Antibody levels to multiple malaria vaccine candidate antigens in relation to clinical malaria episodes in children in the Kasena-Nankana district of Northern Ghana.

Abstract

Considering the natural history of malaria of continued susceptibility to infection and episodes of illness that decline in frequency and severity over time, studies which attempt to relate immune response to protection must be longitudinal and have clearly specified definitions of immune status. Putative vaccines are expected to protect against infection, mild or severe disease or reduce transmission, but so far it has not been easy to clearly establish what constitutes protective immunity or how this develops naturally, especially among the affected target groups. The present study was done in under six year old children to identify malaria antigens which induce antibodies that correlate with protection from Plasmodium falciparum malaria.
In this longitudinal study, the multiplex assay was used to measure IgG antibody levels to 10 malaria antigens (GLURP R0, GLURP R2, MSP3 FVO, AMA1 FVO, AMA1 LR32, AMA1 3D7, MSP1 3D7, MSP1 FVO, LSA-1and EBA175RII) in 325 children aged 1 to 6 years in the Kassena Nankana district of northern Ghana. The antigen specific antibody levels were then related to the risk of clinical malaria over the ensuing year using a negative binomial regression model.
IgG levels generally increased with age. The risk of clinical malaria decreased with increasing antibody levels. Except for FMPOII-LSA, (p = 0.05), higher IgG levels were associated with reduced risk of clinical malaria (defined as axillary temperature ≥37.5°C and parasitaemia of ≥5000 parasites/ul blood) in a univariate analysis, upon correcting for the confounding effect of age. However, in a combined multiple regression analysis, only IgG levels to MSP1-3D7 (Incidence rate ratio = 0.84, [95% C.I.= 0.73, 0.97, P = 0.02]) and AMA1 3D7 (IRR = 0.84 [95% C.I.= 0.74, 0.96, P = 0.01]) were associated with a reduced risk of clinical malaria over one year of morbidity surveillance.
The data from this study support the view that a multivalent vaccine involving different antigens is most likely to be more effective than a monovalent one. Functional assays, like the parasite growth inhibition assay will be necessary to confirm if these associations reflect functional roles of antibodies to MSP1-3D7 and AMA1-3D7 in this population.

Full text

Page 1

RESEARCHOpen Access
Antibody levels to multiple malaria vaccine
candidate antigens in relation to clinical malaria
episodes in children in the Kasena-Nankana
district of Northern Ghana
Daniel Dodoo1*, Frank Atuguba2, Samuel Bosomprah3, Nana Akosua Ansah2, Patrick Ansah2, Helena Lamptey1,
Beverly Egyir1, Abraham R Oduro2, Ben Gyan1, Abraham Hodgson2and Kwadwo A Koram4
Abstract
Background: Considering the natural history of malaria of continued susceptibility to infection and episodes of
illness that decline in frequency and severity over time, studies which attempt to relate immune response to
protection must be longitudinal and have clearly specified definitions of immune status. Putative vaccines are
expected to protect against infection, mild or severe disease or reduce transmission, but so far it has not been
easy to clearly establish what constitutes protective immunity or how this develops naturally, especially among the
affected target groups. The present study was done in under six year old children to identify malaria antigens
which induce antibodies that correlate with protection from Plasmodium falciparum malaria.
Methods: In this longitudinal study, the multiplex assay was used to measure IgG antibody levels to 10 malaria
antigens (GLURP R0, GLURP R2, MSP3 FVO, AMA1 FVO, AMA1 LR32, AMA1 3D7, MSP1 3D7, MSP1 FVO, LSA-1and
EBA175RII) in 325 children aged 1 to 6 years in the Kassena Nankana district of northern Ghana. The antigen
specific antibody levels were then related to the risk of clinical malaria over the ensuing year using a negative
binomial regression model.
Results: IgG levels generally increased with age. The risk of clinical malaria decreased with increasing antibody
levels. Except for FMPOII-LSA, (p = 0.05), higher IgG levels were associated with reduced risk of clinical malaria
(defined as axillary temperature ≥37.5°C and parasitaemia of ≥5000 parasites/ul blood) in a univariate analysis, upon
correcting for the confounding effect of age. However, in a combined multiple regression analysis, only IgG levels
to MSP1-3D7 (Incidence rate ratio = 0.84, [95% C.I.= 0.73, 0.97, P = 0.02]) and AMA1 3D7 (IRR = 0.84 [95% C.I.= 0.74,
0.96, P = 0.01]) were associated with a reduced risk of clinical malaria over one year of morbidity surveillance.
Conclusion: The data from this study support the view that a multivalent vaccine involving different antigens is
most likely to be more effective than a monovalent one. Functional assays, like the parasite growth inhibition assay
will be necessary to confirm if these associations reflect functional roles of antibodies to MSP1-3D7 and AMA1-3D7
in this population.
* Correspondence: DDodoo@noguchi.mimcom.org
1Department of Immunology, Noguchi Memorial Institute for Medical
Research, University of Ghana, P.O. Box LG581, Accra, Ghana
Full list of author information is available at the end of the article
Dodoo et al. Malaria Journal 2011, 10:108
http://www.malariajournal.com/content/10/1/108
© 2011 Dodoo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Page 2

