Plasmodium falciparum: Sequence analysis of the gene encoding the C-terminus region of the merozoite surface protein-1, a potential malaria vaccine antigen, in Iranian clinical isolates

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Abstract

C-terminal region of merozoite surface protein-1 of Plasmodium falciparum (PfMSP-1) isolated from different parts of the world revealed sequence variability, however no data exist on sequence heterogeneity of this region from Iran. To address this question, DNA encoding the carboxyl (C)-terminal region of PfMSP-1 was amplified in 144 Iranian P. falciparum clinical isolates, using allele type-specific primers. In this study both MAD20 (88.2%) and K1 (7.6%) types were detected. Sequence analysis of 33 and 92 fragments corresponding to pfmsp-142 and pfmsp-119 revealed eight (15MAD1–15MAD7 and 15KCH) and five [A1 (E/TSR/L), A2 (Q/KNG/F), A3 (E/KNG/F), A4 (E/TSG/L), and A5 (Q/KNG/L)] distinct haplotypes, respectively. E/TSG/L variant type was the predominant haplotype, and reported only from Thailand and India, but E/KNG/L is widespread in Africa, Asia, and Latin America; but not found among Iranian isolates. In summary, result of this study indicates limited antigenic diversity, and thus support the potential utility of the C-terminal region of PfMSP-1 in designing polyvalent vaccine constructs.

Introduction

Malaria is one of the most threatening parasitic diseases, being endemic in most of the tropical and subtropical countries and having a heavy burden on public health throughout the world. The rapid spread of drug resistant Plasmodium falciparum, the development of resistance to insecticides by anopheles mosquitoes, movement of refugees or populations seeking job, deterioration of socioeconomic conditions and environmental change have been associated with global resurgence of malaria (Krogstad, 1996, Marsh, 1998). Therefore, these factors emphasize the urgent need for the development of an effective vaccine that in combination with other control strategies would combat the disease.

In Iran, malaria was a major health problem in the past and national malaria control programs were initiated in the 1958 and eradication was achieved for the northern Caspian region by 1977 and transmission was reduced in the south-eastern provinces of Sistan and Baluchistan, Hormozgan and tropical region of Kerman (Palmquist and Aldridge, 1954, Zaim, 1987, Beljaev, 1999, Sadrizadeh, 2001). Population movements from Pakistan and Afghanistan have also led to importation of malaria cases from neighboring countries to south-eastern of Iran. This may interfere with control of the disease in the provinces bordering these countries and may also spread the disease to malarious freed areas inside the country. Based on recent reports from Center for Diseases Management and Control, Ministry of Health in 2005, about 20, 000 malaria cases were detected in the whole country; 85% of those were from south-eastern provinces with both Plasmodium vivax (80%) and P. falciparum (20%) species were present (unpublished data).

Plasmodium falciparum is one of the most prevalent of the four malaria parasites of humans and causes most of the severe and lethal infections. The multistage life cycle of malaria parasites provides multiple potential targets for vaccine development that are being evaluated for inclusion in a polyvalent malaria vaccine (Siddiqui et al., 1987, Herrera et al., 1990, Howard and Pasloske, 1993, D’Alessandro et al., 1995). Although several vaccine candidate antigens of P. falciparum have been identified and characterized, the development of a universal malaria vaccine is still a difficult challenge. One reason is the antigenic variation of different geographic isolates of the parasite that is an important concern in the development of malaria subunit vaccines.

