Nucleotide sequences of each variable block in the Plasmodium falciparum merozoite surface ...... Hall R, Hyde JE, Goman M, Simmons DL, Hope IA, Mackay.
Am. J. Trop. Med. Hyg., 59(3), 1998, pp. 474–480 Copyright q 1998 by The American Society of Tropical Medicine and Hygiene
ALLELIC DIVERSITY AT THE MEROZOITE SURFACE PROTEIN-1 LOCUS OF PLASMODIUM FALCIPARUM IN CLINICAL ISOLATES FROM THE SOUTHWESTERN BRAZILIAN AMAZON MARCELO URBANO FERREIRA, QING LIU, OSAMU KANEKO, MASATSUGU KIMURA, KAZUYUKI TANABE, EMILIA AKEMI SHIRAISHI KIMURA, ALEJANDRO MIGUEL KATZIN, SHIN ISOMURA, AND FUMIHIKO KAWAMOTO Department of Parasitology, Institute for Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil; Department of Medical Zoology, Nagoya University School of Medicine, Nagoya, Japan; Department of Medical Zoology and Laboratory of Biophysics, Osaka City University Medical School, Osaka, Japan; Laboratory of Biology, Osaka Institute of Technology, Osaka, Japan
Abstract. Nucleotide sequences of each variable block in the Plasmodium falciparum merozoite surface protein1 gene (PfMSP-1) may be grouped into one of two or three possible allelic types, named after the reference isolates MAD20, K1, and RO33. Allelic diversity at this locus basically results from different combinations of allelic types in variable blocks. We used a polymerase chain reaction (PCR)–based strategy to type the variable blocks 2, 4a, 4b, and 10 of the PfMSP-1 gene of P. falciparum isolates from 54 symptomatic malaria patients living in Rondonia, a hypoendemic area in the southwestern Brazilian Amazon. Ten different PfMSP-1 gene types, defined as unique combinations of allelic types in variable blocks, were identified among the 54 isolates. Twenty-one isolates (39%) harbored more than one gene type and two had at least three genetically distinct clones. Hybrid sequences, with a MAD20-type sequence in the 59 segment (4a) and a K1-type sequence in the 39 segment (4b), were quite common in block 4. Direct sequencing of block 4 PCR products revealed a new putative recombination site in four isolates. In contrast with previous studies, the observed distribution of gene types does not deviate significantly from that expected under the null hypothesis of random association between allelic types detected in each variable block. These contradictory data are discussed with reference to the immunoepidemiologic features prevailing in distinct malariaendemic areas. Extensive polymorphism has been revealed by comparing nucleotide sequences of the merozoite surface protein-1 gene of Plasmodium falciparum (PfMSP-1) that codes for a major malaria vaccine candidate.1 Seven variable blocks, that are separated by either conserved or semiconserved regions, have been described in this molecule (Figure 1a).2 There are two versions of each PfMSP-1 variable block, represented by the sequences of the isolates MAD20 and K1. Most PfMSP-1 polymorphisms may be generated by intragenic recombination between these two representative types at sites near the 59 end of the gene (blocks 3, 4, and 5).2, 3 The only major exception to this dimorphic rule is the variable block 2, which has a third polymorphic form represented by the isolate RO33.4 This third version is relatively infrequent in culture-adapted isolates5 but quite common in field isolates from endemic areas in South America,6, 7 Africa,8 and Oceania.9 Therefore, major PfMSP-1 gene types can be defined as unique combinations of variable regions derived from 1) either of three possible block 2 allelic types, 2) one or both allelic types in block 4 because recombinant types with sequences derived from both MAD20 and K1 alleles have been found in this region,10, 11 and 3) either of two allelic types in variable blocks 6, 8, 10, 14, and 16 in the central and 39 portions of the gene. Immunization experiments in monkeys have been performed with native PfMSP-1 and recombinant peptides derived from different P. falciparum isolates,12–14 but it is unclear whether or not cross-protection can be induced when the immunizing and challenging parasites exhibit high degrees of amino acid diversity. This issue is particularly relevant when considering PfMSP-1 as a malaria vaccine candidate. Because the prevalence of PfMSP-1 polymorphisms is susceptible to both geographic and temporal variation, a particular immunogen may or may not match the most prev-
alent gene types in a given endemic area at a given moment. To address this question, more information is required about the genetic diversity in parasites from geographically diverse regions. We used a recently developed polymerase chain reaction (PCR)–based strategy15 to investigate the extent of PfMSP-1 allelic diversity in P. falciparum isolates from the southwestern Brazilian Amazon. Ten of the 24 possible distinct PfMSP-1 gene types were identified. Most parasites exhibited intragenic recombination within block 4, and a new putative recombination site was found in this variable region. In contrast with previous studies, however, we were unable to detect nonrandom associations between allelic types in this field parasite population. SUBJECTS, MATERIALS, AND METHODS
