Experimental Parasitology 103 (2003) 127–135

www.elsevier.com/locate/yexpr

Plasmodium falciparum: limited genetic diversity of MSP-2in isolates circulating in Brazilian endemic areas

S. Sallenave-Sales,a,* M.F. Ferreira-da-Cruz,a C.P. Faria,a C. Cerruti Jr.,b

C.T. Daniel-Ribeiro,a and M.G. Zalisc,d

a WHO Collaborating Center for Research and Training in the Immunology of Parasitic Diseases, Departamento de Imunologia,

Instituto Oswaldo Cruz/Fiocruz, Avenida Brasil 4365, Manguinhos, CEP 21045-900, Rio de Janeiro, Brazilb Departamento de Medicina Social, Universidade Federal do Esp�ıırito Santo, Rio de Janeiro, Brazil

c Instituto de Biof�ıısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazild Departamento de Medicina Preventiva, Programa de Doenc�as Infecto-Parasit�aarias, Hospital Universit�aario Clementino Fraga Filho,

UFRJ, CEP 21949-000, Rio de Janeiro, Brazil

Received 13 August 2002; received in revised form 24 April 2003; accepted 5 May 2003

Abstract

The genetic polymorphism of the surface merozoite protein 2 (MSP-2) was evaluated in Plasmodium falciparum isolates from

individuals with uncomplicated malaria living in a Brazilian endemic area of Peixoto de Azevedo. The frequency of MSP-2 alleles

and the survival of genetically different populations clones in 104 isolates were verified by Southern blot and SSCP-PCR. Single and

mixed infections were observed in similar frequencies and the rate of detection of FC27 and 3D7 allelic families was equivalent.

Eight alleles were identified and among them, the sequence polymorphism was mainly attributed to variations in the repetitive

region. Interestingly, in three alleles nucleotide polymorphism was identical to that detected in a previous study, conducted in 1992,

in a near Brazilian endemic area. This finding demonstrated the genetic similarity between two isolate groups, besides the certain

temporal stability in the allelic patterns. The implications of these data for studies on the genetic diversity are also discussed.

� 2003 Elsevier Science (USA). All rights reserved.

Index Descriptors and Abbreviations: MSP-2; Plasmodium falciparum; allelic diversity; SSCP-PCR; MSP-2, merozoite surface protein 2; PCR,

polymerase chain reaction; SSCP, single strand conformational polymorphism; SDS, sodium dodecyl sulfate; EDTA, ethylenediaminetetraacetic acid

1. Introduction

The great genetic variability of Plasmodium falcipa-

rum contributes to generate a number of biologicallyand antigenically different parasite populations (Anders

et al., 1993; Babiker et al., 1997; Druilhe et al., 1998).

This genetic diversity can occur at any endemic level and

appears to be related to pathology as well as acquired

immunity, since multiple allelic forms could differ in

their ability to escape recognition by the host�s immuneresponse (Engelbrecht et al., 1995; Gupta et al., 1994;

Muller et al., 2001; Zevering et al., 1994). Presumably,this polymorphism would also restrict the effectiveness

of subunit vaccines against P. falciparum infection if

* Corresponding author. Fax: +55-21-280-1589.

E-mail address: sallenave@ioc.fiocruz.br (S. Sallenave-Sales).

0014-4894/03/$ - see front matter � 2003 Elsevier Science (USA). All rights

doi:10.1016/S0014-4894(03)00091-2

variable proteins are included (Genton et al., 2002).

Additionally, the nature and extent of antigenic diversity

could also give new information on the dynamics of

host–parasite relationships and on parasite evasionmechanisms.

Results from epidemiological investigations using

PCR-based assays and allele specific hybridization

studying P. falciparum polymorphic antigens have given

valuable data on parasite population structure in the

human host. These typing schemes estimate the extent

of antigenic polymorphism by the frequency evaluation

of different circulating allelic variants and by the ge-notype complexity of individual infections (Babiker et

al., 1999; Daubersies et al., 1994; Mercereau-Puijalon,

1996; Ntoumi et al., 1995; Sallenave-Sales et al., 2000).