One of the most extensively characterized vaccine candidate antigens is merozoite surface protein-1 (MSP-1). This protein is a 195 kDa glycoprotein on the merozoite surface of P. falciparum (PfMSP-1), which capable of binding sialic acid (Holder and Freeman, 1982, Hall et al., 1984). As a requirement for merozoite entry into a red cell, MSP-1 undergoes two separate proteolytic cleavage, first at merozoite release from an infected cell, which it is cleaved into four polypeptide fragments (83, 30, 38, and 42 kDa), and the second during invasion of a red blood cell that C-terminal 42 kDa (MSP-142) fragment is cleaved to 33 kDa and 11 (MSP-119) polypeptide fragments (Holder et al., 1987, Holder et al., 1992, Cooper, 1993). The MSP-119 fragment is the only fragment that remains anchored to the merozoite membrane by a glycosylphosphatidylinositol tail attached to the carboxyl (C)-terminal residue after invasion (Blackman et al., 1990, Blackman et al., 1991). This fragment consists of two epidermal growth factor (EGF)-like domains each containing six cysteine residues (Blackman et al., 1991) that are thought to be required in interactions with the erythrocytes (Holder, 1994). Several studies indicate that conformational epitopes created by both EGF-like structures are the target of protective antibodies (Chang et al., 1992, Chappel and Holder, 1993, Ling et al., 1994, Ling et al., 1995, Daly and Long, 1993, Farley and Long, 1995, Calvo et al., 1996) (Fig. 1. The MSP-142 (coded by block 15, 16, 17) is of particular interest among PfMSP-1 fragments and similar MSP-119 fragment (coded by block 17) is the target of immune protection against asexual blood stages, therefore both molecules are among promising antigens to be used as a subunit malaria vaccine (Blackman et al., 1990, Chang et al., 1992, Chappel and Holder, 1993). However, several lines of evidence suggest that C-terminal fragment of MSP-1 of parasites isolated from different parts of the world reveal variability due to point mutations (Ferreira et al., 2003, Kaslow et al., 1994, Lalitha et al., 1999, Qari et al., 1998, Raj et al., 2004, Vijay Kumar et al., 2005), which is one of the main factors potentially limiting the efficacy of any asexual stage vaccine development based on this protein. Therefore, to apply MSP-142 or MSP-119 fragments as part of an effective polyvalent vaccine, it is essential to determine the extent of sequence variation in the wild type parasites from global geographical regions.

Most of the sequence data of C-terminal region of PfMSP-1 have been obtained from Africa, South East Asia, Latin America, and India subcontinent and so far no published data exist on sequence variation of this region of the gene in clinical isolates of P. falciparum from Middle East including Iran and its neighboring countries, which could be used for running field trials and/or developing local studies on possible vaccines and drug formulation against parasite (Miller et al., 1993, Jongwutiwes et al., 1993, Kang and Long, 1995, Tolle et al., 1995, Qari et al., 1998, Lalitha et al., 1999, Raj et al., 2004, Vijay Kumar et al., 2005). As the presence of polymorphism in C-terminal region of PfMSP-1 causes a serious challenge to the use of it in developing a polyvalent vaccine based on MSP-1 protein, and because there is no such information about sequence diversity and also the prevalence of pfmsp-1 alleles in clinical isolates of Iranian P. falciparum, therefore, we considered that it is necessary to define the sequence variations in the C-terminal region of PfMSP-1 comprising of 15th, 16th, and 17th blocks in 33 isolates and part of 16th and the full-length of block 17 in additional 59 isolates of clinical P. falciparum isolates from malaria hypoendemic areas of Iran.

Section snippets

Study area and P. falciparum isolates

Blood samples were collected from 144 P. falciparum infected individuals with symptomatic uncomplicated malaria attending at the Malaria Health Center in Chabahar Public Health Department in Sistan and Baluchistan province, south-eastern Iran during 2001–2005. The data of collection were 2001 (n = 48), 2002 (n = 3), 2003 (n = 10), 2004 (n = 44), and 2005 (n = 39). Malaria endemicity in the study areas is characterized by year-round hypoendemic transmission of both P. falciparum (20%) and P. vivax (80%)

Frequency distribution of diversity in C-terminal region of pfmsp-1

The PCR amplification of the C-terminal region of pfmsp-1 gene was successfully carried out on 144 isolates collected from patients infected with P. falciparum. PCR mediated amplification of C-terminal region of pfmsp-1 using allele specific primers resulted in the amplification of a 1312 bp (MAD20 type) and 1257 bp (K1 type) DNA fragments.

Total 144 samples were amplified by PCR using allele specific primers for amplification of C-terminal region of pfmsp-1 (with 15KF/TR and 15MF/TR primers).

Discussion

C-terminus of PfMSP-1 region is an important malaria vaccine candidate antigen; therefore genetic analysis of this region of protein in clinical isolates from different endemic areas is very important issue. The choice of this gene was motivated because, pfmsp-1 is one of the most extensively studied genes of Plasmodium species and its polymorphisms have been investigated in isolates from diverse endemic regions. Our study would complement this information and would allow comparing the Iranian

Acknowledgments

We thank the Malaria Division, CDC, Iran, for their cooperation. We are grateful for the hospitality and generous collaboration of Zahedan University of Medical Sciences, and the staff of the Public Health Department, Sistan and Baluchistan province, Chabahar district, for their assistance in collecting blood samples from the field. We also thank the patients in Chabahar district for their participation in this study. This investigation received financial support from Pasteur Institute of Iran.

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