Within-population genetic diversity of Plasmodium falciparum

Vaccine 31 (2013) 1334–1339
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Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Within-population genetic diversity of Plasmodium falciparum vaccine candidate
antigens reveals geographic distance from a Central sub-Saharan African origin
Kazuyuki Tanabe a,b,∗ , Toshihiro Mita c , Nirianne M.Q. Palacpac b , Nobuko Arisue b , Takahiro Tougan b ,
Satoru Kawai d , Thibaut Jombart e , Fumie Kobayashi f , Toshihiro Horii b,∗
a
Laboratory of Malariology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
c
Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, Tokyo 113-8421, Japan
d
Department of Tropical Medicine and Parasitology, Dokkyo Medical University, Tochigi 321-0293, Japan
e
MRC Centre for Outbreak Analysis and Modelling, Department of Infectious Disease Epidemiology, School of Public Health, Faculty of Medicine, Imperial College, London W2 1PG, UK
f
Department of Infectious Diseases, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan
i n f o
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Populations of Plasmodium falciparum, the most virulent human malaria parasite, are diverse owing
to wide levels of transmission and endemicity of infection. Genetic diversity of P. falciparum antigens,
within and between parasite populations, remains a confounding factor in malaria pathogenesis as well
as clinical trials of vaccine candidates. Variation of target antigens in parasite populations may arise from
immune pressure depending on the levels of acquired immunity. Alternatively, similar to our study in
housekeeping genes [Tanabe et al. Curr Biol 2010;70:1–7], within-population genetic diversity of vaccine candidate antigens may also be determined by geographical distance from a postulated origin in
Central sub-Saharan Africa. To address this question, we obtained full-length sequences of P. falciparum
genes, apical membrane antigen 1 (ama1) (n = 459), circumsporozoite protein (csp) (n = 472) and merozoite surface protein 1 (msp1) (n = 389) from seven geographically diverse parasite populations in Africa,
Southeast Asia and Oceania; and, together with previously determined sequences (n = 13 and 15 for csp
and msp1, respectively) analyzed within-population single nucleotide polymorphism (SNP) diversity. The
three antigen genes showed SNP diversity that supports a model of isolation-by-distance. The standardized number of polymorphic sites per site, expressed as S , indicates that 77–83% can be attributed by
geographic distance from the African origin, suggesting that geographic distance plays a signiﬁcant role in
variation in target vaccine candidate antigens. Furthermore, we observed that a large proportion of SNPs
in the antigen genes were shared between African and non-African parasite populations, demonstrating
long term persistence of those SNPs. Our results provide important implications for developing effective
malaria vaccines and better understanding of acquired immunity against falciparum malaria.
© 2012 Elsevier Ltd. All rights reserved.
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Article history:
Received 20 September 2012
Received in revised form
15 November 2012
Accepted 14 December 2012
Available online 4 January 2013
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Keywords:
Malaria
Plasmodium falciparum
Vaccine
AMA1
CSP
MSP1
1. Introduction
Malaria is a serious public health problem in the tropics with
estimated 216 million cases and 655,000 malaria deaths in 2010
[1], due mostly to infections of Plasmodium falciparum, the most
virulent human malaria parasite. To effectively contain this deadly
disease, malaria vaccines are decisively needed. A few parasite
∗ Corresponding authors at: Department of Molecular Protozoology, Research
Institute for Microbial Diseases, Osaka University, 3-1 Yamada-Oka, Suita, Osaka
565-0871, Japan. Tel.: +81 6 6879 8279; fax: +81 6 6879 8281.
E-mail addresses: [email protected] (K. Tanabe),
[email protected] (T. Mita), [email protected] (N.M.Q. Palacpac),
[email protected] (N. Arisue), [email protected] (T. Tougan),
[email protected] (S. Kawai), [email protected] (T. Jombart),
[email protected] (F. Kobayashi), [email protected] (T. Horii).
developmental stages have been targeted for malaria vaccine
development. Currently, various vaccine constructs based on surface proteins of sporozoites and merozoites are being developed
[2]. Success in interventions using any of the developmental stages
will require better understanding in genetic variation within and
between parasite populations.