In this context, particular attention has been given to

antigens associated with the surface of merozoite stage,

reserved.

128 S. Sallenave-Sales et al. / Experimental Parasitology 103 (2003) 127–135

such as the 35–56-kDa merozoite surface protein-2(MSP-2) of P. falciparum. The location of this protein,

and its possible role in the invasion process, as well as

the ability of anti-MSP-2 antibodies to inhibit parasite

growth in vitro and in vivo, motivated its use as a

potential malaria vaccine candidate (Genton et al.,

2000; Lawrence et al., 2000). The MSP-2 gene contains

blocks with tandem repeated units that can vary in size

and nucleotide sequence in P. falciparum isolates(Smythe et al., 1988, 1990). The MSP-2 alleles described

generally fall into two allelic families, FC27 and 3D7,

which differ considerably by the dimorphic structure of

the variable central region. The FC27 family shows

varying numbers of structurally conserved R1 (96 bp)

and R2 (36 bp) repetitive regions, in contrast to those

observed in the 3D7 family, where the R1 region is less

conserved being highly variable in length, copy number,and sequence.

Field studies on natural populations of P. falciparum

carried out mainly in hyper- and holoendemic regions

have demonstrated that MSP-2 gene is highly poly-

morphic, showing considerable size and sequence vari-

ation which can reflect in complexes infections with a

mixture of allelic variants belonging to the same and/or

different allelic family (Babiker et al., 1999; Felger et al.,1999; Kyes et al., 1997; Paul et al., 1995). Sequence

variation may also result from the recombination events

between allelic variants of FC27 and 3D7 families within

the mosquito vector (Irion et al., 1997; Keer et al., 1994;

Marshall et al., 1991; Snewin et al., 1991). This extensive

polymorphism raises the important question about the

impact of MSP-2 diversity on the immune response, as

well as the ability of different allelic variants to inducedifferent clinical manifestations (Al-Yaman et al., 1997;

Engelbrecht et al., 1995). In fact, specific effects of a

malaria vaccine containing 3D7 allele of MSP-2 antigen

on parasites with this particular MSP-2 genotype were

demonstrated, besides the vaccination with 3D7 may

have contributed to the increase the rate of FC27-as-

sociated morbidity in vaccinated children (Genton et al.,

2002).MSP-2 alleles and their frequencies have been com-

monly determined by PCR-RFLP and/or Southern blot

hybridization using specific probes and rely on size dif-

ferences between repeat regions. However, such meth-

ods underestimate the true level of diversity and further

most of them cannot identify hybrid recombinant MSP-

2 alleles. The association of restriction analysis methods

to nucleotide sequencing provides a more accurate de-termination of the genetical microheterogeneity of MSP-

2. Considering these aspects together with the fact that

few data are available about the complexity and be-

havior of P. falciparum populations in Brazilian endemic

areas (Creasey et al., 1990; Hoffmann et al., 2001;

Sallenave-Sales et al., 2000), we decided to study the

allelic diversity of MSP-2 gene in P. falciparum Brazilian

isolates from residents of Peixoto de Azevedo village(MT-Brazil).

2. Materials and methods

2.1. Study site

Studies have been carried out between 1995 and 1996in Peixoto de Azevedo village, which is localized in the

Mato Grosso State in the southern part of the Brazilian

Amazon Forest. The population consists mainly of mi-

grants individuals exposed during all the year to the risk

of malaria infection (Duarte and Fontes, 1998). At time

collection, the village had the highest malaria transmis-

sion rates in Brazil with an annual parasitic index (po-

sitive slides/1000 inhabitants) of 284.8 and 263.1 in 1995and 1996, respectively. The number of malaria cases in-

duced by P. falciparum and Plasmodium vivax was very

similar, corresponding to 47 and 52% in 1995 and to 43

and 56% in 1996, respectively. In 1995, the rate of bites/

person/hour by Anopheles darlingi vector was 10. Some

P. falciparum isolates collected in 1992 in a similar study

carried out in Porto Velho, another Brazilian endemic

area, were used in a comparative analysis.

