Necator americanus and helminth co-infections: further down-modulation of hookworm-specific type 1 immune responses.

Abstract

Helminth co-infection in humans is common in tropical regions of the world where transmission of soil-transmitted helminths such as Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenale as well as other helminths such as Schistosoma mansoni often occur simultaneously.
We investigated whether co-infection with another helminth(s) altered the human immune response to crude antigen extracts from either different stages of N. americanus infection (infective third stage or adult) or different crude antigen extract preparations (adult somatic and adult excretory/secretory). Using these antigens, we compared the cellular and humoral immune responses of individuals mono-infected with hookworm (N. americanus) and individuals co-infected with hookworm and other helminth infections, namely co-infection with either A. lumbricoides, Schistosoma mansoni, or both. Immunological variables were compared between hookworm infection group (mono- versus co-infected) by bootstrap, and principal component analysis (PCA) was used as a data reduction method.
Contrary to several animal studies of helminth co-infection, we found that co-infected individuals had a further downmodulated Th1 cytokine response (e.g., reduced INF-γ), accompanied by a significant increase in the hookworm-specific humoral immune response (e.g. higher levels of IgE or IgG4 to crude antigen extracts) compared with mono- infected individuals. Neither of these changes was associated with a reduction of hookworm infection intensity in helminth co-infected individuals. From the standpoint of hookworm vaccine development, these results are relevant; i.e., the specific immune response to hookworm vaccine antigens might be altered by infection with another helminth.

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Necator americanus and Helminth Co-Infections: Further
Down-Modulation of Hookworm-Specific Type 1 Immune
Responses
Stefan Michael Geiger1,2*, Neal Douglas Edward Alexander2,3, Ricardo Toshio Fujiwara4, Simon
Brooker3, Bonnie Cundill3, David Joseph Diemert2, Rodrigo Correa-Oliveira1, Jeffrey Michael Bethony1,2
1Centro de Pesquisas Rene ´ Rachou, Fundac ¸a ˜o Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil, 2Department of Microbiology, Immunology and Tropical Medicine,
Medical Center, The George Washington University, Washington, D.C., United States of America, 3Department of Infectious and Tropical Diseases and Department for
Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London, United Kingdom, 4Departamento de Parasitologia, Instituto de Cie ˆncias
Biolo ´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Abstract
Background: Helminth co-infection in humans is common in tropical regions of the world where transmission of soil-
transmitted helminths such as Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and
Ancylostoma duodenale as well as other helminths such as Schistosoma mansoni often occur simultaneously.
Methodology: We investigated whether co-infection with another helminth(s) altered the human immune response to
crude antigen extracts from either different stages of N. americanus infection (infective third stage or adult) or different
crude antigen extract preparations (adult somatic and adult excretory/secretory). Using these antigens, we compared the
cellular and humoral immune responses of individuals mono-infected with hookworm (N. americanus) and individuals co-
infected with hookworm and other helminth infections, namely co-infection with either A. lumbricoides, Schistosoma
mansoni, or both. Immunological variables were compared between hookworm infection group (mono- versus co-infected)
by bootstrap, and principal component analysis (PCA) was used as a data reduction method.
Conclusions: Contrary to several animal studies of helminth co-infection, we found that co-infected individuals had a further
downmodulated Th1 cytokine response (e.g., reduced INF-c), accompanied by a significant increase in the hookworm-
specific humoral immune response (e.g. higher levels of IgE or IgG4 to crude antigen extracts) compared with mono-
infected individuals. Neither of these changes was associated with a reduction of hookworm infection intensity in helminth
co-infected individuals. From the standpoint of hookworm vaccine development, these results are relevant; i.e., the specific
immune response to hookworm vaccine antigens might be altered by infection with another helminth.
Citation: Geiger SM, Alexander NDE, Fujiwara RT, Brooker S, Cundill B, et al. (2011) Necator americanus and Helminth Co-Infections: Further Down-Modulation of
Hookworm-Specific Type 1 Immune Responses. PLoS Negl Trop Dis 5(9): e1280. doi:10.1371/journal.pntd.0001280
Editor: Maria Yazdanbakhsh, Leiden University Medical Center, Netherlands
Received March 17, 2010; Accepted July 8, 2011; Published September 6, 2011
Copyright: ? 2011 Geiger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors’ fieldwork receives financial support from the Oswaldo Cruz Foundation and from the Human Hookworm Vaccine Initiative (HHVI) of the
Sabin Vaccine Institute. SB is supported by a Research Career Development Fellowship from the Wellcome Trust (#081673). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: stefan@cpqrr.fiocruz.br
Introduction
Helminth co-infection in humans is common in tropical regions
[1,2], where transmission of Ascaris lumbricoides, Trichuris trichiura,
the hookworms (N. americanus or A. duodenale), and schistosomes
often occur concurrently [3,4]. Although co-infection is often the
rule rather than the exception in endemic areas, most previous
immuno-epidemiological studies of human helminth infection
have focused on the immune response to a single helminth species
(mono-infection) rather than the more common situation where an
individual is infected with one or more different helminth species
[5]. At our study site in Northeastern Minas Gerais State, Brazil,
where co-infection with schistosomes and soil-transmitted hel-
minths (STHs) is common [6], we have attempted to study the
epidemiologic, immunologic, and genetic determinants of infec-
tion in individuals resident in these co-endemic areas [7–11].
