B cell epitope mapping and characterization of naturally acquired antibodies to the Plasmodium vivax Merozoite Surface Protein-3α (PvMSP-3 α) in malaria exposed individuals from Brazilian Amazon
The Plasmodium vivax Merozoite Surface Protein-3α (PvMSP-3α) is considered as a potential vaccine candidate. However, the detailed investigations of the type of immune responses induced in naturally exposed populations are necessary. Therefore, we aim to characterize the naturally induced antibody to PvMSP-3α in 282 individuals with different levels of exposure to malaria infections residents in Brazilian Amazon. PvMSP3 specific antibodies (IgA, IgG and IgG subclass) to five recombinant proteins and the epitope mapping by Spot-synthesis technique to full-protein sequence of amino acids (15aa sequence with overlapping sequence of 9aa) were performed. Our results indicates that PvMSP3 is highly immunogenic in naturally exposed populations, where 78% of studied individuals present IgG immune response against the full-length recombinant protein (PVMSP3-FL) and IgG subclass profile was similar to all five recombinant proteins studied with a high predominance of IgG1 and IgG3. We also observe that IgG and subclass levels against PvMSP3 are associated with malaria exposure. The PvMSP3 epitope mapping by Spot-synthesis shows a natural recognition of at least 15 antigenic determinants, located mainly in the two blocks of repeats, confirming the high immunogenicity of this region. In conclusion, PvMSP-3α is immunogenic in naturally exposed individuals to malaria infections and that antibodies to PvMSP3 are induced to several B cell epitopes. The presence of PvMSP3 cytophilic antibodies (IgG1 and IgG3), suggests that this mechanism could also occur in P. vivax.
Vaccine 29 (2011) 1801–1811
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/vaccine
B cell epitope mapping and characterization of naturally acquired antibodies to
the Plasmodium vivax Merozoite Surface Protein-3␣ (PvMSP-3␣) in malaria
exposed individuals from Brazilian Amazon
, J. Jiang
, R.N. Rodrigues-da-Silva
, D.M. Banic
, T.M. Tran
, R.Y. Ribeiro
, S.G. De-Simone
, F. Santos
, A. Moreno
, J.W. Barnwell
, J. Oliveira-Ferreira
Laboratory of Immunoparasitology, Institute Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil
Emory Vaccine Center, Emory University, Atlanta, GA, United States
Laboratory of Malaria Research, Institute Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil
Laboratory of Biochemical of Proteins and Peptides, Institute Oswaldo Cruz, Fiocruz and Department of Biochemistry and Cellular Biology,
Universidade Federal Fluminense, Rio de Janeiro, Brazil
Department of Entomology, LACEN, Porto Velho, RO, Brazil
Division of Parasitic Diseases, CDC/National Center for Infectious Diseases, Atlanta, GA, United States
Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA, United States
Received 10 May 2010
Received in revised form 2 December 2010
Accepted 22 December 2010
Available online 6 January 2011
Merozoite surface protein
B cell epitope
The Plasmodium vivax Merozoite Surface Protein-3␣ (PvMSP-3␣) is considered as a potential vaccine
candidate. However, the detailed investigations of the type of immune responses induced in naturally
exposed populations are necessary. Therefore, we aim to characterize the naturally induced antibody to
PvMSP-3␣ in 282 individuals with different levels of exposure to malaria infections residents in Brazilian
Amazon. PvMSP3 speciﬁc antibodies (IgA, IgG and IgG subclass) to ﬁve recombinant proteins and the
epitope mapping by Spot-synthesis technique to full-protein sequence of amino acids (15aa sequence
with overlapping sequence of 9aa) were performed. Our results indicates that PvMSP3 is highly immuno-
genic in naturally exposed populations, where 78% of studied individuals present IgG immune response
against the full-length recombinant protein (PVMSP3-FL) and IgG subclass proﬁle was similar to all ﬁve
recombinant proteins studied with a high predominance of IgG1 and IgG3. We also observe that IgG and
subclass levels against PvMSP3 are associated with malaria exposure. The PvMSP3 epitope mapping by
Spot-synthesis shows a natural recognition of at least 15 antigenic determinants, located mainly in the
two blocks of repeats, conﬁrming the high immunogenicity of this region. In conclusion, PvMSP-3␣ is
immunogenic in naturally exposed individuals to malaria infections and that antibodies to PvMSP3 are
induced to several B cell epitopes. The presence of PvMSP3 cytophilic antibodies (IgG1 and IgG3), suggests
that this mechanism could also occur in P. vivax.
© 2011 Elsevier Ltd. All rights reserved.
Plasmodium vivax is a leading cause of human malaria and,
together with Plasmodium falciparum, accounts for the majority
of malaria cases worldwide. Although P. falciparum is dominant
in most of Sub-Saharan Africa, P. vivax causes approximately 50%
of all malaria cases in endemic regions outside of Africa, with
2.5 billion inhabitants of the Middle East, Asia, Eastern Africa,
Corresponding author at: Laboratory of Immunoparasitology, Institute Oswaldo
Cruz, Fiocruz, Pavilhao Leonidas Deane, 4th Floor, Av. Brasil 4365, ZIP: 21045-900,
Rio de Janeiro, RJ, Brazil. Tel.: +55 21 3865 8102; fax: +55 21 3865 8198.
E-mail address: lila@ioc.ﬁocruz.br (J. Oliveira-Ferreira).
Central and South America, and Oceania exposed to P. vivax, result-
ing in an estimated 71–391 million cases of vivax malaria each
year [1–3]. Critically, P. vivax causes signiﬁcant economic and
social damage  and evidence of severe illness and death due
to P. vivax is being reported with increasing frequency [4–9].