2.2. Isolates

After signed consent venous blood samples were ta-

ken from 104 symptomatic individuals from the mu-

nicipality of Peixoto de Azevedo (MT-Brazil). The

P. falciparum parasite density was determined by mi-

croscopic examination of Giemsa stained thick bloodsmears. The fresh blood samples were centrifuged

(10min at 350g) to remove the plasma and the leuko-

cytes were depleted by repeated washes with an equal

volume of 0.15M phosphate-buffered saline. The red

blood cell pellet was stored in 50% (v/v) glycerolyte so-

lution (0.9% NaCl/4.2% sorbitol/20% glycerol) in liquid

nitrogen (N2). Samples from Porto Velho were similarly

collected and stored.

2.3. PCR amplification and electrophoresis

One milliliter of red blood cells was lysed by 2–3

washing cycles with distilled water. Free parasites were

resuspended in 5 volumes of TEN buffer (10mM Tris–

HCl, pH 8.0, 1mM EDTA, pH 8.0, 0.15M NaCl, 0.5%

Triton X-100, and 0.5% SDS) and 5mg/ml proteinase Kand incubated at 37 �C for 2–3 h. The DNA was phenol-

extracted, precipitated, and amplified using P. falciparum

oligonucleotides specific for the central polymorphic re-

gion of MSP-2 antigen, as described elsewhere (Conta-

min et al., 1995). Two microliters of DNA was amplified

in a 100 ll reaction volume containing 10 nmol of each

dNTP, 100 pmol of each primer, 2.5U TaqI DNA

Fig. 1. MSP-2 gene polymorphism by PCR in P. falciparum isolates

from Peixoto de Azevedo. I—520 bp; II—500 bp; III—600bp; and IV—

620 bp.

S. Sallenave-Sales et al. / Experimental Parasitology 103 (2003) 127–135 129

Polymerase (Perkin–Elmer), and 5 ll of 10� buffer(Perkin–Elmer, 2.0mM MgCl2). The PCRs were carried

out using a Hybaid automated heating block for 35 cy-

cles (2 s at 94 �C, 1min at 55 �C, and 2min at 72 �C). Tenmicroliters of PCR was loaded onto a 2.5% Nusieve

GTG agarose gel (FCM Bioproduct, USA) in 1� TAE

buffer (0.04M Tris–acetate, 1mM EDTA) in the pres-

ence of ethidium bromide (0.5 lg/ml).

2.4. Hybridization conditions

Following gel analysis, amplified products from

MSP-2 were transferred onto a Nylon membrane (Hy-

bond-N, Amersham), as recommended by suppliers, and

hybridized with specific radioactive probes representing

each one of the two allelic families of the MSP-2 gene:

FC27 and 3D7. The probes were obtained by amplifi-cation of P. falciparum DNA from reference isolates and

clones (Contamin et al., 1995). These probes were la-

beled by Nick Translation (Boehringer–Mannheim,

France) and the unincorporated nucleotides were re-

moved by spin dialysis (Quickspin, Amersham). Hy-

bridizations were performed overnight at 65 �C in the

presence of 6� SSC (0.9M NaCl, 0.09M sodium cit-

rate), 0.1% SDS, 2.5% skimmed milk, and 100 lg/ml ofsheared salmon sperm DNA (Sigma). The membranes

were successively washed at 65 �C in 6� SSC, 2� SSC,

0.5� SSC, and 0.1� SSC. After each wash, the mem-

branes were exposed to an X-OMAT film (Kodak) with

an intensifying screen for 6–48 h at )70 �C.

2.5. Single-strand conformational polymorphism

To assess sequence microheterogeneity within frag-

ments defined as the same genotype by PCR and allelic

typing, each amplified fragment was analyzed by SSCP.