Background
In malaria endemic regions, clinical malaria is responsi-
ble for high morbidity and mortality in less than five
year old children and pregnant women. In these regions,
individuals develop a partial ‘non-sterile’ immunity
against erythrocytic stage disease in an age and exposure
dependent manner and, therefore, older individuals suf-
fer less clinical symptoms and disease complications.
Sero-epidemiological studies show three sequential
phases of development of acquired immunity to malaria:
first, immunity to life-threatening disease; second,
immunity to symptomatic infection; and only then, can
the third phase, partial immunity to parasitization be
achieved [1,2]. Passive transfer of antibodies from
malaria-immune adults have been successfully used in
the treatment of malaria patients [3,4], suggesting a cru-
cial role of antibodies in immunity to malaria.
Several studies have reported associations between
levels of antibody to various malaria parasite specific
antigens and reduced risk of infection [5-9]. However,
as yet, the precise antigenic targets of protective immu-
nity to malaria remain largely unknown as findings from
different correlates of antibody mediated immunity stu-
dies are often conflicting in their conclusions. Thus,
there is presently no single immunological correlate of
protection to clinical malaria, and those described do
not sufficiently account for the overall variation in sus-
ceptibility observed in a population [10].
Several antigens due to their structures and locations
have been deemed of importance in inducing protective
antibodies against clinical malaria of the erythrocytic
stage of the parasite. These include the merozoite sur-
face proteins (MSP1, MSP2, MSP3, etc.) and the apical
membrane antigen - 1 (AMA1), EBA-175 RII and
GLURP [6,7,9,9,11-13], but the mechanism of action of
these antibodies in vivo remains unclear [7].
In this longitudinal study, baseline IgG levels to ten
malaria vaccine candidate antigens, namely, GLURP R0,
GLURP R2, MSP3 FVO, AMA1 FVO, AMA1 LR32,
AMA1 3D7, MSP1 3D7, MSP1 FVO, FMP011 (LSA-1)
and EBA175RII were measured by the multiplex assay
in plasma samples of 1 to 6 year old children, living in a
malaria endemic region and the levels related to the risk
of clinical malaria estimated over a one year period. The
multiplex technique which has been validated and
shown to have high correlation with the traditional
ELISA technique in malaria antibody measurements and
which has a higher detection range [14] was the pre-
ferred assay of choice for this study. In studies involving
infants and children where only small volumes of sam-
ples are obtained and antibody measurements to multi-
ple antigens are required as in this study, the traditional
ELISA method is limited by the large sample volumes
required. This study was aimed at elucidating which of
the antibodies to the various antigens could act indivi-
dually or in a concerted manner to confer immunity to
malaria in the studied population.
Methods
Study site and population
The study was conducted in the Kassena-Nankana Dis-
trict (KND) of the Upper East region of northern
Ghana. This is a savannah region where the people are
mainly subsistence farmers. There are two main seasons,
a dry season from about October to April and a wet
season from approximately May to October. Malaria
transmission occurs throughout the year with distinct
patterns during the two seasons. The estimated malaria
attack rate is approximately 3.5 attacks per child per
year. The district is under the Navrongo Demographic
Surveillance System (NDSS), which administers a com-
plete population census of the population every 90 days.
Details of the study area and population have been pub-
lished elsewhere [15]. The KND is served by the Nav-
rongo Health Research Centre, the Navrongo War
Memorial Hospital and four other health centres. The
study enrolled children between the ages from 1 to 6
years in KND, who planned to remain in the district for
at least the entire duration of the study (one calendar
year) and their parents or guardians consented to the
study.
The study design
The study enrolled 325 children between one and six
years old independent of gender and with no regards to
ethnicity. Children were randomly generated from the
database of the NDSS and subsequently contacted in the
recruitment exercise for enrolment. Only those whose
parents/guardians agreed to be part of the study keeping
all study required protocols were enrolled within the
month of May 2004 (over a period of three weeks).
These children were passively followed over one calen-
dar year (May 2004 to May 2005) during which clinical,
haematological and parasitological data were collected at
the beginning of the study and every two months. Fin-
gerprick blood samples (0.5-1.0 ml) were used to deter-
mine haemoglobin concentrations with a Hemocue Hb
201 (Angelhom, Sweden) and malaria infection. Giemsa-
stained thin and thick malaria blood films (MBFs) were
done and examined by two microscopists independently
for parasites. An enhanced passive follow up approach
was adopted where parents and guardians were encour-
aged to bring study participants to the health service
facilities for any perceived illness. Field workers were
also placed in the community to assist parents and guar-
dians to access medical care. During every visit to the
health facility, the participants were evaluated with a
morbidity questionnaire and a physical examination by
Dodoo et al. Malaria Journal 2011, 10:108
http://www.malariajournal.com/content/10/1/108
Page 2 of 9