While considerably greater investments have been made over the
past 30 years to research and control P. falciparum, there have
been recent attempts to call attention to the need for increased
resources for P. vivax vaccine and drug research and development
. Technological advances enabling the sequencing and anal-
ysis of the P. vivax genome [11,12] and the call for worldwide
malaria eradication , have together placed new emphasis on
the importance of addressing P. vivax as a major public health
0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
1802 J.C. Lima-Junior et al. / Vaccine 29 (2011) 1801–1811
Multiple antigens from the asexual P. vivax parasites have
been identiﬁed and immunologically characterized and a number
of merozoite surface or apical organellar localized proteins have
been receiving the most attention. These include P. vivax Mero-
zoite Surface Protein-1 (PvMSP-1) , the PvMSP-3 family ,
PvMSP-9 , Reticulocyte Binding Protein-1 (PvRBP-1) , Api-
cal Membrane Antigen-1 (PvAMA-1)  and Duffy Binding Protein
(PvDBP) . Among the merozite proteins, those with known
essential functions that can be disrupted by antibodies, represent
the most promising candidates for vaccine development. PvMSP-
3␣ is a merozoite surface protein expressed during schizogony
and it appears to become intimately associated with the surface
of the merozoite [15,20]. Moreover, PvMSP-3␣ is a member of
a multi-gene family , which includes 11 members . The
initially discovered family members, PvMSP-3␣, PvMSP-3␤ and
PvMSP-3␥ share 35–38% identity and 48–53% similarity in pair-
wise comparisons [15,20–22]. Structurally, these proteins lack a
transmembrane domain or a GPI-lipid modiﬁcation to anchor them
in the outer membrane of the merozoite. The bulk of these pro-
teins is an alanine-rich central domain containing a series of heptad
repeats predicted to form a coiled-coil tertiary peptide structure,
which may secure them on the merozoite surface through inter-
action with other surface proteins [15,21]. Due to the remarkable
diversity, particularly noted in the central domain , the PvMSP-
3␣ gene sequence has become a highly regarded polymorphic
marker for population based studies [23–25]; the acidic C-terminal
domain and a smaller hydrophilic N-terminus are relatively con-
served, while the central domain containing two annotated blocks
of coiled-coil heptad repeats (Block I and Block II) is highly poly-
morphic and in some isolates of P. vivax is partially deleted .
PvMSP-3␣ has homologs in the simian malaria Plasmodium
knowlesi [26–28], and in P. falciparum. The initially discovered P.
falciparum MSP-3 contains a small series of alanine-based hep-
tad repeats [29,30]. PfMSP-3 has been of considerable interest as
a vaccine candidate, mainly because anti-PfMSP-3 antibodies sig-
niﬁcantly decrease parasitemia through an antibody-dependent
cellular inhibition mechanism  and partially protected New
World monkeys against lethal P. falciparum infection in a pre-
clinical vaccine trial . PfMSP-3 long synthetic peptides have also
been shown to be safe and immunogenic in a phase I clinical vac-
cine trial [32,33]. The predicted structural importance of PvMSP-3␣
and other PvMSP-3 family members at the surface of merozoites,
the high relative conservation of the C-terminal regions, and the
relationship of PvMSP-3 to a similar merozoite protein which has
been highly regarded as a vaccine candidate in P. falciparum are rea-
sons to investigate these P. vivax antigens as natural immunogens
and possible vaccine candidates. The present study evaluates the
naturally acquired immune response to PvMSP-3␣ in individuals
exposed to malaria infections in Rondonia State, in the Amazon
region of Brazil, and provides important information regarding
PvMSP-3␣ immune responses generated in natural infections in
support of this antigen as a P. vivax vaccine candidate.
2. Material and methods
2.1. Study area and volunteers
A cross-sectional cohort study was conducted involving 282
individuals from communities in the malaria endemic region of
Rondonia State, in the western Amazon region of Brazil, where in
the last 5 years P. vivax malaria accounted for more than 70% of
all malaria cases. The majority of the studied population consists
of rain forest natives or transmigrants from several non-endemic
areas of Brazil that have lived in the region for 10 years or more.
Samples and survey data were collected during the dry months of
June–August, coinciding with the period of increased malaria trans-
mission in Rondonia State. In addition, we also have included as
control subjects 24 naive individuals living in non-endemic regions
of Brazil and the USA (Rio de Janeiro and Atlanta, respectively).
Written informed consent was obtained from all adult donors
or from parents of donors in the case of minors. The study was
reviewed and approved by the Fundac¸ ão Oswaldo Cruz Ethical
Committee and the National Ethical Committee of Brazil.
2.2. Epidemiological survey
In order to evaluate epidemiological factors that may inﬂuence
the immune response against PvMSP-3␣, all donors were inter-
viewed upon informed consent. The survey included questions
related to demographics, time of residence in the endemic area,
personal and family histories of malaria, use of malaria prophy-
laxis, presence of malaria symptoms, and personal knowledge of
malaria. Survey data was entered into a database created with Epi
Info 2002 (Centers for Disease Control and Prevention, Atlanta, GA).
2.3. Malaria diagnosis and blood sampling
Venous peripheral blood was drawn into heparinized tubes
and plasma collected after centrifugation (350 × g, 10 min). Plasma
samples were stored at −20
C and transported to our labora-
tory. Thin and thick blood smears of all donors were examined for
malaria parasites. Parasitological evaluations were done by exam-
ination of 200 ﬁelds at 1000× magniﬁcation under oil-immersion;
all slides were examined by a research expert in malaria diagnosis.
