Am. J. Trop. Med. Hyg., 75(4), 2006, pp. 582–587
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene
ANTIBODIES TO PLASMODIUM VIVAX APICAL MEMBRANE ANTIGEN 1:
PERSISTENCE AND CORRELATION WITH MALARIA TRANSMISSION INTENSITY
CRISTIANE G. MORAIS, IRENE S. SOARES, LUZIA H. CARVALHO, COR J. F. FONTES, ANTONIANA U. KRETTLI,
AND ÉRIKA M. BRAGA*
Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; Departamento de
Análises Clínicas e Toxicológicas, Universidade de Sao Paulo, Sao Paulo, Brazil; Universidade Federal de Mato Grosso, Cuiabá,
Mato Grosso, Brazil; Centro de Pesquisas René Rachou/FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil
Abstract. The antibody responses to the apical membrane antigen 1 of the Plasmodium vivax (PvAMA-1) were
investigated in subjects living in areas of Brazil with different levels of malaria transmission. The prevalence and the
levels of IgG to PvAMA-1 increased with the time of exposure. The frequency of a positive response and the mean IgG
level were higher in areas where malaria prevalence was more intense, especially among non-infected subjects exposed
to moderate transmission over a period of 20 years. The proportions and levels of IgG1and IgG3 isotypes were
significantly higher among those subjects with long-term exposure. Antibodies, mainly IgG1, to PvAMA-1 persisted for
seven years among subjects briefly exposed to malaria in an outbreak outside the Brazilian malaria-endemic area. These
data show the highly immunogenic properties of PvAMA-1 and emphasize its possible use as a malaria vaccine candidate.
In spite of the critical importance of the AMA-1, few seroepidemiologic studies have been conducted to understand
the dynamics of naturally acquired humoral immune responses to this molecule in populations exposed to different
patterns of malaria transmission. Most of those studies have
been conducted in holo-hyperendemic areas and P. falciparum AMA-1 has been used as a target for antibody detection.16–18
Although P. vivax causes less mortality than P. falciparum,
it has an enormous socioeconomic impact, particularly in
South America and Asia. In Brazil, P. vivax was responsible
for approximately 80% of the 459,013 cases of malaria reported in 2004 (Brazilian Ministry of Health). Given the critical importance of understanding naturally acquired immunity
to P. vivax antigens, we conducted an immunoepidemiologic
study focusing on the antibody response to PvAMA-1 in distinct areas within the Brazilian Amazon malaria-endemic region. We also evaluated the persistence of specific antibodies
to PvAMA-1 among subjects briefly exposed to P. vivax
transmission outside the malaria-endemic area.
INTRODUCTION
Antigens of Plasmodium located on the surface or in the
apical organelles of merozoites have been characterized as
targets for protection or as possible vaccines against malaria.
Among several vaccine candidates, apical membrane antigen
1 (AMA-1), an asexual blood stage antigen, is considered to
be an important candidate malaria vaccine antigen.1 This protein is present in all Plasmodium species examined, as well as
in other Apicomplexa.2–4 This antigen is a type I integral
membrane protein and in all characterized Plasmodium vivax
AMA-1 (PvAMA-1) genes 16 invariant Cys residues are encoded in the ectoplasmic region; analysis of the disulfide bond
pattern suggests a division into three distinct domains.5
Analysis of the crystal structure of the ectoplasmic region of
the PvAMA-1 confirmed the division of this region into three
structural domains.6 Comparison of the PvAMA-1 structure
with other known three-dimensional structures showed that
domains I and II are structurally similar to each other and
belong to the PAN module superfamily.6 this superfamily is
related to proteins with diverse adhesion functions binding to
protein or carbohydrates receptors.7
AMA-1 is synthesized late during the development of parasite schizonts as a precursor of an 83-kD protein that is initially located in the micronemes of the apical complex of
merozoites and sporozoites.7 It is processed to a 66-kD form
before its relocation into the surface of mature merozoites.7
Once on the surface, this form is redistributed and undergoes
two C-terminal cleavages, giving rise to 48-kD and 44-kD
soluble forms.8–11 The function of AMA-1 is not well understood, but its stage specificity and location suggest that this
protein is involved in the process of invasion of host erythrocytes.12,13
The inclusion of AMA-1 as malaria vaccine candidate is
supported by evidence from studies of malaria in animal models.14,15 Furthermore, in malaria-endemic African populations, a considerable proportion of individuals has naturally
acquired antibodies and T cell reactivities to AMA-1.16–18
MATERIALS AND METHODS
Study areas, subjects, and blood sample collection. We analyzed four different groups of non-infected adults who had
been exposed to malaria transmission in the Brazilian Amazon area (Table 1). A complete health questionnaire was applied to all study participants. Ethical clearance for this study
was obtained from the institutional review board of the Federal University of Minas Gerais, Brazil (April 15, 1998; #007/
98 and May 16, 2001; #096/01). All participants reported at
least one malaria infection diagnosed by microscopy; the date
and species involved in this most recent episode were recorded. Since most subjects were migrants from areas in Brazil not endemic for malaria, their ages do not correlate with
cumulative exposure to malaria. Past malaria exposure was
therefore estimated as the length of residence or exposure to
malaria in malaria-endemic areas in Brazil. These parameters
were used to classify the four groups.