Page 3

study clinicians. A blood smear was also taken and a
rapid malaria diagnostic test (DiaMed Optimal Rapid
Malaria test) done. Children suffering from uncompli-
cated malaria were treated with chloroquine and sulpha-
doxine-pyrimethamine (SP)(Fansidar®) as the first-line
drugs according to Ghana Ministry of Health policy at
the time. Those with severe forms of the disease were
referred for free treatment with intravenous quinine at
the Navrongo War Memorial Hospital. After discharge,
the subjects were physically and clinically examined by a
study physician and malaria smear and haemoglobin
determination were made after which the subject was
allowed to resume participation in the study. Other
health conditions were managed as per the prevailing
guidelines of Ghana Ministry of Health.
Parasite densities were calculated by enumerating the
parasites against 200 WBC. The baseline blood samples
were used to measure antibody levels to 10 malaria anti-
gens (GLURP R0, GLURP R2, MSP3 FVO, AMA1 FVO,
AMA1 LR32, AMA1 3D7, MSP1 3D7, MSP1 FVO,
LSA-1and EBA175RII) using the multiplex assay. The
immunological data was related to the risk of clinical
malaria within the study period of one year.
Recombinant antigens
The malaria antigens used in this study included a
recombinant GLURP-R0 containing the conserved non-
repeat N-terminal region, (amino acids 25-514), and
GLURP-R2 (amino acids 705-1178) of the carboxy-term-
inal repeat region, all expressed in Escherichia coli [16]
(supplied by Dr. Michael Theisen from State Serum
Institute, Copenhagen). The recombinant C-terminal
MSP1 42 protein of the FVO and 3D7 strains were
expressed in E. coli [17]; and AMA-1 FVO (amino acids
25-545) [18], 3D7 and LR32 strains expressed in yeast
Pichia pastoris, (all donated by Dr. Laura B. Martin
from Malaria Vaccine Development Branch of NIAID,
NIH). The EBA-175 RII expressed in yeast P. pastoris,
containing amino acid residues 144 to 753, was provided
by Division of Microbiology and Infectious Diseases,
(DMID) NIAID, NIH under contract NO1-AI-05421.
USA. The recombinant MSP-3 of the FVO strain was
expressed in E. coli, (supplied by Dr. Richard Shimp
from NIAID, NIH), and the LSA-1 expressed in E. coli
was donated by Dr. David E. Lanar of the Division of
Malaria Vaccine Development, Walter Reed Army Insti-
tute of Research, USA.
Antibody measurements
Antigen specific IgG levels were meseasured against
these antigens using the multiplex assay, a suspension
array technique as described in a previous publication
[14] with modifications as described below. Antigens
were coupled to the microspheres using a modification
of the protocol from Luminex Corp. Carboxylated
microspheres or beads stock (Luminex Corp., Austin,
TX) with different spectral addresses (101, 102, 103,