Recently we showed a strong negative correlation between
within-population genetic diversity of the parasite housekeeping
genes and geographic distance from a postulated origin in Central sub-Saharan Africa over Africa, Asia and Oceania [3]. Neither
regional variation in transmission intensity nor malaria interventions using antimalarial drugs or insecticides appeared to be tightly
associated with the geographic distribution of within-population
parasite genetic diversity. Together with age estimation of parasite populations, the study lends credence to a Central sub-Saharan
African origin of P. falciparum and colonization along with modern
0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2012.12.039
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K. Tanabe et al. / Vaccine 31 (2013) 1334–1339
Table 1
Seven P. falciparum populations analyzed in this study.
Country
Local area
Sampling year
References
Tanzania
Ruﬁji River Delta,
Eastern coast
Winneba, Western
coast
Mae Sot, Northeastern
Thailand
Palawan Island
Wewak, East Sepik,
Northeast coast
Northeastern
Guadalcanal Island
Four islands (Malakula,
Gaua, Santo, Pentecost)
1993, 1998, 2003
[23]
2004
[3]
1995
[19]
1997
2001, 2002
[22]
[3]
1996
[21]
1996–1998
[20]
Ghana
Thailand
Philippines
Papua New Guinea
Solomon Islands
Vanuatu
2.2. DNA sequencing
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To obtain full-length sequences of the P. falciparum AMA1
(ama1), CSP (csp) and MSP1 genes (msp1), genomic DNA was
ampliﬁed by PCR using Takara LA Taq polymerase (Takara Bio,
Japan). Procedures and conditions for PCR ampliﬁcation have been
described [24]. Two sets of primers were used in two successive
reactions. For ama1 ampliﬁcation, the ﬁrst PCR used primers ama1F2 and ama1-R1 (Supplementary Table 1), followed by nested
PCR with primers ama1-F3 and ama1-R1. csp was ampliﬁed with
primers CS.F1 and CS.R0, followed by nested PCR with primers
CS.F1 and CS.1332R. msp1 was ampliﬁed with primers UPF1 and
DWR1, followed by nested PCR with primers UPF3 and DWR3. DNA
sequencing was performed as described elsewhere [24]. Sequences
showing two or more peaks at the same position in an electropherogram were considered to be mixed infections and were excluded
from further analysis. Sequences of msp1 are known to be grouped
into two major allelic types, MAD20- and K1-types in blocks 6–16,
a central 3.5 kb region [25,26]. We obtained very limited numbers
of K1 type in Africa (n = 4); and this scarcity made geographic comparison for K1 type sequences infeasible, and, thus, was excluded
from further analysis.
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human migrations out-of-Africa about 60,000 years ago [4,5]. These
resulted in smooth patterns of reduction in parasite genetic diversity along with geographic distance from the African origin, akin to
the distribution of human genetic diversity. However, P. falciparum
possesses a number of antigens that are targets of host immunity.
Immune target antigen genes are under natural selection [6,7].
In highly endemic areas where repeated infections are common,
parasites are frequently exposed to host immunity, whereas in
low endemic areas where infections are limited, parasites are less
likely to be exposed to strong protective immunity. It is therefore
likely that immune target antigen genes and housekeeping genes
show unique geographic distribution of within-population genetic
diversity. So far very few studies have compared geographic distribution of parasite genetic diversity between antigen genes and
housekeeping genes in parasite populations from wide geographic
regions.