Much of the previous information on the immunology of
helminth co-infections has come from laboratory animal models,
especially experimental rodent models. The majority of these
studies show a competition between the co-infections, with one
infection usually leading to the rapid expulsion of the other [12–
16]. The immune mechanisms behind this effect are hypothesized
to include cross-reactive antibodies (also referred to as ‘‘cross-
protection’’) [13,16], a skewing towards Th2 cytokines (e.g.,
elevated IL-4), increased Th2-type antibody isotypes (e.g., elevated
production of IgG1) [15], and mucosal mast cell activation [13–
15]. However, conflicting animal studies report that co-infection
increases infection intensities by down modulating Th2 cytokine
responses, which in turn reduces intestinal inflammation, leading
to slower worm expulsion and increased worm burdens in co-
infected animals [17]. Possible explanations for these opposite
findings, among others, might be differences in animal models,
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different combinations of parasite infections, and the different
timing of co-infection (by timing of the primary versus the
secondary infection).
The few studies on the human immune response in co-infected
individuals are also contradictory. In one group of studies,
helminth co-infection appeared to result in a synergistic effect
among the infections, with infection with one helminth being
associated with an increased risk of having a high intensity
infection with another helminth [7]. However, other studies imply
a cross-protective effect derived from co-infection: for example,
individuals mono-infected with hookworm or A. lumbricoides
develop antibodies that cross-react with antigens from S. mansoni
[18–20]. In another set of studies, co-infection appeared to skew
the immune response away from the helminth infection under
study, e.g., the humoral and cellular immune responses to
hookworm or Ascaris antigens are diminished in individuals
resident in a schistosomiasis endemic area [21]. Along these same
lines, studies have also demonstrated an upregulation of the
immune response during helminth co-infection; e.g., increased
production of inflammation markers to S. mansoni infection in
children who are also infected with hookworms and/or Entamoeba
species [22]. However, given the contradictory nature of these
outcomes, the central question of whether multiple helminth
infections drive host immune responses towards phenotypes
different from those of a single infection still remains to be
answered [23].
In our previous epidemiological study in Brazil, we showed
synergistic effects among helminth co-infections in terms of egg
counts [7], leading us to expect a similar synergistic effect on immune
responses during helminth co-infection. In keeping with the results
from experimental animal studies [12–16], we further hypothesized
that hookworm co-infections with A. lumbricoides and/or S. mansoni
would significantly alter the immune responses to crude hookworm
antigen extracts, resulting in reduced Th2-type responses (IL-4, IL-5,
IL-13), a reduced inflammatory response (e.g., lower TNF-a
secretion), and an increase in the production of regulatory cytokines
(e.g., IL-10). To test this hypothesis, we compared the cellular and
humoral immune responses of individuals infected with hookworm
alone (mono-infected) and individuals infected with hookworm and
either A. lumbricoides, S. mansoni or both (co-infected).
Materials and Methods
Study site and selection of patients
The study was conducted in an area of the northeastern part of
the state of Minas Gerais in Brazil that is endemic for S. mansoni
and the STH as previously described [7]. The area of American-
inhas is divided into five rural sectors and a central municipality.
The Fundac ¸a ˜o National de Sau ´de (the National Health Founda-
tion) estimates the population to be approximately 1000 in the
urban municipal center and another 1000 in the surrounding rural
areas. Each house was assigned a unique household identification
number (HHID), and each resident, a unique personal identity
number (PID). Only individuals meeting the following inclusion
criteria were included into the study: (1) resident in the study area
over the last 24 months; (2) reporting not to have received
anthelmintic treatment within the last 24 months; and (3) willing
and able to give informed consent to study protocol. Individuals
were not included if they: (1) attended school outside the study
area; (2) worked full-time outside the study area; or (3) tested
positive on a pregnancy test. Females found to be pregnant during
the test were excluded from treatment during their pregnancy and
received treatment for all helminth infections later. For parasito-
logical exams, participants were instructed to deposit one fecal
sample per day into each container and return the container to
one of several collection points, where the sample was stored at
4uC. Fecal samples returned later than 48 h after date of
distribution were not accepted, and new containers were issued.
Presence of infection was determined by using the formalin-ether
sedimentation technique. Individuals positive for any helminth in
the formalin-ether sedimentation technique were asked to
contribute two more samples over the course of two more days
to be analyzed by Kato-Katz technique for assessment of eggs per
gram of feces (infection intensity). Two slides were taken from each
day’s fecal sample for a total of four slides from each individual.
Slides were examined within 45 minutes of slide preparation to
avoid drying of hookworm eggs. The arithmetic means of the four
slides was calculated and then converted to eggs per gram
according to the Kato-Katz method [24].
Out of 1,332 consented participants in the study, two-hundred
and fifty individuals were selected by simple random sampling for
immunological assays. Random sampling was performed on an
age, gender, and infection stratified sampling frame. In brief,
individuals with a negative fecal exam were removed from the
sampling frame; i.e., only persons with a positive fecal exam were
included. The sampling frame was then divided into 10 mutually
exclusive and exhaustive gender-based strata based using the
following age intervals: ,9, 10–19, 20–29, 30–39, and .40 years
of age. Simple random sampling was performed independently in
each stratum. Individuals who refused to enroll in this part of the
study or who were not eligible were replaced by simple random
sampling from the same stratum. The final stratified random
sample was compared to non-participants for age, gender, and
infection intensity, and no statistically significant differences
(p.0.05) were found in terms of those variables between those
individuals included in the survey and those not.