Source of P. falciparum isolates and DNA extraction. The field component of this study was conducted between June and July 1995 at the Center for Tropical Medicine of Rondonia, a 80-bed reference hospital situated in Porto Velho, southwestern Brazilian Amazon, after approval from the scientific and ethical committees of its Division of Research. Venous blood samples were collected after informed consent was obtained from 49 outpatients and 5 inpatients (70% males) eight months to 63 years of age (mean age 5 27 years) with an acute febrile illness and P. falciparum infection that was confirmed by microscopic examination of Giemsa-stained thick blood smears. Blood samples were immediately frozen at 2208C. Parasitemias ranged from 40 to 64,000 parasites/ml of blood, and two patients (an eightmonth-old infant with severe anemia and generalized convulsions and an eight-year-old boy with severe anemia and hemoglobinuria) had severe malaria.16 All patients lived in urban or rural areas in northern Rondonia (municipalities of
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FIGURE 1. a, basic structure of the Plasmodium falciparum merozoite surface protein-1 gene. Conserved, semiconserved, and variable blocks of the gene are shown as open, hatched, and closed boxes, respectively. Block numbers are those of Tanabe and others.2 b, locations and directions of the oligonucleotide primers used to type the variable blocks 2, 4a, 4b, and 10. For details, see Subjects, Materials, and Methods.
Porto Velho, Candeias do Jamari, and Jamari), a hypoendemic region characterized by unstable transmission of both P. falciparum and P. vivax.17 After thawing, 200 ml of clotted blood were lysed in 50 ml of 0.15% saponin in phosphatebuffered saline (PBS, pH 7.4), washed in PBS, and resuspended in 200 ml of TEN buffer (10 mM Tris-HCl, pH 8.0, 5 mM EDTA, and 100 mM NaCl) with 0.5% sodium dodecyl sulfate and 200mg/ml of proteinase K (Boehringer Mannheim, Mannheim, Germany). After overnight incubation at 548C, DNA was extracted using a standard phenolchloroform protocol, precipitated with ethanol at 2808C in the presence of 125 mM sodium acetate (pH 5.2), and resuspended in 50 ml of sterile distilled water. The DNA templates were stored at 2308C until tested.
The PfMSP-1 typing strategy. The four-step procedure may be briefly described as follows, and oligonucleotide primer locations are shown in Figure 2b. First step: block 2 typing. Block 2 was typed by nested PCR essentially as described elsewhere.9 The first amplification was performed with the common primers C1F (59AACTAGAAGCTTTAGAAGATGCAG-39) and C3R (59ACATATGATTGGTTAAATCAAAGAG-39), while the second amplification was performed in three separate reaction tubes with the common reverse primer C3R and either of the type-specific forward primers M2F (59-GGTTCAGGTAATTCAAGACGTAC-39), K2F (59-TCTTAAATGAAGAAGAAATTACTACAAA-39), or R2F (59-TAAAGGATGGAGCAAATACTCAAGT-39).
FIGURE 2. Polymerase chain reaction (PCR)–amplified fragments of the Plasmodium falciparum merozoite surface protein-1 gene variable blocks 2, 10, and 4 as analyzed by 2% agarose gel electrophoresis. Allelic types MAD20, K1, and RO33 are abbreviated respectively as M, K, and R, respectively. Molecular size markers are fX174 DNA fragments digested with Hae III (New England Biolabs, Beverly, MA). (a), block 2 typing. DNA from the isolates R143 (lanes 1–3), R62 (lanes 4–6), and R59 (lanes 7–9) was amplified with either of three type-specific forward primers and a common reverse primer. The MAD20-type (150 basepairs [bp]), RO33-type (270 bp), and K1-type (size range 5 260– 390 bp) PCR products are seen as single bands in lanes 1, 6, and 8, respectively. (b), block 10 typing. DNA from the isolates R75 (lane 1), R45 (lane 2), R134 (lane 3), R70 (lane 4), and R107 (lane 5) was amplified in one reaction with a common forward primer and a mixture of two type-specific reverse primers. Typical MAD20-type products (430 bp) are shown in lanes 1, 2, 3, and 5 and a K1-type product (270 bp) is shown in lane 4. (c), block 4 typing. Two different fragments amplified from the isolate R137 were used as templates for a nested PCR: a 470-bp fragment between blocks 3 and 5 (lane 1) and a 900-bp fragment between blocks 2 and 6 (lane 6). Block 4 was typed by nested PCR in separate reactions with four different combinations of type-specific forward and reverse primers (lanes 2–5 and 7–10). A single 100-bp band, corresponding to MAD20-type sequences in both the 59 and 39 regions of block 4, was amplified from both templates (lanes 2 and 7).