Donors positive for P. vivax and/or P. falciparum at the time of blood
collection were subsequently treated using the chemotherapeutic
regimen recommended by the Brazilian Ministry of Health.
2.4. Cloning, expression and puriﬁcation of recombinant
Five subfragments of the pvmsp3␣ gene, including sequences
encoding the near full length protein (nucleotides 73–2520), the
N-terminal (Nt) region (nucleotides 73–309), Block I (nucleotides
316–1242), Block II (nucleotides 1246–2058), and the C-terminal
(Ct) region (nucleotides 2059–2523), were ampliﬁed from P. vivax
(Belem strain) genomic DNA with the Expand High Fidelity
System (Roche). The ﬁve PCR products were double digested with
Nco I and Xho I, and ligated into the same restriction sites from
the pET24d(+) kanamycin resistant expression vector containing a
C-terminal 6× His Tag comprising the amino acids ELHHHHH for
puriﬁcation purposes. The recombinant expression vectors were
conﬁrmed by Big Dye terminator v3.1 sequencing. All ﬁve recombi-
nant proteins were successfully expressed at high levels in a soluble
state using BL-21 (DE3) or BL21-AI
cells after 3 h induction at
C with 1 mM IPTG. The soluble recombinant proteins were ini-
tially puriﬁed using HisTrap
HP columns under native conditions,
and further puriﬁed by gel-ﬁltration using either a Sephacryl S-200
HR chromatography column or HiLoad Superdex 75 pg column. The
ﬁnal proteins were evaluated on SDS-PAGE gels and via Western
immunoblotting using standard conditions.
2.5. Antibody assays
Plasma samples from study participants were screened for the
presence of naturally acquired antibodies against the ﬁve recom-
binant proteins PvMSP3-FL, PvMSP3-BLI, PvMSP3-BLII, PvMSP3-CT
and PvMSP3-NT by ELISA. Brieﬂy, Maxisorp 96-well plates (Nunc,
Rochester, NY) were coated with 2 g/ml of each recombinant pro-
tein. After overnight incubation at 4
C the plates were washed
with PBS containing 0.05% Tween 20 (PBS–Tween) and blocked
J.C. Lima-Junior et al. / Vaccine 29 (2011) 1801–1811 1803
with PBS–Tween containing 5% non-fat dry milk (PBS–Tween–M)
for 2 h at 37
C. Individual plasma samples diluted with 1:100
PBS–Tween–M were added in duplicate wells and the plates
incubated at room temperature for 1 h. After four washes with
PBS–Tween, bound antibodies were detected with peroxidase-
conjugated goat anti-human IgG (Sigma, St. Louis) followed by
o-phenylenediamine and hydrogen peroxide. The absorbance was
read at 492 nm using an ELISA reader (Spectramax 250, Molecular
Devices, Sunnyvale, CA). The results for total IgG were expressed
as Reactivity Indexes (RIs), which were calculated by dividing the
mean optical density of tested samples by the mean optical density
plus 3 standard deviations of 24 non-exposed control individuals
living in non-endemic areas of malaria. Subjects were scored as
positive for serum IgG to antigen if the RI was higher than 1.
For determination of IgA and IgG subclasses the following perox-
idase conjugated monoclonal mouse anti-human antibodies were
used: clone HP-6001 for IgG1, HP-6002 for IgG2, HP-6050 for IgG3,
HP-6023 for IgG4 and B1524 for IgA (Sigma, St. Louis, MO). This
set of antibodies has been used previously to characterize IgG sub-
classes within a similar cohort of individuals living in Rondonia
. Subclass-speciﬁc prevalence for each antigen was determined
from OD values using 3 standard deviations above the appropriate
mean OD of 24 non-exposed controls as the cut-off for positive reac-
tivity. To adjust the afﬁnity differences between the IgG subclasses,
standard curves were prepared using human IgG kappa myeloma
proteins (Sigma, St. Louis, MO) from each of the four IgG subclasses
and human IgA puriﬁed from human colostrum (Sigma, St. Louis,
MO). In addition, standard curves enable conversion of OD values to
concentration (g/ml) for the comparison of different subclasses.
Puriﬁed human antibodies from each of the four subclasses and
IgA (Sigma, St. Louis, MO) were coated overnight at 4
C in PBS
at 100 l per well onto 96-well plates in 1:2 serial dilutions from
g/ml. After washing four times, the plates were
incubated with the appropriate anti-human IgG subclass-speciﬁc
mAb, washed four times, incubated with peroxidase-labeled goat
anti-mouse antibody, washed a ﬁnal four times, developed with
o-phenylenediamine and hydrogen peroxide, and measured as
described above for subclass-speciﬁc ELISA. Subclass-speciﬁc OD
values were converted to concentration values (g/ml) using sig-
moidal curve-ﬁt equations derived from subclass-speciﬁc standard
B-cell linear epitope mapping was carried out by the synthe-
sis of an overlapping 15-mer peptide library covering the entire
amino acid sequence of P. vivax PvMSP-3␣. The peptides were
simultaneously synthesized by the SPOT-method  on cellulose
membranes with an Ala–Ala linker, for preparation of immobilized
peptides. The assembly of the peptides was carried out utiliz-
ing 9-ﬂuorenylmethoxycarbonyl (Fmoc) chemistry as previously
described [35,36]. The prepared membrane consists of overlapping
15 amino acid peptide residues with an offset of four. For epitope
analysis, a pool of 10 serum samples with high reactivity indices to
all ﬁve recombinant proteins in ELISA test at 1:100 dilutions was
used. Bound antibodies were detected with alkaline phosphatase
(AP)-conjugated secondary antibody (anti-human IgG) followed by
a color reaction with CDP-Star Chemiluminescent Substrate (Sigma,
2.7. Statistical analysis
Statistical analyses were done using Epi Info 2002 (CDC, Atlanta,
GA), Prism 4.0 and Instat (GraphPad Software, San Diego, CA)
applying the statistical test necessary. Differences in medians for
the study population data were tested by the non-parametric
Summary of the epidemiological characteristics of studied individuals enrolled in
Male (n) 158
Female (n) 124
Total (n) 282
Age (mean ± SD) 36.1 + 16.9
Time of residence in malaria endemic area (mean ± SD) 28.5 + 17.0
Number of past malaria infections (mean ± SD) 7.0 + 9.1
Number of malaria infections in the last 6 months (mean ± SD) 0.5 + 1.1
Past months since the last malaria infection (mean ± SD) 41.0 + 50.1
Hospitalization in malaria past infections
Use of prophylactic measures
Previous malaria species contracted
Negative (n) 33/11.8%
P. vivax (n) 56/20%
P. falciparum (n) 34/12.1%
Both species (n) 148/52.9%
Differences in gender proportions were not statistically signiﬁcant.