Individuals found to be infected with hookworm or other
intestinal nematodes were treated with albendazole (400 mg).
Participants with schistosomiasis were treated with praziquantel
(50 mg/kg) under the supervision of the project physician.
In the present study, cellular and humoral immune responses
from individuals with a hookworm mono-infection [9] were
included, as well as from individuals co-infected with (a)
hookworm and A. lumbricoides, (b) hookworm and S. mansoni, or
(c) hookworm, A. lumbricoides and S. mansoni. After parasitological
Author Summary
Parasitic infections in humans are common in tropical
regions and under bad housing and sanitation conditions
multiple parasitic infections are the rule rather than the
exception. For helminth infections, which are thought to
affect almost a quarter of the world’s population, most
common combinations include soil-transmitted helminths,
such as hookworm, roundworm, and whipworm, as well as
extra-intestinal infections by schistosomes. In order to
develop and test a hookworm vaccine in endemic areas,
the understanding of the impact of multiple helminth
infections (co-infection) on the immune response against
hookworm in infected individuals is crucial. The authors
report in their article, that several parameters of the
cellular (T cell markers, cytokines, chemokines) and
humoral immune response (e.g. IgG4 and IgE antibodies)
against hookworm are significantly affected or modulated
in individuals co-infected with hookworm, roundworm
and/or schistosomes. These results imply that the immune
response against components of a hookworm vaccine
might be altered by previous contact with other helminth
species in endemic areas.
Hookworm and Co-Infections
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exams and before anthelminthic treatment, approximately 20 mL
of blood was collected in heparinized tubes from children $6 years
of age and adults for separation of peripheral blood mononuclear
cells (PBMC) and 4 mL of blood in EDTA tubes for the
immunological assays described below. The study was approved
by the ethical review committees of The George Washington
University (GWU, USA), the London School of Hygiene and
Tropical Medicine (UK), the Centro de Pesquisas Rene ´ Rachou
FIOCRUZ and the Brazilian National Committee for Ethics in
Research (CONEP), and all subjects provided written informed
consent to participate in the study, or, in the case of minors,
written informed consent was given by their parents or guardians.
Phenotyping of lymphocytes ex vivo
Phenotyping of lymphocytes was performed as described
elsewhere [9] and the following pairs of monoclonal antibodies
(mAb), either conjugated with phycoerythrin (PE) or fluorescein
isothiocyanate (FITC) were used: CD4(FITC)/CD25(PE), CD4
(FITC)/HLA-DR(PE), CD4(FITC)/CD45RO(PE), CD4(FITC)/
CD45RA(PE), CD8(FITC)/CD28(PE),
(PE),CD8(FITC)/CD45RO(PE),
CD3(FITC)/CD69(PE), and CD19(FITC)/CD27(PE). Mouse
IgG1 antibodies conjugated with FITC or PE served as isotype
controls. Sample acquisition was done on a FACScan flow
cytometer (Becton Dickinson, USA) and results for 10,000 events
were analysed with BD Cell QuestTMsoftware (Becton Dickinson,
USA).
CD8(FITC)/HLA-DR
CD8(FITC)/CD45RA(PE),
Enzyme linked immunosorbant assays (ELISA) for
antigen-specific antibody classes and sub-classes in
serum samples
For the evaluation of humoral and cellular immune responses,
soluble somatic antigen extracts were prepared from third-stage
larvae (L3) and adult worms (AE) of Ancylostoma caninum.
Excretory/secretory (ES) antigens were obtained from cultured
A. caninum adult worms. The preparations were performed as
described elsewhere [9]. For the detection of parasite-specific IgE
antibodies, each of the hookworm antigens were diluted with
carbonate buffer (pH 9.6) to a concentration of 5 mg/ml. High-
binding ELISA plates (NUNC, Maxisorp, Fisher Scientific, USA)
were coated with 100 ml of the diluted antigens and incubated
overnight at 4uC. Plates were washed 5 times with washing buffer
(phosphate buffered saline [PBS]/0.05% Tween-20; pH 7.2–7.4)
and were then blocked for 1 hour at room temperature (RT) with
200 ml of blocking buffer (PBS/ 0.05% Tween-20/ 3% bovine
serum albumin). Individual serum samples were diluted 1:50 in
blocking buffer, 200 ml were added in duplicate to the respective
wells, and plates were incubated overnight at 4uC. On the
following day, plates were washed 10 times with washing buffer. A
1:1,000 dilution of anti-human IgE alkaline phosphatase-conju-
gated antibody (Pharmingen, USA) was prepared in PBS/0.05%
Tween-20 and 100 ml were added to the wells. After another
incubation of 90 minutes at RT, plates were washed 5 times and
then 100 ml of p-nitrophenyl phosphate substrate was added to
each well. Plates were incubated overnight at 4uC and the
following morning the color reaction was read at 405 nm using an
automated ELISA reader (SpectraMax 340 PC, Molecular
Devices, USA) using SOFTmax Pro 5.2 for Windows (Molecular
Devices) for data capture. Reference sera were assayed on each
plate as positive and negative controls.
For detection of parasite-specific IgG subclasses, horseradish
peroxidase-conjugated, anti-human IgG1, IgG3, and IgG4
(Zymed, USA) were used at a dilution of 1:1000, as described
above. As substrate, ortho-phenylene diamine was used and the
color reaction was stopped with H2SO4 after incubation for
30 min at RT in the dark. Plates were read at 490 nm.