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Second step: block 10 typing. A single PCR with the common forward primer C9F (59-TAT(TA)CAAAATAACATACCTG(CT)(TA)ATGTA-39) and the type-specific reverse primers M10R (59-TGTTGTAGAAATGGATTAGG39) and K10R (59-GTAGAATGTGATGTATCATCATTTGC39) was performed to type block 10.15 Third step: preparing the DNA template to type block 4. Two different procedures were used: (3a) amplification of a short fragment between blocks 3 and 5 (about 470 basepairs [bp]) with the common primers C3F (59-TTCGTGCAAATGAATTAGACGTAC-39) and C5R (59-GGATCAGTAAATAAACTATCAATGT-39),11 or (3b) amplification of a long fragment between blocks 2 and 6 (between 900 and 1150 bp) with one of the type-specific forward primers M2F, K2F or R2F, chosen after the first step, and one of the type-specific reverse primers M6R (59-ATTTGAACAGATTTCGTAGGATCTTG-39) or K6R (59-GATATTCATTGGGTTCAG TTGATTC-39), chosen after the second step.15 Since intragenic recombination in the region between blocks 6 and 16 has not been observed,15, 18, 19 the allelic type determined for block 10 (either MAD20 or K1) is considered to be the same for block 6, and this information was used to select typespecific reverse primers for the step 3b. When the first or second step revealed the presence of two or more different allelic types, the reaction 3b was used in the third amplification step. Each parasite subpopulation present in mixed infections could be separately typed, because different DNA templates were prepared to type block 4. Fourth step: block 4 typing. Block 4 was typed by nested PCR in four separate reactions with one of the allelic typespecific forward primers M4F (59-TTGAAGATATAGATAAAATTAAAACAGATG-39) or K4F (59-AATGAAATTAAAAATCCCCCACCGG-39) and one of the allelic typespecific reverse primers M4R (59-TCGACTTCTTTTTTCTTATTCTCAG-39) or K4R (59-TCCTCGATTTTTTTGTTCTTATCAAG-39).11, 15 Both the 59 (block 4a) and the 39 (block 4b) segments of block 4 were typed.11 The 24 gene types distinguishable by the PCR-based strategy used here were assigned numbers (Table 1). Accordingly, a MAD20-like gene type (number 23) has MAD20-type sequences in all variable blocks, while the PfMSP-1 gene type of the isolate Wellcome has a MAD20-type sequence in block 2 and K1-type sequences in all other variable blocks (number 2). Since PfMSP-1 is a single-copy gene in the haploid genome of blood-stage parasites, we believe that isolates harboring more than one gene type represent mixed infections with genetically different P. falciparum subpopulations or clones. Protocols for the PCR. Template DNA (2–10 ml of extracted DNA or 1 ml of the first PCR product diluted 1:10 or 1:40 in distilled water) was amplified in a final reaction volume of 25 ml in the presence of 200 mM of each deoxynucleoside triphosphate (Takara Chemicals, Kyoto, Japan), 10 pmoles of each primer, and 0.8 units of recombinant Taq DNA polymerase (Takara Chemicals) in the buffer supplied by the manufacturer (20 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM 1.4 dithiothreitol, 0.5% Tween 20, 0.5% Nonidet P-40, and 5% glycerol). All reactions were performed in a PJ100 DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). For block 2 typing (first step ), the PCR conditions were one cycle at 948C for 2 min, 40 cycles
TABLE 1 Expected and observed frequencies of Plasmodium falciparum merozoite surface protein-1 gene (PfMSP-1) gene types in 54 P. falciparum isolates from the southwestern Brazilian Amazon Variable block
No. (%) of typed parasites†
Gene type*
2
4a
4b
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
K M R K M R K M R K M R K M R K M R K M R K M R
K K K M M M K K K M M M K K K M M M K K K M M M
K K K K K K M M M M M M K K K K K K M M M M M M
K K K K K K K K K K K K M M M M M M M M M M M M
Expected‡