104, 105, 106, 107, 117, 118, 119 and 120) were centri-
fuged at 2,000 rpm for 5 min. The bead pellets were
then dispersed by sonication and vortexing. A specified
amount of the bead suspension was then centrifuged at
12000 rpm for 2 minutes and supernatant aspirated.
The pellet was washed twice in 80 ul of activation buffer
(0.1 M NaH2PO4, pH 6.2), and then activated with 10 ul
of a 50 mg/ml solution of 1-ethyl 3-(3-dimethylamino-
propyl) carbodiimide hydrochloride (EDC) and 10 ul of
a 50-mg/ml solution of sulfo-N-hyroxysulfosuccinimide
(Sulfo NHS). The microspheres were mixed gently and
incubated at room temperature (RT) ranging from 25°C
to 28°C, in the dark for 20 min. The activated micro-
spheres were then washed twice with 250 ul of coupling
buffer [0.05 M of 2-(N-morpholino) ethanesulfonic acid
or MES, pH 5.0] and re-suspended in 100 ul of coupling
buffer. To determine the optimal concentration of pro-
tein for coupling, 1.6, 4.0, 10, 25, and 62.5 μg/ml of
recombinant proteins and BSA were assessed. Various
concentrations of the recombinant malarial antigens
such as GLURP R2 at (1.6 μg/ml); GLURP-R0, AMA1-
FVO, MSP1-FVO, MSP1-3D7, LSA-1(all at 10 μg/ml),
MSP3 FVO, AMA1 LR32, and EBA175RII (all at 25 μg/
ml); AMA1 3D7 at 62.5 μg/ml were added to the micro-
spheres. They were then mixed gently by vortexing, fol-
lowed by 20 seconds ultrasonic sonication. The total
volume was adjusted to 500 μl by adding the coupling
buffer. The microspheres were incubated at RT in the
dark for 2 hours with rotation, and then washed twice
with 500 μl of storage buffer (phosphate-buffered saline
[PBS], pH 7.2, containing 1% BSA, and 0.02% Tween 20,
and 0.05% sodium azide). The coupled microspheres
were re-suspended in 500 μl of blocking/storage buffer
and stored at 4°C in the dark until ready for use.
Multiplexed assay
Stock suspensions of antigen-coated microspheres were
thoroughly re-suspended by vortexing and sonication
before use. Filter plates (Multiscreen BV; Millipore,
MA) were washed with 200 μl of washing buffer (PBS
plus 0.05% Tween 20) and then washed with 200 μl of
dilution buffer (PBS plus 1% BSA) using a vacuum
manifold (Biorad Laboratories, CA, USA) to pre-wet the
plates. Then, 25 μl of mixed microspheres of the anti-
gens were distributed to microtiter wells (4,000 micro-
spheres/well) and 50 μl of diluted plasma (1:50 and
1:2000 dilutions) was added. The plates were placed on
a Microplate Shaker (Ika-schuttlar, MTS4 Labor-tech-
nik) and incubated at RT in the dark for 1 hr at
500 rpm. The plates were washed five times with 200 ul
washing buffer. A total of 25 ul of 5 ug/ml (1:200) of
Dodoo et al. Malaria Journal 2011, 10:108
http://www.malariajournal.com/content/10/1/108
Page 3 of 9