The PCR sample was previously concentrated with 0.3M

of sodium acetate, resuspended in 30 ll of distilled water,and loaded onto a 2% Nusieve GTG agarose gel (FCM

Bioproduct, USA) in 1� TAE buffer (0.04M Tris–ace-

tate, 1mM EDTA). The amplified fragments were indi-vidually gel-purified using Wizard DNA preps as

recommended by suppliers (Promega) and resuspended

in 40 ll of distilled water. PCR fragments were digested

to completion with RsaI (Promega), then extracted with

phenol–chloroform, and precipitated with ethanol. The

samples were denatured prior to analysis by adding 1 llof 0.5MNaOH/10mM EDTA and then electrophoresed

for 3 h at 10V/cm using 0.5� Tris–borate EDTA (TBE)buffer (pH 8.4). SSCP patterns were visualized by silver

staining as described elsewhere (Gonc�alves et al., 1990).

2.6. DNA sequencing

To verify sequence modifications in the alleles of

MSP-2, the allelic types from Peixoto de Azevedo were

sequenced. In addition, four fragments from PortoVelho (Rondoonia State) that were previously typed

(Sallenave-Sales et al., 2000) were chosen for a com-

parative analysis: two FC27 alleles—PV54 (500 bp) and

PV56 (520 bp) and two 3D7 alleles—PV25 (620 bp) and

PV61 (560 bp). Uncloned amplified fragments were se-

quenced using the di-deoxy chain termination procedure

with Taq polymerase and fluorescently labeled (Dye

terminator kit, Perkin–Elmer) with the primers 50-GAGTAT AAG GAG AAG TAT GG-30 (forward) and 50-CCT GTA CCT TTA TTC TCT GG-30 (reverse). Se-quencing products were separated on an ABI Model

373A automated DNA Sequencer and the sequences

were analyzed using University of Wisconsin Genetics

Computer Group software package. The resulting MSP-

2 allele sequences have been submitted to EMBL/Gen-

Bank Data libraries under Accession Nos. AY137758 toAY137765.

2.7. Statistical analysis

The data were analyzed using the program GrapPad

Instat, version 2.05a. The v2 test was applied for the

analysis of allelic frequencies.

3. Results

3.1. Allelic polymorphism of MSP-2

One hundred and seventeen PCR fragments corre-

sponding to 104 P. falciparum isolates from Peixoto de

Azevedo were obtained from the central polymorphicregion of MSP-2 (Fig. 1). Thirty-four (24%) fragments

that were not visible by ethidium bromide staining were

detected after probing, performing a total of 154 frag-

ments identified in this area. All of them hybridized

against only one of the two probes (either FC27 or

3D7) at low stringency conditions (0.1� SSC) (data not

shown). Ninety (58%) out of 154 fragments were

identified as FC27 family—type I (520 bp), type II(500 bp), and type III (600 bp), whereas the 3D7 family

Fig. 2. Single strand conformational polymorphism analysis of MSP-2

PCR fragments. MSP-2 PCR fragments were digested with RsaI and

analyzed by SSCP in a silver staining 10% polyacrylamide gel: Ia and

Ib—520 bp patterns; II—500 bp pattern; III—600bp pattern; and IVa–

IVd—620bp patterns. The PhiX174 HaeIII (left) and 100 bp (right)

were used as DNA size markers.

130 S. Sallenave-Sales et al. / Experimental Parasitology 103 (2003) 127–135

fragments—type IV (620 bp)—represented about 42%

(64/154). Single (one fragment) and mixed (two frag-

ments) MSP-2 infections were observed in similar fre-

quencies, representing 53 and 47% of the 104 isolates,

respectively. The presence of fragments of both allelic

families in mixed infections was 96%.

A degree of DNA microheterogeneity that was un-

detectable by PCR alone was verified by SSCP analysisof the 117 fragments detected in electrophoresis gel (Fig.