0.23 0.09 0.15 0.95 0.38 0.64 0.05 0.21 0.03 0.21 0.09 0.14 5.67 2.30 3.83 23.42 9.50 15.83 1.26 0.51 0.85 5.20 2.11 3.52
(0.30) (0.12) (0.19) (1.23) (0.49) (0.83) (0.06) (0.27) (0.04) (0.27) (0.12) (0.18) (7.36) (2.99) (4.97) (30.41) (12.34) (20.56) (1.64) (0.66) (1.10) (6.75) (2.74) (4.57)
Observed
0 2 1 0 0 0 0 0 0 0 0 0 7 0 5 25 5 18 0 0 0 5 8 1
(0) (2.60) (1.30) (0) (0) (0) (0) (0) (0) (0) (0) (0) (9.09) (0) (6.49) (32.48) (6.49) (23.38) (0) (0) (0) (6.49) (10.39) (1.30)
* Each gene type is defined as a unique combination of allelic types detected in the variable blocks 2, 4a (59 segment of block 4), 4b (39 segment of block 4), and 10 of the PfMSP-1 gene. Allelic types are named after the reference isolates MAD20 (M), K1 (K), and RO33 (R). † Twenty-one isolates harbored more than one fully typed parasite population; frequencies (%) were computed using the total number of gene types detected in this parasite population (n 5 77) in the denominator. ‡ Expected distribution of PfMSP-1 gene types under the null hypothesis of random association between allelic types.20
(16 cycles for the nested procedure) at 928C for 30 sec, 608C for 1 min, and 708C for 1 min, followed by one cycle at 708C for 5 min. For all other reactions, the PCR conditions were one cycle at 958C for 5 min, 40 cycles (18 cycles in the nested procedure) at 928C for 30 sec (1 min in the three first cycles), 508C for 1 min, and 708C for 1 min, followed by one cycle at 708C for 5 min. The PCR products were analyzed by 2% agarose gel (Gibco BRL, Gaithersburg, MD) electrophoresis in the presence of ethidium bromide. The interpretation of PCR results is shown in Figure 2. DNA sequencing of PCR products. Selected block 4 products were sequenced using an ABI PRISMt Dye Terminator Cycle Sequencing kit (Perkin-Elmer Cetus) following the manufacturer’s protocol (P/N 402078 Revision A, August 1995). Briefly, block 4 fragments amplified by the fourth step were excised from the gels, reamplified with the appropriate primers, and the reactions were loaded onto 2% NuSievet plus 1% SeaKemt agarose gels (FMC BioProducts, Rockland, ME). The products were excised from the gels, purified using the Cleanmix kit (Talent, Trieste, Italy), and sequenced with the forward primers M4F or K4F. Sequences were analyzed by the 373 DNA Sequencing System (Applied Biosystems, Foster City, CA). Data analysis. For the purpose of this study, the frequency of each gene type was computed as its proportion of the total of typed parasite populations among the isolates tested. The proportion of each gene type expected under the null
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477
FIGURE 3. Nucleotide sequences of polymerase chain reaction (PCR)–amplified fragments of the Plasmodium falciparum merozoite surface protein-1 gene from Amazonian isolates, showing hybrid sequences in the variable block 4. Complete block 4 sequences of the isolates MAD20 (Tanabe and others2) and K1 (Kaneko and others11) are given for comparison. Base pairs are numbered after the MAD20 sequence. Identities with the MAD20 sequence are shown as (.) and deletions as (-), while (*) marks a nucleotide position at which the MAD20 sequence differs from all other published MAD20-type sequences.11, 19 Underlined nucleotides correspond to the primers M4F and M4R (MAD20) and K4F and K4R (K1) used to amplify this fragment. Note that isolate R107 has a MAD20-type sequence, isolate R143 has a K1-type sequence, and all other sequenced isolates have hybrid (MAD20 plus K1) sequences. The corresponding gene types, as defined in Table 1, are as follows: R107, type 22; R143, type 2; R39, type 18; and all other isolates, type 16. Vertical arrows indicate the putative region where recombination has occurred, situated between the nearest two informative differences between MAD20-type and K1-type sequences.