With the seven previously collected geographically wide parasite populations from Africa, Asia and Oceania [3], we determined
within-population genetic diversity of P. falciparum vaccine candidate genes and examined its correlation with geographic distance
from a Central sub-Saharan African origin. We speciﬁcally analyze
SNP diversity of three vaccine candidate genes coding for the apical
membrane antigen 1 (AMA1), the circumsporozoite protein (CSP)
and the merozoite surface protein 1 (MSP1). CSP is expressed during the sporozoite and early liver stage [8]. It is involved in the
invasion of the liver cells. Antibodies against this immunodominant
surface antigen inhibit parasite invasion and was associated with
reduced risk of clinical malaria [9,10]. A CSP-based vaccine, RTS,S,
showed 30–50% protection against clinical episodes of malaria
[11]. AMA1 and MSP1, implicated in host erythrocyte invasion, are
leading blood-stage vaccine candidates. AMA1 is localized in the
micronemes of the merozoite [12], an apical organelle that plays
an important role in parasite invasion [13]. Anti-AMA1 antibodies seem to be associated with protection from clinical malaria
[14,15]. Various constructs of AMA1 polypeptides are currently
being pursued for vaccine development/trials [2]. MSP1 is initially
synthesized as a 200-kDa precursor that undergoes processing
during schizont rupture to produce four fragments (83-, 30-, 38and 42-kDa) [16]. Further cleavage of the C-terminal 42-kDa fragment, MSP142 , yields a 19-kDa fragment, MSP119 [17]. Naturally
exposed individuals in malaria-endemic areas have antibodies speciﬁc for MSP119 that correlated with lower risk to clinical malaria
[18]. Intense research efforts have focused on both MSP142 and
MSP119 as vaccine candidates [2]. In this study, we characterized
the geographic distribution of SNPs in these three antigen genes
and compared them to those obtained from housekeeping genes.
Results clearly show a strong negative correlation between withinpopulation genetic diversity of the three vaccine candidates and
geographic distance from the Central sub-Saharan African origin
over Africa, Asia and Oceania.
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2. Materials and methods
2.1. Parasite isolates and DNA collection
A total of 531 P. falciparum isolates were collected from seven
countries: Tanzania, Ghana, Thailand, Philippines, Papua New
Guinea (PNG), the Solomon Islands and Vanuatu (Table 1). These
seven parasite populations were also used for the polymorphism
study in two housekeeping genes, sarcoplasmic/endoplasmic reticulum Ca2+ -transporting ATPase gene (serca) and adenylosuccinate
lyase gene (adsl) [3]. Details of the parasite isolates and procedures
for extraction of the parasite DNA have been described previously
[3,19–23]. In all cases, ethical clearance for sampling was obtained
from relevant ethical committees.
2.3. Statistical analysis
Obtained in this study were 459 ama1, 472 csp and 389 msp1
sequences. We included additional 13 csp and 15 msp1 sequences
from our previous study (Supplementary Table 2) (thus, total n for
csp = 485; msp1 = 404). DNA sequences were aligned as described
[24] and sequence regions that do not allow unambiguous SNP
identiﬁcation such as tandem repeats and insertions/deletions
(indel) were excluded. In csp, the central NANP/NVDP repeat region
and a region containing a 57 bp indel upstream to the central region
were excluded. In msp1, excised regions included blocks 2 and 4,
and short regions with degenerated tandem repeats and indels in
blocks 8 and 16 [26]. Sequences of serca and adsl [3] were used for
comparison.
Nucleotide diversity was estimated as S , the standardized
number of polymorphic sites per site, and , the average pairwise nucleotide difference, as previously described [24]. The mean
numbers of synonymous substitutions per synonymous site (dS)
and non-synonymous substitutions per non-synonymous site (dN)
were estimated [24]. If dN is signiﬁcantly higher than dS, diversifying selection is inferred. Frequency of SNPs was computed using
Arlequin v3.1 [27] and singleton SNPs as well as SNPs with minor
allele frequency (MAF) <5% were calculated. SNPs were classiﬁed
into either geographic area-speciﬁc SNP or those shared by multiple
populations. Difference in frequency of SNPs among various groups
was assessed by the Student’s t-test. Correlation between withinpopulation nucleotide diversity and geographic distance was tested
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K. Tanabe et al. / Vaccine 31 (2013) 1334–1339
Table 2
Polymorphism of P. falciparum vaccine candidate genes and housekeeping genes.