The first group lived in Belém, the capital of Pará State. It
was composed of 59 persons (median age ⳱ 29 years) who
had acquired P. vivax malaria after a brief exposure (a few
* Address correspondence to Érika M. Braga, Departamento de
Parasitologia, Instituto de Ciências Biológicas, Universidade Federal
de Minas Gerais, Av. Antônio Carlos 6627, 31279-901 Belo Horizonte, Minais Gerais, Brazil. E-mail: [email protected]
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ANTIBODIES TO PvAMA-1 IN BRAZIL
TABLE 1
Demographic and epidemiologic data of the subjects exposed to endemic malaria transmission, in the Brazilian Amazon
Group (n)
Characteristics
(sample)
Belém
(59)
Cuiabá
(45)
Terra Nova
do Norte
(68)
Apiacás
(56)
Sex, male (%)
Median of age (years)
Time of malaria exposure (mean ± SD)
Number of previous malaria episodes
Exposure to malaria transmission
34 (59)
29
< 1 month
1*
Low
37 (82)
33
< 1 year
1 to > 10
Sporadic
53 (77)
31
9.8 ± 4.9 years
1 to > 10
Constant
51 (91)
30
19.0 ± 12.7 years
> 10
Constant
* Only cases of Plasmodium vivax were registered.
days) on the island of Cotijuba, which is located 25 km from
Belém.
The second group lived in Cuiabá, the capital of Mato
Grosso, where no malaria transmission occurs. It was composed of 45 persons (median age ⳱ 33 years) who had become infected after short visits to mining and/or agricultural
areas in the northern part of the state where malaria is endemic. They stayed less than one year in the transmission
areas.
The third group was composed of 68 persons (median
age ⳱ 31 years) who had resided for ⱖ 10 years in Terra Nova
do Norte (TNN), a small rural community within the malariaendemic region. These individuals were continuously exposed
to malaria transmission (median time ⳱ 10.5 years).
The fourth group was composed of 56 non-parasitemic persons previously studied who had lived for approximately 20
years in several gold-mining areas in the Brazilian Amazon
basin.19,20 All lived in Apiacás, a municipality located in
northern Mato Grosso State. Apiacás was characterized as a
mesoendemic area because malaria prevalence in 1996, the
study year, was 18%.19
Venous blood samples (2 mL) were drawn into EDTAcontaining tubes and used to prepare thick smears for microscopy (10 ␮L) and to obtain plasma. Plasma samples were
frozen at −20°C for subsequent use.
We also included in our study sera from subjects exposed to
a P. vivax malaria outbreak that occurred in 1988 in a rural
community (Mantena, Minas Gerais) located approximately
1,100 miles from the nearest malaria-endemic area reporting
transmission of P. vivax, as previously described.20–22 Malaria
has never been reported in this area (Brazilian Ministry of
Health). Control measures initially included active search for
acute malaria by collecting thick blood films from all febrile
and symptomatic persons plus their relatives and neighbors
without symptoms who were considered to be exposed to the
risk of infection. The outbreak was controlled by chemotherapy and insecticides because no new cases or relapses
have been reported in this area. Sera from 14 subjects (median age ⳱ 19 years) who had had positive thick blood smears
during the outbreak and 11 of their relatives and/or neighbors
similarly treated despite negative thick blood smears were
collected eight months and seven years after transmission. All
subjects were parasite free at the time of blood collection at
the two time points and were given a questionnaire that included past malaria experience, i.e., eight months and seven
years after the outbreak. Their answers showed that all individuals in our sample were born and raised in the outbreak
area and none reported travel to malaria-endemic regions.