p = 0.2222.
We could not determine whether clinical criteria were compatible with severe
11 (3.2%) individuals did not remember the previous malaria parasite species
Bold typeface indicates that the prevalence was signiﬁcantly higher when com-
pared with all other observations (p < 0.0001).
Were considered as prophylactic measure; i.e., the use of residual insecticide,
bednets or insect repellent.
Mann–Whitney test where appropriate. Student’s t test was used
to compare the means of normally distributed data, or normal-
ized transformations were performed on raw data before testing
by one-way analysis of variance where appropriate. Differences
in proportions were evaluated by the chi-square (
) test. Rela-
tionships between years of residence in the endemic area and the
number of past malaria infections or months since last known
malaria episode were assessed with Spearman’s rank correlation.
Multivariate logistic regression was used to assess the relationship
between antigen-speciﬁc total IgG responses and the independent
variables of gender, age, years of residence in the endemic area,
number of past malaria episodes, and months since last known
3.1. Characteristics of studied population
Among our sample set, the studied population did not differ
signiﬁcantly in gender ratio (
= 1.4201; p = 0.2222). Our epidemi-
ological survey, summarized in Table 1, shows that all individuals
studied are exposed to malaria infections throughout the year. The
time of residence in the malaria endemic area, number of past
malaria episodes and past months since the last infection vary
greatly in studied individuals. However, a signiﬁcant proportion of
studied individuals (52.9%) reported a prior experience with both
P. vivax and P. falciparum malaria when compared with individuals
that had in the past, a single species infection (32.1%) or individuals
that could not recall infections in the past and mentioned that they
never had malaria even though they were born in the endemic area
(p < 0.0001). Among donors with a previous malaria infection(s),
years of residence in the endemic area correlated positively with
the past months since last malaria episode (Spearman r = 0.2344,
p < 0.0001, n = 249). Therefore, we used years of residence in the
endemic area and number of past malaria episodes reported by
donors as indices of malaria exposure, and past months since last
malaria episode and infections during the year of blood collection
as a crude approximation of clinical protection. At the time of blood
1804 J.C. Lima-Junior et al. / Vaccine 29 (2011) 1801–1811
Fig. 1. Expression and puriﬁcation of recombinant PvMSP3␣ proteins in E. coli. (a) Schematic depicting the near-full length, N-terminal (Nt), Block I, Block II and C-terminal
(Ct) PvMSP3␣ segments designed for recombinant protein expression. Each recombinant protein was developed with a C-terminal His tag (with the ELHHHH amino acid
sequence) for protein puriﬁcation purposes. The Block I and Ct recombinant proteins have one additional alanine (A) after the start methionine (M). (b) Puriﬁed recombinant
proteins were separated by SDS-PAGE under reducing and non-reducing conditions, as indicated. The protein molecular weight standards are Precision Plus Protein All Blue
collection 34 (12%) individuals were infected, 25 with P. vivax and
9 with P. falciparum (thus, 74% P. vivax and 26% P. falciparum), con-
sistent with the current local case distribution data for these two
species reported by the Ministry of Health.
3.2. Expression and puriﬁcation of recombinant PvMSP3˛
Five recombinant proteins representing segments of PvMSP3␣
(Fig. 1a) were expressed in the pET24d (+) vector and puriﬁed (see
Section 2), and run in a standard SDS-PAGE gel under reducing
and non-reducing conditions (Fig. 1b). Each recombinant protein
was soluble, and showed a higher molecular weight than projected
from the calculated molecular weight, as observed before for this
family of proteins [15,20]. The near full length (FL) protein, N-
terminus (Nt) protein, Block I (BLI), Block II (BLII), and C-terminus
(Ct) proteins (Fig. 1a) ran at >95%, 116%, 28%, 46% and 79% of their
expected molecular weights under reduced conditions (Fig. 1b).
Additionally, the FL and Block I proteins formed higher molecu-
lar weight structures through predicted disulﬁde bond formation
under the non-reducing conditions (Fig. 1b). The results of Western
immunoblotting with anti-His antibody and anti-PvMSP3␣ anti-
bodies indicated that each of these puriﬁed recombinant protein
bands correspond to the PvMSP3␣ subfragments (not shown). With
the exception of the near full length recombinant PvMSP3␣, which
always appears as two protein bands on a reduced SDS-PAGE gel,
each of the other four recombinant proteins appear as a single pro-
tein band as shown in the reduced SDS-PAGE gel. Based on the
primary amino acid sequences, the recombinant proteins repre-
senting the NT, Block I and the near FL have cysteine residues and
may therefore form higher molecular weight structures through
disulﬁde bond. This seems to be the case for each of these proteins,
as shown in the non-reduced SDS-PAGE gel (Fig. 1b).