2). Alteration sequences were observed only in I (FC27/

520 bp) and IV (3D7/620 bp) types, whereas no mobility

shift was observed for fragments II (FC27/500 bp) and

Table 1

Infection patterns of the 104 P. falciparum isolates analyzed by SSCP-PCR

Complexity infection Fragment (bp) Hybridi

Single infections1

S1 520 FC27

S2 520 FC27

S3 500 FC27

S4 600 FC27

S5 620 3D7

S6 620 3D7

Mixed infections2

M7 520/500 FC27/F

M8 520/600 FC27/F

M9 520/620 FC27/3D

M10 520/620 FC27/3D

M11 520/620 FC27/3D

M12 520/620 FC27/3D

M13 500/620 FC27/3D

1 Isolates with one fragment.2 Isolates with two fragments; bp—base pair

III (FC27/600 bp). Two SSCP patterns were observedfor type I, designated alleles Ia and Ib, and four were

found for type IV—IVa, IVb, IVc, and IVd alleles. The

Ib (59/117) and IVa (28/117) alleles were the most fre-

quent types detected among the 104 isolates. In 4% of

SSCP pattern obtained, we detected additional bands of

low intensity most probably corresponding to the minor

clonal populations that were not discriminated by PCR

technique (data not shown).Considering the size and number of fragments found

by the isolate characterized, the allelic typing, and the

SSCP pattern, we observed nine different MSP-2 infec-

tion profiles in Peixoto de Azevedo (Table 1).

3.2. Sequencing of MSP-2 fragments

The FC27 alleles showed repetitive sequences thatwere related but not identical to each other. The repeat

regions of all compared alleles of this family consist of

one (Ia, Ib, and II alleles) or three (III allele) copies of

96 bp (R1) and two (II allele), three (Ia and Ib alleles) or

none (III allele) copies of 32 bp (R2), thus displaying a

more conserved organization than the 3D7 allelic family

(Fig. 3). Point mutations were spread by the regions�sequences, although some of them were shared by allalleles, such as that which resulted in a non-synonymous

substitution of the six amino acids (SSGNAP) in the E3

region (Fig. 4). Few nucleotide substitutions, spread in

the E1 (family specific region 1), R1, and R2 regions,

account for the difference between the three FC27 alleles

(Ia, Ib, and II), all leading to non-synonymous changes

(Fig. 4). More diversity was observed when referring to

allele III (600 bp) that presented three identical copies of96 bp repeats in R1 region contrasting with the lack of

32 bp repeats in the R2 region. The deletion of two

codons and some nucleotide substitutions were also

and allelic typing

zation SSCP pattern No. of isolates, N (%)

Ia 13 (12)

Ib 50 (48)

II 5 (5)

III 2 (2)

IVa 20 (19)

IVd 1 (1)

C27 Ia/II 1 (1)

C27 Ib/III 1 (1)

7 Ia/IVa 1 (1)

7 Ib/IVa 6 (6)

7 Ib/IVb 1 (1)

7 Ib/IVc 2 (2)

7 II/IVa 1 (1)

Fig. 5. Schematic representation of the predicted 3D7 MSP-2 protein

sequences from Peixoto de Azevedo isolates. The central variable re-

gion of each allelic type is compared to the 3D7 reference strain

(Smythe et al., 1990), which is represented in the upper position. Un-

filled blocks of 3D7 protein sequence indicate conserved regions. The

black blocks represent family-specific regions (E1–3) and the two

blocks of tandem repeats (R1 and R2) are indicated. The numbers

represent amino acid positions corresponding to published sequences

of 3D7 strain. The arrows show the location of the primers used for

PCR (2.1 and 2.2) and sequencing (A and B).

Fig. 3. Schematic representation of the predicted FC27 MSP-2 protein

sequences from Peixoto de Azevedo isolates. The central variable re-

gion of each allele is compared to the FC27 reference strain (Smythe et

al., 1988), which is represented in the upper position. Unfilled blocks of

FC27 protein sequence indicate conserved regions. The dotted blocks

represent family-specific regions (E1–3) and the two blocks of tandem

repeats (R1 and R2) are indicated. The numbers represent amino acid

positions corresponding to published sequence of FC27 strain. The

arrows show the location of the primers used for PCR (2.1 and 2.2)

and sequencing (A and B).

S. Sallenave-Sales et al. / Experimental Parasitology 103 (2003) 127–135 131

detected, but only two of them were situated in positions

not shared by at least two FC27 alleles (Fig. 4). In

contrast, the variability observed in repetitive sequences

of FC27, E2 (family specific region 2) and E3 (family

specific region 3) regions was extremely conserved in all

alleles.