Lymphocyte separation, proliferation assays and
cytokine/chemokine secretion in vitro
The separation of lymphocytes, their stimulation in vitro with
different hookworm antigens and with the mitogen phytohemag-
glutinin (PHA), lymphocyte proliferation, as well as the secretion of
several cytokines and chemokines after in vitro stimulation were
performed as described elsewhere in detail [9]. Here we report the
proliferation of lymphocytes after stimulation with the crude
soluble hookworm antigens L3, AE, and ES. For in vitro cytokine or
chemokine secretion, lymphocyte cultures were stimulated with
the same antigens and with PHA, as described for proliferation
assays, and the following analytes were measured: Interleukin (IL)-
2, IL-4, IL-5, IL-10, IL-13, CXCL10, TNF-a, and IFN-c.
Statistical analyses
The intensity of hookworm infection (as determined by fecal egg
counts) was compared between groups by non-parametric Kruskal-
Wallis test. Associations between Necator intensity of infection and
antibody level against crude antigen extracts or Necator infection
intensity and secreted cytokines/chemokines were analysed by
Spearman’s rank correlation. Analyses of these immune responses
weredoneseparatelyforthedifferentco-infectioncombinationsand
then compared with hookworm mono-infected individuals. As the
results among the different co-infection subgroups were found to be
generally similar (see below, in particular Table 1 and Figure 1), we
merged the various co-infections into a single group. For the
chemokine and cytokine variables, analysis was done on the log-
transformed variables, after replacing any zero values with 1.
Immunological variables were compared by bootstrapping the
geometric mean after adjusting for age by linear regression on the
log-values. For the lymphocyte populations, the untransformed
values were used and hence the arithmetic means were compared.
The immunological variables were summarized using principal
component analysis (PCA), via a projection-pursuit algorithm
Table 1. Demographic characteristics of groups mono- or
co-infected with hookworm.
Patient groupsHW HW+ +ASCHW+ +SM
HW+ +ASC
+ +SM
Number of individuals 25 53 5366
Males/females16 / 921 / 3233 / 20 33 / 33
Median age (range) 53 3642 31
(15–70)(6–76)(8–74) (7–83)
Median HW epg366 528666 909
(range)(3–20,376)(3–15,978)(3–25,698)(3–12,864)
Median ASC epg06,0120 2,403
(range)0 (3–12,024)0(3–12,024)
Median SM epg00 7299
(range)00 (3–1,122)(3–3,774)
Footnotes: Indicated are the total number of participants, numbers of males and
females, median age and median egg counts per gram feces (epg) in individuals
mono- and co-infected with hookworm. Abbreviations: HW: hookworm; ASC: A.
lumbricoides; SM: S. mansoni. Hookworm mono-infected (HW); patients
co-infected with A. lumbricoides (HW+ +ASC); co-infected with S. mansoni
(HW+ +SM); triple-infected patients (HW+ +ASC+ +SM).
doi:10.1371/journal.pntd.0001280.t001
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robust to departures of the data from normality [25,26]. We then
used biplots [27] to simultaneously show i) the contributions of each
of the original variables to the first two principal components (the
‘loadings’), and ii) each person’s value of the principal components
(the ‘scores’). The bivariate score means and their 95% confidence
ellipses [28] were calculated for the mono-infected and co-infected
groups. These means were compared between infection groups by
the multivariate Hotelling’s T2test [29]. PCA analysis was done for
lymphocyte sub-populations, for antibody responses, and for
chemokine and cytokine response to three hookworm antigen
preparations (AE, ES, and L3) and a mitogen (PHA) Pairs of
correlation coefficients by infection group were compared by first
transforming the variable to a standard normal deviate via the
Fisher Z transformation. No adjustment for multiple comparisons
was made in these analsyses. Analyses were performed using S-
PLUSversion6.2orlater(InsightfulCorp,SeattleWA,USA)andR
version 2.10 or later (R Foundation for Statistical Computing,
Vienna, Austria). The PCA analysis used the ‘pcaPP’ package in R.
Results
Of the 250 study participants who were randomly selected, 197
were infected with hookworm and were therefore included in the
immunological assessments. Table 1 shows the demographic
characteristicsofindividualseithermono-infectedwithN.americanus,
co-infected either with A. lumbricoides or S. mansoni, or infected with
all three helminth species. The median age in the co-infected groups
was lower than in the mono-infected group, but the hookworm
parasiteload,estimatedbythe numberofeggspergram offeces,did
not differ significantly between the four groups (Table 1 and
Figure 1). Figure 1 shows the median fecal egg counts for the
different groups, which covered a wide range of infection intensity.
Phenotyping of lymphocytes
We observed a statistically significant increase in CD4/HLA-
DR and CD8/HLA-DR positive T-cells in co-infected individuals
compared to mono-infected individuals. Other comparisons of
surface markers on T and B cells between mono- and co-infected
individuals were not significant (see Table 2). PCA was performed
on these immunological parameters jointly in order to obtain a
more complete and integrated picture of the immunological
pattern and compare the weight of each parameter’s contribution
to the immune response. The first principal component (PC 1) was
dominated by a contrast between CD4+/CD25+(positive loading)
and CD8+/CD282T cells (negative loading). PC 2 is effectively an
average of CD4/CD45RA and CD8/CD45RA positive memory
T cells (see Figure S1).