hypothesis of random association of variable block allelic types was derived from a simple probability model analogous to those used in population genetics to estimate the expected frequency of multi-locus genotypes.20 For instance, the expected proportion of MAD20-like gene types in this population is calculated by multiplying the observed proportions of parasites with MAD20-type sequences in blocks 2 (0.195), 4a (0.805), 4b (0.182), and 6-16 (0.961), while the expected proportion of Wellcome-like gene types is calculated by multiplying the observed proportions of parasites with a MAD20-type sequence in block 2 (0.195) and K1type sequences in blocks 4a (0.195), 4b (0.818), and 6-16 (0.039). To test the null hypothesis of random assortment of allelic types in this parasite population, the expected and observed frequencies were compared using KolmogorovSmirnov statistics.21 The same test was used to compare the distribution of gene types in single and mixed infections. Significance was defined at the 5% level. RESULTS
Ten of 24 possible different PfMSP-1 gene types were identified, and 77 parasite populations were fully typed among 54 isolates. The observed frequency of each type is shown in Table 1. About 95% of all parasites belonged to the seven most frequent gene types. Five PfMSP-1 gene types identified here have been previously characterized in fully or partially sequenced P. falciparum clones or isolates:19 type 2 is Wellcome-like, 13 is CAMP-like or Uganda Palo Alto-like, 16 is RO71-like, 23 is MAD20-like, and 24 is RO33-like. Two other combinations described here were also detected in Thai isolates by Southern blot hybridization18 and partial sequencing:11 types 17 (Thai isolates 807, 827, 828, 836, 837, 842, and 843) and 22 (Thai isolate 946). Moreover, 22 of the 24 types were recently detected in clinical isolates from southern Vietnam by the same PCR-based strategy used here.15 The types 4 and 11 were the only combinations of PfMSP-1 allelic types absent in that endemic area.15 More than one type was found in 21 Amazonian isolates (39%), and two isolates (R3 and R121) harbored at least three genetically distinct clones. Interestingly, the block 4 type described in isolate T9-
101,10 with a MAD20-type sequence in the 59 segment (4a) and a K1-type sequence in the 39 segment (4b), was quite common (62%) in Amazonian isolates. Nevertheless, no parasite population was found to harbor a K1-type sequence in the 59 segment and a MAD20-type sequence in the 39 region of block 4, a recombinant version recently described in isolates from Thailand (3 of 23 sequenced isolates), the Solomon Islands (6 of 23 typed isolates), and Vietnam (29 of 136 typed isolates).11, 15 Furthermore, K1-type sequences in blocks 6-16 were rarely observed here (4%), and no K1-like gene type (number 1 in Table 1) was identified. Table 1 also shows the frequency of each gene type that is expected under the null hypothesis of random association of allelic types. Despite some seemingly divergent data (for instance, gene types 14, 17, and 23), no significant difference was found between expected and observed distributions (d 5 0.076, d0.05 5 0.153, P .. 0.05 by the KolmogorovSmirnov test), and all expected prevalences were included in the 95% confidence interval of the observed data. The same data were also compared by the x2 statistic for goodness of fit, with similar results (x2 5 2.87, degrees of freedom 5 5, P . 0.05). For x2 analysis all cells with expected frequencies , 5 were pooled. We also tested the null hypothesis that gene types were present in single and mixed infections at similar frequencies. For this analysis, we classified as harboring mixed infections all isolates with more than one allelic type detected after the first, second, or fourth step, even if some of the subpopulations present in mixed infections could not be fully typed by all amplification steps. Twenty-six isolates met these criteria. Types 13, 15, and 17 were found only in mixed infections, but once again we were unable to reject the null hypothesis (d 5 0.120, d0.05 5 0.321, P .. 0.05 by the Kolmogorov-Smirnov test). We confirmed the presence of hybrid sequences within block 4 (Figure 3). In two (R39 and R71) of six isolates with recombinant block 4 sequence MAD20 plus K1, the putative site of recombination was the same as that previously described,10, 11 between nucleotides 1129 and 1135 of the MAD20 sequence. However, a new putative site of recombination was found in four isolates (R18, R106, R119, and R134) between nucleotides 1119 and 1124. Nucleotide sequences were further confirmed by analysis of the fragment
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amplified using primers M4F and C5R with the reverse primer C5R applied to the cycle sequencing reaction. Therefore, we propose that the region upstream of nucleotide 1119 may be defined as the 59 segment of block 4 (block 4a), while the region downstream of nucleotide 1135 may be defined as the 39 segment (block 4b). These boundaries are indicated by arrows in Figure 3. DISCUSSION