The sequence modifications of 3D7 antigenic family

were mainly situated in the R1 and E2 regions, i.e., thetwo most polymorphic segments of the molecule in our

study (Fig. 5). However, as described to FC27 alleles,

several nucleotide substitutions occurred in shared

Fig. 4. Alignment of the predicted amino acid sequence of MSP-2 of the FC2

region of allelic types is compared to the reference strain FC27 sequence (

represent no homology residues and have been inserted to optimize the alignm

The family-specific regions (E1–3) and the two tandem repeats (R1 and R2)

points of allelic sequences, demonstrating a non-

randomic pattern of distribution. As expected, the first

repetitive region (R1) showed to be extremely poly-morphic, beginning in all alleles with a short variable

segment, followed by multiple copies of 4–10 amino

acids that differed in number, sequence, and length from

one allele to other (Figs. 5 and 6). The richness of gly-

cine (G), serine (S), and alanine (A) residues, besides

the biased codon usage with an exclusive use of NNT

7 allelic family from Peixoto de Azevedo isolates. The central variable

Smythe et al., 1988). Residues identical are indicated by dots. Gaps

ent of the sequence. The first residue of each unit repeat is underlined.

are indicated.

Fig. 6. Alignment of the predicted amino acid sequence of MSP-2 of the 3D7 allelic family from Peixoto de Azevedo isolates. The central variable

region of allelic types is compared to the reference strain 3D7 sequence (Smythe et al., 1990). Residues identical are indicated by dots. Gaps represent

no homology residues and have been inserted to optimize the alignment of the sequence. The first residue of each unit repeat is underlined. The

family-specific regions (E1–3) and the two tandem repeats (R1 and R2) are indicated.

132 S. Sallenave-Sales et al. / Experimental Parasitology 103 (2003) 127–135

codons characteristic for all 3D7 alleles, was also ob-

served in Brazilian alleles. Due to this exclusive use ofNNT codons, no synonymous substitution was found in

this repetitive region (Fig. 6). The E2 region was the

most polymorphic non-repetitive segment, denoting

deletions of several base pairs, as observed to IVc, up to

insertions of segments with 18 bp (AVASAR), as ob-

served in the case of IVa allele. The other family specific

regions (E1 and E3) presented a limited diversity. All

but IVc allele contained the same E1 nucleotide se-quence, and the polymorphism of E3 region was mainly

due to the deletion of a segment of 33 bp in the IVd

allele, which had also shown the two unique synony-

mous substitutions observed in the Brazilian allelic se-

quences (214 and 220 aas). The poly-threonine stretch

located downstream of the E2 region demonstrated an

example for sequence conservation at nucleotide level.

The nucleotide sequence of IVb allele was not deter-mined due to the ambiguity of sequence probably gen-

erated by a mixture of fragments.

The Brazilian alleles were more closely related among

them than with the FC27 and 3D7 strain sequences

(Smythe et al., 1988, 1990), which differ by the geo-

graphical origin. In the case of FC27 strain, the genetic

diversity was mainly related to copies of repeats, while

to 3D7, this diversity was observed in all regions ofMSP-2 gene in different degrees. Curiously, IVd allele

presents the same R1 repeat sequence as that described

for 3D7 strain, although both sequences differ by nu-

cleotide substitutions and deletions outside the repeats.

4. Discussion

Genetic diversity appears to play an important role in

the acquiring of natural malaria immunity and also with

regard to the development and deployment of control

measures. Here, we outlined the polymorphism of the

important surface antigen MSP-2, in Brazilian malariamesoendemic area and further demonstrated the re-

markable similarity of the genetic characteristics of two

Brazilian group isolates.