Antigen-specific antibodies in mono- and co-infected
patients
In participants either mono-infected or co-infected, we found
positive correlations between individual fecal egg counts and
serum IgG4 antibody levels against all the hookworm crude
Figure 1. Fecal egg counts in hookworm mono- and co-infected individuals. Footnotes: Circles represent individual values for eggs per gram
of feces (epg) and are shown on a logarithmic scale. Boxes indicate the median and the quartiles for each group and the whiskers indicate the 95%
ranges. Groups are split in hookworm mono-infected (HW), co-infected with A. lumbricoides (HW+ASC), co-infected with S. mansoni (HW+SM), and
triple-infected individuals (HW+ASC+SM). Kruskal-Wallis test on differences in hookworm egg counts between groups was not statistically significant
(p=0.523).
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antigen preparations tested: L3, AE and ES. Other isotypes, such
as IgG1, IgG3, and IgE, were not strongly correlated with egg
counts (see Table S1). For individuals with co-infections, the
correlations between fecal hookworm egg counts and hookworm-
specific IgG4 were significant for AE (rho=0.40; p,0.001), ES
(rho=0.21; p=0.007), and L3 (rho=0.26; p=0.001) antigen
preparations.
Optical density values for hookworm-specific serum antibodies
were measured and the age-adjusted ratio between mono- and
co-infected individuals are shown in Table 3, where we observed
significantly higher values for L3-specific IgG3, IgG4, and IgE,
AE-specific IgG1, IgG4, and IgE, and ES antigen specific IgG1
and IgG4 responses in co-infected individuals compared to mono-
infected individuals (Table 3).
Mean PC values for mono-infected and co-infected individuals,
plus their 95% confidence intervals (ellipses), showed distinct
segregation between these infection groups, with the mono-
infected individuals having lower values of PC 1, which was
Table 2. Cell surface markers on PBMC from hookworm mono- and co-infected individuals.
Difference
(adjusted for age)
95% confidence
interval
p-value#
Population (n missing)
Hookworm
mono-infected,
n=25,$
Co-infected,
n=189,$
CD19/CD27 (1)2.8 3.5
20.6(21.8–0.2) 0.17
CD3/CD69 (0) 2.63.4
20.7(21.8–0.5)0.22
CD4/CD25 (11)10.7 9.6 0.6(22.1–3.8) 0.66
CD4/CD45RA (16) 14.4 13.82.0(20.8–5.3) 0.16
CD4/CD45RO (0) 21.618.71.7(21.3–4.8) 0.26
CD4/HLA-DR (0) 1.42.3
21.0(21.5–20.5)
, ,0.001
CD8/CD28 (1) 9.4 10.4
20.4(21.9–0.9) 0.60
CD8/CD28neg(4) 21.016.92.6(21.9–7.4) 0.28
CD8/CD45RA (1)19.818.01.7(21.8–5.4)0.35
CD8/CD45RO (0)6.25.60.1(21.5–1.8)0.92
CD8/HLA-DR (22) 1.52.4
21.1 (21.8–20.3) 0.01
Footnotes:
#Statistically significant differences between groups are highlighted in bold numbers.
$Values indicate the arithmetic mean of the percentage of positive cells.
doi:10.1371/journal.pntd.0001280.t002
Table 3. Comparison of hookworm-specific antibody responses in sera from mono- and co-infected individuals.
Ratio (adjusted
for age)
95% confidence
interval
p-value#
Antigen
Antibody classes
and sub-classes
(n missing)
Hookworm
mono-infected,
n=25,$
Co-infected,
n=195,$
L3IgG1 (17) 0.400.54 0.79 (0.62–1.01) 0.06
IgG3 (17) 0.130.140.91(0.85–0.98)0.02
IgG4 (17)0.18 0.240.76(0.63–0.98)0.04
IgE (7) 0.190.290.65(0.54–0.78)
, ,0.001
AEIgG1 (16)0.140.20 0.75(0.61–0.93)0.01
IgG3 (16) 0.180.24 0.74(0.54–1.05)0.09
IgG4 (16)0.12 0.190.61(0.52–0.74) 0.002
IgE (7) 0.25 0.51 0.51(0.39–0.68)
, ,0.001
ES IgG1 (17)0.130.160.82(0.73–0.93) 0.002
IgG3 (17)0.140.160.86(0.69–1.13) 0.25
IgG4 (17) 0.100.110.87(0.79–0.97) 0.02
IgE (7)0.24 0.290.86(0.67–1.16) 0.30
Footnotes: Indicated are geometric mean optical density values, the age-adjusted ratio between mono-infected and co-infected individuals, the 95% confidence
intervals, and the calculated p-values for statistical differences.
#Statistically significant differences between groups are highlighted in bold numbers.
$Values indicate geometric mean values of optical densities.
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dominated by IgG3 against AE antigen, and IgE against AE and
ES antigens (Figure 2). PC 2 showed a contrast between i) IgG1
and IgG3 against AE antigen (positive loadings) and ii) IgE against
AE and ES antigens (negative loadings).
Lymphocyte proliferation and chemokine and cytokine
responses
Values for lymphocyte proliferation were indicated as stimula-
tion indices, i.e. proliferation of antigen- or mitogen-stimulated
cells divided by the proliferation of unstimulated control cultures.
Analysis of lymphocyte proliferation did not result in any
significant differences between mono- and co-infected groups
(data not shown). Non-parametric correlations between individual
PBMC secreted cytokine or chemokine levels and fecal hookworm
egg counts were strongly negative for IL-10 in mono-infected
participants and significantly different when compared with co-
infected individuals, whether stimulated with L3 or AE (p=0.032
for both comparisons), or ES antigen (p=0.003, Table 4).