n
No. of SNPs
Total
ama1
csp
msp1
Whole regions
42 kDa region
19 kDa region
serca + adsl
a
Nucleotide diversity
Synonymousa
Substitutions rate (per site)
dN > dS
Nonsynonymousa
± SD
S ± SD
dS ± SE
dN ± SE
P value
0.00735 ± 0.00077
0.00760 ± 0.00129
0.00212 ± 0.00129
0.00008 ± 0.00006
0.01530 ± 0.00217
0.00695 ± 0.00198
<10−5
<0.001
459 107
485 41
6
1
83
29
0.01258 ± 0.00008
0.00559 ± 0.00025
404 125
404 22
404
8
453 62
23
2
1
29
99
20
7
30
0.00454
0.00430
0.00811
0.00035
±
±
±
±
0.00005
0.00006
0.00014
0.00002
0.00382
0.00286
0.00427
0.00181
±
±
±
±
0.00035
0.00062
0.00151
0.00023
0.00406
0.00031
0.00115
0.00107
±
±
±
±
0.00130
0.00028
0.00121
0.00049
0.00466
0.00528
0.00978
0.00016
±
±
±
±
0.00076
0.00164
0.00397
0.00006
0.341
0.002
0.019
1.000
Polymorphic sites in complex codons with multiple evolutionary paths were excluded: n = 18 in ama1, n = 11 in csp, n = 3 in msp1, and n = 3 in serca + adsl.
dS in the C-terminal 42-kDa and 19-kDa fragment regions. There
was no evidence for diversifying selection in serca + adsl [3]. Signatures of diversifying selection in ama1, csp, msp1 42-kDa region,
and msp1 19-kDa region were observed in all seven geographically
diverse parasite populations, with few exceptions (Vanuatu in csp,
Solomon in msp1 42 kDa region, and PNG and Solomon in msp1
19-kDa region) (Supplementary Table 3).
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by regression analysis. The coefﬁcient of determination (R2 ) was
calculated. We adopted our previous geographic distances for the
seven P. falciparum populations based on colonization routes along
landmasses from a postulated origin in Central sub-Saharan Africa
[3]. Logarithm of geographic distance was measured as travel cost
over friction routes [3].
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3.1. Signature of diversifying selection in ama1, csp and msp1
A strong negative correlation was noted between withinpopulation nucleotide diversity ( S ) of the three antigen genes
and geographic distance from a postulated origin in Central subSaharan Africa. A signiﬁcant isolation by distance pattern was
observed (R2 = 0.77–0.83; P ≤ 0.01) (Fig. 1A). This indicates that
77–83% of S can be ascribed to geographic distance. Signiﬁcant
negative correlation was also detected for in ama1 and csp
(R2 = 0.67–0.70; P < 0.025), but not for msp1 (Fig. 1B). Regression values for all three antigens were higher in S than in . In serca + adsl,
highly signiﬁcant negative correlation was detected in both S and
(R2 = 0.94 and 0.95, respectively) [3]. There is a steep decline of
S in csp and serca + adsl with geographic distance, compared with
that in ama1 and msp1.
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3. Results
3.2. Negative correlation between genetic diversity and
geographic distance
po
The nucleotide sequences reported in this study have
been deposited in the DDBJ/EMBL/GenBank database (accession
nos.
AB502443–AB502745,
AB502796–AB503151,
AB715434–AB716094) (Supplementary Table 2).
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We identiﬁed 107 SNPs in ama1 (1866 bp), 41 SNPs in csp
(681 bp), and 125 SNPs in msp1 (4740 bp). Nucleotide diversity in
ama1, csp and msp1 was high, compared with that in serca + adsl (62
SNPs in 4943 bp) (Table 3). The three antigen genes have 13- to 36fold and 2- to 4-fold higher and S , respectively, than serca + adsl.