In our analysis, the use of allele specific typing and

PCR technique was fundamental to detect the survival

of genetically different P. falciparum clones within in-

fections. The gain of sensitivity obtained especially in

respect to 620 bp fragment of 3D7 family had greatimpact on the evaluation of the frequency of mixed in-

fections in the area. This aspect is important if we

consider that mixed infections could increase the prob-

ability of crossed gametocyte fertilization between dif-

ferent populations inside the vector, generating new

allelic genotypes (Babiker et al., 1994). Additionally, it

has also been suggested that plasmodial infections con-

taining simultaneously parasites with FC27 and 3D7genotypes could facilitate the intragenic recombination

between the two allelic families, which could provide

allelic hybrid types (Irion et al., 1997; Keer et al., 1994;

Marshall et al., 1991; Snewin et al., 1991). However,

despite the high frequency (44%) of mixed infections

found in Peixoto de Azevedo, the alternative re-

combinant form (hybrid) was not detected by us or by

Hoffmann et al. (2001), in studies conducted in an otherBrazilian state (Rondoonia). In our study, a possible ex-

planation could be the finding that in the majority of

mixed infections parasites carrying 3D7 genotypes were

present in a low density, reducing the possibility of

cross-fertilization between 3D7 and FC27 gametes and,

consequently, the generation of hybrids. Another ex-

planation could be that such hybrids are generated, but

a selective pressure could eliminate the parasites con-taining them. Nevertheless, even in mesoendemic (Kyes

et al., 1997; Zwetyenga et al., 1998) and holoendemic

(Babiker et al., 1997; Daubersies et al., 1996) regions of

S. Sallenave-Sales et al. / Experimental Parasitology 103 (2003) 127–135 133

Africa, where multiclonal infections are prevalent andpresumably the rate sexual reproduction is higher, this

event is rare. Thus, independently of the endemicity of

the area, how frequently new genotypes are introduced

into a population and how they are maintained seem to

be crucial points to better understand the generation of

hybrid types.

The microheterogeneity sequences suggested by the

different degrees of probe homology in the allelic typing(data not shown) were confirmed by the SSCP technique

and sequencing analysis. The two groups of allelic

families presented a sequence polymorphism very simi-

lar to that already described where the inter-allelic MSP-

2 diversity was mainly related to repetitive regions

(Felger et al., 1997; Marshall et al., 1994). One of the

consequences of these variations was the size polymor-

phism, which was less evident among 3D7 alleles, be-cause the number and size alterations of the repeats were

compensated by deletions and insertions in other points

of sequence.

A picture of strong conservation in repeat structure

and nucleotide sequence was observed for FC27 alleles.

It is possible that mechanisms of amplification and/or

unequal recombination during the meiosis could con-

tribute to generate the FC27 allelic variation. These twoprocesses could explain, for example, the duplication

and the deletion of repeats units in R1 and R2 regions of

the III allele, respectively (Fig. 3). In this sense, the II

allele could have evolved from Ia by deleting one repeat

unit or Ia could have derived from II by repeat ampli-

fication. Despite the variability of the two repetitive

units among the isolates, some underlying patterns can

be clearly observed suggesting an ancestral repeat unit.The complete deletion of R2 region (32 bp) seems to be

frequent, as this deletion has also been observed in

isolates from other endemic areas (Felger et al., 1994;

Marshall et al., 1994; Ranford-Cartwright et al., 1996).

This finding led to the conclusion that this repetitive

region is not important to the parasite, since the para-

sites without this region are able to survive. In contrast,

the complete deletion of R1 region (96 bp unit) has notbeen described until now. Moreover, this region con-

tains the epitope STNS that is considered the reactivity

point for blocking monoclonal antibodies, suggesting its

importance in the invasion process (Epping et al., 1988).

The most striking feature of 3D7 nucleotide repeat

sequence was the exclusive presence of NNT codons,

which clearly causes a strong bias in codon usage and

can contribute considerably to increase antigenic diver-sity. It has been suggested by Felger et al. (1997) that

NNT codon exclusivity results from homogenization

mechanisms at nucleotide sequence level, such as un-

equal crossing over and biased gene conversion. In this

case, these proposed events acting only at the DNA level

would not necessarily reflect selection acting on the

amino acid sequence that would not maintain the

homogeneity between repeats. On the other hand, themajor sources of interallelic diversity in 3D7 allelic

family have been proposed to be replication slippage,

causing either duplication or deletion of a hexamer

GGT GCT or multiples of it, and mitotic or meiotic

unequal exchange, which can also increase or reduce the

copy number of repeats (Felger et al., 1997). In this

light, the presence of identical repeats in IVd, PV25, and

PV61 alleles could be explained by amplification mech-anisms, whereas the imperfect nucleotide repeats as seen

in IVa and IVc alleles may be caused by DNA poly-

merase slippage.

