological responses to vaccination [7]. A limited number of stage-specific antigens of the Plasmodium falciparum vaccine candidates have been identified using novel molecular techniques [10]. We have analyzed the genetic diversity of Merozoite surfaceprotein-2 (MSP-2) antigenas a potential vaccine candidate [11]. Plasmodium falciparum MSP-2 antigen is a 45 to 55 KDglycoprotein that is produced during the early stagesschizogony during the parasite life cycle and appears on the surface of the merozoites [12]. MSP-2 gene which is located on chromosome 2 encodes a glycoprotein on the surface of Merozoites that is widely usedin designing new malaria vaccines [7,13]. DNA sequencing has revealed that each copy of the MSP-2 gene is conserved by C-terminal and N-terminal regions (blocks 1 and 5) and two regions of repeated sequences (blocks 2 and 4). There is only acentral polymorphicarea known as Block 3 [14]. In the central polymorphic area of MSP-2 gene, FC27 and 3D7 dimorphic alleles show the greatest diversity [15]. In comparison to FC27 family, 3D7 alleles are much more variable in length and sequence [16]. Furthermore, this antigen is capable of inducing an effective immune response during blood stage [6,11]. MSP-2 is therefore a strong vaccine candidate with limited epidemiologic data; data that areneeded to support continued development along the proposed malaria vaccine roadmap [6,11,15,17]. Iran is located in the Eastern Mediterranean Region, and grouped as low-moderate end emicregion [18]. Sistan and Baluchistan Province, South-East of Iran, is the endemic area of falciparummalaria and is considered as the oriental ecoepidemiological region of malaria [19].

This study investigates allelic variation in the P. falciparum MSP-2gene among samples collected from four different endemic regions in South-East of Iran. Such data are important because the increased frequency of simple infections in such a setting enables us to look at changes in allele frequency over time, which might provide evidence for or against the presence of allele-specific and variant-specific immune responses.

MATERIALS AND METHODS

In this cross-sectional study, 94 individuals suffering from falciparum malaria referring to Malaria-Centers of Chabahar, Iranshahr, Nikshahr and Sarbaz were selected from April 2011to September 2012to characterize allelic variation within P. falciparum MSP-2 in this endemic area. Residence in these regions for over 6 months, no history of consuming anti-malarial drugsduring the last month, and written informed consent were required for inclusion in this study. The presence of P. falciparum infections in the samples were confirmed microscopically using thick and thin Giemsa-stained slides in Department of Parasitology, Zahedan University ofMedical Sciences. Venous whole blood (2 ml) was collected from each consenting patient. The samples were stored at -20[degrees]C until using for DNA extraction. DNA was extracted using Fermentas Genomic DNA Purification Kit (Thermo Fisher Scientific Inc.) from the whole blood samples. All DNA samples were stored at -20[degrees]C before genotyping with a polymerase chain reaction. Nested PCR was used to amplify the central region of P. falciparum MSP-2 gene using the primers mentioned in Table. 1.

The first and second round PCR amplifications were performed in afinal volume of 20 pl using AccuPower TLA PCR Premix (Bioneer, Korea Rep). Cycling conditions for the first PCR cycle were 94[degrees]C for 5 minutes (Initial Denaturation), 94[degrees]C for 1 minutes (Denaturation), 58[degrees]C for 1 minutes (Annealing), and 72[degrees]C for 1 minutes (Extension), followed by a final extension at 72[degrees]C for 5 minutes, for a total of 24 cycles. The second PCR cycles conditions were the same whereas the annealing temperature was considered 61[degrees]C for a total of 30 cycles. Purified DNA from P. falciparum 3D7 (MRA-102G) and FC27 strains were provided by the Malaria Research and Reference Reagent Resource Center, American Type Culture Collection (Manassas, VA) and used as positive control during the amplification reactions. The second amplification products were directly separated by electrophoresis on a 2.0% ethidiumbromide agarose gel and visualized on a Tran illumination Imaging System. Positive controls and a 1000bp Ladder Marker (Bioneer, Korea Rep) were used to interpret the fragments sizes. Total number of gene variants observed in the central region of MSP-2 was 8.Four fragments (280, 300, 380, 400bp) were observed in FC27 alleles and 4 of which (400, 470, 500, and 600bp) were relevant to 3D7 alleles. Multi-clonal infections were defined by the presence of FC27 and 3D7 MSP-2 alleles simultaneously.

RESULTS AND DISCUSION

Results:

The aim of this study was to analyze the polymorphic antigen MSP-2 gene across South-East of Iran among four different districts to identify differences in allele frequency and genetic diversity. Among 94P falciparum samples obtained from the four districts, 85 samples were successfully scored for the presence of MSP-2 gene. Nested PCR on MSP-2 confirmed samples revealed that both 3D7 and FC27 allele classes of P. falciparum MSP-2 were present in the districts of study. The MSP-2 allele classes (FC27 and 3D7 types) showed reasonable prevalence in all districts (Table 2). The frequency of MSP-2 genes in the districts of Chabahar, Iranshahr, Nikshahr and Sarbaz were 34.2%, 23.6%, 23.3% and 3.53% whereas the overall frequencies of FC27 and 3D7 allele classes were 34.2% and 50.5% respectively for each. Among the samples, 13 (15.3%) cases showed multi-clonal infections. The frequency of variants in different areas did not show significant differences.