A signature of diversifying selection was detected for ama1 and csp
by a signiﬁcant excess of dN over dS (Table 2). For the whole msp1,
dN > dS was not signiﬁcant, but dN was signiﬁcantly greater than
ama1 (1866 bp)
No. of SNPs
No. of singletons
No. of SNPs with MAF < 5%*
No. of unique SNPs
SNPs shared with Africa
csp (681 bp)
No. of SNPs
No. of singletons
No. of SNPs with MAF < 5%*
No. of unique SNPs
SNPs shared with Africa
msp1 (4740 bp)
No. of SNPs
No. of singletons
No. of SNPs with MAF < 5%*
No. of unique SNPs
SNPs shared with Africa
serca + adsl (4943 bp)
No. of SNPs
No. of singletons
No. of SNPs with MAF < 5%*
No. of unique SNPs
SNPs shared with Africa
*
Africa
Southeast Asia
Oceania
Southeast Asia + Oceania
Overall
97
10
27
20
–
68
7
14
3
64
77
4
25
6
70
86
9
29
8
74
107
14
60
–
–
34
6
13
15
–
25
4
9
5
19
8
3
5
0
7
26
3
17
6
20
41
9
25
–
–
98
26
52
42
–
64
7
22
12
46
65
3
15
5
52
82
8
31
27
55
125
35
72
–
–
50
32
44
42
–
16
5
12
4
8
13
1
9
4
5
20
6
17
12
8
62
36
58
–
–
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Table 3
Geographical distribution of SNPs in P. falciparum vaccine candidate genes and housekeeping genes.
MAF < 5%, minor allele frequency of less than 5%.
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K. Tanabe et al. / Vaccine 31 (2013) 1334–1339
(A)
(i) ama1
S
(ii) csp
S
0.012
0.012
0.010
0.010
0.008
0.008
0.006
0.006
0.004
0.002
0
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0.004
R2 = 0.765
P = 0.010
0.002
R2 = 0.808
P = 0.006
0
2.5 3.0 3.5 4.0 4.5 5.0 5.5
2.5 3.0 3.5 4.0 4.5 5.0 5.5
ln(distance)
ln(distance)
(iii) msp1
(iv) serca + adsl
S
0.0020
0.003
0.0015
0.002
0.0010
0.0005
(i) ama1
0.010
2.5 3.0 3.5 4.0 4.5 5.0 5.5
ln(distance)
(ii) csp
0.012
0.010
0.008
0.006
0.004
0.002
2.5 3.0 3.5 4.0 4.5 5.0 5.5
C
0
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R2 = 0.671
P = 0.024
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0.015
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(B)
0.005
0
2.5 3.0 3.5 4.0 4.5 5.0 5.5
ln(distance)
R2 = 0.942
P = 0.0003
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0
R2 = 0.834
P = 0.004
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0.001
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0.004
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S
0
R2 = 0.702
P = 0.019
2.5 3.0 3.5 4.0 4.5 5.0 5.5
ln(distance)
ln(distance)
(iii) msp1
(iv) serca + adsl
0.005
0.0006
0.004
0.0004
0.003
0.002
0.001
0
0.0002
R2 = 0.002
P = 0.917
R2 = 0.945
P = 0.0002
0
2.5 3.0 3.5 4.0 4.5 5.0 5.5
ln(distance)
2.5 3.0 3.5 4.0 4.5 5.0 5.5
ln(distance)
Fig. 1. Negative correlation between within-population nucleotide diversity of P. falciparum vaccine candidate genes and geographic distance from a postulated origin in
Central sub-Saharan Africa. (A) The standardized number of polymorphic sites per site ( S ) in (i) apical membrane antigen-1 gene (ama1), (ii) circumsporozoite gene (csp),
(iii) merozoite surface protein-1 gene (msp1), and (iv) sarcoplasmic/endoplasmic reticulum Ca2+ -transporting ATPase gene (serca) and adenylosuccinate lyase gene (adsl).
(B) Average pair-wise nucleotide difference () in (i) ama1, (ii) csp, (iii) msp1, and (iv) serca and adsl. Data of serca + adsl were from Tanabe et al. [3]. The determination
coefﬁcient (R2 ) and P value are given for each gene. Parasite populations are, from left to right, Tanzania, Ghana, Thailand, Philippines, Papua New Guinea, Solomon Islands,
and Vanuatu.