3.3. Frequency of IgG antibodies to recombinant proteins derived
The prevalence of naturally acquired antibodies speciﬁc to the
ﬁve recombinant proteins derived from PvMSP-3 was determined
with plasma of all 282 studied individuals (Fig. 2a). The results
show that IgG antibody reactivity to PvMSP3-FL (full-length pro-
tein) was present in 78% of all 282 donors. Moreover, when we
evaluated the prevalence of IgG antibodies to different regions of
PvMSP-3, we observed that the two blocks of repeats (PvMSP3-BLI,
64% and PvMSP3-BLII; 53%) and the C-terminus (PvMSP3-CT, 54%)
were signiﬁcantly more recognized (p < 0.01) than the N-terminal
region (39%). As expected, all individuals positive for the PvMSP3-
FL recombinant also presented an IgG response to at least one of
the four recombinant proteins. In this context, among the PvMSP3-
FL responders (n = 220) 26.8% also presented an IgG response to all
four recombinant proteins, 25.4% to three of the recombinant pro-
teins (mainly PvMSP3-BLI, PvMSP3-BLII and PvMSP3-CT), 37.7% to
two of the recombinant proteins, and 10% of individuals to one of
the recombinant proteins. Only 22% did not present antibodies to
any of the recombinant proteins.
J.C. Lima-Junior et al. / Vaccine 29 (2011) 1801–1811 1805
Fig. 2. Frequency of IgG responders (a) reactivity indexes and (b) frequency of IgG subclass responders (c) against the ﬁve recombinant proteins tested in the studied
population. Chi squared test for proportions analyses were performed to determine whether a statistical difference existed for each antigen (*). The frequency of IgG
responders to PvMSP3-FL was signiﬁcantly higher when compared with all other recombinants (p < 0.05), while the frequencies to PvMSP3-NT were the lowest when
compared with all others (#) (p < 0.01). The frequency of IgG1 and IgG3 subclasses statistically predominated (*) over IgG2 and IgG4.
3.4. Magnitude of IgG response against the ﬁve PvMSP-3˛
To compare the relative magnitude of antibody responses, we
determined IgG RIs based on negative control cut-off values. As
shown in Fig. 2b, the RI of individuals against the recombinant pro-
teins varied from 0.21 to 8.91. The average RI of responders against
PvMSP3-FL (2.10 ± 1.61), PvMSP3-BLI (2.06 ± 1.45), PvMSP3-BLII
(1,81 ± 1.05) and PvMSP3-CT (1.87 ± 1.37) did not differ signiﬁ-
cantly (p > 0.05). However the average of RI against PvMSP3-NT
(1.27 ± 0.27) was signiﬁcantly lower when compared with all of
the other recombinants tested (PvMSP3-FL: t = 5.365; df = 328;
p < 0.0001; PvMSP3-BLI: t = 5.651; df = 288; p < 0.0001; PvMSP3-
BLII: t = 5.265; df = 258; p < 0.0001; PvMSP3-CT: t = 4.528; df = 260,
p < 0.0001). It is important to mention that all of the serum samples
from the 24 healthy individuals without previous history of expo-
sure to malaria infections were negative for all ﬁve recombinant
3.5. IgG subclass distribution of anti-PvMSP-3˛ antibodies
We assessed the overall subclass distribution of the IgG antibody
responses to each antigen using two different comparative analy-
ses. Firstly, we determined subclass-speciﬁc prevalence of total IgG
positive responders for each antigen using OD cut-offs determined
from ODs of non-exposed controls. Secondly, antigen-speciﬁc IgG1,
IgG2, IgG3, and IgG4 concentrations for total IgG positive respon-
ders were determined from OD values using subclass-speciﬁc
standard curves (Table 2). Therefore, the results of both analyses
were comparable with a noteworthy predominance of IgG1 and
IgG3 cytophilic antibodies. As shown in Fig. 2c and Table 2, the
frequency and concentration respectively of IgG1 and IgG3 antibod-
ies signiﬁcantly predominated over the IgG2 and IgG4 antibodies
against all ﬁve tested PvMSP-3 recombinant proteins (p < 0.01).
3.6. Frequency and concentration of IgA antibodies against
recombinant PvMSP-3˛ antigens
The frequency of positive individuals with IgA antibodies against
the ﬁve recombinant proteins was similar, presenting no sta-
tistically signiﬁcant differences (p > 0.05 in chi-squared test). As
observed in Table 2, the frequencies ranged from 21.3% (PvMSP3-
BLI) to 29.7% (PvMSP3-FL), however the comparison of IgA
concentration with the IgG subclass shows a different proﬁle, with
the concentration of cytophilic antibodies (IgG1 and IgG3) signif-
icantly higher than that of IgA (p < 0.001), on the other hand the
concentration of non-cytophilic antibodies (IgG2 and IgG4) was
similar to IgA concentration.