Venous blood samples (20 mL per individual) were drawn
into Vacutainer威 heparinized tubes (Becton Dickinson, Oxnard, CA). Giemsa-stained thick-blood smears were examined at this point. Control subjects consisted of 40 healthy
adult volunteers living in Belo Horizonte in Minas Gerais
State who had never been exposed to the malaria transmission or visited the malaria-endemic region.
Antigens. Recombinant PvAMA-1 contains the amino acid
sequence (43–487) of the ectodomain of the P. vivax BEL-12
isolate.23 This protein was expressed in Escherichia coli and
purified as described previously.23
Antibody measurement. An enzyme-linked immunobsorbent assay (ELISA) for total IgG antibodies was performed
as previously described.23 The concentration of PvAMA-1
used was 1.0 ␮g/mL. All samples were diluted 1:100 and
evaluated for total IgG using peroxidase-conjugated antihuman IgG antibodies (Sigma, St. Louis, MO).
The ELISA to detect IgG subclasses was performed as previously described.22 The serum dilution used was 1:50 and
mouse monoclonal antibodies to human IgG subclasses used
were clone HP-6012 for IgG1, clone HP-6014 for IgG2, clone
HP-6010 for IgG3, and clone HP-6025 for IgG4 (Sigma) diluted according to the manufacturer’s specifications. Monoclonal antibody binding was detected with peroxidaseconjugated anti-mouse immunoglobulin (Sigma).
The threshold of positivity (cut-off value) was obtained by
testing 40 different negative control sera from individuals not
exposed to malaria from Belo Horizonte. The mean optical
density value at 490 nm (OD490) ± 3 SD for duplicate determinations in negative sera was used as the cut-off value for different subclasses. The thresholds of positivity were 0.32 for IgG,
0.15 for IgG1, 0.13 for IgG2, 0.1 for IgG3, and 0.06 for IgG4.
The reactivity index (RI) was obtained to compare the levels of different subclasses (IgG or IgG isotype) in the same
subject or among distinct groups. The RI value was calculated
by dividing the mean OD value for each test sample assayed
by the cut-off value for each subclass tested using sera from
healthy individuals (control group). Samples with an RI > 1
were considered positive.
Statistical analysis. Data were analyzed using Epi-Info version 6.03 (Centers for Disease Control and Prevention, Atlanta, GA). The chi-square test was used to compare proportions and the Kruskal-Wallis test was used to analyze the
significance of differences between group values. Significance
was set at the 5% level.
RESULTS
Relationship of IgG antibodies to PvAMA-1 and malaria
exposure. The proportions of PvAMA-1 IgG-positive subjects increased with exposure to malaria transmission (P <
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MORAIS AND OTHERS
0.001) (Figure 1). It reached a peak of 95% in those subjects
with long-term exposure in the Brazilian malaria-endemic
area (Apiacás group). However, similar proportions of IgG
antibodies to PvAMA-1 were detected in subjects in the
Belém (59%), Cuiabá (71%), and TNN (71%) groups. Persons in the Belém group, which was composed of subjects who
reported a single P. vivax infection, had a high IgG antibody
response to PvAMA-1 (59%).
The levels of IgG antibodies to PvAMA-1 (measured as
RIs) are shown in Figure 1. High levels of specific IgG (RI ⱖ
5) were observed among 36% of the subjects living in Apiacás
with long-term exposure to malaria. The IgG-positive responses ranged to 3% to 9% in the other groups (P < 0.001).
Proportions and levels of PvAMA-1-specific IgG1 and IgG3
also showed differences related to exposure, being highest for
individuals living in Apiacás (P < 0.05) (Figure 2A and B).
Relationship between most recent malaria episode and antibodies to PvAMA-1. Since subjects from Cuiabá, TNN, and
Apiacás had experienced various numbers of episodes of malaria caused by P. vivax and/or P. falciparum, we analyzed the
effect of the most recent malaria episode on the antibody
responses against PvAMA-1. As shown in Figure 3A, individuals living in Cuiabá whose most recent infection was with
P. vivax showed higher proportions of IgG- and IgG1-positive
responses when compared with those whose most recent malaria episode was caused by P. falciparum. In TNN, the results
showed a non-significant tendency towards increased prevalence of antibodies to PvAMA-1 among individuals whose
most recent malaria episode was caused by P. vivax (P > 0.05)
(Figure 3B). Sera from subjects in the Cuiabá group whose
most recent infection was with P. vivax showed higher levels
of IgG when compared with those whose most recent infection was with P. falciparum.