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4. Discussion
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In the three antigen genes, frequency of singleton SNPs and
SNPs with MAF <5% are relatively low (in ama1, 13% for singleton SNP and 56% for SNPs with MAF; in csp, 22% and 61% and in
msp1, 28% and 58%), as compared with those in serca + adsl (58%
and 94%, respectively) (P < 0.002 for singleton SNPs between each of
three antigen genes and serca + adsl, and P < 0.01 for SNPs with MAF)
(Table 3). Frequency of SNPs unique to African parasite populations
or to non-African parasite populations was low in the three antigen genes (21% (=20/97) and 9% (=8/86), respectively, in ama1; 44%
(=15/34) and 23% (=6/26) in csp; and 43% (=42/98) and 33% (=27/82)
in msp1, compared with those in serca + adsl (84% (=42/50) and 60%
(=12/20), respectively) (P < 0.002 for SNPs unique to Africa between
each of three antigen genes and serca + adsl, and P < 0.05 for SNPs
unique to non-Africa) (Table 3). A large number of SNPs are shared
among African and non-African populations, while such sharing
was limited in serca + adsl (Table 3): 86% (=74/86), 77% (=20/26)
and 67% (=55/82) in ama1, csp and msp1, respectively, compared
with 40% (=8/20) in serca + adsl. Difference in frequency of those
SNPs was signiﬁcant between each of three antigen genes and
serca + adsl (P < 0.05). Together, these show contrasting distribution
of SNPs between antigen genes and housekeeping genes, supporting the emerging conclusion that in antigen genes rare alleles are
low and that most SNPs are shared among Africa and non-Africa,
whereas in housekeeping genes most SNPs are rare and unique to
a continent.
ago [28–31]. Coupled with age estimation for the initial out-ofAfrica expansion of P. falciparum, we have inferred that modern
humans carried the parasite along during their colonization of the
Old World [3], being consistent with earlier reports [32,33]. Serial
bottleneck events during colonization, which resulted in gradual
reduction in the parasite population size, appear to reﬂect the
smooth patterns. The effect of bottleneck on genetic diversity likely
differs among genes: being strong in csp and serca + adsl, but weak in
ama1 and msp1. Steeper decline of regression in csp and serca + adsl
may be ascribed to relatively few numbers of polymorphic sites
(n = 28 and 37, respectively), compared with those in ama1 and
msp1 (n = 81 and 77, respectively). Bottleneck effect could be more
severe at a locus with fewer numbers of polymorphic sites.
This study has important implications for developing malaria
vaccines. Antigen polymorphism is suggested to be involved in
genotype-linked or strain-speciﬁc protective immunity [34–37]
and markedly reduces vaccine efﬁcacy. Indeed, a previously
observed low efﬁciency of the Combination B vaccine has been
attributed partly to genotype-linked protective immunity [38]. The
present study demonstrated reduced antigen diversity in nonAfrican parasite populations, particularly in Oceania populations.
We could therefore expect that a vaccine that covers limited antigen genotypes would be more effective in Oceania than in Africa.
We also observed that a large proportion of SNPs (67–86%) in antigen genes found in non-African populations were shared by African
populations. This suggests that those SNPs have persisted over time
in worldwide parasite populations, extending an earlier ﬁnding of
stable antigen SNPs [39]. Since the timing of out-of-Africa expansion of P. falciparum along with modern humans has been inferred
to be about 60,000 years ago [3], those shared SNPs may have been
stable for at least 60,000 years. This study conﬁrms and extends the
limited observation of sharing identical SNPs between Africa and
Thailand for ama1 [40,41], eba175 [42] and msp3 [43]. A vaccine that
is designed to include stable SNPs would be more effective than
that containing a single genotype. To an extent, selective pressures
on immune target antigens should have driven diversity, allowing immune evasion, but this does not appear to have happened.