1806 J.C. Lima-Junior et al. / Vaccine 29 (2011) 1801–1811
Concentration and 95% conﬁdence intervals of IgG subclass speciﬁc response positive individuals and frequency of IgA responders against PvMSP-3␣ recombinant proteins.
g/ml (CI 95%)
g/ml (CI 95%)
g/ml (CI 95%)
g/ml (CI 95%)
g/ml (CI 95%)
0.525–0.721 0.619–0.947 0.574–0.703 0.419–0.519
IgG2 0.128–0.250 0.168–0.191 0.201–0.299 0.241–0.299 0.107–0.193
IgG3 0.529–0.877 0.360–0.584 0.422–0.507 0.499–0.617 0.411–0.501
IgG4 0.056–0.091 0.041–0.055 0.039–0.061 0.061–0.074 0.041–0.07
0.125–0.293 0.083–0.174 0.120–0.246 0.121–0.276 0.122–0.275
Frequency of IgA responders (n) 28.4% (80) 21.3% (60) 24.5% (69) 22.7% (64) 23.0% (65)
IgG1 and IgG3 predominate over IgG2 and IgG4 in PvMSP3-FL (p < 0.001), PvMSP3-BLI (p < 0.01), PvMSP3-CT (p < 0.001) and PvMSP3-NT (p < 0.0001), however in PvMSP-
3-BLII the IgG1 predominates over all other subclasses (p < 0.05).
Bold typeface indicates the predominant IgG subclass by Mann–Whitney test.
IgA antibody concentrations against all recombinant proteins were signiﬁcantly lower (p < 0.0001) when compared with cytophilic antibodies (IgG1 and IgG3), however
they were similar to the non-cytophilic antibodies IgG2 and IgG4 (p > 0.05).
3.7. PvMSP-3˛ speciﬁc antibody responses and malaria exposure
All humoral immune response data from our 282 studied indi-
viduals (frequency, RI and antibody concentration) were matched
with years of residence in the endemic area. Antibody RIs increased
with years of residence in the endemic area for all recombinant
proteins. However, the analysis by Spearman’s rank correlation
showed that this association was signiﬁcant only with IgG reactivity
indexes against PvMSP3-FL (r = 0.1115; p = 0.0462) and PvMSP3-NT
(r = 0.1147; p = 0.0480). Moreover, the concentration of IgG sub-
class antibodies also correlated with years of residence in endemic
area for PvMSP3-FL (IgG1, r = 0.2432; p = 0.0021; IgG3, r = 0.1231;
p = 0.0325), PvMSP3-BLI (IgG3, r = 0.1672; p = 0.0045) and PvMSP3-
CT (IgG1, r = 0.1990; p = 0.0013). Interestingly, we also observed
an inverse correlation between IgG4 concentration against the
PvMSP3-CT recombinant and years of residence in the endemic area
(r = −0.1213; p = 0.0398).
3.8. PvMSP-3˛ speciﬁc antibody responses and previous malaria
Antibody RIs to PvMSP3-FL (r = 0.1680; p = 0.0048) and PvMSP3-
CT (r = 0.1790; p = 0.0026) were associated with the number of past
malaria episodes. Moreover when we stratiﬁed the group of indi-
viduals according to the number of previous malaria infections
(0, 1–3 or >3) we observed that the total IgG RIs to PvMSP3-
FL, PvMSP3-BLI and PvMSP3-CT increased with the number of
past malaria episodes and were signiﬁcantly (p < 0.05) higher
in individuals who presented three or more previous malaria
infections (Fig. 3). The Spearman’s rank correlation analysis also
showed that the IgG3 antibody concentration against PvMSP3-
FL was correlated with the number of past malaria infections
(r = 0.1401; p = 0.0415) and interestingly, the IgG2 antibody concen-
tration against PvMSP3-CT was inversely correlated (r = −0.1490;
p = 0.0313).
3.9. Humoral response levels and protective immunity
Time (in months) since the last malaria episode and the num-
ber of malaria infections in the last 6 months were used as a
crude means to estimate the donor’s level of protection from clini-
cal malaria. To assess whether IgG and subclass levels correlated
with this estimate, we calculated Spearman’s correlation coefﬁ-
cients between the IgG RI and IgG subclass concentrations and these
two parameters. No association was detected with the total IgG RI.
However, a positive correlation was observed between the IgG3
Epitopes identiﬁed by IgG reactivity detected in the Spot-synthesis assay with serum from positive responders against the PvMSP-3␣ recombinant proteins.
PvMSP-3␣ region Epitopes AA position Position in Spot-synthesis
PvMSP3-Nt EAPNSSRHHLRNGF E
PvMSP3-BLI AEQIQAELQKVKTA A
PvMSP3-BLII EVAKAEVLNAEVK E
PvMSP3-Ct KAAYGLLKTKNQYVL K
J.C. Lima-Junior et al. / Vaccine 29 (2011) 1801–1811 1807
Fig. 3. Stratiﬁcation according to numbers of past malaria infections reported, 0 (n = 33), 1–3 (n = 102) and >3 (n = 138), in the epidemiological interview of donors enrolled
in our study and the reactivity indexes against PvMSP3-FL (a), PvMSP3-BLI (b) and PvMSP3-CT (c). *p < 0.05, **p < 0.01 and ***p < 0.001 by Mann–Whitney rank sum test.
Fig. 4. Stratiﬁcation according to ratio between the concentration of cytophilic
(IgG1 + IgG3) and non-cytophilic antibodies (IgG2 + IgG4) against PvMSP3-FL by past
months since the last malaria infection. *p < 0.05 and ***p < 0.001 by Mann–Whitney
rank sum test.
concentration for PvMSP3-FL (r = 0.1199; p = 0.0495) and IgG1 for
PvMSP3-CT (r = 0.1910; p = 0.01333) and the past months since the
last known malaria episode. Interestingly, as observed in Fig. 4, indi-
viduals with a ratio between the concentration of cytophilic and
non-cytophilic antibodies of at least 3 had a greater time since the
last malaria infection when compared with individuals with a ratio
of 1–3 or less than 1.