The majority of point mutations, observed in both

Brazilian allelic groups, conducted to non-synonymous

substitutions that could contribute to increase the anti-

genic diversity. Several of such mutations occurred in

specific sites of sequences demonstrating a non-randomdistribution pattern that might indicate an immunolog-

ical significance if clustered in putative epitopes involved

in the parasite evasion mechanism. The position-specific

feature of some mutations could suggest the existence of

typical patterns, although these patterns are not exclu-

sive of Brazilian endemic areas (Felger et al., 1994, 1997;

Marshall et al., 1994).

Our data suggest that the P. falciparum parasitepopulations of MSP-2 circulating in two Brazilian

communities are genetically homogeneous and tempo-

rally stable, since isolates from Peixoto de Azevedo and

Porto Velho presented MSP-2 alleles with identical or

similar sequence of central variable regions. In addition,

similar to the data reported by Hoffmann et al. (2000),

there was no remarkable evidence of differences in allelic

family frequencies and infection complexity between thetwo areas, in spite of the fact that P. falciparum isolates

had been collected in two different periods and in geo-

graphically distinct areas. Considering the extensive

polymorphism of MSP-2 gene, the finding of identical

sequences or different combinations of the motifs in

isolates from different geographical areas might be un-

expected. It is possible that all MSP-2 alleles circulating

in wild parasite populations were derived from a com-mon ancestor (Doba~nno et al., 1997) or alternatively the

extensive homoplasy results from unifying selection of

MSP-2 alleles in separate parasite lineages (McCutchan

et al., 1992).

The simplicity of the allelic profiles from isolates of

Peixoto de Azevedo, when compared to those observed

in highly endemic areas such as Senegal (Daubersies

et al., 1996), Tanzania (Babiker et al., 1999; Felger et al.,1999), and Papua New Guinea (Felger et al., 1994), was

consistent with the endemicity of the Brazilian endemic

areas. Thus, diversity is expected to be correlated with

transmission intensity, since in holoendemic areas the

higher complexity of human infections could be a result

of a major exposition to different parasite genotypes

carried by the mosquito vector. On the other hand, the

134 S. Sallenave-Sales et al. / Experimental Parasitology 103 (2003) 127–135

fact that the samples were obtained from symptomaticindividuals should also be considered an alternative or

additional explanation for the limited diversity ob-

served, since minor quantitative and qualitative varia-

tions in the parasite antigen alleles harbored by

symptomatic individuals had been reported (Daubersies

et al., 1994; Sallenave-Sales et al., 2000). In fact, the

higher frequency of a multiplex pattern observed among

asymptomatic infections is claimed to be related topremunition state (Babiker et al., 1997; Daubersies et al.,

1996).

Our molecular epidemiological studies clearly showed

an important, although restricted, genetic diversity of

MSP-2 in two Brazilian endemic areas. Considering that

the genetic diversity would not necessarily reflect selec-

tion acting at protein level, works are in progress to

associate these data with MSP-2 antibody reactivity inorder to evaluate the impact of this polymorphism on

the immune response to MSP-2 antigens. Moreover,

this approach could also open the possibility of investi-

gating the clinical impact of a particular MSP-2 geno-

type, a crucial question in understanding P. falciparum

pathogenesis.

Acknowledgments

We thank Dr. Odile Mercereau Puijalon for the

provision of the MSP-2 probes and all the patients of

Peixoto de Azevedo who volunteered to provide us with

blood samples. We also thank the help of Drs. Adeilton

Alves Brand~aao and Alberto D��AAvila, Department of

Biochemistry and Molecular Biology, and Dr. Rosa,Department of Genetics of Institute Oswaldo Cruz. This

work was supported by CNPq (Brazilian Research

Council) and Fundac�~aao Oswaldo Cruz (Fiocruz).

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