Likewise, strong negative correlations were found for TNF-a in
control cultures from mono-infected individuals or when cells were
stimulated with ES, which were significantly different from the co-
infected group (p=0.002 and p=0.04, respectively, Table 4). In
individuals with co-infection, significant negative correlations
between egg counts and CXCL10 secretion were found in cell
cultures stimulated with L3 (p,0.05) or ES antigen (p,0.01),
however without any significant differences when compared with
mono-infected individuals.
Analysis of cytokine and chemokine production in PBMC after
stimulation with L3 antigen resulted in a significantly higher
production of CXCL10 in mono-infected individuals (Table 5).
Also, in PBMC stimulated either with AE or ES crude antigen
extracts, significantly higher concentrations of TNF-a or IFN-c
were observed in mono-infected individuals when compared with
the co-infected group (Tables 6 and 7). Examples of PCA for
Figure 2. Robust principal component analysis (PCA) of log-transformed serum antibody values in response to hookworm
antigens. Footnotes: The principal component scores for individuals mono- (N) and co-infected (m) with hookworm are shown. The respective mean
values are shown as open symbols, with 95% confidence ellipses (p value for bivariate T2 test is 0.006). The arrows show the strongest loadings, i.e.
contributions of the original variables to the principal components.
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antigen-specific cytokine and chemokine secretion are shown in
Figures S2, S3. For AE, as well as for ES antigen stimulation of
PBMC, the highest loadings for PC1 and PC2 with the same
directions were obtained for both Th1- and Th2-type cytokines or
chemokines.
Discussion
Thisisthefirststudytocomprehensivelyexamine the hookworm-
specific humoral and cellular immune response in individuals who
are co-infected with other helminths in an area of high hookworm
transmission. This is also the first study to examine the effect of co-
infection on the immune response to crude hookworm antigen
extracts from different stages of hookworm development (L3, AE,
ES). Moreover, these effects were analyzed in an epidemiologically
well-characterized group of individuals, where the spatial, genetic
and demographic aspects of hookworm infection and co-infection
have been intensively studied [7,8,10,11]. Apart from non-
parametric methods and comparisons of individual parameters,
we also utilized principal component analysis for comparison of the
immune responses to hookworm crude antigen extracts between
mono- and co-infected individuals, enabling us to examine, and
compare numerous mutually correlated immune variables in
relation to the effects of mono- or co-infection status [30].
Our analyses showed that chronic co-infection with nematode
and trematode species considerably alters the immune response to
hookworm crude antigen extracts. Most interestingly, co-infection
altered to a significant degree the antigen-induced secretion of
inflammatory TNF-a and led to a further diminution of
hookworm-specific IFN-c and CXCL10 secretion, but did not
alter production of IL-10 or the Type-2 cytokines, when compared
to mono-infected individuals. In contrast to our previous study [9],
we found that the immune response to hookworm infection was
increasingly modulated in co-infected individuals, an alteration
that did not lead to expulsion of one parasite species as shown in
experimental co-infections of mice with S. mansoni and Trichuris
muris [15].
These findings are extremely relevant for successful planning of
a hookworm vaccine currently under development [31]. In areas
endemic for hookworm, such as the one studied, co-infections with
other helminth species like A. lumbricoides and Schistosoma are
common. Our results show that Type 1 immune responses to
hookworm are significantly altered by such co-infections, which
might have implications for hookworm vaccine development, with
recent hookworm vaccines focused on inducing a Th1 response
[32] in order avoid problems with hookworm induced IgE.
The major emphasis of our immunological study was on T cells,
i.e., the proliferation of T cells, activation of T cell subpopulations,
Table 4. Correlations (Spearman’s rank test) between individual hookworm egg counts and antigen-induced cytokine/ chemokine
secretions.
Cytokine/chemokine Hookworm antigenMono-infected Co-infected
p-value for differences
between groups
IL-10 Control
20.43
20.060.142
(p-value)(0.075)(0.512)
L3
2 20.55*
20.040.032*
(p-value)(0.018) (0.690)
AE
2 20.49* 0.060.032*
(p-value)(0.042) (0.523)
ES (p-value)
2 20.73**
20.05 0.003**
(p-value)(0.001)(0.594)
CXCL10Control
20.300.00 0.222
(p-value)(0.200) (0.964)
L3
20.10
2 20.21*0.681
(p-value) (0.662)(0.011)
AE 0.21
20.030.337
(p-value)(0.371) (0.703)
ES0.00
2 20.26** 0.350
(p-value) (0.994)(0.003)
TNF-a
Control
2 20.66**
20.090.006**
(p-value)(0.002) (0.288)
L3
20.25
20.140.667
(p-value) (0.298)(0.090)
AE
20.23
20.040.446
(p-value)(0.338) (0.671)
ES
2 20.52*0.00 0.040*
(p-value)(0.033)(0.960)
Footnotes:
*correlation significant at the 0.05 level (2-tailed).
**correlation significant at the 0.01 level (2-tailed).
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and secretion of Th1- and Th2-type cytokines and chemokines.
Changes in CD4 and CD8 T cell counts, together with increased
activation of these T cell subpopulations, have already been
reported for helminth infections [33]. We add to this literature the
finding that percentages of activated CD4+and CD8+T cells
increased with co-infection. We speculate that multiply-infected
individuals have higher percentages of activated CD4+and CD8+
T cells due to ongoing higher antigenic stimulation of the immune
system by different helminth species and cross-reactive antigens.