Longevity of IgG antibodies to PvAMA-1. Twenty-five
persons living in a rural community in Minas Gerais who were
briefly exposed to a P. vivax malaria outbreak outside the
malaria-endemic area were evaluated to measure the persistence of specific antibodies. Sera were tested in parallel in all
samples obtained at two different time points. IgG antibodies
FIGURE 2. Proportions of responders (A) with IgG subclass antibodies to Plasmodium vivax apical membrane antigen 1and antibody
levels (reactivity index) (B) among individuals living in four areas
of the Brazilian Amazon with different levels of malaria transmission (Apiacás > Terra Nova do Norte (TNN) > Cuiabá > Belém).
The horizontal line in B shows the threshold of positivity (reactivity
index ⳱ 1).
to PvAMA-1 were detected in 43% of the subjects who had
had malarial symptoms eight months after transmission (Figure 4). Among the prophylactically treated individuals, 64%
were also positive for IgG antibodies to PvAMA-1. Seven
years later, positive responses were still detected in approximately 36% of both groups. Specific IgG1 was the predominant subclass, regardless of malaria symptoms. It was first
detected in 13 individuals (52%) and was still present in nine
(36%) persons after seven years. Similar distributions of IgG
or IgG1 antibodies to PvAMA were found in symptomatic
and asymptomatic subjects evaluated at the two time points.
DISCUSSION
FIGURE 1. Effect of exposure to malaria transmission on levels of
IgG antibody to Plasmodium vivax apical membrane antigen 1. Antibody responses are expressed as reactivity index (RI) detected by an
enzyme-linked immunosorbent assay in sera from adults living in four
areas of the Brazilian Amazon with different levels of malaria transmission (Apiacás > Terra Nova do Norte (TNN) > Cuiabá > Belém).
Percentage values represent the overall frequency of the IgG response for each group.
Although AMA-1 is an important vaccine candidate antigen, there have been few serologic studies on the nature of
immune responses to this protein in persons living in malariaendemic areas. Previous studies have demonstrated the immunogenicity of P. falciparum AMA-1 among African subjects.16–18 Currently available seroepidemiologic data on the
ANTIBODIES TO PvAMA-1 IN BRAZIL
585
FIGURE 4. Levels of IgG antibody to Plasmodium vivax apical
membrane antigen 1 measured as reactivity indices using sera from
subjects exposed to a P. vivax malaria outbreak eight months and
seven years before who had or did not have malaria symptoms. Percentage values represent the overall frequency of the IgG response
for each group. The horizontal line shows the threshold of positivity
at a reactivity index of 1.
FIGURE 3. Prevalences of antibody responses (IgG and subclasses) to Plasmodium vivax apical membrane antigen 1 detected in
sera from individuals living in Cuiabá (A), Terra Nova do Norte
(TNN) (B), and Apiacás (C) whose last malaria episode was caused
by P. vivax (dark columns) or P. falciparum (white columns). *P <
0.05.
P. vivax homologous protein is limited to a study in Brazil
that showed a significant frequency of positive antibody responses to PvAMA-1 (85%) among individuals with patent P.
vivax infection.24 However, no studies have addressed the
effect of the intensity of malaria exposure on the development of antibodies to PvAMA-1.