We have investigated genetic diversity at the PfMSP-1 locus in field P. falciparum isolates from the Brazilian Amazon. A relatively restricted number of PfMSP-1 gene types (10 of 24 major versions) was identified in this area, and a new putative site of recombination was described within the variable block 4 of this gene. Differential prevalences of dimorphic PfMSP-1 epitopes have been previously investigated by Conway and others in P. falciparum isolates from West Africa (Gambia and Nigeria) and Brazil (eastern Amazon).10 The isolates were serotyped by indirect immunofluorescence with a panel of allelic type-specific monoclonal antibodies after a brief period (24– 48 hr) of in vitro growth.10 The low prevalence of K1-type sequences or epitopes in blocks 6-16 was a common feature of this and our studies, and contrasts with that observed in Thai isolates.18 However, some epitopes were shown to cooccur more frequently than expected in these serotyped parasite populations.10 These findings could be explained by genetic drift if rates of intragenic recombination are low, but Conway and others alternatively suggested that certain epitope associations could confer a selective advantage to the parasites.10 Moreover, there is an indirect statistical evidence, derived from sequence analysis of in vitro cultured strains, for positive selection in blocks 2 and 3.22 Nevertheless, analogous associations between allelic types were not detected in our Amazonian isolates. Our contradictory results may originate from the fact that 1) short-term in vitro growth may have induced selection against particular parasite subpopulations in the serotyped isolates,23 2) typed regions of the PfMSP-1 antigen or gene are not exactly the same in both studies, and 3) different epidemiologic patterns of host-parasite interactions that prevail in each endemic area may affect the genetic structure of malaria parasites. Differences in sample size might theoretically originate the discrepancies between both studies. The number of field isolates typed in Gambia (n 5 445) by far exceeds our sample size, and statistically significant deviations from the null hypothesis of random associations of epitopes are more likely to be detected in such large samples. Negative results, therefore, might be due to a relatively low power of the tests in small samples.20 However, at least some significant associations between dimorphic epitopes were detected by Conway and others in relatively small numbers of field isolates from Nigeria (n 5 60) and the eastern Brazilian Amazon (n 5 62).10 The epitopes recognized by the panel of monoclonal antibodies used by Conway and others are located on the PfMSP-1 blocks 2, 3, 4b, 6-16, and 16-17. The most frequent statistical associations were found between epitopes on blocks 2 and 3 and between epitopes on blocks 3 and 4b.10 In contrast, the present study was focused on the variable
blocks 2, 4a, 4b, and 6-16; semiconserved (block 3) and conserved (block 17) regions were not typed here. As a result, possible nonrandom associations found between epitopes on block 3 and neighboring regions could not be addressed by our methodology. An extensive PCR-based study of P. falciparum isolates from southern Vietnam has provided more information about the genetic diversity and putative selection at the PfMSP-1 locus.15 Laboratory methods and statistical analyses were similar to the ones we used, except that the sampled parasite population in Vietnam is considerably larger (186 genetically distinct parasite populations from symptomatic patients were typed), and that malaria endemicity, as judged by the parasite rate in this region (about 32%),15 is quite high if compared with that usually found in Rondonia (usually , 2%).24, 25 Twenty-two of 24 possible PfMSP-1 gene types were found,15 suggesting a higher degree of genetic diversity in Vietnam than in the Amazon. Nonrandom associations between allelic types were detected between blocks 4 (including both the 59 and 39 regions) and 6 among Vietnamese isolates. Nonrecombinant types (that is, those characterized by the same allelic type, either MAD20 or K1, in blocks 4a, 4b, and 6) were more common than expected by random recombination in this region. It remains unclear whether similar biases tend to be induced by selective pressure in parasite populations from different endemic areas. Possible selection mechanisms for PfMSP-1 polymorphisms in natural parasite populations are still conjectural since the function of PfMSP-1 is unknown.26 The selective role of the host’s immune system remains to be fully investigated. The relative importance of variable versus conserved domains as targets of naturally acquired anti-PfMSP-1 immune responses is controversial, and such responses are likely to differ in both quantitative and qualitative features in human populations exposed to varying levels of malaria transmission.27 It is reasonable to hypothesize that naturally acquired immunity may be more selective in areas of high endemicity than in hypoendemic regions such as the Brazilian Amazon, where effective anti-parasite immune responses rarely develop in the host population. If nonrandom assortment of PfMSP-1 allelic types is essentially a result of immune selection, this phenomenon is expected to be more common under conditions of high malaria transmission. Malaria endemicity in southern Vietnam is considerably lower than in holoendemic Africa, but transmission is still much more intense than in the Brazilian Amazon. Therefore, immune pressure should result in an intermediate pattern in Vietnam (more intense selection than in Brazil but less than in Gambia or Nigeria), which is compatible with the findings by Kaneko and others.15 Further cross-sectional and longitudinal studies of malaria parasites in different endemic areas are required to elucidate this point. The development of molecular methods such as that used here has provided field and laboratory researchers with improved tools to type wild parasite populations. Furthermore, the possible effects of naturally acquired and vaccine-induced immunity against conserved and polymorphic antigens on the genetic structure of natural P. falciparum populations represent a critical issue to be investigated by these methods in the near future.