This is supported by in vitro experiments on naı ¨ve human PBMC
stimulated with soluble egg antigen from S. mansoni (SEA), which
showed an increase in the CD4+/HLA-DR+cell population after
in vitro priming and a further increase during recall responses [34].
Even though mean fecal egg counts in mono-infected patients
were found to be in the range of those from co-infected
individuals, correlations between hookworm egg counts and
hookworm-specific IgG4 responses were stronger in co-infected
patients, which might be attributed to the presence of antibodies
that were cross-reactive with antigens from co-infecting helminth
species [21,35,36]. Chronic infections with multiple helminth
species might induce a stronger and ongoing antigenic stimulation
of the host’s immune system, which may lead to the expansion of
antigen-specific B cells and the secretion of specific IgG4
antibodies, especially in co-infected individuals with increased
hookworm infection. In support of this, a prior study with
volunteers co-infected with hookworm, S. mansoni, and A.
lumbricoides showed an increase in helminth antigen-specific total
IgG antibodies when compared with the respective mono-infected
groups [21]. In hookworm infections, the production of all
antigen-specific IgG subclasses rises with ongoing infection [35]
Table 5. L3 antigen-induced cytokine and chemokine secretion in lymphocyte cultures from individuals mono- or co-infected with
hookworm.
Mean value (pg/ml)Mean value, (pg/ml)
Ratio
(adjusted for age)
95% confidence
interval
p-value#
Cyto- or chemo-kine
(n missing, n below
detection threshold)
Hookworm
mono-infected,
n=23,$
Co-infected,
n=186,$
IL-2 (16, 46)9.010.70.85 (0.43–1.62)0.65
IL-4 (16, 44) 6.98.30.91(0.46–1.71)0.77
IL-5 (46, 22)31.141.20.85(0.30–2.17)0.74
IL-10 (46, 3)363 2401.56(0.91–2.59) 0.11
IL-13 (1, 19) 13698 1.47(0.79–2.32) 0.21
CXCL10 (16, 18)11048 2.29(1.24–4.30) 0.01
TNF-a (16, 10) 56.637.71.61 (0.88–2.91)0.12
IFN-c (47, 19)2141391.56(0.44–4.7)0.46
Footnotes:
$Indicated are geometric mean concentrations (pg/ml) for both groups, together with the age-adjusted ratios between groups and the 95% confidence intervals for L3
antigen preparation.
#Statistically significant differences between groups are highlighted in bold numbers.
doi:10.1371/journal.pntd.0001280.t005
Table 6. Adult worm antigen-induced cytokine and chemokine secretion in lymphocyte cultures from individuals mono- or
co-infected with hookworm.
Mean value, (pg/ml)Mean value, (pg/ml)
Ratio
(adjusted for age)
95% confidence
interval
p-value#
Cyto- or chemo-kine
(n missing, n below
detection threshold)
Hookworm
mono-infected,
n=22,$
Co-infected, n=186,$
IL-2 (15, 42)12.5 12.71.03 (0.51–1.87)0.94
IL-4 (15, 43) 7.1 8.0 0.92(0.47–1.70) 0.78
IL-5 (47, 34)29.9 25.81.33(0.50–3.22)0.56
IL-10 (47, 15)65.460.41.24 (0.39–3.39)0.70
IL-13 (0, 22)65.363.71.12(0.41–2.60) 0.80
CXCL10 (15, 52)27.717.31.61 (0.58–4.31)0.35
TNF-a (15, 19) 46.421.0 2.20(1.38–3.74)
, ,0.001
IFN-c (47, 44)146.3 32.3 4.88(1.33–15.9)0.02
Footnotes:
$Indicated are geometric mean concentrations (pg/ml) for both groups, together with the age-adjusted ratios between groups and the 95% confidence intervals for
adult antigen preparation (AE).
#Statistically significant differences between groups are highlighted in bold numbers.
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and hookworm-specific IgG4 has been proposed as a good marker
for patent and chronic infections [35–37].
Analysis of cytokine and chemokine secretion patterns from
mono-infected volunteers revealed no clear polarization into Th1
or Th2 type immune responses, but rather a mixed pattern [9].
Similar results were recently obtained for individuals co-infected
with A. lumbricoides and T. trichiura [38]. However, in the co-
infected group, we found a decreased TNF-a secretion, together
with a further down-modulation of hookworm-specific IFN-c
production. Another study on co-infection detected elevated levels
of pro-inflammatory cytokines and chemokines in co-infected
children in response to S. mansoni adult worm antigen, whereas
IFN-c and IL-13 secretion patterns revealed no significant
differences between individuals mono- and poly-infected with
schistosomes, hookworm and Entamoeba species [22]. As opposed
to A. lumbricoides and Trichuris trichiura co-infections [38], we were
neither able to detect a positive relationship between hookworm
antigen-induced IL-10 secretion and intestinal worminess, nor to
detect negative associations between IL-10 and Th1/Th2-type
cytokines. These described differences might be due to the
presence of different parasite species and also due to a mixture
of intestinal and extra-intestinal parasites.