It thus seems unlikely that introduction of selective pressure for
diversity in the form of a vaccine would select for new emerging
mutant forms of the parasite which can evade the vaccine. As such,
stable antigen SNP can be taken into account in a positive way for
developing malaria vaccines.
The present study together with previous ﬁndings using
housekeeping genes may lend a parasitological basis for the
epidemiological phenomenon “Paciﬁc enigma” [44]. Malaria
endemicity and transmission levels are comparable in Africa and
Oceania (particularly in PNG and Solomon Islands) [44,45], but
malaria-speciﬁc mortality (or the incidence of severe and complicated malaria) was signiﬁcantly lower in the later than in Africa
[44,46]. In the light of our ﬁndings, genetic diversity is lower in
Oceania [this study, [3]]. It is likely that repeated infections of
limited genotypes in Oceania are common and thus persistent stimulations of immune responses occur through repeated infections.
Protective immunity is likely to develop relatively fast in Oceania
[44]. On the contrary, in highly endemic areas of Africa, protective
immunity is slow to develop owing to diverse genotypes [47].
In conclusion, the present study demonstrated that withinpopulation SNP diversity of three P. falciparum vaccine candidate
genes is primarily determined by geographic distance from a
Central sub-Saharan African origin. The contribution of host
immunity to this diversity appears rather marginal. The ﬁnding has meaningful implications for development of effective
malaria vaccine, reinforcing the importance of investigating
genetic diversity of P. falciparum and evolutionary history of
geographic parasite populations in the ﬁeld of malaria vaccine
development.
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3.3. Distribution of SNPs
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The present study demonstrates that within-population SNP
diversity of the three leading P. falciparum vaccine candidates is
negatively correlated with geographic distance from a Central subSaharan African origin over Africa, Southeast Asia and Oceania. This
pattern of within-population genetic diversity that corresponds to
isolation by distance model is very similar to the one we previously
reported for housekeeping genes [3]. Behavior of SNPs, however,
differs between antigen genes and housekeeping genes in the parasite population studied. Immune target antigens are exposed to
host immunity and thus nonsynonymous SNPs are prone to be
positively selected, probably due to immune evasion, leading to
diversifying selection. In contrast, in housekeeping genes, nonsynonymous SNPs are, in most cases, deleterious and thus tend to be
rapidly eliminated. Consistently, we observed higher SNP diversity in the three vaccine candidate genes, ama1, csp and msp1,
than in housekeeping genes, serca and adsl, in all seven parasite
populations and obtained a signature for diversifying selection in
the three antigen genes. It was therefore unexpected to recover
77–83% of within-population genetic diversity attributed to geographic distance from a Central sub-Saharan African origin in major
immune target antigens showing signature of diversifying selection. At least for these three antigens, selective pressure by host
immunity did not appear to contribute substantially to geographical distribution of SNP, although our study did not speciﬁcally
address the potential for geographic differences in immune pressure as a possible cause of the variation we observed. We do
not completely exclude the contribution of host immune pressure because (i) ama1, csp and msp1 showed somewhat weak
correlation (R2 = 0.77–0.84) compared with that observed for two
housekeeping genes (R2 = 0.94) and (ii) correlation was also weak
in compared with S .
Within-population genetic diversity decreased smoothly with
geographic distance from a Central sub-Saharan African origin and
parallels the one’s described for housekeeping genes and human
population that expanded out-of-Africa about 50,000–60,000 years
14/04/2014
K. Tanabe et al. / Vaccine 31 (2013) 1334–1339
Acknowledgments
We thank Drs. Anna Färnert, Anders Björkman, Masatoshi Nakamura, Kenji Hirayama, Hiroshi Ohmae, and Akira Kaneko for
providing parasite isolates. This work was supported by Grant-inAids for Scientiﬁc Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (18073013, S0991013)
and from the Japan Society for Promotion of Sciences (22406012,
23590498, 23650211, 24249024).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.vaccine.2012.12.039. These data include Google map of the
most important areas described in this article.
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