Page 4

goat anti-human immunoglobulin G (KPL 176-1006,)
was added to each well, and plates were incubated for 1
hr in the dark at RT on a microplate shaker. After the
incubation period, plates were washed five times with
washing buffer, and then 50 ul of strepavidin phycoery-
thrin (1:800) was added and incubated for 30 mins at
RT in the dark on a shaker. The reader (Boiplex system,
Luminex Corp., Austin, TX) was programmed to read a
minimum of 100 microspheres per bead region, and
results were expressed as mean fluorescent intensity
(MFI), which is directly proportional to antibody concen-
tration[14]. For quality control of the assays, a pool of
plasma obtained from the blood bank in Accra and pre-
determined to have high levels of IgG to multiple malaria
antigens was included in each assay as positive control
(PC). In addition, a pool of plasma from non-malaria
exposed US volunteers was included as negative control
(NC) in each assay. The antibody levels detected for the
multiplex assay as mean fluorescent intensity (MFI) in
this study varied with the different antigens used, ranging
from a minimum of 25 MFI to 15, 736 MFI.
The study was reviewed and approved by the IRBs of
both Noguchi Memorial Institute for Medical Research
and the Navrongo Health Research Centre.
Results
Pattern of Plasmodium falciparum infection and clinical
malaria in the study area
Of the total of 325 children, three hundred and fifteen
had adequate samples for the immunological assays and
the data generated from these was included in the analy-
sis. Of this, 63 had at least one episode of clinical
malaria during the one year period of the study and the
incidence rate of clinical malaria decreased with age
(Table 1). Asymptomatic P. falciparum infection was
prevalent all year round; with about 60-80% study
subjects carrying parasites throughout the study period
(Figure 1). However, cases of clinical malaria peaked in
June and dropped gradually to the lower levels typical of
the low transmission season (Figure 1).
Association between antibody levels, age and clinical
malaria
The sensitivity and specificity of defining clinical malaria
cases may vary according to the intensity of malaraia
transmission and the age of residents in malaria ende-
mic areas [19-21]. Case definitions including parasitea-
mias of 2,500/μl blood or 5,000 parasites/ul blood in
addition to reported or measured fever of ≥37.5°C have
been used in previous reports [19,22], although for clini-
cal practice a less strict case definition involving the his-
tory of fever is adopted. In this study involving children
under seven years old in an area with sesonal, but
intense malaria transmission, the case definition used
was measured fever of ≥37.5°C in addition to parasitae-
mia at ≥5,000 parasites/μl and this was used to assess
malaria specific antibody levels in relation to the risk of
clinical malaria. Antibody levels in baseline samples
tended to be lower in children who are negative for
baseline parasitaemia and who are at higher risk to clini-
cal malaria (Table 1). Children were considered to have
a clinical malaria episode if they had axillary tempera-
ture of ≥37.5°C with a parasite density threshold greater
than or equal to 5000/μl.
Antibody titres to most of the antigens significantly
increased with age (0.29≤r≤0.36, p < 0.0001, Figure 2),
except for antibodies to LSA-1, which did not signifi-
cantly correlate with age (r = 0.11, p = 0.051). After
adjusting for the confounding effects of age, the anti-
body levels against all of the antigens tested, except
LSA-1, were significantly associated with reduced risk of
malaria (Table 2).
Table 1 Characteristics of the study population
Characteristics Cumulative incidence of
malaria
Child year at
risk
No. of malaria
episodes
Incidence rate per 100 child year
(95% CI)
Age group
12 <24 months
24 <36 months
36 <48 months
48 <60 months
60 <73 months
52.5% (21/40)
31.5% (17/54)
12.8% (10/78)
10.3% (8/78)
10.8% (7/65)
34.9
46.1
75.1
76.4
62.2
28
24
12
9
7
80 (55, 116)
52 (35, 78)
16 (9, 28)
12 (6, 23)
11 (5, 23)
Baseline P. falciparum parasitemia
level
Negative
Positive
41.4% (24/58)
15.2% (39/257)
52.1
242.7
31
49
59 (42, 84)
20 (15, 27)
TOTAL 20% (63/315) 295.180 27 (22, 34)
Clinical malaria is defined as history of fever or temperature>= 37.5 and parasite density>= 5000 u/l.
Dodoo et al. Malaria Journal 2011, 10:108
http://www.malariajournal.com/content/10/1/108
Page 4 of 9