Discussion:

We used Nested PCR to evaluate allelic variations within the malaria vaccine candidate P. falciparum MSP2 in South-East of Iran. Nested PCR has been shown to possess high sensitivity and specificity of up to 94% in some tests [20] and exhibits a high-through put capacity in comparison to other PCR modifications in the field studies, and is considerably more cost-efficient versus sequencing [18,19]. We allele typed 85 individual P. falciparum infections containing P. falciparum MSP-2genes using Nested PCR and realized that both 3D7 and FC27 allele classes of MSP-2 gene were present in the region. In the present study seasonal frequencies of each allele classes were ignored. It was observed that the MSP-2 gene in this region consists of 8 fragments that shows a higher rate in comparison to a similar study in Columbia [21] and Senegal [22] in which 3 and 7 fragments were demonstrated respectively. Although this extent of allelic diversity is not comparable to 17 variants for MSP-2 gene in Thailand [23]. It seems as if variants distribution is highly affected by geographical region and the gene status and number of fragments can vary from area to area and act as a major contributor to allele variation in small populations.It should be kept in mind that the population structure of Plasmodium Falciparumin different studies has some limitations because of using different molecular methods and differences in experimental conditions [16]. But the comparison of this study with similar studies especially in areas with low endemicity shows that the genetic diversity of these parasites is at higher grade (24). Therefore, in the major endemic foci of Plasmodium Falciparum in Iran we are not dealing with a homogeneous population of parasites and it is likely that patients become contaminated with more than two parasite clones or strains simultaneously (Multi-clonal Infection) [25].

Due to the heterogeneous population in the region multi-stage vaccines should be designed to control malariain different stages of evolution. Therefore, these data are needed to support development of a vaccine based on MSP-2 antigen along the malaria vaccine road map. Also, the results show no remarkable predominance of any allele in the studied area. There should be a comparative analysis in different seasonal peaks to indicate the allelic polymorphism of the MSP-2 over a period of time. These data supports the hypothesis of a biologically important role for MSP-2 in parasite development and highlight the importance of evaluating the distribution of MSP-2 allelic forms in different geographical regions to provide valuable genetic information to design an effective malaria vaccine despite the extensive present genetic diversity.

ARTICLE INFO

Article history:

Received 12 October 2013

Received in revised form 18

December 2013

Accepted 29 December 2013

Available online 25 February 2014

ACKNOWLEDGEMENTS

This research study is based on an original thesis for master degree funded byresearch committee in Zahedan University of Medical sciences. This research group would like to acknowledge all the staff in Parasitology and Mycology Laboratory Department.

REFERENCES

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(1) Fatemeh Namdar, (1) Adel Ebrahimzadeh, (1,2) Saeed Mohammadi

(1) Department of Parasitology, Zahedan University of Medical Sciences, Zahedan, Iran.

(2) Faculty of Advanced Medical Technology, GolestanUniversity of Medical Sciences, Gorgan, Iran.

Corresponding Author: Adel Ebrahimzadeh, Department of Parasitology, Zahedan University of Medical Sciences, Zahedan, Iran.
Table 1: List of Primers and sequences

Primer Name          Sequence Length 5'-3'

MSP-2-OF             GAG TAT AAGGAG AAG TAT GG
MSP-2-OR             CCT GTA CCT TTA TTC TCT GG
[M.sub.2]-FC27-FCF   AAT ACT AAG AGT GTA GGT GCA ATG CTC CA
[M.sub.2]-FC27-FCR   TTT TAT TTGGTG CAT TGC CAG AAC TTG AAC
[M.sub.2]-IC-ICF     AGA AGT ATGGCA GAA AGT AAG CCT CCT ACT
[M.sub.2]-IC-ICR     GAT TGT AAT TCGGGG GAT TCA GTT TGT TCG

Table 2: Merozoite surface protein-2 allele prevalence
in South-East of Iran

District         Chabahar      Iranshahr     Nikshahr

Allele           No.   %       No.   %       No.   %

FC27     280bp   2     2.36    2     2.36    2     2.36
         300bp   3     3.53    2     2.36    2     2.36
         380bp   3     3.23    1     1.20    1     1.20
         400bp   1     1.20    3     3.53    2     2.36
3D7      400bp   1     1.20    2     2.36    1     1.20
         470bp   10    11.80   5     5.90    4     4.70
         500bp   4     4.70    2     2.36    3     3.53
         600bp   0     0.00    1     1.20    2     2.36
FC27 + 3D7       5     5.90    2     2.36    3     3.53
Total            29    34.20   20    23.60   20    23.30

District         Sarbaz       Total

Allele           No.   %      No.   %

FC27     280bp   0     0.00   6     7.10
         300bp   2     2.36   9     10.60
         380bp   2     2.36   7     8.20
         400bp   1     1.20   7     8.20
3D7      400bp   1     1.20   5     5.90
         470bp   2     2.36   21    24.70
         500bp   4     4.70   13    15.30
         600bp   1     1.20   4     4.70
FC27 + 3D7       3     3.53   13    15.3
Total            16    18.9   85    100.00
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Author:Namdar, Fatemeh; Ebrahimzadeh, Adel; Mohammadi, Saeed
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:Jan 1, 2014
Words:2744
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