Considerable antigen-induced IL-10 secretion has been de-
scribed in individuals with hookworm infection [9,39]. In the
current study, IL-10 levels correlated inversely with fecal egg
counts in mono-infected hookworm patients especially in response
to ES. This strong negative correlation was ablated in co-infected
individuals, most probably because A. lumbricoides and S. mansoni
infections induce production of IL-10 themselves [39]. Even
though there was an unexpected negative correlation between
parasite load and IL-10 secretion of lymphocytes, the antigen-
induced IL-10 secretion was significantly associated with mono-
infected individuals, indicating its importance in immune
regulation during hookworm infection.
This study has some important limitations. First, the cross-
sectional study design, in which groups are compared from a single
time point, does not allow causal inferences to be made. In
addition, the small sample size may have limited our ability to
detect small statistical differences between groups. Nor does the
sample size allow for further stratification of the groups in order to
explore other factors which may account for these differences. Age
is likely to be among the most important of such confounding
factors but was included as a covariate when testing for differences
between groups. One positive aspect of the study design was the
population-based sampling which should enhance the generaliz-
ability of the study.
In summary, individuals co-infected with other helminth species
presented with a significantly different immune response when
compared with mono-infected participants. These changes
included a stronger activation of CD4+and CD8+T cells, lower
secretion of Type 1 cytokines, and increased levels of IgG4 and
IgE antibodies against somatic hookworm antigens (L3 and AE).
Furthermore, positive correlations between egg counts and
hookworm-specific IgG4 responses, as well as missing correlations
between egg counts and regulatory (IL-10) and inflammatory
(TNF-a) cytokines in co-infected individuals. This modulation of
hookworm-specific cellular and humoral immune responses by co-
infection with other helminth species will be an important
consideration during clinical trials for hookworm vaccine testing.
Although vaccination is obviously not the same as natural
infection, the immunogenicity of hookworm antigens in a vaccine
might be altered and adversely affected by infections with parasites
such as S. mansoni and A. lumbricoides.
Supporting Information
Figure S1
of ex vivo lymphocyte cell surface markers in PBMCs.
Footnotes: The principal component scores for individuals mono-
(N) and co-infected (m) with hookworm are shown. The respective
mean values are shown as open symbols, with 95% confidence
ellipses (p value for bivariate T2 test is 0.23). The arrows show the
strongest loadings, i.e. contributions of the original variables to the
principal components.
(TIF)
Robust principal component analysis (PCA)
Figure S2
of log-transformed cytokine and chemokine secretion in
PBMCs stimulated with AE antigen. Footnotes: The principal
Robust principal component analysis (PCA)
Table 7. ES antigen-induced cytokine and chemokine secretion in lymphocyte cultures from individuals mono- or co-infected with
hookworm.
Mean value, (pg/ml)Mean value, (pg/ml)
Ratio
(adjusted for age)
95% confidence
interval
p-value#
Cyto- or chemo-kine
(n missing, n below
detection threshold)
Hookworm
mono-infected,
n=16,$
Co-infected,
n=167,$
IL-2 (15, 43) 8.69.8 0.85(0.37–1.80) 0.68
IL-4 (15, 40)5.77.3 0.79(0.38–1.66) 0.55
IL-5 (42, 50) 4.68.2 0.56(0.21–1.88) 0.33
IL-10 (42, 13)175802.34(0.72–5.97) 0.14
IL-13 (0, 37) 26.7 28.7 1.01(0.29–3.20)0.98
CXCL10 (15, 78)3.1 4.50.68 (0.28–1.99)0.44
TNF-a (15, 8) 73.645.31.66(1.05–2.76)0.03
IFN-c (42, 39)69.6 26.7 2.64(1.28–5.37)0.01
Footnotes:
$Indicated are geometric mean concentrations (pg/ml) for both groups, together with the age-adjusted ratios between groups and the 95% confidence intervals for ES
antigen preparation.
#Statistically significant differences between groups are highlighted in bold numbers.
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component scores for individuals mono- (N) and co-infected (m)
with hookworm are shown. The respective mean values are shown
as open symbols, with 95% confidence ellipses (p value for
bivariate T2 test is 0.13). The arrows show the strongest loadings,
i.e. contributions of the original variables to the principal
components.
(TIF)
Figure S3
of log-transformed cytokine and chemokine secretion in
PBMCs stimulated with ES antigen. Footnotes: The principal
component scores for individuals mono- (N) and co-infected (m)
with hookworm are shown. The respective mean values are shown
as open symbols, with 95% confidence ellipses (p value for
bivariate T2 test is 0.08). The arrows show the strongest loadings,
i.e. contributions of the original variables to the principal
components.
(TIF)
Robust principal component analysis (PCA)
Table S1
tibody responses and hookworm egg counts in mono-
Correlations between hookworm-specific an-
and co-infected individuals. Footnotes:
correlation coefficients and calculated p-values for statistical
differences.#Statistically significant correlations in each group
are highlighted in bold numbers.
(DOC)
$
Indicated are
Acknowledgments
We would like to address special thanks to the people in Americaninhas for
their participation in the study and to Renata Diniz and their field team for
their dedication during the epidemiological studies.
Author Contributions
Conceived and designed the experiments: SMG RTF DJD JMB.
Performed the experiments: SMG RTF. Analyzed the data: SMG NDEA
RTF BC JMB. Contributed reagents/materials/analysis tools: DJD RCO
JMB. Wrote the paper: SMG NDEA RTF JMB. Analysis and
interpretation of data: SMG NDEA RTF SB BC DJD RCO JMB.
Drafted the manuscript: SMG RTF NDEA JMB. Revised the manuscript:
SMG NDEA RTF SB BC DJD RCO JMB.
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