We have demonstrated that a recombinant protein containing the ectodomain of P. vivax AMA-1 is naturally immunogenic in Brazilian persons who had distinct degrees of malaria
exposure. The high prevalence of PvAMA-1-specific antibodies could be explained by the fact that the region coding the
variable domain of the PvAMA-1 gene shows limited polymorphism among Brazilian isolates.23
Since the IgG subclasses produced in response to a given
antigen may determine the function of the antibody, we determined the proportions and levels of IgG isotypes of
PvAMA-1. Analyses of IgG subclasses responses showed that
the prevalence and the levels of PvAMA-1-specific IgG1 and
IgG3 were higher among subjects with long-term exposure to
malaria. It is known that IgG1 and IgG3 isotypes have been
implicated in antibody-mediated protective immunity against
Plasmodium blood stages.24–26 Thus, the high level of cytophilic antibodies among subjects who were continuously exposed to malaria for more than 20 years without detectable
parasitemias may be the result of acquisition of partial immunity against malaria, as previously suggested.19,27 High proportion and levels of IgG3 were detected in miners living in
Apiacás who were not infected with Plasmodium. It is important to note that at the time of blood collection, those subjects
were negative for Plasmodium by conventional microscopic
examination of thick blood smears and a nested polymerase
chain reaction for the 18S small subunit ribosomal rRNA
gene.19 Since IgG3 has a relatively short half-life, the antibody response to PvAMA-1 for this isotype may be caused by
frequent re-exposure to the parasite, which is required to
increase levels of these antibodies.28
Long-term antibody production is one hallmark of effective
vaccination and is an important characteristic of immunologic
memory. B cell memory seems to depend on the persistence
of stimulating antigen maintained over long periods by im-
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MORAIS AND OTHERS
mune complex on follicular dendritic cells of germinal centers
in secondary lymphoid organs,29 or an antigen-independent
mechanism such as the limitation of the number of long-lived
plasma cells that allows the immune system to maintain a
stable humoral immunologic memory over long periods.30 We
have shown that that humoral immunity to P. vivax can be
maintained even in the absence of continuous re-exposure to
the parasite.21–23 Based on this finding, in the present study
we also determined the IgG antibody response against
PvAMA-1 in subjects exposed to a previous outbreak of P.
vivax malaria outside the malaria-endemic Amazon region.
Our results showed that eight months after transmission IgG
antibodies to PvAMA-1 were present in six subjects who had
had malarial symptoms during the outbreak and persisted
among five of them. These results are consistent with a oneyear longitudinal study in Kenya in which IgG antibody responses to P. falciparum AMA-1 were persistent.16 Interestingly, eight months after focal malaria transmission, the
prevalence of antibodies to PvAMA-1 was also higher (64%)
among the asymptomatic prophylactically treated subjects.
However, similar distributions of IgG or IgG1 antibodies to
PvAMA-1 were found in both groups regardless of malaria
symptoms.
Since AMA-1 is also localized in the micronemes of the
apical complex of sporozoites,7 it may contribute to the presence of antibodies to PvAMA-1 among those prophylactically
treated subjects that could abolish the infection before detection of the parasites by thick blood smears. Our previous
study conducted in the same area using a circumsporozoite
protein of P. vivax showed a persistent specific antibody response in the absence of reinfection, regardless of malaria
symptoms.21 These data contrast with our previous data for
the same population focusing in blood-stage antigens (Cterminal of P. vivax merozoite surface protein 1)21,22 which
showed that long-term specific antibodies were persistent
only among subjects who had clinical malarial symptoms during the malaria outbreak.
In conclusion, we have found that proportions and levels of
antibodies against PvAMA-1 are correlated with the intensity
of exposure to malaria in areas with low-to-moderate transmission in Brazil. These observations of long-lasting specific
IgG1 indicate the high immunogenicity of PvAMA-1 and emphasize its use as a possible P. vivax malaria vaccine.
Received June 14, 2005. Accepted for publication December 1, 2005.
Financial support: This study was supported by the Fundação
de Amparo à Pesquisa do Estado de Minas Gerais (grant no.
CBB2323/97).
Authors’ addresses: Cristiane G. Morais and Érika M. Braga, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, 31270-901,
Belo Horizonte, Minas Gerais, Brazil. Irene S. Soares, Departamento
de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Av. Prof. Lineu Prestes 580,
Bloco 17, 05508-900, Sao Paulo, Brazil. Luzia H. Carvalho and Antoniana U. Krettli, Laboratório de Malária, Centro de Pesquisas
René Rachou/FIOCRUZ, Av. Augusto de Lima 1715, Barro Preto,
30190-002, Belo Horizonte, Minas Gerais, Brazil. Cor Jesus F. Fontes,
Departamento de Clínica Médica, Hospital Júlio Müller, Universidade Federal de Mato Grosso, Rua L S/N, Jardim Alvorada, 78070150, Cuiabá, Mato Grosso, Brazil.
Reprint requests: Érika M. Braga, Departamento de Parasitologia,
Instituto de Ciências Biológicas, Universidade Federal de Minas
Gerais, Av. Antônio Carlos 6627, 31279-901 Belo Horizonte, Minais
Gerais, Brazil, Telephone: 55-31-3499-2876, Fax: 55-31-3499-2970,
E-mail: [email protected].
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