Page 5

In a final model, including age and all the immunolo-
gical variables that showed evidence of reduced risk to
clinical malaria in the univariate analysis, only MSP1-
3D7 (IRR = 0.84 [95% CI, 0.73, 0.97, p = 0.02)] and
AMA1-3D7 (IRR = 0.8 [95% CI, 0.74, 0.96, p = 0.01])
were shown to be independently associated with
reduced risk of clinical malaria (Table 3).
Discussion
The study found the persistence of asymptomatic P. fal-
ciparum parasitaemia in about 60 - 80% of the study
population throughout the year. The incidence of clini-
cal malaria however varied in parallel with the intensity
of transmission and the onset of the rain as have been
shown in previous studies in the same region [23]. The
study area has been described as a malaria hyperen-
demic area with an entomological inoculation rate (EIR)
of 418 infective bites and the age group used in this
study was shown to have the highest prevalence of
asymptomatic parasitaemia in an earlier study [24]. This
could explain the persistence of asymptomatic parasitae-
mia year round.
The onset of rains increased vector population and
might have allowed the introduction of novel parasites
to which the children had no immunity from previous
infection and, therefore, had the potential to cause more
clinical disease [25]. Except for LSA-1 and MSP1-FVO,
levels of IgG to all the antigens increased with age as
shown in other studies [8,13]. Increasing IgG levels with
age may reflect greater cumulative exposure but could
also be due to older children having a more mature
immune system [26]. The reason for the lack of correla-
tion with age for anti-LSA-1 and MSP1-FVO antibodies,
respectively, is unclear. It may be likely that LSA-1 is
less immunogenic under the pattern of seasonal trans-
mission in the study area in contrast to data from a
study in a holoendemic region of Kenya, where infants
were shown to be capable of mounting and sustaining
strong anti-LSA-1 immune response in the first year of
life [27], suggesting that levels of LSA antibodies might
be more dependent on exposure rather than age. Inter-
estingly, a study in Kenya [28] did not find any signifi-
cant difference between antibody levels to LSA-1 in
children (≤8 years old) and adults (≥ 18 years old)
May-04(baseline)
June-04
July-04
August-04
September-04
October-04
November-04
December-04
January-05
February-05
March-05
April-05
May-05
Prevalence of asymptomatic parasitaemia (%)
0
20
40
60
80
100
Malaria cases
0
5
10
15
20
25
30
35
Parasite prevalence (%)
Malaria cases
Figure 1 Asymptomatic parasitaemia and clinical malaria episodes; The prevalence of asymptomatic parasitaemia for each month is
represented as a bar graph and the pattern of clinical malaria is shown as a line graph.
Dodoo et al. Malaria Journal 2011, 10:108
http://www.malariajournal.com/content/10/1/108
Page 5 of 9