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Acknowledgments: We thank Dr. Photini Sinnis (Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY) for critical reading of the manuscript, Drs. Mauro Shugiro Tada, Juan Miguel Villalobos, and Elza Noronha (Center for Tropical Medicine of Rondonia, Porto Velho, Brazil) and Dr. Luis Marcelo Aranha Camargo (Department of Parasitology, University of Sao Paulo, Sao Paulo, Brazil) for valuable support during field work, Dr. Mian Zhou (Department of Medical Zoology, Nagoya University School of Medicine, Japan) for help in laboratory work, and Dr. Somchai Jongwutiwes (Department of Parasitology, Chulalongkorn University, Bangkok, Thailand) for helpful discussions. Financial support: This work was supported by grants from the UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (Marcelo Urbano Ferreira, Emilia Akemi Shiraishi Kimura, and Alejandro Miguel Katzin), the Program for Malaria Control in the Amazon (PCMAM) of the National Health Foundation (Brazilian Ministry of Health) (Marcelo Urbano Ferreira, Emilia Akemi Shiraishi Kimura, and Alejandro Miguel Katzin), FAPESP (Marcelo Urbano Ferreira), the Japanese Ministry of Education, Science, Sports and Culture (Grant-inAids for Scientific Research on Priority Areas #08281207 and #09270210 to Fumihiko Kawamoto and #08281104 to Kazuyuki Tanabe and for Scientific Research [B] #07457069 to Kazuyuki Tanabe) and the Toyota Foundation (96B3-011 to Fumihiko Kawamoto). Marcelo Urbano Ferreira was supported by a graduate research student scholarship from the Japanese Ministry of Education, Science, Sports and Culture.
8.
9.
10.
11.
12.
13.
14. Authors’ addresses: Marcelo Urbano Ferreira, Emilia Akemi Shiraishi Kimura, and Alejandro Miguel Katzin, Department of Parasitology, Institute for Biomedical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes 1374, 05508-900, Sao Paulo, SP, Brazil. Marcelo Urbano Ferreira, Qing Liu, Shin Isomura, and Fumihiko Kawamoto, Department of Medical Zoology, Nagoya University School of Medicine, 65 Tsuruma, Showa, Nagoya 466, Japan. Osamu Kaneko, Department of Medical Zoology, Osaka City University Medical School, 1-4-54 Asahi-machi, Abeno-ku, Osaka 545, Japan (present address: Laboratory of Malaria Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room B1-38, Bethesda, MD 20892). Masatsugu Kimura, Laboratory of Biophysics, Osaka City University Medical School, 1-4-54 Asahi-machi, Abeno-ku, Osaka 545, Japan. Kazuyuki Tanabe, Laboratory of Biology, Osaka Institute of Technology, 5-16-1 Ohmiya, Asahi-ku, Osaka 535, Japan.
15.
16.
17. 18.
19. REFERENCES
1. Diggs CL, Ballou WR, Miller LH, 1993. The major merozoite surface protein as a malaria vaccine target. Parasitol Today 9: 300–302. 2. Tanabe K, Mackay M, Goman M, Scaife JG, 1987. Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodium falciparum. J Mol Biol 195: 273–287. 3. Kerr PJ, Ranford-Cartwright LC, Walliker D, 1994. Proof of intragenic recombination in Plasmodium falciparum. Mol Biochem Parasitol 66: 241–248. 4. Certa U, Rotmann D, Matile H, Reber-Liske R, 1987. A naturally occurring gene encoding the major surface antigen precursor p190 of Plasmodium falciparum lacks tripeptide repeats. EMBO J 6: 4137–4142. 5. Peterson MG, Coppel RL, Moloney MB, Kemp DJ, 1988. Third form of the precursor to the major surface antigens of Plasmodium falciparum. Mol Cell Biol 8: 2664–2667. 6. Kimura E, Mattei D, di Santi SM, Scherf A, 1990. Genetic diversity in the major merozoite surface antigen of Plasmodium falciparum: high prevalence of a third polymorphic form in strains derived from malaria patients. Gene 91: 57–62. 7. Snewin VA, Herrera M, Sanchez G, Scherf A, Langsley G, Herrera S, 1991. Polymorphism of the alleles of the merozoite surface antigens MSA1 and MSA2 in Plasmodium
20. 21. 22.
23.
24.
25.