3.10. Epitope mapping
To identify the epitopes present in the full-length protein,
pooled serum from IgG responders to the full length protein were
tested against 193 overlapping peptides corresponding to the com-
plete sequence of PvMSP-3␣ synthesized in a solid phase by the
spot method. According to spot-image intensities (Fig. 5), 25 anti-
genic determinants were identiﬁed in the complete sequence of
PvMSP-3␣. The four regions represented in the recombinant pro-
teins present antigenic determinants ranging in size from 10 to
25 amino acids. The region corresponding to PvMSP3-NT presents
three epitopes and PvMSP3-CT presents four. However, the central
domain of PvMSP-3, which comprises the two blocks of tandem
repeats, presents 11 and 7 antigenic determinants in PvMSP3-BLI
and PvMSP3-BLII, respectively (Table 3).
The surface of the P. vivax merozoite is covered with a layer
of proteins organized into a structurally complex coat, and the
antigenic constituents of this surface coat are among the most
frequently discussed vaccine targets [37–39], basically because
antibody-mediated host immunity can limit the success of mero-
zoite invasion into erythrocyte host cells [29,40,41]. However, the
merozoite presents a wide array of proteins that could be included
in a vaccine. Therefore to include a merozoite protein as a vaccine
candidate it is necessary to evaluate several features that include
conservation of structure and function and immune response.
In this context, PvMSP-3␣ has emerged as a potential candidate
mainly because (I) it is expressed during schizogony and appears
to become intimately associated with the merozoite surface; (II) it
contains conserved regions; (III) it is related to P. knowlesi and P.
falciparum MSP-3 proteins , which are classically known as vac-
cine candidates [31,42,43]; and (IV) it induces antibodies that block
merozoite invasion in vitro and induced partial protection in a pre-
liminary trial to examine efﬁcacy in a New World primate model
system (J.W. Barnwell and M.R. Galinski, unpublished results). Con-
sequently, the investigation of naturally acquired anti-PvMSP-3-␣
presented in this work becomes relevant with regards to the con-
tinued consideration of PvMSP-3␣ as a vaccine candidate.
In a cross-sectional study carried out in Porto Velho, Rondo-
nia State, Brazil, we assessed naturally acquired humoral immune
responses against ﬁve recombinant protein constructs represent-
ing the complete amino acid sequences of PvMSP-3␣. The fact that
the majority of studied individuals had reported P. vivax and P. fal-
ciparum infections in the past, suggests that both malaria parasites
circulated in this region in recent decades. It is classically known
that the natural exposure to malaria infections is closely related
with acquisition of antibodies to Plasmodium spp. antigens [44–47].
The broad range of age, number of past malaria episodes, time
of exposure and months since individual’s last reported malaria
episode identiﬁed in our studied population would indicate that the
inhabitants exhibit different degrees of immune reactivity against
P. vivax and consequently to the PvMSP-3-␣ proteins tested here.
Our ﬁrst set of data on antibody responses show that PvMSP-3␣
is a target of the immune response in individuals naturally exposed
1808 J.C. Lima-Junior et al. / Vaccine 29 (2011) 1801–1811
Fig. 5. Reactivity of IgG antibodies from a pool of 10 serum samples from individuals with high reactivity indices to all ﬁve recombinant proteins against peptides covering the full-length sequence of PvMSP-3 (overlapping 15
amino acid peptide residues with an offset of 4).
J.C. Lima-Junior et al. / Vaccine 29 (2011) 1801–1811 1809
to P. vivax malaria transmission. We show a high frequency of IgG
responders against the full length recombinant protein and recom-
binant proteins representing four different regions of the molecule,
indicating that the pET Escherichia coli expression systems success-
fully generated epitopes that share similar antigenic determinants
as the native proteins. The high polymorphism observed in several
regions of the world in the PvMSP-3␣ diverse central domain does
not seem to occur in the studied endemic area, since the frequency
of individuals that recognized the recombinant proteins repre-
senting these domains (PvMSP3-BLI and PvMSP3-BLII) is higher
than the frequency of individuals that recognized the recombi-
nant proteins representing the conserved regions (PvMSP3-NT and
PvMSP3-CT) [21,48,49]. In addition, the frequencies of IgG respon-
ders against PvMSP-3␣ presented similar overall frequencies of
naturally acquired IgG response against classical P. vivax candidate
antigens like PvMSP-1 [50,51], PvAMA-1 , PvMSP-9  and
PvRBP-1 , indicating that PvMSP-3 is highly immunogenic in
naturally exposed individuals. However, recently, it was demon-
strated that the breadth and magnitude of IgG antibody responses
to P. falciparum merozoite antigens (MSP-1, MSP-3 and AMA-1) are
better associated with protection from clinical malaria than only
the frequencies of responders . In agreement with this ﬁnding,
we observed that the RIs were high against the full-length pro-
tein, the two blocks of heptad repeats and the C-terminal region,
while the lowest levels were observed in PvMSP3-NT. The obser-
vation that higher indexes of antibodies against PvMSP3-FL and
PvMSP3-CT were present in individuals with more time of resi-
dence in malaria endemic areas and the IgG antibody levels against
PvMSP3-FL, PvMSP3-BLI and PvMSP3-CT increased accordingly
with the number of past malaria episodes indicate that repeated
P. vivax infections and/or exposure could signiﬁcantly increase,
not only the frequency of responders, but also the magnitude
of IgG antibodies against PvMSP-3␣. This pattern of a cumula-
tive immune response observed here against PvMSP-3␣ was also
observed for several other P. vivax and P. falciparum candidate anti-
The naturally acquired protective immunity against P. vivax
tested with vaccine candidate antigens is poorly explored. The
association of clinical protection with speciﬁc antigens of P. vivax
has been reported in only two prospective cohort longitudinal
studies [59,60]. In the ﬁrst, clinical protection was associated
with IgG3 antibodies against the N-terminus of the Merozoite
Surface Protein-1 (PvMSP-1) in residents of the Brazilian Ama-
zon region of Portuchuelo . In the second, clinical protection
was reported in children from Papua New Guinea  where
the frequency of naturally acquired binding inhibitory antibodies
against the Duffy-binding protein region II (PvDBPII) was asso-
ciated with protection against P. vivax infection. Studies on IgG
responses against P. falciparum antigens have consistently showed
that cytophilic subclasses IgG1 and IgG3 play an important role
in a protective antibody response in humans . The proposed
mechanism involves parasitic inhibition mediated by engagement
of Plasmodium-speciﬁc cytophilic Ig antibodies to Fc␥ receptors on
the surface of monocytes . Interestingly, the ﬁrst observation
of this mechanism was reported by Oeuvray et al. using antibod-
ies against a PvMSP-3 homologue, the originally characterized P.