479
falciparum wild isolates from Colombia. Mol Biochem Parasitol 49: 265–276. Scherf A, Mattei D, Sarthou J-L, 1991. Multiple infections and unusual distribution of block 2 of the MSA1 gene of Plasmodium falciparum detected in West African clinical isolates by polymerase chain reaction analysis. Mol Biochem Parasitol 44: 297–300. Kimura M, Kaneko O, Inoue A, Ishii A, Tanabe K, 1995. Amplification by polymerase chain reaction of Plasmodium falciparum DNA from Giemsa-stained thin blood smears. Mol Biochem Parasitol 70: 193–197. Conway DJ, Rosario V, Oduola AMJ, Salako LA, Greenwood BM, McBride JS, 1991. Plasmodium falciparum: intragenic recombination and nonrandom associations between polymorphic domains of the precursor to the major merozoite surface antigens. Exp Parasitol 73: 469–480. Kaneko O, Jongwutiwes S, Kimura M, Kanbara H, Ishii A, Tanabe K, 1996. Plasmodium falciparum: variation in block 4 of the precursor to the major merozoite surface proteins in natural populations. Exp Parasitol 84: 92–95. Hall R, Hyde JE, Goman M, Simmons DL, Hope IA, Mackay M, Scaife J, Merkli B, Richle R, Stocker J, 1984. Major surface antigen gene of a human malaria parasite cloned and expressed in bacteria. Nature 311: 379–382. Siddiqui WA, Tam LQ, Kramer KH, Hui GSN, Case SE, Yamaga KM, Chang SP, Chan EBT, Kan S-C, 1987. Merozoite surface coat precursor protein completely protects Aotus monkeys against Plasmodium falciparum malaria. Proc Natl Acad Sci USA 84: 3014–3018. Etlinger HM, Caspers P, Matile H, Schoenfeld H-J, Stueber D, Takacs B, 1991. Ability of recombinant or native proteins to protect monkeys against heterologous challenge with Plasmodium falciparum. Infect Immun 59: 3498– 3503. Kaneko O, Kimura M, Kawamoto F, Ferreira MU, Tanabe K, 1997. Plasmodium falciparum: allelic variation in the merozoite surface protein 1 gene in wild isolates from southern Vietnam. Exp Parasitol 86: 45–57. Warrell DA, Molineaux ME, Beasles PF, 1990. Severe and complicated malaria. Trans R Soc Trop Med Hyg 84 (suppl 2): 1–65. World Health Organization, 1996. World malaria situation in 1993. Part II. Wkly Epidemiol Rec 71: 25–29. Jongwutiwes S, Tanabe K, Nakazawa S, Uemura H, Kanbara H, 1991. Coexistence of gp195 alleles of Plasmodium falciparum in a small endemic area. Am J Trop Med Hyg 44: 299–305. Miller LH, Roberts T, Shahabuddin M, McCutchan TF, 1993. Analysis of sequence diversity in the Plasmodium falciparum merozoite surface protein-1 (MSP-1). Mol Biochem Parasitol 59: 1–14. Tibayrenc M, 1995. Population genetics of parasitic protozoa and other microorganisms. Adv Parasitol 36: 47–115. Massay FJ Jr, 1951. The Kolmogorov-Smirnov test for goodness of fit. J Am Stat Assoc 46: 68–78. Hughes AL, 1992. Positive selection and interallelic recombination at the merozoite surface antigen-1 (MSA-1) locus of Plasmodium falciparum. Mol Biol Evol 9: 381–393. Viriyakosol S, Siripoon N, Zhu XP, Jarra W, Seugorn A, Brown KN, Snounou G, 1994. Plasmodium falciparum: selective growth of subpopulations from field samples following in vitro culture, as detected by the polymerase chain reaction. Exp Parasitol 79: 517–525. Camargo LMA, Ferreira MU, Krieger H, Camargo EP, Pereira da Silva LH, 1994. Unstable hypoendemic malaria in Rondonia (Western Amazon Region, Brazil): epidemic outbreaks and work-associated incidence in an agro-industrial rural settlement. Am J Trop Med Hyg 51: 16–25. Camargo LMA, Dal Colletto GMD, Ferreira MU, Gurgel SM, Escobar AL, Marques A, Krieger H, Camargo EP, Pereira da Silva LH 1996. Hypoendemic malaria in Rondonia (Brazil, Western Amazon Region): seasonal variation and risk groups in an urban locality. Am J Trop Med Hyg 55: 32–38.
480
FERREIRA AND OTHERS
26. Holder AA, Blackman MJ, 1994. What is the function of MSP-1 on the malaria merozoite? Parasitol Today 10: 182–184. 27. Ferreira MU, Kimura EAS, Souza JM, Katzin AM, 1996. The
isotype composition and avidity of naturally acquired antiPlasmodium falciparum antibodies: differential patterns in clinically immune Africans and Amazonian patients. Am J Trop Med Hyg 56: 315–323.