falciparum MSP-3 . Nevertheless, only a small number of stud-
ies have investigated cytophilic antibody responses against P. vivax
[47,52,63]. Therefore, in our study, we have demonstrated that
IgG positive individuals against all PvMSP-3␣ recombinant pro-
teins studied here present a high frequency and concentrations of
anti-PvMSP-3 cytophilic antibodies IgG1 and IgG3.
Although we demonstrate a general predominance of anti-
PvMSP-3 cytophilic IgG antibodies, the correlation to clinical
immunity is rather tenuous, based on currently available data.
The cross-sectional design of our study limited the investigation
to retrospective malaria histories, and the best approximation of
an individual’s protection was the estimated amount of time that
had passed since their last malaria episode. We were only able
to observe positive correlations between IgG1 and IgG3 reactivi-
ties against PvMSP3-FL and PvMSP3-CT with the past months since
malaria infection. These results suggest a possible role of PvMSP-3
cytophilic antibodies in protective immunity against P. vivax infec-
tion. However, prospective studies on humoral immune responses
or biologic studies addressing the ability of these antibodies to
inhibit merozoite invasion or the development of blood-stage par-
asites will provide more direct evidence with regards to their
The present work describes for the ﬁrst time the ﬁne B cell
epitope mapping of a full-length protein of P. vivax using Spot-
synthesis. Information at the amino acid level about the epitopes
of proteins recognized by antibodies is important for their use
as biological tools, therapeutic molecules, and for understanding
molecular recognition events in general . In this context, T
and B cell epitope prediction programs are largely used in malaria
research [65,66]. However, the use of chemically prepared arrays
of short peptides has emerged as a powerful tool to identify and
characterize epitopes recognized by antibodies [35,36]. Our results
from the spot synthesis assays suggest that PvMSP-3␣ can present
as many as 25 antigenic determinants to the immune system.
However, several events are important in determining whether an
antibody against a speciﬁc peptide will bind to the native protein
from which the peptide sequence is derived; in this process the
length of the immunizing peptide is critical. To raise antibodies to
a peptide, a minimum length of six amino acids is required, and
peptides of >10 amino acids are generally required for the induc-
tion of antibodies that may bind to the native protein . In this
context, the synthesis of 15 amino acid peptides, with 9 overlap-
ping, has allowed the identiﬁcation of PvMSP-3␣ B-cell epitopes
encompassed in sequences ranging from 10 to 25 amino acids in
length. The majority (18/25) of the sequences containing the B-
cell epitopes are localized in the two blocks of heptad repeats
in the diverse central domain of PvMSP-3␣. On the other hand,
seven epitopes were assigned to sequences in the N and C ter-
minal ﬂanking regions, which are relatively conserved . This
ﬁnding could be important, in a future vaccine composition based
on these conserved regions, rather than the highly polymorphic
central domains, as so far conﬁrmed for PvMSP-3␣ and PvMSP-3␤
[21–25] and which may be the rule for other members of this gene
In conclusion, PvMSP-3␣ is highly immunogenic in individu-
als living in malaria endemic areas of the Brazilian Amazon. This
protein presents several linear B-cell epitopes in its sequence, and
the immune response generated is mainly mediated by cytophilic
antibodies, which are associated with time of exposure to infec-
tions and apparent protective immunity. However, studies on the
functional activity of these antibodies and further characterization
of the B cell epitopes described here are required to further assess
the potential of PvMSP-3␣ as a vaccine candidate. Moreover, the
complexity of the immune response against the surface of the P.
vivax merozoite must be re-considered, particularly if many or all
of the members of the 11-member pvmsp3 gene family  prove
to be similarly diverse, expressed and immunogenic.
This work was supported by Brazilian National Research Coun-
cil – CNPq/PAPES, Fiocruz, National Institute of Health, the Yerkes
National Primate Research Center Base Grant # RR00165 awarded
by the National Center for Research Resources of the National Insti-
tutes of Health, and NIH Grant #RO1 AI0555994 (MRG). Josué da
1810 J.C. Lima-Junior et al. / Vaccine 29 (2011) 1801–1811
Costa Lima Junior was the recipient of a FAPERJ Fellowship. We
are grateful to all individuals that participate in this study for their
cooperation and generous donation of blood, which made this study
possible. We thank Eileen Farnon and Jennie Larson for the assis-
tance during the sample collection and Paloma Napoleão Pêgo for
the Spot-synthesis technique. We thank the Secretary of Health of
Rondonia State and the Laboratorio Central – LACEN of Rondonia
for providing ﬁeldwork support.
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